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
re S18: Seleodiated) im
ected Ion Chmpurities wit
hromatograth a +14 am
ams for varimu mass defe
ious lots of rect.
rapamycin representin
ng