Label for making a charge-transfer fluorescent probe

Details Label for making a charge-transfer fluorescent probe Label for making a charge-transfer fluorescent probe

2000-01-26

2004-11-16

Snay, Jeffrey (Department: 1743)

Chemistry: analytical and immunological testing

Optical result

With fluorescence or luminescence

Reexamination Certificate

active

06818453

ABSTRACT:

The invention pertains to a label which is suitable for making a charge-transfer fluorescent probe, to a charge-transfer fluorescent probe comprising said label, to a diagnostic kit comprising the same, and to a method for detecting a material by using said charge-transfer fluorescent probe.
Donor-bridge-acceptor systems like the interesting fluorescent molecule Fluoroprobe (FP) have already been studied extensively (see, for instance, R. M. Hermant, N. A. C. Bakker, T. Scherer, B. Krijnen, and J. W. Verhoeven,
J.Am.Chem.Soc.
112 (1990) 1214-1221) and used as fluorescent probes (inter alia, J. W. Hofstraat, H. J. Verhey, J. W. Verhoeven, M. U. Kumke, G. Li, S. L. Hemmingsen, and L. B. McGown,
Polymer
38 (1997) 2899-2906). Fluoroprobe shows a characteristic intramolecular charge transfer fluorescence that depends strongly on the polarity and polarizability of the medium; the fluorescence maximum shifts from 407 nm in a non-polar environment, like n-hexane, to 697 nm in polar acetonitrile. The main disadvantage of the Fluoroprobe system lies in its poor absorption characteristics. Fluoroprobe has no significant absorption above 350 nm, which strongly reduces its applicability in (bio)polymer experiments.
It has now been found that these problems can be solved by using a label which is suitable for making a charge-transfer fluorescent probe having a donor-bridge-acceptor structure and a maleimide moiety. These labels exhibit enhanced through-bond interaction between the donor and the acceptor.
The invention therefore relates to a non-fluorescent label which is suitable for making a charge-transfer fluorescent probe having a donor-bridge-acceptor structure, characterized in that the label comprises a maleimide moiety and the donor-bridge-acceptor structure, wherein the bridge is a group which leads to an all-trans orbital coupling of the donor and the acceptor, and the donor-bridge-acceptor structure has a higher energy charge-transfer emissive state than at least one non-emissive state of part of the label comprising the maleimide moiety, which non-emissive state must have a higher energy than the charge-transfer emissive state of the donor-bridge-acceptor structure after coupling of the maleimide moiety to a suitable system.
The maleimide moiety can be any maleimide group which may optionally be substituted or wherein one or both oxygen atoms may be replaced by sulfur. The maleimide moiety can be coupled to a system to obtain a fluorescent probe, preferably a (bio)macromolecular system. Such a system comprises a group which is able to couple to the maleimide moiety, such as a primary or secondary amine, a thiol group, a negatively charged oxygen atom, or an activated oxygen atom having a leaving group attached thereto, a (meth)acrylate, vinyl ether, styrene, or a compound with a diene moiety which is able to react with the maleimide moiety in a Diels-Alder reaction. The maleimide moiety preferably is the unsubstituted N-maleimidoyl group.
Preferably, the acceptor moiety is an unsaturated or aromatic moiety having an electron-withdrawing group. Electron-withdrawing groups are known in the art and comprise, inter alia, nitrile, nitro, carbonyl, and halogen groups. Suitable aromatic groups are phenyl, naphthyl, furanyl, and thienyl groups, which may be substituted or may contain hetero atoms (for phenyl and naphthyl groups). If unsaturated moieties contain more than one unsaturated bond, these bonds are conjugated bonds.
The donor moiety comprises an aromatic moiety with an electron-donating group. The aromatic moiety is, preferably, phenyl or naphthyl, whereas the electron-donating group is a nitrogen, oxygen, sulfur, or phosphorous atom. Preferably the electron donating group is a nitrogen atom. This atom is directly attached to the aromatic moiety. When the electron-donating moiety is a nitrogen atom, the nitrogen atom of the maleimide moiety may be the electron-donating group.
The bridge moiety enables the transfer of charge from the donor moiety to the acceptor moiety. This bridge moiety is a group which leads to an all-trans orbital coupling of the donor and the acceptor. Examples are conjugated unsaturated groups and groups without conjugated unsaturated bonds but with a configuration which leads to the all-trans orbital coupling of orbitals of the donor with orbitals of the acceptor. Such groups lead to a fixed axial orientation between the donor and the acceptor.
A specifically useful label has the formula:
The label of this invention can be coupled to systems comprising nitrogen, sulfur or oxygen atoms, or unsaturated bonds which are able to react with the maleimide moiety. Particularly, (bio)macromolecular systems to obtain a charge-transfer fluorescent probe are useful. Such a probe can be used in diagnostic kits.
The invention further comprises a method for detecting a material by using the above-mentioned charge-transfer fluorescent probe. Excitation of such probes is performed in the 350-850 nm, more preferably in the 365-650 nm, range. Fluorescence is measured in the 370-1100 nm range.


REFERENCES:
Krijnen B. et al. “Effect of Through-Bod Interaction on Conformation and Structure of Some N-Arylpiperidone and N-Aryltropanone Derivatives” J. Am. Chem. Soc. (1989) vol. 111, pp. 4433-4440.*
Goes M. et al. “A Blue Excitable Charge-Transfer Fluroescent Probe and Its Fluorogenic Derivative” Eur. J. Org. Chem. (1998) pp. 2373-2377.*
Verhey H.J. et al “A Fluorogenic Charge-Transfer Polarity Probe for the Derivatization of Thiols and Amines” New Journal of Chemistry, (1996) vol. 20, pp. 809-814.

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