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Reversible Covalent Bond Toolbox

Yunfeng Cheng, Hanjing Peng, and Binghe Wang
Georgia State University, Atlanta, GA, USA

1 Introduction
2 Interactions between the Boronic Acid Unit and
Nucleophiles/Lewis Bases
3 Electron-Deficient Carbonyl Groups and Their
Interactions with Hydroxyl Groups and Amino
Group
4 Metal–Anion/Ligand Interactions
5 Chemosensors Based on the Michael Addition
Reaction
6 Conclusion
Acknowledgments
References

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INTRODUCTION

In supramolecular chemistry, functional group interactions
play a critical role. The use of covalent interactions, however, is not as common as noncovalent interactions mostly
because of their irreversible nature. This chapter covers those covalent interactions that are readily reversible
and are often used in the construction of supramolecular
assemblies.

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INTERACTIONS BETWEEN THE
BORONIC ACID UNIT AND
NUCLEOPHILES/LEWIS BASES

In applying covalent bond formation in supramolecular chemistry, boronic acids stand out as one of the

most commonly used functional groups because of their
ability to reversibly interact with nucleophiles/Lewis bases.
Scheme 1 shows the binding processes involving boronic
acid. The boron atom of boronic acid 1 has an open shell
with only six valence electrons in its trigonal neutral form,
which renders 1 a Lewis acid and endows its reactivity
with nucleophiles/Lewis bases. As a result, 1 can easily
react with a protic solvent leading to the formation of the
anionic tetrahedron form (2), accompanied with the release
of a proton, which is the origin of its Brnsted acidity.
Besides, 1 can also react with other nucleophiles such as
cyanide, fluoride, and hydroxyl groups as well as amino
groups. As a further extension of its ability to interact with
nucleophiles, the boronic acid moiety has a unique ability to bind to compounds with two or three nucleophiles
strategically positioned to give divalent or trivalent binding. As a result, boronic acid is known to interact with
diols, α-aminoacids, alcohols, aminoalcohols, α-hydroxyl
acids, as well as fluoride and cyanide.1–4 For example,
boronic acid is considered as one of the most promising
building blocks for the design of sensors for carbohydrates
in aqueous solution, due to its intrinsic binding affinity
with multiple hydroxyl groups, which exist on most carbohydrates.4 The specific interactions involve covalent bond
formation with two neighboring hydroxyl groups. In some
cases, trivalent interactions are also possible. Such is the
case of sorbitol binding with phenylboronic acid. Because
carbohydrate recognition is the most common applications,
subsequent discussions of the use of the boronic acid moiety
are focused on carbohydrate sensing.
In designing boronic acid-based “receptors” for carbohydrates (and other hydroxyl containing compounds for
various applications, it is important to take into consideration the key factors including the pKa values of the boronic
acid and the diol, the pH, the dihedral angle of the diol, the
buffer and its ionic strength, the solvent, and the stability

Supramolecular Chemistry: From Molecules to Nanomaterials, Online  2012 John Wiley & Sons, Ltd.
This article is  2012 John Wiley & Sons, Ltd.
This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons, Ltd.
DOI: 10.1002/9780470661345.smc014

