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Biologia Química · Probes and bioconjugation

Sondas químicas e inibidores reversíveis

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O que você vai aprender

  • Explain how the spirolactone ⇌ open-form equilibrium of fluorescein and rhodamine dyes switches colour and fluorescence, and use it to design turn-on probes.
  • Calculate brightness, Stokes shifts and FRET efficiencies or distances, and choose donor–acceptor pairs from their spectra.
  • Draw the reactions that make and attach probes: phenol acylation, sulfonamide and amide formation, SNAr labelling with NBD-Cl, carbonyldiimidazole activation and boronate oxidation.
  • Choose affinity tags and photoaffinity groups for pull-down and target-identification experiments, and read the controls that separate specific from background binding.
  • Calculate rates, Ki and IC50 values for reversible inhibitors with the Michaelis–Menten and Cheng–Prusoff equations, and explain why transition-state analogues bind tightly.

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Fluorescein and rhodamine: a ring that switches the dye on and off

Fluorescein and the rhodamines are xanthene dyes with a 2-carboxyphenyl group on the central carbon (C9). That carboxyl can close onto C9 to give a spirolactone. In the lactone (left) C9 is sp³, so the two halves of the xanthene are no longer conjugated: the lactone is colourless and does not fluoresce. In the open form (right) C9 is sp², the xanthene is one planar conjugated chromophore, and the carboxylate hangs off it twisted out of plane. In base a phenoxide oxygen pushes electrons through the ring and breaks the C9–O bond of the lactone. Fluorescein has pKa values of about 4.3 and 6.4; above about pH 8 it is the open dianion, which absorbs near 490 nm (ε ≈ 77 000 M⁻¹ cm⁻¹) and emits near 515 nm with a quantum yield of about 0.93. Below its pKa of 6.4 it loses much of its fluorescence, which makes it a pH indicator but a poor label for acidic organelles. In rhodamines amino groups replace the oxygens: in water the open zwitterion dominates, while non-polar solvents favour the colourless lactone, and acid protonates the carboxylate so the ring cannot close. Acetylating the two phenols of fluorescein locks it as the lactone (fluorescein diacetate), which is neutral, crosses membranes and stays dark until cellular esterases remove the acetyl groups.

Brightness, Stokes shift and FRET

Brightness is the product of the molar absorption coefficient and the fluorescence quantum yield, ε × Φ: a dye that absorbs strongly but emits poorly can be dimmer than one that absorbs less. The Stokes shift is the gap between the absorption and emission maxima; compare dyes in wavenumbers, 10⁷/λabs − 10⁷/λem (λ in nm, result in cm⁻¹), because energy is proportional to wavenumber, not to wavelength. Förster resonance energy transfer (FRET) moves excitation from a donor to a nearby acceptor without emission of a photon. It needs the donor's emission spectrum to overlap the acceptor's absorption spectrum, suitable dipole orientation and a donor–acceptor distance r of roughly 1–10 nm. The efficiency is E = 1/(1 + (r/R₀)⁶), where the Förster distance R₀ (typically 2–8 nm) is the distance at which E = 0.5. The sixth power makes E change steeply near R₀, so FRET reports binding, folding and cleavage events as a spectroscopic ruler. Rearranged, r = R₀ × (1/E − 1)^(1/6).

Turn-on probes and labelling reagents

A turn-on (fluorogenic) probe is dark until a reaction unmasks the fluorophore. Esters of phenolic dyes, such as fluorescein diacetate or 4-methylumbelliferyl acetate, report esterase activity. 7-Hydroxycoumarins fluoresce most strongly as their phenoxides (pKa ≈ 7.8), so end-point assays are stopped with a pH-10 buffer. Aryl boronic acids, such as coumarin-7-boronic acid (left), report hydrogen peroxide. Hydroperoxide adds to boron, the aryl group migrates from boron to oxygen, and hydrolysis releases the phenol, here the anion of 7-hydroxycoumarin (right). Peroxynitrite does the same reaction about a million times faster. Labelling reagents attach a fluorophore to a protein. Dansyl chloride sulfonylates amines; its dimethylaminonaphthalene sulfonamide emits more strongly and at shorter wavelength in non-polar surroundings, so it reports binding sites. NBD-Cl (4-chloro-7-nitrobenzofurazan) undergoes SNAr with amines through a Meisenheimer complex, giving strongly fluorescent push–pull NBD-amines, whereas its thiol adducts are only weakly fluorescent. Amine labelling is done at pH 8.5–9.5 so that some of each lysine ε-amine (pKa ≈ 10.5) is unprotonated, while hydrolysis of the reagent stays slow. Fluorescein itself is made from phthalic anhydride and two equivalents of resorcinol in acid. A Friedel–Crafts acylation gives 2-(2,4-dihydroxybenzoyl)benzoic acid, whose ketone then condenses with the second resorcinol and closes the xanthene ring.

