SUPPLEMENTARY INFORMATION

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1 SUPPLEMETARY IFRMATI DI: /CHEM.1108 A Route to Enantiopure RA Precursors from early Racemic Starting Materials Jason E. Hein, Eric Tse, and Donna G. Blackmond Department of Chemistry The Scripps Research Institute orth Torrey Pines Road La Jolla, CA USA Correspondence to: blackmond@scripps.edu ATURE CHEMISTRY 1

2 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Supplementary Information: Figures and Legends H H H 2 R C 2 H HR' major 2-component reaction products H H H 2 D-ribofuranosyl amino-oxazoline H H H 2 D-arabinofuranosyl amino-oxazoline major 3-component reaction products H R' R H 2 H sugar-amino acid amino-oxazole products 2 Pyridine Fig. S1. Reaction and product acetylation (for ee determination) pathway D-ribo enantiomer L-ribo enantiomer Fig. S2. HPLC trace of racemic acetylated ribofuranosyl derivatives ATURE CHEMISTRY 2

HPLC of racemic acetylated arabinofuranosyl derivatives D-glyceraldehyde

3 DI: /CHEM.1108 SUPPLEMETARY IFRMATI L-arabino enantiomer D-arabino enantiomer Fig. S3. HPLC of racemic acetylated arabinofuranosyl derivatives D-glyceraldehyde DP-hydrazone L-glyceraldehyde DP-hydrazone Fig. S4. HPLC trace of racemic glyceraldehyde DP-hydrazones ATURE CHEMISTRY 3

4 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Fig. S5. 1 H-MR of acetylated ribofuranosyl derivative. ATURE CHEMISTRY 4

5 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Fig. S6. 13 C-APT MR of acetylated ribofuranosyl derivative. ATURE CHEMISTRY 5

6 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Fig. S7. 1 H-MR of acetylated arabinofuranosyl derivative. ATURE CHEMISTRY 6

7 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Fig. S8. 13 C-APT MR of acetylated arabinofuranosyl derivative. ATURE CHEMISTRY 7

8 DI: /CHEM.1108 SUPPLEMETARY IFRMATI H-C1' H-C2' H-C4' H-C3' H 5' 1'' 4' 1' 2'' 5 3' 4 2' H H H-C2' H-C5' H-C5' 1D-selective TCSY Pulse H-C1' H-C2' H-C4' H-C3' H-C5' H-C5' Red Crude 1H-MR, Blue 1D-TCSY centered at 5.2 ppm Fig. S9. 1D- selective TCSY Identification of furanose spin system ATURE CHEMISTRY 8

9 DI: /CHEM.1108 SUPPLEMETARY IFRMATI H-C1 H 5' 1'' 4' 1' 2'' 5 3' 4 2' H H H-C4 H-C2/H-C3 H-C4 H-C1 H-C2 H-C3 H-C2/H-C3 1D-selective TCSY Pulse H-C1 H-C1 H-C2 H-C3 Red Crude 1H-MR, Blue 1D-TCSY centered at 3.65 ppm Fig. S10. 1D- selective TCSY Identification of pyrroline spin system ATURE CHEMISTRY 9

10 DI: /CHEM.1108 SUPPLEMETARY IFRMATI H 5' 1'' 4' 1' 2'' 5 3' 4 2' H H Correlation to 13 C-APT (vertical axis) confirms 1 H resonances at 3.8 and 3.56 ppm are from the methylene (C5 ). Fig. S11. HMQC assignment of H-C3, H-C4 and H-C5 signals ATURE CHEMISTRY 10

11 DI: /CHEM.1108 SUPPLEMETARY IFRMATI H 5' 1'' 4' 1' 2'' 5 3' 4 2' H H H-C2' - H-C4 H-C2' H-C2' - H-C1' H-C2' - H-C4 H-C2' - H-C3' Red Crude 1H-MR, Blue pyrrole ring spin system, Green 1D-GSEY centered at 5.2 ppm. (furanosyl to pyrrole ne highlighted in red, ne contacts within furanose ring in black). Fig. S12. 1D- selective GESY Connectivity of furanose and pyrrole rings ATURE CHEMISTRY 11

12 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Fig. S13. Measurement of rate for matched and mismatched enantiomers. Percentage conversion as a function of time in the reaction of 2-amino-oxazole with D- glyceraldehyde in the presence of D-proline (filled circles) or L-proline (open circles). D- Glyceraldehyde (0.054 g, mmol, 1.00 M), D-proline (0.069 g, mmol, 1.00 M), 2-amino-oxazole (0.061 g, mmol, 1.20 M) in D 2 (0.600 ml). Fig. S14. Efficiency of the kinetic resolution compared to photoresolution. 26,27 ATURE CHEMISTRY 12

