Selective and Reversible Photochemical Derivatization of Cysteine Residues in Peptides and Proteins.

Selective derivatization of solvent-exposed cysteine residues in peptides and proteins is achieved by brief irradiation of an aqueous solution containing 3-(hydroxymethyl)-2-naphthol derivatives (NQMPs) with 350 nm fluorescent lamp. NQMP can be conjugated with various moieties, such as PEG, dyes, carbohydrates, or possess a fragment for further selective derivatization, e.g., biotin, azide, alkyne, etc. Attractive features of this labeling approach include an exceptionally fast rate of the reaction and a requirement for low equivalence of the reagent. The NQMP-thioether linkage is stable under ambient conditions, survives protein digestion and MS analysis. Irradiation of NQMP-labeled protein in a dilute solution (<40 µM) or in the presence of a vinyl ether results in a traceless release of the substrate. The reversible biotinylation of bovine serum albumin, as well as capture and release of this protein using NeutrAvidin Agarose resin beads has been demonstrated.

Sequential "click" - "photo-click" cross-linker for catalyst-free ligation of azide-tagged substrates.

Heterobifunctional linker allows for selective catalyst-free ligation of two different azide-tagged substrates via strained-promoted azide-alkyne cycloaddition (SPAAC). The linker contains an azadibenzocyclooctyne (ADIBO) moiety on one end and a cyclopropenone-masked dibenzocyclooctyne (photo-DIBO) group on the other. The first azide-derivatized substrate reacts only at the ADIBO end of the linker as the photo-DIBO moiety is azide-inert. After the completion of the first SPAAC step, photo-DIBO is activated by brief exposure to 350 nm light from a fluorescent UV lamp. The unmasked DIBO group then reacts with the second azide-tagged substrate. Both click reactions are fast (k = 0.4 and 0.07 M(-1) s(-1), respectively) and produce quantitative yield of ligation in organic solvents or aqueous solutions. The utility of the new cross-linker has been demonstrated by conjugation of azide functionalized bovine serum albumin (azido-BSA) with azido-fluorescein and by the immobilization of the latter protein on azide-derivatized silica beads. The BSA-bead linker was designed to incorporate hydrolytically labile fragment, which permits release of protein under the action of dilute acid. UV activation of the second click reaction permits spatiotemporal control of the ligation process.

Rate determination of azide click reactions onto alkyne polymer brush scaffolds: a comparison of conventional and catalyst-free cycloadditions for tunable surface modification.

The postpolymerization functionalization of poly(N-hydroxysuccinimide 4-vinylbenzoate) brushes with reactive alkynes that differ in relative rates of activity of alkyne-azide cycloaddition reactions is described. The alkyne-derived polymer brushes undergo "click"-type cycloadditions with azido-containing compounds by two mechanisms: a strain-promoted alkyne-azide cycloaddition (SPAAC) with dibenzocyclooctyne (DIBO) and azadibenzocyclooctyne (ADIBO) or a copper-catalyzed alkyne-azide cycloaddition (CuAAC) to a propargyl group (PPG). Using a pseudo-first-order limited rate equation, rate constants for DIBO, ADIBO, and PPG-derivatized polymer brushes functionalized with an azide-functionalized dye were calculated as 7.7 × 10(-4), 4.4 × 10(-3), and 2.0 × 10(-2) s(-1), respectively. The SPAAC click reactions of the surface bound layers were determined to be slower than the equivalent reactions in solution, but the relative ratio of the reaction rates for the DIBO and ADIBO SPAAC reactions was consistent between solution and the polymer layer. The rate of functionalization was not influenced by the diffusion of azide into the polymer scaffold as long as the concentration of azide in solution was sufficiently high. The PPG functionalization by CuAAC had an extremely fast rate, which was comparable to other surface click reaction rates. Preliminary studies of dilute solution azide functionalization indicate that the diffusion-limited regime of brush functionalization impacts a 50 nm polymer brush layer and decreases the pseudo-first-order rate by a constant diffusion-limited factor of 0.233.

Attach, remove, or replace: reversible surface functionalization using thiol-quinone methide photoclick chemistry.