Affinity tags

Biotin binds streptavidin with Kd ≈ 10⁻¹⁴ M, so ΔG° = RT ln Kd ≈ −80 kJ/mol at 25 °C, one of the strongest non-covalent interactions known. Streptavidin is a tetramer with four biotin sites. Biotinylated proteins are captured almost irreversibly, so recovering them needs boiling in SDS or digestion on the beads. Desthiobiotin, which lacks the sulfur ring, binds about 1000 times more weakly (Kd ≈ 10⁻¹¹ M) and is displaced by an excess of free biotin under mild conditions. Every cell contains a few biotinylated carboxylases, such as acetyl-CoA carboxylase and pyruvate carboxylase, so these appear as background bands in every streptavidin pull-down. Biotin is attached through its valerate carboxyl. Carbonyldiimidazole (CDI) converts the acid into an acyl imidazole that acylates amines, for example propargylamine, giving a biotin–alkyne. The His₆ tag binds Ni²⁺–nitrilotriacetic acid (Ni–NTA) through its imidazole nitrogens with roughly micromolar affinity. It is eluted with about 250 mM imidazole, which competes for the nickel sites, or with EDTA or low pH.

Photoaffinity labelling and chemical genetics

A photoaffinity probe is a ligand carrying a photoreactive group and a small handle, usually a terminal alkyne. The probe binds its target, UV light turns the photoreactive group into a reactive species that bonds covalently to the nearest protein, and after lysis a biotin or dye azide is clicked onto the alkyne for enrichment or imaging. The copper(I)-catalysed azide–alkyne cycloaddition (CuAAC) gives only the 1,4-disubstituted 1,2,3-triazole; it is run in lysate rather than in living cells, and the small alkyne keeps the probe close in size and permeability to the parent ligand. Aryl trifluoromethyl diazirines (shown) lose N₂ at about 350 nm to give carbenes that insert into C–H and O–H bonds within nanoseconds; many are quenched by water, so yields are low but labelling is close to the binding site. Benzophenones are excited at 350–365 nm to a triplet n,π* state that abstracts a hydrogen atom from a nearby C–H bond; if none is in reach, the excited state relaxes and can be excited again, so yields are higher, but the group is bulky. Simple aryl azides need light below 300 nm, which damages proteins, and their nitrenes ring-expand to long-lived ketenimines that label nucleophiles away from the site. A protein whose labelling needs light and falls when an excess of the unmodified ligand is added is a specific target; one labelled equally in both experiments binds non-specifically, and bands seen without probe or light are background. Chemical genetics uses small molecules as switchable mutations. Forward chemical genetics screens for a phenotype first and then identifies the target; reverse chemical genetics starts from a chosen protein. In the bump-and-hole strategy a kinase's bulky gatekeeper residue is mutated to glycine or alanine, and a bulky inhibitor such as 1-NM-PP1 then blocks only that analogue-sensitive kinase.

Reversible inhibitors, Ki and transition-state analogues

A competitive inhibitor binds the free enzyme at the active site: v = Vmax[S]/(Km(1 + [I]/Ki) + [S]), so the apparent Km rises by the factor 1 + [I]/Ki while Vmax is unchanged. Its IC50 depends on substrate concentration through the Cheng–Prusoff relation, IC50 = Ki(1 + [S]/Km). For a pure non-competitive inhibitor IC50 = Ki, and for an uncompetitive one, which binds only the enzyme–substrate complex and lowers both Vmax and Km, IC50 = Ki(1 + Km/[S]). On a Lineweaver–Burk plot (1/v against 1/[S]) competitive inhibitors give lines meeting on the 1/v axis, pure non-competitive inhibitors lines meeting on the 1/[S] axis, and uncompetitive inhibitors parallel lines. ATP-competitive kinase inhibitors therefore look much weaker in cells, where ATP is 1–5 mM, than in assays run at [ATP] ≈ Km. Reversible inhibition is recognised because activity returns when the enzyme–inhibitor complex is diluted or dialysed. Enzymes bind the transition state far more tightly than the substrate: K_TS ≈ Km × k_uncat/k_cat. Stable molecules that copy its geometry and charge are therefore potent inhibitors. A trifluoromethyl ketone (shown, on N-acetylphenylalanine) is attacked by the active-site serine of chymotrypsin to give a tetrahedral hemiketal oxyanion that cannot collapse, because CF₃⁻ is no leaving group. Boronic acids take the serine oxygen into boron's empty p orbital, giving a tetrahedral boronate anion. In pepstatin, the statine CH(OH) group mimics the tetrahedral gem-diol intermediate of aspartic proteases such as pepsin. All these adducts mimic the tetrahedral intermediate and release the enzyme on dilution.

