Maurice Goeldner

Dynamic studies in biology : phototriggers, photoswitches and caged biomolecules

Foreword.Preface.List of Authors.1 Photoremovable Protecting Groups Used for the Caging of Biomolecules.1.1 2-Nitrobenzyl and 7-Nitroindoline Derivatives (John E. T. Corrie).1.2 Coumarin-4-ylmethyl Phototriggers (Toshiaki Furuta).1.3 p-Hydroxyphenacyl: a Photoremovable Protecting Group for Caging Bioactive Substrates (Richard S. Givens and Abraham L. Yousef).1.4 Caging of ATP and Glutamate: a Comparative Analysis (Maurice Goeldner).2 Mechanistic Overview of Phototriggers and Cage Release (Richard S. Givens, Mani B. Kotala, and Jong-Ill Lee).2.1 Introduction.2.2 Major Photoremovable Protecting Groups.2.3 Conclusions.Abbreviations.References.3 Caged Compounds and Solid-Phase Synthesis (Yoshiro Tatsu, Yasushi Shigeri, and Noboru Yumoto).3.1 Introduction.3.2 Solid-Phase Synthesis and Photolysis of Peptides.3.3 Synthesis of Caged Peptides.3.4 Synthesis of Other Photoactive Biomolecular Compounds.3.5 Conclusions and Perspective.References.4 Control of Cellular Activity.4.1 Photochemical Release of Second Messengers - Caged Cyclic Nucleotides (Volker Hagen, Klaus Benndorf, and U. Benjamin Kaupp).4.2 Photochemical Release of Second Messengers - Caged Nitric Oxide (Christopher M. Pavlos, Hua Xu, and John P. Toscano).4.3 Photochemical Release of Neurotransmitters - Transient Kinetic Investigations of Membrane-bound Receptors on the Surface of Cells in the Microsecond-to-Millisecond Time Region (George P. Hess).4.4 Caged Neurotransmitters for Probing Neuronal Circuits, Neuronal Integration, and Synaptic Plasticity (Deda C. Gillespie, Gunsoo Kim, and Karl Kandler).5 Photoregulation of Proteins.5.1 Light-activated Proteins: An Overview (Sandra Loudwig and Hagan Bayley).5.2 Photochemical Enzyme Regulation using Caged Enzyme Modulators (Ling Peng and Maurice Goeldner).5.3 The Use of Caged Proteins in Cell-based Systems (John S. Condeelis and David S. Lawrence).6 Photoremovable Protecting Groups in DNA Synthesis and Microarray Fabrication (Michael C. Pirrung and Vipul S. Rana).6.1 Introduction.6.2 Photoremovable Groups used in Conventional Nucleic Acid Synthesis.6.3 The Photolithographic Method for Microarray Fabrication.6.4 The Future.7 Analytical Time-resolved Studies using Photochemical Triggering Methods.7.1 Time-resolved IR Spectroscopy with Caged Compounds: An Introduction (Andreas Barth).7.2 IR Spectroscopy with Caged Compounds: Selected Applications (Vasanthi Jayaraman).7.3 New Perspectives in Kinetic Protein Crystallography using Caged Compounds (Dominique Bourgeois and Martin Weik).8 Multiphoton Phototriggers for Exploring Cell Physiology (Timothy M. Dore).8.1 Introduction and History.8.2 Theory.8.3 The Two-photon Action Cross-section, deltau.8.4 Chromophores for Two-photon Release of Small Organic Ligands or Metal Ions.8.5 Applications.8.6 Conclusion.9 New Challenges.9.1 Laser-Induced T-Jump Method: A Non-conventional Photoreleasing Approach to Study Protein Folding (Yongjin Zhu, Ting Wang, and Feng Gai).9.2 Early Kinetic Events in Protein Folding: The Development and Applications of Caged Peptides (Sunney I. Chan, Joseph J.-T. Huang, Randy W. Larsen, Ronald S. Rock, and Kirk C. Hansen).9.3 Photocontrol of RNA Processing (Steven G. Chaulk, Oliver A. Kent, and Andrew M. MacMillan).9.4 Light Reversible Suppression of DNA Bioactivity with Cage Compounds (W. Todd Monroe and Frederick R. Haselton).9.5 Photoactivated Gene Expression through Small Molecule Inducers (Sidney B. Cambridge).Subject Index.

