Dear Neil,
Below you will see my digest of Arg/FmocSPPS-related info I retrieved
from ABRF archives some time ago for my own needs. I hope you'll find it
useful.
Good luck!
Igor Rodionov
******************For the peptide chemist*****************
I have a problem with the cleavage of a tetrameric MAPS. It contain a
total of 8 Arg (I used Fmoc-Arg(Pbf). I did it on a Perseptive PEG-PS
resin as an amide. When I cleve it (reagent K), using ~5ml for 200 mg of
resin, I found a precipitate in the cleavege mixture. I'm not sure is
the peptide, of if the pbf group can precipitate in TFA.
Anyone with the same experience?
Matteo Villain
------------------------------------------------------------------------
-- Matteo, More than likely what you are seeing in the cleavage solution is precipitated Trt. There maybe some Pbf as well but mostly Trt (of course that assumes you have Trt protected residues in your sequence). You certainly have enough volume of reagent ~25uL/mg. I usually notice the precipitate on rotovaping off the cleavage cocktail prior to workup, but everyones sequences are different so... These will solubilize and wash away in your workup...either extractive or precipitation with ether. Regards, Steven Johnson, B.S. ===================================================*****************PbfArg ****************** Chris- Some time ago you said on the bulletin board that you "had mixed success" with PbfArg: what problems did you have, and did you buy it from Peptides International? David A. Schooley ---------------------------------------------- David- We have been getting our Arg(Pbf) from Sygena. My comment would be that initially we were led to believe that with multiple Arg peptides we could cleave one hour with total deprotection. We've found that in peptides with multiple Args, we still need 4 hours to remove the Pbf. We just made a 23-mer with 4 Args spaced throughout the peptide (not consecutive). A one hour cleave showed only 10% of the totally deprotected peptide. 4 hours yielded about 85% of totally deprotected peptide. We also made some 10-mers with 4-5 Args. Using Pmc was hopeless even after a 24 hour cleave, but the Pbf had total deprotection after 4 hours. As with all things, there are pros and cons and appropiate times to use different derivitives. The Arg(Pbf) has definitely allowed us to make peptides we couldn't with Arg(Pmc), but we've had to keep the extended cleavage times. Chris Lovdahl -------------------------------------------------- Chris, I would be interested in your comments on the cons of using Arg(Pbf)? Gary Davis -------------------------------------------------- Just wanted to let everyone know that I was not specifically told by the company that we could do 1 hour cleavage times with the Pbf and get beautiful results. I think lab personnel may have just made that assumption from the kinetic study that was published. Of course the kinetics for each peptide are going to be different and then we had problems when using such a short cleavage time. Chris Lovdahl
***** 950907 Arg4 containing peptide deprotection - how to? ****** We are trying to synthesize a 9 amino acid peptide with 4 Arg residues. The Arg are Pmc protected and are difficult to deprotect. We use the following cleavage cocktail: 36:2:1:1 TFA: Thioanisole: EDT: H2O on ice for 15 min followed by 4 hrs at room temperature. In two attempts to make this peptide we have only been able to partially deprotect the Arg residues. Any suggestions?? Thanks in advance Paul Motchnik --------------------------------------------------- We recently synthesized a peptide containing arg-arg-arg in the sequence. We have tried Fmoc synthesis a few times and failed to obtain the product. Finally, by using t-Boc synthesis, the peptide was obtained. This is one example where Boc method triumph over Fmoc method. We have also encountered peptides where the Fmoc method gave cleaner products than the Boc method. Anita Hong ---------------------------------------------------- Try using 1 M TMSBr (trimethyl silyl bromide) in TFA, with 1 M thioanisole, 5% thiocresol and 2.5% EDT at ice-bath temperature for 1.5 hours. TMSBr is volatile, and you can get rid of it when you concentrate your cleavage cocktail before precipitating with ether. You can also try 82.5% TFA + 5% thioanisole + 5% thiocresol + 5% water + 2.5% EDT for 1.5 hours at room temp. I was able to remove the PMC from 3 Args in an 11 residue peptide that way. If you have Tyr in the peptide, you can cut the water to 2.5%, and add 2.5% phenol. Angela C. Murphy --------------------------------------------------- Recent e-mail exchange showed that Arginine creates a lot of problems both during the sequencing and synthesis. My experience in Arg- chemistry is moderate. I had 3 Arg in my sequence of 12-mer and I had only Mtr-protection which pronounced to be TFA-labile. Deprotection took 4 days; it was three years ago; most of Mtr are still attached to that peptide. Really good protection. Now I switched to Pmc-protection, but recently I had nesessity to prepare short pentapeptide with still protected Arg (for further fragment condensation) and I choose Mtr. After cleavage of FmocArg(Mtr)-peptide from the resin (45 min) almost all Mtr were lost... This is from the field of emotions. My questions are: 1) If anybody has experience with Sygena FmocArg(Pbf)OH? Ordering information and price? 