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Biologia Química · Click chemistry and probes

Reações click e de ligação

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O que você vai aprender

  • Judge reactions against the click criteria and predict the regiochemistry of thermal, copper-catalysed and ruthenium-catalysed azide–alkyne cycloadditions.
  • Explain strain-promoted azide–cyclooctyne cycloaddition and draw the tetrazine inverse-electron-demand Diels–Alder reaction with loss of N₂.
  • Draw oxime and hydrazone formation and hydrolysis, explain the pH-rate maximum, aniline catalysis and the greater stability of oximes, and follow the Staudinger ligation.
  • Draw thiol–ene radical chains, thiol–maleimide Michael and retro-Michael reactions, active-ester and squarate aminolysis, and explain SuFEx and isothiocyanate couplings.
  • Calculate half-lives, conversions and required concentrations from second-order rate constants, stating when the pseudo-first-order approximation holds.

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What makes a reaction a click reaction

Sharpless defined click reactions as modular, wide in scope and very high yielding. They give only inoffensive by-products that can be removed without chromatography, run under simple conditions that tolerate oxygen and water, and use readily available starting materials. A large thermodynamic driving force (often more than 80 kJ/mol) makes them fast and selective for one product. The two partners must also be orthogonal: they react with each other but ignore the alcohols, amines, carboxylic acids and other functional groups around them. Copper-catalysed azide–alkyne cycloaddition, oxime ligation and thiol–maleimide addition qualify. A Wittig olefination with a non-stabilised ylide does not: it needs strong base under dry conditions, gives E/Z mixtures and leaves stoichiometric Ph₃P=O that usually has to be removed by chromatography.

Azide–alkyne cycloadditions: heat, copper or ruthenium

An organic azide is a 1,3-dipole, and with an alkyne it gives an aromatic 1,2,3-triazole. The uncatalysed Huisgen cycloaddition is concerted but slow; it usually needs 80–120 °C. A terminal alkyne then gives the 1,4- and 1,5-disubstituted triazoles in comparable amounts. Both HOMO–LUMO pairings (azide HOMO with alkyne LUMO and the reverse) contribute, and the orbital coefficients at the two alkyne carbons differ too little to favour one orientation. Copper(I) catalysis (CuAAC) is about 10⁷ times faster and gives only the 1,4-isomer, shown for benzyl azide and phenylacetylene. Copper first forms a σ-acetylide from the terminal C–H, so internal alkynes do not react. The azide then binds copper, and its terminal nitrogen bonds to the β-carbon of the acetylide; this stepwise sequence passes through a copper triazolide, and protonation at C5 releases the triazole. CuSO₄ with sodium ascorbate is the usual catalyst source: ascorbate reduces Cu(II) to Cu(I) and removes dissolved O₂, which would otherwise reoxidise the copper and cause oxidative alkyne homocoupling. Tris(triazolylmethyl)amine ligands such as TBTA and THPTA hold Cu(I) in water, protect it from oxidation and disproportionation, and speed the reaction. Cp*RuCl(PPh₃)₂ (RuAAC) works through a ruthenacycle instead, gives the 1,5-isomer and also accepts internal alkynes.

Strain-promoted cycloaddition

Cyclooctyne is the smallest isolable cycloalkyne. Its C–C≡C angles are bent to about 160°, and the ring holds about 18 kcal/mol (75 kJ/mol) of strain. Because the alkyne is already distorted toward the bent geometry of the transition state, the strain-promoted azide–alkyne cycloaddition (SPAAC) runs at 25 °C without any metal. Second-order rate constants with benzyl azide are about 0.0024 M⁻¹ s⁻¹ for cyclooctyne itself, about 0.08 M⁻¹ s⁻¹ for difluorinated cyclooctynes (the propargylic fluorines lower the alkyne LUMO) and about 0.3 M⁻¹ s⁻¹ for dibenzo-fused azacyclooctynes such as the N-acyl DBCO drawn here. An unstrained alkyne such as hex-3-yne gives no measurable reaction at 25 °C. Unsymmetrical cyclooctynes give regioisomer mixtures, because no metal directs the orientation.

