SUPPLEMENTAL INFORMATION

Figure S1. Interaction of TDZ and its derivatives with Arabidopsis cytokinin receptor CRE1/AHK4 and AHK3. (A) The ability of the compounds to activate the receptor CRE1/AHK4 in an activation assay. Activities of compounds are compared with the activity of tZ and TDZ. The dashed line shows b-galactosidase activity in the non-induced strain (control). Error bars show SD (n = 3). (B, C) Effect of selected compounds on specific binding of 3nM [2-3H]tZ to CRE1/AHK4 (B) and AHK3 (C) in a live-cell binding assay. The concentration of the positive control tZ was 10 nM. To discriminate between the specific and nonspecific binding of [2-3H]tZ on a bacterial membrane, 1 mM tZ was used and this residual value was subtracted from all the data. Adenine (Ade) was used as a negative control. Error bars show SD of two parallel assays, each consisting of two replicates.

Figure S2. Alignment of AtCKX2 (UNIPROT ID: Q9FUJ3) and ZmCKX1 sequence (template structure, PDB ID: 2QKN).

10 20 30 40 50 60 70 80

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

Q9FUJ3 1 MANLRLMITLITVLMITKSSNGIKIDLPK------SLNLTLSTDPSIISAASHDFGNITTVTPGGVICPSSTADISR 71

2QKN 1 ------PWPASLAALALDGKLRTDSNATAAASTDFGNITSALPAAVLYPSSTADLVA 51

90 100 110 120 130 140 150 160

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

Q9FUJ3 72 LLQYAANGKS--TFQVAARGQGHSLNGQASVSGGVIVNMTCITDVV------VSKDKKYADVAAGTLWVDVLKKTAEKGV 143

2QKN 52 LLS-AANSTPGWPYTIAFRGRGHSLMGQAFAPGGVVVNMASLGDAAAPPRINVSADGRYVDAGGEQVWIDVLRASLARGV 130

170 180 190 200 210 220 230 240

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

Q9FUJ3 144 SPVSWTDYLHITVGGTLSNGGIGGQVFRNGPLVSNVLELDVITGKGEMLTCSRQLNPELFYGVLGGLGQFGIITRARIVL 223

2QKN 131 APRSWTDYLYLTVGGTLSNAGISGQAFRHGPQISNVLEMDVITGHGEMVTCSKQLNADLFDAVLGGLGQFGVITRARIAV 210

250 260 270 280 290 300 310 320

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

Q9FUJ3 224 DHAPKRAKWFRMLYSDFTTFTKDQERLISMANDIG----VDYLEGQIF----LSNGVVDTSFFPPSDQSKVADLVKQHG- 294

2QKN 211 EPAPARARWVRLVYTDFAAFSADQERLTAPR----SFGPMSYVEGSVFVNQSLATDLANTGFFTDADVARIVALAGERNA 286

330 340 350 360 370 380 390 400

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

Q9FUJ3 294 -IIYVLEVAKYYDDPN--LPIISKVIDTLTKTLSYLPGFISMHDVAYFDFLNRVHVEENKLRSLGLWELPHPWLNLYVPK 371

2QKN 287 TTVYSIEATLNYDNATAAAAAVDQELASVLGTLSYVEGFAFQRDVAYAAFLDRVHGEEVALNKLGLWRVPHPWLNMFVPR 366

410 420 430 440 450 460 470 480

....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|

Q9FUJ3 372 SRILDFHNGVVKDILLKQKSASGLALLYPTNRNKWDNRMSAMIPEIDEDVIYIIGLL-QSATPKDLPEVESVNEKIIRFC 450

2QKN 367 SRIADFDRGVFKGI-LQGTDIVGPLIVYPLNKSMWDDGMSAATP--SEDVFYAVSLLFSSVAPNDLARLQEQNRRILRFC 443

490 500 510 520 530

....|....|....|....|....|....|....|....|....|....|...

Q9FUJ3 451 KDSGIKIKQYLMHYTSKEDWIEHFG-SKWDDFSKRKDLFDPKKLLSPGQDIF- 501

2QKN 444 DLAGIQYKTYLARHTDRSDWVRHFGAAKWNRFVEMKNKYDPKRLLSPGQDIFN 496


LC-PDA-MS: Ret. time (min), purity, ES+ MS (calculated/found [MH+]) and 1H NMR analyses

Compound 1

LC-PDA-MS: 23.4, 98.6%, 250.28/250.8; 1H NMR (DMSO-d6): 3.83(3H, s, CH3), 6.89(1H, m, ArH), 7.02(2H, m, ArH), 8.00(1H, d, J=8.0, Hz ArH), 8.60(1H, s, thiadiazol-ArH), 8.73(1H, s, NH), 11.25(1H, s, NH).

Compound 2

LC-PDA-MS: 20.8, 97.5%, 236.25/237.1; 1H NMR (DMSO-d6): 6.73(1H, t, J=7.5 Hz, ArH), 6.82(2H, m, ArH), 7.90(1H, d, J=8.0 Hz, ArH), 8.56(1H, s, thiadiazol-ArH), 8.65(1H, s, NH), 10.08(1H, s(br), NH), 11.22(1H, s(br), OH).

