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Author Topic: Amide Bond Formation Between Cyclic Peptide Lysine and Flumequine  (Read 381 times)

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skh1996

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Hi all,
I'm a first year PhD student who is struggling a little with one of my first synthesis problems, which seems like it should be straightforward but ...er....i'm running into quite a few problems.

My issue is with the efficacy of amide bond formation where whilst I do get desired product, both my HPLC and ESI-MS show side products.

https://ibb.co/5vRzLNz

I'm working with a cyclic octapeptide (Lysine (unprotected side chain) - Leu- Trp- Leu - Lysine (Boc)-Leu-Trp-Leu)  which has a lysine residue -NH2 group, which I wish to conjugate to the carboxylic acid terminal of flumequine.

HATU/HCTU conjugation didn't work and led to desired conjugate, plus a lysine/guanidinium side product :(

My supervisor advised me to try DMTMM.BF4 coupling or PyBOP coupling which i've attempted by dissolving my cyclic peptide in DMF (only solvent it dissolves into), activating with base. Separately activating my flumequine with the coupling reagent and small amount of base for 30 mins. Then adding the two solutions together. Once again there is desired product (around 1548 positive mode ESI), plus an unknown side product (this time based on mass spectra i really can't determine what the peak corresponds to based on the reagents i've used and the mechanism). (its' at 1432 in positive mode ESI)

I should note I use 1eq cyclic peptide to 1.2 eq flumequine/1.2 coupling agent.

I thought perhaps the nature of flumequine itself, having an extended delocalisation of electrons possible, it makes flumequine to some extent a better leaving group than intermediate moieties in either of the coupling agent mechanisms?

Is there something fundamental in how I set my reactions up that i'm not doing properly because i've tried all these reactions multiple times and they aren't working, and i'm about close to losing the plot after several weeks of this  ??? :-\ :-[

I wanted to try EDC/NHS coupling but i'm told this can also give side products fairly easily (?)

Many Thanks and apologies if i've not gone into enough detail, i'd be happy to give more :)


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OrganicDan96

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Re: Amide Bond Formation Between Cyclic Peptide Lysine and Flumequine
« Reply #1 on: February 09, 2019, 09:50:23 AM »

how about making pentaflourophenyl ester off flumequine by DCC coupling between flumequine and pentaflouophenol. then reacting this ester with peptide in the presence of DIPEA base.
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wildfyr

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Re: Amide Bond Formation Between Cyclic Peptide Lysine and Flumequine
« Reply #2 on: February 09, 2019, 02:32:52 PM »

On board with PFP ester. It's very specific for amines or even anilines. No need for DIPEA, TEA is fine.
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MOTOBALL

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Re: Amide Bond Formation Between Cyclic Peptide Lysine and Flumequine
« Reply #3 on: February 14, 2019, 12:09:58 PM »

Skh,

Why do you think that your desired product is at m/z 1548?
From the masses shown in the figure, you should expect to see the amide at
m/z 1626.9 for [M+H].
If we take your m/z 1548 as actually m/z 1547.9, this is 79 amu less than the number
I have given above.

Regards,
Motoball
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skh1996

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Re: Amide Bond Formation Between Cyclic Peptide Lysine and Flumequine
« Reply #4 on: February 15, 2019, 06:20:16 AM »

Skh,

Why do you think that your desired product is at m/z 1548?
From the masses shown in the figure, you should expect to see the amide at
m/z 1626.9 for [M+H].
If we take your m/z 1548 as actually m/z 1547.9, this is 79 amu less than the number
I have given above.

Regards,
Motoball

Hi there,
Well, rather stupidly (facepalmm) I THINK I neglected to include a double bond in one of my tryptophan residues, I also mistyped one of my values (i'm off to such a great start here \o/ lmaoo)

I'll start again :
 the new exact mass of the starting material would be : 1380.84
Desired material - 1623.91 (flying with a Na+ and a H+ in mass spec would give the peak at 1647.9) :) I get this peak in my spectra.

My other main peak is at around 1432 (1431.something), which I now think might be the product of DMF induced formylation of my amine group. The exact mass would be 1408.84, flying with sodium Na+ = 1431.84

I've read this can sometimes occur, although this was quite a surprise to others in my research group who have never experienced this. Has anyone else heard about this phenomena? I've seen one example where it has happened with PyBOP, but never with DMTMM.

Next, when i remove my Boc groups with TFA, this peak also shifts -300ish as well as the product peak, which implies it's definitely associated with the cyclic peptide (?).



If that is the case, it's quite difficult as DMF is virtually the only solvent my cyclic peptide will dissolve in...fun times ahead....

(i should just note the mass spectra i get are pretty rubbish due to awful solubility of this compound in acetonitrile or methanol which are the only two solvents im able to use for mass spec)

Also many thanks to to everyone replying :) i'm going to continue with my coupling agents method for the moment but definitely synthesising an active ester sounds good as a last resort.

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MOTOBALL

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Re: Amide Bond Formation Between Cyclic Peptide Lysine and Flumequine
« Reply #5 on: February 15, 2019, 08:59:30 AM »

SKH,

Thanks for coming back!
No need to beat yourself up, it happens very easily.....an interruption here.....an interruption there....

I'll start again :
 the new exact mass of the starting material would be : 1380.84
Desired material - 1623.91 (flying with a Na+ and a H+ in mass spec would give the peak at 1647.9) :) I get this peak in my spectra.


If you had told me that you see flumequine at m/z 284, I would have said OK, you have [RCOOH + Na+]+ and/or [RCOONa + H+]+
But I don't like [M + Na+ + H+]+ at m/z 1467.9 for the product, because
 
1) there is no longer a free -COOH group

2) in my experience, you just do not get addition of two disparate positive charges (Na+, H+) to a molecule

3) You should see m/z 1624.9 for [M+H+]+ and/or m/z 1646.9 for [M+Na+]+; incidentally, if you do see [M+Na+]+ signal for any compound, it will often be accompanied by a much smaller [M+K+]+ signal.   If you see m/z 1647.9, does the MS need re-calibration, or is it just because of a weak/noisy signal due to very low level of analyte?

4) With peptides/proteins under ESI/MS conditions, you can very often see multiply proton-charged signals, e.g. your cyclic peptide might be expected to give [CP +2H+]2+, and that signal would be at m/z 691.4, as well as [CP+H+]+ at m/z 1381.8; also, [CP+3H+]3+ would be at m/z 461.3.

Na is very commonly leached out of glassware, and M+Na+ adducts are commonplace in ESI/MS.

I have seen reference to N-formylation occurring when proteins are treated with NBS/conc. formic acid to induce cleavage between specific residues.

Regards,
Motoball

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