Azide-based ligation reactions

As an alternative, azides have been shown to be especially valuable reporters for bioorthogonal chemistry. In contrary to aldehydes, ketones or thiols, azides do not occur in biological systems. Due to their small size, azides only cause minimal changes in a molecule which is advantageous to create biomolecules that differ merely a little in structure. They can readily be introduced via nucleophilic replacement of a leaving group with an azide ion or via diazo transfer. Being only weak electrophiles, azides are inert to nucleophilic attacks and thus suited for biological applications. Nevertheless, they react readily with phosphines as well as alkynes and are thus suitable reporters for the Staudinger reaction and the azide-alkyne cycloaddition (AAC).

Staudinger ligation

Scheme 8: Non-traceless version of the Staudinger Ligation.

In 1919 STAUDINGER and MEYER reported the reaction of an azide and triphenylphospine forming an iminophosphorane intermediate under the release of nitrogen.^[55] In aqueous media, the reaction is followed by hydrolysis to an amine and triphenylphosphine oxide; the covalent bond between the two molecules is cleaved, thus the reaction is not suitable as a ligation reaction. A modification of this classical Staudinger reaction, developed by BERTOZZI

and SAXON in 2000 allowed the use of this chemistry as a ligation reaction (Scheme 8).^[20,44] By introducing a phosphine bearing an electrophilic trap, the negatively charged nitrogen of the aza-yilide intermediate is trapped by the ester group. The amide bond formed in this reaction is stable in aqueous environment and links the two molecules even after hydrolysis of the P-N bond. This non-traceless Staudinger Ligation fulfills all necessary criteria for the application as a chemoselective, bioorthogonal ligation reaction. With both moieties being abiotic reporters, the method can even be applied in living systems. Furthermore, the reaction can proceed quantitatively, at room temperature and at reasonable speed (up to k=3.8 x 10^-3 M^-1s^-1 CH3CN/KH2PO4: 1/1).^[56] As kinetics can depend on the solvent, it is difficult to compare reaction rates; thus one should pay close attention to the solvent when comparing values

2.4. Bioorthogonal ligation reactions for MGE except DAinv reactions

resulting from different studies. An even more elegant version is the traceless Staudinger ligation (simultaneously published by the groups of BERTOZZI and RAINES) in which the phosphine oxide is eliminated from the final product through hydrolysis.^[57-58] This is an advantage if the ligation is used for a synthetic application, in which a residual phosphine oxide is not desirable. Limitations of the Staudinger Ligation are its rather slow reaction kinetics and the resulting high concentrations of phosphine that are needed. Furthermore, the Staudinger Ligation is limited by the stability of the phosphines. By increasing its nucelophilicity, in order to accelerate the reaction, one also increases the probability for oxidation by air, thus acceleration of the reaction remains challenging.^[59] Despite these limitations, the Staudinger Ligation has successfully been applied for MGE for mammalian as well as bacterial glycans.^[20,60]

Azide-alkyne cycloaddition (click reaction)

Another ligation reaction relying on azides is the 1,3-dipolar azide-alkyne cycloaddition (AAC).

The cycloaddition of azides and alkynes was reported by HUISGEN^[61] long before its potential as ligation reaction was recognized by SHARPLESS and MELDAL^[62], who could achieve the necessary acceleration of the reaction by catalysis with Cu(I).

Scheme 9: Copper-catalyzed (left) and strain-promoted (right) azide-alkyne cycloaddition.

Using alkynes (AAC) rather than phosphines (Staudinger Ligation) as ligation partner for the azide, the reaction rate of the ligation was significantly improved. While the addition of copper accelerated the reaction and enabled the performance at room temperature, copper is toxic for living systems (due to the production of reactive oxygen species and inhibition of enzymes), thus application in living cells is limited. To avoid copper toxicity, strained alkynes can be applied for an accelerated Cu-free click reaction. Building on findings that showed that cyclooctynes react explosively with azides^[63] a cyclooctyne derivative was developed to label azides in cellular glycans. However, the first generation of cyclooctynes (OCT) react relatively slow (k=0.0012M^-1s^-1, CD3CN).^[24] To improve the reactivity, several substituted cyclooctynes (Figure 4) were synthesized and evaluated.

Figure 4: Selected strained alkyne reagents.

2.4. Bioorthogonal ligation reactions for MGE except DAinv reactions The introduction of two fluorine atoms was synthetically challenging, but resulted in a difluorocyclooctyne (DIFO) which has accelerated kinetics (k=0.076 M^-1s^-1, CD3CN)^[64]. Further improvements in the cyclooctyne development led to dibenzocyclooctyne DIBO (k=0.17 M^-1s^-1, MeOH; k=2.3 M^-1s^-1, H2O/CH3CN)^[25] and aza-dibenzocyclooctyne DIBAC (k=0.31 M^-1s^-1, CD3OD)^[65], which are easier to synthesize and react quite well. Another strained alkyne reagent is the 3,3,6,6-tetramethyl-thiacycloalkyne TMTH that is highly reactive (k=4.0 M^-1s^-1, CD3CN) which has so far not been conjugated to a dye.^[66] Both versions of the AAC have successfully been employed for metabolic glycoengineering with azide as well as alkyne modified monosaccharides (Figure 5). The azide reporter has been attached to peracetylated mannosamine (Ac4ManNAz)^[20], galactosamine (Ac4GalNAz)^[44], glucosamine (Ac4GlcNAz^[44] and Ac36GlcNAc^[48]) and fucose (Ac4Fuc6Az)^[22-23]. Recently, inositol derivatives carrying an azido group were applied to label glycosylphosphatidylinositol anchored proteins.^[67] As ligation reactions, the Staudinger ligation or the AAC can be applied in combination with the azide modified sugars. The azide modification is superior to a terminal alkyne, as it allows the performance of the ligation reaction with a strained alkyne (attached to a probe) to avoid the toxicity of the copper. A cyclooctyne attached to a hexosamine is expected to be too bulky to be accepted by the bioenzymatic machinery. Consequently, the ligation of these alkynes has to be copper catalyzed. A way to circumvent the limitation of size is to attach the residue directly to the sialic acid rather than its precursor mannosamine. Next to small terminal alkynes (SiaNAl and Neu5Proc)^[68-70] also the sterically demanding BCN (BCNSia)^[71] was thus successfully used to modify sugars. Alternatively, 9-Az-NeuAc was used to perform strain-promoted azide-alkyne cycloaddition (SPAAC).^[72] So far only terminal alkynes attached as amides (Ac4ManNAlk, Ac4GlcNAlk, Ac4GalNAlk)^[21,73] or carbamates (Ac4ManPoc, Ac4GlcPoc, Ac4GalPoc)^[49] have been attached to hexosamines or fucose (Ac4Fuc6Alk)^[23,73] and were incorporated into glycoconjugates.

In cooperation with Prof. SCHERER we were able to apply GalNAz for the visualization of cupulin in zebrafish. By injecting the modified sugar and monitoring it via the copper catalyzed AAC we could show that the cupula, that is important for the function of the ear, is renewed regularly.

(manuscript in preparation and ^[74])

2.4. Bioorthogonal ligation reactions for MGE except DAinv reactions

Figure 5: Unnatural sugar derivatives modified with reporter groups for click chemistry. The depicted abbreviations correspond to the names used in the original publications and are thus not uniform.

The development and application of azide based ligation reactions has greatly advanced the ligation reaction field. To further expand the panel of ligation reactions, an azide-independent ligation reaction would be advantageous, as it allows the combination of two orthogonal ligation reactions.