“When we promise molecules to our collaborators, we absolutely have to deliver, and we want to deliver quickly. The Biotage® Initiator+ Alstra™ allows us to do that,” says Dr. Andrew Jamieson at the University of Glasgow.
Dr Andrew Jamieson is Senior Lecturer in Chemical Biology at the University of Glasgow, Scotland. He was recently appointed to this position having moved from the University of Leicester. We spoke about his research and how Biotage instruments have improved his peptide synthesis workflow.
What is your field of research?
“Overall chemical biology, peptide chemical biology, specifically the application of synthetic chemistry to conformationally constrained peptides to produce tools and probes for chemical biology applications.”
What made you choose Biotage when deciding to purchase a peptide synthesizer in 2014?
“The cost was a big factor. When we compared with systems that were available commercially, Biotage really stood out as providing a bigger bang for the buck. You got everything all of the other competitors were providing at a much lower cost.”
You recently moved to the University of Glasgow and purchased your second Biotage® Initiator+ Alstra™ system. Having used your first for a couple of years you also had the opportunity to look at other vendors when making an informed decision at Glasgow. What made you choose the Biotage® Initiator+ Alstra™ system again?
“I’ll go right back to when I started my independent academic career at Leicester back in 2010, when there was only one system available from another company. I was a fan of the idea of thermal heating in peptide chemistry so we bought that system. It was fine to some extent, but we learnt there were many issues with the nature of that machine. When Biotage brought the Biotage® Initiator+ Alstra™ onto the market, we very quickly identified that the machine works in a completely different way to the other machine in terms of the robotics – one operates through fluidics, whereas the Biotage® Initiator+ Alstra™ is a syringe delivery system. We had both machines in the lab at this time and were doing comparisons – not so much to do with the chemistry but rather the robotics. The machine with the valves which controlled the fluidic system would get blocked. On every other synthesis, the PhD student working with the machine spent a lot of time at the back of the machine pulling out these valves, unblocking them, putting them back in, and running the synthesis again. Half way through the synthesis it would block, and invariably that would always happen in the middle of the night, so not only are you disadvantaged in terms of the cost of the reagents and the frustration of having to go back to the start, but it was really the time that was lost, it was extremely frustrating. When we compare that to the Biotage® Initiator+ Alstra™ with the syringe delivery, this never happened because fundamentally the machine works differently. So the chemistries, you could argue are comparable, when you actually compare like-for-like chemistries, but it's the robotics for us that makes the big difference. That’s the big advantage for me of the Biotage® Initiator+ Alstra™.”
Why do you need a product like the Biotage® Initiator+ Alstra™?
“We move quite quickly in terms of the ideas we come up with. We generally have meetings with our collaborators and identify a biological problem. We then design molecules that we think can contribute to solving that problem, and so we want to make the molecules quickly. When we promise molecules to our collaborators, we absolutely have to deliver, and we want to deliver quickly. The Biotage® Initiator+ Alstra™ allows us to do that. We can make the molecules that we want to make, and a lot of these are quite complex. There is quite a lot of complex chemistry in addition to the standard peptide chemistry that we use – the macro-cyclization and the like, post-translational modifications. The setup of the Biotage® Initiator+ Alstra™ really allows us to deliver that. We found it incredibly difficult to do some of the chemistry using the other system, and that is really why we bought the second Biotage® Initiator+ Alstra™.”
Can you provide some examples of things the Biotage® Initiator+ Alstra™ does really well?
“The vast majority of peptides that we make are conformation- ally constrained. We make a lot of stapled peptides using Verdine’s chemistry, but we’ve also developed other conformational constraints to induce helicity using ‘click’ chemistry. We can do all this chemistry in the Biotage® Initiator+ Alstra™ very easily. We’ve also made relatively long peptides, moving into making proteo- mimetics as opposed to Pepto mimetics. That means making proteins which have more than one secondary structure where we conformationally constrain one. For instance we’ve just submitted a paper where we conformationally constrained a protein called TPX2, which interacts and activates the kinase Aurora-A, that’s a 43 residue peptide that was relatively difficult to synthesize1. We had to do a lot of development work on the chemistry but we were able to do that in an efficient manner using the Biotage® Initiator+ Alstra™.”
You also use an Isolera™ flash purification system; how does all this fit into your workflow and what advantages do you see of having this kind of set up?
“Yes, we bought an Isolera™ about 2 years ago at Leicester. We used it mainly for purifying small molecules. We do a lot of work synthesizing enantiopure amino acids and used the Isolera™ a lot for that method2. But then at one point we realized that Biotage sold the reversed phase flash cartridges. Some of my students did a lot of complaining about having to do multiple injections on the semi-prep HPLC and I said well, why don't you just get a reversed phase cartridge and use the Isolera™. I think it opened their eyes to stop going with the dogma; that when you are purifying a peptide you’ve got to use HPLC – because it’s not the case. It’s just chromatography! This is chromatography. If you produce a peptide of sufficient purity with no critical impurities, why not use the Isolera™? It’s just as efficient, probably faster in some instances than using HPLC. The thing they like the most is the speed. HPLC always comes with a queue of people waiting to use it because it is an inherently slow process, but the Isolera™ tends to be a lot faster with shorter gradients, so for peptides which were relatively pure after synthesis, it was a really good technique to use for purification.”
What size sequences were you purifying on the Isolera™ in reversed phase?
“We generally make peptides of 10 to 15 residues in length. The longest peptide we’ve purified on the Isolera™ is about 20 residues. We’ve not been using it for that long. It’s the kind of thing that once one student uses it and has a good experience, then word travels fast. As I build up the group, the current students are using the Isolera™, and so I envisage as more people join the group, they will also learn to use the Isolera™ for purification of peptides.”
What kind of quantities of peptides were you purifying?
“We were purifying around 100 mg of peptides on the Isolera™. We have a plan to use it for gram quantities of peptides in the future.”
Were you happy with the recoveries that you were obtaining?
“Yes, I mean ultimately the recoveries are comparable to HPLC. There is what I refer to as the Angel’s share, coming from Scotland, when you put whiskey in bond you lose the Angel’s share. With peptides the Angel’s share is really due to the salts that come through in the synthesis process, so you will always lose the Angel’s share but ultimately the recoveries of the peptides and the yields were good.”
Interview conducted on September 16th 2016
References
- Rennie, Y. K; McIntyre, P. J; Akindele, T; Bayliss, R; Jamieson, A, ACS Chemical Biology, 2016, 11(12), 3383–3390.
- Aillard, B; Robertson, N. S; Baldwin, A. R; Robins, S; Jamieson, A. G, Organic and Biomolecular Chemistry, 2014, 12(43), 8775–8782.
Literature number: PPS439
