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Is liquid-phase peptide synthesis the right strategy for your project?

Liquid-phase peptide synthesis is a hybrid method that combines the advantages of solution-phase and solid-phase chemistry. The approach is best suited for the synthesis of short peptides (up to 20 amino acids) for purity-sensitive applications such as:

  1. Therapy
  2. Cell culture media
  3. Analytical standards or controls

What is liquid-phase peptide synthesis?

Along with conventional solution-phase and solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis (LPPS) is one of the three primary chemical processes employed in the stepwise synthesis of peptides.

In practice, liquid-phase synthesis consists of the elongation of the peptide chain in solution. It differs from conventional and time-consuming solution-phase synthesis because it employs the use of soluble tags. These tags, like polystyrene-based solid supports employed in standard SPPS, simplify the workup after each step of the synthesis.

However, tags need to easily dissolve in aqueous or organic solvents while still being significantly distinct from other reagents and byproducts to simplify their removal by precipitation, filtration, or extraction at the end of each step. For this reason, the selection of adequate soluble tags is the primary criterion for a successful and optimal LPPS strategy.

Even though SPPS remains the method of choice for industrial and pharmaceutical peptide synthesis, interest in its liquid-phase counterpart continues to grow. This is due to the method’s potential for scalability as well as its reduced use of reagents and solvents, which makes it more sustainable and environmentally friendly than any other chemical method of synthesis developed to date.

Liquid-phase peptide synthesis versus solution-phase and solid-phase strategies

Synthesizing peptides in the liquid phase blends the scalability and simplicity of typical SPSS procedures with the sustainability of conventional solution-phase techniques to create the best of both worlds.

Solid-phase synthesis, also known as heterogeneous peptide chemistry, allows the cost-effective preparation of peptides in unparalleled turnaround times. Decades of research have made SPPS the gold standard of industrial-scale production.

Nevertheless, the large excesses of hazardous reagents and solvents required in each step of the synthesis remain a challenge to be solved. Particularly when used to produce longer peptides (>20 amino acids), this strategy often also results in the accumulation of undesired byproducts and erroneous sequences that need to be removed through pricey chromatographic methods to allow their use in purity-sensitive applications.

Solution-phase synthesis, on the other hand, is a method that is both time-consuming and labor-intensive. Because it lacks a stable tag, this strategy requires the isolation of all intermediates at the end of each synthesis step. This results in a significant increase in the purity of the final product, as well as a reduction in isomerization and other unintended side reactions. However, because of its complexity, it remains hard to scale up and incurs significant costs.

Although both this conventional method and liquid-phase synthesis take place in solution, the latter remains more cost-effective and scalable because it employs soluble tags to simplify the separation of peptides from reagents and byproducts.

Common soluble tags used in the liquid-phase synthesis of peptides

The development of soluble tags for the liquid-phase synthesis of peptides is a dynamic and active field of research.

Ideal tags should differ significantly from reagents and byproducts to streamline purification, but they should also be cost-effective in terms of production and easy to cleave from the final product.

The most common soluble tags currently in development include:

Soluble tag Description Advantages Challenges
Polydisperse PEG tags PEGs are versatile supports with a unique solubility profile. In this method, peptide purification is achieved by ultrafiltration or precipitation.
  • Possibility of synthesizing in aqueous media
  • Good yields achieved in tag photocleavage
  • Has been used for the synthesis of peptides up to 10 amino acids
  • The heterogeneity of PEGs makes it hard to use mass spectrometry or chromatography to separate them
Membrane-enhanced peptide synthesis (MEPS) MEPS makes use of monodisperse and branched PEG molecules. Purification is achieved by diafiltration.
  • Synthesis can take place in a single reactor
  • Easy to monitor
  • Automation-friendly
  • Low solvent use
  • Greener solvents and coupling reagents are still in development
Fluorous tags Fluorous tags are soluble in fluorous solvents and thus self-assemble in this phase.
  • The reuse of tags is possible
  • Ideal for the synthesis of peptides with less than 5 amino acids
  • Requires the use of expensive reagents
  • Industrial scale is yet to be achieved
Ionic liquid (IP) tags IPs have high thermal and chemical stability and thus are considered the ideal support for green peptide synthesis chemistry.
  • Zero-waste when IPs are used as solvents
  • Can be used to synthesize peptides with up to 15 residues
  • Industrial scale is yet to be achieved
Polycarbonate Long aliphatic chains of polycarbonates make them easy to precipitate using polar solvents or extract using liquid-liquid methods. In practice, this method is very similar to SPPS. However, these tags are more soluble than conventional polystyrene-based supports employed in SPPS.
  • Easy to scale up and monitor
  • Can be used to synthesize long peptides (>20 amino acids)
  • Cost-effective
  • Still relies on the use of strong, hazardous solvents – greener alternatives are still in development

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