Is sucralose too good to be true?


  • This work is supported by: Susquehanna University.


Student interest in artificial sweeteners can enhance the biochemistry classroom learning experience. This in class, guided-inquiry activity focuses on sucralose and fits into a 50-min biochemistry class for undergraduate science majors. Background knowledge of carbohydrate structure, function, and metabolism as well as familiarity with interpretation of primary literature is assumed. This activity uses short answer questions that stimulate small group discussion. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, 2012

A variety of artificial sweeteners are available in the market [1]. One popular artificial sweetener, sucralose, was chosen for this activity due to its structural similarity to sucrose. Sucralose was created through collaboration between Tate & Lyle and Queen Elizabeth College, University of London, to investigate the perceived sweetness of halogen-substituted sucrose derivatives [2]. Sucralose safety has been reviewed by McNeil Nutritionals, a Johnson & Johnson company that markets Splenda® No Calorie Sweetener [3]. This activity includes analysis of one study cited as an example of sucralose safety in humans [4].

Presented here is a guided-inquiry activity designed for use in a biochemistry course during discussion of sugars and polysaccharides. Students should be familiar with sucrose structure and metabolism before this activity but have not studied sucralose. Thus, the goals of this activity are to introduce students to sucralose structure, examine the “zero Calorie” claim on Splenda® packaging, and explore sucralose metabolism in humans using primary literature data. Students build on prior knowledge (sucrose structure and metabolism) applied to new topics (sucralose structure and metabolism) and progress through Bloom's levels of cognitive thinking (knowledge, comprehension, application, analysis, synthesis, and evaluation) during the activity [5].

Junior and senior biology, biochemistry, and chemistry majors enrolled in a biochemistry course tested this activity. Students worked in small groups (three–four students) with the instructor present to assist when necessary. For the most part, students worked through the activity with each other and without instructor intervention. The activity was completed within a single, 50-min class period. The last few minutes of class were allocated for a groups' spokesperson to answer activity questions in front of the class. Corrections and explanations were given at this time.

Student progress regarding the learning goals was assessed using a 10 question, multiple choice quiz administered before and after the activity. The prequiz mean was 43% ± 20% and the postquiz mean improved to 77% ± 11%. A paired t-test was performed to determine if the differences between pre- and postquiz scores were statistically significant. Student scores increased significantly after the activity (p = 9.65 × 10−4).

The response to piloting of this activity was quite favorable. Students rated several statements using a five-point Likert-like scale (1 = strongly disagree, 2 = disagree, 3 = ambivalent/uncertain, 4 = agree, 5 = strongly agree). The statement “I enjoyed this activity” received an average rating of 4.0. Student rating of “I understand sucralose structure” increased from an average of 1.7 before the activity to 3.6 after the activity. Similar rating increases were reported for “I understand Splenda® Calorie content” (premean = 1.7 and postmean = 4.4) and “I understand sucralose metabolism” (premean = 1.7 and postmean = 3.1). The student ratings combined with the increased quiz scores indicate this activity succeeded in meeting the proposed learning goals.


Many low-Calorie or even no-Calorie (1 Cal = 1 kcal = 1,000 cal) sweeteners are currently available as an alternative to table sugar (sucrose) [1, 6]. This activity focuses on the sweetener sucralose commercially available as Splenda®.

