Application of click chemistry for PET
Scheme 2.13. Structure of one of the tested compounds for peroxisome proliferator
Combinatorial approaches as described above using 18F-labelled azides or alkynes could lead to more rapid identification of potent PET-tracers. In addition, such approaches can increase the role of PET in drug development since suitable PET-tracers will become more readily available.
Click chemistry in Bioconjugation
Click chemistry has become an important tool for bioconjugation procedures in the development of bifunctional molecules. Bioconjugation involves the attachment of small labelled synthons to biomolecules, such as fusing two or more proteins together or linking a carbohydrate with a peptide. Although bioconjugation is applicable to the in vivo labeling of biomolecules, only a few reactions are actually useful. [Sharpless, 2001,2003] The use of click chemistry in bioconjugation was first demonstrated by Tornoe et al. for the preparation of peptidotriazoles via solid state synthesis.[Tonon,2009] The goal was the development of efficient synthetic methods to prepare a range of triazole pharmacophores for potential biologic targets. Various novel functional and/or reporter groups were introduced into peptides and proteins, DNA [Bertozzi,2007] and cell surfaces.[Gierlich,2008]
Wang et al. labeled Cowpea mosaic virus (CPMV) with fluorescein in >95%
yield.[Wang,2003] The labeling was performed by modifying the surface of the viral protein (either lysine or cysteine residues) with azides or alkynes, followed by reaction with
modification of Eschericia coli with an azide-bearing outer membrane protein C (OmpC). The modified cell was then biotinylated by reacting with a biotin alkyne derivative using copper catalysis. [Link,2003]
Most bioconjugation reactions, such as isothiocyanateamine, thiol-maleimide, and amine-carboxylic acid couplings, [Kolk,2008,Francis,2006] cannot be used for labeling in vivo because of competing nucleophiles and non-compatible reaction conditions. Also, condensation reactions between ketones or aldehydes and hydrazines or aminoxy derivatives, are not feasible. The reactions are usually carried out at pH of 5-6, under which condition the resulting hydrazone or oxime bond is not very stable. In addition, other ketones or aldehydes are usually present inside the cells.[ Kolk,2008] Click chemistry overcomes these obstacles by being bioorthogonal and by proceeding irreversibly in water at neutral pH and biocompatible temperatures (25-37°C) without any cytotoxic reagents or byproducts, provided that copper-free conditions can be applied. In vivo copper free click reactions would allow pretargeting strategies using first administration of targeted antibodies contained either an alkyne or an azide functionality with slow pharmacokinetics followed by a second administration of a 18F-labelled small molecule with the click counterpart functional group.
A more reactive compound for labeling biomolecules eliminates the need for toxic metal catalyst [Bertozzi,2007]. To be able to eliminate the copper catalysts would make the cycloaddition biologically friendly and thus useful for labeling biomolecules in cells. It was shown that copper-free click chemistry can label cell surface carbohydrates [Sharpless,2002],, which then move inside the cell. This chemistry helped Bertozzi [Bertozzi,2007] to study dynamic biochemical processes that are otherwise difficult to follow in real time. Of particular interest was the study of glycosylation of proteins. This reaction is difficult to follow over time because the sugar molecules, or glycans, are continuously recycled. But azides can be used as a tag for labeling biomolecules. For in vivo, copper-free application a highly reactive difluorinated cyclooctyne (DIFO) reagent was developed which rapidly reacts with azides in living cells without the need for copper catalysis. The crucial property of the substituted cyclooctyne is the high ring strain and electron-withdrawing fluorine substituents that together promote the [3 + 2] dipolar cycloaddition with azides installed metabolically into biomolecules. This Cu-free click reaction showed comparable kinetics to the Cu-catalyzed reaction and proceeded within minutes in vivo with no apparent
Application of click chemistry for PET
toxicity. Laughlin et al. were able to image glycans in developing zebrafish using click chemistry.[ Laughlin,2008] Embryonic zebrafish were incubated with an azideperacetylated N-azidoacetylgalactosamine derivative (Ac4- GalNAz), which was then reacted with a difluorinated cyclooctyne attached to a dye (scheme. 2.14).
The images obtained by flow cytometry showed high contrast and the derivates were not
Multivalent carbohydrates are attractive synthetic targets as they often bind much stronger to the protein partner than their monovalent equivalents. Multivalency is often used to significantly increase affinity of weakly bound sugars [Kitov, 2000]. Many types of linkages of sugars to biological molecules have been explored and substantial affinity enhancements have been achieved [Fan, 2000- Kitov, 2000]. The copper-catalyzed click reaction has provided an additional tool for the easy construction of multivalent carbohydrates. The click method has already been applied for numerous systems. These
include protected carbohydrates but unprotected carbohydrates have also been used, demonstrating the chemoselective nature of the click reaction.
In carbohydrates an azide group can easily be introduced, either at the anomeric center or via a spacer. Even before copper catalysis was reported, the uncatalyzed click reaction was used by Calvo-Flores group, for conjugating sugars to scaffold molecules aiming at preparing multivalent carbohydrates [Calvo-Flores,2000]. It was needed to perform the reaction for long times (30 h to 6 d) and both the 1,4- and 1,5-linked regioisomeric 1,2,3-triazoles for each linkage were obtained. Prante and co-workers developed 1BF-labelling and glycosylation by click chemistry. A new mannosyl azide precursor was reported for the radiosynthesis of a 'clickable' 1BF-glycosyl donor as a hydrophilic prosthetic group. With this precursor, a simple and reliable click chemistry-based procedure involving !BF-labeling and glycosylation of alkyne-functionalized molecules in high radiochemical yields has been established. The described procedure for the introduction of an 1BF-glucopyranoside label is advantageous due to the stability of the precursor, which is stable for months at -20° C, the hydrophilic nature of the 1BF-labeled prosthetic group, its high-yielding and reliable radiochemistry, and its general applicability for 1BF-labeling and glycosylation of alkyne
bearing molecules.( Prante,2009) (Scheme 2. 15).
OAc O H
� -0 -� -� Cl;ck ,ea<>on Hfl
AR2��N3--- H�;�N3 0