Synthesis and biological evaluation of the ascidian blood-pigment halocyamine A

Abstract

Synthesis of the antimicrobial marine natural product halocyamine A has been achieved utilizing a combination of Sonogashira coupling, ruthenium complex/ytterbium triflate catalyzed hydroamidation and solid-phase peptide synthesis (SPPS) chemistry. The synthetic natural product exhibited only modest levels of antibacterial activities but significant antioxidant activity.

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Introduction

Investigation of the natural product chemistry of blood cells of marine organisms known as ascidians over the years has led to the identification of a number of modified peptides bearing C-terminus decarboxy-enamide moieties.¹ These natural products, collectively known as tunichromes, typically incorporate either a decarboxy-(E)-α,β-dehydro-3,4-dihydroxyphenyl alanine (dcΔDOPA) or a decarboxy-(E)-α,β-dehydro-3,4,5-trihydroxyphenyl alanine (dcΔTOPA) residue at the C-terminus.² While no role has been ascribed with any confidence, potential ecological roles proposed for the tunichromes include iron or vanadium sequestration, cross-linking/tunic formation or as primitive wound repair or clotting agents.³ Unusual members of the tunichrome-family are halocyamines A (1) and B, two DOPA-containing modified tetrapeptides, isolated from the ascidian Halocynthia roretzi (Fig. 1).⁴

Fig. 1 Structure of halocyamine A (1).

NMR and mass spectrometry data were used to characterize the natural products, with acidic hydrolysis, labelling and HPLC analysis used to establish the configuration of the L-His and L-DOPA residues. The structures of the halocyamines were unusual additions to the tunichrome family of modified peptides in that they contained the rare Z-configuration 6-bromoindolic enamide moiety^5,6 at the C-terminus. Biological evaluation of halocyamine A revealed a wide range of activities, including growth inhibition of Gram-positive bacteria,^4a Gram-negative marine bacteria and fish RNA viruses.⁷ As part of our ongoing investigation of the synthesis and biological investigation of tunichromes,⁸ we now report the synthesis and structural confirmation of halocyamine A (1) and present the results of preliminary biological evaluation.

Results and discussion

Prior to attempting the synthesis of halocyamine A, we chose to target the phenylenamide-containing model compound 2 (Fig. 2).

Fig. 2 Target model compound 2.

Established routes for the synthesis of enamides include dehydration⁹ or elimination¹⁰ methods, both of which favour the formation of the E-enamide product. In contrast, hydroamidation coupling of terminal alkynes with primary amides using a ruthenium complex/ytterbium triflate catalyst has been shown to give exclusively the Z-enamide product.¹¹ Thus we envisaged that 2 could be prepared by reaction of the appropriately protected tripeptide L-His-L-DOPA-Gly-NH₂ (3) with phenylacetylene (Scheme 1). Tripeptide 3 was prepared by standard Fmoc solid-phase peptide synthesis procedures using 2-chlorotrityl resin, protected amino acids Fmoc-His(Trt)-OH, Fmoc-DOPA(TBDMS)₂-OH¹² and Fmoc-Gly with HATU as the coupling agent. Cleavage from the resin using 2,2,2-trifluoroethanol afforded the protected tripeptide carboxylic acid 4 in 79% yield over seven steps. Subsequent reaction of 4 with HOBt·NH₃ resulted in smooth conversion to the required tripeptide amide 3 (90% yield). Attempted hydroamidation of 3 and phenylacetylene using 5 mol% bis(2-methylallyl)(1,5-cyctooctadiene)ruthenium(II) heated at 60 °C for 6 h failed to afford the expected enamide-containing product. Unexpectedly, the only products detected were 1,4-disubstituted enynes 5 and 6, isolated as a mixture in a ratio of 1 : 0.8 in 80% yield. Comparison with literature NMR data previously reported for 5 and 6 confirmed their identities.¹³

Scheme 1 Attempted synthesis of model enamide 2.

Based on the outcome of this reaction, we speculated that the steric bulk of tripeptide-amide 3 resulted in crowding at the ruthenium catalytic centre, preventing the progress of the expected hydroamidation reaction.

We next explored an alternative route to model analogue 2: disconnection at the DOPA-Gly amide bond which would require dipeptide 7 and enamide 8 (Fig. 3). Protected dipeptide Fmoc-His(Trt)-DOPA(TBDMS)₂-OH 7 was prepared by SPPS, in a similar manner to that described for 4, in 85% yield over five steps.

Fig. 3 Fragments 7 and 8.

The synthesis of styryl enamide 8 was achieved in two steps, whereby hydroamidation of Fmoc-glycinamide (9) with phenylacetylene gave protected enamide 10 (96% yield, based upon stoichiometry of 9),¹⁴ which upon reaction with 20% piperidine/DMF gave 8 (84% yield) (Scheme 2). Alternatively, 8 could be prepared via hydroamidation of Boc-Gly-NH₂ (11) with phenylacetylene to give 12 (78% yield) which was deprotected cleanly in TFA/TIS/H₂O (95 : 2.5 : 2.5) to give 8 (78% yield).

Scheme 2 Synthesis of styryl enamide 8.

With 7 and 8 in-hand, coupling using HBTU and HOBt in DMF gave 13 in 35% yield (Scheme 3). Stepwise deprotection of the N-terminus using piperidine/DMF (20 min) gave 14 (94% yield), followed by deprotection of the catechol group (triethylamine-trihydrogen fluoride, THF, 55 min) to give 15 (73% yield) and finally, removal of the trityl protecting group (0.01 N HCl/HFIP, 1 h) gave phenyl enamide model compound 2 as the dihydrochloride salt.

Scheme 3 Synthesis of phenyl enamide model 2.

With the successful synthesis of phenylenamide model 2, we chose to employ a similar disconnection methodology for the synthesis of halocyamine A (1), requiring protected dipeptide L-His-L-DOPA (7) and glycyl-indolic enamide 16 (Scheme 4). We have recently demonstrated that the ruthenium catalysed hydroamidation methodology allows rapid synthetic entry to indolic Z-enamides by coupling an appropriately substituted indole-3-alkyne (i.e.17) with a primary amide.¹⁵

Scheme 4 Synthesis of halocyamine A (1).

Entry to 17 was achieved by Boc-protection of the known dihaloindole 18 ^16,17 to give N-Boc-6-bromo-3-iodo indole (19), which, followed by Sonogashira alkynation, gave TMS-protected acetylene 20 in 99% yield (Scheme 4). Subsequent desilylation using TBAF in THF gave terminal acetylene 17 in 72% yield. Hydroamidation of 17 and Fmoc-glycinamide (9) using 5 mol% bis(2-methylallyl)(1,5-cyctooctadiene)ruthenium(II) heated at 70 °C for 24 h afforded glycyl enamide 21 (43%), exclusively as the Z-enamide. Of note was that the reaction product was deprotected at the N-terminus.

A minor (6%) by-product, E-enyne 22 (Fig. 4) was also purified from the product mixture. Detection of a sodiated molecular ion corresponding to C₃₀H₂₈⁷⁹Br₂N₂O₄Na, (observed [M + Na]⁺ 661.0302, calcd 661.0308) as well as two nearly identical sets of ¹H NMR resonances attributable to a 3-substituted N-Boc-6-bromoindole fragment suggested 22 to be a dimer related to the starting material 17. Observation of E-alkene (δ_H 7.10, d, J = 16.4 Hz; δ_H 6.44, d, J = 16.4 Hz) and disubstituted alkyne resonances (δ_C 92.9, 83.0), combined with 2D NMR data analysis identified the minor product as the (E)-1,4-disubstituted enyne 22. Repeating the hydroamidation reaction in the absence of Fmoc-glycinamide afforded 22 in 28% yield. Of note, a number of transition metals are known to promote alkyne dimerization,¹⁸ including ruthenium,¹⁹ though with somewhat variable regio- (head-to-head vs. head-to-tail) and stereoselectivity.

Fig. 4 Structure of E-enyne 22.

Removal of the Boc protecting group of enamide 21 using TFA/CH₂Cl₂ afforded 16 in 80% yield. Peptide coupling (EDC, HOBt, DIPEA, 6 h) of enamide 16 and dipeptide acid 7 gave protected halocyamine A 23 in a disappointing yield of 18%. Efforts to increase the yield of this reaction by altering the coupling agent (HATU or HBTU), reaction time (9 h or 24 h), and the ratio of reactants 7/16 (2 : 1, 1 : 1 or 1 : 2) met with no success (data not shown). Sequential Fmoc deprotection (piperidine, DMF, 20 min) gave 24 (75% yield), followed by desilylation (triethylamine trihydrofluoride, THF, 55 min) to give 25 (63% yield) and removal of the trityl group (HCl/HFIP, H₂O, TIS, 1 h) gave the crude peptide that was purified by reversed-phase C₈ column chromatography [H₂O/MeOH] to afford halocyamine A (1) dihydrochloride salt in 56% yield. NMR (Table S1†) and optical rotation [+3.4 (c 1.07); lit.^4a +5.2 (c 0.50)] data observed for synthetic 1 were in good agreement with those reported for the natural product.^4a

The original reports of the halocyamines noted their abilities to inhibit the growth of Gram-positive bacteria, Gram-negative marine bacteria and fish RNA viruses.^4a,7 Halocyamine A was evaluated against a panel of Gram-positive (Staphylococcus aureus ATCC 25923, Staphylococcus intermedius 1051997), Gram-negative (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Enterococcus faecalis ATCC 29212), and marine Gram-negative bacteria (Vibrio harveyi ATCC 14126, Vibrio alginolyticus ATCC 17749 and Listonella anguillarum ATCC 19264) and for antioxidant activity in the DPPH radical scavenging and oxygen radical absorbance (ORAC) assays. Somewhat at odds with the original isolation report,^4a,7 only modest antibacterial activity was observed for halocyamine A towards P. aeruginosa and E. faecalis, (both MIC 100 μM) and V. harveyi (IC₅₀ 129 μM); no antibacterial activity was observed against the other organisms (MIC > 200 μM). A significant antioxidant activity was observed in the DPPH assay (IC₅₀ of 26.6 ± 2.9 μM; positive control ascorbic acid IC₅₀ 101 ± 8 μM) while in the ORAC assay, halocyamine A (1) was more active (relative ORAC value 1.29 ± 0.09) than Trolox, a water-soluble vitamin E analogue (ORAC value 1) and ascorbic acid (ORAC value 0.61 + 0.06).²⁰

Conclusions

In summary, we have described a total synthesis of the marine natural product halocyamine A (1), making use of a ruthenium-catalysed hydroamidation of an indole acetylene with Fmoc-glycinamide to form the critical Z-enamide moiety. The natural product exhibits only mild levels of antibacterial activity. The relative ease of synthesis of halocyamine A now opens the door for future investigation of the potential ecological roles played by this unusual member of the tunichrome family of marine natural products.

