The Discovery, Development and Demonstration of Three Caged Compounds.

An overview of the history, mechanistic aspects and applications is provided for p-hydroxyphenacyl (pHP) and benzoin photoremovable protecting groups, which release biologically important leaving groups upon photolysis with UV light. Also discussed is (7-diethylaminocoumarin-4-yl)methyl (DEACM), a photoremovable protecting group that absorbs visible light. These are followed by the α-keto amides and naphtho- and benzothiophene-2-carboxanilides as caging groups, which eliminate leaving groups via photochemically produced zwitterionic intermediates. Also covered are amino-1,4-benzoquinones, which upon exposure to green and red wavelengths of light photorearrange to an unstable photoproduct that subsequently eliminates leaving groups in aqueous media. Selected examples are given that use these photoremovable protecting (caging) groups for the light-activated release of biologically important substrates under physiological conditions in cells and tissue as practical applications in biology, biochemistry and physiology. These caging groups have found significant applications because their photochemistry is efficient and a single coproduct is formed in addition to the photoreleased substrate.

Frostbite and Immersion Foot Care.

Historically, cold injury, hypothermia, and frostbite have been severe problems for military units on the battlefield. Kenneth D. Orr and David C. Fainer captured these difficulties in their book, Cold Injuries in Korea During Winter of 1950-51, still cited in military medical readiness training. While not common in modern conflicts, the potential exists for large numbers of these casualties in war and during training.

Reversible Triplet Excitation Transfer in a Trimethylene-Linked Thioxanthone and Benzothiophene-2-Carboxanilide that Photochemically Expels Leaving Group Anions.

The triplet excited state of thioxanthone produced by photolysis undergoes reversible triplet energy transfer with a trimethylene-linked benzothiophene-2-carboxanilide ring system. The ensuing electrocyclic ring closure of the anilide moiety produces a putative zwitterionic intermediate that is capable of expelling leaving groups (LG-) from the C-3 position of the benzothiophene ring. Stern-Volmer quenching studies with cyclohexadiene as quencher furnish the rate constants for the triplet excitation transfer in the forward and reverse directions, which can be expressed as an equilibrium constant K = 0.058. Overall, the rate of the triplet excited state reaction becomes K × kr = 5.7 × 104 s-1 for LG- = Cl-, where kr is the triplet decay rate of the C-3 chloro-substituted benzothiophene-2-carboxanilide, found through Stern-Volmer quenching. The high quantum efficiencies found for the trimethylene-linked systems are due to K × kr being competitive with the triplet excited state decay of the thioxanthone of kd = 7.7 × 104 s-1. On the basis of Φisc = 0.68, the overall expected quantum yield for direct photolysis should be 0.50 for LG- = Cl- as compared to 0.41 at 25 °C experimentally. Φ decreases with increasing basicity of the leaving group (LG- = Cl-, (EtO)2PO2-, PhCH2CO2-, PhS-, and PhCH2S-).

Photochemical electrocyclic ring closure and leaving group expulsion from N-(9-oxothioxanthenyl)benzothiophene carboxamides.

N-(9-Oxothioxanthenyl)benzothiophene carboxamides bearing leaving groups (LG(-) = Cl(-), PhS(-), HS(-), PhCH(2)S(-)) at the C-3 position of the benzothiophene ring system photochemically cyclize with nearly quantitative release of the leaving group, LG(-). The LG(-) photoexpulsions can be conducted with 390 nm light or with a sunlamp. Solubility in 75% aqueous CH(3)CN is achieved by introducing a carboxylate group at the C-6 position of the benzothiophene ring. The carboxylate and methyl ester derivatives regiospecifically cyclize at the more hindered C-1 position of the thioxanthone ring. Otherwise, the photocyclization favors the C-3 position of the thioxanthone. Quantum yields for reaction are 0.01-0.04, depending on LG(-) basicity. Electronic structure calculations for the triplet excited state show that excitation transfer occurs from the thioxanthone to the benzothiophene ring. Subsequent cyclization in the triplet excited state is energetically favourable and initially generates the triplet excited state of the zwitterionic species. Expulsion of LG(-) is thought to occur once this species converts to the closed shell ground state.

Photochemical eliminations involving zwitterionic intermediates generated via electrocyclic ring closure of benzothiophene carboxanilides.

Leaving groups such as carboxylate, thiolate, and phenolate are expelled via zwitterionic intermediates produced upon photochemical electrocyclic ring closure of benzothiophene carboxanilides in the triplet excited state. Chemical yields generally exceed 90%, while quantum yields vary with basicity of the released leaving group.

Photochemical elimination of leaving groups from zwitterionic intermediates generated via electrocyclic ring closure of alpha,beta-unsaturated anilides.

