Supporting Information

Identification of Novel Rapamycin Derivatives as Low-Level Impurities in Active Pharmaceutical Ingredients.

Stephan G. Zech, Michael Carr, Qurish K. Mohemmad, Narayana I. Narasimhan,

Christopher Murray, Leonard W. Rozamus, and David C. Dalgarno

ARIAD Pharmaceuticals, Inc., 26 Landsdowne Street, Cambridge, MA 02139

Experimental Details 1. Analytical HPLC

High Performance Liquid Chromatography (HPLC) analyses of API batches,

impurity pools and isolated individual impurities were carried out on Agilent 1100

and HP1050 quaternary pumping systems running ChemStation software Rev.

A.10.02, each fitted with a degasser, autosampler, and diode array detector

(HP1100) or multi-wavelength detector (HP1050). Each system was programmed

to run the gradient conditions described in Table S1. Due to material limitations,

analytical sample preparations were varied to suit the nature and quantity of the

isolate, and injection volume was limited to 5 µL in 1:1 methanol: buffer. In many

cases, isolates were injected directly as solutions in unbuffered preparative

chromatography eluent. The relative retention times (RRT) of isolates were

established or confirmed by co-injection with an approximately equimolar solution

of a reference sample of rapamycin.

The conditions used in the HPLC analysis are listed in Table S1-A and the mobile

phase gradient is detailed in Table S1-B.

Table S1-A: HPLC Conditions of the Analytical HPLC Method.

Column YMCTM ODS –AQ, 4.6 x 250 mm, 5m, 120Å (Part number: AQ12S052546WT)

Column 50°C

Mobile Phase A 20 mM Ammonium Formate Buffer, pH 4.0

Mobile Phase B Acetonitrile

Detection UV at 280 nm

Flow Rate 1.0 mL/min

Run Time 40 minutes

Retention Time Approximately 13.0 min. for major tautomer of rapamycin

Table S1-B: Mobile Phase Gradient for the Analytical HPLC Method

Time (min) % Mobile Phase A % Mobile Phase B

0.0 28 72

20.0 28 72

25.0 5 95

30.0 5 95

35.0 28 72

40.0 28 72

2. Preparative HPLC

The isolation of individual impurities was carried out interchangeably on two

preparative chromatography instruments:

Varian ProStar Solvent Delivery Module Model 210 running Dynamax

software Version 1.4.6 with UV-Vis Detector Model 320 and Timberline

Instruments TL-105 HPLC column heater / HX-502 Heat Exchanger

Agilent 1200 series Prep Pump / Prep FC / MWD running ChemStation

software Rev. B.02.01 [244]with Timberline Instruments TL-105 HPLC

column heater / HX-502 Heat Exchanger

The two preparative chromatography columns used in this study are:

YMC-Pack ODS-AQ 5μm 120Ǻ, 250x20mm (Part# AQ12S052520WT)

Phenomenex Luna 5μm 100Ǻ C18(2), 250x21.20mm (Part# 00G-4252-

P0)

All preparative chromatographic runs were carried out under isocratic conditions.

The primary method for impurity isolation uses the same ratio of acetonitrile to

(unbuffered) aqueous, as the isocratic portion of the analytical method (see above).

Under these conditions, the retention times of the rapamycin tautomers and

associated impurities are comparable to the analytical method. This preparative

method is generally referred to as Preparative Method 1, and has been used to

generate collections of early and late eluting impurities. In order to further separate

closely eluting or co-eluting impurities, variations of Method 1 are run with a

greater concentration of water to acetonitrile. This has the effect of extending

retention times and, in most cases, enhancing resolution. Unfortunately, as

retention times increase, peaks broaden to an unacceptable point. In such cases,

Preparative Method 2, which uses a different stationary phase, is then employed.

Furthermore, switching the organic component of the solvent system from

acetonitrile to methanol improves resolution or reverses order of elution of closely

eluting species. The choice of preparative method for final isolation of a pure

impurity species usually requires trial and error to optimize conditions.

