Peptide Synthesis Methods: SPPS & Beyond

Every therapeutic peptide --- from semaglutide to BPC-157 to the insulin in a diabetic's pen --- starts as raw amino acids that must be assembled in the correct sequence. How that assembly happens determines the peptide's purity, cost, scalability, and what chemical modifications can be incorporated.

The field has come a long way since the first peptide was laboriously stitched together one amino acid at a time in solution. Bruce Merrifield's invention of solid-phase peptide synthesis (SPPS) in the 1960s transformed peptide chemistry from a grueling multi-month process into something that could be automated and completed in hours. Today, SPPS remains the dominant method, but newer approaches --- native chemical ligation, recombinant expression, automated flow chemistry, and enzymatic synthesis --- are pushing the boundaries of what can be built.

A Brief History: Before Solid Phase
Solid-Phase Peptide Synthesis: The Merrifield Revolution
Boc Chemistry: The Original Approach
Fmoc Chemistry: The Modern Standard
Fmoc vs. Boc: When to Use Which
Solution-Phase Synthesis: Still Relevant
Native Chemical Ligation: Building Bigger
Recombinant Production: Letting Biology Do the Work
Emerging Methods
FAQ
The Bottom Line
References

A Brief History: Before Solid Phase

Solution-phase peptide synthesis was the only game in town for the first half of the 20th century. Chemists would dissolve protected amino acids in organic solvents, form peptide bonds one at a time, purify the product at each step, and repeat. Emil Fischer synthesized the first peptide (glycylglycine) in 1901. Vincent du Vigneaud synthesized oxytocin --- nine amino acids --- in 1953, earning a Nobel Prize for the effort.

The problem was scale and efficiency. Every coupling step required a full purification cycle. Product losses accumulated exponentially. Synthesizing a 20-residue peptide by solution-phase methods could take months and yield milligrams.

The field needed a fundamentally different approach.

Solid-Phase Peptide Synthesis: The Merrifield Revolution

In 1963, Robert Bruce Merrifield, a biochemist at Rockefeller University, published an idea that was initially met with skepticism from the chemistry community. His proposal: anchor the growing peptide chain to an insoluble solid support (a resin bead) and perform all chemical reactions while the peptide remained attached. Excess reagents and byproducts could simply be washed away by filtration, eliminating the need for intermediate purification.

The concept was elegant in its simplicity. The general SPPS procedure works like this:

Attach the first amino acid. The C-terminal amino acid is linked to the resin through a cleavable chemical bond.
Remove the N-terminal protecting group. Each amino acid carries a temporary protecting group on its alpha-amino group to prevent unwanted reactions. This group is selectively removed.
Couple the next amino acid. An activated, protected amino acid is added in excess. It reacts with the free amine of the resin-bound peptide, forming a new peptide bond.
Wash. Unreacted reagents and byproducts are flushed away with solvent.
Repeat. Steps 2 through 4 are cycled for each amino acid in the sequence, building the peptide from C-terminus to N-terminus.
Cleave and deprotect. Once the full sequence is assembled, the peptide is released from the resin and all permanent side-chain protecting groups are removed simultaneously.

The key insight was that driving reactions to completion with excess reagents --- something impractical in solution due to purification requirements --- became trivial when the product was tethered to a solid support. Just add excess, wash, and move on.

Merrifield demonstrated the approach by synthesizing bradykinin (a 9-amino-acid peptide) in 8 days, a task that previously required months. He was awarded the Nobel Prize in Chemistry in 1984.

Boc Chemistry: The Original Approach

Merrifield's original SPPS used the tert-butyloxycarbonyl (Boc) group as the temporary alpha-amino protecting group. This system, more accurately called Boc/benzyl chemistry, uses a graduated acid lability strategy:

Temporary protection (Nα-amino): The Boc group is removed by moderate acid --- typically 25-50% trifluoroacetic acid (TFA) in dichloromethane. This step occurs at every coupling cycle.

Permanent protection (side chains): Amino acid side chains (like the hydroxyl of serine, the amine of lysine, or the carboxyl of aspartate) are protected with benzyl-based groups that are stable to TFA but cleaved by strong acid.

