Supporting Information

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1 Supporting Information Isolation, Synthesis, and Radical-Scavenging Activity of Rhodomelin A, a Ureidobromophenol from the Marine Red Alga Rhodomela confervoides Ke Li,, Ya-Fei Wang,, Xiao-Ming Li, Wei-Jia Wang, Xu-Ze Ai, Xin Li, Sui-Qun Yang, James B. Gloer, Bin-Gui Wang*, and Tao Xu*, Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, and Laboratory of Marine Biology and Biotechnology of Qingdao National Laboratory for Marine Science and Technology, Nanhai Road 7, Qingdao , China Key Laboratory of Marine Drugs, Ministry of Education; School of Medicine and Pharmacy, Ocean University of China, and Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and Technology, 5 Yushan Road, Qingdao , China Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States * wangbg@ms.qdio.ac.cn (B.-G.W.) xutao@ouc.edu.cn (T. X.). 1

2 Table of contents General Information p3 Algal material......p3 Extraction and isolation...p3 Natural rhodomelin A (1)..... p4 Determination of DPPH and ABTS radical-scavenging activity.. p4 Total synthesis of rhodomelin A (1)......p4 References..... p10 NMR spectra. p11 Figure S1. 1 H NMR (500 MHz, DMSO-d 6 ) spectrum of compound 1 p10 Figure S2. 13 C NMR (125 MHz, DMSO-d 6 ) spectrumof compound 1 p11 Figure S3. COSY (DMSO-d 6 ) spectrum of compound 1 p11 Figure S4. HSQC (DMSO-d 6 ) spectrum of compound 1 p12 Figure S5. HMBC (DMSO-d 6 ) spectrum of compound 1 p12 Figure S6. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 5 p13 Figure S7. 13 C NMR (101 MHz, CDCl 3 ) spectrum of 5 p13 Figure S8. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 7 p14 Figure S9. 13 C NMR (101 MHz, CDCl 3 ) spectrum of 7 p14 Figure S10. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 2 p15 Figure S C NMR (101 MHz,CDCl 3 ) spectrum of 2 p15 Figure S12. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 9 p16 Figure S C NMR (101 MHz, CDCl 3 ) spectrum of 9 p16 Figure S14. 1 H NMR (400 MHz, DMSO-d 6 ) spectrum of 10 p17 Figure S C NMR (101 MHz, DMSO-d 6 ) spectrum of synthesized 10 p17 Figure S16. 1 H NMR (500 MHz, DMSO-d 6 ) spectrum of synthesized (R)-rhodomelin A (1) p18 Figure S C NMR (125 MHz, DMSO-d 6 ) spectrum of synthesized (R)-rhodomelin A (1) p18 Figure S18. 1 H NMR (500 MHz, DMSO-d 6 ) spectrum of synthesized (S)-rhodomelin A (1) p19 Figure S C NMR (125 MHz, DMSO-d 6 ) spectrum of synthesized (S)-rhodomelin A (1) p19 Table S1. NMR data for natural and synthesized 1 p20 Scheme S1. Proposed biosynthetic pathway for compound 1 p20 2

3 General Information All reactions were carried out under nitrogen atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Chemicals purchased from commercial sources were used without further purification. Anhydrous THF was distilled from sodium-benzophenone; Dichloromethane (DCM) was distilled from calcium hydride. Toluene was distilled from sodium under nitrogen. Reactions were monitored by thin-layer chromatography (TLC) carried out on 0.20 mm Xinnuo silica gel plates (60F-254) using UV light as visualizing. Xinnuo silica gel (60, particle size mm) was used for flash column chromatography. UV spectra were measured on a Varian Cary 50 UV-vis-NIR spectrophotometer. IR spectra were obtained using a Nicolet NEXUE 470 infrared spectrophotometer. 1D and 2D NMR spectra were recorded on a Bruker Avance 500 or JEOL Advance 400 spectrometers. Mass spectra were acquired on a Thermo Fisher Scientific Exactive ESI+ or VG Autospec 3000 mass spectrometer. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplets, br = broad. Algal material: A sample of the marine red alga Rhodomela confervoides was collected at the Dalian coastline of Liaoning Province, China, in April 2007, and was identified by Prof. B.-M. Xia at the Institute of Oceanology, Chinese Academy of Sciences (IOCAS). A voucher specimen (No. HZ0407R) was deposited in the Key Laboratory of Experimental Marine Biology at IOCAS. Extraction and isolation: The air-dried and ground marine red alga R. confervoides (33 kg) was extracted with 95% EtOH at room temperature (3 72 h). After the solvent was removed under reduced pressure at < 40 o C, a dark residue was obtained. The residue was suspended in H 2 O and then successively partitioned with petroleum ether, EtOAc, and n-butanol. The EtOAc extract (360 g) was chromatographed over silica gel (1500 g) eluting with petroleum ether acetone and CHCl 3 MeOH to give 37 fractions on the basis of TLC analysis. Fr. 34 (8.6 g) was further fractionated by CC on 3

