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Chemistry 2223B Topic 5 - Lipids.pdf

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Western University
Chemistry 2223B
James Wisner

Chemistry 2223b  Winter 2013–14  Western University Topic 5: 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 that is important includes: o Reactions of carboxylic acids and acid derivatives o Reactions of carbonyl compounds o Reactions of the -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. They are divided into two general categories based on what they contain: o A relatively large hydrophobic region and a small hydrophilic region  Triglycerides (fats and oils)  Fatty acids  Phospholipids  Prostaglandins  Leukotrienes o A common structural element  Steroids and derivatives: fused tetracyclic system  Terpenes: isoprene building blocks Lipids  3 B. Fats, Oils, and Fatty Acids 1. Fats and Oils: 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).  In the laboratory, triglycerides can be hydrolyzed under acidic or basic conditions (review the hydrolysis of esters): O CH 2 C R CH 2H O H or OH – 3 RCOOH HC O C R HC OH O CH O C R CH OH 2 2  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.  Fatty acids on their own (not esterified to glycerol) are not found in substantial amounts in the body. Lipids  4 2. Properties of Triglycerides and Fatty Acids  The presence of unsaturation (especially cis C=C bonds) in one or more alkyl chains lowers the melting point of the triglyceride by making the triglyceride harder to pack together. Naturally occurring fatty acids are almost always cis and rarely trans. Completely saturated Cis alkene makes big kink compared to trans alkene  This is why highly saturated fats (such as butter) are solids at room temperature, while highly unsaturated oils (such as olive oil) are liquids at room temperature. Lipids  5  The effect of unsaturation on melting point can be seen even in the individual fatty acids themselves. O Stearic acid (18:0) MP = 70°C OH O Some trans (18:1) MP = 52°C OH O Oleic acid (18:1) MP = 16°C OH O OH Linoleic acid (18:2) MP = –5°C A monounsaturated fatty acid has one C=C bond, while a polyunsaturated fatty acid has two or more C=C bonds. (See food labels) Lipids  6  Fatty acids have both a hydrophilic (polar) head and a hydrophobic (non-polar) tail. O Tail Head OH  Because of the long, hydrophobic tails, fatty acids are practically insoluble in water unless they are ionized (RCOO ). Although fatty acids are essentially carboxylic acids, we don’t call the short ones, such as acetic acid, fatty acids.  The pK oa the carboxyl group is about 5, similar to that acetic acid. When fatty acids are added to water, the majority of the molecules remain unionized. Only a tiny amount is ionized because the ionization decreases the pH of the water, stopping further ionization from taking place. Lipids  7 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 12-24 and most commonly 16 or 18. Furthermore, they are 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. 18C fatty acid has nine2C 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  adds a single carbon (as a cyano group) o Grignard reagent  adds an alkyl group o Aldol condensation  a Nu addition reaction of aldehydes and ketones o Claisen condensation  a Nu acyl substitution reaction using carbanions  We’ll first determine how we could hypothetically synthesize a fatty acid in the lab and then see how these in-the-lab concepts could also be extended to biosynthesis. Lipids  8 1. Hypothetical 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 Na OR (strong base) O O H 3 OR H3C OR -keto ester Very good nucleophile Was electrophilic Was nucleophilic Lipids  9  A series of chemical conversions takes us to a saturated chain. O O multiple steps O OR OR Add another C2unit and do the series of reduction, oxidation, and reduction reactions to a give C ester 6 Lipids  10  The four types of reactions that must occur biologically are exactly the same as those required in the lab synthesis. However, the biosynthesis cannot be identical to the laboratory synthesis, because: o NaBH , 4onc H SO ,2and4H /Pt do2’t exist biologically; o Claisen reactions also have very small equilibrium constants; and  In the lab, we can make the products more favourable by increasing the amount of enolate; this can be done by using stoichiometric amounts of strong base. However, this is difficult to do at physiological pH because the -H is not very acidic.  