Patent Application
20250074944
The present application claims priority to Korean Patent Applications No. 10-2023-0114636, filed 30 August, 2023, and 10-2024-0095918, filed 19 July, 2024, the entire contents of which is incorporated herein for all purposes by this reference.
The contents of the electronic sequence listing (DPH20240316US SEQ.xml; Size: 37,684 bytes; and Date of Creation: Oct. 15, 2024) is herein incorporated by reference in its entirety. The contents of the electronic sequence listing in no way introduces new matter into the specification.
The present invention relates to a peptide having a pharmacologically active motif using acylated tRNA and a ribosome catalyst on a cell-free system, and a method for producing the same.
Research using Flexizyme, a type of aminoacylating ribozyme, has utilized it as an accelerator to expand the candidate group of new drug substances by introducing various types of non-natural amino acids into the peptide. In particular, the strategy of producing macrocyclic peptides through chemical reactions of non-natural amino acids has attracted attention as a key element technology for new drug discovery. Macrocyclic peptides are cyclic peptides composed of about 10 amino acids and are attracting attention due to their high pharmacological effects and stability.
Meanwhile, three major strategies are adopted to introduce a cyclic motif into a peptide. First, there is the formation of a cyclic motif using an enzyme, second, there is the modification of a functional group through a chemical reaction after the introduction of a non-natural amino acid, and third, there is a combined reaction of an enzyme and a chemical reaction.
However, for all strategies, these designs are complex because they require substrates to be designed while considering various biochemical reactivity, and they do not guarantee a wide range of diversity expansion.
Therefore, research is needed on the synthesis of highly efficient ring-shaped motifs that ensure diversity expansion without complicating the process.
In addition, International Publication No. WO 2022/173627 A2 was disclosed as a prior art patent.
The purpose of the present invention is to provide a cell-free protein synthesis system in which non-natural amino acids having ester and hydrazino structures are sequentially introduced into a peptide, and a secondary reaction occurs between residues of the non-natural amino acids to form a square or octagonal structure, or various cyclic compounds, but not limited thereto.
Another purpose of the present invention is to provide a cell-free protein synthesis system in which a highly efficient cyclic motif can be quickly and easily obtained in vitro without going through a complex chemical process.
According to one aspect of the present invention, there is provided an acylated tRNA represented by Structural Formula 1 or Structural Formula 2 below:
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the acylated tRNA may be represented by the Structural Formula 1 and may comprise at least one selected from the group consisting of compounds below:
In addition, the acylated tRNA may be represented by the Structural Formula 2 and may comprise at least one selected from the group consisting of compounds below:
In addition, the acylated tRNA may represented by the Structural Formula 2 and comprises at least one selected from the group consisting of compounds below:
In addition, the acylated tRNA may be represented by the Structural Formula 2 and may comprises at least one selected from the group consisting of compounds below:
According to another aspect of the present invention, there is provided a cyclic compound represented by structural formula 3 below:
wherein X1 and X2 are each independently a hydrogen atom, a substituted or unsubstituted C1-C10 alkyl group, a substituted or unsubstituted C3-C7 cyclic alkyl group,
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the cyclic compound may comprise at least one selected from the group consisting of compounds below,
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the cyclic compound may comprise at least one selected from the group consisting of compounds below,
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the cyclic compound may comprise at least one selected from the group consisting of compounds below,
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
According to another aspect of the present invention, there is provided a method for cyclizing an unnatural substrate comprising a step of conjugating an acylated tRNA represented by Structural Formula 1 and an acylated tRNA represented by Structural Formula 2 in a translation reaction to produce a cyclic compound represented by structural formula 3.
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
The cell-free protein synthesis system of the present invention can obtain a peptide having a highly efficient cyclic motif without going through a complicated chemical process by using an in vitro protein translation system.
In addition, the cyclic motif of the protein synthesized in the cell-free protein synthesis system of the present invention can exhibit higher pharmacological activity than a peptide including a simple non-natural substrate.
In addition, the cyclic motif of the protein synthesized in the cell-free protein synthesis system of the present invention has a high chemical stability because it contains a hydrazinedione group, so it is not easily decomposed even in cells and can maintain its structure for a long time
Since these drawings are for reference in explaining exemplary embodiments of the present invention, the technical idea of the present invention should not be interpreted as limited to the attached drawings.
The arrow was used to indicate bands shifted due to an increase in size increase after acylation. The same reaction conditions were applied to tRNA acylation with the substrate.
Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings so that those skilled in the art can easily implement the present invention.
However, the following description is not intended to limit the present invention to specific embodiments, and in describing the present invention, if it is determined that a detailed description of related known technology may obscure the gist of the present invention, the detailed description will be omitted.
Terms used herein are merely used to describe specific embodiments. This is not intended to limit the present disclosure. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to indicate the presence of features, numbers, steps, operations, components, or a combination thereof described in the specification. Accordingly, the term should be understood as not excluding in advance the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof.
Hereinafter, acylated tRNA, introduction of pharmacologically active motif in peptide using ribosome catalysis and method of preparing same will be described in detail. However, this is presented as embodiments, and the present invention is not limited thereby, and the present invention is only defined by the scope of the claims to be described later.
According to one aspect of the present invention, there is provided an acylated tRNA represented by Structural Formula 1 or Structural Formula 2 below:
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the acylated tRNA may be represented by the Structural Formula 1 and may comprise at least one selected from the group consisting of compounds below:
In addition, the acylated tRNA may be represented by the Structural Formula 2 and may comprise at least one selected from the group consisting of compounds below:
In addition, the acylated tRNA may represented by the Structural Formula 2 and comprises at least one selected from the group consisting of compounds below:
In addition, the acylated tRNA may be represented by the Structural Formula 2 and may comprises at least one selected from the group consisting of compounds below:
According to another aspect of the present invention, there is provided a cyclic compound represented by structural formula 3 below:
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the cyclic compound may comprise at least one selected from the group consisting of compounds below,
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the cyclic compound may comprise at least one selected from the group consisting of compounds below,
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
In addition, the cyclic compound may comprise at least one selected from the group consisting of compounds below,
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
According to another aspect of the present invention, there is provided a method for cyclizing an unnatural substrate comprising a step of conjugating an acylated tRNA represented by Structural Formula 1 and an acylated tRNA represented by Structural Formula 2 in a translation reaction to produce a cyclic compound represented by structural formula 3.
a substituted or unsubstituted C6-C14 aryl group, a substituted or unsubstituted (C1-C8 alkyl) (C6-C14 aryl) group, a substituted or unsubstituted (C6-C14 aryl) (C1-C8 alkyl) group, an amino group, or an amine group represented as —NR′R″, or at least one of X1 and X2 are combined with a carbon in a main chain to form a substituted or unsubstituted C3-C6 ring, wherein R is a hydrogen atom or a C1-C5 alkyl group, and R′ and R″ are each independently a hydrogen atom, a substituted or unsubstituted C1-C8 alkyl group or a substituted or unsubstituted C6-C14 aryl group,
Hereinafter, the present invention will be described with reference to preferred embodiments. However, this is for illustrative purposes only and does not limit the scope of the present invention.
All materials were of the best grade commercially available and used without further purification. NMR solvents (DMSO-d6) were purchased from Sigma-Aldrich or TCI Chemicals. All the oligonucleotides used in this research were purchased from Integrated DNA Technologies (IDT), Bioneer, or Cosmogenetech and used as received according to manufacturer's guidelines.
General synthesis of substrates Preparation of diesters. The diester substrates were synthesized in the following ways: i) The inventors utilized a commercially available molecule containing a carboxylic acid and an ester substituted with R1 as a starting material or an anhydride that would yield an R1 ester upon hydrolysis, ii) Following the formulation of diverse R1 substituents, the inventors carried out esterification reactions to attach FLG for Fx-charging.
Preparation of hydrazine substrate. The hydrazine substrate was synthesized using a methyl lactate in four steps: i) replacement of hydroxyl to hydrazino group (triflation on the hydroxyl group followed by attachment of Boc-NHNH2), ii) conversion of ester to acid by base-catalyzed hydrolysis, iii) attachment of Boc-ABT, and iv) Boc deprotection from the β-nitrogen and ABT.
General synthetic procedure A: Formation of cyanomethyl ester. To a solution of carboxylic acid (1 eq) triethylamine (0.5 eq) and bromoacetonitrile (10 ml) were added and stirred overnight. After stirring for 16 h at room temperature, the reaction mixture was diluted with EtOAc and washed with HCl (1.0 M in water), NaHCO3 (4% (w/v) in water), brine, and dried over MgSO4. The organic phase was concentrated to provide the crude product.
General synthetic procedure B: Formation of dinitrobenzyl esters. To a solution of carboxylic acid (1 eq), dinitrobenzyl alcohol (1.2 eq), 4-dimethylaminopyridine (DMAP) (0.5 eq) and N-(3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC.HCl) (1.2 eq) were added in dichloromethane. After stirring for 16 h at room temperature, the reaction mixture was diluted with EtOAc and washed with HCl (1.0 M in water), NaHCO3 (4% (w/v) in water), brine, and dried over MgSO4. The organic phase was concentrated to provide the crude product.
General synthetic procedure C: Formation of 4-((2-aminoethyl)carbamoyl)benzyl thioates. To a solution of carboxylic acid (1.0 eq), tert-butyl 2-[4-(mercaptomethyl)benzamido]ethyl carbamate (Boc-ABT) (0.3 eq), 4-dimethylaminopyridine (DMAP) (0.5 eq) and N-(3-dimethylaminopropyl) -N′-ethylcarbodiimide hydrochloride (EDC.HCl) (1.0 eq) were added in DCM. After stirring for 16 h at room temperature, the reaction mixture was diluted with EtOAc and washed with HCl (1.0M in water), NaHCO3 (4% (w/v) in water), brine, and dried over MgSO4. The organic phase was concentrated to provide the crude product.
