Degradation of Rapamycin and its Ring-Opened Isomer: Role of Base Catalysis Yuri V. Il ichev*, Lori Alquier, and Cynthia A.

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Degradation of Rapamycin and its Ring-Opened Isomer: Role of Base Catalysis Yuri V. Il ichev*, Lori Alquier, and Cynthia A. Maryanoff Supplementary Information

Table S1. Results of fitting of the kinetic data (A1, t1, y), first-order rate constants (k), and half-lives (t 1/ ). Rapamycin 37 mm ammonium acetate Data Fitting Equation A1 (%) t1 (h) y (%) k (h 1 ) t 1/ (h) Rapamycin 37 mm ammonium acetate Rapamycin Isomer Peak 1 y = A1*exp(-x/t1) + y 9.1 3 3.3E-3 (RT.3 min) a Rapamycin Isomer Peak y = A1*exp(-x/t1) + y 7. 9 3.5E-3 (RT 1.1 min) a Sum of two rapamycin isomer y = A1*exp(-x/t1) + y 9.1 9 3.E-3 1 peaks (RT.3 and 1.1 min) a Product Peak 1 (RT 1.7 min) y = A1*exp(-x/t1) + y -.1 1 3. 5.E-3 17 Product Peak 1 (RT 1.7 min) y = A1*( 1-exp(-x/t1) ). 1 NA.E-3 11 Product Peak (RT 3.7 min) y = A1*exp(-x/t1) + y -. 19 1.1 5.9E-3 117 Product Peak (RT 3.7 min) y = A1*( 1-exp(-x/t1) ). 13 NA.1E-3 113 Sum of two product peaks y = A1*exp(-x/t1) + y -.7 17.3 5.E-3 11 (RT 1.7 and 3.7 min) Sum of two product peaks y = A1*( 1-exp(-x/t1) ) 97 5 NA 3.5E-3 197 (RT 1.7 and 3.7 min) b, c Secorapamycin 37 mm ammonium acetate Sum of two secorapamycin isomer y = A1*exp(-x/t1) + y 77. 1913 5.E- 13 peaks (RT 3.7 and.5 min) a Rapamycin 3.7 mm ammonium acetate Rapamycin Isomer Peak 1 y = A1*exp(-x/t1) + y 9.1 131 7.E- 91 (RT.3 min) a Rapamycin Isomer Peak y = A1*exp(-x/t1) + y 7. 17 7.9E- 3 (RT 1.1 min) a Sum of two rapamycin isomer y = A1*exp(-x/t1) + y 9. 17 7.E- peaks (RT.3 and 1.1 min) a Product Peak 1 (RT 1.7 min) y = A1*exp(-x/t1) + y -9. 559 3.1 1.E-3 3 Product Peak 1 (RT 1.7 min) y = A1*( 1-exp(-x/t1) ).7 39 NA.5E-3 7 Product Peak (RT 3.7 min) y = A1*exp(-x/t1) + y -17. 59 1.3 1.7E-3 1 Product Peak (RT 3.7 min) y = A1*( 1-exp(-x/t1) ) 15. NA.E-3 9 Sum of two product peaks y = A1*exp(-x/t1) + y -7. 57.3 1.7E-3 39 (RT 1.7 and 3.7 min) Sum of two product peaks y = A1*( 1-exp(-x/t1) ) 97 1 NA 7.9E- 7 (RT 1.7 and 3.7 min) b Secorapamycin 3.7 mm ammonium acetate Sum of two secorapamycin isomer y = A1*exp(-x/t1) + y 7.7 5 1.E- 39 peaks (RT 3.7 and.5 min) a a Parameter y was kept fixed at ; b Parameter A1 was kept fixed at 97, which corresponds to the average percentage of the total area for the two isomers of rapamycin found in non-degraded samples; c The first six time points were used for fitting; For these data points, the sum area for the two isomers of rapamycin and two products accounted for >9% of the total area under the curve. S

S 3 % Total Area 5 15 Time (days) Figure S1. %Total area-time profiles showing accumulation of the two degradation products of rapamycin. Rapamycin solutions in 3/7 v/v MeCN/H O mixture contained 3.7 mm (red circles) and 37 mm ammonium acetate (black squares). % Total area was calculated by summing the area under the chromatographic curve for two peaks with RT 1.7 min and RT 3.7 min. Dashed lines show results of fitting with equation y = A1*exp(-x/t1) + y. Solid lines correspond to results of fitting with equation y = A1*(1 - exp(-x/t1), where parameter t1 was varied and parameter A1 was kept constant and equal to 97.%.

