Motivation

As the short chain length fucose derivative Ac₄Fuc6CH₂ (1) reacts poorly in the DAinv reaction, I investigated the nitrile imine cycloaddition, first reported by Huisgen et al.^[161], for its labeling. In the nitrile imine cycloaddition, also known as photo-click reaction, a highly reactive nitrile imine reacts with an alkene in a 1,3-dipolar cycloaddition forming a fluorescent pyrazoline (Scheme 12).

Scheme 12: Photo-click reaction. The nitrile imine can be generated from a tetrazole via UV light or by basic activation of a hydrazonoyl chloride.

Especially electron-deficient alkenes, for example acrylamide (k2 = 46 M^-1s^-1[162]), react fast in the nitrile imine cycloaddition.^[163] In this reaction, the highest occupied molecular orbital (HOMO) of the dipole (nitrile imine) overlaps with the LUMO of the dipolarophile (alkene), thus electron withdrawing groups (EWG) accelerate the reactivity of alkenes with nitrile imines (Figure 23B).^[164]

This is in contrast to the DAinv reaction, were the lowest unoccupied molecular orbital (LUMO) of the diene (tetrazine) interacts with the HOMO of the dienophile (alkene) (Figure 23A). This can be exploited for the application of fucose derivatives 1-3 where the short chain length derivative is expected to react fast, as the double bond is located close to the electron withdrawing ring oxygen and thus electron poor. In addition, the derivative with the short chain length is expected to be well incorporated. Therefore we expect Ac4Fuc6CH2 (1) to be clearly superior, compared to fucose derivatives 2 and 3, as it is likely to be incorporated well and should react fastest in the nitrile imine cycloaddition. This is in contrast to the DAinv reaction, where adverse effects for reactivity and incorporation efficiency occur. Alternatively to the electron poor alkenes, strained alkenes like norbornene^[165] and cyclopropene^{[162, 166]} can be used to accelerate the reaction, with trans cyclooctene^[165] and spiro[2.3]hex-1-ene^[167] being extremely fast.

44 4. Results

Figure 23: Molecular orbital diagram of (A) the DAinv reaction and (B) the nitrile imine cycloaddition.

As nitrile imines are highly reactive, they can react with a variety of molecules, like terminal and strained alkenes as well as alkynes. However, this reactivity also has its drawback, as it is responsible for side reactions that have been reported. Of these side reactions, mainly hydrolysis is observed.^{[168, 169]} In addition, nucleophile addition e.g. of amino acids^[170] as well as dimerization of the nitrile imine to the tetrazine^[171] were reported. Recently, the reaction with tryptophan was described^[172] and tetrazoles were used for photo-crosslinking^[173]. Nevertheless, these studies were all performed in absence of electron poor alkenes which still are the best reaction partners of nitrile imines. As long as a reactive dipolarophile is present these side reactions should be neglectable if the reaction conditions are chosen wisely and the high selectivity between reactive and unreactive alkenes is exploited.

Due to their high reactivity, especially in water,[171, 174, 175]

, nitrile imines are unstable and thus have to be generated in situ. To generate nitrile imines different reactions were reported:

Thermolysis of tetrazoles at 150 – 160°C upon nitrogen release and basic activation of hydrazonoyl-chlorides at room temperature were first reported in 1962.^[161] Basic activation of α-nitro phenylhydrazones^{[176, 177]} as well as photolysis of tetrazoles^[177] were also described in the early 1960s. The Lin lab reinvestigated this nitrile imine cycloaddition in 2008 and applied it as bioorthogonal ligation reaction. They used photo activation of 2,5-diaryl tetrazoles to label functionalized proteins[178, 179]

with the advantage of nitrogen release, which makes the reaction irreversible. Besides this light-induced decomposition of tetrazoles at around 300 nm the nitrile imine intermediates were generated through basic activation of hydrazonoyl chlorides for biological applications (Scheme 12).[163, 180]

In both cases the second step, the reaction with an alkene, is rate determining.^[146]

The photo activation enables spatial and temporal control as the reaction can be started at a defined time point and location. Further, stable pyrazoline products are formed which are fluorescent, enabling direct readout and imaging without a washing step. Theoretically, two

4. Results 45

isomeric pyrazolines can be formed: The 4- and the 5-substituted pyrazolines. In most reactions the 5-substituted pyrazoline is favored which can be explained by electronic effects.^[164] Using mild photo activation conditions only one regioisomer, the 5-substituted one, was detected,[171, 174, 181]

which simplifies analysis. Depending on the structure of the pyrazoline, it can oxidize to the corresponding pyrazole.^[171] All products yield in a covalent linkage between the alkene and the tetrazole, which is important for a bioorthogonal ligation reaction. The only byproduct is nontoxic nitrogen and even though UV light can be toxic to cells, short irradiation times (seconds to minutes) are sufficient for the tetrazole photolysis which minimizes the damage.[162, 182]

Nevertheless the Lin group developed several longer wave length activatable tetrazoles with naphthalene- and thiophene-based scaffolds (Figure 24).[162, 183]

The longest wave length (700 nm) was achieved using two photon absorption.^[184] Concerning the choice of tetrazole derivatives we decided to use simple ones as tuning the photo activation wavelength comes with a laborious synthesis while still relatively short wave lengths (405 nm) are needed for the activation. In addition, diaryl tetrazoles were several times successfully applied for biological applications, mainly for the labeling of proteins. The proteins were labeled by genetically encoding unnatural amino acids with different alkenes, including terminal alkenes[163, 179, 182]

, norbornene^[165,

180], cyclopropene^[162], and cyclooctene^[165]. In addition a tetrazole amino acid was site specifically incorporated into myoglobin^[185] and tetrazole-containing proteins were synthesized via native chemical ligation^[186]. Proteins were also unspecifically modified by linking them to tetrazoles^[178] or cyclopropenes^[166] before the labeling reaction. Using differently substituted methyl cyclopropenes the Prescher lab performed the nitrile imine cycloaddition and the DAinv reaction in the same experiment, showing their compatibility.^[166] Most publications analyze successful incorporation via Western blot analysis or mass spectrometry but microscopy of e.coli[163, 165, 179]

and mammalian cells[162, 182, 186]

was also performed. Besides these protein applications the nitrile imine cycloaddition was also investigated for the labeling of DNA^[187] and RNA^[188].

Figure 24: Selected tetrazoles and their activation wave lengths.[162, 182-184]

So far the reaction was not applied to label carbohydrates. Having the nitrile imine cycloaddition as new bioorthogonal ligation reaction for MGE would be advantageous as small modifications could be incorporated and a spatiotemporal control can be enabled. To establish a method to

46 4. Results

label metabolically engineered carbohydrates, mannosamine derivatives should be applied first as they are known to be well incorporated into membrane glycoconjugates.