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Steviol glycosides are the intensely sweet components of extracts from Stevia rebaudiana. These molecules comprise an invariant steviol aglycone decorated with variable glycans and could widely serve as a low-calorie sweetener. However, the most desirable steviol glycosides Reb D and Reb M, devoid of unpleasant aftertaste, are naturally produced only in trace amounts due to low levels of specific β (1-2) glucosylation in Stevia. Here, we report the biochemical and structural characterization of OsUGT91C1, a glycosyltransferase from Oryza sativa, which is efficient at catalyzing β (1-2) glucosylation. The enzyme's ability to bind steviol glycoside substrate in three modes underlies its flexibility to catalyze β (1-2) glucosylation in two distinct orientations as well as β (1-6) glucosylation. Guided by the structural insights, we engineer this enzyme to enhance the desirable β (1-2) glucosylation, eliminate β (1-6) glucosylation, and obtain a promising catalyst for the industrial production of naturally rare but palatable steviol glycosides., (© 2021. The Author(s).)

Surface active agents derived from the non-toxic sweetener sucralose and fatty acids of different chain length were synthesized. Obtained compounds were characterized chemically and with regard to their properties as emulsifying agents, antimicrobial preservatives and fat-soluble sweeteners. Results show that sucralose-fatty acid esters are possible multi-purpose additives, compatible with both cosmetic and edible emulsions, as well as purely oil-based, waterless formulations. Their relative effectiveness in those applications varies, and is highly dependent on the fatty acid chain length, with hydrophobic/hydrophilic character strongly impacting both emulsifying and antimicrobial properties. While the structural differences between sucrose and sucralose proved to be enough to push all of the newly synthesized compounds out of the detergent/solubilizer category of surfactants, the retention of the substrate's high sweetness is an indication that non-bitter compounds with washing capabilities are possible to obtain., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Luo Han Guo fruit extract ( Siraitia grosvenorii ), mainly composed of mogroside V (50%), could be considered a suitable alternative to free sugars; however, its commercial applications are limited by its unpleasant off-notes. In the present work, a central composite design method was employed to optimize the transglycosylation of a mogroside extract using cyclodextrin glucosyltransferases (CGTases) from three different bacteriological sources ( Paenibacillus macerans , Geobacillus sp., and Thermoanaerobacter sp.) considering various experimental parameters such as maltodextrin and mogroside concentration, temperature, time of reaction, enzymatic activity, and pH. Product structures were determined by liquid chromatography coupled to a diode-array detector (LC-DAD), liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Sensory analysis of glucosylated mogrosides showed an improvement in flavor attributes relevant to licorice flavor and aftereffect. Consequently, an optimum methodology was developed to produce new modified mogrosides more suitable when formulating food products as free sugar substitutes.

A large library of saccharin and acesulfame derivatives has been synthesised and evaluated against four isoforms of human carbonic anhydrase, the two off-targets hCA I/II and the tumour related isoforms hCA IX/XII. Different strategies of scaffold modification have been attempted on both saccharin as well as acesulfame core leading to the obtainment of 60 compounds. Some of them exhibited inhibitory activity in the nanomolar range, albeit some of the performed changes led to either micromolar activity or to its absence, against hCA IX/XII. Molecular modelling studies focused the attention on the binding mode of these compounds to the enzyme. The proposed inhibition mechanism is the anchoring to zinc-bound water molecule. Docking studies along with molecular dynamics also underlined the importance of the compounds flexibility (e.g. achieved through the insertion of methylene group) which favoured potent and selective hCA inhibition.

Mogroside V, a component with high content and sweetness in mogrosides, has many pharmacological activities such as relieving cough, reducing sputum, anti-cancer, anti-oxidation, regulating blood sugar, making it a natural nonsugar sweetener with therapeutic functions, and showing a broad market prospect. However, the limited resources and high extraction costs have restricted its widespread use. The rapid development of synthetic biology has provided a new idea for the production of plant natural products. The low-cost and large-scale production will be realized through the construction of a microbial cell factory for mogroside V. Here, we briefly introduce the structure and pharmacological activity of mogroside V, and review progress in applying synthetic biology for its synthesis, and also discuss the challenges faced by the current research, to provide a reference for further studies on the biosynthesis of mogroside V.

