Iron(III) Coordination Properties of Ladanein, a Flavone Lead with a Broad-Spectrum Antiviral Activity. Supporting Information

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1 Electronic Supplementary Material (ESI) for ew Journal of Chemistry. This journal is The Royal Society of Chemistry and the Centre ational de la Recherche Scientifique 8 Iron(III) Coordination Properties of Ladanein, a Flavone Lead with a Broad-Spectrum Antiviral Activity X. Martin-Benlloch, a A. ovodomska, a,b D. Jacquemin, c E. Davioud-Charvet a,* and M. Elhabiri a,* a. UMR 759 CRS Université de Strasbourg (ECPM), Laboratioey de Chimie Bioorgainque etmédicinale, 5 Rue Becquerel, 6787 Strasbourg, France. (ME) elhabiri@unistra.fr; (EDC) elisabeth.davioud@unistra.fr b. Biochemistry Center (BZH), University of Heidelberg, Heidelberg, Germany c. Ceisam Laboratory, UMR CRS 63. University of antes,, rue de la Houssinière, 443 antes Cedex3, France Supporting Information (pages S-S including this one) S

2 H H FCH 3 (x 4 M - cm - ) 3 LH - LH H H FCH Figure S. Absorption spectrophotometric titration of ladanein (designated as FMe) as a function of ph and electronic absorption spectra of the protonated species of ladanein. Solvent: CH 3 H/H (8/ w/w); I =. M (Et 4 Cl 4 ); T = 5.() C; l = cm; [FMe] tot = 3. x -5 M; () ph = 6.99; () ph = H FP (x 4 M - cm - ) 3 L - LH H FP Figure S. Absorption spectrophotometric titration of the final precursor of ladanein (designated as FP) as a function of ph and electronic absorption spectra of the protonated species of FP. Solvent: CH 3 H/H (8/ w/w); I =. M (Et 4 Cl 4 ); T = 5.() C; l = cm; [FP] tot = 3.5 x -5 M; () ph = 5.5; () ph = H H FH (x 4 M - cm - ) LH LH - H H FH Figure S3. Absorption spectrophotometric titration of negletein FH as a function of ph and electronic absorption spectra of the protonated species of FH. Solvent: CH 3 H/H (8/ w/w); I =. M (Et 4 Cl 4 ); T = 5.() C; l = cm; [FP] tot = 3.8 x -5 M; () ph = 5.3; () ph =.5. S

3 % Species 5 H 3 C H H LH CH 3 LH - % Species 5 H H LH 8 H H H H H LH 7 - LH 6 - LH 5 3- LH 44- H Liver ph ph Liver ph ph Figure S4. Distribution diagrams of the protonated species of ladanein FMe and EGCG as a function of ph. [ligand] = -6 M (close to the anti-hcv EC 5 values); I =. M; T = 5 C CH 3 H 3 C H H at 333 nm CH 3 H 3 C H H Time (min) Figure S5. Absorption spectrophotometric spectra of solution of ladanein FMe as a function of time ( h). Solvent: CH 3 H/H (8/ w/w); T = 5.() C. [FMe] tot = 5. x -5 M; l = cm CH 3 H 3 C H H at 35 nm.6.5 CH 3 H 3 C H H Time (min) Figure S6. Absorption spectrophotometric spectra of solution of ladanein as a function of time ( h) with the presence of base. Solvent: CH 3 H/H (8/ w/w); I =. M Et 4 Cl 4 ; T = 5.() C. [FMe] tot = 4.83 x - 5 M; [Et 4 H] tot = 9.63 x -4 M; [FMe] tot = 4.7 x -5 M; [Et 4 H] tot = 8.75 x -3 M; l = cm. S3

4 Abundance (au) Abundance (au) m/z m/z Figure S7. ESI-MS spectra of ladanein as a function of time ( and 5 minutes) in the presence of base (H 4 H). Solvent: CH 3 H/H (8/ w/w. [FMe] tot = 5 x -5 M; H 4 H ( v/v). Fragmentor = V H 3 C H H (x 4 M - cm - ).5 deprotonated FH xidation product (nm) Figure S8. Variation of the absorption spectrophotometric spectra of solution of FH as a function of time ( h) in the presence of base. Solvent: CH 3 H/H (8/ w/w); I =. M Et 4 Cl 4 ; T = 5.() C. [FH] tot = 4.83 x -5 M; [Et 4 H] tot = 9.63 x -4 M; [FCF 3 ] tot = 4.7 x -5 M; [Et 4 H] tot = 8.75 x -3 M; l = cm.,4,,,8,6,4,, at 3 nm,348,344,34,336, Time (minutes) Figure S9. Absorption spectrophotometric spectra of solution of the final precursor FP as a function of time ( h) with the presence of base. Solvent: CH 3 H/H (8/ w/w); I =. M Et 4 Cl 4 ; T = 5.() C. [FP] tot = 4.7 x -5 M; [Et 4 H] tot = 9.4 x -4 M, l = cm. S4

