Talk:Peptide synthesis/Archive 1

note
To Add: Peptide Purification/Quality Control Peptide Cleavage Chemistry-specific reagent pages Choice of resin/handle Cyclic peptides Phosphorylated peptides

Others are encouraged to add pages in order to clarify terms related to peptide synthesis.

I encourage the merge. Be bold.

I was against it at first, but I am leaning more for it as I think about it. The synopsis in the peptide synthesis page is too brief. Plus, the description of FMOC in the solid-phase page is much more thorough than on the peptide synthesis page. However, I think that to do so, we ought to expand the liquid-phase synthesis area a little. Furthermore (I might do this myself), the initial basic description and definition is a little bit too high-end and needs a bit more on the basic explanation side, I think.


 * is disagree. i needed information regarding solid phase peptide synthesis, and after looking a numerous other sources i was suprised to find it separate from peptide synthesis on wikipedia. this in my opinion was good as it saved me time from having to scroll through all about peptide synthesis, and get straight to what i wanted.


 * On reflection I think the two should be merged. An article on solid phase sythesis (which doesn't seem to link to either of these two) already exists. From a peptide chemistry point of view, the defining feature of the subject is the target of the synthesis, not the method used to reach that target. Therefore, SPPS should be discussed within peptide synthesis as it is the dominant synthetic method employed in the field. The general features and application of solid phase methodology can be better discussed elsewhere.--Charles A Norman 17:26, 28 May 2006 (UTC)

Peptide creation
It states under Peptide Creation that;

''Peptides are synthesized by combining the carboxyl group of one amino acid with the amino group of another. While peptides created biologically are built from the C-terminus (Carboxyl) to the N-terminus (Amine), synthetic peptides are built in the reverse order.''

To my knowledge and experience with peptide synthesis, don't you start building your peptide at the c-terminus? If I am correct, this should be changed. regards-Shannon Lentz

A pretty weak section on peptide synthesis. I will try to edit this when I can. Murray must be rolling over in his grave. Chris Creighton.

Another response
I posted an example of solid phase peptide synthesis before I realized that there was another page dedicated to solid phase synthesis. I firmly believe that these pages should be merged, in order to make the information as ordered, and useful as possible. ~Dr. Faustus

I think that procedure is way too detailed for a page about peptide synthesis overall, and maybe even for an SPSS-specific page. OTOH, if it stays, it needs lots of work...gotta use the degree sign for degrees, subscript the subscript characters, link to relevant regents that have their own page. OT3H, is that really original writing, or is it copied fairly literally from the references? DMacks 05:46, 1 May 2006 (UTC)

It may be too detailed, I won't argue that, but it is original writing; it came out of my doctoral dissertation, with a few minor changes. I posted it mainly as a way to begin a section on the actual procedures of SPPS. Whether or not the procedure stays, it would be useful to give some type of outline for SPPS, including how one chooses the appropriate resin, and handle, as well as a discusion of side-chain protecting groups. As to the links to relevant reagents, the subscripts, degree sign, etc., I am just learning how to use html codes, and the wiki-specific codes for these things. I will attempt to fix these things when I have time, assuming the procedure should stay. I will also simplifying what is there now, to make it a little more succinct. --Dr.Faustus 13:45, 13 May 2006 (UTC)

Protecting groups
The section entitled "Protective Groups" should be divided into two sections, "backbone amine protection, and side-chain protection. --Dr.Faustus 13:45, 13 May 2006 (UTC)

anionic nitrogen with Fmoc
Is it true that Fmoc deprotection yields anionic nitrogen?