8-quinolynylboronic acid-modified thymidine5 -triphosphate (QB-TTP.22 Supramolecular Chemistry: From Molecules to Nanomaterials. peptides.12 Recently. Ltd. 4. the rapid detection and differentiation of different glycosylation patterns in the same protein is not a trivial issue. and proteins scaffolds. Along these lines.4 These areas have been covered in depth in recent reviews3. Since the publication of the seminal papers on boronic acid-based carbohydrate recognition by Czarnik5 and Shinkai6 in the early 1990s.13 Compared to the first-generation of boronic acid-modified TTP (QB-TTP).10. Ltd. the Duggan group studied 4-borono-Lphenylalanine-based PBLs and their affinity for alizarin21 .14. Scheme 2) was also reported by the Wang group. It changes fluorescent properties upon sugar binding (Ka = 73 M−1 with D-fructose) and the fluorescent properties are retained after DNA incorporation (1.8 peptide-based lectin mimics for the recognition of the T-antigen. NB-TTP has its own unique properties.1002/9780470661345. there also have been efforts in making peptidebased boronic acid lectin mimics (PBLs) for glycoproteins. incorporation of a boronic acid into the DNA aptamer would gravitate the selection process toward the glycosylation site. To address this issue. in the modified approach used in the Wang lab.smc014 . selected.9 boronic acid-modified proteins. Glycoprotein is defined as proteins that contain oligosaccharides covalently attached to polypeptide side chains. DNA labeling work. These issues have been comprehensively discussed in a review by Wang and coworkers.5-fold change of fluorescence intensity upon addition of 100 mM D-fructose).4 Earlier efforts were mostly focused on recognition of simple sugars such as mono. and is not dealt with in detail here.19 and Gold. Online  2012 John Wiley & Sons.and disaccharides. 13 Below. the Wang group is working on incorporating boronic acid-modified thymidine into DNA for aptamer selection work for glycoproteins. Tools commonly used include antibodies16 and aptamers.2 Concepts OH B OH O B 3 1 Protic solvent Protic solvent H H HO + − 2H2O HO + 2H2O Protic solvent Protic solvent H H Kapp OH B OH OH O B O OH 2 Scheme 1 O 4 Overall binding equilibrium of phenylboronic acid with a diol. the field has undergone very significant growth.17 Joyce. due to the intrinsic binding affinity of boronic acids with carbohydrates. as well as genomic DNA incorporation. Ltd. It is well known that glycosylation plays a critical role in governing the function and activity of many proteins. a second analog of QB-TTP (NB-TTP. For example. This article is  2012 John Wiley & Sons.17–19 However. Recent efforts have moved to the recognition of biologically important carbohydrate biomarkers and building lectin mimics (boronolectins) using DNA. 15 Although the mechanism of glycosylation is well understood. NB-TTP can be used in applications such as fluorescent DNA aptamer selection. With such fluorescent properties. recent examples are described to illustrate the utility of the boronic acid moiety for recognition of biologically important carbohydrates and in other applications. Recently. there have been efforts in the development of bisboronic acids for the recognition of cell-surface carbohydrate biomarkers in cancer. of the boronic acid. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. there were extensive efforts in the development of boronic acids capable of fluorescent property changes upon binding. The process is also called glycosylation. Such arrays were used for the identification of sucralose from a beverage sample. these methods only focus on the detection of the intact protein without the ability to specifically probe differences in glycosylation. 7 Later on.3 Mentioned below are a few representative examples that show the use of the boronic acid–diol interactions. The central hypothesis is that.18 Specifically. DOI: 10.12. 11 and boronic acid-modified DNA for various applications.1. Scheme 2) was first synthesized and effectively incorporated into DNA through polymerasemediated DNA synthesis. 8 and are not discussed. The general aptamer selection method and idea were developed about 20 years ago by the labs of Szostak. Lavigne and coworkers selected their PBLs for the detection of glycoproteins according to response patterns by arrays of unidentified peptide–boronic acids libraries20 . and the Anslyn group reported a chemosensor array of PBLs for saccharides and their derivatives.