Table S2: Preparative HPLC Methods

Method Column Mobile Phase

Flow Rate Temperature UV Detection

1 YMC MeCN:H2O 20 mL/min 50°C 280 nm

2 Luna MeOH:H2O 20 mL/min 50°C 280 nm

Supplemental Figures and Tables

Figure S1: Full Scan MS1 of the Sodiated Molecular Ion of Rapamycin

Figure S2: Full Scan MS1 of the Sodiated Molecular Ion of Ethyl Rapamycin

Impurities

Rapamycin-030 #6834-7076 RT: 108.15-111.10 AV: 81 NL: 4.08E5F: ITMS + c ESI Full ms [300.00-1100.00]

300 400 500 600 700 800 900 1000 1100

m/z

0

50000

100000

150000

200000

250000

300000

350000

400000

Inte

nsity

936.65

614.44 952.55409.34 453.35 904.59793.50320.24 1026.14731.55

Rapamycin-030 #9953-10171 RT: 156.91-160.36 AV: 73 SB: 2759 170.82-216.72 , 48.22-132.47 NL:F: ITMS + c ESI Full ms [300.00-1100.00]

300 400 500 600 700 800 900 1000 1100

m/z

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

Inte

nsity

950.63

590.85

537.93408.20334.39 670.771071.76

595.80 839.72745.69 912.62

Table S3: m/z Values of Sodiated Product Ions in the MS2 Spectra of Rapamycin

(m/z 936.6) and Ethyl-rapamycin impurities at retention times 159.81 and 166.28

min (m/z 950.6)

Rapamycin (tR = 109.4 min)

Product ions (m/z)

ethyl Rapamycin impurity peak at (tR = 159.81 min)

Product ions (m/z)

ethyl Rapamycin impurity peak at (tR = 166.28 min)

Product ions (m/z)

936 951 951 904 918 918 886 900 900 642 656 642 614 628 628 607 607 621 582 596 596 538 552 552 485 499 499 459 473 473 441 455 455 409 423 423 381 395 395 320 334 320 285 285 299

Figure S3: Proposed MS2 Product Ion Structures of Ethyl-rapamycin impurity at (tR

= 159.81 min)

Figure S4: Proposed MS2 Product Ion Structures of Rapamycin impurity at (tR =

166.28 min)

Figu

ure S5: Topp: Chromato

S

ogram of Im

Scan Mass

mpurity Isol

Spectra of I

late 1, RRT

Isolate 1

1.21-1.22, BBottom: Fulll

Figu

re S6: Top: Chromatoogram of Im

Mass Spe

mpurity Isola

ectra of Isol

ate 2, RRT

late 1

1.23, Bottomm: Full Scaan

Figure S7: Rotamers Around the Peptide Bond Result in Two Conformations of the

Molecule with Different Chemical Shifts

O

OH

O O

ON

O

O

O

OH

OH

OO

O

O

OH

O O

ON

O

O

OOH

OH

OO

O

trans rotamer cis rotamer

Figure S8: 600 MHz Proton NMR Spectrum of Isolate 1 and Proposed Structure of

33-Ethyl-Rapamycin

O

HO

O O

O

N

O

O

O

HO

OH

O

O

O

12

356

78910

11

1213

14

15

2016

17

18

19

21

2223

2425 26

27

28

29

30

31

3233

3435

3637

38

394041

42

43

44

45

46 48

49

50

47

51

452

6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

Figure S9: DQ-COSY Spectrum of Isolate 1 Including Assignments

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

7 7

6 6

5 5

4 4

3 3

2 2

1 1

0 0

1

-1 H

(p

pm

)

41Ha-Hb

34Ha-52H

34Hb-52H

38H-47H12H-37H

45Hb-Ha

44Hb-Ha

8Hb-Ha

52H-34Ha52H-34Hb

31H-51H

42H-41Hb

19Hb-Ha

19Ha-Hb

44Ha-Hb

8Ha-Hb32Ha-33H

37H-12H

47H-38H

41Hb-Ha

45Ha-Hb

32Hb-Ha

32Ha-Hb

31H-32Hb

25H-48H

25’H-48’H

43H-44Ha

7H-8Ha7H-8Hb

9H-8Hb

42H-43H

43H-42H16Ha-Hb

16Hb-Ha

23Hb-Ha

23Ha-Hb

22H-23Ha 22H-23Hb

28H-29H

28’H-29’H

29H-28H

29’H-28’H

2H-1H

4H-5H

5H-4H

1H-2H

2H-3H

3H-2H

25H-26H

48H-25H

10Hb-9H

8Hb-9H

8Ha-7H

44Ha-43H

8Hb-7H

51H-31H

32Hb-31H

48’H-25’H

41Hb-42H

10Hb-Ha

10Ha-Hb

17Ha-Hb

17Hb-Ha

33H-32Ha

20H-19Ha

1H-33H26H-25H

23Ha-22H

23Hb-22H

33H-1H

19Ha-20H

Figure S10: 2D TOCSY Spectrum of Isolate 1

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

7 7

6 6

5 5

4 4

3 3

2 2

1 1

0 0

1

-1 H

(p

pm

)

