Difference between revisions of "Team:Bielefeld-CeBiTec/Results/toolbox/fusing"

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Fusing

Synthesis of N^ε-L-cysteinyl-L-lysine

We used a modified version of the method of Nguyen et al. (2011) to produce N^ε‑L‑cysteinyl‑L‑lysine. To ensure a specific reaction between the amino group of the side chain of lysine (see figure 1) and the carboxyl group of cysteine (see figure 1) in a selective manner; so called protecting groups are introduced. Commonly used protecting groups are tert‑butyloxycarbonyl (Boc), methyl ester and trityl (Trt). They bind reversible to the corresponding functionality and can be easily removed by acids and bases after the coupling 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 carboxyl group of the N‑Boc‑L‑cysteine‑S‑Trt can react with each other. The result is N‑Boc‑L‑lysine[N^ε‑(N‑Boc‑L‑cysteine‑S‑Trt)]‑6‑methyl ester. After removal of ester protection with lithium hydroxide and Boc and Trt with trifluoroacetic acid, N^ε-L-cysteinyl-L-lysine trifluoroacetic acid salt is obtained. Figure 1 shows the schematic reaction.

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

We synthesized N^ε‑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 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.

No.	Reagent	MW / g L^-1	eq.	weight / g	n / mmol
1	NBoc-L-lysine-O-methyl ester acetate salt	320.40	1.00	1.00	3.12
2	N-Boc-L-cysteine-S-Trt	463.60	1.50	2.14	4.62
3	DCC	206.33	1.50	0.95	4.62
4	DMAP	122.17	0.16	0.06	0.49

	Solvent	Volume & mL
5	DMF/THF	10

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[N^ε-(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[N^ε‑(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[N^ε‑(N‑Boc‑L‑cysteine‑S‑Trt)]‑6‑methyl ester.

Removing the methyl ester of the N‑Boc‑L‑lysine[N^ε‑(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.

	First batch	Second batch

No.	Reagent	MW / g L^-1	eq.	weight / g	n / mmol	MW / g L^-1	eq.	weight / g	n / mmol
1	N-Boc-L-lysine[N^ε-(N-Boc-L-cysteine-S-Trt)]-6-methyl ester	704.17	1.00	0.60	0.85	704.17	1.00	0.97	1.42
2	LiOH · H₂O	42.06	1.50	5.36 · 10^-2	1.28	42.06	1.50	0.09	2.13

	Solvent	Volume / mL	Volume / mL
3	THF:H₂O (3:1)	80	100

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[N^ε‑(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[N^ε‑(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[N^ε‑(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.

	First batch	Second batch

No.	Reagent	MW / g L^-1	eq.	weight / g	n / mmol	MW / g L^-1	eq.	weight / g	n / mmol
1	N-Boc-L-lysine[N^ε-(N-Boc-L-cysteine-S-Trt)]	691.67	1.00	0.68	0.98	7691.67	1.00	0.96	1.39
2	Triethylsilane	116.67	4.50	0.52	4.42	116.67	4.50	0.73	6.26

	Solvent	Volume / mL	Volume / mL
3	CH₂Cl₂	30	30

Figure 5: NMR analysis result for the purified N^ε‑L‑cysteinyl‑L‑lysine trifluoroacetatic acid salt.
All peaks of compounds with hydrogen atoms of the N^ε‑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 N^ε‑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 alal. (2011) with 900 mg and 1.89 mmol.

Synthesis of N^γ-cyanobenzothiazolyl-L-asparagine

The synthesis of the novel amino acid N^γ‑cyanobenzothiazolyl‑L‑asparagine (CBT‑asparagine) was a complete new challenge for us. Our idea was to couple the amino group of 6‑amino‑2‑cyanobenzothiazole (ACBT) with the carboxy group of the side chain of aspartic acid. Therefore, it is recommendable to use two different protective groups each bound to the N‑ and C‑terminus of the aspartic acid. Figure 6 and 7 show the schematic synthesis of CBT‑asparagine.

