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Biologia Química · Peptides and nucleic acids

Síntese e ligação de peptídeos

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O que você vai aprender

  • Draw how carbodiimides, additives and mixed anhydrides turn a carboxylic acid into an acylating agent, and predict the N-acylurea and anhydride side paths.
  • Draw Fmoc removal by piperidine (E1cB) and acidolysis of Boc, tert-butyl, trityl and 4-alkoxybenzyl groups, and choose scavengers for the carbocations they release.
  • Plan and troubleshoot the solid-phase cycle: direction of synthesis, coupling tests, loading and yield calculations, cleavage cocktails and diketopiperazine loss.
  • Explain racemization through 5(4H)-oxazolones and aspartimide formation, and predict the by-products and mass shifts they cause.
  • Draw native chemical ligation (transthioesterification, then S→N acyl shift) and explain its selectivity for an N-terminal cysteine.

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Activate the carboxyl group

Mixing a carboxylic acid with an amine gives only an ammonium carboxylate salt: the proton transfer is far faster than any attack on the carbonyl, and a carboxylate is a poor electrophile. Peptide synthesis therefore converts the acid into an acylating agent first. A carbodiimide such as DIC (N,N′-diisopropylcarbodiimide) is protonated by the acid, and the carboxylate adds to its central carbon to give an O-acylisourea, drawn here for Boc-glycine. Its leaving group becomes a urea, so an amine now attacks the acyl carbon easily. DIC is preferred on resin because diisopropylurea stays dissolved in DMF and washes away; the dicyclohexylurea from DCC precipitates. The O-acylisourea has two side paths: a second molecule of acid can attack it to give the symmetric anhydride, and its own imine nitrogen can attack the acyl carbon (an O→N acyl shift) to give an unreactive N-acylurea, a dead end that is worse in polar solvents such as DMF. The N-acylurea is an isomer of the O-acylisourea, with the mass of acid plus carbodiimide, but it never acylates the amine, so long preactivation with a carbodiimide and no additive wastes the amino acid. A mixed carbonic anhydride, made from the carboxylate and isobutyl chloroformate with N-methylmorpholine, is an older alternative: the amine attacks the amino-acid carbonyl, and CO₂ and isobutanol are released.

Additives and active esters

Additives such as HOBt (1-hydroxybenzotriazole, pKa ≈ 4.6), HOAt (pKa ≈ 3.3) and Oxyma (ethyl cyano(hydroxyimino)acetate, pKa ≈ 4.6) intercept the O-acylisourea before it can rearrange or form an oxazolone. They convert it into an active ester such as the OBt ester shown. An active ester is less reactive than the O-acylisourea but still acylates amines quickly, and it racemizes far less. The leaving group gets better as the pKa of its conjugate acid falls: 4-nitrophenol (≈ 7.2) < N-hydroxysuccinimide (≈ 6.0) < HOBt < HOAt. Pentafluorophenyl esters (pentafluorophenol pKa ≈ 5.5) are isolable active esters that react about a hundred times faster than 4-nitrophenyl esters, whereas phenyl (phenol ≈ 10.0) and methyl esters (methanol ≈ 15.5) are far too slow to serve as coupling agents. HOAt is better still than its pKa suggests, because its pyridine nitrogen sits next to the N–O group and helps the incoming amine by hydrogen bonding. Uronium-type reagents such as HBTU and HATU deliver the same esters in one step: the carboxylate, freed by DIPEA, attacks the reagent's central carbon, and the OBt or OAt ester forms together with tetramethylurea. HATU actually exists as a guanidinium N-oxide, but it reacts the same way. The carboxylic acid should always be activated before it meets the resin amine. A free amine attacks excess HATU itself and is capped as a tetramethylguanidinium group that can never be acylated.

