Chemical Synthesis of Peptides

The chemical synthesis of peptides, which are chains of amino acids linked by peptide bonds, is a crucial process in biochemistry, molecular biology, and pharmaceutical development. This post delves into the methodologies, principles, and challenges associated with peptide synthesis.

To indicate how vitally important this synthesis is, the US Food and Drug Administration (FDA) approved 32 different peptides for therapeutic use between 2016 and 2023. That is apparently 8.4% of all drugs approved in that period of time by that agency which had been manufactured by this process. 

The key feature of a peptide that synthesis deals with is that each one is composed of amino acids linked by peptide bonds. Peptide bonds are formed by condensation which means the removal of a molecule of water.  Peptides are not just about combinations of natural amino acids though, they include forms of amino acid that are not found in proteins, they can also contain fatty acids and polyethyleneglycol (PEG).

Peptide synthesis can be subdivided into two types:-

  • solid phase peptide synthesis (SPPS)
  • Liquid phase/solution phase peptide synthesis (LPPS).

One question to be answered is which of these will be chosen.

The other feature that needs to be considered when choosing a synthetic method is that peptides for research are made in milligram quantities but for large number of patients these have to be produced in kg quantities. The scale of difference is extreme!

Basics of Peptide Synthesis

Peptides are chemically synthesized through the formation of amide bonds between the carboxyl group of one amino acid and the amino group of another. The process typically involves:

  1. Protection of Reactive Groups: To prevent undesired reactions, the functional groups of amino acids (amino and carboxyl groups) must be selectively protected. Before starting, the C-terminal (carboxyl) protecting group and the N-terminal (amino group) protecting groups are selected. Before beginning a synthesis it is worth knowing what the C-terminal and N-terminal protecting groups should be.
  2. Activation of the Carboxyl Group: To facilitate bond formation, the carboxyl group of the amino acid is activated.
  3. Coupling Reaction: The activated carboxyl group reacts with the amino group of another amino acid, forming a peptide bond. Be clear about which coupling agent is to be used as this affects subsequent manufacturing.
  4. Deprotection: Protective groups are removed to free up the functional groups for subsequent reactions or for the final application.

Solid-Phase Peptide Synthesis (SPPS)

SPPS, developed by Bruce Merrifield in 1963, revolutionized peptide synthesis (Merrifield, 1963).  He synthesized the nonpeptide, bradykinin in 1962 in 8 days with a 68% yield. He then synthesized ribonuclease of 124 amino-acids in 1969. It tool 369 reactions involving 11,391 steps. He was awarded the Nobel Prize for chemistry in 1984.

Since those early days, the original concept of the SPPS strategy has changed considerably from the early days of demonstrating the principle to one where large-scale demand for peptides must be met.

His method involved anchoring the C-terminal amino acid to a solid resin, facilitating the sequential addition of protected amino acids. The steps in SPPS include:

  1. Attachment to the Resin: The C-terminal amino acid is attached to a solid resin support via its carboxyl group. As we note later, these are readily available resins from a number of suppliers.
  2. Deprotection: The protecting group (usually Fmoc or Boc) on the amino group of the attached amino acid is removed to allow for the next coupling reaction.
  3. Coupling: The next protected amino acid, with its carboxyl group activated, is added and forms a peptide bond with the deprotected amino group.
  4. Repetition: Steps 2 and 3 are repeated until the desired peptide sequence is synthesized.
  5. Cleavage from the Resin: The peptide is cleaved from the resin, usually with a strong acid, and the side-chain protecting groups are removed simultaneously.

Key Reagents and Conditions in SPPS

  • Fmoc and Boc Protecting Groups: These are N-terminal protecting groups.  Fmoc (9-fluorenylmethoxycarbonyl) is base-labile and removed with piperidine, while Boc (tert-butyloxycarbonyl) is acid-labile and removed with trifluoroacetic acid (TFA).
  • C-Terminal Protecting Groups: The carboxyl terminal may have to be protected at times especially in liquid phase peptide synthesis. The most common acid protecting group used in the methyl ester but ethyl esters and allyl esters have been used. The methyl ester protecting group is most stable in a coupling reaction and in deprotection reaction conditions.
  • Coupling or Activating Reagents: Common activating reagents include DCC (N,N’-dicyclohexylcarbodiimide), EDC (N-(3-dimethylaminopropyl)-N’-ethylcarbonate,  HBTU (O-benzotriazole-N,N,N’,N’-tetramethyluronium-hexafluoro-phosphate), TBTU, HATU and PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate).
  • Side Chain Protecting Groups: The amide and amino groups on amino acids such as lysine and arginine need protection during peptide synthesis. An orthogonal Mtt protecting group (Mtt (4-Methyltrityl) is used here.
  • Dye groups: dabcyl moiety is used in quantification – a colour dye attached to amino-acids to help with measuring amounts of peptide produced or whether a reaction has occurred.

