PHD PROPOSAL FOR THE DOCTORAL SCHOOL - " ECOLOGIE, GÉOSCIENCES, AGRONOMIE, ALIMENTATION "
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PhD PROPOSAL FOR THE DOCTORAL SCHOOL
« Ecologie, Géosciences, Agronomie, ALimentation »
GENERAL INFORMATION
Thesis title: Dereplication based on laser desorption Ionization and 13C NMR for biological
fingerprint analyses
Acronym: DEPICt
Disciplinary field 1: Agronomy
Disciplinary field 2: Select an element
Three keywords: Natural Products, NMR, LDI-MS
Research unit : EA921 SONAS
Name of the thesis director: SCHINKOVITZ Andreas
Email address of the thesis director : andreas.schinkovitz@univ-angers.fr
Name of the thesis co-supervisor 1 (if applicable): DERBRE Séverine
Email address of the thesis co-supervisor 1 (if applicable): severine.derbre@univ-angers.fr
Name of the thesis co-supervisor 2 (if applicable):
Email address of the thesis co-supervisor 2 (if applicable):
Thesis grant (funding origin and amount): Doctoral contract: (100%, 91000 €)
Contact(s) (mailing address and E-mail): EA921 SONAS, Campus du végétal ; 42 rue Georges
Morel, 49070 Beaucouzé
Recruitment process: Recruitment process depends on thesis funding. To select the corresponding recruitment
process, please visit the EGAAL website here. This information is needed for proposal publication.
☒ Doctoral school contest ☐ Interview ☐ Other (indicate) :
All sections must be filled. Once filled, please save the proposal form in pdf format using
the following naming: Supervisor Name_Unit_Subject Acronym_EN.pdf
ED EGAAL
Direction : 65 rue de Saint-Brieuc – CS 84215 – 35042 Rennes Cedex – France
Tél : 02 23 48 52 75
Mail : ed-EGAAL@u-bretagneloire.fr
Site Web : https://ed-egaal.u-bretagneloire.fr
SCIENTIFIC DESCRIPTION OF THE PhD PROJECT
Socio-economic and scientific context : (10 lines)
The SONAS laboratory has a long history in natural products (NPs) research comprising the analysis of highly complex mixtures and their biological
activities such as anti-scaling1 [1] and antiAGEs effects [phenolic acids of propolis [2] as well as compounds that stimulate plat defense (alkaloids and
xanthones)] [3]. Within the last five years our lab has developed innovative analytical concepts for the analysis and dereplication of NPs and complex
mixtures based on matrix free laser desorption ionization mass spectrometry (LDI-MS) [4] as well as 13C NMR. Within the process, an in-house NMR
software (MixONat) was created and successfully applied for the characterization of complex mixtures [5]. Moreover, SONAS has recently purchased
a high resolution LC-MS-QTOF, which permits the creation of molecular networks [6]. With this in mind, our overall research strategy is to identify
unknown bioactive NPs within complex mixtures and subfractions thus the avoiding time-consuming fractionation steps by early prioritization.
Assumptions and questions (8 lines)
Polyphenols (Cinnamic acid derivatives, xanthones, PPAPs) represent an important group of NPs and exhibit various beneficial biological effects on
humans and plants. Many important polyphenols were obtained by bioassay-guided fractionation in the past. However this valuable research strategy
also comprises pitfalls. Most importantly, a significant decay in relative biological is often observed when going from crude extracts to single compounds.
In other words, the initial activity of an extract cannot be explained by the overall amount of all confirmed bioactive compounds in this extract. This effect
is well-known but hardly understood and potentially due to synergism as shown for polyphenols from apple [7]. Consequently, a specifically defined
mixture of NPs may exhibit better biological effects. In this context, our preliminary work has shown that LDI-MS allows the statistical grouping (PCA)
of complex propolis samples according to their composition of phenolic compounds. This strategy will be used for associating bioactivity to specific
signal pattern (fingerprint spectra). On the other hand 13C NMR permits the unambiguous identification of major compounds from complex mixtures.
