A New Peptide Isolated from a Marine Derived Streptomyces bacillaris

Abstract

A new peptide, l-O-Lac-l-Val-d-O-Hiv-d-Val (1), consisting of d-valine, l-valine, l-lactic acid, and 3-d-hydroxyisovaleric acid, was isolated from the culture of the marine sediment derived Streptomyces bacillaris. The planar structure of compound 1 was assigned by 1D, 2D NMR and mass spectroscopic analyses. Following acid and base hydrolysis, the absolute configuration of the valine residues in 1 were determined by application of the advanced Marfey’s method and the absolute configurations of hydroxy acids units were determined by a HPLC method based on Mosher’s reagents.

Marine derived actinomycetes have proven to be a valuable resource for drug discovery.¹ A large number of natural products with biological and chemical diversity, such as, salinosporamide A², marinomycins³, cyclomarins⁴ and lomaiviticins⁵ have been found from marine bacteria. As part of our efforts to identify anti-cancer natural products from marine derived actinomycetes, we screened a library of 1500 natural products fractions for their ability to inhibit autophagy. From this screen we obtained a series of fractions that inhibit autophagy from marine derived bacterial strain, SNB-019, which was determined to be Streptomyces bacillaris. Strain SNB-019 was obtained from a sediment collected shallow waters from Galveston Bay, Texas. From these fractions, we obtained a number of bafilomycin analogs⁶ that were responsible for the autophagy inhibitory activity. In addition to the bafilomycin analogs, we identified a small peptide in the active fraction. Herein, we report the isolation and structural elucidation of a new peptide, l-O-Lac-l-Val-d-O-Hiv-d-Val (1).

Compound 1 was isolated as yellow solid. The molecular ions identified in negative ESI-MS at m/z 387 [M – H]⁻ and positive ESI-MS at m/z 389 [M + H]⁺ allowed the deduction of its molecular weight of 388 Da. High-resolution ESI-MS gave an [M + H]⁺ at m/z 389.2288 consistent with a molecular formula of C₁₈H₃₂N₂O₇ (calcd for C₁₈H₃₃N₂O₇, 389.2282) and 4 degrees of unsaturation. The ¹H NMR (600 MHz, in CD₃OD, Table 1) spectrum of 1 exhibited signals for three series of iso-propyl groups at δ 0.92 (d, J = 6.8 Hz, H-4), 0.95 (d, J = 6.8 Hz, H-5), 0.968 (d, J = 6.8 Hz, H-14), 0.973 (d, J = 6.8 Hz, H-9), 0.98 (d, J = 6.8 Hz, H-15), 0.99 (d, J = 6.8 Hz, H-10), 2.16 (m, H-3), 2.22 (m, H-8), and 2.28 (m, H-13), one CH₃-CH group at δ 1.36 (d, J = 6.8 Hz, H-18) and 4.19 (q, J = 6.8 Hz, H-17) and three additional of N- or O- bearing CH groups at 4.18 (d, J = 4.7 Hz), 4.52 (d, J = 6.8 Hz), and 4.94 (d, J = 5.3 Hz). The ¹³C NMR spectrum of 1 (150 MHz, CD₃OD, Table 1) showed 18 carbons signals, including four ester or amide carbonyl (δ 169.2, 170.3, 176.2, 176.4), four N- or O- bearing carbons (δ 57.0, 59.8, 67.7, and 78.9), and seven methyl groups (δ 16.4, 16.9, 17.0, 17.8, 18.3, 18.8, and 19.9). The above NMR data were highly indicative of a peptide-like compound containing four amino acids residues. A combination of HSQC and ¹H-¹H COSY correlations (Figure 2 and Table 1) from H-2/H-3/H-4, H-3/H-5, H-7/H-8/H-9, H-8/H-10, H-12/H-13/H-14, H-13/H-15, and H-17/H-18 suggested that 1 contained two valines (Val), one hydroxyl-isovaleric acid (Hiv) and one lactic acid (Lac). The presence of two valines was further supported by running ¹H NMR in DMSO-d₆ (600 MHz, Table 1), which showed two NH signals at δ 7.71 (d, J = 9.5 Hz) and 7.15 (d, J = 7.4 Hz). Moreover, the two NH signals showed correlations with protons at δ 4.37 (dd, J = 7.4, 9.5 Hz, H-12) and 3.66 (dd, J = 7.4, 3.8 Hz, H-2), respectively.

Table 1.