Experimental

General information and materials

Optical rotations were recorded using a 0.1 dm cell in methanol or dichloromethane. NMR spectra were recorded at either 500 or 400 MHz for ¹H nuclei and 125 or 100 MHz for ¹³C nuclei. Residual solvent signals were used as reference (CD₃OD: δ_H 3.31, δ_C 49.0; CDCl₃: δ_H TMS 0, δ_C 77.16; DMSO-d₆: δ_H 2.50, δ_C 39.52). ¹H NMR data are reported as position (δ), relative integral, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet, br = broad, obs = obscured), coupling constant (J, Hz), and the assignment of the atom. ¹³C NMR data are reported as position (δ) and assignment of the atom. Assignments were based on 2D NMR data acquired using standard pulse sequences. (+)-ESI-MS data were acquired on a micrOTOF Q II mass spectrometer. Column chromatography was carried out with either C₈ reversed-phase or silica gel. All solvents used were distilled analytical grade or better. Chemical reagents were purchased from a commercial supplier and used without purification. 6-Bromo-3-iodo-indole 18 was prepared by a literature method.^16,17

Fmoc-His(Trt)-DOPA(TBDMS)₂-Gly-OH (4). Fmoc-glycine (0.595 g, 2.00 mmol) dissolved in CH₂Cl₂ (15 mL) was added to 2-chlorotrityl chloride resin (loading 0.5 mmol g⁻¹, 4.00 g) followed by DIPEA (0.35 mL, 2.0 mmol). The mixture was agitated for 10 min and DIPEA (0.52 mL, 3.0 mmol) was added and the mixture was further shaken for 1 h. The solution was drained and the resin was washed with DMF (10 mL). A solution of CH₂Cl₂/MeOH/DIPEA (80 : 15 : 5, 25 mL) was added and the mixture agitated for 20 min. The solution was drained and the procedure repeated. The Fmoc-glycine-loaded resin was rinsed with DMF before a solution of piperidine/DMF (10 mL, 1 : 4) was added to the resin and agitated for 10 min. The solution was drained and the procedure was repeated for a further 20 min. The solution was drained off while the remaining resin was washed with DMF (15 mL), isopropanol (15 mL), followed by n-hexane (15 mL). The resin was then extensively dried before storing in the desiccator for 16 h. CH₂Cl₂ (25 mL) was used to swell the resin, which was then drained, before a solution of Fmoc-DOPA(TBDMS)₂-OH (2.59 g, 4.00 mmol), HOBt (1.01 g, 7.50 mmol), HATU (2.85 g, 7.50 mmol) and DIPEA (1.74 mL, 10.0 mmol) in DMF (7.50 mL) was added to the resin and shaken for 2 h. The solution was then drained and the resin was washed with DMF (10 mL). A solution of piperidine in DMF (10 mL, 1 : 4) was added to the Fmoc-DOPA(TBDMS)₂-Gly-loaded resin and agitated. After 10 min, the solution was drained and the procedure was repeated for a further 20 min. The solution was drained and the DOPA(TBDMS)₂-Gly-loaded resin was washed with DMF (10 mL) before a solution of Fmoc-His(Trt)-OH (3.1 g, 5.0 mmol), HOBt (1.01 g, 7.50 mmol), HATU (2.85 g, 7.50 mmol) and DIPEA (1.74 mL, 10.0 mmol) in DMF (7.50 mL) was added. The mixture was agitated for 90 min before the solution was drained. The Fmoc-His(Trt)-DOPA(TBDMS)₂-Gly-loaded resin was washed with DMF (15 mL), isopropanol (15 mL), followed by n-hexane (15 mL) and was extensively dried before storing in the desiccator for 16 h. The protected peptide was cleaved from the resin using a solution of 2,2,2-trifluoroethanol in CH₂Cl₂ (25 mL, 1 : 4) to give the desired product 4 as a yellow solid (1.71 g, 79%). M.p. 130–131 °C; [α]^22.7_D −21.5 (c 0.14, CH₂Cl₂); R_f 0.52 (CH₂Cl₂/MeOH 9 : 1); IR (ATR) ν_max 3278, 3036, 2930, 1662, 1508, 1446, 1251, 1128 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 7.75–7.69 (3H, m, NH-10, 2H-FmocAr), 7.54–7.44 (3H, m, H-6 and 2H-FmocAr), 7.32–7.24 (12H, m, NH-20, 2H-FmocAr, 9H-TrtAr), 7.20–7.18 (2H, m, 2H-FmocAr), 7.01–7.00 (6H, m, 6H-TrtAr), 6.71–6.64 (4H, m, H-8, H-14, H-17 and H-18), 6.40 (1H, br s, NH-1), 4.75 (1H, d, J = 4.4 Hz, H-11), 4.43 (1H, br s, H-2), 4.25–4.13 (3H, m, H₂-21a and CO₂CH₂CH), 4.03 (1H, br s, CO₂CH₂CH), 3.69 (1H, br d, J = 17.4 Hz, H₂-21b), 3.19 (1H, br d, J = 10.3 Hz, H₂-12a), 3.12–3.10 (1H, m, H₂-3a), 2.90–2.86 (1H, m, H₂-12b), 2.80 (1H, br s, H₂-3b), 0.93–0.91 (18H, m, 2SiC(CH₃)₃), 0.14–0.09 (12H, m, 2Si(CH₃)₂); ¹³C NMR (CDCl₃, 125 MHz) δ 174.0 (C-22), 171.7 (C-19), 170.8 (C-9), 156.2 (CO₂CH₂CH), 146.8 (C-15), 145.7 (C-16), 144.0 (C-FmocAr), 143.9 (C-FmocAr), 141.5 (2C-FmocAr or 3C-TrtAr), 141.3 (2C-FmocAr or 3C-TrtAr), 137.5 (C-6), 134.8 (C-4), 129.9 (C-13), 129.7 (6C-TrtAr), 128.6 (3C-TrtAr), 128.4 (6C-TrtAr), 127.8 (2C-FmocAr), 127.2 (2C-FmocAr), 125.5 (C-FmocAr), 125.3 (C-FmocAr), 122.5 (C-14 or C-18), 122.0 (C-14 or C-18), 121.1 (C-17), 120.4 (C-8), 120.0 (2C-FmocAr), 76.5 (CAr₃), 67.2 (COCH₂CH), 55.5 (C-2), 55.1 (C-11), 47.2 (CO₂CH₂CH), 42.5 (C-21), 37.7 (C-12), 31.0 (C-3), 26.1 (2SiC(CH₃)₃), 18.5 (2SiC(CH₃)₃), −4.0 (2Si(CH₃)₂); (+)-HRESIMS [M + H]⁺ 1084.5052 (calcd for C₆₃H₇₄N₅O₈Si, 1084.5070).

Fmoc-His(Trt)-DOPA(TBDMS)₂-Gly-NH₂ (3). DMF (1 mL) was added to 4 (1.0 g, 0.92 mmol) and HOBt·NH₃ (0.280 g, 1.85 mmol), followed by EDC·HCl (0.18, 0.92 mmol). The solution was stirred at r.t. for 18 h under nitrogen. EtOAc (15 mL) was then added and the mixture washed with water (20 mL) followed by brine (20 mL), dried (MgSO₄) and solvent removed in vacuo. Purification by silica gel column chromatography (eluting with n-hexane/EtOAc 1 : 9 to EtOAc) afforded the desired product 3 as a white solid (0.90 g, 90%). M.p. 148–149 °C; [α]^22.7_D −8.1 (c 0.36, CH₂Cl₂); R_f 0.63 (CH₂Cl₂/MeOH 9 : 1); IR (ATR) ν_max 3298, 1721, 1640, 1507, 1250 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 8.88 (1H, br s, NH-20), 7.76–7.73 (2H, m, 2H-FmocAr), 7.55–7.52 (2H, m, 2H-FmocAr), 7.38–7.36 (2H, m, 2H-FmocAr), 7.34–7.32 (10H, m, H-6 and 9H-TrtAr), 7.23–7.20 (2H, m, 2H-FmocAr), 7.09–7.07 (6H, m, 6H-TrtAr), 6.74 (1H, d, J = 7.6 Hz, H-17), 6.67 (1H, d, J = 1.7 Hz, H-14), 6.65 (1H, br s, H-8), 6.64 (1H, br s, NH₂-23a), 6.56 (1H, d, J = 7.6 Hz, H-18), 6.44 (1H, d, J = 5.9 Hz, NH-10), 6.22 (1H, d, J = 5.0 Hz, NH-1), 5.16 (1H, br s, NH₂-23b), 4.49–4.48 (1H, m, H-11), 4.33–4.28 (2H, m, CO₂CH₂CH), 4.23–4.22 (1H, m, H-2), 4.16–4.12 (2H, m, H₂-21a and CO₂CH₂CH), 3.56 (1H, dd, J = 17.0, 5.1 Hz, H₂-21b), 3.09 (1H, dd, J = 14.3, 5.2 Hz, H₂-12a), 3.05–3.01 (2H, m, H₂-3a and H₂-12b), 2.82 (1H, dd, J = 15.4, 5.0 Hz, H₂-3b), 0.95–0.93 (18H, m, 2SiC(CH₃)₃), 0.15–0.14 (12H, m, 2Si(CH₃)₂); ¹³C NMR (CDCl₃, 125 MHz) δ 172.2 (C-9 and C-22), 171.3 (C-19), 156.3 (CO₂CH₂CH), 147.6 (C-15), 146.5 (C-16), 143.8 (2C-FmocAr), 142.2 (3C-TrtAr), 141.4 (2C-FmocAr), 138.8 (C-6), 135.5 (C-4), 129.8 (6C-TrtAr), 129.1 (C-13), 128.5 (3C-TrtAr), 128.4 (6C-TrtAr), 127.9 (2C-FmocAr), 127.2 (2C-FmocAr), 125.2 (2C-FmocAr), 121.9 (C-14 or C-18), 121.8 (C-14 or C-18), 121.5 (C-17), 120.5 (C-8), 120.2 (2C-FmocAr), 75.8 (CAr₃), 67.4 (CO₂CH₂CH), 55.5 (C-2 and C-11), 47.2 (CO₂CH₂CH), 43.1 (C-21), 36.2 (C-12), 30.8 (C-3), 26.0 (2SiC(CH₃)₃), 18.6 (SiC(CH₃)₃), 18.5 (SiC(CH₃)₃), −3.9 (2Si(CH₃)₂); (+)-HRESIMS [M + H]⁺ 1083.5202 (calcd for C₆₃H₇₅N₆O₇Si, 1083.5230).