Biological solution: perform a “modified” Claisen-type condensation using a more-reactive acid derivative and carbanion equivalents. o We’re adding acetate units one at a time, and we need a way to prevent the growing chain from diffusing away until the desired length is reached. Lipids  11 2. Biosynthesis of Fatty Acids a. Modified Claisen condensation  A modified Clasien condensation is used to make the reaction more favourable under physiological conditions. Modification #1: use thioesters instead of oxygen esters  Thioesters are more electrophilic than oxygen esters. O O OR SR smaller  larger  − −  RS is a better leaving group than is RO . (The pKa of a thiol is about 8, while that of an alcohol is about 16.) O O O + SR SR SR Lipids  12 Modification #2: use carbanion equivalents  We rely on the decarboxylation of malonyl thioester to generate the enolate and drive the Claisen reaction to completion in the absence of a strong base. O O –CO 2 O O O SR SR SR malonyl thioester enolate of acetyl thioester  Although we have made the enolate without the use of a base,2the loss of CO is concomitant with nucleophilic attack. In other words, we avoid the enolate completely. i.e. malonyl thioester is the carbanion equivalent. O acetyl2C O O O O –CO2 SR SR O O SR SR SR O SR C4-keto thioester malonyl3C Lipids  13  Malonyl thioester is made from acetyl thioester using 2TP and biotin, a CO carrier. This enzymatic reaction 2ses CO dissolved as bicarbonate. (Note: This is the same CO2that is lost in the modified Claisen reaction). O Biotin O O + HCO3– SR ATP ADP, Pi O SR − o ATP first phosphorylat3sto form carboxyphosphate (mechanism is similar to other phosphorylations), which is then used to form carboxybiotin. O O O O P O HO O P O O O O O O H N NH HO N NH R R R R Lipids  14 o The carboxyl group is subsequently transferred to acetyl CoA. O O O HO N NH HN NH R R R R O O O SR HO SR Lipids  15  In one cycle, acetyl-CoA grows to a four-carbon acyl chain: O O O SR O SR acetyl thioester malonyl thioester (made from acetyl thioester) Once at the butyryl thioester, use it instead of acetyl thioester O O for the next cycle. And repeat... O SR -ketothioester SR butyryl thioester O OH O SR SR unsaturated thioester -hydroxythioester Lipids  16 b. The diffusion problem  Fatty acids are typically synthesized16molecule (additional modifications are possible, such as extension 18 C and other reactions). Because the fatty acid grows 2 C at a time, it must not be allowed to diffuse away until16t reaches C .  The reaction involves the use of an acyl carrier protein (ACP) with a long “arm” made from pantothenic acid and other components, and with a thiol group. O OH C CH O C i 3 3 O p HS O O CH p n N N P l a H H NH p c OH O from pantothenic acid (Vitamin B5)  The reactions are done on a multi-enzyme complex, fatty acid synthase (FAS), which also contains a thiol group. ACP is strongly bound to FAS, so the growing chain does not float away until it is deliberately detached.  Diagrammatically, we have…. Lipids  17 O O O C H 3 SCoA The acetyl group is loaded onto - HS the fatty acid synthase O CH2 SCoA P FAS complex HS A The malonyl-ACP component is bound to an ACP-binding site on FAS. - CoA O Both reactions are catalyzed by C O O H3C S enzymes with transacylase activity - FAS O CH2 S C A The long arm of ACP permits movement for the Claisen- type condensation reaction O - H3C C and allows the product to O access other units of FAS for S O CH 2 subsequent transformations P FAS O A S Lipids  18 O O HS An enzyme catalyzes the H3C CH2 S P FAS Claisen-type condensation A -ketothioester + Reduction: NADPH + H The reduction is OH O HS enzymatically