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Indicator Displacement Assays for Solute Sensing
Julee ByramMecozzi Group
May 10, 2007
2
Chemical Sensors
Detect the presence and quantity of a specific analyte or group of analytes
Industrial, Environmental, and Clinical Applications
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“Desperately Seeking Sensors”
Czarnik, A.W. Chem. Biol. 1995, 2, 7, 423
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“Desperately Seeking Sensors”
Selectivity- specific analyte recognition
Affinity- high Ka value
Spectral properties- detectable signal modulation
Czarnik, A.W. Chem. Biol. 1995, 2, 7, 423
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Traditional Sensing Method
Schematic Reproduced From: Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 20
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Indicator Displacement Assay (IDA)
Schematic Reproduced From: Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 20
7
Commonly Used Indicators
O
OH
OH
OHSO3
pyrocatechol violet
SO3
OHO
xylenol orange
O
O
OH
OH
HNCO2H
CO2
alizarin complexone
N NCO2
N
methyl red
O O
CO2
CO2
5-carboxyfluorescein
RR
R=CH2N(CH2CO2Na)2
O
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IDA Sensing Systems
OOH
CO2O
O
OH
CH2OSO3
NHSO3
n
OP
OO
PO
O
OO
O
O O
NMe H
OMe
O
O
OO N
HOH
OOC COO
COO
OH
OO
O
OH O
OH
OO
O
O
OH
OH
O
OHHO
O
OHOH
OO
O3P
O3P PO3
Citrate Malate Tartrate
2,3-BPG Cocaine Propranolol
Phenyllactic acid HeparinInositol Triphosphate
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Outline
OOC COO
COO
OH
OO
O
O
OH
OO
O
OH O
OH
Applications and Future Work
Designed Sensors Molecularly Imprinted Polymer Sensors
O NH
OH
Designing Synthetic Receptor Systems
Evolved Sensors
NMe H
OMe
O
O
O
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Designing a Receptor
Complimentary functional groups For Binding Diols
Boronic Acids
For Binding CarboxylatesAmmonium GroupsGuanidinium GroupsUrea, ThioureaAmide
Metal Interactions
Pre-organized Cavity
N
H
HH
N
N
N
H
HH
H
HX
N
N
H
HN H
O
BOH
OH
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Boronic Acids as Binding Groups
Complex saccharides and other 1,2- and 1,3-diols
Form reversible covalent bonds with diols, creating boronic esters
Kinetics of interconversion fast when boron tetrahedral
Incorporation of an amine adjacent to the boronic acid creates a tetrahedral sp3 boron at or near neutral pH
B
OH
OHB
OH
OHOH
BOH
OO
n
B
O
On
HO OHn
HO OHn
OH
H
Wiskur, S.L. et al. Organic Letters 2001, 3, 9, 1311Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 16
BOH
OH
N R1R2
BOH
OH
N R1R2
BO
O
N R1R2
HO OH
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Binding Carboxylates
N
H
HH
O
OHN
N
N
O
O
H
HH
H
H
N H
O
O
OH
AmmoniumGuanidinium
Urea, Thiourea
Amide
X
N
N
O
O
H
H
X = O or S
13
Outline
OOC COO
COO
OH
OO
O
O
OH
Applications and Future Work
Molecularly Imprinted Polymer Sensors
OO
O
OH O
OH
O NH
OH
Designing Synthetic Receptor Systems
NMe H
OMe
O
O
O
Evolved SensorsDesigned Sensors
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Synthetic Citrate Receptors
NH
HN
NH
HN NH
HN
HNNH
HN
HN
HN
NH
NH2
H2N
O
O
HN
NH
NH
H2N NH2
O
O
HN
HN
HNNH2
H2N
O
O
OOC COO
COO
OH
Citrate
Guanidinium Groups
(Anslyn)
