© Department of Chemistry, The University of Western Ontario Chem 2223b Intersession 2008: Lipids • This section discusses the properties of triglycerides and fatty acids, the metabolism and biosynthesis of fatty acids and other lipids, soaps and detergents, and vitamins. • Background material, from Chem 2213a or otherwise, that is important includes: o Reactions of carboxylic acids and acid derivatives o Reactions of carbonyl compounds o Reactions of the α-carbon o Dehydration reactions of alcohols o Substitution reactions o Reactions of alkenes and aromatic compounds Lipids 2 A. Classification of Lipids • Lipids are a mixed collection of compounds that are water-insoluble, yet are soluble in less-polar solvents such as dichloromethane, acetone, and diethyl ether. • Therefore, lipids encompass a large assortment of biomolecules, and they are divided into two general categories based on what they contain: o A relatively large hydrophobic region and a smaller hydrophilic region • Fatty acids • Triglycerides (fats and oils) • Phospholipids • Prostaglandins • Fat-soluble vitamins (A, E, D, K) o A common structural element: a fused tetracyclic ring system • Cholesterol, steroids, and other derivatives Lipids 3 B. Fats, Oils, and Fatty Acids 1. Triesters of Glycerol • Fats are solids, while oils are liquids, at room temperature. Aside from this, both fats and oils are triesters of glycerol. Scientists and the medical community refer to these collectively as triglycerides (or triacylglycerides), and they can be hydrolyzed: O CH2 O C R CH2 OH O H+ or OH– CH O C R CH OH 3 RCOOH O CH2 O C R CH2 OH • The R groups are long alkyl chains (same or different) that may contain sites of unsaturation (C=C double bonds). These R groups are part of the fatty acids. • In general, triglycerides containing one or more unsaturated fatty acids have melting points that are lower than those with fully saturated fatty acids. • Fatty acids on their own (not esterified to glycerol) are not found in substantial amounts in the body, but they can be obtained from triglycerides by hydrolysis. Lipids 4 • Mechanism for the acid-catalyzed hydrolysis of an ester: O O H+ RCOR' H ORCOH HOR' 2 • This is the reverse of a Fischer esterification, so it is an equilibrium reaction. Acid- catalyzed hydrolysis of esters does NOT go to completion. Lipids 5 • On the other hand, base hydrolysis (saponification) proceeds to completion. The reaction is stoichiometric, not catalytic, because base is not regenerated. The carboxylate and the alcohol cannot reform the ester. • Homework: work out the mechanism of base hydrolysis (hint: remember that this is simply a nucleophilic acyl substitution, and compare it to amide hydrolysis in base). O O – – RCOR' OH RCO HOR' Lipids 6 2. Properties of Triglycerides and Fatty Acids • The presence of unsaturation (cis C=C bonds) in the fatty-acid portions lowers the melting point of the triglyceride. • Saturated fatty acid has a regular shape, while unsaturated cis fatty acid does not. The introduction of cis fatty acid into the triglyceride makes it more difficult for the triglyceride to pack together. • The cis C=C bond adds kinks to the structure. Thus, triglycerides with more cis unsaturation are even harder to pack, resulting in even lower melting points. (Analogous to the BP of straight and branched alkanes) • Naturally occurring fatty acids are almost always cis and rarely trans. Lipids 7 • The presence of unsaturated fatty acids in triglycerides results in lower melting points, and the same trends apply to the fatty acids themselves. O Stearic acid (18:0) MP = 70°C OH O OH Some trans (18:1) MP = 52°C O OH Oleic acid (18:1) MP = 16°C O Linoleic acid (18:2) MP = –5°C OH A monounsaturated fatty acid has one C=C bond, while a polyunsaturated fatty acid has two or more C=C bonds. (See food labels) • Due to the way they’re synthesized, fatty acids usually occur in integral numbers of two-carbon units, most commonly C16 and C18. Lipids 8 • Fatty acids have both a hydrophilic (polar) head and a hydrophobic (non-polar) tail. O Tail Head OH o In other words, we have a mixture of a highly water-soluble portion (head) with a portion that is highly water-insoluble (tail). • Fatty acids are practically insoluble in water. This is because they are weak acids (pKa ~4.75), and the majority of the molecules will remain unionized. Only the charged, ionized form (RCOO−) is water-soluble. o Although fatty acids are essentially carboxylic acids, we don’t call the short ones, such as acetic acid, fatty acids. Lipids 9 • Like most organic compounds, fatty acids are they are