WO2019147617A1

WO2019147617A1 - Natural and synthetic diterpene glycosides, compositions and methods

Info

Publication number: WO2019147617A1
Application number: PCT/US2019/014678
Authority: WO
Inventors: Indra Prakash; Gil Ma
Original assignee: The Coca-Cola Company
Priority date: 2018-01-23
Filing date: 2019-01-23
Publication date: 2019-08-01

Abstract

Novel diterpene glycosides isolated from Stevia extract as well as diterpene glycosides that are synthetically prepared are provided herein. Compositions and consumables comprising the novel diterpene glycosides are also provided herein. Methods of enhancing the sweetness and/or flavor of consumables using the novel diterpene glycosides, methods of preparing compositions and consumables comprising the novel diterpene glycosides, methods of purifying the novel diterpene glycosides and methods of synthesizing the diterpene glycosides are also provided.

Description

NATURAL AND SYNTHETIC DITERPENE GLYCOSIDES,

COMPOSITIONS AND METHODS

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/620,733, filed January 23, 2018 and U.S. Provisional Patent Application No. 62/666,292, filed May 3, 2018. The contents of the above-referenced applications are incorporated herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to novel diterpene glycosides, compositions (e.g., consumables) comprising said novel diterpene glycosides, and methods for their purification.

BACKGROUND OF THE INVENTION

Natural caloric sugars, such as sucrose, fructose and glucose, are utilized to provide a pleasant taste to beverages, foods, pharmaceuticals, and oral hygienic/cosmetic products. Sucrose, in particular, imparts a taste preferred by consumers. Although sucrose provides superior sweetness characteristics, it is disadvantageously caloric.

Non-caloric or low caloric sweeteners have been introduced to satisfy consumer demand. However, non- and low caloric sweeteners taste different from natural caloric sugars in ways that frustrate consumers. On a taste basis, non-caloric or low caloric sweeteners exhibit a temporal profile, maximal response, flavor profile, mouth feel, and/or adaptation behavior that differ from sugar. Specifically, non-caloric or low caloric sweeteners exhibit delayed sweetness onset, lingering sweet aftertaste, bitter taste, metallic taste, astringent taste, cooling taste and/or licorice-like taste. On a source basis, many non-caloric or low caloric sweeteners are synthetic sweeteners. Consumer desire for natural non-caloric or low caloric sweeteners that tastes like sucrose remains high.

Stevia rebaudiana Bertoni is a perennial shrub of the Asteraceae ( Compositae ) family native to certain regions of South America. Its leaves have been traditionally used for hundreds of years in Paraguay and Brazil to sweeten local teas and medicines. The plant is commercially cultivated in Japan, Singapore, Taiwan, Malaysia, South Korea, China, Israel, India, Brazil, Australia and Paraguay.

The leaves of the plant contain a mixture of diterpene glycosides in an amount ranging from about 10% to 15% of the total dry weight. Structurally, the diterpene glycosides are characterized by a single base, steviol, and differ by the presence of carbohydrate residues at positions C13 and C19. Typically, on a dry weight basis, the four major steviol glycosides found in the leaves of Stevia are dulcoside A (0.3%), rebaudioside C (0.6-1.0%), rebaudioside A (3.8%) and stevioside (9.1%). Other glycosides identified in Stevia extract include rebaudioside B, D, E, and F, steviolbioside and rubusoside. Among these, only stevioside and rebaudioside A are available on a commercial scale.

The use of steviol glycosides has been limited to date by certain undesirable taste properties, including licorice taste, bitterness, astringency, sweet aftertaste, bitter aftertaste, licorice aftertaste, and become more prominent with increase of concentration. These undesirable taste attributes are particularly prominent in carbonated beverages, where full replacement of sugar requires concentrations of steviol glycosides that exceed 600 mg/L. Use of steviol glycosides in such high concentrations results in significant deterioration in the final product taste.

Accordingly, there remains a need to develop natural reduced or non-caloric sweeteners that provide a temporal and flavor profile similar to the temporal and flavor profile of sucrose.

There remains a further need for methods for purifying glycosides from stevia methods of preparing diterpene glycosides synthetically.

SUMMARY OF THE INVENTION

The present invention relates generally to novel diterpene glycosides, and compositions and consumables comprising said novel diterpene glycosides, as well as methods for purifying said novel diterpene glycosides, methods for preparing compositions and consumables comprising said novel diterpene glycosides and methods for enhancing the flavor or sweetness of consumables using the novel diterpene glycosides. In one aspect, the novel diterpene glycoside is isolated from Stevia extract. Exemplary diterpene glycosides are selected from the following:

and

In another aspect, the present invention is a method for purifying the above-referenced diterpene glycosides of the present invention comprising (i) passing a solution comprising a source material comprising a diterpene glycoside of the formulae described herein through a HPLC column and (ii) eluting fractions comprising the diterpene glycoside of the formulae described herein to provide a purified diterpene glycoside of the formulae described herein. The method provides a purified diterpene glycoside of the formulae described herein in a purity greater than about 50% by weight on a dry basis.

The HPLC column can be preparative or semi-preparative. The fractions comprising the diterpene glycoside of interest may be eluted by adding an appropriate eluent. The method may optionally comprise additional steps, such as partial or substantially full removal of solvents and/or further purification steps, e.g. extraction, crystallization, chromatography and distillation.

In still other embodiments, the source material can be one fraction, or multiple fractions, containing the unpurified diterpene glycoside of interest collected from a previous method or HPLC protocol. The material isolated can be subjected to further methods 2, 3, 4 or more times, each time providing a higher level of purity of the diterpene glycoside. The second and subsequent methods may have different HPLC protocols and different steps following elution.

In another aspect, the novel diterpene glycoside is synthetically prepared. Exemplary diterpene glycosides are selected from the following:

In another aspect, the present invention is a method for preparing the above-referenced diterpene glycosides of the present invention synthetically comprising (a) protecting the hydroxyl groups of a starting diterpene glycoside having the following structure:

wherein R¹ and R² are each independently selected from hydrogen, monosaccharide and oligosaccharide,

to provide a protected diterpene glycoside having the following formula:

wherein R³ and R⁴ are each independently selected from hydrogen, monosaccharide and oligosaccharide, and when R³ or R⁴ is a monosaccharide or oligosaccharide, the hydroxyl groups present thereon are also protected;

(b) coupling the protected diterpene glycoside with a functionalized sugar having the following formula:

wherein R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from hydrogen, monosaccharide and oligosaccharide, and when R⁹, R¹⁰, R^uor R¹² is a monosaccharide or oligosaccharide, the hydroxyl groups present thereon are also protected,

to provide a protected, coupled diterpene glycoside of the following formula:

and (c) deprotecting the protected, coupled diterpene glycoside to provide the target glycoside of the following formula:

In a particular embodiment, the diterpene glycoside is isolated and purified.

In a further aspect, the present invention is a composition comprising at least one diterpene glycoside described herein. In a particular embodiment, the present invention is a composition comprising at least one isolated and purified diterpene glycoside described herein. In one embodiment, the present invention is a sweetener composition comprising at least one diterpene glycoside described herein. In a particular embodiment, the present invention is a sweetener composition comprising at least one isolated and purified diterpene glycoside described herein.

In another embodiment, the present invention is a flavor enhancing composition comprising at least one diterpene glycoside described herein, wherein the diterpene glycoside is present in the composition in an amount effective to provide a concentration at or below the flavor recognition threshold of the diterpene glycoside when the flavor enhancing composition is added to a consumable.

In yet another embodiment, the present invention is a sweetness enhancing composition comprising at least one diterpene glycoside described herein, wherein the diterpene glycoside is present in the composition in an amount effective to provide a concentration at or below the sweetness recognition threshold of the diterpene glycoside when the sweetness enhancing composition is added to a consumable.

In yet another embodiment, the present invention is a consumable comprising at least one diterpene glycoside described herein. Suitable consumables include, but are not limited to, liquid-based or dry consumables, such as, for example, pharmaceutical compositions, edible gel mixes and compositions, dental compositions, foodstuffs, beverages and beverage products.

In a particular embodiment, the present invention is a beverage comprising at least one diterpene glycoside described herein. In a particular embodiment, the diterpene glycoside is present in the beverage at a concentration that is above, at or below the threshold sweetness recognition concentration of the diterpene glycoside.

In another particular embodiment, the present invention is a beverage product comprising a diterpene glycoside described herein. In a particular embodiment, the diterpene glycoside is present in the beverage product at a concentration that is above, at or below the threshold flavor recognition concentration of the diterpene glycoside.

In another aspect, the present invention is a method of preparing a consumable comprising (i) providing a consumable matrix and (ii) adding at least one diterpene glycoside described herein to the consumable matrix to provide a consumable. In a particular embodiment, the present invention is a method of preparing a beverage comprising (i) providing a beverage matrix and (ii) adding at least one diterpene glycoside described herein to the beverage matrix to provide a beverage.

In another aspect, the present invention is a method of enhancing the sweetness of a consumable comprising (i) providing a consumable comprising at least one sweet ingredient and (ii) adding at least one isolated and purified diterpene glycoside described herein to the consumable to provide a consumable with enhanced sweetness, wherein the diterpene glycoside is present in the consumable with enhanced sweetness at a concentration at or below the sweetness recognition threshold of the diterpene glycoside. In a particular embodiment, the consumable is a beverage. In certain embodiments, the diterpene glycoside is added in the form of a composition comprising an isolated and purified diterpene glycoside, as described herein.

In a further aspect, the present invention is a method of enhancing the flavor of a consumable comprising (i) providing a consumable comprising at least one flavor ingredient and (ii) adding at least one isolated and purified diterpene glycoside described herein to the consumable to provide a consumable with enhanced flavor, wherein the diterpene glycoside is present in the consumable with enhanced flavor at a concentration at or below the flavor recognition threshold of the diterpene glycoside. In a particular embodiment, the consumable is a beverage. In certain embodiments, the diterpene glycoside is added in the form of a composition comprising an isolated and purified diterpene glycoside, as described herein.

In some embodiments, the compositions of the present invention comprise one or more sweeteners, additives and/or functional ingredients.

In one embodiment, the present invention is a consumable comprising at least one diterpene glycoside of the present invention and one or more sweeteners, additives and/or functional ingredients. In another embodiment, the present invention is a beverage comprising at least one diterpene glycoside of formula of the present invention and one or more sweeteners, additives and/or functional ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Shows a comparison of the taste attributes of CC-00392 and CC-00393 compared to 95% Reb M as determined by a trained sensory panel (Example 13).

DETAILED DESCRIPTION OF THE INVENTION

I. Compounds

In one aspect, the present invention provides a diterpene glycoside of Formula I:

Gic V

Formuia Ϊ wherein R¹ and R² are each independently selected from hydrogen, monosaccharide, disaccharide and oligosaccharide.

Compounds of Formula I are characterized by a l- 3 b-linkage between the rhamnose and Glc V and a l- 6 b-linkage between Glc IV and Glc VII. In exemplary embodiments, each saccharide is selected from the group consisting of glucose, xylose, rhamnose, fructose and 6-deoxy-glucose. In a particular embodiment, each saccharide is glucose.

The linkage between the saccharides in the disaccharide or oligosaccharide can be a-, b or a mixture thereof (if applicable). The linkage between the saccharides (which may be part of a disaccharide or oligosaccharide) at R¹ and Glc II can be a- or b. The linkage between the saccharides (which may be part of a disaccharide or oligosaccharide) at R² and Rha can be a- or b. In exemplary embodiments, both linkages are b.

In a particular embodiment, a diterpene glycoside of Formula I comprises at least four saccharides pendant to C13 (including Glc II, Glc IV and Glc VII), such as, for example, four saccharides, five saccharides or six saccharides. In a particular embodiment, a diterpene glycoside of Formula I comprises from four to six saccharides pendant to C13.

In a particular embodiment, the diterpene glycoside is the following compound:

Glc V

CC-000364

In one embodiment, the present invention provides a diterpene glycoside of Formula II:

Formula II wherein R¹, R², R³ and R⁴ are each independently selected from hydrogen, monosaccharide, disaccharide and oligosaccharide.

In exemplary embodiments, each saccharide is selected from the group consisting of glucose, xylose, rhamnose, fructose and 6-deoxy-glucose. In a particular embodiment, each saccharide is glucose.

The linkage between the saccharides in the disaccharide or oligosaccharide can be a-, b or a mixture thereof (if applicable). The linkage between the saccharides (which may be part of a disaccharide or oligosaccharide) of any of R¹, R², R³ and R⁴ and the neighboring saccharide can be a- or b.

In a particular embodiment, the diterpene glycoside is the following compound:

Glc V CC-00366.

In another aspect, the present invention provides the following diterpene glycoside:

Glc VII

CC-00370.

In still another aspect, the present invention provides the following diterpene glycoside:

CC-00392.

CC-00393 In another aspect, the present invention provides the following diterpene glycoside:

CC-00404.

CC-00405. In another aspect, the present invention provides the following diterpene glycoside:

CC-00375.

CC-00422.

CC-00423.

CC-00424.

CC-00428

In yet another aspect, the present invention provides the following diterpene glycoside:

The compounds described herein have a plurality of stereocenters (R,S). Unless stereochemistry is specifically provided for, all stereochemical configurations are contemplated herein.

In exemplary embodiments, the diterpene glycoside of the present invention is isolated and purified. The term“isolated and purified”, as used herein, means that the compound is about 95% by weight or greater on a dry basis, i.e. is greater than 95% pure. The remainder of the mixture is typically other steviol glycoside and/or Stevia extract. In more specific embodiments, the diterpene glycoside of the formulae described herein has a purity of about 96% or greater, about 97% or greater, about 98% or greater or about 99% or greater. In some embodiments, the compound is enzymatically produced and is in a purity of at least about 95% by weight or greater in a mixture.

In some embodiments, the diterpene glycoside of the present invention is sweet. The sweetness of a given composition is typically measured with reference to a solution of sucrose. See generally "A Systematic Study of Concentration-Response Relationships of Sweeteners," G.E. DuBois, D.E. Walters, S.S. Schiffman, Z.S. Warwick, B.J. Booth, S.D. Pecore, K. Gibes, B.T. Carr, and L.M. Brands, in Sweeteners: Discovery, Molecular Design and Chemoreception , D.E. Walters, F.T. Orthoefer, and G.E. DuBois, Eds., American Chemical Society, Washington, DC (1991), pp 261-276.

The sweetness of a non-sucrose sweetener can be measured against a sucrose reference by determining the non-sucrose sweetener’s sucrose equivalence (SE). Typically, taste panelists are trained to detect sweetness of reference sucrose solutions containing between 1-15% sucrose (w/v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given percent sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, then the sweetener is said to be 10 times as potent as sucrose, and has 10% sucrose equivalence.

In one embodiment, the diterpene glycoside is present in an amount that, when added to a consumable, provides a sucrose equivalence of greater than about 2% (w/v), such as, for example, greater than about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13% or about 14%. The amount of sucrose, and thus another measure of sweetness, in a reference solution may be described in degrees Brix (°Bx). One degree Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by weight (% w/w) (strictly speaking, by mass). In one embodiment, the diterpene glycoside of the present invention is present in an amount that, when added to a consumable, provides a sweetness equivalent from about 0.50 to 14 degrees Brix, such as, for example, from about 5 to about 12 degrees Brix, about 7 to 10 degrees Brix, or above 10 degrees Brix.

In exemplary embodiments, an isolated and purified diterpene glycoside of the present invention has about 30% or more sweetness compared to the partially purified diterpene glycoside or Stevia leaf, such as, for example, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more.

In other exemplary embodiments, an isolated and purified diterpene glycoside of the present invention has at least about 30% less bitterness (the taste stimulated by certain substances such as quinine, caffeine and sucrose octa-acetate) compared the partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, the isolated and purified diterpene glycoside of the present invention has substantially no bitterness. Methods of measuring bitterness of a compound are known in the art

In still other exemplary embodiments, an isolated and purified diterpene glycoside of the present invention has at least about 30% less sweet lingering aftertaste (the intensity of the sweet taste after expectoration) compared to the partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, the isolated and purified diterpene glycoside of the present invention has substantially no sweet lingering aftertaste. Methods of measuring sweet lingering aftertaste are known in the art.

In yet other exemplary embodiments, an isolated and purified diterpene glycoside of the present invention has at least about 30% less metallic taste (taste associated with metals, tinny or iron) compared to the partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, the isolated and purified diterpene glycoside of the present invention has substantially no metallic taste.

In exemplary embodiments, an isolated and purified diterpene glycoside of the present invention exhibits a maximal response (maximum sweetness (%SE) achieved with increasing concentration of compound) that is at least about 30% greater compared to the partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% greater, at least about 50% greater, at least about 60% greater, at least about 70% greater, at least about 80% greater or at least about 90% greater. Methods of measuring the maximal response of a compound are known in the art. In one embodiment, the method is an in vitro cell assay. In some embodiments, the cell is expressing a sweet taste receptor or a dimer of sweet taste receptor.

In other exemplary embodiments, an isolated and purified diterpene glycoside of the present invention exhibits a sweetness onset (the time until maximum sweetness is experienced) that is at least about 30% shorter than the partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% short, at least about 50% shorter, at least about 60% shorter, at least about 70% shorter, at least about 80% shorter or at least about 90% shorter.

Methods of measuring sweetness onset are known in the art. In one embodiment, the method is an in vitro cell assay. In some embodiments, the cell is expressing a sweet taste receptor or a dimer of sweet taste receptor.

II. Compositions

The present invention includes compositions comprising at least one diterpene glycoside of the present invention.“Composition,” as the term is used herein, refers to a mixture of at least one diterpene glycoside of the present invention and at least one other substance, wherein the diterpene glycoside is admixed with the at least one other substance. As used herein,“admix” means to mingle or add to something else, but in any case, requires an active step.

In a particular embodiment, the at least one other substance does not occur and/or is not admixed with the diterpene glycoside in nature, i.e. the Stevia leaf. As such, the compositions contemplated by the present invention do not occur in nature.

In one embodiment, the present invention is a composition comprising at least one diterpene glycoside of the present invention, provided as part of a mixture. In a particular embodiment, the mixture is selected from the group consisting of diterpene glycosides, stevia extract, by-products of other diterpene glycosides’ isolation and purification processes, commercially available diterpene extracts or stevia extracts, by-products of biotransformation reactions of other diterpene glycosides, or any combination thereof.

In one embodiment, the mixture contains at least one diterpene glycoside of the present invention in an amount that ranges from about 1% to about 99% by weight on a dry basis, such as, for example, about 5% to about 99% by weight on a dry basis, from about 10% to about 99%, from about 20% to about 99%, from about 30% to about 99%, from about 40% to about 99%, from about 50% to about 99%, from about 60% to about 99%, from about 70% to about 99%, from about 80% to about 99% and from about 90% to about 99%. In a particular embodiment, the mixture contains at least one diterpene glycoside of the present invention in an amount greater than about 90% by weight on a dry basis, for example, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98% and greater than about 99%.

The term“purified diterpene glycoside”, as used herein, refers to a diterpene glycoside present in at least about 50% by weight in a mixture, e.g. stevia extract, such as, for example, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 97%.

In a particular embodiment, the mixture is an extract of a stevia plant variety. Suitable Stevia varieties include, but are not limited to S. rebaudiana Bertoni and S. rebaudiana Morita.

The stevia extract may contain one or more additional diterpene glycosides, i.e., diterpene glycosides that are not the diterpene glycosides of the present invention, including, but not limited to, stevioside, rebaudioside A, rebaudioside C, dulcoside A, rubusoside, steviolbioside, rebaudioside B, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside M, rebaudioside N, rebaudioside O and combinations thereof.

In one embodiment, the present invention is a composition comprising at least one diterpene glycoside described herein provided as a pure compound, i.e. > 99% purity on a dry basis. The diterpene glycosides of the present invention may be present in the composition in an amount effective to provide a concentration of diterpene glycoside of the present invention from about 1 ppm to about 10,000 ppm when the composition is added to a consumable, such as, for example, from about 1 ppm to about 4,000 ppm, from about 1 ppm to about 3,000 ppm, from about 1 ppm to about 2,000 ppm, from about 1 ppm to about 1,000 ppm, from about 1 ppm to about 600 ppm, from about 1 ppm to about 500 ppm, from about 1 ppm to about 400 ppm, from about 1 ppm to about 300 ppm, from about 1 ppm to about 200 ppm or from about 1 ppm to about 100 ppm..

In another embodiment, the diterpene glycoside of the present invention is present in the composition in an amount effective to provide a concentration of diterpene glycoside of the present invention from about 50 to about 600 ppm when added to a consumable, such as, for example, from about 100 ppm to about 600 ppm, from about 100 ppm to about 500 ppm, from about 100 ppm to about 400 ppm, from about 100 ppm to about 300 ppm or from about 100 ppm to about 200 ppm.

Sweetener Compositions

As noted above, in some embodiments, the diterpene glycoside of the present invention is sweet. Accordingly, the present invention also provides a sweetener composition comprising at least one diterpene glycoside of the present invention.“Sweetener composition,” as the term is used herein, refers to a mixture of at least one diterpene of the present invention and at least one other substance, wherein the at least one diterpene glycoside is admixed with the at least one other substance.

In a particular embodiment, the at least one other substance does not occur and/or is not admixed with the diterpene glycoside in nature, i.e. the Stevia leaf. As such, the sweetener compositions contemplated by the present invention do not occur in nature. In one embodiment, the at least one other substance modulates the taste profile of the at least one diterpene glycoside to provide a composition with a more sucrose-like taste profile compared to the diterpene glycoside in nature and (if applicable) the at least one other substance in nature. For example, in certain embodiments the composition exhibits one or more of the following characteristics: improved sweetness potency, improved mouthfeel, decreased sweetness linger, decreased bitterness and/or decreased metallic taste. In certain exemplary embodiments, the sweetener composition comprises at least one purified diterpene glycoside of this invention.

In one embodiment, the diterpene glycoside of the present invention is the sole sweetener in the sweetener composition, i.e. the diterpene glycoside is the only compound present in the sweetener composition that provides a detectable sweetness.

In further embodiments, the sweetener composition comprising at least one diterpene glycoside of the present invention in combination with at least one additional sweetener. In a particular embodiment, the at least one additional sweetener does not occur with the diterpene glycoside in nature, i.e. Stevia leaf. In a more particular embodiment, a sweetener composition comprises at least one purified diterpene glycoside at least one additional sweetener that does not occur with the diterpene glycoside in nature.

The amount of diterpene glycoside of the present invention in the sweetener composition may vary. In one embodiment, the diterpene glycoside of the present invention is present in a sweetener composition in any amount to impart the desired sweetness when the sweetener composition is added to a sweetenable composition or sweetenable consumable. In a particular embodiment, the diterpene glycoside of the present invention is present in a concentration above its threshold sweetness recognition concentration.

In one embodiment, the diterpene glycoside of the present invention is present in the sweetener composition in an amount effective to provide a sucrose equivalence of greater than about 2% (w/v) when the sweetener composition is added to a sweetenable composition or sweetenable consumable, such as, for example, greater than about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13% or about 14%.

In one embodiment, the sweetener is at least one natural high-potency sweetener. As used herein, the phrase "natural high potency sweetener" refers to any sweetener found naturally in nature and characteristically has a sweetness potency greater than sucrose, fructose, or glucose, yet has less calories. The natural high potency sweetener can be provided as a pure compound or, alternatively, as part of an extract. In another embodiment, the sweetener is at least one synthetic sweetener. As used herein, the phrase "synthetic sweetener" refers to any composition which is not found naturally in nature and characteristically has a sweetness potency greater than sucrose, fructose, or glucose, yet has less calories.

In still other embodiments, combinations of natural high potency sweeteners and synthetic sweeteners are contemplated.

In other embodiments, the sweetener is at least one carbohydrate sweetener. Suitable carbohydrate sweeteners are selected from, but not limited to, the group consisting of sucrose, glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheltulose, octolose, fucose, rhamnose, arabinose, turanose, sialose and combinations thereof.

Other suitable sweeteners include rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, stevia, stevioside, mogroside IV, mogroside V, mogroside VI, Luo ban guo , siamenoside, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein, hemandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin I, abrusoside A, steviolbioside, hesperitin and cyclocarioside I, sugar alcohols such as erythritol, sucralose, potassium acesulfame, acesulfame acid and salts thereof, aspartame, alitame, saccharin and salts thereof, neohesperidin dihydrochalcone, cyclamate, cyclamic acid and salts thereof, neotame, advantame, glucosylated steviol glycosides (GSGs) and combinations thereof.

In a particular embodiment, the sweetener is at least one calorie-providing carbohydrate sweetener. In one embodiment, the sweetener is a caloric sweetener or mixture of caloric sweeteners. In another embodiment, the caloric sweetener is selected from sucrose, fructose, glucose, high fructose corn/starch syrup, a beet sugar, a cane sugar, and combinations thereof. In another embodiment, the sweetener is a rare sugar selected from allulose, sorbose, lyxose, ribulose, xylose, xylulose, D-allose, L-ribose, D-tagatose, L-glucose, L-fucose, L- arabinose, turanose, kojibiose and combinations thereof.

In still another embodiment, the sweetener is a mixture of at least one natural high potency sweeteners and at least one carbohydrate sweetener. In yet another embodiment, the sweetener is a mixture of at least one synthetic sweetener and at least one carbohydrate sweetener. In a further embodiment, the sweetener is at least one natural high potency sweetener, at least one synthetic sweetener and at least one carbohydrate sweetener.

In exemplary embodiments, a sweetener composition comprising at least one purified diterpene glycoside of the present invention has about 30% or more sweetness compared to a corresponding sweetener composition comprising partially purified diterpene glycoside or Stevia, such as, for example, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more.

In other exemplary embodiments, a sweetener composition comprising at least one purified diterpene glycoside of the present invention has at least about 30% less bitterness (the taste stimulated by certain substances such as quinine, caffeine and sucrose octa-acetate) compared to a corresponding composition comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a sweetener composition comprising at least one purified diterpene glycoside of the present invention has substantially no bitterness. Methods of measuring bitterness of a compound are known in the art

In still other exemplary embodiments, a sweetener composition comprising at least one purified diterpene glycoside of the present invention has at least about 30% less sweet lingering aftertaste (the intensity of the sweet taste after expectoration) compared to a corresponding sweetener composition comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a sweetener composition comprising at least one purified diterpene glycoside of the present invention has substantially no sweet lingering aftertaste. Methods of measuring sweet lingering aftertaste are known in the art. In yet other exemplary embodiments, a sweetener composition comprising at least one purified diterpene glycoside of the present invention has at least about 30% less metallic taste (taste associated with metals, tinny or iron) compared to a corresponding sweetener composition comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a sweetener composition comprising at least one purified diterpene glycoside of the present invention has substantially no metallic taste.

