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.
This coupling reaction comes with it’s own set of challenges, which is why many people perform a large scale batch preparation of the pre-loaded resin. But that’s for a later discussion. In today’s post I’ll address a different question. How do you quantify the amount of amino acid loaded onto the resin?
I have certainly encountered groups that choose to couple the first amino acid and move directly into synthesis of the rest of the peptide. And that’s fine. With the increasing use of unnatural, sometimes wildly expensive amino acids, knowing the exact amino acid loading of the resin is important to decrease reagent waste and cost of the synthesis.
Accurately determining the resin loading requires a high degree of precision. While the method is simple in description, sound analytical techniques are required to gain an accurate quantification. This is typically conducted after the amino acid loading reaction is completed and the resin capped, but it can also be used to monitor the progress of the reaction.
We are going to use the fact that the Fmoc-piperidine adduct absorbs UV irradiation strongly at about 300 nm and its removal can therefore be quantified spectrophotometrically using Beer’s Law, Eq 1.
Equation 1: Beer-Lambert Law correlating UV absobance with solution concentration. A = UV absorbance at a specified wavelength (x); ε = molecular extinction coefficient at a specified wavelength (x); l = pathlength through sample (the width of the UV cuvette); c = sample concentration, in units of mol/L.
The following general steps should be performed to determine the amount of amino acid successfully coupled to the Wang linker. Pay special attention to the tips highlighted at each step. These details will contribute greatly to the success of your quantification.
- Execute the amino acid loading protocol outlined here or elsewhere.
- Remove a small quantity of the resin from the reaction vessel to a pre-tared vial.
- Wash the removed resin thoroughly. The goal here is to remove any residual DMF that may be trapped in the resin matrix. Often times, wash cycles with different solvents are completed (for example, alternating between DCM and diethyl ether when using Peg-based resins) to ensure complete DMF removal.
- Dry the resin thoroughly, usually with vacuum. To ensure an accurate quantification the resin MUST be very dry. Can’t let the weight of residual solvent affect the quantification!
- Weigh the dried resin.
- Suspend the resin in a known volume of 20% piperidine in DMF. Accurately delivering the Fmoc-removal solution is critical for an accurate quantification of resin loading.
- Execute the Fmoc deprotection using standard methods.
- Collect ALL of the Fmoc deprotection solution. Just like the accuracy of volume delivery was important in step 6, the accuracy of volume recovered is equally as important here.
- Zero the UV spectrometer with a clean solution of 20% piperidine in DMF. I like to use the wavelength scan feature for these measurement to fully visualize the absorbance peak and the corresponding peak max. Alternatively, you can directly measure the absorbance value at 301 nm.
- Dilute the recovered deprotection solution in neat 20% piperidine in DMF. The dilution amount will depend on the amount of resin used for this analysis as well as the volume of deprotection solution used for the Fmoc removal. Remember, Beer’s law is only linear with absorbance values ranging from about 0.1 to 1 AU.
- Measure the absorabance of your diluted deprotection solution at ~301 nm.
- Calculate the mmol of Fmoc released during the deprotection reaction using Beer’s Law. The extinction coefficient of the Fmoc-piperidine adduct has been reported to be 6000 M-1·cm-1 using this dilution method. Don’t forget to correct for your dilution factor in this calculation! You will need the concentration of the actual solution, not the diluted concentration.
The mmol of Fmoc released into the deprotection solution is directly proportional to the amount of amino acid loaded onto the Wang resin. Using this quantity, you can determine the amino acid loading level via simple division with the mass of dried resin used in the above process.
What other strategies have you used to quantify your first amino acid loading onto Wang resin?
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