5 Table S. ICP-MS data from the most active and the most inactive fractions along ladanein extraction. atural ladanein-enriched fractions extracted from M. atural ladanein peregrinum content % % 89% 83.% Antiviral activity Metal content (mg/kg) LF-MPE SF S3F3 S3F4 Mg Al 3 Cr <5 <5 <5 5 Mn <5 <5 <5 <5 Fe Co <5 <5 <5 <5 i < < 7 5 Cu Zn As <5 <5 <5 <5 Se <5 <5 <5 <5 Sr < < < < Zr <5 <5 <5 <5 Cd < < < < Sn < < < < Ba <5 <5 <5 Hg <5 <5 693 <5 Pb < < 8 I <6 <6 773 <6 Fe-enriched active fraction Cu-enriched active fraction Abolishment of the antiviral activity of synthetic ladanein (BJ486K) after addition of an equimolar amount of Fe(III) chelator ferrioxamine B (DF) + H 3 5 H H H H H CH 3 DF Figure S. Influence of iron chelator DF on antiviral activity of BJ486K. S5

6 H 3 C H H FMe CH 3 (x 4 M - cm - ) 3 H 3 C FMeFe FMe H H FMe CH 3 (x 3 M - cm - ) FMe.Fe L CH 3 H 3 C H FMe H (c) at 633 nm [Fe(III] tot (d) Figure S. Absorption spectrophotometric titration of ladanein FMe with Fe(III) at ph.. Absorption spectra versus [Fe(III)] tot, and (c) absorption electronic spectra of ladanein FMe and its ferric complexes and (d) variation of the absorbance at 633 nm (LMCT) as a function of [Fe(III)] tot. Solvent: CH 3 H/H (8/ by weight); I =. M (Et 4 Cl 4 ); T = 5.() C; ph =.; l = cm; () [FMe] tot = M; () [Fe(III)] tot /[FMe] tot =.6. S6

7 H 3 C H 3C H CH 3 (x 4 M - cm - ) 3 H 3 C H 3C H.Fe CH (x 3 M - cm - ) Fe CH 3 H 3 C H 3C H (c) at 57 nm Fe tot / L tot (d) Figure S. Absorption spectrophotometric titration of salvigenin with Fe(III) at ph.. Absorption spectra versus [Fe(III)] tot, and (c) absorption electronic spectra of salvigenin and its ferric complexes and (d) variation of the absorbance at 57 nm (LMCT) as a function of [Fe(III)] tot. Solvent: CH 3 H/H (8/ by weight); I =. M (Et 4 Cl 4 ); T = 5.() C; ph =.; l = cm; () [salvigenin] tot = M; () [Fe(III)] tot /[salvigenin] tot =... 4 A at 4 nm.5. 5 A at 57 nm 3 x = Fe tot /( FMe tot + Fe tot ) x = Fe tot /( Fe tot + salvigenin tot ) Figure S3. Job plots obtained upon mixing ladanein and Fe 3+ (ΔA/ΔA max at 4 nm) and salvigenin and Fe 3+ (ΔA/ΔA max at 57 nm). Solvent: CH 3 H/H (8/ by weight); I =. M (Et 4 Cl 4 ); T = 5.() C; ph =.; ([FMe] tot + [Fe(III)] tot ) = M; ([salvigenin] tot + [Fe(III)] tot ) = M; l = cm. S7

8 .6 H H.4 Apigenin H H.4. H Apigenin ( 4 M - cm - ).6.8 H Apigenin.Fe Apigenin Figure S4. Absorption spectrophotometric titration of apigenin with Fe(III) at ph.. Absorption spectra versus [Fe(III)] tot and absorption electronic spectra of apigenin and its ferric complexes. Solvent: CH 3 H/H (8/ by weight); I =. M (acl 4 ); T = 5.() C; ph =.; l = cm; () [apigenin] tot =.9-5 M; () [Fe(III)] tot /[apigenin] tot =.6. (from Carrër, C. Chélation de métaux de transition par des polyphénols du régime alimentaire, PhD Thesis from University Louis Pasteur of Strasbourg, January 8 th, 5) CH 3 H 3 C H FMe H (nm) at 633 nm CH 3 H 3 C H FMe H ph (x 3 M - cm - ) FMe.Fe FMe.Fe(H) FMeH.Fe CH 3 H 3 C H FMe H % Species 5 (c) FMe.Fe(H) FMeH.Fe FMe.Fe ph (d) Figure S5. Absorption spectrophotometric titration of the ferric complexes of ladanein as a function of ph. spectral variation of the LMCT recorded, variation of the absorbance at 633 nm as a function of ph, (c) electronic spectra of the ferric complexes and (d) distribution diagrams of the ferric complexes of ladanein. Solvent: CH 3 H/H (8/ by weight); I =. M (Et 4 Cl 4 ); T = 5.() C; l = cm; [FMe] tot = 4.55 x -5 M; [Fe(III)] tot = M;.() ph =.7; () ph = S8