2) If anybody tried to do fragment condensations with unprotected Arg in the middle of the sequence (no solubility problems)? Appreciate any details. Viacheslav Medvedkin ------------------------------------------------------ We have used Pbf protected Arg since it has become available (Sygena 100g $1380). We have had great success with it. It is much more labile than the Pmc as shown by IonSpray analysis of same sequences and same cleavages using the two protections. Steve Wood ------------------------------------------------------ From: Haaseth@brcf.med.umich.edu We have used Fmoc-Arg(Pbf) for repeated syntheses for a customer of a 39mer containing 19 Arg residues, always with some success if the 433 behaves itself. We must be getting the hang of it cause our yield tripled for the last two syntheses of this peptide to a respectable approx. 300 mg purified peptide on a 250 umole scale. We let the cleavage (Reagent R) go overnight. On the other hand, we made RRRRRRRRRRR for another customer, this time using Pmc protection and still got a reasonable if not spectacular yield of purified peptide. Again, an overnight cleavage was used. Ron Haaseth ------------------------------------------------------- No experience with PbfArg (yet), but Carpino claims Pbf is easier to remove than Pmc. Bachem Biosciences, Philadelphia, PA, is selling FmocArg(Pbf) for $60.00, USA, for 5 grams. Angela C. Murphy ---------------------------------------------- several companies sell Pbf. it is somewhat easier to remove than Pmc, but the difference isn't dramatic. mainly, this is a way to get around Ramage Pmc patent. reagent R (depending on who you believe, R stands for either arginine or for the fact that A, B, C, ..through Q didn't work) first published by albericio et al, JOC 55, 3730-3743 (1990) was developed for optimal Arg(Pmc) (and obviously Arg(Pbf) as wel) removal. i don't have the recipe at my fingertips, but it is described in many reviews. derek hudson and alan frankel have a science paper no problems making peptides with lots of arg. by the way, i composed this before having had a chance to read several other very relevant messages that appeared on the subject. George Barany ------------------------------------------------- During the recent discussion about Arg deprotection, I haven't made any comments, because I don't have any special insight into what really works. However, we make a lot of Arg-containing peptides, and held on to doing Boc synthesis for a long time until the Pmc-derivative became available. Some time ago, when we needed to make some 19mers with 5-6 Args clustered in two groups, I was skeptical that we would get decent yields of the correct structures. We just used the standard deprotection conditions for a longer time, and got excellent yields for one or two peptides, and good enough yields for the others. Has anyone analyzed sequence- or composition- dependent parameters which might affect the deprotection step?
Ruth Hogue Angeletti -------------------------------------------------- From: cfields@maroon.tc.umn.edu (Cynthia G. Fields) I highly recommend trying reagent K, which is 2.5% EDT, 5% thioanisole, 5% H2O, 5% (by wt.) phenol, 82.5% TFA. We developed this cleavage cocktail specifically with Arg in the presence of Trp in mind. We do a time course during the cleavage, injecting onto the HPLC every hour or so, to check for peak movement. Stop the cleavage when you've reached equilibrium. Almost always, Pmc groups cause the peptide to elute later. Refer to: King et.al, IJPPR, 1990, 36, 255-266 Fields and Fields, Tet. Lett. 1993, 34, 6661-6664 Stierandova, Sepetov, Lebl, poster presented at the APS in Edmonton, 1993 maybe you can contact Michael Lebl for a reprint or perhaps it's been published by now in full-length. This poster talked about equence-dependent problems on Trp modification by Pmc. Pbf is a good alternative, but personally, I think the cleavage cocktail and time are the key. Cynthia G. Fields, M.S. -------------------------------------------------------- I have tried the Novabiochem cocktail and it was successfull for 36-mer containing 2 Arg(PMC), 1 Cys(Trt) and 1 Trp (no protection). This cocktail is: TFA: 86.5% Phenol: 5% (by weight) Water: 5% EDT: 2.5% TIS: 1% Reaction time: 4 hours