I would say the group is first deprotonated leading to an aromatic intermediate (that's why they use fluorene). then the nitrogen picks up a proton somewhere and the thing breaks apart


 * It's almost certainly not as concerted as it's drawn...initial formation of aromatic fluorene anion makes sense. But under basic conditions it seems less likely that a neutral carbamate N would get protonated on an anionic structure. DMacks


 * I added a new mechanism. It's basically copied out of John Jones, Amino Acid and Peptid Synthesis. I hope that's not a copyright issue. - FelixP 19:16, 11 August 2006 (UTC)

Repetitive
This article seems repetitive. The use of the various protecting groups and coupling agents are repeated in each type of synthesis. This article is almost a collection of several articles than one whole article. I am willing to clean it up, but I'd like to hear opinions on the matter first. --Rifleman 82 16:29, 28 June 2007 (UTC)
 * I thought this was a good way to arrange the article. The categories after the introduction correspond to the chemicals used in peptide synthesis: resins, protective groups, activating agents. I don't think it's repetitive. Maybe a little bit because Boc and Fmoc are first mentioned as general strategies and then as protective groups. FelixP 18:25, 28 June 2007 (UTC)

An example of solid phase peptide synthesis
The following is an outline of the synthetic steps for peptide synthesis on polyamide or polystyrene resin, using the base labile 9H-fluoren-9-ylmethoxycarbonyl (Fmoc) protecting group. Using the techniques outlined below, one will obtain a peptide which is capped on the N-terminus with an acetyl group, and on the C-terminus with a primary amide (CONH2).

Setting up glassware for manual peptide synthesis

Manual peptide synthesis can be accomplished in a fritted-filter reaction vessel with a three-way valve fitted onto a 1 L side arm vacuum flask by way of a 1-hole stopper. One valve is used to bubble nitrogen, which is first passed through a small column of Drierite, and then into the reaction mixture to agitate the solution and mix reagents. The other valve is used to evacuate excess reaction solutions and wash solvent using a vacuum flask. All glass pieces to be used in Solid-phase synthesis should be treated with a silanizing agent (such as 1-5% dimethyldichlorosilane in DCM) prior to use, to avoid accumulation of static charge, which makes the resin very difficult to handle.

Preparation of polyamide-Rink resin

Polyamide (PL-DMA) resin (1g) is treated with ethylene diamine (40 ml) in a 50 ml Falcon tube overnight on a rocker, then filtered, washed with 5x10 ml of 1:1 dimethylformamide (DMF):dichloromethane (DCM) solution, 5x10 ml of 1:1 DCM, and loaded with Fmoc-Rink using Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (3 eq), 1-Hydroxybenzotriazole Hydrate (HOBt) (3 eq), and Diisopropylethylamine (DIPEA) (6 eq) in 1:1 DCM:DMF. It can then be dried under vacuum and stored at −15 °C until needed.

Handling the resin before, and during synthesis

The resin is first swelled for 15 minutes in 10 ml of 1:1 DCM:DMF and drained. The resin is also washed with 5x10 ml of 1:1 DCM and DMF after each completed amino acid coupling.

Fmoc-deprotection

Fmoc deprotection after each amino acid coupling is accomplished using 2x10 ml of 20% piperidine in DMF, with N2 agitation for 10 minutes each treatment. The resin is then washed with 5x10 ml DMF, followed by 5x10 ml of 1:1 DCM:DMF. An alternate treatment is 1% DBU in DMF; this gentler treatment allows removal of Fmoc groups in the presence of other base-labile moieties.

Adding amino acids

An amino acid is coupled to the deprotected N-terminal amine of the resin, or previously coupled amino acid, using a coupling mixture such as the protected amino acid (3 eq), PyBOP (3 eq), HOBt (3 eq), and DIPEA (6 eq) in 1:1 DCM:DMF until the resin is negative to ninhydrin. Another popular set of coupling conditions is amino acid (4.4 eq), HBTU (4 eq), and DIPEA (8 eq) in DMF. Amino acids can also be purchased as the pre-activated ester, in which case coupling agents such as HBTU and PyBOP are unnecessary.

Monitoring the progress of amino acid couplings

The progress of amino acid couplings can be followed using ninhydrin, or p-chloranil. The ninhydrin solution turns dark blue (positive result) in the presence of a free primary amine but is otherwise colorless (negative result). The p-chloranil solution will turn the resin beads dark black or blue in the presence of a primary amine if acetaldehyde is used as the solvent or in the presence of a secondary amine, if acetone is used instead; the beads remain colorless or pale yellow otherwise. (The tests are outlined below)

Testing by ninhydrin (1)

Add 2 drops of 40% phenol in ethanol, 2 drops of 0.014 mol/L KCN in pyridine, and 4 drops of 5% ninhydrin in ethanol to a microcentrifuge tube along with a spatula tip size sample of resin, then vortex the mixture and heat for 5 minutes at 100 °C.