S Rapamycin ; Negative mode 5 9 35 1 7 1 1 1 1 3 ; Positive mode 7 3 17 13 3 1 1 1 1 3 TWA; 19 nm 77 37 19 15 1 79 39 1 1 1 1 3 XIC; Negative mode 1 1 1 1 1 3

No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 31. - - - - - - - - - - Manual 59.5 - - - - - - - - - - Manual 3.5 - - - - - - - - - - Manual 91.5 11937. 15.179-5 1555.1. 79..7 1 91.1 Manual 5 93.5 - - - - - - - - - - Manual 9.5 - - - - - - - - - - Manual S 5 3 1 XIC; Positive mode 1 1 1 1 3 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 33. 15.5 15. - 7 5179.1..7 1. 1.53 Manual 33. 15.5 15. - 7 5179.1..7 1. 1.53 Manual 3 3.5 - - - - - - - - - - Manual 93.5 515959. 15.53-3 179.93 9..91 5. 1 93.1 Manual 5 95.5 - - - - - - - - - - Manual 97.5 - - - - - - - - - - Manual Figure S. RP-HPLC-UV and ESI-MS traces of rapamycin solution in 3/7 MeCN-water, no other additives.

Secorapamycin ; Negative mode S 1 7 1 1 1 1 3 ; Positive mode 135 5 7 1 1 1 1 3 TWA; 19 nm 17 1 73 3 1 1 1 1 3 XIC; Negative mode 15 1 9 3 1 1 1 1 3 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 31. - - - - - - - - - - Manual 59.5 - - - - - - - - - - Manual 3.5 - - - - - - - - - - Manual 91.5 793791. 13. - 199 1771.1. 9.371.1 1 91.59 Manual 5 93.5 - - - - - - - - - - Manual 9.5 - - - - - - - - - - Manual

3 3 XIC; Positive mode S 7 1 1 1 1 1 3 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 33. - - - - - - - - - - Manual 1.5 3175.99 11.39-17 11.99. 7.35.7 1 93.5 Manual 3 3.5 - - - - - - - - - - Manual 93.5 1539.3 13.1-35. 5. 115.9.739 1 93. Manual 5 95.5 17.5 13.1-195 739.. 1..937 1 93. Manual 97.5 - - - - - - - - - - Manual Figure S3. RP-HPLC-UV and ESI-MS traces of secorapamycin solution in 3/7 MeCN-water, no other additives.

Degraded Rapamycin ; Negative mode S 3 1 1 1 1 ; Positive mode 1 1 1 1 1 1 TWA; 19 nm 15 15 75 5 5 1 1 1 1 XIC; Negative mode 75 5 5 1 1 1 1 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 31. - - - - - - - - - - Manual 59.5 15. 11.7-17 9519.75..3.79 1 59.5 Manual 3.5 - - - - - - - - - - Manual 91.5 33.7 13.7-199 995.15..5 1. 1 91.57 Manual 5 93.5 373.5 9.579-1 73.93 3..7. 1 93.1 Manual 9.5 - - - - - - - - - - Manual

XIC; Positive mode S 9 15 5 1 1 1 1 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 33. 193.3 1. - 19 13.15 1..75 3. 1 3.77 Manual 1.5 5775.73 11.399-17 1.7 1. 5.3.15 1 1.9 Manual 3 3.5 - - - - - - - - - - Manual 93.5 173.11 13.3-9.7..17.1 1 35.39 Manual 5 95.5 175..35-155 55.1. 5.5 1.9 1 35.55 Manual 97.5 - - - - - - - - - - Manual Figure S. RP-HPLC-UV and ESI-MS traces of rapamycin solution in 3/7 MeCN-water, 37 mm MeCOONH ; Reaction time ~ months.

