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1

Supporting Information

Driving and photo-regulation of myosin-actin motor at molecular and

macroscopic level by photo-responsive high energy molecules.

Halley M. Menezes,1,2

Md. Jahirul Islam,1,2

Masayuki Takahashi3 and Nobuyuki Tamaoki

1,2*

1Research Institute for Electronic Science, Hokkaido University, Kita 20, Nishi 10, Kita-Ku, Sapporo, Hokkaido,

001-0020, Japan.

2Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido, 060-0810,

Japan.

3Faculty of Science, Hokkaido University, Kita 13, Nishi 8, Kita-Ku, Sapporo, Hokkaido, 060-0810, Japan.

E-mail: [email protected]

Table of contents

1. Synthetic schemes…………………………………………………………………………………S2

2. Photoisomerization of AzoTPs……………………………………………………………………S4

3. Isomerization ratio of cis and trans before and after irradiation………………………………….S5

4. Muscle fibre shortening as a function of AzoTP concentration…………………………………..S6

5. Purity of the AzoTPs by HPLC analysis………………………………………………...…….….S7

6. Mass spectra……………………………………………………………………………..……......S8

7. Supplementary movie information………………………………………………………………S14

8. NMR spectral data (1H,

13C and

31P NMR)……………………………………………………...S15

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2017

mailto:[email protected]

2

1. Synthetic schemes of AzoTP derivatives.

Scheme of AzoethoxyTP (4b)

Scheme of DimethylAzoTP (4c)

(a) mCPBA, EtOAc, 0°C, 3h.

(b) 1,2-phenylenediamine, AcOH, EtOAc, N2- atmosphere, 50 °C, 72h.

(c) Acetoxyactyl chloride, Triethylamine, DCM, RT, 3h.

tert-butyl protected AzoethoxyMP (2b)

a) Ethylene glycol, Pd(OAc)2, PhI(OAc)

2

b) di-tert-butyl N,N-diisopropylphosphoramidite, 1H-tetrazole, dry THF, Ar atmosphere, rt, 6h; then, mCPBA,0°C, 1h;

then rt, 40 min;

c) Trifluoroacetic acid, dry CH2Cl

2, Ar atmosphere, rt, 6 h; then eluting through DEAE Sephadex A-25 anion

exchanger, TEAB;

d) Tributylamine, carbonyldiimidazole, pyrophosphate, dry DMF, Ar atmosphere, rt, overnight.

1b

AzoethoxyTP (4b) AzoethoxyMP (3b)

5 6

DimethylAzoMP (3c) DimethylAzoTP (4c)

1c tert-butyl protected

AzoethoxyMP(2c)

3

(d) K2CO3, MeOH, RT, Overnight.

(e) Di-tert-butyl N,N-Diisopropylphosphoramidite, 1H-tetrazol, dry THF, Ar-atmosphere, RT, 6h, then

mCPBA, 0°C,1h.

(f) TFA, dry DCM, Ar-atmosphere, RT, 6h.

(g) Tributylamine, carbonyldiimidazole, tributylammonium pyrophosphate, dry DMF, Ar-atmosphere, RT,

overnight.

Scheme of AzoethylTP (4)

(a) ArNO, AcOH, Toluene, Ar-atmosphere, 60 °C, 72h.

(b) Di-tert-butyl N,N-Diisopropylphosphoramidite, 1H-tetrazol, dry THF, Ar-atmosphere, RT, 6h, then

mCPBA, 0°C, 1h.

(c) TFA, dry DCM, Ar-atmosphere, RT, 6h.

(d) Tributylamine, carbonyldiimidazole, tributylammonium pyrophosphate, dry DMF, Ar atmosphere, RT,

overnight.

1d

tert-butyl protected AzoethoxyMP (2d)

AzoethylMP (3d) AzoethylTP (4d)

4

2. Photoisomerization of AzoTPs.

200 250 300 350 400 450 500 550 600 650

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

ab

so

rba

nce

wavelength(nm)

Before irradiation

UV pss

Vis pss

Fig. S1. UV-Vis absorption spectra of AzoTP molecules a)DimethylAzoTP (4c), b) AzoethylTP (4d) in BRB-

80 buffer solution at 25 °C; before irradiation (black line), UV PSS (red line), Vis PSS (green line) and c)

AzoethoxyTP (4b) in water at 25 °C, before irradiation (black line), UV PSS (green line), Vis PSS (red line).

