DEMYSTIFYING AND UNRAVELLING THE MOLECULAR STRUCTURE OF THE BIOPOLYMER SPOROPOLLENIN.

RATIONALE We report the unsolved molecular structure of the complex biopolymer sporopollenin exine extracted form Lycopodium clavatum pollen grains. METHODS TOF-SIMS and CID-MS/MS, MALDI-TOF-MS and CID-TOF/TOF-MS/MS were used for the analysis of this complex biopolymer sporopollenin exine extracted from Lycopodium clavatum. Solid-state 1 H- and 13 C-NMR, 2D 1 H-1 H NOESY, Rotor-synchronized 13 C{1 H} HSQC, and 13 C{1 H} multi CP-MAS NMR experiments were used to confirm the structural assigments revealed by MS and MS/MS studies. Finally, high-resolution XPS was used to check for the presence of aromatic components in sporopollenin. RESULTS The combined MS and NMR analyses showed that sporopollenin contained poly (hydroxy acid) dendrimer-like networks with glycerol as a core unit, which accounted for the sporopollenin empirical formula. In addition, these analyses showed that the hydroxy acid monomers forming this network contained a β-diketone moiety. Moreover, MALDI-TOF-MS and MS/MS allowed us to identify a unique macrocyclic oligomeric unit composed of polyhydroxylated tetraketide-like monomers. Lastly, high resolution X-ray photoelectron spectroscopy showed the absence of aromaticity in sporopollenin. CONCLUSION We eport for the first time the two main building units that form the Lycopodium clavatum sporopollenin exine. The first building unit is a macrocyclic oligomer and/or polymer composed of polyhydroxylated tetraketide-like monomeric units, which represents the main rigid backbone of the sporopollenin biopolymer. The second building unit is the poly (hydroxy acid) network upon which their hydroxyl end groups can be covalently attached by ether links to the hydroxylated macrocyclic backbone to form the sporopollenin biopolymer, a spherical dendrimer. Such spherical dendrimers are a typical type of microcapsule that has been used for drug delivery applications. Finally, HR-XPS (high-resolution X-ray photoelectron spectroscopy) indicated the total absence of aromaticity in the sporopollenin exine.

[1]  T. Filley,et al.  Quantitative analysis of diverse sporomorph-derived sporopollenins. , 2019, Phytochemistry.

[2]  J. Warzywoda,et al.  Investigation of the Fate of Proteins and Hydrophilicity/Hydrophobicity of Lycopodium clavatum Spores after Organic Solvent-Base-Acid Treatment. , 2019, ACS applied materials & interfaces.

[3]  Tingting Fu,et al.  Biosynthetic investigation of γ-lactones in Sextonia rubra wood using in situ TOF-SIMS MS/MS imaging to localize and characterize biosynthetic intermediates , 2019, Scientific Reports.

[4]  Vito Pesce,et al.  Administration of Enalapril Started Late in Life Attenuates Hypertrophy and Oxidative Stress Burden, Increases Mitochondrial Mass, and Modulates Mitochondrial Quality Control Signaling in the Rat Heart , 2018, Biomolecules.

[5]  K. Engel,et al.  Recent Developments of Useful MALDI Matrices for the Mass Spectrometric Characterization of Lipids , 2018, Biomolecules.

[6]  J. Weng,et al.  The molecular structure of plant sporopollenin , 2018, Nature Plants.

[7]  A. Dobritsa,et al.  Exine and Aperture Patterns on the Pollen Surface: Their Formation and Roles in Plant Reproduction , 2018, Annual Plant Reviews online.

[8]  Jun Zhu,et al.  The Regulation of Sporopollenin Biosynthesis Genes for Rapid Pollen Wall Formation1[OPEN] , 2018, Plant Physiology.

[9]  N. Abidi,et al.  A chemical treatment method for obtaining clean and intact pollen shells of different species. , 2018, ACS biomaterials science & engineering.

[10]  Tingting Fu,et al.  Tandem Mass Spectrometry Imaging and in Situ Characterization of Bioactive Wood Metabolites in Amazonian Tree Species Sextonia rubra. , 2018, Analytical chemistry.

[11]  M. Tamkun,et al.  Observation of endoplasmic reticulum tubules via TOF-SIMS tandem mass spectrometry imaging of transfected cells. , 2018, Biointerphases.