Our current research project therefore proposes the hyphenated use of LDI-MS and 13C NMR for dereplication purposes as well as a better
understanding of synergistic/antagonistic effects of polyphenols within a well-defined biological test system. The approach will be accompanied by a
systematic evaluation of sample material by LC-MS and the creation of molecular network.
The main steps of the thesis and scientific procedure (10-12 lines)
With regards to structural elucidation, LDI-MS permits detecting a wide range of major and minor NPs while NMR allows the unambiguous assignment
of stereoisomers as well as the identification of major compounds, thus ideally complementing the LDI approach. In a first step, the project will therefore
compare the capacity of dereplication of the proposed methods (LDI-MS, 13C RMN, LC-MS molecular networks) on extracts and subfractions of plants,
which are particularly rich in xanthones and show notable plant defense activating effects [7]. Therefore, 4 chemically well-studied model batches from
one plant will be used [8]. Next, a bioassay-guided fractionation of a different, phytochemically well-studied extract will be performed, this time with focus
on anti-biofilm activities of Candida albicans activities (Biological evaluation will be performed by the team of Pr. C. Imbert and Dr. M. Girardo Université
de Poitiers, laboratoire Ecologie et biologie des interactions-UMR 7267). Each separation step will be systematically monitored by LDI-MS and 13C NMR
1
[1]. Université d’Angers, SAS Esprit d’éthique et EPF Ecole d’ingénieur-e-s. Nouveaux agents antitartre biodégradables. P.
Richomme, S. Derbré, C. Binachon, S. Dejoie, R. Boulmane, O. Horner et H. Cheap-Charpentier. Demande de brevet français n°
FR3072088 Patent Application Publication FR201759383 (Dépôt le 6 octobre 2017).
[2]. A. S. Boisard, A.-M. Le Ray, J. Gatto, M.-C. Aumond, P. Blanchard, S. Derbré, C. Flurin, P. Richomme, J. Agric. Food Chem.
2014, 62, 1344-1351; b. S. Boisard, Y. Shahali, M.-C. Aumond, S. Derbré, P. Blanchard, M. Dadar, A.-M. Le Ray, P. Richomme,
J. Food Sci. Technol. 2020, 55, 453-460.
[3]. Université d’Angers. Potentiating agents for protecting plants from fungal infections. P. Simoneau, T. Guillemette, P.
Richomme, J.-J. Hélesbeux. European Patent Application EP2687097 (Dépôt le 16 juillet 2012, Publication le 22 janvier 2014).
[4]. a. A. Schinkovitz, S. Boisard, I. Freuze, J. Osuga, N. Mehlmer, T. Brück, P. Richomme, Matrix-free laser desorption ionization
mass spectrometry as a functional tool for the analysis and differentiation of complex phenolic mixtures in propolis: a new approach
to quality control. Anal. Bioanal. Chem. 2018, 410, 6187-6195; b. P. Le Pogam, A. Schinkovitz, B. Legouin, A.-C. Le Lamer, J.
Boustie, P. Richomme, Matrix-free UV-laser desorption ionization mass spectrometry as a versatile approach for accelerating
dereplication studies on lichens. Anal. Chem. 2015, 87, 10421-10428; c. Skopikova M, Hashimoto M, Richomme P, Schinkovitz
A. Matrix-Free laser desorption ionization mass spectrometry as an efficient tool for the rapid detection of opiates in crude extracts
of Papaver somniferum. J. Agric. Food. Chem. 2020, 68, 884-891.
[5]. a. A. Bruguière, S. Derbré, C. Coste, M. Le Bot, B. Siegler, S. T. Leong, S. N. Sulaiman, K. Awang, P. Richomme, 13C-NMR
dereplication of Garcinia extracts: Predicted chemical shifts as reliable databases. Fitoterapia 2018, 131, 59-64; b. A. Bruguière,
S. Derbré, J. Dietsch, J. Leguy, V. Rahier, Q. Pottier, D. Bréard, S. Suor-Cherer, G. Viault, A.-M. L. Ray, F. Saubion, P. Richomme,
MixONat, a software for mixtures dereplication based on 13C NMR experiments. Anal. Chem. En révision.