Degraded Secorapamycin ; Negative mode S 3 1 1 1 1 1 ; Positive mode 1 7 1 1 1 1 TWA; 19 nm 19 15 11 7 3 1 1 1 1 XIC; Negative mode 1 1 1 1 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 31. - - - - - - - - - - Manual 59.5 157.99 11.9-17 15. 1. 91.3.3 1 59.3 Manual 3.5 - - - - - - - - - - Manual 91.5 931.7 13. - 1 13.9. 9.3 1.55 1 91.71 Manual 5 93.5 - - - - - - - - - - Manual 9.5 - - - - - - - - - - Manual

XIC; Positive mode S 11 5 1 1 1 1 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 33. - - - - - - - - - - Manual 1.5 97.19 11.517-171 1317.1. 37.57 9.5 1 5.3 Manual 3 3.5 - - - - - - - - - - Manual 93.5 119.7 13.5-1 35.371. 1.91 1. 1 35.59 Manual 5 95.5 - - - - - - - - - - Manual 97.5 - - - - - - - - - - Manual Figure S5. RP-HPLC-UV and ESI-MS traces of secorapamycin solution in 3/7 MeCN-water, 37 mm MeCOONH ; Reaction time ~ months.

S 1 Degraded Rapamycin ; Negative mode 3 5 5 3 7 9 1 1 1 1 3 ; Positive mode 175 15 15 75 5 5 1 1 1 1 3 TWA; 19 nm 3 5 15 11 75 37 1 1 1 1 3 XIC; Negative mode 3 1 1 1 1 1 3 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 31. - - - - - - - - - - Manual 5.5 7.99.5-3.99 1. 5..3 1 5.1 Manual 3 59.5 39.5 11. - 19 73.. 1.31 1.93 1 59. Manual.5 - - - - - - - - - - Manual 5 91.5 19.9 13.7-199 15.99. 3.51.37 1 91.5 Manual 93.5 1757. 9.55-11 31197.31 3. 531. 1.17 1 93.595 Manual 7 9.5 11.51.31-577...3.7 1 9.91 Manual

3 3 XIC; Positive mode S 13 1 1 1 1 1 3 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 33. 1911. 1. - 19 9393.99. 3.13.99 1 33.17 Manual 1.5 177.13 11.379-19 3513.99. 95.9.51 1 1.5 Manual 3 3.5 17.9.35-9 7193.99. 3.13.7 1 3.37 Manual 93.5 19.3 13. - 39. 3. 15.7.95 1 93.7 Manual 5 95.5 55. 9.91-11 3793.99 3. 1..37 1 95. Manual 97.5 115..35-5.9 3. 11.31.33 1 97. Manual Figure S. RP-HPLC-UV and -MS traces of rapamycin solution in 3/7 MeCN-water, 3 mm NaOH; Reaction time 1 h.

Degraded Secorapamycin ; Negative mode S 1 1 1 1 1 1 1 3 ; Positive mode 11 9 3 1 1 1 1 1 3 153 TWA; 19 nm 7 95 71 537 35 179 1 1 1 1 3 XIC; Negative mode 1 9 3 1 1 1 1 3 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 31. - - - - - - - - - - Manual 5.5 9771.5.57-159.1 1. 13.9.155 1 5.57 Manual 3 59.5 513. 11.5-17 5135.. 7.9 1.9 1 59.15 Manual.5 153.5.57-7.. 5.533 1.31 1 5.7 Manual 5 91.5 5997.9 13. - 1337.. 5.77.591 1 91.597 Manual 93.5 9111.9 7.15-5.99. 19.3 1.113 1 93.573 Manual 7 9.5 - - - - - - - - - - Manual

XIC; Positive mode S 15 5 15 5 1 1 1 1 3 No. m/z Area (counts) tr (min) RRT Scan No. Height (counts) FWHM (scans) S / N Asymmetry Number of maxima Base Peak Mass Color Origin 1 33. 135. 3.555-53 13171.3 1. 1.59 5.99 1 33.7 Manual 1.5 793.3 11.39-17 359.953 3. 19.35.9 1 1. Manual 3 3.5 195.5.3-9 533.3 1. 7.19 3.9 1 3. Manual 93. 75.9 13.5-5 759.99 1...53 1 93.75 Manual 5 95.5 39.55.793-1 991.99..97.3 1 95.77 Manual 97.5 171..99-7 71.. 1.77 1.53 1 97. Manual Figure S7. RP-HPLC-UV and -MS traces of secorapamycin solution in 3/7 MeCN-water, 3 mm NaOH; Reaction time 1 h.

