Revealing ligand-receptor interaction
NMR titration
Jan Novotný
BBiEMBl May 7, 2015 1/17
Introduction of reacting partners
h,c.
HaCdoxepin ?
4H 6QH
6
5
HQ.
/
H
H 2
Q
H
1
3H QHq
jS-CD
7
Jan Novotný
Maý 7, 2015        2 / 17
Recommended procedure
H) Assignment of free receptor - ß-cyklodextrine (1D 1H, DQF-COSY) Pj Assignment of free ligand - doxepine (1D 1H, DQF-COSY, NOESY) o Identification of proton resonances of ring A and B o Determination of major and minor conformation of doxepin ^ 1D NMR titration - rearrangement of ß-cyklodextrine resonaces upon
interaction     identification of inner protons H) 1D NMR titration - rearrangement of doxepin resonaces upon complexation
estimation of binding mode [s> ROESY spectrum of complex: ROE intermolecular contacts Fitting the titration isotherm
Jan Novotný
May 7, 2015
3/17
ID 1H of /3-cyklodextrine in D2O
			[4H   OH 1		í				
		(	"Ha iJfV h1 -   J    OH o	7			c,d		
	f				e				
							b	/	a
						u	u	Á u	v L
	o o o o				c c (I o	) >= 0 ,!<•	r c	n n 0 r	to o> o
fl>
co o
co o
o
cm o
2
5.0
4.5
4.0
Jan Novotný
3.5 [ppm]
Maý 7, 2015       4 / 17
DQF-COSY of ^-cyklodextrine in D2O
3.5-
4.0-
E
q. CP
X
I—i
I—i
to
4.5
5.0
5.0
4.5 4.0 82 - XH (ppm)
b,a
3.5
3.5
□ iS1
3.5
4.0
4.5
5.0
Jan Novotný
Maý 7, 2015        5 / 17
ID 1H of /3-cyklodextrine in D2O
			[4H   OH 1	1			
		(	"Ha i^v h1 3H      OH O 7			H6',H	5'
H	11'			H3'			I   1  A |
						H2'	H4
					y	LI	1
1	o o o o			c c (I o	) >= 0 íjc"	0.9391	1.0196 J
co
.0
6. .0
.0
.0
2
5.0
4.5
4.0
Jan Novotný
3.5 [ppm]
Maý 7, 2015       6 / 17
ID 1H of doxep ine in D2O
1 1								h6	
K,l						E	i	h-b1       P \ H-B2^     \{ JI h-bs"55::^      |J ^ h-b4 ha I a	h-a1 nsh-a2 a4
M	J			Fi			f	1 Z- 3 doxepin	1 »C E-
1		H							
J	11					/	1___v	D''	D'
co o O Cl	m oj T- O	Ol co	0.9346	;		c c c c T	0 0 0 1		
7.5 7.0 6.5 6.0 5.5 5.0 [ppm]
Jan Novotný
Maý 7, 2015       8 / 17
NOESY 700ms of doxepine in D2O
Jan Novotný
Maý 7, 2015       9 / 17
NOESY 700ms of doxepine in D2O
8
7
5
r
3
DoxEHb-H4A'
DoxZHb-H4B
/
V
DoxEHg-H4A
E
CL CL
I
3
DoxEH6'-HlA
4
DoxEH6''-HlA
DoxEH6'-HlB
DoxEHa-H4Bt
5
DoxZHa-H4A
8       / \W7 \ MJIH F1
6E1 E2D"
C02 - 1H (ppm)
6
5
D'
C2C
13 A2 B A1
6
4
3
3
4
5
6
Jan Novotný
Maý 7, 2015       9 / 17
NOESY 700ms of doxepine in D2O
6.6
6.8
E 7.0
q. q.
3
7.2
7.4
7.6
Jan Novotný
7.5
7.0
6.5
6.6
M  L K J   I H
7.0-\
C02-1H (ppm)^ 1
Gi F, F
6.8
7.0
7.2
7.4
7.6
6.5
1
Maý 7, 2015       10 / 17
NOESY 700ms of doxepine in D2O
7.5
7.0
6.5
6.6
6.8
E 7.0
q. q.
3
7.2
7.4
7.6
DoxEH3B-H2B
DoxEH3B-H4B
DoxEH4A-H3A
DoxEH2A-HlA
o
—
DoxEHlA-H
DoxEHlB-H2B
7.5 / —r
M  L K J   I H
4
2B
DoxZH4B-H3B
3B
1    7!0    \ -
C02-1H (ppm)° 1 r2
H,C,
mc
Z-doxepin
B-doxepin    y "v <b CH, NH
I
DoxZH2B-HlB
DoxZH2B-H3B
"X"
6.6
6.8
7.0
7.2
7.4
7.6
F1
6.5
□       [51 ► •< -ž
Jan Novotný
Maý 7, 2015       10 / 17
ID 1H NMR titration: /3-cyklodextrine
0
OJ
Jan Novotný
Maý 7, 2015       11 / 17
CM
ID 1H
NMR titration: //-cyklodextrine
doxepin:3CD
4 H OH
6
3.9
	J	
e		
0.9620		
3.0444
d,e
b
a c
3.7
3.6
b
0.9391
a
1.0196
3.4
0
2
[ppm]
CM
Jan Novotný
May 7, 2015
11 / 17
1D 1H
NMR titration: //-cyklodextrine
doxepin:3CD
4 H OH
6
3.9
H3'
H6',H5'
H6 ,H3' H2'
3.7
3.6
H4' H5'
0
CM
3.5 3.4 [ppm]
H2' H4'
Jan Novotný
Maý 7, 2015
11 / 17
CM
ID 1H NMR titration: /3-cyklodextrine
Inner f3-CD protons H3' and H5' are dominantly exposed to shielding induced by
ring current of aromatic ring.
