TỔNG HỢP PEPTIDE PHA RẮN

TỔNG HỢP PEPTIDE PHA RẮN (SOLID PHASE PEPTIDE SYNTHESIS) PEPTIDE SYNTHESIS R H2 N OH O α−Amino acid α-Amino Acids Classical Solution-Phase Methods • Purification and characterization are required after each step • The solubility of the peptide worsens and becomes more unpredictable with increased chain length ► Time consuming ► Require considerable expertise Merrifield Peptide Synthesis First successful synthesis by SPPS was of the tetrapeptide L-Leu-L-Ala-L-Gly-L-Val on nitrochloromethylphenyl polymer resin using benzyloxycarbonyl (Cbz)-protected amino acids Merrifield Peptide Synthesis • Using nitrated chloromethylpolystyrene resin • Cbz-deprotection conditions: 10% HBr (the ester bond linking the peptide to the resin was stable (only 3.2% cleaved after h) Merrifield Peptide Synthesis • Using chloromethylpolystyrene resin (Merrifield resin) • Boc-deprotection conditions: M HCl (without the loss of the peptide from the resin so that nitration of the resin was no longer necessary due to the reduction in acid strength used for amine deprotections, i.e., Boc vs Cbz) SOLID PHASE PEPTIDE SYNTHESIS (SPPS) Two types of strategies for SPPS: Stepwise SPPS: proceeds by repetitive coupling and deprotection steps to introduce individual amino acid Convergent SPPS: efficient stepwise SPPS to create short segments, which are then purified and joined together further to form the target peptide SOLID PHASE PEPTIDE SYNTHESIS (SPPS) Surport for SPPS • Polystyrene, lightly cross-linked (e.g., 1%) with divinylbenzene • Polyethylene glycol (PEG) grafted onto polystyrene Anchoring: • Chloromethyl (Merrifield resin) • Amino • Hydroxyl • Other functionality Solid phase synthesis: building blocks Solid phase synthesis: protecting groups Fmoc Protecting Group • Is cleaved under very mild basic conditions (e.g piperidine) • Stable under acidic conditions Boc Protecting Group • Is removed by a strong acid (trifluoroacetic acid (TFA) in dichloromethane, hydrochloric acid or methanesulfonic acid in dioxane SOLID PHASE PEPTIDE SYNTHESIS SOLID PHASE PEPTIDE SYNTHESIS The Reactive Side-Chain β−Carboxyl side chain O OH HO NH2 O Aspartic acid γ−Carboxyl side chain NH2 HO OH O O Glutamic acid Benzyl (Bzl) type or t-butyl (tBu) groups have most commonly been used for the protection of side-chain carboxylates in SPPS based on t-butyloxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc) Nterminal protection, respectively [...]...Merrifield's Peptide Synthesis Critical issue: The C → N direction in which the peptide is prepared SOLID PHASE PEPTIDE SYNTHESIS (SPPS) Attachment of the first amino acid to a functionalized solid support can be done according to two general strategies: • Direct... approach for the preparation of C-terminal peptide acids with p-alkoxybenzyl (PAB) anchoring and Fmoc chemistry CARBOXYLIC ACID LINKERS FOR SPPS LINKERS FOR SPPS CARBOXAMIDE LINKERS FOR SPPS The continuing need for the synthesis of biologically interesting peptide carboxyl-terminal amides was a driving force for chemists to develop solid-phase syntheses of peptide carboxamides Alcohol linkers Alcohol... addition/elimination, usually on an ester - Base catalysed reaction, such as elimination or cyclization NUCLEOPHILIC LABILE LINKER FOR SPPS COUPLING METHOD: SOLID PHASE FORMATION OF AMIDE AND ESTER • The formation of amides and esters are key steps in the solid phase organic synthesis • Requires activation of the carboxylic group prior to reaction with the amino or alcohol component RACEMIZATION Coupling Reagents... product depends on the acidic reagent (the conjugate acid of the nucleophile) Mitsunobu Mechanism Formation of a strong P=O in the by-product drives this reaction forward ALPHA-AMINO PROTECTING GROUPS • An essential requirement in chemical peptide synthesis is the appropriate selection and use of a temporary protecting group for the a-amino group of amino acids • Choice of Nα -protection • It must withstand... conditions of SPS, cause no side reactions, and be rapidly, efficiently, and selectively cleaved under conditions that leave unaffected any amino acid side-chain (“permanent”) protecting groups Solid phase synthesis: protecting groups