Transcript Indicator Displacement Assays for Solute Sensing

Indicator Displacement Assays
for Solute Sensing
Julee Byram
Mecozzi Group
May 10, 2007
1
Chemical Sensors
 Detect the presence and
quantity of a specific analyte or
group of analytes
 Industrial, Environmental, and
Clinical Applications
2
“Desperately Seeking Sensors”
3
Czarnik, A.W. Chem. Biol. 1995, 2, 7, 423
“Desperately Seeking Sensors”
 Selectivity- specific analyte recognition
 Affinity- high Ka value
 Spectral properties- detectable signal modulation
4
Czarnik, A.W. Chem. Biol. 1995, 2, 7, 423
Traditional Sensing Method
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Schematic Reproduced From: Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 20
Indicator Displacement Assay (IDA)
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Schematic Reproduced From: Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 20
Commonly Used Indicators
O
OH
O
O
OH
OH
O
CO2 H
CO 2
HN
CO2
O
SO3
O
OH
CO2
OH
alizarin complexone
pyrocatechol violet
5-carboxyfluorescein
SO3
R
R
CO 2
N N
HO
O
N
R=CH 2 N(CH 2CO2 Na)2
xylenol orange
methyl red
7
IDA Sensing Systems
OH
OOC
O
COO
Malate
Me
O
O
O
O
P
O
OH
O
O
OH
O
Citrate
O
O
O
COO
O
P
O
O
O
O
OH
O
Tartrate
H
O
N
OMe
O
OH
N
H
O
O
2,3-BPG
Propranolol
Cocaine
CH 2OSO 3
OH
O
OH
OH
Phenyllactic acid
O3 P
O
O
O3 P
HO
OH
OH
O PO
3
Inositol Triphosphate
OH O
O
NHSO3
OCO2
n
Heparin
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Outline
Designing Synthetic Receptor Systems
Designed Sensors
Evolved Sensors
OH
OOC
Me
COO
COO
H
N
Molecularly Imprinted
Polymer Sensors
O
OMe
O
O
OH
O
O
O
OH
O
OH
O
O
O
OH
N
H
O
O
Applications and Future Work
9
Designing a Receptor
 Complimentary functional groups
 For Binding Diols
 Boronic Acids
 For Binding Carboxylates
 Ammonium Groups
 Guanidinium Groups
 Urea, Thiourea
 Amide
OH
B
OH
H
N
H
H
H
N H
H
N H
O
X
N
N H
H
N H
N H
 Metal Interactions
 Pre-organized Cavity
10
Boronic Acids as Binding Groups
 Complex saccharides and other
1,2- and 1,3-diols
OH
B
OH
OH
OH
B
OH
OH
 Form reversible covalent bonds
with diols, creating boronic esters
 Kinetics of interconversion fast
when boron tetrahedral
HO
n
OH
O
n
B O
 Incorporation of an amine
adjacent to the boronic acid
creates a tetrahedral sp3 boron at
or near neutral pH
OH
B OH
OH HO
B OH
N R
1
R2
N R
1
R2
OH
HO
n
OH
n
O
O
B
OH
H
O
B O
N R
1
R2
11
Wiskur, S.L. et al. Organic Letters 2001, 3, 9, 1311
Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 16
Binding Carboxylates
H
N H
H
O
H
N
OH
H
N H
O
O
N H
H
Ammonium
Guanidinium
N H
O
H O
O
X
N H
O
X = O or S
Urea, Thiourea
N H
O
Amide
12
Outline
Designing Synthetic Receptor Systems
Designed Sensors
Evolved Sensors
OH
OOC
COO
Me
COO
O
O
O
OMe
O
O
OH
H
N
O
OH
OH
O
Molecularly Imprinted
Polymer Sensors
OH
N
H
O
O
O
O
O
Applications and Future Work
13
Synthetic Citrate Receptors
