Protein Modification by Strain-Promoted Alkyne–Nitrone Cycloaddition

The bioorthogonal chemical reporter strategy is emerging as a versatile method for the labeling of biomolecules, such as nucleic acids, lipids, carbohydrates, and proteins.1 In this approach, an abiotic chemical functionality (reporter) is incorporated into a target biomolecule and can then react with a complementary bioorthogonal functional group linked to one of a diverse set of probes.


General procedure for cycloaddition of 2 and 4a-f leading to isoxazolines 5a-f.
To a solution of 2 (0.1 mmol) in a 1:1 mixture of CH 3 CN and H 2 O (2 mL) was added 4a-f (0.1 mmol). The mixture was stirred at room temperature until TLC analysis indicated complete consumption of starting materials. The reaction mixture was concentrated in vacuo, and the residue was purified by column chromatography on silica gel.

NMR experiments
Stock solution A: alkyne 2 (79 mg, 0.36 mmol) was dissolved in a mixture of CD 3  Kinetics of the reaction of 2 with nitrone 4a (Table 1, entry 1) were determined by the measuring the decrease of the integral of the signal caused by the nitrone methyl groups, with the integral of the acetonitrile solvent-peak as internal standard. A starting value for the integral of the methyl signals was estimated, due to the fact that cycloaddition had already proceeded significantly by the time of the first measurement. The conversion was determined by dividing the combined integrals of the methyl groups of 5a and 6a by the estimated starting value.
For the reaction of 2 with nitrone 4b (Table 1, entry 2), kinetics were determined by integration of the methyl peaks of the nitrone in starting material and product. The sum of these two integrals was taken as internal standard, due to the fact that the acetonitrile peak was not distinct from other signals. Now, conversion was determined by the relative increase of methyl signals of product 5 and 6, compared to the sum of both signals.
The reaction of 2 with nitrone 4c was too fast to follow by NMR. By the time of the first measurement, the reaction had already reached such a conversion that no reliable kinetic plot could be made. Therefore only a rough estimate was made based on the first measurement.
From the conversion plots thus obtained, the second order rate plots were calculated according to equation (1).
(1) with k = 2 nd order rate constant (M -1 s -1 ), t = reaction time (s), [A] 0 = the initial concentration of substrate A (mmol/mL), [B] 0 = the initial concentration of substrate B (mmol/mL) and [P] = the concentration of product (mmol/mL). As an example, the logarithmic plot of the reaction between nitrone 4b and 2 is shown in Graph S1. Graph S1. Logarithmic plot of the reaction of nitrone 4b with dibenzocyclooctyne 2.

UV-experiments
Stock solution A: alkyne 2, 2b or 3 (110 mg, 0.5 mmol) was dissolved in CH 3  Next, UV-absorption was measured at preset time intervals (in general every five seconds) at 304 nm. The graphs thus obtained showed a decrease of the absorption over time, sloping to a final absorption that was subtracted from the measured absorptions to obtain a relative absorption. To determine conversion, the relative absorption was divided by the difference between the maximal and the minimal absorption. Since the reactions already proceeded before measurement of the first absorption, the maximum absorption was adjusted to an estimated starting point.
From the conversion plots thus obtained, the second order rate plots were calculated according to equation (1).
In Graph S2 the logarithmic plot of nitrone 4e with dibenzocyclooctyne 2 is shown.
Graph S2. Logarithmic plot of the reaction between dibenzocyclooctyne 2 and nitrone 4e. In Graph S3 the logarithmic plots of the reaction of nitrone 4f with dibenzocyclooctyne 2b [5] in different solvent mixtures is visualized. The graph clearly shows an increase of reaction rate upon increase of the polarity of the solvent mixture. When the reaction was performed in the same solvent mixture but at different concentration and with a different ratio between the two reactants (

2,4,7,8,9-Penta-O-acetyl-N-(hydroxyacetyl)-α-D-neuraminic acid methyl ester (G)
To the solution of F (40 mg, 0.06 mmol) in EtOH was added Pd-C (10 wt. % on activated carbon, 5 mg). After which H 2 was bubbled through the solution for 1h followed by stirring under an H 2 atmosphere for 12 h. The reaction mixture was filtered and the mixture was evaporated in vacuo.
Subsequently, 4-MeOPhSH (7.5 mg, 0.053 mmol) was added to give a white cloudy solution, and the new mixture was stirred for 1 h at room temperature, until the reaction mixture turned colourless. p-Anisidine (5.0 mg, 0.040 mmol) was then added and the solution was stirred until dissolved completely (15 min). N-methylhydroxylamine hydrochloride (1.5 mg, 0.018 mmol) and 2