Final cleavage: The peptide is released from the resin and all side-chain protecting groups are removed simultaneously using anhydrous hydrogen fluoride (HF). Scavengers like anisole or cresol must be added to capture reactive carbocations generated during HF cleavage, preventing them from modifying the peptide.

Advantages of Boc Chemistry

Reduced aggregation. The repeated TFA treatments during synthesis disrupt intermolecular hydrogen bonding between growing peptide chains, which helps prevent the on-resin aggregation that can plague long peptide syntheses. For "difficult sequences" prone to folding on the resin, Boc chemistry often outperforms Fmoc.
Base-sensitive targets. Because Boc deprotection uses acid (not base), Boc/benzyl SPPS is preferred for peptides containing base-sensitive functional groups --- depsipeptides, thioesters, and certain modified residues.

Drawbacks

HF handling. Anhydrous HF is extremely corrosive and toxic, requiring specialized Teflon-lined apparatus. This safety concern limits which laboratories can perform Boc chemistry.
Incomplete orthogonality. The graduated acid system means that each cycle of TFA can slowly erode side-chain protecting groups and the resin linkage. Over 40+ cycles, cumulative loss becomes significant.
Automation difficulty. The corrosive reagents make automated instrument design more challenging.

Fmoc Chemistry: The Modern Standard

In the 1970s, R.C. Sheppard at Cambridge University developed an alternative protecting group strategy that solved Boc chemistry's main problems. The fluorenylmethyloxycarbonyl (Fmoc) group is removed by a mild base --- typically 20% piperidine in dimethylformamide (DMF) --- rather than by acid.

This creates a true orthogonal protection scheme:

Temporary protection (Nα-amino): Fmoc, removed by base (piperidine). This occurs at every coupling cycle.

Permanent protection (side chains): tert-Butyl (tBu)-based groups, stable to base but removed by TFA.

Final cleavage: TFA (moderate acid) simultaneously cleaves the peptide from the resin and removes all side-chain protecting groups. No HF required.

The orthogonality is the key advantage. Base removal of Fmoc leaves tBu side-chain groups completely intact. TFA removal of tBu groups has no effect on Fmoc. The two protection systems operate independently, which means no cumulative side-chain deprotection over repeated synthesis cycles.

Why Fmoc Dominates Today

Several practical advantages made Fmoc/tBu the standard for the vast majority of peptide synthesis worldwide:

Milder reagents. No HF, no specialized apparatus. Piperidine and TFA are far safer and easier to handle.

UV monitoring. When piperidine cleaves the Fmoc group, it releases dibenzofulvene, a strong UV-absorbing chromophore. Automated synthesizers measure UV absorbance of the waste stream to confirm complete deprotection at each cycle --- a built-in quality control metric that Boc chemistry lacks.

Easy automation. The milder chemistry and UV monitoring enabled the development of fully automated peptide synthesizers that run unattended, dramatically increasing throughput.

Rich protecting group diversity. The orthogonal acid/base system opened the door to a huge variety of specialized protecting groups for on-resin modifications --- cyclization, branching, selective deprotection of individual side chains --- that would be difficult or impossible with the graduated acid system of Boc chemistry.

Cost. High-quality Fmoc-protected amino acid building blocks are now produced at multi-ton scale, driving costs down through economies of scale.

Today, the vast majority of synthetic peptides --- including commercial therapeutic peptides and research-grade materials --- are made by Fmoc SPPS. Advances in Fmoc SPPS are continually reported, with improvements in coupling reagents, resin technology, and microwave-assisted protocols expanding the range of achievable targets.

Fmoc vs. Boc: When to Use Which

Feature	Boc/Benzyl	Fmoc/tBu
Nα deprotection	TFA (acid)	Piperidine (base)
Final cleavage	Anhydrous HF (strong acid)	TFA (moderate acid)
Orthogonality	Graduated acid lability	True orthogonal (acid/base)
Aggregation during synthesis	Less prone	More prone
Safety profile	HF handling required	Milder, standard fume hood
Automation	More challenging	Easily automated with UV monitoring
Preferred for	Difficult sequences, thioesters, base-sensitive targets	General synthesis, most therapeutic peptides
Current market share	~5-10% of total synthesis	~90-95%

The practical guideline: default to Fmoc. Switch to Boc when synthesizing aggregation-prone sequences longer than 40 residues, when building thioester intermediates for chemical ligation, or when base-sensitive modifications are present.