Insets show the absorbance changes a) at 341 nm, b) at 325 nm and c) at 324 nm after the alternate irradiations

by 365 and 436 nm light for 5 cycles.

b) a)

c)

5

3. Isomerization ratio of cis and trans at UV and Visible PSS

The photo conversion ratio from trans to cis and cis to trans isomers of the AzoTPs corresponding to

irradiation with 365 nm and 436 nm light was measured with 1H NMR (400 MHz, D2O).

2H

1H

3H

1H

1.0 3.08 1.03 1.07 1.36

1.01 0.49 0.456

0.07 0.22 0.09 0.18

3.06

2.06 1.00

1.0

7H for cis (93%)

7H for cis (35%)

7H for trans (7%)

Before irradiation

PSS at 436nm

PSS at 365nm

7H for trans (100%)

7H for trans (65%)

a)

b)

6

Fig. S2.

1H NMR spectra (400 MHz, D2O) showing the trans and cis isomer ratios of a) AzoethoxyTP (4b),

b) DimethylAzoTP (4c) and c) AzoethylTP (4d) before irradiation, at UV PSS and Visible PSS.

4. Muscle fibre shortening as a function of AzoTP concentration

Fig. S3. AzoTP (4a) concentration dependent shortening of muscle fiber. Lines are best fit through the data

trend.

1H 2H

4H

2H

2.0 4.14

1.99 1.11 0.93 1.03

0.30 0.63

0.04 0.94

0.11

0.59

3.08

1.0

Before irradiation

PSS at 436nm

PSS at 365nm

0.11

0.25

2.04 2.02

9H for trans (13%)

9H for cis (87%)

9H for trans(75%)

9H for cis (25%)

9H for trans (100%)

trans

cis

cis

trans

c)

7

5. Purity of the AzoTPs by HPLC analysis.

a)

b)

c)

94.8%

96.7%

96.1%

8

d)

Fig. S4. Reverse phase HPLC chromatograms of a) AzoTP (4a), b) AzoethoxyTP (4b), c) DimethylAzoTP

(4c), d) AzoethylTP (4d) showing > 95% purity. Conditions of the RP-HPLC analysis; Column: CN-80Ts, 4.6

250 mm (TOSOH). Eluent: Eluent - CH3CN/NaPi buffer (pH 6.5). Solvent gradient: 15 – 45% of CH3CN in

NaPi buffer. Monitoring wavelength: λ= 327 nm. Flow rate: 0.5 mL/min at room temperature (23 °C).

Injection volume: 20 μL.

6. Mass spectra of AzoTP molecules.

Fig. S5. ESI mass spectrum of AzoethoxyTP (4b), the peaks at m/z = 504.03, 503.02, 479.02 and 481.04

assigned to the [M-H]-

ions of monosodium tetraprotonated, monosodium triprotonated, diprotonated and

tetraprotonated AzoethoxyTP species respectively. The peaks at m/z = 424.05, 423.05 and 401.07 assigned to

[M-H]- ions of monosodium triprotonated, monosodium diprotonated and triprotonated AzoethoxyDP species

respectively.

m/z

96.4%

9

Fig. S6. ESI mass spectrum of DimethylAzoTP (4c), the peaks at m/z = 668.11, 544.15 and 522.16 can be

assigned to the [M-H]- ions of disodium-mono triethyl ammonium, monosodium and tetra-protonated

DimethylAzoTP species respectively. The peaks at m/z = 464.16 and 442.17 can be assigned to the [M-H]-

ions of monosodium and tri-protonated DimethylAzoDP species respectively.

10

Fig. S7. ESI mass spectrum of AzoethylTP (4d), the peaks at m/z = 487.04, 465.06 and 385.08 can be

assigned to the [M-H]- ions of monosodium, tetra-protonated AzoethylTP and tri-protonated AzoethylDP

species respectively.

Fig. S8. High-resolution mass spectrum of 1b calculated for C14H16N2O2Na [M + Na]+: 265.09475

Fig. S9. High-resolution mass spectrum of 2b calculated for C22H31N2O5PNa [M + Na]+: 457.18628.