[6]. a. P. M. Allard, T. Peresse, J. Bisson, K. Gindro, L. Marcourt, V. C. Pham, F. Roussi, M. Litaudon, J. L. Wolfender, Integration
of molecular networking and in-silico MS/MS fragmentation for natural products dereplication. Anal. Chem. 2016, 88, 3317-3323;
b. M. Wang, J. J. Carver, V. V. Phelan, L. M. Sanchez, N. Garg, Y. Peng, D. D. Nguyen, J. Watrous, C. A. Kapono, T. Luzzatto-
Knaan, C. Porto, A. Bouslimani, A. V. Melnik, M. J. Meehan, W.-T. Liu, et al., Sharing and community curation of mass
spectrometry data with Global Natural Products Social Molecular Networking. Nature Biotechnol. 2016, 34, 828.
1
[7]. Thèse Thomas Charpentier (2019-2022, SONAS-FUNGISEM, Dir. P. Richomme et T. Guillemette)
1
[8]. Collaboration franco-malaysienne [NatProLab (Dir. K. Awang), University of Malaya, Kuala-Lumpur, Malaisie]
in order to identify potential active NPs and specifically active synergistic mixtures. Eventually the activity of crude extracts, bioactive isolated compounds
and reconstituted mixtures of NPs will be thoroughly studied for assigning chemical fingerprint patterns to specifically observed activity.
Methodological and technical approaches considered (4-6 lines)
The project will apply 3 different approaches of dereplication (i.e. LDI-MS, 13C-NMR, LC-MS² molecular networking). Technically this will be facilitated
by a high-resolution (HR) Spiral-TOF (JEOL, Japan) instrument (LDI-MS) a 400 MHz NMR (JEOL, Japan) spectrometer as well as HPLC-HRMS-QTOF
instrument that should be installed by the end of summer 2020. The latter will be used for creating a molecular network. Moreover the project will benefit
from the recently developed in-house software MixONat as well as the Universal Natural Products Database (UNPD),comprising more than 200000
NPs. Biological evaluation on proteins involved in resistance as well and anti-biofilm activities will be statistically evaluated (Origilab, Statbox)
Scientific and technical skills required by the candidate
The candidate should have experience in the chemical analysis of natural product by methods of LC-MS, 1 and 2D NMR and mass spectrometry. This
profile may best apply to a M2 master student who did an internship in a natural products’ lab.
THESIS SUPERVISION2
Unit name: Team name:
EA921 SONAS
Unit director name: Team director name:
Pascal RICHOMME
Mailing address of the unit director: Mailing address of the team director:
pascal.richomme@univ-angers.fr
Thesis director
Surname, first name: SCHINKOVITZ Andreas
Position: Senior Lecturer
Obtained date of the HDR (Habilitation thesis to supervise research): 27/09/2016
Employer: University of Angers
Doctoral school affiliation: EGAAL
Rate of thesis supervision in the present project (%): 50
Total rate of thesis supervision in ongoing theses (supervisions and co-supervisions) (%): 50
Number of current thesis supervisions/co-supervisions: 1
Thesis co-supervisor 1 (if applicable)
Surname, first name: DERBRE Séverine
Position: Assistant Professor
Habilitation thesis to supervise research ☐ yes ☒ no If yes, date diploma received:
Employer: MCU
Doctoral school affiliation: EGAAL
Rate of thesis supervision in the present project (%): 50
Total rate of thesis supervision in ongoing theses (supervisions and co-supervisions) (%): 20
Number of current thesis supervisions/co-supervisions:
Thesis co-supervisor 2 (if applicable)
Surname, first name:
Position:
Habilitation thesis to supervise research ☐ yes ☐ no If yes, date diploma received:
Employer:
Doctoral school affiliation:
Rate of thesis supervision in the present project (%):
Total rate of thesis supervision in ongoing theses (supervisions and co-supervisions) (%):
Number of current thesis supervisions/co-supervisions:
Private partner (if CIFRE funding, private funding,…)
2
In EGAAL Doctoral School, if only one scientist in thesis supervision = 100% of supervision rate; if 2 people involved
in thesis supervision = from 50% to 70% of supervision rate for the director; if 3 people involved in thesis supervision
= 40% / 30% / 30% of supervision rate distribution among supervisors.