In exemplary embodiments, a sweetener composition comprising at least one purified diterpene glycoside of the present invention exhibits a maximal response (maximum sweetness (%SE) achieved with increasing concentration of compound) that is at least about 30% greater compared to a corresponding sweetener composition comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% greater, at least about 50% greater, at least about 60% greater, at least about 70% greater, at least about 80% greater or at least about 90% greater. Methods of measuring the maximal response of a compound are known in the art.

In other exemplary embodiments, a sweetener composition comprising at least one purified diterpene glycoside of the present invention exhibits a sweetness onset (the time until maximum sweetness is experienced) that is at least about 30% shorter than a sweetener composition comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% short, at least about 50% shorter, at least about 60% shorter, at least about 70% shorter, at least about 80% shorter or at least about 90% shorter. Methods of measuring sweetness onset are known in the art.

Sweetness Enhancers

In a particular embodiment, the diterpene glycoside of the present invention is a sweetness enhancer or modifier.“Sweetness enhancer”, as the term is used herein, refers to a compound that enhances, amplifies or potentiates the perception of sweetness of a consumable (e.g. a beverage) when said compound is present in the consumable in a concentration at or below the compound’s sweetener recognition threshold, i.e. in a concentration at which compound does not contribute any noticeable sweet taste in the absence of additional sweetener(s). “Sweetness modifier”, as the term is used herein, refers to a compound that changes the taste properties (such as linger, off-notes, or the like) of sweetness of a consumable (e.g. a beverage) when said compound is present in the consumable in a concentration at or below the compound’s sweetener recognition threshold.

The term "sweetness enhancer" is synonymous with the terms "sweet taste potentiator," "sweetness potentiator," "sweetness amplifier," and "sweetness intensifier."

In a particular embodiment, the additional sweetener(s) does not naturally occur and/or is not admixed with the at least one diterpene glycoside sweetness enhancer in nature, i.e. Stevia leaf. As such, the sweetness-enhanced consumables contemplated by the present invention do not occur in nature.

In one embodiment, a diterpene glycoside of the present invention may be added directly to the consumable, i.e., not provided in the form of a composition but rather as compound, to enhance sweetness. In this embodiment, a diterpene glycoside of the present invention is added to the consumable at a concentration at or below its sweetness recognition threshold concentration, i.e., a sweetness enhancer. In a particular embodiment, a diterpene glycoside of the present invention is added to the consumable at a concentration below its sweetness recognition threshold concentration.

In certain embodiments, a diterpene glycoside of the present invention is a sweetness enhancer or modifier and is added to the consumable in an amount that will provide a concentration of the diterpene glycoside that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50% or more below its sweetness recognition threshold.

In some embodiments, the diterpene glycosides of the present invention enhances the sucrose equivalence (SE) of the consumable by at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4.0% or about 5.0%, when compared to the SE of the consumable in the absence of the diterpene glycoside of the present invention. In other embodiments, at least one diterpene glycoside of the present invention may be added to the consumable in the form of a sweetness enhancing composition. “Sweetness enhancing composition,” as the term is used herein, refers to a composition of the present invention - as described above - wherein the composition enhances, amplifies or potentiates the perception of sweetness of a consumable (e.g. a beverage) when a diterpene glycoside of the present invention is present in the sweetness enhancer composition in an amount that will provide a concentration of the diterpene glycoside that is at or below its sweetness recognition threshold when added to the consumable. In a particular embodiment, the diterpene glycoside of the present invention in an amount that will provide a concentration of the diterpene glycoside of that is below its sweetness recognition threshold.

In certain embodiments, a diterpene glycoside of the present invention is present in the sweetness enhancing composition in an amount effective to provide a concentration of the diterpene glycoside that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50% or more below its sweetness recognition threshold when the sweetness enhancing composition is added to a consumable.

It is contemplated that the sweetness enhancing composition can include one or more sweetness enhancers or modifiers in addition to at least one diterpene glycoside of the present invention. In one embodiment, the sweetness enhancing composition can include one additional sweetness enhancer. In other embodiments, the composition can include two or more additional sweetness enhancers. In embodiments where two or more sweetness enhancers or modifiers are utilized, each one should be present at or below its respective sweetness recognition threshold concentration.

The one or more other sweetness enhancers or modifiers are selected from, but not limited to, the group consisting of 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4- hydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,5- dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6- trihydroxybenzoic acid, 3-aminobenzoic acid, 4-aminobenzoic acid, 4-0-P-D-glucosyl- hesperetin dihydrochalcone, MG isomogrosaide V, 4-hydroxycinnamic acid, 4-methoxycinnamic acid, l-(2-hydroxyphenyl)-3-(4-pyridyl)-l-propanone, 4-ethoxybenzonitrile, 2-methoxy-5- (phenoxymethyl)-phenol, l-(2, 4-dihydroxyphenyl)-2-(3 -m ethoxy -4-hydroxyphenyl)-ethanone, hesperetin, 2,3’,6-trihydroxy-4’-methoxydihydrochalcone, N-(3’-methoxy-4’-hydroxybenzyl)- 2,4,6-trihydroxybenzamide, 3’-7-di hydroxy-4’ -methoxyflavan, FEMA GRAS flavor 4669, FEMA GRAS flavor 4701, FEMA GRAS flavor 4720, FEMA GRAS flavor 4774, FEMA GRAS flavor 4708, FEMA GRAS flavor 4728, FEMA GRAS flavor 4601, FEMA GRAS flavor 4802, 4-amino-5-(cyclohexyloxy)-2-methylquinoline-3-carboxylic acid, rebaudioside M, rebaudioside N, rebaudioside O, rebaudioside C and combinations thereof.

In one embodiment, addition of the sweetness enhancer or modifier increases the detected sucrose equivalence of the at least one sweetener in a consumable compared to the sucrose equivalence of the same consumable in the absence of the sweetness enhancer.

In a particular embodiment, the consumable is a beverage. According to this embodiment, a diterpene glycoside of the present invention and at least one sweetener is added to a beverage, wherein the diterpene glycoside is present in a concentration at or below its sweetness recognition threshold. In a particular embodiment, the detected sucrose equivalence is increased from about 0.2% to about 5.0%, such as, for example, about 1%, about 2%, about 3%, about 4% or about 5%.

Flavor Enhancers

In another particular embodiment, the diterpene glycoside of the present invention is a flavor enhancer. “Flavor enhancer”, as the term is used herein, refers to a compound that enhances, amplifies or potentiates the perceptions of a flavor ingredient (i.e. any substance that provides sweetness, sourness, saltiness, savoriness, bitterness, metallic taste, etc.) when said compound is present in a consumable (e.g. a beverage) in a concentration at or below the compound’s flavor recognition threshold, i.e. in a concentration at which compound does not contribute any noticeable flavor in the absence of any flavor ingredient(s). The term“flavor recognition threshold”, as generally used herein, is the lowest known concentration of a compound that is perceivable by the human sense of taste as the particular flavor. The flavor recognition threshold concentration is specific for a particular compound, and can vary based on temperature, matrix, ingredients and/or flavor system.

The term "flavor enhancer" is synonymous with the terms "flavor potentiator," "flavor amplifier," and "flavor intensifier." In a particular embodiment, the flavor ingredient(s) does not naturally occur and/or is not admixed with the at least one diterpene glycoside sweetness enhancer in nature, i.e. Stevia leaf. As such, the flavor-enhanced consumables contemplated by the present invention do not occur in nature.

In one embodiment, at least one diterpene glycoside of the present invention is added directly to the consumable, i.e., not provided in the form of a composition but rather as a compound, to enhance a flavor. In this embodiment, the diterpene glycoside of the present invention is added to the consumable at a concentration at or below its flavor recognition threshold concentration, i.e., a flavor enhancer. In a particular embodiment, the diterpene glycoside of the present invention is added to the consumable at a concentration below its flavor recognition threshold concentration.

In certain embodiments, a diterpene glycoside of the present invention is a flavor enhancer and is added to the consumable in an amount that will provide a concentration of the diterpene glycoside that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50% or more below its sweetness recognition threshold.

The diterpene glycosides of the present invention enhances the flavor of the consumable by at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 4.0% or about 5.0%, when compared to the flavor of the consumable in the absence of the diterpene glycosides of the present invention.

In other embodiments, at least one diterpene glycoside of the present invention may be added to the consumable in the form of a flavor enhancing composition.“Flavor enhancing composition,” as the term is used herein, refers to a mixture of at least one diterpene glycoside of the present invention and at least one flavor ingredient, wherein the at least one diterpene is admixed with the at least one flavor ingredient - wherein the composition enhances, amplifies or potentiates the perception of the flavor ingredient in a consumable (e.g. a beverage) when the at least one diterpene glycoside of the present invention is present in the flavor enhancer composition in an amount that will provide a concentration of the diterpene glycoside that is at or below its flavor recognition threshold when added to the consumable. Thus, the flavor enhancing compositions contemplated by the present invention do not occur in nature.

Addition of the flavor enhancing composition increases the detected flavor of the at least one flavor ingredient in the consumable compared to the detected flavor of the same ingredient in the consumable in the absence of the flavor enhancer. Without being bound by theory, the flavor enhancing composition likely does not contribute any noticeable taste to the consumable to which it is added because the flavor enhancer is present in the consumable in a concentration at or below the its flavor recognition threshold.

In one embodiment, the flavor enhancing composition comprises at least one diterpene glycoside of the present invention in an amount effective to provide a concentration of the at least one diterpene glycoside that is at or below its flavor recognition threshold when the flavor enhancing composition is added to a consumable.

In a particular embodiment, a diterpene glycoside of the present invention is present in the flavor enhancing composition in an amount effective to provide a concentration of the diterpene glycoside below its flavor recognition threshold when the flavor enhancing composition is added to a consumable.

In certain embodiment, a diterpene glycoside of the present invention is present in the flavor enhancing composition in an amount that, when added to a consumable, is effective to provide a concentration of the compound that is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45% or at least about 50% or more below its flavor recognition threshold.

A person of skill in the art will be able to select the concentration of the diterpene glycoside of the present invention in the flavor enhancing composition so that it may impart an enhanced flavor to a consumable comprising at least one flavor ingredient.

Suitable flavor ingredients include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, citrus, coconut, ginger, viridiflorol, almond, menthol (including menthol without mint), grape skin extract, and grape seed extract.“Flavorant” and“flavoring ingredient” are synonymous and can include natural or synthetic substances or combinations thereof. Flavorants also include any other substance which imparts flavor and may include natural or non-natural (synthetic) substances which are safe for human or animals when used in a generally accepted range. Non-limiting examples of proprietary flavorants include Dohler™ Natural Flavoring Sweetness Enhancer K14323 (Dohler™, Darmstadt, Germany), Symrise™ Natural Flavor Mask for Sweeteners 161453 and 164126 (Symrise™, Holzminden, Germany), Natural Advantage™ Bitterness Blockers 1, 2, 9 and 10 (Natural Advantage™, Freehold, New Jersey, U.S.A.), and Sucramask™ (Creative Research Management, Stockton, California, U.S.A.).

In another embodiment, the flavor enhancing composition comprising at least one diterpene glycoside of the present invention enhances flavors (either individual flavors or the overall flavor) when added to the consumable. These flavors include, but are not limited to, fruit flavors, including tropical fruit flavors, and vanilla-caramel type flavors.

The compositions described herein can be customized to provide the desired calorie content. For example, compositions can be“full-calorie”, such that they impart the desired sweetness when added to a consumable (such as, for example, a beverage) and have about 120 calories per 8 oz serving. Alternatively, compositions can be“mid-calorie”, i.e. have less than about 60 calories per 8 oz serving. In other embodiments, compositions can be“low-calorie”, i.e. have less than 40 calories per 8 oz serving. In still other embodiments, the compositions can be “zero-calorie”, i.e. have less than 5 calories per 8 oz. serving.

Additives

The compositions may comprise, in addition to at least one diterpene glycoside of the present invention, one or more additives and/or functional ingredients, detailed herein below.

Exemplary additives include, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, caffeine, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, plant extracts, flavonoids, alcohols, polymers and combinations thereof.

In one embodiment, the composition further comprises one or more polyols. The term "polyol", as used herein, refers to a molecule that contains more than one hydroxyl group. A polyol may be a diol, triol, or a tetraol which contains 2, 3, and 4 hydroxyl groups respectively. A polyol also may contain more than 4 hydroxyl groups, such as a pentaol, hexaol, heptaol, or the like, which contain 5, 6, or 7 hydroxyl groups, respectively. Additionally, a polyol also may be a sugar alcohol, polyhydric alcohol, or polyalcohol which is a reduced form of carbohydrate, wherein the carbonyl group (aldehyde or ketone, reducing sugar) has been reduced to a primary or secondary hydroxyl group.

Non-limiting examples of polyols in some embodiments include maltitol, mannitol, sorbitol, lactitol, xylitol, isomalt, propylene glycol, glycerol (glycerin), threitol, galactitol, palatinose, reduced isomalto-oligosaccharides, reduced xylo-oligosaccharides, reduced gentio- oligosaccharides, reduced maltose syrup, reduced glucose syrup, and sugar alcohols or any other carbohydrates capable of being reduced which do not adversely affect taste.

Suitable amino acid additives include, but are not limited to, aspartic acid, arginine, glycine, glutamic acid, proline, threonine, theanine, cysteine, cystine, alanine, valine, tyrosine, leucine, arabinose, trans-4-hydroxyproline, isoleucine, asparagine, serine, lysine, histidine, ornithine, methionine, carnitine, aminobutyric acid (a-, b-, and/or d-isomers), glutamine, hydroxyproline, taurine, norvaline, sarcosine, and their salt forms such as sodium or potassium salts or acid salts. The amino acid additives also may be in the D- or L-configuration and in the mono-, di-, or tri-form of the same or different amino acids. Additionally, the amino acids may be a-, b-, g- and/or d-isomers if appropriate. Combinations of the foregoing amino acids and their corresponding salts ( e.g ., sodium, potassium, calcium, magnesium salts or other alkali or alkaline earth metal salts thereof, or acid salts) also are suitable additives in some embodiments. The amino acids may be natural or synthetic. The amino acids also may be modified. Modified amino acids refers to any amino acid wherein at least one atom has been added, removed, substituted, or combinations thereof (e.g., N-alkyl amino acid, N-acyl amino acid, or N-methyl amino acid). Non-limiting examples of modified amino acids include amino acid derivatives such as trimethyl glycine, N-methyl-glycine, and N-methyl-alanine. As used herein, modified amino acids encompass both modified and unmodified amino acids. As used herein, amino acids also encompass both peptides and polypeptides (e.g, dipeptides, tripeptides, tetrapeptides, and pentapeptides) such as glutathione and L-alanyl-L-glutamine. Suitable polyamino acid additives include poly-L-aspartic acid, poly-L-lysine (e.g., poly-L-a-lysine or poly-L-s-lysine), poly-L- ornithine ( e.g ., poly-L-a-omithine or poly-L-s-ornithine), poly-L-arginine, other polymeric forms of amino acids, and salt forms thereof (e.g., calcium, potassium, sodium, or magnesium salts such as L-glutamic acid mono sodium salt). The poly-amino acid additives also may be in the D- or L-configuration. Additionally, the poly-amino acids may be a-, b-, g-, d-, and e- isomers if appropriate. Combinations of the foregoing poly-amino acids and their corresponding salts (e.g, sodium, potassium, calcium, magnesium salts or other alkali or alkaline earth metal salts thereof or acid salts) also are suitable additives in some embodiments. The poly-amino acids described herein also may comprise co-polymers of different amino acids. The poly-amino acids may be natural or synthetic. The poly-amino acids also may be modified, such that at least one atom has been added, removed, substituted, or combinations thereof (e.g., N-alkyl poly- amino acid or N-acyl poly-amino acid). As used herein, poly-amino acids encompass both modified and unmodified poly-amino acids. For example, modified poly-amino acids include, but are not limited to, poly-amino acids of various molecular weights (MW), such as poly-L-a- lysine with a MW of 1,500, MW of 6,000, MW of 25,200, MW of 63,000, MW of 83,000, or MW of 300, 000.

Suitable sugar acid additives include, but are not limited to, aldonic, uronic, aldaric, alginic, gluconic, glucuronic, glucaric, galactaric, galacturonic, and salts thereof (e.g., sodium, potassium, calcium, magnesium salts or other physiologically acceptable salts), and combinations thereof.

Suitable nucleotide additives include, but are not limited to, inosine monophosphate ("IMP"), guanosine monophosphate ("GMP"), adenosine monophosphate ("AMP"), cytosine monophosphate (CMP), uracil monophosphate (UMP), inosine diphosphate, guanosine diphosphate, adenosine diphosphate, cytosine diphosphate, uracil diphosphate, inosine triphosphate, guanosine triphosphate, adenosine triphosphate, cytosine triphosphate, uracil triphosphate, alkali or alkaline earth metal salts thereof, and combinations thereof. The nucleotides described herein also may comprise nucleotide-related additives, such as nucleosides or nucleic acid bases (e.g, guanine, cytosine, adenine, thymine, uracil).

Suitable organic acid additives include any compound which comprises a -COOH moiety, such as, for example, C2-C30 carboxylic acids, substituted hydroxyl C2-C30 carboxylic acids, butyric acid (ethyl esters), substituted butyric acid (ethyl esters), benzoic acid, substituted benzoic acids ( e.g ., 2,4-dihydroxybenzoic acid), substituted cinnamic acids, hydroxyacids, substituted hydroxybenzoic acids, anisic acid substituted cyclohexyl carboxylic acids, tannic acid, aconitic acid, lactic acid, tartaric acid, citric acid, isocitric acid, gluconic acid, glucoheptonic acids, adipic acid, hydroxycitric acid, malic acid, fruitaric acid (a blend of malic, fumaric, and tartaric acids), fumaric acid, maleic acid, succinic acid, chlorogenic acid, salicylic acid, creatine, caffeic acid, bile acids, acetic acid, ascorbic acid, alginic acid, erythorbic acid, polyglutamic acid, glucono delta lactone, and their alkali or alkaline earth metal salt derivatives thereof. In addition, the organic acid additives also may be in either the D- or L-configuration.

Suitable organic acid additive salts include, but are not limited to, sodium, calcium, potassium, and magnesium salts of all organic acids, such as salts of citric acid, malic acid, tartaric acid, fumaric acid, lactic acid (e.g., sodium lactate), alginic acid (e.g, sodium alginate), ascorbic acid (e.g, sodium ascorbate), benzoic acid (e.g, sodium benzoate or potassium benzoate), sorbic acid and adipic acid. The examples of the organic acid additives described optionally may be substituted with at least one group chosen from hydrogen, alkyl, alkenyl, alkynyl, halo, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfo, thiol, imine, sulfonyl, sulfenyl, sulfmyl, sulfamyl, carboxalkoxy, carboxamido, phosphonyl, phosphinyl, phosphoryl, phosphino, thioester, thioether, anhydride, oximino, hydrazino, carbamyl, phosphor or phosphonato.

Suitable inorganic acid additives include, but are not limited to, phosphoric acid, phosphorous acid, polyphosphoric acid, hydrochloric acid, sulfuric acid, carbonic acid, sodium dihydrogen phosphate, and alkali or alkaline earth metal salts thereof (e.g, inositol hexaphosphate Mg/Ca).

Suitable bitter compound additives include, but are not limited to, caffeine, quinine, urea, bitter orange oil, naringin, quassia, and salts thereof.

Suitable flavorants and flavoring ingredient additives include, but are not limited to, vanillin, vanilla extract, mango extract, cinnamon, citrus, coconut, ginger, viridiflorol, almond, menthol (including menthol without mint), grape skin extract, and grape seed extract. “Flavorant” and“flavoring ingredient” are synonymous and can include natural or synthetic substances or combinations thereof. Flavorants also include any other substance which imparts flavor and may include natural or non-natural (synthetic) substances which are safe for human or animals when used in a generally accepted range. Non-limiting examples of proprietary flavorants include Dohler™ Natural Flavoring Sweetness Enhancer K14323 (Dohler™, Darmstadt, Germany), Symrise™ Natural Flavor Mask for Sweeteners 161453 and 164126 (Symrise™, Holzminden, Germany), Natural Advantage™ Bitterness Blockers 1, 2, 9 and 10 (Natural Advantage™, Freehold, New Jersey, U.S.A.), and Sucramask™ (Creative Research Management, Stockton, California, U.S.A.).

Suitable polymer additives include, but are not limited to, chitosan, pectin, pectic, pectinic, polyuronic, polygalacturonic acid, starch, food hydrocolloid or crude extracts thereof (e.g., gum acacia Senegal (Fibergum™), gum acacia seyal, carageenan), poly-L-lysine (e.g., poly-L-a-lysine or poly-L-e-lysine), poly-L-ornithine (e.g., poly-L-a-omithine or poly-L-e- ornithine), polypropylene glycol, polyethylene glycol, poly(ethylene glycol methyl ether), polyarginine, polyaspartic acid, polyglutamic acid, polyethylene imine, alginic acid, sodium alginate, propylene glycol alginate, and sodium polyethyleneglycolalginate, sodium hexametaphosphate and its salts, and other cationic polymers and anionic polymers.

Suitable protein or protein hydrolysate additives include, but are not limited to, bovine serum albumin (BSA), whey protein (including fractions or concentrates thereof such as 90% instant whey protein isolate, 34% whey protein, 50% hydrolyzed whey protein, and 80% whey protein concentrate), soluble rice protein, soy protein, protein isolates, protein hydrolysates, reaction products of protein hydrolysates, glycoproteins, and/or proteoglycans containing amino acids (e.g., glycine, alanine, serine, threonine, asparagine, glutamine, arginine, valine, isoleucine, leucine, norvaline, methionine, proline, tyrosine, hydroxyproline, and the like), collagen (e.g., gelatin), partially hydrolyzed collagen (e.g., hydrolyzed fish collagen), and collagen hydrolysates (e.g., porcine collagen hydrolysate).

Suitable surfactant additives include, but are not limited to, polysorbates (e.g., polyoxyethylene sorbitan monooleate (polysorbate 80), polysorbate 20, polysorbate 60), sodium dodecylbenzenesulfonate, dioctyl sulfosuccinate or dioctyl sulfosuccinate sodium, sodium dodecyl sulfate, cetylpyridinium chloride (hexadecylpyridinium chloride), hexadecyltrimethylammonium bromide, sodium cholate, carbamoyl, choline chloride, sodium glycocholate, sodium taurodeoxycholate, lauric arginate, sodium stearoyl lactylate, sodium taurocholate, lecithins, sucrose oleate esters, sucrose stearate esters, sucrose palmitate esters, sucrose laurate esters, and other emulsifiers, and the like.

Suitable flavonoid additives are classified as flavonols, flavones, flavanones, flavan-3- ols, isoflavones, or anthocyanidins. Non-limiting examples of flavonoid additives include, but are not limited to, catechins (e.g., green tea extracts such as Polyphenon™ 60, Polyphenon™ 30, and Polyphenon™ 25 (Mitsui Norin Co., Ltd., Japan), polyphenols, rutins (e.g., enzyme modified rutin Sanmelin™ AO (San-fi Gen F.F.I., Inc., Osaka, Japan)), neohesperidin, naringin, neohesperidin dihydrochalcone, and the like.

Suitable alcohol additives include, but are not limited to, ethanol.

Suitable astringent compound additives include, but are not limited to, tannic acid, europium chloride (EuCb), gadolinium chloride (GdCb), terbium chloride (TbCb), alum, tannic acid, and polyphenols (e.g., tea polyphenols).

Exemplary functional ingredients include, but are not limited to, saponins, antioxidants, dietary fiber sources, fatty acids, vitamins, glucosamine, minerals, preservatives, hydration agents, probiotics, prebiotics, weight management agents, osteoporosis management agents, phytoestrogens, long chain primary aliphatic saturated alcohols, phytosterols and combinations thereof.

In certain embodiments, the functional ingredient is at least one saponin. As used herein, the at least one saponin may comprise a single saponin or a plurality of saponins as a functional ingredient for the composition provided herein. Saponins are glycosidic natural plant products comprising an aglycone ring structure and one or more sugar moieties. Non-limiting examples of specific saponins for use in particular embodiments of the invention include group A acetyl saponin, group B acetyl saponin and group E acetyl saponin. Several common sources of saponins include soybeans, which have approximately 5% saponin content by dry weight, soapwort plants ( Saponaria ), the root of which was used historically as soap, as well as alfalfa, aloe, asparagus, grapes, chickpeas, yucca, and various other beans and weeds. Saponins may be obtained from these sources by using extraction techniques well known to those of ordinary skill in the art. A description of conventional extraction techniques can be found in LT.S. Pat. Appl. No. 2005/0123662, the disclosure of which is expressly incorporated by reference. In certain embodiments, the functional ingredient is at least one antioxidant. As used herein“antioxidant” refers to any substance which inhibits, suppresses, or reduces oxidative damage to cells and biomolecules. Examples of suitable antioxidants for embodiments of this invention include, but are not limited to, vitamins, vitamin cofactors, minerals, hormones, carotenoids, carotenoid terpenoids, non-carotenoid terpenoids, flavonoids, flavonoid polyphenolics (e.g., bioflavonoids), flavonols, flavones, phenols, polyphenols, esters of phenols, esters of polyphenols, nonflavonoid phenolics, isothiocyanates, and combinations thereof. In some embodiments, the antioxidant is vitamin A, vitamin C, vitamin E, ubiquinone, mineral selenium, manganese, melatonin, a-carotene, b-carotene, lycopene, lutein, zeanthin, crypoxanthin, reservatol, eugenol, quercetin, catechin, gossypol, hesperetin, curcumin, ferulic acid, thymol, hydroxytyrosol, tumeric, thyme, olive oil, lipoic acid, glutathinone, gutamine, oxalic acid, tocopherol-derived compounds, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediaminetetraacetic acid (EDTA), tert-butylhydroquinone, acetic acid, pectin, tocotrienol, tocopherol, coenzyme Q10, zeaxanthin, astaxanthin, canthaxantin, saponins, limonoids, kaempfedrol, myricetin, isorhamnetin, proanthocyanidins, quercetin, rutin, luteolin, apigenin, tangeritin, hesperetin, naringenin, erodictyol, flavan-3-ols (e.g., anthocyanidins), gallocatechins, epicatechin and its gallate forms, epigallocatechin and its gallate forms (ECGC) theaflavin and its gallate forms, thearubigins, isoflavone, phytoestrogens, genistein, daidzein, glycitein, anythocyanins, cyaniding, delphinidin, malvidin, pelargonidin, peonidin, petunidin, ellagic acid, gallic acid, salicylic acid, rosmarinic acid, cinnamic acid and its derivatives (e.g., ferulic acid), chlorogenic acid, chicoric acid, gallotannins, ellagitannins, anthoxanthins, betacyanins and other plant pigments, silymarin, citric acid, lignan, antinutrients, bilirubin, uric acid, R-a-lipoic acid, N-acetylcysteine, emblicanin, apple extract, apple skin extract (applephenon), rooibos extract red, rooibos extract, green, hawthorn berry extract, red raspberry extract, green coffee antioxidant (GCA), aronia extract 20%, grape seed extract (VinOseed), cocoa extract, hops extract, mangosteen extract, mangosteen hull extract, cranberry extract, pomegranate extract, pomegranate hull extract, pomegranate seed extract, hawthorn berry extract, pomella pomegranate extract, cinnamon bark extract, grape skin extract, bilberry extract, pine bark extract, pycnogenol, elderberry extract, mulberry root extract, wolfberry (gogi) extract, blackberry extract, blueberry extract, blueberry leaf extract, raspberry extract, turmeric extract, citrus bioflavonoids, black currant, ginger, acai powder, green coffee bean extract, green tea extract, and phytic acid, or combinations thereof. In alternate embodiments, the antioxidant is a synthetic antioxidant such as butylated hydroxytolune or butylated hydroxyanisole, for example. Other sources of suitable antioxidants for embodiments of this invention include, but are not limited to, fruits, vegetables, tea, cocoa, chocolate, spices, herbs, rice, organ meats from livestock, yeast, whole grains or cereal grains.