Sample No. : C:\Xcalibur\...\170585_azoetho2b_pn2 Instrument : Exactive Mobile phase solvent : MeOHOperator name : yamada miho Sample solvent : submitting solutionDate : 8/28/2017 1:49:55 PMInstrumental method : C:\Xcalibur\methods\HESI_100ul\pn_H110_S45.methInstrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University170585_azoetho2b_pn2 #13-15 RT: 0.21-0.23 AV: 2 NL: 1.31E6T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

440 442 444 446 448 450 452 454 456 458 460 462 464 466 468 470 472 474 476 478 480

m/z

0

20

40

60

80

100

Rela

tive A

bundance

457.18643

458.18993449.36034441.29764

450.36379442.30102 473.18164469.32898459.19291447.29365

170585_azoetho2b_pn2 #15-17 RT: 0.23-0.26 AV: 2 NL: 3.55E6T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

440 442 444 446 448 450 452 454 456 458 460 462 464 466 468 470 472 474 476 478 480

m/z

0

20

40

60

80

100

Rela

tive A

bundance

457.18634

458.18987

449.36022441.29758473.18134459.19263 469.32880

170585_azoetho2b_pn2 #19-22 RT: 0.28-0.30 AV: 2 NL: 6.59E6T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

440 442 444 446 448 450 452 454 456 458 460 462 464 466 468 470 472 474 476 478 480

m/z

0

20

40

60

80

100

Rela

tive A

bundance

457.18633

458.18989

449.36023441.29759 473.18118459.19289 469.32853

11

Fig. S10. High-resolution mass spectrum of 3b calculated for C14H14N2O5P [M - H]-: 321.06458.

Fig. S11. High-resolution mass spectrum of AzoethoxyTP (4b) as tetrasodium salt calculated for

C14H14N2O11Na4P3 [M + H]+: 570.93957

Fig. S12. High-resolution mass spectrum of 5 calculated for C14H16N3 [M + H]+: 226.13387.

Fig. S13. High-resolution mass spectrum of 6 calculated for C18H19N3O3Na [M + Na]+: 348.13186.

Sample No. : C:\Xcalibur\...\170594_azoethox2c_pn Instrument : Exactive NegOperator name : yamada miho Mobile phase solvent : MeOHDate : 8/28/2017 1:39:15 PM Sample solvent : MeOHInstrumental method : C:\Xcalibur\methods\HESI_100ul\pn_H80_S30.methInstrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University170594_azoethox2c_pn #13-16 RT: 0.20-0.23 AV: 2 NL: 9.33E6T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

300 305 310 315 320 325 330 335 340 345 350

m/z

0

20

40

60

80

100

Rela

tive A

bundance

321.06487

322.06811311.16918 325.18440 339.20011337.05986

170594_azoethox2c_pn #15-18 RT: 0.23-0.25 AV: 2 NL: 1.64E7T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

300 305 310 315 320 325 330 335 340 345 350

m/z

0

20

40

60

80

100

Rela

tive A

bundance

321.06483

322.06807

311.16915 325.18435 339.20007337.05981

170594_azoethox2c_pn #23-27 RT: 0.32-0.34 AV: 2 NL: 2.52E7T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

300 305 310 315 320 325 330 335 340 345 350

m/z

0

20

40

60

80

100

Rela

tive A

bundance

321.06478

322.06802

311.16908 325.18427 339.20001337.05975

12

Fig. S14. High-resolution mass spectrum of 1c calculated for C16H17N3O2Na [M + Na]+: 306.12130.

Fig. S15. High-resolution mass spectrum of 2c calculated for C24H34N3O5P [M + Na]+: 498.21283.

Fig. S16. High-resolution mass spectrum of 3c calculated for C16H17N3O5P [M – C6H17N(TEA)]-:362.09113.

Fig. S17. High-resolution mass spectrum of DimethylAzoTP (4c) as tetrasodium salt calculated for

C16H16N3O11Na4P3 [M + H]+: 611.96612.

Sample No. : C:\Xcalibur\...\170587_dimethyl3b_pn2 Instrument : Exactive Mobile phase solvent : MeOHOperator name : yamada miho Sample solvent : MeOHDate : 8/28/2017 12:46:44 PMInstrumental method : C:\Xcalibur\methods\HESI_100ul\pn_H110_S45.methInstrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University170587_dimethyl3b_pn2 #14 RT: 0.23 AV: 1 NL: 1.29E8T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

480 482 484 486 488 490 492 494 496 498 500 502 504 506 508 510 512 514 516 518 520

m/z

0

20

40

60

80

100

Rela

tive A

bundance

498.21246

499.21600

500.21893 514.18652

170587_dimethyl3b_pn2 #16 RT: 0.25 AV: 1 NL: 2.08E8T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

480 482 484 486 488 490 492 494 496 498 500 502 504 506 508 510 512 514 516 518 520

m/z

0

20

40

60

80

100

Rela

tive A

bundance

498.21237

499.21582

500.21887

170587_dimethyl3b_pn2 #20-23 RT: 0.29-0.32 AV: 2 NL: 2.51E8T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

