Caged connections

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caged compounds (engl. cage cage) are chemical compounds which upon irradiation with light of certain wavelengths of releasing a different substance. The German translation of cage connection for these substances is not common in scientific usage.

The main area of ​​application of the caged compounds is biochemical and cell biological research. Biologically active compounds are equipped with a photolabile protective group ("cage") and thus temporarily lose their biological function. The photolabile protective group is irreversibly split off by exposure to light and the previously inactive compound has biological activity again.

Caged compounds are used to release effectors at a specific time at a specific location if their direct application is difficult (e.g. inside a cell) or too slow to achieve the desired concentration at the target location. The inactive caged compound, on the other hand, can also accumulate at the target through slow diffusion and, with subsequent exposure, release a sufficient amount of effector in a short time. By using intense flash lamps or lasers it is possible to detect a biochemical process, e.g. B. an enzymatically catalyzed reaction or a signal transmission to start very quickly (picoseconds to milliseconds).

As the first biochemical work with caged compounds is often a publication on caged - ATP JH Kaplan et al. from 1978, but shortly before that J. Engels et al. the synthesis and application of photo-releasable cAMP described.

Scope of application of caged connections

Today, caged compounds are used for many different purposes.

With them you can u. a.

  • Release substrates for enzymes and follow the subsequent reaction of the enzyme in a time-resolved manner,
  • Release signal molecules in a cell and observe how it reacts to the signal,
  • turn the expression of specific genes on or off.

Requirements for caged connections

Caged compounds are photolyzable compounds; H. they enter into a chemical reaction when exposed to light. For a photolyzable compound to be suitable as a caged compound, it must meet several conditions:

  • Stability. The unexposed substance should be stable. If a caged compound slowly disintegrates during storage or in solution even without exposure, the actually inactive caged compound is often contaminated with the active effector molecule. This can seriously falsify the interpretation of experimental results.
  • Biochemical inactivity. The biochemical function of the effector molecule must be suppressed as completely as possible by linking it to the photolyzable group.
  • Suitable photolysis properties. Three parameters are essential here:
    • Speed. The photolytic release of the effector from the caged compound should take place much faster than the process triggered by the effector molecule in the system under investigation. Only then can a statement be made about the speed of the subsequent process.
    • Specificity. After exposure, if possible, only one photochemical reaction of the caged compound should take place, so that no by-products are formed which do not have the desired or even no effect.
    • Efficiency. The cleavage of the caged compound should occur with as little light as possible. The efficiency of photolysis depends on two factors. On the one hand, as many of the photons as possible that are radiated into the sample should also be absorbed by the caged compound. For this purpose, the extinction coefficient ε of the caged compound must be high for the light wavelength used. On the other hand, every absorbed photon should actually cause a photolytic cleavage, i.e. H. the photochemical quantum yield Φ must also be high. An efficient photolyzable caged compound is therefore characterized by the largest possible product εΦ.
  • Light absorption at wavelengths greater than 300 nm. Many biomolecules, including proteins and nucleic acids, absorb UV light at wavelengths around 260–280 nm. If the caged compound also has to be excited in this range, other sample components already filter some of the for radiation required for photolysis and can suffer photo damage as a result. For these reasons, light absorption in the longer-wave spectral range is desirable.
  • Low reactivity and toxicity of the reaction products. During the photolytic cleavage, not only the desired effector molecule is created, but also a compound that is derived from the photolyzable group itself. If possible, this should not react further and should be non-toxic.
  • Good solubility of the caged compound and the reaction products.

structure

Although caged compounds do not form a uniform class of chemicals, there are structural similarities between them. They usually consist of two parts of the molecule: the target molecule to be released, also known as the effector molecule, and a photolyzable, i.e. H. group that can be split off by light, the " cage ". These photolyzable groups are related and e.g. Partly identical to the photolyzable protective groups used in organic synthesis . The chemical bond between the two parts of the molecule is broken by a photochemical process when irradiated with light of a suitable wavelength.

An example is caged - ATP (adenosine-5'-triphosphate-P 3 - (1- (2-nitrophenyl) ethyl) ester): the nucleotide adenosine triphosphate is modified with the photolabile nitrophenyl-ethyl (NPE) group ( Fig . 1 ). This caged -ATP can now be mixed with the sarcoplasmic calcium-ATPase - a muscle enzyme that requires ATP as a substrate - without reacting with it, because the attached NPE group prevents the enzyme from recognizing the modified ATP. When exposed to UV light, the NPE group is split off and the released ATP can now react with the enzyme. How the enzyme changes in the reaction with the ATP can then be e.g. B. be examined by infrared spectroscopy .

