Source: Brown, Iverson, Anslyn, Foote, Organic Chemistry 8th ed., Chapter 23 + supplementary lecture material (cofactors, proteins, nucleic acids, non-covalent interactions)
Difficulty: Advanced | Prerequisites: Modules 1–3 (aromaticity, spectroscopy, carbonyl chemistry, acid derivatives, enolate chemistry, EAS)
Tags: amines, amine basicity, amine reactions, amino acids, peptides, proteins, peptide bond, cofactors, coenzymes, NAD, FAD, thiamine pyrophosphate, nucleic acids, DNA, RNA, nucleotide, nucleoside, non-covalent interactions, hydrogen bonding, van der Waals, hydrophobic effect
This module brings the organic chemistry of the entire semester into a biological context. You have spent 13 weeks learning how carbonyls react, how enolates form C–C bonds, and how aromatic rings are functionalised. Now you see those same reactions at work inside living cells. Amines are the nitrogen-containing functional group central to amino acids and neurotransmitters. Cofactors are small organic molecules that assist enzymes by performing chemistry (redox, C–C bond formation, group transfer) that amino acid side chains cannot do alone. Proteins are polymers of amino acids linked by amide (peptide) bonds. Nucleic acids store and transmit genetic information through hydrogen-bonded base pairing. Non-covalent interactions explain why proteins fold, why membranes form, and why drugs bind their targets. Everything in this module is a direct application of mechanisms you have already learned.
Amines are organic bases and nucleophiles whose reactivity depends on nitrogen's lone pair. Amino acids are bifunctional (amine + carboxylic acid) and polymerise via peptide bonds. Cofactors carry out the redox, decarboxylation, and C–C bond-forming chemistry of metabolism using mechanisms from Modules 2 and 3. Nucleic acids encode information through complementary hydrogen bonding. Non-covalent interactions (hydrogen bonds, ionic, dipole, van der Waals, hydrophobic effect) collectively govern the three-dimensional shape and function of biomolecules.
Amine
An organic compound containing a nitrogen with a lone pair bonded to one, two, or three carbon groups (primary, secondary, tertiary). Quaternary ammonium salts have four carbon groups on nitrogen and carry a permanent positive charge. Think of amines as the nitrogen-based analogue of alcohols and ethers, but more nucleophilic and more basic.
Basicity of Amines
Amines are Brønsted bases (lone pair on nitrogen accepts a proton). Typical pKb values: aliphatic amines ~3–4 (pKa of conjugate acid ~10–11); aromatic amines (anilines) are much weaker bases (pKa of conjugate acid ~4–5) because the lone pair is delocalised into the ring.
Amino Acid
A molecule containing both an amine group and a carboxylic acid group. The 20 standard alpha-amino acids have both groups attached to the same carbon (the alpha carbon). At physiological pH (~7.4), amino acids exist as zwitterions (internal salt: NH₃⁺ and COO⁻ on the same molecule).
Zwitterion
A molecule carrying both a positive and a negative charge, with a net charge of zero. In simple terms, the amine grabs a proton from the carboxylic acid, so both groups are charged.
Isoelectric Point (pI)
The pH at which an amino acid has no net charge. For amino acids with non-ionisable side chains, pI = (pKa₁ + pKa₂) / 2, where pKa₁ is the carboxyl and pKa₂ is the ammonium.
Peptide Bond
An amide bond linking the carboxyl group of one amino acid to the amino group of the next. Partial double-bond character due to resonance means the bond is planar, shorter than a typical C–N single bond, and restricted in rotation.
Primary Structure
The linear sequence of amino acids in a polypeptide, read from the N-terminus to the C-terminus.
Secondary Structure
Local folding patterns stabilised by backbone N–H···O=C hydrogen bonds. The two major types are the alpha helix and the beta sheet (parallel and antiparallel).
Tertiary Structure
The overall three-dimensional fold of a single polypeptide chain, stabilised by side-chain interactions: hydrogen bonds, ionic (salt bridges), disulfide bonds (covalent S–S between cysteine residues), hydrophobic interactions, and van der Waals forces.
Quaternary Structure
The arrangement of two or more polypeptide subunits into a multi-subunit complex (e.g. haemoglobin, a tetramer).
Cofactor
A non-protein molecule required by an enzyme to carry out its reaction. Can be a metal ion or an organic molecule (coenzyme). Think of cofactors as the chemical tools that enzymes cannot build from amino acids alone.