4 silica gel eluting with a gradient of increasing MeOH (20 100%) in CHCl 3 to yield two sub-fractions. The second sub-fraction was chromatographed twice over Sephadex LH-20 eluting with MeOH H 2 O (1:1) and MeOH respectively to yield compound 1 (7.8 mg). Natural Rhodomelin A (1): N-(2,3-Dibromo-4,5-dihydroxybenzyl)-N'-(5-oxopyrrolidin-2-yl)-γ-ureidobutyric acid, mg, brown powder; [α] D (c 0.09, MeOH); UV (MeOH) λ max (log ε): 215 (4.07), 233 (4.02), 293 (3.60) nm; 1 H- and 13 C-NMR data, see Table 1; IR (KBr) ν max 3747, 3649, 2926, 2846, 1733, 1640, 1534, 1448, 672 cm 1 ; ESIMS m/z 510/508/506 (1:2:1) [M H] ; HRFABMS m/z [M H] (calcd for C 16 H Br 2 N 3 O 6, ). Determination of DPPH and ABTS radical-scavenging activity: The DPPH and ABTS radical-scavenging activity of natural and synthetic compounds were measured using the methods as described in our previous report (Duan, X. J.; Zhang, W. W.; Li, X. M.; Wang, B. G. Food Chem. 2006, 95, 37 43; Li, K.; Li, X. M.; Gloer, J. B.; Wang, B. G. J. Agric. Food Chem. 2011, 59, ). Total Synthesis of Rhodomelin A (1) Synthetic sequence of compound 2 Lanosol aldehyde (4) was synthesized using a literature procedure 1 and the analytical data were in complete agreement with the literature data. 1 4

5 To a solution of compound 4 (5.00 g, mmol) in THF (150 ml) was added TEA (2.34 ml, mmol) and BnBr (2.00 ml, mmol) in THF (20 ml) sequentially. The mixture was stirred for 20 hours at room temperature. The reaction was quenched by addition of EtOAc (80 ml) and 1 N HCl (8 ml). The aqueous phase was extracted with cold EtOAc (30 ml) three times. The combined organic phases were dried over Na 2 SO 4. The solvent was removed under vacuum, and the residue was purified by flash chromatography on silica gel to give compound 5 (3.96 g, 61 % yield) as colorless solid. R f = 0.23 (silica gel, EtOAc/petroleum ether = 1/5); 1 H NMR (400 MHz, CDCl 3 ) δ (s, 1H), 7.48 (s, 1H), , (m, 5H), 5.58 (s, 1H), 5.14 (s, 2H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , , , , , , 76.45; HRMS-ESI Calcd for C 14 H 9 Br 2 O 3 [M-H] - : m/z , , ; Found: , , ; IR (KBr) ν max 3140, 2917, 1672, 750, 732, 698 cm -1 ; mp o C. To a solution of compound 5 (1.86 g, 4.82 mmol) and methyl 4-aminobutyrate hydrochloride (6) ( mg, 5.78 mmol) in anhydrous DCM (60 ml) was added TEA (1.67 ml, mmol). The mixture was stirred for 1 hour at room temperature. To this solution was added (Boc) 2 O (1.33 ml, 5.78 mmol) and Na(OAc) 3 BH (2.55 g, mmol). The reaction was stirred for an additional 4 hours at room temperature and was quenched with saturated aqueous NaHCO 3 solution (20 ml). The aqueous phase was extracted with DCM (40 ml x 3). The combined organic fractions were 5

