Chem 2223b Intersession 2008: Carbohydrates

Transcription

1 Department of Chemistry, The University of Western ntario Chem 2223b Intersession 2008: Carbohydrates This chapter discusses the fundamental chemistry and properties of carbohydrates, emphasizing on monosaccharides. The metabolism of glucose (glycolysis and the citric acid cycle) is also introduced, either descriptively or mechanistically. Background material, from Chem 2213a or otherwise, that is important includes: o Stereochemistry, enantiomers and diastereomers, Fischer projections o Reactions of alcohols, carbonyl compounds, and carboxylic acids ucleophilic addition ucleophilic substitution xidation and reduction

2 A. Biological Significance and Categories of Carbohydrates Carbs 2 Carbohydrates are the most abundant, naturally occurring class of biomolecules: they constitute half of the world s biomass (dry weight) They are made primarily by plants via photosynthesis. In turn, we consume them for energy. ence, they also serve as a mechanism of energy storage. Light energy + C carbohydrates + 2 Carbohydrates can also have structural roles o Cellulose: wood o Chitin: exoskeleton material of insects They are also found in some essential biomolecules, such as nucleic acids.

3 Carbs 3 Carbohydrates are also present as integral components of many biologically active compounds, either natural or synthetic. For example: o Salicin, which contains D-glucose, is an anti-inflammatory from willow bark. When consumed, it is metabolized to salicylic acid. o Adriamycin, which contains a modified sugar, is an anti-cancer agent that is used for breast cancer and was isolated from Streptomyces achromogenes. C 2 C 2 Salicin C 3 3 C 2 Adriamycin

4 Carbs 4 Carbohydrates are found as components of not only small biomolecules, but also in large, high-molecular weight biopolymers. These include: o Glycoproteins mainly protein with some short, branched carbohydrates, e.g. Blood-group determinants (A, B, ) Used for cell-cell interactions and recognition o Proteoglycans long, linear carbohydrates with minor protein portions. These are also referred to as mucopolysaccharides. e.g. Animal connective tissues, such as ligaments Mucus, snot, and lubricating fluid for joints o Peptidoglycans long, linear carbohydrates that are crosslinked by short oligopeptides, as found in rigid and strong bacterial cell walls. Their biosynthesis is inhibited by penicillin antibiotics o Lipopolysaccharides fatty acids linked to carbohydrates, as found in the outer cell envelope of Gram-negative bacteria.

5 From a chemical perspective, the term carbohydrate refers to a diverse class of organic compounds that are mainly composed of C,, and. The empirical formula is that of a hydrated carbon, i.e. C n ( 2 ) n. This is commonly written as C n 2n n, and most simple sugars adhere to this formula. Carbohydrates are simply polyhydroxyaldehydes or polyhydroxyketones Carbs 5 Carbohydrates can also be derivatives of such compounds. These derivatives can contain functional groups (e.g. 2 ) that replace the groups, and some functional groups may be oxidized or reduced.

6 Carbs 6 Carbohydrates are often called saccharides by scientists. This is from the Latin word saccharum, which stands for sugar. A monosaccharide is defined as a carbohydrate that cannot be hydrolyzed in the lab (using + / 2 ) to a simpler carbohydrate. Carbohydrates can also be in a polymeric form (poly = many; mer = parts), i.e. chains of discrete units (monomers) linked together. o The smallest, individual units are the monosaccharides o The term polysaccharide is usually reserved for chains containing many monosaccharides (typically > 10). Usually naturally isolated (hard to make). o ligosaccharide (oligo = short) is used for anything in between. It can be made in the lab and may have useful biological properties.

7 B. Monosaccharides Carbs 7 The study of monosaccharides is fundamental to the understanding of oligo- and polysaccharides, since these polymers are comprised of linked monosaccharides. 1. Classification Monosaccharides can be broadly classified according to their carbon atoms. A prefix indicating the number of carbons is followed by the suffix ose. o i.e. triose, tetrose, pentose, hexose, octose, etc. This can further be broken down depending on the carbonyl group present. o Those with aldehydes are classified as aldose sugars (aldo ) o Similarly, those with ketones are classified as ketose sugars (keto ) owever, aldose and ketose are still relatively broad classifications and do not indicate the number of carbons.

8 Carbs 8 So, we combine the aldose or ketose with the designator indicating the number of carbons. o Glucose is a C 6 sugar with an aldehyde, so it is a hexose, or more accurately, an aldohexose. o Ribulose is a C 5 sugar with a ketone, so it is also a pentose, but more accurately described as a ketopentose. D-glucose D-ribulose Very commonly, the keto is dropped and the modified suffix ulose is used instead. Thus, an ulose compound is the same as a ketose. o e.g. ketopentose = pentulose The most-common ketoses have the carbonyl group at carbon #2, so when the position is not stated, the keto group is assumed to be at that position.