Tetrazine ligation

A 1,2,4,5-tetrazine is a very electron-poor azadiene with a low-lying LUMO. It reacts with strained, electron-rich alkenes such as trans-cyclooctene (TCO) or norbornene by an inverse-electron-demand Diels–Alder (IEDDA) reaction, in which the dienophile HOMO and the diene LUMO interact. Electron-withdrawing substituents on the tetrazine (esters, 2-pyridyl groups) speed the reaction, and donors (alkoxy, amino) slow it. Norbornene, drawn here with 3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine, reacts on its exo face. The bicyclic adduct at once loses N₂ in a retro-Diels–Alder step, which makes the reaction irreversible. An alkene gives a 4,5-dihydropyridazine, which tautomerises in protic solvents to the 1,4-dihydropyridazine and needs an oxidant such as DDQ to become the aromatic pyridazine. An alkyne gives the pyridazine directly. Rate constants with TCO reach 10³–10⁶ M⁻¹ s⁻¹, the fastest of the common ligations.

Oximes and hydrazones

Aminooxy compounds (R–O–NH₂) and hydrazines condense with aldehydes and ketones through a tetrahedral carbinolamine, which then loses water. The rate peaks near pH 4–5. More acid protonates the nucleophile (methoxyamine has conjugate-acid pKa ≈ 4.6), and less acid slows the acid-catalysed dehydration. Aniline (pKa ≈ 4.6) is a nucleophilic catalyst: it rapidly forms a protonated Schiff base (an iminium ion), which is far more electrophilic than the aldehyde, and the aminooxy compound then displaces aniline by transimination. Equilibrium constants for formation are about 10⁴–10⁶ M⁻¹ for hydrazones and more than 10⁸ M⁻¹ for oximes. Oximes also hydrolyse far more slowly: the electronegative oxygen makes the C=N nitrogen less basic, so the N-protonation that starts hydrolysis is less favourable. The (E)-O-methyl oxime of benzaldehyde is shown.

Staudinger ligation

Triphenylphosphine adds to the terminal nitrogen of an azide to give a phosphazide, which loses N₂ to form an iminophosphorane (aza-ylide, R₃P⁺–N⁻R). Water hydrolyses it to an amine and a phosphine oxide (Staudinger reduction). In the Staudinger ligation, the phosphine carries an ortho methyl ester. The aza-ylide nitrogen attacks that ester through a five-membered ring faster than water can hydrolyse it, methoxide leaves, and hydrolysis of the resulting amidophosphonium ion gives an amide that still carries the phosphine oxide, as shown. In the traceless version, a phosphinothioester such as Ph₂PCH₂S–C(O)R transfers the acyl group to nitrogen, and hydrolysis releases a plain amide R–C(O)NHR′ and the phosphine oxide as a separate molecule. The reaction is slow (k ≈ 2 × 10⁻³ M⁻¹ s⁻¹), and the phosphine is slowly oxidised by air.

Thiols, amines and sulfur(VI) fluorides

Thiols add to alkenes by two mechanisms. With unactivated alkenes such as allyl ethers, a radical initiator generates a thiyl radical. It adds to the CH₂ end of the alkene to give the more substituted carbon radical, which takes a hydrogen atom from the next thiol, so the anti-Markovnikov thioether forms in a chain. With electron-poor alkenes such as maleimides and acrylates, a thiolate adds in a Michael reaction. Near pH 7, maleimides react with thiols about 10³ times faster than with amines, and the 3-thiosuccinimide shown forms. That adduct can slowly undergo retro-Michael elimination (E1cB) and exchange its thiol, but once the succinimide ring is hydrolysed the exchange stops. Amines are acylated by N-hydroxysuccinimide (NHS, pKa ≈ 6.0) and pentafluorophenyl (pKa ≈ 5.5) esters. They add to isothiocyanates to give thioureas and to dialkyl squarates by addition–elimination; the first squaramide forms readily and the second needs harsher conditions. In SuFEx, a sulfonyl fluoride resists water and reduction but exchanges fluoride for an aryloxide when it meets an aryl silyl ether and a base such as DBU, because the strong Si–F bond forms.

Rates at low concentration

Ligations are often run at micromolar concentrations, so the second-order rate constant k decides what is practical. If one partner B is in large excess (ten-fold or more), [B] stays nearly constant, and the reaction is pseudo-first-order in the limiting partner, with k′ = k[B], t½ = ln 2/(k[B]) and fraction converted 1 − e^(−k′t). If both partners start at the same concentration c₀, t½ = 1/(k c₀). For example, a cyclooctyne with k = 0.30 M⁻¹ s⁻¹, used in excess at 1.0 mM, gives the limiting azide t½ = 0.693/(3.0 × 10⁻⁴ s⁻¹) ≈ 39 min, whereas a tetrazine–TCO pair with k = 2.0 × 10³ M⁻¹ s⁻¹ at 10 µM each needs only 50 s.

