7KLV - Royal Society of Chemistry · ts. Production and purification of TB-GlfT1 and Ms-GlfT1...
75
S1 Supporting Information for: Synthetic Polyprenol-Pyrophosphate Linked Oligosaccharides Are Efficient Substrates for Mycobacterial Galactan Biosynthetic Enzymes Xiaochao Xue, Ruixiang Blake Zheng, Akihiko Koizumi, Ling Han, John S. Klassen, and Todd L. Lowary* Alberta Glycomics Centre and Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, Alberta T6G 2G2, Canada [email protected] Table of Contents Production and purification of TB-GlfT1 and Ms-GlfT1 S2–S5 Figure S1. Negative ion mode ESI mass spectra of GlfT2-catalyzed elongation reactions with 22 and 23. S6 NMR Spectra for new compounds S7–S75 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is © The Royal Society of Chemistry 2018
Transcript of 7KLV - Royal Society of Chemistry · ts. Production and purification of TB-GlfT1 and Ms-GlfT1...
S1
Supporting Information for:
Synthetic Polyprenol-Pyrophosphate Linked Oligosaccharides Are Efficient
Substrates for Mycobacterial Galactan Biosynthetic Enzymes
Xiaochao Xue, Ruixiang Blake Zheng, Akihiko Koizumi, Ling Han,
John S. Klassen, and Todd L. Lowary*
Alberta Glycomics Centre and Department of Chemistry, University of Alberta,
11227 Saskatchewan Drive, Edmonton, Alberta T6G 2G2, Canada
Table of Contents
Production and purification of TB-GlfT1 and Ms-GlfT1 S2–S5
Figure S1. Negative ion mode ESI mass spectra of GlfT2-catalyzed
elongation reactions with 22 and 23.
S6
NMR Spectra for new compounds S7–S75
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2018
S2
Production and purification of TB-GlfT1 and Ms-GlfT1
PCR and cloning procedure
TB-GlfT1. The GlfT1 genes for Rv3782 from the M. tuberculosis H37Rv genome
were amplified using TB genomic DNA as a template by PCR with Q5® High-Fidelity
DNA Polymerase (New England BioLab Inc.). The sense strand
5ʹ-TTATTAGGATCCATGACTGAATCGGTCTTCGCCG-3ʹ contained a BamHI site at
the 5ʹ-end and the antisense strand 5ʹ-TTATTAGATATCTTATCATTTTTCGAACTGCG
GGTGGCTCCAAGCGCTTGCAGATCCTCCAGGCTTGCCA-3ʹ contained TGATAA
as the stop codon, a strep tag and an EcoRV site at the 3ʹ-end. Following the
manufacturer’s instruction, the PCR profile was 98 °C for 3 s (initial denaturation) and
then 30 cycles of 98 °C for 10 s; 72 °C for 30 s; and 72 °C for 30s. The PCR product was
digested with BamHI and EcoRv and separated from reaction impurities by agarose gel
electrophoresis. The digested DNA was excised from the gel and purified using the
GeneJET Gel Extraction Kit (Thermo Fisher Scientific). The purified Rv3782 cDNA was
ligated into the pMF406 vector (a gift from Professor Xao-Yong Fan, Laboratory of
Tuberculosis & Mycobacteria Shanghai Public Health Clinical Center, Fudan University,
Shanghai China) that had been previously digested with BamHI and EcoRv, yielding an
expression construct for TB-GlfT1. Ca2+ competent DH5α E. coli cells were transformed
with the ligation mix and plated on LB-agar plates containing kanamycin (25 mg/L).
After growing at 37 °C overnight, a single colony was picked to inoculate 5 mL LB
S3
medium containing kanamycin (25 mg/L) for another overnight growth. Plasmid DNA
was purified using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) and
sequenced for verification. Sequence verified pMF406 expression plasmids for GlfT1
were electrotransformed into competent Mycobacterium smegmatis mc2 155 cells and
plated on Middlebrook 7H10 agar plate containing kanamycin (15 mg/L) for growth. A
fresh single colony was used to inoculate 3.5 mL 7H9 broth containing ADC enrichment
cultures and kanamycin (15 mg/L) for 24 h. Sterile glycerol was added into the cultures,
aliquoted and stored at 80 °C freezer for further use.
Ms-GlfT1. The genomic DNA was extracted from the fresh culture of M. smegmatis
mc2155. Similar cloning procedures to that used for TB-GlfT1 provided expression
constructs using the genomic DNA as the template. The sense and antisense sequences
were 5ʹ-TTATTAGGATCCATGACGCACACTGAGGTCGTCTG-3ʹ and 5ʹ-TTATTAGA
ATTCTCAGTGGTGGTGGTGGTGGTGCTCGAGTCGCTGGAACCTTTCGCGTCT-3ʹ,
respectively.
Bacterial protein expression
Single colonies of TB-GlfT1 and Ms-GlfT1 were used to inoculate 100 mL LB cultures
containing kanamycin (15 mg/L), glycerol (2%) and 0.2% Tween 80. The cultures were
incubated at 37 °C with shaking at 200 rpm for 2 days. Fresh LB medium was inoculated
with turbid cultures at a dilution of 1:50. Protein expression was induced by addition of
S4
0.02% acetamide when cell density reached 1.2-1.4 at 600nm. Bacterial cultures were
further grown at 37 °C with shaking at 200 rpm for 24 h. Cell suspensions were
centrifuged at 6500 × g at 4 °C for 15 min and cell pellets were purified immediately or
stored at 80 °C freezer for further use.
Purification
Purification of TB-GlfT1. To purify the Strep-tagged TB-GlfT1, cell pellets (20 g/L
culture) were resuspended in 100 mM Tris-Cl, containing 1 mM EDTA, 150 mM NaCl,
10% Glycerol and 0.1% Triton X-100 (pH 8.0) supplemented with Complete Protease
Inhibitor Cocktail Tablets (Roche). The suspension was passed through a cell disruptor at
35000 psi twice and centrifuged at 100500 g for 60 min. The supernatant was loaded onto
the Strep-Tactin Sepharose column (2.5 mL resin) equilibrated with 100 mM Tris-Cl, 150
mM NaCl, 10% Glycerol (pH 8.0) at a flow rate of 1 mL/min and washed with the same
buffer until the OD280 was at the baseline. The enzyme was eluted with 50 mM MOPS,
0.15 M NaCl, 10% Glycerol and 5 mM Desthiobiotin (pH 7.6) at a flow rate of 1 mL/min.
Fractions were frozen immediately for further use at − 80 °C.
Purification of Ms-GlfT1. To purify the His-tagged Ms-GlfT1, the cell pellet (20 g
/L culture) was resuspended in 50 mM sodium phosphate buffer, pH 7.6, containing 400
mM NaCl, 20 mM imidazole, 10% glycerol and 0.1% Triton X-100 supplemented with
Complete Protease Inhibitor Cocktail Tablets (Roche). The suspension was passed
S5
through a cell disruptor through at 35000 psi twice. The cell lysate was centrifuged at
100500 g for 60 min at 4 °C. The Ms-GlfT1 enzyme was purified from the resulting
supernatant by applying directly to a Ni-NTA Superflow column that has been treated
with an equilibrating buffer of 50 mM sodium phosphate buffer, pH 7.6, containing 400
mM NaCl, 20 mM imidazole, 10% glycerol. The column was washed with equilibrating
buffer until the A280 nm returned to background levels. At this point the column was
treated with the second wash buffer (50 mM sodium phosphate buffer, pH 7.6, containing
400 mM NaCl, 50 mM imidazole, 10% glycerol). The protein was eluted with 50 mM
sodium phosphate buffer, pH 7.6, containing 400 mM NaCl, 250 mM imidazole, 10%
glycerol. A flow rate of 1 mL/min was used through the column equilibration, column
washing and protein elution steps. Fractions were frozen immediately for further use at
− 80 °C.