9 2. Representations Carbs 9 The smallest and simplest monosaccharides are trioses (C 3 sugars). There are only two trioses, one of which is an aldose and the other a ketose. Glyceraldehyde (an aldotriose) * C C C 2 Dihydroxyacetone (a ketotriose/triulose) C 2 C C 2 Since stereocentres are involved, monosaccharides in their open-chain form are often drawn in Fischer projections, which are useful for compounds with multiple stereocentres. By convention, we place the most-oxidized carbon at the top. Recall that in Fischer projections, horizontal bonds are projected to the front of the page, while vertical bonds are projected towards the back. C C C C = = C 2 C 2 C 2 C 2 D-Glyceraldehyde (R enantiomer) L-Glyceraldehyde (S enantiomer)

10 3. D/L System of omenclature Carbs 10 In 1891, Emil Fischer knew that two enantiomeric forms of glyceraldehyde existed, both of which exhibited optical activity when placed into a polarimeter. ne of these enantiomers rotated plane-polarized light (PPL) to the right ([α] = +), while the other rotated PPL to the left ([α] = ). Two enantiomers will each rotate PPL to the same magnitude, but in opposite directions. Realize that the direction of light rotation does not correlate with R/S configuration labels. TE: The optical-activity relationship of diastereomers cannot be predicted, and meso compounds do not rotate PPL.

11 Carbs 11 So how did Fischer know which form of glyceraldehyde rotated PPL to the right, and which one to the left? There were no modern techniques available in 1891 that allowed the elucidation of the structures of enantiomers. o e guessed and assumed that the one with the group on the right-hand side, when drawn in his very own Fischer projection, rotated PPL to the right. This was subsequently called D-glyceraldehyde (D = dextro = right). o Likewise, L-glyceraldehyde (L = levo = left) rotated PPL to the left. o All subsequent scientific work was based on Fischer s assumption, with no definite evidence that the enantiomer rotating PPL to the right had the on the right. Fortunately, half a century later, his assumption was proven correct. C C 2 C C 2 D-Glyceraldehyde [α] = L-Glyceraldehyde [α] = 13.5 Thus, for glyceraldehydes only, D corresponds to [α] = +, while L to [α] =.

12 The D/L nomenclature system was subsequently extended to other monosaccharides, including those with multiple stereocentres, and defined as: Carbs 12 o A D-monosaccharide is one that, when drawn in Fischer projection, has the group on the penultimate carbon on the right. Similarly, an L-monosaccharide has the group on the penultimate carbon on the left. o The word penultimate is defined in an English dictionary as second last. In monosaccharides, the second-last carbon is almost always the last chiral carbon, so in this context, penultimate refers to the last chiral carbon. ote that Fischer s assignment of D/L was based purely on glyceraldehyde. For other sugars, there is CRRELATI between D/L and light rotation. The rotation of light is an experimentally determined parameter. As well, the D/L terms are also used to identify other sugars that are enantiomers. If the common name stays the same, then the D/L forms are enantiomers of each other, and all the chiral centres need to be switched. This is all illustrated in the examples provided on the next page.

13 Carbs 13 C C 2 D-(+)-Xylose C C 2 L-( )-Xylose on penultimate carbon L-( )-ame Common name C change configuration of penultimate carbon C measured PPL ote the D/L relationships, the direction of PPL rotation, and the stereoisomerism (enantiomers/diasteromers) between the four compounds. What is noticeable? C 2 C 2 name? D-( )-Arabinose

14 Carbs 14 Chart of D-aldoses (included on exams... you can use this to draw L-aldoses) C C C 2 Glyceraldehyde C C 2 C 2 C Erythrose C C Threose C C 2 C 2 C 2 C 2 Ribose Arabinose Xylose Lyxose C C C C C C C C C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 Allose Altrose Glucose Mannose Gulose Idose Galactose Talose

15 Carbs 15 Chart of D-ketoses (included on exams... you can use this to draw L-ketoses) C 2 C 2 Dihydroxyacetone C 2 C 2 Ribulose C 2 C 2 Erythulose C 2 C 2 Xylulose C 2 C 2 C 2 C 2 C 2 C 2 C 2 C 2 Psicose Fructose Sorbose Tagulose

16 Carbs 16 Modified monosaccharides are found naturally. While this chart is T provided on exams, you should be able to draw them using the other two charts if the name is provided (e.g. deoxy-amino-d-glucose can be drawn from D-glucose). These are some of the deoxy, amino, acetylamino, and oxidized sugars. C C 2 deoxy-ribose C 2 C 2 deoxy-amino-d-glucose "glucosamine" C CC 3 C 2 deoxy--acetylamino-d-glucose "-acetyl glucosamine" C C D-glucuronic acid 2 C C 2 deoxy-amino-d-mannose "mannosamine" C 2 C 2 deoxy-amino-d-galactose "galactosamine"

17 4. Formation of emiacetals Carbs 17 Monosaccharides have hydroxyl and carbonyl groups that can react together and form hemiacetals. The reaction is exclusively intramolecular, because these reactions are much more favoured over intermolecular ones. Due to the high favourability of intramolecular hemiacetals, monosaccharides exist almost entirely as five- or six-membered cyclic forms. o A five-membered cyclic sugar is called furanose sugar o A six-membered cyclic sugar is called a pyranose sugar o These names originate from compounds furan and pyran The formation of a hemiacetal most commonly involves the on the penultimate carbon linking with the carbonyl. Fischer projections are not convenient for drawing cyclic compounds, so a aworth projection (side view) is used. C C 2

18 a. Drawing aworth projections Carbs 18 C C = 2 C C C 2 C2 2 C 2 C C 2 A new stereocentre is formed a squiggly line indicates undefined stereochemistry.