Almost all peptides of at least 40 amino acids can be manufactured using SPPS. These are produced in ‘peptide synthesizers’. They are machines that have been designed to be automated in terms of the required coupling and then washing cycles.

 The Boc strategy developed by Merrifield requires TFA (trifluoroacetic acid) for the repetitive removal of the Boc groups. Invariably it required hydrofluoric acid (HF) to release the final peptide from the matric or support. HF is too corrosive and toxic to make it routinely accessible to those wishing to manufacture peptides. Hence, the Fmoc strategy is now preferred over the Boc synthesis. The Fmoc group is removed under more benign conditions using secondary amines, most usually a 1:4 solution of piperidine in DMF.

Whichever strategy is used, the Boc strategy relies on graduated acid-lability. This is between the Boc which is removed using TFA and the linkage to the support.

The most commonly used resins are polystyrene based with 1% cross-linking. One important base resin is aminomethyl-polystyrene as well as chloromethyl-polystyrene.  The choice of resin is crucial because it affects the efficiency, yield, and ease of purification of the synthesized peptide.  

The types of resin available are listed here. They all have the appearance of a very fine sand which must be retained within a column if synthesis is to be managed carefully.

1. Merrifield Resin

  • Type: Chloromethyl or hydroxymethyl polystyrene.
  • Functional Group: Chloromethyl or hydroxymethyl.
  • Features:
    • Introduced by Robert Bruce Merrifield, who won the Nobel Prize for this innovation.
    • Widely used due to its robustness and compatibility with a wide range of reactions.
    • The resin is relatively inexpensive and allows for the synthesis of a wide variety of peptides.
  • Applications: General-purpose resin for Fmoc (9-fluorenylmethyloxycarbonyl) and Boc (tert-butyloxycarbonyl) strategies.

2. Wang Resin

  • Type: p-Alkoxybenzyl alcohol polystyrene.
  • Functional Group: p-Hydroxybenzyl alcohol.
  • Features:
    • Suitable for the synthesis of peptides using Fmoc chemistry.
    • Acid-labile, allowing for mild cleavage conditions (typically using trifluoroacetic acid (TFA)).
    • Provides good stability during synthesis and easy cleavage of the peptide.
  • Applications: Fmoc solid-phase peptide synthesis, especially for peptides requiring mild cleavage conditions.

3. Rink Amide Resin

  • Type: Polystyrene-based resin.
  • Functional Group: Rink amide linker.
  • Features:
    • Designed for Fmoc chemistry.
    • Produces C-terminal amides directly upon cleavage.
    • The Rink amide linker is stable under synthesis conditions but cleaves cleanly with TFA. It is the resin for beginners in peptide synthesis because the loading steps are the same as the coupling steps.
  • Applications: Synthesis of C-terminal amide peptides. 

4. HMBA Resin (Hydroxymethylbenzoic Acid Resin)

  • Type: Polystyrene-based resin.
  • Functional Group: Hydroxymethylbenzoic acid.
  • Features:
    • Suitable for Fmoc synthesis.
    • Provides stable linkages during peptide elongation.
    • Acid-labile, allowing for the release of free carboxylic acid peptides.
  • Applications: Fmoc synthesis with the production of peptides having free carboxyl termini.

5. 2-Chlorotrityl Chloride Resin

  • Type: Polystyrene-based resin.
  • Functional Group: 2-Chlorotrityl chloride.
  • Features:
    • Versatile resin for both Fmoc and Boc strategies.
    • Highly acid-labile, providing very mild cleavage conditions.
    • Allows for partial cleavage to monitor the progress of the synthesis.
  • Applications: Synthesis of peptides that are sensitive to harsher cleavage conditions, and for fragment condensation strategies.