  • 1)Sucralose (Fig. 1) is a no-Calorie alternative to sucrose [1]. Sucralose is the disaccharide sucrose (Fig. 2) with three of the hydroxyl groups replaced by chlorine atoms. In this process, what stereochemical changes occur in the glucose portion of the molecule?
  • 2)Sucralose is 600 times sweeter than sugar, but it is available in granulated form as Splenda®, which can be used for equal volume substitution with sugar [8, 9]. Explain.
  • 3)Sugar is available in 3.5 g single serve packets with an energy equivalent of about 15 Cal. Contrary to package labeling, Splenda® single serve packets (1 g) actually contain 3.36 Cal [10]. The Food and Drug Administration allows manufacturers to label an item as “zero Calories” if it contains less than 5 Cal per serving [11]. What is the source of the Calories in Splenda® packets?
  • 4)In a sucralose metabolism study using human volunteers, subjects were administered an oral dose of 14C-sucralose (1 or 10 mg/kg) with subsequent urine and fecal sample analysis [4]. The majority of 14C was eliminated in the feces (average 78.3%), while an average of 14.5% was eliminated in the urine. Overall, the total recovery of radioactivity over 5 days averaged 92.8%. Figure 3 shows the thin layer chromatography (TLC) radiochromatogram profile of urine from one subject. Two different solvent mixtures were used in separate experiments. Solvent A is diethyl ether-methyl ethyl ketone-water (25:25:1, by vol.) and solvent B is ethyl acetate-methanol-water-concentrated ammonia (60:20:10:2, by vol.). The large peak (S) was identified, based on its profile, as sucralose. What are the relative polarities of M1 and M2 when compared with sucralose? What are M1 and M2? Based on these data, is sucralose metabolized in humans?
  • 5)In the same study discussed in question 4, the main 14C urine component was identified as sucralose by both gas chromatography-mass spectrometry and liquid chromatography-thermospray mass spectrometry [4]. M2, representing about 10% of the urine radioactivity, was identified from mass spectra as a glucuronide conjugate of sucralose. A glucuronide is formed by addition of glucuronic acid to a molecule. M1 was hydrolyzed by β-glucuronidase while M2 was hydrolyzed by a mixture of sulfatase and β-glucuronidase [4]. Draw a glucuronide conjugate of sucralose. How does this modification affect the polarity and removal of sucralose from the body? Based on these data, what happens to sucralose after ingestion?
  • 6)The crystal structure of human sucrase-isomaltase (sucrose-α-D-glucohydrolase) C-term, which hydrolyzes the α(1→2)-glycosidic bond in sucrose releasing glucose and fructose, has not been solved. However, the structure of invertase (β-D-fructofuranoside fructohydrolase), a sucrose hydrolase produced by microbes in the intestine, is available. The active site of inactivated invertase from Thermotoga maritima is shown in Fig. 4 [12]. Raffinose (α-D-galactopyranosyl-(1,6)-α-D-glucopyranosyl-β-D-fructofuranoside) is bound in the active site with fructose at the −1 position and glucose at the +1 position (Fig. 4). Table I shows select hydrogen bonding in the active site between the enzyme and its substrate [12]. Would you expect this enzyme to hydrolyze sucralose? Explain.
  • 7)In a laboratory, how would you dispose of sucralose? Would it differ from how you dispose of sucrose? Explain.
  • 8)Based on your analysis of sucralose during this activity, is it a safe, no-Calorie alternative to sucrose or is it “too good to be true”?
Figure 1.

Structure of sucralose (modification of sucrose figure from ref. [7]).

Figure 2.

Structure of sucrose [7].

Figure 3.

TLC radiochromatogram profiles of the 6–12 hours urine from Subject 2. (a) solvent A, (b) solvent B, O: origin, SF: solvent front, S: sucralose. TLC using solvent A Rf values: sucralose = 0.28, M1 = 0.03, and M2 = 0.03. TLC using solvent B Rf values: sucralose = 0.50, M1 = 0.10, and M2 = 0.20 [4].

Figure 4.

Structure and interactions of raffinose bound in the active site of Thermotoga maritima invertase [12]. (a) Ribbon representation of active site with bound raffinose. Raffinose is shown in gold and red (oxygen), catalytic residues in yellow, aromatic residues in dark blue, other residues in green and water molecules as blue balls. (b) Fourier map (2FOFC) showing the trapped substrate in the active site of inactivated invertase (E190D).

Table I. Select hydrogen bonding between substrate and invertase active site residues
Substrate atomEnzyme atomEnzyme residue functionDistance (Å)
  1. Descriptions of enzyme residue functions are based on discussions in ref. [13].

Fructose O1Asp 17 Oδ1Nucleophile2.77
Trp 260 Nϵ1Substrate specificity, recognition3.07
Fructose O2Asp 17 Oδ2Nucleophile3.11
Asn 16 Nδ2Substrate specificity, recognition3.26
Fructose O3Glu 190 Oϵ2General acid/base3.05
Asp 138 Oδ2Substrate binding, recognition2.65
Fructose O4Asp 138 Oδ1Substrate binding, recognition2.78
Fructose O6Trp 41 Nϵ1Substrate specificity, recognition3.07
Gln 33 Oϵ1Substrate specificity, recognition2.74
Asn 16 Nδ2Substrate specificity, recognition3.01
Glucose O1Glu 190 Oϵ1General acid/base2.74
Glu 190 Oϵ2General acid/base2.67
Glucose O2Thr 208 OY1Substrate specificity3.39
Glucose O3Glu 188 Oϵ1Substrate specificity3.23
Glucose O4Arg 137 Nη2Substrate binding, recognition2.86


