"L19K was synthesized by CPC Scientific and comprised the sequence NO2A-PEG4-GGNECDIARMWEWECFERK-CONH2, with Cys-Cys disulfide bridge and polyethylene glycol (PEG4) as a spacer between peptide and chelator. "
Abstract
Imaging agents based on peptide probes have desirable pharmacokinetic properties provided that they have high affinities for their target in vivo. An approach to improve a peptide ligand’s affinity for its target is to make this interaction covalent and irreversible. For this purpose, we evaluated a 64Cu-labeled affinity peptide tag, 64Cu-L19K-(5-fluoro-2,4-dinitrobenzene) (64Cu-L19K-FDNB), which binds covalently and irreversibly to vascular endothelial growth factor (VEGF) as a PET imaging agent. We compared the in vivo properties of 64Cu-L19K-FDNB in VEGF-expressing tumor xenografts with its noncovalent binding analogs, 64Cu-L19K-(2,4-dinitrophenyl) (64Cu-L19K-DNP) and 64Cu-L19K. Methods: The L19K peptide (GGNECDIARMWEWECFERK-CONH2) was constructed with 1,4,7-triazacyclononane-1,4,7-triacetic acid at the N terminus for radiolabeling with 64Cu with a polyethylene glycol spacer between peptide and chelate. 1,5-difluoro-2,4-dinitrobenzene was conjugated at the C-terminal lysine for cross-linking to VEGF, resulting in L19K-FDNB. 64Cu-L19K-FDNB was assayed for covalent binding to VEGF in vitro. As a control, L19K was conjugated to 1-fluoro-2,4-dinitrobenzene, resulting in L19K-DNP. PET imaging and biodistribution studies of 64Cu-L19K-FDNB, 64Cu-L19K-DNP, and the native 64Cu-L19K were compared in HCT-116 xenografts. Blocking studies of 64Cu-L19K-FDNB was performed with a coinjection of excess unlabeled L19K-FDNB. Results: In vitro binding studies confirmed the covalent and irreversible binding of 64Cu-L19K-FDNB to VEGF, whereas 64Cu-L19K-DNP and 64Cu-L19K did not bind covalently. PET imaging showed higher tumor uptake with 64Cu-L19K-FDNB than with 64Cu-L19K-DNP and 64Cu-L19K, with mean standardized uptake values of 0.62 ± 0.05, 0.18 ± 0.06, and 0.34 ± 0.14, respectively, at 24 h after injection (P < 0.05), and 0.53 ± 0.05, 0.32 ± 0.14, and 0.30 ± 0.09, respectively, at 48 h after injection (P < 0.05). Blocking studies with 64Cu-L19K-FDNB in the presence of excess unlabeled peptide showed a 53% reduction in tumor uptake at 48 h after injection. Conclusion: In this proof-of-concept study, the use of a covalent binding peptide ligand against VEGF improves tracer accumulation at the tumor site in vivo, compared with its noncovalent binding peptide analogs. This technique is a promising tool to enhance the potency of peptide probes as imaging agents.
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CPC Scientific at DCAT 2025!
GMP Peptide Manufacturing
What an amazing time we had at DCAT 2025! It was fantastic to meet with colleagues, clients, and friends, and showcase CPC Scientific’s cutting-edge capabilities in synthetic API development and commercial production.
A heartfelt […]
We are excited to share that CPC Scientific Inc. has officially received the Good Manufacturing Practice (GMP) Certificate in accordance with the ISO 22716:2007(E) Cosmetic Guidelines.
This certification highlights our unwavering commitment to delivering the highest quality peptide ingredients for cosmetic use, guaranteeing:
- Superior Quality Control – Striving for excellence in safety, consistency, and purity.
