synthesis of tri-functionalized mmp2 fret probes using a chemo ... · oriana et. al., supplementary...
TRANSCRIPT
Oriana et. al., supplementary information
S1/S23
Synthesis of tri-functionalized MMP2 FRET probes using a chemo-selective and late-stage modification of unprotected peptides
–supporting information–
Sean Oriana, Ye Cai, Jeffrey W. Bode, and Yoko Yamakoshi* Laboratorium für Organische Chemie, ETH-Zürich, Vladimir-Prelog-Weg 3, CH8093 Zürich, Switzerland
1. Synthesis of ketoacid derivative of methoxylcounarin and hydroxylamine Lys deriveative
Fig. S1 1H-NMR spectrum of compound 4 (in CDCl3, 400 MHz).
NH
Boc
4
ON
O
CF3
0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.5f1 (ppm)
-5000
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
60000
65000
70000
75000
80000
85000
9.0
6
2.8
8
2.1
6
2.1
4
2.0
1
1.0
0
1.4
41
.55
1.5
71
.59
1.6
11
.62
1.7
71
.79
1.8
11
.83
1.8
4
2.7
12
.72
2.7
32
.73
2.7
52
.75
3.1
43
.16
3.1
73
.19
4.5
6
5.3
0
6.0
76
.09
6.1
06
.11
7.2
6
6.06.16.2f1 (ppm)
0
50000
1.0
0
6.0
76
.09
6.1
06
.11
2.72.82.93.03.13.23.3f1 (ppm)
0
50000
2.1
4
2.0
1
2.7
12
.72
2.7
32
.73
2.7
52
.75
3.1
43
.16
3.1
73
.19
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2017
Oriana et. al., supplementary information
S2/S23
Fig. S2 13C-NMR spectrum of compound 4 (in CDCl3, 100 MHz).
Fig. S3 19F-NMR spectrum of compound 4 (in CDCl3, 377 MHz).
2030405060708090100110120130140150160170f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
15
.42
22
.42
28
.06
28
.53
29
.59
40
.06
66
.00
77
.16
79
.45
11
8.9
21
21
.71
12
4.5
1
15
6.1
0
16
3.6
6
16
8.9
5
-114-112-110-108-106-104-102-100-98-96-94-92-90-88-86-84-82-80-78-76-74-72-70-68-66f1 (ppm)
-4000
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
34000
36000
13-17-02-borianSample SO-D-157repurifiedgroup bode -
78
.95
-7
5.9
4
Oriana et. al., supplementary information
S3/S23
Fig. S4 IR spectrum of compound 4
Fig. S5 1H NMR spectrum of compound 2 (in CDCl3, 600 MHz).
NH
ON
O
CF3O
OMeO O 2
-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
2.5
1
2.5
0
2.2
2
2.2
1
3.1
9
1.0
4
0.9
91
.08
1.1
6
0.6
80
.90
1.5
51
.73
1.7
51
.78
1.8
61
.89
1.9
1
2.7
62
.77
2.7
9
3.4
93
.50
3.5
13
.52
3.9
2
6.0
96
.10
6.1
1
6.8
76
.87
6.9
46
.94
6.9
56
.95
7.2
6
7.5
87
.59
8.8
08
.81
8.8
3
Oriana et. al., supplementary information
S4/S23
Fig. S6 13C NMR spectrum of compound 2 (in CDCl3, 150 MHz).
Fig. S7 19F NMR spectrum of compound 2 (in CDCl3, 377 MHz).
0102030405060708090100110120130140150160170f1 (ppm)
0
500
1000
1500
2000
2500
3000
22
.85
28
.27
29
.20
39
.49
56
.40
77
.16
77
.16
93
.20
93
.44
93
.67
93
.90
10
0.6
9
11
2.7
9
11
4.4
5
13
1.3
31
31
.47
14
8.7
51
49
.00
15
7.0
3
16
2.2
91
62
.60
16
3.8
81
65
.26
16
9.1
1
-84-83-82-81-80-79-78-77-76-75-74-73-72-71-70-69f1 (ppm)
-40000
-30000
-20000
-10000
0
10000
20000
30000
40000
50000
60000
-7
8.9
0
Oriana et. al., supplementary information
S5/S23
Fig. S8 IR spectrum of compound 2
Fig. S9 1H-NMR spectrum of compound 7 (in CDCl3, 400 MHz).
