Stapled Peptides

Peptides that have been "hydrocarbon stapled" are peptides that have been chemically braced with an all-hydrocarbon staple at a specified position to keep them in their bioactive alpha-helical shape. To address the shortcomings of two major kinds of therapeutic agents (small molecules and protein biologics) in focusing on intracellular protein-protein interactions, the concept of peptide stapling was developed. Small molecules only interact with proteins that have a particular surface characteristic, and the majority of protein biologics do not enter cells. Cells are easily penetrated by stapled peptides because they are locked into a stable -helical structure, which is the most prevalent component of protein secondary structures. Stapled peptides, a rapidly developing class of next-generation pharmaceuticals, are anticipated to combine the three-dimensionality and adaptable target identification capabilities of biologics with the synthetic manipulability and cell penetration of small molecules. The company you should use to manufacture your stapled peptide needs is CPC Scientific because it has extensively developed stapled peptide architectures. You are welcome to contact our technical consultants at any moment to discuss your demands for structural design.

Stapled Peptide Chemistry

The first-generation Grubbs catalyst is a relatively stable ruthenium complex utilized for olefin metathesis in peptides. It has tricyclohexylphosphine (PCy3) ligands and apical-positioned carbene carbon. The creation of a second-generation Grubbs catalyst that is more thermally stable is the result of later research (middle). A third-generation catalyst, also known as the Hoveyda-Grubbs catalyst (right), substitutes benzylidene ligands with chelating ortho-isopropoxy groups on the benzene ring for N-heterocyclic carbene ligands.

lone peptide reaction stapled. A single stapled peptide can be produced by the Grubbs reaction when two alpha-4-n-pentenylalanine (S5) residues are incorporated into a peptide strand. This process is known as ring-closure metathesis. The staple type is referred to as an i, i + 4 when n = 3 (i.e., when there are 3 amino acids between the S5 residues).

In order to insert hydrocarbon building blocks into peptides during olefin metathesis, Grubbs catalysts are frequently utilized. The development of macro-cyclic bridges (conformational constraint) and,-di-substitution (helix nucleation by -methylation) are two different conformational techniques that are used to generate and stabilize a -helical shape.

Phillips and coworkers created the sequence Ac-His-S5-Ile-Leu-His-S5-Leu-Leu-Gln-Asp-Ser-NH2 (olefin bond between S5 and S5) in a stapled peptide made by CPC Scientific, where S5 is alpha-4-n-pentenylalanine before olefin metathesis (figure 2). When the peptide is completely protected and linked to the resin as Ac-His(Trt)-S5-Ile-Leu-His(Trt)-S5-Leu-Leu-Gln(Trt)-Asp(otBu)-Ser(tBu)-Rink Amide, the Grubbs reaction is carried out. MBHA Resin. The peptide resin was mixed with 20 mL of a bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride (1 mg/mL) solution in DCE. The reaction took place for two hours at 50 °C. In this illustration, the peptide is bound to the resin as the stapling process (also known as Grubbs Metathesis) is being carried out. In the image, two distinct kinds of hydrocarbon staples are used to illustrate how a stabilized -helix can be formed in a peptide. I and i+4 and i, i+7 would correspond to roughly one and two revolutions of the helix, respectively. Please consult the Cahn-Ingold-Prelog priority guidelines for naming stereoisomers for details on the R/S descriptors depicted in the picture.

A double stapled peptide was created to mimic the binding domain of the human angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 in a collaboration between the Laboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball Research Institute, New York Blood Center, and CPC Scientific. To cover the 30-amino-acid long binding domain of ACE2, a double staple motif of i + 7 and i + 4 was necessary.

Single Staple Configurations

Grubb’s ring-closure metathesis may result in two types of stapled peptides, i, i + 4 and i, i + 7.

Stapled Peptides in Drug Design

High amounts of -helical content are conferred by the addition of a hydrocarbon staple, which causes:

• A higher target affinity of 5 to 5,000 times

• Cell penetration by endocytic vesicle trafficking and increased proteolytic resistance

• selecting extracellular or intracellular proteins as a target

• protein-protein interactions being hampered

• Non-immunogenicity

• Pharmacokinetics that work and in vivo stability

Protein Targets

Targeting of many proteins involved in diseases like cancer, diabetes, HIV, and atherosclerosis has been researched using stapled peptides. Among these proteins are:

• B-cell lymphoma 2 (Bcl-2)

• B-cell lymphoma-extra large (Bcl-xL)

• Bcl-2-associated X protein (Bax)

• Induced myeloid leukemia cell differentiation(Mcl-1)

• Glucokinase (GK)

• Murine double minute 2 (Mdm2)

• Notch/CSL

• HIV-1 capsid and HIV-1 gp41

• ATP-binding cassette transporter (ABCA1)

• Estrogen receptor

Figure 5. i, i + 4 Stapled peptide (Ac-His-S5-Ile-Leu-His-S5-Leu-Leu-Gln-Asp-Ser-NH2) bound to estrogen receptor. Stapling organizes non-adjacent leucine side chains (Leu-4 and Leu-8) project from the same side into the hydrophobic pocket of estrogen protein.[1]

Modifications of Stapled Peptides

The two types of stapled peptide modifications are fluorescent label and affinity tag, respectively. Fluorescein and biotin are two of the most prevalent moieties added to the N-terminus of stapled peptides, and they can be employed for biophysical characterisation and target interaction studies as well as studies of intracellular absorption. To separate the alteration from the peptide's core, a flexible molecular spacer is typically preferred.

• N-acetylation

• Linker attachment (β-alanine, mini-PEG, etc.)

• Fluorescent labeling (FITC, 5-FAM, etc.)

Template-Assisted Β-Sheet Stapled Peptide

Peptide stapling can also be used to reproduce other secondary protein structures, such as -sheets. In the example below, a template-assisted olefin metathesis reaction using a third-generation Hoveyda-Grubbs catalyst converts terminal olefins into internal olefins. Due to stability of the anti-parallel -sheet configuration created by the six hydrogen bonds, only one conformer is visible.[3]

Stapled Peptides by Click Chemistry

It is possible to easily synthesize triazole-stapled peptides thanks to the high efficiency and benign circumstances of the "click" reaction (Copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction) and the availability of the unnatural amino acids that are required. To make single triazole-stapled peptides, for instance, L-Nle (N3) and D-Pra (D-propargylalanine) can be combined and substituted at the i and i+4 locations, respectively.

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References

1. Phillips, Chris, et al. Journal of the American Chemical Society 133.25 (2011): 9696-9699 (PDB: 2YJA).

2. Curreli, Francesca, Sofia MB Victor, Shahad Ahmed, Aleksandra Drelich, Xiaohe Tong, Chien-Te K. Tseng, Christopher D. Hillyer, and Asim K. Debnath. Mbio 11, no. 6 (2020): e02451-20.

3. Gothard, Chris. Unpublished work in the Nowick Group (2005).