chemical synthesis of peptides peptides, proteins, pseudopeptides, peptidomimetics ---- chemistry,...
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Chemical Synthesis of Peptides
Peptides, proteins, pseudopeptides, peptidomimetics ---- chemistry, biology, biophysicspeptide synthesis ( > 3,000 – 10,000 Da) [protecting group, racemization-free condensation]
Synthetic methodology [solution-phase and solid-phase peptide synthesis]
[1] General Considerations• Development of rapid, highly stereospecific, high yield synthesis• Two major impetuses for peptide synthesis
(1) to improve potency, selectivity, stability, diminution of toxic side effects of the native ligands such as peptides and pseudopeptides
(2) to predict “the second code” for the three-dimensional structure of a peptide/protein
[2] Solution Phase Synthesis(1) Choice of N-protecting groups
peptide synthesis [C --- N synthesis to minimize racemization]temporary –amino protecting groups
[coupling strategy, side chain protection, final deprotection](a) tert-butoxycarbonyl (t-Boc) [deprotection by mild acidic condition](b) 9-fluorenylmethoxycarbonyl (Fmoc) [basic condition for deprotection]
1. no need for acidic condition2. hydrophobic seq. 합성
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(2) Side chain protection orthogonal: stable during the deprotection of the N-protecting group readily removable at the final deprotection step
benzyl group --- orthogonal to the tBoctert-butyl group --- orthogonal to the Fmoc
(3) Coupling methods dicyclohexylcarbodiimide (DCC) 1H-hydroxybenzotriazole (HOBt) BOP (benzotriazole-1-yl-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate) HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)(4) Deprotection strategies 1) Boc-benzyl protecting groups ---- HF or TFMSA (trifluoromethane sulfonic acid) 2) Fmoc-t-butyl protecting groups ---- TFA cf) choice of scavengers to avoid side reactions(5) Protection of the C-terminal carboxyl group C-terminus with carboxyl or amide group --- protection via ester or amide formation(6) Stepwise and Fragment Condensation 1) stepwise addition and N-deprotection 2) ligation of fragments separately synthesized
※ carboxylic group activation prior to aminolysis ---- leading to condensation1) reactive acylating agent2) stable acylating agent
Coupling Methods
(1) Carbodiimide [DCC, diisopropylcarbodiimide, water soluble carbodiimide]• Drawbacks: racemization, dehydration (Asn & Gln), by-products (N-acylurea)• O-acylisourea causes the side reactions.• Trapping agents
a) p-nitrophenol b) pentachloro and pentafluorophenols c) N-hydroxysuccinimide (HOSu) d) N-hydroxybenzotriazole (HOBt)
• DCC/HOBt: no major drawbacks (good pair)
(2) Mixed carbonic anhydride method
a) racemization [5(4H)oxazolone]b) unstable mixed anhydridec) in situ reaction with the amined) exothermic reaction (-15ºC)
Side reactionsa) urethane formationb) racemizationc) appropriate solvents and short reaction
DCC method preferred !!!
(3) Active ester method (part of DCC method)• mixture of N-protected A.a. and DCC --- trapping agent --- active ester ---- amine---- acetylating the amine group
(4) Azide method i) hydrazide formation ii) azide formation iii) aminolysis with the amine component
(5) BOP reagent (Castro’s reagent) i) stable, nonhygroscopic, soluble ii) high yield peptide synthesis
with low racemization iii) difficult coupling iv) on-resin cyclization
via lactam formation v) BOP > DCC/HOBt
vi) carcinogenic hexamethylphosphoric triamide formation (BOP reaction) PyBOP / BrBOP / PyBroP / BroP (6) HBTU reagent [2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate]• side chain to side chain cyclic lactam formation
(7) Amino acid halides• Fmoc protected amino acid chloride
rapid synthesis of short peptidescoupling in immiscible solventshigh efficiencyfastease of synthesis of the activated amino acid at a low cost
• Solid phase peptide synthesisFmoc A.a. chlorides + DIEA (basic coreactant) ---- oxazolone formationHOBt addition (1:1) --- acylation (peptide formation)
• Protected amino acid fluoride with Fmoc, Boc, Zstable and effective for coupling
(8) Urethane protected amino acid N-carboxyanhydrides (UNCAs)• the best coupling reagent (better than BOP or HBTU)• difficult coupling steps (highly sterically hindered amino acid)
• stable ---- stability, reactivity, solubility• Boc, Fmoc, Z derivatives ---- commercially available• highly reactive toward Nu like amines ---- good yield & high purity w/o racemization
(9) Difficult couplings• UNCA, A.a. fluorides, HBTU, PyBroP > BOP or Carbodiimide[hindered peptides or amino acids bearing N-terminal ,’-dialkylation]
