chemistry_of_Briareum_asbestinum

THE CHEMISTRY OF BRIAREUM ASBESTINUM by Melody D Rondeau A Thesis Submitted to the Faculty of the Charles E Schmidt College of Science In Partial Fulfillment of the Requirements for the Degree of Master of Science Florida Atlantic University Boca Raton, FL August 2012 ACKNOWLEDGEMENTS I am profoundly grateful to all the amazing people that contributed to my success as a graduate student First, I would like to thank my advisor, Dr Lyndon M West for his support, guidance and, most of all, patience I would also like to thank my graduate committee, Dr Charles Carraher and Dr Frank Mari; your consideration and input were invaluable I would like to thank Dr Salvatore Lepore for always being available to lend a listening ear Your advice always motivated and encouraged me I am indebted to the members of the West laboratory; Rian Meginley, Tifanie Vansach, Andrew Hall, Rolando Rueda de Leon, Long Zhang, and Yangqing He as well as Dr Prasoon Gupta and Dr Upasana Sharma Your collaboration, advice, and time were vital to the success of this project and are appreciated beyond measure I want to extend a special thank you to my family and friends I really could not have done it without you I am so fortunate to be surrounded by such support Finally, I would like to thank Michael Rondeau for his love and encouragement throughout this journey iii ABSTRACT Author: Melody D Rondeau Title: The Chemistry of Briareum asbestinum Institution: Florida Atlantic University Thesis Advisor: Dr Lyndon M West Degree: Master of Science Year: 2012 Briareum asbestinum, a soft coral, is a rich source of diterpenoid natural products The secondary metabolites of B asbestinum fall into four classes; asbestinins, briarellins, briareolate esters, and briaranes Briareolate esters have been shown to possess biological activity and were previously only reported from Tobago Our group recently isolated briareolate esters from a specimen collected off the coast of Boca Raton, Florida To determine whether location has an impact on the chemistry produced by the organism, a method to discern between chemotypes was sought Several techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and sclerite analysis were employed, with NMR being the most successful method iv By utilizing both 1H and COSY NMR experiments, it is possible to differentiate between the chemotypes of B asbestinum Application of this method allowed analysis of chemical variability with respect to location v To my son, Aidan Patrick May the world be a better place THE CHEMISTRY OF BRIAREUM ASBESTINUM TABLES ix FIGURES x ABBREVIATIONS xiii INTRODUCTION AND BACKGROUND 1.1 Natural Products 1.2 Marine Natural Products 1.3 Briareum asbestinum 1.4 The Chemistry of Briareum asbestinum 1.5 Research Objectives 14 ANALYTICAL METHOD DEVELOPMENT 15 2.1 Sclerite Analysis 15 2.2 Thin Layer Chromatography 17 2.3 High Performance Liquid Chromatography 18 2.4 Differentiation of B asbestinum Chemotypes using NMR spectroscopy 22 2.5 Analytical Method 23 2.6 Analysis of the Briareolate Ester Containing Chemotype 25 2.7 Analysis of the Briarane/Eunicellin Containing Chemotype 31 2.8 Analysis of the Briarane-rich Chemotype 42 2.9 Application to Location 49 vii STRUCTURAL ELUCIDATION OF (7E-11E)-3,4-EPOXY-7,11,15CEMBRATRIENE 52 GENERAL EXPERIMENTAL PROCEDURES 61 4.1 General Experimental Procedures 61 4.2 Collection of Animal Material 61 4.3 Sclerite Analysis 62 4.4 Solid Phase Extraction 62 4.5 Thin Layer Chromatography 62 4.6 Development of High Performance Liquid Chromatography Method 63 4.7 Protocol for NMR Analysis 63 4.8 Isolation of 3,4-epoxy-7,11,15-cembratriene 64 4.9 Protocol for Chemotypic Analysis of B asbestinum by 1H and COSY NMR 64 