SYNTHESIS OF A 3-THIOMANNOSIDE

Supporting Information SYNTHESIS OF A 3-THIOMANNOSIDE María B Comba, Alejandra G Suárez, Ariel M Sarotti, María I Mangione* and Rolando A Spanevello and Enrique D V Giordano Instituto de Química Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario CONICET Suipacha 531, S2002RLK Rosario, Argentina Corresponding authors: *spanevello@iquir-conicetgovar and mangione@iquir-conicetgovar Table of contents Experimental Section S2 Synthesis of Allylic alcohol 4 S2 Synthesis of Allylic xanthate 5 S3 Synthesis of Diol 2 S3 Synthesis of Thiocarbonate 6 S3 Synthesis of Disulfide 7 S4 Synthesis of Acetylated disulfide 8 S4 Synthesis of Triacetate 11 S4 Thiomannopyranoside 1 S5 Selected spectra S6 Figure S-1: 1 H-NMR (300 MHz) of compound 4 S6 Figure S-2: 13 C-NMR (75 MHz) of compound 4 S6 Figure S-3: 1 H-NMR (300 MHz) of compound 5 S7 Figure S-4: 13 C-NMR (75 MHz) of compound 5 S7 Figure S-5: 1 H-NMR (300 MHz) of compound 2 S8 Figure S-6: 13 C-NMR (75 MHz) of compound 2 S8 Figure S-7: 1 H-NMR (300 MHz) of compound 6 S9 Figure S-8: 13 C-NMR (75 MHz) of compound 6 S9 Figure S-9: 1 H-NMR (300 MHz) of compound 7 S10 Figure S-10: 13 C-NMR (75 MHz) of compound 7 S10 S 1

Figure S-11: 1 H-NMR (300 MHz) of compound 8 S11 Figure S-12: 13 C-NMR (75 MHz) of compound 8 S11 Figure S-13: 1 H-NMR (300 MHz) of compound 11 S12 Figure S-14: 13 C-NMR (75 MHz) of compound 11 S12 Figure S-15: 1 H-NMR (300 MHz) of compound 1 S13 Figure S-16: 13 C-NMR (75 MHz) of compound 1 S13 Experimental Section All reagents and solvents were used directly as purchased or purified according to standard procedures Analytical thin layer chromatography was carried out using commercial silica gel plates (Merck, Silica Gel 60 F254) and visualization was effected with short wavelength UV light (254 nm) and p-anysaldehyde solution with subsequent heating Flash column chromatography was performed with silica gel 60 H (Merck) using EtOAc:hexanes mixtures NMR spectra were recorded at 300 MHz for 1 H, and 75 MHz for 13 C on a Bruker Avance-300 DPX spectrometer with as solvent and (CH 3 ) 4 Si ( 1 H) as internal standard Chemical shifts are reported in delta (δ) units in parts per million (ppm) and splitting patterns are designated as s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet Coupling constants are recorded in Hertz (Hz) The structure of the products were determined by a combination of spectroscopic methods such as IR, 1D and 2D NMR (including NOE, COSY, HSQC and HMBC experiments) and HRMS Infrared spectra were recorded on a Shimadzu IR Prestige-21 spectrometer using sodium chloride plate pellets Absorbance frequencies are recorded in reciprocal centimeters (cm 1 ) High resolution mass spectra (HRMS) were obtained on a Bruker microtof-q II LC-MS spectrometer Optical rotations were determined using a JASCO DIP-1000 digital polarimeter in 100 mm cells and the sodium D line (589 nm) at room temperature in the solvent and concentration indicated The melting points were taken on a Leitz Wetzlar Microscope Heating Stage Model 350 apparatus and are uncorrected Levoglucosenone 3 was obtained according to the procedure previously described 16 Synthesis of Allylic alcohol 4 Compound 3 (594 g, 4713 mmoles) was dissolved in MeOH (32 ml) and cooled at 0 ºC CeCl 3 7H 2 O (1756 g, 4713 mmoles) and NaBH 4 (142 g, 377 mmoles) were added and stirred for 25 h The solution was neutralized with HCl 01 N to reach ph = 7 The mixture was diluted with water (5 ml) and extracted several times with 50 ml portions of EtOAc The combined organic extracts were dried (Na 2 SO 4 ) and concentrated The residue was purified by flash chromatography to afford 4 (559 g, 4360 mmoles, 92%) as a white crystalline solid; Rf (60% hexane- EtOAc) 017; mp = 67-68 ºC (ethyl ether) [Lit 67-69 ºC] 29 ; [α] D 25-322 (c 0995, CHCl 3 ) [Lit - 34 (c 100, CHCl 3 )] 29 ; max (KBr) 3445 (OH), 3420, 1628 (C=C), 1122, 1066, 1045 cm -1 ; H (300 MHz ) 612 (1H, dd, J 98, 43 Hz, ), 570 (1H, ddd, J 98, 24, 22 Hz, H-3), 551 (1H, dd, J 27, 22 Hz, ), 466 (1H, dd, J 43, 42 Hz, H-5), 433 (1H, s, H-2), 384 (1H, d, J 66 Hz, endo), 375 (1H, dd, J 66, 42 Hz, exo), 215 (1H, s, OH); C (75 MHz ) 1306 (C-4), 1290 (C-3), 1011 (), 711 (C-2), 705 (C-6), 686 (C-5) S 2