13 DI: /CHEM.1108 SUPPLEMETARY IFRMATI (B) D-ribo enantiomer Fig. S15. Crystal (A) and subsequent HPLC trace (B) of ribofuranosyl 2-amino-oxazoline isolated from EtH enrichment protocol. Fig. S16. HPLC trace of acetylated L-ribofuranosyl 2-amino-oxazoline isolated from CHCl 3 enrichment protocol. ATURE CHEMISTRY 13

14 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Supplementary Methods General. Solvents and chemicals were obtained from commercial sources (Aldrich, TCI- America, AK-Scientific and ros) and used as received. 1 H and 13 C MR spectra were recorded on either a Bruker DRX-600 equipped with a DCH cryoprobe or a Bruker DRX Chemical shifts (δ) are expressed in parts per million relative to residual CHCl 3 or H 2 as internal standards. Proton magnetic resonance ( 1 H MR) spectra were recorded at either 600 or 500 MHz. Carbon magnetic resonance ( 13 C MR) spectra were recorded at 150 or 125 MHz. MR acquisitions were performed at 295 K unless otherwise noted. Abbreviations are: s, singlet; d, doublet; t, triplet; q, quartet; br s, broad singlet. TLC analysis was performed using precoated silica gel 60 F 254 plated from EMD Chemicals Inc. Preparative TLC was carried out using 20 cm X 20 cm X 0.5mm Silica gel 60 F 254 PLC plates from EMD Chemicals Inc. Synthesis of 2-amino-oxazole. 24 Synthesis was accomplished using a modification of the protocol reported by Ashwell et al. Cyanamide (7.00 g, 167 mmol) was dissolved in a mixture of THF (40.0 ml) and water (7.00 ml) and cooled to 0 C. Separately, a solution of glycoaldehyde (10.0 g, 167 mmol) in water (16.0 ml) was prepared and added dropwise to the reaction over 5 minutes. nce the internal temperature reached 4 C an aqueous solution of sodium hydroxide (2.00 M, 16.7 ml, 33.3 mmol) was added dropwise over 25 minutes. The reaction solution was allowed to warm to room temperature over 24 hrs. At this time the solution had become lightly orange in color. The reaction mixture was concentrated under vacuum to remove the majority of the THF. The remaining aqueous solution was extracted with Et (4 200 ml). The combined organic fractions were dried with MgS 4 and evaporated to dryness under vacuum. This gave 2- amino-oxazole (10.1 g, 121 mmol, 72% yield) as a white powder. Physical and chemical properties of this material matched those reported in the literature (1). This protocol yields material of sufficient purity to be used without further purification. Analytically pure material was obtained by sublimation using a standard 100 ml sublimation apparatus (65 85 C, 0.5 mm Hg, cooling with acetone/dry ice). Resolution of rac-glyceraldehyde using L-proline. rac-glyceraldehyde (0.09 g, 1.00 mmol) was dissolved in water (0.600 ml) and treated with L-proline (115 mg, 1.00 mmol). In a separate vial, 2-amino-oxazole (101 mg, 1.20 mmol) was dissolved in water (0.400 ml) and then added to the reaction solution. The sample was allowed to stand at room temperature for 8 hrs and then the water was removed by lyophilization to give a crude powder. Reaction using D-Glyceraldehyde and either L or D-proline. D-Glyceraldehyde (0.054 g, mmol) and D-proline (0.069 g, mmol) were dissolved in D 2 (0.400 ml) and loaded into a standard 5 mm MR tube. A solution of 2-amino-oxazole (0.061 g, mmol) in D 2 (0.200 ml) was added to initiate the reaction. A standardized, sealed glass capillary loaded with a 0.5 M pyridine solution in D 2 was then added to the sample to permit quantification of 1 H MR signals. Spectra were recorded at 2.45 min ATURE CHEMISTRY 14