9 % Ferric Species 5 Fe(Cat) Fe(Cat) Fe(Cat) ph Figure S6. Distribution diagrams of the ferric complexes with catechin (noted Cat). Solvent: H ; I =. M (acl 4 ); T = 5.() C; [Cat] tot =.5 x -4 M; [Fe(III)] tot = 5-5 M. (from Elhabiri, M.; Carrër, C.; Marmolle, F. and Traboulsi, H. Inorg. Chim. Acta 7, 36, 353.) % Species H 3 C H FMe.Fe H CH 3 FMe + H 3 5 H H H DF.Fe H H CH DF tot / FMe.Fe tot Figure S7. Distribution diagrams of the ferric complexes with ladanein FMe and DF showing the competition between these two chelators for Fe(III). Solvent: H ; I =. M; T = 5.() C; [FMe] tot = 5 x -5 M; [Fe(III)] tot = 5-5 M. S9

10 .6 H 3 C..8.4 H H FMe CH 3 (x 4 M - cm - ) FeTA L H 3 C H FMe.FeTA H FMe CH (c) (x 3 M - cm - ) H 3 C H max = 57 nm 57 = M - cm - H FMe CH 3 FeTA FMe.FeTA (d) at 57 nm.3.. CH 3 H 3 C H FMe H FMe tot / FeTA tot (e) Figure S8. Absorption spectrophotometric titration of ladanein FMe with FeTA at ph 7.4. (a and c) Absorption spectra versus [FeTA] tot, (b and d) absorption electronic spectra of ladanein and its ferric ternary complexes with TA and (e) variation of the absorbance at 57 nm (LMCT) as a function of the [FMe] tot /[FeTA] tot ratio. Solvent: CH 3 H/H (8/ by weight); ph = 7.4 (Hepes buffer); I =. M (Hepes); T = 5.() C; l = cm; () [FMe] tot = M; () [FeTA] tot /[FMe] tot =.; () [FeTA] tot =.3-4 M; () [FMe] tot /[FeTA] tot =.5. S

11 . H. H H TA H (x 4 M - cm - ) FeTA H TA H Fe TA at 45 nm TA tot / Fe tot (c) Figure S9. Absorption spectrophotometric titration of TA with Fe(III) at ph 7.4, (c) electronic spectra of the ferric TA complexes and (c) variation of the absorbance at 45 nm suggesting the formation of the monoferric monochelate FeTA complex.. Solvent: CH 3 H/H (8/ by weight); I =. M (Hepes); ph 7.4; T = 5.() C; l = cm; () [TA] tot = 8.37 x -5 M; () [Fe(III)] tot /[TA] tot =.5. Abundance (%) 5 H 3C H Fe FMe.FeTA CH 3 FMe-H + FeTA m/z theo = m/z theo = m/z Abundance (%) m/z.6.4. Theoritical Abundace Figure S. Electrospray mass spectra of ladanein ferric complex in the presence of TA. Solvent: CH 3 H/H, capillary voltage = 4 V. [FMe.FeTA] tot = -4 M; negative mode; Fragmentor = 5 V. S

12 CH 3 H 3 C H 3C H (x 4 M - cm - ) 3 FeTA.FeTA CH 3 H 3 C H 3C H 4 6 at 5 nm 3 H 3 C H 3C FeTA tot / tot H CH 3 (c) Figure S. Absorption spectrophotometric titration of salvigenin with FeTA at ph 7.4. Absorption spectra versus [FeTA] tot, absorption electronic spectra of salvigenin and its ferric ternary complexes with TA and (c) variation of the absorbance at 5 nm (LMCT) as a function of the [FeTA] tot /[salvigenin] tot ratio. Solvent: CH 3 H/H (8/ by weight); ph = 7.4 (Hepes buffer); I =. M (Hepes); T = 5.() C; l = cm; () [FMe] tot = M; () [FeTA] tot /[salvigenin] tot =.54. S