Fig. 1: Photolytic cleavage of NPE- caged -ATP

Today there is a great variety of caged compounds, both in terms of the released effector molecules and the photolyzable groups used.

Effector molecules

The classification of the effector molecules can be based on which chemical functionality is used to attach the photolabile group. Members of a substance class can often be provided with similar photolyzable groups by similar synthesis processes. Common classes of substances are: carboxylic acids , phosphoric acid esters , alcohols , amines , carbamates , thiols , ketones , aldehydes .

A classification according to the function of the released effector molecule is also possible. Some compounds can be classified into several of the following substance classes:

Photolyzable groups

A number of photolyzable groups have proven to be beneficial for the production of caged compounds. These are shown in Fig. 2 . The part of the molecule that is split off as an effector is located at the position marked with E.

  • 2-nitrobenzyl derivatives (a): The advantages of this class of substances are the good synthetic accessibility and the well-understood photochemistry. Disadvantages are a z. Sometimes poor water solubility, an absorption maximum in the short-wave UV range of the spectrum, slow photolysis and reactive by-products (2-nitroso-benzophenones). Despite the disadvantages, most of the commercially available caged compounds are 2-nitrobenzyl compounds.
  • Coumarinyl derivatives (b): The advantages are strong absorption in the longer-wave UV range, e.g. Sometimes even in the visible part of the light spectrum, usually a very fast photolysis and a non-toxic by-product. The disadvantage is an often low photochemical quantum yield. T. is compensated for by the high extinction coefficient.
  • p -Hydroxyphenacyl-Derivate (c): Advantages are the good synthetic accessibility, the good photochemical quantum yield and the fast photolysis. The disadvantage is that these compounds only absorb light significantly below 320 nm, similar to proteins and nucleic acids.
  • Benzoin derivatives (d): Advantages of benzoin derivatives are high quantum yields, rapid photolysis and an inert benzofuran by-product. The disadvantage is the strong fluorescence of the latter, which can interfere with certain optical detection methods. Furthermore, the chirality of benzoin can be problematic because the two isomers of the resulting caged compound can have different properties, e.g. B. in binding to enzymes.
  • 1-Acyl-7-nitroindoline derivatives (e): There are only a few examples of this class of compounds. One advantage of the class is rapid photolysis, disadvantageous is a low quantum yield.
Fig. 2: Frequently used photolyzable groups. E denotes the point at which the effector is attached; additional modifications can be made at the positions denoted by R.

In order to improve the properties of a caged compound, the listed photolyzable groups can be structurally modified. Various substituents at the positions marked with R can, for. B. move the absorption maximum of the compound in a more favorable area of ​​the light spectrum or improve the water solubility. The effector that is to be linked to the photolyzable group also has an influence on the properties of the caged compound that is not always easily predictable .

In addition to the compound classes mentioned, there are also a number of special caged compounds that cannot be classified into them ( Fig. 3 ). These include a .:

  • reversible photo acids, e.g. B. 8-hydroxypyrene-1,3,6-trisulfonic acid (f). When exposed to light, these emit a proton into the solution and take up a proton again after the excitation has subsided. In doing so, they cause a temporary pH shift in the solution.
  • Complex compounds of cobalt (g) serve as caged oxygen.
  • Complex compounds of ruthenium are used as caged electrons (h) or caged nitrogen monoxide.
Fig. 3: Special caged connections. The molecular parts split off during exposure are marked in blue. In the ruthenium complex (h) only the oxidation number of the central ruthenium atom changes.

Mechanisms of photo protection

The reaction of the caged compound triggered by the light can be roughly divided into three steps.

First of all , the caged compound absorbs a photon and thus changes to the electronically excited state .

Then the actual photochemical process of the excited state takes place. The reaction mechanisms that take place are very diverse and differ not only between the individual classes of substances, but sometimes even within a class of substances for different effector molecules or, in extreme cases, for one and the same compound when it is present in different solvents. What they have in common is that in the end the products are back in their basic electronic state. In the simplest case, the effector molecule has already been split off. The primary photochemical processes are often very quick and complete in picoseconds to microseconds.

Fig. 4: Caged calcium. During photolysis, it is primarily the blue-marked part of the molecule that is split off, not the calcium ion itself.

