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Biologia Química · Peptides and nucleic acids

Química e síntese de ácidos nucleicos

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O que você vai aprender

  • Name nucleosides and nucleotides, number their sugar atoms, and recognise the β-N-glycosidic bond, the sugar pucker and the 3′→5′ phosphodiester link with its charge.
  • Explain Watson–Crick pairing, the major tautomers of the bases and their pKa values, and calculate charges, hydrogen-bond counts and protonation fractions.
  • Quantify oligonucleotides by UV absorbance at 260 nm with the Beer–Lambert law, and relate hyperchromicity and melting temperature to base stacking and G·C content.
  • Draw the phosphoramidite cycle: DMT removal, tetrazole activation, coupling, capping and iodine oxidation of P(III) to P(V), and calculate yields from coupling efficiency and the trityl assay.
  • Choose protecting groups for the bases, phosphates and RNA 2′-OH, draw their removal, and explain from the 2′-OH transesterification why RNA is far less stable than DNA in base.

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Nucleosides, nucleotides and their numbering

A nucleoside is a base joined to a sugar by a β-N-glycosidic bond from C1′ of the sugar to N9 of a purine (adenine, guanine) or N1 of a pyrimidine (cytosine, thymine, uracil). Sugar atoms carry primes (C1′ to C5′) so they are not confused with the ring atoms of the base. β means the base lies on the same face of the furanose ring as the C4′–CH₂OH group; the α-anomer, with the base on the opposite face, is not found in natural nucleic acids. RNA uses ribose (adenosine, guanosine, cytidine, uridine); DNA uses 2′-deoxyribose (2′-deoxyadenosine, 2′-deoxyguanosine, 2′-deoxycytidine and thymidine). The furanose ring is not flat: it puckers so that C2′ or C3′ lies out of the plane on the same side as C5′ (C2′-endo or C3′-endo). B-form DNA prefers C2′-endo; RNA duplexes are A-form and prefer C3′-endo, because the 2′-OH favours that pucker. A nucleotide is a phosphate ester of a nucleoside, usually at the 5′-OH. In a chain, each phosphodiester joins the 3′-O of one nucleotide to the 5′-O of the next, and sequences are written 5′→3′. The drawing is the dinucleotide d(ApT): 2′-deoxyadenosine on the left, its 3′-O linked through one phosphodiester to the 5′-O of thymidine. The P–OH of a phosphodiester has pKa ≈ 1, so every internal phosphate carries one negative charge at pH 7, and a linear n-mer with free 5′- and 3′-OH ends carries n − 1 charges.

Base pairing, tautomers and ionization

In Watson–Crick pairs, guanine pairs with cytosine through three hydrogen bonds (O6 to H–N4, N1–H to N3, N2–H to O2) and adenine with thymine or uracil through two (N6–H to O4, N1 to H–N3). Double-stranded DNA therefore has %A = %T and %G = %C. The pairing depends on the major tautomers, the keto (lactam) and amino forms. A rare enol tautomer of guanine or imino tautomer of adenine presents the wrong pattern of donors and acceptors and pairs with T or C instead. The bases are almost neutral at pH 7. The conjugate acids have pKa ≈ 4.2 (cytidine N3), ≈ 3.5 (adenosine N1) and ≈ 2 (guanosine N7), and the N–H acids have pKa ≈ 9.2 (guanosine N1–H), 9.3 (uridine N3–H) and 9.8 (thymidine N3–H). Only the phosphates are charged, and the ribose 2′-OH (pKa ≈ 12.5) is ionized only in strong base. The drawing shows 9-methylguanine and 1-methylcytosine, the base pair with the sugars replaced by methyl groups.

