III. Applications to ligation, photocaging, and DNA-protein crosslinking
3.4. Experimental
57
58
To an aqueous solution containing 24a, 25b and 25c (0.5 μL, 2 mM) suspended in water (78 μL), MES (10 μL, 50 mM, pH 6.0), and Mg(OAc)2 (10 μL, 50 mM) was added an aqueous solution of 26 (1.5 μL, 10 mM in H2O) and mixed thoroughly. The reaction mixture was kept at r.t. for 1 h. The reaction mixture was analyzed HPLC-MS and 15% denaturing PAGE (7 M urea, 20 : 1 = acrylamide : bisacrylamide) for 1 h at 180 V.
3.4.2. Single-step Synthesis of Photocaged ONs
The reaction was performed with several modifications from General Procedure B; 29 in DMSO (1.5 μL, 400 mM), [Rh(COD)Cl]2 in DMSO (0.5 μL, 10 mM), for 1 h.
For ORN-ODN hybrid duplex 22n; water (3.75 μL), Mg(OAc)2 (2 μL, 250 mM), MES buffer (2 μL, 250 mM, pH 6.0), 29 in DMSO (1.25 μL, 800 mM), and [Rh(COD)Cl]2 in DMSO (0.5 μL, 10 mM) Deprotection of photocaged hairpin ssODN 30a: The mixture of 17a and 30a from the alkylation reaction (1 μL) was diluted with water (49 μL), and the solution was irradiated for 10 min at 302 nm.
The reaction mixture was analyzed by HPLC-MS.
3.4.3. DNA-protein Cross-linking
3.4.3.1. DNA-protein conjugation with T7 RNA polymerase
To a solution of dsDNA containing O6-acetonyl G in HEPES buffer was added T7 RNA polymerase (Enzynomics) and incubated for 30 min at 4 ℃. The solution was treated with a stock solution of NaCNBH3 (Final concentration: T7 RNA polymerase (6.8 μM), dsDNA (13.5 μM), NaCNBH3 (0.1 M),
59
HEPES (40 mM), Mg(OAc)2 (5 mM), pH 7.4; Final volume: 15 μL) and incubated at 37 ℃ for 12 h by using SimpliAmp thermocycler (Thermo Fisher Scientific). The mixture was quenched with 4x SDS loading buffer and analyzed by 5% SDS-PAGE (30 : 1 = acrylamide : bisacrylamide, 1x SDS Tris-Cl buffer) with 180 V. The gels were visualized by SYBR Gold staining (Thermo Fisher Scientific) and silver staining by using ChemiDoc (Bio-Rad).
3.4.3.2. Preparation of E. coli lysate
DH5α (Intron Biotechnology, Cat. #15063) was plated on LB-agar plates and incubated overnight. A single colony was incubated in LB medium (20 mL) at 37 ℃ until the culture reached to OD600of 0.6.
The cell pellet was harvested by centrifugation. The pellet was washed with 1x PBS 4 times and lysed by sonication on ice (20 x 15 s) with 15 s intervals. The resultant suspension was centrifuged (17,000 x g, 4 ℃, 20 min), pelleted, and the supernatant was used for the DNA-protein crosslinking in the lysate.
3.4.3.3. DNA-protein conjugation with T7 RNA polymerase in E. coli lysate
To a solution of dsDNA containing O6-acetonyl G in HEPES buffer and E. coli lysate was added T7 RNA polymerase (Enzynomics) and incubated for 30 min at 4 ℃. The solution was treated with a stock solution of NaCNBH3 (Final concentration: T7 RNA polymerase (3.4, 6.8, 13.5 μM), dsDNA (13.5 μM), NaCNBH3 (0.1 M), HEPES (40 mM, pH 7.4), Mg(OAc) (5 mM), 30% E. coli lysate, pH 7.4; Final volume: 15 μL) and incubated at 37 ℃ for 12 h by using SimpliAmp thermocycler (Thermo Fisher Scientific). The mixture was quenched with 4x SDS loading buffer and analyzed by 6% SDS-PAGE (30 : 1 = acrylamide : bisacrylamide, 1x SDS Tris-Cl buffer) with 180 V. The gels were visualized by UV (302 nm) for fluorescence (Rhodamine B), SYBR Gold staining (Thermo Fisher Scientific), and silver staining by using ChemiDoc (Bio-Rad).
3.4.4. Preparation of functionalized oligonucleotides for DNA-protein cross-linking 3.4.4.1. Preparation of ssDNA functionalized with Rhodamine B
60
The reaction was performed with several modifications from General Procedure B; using 31, water (2.5 μL), Mg(OAc)2 (2 μL, 250 mM), MES buffer (2 μL, 250 mM, pH 6.0), diazoacetone (2.5 μL, 400 mM).
After 45 min, diluted with water (20 μL), vigorously washed with ethyl acetate (30 μL, 3 times).
Combined organic layers were discarded, and the aqueous layer was concentrated. The dried crude material was resuspended in water (36.8 μL) and an aqueous solution of 32 (9.2 μL, 50 mM in H2O) was added and mixed thoroughly. The reaction mixture was kept at r.t. for 1 h and was purified by preparative HPLC using a gradient of acetonitrile with 5 mM triethylammonium acetate (pH 7.0) as an eluent. The collected fractions were concentrated with the Thermo Scientific SPD131DDA SpeedVac Concentrator to yield pure 33 (14 nmol, 28%), which was resuspended in water and quantified by using the Thermo Scientific Nanodrop-One at 260 nm.
