Isotope effects in photochemistry: application to chromatic orthogonality

The main challenge in developing new wavelength-specific photolabile protecting groups is the rigorous control of the photolysis rate. This rate is controlled by two factors: the chromophore absorbance and the reaction quantum yield. Fine-tuning the properties by changing substituents or structural features is difficult, because both factors are independently affected. By the use of the kinetic isotope effect, we could tune the quantum yield without altering the absorbance, and hence control the overall reaction rate. We exemplified this approach with chromatically orthogonally protected diesters.     

Brominated hydroxyquinoline as a photolabile protecting group with sensitivity to multiphoton excitation

The synthesis and photochemistry of a new photolabile protecting group for carboxylic acids based on 8-bromo-7-hydroxyquinoline (BHQ) is described. BHQ possesses a greater single photon quantum efficiency than 4,5-dimethoxy-4-nitrobenzyl ester (DMNB) and 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc), and it has sufficient sensitivity to multiphoton-induced photolysis for use in vivo. Its increased solubility and low fluorescence make it quite useful as a caging group for biological messengers. [structure: see text]     

C-O bond fragmentation of 4-picolyl- and N-methyl-4-picolinium esters triggered by photochemical electron transfer

Photochemical reduction of several 4-picolyl- and N-methyl-4-picolinium esters was examined using product analysis, laser flash photolysis, and fluorescence quenching. It is demonstrated that the radical (anions) formed in these reactions readily fragment to yield a carboxylic acid and a 4-pyridylmethyl radical intermediate. The high chemical and quantum yields observed for these photoreactions suggests that these esters can be used as photolabile protecting groups.     

o-nitrobenzyl photolabile protecting groups with red-shifted absorption: syntheses and uncaging cross-sections for one- and two-photon excitation

We evaluated the o-nitrobenzyl platform for designing photolabile protecting groups with red-shifted absorption that could be photolyzed upon one- and two-photon excitation. Several synthetic pathways to build different conjugated o-nitrobenzyl backbones, as well as to vary the benzylic position, are reported. Relative to the reference 4,5-dimethoxy-2-nitrobenzyl group, several o-nitrobenzyl derivatives exhibit a large and red-shifted one-photon absorption within the near-UV range. Uncaging after one-photon excitation was studied by measuring UV-visible absorption and steady-state fluorescence emission on model caged ethers and esters. In the whole series investigated, the caged substrates were released cleanly upon photolysis. Quantum yields of uncaging after one-photon absorption lie within the 0.1-1 % range. We observed that these drop as the maximum wavelength absorption of the o-nitrobenzyl protecting group is increased. A new method based on fluorescence correlation spectroscopy (FCS) after two-photon excitation was used to measure the action uncaging cross section for two-photon excitation. The series of o-nitrobenzyl caged fluorescent coumarins investigated exhibit values within the 0.1-0.01 Goeppert-Mayer (GM) range. Such results are in line with the low quantum yields of uncaging associated with cross-sections of 1-50 GM for two-photon absorption. Although the cross-sections for one- and two-photon absorption of o-nitrobenzyl photolabile protecting groups can be readily improved, we emphasize the difficulty in enlarging the corresponding action uncaging cross-sections in view of the observed trend of their quantum yield of uncaging.     

Photocaging strategy for functionalisation of oligonucleotides and its applications for oligonucleotide labelling and cyclisation

We have used a photocaging strategy to develop novel phosphoramidites and expand the repertoire of protecting groups for modification of oligonucleotides by solid-phase synthesis. We synthesised five photolabile phosphoramidites and four new photolabile controlled pore glasses (CPGs). By using these photolabile phosphoramidites and CPGs, modified oligodeoxynucleotides (ODNs) with phosphate, amine, acid, thiol and carbonyl moieties at 5' and/or 3' ends were readily synthesised. To the best of our knowledge, this is the first report of introducing a carbonyl at the 5' end and thiol groups at both ends of ODNs with photolabile modifiers. Terminal labelling was also easily realised in solution or by on-column solid-phase synthesis. By using the photolabile amine modifier and the photolabile acid CPG, cyclisation of an oligodeoxynucleotide was achieved with good yields. This study provides an alternative way to introduce functional groups into oligonucleotides and expand the scope of oligonucleotide bio-orthogonal labelling.     

