Synthesis of TATs-PEG-PEI
1. NHS-PEG-VS
[edit]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 (TATs) (short form of TAT)
NHS-PEG-VS: α-vinyl sulfone-ω-N-hydroxysuccinimide ester poly(ethylene glycol) 3.4 kDa 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 NHS-PEG-VS to a PEI-solution corresponding to 0.486 µmol.
React 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 the activated PEG-PEI
Stir the mixture for an additional 2 h at room temperature.
The resulting bioconjugates are 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
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.nanoc.net/PEG/Sulfhydryl-reactive-PEG/vinylsulfone-PEG-NHS.htm
http://www.jenkemusa.com/heterobifunctional-pegs
MAL-PEG-SCM, MAL-dPEG 24-NHS ester
[edit]
ThermoFisher SM(PEG)2:
• Reactive groups: NHS ester and maleimide
• Reactive toward: amino and sulfhydryl groups
• Irreversibly crosslink proteins or peptides by flexible PEG spacer arms
• Polyethylene glycol spacer arms help maintain conjugate solubility
• Pure compound with defined structure and molecular weight, ensuring reproducible protein-modification effects
• PEG spacer provides unique advantages, including increased stability, reduced tendency toward aggregation and reduced immunogenicity
• Ideal for performing controlled, two-step protein conjugations (see Sulfo-SMCC)
SM(PEG)n NHS- and maleimide-activated PEG compounds for crosslinking between primary amines (NH2) and sulfhydryl (SH) groups in proteins and other molecules. A complete set of compounds are offered, each having the same heterobifunctional structure (NHS-PEGn-Maleimide) but differing in the number of discrete ethylene glycol units ( n = 2, 4, 6, 8, 12 or 24). The N-hydroxysuccinimide ester (NHS) group reacts specifically and efficiently with lysine and N-terminal amino groups at pH 7-9 to form stable amide bonds. The maleimide group reacts with reduced sulfhydryls at pH 6.5-7.5 to form stable thioether bonds.
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.
maleimides react selectively with the thiols of cysteine residues by Michael addition in the pH range 6.5–7.5.
MAL-dPEG 24-NHS ester can be used for Crosslinking of Peptides (Amine PEGylation) to Thiolated Nanoparticles
Polyethylenimine 25 kDa (PEI) cat. no. 23966 : 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 (PEG/PEI ratios from 4.5 to 30).
PEI 25000: 25 mg-->1 umole
1 ml of 2.5 mg/ml PEI25000==> (2.5 mg)1 umole
For 1:5 PEG/PEI ratio: need 5 umoles of PEG
MAL-PEG-SCM, MW 2K, 2 mg ==>1 umole, 10 mg===> 5 umole
PEG Stock: 25 mg/500 ul===> 5mg/100 ul
Stock: 250 mg PEI25000 in 10 ml 0.1 M Na-borate, 25 mg/1000 ul==>2.5 mg/100 ul
2.5 mg PEI (100 ul PEI stock), + 5 mg PEG2K in 100 ul 0.1 M Na-Borate. react at RT o/n
The activation reaction is 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 unreacted PEG and low molecular weight residues using 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 by a copper assay. 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 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.jenkemusa.com/?s=MAL-PEG-SCM&post_type=product&tags=1&ixwps=1
https://advancedbiochemicals.com/product/mal-peg-scm/
http://www.guidechem.com/trade/pdetail3340243.html
https://www.jk-scientific.com/Product/ProductDetails/27603 1g: $1350
3. OPSS-PEG-NHS (Orthopyridyl disulfide PDP PEG Succinimidyl ester)
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OPSS-PEG-NHS is a linear heterobifunctional PEG reagent with an OPSS and a NHS ester. It is a useful crosslinking reagent with a PEG spacer. OPSS stands for ortho-pyridyl disulfide or ortho pyridyldithio and reacts with thiol, SH, sulfhydryl or mercapto to form a S-S bond. OPSS is sometimes called PDP, pyridyl dithio propionate. NHS ester is an activated carboxylic acid and can react with amine. Can be used to Crosslinking Antibody to Nanoparticles
Orthopyridyl disulfide functionalized polyethylene glycol NHS(NHS-PEG-OPSS) is athiol (-SH) and amino reactive PEG derivative that can be used to modify protein, peptide or any other surfaces via available thiol (sulfulhydryl) group in cysteine residues to generate acleavable disulfide bond. OPSS PEGs react with thiol groups at pH 5.0~6.5.
Heterobifunctional OPSS PEG Succinimidyl Carboxymethyl Ester products from JenKem Technology are generally employed as crosslinking agents or as spacers between two different chemical entities. The PEG moiety in the heterofunctional PEG derivatives provides water solubility, biocompatibility, and flexibility. Thethiol reactive OPSS group offers the ability to form a disulfide bond, while the SCM group reacts with the amine group of lysine(s). Applications are especially geared towards the development of antibody drug conjugates (ADC’s).
PG2-NSOS-3k OPSS-PEG-NHS, MW 3400 $325
PG2-NSOS-2k OPSS-PEG-NHS, MW 2000 $385
http://www.nanocs.com/PEG/SPEG.htm
http://www.creativepegworks.com/product_view.php?cid=8&pid=3&sid=36&id=600
http://www.jenkemusa.com/product/opss-peg-succinimidyl-carboxymethyl-ester