Photochemistry_of_Organic

Special Topic 6.16: Photoaffinity labelling

Photoaffinity labelling is a technique for studying the structures of proteins, DNA and other biomolecules, and also biomolecule–ligand and biomolecule–biomolecule

transient interactions, in order to understand specific biochemical mechan- isms.854,1147,1166–1171 In this technique, a ligand (label, probe), often fluorescent or

radioactive, bears a photoactivatable group (see also Special Topic 6.18), which upon irradiation generates a highly reactive intermediate that appends to the specific site on a biomolecule in its vicinity through a covalent bond (Figure 6.12). Such a modified biomolecule, either directly or after some modifications (such as chemical fragmentation), is examined spectroscopically, radiochemically or by conventional chemical analyses. When the photoactivatable group is a part of another biomolecule, cross-linking between biomolecules may take place.

Figure 6.12 Photoaffinity labelling

Photoactivatable groups should be sufficiently stable under ambient light and the photoreactions should be faster than dissociation of the ligand–receptor complex and be site specific.1147 The reactions of typical photoaffinity groups have already been described: azides (Scheme 6.175) or 3H-diazirines (Scheme 6.169) and diazo compounds (Scheme 6.170) form nitrenes or carbenes, respectively, upon irradiation, whereas excited benzophenone derivatives abstract hydrogen to form ketyl radicals that recombine to form a new bond (Scheme 6.99). Carbenes are generally recognized as being more reactive than nitrenes. A mechanistic study of 3-p-tolyl-3-trifluoromethyl- diazirine (378) photoreactivity has shown, for example, that p-tolyl(trifluoromethyl) carbene (379) formed upon irradiation may add to an unsaturated or aromatic system to

form a cyclopropane intermediate 380 (and subsequent rearrangement products) or insert to an inactivated C H bond to form 381 (Scheme 6.178).1172

Here we show two examples of photoaffinity labels. A diazo group containing immunosuppressant cyclosporin A (382), which binds to protein cyclophilin and undergoes specific cross-linking upon irradiation due to formation of the corresponding aryltrifluoromethycarbene, was used to study signalling pathways involved in immunomodulation.1173 The enzymatically non-cleavable azidoanilido guanosine triphosphate analogue 383 was utilized as an efficient label for the G protein (a protein involved in second messenger cascades).1174 The 32P tag was used for identification and active site mapping.

N-Oxides

Nitrones or heterocyclic N-oxides are examples of photolabile compounds with polarized

N O bonds, where nitrogen is sp2 hybridized. They possess a p,p lowest-energy absorption band with a strong charge-transfer character.1061,1067 Apart from E–Z

isomerization, they undergo a characteristic rearrangement to form oxazirine intermediates (Scheme 6.179), which are photolabile and may react further. For example, azanaphthalene N-oxide (384) affords ring enlargement to benzoxazepine (385) in aprotic solvents or rearranges in the presence of water to an indoline derivative 386, possibly via an oxazirine intermediate (Scheme 6.180).1175

hν

H2O

OH

N

CHO

386

Scheme 6.180

Nitrite Esters

The primary photochemical process of organic nitrites is homolytic fission of the N O bond1176 [DO–N(ethyl nitrite) 176 kJ mol 1].874 When alkyl d-hydrogens are available,

intramolecular d-hydrogen abstraction by the resulting alkoxyl radical to generate a carbon radical, which further combines with the nitric oxide hereby formed and isomerizes to the corresponding oxime, is called the Barton reaction1177 (e.g. nitrite ester 387 photolysis1178 in Scheme 6.181). This reaction provides a unique tool for preparing suitable d-substituted derivatives in steroids (an oxime group is easily transformed into a carbonyl moiety, for example), because the corresponding methyl groups and alkoxyl radicals with a 1,3-diaxial configuration (388) prefer hydrogen abstraction through a six-membered cyclic transition state.1176

Scheme 6.181

Heteroaromatic Compounds

Nitrogen-containing heteroaromatic compounds, such as triazoles, tetrazoles,1111 pyrazoles and 1,2,4-oxadiazoles1117 undergo various photoinduced isomerization and

ring-opening reactions. Photolysis of 1H-benzotriazole (389), for example, leads to fast and efficient, yet reversible, N N bond fission to give the diazo compound 390.1179

Nitrogen elimination products are obtained only upon prolonged irradiation and typically with low chemical yields (Scheme 6.182).