S6
Figure S1. Negative ion mode ESI mass spectra of GlfT2-catalyzed elongation reactions with 22
and 23. Peaks are labeled with the number of additional Galf residues (s) in the product and the
charge state of the ion (superscripted number). The peaks labelled p correspond to the starting
material (22 or 23).
S7
1H NMR of Compound 3 (700 MHz, CD3OD)
S8
31P NMR of Compound 3 (162 MHz, CD3OD)
S9
1H NMR of Compound 4 (500 MHz, D2O)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
61
1.2
73
1.6
46
1.7
12
1.7
41
2.0
50
2.0
65
2.1
00
2.1
23
2.1
35
2.1
47
2.1
64
2.1
78
2.1
92
3.5
62
3.5
82
3.6
06
3.6
93
3.7
08
3.7
27
3.7
36
3.7
72
3.7
97
3.8
08
3.8
22
3.8
31
3.8
41
3.8
61
3.8
82
3.8
88
3.9
01
3.9
08
3.9
64
3.9
71
3.9
75
4.0
83
4.0
89
4.1
04
4.1
09
4.1
23
4.1
29
4.1
36
4.1
68
4.5
09
4.5
16
4.7
80
4.8
72
5.2
07
5.2
18
5.2
32
5.2
79
5.4
62
5.4
90
5.4
96
5.5
04
3.2
57
5.8
55
3.0
29
2.7
85
11
.25
8
2.1
81
10
.88
02
.16
67
.46
30
.98
2
2.0
49
1.0
16
3.1
18
1.0
44
1.1
03
1.0
00
S10
13C NMR of Compound 4 (125 MHz, D2O)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
16
.99
17
.03
22
.18
24
.92
25
.58
25
.80
30
.28
38
.83
53
.28
53
.35
60
.40
61
.35
62
.53
62
.87
63
.10
63
.15
67
.37
68
.20
70
.50
70
.66
71
.03
73
.31
75
.70
76
.41
76
.50
78
.37
79
.58
81
.30
81
.59
81
.62
82
.59
94
.61
94
.66
10
1.4
31
07
.33
10
8.4
21
19
.38
11
9.4
51
24
.29
12
4.5
01
33
.53
13
6.7
31
43
.15
17
4.4
5
S11
31P NMR of Compound 4 (162 MHz, D2O)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-13
.32
8-1
3.1
94
-10
.72
1-1
0.5
88
S12
1H NMR of Compound 5 (700 MHz, CD3OD)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
77
1.2
86
1.2
97
1.3
07
1.9
75
2.0
28
2.0
37
2.0
59
2.0
69
2.0
85
2.0
95
2.1
06
2.1
15
3.1
38
3.1
40
3.1
48
3.1
50
3.3
05
3.3
08
3.3
10
3.3
12
3.3
14
3.6
43
4.0
55
4.1
88
4.1
95
4.2
01
4.2
04
4.2
09
4.2
13
4.2
18
4.2
25
4.2
32
4.2
88
4.2
92
4.2
95
4.2
98
4.8
63
4.9
06
4.9
09
4.9
33
4.9
34
5.0
27
5.0
35
5.0
67
5.0
70
5.0
76
5.0
80
5.0
94
5.1
41
5.1
44
5.1
45
5.2
30
5.2
39
5.3
32
8.8
81
2.7
81
3.1
40
2.8
13
2.9
14
2.9
77
3.2
02
5.5
95
5.3
74
6.3
14
3.3
15
0.9
99
1.0
28
0.9
54
0.9
51
1.0
42
4.8
48
2.0
72
1.1
80
0.9
88
0.9
08
0.9
52
1.0
36
2.9
46
1.0
05
1.9
89
1.0
02
1.0
38
1.0
00
S13
13C NMR of Compound 5 (175 MHz, CD3OD)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
9.2
21
8.5
02
0.6
62
0.7
02
0.7
22
0.7
52
0.7
62
0.7
82
0.9
72
1.4
72
2.9
34
7.3
34
8.6
34
8.6
44
8.7
54
8.7
64
8.8
84
8.8
84
9.0
04
9.0
04
9.1
24
9.1
24
9.2
44
9.2
54
9.3
64
9.3
76
3.1
76
4.0
16
4.4
36
8.9
26
9.8
77
0.7
07
0.7
17
1.2
37
4.2
67
7.0
17
7.7
57
9.3
28
1.9
48
2.8
68
3.3
51
06
.48
10
7.9
21
71
.22
17
1.3
21
71
.54
17
1.5
61
71
.70
17
1.8
41
71
.86
17
2.2
21
72
.33
17
2.6
81
73
.95
S14
31P NMR of Compound 5 (162 MHz, CD3OD)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-1.1
77
S15
1H NMR of Compound 7 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
2.2
77
4.6
32
4.6
43
4.6
46
4.6
52
4.7
21
4.7
26
4.7
85
5.0
02
5.6
33
5.6
40
5.6
55
5.6
89
5.6
92
5.7
12
5.7
14
5.7
75
7.0
39
7.0
51
7.1
90
7.2
01
7.2
12
7.2
39
7.2
50
7.2
60
7.2
69
7.2
88
7.2
99
7.3
10
7.3
34
7.3
45
7.3
56
7.4
02
7.4
06
7.4
13
7.4
21
7.4
32
7.4
53
7.4
63
7.4
73
7.5
08
7.5
60
7.8
23
7.8
34
7.8
56
7.8
67
7.9
02
7.9
13
7.9
56
7.9
67
7.9
94
8.0
05
8.0
17
8.0
27
8.0
37
2.9
65
2.1
47
3.0
68
1.0
64
0.9
88
1.0
30
1.0
31
1.0
02
1.0
28
0.9
99
1.0
02
1.0
00
2.0
33
2.1
88
6.9
96
2.3
35
2.2
71
6.2
60
3.1
02
1.2
13
1.1
63
2.0
66
2.0
09
2.1
58
2.1
79
6.1
56
S16
13C NMR of Compound 7 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
21
.23
63
.93
64
.50
70
.59
73
.56
76
.91
77
.16
77
.41
77
.45
77
.92
81
.64
82
.27
82
.33
82
.46
91
.32
10
5.5
51
28
.35
12
8.3
81
28
.46
12
8.4
71
28
.62
12
8.6
81
28
.89
12
8.9
21
28
.97
12
8.9
81
29
.56
12
9.6
81
29
.71
12
9.7
81
29
.80
12
9.8
21
29
.91
12
9.9
31
29
.98
13
0.0
91
30
.15
13
2.9
51
33
.04
13
3.1
51
33
.23
13
3.4
31
33
.63
13
3.6
81
38
.28
16
5.3
51
65
.45
S17
1H NMR of Compound 8 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
33
0.7
39
0.7
96
0.8
02
1.2
39
1.2
48
1.5
89
1.8
60
1.8
92
1.9
25
2.0
98
2.1
12
3.4
96
3.5
06
3.6
97
3.7
01
3.7
06
3.7
10
4.1
37
4.1
40
4.1
54
4.1
57
4.2
49
4.2
56
4.2
67
4.2
74
4.3
00
4.3
12
4.3
15
4.3
27
4.5
45
4.5
48
4.5
49
4.5
52
4.5
61
4.5
74
4.5
76
4.5
89
4.6
21
4.6
24
4.8
34
4.8
38
4.8
48
4.8
53
5.1
26
5.1
40
