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Stapled Peptide Synthesis for Research Labs: Amino Acid Analysis & QC

· Vertex Labs Editorial Team

The standard laboratory method for hydrocarbon-stapled peptides is Fmoc-based solid-phase peptide synthesis (Fmoc-SPPS) followed by on-resin ruthenium-catalyzed ring-closing metathesis (RCM) to form the all-hydrocarbon staple. Staples placed at i,i+4 or i,i+7 positions reinforce alpha-helical structure, improve resistance to proteolysis, and support better membrane permeability in research models. This guide outlines the chemistry, materials, and quality-control steps our team recommends for laboratory research. For Research Use Only. Not for human or veterinary use.

TL;DR:

  • Hydrocarbon stapling using on-resin ruthenium-catalyzed ring-closing metathesis requires careful reagent sourcing, extended coupling times for hindered amino acids, and optimized reaction conditions for high conversion rates.
  • Staple placement at i,i+4 or i,i+7 positions and matching stereochemistry, especially S5/R8 pairing, are critical for efficient ring closure and stabilizing the desired helical conformation.
  • Catalysts like second-generation Grubbs or Hoveyda-Grubbs variants improve efficiency but demand strict handling and fresh preparation, with solvent choice and vessel design significantly impacting the RCM outcome.
  • Proper characterization involves confirming staple formation through LC-MS, verifying helicity with circular dichroism, and quantifying concentration accurately with amino acid analysis, not just UV.
  • Cost considerations include higher expenses for nonstandard olefinic amino acids, larger scale synthesis reduces unit costs, and designing multiple staple variants upfront can save time and resources during optimization.
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Table of Contents

Why hydrocarbon staples work and how the synthesis workflow unfolds

Unconstrained peptides pay an entropic cost when they fold into a helix to bind a target: the backbone has many rotatable bonds and few stay ordered without help. A hydrocarbon staple, an all-carbon macrocyclic bridge installed between two side chains on the same face of the helix, locks part of that structure in place before binding even occurs. According to a review of stapling chemistry and applications, this preorganization reduces the entropic penalty of folding, increases resistance to proteolytic enzymes, and in many constructs improves cell permeability relative to the unmodified linear peptide.

The workflow that gets a researcher from sequence design to a characterized stapled peptide runs through several distinct stages, each with its own failure modes:

  • Design the sequence and choose staple positions, spacing, and stereochemistry based on the target helix.
  • Perform Fmoc-SPPS, substituting olefin-bearing alpha,alpha-disubstituted amino acids at the chosen positions.
  • Run on-resin ring-closing metathesis with a ruthenium catalyst to close the hydrocarbon bridge.
  • Cleave the peptide from resin and remove protecting groups.
  • Purify by reversed-phase HPLC.
  • Characterize by LC-MS, circular dichroism, and amino acid analysis.

Hydrocarbon stapling earns its place as the default choice when the target is an intracellular protein-protein interaction and helicity, protease resistance, and permeability all matter simultaneously. When the target sits outside the cell or accessibility to nonstandard reagents is limited, other macrocyclization chemistries discussed later in this guide may be more practical starting points.

Materials and reagents checklist and selection notes

Reproducible stapled-peptide synthesis depends as much on reagent selection as on technique. A working checklist for a typical on-resin RCM project includes:

  • Resin: Rink amide or Wang resin at a low substitution level (0.2 to 0.4 mmol/g) to reduce interchain metathesis and aggregation during chain assembly.
  • Olefinic amino acids: Fmoc-protected alpha-methyl, alpha-alkenyl amino acids (commonly the S5 and R8 building blocks) sourced from a peptide chemistry supplier with a certificate of analysis for each lot.
  • Ruthenium catalyst: a Grubbs-type or Hoveyda-Grubbs-type catalyst suitable for on-resin metathesis, stored under inert atmosphere and weighed fresh for each reaction.
  • Solvents: anhydrous 1,2-dichloroethane (DCE) for the metathesis step, along with standard SPPS solvents (DMF, NMP, DCM) at peptide synthesis grade.
  • Coupling reagents: HATU or HBTU with DIPEA, and extended reaction protocols for hindered alpha,alpha-disubstituted residues.
  • Analytical consumables: an analytical HPLC column, LC-MS-compatible buffers, CD-grade cuvettes, and amino acid analysis standards.