Protecting groups: base-labile Fmoc, acid-labile tBu, Boc and Trt

Fmoc/tBu chemistry relies on orthogonal protecting groups. The Nα-Fmoc group (shown on glycine) comes off with 20% piperidine in DMF, and the side-chain groups and the resin link come off together at the end with about 95% TFA. Fmoc falls to base because the fluorene C9–H is acidic (pKa ≈ 23 in DMSO): its conjugate base is an aromatic fluorenyl anion. Piperidine removes that proton, the anion expels the carbamate (an E1cB elimination), the carbamic acid loses CO₂ to free the amine, and a second piperidine adds to the dibenzofulvene by-product. The dibenzofulvene–piperidine adduct absorbs at 301 nm (ε ≈ 7800 L mol⁻¹ cm⁻¹), so measuring it gives the amount of Fmoc released. Acid-labile groups leave as carbocations, and the more stable the cation, the milder the acid needed. Cbz and benzyl groups survive neat TFA and need HF or hydrogenolysis. Boc, tBu ethers and tBu esters give the tert-butyl cation in 25–95% TFA. Trityl cations are more stable still: the 4-methyltrityl (Mtt) group on a lysine amine comes off with 1% TFA, and 4-methoxytrityl (Mmt) comes off with even weaker acid. Trityl on a cysteine thiol is cleaved by the final TFA cocktail but re-forms unless the cation is destroyed. The released cations alkylate Trp, Met, Cys and Tyr unless scavengers catch them first: water captures tert-butyl cations, triisopropylsilane (TIS) transfers a hydride to the trityl cation irreversibly, and 1,2-ethanedithiol protects Cys and Met. Allyl-based groups add a third, independent dimension: Alloc on a lysine amine and allyl esters on Asp or Glu survive both piperidine and TFA, and Pd(PPh₃)₄ with phenylsilane as the allyl scavenger removes them under neutral conditions, so a side chain can be modified or a side-chain lactam closed on the resin.

The solid-phase cycle

Chains are built from the C-terminus toward the N-terminus. Each new residue arrives as an Nα-urethane-protected (Fmoc or Boc) amino acid whose carboxyl is activated, and urethane protection keeps that activation free of racemization. One cycle is: remove Fmoc, wash, couple with an excess (typically 3–5 equiv) of activated amino acid, wash, then test. In the Kaiser (ninhydrin) test, deep blue beads mean free primary amines remain, so the coupling is incomplete; recouple, or cap with acetic anhydride. N-terminal proline gives only a red-brown colour. Errors multiply: if every coupling has yield y, the fraction of full-length chains after n couplings is yⁿ, so 29 couplings at 99.0% give 0.99²⁹ ≈ 0.747. Resin loading comes from the Fmoc test: moles released = A × V ÷ (ε × path length). It can also be estimated from the mass gain: on 2-chlorotrityl chloride resin each attached amino acid adds its own molar mass minus that of the HCl released, and the loading is quoted per gram of the loaded resin. On Wang resin the peptide is an ester of a 4-alkoxybenzyl alcohol, modelled here by a 4-methoxybenzyl ester. TFA cleaves it because the para-oxygen stabilises the benzylic cation. At the dipeptide stage, the newly freed amine can attack the C-terminal ester and release a cyclic diketopiperazine from the resin. This happens most with Pro or Gly in the first two positions. Bulky 2-chlorotrityl resin, or adding the first two residues as one dipeptide unit, suppresses it.

Racemization and aspartimide formation

When the carboxyl of an N-acyl amino acid or peptide is activated, the neighbouring amide oxygen can attack it and close a 5(4H)-oxazolone, shown here for acetyl-alanine. Losing the C4–H gives an aromatic oxazol-5-olate, so even a tertiary amine removes it. Reprotonation happens on either face, so the stereocentre is lost, and an amine then opens the racemic oxazolone to give an epimeric peptide mixture. Carbamate (urethane) protecting groups make oxazolones form slowly, and the 2-alkoxy-oxazolones that do form enolize less. This is why stepwise couplings of Fmoc amino acids keep their configuration, while couplings of peptide segments are at risk unless the activated residue is Gly or Pro. Aspartimide formation is the main base-driven side reaction. Piperidine deprotonates the backbone N–H of the residue after Asp, and that nitrogen attacks the side-chain ester to close a succinimide. Asp-Gly is the worst sequence. The aspartimide is 18 Da lighter than the target peptide, epimerizes easily, and reopens at either carbonyl. Water gives a mixture of α- and β-aspartyl (isoaspartyl) peptides of identical mass, and piperidine gives α- and β-piperidides (+67 Da).

Native chemical ligation

Two unprotected peptides can be joined in water at pH ≈ 7 (with 6 M guanidine·HCl, phosphate buffer and TCEP to keep the thiols reduced) when one ends in a C-terminal thioester and the other begins with cysteine. The cysteine thiolate attacks the thioester (transthioesterification). This exchange is reversible, and any cysteine can take part. Only an N-terminal cysteine has its α-amine positioned to attack the new thioester through a five-membered ring, and this S→N acyl shift irreversibly gives a native amide bond at the junction. With a thiol pKa ≈ 8.3, only 1/(1 + 10^(8.3 − 7.0)) ≈ 4.8% of the cysteine is thiolate at pH 7.0, yet that is enough. The aryl thiol MPAA (pKa ≈ 6.6) first converts slow alkyl thioesters into more reactive aryl thioesters. Lysine ε-amines (pKa ≈ 10.5) are almost fully protonated and have no intramolecular delivery, so they are not acylated. To place alanine at the junction, ligate at cysteine and then desulfurize Cys to Ala, either by a radical method (TCEP, the initiator VA-044 and a thiol) or with Raney nickel. Any other cysteines must be protected, for example as Cys(Acm).