The pJL1 plasmid variants used were prepared as follows using pJL1 (https://www.addgene.org/69496/) shared on Addgene. Primers were designed to have the sequences of pJL1_M-StrepII-ST (MWSHPQFEKST), pJL1 MI-StrepII (MIWSHPQFEK), and pJL1 MR-StrepII-DR (MRWSHPQFEKDR) based on pJL1_M-StrepII (MWSHPQFEK), and the primers were ordered through Bioneer and Cosmogenetech. The ordered primers were dissolved to 100 mg/μl, mixed with the existing pJL1_M-StrepII, and PCR was performed. DNA fragments obtained through PCR were separated into DNA of the desired size through electrophoresis, and then T5 nuclease was added to perform Gibson assembly to anneal matching sequences, and plasmids were connected using polymerase and ligase. Since pJL1 contains a kanamycin resistance gene, the synthesized plasmid was transformed into E. coli and cultured in LB mixed with kanamycin to selectively amplify the plasmid. The base sequence of the manufactured plasmid was confirmed by Sanger sequencing or NGS.
Microhelix acylation. 1 μL of 0.5 M HEPES (pH 7.5) or bicine (pH 8.8), 1 μL of 10 μM microhelix, and 3 μL of nuclease-free water were mixed in a PCR tube with 1 μL of 10 μM eFx, dFx, and aFx, respectively. The mixture was heated for 2 min at 95° C. and cooled down to room temperature over 5 min. 2 μL of 300 mM MgCl2 was added to the cooled mixture and incubated for 5 min at room temperature. Followed by the incubation of the reaction mixture on ice for 2 min, 2 μL of 25 mM activated ester substrate in DMSO was then added to the reaction mixture. The reaction mixture was further incubated for 16-120 h on ice in a cold room at 4° C.
tRNA acylation. 2 μL of 0.5 M HEPES (pH 7.5), 2 μL of 250 μM tRNA, 2 μL of 250 μM of a Fx selected from the respective microhelix experiment and 6 μL of nuclease-free water were mixed in a PCR tube. The mixture was heated for 2 min at 95° C. and cooled down to room temperature over 5 min. 4 μL of 300 mM MgCl2 was added to the cooled mixture and incubated for 5 min at room temperature. Followed by the incubation of the reaction mixture on ice for 2 min, 4 μL of 25 mM activated ester substrate in DMSO was then added to the reaction mixture. The reaction mixture was further incubated for the optimal time determined during the microhelix experiment on ice in cold room.
In vitro synthesis of cyclic hydrazinediones
Ribosome-mediated synthesis of cyclic hydrazinediones. As a reporter peptide, a T7 promoter-controlled DNA template (pJL1_XZ-StrepII) was designed to encode a streptavidin (Strep) tag and additional Met(AUG) and Ile(AUC) codons (XZWHSPQFEKST (strep-tag), where X and Z indicate the position of the diester and Hz substrates, respectively) for N-terminal incorporation of two ncMs. The synthesis was performed using only the 9 amino acids that decode the purification tag without the other 11 amino acids to prevent corresponding endogenous tRNAs from being aminoacylated and participating in translation. The PURExpress™ A (aa, tRNA) kit (NEB, E6840S) was used for in vitro ribosomal synthesis and the reaction mixtures were incubated at 37° C. for 2 h. The synthesized peptides were then purified using Strep-Tactin®-coated magnetic beads (IBA), denatured with a 0.1% (v/v) SDS solution in water, and characterized by MALDI-TOF mass spectrometry.
Multiple syntheses of cyclic hydrazinediones. A new plasmid (pJL1-StrepII-2XZYZ) encoding the XZWHSPQFEKSX′Z was used for multiple ring-closing reactions, where X, X′, and Z indicate the position of the diester (1, 12) and Hz, respectively. The diesters were incorporated into the Met (AUG-X) and Asp (GAC-X′) codons. Simultaneously, Hz was incorporated into the Arg (CGU-Z) codon. The reaction mixtures, containing only the 8 amino acids necessary for decoding the Strep tag, were incubated at 37° C. for 2 hours. The reaction mixtures were incubated at 37° C. for 2 h. The resulting products were purified and characterized by the same method described below.
Purification and characterization of cyclic hydrazinediones
The products containing hydrazinediones were purified using an affinity tag purification technique or a C18 spin column purification. The purified products were characterized by MALDI spectrometry. For MALDI sample preparation, the purified peptide in 1.5 μL of SDS solution was dried with 0.5 μL of the matrix (α-cyano-4-hydroxycinnamic acid in THF, 10 mg·mL−1). The dried sample was characterized on a Bruker Autoflex MALDI-TOF and processed using Compass DataAnalysis (Bruker).
Reaction of hydrazone between hydrazine and aldehyde
The sample was freeze-dried to remove a solvent (water). Then, the solid-state peptide was dissolved by 4-methylbenzaldehyde (TCI, T0259) and incubated for 30 min at 37° C. Afterward, water was added again, and if layer separation occurs, the upper aqueous layer was characterized by MALDI-TOF and processed using Compass DataAnalysis (Bruker).
Reaction of pyrazolidinedione using ribozyme:P1c2UGGU
1000 pmol of P1c2UGGU and was dissolved in 32 μL of 62.5 mM HEPES (pH 7.5). 8 μL of 300 mM MgCl2 was added to the cooled mixture and incubated on ice for 60 min to be self-dimerized. Then, 1000 pmol of tRNAfMet (CAU): (s) and tRNAPro1E2 (GGU): Hz was added and placed at 4° C. for 24 h. The reaction product was treated with 44 μl RNase A solution (RNase A (Favorgen) and 200 mM sodium acetate, pH5.2) to liberate any pyrazolidinedione that might form and incubated for 5 min at room temperature. The treated sample was then transferred to autosampler vials, kept on ice until immediately before LC-MS analysis and returned to ice immediately afterwards. As a negative control, P1c2 was not added to the buffer with the same composition in the first step.
All chemicals were purchased from Sigma-Aldrich, Sejin TCI, and Alpha Aesar, and used as received without further purification. Synthetic DNA oligonucleotides were purchased from IDT Korea, Bioneer (Korea), and Cosmogenetech (Korea). Silica gel flash chromatography was performed using 0.040-0.063 mm, 60 Å silica purchased from Supelco®. Thin layer chromatography was performed using glass silica plates coated with fluorescent indicator (F254) purchased from Supelco®. Sand, sodium chloride, sodium bicarbonate, potassium carbonate, concentrated hydrochloric acid, and sodium hydroxide pellets were purchased from Sigma-Aldrich, Biosesang (Korea), Samchun (Korea) and Daejung (Korea).
MALDI Mass spectra were recorded on a Bruker Autoflex. High-resolution mass spectrometry (HRMS) analysis was performed at the Department of Chemistry at Pohang University of Science and Technology (POSTECH) using the 6560 Accurate Mass Q-TOF LC/MS system from Agilent Technologies. MALDI mass spectra were processed using Compass DataAnalysis Viewer v6.1 (Bruker) and FlexControl v2.0 (Bruker) software utilizing the smoothening and baseline subtraction. 1H and 13C NMR spectra were collected either using a Bruker AVANCE III™ HD 500 MHz cryoprobe or Bruker AVANCE III™ HD 400 MHz cryoprobe NMR spectrometer and processed with TopSpin (4.3.0). Chemical shifts, denoted in ppm, were assigned relative to the residual NMR solvent peaks.
DNA template synthesisExtension: 2.5 μL of 10 μM forward and reverse primer (Ext.) (see List of primers) was added to 10 μL KAPA HiFi 2× polymerase, and mixed with 5 μL dH2O in a PCR tube. The thermocycling conditions were as follows: 5 min at 95° C. followed 37° C. for 10 min and 72° C. for 30 s. PCR amplification: 20 μL of the product was used as an extension template, 20 μL each of 10 μM forward and reverse primer (Amp.) (see List of primers) were added to 250 μL KAPA HiFi 2× polymerase, and mixed with 190 μL dH2O in a PCR tube. The mixture was divided into several PCR tubes, and the DNA was amplified by the following thermocycling conditions: 3 min at 95° C., followed by 25 cycles of 95° C. for 30 s and Tm (either 55° C. or 65° C.) for 30 s, and 72° C. for 1 min. PCR products were assayed on a 1% (w/v) agarose gel.
DNA template precipitation
PCR products were extracted using phenol/chloroform/isoamyl alcohol and precipitated with ethanol. Samples were dried at room temperature for 5 min and resuspended in 100 μL nuclease-free water. DNA concentrations were determined from absorbance measured on a Nabi UV/Vis Nano Spectrophotometer. (MicroDigital Co., Ltd)
In vitro transcription
RNAs were prepared using a HiScribe® T7 quick high-yield RNA synthesis kit (NEB, E2050S). For in vitro transcription, 25 μg of DNA template was used with 50 μL of T7 RNA polymerase mix, 250 μL of NTP buffer mix, and nuclease-free water up to 500 μL. The mixture was incubated at 37° C. overnight.
Digestion of DNA templates
The DNA templates were removed by adding 50 μL of DNase I (NEB, E2050S) into the 500 μL of transcription reaction products. The reaction mixture was incubated at 4° C. for 2 h.