Human sensation for sweet tastes and the thus resulting over-consumption of sugar in recent decades has led to an increasing number of people suffering from caries, diabetes, and obesity. Therefore, a demand for sugar substitutes has arisen, which increasingly has turned towards natural sweeteners over the last 20 years. In the same period, thanks to advances in bioinformatics and structural biology, understanding of the sweet taste receptor and its different binding sites has made significant progress, thus explaining the various chemical structures found for sweet tasting molecules. The present review summarizes the data on natural sweeteners and their most important (semi-synthetic) derivatives until the end of 2019 and discusses their structure-activity relationships, with an emphasis on small-molecule high-intensity sweeteners.

A unique thermostable amylosucrase from Bifidobacterium thermophilum was produced as a recombinant protein with the half-life of 577 h at 50 °C. By adding 1.0 M fructose, turanose yield was improved from 22.7% to 43.3% with 1.0 M sucrose, and from 23.7% to 39.4% with 1.5 M sucrose. Sucrose consumption rate was greatest at 55 °C, but the lowest amount of turanose was produced. Thus, turanose yield from sucrose biomass was inversely proportional to reaction temperature and was highly dependent on [fructose]. Meanwhile, insoluble α-glucan yield was clearly reduced as [fructose] increased. With 1.0 M fructose + 1.0 M sucrose, glucan byproduct yield significantly decreased from 29.4% to 1.1%. Molecular weights of linear glucans were almost identical among various [sucrose]s and were homogenous with very low polydispersity. This unique dual reaction patterns of amylosucrase enzyme would be very useful for massive productions of two different biomaterials simply by changing sucrose biomass concentration., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In recent times, inulinase has emerged as one the most prominent and industrially upcoming enzymes applied to meet the ever increasing demand of d-fructose and fructooligosaccharides (FOS) as sweetener and prebiotics in the food and pharmaceutical industry, respectively. This review deals with types of inulinase and the attempts made to modify it for better thermal stability and shelf life. The ease of immobilization of inulinase has led us to the path of experimenting with different methods of enzyme immobilization since 1979. Several modes of immobilization ranging from simple cross-linking of enzymes onto a polymer support to nanoparticles have been applied over the years. The approach and concept of this review provide a yet unexplored focus on pioneering advances for the development of white biotechnology, for instance production of immobilized inulinase-based reusable biocatalysts and bioreactors designed for their use and for the continuous production of fructose and FOS.

D-Allose is a rare monosaccharide, which rarely appears in the natural environment. D-Allose has an 80% sweetness relative to table sugar but is ultra-low calorie and non-toxic and is thus an ideal candidate to take the place of table sugar in food products. It displays unique health benefits and physiological functions in various fields, including food systems, clinical treatment, and the health care fields. However, it is difficult to produce chemically. The biotechnological production of D-allose has become a research hotspot in recent years. Therefore, an overview of recent studies on the physiological functions, applications, and biotechnological production of D-allose is presented. In this review, the physiological functions of D-allose are introduced in detail. In addition, the different types of D-allose-producing enzymes are compared for their enzymatic properties and for the biotechnological production of D-allose. To date, very little information is available on the molecular modification and food-grade expression of D-allose-producing enzymes, representing a very large research space yet to be explored.

Glucosyl stevioside was synthesized via transglucosylation by dextransucrase from Leuconostoc citreum KM20 (LcDexT), forming α-d-glucosyl stevioside. A production yield of 94% was reached after 5days of LcDexT reaction at 30°C. Glucosyl stevioside induced a 2-fold improved quality of taste and sweetness, compared to stevioside. After 15days of storage at 25°C, 98% of glucosyl stevioside in an aqueous solution was present in a soluble form, compared to only 11% for stevioside or rebaudioside A. Furthermore, glucosyl stevioside exhibited a similar or improved stability in commercially available soft drinks, when compared to stevioside and rebaudioside A. These results suggest that glucosyl stevioside could serve as a highly pure and stable sweetener in soft drinks., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed., Results: The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h)., Conclusions: In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.

Enantiomerically pure 2-substituted-2,5-dihydro-3-(aryl) sulfonyl/sulfinyl furans have been prepared from the easily accessible carbohydrate derivatives. The orientation of the substituents attached at the C-2 position of furans is sufficient to control the diastereoselectivity of the addition of various nucleophiles to the vinyl sulfone/sulfoxide-modified tetrahydrofurans, irrespective of the size of the group. The orientation of the substituents at the C-2 center also suppresses the influence of sulfoxides on the diastereoselectivity of the addition of various nucleophiles. The strategy leads to the creation of appendage diversity, affording a plethora of enantiomerically pure trisubstituted furanics for the first time.