h6
Jan Novotný
Maý 7, 2015        12 / 17
1D 1H NMR titration: doxepin
[ppm]
Jan Novotný
Maý 7, 2015       13 / 17
1D 1H NMR titration: doxepin
doxepin: 5:1
3,75:1 1,75:1 1:1
0,75:1
0,2:1 0:1
BCD
1.0035
1.9605   1.6154 1.1021
K,L J   | 1.139
1
H6
P H-A1
H-B2
7.4
7.2
7.0
6.8
H-B3
Z-
H-A2
H-A3
N I
CH3
'■y /CH3
H3C
E-
doxepin
6.6
[ppm]
Find the most perturbed resonances and estimate the time regime of interaction:
Jan Novotný
Maý 7, 2015        14 / 17
CO
CM
ID 1H 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 1021
K,L J   | 1139
0.9346
3
h-b1 P~
h-a3
'ch3
E-
[ppm]
Find the most perturbed resonances and estimate the time regime of interaction:
Jan Novotný
Maý 7, 2015        14 / 17
h6
h-a2
co
it
CM
7.4
7.2
7.0
6.8
6.6
ID 1H NMR titration: doxepin
doxepin:pCD    H-A2,A3 H-B4 i_i a a
i--TI-1-H-A4
l_l   a 1 ii I Ji
1:0
5:1
3,75:1 1,75:1 1:1
0,75:1 0,2:1
0:1
H-A1H-A2   H-A4 H-B4 H-B2 H-A3
H-B3
H-A2
H-A3
CH3
E-
[ppm]
One set of broaden signals of doxepin     fast/intermediate rate of
exchange.
Jan Novotný
Maý 7, 2015        14 / 17
7.4
7.2
7.0
6.8
6.6
2.0H
2.5H
E
cL
j: 3.0'
i
3
3.5H
4.0H
4.5-1
8
8
7
11111
i i i i i
M  JIH 7G1F1
5
11111
lit
4
3
11111111111
JL
V
y8
-4-&—
I i \^   I I I I i
6 E
5
CO2 - 1H (ppm)
i i | i i i i | i     T i i
4
3
h2.0
h2.5
■au b d
h3.5
h4.0
M.5
Jan Novotný
Maý 7, 2015        15 / 17
6
f
2.0H
2.5H
E
cL
j: 3.0'
i
3
3.5H
4.0H
4.5-1
8
7
11111
DoxEHb-Hj4A
bCDH5'-DoxEH4B
bCDH6'-DoxEH1A
bCDH3'-DoxEH4B
bcDH5'-DoxEH1B bCDH3'-Do|xEH|B
8
5
4
bCDH3'-DoxEH1B bCDH3'-DoxEH3B
■
bCDH3'-DoxEHla
11111
3
11111111111
DoxEHb-Ha
r
I
DoxEHb-Hc DoxECH3-ttC
DoxEHc-Ha
I      bCDH5'-H6bCDH4'-H4' ^\
bCDH3'-H3'
bCDH2'-H1'
lA"!
i ■ /   / —1—1—r^r-—1—1—1—i
M  JIH 7G1F1
6 E
5
©2 - 1H (ppm)
i i | i i i i | i     T i i
4
3
h2.0
h2.5
b
h3.5
h4.0
M.5
Jan Novotný
Maý 7, 2015        15 / 17
6
f
Proposed orientation of doxepine in ///-cyklodextrine
Jan Novotný
Maý 7, 2015       16 / 17
ID 1H NMR titration: determination of stechiometry/K^
R + L <=► RL
[RL] _ [RL]
[R ][L]
(Ro -[RL])(Lo — [RL])
Upon titration we are gradually changing L0 concentration:
or = [RL]örbound + (Ro - [RL])örfree
Titration curve of A5(H1') as a function of increasing concentration of doxepine.
-0.01
-0.02
-0.03
E
Ü -0.04
-0.05
-0.06
-0.07
-0.08
				EXP o FIT-------
\  A8= 0.58A	LR([Ka(cL+cR0)+	1]-sqrt[(Ka(cL+c x , „__0 071	R0)+1)2-4Ka2CR(	)])/[KaCR0]
\		°ALR = 0.0/1 Ka = 2.997e+04		
\				
V				
				
		- 0-------		
			°—-—-*	>
0.001
0.002
0.003
0.004
0.005
Cl [M]
0
0
Jan Novotný
Maý 7, 2015        17 / 17