Chemical Investigation of the Antarctic Marine Invertebrates Austrodoris kerguelenensis

293 Pages·2015·10.48 MB·English

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Graduate Theses and Dissertations Graduate School
2011
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John Alan Maschek
University of South Florida, [email protected]
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SScchhoollaarr CCoommmmoonnss CCiittaattiioonn
Maschek, John Alan, "Chemical Investigation of the Antarctic Marine Invertebrates Austrodoris
kerguelenensis & Dendrilla membranosa and the Antarctic Red Alga Gigartina skottsbergii" (2011).
Graduate Theses and Dissertations.
https://scholarcommons.usf.edu/etd/3236
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Chemical Investigation of the Antarctic Marine Invertebrates Austrodoris

kerguelenensis & Dendrilla membranosa and the Antarctic Red Alga Gigartina

skottsbergii

by

J. Alan Maschek

A dissertation submitted in partial fulfillment
of the requirement for the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Science
University of South Florida

Major Professor: Bill Baker, Ph.D.
Abdul Malik, Ph.D.
Roman Manetsch, Ph.D.
Alberto van Olphen, Ph.D.

Date of Approval:
June 7, 2011

Keywords: organic chemistry, natural products, nudibranch, sponge, red alga,
palmadorin, lectin, bioassay

Copyright © 2011, J. Alan Maschek
Dedication
This dissertation is dedicated to Hollie, Mother & Father

“Science is magic that works” - Kurt Vonnegut, Jr
Acknowledgements

This dissertation would not have been possible without the efforts of several individuals.
Dr. Bill Baker.
Dr. Charles Amsler, Dr. Jim McClintock
Dr. Thushara Diyabalange, Dr. Solomon Welderirma, Dr. Yoshinari Miyata, Dr. Jaime
Noguez, Dr. Matt Lebar, Wai Ma, Ryan Centko, Emily Mevers, Jason Cuce, Jeremy
Beau, Chandan Barhate
Dr. Alberto van Olphen, Cindy Bucher
Dr. Barry O‟Keefe, Carrie Saucedo
The US Antarctic Program, National Science Foundation, Office of Polar Programs,
Palmer Station Personnel
Dr. David Corey, Dr. Rob Bergstrom
Dr. Edward Turos, Dr. Roman Manetsch, Dr. Abdul Malik
Table of Contents

List of Tables .......................................................................................................................v

List of Figures ................................................................................................................... vii

List of Schemes .................................................................................................................. ix

List of Abbreviations ...........................................................................................................x

Abstract ............................................................................................................................. xii

Chapter 1. On natural products, marine drugs and Antarctic chemical ecology ...............1
1.1 A brief history of natural products ...................................................................1
1.2 Conceptual framework of natural products ......................................................4
1.3 Marine natural products as drugs .....................................................................7
1.4 Antarctic chemical ecology ............................................................................12
1.5 Research objectives ........................................................................................16
1.6 References cited .............................................................................................17

