Revealing ligand-receptor interaction
NMR titration
Martin Novák, Jan Novotný May 4, 2016
Martin Novák, Jan Novotný
May 4, 2016 1/17
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 / 17
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 / 17
ID H of /3-cyclodextrine in D2O
ID *H of doxepin in D20
ID :H of doxepin in D20
Martin Novák, Jan Novotný
May 4, 2016       8 / 17
NOESY 700ms of doxepin in D20
Martin Novák, Jan Novotný
May 4, 2016       9 / 17
NOESY 700ms of doxepin in D20
M J IH F
E  E D"
C02 - H (ppm)
C2 C| A2 ^ ^1
Martin Novák, Jan Novotný
May 4, 2016       9 / 17
NOESY 700ms of doxepin in D20
Martin Novák, Jan Novotný
May 4, 2016
10 / 17
NOESY 700ms of doxepin in D20
7.5
6.6
DoxĚ
6.8
DOXEH3B-
E 7.0
Cl Cl
7.2
DOXEH4A-H3Á \
DOXEH2A-H1A
7.4
DOXZH4B-H3
7.6
6.5
1B
O 2B 3B
H3CX
	
	V-/ \
4B	
	
	a   4A 3A
Y	Z-doxepin
■6.6
2A
H,C
■6.8
■7.0
E-doxepin    Y \® ^-CH, NH
DOXZH2B-HÍB
DOXZH2B-H3B
■7.2
■7.4
7<5    /   A \
M   L K J
■7.6
6.5
Martin Novák, Jan Novotný
May 4, 2016
10 / 17
1H NMR titration: /3-cyclodextrine
1H NMR titration: /3-cyclodextrine
doxepin:3CD
lartin Novák, Jan Novotný
[ppm]
May 4, 2016        11 / 17
1H NMR titration: /3-cyclodextrine
ID *H NMR titration: ß-cyclodextrine
Inner /3-CD protons H3' and H5' are dominantly exposed to shielding induced by
ring current of aromatic ring.
Martin Novák, Jan Novotný
May 4, 2016        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   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        14 / 17
ID ľH 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
lartin Novák, Jan Novotný
May 4, 2016        14 / 17
ID XH NMR titration: doxepin
One set of broaden signals of doxepin ^> fast/intermediate rate of
exchange.
ROESY 300ms - doxepin:/3-cyclodextrine=5:l
Martin Novák, Jan Novotný
May 4, 2016        15 / 17
ROESY 300ms - doxepin:/3-cyclodextrine=5:l
Martin Novák, Jan Novotný
May 4, 2016        15 / 17
Proposed orientation of doxepin in /5-cyclodextrine
Martin Novák, Jan Novotný
May 4, 2016        16 / 17
ID H NMR titration: determination of stoichiometry//^
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])SRfreĚ
AÖr = Ör - örfn
tree
* HypNMR2008
• log ka = 4.49(7)
E Q.
CL
C/3
"čo o
E
CD .C
Ü
4.95
4.9
Fitting of H1 induced chemical shift
				
		H1expt	•	-
		— m		* <
		/K	X	-X
12 3 4
Concentration Ratio [DOX]/[CD]
0.02	Ind
	CZ
	o
n	CD
	CL
	O
	ZT
-0.02	CD
	3
	o"
-0.04	P
	in
	zr
-0.06	
	^—^
	"O
-0.08	pm
Martin Novák, Jan Novotný
May 4, 2016        17 / 17