Protecting Groups
Amino acid i) N-terminal amino group ii) C-terminal carboxyl group iii) reactive group in the side chain
Considerationsi) peptide synthesis from C to N-terminus ii) racemization through 5(4H)oxazolone formation
iii) C-terminal protection (solid phase synthesis)
(1) tert-Butyloxycarbonyl (t-Boc) protectioni) acid cleavage (but stable to base, sodium in NH3, catalytic hydrogenation)ii) scavengers against reactive tert-butyl carbocations
anisole, ethanedithioliii) side chain protection [HF with appropriate scavengers ---- deprotection & cleavage]
Arg tosyl groupHis tosyl group HOBt deprotection
dinitrophenyl thiophenol in DMF before HF deprotectionBOM group HF & TFMSA-TFA
Cys p-methylbenzyl groupAsp/Glu cyclohexyl estersLys Z or substituted z group (benzyloxycarbonyl)Met unprotectedMet(O) reduction by low-high HF procedure
Trp formyl group piperidine in DMF or low-high HF procedureSer/Thr/Tyr benzyl ethers Tyr --- benzylation by strong acidTyr 2’,6’-dichlorobenzyl or Br-Z group protection
(2) 9-Fluorenylmethyloxycarbonyl (Fmoc) Group• deprotection under mild basic conditions [dil. liq. ammonia, ethanolamine, morpholine,piperidine]• N-Fmoc amino acid preparation [Fmoc-succinimide (Fmoc-ONSu)]• Deprotection: 20% piperidine in dimethylformamide• Cleavage of peptide from the resin: TFA [side group protecting groups --- basic stable and TFA removable]
Arg PMC group TFA labileAsn & Gln unprotected
trityl or benzhydryl groups (dehydrated)Asp & Glu tert-butyl estersTyr tert-butyl etherCys trityl group TFA
Acm (acetamidomethyl) TFA resistant (S-S bond formation)His trityl groupLys Z or substituted z groupMet no protectionTrp no protection or Boc-indole derivative
(3) N-Allyloxycarbonyl type protection• many undesired side reactions during the deprotection-cleavage steps [carbocations and other active species generated by HF or TFA]
• N-allyoxycarbonyl group for amine and alcohol protection• allyl ester for carboxylic group protection• ethers for aryl alcohol protection [reaction with allyloxycarbonyl chloride]
• easy deprotection via mild hydrogenation [complexation of allyl groups with Pd(0) and tributyl tin as H2 donor]
• all allyl and alloc side chain protected amino acids & N-alloc protected amino acids available
• allylic anchor groups (resin linkers)• peptide cleavage from the resin – neutral and extremely mild conditions
[3] Solid phase peptide synthesis (SPPS)
Scheme 2-13
unreacted A.a. and coupling reagents -- filtration and washing 2x – 4x excess of reagents
-- complete couplingautomated SPPS
Considerations i) protecting groups ii) coupling methods iii) choice of the solid support (resin)
Resins i) linkage of the 1st A.a. ii) functionalization of C-terminal group iii) swelling property iv) pressure v) chemicals
Abs at 570 nm
Kaiser test
(1) Solid support • polymer of styrene crosslinked with m-divinylbenzene (1%)• X-linking
i) rigidity and physical stability of resins ii) swelling property in the solvents iii) accessibility of the reacting groups
• 1% X-linked polystyrene resin with resin handles (spacers) i) swelling property ii) decreasing the interaction between the growing peptides and the resin iii) different anchoring and cleavage techniques
(2) Solid supports and resins for SPPS• handles
i) high yield for peptide synthesized ii) increased optical purity iii) better control of cleavage conditions
※ Resins with different handles a) Wang resin p-alkoxybenzyl ester linkage [Merrifield resin + 4-hydroxybenzyl alcohol] deprotection and cleavage of the peptide with 50% TFA in dichloromethane
b) PAL (peptide amide linker) handle PAL-COOH + p-methylbenzhydrylamine (MBHA) resin cleavage: 70-90% TFA in dichloromethane peptide with a C-terminal primary amide
c) Rink resin [Fmoc-t-butyl strategy] Fully protected (A.a.) peptide 합성 cleavage: mild acidic conditions (10% acetic acid or 0.2% TFA in dichloromethane)
d) Sasrin resin disubstituted phenol with the Merrifield resin protected peptide 합성 cleavage: 0.5% TFA in dichloromethane
e) p-methylbenzhydrylamine resin (Boc-benzyl protection strategy) peptide amide with Boc-protected A.a. cleavage: HF or TFMSA in TFA
f) PAM resin (phenylacetamidomethyl) or Oxymethyl-PAM resin peptide acids high resistance to acidolysis
g) Oxime resin fully protected peptide cyclization of protected peptides on oxime resins cleavage: aminolysis or hydrazinolysis
h) Nitrobenzyl resin cleavage: photolysis at 350 nm
i) Allylic resins [Hycram resin] alloc group for the –amino group protection fully protected peptide 합성 cleavage: neutral and mild conditions
(3) Deprotection methodsremoving the side chain protecting groups and cleavage of the peptide from the resin