Testing by chloranil (2)

Add 5 drops of acetone or acetaldehyde, 5 drops of a saturated solution of p-chloranil in toluene, plus a small spatula-tip-size sample of resin to a microcentrifuge tube, then vortex the mixture and allow to stand at room temperature for 5 minutes. Acetone is used for the detection of secondary amines, where acetaldehyde is used for primary amines.

Continuing peptide extension

Once the coupling of the amino acid is complete, the resin is washed, the Fmoc group deprotected with piperidine, and the resin washed again to prepare it for the next coupling. This process is repeated until all necessary amino acids have been added.

Acetylating the N-terminus

After the peptide sequence is completed, the N-terminal amine can be acetylated with 2 ml of 1:1 acetic anhydride and triethylamine in 10 ml of 1:1 DCM:DMF for 1 hour or until negative to ninhydrin, and the resin then washed with 5x10 ml of 1:1 DCM:DMF, before the peptide is cleaved from the resin. The N-terminus can also be left as the free amine if required.

Cleaving the peptide from the resin

The resin is treated with a cocktail of trifluoroacetic acid (TFA) and cleavage scavenger reagents, such as triisopropylsilane (TIPS), 1,2-ethanedithiol, and water (H2O). The choice of scavengers is dependent on the amino acid sequence. The resin is then filtered away, and the combined filtrates allowed to stand for 1 hour to ensure removal of the acid labile protecting groups.

Workup of peptides after cleavage from the resin

The TFA is evaporated to dryness (or a heavy oil or glass if it does not solidify) on the rotary evaporator, followed by the addition of 5 ml of diethyl ether to the flask to precipitate the peptide, and remove the bulk of the by-products. Once the peptide is precipitated with the ether, filter through a sintered glass funnel and redissolve peptide into 60% AcN/ 0.1 %TFA. The peptide solution is then frozen in a dry ice/ethanol bath and lyophilised ( dried). The result is a crude peptide which is stable for a number of years. The peptide can then be purified by Ion-exhange and Reverse Phase chromatography.

Typically preparative HPLC is used to purify the final product. Mass spectrometry data are obtained to ensure the target peptide was obtained.

A former professor responds, bluntly
This article is little more than strung-together, draft-quality student drivel, which fails utterly as encyclopedic writing. The whole of it should be removed—for its repeated violation of WP:VERIFY, at least, but also because of its unscholarly structure, which is giving rise to further unsouced text dumping.

Otherwise, the only approach is to find 2-3 scholarly sources whose outlines of material can be trusted, then to slowly migrate material into that outline, followed by careful editing to make the material conform to the scholarly sources. As it is now, it is near-to-stream of consciousness content (likely note taking from classroom notes or an uncited book). It cannot stand as an encyclopedic article, and its paragraph upon paragraph of unsourced text makes it utterly impossible to turn into scholarship. It is nothing more than rule-violating, "just trust me" writing. Le Prof 73.211.138.148 (talk) 19:19, 12 November 2016 (UTC)

Here is an outline from a review
Here is an outline from a review, that might be of interest to those wanting to improve the structure of the article. Note that some major sections here, do not even appear in the WP article, while others are overly long and detailed here. WIll provide the citation, shortly."DEVELOPMENT OF SOLID-PHASE PEPTIDE-SYNTHESIS METHODOLOGY

Figure

Table Instruments for Solid-Phase Peptide Synthesis currently available on the market.

THE SOLID SUPPORT

COUPLING REAGENTS

Table Peptide synthesis reagents suppliers list.

SYNTHESIS OF MODIFIED RESIDUES AND STRUCTURES

PROTEIN SYNTHESIS

SIDE-REACTIONS

Table Common problems encountered during peptide synthesis.