6. PAL Resin (Peptide Amide Linker)

  • Type: Polystyrene-based resin.
  • Functional Group: PAL linker.
  • Features:
    • Compatible with Fmoc chemistry.
    • Produces C-terminal amides upon cleavage.
    • Stable during synthesis and easily cleaved under acidic conditions.
  • Applications: Synthesis of C-terminal amide peptides, similar to Rink amide resin but with different linker stability.

Key Considerations in Choosing and Handling a Resin:

  • Cleavage Conditions: The stability of the resin-linker under synthesis conditions and the ease of cleavage are important. Acid-labile resins (e.g., Wang, Rink Amide) allow for mild cleavage.
  • Compatibility with Chemistry: Resins must be compatible with either Fmoc or Boc strategies. Fmoc chemistry is generally preferred for its milder conditions.
  • Type of Peptide: The desired C-terminal functionality (amide or acid) dictates the choice of resin (e.g., Rink Amide for C-terminal amides, Wang for C-terminal acids).
  • Peptide Length and Complexity: Some resins are better suited for longer and more complex peptides due to their stability and efficiency in synthesis.
  • Availability: Many of these resins are readily available from stockists without having to be prepared with linkers.
  • Preparation: Most practitioners will take a ready made resin and swell in a small chromatography plastic column (5ml) with a sinter to retain the fines. The initial setting up of the resin is critical to the overall success. The resin is usually swollen in a separate vial before use. This is about 24 hours beforehand in a cool place using a solvent such as N-methyl-pyrrolidone (NMP) or dimethylformamide. Enough solvent is added to enable complete swelling of the resin. When swollen the beads behave like a slurry similar to silica gel prior to use in liquid chromatography. The slurry is added to the sintered chromatography column. The residue in the vial used for swelling is rinsed with the same solvent – 1 ml, and added to the column containing the resin. The solvent will drip through the resin. This is helped by applying pressure using a 10ml syringe to force solvent from of the resin. Once solvent has been removed the end nozzle is stoppered! 

By carefully selecting the appropriate resin, chemists can optimize the synthesis process, ensuring higher yields, better purity, and more efficient production of the desired peptides.

Detailed Method Of Synthesis Using a RINK Amide resin.

Step 1: Removal of the Fmoc Group (Deprotection of the amine group).

A RINK Amide resin for example starts with an Fmoc group attached. To prepare make a reactant solution of 20% 4-methyl piperidine to 80% N-methyl pyrrolidone (solvent). The piperidine is a base that reacts selectively with the Fmoc group. A small volume of reactant (1ml) is added to the resin. The column is stoppered The resin is agitated by shaking so all the reactant accesses the resin and left for 15 minutes to complete the reaction.

The resin is drained of reactant and the process above repeated. At times the stopper in the column is removed to relieve pressure from the build up of gas evolved from reaction. The resin in this example will turn red but not because of the deprotection reaction. The eluent (reactant) is collected – pressure generated by reactants forces some of that liquid out. To check how much Fmoc is eluted involves UV spectral analysis of the eluent. This is quantified as number of moles of Fmoc generated. Eluted  Fmoc has a spectral peak at 300 nm.

Step 2: Column Washing

 The beads need to be washed thoroughly to remove reagent otherwise remaining chemicals will react and waste further added reactants. The solvent NMP is again used, three times – only a small amount is required and the walls of the chromatography column need to be washed thoroughly. The washing solution is pushed out by the application of pressure using a syringe with each wash stage. The final washing solution is dichloromethane to get rid of remaining reactants. It is less viscous and so can more thoroughly wash the beads.

Step 3: Activation of the Amino Acid to Attach To The Resin.

The amount of amino acid attached is measured/determined by the amount of freely available amine on the resin. That amount had previously been measured from the UV spectral analysis of Fmoc eluted.

A variety of coupling/activating agents are available. DCC, HATU etc. are available. HATU is used because it undergoes a colour change during its reaction. The reagent is made up in NMP solvent.  Included in the reaction mixture is the amino acid of choice, the coupling agent, an activating agent such as DIEA (diisopropyl ethyl amine) which is a base. The amino acid has a dye molecule attached. This amino acid is preactivated with the coupling agent and the base for a few minutes. The reason the resin changes colour is because of the binding of the amino acid to the resin and acts as good indicator of reaction. The  reaction is complete within 5 minutes but generally is left for the reaction to fully complete. Excess activated amino acid is drained from the resin. The resin is successively washed as before with NMP and then dichloromethane. This is checked by observing a colourless wash solution at the end with the resin faintly coloured by the covalently coupled amino acid.

