Strategies for Coupling Molecular Units if ... - ACS Publications

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Strategies for Coupling Molecular Units if Subsequent Decoupling Is Required Roman Bielski*,† and Zbigniew Witczak*,‡ †

Value Recovery, Inc., 510 Heron Drive, Suite 301, Bridgeport, New Jersey 08014, United States Department of Pharmaceutical Sciences, Nesbitt School of Pharmacy, Wilkes University, Wilkes-Barre, Pennsylvania 18766, United States

‡

7.3.5. Formation, Functionalization, and Cleavage of Carbonyl Derivatives 7.3.6. Cleavage of Carbonates, Carbamates, and Heterocarbonates 7.3.7. Decoupling of Alkyl (Aryl) Ethers, Thioethers, and Amines 7.3.8. Formation of and Cleavage of Aza, Diazo, etc. Derivatives 8. Potential Future Applications of CAD Chemistry 9. Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments Abbreviations References

CONTENTS 1. Introduction 2. CAD ChemistryWhen? 3. CAD ChemistryHow? 3.1. Processes in Which the Same Bonds Are Broken during the Coupling and Decoupling 3.2. Introduction of Sacrificial Units 3.3. Reversible versus Sacrificial 4. CAD ChemistryDesirable Properties 5. Things To Consider When Selecting a Specific CAD Chemistry 5.1. Triggers 5.2. Structure of Units to Be Coupled and Decoupled 5.3. Do Both Coupled Units Need To Be Recovered? 5.4. How Well Established Is the Chemistry To Be Used? 6. Existing Cleavable Linkers 6.1. Physically Cleavable Linkers 6.2. Enzymatically Cleavable Linkers 6.3. Chemically Cleavable Linkers 7. Potential Specific Reactions 7.1. Decoupling by Light 7.2. Decoupling by Enzymes 7.2.1. Enzymatic Cleavage of a Double Bond 7.2.2. Cleavage of Acid Derivatives 7.2.3. Dearylation of Arylamino Compounds 7.3. Decoupling by Chemicals 7.3.1. Desulfurization of Thioethers, Dithioacetals, Sulfoxides, and Sulfones 7.3.2. Cleavage of Double Bonds 7.3.3. Carbonyl Group as an Object of CAD Chemistry 7.3.4. Cleavage of Derivatives of Carboxylic and Other Acids © 2012 American Chemical Society

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1. INTRODUCTION Chemists spend a great deal of time developing new methodologies to connect various molecules and add “decorations” to molecules of biological origin. The relevant processes are called bioconjugation, click chemistry, labeling, ligation, tagging, derivatization, etc. and are reported in a number of recent reviews.1−19 Successful tagging or bioconjugation methods tend to produce very strong (usually covalent) interactions. Thus, decoupling molecules is more difficult than coupling, which is a major reason why much less effort has been devoted to decoupling connected units. Additionally, more often than not we do not care what the fate of the connected constructs is after the experiment is complete. The exception is protection/deprotection chemistry, where the original chemical functionality is restored. However, protection/deprotection chemistry is applied almost exclusively to the multistep organic synthesis of small molecules. There are many circumstances when there is a need to couple two or more units and then decouple them later. We propose to call this chemistry coupling and decoupling, or CAD chemistry. The present review discusses applicable strategies and highlights some of the potential approaches and reactions that can be useful when selecting and performing CAD chemistry. It is worth noting that CAD chemistry often applies to situations when at least one molecular unit is a large molecule with many functional groups.

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Received: August 26, 2011 Published: November 15, 2012

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While specific coupling reactions within CAD chemistry may be the same or similar to those used in protection, tagging, labeling, bioconjugation, derivatization, etc., the general approach is different. Coupling such as click chemistry utilizes reactions providing very stable products that are not expected to be decoupled. When selecting the coupling reactions for CAD, one must keep in mind that at a certain point some bonds will have to be broken. One obvious strategy takes advantage of the formation of specific bonds during the coupling (1) and breaking the same bonds during the decoupling (2) reactions (Scheme 1, pathway a). This option

Scheme 2. Example Showing Differences between Deprotection and Decoupling

Scheme 1. Possible Approaches to the Issue of Future Decoupling of Connected Molecular Units

from the original ketone, but only the first procedure can be called deprotection. There is no doubt that decoupling chemistry is much less developed than coupling chemistry. However, there are many situations that call for the use of reliable disconnection reactions. Initially, we will list various circumstances requiring decoupling chemistry. Next, we will discuss the possible strategies for designing coupling procedures that will enable a relatively painless future decoupling. Then, we will review the literature of existing cleavable linkers. Finally, we will discuss specific reactions that have been or can be employed as potential decoupling processes. The selected decoupling reactions are divided into several categories based on the type of employed trigger and functionalities present in the starting material. The specific trigger may be of a chemical, biological (usually enzymatic), or physical (mainly photocleavage) nature. Only some of the disconnection reactions discussed here are already reasonably well developed and practiced, many offer only the potential to become useful, and the scope of most of these reactions is not known. Generating a complete set of reactions for a review such as this is an impossible task. This is because we review not only reactions that have been used in decoupling larger constructs but also reactions that we think have the potential to become applicable to the process. Also, most potentially useful reactions have not been tried on natural macromolecules such as polysaccharides, nucleic acids, proteins, or glycoproteins. Thus, the scope of these reactions is not well established. We tried to choose reactions that are applicable to a large variety of substrates and those which have the potential to become sufficiently general or high yielding. The following examples are compiled from the existing literature in a selective manner and cover only a number of compounds/reactions and methodologies. While CAD chemistry applies mainly to large molecules, the majority of examples reviewed herein come from the chemistry of small molecules, as the number of existing examples of good decoupling reactions is very limited. There is an acute need to develop reliable decoupling processes and determine their scope. The main purpose of this review is to initiate a discussion on the best methodologies applicable to CAD chemistry and to encourage chemists to search for new and better decoupling reactions.

has been successfully employed in various circumstances. However, such reverse reactions are not always sufficiently chemoselective or require conditions that are too harsh for many biomolecules. Therefore, it is often necessary to employ another approach. Another different strategy (Scheme 1; pathway b) requiring the introduction of a sacrificial chemical moiety during the coupling process and the cleavage of this very moiety during the decoupling seems to be particularly suitable. In such a case, different bonds are formed during the coupling procedure (1) and broken during the decoupling (2). There is a major difference between CAD and protection/ deprotection chemistry. While protecting groups are generally small parts of the molecules they are on, the constructs formed in the CAD process may be very large and often consist of two or more macromolecules. Additionally, during deprotection, the original functionality is restored. This is not necessarily the case with CAD. For example, let us assume that a ketone (8) is reacted with 1,2-dithiol (9) containing a substituent R3 to form a cyclic thioacetal (10) (Scheme 2). If the thioacetal formation was to act as a protecting group for the carbonyl group, the ultimate hydrolysis of the thioacetal will re-form the original ketone (8) (pathway a). In the CAD chemistry we can do this, but we may choose to desulfurize the thioacetal to form a product with a methylene group (11) (pathway b). Both procedures (hydrolysis and reduction) decouple the R3 group

2. CAD CHEMISTRYWHEN? When is there a need to decouple connected units? In most cases we discard the pieces, or they become a part of a large (macromolecular) structure. Nevertheless, situations do exist when we need to decouple units and recover a piece or pieces. The following examples describe some of these situations: 1. Therapeutic Delivery. Upon delivering a specific therapeutic to the target and after the selected time, we need 2206


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1) that can be decoupled under specific conditions. Similarly, Kobayashi at al.29 and Matsumura et al.30 show that certain aliphatic polyesters can be cleaved using hydrolytic enzymes such as Candida antarctica to form oligomeric units exhibiting well-defined molecular weight that are good starting materials for polymerization using the same enzyme. This interesting approach to polymer recycling should be expanded to other categories of polymers, such as other polyesters, polyacetals, polyamides, etc. 5. One way to make biodegradable polymers is to introduce units degraded by environmental enzymes. For example, (poly)ester units can be spread over a chain made of acrylic or styrenic polymers. Decoupling in other systems can be designed in a similar way. For example, a microcapsule can be made of a polymer containing sacrificial units (Scheme 1), such as appropriate dipeptides. If the formulated forms of drug contain a microencapsulated active compound, the active compound will be released in the stomach, where there are enzymes operating at low pH capable of degrading the selected dipeptides. 6. Auxiliaries for Convenient Purif ication. We may want to add to the molecules of interest a moiety that helps during the purification process, such as a moiety with ferromagnetic properties. (Ferromagnetism is a macroscopic phenomenon, and therefore, there are no ferromagnetic atoms or small molecules. However, since the units exhibiting ferromagnetic properties are becoming smaller and smaller and have already attained a size of a few nanometers, molecules equipped with ferromagnetic units may soon be exploited as an analytical tool.) After separating out the desired molecules from the mixture, we must then decouple the units used to aid the separation (in this case ferromagnetic units). Similarly, we may want to cleave molecules from the surface of magnetic beads. A related example comes from the use of chiral auxiliaries. We often react the enantiopure units of a chiral auxiliary with the racemic product of a reaction, chromatograph the mixture of the formed diastereoisomers, and decouple the chiral auxiliary from the resolved products. After decoupling the enantiopure auxiliary, we should end up with the resolved racemate components and the chiral auxiliary that can be reused. 7. Quantitation Tools. The quantitation of a specific analyte (A in Scheme 3) may require taking advantage of strong interactions such as biotin−avidin interactions. In this case, biotin is coupled to our compound of interest, and the strong biotin−(strept)avidin interaction is used to select the

to terminate the treatment by triggering the appropriate decoupling chemistry. One option for the delivery of therapeutics is to take advantage of such constructs as prodrugs,20 codugs,21 or polymer therapeutics.22 “Prodrugs are bioreversible derivatives of drug molecules that undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug, which can then exert the desired pharmacological effect”.20 There are several reasons why one may add functionalities to the active compound to render it inactive. The most common reason is to better control the delivery of the active drug. The concept of codrugs is similar. “Codrug or mutual prodrug is an approach where various effective drugs, which are associated with some drawbacks, can be modified by attaching with other drugs of same or different categories directly or via a linkage. More appropriately one can say combining two different pharmacophores with similar or different pharmacological activities elicit synergistic action or help to target the parent drug to specific site/organ/cells respectively. This approach is commonly used to improve physicochemical, biopharmaceutical and drug delivery properties of therapeutic agents”.21 “Polymer therapeutics encompass polymer−protein conjugates, drug−polymer conjugates, and supramolecular drug-delivery systems”.22 Some specific examples of codrug and prodrug applications are discussed later in the text. 2. Targeted Release. Sometimes there is a need to synthesize a compound that is not degraded under specific conditions, such as those in the gastrointestinal system or when crossing the blood/brain barrier. This may be accomplished by introducing a specific moiety that inhibits a potential hydrolysis or masks the active ingredient. At a certain point, the active must be released into the system. Clearly, we need simple decoupling chemistry for the process, where the cleavage must take place inside the living cell.14 3. Decoupling from the Solid Support. Solid phase peptide, glycopeptide, and other solid-based syntheses are very important synthetic methodologies. One of the difficulties in establishing such procedures (particularly when synthesizing oligosaccharides and glycoconjugates) has been the cleavage of the product, i.e. decoupling the synthetic construct from the solid support, without destruction of acid- or base-sensitive functionalities. While many very effective linkers (Wang linker, Rink linker, Rich photolabile linker, HMBA linker, etc.) that can be cleaved from the support have been developed over the last few decades, most of them are applicable only to selected situations and cannot be used for other applications.23−26 To resolve some of these issues, solid phase linkers that can be decoupled under essentially neutral conditions have been developed.27 4. Af fecting Chemical Outcome Using Cleavable Units. Organic chemists have used directing units to control the selectivity of chemical reactions for decades. Often, the directing groups must be removed after the targeted delivery into the compound has been accomplished. Such situations require a reliable decoupling methodology. The use of removable directing groups in synthesis and catalysis has been reviewed recently.28 5. Self-destruction/Recycling. It may be of value to destroy a construct after it has fulfilled its function. For example, sometimes a bioconjugate must be degraded after successful delivery and subsequent release of the active compound. In such a case the bioconjugate should be equipped with a plurality of chemical functionalities (sacrificial unitsScheme

Scheme 3. Example of Using Biotin−Avidin Interactions Followed by Quantitation and Decoupling

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electric field, the molecules become oriented, and this orientation can be frozen by the formation of (at least) two covalent bonds (per molecule) with the surface. After the molecules are attached to the surface, the moieties rendering a large DM, which are no longer useful, may be decoupled prior to moving to the next step in the research.31 10. Controlling Surface of Nanomaterials. A recent paper by Russel, Thayumanavan, et al.32 states, “The repertoire of nanoporous materials will be significantly enhanced, if we were able to decorate the pore walls with reactive functional groups. ... One can envision the introduction of a cleavable group between the two blocks of a diblock copolymer assembly, which can be broken under mild conditions and leave behind a chemically active functional group.” This will create novel applications of this modern technology. Scheme 5 demonstrates the execution of the concept, which uses the S−S bond cleavage, leading to gold-coated nanopores. Interestingly, a similar concept was proposed and executed (using trityl ethers) by another team.33,34 11. Determining the Structure or Taking Advantage of Natural Products. Determining the sequence of connecting units of natural macromolecules is a constant challenge. It usually requires employing some kind of cleavage methods. While the existing protocols applicable to protein and nucleic acids may be deemed sufficient (even if better methods are always welcome), there is a deep need to develop better methodologies for determining a sequence of monosaccharides in a polysaccharide chain. There are many reasons why such knowledge is essential, including gaining a better understanding of receptors and their selectivity, but often we do not have the necessary tools to study their structure. This described case formally does not belong to CAD chemistry, since we do not couple the connecting units. Nevertheless, we need to perform decoupling chemistry, and the selected methodologies may come from the repertoire of the discussed decoupling reactions. Here is another example where we have no influence over the structure of the natural compound, but the use of appropriate decoupling chemistry may become very handy. Let a natural product be available but a portion of that product be useful. For example, let us imagine that β-carotene (12) is easily available but we are interested in vitamin A (retinol 13). There is a clear need for a relevant decoupling method that will split β-carotene at the (15,15′) double bond to form two molecules of the aldehyde that can be reduced to retinol (Scheme 6).35,36 It is worth adding that β-carotene cleavage at double bonds at position 9,10 (and 9′,10′) offers another valuable product: β-ionone (14). Such cleavage can be accomplished using a carotenoid cleavage dioxygenase from Arabidopsis thaliana.37,38

compound of interest. After washing away nonspecific interactions, the analyte molecules then need to be decoupled and measured. While quantitation of the analyte molecules that are still attached to avidin may be performed, if the compound is valuable, it must ultimately be recovered. While it may be unwise to split avidin from biotin, decoupling units connected by well designed CAD chemistry will disconnect biotin from the compound of interest. Many so-called cleavable linkers attached to the biotin−avidin system have been described in the literature and are discussed later in the review. 8. Limited Amount of Compounds for Multiple Experiments. The detection or quantitation of a specific analyte often requires the introduction of a moiety compatible with a selected detection technique (derivatization). This is particularly useful when taking advantage of ultrasensitive detection methods. If we need to perform two or more measurements, we introduce a selected derivatizing agent into each sample and run experiments with each derivatized sample independently, using various instruments. However, if there is very minute amount of an analyte available, we may want to incorporate an easily detectable unit into the analyte, perform measurements employing the selected detection method, and finally decouple the detectable unit. Next, we can couple the analyte to the unit compatible with a second quantitation method and submit it to the second detection methodology. Let us be more specific. Assume that we have 12 mg of a material and need to perform three different experiments, each requiring 9 mg. It seems that the CAD chemistry is our only hope. After each experiment, we isolate the construct of interest, decouple the unit which was compatible with a specific instrument, couple a unit necessary for the next experiment, decouple it after the experiment is over, and so on (Scheme 4). Provided that the available CAD chemistry offers high yields, such an approach may enable use of less of a valuable material and offer an economic advantage. Scheme 4. Multiple Measuarement of Minute Amounts of Analyte