Compound 3

LC-PDA-MS: 17.3, 99.4%, 250.28/250.8; 1H NMR (DMSO-d6): 3.75(3H, s, CH3), 6.66(1H, d, J=6.6 Hz, ArH), 7.02(1H, d, J=8.0 Hz, ArH), 7.17(1H, s, ArH), 7.23(1H, t, J=8.0 Hz, ArH), 8.64(1H, s, thiadiazol-ArH), 9.45(1H, s, NH), 10.83(1H, s, NH).

Compound 4

LC-PDA-MS: 15.0, 98.2%, 236.25/237.1; 1H NMR (DMSO-d6): 6.41(1H, d, J=8.0 Hz, ArH), 6.80(1H, d, J=8.0 Hz, ArH), 7.01-7.07(2H, m, ArH), 8.59(1H, s, thiadiazol-ArH), 9.30(1H, s, NH), 9.40 (1H, s(br),OH), 10.71(1H, s, NH).

Compound 5

LC-PDA-MS: 21.0, 99.9%, 280.31/281.3; 1H NMR (DMSO-d6): 3.71(3H, s, CH3), 3.83(3H, s, CH3), 6.59(1H, dd, J1=3.0 Hz, J2=6.0 Hz, ArH), 6.98(1H, d, J=9.0 Hz, ArH), 7.74(1H, d, J=3.0 Hz, ArH), 8.65(1H, s, thiadiazol-ArH), 8.78(1H, s, NH), 11.32(1H, s, NH).

Compound 6

LC-PDA-MS: 12.6, 97.7%, 252.25/253.3; 1H NMR (DMSO-d6): 6.27(1H, dd, J1=3.0 Hz, J2=6.0 Hz, ArH), 6.65(1H, d, J=9.0 Hz, ArH), 7.58(1H, d, J=3.0 Hz, ArH), 8.61(1H, s, thiadiazol-ArH), 8.63(1H, s, OH), 8.77(1H, s, NH), 9.35(1H, s, OH), 11.29(1H, s, NH).

Compound 7

LC-PDA-MS: 16.7, 98.4%, 250.28/250.8; 1H NMR (DMSO-d6): 2.02(3H, s, CH3), 6.70(1H, dd, J1=1.5 Hz, J2=7.5 Hz, CH), 6.94(1H, d, J=7.5, CH), 7.1(1H, d, J=1.5, CH), 8.59(1H, s, thiadiazol-ArH), 9.24(1H, s, NH), 9.34(1H, s, NH), 10.68(1H, s(br), OH).

Compound 8

LC-PDA-MS: 14.6, 98.2%, 250.28/250.8; 1H NMR (DMSO-d6): 4.51(2H, d, J=5.5 Hz, CH2), 5.53(1H, t, J=5.5 Hz, OH), 7.07(1H, t, J=6.6 Hz, ArH), 7.25(1H, t, J=8.0 Hz ArH), 7.30(1H, d, J=7.5 Hz, ArH), 7.76(1H, d, J=8.0 Hz, ArH), 8.56(1H, s, thiadiazol-ArH), 8.85(1H, s, NH), 11.52(1H, s, NH).

Compound 9

LC-PDA-MS: 14.7, 99.6%, 264.31/264.8; 1H NMR (DMSO-d6): 2.72(2H, t, J= 6.5 Hz, CH2), 3.60(2H, q, J1=6.5 Hz, J2=4.0 Hz, CH2), 5.02(1H, t, J=4.0 Hz, OH), 7.08(1H, t, J=7.5 Hz, ArH), 7.16-7.24(2H, m, ArH), 7.58(1H, t, J=7.5 Hz, ArH), 8.56(1H, s, thiadiazol-ArH), 8.85(1H, s, NH), 11.25(1H, s(br), NH).

Compound 10

LC-PDA-MS: 25.6, 98.7%, 304.25/304.1; 1H NMR (DMSO-d6): 7.0(1H, d, J=7.5 Hz, ArH), 7.38-7.43(2H, m, ArH), 7.62(1H, s(br), ArH, 8.62(1H, s, thiadiazol-ArH), 9.75(1H, s, NH), 10.98(1H, s, NH).

Compound 11

LC-PDA-MS: 25.7, 97.9%, 288.25/288.8; 1H NMR (DMSO-d6): 7.24(1H,d, J=7.5 Hz, ArH), 7.39(1H, t, J=8.0 Hz, ArH), 7.54(1H, d, J=8.0 Hz, ArH), 7.80(1H, s(br), ArH), 8.49(1H, s, thiadiazol-ArH), 9.66(1H, s, NH), 10.90(1H, s, NH).

Compound 12

LC-PDA-MS: 22.9, 99.5%, 289.14/288.7; 1H NMR (DMSO-d6): 7.42(1H, dd, J1=2.5 Hz, J2=8.5 Hz, ArH), 7.67(1H, d, J=2.5 Hz, ArH), 8.03(1H, d, J=9.5 Hz, ArH), 8.67(1H, s, thiadiazol-ArH), 9.00(1H, s, NH), 11.34(1H, s, NH).