A mixture of (Z)-but-1-en-3-yne-1,4-diyldibenzene (5) and (E)-but-1-en-3-yne-1,4-diyldibenzene (6). An oven-dried flask was charged with 3 (51.0 mg, 47.1 μmol), bis(2-methylallyl)(1,5-cyclooctadiene)ruthenium(II) (0.75 mg, 2.36 μmol), 1,4-bis(dicyclohexylphosphino)butane (1.27 mg, 2.83 μmol) and ytterbium triflate (1.17 mg, 1.88 μmol) under an atmosphere of nitrogen. Degassed DMF (0.2 mL) and phenylacetylene (0.01 mL, 94.2 μmol) were added to the flask followed by degassed water (5 μL, 0.28 mmol). The solution was stirred at 60 °C for 6 h, then poured into sat. aqueous NaHCO₃ (10 mL). The resulting mixture was extracted with EtOAc (3 × 20 mL), the combined organic layers were then washed with water (10 mL) and brine (10 mL), dried (MgSO₄) and the solvent removed in vacuo. Purification by silica gel column chromatography (eluting with n-hexane) gave a mixture of 5 and 6 as an orange oil (7.7 mg, 80%). Data for a mixture of 5 and 6: R_f 0.34 (n-hexane); IR (ATR) ν_max 3059, 3025, 1596, 1489, 1447, 1263, 1176 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 7.92 (2H, d, J = 7.5 Hz, 2H-Ar), 7.50–7.46 (4H, m, 4H-Ar), 7.43–7.28 (14H, m, 14H-Ar), 7.04 (1H, d, J = 16.2 Hz, H_E-1), 6.70 (1H, d, J = 12.0 Hz, H_Z-1), 6.39 (1H, d, J = 16.2 Hz, H_E-2), 5.92 (1H, d, J = 12.0 Hz, H_Z-2); ¹³C NMR (CDCl₃, 125 MHz) δ 141.4 (C_E-1), 138.8 (C_Z-1), 136.7 (C_Z–Ar), 136.5 (C_E–Ar), 131.7 (C–Ar), 131.6 (C–Ar), 128.92 (C–Ar), 128.89 (C–Ar), 128.8 (C–Ar), 128.7 (C–Ar), 128.6 (C–Ar), 128.51 (C–Ar), 128.49 (C–Ar), 128.4 (C–Ar), 128.3 (C–Ar), 126.5 (C–Ar), 123.62 (C–Ar), 123.57 (C–Ar), 108.3 (C_E-2), 107.6 (C_Z-2), 96.0 (C_Z-4), 91.9 (C_E-4), 89.0 (C_E-3), 88.4 (C_Z-3); (+)-HRESIMS [M + H]⁺ 205.1001 (calcd for C₁₆H₁₃, 205.1012).

(9H-Fluoren-9-yl)methyl (Z)-(2-oxo-2-(styrylamino)ethyl)carbamate (10). Fmoc-Gly-NH₂ (9) (0.296 g, 1.00 mmol), bis(2-methylallyl)(1,5-cyclooctadiene)ruthenium(II) (0.016 g, 0.050 mmol), 1,4-bis(dicyclohexylphosphino)butane (0.027 g, 0.060 mmol) and ytterbium triflate (0.025 g, 0.040 mmol) were placed in a two neck flask and the air evacuated. The system was then flushed with nitrogen. Degassed DMF (3 mL), phenylacetylene (0.22 mL, 2.0 mmol) and degassed water (0.108 mL, 6.00 mmol) were added. The mixture was stirred under nitrogen at 60 °C for 6 h. The reaction mixture was added into sat. aqueous NaHCO₃ solution (30 mL) and was extracted with EtOAc (4 × 20 mL). The combined organic layers were washed with water (30 mL) and brine (30 mL), dried (MgSO₄), filtered, and the solvent removed in vacuo. Purification using silica gel column chromatography (eluting with n-hexane/EtOAc 9 : 1 to n-hexane/EtOAc 7 : 3) gave the desired product 10 as a yellow foam (0.38 g, 96%). M.p. 54–55 °C; R_f 0.68 (n-hexane/EtOAc 1 : 1); IR (ATR) ν_max 3305, 1686, 1647, 1514, 1481, 1448, 1334, 1253 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 8.31 (1H, br d, J = 9.9 Hz, NH-4), 7.74 (2H, d, J = 7.2 Hz, 2H-FmocAr), 7.53 (2H, d, J = 7.2 Hz, 2H-FmocAr), 7.38 (2H, t, J = 7.2 Hz, 2H-FmocAr), 7.27 (4H, t, J = 7.2 Hz, 2H-FmocAr and 2H-9), 7.22 (2H, d, J = 7.2 Hz, 2H-8), 7.15 (1H, t, J = 7.2 Hz, H-10), 6.89 (1H, dd, J = 9.9, 9.7 Hz, H-5), 5.77 (1H, d, J = 9.7 Hz, H-6), 5.60 (1H, br s, NH-1), 4.36 (2H, d, J = 6.9 Hz, CO₂CH₂CH), 4.15 (1H, t, J = 6.9 Hz, CO₂CH₂CH), 3.87 (2H, br s, H₂-2); ¹³C NMR (CDCl₃, 125 MHz) δ 167.0 (C-3), 156.9 (CO₂CH₂CH), 143.7 (2C-FmocAr), 141.4 (2C-FmocAr), 135.3 (C-7), 129.1 (2C-9), 128.0 (2C-8), 127.9 (2C-FmocAr), 127.2 (2C-FmocAr and C-10), 125.1 (2C-FmocAr), 121.2 (C-5), 120.1 (2C-FmocAr), 111.5 (C-6), 67.5 (CO₂CH₂CH), 47.1 (CO₂CH₂CH), 44.9 (C-2); (+)-HRESIMS [M + Na]⁺ 421.1529 (calcd for C₂₅H₂₂N₂NaO₃, 421.1523).

tert-Butyl (Z)-(2-oxo-2-(styrylamino)ethyl)carbamate (12). Boc-Gly-NH₂ (11) (0.17 g, 1.0 mmol), bis(2-methylallyl)(1,5-cyclooctadiene)ruthenium(II) (0.016 g, 0.050 mmol), 1,4-bis(dicyclohexylphosphino)butane (0.027 g, 0.060 mmol) and ytterbium triflate (0.024 g, 0.040 mmol) were placed under vacuum and then flushed with nitrogen (four times). Subsequently, degassed DMF (3 mL), phenylacetylene (0.22 mL, 2.0 mmol) and degassed water (0.108 mL, 6.00 mmol) were added. The mixture was stirred under nitrogen at 60 °C for 6 h, then poured into sat. aqueous NaHCO₃ (30 mL). The resulting mixture was extracted with EtOAc (4 × 20 mL), the combined organic layers were washed with water (30 mL) and brine (30 mL), dried (MgSO₄) and the solvent removed in vacuo. Purification by silica gel column chromatography (eluting with n-hexane/EtOAc 9 : 1 to n-hexane/EtOAc 7.5 : 2.5) gave the desired product 12 as a yellow oil (0.21 g, 78%). R_f 0.48 (n-hexane/EtOAc 7 : 3); IR (ATR) ν_max 3333, 2977, 1674, 1512, 1453, 1368, 1252 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.42 (1H, d, J = 10.4 Hz, NH-4), 7.37 (2H, t, J = 7.7 Hz, 2H-9), 7.27 (2H, d, J = 7.7 Hz, 2H-8), 7.24–7.22 (1H, m, H-10), 6.93 (1H, dd, J = 10.4, 9.7 Hz, H-5), 5.79 (1H, d, J = 9.7 Hz, H-6), 5.08 (1H, br s, NH-1), 3.83 (2H, d, J = 6.0 Hz, H₂-2), 1.42 (9H, s, CO₂C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) δ 167.6 (C-3), 156.3 (CO₂C(CH₃)₃), 135.5 (C-7), 129.2 (2C-9), 128.0 (2C-8), 127.1 (C-10), 121.3 (C-5), 111.1 (C-6), 80.9 (CO₂C(CH₃)₃), 44.9 (C-2), 28.4 (CO₂C(CH₃)₃); (+)-HRESIMS [M + Na]⁺ 299.1370 (calcd for C₁₅H₂₀N₂NaO₃, 299.1366).

(Z)-2-Amino-N-styrylacetamide (8). A solution of piperidine (0.9 mL, 20% in DMF) was added to 10 (0.086 g, 0.22 mmol) and stirred under nitrogen for 20 min. EtOAc (20 mL) was added and washed with H₂O (5 mL), the organic layer separated and the solvent removed in vacuo. Purification using silica gel column chromatography (eluting with n-hexane to EtOAc to CH₂Cl₂/MeOH 9 : 1) gave the desired product 8 as a yellow oil (0.032 g, 84%).

Alternatively, a solution of TFA/TIS/H₂O (0.5 mL, 95 : 2.5 : 2.5) was added to 12 (0.042 g, 0.15 mmol) and the solution was stirred at 0 °C under nitrogen for 20 min. The reaction was concentrated in vacuo. Purification using silica gel column chromatography (eluting with n-hexane/EtOAc 9 : 1 to EtOAc to CH₂Cl₂/MeOH 9 : 1) gave the desired product 8 as a yellow oil (0.021 g, 78%).

R _f 0.68 (n-hexane/EtOAc 1 : 1); IR (ATR) ν_max 3305, 3023, 1677, 1645, 1503, 1477, 1442 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 9.85 (1H, br s, NH-4), 7.38 (2H, t, J = 7.3 Hz, 2H-9), 7.33 (2H, d, J = 7.3 Hz, 2H-8), 7.24 (1H, t, J = 7.3 Hz, H-10), 6.95 (1H, dd, J = 11.9, 9.6 Hz, H-5), 5.76 (1H, d, J = 9.6 Hz, H-6), 3.43 (2H, s, H₂-2); ¹³C NMR (CDCl₃, 125 MHz) δ 170.7 (C-3), 136.0 (C-7), 129.0 (2C-9), 128.0 (2C-8), 126.9 (C-10), 121.2 (C-5), 110.6 (C-6), 44.6 (C-2); (+)-HRESIMS [M + H]⁺ 177.1023 (calcd for C₁₀H₁₃N₂O, 177.1022).

