Chemistry · Organic chemistry · Year 3

Heterocycles and DNA

Why is pyridine a base while pyrrole hardly is — and what holds the DNA double helix together?

The situation

At the Institute of the Chemistry of Plant Substances in Tashkent, alkaloids are isolated from plants: almost all of them are nitrogen heterocycles. In a neighbouring genomics laboratory, DNA is isolated from cotton seeds and heated in a spectrophotometer — the absorption at 260 nm suddenly rises. Why is the pyridine ring a base, while pyrrole is hardly a base at all? What holds the two strands of DNA together, and why do they “melt” on heating?

Topic

Heterocycles and nucleic acids. Institute of the Chemistry of Plant Substances of the Academy of Sciences of Uzbekistan (the school of alkaloids of Academician S. Yu. Yunusov) and the Genomics Centre: extracting DNA from cottonseed, alkaloid crystals,…

Key ideas

Aromaticity (4n + 2)
Watson–Crick pairs
DNA melting and Tₘ

Formulas

N_{π} = 4 n + 2

Hückel's rule: a planar, continuously conjugated ring with $4 n + 2$ π electrons (n = 0, 1, 2 …) is aromatic.

L_{r}^{+} = E_{π} (ring) - E_{π} (σ -complex)

The localisation energy for electrophilic attack (in units of $| β |$ ): the smaller the L, the more favourable the position.

\frac{A + G}{T + C} = 1, A = T, G = C

Chargaff's rule (double-stranded DNA): the number of purines equals the number of pyrimidines.

T_{m} = 69.3 + 0.41 \cdot (% G C)^{\circ} C

Marmur–Doty: the melting temperature of long DNA in 1×SSC solution depends on the proportion of GC pairs.

A = ε c l; A_{260} = 1 \Leftrightarrow 50 µg/ml DNA

Beer–Lambert: for double-stranded DNA $A_{260} = 1$ corresponds to ≈ 50 µg/mL, and for RNA ≈ 40 µg/mL.

Theory

Heterocycles: aromaticity and naming

A heterocycle is a compound that has a heteroatom such as N, O or S in its ring together with carbon. The five-membered pyrrole, furan and thiophene and the six-membered pyridine are, like benzene, planar, continuously conjugated rings; they have 6 π electrons ( $4 n + 2$ , $n = 1$ ) and so are aromatic. The difference lies in the contribution of the heteroatom: the =N– in pyridine gives one electron and its lone pair stays in the plane of the ring, while the –NH– in pyrrole, the –O– in furan and the –S– in thiophene give their pair to the π sextet. So the five-membered rings are π-excessive (π density on the carbons > 1), while pyridine is π-deficient (< 1 at positions 2 and 4). Aromaticity is measured by the resonance energy (Joule–Mills): benzene ≈ 150, thiophene ≈ 122, pyridine ≈ 117, pyrrole ≈ 90, furan ≈ 67 kJ/mol. Hantzsch–Widman nomenclature: a prefix for the heteroatom (oxa-, thia-, aza-; if there are several, in the order O > S > N, with numbers) + a stem showing the ring size and saturation (saturated / unsaturated): 3-membered -irane / -irene, 4 — -etane / -ete, 5 — -olane / -ole, 6 — -ane / -ine; for nitrogen rings the saturated forms are -iridine (aziridine, azetidine, azolidine), for 6-membered rings -inane (azinane), and the unsaturated 3-membered ring is -irine. Many rings have kept their trivial names: pyrrole, furan, thiophene, pyridine, imidazole, pyrimidine, purine.

Basicity, reactions and synthesis strategies

Basicity depends on where the lone pair is: pyridine adds H⁺ to its in-plane pair and aromaticity is kept — p $K_{a H}$ = 5.2; imidazole — 7.0; pyrimidine — 1.3. In pyrrole the pair is in the π sextet: the proton adds only at C2 and aromaticity is lost — p $K_{a H}$ ≈ −3.8, so pyrrole is hardly a base at all (on the contrary, the N–H is a weak acid). Electrophilic substitution (SEAr) goes through a σ complex (the Wheland intermediate); which position is favoured is decided by the stability of the intermediate — the localisation energy $L^{+}$ — and not by the density in the ground state (in pyrrole the density is slightly greater at C3, but the attack goes to C2). Pyrrole, furan and thiophene react faster than benzene, mainly at position 2; in pyridine SEAr is very difficult (in acid the N is protonated), while nucleophiles attack readily (Chichibabin: NaNH₂ → 2-aminopyridine). Synthesis strategies show from which fragments the ring is assembled: “4 + 1” (Paal–Knorr: 1,4-diketone + NH₃ → pyrrole, + P₄S₁₀ → thiophene; with acid, the diketone's own oxygen closes the ring to give furan), “3 + 2” (1,3-diketone + hydrazine → pyrazole; the Knorr synthesis), “5 + 1” (1,5-dicarbonyl + NH₃ → pyridine), “3 + 3” (malonic ester + urea → barbituric acid — a pyrimidine ring).