Sourcing matters more for the nonstandard olefinic residues than for any other component. Confirm optical purity and identity by certificate before committing an entire synthesis run to a new lot, since a racemized or mislabeled building block will not reveal itself until the RCM step fails to close cleanly.

Pro Tip: Order olefinic amino acids in slightly larger excess than the calculation suggests. Hindered couplings consume more reagent through side reactions, and running short mid-synthesis forces a costly pause.

Step 1: Fmoc-SPPS incorporation of olefinic nonnatural amino acids

Installing the olefin-bearing residues is the step most likely to determine whether the eventual RCM succeeds. The alpha,alpha-disubstituted amino acids used for stapling are sterically hindered relative to proteogenic residues, and standard coupling conditions calibrated for natural amino acids often underperform here.

We recommend the following sequence of operations:

  1. Synthesize the unmodified linear template first. Running the native or wild-type sequence through standard Fmoc-SPPS before touching the stapled variant establishes a baseline yield and purity, and it flags synthesis-difficult stretches (beta-sheet-prone regions, repeated hydrophobic residues) before nonstandard residues and RCM chemistry are added to the problem, a practice reported to save both time and material in practitioner-level optimization notes.
  2. Increase equivalents and coupling time for the olefinic residues. Where a standard coupling might run 30 to 60 minutes at 4 to 5 equivalents, hindered alpha-methyl, alpha-alkenyl residues typically need extended coupling windows and higher molar excess to reach acceptable conversion.
  3. Select an activator suited to hindered couplings. HATU with DIPEA, or a combination that includes HOAt, generally outperforms simpler carbodiimide chemistry for these residues; double coupling is common practice at and immediately after the olefinic position.
  4. Maintain a protecting-group scheme orthogonal to RCM. Side-chain protecting groups elsewhere in the sequence must survive the ruthenium-catalyzed step without interference, so standard acid-labile groups compatible with Fmoc chemistry (Boc, tBu, Trt, Pbf) are the default choice, reserved and removed only at final cleavage.
  5. Verify each critical coupling before moving on. A microcleavage followed by LC-MS after the olefinic residue, and again after the second staple-forming residue, catches a failed or truncated coupling while it is still cheap to repeat, rather than after the full chain and RCM are complete.

Ninhydrin or chloranil tests give a fast qualitative read on coupling completion for secondary amines, but they are not a substitute for LC-MS confirmation at the positions that matter most. A single missed coupling at an olefinic residue does not always show up clearly on a resin test, and it will not be recoverable once the chain has been extended past it.

Extended coupling times add real hours to a synthesis schedule, and researchers new to stapling chemistry sometimes underestimate how much slower these residues run compared to a standard 20-residue peptide. Building the extra time into the schedule from the outset, rather than discovering the need for it mid-run, keeps a multi-day synthesis from becoming a multi-week one.

Step 2: On-resin ring-closing metathesis to close the hydrocarbon bridge

Once both olefinic residues are installed and confirmed, the resin-bound linear precursor is ready for ring-closing metathesis. This is the step that actually forms the hydrocarbon staple, and it is also the step most sensitive to reaction conditions.

RCM is an equilibrium reaction, and the byproduct, ethylene gas, has to leave the system for the equilibrium to favor the closed macrocycle. According to practitioner notes on on-resin metathesis, solvent choice and reaction vessel design matter more to conversion than simply adding more catalyst. Key practices include:

  • Use 1,2-dichloroethane (DCE) as the reaction solvent. Its higher boiling point relative to dichloromethane tolerates the mild heating some labs use to help drive the reaction, and it supports efficient ethylene escape from solution.
  • Allow adequate headspace and gentle agitation. A loosely capped or vented reaction vessel with enough headspace lets ethylene leave the system without requiring a strict inert-atmosphere setup, which simplifies bench logistics for a resin-swelling reaction.
  • Replenish catalyst partway through the reaction rather than dosing once. A second, smaller addition of catalyst after several hours often pushes conversion further than doubling the initial dose, since catalyst degradation over time limits the effective concentration late in the reaction.
  • Monitor conversion by test cleavage and LC-MS at intervals. Removing a small resin sample at the 2 hour, 6 hour, and overnight marks and cleaving it for a quick LC-MS read shows whether conversion is progressing or has plateaued, which tells you whether to add catalyst, extend time, or move on.

