Revealing ligand-receptor interaction
NMR titration
Jan Novotný May 2, 2018
Jan Novotný
May 2, 2018 1/17
Introduction of reacting partners
Jan Novotný
May 2, 2018       2 / 17
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 perturbed 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 2, 2018       3 / 17
ID 1H of ß -cyclodext rin in D2O
DQF-COSY of (3 -cyclodextrin in D20
3.5
4.0-
E
Q. Q_
CO
4.5
5.0-
Jan Novotný
f
5.0
5.0
4.5
e c,d b,a
4.0 3.5
4.5 4.0 82 - -"-H (ppm)
3.5
4.0
4.5
5.0
3.5
□ |3I ►   A =
May 2, 2018       5 / 17
ID 1H of ß -cyclodext rin in D2O
ID :H of doxepin in D20
Jan Novotný
□ i3>
May 2, 2018       8 / 17
NOESY 700ms of doxepin in D20
Jan Novotný
May 2, 2018       9 / 17
NOESY 700ms of doxepin in D20
DokEHb-Ha
-DoxEHb
DoxEHb-H4A»
DoxZHb-E43
DoxZHb-Ha
DoxZHb-Hc
CL
S 4
DoxEHg-H4A
DoxE,Hg-Ha
DoxEH6'-H1A
DoxEH6'-
H6
DoxEH6''-H1A
DoxEH6'-H1B
novF,Ha-H4fí,
DoxZHa-H4A
E-doxepin    v \o ^XH3
"T-
8
/ IWj \
M J I H F,
-7^-1-7—^--r-
6p    p ryi 5
l_1 L_2
C02 - H (ppm)
C2 C| A2 ^ ^1
D'
Jan Novotný
May 2, 2018       9 / 17
NOESY 700ms of doxepin in D20
7.5    /   A \
M   L K J
7~T I H
1   n ©2-1H (ppm)
Jan Novotný
May 2, 2018        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-
DoxZíf4B-H3B[|
3B
DoxEH4A-
DoxEH2A-HlA
Q
6.5
H,C
2B
Z-doxepin
E-doxepin    Y \® ^-CH, NH
I
DoxZH2B-HTB
DoxZH2B-H3B
7:5 / A A TTo
M   LK J   I H^.^G, F3
6.5
6.6
6.8
7.0
7.2
7.4
7.6
Jan Novotný
May 2, 2018
10 / 17
*H NMR titration: ß -cyclodextrin
H NMR titration: (3 -cyclodextrin
doxepimpCD
	J	
e		
0.9620		
3.0444
Jan Novotný
[ppm]
May 2, 2018        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 2, 2018        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 2, 2018        14 / 17
ID *H NMR titration: doxepin
doxepin:(3CD
1:0
5:1
3,75:1
1,75:1
1:1
0,75:1
0,2:1
0:1
1.9605   1.6154    1 1(
K,L J |
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 2, 2018        14 / 17
ID ^ NMR titration: doxepin
One set of broaden signals of doxepin ^> fast/intermediate rate of
exchange.
Jan Novotný
May 2, 2018        14 / 17
ROESY 300ms - doxepin:/3 -cyclodextrin=5:l
Jan Novotný
May 2, 2018        15 / 17
ROESY 300ms - doxepin:/3 -cyclodextrin=5:l
Jan Novotný
May 2, 2018        15 / 17
Proposed orientation of doxepine in (3 -cyclodextrin
Jan Novotný
May 2, 2018        16 / 17
ID H NMR titration: determination of stoichiometry//^
R + L±=> RL
[RL] [RL\
[R][L] - (Ro-[RL])(L0-[RL])
Upon titration we are gradually changing Z_o concentration:
Sr = [RL]SRbound + (Ro - [RL])6Rf,
aór = 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
\				EXP —e— FIT-------
\ A8=0.58A t \v	LR([Ka(cL+cRo)+	1]-sqrt[(Ka(cL+c s , „__n A71	R0)+1)2-4Ka2cR(	)])/[KacR0]
i \^		°ALR - I Ka = 2.997e+04		
				
				
				
		~ 0--------		
				>■— -------
_I_
0.001
_I_
0.002
_I_
0.003
0.004
0.005
Jan Novotný
May 2, 2018        17 / 17