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We used a modified version of the method of Nguyen et al. (2011) to produce N<sup>ε</sup>-L-cysteinyl-L-lysine. To ensure a specific reaction between the amino group of the side chain of lysine (see figure 1) and the hydroxide group of cysteine (see figure 1) in a directed manner, so called protecting groups are introduced. Commonly used groups are tert-butyloxycarbonyl (Boc), methyl ester and triphenylmethane (Trt). They bind reversible to the corresponding groups and can be easily removed by acids and bases after the specific reaction happened. The first step of the synthesis is a coupling reaction of N-Boc-L-lysine-O-methyl ester and N Boc L cysteine-S-Trt. Due to the protected functional groups, only the unprotected amino group of N-Boc-L-lysine-O-methyl ester and the unprotected hydroxide group of the N Boc L cysteine-S-Trt can react with each other. The result is N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester. After removing the protecting groups with lithium oxide, triethylsilane and fluoroacetic acid Nε-L-cysteinyl-L-lysine trifluoroacetic acid salt is left. Figure 1 shows the schematic reaction.
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We used a modified version of the method of Nguyen et al. (2011) to produce N<sup>ε</sup>-L-cysteinyl-L-lysine. To ensure a specific reaction between the amino group of the side chain of lysine (see figure 1) and the hydroxide group of cysteine (see figure 1) in a directed manner, so called protecting groups are introduced. Commonly used groups are tert-butyloxycarbonyl (Boc), methyl ester and triphenylmethane (Trt). They bind reversible to the corresponding groups and can be easily removed by acids and bases after the specific reaction happened. The first step of the synthesis is a coupling reaction of N-Boc-L-lysine-O-methyl ester and N Boc L cysteine-S-Trt. Due to the protected functional groups, only the unprotected amino group of N-Boc-L-lysine-O-methyl ester and the unprotected hydroxide group of the N Boc L cysteine-S-Trt can react with each other. The result is N-Boc-L-lysine[N<sup>ε</sup>-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester. After removing the protecting groups with lithium oxide, triethylsilane and fluoroacetic acid N<sup>ε</sup>-L-cysteinyl-L-lysine trifluoroacetic acid salt is left. Figure 1 shows the schematic reaction.
 
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<h3>Coupling reaction of N-Boc-L-lysine-O-methyl ester and N-Boc-L-cysteine-S-Trt</h3>
 
<h3>Coupling reaction of N-Boc-L-lysine-O-methyl ester and N-Boc-L-cysteine-S-Trt</h3>

Revision as of 16:43, 31 August 2017

Fusing

Synthesis of Nε-L-cysteinyl-L-lysine

We used a modified version of the method of Nguyen et al. (2011) to produce Nε-L-cysteinyl-L-lysine. To ensure a specific reaction between the amino group of the side chain of lysine (see figure 1) and the hydroxide group of cysteine (see figure 1) in a directed manner, so called protecting groups are introduced. Commonly used groups are tert-butyloxycarbonyl (Boc), methyl ester and triphenylmethane (Trt). They bind reversible to the corresponding groups and can be easily removed by acids and bases after the specific reaction happened. The first step of the synthesis is a coupling reaction of N-Boc-L-lysine-O-methyl ester and N Boc L cysteine-S-Trt. Due to the protected functional groups, only the unprotected amino group of N-Boc-L-lysine-O-methyl ester and the unprotected hydroxide group of the N Boc L cysteine-S-Trt can react with each other. The result is N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester. After removing the protecting groups with lithium oxide, triethylsilane and fluoroacetic acid Nε-L-cysteinyl-L-lysine trifluoroacetic acid salt is left. Figure 1 shows the schematic reaction.

Figure 1: Schematic reaction of the synthesis of Nε-L-cysteinyl-L-lysine fluoroacetatic acid salt (Nguyen et al., 2011).
The unprotected hydroxide group of the cysteine (red) and the unprotected amino group of the lysine (green) are highlighted.