Triplet‐Sensitized Photodeprotection of Oligonucleotides in Solution and on Microarray Chips

Conditions and kinetics of triplet sensitization as a method for increasing the light sensitivity of photolabile protecting groups used for the photolithographic synthesis of oligonucleotide microarrays were quantitatively studied with the photolabile 2‐(2‐nitrophenyl)propyl protecting group in homogeneous solutions and on glass substrates by using laser flash photolysis, continuous illumination with HPLC analysis, fluorescence dye labelling, and hybridization. In terms of efficiency and avoidance of chemical side reactions, 9H‐thioxanthen‐9‐one was the most‐suitable sensitizer. Both in solution and on a glass substrate, the photostationary kinetics were quantitatively modelled and the relevant kinetic parameters determined. While the sensitization kinetics was diffusion‐controlled both in solution and on the chip, the photostationary kinetics was essentially of zero order only on the chip because here the triplet‐quenching effect of the released photoproduct 2‐(2‐nitrophenyl)propene was suppressed as a consequence of the inhomogeneous reaction that took place in a narrow diffusion zone above the surface from where the photoproducts could quickly escape. The kinetic simulation allowed quantitative estimate of the density of reactive groups on the surface. It was further demonstrated that, with 9H‐thioxanthen‐9‐one as a sensitizer, high‐density oligonucleotide microarrays of high quality can be produced with one‐third of the normal exposure time.     

Intramolecular sensitization of photocleavage of the photolabile 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protecting group: photoproducts and photokinetics of the release of nucleosides

Novel photolabile protecting groups based on the 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) group with a covalently linked thioxanthone as an intramolecular triplet sensitizer exhibit significantly enhanced light sensitivity under continuous illumination. Herein we present a detailed study of the photokinetics and photoproducts of nucleosides caged with these new protecting groups. Relative to the parent NPPOC group, the light sensitivity of the new photolabile protecting groups is enhanced by up to a factor of 21 at 366 nm and is still quite high at 405 nm, the wavelength at which the sensitivity of the parent compound is practically zero. A new pathway for deprotection of the NPPOC group proceeding through a nitroso benzylalcohol intermediate has been discovered to complement the main mechanism, which involves beta elimination. Under standard conditions of lithographic DNA-chip synthesis, some of the new compounds, while maintaining the same chip quality, react ten times faster than the unmodified NPPOC-protected nucleosides.     

Phototaxis of Oil Droplets Comprising a Caged Fatty Acid Tightly Linked to Internal Convection

We found that novel sub‐millimeter‐sized photoactive oil droplets of oleic acid bearing a photolabile protecting group, 2‐nitrobenzyl oleate (NBO), in basic water exhibited unidirectional motion toward a UV light source. This unidirectional motion can be explained by anisotropic photolysis on a surface of the NBO droplet with low permeability for UV light. Time‐dependent changes of the movement under UV irradiation occurred in a cascade manner (still‐standing, induction, and active stages). The velocity of the UV‐irradiated droplet in the induction stage was small, but it was accelerated sixteen times by the presence of an inner convection structure, which was created by continued photolysis. This characteristic dynamics, which is derived from a supramolecular machinery system towards the external stimulus, may be similar to the phototaxis of a living cell.     

Light-directed radial combinatorial chemistry: orthogonal safety-catch protecting groups for the synthesis of small molecule microarrays

We describe the development of photolabile protecting groups based on the 3,4,5-trimethoxyphenacyl group (TMP). Orthogonal safety-catches were created by introducing an acid-activatible dimethyl ketal (AA-TMP) and an oxidatively activatible 1,3-dithiane (OA-TMP) into the photolabile TMP group. We demonstrate the application of these protecting groups in light-directed synthesis of small molecule microarrays with diversity elements radially attached to a hydroxyproline scaffold.     

Fast and Reliable Automated Synthesis of RNA and Partially 2′-O- Protected Precursors (`Caged RNA') Based on Two Novel,Orthogonal 2′-O-Protecting Groups,Preliminary Communication

Two sets of RNA phosphoramidites, carrying the (fluoride-labile) 2′-O-[(triisopropylsilyl)oxy]methyl (=tom) group and the (photolabile) [(R)-1-(2-nitrophenyl)ethoxy]methyl (=(R)-npeom) group, were prepared (see 1 – 4 and 5 – 8 , resp.). The two protecting groups were completely orthogonal to each other. Three ribozyme-substrate constructs, protected each by a (R)-npeom group, were synthesized; on photolysis, efficient cleavage of this remaining protecting group occurred (Scheme 3). It could be demonstrated that the presence of one (R)-npeom group within a RNA strand has only a minor influence on the pairing properties of corresponding duplexes.     

Convergent solid phase peptide synthesis. v. synthesis of the 1-4, 32-34, and 53-59 protected segments of the toxin ii of androctonus australis hector.