Step 4: Adding A Second Amino Acid

As before the 2nd amino acid is activated in the same way with an RXN mixture. This now includes a small amount of Fmoc linked amino acid with the coupling agent and base. This too has a dye attached which turns a pale yellow colour. The preactivation step takes 5 minutes. The preactivated amino acid is added to the column. Assessing the reaction is lee obvious. When a nucleophile reacts with the HATU-activated amino acid it produces a bright yellow coloured molecule. The colour is check on the reaction and the eluate is yellow. It shows whether a reaction is taking place.

Step 5: Checking Progress

A ninhydrin or Kaiser test is performed.  This is performed using a check on the resin. Some of the beads are added to a test kit. It comes as a ready made test kit. The Kaiser test shows deprotected beads and those which are protected. If the activated amino acid has reacted then there are no more free amine groups for reaction and the test reagent should be colourless.

The addition of each amino acid follows this regimented process. Deprotection, washing, activate amino acid. With such an involved process it will take a number of days to produce a 10 to 15 amino acid peptide.

Once the desired peptide has been generated, the peptide is ready to be cleaved from the resin.

Step 6: Cleavage of Peptide

 The resin is dried out using a vacuum dryer. The resin is treated with a small amount of TFA (95% aqueous solution). This cleaves the peptide from the beads. Having a coloured peptide is a good check on the release of the peptide. It takes a couple of hours to release the peptide and any protecting groups from the peptides. The resins will float and the solution contains the peptide. An HPLC assessment alongside an electrospray mass spectroscopy assessment will indicate the degree of purity. The peptide is dissolved in the TFA solution. This is lyophilised to generate a red powder.  

Liquid-Phase Peptide Synthesis (LPPS)

Although less common than SPPS, LPPS is used even preferred, especially for large-scale production. It involves synthesizing peptides in solution rather than on a solid support. It relies on growing a peptide chain which is connected to a soluble tag such as polyethyleneglycol (PEG). The method is much older than SPPS but because of the issues surrounding side reactions and low yields, it lost out to SPPS.

The process is similar to SPPS but with some differences:

  1. Sequential Addition in Solution: The amino acids are added sequentially in solution, requiring careful purification after each coupling step to remove by-products.
  2. Use of Protecting Groups: Like SPPS, protecting groups are used to prevent side reactions, but the choice may differ based on solubility and purification considerations.
  3. Activation and Coupling: Similar activation methods are used, but the solvent system and reaction conditions can vary significantly.

The new versions of LPPS now cover polydisperse polyethylene glycol (PEG), membrane-enhanced peptide synthesis (MEPS), fluorous technology, ionic liquids (ILs), PolyCarbon, hydrophobic polymers, and group-assisted purification (GAP) (Sharma et al., 2022).

 All the soluble supports used in LPPS have to be:-

  • commercially available or be amenable to preparation in the laboratory
  • have good mechanical and chemical stability. 
  • offer appropriate functional groups for easy attachment of organic groups
  • high solubilizing power to dissolve other molecules with low solubility.
  • they should be supports of variable molecular weight because polymer properties vary with chain length. The molecular weight range should be narrow
  • Ideally, they are solid or crystalline at room temperature but too high to reduce their solubility.
  • A high solubilizing power is desirable to ensure homogeneous reactions and high yields throughout any synthetic process.

Soluble Polymers Used in LPPS

The list of soluble polymers is not extensive and  deserves further research. Various types include:-

  • unlinked polystyrene
  • polyvinyl alcohol
  • polyacrylic acid
  • polyethylene glycol (PEG) plus derivatives
  • polyacrylamide.

PEG is formed from the polymerization of ethylene oxide. Its molecular weight is less than 20 kDa. They are crystalline with loading capacities of 1 to 0.1 mmol/g. The lower molecular weight PEGs are liquids ate room temperature. PEG termini can consist of hydroxyl groups or be selectively functionalized.

Typical examples of LPPS Using PEG include:-

  • homoglycine peptides of 1 to 9 residues on PEG 10 000 for conformational studies (Bonora et al., 1980). Higher homologues could not be obtained because of poor and incomplete coupling. 

Differences Between Liquid (Solution) and Solid Phase Peptide Synthesis

 Liquid phase peptide synthesis is time consuming and requires side chain protecting groups for amino acids such as lysine, aspartate, glutamate and cysteine. In terms of coupling reactions, there is less than 90% conversion. It requires purification after each step but it can be operated at large scale and is cheap to run.

