Tyrosine sulfate can be encrypted by incorporation of sulfate monoesters. Neopentyl is popular with synthetic chemists. I expect you'll want the ester cleaved inside the bacteria, and nP can be difficult to remove. If you're lucky the bacteria may possess an enzyme that can do the trick.

Otherwise you could try a sodium salt of the sulfate, but the sodium may come off too easily in your aqueous media.

You could try polymyxin B nonapeptide (PMBN). It enhances the permeability of Gram negative membranes but doesn't kill the cells. If the unnatural amino acid is a substrate for certain efflux pumps, its permeability might be increased by an efflux pump inhibitor such as phenylalanine-arginine beta-naphthylamide. Both of these substances are commercially available.

Thank you so much for your suggestions. I will eead more detail and protocol to do work.

To demonstrate how the membrane of E. coli BL21(DE3) is permeable to negatively charged unnatural amino acids such as sTyr, here are some steps to take:

1- Introduce or enhance the expression of membrane transporters that can facilitate the uptake of negatively charged amino acids. For example, engineer amino acid permeabilizers or create mutants with altered substrate specificity.

2- Chemical treatments with detergents: Use nonionic detergents (such as Triton X-100) at concentrations below lethal to temporarily permeabilize the membrane, allowing sTyr to enter more easily.

Sodium salts: Treating cells with sodium salts (such as NaCl or KCl) can sometimes enhance membrane fluidity, potentially increasing its permeability.

3- Adjust pH: Change the pH of the growth medium to a level where the charge state of sTyr may be more favorable for membrane diffusion.

4- Use of polyethylene glycol to temporarily increase the permeability of the cell membrane for sTyr uptake.

Mohammed Rafeeq Ali Thank you so much. i am trying to optimize with your suggestions as well.