Phototriggering of Cell Adhesion by Caged Cyclic RGD Peptides

The controlled and selective adhesion of cells to surfaces is an important issue in cell biology and tissue engineering. Different strategies have been reported in which thermally, photochemically, and electrochemically responsive surfaces and materials are used to manipulate cell adhesion. A more generic approach that would be suitable for any system, independent of its chemical constitution, would be advantageous. Such a strategy could not rely on material properties; instead, the molecular interactions involved in cell attachment must be controlled directly. The design of a strategy to trigger the attachment event needs to consider the sensitivity of cells to most triggering sources (electric fields, chemical stimuli, pressure, and temperature jumps). Light of wavelength above 320 nm appears to be a convenient trigger, as its interaction with biomolecular species is negligible. Light-controlled cellular attachment requires the development of photosensitive molecules able to mediate cellular adhesion and whose activity changes upon irradiation. For this study, we selected the RGD cell-adhesive peptide, well known to promote integrin-mediated cell adhesion, and modified it by introducing a photolabile caging group on the carboxylic acid side chain of the aspartic acid residue (Scheme 1). The presence of the caging group may cause steric hindrance, conformational constraint, or changes in the charge distribution of the peptide and thus prevent recognition of the peptide by the integrins. Light irradiation releases the cage from the peptide structure and restores the activity of the peptide to enable in situ site and temporal control of cell attachment. Cell-repellent surfaces modified with the caged peptide (“off” state) can become cell-adhesive (“on” state) upon irradiation with light of the appropriate wavelength and intensity. The selection of the caging position requires previous knowledge of the structural characteristics of the RGD– integrin binding site. In the particular case of the pentapeptide cyclo(-Arg-Gly-Asp-d-Phe-Val-) (cyclo(RGDfK)), a very active and selective ligand of integrin aVb3, [7] it has been shown that the binding site involves two divalent cations, and that the aspartate unit acts as a ligand for one of them. Therefore, we decided to introduce the caging group at this position. It is also known that the amino acid in the fifth position (Lys) does not have significant influence on the activity of the peptide. The free amine group of the Lys residue has been used as anchoring position through which the peptide can be coupled to surfaces. 3-(4,5-Dimethoxy-2-nitrophenyl)-2-butyl ester (DMNPB) was selected as the photolabile caging group (lmax = 346 nm, emax = 4100m 1 cm ). The caged Asp derivative DMNPBAsp-Fmoc (Fmoc= 9-fluorenylmethoxycarbonyl) and the caged peptide cyclo[RGD(DMNPB)fK] were obtained and characterized as described in the Supporting Information. Their UV spectra are shown in Figure 1. The photolytic properties of the caged peptide in solution were then determined quantitatively. Upon exposure for 2 h to light of wavelength 364 nm in neutral buffered solution, up to 70% of cyclo[RGD(DMNPB)fK] disappeared, and up to 93% of the photolytic reaction product obtained was Scheme 1. Chemical structure of cyclo[RGD(DMNPB)fK] (DMNPB in red) attached to the surface through the TEG linker (green). The caging group is released upon irradiation at 351 nm.

Dynamic deconvolution of a pre-equilibrated dynamic combinatorial library of acetylcholinesterase inhibitors.

A dynamic combinatorial library composed of interconverting acylhydrazones has been generated and screened towards inhibition of acetylcholinesterase from the electric ray Torpedo marmorata. Starting from a small set (13) of initial hydrazide and aldehyde building blocks, a library containing possibly 66 different species was obtained in a single operation. Of all possible acylhydrazones formed, active compounds containing two terminal cationic recognition groups separated by an appropriate distance, permitting two‐site binding, could be rapidly identified by using a dynamic deconvolution–screening procedure, based on the sequential removal of starting building blocks. A very potent bis‐pyridinium inhibitor (Ki=1.09 nM, αKi=2.80 nM) was selected from the process and the contribution of various structural features to inhibitory potency was evaluated.

