Solid-phase peptide synthesis
Solid-phase peptide synthesis builds a peptide one residue at a time on a resin support. This guide covers Merrifield's method, Fmoc and Boc strategies, coupling and cleavage, and the impurities that synthesis produces.
The idea behind SPPS
Solid-phase peptide synthesis (SPPS) was introduced by R. Bruce Merrifield in 1963, and the work was recognized with the 1984 Nobel Prize in Chemistry. Its central idea is to attach the first amino acid to an insoluble polymer bead, the resin, and build the chain on it step by step. Because the growing peptide stays bound to the solid support, excess reagents and by-products can be removed simply by washing and filtering, rather than by purifying an intermediate after every step.
This approach made it practical to drive each reaction to near completion with large excesses of reagents, and it lends itself to automation. SPPS remains the standard method for making peptides up to roughly 50 residues, and with optimization, somewhat longer sequences.
The synthesis cycle
Synthesis proceeds from the C-terminus to the N-terminus, the opposite of the direction in which sequences are written. Each amino acid is added with its alpha-amino group temporarily protected and its reactive side chain permanently protected until the end. One cycle has a fixed sequence of steps, repeated for each residue.
- Deprotection: remove the temporary protecting group from the N-terminus of the resin-bound chain.
- Washing: remove deprotection reagent and by-products.
- Coupling: add the next protected amino acid, activated with a coupling reagent such as a carbodiimide (DIC) with an additive like Oxyma, or uronium and phosphonium reagents such as HBTU or HATU.
- Washing: remove excess amino acid and reagents.
- Optional capping: acetylate unreacted chains with acetic anhydride so they cannot grow further.
Fmoc vs Boc chemistry
Two protecting-group strategies dominate. In Boc chemistry, the original Merrifield approach, the alpha-amino group is protected with tert-butyloxycarbonyl (Boc), which is removed with TFA at each cycle. Side-chain protecting groups and the resin linkage are stable to TFA and are removed at the end with a much stronger acid, classically anhydrous hydrogen fluoride, which requires specialized equipment.
In Fmoc chemistry, the alpha-amino group is protected with 9-fluorenylmethoxycarbonyl (Fmoc), which is removed under mild basic conditions, typically with piperidine in DMF. Side chains are protected with acid-labile groups such as tert-butyl and trityl, and the final cleavage uses TFA. Because the temporary and permanent protecting groups are removed by completely different conditions, the strategy is described as orthogonal. Fmoc chemistry avoids HF and is now the more widely used approach, although Boc chemistry remains useful for some difficult sequences.
Resins, cleavage and purification
The choice of resin and linker determines the C-terminus of the final peptide. Wang-type resins release a C-terminal carboxylic acid, while Rink amide resins release a C-terminal amide, which many bioactive peptides carry. 2-Chlorotrityl resins allow very mild cleavage and are useful for protected fragments.
At the end of an Fmoc synthesis, the resin is treated with a TFA cocktail containing scavengers such as water, triisopropylsilane and sometimes thiols. Scavengers trap reactive carbocations released from protecting groups that would otherwise alkylate sensitive residues such as Trp, Met, Cys and Tyr. The crude peptide is precipitated, typically in cold ether, then purified by reversed-phase HPLC and lyophilized. This is why synthetic peptides are often TFA salts; see counter-ions: TFA vs acetate.
Common synthesis impurities
Because each step is less than perfectly efficient, crude peptides contain a family of closely related by-products. Their proportion tends to grow with chain length, since small losses at each step compound. Most are separated by HPLC, but some have similar retention and are detected only by careful LC-MS analysis.
- Deletion sequences: chains missing one or more residues because a coupling was incomplete and the next residue was added to the unreacted chain. They differ in mass by the missing residue and often elute close to the product.
- Truncations: chains that stopped growing, either because capping blocked them deliberately or because the N-terminus became unreactive. Capped truncations carry an N-terminal acetyl group.
- Racemization: conversion of an L-amino acid to its D-form during activation, especially for histidine and cysteine. The product has the same mass and may co-elute, so it needs chiral or high-resolution methods to detect.
- Aspartimide formation: base-catalyzed cyclization at Asp, especially Asp-Gly, during repeated piperidine treatment, leading to isomers and piperidide adducts.
- Incomplete side-chain deprotection: residual protecting groups, visible as characteristic mass additions.
- Oxidation and alkylation: modification of Met, Trp or Cys during cleavage when scavenging is insufficient.
Longer peptides and difficult sequences
Some sequences aggregate on the resin as they grow, forming beta-sheet structures that block reactive sites and cause coupling failures. Strategies to manage this include pseudoproline dipeptides and backbone-protecting groups that disrupt aggregation, elevated temperature or microwave heating, alternative solvents, and lower resin loading.
For peptides and small proteins too long for efficient stepwise synthesis, chemists synthesize shorter segments and join them. Native chemical ligation couples a peptide thioester to a peptide with an N-terminal cysteine to form a native peptide bond. Large-scale manufacture of some approved peptides combines SPPS with solution-phase steps or recombinant expression of a precursor followed by chemical modification, as with lipidated peptides such as semaglutide.
Why synthesis matters when reading a COA
Understanding synthesis explains what analytical data should look for. Deletion sequences and truncations appear as peaks with predictable mass differences, aspartimide products show −18 Da, and incomplete deprotection gives characteristic mass additions. Racemized products are not distinguished by mass at all. A certificate that shows a single clean HPLC peak and a matching mass is reassuring, but for sequences prone to racemization or aspartimide formation, additional methods may be warranted.
Expected masses for the target sequence and for likely deletion products can be calculated with the sequence analyzer. For interpretation of HPLC and MS data, see HPLC and mass spec explained and how to read a COA.
Frequently asked questions
What is the difference between Fmoc and Boc synthesis?
Both are protecting-group strategies for SPPS. Boc chemistry removes the temporary N-terminal group with TFA and needs a very strong acid such as hydrogen fluoride for final cleavage. Fmoc chemistry removes the temporary group with a mild base, usually piperidine, and uses TFA for final cleavage and side-chain deprotection. Fmoc is now the more common approach.
What is a deletion sequence?
A deletion sequence is a peptide missing one or more residues from the intended sequence. It forms when a coupling step does not go to completion and the unreacted chain is extended in the next cycle. Deletion sequences differ from the product by the mass of the missing residue and are a common impurity in synthetic peptides.
Why can racemization be hard to detect?
Racemization converts an L-amino acid into its D-form, producing a diastereomer with exactly the same mass as the intended peptide. Mass spectrometry therefore cannot distinguish it, and it may co-elute in standard reversed-phase HPLC. Detection may require optimized chromatography, chiral amino acid analysis after hydrolysis, or comparison with a reference standard.
Compounds mentioned
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