Fmoc-His(Trt)-DOPA(TBDMS)₂-OH (7). A solution of Fmoc-DOPA(TBDMS)₂-OH¹² (2.00 g, 3.09 mmol) in CH₂Cl₂ (25 mL) was added to 2-chlorotrityl chloride resin (loading at 0.5 mmol g⁻¹, 6.18 g), followed by DIPEA (0.54 mL, 3.09 mmol). After the resin mixture was agitated for 10 min, DIPEA (0.81 mL, 4.63 mmol) was added and the mixture was further shaken for 1 h. The solution was drained off and the resin was washed with DMF (20 mL). A solution of CH₂Cl₂/MeOH/DIPEA (40 mL, 80 : 15 : 5) was added to the mixture and shaken for 20 min. The solution was drained and the procedure was repeated. The resin was then washed with DMF (20 mL). Piperidine in DMF (15 mL, 1 : 4) was added to the resin mixture and shaken for 10 min. The liquid was drained off and the piperidine washing was repeated for another 20 min. The amino acid-loaded resin was thoroughly washed with DMF (20 mL), isopropanol (20 mL) and n-hexane (20 mL). The resin was dried under vacuum for 30 min and stored in a desiccator overnight. CH₂Cl₂ (40 mL) was added to the amino acid-loaded resin which was left to swell for 1 h. The solution was drained and a solution of HBTU (4.39 g, 11.6 mmol), HOBt (1.57 g, 11.6 mmol), Fmoc-His(Trt)-OH (4.79 g, 7.72 mmol) and DIPEA (2.69 mL, 15.4 mmol) in DMF (11.6 mL) was added. The amino acid resin mixture was agitated for 2 h. The solution was then drained and washed with DMF (20 mL), isopropanol (20 mL) and n-hexane (20 mL). The resin was extensively dried and stored in a desiccator overnight. 2,2,2-Trifluoroethanol in CH₂Cl₂ (11.7 mL, 1 : 4) was added to the amino acid-loaded resin and agitated for 1 h. The solution was drained and the organic solvent was removed in vacuo to afford 7 as a yellowish-brown foam (2.70 g, 85% yield). M.p. 129–130 °C; [α]^21.9_D −5.7 (c 0.71, CH₂Cl₂); R_f 0.61 (CH₂Cl₂/MeOH 9 : 1); IR (ATR) ν_max 3320, 2930, 2857, 1723, 1655, 1509, 1446, 1251, 1128 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 7.94 (1H, br s, NH-10), 7.75 (2H, d, J = 7.3 Hz, 2H-FmocAr), 7.55–7.51 (3H, m, H-6 and 2H-FmocAr), 7.38 (2H, t, J = 7.3 Hz, 2H-FmocAr), 7.29–7.28 (2H, m, 2H-FmocAr), 7.26–7.23 (9H, m, 9H-TrtAr), 7.03–7.01 (6H, m, 6H-TrtAr), 6.75 (2H, br s, H-14 and H-17), 6.67–6.65 (2H, m, H-18 and H-8), 6.02 (1H, d, J = 7.8 Hz, NH-1), 4.72–4.66 (2H, m, H-2 and H-11), 4.29 (1H, dd, J = 12.9, 11.0 Hz, CO₂CH₂CH-a), 4.09–4.05 (2H, m, CO₂CH₂CH-b and CO₂CH₂CH), 3.33 (1H, d, J = 12.7 Hz, H₂-3a), 3.07 (1H, dd, J = 13.6, 4.7 Hz, H₂-12a), 3.01–2.97 (1H, m, H₂-12b), 2.64 (1H, dd, J = 12.7, 12.7 Hz, H₂-3b), 0.92–0.89 (18H, m, 2SiC(CH₃)₃), 0.11–0.08 (12H, m, 2Si(CH₃)₂); ¹³C NMR (CDCl₃, 125 MHz) δ 175.6 (C-19), 170.7 (C-9), 155.7 (CO₂CH₂CH), 146.4 (C-16), 145.5 (C-15), 144.1 (C-FmocAr), 143.9 (C-FmocAr), 141.5 (3C-TrtAr), 141.3 (2C-FmocAr), 137.7 (C-6), 134.8 (C-4), 130.6 (C-13), 129.8 (6C-TrtAr), 128.5 (3C-TrtAr), 128.4 (6C-TrtAr), 127.8 (2C-FmocAr), 127.2 (2C-FmocAr), 125.6 (C-FmocAr), 125.3 (C-FmocAr), 122.7 (C-14 or C-18), 122.5 (C-14 or C-18), 120.9 (C-17), 120.3 (C-8), 120.0 (2C-FmocAr), 76.3 (CAr₃), 67.1 (CO₂CH₂CH), 55.3 (C-2 or C-11), 55.0 (C-2 or C-11), 47.3 (CO₂CH₂CH), 38.4 (C-12), 32.4 (C-3), 26.1 (SiC(CH₃)₃), 26.0 (SiC(CH₃)₃), 18.5 (SiC(CH₃)₃), 18.4 (SiC(CH₃)₃), −3.9 (Si(CH₃)₂), −4.0 (Si(CH₃)₂); (+)-HRESIMS [M + H]⁺ 1027.4890 (calcd for C₆₁H₇₁N₄O₇Si₂, 1027.4856).

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-3-(3,4-bis((tert-butyldimethylsilyl)oxy)phenyl)-1-oxo-1-((2-oxo-2-(((Z)-styryl)amino)ethyl)amino)propan-2-yl)amino)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-2-yl)carbamate (13). To a solution of 7 (74.2 mg, 72.4 μmol), HBTU (54.9 mg, 0.140 mmol) and HOBt (19.6 mg, 0.14 mmol) dissolved in DMF (0.50 mL) was added DIPEA (37.8 μL, 0.22 mmol). The mixture was stirred at r.t. under nitrogen for 1 h before a solution of 8 (12.7 mg, 72.4 μmol) in DMF (0.50 mL) was added. The reaction mixture was stirred at r.t. for 26 h before EtOAc (20 mL) was added and washed with H₂O (10 mL), 10% aqueous HCl (10 mL), sat. aqueous NaHCO₃ (10 mL) and brine (10 mL). The organic layer was dried (MgSO₄), filtered and the solvent removed in vacuo to give a yellow oil. Purification using silica gel chromatography (eluting with n-hexane/EtOAc 8 : 2 to n-hexane/EtOAc 6 : 4) gave the desired product 13 as a yellow foam (30.0 mg, 35%). [α]^22.9_D −1.2 (c 0.83, CH₂Cl₂). M.p. 94–96 °C; R_f 0.57 (n-hexane/EtOAc 1 : 1); IR (ATR) ν_max 3301, 3025, 2929, 1652, 1509, 1493, 1252 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 9.26 (1H, br s, NH-20), 8.51 (1H, d, J = 10.7 Hz, NH-23), 7.75 (1H, d, J = 6.5 Hz, H-FmocAr), 7.73 (1H, d, J = 5.5 Hz, H-FmocAr), 7.54 (1H, d, J = 7.5 Hz, H-FmocAr), 7.51 (1H, d, J = 7.5 Hz, H-FmocAr), 7.38–7.34 (4H, m, 2H-28 and 2H-FmocAr), 7.31–7.30 (7H, m, 6H-TrtAr and H-6), 7.26–7.25 (4H, m, 2H-27 and 2H-FmocAr), 7.23–7.19 (3H, m, 3H-TrtAr), 7.07–7.05 (7H, m, H-29 and 6H-TrtAr), 6.75 (1H, dd, J = 10.7, 10.0 Hz, H-24), 6.72 (1H, d, J = 8.1 Hz, H-17), 6.66 (1H, s, H-14), 6.59 (1H, s, H-8), 6.52 (1H, dd, J = 8.1, 1.4 Hz, H-18), 6.15 (1H, d, J = 6.5 Hz, NH-10), 6.03 (1H, d, J = 5.4 Hz, NH-1), 5.68 (1H, d, J = 10.0 Hz, H-25), 4.69 (1H, ddd, J = 6.5, 6.5, 6.5 Hz, H-11) 4.31–4.23 (2H, m, CO₂CH₂CH), 4.18–4.17 (1H, m, H-2), 4.13 (1H, t, J = 7.2 Hz, CO₂CH₂CH), 3.99 (1H, dd, J = 16.3, 5.8 Hz, H₂-21a), 3.84 (1H, dd, J = 16.3, 5.8 Hz, H₂-21b), 3.07 (1H, dd, J = 14.7, 6.5 Hz, H₂-12a), 3.00–2.95 (2H, m, H₂-3a and H₂-12b), 2.69 (1H, dd, J = 14.4, 3.6 Hz, H₂-3b), 0.96–0.94 (18H, m, 2SiC(CH₃)₃), 0.16–0.15 (12H, m, 2Si(CH₃)₂); ¹³C NMR (CDCl₃, 125 MHz) δ 172.3 (C-19), 170.7 (C-9), 167.5 (C-22), 156.1 (CO₂CH₂CH), 147.4 (C-15), 146.2 (C-16), 143.9 (C-FmocAr), 143.8 (C-FmocAr), 142.2 (3C-TrtAr), 141.4 (2C-FmocAr), 138.7 (C-6), 135.6 (C-26), 135.4 (C-4), 129.8 (6C-TrtAr), 129.3 (C-13), 129.1 (2C-28), 128.4 (3C-TrtAr), 128.3 (2C-27 and 6C-TrtAr), 127.9 (2C-FmocAr), 127.2 (2C-FmocAr), 127.1 (C-29), 125.3 (2C-FmocAr), 122.0 (C-18), 121.9 (C-14), 121.4 (C-17 and C-24), 120.7 (C-8), 120.1 (2C-FmocAr), 111.2 (C-25), 75.7 (CAr₃), 67.2 (CO₂CH₂CH), 54.9 (C-2), 54.3 (C-11), 47.3 (CO₂CH₂CH), 44.0 (C-21), 36.3 (C-12), 31.0 (C-3), 26.1 (2SiC(CH₃)₃), 18.6 (2SiC(CH₃)₃), −3.9 (2Si(CH₃)₂); (+)-HRESIMS [M + H]⁺ 1185.5726 (calcd for C₇₁H₈₁N₆O₇Si₂, 1185.5700).