Synthesis of Allylic xanthate 5 To a solution of compound 4 (126 g, 984 mmoles) in 10 ml of anhydrous THF was added a suspension of NaH (60% dispersion in mineral oil) (086 g, 1970 mmoles) in 40 ml of anhydrous THF The mixture was cooled at 0 ºC CS 2 (15 ml, 2460 mmoles) and CH 3 I (215 ml, 3450 mmoles) were added The mixture was stirred during 3 h and then a solution of NH 4 Cl (sat) (2 ml) was added The solution was extracted with EtOAc (100 ml) and then washed with 5 ml of brine The combined organic extracts were dried (Na 2 SO 4 ) and concentrated The residue was purified by flash chromatography to afford 5 (201 g, 922 mmoles, 94%) as a white crystalline solid; Rf (60% hexane- EtOAc) 056; mp = 61-62 ºC (diisopropylether) [α] D 22 +182 (c 1145, CHCl 3 ); max (KBr) 3000, 2968, 2908, 1653 (C=C), 1288, 1208 (C=S), 1120, 882 cm -1 ; H (300 MHz ) 636 (1H, s, H-2), 627 (1H, dd, J 98, 41 Hz, ), 579 (1H, s, ), 576 (1H, d, J 98 Hz, H-3), 473 (1H, dd, J 43, 41Hz, H-5), 402 (1H, d, J 64 Hz, endo), 382 (1H, dd, J 64, 43 Hz, exo), 259 (3H, s, SCH 3 ); C (75 MHz ) 2158 (C=S), 1332 (C-4), 1238 (C-3), 983 (), 793 (C-2), 713 (C-5), 711 (C-6), 191 (SCH 3 ); HRMS (EI, MH + ) MH + found 21901384, C 8 H 11 O 3 S 2 + calcd 21901441 Synthesis of Diol 2 Compound 5 (795 mg, 364 mmoles) was dissolved in acetone:h 2 O 95:5 mixture (257 ml), NMO was added and then a solution of OsO 4 in t BuOH was added (920 mg, 004 mmoles) The mixture was stirred during 72 h and then cooled at 0 ºC Na 2 SO 3 was added and after 10 min the solution was filtered The residue was purified by flash chromatography to afford 2 (861 mg, 342 mmoles, 94%) a white crystalline solid; Rf (60% Hexane-EtOAc) 008; mp = 119-120 ºC (diisopropylether); [α] D 21-1556 (c 1095, CHCl 3 ); max (KBr) 3534 (OH), 3442 (OH), 2973, 2959, 2928, 2896, 1477, 1409, 1332, 1311, 1283, 1208 (C=S), 1088, 915cm -1 ; H (300 MHz ) 563 (1H, dd, J 83, 17 Hz, H-2), 560 (1H, d, J 17 Hz, ), 472 (1H, ddd, J 51, 19, 16 Hz, H-5), 409 (1H, s, H-3), 400 (1H, s, ), 386 (1H, dd, J 81, 51 Hz, exo), 381 (1H, dd, J 81, 16 Hz, endo), 306 (1H, s, OH), 302 (1H, s, OH), 260 (3H, s, SCH 3 ); C (75 MHz ) 2170 (C=S), 983 (), 832 (C-2), 762 (C-5), 705 (C-3), 679 (C-4), 655 (C-6), 193 (SCH 3 ); HRMS (EI MNa + ) found 27500115 C 8 H 12 O 5 S 2 Na + calcd 27500184 Synthesis of Thiocarbonate 6 Xanthate 2 (190 mg, 075 mmoles) was dissolved in AcOH (12 ml) and cooled at 0 ºC in an ice-bath A solution of H 2 SO 4 /AcOH 50 % (78 ml) was added and the mixture was stirred for 6 h at room temperature The mixture was cooled to 0º C in an ice bath and diluted with EtOAc (150 ml) Solid NaHCO 3 (10 g) was added and after 10 minutes of stirring, the base was dissolved with water and the phases were separated The organic layer was washed with 5% aqueous NaHCO 3 (2 x 50 ml), brine (50 ml) and dried (Na 2 SO 4 anh) After filtration, the solvents were concentrated under reduced pressure The residue was purified by flash chromatography to afford compound 6 (1243 mg, 050 mmoles, 67%) as white crystalline solid; Rf (60%, hexane:etoac) 021; [ ] D 23-1124 (c 11, CHCl 3 ); max (KBr) 2981, 2843, 1748 (C=O), 1357, 1223, 1206, 1160, 1023, 841 cm 1 ; H (300 MHz CDC1 3 ) 556 (1H, d, J 23 Hz, H- 1), 500 (1H, s, ), 469 (1H, dd, J 86, 23 Hz, H-2), 466 (1H, dd, J 58 Hz, H-5), 432 (1H, dd, J 86 Hz, H-3), 430 (1H, dd, J 84 Hz, endo), 388 (1H, dd, J 84 Hz, 58 Hz, exo), 215 (3H, s, ); C (75 MHz, ) 1703 (C=O), 1698 ( ), 980 (), 744 (C-5), 742 (C-2), 698 (C-4), 661 (C-6), 453 (C-3), 208 ( ); HRMS (EI, MNa + ): MNa +, found 26900921 C 9 H 10 O 6 SNa + calcd 26900903 S 3