9. Chemistry on Molecules Attached to a Surface. It may be important to take advantage of the fact that essentially all molecules adsorbed on a flat surface are oriented in the same direction (perpendicular to the surface). Thus, we may couple units containing groups that are adsorbed on the surface (say, a thiol group to form a self-assembly on a gold surface) to molecules of interest, perform the selected chemistry on molecules attached to the surface, and remove the molecules from the surface, leaving the adsorbing functionality (thiol) behind. Sometimes a reaction must be performed under very specific conditions. For example, a static electric field orients molecules equipped with a nonzero dipole moment (DM). The degree of orientation depends on the value of E and the substance’s DM. Thus, we may want to introduce a moiety with functionalities that render a prominent DM to the compound. When in an

3. CAD CHEMISTRYHOW? When considering potential coupling and future decoupling reactions, two completely different strategies are conceivable. The first one takes advantage of reactions that are reversible. (The word “reversible” is used here in a broad sense. For example, benzyl ethers are formed usually as a result of a reaction between an alcohol and a benzyl halide in the presence of a base. Debenzylation restores the alcohol functionality but does not restore the halide functionality. Nevertheless, for the purpose of this review, such processes will be called reversible.) The second strategy requires that, during the coupling, an easily cleavable unit is introduced. This unit must not be involved in any reactions until the decoupling. 2208


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Scheme 5. Example of Decoupling in Controlling the Surface of Porous Nanomaterialsa

a

Reprinted with permission from ref 32. Copyright 2009 American Chemical Society.

Scheme 6. Possible Use of CAD Chemistry When Manufacturing Retinol and β-Ionone from β-Carotene

Scheme 7. Using Two Reversible Coupling Reactions Offers a Choice of Conditions during Decoupling

chemistry.39−41 Schubert et al.12 call such processes reverse click reactions. However, at this point the conditions of these retro-Diels−Alder reactions are rather harsh (160 °C, 24 h). Thus, “this approach might be the inspiration for the development of reversible click reactions in the future”.12 Retro-homo-Diels−Alder reactions can also conceivably serve as decoupling processes, but the conditions of the thermal reactions are rarely mild and they are often performed only on small molecules. However, there are exceptions. For example, Deloisy et al. prepared a small β-substituted α,β-ethylenic aldehydes library by solid-phase cleavage using a thermal retroDiels−Alder reaction which required very mild conditions (20 °C or less) and gave usually good yields,42 and Workentin and co-workers prepared maleimide-modified monolayer-protected gold nanoparticles from the protected furan-maleimide via the thermally reversible Diels−Alder reaction (Scheme 8).43

3.1. Processes in Which the Same Bonds Are Broken during the Coupling and Decoupling

There are a number of reactions coupling appropriately functionalized units to form stable products which can be decoupled using reactions that are the reverse of the coupling ones. The approach is shown in Scheme 1, pathway a. The XY connection (the coupling of X and Y) is formed as a result of the coupling chemistry (1), and this very functionality (XY) is split using the decoupling chemistry (2) to produce the original reactants. In other words, the same bonds are formed during the coupling and broken during the decoupling processes. Alternatively, one can introduce two cleavable units (XY and WZ). This approach offers the choice of decoupling conditions, since either XY or WZ can be cleaved (Scheme 7). While the methodology shown in Scheme 1a is broadly used, it is often not applicable to biomolecules with a large number of various functional groups. The most common decoupling reaction, which is reversible, is hydrolysis. Processes forming esters, thioesters, amides, acetals, etc. are usually reversible. Unfortunately, with the exception of some (particularly enzymatic) procedures, hydrolyses of these products are often nonchemospecific and cannot be applied to many natural macromolecules such as proteins or polysaccharides. Other useful processes that can be reversed are protection-like reactions using benzyl, allyl, or propargyl substituents, redox reactions of sulfide/disulfide systems, etc. Also, some (reversible) hetero-Diels−Alder reactions are so easy to perform that they are considered to belong to click

Scheme 8. Example of a Reversible Homo-Diels−Alder Reaction Applicable to the CAD Chemistry

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(represented by single squares) at both ends (plus linkers of varying length). Since click chemistry is an exceptionally useful methodology of connecting molecules, Z and W (Scheme 1, path b) or single squares (Scheme 9, path b) should be functionalities taken from the repertoire of the click chemistry processes. However, it is unlikely to successfully form a cyclic triazole at both ends. If Z and W represent an azide and a triple bond, respectively (Scheme 10; 26), then the main product will

Finally, it is worth mentioning that 1,3-dipolar cycloaddition reactions such as those forming 1,2,3-triazoles can be reversed, but the necessary conditions make the potential use of the reverse reactions rather unrealistic at this point.44 3.2. Introduction of Sacrificial Units

Another approach requires the formation of a construct containing an easily decouplable moiety (AB in Scheme 1b) which is not involved in the formation of the construct. This moiety must survive all the conditions the construct will be exposed to and should be different from all the groups present in the connected units and easy to cleave using selective conditions. One could say that AB is a sacrificial functionality, since it is introduced only to be sacrificed later. Note that in this case entirely different chemical entities are involved in the coupling and decoupling processes. In Scheme 1, pathway b, the formation of XZ and WY (structure 5) represents the coupling chemistry (connecting a circle to a square), while the splitting of AB represents the decoupling chemistry (disconnecting a circle from a square). There are two possible strategies to form the relevant constructs, as shown in Scheme 9. While almost all constructs

Scheme 10. Possibility of Forming a Cyclic Triazole at Both Ends of the Sacrifical Unit

be a polymer containing no molecules with substituents X and Y. Alternatively, if both functionalities attached to the sacrificial group (Z and W) represent the same moiety [either both are triple bonds or both are azide groups as shown in Scheme 10 (27 and 28)], the formation of symmetrically substituted products seems unavoidable. Thus, the Huisgen cycloaddition reaction should not be used at both ends. Of course, there are other good click reactions not producing cyclic triazoles, such as the addition of thiols to various double bonds. Incidentally, the effective introduction of cleavable units taking advantage of two azide-acetylene reactions is possible, but it requires that the two reactions are not performed simultaneously in the same pot. Let us look at some more specific examples. When coupling two proteins (Scheme 11, structures 29 and 30), one can use a

Scheme 9. Synthesis of a Construct Containing a Sacrificial Unit AB

Scheme 11. Use of Appropriately Substituted Sucrose as the Sacrificial Unit and Invertase for Decoupling

of this type described in the literature have been formed using the approach more or less following pattern a, pattern b is clearly advantageous in most cases. It requires taking advantage of two coupling reactions, but they can usually be performed simultaneously. In the case of constructs containing large, sensitive, and multifunctional units, this pathway seems to be the strategy of choice provided that we have a library of good, easily cleavable, AB containing units. While the decoupled pieces differ slightly from the original molecules, almost always it is not the exact structure but the splitting of both macromolecules (represented by a circle and a square) in which we are interested. We expect a plurality of compounds represented by structure 24 to be available in the near future. They will be equipped with easily cleavable units AB in the middle and various moieties

selected disaccharide moiety such as a conveniently substituted sucrose (31) to connect (appropriate linkers may be necessary) the proteins and take advantage of a specific enzyme such as invertase (β-fructofuranosidase) to decouple the proteins when needed. 2210


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While decoupling using enzymatic triggers is appealing, other options, such as specific physical and chemical triggers, can be equally useful. It should be noted that this strategy does not fulfill the atom-economy45 and step-economy46 ideals.

Equally attractive are similar schemes utilizing (appropriately substituted) maltose (35) as a sacrificial unit (Scheme 12) with Scheme 12. Use of Appropriately Substituted Maltose as the Sacrificial Unit and Amylase for Decoupling

3.3. Reversible versus Sacrificial

In Table 1 the selected examples of potential coupling/ decoupling chemistries are presented with some comments on selectivity and usefulness within different classes of derivatives. This compilation is not intended to limit the scope of the presented methodologies and can be expanded to many different and new classes of reactions. α-amylase acting as the decoupling agent or substituted oligopeptides connecting proteins, polysaccharides, or nucleic acids, with relevant peptidases acting as the decoupling agents (Schemes 13 and 14).

4. CAD CHEMISTRYDESIRABLE PROPERTIES Most of the requirements for the specific conditions of the acceptable reactions are rather trivial. The yields of both the connection and disconnection processes should be very high at least 85% for both processesbut hopefully significantly better. All the conditions and reactants should be safe, ideally avoiding heavy metals, flammable or harmful solvents, and high pressures and temperatures. If it is necessary to use heavy metals, harmful solvents, etc., they should be fully separable and reusable, practically in 100% of cases. All reactants should be such that waste concerns are nonexistent. Furthermore, reactants should be able to survive water, light, and air. Ideally, CAD chemistry should exclusively utilize green reactions.47−49 CAD chemistry should take advantage of nontypical reactants and conditions, so the decoupling will not happen too early. The coupling chemistry should be such that functional groups not involved in the ligation reactions remain unchanged. It would be useful if the CAD chemistry could survive physiological conditions and that the atoms added during the coupling process produce only exotic signals in NMR, IR, etc. The products should be easy to purify by simple crystallization, if possible, and able to survive chromatography, if necessary. Sometimes the coupling products must be GRAS (generally recognized as safe); in some applications the coupling products should be administrable as drugs or used in vitro and/or in vivo. In most cases the enantio- and diastereoselectivity of CAD chemistry is of no importance. However, if diastereisomers are formed, it is usually critical that only one diastereoisomer is formed. Regioselectivity is often required, and the processes must almost always be chemoselective, being one of the most challenging requirements of CAD chemistry. This is because it is difficult to find reactants that will perform the coupling and

Scheme 13. Use of D-Oligopeptide as the Sacrificial Unit and Relevant Hydrolase for Decoupling

Scheme 14. Use of L-Oligopeptide as the Sacrificial Unit and Hydrolase for Decoupling

Table 1. Summary of the Reactions Applicable to CAD Chemistry coupling chemistry 1

Formation of esters, amides, etc.

2

Formation of ethers, alkylated amines, etc. Formation of thio-ethers, sulfoxides, etc.

3 4 5 6 7 8

Formation of acetals and other carbonyl derivatives Formation of double bonds Formation of aza, diazo compounds Formation of carbonates, heterocarbonates, etc. Introduction of sacrificial units via click chemistry (if possible).

potential decoupling chemistry

comments

Hydrolysis or its equivalent to form alcohols, amines, etc. Dealkylation

Useful but chemo- and regioselectivity of the reverse procedures render this chemistry often not practical. As above

Desulfurization (reduction) to form hydrocarbons Hydrolysis to form carbonyl compounds

Useful but often not applicable to many proteins with thiol moiety.

Ozonolysis + equivalents, diols formation followed by C−C cleavage. Oxidative or reductive cleavage Hydrolysis to alcohols or amines.

Doubtful when applied to polysaccharides; chemoselectivity is of concern in other biomolecules. Robust methodologies since double bonds are not typical components of biological macromolecules. Chemo- and/or regioselectivity may often be a problem. Highly useful where applicable; may interfere with other hydrolyses.

Enzymatic, photopromoted, or chemical degradation of sacrificial units.

Requires two different coupling reactions but offers a plurality of highly selective decoupling processes.

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reactions, or hydrolyses that employ even moderate conditions. Such constructs as nucleoproteins are labile under both acidic and basic conditions.51 This does not mean that CAD methods can never be applied to molecules of biological origin, but one must be aware of potential degradation of the sophisticated molecular constructs. Since fragile compounds require extremely specific methods, targeted enzymes are particularly appropriate in many circumstances. The molecular structure is not the only key element we should take into account. The potential presence and stability of protecting groups and their deprotection requirements (if the deprotection takes place before the decoupling) must also be considered. It was mentioned earlier that protecting groups are mostly used during multistep organic synthesis. Often the final product is protected, and this protection may be conveniently carried to the coupling process. Protecting groups may be present in one or more coupled units. In most cases we do not want these protected groups to be deprotected, and consequently, we must use the appropriate CAD chemistry. One must also take into account the specific experimental conditions, such as high magnetic fields, high pressures, or high pH to which the intermediates will be exposed. The coupled molecules must be able to withstand these specific conditions.

decoupling chemistry on complex proteins or polysaccharaides without affecting the delicate macromolecular structure of the biomolecule. Recently, Afagh and Yudin50 published an excellent review on chemoselectivity where they say that “chemoselectivity has been the Achilles′ heel of chemical synthesis”.