performed H3C CH2 S P FAS C -hydroxythioester A Dehydration Dehydration is also O HS enzymatic H C S P FAS 3 A -unsaturated thioester Lipids  19 O HS Enzymatic reduction FAS H 3 S C of C=C bond to give A a fully reduced tail swap positions O H C S 3 growing chain moves HS FAS to spot of original C electrophile for next A ACP controls reaction cycle new electrophile O add new malonyl thioester Nu S Ready for next cycle H3C O O FAS O CH2 S Same as first step Lipids  20  Note that the growing fatty acid is always attached to either ACP, which is in turn attached to FAS, or directly to one of the enzymes in the FAS complex. o The electrophile is always the growing chain, and it grows at the thioester end. o The nucleophile is always malonyl thioester, regardless of the length of the growing chain. Remember that the use of malonyl thioes3er, a C compound, only adds an acetate uni2 (C ), because one carbon is l2st as CO . o There are two reductions and one dehydration per cycle.  After seven cycles, we will have made seven new C-C bonds a16 a C fatty acid. 7 new C-C bonds O C C C C C C C C C C C C C C C C SR acetyl malonyl-derived2C units derived  Because the malonyl-derived units are made from acetyl thioe2ter + CO , and the CO 2s later lost, we can say 16e C compound originates from eight acetyl units. Lipids  21 c. Reduction and dehydration reagents  How do we perform the reduction, dehydration, and reduction reactions? 1. Reduction of ketone to alcohol by NADPH O O OH O SR SR -ketothioester -hydroxythioester 2. Dehydration of alcohol to alkene OH O O SR SR -hydroxythioester -unsaturated thioester  Compare this reaction to the formation of phosphoenolpyruvate in glycolysis. Lipids  22 3. Reduction of alkene to alkane by NADPH O O SR SR -unsaturated thioester saturated tail  Alkenes normally cannot be reduced by hydride reagents (Na4H , NADH, NADPH, etc.) because hydrides do not attack nonpolar C=C bonds. However, it works in this case because the alkene is conjugated to the C=O. A resonance structure shows that the -carbon has positive character and is therefore electrophilic. Polarized to be more O because of resonanceO SR SR  Reaction with NADPH would generate the enol form, which tautomerizes to the product. The addition of a nucleophile to the  carbon of an ,-unsaturated carbonyl is termed conjugate addition and has the name Michael Addition. Lipids  23 d. Other fatty acids  The normal product of fatty-acid biosynthesis in animals is palmitic acid (16).  Separate elongase and desaturase enzymes are responsible for elongating this fatty acid and producing monounsaturated fatty acids, respectively. Desaturase enzymes are remarkable in that they introduce a cis alkene using atmospheric oxygen as the oxidizing agent. (We won’t look at these details). Palmitic acid (C16 O elongase OH Stearic acid (C18 Oleic acid C 18 9 desaturase We can’t desaturate any further than delta 9 Lipids  24  There are some polyunsaturated fatty acids (PUFA’s) that humans cannot synthesize and must obtain in their diet. These two are the “parent” PUFA’s, as they can be converted to other necessary PUFA’s by the liver when consumed. They have desaturation further than 9 OH Linoleic acid 189,12 An omega-6 fatty acid, which has a double bond six carbons from the omega end (methyl) O OH 9,12,15 Alpha linolenic acid (ALA) 18 An omega-3 fatty acid, which has a double bond three carbons from the omega end (methyl) Lipids  25 C. Catabolism of Triacylglycerides and Fatty Acids 1. Hydrolysis of Triacylglycerides  Triacylglycerides are poorly absorbed from the digestive tract, but the hydrolysis products can be absorbed. Hydrolysis is performed by lipase enzymes.  