Advancements to Sustainability in Peptide Synthesis: The Way to Greener Chemistry

Recent developments in the peptide synthesis field are gearing towards more sustainable and greener practices. The conventional methods used for peptide synthesis often involve large amounts of organic solvents, high energy consumption, and hazardous waste products. 

Did you know that for every pound of peptide drugs, tons of solvents and reagents are exhausted? A study published by Green Chemistry and sponsored by Italy’s Department of Chemical and Pharmaceutical Science asserts that 80-90% of the waste generated during peptide synthesis is due to the multiple washes and purifications required.

This post reviews the innovative approaches developed to address the sustainability concerns in the peptide synthesis process. Let’s start by understanding the underlying principles of peptide synthesis and why making it a greener process has been challenging.

Understanding Peptide Synthesis

You may need a quick introduction to peptide synthesis for more context, but let’s go on.

Peptides are formed when amide bonds connect amino acids in short chains. Peptides function as hormones, enzymes, signaling molecules, or components of structures like collagen or keratin. Here’s more information on what peptides are.

In the pharmaceutical industry, peptides have gained significant attention for their potential use because they’re precise and have minimal toxicity compared to regular small-molecule drugs.

Let’s discuss two of the main approaches used for peptide synthesis: solid-phase peptide synthesis (SPPS) and liquid-phase peptide synthesis (LPPS). In both methods, the goal is to build the peptide chain one amino acid at a time.

Chemical protection groups help to temporarily “shield” the other end of the peptide chain to start with the C-terminus. Generally, SPPS is considered more efficient and less prone to side reactions than LPPS.

Solid-Phase Peptide Synthesis (SPPS)

In SPPS, the peptide chain is attached to an insoluble resin support and assembled step by step using protected amino acids. An Fmoc group helps attach the first amino acid to the insoluble resin while simultaneously protecting the carboxyl group.

Washing occurs after every SPPS step, so the Fmoc group is washed away before coupling reagents are added to link the next amino acid in the chain.

This process is repeated until the entire peptide has been synthesized. Finally, a cleavage agent removes the peptide from the resin support, and all side chains are deprotected.

One of the main benefits of SPPS is its higher yield, purity, and easier purification compared to LPPS. However, this process still relies heavily on organic solvents and reagents, which harm human health and negatively impact the environment.

Moreover, over 80-90% of the reagents and solvents in the SPPS process are wasted, which is a major challenge for sustainability.

Liquid-Phase Peptide Synthesis (LPPS)

In LPPS, the peptide chain is synthesized entirely in solution using protected amino acids and coupling reagents. Compared to SPPS, LPPS has the main advantage of being simple and requiring fewer steps.

However, this method usually has lower yield, purity, and longer reaction times than SPPS. It also requires more purification steps as coupling reactions are less efficient in solution.

Another disadvantage of LPPS is that it often results in peptides with higher toxicity due to incomplete removal of protecting groups and side reactions.

The Sustainability Innovations in Peptide Synthesis

Some of the efforts to make peptide synthesis more sustainable are anchored in some of the following principles:

  1. Moving from linear synthesis to convergent synthesis, where shorter peptide fragments are synthesized and combined to form the final peptide. It reduces the overall number of steps and waste generated.
  2. Using more environmentally friendly reagents and solvents like water-based systems or greener alternatives like ether instead of potentially harmful organic solvents.
  3. Moving away from batch processes to continuous flow systems reduces the amount of excess reagents and solvents used.
  4. Embracing green solvent methods, such as microwave and ultrasound-assisted synthesis, reduces reaction times and improves yields.
  5. Reducing the number of washes and purification steps required by optimizing reaction conditions and using more efficient reagents.
  6. Using TFA-free protocols for peptide cleavage avoids the generation of hazardous waste.
  7. Moving away from piperidine to 20% 4-methylpiperidine (4-MP) in the deprotection step can lead to improved product purity and less hazardous waste.

Minimizing the Wash Steps: 3-Step in-situ Fmoc Removal

Mthethwa, Ndumiso & Kp, and Nandhini & Kumar, among others, worked on a study and published a paper titled: Toward Sustainable Solid-Phase Peptide Synthesis Strategy – in situ Fmoc Removal.