5.1
48
5.1
53
5.1
60
7.2
60
7.7
18
7.7
22
7.7
25
7.7
30
7.8
75
9.7
95
1.1
33
1.3
61
3.6
25
3.0
45
3.0
52
1.1
66
3.2
49
3.2
37
1.0
18
1.0
95
1.0
86
1.1
05
1.0
93
1.0
59
1.0
85
1.0
85
0.9
95
1.0
00
2.1
67
2.2
02
1.9
96
S18
13C NMR of Compound 8 (175 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.3
9
17
.49
17
.92
20
.74
20
.76
20
.98
21
.32
55
.64
62
.45
67
.33
70
.09
70
.34
70
.63
71
.17
71
.58
72
.15
76
.98
77
.16
77
.34
78
.98
97
.90
99
.80
13
1.8
01
34
.08
16
9.5
51
69
.71
17
0.3
81
71
.01
S19
1H NMR of Compound 9 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.5
94
4.7
17
4.7
31
4.7
41
4.7
55
4.7
89
4.7
98
4.8
13
4.8
22
4.9
11
4.9
18
4.9
25
5.6
65
5.6
78
5.6
91
5.6
99
5.9
88
6.0
90
6.0
99
6.1
04
6.1
12
6.1
20
7.2
60
7.2
66
7.2
82
7.2
98
7.3
10
7.3
25
7.3
41
7.3
57
7.3
72
7.4
30
7.4
46
7.4
62
7.4
95
7.5
09
7.5
24
7.5
39
7.5
41
7.5
80
7.5
95
7.6
10
7.8
77
1.0
72
1.0
18
1.0
00
1.0
32
0.9
93
0.4
91
1.5
31
6.3
36
2.1
09
3.1
80
1.0
57
2.0
11
2.0
37
4.1
19
S20
13C NMR of Compound 9 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm6
3.5
17
0.3
67
6.6
57
6.9
17
7.1
67
7.4
18
0.9
38
1.2
58
4.9
4
11
1.6
51
13
.45
12
8.4
71
28
.50
12
8.5
81
28
.64
12
8.6
81
28
.79
12
9.4
51
29
.60
12
9.8
51
30
.04
13
0.1
41
33
.27
13
3.5
01
33
.79
13
3.8
91
65
.30
16
5.7
31
65
.81
16
6.1
6
S21
1H NMR of Compound 11 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
2.3
35
2.5
50
2.5
59
2.5
62
2.5
74
2.5
84
2.5
96
2.6
11
2.6
51
2.6
62
2.6
66
2.6
77
2.6
82
2.6
96
2.7
04
2.7
16
2.7
28
2.8
40
2.8
55
2.8
58
2.8
70
2.8
78
2.8
90
2.8
97
2.9
05
4.1
73
4.1
86
4.1
94
4.1
99
4.2
06
4.2
10
4.2
15
4.2
19
4.2
25
4.2
31
4.3
41
4.3
51
4.8
98
4.9
13
4.9
18
4.9
33
5.6
78
7.1
22
7.1
38
7.2
60
7.3
51
7.3
68
2.9
66
2.9
47
2.9
02
2.8
82
2.9
30
1.0
77
2.0
73
1.1
02
2.0
11
1.0
25
1.0
26
1.0
00
1.9
54
1.9
57
S22
13C NMR of Compound 11 (125 MHz, CDCl3)
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
16
.96
21
.28
26
.68
27
.85
28
.13
29
.94
38
.07
65
.69
75
.11
75
.64
76
.61
76
.91
77
.16
77
.41
84
.19
11
0.1
2
12
9.4
91
30
.04
13
2.6
81
38
.19
17
2.1
6
20
6.4
9
S23
1H NMR of Compound 15 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
30
0.5
94
0.6
07
1.0
35
1.3
08
1.5
63
2.1
51
2.5
06
2.5
19
2.5
21
2.5
34
2.5
37
2.5
49
2.6
41
3.5
12
3.6
65
3.6
70
3.6
78
3.6
83
3.6
86
3.7
77
3.7
87
3.8
19
3.9
77
3.9
88
3.9
93
4.2
50
4.2
67
4.2
71
4.2
88
4.5
73
4.5
89
4.5
93
4.6
09
4.7
24
5.3
04
5.3
22
5.5
51
7.2
59
7.3
48
7.3
50
7.3
54
7.3
63
7.4
81
7.4
84
7.4
95
7.5
00
7.7
44
7.7
50
7.7
55
7.7
61
7.8
61
7.8
67
7.8
72
7.8
78
9.0
21
3.0
03
1.1
76
1.0
21
3.0
33
3.0
04
3.0
98
3.2
47
1.1
59
1.1
17
1.9
13
2.9
27
1.0
98
1.0
25
1.0
58
1.0
75
1.9
79
0.9
92
1.0
38
1.0
00
2.9
21
2.0
31
1.9
91
1.9
61
S24
13C NMR of Compound 15 (125 MHz, CDCl3)
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.4
0
16
.29
18
.05
26
.18
27
.54
28
.04
29
.94
38
.00
57
.03
64
.73
66
.59
67
.64
68
.98
74
.41
74
.61
75
.60
75
.98
76
.91
77
.16
77
.41
80
.83
97
.64
98
.36
10
2.2
41
09
.56
12
3.7
81
26
.53
12
8.3
81
29
.41
13
1.5
81
34
.53
13
7.1
8
17
1.9
8
20
6.3
1
S25
1H NMR of Compound 16 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
34
0.8
09
1.2
35
1.2
48
1.6
17
2.1
92
2.5
18
2.5
31
2.5
35
2.5
42
2.5
45
2.5
50
2.5
61
2.8
05
2.8
15
2.8
26
2.8
37
2.8
41
3.3
11
3.3
18
3.4
93
3.4
99
3.5
13
3.5
32
3.5
50
3.6
09
3.9
21
3.9
32
4.0
07
4.1
44
4.1
61
4.1
66
4.1
83
4.2
89
4.3
05
4.3
11
4.3
27
4.3
91
4.3
93
4.8
30
4.8
33
4.8
48
4.8
67
4.8
86
5.2
01
5.2
18
7.2
60
7.7
52
7.7
58
7.7
63
7.7
69
7.8
59
7.8
65
7.8
69
7.8
75
9.0
17
1.1
76
1.0
80
3.0
04
1.0
55
3.0
03
2.9
53
2.1
45
1.0
87
3.0
57
1.0
26
5.0
43
1.0
45
1.0
36
0.9
32
0.9
15
0.9
87
1.0
00
2.0
25
1.9
46
S26
13C NMR of Compound 16 (125 MHz, CDCl3)
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.3
8
17
.46
18
.00
28
.38
29
.91
38
.55
55
.02
62
.75
67
.43
67
.44
69
.50
71
.00
71
.36
74
.84
75
.29
76
.91
77
.16
77
.41
83
.70
97
.92
10
1.1
1
12
3.7
7
13
1.5
413
4.5
0
17
3.3
3
20
7.9
1
S27
1H NMR of Compound 17 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
30
1.0
99
1.1
08
1.5
71
1.7
85
1.9
20
2.0
89
2.1
12
2.1
51
2.4
57
2.4
60
2.4
67
2.4
76
2.4
78
2.6
82
2.6
85
2.6
91
2.6
95
3.5
01
3.5
10
4.1
37
4.1
51
4.1
55
4.2
45
4.2
52
4.2
63
4.3
15
4.3
27
4.3
31
4.3
43
4.5
35
4.5
38
4.5
40
4.5
43
4.5
70
4.5
82
4.5
85
4.5
98
4.6
38
4.6
41
4.8
75
4.8