19 Carbs 19 While the previous page explains the conversion in detail, it is desirable to be able to convert a Fischer to a aworth quickly and skipp the details. e.g. D-glucose C C 2 2 C C (link C= to boxed ) C 2 ow did we do this??? C 2 (ydrogens implied)

20 Carbs 20 The same can be done with an L-sugar, e.g. L-glucose. C C 2 2 C C C 2 Even in the aworth projections, it can be seen that L- and D-glucose are enantiomers. C 2 C 2 The aworth projections also easily show the cis/trans relationships of the ring substituents. D-Glucose C 2 L-Glucose turn over L-Glucose

21 Mechanism of acid-catalyzed hemiacetal formation (reversible): Carbs 21

22 Mechanism of base-catalyzed hemiacetal formation (also reversible): Carbs 22

23 Carbs 23 b. The anomeric carbon The new stereocentre formed in the hemiacetal originates from the carbonyl carbon, and this carbon is called the anomeric carbon. ther stereocentres are unchanged. Since the carbonyl group is sp 2 (flat) and freely rotates about the C-C bond, nucleophilic attack by the penultimate results in two possibilities: α or β forms. C 2 C 2 β anomer C 2 C 2 α anomer These two compounds, which differ only by configuration at the anomeric carbon, are called anomers. They are also a pair of diastereomers.

24 In the diagrams on the previous page, the α anomer has the hemiacetal group down, while β anomer has the hemiacetal up. Carbs 24 Frequently, the up/down relationship of the hemiacetal group is used to assign α or β. owever, this is incorrect because whether it is up or down depends on how you look at the ring. Thus, a better way is to compare it to a point of reference: o α if on anomeric carbon is trans to the terminal C 2 o β if on anomeric carbon is cis to the terminal C 2 o This method of assigning α/β works only if the penultimate is involved in hemiacetal formation. ther groups can give rise to α/β as well, but we won t examine the details on assigning α/β to non-penultimate groups. C 2 α-d-glucose (α-d-glucopyranose) α-d-glucose β-d-glucose (conformational representations)

25 Carbs 25 Ketoses can be drawn and assigned α/β exactly the same way as aldoses: C 2 C 2 D-Fructose Since anomers are diastereomers, they have different properties. Anomers do not interconvert in the solid phase, because water is required for the reaction. α-d-glucose [α] =+112 β-d-glucose [α] =+18.7

26 c. Mutarotation Carbs 26 owever, anomers interconvert in solution phase, because hemiacetals readily revert back to their carbonyl and alcohol components. Even at neutral p, there is sufficient acid present to catalyze the reaction. Thus, when one anomer is dissolved, an equilibrium mixture of both will form. Since process this can be monitored by optical rotation, and it is called mutarotation. ote that the rotations cannot be predicted and must be experimentally measured. Monosaccharide pure anomer [α] [α] after mutarotation % present at equilibrium α-d-glucose β-d-glucose (why more β?) α-d-galactose β-d-galactose

27 5. Formation of Glycosides (Acetals of Sugars) Carbs 27 emiacetals can react with another alcohol to form glycosides, which are acetals of sugars. The alcohol is an oxygen nucleophile, so these are also called -glycosides. C 2 C 2 + / C 3 C 2 C 2 Me Me Methyl β-dglucopyranoside Methyl α-dglucopyranoside Glycosides are named by listing the name of the group attached to the oxygen, followed by the name of the carbohydrate, but ending in ide. These glycoside linkages are used to join monosaccharides together (more later).

28 Carbs 28 The mechanism of hemiacetal to glycoside conversion is S 1, which involves a carbocation intermediate. i.e. a mixture of α and β glycosides is formed. C 2 α and/or β + / C 3 C 2 Me Both α and β

29 Carbs 29 ote: Glycosides (acetals) are stable in basic and neutral p. They will T revert to their open-chain forms unless either acid or a glycosidase enzyme is present. Glycoside linkages are found in drugs (page 1) and natural products. Examples: Me coniferin is the main glycoside found in the sap of conifer trees The anomeric carbon of a cyclic hemiacetal can also react with amines to form -glycosides. This forms in a mechanistically similar way to -glycosides. 2 -glycosides are found in nucleosides components of nucleic acids. ote that in both - and -glycosides, the anomeric C is attached to two heteroatoms.

30 Carbs 30 Up to this point, some of the concepts we ve covered include: o omenclature and categorization o Fischer and aworth projections o Conformational representations o Formation of hemiacetals and glycosides (acetals) o Mutarotation Using the carbohydrate structures provided in the lab manual, attempt: o Practice problems: 1 20 o 2006 midterm: 1, 3, 5, 10, 11 o 2006 intersession midterm: 1, 2, 3, 4, 6, 13, 15 o 2007 term test #1: 1 10 o 2008 midterm: 23 28

31 6. xidation and Reduction Reactions Carbs 31 Since carbohydrates contain carbonyl groups and alcohols, they can undergo oxidation/reduction reactions. a. xidation to Aldonic Acids The aldehyde group of aldoses can be oxidized to carboxylic acids. Sugars with this C C oxidation are referred to as aldonic acids. Realize that because hemiacetals are in equilibrium with the open-chain form, they are also oxidized if the hemiacetal is derived from an aldehyde. C xidizing Agent C D-Gluconic acid C 2 C 2 nly the C is selectively oxidized by weak oxidizing agents. Several methods can perform this task without oxidizing any of the alcohol groups.

