Native Chemical Ligation for Long Peptides: NCL vs SPPS

Solid-phase peptide synthesis is highly effective for many research peptides, but adding more coupling cycles is not always the best way to reach a longer or more complex target.

As peptide length increases, incomplete coupling, aggregation, deletion sequences, difficult cleavage, and poor crude resolution can accumulate. A 2025 study discussing backbone protection in Fmoc-SPPS noted that resin-bound aggregation remains a major limitation for routine synthesis of peptides above roughly 40 residues, although sequence behavior matters more than any fixed length cutoff.

For some targets, the better strategy is to stop treating the molecule as one long linear synthesis and instead prepare shorter peptide fragments that are joined later.

Native chemical ligation (NCL) is one of the most established ways to do this.

What Native Chemical Ligation Actually Solves

Classical NCL joins an unprotected peptide bearing a C-terminal thioester with a second peptide bearing an N-terminal cysteine. The reaction ultimately forms a native peptide bond at the ligation site.

The concept was introduced by Dawson, Muir, Clark-Lewis and Kent in 1994 and demonstrated through chemical synthesis of a disulfide-containing cytokine.

Its importance is not simply that two peptides can be connected.

NCL changes the synthesis architecture from a long sequential process into a convergent process.

Instead of asking one resin-bound chain to tolerate every coupling required for a 60-, 80- or 100-residue target, shorter fragments can be synthesized and characterized separately before assembly.

That distinction becomes important when one problematic region compromises the crude quality of an otherwise accessible peptide.

There Is No Universal “Maximum Length” for SPPS

Researchers frequently ask whether a 40-, 50- or 70-residue peptide should be synthesized directly or divided into fragments.

There is no scientifically useful universal cutoff.

A 55-residue hydrophilic sequence may synthesize more cleanly than a 30-residue peptide containing a strongly aggregation-prone region. Resin loading, hydrophobicity, protecting groups, secondary structure, modification chemistry, and the location of difficult residues all affect the result.

For initial troubleshooting of linear synthesis, our SPPS Practical Guide and Common Side Reactions in SPPS discuss aggregation and sequence-dependent failure in more detail.

When Fragment Assembly Becomes Worth Considering

We consider sequence behavior more informative than peptide length alone when deciding whether ligation should enter the synthesis plan.

The Ligation Junction Is a Design Variable

Classical NCL requires an N-terminal cysteine on one fragment. That initially appears restrictive because many target sequences do not contain cysteine at a useful fragment boundary.

Modern ligation chemistry has substantially expanded this sequence space.

Post-ligation desulfurization can convert cysteine at the ligation site into alanine. Early work demonstrated this strategy in targets including a 56-residue protein G domain and a 110-residue barnase variant, showing that NCL could be extended to proteins without relying on a native cysteine at the final junction.

Subsequent approaches have extended ligation chemistry to additional residue environments and introduced thiolated amino-acid surrogates, auxiliary-mediated reactions, and improved desulfurization methods. A 2026 review describes continued development of chemoselective desulfurization beyond conventional VA-044-based approaches.

This means sequence segmentation should not be based simply on finding the first cysteine.

A good junction should also consider fragment solubility, thioester reactivity, steric environment, side-chain chemistry, downstream desulfurization, and how easily each fragment can be purified.

Not Every Junction Is Equally Efficient

β-Branched residues and sterically congested junctions can react more slowly. C-terminal Val, Ile, Thr, and Pro environments can be particularly challenging in conventional NCL chemistry.

Recent methods continue to address these limitations. For example, β-lactone-mediated ligation was reported as a strategy for difficult Thr-containing junctions while controlling epimerization.

We think junction selection should be made before fragment synthesis begins, not after two independently synthesized fragments happen to be available.

That single decision can influence the success of the entire project.

Real Data: Multi-Segment Ligation Still Has Competing Reactions

Modern templating strategies can improve reactions at low peptide concentration and difficult junctions, but multi-fragment assembly is not automatically quantitative.

A 2025 Angewandte Chemie study reported a three-segment templated NCL experiment in which the desired ligated product reached 70% RP-HPLC yield, while cross-ligation and peptide cyclization accounted for 6.5% and 18%, respectively. The authors subsequently developed controlled activation approaches to reduce competing reactions during multi-segment assembly.

Figure 1. Product Distribution in a Reported Three-Segment Templated NCL

Data reproduced as a visualization of values reported by Spaltenstein et al., Angew. Chem. Int. Ed. 2025. Values represent RP-HPLC yields for the reported simultaneous three-segment templated NCL experiment.

This result highlights an important practical point: detecting full-length ligated product is not the same as having a process that is ready for routine production.

The impurity architecture after ligation matters just as much as crude purity after SPPS.

Intermediate Purification Can Become the Hidden Yield Problem

A multi-fragment strategy creates a new question: how many times should intermediates be isolated and purified?

Repeated purification can remove side products, but every isolation step also consumes time and peptide.

This is one reason recent research has moved toward one-pot and telescoped ligation strategies.

Controlled Activation of Peptides for Templated NCL, or CAPTN, was developed partly to enable multi-segment assembly without repeated intermediate purification. The method was demonstrated in synthesis of the E. coli ribosomal proteins S16 and S17 and was designed to support difficult junctions and low peptide concentrations.

Other 2025–2026 studies have explored ruthenium-controlled one-pot ligation, orthogonal cysteine protection, and direction-switching multi-segment assembly. A 2026 study used five peptide segments in one pot for semisynthesis of a multiply modified histone H3 construct.