Purification of in vitro transcribed RNA
The digested transcription reactions were mixed with 500 μL 2× RNA loading dye and loaded onto a 15% TBE-Urea gel (24 g of Urea, 18.75 mL of 40% Acrylamide/Bis solution 19:1 (Bio-Rad, #1610144), and 5 mL of 3 M NaOAc, 10× TBE buffer up to 50 mL using water). The gel was run in 0.5× TBE buffer at 130V for 3 h at room temperature. The gel was placed on a silica plate and the transcribed RNAs were visualized by irradiation via a UV lamp (260 nm). The RNA products were excised from the gel and added to 5 mL of water. The gels were crushed and then placed in the cold room for 1 h. The gels were transferred to a HiGene™ HiFilter with a collection tube (Biofact) and centrifuged at 4000×g for 2 min. The flow-through was collected and added to the solution of 0.3 mL of 5M NaCl and 10 mL of 100% ethanol. The solution was placed at −20° C. for 16 h and centrifuged at 13,000×g for 30 min at 4° C. The supernatant was removed, and the pellet was dried for 5 min at room temperature. The dried RNA pellet was dissolved in nuclease-free water and the concentration was determined using a spectrophotometer.
Gel electrophoresis
10.8g of UREA, 15 mL of 40% Acrylamide/Bis Solution 19:1 (Bio-Rad, #1610144), and 500 μL of 3 M NaOAc were mixed in a 50 mL tube. Water added to a total volume of 30 mL. After the urea was completely dissolved, 300 μL of 10% (wt/vol) APS and 24 μL of TEMED were added to the solution. The mixture solution was casted into a gel cassette immediately using a serological pipette and the gel was allowed to polymerize overnight.
1 μL of prepared sample was mixed with 5 μL of acidic-loading dye (3 M NaOAc (pH 5.2), 20 μL of 0.5 M EDTA (pH 8.0), 980 μL of 2% (wt/vol) formamide, and 8 μL of Bromophenol Blue) and loaded onto a prepared 10% acidic urea gel. The gel was run in 50 mM NaOAc buffer at 135V for 2 h at 4° C. in a cold room. After electrophoresis, gel was stained by 2.5 uL of SYBR™ Gold Nucleic Acid Gel Stain (Invitrogen, S11494) in 50 mL of water for 10 min.
The gel was visualized on a Bio-Rad gel documentation system (GelDoc Go).
Methyl 3-((4-((2-aminoethyl)carbamoyl)benzyl)thio)-3-oxopropanoate (m): Prepared according to the general procedure A using methyl hydrogen malonate (341.1 μl, 3 mmol), Boc-ABT (155.2 mg, 0.5 mmol), DMAP (61.1 mg, 0.5 mmol), EDC-HCl (575.1 mg, 3 mmol) and CH2Cl2. (10 mL). Purification by flash column chromatography (60% EtOAc in n-Hex) yielded the Boc-protected product as a white solid (82.9 mg, 40.4%). The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane for 30 min at room temperature, and the resulting product was used without further purification. : 1H NMR (500 MHz, DMSO-d6) δ 8.72 (t, J=5.28 Hz, 1H), 8.01 (br s, 3H), 7.85 (d, J=8.19 Hz 2H), 7.41 (d, J=8.26 Hz, 2H),4.22 (s, 2H), 3.84 (s, 2H), 3.64 (s, 3H), 3.51 (q, J=5.90 Hz, 2H), 2.98 (q, J=5.69 Hz, 2H), 13C NMR (126 MHz, DMSO-d6) δ 191.4 (1C, O═C—S), 166.9 (1C, O═C—O), 166.9 (1C, O═C—NH), 141.3 (1C, Ph), 133.4 (1C, Ph), 129. 1 (2C, Ph), 128.1 (2C, Ph), 52.7 (1C, H3C—O), 49.1 (1C, CH2), 39.1 (1C, CH2NH3+), 37.5 (1C, NHCH2), 32.8 (1C, SCH2). HRMS (ESI): Exact mass calc. for C14H19N2O4S+[M+H]+=311.1060, ; Found 311.1089.
3,5-dinitrobenzyl methyl malonate (m-2) Prepared according to the general procedure B using methyl hydrogen malonate (104.7 μL, 1 mmol), 3,4-dinitrobenzyl alcohol (198.1 mg, 1 mmol), DMAP (122.2 mg, 1 mmol), EDC-HCl (191.7 mg, 1 mmol) and CH2Cl2 (10 ml). Purification by silica gel flash chromatography (30% EtOAc in n-Hex) yielded a yellow solid. (46.9 mg, 15.7%): 1H NMR (500 MHz, DMSO-d6) δ 8.80 (t, J=2.20 Hz, 1H), 8.67 (d, J=1.84 Hz, 2H), 5.43 (s, 2H), 3.69 (s, 2H), 3.68 (s, 3H), 13C NMR (126 MHz, DMSO-d6) δ 169.3 (1C, O═C), 168.7 (1C, O═C), 150.7 (2C, C—N(═O)═O), 142.7 (1C, Ph), 130.4 (2C, Ph), 120.5 (1C, Ph), 66.6 (1C, OCH2), 54.8 (1C, O—CH3), 43.1 (1C, CH2, Overlapping Solvent peak). HRMS (ESI): Exact mass cal. for C11H10N2O8Na+[M+Na]+=321.0329; Found 321.0321.
tert-butyl 3-((4-((2-aminoethyl)carbamoyl)benzyl)thio)-3-oxopropanoate (0): Prepared according to the general procedure A using mono-tert-butyl malonate (710.0 mg, 5 mmol), Boc-ABT (310.4 mg, 1 mmol), DMAP (122.2 mg, 1 mmol), EDC-HCl (383.4 mg, 2 mmol) and CH2Cl2. (10 mL). Purification by flash column chromatography (30% EtOAc in n-Hex) yielded Boc-protected product as a white solid. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane at 0° C. for 30 min, and the resulting product was used without further purification. The product was obtained as a white solid. (32.8 mg, 8.4%). : 1H NMR (500 MHz, DMSO-d6) δ 8.79 (t, J=4.94 Hz, 1H), 8.15 (br s, 3H), 7.94-7.84 (m, 2H), 7.43 (d, J=8.03 Hz, 2H), 4.22 (s, 2H), 3.69 (s, 2H), 3.56-3.49 (m, 2H), 2.99-2.93 (m, 2H), 1.39 (s, 9H)13C NMR (126 MHz, DMSO-d6) δ 191.6 (1C, O═C—S), 166.9 (1C, O═C—O), 165.6 (1C, O═C—NH), 141.4 (1C, Ph), 133.3 (1C, Ph), 129.0 (2C, Ph), 128.1 (2C, Ph), 82.0 (1C, C—O), 50.8 (1C, CH2), 39.0 (1C, CH2NH3+), 37.6 (1C, NHCH2), 32.7 (1C, SCH2), 28.0 (3C, (CH3)3). HRMS (ESI): Exact mass cal. for C17H25N2O4S+[M+H]+=353.1530; Found 353.1555.
1-(3,5-dinitrobenzyl) 3-ethyl 2-aminomalonate (1) Prepared according to the general procedure B using 2-((tert-butoxycarbonyl) amino)-3-ethoxy-3-oxopropanoic acid (100 mg, 0.4 mmol), 3,4-dinitrobenzyl alcohol (198.13 mg, 1 mmol), DMAP (122.2 mg, 1 mmol), EDC-HCl (191.7 mg, 1 mmol) and CH2Cl2 (10 mL). The product was successfully obtained. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane for 30 min at room temperature, and the resulting product was washed with diethyl ether to remove 3,4-dinitrobenzyl alcohol. The solvent was concentrated under vacuum, and the product was obtained as a yellow solid (163.6 mg, 45.0%). : 1H NMR (500 MHz, DMSO-d6) δ 9.23 (br s, 3H), 8.84 (t, J=2.21 Hz, 1H), 8.74 (d, J=2.10 Hz, 2H), 5.58 (q, J=16.03 Hz, 2H), 5.26 (s, 1H), 3.56 (s, 2H), 1.22 (t, J=7.08 Hz, 3H)13C NMR (126 MHz, DMSO-d6) δ 164.0 (2C, O═C), 148.5 (2C, C—N(═O)═O), 139.7 (1C, Ph), 128.7 (2C, Ph), 119.0 (1C, Ph), 66.8 (1C, O—CH2-Ph), 66.2 (1C, CH—NH3+), 63.6 (1C, OCH2), 14.2 (1C, CH3). HRMS (ESI): Exact mass cal. for C12H14N3O8+[M+H]+=328.0775; Found 328.0803.
Methyl 1-(((4-((2-aminoethyl)carbamoyl) benzyl)thio) carbonyl) cyclopropane-1-carboxylate (2) To a stirred solution of 1-(methoxycarbonyl)cyclopropane-1-carboxylic acid (200 mg, 1.39 mmol) in CH2Cl2 (5 mL) at 0° C. in an inert atmosphere, oxalyl chloride (1.2 mL, 13.9 mmol) was added dropwise and continued stirring for 5 h at room temperature. Removed the solvent and oxalyl chloride under reduced vacuum and redissolved the resulting oily residue in CH2Cl2. Added triethylamine (968.0 μl, 6.94 mmol) followed by Boc-ABT (344.5 mg, 1.11 mmol) in CH2Cl2 at 0° C. Allowed the reaction mixture to continue stirring under an inert atmosphere at room temperature for 5 h. Progress of the reaction was monitored using thin-layer chromatography (TLC). Purification of the crude material by flash column chromatography (petroleum ether/ethyl acetate, 95:5 to 85:15) furnished white solid product (92.9%, 450 mg). The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane for 30 min at room temperature, and the resulting product was used without further purification. : 1H NMR (500 MHz, DMSO-d6) δ 8.79 (t, J=5.16 Hz 1H), 8.15 (br s, 3H), 7.88 (d, J=8.16 Hz, 2H), 7.39 (d, J=8.18 Hz, 2H), 4.18 (s, 2H), 3.66 (s, 3H), 3.53 (q, J=5.85 Hz, 2H), 2.99 (m, 3H), 1.54 (m, 4H), 13C NMR (126 MHz, DMSO-d6) δ 194.6 (1C, O═C—S), 170.0 (1C, O═C—O), 166.8 (1C, O═C—NH), 141.4 (1C, Ph), 133.3 (1C, Ph), 129.1 (2C, Ph), 128.1 (2C, Ph), 53.0 (1C, OCH3), 39.0 (1C, NH3+—CH2), 37.6 (1C, NH-CH2), 35.7 (1C, C), 33.0 (1C, SCH2), 19.1 (2C, CH2—CH2). HRMS (ESI): Exact mass cal. for C16H21N2O4S+[M+H]+=337.1217; Found 337.1247.