Solution-Phase Synthesis: Still Relevant

Solution-phase synthesis has not disappeared. It occupies two niches where it outperforms SPPS:

Large-scale manufacturing. For short peptides (under 10-15 residues) produced at kilogram to ton scale, solution-phase fragment condensation can be more economical than SPPS. The reason: each intermediate can be crystallized and purified to high purity before the next coupling, reducing the exponential error accumulation that plagues long SPPS runs. Pharmaceutical companies often use hybrid approaches --- synthesizing 5- to 10-residue fragments by SPPS, then joining them in solution.

Segment condensation. For very long peptides (over 50 residues), SPPS alone may not achieve sufficient purity. The strategy is to synthesize two or more segments by SPPS, purify each segment individually, and then couple the purified fragments in solution. This convergent approach combines the speed of SPPS with the purification advantages of solution chemistry.

Native Chemical Ligation: Building Bigger

Standard SPPS reaches practical limits around 40 to 50 amino acids. Beyond that, cumulative side reactions, deletions, and on-resin aggregation erode product purity to the point where the target peptide becomes difficult to isolate. Proteins, which routinely exceed 100 residues, seemed out of reach for chemical synthesis.

Native chemical ligation (NCL), introduced by Philip Dawson and Stephen Kent in 1994, solved this problem. The concept: synthesize two unprotected peptide segments separately by SPPS, then join them in aqueous solution through a chemoselective reaction that forms a native peptide bond.

How NCL Works

The reaction requires two components:

Peptide 1 carries a C-terminal thioester group.
Peptide 2 has an N-terminal cysteine residue.

In aqueous buffer at neutral pH, the thiol of cysteine attacks the thioester through a reversible transthioesterification. This brings the two peptide segments into proximity. An irreversible S-to-N acyl shift then occurs spontaneously, forming a native amide (peptide) bond at the ligation junction. The result is a full-length, unprotected polypeptide with a native peptide backbone.

Expanding Beyond Cysteine

The original NCL required cysteine at the ligation site, which limited where peptides could be joined. Two developments expanded the scope:

Desulfurization. Researchers discovered that the cysteine at the ligation junction could be converted to alanine through radical desulfurization after ligation. Since alanine is the second most common amino acid in proteins, this dramatically increased the number of viable ligation sites. Similar thiol-containing analogs were developed for other amino acids (valine, threonine, leucine), enabling ligation at almost any position.

Extended ligation methods. Recent advances in NCL include KAHA (alpha-ketoacid-hydroxylamine) ligation, which uses a completely different chemical reaction to join peptide segments and does not require cysteine at all. A 2025 paper in JACS described cyclic hydroxylamine building blocks that form canonical amino acids at ligation junctions --- including difficult sequences like Leu-Ile --- for the first time.

Templated NCL. A 2025 approach called CAPTN (Controlled Activation of Peptides for Templated NCL) enables one-pot assembly of multiple peptide segments by selectively activating thioester precursors in sequence, minimizing cross-reactions and enabling efficient multi-segment protein synthesis.

Using NCL and its extensions, chemists have synthesized proteins exceeding 300 amino acids, including enzymes with full catalytic activity. Kent's lab synthesized a 166-residue protein (HIV-1 protease) entirely by chemical synthesis, demonstrating that the products are functionally identical to their recombinant counterparts.

Recombinant Production: Letting Biology Do the Work

Not every peptide needs to be made by chemical synthesis. Recombinant DNA technology enables living cells to produce peptides and proteins using the cell's own ribosomal machinery.

The approach: insert the gene encoding the desired peptide into an expression vector, transform a host organism, grow the culture, and harvest the peptide from the cells or culture medium. The most common expression hosts are:

Escherichia coli --- fast growth, well-understood genetics, high yield for many peptides
Saccharomyces cerevisiae (baker's yeast) --- better at folding complex proteins and performing post-translational modifications
Pichia pastoris --- a methylotrophic yeast with strong secretory pathways, widely used for industrial peptide production

The first peptide ever produced recombinantly was insulin, in 1978. Genentech inserted synthetic human insulin genes into E. coli, producing the A and B chains separately, then combined them chemically. Today, virtually all therapeutic insulin is made recombinantly.