Measuring nucleic acids by UV

The aromatic bases absorb strongly near 260 nm, so nucleic acids are quantified with the Beer–Lambert law, A = εcl. For an oligonucleotide, ε₂₆₀ is roughly the sum of its nucleotides' values (on the order of 10⁴ M⁻¹ cm⁻¹ per base), so a 20-mer has ε₂₆₀ near 2 × 10⁵ M⁻¹ cm⁻¹. A diluted sample must be multiplied back by the dilution factor. Stacked bases in a duplex absorb less than the same bases unstacked. When a duplex melts into single strands, A₂₆₀ rises (hyperchromicity, typically 10–40%), and the percentage is (A_melted − A_duplex)/A_duplex × 100. The melting temperature, Tm, is the temperature at which half of the duplex has melted, the midpoint of the rise. Tm increases with G·C content (three hydrogen bonds and stronger stacking), with length, and with salt concentration, because cations screen the repulsion between the two negatively charged backbones.

The phosphoramidite cycle

Oligonucleotides are made from the 3′-end toward the 5′-end on a solid support, with the first nucleoside attached through a 3′-O-succinyl ester. Each building block is a 5′-O-DMT nucleoside 3′-O-(2-cyanoethyl N,N-diisopropylphosphoramidite), drawn here for thymidine; it is made from the nucleoside's 3′-OH and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite with a hindered amine base. One cycle has four steps. Detritylation: 3% dichloroacetic acid (pKa ≈ 1.3) or trichloroacetic acid (pKa ≈ 0.7) in CH₂Cl₂ releases the orange DMT cation (λmax ≈ 498 nm). Coupling: an activator such as 1H-tetrazole (pKa ≈ 4.9) or the more acidic 5-(ethylthio)-1H-tetrazole (ETT, pKa ≈ 4.3) protonates the diisopropylamino nitrogen, and the azolide displaces the amine. The free 5′-OH then attacks the tetrazolyl phosphite to give a phosphite triester. The solvent must be dry, because water turns the activated amidite into an unreactive H-phosphonate. Capping: acetic anhydride with N-methylimidazole forms an N-acetylimidazolium ion that acetylates any 5′-OH that failed to couple, so that chain cannot grow and give a deletion product missing one nucleotide (n − 1). Oxidation: I₂ in water/pyridine converts the acid-labile P(III) phosphite triester into a stable P(V) phosphotriester before the next acid step. If each coupling has yield y, the full-length fraction of an n-mer is y^(n − 1). The trityl assay measures y: capped chains release no more DMT, so the DMT absorbance falls by a factor y per cycle, and y = (A_late/A_early)^(1/k) for k couplings between the two readings.

Protecting groups and final deprotection

Every nucleophile except the 5′-OH must be masked during coupling. The exocyclic amino groups of A, C and G are protected as amides (N6-benzoyl-dA, drawn here, N4-benzoyl- or N4-acetyl-dC and N2-isobutyryl-dG); thymine and uracil have no amino group and need none. Amides survive the acid, activator and iodine of every cycle but are removed at the end. Concentrated aqueous ammonia at about 55 °C does all the final work at once: it cleaves the succinyl ester that links the chain to the support, removes each 2-cyanoethyl group from phosphorus by β-elimination (releasing acrylonitrile and the phosphodiester anion), and removes the amides by aminolysis. The 5′-O-DMT ether is stable to base, so a chain can be left 'DMT-on' to help purification and detritylated afterwards with dilute acid. RNA synthesis also protects the 2′-OH, usually as a tert-butyldimethylsilyl (TBS) ether. It survives the ammonia step and is removed last with fluoride, for example Et₃N·3HF, which is not basic enough to cleave RNA.

The 2′-OH and why DNA lasts

RNA is cleaved by base because its 2′-OH sits next to each 3′-phosphodiester. Hydroxide removes the 2′-OH proton, and the 2′-alkoxide attacks phosphorus in line with the 5′-oxygen of the next nucleotide, which leaves. The product is a five-membered 2′,3′-cyclic phosphate (drawn for a ribose with the base removed). Hydroxide then opens that ring at either P–O bond, so alkaline hydrolysis gives a mixture of 2′- and 3′-monophosphates. Intramolecular attack through a five-membered ring is far faster than attack by external hydroxide, which is also repelled by the negative charge on the phosphodiester. DNA has no 2′-OH, so its phosphodiesters are hydrolysed only extremely slowly, and 2′-O-methyl RNA, whose 2′-oxygen carries no proton to lose, resists alkali for the same reason. This is also why the 2′-O-TBS group must stay on until the basic deprotection steps are over.

