Aspartame and Glucose: The Role of Properties in Taste

Aneka Vo, Jen Choi, and Mollie Holmberg CHEM 165 May 25, 2011

Introduction Of the artificial sweeteners approved for human consumption, aspartame is one of the most popular. It was discovered by accident in 1965 by James Schlatter. At the time, Schlatter and a group of others were working to develop an ulcer drug. Schlatter synthesized aspartylphenylalanine-methyl-ether and accidently got some on his fingers. It was only later when he licked his finger to pick up a piece of paper that he discovered the sweet tasting chemical aspartame.1 Aspartame and glucose, a natural simple sugar, both taste sweet because they bind to the same taste receptors. However, the two substances vary in sweetness because the strength of their bonds to these receptors varies. The artificial processes used to synthesize aspartame rely on different kinds of chemical reactions than those in the Calvin Cycle by which plants produce glucose. These produce two distinct kinds of molecules. Since aspartame and glucose look so different, the body processes both of them via different pathways using specialized enzymes. This explains why people extract energy from glucose but not aspartame, even though both molecules taste sweet. The properties of glucose and aspartame are investigated to explain how molecules with such different properties can both be perceived as sweet by taste bud receptors. Sensing Sweetness People are able to taste sweetness when the molecules in certain substances bond to their sweet taste receptors. These protein taste receptors are located as bundles called taste buds in the upper esophagus, soft palate, and most prominently, the tongue. Each taste bud houses between 50 to 100 receptors, and an average person has 2000 to 8000 taste buds. Though many people believe otherwise, receptors for different tastes are not located in different parts of the tongue;

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receptors for sweetness are located in the same areas as receptors for bitter, salty, umami, and sour tastes.2

Figure 1. Diagrams of taste buds and tongue maps show where taste receptors are located on the tongue. Taste buds 2,3 for all types of taste are located in the same areas of the tongue.

Once the sweetener binds to the protein receptor, signals must be sent to the brain to create a sweet sensation; however, there are disputes on how these signals are transmitted. One proposed method is the labeled-line model, in which there are specific nerve fibers that connect the sweet sensitive cells to the brain. Another proposed method is the across-fiber model in which nerve receptor cells connect multiple different taste cells with the brain, so that the same nerve receptor fibers carry information for multiple different tastes to the brain.4 There are two sub types of the across-fiber model; in the first, taste receptor cells can receive all different types of taste and the resulting nerve fibers are able to carry information from all types of taste, whereas the second is more in tune with the most widely accepted method of taste reception, in which taste receptors only bind with certain flavored substances, but the nerve fibers can carry information for all tastes.2

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Figure 2. Diagrams of taste buds show the different methods by which signals of taste are sent to the brain.2

Taste Receptor Binding The most important factor that determines taste when food is consumed is binding; in order for a person to taste something, molecules from the food must bind to taste receptors. These receptors are made of proteins, called G-protein-coupled receptors (GPCRs). A family of these GPCRs is T1R, or taste receptor type 1, molecules, and although many of them are associated with taste, only T1R2 and T1R3, the second and third members of the T1R family, are associated with sensing sweetness.5 A complex of one T1R2 and one T1R3 molecule forms a heterodimer that serves as a receptor for a wide range of sweet molecules, both natural and artificial. Both of the molecules are necessary to sense all types of sweetness; research supports that a homodimer of two T1R3 molecules only acts as a receptor for high concentrations of natural sugars and that each protein alone does not have a great effect in sensing sweetness.4 Most sugars and artificial sweeteners bind to the T1R2 unit of the heterodimer6 at what is named the Venus flytrap module (VFTM).7

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Figure 3. A model of the T1R2 and T1R3 heterodimer shows the bonding site for sugars, including glucose, and aspartame.7

The bonding site is important because the ability for sensing sweetness is based upon the strength of the hydrogen bond between the glycol unit of the sweet tasting molecule and the taste receptor site. The distance between the proton donor and proton acceptor, AH and B, respectively, is much more important than the distance between simply A and B, which are electronegative atomsoxygen and nitrogen in glucose and aspartame, but are sometimes carbon and chlorine in other molecules. This distance must be approximately three angstroms for molecules that taste sweet because it makes intramolecular bonding impossible, which otherwise would inhibit bonding to the taste receptor.