Chapter 2. Chemical investigation of Austrodoris kerguelenensis ..................................22
2.1 Introduction to nudibranchs ............................................................................22
2.2 Chemistry of Doridae .....................................................................................23
2.2 Chemistry of Austrodoris kerguelenensis ......................................................25
2.3 Results and discussion for new palmadorins D - S (2.32 - 2.47) ...................28
2.3.1 Characterization of substructure A ................................................34
2.3.2 Characterization of substructures B - E .........................................35
2.3.3 Characterization of palmadorin D (2.32) .......................................36
2.3.4 Characterization of palmadorin E (2.33) .......................................37
2.3.5 Characterization of palmadorin F (2.34) ........................................38
2.3.6 Characterization of palmadorin G (2.35) .......................................39
2.3.7 Characterization of palmadorin H (2.36) .......................................41
2.3.8 Characterization of palmadorin I (2.37) .........................................41
2.3.9 Characterization of palmadorin J (2.38) ........................................42
2.3.10 Characterization of palmadorin K (2.39) .......................................43
2.3.11 Characterization of palmadorin L (2.40) .......................................43
i
2.3.12 Characterization of palmadorin M (2.41) ......................................45
2.3.13 Characterization of palmadorin N (2.42) .......................................48
2.3.14 Characterization of palmadorin O (2.43) .......................................48
2.3.15 Characterization of palmadorins P (2.44) and Q (2.45) .................49
2.3.16 Characterization of palmadorin R (2.46) .......................................51
2.3.17 Characterization of palmadorin S (2.47) ........................................53
2.4 Metabolomic investigation of A. kerguelenensis……………. ......................53
2.4.1 Genetic studies to establish phylogroups .......................................55
2.4.2 Generation of LC/MS chromatogram library ................................56
2.4.3 Data clustering ...............................................................................59
2.4.4 Discussion of outliers within clustering results .............................62
2.4.5 Investigation of phylogroups 4, 20, 29 and 32...............................64
2.5 Research summary and future directions .......................................................68
2.6 References cited ............................................................................................69

Chapter 3. Chemical investigation of Gigartina skottsbergii ..........................................75
3.1 Influenza ........................................................................................................75
3.1.1 Conceptual framework of influenza virus ......................................77
3.1.2 Drugs for influenza ........................................................................80
3.2 Proteins as therapeutics ..................................................................................82
3.2.1 Lectins ............................................................................................83
3.2.2 Potent anti viral lectins...................................................................84
3.3 Marine red algae: a dominant source of natural products ..............................86
3.3.1 Chemistry of the genus Gigartina..................................................88
3.4 Results and discussion ....................................................................................90
3.4.1 Primary and secondary screening of extract library ......................90
3.4.2 Initial purification of active extract ................................................93
3.4.3 Hemagglutination assay and drug combination studies .................95
3.4.4 Bioassay guided isolation at NCI-Frederick ..................................97
3.4.5 Immunoblot assay ........................................................................101
3.4.6 Characterization of Gigartinin .....................................................102
3.5 Research summary and future directions .....................................................104
3.6 References Cited ........................................................................................105

Chapter 4. Chemical investigation of Dendrilla membranosa ......................................113
4.1 Introduction ..................................................................................................113
4.2 Background ..................................................................................................115
4.3 Spongian diterpenoids from D. membranosa ...............................................116
4.4 Results and discussion on the characterization of the membranolides ........119
4.4.1 Characterization of membranolide E (4.22) .................................122
ii
4.4.2 Characterization of membranolide F (4.23) .................................123
4.4.3 Characterization of fused furan membranolides ..........................124
4.4.4 Characterization of membranolide G (4.24) ................................125
4.4.5 Characterization of membranolide H (4.25) ................................126
4.4.6 Reinvestigation of membranolides C and D ................................128
4.4.7 Revision of membranolide C (4.26).............................................130
4.4.8 Revision of membranolide D (4.27) ............................................132
4.4.9 Characterization of membranolides as artifacts from
extraction......................................................................................134
4.5 Results and discussion for feeding assay .....................................................137
4.6 Research summary and future directions .....................................................138
4.7 References Cited ........................................................................................139