S 1 1 1 Absorbance(7 nm) 37 mm MeCOONH (a) Absorbance(7 nm) 3.7 mm MeCOONH (b) 1 1 Retention time / min Figure S. RP-HPLC-UV profiles for degraded rapamycin solutions containing 37 mm MeCOONH s s wph =7.7 (a) or 3.7 mm MeCOONH, wph =7. (b). Reaction time (time elapsed between solution mixing and sample injection) was ~ months. Mobile phase program: 5/5 MeCN/HO to 9/ MeCN/H O over 5 min; Flow rate 1 ml/min; Column temperature o C. Mobile phase contained.1 vol% HCOOH.

Degraded Rapamycin Sample-3_1_- c [ 1.-1.] ESI Q1MS._ RT 13.5 min 91. (a) S 17 Intensity 39. 913.5 91. 3 35 5 5 55 5 7 m/z 75 5 9 95 5 Degraded Rapamycin Sample-3_1_- c [ 1.-1.] ESI Q1MS._ 91. RT 13.9 min (b) Intensity 395.1 7. 913. 3 35 5 5 55 5 7 m/z 75 5 9 95 5 5 Degraded Secorapamycin Sample-_1_- c [ 1.-1.] ESI Q1MS._ 91.7 913. RT 13.5 min (c) Intensity 3 3. 1.1 3 35 5 5 55 5 7 m/z 75 5 9 95 5 Sample-_1_- c [ 1.-1.] ESI Q1MS._ Degraded Secorapamycin 91. RT 13.9 min (d) Intensity 3 1 1.5. 395.1 53.7 53.7 913. 91. 3 35 5 5 55 5 7 m/z 75 5 9 95 5 Figure S9. Negative ESI mass spectra acquired at RT 13.5 min (a, d) and 13.9 min (c, d) for rapamycin (a, b) and secorapamycin (c, d) solutions stored for ~ months. HPLC-UV and MS traces are presented in Figures S3 and S. Solvent: 3/7 MeCN-water, 37 mm MeCOONH.