The solid phase peptide synthesis method is much quicker. All the side chain protecting groups need protection but at least coupling is over 99.5% conversion. Purification occurs at the end and it unfortunately it is still small scale. It is also very expensive.

LPPS does reduce the use of excess reagents and solvents. It is a much ‘greener’ synthesis (Sharma et al., 2022).

Challenges in Peptide Synthesis

  • Racemization: During activation, amino acids can racemize, leading to the formation of D-isomers, which can affect the biological activity of the peptide. Careful selection of coupling reagents and conditions can minimize this.
  • Side Reactions: Unwanted side reactions, such as aspartimide formation, can occur. Optimizing reaction conditions and using appropriate protecting groups can mitigate these issues.
  • Purification: Peptide synthesis often results in mixtures containing truncated sequences and by-products. High-performance liquid chromatography (HPLC) is typically used to purify the final product. Other techniques of downstream processing include phase partitioning, affinity chromatography, ion-exchange chromatography, 
  • Yield and Efficiency: Each coupling and deprotection step can result in some loss of material. Automated synthesizers and optimized protocols aim to maximize yield and efficiency.

Recent Advances in Peptide Synthesis

  1. Microwave-Assisted Peptide Synthesis: Microwave irradiation accelerates reaction rates, leading to shorter synthesis times and often higher yields and purities.
  2. Native Chemical Ligation (NCL): This method involves chemoselective ligation of unprotected peptide segments, facilitating the synthesis of longer peptides and proteins.
  3. Automated Synthesis: Advanced synthesizers with better control over reaction conditions and real-time monitoring have improved the efficiency and scalability of peptide synthesis.
  4. Green Chemistry Approaches: Efforts to reduce the environmental impact of peptide synthesis involve the use of less hazardous solvents, recyclable reagents, and more efficient methodologies. SPPS is possibly the process of biggest concern now because excess amounts of solvents are needed and is one of the main impediments to development of an eco-process or green process. The main solvent is DMF which is highly reprotoxic and classified as a Substance of Very High Concern (SVHC). It is extremely difficult to replace.

Applications of Peptide Synthesis

Solid phase peptide synthesis in particular is now the ‘go to’ method for generating peptides for both research and industrial scale use.

Peptide synthesis is critical in various fields, including:

  1. Pharmaceuticals: Synthetic peptides are used as drugs for conditions like diabetes (e.g., insulin analogs), cancer, and infectious diseases. Peptides such as glucagon-like peptide-1 (GLP-1) analogs are used for diabetes treatment.
  2. Research Tools: Peptides are used as probes, inhibitors, and antigens in biochemical research to study protein interactions, enzyme functions, and immune responses.
  3. Vaccines: Synthetic peptides can be used to develop epitope-based vaccines, providing a targeted immune response with minimal side effects.
  4. Diagnostics: Peptides are used in diagnostic assays, such as ELISA, for detecting antibodies and pathogens.
  5. Biotechnology: Peptides are employed in developing biosensors, biomaterials, and novel delivery systems for therapeutic agents.

Specific Methodologies and Techniques in Peptide Synthesis

Protecting Groups

Protecting groups are crucial in peptide synthesis to prevent side reactions. The two main strategies involve orthogonal protecting groups and selective deprotection. Some common protecting groups include:

  • Fmoc (9-fluorenylmethoxycarbonyl): Removed with piperidine, widely used due to its mild deprotection conditions (Li et al., 2019).
  • Boc (tert-butyloxycarbonyl): Removed with TFA (trifluoroacetic acid), useful for its stability in certain synthesis environments.
  • tBu (tert-butyl): Often used for side-chain protection, stable under basic conditions but removable under acidic conditions.

Coupling Reagents

Coupling reagents activate the carboxyl group for peptide bond formation.

Some widely used coupling reagents include:

  • DCC (dicyclohexylcarbodiimide): Forms active esters but may lead to racemization and by-products (Kvasnica, 2007).
  • HBTU (O-benzotriazole-N,N,N’,N’-tetramethyluronium-hexafluoro-phosphate): Efficient and widely used in SPPS, reducing racemization risks.
  • PyBOP (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate): Similar to HBTU, often preferred for its efficiency and low side reaction profile.

