Richard S. Givens

Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

The review covers the knowledge on photoremovable protecting groups and includes all relevant chromophores studied in the time period of 2000–2012; the most relevant earlier works are also discussed.

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.

Deoxycholate Interacts with IpaD of Shigella flexneri in Inducing the Recruitment of IpaB to the Type III Secretion Apparatus Needle Tip

Type III secretion (TTS) is an essential virulence function for Shigella flexneri that delivers effector proteins that are responsible for bacterial invasion of intestinal epithelial cells. The Shigella TTS apparatus (TTSA) consists of a basal body that spans the bacterial inner and outer membranes and a needle exposed at the pathogen surface. At the distal end of the needle is a “tip complex” composed of invasion plasmid antigen D (IpaD). IpaD not only regulates TTS, but is required for the recruitment and stable association of the translocator protein IpaB at the TTSA needle tip in the presence of deoxycholate or other bile salts. This phenomenon is not accompanied by induction of TTS or the recruitment of IpaC to the Shigella surface. We now show that IpaD specifically binds fluorescein-labeled deoxycholate and, based on energy transfer measurements and docking simulations, this interaction appears to occur where the N-terminal domain of IpaD meets its central coiled-coil, a region that may also be involved in needle-tip interactions. TTS is initiated as a series of distinct steps and that small molecules present in the bacterial milieu are capable of inducing the first step of TSS through interactions with the needle tip protein IpaD. Furthermore, the amino acids proposed to be important for deoxycholate binding by IpaD appear to have significant roles in regulating tip complex composition and pathogen entry into host cells.

Applications of p-hydroxyphenacyl (pHP) and coumarin-4-ylmethyl photoremovable protecting groups

Most applications of photoremovable protecting groups have used o-nitrobenzyl compounds and their (often commercially available) derivatives that, however, have several disadvantages. The focus of this review is on applications of the more recently developed title compounds, which are especially well suited for time-resolved biochemical and physiological investigations, because they release the caged substrates in high yield within a few nanoseconds or less. Together, these two chromophores cover the action spectrum for photorelease from >700 nm to 250 nm.

Astrocytic Connectivity in the Hippocampus

Little is known about the functional connectivity between astrocytes in the CNS. To explore this issue we photo-released glutamate onto a single astrocyte in murine hippocampal slices and imaged calcium responses. Photo-release of glutamate causes a metabotropic glutamate receptor (mGluR)-dependent increase in internal calcium in the stimulated astrocyte and delayed calcium elevations in neighboring cells. The delayed elevation in calcium was not caused by either neuronal activity following synaptic transmission or by glutamate released from astrocytes. However, it was reduced by flufenamic acid (FFA), which is consistent with a role for adenosine triphosphate (ATP) release from astrocytes as an intercellular messenger. Exogenous ligands such as ATP (1 mircoM) increased the number of astrocytes that were recruited into coupled astrocytic networks, indicating that extracellular accumulation of neurotransmitters modulates neuronal excitability, synaptic transmission and functional coupling between astrocytes.

p-Hydroxyphenacyl ATP1: A new phototrigger

Abstract A new photoactivated “caged” ATP, p -hydroxyphenacyl ATP ( 4 ), is introduced to replace the o -nitrophenethyl and desyl analogues as a more efficient, rapid release phototrigger for ATP.

New photoprotecting groups: desyl and p-hydroxyphenacyl phosphate and carboxylate esters.

Abstract This chapter introduces two new phototriggers for caging nucleotides, amino acids, peptides, and related functional group derivatives, potentially including higher-order homologs. These new phototriggers have several advantages that favor them over the conventional o-nitrobenzyl derivatives, including a more rapid release of the substrate by a primary photochemical fragmentation process; adequate to excellent aqueous solubility; and stable, benign photoproducts. Added attractive features for the p-hydroxyphenacyl phototrigger include its rearrangement to a phenylacetic acid, thus shifting the chromophore absorption to a shorter wavelength, eliminating its interference with the incident radiation. Finally, the absence of a chiral center eliminates diastereomeric mixtures during synthesis. It is anticipated that these features will result in added demand for use of α-keto phototriggers for many new applications in biochemistry and physiological studies.

The photo-Favorskii reaction of p-hydroxyphenacyl compounds is initiated by water-assisted, adiabatic extrusion of a triplet biradical.

The p-hydroxyphenacyl group 1 is an effective photoremovable protecting group, because it undergoes an unusual photo-Favorskii rearrangement concomitant with the fast release (<1 ns) of its substrates in aqueous solution. The reaction mechanism of the diethyl phosphate derivative 1a was studied by picosecond pump−probe spectroscopy, nanosecond laser flash photolysis, and step−scan FTIR techniques. The primary photoproduct is a triplet biradical, 33, with a lifetime of about 0.6 ns. The release of diethyl phosphate determines the lifetime of the triplet state T1(1a), τ(T1) = 60 ps in wholly aqueous solution. Formation of a new photoproduct, p-hydroxybenzyl alcohol (6), was observed at moderate water concentrations in acetonitrile. It is formed by CO elimination from the elusive spirodione intermediate (4), followed by hydration of the resulting p-quinone methide (5). Computational studies show that CO elimination from the spirodione is a very facile process.