Cyanomethyl phenyl malonate (3): 2,2-Dimethyl-1,3-dioxane-4,6-dione (721 mg, 5 mmol) and phenol (615.7 μl, 7 mmol) were dissolved in toluene (10 mL). The reaction proceeded under reflux for 4 h. The resulting mixture was then added to saturated NaHCO3. The layers were separated, and the aqueous layer was washed with toluene. The water layer was acidified with 1 M HCl, and the organic layer was separated using ethyl acetate, dried over MgSO4, filtered, and concentrated under vacuum. Prepared according to the general procedure C using the resulting product, bromoacetonitrile (5 mL, 718 mol, excess) and triethylamine (139 μl, 1 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting solution was washed with 1 M HCl, 4% NaHCO3, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The product was obtained as a colorless liquid (217 mg, 19.8%). : 1H NMR (500 MHz, DMSO-d) δ 7.46 (m, 2H), 7.30 (m, 1H), 7.19 (m, 2H), 5.12 (s, 2H), 3.99 (s, 2H), 13C NMR (126 MHz, DMSO-d6) δ 167.9 (1C, O—O═C), 167.2 (1C, O═C—O), 152.4 (1C, Ph), 132.0 (2C, Ph), 128.6 (1C, Ph), 123.9 (2C, Ph), 117.6 (1C, C—N), 52.1 (1C, OCH2), 41.8 (1C, CH2, Overlapping Solvent peak). HRMS (ESI) Exact mass cal. for C11H9NO4Na+[M+Na]+=242.0424; Found 242.0428.
Methyl 4-((4-((2-aminoethyl)carbamoyl)benzyl)thio)-4-oxobutanoate (s): Prepared according to the general procedure A using monomethyl succinate (341 μl, 3 mmol), Boc-ABT (155.2 mg, 0.5 mmol), DMAP (61.1 mg, 0.5 mmol), EDC-HCl (575.1 mg, 3 mmol) and CH2Cl2. (10 mL). The deprotection was achieved upon treatment with 4M solution of HCl in 1,4-dioxane for 30 min at room temperature and the product was obtained as a white solid. (102.4 mg, 56.8%): 1H NMR (500 MHz, DMSO-d6) δ 8.78 (t, J=5.45 Hz, 1H), 8.14 (br s, 3H), 7.85 (d, J=8.26 Hz, 2H), 7.38 (d, J=8.25 Hz, 2H), 4.18 (s, 2H), 3.59 (s, 3H), 3.53 (q, J=5.92 Hz, 2H), 2.98 (m, 2H), 2.90 (t, J=6.53 Hz, 2H), 2.62 (t, J=6.53 Hz, 2H), 13C NMR (126 MHz, DMSO-d6) δ 197.4 (1C, O═C—S), 172.4 (1C, O═C—O), 166.8 (1C, O═C—NH), 141.8 (1C, Ph), 133.3 (1C, Ph), 129.0 (2C, Ph), 129.1 (2C Ph), 52.0 (1C, CH3O), 39.03 (1C, CH2NH3+), 38.3 (1C, CH2NH), 37.6 (1C, CH2), 32.3 (1C, SCH2), 28.9 (1C, CH2). HRMS (ESI): Exact mass cal. for C15H21N2O4S+[M+H]+=325.1217, Found 325.1242.
4-(tert-butyl) 1-(3,5-dinitrobenzyl) L-aspartate (4) Prepared according to the general procedure B using N-Boc-L-aspartic acid 4-tert-butyl ester (289.3 mg, 1.0 mmol), 3,4-dinitrobenzyl alcohol (237.8 mg, 1.2 mmol), DMAP (122.2 mg, 1.0 mmol), EDC-HCl (230.0 mg, 1.2 mmol) and EtOAc (10 mL). Boc-protected product was successfully obtained. The deprotection was achieved upon treatment with 4M solution of HCl in 1,4-dioxane at 0° C. for 30 min, and the resulting product was washed with diethyl ether to remove 3,4-dinitrobenzyl alcohol. The solvent was concentrated under vacuum, and the product was obtained as a yellow solid. (101.3 mg, 20.5%) 1H NMR (500 MHz, DMSO-d6) δ 8.83 (t, J=2.12 Hz, 1H), 8.82 (br s, 3H, overlapped other peaks), 8.76 (d, J=2.11,2H), 5.51 (q, J=4.57 Hz, 2H), 4.42 (t, J=5.30 Hz, 1H), 3.00 (m, 2H), 1.36 (s, 9H)13C NMR (126 MHz, DMSO-d) δ 168.9 (1C, O═C), 168.7 (1C, O═C), 148.5 (2C, O═C—NH), 140.0 (1C, Ph), 129.1 (2C, Ph), 118.9 (1C, Ph), 82.1 (1C, OC), 66.8 (1C, OCH2), 48.9 (1C, CHNH3+), 35.5 (1C, CH2), 28.0 (3C, CH3). HRMS (ESI): Exact mass cal. for C15H20N3O8+[M+H]+=370.1245, Found 370.1245.
Ethyl 4-((4-((2-aminoethyl)carbamoyl)benzyl)thio)-4-oxobutanoate (5): Succinic anhydride (1.00 g, 10.0 mmol) was dissolved in ethanol (50 mL). The reaction proceeded under reflux for 3 h. The resulting mixture was evaporated to remove ethanol. The monoethyl succinate was obtained. Then, general procedure A using monomethyl succinate (146.1 mg, 1.0 mmol), Boc-ABT (155.2 mg, 0.5 mmol), DMAP (122 mg, 1.0 mmol), EDC-HCl (191.7 mg, 1.0 mmol) and EtOAc (10 mL) was carried out. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane at room temperature for 30 min, and the product was obtained as a white solid (163.1 mg, 87.0%). : 1H NMR (500 MHz, DMSO-d6) δ8.78 (t, J=5.29 Hz, 1H), 8.11 (br s, 3H), 7.85 (d, J=8.18 Hz, 2H), 7.37 (d, J=8.15 Hz, 2H), 4.17 (br s, 2H), 4.03 (q, J=7.12, 2H), 3.54-3.52 (m, 2H, Overlapping Solvent peak), 3.01-2.95 (br t, 2H), 2.88 (t, J=6.43 Hz, 2H), 2.63-2.57 (br t, 2H), 1.15 (t, J=7.10 Hz, 3H), 13C NMR (126 MHz, DMSO-d6) δ197.5 (1C, O═C—S), 172.0 (1C, O═C—O), 166.9 (1C, O═C—NH), 141.8 (1C, Ph), 133.2 (1C, Ph), 129.0 (2C, Ph), 128.1 (2C Ph), 60.6 (1C, CH2O), 39.02 (1C, CH2NH3+), 38.3 (1C, CH2NH), 37.6 (1C, CH2), 32.2 (1C, SCH2), 29.1 (1C, CH2), 14.5 (1C, CH3). HRMS (ESI): Exact mass cal. for C16H23N2O4S+[M+H]+=339.1373; Found 339.1393.
1-(3,5-dinitrobenzyl) 4-methyl L-aspartate (6) Prepared according to the general procedure B using (S)-2-((tert-butoxycarbonyl) amino)-4-methoxy-4-oxobutanoic acid (247.2 mg, 1 mmol), 3,4-dinitrobenzyl alcohol (198.1 mg, 1.2 mmol), DMAP (122.1 mg, 1 mmol), EDC-HCl (237.8 mg, 1.2 mmol) and CH2Cl2 (10 mL). Boc-protected product was successfully obtained. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane at 0° C. for 30 min, and the resulting product was washed with diethyl ether to remove 3,4-dinitrobenzyl alcohol.
The solvent was concentrated under vacuum, and the product was obtained as a yellow solid. (73.7 mg, 20.3%) 1H NMR (500 MHz, DMSO-d6) δ 8.90-8.51 (br, 3H), 8.82 (t, J=1.90 Hz, 1H), 8.74 (d, J=1.90 Hz, 2H), 5.50 (q, J=11.73 Hz, 2H), 4.49 (t, J=5.43 Hz, 1H), 3.62 (s, 3H), 3.12 (m, J=7.59 Hz, 2H)13C NMR (126 Hz, DMSO-d6) δ 170.1 (1C, O═C), 168.5 (1C, O═C), 148.5 (2C, C—N(═O) ═O)), 140.0 (1C, Ph), 128.9 (2C, Ph), 118.8 (1C, Ph), 65.6 (1C, 5° C. H2), 52.6 (1C, CH3O), 48.9 (1C, CHNH3+), 34.4 (1C, CH2). HRMS (ESI): Exact mass cal. for C12H14N3Os[M+H]+=328.0775; Found 328.0815.