S 1 Table S. 1 H and 13 C NMR chemical shifts (ppm) of rapamycin and secorapamycin salt with triethylamine (TEA) in 1:1 THF-d /D O a Rapamycin Secorapamycin Secorapamycin,.5% TFA No. 13 C chemical shift (ppm) b 1 H chemical shift (ppm) c 13 C chemical shift (ppm) b 1 H chemical shift (ppm) c 13 C chemical shift (ppm) b 1 H chemical shift (ppm) c 1 17.3 --- 175. --- 17.7, 173. --- 51.7 (t), 57.1 (c).99 (t),.1 (c) 5. (t), 59. (c). (t),.1 (c) 51. (t), 5.7 (c) 5. (t),.31 (c) 3 7..1..19, 1.3.7; 7.9., 1.5;., 1.5.9 1. 1. 1.5, 1. 1.1 1., 1.3 5 5.1 1.5, 1.33 5. 1.3, 1.1 5.3 1.57, 1.37.5 3.1, 3..9 (t); 39. (c) 3., 3.31 (t);.1,.3 (c).9 (t); 39. (c) 3.3, 3. (t);.5,.9 (c) 1. --- 17.5 --- 17.9, 17.3 --- 9 19. ---. --- 175.9 --- 99. ---., 99.1 ---., 99. --- 11 35.1. 35. 1.95 3.7 1.9 1.9 1.5 7.1 1.5 7. 1.5 13 3. 1.7, 1.19 3.7 1.59,.3 3.7 1.5,.1 1 7.3 3.95 7.5 3., 3.9 7., 7. 3.77, 3.9 15.5 1.7, 1.. 1.7, 1.3.1 1.7, 1. 1 3.7 3.5 3.9 3.. 3.59 17 13. --- 137. --- 13.7 --- 1 1.1 5.9 1.. 1.5.7 19 17..31 17.1. 1.9.31 133..17 133.3.1 133..15 1 13.9.3 13.5.3 13.5. 139.9 5.3 139.7 5.7 139.9 5.5 3 35..1 35.1.17 35.1.1,..3 1.3, 1. 39.7 1.5, 1.7 39.; 39.3 1., 1.7; 1.73, 1.5 5 1..3.9, 1..75,.9 1.,.9.7,.7 15.3, 13. --- 15., 1. --- 1.5, 15. --- 7 5.9, 7.3 3.99, 3.1 7., 7. 3., 3.79 7.5,.9 3.7, 3. 7..1, 3.9 7..9 7..7 9 137. --- 137.7 --- 137.7 --- 3 15.7 5.1 1., 17. 5., 5. 17., 1.7 5., 5.5 31. 3.37, 3.1. 3.5. 3.5 3 9. --- 1.9 ---., 1. --- 33.7.5,.3 1.7. 19.1 5.9 3 75.1.99 153.3, 153..7,.3 153.3.5,.3 35 3.1 1.71 3..3 3..33 3 39. 1.5,.97 3. 1.19 3.1 1.1 37 33.3 1.7 33.7 1. 33. 1.5 3 35.5 1.9,. 3.1 1.95,.7 3. 1.9,.9 39. 3.5,.9.3 3.7,.. 3.5,.

Table S.(continued) Rapamycin Secorapamycin Secorapamycin,.5% TFA S 19 No. 13 C chemical shift (ppm) 1 H chemical shift (ppm) 13 C chemical shift (ppm) 1 H chemical shift (ppm) 13 C chemical shift (ppm) 1 H chemical shift (ppm) 1 3.5 1., 1.1 3. 1., 1.15 3.5 1.1, 1.1 31. 1.5,. 31.1 1.55, 1.3 31. 1.51, 1.7 3 15.5.7 15..71 15.7, 15..75,.71.3 1.5.5 1.5.5 1.5 5 1..93 1.5.9 1.3.91 13.1. 1., 1.7.,. 1.7, 1.3.7,. 7 13.7 1.7 1.5, 1.9 1., 1. 1.9, 1.5 1.3, 1. 15.5.7 1. 1. 1.5, 1..99,.9 9 15.1.7 19..93 19.5.93 5 55. 3.1 55.3 3.3 55.3 3. 51 57.7, 5.3 3.13, 3.1 5.3, 5.1 3.1, 3.1 5., 5.1 3., 3.1 5 5.9 3.31 5. 3.9 5. 3.9 a The chemical shifts were obtained from DEPT-edited ghsqc and ghmbc experiments. The chemical shifts for carbon No. 9 were obtained from a 13 C inverse-gated decoupled NMR experiment for rapamycin and from an additional ghmbc experiment with a smaller n J CH value for secorapamycin. Resonances corresponding to trans- and cis-rotamers were labeled with letters t and c, respectively. For secorapamycin, the measurements were performed in the absence and in the presence of.5% trifluoroacetic acid (TFA); b Relative to the residual proton signal of THF-d assigned to 3.5 ppm; c Relative to the signal of THF-d assigned to 7.57 ppm;

Figure S. 1 H-NMR spectrum of rapamycin in 1:1 THF-d /D O. Resonances corresponding to transand cis-rotamers were labeled with letters T and C, respectively. S

S 1 Figure S11. 1 H-NMR spectrum of secorapamycin in 1:1 THF-d /D O. Resonances corresponding to trans- and cis-rotamers were labeled with letters T and C, respectively.

S Figure S1. 1 H-NMR spectrum of secorapamycin in 1:1 THF-d /D O containing.5% trifluoroacetic acid. Resonances corresponding to trans- and cis-rotamers were labeled with letters T and C, respectively.

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