Synthesis and Cytotoxicity of Cyclic Octapeptide Surugamides with Varied N-Acyl Moieties

Abstract

Surugamides are a group of non-ribosomal peptides produced by Streptomyces spp. Several derivatives possess acyl groups, which are proposed to be attached to a lysine side chain after backbone-macrocyclization during biosynthesis. To date, five different acyl groups have been identified in nature, yet their impacts on biological activity remain underexplored. Here we synthesized surugamide B derivatives with varied acyl moieties. Biological evaluations revealed that larger hydrophobic acyl groups on lysine ε-NH₂ enhance cytotoxicity.

Introduction

Surugamides are growing group of non-ribosomal cyclic peptides originally isolated from marine-derived Streptomyces sp. JAMM992,¹⁾ and later identified from several related and taxonomically unique species.^2–17) Surugamides consist of two groups: the cyclic octapeptides (1–21) represented by surugamide A (1)¹⁾ (Fig. 1a) and the decapeptides (i.e., cyclosurugamide F (22)¹⁸⁾ and its linear derivatives 23–25^3,19)) (Fig. 1b). The octapeptidyl group is linked to moderate, yet diverse biological activities, including inhibition of cathepsin B,^1,3) antibacterial activity against Staphylococcus aureus,²⁾ antifungal activity against Saccharomyces cerevisiae,³⁾ cyclin-dependent kinase (CDK)-modulating activity¹²⁾ and anthelmintic activity against Dirofilaria immitis.²⁰⁾ In contrast, the decapeptidyl group has not yet been associated with any biological activity. Although structurally unrelated, the two groups of surugamides are closely related in terms of biosynthesis, as the non-ribosomal peptide synthetases (NRPSs) for both are encoded in a single biosynthetic gene cluster.¹⁹⁾ After the elongation of linear intermediates on two assembly lines, the single, stand-alone peptide cyclase SurE acts in trans with both assembly lines to release the two types of surugamides.^5,18)

Fig. 1. Naturally Occurring and Synthetic Surugamides

(a) Structures of naturally occurring octapeptidyl surugamides. Note: According to the structure revision of 1, where the originally assigned D-Ile at p4 was revised to D-allo-Ile,²¹⁾ D-Ile* in the original structures^1,3) should also require revisions to D-allo-Ile. (b) Structures of decapeptidyl surugamides. Note: According to the structure revision of 23, where the originally assigned (S)-AMPA at p5 was revised to (R)-AMPA,²²⁾ (S)-AMPA* in the original structures^3,19) should also require revisions to (R)-AMPA. (c) Structures of the synthetic acyl-surugamides 26–28 in the previous study²⁰⁾ and the new derivatives 29–32 in this study.

To date, various derivatives of octapeptidyl surugamides have been identified (Fig. 1a). The structural variation can be generated in two ways. One is varying the aliphatic residues, where one or two aliphatic residues (i.e., L-Ile, D-allo-Ile and D-Leu) are substituted with L/D-Val, as in the cases of surugamides B–E (2–5)¹⁾ and G–J (6–9)³⁾ (Fig. 1a). This type of variation likely arose from the promiscuity of adenylation domains, which are responsible for specific adenylation and thioesterification of amino acid building blocks for assembly. The other source of structural variation is modification of the lysine ε-NH₂: methylation and acylation generate surugamide K (10) and acyl-surugamides 11–15, respectively.^3,16,20) The lysine ε-NH₂ can even be modified by isoquinoline quinone to generate albucyclones (16–21)³⁾ (Fig. 1a). Although the exact mechanism remains elusive, modification of the lysine ε-NH₂ in the biosynthesis of 10–15 most likely occurs in a post-assembly rather than pre-assembly manner, given the generally high specificity of the adenylation domain. Notably, the acyl units at the lysine ε-NH₂ reportedly play an important role in enhancing the biological activities of surugamides. In stark contrast to the lack of or subtle activity of many non-acylated surugamides in cell-based or whole-organism assays, the biological evaluation of acyl-surugamides and the synthetic derivatives 26–28 (Fig. 1c) showed that 11 exhibits antifungal activity, while 13 and 28 possess anthelmintic activity.^3,20) These findings warrant further evaluation of the biological activities of other acylated derivatives, which remain underexplored.