  • 1)There is inversion of configuration at C4 from the gluco- to galacto-analog.
  • 2)Splenda® is mixed with filler (dextrose and/or maltodextrin) for equal volume substitution with sugar.
  • 3)Dextrose and maltodextrin are the sources of Calories in Splenda®. Dextrose is glucose which is metabolized by humans for energy via glycolysis [7]. Maltodextrin, a polysaccharide of D-glucose, can be produced by heating cornstarch in an acidic environment resulting in the formation of nondigestible linkages such as β(1→2), β(1→3), β(1→4), and β(1→6) in addition to the digestible α(1→4) and α(1→6) linkages [13, 14]. This process results in a form of maltodextrin resembling dietary fiber that is only partially hydrolyzed by human digestive enzymes [14]. Thus, the main source of Calories in Splenda® packets is dextrose.
  • 4)M1 and M2 are more polar than sucralose as indicated by a shift to the left on the TLC profiles (Fig. 3) [4]. At this point, the exact identities of M1 and M2 are unknown. However, as M1 and M2 are detected separately from S on the TLC radiochromatogram, this indicates they contain 14C-sucralose in a modified (more polar) form. The presence of M1 as distinguishable from M2 (Fig. 3b) indicates modification of sucralose involves more than one change affecting sucralose polarity. Based on this data, a small portion of ingested sucralose appears to be metabolized in humans.
  • 5)Students should look up the structure of glucuronic acid in their class text [7] and draw β-D-glucopyranuronic acid (M1 and M2 were hydrolyzed in the presence of β-glucuronidase) attached from C1 to a hydroxyl group (most likely to the primary hydroxyl group at C6 of the 4-chlorogalactopyranosyl moiety which is the most reactive group) of sucralose [15]. The addition of glucuronic acid increases the polarity of sucralose. With increased polarity, sucralose will be more water soluble and more easily eliminated in the urine. As learned from the previous question, most sucralose is evacuated in the feces passing through the digestive system unaltered. Due to the water solubility of sucralose, some is absorbed by the body. A small portion of the absorbed sucralose is glucuronated and thus becomes more water soluble before being eliminated in the urine.
  • 6)It is unlikely that sucralose would be hydrolyzed by invertase. The hydrogen bonds important for substrate specificity and recognition (including Trp 260 to fructose O1, Arg 137 to glucose O4, and several to fructose O6) are listed on Table I. In sucralose, fructose O1, glucose O4, and fructose O6 are replaced with chlorine atoms. Chlorine cannot participate by donating a hydrogen bond at these positions potentially preventing recognition and decreasing binding affinity for sucralose to the active site of invertase. Additionally, ref. [12] describes the substitution of Glu with Asp at position 190 (general acid/base) as helping to maintain the electronegative microenvironment of the active site necessary for substrate binding while reducing enzymatic activity to 14%. Sucralose, with three highly electronegative chlorine atoms replacing hydroxyl groups, could be repulsed by the electronegative amino acids in the active site preventing binding. Students should also take into consideration evidence from the study discussed in Questions 4 and 5 where an average of 92.8% of radioactivity from 14C-sucralose was recovered within 5 days indicating the only metabolism of sucralose in humans involves glucuronidation [4]. These results have been supported by studies in dogs, mice, and rats [15–17].
  • 7)Sucralose is a halogenated compound. According to prior hazardous waste guidelines, sucralose would need to be disposed of as halogenated organic waste. However, recent Environmental Protection Agency (EPA) guidelines specify spent halogenated solvents [18]. Thus, sucralose would not be classified as hazardous waste and could be disposed of in the trash similar to sucrose.
  • 8)Answers may vary. Some students may argue the experimental results indicate sucralose is predominantly removed from humans unaltered, thus supporting the statement that it is a safe, no-Calorie alternative to sugar. Others may be hesitant to declare sucralose safe and want more information from other studies before making a decision.


  • 1)To aid students with Question 2, I set out a box of Splenda® packets for students to read the ingredients.
  • 2)To expand Question 6 or as a postactivity exercise [19], instructors may ask students to examine invertase in a protein visualization program such as RasMol or Visual Molecular Dynamics [20, 21]. Students can load the crystal structure as Protein Data Bank (PDB) entry 1W2T [12], alter coloring, drawing method, zoom, rotate and use other manipulations to better understand the physical properties and interactions within invertase and its active site. As PDB entry 1W2T contains six identical subunits, instructors may ask students to display only the first subunit (residues 1–431) to focus on a single active site. If instructors prefer, the crystal structure of T. maritima invertase with sucrose modeled into the active site is also available from PDB as entry 1UYP [22]. This crystal structure was solved with glycerol in the active site mimicking the presence of hydroxyl groups O4 and O6 of the fructose moiety of sucrose.
  • 3)Answers to the questions are included for instructor use. Please be mindful of the ease with which students could obtain these answers via the internet. This activity is designed for use in-class, with the instructor present.


The author wishes to thank Dr. Wade Johnson, Dr. Catherine Dent, and referees for critical reading of the article.