480 482 484 486 488 490 492 494 496 498 500 502 504 506 508 510 512 514 516 518 520

m/z

0

20

40

60

80

100

Rela

tive A

bundance

498.21227

499.21579

500.21872

Sample No. : C:\Xcalibur\...\170588_dimethyl3c_pn Instrument : Exactive NegOperator name : yamada miho Mobile phase solvent : MeOHDate : 8/28/2017 12:38:05 PM Sample solvent : MeOHInstrumental method : C:\Xcalibur\methods\HESI_100ul\pn_H80_S30.methInstrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University170588_dimethyl3c_pn #14-16 RT: 0.20-0.23 AV: 2 NL: 4.04E6T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

350 352 354 356 358 360 362 364 366 368 370 372 374 376 378 380

m/z

0

20

40

60

80

100

Rela

tive A

bundance

362.09148

363.09487

364.09759

170588_dimethyl3c_pn #16-18 RT: 0.23-0.25 AV: 2 NL: 6.94E6T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

350 352 354 356 358 360 362 364 366 368 370 372 374 376 378 380

m/z

0

20

40

60

80

100

Rela

tive A

bundance

362.09139

363.09473

364.09735

170588_dimethyl3c_pn #23-26 RT: 0.32-0.35 AV: 2 NL: 9.23E6T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

350 352 354 356 358 360 362 364 366 368 370 372 374 376 378 380

m/z

0

20

40

60

80

100

Rela

tive A

bundance

362.09140

363.09471

364.09744

13

Fig. S18. High-resolution mass spectrum of 1d calculated for C14H14N2ONa [M + Na]+: 249.09983.

Fig. S19. High-resolution mass spectrum of 2d calculated for C22H31N2O4PNa [M + Na]+: 441.19137.

Fig. S20. High-resolution mass spectrum of 3d C14H14N2O4P [M - C6H16N(TEA)]-: 305.06967.

Fig. S21. High-resolution mass spectrum of AzoethylTP (4d) as tetrasodium salt calculated for

C14H14N2O10Na4P3 [M + H]+: 554.94466.

Sample No. : C:\Xcalibur\...\170421_Azoethyl_2_pn2 Instrument : Exactive Mobile phase solvent : MeOHOperator name : yamada miho Sample solvent : MeOHDate : 7/19/2017 3:11:18 PMInstrumental method : C:\Xcalibur\methods\HESI_100ul\pn_H110_S45.methInstrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University170421_Azoethyl_2_pn2 #14-17 RT: 0.22-0.24 AV: 2 NL: 1.37E8T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

410 415 420 425 430 435 440 445 450 455 460 465 470 475 480

m/z

0

20

40

60

80

100

Rela

tive A

bundance

441.19107

442.19462

419.20943443.19760 457.16509

170421_Azoethyl_2_pn2 #17-19 RT: 0.24-0.27 AV: 2 NL: 1.84E8T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

410 415 420 425 430 435 440 445 450 455 460 465 470 475 480

m/z

0

20

40

60

80

100

Rela

tive A

bundance

441.19100

442.19456

419.20942 443.19759

170421_Azoethyl_2_pn2 #21-23 RT: 0.29-0.31 AV: 2 NL: 2.22E8T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

410 415 420 425 430 435 440 445 450 455 460 465 470 475 480

m/z

0

20

40

60

80

100

Rela

tive A

bundance

441.19109

442.19457

419.20949 443.19757

Sample No. : C:\Xcalibur\...\170595_azoethyl4c_pn Instrument : Exactive NegOperator name : yamada miho Mobile phase solvent : MeOHDate : 8/29/2017 10:13:49 AM Sample solvent : submitting solutionInstrumental method : C:\Xcalibur\methods\HESI_100ul\pn_H80_S30.methInstrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University170595_azoethyl4c_pn #10-14 RT: 0.14-0.20 AV: 3 NL: 6.05E5T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

290 292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

305.07001

307.08550

325.18453311.16887

308.08880297.15328 326.18798312.17223299.25950293.17975

170595_azoethyl4c_pn #14-18 RT: 0.20-0.24 AV: 3 NL: 2.22E6T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

290 292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

305.06991

307.08536

325.18438311.16864

308.08868297.15316 326.18778312.17205299.25940293.17962

170595_azoethyl4c_pn #71-74 RT: 0.96-0.98 AV: 2 NL: 2.35E5T: FTMS {1,2} - p ESI Full ms [150.00-2000.00]