(S)-2-Amino-N-((S)-3-(3,4-bis((tert-butyldimethylsilyl)oxy)phenyl)-1-oxo-1-((2-oxo-2-(((Z)-styryl)amino)ethyl)amino)propan-2-yl)-3-(1-trityl-1H-imidazol-4-yl)propanamide (14). Piperidine (0.32 mL, 20% in DMF) was added to 13 (0.078 g, 0.066 mmol) and stirred at r.t. under nitrogen atmosphere. After 1 h, the brown solution was added to EtOAc (20 mL) and washed with water (10 mL). The aqueous layer was further washed with EtOAc (2 × 20 mL), the organic layers were combined and dried in vacuo. Purification by silica gel column chromatography (CH₂Cl₂/MeOH 9 : 1), afforded the desired product 14 as a yellow oil (59.4 mg, 94%). [α]^21.9_D −24.2 (c 0.91, CH₂Cl₂); R_f 0.49 (CH₂Cl₂/MeOH 9 : 1); IR (ATR) ν_max 3312, 2929, 2857, 1650, 1508, 1444, 1252, 1128 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.60 (1H, dd, J = 5.7, 5.6 Hz, NH-20), 8.51 (1H, d, J = 10.8 Hz, NH-23), 7.38–7.27 (15H, m, 9H-TrtAr, H-6, NH-10, 2H-27 and 2H-28), 7.20 (1H, t, J = 7.4 Hz, H-29), 7.09–7.06 (6H, m, 6H-TrtAr), 6.78 (1H, dd, J = 10.8, 9.8 Hz, H-24), 6.71 (1H, d, J = 8.1 Hz, H-17), 6.65 (1H, d, J = 2.0 Hz, H-14), 6.59 (1H, s, H-8), 6.54 (1H, dd, J = 8.1, 2.0 Hz, H-18), 5.71 (1H, d, J = 9.8 Hz, H-25), 4.45 (1H, ddd, J = 7.1, 7.1, 7.1 Hz, H-11), 3.93 (1H, dd, J = 16.3, 5.6 Hz, H₂-21a), 3.87 (1H, dd, J = 16.3, 5.7 Hz, H₂-21b), 3.40 (1H, dd, J = 5.5, 5.5 Hz, H-2), 3.09 (1H, dd, J = 14.1, 7.1 Hz, H₂-12a), 2.98 (1H, dd, J = 14.1, 7.1 Hz, H₂-12b), 2.82 (1H, dd, J = 14.9, 5.5 Hz, H₂-3a), 2.71 (1H, dd, J = 14.9, 5.5 Hz, H₂-3b), 0.97–0.96 (18H, m, 2SiC(CH₃)₃), 0.17–0.16 (12H, m, 2Si(CH₃)₂); ¹³C NMR (CDCl₃, 125 MHz) δ 174.3 (C-9)^a, 172.5 (C-19), 167.6 (C-22), 147.0 (C-15), 145.9 (C-16), 142.3 (3C-TrtAr), 138.8 (C-6), 136.4 (C-4), 135.5 (C-26), 130.2 (C-13), 129.8 (6C-TrtAr), 129.1 (2C-28), 128.3 (9C-TrtAr and 2C-27), 127.0 (C-29), 122.2 (C-18), 122.0 (C-14), 121.4 (C-24), 121.2 (C-17), 120.2 (C-8), 111.4 (C-25), 75.6 (CAr₃), 55.2 (C-11), 54.7 (C-2), 44.0 (C-21), 36.1 (C-12), 32.3 (C-3), 26.1 (2SiC(CH₃)₃), 18.6 (SiC(CH₃)₃), 18.5 (SiC(CH₃)₃), −3.9 (2Si(CH₃)₂); (+)-HRESIMS [M + H]⁺ 963.5055 (calcd for C₅₆H₇₁N₆O₅Si₂, 963.5019). ^a assignment by HMBC correlation.

(S)-2-Amino-N-((S)-3-(3,4-dihydroxyphenyl)-1-oxo-1-((2-oxo-2-(((Z)-styryl)amino)ethyl)amino)propan-2-yl)-3-(1-trityl-1H-imidazol-4-yl)propanamide (15). Compound 14 (54.8 mg, 56.9 μmol) was dissolved in THF (0.50 mL) under nitrogen atmosphere and the resulting yellow solution was stirred in an ice bath. Triethylamine trihydrofluoride (27.8 μL, 0.170 mmol) was then added dropwise to the yellow solution and continued to stir for 45 min. The reaction mixture was dried under a stream of N₂ gas, after which, water (15 mL) was added and the aqueous layer was extracted with CH₂Cl₂ (4 × 20 mL), the organic layers were combined and solvent was removed in vacuo to give a yellow foam. Purification by silica gel column chromatography (eluting with EtOAc to MeOH/CH₂Cl₂, 1 : 9), afforded 15 as a yellow oil (30.5 mg, 73%). [α]^22.7_D −21.9 (c 1.42, CH₂Cl₂); R_f 0.26 (CH₂Cl₂/MeOH 9 : 1); IR (ATR) ν_max 3277, 3057, 2926, 1651, 1508, 1486, 1260 cm⁻¹; ¹H NMR (CD₃OD, 500 MHz) δ 7.42 (1H, d, J = 1.0 Hz, H-6), 7.36–7.33 (13H, m, 2H-27, 2H-28 and 9H-TrtAr), 7.20–7.17 (1H, m, H-29) 7.14–7.12 (6H, m, 6H-TrtAr), 6.74 (1H, d, J = 9.7 Hz, H-24), 6.70 (1H, s, H-8), 6.64 (1H, d, J = 1.9 Hz, H-14), 6.58 (1H, d, J = 8.1 Hz, H-17), 6.44 (1H, dd, J = 8.1, 1.9 Hz, H-18), 5.77 (1H, d, J = 9.7 Hz, H-25), 4.49 (1H, dd, J = 9.0, 5.6 Hz, H-11), 3.98 (1H, d, J = 16.8 Hz, H₂-21a), 3.84 (1H, d, J = 16.8 Hz, H₂-21b), 3.72 (1H, t, J = 5.6 Hz, H-2), 2.98 (1H, dd, J = 13.8, 5.6 Hz, H₂-12a), 2.78 (1H, dd, J = 14.8, 5.6 Hz, H₂-3a), 2.73–2.66 (2H, m, H₂-3b and H₂-12b); ¹³C NMR (CD₃OD, 125 MHz) δ 174.3 (C-9 or C-19), 174.2 (C-9 or C-19), 169.7 (C-22), 146.3 (C-15), 145.3 (C-16), 143.6 (3C-TrtAr), 140.0 (C-6), 136.9 (C-4), 136.7 (C-26), 130.9 (6C-TrtAr), 129.8 (3C-TrtAr), 129.5 (C-13), 129.4 (2C-27 or 2C-28), 129.34 (2C-27 or 2C-28), 129.28 (6C-TrtAr), 128.0 (C-29), 122.1 (C-24), 121.64 (C-8 or C-18), 121.57 (C-8 or C-18), 117.4 (C-14), 116.3 (C-17), 113.4 (C-25), 76.9 (CAr₃), 56.3 (C-11), 55.2 (C-2), 43.7 (C-21), 38.1 (C-12), 32.7 (C-3); (+)-HRESIMS [M + H]⁺ 735.3292 (calcd for C₄₄H₄₃N₆O₅, 735.3289).

4-((S)-2-Ammonio-3-(((S)-3-(3,4-dihydroxyphenyl)-1-oxo-1-((2-oxo-2-(((Z)-styryl)amino)ethyl)amino)propan-2-yl)amino)-3-oxopropyl)-1H-imidazol-3-ium dichloride (2). A solution of 0.01 N HCl/HFIP–TIS/H₂O (1 mL, 95 : 2.5 : 2.5) was added to 15 (35.0 mg, 47.7 μmol) and the solution was stirred at r.t. for 1 h. The solution was then dried under nitrogen and the crude product was purified by C₈ column chromatography (eluting with H₂O to H₂O/MeOH 6 : 4) to afford the desired product 2 as a white solid (18.0 mg, 77%). M.p. 240 °C (decomposed); [α]^20.9_D −5.7 (c 3.12, MeOH); R_f 0.66 (butan-1-ol/acetic acid/water 2 : 1 : 1); IR (ATR) ν_max 3023, 2924, 1653, 1517, 1493, 1445, 1260, 1078, 1031 cm⁻¹; ¹H NMR (DMSO-d₆, 500 MHz) δ 9.54 (1H, d, J = 10.2 Hz, NH-23), 8.65 (1H, br s, H-20), 8.08 (1H, br s, NH-10), 7.54 (1H, s, H-6), 7.39–7.34 (4H, m, 2H-27 and 2H-28), 7.22 (1H, tt, J = 6.8, 1.8 Hz, H-29), 6.81 (1H, br s, H-8), 6.76 (1H, dd, J = 10.2, 10.0 Hz, H-24), 6.60–6.59 (2H, m, H-14 and H-17), 6.41 (1H, dd, J = 8.2, 2.0 Hz, H-18), 5.69 (1H, d, J = 10.0 Hz, H-25), 4.44 (1H, br s, H-11), 3.96 (1H, dd, J = 16.7, 5.4 Hz, H₂-21a), 3.89 (1H, dd, J = 16.7, 5.4 Hz, H₂-21b), 3.34 (1H, dd, J = 8.2, 4.1 Hz, H-2), 2.90 (1H, dd, J = 13.9, 4.3 Hz, H₂-12a), 2.78 (1H, dd, J = 14.4, 4.1 Hz, H₂-3a), 2.67 (1H, dd, J = 13.9, 9.0 Hz, H₂-12b), 2.48 (1H, d, J = 8.2 Hz, H₂-3b); ¹³C NMR (DMSO-d₆, 125 MHz) δ 174.1 (C-9), 171.9 (C-19), 168.1 (C-22), 144.8 (C-15), 143.7 (C-16), 135.4 (C-26), 134.9 (C-6), 128.6 (2C-28), 128.4 (C-13), 128.2 (2C-27), 126.5 (C-29), 121.7 (C-24), 120.0 (C-18), 116.6 (C-14), 115.2 (C-17), 110.2 (C-25), 54.9 (C-2), 53.7 (C-11), 42.4 (C-21), 37.1 (C-12), 32.1 (C-3); (+)-HRESIMS [M + H]⁺ 493.2182 (calcd for C₂₅H₂₉N₆O₅, 493.2194).

