β-Amino Acids Reduce Ternary Complex Stability and Alter the Translation Elongation Mechanism

Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to expand the chemical space available to biological therapeutics and materials, but existing technologies are still limiting. Addressing these limitations requires a deeper understanding of the mechanism of protein synthesis and how it is perturbed by nnAAs. Here we examine the impact of nnAAs on the formation and ribosome utilization of the central elongation substrate: the ternary complex of native, aminoacylated tRNA, thermally unstable elongation factor, and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (R)- and (S)-β2 isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-β3-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (R)- and (S)-β2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-β3-Phe stereoisomers were utilized inefficiently. (R)-β3-Phe but not (S)-β3-Phe also exhibited order of magnitude defects in the rate of translocation after mRNA decoding. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include the consideration of the efficiency and stability of ternary complex formation.


Figure S3
Figure S3.Ensemble FRET assay using mutant tRNA Phe (C49A G65U) and/or EF-Tu (N273A).A) Sequences of WT (left) and mutant (right) E coli tRNA Phe used for stopped-flow kinetic experiments.B) Structure of EF-Tu (PDB: 1OB2) with the N273 amino acid residue in the amino acid binding pocket shown next to the Phe (gray) aminoacylated to the A76 on the 3'end of tRNA Phe (yellow).C-E) Stopped-flow ternary complex formation assays using Cy3Blabeled mutant aa-tRNA Phe (MUT) with the indicated monomers inset.F-H) Stopped-flow ternary complex assays with both tRNA Phe MUT and mutant EF-Tu N273A with the indicated monomers inset.Error bars represent S.D. of 3-5 replicates.

Figure S4
Figure S4.Kinetic simulations of ternary complex formation.A) Minimal reaction scheme for kinetic simulations.Contour plots of B) L-α-Phe and C) (S)-β 3 -Phe ternary complex abundance simulated at different physiologically relevant aa-tRNA and ET-Tu/Ts concentrations.Simulations were done using measured rate constants from experiments reported here and from the literature.Ternary complex fraction was calculated as [aa-tRNA]bound/[aa-tRNA]total  Subsequently, the 3D atomic models and RNA-peptide links were constructed using JLigand 80 .
The figures were generated using PYMOL, the PyMOL Molecular Graphics System, Version 2.5.7 Schrödinger, LLC.

Synthesis of Boc-Protected β 2 -amino acids.
Note: The enantiomer of Intermediate D was prepared from Boc-L-Val-OMe.SuperQuat and N-acryloyl SuperQuat were prepared according to literature procedure (Asymmetric synthesis of b2-amino acids: 2-substituted-3-aminopropanoic acids from N-acryloyl SuperQuat derivatives.Org.Biomol. Chem., 2007, 5, 2812-2825.)Intermediate A and B were also prepared according to the same literature.For Intermediate A, we used a different eluent system (hexanes:EtOAc 10 :1 to 7:1, instead of hexanes:Et2O 20:1 in the literature condition) for the SiO2 column chromatographic purification to afford Intermediate A as pale yellow oil (980 mg, 1.96 mmol) in 28% yield (from 7 mmol N-acryloyl SuperQuat).For Intermediate B, same literature procedure was followed to afford Intermediate B as colorless oil (650 mg,1.74 mmol) in 89% yield (from 1.96 mmol of A).
Note: The enantiomer of Intermediate A and B were prepared from Boc-L-Val-OMe as the starting material by the same procedure.
Intermediate B (650 mg, 1.74 mmol) was dissolved in THF/t-BuOH (v:v, 3:1, total 16 mL, ca.0.1 M) and AcOH (1.6 mL).Pd(OH)2 (20% on carbon) (65 mg) was added to the solution.The reaction was degassed, connected to a H2 balloon and stirred at rt for overnight before filtration over Celite.The filtrate was concentrated under reduced pressure to afford Intermediate C as oil (364 mg, 1.44 mmol, 83%) which was subjected to next step without further purification.Same experimental sequence was carried out to prepare the enantiomer of Intermediate C.

General procedure for synthesis of β-amino acid-cyanomethyl ester-formate salt
To a stirred solution of Boc-protected-amino acid (0.5 mmol, 1 equiv.)was added dry DMF (1 mL), ClCH2CN (0.75 mmol, 1.5 equiv.)and dry DIPEA (1 mmol, 2 equiv.), the solution was stirred at rt for 16 h before dilution with EtOAc (10 mL).The organic phase was washed successively with 10% citric acid solution, 5 % LiCl aq.solution and brine.The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford the corresponding Boc-protected-Amino Acid-Cyanomethyl ester, which was used without further purification.Note: The cyanomethyl ester is very sensitive to aqueous basic medium.
The Boc-protected-Amino Acid-Cyanomethyl ester (ca 0.5 mmol) was treated with neat formic acid (2 mL).The solution was stirred at rt for 12 h before removing all the formic acid under reduced pressure by azeotropic distillation with CHCl3 to afford a pale-yellow oil.The oil was dissolved in minimum amount of THF (ca. 2 mL), triturated with excess MTBE or Et2O until white solid was formed persistently.All the residual solvent was removed under reduced pressure.The white solid was crushed into fine powder, rinsed thoroughly with Et2O (10 mL) and dried over vacuum for overnight.The typical yield over two steps was 50%.Note: The final product is very sensitive to water and alcohol which cause saponification or transesterification.

Procedure for anion metathesis with chloride
After removal of residual formic acid from the Boc deprotection, the oily form product (formate salt) was dissolved in minimum amount of THF (ca 2 mL).HCl (1M in Et2O) (1.5 mL, 3 equiv.)was added.The solution was stirred for 0.5 h before concentration to dryness, and repetitively triturated with excess MTBE or Et2O until white solid was formed persistently.All the residual solvent was removed under reduced pressure.The white solid was crushed into fine powder, rinsed thoroughly with Et2O (10 mL) and dried over vacuum for overnight.

Synthesis of α-amino acid-cyanomethyl esters
To a 5-mL round-bottom flask, N-Boc protected amino acid (0.5 mmol) was dissolved in 1 mL of tetrahydrofuran.Flask was then charged with 315 μL of chloroacetonitrile (5.0 mmol, 10 eq.), followed by addition of 100 L of N,N-diisopropylethylamine (0.6 mmol, 1.2 eq.).Flask was capped with septa and stirred at room temperature overnight, 16 hours.Solvent was removed via rotary evaporation then the crude material was purified by reverse-phase flash chromatography, 0-100% acetonitrile in water, holding at 60% acetonitrile until product was collected.Solvent removed via rotary evaporation, where the resulting oil was dissolved in 1 mL of tetrahydrofuran for deprotection.To the resulting solution, 1.9 mL of trifluoroacetic acid (25 mmol, 50 eq.)was added, and allowed to stir at room temperature for 2 hours.Upon completion, the solvent was removed followed by purification by reverse-phase flash chromatography utilizing a 2% acetonitrile in water mobile phase.Solvent was removed by lyophilization to yield target materials as colorless oils at 54% and 49% yield for phenylalanine and 4-azidophenylalanine derivatives respectively.Note: The final product is very sensitive to water and alcohol which cause saponification or transesterification.

Figure S2 Figure
Figure S2