tboc fmoc protocol in solid phase peptide synthesis
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This document discusses strategies for solid phase peptide synthesis (SPPS) using different protecting groups. It compares the t-Boc and Fmoc protection methods, noting the advantages of Fmoc such as using milder acidic conditions for deprotection and cleavage from the resin. Protocols are provided for various steps in Fmoc SPPS including resin loading, amino acid coupling and deprotection, and final cleavage and deprotection. Potential side reactions are also described such as diketopiperazine formation and aspartimide formation, along with ways to prevent these reactions.
tboc fmoc protocol in solid phase peptide synthesis
- 1. Presented by: SantoshSantosh KumarKumar SahooSahoo MC-Ph. D/2016/08
- 2. Protecting groups Strategies Comparison Procedure Approaches in synthesis Protocol Side reactions 10/26/2016 2niper_H Flow of Presentation
- 3. 10/26/2016 3niper_H Solid Phase Peptide Synthesis Principle
- 4. Transient protecting groups for amino groups that form the peptide bond Permanent protecting groups for functional groups within the amino acid side chains tBoc Fmoc 1963: Merrifield acid labile, Nα-protecting group 1970: Carpino & Han a base labile Nα-protecting group Synthesis, 1979, 955.Biochemistry, 1964, 3, 1385. Protecting groups 10/26/2016 4niper_H Transient protecting groups tBoc Protection
- 5. 10/26/2016 niper_H 5 tBoc Protection tBoc deprotection
- 6. 10/26/2016 6 Fmoc protection Fmoc deprotection Based upon the graduated acid lability of the side-chain protecting groups. In this approach, • Boc group is removed by neat TFA or TFA in DCM • Side-chain protecting groups • peptide–resin linkages tBoc/ Bzl Fmoc/tBu Removed by strong acid like HF •Use of highly toxic HF •Need for PTFE-lined apparatus •Specialists job •Use of strongly acidic conditions can damage peptides In this approach, •Base labile Fmoc group is used for protection •Acid labile side-chain protecting groups •Acid labile linkers that constitute the C-terminal amino acid protecting group Advantage: Temporary / permanent orthogonal protections are removed by different mechanisms allowing the use of milder acidic conditions for final deprotection and cleavage of the peptide from the resin. For all these reasons, Fmoc-based SPPS Method is now the method of choice for the routine synthesis of peptides Based upon an orthogonal protecting group strategy But not generally used, Why? Strategies Mol. Biotech., 2006, 33, 239-54.
- 7. 10/26/2016 7 Boc / Bn → Based upon the graduated acid lability of the side-chain protecting groups. Fmoc / tBu → Based upon an orthogonal protecting group strategy niper_H Strategies
- 8. 8 10/26/2016 niper_H Comparison at synthesis level
- 9. 10/26/2016 niper_H 9 Resin + BOC AA (Cs/ TEA salt) Carboxamide resin by DCC/DMAP Benzoylation / acetylation of unreacted –OH group of PAM BOC deprotection by TFA / DCM + Dithioerythritol (DTE) scavenger for Cys, Met, Trp Washing DCM, IPA AA as Trifluoro acetate + TEA / DCM neutralization Washing DCM, IPA Coupling of activated BOC AA Deprotection, Neutralization, coupling Dinytrophenylethyl (DNPE) PG of His, Formyl PG of Trp removed by Piperidine / DMF Cleavage from resin by HF Anisole, Thiocresol, Dimethyl sulfide as Scavenger for side chain PG alkylating agent BOC-SPPS
- 10. 10/26/2016 niper_H 10 PAM resin Phenyl acetamido methyl linker BHA resin MBHA resinMerrifield resin Partial cleavage during deprotection of BOC Balances Stability to TFA, lability to HF N H R O H N O H+ HO X N H R HO HN O O X H+ N H R NH3 O XO O X = H or CH3 Acid catalyzed acyl NO migration Side Reactions for BOC-PSSP Resins BOC-SPPS Diketopiperazine Formation Protonated N → Less prone Reversed by Base Aspartamide Formation Peptide having Asp-Gly Asp-Ala Asp-Ser
- 11. 10/26/2016 niper_H 11 HN NH Asp-Pro cleavage with HF Homoserine Lactone Formation C-terminal Met cyclize to homoserine lactone γ-COOH lose water in acid ↓ acylium ion ↓ cyclization Side chain involving Glu
- 12. 10/26/2016 niper_H 12 Resins for peptide amide synthesis. Resins for peptide acid synthesis. (Wang) resin: Fmoc-Aaa(X)-OH:DIC:DMAP 2:2:0.2 equiv to the resin OH content) in DMF diketopiperazine formation side reaction SASRIN (Super Acid Sensitive ResIN) Peptide is cleavable with 0.5-1.0% TFA in DCM 1 g ClTrt-resin + 2 mmol Fmoc-Aaa(X)-OH + 8 mmol DIEA in 3-5 mL DCM, for 1.5 h 0.8 mL MeOH to block unreacted groups washing with DCM, iPrOH, MeOH, ether prevents the diketopiperazine formation Attachment of Cys and His derivative to the resin is free from enantiomerisation Rink Amide-AM (Aminomethyl) and Rink Amide-MBHA (4-methylbenz hydrylamine) resins. Fmoc-SPPS