tert-Butyl 6-bromo-3-iodo-1H-indole-1-carboxylate (19). 6-Bromo-3-iodo-indole 18 ^16,17 (0.66 g, 2.0 mmol), DMAP (0.02 g, 0.2 mmol) and di-tert-butyl dicarbonate (0.67 g, 3.1 mmol) were dissolved in CH₂Cl₂ (4 mL) and stirred for 75 min at r.t. under nitrogen. 10% aqueous HCl (20 mL) was added and extracted with CH₂Cl₂ (3 × 30 mL). The organic layers were combined, dried (MgSO₄) and filtered. The solvent was then removed in vacuo. The crude product was subjected to silica gel column chromatography (n-hexane) to yield 19 as an orange solid (0.86 g, 71% yield). M.p. 148–149 °C; R_f 0.70 (n-hexane/EtOAc 9 : 1); IR (ATR) ν_max 2986, 1732, 1602, 1427, 1365, 1244, 1115 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.35 (1H, br s, H-7), 7.68 (1H, s, H-2), 7.42 (1H, dd, J = 8.3, 1.8 Hz, H-5), 7.25 (1H, d, J = 8.3 Hz, H-4), 1.67 (9H, s, CO₂C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) δ 148.4 (CO₂C(CH₃)₃), 135.5 (C-7a), 131.2 (C-3a), 130.7 (C-2), 126.8 (C-5), 122.8 (C-4), 119.5 (C-6), 118.4 (C-7), 85.1 (CO₂C(CH₃)₃), 65.0 (C-3), 28.2 (CO₂C(CH₃)₃); (+)-HRESIMS [M + Na]⁺ 443.9060 (calcd for C₁₃H₁₃⁷⁹Br¹²⁶INNaO₂, 443.9067).

tert-Butyl 6-bromo-3-((trimethylsilyl)ethynyl)-1H-indole-1-carboxylate (20). Triethylamine (1.40 mL) was added to a solution of tert-butyl 6-bromo-3-iodo-1H-indole-1-carboxylate (19) (0.59 g, 1.40 mmol) in DMF (1.40 mL) and was degassed in a sonic bath for 30 min, under nitrogen. Bis(triphenylphosphine)palladium(II) dichloride (19.7 mg, 28.1 μmol), copper(I) iodide (10.7 mg, 56.2 μmol) and ethynyltrimethylsilane (174.6 μL, 1.26 mmol) were added to the solution. After the solution was stirred at 60 °C for 2 h, it was quenched with water (30 mL) and extracted with EtOAc (3 × 25 mL). The organic layers were combined and dried (MgSO₄). The organic solvent was then removed in vacuo. The crude product was passed through a short plug of silica (n-hexane/EtOAc 20 : 1) to afford 20 as a brown oil (0.489 g, 99% yield). R_f 0.93 (n-hexane/EtOAc 9 : 1); IR (ATR) ν_max 2980, 2162, 1734, 1432, 1364, 1247, 1154, 1092 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.35 (1H, br s, H-7), 7.72 (1H, s, H-2), 7.51 (1H, d, J = 8.4 Hz, H-4), 7.41 (1H, dd, J = 8.4, 1.9 Hz, H-5), 1.66 (9H, s, CO₂C(CH₃)₃), 0.28 (9H, s, Si(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) δ 148.8 (CO₂C(CH₃)₃), 135.3 (C-7a), 129.9 (C-2), 129.5 (C-3a), 126.6 (C-5), 121.4 (C-4), 119.1 (C-6), 118.6 (C-7), 103.6 (C-3), 98.8 (C-9), 96.2 (C-8), 85.0 (CO₂C(CH₃)₃), 28.2 (CO₂C(CH₃)₃), 0.2 (Si(CH₃)₃); (+)-HRESIMS [M + Na]⁺ 414.0495 (calcd for C₁₈H₂₂⁷⁹BrNNaO₂Si, 414.0495).

tert-Butyl 6-bromo-3-ethynyl-1H-indole-1-carboxylate (17). tert-Butyl 6-bromo-3-((trimethylsilyl)ethynyl)-1H-indole-1-carboxylate 20 (0.54 g, 1.38 mmol) in THF (30 mL) was stirred in ice bath for 10 min before tetrabutylammonium fluoride (0.44 g, 1.68 mmol) was added and was further stirred in ice bath for 20 min, under nitrogen. Sat. aqueous NH₄Cl (25 mL) was added and extracted with diethyl ether (4 × 20 mL), dried (MgSO₄) and concentrated in vacuo. The crude brown oil was purified by silica gel column chromatography eluting with n-hexane to give 17 as brown solid (0.32 g, 72% yield). M.p. 115–117 °C; R_f 0.64 (n-hexane/EtOAc 9 : 1); IR (ATR) ν_max 3292, 2987, 1735, 1456, 1364, 1312, 1251 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.31 (1H, br s, H-7), 7.73 (1H, s, H-2), 7.47 (1H, d, J = 8.3 Hz, H-4), 7.37 (1H, dd, J = 8.3, 1.8 Hz, H-5), 3.23 (1H, s, H-9), 1.66 (9H, s, CO₂C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) δ 148.6 (CO₂C(CH₃)₃), 135.2 (C-7a), 130.2 (C-2), 129.3 (C-3a), 126.6 (C-5), 121.1 (C-4), 119.1 (C-6), 118.5 (C-7), 102.3 (C-3), 85.0 (CO₂C(CH₃)₃), 81.2 (C-9), 75.2 (C-8), 28.1 (CO₂C(CH₃)₃); (+)-HRESIMS [M + Na]⁺ 342.0099 (calcd for C₁₅H₁₄⁷⁹BrNNaO₂, 342.0100).

tert-Butyl (Z)-3-(2-(2-aminoacetamido)vinyl)-6-bromo-1H-indole-1-carboxylate (21) and di-tert-butyl 3,3′-(but-1-en-3-yne-1,4-diyl)(E)-bis(6-bromo-1H-indole-1-carboxylate) (22). Fmoc-Gly-NH₂ (9) (0.287 g, 0.97 mmol), tert-butyl 6-bromo-3-ethynyl-1H-indole-1-carboxylate 17 (0.618 g, 1.94 mmol), bis(2-methylallyl)(1,5-cyclooctadiene)ruthenium(II) (15.0 mg, 47.0 μmol), 1,4-bis(dicyclohexylphosphino)butane (26.0 mg, 58.0 μmol) and ytterbium triflate (24.0 mg, 38.7 μmol) were placed under vacuum and then flushed with nitrogen (four times). Subsequently, degassed DMF (3.00 mL) was added, followed by degassed water (105.0 μL, 5.82 mmol) and the mixture was further stirred under nitrogen at 70 °C for 24 h. The reaction mixture was added to sat. aqueous NaHCO₃ (30 mL) and the resulting mixture was extracted with EtOAc (5 × 20 mL). The organic layers were combined and washed with water (30 mL) then brine (30 mL), dried (MgSO₄), filtered, and the solvent removed in vacuo. Purification using silica gel column chromatography (eluting with n-hexane to n-hexane/EtOAc 9 : 1) gave enyne 22 as a brown oil (37.2 mg, 6% yield) and (eluting with n-hexane/EtOAc 9 : 1 to EtOAc) 21 as a yellow oil (0.164 g, 43% yield).

tert-Butyl (Z)-3-(2-(2-aminoacetamido)vinyl)-6-bromo-1H-indole-1-carboxylate (21). R _f 0.14 (EtOAc); IR (ATR) ν_max 3333, 2979, 1732, 1694, 1497, 1370, 1250, 1155 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 9.70 (1H, d, J = 11.3 Hz, H-4), 8.35 (1H, s, H-10), 7.71 (1H, s, H-8), 7.44–7.38 (2H, m, H-12 and H-13), 7.06 (1H, dd, J = 11.3, 9.6 Hz, H-5), 5.79 (1H, d, J = 9.6 Hz, H-6), 3.47 (2H, s, H₂-2), 1.68 (9H, s, CO₂C(CH₃)₃); ¹³C NMR (CDCl₃, 100 MHz) δ 170.4 (C-3), 149.3 (CO₂C(CH₃)₃), 135.8 (C-9a), 128.7 (C-13a), 126.2 (C-12), 122.9 (C-5), 122.3 (C-8), 120.6 (C-13), 119.0 (C-11), 118.6 (C-10), 115.7 (C-7), 99.7 (C-6), 84.6 (CO₂C(CH₃)₃), 44.6 (C-2), 28.3 (CO₂C(CH₃)₃); (+)-HRESIMS [M + H]⁺ 394.0751 (calcd for C₁₇H₂₁⁷⁹BrN₃O₃, 394.0761).

Di-tert-butyl 3,3′-(but-1-en-3-yne-1,4-diyl)(E)-bis(6-bromo-1H-indole-1-carboxylate) (22). R _f 0.59 (n-hexane/EtOAc 9 : 1); IR (ATR) ν_max 2980, 2934, 2860, 1736, 1602, 1431, 1360, 1249, 1081 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 8.35 (2H, d, J = 9.9 Hz, H-7 and H-15), 7.72 (1H, s, H-13), 7.65 (1H, s, H-2), 7.59 (1H, d, J = 8.4 Hz, H-4), 7.54 (1H, d, J = 8.3 Hz, H-18), 7.40 (1H, dd, J = 8.3, 1.7 Hz, H-17), 7.39 (1H, dd, J = 8.4, 1.7 Hz, H-5), 7.10 (1H, d, J = 16.4 Hz, H-8), 6.44 (1H, d, J = 16.4 Hz, H-9), 1.68 (18H, s, 3H₃-21 and 3H₃-24); ¹³C NMR (CDCl₃, 100 MHz) δ 149.0 (C-19), 148.7 (C-22), 136.7 (C-7a), 135.4 (C-14a), 132.2 (C-8), 129.3 (C-18a), 128.9 (C-13), 127.0 (C-3a), 126.6 (C-5 and C-17), 126.5 (C-5 and C-17), 125.0 (C-2), 121.3 (C-18), 121.0 (C-4), 119.1 (C-6 or C-16), 118.8 (C-3 and C-7 or C-15), 118.6 (C-7 or C-15), 118.3 (C-6 or C-16), 108.3 (C-9), 103.7 (C-12), 92.9 (C-10), 85.0 (C-20 or C-23), 84.9 (C-20 or C-23), 83.0 (C-11), 28.2 (3C-21 and 3C-24); (+)-HRESIMS [M + Na]⁺ 661.0302 (calcd for C₃₀H₂₈⁷⁹Br₂N₂O₄Na, 661.0308).