Methyl 4-((4-((2-amninoethyl)carbamoyl)benzyl)thio)-2-methyl-4-oxobutanoate+Methyl 4(4(2 aininoethyl)carbamoyl) benzyl) thia)-3-methyl-4-oxobutanoate (7): Methyl succinic anhydride (280.6 μL, 3 mnmol) was dissolved in methanol (5 mL). The reaction proceeded under reflux for 3 h. The resulting mixture was evaporated to remove methanol, and the methoxy methyloxobutanoic acid was obtained. Then, general procedure A using methoxy methyloxobutanoic acid (438.4 mg, 3.0 mmol), Boc-ABT (341.4 mg, 1.1 mmol), DMAP (61.1 mg, 0.5 mmol), EDC-HCl (230 mg, 1.2 mmol) and CH2Cl2. (10 mL) was carried out. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane at room temperature for 30 min, and the product was obtained as a white solid (406.9 mg, 98.7%). As a result of the NMR analysis, two products were made. (Methyl 4-((4-((2-aminoethyl)carbamoyl)benzyl)thio)-2-methyl-4-oxobutanoate) (50%): 1H NMR (500 MHz, DMSO-d6) δ 8.82 (t, J=5.25 Hz, 1H), 8.19 (br s, 3H), 7.89 (d, J=8.08 Hz, 2H), 7.39 (d, J=7.85 Hz, 2H), 4.19 (s, 2H), 3.59 (s, 3H), 3.57-3.51 (m, 2H), 3.12-2.55 (m, 5H), 1.17 (d, J=7.07, 3H). 13C NMR (126 Hz, DMSO-d6) δ 197.0 (1C, O═C—S), 175.1 (1C, O═C—O), 166.8 (1C, O═C—NH), 141.8 (1C, Ph), 133.3 (1C, Ph), 129.1 (2C, Ph), 129.0 (2C, Ph), 52.2 (1C, CH3O), 46.5 (1C, CH2), 39.0 (1C, CH2NH3), 37.6 (1C, CH2NH) 35.9 (1C, CH), 32.1 (1C, S—CH2), 16.9 (1C, CH3) Methyl 4-((4-((2-aminoethyl)carbamoyl)benzyl)thio)-3-methyl-4-oxobutanoate (50%): 1H NMR (500 MHz, DMSO-d6) δ 8.82 (t, J=5.25 Hz, 1H), 8.19 (br s, 3H), 7.89 (d, J=8.08 Hz, 2H), 7.39 (d, J=7.85 Hz, 2H), 4.19 (s, 2H), 3.60 (s, 3H), 3.57-3.51 (m, 2H), 3.12-2.55 (m, 5H), 1.12 (d, J=6.12, 3H). 13C NMR (126 Hz, DMSO-d6) δ 201.5 (1C, O═C—S), 171.8 (1C, O═C—O), 166.8 (1C, O═C—NH), 141.8 (1C, Ph), 133.3 (1C, Ph), 129.1 (2C, Ph), 129.0 (2C, Ph), 52.0 (1C, CH3O), 44.2 (1C, CH), 39.0 (1C, CH2NH3+), 37.6 (1C, CH2NH) 37.3 (1C, CH2), 32.3 (1C, S—CH2), 18.1 (1C, CH3). HRMS (ESI): Exact mass cal. for C16H23N2O4S+[M+H]+=339.1373; Found 339.1392.
1-(3,5-dinitrobenzyl) 4-methyl 3-methylsuccinate+1-(3,5-dinitrobenzyl) 4-methyl 2-methylsuccinate (7-2): Methyl succinic anhydride (280.6 μl, 3 mmol) and NaOH (40.0 mg, 1 mmol) was dissolved in methanol (5 mL). The reaction proceeded under reflux for 3 h. The resulting solution was washed with 1 M HCl and brine. Prepared according to the general procedure B using resulting product, 3,4-dinitrobenzyl alcohol (594.4 mg, 3 mmol), DMAP (122.2 mg, 1 mmol), EDC-HCl (575.1 mg, 3 mmol) and CH2Cl2 (10 ml). Purification by silica gel flash chromatography (30% EtOAc in n-Hex) yielded a yellow solid. (314.8 mg, 32.2%) As a result of the NMR analysis, two products were made. : 1-(3,5-dinitrobenzyl) 4-methyl 2-methylsuccinate (50%) 1H NMR (500 MHz, DMSO-d6) δ 8.81-8.78 (m, 1H), 8.64 (t, J=2.42 Hz, 2H), 5.36 (s, 2H), 3.57 (s, 3H), 3.01-2.80 (m, 1H), 2.74-2.58 (m, 2H), 1.21-1.12 (m, 3H), 13C NMR (126 MHz, DMSO-d6) δ 174.2 (1C, O═C), 173.5 (1C, O═C), 150.4 (2C, C—N(═O)═O), 143.0 (1C, Ph), 130.4 (1C, Ph), 130.2 (1C, Ph), 120.5 (1C, Ph), 66.0 (1C, OCH2), 54.0 (1C, CH3O), 39.1 (1C, CH2), 37.5 (1C, CH), 19.0 (1C, CH3), 1-(3,5-dinitrobenzyl) 4-methyl 3-methylsuccinate (50%): 1H NMR (500 MHz, DMSO-d6) δ 8.81-8.78 (m, 1H), 8.64 (t, J=2.42 Hz, 2H), 5.37 (s, 2H), 3.58 (s, 3H), 3.01-2.80 (m, 1H), 2.98-2.59 (m, 2H), 1.21-1.12 (m, 3H), 13C NMR (126 MHz, DMSO-d6) δ 174.2 (1C, O═C), 173.5 (1C, O═C), 150.4 (2C, 2C, C—N(═O)═O), 142.9 (1C, Ph), 130.4 (1C, Ph), 130.2 (1C, Ph), 120.4 (1C, Ph), 66.0 (1C, OCH2), 53.8 (1C, CH3O), 38.9 (1C, CH2), 37.5 (1C, CH), 18.9 (1C, CH3). HRMS (ESI): Exact mass cal. for C13H14N2O8Na+[M+Na]+=349.0642; Found 349.0641.
1-(cyanomethyl) 4-methyl 3-phenylsuccinate+1-(cyanomethyl) 4-methyl 2-phenylsuccinate (8): Phenyl succinic anhydride (528.5 mg, 3 mmol) was dissolved in methanol (5 mL). The reaction proceeded under reflux for 5 h. The resulting mixture was evaporated to remove methanol. methoxy phenyloxobutanoic acid was obtained. Prepared according to the general procedure C using the resulting product, bromoacetonitrile (348.3 μl, 5 mmol) and triethylamine (139.5 μl, 1 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting solution was washed with 1 M HCl, 4% NaHCO3, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The product was obtained as a colorless liquid (32.8 mg, 4.42%). As a result of the NMR analysis, two products were made: 1-(cyanomethyl) 4-methyl 3-phenylsuccinate (33%): 1H NMR (500 MHz, DMSO-d6) δ 7.39-7.25 (m, 5H), 4.95 (s, 2H), 4.11-4.06 (i, 1H) 3.57 (s, 3H, Overlapping Solvent peak), 3.22-2.67 (n, 2H), 13C NMR (126 MHz, DMSO-d6) δ 173.4 (1C, O═C), 173.2 (1C, O═C), 137.7 (1C, Ph), 129.3 (2C, Ph), 128.3 (2C, Ph) 128.0 (1C, Ph), 116.2 (1C, C═N), 52.6 (1C, OCH3), 49.5 (1C, OCH2), 46.7 (1C, CH), 36.9 (1C, CH2), 1-(cyanomethyl) 4-methyl 2-phenylsuccinate (67%): 1H NMR (500 MHz, DMSO-d6) δ 7.39-7.25 (i, 5H), 4.97 (s, 2H), 4.19-4.13 (i, 1H), 3.59 (s, 3H, Overlapping Solvent peak), 3.22-2.67 (i, 2H), 13C NMR (126 MHz, DMSO-d6) δ 172.1 (1C, O═C), 171.8 (1C, O═C), 137.1 (1C, Ph), 129.4 (2C, Ph), 128.2 (2C, Ph), 128. 1 (1C, Ph), 116.0 (1C, C═N), 52.2 (1C, OCH3), 49.8 (1C, OCH2), 46.4 (1C, CH), 37.3 (1C, CH2). HRMS (ESI): Exact mass cal. for C13H13NO4Na+[M+Na]+=270.0737; Found 270.0750.
4-benzyl 1-(3,5-dinitrobenzyl) L-aspartate (9) Prepared according to the general procedure B using N-Boc-L-aspartic 4-benzyl ester (323.3 mg, 1.0 mmol), 3,4-dinitrobenzyl alcohol (237.8 mg, 1.2 mmol), DMAP (122.2 mg, 1.0 mmol), EDC-HCl (230.0 mg, 1.2 mmol) and EtOAc (10 mL). Boc-protected product was successfully obtained. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxanev at 0° C. for 30 min, and the resulting product was washed with diethyl ether to remove 3,4-dinitrobenzyl alcohol. The solvent was concentrated under vacuum. The product was obtained as a yellow solid (182.5 mg, 41.5%). 1H NMR (500 MHz, DMSO-d6) δ 8.86 (br, 3H), 8.80 (t, J=2.10 Hz, 1H), 8.71 (d, J=2.10 Hz, 2H), 7.35-7.30 (m, 5H), 5.46 (s, 2H), 5.12 (s, 2H), 4.53 (t, J=5.51 Hz, 1H), 3.23-3.10 (m, J=5.51 Hz, 2H) 13C NMR (126 MHz, DMSO-d6) δ 169.6 (1C, O═C), 168.5 (1C, O═C), 148.5 (2C, C—N(═O)═O), 140.0 (1C, Ph), 135.9 (1C, Ph), 128.9 (2C, Ph), 128.8 (2C, Ph), 128.6 (1C, Ph), 128.5 (2C, Ph), 118.8 (1C, Ph), 66.8 (1C, OCH2), 65.6 (1C, OCH2), 49.0 (1C, CHNH3+), 34.6 (1C, CH2). HRMS (ESI): Exact mass cal. for C18H18N3O+[M+H]+=404.1088; Found 404.1094.