The new platinum(II) complexes cis-[Pt(sac)2(NH3)2] (sac=saccharinate) and cis-[Pt(tsac)2(NH3)2] (tsac=thiosaccharinate) have been prepared, the X-ray crystal structure of cis-[Pt(sac)2(NH3)2] x H2O reveals that both saccharinate anions are N-bound in a cis-arrangement being inequivalent in both the solid-state and in solution at room temperature. Preliminary anti-cancer activity has been assessed against A549 human alveolar type-II like cell lines with the thiosaccharinate complex showing good activity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Polyols as sugar substitutes, intense sweeteners and some new carbohydrates are increasingly used in foods and beverages. Some sweeteners are produced by fermentation or using enzymatic conversion. Many studies for others have been published. This chapter reviews the most important sweeteners.

As a continuation of our studies on the relationship between structure and sweet taste of isovanilline-type sweeteners, (3-hydroxy-4-methoxy)benzyl-benzoate (17) and -salicylate (17c), analogues of dihydrochalcone-type sweetener analogues of (+)-phyllodulcin [(+)-5] and CH-401 (14c) have been synthesized. Surprisingly, 17c has been found to be tastless, while 17e was slightly sweet. These observations could be explained by the current hypothesis on the receptor model for isovanilline-type sweet derivatives.

The potential for dynamic chirality of an azapeptide nitrogen was examined by substitution of nitrogen for the α-carbon of the aspartate residue in the sweetener S,S-aspartame. Considering that S,S- and R,S-aspartame possess sweet and bitter tastes, respectively, a bitter-sweet taste of aza-aspartame 9 could be indicative of a low isomerization barrier for nitrogen chirality inter-conversion. Aza-aspartame 9 was synthesized by a combination of hydrazine and peptide chemistry. Crystallization of 9 indicated a R,S-configuration in the solid state; however, the aza-residue chiral center was considerably flattened relative to its natural amino acid counterpart. On tasting, the authors considered aza-aspartame 9 to be slightly bitter or tasteless. The lack of bitter sweet taste of aza-aspartame 9 may be due to flattening from sp2 hybridization in the urea as well as a high barrier for sp3 nitrogen inter-conversion, both of which may interfere with recognition by taste receptors.

Catalytic hydrogenation of the three ent-kaurane diterpene glycosides isolated from Stevia rebaudiana, namely rubusoside, stevioside, and rebaudioside-A has been carried out using Pd(OH)₂ and their corresponding dihydro derivatives have been isolated as the products. Synthesis of reduced steviol glycosides was performed using straightforward chemistry and their structures were characterized on the basis of 1D and 2D NMR spectral data and chemical studies. Also, we report herewith the sensory evaluation of all the reduced compounds against their corresponding original steviol glycosides and sucrose for the sweetness property of these molecules.

The structure activity relationship between the C₁₆-C₁₇ methylene double bond on the aglycone of steviol glycosides and the corresponding impact on their sweet taste has been reported here for the first time. It has been observed that converting stevioside and rebaudioside A to their corresponding ketones by switching the doubly bonded methylene on C-17 for a ketone group actually removes the sweet taste properties of these molecules completely. Regenerating the original molecules tends to restore the sweet taste of both the steviol glycosides. Thus this C₁₆-C₁₇ methylene double bond in rebaudioside A and stevioside can be regarded as a pharmacophore essential for the sweetness property of these molecules.

Background: Difructose anhydride (DFA) III is a natural and low-calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III., Results: IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over-expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL(-1) . This value was 4.1-fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 °C, and retained 81.3% of its initial activity after incubation at 60 °C for 4 h., Conclusion: IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over-expressed in E. coli. IFTase was reported for the first time to be over-expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production., (Copyright © 2011 Society of Chemical Industry.)

Synthesis of two ent-kaurane diterpene glycosides, steviol 19-O-β-D-glucopyranosiduronic acid (steviol glucuronide, 5), and 13-hydroxy ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (7) has been achieved from a common starting material, steviol, using phase transfer catalyst. Also, synthesis of an additional 17-nor-ent-kaurane glycoside, namely 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester (10) was performed using the starting material isosteviol and similar synthetic methodology. Synthesis of all three steviol glycosides was performed using straightforward chemistry and their structures were characterized on the basis of 1D and 2D NMR as well as mass spectral (MS) data.