[…]
In Part 1 of our Minimal Protection Group Strategies for SPPS, we discussed methods for eliminating sidechain protection on hydroxy-bearing amino acids such as serine, threonine, tyrosine, and hydroxyproline. By omitting t-butyl protection, we enhanced atom economy and avoided the use of hazardous solvents typically required to remove these protection groups. In Part 2, we present a new case study, expanding our approach to include the unprotected side chains of histidine, tryptophan, and arginine. We demonstrate the synthesis of a Goserelin peptide API impurity, showcasing how a convergent peptide fragment strategy can be used to eliminate the need for TFA and diethyl ether, eliminate side chain protection of Arginine, Histidine, and Tryptophan.
The synthesis of the linear RP-182 analog, bicyclo[6.1.0]non-4-yn-9-ylmethyloxycarbonyl-PEG2-Lys-Phe-Arg-Lys-Ala-Phe-Lys-Arg-Phe-Phe-Lys(azido-PEG)-NH2, was achieved using standard solid-phase peptide synthesis (SPPS) protocols. After cleaving the linear peptide from the resin, macrocyclization was performed in the liquid phase through a strain-promoted click reaction. BCN introduces extra ring strain due to its fused cyclopropane structure. The combined effect of ring strain, the selection of BCN, and copper catalysis significantly increases the macrocyclization efficiency of longer peptides like RP-182.
Nakagawa, Mayumi, Teresa Evans, Milan Bimali, Hannah Coleman, Jasmine Crane, Nadia Darwish, Jennifer L. Faulkner et al. MedRxiv (2025): 2025-01.
The vaccine consisted of four current good manufacturing production-grade synthetic peptides covering the HPV 16 E6 protein [amino acid (aa)1-45, 46-80, 81-115, and 116-158] (CPC Scientific, San Jose, CA [..]
Stapled peptides have emerged as a powerful tool in drug discovery and therapeutic development due to their ability to overcome the limitations associated with traditional peptide drugs, such as poor stability and low cell permeability. By introducing staples into the peptide backbone, researchers can stabilize peptide conformations and enhance their interactions with target proteins, resulting in improved efficacy and specificity. This approach not only addresses the challenges of peptide drug design but also opens new avenues for targeting challenging biomolecular interactions that are difficult to modulate with small molecules or antibodies. The development of stapled peptides has led to significant advancements in targeting protein-protein interactions, addressing previously intractable diseases, and enhancing the precision of therapeutic interventions.
Peptide receptor radionuclide therapy (PRRT) is a targeted therapeutic approach that utilizes peptides to deliver cytotoxic radiation to specific receptors overexpressed on cancer cells. Peptides offer several advantages as therapeutic vectors in PRRT due to their small size, favorable pharmacokinetics, high binding affinity, low immunogenicity and toxicity, and minimal off-target effects. Tumor-targeting peptides conjugated to radionuclide chelates represent a promising class of cancer therapeutics.
Singh, S.S., Calvo, R., Kumari, A., Sable, R.V., Fang, Y., Tao, D., Hu, X., Castle, S.G., Nahar, S., Li, D. and Major, E. Molecular Cancer Therapeutics (2024).
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[..] assembling the peptide on the Rink Amide resin and attaching the PEG azide moiety to the N-terminal Lys, the Dde group was removed as previously shown and coupled to the Fmoc-PEG2-acid. Removal of the Fmoc followed by simultaneously click/coupling to bicyclo[6.1.0]non-4-yn-9-ylmethyl (2,5-dioxopyrrolidin-1-yl) carbonate gave 1c which was deprotected and cleaved from the resin to give 1c.
Most proteinogenic peptides, except for certain hydrophobic sequences, can be synthesized in a linear fashion using solid-phase peptide synthesis (SPPS) methods. However, longer sequences, particularly those exceeding 70 amino acids, often require alternative techniques due to challenges like steric hindrance. Other issues, such as poor solvation of the protected peptide during synthesis and the formation of intermolecular hydrogen bonds (e.g., β-sheets) between fragments, can also result in inefficient coupling and deprotection.