[12]  A. Schnapp,et al.  New Insights into the Wavelength Dependence of MALDI Mass Spectrometry. , 2017, Analytical chemistry.

[13]  K. Iyer,et al.  Supramolecular Assemblies of Dendrimers and Dendritic Polymers in Nanomedicine , 2017 .

[14]  G. Fisher,et al.  The Composition of Poly(Ethylene Terephthalate) (PET) Surface Precipitates Determined at High Resolving Power by Tandem Mass Spectrometry Imaging , 2017, Microscopy and Microanalysis.

[15]  R. Heeren,et al.  A New Method and Mass Spectrometer Design for TOF-SIMS Parallel Imaging MS/MS. , 2016, Analytical chemistry.

[16]  S. Atkin,et al.  Sporopollenin, The Least Known Yet Toughest Natural Biopolymer , 2015, Front. Mater..

[17]  T. D. Fridgen,et al.  The in situ gas-phase formation of a C-glycoside ion obtained during electrospray ionization tandem mass spectrometry. A unique intramolecular mechanism involving an ion-molecule reaction. , 2015, Rapid communications in mass spectrometry : RCM.

[18]  E. Balan,et al.  Evolution of the macromolecular structure of sporopollenin during thermal degradation , 2015, Heliyon.

[19]  J. Dimmock,et al.  The unexpected formation of [M - H]+ species during MALDI and dopant-free APPI MS analysis of novel antineoplastic curcumin analogues. , 2014, Journal of mass spectrometry : JMS.

[20]  M. Sephton,et al.  Pollen and spores as a passive monitor of ultraviolet radiation , 2014, Front. Ecol. Evol..

[21]  K. Schmidt-Rohr,et al.  Quantitative solid-state 13C NMR with signal enhancement by multiple cross polarization. , 2014, Journal of magnetic resonance.

[22]  Amit Arora,et al.  Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology , 2013, Clinical Microbiology Reviews.

[23]  R. Knochenmuss.  MALDI and Related Methods: A Solved Problem or Still a Mystery? , 2013, Mass spectrometry.

[24]  R. Caprioli,et al.  Matrix precoated targets for direct lipid analysis and imaging of tissue. , 2013, Analytical chemistry.

[25]  C. Douglas,et al.  Sporopollenin monomer biosynthesis in arabidopsis , 2013, Journal of Plant Biology.

[26]  J. Watson,et al.  Formation of a polyalkyl macromolecule from the hydrolysable component within sporopollenin during heating/pyrolysis experiments with Lycopodium spores , 2012 .

[27]  Mark A. Smith,et al.  Rapid identification of triacylglycerol-estolides in plant and fungal oils , 2012 .

[28]  P. Chaurand,et al.  Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition. , 2012, Analytical chemistry.

[29]  P. Traldi,et al.  The double nature of 1,5-diaminonaphthalene as matrix-assisted laser desorption/ionization matrix: some experimental evidence of the protonation and reduction mechanisms. , 2011, Rapid communications in mass spectrometry : RCM.

[30]  C. Wesdemiotis,et al.  Fragmentation pathways of polymer ions. , 2011, Mass spectrometry reviews.

[31]  T. Ariizumi,et al.  Genetic regulation of sporopollenin synthesis and pollen exine development. , 2011, Annual review of plant biology.

[32]  Bernd Ondruschka,et al.  Ball milling in organic synthesis: solutions and challenges. , 2011, Chemical Society reviews.

[33]  J. Wadhawan,et al.  Viability of plant spore exine capsules for microencapsulation , 2011 .

[34]  C. Douglas,et al.  LAP6/POLYKETIDE SYNTHASE A and LAP5/POLYKETIDE SYNTHASE B Encode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis in Arabidopsis thaliana[C][W][OA] , 2010, Plant Cell.

[35]  C. Douglas,et al.  Analysis of TETRAKETIDE α-PYRONE REDUCTASE Function in Arabidopsis thaliana Reveals a Previously Unknown, but Conserved, Biochemical Pathway in Sporopollenin Monomer Biosynthesis[C][W] , 2010, Plant Cell.

[36]  M. Galésio,et al.  Comparative study of matrices for their use in the rapid screening of anabolic steroids by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. , 2009, Rapid communications in mass spectrometry : RCM.

[37]  Charles L. Wilkins,et al.  Developments in MALDI Mass Spectrometry: The Quest for the Perfect Matrix , 2008 .