Surname, first name: Position: Employer: Rate of thesis supervision in the present project (%): Total rate of thesis supervision in ongoing theses (supervisions and co-supervisions) (%): Number of current thesis supervisions/co-supervisions: International partner (if Cotutelle thesis) Surname, first name: Position: Employer: Rate of thesis supervision in the present project (%): Total rate of thesis supervision in ongoing theses (supervisions and co-supervisions) (%): Number of current thesis supervisions/co-supervisions: Professional status of previous PhD students supervised by both director and co-supervisors (from 5 years) Please provide the following information for each PhD students supervised Andreas Schinkovitz (Head supervisor, Director) Surname, first name: JABER Ali Date of PhD beginning and PhD defence: 01/11/2012-12/07/2017 Thesis supervision: SERAPHIN Denis Professional status and location: Maître de conférences en chimie synthétique, Lebanese University, Beirut | Faculty of Science (Beyrouth, Liban). Contract profile (post-doc, fixed-term, permanent): permanent List of publications from the thesis work:2 Jaber A, Séraphin D, Guilet D, Osuga J, Cheble E, Ibrahim G, Richomme P, Schinkovitz A. Bithiophenic MALDI matrices as valuable leads for the selective detection of alkaloids. Anal Bioanal Chem. 2017; 409, 6791-6801. doi : 10.1007/s00216-017-0634-x Patent application: Séraphin D, Guilet D, Schinkovitz A, Richomme P, Dias M, Levillain E, Jaber A. Matrix for specific detection of alkaloids by MALDI-TOF mass spectrometry. Brevet d'invention mondial Oct. 18, 2018 WO 2018189484 A1 20181018, FR 3065214 A1 20181019 Surname, first name: ŠKOPÍKOVÁ Michaela Date of PhD beginning and PhD defence: since 01/10/2017 Thesis supervision: SCHINKOVITZ Andreas Professional status and location: PhD student EGAAL Contract profile (post-doc, fixed-term, permanent): fixed-term List of publications from the thesis work: Skopikova M, Hashimoto M, Richomme P, Schinkovitz A. Matrix-Free laser desorption ionization mass spectrometry as an efficient tool for the rapid detection of opiates in crude extracts of Papaver somniferum. J Agric Food Chem. 2020; 68, 884-891. doi : 10.1021/acs.jafc.9b05153 Five main recent publications of the supervisors on thesis subject: Head supervisor (Andreas Schinkovitz) 1. Skopikova M, Hashimoto M, Richomme P, Schinkovitz A. Matrix-Free laser desorption ionization mass spectrometry as an efficient tool for the rapid detection of opiates in crude extracts of Papaver somniferum. J Agric Food Chem. 2020; 68, 884-891. doi : 10.1021/acs.jafc.9b05153. 2. Schinkovitz A, Le Pogam P, Derbré S, Roy-Vessieres E, Blanchard P, Thirumaran SL, Breard D, Aumond MC, Zehl M, Urban E, Kaur A, Jäger N, Hofer S, Kopp B, Stuppner H, Baglin I, Séraphin D, Tomasi S, Henrion D, Boustie J, Richomme P. Secondary metabolites from lichen as potent inhibitors of advanced glycation end products and vasodilative agents. Fitoterapia, 2018, 131, 182-188. doi : 10.1016/j.fitote.2018.10.015, 3. Schinkovitz A, Boisard S, Freuze I, Osuga J, Mehlmer N, Brück T, Richomme P. Matrix-free laser desorption ionization mass spectrometry as a functional tool for the analysis and differentiation of complex phenolic mixtures in propolis: a new approach to quality control. Anal Bioanal Chem, 2018, 410, 6187– 6195. doi : 10.1007/s00216-018-1225-1 4. Le Pogam P., Boustie J, Richomme P, Denis A, Schinkovitz A. The inherent matrix properties of lichen metabolites in matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom, 2017, 31, 1993-2002. doi : 10.1002/rcm.7980. 