REFERENCES 66 viii TABLES Table 1: Location and Chemotype………………………………………………… 51 Table 2: 1H and 13C NMR Data for 36……………………………………………….53 Table 3: Selected 1H-1H COSY and 1H-13C HMBC Correlations for 36………….56 ix FIGURES Figure 1: Taxonomy of Briareum asbestinum Figure 2: Briareum asbestinum Figure 3: Mevalonate Pathway Figure 4: Deoxyxylulose PhosphatePathway 10 Figure 5: Cembrane Formation 11 Figure 6: Briarane Formation 13 Figure 7: Asbestinin and Briarellin Formation 13 Figure 8: Eunicellin Sclerite 16 Figure 9: Briareolate Ester Sclerite 16 Figure 10: TLC Chromatogram 17 Figure 11: Eunicellin HPLC Chromatogram 21 Figure 12: Briareolate Ester HPLC Chromatogram 21 Figure 13: Briarane HPLC Chromatogram 22 Figure 14: Typical 1H NMR spectrum of the 75% acetone/water fraction of the briareolate ester containing B asbestinum chemotype (400MHz, CD3OD) 26 Figure 15: 1H NMR spectrum of briareolate ester G (31) (400MHz, CD 3OD) 27 Figure 16: 1H NMR spectrum of briareolate ester L (32) (400MHz, CD3OD) 28 Figure 17: 1H NMR Spectrum Overlay, Briareolate Esters (400 MHz, CD3OD) 29 Figure 18: Typical 1H-1H COSY NMR spectrum of the 75% acetone/water fraction of the briareolate ester containing B asbestinum chemotype x (7E-11E)-3,4-epoxy-7,11,15-cembratriene was obtained as a white powder and had a molecular formula of C20H32O as determined by HRESIMS of the [M + Na]+ ion at m/z 311.2, indicating five degrees of unsaturation Analysis of the 13C NMR data revealed three C-C double bonds accounting for three of the five degrees of unsaturation The other two double bond equivalents could be attributed to the presence of two rings A trisubstiuted epoxide containing a methyl substituent was elucidated from the signals of an oxymethine, a quaternary oxygen-bearing carbon, and a methyl singlet The presence of 20 carbons suggests a diterpene, a common skeleton produced by B asbestinum Therefore, it was deduced the molecule was a cyclic diterpene containing three double bonds and an epoxide 53 Table 2: 1H and 13C NMR Data for 36 Position 13 2.07 m 1.95 dd 1.30 m 2.84 t 48.12 (s) 39.84 (s) 10 11 12 13 14 15 16 17 18 19 20 H C 1.72 t (2H) 2.18 t 2.26 m 5.26 t 2.00 t 2.03 m 1.89 dt 1.48 m 5.16 t 64.38 (s) 62.76 (s) 28.27 (s) 37.63 (s) 136.58 (s) 126.33 (s) 34.01 (s) 24.16 (s) 2.02 t 1.74 t 1.52 m (2H) 4.67 d 5.14 d 1.77 s (3H) 1.33 s (3H) 1.69 s (3H) 1.57 s (3H) 124.21 (s) 136.92 (s) 36.73 (s) 30.28 (s) 150.81 (s) 111.31 (s) 20.19 (s) 18.19 (s) 17.15 (s) 17.88 (s) The structure of 36 was determined by a detailed analysis of the NMR data The HSQC experiment showed a correlation from each proton to its respective carbon atom From the H-1H COSY experiment (Table 2), correlations were found between H-1 and H-2, H-1 and H-14 Additional correlations aided in the establishment of a molecular framework; H-5 to H-4 and 54 H-6, H-8 to H-9, H-9 to H-10, H-10 to H-11, and H-13 to H-14 Furthermore, HMBC correlations between H-4 and C-3, H-9 to C-7 and C-8, and H-13 to C-14 established the connectivity of the 14-member ring The position of the methyl group at position 20 was verified by HMBC correlations from H-20 to C-13 and C11 and a COSY correlation from H-20 to H-11 The methyl substituent at position was demonstrated by correlations in both the COSY and HMBC experiments; H-19 to C-6, C-7, C-8, and C-9 and H-7 to H-19 The final methyl group was placed at C-12 and was verified by correlations between H-20 and H-11 as well as H-20 to C-11 and C-13 The isopropene substituent was placed at C-1 due to COSY correlations from H-14 and H-16 to H-17 HMBC correlations between H14 and C-15, H-16 to C-15 and C-17, and H-17 to C-15 and C-16 also verified the connectivity The epoxide was found to be at C-3 and C-4 due to a COSY