Online  2012 John Wiley & Sons. As a specific example.23. this is an O B OH O B HO O O H2N R N H HN H N O O HN O N H CH3O O H N O N H O B O Building block: 6 Figure 1 HO 5 Structures of compound 5 and PBL 6. Ltd. the Schultz lab also reported boronic acid-modified proteins. As a prelude. Specifically.1002/9780470661345.smc014 . the Hall lab first reported ortho-hydroxymethyl phenylboronic acid (5. Ltd. in 2006. Figure 1). 24 This is especially important. Ltd. glycolipids. DOI: 10. and lipopolysaccharides are almost always six-membered ring sugars and linear diols. compound 6 was selected as the best receptor for the Thomsen–Friedenreich (TF) antigen (Kd = 0. 7. the Hall group recently developed a library of PBL by incorporating 5 as hexopyranoside-binding agents. a recent study by the Hall lab is used for in-depth discussions. the Schultz lab successfully incorporated a boronic acid-modified phenylalanine (p-boronophenylalanine. This article is  2012 John Wiley & Sons. Compound 5 binds methyl α-D-glucopyranoside with a Ka of 22 M−1 in neutral aqueous media by complexing hexopyranosides primarily using their 4. since carbohydrates found in glycoproteins. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. Figure 2) into protein for the selection of lectin mimics with Supramolecular Chemistry: From Molecules to Nanomaterials. excellent example developing PBLs for carbohydrate-based biomarkers.Reversible covalent bond toolbox 3 O N3 N H O N N N O or N3 O HO B OH QB NB HN H N O NH O HO N OH OH OH O O O P P P O O O N N N O R= O O QB-TTP O O NH O OH OH OH O O O P P P O O O N N HO B OH OH H N R HO OH B OH N N H O O O OH N O R= NB-TTP HN Scheme 2 N N N OH B OH Synthesis of BQ-TTP and NB-TTP. while most boronic acids normally show a preference for furanose sugars in binding. which is implicated in cancer. In addition to PBLs mentioned above. which has a weak but encouraging binding with a model of hexopyranosides (methyl α-D-glucopyranoside). Having this compound on hand.6-diol.9 mM in neutral water).9 After competitive enzyme-linked immunosorbent assay (ELISA) screening of the library. Although further studies such as exploiting the possibility of selectively labeling TF-specific tumor cells is needed to demonstrate full utility.

11 This strategy can also be extended to other uses such as purification of native protein sequences by a one-step scarless affinity procedure as well as in vivo labeling of boronate-containing proteins. there have been efforts in the development of chemosensors based on reversible covalent interactions between electron-deficient carbonyl groups and different nucleophiles. Scheme 3). DOI: 10.25. Supramolecular Chemistry: From Molecules to Nanomaterials. Ltd.43 and cyanides. Ltd.31 Good 7 Structure of p-boronophenylalanine 7. 48 For example. Along this line.25–27 amino groups. It readily reacts with various nucleophiles.1002/9780470661345. a coumarin aldehydebased sensor for amines was developed by the Glass group (8. such as hydroxyl groups.41 sulfide.10. Ltd. Bn N O N O N H g Amino acid sensors based on imine formation. 29–38.smc014 .1 O Interactions with amino groups–reversible Schiff base formation OH HO Figure 2 B OH NH2 Reversible covalent interactions between a carbonyl group and an amino group result in the formation of a Schiff base (imine) or an aminol. enhanced affinities for carbohydrates. A series of similar sensors were also developed by the Glass group using dimers of a quinolone aldehyde chromophore for the recognition of diamines.25. 3 ELECTRON-DEFICIENT CARBONYL GROUPS AND THEIR INTERACTIONS WITH HYDROXYL GROUPS AND AMINO GROUP The carbonyl group has always been an important functional group in molecular recognition.4 Concepts 3.42 hydrozine. It is believed that the hydrogen bond between the iminium ion and the carbonyl group of the coumarin effectively modulates the coumarin fluorescence.44–47 R O H3 N Bu Bu H O O N O O N O 8 X S S Bn N O N H N O H2N O O OH X S S H N N N O Bn 11 10 or or a b e Scheme 4 O or c or or O N H N O Bn X= d or f O 9 NH2 H O Scheme 3 Reversible interaction between coumarin aldehyde 8 and amino acid. This article is  2012 John Wiley & Sons. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. Online  2012 John Wiley & Sons. among which some are reversible. 28–40 bisulfite.35 The coumarin aldehyde 8 displayed a large (34 nm) red shift in absorption and a strong fluorescence increase when exposed to glycine. There have been a number of research reports on taking advantage of such reactions for sensing. 40. thus inducing the change in its spectrometric property.