HN OFmoc
HN
O
Boc7
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)
-20000
-10000
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
110000
120000
130000
140000
150000
160000
170000
180000
190000
200000
210000
22000015-23-10-borianSample SO-32-14group bodePRO CDCl3 /opt/v borian 15
1.9
89
.11
2.1
1
2.0
6
0.9
2
1.9
0
1.0
6
3.1
8
0.6
5
1.8
3
2.9
4
0.9
8
2.0
8
2.0
9
2.0
4
2.0
4
1.2
41
.26
1.2
61
.28
1.3
51
.35
1.3
71
.38
1.3
91
.39
1.4
31
.49
1.5
01
.52
1.5
31
.66
1.6
71
.69
1.7
11
.72
1.7
31
.74
1.7
51
.84
1.8
51
.86
1.8
81
.89
1.9
01
.91
2.0
02
.04
3.0
53
.09
3.1
13
.12
3.1
4
4.0
54
.06
4.0
84
.11
4.1
34
.21
4.2
34
.24
4.3
54
.36
4.3
84
.40
4.4
04
.42
4.4
34
.45
4.4
94
.56
4.6
14
.64
4.6
55
.05
5.0
75
.25
5.2
55
.28
5.2
85
.31
5.3
65
.36
5.8
65
.88
5.8
95
.90
5.9
25
.93
5.9
45
.96
7.2
6 C
DC
l37
.26
7.2
97
.30
7.3
07
.31
7.3
27
.32
7.3
37
.33
7.3
47
.38
7.4
07
.42
7.5
67
.57
7.5
97
.60
7.6
17
.62
7.7
67
.77
Oriana et. al., supplementary information
S6/S23
Fig. S10 13C-NMR spectrum of compound 7 (in CDCl3, 100 MHz).
Fig. S11 IR spectrum of compound 7.
-100102030405060708090100110120130140150160170180190200210f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
550015-23-10-borianSample SO-32-14group bodeCAR CDCl3 /opt/v borian 15
22
.50
28
.56
29
.74
32
.34
40
.20
47
.32
53
.92
66
.14
67
.16
76
.84
77
.16
77
.16
77
.36
77
.48
79
.34
11
9.1
81
20
.10
12
0.1
2
12
5.2
31
27
.20
12
7.8
41
31
.63
14
1.4
41
43
.89
14
4.0
4
15
6.1
11
56
.19
17
2.2
7
Oriana et. al., supplementary information
S7/S23
Fig. S12 1H-NMR spectrum of compound 8 (in CDCl3, 400 MHz).
Fig. S13 13C-NMR spectrum of compound 8 (in CDCl3, 100 MHz).
HN OFmoc
NH2
O
8
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
5000054-03-02-borianSample SO-33-15-purifiedgroup bodePRO CDCl3 /opt/v borian 54
0.8
1
1.4
4
0.7
3
0.0
8
0.5
0
0.9
7
0.7
2
0.7
3
0.2
7
0.3
7
0.0
9
1.6
7
0.7
3
0.7
2
1.0
0
1.3
51
.37
1.3
81
.40
1.4
21
.43
1.4
31
.45
1.6
21
.65
1.6
61
.67
1.6
91
.71
1.8
11
.83
2.8
82
.91
2.9
32
.94
2.9
5
3.4
8
4.0
34
.16
4.1
84
.19
4.3
04
.31
4.3
34
.34
4.3
44
.36
4.3
64
.38
4.4
04
.53
4.5
94
.61
5.2
05
.23
5.2
75
.31
5.6
25
.64
5.8
25
.84
5.8
55
.86
5.8
85
.88
5.8
9
6.2
7
7.2
67
.26
7.2
67
.27
7.2
87
.29
7.3
07
.35
7.3
57
.37
7.3
77
.38
7.3
97
.49
7.5
17
.51
7.5
27
.56
7.5
87
.72
7.7
47
.99
-100102030405060708090100110120130140150160170180190200210f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
54-03-02-borianSample SO-33-15-purifiedgroup bodeCAR CDCl3 /opt/v borian 54
22
.26
27
.01
31
.96
39
.61
47
.21
53
.80
66
.30
67
.30
76
.84
77
.16
77
.16
77
.36
77
.48
11
9.2
21
20
.13
12
5.2
41
27
.23
12
7.8
81
31
.51
14
1.4
1
14
3.8
8
15
6.3
4
17
2.1
7
Oriana et. al., supplementary information
S8/S23
Fig. S14 IR spectrum of compound 8.