Jean Lefebvre -------------------------------------------------- Reading the discussion about deprotection coctails: Scintie Fields recommend deprotection coctail Reagent K. Jean Lefevbre has very good experience with Nova-Biochem receipt (TIS instead of thioanisol). But: How to avoid this TERRIBLE odour from ethaneditiol? In Russia we ALWAYS substitute ethanedithiol with dithiothreitol (DTT). The first reason was that we simply had no EDT; the second, but not the last was the awful odour from EDT. >From my experience DTT and products of its oxidation are easily removed during the ether washes and it works perfect in our experience. No systematic comparative search were made and and I suggest to all ABRF- friends: try DTT and save your health!! I call this coctail "reagent KM". The weight proportions were made the same as for EDT -about 2,5%. Viacheslav Medvedkin --------------------------------------------- see Van Abel et al., ijppr 45, 401-409 (1995), synthesis of indolicidin which is a c-terminal peptide amide of 13 residues discovered by Michael Selsted, that includes 5 trp and 2 arg. best cleavages (fewest side reactions) with reagent K = TFA-phenol-water-thioanisole-EDT, 82.5 : 5 : 5 : 5: 2.5 reagent B = TFA-phenol-water-triisopropylsilane, 88 : 5 : 5 : 2 note that when triisopropylsilane is used, there is no need for odiferous EDT or thioanisole. if no Trp, triethylsilane can be used, but as Pearson first showed and we confirmed, triethylsilane will reduce the Trp indole. the above-referenced paper also provides a comprehensive literature review of scavenger and cocktails proposed for Fmoc SPPS. George Barany
***********Subj: Cleavage of RRRRRRRRRRR*************** Date: Wed, 20 Sep. 1995 Follow-up to my earlier message about cleavage of R-containing peptides. We did not do the cleavage of RRRRRRRRRRR overnight, but for three hours and used Reagent Z. We compared four cleavage cocktails for this peptide and "Z" won. The others we tried were: Reagent R, Reagent K, and 95% TFA/5% water. The recipe for Reagent Z: 85% TFA - 10% thioanisole - 5% ethanedithiol Ron Haaseth *****************TMSBr.txt******************** Mark, We have just published the following procedure modified from that of Hughes and Leopold in the next issue of Peptide Research. It works as well as HF to cleave and deprotect peptides in Boc/benzyl chemistry and should work with your 4-methyl-benzyl-3-thio propionyl group.
====TMSBr/TFA Resin Cleavage Procedure======
One gram (1g) of peptidyl-resin is placed in a dry 100 ml round bottom flask protected with a CaSO4 drying tube. The resin is stirred as 11.74 ml of thioanisole and 0.2 ml of ethanedithiol are added followed by 50 ml of trifluoroacetic acid. The cleavage mixture is cooled to -10 C with stirring; 6.6 ml of trimethylsilylbromide is added and the stirring at -10 C continued for 10 min. The flask is then transfer to a stirrer at room temp. and connected to a N2 source. Stirring is continued for 2 1/2 hr. at room temp under N2 and the flask protected from light with Al foil. The reaction mixture is then filtered into a 250 ml r. b. flask and the flask, filterand resin washed with 2 X 10 ml of TFA. The excess HBr is removed on the rotary evaporator under house vacuum to prevent bumping and then the evaporator transferred to the vacuum pump to remove the TFA at 35 C. The peptide is precipitated with ether, filtered and washed with ether. The dry powder is dissolved is water or 6 M GuHCl, 1 M Tris; the peptide can be desalted or purified directly depending on whether the residual thioanisole/EDT interfers with the HPLC separation. For extended HPLC column life, we usually desalt our crude peptides on BioGel P-2 in 0.1 M ammonium bicarbonate or 5% acetic acid depending on the pI of the peptide. Jim Sparrow ------------------------------------------------------------------------
**********98/08********** I have been cursed with a peptide which has some residual Pbf protecting group left on an Arg residue (it is a 21-mer). The peptide synthesizes beautifully, one peak on HPLC and usually one peak on MALDI-TOF but the removal of the Pbf is somewhat hit and miss. I have never encountered a problem with its removal before so I have been surprised to see it on this peptide. I make a bunch of this stuff and the cleavage either goes to completion or it gives me this portion of still Pbf protected peptide even when batches are cleaved side by side. As it happens, the Pbf protected bits elute at roughly the same % as the clean stuff so I had hoped to try a rescue measure and re-cleave the already cleaved peptide. I looked through the ABRF archive and could not find any instance of this being attempted before so here I am, asking for your assistance. I have had excellent results removing residual Trt from cleaved peptides by stirring them in 99% TFA and 1% TIS, anyone have any experience with Pbf removal in the same general fashion? I was thinking of using 95% TFA + 2.5% EDT + 2.5% TIS. Jane A. Nagel MSc ---------------------------------------------------------------------- Jane, Two suggestions: Are you sure that the Pbf has stayed with the Arg residue and not added somewhere else in the peptide? Have you tried adding the amino acid Arg to the cleave cocktail as an additional scavenger? Dr. Jim Farmar ---------------------------------------------------------------------- Jane: We have often re-cleaved crude peptides which are unsatisfactory. This often improves their quality. I usually use reagent K for everything (recently I discovered using too little gives poor