6 washed with brine (20 ml) and were dried over Na 2 SO 4. The organic fractions were concentrated under reduced pressure. The crude residue was purified by flash chromatography to afford compound 7 (1.81g, yield 72 %) as colorless solid. R f = 0.3 (silica gel, EtOAc/petroleum ether = 1/5); 1 H NMR (400 MHz, CDCl 3 ) δ 7.47 (d, J = 4.3, 2H), (m, 3H), 6.73 (s, 1H), 5.88 (s, 1H), 5.02 (d, J = 20.0 Hz, 2H), 4.44 (d, J = 25.0 Hz, 2H), 3.66 (s, 3H), 3.24 (t, J = 8.0 Hz, 2H), 2.32 (m, 2H), 1.87 (t, J = 7.2 Hz, 2H), 1.44 (s, 9H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , , , , , , , 80.53, 75.84, 51.75, 46.45, 31.27, 28.38, 23.53; HRMS-ESI Calcd for C 24 H 28 Br 2 NO 6 [M-H] - : m/z , , ; Found: , , ; IR (KBr) ν max 2972, 1735, 1664, 1417, 732, 700, 614 cm -1 ; mp: o C. To a solution of compound 7 (1.00 g, 1.70 mmol) in DCM (20 ml) was added slowly trifluoroacetic acid (1.26 ml, mmol) at 15 o C. The mixture was stirred overnight at 0 o C and was quenched with saturated aqueous NaHCO 3 solution (30 ml). The aqueous phase was extracted with DCM (20 ml x 3). The combined organic fractions were washed with brine (15 ml) and were dried over Na 2 SO 4. The organic fractions were concentrated under reduced pressure to give crude compound 2 as light yellow solid (828.2 mg) quantitatively. The crude compound 2 was used directly in the next step without further purification. R f = 0.20 (silica gel, EtOAc/petroleum ether = 1/1) ; 1 H NMR (400 MHz, CDCl 3 ) δ (d, J = 4.0 Hz, 2H), (m, 3H), 6.93 (s, 1H), 4.93 (d, J = 2.0 Hz, 2H), 3.80 (d, J = 1.8 Hz, 2H), 3.65 (s, 3H), (t, J = 4.0 Hz, 2H), 2.26 (m, 2H), 1.74 (t, J = 2.5 Hz, 1H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , , , , , , 74.21, 53.95, 51.79, 48.01, 31.46, 23.41; HRMS-ESI Calcd for C 19 H 20 Br 2 NO 4 [M-H] - : m/z , 6

7 , ; Found: , , ; IR (KBr) ν max 3310, 2950, 1733, 1663, 1440, 737, 696, 603 cm -1 ; mp o C. Compound 8 was synthesized according to a reported procedure. 2 The analytical data of compound 8 was in complete agreement with the literature. 2 To a solution of compound 8 (0.10 g, 0.38 mmol) in THF (5 ml) was added TEA (0.05 ml, 0.38 mmol) and ethyl chloroformate (0.036 ml, 0.38 mmol) under N 2 at -15 C sequentially. After 10 min, the reaction mixture was transferred to an ice bath and aqueous solution of NaN 3 (8 N, 3.52 ml, mmol) was added via syringe. Stirring was continued at 0 o C for 30 mins. The aqueous phase was extracted with cold EtOAc (10 ml 3). The combined EtOAc extracts were washed with brine (10 ml) and dried over Na 2 SO 4 at 0 C. The solvent was removed under reduced pressure to give the acyl azide, which was redissolved in toluene (5 ml). The stirred solution of the acyl azide was heated 80 C under N 2, until complete conversion (about 5 min). Compound 2 ( mg, 0.76 mmol) was added to the above solution, and the mixture was stirred under N 2 for 30 min at 80 C and additional 5 hours at 60 C. The reaction mixture was cooled, and evaporated to give an oil. The crude residue was purified by flash chromatography to afford compound 9 (75 mg, yield 75%) as a colorless solid. R f = 0.20 (EtOAc/petroleum ether = 1/1); 1 H NMR (400 MHz, CDCl 3 ) δ (d, J = 4.0 Hz, 2H), (m, 5H), (m, 3H), 6.78 (s, 1H), (s, 1H), (d, J = 8.0 Hz, 1H), (d, J = 12.0 Hz, 1H), 5.00 (s, 2H), 4.48 (s, 2H), 3.64 (s, 3H), (m, 3H), (m, 2H), (m, 2H), 1.62 (m, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ , , , , , , , , , , , , , , , 75.06, 68.00, 66.39, 52.19, 51.51, 45.94, 30.87, 29.80, 26.07, 22.89; HRMS-ESI Calcd for C 32 H 32 Br 2 N 3 O 8 [M-H] - : m/z , , ; 7