Aim: To evaluate the efficacy and safety of 2 analogs of L-carnitine on rats made insulin resistant by a high-fructose diet., Methods: Using rats made insulin resistant by a high-fructose diet, we investigated the impact of 2 analogs of L-carnitine (25 mg/kg) and L-carnitine (250 mg/kg) on glucose, triglycerides and cholesterol blood levels, and liver glycogen. We also evaluated the safety of both analogs by the assessment of some biochemical and hematological parameters, a histological analysis and a study of embryotoxicity., Results: Both analogs reduced the levels of triglycerides in the liver and plasma, but only analog 2 reduced the cholesterol levels in insulin-resistant rats. No changes were observed in glycogen content. Safety evaluations revealed alterations in blood lymphocytes and embryotoxicity data., Conclusion: This study demonstrated that the 2 analogs maintain the pharmacological properties of L-carnitine but have a different efficacy, potency and toxicity., (Copyright © 2011 S. Karger AG, Basel.)

Acesulfame is found to exist in two crystalline forms of which Form I (needles) shows bending upon mechanical stress. Crystal structures explain their mechanical response. This is the first case of aliphatic organic compounds featuring a bending phenomenon. Form I is physically more stable than Form II in ambient conditions.

2,3-dihydrobenzo[b][1,4]oxathiine represents a valuable pharmacophoric heterocyclic nucleus known since very long time. Initially, together with some patents reporting the use of these compounds as herbicides or lipogenesis inhibitors, several papers reported their ability as melatonin, histamine and serotonin receptor ligands, alpha-adrenoreceptor blockers as well as non-glycoside sweeteners. This wide range of biological activities has been recently further improved by studies stating their activity as antimycotics, multi-defense antioxidants and estrogen receptor ligands. The last insights regarding the preparation, the biological activity and the structure activity relationship (SAR) of derivatives containing the dihydrobenzoxathiine skeleton will be discussed in this review.

A total of 28 new five-membered aromatic ring thiazolyl-, benzothiazolyl-, and thiadiazolylsulfamates, as their sodium salts, have been synthesized and combined with 30 known similar heterocyclic sulfamates to create a database for the study of structure-activity (taste) relationships (SARs) in this heterocyclic subgroup, which is known to contain a somewhat disproportionate number of sweet compounds compared to other groups of tastants. A series of nine parameters (descriptors) to describe the properties of the sulfamate anions were calculated in Spartan Pro and HyperChem programs. These are the highest occupied molecular orbital (HOMO), lowest unoccupied molecular orbital (LUMO), length of the molecule, dipole moment, area, volume, E(solv), sigma (from the literature), and log P. The taste data for all 58 compounds were categorized into three classes, namely, sweet (S), nonsweet (N), and nonsweet/sweet (N/S). Discriminant analysis only classified 44 of the 58 compounds correctly. Classification and regression tree analysis (CART) using the S_ Plus program proved highly effective, in that the derived tree correctly classified 46 compounds from a training set of 48 and, from a computer randomly selected test set of 10 compounds, 7 had their taste correctly predicted. A second tree was grown using the additional taste category N/S, and this tree also performed extremely well, with 8 of the 10 compounds in the test set correctly classified. These trees should be very reliable for predicting the tastes of other heterocyclic sulfamates, which belong to the subset used here.

Within a research project aimed at the design of new sweeteners, the tetrazole moiety was introduced to arylsulfonylalkanoic acids (ASA) as a bioisostere of the carboxyl group. The crystal structures of four newly synthesized tetrazole derivatives and one intermediate product of the reaction were determined in order to explain the bitter taste of these compounds. Three chiral compounds crystallize as racemic mixtures in centrosymmetric space groups of the monoclinic system, whereas the non-chiral compound, with a higher dipole moment, crystallizes in the polar space group Cc. Intermolecular N-H...N hydrogen bonds between tetrazole moieties were observed in all four structures and are compared with the analogous interactions observed in tetrazole derivatives deposited in the Cambridge Structural Database (CSD). Specifically, the typical N1-H...N4 as well as N1-H...N3 interactions, which are less abundant in the CSD, are described. The formation of the latter interaction type can be hypothetically explained by an asymmetry of pi-electron distribution in the tetrazole rings caused by the crystalline environment. Important features of the crystal architecture are the chains of molecules linked by N-H...N bonds. A possible reason for the lack of a sweet taste of the tetrazoles investigated may be the improper position of the tetrazole H atom, and the mutual orientation of the proton donor and acceptor in their molecules. This orientation does not allow the tetrazoles to interact with the sweet-taste receptor in a way similar to that of ASA. The bitter taste of the investigated compounds needs further study.

