For anyone unfamiliar with solid-phase peptide synthesis, what is it?
By breaking a peptide down into individual monomers, be that natural or unnatural building blocks/amino acids, solid-phase peptide synthesis (SPPS) enables the sequential construction of a peptide on a solid support.
Imagine three bricks, green (the solid support), red (a monomer/building block) and blue (a protecting group). The green brick forms the foundation of what we will build. Place the green brick down as the base; next you have two stacked bricks, the top brick is blue and the bottom brick is red; place blue and red bricks on top of the green brick.
Next, remove the blue brick which frees up the red brick, and add a second blue/red unit (another Fmoc protected amino acid) on top of the red, you then slowly build your red tower or, in this case, peptide until it is complete.
Finally, you take your red tower (peptide) off the green brick (solid support) resulting in the desired peptide. The chemistry starts to become very interesting when instead of just blue bricks for protections you have a rainbow to choose from depending on what you’re doing
Building blocks is a great way to describe it. What is the advantage of solid-phase over solution-phase peptide chemistry?
When you bind building blocks on to a resin, the resin is never in solution, but the attached building blocks are exposed to the solvent. Typically an excess of reagents are used which drive the reaction to completion. The key advantage of the solid support is that it enables purification by filtration, with multiple washings removing the excess reagents used while the solid support peptide remains on your filter, enabling you to easily move on to the next step immediately without additional purification. The washing process generally takes seconds to minutes depending on the type of SPPS you are performing.
Whereas in solution-phase chemistry when a reaction is complete it typically requires a labour intensive and time-consuming purification process, such as column chromatography, which significantly slows the process.
Why would you use a peptide synthesiser over manual synthesis?
One of the big advantages of using automation for anything is they don’t sleep; they just don’t stop.
Solid-phase by hand has been done for years, it will take you three hours do a coupling then another 30 minutes to complete the deprotection. But the PurePep Chorus allows us heat the individual reactor vessels. All of a sudden that three hour coupling time has reduced into two to three minutes. There is also the option to heat the deprotection step shortening this too.
In addition, this instrument enables you to synthesis four peptides in at the same time. If you wanted to you could do four peptides that were 20 amino acids in length overnight and you are finished. By hand that would be weeks of works. It’s a significant time-saver when we use it on client projects.
What Scale can you do on the peptide synthesiser?
The manufacturer typically recommends 0.005 mmol up to 1 mmol, but depending on the loading of the resin we use and length of the peptides we can increase that to 2.5 mmol. The PurePep Chorus is mainly focused towards small scale synthesis, but it’s brilliant for high throughput as you can do four different peptides at the same time.
How unusual is it for a CRO to have peptide experience and supporting technology?
A client said it was very rare to get a CRO with a high level of peptide experience. Although I’m the lead for this technique we have others in the business with experience and are also investing in developing colleagues in this area.
What types of peptides can we synthesise in-house?
With regard types of SPPS, our focus with the PurePep Chorus is Fmoc-SPPS but the instrument is also listed as Boc-SPPS compatible by the manufacturer. The system complements our existing in-house SPPS equipment which enables us to perform manual SPPS on a range of scales.
When considering functionality, the Chorus has four reactor slots, 27 amino acid bottles and six slots for single use amino acids, as well as eight solvent/reagent bottles which enables us to access significant complexity. Whilst the primary focus of automated synthesisers is generally natural peptides via Fmoc-SPPS, we also have the option to synthesise unnatural peptides as well which, as a CRO, is how we commonly use the machine. With appropriate protections, such as Teoc, Dmab, Allyl, Alloc etc, we can also do side chain modifications allowing us to access complex linear and cyclic peptides of varying sizes. Alternatively, we can even switch to PNA synthesis.
In terms of the length, we wouldn’t go above a chain of 30 amino acids. If you wanted to go beyond 30 to make long peptides, you would make a portion of the desired peptide of a certain length, cleave it then use the resultant peptide as a building block onto another segment of the desired peptide. When accessing particularly long peptides, secondary effects of the peptides prevent the standard step wise approach, such as coiling of the peptide onto themselves which can make them collapse which prevents further reactions.
We also have experience with an array of different resins which allows us to tailor the synthesis to meet the needs of our clients. Additionally, we also have experience making complex analogues of peptides off resin.
So what is the likelihood of success using peptide synthesis?
With peptide synthesis everything depends on your sequence, there are problem amino acid, but these are well known. With natural peptide synthesis the chances of success are very high, it’s a well ironed out process. When you start to introduce unnatural amino acids or bifurcated peptides, things start to get more interesting and challenging but that doesn’t mean you can’t make them.
What is the purification of the peptides like?
Once you have built your chain, you go through a couple of steps. The first is filtration, just washing with excess dimethylformamide (DMF) is the first round of purification, this happens all the way through the process, after each of the building blocks are attached. With the desired peptide constructed you then cleave the peptide off the resin itself and we can automate this on the PurePep Chorus, by setting a time for completion it means that your peptide is not allowed to sit in trifluoro acetic acid (TFA), which can cause degradation. It also makes the process more efficient, aligning the cleavage to people’s working hours.
Next step is to triturate in cold ether, any TFA salts of your peptide will crash out during this stage, and we remove them by centrifuging to obtain a white solid of quite a high purity. If you are working with natural amino acids you are generally able to attain reasonably high purity at this stage (70-90%) without the need for further purification. If working with peptides that don’t precipitate from cold ether, then we would concentrate and purify.
If further purification is required then it depends on the peptide how we would proceed, it is fairly routine at Charnwood Discovery to purify peptides via reverse phase flash column chromatography on our in lab CombiFlash Teledyne systems, or use our in-house purification team’s expertise who will purify on one of our HPLC systems. Having an in-house purification team is a huge advantage for us, as they save us a lot of time not only in shipping, but the collaboration between the synthetic chemist and the purification team enables a rapid turnaround time.
Why does the PurePep Chorus give us the advantage over other peptide synthesisers?
One of the things that I really like about it is you can do multiple peptides at once. To accelerate your reactions, you are looking at heat profiles. There are two ways to heat by: convention; and microwave. Most microwave reactors can do one peptide at a time, there is a way around this as you have flow systems dedicated to moving resins but with difficult to handle resins this can potentially become problematic. Personally, I prefer having the resin in a single reactor vessel where all the chemistry occurs, which the PurePep Chorus enables us to do.
Why is having access to a peptide synthesizer important for our work with clients?
We can access complex peptides or perform high throughput peptide synthesis easily, and most importantly in a time efficient manner.
The value to our clients is the flexibility and time-saving the PurePep Chorus provides. We are always looking at how we can advance clients’ drug discovery projects as efficiently as possible using a combination of our in-house expertise and intellectual input combined with the relevant technology.
I’m delighted that Charnwood Discovery has invested in the technology so our clients with peptide projects really see the benefits.
Read more about our resident peptide experts in our blog Discover More About… Our Scientists’ Experience of Peptide Synthesis.
