Systematic Analysis and Industrial Application Research of Stapled Peptide Synthesis Technology
Chapter 1 Overview and Development History of Stapled Peptides
Stapled peptides are a class of modified peptides that stabilize the α-helical structure through an all-carbon scaffold. The concept was first proposed and synthesized by Verdine's research team in 2000. This technology emerged from breakthrough thinking about the limitations of traditional peptide drugs, providing innovative solutions to target protein-protein interactions (PPI).
From a molecular structural perspective, stapled peptides introduce two non-natural amino acids containing α-methyl and α-alkene into the α-helical peptide sequence, forming an all-carbon 'staple' structure via ring-closing metathesis (RCM). This unique molecular design endows stapled peptides with three significant advantages: firstly, their stability in the α-helical conformation is enhanced by 3-5 times compared to natural peptides; secondly, their cell membrane penetration efficiency can reach 10-100 times that of ordinary peptides; thirdly, their half-life in serum can be extended to over ten times that of natural peptides.
After more than twenty years of development, stapled peptide technology has gradually transitioned from initial laboratory research to industrial application. Particularly after 2010, with continuous optimization of synthetic processes and the emergence of innovative cyclization techniques, stapled peptides have shown groundbreaking application prospects in fields such as anti-tumor and anti-viral therapies. Currently, dozens of stapled peptide drugs have entered clinical research stages globally; among them is ALRN-6924 targeting p53-MDM2 interaction inhibitors which has entered Phase II clinical trials—marking a key step towards practical medical applications for this technology.
Chapter 2 Molecular Design Principles and Structural Features of Stapled Peptides
2.1 Core Structure Design Principles The molecular design principles for stapled peptides adhere strictly to structure-activity relationship principles. In terms of position selection, the insertion sites for two non-natural amino acids are typically spaced i,i+3/i+4/i+7 residues apart corresponding to one or two turns within an α-helical spatial structure. This spacing ensures that the 'staple' effectively maintains secondary structures without causing significant conformational distortion. Studies indicate that i/i+4 spacing exhibits optimal conformational stability and biological activity balance across most applications. In charge modulation aspects, designers often introduce positively charged amino acids (such as lysine) at the N-terminal to enhance transmembrane capability while introducing negative charges at C-terminal positions neutralizes inherent dipole moments associated with alpha helices. Such charge modification strategies not only improve cellular permeability but also significantly increase helical structural stability. A typical sequence structure for a staped peptide may be represented as: Ac-Leu-Glu-X₁-Gln-Ala-X₂-Lys-Leu-NH₂ where X₁ and X₂ represent specially modified non-natural amino acids. 2.2 Progress on Structure-Activity Relationship Studies In recent years，with advancements in structural biology & computational chemistry，notable progress has been made regarding studies on staple-peptide relationships。Through x-ray crystallography & nuclear magnetic resonance technologies，researchers found staples not only stabilized alpha helix structures but could also enhance binding specificity toward target proteins via conformational selection mechanisms。Molecular dynamics simulations suggest ideal lengths for staple bridges typically range between eight - twelve atoms；this spatial scale guarantees sufficient conformational constraint without inducing excessive rigidity。 It’s particularly noteworthy that biological activity exhibited by these staples shows nonlinear correlation with its helical content；excessively high levels may actually lead reduced affinity towards target proteins indicating fine tuning based upon specific targets characteristics during actual applications is necessary。Recent AI-assisted design platforms now accurately predict how different staple placements affect binding activities greatly improving rational designs success rates。
Chapter 3 Traditional Synthetic Routes & Technical Details