3.4.4.2. Preparation of Rhodamine B & ketone functionalized dsDNA
61
The reaction was performed in PCR tubes in a total volume of 2.8 μL. An aqueous solution of 21 (S1- RB) (14 nmol) and S2 (14 nmol) in a PCR tube was concentrated, and the ON resuspended in water (0.6 μL) was treated with an aqueous solution of Mg(OAc)2 (0.6 μL, 250 mM), MES buffer (0.6 μL, 250 mM, pH 6.0), and an aqueous solution of diazoacetone (0.7 μL, 400 mM). A solution of [Rh(COD)Cl]2 in THF (0.3 μL, 5 mM) was added and mixed thoroughly. The reaction mixture was incubated at r.t. for 45 min and analyzed by HPLC-MS. The mixture was diluted with water (30 μL) and vigorously washed with ethyl acetate (30 μL x 3). Combined organic layers were discarded, and the aqueous layer was concentrated.
Derivatization of the acetonylated products by oxime ether conjugation with benzyloxyamine was performed as partial separation of HPLC peaks was observed. The crude material from the reaction was diluted with water (20 μL), extracted with ethyl acetate (30 μL x 3), and the resulting aqueous layer was concentrated. The crude material from aqueous layer was dissolved in water, and a small portion (5 nmol of dsDNA) was diluted with water to a final volume of 4 μL. The solution was treated with benzyloxyamine (1 μL, 50 mM in DMSO), kept at r.t. for 1 h (unless otherwise noted), and analyzed by HPLC-MS.
The rest of the crude material from aqueous layer was further purified with the Glen-Park DNA purification cartridge. Concentrations of purified alkylated oligonucleotides were analyzed with the Thermo Scientific Nanodrop-One at 260 nm to give 34a (70% acetonylated, calculated from oxime ether conjugation). Fluorescence emission was measured by excitation at 570 nm.
62
Appendix. HPLC-MS Spectra of reactions
Figure A-1. Selected reaction in Table 2- 1 Figure A-2 ~ A-5. Selected reactions in Table 2-2
Figure A-10. Comparison of the reactivity between deoxyguanosine and deoxyinosine Figure A-11. Modification of 7-deaza-dG with Rh(I)-catalysis
Figure A-12 – A-15. Selected reactions in Supplementary Table 2-4 Figure A-16 ~ A-24. Selected reactions in Table 2-5
Figure A-25 ~ A-38. Selected reactions in Table 2-6
Figure A-39 and A-40. Modifications under high dilution conditions Figure A-41 and A-42. Chemical ligation of ODNs
Figure A-43 ~ A-47. Synthesis of photocaged ONs and decaging
Figure A-48. Preparation of Rhodamine B and ketone functionalized oligonucleotides
63 Figure A-1. Selected reaction in Table 2- 1
Table 2-1, Entry 3
before reaction
at 30 min
Figure A-1. HPLC-MS analysis of Entry 3 of Table 1. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
0 3 6 9
0 100 200
mAU
min
1.786 3.226 4.393 5.426 5.866
0 3 6 9
0 100 200
mAU
min
2.029 4.209 5.309 5.775 6.569
dC
dA dG
dT
dA dG
dT
dC 6
208.0 346.0
230.0 324.0
calcd. 230.1 found 230.0 calcd. 208.1
found 208.0
[M+H]+ calcd. 324.1 found 324.0 [M+Na]+ calcd. 346.1 found 346.0
64 Figure A-2 ~ A-5. Selected reactions in Table 2-2
Table2-2, Entry 1
before reaction at 1.5 h
Figure A-2. HPLC-MS analysis of Entry 1 of Supplementary Table 2. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
Table 2-2, Entry 2
before reaction at 1.5 h
Figure A-3. HPLC-MS analysis of Entry 2 of Supplementary Table 2. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
0 3 6 9
0 300 600
mAU
min
3.487 5.440
0 3 6 9
0 100 200
mAU
min
6.648
5.588
0 3 6 9
0 300 600
mAU
min
3.508 5.448
0 3 6 9
0 100 200
mAU
min
6.564
1 6
1 6
1
65 Table 2-2, Entry 3
before reaction at 10 min
Figure A-4. HPLC-MS analysis of Entry 3 of Supplementary Table 2. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
Table 2-2, Entry 4
before reaction at 10 min
Figure A-5. HPLC-MS analysis of Entry 4 of Supplementary Table 2. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
0 3 6 9
0 300 600
mAU
min
3.508 5.448
0 3 6 9
0 50 100
mAU
min
6.639
5.599
0 3 6 9
0 200 400
mAU
min
4.468 5.655
0 3 6 9
0 50 100
mAU
min
5.656 6.696
1
6
1 6
1 1
66 Figure A-6 ~ A-9. Selected reactions in Table 2-3
Table 2-3, Entry 1
before reaction at 10 min
Figure A-6. HPLC-MS analysis of Entry 4 of Table 1. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
Table 2-3, Entry 2
before reaction o/n
Figure A-7. HPLC-MS analysis of Entry 5 of Table 1. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
0 3 6 9
0 200 400
mAU
min
2.719 4.225 5.492 5.619 5.932
0 3 6 9
0 100 200
mAU
min
2.926 5.519 5.946 6.693
0 3 6 9
0 200 400
mAU
min
1.913 3.666 5.299 5.499 5.859
0 3 6 9
0 150 300
mAU
min
1.924 3.691 5.3115.511 5.871 6.584
dC
dA dG
dT 6
dC
dA dG
dT 6
dT dC
dG dT dC
dA
dA
67 Table 2-3, Entry 3
before reaction o/n
Figure A-8. HPLC-MS analysis of Entry 6 of Table 1. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
Table 2-3, Entry 4
before reaction o/n
Figure A-9. HPLC-MS analysis of Entry 7 of Table 1. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-3/25-7/25-9/90-10/0 (time [min]/solvent B).
0 3 6 9
0 200 400
mAU
min
1.899 3.666 5.299 5.499 5.866
0 3 6 9
0 150 300
mAU
min
2.005 3.732 5.378 5.552 5.892 6.618
0 3 6 9
0 200 400
mAU
min
1.913 3.666 5.299 5.499 5.866
0 3 6 9
0 150 300
mAU
min
2.005 3.745 5.385 5.558 6.611
5.891
dC
dA dG
dT 6
dC
dA dG dT
6 dC
dA dT
dC
dG dT dG
dA
68
Figure A-10. Comparison of the reactivity between deoxyguanosine and deoxyinosine
before reaction
at 30 min
Figure A-10. HPLC-MS analysis of comparison of the reactivity between deoxyguanosine and deoxyinosine.
