electronic supplementary information revised s1-s17 · cameron c. ellis, nishat tasnim, supriyo...
TRANSCRIPT
ElectronicSupplementaryInformation
Synthesis and Characterization of a Photocleavable Collagen-Like Peptide Alfredo Ornelas, Kaitlyn N. Williams, Kevin A. Hatch, Aurelio Paez, Angela C. Aguilar, Cameron C. Ellis, Nishat Tasnim, Supriyo Ray, Carl W. Dirk, Thomas Boland, Binata Joddar, Chunqiang Li,* and Katja Michael* Table of content Collagen-like peptide 1 HR ESI-TOF-MS ………………………………………………………………… S3 Comparison of actual MS and isotopic simulation …………………………….. S4 FPLC profile ……………………………………………………………………… S5 Amino acid building block 6 HR ESI-TOF-MS ………………………………………………………………… S6 1H NMR spectrum ……………………………………………………………….. S7 13NMR spectrum …………………………………………………………………. S8 Peptide building block 7 HR ESI-TOF-MS …………………………………………………………………. S9 Comparison of actual MS and isotopic simulation …………………………….. S10 FPLC profile ……………………………………………………………………… S11 N-Fmoc-glycyl-5-bromo-7-nitroindoline (13) HR ESI-TOF-MS …………………………………………………………………. S12 1H NMR spectrum ………………………………………………………………… S13 13NMR spectrum ………………………………………………………………….. S14 Allyl N-(Fmoc-glycyl)-5-carboxylate-7-nitroindoline (16) HR ESI-TOF-MS …………………………………………………………………. S15 1H NMR spectrum ………………………………………………………………… S16 13NMR spectrum ………………………………………………………………….. S17
S1
Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2018
Liquid Chromatography-Mass Spectrometry (LC-MS) and MS/MS analysis of the photodecomposition products of peptide 1 obtained upon illumination with UV light at 350 nm in water TotalIonCount(TIC)chromatogramofthephotodecomositionproducts ofpeptide1.................................................................................................................................... S18 MSofpeptide9elutingat40.94min…………………………………………………………….S19 MSofpeptide9elutingat41.38min…………………………………………………………….S20 MSofpeptide17elutingat41.92min………………………………………………………….S21 MSofpeptides9and17elutingat41.96min……………………………………………….S22 MSofpeptide18elutingat45.47min………………………………………………………... S23 MS/MSofpeptide18…………………………………………………………………………………. S24 MSofpeptide19elutingat45.89min………………………………………………………... S25 MS/MSofpeptide19…………………………………………………………………………………. S26 MSofpeptide11elutingat50.98min………………………………………………………... S27 MSofpeptides10and12elutingat51.37min…………………………………………….S28 MSofpeptides10and12elutingat51.47min…………………………………………….S29 MSofpeptide17elutingat51.76min………………………………………………………….S30 MSofpeptide20elutingat52.13min………………………………………………………….S31 MS/MSofpeptide20…………………………………………………………………………………. S32
S2
HRESITOFMSofpeptide1. m/z [M+2H]2+ calcd. 1685.7205 (most abundant species), obs. 1685.7175; [M+3H]3+ calcd. 1123.8153 (monoisotopic species), obs. 1123.8211.
S3
H-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-NH2
N
O2NHN
ON
ON
HO
ONH
ON
ONO
O HN
ON
ON
HO
ONH
ON
ON
ON
O2NHN
ON
ON
HO
ONH
ON
ON
O
5-carboxy-7-nitroindolinylmoiety
5-carboxy-7-nitroindolinylmoiety
O
1
Actual (top) and simulated mass spectrum (bottom) of peptide 1 m/z [M+2H]2+ (most abundant peak) calcd. 1685.7205, obs. 1685.7175.
Actual (top) and simulated mass spectrum (bottom) of peptide 1 (m/z)[M+3H]3+ calcd. 1123.8061 (monoisotopic species), obs. 1123.8211.