5. THINGS TO CONSIDER WHEN SELECTING A SPECIFIC CAD CHEMISTRY CAD chemistry should require conditions or reagents that are not common. An ideal decoupling chemistry would be performed only in the presence of minute amounts of a very unnatural compound(s) (belonging to, say, quaternary phosphonium salts) that can be attached to a solid support. Additionally, the reaction would be instantaneous, the pH would be more or less neutral, the temperature would be between 10 and 40 °C, the solvents would be water or acetonitrile, and only the functional group of interest would be cleaved when employing the magic decoupling agent. The number of processes that can selectively couple and decouple various moieties is rather limited. Later in the text, we review some of the potential options. Factors which must be considered when selecting specific reactions, reagents, and conditions include high reactivity toward functionalization, nondependence on the chirality of a molecule, stability under selected condition (enzymatic or photolytic/photochemical), and finally convenient separation of products from coproducts and substrates.

5.3. Do Both Coupled Units Need To Be Recovered?

Usually the coupling process connects two molecular units. The strategy may be different depending on whether we want to recover one or both involved units. The latter case requires methods and chemistries that will keep both units intact. However, when only one unit is valuable, we will not care about the fate of the other unit. Let us imagine that the coupling is between a protein and a polysaccharide. If only the polysaccharide is of value, after the desired experiments have been performed, decoupling by an appropriate peptidase may be the best mode of action. Alternatively, when only the protein is of interest, we may select an appropriate enzyme that hydrolyzes the acetal bonds of the carbohydrate. Analogously, we may choose photocleavage conditions, degrading the sacrificial functional unit along with the unwanted macromolecule.

5.1. Triggers

A major factor in choosing specific CAD chemistries is the type of triggers and conditions needed to induce the coupling and decoupling processes. It is paramount that no other functionality on the molecules of interest is affected (with the exception of the target functionality). We consider three types of triggers: physical (elevated temperature, ultrasound, photocleavage, laser or electron beam, etc.), biological (biocatalysts such as enzymes or antibodies), and chemical (pH change, presence of specific chemicals such as nucleophiles or electrophiles, redox reactants, etc.). Again, it is much easier to find an efficient coupling chemistry, since it is almost always performed under well controlled laboratory conditions. Thus, we can use a variety of reactants and physical conditions that can cause the selected reactions to take place. Triggers such as visible or UV light, microwave, laser beam, or elevated temperatures up to 100 °C may be acceptable. While the decoupling chemistry may employ the same conditions, some decoupling reactions must be performed in vivo. The number of satisfactory triggers for chemistries to take place in vivo is rather limited; however, physical triggers, such as ultrasound, and certain biological or chemical triggers, such as (moderately) increased or decreased pH, can be acceptable.

5.4. How Well Established Is the Chemistry To Be Used?

While many of the decoupling chemistry reactions are expected to be robust, this has not always been sufficiently documented. For example, a benzyl group alone or with a variety of substituents both in the ring and in the α-position has been used successfully as a protective group for alcohols, phenols, thiols, carboxylic acids, and amines. Nevertheless, there is no literature describing the cleavage of a benzyl group directly connecting a protein or nucleic acid to another macromolecular unit. The lack of pertinent literature examples applies to other protection methodologies (using specific esters, amides, thioesters, thiourethanes, etc).

5.2. Structure of Units to Be Coupled and Decoupled

6. EXISTING CLEAVABLE LINKERS Molecules that can serve as connectors of molecular units are often called linkers. Consequently, those linkers that carry functionalities capable of being disconnected to two or more pieces are cleavable linkers. They have been used for more than two decades, mainly for the cleavage of synthesized oligomers or polymers from solid supports such as Merrifield and similar resins. The methodology we propose employing sacrificial units (Scheme 9, path b) takes advantage of ready to use substrates (24) that can be coupled to two (or more) molecular units in a

The structure of the units to be coupled and decoupled is probably the most important factor to consider, when selecting a specific CAD chemistry, because it determines what conditions and reactants will be acceptable. Specifically, one must take into account the presence of fragile chiral centers, the solubility and stability of both units in the selected solvent(s), their stability at the pH and temperature of the chosen reactions, their stability against UV and visible light, etc. For example, many proteins or polysaccharides will not survive the oxidative or reductive conditions used in some decoupling 2212


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a). The authors “examined the complexation of BDQB (biotindopamine-QB) with avidins and an anti-dopamine antibody (IgG1) and the photorelease of dopamine−IgG1 complexes by ELISA, which was also confirmed by Western blot. These methods may provide effective strategies for the recovery of intact ligand−receptors complexes under mild conditions, without the need for damaging chemical reagents”. Recently, A.B. Smith, III, Hochstrasser, and their coworkers53 have shown that a disulfenyl tetrazine system can be readily incorporated within a bis-thiolate peptide motif by treatment with dichloro-s-tetrazine (Scheme 16). The photo-

one pot reaction. Almost all existing constructs containing cleavable linkers have been synthesized as a result of a multistep synthesis (Scheme 9, path a); that is, one large molecule was reacted with an appropriate reagent followed by several steps and, finally, by an introduction of a second large molecule. Many of these linkers are suitable only for a specific application. We refer interested readers to reviews on linkers applicable to solid-phase synthesis23−26 which discuss multiple types of cleavable linkers. In recent years a dramatic increase in the number of papers describing novel cleavable linkers has been observed. In the following we review several more interesting and useful linkers, including those described in patents and published patent applications. Since the U.S. PTO database is the easiest to access, we refer mostly to U.S. Patent Office applications, but most discussed patents were also filed with WIPO. The section is divided according to the type of trigger causing the cleavage.

Scheme 16. Tetrazine Phototriggers Developed by Smith, Hochstrasser, et al.53

6.1. Physically Cleavable Linkers

The most often used physical trigger is a phototrigger. We begin reviewing the photocleavable linkers with an elegant example of CAD chemistry. Japanese scientists52 designed and synthesized a photocleavable biotin-linker (40) for the photoisolation of ligand−receptor complexes based on the photolysis of 8-quinolinyl sulfonates in aqueous solution (Scheme 15). Scheme 15. Photocleavable Biotin-Linker Designed and Synthesized by Aoki et al.

lysis rate of the resulting construct was demonstrated to occur on the picosecond time scale, and the photolysis rate and yield of the tetrazine trigger are similar to those of acyclic versions. This proves that the mechanism of dissociation is not influenced by the rigidity of the peptide system or the presence of other side chain functionalities. Ju et al.54 synthesized three single-stranded DNA molecules of different lengths. Each molecule contained a fluorescent dye (6-carboxyfluorescein) connected to the 5′ end via a photocleavable 2-nitrobenzyl linker and a biotin moiety at the 3′ end. UV irradiation (340 nm) of solutions containing these fluorescent DNA molecules caused the complete cleavage of the nitrobenzyl linker, separating the fluorophore from the DNA. Experimental results indicated that the proximity of the chromophore 6-carboxy-fluorescein to the 2-nitrobenzyl linker did not hinder the quantitative photocleavage of the linker in the DNA molecules. The biotin moiety allowed immobilization of the fluorescent DNA on streptavidin-coated glass chips. The photocleavage of the immobilized DNA was investigated directly by fluorescence spectroscopy. Scheme 17 illustrates the methodology. Sieber and Orth55 developed a photocleavable linker for a selective release of enriched biomolecules from solid support in proteomic and metabolomic studies. Scheme 18 shows the structure of the photocleavable linker and principal workflow of the metabolite capture, enrichment, and cleavage procedure. Note a very skillful exploitation of the (triazole forming) click chemistry procedures. Finn and co-workers56 developed a cleavable linker for porous silicon-based MS. They take advantage of a laser pulse

The structure of the linker is shown. The biotin and dopamine units are coupled to each other using the photocleavable (300−330 nm) element, which is connected to the biotin unit via the amide bond and to the dopamine unit via the 1,2,3-triazole. The general structure is very similar to the one discussed earlier in this paper (Scheme 1). It includes a unit (which we call a sacrificial unit) whose only purpose is to serve as an easy photocleavable moiety (AB in the Scheme 1) that can be decoupled when needed. It should be emphasized that while the construct composed of dopamine and biotin looks like the one formed as a result of employing a sacrificial unit, it was synthesized on a step by step basis (Scheme 9; path 2213


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Scheme 17. 5′-Fam-linker-(T) 5,10,20-Biotin-3′ and Their Photocleavage under Irradiation with near UV Light Developed by Ju et al.54

Scheme 18. Structure of the Photocleavable Orth−Sieber Linker and Principal Workflow of the Metabolite Capture, Enrichment, and Cleavage Procedurea

a

Reproduced with permission from ref 55. Copyright 2009 American Chemical Society.

as a physical trigger to accomplish retro-Diels−Alder reaction of an isobenzofuran−maleimide system during the DIOSMS (desorption/ionization on silicon/MS). In one of the described

procedures for coupling of the triazine derivative carrying the DA product to silicon, the authors employ a click chemistry (CACC) to form a triazole ring. 2214


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Scheme 19. Use of Electrochemical Trigger during Immobilization of Cysteinyl Biomolecules by Lee, Chung, and Co-workersa

a

Reproduced with permission from ref 57. Copyright 2012 American Chemical Society.

6.2. Enzymatically Cleavable Linkers

Another rare physical trigger was exploited by Korean scientists.57 They cleaved the azo functionality electrochemically as shown in Scheme 19. A subsequent oxidation of paminophenol to p-quinoneimine was followed by a reaction (Michael addition) with the cysteine moiety to immobilize it. Chen, Sinha, Shestopalov, and Ouyang58 developed bifunctional linkers for two amino functionalities (Scheme 20). The

The subject of enzymatically cleavable linker groups in polymer-supported synthesis was reviewed by Reents, Jeyaraj, and Waldmann.59 Cravatt and Speers60 developed a tandem orthogonal proteolysis (TOP) strategy for activity-based protein profiling and post-translational modifications which uses tobacco etch virus protease (TEV) as an enzyme triggering the cleavage. Scheme 21 shows the general concept, its execution, and the cleavage site and illustrates the use of click chemistry (CC) during the synthesis. The experimental results underscore the value of TOP methods, which provide two independent data sets (whole protein digests and probe-labeled peptides) for the verification of targets of chemical probes. Flitsch and co-workers61 have shown that linkers deriving from hydroxymethyl-phenoxyacetic (HMPAA) and hydoxymethylbenzoic (HMBA) acid, commonly used in solid phase peptide synthesis, are surprisingly susceptible to cleavage by the protease chymotrypsin. The reactions are very effective and take place under neutral conditions with a broad range of amino acid residues being tolerated at the scissile bond. The authors applied the enzyme-cleavable linker system to peptide and glycopeptide synthesis (Scheme 22). They rightfully emphasize that the chymotrypsin catalysis is a useful complementary method to the cleavage of these common ester linkers which are normally cleaved under acidic or basic

Scheme 20. General Structure of Bifunctional Photocleavable Linkers Developed by Chen et al.

reactive groups at both ends are independently selected from a group containing succinimide, isothiocyanate, propargyl, iodoacetamide, maleimide, azide, and terminal alkene. Several photocleavable moieties (1,2-dimethoxy-4-nitrobenzyl, 1,2dimethoxy-2-nitrobenzyl, nitrobenzyl, 6-bromo-7-hydroxycoumarin-4-ylmethyl, 8-bromo-7-hydroxy-quinolyl, nitrodibenzofuran) are claimed. This patent application clearly exploits sacrificial units that can be reacted in one pot with two different (macro)molecules.

Scheme 21. Cravatt and Speers Tandem Orthogonal Proteolysis Strategy for Activity-Based Protein Profiling (TOP-ABPP)a

a

Reproduced with permission from ref 60. Copyright 2005 American Chemical Society. 2215


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cleave a specific linker, resulting in the release of radio-metal from the circulating radioimmuno-conjugate (RIC) in a form that will have rapid renal clearance through urine.” While there are many patents claiming the use of enzymes for effective cleavage, we want to highlight only three. Zhao, Kozlov, and Shults64 developed a novel method of sequencing nucleotides which takes advantage of an enzymatically cleavable peptide linker attaching a dye to nucleosides and nucleotides. Roberts, Lebowitz, and Ghanbari65 disclose peptide linkers for conjugating drugs to ligands which employ furin (a protein encoded by the FURIN gene) as a cleaving agent. Sufi et al.66 disclose novel drug−ligand conjugates employing such cleavable linkers as peptidyl and disulfide.

Scheme 22. Enzymatic Hydrolysis To Form FmocPheAsp and the Structure of One of the Synthesized Glycopeptides61

6.3. Chemically Cleavable Linkers

There are a few different triggers belonging to a chemical category. They include pH change and oxidative, reductive, nucleophilic, and electrophilic cleavage. Let us start with oxidatively cleaved linkers. Charych, Zuckerman, and coworkers67 developed an attractive tartaric acid based linker (54) which was used in the synthesis of a number of peptoids. Its hydrophilicity helps reduce nonspecific binding to biological samples, and its oxidative (periodate) cleavage generates a Cterminal aldehyde that can be used for various derivatizations. Scheme 24 shows the structure and oxidative cleavage forming a simple pentamer.

conditions. Scheme 22 shows an example of the enzymatic hydrolysis of solid-supported FmocPheAsp (45) to form peptide (46) and a structure of one (48) of the synthesized glycopeptides. Bertozzi et al.62 recently described a very interesting application of their Cu-free click chemistry using difluorinated cyclooctyne (DIFO) for selective imaging of glycans. The cleavage was accomplished by prostate-specific antigen (PSA) protease. The concept execution [Mu-HSSKLY (Mu) morpholino ureidyl represents a hexapeptide] is presented in Scheme 23.

Scheme 24. Example of Oxidative Cleavage Forming a Simple Test Peptoid Pentamer67

Scheme 23. Bertozzi et al.62 Cleavage Using PSA Protease

Bogyo and co-workers68 developed the diazobenzene system (56) that can be (reductively) cleaved under relatively very mild conditions (phosphate buffer plus Na2S2O4) (Scheme 25). The concept is also the subject of a patent application.68b Hulme and collaborators69 developed a simple and elegant, new azobenzene linker (61) for affinity chromatography applications. It is also reductively cleaved with sodium dithionite (Na2S2O4). The linker was designed with orthogonal reactive functionalities: an amine for linking to a matrix support, and an azide for the copper assisted reactions with the propargyl functionalized ligands of interest (Scheme 26). The

Kumaresan, Luo, and Lam63 offered a pragmatic but important argument why they selected TNKase, a serine protease tissue plasminogen activator, as the cleavage agent. It is “because it has already been in clinical use for the treatment of myocardial infarction and is therefore readily available.” “After radio-targeting agents have accumulated in the tumor, a cleaving agent (protease) can be administered “on demand” to 2216


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first generation cleavable affinity tags are presented in Scheme 27.