- 13. 10/26/2016 niper_H 13 Linkers are bifunctional molecules anchoring the growing peptide to the insoluble carrier. Linkers may be coupled to any carrier suitable for SPPS, an important option if alternatives to polystyrene-based resins (PS-DVB) have to be considered. Ramage linker Rink linker HMP linker 4-Formyl-3-methoxy Phenoxyacetic acid LinkersFmoc-SPPS
- 14. 10/26/2016 niper_H 14 Mechanism of base-catalyzed racemization during activation. More potent coupling reagents such as HATU or very active Fmoc amino acid derivatives such as the acid fluorides may drive the coupling to completion. Path A Base O X H N O R' BH+ O X H N H RO R' R O N H H N H RO R' H2N Coupling Reagents
- 15. 10/26/2016 15niper_H O OR1 N N R' R' H Carbodiimide N H N R' R' O O R1 O-acylisourea N N DCC O OXtR1 HOXt HOXt R2 -NH2 N N N OHHOBt Problems with Carbodiimides Coupling Reagents
- 16. 10/26/2016 niper_H 16 Carbodiimide reagents: Additives: N N N N N N N HCl N N N N N NN N N N O O O O N NN N DCC DIC EDC CIC BMC BEMCPC BDDC PECPIC N N N OH HOBt N N N OH O2N 6-NO2-HOBt N N N OH F3C 6-CF3-HOBt N N N N OH HOAt N N N OH Cl 6-Cl-HOBt N N N O OH HODhbt N N N N O OH HODhat N N OH Ph HOBI N O O OH HOSu N NC OH O O Oxyma
- 17. 10/26/2016 niper_H 17 HOXt-based coupling reagents
- 18. Resin beads + DCM filtration under vacuum Wash reaction vessel, resin with DMF → MeOH → DCM→ DMF Desired Fmoc-amino-acid in dry DCM + DMAP in DMF ↓ Cap the remaining hydroxyl groups by adding benzoic or acetic anhydride And pyridine in DMF. Mol. Biotech., 2006, 33, 239-54. 10/26/2016 18niper_H Protocol 1: Resin swelling Protocol 3: Attachment to hydroxy methyl based resin Protocol 2: Standard washing procedures Fmoc amino acid + DIPEA in dry DCM ↓ Wash the resin with DMF ↓ + DCM/MeOH/ DIPEA (80:15:5) to cap remaining reactive chloride group. ↓ Wash with DMF and DCM After drying in vacuum, the Substitution can be measured from Fmoc release. Protocol 4: Attachment to trityl based resin Fmoc-SPPS
- 19. 10/26/2016 niper_H 19 Loading 2-chlorotrityl chloride resin. capping of 2-chlorotrityl chloride resin. Loading Rink Amide resin. Capping unreacted sites on Rink Amide resin.
- 20. 10/26/2016 20 X X = Cl, OH, NH2 N H O OH R1 PG1 Fmoc N H O O(NH) R1 PG1 Fmoc piperidine H N H2N O O(NH) R1 PG1 Fmoc H N O OH R2 PG2 coupling reagent Fmoc H N O O R2 PG2 Y Y: activating group N H O O(NH) R1 PG1 Fmoc H N O R2 PG2 Fmoc deprotection Fmoc-Amino acid coupling repetitive Fmoc deprotection and AA coupling N H O O(NH) R1 PG1 H N O R2 PG2 Peptide segment PG3 PG4 O PGn-1 H2N Rn PGn TFA/Scavenger N H O OH(NH2) R1 H N O R2 Peptide segment O H2N Rn Loading Cleavage and Global deprotection Peptide Assembly Fmoc-SPPS
- 21. wash the resin with DMF + N-α Fmoc protected amino acid ↓ + HBTU / HCTU coupling → filtration → Wash the resin. 10/26/2016 21niper_H Protocol 5: Standard coupling procedure Activating amino acid Coupling amino acid Swell the resin in DCM → filtration+ 50/50 DCM/acetic anhydride solution Remove the capping solution by filtration → Wash with DCM Check the disappearance of free amino groups by colorimetry Resin is washed once with DMF ↓ 80/20 DMF/piperidine solution ↓ Filtration → Wash the resin. Protocol 6: Capping Protocol 7: Removal of Nα Fmoc protection
- 22. (Scavengers) Trifluoroacetic acid/water/triisopropyl silane 95/2.5/2.5 per 100 mg of resin Filter + MTBE to precipitate the peptide. Solubilize the peptide in acetonitrile/water/ TFA 50/50/0.1 and lyophilize. Dissolve the lyophilized peptide in a 5% acetic acid solution + 10% volume of DMSO pre-adjusted pH to 6.0 - 7.0 with a 0.5 M NH4OAc Disulfide bridge formation should be monitored by reverse phase HPLC. Dissolve linear peptide in buffer (0.1–0.2 M Tris-HCl, pH 7.7–8.7) + 1 mM EDTA and reduced (1–10 mM) and oxidized (0.1–1 mM) glutathione Lyophilize the solution after acidification with a TFA solution (pH 2.0) 10/26/2016 22niper_H Protocol 8: Standard TFA cleavage Protocol 9: Standard formation of disulfide bridges by air oxidation Protocol 10: Formation of disulfide bridges using oxidative folding in redox buffer
- 23. 10/26/2016 niper_H 23 Cleaving a side-chain protected peptide form 2- chlorotrityl chloride resin with HFIP Formation of disulde bond with DMSO.