Benzyl 4-((4-((2-aminoethyl)carbamoyl)benzyl)thio) -4-oxobutanoate (10): Succinic anhydride (1.00 g, 10 mmol) was dissolved in benzyl alcohol (50 mL). The reaction proceeded under reflux for 3 h. The resulting mixture was evaporated to remove ethanol. The monoethyl succinate was obtained. Prepared according to the general procedure C using monobenzyl succinate (208.2 mg, 1.0 mmol), Boc-ABT (155.2 mg, 0.5 mmol), DMAP (122.2 mg, 1.0 mmol), EDC-HCl (191.7 mg, 1.0 mmol) and EtOAc. (10 mL). The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane at 0° C. for 30 min, and the product was obtained as a white solid (171.8 mg, 78.6%). : 1H NMR (500 MHz, DMSO-d6) δ 8.78 (t, J=5.27 Hz, 1H), 8.12 (br, 3H), 7.87 (d, J=8.15 Hz, 2H), 7.41-7.31 (m, 7H), 5.09 (s, 2H), 4.18 (s, 2H), 3.52 (q, J=3.52, 2H), 2.98 (m, 2H), 2.93 (t, J=6.47 Hz, 2H), 2.69 (t, J=6.47 Hz, 2H), 13C NMR (126 Hz, DMSO-d6) δ 197.4 (1C, O═C—S), 171.9 (1C, O═C—O), 166.8 (1C, O═C—NH), 141.7 (1C, Ph), 136.5 (1C, Ph), 133.3(1C, Ph), 129.0 (2C, Ph), 128.9 (2C Ph), 128.5 (1C, Ph), 128.4 (2C, Ph), 128.1 (2C, Ph), 66.2 (1C, CH2O), 39.04 (1C, CH2NH3+), 38.3 (1C, CH2NH), 37.6 (1C, CH2), 32.3 (1C, SCH2), 29.1 (1C, CH2). HRMS (ESI): Exact mass cal. for C21H25N2O4S+[M+H]+=401.1530; Found 401.1549.
Cyanomethyl phenyl succinate (11) Succinic anhydride (400.3 mg, 4 mmol) and phenol (615.7 μL, 7 mmol) was dissolved in 1 M NaOH (10 mL). The reaction mixture was stirred at room temperature for 6 h. The resulting mixture was treated with cold 1 M HCl. The product solidified, and the neutralizing solution was removed by filtration. Remaining phenol was removed with an additional wash using cold water. Prepared according to the general procedure C using resulting product, bromoacetonitrile (5 mL, 71.8 mmol, excess) and triethylamine (139.5 μL, 1 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting solution was washed with 1 M HCl, 4% NaHCO3, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The product was obtained as a colorless liquid. (182.0 mg, 19.5%): 1H NMR (500 MHz, DMSO-d6) δ 7.44-7.38 (m, 2H), 7.30-7.23 (m, 1H), 7.15-7.09 (m, 2H), 7.37 (d, J=8.15 Hz, 2H), 4.78 (s, 2H), 2.99-2.91 (m, 2H), 2.90-2.82 (m, 2H), 13C NMR (126 MHz, DMSO-d6) δ 170.7 (1C, O═C), 170.5 (1C, O═C), 150.5 (1C, Ph), 129.5 (2C, Ph), 126.1 (1C, Ph), 121.4 (2C, Ph), 114.2 (1C, C═N), 48.6 (1C, OCH2), 28.9 (1C, CH2), 28.5 (1C, CH2). HRMS (ESI) Exact mass cal. for C12H11NO4Na+[M+Na]+=256.0580; Found 256.0584.
3,5-dinitrobenzyl phenyl succinate (11-2) Succinic anhydride (500.4 mg, 5 mmol), 3,4-dinitrobenzyl alcohol (990.7 mg, 5 mmol) NaOH (40.0 mg, 1 mmol) was dissolved in EtOAc (10 mL). The reaction proceeded under reflux for 16 h. The resulting solution was washed with 1 M HCl, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. Purification by silica gel flash chromatography (50% EtOAc in n-Hex) yielded a product. The product was dissolved in EtOAc (10 mL) and treated with phenol (263.9 μL, 3 mmol), EDC-HCl (575.1 mg, 3 mmol) and DMAP (122.2 mg, 1 mmol). The reaction mixture was stirred at room temperature for 16 h. The resulting solution was washed with 1 M HCl, 4% NaHCO3, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. Purification by silica gel flash chromatography (50% EtOAc in n-Hex) yielded a yellow solid. (26.3 mg, 2.34%). : 1H NMR (500 MHz, DMSO-d6) δ 8.78 (t, J=2.10, 1H), 8.67 (d, J=2.10, 2H), 7.41-7.37 (m, 2H), 7.27-7.23 (m, 1H), 7.06-7.02 (m, 2H), 5.40 (s, 2H), 2.91-2.87 (m, 2H), 2.83-2.80 (m, 2H), 13C NMR (126 MHz, DMSO-d6) δ 172.2 (1C, O═C), 171.3 (1C, O═C), 150.8 (1C, O-Ph(C)), 148.5 (2C, C—N(═O)═O), 141.1 (1C, Ph), 129.9 (2C, Ph), 128.7 (2C, Ph), 126.3 (1C, Ph), 122.0 (2C, Ph), 118.6 (1C, Ph), 64.3 (1C, OCH2), 49.0 (1C, CH), 29.1 (1C, CH2). HRMS (ESI): Exact mass cal. for C12H14N2ONa+[M+Na]+=397.0642; Found 397.0633.
1-(3,5-dinitrobenzyl) 4-phenyl L-aspartate (12): Prepared according to the general procedure B, using N-Boc-L-aspartic acid 4-tert-butyl ester (1.45 g, 5.0 mmol), 3,4-dinitrobenzyl alcohol (1.19 g, 6.0 mmol), DMAP (610.9 mg, 5.0 mmol), EDC-HCl (1.15 g, 6 mmol) and EtOAc (50 mL). Boc and tert-butyl protected product was successfully obtained. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane at room temperature for 1 h, and the resulting product was washed with diethyl ether to remove 3,4-dinitrobenzyl alcohol. The resulting product was dissolved in dioxane (20 mL) and water (20 mL). The solution was added to 1 M NaOH (10 mL). The mixture was cooled to 0° C., Boc2O (1.09 g, 5 mmol) was slowly added, and the reaction was stirred for 6 h at room temperature. Subsequently, the reaction mixture was diluted with EtOAc (10 mL) and acidified with 1 M HCl. The layers were separated, and the aqueous fraction was extracted with EtOAc. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The resulting product and DCC (1.03 g, 5 mmol) were dissolved in THF (10 mL) and treated with pyridine (402.7 μL, 5.0 mmol). The reaction was stirred for 18 h at room temperature. The resulting solution was filtered to remove precipitants. The resulting mixture was then added to 4% NaHCO3. The layers were separated, and the aqueous layer was washed with EtOAc. The water layer was acidified with 1 M HCl, and the organic layer was separated using EtOAc, dried over MgSO4, filtered, and concentrated under vacuum. The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane at 0° C. for 30 min, and the product was obtained as a white solid (381.7 mg, 17.9%). 1H NMR (500 MHz, DMSO-d6) δ 8.80 (t, J=2.08 Hz, 1H), 8.76 (d, J=1.90 Hz, 2H), 8.59 (br, 3H), 7.41-7.37 (m, J=7.80 Hz, 2H), 7.30-7.21 (m, 1H), 7.11-7.04 (m, 2H), 5.51 (s, 2H), 4.68 (t, J=5.49 Hz, 1H), 3.41-2.85 (m, 2H)13C NMR (126 MHz, DMSO-d6) δ 171.3 (1C, O═C), 168.4 (1C, O═C), 150.4 (1C, Ph), 148.5 (2C, C—N(═O)—O)), 140.0 (1C, Ph), 129.9 (2C, Ph), 129.2 (2C, Ph), 126.6 (1C, Ph), 121.9 (2C, Ph), 118.9 (1C, Ph), 65.4 (1C, OCH2), 49.0 (1C, CHNH3+), 34.9 (1C, CH2). HRMS (ESI) Exact mass cal. for C17H16N3O+[M+H]+=390.0932; Found 390.0946.
3,5-dinitrobenzyl methyl maleate (13) Maleic anhydride (196.1 mg, 2 mmol) was dissolved in 4 ml of methanol. The reaction proceeded under reflux for 3 h. The resulting solution was washed with 1 M HCl and brine. Then, general procedure B using resulting product, 3,4-dinitrobenzyl alcohol (594.4 mg, 3 mmol), DMAP (122.2 mg, 1 mmol), EDC-HCl (575.1 mg, 3.0 mmol) and CH2Cl2 (10 mL) was carried out. Purification by silica gel flash chromatography (50% EtOAc in n-Hex) yielded a yellow solid. (140.4 mg, 22.6%): 1H NMR (500 MHz, DMSO-d6) δ 8.81 (t, J=4.14 Hz, 1H), 8.73 (t, J=2.07 Hz, 2H), 6.89 (d, J=6.88 Hz, 2H), 5.49 (s, 2H), 3.76 (s, 3H), 13C NMR (126 MHz, DMSO-d6) δ 165.2 (1C, O═C—O), 164.5 (1C, O═C—O), 148.6 (2C, C—N(═O)═O), 140.4 (1C, Ph), 134.1 (1C, ═CH), 133.2 (1C, ═CH), 128.9 (2C, Ph), 118.7 (1C, Ph), 65.0 (1C, CH2O), 52.8 (1C, CH3O). HRMS (ESI): Exact mass cal. for C12H10N2O8Na+[M+Na]+=333.0329; Found 333.0320.