25. a series of amine sensors have been developed by Mohr and coworkers (Figure 3. 30 Recently. The imprinted polymer selectively binds to the aliphatic diamine with a C3 or C4 carbon chain between the two amino groups (Scheme 7) with an apparent association constant (Kapp = 2. Supramolecular Chemistry: From Molecules to Nanomaterials.smc014 .Reversible covalent bond toolbox in the detection of amines with a distinct color change upon binding (∼50 nm blue shift). propylamine).. 1. ETHT 4001–4014).4-diaminobutane was imprinted into polymer prepared using a dendrimer dye as the monomeric unit.51 The sensor system shows good reversibility and fast response to the exposure of amines (e. Online  2012 John Wiley & Sons. O C6H13 C6H13 O O N N NH2 O O R1 16 (Non-fluorescent) Scheme 6 5 R2 O O R2 N N N O O C6H13 C6H13 R1 17 (Fluorescent) Reversible interaction of compound 16 with aldehyde.4-diaminobutane was removed. 29. 40 and trifluoroacetylazobenzene29 have used NH N B OH B(OH)2 O N O O 12 HO NH2 N O O N H 13 HO O N O N B(OH)2 B O OH NH 14 Scheme 5 15 Catecholamine sensors utilizing both amine–aldehyde and boronic acid–diol interactions. Different linker lengths were utilized to optimize selectivity and binding with various diamines (10a–g. The trifluoroacetyl–amine interaction has also been used in creating artificial receptors using the molecular imprinting method. A similar design from the Yoon group describes an anthracene bearing a phenylboronic acid moiety and an aldehyde carbonyl group for sensing of dopamine. This article is  2012 John Wiley & Sons. the intrinsic affinity of the boronic acid toward the catechol diol group. 48 After the dendrimer was cross-linked using the Grubbs catalyst. DOI: 10. which presumably left behind a complementary cavity. a sol–gelbased optical sensor for the detection of amines in aqueous solution has been developed using ETHT 4001 as the indicator dye.36 The reversible trifluoroacetophenone–amine interaction50 has been extensively studied. Scheme 4). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. For example. there is one that takes advantage of an electron-deficient carbonyl group and a phenylboronic acid moiety in a bifunctional fluorescent sensor (Scheme 5). and has potential applications in the food industry. On the basis of this type of interactions. Ltd. which is 200-fold higher than that for butylamine. and the specifically designed threedimensional arrangement. 28. Ltd. fluorescent compounds such as trifluoroacetylstilbene27.1002/9780470661345.49 Similar types of interactions can also be used in the detection of aldehydes. A fluorescent chemosensor reported by the Mohr group exhibits a “turn-on” fluorescence property upon exposure to an aldehyde (Scheme 6).32 The boronic acid-containing coumarin aldehyde 12 binds selectively with catecholamines such as dopamine and norepinephrine as a result of Schiff base formation. Among these series of sensors. binding constants and large fluorescence changes (∼30 nm red shift) were observed. which included trifluoroacetyl azobenzene as the chromogenic reporter for amine.7 × 104 M−1 ). Ltd. Thus. template 1.34.g.

where hydrogen sulfite is used to stabilize wine during/after fermentation. ETHT 4003 OC11H22 24 R1 = R2 = . Ltd.1002/9780470661345. such as sulfur nucleophiles42. which is easily detectable by the naked eye.3 Interactions with sulfur nucleophiles Diamine O CF3 O N N O N Scheme 7 A cartoon description 1. This is explained by the formation of the thiopyrylium ring (27).42 This reaction is shown in Scheme 8. Ltd. there have been only a limited number of studies of such sensors based on carbonyl groups. ETHT 4014 O O R2 = C2H5 21 R1 = OC11H22 Polymer O R2 = C2H5 Figure 3 Structures of compounds 18–24. the colorimetric sensor for hydrogen sulfite developed by the Mohr group52 possesses an alkylamino azobenzene backbone.4. R2 = C2H5 OC11H22 . The Martinez–Manez lab has developed a highly selective colorimetric sensor for sulfide anion by taking advantage of the pyrylium cycle in a 2. DOI: 10.4-diaminobutane binding to imprinted polymer. After the addition of sulfide at only 5 × 10−5 M. Therefore.2 Interactions with hydroxyl groups Similar to amino groups. Instead of the trifluoroacetyl group. Supramolecular Chemistry: From Molecules to Nanomaterials. which is similar to the amine sensors developed by the same group. This article is  2012 John Wiley & Sons. As an example. 53 3. some sensors developed for amines have similar responses to alcohols. ETHT 4001 19 R1 = C11H22OH. Online  2012 John Wiley & Sons. The reversible covalent interactions between an aldehyde and hydrogen sulfite are useful for detection applications in the wine industry.25–27 The same strategy has been extended to chemosensors for other nucleophiles. 52 and cyanide.6-triphenylpyrylium analog.6 Concepts O R1 N R2 N N CF3 18 R1 = R2 = C8H17. it is conceivable that carbonylbearing fluorophores could also be used in the development of sensors for molecules containing hydroxyl groups. However. the color of the solution changes dramatically from magenta to blue. leading to a color change from pink to orange (Scheme 9). The pyrylium ring is formed when the pH of the solution is adjusted to acidic conditions. thus posing an interference problem. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. 3. Ltd. However. N N N F3C O O O compounds containing hydroxyl groups. hydroxyl groups can also engage in reversible covalent interactions with electron-deficient carboxyl groups. formyl group is used to bind with the bisulfite anion. ETHT 4012 20 R1 = O R3 N R4 CF3 22 R3 = R4 = C8H17. ETHT 4004 23 R1 = R2 = C4H9. due to the wide use of boronic acids in sensing Detection of sulfide in an aqueous solution is very useful in the food industry.smc014 . A 36-nm red shift (from 524 to 488 nm) was observed when the compound binds to bisulfite anion.45–47.

