I did synthesis of 2 peptides (1-20 amino acids) using MDHA Rink resin, I used HBTU (3.7g in 20ml (DMF)), HOBT (1.35g (29ml) NMP)
to get 0.5 M.
For each reaction I used 1 mL HBTU solution, 1ml HOBT and 890 mkl DIPEA, amount or resin was 0.1 mM amd Fmoc-amino acids 5 time excess i.e. 0.5 mM (example C I weight 0.293 g etc.).
Reaction time was 1.5 h, and 20min in (20% piperidine) each step I controlled with ninhydrin test and was fine.
Can anyone suggest what could be reason that I got very low yield?
In my experience, solid phase peptide synthesis tends to be fairly variable in any case. Similar procedures would lead to between 10% and 90% yield, depending on the exact reagents used. Bulkier residues tend to couple more poorly to one another, for instance. That being said, two things to note:
1) The kaiser test/ninhydrin test can be a little flaky. You'll get a purple color if you have ANY amine present, so it doesn't really reflect total deprotection. If your yield is really too low, you might look for a test that gives a positive only if there's no amine present. Not sure what one would be off the top of my head, though.
2) Most procedures I've seen do each step twice. e.g. couple the peptide, wash everything away, add the coupling reagent and next peptide again. Then deprotect, wash, deprotect again. It's sort of like extractions: multiple repetitions with lower volumes gives you better total extraction than one trial with a single larger volume.
analyze the crude and see if there are fragments, or smaller peptides tha expected, so you can see which steps was failing and repeat that coupling two or three times. However, mw irradiation could help, temperature, you can also change hbtu with other coulpling reagent.
If possible try with higher temperatures by changing the coupling reagent and base. I guess reduces the fmoc amino acids instead of 5 times to two times to get better yeild.
Definitely, for long peptides (> 6-8 amino acids) automated microwaved SPPS would be the best choice.
For manual couplings, I would recommend for 0.1 mmol resin to use 2-2.5 equivalents of Fmoc-amino acid, 0.9 equiv. of HBTU, 0.25 mL of DIPEA (2.0 M in NMP, better if freshly made) in 2 mL DMF. 30-40 min per coupling at room temp. should be fine. You could also add more equiv. of the amino acid to reduce reaction time. I would also recommend vigorous shaking.
In case of hindered amino acids you may need to increase reaction time. And, as has been mentioned, you can check coupling performance by Kaiser test. If not completed, rinse your reaction vessel and proceed with a double coupling under the same conditions.
Hope it helps!
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