A very facile reaction between photochemically generated o-naphthoquinone methides (oNQMs) and thiols is employed for reversible light-directed surface derivatization and patterning. A thiol-functionalized glass slide is covered with an aqueous solution of a substrate conjugated to 3-(hydroxymethyl)-2-naphthol (NQMP). Subsequent irradiation via shadow mask results in the efficient conversion of NQMP into reactive oNQM species in the exposed areas. The latter react with thiol groups on the surface, producing thioether links between the substrate and the surface. Unreacted oNQM groups are rapidly hydrated to regenerate NQMP. The short lifetime of oNQM in aqueous solution prevents its migration from the site of irradiation, thus allowing for the spatial control of the surface derivatization. A two-step procedure was employed for protein patterning: photobiotinylation of the surface with an NQMP-biotin conjugate followed by staining with FITC-avidin. The orthogonality of oNQM-thiol and azide click chemistry allowed for the development of a sequential click strategy, which might be useful for the immobilization of light-sensitive compounds. The thioether linkage produced by the reaction of oNQM and a thiol is stable under ambient conditions but can be cleaved by UV irradiation, regenerating the free thiol. This feature allows for the removal or replacement of immobilized substrates.

Preparation and photophysical properties of a caged kynurenine.

We have prepared l-kyurenine 4-hydroxyphenacyl ester, a caged derivative of L-kynurenine. N(α)-tBOC-L-tryptophan was reacted with 4-hydroxyphenacyl bromide in DMF with K(2)CO(3) as the base to give the N(α)-tBOC 4-hydroxyphenacyl ester. The ester was then treated with O(3) in MeOH at -20°C, followed by trifluoroacetic acid in CH(2)Cl(2), then aqueous HCl to obtain the caged kynurenine as the dihydrochloride salt. The caged kynurenine is stable as a dry solid in the dark at -78°C, but in aqueous solutions in phosphate buffer at pH 7-8 hydrolyzes rapidly (t(1/2) ∼5 min). Solutions in Tris at pH 7 are more stable (t(1/2) >30 min), and solutions in 1mM HCl are stable for several hours. As expected, the ester is cleaved in microseconds with laser pulses at 355 nm. The caged kynurenine may be useful for preparation of substrate complexes for crystallography or in biological studies on kynurenine.

Photoreactive polymer brushes for high-density patterned surface derivatization using a Diels-Alder photoclick reaction.

Reactive polymer brushes grown on silicon oxide surfaces were derivatized with photoreactive 3-(hydroxymethyl)naphthalene-2-ol (NQMP) moieties. Upon 300 or 350 nm irradiation, NQMP efficiently produces o-naphthoquinone methide (oNQM), which in turn undergoes very rapid Diels-Alder addition to vinyl ether groups attached to a substrate, resulting in the covalent immobilization of the latter. Any unreacted oNQM groups rapidly add water to regenerate NQMP. High-resolution surface patterning is achieved by irradiating NQMP-derivatized surfaces using photolithographic methods. The Diels-Alder photoclick reaction is orthogonal to azide-alkyne click chemistry, enabling sequential photoclick/azide-click derivatizations to generate complex surface functionalities.

Bichromophoric fluorescent photolabile protecting group for alcohols and carboxylic acids.

Novel bichromophoric fluorescent photolabile protecting group, (5-dansyloxy-3-hydroxynaphthalen-2-yl)methyl (DNS-NQMP), allows for the independent photochemical release and fluorescent imaging of caged substrates. Irradiation of DNS-NQMP-caged alcohols and carboxylic acids with 300 or 350 nm light results in fast (k(release) ~ 10(5) s(-1)), efficient (Φ = 0.2), and quantitative release of the substrates. This uncaging chemistry is compatible with aqueous media and DNS-NQMP-protected hydroxy compounds are hydrolytically stable at neutral pH. Upon excitation with 400 nm light, caged compounds show intense green emission (λ(max) = 559 nm) with 21% fluorescence quantum yield. Fluorescent readout conducted using 400 nm or longer wavelengths does not cause substrate release. The DNS-NQMP chromophore retains its fluorescent properties after photo-uncaging reaction.

[18F]azadibenzocyclooctyne ([18F]ADIBO): a biocompatible radioactive labeling synthon for peptides using catalyst free [3+2] cycloaddition.