Methacrylanilides, ArN(CH3)COC(CH2LG)=CH2, with allylic leaving groups (LG(-) = BocAla, PhCO2(-), PhCH2CO2(-), PhO(-)) undergo photochemical electrocyclic ring closure to produce a zwitterionic intermediate. Further reaction of the intermediate results in expulsion of the leaving group to give an alpha-methylene lactam as the major product. In addition, a lactam product that retains the leaving group is formed via a 1,5-H shift in the intermediate. Elimination of the leaving group is generally preferred, even for LG(-) = PhO(-), although in benzene as the solvent the lactam retaining the phenolate group becomes the sole photoproduct. The electrocyclic ring closure occurs in the singlet excited-state for the para-COPh-substituted anilide derivative and is not quenched by 0.15 M piperylene or 0.01 M sodium 2-naphthalenesulfonate (2-NPS) as triplet quenchers. Comparable concentrations of 2-NPS strongly quench the transient absorption of the triplet excited state observed at 450-700 nm according to laser flash photolysis experiments. In aqueous media, quantum yields for total products are insensitive to leaving group ability, and Phi(tot)(para-CO2CH3) = 0.04-0.06 at 310 nm and Phi(tot)(para-COPh) = 0.08-0.1 at 365 nm, for which Phi(isc) = 0.15.

Photoactivation of amino-substituted 1,4-benzoquinones for release of carboxylate and phenolate leaving groups using visible light.

Upon exposure to visible light, 2-pyrrolidino-substituted 3,6-dimethyl-1,4-benzoquinones photocyclize to give benzoxazolines with quantum yields of 0.07-0.10 in CH2Cl2, 0.02-0.04 in CH3CN, and <0.01 in 30% aq CH3CN. With carboxylate or phenolate leaving groups incorporated via coupling to a 5-hydroxymethyl group of the quinones, the photocyclizations give benzoxazolines that eliminate the leaving groups in a dark reaction. Lifetimes for elimination of 4-YC6H4OH in 30% phosphate buffer in CD3CN (pD 7) at 17 degrees C are 13.1, 0.54, and 0.13 h for Y = H, CF3, and CN, respectively, and the linear equation log k (h(-1)) = 0.998(-pKa) + 8.80 gives a best fit to the data. Carboxylate leaving groups are rapidly eliminated upon photolysis of the quinones in aq CH3CN to produce an o-quinone methide intermediate that is trapped by 4 + 2 cycloaddition with unreacted starting material or with added 3-(dimethylamino)-5,5-dimethyl-2-cyclohexen-1-one. The ortho-quinone methide is observed at 339 and 455 nm by conventional absorption spectroscopy and gives a pseudo-first-order fit of the decay kinetics with tau1/2 = 34.9 min in 30% phosphate buffer in CH3CN at 20 degrees C.

Photochemical cleavage and release of para-substituted phenols from alpha-keto amides.

In aqueous media alpha-keto amides 4-YC6H4OCH2COCON(R)CH(R')CH3 (5a, R = Et, R' = H; 5b, R = iPr, R' = Me) with para-substituted phenolic substituents (Y = CN, CF3, H) undergo photocleavage and release of 4-YC6H4OH with formation of 5-methyleneoxazolidin-4-ones 7a,b. For both 5a,b quantum yields range from 0.2 to 0.3. The proposed mechanism involves transfer of hydrogen from an N-alkyl group to the keto oxygen to produce zwitterionic intermediates 8a-c that eliminate the para-substituted phenolate leaving groups. The resultant imminium ions H2C=C(OH)CON+(R)=C(R')CH3 9a,b cyclize intramolecularly to give 7a,b. The quantum yields for photoelimination decrease in CH3CN, CH2Cl2, or C6H6 due to competing cyclization of 8a,b to give oxazolidin-4-one products which retain the leaving group 4-YC6H4O- (Y = H, CN). A greater tendency to undergo cyclization in nonaqueous media is observed for the N,N-diethyl amides 5a than the N,N-diisopropyl amides 5b. With para electron releasing groups Y = CH3 and OCH3 quantum yields for photoelimination significantly decrease and 1,3-photorearrangment of the phenolic group is observed. The 1,3-rearrangement involves excited state ArO-C bond homolysis to give para-substituted phenoxyl radicals, which can be observed directly in laser flash photolysis experiments.

Photochemical cyclization with release of carboxylic acids and phenol from pyrrolidino-substituted 1,4-benzoquinones using visible light.

Visible light irradiation of 5-acyloxymethyl- and 5-phenoxymethyl-2-pyrrolidino-1,4-benzoquinones effects photoisomerization to labile oxazolidines, which undergo elimination of carboxylate or phenolate leaving groups in high yields to generate trappable o-quinone methide intermediates. [reaction: see text]

Photochemical cleavage and release of carboxylic acids from alpha-keto amides.