correlation from H-4 to C-3 and HMBC correlations from H-18 to C-3 and C-4 Overall, various NMR experiments allowed the structure of 36 to be elucidated unambiguously 55 55 Table 3: Selected 1H-1H COSY and 1H-13C HMBC Correlations for 36 Position H-2 H-4 H-5 H-6 H-7 H-9 H-10 H-13 COSY H-1 H-14 H-16 H-17 H-18 H-19 H-20 H-1, H-17 H-17 H-4, H-6 H5 H-9, H-19 H-10 H-11 H-14 H-2 H-11 56 HMBC C-18 C-3 C-6, C-19 C-7, C-8, C-11 C-11 C-1, C-12,C-14, C-20 C-1, C-15 C-1, C-15, C-17 C-1, C-15, C-16 C-2, C-3, C-4 C-6, C-7, C-8, C-9 C-11, C-13 Figure 33: 13C NMR Spectrum of 36 (400 MHz, CD3OD) 57 Figure 34: 1H-1H COSY NMR Spectrum of 36 (400 MHz, CD3OD) 58 Figure 35: HSQC NMR Spectrum of 36 (400 MHz, CD3OD) 59 Figure 36: HMBC NMR Spectrum of 36 (400 MHz, CD3OD) 60 GENERAL EXPERIMENTAL PROCEDURES 4.1 General Experimental Procedures All 1D and 2D NMR spectra were obtained on a Varian MercuryPlus 400 MHz NMR spectrometer, and all chemical shifts are expressed in parts per million (δ) relative to tetramethylsilane Solid phase extraction (SPE) was performed using HP20 resin (3.0 g) in a 25 mL syringe-barrel column using an 10 port manifold Semi-preparative HPLC separations were carried out on a Shimadzu HPLC system comprised of a Shimadzu D6U-20AF online degasser, Shimdazu LC-20AT quaternary solvent delivery system, Shimadzu ELSD- LTII detector, Shimadzu SPDA-M20A photo diode array detector, and a Shimadzu FRC-10A fraction collector The flow was split 1:20 to the ELSD and fraction collector The system was controlled using EZStart chromatography software 4.2 Collection of Animal Material Samples of Briareum asbestinum were collected off the coast of southeast Florida and the Florida Keys at depths ranging from 45 to 75 ft approximately mile from the coast between August 2009 and June 2011 Immediately following collection, the samples were frozen 61 4.3 Sclerite Analysis Sclerite analysis began with 0.5 g (dry weight) of animal material This material was placed in a test tube with 20.0 mL 0.1M nitric acid This mixture was gently heated in a warm water bath until all solid material had dissolved, resulting in a yellow solution This was allowed to settle and the bottom layer was removed for microscopic analysis 4.4 Solid Phase Extraction The organic extracts were fractionated using a solid phase extraction 10port vacuum manifold This extract was concentrated onto polymeric HP20 using a savant vacuum centrifuge system The HP20 was transferred to a 25 mL syringe-barrel SPE column, and, after washing the column with water (20 mL), the column was eluted drop-wise with 15 mL fractions of: 1) 40% acetone/water 2) 75% acetone/water, and 3) 100% acetone The eluent was collected in 20 mL scintillation vials and dried in a vacuum centrifuge 4.5 Thin Layer Chromatography Thin layer chromatography began with extracts of the raw animal material from four different specimens of Briareum asbestinum with known chemotype The 75% and 100% fractions were dissolved in MeOH to a concentration of 20 mg/mL The solution was then spotted onto alumina backed silica gel plates and allowed to dry The first system used for separation was 20% EtOAc/hexane The 62 next system employed was 5% MeOH/CH2Cl2 Finally, a 10% MeOH/CH2Cl2 system gave satisfactory separation Each time, the TLC was recovered using a 10% H2SO4 spray with applied heat 4.6 Development of High Performance Liquid Chromatography Method Semi-preparative HPLC separations were performed on a Shimadzu HPLC system comprised of a Shimadzu LC-20AT quaternary solvent delivery system, evaporative light scattering detector, and photodiode array detector A sample of the 75% acetone/water fraction (5 mg) was subjected to semipreparative HPLC separation on reversed phase columns