(Z)-2-Amino-N-(2-(6-bromo-1H-indol-3-yl)vinyl)acetamide (16). A solution of 21 (0.167 g, 0.425 mmol) in CH₂Cl₂ (1.30 mL) was stirred in a salted ice bath under nitrogen for 5 min before TFA (1.30 mL, 17.0 mmol) was added dropwise. The solution was stirred for 8 h in an ice bath. Sat. aqueous NaHCO₃ (20 mL) was added and the mixture was extracted with EtOAc (4 × 20 mL). The organic layers were combined and dried in vacuo. The crude black oil was subjected to silica gel column chromatography (eluting with EtOAc to CH₂Cl₂/MeOH 9 : 1) to give 16 as a black oil (0.10 g, 80% yield). R_f 0.90 (CH₂Cl₂/MeOH 9 : 1); IR (ATR) ν_max 3209, 2931, 1652, 1532, 1455, 1227, 1002 cm⁻¹; ¹H NMR (DMSO-d₆, 400 MHz) δ 11.73 (1H, br s, NH-9), 7.69 (1H, br, s, H-8), 7.62 (1H, d, J = 1.8 Hz, H-10), 7.56 (1H, d, J = 8.5 Hz, H-13), 7.17 (1H, dd, J = 8.5, 1.8 Hz, H-12), 6.73 (1H, d, J = 9.4 Hz, H-5), 5.95 (1H, d, J = 9.4 Hz, H-6), 3.53 (2H, br s, H₂-2); ¹³C NMR (DMSO-d₆, 100 MHz) δ 168.6 (C-3), 136.7 (C-9a), 125.7 (C-13a), 124.5 (C-8), 122.0 (C-12), 120.3 (C-13), 118.3 (C-5), 114.6 (C-11), 114.3 (C-10), 110.0 (C-7), 102.2 (C-6), 42.8 (C-2); (+)-HRESIMS [M + Na]⁺ 316.0060 (calcd for C₁₂H₁₂⁷⁹BrN₃NaO, 316.0056).

(9H-Fluoren-9-yl)methyl((S)-1-(((S)-3-(3,4-bis((tert-butyldimethylsilyl)oxy)phenyl)-1-((2-(((Z)-2-(6-bromo-1H-indol-3-yl)vinyl)amino)-2-oxoethyl)amino)-1-oxopropan-2-yl)amino)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-2-yl)carbamate (23). Fmoc-His(Trt)-DOPA(TBDMS)₂-OH 7 (0.267 g, 0.26 mmol), EDC·HCl (54 mg, 0.28 mmol), HOBt (51.9 mg, 0.38 mmol) and enamide 16 (76.2 mg, 0.26 mmol) were dissolved in CH₂Cl₂ (3 mL). DIPEA (0.22 mL, 1.28 mmol) was added to the solution and stirred at r.t. under a nitrogen atmosphere for 6 h. EtOAc (20 mL) was added and washed with H₂O (10 mL). The aqueous layer was washed with EtOAc (3 × 15 mL) and combined. The organic layers were dried (MgSO₄), filtered and the solvent removed in vacuo to give a yellow crude oil. Purification using silica gel chromatography (eluting with n-hexane/EtOAc 8 : 2 to n-hexane/EtOAc 4 : 6) gave 23 as a yellow oil (61.2 mg, 18% yield). R_f 0.61 (CH₂Cl₂/MeOH 9 : 1); [α]^23.0_D +12.6 (c 1.03, CH₂Cl₂); IR (ATR) ν_max 3317, 2930, 2857, 1657, 1506, 1446, 1251, 1229, 1158, 1129, 1041 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 9.06 (1H, br s, NH-28), 9.00 (1H, br s, NH-20), 8.21 (1H, d, J = 10.7 Hz, H-23), 7.74 (1H, d, J = 7.7 Hz, H-FmocAr), 7.73 (1H, d, J = 7.7 Hz, H-FmocAr), 7.52 (1H, d, J = 7.7 Hz, H-FmocAr), 7.50 (1H, d, J = 7.7 Hz, H-FmocAr), 7.49 (1H, br s, H-29), 7.41 (1H, d, J = 8.3 Hz, H-32), 7.37 (2H, t, J = 7.7 Hz, 2H-FmocAr), 7.32 (1H, s, H-6), 7.28–7.23 (11H, m, 2H-FmocAr, 9H-TrtAr), 7.21 (1H, d, J = 8.3 Hz, H-31), 7.16 (1H, br s, H-27), 6.99–6.98 (6H, m, 6H-TrtAr), 6.75–6.68 (3H, m, H-14, H-17 and H-24), 6.58 (1H, br s, H-8), 6.54 (1H, d, J = 7.6 Hz, H-18), 6.30 (1H, br s, NH-10), 5.99 (1H, br s, NH-1), 5.79 (1H, d, J = 9.1 Hz, H-25), 4.78–4.76 (1H, m, H-11), 4.33–4.30 (3H, m, H-2 and CO₂CH₂CH), 4.23–4.18 (1H, m, H₂-21a), 4.15 (1H, t, J = 6.9 Hz, CO₂CH₂CH), 3.70 (1H, dd, J = 16.7, 4.2 Hz, H₂-21b), 3.11 (1H, dd, J = 14.1, 6.1 Hz, H₂-12a), 2.98 (1H, dd, J = 14.8, 4.2 Hz, H₂-3a), 2.94 (1H, dd, J = 14.1, 6.1 Hz, H₂-12b), 2.77 (1H, dd, J = 14.8, 4.2 Hz, H₂-3b), 0.96–0.95 (18H, m, 2SiC(CH₃)₃), 0.16 (12H, s, 2Si(CH₃)₂); ¹³C NMR (CDCl₃, 125 MHz) δ 172.0 (C-19), 170.8 (C-9), 166.6 (C-22), 156.3 (CO₂CH₂CH), 147.4 (C-15), 146.4 (C-16), 143.8 (C-FmocAr), 143.7 (C-FmocAr), 142.0 (3C-TrtAr), 141.4 (2C-FmocAr), 138.8 (C-6), 136.6 (C-28a), 135.2 (C-4), 129.7 (6C-TrtAr), 129.0 (C-13), 128.3 (9C-TrtAr), 128.0 (2C-FmocAr), 127.2 (2C-FmocAr), 126.0 (C-32a), 125.1 (2C-FmocAr), 123.4 (C-27), 123.2 (C-31), 122.3 (C-18), 122.0 (C-14), 121.4 (C-17), 120.8 (C-8), 120.2 (2C-FmocAr and C-32), 119.3 (C-24), 116.3 (C-30), 114.4 (C-29), 111.2 (C-26), 102.8 (C-25), 75.8 (CAr₃), 67.3 (CO₂CH₂CH), 54.9 (C-2), 54.0 (C-11), 47.3 (CO₂CH₂CH), 43.9 (C-21), 36.6 (C-12), 30.9 (C-3), 26.0 (2SiC(CH₃)₃), 18.6 (2SiC(CH₃)₃), −3.9 (2Si(CH₃)₂); (+)-HRESIMS [M + H]⁺ 1302.4954 (calcd for C₇₃H₈₁⁷⁹BrN₇O₇Si₂, 1302.4914).

(S)-2-Amino-N-((S)-3-(3,4-bis((tert-butyldimethylsilyl)oxy)phenyl)-1-((2-(((Z)-2-(6-bromo-1H-indol-3-yl)vinyl)amino)-2-oxoethyl)amino)-1-oxopropan-2-yl)-3-(1-trityl-1H-imidazol-4-yl)propanamide (24). Piperidine (20% in DMF, 0.50 mL) was added to protected halocyamine A 23 (25.0 mg, 19.2 μmol) and was stirred under N₂ at r.t. for 20 min. EtOAc was added to the reaction solution and the mixture was washed with water (5 mL). The aqueous layer was further washed with EtOAc (3 × 15 mL) and the organic layers were then combined and dried in vacuo. Purification by silica gel column chromatography (eluting with EtOAc to CH₂Cl₂/MeOH 9 : 1), afforded 24 as a yellow oil (15.6 mg, 75% yield). R_f 0.59 (CH₂Cl₂/MeOH 9 : 1); [α]^20.4_D −20.5 (c 1.07, CH₂Cl₂); IR (ATR) ν_max 3257, 2929, 1663, 1509, 1445, 1252, 1202, 1129, 1023 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) δ 9.58 (1H, br s, NH-28), 8.70 (1H, t, J = 5.9 Hz, NH-20), 8.22 (1H, d, J = 10.7 Hz, NH-23), 7.50 (1H, d, J = 1.6 Hz, H-29), 7.42 (1H, d, J = 8.5 Hz, H-32), 7.34 (1H, d, J = 0.9 Hz, H-6), 7.30–7.25 (9H, m, 9H-TrtAr), 7.22–7.19 (2H, m, H-27 and H-31), 7.01–6.99 (6H, m, 6H-TrtAr), 6.88 (1H, d, J = 7.5 Hz, NH-10), 6.73 (1H, d, J = 8.2 Hz, H-17), 6.71 (1H, dd, J = 10.7, 9.2 Hz, H-24), 6.67 (1H, d, J = 2.1 Hz, H-14), 6.60 (1H, d, J = 0.9 Hz, H-8), 6.56 (1H, dd, J = 8.2, 2.1 Hz, H-18), 5.80 (1H, d, J = 9.2 Hz, H-25), 4.68 (1H, ddd, J = 7.5, 7.5, 7.5 Hz, H-11), 4.14 (1H, dd, J = 16.9, 5.9 Hz, H₂-21a), 3.81 (1H, dd, J = 16.9, 5.9 Hz, H₂-21b), 3.54 (1H, dd, J = 5.2, 5.2 Hz, H-2), 3.08 (1H, dd, J = 14.1, 7.5 Hz, H₂-12a), 2.98 (1H, dd, J = 14.1, 7.5 Hz, H₂-12b), 2.88 (1H, dd, J = 14.8, 5.2 Hz, H₂-3a), 2.74 (1H, dd, J = 14.8, 5.2 Hz, H₂-3b), 0.97–0.96 (18H, m, 2SiC(CH₃)₃), 0.17–0.16 (12H, m, 2Si(CH₃)₂); ¹³C NMR (CDCl₃, 125 MHz) δ 174.7 (C-9), 172.3 (C-19), 166.6 (C-22), 147.1 (C-15), 146.1 (C-16), 142.0 (3C-TrtAr), 138.9 (C-6), 136.4 (C-28a), 135.7 (C-4), 129.6 (9C-TrtAr), 129.4 (C-13), 128.2 (6C-TrtAr), 125.9 (C-32a), 123.6 (C-27), 123.0 (C-31), 122.1 (C-18), 121.9 (C-14), 121.2 (C-17), 120.6 (C-8), 120.1 (C-32), 119.0 (C-24), 116.0 (C-30), 114.2 (C-29), 110.9 (C-26), 102.9 (C-25), 75.5 (CAr₃), 54.4 (C-11), 54.2 (C-2), 43.7 (C-21), 36.5 (C-12), 33.0 (C-3), 25.9 (2SiC(CH₃)₃), 18.5 (SiC(CH₃)₃), 18.4 (SiC(CH₃)₃), −4.1 (2Si(CH₃)₂); (+)-HRESIMS [M + H]⁺ 1080.4262 (calcd for C₅₈H₇₁⁷⁹BrN₇O₅Si₂, 1080.4233).