Forty-two new disubstituted phenylsulfamates have been synthesized, and 30 of these have been combined with 40 already available from earlier work to create a training database of 70 compounds. On the basis of panel taste data these were divided into three categories, N (nonsweet), N/S (nonsweet/sweet), and (S) sweet, and a "sweetness value" or weighting was also calculated for each compound. Using these 70 compounds as a training set and a series of nine predictors derived from Corey-Pauling-Koltun (CPK) models, calculated from the PC SPARTAN PRO program and Hammett sigma values taken from the literature, a classification and regression tree analysis (CART) was carried out leading to a regression tree that correctly classified 62 of the 70 compounds (89% overall correct classification). The tree's predictive ability varies for the different taste categories, and for nonsweet compounds it is virtually 100%; for nonsweet/sweet compounds it is 66%, and for sweet compounds it is approximately 75%. This tree correctly predicted taste categories for 10 compounds from a test set of 12 randomly selected from among the 42 new compounds (83% correct classification). Therefore, it can be used with a good degree of confidence to predict the tastes of disubstituted phenylsulfamates. For the design of new sweeteners, appropriate values or ranges of the descriptors are derived.

The binding mechanism of sweet proteins to their receptor, a G-protein-coupled receptor, is not supported by direct structural information. In principle, the key groups responsible for biological activity (glucophores) can be localized on a small structural unit (sweet finger) or spread on a larger surface area. A recently proposed model, called "wedge model", implies a large surface of interaction with the receptor. To explore this model in greater detail, it is necessary to examine the physicochemical features of the surfaces of sweet proteins, since their interaction with the receptor, with respect to that of small sweeteners, is more dependent on general physicochemical properties of the interface, such as electrostatic potential and hydration. In this study, we performed exhaustive molecular dynamics simulations in explicit water of the sweet protein MNEI and of its structural mutant G-16A, whose sweetness is one order of magnitude lower than that of MNEI. Solvent density and self-diffusion calculated from molecular dynamics simulations suggest a likely area of interaction delimited by four stretches arranged as a tetrahedron whose shape is complementary to that of a cavity on the surface of the receptor, in agreement with the wedge model. The suggested area of interaction is amazingly consistent with known mutagenesis data. In addition, the asymmetric hydration of the only helix in both proteins hints at a specific role for this secondary structure element in orienting the protein during the binding process.

We report here a range of new sucrose derivatives obtained from '3-ketosucrose' in aqueous medium with few reaction steps. As an intermediate, 3-amino-3-deoxy-alpha-D-allopyranosyl beta-D-fructofuranoside (1) was obtained via the classical route of reductive amination with much improved yield and high stereoselectivity. Building blocks for polymerization were synthesized by introduction of acrylic-type side chains, for example, with methacrylic anhydride. Corresponding polymers were synthesized. Aminoacyl and peptide conjugates were obtained through conventional peptide synthesis with activated and protected amino acids. Deprotection yielded new glycoderivatives having an unconventional substitution pattern, namely 3-(aminoacylamino) allosaccharides. Both mono- and di-peptide conjugates of allosucrose have been synthesized.

We report here the synthesis and the conformation analysis by 1H NMR spectroscopy and computer simulations of six potent sweet molecules, N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-alpha-L-aspartyl-S-tert-butyl-L-cysteine 1-methylester (1; 70 000 times more potent than sucrose), N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-alpha-L-aspartyl-beta-cyclohexyl-L-alanine 1-methylester (2; 50 000 times more potent than sucrose), N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-alpha-L-aspartyl-4-cyan-L-phenylalanine 1-methylester (3; 2 000 times more potent than sucrose), N-[3,3-dimethylbutyl]-alpha-L-aspartyl-(1R,2S,4S)-1-methyl-2-hydroxy-4-phenylhexylamide (4; 5500 times more potent than sucrose), N-[3-(3-hydroxy-4-methoxyphenyl)propyl]-alpha-L-aspartyl-(1R,2S,4S)-1-methyl-2-hydroxy-4-phenylhexylamide (5; 15 000 times more potent than sucrose), and N-[3-(3-hydroxy-4-methoxyphenyl)-3-methylbutyl]-alpha-L-aspartyl-(1R,2S,4S)-1-methyl-2-hydroxy-4-phenylhexylamide (6; 15 000 times more potent than sucrose). The "L-shaped" structure, which we believe to be responsible for sweet taste, is accessible to all six molecules in solution. This structure is characterized by a zwitterionic ring formed by the AH- and B-containing moieties located along the +y axis and by the hydrophobic group X pointing into the +x axis. Extended conformations with the AH- and B-containing moieties along the +y axis and the hydrophobic group X pointing into the -y axis were observed for all six sweeteners. For compound 5, the crystal-state conformation was also determined by an X-ray diffraction study. The result indicates that compound 5 adopts an L-shaped structure even in the crystalline state. The extraordinary potency of the N-arylalkylated or N-alkylated compounds 1-6, as compared with that of the unsubstituted aspartame-based sweet taste ligands, can be explained by the effect of a second hydrophobic binding domain in addition to interactions arising from the L-shaped structure. In our examination of the unexplored D zone of the Tinti-Nofre model, we discovered a sweet-potency-enhancing effect of arylalkyl substitution on dipeptide ligands, which reveals the importance of hydrophobic (aromatic)-hydrophobic (aromatic) interactions in maintaining high potency.