Synthesis of Disulfide 7 Compound 6 (88 mg, 036 mmoles) was dissolved in anhydrous CH 3 OH (12 ml) and anhydrous K 2 CO 3 (86 mg, 062 mmoles) was added The mixture was stirred for 3 h at room temperature under argon atmosphere After completion (according to TLC analysis) the reaction mixture was filtered over a Celite pad and washed with aliquots of CH 3 OH The combine filtrates were concentrated under reduced pressure obtaining colorless oil This compound was used without further purification in the next reaction step For structural characterization an aliquot of crude product 7 was suspended in methyl tert-butyl ether and stirred overnight to obtain compound 7 as an off-white solid; max (KBr) 3342 (OH), 2926, 1635, 1558, 1386, 1078 cm 1 ; H (300 MHz, H 2 O) 532 (1H, s, ), 455 (1H, d, J 51 Hz, H- 5), 443 (1H, d, J 81 Hz, endo), 430 (1H, s, ), 411 (1H, d, J 67 Hz, H-2), 366 (1H, dd, J 51, 77 Hz, exo), 336 (1H, d, J 67 Hz, H-3), 327 (1H, s, OH), 183 (1H, s, OH); C (75 MHz, H 2 O) 1012 (), 768 (C-5), 713 (C-4), 671 (C-2), 656 (C-6), 568 (C-3), HRMS (EI, MK + ): MK +, found 39300692 C 12 H 18 KO 8 S 2 + calcd 39300747 Synthesis of Acetilated disulfide 8 Crude product 7 (88 mg) was dissolved in anhydrous CH 2 Cl 2 (18 ml) and the distilled pyridine (08 ml) was added at room temperature Ac 2 O (04 ml, 357 mmoles) and DMAP (11 mg, 009 mmoles) were added and the solution was stirred overnight under argon atmosphere The mixture was treated with HCl 50% (1 ml) and extracted with EtOAc (3 x 30 ml) The combined organic layers were dried (Na 2 SO 4 anh) and concentrated under reduce pressure The resulting residue was purified by flash chromatography to afford compound 8 (71 mg, 020 mmoles, 55% two steps) as a white crystalline solid; 8: R f (60% hexane/ EtOAc) 032; [ ] D 28 +1348 (c 11, ); mp 199 200º C (CH 2 Cl 2 /hexane); max (KBr) 1734 (C=O), 1375, 1361, 1247, 1236, 1153 cm 1 ; H (300 MHz, ) 541 (1H, bs, ), 524 (1H, s, ), 488 (1H, dd, J 13, 70 Hz, H-2), 463 (1H, d, J 51 Hz, H-5), 449 (1H, d, J 80 Hz, endo), 377 (1H, dd, J 51, 80 Hz, exo), 359 (1H, d, J 70, 80 Hz, H-3), 217 (3H, s, ), 208 (3H, s, ); C (75 MHz, ) 1702 (COCH3), 1701 (COCH3), 996 (), 751 (C-5), 724 (C-4), 704 (C-2), 659 (C-6), 486 (C-3), 211 ( ), 205 ( ); HRMS (EI, MNa + ): MNa +, found 54507622 C 20 H 26 NaO 12 S 2 + calcd 54507579 Synthesis of Triacetate 11 Compound 8 (25 mg, 005 mmoles) was dissolved in anhydrous THF (6 ml) and cooled to 0º C LiAlH 4 (18 mg, 050 mmoles) was added and the resulting suspension was stirred at room temperature for 16 h The mixture was cooled to 0º C, diluted with EtOAc (25 ml) and the excess of reducing agent was destroyed by addition of methanol (25 ml) Finally, the mixture was neutralized with AcOH and solvents were concentrated under reduced pressure The resulting residue was dissolved in anhydrous pyridine (07 ml) and Ac 2 O (07 ml) and catalytic amount of DMAP was added The mixture was stirred for 16 h at room temperature The reaction was concentrated and the residue was dissolved in CH 2 Cl 2 (20 ml) and washed with water (2 x 10 ml) The organic layer was dried (Na 2 SO 4 anh) S 4