Reaction times for on-resin RCM commonly run from several hours to overnight, and a plateau in conversion after the first catalyst addition is common rather than exceptional. Ring size, tether length, and steric environment around the olefins all affect how readily the macrocycle closes, which is part of why the i,i+4 and i,i+7 spacings dominate the literature: they consistently give tether geometries the catalyst can close efficiently.

Pro Tip: When conversion stalls below 80 percent by test-cleavage LC-MS, try a fresh catalyst aliquot with a brief solvent exchange before assuming the sequence itself is the problem.

Step 3: Cleavage, purification, and characterization

After RCM reaches an acceptable conversion by test cleavage, the full resin batch is cleaved and carried through purification and analytical characterization.

  1. Cleave with a standard TFA-based cocktail. A mixture of trifluoroacetic acid with scavengers such as triisopropylsilane and water (commonly in a 95:2.5:2.5 ratio) removes side-chain protecting groups and releases the peptide from resin; cold ether precipitation typically follows to isolate crude product.
  2. Purify by reversed-phase HPLC. Stapled peptides tend to run more hydrophobic than their linear counterparts because of the added hydrocarbon bridge, so gradient conditions optimized for the unmodified template usually need adjustment, and the stapled and unstapled species can co-elute closely enough to require careful fraction collection, a challenge noted directly in practitioner guidance on RCM purification. Our guide to peptide purification methods walks through gradient optimization for closely eluting species in more depth.
  3. Confirm mass and staple closure by LC-MS. The closed staple shows a mass loss consistent with the ethylene byproduct relative to the unclosed diene precursor, and LC-MS is the fastest way to distinguish fully stapled product from partially reacted or ring-opened side products.
  4. Assess helicity by circular dichroism. A CD spectrum with the characteristic double minimum near 208 and 222 nanometers indicates alpha-helical content, letting you compare stapled and unstapled versions of the same sequence directly.
  5. Quantify concentration by amino acid analysis rather than UV alone. UV absorbance depends on aromatic residue content and can misrepresent concentration for peptides with few or no tryptophan or tyrosine residues; amino acid analysis on duplicate samples gives a more reliable figure for downstream binding or stability assays. Our overview of peptide sequence characterization methods covers how these techniques fit together for a full analytical package.

Staple design and placement: choosing positions and stereochemistry

Staple placement is a design decision, not an afterthought, and it determines whether the finished construct actually helps or hurts helicity and binding.

  • i,i+4 spacing bridges one helical turn and is commonly used for stabilizing shorter helical segments or for stapling near a helix terminus.
  • i,i+7 spacing bridges two turns and is the more common choice for longer helices where broader stabilization across the binding face is needed; i,i+3 and i,i+11 spacings appear less frequently for specialized geometries, per the staple-position guidance in the literature.
  • Stereochemistry pairing matters as much as spacing. The S5/R8 combination (an S-configured residue at the i position paired with an R-configured residue at i+7) is a widely used pairing that supports efficient ring closure and stable helical geometry; mismatched stereochemistry can prevent RCM from closing efficiently or distort the resulting helix.
  • Use structural data to guide placement. A solved or modeled structure of the peptide-target complex, or alanine-scanning data identifying residues that tolerate substitution, points to which face of the helix is free for staple placement without disrupting the binding interface.
  • Design a small panel rather than a single construct. Two or three staple positions and stereochemistry combinations, synthesized and screened in parallel, are more likely to yield a working construct than committing all resources to one design, since small changes in spacing can swing helicity and binding substantially.

Optimization and troubleshooting for higher conversion and cleaner product

Most problems in stapled-peptide synthesis trace back to a handful of recurring issues, and most of them are preventable with the right sequence of checks.

  • Benchmark against the unmodified peptide. A poor yield on the stapled variant is easier to interpret once you know whether the linear template itself synthesizes cleanly.
  • Manage ethylene removal actively during RCM. Solvent choice, headspace, and mild agitation do more for conversion than catalyst quantity alone, as covered in the RCM section above.
  • Replenish catalyst rather than overloading it up front. A staged addition strategy generally outperforms a single large dose for reactions that plateau early.
  • Separate stapled from unstapled species with a shallow HPLC gradient. A shallow gradient across the region where the two species elute gives better resolution than a fast, broad gradient built for standard peptides.
  • Run amino acid analysis in duplicate at different dilutions. This catches pipetting or dilution errors before a mis-quantified stock peptide propagates into every downstream assay.