Neohesperidin dihydrochalcone (NHDC) is a seminatural, safe, low-calorie sweetener, bitterness blocker, and flavor enhancer with unique properties and applications for the food, beverage, pharmaceutical, and animal feed industries. Current production is limited by the availability of the substrate neohesperidin, a flavonoid that accumulates to significant levels only in the inedible bitter citrus species. We propose a process to convert hesperidin, a tasteless flavonoid extracted from orange peels that are abundant byproducts of the vast orange juice industry, into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin from orange peels, (ii) hydrolysis of sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. We overcame the current technological bottleneck in biotransformation of hesperidin hydrolysis products into neohesperidin using metabolically engineered plant cell cultures expressing a recombinant flavanone-7-O-glucoside-2-O-rhamnosyltransferase. A small-scale production experiment established the feasibility of the proposed process.

The crystal structures of the murine monoclonal IgG2b(kappa) antibody NC6.8 Fab fragment complexed with high-potency sweetener compound SC45647 and nontasting high-affinity antagonist TES have been determined. The crystal structures show how sweetener potency is fine-tuned by multiple interactions between specific receptor residues and the functionally different groups of the sweeteners. Comparative analysis with the structure of NC6.8 complexed with the super-potency sweetener NC174 reveals that although the same residues in the antigen binding pocket of NC6.8 interact with the zwitterionic, trisubstituted guanidinium sweeteners as well as TES, specific differences exist and provide guidance for the design of new artificial sweeteners. In case of the nonsweetener TES, the interactions with the receptor are indirectly mediated through a hydrogen bonded water network, while the sweeteners bind with high affinity directly to the receptor. The presence of a hydrophobic group interacting with multiple receptor residues as a major determinant for sweet taste has been confirmed. The nature of the hydrophobic group is likely a discriminator for super- versus high-potency sweeteners, which can be exploited in the design of new, highly potent sweetener compounds. Overall similarities and partial conservation of interactions indicate that the NC6.8 Fab surrogate is representing crucial features of the T1R2 taste receptor VFTM binding site.

In the mid-1970s, Dr. Murray Goodman was interested in a reversed peptide bond as a surrogate to understand the functional role of the amide bond in aspartame, a dipeptide sweetener. Very soon, realizing the breath and potential of this modification, Murray expanded this activity into a full program and I was fortunate to be part of it. Together we formulated new concepts such as the partially modified retro-inverso and end-group modified retro-inverso transformations, tested hypotheses, generated novel nomenclature, developed synthetic routes, characterized the preferred conformations of the unique building blocks employed in this modification, the gem-diaminoalkyl and the C2-substituted malonyl residues, and studied the biological activity of retro-inverso isomers of bioactive peptides. In the early 1980s several laboratories initiated extensive research targeted at the retro-inverso modification. The revival of this field led to new applications, new methods of synthesis, and new insights on the conformational and topological properties of the retro-inverso modification. Among the fields that embraced the retro-inverso concept were immunology as pertains to subjects such as synthetic vaccines, immunomodulators, and diagnostic tools, and drug delivery field as pertains to targeted and nontargeted cell permeation vectors loaded with bioactive cargo. Doctor Murray Goodman's sudden death leaves behind not only family, friends, and colleagues, but also an impressive record of scientific achievements among which is the revival of the modern era of the retro-inverso transformation. Murray's numerous contributions, excellent leadership, enthusiastic promotion, and outstanding teachings in this field will carry and illuminate his memory far into the future., (Copyright 2005 Wiley Periodicals, Inc)

Four different routes for the synthesis of saccharin containing peptides were studied. While reactions between saccharin sodium and alpha-halogeno acids were limited to two examples, reactions of sulfobenzoic anhydride (4) or saccharin-N-carboxylate (6) with amino acid esters yielded the ring opened products 5 and 7. Finally, we found, that the reaction between the benzoxathiol derivative 8 and amino acid derivatives represents a versatile route to the peptidic compounds 9 and 11. Hydrolysis and hydrogenolysis were studied, and by combination of the different routes the "saccharin tripeptides" 18 were obtained. Structures and stereochemistry were elucidated by spectroscopic and chromatographic methods. Selected compounds were tested as sweeteners or as inhibitors of elastase, but no exiting results were found.