Figure 4. A model of the taste receptor and sweet unit shows the relative interaction between the two sites.

In order to obtain this distance, the vicinal hydroxyl groups need to be in the gauche or staggered conformations; those that are not create too great of a distance to bond to the sweet

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taste receptor. The eclipsed formation inhibits interaction of the glycol unit with the receptor site because it causes intramolecular hydrogen bonds.8

Figure 5. Conformations of sweet and non-sweet glycol units

There is ongoing research that may support multiple binding sites per taste receptor that could explain why the distance between AH and B vary from 3.5 to 5.5 angstroms for certain molecules.9 Exact details of the sweet taste receptors are difficult to obtain because there are no crystal structures available for it.10 This is due to the difficulty in obtaining T1R molecules in large enough quantities for biochemical or structural studies due to their low levels in taste receptor cells. Additionally, T1R molecules do not express well in heterogeneous expression systems and they have low binding affinities, making it difficult to use conventional methods of binding analysis.7 Glucose is a monosaccharide, which is the simplest form of a sugar,11 while aspartame, a commonly used artificial sweetener, contains no saccharides and is a methyl ester of the dipeptide of two amino acids, L-aspartic acid and L-phenylalanine, bounded by a peptide, or amide, bond.1 These substances clearly have different structures but both induce a sweet sensation.

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Figure 6. Structures of glucose and aspartame

The relative sweetness of sweeteners is measured against the sweetness of sucrose, which is table sugar. Although glucose bonds with the protein receptors, aspartame is able to bond much more strongly. While glucose is only 70 to 80 percent as sweet as sucrose, aspartame is approximately 180 times as sweet as sucrose.12 This is why the amount of sugar substitute used is often less than the amount of sugar that would have been needed to achieve the same intensity of sweetness. Synthesis of Glucose The process that makes glucose is carried out in photosynthetic plants, algae, and cyanobacteria. In photosynthesis, carbon dioxide, water, and light photons form glucose and oxygen. The overall chemical equation is as follows 6 H2O + 6 CO2 + light energy C6H12O6 + 6 O2 [1]

The process starts with the light reactions, in which chlorophyll absorbs photons of light. This energy allows the chlorophyll to lose an electron (which has become unstable due to the absorbance of the light energy). This electron is passed to the electron transport chain, which eventually leads to the reduction of nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. Also, adenosine triphosphate (ATP), which functions as chemical energy, is produced during these reactions.13

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Next, the light independent reactions, collectively known as the Calvin Cycle, begin. This is where carbon fixation happens. To do this, a carbon dioxide molecule combines with an intermediate called ribulose 1,5-bisphosphate (RuBP) which makes two molecules of glycerate 3-phosphate (GP), a three carbon compound. The ATP and NADPH from the light reactions reduce the GP to glyceraldehyde-3-phosphate (G3P). The G3P either goes to regenerate the RuBP (to allow the cycle to continue) or goes on to condense into hexose phosphates of sucrose, starch, or cellulose. Starch and cellulose are both polymers of glucose molecules.13

Figure 7. An overview of the basic elements of Photosynthesis. Light, water, and oxygen go in and ATP and NADPH are created. Then, carbon is fixed in the light independent reactions, creating G3P. 14

Synthesis of Aspartame The synthesis of aspartame is very different from that of glucose; aspartame is not made in nature and must be artificially synthesized. Aspartame is made from two amino acids, aspartic acid and phenylalanine. Both of the amino acids are the L isomers.15 Using the D isomers does not produce a sweet product. Aspartic acid and phenylalanine do not have sweet tastes alone, but Page 7 of 15