6-disulfonic acid) sensor showed a significant intensity change in absorption in the 433–533 nm range upon binding to cyanide. sulfide..44–47 For example. no response was observed toward other reference anions. In the first example.45 oligothiophene was used as the fluorophore. Ltd. Interactions with cyanides The use of trifluoroacetophenone–anion interactions is not limited to the sensing of amines. and H2 PO4 2− are stabilized by solvation effect.4-dinitrophenylazo)-1-naphthol-3. the convenient detection by naked eye in aqueous media make this agent very useful. leading to an orange-red color in the aqueous solution (H2 O–CH3 OH). shows strong nucleophilicity and attacks the carbonyl group more readily. and bromide. DOI: 10.g. which is a poor H-bond acceptor. chloride. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. acetate) often leads to fluorescence quenching rather than enhancement. Though these four sensors take advantage of different types of chromophores and fluorophores. with a large fluorescence enhancement factor (FEF) of 120.Reversible covalent bond toolbox N N OH SH−. Ltd. thiocyanide.smc014 . The third44 and fourth46 Supramolecular Chemistry: From Molecules to Nanomaterials. This is explained by the fact that anions such as F− . Online  2012 John Wiley & Sons. such as fluoride. H+ OH− OH− 25 Scheme 8 N H+ O O S 26 (Magenta) 27 (Blue) Sulfide anion sensor based on the pyrylium cycle.4 OH SO3H H N N N C8H17 28 (Pink) Scheme 9 7 29 (Orange) Bisulfite sensing using an azobenzene derivative. Chemosensors for cyanides have also been developed on the basis of the trifluoroacetophenone structure. in most others cases. In contrast. Ltd. The high selectivity toward cyanide and S S S S S S CN O O H N O H N O F3C CN F3C 30 (Non-fluorescent) 31 (Fluorescent) NO2 NO2 H N O2N O CF3 H N CN O2N O CF3 CN O O 32 (Colorless) 33 (Orange-red) N N CN H O S O O N O S H O O N CF3 CF3 CN 34 35 (Increased fluorescence intensity) O Et N CN H O CF3 36 Scheme 10 O CN Et N CN H O NC CF3 37 (Decreased fluorescence intensity) Trifluoroacetophenone-based sensors for cyanide. acetate.1002/9780470661345. This article is  2012 John Wiley & Sons. whereas CN− . In the second example in Scheme 10. Such findings are very valuable because. phosphate. the Ahn group has reported four different types of sensors for cyanides (Scheme 10).47 the DNPA (2-(2. leading to the consequent spectrometric property changes. they all include the o-(carboxamido) trifluoroacetophenone moiety in their structures. AcO− . The enhancement in this case is due to the elimination of the quenching processes on the fluorophore by the addition of the anionic guest. the nonfluorescent compound 30 became fluorescent (31). binding of an anion (e. O2N C8H17 C8H17 N N N O2 N O H HSO3− C8H17 3. Upon exposure to cyanide or acetate.

