Revealing ligand-receptor interaction
NMR titration
Martin Novák, Jan Novotný May 4, 2016
Martin Novák, Jan Novotný
May 4, 2016 1/13
Introduction of reacting partners
cdoxepin ?
CH3
Martin Novák, Jan Novotný
Recommended procedure
O Assignment of free receptor - /3-cyclodextrine (ID H, DQF-COSY) O Assignment of free ligand - doxepin (ID 1H, DQF-COSY, NOESY)
• Identification of proton resonances of ring A and B
• Determination of major and minor conformation of doxepin
O ID NMR titration - rearrangement of /3-cyclodextrine resonaces upon
interaction     identification of inner protons O ID NMR titration - rearrangement of doxepin resonaces upon complexation
estimation of binding mode Q ROESY spectrum of complex: ROE intermolecular contacts O Fitting the titration isotherm
Martin Novák, Jan Novotný
May 4, 2016       3 / 13
ID H of /3-cyclodextrine in D2O
DQF-COSY of /3-cyclodextrine in D20
E
Q_ Q.
CO
f
5.0
e c,d     b,a
4.0 3.5
4.5 4.0 82 - -"-H (ppm)
lartin Novák, Jan Novotný
May 4, 2016       5 / 13
ID *H of doxepin in D20
ID :H of doxepin in D20
Martin Novák, Jan Novotný
May 4, 2016       7 / 13
NOESY 700ms of doxepin in D20
Martin Novák, Jan Novotný
May 4, 2016       8 / 13
NOESY 700ms of doxepin in D20
Martin Novák, Jan Novotný
May 4, 2016
9/13
1H NMR titration: /3-cyclodextrine
*H NMR titration: doxepin
ID *H NMR titration: doxepin
1.9605 1.6154
doxepin:(3CD 5:1
3,75:1
1,75:1
1:1
0,75:1
0,2:1
0:1
1.1021
1.0035
K,L J   i '
1394
0.7815
H6
H-A2
7.4
7.2
7.0
6.8
A
6.6
[ppm]
Find the most perturbed resonances and estimate the time regime of interaction
lartin Novák, Jan Novotný
May 4, 2016        12 / 13
ROESY 300ms - doxepin:/3-cyclodextrine=5:l
Martin Novák, Jan Novotný
May 4, 2016        13 / 13