The paper was featured in Green Chemistry Letters and Reviews, and this is an attempt to explain some of the key takeaways from the publication on using 20% 4-MP in DMF instead of piperidine to minimize wash steps.

As discussed earlier, the traditional solid-phase peptide synthesis (SPPS) process involves multiple deprotection and wash steps. This results in significant solvent waste and increased production costs.

The use of piperidine as a deprotection reagent has been a common practice in SPPS thanks to:

  • High deprotection efficiency.
  • Low cost.
  • Readily available.
  • Ease of handling.

Although piperidine’s potential use in illegal drug production can be a contributing factor to restrictions, its primary drawbacks are its hazardous nature and the availability of greener alternatives. Because of this, researchers sought a suitable replacement. They found that 4-MP in DMF is just as effective as piperidine for removing the Fmoc protecting group from amino acids.

Mthethwa et al. used a three-step in-situ protocol using 20% 4-MP in DMF for peptide cleavage in this study. However, they had to use 1% OxymaPure as it’s mildly acidic to eliminate any unwanted remnants of the basic additive 4-MP.

This protocol helped to minimize wash steps, from the usual 4-step process to just 3-step, improving product purity and reducing hazardous waste.

The study showed that using 20% 4-MP in DMF had a negligible impact on the overall synthesis time. They found that this new method helped save 60% of the solvents typically used in the traditional SPPS process while achieving comparable crude peptide purity.

Furthermore, the researchers also leveraged other Green Chemistry principles to refine the process further. Let’s discuss some of them separately, including green solvents and reagents, continuous processing, and the (SPPS) improvements we’ve discussed.

Green Solvents and Reagents

Green solvents and reagents are those that have a minimal environmental impact during their use. They can be readily biodegradable, non-toxic, or produced from renewable resources. In the case of SPPS, researchers have explored alternatives to commonly used organic solvents like DMF and DCM.

One such example is the use of water as a solvent in SPPS reactions. Water is environmentally friendly and reduces hazardous waste production. Additionally, it may serve as a nucleophile and facilitate certain reactions without the need for harsh reagents.

Studies have shown that using ethanol as a solvent can improve the yield and purity of synthesized peptides. It’s a sustainable alternative for reducing waste production and improving safety in the laboratory.

Continuous Processing

Continuous processing is a principle of Green Chemistry that aims to minimize or eliminate the need for separation and purification steps. In traditional SPPS, multiple steps are required to remove excess reagents and by-products and protect groups from the crude peptide product. 

It often results in high solvent consumption and hazardous waste generation. To tackle this issue, researchers have explored using continuous flow reactors instead of traditional batch reactors. 

Continuous flow systems allow for precise control of reaction conditions and can achieve higher conversion rates with less waste production. So, instead of running a loop of batch reactions, continuous processing enables the production of peptides in a single, uninterrupted process. 

For example, if the peptide is to have a sequence of A-B-C, the first step in batch processing would be to couple A and B together, followed by deprotection and then coupling with C. Continuous flow processing is achievable by having two reactors connected in series, each containing the reactants for one step. 

It would allow for the sequential reaction of A and B, followed by deprotection and coupling with C in a continuous flow. Overall, this approach reduces the number of steps required, minimizes waste generation, and improves the efficiency of peptide synthesis.

Let’s Embrace Sustainability in Peptide Synthesis

SPPS has been evolving over the last half-decade, and some of the recent efficiency tweaks on this method were as recent as 2020. The push towards sustainably produced peptides has also led to the emergence of alternative methods.

All the developments towards sustainability in peptide synthesis have been targeted at minimizing waste production and optimizing reagent consumption. Let’s embrace new, proven methods like green chemistry, continuous flow reactors, and other continuous processing technologies.

At Advanced ChemTech, we seek out and embrace the most cutting-edge technologies. As a result, we offer a wide range of sustainable peptide pool products. Call us today to learn more about how we can assist you in producing high-quality, sustainable peptides.