DOI: 10. peptides.1002/9780470661345. Figure 4). The comparatively large and complicated structure of anions also makes selective and efficient sensing difficult. indicator displacement mechanism and fluorescence quenching mechanism. Because of the enormous importance of protein posttranslational phosphorylation.54–64 These sensors are composed of a fluorescent or nonfluorescent multivalent ligand. Many of the sensors based on dimetallic coordination complexes take advantage of the dipicolylamine (DPA) ligand. and phospholipids.65. giving significant fluorescence increase (4–5fold). This versatility in modification and modulation offers the possibility of developing new sensors with diverse structural and spectroscopic properties. namely. anion recognition in aqueous solution has always been challenging because the dominant solvation effect keeps all anions strongly hydrated. The first example is from the Hamachi group (38. Online  2012 John Wiley & Sons.64 This fluorescent chemosensor selectively binds to phosphorylated peptides with both high affinity (107 M−1 ). This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. indicating that the anion-H-bond stabilization process can affect the electron density distribution on both parts. Such interactions have also been used by scientists in the development of binders for anions with impressive results. Ltd. Along a similar line. This is because most of the important biomolecules. examples in Scheme 10 are similar in terms of the binding mechanism. and Cd2+ . and the phenolate oxygen is utilized to bind with Zn and establish a connection between the pyrophosphate binding and the electron density in the fluorophore. are anionic. this type of phosphate sensors has become very useful in the detection and/or understanding of protein phosphorylations. Besides. such as nucleotides. such as Tb and Eu have also been used in some sensors. 4 Zn N 38 Figure 4 Zn2+ O METAL–ANION/LIGAND INTERACTIONS The selective recognition of anions is an important research field. fluorescent lanthanides. This article is  2012 John Wiley & Sons. 66 The two sensors in Figure 5 are both based on the dimetallic complex. Although reversible interactions between electron-deficient carbonyl groups and anions have been utilized in sensor design. Ltd. there are two major types of fluorometric or colorimetric sensing mechanisms. Nature has employed metal–anion/ligand interactions in molecular recognition involving proteins/enzymes. Cu2+ .smc014 .54–56. which could form stable complexes with various metal cations. and some carbohydrates. 61 In terms of detection. In contrast. it does not respond to nonphosphorylated peptides. which coordinates with one or two metal cations. much more work is needed in order to achieve high sensitivity and selectivity. Compound 39 is a colorimetric sensor that shows a red shift of about 50 nm (color from yellow to red) upon addition of NO2 N Zn2+ O− N Zn2+ N N Figure 5 phate. OO N P O O P O O 39 O N N Zn2+ O− N Zn2+ N N N OO N P O O P O O O 40 Colorimetric and fluorometric sensors for pyrophos- Supramolecular Chemistry: From Molecules to Nanomaterials. Ltd. nucleic acids.8 Concepts (NO3−)4 Zn2+ N N N Zn2+ N Zn2+ O P O O N 2+ O O P O O Zn2+ Phosphorylated peptide Phosphorylated peptide Sensor for phosphorylated site of peptide based on DPA–Zn. However. to form a ligand–metal complex. the Hong group designed a series of Zn–DPA-based sensors for pyrophosphate (Figure 5). such as Zn2+ . especially those made by the Gunnlaugsson group. An interesting aspect in these examples is that the fluorophore substitution on the carboxamido group and the phenyl ring can both result in spectrometric property modulation. phospholipids.