Coenzyme
An organic cofactor. Many are derived from vitamins.
NAD⁺ / NADH (Nicotinamide Adenine Dinucleotide)
A redox coenzyme. NAD⁺ is the oxidised form (accepts a hydride, H⁻); NADH is the reduced form. The nicotinamide ring undergoes 1,4-addition of hydride. Derived from niacin (vitamin B₃).
FAD / FADH₂ (Flavin Adenine Dinucleotide)
A redox coenzyme. FAD accepts two hydrogens (two electrons + two protons); FADH₂ is the reduced form. The isoalloxazine ring system is the reactive part. Derived from riboflavin (vitamin B₂).
Thiamine Pyrophosphate (TPP)
A coenzyme that facilitates decarboxylation of alpha-keto acids and transketolase reactions. The thiazolium ring generates a stabilised carbanion (an ylide) that attacks the carbonyl of the substrate. Derived from thiamine (vitamin B₁).
Coenzyme A (CoA-SH)
A thiol coenzyme that forms thioester linkages with acyl groups (acetyl-CoA is the most important example). Thioesters are more reactive than oxygen esters because sulfur is less effective at resonance donation into the carbonyl, making the carbonyl more electrophilic. Derived from pantothenic acid (vitamin B₅).
Pyridoxal Phosphate (PLP)
A coenzyme involved in amino acid transformations: transamination, decarboxylation, racemisation, and elimination. Forms an imine (Schiff base) with the amino acid substrate, and the pyridinium ring acts as an electron sink. Derived from pyridoxine (vitamin B₆).
Nucleotide
The monomer of nucleic acids. Composed of a nitrogenous base, a five-carbon sugar (ribose in RNA, deoxyribose in DNA), and one or more phosphate groups.
Nucleoside
A nucleotide without the phosphate group: just the base and the sugar.
Purine Bases
Adenine (A) and guanine (G). Two-ring aromatic heterocyclic systems.
Pyrimidine Bases
Cytosine (C), thymine (T, in DNA), and uracil (U, in RNA). Single-ring aromatic heterocyclic systems.
Complementary Base Pairing
A pairs with T (or U) through two hydrogen bonds; G pairs with C through three hydrogen bonds. This specificity is the basis of DNA replication and transcription.
Phosphodiester Bond
The linkage between nucleotides in a nucleic acid strand: a phosphate group esterified to the 3' –OH of one sugar and the 5' –OH of the next.
Hydrogen Bond
An attractive interaction between an electronegative atom bearing a lone pair (acceptor) and a hydrogen bonded to another electronegative atom (donor). Typical strength: 10–40 kJ/mol. In simple terms, it is the strongest of the dipole-based interactions and the one that holds DNA strands together and shapes protein folds.
Van der Waals (London Dispersion) Forces
Weak, short-range attractive forces arising from temporary fluctuations in electron distribution (induced dipoles). Present in all molecules. Strength increases with molecular size and surface area.
Hydrophobic Effect
The tendency of nonpolar molecules or nonpolar parts of molecules to aggregate in aqueous solution, driven primarily by the entropy gain when ordered water molecules around individual nonpolar surfaces are released. This is the dominant force driving protein folding and membrane assembly.
Ionic Interaction (Salt Bridge)
The electrostatic attraction between a full positive charge and a full negative charge. In proteins, this occurs between charged side chains (e.g. Lys NH₃⁺ and Asp COO⁻).