- 24. 10/26/2016 niper_H 24 Side-chain Protecting Groups for Fmoc Amino Acids Acm: acetamidomethyl Al: allyl Aloc: allyloxycarbonyl Boc: t-butyloxycarbonyl Fmoc: 9-fluorenyl methoxycarbonyl Mbh: 4,4-dimethyloxybenzhydryl Mtr: methoxytrimethyl benzene sulfonyl OAl: allyl ester OtBu: t-butyl ester Pbf: 2,2,4,6,7-pentamethyl- dihydrobenzofuran-5-sulfonyl Pmc: 2,2,5,7,8-pentamethyl- chroman-6-sulfonyl chloride tBu: t-butyl ether Tmob: 2,4,6-trimethoxybenzyl Trt: trityl or triphenyl
- 25. Diketopiperazine formation after deprotection of the penultimate amino acid N-Terminal guanidinylation by the coupling reagent 10/26/2016 25niper_H Reaction occurs during couplings mediated by uronium/ aminium reagents or carbodiimides. Avoided by preactivation of the amino acid (i.e. the coupling reagent is consumed). The side reaction can’t occur when activating with phosphonium salts (Bop, PyBop). By using 2-chlorotrityl chloride resin or other bulky resins such as DHPP-Resin. By coupling the appropriate Fmoc-dipeptide in lieu of the penultimate amino acid. By coupling the appropiate Trt-amino acid → Deblocking with dilute TFA yields the protonated dipeptide Fmoc-SPPS – Side chain reactions
- 26. Base-catalyzed aspartimide Formation and subsequent reactions. Chem. Commun., 1994, 20, 2343–2344.10/26/2016 26
- 27. 10/26/2016 niper_H 27 Application of other cleavage reagents (DBU, TBAF, DEA, morpholine) eliminate the piperidide formation, but not the succinimide formation. Addition of HOBt to the cleavage mixture can reduce the succinimide ring closure. But the best results may get with the use of Fmoc-(Hmb)- amino acid derivatives: Hmb: 2-hydroxy-4-methoxybenzyl (removable with TFA) NH CH C O N CH2 C O CH2 C OtBu O (Hmb)amino acid derivatives are secundary amines: Removal of Fmoc group and the attachement of the next Asp derivative is difficult, needs longer time. Ninhydrin test can’t detect the efficacy of the coupling. Fmoc-(Fmoc-Hmb)Gly-OH The increasing of the solubility of protected peptide fragments as well as preventing of aggregation of ”difficult” sequences can be reach by the application of Hmb groups.
- 28. N-O shift involving serine or threonine Base-induced β-elimination of C- terminal cysteine J. Pept. Sci., 2005, 11, 441.10/26/2016 28niper_H Reversal is induced by bases, e.g. aq. NH3. This side reaction is minimized (but not avoided!) when trityl is used for the protection of the C-terminal Cys. Deguanidination side reaction on Arg If the guanidino moiety from Arg side chain is acylated by amino acid derivatives, it could be decomposed into Orn side product.
- 29. 10/26/2016 niper_H 29 Boc Fmoc It is better for avoiding DKP formation; There is no problem with the Boc cleavage, so it is better in case of peptides that aggregate easily. Aggregates are destroyed in every TFA cleavage step; Because of the extra neutralisation step, the synthetic cycle takes longer time; Resins for Boc-strategy are available for Fmoc-chemistry, too. Two steps cleavage procedure may results in better crude product. First step TFA cleavage (side chain protecting groups) then HF (peptide-resin bond). More suitable for preparation of branched peptides. ClTrt resin must be used to prevent DKP formation; Incomplete Fmoc deprotection in case of aggregating peptides; It is better for acid sensitive peptides (Trp, Met), oxidation, alkylation can be avoided. Asp-Pro bond is highly acid sensitive. especially recommended for O-glycosylated or sulfated peptides; Because of the orthogonality of N and side chain protecting groups fully protected sequences can be prepared. Comparision