Scheme 25. Example of a Probe with the Benzenediazonium Linker and Its Chemoselective Cleavage68

Scheme 27. Concept and Structure of the Azobenzene Linker Developed by Hang et al.70

structure of the linker and its cleavage products (62 and 63) are shown in Scheme 26. Hang et al.70 also synthesized several clickable affinity tags for bioorthogonal proteomics with linkers that are cleavable using sodium dithionite (Na2S2O4). The authors show that the presence of the hydroxyl group in the ortho position affects the rate of cleavage of the azobenzene moiety. The structures of the Scheme 26. Example of the Hulme at al. DithioniteCleavable Linker for Affinity Chromatography69

Gygi et al.71 developed another very interesting, reductively cleaved linker. The unique disulfide moiety (Scheme 28) is stable to reductive conditions employed during sample labeling (DTT) but is readily cleaved under mild conditions using tris(2-carboxyethyl)phosphine (TCEP). The jagged line adjacent to the disulfide group denotes a mixture of isopropyl Scheme 28. Novel Disulfide Cleavable Linker Developed by Gygi at al.

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stereoisomers. The asterisks represent carbon atoms that carry isotopic density (13C) in the reagent’s heavy version. The authors demonstrate the stability of the linker under two different reducing conditions and show how this “catch-andrelease (CAR)” reagent can be used to quantitatively compare protein abundances from two distinct cellular lysates. Starting with only 40 μg of protein from each sample, 1840 individual proteins were identified in a single experiment. Gothelf et al.72 needed a cleavable amino-thiol cross-linker for reversible linking of amines to DNA. It had to fulfill the following requirements: (i) be capable of coupling an amino group to thiol-modified DNA sequence, (ii) be cleavable under DNA compatible conditions to release a free amino group, and (iii) leave a nonreactive group at the DNA strand. To accomplish their goal, they developed a linker they call SVEC {succinimidyl 2-(vinylsulfonyl)ethyl carbonate}. Scheme 29

Scheme 30. Example of a Multifunctional Probe with an Acid-Cleavable Linker Developed by Liskamp at al.73

proteomic expression profiling analysis. The linker (79) contains a mild, acid-cleavable carbamate moiety (Scheme 31). Scheme 31. Schematic of Two Variants of ICAT Reagents Developed by Fauq and Co-workers74

Scheme 29. Demonstration of the Attachment of Leucine to a Thiol-Modified DNA Sequence and Subsequent Cleavage of the Linker

shows the structure of the cleavable linker (73) and demonstrates coupling and decoupling of leucine (71) to a thiol-modified DNA sequence. Note that the coupling of DNA represents a click chemistry reaction not involving an alkyne− azide reaction (addition of a thiol to vinyl sulfone) and that the cleavage produces unmodified leucine. Liskamp and co-workers73 have designed a probe (75) consisting of four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The structure of the probe is shown (Scheme 30). Fauq et al.74 designed and synthesized new acid-cleavable light isotope-coded affinity tags (ICAT-L) for potential use in

Bertrand and Gesson75 encountered unexpected difficulties when synthesizing the triazole (82) from a compound containing a dimethylpropargyl group (81). The formation of unexpected products was attributed to the lability of the Opropargylcarbamate group in the presence of Cu(I) salts. The use of nonpolar solvent such as toluene with amines or Cu(I) chelating agents highlighted the importance of the solvating effect for this unwanted result. Changing the reaction medium to pyridine as solvent effectively addressed the problem. The synthesized compounds could be easily hydrolyzed at low pH (Scheme 32). Park, Liu, and Kohn76 designed and synthesized an acylhydrazone-based, high-yielding cleavable linker which contained an acylhydrazone cleavage site, a biotin unit, a terminal azide to permit capture with an alkyne-modified protein using a copper(I)-mediated cycloaddition reaction, and 2218


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Scheme 32. Acid-Cleavable Linker Developed by Bertrand and Gesson75

Scheme 34. Decoupling of the Pyridine Unit Using Chmielewski’s Click-Clack Chemistry77

a polyethylene glycol linker to increase its water solubility and to minimize adverse steric interactions with the immobilized streptavidin during protein capture. Scheme 33 shows the structure (85) of the linker and its efficient reaction with acetylhydrazide. Scheme 33. Decoupling of Kohn et al. Acylhydrazone-Based Cleavable Linkers

Scheme 35. Wade et al. Base-Cleavable Linker78

An entirely different, novel approach to protection/ deprotection chemistry that can be utilized in some coupling/ decoupling processes was recently developed by Chmielewski.77 It was found that during intramolecular cyclization of the 2-pyridyl type of thermolabile protecting group (TPG) the thermally stable 3-pyridyl[1,3,2]oxazaphospholidine ring was formed and thermolabile properties were reduced. Thermolability could be recovered upon hydrolytic ring-opening of a 3pyridyl[1,3,2]oxazaphospholidine (Scheme 34). The author calls the process a click-clack approach. Wade and co-workers78 have developed a linker that is cleaved by an (amine) base. During the solid phase synthesis, the sulfide bond acts as a linker. After the synthesis is complete, the linker is oxidized with peracid to sulfone (98), which can undergo elimination in the presence of the dimethylamine either in solution or in the gaseous phase. The final synthetic steps including the cleavage are shown (Scheme 35). Sharma and Moses79 have developed a new alkynylsilane “click linker” (and a protecting group)the ethynyldiisopropylsilyl group. The new silyl-based reagent can serve for “catch and release” immobilization, combining click chemistry with silyl protection. It clearly represents the CAD chemistry

example. Scheme 36 shows the structure of this simple linker and its coupling and decoupling reactions and conditions. Overkleeft et al.80 developed a cleavable linker based on the levulinoyl ester for activity-based protein profiling. It is cleaved chemoselectively with hydrazine. While it is a very useful linker for the specific purpose, one has to notice that its synthesis is rather tedious. Scheme 37 shows the structure and application of the linker attached to the system. 2219


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Scheme 38. Biotin Probes Developed by Tirrell et al.81

Scheme 36. Coupling and Decoupling Using a Sharma and Moses Alkynylsilane Linker79

Scheme 37. Structure of Target Proteasome Probe Containing the Cleavable Linker Based on Levulinoyl Ester80

Scheme 39. Transimination of a Linker Containing Bisaryl Hydrazone with Aniline in the Presence of NH2OH82

Tirrell and co-workers81 recently published a pivotal paper describing as many as five different cleavable biotin probes for labeling of biomolecules via azide−alkyne cycloaddition. Their cleavage employs such diverse conditions as 50 mM Na2S2O4, 2% HOCH2CH2SH, 10% HCO2H, 95% CF3CO2H, or irradiation at 365 nm. The following Scheme 38 shows the structure of the developed linkers. Probe 108, designed around the acid-sensitive dialkoxydiphenylsilane (DADPS) motif, was found to be the most selective. Efficient cleavage (>98%) under mild conditions (10% formic acid, 0.5 h) and the small (143 Da) molecular fragment left on the labeled protein following cleavage make this probe especially attractive for use in proteomic studies. Importantly, no significant cleavage of this probe was observed upon treatment with aqueous KF solutions of concentrations up to 5 M. Dawson et al.82 took advantage of a linker containing bisaryl hydrazone (111) as a cleavable unit for enrichment using affinity purification on (strept)avidin beads with biotin as an affinity tag. The cleavage caused by transimination required very mild conditions. The concept’s execution is shown in the Scheme 39. Wittmann and co-workers83 developed an elegant methodology enabling a temporary coupling of a monosaccharide and a cyclopeptide. The structures and employed chemicals are shown in Scheme 40. 2220


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reduction) is nobody’s favorite reaction. Nevertheless, we included it in the list of our examples (as a method to cleave double bonds) because it offers high yields, produces no hazardous byproducts, can be performed very safely, and decouples a double bond, which is a rather exotic functionality for a large majority of natural biomacromolecules. Moreover, we think that a plurality of methods better than ozonolysis for cleaving double bonds will be developed provided that there is a clear need for it. Coupling strategies for various molecular units are usually well-known. Thus, we emphasize the decoupling part of CAD chemistry and discuss them in more detail. Specific methods are divided into three categories of triggers: physical, biological, and chemical.

Scheme 40. Temporary Coupling and Decoupling of a Carbohydrate Unit to a Peptide83

7.1. Decoupling by Light

Photochemistry can provide extremely specific and convenient methods of connecting and disconnecting various molecules. Reactions causing decoupling by the use of photons often offer high yields, rates, and quantum efficiencies. It is usually important that the process takes place at a specific wavelength (not affecting other chemical groups in the system) and that the photocleavage products are soluble in the employed buffer. While a specific enzyme usually connects or cleaves only a single chemical functionality, photocleavable units are unique in the fact that a specific photocleavable group can be applicable to several functionalities. For example, Hagen and collaborators90 developed a coumarine-based photocleavable group that is applicable to caging of such diverse groups as carboxylic acids, phenols, alcohols, amines, and carbonyl compounds. There are a few reviews on the mechanism and applications of photocleavable units. They include a review limited to carbohydrate chemistry,91 reviews on photolabile protecting groups,92,93 a review limited to the p-hydroxyphenacyl group,94 a review on photoremovable protecting groups based on electron transfer chemistry,95 and reviews of photochemistry related to the DNA research.96,97 Hess and colleagues98,99 developed a caging group, 7(diethylamino)coumarin (DECM), a protecting group for a carboxylic functionality which is cleaved by visible light. Its usefulness has been shown for the neurotransmitters glutamate and glycine. Scheme 41 shows the coupling between the carboxylic group of glycine and the hydroxymethyl unit of DECM, followed by Boc deprotection and photocleavage (at 400 nm) to release glycine with a half-life of ∼2.5 μs and a quantum yield of 0.12. Importantly, under physiological conditions, DECM-caged glycine is water-soluble and stable. Schultz et al.100 “have demonstrated that 2-nitrophenylalanine (2-NPA), when incorporated chemically or biosynthetically into a polypeptide, is able to site-specifically cleave the polypeptide backbone upon irradiation with 365 nm light (Scheme 42). In a model peptide (127), the photocleavage reaction yields a C-terminal carboxylate group and a N-terminal cinnoline group as the major cleavage products in excellent overall yields. A maximum cleavage efficiency of ∼30% was achieved when the 2-NPA group was biosynthetically incorporated into proteins, which is likely due to competing intramolecular cyclization reactions with nearby nucleophilic groups. Because 2-NPA is a close analog of phenylalanine, its incorporation into peptides or proteins is expected to minimally perturb their structure and function. In the case of proteins, this methodology will be most useful for generating biologically active species from inactive precursors” (Scheme 42).

Most of the chemically cleavable linkers claimed in patents are those well-known from the publications. We want to highlight a few novel linkers and uses. Scientists at CombiMatrix Corporation developed a series of microarrays having phosphoramidite base cleavable linkers84 and a sulfonylcontaining base cleavable linker.85 The patent application by Church and Sismour86 describes the use of phosphoramidite cleavable linker in a novel method of nucleotide sequencing. The conditions causing the cleavage include the use of silver salts. Dellinger et al.87 claim novel compositions having a polynucleotide bound to a substrate via a cleavable linker. The cleavage is caused by a buffered hydrogen peroxide solution. Ju et al.88 claim a process for sequencing nucleic acids that includes photocleavable and chemically cleavable linkers. One of the claimed triggers is tris(2-carboxyethyl)phosphine. Kratz and Merfort89 disclose certain prodrugs, wherein two or more cleavable linkers can independently be cleaved hydrolytically and/or enzymatically and/or pH-dependently. Interestingly, for these authors “the expression 'cleavable linker' means any linker which can be cleaved physically and chemically. Examples for physical cleavage may be cleavage by light, radioactive emission or heat, while examples for chemical cleavage include cleavage by redox-reactions, hydrolysis, pH-dependent cleavage or cleavage by enzymes.”

7. POTENTIAL SPECIFIC REACTIONS The execution of the concept of sacrificial units requires that there is a large repertoire of reliable decoupling reactions. In the following part of the review, we discuss reactions either that have been shown to belong to CAD chemistry or that we believe are potentially useful but have not been tried or their scope is not sufficiently known. We believe that less harsh conditions, milder reagents, and better catalysts for some of these reactions will be soon developed to make them more efficient. For example, ozonolysis (followed by the ozonide 2221


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Scheme 41. Protection and Deprotection of Glycine Using DECM Developed by Hess at al.98,99

Scheme 43. High Yield Example of a Selective and Orthogonal Photodecoupling Developed by Klán et al.101

cleavage by irradiation in MeOH/HEPES buffer solution (80:20), in a photochemical reactor at different wavelengths (254, 300, 350, and 419 nm). Scheme 44 shows the structures of the studied fluorophores, the irradiation times, and photochemical quantum yields (10−3Φphot). Scheme 44. Structures of Fluorophores Studied by Costa et al.102 and Irradiation Times with Photochemical Quantum Yields

Scheme 42. Cleavage Employing 2-NPA Developed by Schultz at al.100

Klán et al.101 have proposed the 4-acetyl-2-nitrobenzyl moiety, substituted in both benzylic and phenacyl positions with leaving groups, as a monochromophoric photocleavable linker (130). The attached groups can be disconnected selectively and orthogonally upon irradiation. Scheme 43 shows the accomplished yields. Costa et al. 102 performed a comparative study of polyaromatic and polyhetero-aromatic fluorescent photocleavable protecting groups. They prepared several fluorescent conjugates of N-benzyloxycarbonyl protected γ-aminobutyric acid (GABA) and evaluated their behavior toward photo-

The authors offer the following conclusions of their study: “In summary, all labels considered lead to conjugates, which required low irradiation times for photocleavage to occur, making them appropriate to use as photolabile protecting groups for organic molecules, including amino acids and other relevant biomolecules, in addition to their usefulness in fluorescent labeling due to the high Stokes’ shifts and moderate 2222


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to excellent fluorescence quantum yields. Regarding the photocleavage studies of the fluorescent conjugates, in methanol/HEPES buffer solution (80:20), at 254, 300, and 350 nm, it was possible to conclude that the irradiation time depended on the structure of the label. Depending on the stability to radiation of the analyte, a choice of the protecting group can be made based on the wavelength of irradiation, i.e., at 350 nm, benzo[f ]benzopyran (Bba), at 300 nm naphthalene (Npm), and at 254 nm pyrene (Pym). Irradiation times at 419 nm were too long and not convenient for practical applications”.

