Supplementary Material Synthesis and evaluation of D

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: [email protected]

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




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

mailto:[email protected]


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


200 400 600 8000

20

40

60D-Thioluciferin


Wavelength (nm)

Inte

nsit

y (

RU

)



200 400 600 8000

20

40

60D-Thioluciferin


Wavelength (nm)

Inte

nsit

y (

RU

)



Page S3 ©AUTHOR(S)


200 400 600 8000

20

40

60D-Thioluciferin


Wavelength (nm)

Inte

nsit

y (

RU

)


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

)


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 -


Page S5 ©AUTHOR(S)

S9. 1H NMR Spectrum of benzyl propiolate

S10. 13C NMR Spectrum of benzyl propiolate


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)


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)


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)


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)


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)


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

Documents

Supplementary Material Synthesis and evaluation of D