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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
5
Schematic Reproduced From: Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 20
Indicator Displacement Assay (IDA)
6
Schematic Reproduced From: Wiskur, S.L., Ph.D. thesis, University of Texas at Austin, Austin, 2003, 20
Commonly Used Indicators
O
OH O O O
O OH
OH
CO2 H
HN CO 2
CO2
O
SO3 OH
CO2
OH
pyrocatechol violet alizarin complexone 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 O OH O OH
OOC COO O O
O O
COO
O OH O
Citrate Malate Tartrate
H
O Me
N O
O O N
P O O OMe H
O O P OH
O
O O
O O
O
2,3-BPG Cocaine Propranolol
CH 2OSO 3
OH
O
O3 P OH
OH
OH O OH O NHSO3
O3 P O O PO O
OH 3
HO
OCO2
n
Phenyllactic acid Inositol Triphosphate Heparin
8
Outline
Designing Synthetic Receptor Systems
Designed Sensors Evolved Sensors Molecularly Imprinted
OH Polymer Sensors
H
OOC COO Me
N O
COO OMe
O N
O OH O OH O H
OH
O O
O O O
OH O O
Applications and Future Work
9
Designing a Receptor
Complimentary functional groups
For Binding Diols OH
B
Boronic Acids OH
For Binding Carboxylates H
H N H O N H
H
Ammonium Groups N X
H N
Guanidinium Groups H N H
N H
N H
Urea, Thiourea H
Amide
Metal Interactions
Pre-organized Cavity
10
Boronic Acids as Binding Groups
Complex saccharides and other
1,2- and 1,3-diols OH OH
OH
B B
OH OH OH
Form reversible covalent bonds
with diols, creating boronic esters
n n
Kinetics of interconversion fast HO OH HO OH
when boron tetrahedral
O O n
n O
B O B
Incorporation of an amine H OH
adjacent to the boronic acid
creates a tetrahedral sp3 boron at
or near neutral pH
OH OH HO O
OH
B OH B OH B O
N R N R N R
1 1 1
R2 R2 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
H O N H O
H
N H N
H OH N H O
H
Ammonium
Guanidinium
N H O
X O H O
N H O
N H O
X = O or S
Amide
12
Urea, Thiourea
Outline
Designing Synthetic Receptor Systems
Designed Sensors Evolved Sensors Molecularly Imprinted
OH Polymer Sensors
OOC COO H
Me
COO N O
OMe O N
H
O OH OH
O OH O
O
O O
O
OH O O
O
Applications and Future Work
13
Synthetic Citrate Receptors
OH
OOC COO 1.3.5-2,4,6-Functionalized
COO Facially Segregated Benzene
Scaffold
Citrate
NH2
O
H 2N
H2 N NH2 N
H
NH HN
HN O H2N
H NH NH2
N HN N HN O HN
H
NH H H
N H HN O
N N N
H HN O
N O
H
Guanidinium Groups Guanidinocarbonyl Pyrrole Groups
14
(Anslyn) (Schmuck)
Citrate Binding
Using Guanidinium Groups
Br
N3 NH 3
1. PPh3
NaN3 THF/H 2O
Br
DMF N3 2. HCl H 3N
Br
N3 NH3
SMe HN
NH3
H N
HN N N HN
H
NH H
H3 N N N
NH 3
H HN
N
H
yield 63%
Metzger, A.; Lynch, V.M.; Anslyn, E.V. Angew. Chem. Int. Ed. 1997, 36, 862
15
Hennrich, G.; Anslyn, E.V. Chem. Eur. J. 2002, 8, 2218
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Citrate Binding
Using Guanidinium Groups
O
H HO
N O O O O
H
H N O O
N N
H O
NH H O
N H N CO 2
H N
N
H
CO 2 O
HO
O O O O
O H
O
O N
H O H
N H N N CO2
H
N H
N H N
H N
N CO 2
H
16
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
Citrate Binding
Using Guanidinium Groups
Kassoc (H●C)
HN
H
N HN N
H
H●I
N
NH H
N
6.9 x 103 M-1
H HN
N
H
HN
H HN N
N H
N
NH H
N 2.4 x 103 M-1
H HN
N
H
H●C + I
NH 3
H 3N
NH 3 3.0 x 103 M-1
17
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
Citrate Binding
Using Guanidinium Groups
Guest Binding Constant (M-1)
OH
OH citrate 6.9 x 103 OOC COO
OOC COO O COO
COO
O
tricarballate 7.3 x 103 OOC COO
O COO
O
O O
O succinate 2.1 x 102 O
O
O
OH glutarate 2.2 x 102 O O
OOC COO
O O
COO
OH
OOC COO
acetate <10 O
COO O
525 nm NH2
ATP4- 1.2 x 103 N N
O O O N N
O P O P O P O
O O O O
H H
H H 18
Metzger, A.; Lynch, V.M.; Anslyn, E.V. Angew. Chem. Int. Ed. 1997, 36, 862 OH OH
Metzger, A.; Anslyn, E.V. Angew. Chem. Int. Ed. 1998, 37, 649
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
19
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
Citrate Binding
Using Guanidinocarbonyl Pyrrole Groups
Br NHBoc NH3 Cl
1. conc. NH3 1. TFA/DCM
Br BocHN
yield 63%
2. Boc2 O 2. HCl/H2 O ClH3 N
Br NHBoc NH3 Cl
OH
O
HN O
H O
NH3 HN N