Elution method (solvent A: 5 mM TEAA, solvent B: acetonitrile): 0/0-9/10-10/90-14.5/90-15.5/0 (time [min]/solvent B).
0 5 10 15
0 300 600
mAU
min
3.040 6.500 6.947
0 5 10 15
0 100 200
mAU
min
6.432 6.859 12.585 13.172
2
12a 2a 12
calcd. 230.1 found 230.0
2a
[M+CH3CO2]- calcd. 382.1 found 382.0
12 2
12a [M+CH3CO2]- calcd. 367.1 found 367.0 367.0
382.0
69 Figure A-11. Modification of 7-deaza-dG with Rh(I)-catalysis
before reaction at 10 min
Figure A-11. HPLC-MS analysis of the reaction of 7-deaza-dG with Rh(I)-catalysis. Elution method (solvent A: 0.1% formic acid in H2O, solvent B: acetonitrile): 0/0-4/20-5/90-9/90-10/0 (time [min]/solvent B).
0 4 8
0 300 600
mAU
min 3.289
6.689
0 4 8
0 200 400
mAU
min 6.738
13 13
70
Figure A-12 – A-15. Selected reactions in Supplementary Table 2-4
before reaction 15a
at 30 min 16a
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-H]- [M-2H]2-
15a 1173.2 1172.2 585.6
16a 1229.3 1228.2 613.6
before reaction found m/z
sequence LC RTa [M-H]- [M-2H]2-
15a 9.268 1171.8 585.6
at 30 min found m/z
sequence LC RT [M-H]- [M-2H]2-
16a 10.437 1228.8 613.6
Figure A-12. HPLC-MS analysis of acetonylation of 15a. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-14/20-15/90-19/90-20/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20
0 200 400
mAU
min
2.595 9.268
0 10 20
0 100 200
mAU
min
9.310 10.437
15a
16a
[M-H]- 1228.8 [M-2H]2-
613.6 15a
[M-2H]2- 585.6
[M-H]- 1171.8
71
before reaction 15b
at 1 h S1-act or S1-act‘ (13.993 & 14.222 min) 16b
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3-
15b 3010.6 1504.8 1002.8
S1-act/
S1-act’ 3066.6 1532.8 1021.5
16b 3122.6 1560.8 1040.2
before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3-
15b 13.286 1504.6 1002.8
at 1 h found m/z
sequence LC RT [M-2H]2- [M-3H]3-
S1-act/
S1-act’ 13.893,
14.146 1533.4, 1532.4 1021.8, 1021.4
16b 14.766 1560.6 1040.2
Figure A-13. HPLC-MS analysis of acetonylation of 15b. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-18/15-19/90-25/90-26/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 6 12 18
0 300 600
mAU
min
2.933 13.286
0 6 12 18
0 200 400
mAU
min
13.893 14.146 14.766
15b
S1-act or S1-act’ 16b
[M-3H]3- 1002.8
[M-2H]2- 1504.8
[M-3H]3- 1021.8
[M-2H]2- 1532.4
[M-3H]3- 1040.2
[M-2H]2- 1560.6 [M-2H]2-
1533.4 [M-3H]3- 1021.4
72
before reaction 15c
at 30 min 15c
Calculated and found MS data
calculated mass calculated m/z
sequence MW/EM [M-3H]3- [M-4H]4- [2M-5H]5-
15c 5058.9 1685.6 1264.0 2023.1
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4- [2M-5H]5-
15c 15.233 1685.8 1264.0 2023.0
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4- [2M-5H]5-
15c 15.346 1685.8 1264.2 2024.0
Figure A-14. HPLC-MS analysis of acetonylation of 15c. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-18/15-19/90-25/90-26/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20
0 400 800
mAU
min
3.120 15.233
0 10 20
0 400 800
mAU
min
15.346
15c
15c
[M-4H]4-
1264.0 [M-3H]3- 1685.8
[2M-5H]5- 2023.0
[M-4H]4-
1264.2 [M-3H]3- 1685.8
[2M-5H]5- 2024.0
73
before reaction 15d
at 30 min 16d
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H+Na+]2- [M-4H+Na+]3- [M-5H+Na+]4-
15d 3080.4 1550.7 1033.5 774.8
16d 3136.4 1578.7 1052.1 788.9
before reaction found m/z
sequence LC RTa [M-3H+Na+]2- [M-4H+Na+]3- [M-5H+Na+]4-
15d 10.313 1551.0 1033.6 775.4
at 30 min found m/z
sequence LC RT [M-3H+Na+]2- [M-4H+Na+]3- [M-5H+Na+]4-
16d 10.831 1578.8 1052.2 788.8
Figure A-15. HPLC-MS analysis of acetonylation of 15d. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-20/20-21/90-25/90-26/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20
0 200 400
mAU
min
10.313
2.393
0 10 20
0 200 400
mAU
min
10.305 10.831
15d
15d 16d
[M-4H+Na]3- 1033.6
[M-3H+Na]2- 1551.0 [M-5H+Na]4-
775.4
[M-4H+Na]3- 1052.2
[M-3H+Na]2- 1578.8 [M-5H+Na]4-
788.8
74 Figure A-16 ~ A-24. Selected reactions in Table 2-5
before reaction 17a
at 30 min 18a
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7- [M-8H]8- [M-9H]9-
17a 7120.2 1759.5 1407.4 1172.7 1005.0 879.3 781.5
18a 7176.2 1773.6 1418.6 1182.0 1013.0 886.2 787.7
before reaction found m/z
sequence LC RTa [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7- [M-8H]8- [M-9H]9-
17a 18.635 1759.4 1407.2 1173.0 1004.8 879.2 781.2
at 30 min found m/z
sequence LC RT [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7- [M-8H]8- [M-9H]9-
18a 18.975 1773.2 1418.4 1182.6 1012.6 886.0 787.6
