Solid phase peptide synthesis (SPPS), strategies, resins and comparison with Fmoc-strategy General scheme of SPPS
attach to linker
deprotect amino function
couple
n times deprotection and coupling cleave N-α-protecting groups
• two mainly used N-α-protecting groups
Fmoc Boc Boc-protecting group
Boc = tert. Butyloxycarbonyl or tert. Butoxycarbonyl
stable to bases and nucleophiles unaffected by catalytic hydrogenation deprotection with TFA is rapid Introduction of the Boc-group
Di-tert-butyl-dicarbonate 2-(tert. Butoxycarbonyl-
= Boc-anhydride = (t-boc)2O oxyimino)-2-phenylacetonitril = Boc-NO
O O O O O O O O N
N
• both commercially available • storage in refrigerator for extended periods
•(t-boc)2O is more expensive • preparation: „Kates S. A., Albericio F. (ed): Solid-Phase Synthesis, A practical guide, Marcel Dekker Inc. 2000, p. 105-107“ N-α-Boc protected amino acids
•alreadyN-α-Boc protected amino acids can simply be bought from firms like „Novabiochem“
O e.g. Boc-Ala-OH O N COOH H Cleavage of the Boc-group
TFA
E1-elimination
CO2 cleavage
TFA is volatile and can be easily removed in vacuum! Resins for Boc SPPS Resins for preparing peptide acids
Merrifield (Chloromethylstyrene-divinylbenzene) - was the standard support for the synthesis of peptide acids by Boc SPPS - now only used in the synthesis of small to medium sized peptides, because the benzylic ester resin linkage is not completely stable towards repetitive treatment with TFA O H Cl , DMF N boc O - + + BocAS Cs ,KI R
- attachment of the C-terminal residue is achieved by heating the resin in DMF with the appropiate amino acid cesium salt in the presence of KI - cleavage is affected by treatment of resin with HF or TFMSA, or by hydrogenolysis
- alcohols can be released using reducing agents like DIBALH or LiBH4 - methyl esters can be produced by transesterification with NaOMe Resins for Boc SPPS Resins for preparing peptide acids
PAM (4-Hydroxymethylphenylacetamidomethyl) - also a standard support for Boc SPPS - stabilizing effect of the phenylacetamidomethyl function on the ester linkage reduction of losses during repetitive TFA acidolysis
O H N boc O O R N coupling: H a) first addition of the PAM-linker on to the aminomethyl resin and then coupling of the Boc-protected amino acid b) first coupling of the Boc-protected amino acid to the PAM-linker and then reaction with the aminomethyl resin followed by end-capping of unreacted aminomethyl groups cleavage: - treatment with HF or TFMSA releases the peptide acid Resins for Boc SPPS Resins for preparing peptide acids
Brominated Wang (Brominated α-Methylphenylacyl resin)
O R O Br , DMF boc O N + BocAS-Cs+,KI H O
hν (350 nm)
O H N boc OH R Resins for Boc SPPS Resins for preparing peptide amides
BHA / MBHA (Benzhydrylamine / 4-Methylbenzhydrylamine) - used for the synthesis of peptide amides by Boc SPPS - attachment of the first amino acid with standard methods of amide bond formation - cleavage of the carboxamides with HF or TFMSA - MBHA is more acid sensitive and the peptide amide can be released with HF or TFMSA under less drastic conditions
NH2 NH2 Resins for Boc SPPS Resins for preparing C-terminally modified peptide fragments
Brominated PPOA (Brominated [4-Propionylphenoxy]-acetic acid) - versatile resin for the Boc SPPS of peptide acids, esters and hydrazides by photolytic or nucleophilic cleavage
O O O O N , DMF O N H H H Br - + N + BocAS Cs ,KI boc O O R O
ν h or NaOH in NEt3 in NH2NH2/ dioxane methanol/ DMF dioxane
O O O H H H N N N NH boc O boc OMe boc N 2 H R R R Resins for Boc SPPS Resins for preparing C-terminally modified peptide fragments
Oxime resin - attachment of the first amino acid with DCC - afterwards acetylation of unreacted oxime groups
- cleavage from the support by various nucleophiles like NaOH, NH3, R1NH2, NaBH4, MeOH, NH2NH2
NO 2 NO2
amino acid, DCC H HO N O N R N O General aspects of Boc strategy
• cleavage of the N-α-Boc-protection group with TFA (usually 25-50% (v/v) in DCM) • side chain protecting groups must be orthogonal(!), that means: • stable against TFA during N-α-Boc deprotection • removable at the end of peptide synthesis • release of the peptide from the resin by treatment with HF Side chain protecting groups for Boc strategy
Arg: Toluolsulfonyl- (Tos) or Mesitylen-2-sulfonyl-group (Mts) cleavage: HF/anisole (Tos) thioanisole (Mts) Ser, Thr, Tyr: Benzyl (Bzl) O cleavage: HF R
Asp, Glu: Bzl boc N COOH H O O O H Lys: Fmoc or Cl O N H OH O N O HN 2-Chlorobenzyloxycarbonyl (2ClZ) boc OH HN cleavage: piperidine (Fmoc), TFA (2ClZ) boc
O S Cys: Acetamidomethyl (Acm) or OH MeO NH 4-Methoxybenzyl (MeOBzl) boc cleavage: Hg2+-or Ag+-salts (Acm), HF (MeOBzl)
His: Dinitrophenyl-group (DNP) cleavage: thioles DNP Advantages and disadvantages of Boc- and Fmoc-strategy
Advantages Boc Disadvantages easy to introduce temporary and permanent (side chain) Boc-amino acids are stable at room protecting groups are both acid labile side chain deprotection during repeated temp. for extended periods (but TFA treatment can occur storage at 4°C is recommended) repeated TFA-mediated N-α-deprotection deprotection with TFA is rapid over the course of a long synthesis may successful strategy for many peptide lead to modification and/or degradation of synthesis applications sensitive peptide sequences good coupling results difficulties for fragile peptides that don‘t survive the relatively harsh final HF cleavage Boc-strategy requires the use of “dangerous“ HF and expensive laboratory apparates side reactions are possible: t-Bu+ reacts with nucleophilic side chains like trp, tyr, met, his side chain protecting groups / adding of scavengers (1,2-Ethanedithiole) to the deprotection reagent Advantages and disadvantages of Boc- and Fmoc-strategy Fmoc Advantages Disadvantages
orthogonal protection sheme Piperidine: harmful vapor, toxic Fmoc-amino acids are easy to side reactions: prepare in crystalline form in high • aspartimide formation at Asp- yield and stable when stored at 4°C X residues like Asp-Gly, -Ser, milder reaction conditions: -Thr, -Asn, -Gln milde base (piperidine) for N-α • linker-bound C-terminal Cys deprotection, TFA only for the final undergoes significant resin cleavage and deprotection racemisation (ca. 0,5%) with progress of each deprotection each cycle of Piperidine- reaction can be followed by real time treatment spectrophotometric monitoring the release of the cleaved Fmoc-group at 300-320 nm Literature
-Novabiochem 2002/3 Catalog -www.cup.unimuenchen.de/oc/carell/lehre/peptide2.pdf -Kates S. A., Albericio F. (ed): Solid-Phase Synthesis, A practical guide, Marcel Dekker Inc. 2000