Research Report

Md Jahirul Islam2015-12-15

Azo-triphosphate MoleculesAmide sub. AzoTPAzoTP (alkyl chain)AzoTP

Amide sub. AzoMP-PNP

AzoMP-PCP

AzoMP-PNP

Dimethyl sub. AzoTP Dimethyl sub. AzoMP-PCP

AzoMP-PNP PurificationIn

ten

sity

(au

)In

ten

sity

(au

)

Retention Time (min)

Retention Time (min)

Batch-1

Batch-2

Preparative HPLC Profile

Column : TSK-GEL (TOSH BIOSCIENCE)Eluent: TEAB buffer (pH-7.33) 82% - 75% to 10%

and ACN 18% -25% to 90%

Column : TSK-GEL (TOSH BIOSCIENCE)Eluent: TEAB buffer (pH-7.33) 82% - 75% to 10%

and ACN 18% -25% to 90%

AzoTP

AzoMP-PNP

AzoMP-PNHAzoMP

AzoMP-PNP Purification

Analytical HPLC Profile

Retention Time (min)

Retention Time (min)

Inte

nsi

ty (

au)

Inte

nsi

ty (a

u) Column : Mightysil RP-18 (Cica)

Eluent: TEAB buffer (pH-7.33) 85% - 75% to 20%and ACN 20% -25% to 80%

Column : Mightysil RP-18 (Cica)Eluent: Na-Phospahte buffer (pH-6) 85% - 75% to 20%

and ACN 15% -25% to 80%

* Detection wavelength: 327nm and 282nm

Batch-2, Peak-31

Synthesis of Dimethyl sub. AzoTP and AzoMP-PCP

a b c d

gfe

a) mCPBA, ice bath, 3h, EtOAc b) 1,2-phenylenediamine , AcOH, 50 °C, 72h, EtOAc c) acetoxyactyl chloride, TEA, RT, 3h,

DCM d) K2CO3, RT, Overnight, MeOH (with little DMSO), e) Di-tert-butyl N,N-Diisopropylphosphoramidite, 1H-tetrazol,

RT,6h, then mCPBA, ice bath, 1h , dry THF , Ar atm f) TFA, 6h, Ar atm, dry DCM than ion exchange column with TEAB g) Tributyl amine, dry MeOH

y. 30-40% y. 89% y. 90%

y. 42% y. 94% y. quantitative

+

+

*

*

h

h

Synthesis of Dimethyl sub. AzoTP and AzoMP-PCP

y. 95%

y. 92%

h) carbonyldiimidazole, dry DMF, Ar atmosphere, rt, overnight, then ion exchange ( and purification) with SephadexA-25, TEAB as eluent and finally convert into Na salt

NMR of Dimethyl sub. AzoMP as triethyl salt

NMR of Dimethyl sub. AzoTP

NMR of Dimethyl sub. AzoMP-PCP

Purity of Dimethyl sub. AzoTP and AzoMP-PCP

Analytical HPLC Profile Column : Mightysil RP-18 (Cica)Eluent: Na-Phospahte buffer (pH-6) 85% - 75% to 30%

and ACN 15% -25% to 70%

Dimethyl sub. AzoTP

Dimethyl sub. AzoMP-PCP

Retention Time (min)

Retention Time (min)

Inte

nsi

ty (a

u)

Inte

nsi

ty (a

u)

* Detection wavelength: 327nm and 282nm

Future Plan

1. Study the efficiency of newly synthesized (purified) Dimethyl sub. AzoTP in motility asssay

2. Study Dimethyl sub. AzoMP-PCP molecule in motor protein assay as competitive inhibitor

3. Try to use these non-hydrolysable Dimethyl sub. AzoMP-PCP as energy molecule in the kinesin-microtubule system in non-hydrolysable manner

Thank you for kind attention