Figure A-16. HPLC-MS analysis of acetonylation of 17a. Elution method (solvent A: 20 mM TEAA, solvent B:
acetonitrile): 0/0-33/15-35/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20
0 400 800
mAU
min
2.481 18.635
0 10 20
0 400 800
mAU
min
18.975
17a
18a s
[M-8H]8-
879.2 [M-5H]5-
1407.2 [M-4H]4- 1759.4
[M-6H]6- 1173.0 [M-7H]7- 1004.8 [M-9H]9- 781.2
[M-8H]8- 886.0
[M-5H]5-
1418.4 [M-4H]4- 1773.2
[M-6H]6- 1182.6 [M-7H]7- 1012.6 [M-9H]9- 787.6
75
before reaction 17b
at 30 min 18b
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4-
17b 5493.9 1830.6 1372.7
18b 5550.0 1849.3 1386.7
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4-
17b 14.490 1830.8 1372.6
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4-
18b 14.790 1849.4 1387.0
Figure A-17. HPLC-MS analysis of acetonylation of 17b. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-18/15-19/90-22/90-23/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 6 12 18
0 400 800
mAU
min
3.356 14.490
0 6 12 18
0 400 800
mAU
min
14.557 14.790
17b
18b
[M-4H]4- 1372.6
[M-3H]3- 1830.8
17b
[M-4H]4- 1387.0
[M-3H]3- 1849.4
76
before reaction 17c
at 30 min 18c
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
17c 6427.2/6424.1 2141.0 1605.5 1284.2 1070.0
18c 6483.3/6480.1 2159.7 1619.5 1295.4 1079.3
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
17c 10.358 2141.0 1605.0 1283.8 1069.4
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
18c 10.579 2159.2 1619.2 1295.0 1079.0
Figure A-18. HPLC-MS analysis of acetonylation of 17c. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-14/20-15/90-19/90-20/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 6 12 18
0 400 800
mAU
min
2.658 10.358
0 6 12 18
0 200 400 600
mAU
min
10.393 10.579
17c
18c
[M-4H]4- 1605.0
[M-3H]3- 2141.0 [M-5H]5-
1283.8 [M-6H]6- 1069.4
[M-4H]4- 1619.2
[M-3H]3- 2159.2 [M-5H]5-
1295.0 [M-6H]6- 1079.0 17c
77 before reaction 17d
at 30 min S1-act or S1-act’ (13.878 and 14.050 min) 18d
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4-
17d 6136.0 2045.0 1533.5
S1-act
S1-act’ 6192.1 2063.7 1547.5
18d 6248.1 2082.4 1561.5
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4-
17d 13.557 2044.8 1533.4
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4-
S1-act
S1-act’ 13.782
14.005 2064.8, 2063.6 1547.6, 1547.2
18d 14.265 2082.0 1561.6
Figure A-19. HPLC-MS analysis of acetonylation of 17d. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-18/15-19/90-25/90-26/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20
0 500 1000
mAU
min
3.038 13.558
0 10 20
0 300 600
mAU
min
13.561 13.80114.001 14.267
17d
18d S1-act
or S1-act’
17d
[M-4H]4- 1533.4
[M-4H]4- 1547.8
[M-3H]3- 2044.8
[M-3H]3- 2064.8
[M-3H]3- 2063.6 [M-4H]4- 1547.2
[M-3H]3- 2082.0 [M-4H]4-
1561.6
78
before reaction 17e
at 30 min 18e
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4- [M-5H]5-
17e 6012.8 2003.6 1502.4 1201.8
18e 6068.8 2022.3 1516.5 1213.0
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4- [M-5H]5-
17e 13.069 2004.6 1502.6 1202.0
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4- [M-5H]5-
18e 13.546 2022.0 1516.8 1212.2
Figure A-20. HPLC-MS analysis of acetonylation of 17e. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-34/20-35/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20
0 500 1000
mAU
min 3.002
13.069
0 10 20
0 500 1000
mAU
min 12.926
13.546
17e
17e 18e
[M-4H]4- 1502.6
[M-3H]3- 2004.6 [M-5H]5-
1202.0
[M-3H]3- 2022.0 [M-4H]5- 1516.8
[M-5H]6- 1212.2
79
before reaction 17f
at 30 min 18f
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4- [M-5H]5-
17f 6012.8 2003.6 1502.4 1201.8
18f 6068.8 2022.3 1516.5 1213.0
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4- [M-5H]5-
17f 12.872 2003.6 1502.8
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4- [M-5H]5-
18f 13.586 2022.6 1516.6 1212.2
Figure A-21. HPLC-MS analysis of acetonylation of 17f. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-34/20-35/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20
0 500 1000
mAU
min 3.259
12.872
0 10 20
0 500 1000
mAU
min 13.106
13.586
17f
17f 18f
[M-4H]4- 1502.8
[M-3H]3- 2003.6
[M-3H]3- 2022.6 [M-4H]5- 1516.6
[M-5H]6- 1212.2
80
S1 S1-act
S2 S2
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4-
S1 6006.0 2001.7 1501.0
S2 5304.9 1767.6 1325.5
S1-act 6062.0 2020.3 1515.0
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4-