In aqueous media, alpha-keto amides LGCH(2)COCON(R)CH(R')CH(3) (1a, R = Et, R' = H; 1b, R = (i)()Pr, R' = Me; 1c, R = Ph, R' = H) with various carboxylate leaving groups (LG) at the C-3 position undergo photocleavage and release of carboxylic acids with formation of diastereomeric 5-hydroxyoxazolidin-4-ones 2a,c in the cases of 1a,c or 5-methyleneoxazolidin-4-ones 3b in the case of 1b. For 1a,b, Phi(photocleavage) = 0.24-0.38, whereas Phi(photocleavage) = ca. 0.05 for 1c. The proposed mechanism involves transfer of hydrogen from an N-alkyl group to the keto oxygen to produce zwitterionic intermediates 4a-c that eliminate carboxylate anions. The resultant imminium ions, H(2)C=C(OH)CON(+)(R)=C(R')CH(3) 5a-c, cyclize intramolecularly to 3b or undergo intermolecular addition of water followed by tautomerization and cyclization to give 2a,c. These inter- or intramolecular trapping reactions of 5 release protons that decrease the pH and cause bleaching of the 620 nm band of the pH indicator, bromocresol green. Determination of the bleaching kinetics by laser flash photolysis methods in the case of 1a gives time constants of 18-137 mus, depending on the leaving group ability of the carboxylate anion, whereas amides 1b show only a small leaving group effect. For 1a, the large leaving group effect is consistent with rate-limiting carboxylate elimination from 4a, whereas the proton release step would be largely rate determining for 1b. Photolyses of 1a (LG = CH(3)CO(2)(-), PhCH(2)CO(2)(-)) in neat CH(3)CN results in carboxylate elimination to form imminium ion 5a, followed by internal return to give aminals.

Substituent effects on competitive release of phenols and 1,3-rearrangement in alpha-keto amide photochemistry.

[reaction: see text] Photolysis of alpha-keto amides bearing 4-YC(6)H(4)O leaving groups at the position alpha to the keto group efficiently produces high yields of phenols when Y is an electron-withdrawing group or H. The photoelimination likely involves cleavage of zwitterionic intermediates produced via excited-state hydrogen transfer. When Y is an electron-donating group, competing excited-state ArO-C(alpha) bond scission to radicals occurs, followed by recombination to give 1,3-photorearrangment products.

Photochemical cleavage and release of carboxylic acids from alpha-keto amides.

Carboxylate groups incorporated at the position alpha to the keto carbonyl of alpha-keto amides 1 were photochemically cleaved in aqueous media to give carboxylic acids in 70-90% yields with quantum yields of 0.3. The cleavage coproducts were diastereomeric hemiacetals 2. Prompt release of acetate and gamma-aminobutyrate (GABA) in buffer was observed by difference FT-IR spectroscopy upon 355 nm laser flash photolysis. The time-constant for release of GABA was <30 ms. [reaction--see text]

Photolysis of gamma-(alpha-carboxy-2-nitrobenzyl)-L-glutamic acid investigated in the mcrosecond time scale by time-resolved FTIR.

The photolytic release of substrates from caged substrates has proven to be an excellent method to generate concentration jumps for kinetic measurements in the microsecond time scale. In this report we use time-resolved FTIR in the step-scan mode to probe the photolysis mechanism of one such caged compound, namely gamma-(alpha-carboxy-2-nitrobenzyl)glutamate, and to obtain a direct measure of the rate of photorelease of the substrate glutamate. The time-resolved difference FTIR spectra exhibit specific signals that can be assigned to the reactant caged glutamate, photolytically released product glutamate, as well as to the aci-nitro intermediate, the key intermediate of the photolysis reaction. Therefore these signals allow the characterization of the kinetics of formation and decay of the intermediate and products. This is the first such report that provides a direct determination of the rate of formation of the photolysis product from a caged compound in the microsecond time scale. Furthermore, the results presented provide a good basis for further time-resolved FTIR studies of molecular reaction mechanisms, such as ligand protein interactions, in the microsecond time scale through the photolytic release of substrates from caged compounds.

Photochemistry of a Trisilane Substituted by a Pendant p-Cyanostilbene Electron Acceptor Chromophore.

Photolysis of (E)-4-cyano-4'-(2-heptamethyltrisilanyl)stilbene (E)-3 in a medium polarity solvent (CH(2)Cl(2)) results in efficient intramolecular electron transfer, which converts the initial pi,pi excited state ((1)LE state) to the charge transfer (CT) excited state. The CT excited state fluorescesces, undergoes E,Z photoisomerization, and reacts with MeOH to produce hydrosilane 4 via Si-Si bond cleavage and protonation of the central silicon, as shown by deuterium labeling. The CT excited state assignments are consistent with the observed quadratic plots of 1/Phi(f) versus [MeOH] and 1/Phi(EZ)() versus [MeOH], which indicate that both the initial (1)LE state and the CT excited state are quenched by MeOH, with the CT excited state serving as the emissive state and the state primarily responsible for the E,Z photoisomerization. Although biacetyl triplet sensitized photolysis results in efficient E,Z isomerization, quenching of direct photolyses by azulene shows (Z)-3 is not a product of the lowest energy triplet excited state, populated from the CT state by back electron transfer. The azulene quenching also shows that hydrosilane 4 is formed from the same excited state that gives (Z)-3. The intermediacy of a complex of the CT excited state with MeOH accounts for the observed upward curvature in the plot of 1/Phi(SiH) vs 1/[MeOH]. The linear behavior of Phi(EZ)()/Phi(SiH) x [MeOH] versus 1/[MeOH] further suggests that this complex reacts with uncomplexed MeOH to give hydrosilane 4.