Chemistry: analytical and immunological testing – Optical result
Reexamination Certificate
2000-05-02
2003-07-01
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
C436S172000, C552S101000, C564S305000
Reexamination Certificate
active
06586256
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to compounds for quantifying analytes. In particular, the present invention relates to small molecule compounds that have analyte-binding moieties functionally linked to reporter moieties. The reporter moieties of these molecules emit a measurable signal when the analyte-binding moieties interact with the analyte.
BACKGROUND OF THE INVENTION
Chemical sensors or chemosensors are small synthetic molecules that produce a measurable signal upon interaction with a specific analyte. They are used to determine the concentration of an analyte without involving complicated analytical techniques or having to “disturb” the system being analyzed. Numerous uses for chemosensors exist. For example, in the biochemical community, they have been used as sensitive, nondestructive probes for quantifying the amount of a particular analyte in living cells. In the medical industry, chemosensors are used for quickly quantifying the amount of certain analytes in bodily fluids such as blood or urine. Myriad other applications for chemosensors exist, including, for example, monitoring pollutants in waste water and quantifying contaminants in chemical compositions. See,
Chemosensors of Ion and Molecule Recognition
, Desvergne, J- P.; Czarnik, A. W., Eds.; NATO ASI Series C: 492; Kluwer: New York, 1997; “Signaling Recognition Events with Fluorescent Sensors and Switches” de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson T.; Huxley, A. J. M.; McCoy, C. P.; Radermacher, J. T.; Rice, T. E.
Chem. Rev
. 1997, 97, 1515; “Desperately Seeking Sensors” Czamik, A. W.,
Chem. and Bio
. 1995, 2, 423
; Fluorescent Chemosensors for Ion and Molecule Recognition
, Czarnik, A. W. (Ed.), ACS Symp. Set. 538; ACS: Washington D.C., 1993.
Several molecules that can serve as chemosensors have previously been developed (see, e.g., http://www.molecularprobes.com). These compounds are typically designed to have an analyte-binding moiety functionally connected to a fluorescent reporter moiety. Binding of the analyte of interest by the analyte-binding moiety causes a conformational change in the chemosensor molecule that results in the modulation of the fluorescence of the reporter moiety. For example, fluorescence resonance energy transfer (FRET) may result when two fluorescent moieties are brought into close proximity by the steric change of a chemosensor molecule caused by analyte-binding. FRET alters the fluorescence emission of the reporter moiety, resulting in a measurable signal from which the analyte concentration can be extrapolated. Other chemosensor molecules produce a visible color change upon binding an analyte. Analyte-binding by these molecules alters their conformation so that in solution they absorb visible light differently and thereby cause a color change.
Three examples of chemical sensors are shown below.
Compound A acts as a calcium sensor by exhibiting increased fluorescence intensity upon binding free Ca
2+
. “Desperately Seeking Sensors” Czarnik, A. W.,
Chem. and Bio
. 1995, 2, 423. Compound B is a fluorescent sensor for &ggr;-amino butyrate, which functions in aqueous methanol. “Fluorescent Signaling of the Brain Neurotransmitter &ggr;-Aminobutyric Acid and Related Amino Acid Zwitterions.” de Silva, A. P.; Gunaratne, H. Q. N.; McVeigh, C.; Maguire, G. E. M.; Maxwell, P. R. S.; O'Hanlon. E.
Chem. Commun
. 1996, 2191. See also: “Synthesis of an Abiotic Ditopic Receptor Molecule.” Schmidtchen, F. P.
Tetrahedron Lett
. 1984, 25, 4361. Compound C is a selective sensor that changes color upon binding creatinine.
Each particular chemosensor has various advantages and drawbacks that make it suitable for some applications and inappropriate for others. Many compounds are not particularly selective and/or sensitive in analyte-binding. For example, if one prepares a calcium ion sensor that also produces a signal in the presence of magnesium ions, it is not a useful sensor in systems that contain high concentrations of magnesium ions. As another example, because Compound B is a photoinduced electron transfer (PET)-based sensor, it can be activated by molecules aside from amino-butyric acid, including anything that sequesters the lone pair of the crown ether nitrogen (e.g., simple protonation).
Although the need for sensors which recognize small molecule targets is great, few have reached the state of practical utility. A common drawback of many existing chemosensors is that their molecular framework does not allow them to be easily adapted for use with different analytes or reporter readout devices. Thus, chemosensors having a molecular framework to which different moieties can be easily attached would be advantageous. For example, molecules such as, Compound D, bistrityl acetylene (“Uber einige neue Abkommlinge des Triphenylmethans.” Wieland, H.; Kloss, H. Justus Liebigs Ann. Chem. 1929, 4 70, 202-21) or, Compound E, bistrityl butadiyne (“Molecular Design for Hosts in Crystalline Host-Guest Complexes.” Hart, H.; Lin, L.-T. W.; Ward, D. L.
J. Am. Chem. Soc
. 1984,106, 4043-4045) would be useful for preparing frameworks for chemosensor molecules.
SUMMARY OF THE INVENTION
The invention relates to chemical sensors having analyte-binding moieties and reporter moieties covalently attached to a framework composed of two trityl groups connected by a linear spacer such as an ethyne or butadiyne. Each of the three phenyls of the trityl group is substituted at the meta position, two with analyte-binding moieties and one with a reporter moiety. In some cases, one or more of the phenyl groups of each trityl group are also substituted with another chemical group such as a methoxy group. The architecture of the framework allows pre-selected analyte-binding moieties and reporter moieties to be bonded to the framework by simple chemical reactions to form chemosensors useful for quantifying predetermined analytes using predetermined reporter measuring devices. Thus, chemosensors with predetermined characteristics can be readily fashioned by simply reacting appropriate analyte-binding and reporter molecules with the framework.
Each sensor molecule is capable of binding two analyte molecules by chelation of each analyte molecule between each pair of analyte-binding moieties across the linear spacer (e.g., the acetylene axis). An important feature of the invention is that in the absence of analyte, the trityl groups of the sensor molecule rotate freely about the linear spacer. In the presence of analyte, the analyte-binding moieties engage the analyte, thereby stabilizing the molecule in an eclipsed rotamer conformation. This conformation also causes the reporter moieties to be in an eclipsed geometry, a geometry which causes the reporter moieties to signal (e.g., for fluorescent reporters, the signal is modulated fluorescence intensity or a shift in the fluorescence spectrum such as that caused by excimer fluorescence).
Chemosensor molecules within the invention feature two pairs of analyte-binding moieties that can cooperatively bind an analyte. This cooperative recognition enhances the overall affinity of the sensor for the analyte as the first analyte-binding event preorganizes the chemosensor molecule for the second analyte-binding event. Moreover, this double analyte-binding moiety design enhances the selectivity of these the chemosensors of the invention compared to those sensors having other designs.
Accordingly, the present invention features compounds having the structure:
wherein B is an analyte-binding moiety and RM is a reporter moiety. A is the analyte shown interacting with the compounds. In various such compounds, B is a chemical group selected from ethylene diamine, trimethyl ethylenediamine, guanidinium, and the chemical groups shown in
FIGS. 11-15
. In some of these compounds, RM is a chemical group selected from napthyl, pyrenyl acetyl, and the chemical groups shown in FIG.
10
. In a preferred aspect of the invention, B is ethylene diamine and RM is napthyl. In another preferred aspect of the invention, B is guanidinium and RM is napthyl. In ye
Glass Timothy E.
Moran Ricardo
Raker Joseph
Akerman & Senterfitt
Cross Latoya
Kim Stanley A.
The Penn State Research Foundation
Warden Jill
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