Advantages of Recombinant Production

Scalability. Fermentation can produce grams to kilograms of peptide from bacterial cultures.
Cost for long sequences. For peptides over 40-50 amino acids, recombinant production is typically cheaper than SPPS.
Correct folding. Eukaryotic expression systems can fold complex proteins and add post-translational modifications (glycosylation, disulfide bonds) that chemical synthesis cannot easily replicate.

Limitations

Natural amino acids only. The ribosome can only incorporate the 20 standard amino acids (with some expanded genetic code exceptions). Non-natural amino acids, D-amino acids, and chemical modifications like PEGylation or lipidation require post-expression chemical modification or alternative methods.
Purification complexity. Recombinant peptides must be separated from host cell proteins, endotoxins (in E. coli), and other contaminants.
Expressed protein ligation. A hybrid approach where one segment is produced recombinantly and the other by SPPS, then joined by NCL. This allows incorporation of non-natural modifications into an otherwise recombinant protein.

Emerging Methods

Automated Flow Peptide Synthesis (AFPS)

In 2020, a team at MIT led by Bradley Pentelute reported an automated fast-flow system that performs amide bond formation in seconds rather than minutes. The machine uses continuous-flow chemistry with rapid heating to drive each coupling and deprotection step to completion at high speed. The result: a 164-amino-acid protein chain synthesized through 327 consecutive reactions in hours, not days.

The team demonstrated the synthesis of nine different protein chains --- enzymes, structural proteins, and regulatory factors --- that displayed biophysical and enzymatic properties comparable to biologically expressed versions. This proof-of-concept showed that chemical synthesis can produce functional single-domain proteins without the ribosome, though the technology is still being refined for broader adoption.

Microwave-Assisted SPPS

Microwave irradiation accelerates coupling and deprotection reactions, reducing cycle times and often improving yields for difficult sequences. The thermal energy disrupts on-resin aggregation, a common problem in conventional room-temperature Fmoc SPPS. Many modern automated synthesizers incorporate microwave heating as standard.

Chemo-Enzymatic Synthesis

Enzymes like sortase A, butelase 1, peptiligase, and omniligase catalyze peptide bond formation with high specificity under mild aqueous conditions. These ligases can join unprotected peptide fragments, cyclize linear peptides, and introduce site-specific modifications without the need for protecting group chemistry.

The Dutch company EnzyPep B.V. has demonstrated industrial-scale chemo-enzymatic synthesis of cyclic peptides and fragment condensation products. The approach is particularly appealing for medium to large peptides (15-60 residues) where SPPS purity begins to decline and the mild reaction conditions avoid the harsh reagents of chemical ligation.

Hybrid Flow-Enzymatic Approaches

A 2024 study in JACS combined flow-based chemical synthesis of precursor peptides with in vitro enzymatic maturation to produce lasso peptides --- a class of natural products with a threaded ring-and-tail topology that resists chemical synthesis by conventional means. The hybrid approach synthesized chemically modified 57-amino-acid precursors by fast-flow SPPS, then used recombinant maturation enzymes to fold and thread them into the final lasso structure.

AI and Computational Design

Computational tools are increasingly integrated into peptide synthesis planning. Machine learning models can predict which sequences will be difficult to synthesize (aggregation-prone, racemization-prone) and suggest optimized synthesis conditions or alternative routes. While these tools do not replace experimental chemistry, they reduce the trial-and-error required to successfully synthesize challenging targets.

FAQ

What is solid-phase peptide synthesis (SPPS)?

SPPS is a method for building peptides by anchoring the growing chain to an insoluble resin bead and performing sequential amino acid coupling reactions. Excess reagents are washed away after each step, eliminating intermediate purification. The completed peptide is cleaved from the resin at the end. It was invented by Bruce Merrifield in 1963 and earned him the Nobel Prize.

What is the difference between Fmoc and Boc chemistry?

The main difference is the protecting group strategy. Boc chemistry uses acid (TFA) for temporary deprotection and strong acid (HF) for final cleavage. Fmoc chemistry uses base (piperidine) for temporary deprotection and moderate acid (TFA) for final cleavage. Fmoc is safer, easier to automate, and used for roughly 90% of peptide synthesis today.

How long of a peptide can SPPS produce?