N-Terminally azido-modified peptides were labeled with the novel prosthetic labeling synthon [(18)F]azadibenzocyclooctyne ([(18)F]ADIBO) using copper-free azide-alkyne [3+2]-dipolar cycloaddition in high radiochemical yields (RCYs). (18)F-Labeled [(18)F]ADIBO was prepared by nucleophilic substitution of the corresponding tosylate in 21% overall RCY (EOB) in a fully automated synthesis unit within 55 min. [(18)F]ADIBO was incubated with azide-containing peptides at room temperature in the absence of toxic metal catalysts and the formation of the triazole conjugate was confirmed. Finally, the azide-alkyne [3+2]-dipolar cycloaddition was shown to proceed with 95% radiochemical yield in ethanol within 30 min, allowing for a development of a kit-like peptide labeling approach with [(18)F]ADIBO.

Patterned surface derivatization using Diels-Alder photoclick reaction.

The utility of photochemically induced hetero-Diels-Alder reaction for the light-directed surface derivatization and patterning was demonstrated. Glass slides functionalized with vinyl ether moieties are covered with aqueous solution of substrates conjugated to 3-(hydroxymethyl)-2-naphthol (NQMP). Subsequent irradiation via shadow mask results in an efficient conversion of the latter functionality into reactive 2-napthoquinone-3-methide (oNQM) in the exposed areas. oNQM undergoes very facile hetero Diels-Alder addition (k(D-A) ≈ 4 × 10(4) M(-1)s(-1)) to immobilized vinyl ether molecules resulting in a photochemically stable covalent link between a substrate and a surface. Unreacted oNQM groups are rapidly hydrated to regenerate NQMP. The click chemistry based on the addition of photochemically generated oNQM to vinyl ether works well in aqueous solution, proceeds at high rate under ambient conditions, and does not require catalyst or additional reagents. This photoclick strategy represents an unusual paradigm in photopatterning: the surface itself is photochemically inert, while photoreactive component is present in the solution. The short lifetime (τ ≈ 7 ms in H(2)O) of the active form of a photoclick reagent in aqueous solution prevents its migration from the site of irradiation, thus allowing for the spatial control of surface derivatization. Both o-napthoquinone methide precursors and vinyl ethers are stable in dark and the reaction is orthogonal to other derivatization techniques, such as acetylene-azide click reaction.

Light-induced hetero-Diels-Alder cycloaddition: a facile and selective photoclick reaction.

2-Napthoquinone-3-methides (oNQMs) generated by efficient photodehydration (Φ=0.2) of 3-(hydroxymethyl)-2-naphthol undergo facile hetero-Diels-Alder addition (k(D-A)∼ 4×10(4) M(-1) s(-1)) to electron-rich polarized olefins in an aqueous solution. The resulting photostable benzo[g]chromans are produced in high to quantitative yield. The unreacted oNQM is rapidly hydrated (k(H2O) ∼145 s(-1)) to regenerate the starting diol. This competition between hydration and cycloaddition makes oNQMs highly selective, since only vinyl ethers and enamines are reactive enough to form the Diels-Alder adduct in an aqueous solution; no cycloaddition was observed with other types of alkenes. To achieve photolabeling or photoligation of two substrates, one is derivatized with a vinyl ether moiety, while 3-(hydroxymethyl)-2-naphthol is attached to the other via an appropriate linker. The light-induced Diels-Alder "click" strategy permits the formation of either a permanent or hydrolytically labile linkage. Rapid kinetics of this photoclick reaction (k=4×10(4) M(-1) s(-1)) is useful for time-resolved applications. The short lifetime (τ ∼7 ms in H(2)O) of the active form of the photoclick reagent prevents its migration from the site of irradiation, thus, allowing for spatial control of the ligation or labeling.

Metal-free sequential [3 + 2]-dipolar cycloadditions using cyclooctynes and 1,3-dipoles of different reactivity.