Water-Soluble, Donor–Acceptor Biphenyl Derivatives in the 2-(o-Nitrophenyl)propyl Series: Highly Efficient Two-Photon Uncaging of the Neurotransmitter γ-Aminobutyric Acid at λ=800 nm†

By using two-photon (TP)[1] photoreleasable neurotransmitters like glutamate (Glu) or γ-aminobutyric acid (GABA), neuronal processes can be activated or inhibited with high temporal and spatial control (with around one micron three-dimensional precision) with reduced photodamage to cells or organs, and with deeper penetration of the light beam into living tissue compared to that of UV photoactivation.[2]

Two-photon uncaging: New prospects in neuroscience and cellular biology.

An uncaging process refers to a fast and efficient release of a biomolecule after photochemical excitation from a photoactivatable precursor. Two-photon excitation produces excited states identical to standard UV excitation while overcoming major limitations when dealing with biological materials, like spatial resolution, tissue penetration and toxicity and has therefore been applied to the uncaging of different biological effectors. A literature survey of two-photon uncaging of biomolecules is described in this article, including applications in cellular- and neurobiology.

Small photoactivatable molecules for controlled fluorescence activation in living cells.

The search for chemical probes which allow a controlled fluorescence activation in living cells represent a major challenge in chemical biology. To be useful, such probes have to be specifically targeted to cellular proteins allowing thereof the analysis of dynamic aspects of this protein in its cellular environment. The present paper describes different methods which have been developed to control cellular fluorescence activation emphasizing the photochemical activation methods known to be orthogonal to most cellular components and, in addition, allowing a spatio-temporal controlled triggering of the fluorescent signal.

Molecular Engineering of Photoremovable Protecting Groups for Two-Photon Uncaging†

The photochemical release of theactive molecule is usually induced by an initial one-photonabsorption process, leading to a limited spatial localization ofthe released substance. To overcome this obstacle, two-photon (TP) excitation has recently emerged as a verypromising technique to obtain spatial control.

Live-Cell One- and Two-Photon Uncaging of a Far-Red Emitting Acridinone Fluorophore

Total synthesis and photophysical properties of PENB-DDAO, a photoactivatable 1,3-dichloro-9,9-dimethyl-9H-acridin-2(7)-one (DDAO) derivative of a far-red emitting fluorophore, are described. The photoremovable group of the DDAO phenolic function comprises a donor/acceptor biphenyl platform which allows an efficient (> or = 95%) and rapid (< 15 micros time-range) release of the fluorescent signal and displays remarkable two-photon uncaging cross sections (delta(a) x Phi(u) = 3.7 GM at 740 nm). PENB-DDAO is cell permeable as demonstrated by the triggering of cytoplasmic red fluorescent signal in HeLa cells after one-photon irradiation (lambda(exc) around 360 nm) or by the generation of a red fluorescent signal in a delineated area of a single cell after two-photon photoactivation (lambda(exc) = 770 nm).

Photolabile Glutamate Protecting Group with High One- and Two-Photon Uncaging Efficiencies

A π‐extended [2‐(2‐nitrophenyl)propoxy]carbonyl (NPPOC) derivative has been prepared as an efficient UV and near‐IR photolabile protecting group for glutamate. This glutamate cage compound exhibits efficient photorelease upon one‐photon excitation (εΦ=990 M−1 cm−1 at 315 nm). In addition, it also shows efficient photorelease in activation of glutamate receptors in electrophysiological recordings. Combined with a high two‐photon uncaging cross‐section (δΦ=0.45 GM at 800 nm), its overall properties make this new cage—3‐(2‐propyl)‐4′‐methoxy‐4‐nitrobiphenyl (PMNB)—for glutamate a very promising tool for two‐photon neuronal studies.

Photochemical tools to study dynamic biological processes.

Light‐responsive biologically active compounds offer the possibility to study the dynamics of biological processes. Phototriggers and photoswitches have been designed, providing the capability to rapidly cause the initiation of wide range of dynamic biological phenomena. We will discuss, in this article, recent developments in the field of light‐triggered chemical tools, specially how two‐photon excitation, “caged” fluorophores, and the photoregulation of protein activities in combination with time‐resolved x‐ray techniques should break new grounds in the understanding of dynamic biological processes.