90
4.9
04
4.9
92
4.9
97
5.0
06
5.0
11
5.1
23
5.1
39
5.1
51
7.2
59
7.7
10
7.7
15
7.7
18
7.7
23
9.0
78
1.0
28
1.2
82
3.0
89
2.9
79
3.0
17
3.0
18
3.0
50
3.0
92
2.0
31
2.0
35
1.0
05
1.0
08
1.0
01
1.0
08
1.0
07
1.0
05
1.0
01
0.9
86
1.0
24
0.9
96
0.9
92
1.0
00
2.0
16
2.0
29
1.8
09
S28
13C NMR of Compound 17 (175 MHz, CDCl3)
210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.3
9
17
.24
17
.93
20
.52
20
.82
20
.98
21
.34
28
.06
29
.85
37
.80
55
.45
62
.43
67
.34
67
.88
68
.25
70
.06
70
.69
70
.89
72
.12
76
.98
77
.16
77
.34
79
.57
97
.94
10
0.0
2
13
1.7
71
34
.06
16
9.2
21
69
.68
16
9.7
31
70
.99
17
1.9
1
20
6.1
4
S29
1H NMR of Compound 18 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
20
1.1
45
1.1
54
1.6
53
1.6
99
1.8
88
1.9
74
2.1
12
4.4
99
4.6
00
4.6
71
4.6
75
4.6
94
4.7
64
5.1
20
5.1
26
5.1
31
5.3
38
5.3
59
5.6
05
5.6
11
5.6
99
5.7
83
5.8
57
5.8
62
7.1
14
7.1
24
7.1
35
7.2
27
7.2
39
7.2
51
7.2
60
7.3
20
7.3
24
7.3
45
7.3
56
7.4
10
7.4
20
7.4
30
7.4
63
7.4
73
7.4
83
7.5
90
7.7
09
7.7
38
7.7
49
7.8
14
7.8
24
7.8
98
7.9
10
7.9
21
7.9
84
7.9
94
8.0
30
8.0
40
9.3
65
1.0
22
1.1
75
3.0
34
3.1
14
3.1
03
2.9
98
3.1
01
1.0
56
0.9
96
1.0
28
1.0
14
1.0
00
1.0
22
1.0
14
0.9
96
1.0
48
3.1
49
3.1
76
2.0
56
1.0
48
3.1
80
0.9
82
0.9
61
0.9
97
0.9
84
1.0
54
0.9
83
1.0
00
2.1
26
7.9
35
5.6
58
7.9
69
1.0
50
2.1
67
2.0
90
2.5
13
5.2
12
4.4
72
2.2
00
S30
13C NMR of Compound 18 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.4
01
7.8
41
7.9
32
0.5
72
0.7
92
0.9
72
1.2
65
5.3
26
2.5
36
7.2
96
8.4
87
0.7
37
2.1
47
6.9
17
7.1
67
7.4
17
9.4
59
8.0
11
00
.26
10
5.0
51
06
.95
12
8.2
31
28
.37
12
8.4
71
28
.48
12
8.7
11
28
.75
12
8.8
31
28
.85
12
8.9
61
28
.98
12
9.6
81
29
.70
12
9.7
21
29
.74
12
9.7
91
29
.82
12
9.9
61
30
.10
13
0.1
51
33
.09
13
3.3
21
33
.93
16
5.1
01
65
.37
16
5.6
71
65
.83
16
5.9
81
66
.09
16
6.2
31
69
.00
16
9.5
81
69
.69
17
0.9
8
S31
1H NMR of Compound 19 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
0.0
05
1.2
19
1.2
27
1.9
74
1.9
99
2.0
28
2.0
59
2.0
63
2.0
86
2.0
91
2.1
00
2.1
21
3.6
41
3.6
55
4.0
69
4.0
72
4.1
67
4.1
73
4.1
79
4.1
84
4.2
00
4.2
04
4.2
08
4.2
25
4.2
32
4.3
25
4.3
30
4.3
95
4.3
99
4.4
04
4.5
49
4.6
69
4.9
33
4.9
35
4.9
43
5.0
29
5.0
33
5.0
38
5.0
42
5.1
38
5.1
41
5.1
83
5.1
96
5.2
04
5.2
06
5.2
13
5.2
25
5.2
78
5.3
07
5.3
59
5.3
65
5.3
70
6.1
93
6.2
02
7.2
60
8.4
33
1.2
09
0.9
77
2.8
15
2.7
99
2.9
33
2.9
70
11
.09
61
1.0
17
3.1
80
0.9
23
0.9
73
2.1
27
0.9
42
1.0
50
0.9
47
3.7
26
1.0
22
1.8
93
0.9
41
0.9
26
0.9
14
1.8
37
0.9
72
0.9
30
2.7
83
0.9
27
0.9
55
0.9
57
1.0
00
0.8
90
S32
13C NMR of Compound 19 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
20
.75
20
.79
20
.85
20
.86
20
.92
20
.93
20
.97
20
.98
21
.31
23
.60
59
.44
62
.53
63
.00
63
.24
67
.58
68
.51
69
.21
69
.81
70
.77
71
.49
72
.27
72
.29
74
.86
76
.46
76
.49
76
.91
77
.16
77
.41
80
.43
80
.59
81
.55
81
.87
82
.09
98
.39
10
0.6
81
05
.06
10
6.5
5
16
9.5
61
69
.64
16
9.8
61
69
.95
17
0.1
51
70
.22
17
0.3
31
70
.60
17
0.6
31
70
.93
17
1.9
5
S33
1H NMR of Compound 20 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
15
1.2
24
1.9
77
2.0
34
2.0
61
2.0
69
2.0
73
2.0
87
2.1
03
2.1
11
2.1
24
3.6
43
4.0
66
4.0
70
4.0
73
4.0
78
4.0
84
4.0
87
4.1
48
4.1
64
4.1
74
4.1
81
4.1
91
4.1
99
4.2
03
4.2
06
4.2
10
4.3
21
4.3
27
4.3
93
4.3
98
4.4
01
4.4
07
4.7
46
4.9
29
4.9
31
5.0
31
5.0
34
5.0
39
5.0
43
5.1
05
5.1
20
5.1
23
5.1
28
5.1
39
5.1
42
5.1
90
5.1
92
5.1
97
5.1
99
5.2
79
5.2
90
5.3
09
5.3
65
7.2
60
3.4
66
3.1
06
3.4
39
10
.31
56
.56
21
2.4
60
3.6
42
1.1
37
1.1
19
4.4
03
5.8
25
2.2
75
1.1
43
0.9
95
1.0
80
1.0
84
2.3
35
3.3
18
1.0
72
2.1
63
1.1
18
1.1
58
1.0
00
S34
13C NMR of Compound 20 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
20
.75
20
.81
20
.86
20
.87
20
.88
20
.88
20
.93
20
.96
21
.06
21
.35
23
.37
54
.14
62
.34
62
.97
63
.32
67
.96
68
.32
69
.24
70
.24
70
.55
71
.92
72
.29
75
.22
76
.46
76
.48
76
.91
77
.16
77
.41
79
.18
80
.59
81
.57
81
.89
81
.92
92
.62
10
0.1
01
05
.07
10
6.5
8
16
9.4
51
69
.67
16
9.7
51
69
.98
17
0.0
21
70
.17
17
0.3
91
70
.56
17
0.6
61
71
.04
17
2.2
5
S35
1H NMR of Compound 21 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
03
1.2
12
1.8
99
1.9
69
2.0
03
2.0
36
2.0
61
2.0
70
2.0
83
2.1
04
2.1
06
2.1
27
2.1
38
3.6
25
3.6
39
3.8
91
4.1
76
4.1
82
4.1
91
4.1
97
4.2
02
4.3
17
4.3
23
4.3
99
4.4