We consider purification frequency one of the most underestimated variables in fragment-based peptide synthesis.

A theoretically elegant route can become inefficient if every ligation requires another low-recovery preparative HPLC step.

Tirzepatide Shows Why Ligation Is Becoming a Process Strategy

The relevance of fragment assembly is no longer restricted to academic chemical protein synthesis.

In 2026, researchers from Eli Lilly reported a convergent strategy for gram-scale tirzepatide synthesis. Their work included a four-fragment hybrid SPPS/LPPS route and a separate two-fragment approach using NCL followed by desulfurization.

Tangential flow filtration was used between ligation and desulfurization to process intermediates while avoiding more solvent-intensive purification operations.

This does not mean NCL should replace linear SPPS for routine custom peptides.

It demonstrates something more useful: ligation can become part of process design when peptide complexity and manufacturing scale make a purely linear strategy inefficient.

For a scientist ordering 5 mg of a 25-residue peptide, this may be irrelevant.

For a development group evaluating a long modified peptide that may later require hundreds of milligrams or grams, route architecture can matter from the beginning.

What Should Be Defined Before Requesting a Ligation Project?

For scientists and lab managers, the most useful starting information is the complete target sequence together with modifications, desired termini, disulfide requirements, final quantity, purity, and downstream application.

The supplier should then evaluate where the sequence should be divided rather than asking the customer to arbitrarily specify fragments.

Procurement teams should also distinguish between fragment specifications and final-product specifications.

A project may involve several internally produced fragments at process-appropriate purity followed by ligation, final purification, and final QC. Requiring every intermediate to meet the same high purity as the finished peptide can increase cost without necessarily improving the final material.

This is particularly relevant when expensive Fmoc-protected amino acids, specialty residues, or difficult modifications are involved.

Questions Worth Asking a Supplier

Ligation Does Not Remove the Need for Good SPPS

Native chemical ligation depends on peptide fragments, and those fragments still have to be made.

Poorly synthesized fragments do not become good peptides simply because they are ligated.

Fragment design should therefore make SPPS easier: shorter segments, improved solubility, manageable hydrophobic regions, accessible purification, and appropriate reactive termini.

The same principles discussed in Peptide Purification by RP-HPLC still apply. Peak separation, impurity identity, recovery, and final QC remain important after ligation.

In some sequences, specialized building blocks such as pseudoproline derivatives may improve the individual SPPS fragments before ligation is considered.

The useful distinction is therefore not SPPS versus NCL.

It is whether the complete target should be built linearly or assembled from independently manageable synthetic units.

What This Means for Custom Peptide Projects

For most routine peptides, direct Fmoc-SPPS remains the simplest option.

Fragment ligation becomes more attractive when the target combines length, aggregation, difficult modifications, protein-like architecture, or a scale at which repeated linear synthesis produces poor process efficiency.

The decision should be made from the complete sequence and final experimental requirement.

A researcher may need only a few milligrams for an initial assay. A lab manager may need reproducibility across several batches. Procurement may care about whether the same synthetic route can support a larger follow-up order.

Those are different questions, but they should be considered within the same synthesis plan.

Alan Scientific supports custom peptide synthesis, peptide building blocks, purification, analytical QC, and evaluation of challenging or modified peptide sequences.

For long or difficult targets, customers can submit the complete sequence rather than designing the fragment strategy themselves. The synthesis route can then be evaluated according to sequence behavior, modification chemistry, required quantity, final purity, and downstream research use.

Conclusion

Native chemical ligation is most valuable when it solves a specific limitation of linear synthesis.

Its advantage is not simply the ability to make a longer peptide. It allows chemists to redesign how a difficult molecule is assembled.

Recent work from multi-segment templated NCL to gram-scale tirzepatide manufacturing shows the same direction: peptide synthesis is becoming more convergent, more sequence-aware, and less dependent on forcing every target through one linear workflow.

For researchers, the practical question is therefore not:

“Is my peptide too long for SPPS?”

It is:

“Which synthesis architecture gives the cleanest, most reproducible route to the peptide required for my experiment?”

References

1. Dawson PE, Muir TW, Clark-Lewis I, Kent SBH. Synthesis of proteins by native chemical ligation. Science. 1994;266:776–779. DOI: 10.1126/science.7973629.

2. Yan LZ, Dawson PE. Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J Am Chem Soc. 2001.

3. Paravizzini SJ, Hutton CA, Karas JA. Tetrahydropyranyl Backbone Protection for Enhanced Fmoc Solid-Phase Peptide Synthesis. Chem Eur J. 2025. DOI: 10.1002/chem.202501510.

4. Spaltenstein P, et al. Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations. Angew Chem Int Ed. 2025;64:e202413644. DOI: 10.1002/anie.202413644.

5. Jalan A, et al. A Convergent Hybrid Gram-Scale Synthesis of Tirzepatide: Tangential Flow Filtration Assisted Native Chemical Ligation-Desulfurization Approach. Angew Chem Int Ed. 2026;65:e20060. DOI: 10.1002/anie.202520060.

6. Pallava R, Bisher S, Brik A. Total Chemical Synthesis of RNF4 by Sequential Native Chemical Ligation: C-To-N Versus N-To-C Strategies. J Org Chem. 2026;91:2965–2972.

7. Ding X, et al. Recent advances in chemoselective protein desulfurization. Org Biomol Chem. 2026;24:5583–5598.