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Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S1 ©AUTHOR(S)
Supplementary Material
Synthesis and evaluation of D-thioluciferin, a bioluminescent 6’-thio analog of D-luciferin
Marwaan Rylands and Anwar Jardine*
Department of Chemistry, University of Cape Town, Rondebosch 7701, Cape Town, South Africa
Email: anwar.jardine@uct.ac.za
Table of Contents
S1. Excitation and emission spectra of D-thioluciferin (3) ................................................................................ S2
S2. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 360 nm ............................. S2
S3. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 390 nm ............................. S2
S4. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 450 nm ............................. S2
S5. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 520 nm ............................. S3
S6. Inhibition study. ........................................................................................................................................... S3
S7. Michaelis-Menton Kinetics .......................................................................................................................... S3
S8. Table (Luminescence data for luciferins)1-4 ................................................................................................. S4
S9. 1H NMR Spectrum of benzyl propiolate ...................................................................................................... S5
S10. 13C NMR Spectrum of benzyl propiolate ..................................................................................................... S5
S11. 1H NMR spectrum of benzyl (Z)-3-((4-aminophenyl)thio)acrylate (Id) ....................................................... S6
S12. 13C NMR Spectrum of benzyl (Z)-3-((4-aminophenyl)thio)acrylate (Id) ...................................................... S6
S13. 1H NMR spectrum of benzyl (Z)-3-((4-(((Z)-4-chloro-5H-1,2,3-dithiazol-5-
ylidene)amino)phenyl)thio)acrylate ............................................................................................................ S7
S14. 13C NMR spectrum of benzyl (Z)-3-((4-(((Z)-4-chloro-5H-1,2,3-dithiazol-5-
ylidene)amino)phenyl)thio)acrylate ............................................................................................................ S7
S15. 1H NMR Spectra of benzyl (Z)-3-((4-((cyanocarbonothioyl)amino)phenyl)thio)acrylate (IIId) ................... S8
S16. 13C NMR Spectra of benzyl (Z)-3-((4-((cyanocarbonothioyl)amino)phenyl)thio)acrylate (IIId) .................. S8
S17. 1H NMR Spectra of benzyl (Z)-3-((2-cyanobenzo[d]thiazol-6-yl)thio)acrylate (IVd) ................................... S9
S18. 13C NMR Spectra of benzyl (Z)-3-((2-cyanobenzo[d]thiazol-6-yl)thio)acrylate (IVd) .................................. S9
S19. 1H NMR Spectra of (S,Z)-2-(6-((3-(benzyloxy)-3-oxoprop-1-en-1-yl)thio)benzo[d]thiazol-2-yl)-4,5-
dihydrothiazole-4-carboxylic acid (IX) ....................................................................................................... S10
S21. Accurate mass spectrum of D-Thioluciferin (3) ......................................................................................... S11
References ........................................................................................................................................................... S11
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S2 ©AUTHOR(S)
Experimental Procedures
Excitation/Emission Spectra of D-thioluciferin
200 400 600 8000
20
40
60
ex 490 nm
em 520 nm
Wavelength (nm)
Inte
nsit
y (
RU
)
S1. Excitation and emission spectra of D-thioluciferin (3)
Emision Spectra of D-Thioluciferin andD-Thioluciferinvinylsulfide at 360 nm
200 400 600 8000
10
20
30
40
50D-Thioluciferin
D-Thioluciferinvinylsulfide
Wavelength (nm)
Inte
nsit
y (
RU
)
S2. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 360 nm
Emision Spectra of D-Thioluciferin andD-Thioluciferinvinylsulfide at 390 nm
200 400 600 8000
20
40
60D-Thioluciferin
D-Thioluciferinvinylsulfide
Wavelength (nm)
Inte
nsit
y (
RU
)
S3. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 390 nm
Emision Spectra of D-Thioluciferin andD-Thioluciferinvinylsulfide at 450 nm
200 400 600 8000
20
40
60D-Thioluciferin
D-Thioluciferinvinylsulfide
Wavelength (nm)
Inte
nsit
y (
RU
)
S4. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 450 nm
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S3 ©AUTHOR(S)
Emision Spectra of D-Thioluciferin andD-Thioluciferinvinylsulfide at 520 nm
200 400 600 8000
20
40
60D-Thioluciferin
D-Thioluciferinvinylsulfide
Wavelength (nm)
Inte
nsit
y (
RU
)
S5. Emission spectra of D-thioluciferin (3) and D-thioluciferin vinylsulfide (IX) at 520 nm
D-th
iolu
cife
rin
M s
ulfide
+ 0.
1 M
sulfi
de
+ 1
M s
ulfide
+ 10
0
50
100
150
Rela
tive L
igh
t U
nit
s (
RL
U)
S6. Inhibition study. Inhibition of firefly luciferase by D-thioluciferin thioacrylate (sulfide) (IX). D-
thioluciferin 3 (10 µM) was treated with purified firefly luciferase (10 nM) expressed from E. coli and
the luminescence recorded in the absence and presence of (IX).