Scheme 45. Enzymatic Cleavage of Double Bonds Using Tremetes hirsuta118

7.2. Decoupling by Enzymes

As was already discussed, enzymes due to their selectivity, mild reaction conditions, and versatility become exceptionally useful in promoting CAD chemistry. Unfortunately, at this point, some enzymatic processes are clearly not ready to become the method of choice for many CAD reactions. The number of biocatalysts that can be purchased is not satisfactory, and the scope of their action is often unknown. It is still often a guess if given enzymes can be applied to coupling or decoupling of specific biomolecules. The references include useful reviews describing the use of enzymatic methods in both coupling and decoupling processes. In particular, reviews have been published on the use of enzymes in organic synthesis,103−106 enzymatic protecting group techniques,107 enzymatic oxidations,37 and enzymatic and nonenzymatic syntheses of sucrose based esters,108 enzymatic acylation of oligosaccharides with fatty acids,109 regioselective enzymatic acylation of polyhydroxyl compounds,110 chemo-enzymatic syntheses of nucleosides and nucleotides,111 enzyme-catalyzed synthesis and degradation of biopolymers,112 and the use of specific enzymes such as Candida antarctica lipase B113 and fungal laccases.114 7.2.1. Enzymatic Cleavage of a Double Bond. The enzymatic cleavage of a double bond is very attractive because “enzymes might have a significant advantage over chemical catalysts, namely the radical intermediates are shielded in the active site of the enzyme until the catalytic cycle is completed, therefore minimizing possible side reactions, such as epoxidation, rearrangements, polymerization”.115 Recently, Austrian researchers115 have shown the enzyme preparation of Trametes hirsuta cleaves alkenes following neither the classical dioxygenase116 nor the monooxygenase117 mechanism. At this point the only viable enzymatic cleavage of olefins has been presented by Kroutil et al.,118 who employed lyophilized cells of the fungus Trametes hirsuta GFCC047 to conjugated aryl alkenes (Scheme 45). While the yields are generally not impressive, they can be as high as 80%. Moreover, the methodology uses oxygen as the only oxidizing agent, employs no metals, is very safe and reasonably chemoselective, produces no byproducts, and offers the best possible atom economy. 7.2.2. Cleavage of Acid Derivatives. In this section our attention is directed toward hydrolysis and aminolysis of esters and hydrolysis of amides promoted by enzymes. A variety of hydrolases is available for such processes, but lipases such as porcine pancreatic lipase (PPL) or lipases from microorganisms (for example, from Candida cylindracea {lipase-CC} or Pseudomonas f luorescens) seem particularly effective.119 Enzymes’ chemo- and regioselectivity is of particular appeal. For example, Stein and Toogood120 studied enzymes as catalysts of regioselective hydrolysis of aspartate diesters (148). From nine enzymes tried, porcine liver esterase (PLE)

offered the highest selectivity (R = Me, 98:2; 30 min) toward the formation of the β-alkyl ester. The yields were high, and the methyl, ethyl, allyl, benzyl, and tert-butyl esters were successfully hydrolyzed to the corresponding β-alkyl ester (Scheme 46): Scheme 46. Regioselectivity in Enzymatic Hydrolysis of Diesters Using Pork Liver Esterase (PLE)120

Greek and German scientists have recently demonstrated the successful application of an esterase from Bacillus subtilis and a lipase from Candida antarctica to the hydrolysis of tert-butyl esters,121 methyl and benzyl esters,122 and allyl and chloroethyl esters123 from a variety of substrates, including N-protected amino acids. The conditions are mild, and the yields are high or very high in most cases. The series of publications describe a novel methodology to remove specific protecting groups. Paterson and Miller124 were able to hydrolyze exclusively the acetyl group from a compound containing additional amide and tert-butyl ester functionalities (Scheme 47). It is possible to selectively acetylate one hydroxyl group from a number of other hydroxyl groups within a compound, and to hydrolyze only one out of a few acetyloxy groups in a compound. The publication by Nazir et al.125 is an excellent illustration of such an accomplishment. These authors were able to synthesize both possible monoacetates of a compound with two phenolic hydroxyl groups (153) (Scheme 48). Khan et al.126 disconnected only an anomeric acyl group from several disaccharides such as cellobiose equipped with eight acyl groups (157). If one takes into account the selectivity of the process, the yield is spectacular. A prolonged reaction 2223


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Scheme 47. Selective Hydrolysis using Candida Antarctica124

Scheme 49. Example of a Regioselective Deacetylation from a Disaccharide Promoted by a Specific Lipase126

Scheme 48. Example of a Synthesis of Both Possible Monoacetates of a Compound with Two Phenolic Hydroxyl Groups125 Scheme 50. Selective Deacetylation of Peracetylated Nucleosides Using a Specific Lipase127

Immobilized catalytic antibodies are another type of biocatalysts applicable to regio- and chemoselective decoupling. Kondo and co-workers studied the reaction properties of immobilized catalytic antibodies in aqueous dimethyl sulfoxide.129 The specific antibody, 17E11, was used as a model system. The immobilized catalytic antibodies were stable and repeatedly used for the hydrolysis of the diacylated substrate in 10% DMSO/50 mM Tris-HCI, pH 8.2, without detectable activity reduction, indicating a high durability. The immobilized catalytic antibodies are advantageous for quick production and separation of products that are unstable under the reaction conditions. Model calculations based on the kinetic parameters obtained experimentally showed that unstable monosaccharide product (163) (with a free hydroxyl group at position 4) can be produced with a high yield using an appropriate amount of immobilized 17Ell (Scheme 51). As already mentioned, prodrugs and codrugs are transformed into the active forms as a result of enzymatic hydrolysis. For example, scientists at Psivida company developed dozens of codrugs connected as esters or carbonates. Here are a few examples showing that while the chemical hydrolysis does take place at buffered pH, the enzymatic hydrolysis is much faster (Scheme 52). 7.2.3. Dearylation of Arylamino Compounds. Most organic chemists are surprised when they learn that the removal of an aryl group from arylamino compounds can be accomplished using enzymes. Rutjes et al.133 studied laccase-

time enabled this group to isolate the single hexaacetylated product (158) (Scheme 49). Italian researchers127 compared recombinant and nonrecombinant lipases from Candida rugosa for regioselective hydrolysis of peracetylated nucleosides (Scheme 50). One of the recombinant lipases, rCRL1, was shown to be far more stable in the presence of acetonitrile (30%, v/v) than the commercial CRL Sigma immobilized on the same support. The recombinant lipases also gave better yields of the deacetylated product. Almost all the studied lipases exhibited good to excellent selectivities (conversion of 94% or more; yield between 65 and 96% in most cases) in cleaving the primary acetoxy group. Amino acid phenylhydrazides are easily formed, and the yields of their formation are usually high. Waldmann et al.128 found that peptides protected at their C-terminal end as phenylhydrazides can be cleaved using the enzyme tyrosinase derived from mushrooms. The conditions are exceptionally mild (oxygen, pH 7, RT), the process is highly regio- and chemoselective, and the yields of the cleavage are very high. The mild nature of the process is confirmed by the fact that a delicate amino acid, methionine, is not oxidized under the cleavage conditions. The presented data suggest that the process is truly free radical. 2224


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products which were not produced in the mediators’ absence. Scheme 53 shows more impressive examples:

Scheme 51. Selective Deacetylation Using Catalytic Antibody129

Scheme 53. Removal of Aryl Substituents from Amines Using Laccases133

mediated oxidative deprotection of amines protected with a pmethoxyphenyl (PMP) group. A variety of PMP-protected amines were successfully deprotected under mildly acidic conditions using commercially available laccases (from Trametes versicolor {laccase T} or Agaricus bisporus {laccase AB}). The rate of deprotection was solvent and pH-dependent, and the reaction scope could be increased by using such mediators as 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). In some cases the addition of mediators did increase the conversion or even enabled the formation of

7.3. Decoupling by Chemicals

While enzymatic and photocleavage processes are considered to be substantially more specific than the chemical ones, the literature provides many more examples of the selective CAD chemistry performed using specific chemicals rather than enzymes or photons as triggers. As before, the presented reactions are meant to serve only as examples of potential

Scheme 52. Decoupling of Selected Codrugs: Comparison of Kinetics of Chemical and Enzymatic Reactions

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methods applicable to the decoupling processes. Usually, the literature data do not describe the scope and limitations of relevant methods, and their usefulness for a specific situation must be an educated guess only. Some help can be found in specific reviews referenced in the text. 7.3.1. Desulfurization of Thioethers, Dithioacetals, Sulfoxides, and Sulfones. The reaction discussed here is a reductive cleavage of a sulfur−carbon bond to form a carbon− hydrogen bond. Additionally, we included one example which gives a similar result, but the final product comprises a carbon− oxygen (and not a carbon−hydrogen) bond. The starting materials (moieties of coupled compounds) include sulfides, disulfides, sulfoxides, sulfones, sulfonates, and (di) thioacetals (thioketals) (Scheme 54).

Scheme 55. Mild Desulfurization without the Loss of Optical Activity Using a Raney Ni/Sodium Hypophosphite Combination139,140

Scheme 54. Scope of Discussed Desulfurization Reactions

Scheme 56. Specific Examples of Raney Ni/NaPH2O2 Desulfurizations139,140

There is a plurality of thioethers forming reactions, and many of them require very mild conditions and often give excellent yields with or without a catalyst. The possible processes include nucleophilic, free radical, and electrophilic addition of thiols to double bonds and the sulfur equivalent of the Williamson reaction. Relevant processes producing a carbon−sulfur bond containing compounds other than thioethers are not that abundant but often offer good yields and mild conditions. Since some sulfide forming reactions are considered to belong to the click chemistry, it must be noted that the thiol-click chemistry has been recently reviewed by Bowman et al.134 Importantly, mild desulfurizations give decoupled products usually in high yields. Such reactions are often highly chemoselective; that is, they can be performed in the presence of various chemical functionalities, including such protective groups as a benzyl group. In the 1970, there were practically only two methods of desulfurization available, and both were not applicable to many compounds: Raney nickel reduction135 and sodium amalgam reduction.136 Twenty years later, the number of available methods was substantially expanded to include those mediated by transition metals137 and sodium boride formed in situ.138 Since then, several new mild methods have been developed. Some examples are presented below. Node and co-workers139,140 described a reductive desulfurization method using a Raney nickel−sodium hypophosphite combination system that is sufficiently mild to reduce thioethers and sulfoxides without racemization of a secondary alcohol. Importantly, the yields are impressive and the benzyl ethers are not affected by this method. Schemes 55 and 56 illustrate the scope of the methodology. Park and co-workers141 developed a methodology enabling a high yield hydrogenolysis of arenesulfonates (187) using secondary alkyl Grignard reagents and a nickel catalyst. 1,1′Bis[(diphenylphosphino)ferrocene] dichloronickel (dppfNiCl2) proved to be best for the hydrogenolysis among selected nickel catalysts. While the yields are very high, the procedure has not been performed with compounds containing labile moieties (Scheme 57).

Scheme 57. High Yield Desulfurization Using Grignard Reagent and a Nickel Catalyst141

Jeong et al.142 desulfurized fluorinated dithioketals (190) to form 1H,1H-perfluoroalkyl aromatics using 2.2 equiv of tributyltin hydride and AIBN (10 mol %) at 80−90 °C without a solvent (Scheme 58). The isolated yields are between 73 and 96%. Yoda and co-workers143 offered an entirely different approach to the desulfurization process. They treated a variety of alicyclic and aromatic γ-phenylthio-substituted lactams with Lewis acids such as cuprous or cupric halides in aqueous solution at room temperature to accomplish a very high yielding tandem desulfurization and hydroxylation reactions to generate γ-hydroxylated lactams. Scheme 59 shows a few 2226


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the reduction of (205) with 3H-borohydride. The yield was about 85−90% (Scheme 61).

Scheme 58. Desulfurization of Thioketals Using Tributyltin Hydride142

Scheme 61. Example of Desulfurization Using Raney Ni145

examples of the processes in which the N-benzyl and O-benzyl groups are not affected: Scheme 59. Tandem Desulfurization/Hydroxylation Reactions Using Cuprous and Cupric Halides143

7.3.2. Cleavage of Double Bonds. The formation of carbon−carbon bonds, particularly the olefinic bonds, is one of the pillars of modern organic synthesis. The double bond forming reactions include the Diels−Alder, Wittig−Horner, Heck, metathesis, Peterson alkenylation, Julia−Kocienski olefination, Sonogashira, Suzuki, Negishi, Hiyama, Kumada− Corriu, and Stille coupling reactions. What is perhaps even more important from the standpoint of this review is that there is a variety of decent methods for cleaving double bonds. Moreover, many if not most biologically important molecules do not include double bonds. Thus, one can expect to find chemoselective methods of cleaving double bonds that lead to a successful cleavage of large, complex macromolecules. Usually, a decoupling, that is, a complete cleavage of the double bond, requires two or more steps, but often they can be performed in one reactor. The overall process is an oxidation. Here are some of the possibilities: 7.3.2.1. Ozonolysis Followed by the Reduction of the Ozonide (1,2,4-Trioxolane). The use of ozonolysis in drug synthesis has been reviewed recently.146 One of the virtues of the “decoupling by ozonolysis” is that the structure of the final product(s) depends on the conditions of the workup. Reductive work-ups of the ozonide can afford aldehydes, ketones, or alcohols, but choosing nonreductive workup conditions enables the extension of the potential products to acetals, carboxylic acids, lactones, and other products.147 Many functional groups stay unaffected during the ozonolysis and related workup. For example, Danishefsky et al.148 synthesized the ketone 209 (Scheme 62) as a result of the high yield ozonolysis of the double bond in the presence of unsaturated acid, lactam, and lactone functionalities during the total synthesis of camptothecin. 7.3.2.2. Equivalents of Ozonolysis Using Other Oxidizing Agents. Double bonds can also be transformed into the corresponding carbonyl compounds with ruthenium tetroxide in the organic/water solvent system. For example, Yang and Zhang149 used RuCl3/oxone (potassium peroxymonosulfate) or RuCl3/NaIO4 to accomplish a high yielding oxidation in the presence of such functionalities as benzyl and ester groups (Scheme 63). Oget and co-workers150 described a procedure enabling the effective, high yield cleavage employing ruthenium tetroxide