3.1 Non-Natural Amino Acid Synthesis Processes nThe primary step involved within traditional synthesis involves preparing critical building blocks —non-natural amino acids.This process entails complex chiral control usually employing(1R ,2S)-2-amino -1 ,2-diphenylethanol as chiral auxiliary.The specific synthetic route includes：firstly allowing auxiliary react with ethyl bromoacetate undergo nucleophilic substitution reaction then protecting amines using Boc group followed catalytic cyclization under para-toluenesulfonic acid conditions.Under strictly controlled low-temperature alkaline conditions stereoselectively introducing allyl etc side chains finally deprotecting lithium amide removing auxiliaries yielding desired product Fmoc-AA(alkene)-OH . nChallenges faced include difficulty controlling chirality along high optical purity requirements.Due expensive chiral auxiliary reagents used multi-step reactions overall yields tend hover around fifty-seventy percent which remains major bottleneck constraining scalable production.In recent years researchers developed various improvements including utilizing cheaper sources optimizing reaction conditions reducing racemization yet fundamentally addressing cost issues still proves challenging . n **3 .2 Solid-phase Peptide Chain Assembly Techniques ** nUpon obtaining non-natural amino acid building blocks next step involves assembling peptide chains through solid phase peptide synthesis(SPPS )techniques.Fmoc protection strategy currently stands out amongst methods whereby each individual unit sequentially attaches onto resin supported carriers.This process necessitates precise control over reaction parameters especially when inserting both types required spaces consideration must factor steric hindrance affecting coupling efficiencies.Typical synthesis workflow comprises :initial anchoring Fmoc protected C terminal residue Wang resin Rink amide resins then iteratively performing deprotection（20% piperidine/ DMF）coupling （HBTU/HOBt activation cycles）。For locations featuring non-naturals extending timeframes higher efficacy coupling agents like PyBOP might need employed.Completed linear chain assembly requires additional protective modifications e.g., acetylation at N terminus ensuring readiness subsequent cyclizations reactions 。 n **3 .3 Olefin Metathesis Cyclization Reactions ** nCyclizations form crucial steps synthesizing staples generally adopting Grubbs second generation ruthenium catalysts(e.g.H₂IMesCl₂Ru=CHPh)to catalyze intramolecular olefin metathesis(RCM ).Reactions occur under rigorously oxygen-free inert atmospheres solvents predominantly selected dichloromethane(DCM)or dichloroethane(DCE),peptide concentrations maintained within ranges zero point one-one mM catalyst loadings set five-twenty mol%.Mechanisms involve dual alkenyl sidechains undergoing formation metal-cyclobutane intermediates subsequently leading β-hydride elimination generating new carbon-carbon double bonds ultimately resulting stable eight-twelve membered cyclic-staples structures.Reaction durations commonly span between two twenty-four hours monitored via HPLC tracking progress.Notably,coupling efficiencies impacted multiple factors including length composition solvent polarity temperature requiring systematic optimizations yield best results. n ### Chapter Four Innovative Breakthroughs In Synthetic Technologies ** n **4 .1 Olefin Thioether-Based Stabled-Peptides Technologies ** To reduce production costs researchers devised syntheses routes centered L-cysteine thioether-based systems leveraging inexpensive readily available cysteine replacing costly unnatural ones thus facilitating thiolation reactions basic mediums involving halogenated olefins(C₃-C₅)(e.g., four-bromobutylene )yielding intermediate products Fmoc-Cys(C₃-C₅ alkene)-OH.Later conventional solid-phase methodologies assemble final sequences culminating RCM cyclizations relative simplicity yields considerable economic advantages overall costs dropping sixty percent plus enhancing output stabilities exceeding eighty percentages whilst entirely avoiding complexities tied managing chirality concerns streamlining operations.Currently achieving hundred-kilogram scales confirms feasibility supporting commercial endeavors surrounding this approach creating robust foundations underpinning broader utilization potentialities across industries! ... [Content truncated] ...
Chapter Six Prospects Applications Trends Developments
6.[Omitted sections]...
[Disclaimer] Article derived primarily published scientific literature industry practices presented herein technical parameters cases merely serve reference purposes exact experiments productions should comply relevant regulations standards operating protocols expressed opinions solely reflect academic discussions do not constitute investment medical advice inquiries pertaining technologies recommend consulting professional institutions domain experts.