Recently there has been substantial motivation to consider and evaluate alternative, more environmentally friendly solvents. Some countries have even gone so far as to ban some of the more toxic, yet commonly used solvents. In addition to general toxicity, additional consideration in the green chemistry movement is the volume of solvent used in any particular application. In this regard, purification solvent selection is closely monitored as they are often used in large quantities.
In peptide purification, sample loading onto the column is rarely considered. Most, if not all, HPLC instruments come equipped with a sample injection loop which demands a liquid injection of the sample for purification. If you decide to use flash chromatography to purify your peptides though, liquid injection is no longer the exclusive method for sample introduction to the column. Alternatively, dry loading crude material is a strategy often used in small molecule purification, particularly when sample solubility concerns arise.
You’ve just finished a peptide synthesis and now it’s time to cleave the peptide from the resin. You’ve selected a specific cleavage cocktail, performed the reaction and now what? The vast majority of peptide chemists will precipitate their peptide using an ether solution, lyophilize, and move on to purification. But is that the only option?
In today’s post I’ll highlight an alternative strategy that saves both processing time, potentially dangerous reagents, all without compromising the integrity of the recently synthesized peptide.
In a previous post, I did some work evaluating the efficiency of alloc removal with tetrakis palladium using microwave assistance and atmospheric conditions, which worked beautifully. Given the known sensitivity of palladium catalysts (see Derek Lowe's post for a humorous dialogue), I sought to further explore the sensitivity of palladium towards the alloc removal in the context of a peptide.
In this post, I'll explore a variety of atmospheric, room temperature alloc deprotection conditions aimed at evaluating the catalytic lifetime of palladium tetrakis for effective alloc removal.
As a chemist new to the peptide community, there are many choices that have to be made. Which coupling reagents to use? Heat or no heat to promote chemistry? And most importantly, which resin? I have talked previously about resin choices, from loading levels to swelling capacity and how they affect the synthesis outcome. But I haven't addressed yet a fundamental feature of commercially available resins, and that's the functional handle to which the peptide chain is conjugated.
In today's post, I'll describe some, and I mean only some, of the most commonly used chemical functionalities for Fmoc-based solid phase peptide synthesis and some scenarios in which you would choose one resin type over another.
Chemical synthesis of peptides, and even proteins, offers the possibility to expand the functionality and stability imbued by nature. However, chemical synthesis of very long peptides and small proteins remains today an exceedingly difficult task. Several ligation strategies have been developed that help to alleviate this challenge. These strategies though, require a purified, yet fully protected peptide fragment.
Orthogonal amino acid protecting groups effectively expand the chemical tool kit available to peptide chemists allowing for synthesis of much more complex molecules. Often times, orthogonal protecting groups are used in Fmoc-based chemistry to facilitate post-synthesis modifications of peptides, like the addition of small molecule fluorophores and more commonly now, peptide cyclization efforts.
There are several strategies often employed to improve peptide purity achieved using reversed phase HPLC. These strategies can include, changing column length, particle size, particle functionality (C4 vs C18). I have experimented a bit with some of these criteria while purifying peptides using reversed phase flash chromatography but one obvious change that I have not yet explored is the length of column.
In today's post, I'll explore how the length of the cartridge affects the overall resolution and purification efficiency using reversed phase flash column chromatography.
There are several strategies employed when a peptide synthesis requires optimization. Typically, the first thing considered is whether or not to double couple specific amino acids within the sequence. This is somewhat of a change in mentality from traditional room temperature synthesis strategies where double coupling is frequently used for the entire peptide sequence.
As the complexity of peptides continues to grow, so does the use of amino acids with side chain protecting groups that can be selectively removed, leaving the peptide on resin and the remaining side chain protecting groups intact. While there are protocols to be found in the literature, they may not work to the highest level of efficiency every single time. This can lead to disasterous results for any subsequent chemistry.
While resins loaded with the natural 20 amino acids are commercially available these days, there may be times when loading the first amino acid onto the resin in house may be necessary. And unlike loading the first amino acid onto amide-leaving resins, the first coupling reaction for C-terminal acids can be chemically more challenging.
While many of the standard amino acids can be purchased pre-loaded onto Wang type resins, there are still cases where coupling the first amino acid onto Wang resin manually is necessary. In my case, an unnatural amino acid was required on the C-terminus so there was not a commercially available source.
In today’s post I’ll answer the above question by comparing the crude purity of peptides synthesized using amino acid stock solutions or freshly dissolved amino acids.
What is the main goal of a peptide chemist? Elizabeth Denton, Ph.D., explains how Biotage sees the peptide synthesis workflow and how we focus on shortening the process time for scientists.
The diversity of amino acid side chain functionalities, coupled with secondary structure, gives peptides and proteins their unique properties and activities. However, when it comes to chemically synthesizing peptides or even small proteins, the side chain functionalities can do more harm than good.
Our seasoned peptide chemist Dr. Elizabeth Denton has tried it all when it comes to peptides. In this short video she shows that using DMSO (dimethyl sulfoxide) in a peptide synthesis workflow is perfectly fine together with the Biotage® V-10 Touch evaporator. Watch her evaporate it in just over eight minutes.