15 DI: /CHEM.1108 SUPPLEMETARY IFRMATI intervals (Bruker DRX-500) over the entire course of the reaction. Fractional conversion was calculated by measuring the combined total integral of the anomeric proton (H-C1 ) from all furanosyl reaction products (δ = ppm ) relative to the pyridine standard. Determination of enantiomeric excess of ribo- and arabinofuranosyl aminooxazolines. The crude lyophilized material was dissolved in acetic anhydride (4.50 ml, 47.7 mmol) and treated with pyridine (0.250 ml, 3.09 mmol) and then allowed react at room temperature for 4 hrs. The volatile components were removed under high vacuum to give the acetylated products as a crude mixture. This material was dissolved in a minimum of CH 2 Cl 2 and purified by preparative TLC (7:3 Et:Hexane). UV-active bands corresponding to the per-acetylated ribofuranosyl amino-oxazoline (R f = 0.41) and arabinofuranosyl amino-oxazoline (R f = 0.35) were separately isolated. The purified diastereomers were extracted from the silica gel using CH 2 Cl 2 (4 25 ml) and the combined organic fractions were evaporated to dryness. The resulting oils were separately dissolved in EtH (100 μl) and analyzed by HPLC. Analysis was performed using an Eksigent ExpressLC Ultra micro-lc equipped with a CTC PAL autoinjector and nm variable wavelength, diode array detector configured with a 90nL flow cell. (150 nl injection, Chiracel D-H, i-prh:hexanes 10:90 23:77 linear gradient over 45 min, 13 μl/min, UV 220 nm). Characterization of standard per-acetylated furanosyl amino-oxazolines. rac-2-(,-diacetyl)-4,5-dihydro-(3,5-di--acetyl-1,2,-dideoxy-α-ribofuranoso)[1,2-d]- 1,3-oxazole: rac-glyceraldehyde (0.900 g, 10.0 mmol) was dissolved in water (10 ml) and treated with 2-amino-oxazole (1.01 g, 12.0 mmol). The sample was allowed to stand at room temperature for 8 hrs and then the ph was adjusted to 10 using 1M KH. The reaction solution became cloudy and rac-ribofuranosyl amino-oxazoline began crystallizing as a fine powder. The suspension was cooled to 4 C for 4 hrs to maximize the precipitation of rac-ribofuranosyl amino-oxazoline product. The solid material was isolated by filtration to give rac-ribofuranosyl amino-oxazoline (366 mg, 2.10 mmol) as a white powder. This material was reacted according to the general acetylation protocol. Physical and spectroscopic characteristics were identical to the racemic material; HPLC (Chiracel D-H) (D-enantiomer) = min, (L-enantiomer) = min. 2-(,-diacetyl)-4,5-dihydro-(3,5-di--acetyl-1,2,-dideoxy-β-D-arabinofuranoso)[1,2- d]-1,3-oxazole. R f : = 0.35 (7:3, Et:Hexanes); 1 H MR (600 MHz, DMS) δ 6.34 (d, J = 5.4 Hz, 1H), 5.23 (d, J = 2.0 Hz, 1H), 4.97 (d, J = 5.4 Hz, 1H), 4.32 (ddd, J = 5.6, 5.3, 2.1 Hz, 1H), 4.23 (dd, J = 11.9, 6.0 Hz, 1H), 4.08 (dd, J = 11.9, 5.3 Hz, 1H), 2.51 (s, 3H), 2.20 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H); 13 C MR (151 MHz, CDCl 3 ) δ 181.5, 171.4, 170.4, 170.2, 145.8, 88.6, 84.6, 83.9, 77.5, 63.5, 27.4, 25.3, 21.4, 21.4, 21.2; HRMS (m/z) [M-H + ] calcd for C 14 H , ; found, ; HPLC (Chiracel D-H ) = min. 2-(,-diacetyl)-4,5-dihydro-(3,5-di--acetyl-1,2,-dideoxy-β-L-arabinofuranoso)[1,2-d]- 1,3-oxazole. Physical and spectroscopic properties were identical to D-compound; HPLC (Chiracel D-H) = min. ATURE CHEMISTRY 15