5. Jaber A, Séraphin D, Guilet D, Osuga J, Cheble E, Ibrahim G, Richomme P, Schinkovitz A. Bithiopheic MALDI matrices as valuable leads for the selective detection of alkaloids. Anal Bioanal Chem, 2017 409, 6791-801. doi : 10.1007/s00216-017-0634-x,
Professional status of previous PhD students supervised by both director and co-supervisors (from 5 years) Please provide the following information for each PhD students supervised DERBRE severine (Co-supervisor) Surname, first name: ROUGER Caroline Date of PhD beginning and PhD defence: 01/10/2012-11/12/2015 Thesis supervision: RICHOMME Pascal Professional status and location: Maître de conférences en pharmacognosie, Faculté de pharmacie, Université de Bordeaux Contract profile (post-doc, fixed-term, permanent): permanent List of publications from the thesis work: 3 Rouger, C., Pagie, S., Derbré, S.*, Le Ray, A.-M., Richomme, P., Charreau, B. Prenylated polyphenols from Clusiaceae and Calophyllaceae with immunomodulatory activity on endothelial cells. PLoSOne 2016, 11, e0167361. Dang, B. T., Rouger, C., Litaudon, M., Séraphin, D., Richomme, P., Derbré, S.* Identification and purification of minor benzoylated 4-phenylcoumarins from an anti-AGE M. neurophylla bark extract. Molecules 2015, 20, 17735-17746. Rouger, C., Derbré, S.*, Charreau, B., Pabois, A., Cauchy, T., Litaudon, M., Awang, K., Richomme, P. Lepidotol A from Mesua lepidota inhibits inflammatory and immune mediators in human endothelial cells. J. Nat. Prod. 2015, 78, 2187–2197.Surname, first name: ŠKOPÍKOVÁ Michaela Surname, first name: DEJOIE Stéphane Date of PhD beginning and PhD defence: 01/10/2014-08/12/2017 Thesis supervision: RICHOMME Pascal Professional status and location: Researcher at Schweitzer-Mauduit International Inc (Le Mans) Contract profile (post-doc, fixed-term, permanent): fixed-term List of publications from the thesis work: Patent application : Université d’Angers, SAS Esprit d’éthique et EPF Ecole d’ingénieur-e-s. Nouveaux agents antitartre biodégradables. P. Richomme, S. Derbré, C. Binachon, S. Dejoie, R. Boulmane, O. Horner et H. Cheap-Charpentier. Demande de brevet français n° FR3072088 Patent Application Publication FR201759383 (Dépôt le 6 octobre 2017). Surname, first name: BRUGUIERE Antoine Date of PhD beginning and PhD defence: 01/10/2016-08/12/2019 Thesis supervision: RICHOMME Pascal Professional status and location: ATER in pharmacognosie at Université de Bourgogne, Faculté de pharmacie Contract profile (post-doc, fixed-term, permanent): fixed-term List of publications from the thesis work: Bruguière, A. Derbré, S.*, Dietsch, J., Leguy, J., Rahier, V., Pottier, Q., Bréard, D., Suor-Cherer, S., Viault, G., Le Ray, A.-M., Saubion, F., Richomme, P. MixONat, a software for mixtures dereplication based on 13C NMR experiments. Anal. Chem. Soumise. Bruguière, A., Derbré, S.*, Coste, C., Le Bot, M., Siegler, B, Leong, S. T., Sulaiman, S. N., Awang, K., Richomme, P. 13C-NMR dereplication of Garcinia extracts: predicted chemical shifts as reliable databases. Fitoterapia 2018, 131, 59-64. Bréard, D., Viault, G. Mezier, M.