PURIFICATION AND ANALYSIS OF SYNTHETIC PEPTIDES"

Le Prof 73.211.138.148 (talk) 21:33, 12 November 2016 (UTC)

[Purported] Attention banner spam
It is unnecessary, excessive, and completely counter productive to tag multiple sections with the same issue. A single tag at the top of the page is sufficient. Why is the following repeatedly the following to multiple sections:


 * As noted above and below, * * * the same tags are not used in every section or subsection, only in those where it applies. In others, where the problems differ, either a different ordering of tags appears, or with one or both being different tags. Please do not misrepresent the case to which you object. Reductio absurdum does not work. THe tags in place are accurate, and reasonable given the state of the article. 73.211.138.148 (talk) 21:55, 12 November 2016 (UTC)

This is insulting the intelligence of editors. Yes, we can figure out that certain sections do not contain sufficient references. A single banner at the top of the article is sufficient. Adding the same banner to multiple sections is disruptive

WP:OVERTAG is especially relevant. Redundant tags: Especially avoid adding two tags to the same article, section or passage that essentially mean the same thing, in whole or in part. Boghog (talk) 20:48, 12 November 2016 (UTC)

Please see all further responses here
Meanwhile, please try to make constructive edits to the article. For instance, I have been looking for a citation, that covers the table that provides coupling efficiencies at various levels, depending on chain length. Do you know of a source for this? If you can help find a source, it will remove two tags (one combined section tag). Please assist in improving the article, and not focusing on appearances, and the history of your animosity toward me. Our philosophies differ. Mine is client (reader focused); yours is editor and appearance focused. We will not reconcile these. But you can, if you insist on following me to articles, rest assured I do not stop and work (a) if I have not professionally worked and published in a areas, and (b) if the article is not awfully deserving of critical attention. Please make the article better. Leave the philosophical differences off table, when 5 mins of work on your esteemed part, can likely remove a tag in a way we would both agree on. Now, how about a source for that table? Le Prof 73.211.138.148 (talk) 21:29, 12 November 2016 (UTC)
 * How can I respond on your talk page when you keep deleting my questions? Also, why are you adding the same attention banner to the multiple sections of the same article? I am still waiting for an answer.  Boghog (talk) 21:35, 12 November 2016 (UTC)
 * Please respond to the call for constructive engagements and edits. As for the "why re you…", this has been asked, and answered. All further communications just one place, here. See inset message below your box, above. Not all are the same. 73.211.138.148 (talk) 21:56, 12 November 2016 (UTC)
 * Please respond to the call for responsible tagging. And no, you have not responding to my question. There is no reason to add the same tag to a large number of individual sections. If multiple sections have the same problem, then it would be appropriate to add one tag at the beginning of the article. Boghog (talk) 22:11, 12 November 2016 (UTC)
 * Please see here * * * and here * * * and here * * *. Startng from the top of the section, the matter has been addressed three times. Leprof 7272 (talk) 22:23, 12 November 2016 (UTC)

Content from Leprof_7272 Talk page
As I have addressed before, * * * the article tags have one function—to call attention to need for the article as a whole, and to place it at places of notice, for other editors, so that they might look to improve the article. The tags at sentences, and sections—which in this case, are NOT all the same, but are tailored to each section or subsection WHERE MAJOR ISSUES OCCUR—are there to let people know where the most egregious cases of WP:VERIFY occur. When one jointly edits a document, it is appropriate to call other editor's attention to the points of tha article where attention is needed (whether in an FDA submission, jointly submitted manuscript, or WIkipedia article).

The larger question is, why do you follow to articles, only to revert tags? Why do you not find sources, or take the bold action of deleting whole sections that are plagiarised? Why do you focus on IMAGE / APPEARANCE, RATHER THAN SUBSTANCE? Why not improve the article? Whether you like it or not, the user feedback I receive is this: it is helpful for student readers to know whether the material they are reading is of high enough quality to justify their time.

Do we disagree, that this article is subpar? Why not begin adding quality content, instead of wasting time, asking that poor quality be allowed to remain throughout a ridiculously long, plagiarized, and otherwise WP:VERIFY-ignoring article? Le Prof 73.211.138.148 (talk) 21:06, 12 November 2016 (UTC)

Purported attention banner spam [and editor stalking]
Please see above. There is no need to create multiple sections to raise your objection. Stop reverting good faith edits to make appearances suit your tastes. Fix the article problems.