S4
ReversedPhaseFPLCprofileofpeptide1.Stationaryphase:15 µm diameter polystyrene/divinyl benzene beads; Solvent A: 2% CH3CN and 0.1% TFA in water, solvent B: 85% CH3CN and0.1%TFAinwater;Gradient(green):0-90%B.Detectionat250nm(blue),350nm(red),and430nm(pink).
S5
H-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-Ind-Pro-Pro-Gly-Hyp-Pro-Gly-NH2
N
O2NHN
ON
ON
HO
ONH
ON
ONO
O HN
ON
ON
HO
ONH
ON
ON
ON
O2NHN
ON
ON
HO
ONH
ON
ON
O
5-carboxy-7-nitroindolinylmoiety
5-carboxy-7-nitroindolinylmoiety
O
1
HR ESI TOF MS of amino acid building block 6, m/z [M-H]- calcd. 486.1301; obs. 486.1327.
S6
NNO2
HOOC
O
NHFmoc6
1HNMRspectrumofcompound6.
S7
NNO2
HOOC
O
NHFmoc6
13CNMRspectrumofcompound6.
S8
NNO2
HOOC
O
NHFmoc6
HRESITOFMSofpeptide7,m/z [M+H]+ calcd.: 1005.4358; obs. 1005.4334; [M+Na]+ calcd. 1027.4178; obs. 1027.4150.
S9
GlyProHypGlyProProFmoctBu
7
COOH
NNO2
Actual (top) and simulated mass spectrum (bottom) of peptide 7, m/z [M+H]+ calcd.: 1005.4358; obs. 1005.4334.
S10
ReversedPhaseFPLCprofileofpeptide7.Stationaryphase:15 µm diameter polystyrene/divinyl benzene beads; Solvent A: 2% CH3CN and 0.1% TFA in water, solvent B: 85% CH3CN and0.1%TFAinwater;Gradient(green):0-90%B.Detectionat250nm(blue),350nm(red),and430nm(pink).
S11
GlyProHypGlyProProFmoctBu
7
COOH
NNO2
HR ESI TOF MS of compound 13; m/z [M+NH4]+ calcd. 539.0930 and 541.0913, obs. 539.0904 and 541.0937; [M+Na]+ calcd. 544.0484 and 546.0467, obs. 544.0489 and 546.0463.
S12
NNO2
Br
O
NHFmoc13
1H NMR spectrum of compound 13.
S13
NNO2
Br
O
NHFmoc13
13CNMRspectrumofcompound13.
S14
NNO2
Br
O
NHFmoc13
HR-ESI-TOF-MS of compound 16; m/z [M+Na]+ calcd. 550.1590, obs. 550.1597; [M+K]+ calcd. 566.1330, obs. 566.1333.
S15
N
AllOOC
NO2
16
O
NHFmoc
1HNMRspectrumofcompound16.
S16
N
AllOOC
NO2
16
O
NHFmoc
13CNMRspectrumofcompound16.
S17
N
AllOOC
NO2
16
O
NHFmoc
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 0.00 - 60.00 SM: 3B
0 5 10 15 20 25 30 35 40 45 50 55 60Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
40.98
41.33
45.8442.30
51.5142.97 51.59
51.8153.1151.04
45.48
47.71
49.28
40.3856.08
8.598.31 33.10 56.419.166.58 34.06 37.584.85 27.7426.78 28.8719.793.12 24.1117.9715.9513.87
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
Total Ion Count (TIC)-based Nano-UHPLC chromatogram of the peptide fragments obtained after irradiation of the full length peptide 1 with 350 nm light in water.
S18
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 9 eluting at 40.94 min: m/z [M+H]
+ calcd. 537.2673, obs. 537.2678.
S19
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #12892 RT: 40.94 AV: 1 NL: 1.49E10T: FTMS + p NSI Full ms [300.0000-1600.0000]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
537.2678
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 9 eluting at 41.38 min: m/z [M+H]
+ calcd. 537.2673, obs. 537.2676.