Subsequent reactions sometimes follow, namely when the products created by photolysis are themselves not chemically stable. This effect is z. B. for the production of caged amines quite desirable. Caged compounds are difficult to produce with the amines themselves, which is why the corresponding carbamates are used . If the carbamate was released from the caged compound after exposure , it spontaneously decomposes further into CO 2 and the desired amine. The decomposition of the carbamate is slower than its release from the caged compound and thus limits the rate of formation of the amine.

Such a secondary reaction is also used for the often used caged calcium ( Fig. 4 ). In the caged compound itself, the calcium ion is bound as a chelate complex . The exposure does not release the ion directly, but primarily only leads to the cleavage of a bond in the chelate ligand. The "defective" ligand can no longer bind the calcium tightly enough: it breaks down into two parts and releases the ion into the solution.

literature

Individual evidence

  1. ↑ a b c G. CR Ellis-Davies (2007) Caged compounds: photorelease technology for control of cellular chemistry and physiology. Nat. Methods 4 , 619-628, doi : 10.1038 / NMETH1072 .
  2. ^ SR Adams, RY Tsien (1993) Controlling cell chemistry with caged compounds. Annu. Rev. Physiol. 55 , 755-784, doi : 10.1146 / annurev.ph.55.030193.003543 .
  3. ↑ A. Deiters (2009) Light activation as a method of regulating and studying gene expression. Curr. Opin. Chem. Biol. 13 , 678-686, doi : 10.1016 / cbpa.2009.09.026 .
  4. ↑ JH Kaplan, B. Forbush III, JF Hoffman (1978) Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: Utilization by the Na: K pump of human red blood cell ghosts. Biochemistry 17 , 1929-1935, doi : 10.1021 / bi00603a020 .
  5. ↑ J. Engels, Ernst-Jürgen Schlaeger (1977) Synthesis, structure, and reactivity of adenosine cyclic 3 ', 5'-phosphate benzyl triesters. J Med Chem 20 , 907-911, doi : 10.1021 / jm00217a008 .
  6. ↑ C. Zscherp, A. Barth (2001) Reaction-induced infrared difference spectroscopy for the study of protein reaction mechanisms. Biochemistry 40 , 1875-1883, doi : 10.1021 / bi002567y .
  7. ↑ L. Kröck, A. Heckel (2005) Light-induced transcription with temporarily mismatched oligonucleotides. Angew Chemie 117 , 475-477, doi : 10.1002 / anie.200461779 .
  8. ↑ a b c d A. P. Pellicioli, J. Wirz (2002) Photoremovable protecting groups: reaction mechanisms and applications. Photochem. Photobiol. Sci. 1 , 441-458, doi : 10.1039 / b200777k .
  9. ↑ TW Green, PGM Wuts. Protective Groups in Organic Synthesis. Wiley-Interscience, New York, 2006, ISBN 978-0471697541 .
  10. ↑ A. Barth, W. Mäntele, W. Kreutz (1991) Infrared spectroscopic signals arising from ligand binding and conformational changes in the catalytic cycle of sarcoplasmic reticulum calcium ATPase. Biochim Biophys Acta 1057 , 115-123, doi : 10.1016 / S0005-2728 (05) 80091-X .
  11. ^ JA McCray, DR Trentham (1989) Properties and uses of photoreactive caged compounds. Annu. Rev. Biophys. Biophys. Chem. 18 , 239-270, doi : 10.1146 / annurev.bb.18.060189.001323 .
  12. ^ DB Spry, MD Fayer (2008) Charge redistribution and photoacidity: Neutral versus cationic photoacids. J. Chem. Phys 128 , 084508-1-084508-9, doi : 10.1063 / 1.2825297 .
  13. ↑ R. MacArthur, A. Sucheta, FF Chong, O. Einarsdottir (1995) Photodissociation of a (μ-peroxo) (μ-hydroxo) bis [bis (bipyridyl) cobalt (III)] complex: a tool to study almost biological reactions involving O 2 . Proc. Natl. Acad. Sci. USA 92 , 8105-8109, abstract .
  14. ↑ R. Nakano, H. Sato, T. Shimizu (1996) Tris (2,2'-bipyridyl) ruthenium (II) -mediated photoinduced electron transfer of engineered cytochrome P450 1A2. J. Photochem. Photobiol. B 32 , 171-176, doi : 10.1016 / 1011-1344 (95) 07230-6 .