Editing Synthesis of TATs-PEG-PEI (section)

==  ==

MAL-PEG-SCM is a linear heterobifunctional PEG reagent with a maleimide and a succinimidyl NHS ester group. It is a useful crosslinking reagent with a PEG spacer. Maleimide reacts with thiol, SH, sulfhydryl or mercapto, and SCM stands for succinimidyl carboxyl methyl ester and can react with '''primary amine'''. Maleimide contains a reactive C=C double bond and is light or oxygen sensitive.

<span style="color: rgb(0, 0, 0); font-family: Helvetica, Arial, sans-serif; font-size: 14px;">maleimides&nbsp;react selectively with the thiols of cysteine residues by Michael addition in the pH range 6.5–7.5.</span>



MAL-dPEG 24-NHS ester can be used for Crosslinking of Peptides (Amine PEGylation) to '''Thiolated Nanoparticles'''

Polyethylenimine 25 kDa (PEI) cat. no. 23966&nbsp;: Polysciences (Warrington, PA).

Heterobifunctional pegylation crosslinker polyethylene glycol MAL-dPEG 24-NHS ester (PEG), cat. no. 10314 from Quanta BioDesign (Powell, OH).



TAT-peptide, GRKKKRRQRC

Block-copolymer syntheses:

'''Activation of PEI.'''

Weigh Bifunctional PEG containing both a maleimide and a N-hydroxysuccinimide ester groups into a glass flask.

1.55g boric acid, add 250 ml H2O, add 50 ul 10N NaOH===> 0.1 M, pH 5.5

Add PEI solution (2.83 mg/ml) in 0.1 mol/l borate buffer at pH 5.5 &nbsp;(PEG/PEI ratios from 4.5 to 30).

PEI 25000:&nbsp;25 mg-->1 umole

1 ml of 2.5 mg/ml PEI25000==> &nbsp;(2.5 mg)1 umole

For 1:5 PEG/PEI ratio:&nbsp;need 5 umoles of PEG

MAL-PEG-SCM, MW 2K, &nbsp;2 mg ==>1 umole, 10 mg===> 5 umole

PEG Stock: 25 mg/500 ul===>&nbsp;5mg/100 ul

Stock: 250 mg PEI25000 in 10 ml 0.1 M Na-borate, 25&nbsp;mg/1000 ul==>2.5 mg/100 ul

2.5 mg PEI (100 ul PEI stock), + 5&nbsp;mg PEG2K in 100 ul 0.1 M Na-Borate. react at RT o/n



The activation reaction is&nbsp;carried out for 4 hours at room temperature

Adjust pH adjustment to 7 with 10 mol/l NaOH.

'''Conjugation of the oligopeptide with the activated PEG–PEI.'''

Dissolve TAT-peptide in MilliQ (12 mg/ml concentration).

Add dissolved oligopeptide to the activated PEG–PEI for the reaction of maleimide residues of the bifunctional PEG with cystein sulfhydryl groups of TAT-peptide.

Stir the mixture for 2 hours at room temperature and kept in a refrigerator overnight at 4 °C with constant stirring.

'''Purification of TAT–PEG–PEI.'''

Remove&nbsp;unreacted PEG and low molecular weight residues using&nbsp;an ultrafiltration cell Amicon Ultra Ultracel-10k (Millipore, Bedford, MA) equipped with a 10 kDa molecular weight cutoff membrane and 0.1 mol/l borate buffer at pH 7.5 as an eluent.

Determine PEI concentration of the conjugate &nbsp;by a copper assay.&nbsp;The ratios of TAT/PEI in the conjugates were from 0.40 to 0.95.

Perrine, TD and Landis, WR (1967). Analysis of polyethylenimine by spectrophotometry of its copper chelate. J Polym Sci A1 5: 1993–2003.

Kunath, K, von Harpe, A, Petersen, H, Fischer, D, Voigt, K, Kissel, T et al. (2002). The structure of PEG-modified poly(ethylene imines) influences biodistribution and pharmacokinetics of their complexes with NF-kappaB decoy in mice. Pharm Res 19: 810–817

'''Polyplex formation.'''

Polyplexes consisting of plasmid DNA and TAT– PEG–PEI were prepared in sterile isotonic glucose solution at pH 7.4. Briefly, the polymer solution was added rapidly to the DNA and mixed by vortex followed by 20 minutes incubation at room temperature prior to use. When various PEI nitrogen to DNA phosphate ratios (N/P) were investigated, the concentration of the TAT–PEG–PEI solution was adjusted to the amount of DNA 20 μg/ml polyplex solution in order to obtain N/P ratios between 10 and 40.

[http://www.creativepegworks.com/product_view.php?cid=8&pid=2&sid=12&id=550 http://www.creativepegworks.com/product_view.php?cid=8&pid=2&sid=12&id=550] MAL-PEG-SCM, MW 3.4k MAL-PEG-SCM (Maleimide PEG Succinimidyl NHS acid ester)

PHB-951, PHB-950

MAL-PEG-SCM is a linear heterobifunctional PEG reagent with a maleimide and a succinimidyl NHS ester group. It is a useful crosslinking reagent with a PEG spacer. Maleimide reacts with thiol, SH, sulfhydryl or mercapto, and SCM stands for '''succinimidyl carboxyl methyl''' ester and can react with primary amine. Maleimide contains a reactive C=C double bond and is light or oxygen sensitive.

[http://www.polysciences.com/default/maleimide-peg-nhs-3000 http://www.polysciences.com/default/maleimide-peg-nhs-3000]



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

[http://www.jenkemusa.com/?s=MAL-PEG-SCM&post_type=product&tags=1&ixwps=1 http://www.jenkemusa.com/?s=MAL-PEG-SCM&post_type=product&tags=1&ixwps=1]

[https://advancedbiochemicals.com/product/mal-peg-scm/ https://advancedbiochemicals.com/product/mal-peg-scm/]

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

[http://www.guidechem.com/trade/pdetail3340243.html http://www.guidechem.com/trade/pdetail3340243.html]

[https://www.jk-scientific.com/Product/ProductDetails/27603 https://www.jk-scientific.com/Product/ProductDetails/27603] 1g: $1350