S20
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #13013 RT: 41.38 AV: 1 NL: 3.92E9T: FTMS + p NSI Full lock ms [300.0000-1600.0000]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
537.2676
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 17 eluting at 41.92 min: m/z [M+NH4]
+ calcd. 744.3317, obs.
744.3313; m/z [M+H+NH4]2+
calcd. 372.6697, obs. 372.6693.
S21
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #13248 RT: 41.92 AV: 1 NL: 6.23E8T: FTMS + p NSI Full lock ms [300.0000-1600.0000]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
762.3417
372.6693
744.3313381.6744
780.3522535.2512 705.3204344.1585 391.6424
Peptide 17
unidentified
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptides 9 and 17 eluting at 41.96 min: 9, m/z [M+H]
+ calcd. 537.2673, obs.
537.2663; 17, m/z [M+NH4]+ calcd. 744.3317, obs. 744.3303; m/z [M+H+NH4]
2+ calcd.
372.6697, obs. 372.6690.
S22
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #13263 RT: 41.96 AV: 1 NL: 7.03E8T: FTMS + p NSI Full lock ms [300.0000-1600.0000]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
372.6690z=2
762.3408z=2537.2663
z=1
381.6738z=2
744.3303z=1
780.3510z=?
569.2560z=1
391.6419z=2
705.3196z=1
344.1581z=2
Peptide 9
Peptide 17
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 18 eluting at 45.47 min: m/z [M+H]+ calcd. 710.3262, obs. 710.3259; m/z [M+2H]2+ calcd. 355.6670, obs. 355.6664.
S23
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #14889 RT: 45.47 AV: 1 NL: 1.91E9T: FTMS + p NSI Full lock ms [300.0000-1600.0000]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
710.3259z=2
355.6664z=2
579.2774z=1
MS/MS spectrum of peptide 18: N-terminal nitrosoindoline fragment, C9H7N2O2, m/z [M]+
calcd. 175.0508, obs. 175.0501; protonated C-terminal fragment, C24H38N7O7, m/z [M+H]+ calcd. 536.2833, obs. 536.2835.
S24
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
CLP_Irradiated_Alfredo_DrMichael_171208195110 #14566-16057 RT: 45.31-47.11 AV: 2 RF: 6.00,3 NL: 1.75E7F: FTMS + c NSI d Full ms2 710.3259
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
175.0501z=1
536.2825z=1
272.1025z=1
286.1380z=?
244.1077z=1
439.2294z=1
159.0552z=? 342.1774
z=? 519.2554z=?
559.2510z=1
98.0604z=? 710.2731
z=1866.3157
z=11117.4409
z=?
N
NN
NO
O
NHO
HO
O O
O
NH2
HN
O
NH
NO
18
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 19 eluting at 45.89 min: m/z [M+H]+ calcd. 711.3102, obs. 711.3093; m/z [M+H]2+ calcd. 356.1590, obs. 356.1580.
S25
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #15087 RT: 45.89 AV: 1 NL: 2.82E9T: FTMS + p NSI Full ms [300.0000-1600.0000]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
711.3093z=2
356.1580z=2
MS/MS spectrum of peptide 19: N-terminal nitrosoindoline fragment, C9H7N2O2, m/z [M]+
calcd. 175.0508, obs. 175.0501; protonated C-terminal fragment, C24H37N6O8, m/z [M+H]+ calcd. 537.2673, obs. 537.2672.
S26
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
CLP_Irradiated_Alfredo_DrMichael_171208195110 #14559-16129 RT: 45.67-46.60 AV: 3 RF: 6.00,3 NL: 2.51E7F: FTMS + c NSI d Full ms2 711.3093
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
175.0501z=1
272.1025z=1 537.2672
z=1
244.1077z=1
440.2130z=1
343.1603z=?
155.0813z=?
519.2557z=?
713.2845z=2
598.2021z=?
851.3367z=?
1102.4276z=?
1036.3981z=?
N
NN
NO
O
NHO
HO
O O
O
OH
HN
O
NH
NO
19
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 11 eluting at 50.98 min: m/z [M+H]+ calcd. 708.3105, obs. 708.3095.