32 Carbs 32 Bromine dissolved in water ( bromine water ): o Relatively gentle technique and selective for aldehydes o In the reaction, molecular Br 2 is reduced to two Br ions o Bromine water is also slightly acidic, and lactones are formed via a Fischer esterification. owever, it is not acidic enough to hydrolyze acetals. o Recall that a lactone is a cyclic ester, an acid derivative. It can be hydrolyzed back to the corresponding carboxylic acid (or acid salt) and alcohol components in a separate step. C 2 Br 2 / 2 C 2 a / 2 C 2 a

33 Carbs 33 Metal ions: Tollen s Reagent Ag( 3 ) 2 in aqueous ammonia (functions as Ag + ) xidation of aldehyde to acid is indicated by Ag (s) formation (silver mirror) Benedict s or Fehling s Reagents Cu 2+ in citrate (Benedict s) or tartrate (Fehling s) buffer xidation of aldehyde to acid is indicated by Cu 2 (s) formation (red solid) These are useful qualitative tests for aldehyde sugars (Experiment 1). With both bromine and metal-ion reagents, the reagents are themselves reduced. Because the aldehyde group of the sugar reduces the oxidant, these sugars are called reducing sugars. R C R xidant Reduced oxidant C

34 Carbs 34 b. xidation to Aldaric Acids If nitric acid ( 3 ), a stronger oxidizing agent, is used, both the aldehyde and the primary alcohol are oxidized. owever, 3 is too weak for secondary alcohols. Analysis of the oxidation product can provide useful structural information on the original sugar. D-Glucose D-Glucaric acid D-Galactose D-Galactaric acid C C 3 3 C 2 C 2 optically active optically active Remember, meso compounds are optically inactive.

35 Carbs 35 c. xidation to Uronic Acids What if we wanted to leave the aldehyde alone, but oxidize only the primary alcohol to a carboxylic acid? There is no easy way in the lab, but enzymes could do it. Glucuronic acid is used by the liver to detoxify toxic substances containing hydroxyl groups. This is done by combining glucoronic acid and the substance to form glucuronides, which may then be excreted in the urine. (The carboxylic acid functional group increases the water solubility of the glucuronide). C enzyme C C C 2 D-Glucose C D-Glucuronic acid forms glycoside with toxic substance Some drugs, such as morphine, TC, and anabolic steroids, are excreted as metabolites bound to glucuronic acid. Drug testing looks for these compounds.

36 Carbs 36 d. Reduction to Alditols Carbonyl groups can be reduced to alcohols by a variety of reducing agents, including catalytic hydrogenation ( 2 / metal), ab 4, and LiAl 4. C C 2 D-Glucose Reductant C 2 C 2 D-Glucitol (D-Sorbitol) These sugar alcohols are poorly absorbed and metabolized by the body o Sorbitol is commonly found in sugar free candy o Xylitol is found in sugar free gum Reduction may result in stereocentres if the reduced carbon is bonded to four different substituents (work out the reduction of D-fructose at home).

37 7. Reactions of the α-carbon Carbs 37 Aldehydes and ketones featuring a hydrogen on the α-carbon can form enolate anions. The formation of enolates destroys stereochemistry at the α-carbon. C B C C a. Epimierzation and Isomerization Example: if any one of D-glucose, D-mannose, or D-fructose is placed in a basic solution, the same equilibrium mixture of all three is formed. C C 2 D-Glucose 57% C C 2 D-Mannose 3% C 2 C 2 D-Fructose 28%

38 Carbs 38 Epimers are diastereomers that differ in the configuration of any one stereocentre. Whereas, D-mannose and D-fructose are isomers. Although these are shown in the open-chain form, recall that the open-chain forms are also in equilibrium with the cyclic hemiacetals. Mechanism of epimerization: R R

39 Carbs 39 Isomerization also involves the α-carbon, but proceeds through an ene-diol rearrangement (the ene-diol is a tautomer of the aldehyde/ketone): R R C 2 R Since Tollen s Reagent is alkaline, ketoses will slowly isomerize to aldehydes and show a positive reducing-sugar test. The enzyme triose phosphate isomerase interconverts dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in glucose metabolism.

40 b. Aldol Reactions Carbs 40 Enolates can attack other aldehydes or ketones in a reaction known as the aldol reaction. This is simply a nucleophilic addition reaction, and it is one that forms a new carbon-carbon bond. Example with acetone: A β-hydroxy carbonyl compound is formed, where the C with the is the point of connection between the two molecules. The nucleophile retains the C=, while the electrophile becomes the alcohol.

41 Carbs 41 Biologically, the enzyme aldolase catalyzes the reversible formation of fructose- 1,6-bisphosphate in glucose biosynthesis. ne postulated mechanism: dihydroxyacetone phosphate C 2 P 3 C Enz-B glyceraldehyde- 3-phosphate C 2 P 3 C C 2 P 3 + from enzyme C 2 P 3 C 2 P 3 β-hydroxy group fructose-1,6-bisphosphate The reverse reaction (glycolysis) is called a retro-aldol reaction. A deficiency of the enzyme aldolase has serious clinical manifestations.

42 8. Acylation of Groups (Esterification) Carbs 42 The alcohol groups on can be converted into esters using an acid derivative that is more reactive than the ester, such as acetic anhydride. Acylation refers to the addition of acyl groups to the alcohols to form, in this case, acetyl esters, by a nucleophilic acyl substitution reaction. While the anhydride is quite reactive, the presence of acid accelerates the reaction. C 3 + C 3 Methyl β-d-glucopyranoside In the lab, you ll be preparing cellulose acetate by the acetylation of cellulose.