Otherwise, please stop stalking me. Address problems, don't make them. Le Prof Leprof 7272 (talk) 21:09, 12 November 2016 (UTC)


 * I repeat, why are you adding the same attention banner to the multiple sections of the same article? I am still waiting for an answer. Boghog (talk) 21:20, 12 November 2016 (UTC)
 * Also I know it is your right to delete comments on your talk page, but why provide answers to deleted questions? Are you that insecure? Boghog (talk) 21:25, 12 November 2016 (UTC)
 * Asked and answered, see above. * * * Each tag is unique to section vis-a-vis entries and order. It cannot be helped that there are commonalities, because the rampant plagiarism that characterizes the article has common themes throughout. But there are sections with no citations, only one source for a large block, only primary sources for a whole section, etc. So not all subsections under a section need the same attention. To focus editor attention to the most egregious problems has been the historical point of tags, and is the point here. See below re: plagiarism. Leprof 7272 (talk) 22:23, 12 November 2016 (UTC)


 * Per WP:BRD, you made an edit, I have objected and stated the reasons for my objection on the article's talk page. Now it is time to discuss.  So far you have not responded on the talk page. Finally your charge of editor stalking is unfounded.  The article in question has been on my watch list for a very long time. Boghog (talk) 21:31, 12 November 2016 (UTC)
 * So you have allowed an article to persist, with rampant WP:VERIFY and plagiarism violations, and you ask me why need to tag? Simply put, because editors knowledgable about this subject need to be drawn in, to address egregious problems. See below. Otherwise, what is critical, is not how many articles you have watch listed, but how quickly you respond to my edits (always before I finish) and how you respond to them (reverting at knee-jerk, finding nothing of value). Physician, heal thyself (rather than always spouting self-justifying wikilawyer references to me). I care about two things, that content be sources, and that it be sourced to quality content that reflects the preponderant views in a field. Who gets it there is of no matter to me. But merde is merde, even when you remove the signs that say "Watch where you step." Leprof 7272 (talk) 22:23, 12 November 2016 (UTC)


 * My first edit on the article in question was made on 14 June 2010. Your first edit was apparently on 12 November 2016. Boghog (talk) 21:46, 12 November 2016 (UTC)
 * Congratualtions. And knowing you, I presume it did have a citation. But this has not saved the article from what it is. A rampant poster-child of a case, for the failure of Wikipedia to call attention to and to respond to plagiarism and WP:VERIFY violations. As for the plagiarism, please note that another editor has come and gone form the page, responding to a tag, and deleted the whole plagiarised section. This is important for two reasons. (i) It shows another editors response to the same situation, making your reversions appear all the more unreasonable, and (ii) It shows that others do respond to tags, buy making constructive changes to the text. Deleted any large plagiarised section. I will not complain. Replace it with a short two sentence paragraph, with sourced content, stubbing in the beginning of a future well-sourced section. But do not revert tags when the sections have clear issues remaining.


 * Otherwise, please note, there was, earlier, a section that was marked as plagiarism. But another editor removed it. That section was one I invited you to source (and that I tried, unsuccessfully to, only being able to find evidence of cut and paste). Why not constructively begin rewriting and restricting the article? My objection is to merde, not to quality that is not my own (except, as our past has shown, when you can see no quality in toe work of those other than yourself, that is, when you take over ownership of articles). Leprof 7272 (talk) 22:23, 12 November 2016 (UTC)


 * You have copied and pasted identical attention banners into multiple sections. By assuming that we couldn't figure out which sections are not adequately sourced insults our intelligence. Boghog (talk) 23:39, 12 November 2016 (UTC)
 * The copypaste banner was not redundant and consequently I did not remove it. However, why didn't you simply delete the infringing material instead of leaving an attention banner behind and expect someone else to delete it? This is inefficient. Boghog (talk) 07:13, 13 November 2016 (UTC)


 * Once again, you conveniently ignore when parts of your arguments are addressed—never acknowledging what it is you have raised, then immediately tacking a new course of objections.
 * My brief response to your latest is simple to restate what I have already said. The tags for each section, while repetitive, are not identical, because the issues in each section vary, from having no citations, to having one, to having only primary citations, etc. The fact that they are somewhat redundant is the fault of original editors that committed repeated errors, and not of this editor, calling attention to those repeated, WP:VERIFY and WP:OR-defying edits. I will check one last time, but there has to be a way to both call the article to others' attention, and save other editors the time of figuring our where first to put their efforts.