S27
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #17473 RT: 50.98 AV: 1 NL: 2.56E9T: FTMS + p NSI Full ms [300.0000-1600.0000]
675 680 685 690 695 700 705 710 715 720 725 730 735 740 745 750 755 760 765m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
708.3095z=1
709.3121z=1
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptides 10 and 12 eluting at 51.37 min: 10, m/z [M+H]+ calcd. 709.2946, obs. 709.2946; 12, m/z [M+H]+ calcd. 726.3211, obs. 726.3209.
S28
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #17665 RT: 51.37 AV: 1 NL: 1.30E9T: FTMS + p NSI Full lock ms [300.0000-1600.0000]
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
726.3209z=1
709.2946z=1
Peptide 10
Peptide 12
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptides 10 and 12 eluting at 51.47 min: 10, m/z [M+H]+ calcd. 709.2946, obs. 709.2943; 12, m/z [M+H]+ calcd. 726.3211, obs. 726.3203.
S29
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
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Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #17711 RT: 51.47 AV: 1 NL: 2.45E9T: FTMS + p NSI Full ms [300.0000-1600.0000]
350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400m/z
0
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Re
lative
Ab
un
da
nce
709.2943z=1
726.3203z=1
Peptide 10
Peptide 12
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 17 eluting at 51.76 min: m/z [M+H]+ calcd. 727.3051, obs. 727.3049; m/z [M+NH4]+ calcd. 744.3317, obs. 744.3312.
S30
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #17827 RT: 51.76 AV: 1 NL: 1.92E9T: FTMS + p NSI Full ms [300.0000-1600.0000]
350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400m/z
0
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Re
lative
Ab
un
da
nce
727.3049z=1
744.3312z=?
Top: TIC chromatogram of photolysis products of peptide 1, irradiated with near UV light (350 nm) in water. Bottom: HRMS of peptide 20 eluting at 52.13 min: m/z [M+H]+ calcd. 725.2895, obs. 725.2885; m/z [M+NH4]+ calcd. 742.3160, obs. 742.3148.
S31
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
RT: 35.00 - 60.00 SM: 3B
35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
40.9840.91
41.07
41.11
41.22
41.3345.8442.30
51.5142.3941.4651.81 53.1143.07 51.04
45.4843.1447.7147.3843.91 44.26 49.28 49.6648.7640.38 46.3840.18 56.0853.68 56.4154.12 55.72 59.9858.3139.3837.5837.1736.39
NL: 2.78E10TIC F: FTMS + p NSI Full ms [300.0000-1600.0000] MS CLP_Irradiated_Alfredo_DrMichael_171208195110
CLP_Irradiated_Alfredo_DrMichael_171208195110 #17996 RT: 52.13 AV: 1 NL: 8.76E8T: FTMS + p NSI Full lock ms [300.0000-1600.0000]
400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450m/z
0
10
20
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Re
lativ
e A
bu
nd
an
ce
725.2885z=1
742.3148z=1
MS/MS spectrum of peptide 20: N-terminal nitroindole fragment, C9H5N2O3, m/z [M]+ calcd. 189.0300 , obs. 189.0293; protonated C-terminal tripeptide fragment, C12H20N3O5, m/z [M+H]+
calcd. 286.1403, obs. 286.1391.
S32
CLP_Irradiated_Alfredo_DrMichael_1712... 12/08/17 19:55:45
CLP_Irradiated_Alfredo_DrMichael_171208195110 #17670-18477 RT: 51.96-52.75 AV: 3 RF: 6.00,3 NL: 9.50E7F: FTMS + c NSI d Full ms2 725.2885
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e A
bund
ance
258.0870z=?
189.0293z=?
286.1391z=?
173.0920z=?
440.2129z=1
86.0605z=?
155.0813z=?
343.1600z=?
509.1567z=?
663.2312z=1
610.1680z=1
726.7686z=2
845.2963z=1
930.3410z=?
1112.3971z=?
N
NN
NO
O
NHO
HO
O O
O
OH
HN
O
NH
NO2
20