41Ha-Hb

34Ha-52H

34Hb-52H

38H-47H12H-37H

45Hb-Ha

44Hb-Ha

8Hb-Ha

52H-34Ha52H-34Hb

31H-51H

42H-41Hb

19Hb-Ha

19Ha-Hb

44Ha-Hb

8Ha-Hb

37H-12H

47H-38H

41Hb-Ha

45Ha-Hb

32Hb-Ha

32Ha-Hb

25H-48H

25’H-48’H

43H-44Ha

7H-8Ha7H-8Hb

9H-8Hb

42H-43H

43H-42H16Ha-Hb

16Hb-Ha

23Hb-Ha

23Ha-Hb

22H-23Ha 22H-23Hb

28H-29H

28’H-29’H

29H-28H29’H-28’H

2H-1H

4H-5H

5H-4H

1H-2H

2H-3H

3H-2H

25H-26H

48H-25H

10Hb-9H

8Hb-9H

8Ha-7H

44Ha-43H

8Hb-7H

51H-31H

48’H-25’H

41Hb-42H

10Hb-Ha

10Ha-Hb

17Ha-Hb

17Hb-Ha

20H-19Ha

26H-25H

23Ha-22H

23Hb-22H

33H-1H

19Ha-20H 11Ha-37H

39Ha-47H

47H-39Ha

32Hb-51H

32Ha-51H

19Hb-18Ha

7H-9H

9H-7H

20H-19Hb

26H-48H

26’H-48’H

5H-1H

3H-4H

4H-3H

5H-2H

1H-5H1H-3H

5H-3H

3H-5H3H-1H

19Hb-20H

18Ha-20H

48H-26H

35H-5H

49H-26H

11Ha-9H

19Ha-16Ha

41Ha-43H 41Ha-42H

44Hb-42H44Hb-43H

37H-11Ha

17Ha-18Hb

11Ha-12H

18Ha-19Hb

42H-41Ha

43H-44Hb 16Ha-19Ha

9H-11Ha

16Hb-18Ha16Hb-19Ha

20H-18Ha

4H-2H

52H-33H

33H-52H

12H-11Ha

51H-32Ha

44Ha-45Hb44Ha-42H

18Ha-16Hb

19Ha-16Hb

17Ha-16Ha

16Ha-17Ha

Figure S11: Multiplicity-edited* 1H-13C HSQC Spectrum of Isolate 1

* The peaks for CH3 and CH groups are shown in red; peaks for CH2 groups are in green.

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

140 140

120 120

100 100

80 80

60 60

40 40

20 20

1

-13

C (

pp

m)

52C-H

1C-H

2C-H3C-H

4C-H

5C-H

32C-Hb32C-Ha

51C-H

35C-H

41C-Hb41C-Ha

42C-H

43C-H

10C-Hb

10C-Ha 45C-Ha45C-Hb

40C-H

39C-Ha39C-Hb

33C-H

37C-H

22C-H

23C-Hb23C-Ha

16C-Hb16C-Ha

47C-H

25C-H

26C-H

29C-H

28C-H

7C-H

9C-H

48C-H

38C-H

11C-Ha

17C-Hb 17C-Ha

20C-H

19C-Hb19C-Ha

18C-Hb 18C-Ha

31C-H

12C-H

8C-Ha8C-Hb

36C-H46C-H

50C-H

49C-H

20’C-H

48’C-H

25’C-H

9’C-H

22’C-H28’C-H

50’C-H

29’C-H

7’C-H

51’C-H

26’C-H

5’C-H

4’C-H

37’C-H

44C-Hb

44C-Ha

34C-Hb 34C-Ha

ALK1C-H

ALK3C-H

Figure S12: 1H-13C HMBC Spectrum of Isolate 1

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

200 200

150 150

100 100

50 50

1

-13

C (

pp

m)