What to carry forward

Treat a probe as a switch plus a handle. The switch is a reaction or an equilibrium, such as spirolactone opening, ester or boronate unmasking, or SNAr onto NBD, that changes conjugation and therefore colour and fluorescence. The handle is a tag, such as biotin, His₆ or an alkyne, or a photoreactive group that lets you capture or image the target. For inhibitors, separate the intrinsic constant Ki from the assay-dependent IC50, and remember that the tightest reversible inhibitors copy the transition state.

Problemas de prática (40)

Praticar é grátis com uma conta.

Desenhe o mecanismo 12

Affinity tags and target identification

  • Biotinyl imidazole acylates propargylamine to give a biotin–alkyneDraw the aminolysis of biotinyl imidazole by propargylamine through a tetrahedral intermediate, giving the biotin–alkyne amide and imidazole.
  • Carbonyldiimidazole activates biotin as its acyl imidazoleDraw how carbonyldiimidazole converts the carboxylic acid of biotin into an acyl imidazole, with loss of CO₂ and imidazole, through an acyl imidazole-1-carboxylate mixed anhydride.

Turn-on probes, labels and FRET

  • Hydrogen peroxide turns coumarin-7-boronic acid into fluorescent 7-hydroxycoumarinDraw the oxidation of an aryl boronic acid probe by hydroperoxide: addition to boron, 1,2-aryl migration from boron to oxygen with loss of hydroxide, and hydrolysis of the aryl borate that releases the fluorescent phenoxide.
  • Dansyl chloride labels the amino group of glycineDraw the sulfonylation of glycinate by dansyl chloride and the deprotonation that gives the fluorescent dansyl sulfonamide.
  • An esterase releases fluorescent 4-methylumbelliferone from its acetateDraw the acylation of an esterase active-site serine by 4-methylumbelliferyl acetate, which releases the fluorescent 4-methylumbelliferone.
  • NBD-Cl labels an amine by nucleophilic aromatic substitutionDraw the addition–elimination (SNAr) reaction of NBD-Cl with butylamine through a Meisenheimer complex stabilised by the nitro group, giving the fluorescent NBD-amine.

Reversible inhibitors and kinetics

  • A boronic acid inhibitor forms a tetrahedral boronate with the active-site serineDraw how the active-site serine adds to the empty p orbital of a boronic acid and how histidine then removes its proton, giving a tetrahedral boronate that mimics the tetrahedral intermediate of acyl transfer.
  • A trifluoromethyl ketone traps the active-site serine of chymotrypsin as a hemiketalDraw how the active-site serine, activated by histidine, adds to a trifluoromethyl ketone to give a tetrahedral hemiketal oxyanion that mimics the tetrahedral intermediate of peptide hydrolysis.

Fluorophores and spirolactone switches

  • Friedel–Crafts acylation of resorcinol by phthalic anhydride, the first stage of the fluorescein synthesisDraw how methanesulfonic acid opens phthalic anhydride to an acylium ion and how resorcinol is acylated para to one hydroxyl group, giving 2-(2,4-dihydroxybenzoyl)benzoic acid.
  • Acetylation of fluorescein to the cell-permeant diacetateDraw the acetylation of both phenol groups of fluorescein by acetic anhydride in pyridine, giving fluorescein diacetate locked in the spirolactone form.
  • Fluorescein opens from the colourless spirolactone to the fluorescent dianionDraw how base converts the colourless fluorescein spirolactone into the open, fluorescent dianion: deprotonation of a phenol, a push from the phenoxide that breaks the C9–O lactone bond, and deprotonation of the second phenol.
  • Tetramethylrhodamine closes to its colourless spirolactone in a non-polar solventDraw the intramolecular addition of the carboxylate of tetramethylrhodamine to C9 of the xanthene that converts the coloured zwitterion into the colourless spirolactone.

