Editing Synthesis of TATs-PEG-PEI (section)

== 1. NHS-PEG-VS ==

N-hydroxysuccinimide (NHS) esters are reactive toward amines at physiological pH.

Vinylsulfone (VS) functionalized polyethylene glycol (VS PEG) is a thiol (-SH) group reactive PEG derivative that can be used to modify biomolecules or other materials via their available thiol groups. VS group react with free thiol groups easily in aqueous buffer between pH 6.5~8.5 at room temperature.



GRKKRRQRRRC (TAT)

GRKKRRQRC&nbsp;(TATs) (short form of TAT)

NHS-PEG-VS: α-vinyl sulfone-ω-N-hydroxysuccinimide ester poly(ethylene glycol) 3.4 kDa &nbsp;from Nektar Therapeutics (Huntsville, AL, USA).

Polyethylenimine 25 kDa (PEI): BASF

Synthesis was performed as previously described [18].

Dissolve NHS-PEG-VS (5.65 µmol) in 0.1 M borate buffer

Add 5.65 µmol of&nbsp;NHS-PEG-VS to a PEI-solution corresponding to 0.486 µmol.

React &nbsp;at pH 5.5 for 4 h at room temperature

Remove unreacted NHSPEG-VS by an ultrafiltration cell (Amicon, Bedford, MA, USA) using a 10-kDa molecular weight cutoff membrane (Millipore, Bedford, MA, USA).

Adjust pH to 7.

Add Tat to &nbsp;the activated PEG-PEI

Stir&nbsp;the mixture for an additional 2 h at room temperature.

The resulting bioconjugates are&nbsp;purified 15 times from unreacted low molecular weight residues, similar to the peptides, as described above.

Determine amine concentration of the bioconjugates by a recently described copper assay [19].

18. Kleemann E, Neu M, Jekel N, et al. Nano-carriers for DNA delivery to the lung based upon a TAT-derived peptide covalently coupled to PEG-PEI. J Control Release 2005; 109: 299–316.

19. Kunath K, von Harpe A, Petersen H, et al. The structure of PEGmodified poly (ethylenimines) influences biodistribution and pharmacokinetics of their complexes with NF-kB decoy in mice. Pharm Res 2002; 19: 810–817.



[http://www.nanocs.com/PEG/SPEG.htm http://www.nanocs.com/PEG/SPEG.htm]

PG2-NSVS-2k Vinyl sulfone PEG NHS, VS-PEG-NHS, MW 2000 $385

PG2-NSVS-3k Vinyl sulfone PEG NHS, VS-PEG-NHS, MW 3400 $325

[http://www.broadpharm.com/ http://www.broadpharm.com/]

[http://www.nanoc.net/PEG/Sulfhydryl-reactive-PEG/vinylsulfone-PEG-NHS.htm http://www.nanoc.net/PEG/Sulfhydryl-reactive-PEG/vinylsulfone-PEG-NHS.htm]

[http://www.jenkemusa.com/heterobifunctional-pegs http://www.jenkemusa.com/heterobifunctional-pegs]



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