 * That we disagree about how to do this is clear, and is something that two well-trained scholars should be able to agree to disagree about. It is not your, or my encyclopedia. And we apparently continue to disagree about whether your time is best spent chasing me about and attacking my edits, versus actually getting busy responding to the tags, and either deleting plagiarised content, or doing hard edits to replace material that is at the same time dishonest in its wholesale cribbing of content (because ideas and data, unsourced, are still plagiarism), and for the most part sophomoric in its structure, quality of explanation, and level of communicated ideas.


 * Regarding the copypaste, I always first place the tag, to give the original or concerned editors the time to source the apparent cribbed material. I can always be wrong—material that appears cribbed may have passed to free use without my knowledge. Or, it may be readily computed information (as we both know this table is), but as such it is information that has to be computed somewhere else, so that we can cite that somewhere else. That you muddled about with the small issues, rather than assisting with the real problems, is part of my longstanding objection to your following. (That, and your predisposition to heavy-handed-I-am-always-and-alone-right way of approaching other editors, as others have also note to you.)


 * Otherwise, I have had my fill of this interaction, and should you choose to ignore my explanations (there is nothing new in this fourth version of explaining to you), and go back to reverting, I will probably bow out, and leave this another of "Boghog's owned articles". If I am left to do this, however, it will also be referred to a third party that has, since the demise of the WP-provided article grading, begun cataloging historical and traditional poor quality articles, with a focus on academic dishonesty. Articles with problems do not appear on that app, at least not front-and-center. Only articles with problems where there is commitment to keep the reality from WP readers. (The app is directed to academic users.)


 * Cheers, I hope we can fix the article, and leave it honestly marked, until it is no longer bad, and so patently a mass of poor and dishonest work.  73.211.138.148 (talk) 21:14, 13 November 2016 (UTC)
 * LeProf, consider this your WP:AGF warning regarding interactions with other editors. Further assumptions or insinuations that others are being dishonest, or are out to get you, or that there is some cabal working against you, will lead to your being blocked. WP:CIVIL is a site policy. DMacks (talk) 21:48, 13 November 2016 (UTC)

[Purported] Attention banner spam, Part 2
Please continue discussion here, last section is getting too long to navigate. Bottom line, I have addressed the objection that all tags are identical (similar but not identical, as required by similar but not identical problems section by section), and I maintain—as one who has collaborated on multi-author published manuscripts, FDA documents submissions, multiparty consulting documents, etc.—that it is never the case that contributing minds simply state once at top of a complex document, "There are problems herein, fix them," rather they always have such top-of-document attention getters, following by specific flags points throughout the complex document (manuscript, FDA submission, report, or wikipedia article) making clear what the problems are, and where the priority attention and fixes are needed.

I will grant that the tags available for highlighting specific problems are not optimal—in real professional collaborative work, the constraints are lifted, and statements of the real issues can be penned into marginal notes (e.g., in SharePoint). But I know, were I to use "cleanup" tags that allow for Le Prof prose, though more specific, they would be less acceptable to the single objecting editor here.

Finally, the acquiescences not to leave my own, prose-filled tags (though they are more helpful in being more specific), and not to leave inline tags alongside section tags (except in cases where a single or few citations left the impression of a section being done, when indeed that was far from the actual case), are two ways in which I have "given in" to this Leprof_7272-following editor. Le Prof 73.211.138.148 (talk) 21:24, 13 November 2016 (UTC)
 * The WP:TAGBOMBing pattern of LeProf's edits has already gotten notice at WP:ANI, with no support as being helpful. Regardless of how you act on other editorial projects, and whether or not that behavior is within their norms, is not relevant here. Here, it appears others do not find it useful or proper. DMacks (talk) 21:55, 13 November 2016 (UTC)