when chemically combined, they do. If the wrong isomers of aspartic acid and phenylalanine are used for the synthesis of aspartame, the resulting product will not have the right geometry and cannot attach to the binding sites of the sweet receptors on taste buds. The synthesis of aspartame begins with the production of aspartic acid and phenylalanine. Bacteria that produce these amino acids are grown and allowed to thrive in a fermentation tank until the desired amount of aspartic acid and phenylalanine are produced. Centrifugal separation techniques are used to isolate the bulk of the amino acids from the other impurities. Then, the amino acids are further purified using ion exchange chromatography. Ion exchange chromatography uses charge-charge interactions between the amino acids and the resin along with different solvents to separate the different components of the sample. Aspartic acid is negatively charged at most pHs while phenylalanine is a zwitterion that is negatively charged at low pHs. Using these charges, ion exchange can purify the samples from impurities formed during synthesis.15 Once the amino acids have been made, they are modified before use in aspartame synthesis. First, phenylalanine is modified using methanol, which results in L-phenylalanine methyl ester.16 This reaction, also known as a Fischer esterification reaction, is done in the presence of HCl to catalyze the reaction. Depending on the method of synthesis, the aspartic acid is reacted with different chemicals to allow only the desired carboxyl group to react with the Lphenylalanine methyl ester. Aspartic acid has two carboxyl groups and an amine group which tend to react with other aspartic acid molecules because they all contain the necessary groups for reaction.17 Because the amine group causes most of these reactions, the aspartic acid is modified to either N-carbobenzoxy-aspartic anhydride or N-formyl-aspartic anhydride to protect the amine group from reacting during the actual synthesis.16

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After preparing the two amino acids for reaction, the actual synthesis of aspartame can begin. The modified phenylalanine and aspartic acid are mixed and allowed to react at room temperature for one day. The temperature is then increased to 65 degrees Celsius for another 24 hours. Lastly, the solution is cooled, diluted in solvents, and then brought to 0 degrees Celsius to encourage crystallization. The crystals are then collected and reacted with acetic acid to produce the final product, using palladium as a catalyst.15 During the synthesis, the amine group on the phenylalanine reacts with one of the carboxyl groups on the aspartic acid through a condensation reaction, forming the dipeptide aspartame. The bond that forms is also known as a peptide bond.18 After synthesis, the product is filtered to remove the catalysts and distilled, leaving a solid. Lastly, it is redissolved in ethanol and then crystallized again, leaving the pure product.15

Figure 8. The reaction of L-aspartic acid and L-phenylalanine forms a peptide bond

Like most reactions, the yield when synthesizing aspartame can vary greatly. The best way to increase the yield is to use the correct isomers of both amino acids. Using racemic mixtures of both acids will automatically decrease the maximum yield to 25 percent. Various catalysts also help to increase product yields. Also, recently, a thermolysin from the bacteria Bacillus thermoproteolyticus has been used recently to help direct the reaction due to its selectivity. This increases the yield by preventing the beta-form of aspartame (which is bitter and, therefore, undesirable) from being made and makes aspartame a better artificial sweetener because it tastes more like sugar. 16

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Health Effects Though aspartame seems like a great alternative to calorie-filled sugar, which does not have any potential side effects when consumed, it has been met with controversy as to whether it is really safe or not. One study published in March 2006 conducted a long-term test of the effects of regular aspartame consumption on rats. From the study, they concluded that aspartame is a multipotential carcinogenic agent at levels much lower than the recommended daily intake limit.19 In particular, the study found that rates of lymphoma, leukemia, neoplastic lesions, and brain tumors increased significantly. However, later in 2006, a review by the European Food Safety Authority concluded that the increased occurrence of cancer was unrelated to consumption of aspartame and that the previous study had many flaws.20 Though it is known that aspartame eventually metabolizes into toxic substances, the amount is much less than other natural sources of these chemicals that may be consumed.21 Aspartame breaks down into its componentsphenylalanine, aspartic acid, and methanolalmost as soon as it enters the body. This occurs via hydrolysis (Equation 2) due to the amine and ester linkages between the component molecules of aspartame. Methanol can then be oxidized to formaldehyde and then formic acid, as shown in Equations 3 and 4.