What to carry forward

Nucleic acid chemistry is phosphate and glycosidic-bond chemistry controlled by a few functional groups. The 2′-OH makes RNA self-cleaving in base, and its absence makes DNA durable. Phosphoramidite synthesis builds chains 3′→5′ by activating P(III) with a weak acid, capping failures, then oxidizing P(III) to P(V) before the next acid step; protecting groups are chosen so that one ammonia treatment removes the amides, the cyanoethyl groups and the support link, while DMT and 2′-O-TBS come off under their own conditions. Track the charges: one per phosphodiester, essentially none on the bases at pH 7, and count molecules with A = εcl.

Problemas de prática (40)

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Desenhe o mecanismo 12

Phosphoramidite synthesis

  • The 5′-OH couples with the activated phosphoramiditeDraw the attack of the chain's free 5′-OH on the tetrazolyl phosphite and the proton transfer that gives the phosphite triester and regenerates tetrazole.
  • Water destroys the activated amidite as an H-phosphonateDraw the hydrolysis of an activated phosphoramidite by water and the tautomerization of the resulting dialkyl phosphite into an unreactive H-phosphonate diester.
  • Tetrazole activates a nucleoside phosphoramiditeDraw how 1H-tetrazole protonates the diisopropylamino nitrogen of a phosphoramidite and how tetrazolide then displaces diisopropylamine to give the reactive tetrazolyl phosphite.
  • Acetic anhydride and N-methylimidazole cap an unreacted 5′-OHDraw how N-methylimidazole turns acetic anhydride into an N-acetylimidazolium ion and how that ion acetylates a 5′-OH that failed to couple.
  • Dichloroacetic acid removes the 5′-O-DMT groupDraw the acid-catalysed ionization that releases the 5′-OH and the 4,4′-dimethoxytrityl cation, and explain why the cation forms so easily.
  • Iodine and water oxidize the phosphite triester to a phosphotriesterDraw the oxidation of a P(III) phosphite triester by iodine and water, with pyridine as the base, to the P(V) phosphotriester.

Protection and deprotection

  • Ammonia removes the 2-cyanoethyl group by β-eliminationDraw the β-elimination in which ammonia removes the acidic hydrogen next to the nitrile, releasing acrylonitrile and the negatively charged phosphodiester.
  • Making a phosphoramidite from the nucleoside 3′-OHDraw how the 3′-OH of a protected nucleoside displaces chloride from 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, and explain why the diisopropylamino group stays on phosphorus.
  • Ammonia releases the chain from a succinyl-linked supportDraw the aminolysis of the 3′-O-succinyl ester that releases the oligonucleotide with a free 3′-OH and leaves a succinamide on the support.
  • Fluoride removes the 2′-O-TBS groupDraw how fluoride attacks silicon to release the 2′-OH of RNA from its tert-butyldimethylsilyl ether, with HF from the reagent as the proton source.

RNA versus DNA

  • Hydroxide opens the 2′,3′-cyclic phosphateDraw the attack of hydroxide on a 2′,3′-cyclic phosphate that opens the ring to a nucleoside 2′-phosphate, and explain why alkaline hydrolysis gives both 2′- and 3′-phosphates.
  • Hydroxide cleaves RNA through its 2′-OHDraw the hydroxide-catalysed transesterification in which the 2′-alkoxide of RNA attacks the adjacent phosphorus, expels the 5′-oxygen of the next nucleotide and forms a 2′,3′-cyclic phosphate.

Preveja o produto 4

Protection and deprotection

  • What survives the ammonia step?Predict which protecting groups of a DMT-on RNA synthesis survive the ammonia deprotection and which are removed.

Pairing, ionization and UV

  • Guanine's Watson–Crick partnerPredict guanine's Watson–Crick partner from the donor and acceptor pattern of its pairing edge.

RNA versus DNA

  • RNA and DNA in alkaliPredict the different fates of RNA and DNA in dilute aqueous alkali from the presence or absence of the 2′-OH.
  • Which link survives alkali?Predict which phosphodiester in a partly 2′-O-methylated RNA resists alkaline cleavage.

