What are cell penetrating peptides?
Cell Penetrating Peptides (CPPs) are short peptides that have the ability to cross biological membranes and enter cells. They are able to traverse the cell membrane by directly translocating across the lipid bilayer or by exploiting endocytic pathways.
CPPs are generally composed of 5-30 amino acids and are known for their high cell-penetrating efficiency and low toxicity. They can transport a wide variety of cargo molecules, such as small molecules, peptides, proteins, nucleic acids, and nanoparticles, into cells. This property makes CPPs valuable tools in various biomedical applications, including drug delivery, gene therapy, and molecular imaging.
The exact mechanism by which CPPs enter cells is not fully understood, but several theories have been proposed. One hypothesis suggests that CPPs interact with cell surface glycosaminoglycans or lipid rafts, leading to membrane destabilization and subsequent internalization. Other mechanisms involve direct membrane translocation through a non-specific, energy-dependent process or receptor-mediated endocytosis.
CPPs have been extensively studied and numerous sequences have been identified, with examples including penetratin, TAT peptide, and polyarginine. These peptides can be modified to improve their efficiency, stability, and specificity by incorporating different amino acid residues or chemical modifications.
How to synthesize cell penetrating peptides in 10 steps -- a general strategy.
1. Design the peptide sequence: Determine the amino acid sequence of the CPP based on the desired cellular target and cargo delivery.
2. Select protected amino acids: Choose the appropriate Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonyl) protected amino acids for SPPS. These protected amino acids ensure the desired peptide sequence is synthesized correctly.
3. Prepare the resin: Attach the first amino acid, typically Fmoc-protected, to a solid support resin. Popular resin choices include Wang, Rink amide, or 2-chlorotrityl chloride resin.
4. Fmoc deprotection: Remove the Fmoc protecting group from the attached amino acid using a deprotection reagent, such as 20% piperidine in dimethylformamide (DMF).
5. Coupling: Activate the next Fmoc-protected amino acid and couple it to the growing peptide chain using a coupling reagent, such as 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/ N,N-diisopropylethylamine (DIEA) or N,N'-diisopropylcarbodiimide (DIC) /HOBt (1-hydroxybenzotriazole).
6. Repeat steps 4 and 5: Alternate between deprotection and coupling until the desired peptide sequence is synthesized.
7. Final deprotection: Remove all remaining protecting groups from the fully synthesized peptide using a cocktail of cleavage reagents, such as trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water.
8. Purification: Purify the synthesized peptide by reverse-phase high-performance liquid chromatography (HPLC) using a suitable solvent system and detect the peptide using UV absorption at 214 nm.
9. Characterization: Analyze the purified peptide by various techniques, such as matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or electrospray ionization mass spectrometry (ESI-MS), to confirm the correct structure and purity of the synthesized CPP.
10. Cargo labeling: If necessary, conjugate the cargo molecule to the CPP using appropriate bioconjugation chemistry, such as maleimide-thiol or click chemistry, to enable efficient cargo delivery.
It is important to note that specific modifications and variations to this general strategy may be required depending on the specific CPP sequence and desired experimental application. More strategies for custom peptide synthesis will be introduced in the next few posts.
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