25C-48H 33C-52H

34C-52H

39C-47H

31C-51H

32C-51H

38C-47H

11C-37H

12C-37H

22C-47H

13C-37H

30C-51H

24C-48H

26C-48H26C-49H

5C-35H

6C-35H27C-49H

7C-35H

28C-49H

42C-36H29C-50H

42C-46H

22C-23Hb

29C-28H

28C-29H

5C-7H

26C-28H

27C-28H

30C-28H

13’C-37’H

25’C-48’H

42C-41Hb

26’C-48’H

24’C-48’H

30’C-51’H

24C-23Hb24C-23Ha

21C-20H

2C-3H

4C-2H

3C-5H 3C-1H

28C-26H

7C-5H

33C-2H

19C-20H

18C-20H

35C-5H

33C-1H

35C-7H

8C-7H

24C-25H

7C-8Hb

ALKC-ALK1H

Figure S13: 600 MHz Proton NMR Spectrum of Isolate 2 and Proposed Structure

of 12-Ethyl-Rapamycin

O

HO

O O

O

N

O

O

O

HO

OH

O

O

O

12

356

78910

11

1213

14

15

2016

17

18

19

21

2223

2425 26

27

28

29

30

31

3233

3435

3637

38

394041

42

43

44

45

46 48

49

50

47

51

4

52

7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

Figure S14: DQ-COSY Spectrum of Isolate 2 Including Assignments

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

7 7

6 6

5 5

4 4

3 3

2 2

1 1

0 0

1

-1 H

(p

pm

)

41Ha-Hb

42H-41Hb

31H-51H

31’H-51’H

31H-32Hb

33H-34H

38H-47H

37Ha-52H

45Hb-Ha

19Hb-Ha

19Ha-Hb

25H-48H43H-44Ha

16Ha-17Ha

9H-8Hb

41Hb-Ha

47H-38H 52H-37Ha

8Ha-Hb

8Hb-Ha

44Hb-Ha

32Ha-Hb

51H-31H

48H-25H51’H-31’H

32Hb-31H

34H-33H

8Hb-9H

44Ha-43H

16’Ha-Hb

23Hb-Ha

23Ha-Hb

42H-43H

43H-42H

16’Hb-Ha

29’H-28’H

28H-29H

28’H-29’H

23Hb-22H

23Ha-22H

2H-1H

4H-5H

5H-4H 2H-3H

3H-2H

3H-4H

4H-3H

1H-2H

25H-26H

33H-1H

19Ha-20H

25’H-48’H

20H-19Ha

22H-23Hb

26H-25H

33H-32Ha

32Ha-33H8Ha-7H

8Hb-7H

41Hb-42H

38H-39Ha

32Hb-Ha

44Ha-Hb

7H-8Ha7H-8Hb

1H-33H26’H-25’H

22H-23Ha

29H-28H

25’H-26’H

2’H-1’H

1’H-2’H

4’H-5’H

5’H-4’H

16Ha-Hb

16Hb-Ha

17Ha-16Ha

44Hb-43H

45Ha-Hb

Figure S15: 2D TOCSY Spectrum of Isolate 2

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

7 7

6 6

5 5

4 4

3 3

2 2

1 1

0 0

1

-1 H

(p

pm

)

