Article pubs.acs.org/JPCB
Photophysics and Release Kinetics of Enzyme-Activatable Optical Probes Based on H‑Dimerized Fluorophores on Self-Immolative Linkers Jagoda Sloniec, Ute Resch-Genger, and Andreas Hennig* BAM Federal Institute for Materials Research and Testing, D-12489 Berlin, Germany S Supporting Information *
ABSTRACT: A series of activatable optical probes for the model enzyme penicillin G amidase based on intramolecular formation of non-fluorescent H-dimer between two identical dyes were synthesized. The probes are based on a selfimmolative linker, which allows positioning both dyes in close spatial proximity to ensure efficient quenching of probes with absorption and fluorescence emission in the near-infrared (NIR) range. A detailed photophysical investigation of the novel optical probes led to a revision of a previously anticipated quenching mechanism and revealed their potential for optimizing the performance of activatable probes based on H-dimer formation. A kinetic analysis indicated that the fluorescence progress curves can be used to qualitatively extract enzyme kinetic parameters.
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fluorescence resonance energy transfer (FRET), which has been established for the visible range22,23 and is currently extended to the NIR.19−21 However, the shortcomings of FRET-based probes are well-documented and include their less efficient quenching due to direct excitation of the acceptor as well as an imperfect overlap of donor fluorescence and acceptor absorption leading to residual donor fluorescence.24−30 As an alternative, contact-based quenching mechanisms such as Hdimer or H-aggregate formation,29,31 photoinduced electron transfer (PET),29,30,32,33 exciplex formation,26−28 and hydrogen atom abstraction27,28 have been proposed. H-aggregate formation and PET are commonly based on the formation of non-f luorescent ground state complexes,29,31 such that contactbased quenching mechanisms can potentially become much more efficient than FRET; this would theoretically allow elimination of virtually any residual background fluorescence from the inactivated probe.13,24−30,32−35 However, to efficiently utilize a contact-based quenching mechanism, the substrate length, conformation, and flexibility of the peptide plus linker need to be highly optimized to enable optimal dye−quencher interaction. This challenging and time-consuming task must be performed for every new substrate.13,26−30,32−35 Another strategy toward molecularly well-defined probes are fluorogenic probes, in which the fluorescence change is directly coupled to a functional group interconversion; for example, the hydrolysis of an amide into an amine significantly alters the chromophore as such and substrate and product will thus have different spectroscopic properties.36,37 However, also in this case, only a
INTRODUCTION The highly sensitive and specific identification of diseases such as cancer and inflammation at an early stage by fluorescence methods involves contrast agents, which cause pathological tissue to brightly fluoresce in front of a dark background. This requires probes, which absorb and fluoresce in the near-infrared (NIR) spectral region, to bypass concerns related to background absorption, scattering, and autofluorescence and thus allow deep tissue penetration. Conventional optical probes accumulate in pathological tissue either by their physicochemical properties or by a ligand or an antibody targeted against a disease-specific biomarker.1 As an alternative, activatable optical probes have been proposed, which are typically designed to be activated by hydrolytic cleavage by disease-related proteases, e.g., by cathepsins, caspases, or matrix metalloproteinases (MMP). This proteolytic cleavage liberates a previously quenched fluorescent dye into its unquenched, brightly fluorescent state, which leads to an increased signal-tobackground ratio compared with conventional probes.2−8 Activatable optical probes based on nanometer-sized polymeric or colloidal carriers have been developed,9−17 but these probes suffer from several shortcomings, for example, from a reduced bioactivity8 and from limited possibilities for fine-tuning their biodistribution,2 which called for the construction of simpler and molecularly more defined probes.18−20 Small molecularly defined activatable probes are commonly designed such that the peptide recognition sequence is flanked on both ends by a fluorescent dye and a quencher, which diffuse apart after proteolytic cleavage of the peptide backbone. One of the challenges in designing molecularly defined probes involves establishing an efficient quenching of the NIR fluorescent dyes.8,16,19−21 The fluorescence quenching mechanism in small molecularly defined probes commonly involves © 2013 American Chemical Society
Received: September 19, 2013 Revised: October 18, 2013 Published: October 21, 2013 14336
dx.doi.org/10.1021/jp409388b | J. Phys. Chem. B 2013, 117, 14336−14344
The Journal of Physical Chemistry B
Article
appropriate path lengths were used to ensure an absorbance