Particular antioxidants belong to the class of phytonutrients called polyphenols (also known as “polyphenolics”), which are a group of chemical substances found in plants, characterized by the presence of more than one phenol group per molecule. A variety of health benefits may be derived from polyphenols, including prevention of cancer, heart disease, and chronic inflammatory disease and improved mental strength and physical strength, for example. Suitable polyphenols for embodiments of this invention include catechins, proanthocyanidins, procyanidins, anthocyanins, quercerin, rutin, reservatrol, isoflavones, curcumin, punicalagin, ellagitannin, hesperidin, naringin, citrus flavonoids, chlorogenic acid, other similar materials and combinations thereof.

In particular embodiments, the antioxidant is a catechin such as, for example, epigallocatechin gallate (EGCG). In another embodiment, the antioxidant is chosen from proanthocyanidins, procyanidins or combinations thereof. In particular embodiments, the antioxidant is an anthocyanin. In still other embodiments, the antioxidant is chosen from quercetin, rutin or combinations thereof. In yet other embodiments, the antioxidant is reservatrol. In still further embodiments, the antioxidant is an isoflavone. In yet further embodiments, the antioxidant is curcumin. In other embodiments, the antioxidant is chosen from punicalagin, ellagitannin or combinations thereof. In still other embodiments, the antioxidant is chlorogenic acid.

In certain embodiments, the functional ingredient is at least one dietary fiber source. Numerous polymeric carbohydrates having significantly different structures in both composition and linkages fall within the definition of dietary fiber. Such compounds are well known to those skilled in the art, non-limiting examples of which include non-starch polysaccharides, lignin, cellulose, methylcellulose, the hemicelluloses, b-glucans, pectins, gums, mucilage, waxes, inulins, oligosaccharides, fructooligosaccharides, cyclodextrins, chitins and combinations thereof. Although dietary fiber generally is derived from plant sources, indigestible animal products such as chitins are also classified as dietary fiber. Chitin is a polysaccharide composed of units of acetylglucosamine joined by b(1-4) linkages, similar to the linkages of cellulose.

In certain embodiments, the functional ingredient is at least one fatty acid. As used herein,“fatty acid” refers to any straight chain monocarboxylic acid and includes saturated fatty acids, unsaturated fatty acids, long chain fatty acids, medium chain fatty acids, short chain fatty acids, fatty acid precursors (including omega-9 fatty acid precursors), and esterified fatty acids. As used herein,“long chain polyunsaturated fatty acid” refers to any polyunsaturated carboxylic acid or organic acid with a long aliphatic tail. As used herein,“omega-3 fatty acid” refers to any polyunsaturated fatty acid having a first double bond as the third carbon-carbon bond from the terminal methyl end of its carbon chain. In particular embodiments, the omega-3 fatty acid may comprise a long chain omega-3 fatty acid. As used herein, “omega-6 fatty acid” any polyunsaturated fatty acid having a first double bond as the sixth carbon-carbon bond from the terminal methyl end of its carbon chain.

Suitable omega-3 fatty acids for use in embodiments of the present invention can be derived from algae, fish, animals, plants, or combinations thereof, for example. Examples of suitable omega-3 fatty acids include, but are not limited to, linolenic acid, alpha-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, stearidonic acid, eicosatetraenoic acid and combinations thereof. In some embodiments, suitable omega-3 fatty acids can be provided in fish oils, (e.g., menhaden oil, tuna oil, salmon oil, bonito oil, and cod oil), microalgae omega-3 oils or combinations thereof. In particular embodiments, suitable omega-3 fatty acids may be derived from commercially available omega-3 fatty acid oils such as Microalgae DHA oil (from Martek, Columbia, MD), OmegaPure (from Omega Protein, Houston, TX), Marinol C-38 (from Lipid Nutrition, Channahon, IL), Bonito oil and MEG-3 (from Ocean Nutrition, Dartmouth, NS), Evogel (from Symrise, Holzminden, Germany), Marine Oil, from tuna or salmon (from Arista Wilton, CT), OmegaSource 2000, Marine Oil, from menhaden and Marine Oil, from cod (from OmegaSource, RTP, NC).

Suitable omega-6 fatty acids include, but are not limited to, linoleic acid, gamma- linolenic acid, dihommo-gamma-linolenic acid, arachidonic acid, eicosadienoic acid, docosadienoic acid, adrenic acid, docosapentaenoic acid and combinations thereof. Suitable esterified fatty acids for embodiments of the present invention may include, but are not limited to, monoacylgycerols containing omega-3 and/or omega-6 fatty acids, diacylgycerols containing omega-3 and/or omega-6 fatty acids, or triacylgycerols containing omega-3 and/or omega-6 fatty acids and combinations thereof.

In certain embodiments, the functional ingredient is at least one vitamin. Suitable vitamins include vitamin A, vitamin D, vitamin E, vitamin K, vitamin Bl, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B7, vitamin B9, vitamin B 12, and vitamin C.

Various other compounds have been classified as vitamins by some authorities. These compounds may be termed pseudo-vitamins and include, but are not limited to, compounds such as ubiquinone (coenzyme Q10), pangamic acid, dimethylglycine, taestrile, amygdaline, flavanoids, para-aminobenzoic acid, adenine, adenylic acid, and s-methylmethionine. As used herein, the term vitamin includes pseudo-vitamins. In some embodiments, the vitamin is a fat- soluble vitamin chosen from vitamin A, D, E, K and combinations thereof. In other embodiments, the vitamin is a water-soluble vitamin chosen from vitamin Bl, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, biotin, pantothenic acid, vitamin C and combinations thereof.

In certain embodiments, the functional ingredient is glucosamine, optionally further comprising chondroitin sulfate.

In certain embodiments, the functional ingredient is at least one mineral. Minerals, in accordance with the teachings of this invention, comprise inorganic chemical elements required by living organisms. Minerals are comprised of a broad range of compositions (e.g., elements, simple salts, and complex silicates) and also vary broadly in crystalline structure. They may naturally occur in foods and beverages, may be added as a supplement, or may be consumed or administered separately from foods or beverages.

Minerals may be categorized as either bulk minerals, which are required in relatively large amounts, or trace minerals, which are required in relatively small amounts. Bulk minerals generally are required in amounts greater than or equal to about 100 mg per day and trace minerals are those that are required in amounts less than about 100 mg per day. In one embodiment, the mineral is chosen from bulk minerals, trace minerals or combinations thereof. Non-limiting examples of bulk minerals include calcium, chlorine, magnesium, phosphorous, potassium, sodium, and sulfur. Non-limiting examples of trace minerals include chromium, cobalt, copper, fluorine, iron, manganese, molybdenum, selenium, zinc, and iodine. Although iodine generally is classified as a trace mineral, it is required in larger quantities than other trace minerals and often is categorized as a bulk mineral.

In a particular embodiment, the mineral is a trace mineral, believed to be necessary for human nutrition, non-limiting examples of which include bismuth, boron, lithium, nickel, rubidium, silicon, strontium, tellurium, tin, titanium, tungsten, and vanadium.

The minerals embodied herein may be in any form known to those of ordinary skill in the art. For example, in a particular embodiment the minerals may be in their ionic form, having either a positive or negative charge. In another particular embodiment the minerals may be in their molecular form. For example, sulfur and phosphorous often are found naturally as sulfates, sulfides, and phosphates.

In certain embodiments, the functional ingredient is at least one preservative. In particular embodiments of this invention, the preservative is chosen from antimicrobials, antioxidants, antienzymatics or combinations thereof. Non-limiting examples of antimicrobials include sulfites, propionates, benzoates, sorbates, nitrates, nitrites, bacteriocins, salts, sugars, acetic acid, dimethyl dicarbonate (DMDC), ethanol, and ozone. In one embodiment, the preservative is a sulfite. Sulfites include, but are not limited to, sulfur dioxide, sodium bisulfite, and potassium hydrogen sulfite. In another embodiment, the preservative is a propionate. Propionates include, but are not limited to, propionic acid, calcium propionate, and sodium propionate. In yet another embodiment, the preservative is a benzoate. Benzoates include, but are not limited to, sodium benzoate and benzoic acid. In a still further embodiment, the preservative is a sorbate. Sorbates include, but are not limited to, potassium sorbate, sodium sorbate, calcium sorbate, and sorbic acid. In a yet further embodiment, the preservative is a nitrate and/or a nitrite. Nitrates and nitrites include, but are not limited to, sodium nitrate and sodium nitrite. In another embodiment, the at least one preservative is a bacteriocin, such as, for example, nisin. In a further embodiment, the preservative is ethanol. In still another embodiment, the preservative is ozone. Non-limiting examples of antienzymatics suitable for use as preservatives in particular embodiments of the invention include ascorbic acid, citric acid, and metal chelating agents such as ethylenediaminetetraacetic acid (EDTA).

In certain embodiments, the functional ingredient is at least one hydration agent. In a particular embodiment, the hydration agent is an electrolyte. Non-limiting examples of electrolytes include sodium, potassium, calcium, magnesium, chloride, phosphate, bicarbonate, and combinations thereof. Suitable electrolytes for use in particular embodiments of this invention are also described in U.S. Patent No. 5,681,569, the disclosure of which is expressly incorporated herein by reference. In one embodiment, the electrolyte is obtained from their corresponding water-soluble salt. Non-limiting examples of salts for use in particular embodiments include chlorides, carbonates, sulfates, acetates, bicarbonates, citrates, phosphates, hydrogen phosphates, tartrates, sorbates, citrates, benzoates, or combinations thereof. In other embodiments, the electrolytes are provided by juice, fruit extracts, vegetable extracts, tea, or teas extracts.

In particular embodiments of this invention, the hydration agent is a carbohydrate to supplement energy stores burned by muscles. Suitable carbohydrates for use in particular embodiments of this invention are described in U.S. Patent Numbers 4,312,856, 4,853,237, 5,681,569, and 6,989,171, the disclosures of which are expressly incorporated herein by reference. Non-limiting examples of suitable carbohydrates include monosaccharides, di saccharides, oligosaccharides, complex polysaccharides or combinations thereof. Non-limiting examples of suitable types of monosaccharides for use in particular embodiments include trioses, tetroses, pentoses, hexoses, heptoses, octoses, and nonoses. Non-limiting examples of specific types of suitable monosaccharides include glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, ribose, xylose, ribulose, xylulose, allose, altrose, galactose, glucose, gulose, idose, mannose, talose, fructose, psicose, sorbose, tagatose, mannoheptulose, sedoheltulose, octolose, and sialose. Non-limiting examples of suitable disaccharides include sucrose, lactose, and maltose. Non-limiting examples of suitable oligosaccharides include saccharose, maltotriose, and maltodextrin. In other particular embodiments, the carbohydrates are provided by a corn syrup, a beet sugar, a cane sugar, a juice, or a tea.

In another particular embodiment, the hydration agent is a flavanol that provides cellular rehydration. Flavanols are a class of natural substances present in plants, and generally comprise a 2-phenylbenzopyrone molecular skeleton attached to one or more chemical moieties. Non limiting examples of suitable flavanols for use in particular embodiments of this invention include catechin, epicatechin, gallocatechin, epigallocatechin, epicatechin gallate, epigallocatechin 3 -gallate, theaflavin, theaflavin 3 -gallate, theaflavin 3’ -gallate, theaflavin 3,3’ gallate, thearubigin or combinations thereof. Several common sources of flavanols include tea plants, fruits, vegetables, and flowers. In preferred embodiments, the flavanol is extracted from green tea.

In a particular embodiment, the hydration agent is a glycerol solution to enhance exercise endurance. The ingestion of a glycerol containing solution has been shown to provide beneficial physiological effects, such as expanded blood volume, lower heart rate, and lower rectal temperature.

In certain embodiments, the functional ingredient is chosen from at least one probiotic, prebiotic and combination thereof. The probiotic is a beneficial microorganisms that affects the human body’s naturally-occurring gastrointestinal microflora. Examples of probiotics include, but are not limited to, bacteria of the genus Lactobacilli , Bifidobacteria , Streptococci , or combinations thereof, that confer beneficial effects to humans. In particular embodiments of the invention, the at least one probiotic is chosen from the genus Lactobacilli. According to other particular embodiments of this invention, the probiotic is chosen from the genus Bifidobacteria. According to still other particular embodiments of this invention, the probiotic is chosen from the genus Streptococcus.

Probiotics that may be used in accordance with this invention are well-known to those of skill in the art. Non-limiting examples of foodstuffs comprising probiotics include yogurt, sauerkraut, kefir, kimchi, fermented vegetables, and other foodstuffs containing a microbial element that beneficially affects the host animal by improving the intestinal microbalance.

Prebiotics, in accordance with the teachings of this invention, include, without limitation, mucopolysaccharides, oligosaccharides, polysaccharides, amino acids, vitamins, nutrient precursors, proteins and combinations thereof. According to a particular embodiment of this invention, the prebiotic is chosen from dietary fibers, including, without limitation, polysaccharides and oligosaccharides. Non-limiting examples of oligosaccharides that are categorized as prebiotics in accordance with particular embodiments of this invention include fructooligosaccharides, inulins, isomalto-oligosaccharides, lactilol, lactosucrose, lactulose, pyrodextrins, soy oligosaccharides, transgalacto-oligosaccharides, and xylo-oligosaccharides. In other embodiments, the prebiotic is an amino acid. Although a number of known prebiotics break down to provide carbohydrates for probiotics, some probiotics also require amino acids for nourishment.

Prebiotics are found naturally in a variety of foods including, without limitation, bananas, berries, asparagus, garlic, wheat, oats, barley (and other whole grains), flaxseed, tomatoes, Jerusalem artichoke, onions and chicory, greens (e.g., dandelion greens, spinach, collard greens, chard, kale, mustard greens, turnip greens), and legumes (e.g., lentils, kidney beans, chickpeas, navy beans, white beans, black beans).

In certain embodiments, the functional ingredient is at least one weight management agent. As used herein,“a weight management agent” includes an appetite suppressant and/or a thermogenesis agent. As used herein, the phrases“appetite suppressant”,“appetite satiation compositions”,“satiety agents”, and“satiety ingredients” are synonymous. The phrase“appetite suppressant” describes macronutrients, herbal extracts, exogenous hormones, anorectics, anorexigenics, pharmaceutical drugs, and combinations thereof, that when delivered in an effective amount, suppress, inhibit, reduce, or otherwise curtail a person’s appetite. The phrase “thermogenesis agent” describes macronutrients, herbal extracts, exogenous hormones, anorectics, anorexigenics, pharmaceutical drugs, and combinations thereof, that when delivered in an effective amount, activate or otherwise enhance a person’s thermogenesis or metabolism.

Suitable weight management agents include macronutrient selected from the group consisting of proteins, carbohydrates, dietary fats, and combinations thereof. Consumption of proteins, carbohydrates, and dietary fats stimulates the release of peptides with appetite- suppressing effects. For example, consumption of proteins and dietary fats stimulates the release of the gut hormone cholecytokinin (CCK), while consumption of carbohydrates and dietary fats stimulates release of Glucagon-like peptide 1 (GLP-l).

Suitable macronutrient weight management agents also include carbohydrates. Carbohydrates generally comprise sugars, starches, cellulose and gums that the body converts into glucose for energy. Carbohydrates often are classified into two categories, digestible carbohydrates (e.g., monosaccharides, disaccharides, and starch) and non-digestible carbohydrates (e.g., dietary fiber). Studies have shown that non-digestible carbohydrates and complex polymeric carbohydrates having reduced absorption and digestibility in the small intestine stimulate physiologic responses that inhibit food intake. Accordingly, the carbohydrates embodied herein desirably comprise non-digestible carbohydrates or carbohydrates with reduced digestibility. Non-limiting examples of such carbohydrates include polydextrose; inulin; monosaccharide-derived polyols such as erythritol, mannitol, xylitol, and sorbitol; disaccharide- derived alcohols such as isomalt, lactitol, and maltitol; and hydrogenated starch hydrolysates. Carbohydrates are described in more detail herein below.

In another particular embodiment weight management agent is a dietary fat. Dietary fats are lipids comprising combinations of saturated and unsaturated fatty acids. Polyunsaturated fatty acids have been shown to have a greater satiating power than mono-unsaturated fatty acids. Accordingly, the dietary fats embodied herein desirably comprise poly-unsaturated fatty acids, non-limiting examples of which include triacylglycerols.

In a particular embodiment, the weight management agent is an herbal extract. Extracts from numerous types of plants have been identified as possessing appetite suppressant properties. Non-limiting examples of plants whose extracts have appetite suppressant properties include plants of the genus Hoodia , Trichocaulon , Caralluma , Stapelia , Orbea, Asclepias, and Camelia. Other embodiments include extracts derived from Gymnema Sylvestre, Kola Nut, Citrus Auran tium, Yerba Mate, Griff onia Simplicifolia, Guarana, myrrh, guggul Lipid, and black current seed oil.

The herbal extracts may be prepared from any type of plant material or plant biomass. Non-limiting examples of plant material and biomass include the stems, roots, leaves, dried powder obtained from the plant material, and sap or dried sap. The herbal extracts generally are prepared by extracting sap from the plant and then spray-drying the sap. Alternatively, solvent extraction procedures may be employed. Following the initial extraction, it may be desirable to further fractionate the initial extract (e.g., by column chromatography) in order to obtain an herbal extract with enhanced activity. Such techniques are well known to those of ordinary skill in the art.

In a particular embodiment, the herbal extract is derived from a plant of the genus Hoodia , species of which include H. alstonii , H. currorii , H. dregei , H. flava, H. gordonii, H. jutatae , H. mossamedensis, H. officinalis , H. parviflorai , H. pedicellata , H. pilifera , H. ruschii , and H. triebneri. Hoodia plants are stem succulents native to southern Africa. A sterol glycoside of Hoodia , known as P57, is believed to be responsible for the appetite-suppressant effect of the Hoodia species. In another particular embodiment, the herbal extract is derived from a plant of the genus Caralluma , species of which include C. indica , C. fimbriata , C. attenuate , C. tuberculata , C. edulis , C. adscendens , C. stalagmifera , C. umbellate , C. penicillata , C. russe liana, C. retrospicens , C. Arabica , and C. lasiantha. Carralluma plants belong to the same Subfamily as Hoodia , Asclepiadaceae. Caralluma are small, erect and fleshy plants native to India having medicinal properties, such as appetite suppression, that generally are attributed to glycosides belonging to the pregnane group of glycosides, non-limiting examples of which include caratuberside A, caratuberside B, bouceroside I, bouceroside II, bouceroside III, bouceroside IV, bouceroside V, bouceroside VI, bouceroside VII, bouceroside VIII, bouceroside IX, and bouceroside X. In another particular embodiment, the at least one herbal extract is derived from a plant of the genus Trichocaulon. Trichocaulon plants are succulents that generally are native to southern Africa, similar to Hoodia , and include the species T. piliferum and T. officinale. In another particular embodiment, the herbal extract is derived from a plant of the genus Stapelia or Orbea, species of which include S. gigantean and 0. variegate, respectively. Both Stapelia and Orbea plants belong to the same Subfamily as Hoodia , Asclepiadaceae. Not wishing to be bound by any theory, it is believed that the compounds exhibiting appetite suppressant activity are saponins, such as pregnane glycosides, which include stavarosides A, B, C, D, E, F, G, H, I, J, and K. In another particular embodiment, the herbal extract is derived from a plant of the genus Asclepias. Asclepias plants also belong to the Asclepiadaceae family of plants. Non-limiting examples of Asclepias plants include A. incarnate , A. curassayica, A. syriaca, and A. tuberose. Not wishing to be bound by any theory, it is believed that the extracts comprise steroidal compounds, such as pregnane glycosides and pregnane aglycone, having appetite suppressant effects.

In a particular embodiment, the weight management agent is an exogenous hormone having a weight management effect. Non-limiting examples of such hormones include CCK, peptide YY, ghrelin, bombesin and gastrin-releasing peptide (GRP), enterostatin, apolipoprotein A-IV, GLP-l, amylin, somastatin, and leptin. In another embodiment, the weight management agent is a pharmaceutical drug. Non limiting examples include phentenime, diethylpropion, phendimetrazine, sibutramine, rimonabant, oxyntomodulin, floxetine hydrochloride, ephedrine, phenethylamine, or other stimulants.

In certain embodiments, the functional ingredient is at least one osteoporosis management agent. In certain embodiments, the osteoporosis management agent is at least one calcium source. According to a particular embodiment, the calcium source is any compound containing calcium, including salt complexes, solubilized species, and other forms of calcium. Non-limiting examples of calcium sources include amino acid chelated calcium, calcium carbonate, calcium oxide, calcium hydroxide, calcium sulfate, calcium chloride, calcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium citrate, calcium malate, calcium citrate malate, calcium gluconate, calcium tartrate, calcium lactate, solubilized species thereof, and combinations thereof.

According to a particular embodiment, the osteoporosis management agent is a magnesium soucrce. The magnesium source is any compound containing magnesium, including salt complexes, solubilized species, and other forms of magnesium. Non-limiting examples of magnesium sources include magnesium chloride, magnesium citrate, magnesium gluceptate, magnesium gluconate, magnesium lactate, magnesium hydroxide, magnesium picolate, magnesium sulfate, solubilized species thereof, and mixtures thereof. In another particular embodiment, the magnesium source comprises an amino acid chelated or creatine chelated magnesium.

In other embodiments, the osteoporosis agent is chosen from vitamins D, C, K, their precursors and/or beta-carotene and combinations thereof.

Numerous plants and plant extracts also have been identified as being effective in the prevention and treatment of osteoporosis. Non-limiting examples of suitable plants and plant extracts as osteoporosis management agents include species of the genus Taraxacum and Amelanchier , as disclosed in U.S. Patent Publication No. 2005/0106215, and species of the genus Lindera , Artemisia , Acorus , Carthamus , Carum , Cnidium , Curcuma , Cyperus , Juniperus , Prunus , Iris , Cichorium , Dodonaea, Epimedium , Erigonoum , Soya , Mentha , Ocimum , thymus , Tanacetum , Plantago , Spearmint , Bixa, Vitis, Rosemarinus , Rhus, and Anethum , as disclosed in U.S. Patent Publication No. 2005/0079232.

In certain embodiments, the functional ingredient is at least one phytoestrogen. Phytoestrogens are compounds found in plants which can typically be delivered into human bodies by ingestion of the plants or the plant parts having the phytoestrogens. As used herein, "phytoestrogen" refers to any substance which, when introduced into a body causes an estrogen like effect of any degree. For example, a phytoestrogen may bind to estrogen receptors within the body and have a small estrogen-like effect. Examples of suitable phytoestrogens for embodiments of this invention include, but are not limited to, isoflavones, stilbenes, lignans, resorcyclic acid lactones, coumestans, coumestrol, equol, and combinations thereof. Sources of suitable phytoestrogens include, but are not limited to, whole grains, cereals, fibers, fruits, vegetables, black cohosh, agave root, black currant, black haw, chasteberries, cramp bark, dong quai root, devil's club root, false unicorn root, ginseng root, groundsel herb, licorice, liferoot herb, motherwort herb, peony root, raspberry leaves, rose family plants, sage leaves, sarsaparilla root, saw palmetto berried, wild yam root, yarrow blossoms, legumes, soybeans, soy products (e.g., miso, soy flour, soymilk, soy nuts, soy protein isolate, tempen, or tofu) chick peas, nuts, lentils, seeds, clover, red clover, dandelion leaves, dandelion roots, fenugreek seeds, green tea, hops, red wine, flaxseed, garlic, onions, linseed, borage, butterfly weed, caraway, chaste tree, vitex, dates, dill, fennel seed, gotu kola, milk thistle, pennyroyal, pomegranates, southernwood, soya flour, tansy, and root of the kudzu vine (pueraria root) and the like, and combinations thereof.

Isoflavones belong to the group of phytonutrients called polyphenols. In general, polyphenols (also known as "polyphenolics"), are a group of chemical substances found in plants, characterized by the presence of more than one phenol group per molecule.

Suitable phytoestrogen isoflavones in accordance with embodiments of this invention include genistein, daidzein, glycitein, biochanin A, formononetin, their respective naturally occurring glycosides and glycoside conjugates, matairesinol, secoisolariciresinol, enter olactone, enterodiol, textured vegetable protein, and combinations thereof.

Suitable sources of isoflavones for embodiments of this invention include, but are not limited to, soy beans, soy products, legumes, alfalfa sprouts, chickpeas, peanuts, and red clover. In certain embodiments, the functional ingredient is at least one long chain primary aliphatic saturated alcohol. Long-chain primary aliphatic saturated alcohols are a diverse group of organic compounds. The term alcohol refers to the fact these compounds feature a hydroxyl group (-OH) bound to a carbon atom. Non-limiting examples of particular long-chain primary aliphatic saturated alcohols for use in particular embodiments of the invention include the 8 carbon atom l-octanol, the 9 carbon l-nonanol, the 10 carbon atom l-decanol, the 12 carbon atom l-dodecanol, the 14 carbon atom l-tetradecanol, the 16 carbon atom l-hexadecanol, the 18 carbon atom l-octadecanol, the 20 carbon atom l-eicosanol, the 22 carbon l-docosanol, the 24 carbon l-tetracosanol, the 26 carbon l-hexacosanol, the 27 carbon l-heptacosanol, the 28 carbon l-octanosol, the 29 carbon l-nonacosanol, the 30 carbon l-triacontanol, the 32 carbon 1- dotriacontanol, and the 34 carbon l-tetracontanol.