41Ha-Hb

42H-41Hb

31H-51H

31’H-51’H

31H-32Hb

33H-34H

38H-47H

37Ha-52H

45Hb-Ha

19Hb-Ha

19Ha-Hb

25H-48H43H-44Ha

9H-8Hb

41Hb-Ha

47H-38H52H-37Ha

8Ha-Hb

8Hb-Ha

44Hb-Ha

32Ha-Hb

37Ha-48H

51H-31H

48H-25H51’H-31’H

32Hb-31H

34H-33H

8Hb-9H

44Ha-43H

23Hb-Ha

23Ha-Hb

42H-43H

43H-42H

29’H-28’H

28’H-29’H

23Hb-22H

23Ha-22H

2H-1H

4H-5H

5H-4H

2H-3H

3H-2H

3H-4H

4H-3H

1H-2H

25H-26H

33H-1H

19Ha-20H

25’H-48’H

20H-19Ha

22H-23Hb

26H-25H

33H-32Ha

32Ha-33H8Ha-7H

8Hb-7H

41Hb-42H

38H-39Ha

32Hb-Ha

44Ha-Hb

43H-41Hb43H-45Hb16Ha-17Ha

16Hb-17Ha16Hb-17Hb

7H-8Ha

9H-8Ha

16Ha-19Hb43H-44Hb

42H-41Ha 42H-45Hb

42H-44Hb

16Ha-Hb

16Hb-Ha

7H-9H

9H-7H

28H-29H

29H-28H

41Ha-42H

41Ha-43H

45Hb-43H

17Ha-16Hb

49H-16Hb

8Ha-9H

41Hb-43H

45Hb-42H

19Hb-16Hb 19Hb-16Ha

18’Hb-20’H

11Ha-9H

18’Ha-16’Hb

19Hb-20H

1H-5H1H-3H

5H-1H

3H-1H

3H-5H

4H-2H

5H-2H5H-3H

34H-2H

48H-26H

48’H-26’H

35H-5H

18Ha-20H

44Hb-43H 44Hb-42H

26H-48H

20H-18Ha

22H-23Ha

20H-19Hb

2H-34H

26’H-48’H

16’Hb-18’Hb

9H-11Ha16Hb-19Hb

42H-40H

40H-42H

52H-12H

41Hb-44Hb41Hb-45Hb

39Hb-33H

34H-1H

16’Ha-Hb

31H-32Ha

2H-4H

1H-4H

49H-26H

47H-22H

18’Hb-16’Hb

16’Hb-18’Ha

20H-18Hb

5H-35H

1H-34H

22H-47H

44Hb-45Hb

3H-35H

4H-35H

26H-49H

48’H-25’H

7H-8Hb

37Hb-12H

12H-37Hb

37Ha-12H

12H-37Ha

16Ha-17Hb

12H-52H

32Ha-31H

16Hb-20H

16Ha-20H

33H-2H

4’H-5’H

3’H-5’H

5’H-4’H 5’H-3’H

4’H-3’H

33H-32Hb

43H-40H

Figure S16: Multiplicity-edited* 1H-13C HSQC Spectrum of Isolate 2

* The peaks for CH3 and CH groups are shown in red; peaks for CH2 groups are in green.

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

140 140

120 120

100 100

80 80

60 60

40 40

20 20

1

-13

C (

pp

m)

1C-H

2C-H

3C-H

4C-H

5C-H

34C-H

32C-Hb32C-Ha

51C-H

35C-H

41C-Hb

41C-Ha

42C-H

43C-H

10C-Hb10C-Ha45C-Ha45C-Hb

40C-H

39C-Ha39C-Hb33C-H

22C-H

23C-Hb23C-Ha

16C-Hb16C-Ha

47C-H

25C-H

26C-H

29C-H

28C-H

7C-H

9C-H

48C-H

38C-H

11C-Ha

17C-Hb 17C-Ha

20C-H

19C-Hb 19C-Ha

18C-Hb 18C-Ha

31C-H 12C-H

8C-Ha

8C-Hb

36C-H46C-H

50C-H

49C-H

20’C-H

48’C-H

25’C-H

9’C-H

22’C-H

28’C-H

50’C-H

29’C-H

7’C-H

51’C-H

26’C-H

5’C-H

44C-Hb

44C-Ha

37C-Hb 37C-Ha

52C-H

ALK1C-H

ACNC-H

ALK2C-H

ALK3C-H

1’C-H

2’C-H

16’C-Hb 16’C-Ha

3’C-H

Figure S17: 1H-13C HMBC Spectrum of Isolate 2

7

7

6

6

5

5

4

4

3

3

2

2

1

1

0

0

2 - 1H (ppm)

200 200

150 150

100 100

50 50

1

-13

C (

pp

m)

25C-48H

38C-47H

39C-47H33C-34H

32C-34H

37C-52H

12C-52H

22C-47H28C-49H

7C-35H

26C-48H

1C-34H

30C-51H

24C-48H

6C-35H

5C-35H

27C-49H

26C-49H

24C-23Hb

24C-23Ha

7C-36H

29C-50H42C-46H

22C-23Hb

32C-51H

31C-51H

38C-47H

29C-28H

26C-28H

21C-20H

3C-1H

ALK2C-ALK3H

25’C-48’H

ALK1C-H

27C-28H

30C-28H

28C-26H

7C-5H

3C-5H

2C-4H5C-3H

3C-2H3C-4H

5C-7H

27C-29H

30’C-51’H

24’C-48’HACN2C-ACNH

28C-29H 28’C-29’H

8C-7H

35C-7H

26’C-48’H

35C-5H

49C-26H

18C-20H

19C-20H

Table S4: 13C and 1H chemical shift data of Isolate 1 (33-Ethyl-Rapamycin) in

CDCl3 at 300 K

(n.o. = not observed). The ‘ indicates the shifts for the minor cis rotamer.