crudes; more is better assuming you can precipitate the product). Your choices of reagents are probably sound. Are you sure the Pbf group is on Arg and hasn't migrated to a sensitive residue (Trp, Tyr, others)? Ms/Ms sequencing (difficult of a 21 mer; you probably don't want to bother with trypsin affording smaller peptides) or Edman (close enough to the N-terminus to get you info?) should tell you exactly what's modified. If you re-cleave and see no changes, I'd bet on migration (Friedel-crafts acylation) rather than incomplete deprotection. If you need to remove the Pbf peptide, (assuming it's still on Arg), ion exchange should work well. You now have 2 different zeta potentials at acidic pH. David H. Singleton ---------------------------------------------------------------------- You don't mention which cleavage mixture you use for the original cleavage from the resin, so I can't comment on whether that is part of the problem. I think it is OK to try to cleave the residual Pbf, but I would use a mixture of: 85% TFA/5% thioanisole/5% p-thiocresol/2.5% water/2.5% DTT (very smelly, but not as smelly as with EDT). TIS is a great scavenger for Trt and Boc carbocations, but I'm not sure it would be very effective with Pbf. I have had good luck with the mixture above, cleaving not only Pbf, but also Pmc, even with multiple Arginines in a sequence. The thio- anisole/thiocresol combination is also supposed to prevent sulfate from being attached to Arg or other residues (happens when there is cleavage at the sulfur of the sulfonyl part of Pbf or Pmc). Angela C. Murphy ---------------------------------------------------------------------- Jane, I use a cocktail of 90%TFA, 8.5% Water, and 1.5% TIS for a 34-mer with 3 Arg's in a row with out any noticible Pbf peaks by mass spec. I also found that this seems to suppress linker cleavage as well...+163 w/o the Nle spacer and +275 with the spacer on MBHA amide resin. Best part is...no smellies!!! But, if you have any Met or Cys in your peptide then they are a necessary evil. Steven Johnson ---------------------------------------------------------------------- Thank you to everyone who took the time to respond to my query. I re-cleaved the peptide over the weekend (with TFA 97.5% and TIS 2.5%) for one hour and got an excellent result. Although the resulting peptide would not precipitate when poured directly into cold ether, after I roto-vapped (my old adviser who flip that I just used "roto-vap" as a verb) off the TFA etc., the peptide formed a large yield of crystals with excellent purity. It is amazing that we all still have our favourite cocktails and we all believe them to be the best. Just goes to show you that there is no true right or wrong in peptide synthesis! Jane ----------------------------------------------------------------------
**********************Re: -85 AMU 09 Dec_1997******************** Mark Lively wrote:
We have encountered difficulties making the following peptide: ARRTRRLMLRAVRIIAE Theo. mass = 2081.5 After purification we have a 70:30 mixture of two peaks by hplc with mass 2081.14 and 1996.0. Unfortunately, the Mass 1996 appears to be the major peak and is 85.1 amu smaller than it should be. I am unable to explain its structure. If one assumes the presence of an Ala in place of one Arg, the mass difference could be explained. Since we do have the desired 2081 amu product it is hard to explain an Ala for Arg substitution as a synthesis error (unless one of our Arg cartridges contained some Ala!). Is it possible to cause cleavage of the Arg side chain AFTER the beta carbon during workup and/or mass analysis? I can't think of how this could happen. Mass data were obtained using a MicroMass Quatro II with an electrospray interface. Is there another explanation for a difference of 85 amu? Mark Lively ---------------------------------------------- On Tue, 9 Dec 1997, alessandro tossi wrote: Some time ago, I sent a help request to the abrf newsletter asking if anyone had any information about what cuased a -85 AMU peak in several of our peptides. Like Mark's, our peptides are very rich in Arg, and sometimes we have up to 30% of the lighter peptide, although we have never had it so bad as he has. It was the only time I asked for help and no one could provide a solution. The only possibly useful suggestion was made by Prof. Andreu, I think, who wondered if it could not be dehydroalanine resulting from loss of part of the side chain in Arg. We have excluded an ES MS artefact as the lighter peptide does not quite co-elute in RF-HPLC with the correct peptide, allowing us to separate the peaks. The fact that Arg is always present in the peptides makes me think it is at the level of this residue. Furthermore, it is a single modification (ie not 2 deguanylations, = 2 x 44) and we have sometimes observed -170 (2 x - 85). I suppose an ionic exchange chromatography might indicate if the modification is on Arg better than RP-HPLC. I should say that this modification has been observed in peptides synthesized over several years, starting with AA-oPfp/HOBT, through AA-OH/TBTU/HOBT to AA-OH/HATU, with cleavage conditions ranging from TFA/EDT/H2O to reagent K with TIPS, and using both Arg(Mtr) or Arg(Pmc). I need to check if we got it with Arg(Pbf) as well. It would be really useful if we could solve this problem, and I am glad we are no longer alone. Bye, Alex Tossi ------------------------------------------------------------------------ Reply from Katheryn Resing ( I hope she will not mind my posting it without asking)