Caubere et al.144 have shown that nickel-containing complex reducing agents alone or in the presence of 2,2′-bipyridine (NiCRA and NiCRA-bpy, respectively) are very efficient in the desulfurization of sulfur-containing organic compounds such as sulfides and disulfides. The reactions are performed under mild conditions, require 2 h or less, and offer excellent yields (Scheme 60). Desulfurization using a traditional Raney nickel can be very useful as well. Uchida and Stradtman145 cleaved a construct (206) containing a protein and a radioactive unit derived from Scheme 60. High Yield Desulfurization Using NickelContaining Complex Reducing Agent144

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Scheme 62. High Yield Selective Ozonolysis148

osmium, ruthenium, or manganese. Yields are good to excellent. However, most examples derive from cyclic compounds; that is, the product(s) are the result of a double bond cleavage, but the oxidized functionalities stay within the same molecule. This means that, formally, they are not decoupled. Scheme 64 shows a few examples of the decoupling chemistry using iodomesitylene. Scheme 64. Effective Decoupling of the Double Bond by Oxidation with MCPBA and Iodomesitylene as a Catalyst155

Scheme 63. Examples of Ozonolysis’ Equivalents using RuCl3 plus Oxone or Periodate149

7.3.2.3. Decoupling of Double Bonds via the Formation of Vicinal Diols and Further Oxidation. Vicinal diols can be formed from olefins as a result of cis or trans hydroxylation. cisHydroxylation products can be produced using osmium tetroxide, alkaline potassium permanganate, and similar oxidations. The trans-hydroxylation can be executed by epoxidation (m-CPBA, nitrile + hydrogen peroxide, etc.) followed by the epoxide ring-opening with water in a basic or acidic medium. The oxidative cleavage of the 1,2-diol can be accomplished by reactions with such oxidants as lead tetraacetate, periodic acid, or Dess−Martin periodinane (DMP)157,158 and iodoxybenzoic acid (IBX).159 The second step in the discussed process is a cleavage of the carbon−carbon bond of vicinal diols. It has to be noted that the vicinal diol structure is typical for carbohydrates, and thus, the same methods can often be taken advantage of when cleaving many mono- and polysaccharides as well as diols formed directly from olefins. Recently, Wang and Jiang160 developed a very interesting method of oxidizing double bonds with molecular oxygen as the sole oxidant in the presence of palladium acetate as a catalyst. The reactions required elevated pressure (8 atm) and produced diols when the medium was basic (sodium carbonate) or carbonyl compounds in the acidic medium (ptoluenesulfonic acid). 7.3.2.4. Decoupling Using Retro Aldol Condensation. While this decoupling methodology does not necessarily call for the presence of the double bond, it usually starts with an olefinic system. For example, in the spectacular retro-aldol cleavage161,162 which was utilized by Avery and co-workers in their total synthesis of (+)artemisin,162 the double bond of (R)(+)-pulegone (218) is epoxidized to form an α,β-epoxyketone (219). The ring-opening with thiophenolate produces an alcohol (220) that gives the retro-aldol product (221) in a very high yield, as shown in Scheme 65. The enol of (221) can be easily alkylated and transformed into an α,β-unsaturated ketone. 7.3.2.5. Epoxidation of α,β-Unsaturated Ketones Followed by the Formation and Degradation of the Tosylhydrazone (Eschenmoser−Tanabe Fragmentation). This strategy requires that the double bond of an α,β-unsaturated ketone is oxidized to the corresponding epoxide but the epoxide is not transformed into the diol (as in section 6.3.2.3) or phenyl sulfide (as in section 6.3.2.4), but its ketone group is reacted

(from RuCl3 and NaIO4) in the absence of an organic solvent using ultrasonic irradiation and Aliquat 336 as an emulsifier. Similar results were achieved by Borhan et al. using catalytic amounts of osmium tetroxide and oxone (4 equiv)151 or other osmium sources and oxone.152 Mono- and vicinal disubstituted alkenes form corresponding carboxylic acids as cleavage products, and higher order alkenes including geminal disubstituted olefins form the corresponding ketones. The yields are very high. Additionally, there are several reagents capable of cleaving double bonds that take advantage of potassium permanganate. The options include the Lemieux−von Rudloff reagent153 (periodic acid plus traces of permanganate) or the use of potassium permanganate at acidic or neutral pH or in the presence of an appropriate phase transfer catalyst.154 It must be emphasized that high-valent oxo-metals such as ruthenium and osmium tetroxides in combination with such oxygen donors as sodium periodate or oxone are very strong oxidizing agents capable of oxidizing various functional groups and, thus, are not applicable to some natural products. Additionally, these compounds are not environmentally friendly. Ochiai et al.155,156 accomplished comparable results without any metal catalysts. They used iodomesitylene155 or iodosylbenzene156 as an effective organocatalyst and m-chloroperoxybenzoic acid as an oxidant. The method is a safe alternative for both ozonolysis and oxidations using reagents containing 2228


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Scheme 65. Specific Example of the Decoupling Caused by a Retro Aldol Reaction161,162

Scheme 67. Decoupling of Furans via Oxidation to Substituted Olefins

with sulfonylhydrazine to produce the corresponding tosylhydrazone. The fragmentation affords a compound with a triple bond (226) and a ketone (227) (Scheme 66). While the yields

• Cyclization to form a furan ring: The effective methods include those starting from alkynes and using a Ru(II)or Cu(II)-catalyzed process,173 transition metal catalyzed transformation of allenyl ketones,174 and Ag(I)-catalyzed cyclization and dehydration of 3,4-dihydroxy-5-alkynylcarboxylic acids to 5-substituted 2-furylacetic acids.175 One way to achieve a furan cleavage is by ozonolysis, which with furans can be problematic. Nevertheless, recently Pearlman et al.176 introduced a novel approach to furan degradation involving opening/isomerization to the trans-enedione, ozonization/reduction to the ketoaldehyde, and Baeyer−Villiger oxidation to the carboxylic acid. 2,5-Disubstituted furans can be efficiently oxidized with m-CPBA acid to (Z)-2-butene-1,4diones.177,178 Further oxidation (perhaps it can be a one pot process) of the double bond will disconnect the furan substituents from positions 2 and 5. Another method of furan degradation is applicable only to furfuryl alcohols (232). They can be submitted (bromine in methanol followed by acid hydrolysis or oxidation with m-CPBA) to the Achmatowicz reaction179,180 to form dihydropyran derivatives (233) that can be cleaved using one of the olefinic bond cleavage methods (Scheme 68).

Scheme 66. Decoupling Utilizing the Eschenmoser−Tanabe Fragmentation

of the fragmentation are not always satisfactory, it is a beautiful reaction, and hopefully, methods to improve the yields will be found in the near future. Recently, the fragmentation was taken advantage of in a very interesting total synthesis of the Galbulimima Alkaloid.163 α,β-Epoxyketones can be formed in good yield from allylic alcohols or α,β-unsaturated ketones by one pot palladiumcatalyzed epoxidation−oxidation,164 microwave accelerated epoxidation of α,β-unsaturated ketones with urea-hydrogen peroxide,165 polyvinyl-pyrrolidone supported oxone and tBuOOH,166 or lanthanoid catalyzed cumene hydroperoxide oxidation.167 7.3.2.6. Cleavage of Furans and Similar Compounds. Furans can be considered masked double bonds. They can be oxidized to form 2-ene-1,4-diketones or their equivalents (Scheme 67). Of course, not every substituted furan is of interest to us. Only 2,5- (and possibly 2,4- and 3,4-) disubstituted furans can be cleaved to offer a meaningful decoupling: There are many reactions forming 2,5-disubstituted furans. Here are recent reviews on the subject.168,169 Essentially, two approaches have been successful: • Functionalization of furans: Most methods which start with furan or substituted furan are not effective due to the furan degradation. However, some methods are sufficiently mild. For example, halofurans can be transformed to the required products via the halogen− magnesium exchange.170−172

Scheme 68. Decoupling of Furfuryl Alcohols via the Achmatowicz Reaction179,180

7.3.3. Carbonyl Group as an Object of CAD Chemistry. Carbonyl groups (containing α-hydrogen atoms) can be degraded in a specific way to form the decoupling products. There is a variety of ketone producing reactions. We want to highlight a particularly interesting one. Many heterocyclic systems (five or six member rings) can be conveniently assembled in a multiple manner connecting two different and chemically diversified substrates, which later on will be 2229


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functionalized to new molecules having different connecting units. The 1,3 dipolar cycloaddition reaction is a classical example of assembling complex molecules starting from relatively small starting templates. In this way C-disaccharide haw also been assembled181 (Scheme 69).

Scheme 70. Specific Example of Decoupling of Enol Ethers184

Scheme 69. Creative Way of the Formation of CDisaccharides182,183

of a catalytic amount of peroxotungstophosphate (PCWP) under phase-transfer conditions. The yields are good for ethers deriving from aldehydes and only modest for ethers deriving from ketones (adipic acid was isolated in 48% yield from the reaction of silyl ether formed from cyclohexanone). Yang and Zhang,149 who developed three different protocols for oxidation of double bonds with ruthenium-containing systems, have shown that one of their protocols gave a very decent yield of the oxidative cleavage when the enol ether (245) was a starting material (Scheme 71). 7.3.4. Cleavage of Derivatives of Carboxylic and Other Acids. The compounds discussed in this chapter are derivatives of carboxylic acids in which the carboxylic hydroxyl group was replaced with any of the following groups, OR, NR1R2 (one of the R1 and R2 substituents can be H), HNOR, NRNR2 (R may represent H), or SR, or the carboxylic

The methodology reported by Paton et al.182,183 explored the cycloaddition of carbohydrate nitrile oxide (236) to the reactive carbohydrate ene (237) with the formation of two isomers (of total of four possible isomers). Reductive hydrolysis gave after reacetylation the D-xyl-C-(1−3)-α-D-glc-OEt linked C-disaccharide. The linker is now a carbonyl group in lieu of the interglycosidic oxygen atom. It is important to note that the cycloaddition occurred exclusively from the top b-face opposite the substituents at C-4 and C-1. Alternatively, attachment of the nitrile to the unsaturated sugar via temporary connection may increase the regioselectivity by forcing the cycloaddition to proceed in an intramolecular fashion. The reported methodology is very efficient, and C-disaccharides are quickly assembled from accessible and properly functionalized precursors. The number of possibilities when considering the decoupling of the C−C bond of C-disaccharides is rather limited. The most advantageous option seems to be the oxidative cleavage of the corresponding enol ethers. For example, Martin and Miller184 oxidized the silylated enol ether with osmium tetroxide to form the hydroxyketone and then used lead tetraacetate to achieve the desired hydroxyester. Alternatively, they applied Johnson− Lemieux conditions (sodium periodate plus a catalytic quantity of osmium tetroxide) to successfully transform the starting enol ether into aldehyde/carboxylic acid (the yield of the corresponding lactone formed as a result of a subsequent reduction with borohydride was 55%; see Scheme 70). Ishii et al.185 performed an oxidative cleavage of silyl enol ethers, derived from aldehydes and ketones and trimethylchlorosilane, using aqueous hydrogen peroxide in the presence

Scheme 71. Decoupling by Degradation of Enol Ether via Ruthenium-Catalyzed Oxidation149

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carbonyl group was replaced with SC or RNC. Additionally, derivatives of sulfonic and phosphoric acids belong to this category. The presented examples are meant to be representative and to show that there are many acid derivatives’ reactions that are applicable to the CAD chemistry. In particular, many processes of connecting the relevant units are reversible and there are instances when the divorce conditions are relatively mild. It must be noted that ester and amide functionalities are present in such important natural compounds as fats, proteins, and many polysaccharides. On one hand, this means that a plurality of methods of forming esters and amides has been developed, but on the other hand, it creates a challenge to find the decoupling methodologies that are sufficiently selective. It is relatively easy to deprotect Boc substituted amines in the presence of carboxylic groups protected as esters. Marcantoni, Bartoli, and co-workers186 developed a method to cleave tertbutyl esters in the presence of amino groups protected by Boc using cerium trichloride/sodium iodide in acetonitrile. Other protective groups such as methyl esters or Cbz are not affected. The reaction requires usually a few hours to reach completion. The yields in the shown examples represent isolated yields (Scheme 72).

