This discussion will assume that the reader has an automated peptide synthesizer which is maintained in pristine condition. Even if you perform your syntheses manually (i.e., RaMPS or Tea-Bag method) most of these tips will be useful. Over the years, we have found that regular (weekly) instrument maintenance and a daily logbook have been invaluable for preventing and tracking down intermittent instrument failures. Labor spent in this area more than pays for itself. We have instruments from two different manufacturers and have greatly improved our throughput by changing filters at twice the suggested frequency, adjusting manifold pressures regularly and getting preventative maintenance quarterly (the service representative can teach you the basics if visits are too expensive). The other critical aspect is reagents: buy high quality amino acids and store them and all peptide resins at 4 degrees Celsius to prevent degradation. At first, I scrupulously attended to my DMF purity, especially for FMOC chemistry, but over the years I have found that a little dimethylamine contaminant found in reagent-grade DMF actually accelerates my couplings and doesn't touch the FMOC group in the time frame of coupling. If you use commercially available HOBT solutions, store them at 4 degrees C. or less, as they degrade in ~4 weeks in DMF and in ~8 weeks in NMP at 25 degrees C (1).
To avoid unnecessary labor and lost yield in subsequent steps, the chain extension step deserves your undivided attention. In my opinion, about 90% of all linear peptides shorter than 30 residues are simple to produce in high purity and yield, but the last 10% can ruin your year. Several of the new synthesizers can monitor the extent of coupling by absorbance or conductivity; if you have one of these units, use this feature religiously. If your synthesizer can automatically withdraw resin from the reaction vessel, the resins can be easily assayed by washing the resin with methanol and adding 2 ml of a 5 mg/ml bromophenol blue (BPB) solution in DMF. A yellow color for the resulting suspension is obtained when the coupling is complete whereas a blue to green color indicates the need for re-coupling. Alternatively, you can add BPB directly to the coupling mixture (at I mg/ml) and monitor the coupling in real time (even with BOP or HBTU couplings; but they never go to yellow, only a light green). The BPB method (2) works well, but it sometimes gives false positives (i.e., blue color when the coupling is actually complete, especially in the presence of NMP) which induces me to re-couple several times (improving my purity) before I double check the results with a Kaiser test (3). Obviously, these methods will also work well even if you have to personally reach into the vessel to remove resin.
If you are in the process of purchasing a new instrument, consider flexibility to be as important as reliability. If you are lucky enough to be both automated and flexible, then these suggestions should be straightforward to implement. When couplings do not go to completion, I assume that the amino group is unreactive due to the presence of a counterion or to steric effects. The reason is irrelevant; I need the peptide. I have been able to save such syntheses by the following methods.
* When the BPB test is positive (blue or green), I add one equivalent (based on synthesis scale) of diisopropylethylamine (175 ul/mmole) to the reaction slurry to complete the coupling in ~ 15 minutes.
* If this fails, I wash the resin with a 10% solution of diisopropylethylamine (v/v in DMF) and re-couple using a different coupling method than was used during the machine's attempt. As a rule, if the synthesizer used BOP or HBTU in DMF, NMP or DMSO, I re-couple with an HOBT ester or an anhydride in methylene chloride, invert this rule to fit your machine's chemistry. This exposes the amino group to a differently shaped activated amino acid in a solvent of vastly differing polarity during recoupling and usually improves the coupling yield drastically. Also, don't be cheap, use a 10-fold excess of the protected amino acid; the real money is spent on labor, synthesis solvents and waste disposal.
If this gentle re-coupling method doesn't do it, I resort to mechanical methods (beatings!) to couple the amino acid. Some of my favorite, less damaging methods:
* Neutralize the resin with 10deg.70 DIEA in DMF/DMSO (1:1) and then wash the resin three times with DMF/DMSO. Re-couple using BOP in neat DMSO using a 10-fold excess of activated amino acid. I avoid BOP because of its toxicity, but it is a wonderful coupling agent. Check the coupling on a washed aliquot of the resin, don't drain the whole batch!
* If the coupling is not complete, push a little harder by immersing your sealed reaction vessel in a 40 C sonicator bath for 30 minutes. I don't recommend this for peptides containing Cys, Met or Trp; the possibility of side reactions scares me.
* I have also used stronger hydrogen-bond disrupting solvents, such as hexamethylphosphoramide and hexafluoroisopropanol added to ongoing couplings with great success, but they should be used with extreme care due to their toxicity.
The vast majority of methods required for the production of pure peptides have been left out due to space constraints, but these simple guidelines should take most users a long way along the path to pure compounds.
1. Wolfe, H., Unpublished GC-MS studies on the stability of HOBT in DMF and NMP.
2. Krchnak, V., et al, Coll. Czech., Chem. Comm., 53: 2542-2548 (1988).
3. Kaiser, E., et al, Analyl. Biochem., 34: 595-598 (1970).
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