02
4.4
07
4.7
03
4.7
05
4.9
23
4.9
25
5.0
22
5.0
26
5.0
31
5.0
35
5.0
39
5.0
51
5.0
69
5.0
82
5.0
91
5.0
96
5.0
98
5.1
02
5.1
40
5.1
43
5.2
71
5.3
11
7.2
60
7.3
43
7.3
48
7.3
53
7.3
64
7.3
72
7.3
76
7.3
84
7.3
86
3.2
23
3.1
03
3.2
49
3.3
41
6.2
58
3.4
93
3.1
02
6.4
35
3.4
81
2.9
65
2.1
84
1.0
72
2.0
75
1.0
73
4.3
01
2.1
81
2.0
79
1.0
20
1.0
44
8.7
09
1.0
87
1.0
26
1.0
61
1.0
67
1.1
32
1.0
00
1.0
00
11
.17
6
S36
13C NMR of Compound 21 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
17
.69
20
.69
20
.75
20
.80
20
.82
20
.86
20
.88
20
.93
21
.08
21
.23
23
.03
51
.85
51
.91
61
.60
62
.95
63
.35
68
.32
69
.25
69
.50
70
.04
70
.08
70
.12
71
.64
72
.31
76
.91
77
.16
77
.41
80
.58
81
.58
81
.72
81
.98
97
.01
99
.73
10
5.0
81
06
.63
12
8.2
51
28
.31
12
8.9
41
28
.96
12
9.1
21
35
.40
13
5.4
51
35
.59
13
5.6
41
69
.33
16
9.4
31
69
.58
16
9.6
51
69
.97
17
0.1
51
70
.38
17
0.6
31
70
.65
17
0.7
9
S37
31P NMR of Compound 21 (162 MHz, CDCl3)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-2.3
84
S38
1H NMR of Compound 22 (500 MHz, CD3OD)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
37
1.2
50
1.5
99
1.6
06
1.6
68
1.7
16
1.9
59
1.9
73
1.9
89
2.0
46
2.0
61
2.0
77
2.0
92
2.1
04
2.1
17
2.1
32
3.3
03
3.3
07
3.3
10
3.3
13
3.3
17
3.3
54
3.3
73
3.3
92
3.4
48
3.4
64
3.6
25
3.6
28
3.6
32
3.6
44
3.6
51
3.7
20
3.7
46
3.7
49
3.8
10
3.9
29
3.9
38
3.9
42
3.9
49
3.9
56
3.9
61
3.9
69
4.1
22
4.1
42
4.5
78
4.8
43
4.8
65
5.0
89
5.0
92
5.0
95
5.1
06
5.1
09
5.1
12
5.1
26
5.4
35
3.5
26
4.5
93
2.5
73
2.2
36
10
.02
4
1.3
70
1.4
97
1.4
17
2.5
67
1.4
76
3.6
82
1.4
09
1.5
87
0.9
51
1.8
22
1.0
00
1.4
09
S39
13C NMR of Compound 22 (125 MHz, CD3OD)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
17
.74
17
.87
17
.89
22
.89
22
.96
25
.89
27
.43
27
.80
40
.64
40
.85
48
.49
48
.66
48
.83
49
.00
49
.17
49
.34
49
.51
62
.51
62
.55
64
.42
70
.34
70
.59
70
.67
72
.14
72
.58
72
.59
73
.86
75
.19
75
.27
81
.65
81
.79
96
.61
96
.66
10
3.4
71
03
.55
12
1.7
91
25
.16
12
5.4
31
32
.10
13
6.2
61
41
.71
17
3.9
4
S40
31P NMR of Compound 22 (162 MHz, CD3OD)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-11
.73
7-9
.22
9
S41
1H NMR of Compound 23 (700 MHz, CD3OD)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
57
1.2
66
1.5
99
1.6
06
1.6
68
1.7
08
1.9
63
1.9
73
1.9
84
2.0
28
2.0
38
2.0
57
2.0
73
2.1
02
2.1
13
2.1
24
3.3
05
3.3
08
3.3
10
3.3
12
3.3
15
3.5
54
3.5
68
3.6
11
3.6
16
3.6
19
3.6
25
3.7
14
3.7
18
3.7
27
3.7
30
3.7
97
3.8
02
3.8
11
3.8
16
3.9
44
3.9
49
3.9
54
3.9
58
3.9
91
3.9
93
3.9
97
3.9
99
4.0
11
4.0
17
4.0
20
4.0
26
4.5
40
4.8
43
4.8
60
5.0
90
5.0
92
5.1
26
5.2
75
5.2
77
3.1
63
6.1
61
3.2
06
3.0
45
2.1
39
7.1
50
2.1
00
0.9
86
1.0
92
2.1
78
0.8
95
2.1
32
1.9
36
0.9
16
1.1
84
3.9
07
1.0
28
1.7
11
0.8
28
1.0
22
0.9
86
1.0
00
1.0
07
0.8
26
S42
13C NMR of Compound 23 (125 MHz, CD3OD)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
16
.09
16
.64
17
.76
18
.39
23
.03
25
.90
27
.48
27
.80
40
.66
40
.86
62
.79
64
.04
64
.77
68
.93
70
.83
72
.27
72
.58
72
.82
75
.15
78
.50
79
.11
81
.54
83
.49
84
.35
96
.38
10
3.3
0
11
0.1
8
12
2.1
61
25
.20
12
5.4
31
32
.10
13
6.2
2
14
1.1
8
17
4.0
1
S43
31P NMR of Compound 23 (202 MHz, CD3OD)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-12
.83
-10
.41
S44
1H NMR of Compound 25 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
38
0.5
82
0.5
94
1.5
70
1.7
80
1.9
05
1.9
41
3.4
76
3.4
89
3.7
14
3.7
32
3.8
34
3.8
54
4.2
81
4.2
98
4.3
01
4.3
18
4.4
13
4.4
24
4.5
18
4.5
21
4.5
95
4.6
13
4.6
15
4.6
33
4.7
08
4.7
11
4.7
15
4.7
18
4.7
92
4.8
12
4.8
32
5.2
13
5.2
20
5.2
33
5.2
39
5.2
56
5.5
77
7.2
60
7.3
26
7.3
30
7.3
33
7.3
35
7.3
39
7.4
90
7.4
95
7.4
98
7.5
01
7.5
03
7.5
09
7.7
19
7.7
25
7.7
30
7.7
36
7.8
55
8.9
40
2.9
62
1.0
49
1.2
13
3.0
04
3.0
56
2.9
74
1.0
08
1.0
45
1.0
28
2.0
22
1.0
16
1.0
10
1.0
04
0.9
83
0.9
93
1.0
09
0.9
96
1.9
98
1.0
00
3.0
09
2.0
54
2.0
44
1.8
35
S45
13C NMR of Compound 25 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.4
1
16
.57
18
.04
20
.66
20
.79
20
.86
56
.57
66
.54
66
.59
67
.59
68
.48
69
.00
70
.45
71
.44
74
.80
76
.91
77
.16
77
.41
80
.59
97
.53
98
.45
10
2.2
5
12
3.7
61
26
.63
12
8.2
81
29
.35
13
1.7
81
34
.17
13
7.3
1
16
9.4
61
69
.79
17
0.0
6
S46
1H NMR of Compound 26 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
27
0.7
42
0.7
53
0.7
62
0.8
11
0.8
19
0.8
32
1.2
