[vitaminvater]

So I am synthesizing a 13-mer peptide on a microwave assisted spps machine using fmoc-protected amino acids. I have been getting two major species: my correct sequence and the sequence with a +44 mass change. This +44 mass change is also present for other minor peptide species.

Relevant info:
Peptide contains two unnatural amino acids: a disubstituted alkenyl and a mono substituted azide.
The rest of the amino acids are as follow: arginine, asparigine, aspartate, leucine, phenylalanine, glutamate, threonine, and tryptophan. All the amino acids with reactive side chains are of course protected during SPSS.
Using Rink amide resin.

Troubleshooting I have done already:
Cleavage from resin- I am using reagent B as my cleavage cocktail. I ran an experiment to see if cleavage time had an affect on the ratio of correct masses and +44 masses. Taking aliquots at 30 min, 1h, 2h, and 3h had no discernible effect on such a ratio
Acetylation-I remove my peptide from the machine without cleaving the final fmoc. I then do the final deprotection and acetylation by hand. It seems the +44 masses are present from the moment I take the peptide out of the machine, so I do not think my deprotection or acetylation procedures are at fault.


I have not been able to find any literature on +44 mass impurities in SPPS. If anyone has encountered this problem before or has any suggestions, I would truly appreciate them.

Thank you!

[kriggy]

Could it be that after removal of fmoc, you have part of the amino group as a carbamate? COO- is exactly 44

[vitaminvater]

Could it be that after removal of fmoc, you have part of the amino group as a carbamate? COO- is exactly 44

Well that seems rather unlikely since +44 masses are present even before removal of the fmoc group.

[TheUnassuming]

You say the M+44 is showing up for your minor/incomplete peptides as well.  All of them or just some? If it seems to arrive after a certain residue is attached you can get better idea of where it is happening.

[kriggy]

Also, rather (super) stupid idea but did you calculate the mass of the product right? (talking from experience, please take no offense)

[vitaminvater]

You say the M+44 is showing up for your minor/incomplete peptides as well.  All of them or just some? If it seems to arrive after a certain residue is attached you can get better idea of where it is happening.

The minor peptide products come after the 10th AA, which is the disubstituted alkenyl AA. The truncated sequence problems have been fixed by doing some manual coupling but even after that I am still getting M+44 at an almost 1:1 ratio.

Also, rather (super) stupid idea but did you calculate the mass of the product right? (talking from experience, please take no offense)

Haha no offense at all, I've actually been very careful about that. The mass has been calculated using chemdraw and I've double checked every residue and structure.

[kriggy]

Maybe the double bond is reacting? What about trying to imobilize just the alkene AA and then cleave it if you also get the +44 product?

[vitaminvater]

Maybe the double bond is reacting? What about trying to imobilize just the alkene AA and then cleave it if you also get the +44 product?

Could you clarify what you mean by "imobilize just the alkene AA"?

[kriggy]

My idea was to imobilize the amino acid with alkene substituent to resin as if you were making a peptide with this amino acid 1st in sequence and then try to cleave it from resin using your conditions

[vitaminvater]

My idea was to imobilize the amino acid with alkene substituent to resin as if you were making a peptide with this amino acid 1st in sequence and then try to cleave it from resin using your conditions

Ahh ok understood. Seems like something worthwhile to try, will report back!

[MOTOBALL]

Remember Occam's Razor; do you possibly have the Na+ salts of the aspartate/glutamate ??

Regards,

Motoball

[kriggy]

Probably not, because Na adds 23 so two sodium atoms are +46 and even than, it would change the ratio of M/z because you then have 2 charges on the molecule

[Doc Oc]

Are you analyzing by LC/MS or direct inject MS? If by LC/MS, do you have formic acid in the eluent? That can often form salts that add 44 to the mass.

[vitaminvater]

Are you analyzing by LC/MS or direct inject MS? If by LC/MS, do you have formic acid in the eluent? That can often form salts that add 44 to the mass.

I'm analyzing by LCMS and yes it has formic acid in the eluent. However, under HPLC, which uses TFA, there is still a different peak appearing for what I presume to be the M+44 masses. Also, the LCMS system was the same one used by a different group that made the same sequence, and they did not see any M+44 masses.



Something my friend has suggested is contamination from PEG leaching.
When I remove the completed peptide from the synthesizer, I do the final deprotection and acetylation in a plastic frit (ones used for automated columns;empty of silica of course), and I do my cleavages in PCR tubes. Could this be a potential source?

[MOTOBALL]

Probably not, because Na adds 23 so two sodium atoms are +46 and even than, it would change the ratio of M/z because you then have 2 charges on the molecule

Not quite----2 x H are replaced, so +44.

Also, (RCOO-Na+)Na+ is singly charged, as is AspCOONaGluCOONa)H+