Pro Tip: Keep a synthesis log that records catalyst lot, batch, and addition timing alongside conversion by test cleavage. Patterns across projects often reveal that one catalyst lot or supplier consistently underperforms.

Costs, scale, and project planning for custom stapled peptides

Budgeting for a stapled-peptide project starts with the baseline cost of standard peptide synthesis and then adds the premium for nonstandard residues and the RCM step itself. Academic core facility pricing for a standard peptide up to about 25 residues often starts in the low hundreds of dollars and increases with sequence length; nonstandard amino acids and on-resin RCM add cost on top of that baseline and typically require an individualized quote rather than a standard price list entry.

A few planning points worth building into a proposal or purchase request:

  • Scale affects per-milligram cost more than most researchers expect. Small research-scale batches carry a higher effective cost per milligram than bulk synthesis runs, since fixed setup and QC costs are spread across less product.
  • Analytical requirements belong in the quote, not as an afterthought. HPLC purification to a stated purity threshold, LC-MS confirmation, and amino acid analysis each add turnaround time and cost, and specifying them up front avoids a surprise change order later.
  • Panels of staple variants multiply cost linearly but often save time overall. Requesting quotes for a panel of two or three staple positions at once is usually more efficient than iterating one design at a time. Our breakdown of peptide synthesis cost walks through the dollar-per-milligram math in more detail.

Alternative stapling chemistries and when to use them

Hydrocarbon stapling via RCM is not the only macrocyclization route, and other chemistries fit certain projects better.

  • Lactamization (amide bond formation between side chains) uses only proteogenic residues and standard SPPS chemistry, making it accessible without specialized building blocks, though the resulting staples generally show more limited cell penetration than hydrocarbon staples.
  • CuAAC click stapling links azide- and alkyne-bearing side chains and offers a two-component approach useful when olefinic amino acids are hard to source or when a triazole-based tether suits the target geometry.
  • Thioether and disulfide stapling are straightforward to install but generally carry stability trade-offs; disulfides in particular are reducible in cellular environments, which limits their use for intracellular targets.
  • According to a review of stapled-peptide chemistries, hydrocarbon stapling remains the preferred route for intracellular protein-protein interaction targets, while the alternative chemistries above often suit extracellular or membrane-bound targets better.

Vertex Labs resources and how we support stapled-peptide workflows

The company supplies peptides and custom peptide synthesis services for laboratories designing hydrocarbon-stapled constructs, with documentation provided for each batch. For Research Use Only. Not for human or veterinary use.

To request a quote, submit your target sequence, staple positions, and desired scale and purity. In return, you receive documentation covering HPLC purity, LC-MS mass confirmation, and the COA tied to that specific batch, the same documentation standard described in our guide to peptide manufacturing quality benchmarks.

Detailed protocols for synthesis including reagent preparations and step-by-step procedures

A working bench protocol for a stapled-peptide project generally follows this sequence: swell the resin in DMF for 30 minutes, perform standard Fmoc deprotection with 20 percent piperidine in DMF, and couple each residue using HATU/DIPEA activation, extending coupling time and equivalents specifically at the olefinic positions as described earlier. After the second olefinic residue is confirmed by microcleavage, exchange the resin into anhydrous DCE, add the ruthenium catalyst under gentle agitation, and monitor conversion at intervals by test cleavage and LC-MS.

Stapled peptide synthesis protocol workflow

Reagent preparation deserves the same rigor as the reaction steps themselves. Activator solutions should be made fresh each day, since HATU and HBTU degrade in solution over time and stale activator is a common, underdiagnosed cause of poor coupling yields. Catalyst stock solutions are best prepared immediately before use and kept under inert atmosphere until the moment of addition, since ruthenium catalysts of the Grubbs and Hoveyda-Grubbs type are air- and moisture-sensitive in solution even when the solid form tolerates brief bench handling.

Documenting each step, including exact equivalents, coupling times, and catalyst lot numbers, turns a one-off synthesis into a repeatable protocol and makes troubleshooting a failed batch far faster the second time around.

Tips for improving yield and purity beyond standard optimizations

Beyond the core RCM and coupling optimizations already covered, a few additional practices consistently improve outcomes on difficult sequences. Capping unreacted amine after each coupling step with acetic anhydride prevents deletion sequences from accumulating and complicating purification later, a step some labs skip on standard peptides but should not skip on stapled constructs given how much harder deletion products are to separate from a stapled product of similar hydrophobicity.