43 Mechanism of acid-catalyzed acetylation using acetic anhydride: Carbs 43

44 9. Alkylation of Groups (Etherification) Carbs 44 Alcohols can be converted to alkoxides in the presence of a base. Alkoxides are good nucleophiles and can react with alkyl halides to form ethers, in a reaction mechanism known as: These reactions prefer polar, aprotic solvents. Recall that these are solvents that cannot hydrogen bond. ne of these is dimethylsulfoxide, abbreviated DMS. Sodium hydride is commonly used as a base, as the 2 byproduct bubbles away. R 1. a / DMS 2. C 3 I Me Me Me Me R

45 10. Kiliani-Fischer Synthesis of Monosaccharides Carbs 45 This is a carbon-chain lengthening procedure that extends the monosaccharide by one carbon. The procedure is used for the synthesis of rare sugars that are difficult to isolate, and it is also historically important in the determination of sugar structure. A key step in the reaction is the formation of a carbon-carbon bond via the nucleophilic addition of cyanide to the aldehyde, forming a cyanohydrin. This results in the formation of a new stereocentre. C C R R R C C C R R Because a new stereocentre is formed, the lengthening of one aldose results in two epimeric products. i.e. two diastereomers are present in one flask. There are two synthetic routes: a traditional method and a modern one.

46 Traditional method (synthesis of D-talose and D-galactose from D-lyxose shown): Carbs 46 C C 2 ac C C 3 + C 2 2 C C C C C 2 C 2 C - 2 a + C a C C 2 C C C - 2 a + C C C C C 2 C 2 C 2 C 2 The carboxylic acid spontaneously forms a lactone, which can then be hydrolyzed with base. This yields two diastereomeric salts in one flask, and they can be relatively easily separated by recrystallization (diastereomers have diff. properties).

47 Carbs 47 The separated diastereomers are then placed in separate flasks, converted back to the lactones with acid, and reduced to an aldehyde with a a/g amalgam. i.e. the point of the carboxylic acid salt formation was separation. ote that C-1 in the original sugar became C-2 in the two new sugars. Flask C 2 a C C C C C 2 a/g C C C 2 D-Galactose Flask C 2 a C 3 + C C a/g C C D-Talose C 2 C 2 C 2

48 Modern method: uses a catalyst to reduce the nitrile to an imine. Subsequent hydrolysis of the imine (recall amino acids section) results in the aldehyde. There are fewer steps and fewer toxic reagents. Example: lengthening of D-erythrose Carbs 48 C C C C C 2 ac C 2 + C C Pd / BaS 4 C + / 2 C 2 + C C 2 C 2 C 2 The pair of diasteromeric products could be separated by modern PLC.

49 Carbs 49 Up to now, some of the concepts we ve covered include: o xidation: aldonic, aldaric, and uronic acids o Reduction: alditols o Epimerization and isomerisation, aldol reactions o Acetylation and substitution reactions Using the carbohydrate structures provided in the lab manual, attempt: o Practice problems: o 2006 midterm: 2, 4, 6, 7, 9 o 2006 intersession midterm: 5, 7, 10, 12 o 2007 term test #1: o 2008 midterm: 29 35

50 C. Disaccharides Carbs 50 Disaccharides are composed of two monosaccharides (aldoses or ketoses) connected using an acetal linkage, otherwise known as a glycoside. At least one of the anomeric carbons in the disaccharide must be involved in the acetal linkage, but sometimes, both may be involved. (e.g. lactose vs. sucrose) Acetals are formed and degraded in acidic conditions, and but unlike hemiacetals, they are stable under neutral or basic conditions. Since acetals do not readily interconvert with the open-chain forms, except in the presence of acid, acetals do not mutarotate, nor are they reducing. o If one part of the molecule is a hemiacetal, then the whole molecule, collectively, will mutarotate and be a reducing sugar.

51 1. Disaccharides of D-Glucose: Maltose and Cellobiose Carbs 51 Maltose is found in malt, the juice of sprouted barley and other cereal grains. It is linked by an α(1,4) glycoside bond, and because the compound also has a hemiacetal, it is. 1 4 Cellobiose is derived from the partial hydrolysis of cellulose, and the two monomers are linked β(1,4). 1 4 ote that these two disaccharides differ only in the configuration of the anomeric carbon involved in the glycoside bond.

52 Carbs Lactose Lactose is D-galactose joined β(1,4) to D-glucose. Galactose differs from glucose only in the configuration of C-4. (So, galactose and glucose are a pair of epimers). 1 4 Lactose is commonly referred to as milk sugar, as it comprises about 5% of the weight of milk. The enzyme lactase (also called β-galactosidase) cleaves the β-galactose linkage. Lactose-intolerant individuals exhibit are deficient in this enzyme.

53 3. Sucrose Carbs 53 Known as table sugar, sucrose is obtained from sugar cane and the sugar beet. It is comprised of D-glucose and D-fructose linked α(1,2). This linkage is unusual because it involves the anomeric carbons of both monomers. There are no hemiacetals, so sucrose does not 1 And, sucrose is 2 C 2 ydrolysis of sucrose yields one equivalent each of D-glucose and D-fructose. This is industrially important for two reasons, because this mixture: o Tastes sweeter than the same weight of sucrose o Is less likely to crystallize at high concentration C 2

54 Carbs 54 Because the mixture of D-glucose and D-fructose has a sweeter taste than sucrose, a smaller amount of the mixture is required to achieve a desired tasted. Bees collect sucrose from flowers and then hydrolyze and concentrate it. Compound Relative sweetness ther sweeteners D-fructose 170 oney = 97 D-glucose 74 Molasses = 74 D-galactose 0.22 Corn syrup = 74 Sucrose 100 Lactose 0.16 (does milk taste sweet?) Many beverages, included soft drinks and sweetened juices, use a mixture of D-glucose and D-fructose because of the mixture s higher sweetness. This 1:1 mixture can simply be labelled on the ingredients as glucose and fructose, or it can be called invert sugar. What is the origin of this name?