Classification:
Primary (RNH₂), secondary (R₂NH), tertiary (R₃N), quaternary ammonium (R₄N⁺)
Note: this classification is based on carbons on nitrogen, unlike alcohol classification (which counts carbons on the carbon bearing –OH)
Basicity trends:
Alkylamines (pKb ~3–4) are stronger bases than ammonia (pKb ~4.7) because alkyl groups donate electron density to nitrogen
Arylamines (anilines, pKb ~9–10) are much weaker bases because the nitrogen lone pair is delocalised into the aromatic ring
Electron-withdrawing groups on the ring (–NO₂) further reduce aniline basicity
Electron-donating groups on the ring (–OCH₃) increase aniline basicity
Amides are essentially non-basic (pKa of conjugate acid ~ −1) because the lone pair is delocalised into the carbonyl
Preparation of amines:
Reduction of nitro groups (catalytic hydrogenation, Sn/HCl, or Fe/HCl): ArNO₂ → ArNH₂
Reduction of amides with LiAlH₄: RCONR'₂ → RCH₂NR'₂
Reduction of nitriles with LiAlH₄ or catalytic H₂: RCN → RCH₂NH₂
Reductive amination: carbonyl + amine + reducing agent (NaBH₃CN) → amine via imine intermediate
Gabriel synthesis: phthalimide salt + alkyl halide → N-alkylphthalimide → primary amine (after hydrazine or base cleavage)
Reactions of amines:
As bases: protonation by acids to form ammonium salts
As nucleophiles: alkylation (SN2 with alkyl halides), acylation (react with acid chlorides, anhydrides to form amides), reaction with aldehydes/ketones to form imines or enamines
Reaction with nitrous acid (HNO₂):
Primary aliphatic amines → unstable diazonium ions → loss of N₂ → mixture of products (not synthetically useful)
Primary aromatic amines → relatively stable aryl diazonium salts (ArN₂⁺) at 0–5 °C → useful for Sandmeyer reactions and azo coupling
Sandmeyer reactions: ArN₂⁺ + CuCl → ArCl; + CuBr → ArBr; + CuCN → ArCN; + H₃PO₂ → ArH; + HBF₄ then heat → ArF
Azo coupling: ArN₂⁺ + activated aromatic ring (phenol or amine) → azo dye (Ar–N=N–Ar')
Hofmann elimination: quaternary ammonium hydroxide heated → least substituted alkene (Hofmann product), opposite to the usual Zaitsev selectivity
The 20 standard amino acids:
All are alpha-amino acids (amino and carboxyl on the same carbon)
All except glycine are chiral; biological amino acids are L-configuration (S at the alpha carbon, except for cysteine which is R due to priority rules)
Side chains determine properties: nonpolar (Ala, Val, Leu, Ile, Pro, Phe, Trp, Met), polar uncharged (Ser, Thr, Cys, Tyr, Asn, Gln), positively charged at pH 7.4 (Lys, Arg, His), negatively charged at pH 7.4 (Asp, Glu)
Peptide bond formation:
Condensation reaction between the carboxyl of one amino acid and the amino of another, losing water
The resulting amide bond has partial double-bond character (C–N bond order ~1.5)
Rotation around the C–N bond is restricted: the six atoms of the peptide unit (Cα–CO–NH–Cα) are coplanar
Convention: the chain is written and read from N-terminus (free NH₃⁺) to C-terminus (free COO⁻)
Protein structure levels:
Primary: amino acid sequence
Secondary: alpha helices (right-handed, 3.6 residues per turn, H-bonds between C=O of residue i and N–H of residue i+4) and beta sheets (H-bonds between extended strands, parallel or antiparallel)
Tertiary: the complete 3D fold, driven by hydrophobic collapse and stabilised by all types of non-covalent interactions plus disulfide bonds
Quaternary: multi-subunit assembly
NAD⁺/NADH:
Oxidation of alcohols to carbonyls (and reverse): NAD⁺ accepts a hydride (H⁻) at the 4-position of the nicotinamide ring
Mechanism: direct hydride transfer (no radical intermediates)
Stereospecific: enzymes deliver hydride to a specific face of NAD⁺
FAD/FADH₂:
Two-electron, two-proton reduction of the isoalloxazine ring
Involved in oxidation of C–C single bonds to C=C double bonds (e.g. succinate to fumarate in the citric acid cycle)
Can also participate in one-electron (radical) pathways via the semiquinone form
Thiamine pyrophosphate (TPP):
The C-2 of the thiazolium ring is deprotonated to form a nucleophilic carbene/ylide
This carbon attacks the carbonyl of an alpha-keto acid (e.g. pyruvate), and subsequent decarboxylation removes CO₂
Key reaction: pyruvate decarboxylation (pyruvate → acetaldehyde + CO₂, catalysed by pyruvate decarboxylase)
Coenzyme A:
The thiol (–SH) forms thioesters (R–CO–S–CoA)
Thioesters are more reactive than oxygen esters toward nucleophilic acyl substitution because sulfur is a poor resonance donor, leaving the carbonyl more electrophilic
Acetyl-CoA is the central metabolic hub: fatty acid oxidation produces it, it enters the citric acid cycle, and it is the building block for fatty acid and steroid biosynthesis