and Cu2+ . especially biomolecules containing the phosphate group. K+ . It is believed that the binding of PPi to the Zn cation weakens the bond between Zn and the phenolate oxygen. Online  2012 John Wiley & Sons. sensors with higher selectivity and sensitivity for similar or other biologically important targets are still very much needed. Two different sensors have been developed by the Kikuchi and the Mohr labs. However. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. and inorganic phosphate (Pi). that is. upon expression. Cd2+ .70 In this approach. fluorescence is greatly enhanced as a result of a decrease in quenching resulting from photo-induced electron transfer (PET). Compound 40 is a fluorometric sensor that shows a 9.3diazol) based fluorescent probe (43. Supramolecular Chemistry: From Molecules to Nanomaterials. this sensor might be useful in monitoring the activity of phosphodiesterase. From the above examples. This sensor also displays high selectivity for PPi compared to other anions including Pi. ADP. Ltd. respectively. AMP. Another very interesting example came from the Wang group.1002/9780470661345. one can see that metal–anion interactions have proven to be very useful in the development of sensors for anionic species. or they may be independent fluorescent molecules. 43 also showed very good selectivity against other represented metal ions in living cells. DOI: 10. which then becomes more negatively charged and induces colorimetric changes in the azobenzene fluorophore. Ltd. immobilized Ni2+ absorption chromatography has been used to purify histidine-tagged proteins. and gives a 39 nm red shift in fluorescence upon binding (Figure 6). When the ATP anion comes to bind with the Zn to displace the imine. the protein of interest that needs to be purified is modified using a His-6 label or His-tag. Mg2+ .Reversible covalent bond toolbox F3C O O R N O N O N O 41 Figure 6 N (ClO4−)2 N 43 (Weakly fluorescent) Scheme 11 inorganic pyrophosphate (PPi).6 µM) with 25-fold maximum fluorescence intensity change in phosphate based saline (PBS) buffer (pH = 7. Ni2+ . More dimetallic chemosensors are reviewed in detail by Smith. Co2+ . Ca2+ . Ltd.67 The indicator displacement principle has been utilized in many sensor designs. a polyhistidine residue tagged on the amino or carboxyl terminus. Because of this interesting character. which binds to AMP much more strongly than cAMP. such as Na+ . Besides. The protein of interest can be recovered in a pure state (>95%) by washing the resin with high concentrations (50–100 mM) of imidazole (Figure 7). NH O N Zn2+N N 2+ Zn N N O N 42 Target protein Zn2+ HN NH NH N Cd NH N H N OH R= HN N 9 Ni Target protein Imidazole elution Affinity purification of protein based on Ni–His-tag interaction. The indicator(s) can be incorporated into the sensor itself. This is among the most popular and successful applications of reversible metal–anion interactions in molecular recognition. which is further linked to a naphthalimide fluorophore. Binding washing Figure 7 N NO2 Indicator displacement sensors for AMP and ATP. Reversible metal–anion interactions have also been used in affinity purification of proteins.69 This probe is water-soluble and very sensitive to Zn(II) (Kd = 4.smc014 . The sensor recently reported by the Mohr group68 has a Zn(II) core coordinated by a DPA unit.3). Such proteins can be easily separated from the cell lysate mixture through reversible His-6-Ni interactions. which converts cAMP to AMP. which developed an NBD (7-nitrobenzo-2-oxa-1. This article is  2012 John Wiley & Sons. Mn2+ . Fe3+ . Target protein Ni NO2 44 (Strongly fluorescent) NBD-based sensor for Zn(II). Fe2+ . Hg2+ . The sensor developed by the Kikuchi group63 is a Cd(II) complex. Scheme 11) for zinc ion. For example.5fold fluorescence enhancement upon addition of PPi and good selectivity for PPi compared to ATP.

