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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