Peptide Synthesis

Solid-phase peptide synthesis and the science of building amino acid chains.

FOR RESEARCH USE ONLY. This material is sold for laboratory research purposes only. Not for human consumption, veterinary use, or any diagnostic/therapeutic applications.

Peptide Synthesis: From Amino Acids to Research-Grade Compounds

Modern peptide synthesis enables the production of defined amino acid sequences with high fidelity and purity. Understanding how research peptides are manufactured provides essential context for evaluating peptide quality and interpreting experimental results.

Solid-Phase Peptide Synthesis (SPPS)

Virtually all research-grade peptides are produced using solid-phase peptide synthesis, a method introduced by Robert Bruce Merrifield in 1963 (for which he received the Nobel Prize in Chemistry in 1984). SPPS builds the peptide chain on an insoluble polymer resin, one amino acid at a time, from the C-terminus to the N-terminus.

The SPPS cycle consists of four repeated steps:

Deprotection: The temporary protecting group on the N-terminus of the growing chain is removed, exposing the free amino group for the next coupling
Activation: The incoming amino acid’s carboxyl group is activated using coupling reagents (HBTU, HATU, or DIC/HOBt), making it reactive toward nucleophilic attack
Coupling: The activated amino acid reacts with the free amino group of the resin-bound chain, forming a new peptide bond. This step typically proceeds to >99% completion per cycle
Washing: Excess reagents and byproducts are washed away with solvent, leaving only the resin-bound peptide

Fmoc vs. Boc Chemistry

Two major protecting group strategies dominate SPPS:

Fmoc (9-fluorenylmethoxycarbonyl): The most widely used strategy today. Fmoc is removed with piperidine in DMF (base-labile). Side-chain protecting groups are removed with TFA during final cleavage. Fmoc chemistry is preferred for its compatibility with acid-sensitive sequences and automated synthesizers
Boc (tert-butyloxycarbonyl): The original Merrifield strategy. Boc is removed with TFA (acid-labile), and side-chain protecting groups require strong acid (HF) for removal. Boc chemistry is still used for certain challenging sequences and for in situ neutralization protocols

Cleavage and Side-Chain Deprotection

After the complete sequence has been assembled on the resin, the peptide is cleaved from the solid support and all side-chain protecting groups are simultaneously removed. In Fmoc chemistry, this is accomplished using a TFA-based cleavage cocktail containing scavengers (water, triisopropylsilane, ethanedithiol) that trap the reactive carbocations generated during deprotection.

Challenges in Synthesis

Several factors can complicate peptide synthesis:

Difficult sequences: Some peptide sequences aggregate on the resin, reducing coupling efficiency. Beta-sheet-forming sequences are particularly problematic
Long peptides: Each coupling step has a finite yield (<99.5%), and for a 40-residue peptide, even 99.5% efficiency per step yields only 82% of the correct full-length product
Post-synthetic modifications: Cyclization, disulfide bond formation, PEGylation, and other modifications add complexity and additional purification steps

Quality Control

After synthesis and purification, research peptides undergo quality control testing:

HPLC: Confirms purity percentage
Mass spectrometry (MS): Confirms molecular weight matches the expected value for the target sequence
Amino acid analysis (AAA): Verifies the amino acid composition (used selectively)
Peptide content: Determines the actual peptide fraction of the lyophilized material (typically 60-85%, with the remainder being counter-ions and water)

Peptide Synthesis