55 Carbs 55 Sucrose is optically active, with a specific rotation [α] = +66. ote that this number is different than the sum of the α values of the constituent monosaccharides. owever, a hydrolysis mixture comprised of 1:1 D-glucose and D-fructose rotates plane-polarized left to the left. The origin of the name invert sugar is due to the switching of the direction of optical rotation of sucrose before and after hydrolysis. Realize that sucrose is a non-reducing sugar, but the resulting mixture of hydrolysis products is reducing (you ll be hydrolyzing sucrose in Experiment 1). 2 C C 2 Acid or enzyme 2 C C 2 Interestingly, D-glucose was known as dextrose (still used on ingredient labels), because an equilibrium mixture of the α/β forms had [α] = Similarly, D-fructose was known has levulose, with an equilibrium [α] = 92.

56 D. Polysaccharides Carbs 56 Due to their complexity, polysaccharides cannot be easily synthesized in the lab. 1. Starch Starch is used for energy storage in plants and can be divided into two types, amylose and amylopectin. Both are polymers containing only D-glucose. Interestingly, based on calories, 4/5 th of the world s food is provided by three grain crops (maize, wheat, and rice) and three tuber crops (potato, yam, and cassava). The dry weight of these crops contains 60-90% starch. Amylose is composed of continuous, unbranched chains of up to 4000 D-glucose units joined α(1,4) glycoside linkages. It forms a hollow, helical structure, where other molecules can fit inside. atural curve

57 Carbs 57 Amylopectin, on the other hand, is a branched polymer of D-glucose. In addition to α(1,4) glycoside linkages, it also has some α(1,6) branches about every units running along the α(1,4) chain

58 2. Glycogen Carbs 58 While plants use starch for carbohydrate storage, animals use glycogen. A typical, well-nourished adult human contains about 350 g of glycogen, almost equally divided between the liver and muscle. Similar to amylopectin, glycogen is a branched polymer of D-glucose. It also contains α(1,4) and α(1,6) glycoside linkages, but it is much more branched.

59 3. Cellulose Carbs 59 Cellulose is used primarily for structural functions and is comprised of a linear polymer of D-glucose linked β(1,4). It is thus similar to amylose except for the configuration of the anomeric carbon. The average length of one polymer chain is about 2800 units, with a range of about units. Because the configuration is β, cellulose can adopt a straight chain (not curved). Every 2 nd unit is rotated to allow extra hydrogen-bonding to occur. Multiple polymer chains associate together very tightly by hydrogen bonding. As a result, cellulose is very insoluble in water. o Since it is insoluble, organisms that degrade cellulose must excrete cellulase enzymes and that break it down outside of the cell (extracellular degradation).

60 Carbs 60 nly microorganisms are known to produce cellulase enzymes. Wood rot is often caused by fungi and bacteria. umans cannot obtain their carbohydrates from cellulose, because humans do not excrete cellulase enzymes. owever, some animals can indeed degrade cellulose into D-glucose, but these animals do not produce their own cellulase enzymes. Such animals are in a symbiotic relationship with cellulase-producing microorganisms.

61 Carbs 61 Cellulose forms a supramolecular structure, where the individual chains bundle up, form microfibrils, fibrils, and then fibres. This is found in wood, cotton, paper, etc. Chain Bundle Polymer chains line up in parallel and hydrogen bond to each other, forming a "crystalline arrangement" Microfibril Chain bundles with microcapillaries between them Fiber Fibril Microfibrils with coarse capillaries between them Fibrils intertwined and overlapping

62 Carbs 62 Up to now, some of the concepts we ve covered include: o Linkages in disaccharides and polysaccharides o Reactivity of hemiacetals and acetals under acidic, neutral, and basic conditions o Invert sugar o Structures of common polysaccharides Using the carbohydrate structures provided in the lab manual, attempt: o Practice problems: o 2006 midterm: 8 and lab question 28 o 2006 intersession midterm: 18 o 2007 term test #1: 18 o 2008 midterm: 36

63 D. Metabolism of Glucose Carbs 63 Its metabolism into C 2 is one of the most important pathways for energy generation, and we will mechanistically examine glycolysis and the pyruvate dehydrogenase reaction, plus aminotransferase reactions. 1. Glycolysis In the net reaction below, glucose is converted into pyruvate over 10 steps. It is an anaerobic process (no oxygen used). C AD+ + 2 P ADP 2 C C 3 Pyruvate + 2 AD + 2 ATP ote the origin of the carbons in pyruvate. C3 and C4 of glucose become C1.