1D and 2D NMR data of 1 in CD₃OD and DMSO-d₆ (600 MHz)

no.	δH (J in Hz)a	δCa	COSYa	HMBCa	δH (J in Hz)b	δCb	COSYb	HMBCb
1		176.2 C				172.2 C
2	4.18, d (4.8)	59.8 CH	3	1, 3, 6	3.66, dd (7.4, 3.9)	59.5 CH	3, NH-2
3	2.16, m (6.8, 4.8)	31.5 CH	2, 4,5	2	2.00, m (6.8, 3.9)	31.5 CH	2, 4, 5
4	0.92, d (6.8)	17.0 CH3	3	2, 3	0.76, d (6.8)	18.6 CH3	3	2, 3
5	0.95, d (6.8)	18.8 CH3	3	2, 3	0.78, d (6.8)	20.2 CH3	3	2
6		169.2 C				167.3 C
7	4.94, m (5.3)	78.9 CH	8	6, 8, 11	4.80, m (4.1)	78.7 CH	8	6, 8
8	2.22, m (6.8, 5.3)	30.5 CH	7, 9, 10	7	2.10, m (6.8, 4.1)	30.4 CH	7, 9, 10
9	0.97, d (6.8)c	16.4 CH3	8	7, 8	0.88, d (6.8)	17.2 CH3	8	8, 10
10	0.99, d ( 6.8)	17.8 CH3	8	7	0.90, d (6.8)	19.1 CH3	8	7, 8
11		170.3 C				170.9 C
12	4.52, d (6.8)	57.0 CH	13	11, 13, 16	4.37, dd (7.4, 9.5)	56.5 CH	13, NH-12	11, 13
13	2.28, m (6.8)	30.5 CH	12, 14, 15	12	2.10, m (6.8, 7.4)	30.0 CH	12, 14, 15	12
14	0.97, d (6.8)c	16.9 CH3	13	12, 13	0.84, d (6.8)	18.1 CH3	13	6, 12, 13
15	0.98, d (6.8)	18.3 CH3	13	13	0.88, d (6.8)	19.6 CH3	13	12
16		176.4 C				175.8 C
17	4.19, q (6.8)	67.7 CH	18		4.00, t (6.9)	67.9 CH	18	18
18	1.36, d (6.8)	19.9 CH3	17	16, 17	1.21, d (6.8)	21.1 CH3	17	16, 17
NH-12					7.71, d (9.5)		12	12
NH-2					7.15, d (7.4)		2

^(a)in CD₃OD;

^(b)in DMSO-d₆;

^(c)methyl groups can be distinguished by ¹H chemical shift.

Figure 2.

Key COSY and HMBC correlations for 1 in CD₃OD.

Sequencing the amino acid residues in 1 was accomplished by an HMBC experiment. HMBC correlations (in CD₃OD Figures 2) from H-18 (δ 1.36) to C-16 (δ 176.4) and C-17 (δ 67.7); from H-12 (δ 4.52) to C-16 and C-11 (δ 170.3); from H-7 (δ 4.94) to C-11 and C-6 (δ 169.2), and from H- 2 (δ 4.18) to C-6 and C-1 (δ 176.2) established the connection of the four amino acid units. Herein, the planar structure of compound 1 was assigned as O-Lac-Val-O-Hiv-Val.

The absolute configurations of the two valine (Val) units in 1 were determined by application of a modified advanced Marfey’s method ⁷ on the basis of both alkali hydrolysis and acid hydrolysis. Complete acid hydrolysis of 1 with 6 N HCl, followed by treatment with Marfey’s reagent (N-α-(2,4-dinitro-5-fluorophenyl)-l-alaninamide) and analysis by LC-MS indicated the presence of both l-Val and d-Val, based on comparison to authentic standards. To differentiate these two Val moieties, alkali hydrolysis of 1 was applied, which yielded two dipeptides 1a and 1b (Figure 3), which separated the two valine residues onto different compounds. After purification by HPLC, dipeptides 1a and 1b were then hydrolyzed with 6 N HCl and subjected to the advanced Marfey’s method, in separate experiment, revealing the absolute configuration of Val was L in 1a, and D in 1b. On this basis, the absolute configuration of the terminal valine unit in 1 was determined to be D and the valine residue flanked by lactic acid and hydroxyisovaleric acid was determined to be l.

Figure 3.

Principle (a) and procedure (b) for determining the absolute configuration of compound 1.

The absolute configurations of Lac and Hiv units in 1 were determined by a modified method based on Mosher’s reagents.⁸ Treatment of the hydrolysate of 1 with R-(–)-α-methoxy-α-(trifluoro-methyl) phenylacetyl chloride (R-MTPA-Cl) yield S-MTPA esters that were analyzed by LC-MS under negative-mode electrospray ionization. On the base of comparison of the retention times, molecular weights, and UV spectra with those of the appropriate S-MTPA esters of d- and l- hydroxyl amino acid standards, the determination of absolute configuration for the Lac and Hiv moieties in 1 was accomplished. The retention times (t_R in min) and ESIMS product ions of the S-MTPA esters of standard hydroxyl amino acids were observed to be 28.17 (l-Lac), 27.11 (d-Lac), 37.19 (l-Hiv), and 35.67 min (d-Hiv). The retention times of the MTPA-Cl derivatized hydrolysate of 1 were 28.17 and 35.67 min, indicating the presence of l-Lac and d-Hiv in 1. As a result, the absolute configuration of compound 1 could be assigned as 2R, 7R, 12S, 17S.