Standard SPPS can reliably produce peptides up to about 40-50 amino acids with high purity. Beyond that, cumulative side reactions reduce quality. Peptides up to 100 residues are achievable but require optimized conditions (microwave assistance, pseudoproline dipeptides, Boc chemistry for aggregation-prone sequences). For longer targets, chemical ligation or recombinant methods are used.

What is native chemical ligation?

NCL is a method for joining two unprotected peptide segments in aqueous solution to form a native peptide bond. One segment has a C-terminal thioester and the other has an N-terminal cysteine. The reaction is chemoselective and produces full-length proteins with native backbone structure. It enables chemical synthesis of proteins exceeding 300 amino acids.

When is recombinant production preferred over chemical synthesis?

Recombinant production is generally preferred for peptides longer than 50 amino acids, when only natural amino acids are needed, and when large quantities are required. Chemical synthesis is preferred for shorter peptides, when non-natural amino acids or chemical modifications are needed, and when specific isotope labeling or backbone modifications are required.

What is the future of peptide synthesis?

The field is moving toward faster, greener, and more versatile methods. Automated flow synthesis promises rapid access to full-length proteins. Chemo-enzymatic approaches reduce waste and enable mild-condition synthesis. Hybrid methods combining chemical and biological approaches can access structures that neither method could produce alone. For the fundamentals of peptide chemistry, see our guide to amino acids, peptide bonds, and biochemistry basics.

The Bottom Line

Peptide synthesis has evolved from Fischer's painstaking solution-phase work through Merrifield's resin-based revolution to a modern toolkit that includes Fmoc and Boc SPPS, native chemical ligation, recombinant expression, automated flow chemistry, and enzymatic methods. Each approach has its sweet spot: Fmoc SPPS for peptides under 50 residues, NCL for assembling larger proteins from fragments, recombinant methods for long natural-sequence peptides at scale, and emerging hybrid approaches for targets that straddle the boundaries.

For anyone working with therapeutic peptides --- whether studying their biology or considering their clinical applications --- understanding how they are made provides context for their purity, cost, scalability, and the types of modifications that are feasible. The synthesis method determines what is chemically possible, and what is possible determines what reaches the clinic.

References

Merrifield, R.B. (1963). Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. Journal of the American Chemical Society, 85(14), 2149-2154.
Behrendt, R., White, P., & Offer, J. (2016). Advances in Fmoc solid-phase peptide synthesis. Journal of Peptide Science, 22(1), 4-27. PMC4745034
Kent, S.B.H. (2009). Total chemical synthesis of proteins. Chemical Society Reviews, 38(2), 338-351.
Dawson, P.E., et al. (1994). Synthesis of proteins by native chemical ligation. Science, 266(5186), 776-779.
Kulkarni, S.S., et al. (2018). Rapid and efficient protein synthesis through expansion of the native chemical ligation concept. Nature Reviews Chemistry, 2, 0122. doi:10.1038/s41570-018-0122
Agouridas, V., et al. (2019). Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations. Chemical Reviews, 119(12), 7328-7443. doi:10.1021/acs.chemrev.8b00712
Spaltenstein, P., et al. (2025). Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations. Angewandte Chemie International Edition. doi:10.1002/anie.202413644
Hartrampf, N., et al. (2020). Synthesis of proteins by automated flow chemistry. Science, 368(6494), 980-987. doi:10.1126/science.abb2491
Koh, M., et al. (2024). Merging Flow Synthesis and Enzymatic Maturation to Expand the Chemical Space of Lasso Peptides. Journal of the American Chemical Society. doi:10.1021/jacs.4c03898
Chen, M., et al. (2025). Cyclic Hydroxylamines for Native Residue-Forming Peptide Ligations: Synthesis of Ubiquitin and Tirzepatide. Journal of the American Chemical Society. doi:10.1021/jacs.5c11881
Nuijens, T., et al. (2016). Natural occurring and engineered enzymes for peptide ligation and cyclization. Frontiers in Chemistry, 7, 829.
ProteogeniX. Challenges in chemical and recombinant peptide production processes. proteogenix.science
Bachem. Solid Phase Peptide Synthesis Explained. bachem.com
Introduction to Peptide Synthesis. (2013). Current Protocols in Protein Science. PMC3564544

Table of Contents