In a particularly desirable embodiment of the invention, the long-chain primary aliphatic saturated alcohols are policosanol. Policosanol is the term for a mixture of long-chain primary aliphatic saturated alcohols composed primarily of 28 carbon l-octanosol and 30 carbon 1- triacontanol, as well as other alcohols in lower concentrations such as 22 carbon l-docosanol, 24 carbon l-tetracosanol, 26 carbon l-hexacosanol, 27 carbon l-heptacosanol, 29 carbon 1- nonacosanol, 32 carbon l-dotriacontanol, and 34 carbon l-tetracontanol.

In certain embodiments, the functional ingredient is at least one phytosterol, phytostanol or combination thereof. As used herein, the phrases“stanol”,“plant stanol” and“phytostanol” are synonymous. Plant sterols and stanols are present naturally in small quantities in many fruits, vegetables, nuts, seeds, cereals, legumes, vegetable oils, bark of the trees and other plant sources. Sterols are a subgroup of steroids with a hydroxyl group at C-3. Generally, phytosterols have a double bond within the steroid nucleus, like cholesterol; however, phytosterols also may comprise a substituted side chain (R) at C-24, such as an ethyl or methyl group, or an additional double bond. The structures of phytosterols are well known to those of skill in the art.

At least 44 naturally-occurring phytosterols have been discovered, and generally are derived from plants, such as corn, soy, wheat, and wood oils; however, they also may be produced synthetically to form compositions identical to those in nature or having properties similar to those of naturally-occurring phytosterols. According to particular embodiments of this invention, non-limiting examples of phytosterols well known to those or ordinary skill in the art include 4-desmethyl sterols (e.g., b-sitosterol, campesterol, stigmasterol, brassicasterol, 22- dehydrobrassicasterol, and A5-avenasterol), 4-monomethyl sterols, and 4,4-dimethyl sterols (triterpene alcohols) (e.g., cycloartenol, 24-methylenecycloartanol, and cyclobranol).

As used herein, the phrases“stanol”,“plant stanol” and“phytostanol” are synonymous. Phytostanols are saturated sterol alcohols present in only trace amounts in nature and also may be synthetically produced, such as by hydrogenation of phytosterols. According to particular embodiments of this invention, non-limiting examples of phytostanols include b-sitostanol, campestanol, cycloartanol, and saturated forms of other triterpene alcohols.

Both phytosterols and phytostanols, as used herein, include the various isomers such as the a and b isomers (e.g., a-sitosterol and b-sitostanol, which comprise one of the most effective phytosterols and phytostanols, respectively, for lowering serum cholesterol in mammals).

The phytosterols and phytostanols of the present invention also may be in their ester form. Suitable methods for deriving the esters of phytosterols and phytostanols are well known to those of ordinary skill in the art, and are disclosed in U.S. Patent Numbers 6,589,588, 6,635,774, 6,800,317, and U.S. Patent Publication Number 2003/0045473, the disclosures of which are incorporated herein by reference in their entirety. Non-limiting examples of suitable phytosterol and phytostanol esters include sitosterol acetate, sitosterol oleate, stigmasterol oleate, and their corresponding phytostanol esters. The phytosterols and phytostanols of the present invention also may include their derivatives.

Generally, the amount of functional ingredient in the composition varies widely depending on the particular composition and the desired functional ingredient. Those of ordinary skill in the art will readily ascertain the appropriate amount of functional ingredient for each composition.

In one embodiment, a method for preparing a composition comprises combining at least one target steviol glycoside and at least one sweetener and/or additive and/or functional ingredient.

In a particular embodiment, a method for preparing a composition comprises combining at least one target steviol glycoside and at least one additional sweetener and/or additive and/or functional ingredient. In a particular embodiment, a method for preparing a composition comprises combining at least one diterpene glycoside of the present invention and at least one sweetener and/or additive and/or functional ingredient, wherein the at least one sweetener and/or additive and/or functional ingredient does not exist with (is not admixed with) the at least one diterpene glycoside in nature, i.e. Stevia leaf, and the composition provides a more sucrose-like taste profile compared to the diterpene glycoside in nature and (if applicable) the at least one sweetener and/or additive and/or functional ingredient in nature. For example, in certain embodiments the composition exhibits one or more of the following characteristics: improved sweetness potency, improved mouthfeel, decreased sweetness linger, decreased bitterness and/or decreased metallic taste.

Consumables

In one embodiment, the present invention is a consumable comprising at least one diterpene glycoside of the present invention, or a composition comprising at least one diterpene glycoside of the present invention. In a particular embodiment, the at least one diterpene glycoside is isolated and purified.

The diterpene glycoside(s) of the present invention, or a composition comprising the same, can be admixed with any known edible or oral composition, referred to herein as a “consumable”. Consumables, as used herein, mean substances which are contacted with the mouth of man or animal, including substances which are taken into and subsequently ejected from the mouth and substances which are drunk, eaten, swallowed or otherwise ingested, and are safe for human or animal consumption when used in a generally acceptable range.

Exemplary consumables include pharmaceutical compositions, edible gel mixes and compositions, dental compositions, foodstuffs (confections, condiments, chewing gum, cereal compositions baked goods dairy products, and tabletop sweetener compositions) beverages and beverage products. The consumables of the present invention require admixing and, as such, do not occur in nature.

For example, a beverage is a consumable. The beverage may be sweetened or unsweetened. The diterpene glycoside(s) of the present invention, or a composition comprising the same, may be added to a beverage or beverage matrix to sweeten the beverage or enhance its existing sweetness or flavor. In one embodiment, the present invention is a consumable comprising at least one diterpene glycoside of the present invention. In particular embodiments, a diterpene glycoside of the present invention is present in the consumable in a concentration greater than about 1 ppm, such as, for example, from about 1 ppm to about 1,000 ppm, from about 25 ppm to about 1,000 ppm, from about 50 ppm to about 1,000 ppm, from about 75 ppm to about 1,000 ppm, from about 100 ppm to about 1,000 ppm, from about 200 ppm to about 1,000 ppm, from about 300 ppm to about 1,000 ppm, from about 400 ppm to about 1,000 ppm, from about 500 ppm to about 1,000 ppm or from about 50 ppm to about 600 ppm.

In other particular embodiments, a diterpene glycoside of the present invention is present in the consumable in a purity of at least about 5% with respect to a mixture of diterpene glycosides or stevia extract, such as, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 97%. In still other embodiments, a diterpene glycoside of the present invention is present in the consumable in >99% purity.

The consumable can optionally include additives, additional sweeteners, functional ingredients and combinations thereof, as described herein. Any of the additive, additional sweetener and functional ingredients described above can be present in the consumable.

In exemplary embodiments, a consumable comprising at least one purified diterpene glycoside of the present invention has about 30% or more sweetness compared to a corresponding consumable comprising partially purified diterpene glycoside or Stevia, such as, for example, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more.

In other exemplary embodiments, a consumable comprising at least one purified diterpene glycoside of the present invention has at least about 30% less bitterness (the taste stimulated by certain substances such as quinine, caffeine and sucrose octa-acetate) compared to a corresponding consumable comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a consumable comprising at least one purified diterpene glycoside of the present invention has substantially no bitterness. Methods of measuring bitterness of a compound are known in the art

In still other exemplary embodiments, a consumable comprising at least one purified diterpene glycoside of the present invention has at least about 30% less sweet lingering aftertaste (the intensity of the sweet taste after expectoration) compared to a corresponding consumable comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a consumable comprising at least one purified diterpene glycoside of the present invention has substantially no sweet lingering aftertaste. Methods of measuring sweet lingering aftertaste are known in the art.

In yet other exemplary embodiments, a consumable comprising at least one purified diterpene glycoside of the present invention has at least about 30% less metallic taste (taste associated with metals, tinny or iron) compared to a corresponding consumable comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a consumable comprising at least one purified diterpene glycoside of the present invention has substantially no metallic taste.

In exemplary embodiments, a consumable comprising at least one purified diterepene glycoside of the present invention exhibits a maximal response (maximum sweetness (%SE) achieved with increasing concentration of compound) that is at least about 30% greater compared to a corresponding consumable comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% greater, at least about 50% greater, at least about 60% greater, at least about 70% greater, at least about 80% greater or at least about 90% greater. Methods of measuring the maximal response of a compound are known in the art.

In other exemplary embodiments, a consumable comprising at least one purified diterpene glycoside of the present invention exhibits a sweetness onset (the time until maximum sweetness is experienced) that is at least about 30% shorter than a consumable comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% short, at least about 50% shorter, at least about 60% shorter, at least about 70% shorter, at least about 80% shorter or at least about 90% shorter. Methods of measuring sweetness onset are known in the art.

In another embodiment, the present invention is a beverage or beverage product comprising a composition that comprises at least one diterpene glycoside of the present invention. In a particular embodiment, the beverage or beverage product comprises a composition comprising at least one purified diterpene glycoside of the present invention.

As used herein a “beverage product” is a ready-to-drink beverage, a beverage concentrate, a beverage syrup, or a powdered beverage. Suitable ready-to-drink beverages include carbonated and non-carbonated beverages. Carbonated beverages include, but are not limited to, enhanced sparkling beverages, cola, lemon-lime flavored sparkling beverage, orange flavored sparkling beverage, grape flavored sparkling beverage, strawberry flavored sparkling beverage, pineapple flavored sparkling beverage, ginger-ale, soft drinks, root beer and frozen carbonated beverages. Non-carbonated beverages include, but are not limited to fruit juice, fruit- flavored juice, juice drinks, nectars, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, enhanced water drinks, enhanced water with vitamins, near water drinks (e.g., water with natural or synthetic flavorants), coconut water, tea type drinks (e.g. black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverage containing milk components (e.g. milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages), beverages containing cereal extracts, smoothies and combinations thereof.

Beverage concentrates and beverage syrups are prepared with an initial volume of liquid matrix (e.g. water) and the desired beverage ingredients. Full strength beverages are then prepared by adding further volumes of water. Powdered beverages are prepared by dry-mixing all of the beverage ingredients in the absence of a liquid matrix. Full strength beverages are then prepared by adding the full volume of water.

Beverages comprise a matrix, i.e. the basic ingredient in which the ingredients - including the compositions of the present invention - are dissolved. In one embodiment, a beverage comprises water of beverage quality as the matrix, such as, for example deionized water, distilled water, reverse osmosis water, carbon-treated water, purified water, demineralized water and combinations thereof, can be used. Additional suitable matrices include, but are not limited to phosphoric acid, phosphate buffer, citric acid, citrate buffer and carbon-treated water. In one embodiment, the present invention is a beverage comprising at least one diterpene glycoside of the present invention.

In a further embodiment, the present invention is a beverage product comprising at least one diterpene glycoside of the present invention.

The at least one diterpene glycoside can be provided as a single compound or as part of any composition described above. In an exemplary embodiment, the at least one diterpene glycoside is purified.

In a particular embodiment, a beverage or beverage product comprises at least one diterpene glycoside of the present invention in purified form and at least one other substance that does not occur with the diterpene glycoside in nature, i.e. Stevia leaf. In one embodiment, the at least one additional substance modulates the taste profile of the at least one diterpene glycoside to provide a beverage with a more sucrose-like taste profile compared to the diterpene glycoside in nature and (if applicable) the at least one other substance in nature. For example, in certain embodiments the beverage exhibits one or more of the following characteristics: improved sweetness potency, improved mouthfeel, decreased sweetness linger, decreased bitterness and/or decreased metallic taste.

The concentration of the diterpene glycoside of the present invention in the beverage may be above, at or below the threshold sweetness or flavor recognition concentration of the diterpene glycoside of the present invention.

In one embodiment, a diterpene glycoside of the present invention is present in the beverage in a concentration greater than about 1 ppm, such as, for example, from about 1 ppm to about 1,000 ppm, from about 25 ppm to about 1,000 ppm, from about 50 ppm to about 1,000 ppm, from about 75 ppm to about 1,000 ppm, from about 100 ppm to about 1,000 ppm, from about 200 ppm to about 1,000 ppm, from about 300 ppm to about 1,000 ppm, from about 400 ppm to about 1,000 ppm or from about 500 ppm to about 1,000 ppm.

In a more particular embodiment, a diterpene glycoside of the present invention is present in the beverage in a concentration from about 25 ppm to about 600 ppm, such as, for example, from about 25 ppm to about 500 ppm, from about 25 ppm to about 400 ppm, from about 25 ppm to about 300 ppm, from about 25 ppm to about 200 ppm, from about 25 ppm to about 100 ppm, from about 50 ppm to about 600 ppm, from about 50 ppm to about 500 ppm, from about 50 ppm to about 400 ppm, from about 50 ppm to about 300 ppm, from about 50 ppm to about 200 ppm, from about 50 ppm to about 100 ppm, from about 100 ppm to about 600 ppm, from about 100 ppm to about 500 ppm, from about 100 ppm to about 400 ppm, from about 100 ppm to about 300 ppm, from about 100 ppm to about 200 ppm, from about 200 ppm to about 600 ppm, from about 200 ppm to about 500 ppm, from about 200 ppm to about 400 ppm, from about 200 ppm to about 300 ppm, from about 300 ppm to about 600 ppm, from about 300 ppm to about 500 ppm, from about 300 ppm to about 400 ppm, from about 400 ppm to about 600 ppm, from about 400 ppm to about 500 ppm or from about 500 ppm to about 600 ppm.

In other particular embodiments, a diterpene glycoside of the present invention is present in the beverage in a purity of at least about 5% with respect to a mixture of diterpene glycosides or stevia extract, such as, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 97%. In still other embodiments, a diterpene glycoside of the present invention is present in the beverage in >99% purity.

The beverage can include one or more sweeteners. Any of the sweeteners detailed herein can be used, including natural, non-natural, or synthetic sweeteners. These may be added to the beverage either before, contemporaneously with or after the diterpene glycoside(s) of the present invention. In a particular embodiment, the sweetener does not occur with the at least one diterpene glycoside in nature, i.e. Stevia leaf.

The consumable can optionally include additives, functional ingredients and combinations thereof, as described herein. Any of the additives and functional ingredients described above can be present in the consumable. In certain embodiments, the additive and/or functional ingredient modulates the taste profile of the at least one diterpene glycoside to provide a composition with a more sucrose-like taste profile compared to the diterpene glycoside in nature and (if applicable) the additive and/or functional ingredient in nature. For example, in certain embodiments the composition exhibits one or more of the following characteristics: improved sweetness potency, improved mouthfeel, decreased sweetness linger, decreased bitterness and/or decreased metallic taste. It is contemplated that the pH of the consumable, such as, for example, a beverage, does not materially or adversely affect the taste of the sweetener. A non-limiting example of the pH range of the beverage may be from about 1.8 to about 10. A further example includes a pH range from about 2 to about 5. In a particular embodiment, the pH of beverage can be from about 2.5 to about 4.2. On of skill in the art will understand that the pH of the beverage can vary based on the type of beverage. Dairy beverages, for example, can have pHs greater than 4.2.

The titratable acidity of a beverage may, for example, range from about 0.01 to about 1.0% by weight of beverage.

In one embodiment, the sparkling beverage product has an acidity from about 0.01 to about 1.0% by weight of the beverage, such as, for example, from about 0.05% to about 0.25% by weight of beverage.

The carbonation of a sparkling beverage product has 0 to about 2% (w/w) of carbon dioxide or its equivalent, for example, from about 0.1 to about 1.0% (w/w).

The temperature of a beverage may, for example, range from about 4°C to about 100 °C, such as, for example, from about 4°C to about 25°C. The beverage can also be a frozen carbonated beverage.

The beverage can be a full-calorie beverage that has up to about 120 calories per 8 oz serving. The beverage can be a mid-calorie beverage that has up to about 60 calories per 8 oz serving. The beverage can be a low-calorie beverage that has up to about 40 calories per 8 oz serving. The beverage can be a zero-calorie that has less than about 5 calories per 8 oz. serving.

In one embodiment, the beverage comprises natural sweetener(s) only, i.e. the only type of sweetener(s) are naturally-occurring.

In exemplary embodiments, a beverage comprising at least one purified diterpene glycoside of the present invention has about 30% or more sweetness compared to a corresponding beverage comprising partially purified diterpene glycoside or Stevia, such as, for example, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more or about 90% or more.

In other exemplary embodiments, a beverage comprising at least one purified diterpene glycoside of the present invention has at least about 30% less bitterness (the taste stimulated by certain substances such as quinine, caffeine and sucrose octa-acetate) compared to a corresponding beverage comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a beverage comprising at least one purified diterpene glycoside of the present invention has substantially no bitterness.

In still other exemplary embodiments, a beverage comprising at least one purified diterpene glycoside of the present invention has at least about 30% less sweet lingering aftertaste (the intensity of the sweet taste after expectoration) compared to a corresponding beverage comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a beverage comprising at least one purified diterpene glycoside of the present invention has substantially no sweet lingering aftertaste.

In yet other exemplary embodiments, a beverage comprising at least one purified diterpene glycoside of the present invention has at least about 30% less metallic taste (taste associated with metals, tinny or iron) compared to a corresponding beverage comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90%. In a particular embodiment, a beverage comprising at least one purified diterpene glycoside of the present invention has substantially no metallic taste.

In exemplary embodiments, a beverage comprising at least one purified diterpene glycoside of the present invention exhibits a maximal response (maximum sweetness (%SE) achieved with increasing concentration of compound) that is at least about 30% greater compared to a corresponding beverage comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% greater, at least about 50% greater, at least about 60% greater, at least about 70% greater, at least about 80% greater or at least about 90% greater.

In other exemplary embodiments, a beverage comprising at least one purified diterpene glycoside of the present invention exhibits a sweetness onset (the time until maximum sweetness is experienced) that is at least about 30% shorter than a beverage comprising partially purified diterpene glycoside or Stevia leaf, such as, for example, at least about 40% short, at least about 50% shorter, at least about 60% shorter, at least about 70% shorter, at least about 80% shorter or at least about 90% shorter.

III. Methods of Use

The compounds and compositions of the present invention can be used to impart sweetness or to enhance the flavor or sweetness of consumables or other compositions.

In one aspect, the present invention is a method of preparing a sweetened consumable comprising (i) providing a consumable and (ii) adding at least one diterpene glycoside of the present invention to the consumable to provide a sweetened consumable.

In a particular embodiment, a method of preparing a sweetened consumable comprises (i) providing an unsweetened consumable and (ii) adding at least one diterpene glycoside of the present invention to the unsweetened consumable to provide a sweetened consumable.

In a particular embodiment, the present invention is a method of preparing a sweetened beverage comprising (i) providing a beverage and (ii) adding at least one diterpene glycoside of the present invention to the beverage to provide a sweetened beverage.

In a particular embodiment, the present invention is a method of preparing a sweetened beverage comprising (i) providing an unsweetened beverage and (ii) adding at least one diterpene glycoside of the present invention to the unsweetened beverage to provide a sweetened beverage.

In the above methods, the diterpene glycoside(s) of the present invention may be provided as such, i.e., in the form of a compound, or in form of a composition. When provided as a composition, the amount of diterpene glycoside in the composition is effective to provide a concentration of the diterpene glycoside that is above, at or below its flavor or sweetness recognition threshold when the composition is added to the consumable (e.g., the beverage). When the diterpene glycoside(s) of the present invention is not provided as a composition, it may be added to the consumable at a concentration that is above, at or below its flavor or sweetness recognition threshold.

In one embodiment, the present invention is a method for enhancing the sweetness of a consumable comprising (i) providing a consumable comprising at least one sweet ingredient and (ii) adding at least one diterpene glycoside of the present invention, or a composition comprising the same, to the consumable to provide a consumable with enhanced sweetness, wherein the diterpene glycoside of the present invention is added to the consumable at a concentration at or below its sweetness recognition threshold. In a particular embodiment, a diterpene glycoside of the present invention is added to the consumable at a concentration below its sweetness recognition threshold.

In a particular embodiment, the present invention is a method for enhancing the sweetness of a beverage comprising (i) providing a beverage comprising at least one sweet ingredient and (ii) adding at least one diterpene glycoside of the present invention, or a composition comprising the same, to the beverage to provide a beverage with enhanced sweetness, wherein the diterpene glycoside is added to the beverage at a concentration at or below its sweetness recognition threshold. In a particular embodiment, the diterpene glycoside of the present invention is added to the consumable at a concentration below its sweetness recognition concentration threshold.

In another embodiment, the present invention is a method for enhancing the flavor of a consumable comprising (i) providing a consumable comprising at least one flavor ingredient and (ii) adding at least one diterpene glycoside of the present invention, or a composition comprising the same, to the consumable to provide a consumable with enhanced flavor, wherein the diterpene glycoside of the present invention is added to the consumable at a concentration at or below its flavor recognition threshold. In a particular embodiment, the diterpene glycoside of the present invention is added to the consumable at a concentration below its flavor recognition threshold.

In a particular embodiment, a method for enhancing the flavor of a beverage is provided that comprises (i) providing a beverage comprising at least one flavor ingredient and (ii) adding at least one diterpene glycoside of the present invention, or a composition comprising the same, to the beverage to provide a beverage with enhanced flavor, wherein the diterpene glycoside is added to the beverage at a concentration at or below the flavor recognition threshold of the diterpene glycoside. In a particular embodiment, the diterpene glycoside of the present invention is added to the consumable at a concentration below its flavor recognition threshold. The present invention also includes methods of preparing sweetened compositions (e.g., sweetened consumables) and flavor enhanced compositions (e.g., flavored enhanced consumables) by adding at least one diterpene glycoside of the present invention or a composition comprising the same to such compositions/consumables.

IV. Methods of Purification

The present invention also extends to methods of purifying a diterpene glycoside of the present invention.

In one embodiment, the present invention is a method for purifying a diterpene glycoside of the present invention comprising (i) passing a solution comprising a source material comprising a diterpene glycoside of the present invention through a HPLC column and (ii) eluting fractions comprising a diterpene glycoside of the present invention to provide purified diterpene glycoside of the present invention. The HPLC column can be any suitable HPLC preparative or semi-preparative scale column.

As used herein, the term "preparative HPLC" refers to an HPLC system capable of producing high (500 or more) microgram, milligram, or gram sized product fractions. The term "preparative" includes both preparative and semi-preparative columns, but is not intended to include analytical columns, which provide fractions in the nanogram to low microgram range.

As used herein, an "HPLC compatible detector" is a detector suitable for use in an HPLC system which is capable of providing a detectable signal upon elution of a compound peak. For example, a detector capable of generating a signal when a compound elutes from the compound is an HPLC compatible detector. Where component absorbance varies widely, it may be necessary to utilize more than one detector. A detector capable of detecting a desired component is not an "incompatible" detector due to its inability to detect a non-desired peak.

An HPLC device typically includes at least the following components: a column, packed with a suitable stationary phase, a mobile phase, a pump for forcing the mobile phase through the column under pressure, and a detector for detecting the presence of compounds eluting off of the column. The devices can optionally include a means for providing for gradient elution, although such is not necessary using the methods described herein. Routine methods and apparatus for carrying out HPLC separations are well known in the art.

Suitable stationary phases are those in which the compound of interest elutes. Preferred columns can be, and are not limited to, normal phase columns (neutral, acidic or basic), reverse phase columns (of any length alkyl chain), a synthetic crosslinked polymer columns (e.g., styrene and divinylbenzene), size exclusion columns, ion exchange columns, bioaffmity columns, and any combination thereof. The particle size of the stationary phase is within the range from a few pm to several 100 pm.

Suitable detection devices include, but are not limited to, mass spectrometers, UV detectors, IR detectors and light scattering detectors. The methods described herein use any combination of these detectors. The most preferable embodiment uses mass spectrometers and UV detectors.

“Source material”, as used herein, refers to the material being purified by the present method. The source material contains a diterpene glycoside of the present invention in a purity less than the purity provided by the present purification method. The source material can be liquid or solid. Exemplary source materials include, but are not limited to, mixtures of diterpene glycosides, stevia extract, Stevia plant leaves, by-products of other diterpene glycosides’ isolation and purification processes, commercially available diterpene extracts or stevia extracts, by-products of biotransformation reactions of other diterpene glycosides, or any combination thereof.

As understood by persons skilled in the art, any solid source materials must be brought into solution prior to carrying out the HPLC method.

In one embodiment, a representative analytical HPLC protocol is correlated to a preparative or semi-preparative HPLC protocol used to purify a compound.

In another embodiment, appropriate conditions for purifying a diterpene glycoside of the present invention can be worked out by route scouting a representative sample for a given analytical HPLC column, solvent system and flow rate. In yet another embodiment, a correlated preparative or semipreparative HPLC method can be applied to purify a diterpene glycoside of the present invention with modifications to the purification parameters or without having to change the purification parameters.

In some embodiments, the eluent (mobile phase) is selected from the group consisting of water, acetonitrile, methanol, 2-propanol, ethyl acetate, dimethylformamide, dimethylsulfide, pyridine, triethylamine, formic acid, trifluoroacetic acid, acetic acid, an aqueous solution containing ammonium acetate, heptafluorobutyric acid, and any combination thereof.

In one embodiment, the HPLC method is isocratic. In another embodiment, the HPLC method is a gradient. In still another embodiment, the HPLC method is step-wise.

In one embodiment, impurities are eluted off of the HPLC column after eluting one or more fractions containing a diterpene glycoside of the present invention. In another embodiment, impurities are eluted off of the HPLC column before eluting one or more fractions containing a diterpene glycoside of the present invention.

The method can further include removal of solvent from the eluted solution, i.e. drying. In one embodiment, the method further comprises partial removal of solvents from the eluted solution to provide a concentrate comprising a diterpene glycoside of the present invention. In another embodiment, the method further comprises removing substantially all the solvent from the eluted solutions to provide substantially dry material comprising a diterpene glycoside of the present invention.

Removal of solvent can be performed by any known means to one of skill in the art including, but not limited to, evaporation, distillation, vacuum drying and spray drying.

The resulting purified fractions comprising a diterpene glycoside of the present invention can be further purified by other methods to increase purity. Suitable methods include, but are not limited to, crystallization, chromatography, extraction and distillation. Such methods are well- known to persons skilled in the art.

The source material can be one fraction, or multiple fractions, containing a diterpene glycoside of the present invention collected from at least one previous method or HPLC protocol. In one embodiment, multiple fractions from the same, previous methods or HPLC protocols are pooled and optionally, solvents are removed, prior to re-subjecting the source material to another method. In other embodiments, fractions from different, previous methods or HPLC protocol are pooled, and optionally, solvents are removed, prior to re-subjecting the source material to another method.