1. PyBOP, NMM, DMAP
2. H 2, Pd/C, THF/MeOH O OH NH
H3 N 3 HO2 C CO2 Bz N
N H 3
H 83% H 2N NHBoc
NH3 NH
O NH2
O
NH H 2N
H2 N NH2 N
HO H
O
NH HN
O H2N
1. PyBOP, NMM NH NH2
HN O HN
2. TFA H
N H HN O
72% N
O
O
20
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
Citrate Binding
Using Guanidinocarbonyl Pyrrole Groups
+ +
Kassoc (H●C)
OH
1.6 x 105 M-1
NH2 OOC COO
O COO
H 2N
H2 N NH2 N O OH
H
O
NH HN O
O H 2N O
O OH O
NH NH2
HN O HN O
O O
H
N H HN O OH O
N
O
O
518 nm
21
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
Citrate Binding
Using Guanidinocarbonyl Pyrrole Groups
O OH OH
O
O OOC COO
O
O COO
O
22
Schmuck, C.; Schwegmann, M. J. Am. Chem. Soc. 2005, 127, 3373
Schmuck, C.; Schwegmann, M. Org. Biol. Chem. 2006, 4, 836
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
23
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 Output
Sensor Sensor Sensor
1 2 3
26
Greene, N.T.; Morgan, S.L.; Shimizu, K.D. Chem. Commun. 2004, 10, 1172
Receptors for Tartrate and Malate Sensing
HO O OH
OH OH
HN HO B O
B OH B OH O Tartrate
HN N H N OH O
H
N H N H
H N H N
HN O OH
HN Malate
N N O
H H O
O
Similar affinity for both Greater affinity for tartrate
O
O O
O H N O HN
B O O H B O O
N N HN N
O H OH O H
N N H
H H H N H H N
N N
N N
H H
Predicted Tartrate Binding Actual Tartrate Binding
27
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
Combined Sensing of Tartrate and Malate
Kassoc (H●A)
O OH
OH HN
B OH O
O Tartrate 5.5 x 104 M-1 O OH
HN N
H OH O OH
N CO2 H
H HN
H N
HN CO2
O OH
N Malate 4.8 x 104 M-1 O
H O
O Alizarin Complexone
Similar affinity for both O
H●I H●A + I
28
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
Combined Sensing of Tartrate and Malate
O OH OH
O OH O
O O
O
O O
OH O HO OH O
Tartrate () Ascorbate (◊) Succinate (▲)
H OH
O OH O
HO
O O HO
O HO H
H OH
OH
O H OH
Malate (○) Lactate (●) Glucose (■)
450 nm 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
29
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
Differential Sensing of Tartrate and Malate
HO O O
OH OH
HN HO B OH Br OH
B OH B OH
HN N H N OH
H OH
N H N H OH
H N H N
HN HN SO3 OH SO3 OH
N N OH Br
H H
pyrocatechol violet bromopyrogallol red
λmax = 445 nm λmax = 567 nm
O OH
O
O Tartrate
OH O
O OH
O Malate
O
O
30
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
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
31
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
Outline
Designing Synthetic Receptor Systems
Designed Sensors Evolved Sensors Molecularly Imprinted
OH Polymer Sensors
OOC COO
H
COO Me
N O
OMe O N
H
O OH O OH OH
O O
O
O O
OH O 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 Q
T Q
F
G
518 nm G G
T G G C
A A
H T
F G
A
Me
N O A
G T
G A
472 nm OMe G G G
A C T
G A
C O
C A
T O A T
A T C A A
A T
A T
G C
G C
G C
G C
Kd ~ 100 μM A T
A T
Cocaine concentration in serum T A
T A
10-4000 μM A A
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 Kd < 5 μM
H
G C Me
N O G C
OMe
A T A T
O
G G G T G A G
C O C G G T G A
A A
A G A T C A A T G
T C A A T
A A T
T
G G C
C
G G
C N N
C
A A
T S S T
T T
A A
A A A
A 35
Stojanovic, M.N.; Landry, D.W. J. Am. Chem. Soc. 2002, 124, 9678
Aptamer-Based Sensor for Cocaine
H H H
Me Me Me
N O O N O
N
O OMe OMe
O OH O
O O
3 4 C
0 = blank control
36
Stojanovic, M.N.; Landry, D.W. J. Am. Chem. Soc. 2002, 124, 9678
Outline
Designing Synthetic Receptor Systems
Designed Sensors Evolved Sensors Molecularly Imprinted
OH Polymer Sensors
OOC COO H
Me
COO N O
OMe O N
H
OH
O OH O OH O
O O
O O O
OH 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
P0 none P4 A4
O N
H
OH NH 2
P1 A1 P5 A5
OH
H
N
NH 2
P2 A2 P6 A6
OH
H
N NH2
P3 A3 A7
N
HN
N Benzofurazan-based Amine Dye
O
N λmax 460 nm 40
NO 2
Greene, N.T.; Shimizu, K.D. J. Am. Chem. Soc. 2005, 127, 5695
Outline
Designing Synthetic Receptor Systems
Designed Sensors Evolved Sensors Molecularly Imprinted
OH Polymer Sensors
OOC COO H
Me
COO N O
OMe O N
H
OH
O OH O OH O
O O
O O O
OH 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