S1 17.912 2001.6 1501.0
S2 14.612 1768.0 1325.4
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4-
S1-act 18.315 2019.8 1515.0
S2 14.575 1768.2 1325.6
Figure A-22. HPLC-MS analysis of acetonylation of 17g. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-18/15-19/90-25/90-26/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 5 10 15 20
0 200 400
mAU
min
14.575 15.102 17.989 18.315
0 5 10 15 20
0 200 400 600
mAU
min
3.285 14.612 17.912
S1 S2
S2 S1
S1-act S2-act
[M-4H]4- 1501.0
[M-3H]3- 2001.6
[M-4H]4- 1325.4
[M-3H]3- 1768.0
[M-4H]4-
1325.6 [M-3H]3- 1768.2 [M-4H]4-
1515.0
[M-3H]3- 2019.8
before reaction
at 30 min
before reaction at 30 min
81
0 10 20 30
0 150 300
mAU
min
24.64023.47422.580
S1 S1-act
S2 S2
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4-
S1 4924.8 1640.9 1230.5
S2 4542.8 1513.6 1134.9
S1-act 4980.9 1659.6 1244.5
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4-
S1 22.811 1641.0 1230.4
S2 22.078 1514.2 1134.8
at 2 h found m/z
sequence LC RT [M-3H]3- [M-4H]4-
S1-act 24.640 1659.2 1244.4
S2 22.580 1513.2 1134.8
Figure A-23. HPLC-MS analysis of acetonylation of 17h. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-33/15-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 10 20 30
0 150 300
mAU
min
22.078 22.811
S2 S1
S1 S2
S1-act
[M-4H]4- 1134.8
[M-3H]3- 1514.2
[M-4H]4- 1134.8
[M-3H]3- 1513.2 [M-4H]4-
1230.6
[M-3H]3- 1641.0
[M-4H]4- 1244.4
[M-3H]3- 1659.2
before reaction at 2 h
before reaction
at 30 min
82
S1 S1
S2 S2-act
0 10 20
0 200 400 600
mAU
min
21.094 24.454 24.847
0 10 20
0 400 800
mAU
min
2.652 21.172 24.152
S1 S2
S1 S2-act S2
[M-7H]7- 746.4
[M-6H]6- 870.8
[M-5H]5- 1045.8
[M-4H]4- 1307.2
[M-3H]3- 1743.2
[M-11H]11- 745.2
[M-10H]10- 819.8
[M-4H]4- 2051.6 [M-9H]9-
911.0 [M-6H]6- 1367.0 [M-7H]7- 1171.4
[M-4H]4- 1307.0
[M-5H]5- 1641.2 [M-8H]8-
1025.4
[M-7H]7- 746.4
[M-6H]6- 871.0
[M-5H]5- 1045.2
[M-3H]3- 1742.4
[M-5H]5- 1651.8 [M-11H]11-
750.2
[M-10H]10- 825.4
[M-8H]8- 1031.8
[M-4H]4- 2065.2 [M-9H]9-
917.4
[M-6H]6- 1376.4 [M-7H]7- 1179.2
before reaction
at 30 min
before reaction at 30 min
83 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
S1 5231.9 1743.3 1307.2 1045.6 871.1 746.6
sequence exact mass
S2 8208.4 [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7- 2051.6 1641.1 1367.4 1171.9 [M-8H]8- [M-9H]9- [M-10H]10- [M-11H]11-
1025.3 911.3 820.0 745.4
S2-act 8264.4 [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7- 2065.6 1652.3 1376.7 1179.9 [M-8H]8- [M-9H]9- [M-10H]10- [M-11H]11-
1032.3 917.5 825.6 750.5
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
S1 21.172 1743.2 1307.2 1045.8 870.8 746.4
S2 24.152 [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
2051.6 1641.2 1367.0 1171.4 [M-8H]8- [M-9H]9- [M-10H]10- [M-11H]11-
1025.4 911.0 819.4 745.2
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
S1 21.094 1742.8 1307.0 1045.2 870.8 746.4
S2-act 24.847 [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
2065.2 1651.8 1376.4 1179.2 [M-8H]8- [M-9H]9- [M-10H]10- [M-11H]11-
1031.8 917.4 825.4 750.2
Figure A-24. HPLC-MS analysis of acetonylation of 17i. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-33/15-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
84 Figure A-25 ~ A-38. Selected reactions in Table 2-6
before reaction 22a
at 30 min 23a
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
22a 7354.3 2451.1 1838.1 1470.2 1225.0
23a 7426.3 2469.8 1852.1 1481.4 1234.4
before reaction found m/z
sequence LC RTa [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
22a 10.718 2452.0 1838.0 1470.0
at 30 min found m/z
sequence LC RT [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
23a 10.991 1852.4 1481.0 1233.8
Figure A-25. HPLC-MS analysis of acetonylation of 22a. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-14/20-15/90-19/90-20/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
0 6 12 18
0 300 600
mAU
min
2.685 10.718
0 6 12 18
0 400 800
mAU
min
10.711 10.991
22a
22a 23a
[M-4H]4- 1838.0
[M-3H]3- 2452.0 [M-5H]5-
1470.0
[M-4H]4- 1852.4 [M-5H]5- 1461.0 [M-6H]6- 1233.8
85
0 10 20
0 200 400
mAU
min
2.476 22.136 24.543
0 10 20
0 200 400
mAU
min
23.092 24.352
22.525
before reaction
acetonylation at 30 min
before reaction acetonylation at 30 min
S1 S1-act
S2 S2
S1 S2
S1-act S2
[M-4H]4- 1504.8
[M-3H]3- 2176.4 [M-4H]4- 1631.6 [M-5H]5- 1305.0
[M-6H]6- 1087.8
[M-3H]3- 2006.8 [M-5H]5-