In one embodiment, the source material re-subjected to additional method(s) comprises liquid fractions obtained from one or more previous (and optionally, different) methods mixed with substantially dry material obtained via drying of fractions obtained from one or more previous (and optionally, different) methods. In another embodiment, the source material re- subjected to additional method(s) comprises substantially dry material obtained via drying of fractions obtained from one or more previous (and optionally, different) methods, where said source material is brought into solution prior to passing the solution through the next HPLC column.

The second and subsequent methods may have different HPLC protocols (e.g. solvent systems, columns, methods) and different steps following elution (e.g. partial removal of solvent, complete removal of solvent, elution of impurities, use of crystallization or extraction).

The material isolated can be subjected to further methods 2, 3, 4 or more times, each time providing a higher level of purity of purified diterpene glycoside of the present invention.

In one embodiment, the method provides a purified diterpene glycoside of the present invention in a purity of about 50% by weight or greater on a dry basis, such as, for example, about 60% or greater, about 65% or greater, about 70% or greater, about 75% or greater, about 80% or greater, about 85% or greater, about 90% or greater, about 95% or greater and about 97% or greater. In a particular embodiment, the method provides a diterpene glycoside of the present invention in a purity greater of about 99% or greater by weight on a dry basis.

V. Methods of Synthesis

The present invention also provides methods of synthetically preparing certain diterpene glycosides disclosed herein.

In one embodiment, target diterpene glycosides can be prepared by the following general method:

Starting Glycoside

target diterpene glycoside

The starting diterpene glycoside has a free C-19 carboxylic acid, and can be ultimately functionalized at that position. In the starting glycoside, R¹ and R² are each independently selected from hydrogen, monosaccharide and oligosaccharide. The hydroxyl groups of the C-13 glycoside (including those on the optional R'/R² saccharide(s)) are then protected. In one embodiment, the hydroxyl groups are protected with acetate groups, e.g. by using acetic anhydride and trimethylamine. R³ and R⁴ represent protected forms of R¹ and R², e.g. -OAc or mono/oligosaccharide wherein the -OHs are transformed to -OAc.

The protected diterpene glycoside is then coupled at the C-19 carboxylic acid with a functionalized sugar having protected hydroxyl groups to provide a coupled, protected diterpene glycoside. R⁹, R¹⁰, R¹¹ and R¹² of the functionalized sugar are each independently selected from hydrogen, monosaccharide and oligosaccharide. If R⁹, R¹⁰, R¹¹ and R¹² are saccharide(s) or oligosaccharides, the hydroxyl groups of these moieties are also protected (see scheme below). Any suitable coupling method can be used. In one embodiment, base (e.g. K2CO3) and a catalytic amount of TB AB are used. The coupled, protected diterpene glycoside is then globally deprotected to provide the target diterpene glycoside. R⁵, R⁶, R⁷ and R⁸ of the target diterpene glycoside represent the unprotected form of the sugar, or hydrogen (if the corresponding R⁵, R⁶, R⁷ and R⁸ was also hydrogen). The deprotection can be performed by various methods known in the art, depending on the respective protecting groups. In one embodiment, the deprotection is carried out with sodium methoxide (NaOMe).

The functionalized sugar, in turn, is prepared by first protecting the hydroxyl groups of the sugar. R⁵, R⁶, R⁷ and R⁸ are each independently selected from hydrogen, monosaccharide and oligosaccharide. In one embodiment, the hydroxyl groups are protected with acetate groups using acetic anhydride and trimethylamine. The protected sugar is then brominated, e.g with HBr AcOH at the anomeric position: protection R¹²Q R₁₂0- bromination

R _R¾3å: --— — OAC

OR¹¹ ^R R¾ 0^ ¾¾ ^B1^1r

If the starting sugar comprises a saccharide in the a- configuration (e.g. at R⁵, R⁶, R⁷ or R⁸), then the resulting target diterpene glycoside will also have the a- configuration at that position.

Specific methods of preparing the diterpene glycosides of the method are provided in the Examples.

EXAMPLES

EXAMPLE 1: Isolation and Characterization of CC-00364

Materials. The material used for the isolation of CC-00364 was a Stevia extract.

HPLC Analysis. HPLC analyses were performed on a Waters 2695 Alliance System coupled to a Waters 996 Photo Diode Array (PDA) detector. In addition, sample purities were assessed using an ESA Corona Charged Aerosol Detector (CAD). Sample analyses were performed using the method conditions described in Tables 1-4.

Table 1 : Analytical HPLC Conditions for Fraction Analysis in Primary Process

Table 2: Analytical HPLC Conditions for Fraction Analysis in Secondary Process

Table 3: Analytical HPLC Conditions for Fraction Analysis in Tertiary Process

Table 4: Analytical HPLC Conditions for Final Purity Analysis

Primary Preparative HPLC. Primary processing was performed using either a pre-packed Waters XBridge RP18 column (50 x 250 mm, 7 pm) or Phenomenex Luna Cl 8 (2) column (50 x 250 mm). The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV-Vis detector. Details of the preparative method are summarized in Table 5. Approximately 300 g of Stevia extract was processed. The peak at approximately 14 mins was collected for further processing.

Table 5: Conditions for Primary Preparative HPLC Method.

Secondary Preparative HPLC Method. Secondary processing was performed using a Waters XB ridge Phenyl (19 x 250 mm, 5 pm, PN 186004024, SN 1251130651II02) column. The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV-Vis detector. Details of the preparative method are summarized in Table 6.

Fraction Lot# KHA-A-35(3C) (from primary preparative processing) was reprocessed with the conditions summarized in Table 6. Fractions were analyzed using the analytical method summarized in Table 2. Fraction Lot# RAD-C-97(28) was selected for reprocessing, retention time approximately 42.000 min on the preparative trace. Table 6: Conditions for Secondary Preparative HPLC Method.

Tertiary Processing Method. Tertiary processing was performed using a Waters XBridge Amide (19 x 250 mm, 5 pm, PN 186006606, SN 0107341600112 02) column. The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV-Vis detector. Details of the preparative method are summarized in Table 7.

Fraction Lot# RAD-C-97(28) (from secondary preparative processing) was reprocessed with the conditions summarized in Table 7. Fractions were analyzed using the analytical method summarized in Table 3. Fraction Lot# CJP-F-l26(37) was found to be sufficiently pure for NMR analysis, retention time approximately 41.000 min on the preparative trace.

Table 7: Conditions for Tertiary HPLC Process.

Isolation Procedure. The fractions were filtered through a stainless steel sieve and concentrated in vacuo using a Buchi^® Rotary Evaporator, Model R-l 14. The concentrated solution was dried for 48 - 72 h using the Kinetics Flexi-Dry Personal Freeze Dryer.

Mass Spectrometry The ESI-TOF mass spectrum acquired by infusing a sample of CC-00364 showed a [M-H] ion at m/z 1597.5939. The mass of the [M-H] ion was in good agreement with the molecular formula C68H110O42 (calcd for C69H109O42: 1597.6393, error: -3.4 ppm) expected. The MS data confirmed that CC-00364 has a nominal mass of 1598 Daltons with the molecular formula, C68H110O42.

The MS/MS spectrum of CC-00364, selecting the [M-H] ion at m/z 1597.0 for fragmentation, indicated sequential loss of three glucose units at m/z 1435.5928, 1273.4994 and 1111.4652 and loss of one rhamnose unit at m/z 965.3979 followed by sequential loss of four glucose units at m/z 803.3578, 641.2988, 479.2723 and 317.2072. The data thus indicated the presence of seven glucose units and one rhamnose unit in the structure.

NMR Spectroscopy. A series of NMR experiments including ¾ NMR (600 MHz, CD3OD at 300 K), ¹³C NMR (150 MHz, CD3OD at 300 K), ¾-¾ COSY (600 MHz, CD3OD at 300 K),

HSQC-DEPT (600 MHz, CD3OD at 300 K) and HMBC (600 MHz, CD3OD at 300 K), ROESY (600 MHz, CD3OD at 300 K) were acquired to allow assignment. The following 1D TOCSY (all 500 MHz, CD30D, 300K) spectra were also acquired: the Glcl anomeric proton (6H 5.70) over a range of mixing times (40-140 msec), the Rha anomeric proton (6H 5.20) over a range of mixing times (40-140 msec), the GlcV anomeric proton (6H 4.57) over a range of mixing times (40-140 msec) and GlcVI anomeric proton (6H 4.50) over a range of mixing times (40-140 msec).

A summary of the ¾ and ¹³C chemical shifts for the aglycone are found in Table 8:

Table 8. ¾ and ¹³C NMR (600 and 150 MHz, CD3OD), assignments of the CC-00364 aglycone.

^€Resonance overlapped with CD3OD, assignment based on HSQC- DEPT data.

^†Resonance overlapped with H2O, assignment based on HSQC- DEPT data.

Resonances overlapped.

A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-19 are found in Table 9:

Table 9. ¾ and ¹³C NMR (600 and 150 MHz, CD3OD), assignments of the CC-00364 C-19 glycoside.

^§Eleven carbon resonances in the range of 77.5-78.4 ppm (77.45, 77.55, 77.60, 77.68, 77.89, 78.04, 78.17, 78.25, 78.37 and 78.41 ppm; two carbons overlapped at 77.60), hence chemical shifts could not be unequivocally assigned.

^Four carbon resonances in the range of 62.6-62.7 ppm (62.61 , 62.64, 62.69 and 62.71 ppm), hence chemical shifts could not be unequivocally assigned.

^ATwo carbon resonances at 70.2 ppm (70.17 and 70.24 ppm).

^¥Three carbon resonances in the range of 75.3-75.4 ppm (75.26 and 75.44 ppm; two carbons overlapped at 75.26 ppm), hence chemical shifts could not be unequivocally assigned. A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-13 are found in Table 10:

Table 10. ¾ and ¹³C NMR (600 and 150 MHz, CD₃OD), assignments of the CC-00364 C-13 glycoside.

^ATwo carbon resonances at 70.2 ppm (70.17 and 70.24 ppm).

HFour carbon resonances in the range of 62.6-62.7 ppm (62.61 , 62.64, 62.69 and 62.71 ppm), hence chemical shifts could not be unequivocally assigned.

^¥Three carbon resonances in the range of 75.3-75.4 ppm (75.26 and 75.44 ppm; two carbons overlapped at 75.26 ppm), hence chemical shifts could not be unequivocally assigned.

^§ Resonance overlapped with H2O, assignment based on HSQC-DEPT data.

CC-00364 was determined to be l3-[(2-0^-D-glucopyranosyl-3-0^-D-glucopyranosyl- (6-0- -D-glucopyranosyl)- -D-glucopyranosyl)oxy] ent- kaur- 16-en- 19-oic acid-[(2-0-a-L- rhamnopyranosyl-(3-0- -D-glucopyranosyl)-3-0- -D-glucopyranosyl- -D-glucopyranosyl) ester], a new glycoside containing seven glucose units and one rhamnose unit in which Glcv is attached to Rha via a l- 3 b-linkage and Glcvn is attached to Glciv via a l- 6 b-linkage.

EXAMPLE 2: Isolation and Characterization of CC-00366

Materials. The material used for the isolation of CC-00366 was a Stevia extract.

HPLC Analysis. HPLC analyses were performed with the same equipment as Example 1. Sample analyses were performed using the method conditions described in Tables 1-4 in Example 1, with the additional method for fraction analysis in the quaternary process:

Table 11 : Analytical HPLC Conditions for Fraction Analysis in Quaternary Process

Primary Preparative HPLC. Primary processing was performed using either a pre-packed Waters XBridge RP18 column (50 x 250 mm, 7 pm) or Phenomenex Luna Cl 8 (2) column (50 x 250 mm). The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV-Vis detector. Details of the preparative method are summarized in Table 5, Example 1.

Approximately 300 g of Stevia extract was processed. Collected fractions (retention time 18-30 mins) were analyzed by LC-MS using the analytical method summarized in Table 1, Example 1.

Secondary Preparative HPLC. The secondary processing was performed using a Waters XBridge Phenyl (19 x 250 mm, 5 pm, PN 186004024, SN 1251130651II02) column. The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV- Vis detector. Details of the preparative method are summarized in Table 6, Example 1.

Fractions from primary processing were reprocessed. Collected fractions (retention time ~30 minutes) were analyzed using the analytical method summarized in Table 2, Example 1.

Tertiary Preparative HPLC. The tertiary processing was performed using a Waters XBridge Amide (19 x 250 mm, 5 pm, PN 186006606, SN 0107341600112 02)) column. The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV- Vis detector. Details of the preparative method are summarized in Table 7, Example 1.

Fractions from secondary preparative processing were reprocessed with conditions summarized in Table 7, Example 1. The collected fractions had a retention time of -30-40 mins. Quaternary Processing Method. The quaternary processing was performed using a Waters XBridge Amide (19 x 250 mm, 5 pm, PN 186006606, SN 0107341600112 02)) column. The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV-Vis detector. Details of the preparative method are summarized in Table 12.

Collected fractions from tertiary preparative processing were reprocessed with conditions summarized in Table 12. Fraction RAD-D-69(l) was sufficiently pure for NMR analysis and had a retention time of 23.207 min in the analytical HPLC method of Table 4, Example 1 (Final Purity Analysis).

Table 12: Conditions for Quaternary HPLC Process.

Isolation Procedure. Fraction Lot# RAD-D-69(l) was concentrated by rotary evaporation. The concentrated solution was further dried via lyophilization for 48 hours. The final yield of the batch RAD-D-69(l) was 15.1 mg with a sample purity of >99%.

Mass Spectrometry. The ESI-TOF mass spectrum acquired by infusing a sample of CC-00366 showed a [M-H] ion at m/z 1581.6129. The mass of the [M-H] ion was in good agreement with the molecular formula C68H110O41 (calcd for C68H109O41: 1581.6444, error: -3.3 ppm) expected. The MS data confirmed that CC-00366 has a nominal mass of 1582 Daltons with the molecular formula, C68H110O41. The ion observed at m/z 1695.6654 is most likely due to [M+CF3COOH- H] adduct.

The MS/MS spectrum of CC-00366, selecting the [M-H] ion at m/z 1581.0 for fragmentation, indicated loss of one glucose units at m/z 1419.5906 followed by loss of one rhamnose unit at m/z 1273.6028 and successive loss of three glucose units at m/z 1111.4652, 949.3948 and 787.3282 which is followed by loss of one rhamnose unit at m/z 641.3233 and loss of two glucose units at m/z 479.2617 and 317.2158 as a major fragmentation ions. Alternative fragmentation pathways are also observed in the spectrum. For example, the ion at m/z 1257.4368 would correspond to loss of one glucose from m/z 1419.5906 followed by loss of one rhamnose unit to give m/z 1111.4652. Similarly, the ion at m/z 625.2698 would correspond to loss of one glucose from m/z 787.3282 followed by loss of one rhamnose unit to give m/z 479.2617. The data thus indicated the presence of six glucose and two rhamnose units in the structure.

NMR Spectroscopy. A series of NMR experiments including 'H NMR (500 MHz, CD3OD at 300 K), ¹³C NMR (125 MHz, CD3OD at 300 K), ¾-¾ COSY (500 MHz, CD3OD at 300 K),

HSQC-DEPT (500 MHz, CD3OD at 300 K), HMBC (500 MHz, CD3OD at 300 K), ROESY (500 MHz, CD3OD at 300 K), and 1D TOCSY were acquired to allow assignment. The following 1D TOCSY (all 500 MHz, CD30D, 300K) spectra were also acquired: the Glcl anomeric proton (6H 5.65) over a range of mixing times (40-140 msec), the Rha anomeric proton (6H 5.20) over a range of mixing times (40-140 msec), the GlcV/GlcII anomeric proton (6H 4.60) over a range of mixing times (40-140 msec) and GlcVI anomeric proton (6H 4.51) over a range of mixing times (40-140 msec).

A summary of the ¾ and ¹³C chemical shifts for the aglycone are found in Table 13: Table 13. ¾ and ¹³C NMR (500 and 125 MHz, CD3OD), assignments of the aglycone.

^¥Resonance obscured by CD3OD, assignment based on HSQC- DEPT data.

Resonance partially overlapped with Rhai H-1 , assignment based on HSQC-DEPT data.

A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-19 are found in Table:

Table 14. ¾ and ¹³C NMR (500 and 125 MHz, CD₃OD), assignments of the C-19 glycoside.

^§Nine carbon resonances in the range of 77.4-78.2 ppm (77.42, 77.60, 77.65, 77.67, 77.87, 78.03, 78.17 and 78.24 ppm; two carbons resonated at 78.24 ppm), hence chemical shifts could not be unequivocally assigned.

€Six carbon resonances in the range of 62.5-62.8 ppm (62.49, 62.56, 62.63, 62.70 and 62.78 ppm; two carbons resonated at 62.63 ppm), hence chemical shifts could not be unequivocally assigned.

^¥Two carbon resonances observed at 70.2 ppm (70.18 and 70.24 ppm).

^†Three carbon resonances observed at 71 .6 ppm (71 .57 and 71 .61 ppm; two carbons overlapped at 71 .57 ppm).

^Partially overlapped with Rhan H-6, but observed as a brd.

A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-13 are found in Table 15: Table 15. ¾ and ¹³C NMR (500 and 125 MHz, CD₃OD), assignments of the C-13 glycoside.

^¥Two carbon resonances observed at 70.2 ppm (70.18 and 70.24 ppm).

^€Six carbon resonances in the range of 62.5-62.8 ppm (62.49, 62.56, 62.63, 62.70 and 62.78 ppm; two carbons resonated at 62.63 ppm), hence chemical shifts could not be unequivocally assigned.

^Partially overlapped with Rhai H-6, but observed as a brd.

CC-00366 was determined to be l3-[((2-0-a-L-rhamnopyranosyl-(3-0- -D- glucopyranosyl)-3 -0- -D-glucopyranosyl)- -D-glucopyranosyl)oxy] ent- kaur- 16-en- 19-oic acid-[((2-0-a-L-rhamnopyranosyl-(3-0- -D-glucopyranosyl)-3-0- -D-glucopyranosyl)- -D- glucopyranosyl) ester], a new glycoside containing six glucose and two rhamnose units in which Rhal has an a-configuration, GlcV is attached to Rhal via a l- 3 linkage, an a- configuration for Rhall and a 1 ->3 linkage between Glcm and Rhaii.

EXAMPLE 3: Isolation and Characterization of CC-00370

Materials. The material used for the isolation of CC-00370 was a Stevia extract.

HPLC Analysis. HPLC analyses were performed with the same equipment as Example 1. Sample analyses were performed using the method conditions described in Tables 1-4 in Example 1, with the quaternary fraction analysis process of Table 11 in Example 2.

Approximately 300 g of Stevia extract was processed. Collected fractions (retention time -18-30 mins) were selected for reprocessing.

Secondary Preparative HPLC. Fraction Lot# KHA-A-35(4C, 5C, 6C) (from primary preparative processing) was pooled into RAD-C-l83(l) and reprocessed with conditions summarized in Table 6, Example 1. Collected fractions were analyzed using the analytical method summarized in Table 2, Example 1. Fraction Lot# RAD-C- 184(27) was selected for further processing, retention time approximately 35.000 min on the preparative trace.

Tertiary Preparative HPLC. The tertiary processing was performed using a Waters XBridge Amide (19 x 250 mm, 5 pm, PN 186006606, SN 0107341600112 02) column. The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV- Vis detector. Fraction Lot# RAD-C-l 84(27) (from secondary processing) was reprocessed with conditions summarized in Table 7, Example 1. Collected fractions were analyzed using the analytical method summarized in Table 3, Example 1. Fraction Lot# RAD-D-66(6) was selected for further processing, retention time approximately 25.000 min on the preparative trace.

Quaternary Preparative HPLC. The quaternary processing was performed using a Waters XB ridge Amide (19 x 250 mm, 5 pm, PN 186006606, SN 0107341600112 02) column. The purification process was performed with a Waters Delta Prep LC Model 2000/4000 system coupled to a UV-Vis detector.

Fraction Lot# RAD-D-66(6) (from tertiary preparative processing) was processed with conditions summarized in Table 12, Example 2. Collected fractions were analyzed using the analytical method summarized in Table 11, Example 2, and fraction Lot# RAD-D-73(l) was found to be sufficiently pure for NMR analysis, retention time approximately 38.000 min on the preparative trace.

Isolation Procedure. Fraction Lot# RAD-D-73(l) was concentrated by rotary evaporation and further dried via lyophilization for 48 hours. The final yield of the batch, RAD-D-73(l), was 1.5 mg. The final purity was determined using the analytical method summarized in Table 4, Example 1 (Final Purity Analysis) and found to be >99.0% (AUC, UV) with a retention time of 27.030 min.

Mass Spectrometry The ESI-TOF mass spectrum acquired by infusing a sample of CC-00370 showed a [M-H] ion at m/z 1451.5789. The mass of the [M-H] ion was in good agreement with the molecular formula C62H100O38 (calcd for C62H99O38: 1451.5814, error: -1.1 ppm) expected. The MS data confirmed that CC-00370 has a nominal mass of 1452 Daltons with the molecular formula, C62H100O38. The ions observed at m/z 1549.5634 and 1565.5531 are most likely due to [M-H+H3PO4] and [M-H+CF3COOH] , respectively.

The MS/MS spectrum of CC-00370, selecting the [M-H] ion at m/z 1451.0 for fragmentation, indicated sequential loss of seven glucose units at m/z 1289.5464, 1127.5284, 965.4500, 803.3655, 641.3157, 479.2735 and 317.2176. The data thus indicated the presence of seven glucose units in the structure. NMR Spectroscopy. A series of NMR experiments including 'H NMR (500 MHz, CD3OD at 300 K), ¹³C NMR (125 MHz, CD3OD at 300 K), ¾-¾ COSY (500 MHz, CD3OD at 300 K), HSQC-DEPT (500 MHz, CD3OD at 300 K), HMBC (500 MHz, CD3OD at 300 K), ROESY (500 MHz, CD3OD at 300 K), and 1D TOCSY were acquired to allow assignment. The following 1D TOCSY (all 500 MHz, CD3OD, 300K) spectra were also acquired: the Glcl anomeric proton (6H 5.48) over a range of mixing times (40-140 msec), the GlcV anomeric proton (6H 5.10) over a range of mixing times (40-140 msec), the GlcVII anomeric proton (6H 4.66) over a range of mixing times (40-140 msec), GlcVI anomeric proton (6H 5.01) over a range of mixing times (40- 140 msec), the GlcII anomeric proton (6H 4.63) over a range of mixing times (40-140 msec), the

Glen H-3 (6H 4.15) over a range of mixing times (40-140 msec), the GlcIII anomeric

proton (6H 4.75) over a range of mixing times (40-140 msec), and the GlcIV anomeric proton (6H 4.59) over a range of mixing times (40-140 msec).

A summary of the ¾ and ¹³C chemical shifts for the aglycone are found in Table 16:

Table 16. ¾ and ¹³C NMR (500 and 125 MHz, CD3OD), assignments of the aglycone.

^Resonance obscured by H2O, assignment based on HSQC-DEPT data.

A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-19 are found in Table 17:

Table 17. ¾ and ¹³C NMR (500 and 125 MHz, CD3OD), assignments of the C-19 glycoside.

^§Eleven carbon resonances in the range of 77.6-78.9 ppm (77.58, 77.76, 77.89, 77.92, 78.07, 78.26, 78.27, 78.42, 78.49 and 78.87 ppm; one additional carbon overlapped in this range), hence chemical shifts could not be unequivocally assigned.

^¥Five carbon resonances in the range of 75.3-75.9 ppm (75.29, 75.52, 75.62, 75.68 and 75.87 ppm), hence chemical shifts could not be unequivocally assigned.

^†Four carbon resonances in the range of 71 .3-72.0 ppm (71 .28, 71 .59, 71 .61 and 72.01 ppm), hence chemical shifts could not be unequivocally assigned.

^€Four carbon resonances in the range of 62.7-63.0 ppm (62.74, 62.85, 62.87 and 63.01 ppm), hence chemical shifts could not be unequivocally assigned.

^GIci H-3 and Glcvn H-1 are partially overlapped.

A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-13 are found in Table 18: Table 12. ¾ and ¹³C NMR (500 and 125 MHz, CD₃OD), assignments of the C-13 glycoside.

€Four carbon resonances in the range of 62.7-63.0 ppm (62.74, 62.85, 62.87 and

63.01 ppm), hence chemical shifts could not be unequivocally assigned.

†Four carbon resonances in the range of 71 .3-72.0 ppm (71 .28, 71 .59, 71 .61 and

72.01 ppm), hence chemical shifts could not be unequivocally assigned. CC-00370 was determined to be l3-[((2-0- -D-glucopyranosyl-3-0- -D- glucopyranosyl)- -D-glucopyranosyl)oxy] ent-kaur-l6-en-l9-oic acid-[((2-0- -D- glucopyranosyl-(3-0- -D-glucopyranosyl)-3-0- -D-glucopyranosyl)- b-D-glucopyranosyl) ester], a new glycoside containing seven glucose units.

EXAMPLE 4: PREPARATION OF CC-00392

All reactions were performed under a dry atmosphere of nitrogen unless otherwise specified. Indicated reaction temperatures refer to the reaction bath, while room temperature (rt) is noted as 25 °C. Commercial grade reagents and anhydrous solvents were used as received from vendors and no attempts were made to purify or dry these components further. Removal of solvents under reduced pressure was accomplished with a Buchi rotary evaporator at approximately 28 mm Hg pressure using a Teflon-linked KNf vacuum pump. Flash column chromatography was carried out using a Teledyne Isco combiflash companion unit with redisep Rf silica gel columns. Proton NMR spectra were obtained on a 300 MHz and 400 MHz Bruker Nuclear Magnetic Resonance Spectrometer. Chemical shifts (d) are reported in parts per million (ppm) and coupling constants {J) values are given in Hz, with the following spectral pattern designations: singlet (s), doublet (d), triplet (t), quartet (q), doublet of doublet (dd), multiplet (m), broad singlet (brs). Tetramethylsilane was used as an internal reference. Mass spectroscopic analyses were performed using positive and negative mode electron spray ionization (ESI) on an Agilent 1200 system. High pressure liquid chromatography (HPLC) purity analysis was performed using a Varian Pro Star HPLC system with a binary solvent system A and B using a gradient elution:

A) Preparative HPLC Method:Gemini@l0pm NX (250 x 4.6 mm, 10 pm); mobile phase, A= H2O and B= CH3CN; Flow rate: 32 mL/min, Injection volume: 500 pL, Runtime: 40 min, gradient: 10-80%A, 20-90% B (0.0-58 min); UV detection at 210 nm.