Pseudoproline dipeptides at aggregation-prone junctions reduce on-resin aggregation, which otherwise slows coupling kinetics and lowers overall yield on longer helical sequences. For the RCM step specifically, a brief resin wash and solvent exchange immediately before catalyst addition removes residual DMF or piperidine traces that can otherwise poison the ruthenium catalyst and stall the reaction before it starts.

Finally, purity assessment should use more than one analytical method before a batch is called clean. A peptide that looks pure by analytical HPLC can still contain a diastereomer or regioisomer that only resolves on a different column chemistry or a CD comparison against a reference standard.

Comparison of different ring-closing metathesis catalysts and their advantages and disadvantages

Catalyst choice for on-resin RCM generally comes down to a small set of ruthenium carbene catalysts from the Grubbs and Hoveyda-Grubbs families. First-generation Grubbs catalysts are more tolerant of trace moisture but generally show lower activity toward the hindered, alpha,alpha-disubstituted olefins used in stapling chemistry, which can leave conversion low even with extended reaction times.

Second-generation Grubbs and Hoveyda-Grubbs catalysts, bearing an N-heterocyclic carbene ligand, show markedly better activity on hindered olefins and are the more common choice for stapling projects, though they are more sensitive to air and moisture and typically cost more per gram. Hoveyda-Grubbs variants, with their chelating isopropoxybenzylidene ligand, offer somewhat easier handling and storage stability compared to standard Grubbs catalysts, which can matter for labs running stapling projects infrequently and needing a catalyst that tolerates longer shelf storage between uses.

The practical trade-off for most research labs is activity versus handling convenience: a more active, more moisture-sensitive catalyst closes difficult staples faster but demands more careful technique, while a more robust catalyst may need a longer reaction time or a second catalyst addition to reach comparable conversion.

Analytical techniques beyond LC-MS and CD for stapled peptide characterization

LC-MS and circular dichroism cover mass confirmation and helicity, but a complete characterization package for a stapled peptide often draws on additional techniques. Nuclear magnetic resonance (NMR) spectroscopy, while more resource- and sample-intensive than LC-MS, can resolve stereochemical questions that mass spectrometry cannot, including confirming which diastereomer formed at the staple-forming residues when more than one is chemically possible.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry offers a fast, complementary mass confirmation alongside LC-MS, particularly useful for a quick check of crude cleavage product before committing it to a full HPLC purification run. Because MALDI-TOF ionization behaves differently from electrospray ionization used in most LC-MS setups, running both provides a useful cross-check when a peptide gives an ambiguous or unexpected mass on one platform.

For labs studying binding interactions rather than synthesis alone, techniques such as circular dichroism thermal denaturation (to assess helix stability across temperature) and analytical ultracentrifugation or size-exclusion chromatography (to rule out aggregation) round out a characterization package appropriate for a stapled peptide headed into a binding or stability assay.

Applications of stapled peptides in research contexts

Understanding why researchers invest in stapling chemistry in the first place helps clarify which design choices matter most for a given project. Hydrocarbon-stapled peptides are studied extensively as tools for probing intracellular protein-protein interactions, since the reinforced helix can engage binding grooves that linear peptides struggle to occupy stably, according to the mechanistic rationale summarized in stapling literature.

Beyond binding studies, stapled peptides serve as research tools for probing structure-activity relationships in a helix-mediated interaction, since a panel of staple positions and stereochemistries lets researchers map which residues and which face of the helix are essential for a given interaction. Their improved resistance to proteolytic degradation also makes them useful for extended in vitro concentration-response studies where a linear peptide would degrade before an experiment concludes, letting researchers observe effects across a longer assay window without the confound of peptide breakdown. These properties make stapled peptides a recurring tool in structural biology and chemical biology research settings focused on protein interaction mapping.

Storage and stability considerations for synthesized stapled peptides

A stapled peptide’s improved proteolytic resistance does not eliminate the need for careful storage, and the hydrocarbon bridge itself is generally stable under standard peptide storage conditions. Lyophilized stapled peptide is typically stored at negative 20 degrees Celsius or colder, protected from light and moisture, and brought to room temperature before opening the container to avoid condensation.