Two protected peptide segments corresponding to the sequence 32-34 and 53-59 of toxin II of the north African scorpion Androctonus australis Hector have been synthesized on a photolabile Nbb-resin using the TFA-labile Boc -amino protection and HF-labile side chain protecting groups. A third protected peptide corresponding to segment 1-4 has been synthesized on the same resin but with a t-butyl group for β protection of aspartic acid and a Z group on the lysine side chain. For this last segment a combination of Boc and Fmoc groups for -amino protection has been used successfully on the Nbb-resin. After photolysis the crude peptides have been treated by solvent extraction and semi-preparative HPLC to yield highly purified segments. These syntheses show the flexibility of the convergent solid phase approach and how segments with different and independent protecting groups can be assembled by solid phase peptide procedure.     

Toward the development of new photolabile protecting groups that can rapidly release bioactive compounds upon photolysis with visible light

The synthesis and characterization of a new photolabile protecting group (caging group) for carboxylic acids, the 2-(dimethylamino)-5-nitrophenyl (DANP) group, is described. This compound has a major absorption band in the visible wavelength region with a maximum near 400 nm (epsilon400 = 9077 M(-1) cm(-1) at pH 7.4 and 21 degrees C). The caging group is attached through an ester linkage to the carboxyl functionality of beta-alanine, which activates the inhibitory glycine receptor in the mammalian central nervous system. Such caged compounds play an important role in transient kinetic investigations of fast cellular processes. Upon photolysis of DANP-caged beta-alanine, the caging group is released within 5 micros. Quantum yields of 0.03 and 0.002 were obtained in the UV region (308 and 360 nm) and the visible region (450 nm), respectively. Laser-pulse photolysis experiments, using 337 or 360 nm light, were performed with the caged compound equilibrated with HEK 293 cells transiently transfected with cDNA encoding the alpha1 homomeric, wild-type glycine receptor. The experiments demonstrated that neither DANP-caged beta-alanine nor its byproducts inhibit or activate the glycine receptors on the cell surface. Under physiological conditions, the DANP-caged beta-alanine is water-soluble and stable and can be used for transient kinetic measurements.     

A photolabile protection strategy for terminal alkynes

We present a strategy for photolabile protection of terminal alkynes. Several photo-caged alcohols were synthesized via mild copper(II)-catalyzed substitution between tertiary propargylic alcohols and 2-nitrobenzyl alcohol to build up robust, base stable o-nitrobenzyl (NB) photo-cleavable compounds. We compare the new photolabile protecting group with the commonly used alkyne protecting group, 2-methyl-3-butyn-2-ol and the results show that NB ethers are stable under the cleaving conditions for the cleavage of methylbutynol protected alkynes. Additionally, we present the synthesis of photo-cleavable NB derivatives containing thiol groups that can serve as agents for photoinduced surface functionalization reactions.     

Rapid Generation of Hierarchically Porous Metal–Organic Frameworks through Laser Photolysis

Hierarchically porous metal–organic frameworks (HP-MOFs) facilitate mass transfer due to mesoporosity while preserving the advantage of microporosity. This unique feature endows HP-MOFs with remarkable application potential in multiple fields. Recently, new methods such as linker labilization for the construction of HP-MOFs have emerged. To further enrich the synthetic toolkit of MOFs, we report a controlled photolytic removal of linkers to create mesopores within microporous MOFs at tens of milliseconds. Ultraviolet (UV) laser has been applied to eliminate “photolabile” linkers without affecting the overall crystallinity and integrity of the original framework. Presumably, the creation of mesopores can be attributed to the missing-cluster defects, which can be tuned through varying the time of laser exposure and ratio of photolabile/robust linkers. Upon laser exposure, MOF crystals shrank while metal oxide nanoparticles formed giving rise to the HP-MOFs. In addition, photolysis can also be utilized for the fabrication of complicated patterns with high precision, paving the way towards MOF lithography, which has enormous potential in sensing and catalysis.     

The Development of "Caged" Substrates for the Studies of Rapid Electron Transfer and Early Events in Protein Folding

In recent years,the applications of photolabile compounds, or compounds that unmask a function group upon absorption of light, have become more diverse. Since such compounds can provide an effective means of orthogonal protection, they have been used to facilitate the synthesis of complex, polyfunctional organic molecules. These materials have also been used to "cage" compounds by protection of an essential functional group, so that a chemical reaction may be initiated by a pulse of light. In this manner, mixing difficulties can be circumvented in kinetics measurements. Applications of this method include the photolysis of caged ATP for studies of muscle fiber contraction, where diffusion of ATP into the muscle fiber is, slow; of caged fluorescent probes that only emit light after photolysis; and even of caged enzymes by incorporating photolabile groups on essential side chains.     