OH
OOC
1.3.5-2,4,6-Functionalized
Facially Segregated Benzene
Scaffold
COO
COO
Citrate
NH2
H2 N
O
HN
H
N
HN
NH
N
H
N
H
H
N
NH2
NH
H 2N
O
N
H
HN
NH
HN
H
N
O
H
N
O
HN
H2N
NH2
HN
HN
O
O
N
H
Guanidinium Groups
Guanidinocarbonyl Pyrrole Groups
(Anslyn)
(Schmuck)
14
Citrate Binding
Using Guanidinium Groups
Br
N3
NH 3
1. PPh3
THF/H 2O
NaN3
Br
DMF
N3
2. HCl
Br
H 3N
N3
NH3
HN
SMe
NH3
HN
N
H
N
HN
NH
H3 N
NH 3
N
H
N
H
H
N
HN
N
H
yield 63%
Metzger, A.; Lynch, V.M.; Anslyn, E.V. Angew. Chem. Int. Ed. 1997, 36, 862
Hennrich, G.; Anslyn, E.V. Chem. Eur. J. 2002, 8, 2218
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
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Citrate Binding
Using Guanidinium Groups
H
N
H
N
H
N
NH
N
H
O
HO
O
O
O
O
O
O
N
H
H
H
N
N
N
H
O
O
CO 2
CO 2
O
HO
O
O
H
N
N
H
O
O
H
N
O
O
H
O
O
N
H
N
H
N
N
H
H
N
N
H
Metzger, A.; Lynch, V.M.; Anslyn, E.V. Angew. Chem. Int. Ed. 1997, 36, 862
Metzger, A.; Anslyn, E.V. Angew. Chem. Int. Ed. 1998, 37, 649
CO2
CO 2
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Citrate Binding
Using Guanidinium Groups
Kassoc (H●C)
HN
H
N
NH
N
H
H●I
N
H
H
N
HN
6.9 x 103 M-1
HN
N
H
HN
H
N
N
H
H
N
HN
NH
N
H
2.4 x 103 M-1
HN
N
H
H●C + I
NH 3
H 3N
NH 3
3.0 x 103 M-1
Metzger, A.; Lynch, V.M.; Anslyn, E.V. Angew. Chem. Int. Ed. 1997, 36, 862
Metzger, A.; Anslyn, E.V. Angew. Chem. Int. Ed. 1998, 37, 649
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Citrate Binding
Using Guanidinium Groups
Guest
citrate
OH
OOC
6.9 x
OH
103
OOC
COO
COO
O
COO
COO
Binding Constant (M-1)
O
tricarballate
7.3 x 103
OOC
COO
O
O
COO
O
O
succinate
O
2.1 x 102
O
O
O
OH
OOC
COO
glutarate
2.2 x 102
OH
COO
O
O
COO
OOC
O
acetate
O
<10
O
COO
O
525 nm
ATP4-
NH2
1.2 x 103
N
O
O
O
O P O P O P O
O
O
O
Metzger, A.; Lynch, V.M.; Anslyn, E.V. Angew. Chem. Int. Ed. 1997, 36, 862
Metzger, A.; Anslyn, E.V. Angew. Chem. Int. Ed. 1998, 37, 649
N
N
H
H
OH OH
18
H
H
N
O
Citrate Binding
Using Guanidinium Groups
Concentrations of
Citrate in Beverages
(mM)
NMR
Absorption
Emission
43.1
44.1
44.7
Gatorade
15.95
15.05
15.1
Powerade
12.4
11.1
11.3
7.4
7.1
8.1
7.95
5.5
5.4
Tonic Water
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21.15
20.8
Coca Cola
0
0
<0.5
Diet Coke
<0.2
<0.4
<0.7
Orange Juice
All Sport
Mountain Dew
Metzger, A.; Lynch, V.M.; Anslyn, E.V. Angew. Chem. Int. Ed. 1997, 36, 862
Metzger, A.; Anslyn, E.V. Angew. Chem. Int. Ed. 1998, 37, 649
19
Citrate Binding
Using Guanidinocarbonyl Pyrrole Groups
Br
NHBoc
1. conc. NH3
Br
2. Boc2 O
NH3 Cl
1. TFA/DCM
BocHN
2. HCl/H2 O
Br
yield 63%
ClH3 N
NHBoc
NH3 Cl
OH
O
O
HN
NH3
H3 N
HN
1. PyBOP, NMM, DMAP
3 HO2 C
N
H
CO2 Bz
2. H 2, Pd/C, THF/MeOH
O
H
N
O
OH
N
H
NH
3
H 2N
83%
NH3
NHBoc
NH
NH2
O
NH
HO
H2 N
H 2N
O
O
1. PyBOP, NMM
2. TFA
NH2
NH
HN
NH
HN
H
N
72%
O
N
H
O
H2N
O
H
N
NH2
HN
HN
O
O
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
20
Citrate Binding