Biological Claisen condensation: two acetyl-CoA units condense to form acetoacetyl-CoA (the first step of fatty acid and cholesterol biosynthesis)
Pyridoxal phosphate (PLP):
Forms an imine (Schiff base) with the alpha-amino group of the substrate amino acid
The pyridinium ring acts as an electron sink, stabilising carbanion intermediates at the alpha carbon
Catalyses: transamination (swaps amino and keto groups between an amino acid and an alpha-keto acid), decarboxylation (removes CO₂ from amino acids), racemisation, and beta-elimination
DNA vs. RNA:
Sugar: deoxyribose (DNA) vs. ribose (RNA)
Bases: A, G, C, T (DNA) vs. A, G, C, U (RNA)
Structure: double helix (DNA) vs. usually single-stranded with secondary structure (RNA)
Nucleosides: base + sugar (no phosphate). Named adenosine, guanosine, cytidine, thymidine, uridine
Nucleotides: nucleoside + phosphate group(s). Named as monophosphates (AMP), diphosphates (ADP), triphosphates (ATP)
Phosphodiester linkages: connect 3' –OH of one nucleotide to 5' –OH of the next via a phosphate bridge
The backbone is sugar-phosphate-sugar-phosphate; the bases project inward
Directionality: strands run 5' → 3'
Base pairing (Watson-Crick):
A–T: two hydrogen bonds
G–C: three hydrogen bonds
Strands are antiparallel (one runs 5'→3', the other 3'→5')
Higher G–C content means higher melting temperature (more H-bonds to break)
Types (roughly in order of decreasing strength for typical biological examples):
Ionic interactions / salt bridges (~40–400 kJ/mol in vacuum, much weaker in water due to solvation): full charge–charge attraction
Hydrogen bonds (~10–40 kJ/mol): N–H···O, O–H···N, O–H···O, N–H···N
Dipole-dipole (~5–25 kJ/mol): partial charge interactions between polar bonds
Cation-pi (~5–80 kJ/mol): positively charged group (Lys, Arg) attracted to the electron-rich face of an aromatic ring (Phe, Trp, Tyr)
Van der Waals / London dispersion (~2–10 kJ/mol per contact): temporary dipole–induced dipole. Weak individually but sum over large surfaces
Hydrophobic effect: not a direct attractive force between nonpolar groups, but an entropy-driven aggregation caused by release of ordered water. The dominant force in protein folding and membrane formation
In biological context, these interactions are:
Individually weak compared to covalent bonds (~350–500 kJ/mol)
Collectively powerful when many act together (protein folding involves thousands)
Reversible and dynamic, allowing molecular recognition, signal transduction, and enzyme catalysis
Amine chemistry underpins many pharmaceuticals: antihistamines, local anaesthetics (lidocaine), and antidepressants (SSRIs) all contain amine functional groups whose basicity and hydrogen-bonding capacity determine drug-receptor interactions. The understanding of non-covalent interactions is central to rational drug design: medicinal chemists optimise hydrogen bonds, hydrophobic contacts, and salt bridges between a drug molecule and its protein target. DNA base pairing is the basis of PCR (polymerase chain reaction), genetic sequencing, and CRISPR gene editing.
Students often confuse amine classification with alcohol classification. For amines, the classification (primary, secondary, tertiary) counts the number of carbons bonded to nitrogen. For alcohols, it counts the carbons bonded to the carbon bearing the –OH. A tertiary amine (R₃N) and a tertiary alcohol (R₃C–OH) are classified by different rules.
A common error is assuming that amides are basic like amines. The nitrogen lone pair in an amide is delocalised into the carbonyl, making amides essentially non-basic (pKa of conjugate acid around −1).
Students sometimes think the hydrophobic effect is a direct attraction between nonpolar molecules. It is better understood as an entropy-driven process: when nonpolar surfaces aggregate, the highly ordered shell of water molecules around them is released, increasing the entropy of the system.
Students frequently miscount hydrogen bonds in base pairs: A–T has two (not three), G–C has three (not two). This matters for predicting DNA melting temperature.
⚠️ Amine basicity trends are heavily tested. Know how alkyl groups, aryl groups, and resonance with adjacent carbonyls (amides) affect basicity, and be able to rank a set of amines by pKb.
⚠️ Diazonium chemistry (Sandmeyer reactions) often appears in multi-step synthesis problems. It is the only way to replace –NH₂ on an aromatic ring with –Cl, –Br, –CN, –F, –OH, or –H after using the amine as a directing group.