Development of a photolabile carbonyl-protecting group toolbox

New salicyl alcohol derived photolabile carbonyl protecting groups have been developed, and the effect of substituents on the photochemical properties of photolabile protecting groups (PPGs) has been studied. The 3-(dimethylamino)phenyl groups at the α position prove to be important to the efficiency of the deprotection reactions, as shown in the photo reactions of the acetal 9. On the other hand, expansion of the salicyl alcohol's benzene skeleton to naphthalene does not improve the photochemical properties of PPGs. A neutral protecting protocol has been generalized to new PPGs with α,α-diaryl salicyl alcohol backbone. Thus, installation of PPGs onto aldehydes is readily achieved at 140 °C without using any other chemical reagents. These PPGs are stable under acidic conditions typical for hydrolyzing acetals and constitute orthogonal protecting groups with traditional 1,3-dioxane/1,3-dioxolane for carbonyl compounds. Highly efficient release of carbohydrate molecules is demonstrated, which can be potentially useful in site-specific release and immobilization of carbohydrates for preparation of high-density microarrays. With the enriched PPG toolbox, PPGs are divided into three subgroups based on their UV absorption profiles. PPGs from different subgroups can be sequentially removed by using different UV irradiation wavelengths. For PPGs absorbing UVA (λ >315 nm), photochemical deprotection can be carried out with sunlight in high yields.     

Isotope effects in photochemistry. 1. o-nitrobenzyl alcohol derivatives

The photolysis of o-nitrobenzyl alcohol derivatives shows a strong kinetic isotope effect (KIE up to 8.3) at the benzylic center. For some derivatives, the KIE is wavelength dependent, suggesting the involvement of higher excited states. In addition to the important mechanistic consequences, isotopic substitution is a convenient way to alter the quantum yield without changing the absorbance, as would the introduction of substituents. This paves the way for a subtle tuning of the reaction rates in photochemical reactions. We illustrated this feature in the sequential deprotection of a diester protected at both termini with photolabile groups.     

Photolytic control of peptide self-assembly

Herein, we present a methodology that allows for the temporal control of fibrillization of amyloidogenic peptides. This general approach implements a photolabile linker that connects the amyloidogenic peptide to a fibril-inhibitory unit, in this case, a pentamer of amino acids modified with the solubilizing N,N-dimethylethylenediamine (DMDA) units. Upon photolysis, the linker can be dissociated to afford the intact and native amyloidogenic peptide. This methodology should be of value in a variety of studies where spatial and temporal control of supramolecular association processes is desired.     

Rapid Generation of Hierarchically Porous Metal–Organic Frameworks through Laser Photolysis

Hierarchically porous metal–organic frameworks (HP‐MOFs) facilitate mass transfer due to mesoporosity while preserving the advantage of microporosity. This unique feature endows HP‐MOFs with remarkable application potential in multiple fields. Recently, new methods such as linker labilization for the construction of HP‐MOFs have emerged. To further enrich the synthetic toolkit of MOFs, we report a controlled photolytic removal of linkers to create mesopores within microporous MOFs at tens of milliseconds. Ultraviolet (UV) laser has been applied to eliminate “photolabile” linkers without affecting the overall crystallinity and integrity of the original framework. Presumably, the creation of mesopores can be attributed to the missing‐cluster defects, which can be tuned through varying the time of laser exposure and ratio of photolabile/robust linkers. Upon laser exposure, MOF crystals shrank while metal oxide nanoparticles formed giving rise to the HP‐MOFs. In addition, photolysis can also be utilized for the fabrication of complicated patterns with high precision, paving the way towards MOF lithography, which has enormous potential in sensing and catalysis.     

Photodecomposition of polystyrene on long-wave ultraviolet irradiation: A possible mechanism of initiation of photooxidation

The long-wave (λ < 3000 Å) photo-oxidation of polystyrene in solution at 25°C has been studied osmometrically. Two types of chain scission have been observed: a purely photo process which occurs completely independently of oxygen and which is attributed to fission of photolabile groups in the polymer, and another process associated with random photolyses of the products of oxidation Scavenger experiments with ¹³¹I₂ have shown that approximately two iodine atoms are incorporated per chain scission when photolysis is carried out in solution (benzene, hexafluorobenzene, methylene chloride) under high vacuum conditions in the presence of ¹³¹I₂. No iodine incorporation or chain scission occurs when ionically prepared polystyrenes are treated similarly. The nature of the photolabile bond has been discussed, and there is some evidence for a peroxidic linkage arising from oxygen copolymerization in the chains. It is suggested that fission of the photolabile groups contributes to the initiation of the long-wave photooxidation of the polymer.