Although metal-free cycloadditions of cyclooctynes and azides to give stable 1,2,3-triazoles have found wide utility in chemical biology and material sciences, there is an urgent need for faster and more versatile bioorthogonal reactions. We have found that nitrile oxides and diazocarbonyl derivatives undergo facile 1,3-dipolar cycloadditions with cyclooctynes. Cycloadditions with diazocarbonyl derivatives exhibited similar kinetics as compared to azides, whereas the reaction rates of cycloadditions with nitrile oxides were much faster. Nitrile oxides could conveniently be prepared by direct oxidation of the corresponding oximes with BAIB, and these conditions made it possible to perform oxime formation, oxidation, and cycloaddition as a one-pot procedure. The methodology was employed to functionalize the anomeric center of carbohydrates with various tags. Furthermore, oximes and azides provide an orthogonal pair of functional groups for sequential metal-free click reactions, and this feature makes it possible to multifunctionalize biomolecules and materials by a simple synthetic procedure that does not require toxic metal catalysts.

Dual reactivity of hydroxy- and methoxy- substituted o-quinone methides in aqueous solutions: hydration versus tautomerization.

4-Hydroxy-6-methylene-2,4-cyclohexadien-1-one (1) and 4-methoxy-6-methylene-2,4-cyclohexadien-1-one (2) were generated by efficient (Φ = 0.3) photodehydration of 2-(hydroxymethyl)benzene-1,4-diol (3a) and 2-(hydroxymethyl)-4-methoxyphenol (4a), respectively. o-Quinone methides 1 and 2 can be quantitatively trapped as Diels-Alder adducts with ethyl vinyl ether or intercepted by good nucleophiles, such as azide ion (k(N3)(1) = 3.15 × 10(4) M(-1) s(-1) and k(N3)(2) = 3.30 × 10(4) M(-1) s(-1)). In aqueous solution, o-quinone methide 2 rapidly adds water to regenerate starting material (τ(H(2)O)(2) = 7.8 ms at 25 °C). This reaction is catalyzed by specific acid (k(H(+))(2) = 8.37 × 10(3) s(-1) M(-1)) and specific base (k(OH(-))(2) = 1.08 × 10(4) s(-1) M(-1)) but shows no significant general acid/base catalysis. In sharp contrast, o-quinone methide 1 decays (τ(H(2)O)(1) = 3.3 ms at 25 °C) via two competing pathways: nucleophilic hydration to form starting material 3a and tautomerization to produce methyl-p-benzoquinone. The disappearance of 1 shows not only specific acid (k(H(+))(1) = 3.30 × 10(4) s(-1) M(-1)) and specific base catalysis (k(OH(-))(1) = 3.51 × 10(4) s(-1) M(-1)) but pronounced catalysis by general acids and bases as well. The o-quinone methides 1 and 2 were also generated by the photolysis of 2-(ethoxymethyl)benzene-1,4-diol (3b) and 2-(ethoxymethyl)-4-methoxyphenol (4b), as well as from (2,5-dihydroxy-1-phenyl)methyl- (3c) and (2-hydroxy-5-methoxy-1-phenyl)methyltrimethylammonium iodides (4c). Short-lived (τ(25°)(C) ≈ 20 µs) precursors of o-quinone methides 1 and 2 were detected in the laser flash photolysis of 3a,b and 4a,b. On the basis of their reactivity, benzoxete structures have been assigned to these intermediates.

High density orthogonal surface immobilization via photoactivated copper-free click chemistry.

Surfaces containing reactive ester polymer brushes were functionalized with cyclopropenone-masked dibenzocyclooctynes for the light activated immobilization of azides using catalyst-free click chemistry. The photodecarbonylation reaction in the amorphous brush layer is first order for the first 45 s with a rate constant of 0.022 s(-1). The catalyst-free cycloaddition of surface bound dibeznocyclooctynes proceeds rapidly in the presence of azides under ambient conditions. Photolithography using a shadow mask was used to demonstrate patterning with multiple azide containing molecules. This surface immobilization strategy provides a general and facile platform for the generation of multicomponent surfaces with spatially resolved chemical functionality.

Photochemical generation and the reactivity of o-naphthoquinone methides in aqueous solutions.