Guanidinocarbonyl Pyrrole Groups
(Schmuck)
1.3.5-2,4,6-Functionalized Facially Segregated Benzene
Scaffold
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Citrate Binding Using Guanidinium Groups
Br
Br
Br
N3
N3
N3
NH3
NH3
H3N
THF/H2O
1. PPh3
2. HCl
NaN3
DMF
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
NH
HN
NH
HN NH
HN
HNNH
HN
NH3
NH3
H3N
NHN
SMe
yield 63%
16
Citrate Binding Using Guanidinium Groups
NH
HN
NH
N NH
N
NNH
N
NN
NH
N NH
N
NNH
HN
H
HH
H
HOO
O O
O
O
O
HOO
O O
O
O
O
HH
H
H
H
CO2
CO2
OO O
CO2
CO2
OO 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
17
Citrate Binding Using Guanidinium Groups
NH
HN
NH
HN NH
HN
HNNH
HN
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
H●I
H●C + I
6.9 x 103 M-1
2.4 x 103 M-1
NH
HN
NH
HN NH
HN
HNNH
HN
H3N
NH3
NH3 3.0 x 103 M-1
Kassoc (H●C)
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Citrate Binding Using Guanidinium Groups
OOC COO
COO
OH
OOC COO
COO
OH
O
O
OOC COO
COO
OH
OO
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
OO
O
O
OOC COO
COO
OH
OOC COO
COO
O
O
O
O
O
O
N
NN
N
NH2
O
OHOH
HHHH
OPO
O
O
PO
O
O
PO
O
O
Guest Binding Constant (M-1)
citrate 6.9 x 103
tricarballate 7.3 x 103
succinate 2.1 x 102
glutarate 2.2 x 102
acetate <10
ATP4- 1.2 x 103525 nm
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Citrate Binding Using Guanidinium Groups
Concentrations of Citrate in Beverages (mM) NMR Absorption Emission
Orange Juice 43.1 44.1 44.7
Gatorade 15.95 15.05 15.1
Powerade 12.4 11.1 11.3
All Sport 7.4 7.1 8.1
Mountain Dew 7.95 5.5 5.4
Tonic Water 21 21.15 20.8
Coca Cola 0 0 <0.5
Diet Coke <0.2 <0.4 <0.7
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
20
Citrate BindingUsing Guanidinocarbonyl Pyrrole Groups
H3N
NH3
NH3
NH
HO2C CO2Bz3
1. PyBOP, NMM, DMAP
2. H2, Pd/C, THF/MeOH NH
HN
NH
HN
OH
OO
NH
HO
O
O
HN
OH
O
O
3NH
H2N NHBoc83%
HN
HN
NH
NH2
H2N
O
O
HN
NH
NH
H2N NH2
O
O
HN
HN
HNNH2
H2N
O
O
Br
Br
Br
NHBoc
NHBoc
BocHN
NH3Cl
NH3Cl
ClH3N
1. conc. NH3
2. Boc2O
1. TFA/DCM
2. HCl/H2O
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
1. PyBOP, NMM
2. TFA
72%
yield 63%
21
Citrate BindingUsing Guanidinocarbonyl Pyrrole Groups
HN
HN
NH
NH2
H2N
O
O
HN
NH
NH
H2N NH2
O
O
HN
HN
HNNH2
H2N
O
O
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
+
OO
O
O
OH
OO
O
OH O
OH
OOC COO
COO
OH
O
O
+
1.6 x 105 M-1
Kassoc (H●C)
518 nm
22
Citrate BindingUsing Guanidinocarbonyl Pyrrole Groups
OO
O
O
OH
O
OOOC COO
COO
OH
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
23
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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25
Principle Component Analysis (PCA)
Buryak, A.; Severin, K. J. Am. Chem. Soc. 2005, 127, 3700
26
Artificial Neural Network (ANN)Multi-Layer Perceptron (MLP)
Sensor
1
Sensor
2
Sensor
3
Input OutputHidden
Greene, N.T.; Morgan, S.L.; Shimizu, K.D. Chem. Commun. 2004, 10, 1172
27
Receptors for Tartrate and Malate Sensing
N
N NH
N
NNH
N
BO
O
H
O
O
O
O
HH
HHN
HN NH
HN
NNH
N
BO
O
H
O
O
O
HH
OH
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
N
HN NH
HN
HNNH
HN
BOH
OH
H
N
BHOHO
N
HNNH
HN
BOH
OH
H
H
OO
O OH
O
OO
O
OH
OH
O
Similar affinity for both Greater affinity for tartrate
Tartrate
Malate
Actual Tartrate BindingPredicted Tartrate Binding
28
Combined Sensing of Tartrate and Malate