Anonymous peptide vials in cold storage

Once reconstituted into solution for an assay, stability depends heavily on solvent, pH, and temperature, and a stapled peptide in aqueous buffer should generally be used within the timeframe validated for that specific construct rather than assumed stable indefinitely. Repeated freeze-thaw cycles degrade peptide solutions generally, and stapled peptides are not an exception, so aliquoting a stock solution into single-use volumes before freezing avoids unnecessary degradation from repeated thawing.

Documenting the storage conditions and reconstitution date alongside the COA for each batch gives downstream users of the peptide a clear record for interpreting any drift in assay results over time, an approach consistent with the documentation standards described in our guide to laboratory quality control best practices.

What separates a reliable stapling protocol from a fragile one

The literature on stapled-peptide synthesis often reads as though RCM conversion is the hard part. In practice, the harder part is discipline earlier in the process: running the unmodified template first, confirming each hindered coupling before moving on, and quantifying the final product by amino acid analysis instead of trusting a UV reading calibrated for a different sequence. Labs that skip these steps do not usually fail outright, they just spend more time troubleshooting a batch that looked fine on paper. Treat every stapled construct as a small research project with its own baseline, its own QC checkpoints, and its own realistic timeline, and start with a small-scale batch before scaling up procurement once the analytics confirm the design works. For Research Use Only. Not for human or veterinary use.

— Vertex Labs Editorial Team

Getting support for your stapled peptide project

Vertex Labs

Designing and validating a hydrocarbon-stapled peptide takes real bench time, and sourcing the olefinic building blocks and catalyst reliably is often the slower part of getting a project started. Research laboratories can source peptides, custom synthesis services, and bulk procurement options, typically accompanied by batch-specific Certificates of Analysis to support documentation requirements.

To get an accurate quote for a stapled-peptide project, include:

  • The target sequence with staple positions and stereochemistry marked.
  • Desired scale (milligrams) and target purity.
  • Any specific analytical documentation needed beyond standard HPLC and LC-MS.

Visit our shop to review available research peptides and compounds, or start at our main page to submit a custom sequence request and see current COA practices for every batch. For Research Use Only. Not for human or veterinary use.

Authoritative protocols and reviews for deeper reading

For stepwise protocol detail beyond this guide, Nature Protocols publishes the Kim and Verdine method for Fmoc-SPPS incorporation and on-resin RCM, a widely cited procedural reference for this chemistry. The review of stapling principles and applications, practitioner notes on RCM optimization, and recent advances in stapling technologies cover the chemistry and design considerations in greater depth.

Sources

FAQ

Can you synthesize your own stapled peptides in a standard lab?

A lab equipped for standard Fmoc-SPPS can perform stapled-peptide synthesis if it also has access to a ruthenium metathesis catalyst, olefinic amino acid building blocks, and LC-MS or equivalent characterization equipment. Many research groups instead order custom stapled sequences from a supplier to avoid the equipment and optimization overhead of setting up RCM chemistry for a single project.

What analytical methods confirm a peptide has stapled correctly?

Mass confirmation by LC-MS shows the mass loss consistent with ethylene release that indicates successful ring closure, while circular dichroism confirms the resulting alpha-helical content. Amino acid analysis is recommended for accurate concentration determination since UV-based quantitation can misrepresent concentration depending on aromatic residue content.

How much does custom stapled peptide synthesis typically cost?

Standard peptide synthesis at an academic core facility for sequences up to about 25 residues often starts in the low hundreds of dollars, with nonstandard amino acids and on-resin RCM adding cost on top of that baseline. Custom stapled peptides generally require an individualized quote because scale, purity target, and analytical requirements all affect the final price.

What is the difference between i,i+4 and i,i+7 staple positions?

An i,i+4 staple bridges one turn of the helix and suits shorter helical segments, while an i,i+7 staple bridges two turns and is more common for stabilizing longer helices. The choice affects tether length, stereochemistry pairing, and how efficiently the ring-closing metathesis step closes the bridge.

Are there alternatives to hydrocarbon stapling for peptide stabilization?

Yes, lactamization, CuAAC click chemistry, and thioether or disulfide stapling are all used as alternative macrocyclization strategies. According to a review of stapling chemistries, hydrocarbon stapling remains the preferred approach for intracellular protein-protein interaction targets, while the alternatives often suit extracellular or membrane-bound targets better.

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