Using Guanidinocarbonyl Pyrrole Groups
+
+
Kassoc (H●C)
1.6 x 105 M-1
OH
OOC
NH2
H2 N
O
NH2
NH
COO
O
N
H
O
HN
H
N
OH
O
O
HN
NH
O
H 2N
COO
H 2N
O
H
N
O
HN
O
NH2
HN
OH
O
O
OH
O
O
O
O
O
518 nm
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
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Citrate Binding
Using Guanidinocarbonyl Pyrrole Groups
O
O
O
OH
OH
O
O
O
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
OOC
COO
COO
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Multi-analyte Differential Sensing
 Nature often does not use highly selective
receptors
 “Differential” receptors used in arrays
 Response from each of these receptors for a
particular mixture of stimuli creates a pattern
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24
Principle Component Analysis (PCA)
25
Buryak, A.; Severin, K. J. Am. Chem. Soc. 2005, 127, 3700
Artificial Neural Network (ANN)
Multi-Layer Perceptron (MLP)
Hidden
Input
Sensor
1
Sensor
2
Output
Sensor
3
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Greene, N.T.; Morgan, S.L.; Shimizu, K.D. Chem. Commun. 2004, 10, 1172
Receptors for Tartrate and Malate Sensing
OH
B OH
HN
N
H
N
H
H
N
HO
HO B
OH
B OH
HN
O
OH
O
H N
N
OH
H
N
H
O
HN
HN
N
H
N
H
O
Tartrate
O
Malate
O
OH
O
O
Similar affinity for both
Greater affinity for tartrate
O
O
O
B O
N
H
H N
O H
N
N
H
O
H H N
N
N
H
Predicted Tartrate Binding
O
O
B O
N
H
HN
O
N
HN
OH O
H
H
H
N
N
N
H
Actual Tartrate Binding
Wiskur, S.L. et al. Angew. Chem. Int. Ed. 2003, 42, 2070
Lavigne, J.L.; Anslyn, E.V. Angew. Chem. Int. Ed. 1999, 38, 3666
27
Combined Sensing of Tartrate and Malate
OH
B OH
O
HN
HN
N
H
O
O
N
H
H
N
Kassoc (H●A)
OH
OH
Tartrate
5.5 x 104 M-1
O
OH
O
OH
CO2 H
HN
HN
O
N
H
OH
O
O
Similar affinity for both
Malate
4.8 x 104 M-1
O
CO2
Alizarin Complexone
O
H●I
H●A + I
Wiskur, S.L. et al. Angew. Chem. Int. Ed. 2003, 42, 2070
Lavigne, J.L.; Anslyn, E.V. Angew. Chem. Int. Ed. 1999, 38, 3666
28
Combined Sensing of Tartrate and Malate
O
OH
OH
O
O
OH
O
O
OH
Ascorbate (◊)
O
450 nm
O
Succinate (▲)
O
Lactate (●)
HO
HO
HO
OH
O
Malate (○)
O
H OH
O
OH
O
O
OH
O
HO
Tartrate ()
O
O
H
H
H
OH
OH
Glucose (■)
Concentrations of Tartrate and Malate
in Beverages (mM)
NMR
UV/Vis
Ernest & Julio Gallo Sauvignon Blanc
35.6
32.9
Ste. Genevieve Chardonnay
34.1
36.3
Henri Marchant Spumante
26.5
24.9
Talus Merlot
19.5
20.3
Santa Cruz organic white grape juice
43.6
42.3
Welch's grape juice
69.4
71.3
Wiskur, S.L. et al. Angew. Chem. Int. Ed. 2003, 42, 2070
Lavigne, J.L.; Anslyn, E.V. Angew. Chem. Int. Ed. 1999, 38, 3666
29
Differential Sensing of Tartrate and Malate
OH
B OH
OH
B OH
HN
HN
N
H
N
H
H
N
N
H
Br
OH
OH
OH
OH
H N
N
H
N
H
HN
HN
O
O
HO
HO B
N
H
SO3
OH
SO3
OH
OH
OH
Br
pyrocatechol violet
bromopyrogallol red
λmax = 445 nm
λmax = 567 nm
O
OH
O
O
OH
O
Tartrate
O
OH
O
O
Malate
O
Wiskur, S.L. et al. Angew. Chem. Int. Ed. 2003, 42, 2070