Four isomers of the L-aspartyl-D-alanine fenchyl esters were prepared as potential peptide sweeteners. L-Aspartyl-D-alanine (+)-alpha-fenchyl ester and L-aspartyl-D-alanine (-)-beta-fenchyl ester showed sweetness with potencies 250 and 160 times higher than that of sucrose, respectively. In contrast, L-aspartyl-D-alanine (+)-beta-fenchyl ester and L-aspartyl-D-alanine (-)-alpha-fenchyl ester had the highest sweetness potencies at 5700 and 1100 times that of sucrose, respectively. In particular, L-aspartyl-D-alanine (-)-alpha-fenchyl ester had an excellent sweetness quality; but L-aspartyl-D-alanine (+)-beta-fenchyl ester did not have an excellent quality of sweetness because it displayed an aftertaste caused by the strong sweetness.

The use of Kohonen feature maps for the visualization of various aspects of molecular similarity is briefly reviewed and illustrated. It is shown that a specific feature of self-organizing maps (SOM) makes them of special interest for the screening of compounds. In particular, these methods were used to design candidates for new sweeteners, which were then synthesized.

We used arylsulfonylalkanoic acids as parent structures for designing new potential sweeteners. The Kohonen maps of the molecular electrostatic potential of the possible bioisosteric replacements of carboxylic function have been simulated and used for the selection of the potential synthetic targets which are now under synthesis.

The hydrogenation of D-fructose on Ru/C catalysts was studied. Under the conditions applied (1 bar H2, 72 degrees C), the furanose forms of D-fructose react, while the pyranose forms do not. However, all anomers adsorb with comparable strength on the surface. The reaction rate is controlled by product inhibition. The selectivity to D-mannitol can be increased from 47 to 63% by promotion of Pd/C and Pt/C catalysts with Sn.

The naturally occurring sweetener Monatin, a diastereomer of Monatin, and a phenyl analogue of Monatin have been prepared and isolated in their enantiomerically pure forms.

The glucose isomerase from Streptomyces olivaceoviridis E-86 was purified by chromatographic procedures, showing one single protein band in the SDS-PAGE. The enzyme had high acid stability, and there was no loss in enzyme activity at pH 5.0 after incubation at 60 degrees C for 30 hr. The enzyme had sufficients activity at 60 degrees C, pH 5.5, (which is the reaction condition for a single-step process with a glucoamylase from A. niger), and at 58 degrees C, pH 6.0, (condition with a glucoamylase from R. niveus). By using this acid-stable glucose isomerase, a single-step process to produce high-fructose corn sweetener (HFCS) from liquefied starch was formed without any reductant or other reagents for enzyme stabilization. The HFCS produced was about fifty percent fructose and less than 1.5% unknown oligosaccharides.

Monellin, a sweet protein, consists of two noncovalently associated polypeptide chains: an A chain of 44 amino acid residues and a B chain of 50 residues. Microbial transglutaminase (MTGase) was used for ligation of the monellin subunits without any protecting groups, and without activation of the C alpha-carboxyl group at the C-terminus. Since a peptide fragment LLQG is a good substrate for MTGase to form an amide bond between the gamma-amide group of the Gln residue and the epsilon-amino group of Lys, a monellin B chain analogue in which LLQG was elongated at the C-terminus (B-LLQG) was synthesized by solid-phase synthesis. The monellin A chain analogue in which KGK was elongated at the N-terminus (KGK-A) was synthesized by the same method as that of the B chain analogue. The KGK-A chain and the B-LLQG chain were coupled by MTGase to give single-chain analogue of monellin. The single-chain analogue of monellin was characterized by analytical reverse phase high performance liquid chromatography, electrospray ionization, and amino acid analyses. All analyses gave satisfactory results. The single-chain analogue of monellin was more heat stable than natural monellin.