B) HPLC Method: Phenomenex Hydro RP (250 x 4.6 mm, 5 pm); mobile phase, A= H2O with 0.0284% NH₄OAC and 0.0116% Acetic acid and B= CH3CN; gradient: 15-70% B (0.0-58 min); UV detection at 210 nm. Preparation of 2:

To a stirred solution of 1 (200 mg) in CH2CI2 (5 mL) was added HBr in acetic acid (0.5 mL) at 0 ^°C for 30 min. The reaction mixture was stirred at room temperature (25 °C) for 4 h. After completion of the (TLC monitored) reaction, the reaction mixture was quenched with ice cooled water and extracted the compound with CH2CI2 (3 x 5 mL). The combined organic layer was washed with saturated bicarbonate solution (2 x 10 mL), dried over (Na2S0₄) and concentrated under reduced pressure to afford the compound 2 (205 mg) as a light yellow solid (Note: crude product was directly used in the next step). ¾ NMR (400 MHz, CDCh): d 6.50 (d, J= 4 Hz, 1H), 5.61 (t, J= 9.2 Hz, 1H), 5.43-5.35 (m, 2H), 5.08 (t, 7= 10 Hz, 1H), 4.87 (dd, J = 4, 10.4 Hz, 1H), 4.7l7(dd, 7= 4, 9.6 Hz, 1H) , 4.55-4.50(m, 1H) , 4.28-4.23 (m, 3H), 4.09-3.93 (m, 3H), 2.15-2.01 (m, 21H).

Preparation of CC-0392:

Si?"

CC-00392

To a stirred solution of 2 (18 g, 22.39 mmol) in acetic anhydride (31.7 mL, 335.85 mmol) was added DMAP (cat) and trimethylamine (9.36 mL, 67.2 mmol) at room temperature (25 °C). The reaction mixture was stirred at 60 ^°C for 3 h. After completion of the reaction (TLC monitored), the reaction mixture was cooled to room temperature, quenched with water and extracted with CH2CI2 (3 ^c 100 mL). The combined organic layer was separated, dried over (Na2S0₄) and concentrated under reduced pressure. The residue was dissolved in MeOH (10 mL) and acidified with 1% aq HC1 (3 mL) was added drop wise. The reaction mixture was stirred for 4 h and basified with 2 M KOH to get the pH 4-5. The solvent was evaporated under reduced pressure to afford the compound 3 (26 g, 94%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.25-4.80 (m, 10H), 4.51 (d, J= 7.6Hz, 1H), 4.43 (dd, 7 = 12.4, 4.4 Hz, 1H), 4.18 (dd, J= 12.4, 6.0 Hz, 1H), 4.12-3.98 (m, 4H), 3.94-3.81 (m, 2H), 3.73-3.63 (m, 2H), 3.55-3.48 (m, 1 H), 2.23- 1.98 (m, 32H), 1.98-1.72 (m, 6H), 1.68-1.50 (m, 3H), 1.50-1.43 (m, 3H), 1.34-1.25 (m, 2H), 1.23 (s, 3H), 1.16-1.09 (m, 1H), 1.02 (s, 3H), 0.99-0.95 (m, 1H), 0.91-0.82 (m, 2H).

To a stirred solution of 4 (0.3 g, 0.245 mmol) and 2 (0.205 g, 0.294 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (0.130 g, 2.45 mmol) and TBAB (0.07 mg, 0.0024 mmol) at room temperature (25 °C). The reaction mixture was stirred at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water and extracted with CH2CI2 (3 x 30 mL). The organic layer was separated, dried over (Na2S0₄) and

concentrated under reduced pressure. The residue was purified by silica gel chromatography (70 - 80 % ethyl acetate in hexanes) to afford compound 5 (150 mg, 40%) as a white solid; 'H NMR (400 MHz, CD3OD): d 5.93 (d, J=4A Hz,IH), 5.42 -5.34 (m, 3H), 5.27-5.19 (m, 2H), 5.08-4.93 (m, 8H), 4.83-4.76 (m, 4H), 4.60-4.55 (m, 2H), 4.45 (dd, J=4.4, 12.4 Hz, 1H), 4.33-4.21 (m,

4H), 4.14-4.01 (m, 8H), 3.84-3.73 (m, 4H), 2.33-1.98 (m, 55H), l.94-l.76(m, 6H), 1.70-1.41 (m, 6H), 1.16 (s, 3H), 1.13-0.97 (m, 3H), 0.91-0.85 (m, 4H).

To a stirred solution of 5 (150 mg, 0.0814 mmol) in MeOH (2 mL) NaOMe (1 mg, 0.018 mmol) was added at 0 ^°C and stirred for 5 min. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (TLC monitored), the solvent was removed under reduced pressure. The crude residue was triturated with n-pentane (2 ^ 5 mL), the solvent was decanted and dried the compound under reduced pressure. The crude compound was purified by prep. HPLC (Method A) to afford CC-00392 (52 mg, 56%) as a white fluffy solid; UPLC-MS m/z 1128 [M-H]⁺; ¾ NMR (400 MHz, CDCh): d 5.30 (d, J= 8.0 Hz, 1H), 5.l5(brs, 1H), 5.11 (d, J= 4.0 Hz, 1H), 4.92-4.87 (m, 1H), 4.57 (d, J= 4.0 Hz, 1H), 4.57-4.44 (m, 5H), 3.82-3.70 (m, 6H), 3.67-3.45 (m, 10H), 3.42-3.24 (m, 6H), 3.18-3.03 (m, 5H), 2.14-1.69 (m, 10H), 1.58-1.30 (m, 6H), 1.12 (s, 3 H), 1.07-0.92 (m, 3H), 0.87 (s, 3H), 0.79-0.73 (m, 1H).

HPLC 98.05% (AUC), (Method B), t_R = 36.79 min.

Structural Elucidation:

CC-00392 (~4 mg) was dissolved in methanol-d4 and a suite of 1D and 2D (COSY, HSQC, HSQC-TOCSY, and HMBC) homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Table 19 summarizes the ¾ and ¹³C NMR assignments for CC-00392:

Table 19. ¾ NMR (500.13 MHz, CD3OD) and ¹³C NMR (125 MHz) assignments of CC-00392 Sugar Position ¹H Chemical Shift ¹³C Chemical Shift

I 1 5.40 (d), ³J=8.3 Hz 95.7

2 3.40 (t) 73.8

3 3.73 (t) 78.3

4 3.63 (t) 80.2 5 3.49 (m) 77.4

6 3.84 (m) 61 .8

II V 4.60 (d), ³J=7.9 Hz 97.5

2 3.63 (t) 80.0 3’ 3.72 (t) 87.7 4’ 3.35 (t) 70.6 5’ 3.26 (m) 77.8

6 3.87, 3.64 (m) 62.8

III 1” 4.85 103.8

2 3.22 75.9 3” 3.33 78.3 4” 3.16 72.3 5” 3.28

6 3.58 (d), 3.83 (d) 63.6

IV 1’” 4.67 104.3

2 3.27

3’”

4’”

5’”

6

V 1’” 5.21 102.7

2 3.44 74.2 3’” 3.61 75.1 4’” 3.26 71 .6 5’” 3.77 73.4

6 3.83

It was determined that CC-00392 is (l3-[(2-0-P-D-glucopyranosyl-3-0-P-D- glucopyranosyl-P-D-glucopyranosyl)oxy] enl- aux- 16-en- l 9-oic acid-[(4-0-a-D-glucopyranosyl -b-D-glucopyranosyl) ester], a diterpene glycoside where Glc I, Glc II, Glc III, and Glc IV are in the b-configuration and Glc V is in the a-configuration. Glc I and Glc V are connected via a l- 6 linkage.

EXAMPLE 5: PREPARATION OF CC-00393

Preparation of 8:

Nigerosc

To a stirred solution of 6 (0.3 g, 0.877 mmol) in 50 mL flask add pyridine (3 mL) and AC₂0 (1.5 mL, 13.14 mmol) stirred at room temperature for 16 h. After completion of the reaction, the reaction was co-distilled with toluene (2 ^c 30 mL) and CH2CI2 (30 mL) at 45^°C to afford compound 7 (0.35 g, 59%) as a white solid (Note: crude compound was directly used in the next step); ¾ NMR (400 MHz, CDCh): d 6.33 (d, J= 3.6 Hz, 0.5H), 5.62 (d, J= 8.4 Hz, 0.5H), 5.37-5.15 (m, 4H), 5.09-5.03 (m, 1.5H), 4.81-4.76 (m, 1H), 4.22-3.89 (m, 6H), 3.76-3.71 (m, 0.5H), 2.17-1.98 (m, 24H).

To a stirred solution of 7 (0.35 g, 2.21 mmol) in CH2CI2 (10 mL) was added HBr in acetic acid (1 mL) at 0 ^°C for 30 min. The reaction mixture was stirred at room temperature for 4 h. After completion of the (TLC monitored) reaction, the reaction mixture was quenched with ice cooled water and extracted the compound with CH2CI2 (3 ^ 10 mL). The combined organic layer was washed with saturated bicarbonate solution (2 ^c 30 mL), dried over (Na2S0₄) and concentrated under reduced pressure to afford the compound 8 (0.25 g, 69%) as a light yellow solid (Note: crude product was directly used in the next step). ¾ NMR (400 MHz, CDCh): d 6.65 (d, J= 4.0 Hz, 1H), 5.40-5.26 (m, 3H), 5.02 (t, J= 10.0 Hz, 1H), 4.82-4.77 (m, 2H), 4.40- 4.05 (m, 7H) 2.13 (s, 3H), 2.12 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H).

Preparation of CC-00393:

To a stirred solution of 9 (3.5 g, 4.35 mmol) in acetic anhydride (6.2 mL, 65.3 mmol) was added DMAP (cat) and trimethylamine (1.8 mL, 13.05 mmol) at room temperature. The reaction mixture was stirred at 60 ^°C for 3 h. After completion of the reaction (TLC monitored), the reaction mixture was cooled to room temperature, quenched with water and extracted with CH2CI2 (3 x 10 mL). The combined organic layer was separated, dried over (Na2S0₄) and concentrated under reduced pressure. The residue was dissolved in MeOH (10 mL) and acidified with 1% aq HC1 (3 mL) was added drop wise. The reaction mixture was stirred for 4 h and basified with 2 M KOH to get the pH 4-5. The solvent was evaporated under reduced pressure to afford the compound 10 (4 g, 75%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.25-4.80 (m, 10H), 4.51 (d, J= 7.6Hz, 1H), 4.43 (dd, 7=12.4, 4.4 Hz, 1H), 4.18 (dd, J= 12.4, 6.0 Hz,

1H), 4.12-3.98 (m, 4H), 3.94-3.81 (m, 2H), 3.73-3.63 (m, 2H), 3.55-3.48 (m, 1 H), 2.23-1.98 (m, 32H), 1.98-1.72 (m, 6H), 1.68-1.50 (m, 3H), 1.50-1.43 (m, 3H), 1.34-1.25 (m, 2H), 1.23 (s, 3H), 1.16-1.09 (m, 1H), 1.02 (s, 3H), 0.99-0.95 (m, 1H), 0.91-0.82 (m, 2H).

To a stirred solution of 10 (0.45 g, 0.367 mmol) and 8 (0.25 g, 0.441 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (0.203 g, 1.468 mmol) and TBAB (0.12 g, 0.036 mmol) at room temperature. The reaction mixture was stirred at 60 °C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water and extracted with CH2CI2 (3 x 30 mL). The organic layer was separated, dried over (Na2S0₄) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (70 - 80 % ethyl acetate in hexanes) to afford the compound 11 (250 mg, 37%) as a white solid; ¾ NMR (400 MHz, CD3OD): d 5.74 (d, J= 8.4 Hz, 1H), 5.32-5.18 (m, 6H), 5.l5-5.0l(m, 7H), 4.97-4.90 (m, 4H), 4.84-4.75 (m, 3H) 4.20 (dd, J= 4.4, 8.0 Hz, 1H), 4.34-4.02 (m, 10H), 3.97-3.91 (m, 1H), 3.89-3.81 (m, 2H), 3.79-3.67 (m, 2H), 2.21-1.98 (m, 55H), 1.96-1.77 (m, 6H), 1.74-1.64 (m, 1H), 1.58-1.42 (m, 5H), 1.16 (s, 3H), 1.13-0.97 (m, 3H), 0.90 (s, 3H), 0.87-0.82 (m, 1H).

To a stirred solution of 11 (250mg, 0.135 mmol) in MeOH(3 mL) NaOMe (2 mg, 0.013 mmol) was added at 0 ^°C and stirred for 5 min. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (TLC monitored), the solvent was removed under reduced pressure. The crude residue was triturated with n-pentane (2 ^ 5 mL), the solvent was decanted and dried the compound under reduced pressure. The crude residue was purified by preparative HPLC (Method A, Example 4) to afford CC-00393 (140 mg, 32%) as a white fluffy solid; UPLC-MS m/z 1147 [M+NH₄]⁺; ¾ NMR (400 MHz, CDCh): d 5.33 (d, J= 8.4 Hz, 1H), 5.16 (m, 2H), 4.55 (dd, J= 7.6, 16.4 Hz, 2H), 3.87-3.71 (m, 6H), 3.65-3.35 (m, 13H), 3.34- 3.22 (m, 6H), 3.19-3.08 (m, 6H), 3.01 (t, J= 9.2 Hz, 1H) 2.16-1.69 (m, 10H), 1.62-1.28 (m, 6H), 1.12 (s, 3H), 1.06-0.89 (m, 3H), 0.87 (s, 3H), 0.83-0.73 (m, 1H). HPLC 99.34% (AUC),

(Method B, Example 4), Rt= 35.22 min

Structural Elucidation:

CC-00393 (~4 mg) was dissolved in methanol-d4 and a suite of 1D and 2D (COSY, HSQC, HSQC-TOCSY, and HMBC) homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Table 20 summarizes the ¾ and ¹³C NMR assignments for CC-00393: Table 20. ¾ NMR (500.13 MHz, CD₃OD) and ¹³C NMR (125 MHz) assignments of CC-00393

Sugar Position ¹H Chemical Shift ¹³C Chemical Shift

I 1 5.43 (d), ³J=8.1 Hz 95.8

2 3.50 (t) 72.9

3 3.73 (t) 78.3

4 3.64 (t) 87.3

5

6

1 4.63 97.2 2 3.63 80.0 3’ 3.72 87.6 4’ 3.38 70.6 5’ 3.26

77.8

6 3.84, 3.66 (m)

1 4.85 103.8 2 3.22 76.0 3” 3.33 78.3 4” 3.16 72.5 5” 3.27

6 3.58 (d), 3.83 (d) 63.6

IV 1 4.67 104.3

2 3.26

75.5

3.37 (t) 78.3

4”

5”

6

V 1 5.27 (d), ³J=3.8 Hz 101 .3

2 3.47 (t) 74.1

3” 3.69 (t) 75.0

4” 3.29 (t) 71 .9

5” 3.93 (m) 74.3

6 3.65, 3.90 (d) 62.7

It was determined that CC-00393 is (l3-[(2-0-P-D-glucopyranosyl-3-0-P-D- glucopyranosyl-P-D-glucopyranosyl)oxy] enl- aux- 16-en- l 9-oic acid-[(3-0-a-D-glucopyranosyl -b-D-glucopyranosyl) ester], a diterpene glycoside where Glc I, Glc II, Glc III, and Glc IV are in the b-configuration and Glc V is in the a-configuration. Glc I and Glc V are connected via a l - 3 linkage.

EXAMPLE 6: PREPARATION OF CC-00404

Preparation of 14:

To a stirred solution of 12 (0.5 g, 1.46 mmol) in pyridine (10 mL), was added AC2O (2.23 mL, 21.91 mmol, 15 eq) and the reaction mixture was stirred at room temperature for 16 h. The mixture was azeotrope with toluene (30 mL x 2) followed with CH2CI2 (30 mL x 2) and dried under vacuum to obtain compound 13 (0.65 g, 65%) as a white solid. The crude compound was directly used in the next step. ¾ NMR (300 MHz, DMSO-D6): d 6.32 (d, J= 3.0 Hz, 1H), 5.35-5.25 (m, 2H), 5.15 (t, J= 9.9 Hz, 1H), 4.97 (t, J= 9.9 Hz, 2H), 4.87-4.82 (dd, J= 10.5, 3.6 Hz, 1H), 4.21-3.96 (m, 6H), 3.91-3.87 (m, 1H), 2.18 (s, 3H), 2.09-1.90 (m, 21H).

To a stirred solution of 13 (0.65 g, 0.958 mmol) in CH2CI2 (30 mL) at 0 °C, was added HBr in acetic acid (2 mL) in a drop-wise manner over a period of 30 min. After complete addition, the reaction mixture was warmed to room temperature and stirred for 4 h. After completion of the reaction (by TLC), the reaction mixture was quenched with ice cooled water and extracted with CH2CI2 (3 ^c 30 mL). The combined organic extract was washed with saturated bicarbonate solution (2 ^c 50 mL), dried (Na2S0₄) and concentrated under reduced pressure to obtain compound 14 (0.41 g, 61%) as a light yellow solid. The crude product was directly used in the next step. ¾ NMR (400 MHz, CDCh): d 6.43 (d, J= 3.6 Hz, 1H), 5.52 (t, J = 9.2 Hz, 1H), 5.38 (t, J= 9.2 Hz, 1H), 5.22 (d, J= 3.6 Hz, 1H), 5.10-5.03 (m, 2H), 4.79 (dd, J = 10.0, 3.2 Hz, 1H), 4.37-4.29 (m, 2H), 4.20 (brs, 2H), 4.12-4.04 (m, 2H), 3.85 (dd, J= 9.6 ,3.6 Hz, 1H), 2. l2-2.0l(m, 21H). Preparation of CC-00404:

18 CC-00404 To a stirred solution of 15 (1.0 g, 1.55 mmol) in methanol (20 mL) NaOH (0.63 g, 15.57 mmol) was added in one lot at room temperature and stirred at reflux condition for 6 h. After completion of the reaction (TLC monitored), reaction mixture was cooled to room temperature, acidified with 1 N HC1 (pH 4.0-5.0) at 10 ^°C, the solvent was evaporated under reduced pressure and compound was extracted with n-butanol (3 x 50 mL). The combined organic layer was washed with water and concentrated under reduced pressure. The crude compound was azeotrope with methanol: acetonitrile (1 : 1) (3 ^c 30 mL) to afford 16 (0.8 g) as a white solid.

(Note: crude product was directly used in the next step). ESI MS: m/z 503.3 [M+Na]⁺.

To a stirred solution of compound 16 (0.8 g, 1.666 mmol) in pyridine (5 mL) acetic anhydride (1.7 mL, 16.66 mmol) was added at room temperature under nitrogen atmosphere for overnight. After completion of the reaction (TLC monitored), the reaction mixture was azeotrope with toluene (3 ^c 30 mL), CH2CI2 (100 mL) the solvent was evaporated under reduced pressure. The crude compound was purified by silica gel chromatography (eluted with 60 - 70 % ethyl acetate in hexanes) to afford the compound 17 (0.6 g, 55%) as a white solid. ¾ NMR (400 MHz, CDCb): d 5.26-5.18 (m, 1H), 5.12-4.97 (m, 2H), 4.87 (brs, 1H), 4.76-4.6l(m, 2H), 4.20-4.09 (m, 2H), 3.74-3.65 (m, 1H), 2.21-1.95 (m, 16H), 1.96-1.44 (m, 12H), 1.24 (s, 3H), 1.12-0.97 (m,

3H), 0.93 (s, 3H), 0.89-0.78 (m, 1H).

To a solution of 17 (600 mg, 0.924 mmol) and 14 (774 mg, 1.109 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (510 mg, 1.02 mmol), TBAB (28 mg, 0.092 mmol) at room temperature and stirred in sealed tube at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water (20 mL) and extracted the compound with CH2CI2 (3 x 10 mL). The organic layer was dried over (Na2S0₄) and

concentrated under reduced pressure. The crude compound was purified by silica gel

chromatography (70-80 % ethyl acetate in hexanes) to afford 18 (300 mg, 56%) as a white solid. ¾ NMR (400 MHz, CD3OD): d 5.70 (d, J= 8.0 Hz, 1H), 5.34-5.14 (m, 4H), 5.03-4.81 (m, 6H), 4.72 (brs, 1H), 4.24 (dd, J= 12.4, 2.4 Hz, 1H), 4.18-3.80 (m, 10H), 2.12-1.85 (m, 37H), 1.85-1.32 (m, 12H), 1.13 (s, 3H), 1.07-0.87 (m, 3H), 0.80 (s, 3H), 0.78-0.72 (m, 1H).

To a stirred solution of 18 (300 mg, 0.236mmol) in MeOH(2 mL) NaOMe (2 mg, 0.23 mmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h, after completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was triturated in n-pentane (2 x 5 mL), the solvent was decanted and dried the compound under reduced pressure. The crude compound was purified by prep. HPLC (Method A, Example 4) to afford CC-00404 (72 mg, 38%) as a white puffy solid; UPLC MS: m/z 803 [M-H]⁺; Ή NMR (400 MHz, CD3OD): d 5.51 (d, J= 7.6 Hz, 1H), 5.08 (s, 1H), 5.04 (d, J= 4.0 Hz, 1H), 4.89-4.85 (m, 3H), 4.75-4.72 (m, 2H), 4.39 (d, J= 8.0 Hz, 1H), 4.02-3.97 (m, 1H), 3.74-3.22 (m, 7H), 3.22-3.17 (m, 4H), 3.13-3.02 (m, 2H), 2.17-1.68 (m, 10H), 1.66-1.27 (m, 6H),l. l6 (s, 3H), 1.03-0.87 (s, 3H), 0.86 (s, 3H), 0.81- 0.75 (m, 1H). HPLC 98.38% (AUC), (Method B, Example 4), R_t = 33.01 min.

Structural Elucidation:

CC-00404 (~4 mg) was dissolved in methanol-d4 and a suite of 1D and 2D (COSY, HSQC, HSQC-TOCSY, and HMBC) homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Table 21 summarizes the ¾ and ¹³C NMR assignments for CC-00404: Table 1. ¾ NMR (500.13 MHz, methanol-d4) and ¹³C NMR (125 MHz) assignments of CC-

00404

Sugar Position ¹H Chemical Shift ¹³C Chemical Shift

I 1 5.61 (d), ³J=7.9 HZ 95.5

2 3.59 (t) 78.9

3 3.53 (t) 76.9

4 3.43 (t) 71.6

5 3.37 (m) 78.4

6 3.70 (m), 3.83 (m) 62.6

II 1 4.49 (d), ³J=7.8 HZ 99.3

2 3.18 (t) 75.3

3 3.35 (t) 78.2

4 3.30 (t) 71.7

5 3.19 (m) 77.86

6 3.65 (m), 3.80 (m) 62.8

III 1 5.15 (d), ³J=3.9 HZ 100.3

2 3.36 (t) 73.5

3 3.63 (m) 75.2

4 3.37 (t) 71.3

5 4.09 (m) 73.4

6 3.71 (m), 3.80 (m) 62.2

It was determined that CC-00404 is l3-(P-D-glucopyranosyl-P-D-glucopyranosyloxy) t'///-kaur- 16-en- l 9-oic acid-[(2-0-a-D-glucopyranosyl -b-D-glucopyranosyl) ester], a diterpene glycoside where Glc I and are in the b-configuration and Glc III is in the a-configuration.

EXAMPLE 7: PREPARATION OF CC-00405

To a stirred solution of 19 (5.0 g, 6.21 mmol) in methanol (150 mL) NaOH (9.44 g, 236.06 mmol) was added in one lot at room temperature and stirred at reflux condition for 16 h. After completion of the reaction (TLC monitored), reaction mixture was cooled to room temperature, Acidified with 1 N HC1 (pH 4.0-5.0) at 10 ^°C, the solvent was evaporated under reduced pressure and compound was extracted with n-butanol (3 ^c 50 mL). The combined organic layer was washed with water and concentrated under reduced pressure. The crude compound was azeotrope with methanol: acetonitrile (1 : 1) (3 ^c 30 mL) to afford 20 (3.2 g) as a white solid. (Note: crude product was directly used in the next step). ESI MS: m/z 665 [M+Na]⁺; ¾ NMR (400 MHz, DMSO-de): d 5.68 (s, 1H), 5.41 (d, J= 3.2Hz, 1H), 5.12-4.88 (m, 4H), 4.74 (s, 1H), 4.48 (d, J= 7.6 Hz, 1H), 4.43-4.38 (m, 1H), 4.36 (d, J= 7.6Hz, 1H), 4.25-4.17 (m, 1H), 4.15-4.10 (m, 1H), 3.63-3.53 (m, 2H), 3.51-3.37 (m, 3H), 3.27-2.96 (m, 6H), 2.13-1.65 (m, 9H), 1.54-1.28 (m, 7H), 1.10 (s, 3H), 1.03-0.90 (m, 3H), 0.87 (s, 3H), 0.83-0.72 (m, 1H).

To a stirred solution of compound 20 (3.2 g, 4.98 mmol) in pyridine (30 mL) acetic anhydride (5.08 mL, 49.84 mmol) was added at room temperature under nitrogen atmosphere for overnight. After completion of the reaction (TLC monitored), the reaction mixture was azeotrope with toluene (3 ^c 50 mL), CH2CI2 (100 mL) the solvent was evaporated under reduced pressure. The crude compound was purified by silica gel chromatography (eluted with 60 - 70 % ethyl acetate in hexanes) to afford the compound 21 (3.5 g, 75%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.20-5.09 (m, 3H), 5.00-4.82 (m, 4H), 4.65 (d, J= 8.0 Hz, 1H), 4.58 (d, J= 8.0 Hz, 1H), 4.22-4.05 (m, 4H), 3.81-3.60 (m, 3H), 2.26-2.12 (m, 3H), 2.09(s, 6H), 2.07 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 1.98 (s, 6H), 1.97-1.38 (m, 13H), 1.23 (s, 3H), 1.17-1.09 (m, 1H), 1.03 (s, 3H), 1.02-0.95 (m, 2H), 0.90-0.80 (m, 1H).