64 In the first five steps, glucose and 2 ATP are used to make 2 G-3-P. This is known as the energy consumption phase of glycolysis (2 ATP used). C 2 C 2 P ATP ADP 3 C 2 P 3 C 2 hexokinase phosphoglucose isomerase glucose glucose-6-phosphate fructose-6-phosphate Carbs 64 dihydroxyacetone phosphate triosephosphate isomerase glyceraldehyde- 3-phosphate C 2 P 3 C 2 C 2 P 3 aldolase C 2 P 3 C 2 P 3 phosphofructokinase ATP ADP C 2 P 3 C 2 P 3 fructose-1,6- bisphosphate

65 Carbs 65 Finally, glyceraldehyde-3-phosphate is converted to pyruvate (2 from glucose). This is known as the energy production phase of glycolysis (4 ATP from 2 G-3-P). C 2 P 3 AD + P 4 3- AD + G-3-P dehydrogenase P 3 C 2 P 3 ADP ATP phosphoglycerate kinase 3-phosphoglycerate C 2 P 3 glyceraldehyde- 3-phosphate 1,3-bisphosphoglycerate phosphoglycerate mutase C 3 ATP ADP pyruvate kinase C 2 P 3-2 enolase P 3 C 2 phosphoglycerate pyruvate (two from one glucose) phosphoenolpyruvate (high energy, unstable) The majority of these enzymatic reactions can be explained with chemical concepts that we are already familiar with. Learn the chemical reactions. There is no need to memorize the names of enzymes or compounds.

66 Carbs 66 a. exokinase (glucose glucose-6-phosphate) The hexokinase reaction is mechanistically similar to aminoacyl-tra synthetases, except phosphate is added to glucose, and ADP is the leaving group. Mg 2+ needed. C 2 P P P 2 2 C 2 P 3 P P

67 b. Phosphoglucose isomerase (Frc-6-phosphate fructose-1,6-bisphosphate) The enzyme converts the hemiacetal to the aldehyde, after which it isomerizes the aldehyde to the ketone via an ene-diol. Carbs 67 C 2 P 3 C 2 P 3 C 2 P 3 C 2 C 2 P 3 C 2 P 3 C 2

68 Carbs 68 c. Phosphofructokinase (Frc-6-phosphate fructose-1,6-bisphosphate) Interestingly, the enzyme acts only on the β anomer, which is in equilibrium with the α anomer. Mechanistically identical to the hexokinase reaction. C 2 P 3 C 2 C 2 P 3 ATP ADP C 2 P 3 C 2 C 2 P 3 Phosphofructokinase is an important regulator of glycolysis.

69 Carbs 69 d. Aldolase (fructose-1,6-bisphosphate DAP and G-3-P) We have previously seen the mechanism of aldolase on page 41. In the forward direction, two C3 compounds are joined to produce a β-hydroxy carbonyl compound. A retro-aldol reaction is the reverse reaction. C 2 P 3 C 2 P 3 C C 2 P 3 C 2 C 2 P 3 C 2 P 3 In the above reaction, the C= is the electron acceptor. This is the case for simple organisms, such as fungi and bacteria, but in plants and animals, the aldolase enzyme is more efficient and takes a modified route by using a positively charged nitrogen as the electron acceptor.

70 Carbs 70 This is accomplished by forming an iminium ion (Schiff base) with a lysine side chain (recall the reactions of aldehydes and ketones with amines to form imines). C 2 P 3 C 2 P 3 -Enz C 2 2 C 2 P 3 C 2 C 2 P 3 Enz- 2 C 2 P 3 C 2 P 3 -Enz -Enz C C 2 P 3 C 2 P 3

71 Carbs 71 e. Triosephosphate isomerase (dihydroxyacetone phoshate G-3-P) omework: propose a mechanism for the following reaction: C 2 P 3 C 2 C 2 C 2 P 3 C3 of DAP came from C3 of glucose, which turnsintoc1ofg-3-pandthenc1ofpyruvate C 2 P 3

72 Carbs 72 f. G-3-P dehydrogenase (G-3-P 1,3-bisphosphoglycerate) This enzyme oxidizes and phosphorylates G-3-P (oxidative phosphorylation). Before we can understand how the enzyme works, it is important to know how AD and AD + act as reducing and oxidizing agents, respectively. Consider the reduction of acetone with ab 4, a nucleophilic addition reaction. In biology, the coenzyme AD is the reducing agent and hydride donor. In the reverse direction, AD + would act as the oxidizing agent and hydride acceptor. The active part of the coenzyme is the nicotinamide ring. C 2 C 2 R R

73 Carbs 73 With G-3-P, oxidation is actually carried out on the hemithioacetal, the sulfur version of a hemiacetal (an aldehyde derivative), to form a thioester (an acid derivative). The thioester is subsequently converted into a mixed anhydride via a nucleophilic acyl substitution using phosphate as the nucleophile. Enz-S S-Enz C 2 C 2 P 3 C 2 P 3 R P 3 P 4 3- S-Enz Enz-S C 2 P 3 C 2 P 3

74 g. Phosphoglycerate kinase (1,3-bisphosphoglycerate 3-phosphoglycerate) In this reaction, the phosphate group is transferred to ADP, forming ATP. Carbs 74 P C 2 P 3 P P 2 2 P P P C 2 P 3

75 Carbs 75 h. Phosphoglycerate mutase (3-phosphoglycerate phosphoglycerate) In animals, the movement of phosphate from C3 to C2 involves the introduction of a new phosphate at C2, using histidine that has been phosphorylated by ATP, followed by the removal of the old phosphate at C3. C C 2 P P Enz C P 3 C 2 P Enz C P 3 C 2 P Enz In plants, the C3 phosphate is removed by is, and the same one is returned to C2.