less dense than water, so they will form a layer at the surface if mixed with water. o A portion of the molecule wants to be with water (the hydrophilic head), and a portion that wants to be away from water (the hydrophobic tail). o The best arrangement is a monolayer, where the fatty-acid heads face the water and the tails are exposed to the air. air hydrophobic interactions (van der Waals forces) between tails H-bonding and polar interactions between heads and with water water surface o Note that the monolayer is in equilibrium with the tiny amount of ionized fatty acid that is dissolved in the water. Lipids 10 B. In-the-lab Preparation and Biosynthesis of Fatty Acids • In natural systems, fatty acids almost always have an even number of carbon atoms, usually in the range of 12-20 and most commonly 16 or 18. They are also biosynthesized not as acids, but as esters. • This even-number phenomenon arises from an abundant carbon source used for biosynthesis: a derivative of acetic acid. Long fatty acids are generated by joining multiple acetyl units together, e.g. a C18 fatty acid has nine C2 units. O # new C-C bonds? OH • A crucial step in fatty-acid synthesis is the formation of a new C-C bond between two acetate-ester units. How many C-C bond forming reactions do you know of? o Cyanide as a nucleophile Å cyanide in a biological system? o Grignard reagent Å present biologically? o Aldol condensation Å works with aldehydes and ketones o Claisen condensation Å an α-carbon reaction that works with esters • We’ll first determine how we could synthesize a fatty acid in the lab and then see how these in-the-lab concepts could also be extended to biosynthesis. Lipids 11 1. In-the-lab Synthesis of Fatty Acids • The key step is the formation of a new C-C bond by joining two acetate esters together via a Claisen condensation. This is a nucleophilic acyl substitution, where the enolate of one ester acts as a carbon nucleophile, replacing the OR group. O O O Na OR (strong base) H3C OR H3C OR β-keto ester Lipids 12 • A series of chemical conversions takes us to a saturated chain. O O multiple steps O OR OR repeat Claisen to yield C6 ester Lipids 13 • The four types of reactions that must occur biologically are exactly the same as those required in the lab synthesis. 1. Formation of C-C bond, resulting in a β-ketoester 2. Reduction of carbonyl group to produce a β-hydroxyester 3. Dehydration of alcohol to form an α,β-unsaturated ester 4. Reduction of C=C, which leads to the saturated alkyl chain • The biosynthesis cannot be identical to the laboratory synthesis, because: o Claisen reactions also have equilibrium constants that do not favour the products. One way to increase the products is to increase the amount of enolate, but this is difficult at physiological pH, as the α-H is not very acidic. Biological solution: modify it and perform a Claisen-type condensation o Since we’re adding acetate units one at a time, we need a way to prevent the growing chain from diffusing away until the desired length is reached. o NaBH4, conc H2SO4, and H2/Pt don’t exist biologically Lipids 14 • Up to this point, some of the concepts we’ve covered include: o Properties of fats, oils, and fatty acids o Mechanisms of ester hydrolysis (nucleophilic acyl substitution) o Claisen Condensation o Chemical steps needed for fatty-acid synthesis in the lab • Attempt: o Practice problems in the lab manual: 1 – 7 o 2006 Midterm: 19 o 2007 Term Test #2: 13 • It is crucial at this stage that you thoroughly understand the Claisen condensation. Lipids 15 2. Biosynthesis of Fatty Acids a. Modified Claisen condensation • A modified Clasien condensation is used so that the reaction favours the products under physiological conditions. This is done by: o Using a thioester (RCOSR’) instead of a regular oxygen ester (RCOOR) o Using a α-carbanion equivalent instead of a true carbanion 1. Thioesters • Thioesters (an acid derivative) are more reactive than their oxygen counterparts. o The first reason is because RS− is a better leaving group than is RO−. O O O + SR SR SR Lipids 16 o The second reason is because the reactivity of an acid derivative O depends on the amount of δ+ charge on the carbon of the C=O. How likely the C is going to be attacked by a Nu depends on its δ+. δ+ X For example, O esters are more reactive than amides, because nitrogen is a better resonance donor and can stabilize the δ+ charge. Similarly, S esters are more reactive than O O O esters, because S cannot donate very well by resonance: it cannot overlap a 3p orbital with 2p. OR SR + i.e. the δ charge is larger in thioesters.