S7. Michaelis-Menton Kinetics Kinetic reaction curves with purified firefly luciferase (luciferase from
Photinus Pyralis, Sigma-Aldrich) were recorded at a 10 nM final enzyme concentration. All the assays
were performed in triplicate and measurements were taken every 5 s post-addition for 20 min.
0 2 4 6 8 100.0
0.2
0.4
0.6
0.8
Km 0.09801 M
[S]
V (
RL
U/s
)
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S4 ©AUTHOR(S)
S8. Table (Luminescence data for luciferins)1-4
D-Luciferin D-Aminoluciferin D-Thioluciferin
λmax 553 nm 597 nm 599 nm
λmax (lit.) 551 nm 594 nm -
Vmax 57 + 13 RLU/s 13 + 2.2 RLU/s 0.7597 + 0.1071 RLU/s
0.6661+ 0.1522 RLU/s
Vmax (lit.) 1626 + 24 photons/min
1.027 ± 0.036 x 10-5 RLU/s 169 + 2.6 photons/min -
Km (App.) 8.3 + 0.5 µM 0.39 + 0.08 µM 0.1169 + 0.01163 µM
0.098 + 0.01471 µM
Km (lit.) 9.15 ± 1.32 µM
16 ± 1 µM
14.8 ± 1.9 µM
7 µM
0.62 + 0.05 µM -
Fluorescence - - 500 nm
Fluorescence (lit.) 528 nm 517 nm -
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S5 ©AUTHOR(S)
S9. 1H NMR Spectrum of benzyl propiolate
S10. 13C NMR Spectrum of benzyl propiolate
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S6 ©AUTHOR(S)
S11. 1H NMR spectrum of benzyl (Z)-3-((4-aminophenyl)thio)acrylate (Id)
S12. 13C NMR Spectrum of benzyl (Z)-3-((4-aminophenyl)thio)acrylate (Id)
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S7 ©AUTHOR(S)
S13. 1H NMR spectrum of benzyl (Z)-3-((4-(((Z)-4-chloro-5H-1,2,3-dithiazol-5-
ylidene)amino)phenyl)thio)acrylate (IId)
S14. 13C NMR spectrum of benzyl (Z)-3-((4-(((Z)-4-chloro-5H-1,2,3-dithiazol-5-
ylidene)amino)phenyl)thio)acrylate (IId)
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S8 ©AUTHOR(S)
S15. 1H NMR Spectra of benzyl (Z)-3-((4-((cyanocarbonothioyl)amino)phenyl)thio)acrylate (IIId)
S16. 13C NMR Spectra of benzyl (Z)-3-((4-((cyanocarbonothioyl)amino)phenyl)thio)acrylate (IIId)
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S9 ©AUTHOR(S)
S17. 1H NMR Spectra of benzyl (Z)-3-((2-cyanobenzo[d]thiazol-6-yl)thio)acrylate (IVd)
S18. 13C NMR Spectra of benzyl (Z)-3-((2-cyanobenzo[d]thiazol-6-yl)thio)acrylate (IVd)
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S10 ©AUTHOR(S)
S19. 1H NMR Spectra of (S,Z)-2-(6-((3-(benzyloxy)-3-oxoprop-1-en-1-yl)thio)benzo[d]thiazol-2-yl)-4,5-
dihydrothiazole-4-carboxylic acid (IX)
S20. 1H NMR Spectra of D-thioluciferin (3)
Regional Issue 'Organic Chemistry in South Africa' ARKIVOC 2020, v, S1-S11
Page S11 ©AUTHOR(S)
S21. Accurate mass spectrum of D-Thioluciferin (3)
References
1. Shinde, R.; Perkins, J.; Contag, C. H., Biochemistry 2006, 45 (37), 11103-12. '10.1021/bi060475o'
2. Kiyama, M.; Iwano, S.; Otsuka, S.; Lu, S. W.; Obata, R.; Miyawaki, A.; Hirano, T.; Maki, S. A., Tetrahedron
2018, 74 (6), 652-660.
https://doi.org/10.1016/j.tet.2017.11.051'
3. Pirrung, M. C.; Carlson, A. D.; De Howitt, N.; Liao, J., Bioorganic & medicinal chemistry letters 2019, 29 (19),
126591. '10.1016/j.bmcl.2019.07.050'
4. da Silva, L. P.; da Silva, J. C. E., Photochemical & Photobiological Sciences 2011, 10 (6), 1039-1045.
B66.1
m/z250 255 260 265 270 275 280 285 290 295 300 305 310 315 320 325 330 335 340 345 350 355 360 365 370 375 380 385 390 395 400 405 410 415 420 425
%
0
100
MS_Direct_170809_SS20 17 (0.091) Cm (14:24) 1: TOF MS ES+ 1.72e4308.9716
292.9486
290.9697
276.9669256.9594
274.9887258.9567
260.9630
261.9624
278.9594
282.9016
292.9939
294.9907
302.1056
310.9688
370.9521319.0002
354.9663
338.9894
320.9986
334.9144330.9018
344.9371
368.9829
376.8943422.8916386.9097 418.8800
406.9081402.8690
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