Caged Thiophosphotyrosine Peptides

Caged reagents are molecules from which biological effectors are released by photolysis.[1, 2] In recent studies, caged peptides and proteins have received attention because of their potential importance in investigations of processes such as cell signaling.[3, 4] Although methods suited to specific situations have been explored,[5±10] we have focused our attention on general procedures to cage unprotected peptides and proteins in aqueous solutions. Because of the relative ease of nucleophilic substitution on cysteine, this amino acid has been introduced into peptides by chemical synthesis and into proteins by mutagenesis as a site for modification by caging reagents.[3, 11±13] We recognized that most proteins involved in cell signaling are regulated by phosphorylation. Indeed, up to 30 % of proteins in mammalian cells are phosphorylated and well over one percent of mammalian proteins are protein kinases.[14±16] Therefore, we reasoned that thiophosphoryl groups in peptides and proteins might also be utilized as sites for caging. This idea was readily demonstrated with thiophosphorylserine residues in peptides, by reaction with 2-nitrobenzyl bromide (NBB) to generate caged peptides that could be converted back into the thiophosphoryl peptides by near-UV light.[17] Unfortunately, we encountered difficulties with thiophosphoryltyrosine residues, which was especially troublesome because of the pivotal role of tyrosine phosphorylation in cell signaling.[18±23] The initial difficulty centered around the enzymatic thiophosphorylation of model peptides. Herein, we show that tyrosine-containing peptides can be thiophosphorylated with cognate kinases by using divalent transition metal ions instead of MgII in the reaction buffer. The thiophosphorylated peptides can then be caged by reaction with electrophilic reagents such as NBB or p-hydroxyphenacyl bromide (HPB). The p-hydroxyphenacyl group has been used previously to cage molecules, including amino acids and oligopeptides, on carboxylate groups[24±27] and, recently, a protein phosphatase on a cysteine residue.[28] The mechanism of photorelease of substrates from p-hydroxyphenacyl carboxylate and phosphate esters has recently been examined.[27, 29, 30] In the present context, HPB is shown to be superior to the prevailing 2-nitrobenzyl reagents. In attempting to prepare thiophosphotyrosine peptides, we were, in most cases, unable to detect high extents of thiophosphorylation with ATP(g)S by using tyrosine kinases under conditions that work well when ATP itself is used.[31±33] For example, Abl, Src, and EGF and insulin receptor kinases[23] failed to thiophosphorylate peptide substrates. To explore this problem more thoroughly, we focused on EPQYEEIPILG. A related peptide, EPQYEEIPIYL, binds to Src homology (SH2) domains when phosphorylated and phosphorylated EPQYEEIPIA acts as an activator of Src kinase.[34] Divalent metal ions other than MgII have proved to be useful for promoting thiophosphorylation in other circumstances. For example, Cole and colleagues showed that kcat for the phosphorylation of poly(Glu, Tyr) by C-terminal Src kinase (Csk) is hardly reduced for ATP(g)S versus ATP in the presence of NiII and CoII, while ATP(g)S is far less effective in the presence of MgII and MnII.[33] Following this lead, we found that EPQYEEIPILG could be thiophosphorylated with Hck kinase in the presence of CoII (Scheme 1).[35]

Oxazole-based tagging reagents for analysis of secondary amines and thiols by liquid chromatography with fluorescence detection

The reactions of three fluorescent tagging reagents, 2-chloro-4,5-diphenyloxazole (DICLOX), 2-fluoro-4,5-diphenyloxazole (DIFOX) and 2-chloro-4,5-bis(p-N,N-dimethylaminosulfonylphenyl)oxazole (SAOX-CI), with thiols and amines are reported. Emission maxima for the diphenyloxazole (DIOX) and SAOX derivatives of amines were 420 nm (λex 320 nm) and 485 nm (λex 360 nm), respectively. The emission wavelengths for the DIOX- and SAOX-thiols are 390 nm (λex 310 nm) and 425 nm (λex 330 nm), respectively. In all cases, the derivatives exhibited strong fluorescence whereas the reagents themselves exhibited only weak fluorescence. The labelled derivatives are very stable, less than 5% decomposition occurs after heating at 60 °C for 2 h. Fluorescence intensities of the amine derivatives were higher in neutral and alkaline than in acidic solutions and were virtually independent of solvent polarity. The thiol derivatives exhibited fluorescence intensities that were relatively constant under all conditions studied. The relative reaction rate toward both thiols and amines was DIFOX > SAOX-CI > DICLOX. The reaction of proline with DIFOX was complete after 60 min at room temperature at pH 9.3. However, the yield with SAOX-CI was only 70% at 60 °C after 3 h, and only a small amount of proline could be derivatized with DICLOX (less than 3%). Thiols, on the other hand, reacted relatively rapidly with SAOX-CI. Therefore, SAOX-CI was used for the determination of thiols and DIFOX was employed for amines in all subsequent studies. Detection limits (signal-to-noise ratio = 2) for authentic DIOX-amines ranged from 3.7 to 28.4 fmol, and SAOX-thiols ranged from 1.2 to 1.9 fmol.