Compound 13

LC-PDA-MS: 19.8, 97.5%, 250.28/250.8; 1H NMR (DMSO-d6): 4.29(2H, d, J=6.6 Hz, CH2), 6.75(1H, t, J=7.0 Hz, ArH), 6.81(1H, d, J=8.0 Hz, ArH), 7.05-7.13(2H, m, ArH), 7.31(1H, t, J=6.6 Hz, NH), 8.50(1H, s, thiadiazol-ArH), 9.63(1H, s, OH), 10.79(1H, s, NH).

Compound 14

LC-PDA-MS: 22.3, 99.7%, 248.31/249.2; 1H NMR (DMSO-d6): 2.24(3H, s, CH3), 4.29(2H, d, J=5.5 Hz, CH2), 7.09-7.13(3H, m, ArH), 7.17(1H, t, J=3.5 Hz, ArH), 7.45(1H, t, J=5.5 Hz, NH), 8.47(1H, s, thiadiazol-ArH), 10.73(1H, s, NH).

Compound 15

LC-PDA-MS: 17.1, 98.2%, 250.28/250.8; 1H NMR (DMSO-d6): 4.27(2H, d, J=6.6 Hz, CH2), 6.61-6.70(3H, m, ArH), 7.11(1H, t, J=8.0 Hz, ArH), 7.55(1H, t, J=6.6 Hz, NH), 8.50(1H, s, thiadiazol-ArH), 9.36(1H, s, OH), 10.76(1H, s, NH).

Compound 16

LC-PDA-MS: 23.2, 98.4%, 268.73/269.1; 1H NMR (DMSO-d6): 4.36(2H, d, J=6.6 Hz, CH2), 7.23-7.40(4H, m, ArH), 7.65(1H, t, J=6.6 Hz, NH), 8.50(1H, s, thiadiazol-ArH), 10.95(1H, s, NH).

Compound 17

LC-PDA-MS: 21.0, 97.9%, 252.27/253.1; 1H NMR (DMSO-d6): 4.37(2H, d, J=6.6 Hz, CH2), 7.05-7.15(3H, m, ArH), 7.37(1H, q, J=6.6 Hz, ArH), 7.64(1H, t, J=6.6 Hz, NH), 8.50(1H, s, thiadiazol-ArH), 10.94(1H, s, NH).

Compound 18

LC-PDA-MS: 5.6, 98.5%, 264.31/264.8; 1H NMR (DMSO-d6): 3.57(3H, s, CH3), 3.78(2H, d, J=5.5 Hz, CH2), 6.74(1H, d, J=6.5 Hz, ArH), 6.84(1H, d, J=7.5 Hz, ArH), 6.94(1H, s, ArH), 7.12(1H, t, J=8.0 Hz, ArH), 7.35(1H, t, J=5.5 Hz, NH), 8.48(1H, s, thiadiazol-ArH), 10.08(1H, s, NH).

Compound 19

LC-PDA-MS: 22.0, 98.6%, 264.31/264.8; 1H NMR (DMSO-d6): 1.95(3H, s, CH3), 4.10(2H, d, J=5.5 Hz, CH2), 6.49(1H, t, J=8.0 Hz, ArH), 6.77(2H, t, J=6.0 Hz, ArH), 7.16(1H, t, J=5.5 Hz, NH), 8.27(1H, s, thiadiazol-ArH), 8.41(1H, s, OH) 10.63(1H, s, NH).

Compound 20

LC-PDA-MS: 21.3, 97.7%, 294.33/295.2; 1H NMR (DMSO-d6): 3.62(3H, s, CH3), 3.72(3H, s, CH3), 4.25(2H, d, J=6.0 Hz, CH2), 6.72(1H, d, J=3.5 Hz, ArH), 6.76(1H, dd, J1=9.0 Hz, J2=3.5 Hz, ArH), 7.32(1H, t, J=6.0 Hz, NH), 8.46(1H, s, thiadiazol-ArH), 10.77(1H, s, NH).

Figure S3. 1H NMR (DMSO-d6) spectrum of 3FMTDZ.

Figure S4. 1H NMR (DMSO-d6) spectrum of HETDZ.

Figure S5. MS/MS spectra of HETDZ (A) and 3FMTDZ (B) in graphical form

Supplemental experimental procedure. The mass spectrometry measurements of HETDZ and 3FMTDZ were done on a tandem mass analyser Quatro Micro API with MassLynx data system software (Waters, Manchester, UK). Compounds were diluted from a stock solution of 1 mM in DMSO to 1 µM concentration in methanol. The samples were applied in the form of an in-line spray at the injection rate of 10 μl/min. In the full scan mode, the data acquisition was done in the range 50-500 Da with the scan time 0.5 s, cone voltage 20 V. The electrospray ionization in the positive mode was done using following parameters: source block/desolvation temperature 80/150 °C, capillary voltage 2500 V, spray/cone gas flow (nitrogen) 50/500 l/h. For the MS/MS experiments, the fragmentation was done in argon gas-filled collision cell with the collision energy 20 eV. Other parameters were the same as in the simple MS experiments.

1