What to carry forward

Every peptide bond is an acyl substitution on an activated carboxyl. Choose the activation (carbodiimide plus HOBt, HOAt or Oxyma, or a uronium salt) that is fast but avoids oxazolone formation. Pair a base-labile Nα group with acid-labile side-chain groups, and scavenge every carbocation released at cleavage. Watch for the predictable side reactions: N-acylurea, guanidinylation, diketopiperazine and aspartimide. For long chains, join unprotected segments by native chemical ligation through an N-terminal cysteine.

Problemas de prática (40)

Praticar é grátis com uma conta.

Desenhe o mecanismo 18

Racemization, side reactions and ligation

  • Piperidine turns Asp(OtBu)-Gly into an aspartimideDraw how the backbone nitrogen of the residue after aspartate attacks the side-chain tert-butyl ester under piperidine treatment, closing the five-membered aspartimide ring.
  • Piperidine opens an Asp-Gly aspartimide to the β-piperidideDraw how piperidine opens an aspartimide at the side-chain carbonyl to give the piperidide by-product with the normal α-peptide backbone.
  • Native chemical ligation, part 2: the S→N acyl shift forms the native amideDraw the intramolecular S→N acyl shift through a five-membered cyclic tetrahedral intermediate that turns the cysteine thioester into the native peptide bond Ac-Ala-Cys-Gly-NH₂.
  • Native chemical ligation, part 1: the cysteine thiolate captures the thioesterDraw the transthioesterification in which an N-terminal cysteine thiolate exchanges with a peptide thioester to give the S-acyl cysteine intermediate.
  • Glycine methyl ester opens 2,4-dimethyl-5(4H)-oxazoloneDraw how an amine opens a 5(4H)-oxazolone at the C5 carbonyl to give the dipeptide, and explain why any racemization of the oxazolone is carried into the peptide.
  • Ac-Ala activated with DIC closes to a 5(4H)-oxazoloneDraw how the amide oxygen of an activated N-acetylalanine attacks its own activated carbonyl to form the 2,4-dimethyl-5(4H)-oxazolone.
  • Triethylamine racemizes (4S)-2,4-dimethyl-5(4H)-oxazoloneDraw how a tertiary amine removes the C4–H of an oxazolone to form an aromatic enolate, and how reprotonation from the other face inverts the stereocentre.

Protecting groups and deprotection

  • TFA removes trityl from Ac-Cys(Trt)-NH₂ and triisopropylsilane destroys the trityl cationDraw the acid cleavage of an S-trityl cysteine and the hydride transfer from triisopropylsilane that converts the trityl cation into triphenylmethane.
  • Piperidine traps dibenzofulvene as 1-(9H-fluoren-9-ylmethyl)piperidineDraw the conjugate addition of piperidine to dibenzofulvene and the proton transfers that give the stable fulvene–piperidine adduct.
  • Fmoc-Cl protects L-alanine under Schotten–Baumann conditionsDraw how the amine of alaninate attacks 9-fluorenylmethyl chloroformate, how chloride leaves, and how carbonate removes the last proton to give Fmoc-alaninate.
  • Piperidine removes the Fmoc group from Fmoc-Gly-OMe by E1cBDraw the E1cB removal of an Fmoc group by piperidine: deprotonation at C9, loss of the carbamate to give dibenzofulvene, and decarboxylation that frees the amine.
  • TFA cleaves the tert-butyl ether of Ac-Ser(tBu)-NH₂, and water scavenges the cationDraw the acid-catalysed cleavage of a serine tert-butyl ether through the tert-butyl cation, and its capture by water in the cleavage cocktail.

Activating the carboxyl group

  • A second Boc-alanine turns the O-acylisourea into the symmetric anhydrideDraw how a second molecule of Boc-alanine attacks the O-acylisourea from DIC to give Boc-alanine anhydride and N,N′-diisopropylurea.
  • HOBt converts the Boc-Ala O-acylisourea into the OBt active esterDraw how 1-hydroxybenzotriazole intercepts the O-acylisourea of Boc-alanine to give the OBt active ester and N,N′-diisopropylurea.
  • Mixed-anhydride coupling of Boc-Ala-OH and H-Gly-OMe with isobutyl chloroformateDraw how isobutyl chloroformate and N-methylmorpholine activate Boc-alanine as a mixed carbonic anhydride, and how glycine methyl ester then forms the peptide bond with release of CO₂ and isobutanol.
  • The O-acylisourea from Boc-Ala-OH and DIC rearranges to an N-acylureaDraw the intramolecular O→N acyl shift that turns the O-acylisourea of Boc-alanine into the unreactive N-acylurea.