Irradiation of 3-hydroxy-2-naphthalenemethanol (3a) and 2-hydroxy-1-naphthalenemethanol (4a) results in efficient (Phi(254) = 0.17 and 0.20) dehydration and the formation of isomeric naphthoquinone methides, 2,3-naphthoquinone-3-methide (1) and 1,2-naphthoquinone-1-methide (2), respectively. In aqueous solution, naphthoquinone methides 1 and 2 undergo rapid hydration to regenerate starting materials (tau(H2O) (1) = 7.4 ms and tau(H2O) (2) = 4.5 ms at 25 degrees C). The hydration reaction is strongly catalyzed by the hydroxide ion but shows acid catalysis only at pH < 1. Reactive intermediates 1 and 2 can be intercepted by other nucleophiles, such as the azide ion (k(N3)(1) = 2.0 x 10(4) M(-1) s(-1) and k(N3)(2) = 3.0 x 10(4) M(-1) s(-1)) or thiol (k(SH)(1) = 2.2 x 10(5) M(-1) s(-1) and k(SH)(2) = 3.3 x 10(5) M(-1) s(-1)). Ethyl vinyl ether readily reacts with 1 and 2 (k(DA)(1) = 4.1 x 10(4) M(-1) s(-1) and k(DA)(2) = 6.0 x 10(4) M(-1) s(-1)) to produce Diels-Alder adducts in excellent yield. o-Naphthoquinone methides 1 and 2 were also generated by photolysis of 3-ethoxymethyl- (3b) and 1-(ethoxymethyl)-2-naphthols (4b), as well as from (2-hydroxy-3-naphthyl)methyl- (3c) and [(2-hydroxy-1-naphthyl)methyl] trimethylammonium iodides (4c). Laser flash photolysis of 3a,b and 4a,b allows the detection of short-lived (tau(25 degrees C) approximately 12 micros) precursors of naphthoquinone methides 1 and 2. On the basis of the precursor reactivity and the results of DFT calculations, 2H-naphthoxete structure was assigned to these species.

Photolabile protection of alcohols, phenols, and carboxylic acids with 3-hydroxy-2-naphthalenemethanol.

Irradiation of alcohols, phenols, and carboxylic acids "caged" with the (3-hydroxy-2-naphthalenyl)methyl group results in fast (k(release) approximately = 10(5) s(-1)) release of the substrates with good quantum (Phi = 0.17-0.26) and chemical (>90%) yields. The initial byproduct of the photoreaction, 2-naphthoquinone-3-methide, reacts rapidly with water (k(H2O) = 144 +/- 11 s(-1)) to produce parent 3-hydroxy-2-naphthalenemethanol. The o-quinone methide intermediate can be also trapped by other nucleophiles or converted into a photostable Diels-Alder adduct with ethyl vinyl ether.

Alkali ion exchanged Nafion as a confining medium for photochemical reactions.

Methanol-swollen Nafion beads were used as microreactors to control the photochemical reaction pathways. Product selectivity in three unimolecular reactions, namely, the photo-Fries rearrangement of naphthyl esters, Norrish Type I reaction of 1-phenyl-3-p-tolyl-propan-2-one and Norrish Type I and Type II reactions of benzoin alkyl ethers were examined. The influence of cations over the photodimerization of acenaphthylene and cross-photodimerization between acenaphthylene and N-benzyl maleimide included within Nafion were also examined. The photochemical behaviors of the above substrates were significantly altered within Nafion compared with their solution photochemistry. Of particular interest, the product distributions were found to depend on the counter cations of Nafion.

Amphiphilic homopolymer as a reaction medium in water: product selectivity within polymeric nanopockets.

A styrene-based water-soluble polymer has been explored for its use as a host for lipophilic substrates in aqueous medium. Unimolecular reactions, namely, photo-Fries rearrangement of naphthyl esters, alpha-cleavage reaction of 1-phenyl-3-p-tolyl-propan-2-one, and Norrish type I and type II reactions of benzoin alkyl ethers were examined. We find that the hydrophobic domains generated by the polymer not only restrict the mobility of the radicals but also modestly incarcerate the substrate, intermediates, and products during the time scale of the reactions. Comparative studies of the same photoreactions in micelles formed from small molecule surfactants and an amphiphilic diblock copolymer demonstrate that the styrene-based water-soluble polymer aggregates in aqueous medium offer better selectivity.