Chapter 5. Experimental ................................................................................................143
5.1 General procedures .......................................................................................143
5.1.1 Collection of biological materials ................................................144
5.2 Experimental supporting Chapter 2 ..............................................................144
5.2.1 Collection protocol for nudibranchs ............................................144
5.2.2 Bulk isolation of palmadorins D - S from the Antarctic
nudibranch Austrodoris kerguelenensis .......................................145
5.2.3 Collection of LC/MS data ............................................................153
5.3 Experimental supporting Chapter 3 ..............................................................154
5.3.1 Preparation of fraction PSC07-52-A-I .........................................154
5.3.2 Preparation of fraction PSC08-8-3...............................................155
5.3.3 Preparation of 319-A fractions.....................................................155
5.3.4 Chromatography on POROS PE column .....................................156
5.3.5 Chromatography on POROS HQ column ....................................156
5.3.6 Chromatography on Hamilton PRP-3 RP protein HPLC ............156
5.3.7 MALDI-TOF analysis ..................................................................156
5.3.8 LC/MS/MS chromatography of 319A-103E ...............................157
5.3.9 SDS-PAGE experiments ..............................................................158
5.3.10 Immunoblotting and gp120 dot blot assay ...................................158
5.4 Experimental supporting Chapter 4 ..............................................................159
5.4.1 Purification of membranolides in the field laboratory .................159
5.4.2 Amphipod feeding bioassays .......................................................160
5.4.3 Statistical analyses .......................................................................161
5.4.4 Isolation of membranolides from the Antarctic sponge
Dendrilla membranosa ................................................................161
5.4.5 Collection of LC/MS data ............................................................162

iii
Appendices .......................................................................................................................163
Appendix A: Spectroscopic and spectrometric data supporting Chapter 2 ........164
Appendix B: Spectroscopic and spectrometric data supporting Chapter 4.........247

iv
List of Tables

Table 1.1. Potential therapeutics from marine sources .....................................................10

Table 2.1. Palmadorins D - S (2.32 - 2.47) .....................................................................31

Table 2.2. 13C NMR Data and Multiplicity of palmadorins D - S (2.32 - 2.47) ...............32

Table 2.3. Averaged 1H and 13C NMR Data of Substructure A ........................................34

Table 2.4. HMBC and COSY Correlations and Averaged 1H and 13C NMR Data
Among the Glycercerol Substructres B - E................................................36

Table 2.5. LC/MS Palmadorin Retention Times ..............................................................57

Table 2.6. LC/MS Data Results for Selected PSC10-10 specimens ................................64

Table 3.1. Examples of Bioactive Proteins on the Market ...............................................83

Table 4.1. NMR Data (CDCl ) of Membranolides E (2.23) and F (2.23) ......................123
3

Table 4.2. Comparison of 1H and 13C NMR Data of Aplysulphurin (4.21) and
AM5090D2 in CDCl ..............................................................................136
3

v
List of Figures

Figure 1.1. Illustration of Salix and Aspirin. ©Dov Bock. Used with
permission ....................................................................................................1

Figure 1.2. Biosynthetic origin of the major classes of natural products .......................7

Figure 1.3. Selected marine natural products that have advanced as
therapeutics ..................................................................................................9

Figure 1.4. Illustration of allopatric (geographic) speciation whereby a) an
original population experiences b) geographic isolation (e.g.,
period of glaciation). If kept separated long enough, c) a new
species may evolve forming two distinct species once d) the
barriers are removed (retreat of icesheets) .................................................14

Figure 2.1. Austrodoris kerguelenensis and its representative chemistry ....................22

Figure 2.2. 1H and 13C NMR illustrating characteristic shifts for substructure A ........34

Figure 2.3. 13C NMR data comparison of 2.32 versus 2.23 .........................................37

Figure 2.4. 13C NMR data comparison of 2.33 versus 2.48 .........................................38

Figure 2.5. 13C NMR data comparison of 2.34 versus 2.25 .........................................39

Figure 2.6. 13C NMR data comparison of 2.35 versus 2.49 .........................................40

Figure 2.7. 13C NMR data comparison of 2.37 versus 2.50 .........................................43

Figure 2.8. Key ROESY correlations for 2.36 .............................................................45

Figure 2.9. Key NOE enhancements and ROESY correlations for palmadorin
2.41.............................................................................................................47

Figure 2.10. 13C NMR data comparison of 2.41 versus 2.51 .........................................47

Figure 2.11. 13C NMR data comparison of 2.44 versus 2.52 .........................................50

Figure 2.12. 13C NMR data comparison of 2.45 versus 2.52 .........................................51

vi