[38]  R. Cecchelli,et al.  Structural determination of the novel fragmentation routes of morphine opiate receptor antagonists using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. , 2005, Rapid communications in mass spectrometry : RCM.

[39]  H. Neubert,et al.  MALDI post-source decay and LIFT-TOF/TOF investigation of α-cyano-4-hydroxycinnamic acid cluster interferences , 2004, Journal of the American Society for Mass Spectrometry.

[40]  C. Borchers,et al.  Pseudo-MS3 in a MALDI orthogonal quadrupole-time of flight mass spectrometer , 2002, Journal of the American Society for Mass Spectrometry.

[41]  J. Terracciano,et al.  Structure of sch 419560, a novel alpha-pyrone antibiotic produced by Pseudomonas fluorescens. , 2002, The Journal of antibiotics.

[42]  A G Marshall,et al.  Kendrick mass defect spectrum: a compact visual analysis for ultrahigh-resolution broadband mass spectra. , 2001, Analytical chemistry.

[43]  K. Hartfelder,et al.  Identification of oxygen containing volatiles in cephalic secretions of workers of Brazilian stingless bees , 2000 .

[44]  H. Bubert,et al.  The Nature of Oxygen in Sporopollenin from the Pollen of Typha angustifolia L. , 2000, Zeitschrift fur Naturforschung. C, Journal of biosciences.

[45]  Jen-kun Lin,et al.  Biotransformation of curcumin through reduction and glucuronidation in mice. , 1999, Drug metabolism and disposition: the biological fate of chemicals.

[46]  D. Murphy,et al.  Biogenesis and function of the lipidic structures of pollen grains , 1998, Sexual Plant Reproduction.

[47]  T. Osawa,et al.  Involvement of the beta-diketone moiety in the antioxidative mechanism of tetrahydrocurcumin. , 1996, Biochemical pharmacology.

[48]  Angela N. García,et al.  Pyrolysis of Polyethylene in a Fluidized Bed Reactor , 1994 .

[49]  C. Saiz-Jimenez.  Production of alkylbenzenes and alkylnaphthalenes upon pyrolysis of unsaturated fatty acids , 1994, Naturwissenschaften.

[50]  C. Saiz-Jimenez.  Production of alkylbenzenes and alkylnaphthalenes upon pyrolysis of unsaturated fatty acids. A model reaction to understand the origin of some pyrolysis products from humic substances , 1994 .

[51]  V. Bertolasi,et al.  Resonance-assisted hydrogen bonding. III: Formation of intermolecular hydrogen-bonded chains in crystals of β-diketone enols and its relevance to molecular association , 1993 .

[52]  J. Boon,et al.  p-coumaric acid — a monomer in the sporopollenin skeleton , 1989, Planta.

[53]  R. Winans,et al.  Chemical alteration of a biological polymer Sporopollenin during coalification: origin, formation, and transformation of the coal maceral sporinite , 1988 .

[54]  G. Bergström,et al.  Volatile Secretions in Three Species of Dufourea (Hymenoptera: Halictidae) Bees: Chemical Composition and Phylogeny , 1985 .

[55]  J. BROOKS,et al.  Chemical Structure of the Exine of Pollen Walls and a New Function for Carotenoids in Nature , 1968, Nature.

[56]  T. Bier,et al.  Lignin , 2020, Springer Series on Polymer and Composite Materials.

[57]  Jun Zhu,et al.  Molecular Cell Biology of Pollen Walls , 2014 .

[58]  S. Atkin,et al.  Pollen and Spore Shells—Nature’s Microcapsules , 2014 .

[59]  J. Fréchet,et al.  Dendrimers and dendritic polymers in drug delivery. , 2005, Drug discovery today.

[60]  J. D. de Leeuw,et al.  Biomacromolecules of Algae and Plants and their Fossil Analogues , 2005, Plant Ecology.

[61]  E. Domínguez,et al.  Isolation of intact pollen exine using anhydrous hydrogen fluoride , 1998 .

[62]  G. Shaw,et al.  The Post-Tetrad Ontogeny of the Pollen Wall and the Chemical Structure of the Sporopollenin of Lilium Henryi , 1968 .

[63]  F. Zetzsche,et al.  Untersuchungen über die Membran der Sporen und Pollen V. 4. Zur Autoxydation der Sporopollenine , 1931 .