-C., Pagie, S., Bruguière, A., Richomme, P., Charreau, B., Derbré, S.* Additional insights into Hypericum perforatum content: Isolation, total synthesis, and absolute configuration of Hyperbiphenyl A and B from immunomodulatory root extracts. J. Nat. Prod. 2018, 81, 1850-1859. Five main recent publications of the supervisors on thesis subject: Co-supervisor (Severine Derbre) 1. Bruguière, A. Derbré, S.*, Dietsch, J., Leguy, J., Rahier, V., Pottier, Q., Bréard, D., Suor-Cherer, S., Viault, G., Le Ray, A.-M., Saubion, F., Richomme, P. MixONat, a software for mixtures dereplication based on 13C NMR experiments. Anal. Chem. En révision. 2. Rouger, C., Derbré, S.*, Richomme, P. Mesua sp.: Chemical aspects and pharmacological relevance of prenylated polyphenols Phytochemistry reviews 2019, 18, 317-342. 3. Schinkovitz, A., Le Pogam, P., Derbré, S. Vessieres, E., Blanchard, P. Thirumaran, S.-L., Breard, D., Aumond, M.-C., Zehl, M., Urban, E., Kaur, A., Jäger, N., Hofer, S., Kopp, B., Stuppner, H., Baglin, I., Seraphin, D., Tomasi, S., Henrion, D., Boustie, J., Richomme, P. Secondary metabolites from lichen as potent inhibitors of advanced glycation end products and vasodilative agents Fitoterapia 2018, 131, 182- 188.
4. Bruguière, A., Derbré, S.*, Coste, C., Le Bot, M., Siegler, B, Leong, S. T., Sulaiman, S. N., Awang, K., Richomme, P. 13C-NMR dereplication of Garcinia extracts: predicted chemical shifts as reliable databases. Fitoterapia 2018, 131, 59-64. 5. Bréard, D., Viault, G. Mezier, M.-C., Pagie, S., Bruguière, A., Richomme, P., Charreau, B., Derbré, S.* Additional insights into Hypericum perforatum content: Isolation, total synthesis, and absolute configuration of Hyperbiphenyl A and B from immunomodulatory root extracts. J. Nat. Prod. 2018, 81, 1850-1859. 6. Rouger, C., Pagie, S., Derbré, S.*, Le Ray, A.-M., Richomme, P., Charreau, B. Prenylated polyphenols from Clusiaceae and Calophyllaceae with immunomodulatory activity on endothelial cells. PLoSOne 2016, 11, e0167361. 7. Dang, B. T., Rouger, C., Litaudon, M., Séraphin, D., Richomme, P., Derbré, S.* Identification and purification of minor benzoylated 4-phenylcoumarins from an anti-AGE M. neurophylla bark extract. Molecules 2015, 20, 17735-17746. 8. Rouger, C., Derbré, S.*, Charreau, B., Pabois, A., Cauchy, T., Litaudon, M., Awang, K., Richomme, P. Lepidotol A from Mesua lepidota inhibits inflammatory and immune mediators in human endothelial cells. J. Nat. Prod. 2015, 78, 2187–2197. 9. Dang, B. T., Guitton, Y., Freuze, I., Grovel, O., Litaudon, M., Richomme, P., Séraphin, D., Derbré, S.* Dereplication of Mammea neurophylla metabolites to isolate original 4-phenylcoumarins. Phytochem. Lett. 2015, 11, 61-68. THESIS FUNDING Origin(s) of the thesis funding: UA Angers Gross monthly salary: Selon la grille salariale officielle (pour information 1769 € brut mensuel en 2020) Thesis funding state : Non acquired Funding beginning date/Funding ending date: 01/10/2020 – 3 years Date: 14 avril 2020 Name, signature of unit director: Pascal Richomme Name, signature of team director: Pascal Richomme Name, signature of thesis project director: Andreas Schinkovitz
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