Final Report Summary - MOLFACTORY (Towards a ‘molecular factory’: Processive sequence-selective synthesis with a synthetic molecular machine)
The aim of this project is to construct a synthetic molecular machine that is capable of performing a complex task - chemical synthesis - far beyond the current state-of-the-art. Our target is to mimic translation, the process through which protein is synthesized on the mRNA template by the ribosome. To achieve this ambitious task we propose to utilise a [2]rotaxane in which the macrocycle component acts as both a catalyst and a molecular transporter, abstracting bulky aromatic amino acid substituents from a sequence-specific ‘thread’ and transporting them in turn to the next amino acid fragment before mediating the formation of a new amide bond between them. The design is such that the macrocycle is forced to approach each amino acid in sequence, and is unable to pass until the amino acid unit is transferred, imparting sequential integrity to the oligopeptide synthesis. The mechanically interlocked nature of the rotaxane ensures processivity during the machine’s operation. Although biology uses threaded molecular architectures to transfer chemical information during sequence-specific oligomer and polymer assembly, such effects are yet to be improved in artificial small-molecule systems.
The proposed artificial molecular machine for sequence-specific peptide synthesis incorporates several key components that have analogues in natural systems: a thread carries reactive building blocks in a specific sequence that ensures the monomer units are assembled in the required order (the role played by mRNA in protein synthesis by the ribosome), and a macrocycle that has the same function as the clamp in DNA polymerase III. The macrocycle bears the catalytic group for removing the amino acid from the thread which fulfils the role of the aminoacyl tRNA synthetase active site in biology. In order to report the outcome of the operation, a dye molecule is planned to be tethered at the end of the track through ester linkage which is expected to change fluorescence intensity upon removal by the machine arm.
The synthetic route towards the macrocycle and sub-components required for the sequence specific peptide synthesis machine (stoppers, aminoacid barriers, catalytic unit) was optimized and the chemical structure of the backbone of the machine was determined. Alkyne and azide functionalized tyrosine barriers (each having different amino acids) were synthesized and sequential copper-catalysed Huisgen cycloaddition and amide coupling reactions were performed. In order to increase the UV-activity of the products and thereby facilitate the monitoring of the progress of the reactions and operation, model nitrophenyl derivatives were used as the ester load on the tyrosine moiety. ‘Active metal template’ strategy was used successfully to assemble rotaxanes having one or more amino ester barriers and the products were characterized by NMR, High Resolution Mass Spectrometry. Various catalytic units (4-N-methylaminomethylbenzamide, 4H-1,2,4-triazole) on the macrocycle arm were screened in order to achieve efficient acyl transfer reaction during the operation. The desired conversion with the catalyst was verified with model systems as well as rotaxane systems with model esters. It was also demonstrated that overreaching to the ester on the remoter tyrosine did not occur, indicating that the synthesis was sequence specific. Operation conditions were screened by taking stability of the barriers and the machine into consideration (machine activation by removal of the protecting groups followed by operation, choice of acid and base, reaction temperature and duration were screened). Attaching a dye molecule to the rotaxane was attempted in order to monitor the operation using single molecule fluorescence techniques.
The proposed artificial molecular machine for sequence-specific peptide synthesis incorporates several key components that have analogues in natural systems: a thread carries reactive building blocks in a specific sequence that ensures the monomer units are assembled in the required order (the role played by mRNA in protein synthesis by the ribosome), and a macrocycle that has the same function as the clamp in DNA polymerase III. The macrocycle bears the catalytic group for removing the amino acid from the thread which fulfils the role of the aminoacyl tRNA synthetase active site in biology. In order to report the outcome of the operation, a dye molecule is planned to be tethered at the end of the track through ester linkage which is expected to change fluorescence intensity upon removal by the machine arm.
The synthetic route towards the macrocycle and sub-components required for the sequence specific peptide synthesis machine (stoppers, aminoacid barriers, catalytic unit) was optimized and the chemical structure of the backbone of the machine was determined. Alkyne and azide functionalized tyrosine barriers (each having different amino acids) were synthesized and sequential copper-catalysed Huisgen cycloaddition and amide coupling reactions were performed. In order to increase the UV-activity of the products and thereby facilitate the monitoring of the progress of the reactions and operation, model nitrophenyl derivatives were used as the ester load on the tyrosine moiety. ‘Active metal template’ strategy was used successfully to assemble rotaxanes having one or more amino ester barriers and the products were characterized by NMR, High Resolution Mass Spectrometry. Various catalytic units (4-N-methylaminomethylbenzamide, 4H-1,2,4-triazole) on the macrocycle arm were screened in order to achieve efficient acyl transfer reaction during the operation. The desired conversion with the catalyst was verified with model systems as well as rotaxane systems with model esters. It was also demonstrated that overreaching to the ester on the remoter tyrosine did not occur, indicating that the synthesis was sequence specific. Operation conditions were screened by taking stability of the barriers and the machine into consideration (machine activation by removal of the protecting groups followed by operation, choice of acid and base, reaction temperature and duration were screened). Attaching a dye molecule to the rotaxane was attempted in order to monitor the operation using single molecule fluorescence techniques.