25
1.2
38
1.6
00
1.8
42
1.9
31
2.0
29
2.0
98
3.5
07
3.5
19
3.5
26
3.5
35
3.5
39
3.6
54
3.8
03
3.8
08
3.8
99
3.9
19
3.9
30
4.0
82
4.0
95
4.1
01
4.2
05
4.2
22
4.2
26
4.2
43
4.3
08
4.3
24
4.3
29
4.6
97
4.7
01
4.7
07
4.7
29
4.7
34
4.9
69
4.9
89
5.0
08
5.0
67
5.0
74
5.0
87
5.0
93
5.2
08
5.2
25
7.2
60
7.7
26
7.7
32
7.7
37
7.7
42
7.8
84
8.9
98
1.1
43
1.0
81
3.0
00
3.0
05
3.0
01
3.0
01
1.0
79
1.9
79
1.0
62
0.9
41
1.0
03
1.0
48
0.9
90
1.1
24
1.0
64
1.0
98
0.9
57
0.9
38
1.0
56
1.0
29
1.0
00
2.0
48
2.0
82
S47
13C NMR of Compound 26 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.3
8
17
.64
18
.03
20
.64
20
.74
20
.88
54
.95
62
.66
67
.44
68
.19
68
.53
69
.67
70
.67
71
.20
75
.30
76
.91
77
.16
77
.41
83
.28
98
.02
99
.18
12
3.8
0
13
1.6
813
4.2
4
16
9.2
816
9.6
617
0.0
0
S48
1H NMR of Compound 27 (700 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
0.0
11
0.8
96
0.8
99
0.9
04
1.1
41
1.1
50
1.9
68
2.0
05
2.0
36
2.0
70
2.0
74
2.1
15
2.9
40
2.9
42
2.9
54
3.5
68
3.5
78
3.6
41
3.6
44
3.6
51
3.6
55
3.8
74
3.8
79
3.8
88
3.9
45
3.9
53
4.0
58
4.0
61
4.0
76
4.0
79
4.2
40
4.2
47
4.2
57
4.2
64
4.5
68
4.5
83
4.5
96
4.7
30
4.9
55
4.9
70
4.9
83
5.0
21
5.0
35
5.0
49
5.1
57
5.1
61
5.1
72
5.1
76
5.1
82
5.1
94
5.2
01
5.2
05
5.2
08
6.0
79
6.0
89
9.4
08
1.1
81
1.1
06
3.1
83
3.2
00
3.1
43
3.1
88
6.2
69
3.2
86
1.0
13
1.0
70
1.0
35
1.0
46
1.0
78
1.0
49
1.0
46
1.0
20
1.0
20
1.0
32
1.0
67
3.0
99
1.0
00
S49
13C NMR of Compound 27 (175 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.2
4
17
.50
18
.18
20
.95
20
.95
21
.02
21
.31
23
.65
59
.47
62
.52
67
.60
68
.05
69
.56
69
.61
70
.42
70
.61
71
.56
76
.98
77
.16
77
.34
80
.47
98
.38
10
0.5
5
16
9.8
71
70
.09
17
0.3
21
70
.66
17
0.9
31
71
.89
S50
1H NMR of Compound 28 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.1
33
1.1
45
1.9
75
2.0
27
2.0
83
2.0
88
2.1
32
3.8
51
3.8
63
3.8
76
3.8
83
3.8
95
3.9
08
3.9
47
3.9
67
3.9
86
4.0
66
4.0
95
4.1
03
4.1
08
4.1
16
4.1
24
4.1
63
4.1
68
4.1
87
4.2
01
4.2
20
4.2
26
4.7
99
5.0
04
5.0
24
5.0
44
5.0
65
5.0
82
5.1
31
5.1
51
5.2
33
6.1
89
7.2
60
2.9
22
2.8
08
2.7
97
8.4
57
2.9
08
1.0
00
0.8
41
3.7
34
0.9
33
2.8
27
1.0
54
0.8
57
0.6
58
S51
13C NMR of Compound 28 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
17
.41
20
.79
20
.89
20
.91
20
.99
21
.07
21
.28
21
.33
23
.23
53
.50
62
.33
67
.77
67
.92
69
.48
70
.20
70
.54
92
.15
10
0.1
3
16
9.7
617
0.2
217
0.6
817
1.2
117
2.2
3
S52
1H NMR of Compound 29 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.1
23
1.1
36
1.8
86
1.9
41
2.0
11
2.0
68
2.1
53
3.6
28
3.6
48
3.6
68
3.8
25
3.8
38
3.8
44
3.8
57
3.8
69
3.8
73
3.8
85
3.8
88
3.8
93
3.8
97
3.9
10
3.9
14
4.0
44
4.0
52
4.0
69
4.0
77
4.4
29
4.4
35
4.7
46
5.0
01
5.0
16
5.0
20
5.0
28
5.0
33
5.0
39
5.0
43
5.0
50
5.0
56
5.0
67
5.0
85
5.0
91
5.1
05
5.1
09
5.1
28
5.5
72
5.5
93
5.6
01
5.6
06
5.6
12
7.2
60
7.3
40
7.3
54
7.3
62
7.3
75
7.3
84
3.1
19
3.0
60
3.0
47
6.2
87
3.2
64
3.1
99
1.0
38
3.0
66
1.0
54
1.0
11
1.0
02
8.5
06
2.0
00
10
.62
7
S53
13C NMR of Compound 29 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
17
.35
20
.77
20
.79
20
.91
21
.10
21
.20
23
.04
51
.91
51
.97
61
.53
67
.73
68
.98
69
.25
70
.07
70
.11
70
.15
70
.64
76
.91
77
.16
77
.41
79
.11
97
.03
97
.08
99
.74
12
8.2
512
8.3
212
8.9
412
8.9
612
9.1
413
5.3
513
5.4
013
5.5
613
5.6
0
16
9.4
116
9.6
117
0.1
617
0.5
217
0.7
817
0.8
9
S54
31P NMR of Compound 29 (162 MHz, CDCl3)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-2.2
96
S55
1H NMR of Compound 30 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
57
1.2
72
1.2
86
1.9
26
2.0
30
2.0
41
2.0
56
2.0
82
2.1
19
3.0
66
3.0
80
3.0
94
3.1
09
3.3
07
3.3
10
3.3
13
3.8
36
3.8
48
3.8
61
3.8
68
3.8
80
3.8
93
4.0
40
4.0
61
4.0
80
4.1
02
4.1
09
4.2
04
4.2
24
4.8
46
4.9
44
4.9
48
4.9
61
4.9
81
5.0
79
5.0
98
5.1
01
5.1
15
5.1
21
5.1
60
5.1
64
5.1
70
5.4
31
5.4
38
2.9
25
8.9
88
3.1
03
3.1
10
3.0
02
3.0
06
3.0
96
3.0
61
6.0
73
1.1
53
2.1
10
2.9
71
2.0
04
2.0
46
1.0
00
1.1
45
S56
13C NMR of Compound 30 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
9.4
117
.74
20
.55
20
.63
20
.65
20
.73
21
.37
22
.96
47
.37
48
.49
48
.66
48
.83
49
.00
49
.17
49
.34
49
.51
54
.59
54
.65
63
.21
68
.39
69
.86
70
.42
71
.18
71
.28
72
.13
80
.00
95
.20
95
.24
10
0.7
5
17
1.1
417
1.4
617
1.5
717
1.7
317
2.6
817
4.0
4
S57