4-(3,5-dinitrobenzyl) 1-methyl 2-methylmaleate+1-(3,5-dinitrobenzyl) 4-methyl 2-methylmaleate (14) Citraconic anhydride (179.8 μl, 2 mmol) and NaOH (20.0 mg, 0.5 mmol) was dissolved in 4 ml of methanol. The reaction proceeded under reflux for 3 h. The resulting solution was washed with 1 M HCl and brine. General procedure B using the resulting product, 3,4-dinitrobenzyl alcohol (594.4 mg, 3 mmol), DMAP (122.2 mg, 1 mmol), EDC·HCl (512.3 mg, 1.2 mmol) and CH2Cl2 (10 mL) was carried out. Purification by silica gel flash chromatography (50% EtOAc in n-Hex) yielded a yellow solid. (60.5 mg, 9.32%) As a result of the NMR analysis, two products were made. : 4-(3,5-dinitrobenzyl) 1-methyl 2-methylmaleate (25%) 1H NMR (500 MHz, DMSO-d6) δ 8.79 (m, 1H), 8.66 (d, J=3.45 Hz, 2H), 6.22 (s, 1H), 5.41 (s, 2H), 3.66 (s, 3H), 2.04 (s, 3H), 13C NMR (126 MHz, DMSO-d6) δ 168.3 (1C, O═C), 165.5 (1C, O═C), 148.5 (2C, C—N(═O)═O, 145.2 (1C, C═), 140.4 (1C, Ph), 129.7 (2C, Ph), 120.9 (1C, HC═) 118.9 (1C, Ph), 64.4 (1C, OCH2), 52.2 (1C, CH3O), 20.3 (1C, CH3), 1-(3,5-dinitrobenzyl) 4-methyl 2-methylmaleate (75%): 1H NMR (500 MHz, DMSO-d6) δ 8.81 (m, 1H), 8.68 (t, J=3.45 Hz, 2H), 6.18 (s, 1H), 5.48 (s, 2H), 3.62 (s, 3H), 2.07 (s, 3H), 13C NMR (126 MHz, DMSO-d6) δ 168.3 (1C, O═C), 165.5 (1C, O═C), 148.5 (2C, C—N(═O)═O), 145.2 (1C, C═),140.4 (1C, Ph), 128.8 (2C, Ph), 121.7 (1C, HC═) 118.7 (1C, Ph), 64.8 (1C, OCH2), 52.6 (1C, CH3O), 20.2 (1C, CH3). HRMS (ESI): Exact mass cal. for C13H12N2O8Na+[M+Na]+=347.0486; Found 347.0485.
Cyanomethyl methyl phthalate (15) Prepared according to the general procedure C using monomethyl phthalate (95.5 mg, 0.53 mmol), bromoacetonitrile (1.0 mL, 14.4 mmol) and triethylamine (1.0 mL, 7.17 mmol).
The reaction mixture was stirred at room temperature for 2 h. The resulting solution was washed with 1 M HCl, 4% NaHCO3, and brine. The organic layer was dried over MgSO4, filtered, and concentrated under vacuum. The product was obtained as a colorless liquid (109.5 mg, 94.3%). : 1H NMR (500 MHz, DMSO-d6) δ 7.73-7.84 (m, 4H), 5.22 (s, 2H), 3.85 (s, 3H), 13C NMR (126 MHz, DMSO-d6) δ 167.3 (1C, O═C), 166.5 (1C, O═C), 132.8 (1C, Ph), 132.5 (1C, Ph), 131.6 (1C, Ph), 130.3 (1C, Ph), 129.6 (1C, Ph), 129.5 (1C, Ph), 116.1 (1C, C--N), 53.3 (1C, OCH3). 50.6 (1C, OCH2). HRMS (ESI): Exact mass cal. for Cn11H9NO4Na+[M+Na]+=242.0424; Found 242.0425.
Tables 1 and 2 below show the cyclization efficiency.
S-(4-((2-aminoethyl)carbamoyl)benzyl) 2-hydrazineylpropanethioate (Hz) (+) -Methyl D-lactate was obtained from a commercial supplier (Sigma-Aldrich) and used as received: To a solution of (+)-methyl D-lactate (1.43 mL, 15.0 mmol) in DCM (45 mL) was added trifluoromethanesulfonic anhydride (3.28 mL, 19.5 mmol) and 2,6-lutidine (3.47 mL, 30.0 mmol) at 0° C., and the reaction was stirred at the same temperature until full consumption of the starting material (confirmed by TLC). To this was then added tert-butyl carbazate (3.96 g, 30.0 mmol), and the resulting mixture was further stirred at 0° C. for 4 h, then at room temperature for 16 h. The reaction mixture was diluted with DCM and washed with H2O, brine and 1 M HCl. The organic layer was then dried over anhydrous MgSO4, concentrated under reduced pressure, and purified by flash column chromatography (30% EtOAc/n-Hex) to furnish [(tert-butoxycarbonyl)amino]-L-alanine methyl ester as a yellow oil (2.64 g, 81%).
The methyl ester (2.44 g, 11.2 mmol) obtained above was then dissolved in 1:1 mixture of THF/HO (24 mL) and treated with LiOH-H2O (940 mg, 22.4 mmol). After stirring at room temperature for 3 h, the mixture was concentrated under reduced pressure and the remaining aqueous layer was washed with EtOAc. The aqueous layer was then acidified to pH ˜1 using 1M HCl (aq, extracted with EtOAc, dried over anhydrous MgSO4, and concentrated under reduced pressure to give [(tert-butoxycarbonyl)amino]-L-alanine as a thick colorless oil (2.08 g, 91%).
Prepared according to General Procedure C using [(tert-butoxycarbonyl)amino]-L-alanine. (428 mg, 2.1 mmol), Boc-ABT (465 mg, 1.5 mmol), DMAP (512 mg, 4.2 mmol), EDC·HCl (803 mg, 4.2 mmol) and DCM (10 mL). Purification by flash column chromatography (60% EtOAc in n-Hex) afforded the corresponding Boc-protected product as a colorless oil (338 mg, 45%). The deprotection was achieved upon treatment with 4 M solution of HCl in 1,4-dioxane, and the resulting product was used without further purification and characterization. (Boc)2-(Hz): 1H NMR (500 MHz, DMSO-d6) δ 8.40 (t, J=5.11 Hz, 1H), 8.31 (br s, 1H), 7.76 (d, J=7.95 Hz, 2H), 7.37 (d, J=8.15 Hz, 2H), 6.89 (t, J=5.38 Hz, 1H), 4.06 (s, 2H), 3.68 (d, J=5.53 Hz, 1H), 3.28 (q, J=6.04 Hz, 2H), 3.10 (q, J=6.06 Hz, 2H), 1.38 (s, 9H), 1.37 (s, 9H), 1.15 (d, J=6.96 Hz, 3H)13C NMR (126 MHz, DMSO-d6) δ 204.0 (1C, O═C—S), 166.5 (1C, O═C—NH), 156.2 (2C, O═C—O), 141.8 (1C, Ph), 133.6 (1C, Ph), 129.0 (2C, Ph), 127.8 (2C, Ph), 79.3 (1C, O—C), 78.1 (1C, O—C), 65.5 (1C, NH-CH), 42.2 (1C, NH—CH2—CH2-NH, Overlapping Solvent peak), 31.8 (1C, S—CH2), 28.7 (3C, (CH3)3), 28.6 (3C, (CH3)3), 17.3 (1C, CH3). HRMS (ESI): Exact mass cal. for C23H37N4O6S+[M+H]+=497.2428; Found 497.2428.