Here, by employing a previously established biomimetic synthetic route for octapeptidyl surugamides,⁵⁾ we synthesized the new derivatives of acyl-surugamides 29–32, including those with previously unseen acyl groups: octanoyl and 4-phenylbenzoyl groups (Fig. 1c). Cytotoxic evaluation showed that the new derivatives are more potent than those used as positive controls (cytarabine and fluorouracil), highlighting the potential of acyl-surugamides as leads for further structure optimization toward developing antitumor agents.

To avoid the use of the expensive building block Fmoc-D-allo-isoleucine, we chose surugamide B (2), in which the D-allo-isoleucine in surugamide A is substituted with D-valine, as the cyclic peptide scaffold. Compound 2 was synthesized through the scheme established previously⁵⁾ (Fig. 2a). Briefly, the tert-butoxycarbonyl (Boc)-protected linear octapeptide 35 was synthesized from Fmoc-D-leucine tethered on 2-chlorotrityl resin after seven rounds of conventional solid-phase peptide synthesis using DIC/Oxyma. After hexafluoro-2-propanol (HFIP)-mediated resin cleavage, head-to-tail macrolactamization was performed using PyBOP, HOAt and 2,4,6-collidine, and then the Boc group on the L-lysine residue was removed by a deprotection cocktail (trifluoroacetic acid (TFA)/i-Pr₃SiH/H₂O) to afford 2, which was subjected to successive acylation.

Fig. 2. Synthesis of 2 Derivatives

a. Synthetic scheme of 2. b. Acylations of 2 to generate 29–32.

Acylation was performed in anhydrous tetrahydrofuran (THF) or dichloromethane (DCM) with triethylamine (Fig. 2b). Compound 2 was treated with acetic anhydride, butyryl chloride, octanoyl chloride, or 4-phenylbenzoyl chloride. After purification by ODS HPLC, acyl-surugamide B2 (29), acyl-surugamide B (30), acyl-surugamide BS4 (31) and acyl-surugamide BS5 (32) were obtained with synthetic yields ranging from 38–69% from 2 (Fig. 2b).

The natural product 2 and its synthetic acyl-derivatives 29–32 were tested for cytotoxicity against four human cancer cell lines (MT-4, RPMI8226, A549, and SW-13). These derivatives exhibited moderate to strong cytotoxicity, and those with higher hydrophobicity tended to exhibit stronger cytotoxicity (Table 1). While 31 and 32 were more potent against several cell lines than positive controls such as cytarabine (araC) and fluorouracil (5-FU), 32, with its bulkier, more hydrophobic biphenyl group, was less cytotoxic to the SW-13 cell line than 31, with a smaller acyl substituent. This suggests that the appropriate size of the acyl substituent is important for the cytotoxicity, and the cytotoxicity likely arises from binding and inhibiting specific molecular targets, rather than simple membrane disruption. This result aligns with a previous study,²⁰⁾ which reported that acyl-surugamide A3 (14) and acyl-surugamide AS3 (28) specifically inhibit D. immitis microfilarial motility, while other derivatives with different acyl substituents showed only negligible activity.

Table 1. Cytotoxicity (CC₅₀ [µM]) of Surugamide B (2) and Its Acyl-Derivatives 29–32 against Five Human Cancer Cell Lines

Compounds	MT-4	RPMI8226	A549	SW-13
2	25.5 ± 6.6	>50	>50	>50
29	34.5 ± 9.5	26.3 ± 4.0	29.3 ± 9.6	>50
30	10.4 ± 4.0	16.3 ± 1.6	14.0 ± 2.9	>50
31	0.2 ± 0.1	0.7 ± 0.1	5.4 ± 3.2	12.6 ± 0.4
32	0.1 ± 0.0	0.7 ± 0.0	7.0 ± 6.7	>46.4*
araC	0.3 ± 0.1	5.5 ± 1.6	20.8 ± 8.4	1.4 ± 0.1
5-FU	1.8 ± 0.1	2.4 ± 1.0	32.2 ± 8.7	N.D.