8 Found: , , ; IR (KBr) ν max 3854, 3744, 2948, 2361, 1734, 1496, 754, 697, 668 cm -1 ; mp o C. To a solution of compound 9 (500.0 mg, 0.67mmol) in dry MeOH/THF (5 ml/5 ml) was added 10%w Pd/CaCO 3 (50.0 mg, 0.47mmol). The reaction was degassed and purged with an H 2 balloon. The mixture was stirred for 2 hours at room temperature under an H 2 balloon. Aqueous phase was filtered and the filtrate was concentrated under reduced pressure. The crude residue was purified by flash chromatography to afford 10 (287.4 mg, yield 82 %) as a colorless solid. R f = 0.60 (DCM/MeOH = 5/1); 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.71 (s, 1H), 7.84 (s, 1H), 6.90 (d, J = 8.1 Hz, 1H), 6.58 (s, 1H), (m, 1H), (dd, J = 16.0, 8.0 Hz, 2H), 3.53 (s, 3H), 3.11 (t, J = 4.0 Hz, 2H), (m, 4H), (m, 1H), 1.79 (m, 1H), 1.67 (m, 2H); 13 C NMR (101 MHz, DMSO-d 6 ) δ , , , , , , , , , 62.52, 51.87, 50.89, 51.51, 45.74, 31.00, 29.72, 28.13, 23.74; HRMS-ESI Calcd for C 17 H 20 Br 2 N 3 O 6 [M-H] - : , , ; Found: , , ; IR (KBr) ν max 3198, 2953, 1685, 1532, 771, 734, 648 cm -1 ; mp o C. To a solution of compound 10 (100.0 mg, 0.19 mmol) in MeOH (10 ml) was added lithium hydroxide (80 mg, 1.90 mmol ) in water (1 ml) and the mixture was stirred for 30 min at room temperature. The solution was evaporated and the residue 8

9 was acidified with 1 N HCl (1.90 ml) to ph 7. The mixture was extracted with EtOAc (4 ml 3). The combined organic fractions were washed with brine (10 ml), dried over Na 2 SO 4, and concentrated under reduced pressure. The crude residue was purified by preparative HPLC to afford (R)-rhodomelin A (1) (29.0 mg, yield 30%) as a colorless solid. R f = 0.20 (DCM/MeOH = 5/1); 1 H- and 13 C-NMR (500 and 125 MHz, respectively, DMSO-d 6 ), see Table S1; HRMS (ESI) calcd for C 16 H 18 Br 2 N 3 O 6 [M-H] - : , , Found: m/z , , ; IR (KBr) ν max 3747, 3649, 2926, 2846, 1733, 1640, 1534, 1448 cm 1. Synthesis of (S)-rhodomelin A (1): (S)-rhodomelin A was synthesized following the same procedure as that of (R)-rhodomelin A from (R)-Cbz glutamic acid to afford 32.9 mg of product in a 10% overall yield. R f = 0.20 (DCM/MeOH = 5/1); 1 H- and 13 C-NMR (500 and 125 MHz, respectively, DMSO-d 6 ), see Table S1; HRMS (ESI) calcd for C 16 H 18 Br 2 N 3 O 6 [M-H] - : , , ; Found: m/z , , ; IR (KBr) ν max 3747, 3649, 2926, 2846, 1733, 1640, 1534, 1448 cm 1. References: (1) (a) Lee, A. H. F.; Chen, J.; Liu, D.; Leung, T. Y. C.; Chan, A. S. C.; Li, T. J. Am. Chem. Soc. 2002, 124, ; (b) Shoeib, N. A.; Bibby, M. C.; Blunden, G.; Linley, P. A.; Swaine, D. J.; Wheelhouse, R. T.; Wright, C. W. J. Nat. Prod. 2004, 67, (2) Chorev M.; Rubini, E.; Gilon, C.; Wormser, U. Selinger, Z. J. Med.Chem. 1983, 26,

10 NMR Spectra Figure S1. 1 H NMR (500 MHz, DMSO-d 6 ) spectrum of compound 1 10

11 Figure S2. 13 C NMR (125 MHz, DMSO d 6 ) spectrum of compound 1 Figure S3. COSY (DMSO d 6 ) spectrum of compound 1 11

12 Figure S4. HSQC (DMSO d 6 ) spectrum of compound 1 Figure S5. HMBC (DMSO d 6 ) spectrum of compound 1 12

13 Figure S6. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 5 Figure S7. 13 C NMR (101 MHz,, CDCl 3 ) spectrum of 5 13

14 Figure S8. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 7 Figure S9. 13 C NMR (101 MHz,, CDCl 3 ) spectrum of 7 14

15 Figure S10. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 2 Figure S C NMR (101 MHz,CDCl 3 ) spectrum of 2 15

16 Figure S12. 1 H NMR (400 MHz, CDCl 3 ) spectrum of 9 Figure S C NMR (101 MHz, CDCl 3 ) spectrum of 9 16

17 Figure S14. 1 H NMR (400 MHz, DMSO-d 6 ) spectrum of 10 Figure S C NMR (101 MHz, DMSO-d 6 ) spectrum of synthesized 10 Br HO Br OH N 10 O N H HN CO 2 Me O 17