Scheme 73. Highly Selective Hydrolysis of Tosylamides and Tosylesters188

chemistry. Palladium-catalyzed deprotection of DMA esters was shown to be compatible with tert-butyl, benzyl, and Fmoc protecting groups, and Fmoc deprotection could be carried out selectively in the presence of DMA esters. In a later paper, an alternative method of synthesizing DMA esters was presented.190 7.3.5. Formation, Functionalization, and Cleavage of Carbonyl Derivatives. The carbonyl group linker can function as an effective and reactive chemical entity which can be further functionalized (e.g., via oximes, hydrazones) and then effectively cleaved into single fragment molecules. One of the extremely important and synthetically valuable methodologies is the classical conversion of keto functionality into substituted 1,3-dithiane derivatives191 originally developed by Seebach and Corey.192−195 This approach of metalation (lithiation) of 1,3-dithianes leads to protected acyl-lithium derivatives, which are synthetically equivalent to acyl-anion. These sulfur-stabilized anionic reagents can be used quite effectively to reverse the electrophilicity of a carbonyl carbon known as reversible umpolung. Note that the formation of the dithioacetal here can be considered a coupling between R and R1 (of course R1 cannot be a hydrogen atom as it was in the Seebach-Corey process). The structure of the formed dithioacetal can be exploited to introduce a variety of substituents, and finally, R and R1 can be decoupled as a result of Raney Ni reduction or hydrolysis (Scheme 74). Seebach’s original concept of utilization of 1,3-dithiane classifies many types of selective protection of keto functionality in the presence of other mildly removable protecting groups. This important aspect of selective functionalization of keto functionality creates enormous synthetic applications of this methodology. 1,3-Dithianes are conveniently produced from aldehydes (method A) or acetals (method B). While such reactions as those using butyllithium may not be applicable to most natural biomacromolecules, the 1,3-dithiane alkylation and hydroxyalkylation and follow up desulfurization process of thioacetals are perfectly fitting our CAD chemistry strategy and can be effectively utilized in many synthetic approaches which require C−C bond formation via chain elongation or connecting two different molecules with follow up of removal of a sacrificial unit. Horton and Priebe196 utilized the 2-lithio-1,3-dithiane approach to the synthesis of higher sugars starting from fully protected (silylated) lactones. This particular method was

Scheme 72. Highly Chemoselective Decoupling of tert-Butyl Esters186

Lubell et al. studied the deprotection of tBu esters in the presence of other protective groups using a mild Lewis acid− zinc bromide in dichloromethane.187 When amino acids with the carboxyl group protected as the tBu ester and the amino group protected as the t-Boc were subjected to the selected conditions, both groups were cleaved. However, the method offers selective deprotection of tert-butyl esters in the presence of N-(PhF) amines and provides an effective means for obtaining N-(PhF) amino acids possessing a wide range of functional group diversity. Swedish researchers188 developed a mild method of rapid deprotection of tosylamides and esters using SmI2/amine/ water. The method leaves such protecting groups as N-Boc, cyclic acetals, and benzyl ethers intact. The other product of the reductive cleavage is p-methylthiophenol. Scheme 73 shows a few examples. The Lipton group189 developed a novel method of synthesizing and deprotecting 1,1-dimethylallyl (DMA) esters in the presence of other protective groups used in the protein 2231


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carbamates with different heteroatoms. The heteroatoms include sulfur, oxygen, nitrogen, selenium, and phosphorus. Thus, the compounds of interest are carbonates, thiolo- and thionocarbonates, xanthates (dithiocarbonates), trithiocarbonates, selenocarbonates, tellurocarbonates, carbamates, thiocarbamates, selenocarbamates, etc. There are reactions of the classical organic chemistry that cleave certain bonds in such compounds as xanthates. Let us just mention such reactions as Czugaev degradation of xanthates, which enables cleavage of the thioalkyl group (it usually is the thiomethyl group, but there are no good reasons for other substituents such as substituted benzyl or propargyl198 to be employed) from the alkoxy part. Another good example is the Barton−McCombie reaction when applied to xanthates or thionocarbonates.199 The following text offers a few examples of the possible decoupling chemistry within the “carbonate” category. Chandrasekaran et al.200 showed that propargyl carbonates and carbamates can be synthesized in very high yields. The researchers took advantage of a significant difference of the cleavage rate of comparable propargyl carbonates and propargyl carbamates when using 1 equivalent of benzyltriethylammonium tetrathiomolybdate (TTM) as a cleaving agent (Scheme 76).

Scheme 74. Seebach’s Approach to 1,3-Dithianyl Functionalization of Carbonyl Groups192−195

Scheme 76. Significant Diferenece in Kinetics of Propargyl Carbonate and Carbamate Hydrolyses200

proven to be highly efficient to produce C-glycosides and Cglycosyl compounds (Scheme 75). Similar utilization of 2-lithio-1,3-dithiane addition to fully protected 2,3;5,6-di-O-isopropylidene-D-mannofuranose was reported by Krohn and Borner.197 This approach is proceeding through activation of intermediate 1,3-dithianyl derivative (as tosylate or epoxide) and can successfully undergo intramolecular cyclizations to carbocycles. 7.3.6. Cleavage of Carbonates, Carbamates, and Heterocarbonates. We are not aware of an adequate general name for compounds containing a carbon atom bonded to three heteroatoms with one of these bonds being a double bond. To keep the names of familiar functionalities unchanged, we use the word “carbonate” when the three heteroatoms are oxygen atoms and “carbamate” when one of the noncarbonyl oxygen atoms in carbonate was replaced with nitrogen. We call “heterocarbonates” all compounds related to carbonates and

The examples in Scheme 77 show selective cleavage of carbonates in the presence of carbamates. Bennasar et al.201 cleave benzyl (and tert-butyl) carbamates deriving from amides or nitrogen containing heteroaromatic

Scheme 75. 1,3-Dithianyl Anion Synthetic Route to C-Glycosyl Compounds196

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and carbonate resins. The following examples (Scheme 79) show very high yield of the reaction(s).

Scheme 77. Decoupling of Propargyl Carbonates in the Presence of Propargyl Carbamates200

Scheme 79. Examples of Zinc Bromide Promoted Decoupling of Carbonates and Carbamates from the Merrifield Resin202

rings in the presence of various functionalities using tributylstannyl radicals. Interestingly, these radical conditions do not affect N-Cbz derivatives of basic amines. Scheme 78 shows a few examples. Li and co-workers202 have developed a new cleavage strategy for Merrifield resin-bound benzyloxycarbonyl linkers using a mild Lewis acid (ZnBr2). This new one-pot release protocol has proven to be a general reaction for the cleavage of carbamate In other examples, the yields are substantially lower but the calculated yields are based on the loading level of the hydroxymethylated resin and include more than the single reaction shown. Zard et al.203 developed an interesting and practical method for the reductive cleavage of sulfide bonds in such heterocarbonates as xanthates. They heat a xanthate in 2propanol in the presence of equimolar amounts of dilauroyl peroxide, added in small portions. The process gives good results, mainly with secondary compounds. While many examples are for methyl and ethyl xanthates, the authors have shown that tert-butylmethyl xanthates are at least equally acceptable as substrates, suggesting that almost any alkyl group can be taken advantage of. The following examples (Scheme 80) show typical reaction yields and functionalities that are not affected by the reaction conditions. 7.3.7. Decoupling of Alkyl (Aryl) Ethers, Thioethers, and Amines. The compounds discussed here are represented by the following formula: Ra−X−Rb (X = O, S, NRc). They are usually formed from alcohols, thiols, and amines which become protected as alkyl ethers, thioethers, or alkyl (aryl) amines. There are many deprotection procedures that are clearly applicable to the CAD chemistry. When selecting the examples, we put emphasis on methods that offer high yields and moderate conditions and were shown to be highly chemo- and/ or regioselective. They are to serve as examples of potential use of substituted and nonsubstituted benzyl, trityl, allyl, etc. ethers, thioethers, and amines in the CAD chemistry. Those interested in more exhaustive coverage of protection and deprotection of alcohols are encouraged to read the relevant reviews. Recently, Weissman and Zewge204 reviewed the dealkylation of ethers, Shashidhar et al.205 reviewed the regioselectivity in protection and deprotection of the hydroxyl group in inositols, Falvey and Sundararajan95 reviewed the photoremovable protecting groups

Scheme 78. Highly Selective Decoupling of Benzyl Carbamates Using Tributyltin Hydride201

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examples yet of the use of silyl derivatives comprising very large molecules. It seems that there are three major reasons for the popularity of benzyl protective groups: (a) Conditions involved in their removal are usually very mild and such that they do not affect other protective groups and most functionalities typically present in proteins and polysaccharides. This applies particularly to catalytic hydrogenolysis but also to other procedures. (b) There are several types of cleavage procedures each using a very different set of conditions. Reductive, oxidative, Lewis acid, or sodium hydroxide containing conditions can be effective. Thus, conditions enabling introduction and removal of benzyl and similar groups can be found for almost any organic construct. (c) The benzylated products are usually formed very easily and the yields are typically excellent. Additionally, variously substituted benzyl groups (such as pmethoxy) have been used for the protection of the same functionalities. The rates and conditions of deprotection for various benzyl groups are not necessarily the same or even similar. This fact can be taken advantage of to sequentially deprotect various benzyl substituents. Here are a few selected deprotection methods we hope can be applied to the CAD chemistry: Spencer and colleagues developed a methodology to sequentially remove various substituted benzyl groups from benzyl ethers by catalytic hydrogenolysis.208 They demonstrate that the rates of deprotection of benzyl groups with different substituents at the para position differ substantially. Scheme 81 shows the relative rates.

Scheme 80. Selective Decoupling of Xanthates by Cleavage of the Carbon−Sulfur Bond203

based on electron transfer chemistry, and Lukáč et al.206,207 reviewed the deprotection and protection of alcohols and phenols substituted with a methoxybenzyl group. The most popular protective group for alcohols, amines, and thiols is arguably the benzyl group. While there is a plethora of papers on the formation and cleavage of benzyl, trityl, and similar derivatives, there are no papers describing the formation and cleavage of benzyl derivatives in which the aromatic ring (or the α carbon atom) is substituted with a large moiety, such as a steroid, polysaccharide, nucleic acid, or protein. Nevertheless, when one considers mechanisms of the relevant reactions, there are good reasons to believe that often the scission can be accomplished equally easily when the substituents of benzyl and other similar groups comprise large or very large moieties. There are many examples of the successful use in protection/deprotection chemistry of benzyl groups equipped with both electron withdrawing and electron donating groups. However, one cannot overstate the fact that steric hindrance may dramatically affect (kinetics of) many processes discussed here. Furthermore, when using the enzymatic method of cleavage, one must remember that the requirements are often very stringent. The same applies to other protective groups for alcohols (and other functional groups) such as allyl, methallyl, propargyl, etc. We decided not to use examples of the protection/ deprotection chemistry of methyl or ethyl groups. While there are better and better chemistries of cleavage of methyl ethers, it seems that the chemistry of cleavage of methyl ethers, in which one of the hydrogen atoms was replaced with a large moiety comprising an oligo- or polysaccharide or peptide, is not established yet. One can argue that the same may be said about the benzyl ethers. This is correct, but we decided that the extrapolation of benzyl group properties to the properties of the benzyl group substituted with a large substituent is significantly less overreaching. Another category of very useful protective groups that is omitted in the review is silyl derivatives. While they have been very successfully used for protection and deprotection of alcohols, phenols, and other functionalities, there are no

Scheme 81. Kinetics of Hydrogenation of Benzyl Groups with Various Substituents208

The electronic effect is not the only factor determining the reaction rates. The authors found that the 2-naphthylmethyl group (NAP) is particularly easily removed when palladium is a catalyst. Thus, the NAP group can be cleanly cleaved from primary or secondary hydroxyl groups in the presence of single or multiple benzyl ethers. The yields are very high (Scheme 82). Interestingly, Matta with co-workers209 and Lipták with coworkers210 developed an alternative protocol for the decoupling of the NAP group using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) which offers equally impressive yields. Recently, Boons et al.211 have shown the exceptional usefulness 2234


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Indian scientists215 developed a mild, highly chemoselective and efficient protocol for detritylation using a catalytic amount of BiCl3 in acetonitrile. The cleavage proceeds at high yields (86−95%) in the presence of various acid/base sensitive groups, such as tetrahydropiranyl, TBDPS, Piv, benzyl, pmethoxybenzyl, acetyl, tosyl, isopropylidene, allyl, prenyl, and NHBoc. The following carbohydrate chemistry examples (Scheme 84) show the typical conditions and yields.

Scheme 82. Selective Decoupling of the NAP Group in the Presence of Other Benzyl Groups208

Scheme 84. Mild, Chemoselective Decoupling of Trityl Groups Using Bismuth Chloride215

of this protocol during the synthetic studies of an unusual phospho-glycopeptide derived from α-dystroglycan. Sajiki212 proposes an entirely different approach toward the chemoselectivity issue. He shows that ammonia, pyridine, and ammonium acetate are extremely effective as inhibitors of Pd/C catalyzed benzyl ether hydrogenation. Olefin, Cbz, azide, and benzyl ester functionalities were hydrogenated smoothly in the presence of the benzyl ether removal inhibitors. The yields of the recovery of nondeprotected benzyl ethers were very high. An alternative to using a hydrogen gas in catalytic hydrogenolyses of benzyl groups is to take advantage of a catalytic transfer hydrogenation (CTH) using such compounds as cyclohexene, cyclohexadiene, hydrazine, or formate salts. Recently, Pohl et al.213 used ammonium formate as a hydrogen donor in a microwave assisted debenzylation in the presence of 10% Pd−C as a catalyst. The reaction required only 5 min to give 90% yield of the deprotected alcohol as compared with traditional hydrogenation with hydrogen in ethanol, which required 12 h and gave 95% yield of the alcohol. Prasad et al.214 cleaved benzylamine, benzyloxycarbonyl, and benzyl ester using a few equivalents of formic acid and a catalytic amount of triethylamine. The yields are high. Scheme 83 shows an example of debenzylation of the tertiary benzylamine (319).

Vogel and Marković216 developed a very mild and efficient reagent for selective cleavage of allyl, methallyl, prenyl, and methylprenyl ethers under neutral conditions. The method utilizes diphenyldisulfone (10 mol %) at 80 °C. Such protective groups as benzyl, acetyl, and t-BuMe2Si are not affected, and yields of isolated products can be as high as 96%. The rate of cleavage differs very significantly for various alkenyl groups. Scheme 85 gives approximate half-lives of menthol derivatives with its hydroxyl group protected with selected alkenyl groups under comparable conditions. Scheme 85. Kinetics of Decoupling of Substituted Allyl Groups216

Scheme 83. Example of Pd-Assisted Debenzylation Using Formic Acid and Triethylamine214 The authors note that, in comparable deprotection reactions assisted by such metals as Pd, Rh, and Ir, the higher the degree of substitution of the allyl ether, the slower is the transition metal catalyzed isomerization of the allyl to alkenyl ether. The isomerization is followed by acidic hydrolysis, which liberates the deprotected alcohol. In the presence of catalytic amounts of diphenyldisulphone, methallyl, prenyl, and methylprenyl ethers are cleaved readily, and what is truly remarkable, the fastest reaction occurs with the most substituted allyl systems. This is clearly extremely important from the standpoint of CAD processes, where there is always a concern that the presence of additional (large) substituents may slow down many reactions. The authors have shown several examples in which differences in reaction rates were exploited to sequentially cleave sugar hydroxyl groups protected as various substituted allyl ethers. 2235


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The following example (Scheme 86) shows the method’s potential.

Scheme 88. Mild and Highly Chemoselective Decoupling of tert-Butyl Ethers218

Scheme 86. Sequential Decoupling of Various Allyl Groups216

Akao et al.217 studied debenzylation of benzyl thioethers. They developed a highly effective and relatively mild deprotection method using dibutylmagnesium in the presence of catalytic amounts of titanocene dichloride. The methodology is applicable to aliphatic and aromatic thioethers and does not affect benzylethers (see the reaction of 334) and benzylamines (see the reaction of 336). A few examples are shown in Scheme 87.

