Hi Antonia,

My best guess is that which way is best, depends on the sequence. we tried both and got mixed results. I recommend to consider several additional aspects: do you just need the compound once for testing purposes, or do you need to scale up later? How much effort is SPPS for you: do you hava a peptide synthesizer at hand to screen synthesis conditions/Protection group schemes? How much effort for purification can you tolerate? Is prep. HPLC/size exclusion/ an option to you? Resin has the advantage of pseudo-dilution, but rink-resin makes controlling of the reaction harder (depending on functional side chains present in your sequence. You might want to consider 2Cl-Trt for easier cleavage from resin, making reaction control much easier. What I didn't get from your description if you have one or two disilfide bridges to form? if you want to have two sequential disulfide bridges to form, I suggest puting higher emphasis on controlling your reaction. Hope that helps?

Kind regards, Marc

I have done such intramolecular disulfide bridging to prepare cyclic peptides on Rink Amide AM resin. In my opinion the best way would be to assemble the peptide with Fmoc-Cys(Acm)-OH, perform a global deprotection to get the protecting group free peptide with only two Acm groups and finally try a solution phase oxidation via the Iodine mediated Acm deprotection-disulfide formation in one-pot fashion. This has worked for me quite nicely and would work for you as well (assuming you don't have any oxidation sensitive special amino acids).

Generally, formation of disulfide bonds tends to work better in solution. That way it is also easier to monitor the reaction and optimise the conditions if need be. There are several methods and cysteine protecting groups to choose from. How many cysteines does your peptide have, and do you know the disulfide connectivity you want to achieve (i.e. which pairs of Cys do you want to connect)? It would also be helpful to know the sequence of your peptide before deciding on which strategy is best.

Thank you for your help. We wanted only to try both: disulfide bond formation in solution and on Rink Amide AM resin to know what methodology works better. There are two Cys residues in our Fmoc-octapeptide, one is Acm-, the other one Trt- protected. By disulfide bond formation on resin (like Somnath Mukherjee proposed), do you know, there are some troubless with the final cleavage of disulfide from the resin?

That's an unusual protecting group combination for making a single disulfide bond, but the regular iodine oxidation that you'd use for Acm removal should work.

On resin: Add 10 equivalents of iodine (dissolved in DMF), to your resin bound peptide (pre-swollen in DMF), leave for 1 hour with shaking occasionally. Wash the resin several times with DMF, then DMF/water +ascorbic acid, then DMF again to remove all the iodine. Do a test cleavage to check. Disulfide bonds are stable to TFA, but it's best not to use thiol scavengers. TIPS/H2O should be enough. It is possible that you could get dimers/polymers of your peptide - that will depend on the resin loading and sequence.

In solution: Dissolve the lyophilised peptide (~1 mg/mL) in 50% MeOH / 50% H2O with 25mM HCl. Add 10 equivalents of iodine (dissolved in MeOH) to a stirring solution of the peptide, leave for 1 hour. Add ascorbic acid to reduce the iodine before analysis by LC-MS.

If the oxidation is incomplete, add more iodine and/or let it go for longer. Met and Trp are susceptible to oxidation, and Tyr could get iodinated amongst other possible side reactions. If you see multiples of +16 or +126 Da, reduce the amount of iodine and/or the reaction time.

The best condition will depend on your particular peptide sequence, so only do small scale trials until you're confident in your conditions. You can play with the amount of iodine and the reaction time to get the best result. Changing solvent can also influence the rate of the reaction and formation of side products. There are also alternatives to iodine, but it is the most commonly used reagent.

In all cases, always handle the peptide in acidic solution (e.g. 0.1% TFA) to prevent dimerisation through the free Cys.

Thank you Zoltan for your advice. We will try it.

I have found that in the case, there are present some oligomers or polymers of the peptide after the disulfide bond formation, the best possibility to get they lost is to use the shott filter 40 (15-40 micrometer) for the first filtration to hold there the polymer particles and then the shott filter 16 (5-15 micrometers) to hold the desired peptid from the solution (after cleavage from the resin). And this way it is very easy to remove the undesired oligomers and polymers, if there are some.

I hope it can be also helpful for you (also for HPLC analysis of product is good, you don`t need to wash the column after the analysis from polymers...