Atom* 13C (ppm) 1H (ppm)01 139.2 5.41 02 131.7 6.13 03 133.7 6.30 04 126.3 6.37 04' 126.1 6.30 05 129.7 5.94 05' 129.4 5.89 06 135.7 n.o. 07 84.4 3.63 07' 84.4 3.59 08 39.0 1.49, 1.8509 67.1 3.86 09' 67.8 3.77 10 31.3 1.62, 1.2911 27.3 1.59, 1.5912 33.6 1.98 13 98.5 n.o. 13' 99.0 n.o. 16 44.2 3.40, 3.5617 25.3 1.46, 1.7418 20.7 1.46, 1.7719 26.9 1.74, 2.3320 51.3 5.26 20' 56.3 4.25 21 169.4 n.o. 22 75.6 5.14 22' 75.9 5.08 23 40.6 2.77, 2.5624 208.2 n.o. 24' 207.9 n.o. 25 46.5 3.30 25' 46.1 3.40 26 127.0 5.38 26' 126.9 5.46 27 136.3 n.o. 28 77.2 4.13 28' 77.1 4.19 29 85.0 3.65 29' 86.5 3.65 30 216.3 n.o. 30' 215.9 n.o. 31 40.9 2.75 32 38.3 1.49, 1.2233 43.1 2.03 34 28.8 1.27, 1.38

35 10.1 1.65 36 55.9 3.12 37 16.2 0.93 37' 16.0 0.81 38 32.9 1.97 39 38.3 1.11, 1.1840 33.2 1.37 41 34.2 2.09, 0.6542 84.5 2.92 43 73.8 3.37 44 31.2 1.33, 1.9845 31.6 0.98, 1.6846 56.6 3.39 47 16.1 0.90 48 16.1 1.08 48' 16.5 1.13 49 13.0 1.72 50 59.5 3.32 50' 59.3 3.39 51 13.8 0.97 51' 15.1 1.04 52 12.0 0.86 ALK 29.9 n.o. ALK1 29.8 1.24 ALK3 14.1 0.88 * ALKx denotes resonances arising from a long chain alkane, attributed to residual HPLC column material used during purification.

Table S5: 13C and 1H chemical shift data of Isolate 2 (12-Ethyl-Rapamycin) in

CDCl3 at 300 K

(n.o. = not observed). The ‘ indicates the shifts for the minor cis rotamer.

Atom* 13C (ppm) 1H (ppm)01 140.1 5.50 01' 139.2 5.40 02 130.0 6.12 02' 131.5 6.12 03 133.6 6.28 03' 133.5 6.22 04 126.3 6.37 04' n.o. 6.36 05 129.4 5.94 05' 129.5 5.87 06 135.8 n.o. 07 84.2 3.63 07' 84.4 3.57 08 39.0 1.47, 1.8309 67.3 3.86 09' 68.0 3.78 10 31.3 1.62, 1.2711 27.2 1.59, 1.5912 40.5 1.73 16 44.2 3.39, 3.5616' 39.0 3.20, 4.4117 25.3 1.45, 1.7218 20.6 1.46, 1.7618' n.o. 1.45, 1.7719 26.9 1.73, 2.3220 51.3 5.25 20' 56.2 4.28 21 169.3 n.o. 22 75.6 5.14 22' 75.6 5.08 23 40.6 2.70, 2.5524 208.1 n.o. 24' 207.7 n.o. 25 46.6 3.29 25' 46.1 3.40 26 126.7 5.38 26' 126.9 5.47 27 136.0 n.o. 28 77.2 4.15 28' 77.2 4.18 29 84.8 3.72 29' 86.4 3.64 30 215.3 n.o. 30' 215.4 n.o. 31 41.6 2.69

31' n.o. 2.84 32 40.3 1.47, 1.1833 35.2 2.30 34 21.5 1.03 35 10.2 1.63 36 55.8 3.11 37 23.6 1.54, 1.8438 33.4 1.95 39 38.4 1.09, 1.2040 33.3 1.36 41 34.1 2.08, 0.6542 84.5 2.92 43 74.0 3.36 44 31.3 1.31, 1.9745 31.4 0.97, 1.6846 56.7 3.38 47 16.0 0.90 48 16.1 1.07 48' 16.5 1.13 49 13.1 1.73 50 59.3 3.31 50' 59.2 3.37 51 13.7 0.97 51' 15.2 1.04 52 11.7 0.86 ACN 30.9 2.14 ACN2 207.0 n.o. ALK1 29.7 1.23 ALK2 25.9 0.86 ALK3 14.0 0.86

* ALKx denotes resonances arising from a long chain alkane, attributed to residual HPLC column material used during purification. ACNx = Acetone.

Figurall (s

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rapamycin representin

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