We synthesized Nε-L-cysteinyl-L-lysine in two batches to ensure that the method of Nguyen et al. (2001) is successful. For the first batch, we used dry dimethylformamide (DMF) as solvent for the coupling reaction as described by Nguyen et al. (2011). Due to low yield using DMF compared to Nguyen et al., we used less reactive tetrahydrofuran (THF) for the second batch.

Coupling reaction of N-Boc-L-lysine-O-methyl ester and N-Boc-L-cysteine-S-Trt

Table 1 shows the used quantity of reactants and solvents for both batches.

Table 1: List of used reactants and solvents for the coupling.
In both batches, we used the same quantity of reactants and solvents for the coupling reaction.

Figure 2: Result of the TLC analysis after the coupling reaction.
A: N-Boc-L-lysine-O-methyl ester; B: N Boc L cysteine-S-Trt; C: N-Boc-L-lysine-O-methyl ester, N Boc L cysteine-S-Trt and the reaction mixture after the coupling reaction; D: the reaction mixture after the coupling reaction.

The thin layer chromatography (TLC) analysis of the reaction mixture shows that after the coupling reaction no N-Boc-L-lysine-O-methyl ester was left (see figure 2). This indicates that the N-Boc-L-lysine-O-methyl ester completely reacted. The two spots on the top of C and D are the product – the N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester (lower spot) – and a byproduct of the reaction (upper spot).

Figure 3: Nuclear magnetic resonance (NMR) analysis result for the purified reaction mixture after the coupling reaction.
The signals for the hydrogen bonds of the protecting groups were highlighted because they are characteristic for the estimated product – N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester.

The NMR analysis of the purified reaction mixture of the coupling reaction shows that the hydrogen atoms of all protecting groups are present (see figure 3). The Tritylphenylmethane (Trt) at 7.2 ppm is part of the N-Boc-L-cysteine-S-Trt and the methyl ester at 3.6 ppm is originating from the N-Boc-L-lysine-O-methyl ester. The tert-Butyloxycarbonyl protecting group is part of both educts. In this reaction, should be no protecting groups split off so that you can see here the NMR analysis for N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester.

Removing the methyl ester of the N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester

Table 2 shows the used quantity of reactants and solvents for both batches.

Table 2: List of used reactants and solvents for the reaction to remove methyl ester of the first and the second batch.

Figure 4: Result of the TLC analysis after removing the methyl ester.
KC2: the reaction mixture after the coupling reaction; KC3: the reaction mixture after removing the methyl ester.

After removing the methyl ester, the product is more polar than before. The result is that the N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)] is not soluble in the EtOAc:PE solution. The dark spot at the TLC plate for the sample KC3 is the N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)] and the lighter spot is the removed methyl ester (see figure 4).

Removing tert-Butyloxycarbonyl protecting group (Boc) and Triphenylmethane (Trt) of the N-Boc-L-lysine[Nε-(N-Boc-L-cysteine-S-Trt)]

Table 3 shows the used quantity of reactants and solvents for both batches.

Table 3: List of used reactants and solvents for the reaction to remove Boc and Trt of the first and the second batch.

Figure 5: NMR analysis result for the purified Nε-L-cysteinyl-L-lysine trifluoroacetatic acid salt.
All peaks of compounds with hydrogen atoms of the Nε-L-cysteinyl-L-lysine were highlighted because they are characteristic for this molecule.

The NMR analysis shows that all estimated hydrogen atoms are present and that the synthesis was successful (see figure 5).
In the first batch, we got 400 mg of Nε-L-cysteinyl-L-lysine trifluoroacetic acid salt and in the second batch 500 mg. This correspond to 0.84 mmol for the first batch and 1.05 mmol for the second batch. This equals to the half of the yield of Nguyen et al. (2011) with 900 mg and 1.89 mmol.


Nguyen, D.P., Elliott, T., Holt, M., Muir, T.W., Chin, J.W., 2011. Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation. J. Am. Chem. Soc. 133, 11418–11421. doi:10.1021/ja203111c