Abrusoside A methyl ester was prepared from abrusogenin through methylation (CH2N2) and a subsequent coupling reaction with 1-chloro-2,3,4,6-tetra-O-acethylglucopyranose in the presence of AgOTf and TMU in CH2Cl2, followed by deacetylation using K2CO3 in MeOH-H2O.

The sweet protein mabinlin II isolated from the seeds of Capparis masaikai consists of the A chain with 33 amino acid residues and the B chain composed of 72 residues. The B chain contains two intramolecular disulfide bonds and is connected to the A chain through two intermolecular disulfide bridges. The A chain was synthesized by the stepwise fluoren-9-ylmethoxycarbonyl (Fmoc) solid-phase method in a yield of 5.9%, while the B chain was synthesized by a combination of the stepwise Fmoc solid-phase method and fragment condensation in a yield of 6.0%. Disulfide formation and combination of the A and B chains followed by purification by ion-exchange high-performance liquid chromatography (HPLC) gave mabinlin II in a yield of 47.4%. The characterization of the synthetic mabinlin II by HPLC, electrospray ionization mass spectrometry, amino acid analysis, and disulfide bond determination fully supported the expected structure. A 0.1% solution of the synthetic mabinlin II had an astringent-sweet taste.

The activity of enzymes to form a peptide bond in organic solvent was greatly influenced by observed pH and water content. The precursors of two sweeteners, P-Asp-Xaa-OR (P=Z or For, Xaa-OR=Phe-OMe or Ala-OcHex), were synthesized by enzyme, and the reaction conditions were studied systematically. Z-Asp-OH was coupled with H-Phe-OMe or H-Ala-OcHex by thermolysin in tert-amyl alcohol. The best coupling results were obtained when the optimized observed pH was 8 or 9, and the water content was about 6% (V/V). The protecting group Z is better than For under the reaction conditions and H-Phe-OMe is a better nucleophile than H-Ala-OcHex. The expected optically pure peptides were obtained when the racemic amino acids were used as amino components in the starting materials. The physical constants of P-Asp-Xaa-OR synthesized by thermolysin are identical with those of peptides synthesized by chemical method.

Single-chain monellin, a sweet protein, was synthesized by stepwise solid-phase synthesis. After deprotection, and purification with HPLC, single-chain monellin was obtained in a yield of 0.3% based on the starting resin.

A synthesis of a biotinylated-, coumarin-substituted-N,N'-diarylguanidine 1 is reported. This ligand has structural features conducive to studying sweet taste including a fluorescent tag to facilitate spectroscopic studies of binding to protein-receptors for sweet ligands and an anchor for affinity purification.

Four potent sweet-tasting molecules, N-(3,3-dimethylbutyl)-L-aspartyl-L-phenylalanine methylester 1 (7000 times more potent than sucrose), N-(3,3-dimethylbutyl)-L-aspartyl-D-valine (S)-alpha-ethylbenzylamide 2 (3000 time more potent than sucrose), L-aspartyl-D-valine (R)-alpha-methoxymethylbenzylamide 3 (1350 times more potent than sucrose and L-aspartyl-(1R,2S,4S)-1-methyl-2-hydroxy-4-phenylhexylamide 4 (2500 times more potent than sucrose) were studied by 1H NMR and computer simulations. These flexible molecules adopt multiple conformations in solution. The "L-shaped" structure, which we believe to be responsible for sweet taste is accessible to all four compounds in solution. Extended conformations with the AH and B-containing moieties in the +y-axis and the hydrophobic group X pointing in the y-axis have also been observed for all four sweeteners. For compounds 1 and 3, the solid-state conformations were determined by X-ray diffraction studies. These results demonstrate that compounds 1 and 3 adopt an "L-shaped" structure even in the crystalline state. The extraordinary potency of the N-alkylated compound 1 compared with the unsubstituted Asp-Phe-OMe may be explained by the effect of a second hydrophobic binding domain in addition to interactions arising from the "L-shaped" structure.

A dipeptide taste ligand L-aspartyl-D-2-aminobutyric acid-(S)-alpha-ethylbenzylamide was found to be about 2000 times more potent than sucrose. To investigate the molecular basis of its potent sweet taste, we carried out conformational analysis of this molecular and several related analogues by NMR spectroscopy, computer simulations and X-ray crystallographic studies. The results of the studies support our earlier model that an L-shape molecular array is essential for eliciting sweet taste. In addition, we have identified an aromatic group located between the stem and the base of the L-shape, which is responsible for enhancement of sweetness potency. In this study, we also assessed the optimal size of the essential hydrophobic group (X) and the effects of the chirality of the second residue toward taste.