and the solvent was concentrated under reduced pressure The residue was purified by flash chromatography to give compound 11 (176 mg, 006 mmoles, 57% two steps) as a colorless oil; R f (60% hexane/etoac) 039; [ ] D 26-858 (c 06, ); max (liquid film) 1735 (C=O), 1691, 1373, 1217, 1062 cm 1 ; H (300 MHz, ) 545 (1H, s, ), 515 (1H, dd, J 16, 71 Hz, H-2), 486 (1H, bs, ), 457 (1H, d, J 54 Hz, H-5), 429 (1H, d, J 71 Hz, H-3), 427 (1H, d, J 84 Hz, endo), 378 (1H, dd, J 54, 84 Hz, exo), 235 (3H, s, ), 217 (3H, s, ), 210 (3H, s, ); C (75 MHz, ) 1929 ( ), 1688 ( ), 1682 ( ), 988 (), 737 (C-5), 731 (C-4), 675 (C-2), 649 (C-6), 411 (C-3), 292 ( ), 201 ( ), 196 ( ); HRMS (EI, MNa + ): MNa +, found 32704975 C 12 H 16 NaO 7 S + calcd 32705089 Synthesis of Thiomannopyranoside 1 Compound 11 (55 mg, 018 mmoles) was dissolved in Ac 2 O (18 ml) under argon atmosphere The solution was cooled at 0 ºC and 2 drops of TMSOTf were added The mixture was stirred for 1 h at room temperature Then, the reaction mixture was treated with a saturated solution of NaHCO 3 (1 ml) and extracted with EtOAc (3 x 10 ml) The combine organic layers were dried (Na 2 SO 4 anh) and the solvent was concentrated under reduce pressure The residue was purified by flash chromatography to give 1 (576 mg, 016 mmoles, 88%) as a colorless oil; R f (60% hexane/etoac) 06; [a] D 22 +284 (c 11, ); max (liquid film) 1749 (C=O), 1371, 1217 cm 1 ; H (300 MHz, ) 601 (1H, d, J 17 Hz, ), 517 (1H, dd, J 97, 114 Hz, ), 500 (1H, dd, J 17, 30 Hz, H-2), 426 (1H, dd, J 30, 114 Hz, H-3), 422 (1H, m, ), 408 (1H, m, H-5), 405 (1H, m, ), 231 (3H, s, ), 217 (3H, s, ), 213 (3H, s, ), 205 (3H, s, ); 201 (3H, s, ); C (75 MHz, ) 1934 ( ), 1706 ( ), 1696 (2 ), 1682 ( ), 896 (), 713 (C-5), 708 (C-2), 656 (C-4), 623 (C-6), 437 (C-3), 305 ( ), 209 ( ), 207 ( ), 206 ( ), 205 ( ); HRMS (EI, MNa + ): MNa +, found 42908385 C 16 H 22 NaO 10 S + calcd 42908259 S 5