31P NMR of Compound 30 (162 MHz, CDCl3)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-0.4
34
S58
1H NMR of Compound 32 (400 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
29
0.5
48
0.5
64
0.7
33
0.7
45
0.7
92
0.8
02
1.0
48
1.3
26
1.5
65
1.9
98
3.4
98
3.5
15
3.6
66
3.6
78
3.6
84
3.6
92
3.7
85
3.7
99
3.8
10
3.8
33
3.9
12
3.9
82
3.9
95
4.0
01
4.0
15
4.2
47
4.2
68
4.2
72
4.2
94
4.5
74
4.5
93
4.5
99
4.6
19
4.7
23
5.3
06
5.3
27
5.5
44
7.2
60
7.3
26
7.3
33
7.3
42
7.4
78
7.4
84
7.4
89
7.4
93
7.5
02
7.7
46
7.7
54
7.7
60
7.7
67
7.8
62
7.8
70
7.8
76
7.8
84
9.4
06
2.9
92
1.1
01
1.2
91
3.0
72
3.0
50
3.0
11
1.0
88
2.1
88
3.1
47
1.1
02
1.0
31
1.0
49
1.0
71
2.0
90
1.0
08
1.0
54
1.0
00
3.2
05
2.1
94
2.1
56
2.1
21
S59
13C NMR of Compound 32 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.4
3
16
.18
18
.01
21
.06
26
.16
27
.50
56
.98
64
.64
66
.54
67
.60
68
.94
74
.23
74
.25
75
.60
75
.93
76
.91
77
.16
77
.41
80
.81
97
.57
98
.34
10
2.2
31
09
.53
12
3.7
41
26
.56
12
8.2
51
29
.31
13
1.5
31
34
.52
13
7.2
2
17
0.0
4
S60
1H NMR of Compound 33 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
36
0.6
95
0.7
00
0.7
07
0.7
17
0.7
28
0.7
38
1.0
33
1.2
55
2.1
43
2.1
52
3.4
97
3.5
10
3.6
59
3.6
64
3.6
72
3.6
77
3.6
83
3.6
91
3.7
69
3.7
81
3.8
26
3.8
67
3.8
79
3.8
82
3.8
93
3.9
11
3.9
20
4.2
44
4.2
61
4.2
65
4.2
82
4.6
78
5.3
08
5.3
25
5.5
35
7.2
60
7.3
19
7.3
25
7.3
28
7.3
34
7.3
39
7.4
79
7.4
83
7.4
90
7.4
93
7.4
99
7.7
29
7.7
35
7.7
40
7.7
46
7.8
49
7.8
55
7.8
60
7.8
66
9.0
71
4.0
39
1.0
98
3.0
53
3.2
72
0.8
88
1.0
04
1.0
38
3.1
05
1.0
50
1.0
53
2.0
91
1.0
08
1.0
19
1.0
27
0.9
99
1.0
00
1.0
01
3.1
07
2.0
88
2.0
55
2.0
34
S61
13C NMR of Compound 33 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.4
3
16
.66
17
.99
25
.89
27
.83
57
.01
66
.37
66
.60
67
.54
68
.91
74
.52
74
.60
75
.89
76
.91
77
.16
77
.41
78
.22
80
.80
97
.96
98
.31
10
2.2
31
09
.18
12
3.7
01
26
.57
12
8.3
11
29
.28
13
1.5
51
34
.44
13
7.1
8
S62
1H NMR of Compound 34 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
22
0.7
82
0.7
95
0.9
83
1.2
74
3.6
80
3.6
85
3.6
96
3.7
63
3.7
74
4.5
45
4.6
61
4.6
70
4.6
75
4.7
41
5.3
08
5.3
25
5.4
66
5.5
44
5.5
57
5.6
92
7.1
78
7.1
93
7.2
60
7.2
80
7.2
95
7.3
12
7.3
29
7.3
45
7.3
93
7.4
09
7.4
25
7.4
62
7.4
77
7.4
92
7.5
05
7.5
18
7.7
31
7.7
37
7.7
42
7.7
48
7.8
60
7.8
66
7.8
71
7.8
77
7.9
04
7.9
07
7.9
10
7.9
18
7.9
20
7.9
24
7.9
84
7.9
99
8.0
30
8.0
45
9.0
01
5.0
28
3.0
00
3.1
76
1.0
31
1.0
77
2.0
54
1.0
25
2.0
22
1.0
52
1.0
31
1.0
22
1.0
45
1.0
15
3.0
26
1.0
24
0.9
90
1.0
03
1.9
52
1.0
00
1.0
04
1.1
36
2.0
77
6.9
41
2.1
97
5.0
80
1.0
86
2.0
49
2.0
95
3.8
92
2.0
75
2.0
50
S63
13C NMR of Compound 34 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.4
21
7.2
11
8.0
12
6.0
82
7.6
75
7.0
16
3.4
26
4.7
86
6.6
16
7.5
66
8.9
17
0.4
57
4.5
57
6.0
77
6.3
27
6.9
17
7.1
67
7.4
17
7.7
87
8.1
58
0.8
08
1.5
58
2.2
79
7.8
49
8.3
51
01
.99
10
4.0
41
09
.33
12
3.7
31
26
.42
12
8.1
61
28
.45
12
8.4
71
28
.48
12
8.5
91
29
.12
12
9.1
51
29
.16
12
9.5
71
29
.60
12
9.7
81
29
.99
13
0.0
11
30
.05
13
1.5
51
33
.17
13
3.2
91
33
.40
13
3.6
01
34
.47
13
7.1
41
65
.41
16
5.7
6
S64
1H NMR of Compound 35 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
-0.1
30
1.3
20
1.3
33
1.6
27
2.3
74
2.3
77
3.5
01
3.5
07
3.5
91
3.9
28
3.9
38
3.9
83
4.4
98
4.5
00
4.7
93
4.7
95
5.2
14
5.2
31
5.3
15
5.3
20
5.5
20
7.2
60
7.3
02
7.3
18
7.3
34
7.3
50
7.3
66
7.3
84
7.4
00
7.4
16
7.4
33
7.4
49
7.5
32
7.5
35
7.5
43
7.5
44
7.5
47
7.5
85
7.7
53
7.7
59
7.7
64
7.7
70
7.8
75
7.8
89
7.8
92
7.9
06
7.9
59
7.9
62
7.9
76
8.0
20
8.0
23
8.0
37
8.0
53
8.0
55
8.0
70
9.0
53
1.0
13
1.2
44
3.1
42
0.9
81
0.9
78
5.0
99
1.0
11
5.0
93
1.0
19
1.0
18
0.9
94
1.0
15
1.0
64
0.9
93
1.0
11
0.9
96
1.0
21
0.9
99
1.0
01
1.0
00
4.1
89
4.1
55
4.1
19
2.0
76
4.0
80
2.0
57
4.0
39
S65
13C NMR of Compound 35 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.3
8
17
.83
18
.00
55
.01
62
.81
63
.16
67
.41
68
.10
70
.36
70
.94
71
.43
71
.49
75
.29
76
.91
76
.98
77
.16
77
.41
80
.70
81
.04
83
.75
85
.09
97
.94
10
1.2
61
08
.05
12
8.4
81
28
.57
12
8.6
01
28
.67
12
8.7
01
28
.76
12
9.4
81
29
.59
12
9.8
61
30
.09
13
0.1
11
30
.18
13
1.5
61
33
.37
13
3.5
11
33
.87
13
4.0
41
34
.47
16
5.7
01
65
.81
16
6.1
31
67
.34
S66
1H NMR of Compound 36 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
0.0
01
1.2
20
1.2
32
1.9
72
1.9
95
2.0
45
2.0
60
2.0
62
2.0