>I have seen loss of 85 Da from several peptides with arg-arg sequences >during ms/ms and it occurs more readily with more highly charged ion >isoforms. An organic chemist I consulted about this seemed to think it was >reasonable because the nitrogen at the side group can cyclize to form a >five membered ring. He drew what he said were acceptable mechanism for the >loss of the side-chain. In fact in an ms/ms spectrum of small >arginine-containing peptides, you often see an ion of 86 Da, corresponding >to the charged form of this cyclized product. Arginine can also cyclize >with other sites in peptides by a similar mechanism, this makes >interpretation of those ms/ms spectra complicated. There was a paper on >this type of cyclization in substance p a few years ago in JASMS. If you >want, I can dig it up. > The organic chemist said he thought it would occur in acid cleavage >conditions in solution, but he seemed to think it wouldn't represent too >high a percentage. However, you have two sets next to each other and >nothing would surprise me when it comes to arginine. > Katheryn Resing ------------------------------------------------------------------------ Update from Lively on 10 December.
So, it looks like this is a problem that has been observed before by others in this group so it may be useful to follow up to find an answer. I have additinal data from my peptide that is consistent with the proposed cylcization described by Resing. To fill in details requested by David Schooley, our peptide was made using standard ABI FastMoc chemistry with PMC-Arg, O-tBu-Glu and tBu-Thr. A mass analysis of the initial crude product (deprotection an cleavage with reagent K for 1.5 h) suggested incomplete deprotection (I think the mass analysis was erroneous, though) so I subjected the purified peptide (one peak by hplc and unfortunately we do not have ms analysis of that peak) to a second 1.5 h acid treatement. HPLC of the crude product of the second Reagent K treatment showed three peaks with masses 2081, 1996, and 1911. The mass difference in each case is -85. So, as Alex Tossi noted, we have also seen -170 amu. As suggested by one ABRF member, we are sequencing the three peaks to see which Arg is missing, if in fact that is the answer. So, here is the QUESTION: It now seems more likely that the -85 amu is associated with acid treatment of peptides that contain multiple Arg residues or at least paired Arg. Can any of the chemists out there suggest a mechanism for the cleavage of the Arg side chain after the beta-carbon? Mark O. Lively, Ph.D. ------------------------------------------------------------------------ --
Hi Mark, The older peptide synthesis literature contains several articles on conversion of arginine to ornithine when Boc-nitroarginine was used. Ornithine was easily generated from nitroarginine by base cleavage and also reported in lesser amounts under other cleavage conditions. I am not familiar with possible side reactions when PMA-Arg is used.
Arginine to ornithine results in a loss of 42 amu. Is it possible that adjacent arginines can result in the loss of 84+1? Must be interesting chemistry if it happens, but if ornithines are formed -why not acetylate the peptide and check for a mass increase of 42 for each amine group? I also like Sandy Kielland's approach, a good amino acid analysis. -Lowell H. Ericsson -------------------------------------------- Mark, Alex, et al.: I was wondering if anyone tried MS/MS; although the MS/MS spectrum of the mass deficient (-85 u) product might not be easily interpretable, comparing the tandem mass spectra of the two peptides (that with the expected MW and the one 85 u lower) might clarify the situation by confirming what other evidence seems to indicate, namely that the loss of 85 u has occured at Arg. Once this is unequivocally established, it should be fairly straight forward to come up with structures which make sense chemicaly and also account for the mass deficiency. Ioannis Papayannopoulos -------------------------------------------- Mark Lively and A. Tossi brought up the problem of identifying the reaction(s) responsible for an 85 Da loss from Arg-rich peptides. One experiment that might prove useful is an accurate (+ or - a few ppm) measurement of the mass difference. This should tie down the elemental composition of the lost portion, which would limit the possibilities as far as the chemistry that might be involved. This should not be a difficult experiment with a good MALDI-TOF instrument. Arnie Falick, PerSeptive Biosystems
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