(S)-2-Amino-N-((S)-1-((2-(((Z)-2-(6-bromo-1H-indol-3-yl)vinyl)amino)-2-oxoethyl)amino)-3-(3,4-dihydroxyphenyl)-1-oxopropan-2-yl)-3-(1-trityl-1H-imidazol-4-yl)propanamide (25). To a solution of 24 (0.113 g, 0.105 mmol) in THF (2 mL) cooled in an ice bath, was added dropwise triethylamine trihydrofluoride (51 μL, 0.31 μmol). The solution was stirred at 0 °C under a nitrogen atmosphere for 1 h. The reaction was then dried under nitrogen and dissolved in H₂O (15 mL). The crude product was extracted from the aqueous layer with EtOAc (5 × 20 mL) and the organic layers were combined. Removal of solvent in vacuo gave the crude product as a yellow oil. Purification by silica gel column chromatography (eluting with EtOAc to MeOH/CH₂Cl₂, 1 : 9), afforded 25 as a yellow oil (56.5 mg, 63% yield). R_f 0.10 (CH₂Cl₂/MeOH 4 : 1); [α]^27.3_D −8.8 (c 1.96, CH₂Cl₂); IR (ATR) ν_max 3282, 2929, 1655, 1532, 1446, 1338, 1041 cm⁻¹; ¹H NMR (CD₃OD, 500 MHz) δ 7.52 (1H, d, J = 1.7 Hz, H-29), 7.43–7.41 (2H, m, H-27 and H-32), 7.36 (1H, br s, H-6), 7.32–7.30 (9H, m, 9H-TrtAr), 7.13 (1H, dd, J = 8.6 1.7 Hz, H-31), 7.08–7.06 (6H, m, 6H-TrtAr), 6.69 (1H, br s, H-8), 6.65–6.62 (3H, m, H-14, H-17 and H-24), 6.49 (1H, dd, J = 8.0, 1.9 Hz, H-18), 5.92 (1H, d, J = 9.9 Hz, H-25), 4.55 (1H, dd, J = 9.1, 5.2 Hz, H-11), 3.90 (1H, d, J = 16.7 Hz, H₂-21a), 3.86 (1H, d, J = 16.7 Hz, H₂-21b), 3.53 (1H, dd, J = 5.9, 5.9 Hz, H-2), 3.02 (1H, dd, J = 14.0, 5.2 Hz, H₂-12a), 2.79–2.68 (3H, m, H₂-3 and H₂-12b); ¹³C NMR (CD₃OD, 125 MHz) δ 175.9 (C-9), 174.6 (C-19), 169.3 (C-22), 146.3 (C-15), 145.2 (C-16), 143.6 (3C-TrtAr), 139.8 (C-6), 138.3 (C-28a), 137.6 (C-4), 130.8 (6C-TrtAr), 129.7 (C-13), 129.24 (3C-TrtAr), 129.22 (6C-TrtAr), 127.2 (C-32a), 125.5 (C-27), 123.6 (C-31), 121.7 (C-18 or C-8), 121.6 (C-18 or C-8), 121.0 (C-32), 119.5 (C-24), 117.3 (C-14), 116.4 (C-30), 116.3 (C-17), 115.3 (C-29), 111.3 (C-26), 105.4 (C-25), 76.8 (CAr₃), 56.3 (C-11), 55.4 (C-2), 44.0 (C-21), 37.9 (C-12), 33.9 (C-3); (+)-HRESIMS [M + H]⁺ 852.2518 (calcd for C₄₆H₄₃⁷⁹BrN₇O₅, 852.2504).

Halocyamine A dihydrochloride (1). A cocktail solution of 0.01 N HCl/HFIP–TIS/H₂O (2 mL, 95 : 2.5 : 2.5) was added to 25 (29.0 mg, 34.1 μmol). The reaction was stirred at r.t. for 1 h, after which, the solution was dried under a stream of nitrogen. Purification by C₈ column chromatography (eluting with H₂O to H₂O/MeOH 60 : 40) afforded 1 as a white solid (13.09 mg, 56% yield). M.p. 280 °C (decomposed); R_f 0.72 (butan-1-ol/acetic acid/H₂O 2 : 1 : 1); [α]^22.3_D +3.4 (c 1.07, MeOH) (lit.^4a [α]^22.3_D +5.2 (c 0.5, MeOH)); ¹H NMR (DMSO-d₆, 500 MHz) δ 11.51 (1H, br s, NH-28), 9.08 (1H, d, J = 10.0 Hz, NH-23), 8.88 (1H, br s, NH-20), 8.40 (1H, br s, NH-10), 7.72 (1H, s, H-27), 7.60 (1H, s, H-6), 7.58 (1H, d, J = 1.6 Hz, H-29), 7.56 (1H, d, J = 8.3 Hz, H-32), 7.16 (1H, dd, J = 8.3, 1.6 Hz, H-31), 6.86 (1H, s, H-8) 6.67 (1H, dd, J = 10.0, 9.7 Hz, H-24), 6.64 (1H, d, J = 1.5 Hz, H-14), 6.59 (1H, d, J = 8.0 Hz, H-17), 6.45 (1H, dd, J = 8.0, 1.5 Hz, H-18), 5.92 (1H, d, J = 9.7 Hz, H-25), 4.44 (1H, br s, H-11), 3.99 (1H, dd, J = 16.6, 5.8 Hz, H₂-21a), 3.93 (1H, dd, J = 16.6, 5.8 Hz, H₂-21b), 3.61 (1H, br s, H-2), 2.95 (1H, dd, J = 14.0, 4.1 Hz, H₂-12a), 2.91–2.89 (1H, m, H₂-3a), 2.73 (1H, dd, J = 14.3, 7.5 Hz, H₂-3b), 2.66 (1H, dd, J = 14.0, 9.6 Hz, H₂-12b); ¹³C NMR (DMSO-d₆, 125 MHz) δ 171.9 (C-19), 171.6 (C-9), 167.5 (C-22), 144.9 (C-15), 143.7 (C-16), 136.4 (C-28a), 135.1 (C-6), 133.5 (C-4), 128.5 (C-13), 125.7 (C-32a), 124.9 (C-27), 121.9 (C-31), 120.1 (C-32), 119.9 (C-18), 118.6 (C-24), 116.6 (C-14), 115.3 (C-17), 114.3 (C-30), 114.1 (C-29), 109.7 (C-26), 102.2 (C-25), 54.5 (C-11), 53.8 (C-2), 42.6 (C-21), 36.7 (C-12), 30.5 (C-3); (+)-HRESIMS [M + H]⁺ 610.1393 (calcd for C₂₇H₂₉⁷⁹BrN₇O₅, 610.1408).

Antibiotic susceptibility testing

The susceptibility of bacterial strains to antibiotics and compounds was determined in microplates using the standard broth dilution method in accordance with the recommendations of the Comité de l'AntibioGramme de la Société Française de Microbiologie (CA-SFM).²¹ Briefly, the minimal inhibitory concentrations (MICs) were determined with an inoculum of 10⁵ CFU in 200 μL of MH broth containing two-fold serial dilutions of each drug. The MIC was defined as the lowest concentration of drug that completely inhibited visible growth after incubation for 18 h at 37 °C. To determine all MICs, the measurements were independently repeated at least three times. Minimum inhibitory concentration of positive control: colistin [P. aeruginosa (1 μM), E. coli (2 μM)], streptomycin [P. aeruginosa (21.5 μM), E. coli (21.5 μM), S. aureus (21.5 μM), S. intermedius (10.7 μM) and E. faecalis (21.5 μM)] and chloramphenicol [S. aureus (1.5–3 μM), S. intermedius (3–6 μM) and E. faecalis (1.5–3 μM)].

Marine bacteria susceptibility testing

The antibacterial activity assay was performed on the marine environmental bacterial strains Gram-negative V. harveyi ATCC 14126, V. alginolyticus ATCC 17749 and L. anguillarum ATCC 19264 by the liquid growth inhibition in 96-well microplates. A pre-culture of 5 mL marine broth (MB) was prepared by inoculating a colony of each bacterial strain and was incubated at 30 °C with stirring overnight. The concentration of the pre-culture was assessed by measuring the optical density (OD) at 620 nm and was adjusted by dilution in order to obtain a suspension of 0.03 OD. An aliquot of 200 μL of the bacterial suspension was distributed in each well and 10 μL of a serial dilution in DMSO of the pure compound were added in triplicate. The 96-well microplates were incubated at 30 °C overnight with shaking (450 rpm). The optical density of the wells was measured at 620 nm with a microplate reader and the inhibition (IC₅₀) was calculated and plotted versus test concentrations.

Antioxidant testing

Quantitative ORAC assay was run as previously described.²² The result is expressed as relative Trolox (6-hydroxy-2,5,7,8-tetramethyl chroman-2-carboxylic acid) equivalents.

DPPH free radical scavenging assay

The potential antioxidant activity was evaluated in a 96-well microplate format assay whereby an aliquot of 10 μL of a serial dilution in MeOH of the pure compound and 190 μL of DPPH (200 μM, MeOH) were added in triplicate. After 1 h at room temperature in the dark, the absorbance was recorded at 510 nm in a microplate reader (DPPH radical has a characteristic absorption in MeOH at 510 nm, which disappears with acceptance of an electron from the antioxidant sample). Ascorbic acid was used as a positive control (IC₅₀ 101 ± 8 μM). The antioxidant activity of the tested compounds was evaluated by the IC₅₀, which represents the sample concentration required to scavenge 50% of the DPPH free radical.

Acknowledgements

We thank Dr M. Schmitz for assistance with NMR data acquisition, and Mr T. Chen for MS data. We gratefully acknowledge funding from the University of Auckland.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H and ¹³C spectral data. See DOI: 10.1039/c7ob01122a

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