a) deprotection and cleavage at different timefragment condensation (cleavage without deprotection)
orthogonal synthetic – deprotection strategyb) deprotection and cleavage at the same time
Boc-benzyl protected A.a. (HF, TFMSA in TFA, HBr in HOAc)Fmoc-t-butyl protection scheme
(TFA in dichloromethane at the last step, Scavengers to reduce carbocation)
cf) HF strong acid in the cleavage and deprotection step --- milder methods preferred
[4] Some current topics
(1) peptide libraries• peptides and proteins: difficult in practical applications
i) complex structures with multiple functions ii) little knowledge iii) complexity in structures
• Combinatorial chemistry: good quality control [complex mixture of peptides: “problem” rather than “opportunity”]
※ Construction of large diverse peptide libraries(a) Multiple synthetic method
peptide synthesis on the head of polyacrylic and grafted polyethylene rods(b) Tea bag method
porous polyprolylene containers with a small amount of a solid phase resinseparate coupling and combined deprotection
(c) The one peptide, one bead methodrapid synthesis of large diverse mixtures [proportioning-mixing or split synthesis]Mixing – Deprotection – Separation – Coupling – Mixing – etc…. (Fig. 2-17)
(d) SPPS with photolithography
• chemically diverse peptide mixtures• chemical structures and reactivities• unusual amino acid incorporation
(2) Stepwise and Fragment (Segment) condensation strategies
Stepwise strategy [short peptide synthesis] disadvantage: i) insolubility of the growing peptide
ii) difficult purificationiii) aggregation of the peptide (poor coupling and deprotection)iv) by-products
Fragment condensation strategy [protected peptides --- coupling] disadvantage: i) poor coupling
ii) racemizationiii) low solubility
Examples of coupling reagentsDCC/HOBtCuCl2 in DCC/HOBtBOP and HOBtDCC and ethyl-2-(hydroxoimino)-2-cyanoacetatepapain and -chymotrypsin
(3) Cyclization of peptides in solution and on solid supports[disulfide, lactam (cyclic amide), other functional groups]
• intramolecular cyclization • selective cyclization [orthogonal protection of the different reactive groups]• solution cyclization
[under highly diluted condition to prevent intermolecular reaction]
ex) monocyclic disulfide analogues of dynorphin A [MBHA resin, Boc-benzyl protected A.a., Cleavage with HF (+scavengers)dilution with water, oxidation, removal with ion-exchanger, lyophilization]
• cyclization on a solid support
advantage: i) high yield
ii) full automation
iii) pseudo-dilution effect
ex) mixed Boc-Fmoc strategy
for on-resin lactam formation
N-Boc protected A.a.
Fmoc or Ofm esters
side chains for the lactam bridge
HF for the final deprotection-cleavage step
ex) Oxime or Kaiser’s resin
ex) On-resin disulfide bridge formation
by using N-halosuccimides
(Scheme 2-17)
(4) Orthogonal protection and synthesis Orthogonality
N-Fmoc; basic condition (piperidine)tert-butyl or trityl group; TFAhandle cleavage; chemical hydrogenation
• Bicyclic (lactam and disulfide bridge) analogue of oxytocin MBHA resin (HF), N-Boc A.a. (TFA), benzyl-group for side chain (HF) side chains for the lactam formation [Glu/Asp and Lys/Ornithine with Fmoc or Ofm] ---- dilute piperidine treatment peptide cleavage from the resin with HF deprotection with HF S-S bond formation with Fe3(CN)6
• Parallel dimer of deamino-oxytocin (one cysteine and L--mercaptopropionic acid) cysteine – Acm (thallium trifluoroacetate), -Mpa – Tmob (7% TFA) PAL-resin (TFA), Fmoc-A.a. (piperidine)
• Mild three-dimensional orthogonal protection scheme dithiasuccinoyl (Dts) group protected A.a. (thiols) tert-butyl group protected side chains (TFA) 1st A.a. to the resin via o-nitrobenzyl ester linkage (photolysis at 350 nm)
(5) Pseudopeptides: amide bond replacements• backbone modification [CH group]
i) stability of peptides toward proteasesii) receptor selectivityiii) peptide antagonist
Ψ[CH2-NH] : the reduced peptide bondamino acid aldehyde and Schiff base formation with NaCNBH3
--- biological activity
(6) Modifications at the CH group Azapeptide: replacement of CH with nitrogen atom
(7) Allyl-UNCA strategy UNCA as powerful acylating agents allyl-based -amino and side chain protecting groups and resin handles
ex) allylic handle and A.a. as Fmoc or Boc-protected NCAs
(8) Continuous flow synthesis flow stable polyethyleneglycol dimethylacrylamide (PEGA) copolymer [chemical stability, swelling property, spectrophotometric monitoring] Fmoc-A.a. Rink linker (4-Fmoc-amino[2,4-dimethoxyphenyl] methylphenoxyacetic acid] Cleavage with conc. TFA (+ scavengers)
※ No peptide is ever trivial to synthesize !