Team:Bielefeld-CeBiTec/Results/toolbox/fusing

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: Proton nuclear magnetic resonance (¹H 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 ¹H 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	0.05	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 the 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: ¹H 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 ¹H 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 protection 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 over 80 ppm correspond to the estimated spectrum of NBT‑CN. The peak at 77 ppm shows the carbon atom of the used solvent - deuterated chlorofom.

The ¹³C NMR spectrum of the pure product shows that the nucleophilic substitution was successful (see figure 9). The peak at 77 ppm corresponds 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. The signal at 112 pm is here deciding. It shows that the chlorine atom was successful substituted by a cyano group. Additionally we analyzed the NBT‑CN by proton NMR (see figure 10).

Figure 10: The ¹H NMR analysis result of the product of the nucleophilic substitution.
All peaks over 8 ppm correspond to the estimated spectrum of NBT‑CN.

The three signals over 8 ppm show the three hydrogen atoms of the aromatic ring of NBT-CN. The other signals originate from excessive water, ethyl acetate and acetonitrile.

Reduction of the nitro group to an amino group

The product of the nucleophilic substitution was used for the reduction of the nitro group to an amino group. Therefore, we used iron and acetic acid to create an excess of protons. After oxidation of the iron by the oxygen of the nitro group followed by the reduction of the nitro group to an amino group we got 1.5 g of ACBT. The proton NMR shows that the nucleophilic substitution was successful (see figure 10).

Figure 10: The ¹H NMR analysis result of the product of the reduction of the nitro group of NBT-CN.
Despite of the impurities, you can see all peaks of the hydrogen atoms of ACBT.

Compared to the proton NMR of the NBT-CN (see figure 9), there is now an additional peak at 5.7 ppm which originates from the amino group. Additionally, you can see the hydrogen atoms of several impurities with resonance frequencies under 5.7 ppm like ethyl acetate, water and byproducts of the reduction with iron and acetic acid.

Coupling Reaction of ACBT and N‑Fmoc‑aspartic acid‑OAll

For this reaction, we used the method of Yuan et al. (2016). They used it to synthesize (D‑Cys‑Lys‑CBT)₂ which in turn was used to build Cyclic D-Luciferin Nanoparticles. This method enables the coupling of ACBT with a carboxyl group. We needed several attempts to couple successfully ACBT to the carboxy group of the side chain of aspartic acid. Due to the high reactivity of the previous reaction with iron and acetic acid, the crude product of the reduction of the nitro group of NBT-CN to the amino group of ACBT contains different impurities. For the first attempt, we tried to use N‑Boc‑aspartic acid‑OBzl and the crude product as reagen. Figure 11 shows the proton NMR of the product.

Figure 11: The ¹H NMR analysis result of the product of the first attempt of the coupling reaction of ACBT and aspartic acid.
There are no peaks corresponding to the resonance frequencies of the hydrogen bonds of the ACBT. You can only see signals for the hydrogen atoms of the protected aspartic acid.

The peaks at 1.2 ppm, 2.0 ppm and 4.0 ppm are originating from excessive ethyl acetate. All other peaks correspond to the resonance frequencies of the N‑Boc‑aspartic acid‑OBzl. Thus, the first attempt of the coupling reaction failed. After purification of the crude product of the reduction reaction by column chromatography we got with N‑Fmoc‑aspartic acid‑OAll following liquid chromatography‑mass spectroscopy (LC‑MS) signal (figure 12).

Figure 12: The LC-MS result of the product of the second attempt of the coupling reaction of ACBT and aspartic acid
The peak at the position 553.15 shows that the coupling reaction was successful.

With the mass-to-charge ratio, we have got with the signal at the position 553.15 a positive signal for N‑Fmoc‑N^γ‑cyanobenzothiazolyl‑aspartic acid‑OAll which has a molecular weight of 552.61 g mol^‑1.

Removing Fluorenylmethyloxycarbonyl Fmoc and OAll

According to Goodman et al., (2004), we chose morpholine as base to remove the OAll protection group. Tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) serves as catalyst. The applied reactants and solvents for removing the protecting groups Fmoc and OAll are listed in table 6.

Table 6: List of used reactants and solvents for the deprotection of N‑Fmoc‑N^γ‑cyanobenzothiazolyl‑asparagine‑OAll.

No.	Reagent	MW / g L^-1	eq.	weight / g	n / mmol
1	Fmoc-CBT-asparagine-OAll	552.61	1.00	1.40	2.53
2	Tetrakis(triphenylphosphine)palladium(0)	1,155.59	0.10	0.29	0.25
3	Morpholine	87.10	3.00	0.65	7.59

	Solvent	Volume & mL
4	THF	50

Normally the acidity of morpholine with a pK_a of 8.36 is not enough to remove the protecting group Fmoc. But with the 3x equivalent of morpholine to the Fmoc‑CBT‑asparagine‑OAll and using Pd(PPh³)⁴ it is possible to remove even Fmoc resulting in the free variant or the morpholine salt of the amino acid. In our case we got both the free amino acid and the morpholine salt of CBT‑asparagine.

References

Goodman, M. Toniolo, C., and Moroder, L. (2004). Methods of Organic Chemistry: Synthesis of Peptide and Peptidomimetics 4th ed. (Thieme). 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.