What to carry forward

Pick a ligation by what the two partners must ignore and how fast it must be. CuAAC gives the 1,4-triazole and RuAAC the 1,5-triazole; strain lets cyclooctynes react without metal; tetrazines plus strained alkenes are fastest and irreversible because N₂ leaves. Oximes beat hydrazones in equilibrium and hydrolytic stability, and aniline speeds both through an iminium ion. Thiols add to maleimides by Michael addition and to plain alkenes by a radical chain. Active esters, isothiocyanates and squarates acylate amines. Under pseudo-first-order conditions, t½ = ln 2/(k[B]).

Problemas de prática (40)

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Desenhe o mecanismo 12

Oxime, hydrazone and Staudinger ligations

  • Acid-catalysed hydrolysis of an acylhydrazoneDraw the acid-catalysed hydrolysis of an acylhydrazone through N-protonation, water addition, a carbinolamine and loss of the hydrazide, and explain why the leaving nitrogen must first be protonated.
  • Aniline and benzaldehyde form the Schiff base used in aniline catalysisDraw the acid-catalysed condensation of aniline with benzaldehyde through a carbinolamine and an iminium ion to the N-phenyl imine that serves as the activated intermediate in aniline-catalysed oxime ligation.
  • Aniline-catalysed oxime formation by transiminationDraw how methoxyamine displaces aniline from the protonated Schiff base of benzaldehyde to give the (E)-O-methyl oxime and regenerate the aniline catalyst.
  • Staudinger ligation: intramolecular acyl transfer from an ortho esterDraw how the iminophosphorane from a Staudinger reaction attacks an ortho methyl ester, expels methoxide and is hydrolysed to an amide that carries a phosphine oxide.

Click criteria and azide–alkyne cycloadditions

  • Protonolysis of the copper triazolide releases the 1,4-triazoleDraw the final step of the copper-catalysed azide–alkyne cycloaddition, in which triethylammonium protonates the copper(I) triazolide at C5 to release 1-benzyl-4-phenyl-1H-1,2,3-triazole and regenerate Cu(I).

Thiol, amine and SuFEx couplings

  • Retro-Michael release of a thiol from a thiosuccinimideDraw the base-promoted E1cB elimination that returns a 3-thiosuccinimide to the maleimide and thiol, and predict the outcome when another thiol is present in excess.
  • Pentafluorophenyl ester aminolysis installs an alkyne handleDraw the addition–elimination aminolysis of a pentafluorophenyl ester by benzylamine and relate its speed to the pKa of pentafluorophenol.
  • Diethyl squarate and butylamine: first aminolysisDraw the addition–elimination of an amine at a squarate ester carbon and explain why the second ethoxy group reacts much more slowly.
  • Radical thiol–ene coupling of a thiol with allyl alcoholDraw initiation, anti-Markovnikov thiyl-radical addition and hydrogen-atom transfer from a second thiol in the radical thiol–ene coupling of methyl 3-mercaptopropanoate with allyl alcohol.
  • Thiol–Michael addition of phenylmethanethiol to N-ethylmaleimideDraw the base-catalysed conjugate addition of a thiolate to N-ethylmaleimide through an enolate to the 3-thiosuccinimide.

Strain-promoted and tetrazine cycloadditions

  • Strain-promoted cycloaddition of benzyl azide and cyclooctyneDraw the concerted [3+2] cycloaddition of benzyl azide with cyclooctyne and predict the regiochemistry of strain-promoted cycloadditions, which have no metal to direct them.
  • Tetrazine–norbornene ligation: Diels–Alder, then loss of N₂Draw the inverse-electron-demand Diels–Alder reaction of a tetrazine with norbornene on its exo face and the retro-Diels–Alder loss of N₂ that gives a 4,5-dihydropyridazine.

Preveja o produto 6

Click criteria and azide–alkyne cycloadditions

  • Product of a copper-catalysed click reactionPredict the single regioisomer formed in a copper(I)-catalysed azide–alkyne cycloaddition.
  • Benzyl azide and phenylacetylene without a catalystPredict the regiochemical outcome of an uncatalysed azide–terminal alkyne cycloaddition.
  • Benzyl azide and phenylacetylene with a ruthenium catalystPredict the triazole regioisomer formed under ruthenium catalysis and contrast it with copper catalysis.