18 Figure S16. 1 H NMR (500 MHz, DMSO-d 6 ) spectrum of synthesized (R)-rhodomelin A (1) Br Br O HN N N H O HO OH CO 2 H (R)-rhodomelin A (1) Figure S C NMR (125 MHz, DMSO d 6 ) spectrum of synthesized (R)-rhodomelin A (1) Br Br O HN N N H O HO OH CO 2 H (R)-rhodomelin A (1) 18

19 Figure S18. 1 H NMR (500 MHz, DMSO d 6 ) spectrum of synthesized (S)-rhodomelin A (1) Figure S C NMR (125 MHz, DMSO d 6 ) spectrum of synthesized (S)-rhodomelin A (1) 19

20 Table S1. NMR Data for Natural and Synthesized 1 (Recorded in DMSO-d 6 ). a Natural 1 Synthesized (R)-1 Synthesized (S)-1 no. δ H δ C δ H δ C δ H δ C , C 174.4, C 175.1, C , t (7.3) 30.8, CH , t (7.0) 30.7, CH , t (6.1) 31.0, CH , m 23.2, CH , m 23.2, CH , m 23.4, CH , m 45.3, CH , m 45.3, CH , m 45.4, CH , C 156.5, C 156.6, C 1' 129.2, C 129.2, C 129.1, C 2' 113.5, C 113.3, C 113.6, C 3' 112.8, C 112.8, C 112.7, C 4' 143.5, C 143.4, C 143.8, C 5' 145.4, C 145.4, C 145.6, C 6' 6.63, s 113.3, CH 6.64, s 113.3, CH 6.61, s 113.4, CH 7' 4.39, d (16.7) 50.4, CH , d (16.8) 50.4, CH , d (16.6) 50.4, CH , d (16.7) 4.32, d (16.8) 4.31, d (16.6) 1'' 5.37, m 62.0, CH 5.36, m 62.0, CH 5.36, m 61.9, CH 2'' 2.29, m 27.5, CH , m 27.6, CH , m 27.5, CH , m 1.85, m 1.87, m 3'' 2.30, m 29.1, CH , m 29.1, CH , m 29.2, CH , m 2.05, m 2.04, m 4'' 176.1, C 176.1, C 176.1, C NH , d (8.1) 7.00, d (8.0) 7.17, br s NH , br s 7.93, br s 7.91, br s a Recorded in DMSO-d 6 at 500 MHz for 1 H and 125 MHz for 13 C, δ in ppm, J in Hz. Scheme S1. Proposed biosynthetic pathway for compound 1: 20

21 As shown above in Scheme S1, 3,4-dibromo-5-hydroxymethylbenzene-1,2-diol (lanosol, I), a dibromophenolic metabolite that was identified from R. confervoides in our previous report i as well as from several other species of the marine red algal family Rhodomelaceae, ii is presumably as the biosynthetic precursor for 1. To form the ureido unit, grateloupine (II), a ureido amino acid that has been identified from the marine red alga Grateloupia filicina, iii is a reasonable additive with I to give the key intermediate III, which further coupled with pyrrolidin-2-one (IV) that might be derived by cyclization of 4-aminobutanoic (VII, an amino acid that commonly detected in marine environment), iv to yield rhodomelins A (1) via condensation and rearrangement of the pyrrolidone moiety, probably by enzyme catalytic/controlled step(s). References: (i) Li, K.; Li, X. M.; Gloer, J. B.; Wang, B. G. J. Agric. Food Chem. 2011, 59, (ii) (a) Kurata, K.; Taniguchii, K.; Takashima, K.; Suzuki, M. Phytochemistry 1997, 45, ; (b) Shoeib, N. A.; Bibby, M. C.; Blunden, G.; Linley, P. A.; Swaine, D. J.; Wheelhouse, R. T.; Wright, C. W. J. Nat. Prod. 2004, 67, ; (c) Popplewell, W. L.; Northcote, P. T. Tetrahedron Lett. 2009, 50, ; (d) Park, S.-H.; Song, J.-H.; Kim, T.; Shin, W.-S.; Park, G. M.; Lee, S.; Kim, Y.-J.; Choi, P.; Kim, H.; Kim, H.-S.; Kwon, D.-H.; Choi, H. J.; Ham, J. Mar. Drugs 2012, 10, (iii) Wakamiya, T.; Kobayashi, Y.; Shiba, T.; Setogawa, K.; Matsutani, H. Tetrahedron 1984, 40, (iv) Wang, L. L.; Hu, J. F.; Tang, J. H. Acta Oceanol. Sinica (Haiyang Xuebao) 2009, 31,