16 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Measurement of enantiomeric excess of residual glyceraldehyde. rac-glyceraldehyde (0.180 g, 2.00 mmol) was dissolved in water (2.00 ml) and treated with L-proline (0.691 g, 6.00 mmol). The reaction was then initiated by adding 2-amino-oxazole (0.202 g, 2.40 mmol). Aliquots (10.0 μl) were then removed at fixed time points and quenched into a saturated solution of dinitrophenylhydrazine in EtH (1.00 ml) and acidified with glacial acetic acid (20.0 μl). These aliquots were allowed to develop at room temperature for 35 minutes and then analyzed by HPLC (Chiralpak -IA, i-prh:hexanes 28:72 isocratic 21 min, 10 μl/min, UV 350 nm); D-DP-glyceraldehyde = min, L-DP-glyceraldehyde = min. Characterization of the three-component product. Compound was characterized using the crude, unpurified reaction between rac-glyceraldehyde, H 5' 1'' 2-amino-oxazole and L-Proline using ESI-MS and 1D- and 4' 1' 2-1 H/ 13 C MR (Bruker-DRX 600, DCH cryoprobe). 1 H 2'' 5 3' MR (600 MHz, D 2 ) δ 5.73 (d, J = 5.1 Hz, 1H, H-C1 ), 4 2' H H (dd, J = 5.1, 5.2 Hz, 1H, H-C2 ), 3.89 (ddd, J = 9.9, , 2.1 Hz, 1H, H-C4 ), 3.80 (dd, J = 13.1, 1.9 Hz, 1H, HH- 3 C5 ), 3.65 (dd, J = 9.2, 4.2 Hz, 1H, H-C4), 3.56 (dd, J = 13.2, 4.0 Hz, 1H, HH-C5 ), 3.50 (dd, J = 9.9, 5.1 Hz, 1H, H-C3 ), (m, 1H, HH- C1), 2.99 (dd, J = 16.7, 7.6 Hz, 1H, HH-C1), (m, 1H, HH-C2), (m, 3H, HH-C2, HH-C3). ESI-MS [M+H]:= 272 (calc for C 11 H = ). Connectivity and substitution was assigned using a series of 1D-selective and 2D techniques. Crystallization of enantiopure ribofuranosyl 2-amino-oxazoline using mixtures of amino acids. L-Proline (2.88 mg, mmol), L-histidine (3.88 mg, mmol), L- alanine (2.23 mg, mmol), L-tryptophan (4.53 mg, mmol), L-isoleucine (3.28 mg, mmol), L-leucine (3.28 mg, mmol), L-aspartic acid (3.97 mg, mmol) were dissolved in water (1.00 ml) and treated with rac-glyceraldehyde (180 mg, 2.00 mmol) in a 2 ml screw cap vial. The sample was stirred until a homogenous solution was obtained, then the reaction was initiated by adding 2-amino-oxazole (202 mg, 2.40 mmol). The sample was allowed to stand capped for 24 hrs. The vial was uncapped and allowed to stand at room temperature and slowly evaporate. After 6 days a single bundle of crystals had begun to grow at the meniscus of the solution. The liquid was carefully pipetted away and the crystals were washed with distilled water (3 100 μl). The crystalline ribofuranosyl 2-amino-oxazoline was acetylated using the general acetylation protocol. Analysis by HPLC confirmed that the crystalline material was enantiopure D-ribofuranosyl 2-amino-oxazoline. Crystallization of enantiopure ribofuranosyl 2-amino-oxazoline beginning with 1% e.e. proline. From EtH. L-Proline (50.0 mg) was dissolved in 4 ml of EtH. The solution was then treated with D/L-proline (4.95 g) to produce a thick suspension. This heterogenous system was stirred at room temperature overnight. The supernatant was drawn off and filtered through a 0.45 μm PTFE HPLC filter. The solution was evaporated to dryness to give solid proline. The enantiomeric excess of this recovered proline was 36.6% (L-proline) as measure by optical rotation. ATURE CHEMISTRY 16

17 DI: /CHEM.1108 SUPPLEMETARY IFRMATI Enantioenriched proline (35.0 mg, mol) was dissolved in water (0.600 ml) and treated with rac-glyceraldehyde (54.0 mg, mmol) in a 2 ml screw cap vial. The sample was stirred until a homogenous solution was obtained, then the reaction was initiated by adding 2-amino-oxazole (76.0 mg, mmol). The sample was allowed to stand capped for 24 hrs. Crystallization, isolation and analysis of the crystalline ribofuranosyl 2-amino-oxazoline was carried out as previously described. From CHCl 3. D/L-Proline (9.90 g) was suspended in CHCl 3 (100 g). The solution was stirred at room temperature for 20 minutes and then treated with D-proline (0.100 g). This heterogenous system was stirred at room temperature overnight. The mixture was filtered using a 250 ml Büchner funnel. The resultant filtrate was evaporated to dryness to give 39.2 mg solid proline. The resulting enantioenriched proline (12.0 mg, mol) was dissolved in water (0.600 ml) and treated with rac-glyceraldehyde (54.0 mg, mmol) in a 2 ml screw cap vial. The sample was stirred until a homogenous solution was obtained, then the reaction was initiated by adding 2-amino-oxazole (76.0 mg, mmol). The sample was allowed to stand capped for 24 hrs. Crystallization, isolation and analysis of the crystalline ribofuranosyl 2-amino-oxazoline was carried out as previously described. Multicomponent reaction mixtures containing multiple amino acids. The following combinations of amino acids yielded crystals, which upon re-dissolution and acetylation gave ribo-amino-oxazoline ee values > 90% ee. Pro Pro, His Pro, His, Ala, Trp Pro, His, Ala, Trp, Leu, Ile Pro, His, Ala, Trp, Leu, Ile, Asp Pro, His, Ala, Trp, Leu, Ile, Asp, Asn, Glu, Gln, Val, Tyr Pro, His, Ala, Trp, Leu, Ile, Asp, Asn, Glu, Gln, Val, Tyr, Phe, Thr ATURE CHEMISTRY 17

Electronic Supplementary Material (ESI) for Chemical Communications This journal is The Royal Society of Chemistry 2012

Electronic Supplementary Material (ESI) for Chemical Communications This journal is The Royal Society of Chemistry 2012 Ring Expansion of Alkynyl Cyclopropanes to Highly substituted Cyclobutenes via a N-Sulfonyl-1,2,3-Triazole Intermediate Renhe Liu, Min Zhang, Gabrielle Winston-Mcerson, and Weiping Tang* School of armacy,