Fig. S15 1H-NMR spectrum of compound 9 (in CDCl3, 400 MHz).
HN OFmoc
HN
O
OBz9
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
2000045-21-12-borianSample SO34-11group bodePRO CDCl3 /opt/v borian 45
6.1
5
1.8
4
1.0
7
3.0
4
1.9
8
1.7
1
0.9
9
0.8
3
1.0
0
2.1
12
.17
2.1
13
.21
2.1
4
2.0
3
7.2
6
Oriana et. al., supplementary information
S9/S23
Fig. S16 13C-NMR spectrum of compound 9 (in CDCl3, 100 MHz).
Fig. S17 IR spectrum of compound 9
102030405060708090100110120130140150160170f1 (ppm)
-2000
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
22000
24000
26000
28000
30000
32000
45-21-12-borianSample SO34-11group bodeCAR CDCl3 /opt/v borian 45
22
.77
26
.81
32
.39
47
.16
52
.13
53
.81
65
.97
66
.97
77
.16
11
8.9
41
19
.97
12
5.0
81
27
.05
12
7.6
91
28
.36
12
8.5
31
29
.31
13
1.5
21
33
.35
14
1.2
81
43
.75
14
3.8
9
15
5.9
7
16
6.8
4
17
2.1
5
Oriana et. al., supplementary information
S10/S23
Fig. S18 1H-NMR spectrum of compound 10 (in CDCl3, 400 MHz).
HN OFmoc
N
O
OBzBoc10
1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
11-25-09-borianSample SO-NB-B-77-Lys-Boc-OBzgroup bodePRO CDCl3 /opt/v borian 11
0.2
5
5.8
2
1.7
7
0.4
5
0.1
6
1.0
2
0.1
6
0.5
2
1.4
6
1.0
3
2.5
8
0.4
8
1.0
01
.02
1.0
31
.46
1.0
1
1.0
0
1.2
41
.25
1.2
71
.30
1.4
31
.44
1.4
61
.49
1.5
01
.51
1.5
11
.52
1.5
31
.53
1.5
41
.60
1.6
11
.62
1.6
31
.64
1.6
61
.68
1.7
01
.72
1.7
31
.74
1.7
51
.78
1.8
01
.88
1.8
91
.90
1.9
11
.93
1.9
42
.04
2.0
53
.69
3.7
13
.73
4.0
94
.11
4.1
34
.20
4.2
24
.24
4.3
44
.36
4.3
74
.39
4.4
14
.42
4.4
34
.44
4.6
44
.65
5.2
35
.23
5.2
65
.26
5.2
85
.30
5.3
15
.35
5.3
55
.42
5.4
45
.85
5.8
75
.88
5.8
95
.91
5.9
25
.94
7.2
67
.28
7.2
97
.30
7.3
17
.32
7.3
37
.37
7.3
97
.41
7.4
57
.45
7.4
67
.48
7.4
87
.58
7.5
97
.59
7.6
07
.60
7.6
17
.61
7.6
27
.62
7.6
27
.63
7.7
57
.77
8.0
68
.06
8.0
78
.08
8.0
88
.08
8.0
9
Oriana et. al., supplementary information
S11/S23
Fig. S19 13C-NMR spectrum of compound 10 (in CDCl3, 100 MHz).
Fig. S20 IR spectrum of compound 10.
2030405060708090100110120130140150160170f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
280011-25-09-borianSample SO-NB-B-77-Lys-Boc-OBzgroup bodeCAR CDCl3 /opt/v borian 11
22
.39
26
.94
28
.21
32
.22
47
.24
50
.26
53
.52
53
.90
66
.06
67
.10
76
.84
77
.16
77
.16
77
.48
82
.47
11
9.0
21
20
.03
12
5.1
91
27
.14
12
7.6
71
27
.76
12
8.7
11
29
.93
13
1.6
01
33
.91
14
1.3
61
43
.84
14
3.9
8
15
4.9
11
56
.01
16
4.7
8
17
2.1
8
Oriana et. al., supplementary information
S12/S23
Fig. S21 1H-NMR spectrum of compound 1 (in CDCl3, 400 MHz).