⚠️ Know the connection between cofactor chemistry and the organic reactions from earlier modules: NAD⁺ does hydride chemistry (like NaBH₄ in reverse), CoA does thioester acyl substitution, TPP does decarboxylation of alpha-keto acids, and PLP does imine chemistry with amino acids.
⚠️ Base-pairing rules and the distinction between DNA and RNA components are standard final exam material. Practise drawing the hydrogen-bonding pattern for an A–T pair and a G–C pair.
⚠️ Non-covalent interactions appear on the final in the context of protein folding: be able to identify which type of interaction stabilises a given structural feature (hydrophobic core, salt bridge between Lys and Asp, H-bond in alpha helix, disulfide bond between Cys residues).
True or false: A tertiary amine has three hydrogen atoms bonded to nitrogen.
Fill in the blank: At physiological pH, amino acids exist primarily as ____.
True or false: NAD⁺ accepts a hydride ion, becoming NADH.
Fill in the blank: Adenine pairs with thymine through ____ hydrogen bonds.
True or false: The hydrophobic effect is primarily an enthalpy-driven process.
Answers: 1. False (a tertiary amine has three carbon groups and no hydrogens on nitrogen). 2. Zwitterions. 3. True. 4. Two. 5. False (it is primarily entropy-driven).
Q: Rank the following in order of decreasing basicity: aniline, cyclohexylamine, acetamide.
A: Cyclohexylamine > aniline > acetamide. Cyclohexylamine is a typical aliphatic amine (pKa of conjugate acid ~10.6). Aniline's lone pair is delocalised into the aromatic ring (pKa ~4.6). Acetamide's lone pair is delocalised into the carbonyl (pKa ~ −1), making it essentially non-basic.
Q: What type of reaction does thiamine pyrophosphate (TPP) facilitate, and what is the key mechanistic feature?
A: TPP facilitates the decarboxylation of alpha-keto acids. The key feature is that the C-2 of the thiazolium ring forms a nucleophilic carbanion (ylide) that attacks the carbonyl of the substrate. After decarboxylation, the thiazolium ring stabilises the resulting carbanion intermediate (acting as an electron sink).
Q: Describe the four levels of protein structure and name the dominant stabilising interaction at each level.
A: Primary structure is the amino acid sequence, held by covalent peptide (amide) bonds. Secondary structure consists of alpha helices and beta sheets, stabilised by backbone N–H···O=C hydrogen bonds. Tertiary structure is the overall 3D fold, stabilised by a combination of hydrophobic interactions (dominant driving force), hydrogen bonds, salt bridges, van der Waals forces, and disulfide bonds. Quaternary structure is the arrangement of multiple polypeptide subunits, stabilised by the same non-covalent forces as tertiary structure.
Q: Why are thioesters (like acetyl-CoA) more reactive than oxygen esters toward nucleophilic acyl substitution?
A: Sulfur is larger and its 3p lone pair overlaps poorly with the 2p orbital of the carbonyl carbon, so resonance donation into the C=O is much weaker than in an oxygen ester. This leaves the carbonyl carbon more electrophilic and the thiolate (RS⁻) a better leaving group than an alkoxide (RO⁻), both of which increase reactivity.
Q: Draw the hydrogen bonding pattern between guanine and cytosine and explain why G–C base pairs contribute more to DNA thermal stability than A–T pairs.
A: G–C pairs have three hydrogen bonds (G donates from N–H at position 1, accepts at C=O at position 6, donates from N–H₂ at position 2; C accepts at N-3, donates from N–H₂ at position 4, accepts at C=O at position 2). A–T pairs have only two hydrogen bonds. More hydrogen bonds per base pair means more energy is required to separate the strands, so DNA with higher G–C content has a higher melting temperature.
Amine chemistry ties back to Modules 2 and 3: amines react with carbonyls to form imines (Module 2), and aromatic amines direct EAS ortho/para (Module 3). Diazonium salt chemistry is a bridge between amine chemistry and aromatic substitution. Cofactor mechanisms recapitulate nearly every organic reaction type from the semester: hydride transfer (NaBH₄/LiAlH₄ analogy for NAD⁺), thioester acyl substitution (Module 2), imine formation (Module 2), decarboxylation (Module 2 beta-keto acids), and Claisen condensation (Module 3). The final exam is comprehensive, so viewing cofactor chemistry as "biological versions of reactions you already know" is the most efficient study strategy.
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