Although compound 1 was isolated from the active fraction, when tested as pure compounds, it showed no activity as an inhibitor of autophagy up to 20µM concentration. Further biological evaluation of the compound is on-going.

Experimental

General

Optical rotations were recorded with an AUTOPOL AP IV-6W polarimeter equipped with a halogen lamp (589 nm). UV spectra were recorded on a Shimadzu UV-1601 UV–VIS spectrophotometer. ¹H and 2D NMR spectral data were recorded at 600 MHz in CD₃OD or DMSO-d₆ solution on Varian System spectrometer, and chemical shifts were referenced to the corresponding residual solvent signal. ¹³C NMR spectra were acquired at 150 MHz on a Varian System spectrometer. High resolution ESI-TOF mass spectra were provided by The Scripps Research Institute, La Jolla, CA. Low-resolution LC/ESI-MS data were measured using an Agilent 1200 series LC/MS system with a reversed- phase C18 column (Phenomenex Luna, 150 mm × 4.6 mm, 5 µm) at a flow rate of 0.7 mL/min. Preparative HPLC was performed on an Agilent 1200 series instrument with a DAD detector, using a C18 column (Phenomenex Luna, 250 ×10.0 mm, 5 µm). Sephadex LH-20 (GE Healthcare, Sweden) and ODS (50 µm, Merck) were used for column chromatography.

Collection and phylogenetic analysis of strain SNB-019

The marine-derived bacterium, strain SNB-019, was isolated from a sediment sample collected from the brackish waters of Galveston Bay, Galveston Texas (N 29° 42.419’, W 94° 49165’ W). The strain was isolated on A1 agar media (10 g starch, 4 g yeast extract, 2 g peptone, 1 L sH₂O, 10 g agar) Genomic DNA of strain SNB-019 was isolated using standard methods and was amplified using PCR with the Universal 16S rRNA primers FC27 and RC 1492 using the method of Mincer.⁹ The partial 16S rRNA sequence (1462 of 1492 bp) was compared to sequences in available databases using the Basic Local Alignment Search Tool and strain SNB-019 determined to be 99% identical to Streptomyces bacillaris.

Cultivation and Extraction

The bacterium (strain SNB-019) was cultured in 20 2.8 L Fernbach flasks each containing 1 L of a seawater based medium (10 g starch, 4 g yeast extract, 2 g peptone, 1 g CaCO₃, 40 mg Fe₂(SO₄)₃·4H₂O, 100 mg KBr) and shaken at 200 rpm at 27 °C. After seven days of cultivation, sterilized XAD-7-HP resin (20 g/L) was added to adsorb the organic products, and the culture and resin were shaken at 200 rpm for 2 h. The resin was filtered through cheesecloth, washed with deionized water, and eluted with acetone. The acetone soluble fraction was dried in vacuo to yield 3.0 g of extract.

Isolation

The extract (3.0 g) was fractionated by open column chromatography on ODS (50 µm, 80g), eluting with a step gradient of MeOH and H₂O (10:90 –100:0), and 16 fractions (Fr.1~ Fr.16) were collected. Fraction Fr.6 (73 mg) was purified by Sephadex LH-20 (25 g, 1.5 × 65 cm, eluted with MeOH) and HPLC (Phenomenex Luna, Phenyl-Hexyl, 250 × 10.0 mm, 2.5 mL/min, 5 µm, UV = 210 nm) using a gradient solvent system from 20% to 50% CH₃CN (0.1% formic acid) over 30 min to give 1 (6.0 mg).

l-O-Lac-l-Val-d-O-Hiv-d-Val (1)

yellow solid

[α]²⁰ – 6 (c 0.1 MeOH)

UV λ_max(MeCN) nm (log ε): 200 (3.8).

¹H NMR and ¹³C NMR see Table 1.

ESI-MS [M – H]⁻ m/z 387.2.

HRESIMS [M + H]⁺ m/z 389.2288 (C₁₈H₃₃N₂O₇, calcd 389.2282) and [M + Na]⁺ 411.2122(C₁₈H₃₂N₂O₇Na, calcd 411.2102).