Advances in Automation and Techniques

The development of automated peptide synthesizers has greatly improved the efficiency and scalability of peptide synthesis. These machines precisely control the addition of reagents, washing steps, and reaction times, reducing human error and variability. Key features of modern peptide synthesizers include:

  1. Automated Control: Advanced software interfaces allow for precise control over synthesis parameters, optimizing reaction conditions.
  2. Real-Time Monitoring: Techniques like in situ UV monitoring enable real-time tracking of coupling efficiency and reaction progress.
  3. Parallel Synthesis: High-throughput synthesizers can produce multiple peptides simultaneously, useful for screening libraries and combinatorial chemistry.

Scale-Up Issues with Peptide Synthesis Particularly SPSS

One of the main issues associated with all peptide synthesis is how to translate synthetic methods into ones that meet consumer (patient) demand on an industrial scale. A great deal of attention is being placed on SPSS at the moment because it is still the main method of manufacture.

The current SPSS methods have notable drawbacks and issues associated with them. There is a need for a large enough reactor volume to generate products at scale. Wastage of chemicals and the environmental drawbacks that come with scale-up are also evident. It is clear that manufacturing synthetic peptides is one of the most expensive industrial operations out there. It also explains why drugs are some of the most expensive molecules available and are seriously challenging the budgets of many health services.  Some pharmaceutical manufacturers are clearly seeking opportunities to reduce these costs otherwise manufacture will become economically unviable irrespective of the good they do.

SPPS has a considerably higher Process Mass Intensity (PMI) of 13,000 compared to conventional small molecule manufacture of between 168 and 308.

To increase production capacity as in all scale-up activity is to increase the reactor volume which means more reactors which are larger. By increasing volumetric capacity you can get more APIs from a given reactor volume. The problem here is that size means extra cost and just building bigger is not the most cost-effective or efficient solution.

We cannot overlook quality especially when it comes to the nature of the product generated. All SPSS systems need to accommodate a range of polypeptides – they may be short, intermediate or long  and they could be conjugated.

The capacity of the solid-phase support is critical because it dictates the capacity for peptide manufacture. Any modified linkers selected for example with increased binding sites must generate ‘translatable’ binding sites with the linker to the last amino acid.

All resins used in SPSS swell. The better the swelling factor the better the quality of peptide produced. If the swelling factor is too high, it impacts the size of the reactor needed to accommodate this swollen resin and the amount of solvent used.  Finally, the chemistry of synthesis has to be compatible with conventional Fmoc (SPSS) chemistry and reactor performance.

The ideal criteria is that a resin is high capacity because of many binding sites per bead, and with a low swelling resin that can accommodate long and complex peptides. It means more peptide is generated per given volume.

Conventional synthesis is usually based on a resin bead having a low number of linkage points. When the binding capacity of the resin is raised,  we increase the number of linkage points available per bead. The limitations than become steric hindrance in terms of volume available around the bead for peptide manufacture. Another consequence is that the quality of the peptide drops because there are chemical as well as physical interactions occurring between the peptides. So, increasing the degree of substitution on the resin itself beyond what is normal for manufacture leads to poor quality peptides and slower kinetics of reaction.   

A couple of businesses have started using multiple dendrimic constructs for use in SPSS. There are also novel multi-branching arrangements being developed concomitantly with spacers that have demonstrated that product yield can be raised up to 10 fold compared to traditional methods of SPSS.

The PolyPeptide Group have started using a novel branching concept with their resins. They have triple branching linkers which can generate peptides in a more orderly fashion rather than in the congested state that seemed to be occurring. Their branching construct linker has increased the binding site ‘micro-environment’ which means the available space is now more amenable to peptide size. It also means that synthesis of longer peptides is possible – almost beyond 50 amino acids and a cleaner peptide can be produced.

The concept then is to introduce to the resin core matrix, a spacer to which is then attached a branching agent that has at least two binding sites. PolyPetide have termed this ‘Generation 1’. They then extended the concept further by adding a spacer to the binding sites of the first linked branch which expands the number of binding sites to four. This is now termed ‘Generation 2’. The number of binding sites is raised to 8 by repeating the spacer process and this becomes ‘Generation 3’. Having got to that level of expansion, the linker is attached which now has 8 binding sites for amino-acid binding. The linker is the Fmoc-Rink. Generally, the actual number of binding sites available in reality compared to the theoretical situation is very close. Similarly the peptide yield is consistent but slightly less than the theoretical level. The other major consequence is that polypetide quality is better for the reactor volume used.