12899 13061 10113 6863 7053 7106 100 098 099 100 099 104 102 113 373 375 376 377 383 385 433 464 466 467 551 552 552 569 569 570 572 573 573 609 611 612 614 207 208 210 Selected spectra H-5 H-3 H- TMS OH 8 7 6 5 4 3 2 1 0 ppm Figure S-1: 1 H spectrum of 4 in C-4 C-3 C-2 C-6 C-5 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm Figure S-2: 13 C spectrum of 4 in S 6

215804 133161 123757 98248 71143 71281 79261 19130 101 097 197 100 105 105 309 381 383 383 384 401 403 472 473 474 573 574 575 576 577 578 579 579 624 625 625 626 626 626 628 628 628 629 629 629 636 259 SCH 3 H-3 H-2 H-5 TMS 8 7 6 5 4 3 2 1 0 ppm Figure S-3: 1 H spectrum of 5 in C-4 C-3 C-5 C-2 C-6 SCH 3 C=S 200 180 160 140 120 100 80 60 40 20 0 ppm Figure S-4: 13 C spectrum of 5 in S 7

21717 9839 8337 6564 6804 7067 7633 1942 191 100 205 098 103 185 290 302 307 383 383 384 386 401 408 409 409 411 472 473 473 561 561 562 565 565 260 SCH 3 H-2 H-5 H-3 OH TMS 8 7 6 5 4 3 2 1 0 ppm Figure S-5: 1 H spectrum of 2 in C-3 C-4 C-2 C-5 C-6 SCH 3 C=S 200 180 160 140 120 100 80 60 40 20 0 ppm Figure S-6: 13 C spectrum of 2 in S 8

16983 17035 9805 6612 6981 7426 7445 4530 2087 100 104 206 209 106 324 386 388 389 391 430 430 430 431 432 433 433 434 466 468 469 471 471 500 556 556 215 H-2 H-5 H-3 TMS 8 7 6 5 4 3 2 1 0 ppm Figure S-7: 1 H spectrum of 6 in C-5 C-2 C-4 C-6 C-3 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm Figure S-8: 13 C spectrum of 6 in S 9

10119 7681 6563 6714 7131 5680 200 235 183 202 249 203 234 207 532 327 335 338 364 366 369 409 412 430 441 444 454 456 183 -OH -OH H-2 H-3 H-5 7 6 5 4 3 2 1 0 ppm Figure S-9: 1 H spectrum of 7 in D 2 O C-5 C-4 C-2 C-6 C-3 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm Figure S-10: 13 C spectrum of 7 in D 2 O S 10

17009 17018 9956 6592 7042 7239 7507 4857 2048 2114 197 200 204 202 202 205 205 642 620 358 361 374 376 377 379 448 450 460 462 486 487 489 489 523 541 208 216 CDCl H-2 H-5 H-3 TMS 7 6 5 4 3 2 1 0 ppm Figure S-11: 1 H spectrum of 8 in C-4 C-5 C-2 C-6 C-3 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm Figure S-12: 13 C spectrum of 8 in S 11

19296 16858 16888 9876 6496 6746 7312 7372 4111 2923 1963 2003 100 100 099 104 194 106 319 325 298 376 377 378 380 425 428 431 457 459 486 513 514 516 516 545 208 217 235 H-3 H-2 H-5 7 6 5 4 3 2 1 0 ppm Figure S-13: 1 H spectrum of 11 in C-5 C-4 C-2 C-6 C-3 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm Figure S-14: 13 C spectrum of 11 in S 12

19341 16816 16958 17061 8959 6234 6485 7076 7133 4365 2056 2065 2068 2087 3051 100 101 105 218 220 310 320 298 311 322 400 401 405 410 420 422 422 423 424 426 427 499 500 500 501 514 517 518 521 601 601 201 205 213 217 231 H-2 H-5 H-3 7 6 5 4 3 2 1 0 ppm Figure S-15: 1 H spectrum of 1 in C-5 C-2 C-4 C-6 C-3 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm Figure S-16: 13 C spectrum of 1 in S 13