389390

Scheme 6.182

Photochemical transposition reactions of some heteroaromatic compounds have already been discussed in Section 6.2.1. Scheme 6.183 shows the photoisomerization of 1-methylpyrazole (391), which may involve competition between electrocyclic ring

closure and cleavage of the N1–N2 bond to give the same product (1-methylimidazole,

392).1117,1180

Scheme 6.183

Special Topic 6.17: Photochemistry on early Earth and in interstellar space

The primitive atmosphere on early Earth was composed of nitrogen, methane, ammonia, carbon dioxide and other simple inorganic and organic molecules.1181,1182

As a heterogeneous system of dust, aerosol particles and water droplets, it was exposed to high-energy radiation (<250 nm) from the young Sun. Complex abiogenic processes presumably produced biologically important compounds essential for emerging life. Some of them, for example guanine and cytosine base pairs of DNA, possess extraordinary photostability (Special Topic 6.7), which could have been an important selective factor in determining the eventual chemical composition of biomolecules.

Laboratory studies have provided evidence that photochemical and photocatalytic (Section 6.8) steps might play an important role in the formation of amino acids or various heterocyclic compounds from very simple molecules. For example, UVC irradiation of acetonitrile–ammonia–water mixture produces hexamethylenetetramine, a potential precursor of amino acids, via two-step photoinitiated fragmentation of acetamide (formed by acetonitrile hydrolysis) to give carbon oxide, which undergoes further photochemical and dark reactions (Scheme 6.184).1183

Scheme 6.184

Infrared observations, combined with laboratory simulations, have also advanced the understanding of chemical processes occurring in comets in interstellar space.1184 Comets are ices made of simple molecules, such as H2O, CH3OH, NH3, CO and CO2, although more complex species, including nitriles, ketones, esters or aromatic hydrocarbons, can also be present. Chemical changes can be promoted due to penetrating cosmic radiation or absorbed solar radiation. In the laboratory, UV photolysis (usually by a hydrogen-flow discharge lamp producing Lyman-a emission, lirr < 200 nm, in a high vacuum) of cometary ice analogues at temperatures below 50 K

gives moderately complex organic molecules, such as ethanol, formamide, acetamide, nitriles,1185 and even amino acids (Scheme 6.185).1186 The subsequent delivery of extraterrestrial matter to Earth is suggested to have been an alternative source of prebiotic organic molecules.

Scheme 6.185

6.4.3Nitro Compounds: Photofragmentation and Photoreduction

Recommended review article.1187

Selected theoretical and computational photochemistry references.1188–1191

Simple nitroalkanes absorb below 350 nm and are excited to the lowest singlet n,p state, which efficiently intersystem crosses to T1. Homolytic photocleavage is the principal primary process in both the gas phase and solution to produce alkyl radicals and NO2 (Scheme 6.186).1187 Apart from subsequent recombination of the radical intermediates to form nitrites (Section 6.4.2), competing hydrogen abstraction (photoreduction) involving an excited nitro compound and a hydrogen-atom donor may take place.

hν,

[H]photoreduction

OH

R N

O

Scheme 6.186

Nitroarenes display strong absorption in the near-UV region and are efficiently photoreduced.1187 An intermolecular version of this reaction is depicted in Scheme 6.187. The photoreduction is initiated by hydrogen atom abstraction. Substituted nitrobenzenes 393, where X is an electron-donating group (X ¼ p-Me, p-OMe) or nitrobenzene itself (X ¼ H) are photoreduced in the presence of propan-2-ol to the corresponding

Special Topic 6.18: Photoactivatable compounds

In general, photoactivatable compounds (also called caged compounds) are those which, upon photoactivation, either (1) irreversibly release a species (A; Scheme 6.189) possessing desirable physical, chemical, or biological qualities; in such a case, they are called photochemical triggers, and the groups that are responsible for the photoprocess are referred to as photoremovable, photoreleasable or photolabile; or

(2) reversibly induce physical or chemical changes in another, covalently or noncovalently bound moiety (B and C in Scheme 6.190a), modify the affinity for another molecule (D in Scheme 6.190b) or exchange protons or electrons; in this case, they are called photochemical switches and the process is usually photochromic (Special Topic 6.15).

Scheme 6.189

Scheme 6.190

Today, photoactivatable compounds are of great interest in connection with biochemical and biological applications (e.g. photoregulation of proteins and enzyme

activity, neurotransmitters, ATP and Ca2 þ delivery or photoactivatable fluorophores), 1015,1034,1035,1197,1198 organic synthesis (e.g. photoremovable protecting groups; solidphase synthesis; microarray fabrication),1015,1035,1199,1200 nanotechnology (prospective molecular machines and computers; see Special Topic 6.19),1103,1104 or even

cosmetics (photoactivatable fragrances).1201 A great advantage of photochemical activation over other stimuli is the ability to control precisely the processes in time and space.

Photochemical triggers irreversibly release free target molecules and also photoproduct(s) formed by the transformation of photoremovable groups (Scheme 6.189). Their design must fulfil several requirements; for example, the side-products should be chemically and photochemically stable and nontoxic (in biological applications) and the photoremovable moiety should absorb at wavelengths