Preveja o produto 2

Affinity tags and target identification

  • Product of a copper-catalysed click reactionPredict the regiochemistry of the triazole formed in a copper(I)-catalysed azide–alkyne cycloaddition.

Turn-on probes, labels and FRET

  • Which NBD product glows?Predict which product of NBD-Cl labelling is strongly fluorescent.

Classificar 4

Affinity tags and target identification

  • What a diazirine makes in the lightIdentify the reactive intermediate formed when an aryl trifluoromethyl diazirine is irradiated.

Fluorophores and spirolactone switches

  • Fluorescein at pH 9Identify the predominant form of fluorescein in mildly basic buffer from its pKa values.

Turn-on probes, labels and FRET

  • What a FRET acceptor needsIdentify the spectral requirement for a FRET donor–acceptor pair.

Reversible inhibitors and kinetics

  • Parallel double-reciprocal linesClassify an inhibitor that gives parallel Lineweaver–Burk lines and state which enzyme form it binds.

Comparar 2

Fluorophores and spirolactone switches

  • Rank fluorophores by brightnessRank fluorophores by brightness using the product of absorption coefficient and quantum yield.

Reversible inhibitors and kinetics

  • Rank measured IC50 valuesRank inhibitors by the IC50 measured at high substrate from their Ki values and mechanisms.

Calcular 9

Reversible inhibitors and kinetics

  • Apparent Km with a competitive inhibitorCalculate the apparent Michaelis constant in the presence of a competitive inhibitor.
  • Ki of a competitive inhibitor from its IC50Convert the IC50 of a competitive inhibitor into Ki with the Cheng–Prusoff equation.
  • Remaining activity with a competitive inhibitorCalculate the fraction of activity left when a competitive inhibitor is present at a multiple of its Ki and the substrate is at Km.
  • IC50 of an uncompetitive inhibitorCalculate the IC50 of an uncompetitive inhibitor from Ki, Km and the substrate concentration.

Affinity tags and target identification

  • Binding free energy of biotin and streptavidinConvert a dissociation constant into a standard binding free energy.

Fluorophores and spirolactone switches

  • How much fluorescein is the bright dianion at pH 6.0?Calculate the fraction of fluorescein present as its fluorescent dianion at a pH near its pKa.
  • Stokes shift of fluorescein in wavenumbersConvert absorption and emission maxima into a Stokes shift in wavenumbers.

Turn-on probes, labels and FRET

  • Distance from donor quenchingCalculate a donor–acceptor distance from the quenching of donor fluorescence and R₀.
  • FRET efficiency at a known distanceCalculate a FRET efficiency from the donor–acceptor distance and the Förster distance.

Interprete as evidências 3

Turn-on probes, labels and FRET

  • A dansyl ligand binds its proteinInterpret a blue shift and intensity increase in dansyl emission as binding in a non-polar site.

Reversible inhibitors and kinetics

  • Reading a Lineweaver–Burk plotIdentify competitive inhibition from a family of double-reciprocal lines.

Affinity tags and target identification

  • Which band is the real target?Interpret no-UV and competition controls to identify the specific target of a photoaffinity probe.

Trabalhe de trás para frente 1

  • Building blocks of fluoresceinWork backwards from the fluorescein skeleton to its two classical starting materials.

Planeje uma síntese 3

Affinity tags and target identification

  • Why tag a photoaffinity probe with an alkyne, not biotinPlan a two-step labelling route that adds the biotin reporter after photo-crosslinking in live cells.
  • Recovering a native protein from streptavidin beadsChoose an affinity-tag and elution combination that releases a captured protein from streptavidin under mild conditions.

Turn-on probes, labels and FRET

  • Choose a probe for esterase activity in live cellsChoose the fluorescein derivative that reports intracellular esterase activity as a turn-on signal.

Explique por quê 4

Affinity tags and target identification

  • Why benzophenone probes tolerate long irradiationExplain why benzophenone photoaffinity groups are not used up by water and can be excited repeatedly.
  • Eluting a His-tagged proteinExplain how imidazole releases a His₆-tagged protein from a nickel–NTA resin.

Fluorophores and spirolactone switches

  • Why silicon rhodamine probes light up on their targetExplain how a shifted spirolactone equilibrium makes silicon rhodamine probes fluorogenic.

Reversible inhibitors and kinetics

  • Why pepstatin binds pepsin so tightlyExplain how a hydroxyl-bearing sp³ carbon in a peptide mimics the tetrahedral intermediate of aspartic protease catalysis.

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