Initially, a Hamilton PRP reverse phase semi-preparative column, 10 x 250 mm (part number 79496) was used This was followed by a Phenomenex C18, 10 x 250 mm (part number 00G-4041-N0) Finally, the column selected was a Phenomenex Curo-Sil-PFP, 10 x 250 mm (part number 00G-4012-N0) A post column fixed flow splitter was used to split the flow in a ratio of 1:20 to the ELSD and fraction collector, respectively 4.7 Protocol for NMR Analysis NMR spectra were obtained on a Varian Inova 400 NMR spectrometer at 400 MHz in CD3OD Proton chemical shifts were referenced to the residual CD3OD signals ( δH 4.98 and 3.31) and carbon chemical shifts were referenced to the center peak of CD3OD at δC 49.15 63 After extraction and fractionation as described previously, the resulting fractions were diluted in 600 µL CD3OD and placed in a mm NMR tube for analysis 4.8 Isolation of 3,4-epoxy-7,11,15-cembratriene Using the previously described extraction technique, 0.5657 g crude extract was obtained A #11 MPLC column was packed with HP20 and washed with acetone The crude extract was dissolved in MeOH and loaded onto the column At a flow rate of 8.0 mL/min, the following MeCN/H 2O gradient was employed; from – 10 mins 60%, increasing to 100% from 10 – 70 mins, and isocratic for an additional 10 mins 88 tubes were obtained and TLC was carried out on each tube These tubes were pooled in the following manner giving rise to fractions A – S; A: 12 – 13, B: 14 – 16, C: 17, D: 18 – 19, E: 20 – 22, F: 23 – 31, G: 32 – 35, H: 36 – 37, I: 38 – 41, J: 42 – 46, K: 47 – 48, L: 49 – 52, M: 53 – 56, N: 57 – 61, O: 62 – 66, P: 67 – 76, Q: 77 – 83, R: 84 – 85, S: 86 – 88 Each fraction was dissolved in 600 µL CD3OD and 1H NMR was performed Fraction R contained 3,4-epoxy-7,11,15-cembratriene 4.9 Protocol for Chemotypic Analysis of B asbestinum by 1H and COSY NMR (1) Approximately g of the frozen B asbestinum specimen was lyophilized (24 h) to dryness (2) The dry coral tissue was ground to a homogenous powder with a mortar and pestle 64 (3) Approximately g of the powdered coral material was accurately weighed and extracted with MeOH (2 mL, 10 mins) with sonication, filtered into a 20 mL scintillation vial containing g HP20, and concentrated to dryness (4) The HP20 was transferred to a 25 mL syringe-barrel SPE column, and, after washing the column with water (20 mL), the column was eluted drop-wise with 15 mL fractions of: 1) 40% acetone/water 2) 75% acetone/water, and 3) 100% acetone The eluent was collected in 20 mL scintillation vials and dried in a vacuum centrifuge (5) The 75% fraction was dissolved in 600 µL CD 3OD and transferred to a mm NMR tube (6) The NMR is performed using the acquisition and processing parameters: Temperature: 25˚C/298.1 K Relax Delay 1.0 sec Pulse 45.0 degrees Acquisition time: 1H: sec, COSY: 0.213 sec Repetitions: 1H: 32, COSY: Line broadening: 1.5 Hz Total time: 1H: min, 48 sec, COSY: 54 min, sec 65 REFERENCES Bioactive Natural Products Detection, Isolation, and Structural Determination Editors Steven M Colegate, Russell J Molyneux CRC Press, Boca Raton, FL 2008 Hill, R A., Annual Reports of the Progress of Chemistry, 2011, 107, 138-156 Dewick, P M., Nat Prod Rep., 2002, 19, 181-222 Saxena, S., Pant, N., Jain, D.C., Bhakuni, R.S., Curr Sci., 2003, 85, 13141329 Fundamentals of Heterocyclic Chemistry, Louis D Quin, John A Tyrell, John Wiley and Sons, Inc., New Jersey, 2010 Wall, M.E., Wani, M.C., Cancer Res., 1995, 55, 753-760 Molinski, T.F., et al., Nature Reviews, 2009, 8, 69-85 Sebens, K.P., Miles, J.S., Biol Bull., 1988, 175, 378-387 Ehrlich, H., Biologically Inspired Systems Biological Materials of Marine Origin Invertebrates, Vol 1, 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