-Custom Synthesis of Unnatural Aminoacyl-tRNA-
 

[SYNTHETIC SCHEME]
 

Flowchart of Custom Synthesis of Unnatural Aminoacyl-tRNA
 
  Step (i) : An unnatural amino acid (Xaa) is bound to a pdCpA (dinucleotide constituted deoxycytidine and adenosine) by chemical reaction. Step (ii) :The produced Xaa-pdCpA is ligated into a tRNA lacked 3’ terminal pCpA (dinucleotide including cytidine and adenosine) by T4 RNA ligase. The each steps of Xaa-tRNA synthesis are introduced below


Step (i) : Syntheses of Xaa-pdCpA

(Case.1) Fluorescent dyes or functional groups are bond at p-NH2 position of the aminophenyl-alanine-pdCpA (AF-pdCpA).
 
Scheme 1. Syntheses of Fluorescent Labeled AF-pdCpA
 
  The (Epsilon)- position of the lysine is also able to use for same reaction. The following figure shows binding between fluorescent dye-N-hydroxysuccinimide (NHS) ester and aminophenyl-alanin-pdCpA (AF-pdCpA) as example.

 Desired fluorescent dyes or functional groups with NHS ester conjugate to AF-pdCpA.
Basically, NHS esters are used in the reaction.

  We can synthesize Xaa-pdCpA based on chemical structure of other amino acid. Furthermore, we can take on coupling carboxyl compounds for labeling (fluorescent dye carbonates, biotin carbonate, etc.) with succinimidyl ester or other functional group. See detail in Case 2.


(Examples of achievement in Case.1)

Fluorescent Labeling
          Fluorescein, Rhodamine

Biotin Labeling
          Biotin and Biotin-X (Biotin coupled with C6 alkyl spacer), etc.


(Case.2) Alternative reaction-flow to Case.1 showed in the following figure is performed in multi-step process to produce Xaa-pdCpA
 
Scheme 2. Synthesis of unnatural amino acid-pdCpA

  We will propose the synthetic pathways of constructing a Xaa-pdCpA from souse compound that you will select. Then, we will start the synthesis of a Xaa-pdCpA.

(Examples of achievement in Case.2)

For photo-crosslink
        p-Benzolylphenylalanine
        p-Acetylphenylalanine

For imitation of post-transcriptional modification
        (Epsilon)-Methyl-Lysine
        (Epsilon)-Dimethyl-Lysine
        (Epsilon)-Acetyl-Lysine

For modification of polyethylene glycol


Step (ii) : Production of Xaa-tRNA

A Xaa-pdCpA synthesized in Step (i) is ligated into a tRNA lacked 3’ terminal pCpA by T4 RNA ligase.

(*)  We have developed two tRNAs coded with four bases (CGGG) or amber (UAG) anticodon for efficient incorporation into polypeptide translated in in vitro protein synthesis. In this reaction, it is strongly recommended to use the two patterns of tRNAs.


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