Lipases use the same catalytic triad that is found in chymotrypsin (Asp-His-Ser), and may act on one, two, or all three of the esters. The reaction for the hydrolysis of all three esters is shown below. O CH2O C R CH2OH O lipases HC O C R 3 H2O HC OH 3 RCOOH O CH2O C R CH2OH Lipids  26 2. Phosphorylation and Oxidation of Glycerol  Glycerol, a three-carbon compound, is readily converted into a glycolysis intermediate via a phosphorylation followed by an oxidation. CH OH CH OH CH OH 2 ATP 2 NAD + 2 H OH H OH O CH 2H CH 2PO 32- CH 2PO 32- dihydroxyacetone phosphate 3. -Oxidation of Fatty Acids  Fatty acids are degraded into acetyl CoA via a pathway known as -oxidation, where the  carbon is oxidized from an alkyl carbon to a ketone. The pathway is quite similar to the reverse of fatty-acid biosynthesis, but with some differences. Lipids  27  First, the fatty acid is enzymatically converted to the CoA thioester. O O ATP O O AMP HS-CoA If we start wi18 a C O fatty acid, we will obtain 9 acetyl CoA. Where are the 9 units, and which SCoA carbons need to be oxidized? Lipids  28  FAD then oxidizes the saturated tail, resulting in an ,-unsaturated thioester. (Note that in biosynthesis, NADPH was used to reduce the C=C). O SCoA O N NH N N O R O SCoA H O N NH N N O R H Lipids  29  Conjugate addition (Michael addition) of water to the ,-unsaturated thioester forms + a -hydroxy thioester, which is then oxidized by NAD to give a -keto thioester. These two steps are mechanistically opposite to those in biosynthesis. H OH O SCoA OH O SCoA + NAD O O SCoA Lipids  30  The last step is a C-C bond-breaking reaction that uses cysteine residue to expel acetyl CoA as a leaving group. The enolate (-carbanion) of acetyl CoA is protonated as it departs (compare this to a retro-aldol reaction). O O SCoA Enz S H O O S-Enz SCoA HS-CoA O and repeat.... oxidation, hydration SCoA oxidation, cleavage Lipids  31 Summary up to now  Textbook Chapter 19: o Pages covered: 649–653 o Suggested Quick Quiz questions: 2, 5, 10, 14, 16 o Suggested problems: 3, 4, 5, 6, 9, 10, 12  Textbook Chapter 21: o Pages covered: 704–706, 713–716 o Suggested Quick Quiz questions: 1, 5, 9, 10 o Suggested problems: 18, 20  Lab manual: o Practice problems: 1–21 o 2009 Final Exam: 20–26 o Suggestions for the 2011 Final Exam are not included – save that exam to evaluate your performance when preparing for the Final Exam. Lipids  32 D. Soaps and Detergents  Both soaps and detergents perform the same task, but soaps are made from naturally occurring materials, while detergents are considered to be synthetic. 1. Soaps  Soaps are metal salts of fatty acids, prepared by the base hydrolysis of triglycerides. O CH O C R CH OH 2 2 O NaOH O CH O C R HC OH 3R C O Na O CH2O C R CH 2H  The sodium salts are soluble at low concentrations. However, at very high [Na ], the equilibrium below is forced to the left, causing the salt to precipitate (this is useful for isolating and purifying soap). O O R C O Na R C O Na Lipids  33  Individual soap molecules aggregate to form spherical structures known as micelles. These structures form because the hydrophobic tails are attracted to each other via hydrophobic forces.  However, the tails do not pack together tightly. This is because the heads (which also electrostatically repel each other if they have a net charge) are solvated and require a certain amount of space. Micelles are therefore leaky; molecules can enter and exit the micelle. Yet, as a cleaning agent, this is essential so that hydrophobic grease and dirt can enter into the hydrophobic interior. Lipids  34 2. Detergents  Soaps are easily made from natural sources, but there is one major problem with + + them. The Na and K salts of carboxylates are q2+te s2+ubl3+ but the carboxylates precipitate in the presence of polyvalent ions (Ca , Mg , Fe , etc.). O R C O M2+  These polyvalent ions are those found in hard water. Soaps therefore precipitate out and perform poorly in hard water.  One solution is to use synthetic detergents that do not precipitate out in the presence of polyvalent ions. These detergents contain a hydrophilic head that is not a carboxylate group.  Many different detergents are commercially available, and they can be categorized according to the nature of the hydrophilic head. Lipids  35 1. Anionic  One example would be the first detergents developed in the 1940s, and these are the alkylbenzene sulfonate
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