[2]

CH3OH CH2O CH2O CHOOH

[3] [4]

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Alcohol dehydrogenase, an enzyme in the liver, catalyzes the conversion of methanol to formaldehyde, which the body breaks down to use in the synthesis of DNA and various proteins.22 Any formaldehyde the body cannot use becomes formic acid via the reaction shown in Equation 4. Formic acid then breaks down into carbon dioxide and water or is excreted in urine. Methanol and its products can all be toxic if they become concentrated enough in the body because they damage the optic nerve, leading to blindness and death in extreme cases.23 However, studies at the University of Iowa have shown that consuming high levels of aspartame does not cause methanol and its products to reach harmful levels in the bloodstream.24 The other byproducts of aspartame, aspartic acid and phenylalanine, also have potentially neurotoxic properties if they build up in the brain. Usually the body breaks down or utilizes these amino acids before they accumulate to toxic levels. However, people with phenylketonuria, a genetic defect preventing the proper processing of phenylalanine, cannot consume aspartame products because the amino acid will accumulate in their brain and cause mental retardation and other symptoms.25 Although aspartame decomposes into a number of potentially toxic products in the body, consuming fifty times the average daily dose will not produce high enough concentrations of these chemicals to poison the body.22,24 The body extracts energy from glucose via several chemical pathways. Most glucose enters the body as sucrose, a disaccharide that is hydrolyzed into the monosaccharides fructose and glucose. Glucose then enters a process called glycolysis where one molecule of glucose is oxidized to two molecules of pyruvate in a series of ten chemical reactions as depicted in Figure 9.26 Note that a different enzyme catalyzes each reaction. Enzyme catalysts are important because they lower the energy input needed to initiate each reaction. In order for this process to

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produce, rather than consume, energy, it is critical that each reaction requires as little energy input as possible.

Figure 9. The ten steps of glycolysis with enzyme catalysts for each reaction are shown above.27

After glycolysis, pyruvate enters one of two pathways. If oxygen is present, pyruvate will enter the Krebs Cycle by binding to acetyl coenzyme A. In the Krebs Cycle, pyruvate is completely oxidized to carbon dioxide in a series of reactions which produce ATP as well as other molecules that generate ATP via the electron transport chain. If oxygen is not present, lactic acid fermentation occurs where the body reduces pyruvate to lactic acid in order to produce NAD+, nicotinamide adenine dinucleotide. Unlike the Krebs Cycle, lactic acid fermentation does not produce energy, but the regenerated NAD+ drives further energy production in glycolysis.28 Figure 10 demonstrates the relationship between glycolysis and

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fermentation. From this, it can be seen that production of energy from food depends on complex processes composed of tightly regulated steps in the body.

Figure 10. Lactic acid fermentation fuels glycolysis.28

The metabolism of sugars and aspartame have several similarities. Hydrolysis breaks both of these chemicals down into their most important forms in the body. Both can produce small quantities of carboxylic acidsformic acid and lactic acidwhich become toxic to the body at high concentrations. Lactic acid is responsible for the burning sensation people sometimes experience during or after rigorous exercise. However, in both cases, the body has developed ways to dispose of these toxins before they become harmful.24,28 Aspartame differs from glucose because its structure does not allow it to break down via the same pathways as glucose. At least one of the products of aspartame would need to bind to the enzymes regulating glycolysis in order to metabolize like glucose. For example, consider hexokinase, the enzyme responsible for the first reaction in glycolysis. Hexokinase has an active site tailored to recognize the structure of glucose and alter it in a very specific way. Scientists have found that hexokinase recognizes glucose via certain hydrogen bonds formed with the amino acids at the active site on the enzyme.29 This prevents aspartame from even beginning Page 13 of 15

glycolysis. Even if, hypothetically, hexokinase could accept and transform the byproducts of aspartame, it is unlikely that every enzyme in this chemical assembly line would be able to do likewise. Without the proper enzymes, the body does not gain energy by transforming the byproducts of aspartame into pyruvate. Conclusion Though glucose and aspartame both bind to sweet taste receptors and give a sweet taste, their actual properties are completely different. Glucose is a naturally occurring monosaccharide while aspartame is a dipeptide of two man-made amino acids. Their synthesis processes are also completely different, glucose being made naturally by photosynthetic plants and bacteria while aspartame must be artificially synthesized from two unnatural isomers of aspartic acid and phenylalanine. Lastly, the pathways and enzymes used to break them down are different, which makes it possible to obtain energy from the breakdown of glucose but not from aspartame. Despite the differences in synthesis, structure, and metabolism of glucose and aspartame, the body recognizes both as sweet compounds

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