Revealing ligand-receptor interaction
NMR titration
Jan Novotný May 3, 2017
Jan Novotný
May 3, 2017 1/17
Introduction of reacting partners
Recommended procedure
O Assignment of free receptor: ß -cyklodextrine (ID H, DQF-COSY) O Assignment of free ligand: doxepine (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 ß -cyclodextrin 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
Jan Novotný
May 3, 2017       3 / 17
ID 1H of ß -cyclodext rin in D2O
DQF-COSY of (3 -cyclodextrin 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)
□ |3I ►   < S
Jan Novotný
May 3, 2017       5 / 17
ID 1H of ß -cyclodext rin in D2O
ID *H of doxepin in D20
ID :H of doxepin in D20
Jan Novotný
May 3, 2017       8 / 17
NOESY 700ms of doxepin in D20
Jan Novotný
May 3, 2017       9 / 17
NOESY 700ms of doxepin in D20
M J IH F
E  E D"
l_1    L_2 f-J
C02 - H (ppm)
C2 C| A2 ^ ^1
Jan Novotný
May 3, 2017       9 / 17
NOESY 700ms of doxepin in D20
7.5    /   a \
M   L K J
Jan Novotný
May 3, 2017        10 / 17
NOESY 700ms of doxepin in D20
7.5
6.6
6.8
E 7.0
Cl Cl
7.2
7.4
7.6
Dox
DoxEH3B-
DoxEH4A-
DoxEH2A-HlA
q
DoxZíf4B-H3B[|
3B
6.5
H,C
2B
Z-doxepin
E-doxepin    y \^CH3
DoxZH2B-HŤB
DoxZH2B-H3B
7:5 / a \
M   L K J
6.6
6.8
7.0
7.2
7.4
7.6
6.5
□ |3I ►   < s
Jan Novotný
May 3, 2017        10 / 17
*H NMR titration: ß -cyclodextrin
H NMR titration: (3 -cyclodextrin
doxepimpCD
	J	
e		
0.9620		
3.0444
Jan Novotný
[ppm]
May 3, 2017        11 / 17
*H NMR titration: ß -cyclodextrin
ID *H NMR titration: (3 -cyclodextrin
Inner /3-CD protons H3' and H5' are dominantly exposed to shielding induced by
ring current of aromatic ring.
Jan Novotný
May 3, 2017        12 / 17
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   | '
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
Jan Novotný
May 3, 2017        14 / 17
ID *H NMR titration: doxepin
1.9605 1.6154
doxepin:(3CD
1:0
5:1
3,75:1
1,75:1
1:1
0,75:1
0,2:1
0:1
K,L J i
1.1394
D.9346
7.4
7.2
7.0
6.8
6.6
[ppm]
Find the most perturbed resonances and estimate the time regime of interaction
Jan Novotný
May 3, 2017        14 / 17
ID XH NMR titration: doxepin
One set of broaden signals of doxepin ^> fast/intermediate rate of
exchange.
ROESY 300ms - doxepin:/3 -cyclodextrin=5:l
Jan Novotný
May 3, 2017        15 / 17
ROESY 300ms - doxepin:/3 -cyclodextrin=5:l
Jan Novotný
May 3, 2017        15 / 17
Proposed orientation of doxepine in ß -cyclodextrin
ID H NMR titration: determination of stoichiometry/K^
R + L±=> RL
[rl] [rl]
[r][l] - (r0-[rl])(l0-[rl])
Upon titration we are gradually changing Z_o concentration:
Sr = [RL]SRbound + (Ro - [RL])SRfi
ASr = Sr — órfree
ree
Titration curve of A5(H1') as a function of increasing concentration of doxepine.
-0.01 -
-0.02
-0.03
E
§; -0.04
-0.05
-0.06
-0.07
-0.08
0.001
				EXP —e— FIT-------
\ A8=0.58á \\ \\ V	LR([Ka(cL+cR0) +	1]-sqrt[(Ka(cL+c 5ALR =-0.071 Ka = 2.997e+04	R0)+1)2-4Ka2cRC	)])/[KacR0]
\\ V				
				
				
				
				
0.002 0.003 cL [M]
0.004
0.005
Jan Novotný
May 3, 2017        17 / 17