N.D: not determined. Values are the average of three or six measurements. *32 showed a slight cytotoxic effect against SW-13 cells at a concentration of 46.4 µM. At this concentration, the viability of SW-13 cells treated with 32 was 64.5 ± 0.3%.

Discussion

Octapeptidyl surugamides represent a unique group of non-ribosomal peptides with structures diversified via two distinct modes: variations in sequence and post-assembly modification of the lysine ε-NH₂. Our structure–activity relationship study focusing on the modification of the lysine ε-NH₂ identified the new derivatives 31 and 32 with sub-micromolar potencies, and demonstrated that the appropriate size of the acyl substituent is important for the activity. A remaining enigma is the impact of sequence variation on biological activity. We previously established an efficient chemoenzymatic strategy that enables rapid diversification of the backbone sequence of cyclic peptides.²³⁾ With the optimized acyl substituent on the lysine residue, the screening of surugamide variants for improved biological activity is currently underway.

Experimental

General Remarks

¹H- and ¹³C-NMR spectra were recorded on a JEOL ECZ500 (500 MHz for ¹H-NMR) spectrometer. Chemical shifts are denoted in δ (ppm) relative to residual solvent peaks as internal standards (dimethyl sulfoxide (DMSO)-d₆, δ_Η 2.50, δ_C 39.5). Electrospray ionization (ESI)-MS spectra were recorded on a Thermo Scientific Exactive mass spectrometer. Optical rotations were recorded on a JASCO P-1030 polarimeter.

Chemical Synthesis

Surugamide B (2) was synthesized in 20% yield in 18 steps from 33, by following the scheme previously reported.⁵⁾

Cyclic peptide 29: To peptide 2 (35.6 mg, 0.04 mmol) were added acetate anhydrate (18.9 µL, 5 equivalent (equiv.)) and triethylamine (27.8 µL, 5 equiv.) in 5.0 mL of super dehydrated THF. The reaction mixture was stirred for 30 min, then solvent was removed in vacuo. Residue were purified by reverse-phased HPLC (COSMOSIL MS-II 20 mm I.D. × 250 mm) eluted by 59% acetonitrile + 0.05% TFA with flowrate at 10 mL/min to afford 29 (25.4 mg, 68% from 2).

29: [α]_D²⁰ + 20.8 (c = 1.67, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): see Supplementary Fig. S1; ¹³C-NMR (125 MHz, DMSO-d₆): see Supplementary Fig. S2; high resolution (HR)-MS (ESI) Calcd for C₄₉H₈₁O₉N₉Na [M + Na]⁺ 962.60495. Found 962.60320 (Supplementary Fig. S9)

Cyclic peptide 30: To peptide 2 (9.0 mg, 0.01 mmol) were added butyryl chloride (1.55 µL, 1.5 equiv.) and triethylamine (2.05 µL, 1.5 equiv.) in 1.0 mL of super dehydrated DCM. The reaction mixture was stirred for 60 min, then solvent was removed in vacuo. Residue were purified by reverse-phased HPLC (COSMOSIL MS-II 20 mm I.D. × 250 mm) eluted by 73% acetonitrile + 0.05% TFA with flowrate at 10 mL/min to afford 30 (3.7 mg, 39% from 2).

30: [α]_D²¹ + 12.9 (c = 0.20, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): see Supplementary Fig. S3; ¹³C-NMR (125 MHz, DMSO-d₆): see Supplementary Fig. S4; HR-MS (ESI) Calcd for C₅₁H₈₅O₉N₉Na [M + Na]⁺ 990.63625. Found 990.63422 (Supplementary Fig. S10)

Cyclic peptide 31: To peptide 2 (35.6 mg, 0.04 mmol) were added octanoyl chloride (10.3 µL, 1.5 equiv.) and triethylamine (8.36 µL, 1.5 equiv.) in 1.0 mL of super dehydrated DCM. The reaction mixture was stirred for 60 min, then solvent was removed in vacuo. Residue were purified by reverse-phased HPLC (COSMOSIL MS-II 20 mm I.D. × 250 mm) eluted by 65% acetonitrile + 0.05% TFA with flowrate at 10 mL/min to afford 31 (20.2 mg, 49% from 2).

