摘要:

IntroductionAn explosion of techniques has energized the bioorthogonal ligation chemistry field, with new methodologies involving oxime and hydrazone formation, Staudinger ligation, and azide–alkyne [3+2] cycloaddition.1Although many areas of chemistry benefit from click chemistry, the toxicity of the Cu catalyst in the [3+2] azide–alkyne cycloaddition has prohibitedin vivoapplications.1d,1eThus, room-temperature labeling reagents that avoid using copper are highly desirable.Similarly, click reactions provide a means for unique surface functionalization and labelling strategies with application in nanotechnology and surface science. For example, the Heath group and others have developed strategies for functionalizing Si and Au surfaces with azides, which could be attached to alkyne-terminated labels or rotaxanes for sensors or nanomechanical systems.2Here it would be most useful to avoid the requirement of copper catalysts while accelerating the reactions to be fast at room temperature.Recently, the Bertozzi group3demonstrated a new strain-release cyclooctyne labelling reagent (1,Scheme 1) that proceeds in the absence of a Cu catalyst at physiological temperatures, although with aca.52-fold reduction in reaction rate.1aThe increased reactivity of cyclooctyne toward [3+2] cycloaddition has been attributed to strain release, and estimates of the cyclooctyne ring strain range from 10–19 kcal/mol.4The ground-state destabilizing effect of the triple bond is thought to drive the bioorthogonal ligation reactions (e.g.attachment of chemical reporter molecules) in the absence of a catalyst; however, this has been challenged with the concept of dipole distortion.4d,9Bertozzi demonstrated thatgem-difluoro substitution α to the alkyne leads to a reaction rate 63 times faster than the simple cyclooctyne, which has been attributed to the greater LUMO-lowering effect of the fluoro substituent.3cIn addition, dibenzocyclooctyne reagents have allowed visualization of glycoconjugates in living cells using confocal microscopy while a difluorinated cyclooctyne derivative has proven useful for investigatingin vivoglycan trafficking dynamics.3c,5Strain-release azide–alkyne cycloaddition reactions.First, we investigated the best current bioorthogonal cyclooctyne-based labelling reagents to gain insight into reactivity patterns associated with electronic, steric, and strain effects by assessing trends in the activation energies, thus providing a basis of comparison for potential monobenzocyclooctyne reagents. In addition, we investigated the decomposition mechanism observed for a monofluorinated cyclooctyne reagent. Next, we assessed dibenzocyclooctyne- and azacyclooctyne-based reagents and compared the calculated barriers to the cyclooctynes. Finally, we investigated new monobenzocyclooctynes and compared the calculated barriers to that of cyclooctyne and dibenzocyclooctyne reagents. To date, these monocyclooctynes have not been used in bioorthogonal ligation chemistry but provide improved reactivity compared to previously reported cyclooctyne-based reagents.

ISSN:1477-0520    

DOI:10.1039/b911482c

出版商:RSC    

年代:2009

数据来源: RSC

摘要:

IntroductionThe rational design of molecules that exhibit fluorescence properties has gained much interest over the past decade. Among them, luminescent lanthanide complexes with remarkable physical and chemical properties have been developed and found many applications, especially in the field of bioanalytical chemistry.1,2For instance, the time-resolved fluorescence resonance energy transfer (TR-FRET) technique takes advantage of the attracting properties of lanthanide chelates. Indeed, their long-lived excited states along with characteristic narrow emission bands make them ideal for fluorescent background discrimination and delayed emission signal detection. Long decay times result from forbidden transitions involving 4f orbitals and, as a consequence, the molar absorption coefficient of these complexes are very low and typically less than 1 M−1cm−1.3,4Most of the time, effective excitation cannot be performed directly, butviaa light-harvesting antenna that acts as a sensitizer. During that process (termed the “antenna effect”), UV/visible light energy is collected by an allowed antenna-centered absorption. Antenna excitation is followed by a non-radiative intramolecular energy transfer from the excited states of the antenna to Ln(iii), resulting in a radiative metal-centered luminescence.5,6The luminescent properties of lanthanide ions, such as characteristic narrow-line-like emission bands, depend on how efficiently their excited state(s) can be populated and non-radiative deactivation paths minimized. Excitation mainly depends on antenna efficiency (high extinction coefficient, efficient S1→T1intersystem crossing rates, proximity to the metal centre) whereas deactivation is a complex phenomenon. A main route of non-radiative decay occurs by energy transfer from the excited state of Ln(iii) to the O–H vibrational oscillator.7Thus a good shielding of the metal from the surrounding O–H oscillators of the solvent is key for building an efficient luminescent probe that can be finely tuned by ligand design. The lanthanide ions usually possess high coordination requirements and, for Eu(iii) and Tb(iii), a coordination number of nine is very common. Although such high coordination is difficult to fulfil using a single coordinating ligand, the use of functionalized macrocyclic ligands has come close.8-13In such complexes, the antenna is tethered to the chelate so as it can efficiently sensitize Ln(iii).In the course of our efforts to develop portable point-of-care testing devices (POCT),14,15we have been interested in designing lanthanide-based probes to be excited by light-emitting diodes (LEDs) for steady-state and time-resolved fluorescence spectroscopy. The luminescent lanthanide chelate is ultimately expected to play the role of FRET donor in homogenous time-resolved fluorescence (HTRF) assays.2,16-18LEDs as an excitation source offer excellent stability, signal-to-noise ratio, power efficiency and economy, and thus are especially suitable for portable instrumentation manufacturing. Although commercially available LEDs cover the whole visible range, only a few can be found with emission wavelengths below 360–370 nm and specific fluorophores are required. At the end of the 1990s, Selvin developed quinolinone-based lanthanide complexes that proved to be suitable for bioanalytical applications but required excitation at 337 nm.19-22More recently, azaxanthone chromophores have been described as europium sensitizers to be excited at 360–405 nm.23-26Coumarins may also be interesting chromophores with regards to their photochemical and photophysical properties. With emission maximum in the range 350–450 nm, they are ideally suited for the sensitization of lanthanides.27–30Though coumarins so far have scarcely been used in the design of lanthanide chelates, some reports describe their conjugation with crown ethers and cryptands or aminopolycarboxylic chelates and their use as fluorescent probes for applications in metal ion detection,31,32time-resolved fluoroimmunoassays,33–35site-specific protein labeling,36distance measurements in biomolecules,37DNA sensing,38or enzyme activity reporting.39Most of these coumarin-based probes suffer from poor stability and relatively low emission quantum yield in aqueous media. This stresses the necessity to develop additional work based on ligand design for improving their properties, and for allowing their use in bioanalytical applications.Recent work by Lakowiczet al.validated the use of LEDs as light sources in bioanalytical studies.30The authors used compound [Eu(1)], a coumarin-sensitized europium probe resulting from the consecutive functionalization of diethylenetriamine pentaacetic acid dianhydride with 7-amino-4-(trifluoromethyl)coumarin, the metal sensitizer, and dodecylamine, the membrane anchor required for their study (Fig. 1). Thus, the efficiency of a microscope-based fluorimeter with UV/visible LED excitation could be demonstrated. These results prompted us to design compound [Eu(2)] which incorporates a metal chelator, a coumarin core as a sensitizer, and a functionalized spacer.34Functionalization of the spacer with a chemically reactive group (azide) was necessary to allow further conjugation under biocompatible conditions with the adequate biomolecule (AZT derivative) for final assay setup. Although compound [Eu(2)] can be satisfactorily excited at 360–370 nm and proved to be fairly stable in water and human plasma, the fluorescence quantum yield was modest. This particular result is attributed to the likely longer distance between the coumarin moiety and the metal centre in [Eu(2)], compared to [Eu(1)].Structure of 7-amino-4-(trifluoromethyl)coumarin-based europium sensitizers.In order to develop HTRF donor probes with improved properties, we planned the relocation of an adequately functionalized coumarin core in the close proximity of the metal centre. Herein we describe our results concerning the design, synthesis, and evaluation of photophysical properties of a series of new functionalized chelating coumarins and of their corresponding europium complexes.

ISSN:1477-0520    

DOI:10.1039/b907579h

出版商:RSC    

年代:2009

数据来源: RSC

摘要:

1979 University of Heidelberg, Dr. rer. nat., Supervisor Professor Heinz A. Staab1979–1981 University of California at Los Angeles (UCLA) Postdoctoral fellow, Supervisor Professor Orville L. Chapman1981–1985 Research Associate, Max-Planck-Institut für medizinische Forschung, Heidelberg1985 University of Heidelberg, Dr. habil.1985 UCLA, Acting Associate Professor1987 UCLA, Associate Professor1989 UCLA, Full Professor of Organic and Bioorganic Chemistry1992 ETH Zürich, Professor of Organic Chemistry2002 to date ETH Zürich, Head of the new Department of Chemistry and Applied Biosciences

ISSN:1477-0520    

DOI:10.1039/b300290j

出版商:RSC    

年代:2003

数据来源: RSC