HN OHFmoc
N
O
OBzBoc1
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
37-01-10-borianSample SO-NB-B-80group bode
0.3
1
6.6
8
1.6
0
0.4
9
0.1
0
1.5
1
2.7
1
0.6
6
1.5
3
0.1
0
0.6
0
1.0
81
.04
1.0
71
.36
1.0
2
1.0
0
0.0
7
1.2
61
.28
1.3
41
.42
1.4
31
.44
1.4
81
.50
1.5
21
.60
1.6
61
.68
1.7
51
.79
1.9
22
.09
2.1
7
2.9
7
3.4
9
3.6
83
.70
3.7
23
.72
4.0
44
.19
4.2
14
.22
4.3
74
.37
4.3
84
.38
4.3
94
.47
5.3
0
5.4
65
.48
5.6
5
7.2
6 C
DC
l37
.26
7.2
77
.29
7.3
17
.36
7.3
87
.40
7.4
37
.44
7.4
57
.47
7.4
77
.58
7.5
87
.58
7.5
97
.60
7.6
07
.61
7.6
17
.62
7.7
37
.75
8.0
48
.04
8.0
58
.06
8.0
68
.06
Oriana et. al., supplementary information
S13/S23
Fig. S22 Expanded 13C-NMR spectrum of compound 1 (in CDCl3, 100 MHz).
Fig. S23 IR spectrum of compound 1
2030405060708090100110120130140150160170f1 (ppm)
-20
0
20
40
60
80
100
120
140
160
180
200
220
240
260
280
300
320
37-01-10-borianSample SO-NB-B-80group bode 2
2.4
0
26
.96
28
.28
31
.87
47
.30
50
.28
50
.97
53
.84
67
.28
76
.84
77
.16
77
.16
77
.36
77
.48
82
.72
12
0.1
0
12
5.2
71
27
.23
12
7.6
91
27
.85
12
8.7
91
30
.02
13
4.0
1
14
1.4
31
43
.87
14
4.0
0
15
5.0
9
16
4.9
1
Oriana et. al., supplementary information
S14/S23
2. Test KAHA reaction of 4 and 9
Fig. S24 LC trace of test KAHA ligation reaction mixture (23.5 hours).
(a)
(b)
Fig. S25 ESI mass spectra of peaks with retention time at (a) 4.4 (m/z 608, corresponding to [M+H] of 11), (b) 4.9 (m/z 529, corresponding to [M+H] of 9.
NH
Boc
4
ON
O
CF3
+
HN OFmoc
NH
O
O
HN OFmoc
O
9
NHBzO
BocHN
11
tBuOH/0.1M Oxalic buffer (pH 1.25)1:1, 50οC, 23.5 h
Oriana et. al., supplementary information
S15/S23
3. Solid phase peptide synthesis (SPPS)
Fmoc-GABA-Gly-Pro-Leu-Gly-Val-Arg(Pbf)-Gly-Cys(Trt)-Ala-NH2 on MBHA rink amide
resin12.
Fig. S26 MALDI spectrum of crude compound 12 (Matrix: HCCA)
Fmoc-Lys(OBz)-GABA-Gly-Pro-Leu-Gly-Val-Arg(Pbf)-Gly-Cys(Trt)-Ala-NH2 on MBHA rink
amide resin 13.
Fig. S27 MALDI spectrum of crude compound 13 (Matrix: HCCA)
Gly ValPro Leu Gly Arg GlyHN N
H
HN
OS
O
O
Trt
rink amide MBHA resin12
HNN
HFmoc
O
GABA
Gly ValPro Leu Gly Arg GlyHN N
H
HN
OS
O
O
Trt13
HNN
H O
OHNFmoc
NBzO
Boc
Oriana et. al., supplementary information
S16/S23
MPEG12-Fmoc-Lys(OBz)Boc-GABA-Gly-Pro-Leu-Gly-Val-Arg(Pbf)-Gly-Cys(Trt)-Ala-NH2 on
MBHA rink amide resin 14.
Fig. S28 MALDI spectrum of crude compound 14 (Matrix: HCCA)
MPEG12-Fmoc-Lys(OBz)-GABA-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-Ala-NH2 15.
Fig. S29 MALDI-TOF-MS spectrum of compound 15 purified by HPLC (Matrix: HCCA)
Gly ValPro Leu Gly Arg GlyHN N
H
HN
OS
O
O
Trt14
HNN
H O
OHN
NBzO
Boc
PEG
O
Gly ValPro Leu Gly Arg GlyHN N
HNH2
OHS
O
O15
HNN
H O
OHN
NHBzO
PEG
O
Oriana et. al., supplementary information
S17/S23
4. Peptide modification with FRET acceptor, donor, and polymer
MPEG12-Fmoc-Lys(MC)-GABA-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys-Ala-NH2 16.