Alkali hydrolysis for compound 1

Compound 1 (2.0 mg) was hydrolysed with 1.2 N KOH (1.25 mL) at 60 °C for 1.5 h. The hydrolysate was neutralized with 6N HCl (250 µL) and diluted with 2.0 mL H₂O. The resulted mixture was subjected to a C₁₈ SEP-PAK (0.5 × 1.0 cm Waters) and eluted with 4 mL H₂O, followed by 4 mL 80% MeOH/H₂O. The 80% MeOH/H₂O elution was dried and then purified by semi-preparative HPLC (Phenomenex Luna C₁₈, 150 × 4.6 mm, 5µm) with a gradient solvent system (aqueous CH₃CN containing 0.1% formic acid, 10-50% for 30 mins) at 2.5 mL/min flow rate and UV detection of 210 nm. Dipeptides 1a (0.6 mg) and 1b (0.7 mg) were eluted at 14.3 and 22.8 min, respectively.

l-O-Lac-l-Val (1a)

¹H NMR (δ, 600 MHz, CD₃OD) 4.34 (d, J = 5.1 Hz, 1H), 4.15 (q, J = 6.8 Hz, 1H), 2.21 (m, 1H), 1.36 (d, J = 6.8Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H).

ESI-MS: m/z 188.1 [M – H]⁻.

d-O-Hiv-d-Val (1b)

¹H NMR (δ, 600 MHz, CD₃OD) 4.37 (d, J = 4.8 Hz, 1H), 3.89 (d, J = 3.5 Hz, 1H), 2.22 (m, 1H), 2.11 (m, 1H), 1.02 (d, J = 6.9 Hz, 3H), 0.98 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.9 Hz, 3H), 0.87 (d, J = 6.9 Hz, 3H).

ESI-MS: m/z 216.2 [M – H]⁻.

Acid hydrolysis and Advanced Marfey Analysis

The resulted dipeptides 1a and 1b were subjected respectively to acid hydrolysis at 110 °C for 16 h with 6 N HCl (360 µL), and then the hydrolysates were dried under a steam of N₂ gas and redissolved in H₂O (200 µL). To one portion (100 µL) was added 20 µL 1M NaHCO₃ and 100 µL of a 1% (v/v) N-α-(2,4-Dinitro-5-fluorophenyl)-l-alaninamide (FDAA), after which the mixtures were heated at 80 °C for 4 min. The reaction mixtures were cooled, neutralized with 1N HCl (20 µL) and diluted with 300 µL CH₃CN. About 150 µL of each solution of FDAA derivatives was analyzed by LC-MS using a C₁₈ column (Luna, 4.6 × 150 mm, 5µ). Aqueous CH₃CN containing 0.1% formic acid was used as mobile phase with linear gradient elution (30-70% for 40 min) at a flow rate of 0.7 mL/min. FDAA-derivatived amino acids were detected by absorption at 340 nm. An Agilent 1200 series MSD mass spectrometer was used for detection in API-ES (negative, mass range 150- 1400 Da) mode. The retention times (min) of the FDAA-derivatized standards were 13.57 min for l-Val) and 17.10 min for d-Val. The retention times of the FDAA-derivatized hydrolysate of 1a and 1b were 13.57 min (l-val) and 17.10 min (d-val), respectively.

Absolute configuration of Lac and Hiv moieties in 1

To determine the absolute configuration of Lac and Hiv residues in 1, a modified method based on Mosher’s reagents and LC-MS analysis was applied. To the hydrolysate of 1, which was resulted from acid hydrolysis at 110 °C for 16 h with 6 N HCl, dry pyridine (0.5 mL) and α-methoxy-α-trifluoromethylphenylacetic acid (R-MTPA-Cl) (2 µL) were added. The reaction was carried out for 10h at rt, and the solvent was evaporated under N₂. In a similar manner, each isomer of Hiv and Lac (1.0 mg each) was derivatized with R-MTPA-Cl (2 µL each in 0.5 mL pyridine). All derivatives samples were analyzed by LC-MS using a C₁₈ column (Luna, 4.6 × 150 mm, 5µ). Aqueous CH₃CN containing 0.1% formic acid was used as mobile phase with linear gradient elution (30-70% for 50 min) at a flow rate of 0.7 mL/min. An Agilent 1200 series MSD mass spectrometer was used for detection in API-ES (negative, mass range 150-1400 Da) mode. The retention times (t_R in min) and ESIMS product ions (m/z [M – H]⁻) of the R-MTPA-Cl monoderivatized standard hydroxyl amino acids were observed to be l-Lac (28.17 min, 305.1 [M – H]⁻), d-Lac (27.11 min, 305.1 [M – H]⁻), l-Hiv (37.19 min, 333.1 [M – H]⁻), and d-Hiv (35.67 min, 333.1 [M – H]⁻). The retention times of the R-MTPA-Cl derivatized hydrolysate of 1 were l-Lac (28.17 min) and d-Hiv (35.67 min).

Figure 1.

Structure of compound 1