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.
There are several protocols published both in the literature as well as in technical notes from many peptide reagent and instrument suppliers, but they typically occur at room temperature over extended periods of time (3-24 hours and repeated). In today’s post, I’ll evaluate several conditions suitable for efficiently loading the first amino acid onto Wang-type resin.
I wanted to see if the enhancements that have been observed for SPPS using microwaves could also be translated to the first amino acid coupling as well. While many protocols I found during a quick literature search use anhydrous DCM as the solvent, the Kappe group used DMF as the primary solvent though, so I decided to start there.
Please note: DO NOT USE THIS METHOD FOR LOADING CYS OR HIS ONTO WANG RESIN. These two amino acids have a propensity for side chain racemization and should be avoided.
I decided to load Trp onto plain Wang resin for a couple of reasons:
- any steric occlusion could be readily identifiable due to low yields
- a significant change in mass would be observed with increasing resin load
- the fluorogenic side chain could come in handy if I decided to use LC-MS as an alternative method to quantify the resin loading (more on that in a later post)
In addition to the 5 equivalents of amino acid, I added DIC, HOBt and DMAP dissolved in DMF in a 1:1:1:0.1 stoichiometries relative to the amino acid. Using my Biotage® Initiator+ Alstra™ microwave peptide synthesizer, I loaded my ChemMatrix® Wang resin with 1, 2, or 3 coupling attempts each at 75°C for 5 minutes.
One significant difference in the protocol I chose relative to others that have been published lies in the number of amino acid equivalents used during the loading reaction. I chose to use 5 equivalents, significantly more than the 1.5-2 equivalents commonly used in this reaction. The primary reason I chose such a high number was to drive loading efficiency. My resin was only loaded with 0.47 mmol/g reactive sites at the manufacturer. For some groups this may seem like a high loading amount, but with the large swelling propensity for the ChemMatrix® resins I wanted to retain as much of this functionality as possible.
I have described previously a quantitative strategy to determine the resin loading level after this type of reaction is complete. Unfortunately, I don’t have access to a UV spectrometer, so I decided to quantify my resin loading by calculating the difference in mass. For this strategy to be most accurate, the resin must be as dry as possible. I took care after each protocol to wash the resin extensively with DCM to remove any DMF trapped within the polymer network and finished drying the resin under vacuum with my Biotage® V-10 evaporation system.
While the results weren’t as awesome as I’d hoped, I was still able to achieve a 60% loading efficiency after only 3 coupling attempts requiring, about an hour total time (including drying), Table 1.
|Coupling Reactions||difference in MW||Percent Yield||final loading|
|1||6.1 mg||49.2%||23.1 mmol/g|
|2||9.3 mg||67.2%||32.5 mmol/g|
|3||7.8 mg||62.4%||29.3 mmol/g|
The above-mentioned loading level is quite acceptable in most cases. Interestingly, both the second and third experiments (2 and 3 couplings respectively) had similar outcomes. For me though, my goal was to achieve near stoichiometric loading. Rather than continuing with more and more reactions, I think I’ll try changing the number of equivalents used to improve the overall loading efficiency. Stay tuned for those results!
Have you ever used microwave heating to load the first amino acid onto your Wang resin?