Fig. S30 MALDI spectrum of HPLC purified compound 16 (Matrix: HCCA)
MPEG12-Fmoc-Lys(MC)-GABA-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys(DNP)-Ala-NH2 Probe A
17.
16
HN N
HNH2
O
O
O
HNN
H O
OHN
NH
PEG
O
O
HN
O
OO
MeO
Pro GlyGly ValLeuGly Arg
HS
Gly ValPro Leu Gly Arg GlyHN N
HNH2
O
S
O
O
17N OO
NH
NO2O2N
HNN
H O
OHN
NH
PEG
O
O
HN
O
OO
MeO
Oriana et. al., supplementary information
S18/S23
Fig. S31 Analytical HPLC trace of purified compound 17 (probe A) at 220 nm.
Fig. S32 High resolution MALDI spectrum of purified compound 17 (probe A).
MPEG12-Fmoc-Lys(MC)-GABA-Gly-Val-Arg-Leu-Gly-Pro-Gly-Cys(DNP)-Ala-NH2 Probe B.
Fig. S33 Analytical HPLC trace of purified probe B at 220 nm.
Fig. S34 High resolution MALDI spectrum of purified probe B.
(MALDI-TOF m/z calcd. 2219.0788 for C98H156N21O35S, found 2219. 0797 ([M+H]+)
Oriana et. al., supplementary information
S19/S23
5. Enzyme test using MMP2 Probe A MPEG12-Fmoc-Lys(MC)-GABA-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys(DNP)-Ala-NH2 Probe B MPEG12-Fmoc-Lys(MC)-GABA-Gly-Val-Arg-Leu-Gly-Pro-Gly-Cys(DNP)-Ala-NH2
Table S1. Reaction set up for Enzyme cleavage experiment run Substrate MMP2 Inhibitor Reaction time [min]
(MS spectra) Reaction time [min] (fluorescent spectra)
R1 probe A – – 0, 120, 200 min (Fig. S35a-c) 0, 240 min (Fig. S42) R2 probe A + – 0, 120, 200 min (Fig. S36a-c) 240 min (Fig. S43) R3 probe A + + 0, 120, 200 min (Fig. S37a-c) 240 min (Fig. S44) R4 probe B – – 0, 120, 200 min (Fig. S38a-c) 0, 240 min (Fig. S45) R5 probe B + – 0, 120, 200 min (Fig. S39a-c) 240 min (Fig. S46) R6 probe B + + 0, 120, 200 min (Fig. S40a-c) 240 min (Fig. S47)
(a)
(b)
(c)
Fig. S35 MALDI-TOF-MS spectra of R1 (a) 0 min, (b) 120 min, (c) 200 min.
Oriana et. al., supplementary information
S20/S23
(a)
(b)
(c)
Fig. S36 MALDI-TOF-MS spectra of R2 (a) 0 min, (b) 120 min, (c) 200 min.
(a)
(b)
(c)
Fig. S37 MALDI-TOF-MS spectra of R3 (a) 0 min, (b) 120 min, (c) 200 min.
Oriana et. al., supplementary information
S21/S23
(a)
(b)
(c)
Fig. S38 MALDI-TOF-MS spectra of R4 (a) 0 min, (b) 120 min, (c) 200 min.
(a)
(b)
(c)
Fig. S39 MALDI-TOF-MS spectra of R5 (a) 0 min, (b) 120 min, (c) 200 min.
Oriana et. al., supplementary information
S22/S23
(a)
(b)
(c)
Fig. S40 MALDI-TOF-MS spectra of R6 (a) 0 min, (b) 120 min, (c) 200 min.
Fig. S41 Overlaid Fluorescence Spectra of R1-R6 (4 hr)
Oriana et. al., supplementary information
S23/S23
(a) (b)
Fig. S42 Fluorescence Spectra of R1 (a) 0 hr (b) 4 hrs.
Fig. S43 Fluorescence Spectra of R2 (4 hr).
Fig. S44 Fluorescence Spectra of R3 (4 hr).
(a) (b)
Fig. S45 Fluorescence Spectra of R4 (a) 0 hr (b) 4 hrs.
Fig. S46 Fluorescence Spectra of R5 (4 hr). Fig. S47 Fluorescence Spectra of R6 (4 hr).