Natural heterocycles: alkaloids, bases, nucleotides, coenzymes

Alkaloids are the nitrogenous bases of plants: nicotine (pyridine + pyrrolidine), anabasine (Anabasis aphylla — a plant of Central Asia), harmine (Syrian rue — Peganum harmala), caffeine (a purine). Academician S. Yu. Yunusov (1909–1995) founded the Institute of the Chemistry of Plant Substances in Tashkent in 1956; his school isolated many new alkaloids from the plants of Central Asia and determined their structures. The bases of nucleic acids: purines — adenine (A), guanine (G); pyrimidines — cytosine (C), thymine (T, in DNA only), uracil (U, in RNA). The bases can exist in tautomeric forms: lactam ⇌ lactim (keto ⇌ enol), amino ⇌ imino; in DNA the keto and amino forms strongly predominate, and a rare tautomer can form a wrong pair and lead to a point mutation. A nucleoside = a base + a pentose (ribose or 2-deoxyribose) joined by a β-N-glycosidic bond (through N9 in a purine, N1 in a pyrimidine); a nucleotide = the phosphate ester of a nucleoside (5′-AMP, ATP); in the cyclic nucleotide cAMP the phosphate links the 3′- and 5′-O into a ring — the second messenger of a hormone signal. Coenzymes: NAD⁺ and NADP⁺ (the nicotinamide ring accepts H⁻ at C4 and becomes NAD(P)H), FAD (isoalloxazine adds 2H at N1 and N5). Nucleotides are joined by 3′,5′-phosphodiester bonds into oligonucleotides (up to a few dozen) and polynucleotides.

The structure of DNA and RNA

The primary structure is the sequence of bases, written in the 5′→3′ direction. The secondary structure is the Watson–Crick double helix (1953): two antiparallel strands, with A·T (2 hydrogen bonds) and G·C (3) pairs inside and a sugar–phosphate “backbone” outside. Chargaff's rule: A = T, G = C, (A + G) = (T + C). The B form (the main one in aqueous solution): a right-handed helix, ≈ 10.5 pairs per turn, a rise of 0.34 nm, a turn height of ≈ 3.4–3.6 nm, a diameter of ≈ 2 nm, with major and minor grooves. The A form (on dehydration; RNA double helices and DNA–RNA hybrids): 11 pairs per turn, the pairs displaced from the axis and tilted, a diameter of ≈ 2.3 nm. The C form is close to B, at low humidity: ≈ 9.3 pairs per turn. The Z form is a left-handed helix with 12 pairs per turn, in alternating GC sequences (Rich, 1979). Tertiary structure: circular and closed DNA supercoils; in eukaryotes DNA wraps 1.65 turns (147 pairs) around an octamer of histones to form a nucleosome, and the nucleosomes are assembled into a chromatin fibre and then a chromosome. Types of RNA: mRNA (a copy of the protein sequence), tRNA (≈ 76 nucleotides, a “cloverleaf” secondary structure and an L-shaped tertiary structure), rRNA (the basis of the ribosome). Inherited diseases are changes in DNA: for example, in sickle-cell anaemia GAG → GTG in the 6th codon of the β-globin gene, and the protein has valine in place of glutamate.

Lab work: isolation, hydrolysis and the melting curve

Isolating DNA from seeds (L12): grinding → lysis buffer (detergent, NaCl, EDTA, 60 °C) → removing the proteins with chloroform and centrifuging → cold ethanol over the aqueous phase: the DNA comes out as a white thread and is wound onto a rod. RNA from sprouted mung beans (L11) — extraction with acidic phenol, precipitation with ethanol; RNases are active, so everything is done on ice. Hydrolysis (L13): in alkali RNA breaks down quickly with the help of the 2′-OH, while DNA is resistant; in acid the purines are released. Qualitative reactions: diphenylamine (Dische) — deoxyribose, blue; orcinol (Bial) — ribose, green; ammonium molybdate — phosphate, a yellow precipitate; AgNO₃ + NH₃ — a precipitate of purines. Purity: A₂₆₀/A₂₈₀ ≈ 1.8 (DNA), ≈ 2.0 (RNA); protein contamination lowers the ratio. Melting: on heating, the hydrogen bonds and the stacking are broken, the strands separate and the absorption at 260 nm rises by ≈ 40 % (the hyperchromic effect). The midpoint of the curve is $T_{m}$ . A G·C pair is more stable because of its three hydrogen bonds and stronger stacking: $T_{m} = 69.3 + 0.41 \cdot % G C$ (1×SSC). If the salt concentration falls, $T_{m}$ falls (the repulsion of the phosphates increases): by ≈ 16.6 °C when Na⁺ falls 10-fold; that is why GC-rich DNA (M. luteus, 73 %: ≈ 99 °C in 1×SSC) is measured in 0.1×SSC. For the method of spectrophotometry, see the “Optical methods of analysis” experiment. Safety: phenol and chloroform are poisonous — wear gloves and goggles and work in a fume cupboard; keep ethanol away from flames; if alkali or acid gets into the eyes, rinse with plenty of water.

About the model

The π densities and localisation energies of the rings come from the simple Hückel model (Streitwieser's parameters; the parameters for the S of thiophene are model values); it gives the trends correctly, but not exact energies. The 3D models of the molecules are assembled from typical bond lengths and angles (not a quantum-chemical optimisation). DNA: the bases use the standard 3DNA coordinates; the sugar–phosphate chain is fitted using ideal bonds and the helical parameters of the A/B forms (the Z form is a simplified uniform left-handed helix; the true zigzag dinucleotide repeat is not shown). Chromatin is schematic. The melting curve is a two-state model (a width of ≈ 5 °C), with $T_{m}$ from the Marmur–Doty formula; the GC content of the “cotton seed” sample is a teaching sample (different each time), not the real value for the genome.

Did you know?

Watson and Crick published the double-helix model on 25 April 1953 in the journal Nature; the X-ray photograph (“Photo 51”) taken by Rosalind Franklin and Raymond Gosling was important evidence for the helical structure.
If the DNA of a single human cell (≈ 6.4 billion pairs) were stretched out in a line, it would be about 2 m long — it fits into a nucleus a few micrometres in diameter with the help of nucleosomes and chromatin fibres.

Inside the experiment

The experiment starts from a real-life situation: you predict the result, change the controls, run the experiment and draw a conclusion. Inside are missions, theory, a quiz and a guided tour in which the camera moves and explains the key points.

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Physics of macromolecules — Year 3
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