Yan. D. 96. 71 Specifically. N. 513. G. Org. Ltd. X. C.. Pal. et al.smc014 . Berube. 48.. Bushey. Dwek. Bioorg. Scheme 12). Conroy. Hearty. Reid. M.. v/v) at pH 7. 20. 8220. X. 2. Rev. Especially important are the reversible ones including (i) boronic acid interactions with diols 15. et al. 490. G. Angew. 16. and Nbutylamino-1. Int.61 after addition of a thiol. F. Commun. Rev.. 346. Chem. 2009. 14. H. Top. Gold and C. et al. Chem. 2007. Ltd. D. 23. Chem. 7. M. The extraordinary strength and their readily reversible nature put these covalent interactions in a very unique position for the construction of receptors of high affinity and specificity. Chem. The N-butylamino-1. 2010. J. Szostak. A. Jin. 5.. Shinkai. R. 6 CONCLUSION In conclusion. C. 2008. Gao. C.. 2002. 10.1002/9780470661345. W. Struct. For example. 2002. Curr. 13. and G. M. 159. and (iii) metal– anion/ligand interactions. Y. Nature. 30. 218. 505. 47. Ellington and J. the maleimide group was used as a Michael acceptor. Proc.. J. E. Leonard. The precursor 45 was able to quench fluorescence because of the excited state electron transfer between the fluorophore and the electron-deficient alkene. Cheng. and B.19 to 0. The responses to 2-mercaptoethanol. A. 8. 4. 2005. Work conducted in the authors’ lab was supported by the National Institutes of Health (GM086925 and GM084933) and Georgia State University Molecular Basis of Disease (MBD) program (YFC). with 0. M. Curr. W. Fang. Lett. Jackson.. or ACKNOWLEDGMENTS NH Scheme 12 5 Reactions of sensor 45 with a thiol. P. et al. 1. 1992. Rev. Yoon and A. 18. and L-cysteine were studied in CH3 OH : H2 O (1 : 1. S. Ed. J. Gao. Wang. Shiomi. Lin. 2175. 9. It is well suited for optical sensing since both absorbance and emission wavelengths are in the visible spectral range (λex : 443 nm. Cell Dev. T.. Yang.. 17. C. Tetrahedron.A. Science. 5874. 2007. M. 2003. W.10 Concepts O R'SH R'S O N N R R O 45 O 46 O R= or Bu N O and other nucleophiles/Lewis bases. in these specific cases. Gunnlaugsson and coworkers developed several Michael addition–based “sensors” for alcohols and thiols (45.. 1285. F. Yan. Med. 12. Med. DOI: 10. 171. 249.. Nucleic Acids Res. W. Biol. Chem. nbutanethiol. 10. Engl. 1492. Int. 114. J. and B. Y. et al. and B. Ed. Acad. S. J. However.54–0. et al. 1996. Natl. which results in a fluorescence intensity increase. This article is  2012 John Wiley & Sons. A. J.S.. Rev. 2002. Saisho. the sensors behave more likely as “reagents” than “sensors” because Michael addition–based sensing often depends on simple irreversible reactions. L. K. Curr. Biol. V. Lee. Wang. Hall. S. 1990. Res. Chem. the alkene double bond is saturated after reaction with a thiol (Michael addition). 2010. 17.. Soc. A. Brustad.8-naphthalimide analog stands out as the most sensitive one with fluorescence quantum yield changing from 0. Yang. L. Rabinovich. A. 17688. phenanthrene. Mack. 49. Wang. 105. Opin. Supramolecular Chemistry: From Molecules to Nanomaterials. et al. Ltd. λem : 525 nm). Sci. A. Angew. Yang. and R. 6. R. CHEMOSENSORS BASED ON THE MICHAEL ADDITION REACTION REFERENCES Michael addition is one chemical reaction that has been used for the development of chemosensors. Gao. 25. 3. Tsao. Chem.02 M 4-morpholinopropane sulfonic acid as the buffer.. O’Kennedy. 46. Res. Online  2012 John Wiley & Sons. A. Gao. Res. X. James and S. Tuerk. 683. J. Toscanol. S. 8239.. Dai. Czarnik. 1222.1 M KCl for ionic strength control and 0.. S.39. Wang. 346. U. Med. and D. Semin. Dayan. Liu. In reality. 1992. 12. Z. 35. W. 1990. covalent interactions have proven to be very valuable in the construction of supramolecular assemblies and sensors. X. S. 2010. which is connected to different fluorophores (pyrene. Med.8-naphthalimide) via an aliphatic hydrocarbon spacer. This article was published in the Supramolecular Chemistry: From Molecules to Nanomaterials in 2012 by John Wiley & Sons. (ii) interactions of electron-deficient carbonyl groups with hydroxyl groups and amino group or other nucleophiles. Engl. Huang. L. Chem. 818. 1073. P. 11. W.2. 2008. 6. Kondo. Vasta. Am.

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