N
HN NH
HN
HNNH
HN
BOH
OH
H
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
H●A + IH●I
O
O
OH
OH
HNCO2H
CO2
Alizarin ComplexoneOO
O OH
O
OO
O
OH
OH
O
Tartrate
Malate
Similar affinity for both
Kassoc (H●A)
5.5 x 104 M-1
4.8 x 104 M-1
29
Combined Sensing of Tartrate and Malate
Concentrations of Tartrate and Malatein 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
OO
HO OH
OHOH
O
OH
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
OO
O OH
O
OO
O
OH
OH
O
Tartrate ()
Malate (○)
Ascorbate (◊)
Lactate (●)
O
H
HO
H
HO
H
OHOHH H
OH
Glucose (■)
OO
O
O
Succinate (▲)
450 nm
30
Differential Sensing of Tartrate and Malate
O
OH
OH
OHSO3
pyrocatechol violet
N
HN NH
HN
HNNH
HN
BOH
OH
H
N
BHOHO
N
HNNH
HN
BOH
OH
H
H
O
OH
Br
OHSO3
bromopyrogallol red
Br
OHOH
OH
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
OO
O OH
O
OO
O
OH
OH
O
Tartrate
Malate
λmax = 445 nm λmax = 567 nm
31
Differential Sensing of Tartrate and 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
0.6 mM Tartrate
0.2 mM Malate
0.2 mM Tartrate
0.6 mM Malate
Training Set Data
32
Outline
OOC COO
COO
OH
OO
O
O
OH
Applications and Future Work
Molecularly Imprinted Polymer Sensors
OO
O
OH O
OH
O NH
OH
Designing Synthetic Receptor Systems
NMe H
OMe
O
O
O
Evolved SensorsDesigned Sensors
33
Systematic Evolution of Ligands by Exponential Enrichment (SELEX)
Schematic Reproduced From: http://surgery.duke.edu/wysiwyg/images/surgery_SELEX.jpg
34
C
A
T
C
GG
A T C
A T
G C
G C
A T
T
A A
A
Q
G T GA
A
G
TAA
GF
G C
A T
CG G
A TC
A T
G C
G C
A T
T
A AA
G T GA
AG
TAA
QF
Aptamer-Based Sensor for Cocaine
NMe H
OMe
O
O
O
Stojanovic, M.N.; Prada, P.; Landry, D.W. J. Am. Chem. Soc. 2001, 123, 4928
518 nm
472 nm
Kd ~ 100 μM
Cocaine concentration in serum 10-4000 μM
35
G A
G C
G A
A G
G C
A T
C G G
A T CA T
GC
GC
AT
T
A AA
G T G AA
GTAA
G A
G C
G A
A G
G C
A T
C GG
A T C
A TG C
GC
AT
T
A AA
G T G AA
GTAA
Aptamer-Based Sensor for Cocaine
NMe H
OMe
O
O
O
S
N
S
N
Stojanovic, M.N.; Landry, D.W. J. Am. Chem. Soc. 2002, 124, 9678
Kd < 5 μM
36
Aptamer-Based Sensor for Cocaine
NMe H
OMe
OH
ONMe H
O
O
O
O NMe H
OMe
O
O
O
Stojanovic, M.N.; Landry, D.W. J. Am. Chem. Soc. 2002, 124, 9678
3 4 C
0 = blank control
37
Outline
OOC COO
COO
OH
OO
O
O
OH
Applications and Future Work
Molecularly Imprinted Polymer Sensors
OO
O
OH O
OH
O NH
OH
Designing Synthetic Receptor Systems
NMe H
OMe
O
O
O
Evolved SensorsDesigned Sensors
38
Molecularly Imprinted Polymer (MIP) Sensor Array
Greene, N.T.; Shimizu, K.D. J. Am. Chem. Soc. 2005, 127, 5695
39
Molecularly Imprinted Polymer (MIP) Sensor Array
Stephenson, C.J.; Shimizu, K.D. Polym. Int. 2007, 56, 482
40
Molecularly Imprinted Polymer (MIP) Sensor Array
Greene, N.T.; Shimizu, K.D. J. Am. Chem. Soc. 2005, 127, 5695
NO
N
NO2
HNN
O NH
OH NH2
NH2
HN
HN
OH
OH
NH2
NH2
A1
A2
A3
A4
A5
A6
A7
Polymer Template Polymer Template
P0 none P4
P1 P5
P2 P6
P3
Benzofurazan-based Amine Dye
λmax 460 nm
41
Outline
OOC COO
COO
OH
OO
O
O
OH
Applications and Future Work
Molecularly Imprinted Polymer Sensors
OO
O
OH O
OH
O NH
OH
Designing Synthetic Receptor Systems
NMe H
OMe
O
O
O
Evolved SensorsDesigned Sensors
42
Applications and Future
Electronic Tongue
Medical Tests
Food Science
Chemical Warfare
43
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
