Selective Desulfurization Significantly Expands Sequence Variety of 3′-Peptidyl–tRNA Mimics Obtained by Native Chemical Ligation

RNA–peptide conjugates that mimic acylated tRNA termini are valuable compounds for structural and functional studies of the ribosomal elongation cycle, particularly if they contain a hydrolysis-resistant linkage between the RNA and the peptide moiety.1 In a very reduced form, puromycin ((S)-3′-((2-amino-3-(4-methoxyphenyl)-1-oxopropyl)amino)-3′-desoxy-N,N-dimethyladenosine), which possesses an amide instead of an ester junction, represents such a stable conjugate.2 This mimic, for example, was positioned as a substrate in the P-site of the ribosomal peptidyl transferase center (PTC) to capture snapshots along the route to peptide bond formation, as analyzed by X-ray crystallography.3 A more recent example made use of short, stable, 3′-aminoacyl-RNA conjugates to explore how the nascent peptide chain triggers ribosomal stalling, as analyzed by a variety of biochemical methods.4 Straightforward experimental approaches to synthesize this type of bioconjugate are expected to stimulate further investigations and functional characterization of the different states along the ribosomal elongation cycle.5


N-Allyloxycarbonyl-L-glycine
L-Glycine (1 g, 13.3 mmol) and 5.5 eq Na 2 CO 3 (7.8 g, 73.3 mmol) were suspended in 60 mL tetrahydrofuran (THF)/H 2 O (1/2) and cooled to 0°C. Then 1.2 eq diallyl pyro carbonate (2.6 mL, 16.0 mmol) were added and the reaction mixture stirred over night at room temperature. After washing the reaction mixture three times with diethyl ether, the aqueous phase was acidified with concentrated HCl to pH 1 and extracted three times with CH 2 Cl 2 . The organic phase was dried over Na 2 SO 4 . The product was obtained as colorless oil. 1 H NMR (300 MHz, DMSO):

Deprotection of the N-9-(fluorenyl)methoxycarbonyl (Fmoc) group after RNA synthesis on solid support rA-Pen·3
After RNA synthesis, the solid support was rinsed with 20 mL of 20% piperidine in acetonitrile to remove Fmoc and cyanoethyl groups followed by washing with 20 mL acetonitrile.

Deprotection and cleavage of DNA-Cys(StBu) conjugates
After DNA synthesis, the solid support was rinsed with 20 mL of 20% piperidine in acetonitrile followed by 20 mL acetonitrile to remove cyanoethyl groups. 3'-Cysteinyl-DNA was deprotected with NH 3 in H 2 O (32%, 0.6 mL) and EtOH (0.2 mL) for 16 hours at 55°C. After filtration and evaporation to dryness, the crude 3'-cysteinyl-DNA was dissolved in 1 mL of nanopure H 2 O.

Mass spectrometry of 3'-aminoacyl-oligonucleotides
The purified 3'-aminoacyl-oligonucleotides were characterized by mass spectrometry on a Finnigan

Solid phase peptide synthesis
The peptide was synthesized manually in a 2 mL syringe with a filter membrane following standard Peptide thioesters MFFG-ABT, MLLT-ABT, MRVL-ABT, and MRVW-ABT were synthesized as described in reference 4. Leu-ABT was prepared as described in reference 9.

Synthesis of peptide thioester MRFF-ABT
For the synthesis of the protected peptide acid, phenylalanine-loaded 2-chlorotrityl resin (H-L-Phe-2chlorotrityl resin, 200-400 mesh, loading: 0.87 mmol/g, Iris Biotech) was used. After manual Fmoc/tBu solid phase peptide synthesis, the resin was treated with trifluoroethanol/CH 2 Cl 2 (2/8, 15 mL cleavage solution per gram resin) for 45 minutes at room temperature to cleave the crude protected peptide acid. 8 The filtrate was collected and the resin was washed twice with cleavage solution and once with DMF. The united filtrates were concentrated, precipitated in a mixture of cold diethyl ether/hexane (2/1, 30 mL per gram resin), and centrifuged at 4°C for 30 minutes (Hermle Z300K, 6000 rpm). The precipitate was washed twice with cold diethyl ether and then lyophilized out of acetonitrile/H 2 O (1/1).
The crude protected peptide acid was analyzed by RP-HPLC and ESI-MS, and used without further purification.
The ABT group was synthesized in five steps according to the literature. 9 The ABT group (1.4 eq) and the crude protected peptide acid were dissolved with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Analysis and purification of peptides
Analysis of crude products was performed by reversed phase (RP) chromatography on a GE within 20 column volumes; UV-detection at 210 nm. The crude product was purified on the same column using an optimized gradient. Fractions containing peptide were collected and lyophilized.  The reaction solution was exposed to ultrasound for 10 seconds and kept under argon atmosphere. Reactions were carried out at 37°C for 6 (short conjugates) to 12 hours (longer conjugates).

Analysis and purification of 3'-peptidyl-oligonucleotides after native chemical ligation/desulfurization
An aliquot of the reaction mixture was injected for anion-exchange HPLC analysis onto a Dionex Combined fractions of the desulfurized 3'-peptidyl-oligonucleotides were lyophilized.

Mass Spectrometry of 3'-peptidyl-oligonucleotides
The purified 3'-peptidyl-oligonucleotides were characterized by mass spectrometry on a Finnigan LCQ Advantage MAX ion trap instrumentation connected to an Amersham Ettan micro LC system