Lavigne, J.L.; Anslyn, E.V. Angew. Chem. Int. Ed. 1999, 38, 3666
30
Differential Sensing of Tartrate and Malate
Training Set Data
0.2 mM Tartrate
0.6 mM Malate
0.6 mM Tartrate
0.2 mM Malate
Wiskur, S.L. et al. Angew. Chem. Int. Ed. 2003, 42, 2070
Lavigne, J.L.; Anslyn, E.V. Angew. Chem. Int. Ed. 1999, 38, 3666
31
Outline
Designing Synthetic Receptor Systems
Designed Sensors
Evolved Sensors
OH
OOC
COO
Me
COO
Molecularly Imprinted
Polymer Sensors
H
N
O
OMe
O
OH
O
O
O
OH
O
OH
OH
O
O
O
O
N
H
O
O
Applications and Future Work
32
Systematic Evolution of Ligands by
Exponential Enrichment (SELEX)
33
Schematic Reproduced From: http://surgery.duke.edu/wysiwyg/images/surgery_SELEX.jpg
Aptamer-Based Sensor for Cocaine
C
T
G
518 nm
F
472 nm
Q
G G
T G
G
A
A
Me
H
N
T
A
G
T
C
OMe
O
T
O
A
T
G
C
G
C
A
T
C
G
C
A
T
Kd ~ 100 μM
A
T
A
A A
A
T
G
A
T
G
G G
G
T
C
A
C A
A T
A
O
G
C
C
G
A
A
Q
F
A
Cocaine concentration in serum
10-4000 μM
A
T
A
A
34
Stojanovic, M.N.; Prada, P.; Landry, D.W. J. Am. Chem. Soc. 2001, 123, 4928
Aptamer-Based Sensor for Cocaine
G
A
G
A
G
C
G
C
G
A
G
A
A
G
A
G
G
C
G
C
A
T
A
T
C
G
A
T
Me
H
N
O
OMe
C
G G G
A
T
A
T
T
A
A
A
A
O
A
A
T
G
A
G
N
N
S
T G A
C
A T
A
A
G
C
C
A
T
S
T
A
G G
T
G
C
C
A
A
G
T
G
G
C
T
O
Kd < 5 μM
A
A
Stojanovic, M.N.; Landry, D.W. J. Am. Chem. Soc. 2002, 124, 9678
35
Aptamer-Based Sensor for Cocaine
Me
H
O
N
Me
O
H
O
N
Me
H
N
O
OMe
OMe
O
O
OH
O
3
O
4
C
0 = blank control
Stojanovic, M.N.; Landry, D.W. J. Am. Chem. Soc. 2002, 124, 9678
36
Outline
Designing Synthetic Receptor Systems
Designed Sensors
Evolved Sensors
OH
OOC
COO
Me
COO
Molecularly Imprinted
Polymer Sensors
H
N
O
OMe
O
OH
O
O
OH
O
O
OH
O
OH
N
H
O
O
O
O
O
Applications and Future Work
37
Molecularly Imprinted Polymer (MIP)
Sensor Array
38
Greene, N.T.; Shimizu, K.D. J. Am. Chem. Soc. 2005, 127, 5695
Molecularly Imprinted Polymer (MIP)
Sensor Array
39
Stephenson, C.J.; Shimizu, K.D. Polym. Int. 2007, 56, 482
Molecularly Imprinted Polymer (MIP)
Sensor Array
Polymer
Template
Polymer
Template
NH 2
none
P0
OH
A1
OH
P2
P3
P5
A5
P6
A6
H
N
A2
OH
A4
N
H
O
P1
P4
H
N
NH 2
NH 2
NH2
A7
A3
HN
N
N
Benzofurazan-based Amine Dye
N
λmax 460 nm
O
NO 2
Greene, N.T.; Shimizu, K.D. J. Am. Chem. Soc. 2005, 127, 5695
40
Outline
Designing Synthetic Receptor Systems
Designed Sensors
Evolved Sensors
OH
OOC
COO
Me
COO
Molecularly Imprinted
Polymer Sensors
H
N
O
OMe
O
OH
O
O
OH
O
O
OH
O
OH
N
H
O
O
O
O
O
Applications and Future Work
41
Applications and Future
 Electronic Tongue
 Medical Tests
 Food Science
 Chemical Warfare
42
Acknowledgements
 Professor Sandro Mecozzi
 Mecozzi Group Members
 Peter Anderson
 Jonathan Fast
 Andrew Razgulin
 Practice Talk Attendees










Becca Splain
Maren Buck
Katherine Traynor
Matt Windsor
Claire Poppe
Alex Clemens
Richard Grant
Jessica Menke
Lauren Boyle
Margie Mattmann
 God, Family, and Friends
43