31: [α]^D₂₀ + 18.3 (c = 0.14, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): see Supplementary Fig. S5; ¹³C-NMR (125 MHz, DMSO-d₆): see Supplementary Fig. S6; high resolution (HR)-MS (ESI) Calcd for C₅₅H₉₃O₉N₉Na [M + Na]⁺ 1046.69885. Found 1046.69713 (Supplementary Fig. S11)

Cyclic peptide 32: To peptide 2 (18.0 mg, 0.02 mmol) were added 4-phenylbenzoyl chloride (6.50 mg, 1.5 equiv.) and triethylamine (4.10 µL, 1.5 equiv.) in 1.0 mL of super dehydrated DCM. The reaction mixture was stirred for 180 min, then solvent was removed in vacuo. Residue were purified by reverse-phased HPLC (COSMOSIL MS-II 20 mm I.D. × 250 mm) eluted by 80% acetonitrile + 0.05% TFA with flowrate at 10 mL/min to afford 32 (8.2 mg, 38% from 2).

32: [α]^D₂₀ + 7.25 (c = 0.30, DMSO); ¹H-NMR (500 MHz, DMSO-d₆): see Supplementary Fig. S7; ¹³C-NMR (125 MHz, DMSO-d₆): see Supplementary Fig. S8; high resolution (HR)-MS (ESI) Calcd for C₆₀H₈₇O₉N₉Na [M + Na]⁺ 1100.65190. Found 1100.65057 (Supplementary Fig. S12)

Cells

MT-4 cells (a human T-cell leukocyte virus type I transformed cell line), and RPMI8226 cells (a human multiple myeloma cell line) were maintained in RPMI-1640 Medium (Sigma-Aldrich, St. Louis, MO, U.S.A.), and A549 cells (a human lung adenocarcinoma cell line) were in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma-Aldrich). Both media were supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin G, and 50 µg/mL streptomycin (complete media). SW-13 (CCL-105; American Type Culture Collection [ATCC]) cells were cultured in DMEM (Sigma-Aldrich), supplemented with 10% heat-inactivated fetal calf serum and antibiotics (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

Cytotoxicity

Cytotoxicity was determined in 10-fold serial dilution (0.01, 0.1, 1, 10, and 100 µM) by cell viability using 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. Cells and test compounds were cultured in a 96-well microplate with complete DMEM or complete RPMI-1640 at 37 °C and 5% CO₂. After 4 d culture, the MTT reagent (Sigma-Aldrich) was added to dye living cells. The microplate was scanned using a BioTek 800TS microplate reader (Agilent Technologies Inc., Santa Clara, CA, U.S.A.) at a wavelength of 561 nm. All assays to determine 50% cytotoxicity concentration (CC₅₀) values were conducted in triplicate.

Cytotoxic effect to SW-13 cells was measured using CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI, U.S.A.) following the manufacturer’s protocol.²⁴⁾ Briefly, SW-13 cells were seeded in white walled 96 well plates (Clontech) and incubated with each compound 37 °C. After 4 d, luminescence was measured using SpectraMax iD5 (Molecular Devices).

Acknowledgments

This work was partly supported by Hokkaido University, Global Facility Center (GFC), Pharma Science Open Unit (PSOU), funded by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) under “Support Program for Implementation of New Equipment Sharing System,” Global Station for Biosurfaces and Drug Discovery, a project of Global Institution for Collaborative Research and Education in Hokkaido University, Japan Foundation for Applied Enzymology, TERUMO Life Science Foundation, the Japan Agency for Medical Research and Development JP24gm1610007, JP24ak0101163, JP24ama121039, Grants-in-Aid from MEXT, the Japan Science and Technology Agency (JST Grant Number: ACT-X JPMJAX201F), JSPS KAKENHI (Grant Numbers: JP21H02635, JP22K15302, JP22H05128, JP23K07918, JP23K17410, JP24K01659, and JP24KJ0292).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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