The ribosome, an RNA-based catalyst, facilitates the formation of peptide (amide) bonds through a mechanism known as entropy trapping. By positioning amino-acyl tRNA molecules in close proximity within a confined space, the ribosome reduces their conformational freedom, thereby enhancing the efficiency of peptide bond formation. This approach contrasts with typical catalytic strategies in synthetic chemistry, such as acid-base catalysis, and highlights the ribosome's ability to synthesize biopolymers (e.g., peptides or proteins). Uniquely, the ribosome, working in conjunction with other cellular machinery, can achieve sequence-defined, template-directed polymerization with high fidelity at speeds up to 17 monomers/second. The core reaction, aminolysis, involves the nucleophilic α-amino group of an amino acid linked to the transfer RNA (tRNA) at the ribosome's A-site attacking the electrophilic carbonyl carbon in the ester linkage of the tRNA in the ribosome's P-site (
Building on previous efforts to expand the range of substrates, the field has advanced in developing peptide isosteres by leveraging the ribosome's capability for synthesizing linear polypeptides. The replacement of amino acids with structural analogs such as hydroxy-, thio-, and aminocarbothio-acid has demonstrated that the ribosome can directly produce polyesters, polythioesters, and polythioanides, respectively. Another significant advancement that challenges the natural functions of the ribosome is the introduction of cyclic backbones into peptides. These cyclic motifs are important for peptide-based natural products as they enhance proteolytic resistance, structural rigidity, and specificity towards target proteins, often resulting in increased drug efficacy. Such examples include penicillin V (dipeptide of Cys-Val), goadsporin (polyazole antibiotics), and patellamide C (macrocyclic antimicrobial peptides) (
In this invention, the inventors aim to show the ribosome's ability to directly synthesize new and diverse cyclic structures, broadening the spectrum of non-canonical backbones that can be used towards synthesis and discovery of new and improved medicines. To achieve this goal: (i) the inventors design two sets of bifunctional substrates, each containing two electrophiles and nucleophiles within the molecule, respectively (
The synthesis of cyclic backbones using the ribosome requires ncM-acylated tRNAs (
Diesters and hydrazines have been used as substrates to produce heterocyclic structures, where the diester serves as a scaffold for the cyclic structure and react with the hydrazine via a nucleophilic substitution reaction at the two reactive groups. This reaction, however, typically requires high temperatures and a catalyst (acid or base),42, 43 along with an excess of one of the reactants. Utilizing the ribosome as a catalyst for synthesizing this motif would provide a novel platform, enabling rapid synthesis in an aqueous solution under moderate temperatures (30-37° C.) as well as taking advantage of the ribosome's template-guided sequence-specific polymerization capability. To design a diester substrate compatible with Fx, the inventors first converted an acid moiety to an ester containing an FLG (
Fx-mediated acylation of ncMs and ribosome-mediated synthesis of cyclic motifs
The inventors conducted Fx-mediated reaction in two stages to charge ncMs onto tRNAs. Initially, the inventors used a short microhelix tRNA (mihx), a 22-nucleotide tRNA analog, to determine the optimal conditions for acylation. This method offers rapid analysis because acylated mihx, with its increased molecular weight, can be distinguished from unacylated mihx due to their differing mobility on a polyacrylamide gel (
The inventors first charged substrate m (a malonic ester derivative) and Hz to microhelix (
To achieve this, the inventors performed a Fx-mediated acylation reaction with substrate m to tRNAmet(CAU). For ribosome-mediated biosynthesis, the inventors purified the acylated tRNA through ethanol precipitation to remove any unreacted substrate m. The resulting m: tRNAfmet (CAU) conjugate was then re-suspended in an aqueous buffer and introduced into PURExpress™ (NEB), a reconstituted in vitro protein synthesis system. To facilitate the analysis of the products, the inventors designed a reporter gene producing a Strep tag (XWHSPQFEK) on a T7 promoter-controlled DNA plasmid (pJL1_X-StrepII), where X indicates the position to which Fx-charged m is incorporated. The inventors selected the Strep tag due to its high affinity for Strep-Tactin and the ability to elute target peptides under mild denaturing conditions (0.1% SDS). The reaction was carried out at 37° C. for 2 h in the presence of the other eight amino acids required for synthesis of the peptide containing a Strep tag. The reaction products were purified, eluted, and subsequently characterized via MALDI-TOF mass spectrometry. The mass spectrum analysis identified a peak corresponding to the theoretical mass ([M+H]+=1212.5; [M+Na]+=1234.5; [M+2Na—H]+=1256.5) of the product containing m at the N-terminus (
For substrate Hz, the inventors carried out Fx-mediated reaction for tRNAPro1E2(GAU) using the same method. An engineered proline-tRNA was chosen for its efficient incorporation of ncMs into peptides in previous studies. The resulting Hz:tRNAPro1E2(GAU) conjugate was introduced to a PURExpress reaction containing m:tRNAfMet (CAU) and a DNA plasmid (pJL1_XY-StrepII). This template encodes a Strep tag (XZWHSPQFEK), containing the two ncMs at positions X and Z (
Exploring further, the inventors next tested ribosomal synthesis utilizing a wide spectrum of monomers containing diverse non-canonical backbones for incorporation into a growing polymer by the ribosome (
Intriguingly, the mass spectrum (
The inventors also considered the scenario in which the appropriate substrates are incorporated into the peptide, but the ring closing reaction is not efficiently carried out, resulting in a linear product (
Mechanistically, the formation of cyclic structure between a diester and a hydrazinoester, two sequential aminolysis reactions must occur within the ribosome. This involves release of both tRNA and alkoxide (R1O−) from the two carbonyls of diester substrates. In the two reactions using substrates m and s, methoxide (CH3O−), a conjugate base of methanol, with high basicity (pKa of ˜16) is expelled as a leaving group. The inventors hypothesized that high basicity makes methoxide less prone to dissociate and that altering the R1 substituent to groups with different pKa may affect the kinetics of ring-closure reaction. To test this hypothesis, the inventors designed malonate and succinate derivatives with various R1 groups (tert-butyl, ethyl, benzyl and phenyl) and various pKas (
The cyclization kinetics are influenced by the R1 substituents.
First, the inventors synthesized a range of malonic ester derivatives (substrates 1-3), varying the R1 group (ethyl (1), methyl (2), and benzyl (3)) to evaluate its impact on forming 5-membered rings (
The inventors next examined the impact of R1 substituents on the formation of 6-membered rings. To explore this, the inventors used nine succinic ester derivatives (substrates 4-12) with different R1 groups (
Cyclization does not occur in solution spontaneously.
Our data demonstrate the ribosome's ability to form two novel cyclic hydrazinedione derivatives (
Ribosomal catalysis is required for cyclization.
To directly demonstrate that the cyclic hydrazinedione motif results from the ribosome's catalytic activity, the inventors conducted an additional experiment using a functional RNA molecule that mimics the peptidyl transferase center (PTC) components of the E. coli 23S ribosomal RNA (
The ribosome naturally prefers L-α-amino acids for peptide bond formation. Several pioneering studies have shown that the ribosome can incorporate β-amino acids, albeit with considerably reduced efficiency compared to α-amino acids. This reduced efficiency is presumably due to the extended carbon chain of β-amino acids, which hinders the optimal alignment of the β-nitrogen within the ribosome's peptidyltransferase center for an effective nucleophilic attack on the P-site tRNA. Substrate Hz, which has an NH2 group linked to another NH2 group at both the a and B positions, presents unique properties. In reactions involving hydrazine with carboxylic acid derivatives, the β-nitrogen is the primary atom used for bond-forming reactions due to steric hindrance around the α-nitrogen (
The cyclic structures explored in this work have two possible regioisomers. In one scenario, the α-nitrogen atom of the incoming hydrazine ester attacks the ester of the P-site tRNA, while the β-nitrogen targets the ester carbonyl substituted with R1 (
Direct production of non-ribosomal peptide-like materials may significantly enhance the diversity of peptide drug candidates in a library, particularly when prepared for screening via drug discovery platforms. For example, the RaPID (Random non-standard Peptides Integrated Discovery) system leverages the advantages of diverse cyclic structures, which are crucial for drug efficacy, with template-guided synthesis, inherent to ribosome-based synthesis. These structures contribute to improved thermodynamic stability, resistance to proteolytic degradation, and protein-protein interactions important in therapeutics. To test the ribosome's ability to produce non-ribosomal peptide-like materials with multiple alternative backbones in a peptide, the inventors designed an additional plasmid (pJL1-XZ-StrepII-X′Z) that allows site-specific, alternating incorporations of diester substrates and Hz. In a separate tube containing PURExpress™ (NEB) reaction materials, the inventors supplemented Fx-charged tRNAfMet (CAU): 1 and tRNAPro1E2 (GUC): 12, with tRNAPro1E2 (ACG):Hz, respectively, for multiple ring-closing reactions. The mass spectra (
In this work, the inventors demonstrate ribosome-mediated biosynthesis of non-standard cyclic motifs using rationally designed diester and hydrazine substrates (non-canonical monomers, ncMs). These ncMs were charged to tRNA by an acylating-ribozyme then consecutively incorporated into a peptide, with the ribosome catalyzing multiple aminolysis reactions in its catalytic site. Through a series of chemical reactions and mass spectrometric analysis, the inventors revealed that the α-nitrogen atom of the hydrazine monomer extends the monomer onto the nascent peptide chain, while the β-nitrogen undergoes a ring-closing reaction in the ribosome. By varying the length of the monomer scaffolds, the inventors achieved the formation of diverse five- and six-membered cyclic backbones on a peptide. Cyclization efficiencies between 0 to 100% were observed through rational design of ester substituents.
The inventors believe this breakthrough bridges the gap between the chemical advantages of cyclic motifs in drug-like peptides with the ease of synthesis using the translation machinery, catalyzed by the ribosome. To our knowledge, this is the first demonstration of the ribosome's ability to modulate ring-closing reactions, leveraging the inherent biochemical properties of ncMs. Looking ahead, this platform could greatly accelerate the synthesis of heterocyclic motifs. For instance, a previous study showed that incorporating a 6-membered tetrahydropyridazinedione into a peptide required a seven-step process under highly acidic conditions.60 In contrast, our approach using the ribosome in vitro produces the same motif in an aqueous buffer within 2 h at 37° C. This capability could transform the synthesis of the five-membered pyrazolidine motif, already used in small-molecule drugs.61-65 It could also enhance the production of novel peptides library used for drug discovery platforms like RaPID. By integrating diverse cyclic structures into the range of peptidomimetics, this approach holds potential to accelerate the development of new therapeutics.
Interestingly, the reactions occurring within the ribosome appear to follow conventional chemical principles found in solution. For example, five-membered ring formation is faster than six-membered ring formation, and cyclization yield depends on the stability of the leaving group. However, kinetic studies (e.g., entropy, enthalpy, and reaction rates for the ribosome) remain largely unexplored. The inventors expect further kinetic investigations to improve our understanding of the ribosome's catalytic core, enabling the in vitro synthesis of an even greater array of cyclic structures. Non-canonical monomer polymerization efficiency could potentially be improved by incorporating an updated set of translational components, such as EF-P, EF-Tu, tRNAs, and ribosomes specially tailored to accommodate ncMs. Future developments in this field may expand the spectrum of possible chemical reactions, potentially transforming our understanding of polymerization chemistry and producing the next generation of medicinal molecules.
The scope of the present invention is indicated by the claims described later rather than the detailed description above. All changes or modified forms derived from the meaning and scope of the patent claims and their equivalent concepts should be construed as being included in the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2023-0114636 | Aug 2023 | KR | national |
| 10-2024-0095918 | Jul 2024 | KR | national |