1203.4
[M-3H]3- 2194.6 [M-5H]5-
1316.2
[M-4H]4- 1646.0
[M-3H]3- 2006.6 [M-4H]4- 1504.8
[M-5H]5- 1203.8
S1
86 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 6529.1 3264.6 2176.0 1631.8 1305.2 1087.5
S2 6022.0 3011.0 2007.0 1505.0 1203.8 1003.0
S1-act 6585.2 3292.6 2194.7 1645.8 1316.4 1096.9
before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 22.136 2176.4 1631.6 1305.0 1087.8
S2 24.543 2006.8 1504.8 1203.4
acetonylation at 30 min found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-act 23.092 2194.6 1646.0 1316.2
S2 24.352 2006.6 1504.8 1203.8
Figure A-26. HPLC-MS analysis of acetonylation of 22b. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-33/15-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
87
0 10 20
0 300 600
mAU
min
2.860 17.147 17.627
0 10 20
0 500 1000
mAU
min
17.161 17.987
before reaction
acetonylation at 30 min
before reaction acetonylation at 30 min
S1 S1-act
S2 S2
Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 7947.3 3973.7 2648.8 1986.3 1588.9 1323.9
S2 7694.3 3847.2 2564.4 1923.1 1538.3 1281.7
S1-act 8003.4 4001.7 2667.5 2000.3 1600.1 1333.2
Before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 17.627 1986.4 1588.8
S2 17.147 2565.0 1922.4 1537.8
at 30 min found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-act 17.987 2000.2 1600.0
S2 17.161 1922.8 1537.6
Figure A-27. HPLC-MS analysis of acetonylation of 22c. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-30/20-35/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time
S2 S1
S1-act S2
[M-4H]4- 1986.4 [M-5H]5-
1588.8
[M-4H]4- 1922.4
[M-4H]4- 2000.2 [M-5H]5- 1600.0
[M-5H]5-
1537.8 [M-3H]3-
2565.0
[M-4H]4- 1922.8 [M-5H]5-
1537.6
88
at 30 min
0 10 20
0 200 400
mAU
min
2.139 21.819 22.879
0 10 20
0 200 400
mAU
min
21.813 23.293
S1 S1-act
S2 S2
S2 S1
S1-act S2
[M-4H]4- 1986.6 [M-5H]5- 1589.2
[M-6H]6- 1323.0
[M-4H]4- 1923.0 [M-5H]5- 1538.2 [M-6H]6- 1281.8
[M-4H]4- 2000.2 [M-5H]5- 1600.0 [M-6H]6-
1333.0
[M-4H]4- 1923.0 [M-5H]5- 1538.4 [M-6H]6-
1281.6
before reaction
before reaction at 30 min
89 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 7941.3 3973.7 2648.8 1986.3 1588.9 1323.9
S2 7694.3 3847.2 2564.4 1923.1 1538.3 1281.7
S1-act 8003.4 4001.7 2667.5 2000.3 1600.1 1333.2
before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 22.879 1986.6 1589.2 1323.0
S2 21.819 1923.0 1538.2 1281.8
at 30 min found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-act 23.293 2000.2 1600.0 1333.0
S2 21.813 1923.0 1538.4 1281.6
Figure A-28. HPLC-MS analysis of acetonylation of 22d. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-33/15-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
90
0 10 20
0 200 400
mAU
min
2.231 21.665 22.258
0 10 20
0 200 400 600
mAU
min
22.027
0 10 20
0 200 400
mAU
min
22.034 24.634
before reaction
acetonylation at 30 min
oxime ether at 1 h
before reaction acetonylation at 30 min oxime ether at 1 h
*Derivatized to the oxime ether for the separation of overlapping peaks
S1 S1-act S1-OX
S2 S2 S2
S1 S2
S1-act, S2
S2 S1-OX
[M-4H]4- 1625.4 [M-5H]5- 1300.4 [M-6H]6- 1083.6
[M-4H]4- 1511.4 [M-5H]5- 1208.6 [M-6H]6- 1007.4
[M-4H]4- 1640.2 [M-5H]5-
1311.6
[M-6H]6- 1007.0
[M-4H]4- 1511.4
[M-5H]5- 1208.8
[M-6H]6- 1110.2
[M-4H]4- 1666.0 [M-5H]5-
1332.8
[M-6H]6- 1006.8
[M-4H]4- 1511.4 [M-5H]5- 1208.4
91 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 6506.1 3253.1 2168.4 1626.0 1300.6 1083.7
S2 6048.0 3024.0 2015.7 1511.5 1209.0 1007.3
S1-act 6562.2 3281.1 2187.0 1640.0 1311.8 1093.0
S1-OX 6667.2 3333.6 2222.1 1666.3 1332.8 1110.5
before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 21.665 2168.8 1625.4 1300.4 1083.6
S2 22.258 1511.4 1208.6 1007.4
at 30 min found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-act 22.027 1640.2 1311.6
S2 22.027 1511.4 1208.8 1007.0
oxime ether at 1 h found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-OX 24.634 1666.0 1332.8 1110.2
S2 22.034 1511.4 1208.8 1006.8
Figure A-29. HPLC-MS analysis of acetonylation of 22e. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-33/20-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
92
before reaction
acetonylation at 30 min
oxime ether at 1 h
before reaction acetonylation at 30 min oxime ether at 1 h
*Derivatized to the oxime ether for the separation of overlapping peaks
S1 S1-act S1-OX
S2 S2 S2
0 10 20 30
0 200 400
mAU
min
23.490 26.243
0 10 20 30