Recently, Pale and co-workers219 developed a new type of a protecting groupa bis(4-methoxyphenyl)methyl group. Copper(II) bromide acts as an efficient catalyst for the selective protection (in acetonitrile) and deprotection (in ethanol) of alcohols as bis(4-methoxyphenyl) methyl ethers. Both the protection and deprotection are orthogonal to other methods and fully compatible with other functional groups. The yields are high (Scheme 89). 7.3.8. Formation of and Cleavage of Aza, Diazo, etc. Derivatives. Among the specific examples of aza-functionalized derivatives, phenylhydrazides are excellent moieties for masking amino acids in good to excellent yields using a carbodiimide-mediated coupling methodology. The decoupling protocols, however, can be particularly important. They may require that protecting groups removable under mild conditions are utilized. While the classical chemical hydrolysis can be applicable to many conditions, it seems that the enzymatic approach is a method of choice when the hydrolyzed molecules contain multiple peptide bonds. Most commonly, the applied enzymes are hydrolases that directly attack a carbonyl group. The substrate specificity for the individual enzyme, in particular the possibility of undesired peptide bond hydrolysis, has to be taken into account to exclusively guarantee the required chemo- and regioselectivity. An alternative approach is to use a different enzymatic transformation during which an otherwise stable precursor is conveniently converted into a labile intermediate that subsequently hydrolyzes spontaneously under the reaction conditions. Specifically, a required transformation could be an oxidation reaction, which would allow for orthogonality to different hydrolase-labile protecting groups and to other classical chemical blocking moieties, as reported by Muller and Waldmann.220 Note that the enzymatic reaction (oxidation) produces the diazo-compound, which undergoes rapid chemical hydrolysis, which constitutes the final cleavage (Scheme 90). In this way the oxidation of N-terminally protected amino acid phenylhydrazides has previously been used for acylation reaction in the synthesis of peptides.221,222 The oxidation can

Scheme 87. Specific Examples of the Selective Decoupling of Benzyl Thioethers217

Bartoli, Sambria, and co-workers218 developed a novel general, mild, and chemoselective method enabling the effective cleavage of aliphatic and aromatic tert-butyl ethers using cerium chloride and sodium iodide in acetonitrile (the same reagent as for cleavage of tert-butyl esters186). The yields are often practically quantitative, and many other functional and protective groups including acetate, a triisopropylsilyl group, and benzyl are unaffected (Scheme 88). 2236


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concern that some other functionalities may not survive the strong oxidant. Alternatively, the reductive cleavage of the −NN− bond via catalytic transfer hydrogenation (CTH) using iron incorporated hexagonal mesoporous aluminiphosphate (FeHMA) catalyst has been reported.226 The catalyst can be reused several times, but yields are not always satisfactory. Also, for that particular synthetic application, sodium dithionite as a reducing agent has been used successfully.227 Alternative selective reductive cleavage by commercial zinc dust using ammonium formate or formic acid has been reported in the literature.228 The cleavage is high yielding, very fast, and clean, and it occurs without affecting reducible moieties, such as −OH, −OCH3, −COOH, −COCH3, halogen, etc.

Scheme 89. Mild and Selective Removal of a Bis(4methoxyphenyl)methyl Group219

8. POTENTIAL FUTURE APPLICATIONS OF CAD CHEMISTRY From the several approaches and schematic strategies of CAD chemistry presented in this review, it is clear that the choice of the specific decoupling processes is of utmost importance. In particular, enzymatic and pH controlled conversions will be valuable choices in leading (in vivo) applications. A few applications discussed earlier will incorporate CAD chemistry to the relatively new concept of codrugs. CAD will play an important role in the drug delivery methodologies and subsequent controlled release into the specific target. Additionally, CAD chemistry strategies will be extremely useful in the design of soft-drugs, that is, active therapeutic agents with a chemical structure specifically designated to allow predictable metabolism into totally inactive metabolites after exerting the desired therapeutic effect. It must be noted that strategic differences between prodrugs and soft-drugs exist in the way of activation of pro-drugs in vivo and, consequently, forming active metabolites. The combination of pro-soft-drug, which is an inactive prodrug of a soft-drug, can be constructed quite effectively by taking advantage of CAD chemistry approaches. This can be achieved via the careful design of specifically selected functional groups to undergo the desirable metabolic transformations to form the pharmacologically active form of the drug. Most physical triggers are not applicable to in vivo processes. Nevertheless, CAD chemistry approaches utilizing ultrasound could be used in various drug delivery applications to significantly enhance the delivery of bioactive therapeutic agents to specific target tissues. This type of strategy is known as Acoustic Targeted Drug Delivery, and it is employed to enhance chemotherapy treatment to brain cancer cells, which also reduces the time necessary for the drug to work. Ultrasound has also been useful in a much different clinical application, mainly in thrombi, or blood clots, which can form in veins of patients with poor circulation. Therapeutic agents called thrombolytics, which are normally given to break up these clots, are made dramatically more effective when lowintensity ultrasound is applied to the clot during their application. While the mode of action of the ultrasound enhancement is not well understood, there is no doubt that ultrasound causes the decoupling. Some examples of applying ultrasound to the drug delivery of therapeutic agents are reported in the literature.229 The application of ultrasonic techniques in the organic synthesis230 with or without phase transfer catalysts has also been reported.231,232 Well-defined methods of CAD

Scheme 90. Specific Example of Coupling and Decoupling of Azo Compounds220

be performed using N-bromosuccinimide (NBS). Alternatively, phenylhydrazides could be oxidized by such enzymes as laccase, horseradish peroxidase, and mushroom tyrosinase.223,224 On another note, many functionalized hydrazones undergo highly functional transformations and can be hydrolyzed back to a keto functionality by simple chemistry without formation of byproducts and other decomposition products. It is worth mentioning that tosylhydrazones can be hydrolyzed with copper sulfate hydrate or bleach. Carmeli and co-workers225 used HOF·CH3CN as an oxidizing agent to form corresponding aldehydes or ketones from azines, hydrazones, and oximes. The reaction gives very good yields and is complete in seconds; however, there is a 2237


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that this review constitutes the first pioneering compilation of potentially very useful techniques in general organic synthesis. We expect that the arsenal of specific functional groups and availability of sacrificial units used in CAD chemistry will be steadily developed for many years to come.

chemistry triggered by ultrasound will certainly be developed in the near future. All the potential applications of CAD chemistry discussed here are related to the design of drug delivery platforms, but they could also be extended to the specificity and selectivity in bacterial membrane penetration either through simple diffusion or drug transporter systems. This particular strategy should be extremely helpful for combating drug resistance, including MRSA (Methicillin Resistant Staphylococcus aureus) and VRE (Vancomycin Resistant Enterococcus). The discussed approach to specific selection and design of manipulation of functional groups in assembling small and large molecules via CAD chemistry, and subsequent application to various areas in biomedical science will grow steadily. The particularly important field to be explored is again drug delivery platform, which will play a significant role in many areas of modern pharmacotherapy. Recently, Bielawski and co-workers233 published a very interesting paper on mechanically facilitated 1,3-dipolar cycloreversions. The authors show that two poly(methyl acrylate) units connected via a 1,2,3-triazole moiety can be “unclicked’ using ultrasound in the presence of copper(I) iodide. While this is a very important result, we do not expect such cycloreversions to become the method of choice for decoupling molecular units.

AUTHOR INFORMATION Corresponding Author

*E-mail addresses: R.B., [email protected]; Z.W., zbigniew. [email protected]. Notes

The authors declare no competing financial interest. Biographies

9. CONCLUSIONS Coupling and decoupling (CAD) chemistry is a methodology applicable to situations when large molecular pieces need to be connected, worked with, and ultimately disconnected. Many reactions applicable to coupling various functionalities have been developed and are very useful in CAD chemistry. However, the arsenal of available decoupling reactions is rather underdeveloped. Moreover, the scope and limitations of many such reactions are not known. For example, many effective reactions have been shown to work in the synthesis of small molecules, but it is not known how chemospecific the processes are, that is, whether larger peptide, oligosaccharide, or nucleic acid units equipped with a variety of functional groups will survive the decoupling conditions. Particularly attractive seems to be a methodology of introducing a sacrificial functionality into the products formed exclusively in the click processes. These sacrificial functionalities should be unaffected by almost any conditions the coupled products may be exposed to. Finally, when needed, the sacrificial functionality must be cleaved under very specific conditions. Usually, these conditions must be such that they do not affect any other components within the connected units. It seems that biocatalytic cleavage of the sacrificial units is particularly useful and highly recommendable. At this point, the scope and chemo-selectivity of many excellent enzymatic processes is not well-known and, therefore, should not be considered as a general methodology. In addition to the use of sacrificial units, CAD chemistry takes advantage of reversible (in the sense discussed earlier) processes, when possible, as long as they are chemoselective, There is a clear need for more research in establishing good sacrificial functional groups, determining their limitations, and optimizing the decoupling process conditions. Additionally, there are hardly any conveniently substituted sacrificial functional groups in commercially available reactants. We are aware that this review does not cover all potential coupling and decoupling strategies and applications. Nevertheless, we think

Roman Bielski was born in Katowice, Poland. He graduated (majoring in chemical engineering) from the Warsaw University of Technology and received his Ph.D. in organic chemistry (under the direction of Prof. Osman Achmatowicz) in 1974. He carried out his postdoctoral research with Sir Derek Barton at Imperial College in London. After spending several years in academia (Warsaw Agricultural University, Lehigh University, and Cornell University), he worked in and cofounded a few small biotech companies. His research interests include the origin of homochirality (together with M. Tencer, he discovered a novel chiral influencea set of three orthogonal orienting factors), environmental solutions, and sustainable materials. He published about three dozen papers and coauthored sixteen patents.

Zbigniew J. Witczak received M.S. in Pharmaceutical Chemistry in 1973 and his Ph.D. degree in Natural Products Chemistry in 1979 from the Faculty of Pharmacy at the Medical University of Lodz, Poland. After postdoctoral work with Roy L. Whistler at Purdue University, he was appointed in 1991 as an Assistant Professor at the University of Connecticut, School of Pharmacy. Since 2000 he has 2238


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γ-aminobutyric acid generally recognized as safe 2−4-(2-hydroxyethyl)piperazin-1-ylethanesulfonic acid HMBA 4-hydroxymethylbenzoic acid HMPAA hydroxymethylphenoxyacetic acid IBX iodoxybenzoic acid ICAT isotope-coded affinity tags IgG1 immunoglobulin G IR infrared LC liquid chromatography Lipase-CC lipase from Candida cylindracea MCPBA m-chloroperbenzoic acid Me methyl MRSA methicillin resistant Staphylococcus aureus MS mass spectrometry NAP 2-naphthylmethyl NBS N-bromosuccinimide NiCRA nickel-containing reducing agent NiCRAbpy NiCRA−2.2′-bipyridine NMR nuclear magnetic resonance 2-NPA 2-nitrophenylalanine PBS phosphate buffered saline PC-linker photocleavable linker PEGA polyethylene glycol-amine PEO polyethyleneglycol Ph phenyl PhF 9-phenylfluoren-9-yl Piv pivaloyl PMP p-methoxyphenyl PLE porcine liver esterase Poc propargyloxycarbonyl PPL pancreatic lipase PS polystyrene PSA prostate-specific antigen PSL lipase from Pseudomonas cipacia PSWP peroxotungstophosphate PTSA p-toluenesulfonic acid Py pyridine QB quinolinyl benzenesulfonate Rho rhodamine RIC radioimmunoconjugate SDS sodium dodecyl sulfate SVEC succinimidyl 2-(vinylsulfonyl)ethyl carbonate TBDMS tert-butyldimethylsilyl TCEP tris(2-carboxyethyl)phosphine TEA triethylamine TEV tobacco etch virus TFA trifluoroacetic acid THF tetrahydrofuran THP tetrahydropiranyl TIPS triisopropylsilyl TMS trimethylsilyl TOP-ABP tandem orthogonal proteolysis strategy for activity-based protein profiling TPG thermolabile protecting group Tris tris(hydroxymethyl)aminomethane Ts tosyl TTM tetrathiomolybdate USPTO United States Patent and Trademark Office UV ultraviolet VRE vancomycin resistant enterococcus WIPO World Intellectual Property Organization GABA GRAS HEPES

been Professor of Medicinal Chemistry at Nesbitt School of Pharmacy at Wilkes University, PA. His current research interests include thioclick chemistry, natural products and levoglucosenone chemistry, and biochemistry of thio sugars as new glycomimetics and inhibitors of galectin-3. He has published almost one hundred research papers, edited six books, and holds six patents. Recently, he was selected as one of the 2011 ACS Fellows.

ACKNOWLEDGMENTS We wish to express our sincere thanks to our friends and colleagues who read and constructively criticized the manuscript. Their valuable comments substantially helped to improve it. In particular, we want to thank Agata Bielska, Dr. Mike Bigwood, Dr. Ron Bihovsky, Dr. Tadeusz Cynkowski, Dr. John Newport, and Dr. Michal Tencer. ABBREVIATIONS ABP activity-based profiling probe ABTS 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) Ac acetyl AIBN azobisisobutyronitrile Aliquat 336 tricaprylylmethylammonium chloride An analyte Ar aryl Bal β-alanine BDQ biotin-dopamine-quinolinyl-benzenesulfonate Boc butoxycarbonyl Bn benzyl Bu butyl CACC copper-assisted click chemistry CAD coupling and decoupling CAR “catch and release” Cbz carbobenzyloxy CC click chemistry CTH catalytic transfer hydrogenation DA Diels−Alder Dab 2,4-diaminobutyric acid DADPS dialkoxydiphenylsilane DCC dicyclohexylcarbodiimide DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone DECM 7-(diethylamino)coumarin DIFO difluorinated cyclooctyne DIOSMS desorption/ionization on silicon MS DIPEA diisopropylethylamine DM dipole moment DMA 1,1-dimethylallyl DMAP 4-dimethylaminopyridine DME 1,2-dimethoxyethane DMF dimethylformamide DMP Dess−Martin periodinane DMSO dimethyl sulfoxide DNA DNA dppfNiCl2 1,1′-bis(diphenylphosphino)ferrocene dichloronickel Dpr 2,3-diaminopropionic acid DTT dithioerythritol ELISA enzyme-linked immunosorbent assay Et ethyl FeHMA iron (incorporated) hexagonal mesoporous aluminophosphate Fmoc fluorenylmethyloxycarbonyl 2239


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