Then it is feasible to generate peptides of 50 amino acids in length with better purity than used to be possible. It also means that reactor size can be reduced as well as reducing the amount of waste.

Peptide Modification

Whilst peptides are the main focus of this article, it is possible to take a peptide and modify side chains on the amino acid to produce new variants. One example is the lactosylation of peptides by adding a lactose sugar to the peptide. In this instance it uses solid state synthesis but without solvents.

In the example, the peptides were synthesized on a ChemMatrix Rink Resin using an Fmoc protocol with ultrasonic agitation (Wolczanski et al., 2019). This used a mild base of DIEA (N,N-diisopropylethylamine) (6 eq) and TBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate) (3 eq) as a coupling reagent. When the synthesis was performed, acetylation of the N-terminal was achieved with a mixture of Ac2O (acetic anhydride): DIEA (N,N-diisopropylethylamine): DMF (N, N-dimetylformamide) (0.9:1.7:7.4, v:v:v) for 15 minutes in an ultrasonic bath. A 4-methyltrtyl group was removed with 1% TFA (trifluoracetic acid) in DCM (dichloromethane).

The mixture peptydyl resin (20mg) was swelled in DMF then mixed with lactose and microwave irradiated at 40W at 100 C for 25 minutes. When lactosylation was completed, the resin was washed with H2O, DMF, DCM (dichloromethane), THF (tetrahydrofuran), and Et2O (diethyl ether) (each 3 × 1 min) and dried. As in conventional SPPS, the peptide was split from the resin using a TFA: H2O: TIS (triisopropylsilane) (95:2.5:2.5) mixture. These were analysed using liquid chromatography coupled to mass spectroscopy (LC-MS/MS).

The sample of unmodified and lactosylated peptides are purified using a functionalised resin based on boronate affinity chromatography- (PhB-Lys(PhB)-CMRR) (Kijewska et al., 2020). In tis instance a functionalised resin is PhB-Lys(PhB)-ChemMatrix® Rink. This is swelled in a chromatography column with 50mM ammonium bicoarbonate buffer (H2O: ACN, 50:50, pH = 8). The same buffer is used to wash the resin with all fractions collected and then lyophilized to determine how much unreacted peptide there would be. The eluent of 1% HCOOH (H2O: ACN, 50:50) was added to recover the lactosylated peptide (Kijewska et al., 2024).

Future Perspectives in Peptide Synthesis

The field of peptide synthesis is continuously evolving, driven by the need for more efficient, cost-effective, and environmentally friendly methods. Some promising directions include:

  1. Enzymatic Synthesis: Using enzymes like proteases and ligases for peptide bond formation offers a green alternative with high specificity and low energy consumption (You-Shang, 1983; Haddoub et al., 2009). Normally proteases cleave proteins but these reactions are reversible. Where a hydrolytic cleavage of a peptide bond is an exergonic reaction it is possible to choose amino acids with a high concentration and manipulate kinetic conditions to generate peptides.
  2. Flow Chemistry: Continuous flow synthesis can enhance reaction efficiency, reduce waste, and allow for better control over reaction conditions.
  3. Photochemical Methods: Light-driven reactions can provide precise control over reaction initiation and termination, reducing side reactions.
  4. Peptide Mimetics: Developing peptidomimetics, which mimic peptide structures but are more stable and bioavailable, can lead to new therapeutic agents.

Ultra-Efficient Solid Phase Peptide Synthesis (UE-SPPS)

Ultra-Efficient Solid Phase Peptide Synthesis (UE-SPPS) has been touted by some businesses as a new green approach in SPPS. It means the removal of the resin washing steps that are needed in more traditional approaches to peptide production. Washing is eliminated from the process by combining in-situ quenching of excess activated amino acid monomers with controlled evaporation of excess deprotection base. Particular sets of reactions such as coupling and deprotection is enhanced through microwave irradiation. The claim is that up to 100 amino acids can be linked together.

Making Peptide Synthesis More Environmentally Friendly

Making solid-phase peptide synthesis (SPPS) more environmentally friendly involves reducing the environmental impact of the synthesis process, which traditionally consumes large amounts of solvents and reagents. Here are several strategies to achieve greener SPPS which we can bullet point here. We’ve already highlighted one approach in suing branching spacers.