To a solution of 21 (500 mg, 0.534 mmol) and 14 (447.4 mg, 0.641 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (295 mg, 2.138 mmol), TBAB (16.18 mg, 0.05 mmol) at room temperature and stirred in sealed tube at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water (20 mL) and extracted the compound with CH2CI2 (3 x 10 mL). The organic layer was dried over (Na2S0₄) and

chromatography (70-80 % ethyl acetate in hexanes) to afford 22 (250 mg, 30%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.74 (d, J= 7.6 Hz, 1H), 5.35-5.03 (m, 12H), 4.95-4.86 (m, 4H), 4.79 (brs, 1H), 4.73 (d, J= 7.6 Hz, 1H), 4.67 (d, J= 8.0 Hz, 1H), 4.36-4.31 (m, 1H), 4.30-4.25 (m, 1H), 4.24-4.16 (m, 3H), 4.08-4.04 (m, 2H), 3.97 (t , j= 8.9 Hz, 1H), 3.92-3.87 (m, 1H), 3.81- 3.71 (m, 2H), 3.68-3.63 (m, 1H), 2.23-1.95 (m, 46H), 1.90-1.73 (m, 6H), 1.67-1.37 (m, 6H), 1.20 (s, 3H), 1.13-0.87 (m, 3H), 0.83 (s, 3H), 0.82-0.78(m, 1H).

To a stirred solution of 22 (250 mg, 0. l6mmol) in MeOH (2 mL) NaOMe (2 mg, 0.016 mmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h, after completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was triturated in n-pentane (2 x 5 mL), the solvent was decanted and dried the compound under reduced pressure. The crude compound was purified by prep. HPLC (Method A, Example 4) to afford CC-00405 (70 mg, 45%) as a white puffy solid; UPLC MS: m/z 965.50 [M-H]⁺; ¾ NMR (400 MHz, CD3OD): d 5.53 (d, j= 8.0 Hz, 1H), 5. l0(s, 1H), 5.04 (d, j= 3.6 Hz, 1H), 4.51 (dd, j= 14.0, 7.6 Hz, 2H), 4.01-3.96 (m, 1H), 3.78-3.68 (m, 4H), 3.64-3.34 (m, 9H), 3.33-3.23 (m, 6H), 3.18-3.07 (m, 5H), 2.19-1.68 (m, 10H), 1.60-1.28 (m, 6H), 1.17 (s, 3H), 1.03-0.88 (m, 3H), 0.85 (s, 3H), 0.80-0.72 (m, 1H). HPLC 99.03% (AUC), (Method B, Example 4), Rt = 27.23 min. Structural Elucidation:

CC-00405 (~ 2 mg) was dissolved in methanol-d4 and a suite of 1D and 2D (COSY, HSQC, HSQC-TOCSY, and HMBC) homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Table 22 summarizes the ¾ and ¹³C NMR assignments for CC-00405:

Table 22. ¾ NMR (500.13 MHz, methanol-d4) and ¹³C NMR (125 MHz) assignments of CC-

00405

Sugar Position ¹H Chemical Shift ¹³C Chemical Shift

I 1 5.63 (d), ³J=7.9 HZ 95.4

2 3.59 (t) 78.9

3 3.54 (f) 76.9

4 3.41 (t) 71.7

5 3.41 (m) 78.4

6 3.70 (m), 3.85 (m) 62.7

II 1 4.63 (d), ³J=7.7 HZ 97.6

2 3.47 (t) 82.5

3 3.56 (t) 78.2

4 3.32 (t) 71.6

5 3.21 (m) 77.6

6 3.64 (m), 3.81 (m) 62.7

III 1 4.60 (d), ³J=7.7 HZ 105.2

2 3.25 (t) 76.4

3 3.36 (m) 77.9

4 3.27 (t) 71.8

5 3.26 (m) 78.4

6 3.64 (m), 3.81 (m) 63.0

IV 1 5.14 (d), ³J=3.9 HZ 100.3

2 3.36 (t) 73.5

3 3.63 (m) 75.2

4 3.37 (t) 71.4

5 4.08 (m) 73.4

6 3.71 (m), 3.80 (m) 62.2

It was determined that CC-00405 is is l3-[(2-0-P-D-glucopyranosyl-P-D- glucopyranosyl)oxy] enl- aux- 16-en- l 9-oic acid-[(2-0-a-D-glucopyranosyl -b-D- glucopyranosyl) ester], a diterpene glycoside where Glc I, Glc II and Glc III and are in the b- configuration and Glc IV is in the a-configuration. EXAMPLE 8: PREPARATION OF CC-00422

Preparation of 31:

Act)·

--o BnOH NaOCH₃, CH₃OH

AcO^' Br

AcO Ag₂C03, h (cat), CH2CI2 rt, 4 h

OAc

4 A MS, rt, 24 h

To a stirred solution of benzyl alcohol (19.70 g, 182.4 mmol) in CH2CI2 (30 mL) was added 4Ά MS, iodine (50 mg), and silver carbonate (25.15 g, 91.2 mmol) at room temperature in the dark. Reaction mixture was stirred for 15 min at room temperature under inert atmosphere in dark. Solution of (2R,3R,5R)-2-(acetoxymethyl)-6-bromotetrahydro-2H-pyran-3,4,5-triyl triacetate 23 (15.0 g, 36.4 mmol) in CH2CI2 (30 mL) was added slowly drop wise to the reaction mixture for 15 min. After complete addition, the reaction mixture was stirred for 16 h. After completion of the reaction (by TLC), the reaction mixture was diluted with CH2CI2 (50 mL) and filtered through celite and concentrate the filtrate and purified by silica gel chromatography (25- 30 % ethyl acetate in hexanes) to afford 24 (18.0 g, 58%) as a light yellow solid. ¹HNMR (400 MHz, CDCh): d 7.37-7.26 (m, 5H), 5.17-5.04 (m, 3H), 4.90 (d, J= 12.4 Hz, 1H), 4.62 (d, J= 12 Hz, 1H), 4.54 (d, J= 8 Hz, 1H), 4.30-4.25 (m, 1H), 4.18-4.15 (m, 1H), 3.86-3.82 (m, 0.2H), 3.69-3.65 (m, 0.8H), 2.11 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H).

To a stirred solution of 24 (4.0 g, 0.009 mmol) in MeOH(30 mL) NaOMe (0.04 mg, 0.0009 mmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h, after completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure to obtain compound 25 (2.5 g, 93%) as a white solid. The crude compound was directly used in the next step. ¹HNMR (400 MHz, CD3OD): d 7.33- 7.31 (m, 2H), 7.24-7.16 (m, 3H), 4.83(d, 7 = 11.6 Hz, 2H), 4.56 (d , J= 11.6 Hz, 1H), 4.25 (d, J =

7.6 Hz, 1H), 3.80 (dd, J= 12.0, 2.0 Hz, 1H), 3.59 (dd, J= 12.0, 5.6 Hz, 1H), 3.21-3.20 (m, 4H).

To a stirred solution of 25 (13.0 g, 48.14 mmol) in DMF (50 mL) benzaldehyde dimethyl acetal (2l.98g, 14.44 mmol) and /2TSA H2O was added at room temperature and stirred at 60 ^°C for 6 h and depressurizing it by removal of methanol by using flushing of nitrogen. After completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 26 (7.5 g, 51%) as a white solid. ESI MS: m/z 359 [M+H]⁺; ¹HNMR (400 MHz, CDCh): d 7.50-7.48 (m, 2H), 7.40-7.33 (m, 8H), 5.55 (s, 1H), 4.94 (d, J =

11.6 Hz, 1H), 4.64 (d, 7= 11.6 Hz, 1H), 4.51 (d, J= 7.6 Hz, 1H), 4.38 (dd, J= 10.4, 5.2 Hz, 1H), 3.84-3.79 (m, 2H), 3.61-3.55 (m, 2H), 3.52-3.38 (m, 1H).

A mixture of 26 (1.20 g, 3.35 mmol) and 27 (4.2 g, l0.05mmol) was azeotrope in toluene (30 mL x 2) and dried under reduced pressure for 30 min. To that mixture in CH2CI2 (10 mL) was added activated 4 A MS and stirred at room temperature for 30 min under inert atmosphere. Trimethyl silyl trifluoromethanesulfonate (0.02g, 0.1 mmol) in CH2Cl2 (l.O mL) was added drop wise at -60°C. Reaction mixture was stirred at -40 °C for 2 h and allowed to stir at room temperature slowly for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with Et₃N diluted CH2CI2 (10 mL) and filtered through celite pad. The filtrate was concentrated and purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 28 (2.1 g, 72 %) as a white solid. ESI MS: m/z 892 [M+NH₄]⁺; ¹HNMR (400 MHz, CDCh): d 7.47-7.44 (m, 2H), 7.38-7.33 (m, 8H), 5.49 (s, 1H), 5.18 (t, J= 8.0 Hz, 1H), 5.07-5.03 (m, 1H), 4.97-4.86 (m, 7H), 4.64 (d, J= 12.0 Hz, 1H), 4.53 (d, J= 7.2 Hz, 1H), 4.37- 4.33 (m, 1H), 4.15-4.09 (m, 2H), 4.03 (t, J= 8.4 Hz, 1H), 3.91-3.87 (m, 1H), 3.76 (t, J= 12 Hz, 1H), 3.64 (t, J= 9.6 Hz, 1H), 3.45-3.38 (m, 1H), 3.28 (dd, J= 11.6, 8.0 Hz, 1H), 3.18 (dd, J = 12.0, 7.2 Hz, 1H), 2.11 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H).

To a stirred solution of 28 (1.9 g, 2.17 mmol) in ethylacetate (40 mL), was added

Pd(OH)₂/C (400 mg) and the reaction mixture was stirred under H₂ bladder at room temperature for 3 h. After completion of the reaction (TLC monitored), the reaction mixture was filtered through celite pad and dried under vacuum to obtain compound 29 (1.0 g) as a white solid. The crude compound was directly used in the next step. ESI MS: m/z 714 [M+NH₄]⁺; ¹HNMR (300 MHz, CD3OD): d 5.14- 4.81 (m, 8H), 4.71 (d, J= 6.3 Hz, 1H), 4.15-4.03 (m, 2H), 3.90 (t, J= 9.0 Hz, 1H), 3.68-3.24 (m, 7H), 2.07 (s, 3H), 2.04 (s, 3H), 1.95-1.91 (m, 12H).

To a stirred solution of 29 (1.0 g, 1.43 mmol) in pyridine (15 mL), was added Ac₂0 (1.43 mL, 14.36 mmol,) and the reaction mixture was stirred at room temperature for 16 h. The mixture was azeotrope with toluene (30 mL x 2) followed with CH₂Cl₂ (30 mL x 2) and dried under vacuum to obtain compound 30 (1.0 g) as a white solid. The crude compound was directly used in the next step. ESI MS: m/z 840 [M+NH₄]⁺; ¹HNMR (400 MHz, CDCh): d 6.52-6.50 (m, 0.4H), 6.24 (d, J= 3.6 Hz, 0.6H), 5.12 (t, J= 8.4 Hz, 1H), 5.06-5.00 (m, 2H), 4.98-4.86 (m, 3H), 4.85-4.78 (m, 2H), 4.72 (d, J= 6.4 Hz, 1H), 4.21-4.00 (m, 5H), 3.97-3.92 (m, 1H), 3.91-3.87 (m, 1H), 3.49-3.44 (m, 1H), 3.41-3.36 (m, 1H), 2.17-2.02 (m, 27H).

To a stirred solution of 30 (0.60 g, 0.72 mmol) in CH₂Cl₂ (15 mL) at 0 °C, was added HBr in acetic acid (3 mL) in a drop-wise manner over a period of 30 min. After complete addition, the reaction mixture was warmed to room temperature and stirred for 4 h. After completion of the reaction (by TLC), the reaction mixture was quenched with ice cooled water and extracted with CH₂Cl₂ (3 ^c 30 mL). The combined organic layer was washed with saturated bicarbonate solution (2 ^c 50 mL), dried (Na₂S0₄) and concentrate under reduced pressure to obtain compound 31 (0.49 g) as a light yellow solid. The crude product was directly used in the next step. ¹HNMR (400 MHz, CDCh): d 6.58 (d, J= 4.0 Hz, 0.3H), 6.43 (d, J= 4.0 Hz, 0.7H), 5.12 (t, J= 8.0 Hz, 1H), 5.08-4.99 (m, 3H), 4.96-4.92 (m, 1H), 4.85-4.75 (m, 4H), 4.29-4.16 (m, 4H), 4.13-4.03 (m, 2H), 3.75-3.71 (m, 1H), 3.51-3.39 (m, 2H), 2.17 (s, 3H), 2.12 (s, 3H), 2.08- 2.03 (m, 18H).

Preparti on of CC-00422:

To a stirred solution of 32 (2.0 g, 2.48 mmol) in acetic anhydride (3.5 mL, 37.3 mmol) was added DMAP (cat) and trimethylamine (1.04 mL, 7.44 mmol) at room temperature. The reaction mixture was stirred at 60 ^°C for 3 h. After completion of the reaction (TLC monitored), the reaction mixture was cooled to room temperature, quenched with water and extracted with CH2CI2 (3 x 10 mL). The combined organic layer was separated, dried over (Na2S0₄) and concentrated under reduced pressure. The residue was dissolved in MeOH (10 mL) and acidified with 1% aq HC1 (3 mL) was added drop wise. The reaction mixture was stirred for 4 h and basified with 2 M KOH to get the pH 4-5. The solvent was evaporated under reduced pressure to afford the compound 33 (700 mg, 23%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.25- 4.80 (m, 10H), 4.51 (d, J= 7.6Hz, 1H), 4.43 (dd, 7=12.4, 4.4 Hz, 1H), 4.18 (dd, J =12.4, 6.0 Hz, 1H), 4.12-3.98 (m, 4H), 3.94-3.81 (m, 2H), 3.73-3.63 (m, 2H), 3.55-3.48 (m, 1 H), 2.23-1.98 (m, 32H), 1.98-1.72 (m, 5H), 1.68-1.43 (m, 11H), 1.23 (s, 3H), 1.16-1.09 (m, 1H), 1.02 (s, 3H), 0.99- 0.95 (m, 1H).

To a solution of 33 (600 mg, 0.49 mmol) and 31 (454.2 mg, 0.53 mmol) in CH2CI2 (10 mL), H2O (2 mL), were added K2CO3 (202.9 mg, 1.47 mmol), TBAB (15.78 mg, 0.05 mmol) at room temperature and stirred in sealed tube at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water (20 mL) and extracted the compound with CH2CI2 (3 x 10 mL). The organic layer was dried over (Na2S0₄) and

chromatography (70-80 % ethyl acetate in hexanes) to afford 34 (480 mg, 49%) as a white solid. ¹HNMR (400 MHz, CDCh): d 5.57 (d, J= 9.2 Hz, 1H), 5.17 (t, J= 9.2 Hz, 3H), 5.12- 5.06 (m, 6H), 5.02-4.94 (m, 6H), 4.86-4.79 (m, 6H), 4.56 (d, J= 7.6 Hz, 1H), 4.44-4.39 (m, 1H), 4.20- 3.91 (m, 13H), 3.85-3.79 (m, 1H), 3.72-3.64 (m, 3H), 3.60-3.55 (m, 1H), 2.l6-2.l3(m, 6H), 2.10- 2.04 (m, 37H), 2.01-2.00 (m,l3H), 1.85-1.78 (m, 6H), 1.58-1.43 (m, 6H), 1.22 (s, 3H), 1.05-0.96 (m, 4H), 0.89-0.86 (m, 2H), 0.82 (s, 3H).

To a stirred solution of 34 (470 mg, 0.236mmol) in MeOH(8 mL) NaOMe (1.2 mg,

0.023 mmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h, after completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was purified by prep. HPLC (Method A, Example 4) to afford CC-00422 (201.7 mg, 69%) as a white puffy solid. UPLC MS: m/z 1229.65 [M+NH₄]⁺; ¹HNMR (400 MHz, CD3OD): d 5.43(d, J= 8.0 Hz, 1H), 5.16 (brs, 1H),

4.85 (d, J= 8.0 Hz, 1H), 4.81-4.83 (m, 1H), 4.73-4.65 (m, 6H), 4.24-4.19 (m, 1H), 4.02 (t, j =

9.2 Hz, 1H), 3.86-3.63 (m, 7H), 3.61-3.22 (m, 17H), 3.18-3.15 (m, 4H), 3.08 (t, j= 12 Hz, 1H), 2.94-2.89 (m, 1H), 2.19-2.03 (m, 3H), 2.0-1.96 (m, 3H), 1.84-1.72 (m, 3H), 1.69-1.64 (m, 1H), 1.61-1.43 (m, 3H), 1.42-1.29 (m, 3H), 1.10 (s, 3H), 0.99-0.97 (m, 2H), 0.86 (s, 3H), 0.80-0.74 (m, 2H). HPLC 98.48% (AUC), (Method B, Example 4), Rt = 34.96 min.

Structural Elucidation:

CC-00422 (~5 mg) was dissolved in methanol-d4 and a suite of 1D and 2D (COSY, HSQC, HSQC-TOCSY, and HMBC) homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Table 23 summarizes the ¾ and ¹³C NMR assignments for CC-00422: Table 23. ¾ NMR (500.13 MHz, CD₃OD) and ¹³C NMR (125 MHz) assignments of CC-00422 Sugar Position ¹H Chemical Shift ¹³C Chemical Shift

1 1.89 (m), 0.87 (m) 41.1

2 2.13 (m), 1.44 (m) 20.2

3 2.08 (d), 1.08 (m) 39.0

4 45.1

5 1.08 (d) 58.3

6 1.92 (m), 1.86 (m) 24.3

7 1.59 (m), 1.44 (m) 43.1

8 41.9 9 0.96 (d) 55.2

10 40.6

11 1.77 (m), 1.61 (m) 20.6

12 1.92.18 (m), 1.48 (m) 39.0

13 89.4

14 2.24 (d), 1.58 (d) 43.9

15 2.10 47.1

16 152.6

17 5.26 (s), 4.82 (s) 105.5

18 1.21 (s) 28.4

19 178.5

20 0.97 (s) 17.2

Glc l 1 5.53 (d), ³JHH=8.2 HZ 95.2

2 3.76 (t) 77.6

3 4.32 (t) 87.8

4 3.45 (t) 69.9

5 3.45 (m) 78.3

6 3.81,3.67 (m) 62.2

Glc II r 4.79 (d), ³JHH=7.9 HZ 96.0

2 3.50 (t) 81.0

3’ 4.13 (t) 87.5

4’ 3.40 (t) 70.5

5’ 3.28 (m) 77.9

6 3.79 (m), 3.65 62.8

Glc III 1 4.76 (d), ³JHH=7.7 Hz 104.7

2 3.35 (t) 75.9

3 3.30 (t) 78.3

4

5

6

Glc IV 1 4.80 (d), ³JHH=7.7 Hz 103.9

2 3.28 (t) 77.9

3

4

5 6

Xyl I 1” 4.95 (d), ³JHH=7.9 HZ 104.8

2 3.36 (t) 75.7 3” 3.30 (t) 78.3 4” 3.57 71.7 5” 3.91 3.42 66.7

Xyl II 1”’ 4.79 (d), ³JHH=7.9 Hz 104.7

2 3.28 (t)

3’”

4 3.55 71.0

5”’ 3.91, 3.42 67.2

It was determined that CC-00422 is l3-[(2-0-P-D-glucopyranosyl-3-0-P-D- glucopyranosyl-P-D-glucopyranosyl)oxy] t7//-kaur- 16-en- l 9-oic acid-[(2-0-P-D-xylopyranosyl- 3-0-P-D-xylopyranosyl-P-D-glucopyranosyl) ester], a diterpene glycoside containing four glucose and two xylose units. All sugars exist as b-anomers.

EXAMPLE 9: PREPARATION OF CC-00423

To a stirred solution of 35 (5.0 g, crude) in methanol (150 mL) NaOH (9.44 g, 236.06 mmol) was added in one lot at room temperature and stirred at reflux condition for 16 h. After completion of the reaction (TLC monitored), reaction mixture was cooled to room temperature, Acidified with 1 N HC1 (pH 4.0-5.0) at 10 ^°C, the solvent was evaporated under reduced pressure and compound was extracted with n-butanol (3 X 50 mL). The combined organic layer was washed with water and concentrated under reduced pressure. The crude compound was azeotrope with methanol: acetonitrile (1 : 1) (3 X 30 mL) to afford 36 (5 g, crude) as a white solid. (Note: crude product was directly used in the next step). ESI MS: m/z 665 [M+Na]⁺; 'H NMR (400 MHz, DMSO-de): d 5.68 (s, 1H), 5.41 (d, J= 3.2Hz, 1H), 5.12-4.88 (m, 5H), 4.74 (s, 1H), 4.48 (d, J= 7.6 Hz, 1H), 4.43-4.38 (m, 1H), 4.36 (d, J= 7.6Hz, 1H), 4.25-4.17 (m, 1H), 3.63- 3.53 (m, 2H), 3.51-3.37 (m, 3H), 3.27-2.96 (m, 6H), 2.13-1.65 (m, 9H), 1.54-1.28 (m, 7H), 1.10 (s, 3H), 1.03-0.90 (m, 3H), 0.87 (s, 3H), 0.83-0.72 (m, 1H).

To a stirred solution of compound 36 (5.0 g, 7.77 mmol) in pyridine (50 mL) acetic anhydride (7.94 g, 77.79 mmol) was added at room temperature under nitrogen atmosphere for overnight. After completion of the reaction (TLC monitored), the reaction mixture was azeotrope with toluene (3 X 50 mL), CH2CI2 (100 mL) the solvent was evaporated under reduced pressure. The crude compound was purified by silica gel chromatography (eluted with 60 - 70 % ethyl acetate in hexanes) to afford the compound 37 (7.0 g, 97%) as a white solid. ESI MS: m/z 959 [M+NH₄]⁺; ¾ NMR (400 MHz, CDCh): d 5.20-5.09 (m, 3H), 5.00-4.82 (m, 4H), 4.65 (d, J =

8.0 Hz, 1H), 4.58 (d, J= 8.0 Hz, 1H), 4.22-4.05 (m, 4H), 3.81-3.60 (m, 3H), 2.26-2.12 (m, 3H), 2.09 (s, 6H), 2.08 (s, 3H), 2.05 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H), 1.98 (s, 6H), 1.97-1.38 (m, 13H), 1.23 (s, 3H), 1.17-1.09 (m, 1H), 1.03 (s, 3H), 1.02-0.95 (m, 2H), 0.90-0.80 (m, 1H).

To a solution of 37 (300 mg, 0.32 mmol) and 31 (286 mg, 0.32 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (1329 mg, 0.96 mmol), TBAB (10.3 mg, 0.03 mmol) at room temperature and stirred in sealed tube at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water (20 mL) and extracted the compound with CH2CI2 (3 x 10 mL). The organic layer was dried over (Na2S0₄) and concentrated under reduced pressure. The crude compound was purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 38 (247 mg, 45%) as a white solid. UPLC-MS: m/z 1717.68 [M+NH ]⁺; ¹HNMR (400 MHz, CDCh): d 5.57 (d, J= 7.2 Hz, 1H), 5.18 (t, J= 9.6 Hz, 1H), 5.19-5.04 (m, 5H), 5.00-4.98 (m, 2H), 4.92-4.86 (m, 3H), 4.86-4.77 (m, 5H), 4.69 (dd, J= 8.0, 2.4 Hz, 2H), 4.28-4.14 (m, 4H), 4.12-4.03 (m, 6H), 3.78-3.63 (m, 4H), 3.53-3.45 (m, 2H), 2.37 (t, 7= 8.4 Hz, 1H), 2.14 (d, J= 4.4 Hz, 6H), 2.08-2.05 (m, 25H), 2.00-1.98 (m, 15H), 1.89-1.77 (m, 6H), 1.65-1.60 (m, 2H), 1.52-1.41 (m, 5H), 1.22 (s, 3H), 1.07-1.02 (m, 2H), 0.98-0.95 (m, 2H), 0.83 (s, 3H), 0.82-0.77 (m, 1H).

To a stirred solution of 39 (247 mg, 0. l45mmol) in MeOH (4 mL) NaOMe (0.78 mg, 0.014 mmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h, after completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was purified by prep. HPLC (Method A, Example 4) to afford CC-00423 (91 mg, 60%) as a white puffy solid. UPLC-MS: m/z 1067.83 [M-H]⁺; ¹HNMR (400 MHz, CD3OD): d 5.48 (d, J= 8.0 Hz, 1H), 5.12 (brs, 1H), 4.56 (dd, J= 8.0, 2.4 Hz, 2H), 4.49 (d, J= 7.6 Hz, 1H), 3.85-3.79 (m, 2H), 3.77-3.49 (m, 10H), 3.47-

3.30 (m, 6H), 3.28-3.24 (m, 3H), 3.18-3.02 (m, 9H), 2.22-2.12 (m, 2H), 2.05-1.68 (m, 8H), 1.50-

1.30 (m, 5H), 1.12 (s, 3H), 1.00-0.87 (m, 3H), 0.85 (s, 3H), 0.82-0.70 (m, 2H), HPLC 99.43% (AUC), (Method B, Examplr 4), R_t = 34.03 min.

Structural Elucidation:

CC-00423 (~5 mg) was dissolved in methanol-d4 and a suite of 1D and 2D (COSY, HSQC, HSQC-TOCSY, and HMBC) homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Table 24 summarizes the ¾ and ¹³C NMR assignments for CC-00423:

Table 24. ¾ NMR (500.13 MHz, CD3OD) and ¹³C NMR (125 MHz) assignments of CC-00423

Sugar Position ¹H Chemical Shift ¹³C Chemical Shift

1 1.88 (m), 0.86 (t) 41.5

2 2.02 (m), 1.41 (m) 20.4

3 2.29 (d), 1.03 (m) 38.3

4

5 1.08 (d) 58.6

6 1.90 (m), 1.88 (m) 23.2

7 1.57 (m), 1.45 (m) 42.8

8

9 0.99 (d) 55.1

10 40.6

11 1.81 (m), 1.65 (m) 21.1

12 2.01 (m), 1.54 (m) 38.6

13 88.3

14 2.25 (d), 1.52 (d) 45.2

15 2.12, 2.08 48.3 16 153.7

17 5.22 (s), 4.84 (s) 105.5

18 1.22 (s) 29.1

19 177.5

20 0.95 (s) 17.3

Glc I 1 5.58 (d), ³ IHH=8.1 Hz 94.1

2 3.75 (t) 78.5

3 3.91 (t) 87.6

4 3.44 (t) 69.6

5 3.45 (m) 78.3

6 3.85, 3.70 (m) 62.3

Glc II r 4.67 (d), ³JHH=7.8 HZ 97.2

3.44 (t) 82.5

3’ 3.65 (t) 78.1

4’ 3.32 (t) 71.6

5’ 3.21 (m) 77.9

6 3.80 (m), 3.64 62.8

Glc III 1 4.60 (d), ³JHH=7.7 HZ 105.3

2 3.31 (t) 76.1

3” 3.37 (t) 77.9

4” 3.20 (t) 72.1

5” 3.25 (m) 78.5

6 3.82, 3.62 63.3

Xyl l 1 4.84 (d), ³JHH=7.9 Hz 104.5

2 3.15 (t) 75.8

3 3.29 (t) 78.4

4 3.47 71.4

5 3.81. 3.20 66.8

Xyl ll V 4.66 (d), ³JHH=7.6 HZ 104.9

2 3.26 (t) 75.3 3’ 3.30 (t) 78.4 4’ 3.52 71.1 5’ 3.91. 3.30 67.2

It was determined that CC-00423 is l3-[(2-0-P-D-glucopyranosyl-P-D- glucopyranosyl)oxy] enl- aux- 16-en- l 9-oic acid-[(2-0-P-D-xylopyranosyl-3-0-P-D- xylopyranosyl-P-D-glucopyranosyl) ester], a diterpene glycoside containing five glucose and two xylose units. All sugars exist as b-anomers.