Classificar 6

Nucleoside structure

  • Sugar pucker in an RNA duplexIdentify the furanose pucker adopted by ribose in an A-form RNA duplex and contrast it with B-form DNA.
  • Pick the natural β-anomerRecognise the β-D anomeric configuration of a natural 2′-deoxynucleoside from the relative positions of the base and the C4′ side chain.
  • Find 2′-deoxyadenosineRecognise 2′-deoxyadenosine among ribo-, 3′-deoxy- and guanine nucleosides from its sugar oxygenation and base.
  • Name the nucleotideName a ribonucleotide from its base, its sugar and the position of its phosphate.
  • Which nucleotide belongs to RNA?Classify nucleoside 5′-monophosphates as RNA or DNA building blocks from their sugar and base.

Pairing, ionization and UV

  • The tautomer that pairs guanine with thymineIdentify the rare guanine tautomer whose hydrogen-bonding edge complements thymine.

Comparar 3

Pairing, ionization and UV

  • Rank the nucleosides as basesOrder the most basic ring nitrogens of three nucleosides by basicity using their conjugate-acid pKa values.
  • Rank the acidic protons of nucleic acidsOrder the acidic O–H and N–H groups found in nucleotides by acidity from their pKa values and relate them to charge at pH 7.
  • Rank duplexes by melting temperatureOrder DNA duplexes of equal length by melting temperature from their G·C content.

Calcular 7

Pairing, ionization and UV

  • Oligonucleotide concentration from A₂₆₀Calculate the concentration of an oligonucleotide stock from the absorbance of a diluted sample with the Beer–Lambert law.
  • Guanine content from Chargaff's rulesUse Chargaff's base-pairing rules to calculate the guanine percentage of a double-stranded DNA.
  • How much cytidine is protonated at pH 5?Calculate the fraction of cytidine protonated at N3 in a mildly acidic buffer from its pKa.
  • Count the base-pair hydrogen bondsCount the Watson–Crick hydrogen bonds in a short DNA duplex from its G·C and A·T content.
  • Hyperchromicity on melting a duplexCalculate the percentage hyperchromicity when a DNA duplex melts from its A₂₆₀ before and after melting.
  • Net charge of a DNA 20-merCalculate the net charge of a linear oligonucleotide at pH 7 from its number of phosphodiester links.

Phosphoramidite synthesis

  • Full-length yield of a 25-merCalculate the full-length fraction of an oligonucleotide from its stepwise coupling efficiency and the number of couplings.

Encontre o erro 2

Phosphoramidite synthesis

  • A synthesizer skips the iodine stepDiagnose why omitting the iodine oxidation in each cycle ruins an oligonucleotide synthesis.

RNA versus DNA

  • Desilylating too earlyDiagnose why removing the 2′-O-TBS groups before the basic deprotection step destroys a synthetic RNA.

Interprete as evidências 2

Nucleoside structure

  • Read a dinucleotide 5′→3′Assign the 5′→3′ sequence of a dinucleotide from which sugar oxygens carry the phosphodiester.

Phosphoramidite synthesis

  • Average coupling yield from the trityl assayCalculate the average stepwise coupling yield of an oligonucleotide synthesis from DMT-cation absorbances.

Planeje uma síntese 2

Protection and deprotection

  • Protect the amino group of dAChoose a protecting group for the exocyclic amine of 2′-deoxyadenosine that survives every synthesis cycle and is removed by the final ammonia treatment.

Phosphoramidite synthesis

  • Order one synthesis cyclePut the four steps of one phosphoramidite synthesis cycle in order, starting from a 5′-O-DMT chain on the support.

Explique por quê 2

Phosphoramidite synthesis

  • Why use ETT for RNA amidites?Explain why a more acidic tetrazole activator speeds the coupling of hindered 2′-O-TBS ribonucleoside phosphoramidites.

RNA versus DNA

  • Why DNA phosphodiesters resist hydrolysisExplain the resistance of the DNA phosphodiester backbone to hydrolysis in terms of its charge and the lack of an internal nucleophile.

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