Short summary
Terminus independent specific fusion of two or more peptides is a major challenge in synthetic biology and beyond.
Inspired by the highly specific condensation reaction of D‑luciferin from the firefly Photinus pyralis, we came up
with a sophisticated solution, combining organic chemistry, computational modelling, and molecular biology. Based on
our own design, we synthesized the novel synthetic amino acid Nγ‑cyanobenzothiazolyl‑L‑asparagine‑(CBT‑asparagine).
The cyano group of CBT‑asparagine undergoes a condensation reaction with the 1,2‑aminothiol group of Nε‑L‑cysteinyl‑L‑lysine (CL).
Through in silico simulation, we predicted different aminoacyl tRNA synthetase sequences to incorporate CBT‑asparagine into
proteins of interest. This system offers a new way for the production of fusion proteins and polymerized polypeptides.
Searching for Interesting Non-Canonical Amino Acids
During our search for interesting non‑canonical amino acids which we could use for our project, we found CL. It was first synthesized
and described by Nguyen et al. (2011). It is proposed that CL enables the highly specific binding between peptides and ligands containing
a cyanobenzothiazole residue. CL itself consists of L‑cysteine coupled by its carboxy group to the amino group of the side chain of
L‑lysine. As a result, it contains a free 1,2‑aminothiol group at its side chain. According to the highly specific condensation reaction
of 1,2‑aminothiols and cyanobenzothiazole‑derivatives (CBT-derivatives), which is common for the biosynthesis of D‑Luciferin (see Figure
1), the substrate of the firefly luciferase of P. pyralis, CL can undergo the same condensation reaction with CBT‑derivatives (see
Figure 2).
So far, CL was used to label proteins with CBT‑derivatives bound to fluorescent chemicals and proteins. Inspired by the rapid and
highly specific condensation reaction which takes place under physiological conditions, we thought about a possibility to synthesize
a novel amino acid which enables the highly specific binding between peptides using CBT as a side chain and CL as the counterpart.
Designing a Novel Amino Acid
To synthesize a novel amino acid, we needed the right reagents for the reaction. First, it was important to find a CBT‑derivative
which could be coupled to the side chain of a canonical amino acid. To use common methods of peptide synthesis, we needed two
educts one containing a free carboxy group and one containing a free amino group. There are different canonical amino acids containing
one of these two groups at their side chains. All canonical eligible amino acids with a free amino group at their side chain are lysine,
asparagine and glutamine. Because of the long carbon chain at the side chain of lysine it would be too difficult to generate a well
working aminoacyl‑tRNA synthetase for a novel amino acid consisting of lysine and a CBT‑derivative. To get an amino acid as small as
possible containing a CBT-derivative at its side chain, asparagine and its derivative aspartic acid which contains a free carboxy group
at its side chain were the best choices for an educt for the synthesis of a novel amino acid. As CBT-derivative we chose
6‑amino‑2‑cyanobenzothiazole (ACBT) which has a free amino group at its benzene ring (see Figure 3). Its free amino group ACBT
requires an aspartic acid for the coupling reaction to synthesize the novel amino acid.
Synthesizing a Novel Amino Acid
Due to the high price of ACBT, we synthesized it first on ourselves using 2‑chloro‑6‑nitrobenzothiazole (Cl-NBT) as educt. According
to Hauser et al. (2016), we produced ACBT by the nucleophilic substitution of the chlorine atom with a cyano group and the reduction of
the nitro group to an amino group. Figure 4 shows the schematic synthesis of ACBT with Cl‑NBT as the educt.
With ACBT and N‑Fmoc‑aspartic acid‑OAllyl ester we could synthesize the novel amino acid CBT‑asparagine.
Figure 5 and 6 show the two steps of the synthesis.
With the novel amino acid CBT‑asparagine and the CL we provide a new way of rapid highly specific binding of peptides and enzymes
under physiological conditions. First
in vitro tests show that the condensation reaction
between CBT‑asparagine and CL (see Figure 7) takes place.
Modeling New Aminoacyl-tRNA Synthetases by in silico Simulation
Additionally, we generated several new aminoacyl‑tRNA synthetases based on the tyrosyl‑tRNA synthetase of
Methanococcus jannaschii
using the protein design software ROSETTA. All of the three tested synthetases were able to incorporate the CBT‑asparagine (for
further information visit our
modeling page .
Conclusion
We provide a new way to produce fusion proteins and polymerized peptides using the terminus independent, rapid and highly specific
binding ability of CL and the novel amino acid CBT‑asparagine. With this method, it is possible to fuse peptides and enzymes using
an environmentally friendly condensation reaction which is inspired by natural systems.
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.
Liang, G., Ren, H., and Rao, J. (2010). A biocompatible condensation reaction for controlled assembly of nanostructures in living cells. Nat. Chem. 2: 54–60.
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.