Thiol, amine and SuFEx couplings

  • Amine plus isothiocyanatePredict the thiourea formed when a primary amine adds to an isothiocyanate.

Strain-promoted and tetrazine cycloadditions

  • Tetrazine plus cyclooctynePredict the product of a tetrazine Diels–Alder reaction with a strained alkyne.

Oxime, hydrazone and Staudinger ligations

  • Traceless Staudinger ligationPredict the product of a traceless Staudinger ligation between a phosphinothioester and an azide.

Classificar 2

Click criteria and azide–alkyne cycloadditions

  • Which reaction is not a click reaction?Apply the click criteria to classify common bond-forming reactions.

Thiol, amine and SuFEx couplings

  • Radical or Michael?Classify alkenes by whether they add thiols through a radical chain or through a Michael addition.

Comparar 3

Strain-promoted and tetrazine cycloadditions

  • Rank alkynes by rate of reaction with benzyl azideRank alkynes by their rate of metal-free cycloaddition with benzyl azide and relate the order to strain and alkyne electronics.
  • Which tetrazine reacts fastest?Predict how tetrazine substituents change the rate of inverse-electron-demand Diels–Alder reactions.

Oxime, hydrazone and Staudinger ligations

  • Rank C=N conjugates by hydrolytic stabilityRank hydrazones, a semicarbazone and an oxime of the same aldehyde by hydrolytic stability from relative rate data and nitrogen basicity.

Calcular 6

Rates, equilibria and half-lives

  • How much methoxyamine is free to react?Calculate the fraction of an aminooxy nucleophile present as the free base at a given pH.
  • How much cyclooctyne for a 10-minute half-life?Work backwards from a target half-life to the reagent concentration needed in a pseudo-first-order ligation.
  • Equilibrium extent of a hydrazone ligationCalculate the equilibrium conversion of an aldehyde to a hydrazone from the formation constant and the excess reagent concentration.
  • Half-life of a strain-promoted ligationCalculate the half-life of a strain-promoted azide–cyclooctyne reaction under pseudo-first-order conditions.
  • How far does a Staudinger ligation get in 6 hours?Calculate the conversion of a slow pseudo-first-order ligation after a given time.
  • Half-life of a tetrazine–TCO ligation at equal concentrationsCalculate the half-life of a second-order ligation when both partners start at the same concentration.

Encontre o erro 1

  • A click reaction that does nothingDiagnose why an internal alkyne fails in CuAAC and choose a catalyst that accepts it.

Interprete as evidências 1

  • Reading a bell-shaped pH–rate profileInterpret the pH–rate maximum of oxime formation in terms of nucleophile protonation and acid-catalysed dehydration.

Planeje uma síntese 1

  • Finishing a tetrazine–alkene ligationChoose a reagent that converts a dihydropyridazine from a tetrazine–alkene ligation into the aromatic pyridazine.

Explique por quê 8

Click criteria and azide–alkyne cycloadditions

  • Why add sodium ascorbate?Explain why sodium ascorbate is added to copper(II) sulfate in CuAAC reactions.
  • What does a TBTA-type ligand do?Explain the role of a tris(triazolylmethyl)amine ligand in copper-catalysed azide–alkyne cycloaddition.
  • Why thermal azide–alkyne cycloaddition is unselectiveExplain with frontier-orbital arguments why the uncatalysed azide–alkyne cycloaddition gives both triazole regioisomers.

Strain-promoted and tetrazine cycloadditions

  • Why strain speeds up the azide reactionExplain how ring strain accelerates the azide–cyclooctyne cycloaddition.
  • Which orbitals control the tetrazine Diels–Alder reaction?Use frontier-orbital arguments to explain which tetrazines and alkenes react fastest in inverse-electron-demand Diels–Alder reactions.

Oxime, hydrazone and Staudinger ligations

  • Why oximes outlast hydrazonesExplain why an oxime is more hydrolytically stable than a hydrazone of the same aldehyde.

Thiol, amine and SuFEx couplings

  • Why sulfonyl fluorides clickExplain the stability and the selective reactivity of sulfonyl fluorides in SuFEx couplings with silyl ethers.
  • Ring opening stops thiol exchangeExplain why hydrolysing a thiosuccinimide ring prevents later thiol exchange.

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