290 292 294 296 298 300 302 304 306 308 310 312 314 316 318 320 322 324 326 328 330

m/z

0

20

40

60

80

100

Rela

tive A

bundance

305.07003

325.18460311.16901307.08566

297.15327326.18800312.17229308.08896293.17954 314.96846299.25952 323.10678 329.27021301.23859

Sample No. : C:\Xcalibur\...\170415_AzoethylTP_pn2 Instrument : Exactive Mobile phase solvent : MeOHOperator name : yamada miho Sample solvent : MeOHDate : 7/20/2017 10:09:53 AMInstrumental method : C:\Xcalibur\methods\HESI_100ul\pn_H80_S30.methInstrumental Analysis Division, Global Facility Center, Creative Research Institution, Hokkaido University170415_AzoethylTP_pn2 #16 RT: 0.22 AV: 1 NL: 2.23E6T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

530 535 540 545 550 555 560 565 570 575 580 585 590

m/z

0

20

40

60

80

100

Rela

tive A

bundance

554.94507

548.92822532.96320 555.94867 580.95453

549.93158 564.97668534.97900 547.36151 556.94965 581.95801572.89526

170415_AzoethylTP_pn2 #18-21 RT: 0.25-0.27 AV: 2 NL: 2.37E6T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

530 535 540 545 550 555 560 565 570 575 580 585 590

m/z

0

20

40

60

80

100

Rela

tive A

bundance

554.94499

548.92830555.94864532.96322 580.95441

547.36123 549.93164 564.97669534.97899 556.94949 581.95826562.92757 576.92682

170415_AzoethylTP_pn2 #23-25 RT: 0.29-0.31 AV: 2 NL: 2.14E6T: FTMS {1,1} + p ESI Full ms [150.00-2000.00]

530 535 540 545 550 555 560 565 570 575 580 585 590

m/z

0

20

40

60

80

100

Rela

tive A

bundance

554.94501

548.92823555.94864532.96321 580.95436

564.97681534.97926 549.93216547.36121 556.94912 562.92747 581.95841576.92696

14

7. Supplementary Movies

Mv01: Movie demonstrates the in-situ photoregulation of F-actin motility by irradiating alternatingly with

365nm(3s) and 510nm (5s). The change in the velocity of fluroscently labelled actin filaments corresponding

to the light irradiation could be seen. After UV irradiation the filaments move slowly and after shining Visible

light the speed increses drastically. The concentration of 4a is 40 µM.

Mv02: This movie demonstrates the effect of trans and cis isomer of 4a on the glycerinated muscle fibre. cis-

4a was added to one fibre and trans-4a added to the other. Fibre shortens after addition of trans-4a solution

while the fibre infused with cis-4a doesn’t show any change in the length. cis-4a was pregenerated before

addition on muscle fibre. Concentration of 4a is 3 mM (total).

Mv03: This movie demonstrates that the photo-induced shortening of muscle fibre is due to the photo-

generated trans isomer and not due to the heat of irradiation. Buffer solution without any 4a is added to one

fibre and to the fibre cis-4a is added. Both the fibres don’t shorten even in 20s after addition of buffer and cis-

4a solution respectively, unlike as seen in the Mv01 where the fibre shortens significantly in 20s duration

after addition of trans-4a. So this again reconfirms that cis-4a doesn’t induce muscle fibre shortening. Both

the fibres are irradiated with 510nm light for 10s. Muscle fibre infused with cis-4a shortenes about 38% of its

initial length while the buffer solution infused fibre doesn’t show any change in the length. Concentration of

4a is 3 mM (total).

Mv04: This movie demostrates the photoinduced shortening of muscle fibre infused in inactive cis –4a

solution. To both the muscle fibres cis-4a solution was added and one fibre was irradiated with 510nm light

for 10s. The irradiated fibre shortens about 31% of its length before irradiation whereas the non-irradiated

muscle fibre exhibits no change in length as shown in the snapshots of the video below.

15

1H NMR and 13C NMR spectra of 1b

(AzoethoxyTP) synthetic steps

8. NMR spectral data

16



17



18



19

1H NMR and 13C NMR spectra of 1d

(AzoethylTP) synthetic steps

20



21



22


23

1H NMR and 13C NMR spectra of 5

24

1H NMR and 13C NMR spectra of 6

25

1H NMR and 13C NMR spectra of 1c

26



27


28


29

31P NMR spectra of 4c, 4d, 4b.

30

5. Mass spectra of AzoTP

molecules

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