How To Understand The Chemistry Of A Compound From Aqueous Organic Chemistry

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1 Supporting Information Polysulfated Xanthones: Multipathway Development of a New Generation of Dual Anticoagulant/Antiplatelet Agents Marta Correia-da-Silva, Emília Sousa, Bárbara Duarte, Franklim Marques, Félix Carvalho, Luís M. Cunha-Ribeiro #, and Madalena M. M. Pinto * TABLE OF CONTENTS Title page s1 Structural characterization s2 Factor Xa progress curves...s3 S1

2 Structural characterization The IR spectra of sulfated derivatives 6, 7, and 13 showed the presence of two strong bands corresponding to the S=O (ca cm -1 ) and C-O-S (ca cm -1 ) and a band around cm -1 corresponding to the S-O. The high resolution mass spectrometry allowed the elucidation of the number of sulfate groups, eight sulfate groups for compound 13, seven for compound 7, and only four for compound 6. The assignments of the protonated carbons were achieved from HSQC experiments and the chemical shifts of the carbon atoms not directly bonded to proton atoms were deduced from HMBC correlations. The cross peak from glycosyl H-1 (5.18 ppm) to xanthone C-3/6 (162.5 ppm) in the HMBC spectra confirmed the glycosylation at the C-3/6 of xanthone 12 (Figure S1). Furthermore, when the carbon chemical shifts from the 13 C NMR spectra of glycosylated xanthone 12 were compared with those of the respective building block 9, it was possible to observe shielded aromatic carbons for compound 12 (Table S1) corresponding to C- 3/6 O-glycosylated carbons (ca -0.9 ppm). The proton coupling (J = 8.8 Hz) observed for the anomeric proton of glycoside residue demonstrates that the saccharide has the expected β linkage to the xanthone aglycone 12 (Figure S1). Figure S1. Main connectivities found in the HMBC for glycosylated xanthone 12. The 1 H NMR and 13 C NMR data of sulfated compounds 6, 7, and 13 allowed an unambiguous establishment of the sulfate groups positions. When the carbon and proton chemical shifts from the 1 H NMR and 13 C NMR spectra of sulfated compounds 6, 7, and 13 were compared with those of their building block 12 (for compound 13) and 5 (for compounds 6 and 7), it was possible to observe shielded aromatic carbons (C-3, C-6, and C-7) in the 13 C NMR spectra of compound 7 (Table S1) and deshielded protons (H-4, H-5, and H-8, ortho sulfated proton resonances) in the 1 H NMR spectra of compound 7. Furthermore, while all the aromatic OH signals were present in sulfated compound 6, only 1-OH was present in sulfated compound 7 (Table S2). All sugar protons were deshielded for sulfated xanthones 13, 6, and 7 and the OH sugar resonances were only present in the building blocks (Table S2). S2

3 Table S1. 13 C NMR chemical shifts observed after glycosylation of compound 9 (to 12) and after sulfation of compounds 12 (to 13) and 5 (to 6 and 7). a 13 C NMR 12 b * δ 12 -δ 9 13 * δ 13 -δ 12 6 b * δ 6 -δ 5 7 b * δ 7 -δ a a a a a Values in parts per million (δ C) and measured in DMSO- d6 at MHz. *δs-δa, refers to δ sulfate ester comparative to δ aglycone ; negative numbers mean shielded; positive numbers mean deshielded. b Assignments were confirmed by HSQC and HMBC experiments. S3

4 Table S2. 1 H NMR chemical shifts observed after glycosylation of compound 9 (to 12) and after sulfation of compounds 12 (to 13) and 5 (to 6 and 7). a 1 H NMR 9 b 12 c,d 13 b 5 b 6 d 7 d d (J=8.7) 8.10 d (J=8.8) 8.11 d (J=8.9) dd (J=8.7, 2.2) 7.11 dd (J=8.8, 2.2) 7.10 dd (J=8.9, 2.1) d 7.22 d 7.14 d 6.38 s 6.32 s 7.23 s (J=2.2) (J=2.2) (J=2.1) d 7.22 d 7.14 d 6.87 s 6.87 s 7.71 s (J=2.2) (J=2.2) (J=2.1) dd (J=8.7, 2.2) 7.11 dd (J=8.8, 2.2) 7.10 dd (J=8.9, 2.1) d 8.10 d 8.11 d 7.38 s 7.39 s 8.26 s (J=8.7) (J=8.8) (J=8.9) 1-OH s s s 3-OH s OH s OH d 5.64 d 4.59 d 4.85 d 4.76 brd (J=8.8) (J=3.7) (J=9.8) (J= 5.5) t 4.01 brt 4.05 t 4.77 brt 4.77 brt (J= 8.3) (J=8.9) t (J= 8.8) 4.05 brt m m 5.39 t (J= 7.4) t 3.90 t m 4.49 m 4.73 m (J= 8.8) (J= 9.5) m m 4.12 m 3.90 m (under water) 6 a m 4.65 dd (J=8.3, 2.9) 3.69 d (J=11.1) under water 4.09 d (J=9.9) 6 b (under 4.44 t (J= 2.9) under water under water 3.76 t (J=9.6) water) 2 -OH d - - (J=9.3) 3 -OH OH OH a J values (Hz) are shown in parenthesis; s, singlet; dd, double doublet; d, doublet; t, triplet; b Measured in DMSO- d6 at MHz; c Measured in DMSO- d6 at MHz. d Assignments were confirmed by HSQC and HMBC experiments. S4

5 Factor Xa progress curves Figure S2. Example of progress curves demonstrating the direct factor Xa (FXa) inhibition of A) rivaroxaban (positive control) and B) compound 7. The tracings were initiated by the addition of CBS31.39 (125µM), to a preincubated assay mixture of FXa (0.5U/mL) and positive control rivaroxaban ( M) or compound 7 at different concentrations. S5