0 200 400
mAU
min
23.772 23.952
0 10 20 30
0 200 400
mAU
min
2.380 22.833 23.046
S1 S2
S1-act
S2 S1-OX S2
[M-6H]6- 1064.6
[M-4H]4- 1597.8 [M-5H]5- 1278.0
[M-3H]3- 2130.4
[M-6H]6- 1025.8
[M-4H]4- 1538.8 [M-5H]5- 1230.8
[M-4H]4- 1611.8
[M-5H]5- 1289.0
[M-3H]3- 2149.4
[M-3H]3- 2051.4 [M-4H]4-
1538.8
[M-4H]4- 1638.4 [M-5H]5-
1309.8
[M-3H]3- 2184.0
[M-6H]6- 1025.4
[M-4H]4- 1538.6 [M-5H]5- 1230.4
93 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 6494.1 3197.1 2131.0 1598.0 1278.2 1065.0
S2 6158.1 3079.0 2052.3 1539.0 1231.0 1025.7
S1-act 6450.2 3225.1 2149.7 1612.0 1289.4 1074.4
S1-OX 6555.2 3277.6 2184.7 1638.3 1310.4 1091.9
before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 22.833 2130.4 1597.8 1278.0 1064.6
S2 23.046 1538.8 1230.8 1025.8
acetonylation at 30min found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-act 23.952 2149.4 1611.8 1289.0
S2 23.722 2051.4 1538.8
Oxime ether at 1 h found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-OX 26.243 2184.0 1638.4 1309.8
S2 23.490 1538.6 1230.4 1025.4
Figure A-30. HPLC-MS analysis of acetonylation of 22f. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-33/20-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
94
0 10 20
0 200 400
mAU
min
22.344 24.324
21.951
S1 S1-act
S2 S2
0 10 20
0 200 400
mAU
min
2.573 21.920 24.253
S2 S1
S2 S1-act
[M-6H]6-
1087.4 [M-3H]3-
2175.8 [M-5H]5-
1305.0
[M-4H]4- 1631.6
S1
[M-3H]3- 2006.6 [M-5H]5-
1203.2
[M-4H]4- 1504.8
[M-3H]3- 2194.8 [M-5H]5-
1316.4
[M-4H]4- 1645.4
[M-4H]4- 1505.2
[M-3H]3- 2007.0 [M-5H]5-
1203.6 [M-6H]6- 1002.6
before reaction
at 30 min
before reaction at 30 min
95 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 6529.1 3264.6 2176.0 1631.8 1305.2 1087.5
S2 6022.0 3011.0 2007.0 1505.0 1203.8 1003.0
S1-act 6585.2 3292.6 2194.7 1645.8 1316.4 1096.9
before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1 21.920 2175.8 1631.6 1305.0 1087.4
S2 24.253 2006.6 1504.8 1203.2
at 30 min found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6-
S1-act 22.344 2194.8 1645.4 1316.4
S2 24.324 2007.0 1505.2 1203.6 1002.6
Figure A-31. HPLC-MS analysis of acetonylation of 22g. Elution method (solvent A: 5 mM TEAA, solvent B:
acetonitrile): 0/0-33/15-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
96
0 10 20
0 1000 2000
mAU
min
2.655 18.982 19.422
0 10 20
0 1000 2000
mAU
min
19.458 19.851
0 10 20
0 400 800
mAU
min
21.689 21.869
18.969 19.362
before reaction
acetonylation at 30 min
oxime ether at 1 h
before reaction acetonylation at 30 min oxime ether at 1 h
*Derivatized to the oxime ether for the separation of overlapping peaks
S1 S1-act S1-OX
S2 S2-act S2-OX
S2 S1
S2-act S1-act
S2-OX S1-OX
[M-6H]6- 1577.4
[M-5H]5- 1893.6
[M-6H]6- 1596.8
[M-5H]5- 1917.0
[M-6H]6- 1587.6
[M-5H]5- 1905.0
[M-5H]5- 1928.0
[M-6H]6- 1604.8
[M-5H]5- 1925.6
[M-6H]6- 1623.8
[M-5H]5- 1948.8
S2 S1
97 Calculated and found mass data
calculated mass calculated m/z
sequence exact mass [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
S1 9470.6 4735.8 3156.9 2367.4 1893.7 1577.9 1352.4
S2 9586.7 4793.8 3195.5 2396.4 1916.9 1597.3 1368.9
S1-act 9526.6 4763.8 3175.3 2381.4 1904.9 1587.3 1360.4
S1-OX 9631.7 4816.3 3210.6 2407.7 1925.9 1604.8 1375.4
S2-act 9642.7 4821.8 3214.2 2140.4 1928.1 1606.6 1376.9
S2-OX 9747.7 4874.4 3249.2 2436.7 1949.1 1624.1 1392.0
before reaction found m/z
sequence LC RTa [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
S1 18.982 1893.6 1577.4
S2 19.422 1917.0 1596.8
acetonylation at 30 min found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
S1-act 19.851 1905.0 1587.6
S2-act 19.458 1928.0
oxime ether at 1 h found m/z
sequence LC RT [M-2H]2- [M-3H]3- [M-4H]4- [M-5H]5- [M-6H]6- [M-7H]7-
S1-OX 21.869 1925.6 1604.8
S2-OX 21.689 1948.8 1623.8
Figure A-32. HPLC-MS analysis of acetonylation of 22h. Elution method (solvent A: 20 mM TEAA, solvent B:
acetonitrile): 0/0-33/20-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
98
0 10 20
0 400 800
mAU
min
2.712 19.239 19.319
0 10 20
0 400 800
mAU
min
2.724 19.124
0 10 20
0 400 800
mAU
min
18.782 21.029
acetonylation at 30 min
oxime ether at 1 h
*Derivatized to the oxime ether for the separation of overlapping peaks
S1 S1-act S1-OX
S2 S2-act S2-OX
before reaction
S1, S2
S1-act, S2-act
S1-OX, S2-OX
[M-5H]5- 2279.2 [M-6H]6-
1899.4
[M-6H]6- 1893.4
[M-5H]5- 2272.8
[M-6H]6-
1908.4 [M-5H]5- 2290.6
[M-7H]7- 1631.0