The solid-phase cycle

  • H-Ala-Pro-OMe cyclizes to the diketopiperazine cyclo(Ala-Pro)Draw how the free N-terminal amine of a resin-bound dipeptide attacks its own C-terminal ester, releasing the cyclic dipeptide (2,5-diketopiperazine) from the support.
  • TFA releases Ac-Gly-OH from a 4-methoxybenzyl (Wang-type) esterDraw the acid cleavage of a 4-alkoxybenzyl ester through the resonance-stabilised benzylic cation, as happens when a peptide is cleaved from Wang resin.

Preveja o produto 3

Racemization, side reactions and ligation

  • Find the β-aspartyl peptidePredict the connectivity of the β-aspartyl (isoaspartyl) peptide formed when an aspartimide is hydrolysed.

Activating the carboxyl group

  • The first activated intermediate with DICIdentify the O-acylisourea formed when a protected amino acid reacts with a carbodiimide.

Protecting groups and deprotection

  • Which survives piperidine?Predict which fluorenylmethyl- or tert-butyl-protected glycine derivative survives Fmoc-removal conditions.

Classificar 1

  • Remove only the Nα groupClassify deprotection reagents by which Fmoc/tBu protecting groups they remove.

Comparar 3

Protecting groups and deprotection

  • Rank Lys protecting groups by acid labilityRank amine protecting groups by acid lability and relate the order to the stability of the carbocation released.

Activating the carboxyl group

  • Rank Boc-glycine esters by aminolysis rateRank Boc-glycine esters by their rate of aminolysis from the pKa of the alcohol or phenol each releases.
  • Rank active-ester leaving groupsRank active-ester leaving groups from the pKa of their conjugate acids.

Calcular 4

The solid-phase cycle

  • Loading of 2-chlorotrityl resin from the mass gainCalculate the loading of an amino acid on 2-chlorotrityl chloride resin from the mass gain, accounting for the HCl lost and the mass of the loaded resin.
  • Resin loading from the Fmoc testCalculate resin loading in mmol/g from the absorbance of the dibenzofulvene–piperidine adduct.
  • How many chains are full length?Calculate the fraction of full-length chains from the per-coupling yield and the number of couplings.

Racemization, side reactions and ligation

  • Mass of a piperidide by-productCalculate the mass shift of an aspartimide-derived piperidide relative to the target peptide.

Encontre o erro 2

Activating the carboxyl group

  • Why did the valine coupling fail?Diagnose a failed carbodiimide coupling from the mass and inertness of the species that builds up during additive-free preactivation.
  • A capped chain after a HATU couplingDiagnose N-terminal guanidinylation caused by exposing a free amine to HATU before the acid is activated.

Interprete as evidências 1

  • Blue beads after couplingInterpret a positive Kaiser test after a coupling step and choose the next action.

Trabalhe de trás para frente 1

  • Which acid gave this oxazolone?Work backward from a 5(4H)-oxazolone to the N-acyl amino acid that formed it.

Planeje uma síntese 4

The solid-phase cycle

  • Choose the cleavage cocktailChoose a TFA cleavage cocktail whose scavengers protect Trp, Met and Cys from carbocation side reactions.
  • Prevent diketopiperazine lossPlan a change that prevents diketopiperazine loss at the dipeptide stage.

Activating the carboxyl group

  • Choose the coupling reagents for a routine SPPS stepChoose a carbodiimide and additive for routine on-resin Fmoc couplings from by-product solubility and side-reaction control.

Protecting groups and deprotection

  • Protecting groups for an on-resin side-chain lactamPlan a third orthogonal protecting-group pair that lets a Lys–Glu side-chain lactam be closed on the resin during Fmoc/tBu synthesis.

Explique por quê 3

The solid-phase cycle

  • Why build from the C-terminus?Explain why chemical peptide synthesis proceeds from the C-terminus to the N-terminus.

Racemization, side reactions and ligation

  • Why segments racemize but Fmoc amino acids do notExplain why urethane-protected amino acids couple without racemization while N-acyl peptide acids racemize.

Activating the carboxyl group

  • Why the acid must be activatedExplain why a carboxylic acid and an amine form a salt rather than an amide at room temperature.

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