76 i. Enolase (phosphoglycerate phosphoenolpyruvate) Carbs 76 Recall the acid-catalyzed dehydration of an alcohol via an E2 mechanism. The enolase reaction is functions similarly. C P 3 2 C C C 2 P 3 Enols are generally higher in energy than their keto counterparts, which is why enols exist in very small concentrations at equilibrium. Yet, phosphoenolpyruvate, cannot revert to the keto from because the enol oxygen has been phosphorylated (no ). The energy that is stored in this enol is subsequently used to make ATP.

77 Carbs 77 j. Pyruvate kinase (phosphoenolpyruvate pyruvate) Transfer of the phosphate to ADP forms ATP via the usual phosphate-transfer mechanism. The resulting enol tautomerizes to the more-stable keto form. C ADP ATP C C P 3 C 2 C 2 C 3 Glycolysis study hints: o Do not try to memorize the structures of the intermediate compounds! o Learn to work out the mechanisms... don t just memorize them! o Most of these reactions are based on concepts and mechanisms that you are already familiar with, but applied to different molecules. If you treat these as independent reactions, it is a lot of material. Yet, if you can see the chemical similarities and the big picture, it will be much easier.

78 2. Pyruvate dehydrogenase complex Carbs 78 Pyruvate can be converted to acetyl CoA, the central molecule in metabolism, via an oxidative decarboxylation, where there is an oxidation and a loss of C 2. These reactions are performed by the pyruvate dehydrogenase complex. SCoA Two coenzymes are required: thiamine pyrophosphate (TPP) and lipoic acid. Most decarboxylation reactions require an electron acceptor, such as a β-keto group or similar (recall the decarboxylation reactions in the amino acids section). Where there is an α-keto group, as in pyruvate, TPP is often used to remove C 2. The active part of TPP is the thiazolium ring, which is weakly acidic and forms an ylide (an overall neutral compound with adjacent positive and negative charges). R R pka ~18 TPP ylide S R' S R'

79 Carbs 79 The ylide is adds to pyruvate, forming an iminium ion that is β to the carboxylate group. This iminium group accepts the electrons generated in the decarboxylation, which results in the formation of an enamine. R R R' C C C 3 S R' C C S C 3 C 2 R C S R' C 3 The carbon that will later on be attached to CoA needs to be converted to the proper oxidation number via an oxidation. Lipoic acid (also called lipoamide, since it is attached to the enzyme as an amide) in the oxidized form is used.

80 In an S like reaction, the enamine attacks lipoic acid, displacing one sulfur. Carbs 80 R C C 3 S R' S S R'' A hemithioacetal is formed. Subsequent elimination of the ylide results in a thioester, which can then be converted to acetyl CoA by nucleophilic acyl substitution. R C C 3 S S R' S R'' R C S S R' S S S R S R' C 3 R'' R''

81 The reduced lipoic acid is converted back to the oxidized form by FAD, another biological oxidizing agent. FAD, like AD +, is a hydride acceptor (reversible). Carbs 81 S S S S R'' R'' R R flavin ring of FAD FAD 2 omework: FAD 2 can react with AD + to form FAD and AD, and vice versa. Propose reasonable enzyme-catalyzed mechanisms for the reactions.

82 3. Aminotransferase (transaminase) reactions Carbs 82 Pyruvate, an α-keto acid, is an important link between the metabolic pathways of carbohydrates and amino acids. Aminotransferase enzymes moves the amino group between α-keto acids and α-amino acids, e.g. alanine aminotransferase (ALT) 2 2 C C C C C pyruvate glutamate alanine α-ketoglutarate Another important one is aspartate aminotransferase (AST), which uses oxaloacetate in place of pyruvate, and aspartate in place of alanine. C The levels of ALT and AST in the blood are extremely important markers for several medical conditions, especially acute liver damage, where the levels generally rise. ALT and AST are usually measured during routine tests. These reactions require pyridoxal phosphate (PLP), a coenzyme that functions as an amino-group carrier, and are simply based on imine chemistry. P 3

83 Carbs 83 Although shown in the aldehyde form on the previous page, PLP is normally bound to the enzyme via an imine using a lysine side chain. The lysine can be replaced with the α-amino acid undergoing the reaction. Enz P 3 2 R C C R 2 P 3 Enz imine of PLP imine of PLP ote the position of the imine double bond (C=). ere, the double bond is between the carbon of PLP and the nitrogen of the amino acid. Therefore, it is the imine of PLP, since the carbon participating in the imine linkage belongs to PLP. After the next two steps, the C= will be between the α-carbon of the amino acid and the nitrogen of the amino acid. That is, it will be the imine of the α-keto acid.

84 Carbs 84 otice that the top-left and the bottom-right structures are just tautomers. C R 2 P 3 Enz C R 2 P 3 Enz imine of PLP imine α-keto acid C R 2 P 3 Enz imine α-keto acid

85 Carbs 85 Subsequent hydrolysis of the imine forms the α-keto acid and pyridoxamine (PMP). C R P R P 3 C imine of α-keto acid pyridoxamine (PMP) Thus, if glutamate was the initial amino acid used, α-ketoglutarate would be released. Pyruvate could then bind, and in the reverse reaction with PMP, it is converted to alanine, regenerating PLP. All of the enzyme-catalyzed reactions discussed since page 62 are vital processes. Deficiencies or defects in each one of these enzymes has been associated with serious medical issues. Many of these are autosomal recessive disorders, so they are most prevalent in geographically isolated populations due to inbreeding or the founder effect. In Canada, individuals from the Saguenay-Lac-Saint-Jean region have an exceedingly high incidence of recessive diseases. n a positive note, they are excellent contributors to medical genetics.