67
2.0
81
2.0
88
2.1
16
3.5
60
3.5
72
3.6
24
3.6
39
3.6
44
4.0
45
4.0
50
4.0
69
4.0
74
4.2
06
4.2
19
4.2
29
4.2
44
4.2
51
4.2
60
4.2
70
4.2
74
4.5
27
4.5
46
4.5
47
4.5
66
4.6
68
4.6
71
4.9
15
4.9
18
4.9
24
4.9
44
4.9
62
4.9
98
5.0
02
5.0
10
5.0
13
5.1
74
5.1
79
5.1
83
5.1
90
5.2
08
5.2
25
5.2
64
5.3
60
6.1
32
6.1
45
7.2
60
8.9
23
1.0
89
1.0
61
3.1
47
2.9
52
2.9
21
2.9
48
8.7
57
6.0
38
3.0
63
1.0
00
1.0
26
2.0
45
0.9
99
1.0
56
1.0
24
4.0
13
0.9
70
0.9
78
1.0
01
0.9
69
1.0
32
2.0
13
0.9
76
1.0
03
1.0
00
0.9
49
S67
13C NMR of Compound 36 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
-1.2
61
7.8
51
8.1
42
0.7
82
0.7
92
0.8
22
0.9
12
0.9
22
0.9
52
0.9
82
1.3
12
3.6
05
9.4
56
2.4
56
2.5
16
7.5
66
8.5
06
9.4
16
9.8
07
0.7
57
1.4
87
2.1
77
5.1
07
6.4
27
6.9
17
7.1
67
7.4
18
0.4
08
0.7
98
1.6
19
8.3
71
00
.65
10
6.5
5
16
9.4
31
69
.84
17
0.0
41
70
.16
17
0.5
31
70
.56
17
0.9
11
71
.92
S68
1H NMR of Compound 37 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.1
98
1.2
10
1.9
53
2.0
35
2.0
41
2.0
55
2.0
63
2.0
68
2.0
86
2.1
01
3.6
10
3.6
30
3.9
90
4.0
08
4.0
34
4.0
58
4.0
66
4.0
74
4.0
86
4.0
94
4.0
99
4.1
21
4.1
28
4.1
44
4.1
68
4.1
92
4.2
06
4.2
39
4.2
43
4.2
46
4.2
50
4.2
60
4.7
44
4.8
92
4.9
75
4.9
77
4.9
85
4.9
88
5.0
31
5.0
50
5.0
61
5.0
68
5.0
81
5.0
87
5.1
04
5.2
30
5.2
36
5.2
48
5.3
47
5.3
50
5.3
54
5.3
58
6.2
16
6.2
34
7.2
60
2.9
08
2.9
34
5.8
54
8.9
77
5.9
84
3.1
72
1.0
35
0.9
80
0.9
41
5.1
04
2.0
50
0.9
21
1.0
33
0.6
89
1.0
57
1.9
64
0.9
73
0.9
15
1.0
09
1.0
00
0.8
46
S69
13C NMR of Compound 37 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
17
.65
20
.74
20
.77
20
.80
20
.82
20
.89
20
.91
21
.02
21
.31
23
.24
53
.53
62
.33
62
.44
67
.82
68
.14
69
.39
70
.30
70
.48
71
.76
75
.47
76
.36
76
.91
77
.16
77
.41
79
.27
80
.86
81
.35
92
.24
99
.92
10
6.5
7
16
9.3
01
69
.72
16
9.8
51
70
.06
17
0.2
21
70
.48
17
0.5
91
71
.07
17
1.9
3
S70
1H NMR of Compound 38 (500 MHz, CDCl3)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
04
1.2
17
1.8
90
1.9
66
1.9
97
2.0
49
2.0
61
2.0
91
2.1
14
2.1
27
3.6
14
3.6
34
3.6
46
3.8
83
3.8
86
3.8
91
3.9
07
3.9
11
4.2
03
4.2
17
4.2
39
4.2
50
4.2
55
4.2
65
4.7
02
4.7
05
4.8
98
4.9
01
4.9
87
4.9
90
4.9
98
5.0
00
5.0
09
5.0
16
5.0
26
5.0
32
5.0
35
5.0
48
5.0
58
5.0
67
5.0
79
5.0
87
5.0
98
5.2
57
5.5
02
5.5
21
5.6
13
5.6
18
7.2
60
7.3
33
7.3
40
7.3
47
7.3
54
7.3
67
7.3
76
2.9
14
2.8
53
2.9
49
3.0
41
9.2
64
3.2
71
3.1
92
2.9
61
2.0
18
0.9
97
1.9
15
0.9
95
1.0
38
2.1
08
0.9
55
0.9
24
0.9
95
7.9
82
1.0
08
1.0
00
0.9
23
0.9
42
9.4
89
S71
13C NMR of Compound 38 (125 MHz, CDCl3)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
17
.64
20
.77
20
.78
20
.80
20
.82
20
.92
21
.06
21
.22
23
.01
51
.82
51
.88
61
.55
62
.47
68
.27
69
.42
69
.45
70
.01
70
.05
70
.09
70
.22
71
.53
75
.56
76
.36
76
.91
77
.16
77
.41
78
.67
80
.91
81
.26
96
.97
97
.02
99
.68
10
6.6
21
28
.23
12
8.2
81
28
.91
12
8.9
31
29
.09
12
9.1
01
35
.37
13
5.4
21
35
.56
13
5.6
11
69
.19
16
9.4
01
69
.55
17
0.0
31
70
.19
17
0.3
71
70
.56
17
0.7
61
70
.90
S72
31P NMR of Compound 38 (202 MHz, CDCl3)
-80-60-40-2080 60 40 20 0 ppm
-2.4
22
S73
1H NMRof Compound 39 (700 MHz, CD3OD)
10.0 9.5 9.0 8.5 8.0 7.5 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 0.0 ppm
1.2
07
1.2
17
1.2
28
1.2
61
1.2
70
1.9
76
2.0
35
2.0
38
2.0
57
2.0
77
2.0
85
2.0
92
2.1
00
2.1
06
2.9
38
2.9
48
2.9
58
2.9
69
3.3
05
3.3
08
3.3
10
3.3
12
3.3
15
3.6
32
3.6
46
4.0
79
4.2
03
4.2
14
4.2
21
4.2
31
4.2
97
4.3
04
4.3
14
4.3
16
4.3
23
4.3
30
4.8
42
4.9
08
4.9
10
4.9
19
4.9
21
5.0
20
5.0
22
5.0
27
5.0
29
5.0
71
5.0
85
5.0
99
5.1
35
5.1
38
5.1
39
5.1
42
5.2
35
5.3
49
5.3
59
8.8
51
3.1
29
3.1
41
5.9
60
3.1
34
3.1
62
3.1
89
3.4
56
3.1
78
3.8
47
5.3
56
1.0
48
1.1
41
1.0
32
1.0
93
4.1
53
2.1
77
0.9
21
0.9
92
1.0
40
2.0
49
1.0
85
1.0
12
1.1
10
1.0
00
S74
13C NMR of Compound 39 (125 MHz, CD3OD)
200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm
20
.59
20
.60
20
.65
20
.76
20
.77
20
.93
21
.48
22
.97
47
.12
48
.49
48
.66
48
.83
49
.00
49
.17
49
.34
49
.51
54
.76
54
.82
63
.25
63
.57
68
.84
69
.79
71
.03
71
.33
71
.37
72
.93
77
.12
77
.87
79
.30
82
.46
82
.69
95
.11
95
.16
10
0.4
41
08
.01
17
1.1
41
71
.32
17
1.5
11
71
.60
17
1.7
41
71
.85
17
2.1
81
72
.76
17
4.0
3
S75
31P NMR of Compound 39 (202MHz, CD3OD)
-80-70-60-50-40-30-20-1080 70 60 50 40 30 20 10 0 ppm
-1.0
07