Figure 6: Schematic coupling reaction of ACBT and Fmoc‑asparagine‑OAll giving Fmoc‑CBT‑asparagine‑OAll as product.
The amino group of ACBT and the carboxy group of the side chain of aspartic acid get linked under water loss.

Figure 7: Schematic deprotection reaction of Fmoc‑CBT‑asparagine‑OAll.
After the coupling reaction, the deprotection removes the protecting groups to give the free amino acid or a salt of the amino acid regarding to the method.

Due to the high price of 6‑amino‑2‑cyanobenzothiazole (ACBT), we synthesized it on ourselves with 2‑chloro‑6‑nitrobenzothiazole (Cl‑NBT) as starting material following the procedure of (Hauser et al., 2016). Figure 8 shows the schematic synthesis of ACBT.

Figure 8: Schematic synthesis of ACBT. The synthesis contains two steps.
The first step (i) is the nucleophilic substitution of the chlorine atom with a cyano group and the second step (ii) is the reduction of the nitro group to an amino group.

First, we changed the chlorine atom of the Cl‑NBT for a cyano group by nucleophilic substitution resulting in 6‑nitrobenzothiazole‑2‑carbonitrile (NBT‑CN). The second step of the synthesis of ACBT is the reduction of the nitro group of the NCBT to an amino group using iron and acetic acid. The last step is troublesome. Due to the reactivity of the iron and the acetic acid, the reaction mixtures contains a large amount of impurities. These impurities can disturb the coupling reaction between ACBT and aspartic acid. After several attempts to get an ACBT with sufficient purity for the coupling reaction of the amino group of ACBT and the carboxy group of the side chain of aspartic acid we got following results.

Nucleophilic Substitution of the Chlorine Atom with a Cyano Group

The applied reactants and solvents for the nucleophilic substitution of the chlorine atom to the cyano group are listed in table 4.

Table 4: List of used reactants and solvents for the nucleophilic substitution of the chlorine atom to the cyano group.

No.	Reagent	MW / g L^-1	eq.	weight / g	n / mmol
1	2-Chloro-6-nitrobenzothiazole	214.63	1.00	5.00	23.29
2	1,4-diazabicyclo[2.2.2]octane	112.17	0.15	0.37	3.33
3	NaCN	49.01	1.05	1.20	24.48

	Solvent	Volume & mL
4	MeCN	500

Figure 9: The ¹³C NMR analysis result of the product of the nucleophilic substitution.
All peaks correspond to the estimated spectrum of NBT‑CN.

The ¹³C NMR spectrum of the pure product shows that the nucleophilic substitution was successful (see figure 9). The peak between 80 and 75 ppm correspond to the resonance frequency of deuterated chloroform, the chosen solvent for the ¹³C NMR analysis. The resonance frequency of the carbon of the cyano group is 112 ppm. The carbon atom at 141 ppm is part of the thiazole residue and the six carbon atoms at 118 ppm, 123 ppm, 126 ppm, ppm, 147 ppm and 155 ppm are originated from the benzene ring.

Reduction of the nitro group to an amino group

References

Hauser, J.R., Beard, H.A., Bayana, M.E., Jolley, K.E., Warriner, S.L., and Bon, R.S. (2016). Economical and scalable synthesis of 6-amino-2-cyanobenzothiazole. Beilstein J. Org. Chem. 12: 2019–2025.
Nguyen, D.P., Elliott, T., Holt, M., Muir, T.W., and 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.
Yuan, Y., Wang, F., Tang, W., Ding, Z., Wang, L., Liang, L., Zheng, Z., Zhang, H., and Liang, G. (2016). Intracellular Self-Assembly of Cyclic d -Luciferin Nanoparticles for Persistent Bioluminescence Imaging of Fatty Acid Amide Hydrolase. ACS Nano 10: 7147–7153.

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 			<p class="figure subtitle"><b> Figure 9: The <sup>13</sup>C NMR analysis result of the product of the nucleophilic substitution.</b><br>All peaks correspond to the estimated spectrum of NBT&#x2011;CN.</p>