[M-5H]5- 2284.0
[M-6H]6- 1925.6
[M-6H]6- 1920.6
S1, S2
before reaction acetonylation at 30 min oxime ether at 1 h
99 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-5H]5- [M-6H]6- [M-7H]7- S1 11396.9 2279.0 1899.0 1627.6 S2 11366.0 2273.0 1894.0 1623.3 S1-act 11453.0 2290.4 1908.3 1635.6 S1-OX 11558.0 2311.2 1925.8 1650.6 S2-act 11422.0 2284.0 1903.2 1631.1 S2-OX 11527.0 2305.2 1920.8 1646.3 before reaction found m/z
sequence LC RTa [M-5H]5- [M-6H]6- [M-7H]7-
S1 19.239 2279.2 1899.4
S2 19.239 2272.8 1893.4
acetonylation at 30 min found m/z
sequence LC RT [M-5H]5- [M-6H]6- [M-7H]7- S1-act 19.124 2290.6 1908.4 1635.4
S2-act 19.124 2284.0 1631.0
oxime ether 1 h found m/z
sequence LC RT [M-5H]5- [M-6H]6- [M-7H]7-
S1-OX 21.029 1925.6
S2-OX 21.029 1920.6
Figure A-33. HPLC-MS analysis of acetonylation of 22i. Elution method (solvent A: 20 mM TEAA, solvent B:
acetonitrile): 0/0-33/20-35/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
100
0 10 20
0 1000 2000
mAU
min
2.587 18.614 19.060
0 10 20
0 400 800
mAU
min
18.937 19.210
0 10 20
0 400 800 1200
mAU
min
18.992 21.112 21.239
before reaction
acetonylation at 30 min
oxime ether at 1 h
before reaction acetonylation at 30 min oxime ether at 1 h
*Derivatized to the oxime ether for the separation of overlapping peaks
S1 S1-act S1-OX
S2 S2-act S2-OX
Calculated and found MS data
S2 S1
S2-act S1-act
S2-OX, S1-OX S1, S2
[M-5H]5- 1907.8 [M-6H]6- 1589.4
[M-5H]5- 1902.4
[M-6H]6- 1585.6 [M-7H]7- 1358.8
[M-5H]5- 1918.8
[M-6H]6- 1599.6
[M-5H]5- 1913.8 [M-6H]6-
1595.0 [M-7H]7- 1367.0
[M-5H]5- 1940.2
[M-7H]7- 1385.6
[M-5H]5- 1935.0
101 calculated mass calculated m/z
sequence exact mass [M-5H]5- [M-6H]6- [M-7H]7-
S1 9541.6 1907.9 1589.8 1362.5
S2 9515.7 1902.7 1585.4 1358.8
S1-act 9597.6 1919.1 1599.1 1370.5
S1-OX 9702.7 1940.1 1616.6 1385.5
S2-act 9571.7 1913.9 1594.8 1366.8
S2-OX 9676.7 1934.9 1612.3 1381.8
before reaction found m/z
sequence LC RTa [M-5H]5- [M-6H]6- [M-7H]7-
S1 19.060 1907.8 1589.4
S2 18.614 1902.4 1585.6 1358.8
acetonylation at 30 min found m/z
sequence LC RT [M-5H]5- [M-6H]6- [M-7H]7-
S1-act 19.210 1918.8 1599.6
S2-act 18.937 1913.8 1595.0 1367.0
oxime ether at 1 h found m/z
sequence LC RT [M-5H]5- [M-6H]6- [M-7H]7-
S1-OX 21.239 1940.2 1385.6
S2-OX 21.112 1935.0
Figure A-34. HPLC-MS analysis of acetonylation of 22j. Elution method (solvent A: 20 mM TEAA, solvent B:
acetonitrile): 0/0-33/20-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
102
0 10 20
0 500 1000
mAU
min
2.660 18.507 18.760
0 10 20
0 400 800 1200
mAU
min
18.973 19.106
0 10 20
0 500 1000 1500
mAU
min
18.794 21.174
before reaction
acetonylation at 30 min
oxime ether at 1 h
before reaction acetonylation at 30 min oxime ether at 1 h
*Derivatized to the oxime ether for the separation of overlapping peaks
S1 S1-act S1-OX
S2 S2-act S2-OX
S2 S1
S2-act S1-act
S1-OX, S2-OX S1, S2
[M-6H]6- 1572.2
[M-5H]5- 1887.8
[M-7H]7- 1347.8
[M-5H]5- 1923.2 [M-6H]6- 1602.2
[M-5H]5- 1898.4
[M-6H]6- 1582.0
[M-5H]5- 1934.2
[M-6H]6- 1611.8 [M-7H]7- 1381.8
[M-5H]5- 1919.8
[M-7H]7- 1370.2
[M-5H]5- 1955.2 [M-6H]6- 1628.8
103 Calculated and found MS data
calculated mass calculated m/z
sequence exact mass [M-5H]5- [M-6H]6- [M-7H]7-
S1 9439.6 1887.5 1572.8 1347.9
S2 9617.7 1923.1 1602.4 1373.4
S1-act 9495.6 1898.7 1582.1 1355.9
S1-OX 9600.7 1919.7 1599.6 1370.9
S2-act 9673.7 1934.3 1611.8 1381.4
S2-OX 9778.7 1955.3 1629.3 1396.4
before reaction found m/z
sequence LC RTa [M-5H]5- [M-6H]6- [M-7H]7-
S1 18.760 1887.8 1572.2 1347.8
S2 18.507 1923.2 1602.2
acetonylation at 30 min found m/z
sequence LC RT [M-5H]5- [M-6H]6- [M-7H]7-
S1-act 19.106 1898.4 1582.0
S2-act 18.973 1934.2 1611.8 1381.8
oxime ether at 1 h found m/z
sequence LC RT [M-5H]5- [M-6H]6- [M-7H]7-
S1-OX 21.174 1919.8 1370.2
S2-OX 21.174 1955.2 1628.8
Figure A-35. HPLC-MS analysis of acetonylation of 22k. Elution method (solvent A: 20 mM TEAA, solvent B:
acetonitrile): 0/0-33/20-34/90-38/90-40/0 (time [min]/Solvent B%) with flowrate 0.3 mL/min. aRT = retention time.
104
before reaction
acetonylation at 30 min
oxime ether at 1 h
0 10 20
0 200 400
mAU
min
2.268 23.335
0 10 20
0 200 400
mAU
min
23.837
0 10 20
0 200 400
mAU
min
23.719 26.126
*Derivatized to the oxime ether for the separation of overlapping peaks
22l 23l 23l-OX
22l
23l
23l-OX 22l
[M-5H]5- 2025.4 [M-6H]6- 1687.4
[M-5H]5- 2036.4
[M-6H]6- 1696.8
[M-5H]5- 2057.8 [M-6H]6-
1714.2