1. Reduce Solvent Usage

  • Solvent Recycling: Implement solvent recovery systems to recycle and reuse solvents like DMF (dimethylformamide), DCM (dichloromethane), and NMP (N-methyl-2-pyrrolidone). 
  • High Efficiency Mixing: Use advanced mixing techniques to ensure effective resin swelling and peptide coupling, which can reduce the amount of solvent needed.
  • Minimal Volume Protocols: Develop and utilize protocols that minimize the volume of solvents required for washing and deprotection steps.

2. Switch to Greener Solvents

  • Less Toxic Solvents: Replace traditional solvents with less toxic and more environmentally benign alternatives. For example, replace DMF with alternatives like dimethyl sulfoxide (DMSO) or ethyl acetate where feasible. Other solvents to be considered include by γ-valerolactone (GVL), which is a biomass derived solvent (Jad et al., 2018).
  • Water and Aqueous Solvents: Explore the use of water and aqueous solutions for certain steps of the synthesis, such as coupling reactions and wash steps, if compatible with the chemistry.
  • Diethyl ether (DEE) can be substituted by cyclopentyl methyl ether (CPME) in the precipitation step.

3. Optimizing Reagent Usage

  • Coupling Reagents: Use more efficient coupling reagents that require smaller quantities and generate less waste. For example, newer reagents like COMU (ethyl 2-cyano-2-(hydroxyimino)acetate) or HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate) can be more efficient than traditional reagents.
  • Atom Economy: Design reactions and choose reagents that maximize atom economy, minimizing the amount of waste produced per reaction.

4. Waste Management and Reduction

  • In-line Purification: Implement in-line purification systems to reduce the need for extensive washing and purification steps, thus reducing solvent and reagent consumption.
  • Solid Waste Minimization: Develop resins and linkers that allow for cleaner cleavage, reducing the need for extensive purification and waste generation.

5. Improved Process Design

  • Microwave-Assisted SPPS: Use microwave-assisted peptide synthesis to increase reaction rates and reduce reaction times, which can lead to lower solvent and reagent consumption.
  • Automated Synthesis Systems: Employ advanced automated synthesizers that optimize reagent usage, minimize solvent volumes, and enhance overall efficiency.
  • Sonicated peptide synthesis for SPPS. Sonication accelerates various reactions and is claimed to produce peptides of higher purity. It does not cause racemization of sensitive residues such as cysteine and histidine. It reduces reaction times.

6. Green Chemistry Principles

  • Use of Renewable Resources: Where possible, use starting materials and reagents derived from renewable resources.
  • Energy Efficiency: Optimize reaction conditions to occur at ambient temperature and pressure to reduce energy consumption.
  • Catalysis: Use catalytic instead of stoichiometric amounts of reagents to increase efficiency and reduce waste.
  • Microwave and ultrasound: these are green activation techniques. Both methods rely on generating heat. Microwave technology is better understood than sonication because it has not yet been possible to understand how pressure waves operate in reactions.

7. Bio-based and Degradable Resins

  • Renewable Resins: Develop and use resins made from renewable sources that can degrade more easily after use.
  • Recyclable Resins: Use resins that can be regenerated and reused for multiple cycles of synthesis, reducing the amount of solid waste.

8. Minimize Hazardous Chemicals

  • Safer Deprotection Methods: Employ milder deprotection strategies that avoid hazardous chemicals like TFA (trifluoroacetic acid), where possible. For example, using photolabile protecting groups that can be removed by light.
  • Non-toxic Catalysts: Use non-toxic catalysts and reagents that reduce the risk of exposure to harmful chemicals.

9. Sustainable Scale-Up

  • Process Intensification: Develop methods that allow for more concentrated reactions, reducing solvent usage and improving reaction efficiency.
  • Flow Chemistry: Implement continuous flow techniques for peptide synthesis, which can offer better control over reaction conditions and reduce solvent and reagent consumption.

The chemical synthesis of peptides is a cornerstone of modern biochemistry and pharmaceutical science. From solid-phase synthesis to advanced automated techniques, the methods for constructing peptide chains have become increasingly sophisticated and efficient. Despite challenges such as racemization and side reactions, ongoing innovations promise to further enhance the capabilities and applications of peptide synthesis, paving the way for new discoveries and therapies in medicine and biotechnology. The integration of new technologies and methodologies will continue to expand the horizons of peptide chemistry, making it an even more vital tool in scientific research and industrial applications.


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