EXAMPLE 10: PREPARATION OF CC-00424

To a stirred solution of 40 (1.0 g, 1.55 mmol) in methanol (20 mL) NaOH (0.63 g, 15.57 mmol) was added in one lot at room temperature and stirred at reflux condition for 6 h. After completion of the reaction (TLC monitored), reaction mixture was cooled to room temperature, Acidified with 1 N HC1 (pH 4.0-5.0) at 10 ^°C, the solvent was evaporated under reduced pressure and compound was extracted with n-butanol (3 x 50 mL). The combined organic layer was washed with water and concentrated under reduced pressure. The crude compound was azeotrope with methanol: acetonitrile (1 : 1) (3 ^c 30 mL) to afford 41 (0.8 g) as a white solid. (Note: crude product was directly used in the next step). ESI MS: m/z 503.3 [M+Na]⁺.

To a stirred solution of compound 41 (0.8 g, 1.666 mmol) in pyridine (5 mL) acetic anhydride (1.7 mL, 16.66 mmol) was added at room temperature under nitrogen atmosphere for overnight. After completion of the reaction (TLC monitored), the reaction mixture was azeotrope with toluene (3 ^c 30 mL), CH2CI2 (100 mL) the solvent was evaporated under reduced pressure. The crude compound was purified by silica gel chromatography (eluted with 60 - 70 % ethyl acetate in hexanes) to afford the compound 42 (0.6 g, 55%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.26-5.18 (m, 1H), 5.12-4.97 (m, 2H), 4.87 (brs, 1H), 4.76-4.6l(m, 2H), 4.20-4.09 (m, 2H), 3.74-3.65 (m, 1H), 2.21-1.95 (m, 16H), 1.96-1.44 (m, 12H), 1.24 (s, 3H), 1.12-0.97 (m,

3H), 0.93 (s, 3H), 0.89-0.78 (m, 1H). To a solution of 42 (200 mg, 0.308 mmol) and 31 (311 mg, 0.37 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (170 mg, 1.23 mmol), TBAB (10 mg, 0.03 mmol) at room temperature and stirred in sealed tube at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water (20 mL) and extracted the compound with CH2CI2 (3 x 10 mL). The organic layer was dried over (Na2S0₄) and concentrated under reduced pressure. The crude compound was purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 43 (160 mg, 37%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.51 (d, J= 8.0 Hz, 1H), 5.14 (t, J= 9.6 Hz, 1H), 5.03-4.90 (m, 8H), 4.80-4.71 (m, 4H), 4.60 (d, J= 8.0 Hz, 1H), 4.14-3.92 (m, 8H), 3.68-3.59 (m, 2H), 3.47-3.36 (m, 3H), 2.20- 2.12 (m, 2H), 2.09-1.90 (m, 37H), 1.84-1.75 (m, 6H), 1.61-1.54 (m, 1H), 1.48-1.41 (m, 2H), 1.40-1.31 (m, 4H), 1.15 (s, 3H), 0.90-0.88 (m, 3H), 0.83-0.78 (m, 1H), 0.75 (s, 3H).

To a stirred solution of 43 (150 mg, 0. l06mmol) in MeOH(2 mL) NaOMe (2 mg, 0.01 mmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h, after completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was triturated in n-pentane (2 x 5 mL), the solvent was decanted and dried the compound under reduced pressure. The crude compound was purified by prep. HPLC (Method A, Example 4) to afford CC-00424 (31 mg, 32%) as a white puffy solid; UPLC MS: m/z 924 [M+NH₄]⁺; Ή NMR (400 MHz, CD3OD): d 5.44 (d, J= 8.0 Hz, 1H), 5.11 (s, 1H), 4.83-4.79 (m, 1H), 4.75-4.71 (m, 2H), 4.52 (d, J= 7.6 Hz, 1H), 4.41 (d, J= 8.0 Hz, 1H), 3.85-3.79 (m, 1H), 3.76-3.65 (m, 5H), 3.63-3.52 (m, 2H), 3.46- 3.34 (m, 3H), 3.31-3.23 (m, 3H), 3.19-2.97 (m, 7H), 2.26-2.18 (m, 2H), 2.11-2.03 (m, 2H), 1.99- 1.68 (m, 7H), 1.62-1.53 (m, 1H), 1.51-1.41 (m, 3H), 1.38-1.27 (m, 2H), 1.12 (s, 3H), 1.01-0.96 (m, 1H), 0.94-0.83 (m, 1H), 0.84 (s, 3H), 0.81-071 (m, 2H). HPLC 95.2% (AUC), (Method B, Example 4), R_t = 37.12 min.

Structural Elucidation:

CC-00424 (~2 mg) was dissolved in methanol-d4 and a suite of 1D and 2D (COSY, HSQC, HSQC-TOCSY, and HMBC) homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Table 25 summarizes the ¾ and ¹³C NMR assignments for CC-00424: Table 25. ¾ NMR (500.13 MHz, CD₃OD) and ¹³C NMR (125 MHz) assignments of CC-00424 Sugar Position ¹H Chemical Shift ¹³C Chemical Shift

1 1.88 (m), 0.85 (m) 41.7

2 1.97 (m), 1.40 (m) 20.4

3 2.32 (d), 1.02 (m) 38.3

4 45.2

5 1.09 (d) 58.7

6 1.91 (m), 1.87 (m) 22.9

7 1.57 (m), 1.45 (m) 42.7

8 43.0 9 1.00 (d) 55.3

10 40.6

11 1.82 (m), 1.66 (m) 21.3

12 1.96 (m), 1.52 (m) 38.9

13 87.8

14 2.18 (d), 1.58 (d) 45.3

15 2. l7(m), 2.05(m) 49.2

16 153.9

17 5.21 (s), 4.86 (s) 105.6

18 1.22 (s) 29.3

19 177.4

20 0.94 (s) 16.7

Glc l 1 5.55 (d), ³JHH=7.9 HZ 93.9

2 3.78 (t) 87.7

3 3.77 (t) 78.4

4 3.48 (t) 69.3

5 3.39 (m) 78.5

6 3.82,3.71 (m) 62.2

Glc II r 4.51 (d), ³JHH=7.9 HZ 99.2

2 3.17 (t) 75.4

3’ 3.37 (t) 78.1

4’ 3.31 (t) 71.7

5’ 3.19 (m) 77.6

6 3.80 (m), 3.65 62.8

Xyl I 1 4.63 (d), ³JHH=7.7 HZ 105.0

2 3.25 (t) 75.3

3” 3.32 (t) 78.3

4” 3.53 (m) 71.0

5” 3.92 (m), 3.27 (m) 67.2

Xyl II 1 4.83 (d), ³JHH=7.7 HZ 104.4

2 3.10 (t) 75.7

3’” 3.30 (t) 78.3

4 3.48 (m) 71.4

5”’ 3.81, 3.20 66.9 It was determined that CC-00424 is l3-(P-D-glucopyranosyloxy) e«/-kaur-l6-en-l9-oic acid-[(2-0-P-D-xylopyranosyl-3-0-P-D-xylopyranosyl-P-D-glucopyranosyl) ester], a diterpene glycoside containing two glucose and two xylose units. All sugars exist as b-anomers.

EXAMPLE 11: PREPARATION OF CC-00428

To a stirred solution of 44 (15 g, 83.3 mmol) in DMF (150 mL), was added PhCH(OCH3)2 (19 mL, 91.6 mmol, 1.1 eq) p-TSA (0.200 g, 8.33 mmol) and the reaction mixture was stirred at 60 °C for 6 h. The mixture was dried under vacuum to obtain compound 45 (10 g, 45%) as a white solid. The combined organic layer was concentrated under reduced pressure crude compound was obtained purified with combi flash eluted with 60% ethyl acetate: Hexane. ¾ NMR (400 MHz, CD₃OD): d 7.45-7.36 (m, 2H), 7.28-7.21 (m, 3H), 5.46 (s, 0.7H), 5.04 (s, 0.3H), 4.49 (dd, J = 10.8, 4.4 Hz, 0.3H), 4.07 (dd, J = 10.8, 4.4 Hz, 0.7H), 3.90-3.83 (m, 0.7H), 3.77 (t, j = 9.2 Hz, 0.7H), 3.68-3.57 (m, 1.3H), 3.52 (t, j = 9.2 Hz, 0.3H), 3.40-3.30 (m, 2H), 3.17-3.12 (m, 0.3H).

To the solution of compound 45 (6 g, 22.38 mmol) in pyridine (40 mL) acetic anhydride (12.7 g, 134.32 mmol) was added at room temperature under nitrogen atmosphere for overnight. After completion of the reaction, reaction mass was azeotroped with toluene and CH2CI2 of each (3 X 10 mL). The mixture was dried under vacuum to obtain compound 46 (6.5 g, 73%) as a white solid. The combined organic layer was concentrated under reduced pressure crude compound was obtained purified with combi flash eluted with 40%-50% ethyl acetate: Hexane ¾ NMR (400 MHz, CDCb): d 7.45-7.40 (m, 2H), 7.38-7.33 (m, 3H), 6.31 (d, J= 3.6 Hz, 0.3H), 5.79 (d, J= 8.0 Hz, 0.7H), 5.59 (t, J= 10.0 Hz, 0.3H), 5.52 (s, 0.3H), 5.50 (m, 0.7H), 5.37 (t, J = 9.2 Hz, 0.7H), 5.16-5.10 (m, 1H), 4.39 (dd, J = 10.0, 4.4 Hz, 0.7H), 4.32 (dd, J = 10.4, 4.8 Hz, 0.3H), 4.07-3.99 (m, 0.3H), 3.80-3.63 (m, 2.7H), 2.11-2.02 (m, 9H).

To a stirred solution of 46 (9.50 g, 24.11 mmol) in CH2CI2 (100 mL) Et₃SiH (3.35 g, 28.93 mmol) was added slowly at room temperature and stirred at same temperature for 5 min. The reaction mass was cooled to -78 °C for 45 min and EtAlCb was added dropwise at -78 °C for 15 min; maintained at same temperature for 40 min. After completion of the reaction (TLC monitored), reaction mixture was cooled to 0 °C, quenched with saturated NaHCCh solution (50 ml) and extracted with CH2CI2 (3 x 50 mL). The combined organic layer was concentrated under reduced pressure. The crude compound obtained purified with combi flash eluted with 60% ethyl acetate: hexane to afford 47 (6.0 g, 63%) as a thick liquid. ESI MS: m/z 414 [M+H]⁺, 'H NMR (400 MHz, CDCb): 7.37-7.28 (m, 5H), 6.29 (d, J= 3.6 Hz, 0.5H), 5.68 (d, J= 8 Hz, 0.5H), 5.33 (t, j= 10 Hz, 0.5H), 5.12-5.01 (m, 1.5H), 4.64-4.51 (m, 2H), 3.96-3.92 (m, 0.5H), 3.86-3.65 (m, 3.5H), 2.93-2.92 (dd, J= 6.4 Hz, 3.6 Hz, 1H), 2.15-1.95 (m, 9H).

A mixture of 47 (2 g, 5.05 mmol) and 48 (4.89 gm, 6.565mmol) was azeotroped in toluene (30 mL x 2) and dried under reduced pressure for 30 min. To that mixture in CH2CI2 (20 mL) was added activated 4 A MS and stirred at room temperature for 30 min under inert atmosphere. Trimethylsilyl trifluoromethanesulfonate (0.034g, 0. l5mmol) in CH2CI2 (1.0 mL) was added drop wise at -40°C. The reaction mixture was stirred at -40 °C for 2 h and allowed to stir at room temperature slowly for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with EtiN diluted CH2CI2 (10 mL) and filtered through celite pad. The filtrate was concentrated and purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 49 (3 g, 61%) as a white solid. ESI MS: m/z 993 [M+H]+.

To a stirred solution of 49 (3 g, 3.08 mmol) in ethyl acetate (30 mL), was added Pd(OH)2/C (770 mg) and the reaction mixture was stirred under H2 bladder at room temperature for 5 h. After completion of the reaction (TLC monitored), the reaction mixture was filtered through celite pad and dried under vacuum to obtain compound 50 (730 mg, 26%) as a white solid. The crude compound was obtained purified with combi flash eluted with 60% ethyl acetate: Hexane. UPLC MS: m/z 902 [M+H]⁺; ¹HNMR (400 MHz, CDCh): d 8.07-7.77 (m, 8H), 7.60-7.28 (m, 12H), 6.22 (d, J = 3.6 Hz, 1H), 5.92-5.86 (m, 1H), 5.70-5.45 (m, 3H), 5.26 (t, J = 9.6 Hz, 1H), 5.05-4.93 (m, 2H), 4.69-4.64 (m, 1H), 4.46-4.40 (m, 1H), 4.19-4.01 (m, 2H), 3.79- 3.63 (m, 3H), 2.10-1.92 (m, 9H).

A mixture of 50 (700 mg, 0.791 mmol) and 51 (0.506 g, 1.029 mmol) was azeotroped in toluene (30 mL x 2) and dried under reduced pressure for 30 min. To that mixture in CH2CI2 (20 mL) was added activated 4 A MS and stirred at room temperature for 30 min under inert atmosphere. Tert butyldimethylsilyl trifluoromethanesulfonate (0.104 g, 0.395mmol) in CH2CI2 (1.0 mL) was added drop wise at -40°C. The reaction mixture was stirred at -40 °C for 2 h and allowed to stir at room temperature slowly for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with Et₃N diluted CH2CI2 (10 mL) and filtered through celite pad. The filtrate was concentrated and purified by silica gel chromatography (70- 80 % ethyl acetate in hexanes) to afford 52 (600 mg, 62 %) as a white solid. UPLC MS: m/z 1290 [M-H]⁺; ¾ NMR (400 MHz, CDCh): d 8.07-7.77 (m, 8H), 7.61-7.27 (m, 12H), 6.21 (d, J = 4.0 Hz, 0.5H), 5.91 (t, 7 = 9.6 Hz, 0.5H), 5.70-5.56 (m, 0.5H), 5.56-5.45 (m, 3.5H), 5.26 -5.19 (m, 1H), 5.09-4.91 (m, 5H), 4.76-4.66 (m, 1H), 4.49-4.45 (m, 1H), 4.32-4.22 (m, 3H), 4.18-4.10 (m, 1H), 3.99-3.87 (m, 2H), 3.84-3.48 (m, 2H), 2.95-2.94 (dd, J= 3.6 Hz, 1.2 Hz, 1H), 2.26-1.84 (m, 21H).

To a stirred solution of 52 (0.700 g, 0.576 mmol) in CH2CI2 (30 mL) at 0 °C, was added HBr in acetic acid (0.7 mL) in a drop-wise manner over a period of 10 min. After complete addition, the reaction mixture was warmed to room temperature and stirred for 4 h. After completion of the reaction (by TLC), the reaction mixture was quenched with ice cooled water and extracted with CH2CI2 (3 / 30 mL). The combined organic extract was washed with saturated bicarbonate solution (2 ^c 50 mL), dried (Na2S0₄) and concentrated under reduced pressure to obtain compound 53 (0.350 g, crude) as a light yellow solid. The crude product was directly used in the next step. UPLC MS: m/z 1254 [M+H]⁺.

CC-00428

To a stirred solution of 33 (0.35 g, 0.285 mmol) and 53 (0.422 g, 0.343 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (0.157 g, 1.140 mmol) and TBAB (0.086 mg,

0.0028mmol) at room temperature. The reaction mixture was stirred at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water and extracted with CH2CI2 (3 X 30 mL). The organic layer was separated, dried over (Na2S0₄) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (70 - 80 % ethyl acetate in hexanes) to afford the compound 54 (250 mg, 38%) as a white solid; UPLC MS: m/z 2399 [M+H]⁺; ¾ NMR (400 MHz, CD3OD): d 7.98-7.18 (m, 20H), 5.90-5.76 (m, 2H), 5.60-5.54 (m, 1H), 5.36-5.32 (m, 1H), 5.29-5.21 (m, 1H), 5.19-5.08 (m, 3H), 5.04-4.93 (m, 7H), 4.90-4.80 (m, 4H), 4.42-4.04 (m, 12H), 3.95-3.58 (m, 11H), 3.61-3.31 (m, 2H), 2.08- 1.72 (m, 51H), 1.85-1.75 (m, 5H), 1.67-1.55 (m, 1H), 1.52-1.33 (m, 6H), 1.09 (s, 3H), 0.91 (s, 3H), 0.81 (s, 3H), 0.69 (s, 2H). To a stirred solution of 53 (0.250 gm, 0.l05mmol) in MeOH (5 mL) NaOMe (25 mg, O.OlOmmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was triturated in n-pentane (2 x 5 mL), the solvent was decanted and dried the compound under reduced pressure. The crude compound was purified by prep. HPLC (Method A, Example 4) to afford CC-00428 (20 mg, 7.5%) as a white solid UPLC MS: m/z 1290 [M-H]⁺; ¾ NMR (400 MHz, CD3OD): d 5.29 (d, J = 8.4 Hz, 1H), 5.14 (brs, 1H), 4.83 (d, J= 4.8 Hz, 2H), 4.57 (d, J= 8.0 Hz, 1H), 4.50 (t, J= 8.4 Hz, 2H), 4.31 (d, J= 8.0 Hz, 1H), 4.07 (d, J= 9.2 Hz, 1H), 3.87-3.69 (m, 9H), 3.62-3.45 (m, 13H), 3.33-3.24 (m, 7H), 3.16-3.03 (m, 6H), 2.15-1.70 (m, 10H), 1.61-1.28 (m, 6H), 1.07-0.89

(m, 2H), 0.87 (s, 3H), 0.83-0.70 (m, 2H), HPLC 96% (AUC), (Method B, Example 4), R_t =

32.21 min.

It was determined that CC-00428 is [(l3-[(2-0-P-D-glucopyranosyl-3-0-P-D- glucopyranosyl-P-D-glucopyranosyl)oxy] ent- kaur- 16-en- 19-oic acid-[(4-0-P-D- glucopyranosyl-6-0-P-D-glucopyranosyl-P-D-glucopyranosyl) ester].

EXAMPLE 12: PREPARATION OF CC-00432

62 a

To a stirred solution of benzyl alcohol (19.70 g, 182.4 mmol) in CH2CI2 (30 mL) was added 4Ά MS, iodine(50 mg), and silver carbonate (25.15 g, 91.2 mmol) at room temperature in the dark. The reaction mixture was stirred for 15 min at room temperature under inert atmosphere in the dark. A solution of (2R,3R,5R)-2-(acetoxymethyl)-6-bromotetrahydro-2H- pyran-3,4,5-triyl triacetate 54 (15.0 g, 36.4 mmol) in CH2CI2 (30 mL) was added slowly drop wise to the reaction mixture for 15 min at room temperature. After complete addition, the reaction mixture was stirred for 16 h. After completion of the reaction (by TLC), the reaction mixture was diluted with CH2CI2 (50 mL) and filtered through celite and concentrated. The filtrate was purified by silica gel chromatography (25-30 % ethyl acetate in hexanes) to afford 55 (18.0 g, 58%) as a light yellow solid. ¹HNMR (400 MHz, CDCh): d 7.37-7.26 (m, 5H), 5.17- 5.04 (m, 3H), 4.90 (d, J = 12.4 Hz, 1H), 4.62 (d, j = 12 Hz, 1H), 4.54 (d, J = 8 Hz, 1H), 4.30- 4.25 (m, 1H), 4.18-4.15 (m, 1H), 3.86-3.82 (m, 0.2H), 3.69-3.65 (m, 0.8H), 2.11 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H). To a stirred solution of 55 (4.0 g, 0.009 mmol) in MeOH (30 mL) NaOMe (0.04 mg, 0.0009 mmol) was added at 0 ^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure to obtain compound 56 (2.5 g, 93%) as a white solid. The crude compound was directly used in the next step. ¹HNMR (400 MHz, CD3OD): d 7.33-7.31 (m, 2H), 7.24-7.16 (m, 3H), 4.83(d, J = 11.6 Hz, 2H), 4.56 (d, J = 11.6 Hz, 1H), 4.25 (d, J = 7.6 Hz, 1H), 3.80 (dd, J = 12.0, 2.0 Hz, 1H), 3.59 (dd, J = 12.0, 5.6 Hz, 1H), 3.21-3.20 (m, 4H).

To a stirred solution of 56 (13.0 g, 48.14 mmol) in DMF (50 mL) benzaldehyde dimethyl acetal (21.98 g, 14.44 mmol) and /2TSA H2O was added at room temperature and stirred at 60 ^°C for 6 h and depressurizing it by removal of methanol by using flushing of nitrogen. After completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 57 (7.5 g, 51%) as a white solid. ESI MS: m/z 359 [M+H]⁺; ^lHNMR (400 MHz, CDCh): d 7.50-7.48 (m, 2H), 7.40-7.33 (m, 8H), 5.55 (s, 1H), 4.94 (d, j = 11.6 Hz, 1H), 4.64 (d, J= 11.6 Hz, 1H), 4.51 (d, J= 7.6 Hz, 1H), 4.38 (dd, J= 10.4, 5.2 Hz, 1H), 3.84-3.79 (m, 2H), 3.61-3.55 (m, 2H), 3.52-3.38 (m, 1H).

A mixture of 57 (1.50 g, 4.18 mmol) and 58 (2.0 g, 4.18 mmol) was azeotroped in toluene (30 mL x 2) and dried under reduced pressure for 30 min. To that mixture in CH2CI2 (20 mL), was added activated 4 A MS and stirred at room temperature for 30 min under inert atmosphere. /f/T-Butyl di methyl si lyl trifluoromethanesulfonate (0.44g, l .67mmol) in CH2Cl2 (2.0 mL) was added drop wise at -60°C. The reaction mixture was stirred at -40 °C for 2 h and allowed to stir at room temperature slowly for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with Et3N diluted CH2CI2 (10 mL) and filtered through celite pad. The filtrate was concentrated and purified by silica gel chromatography (70- 80 % ethyl acetate in hexanes) to afford 59 and 59a (1.2 g, 41 %) as a white solid. ESI MS: m/z 706.3 [M+l8]^{+ 1}HNMR (400 MHz, CDCh): d 7.49-7.45 (m, 2H), 7.38-7.32(m, 8H), 5.55-5.54 (m, 1H), 5.19-5.14 (m, 1H), 5.09-5.87 (m, 4H), 4.79 (d, J = 8.0 Hz, 0.4H), 4.68-4.63 (m, 1.6H), 4.37-4.33 (m, 1H), 4.12-4.05 (m, 1H), 3.89-3.77 (m, 3H), 3.65-3.52 (m, 2H), 3.52-3.37 (m, 2H), 2.70 (d, J= 2.8 Hz, 0.5H), 2.57 (d, J= 2.8 Hz, 0.3H), 2.04-1.94 (m, 12H). A mixture of 59 and 59a (1.20 g, 1.74 mmol) and 60 (1.10 g, 2.61 mmol) was azeotroped in toluene (30 mL x 2) and dried under reduced pressure for 30 min. To that mixture in CH2CI2 (15 mL) was added activated 4Ά MS and stirred at room temperature for 30 min under inert atmosphere. Trimethylsilyl trifluoromethanesulfonate (O.Olg, 0.05 mmol) in CH2CI2 (1.0 mL) was added drop wise at -60°C. The reaction mixture was stirred at -40 °C for 2 h and allowed to stir at room temperature slowly for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with Et₃N diluted CH2CI2 (10 mL) and filtered through celite pad. The filtrate was concentrated and purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 61 and 61a (0.96 g, 58 %) as a white solid. ESI MS: m/z 964.3 [M+l8]⁺

To a stirred solution of 61 and 61a (1.1 g, 1.05 mmol) in ethylacetate (15 mL), was added Pd(OH)2/C (100 mg) and the reaction mixture was stirred under EL bladder at room temperature for 3 h. After completion of the reaction (TLC monitored), the reaction mixture was filtered through celite pad and dried under vacuum to obtain compound 62 and 62a (0.7 g, 78%) as a white solid. The crude compound was directly used in the next step. ESI MS: m/z 786.2 [M+l8]⁺ ¾NMR (400 MHz, CDCh): d 5.36- 5.30 (m, 1H), 5.22-4.90 (m, 7H), 4.52-4.69 (m, 2H), 4.26- 4.15 (m, 3H), 3.97-3.33 (m, 11H), 2.20-2.17 (m, 5H), 2.10-2.09 (m, 3H), 2.06-2.0l(m, 13H).

To a stirred solution of 62 and 62a (0.7 g, 0.91 mmol) in pyridine (15 mL), was added AC2O (0.86 mL, 9.11 mmol,) and the reaction mixture was stirred at room temperature for 16 h. The mixture was azeotroped with toluene (30 mL x 2) followed with CH2CI2 (30 mL x 2) and dried under vacuum. The crude compound was purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 63 and 63a (0.71 g, 87%) as a white solid. ESI MS: m/z 912.3 [M+l8]⁺

To a stirred solution of 63 and 63a (0.20 g, 0.22 mmol) in CH2CI2 (5 mL) at 0 °C, was added HBr in acetic acid (3 mL) in a drop-wise manner over a period of 30 min. After complete addition, the reaction mixture was warmed to room temperature and stirred for 4 h. After completion of the reaction (by TLC), the reaction mixture was quenched with ice cooled water and extracted with CH2CI2 (3 ^c 30 mL). The combined organic layer was washed with saturated bicarbonate solution (2 ^c 50 mL), dried (Na2S0₄) and concentrate under reduced pressure to obtain compound 64 and 64a (0.16 g) as a light yellow solid. The crude product was directly used in the next step. ESI MS: m/z 932.1 [M+l8]⁺

CC-00278 CC-00432 To a stirred solution of 65 (2.0 g, 2.48 mmol) in acetic anhydride (3.5 mL, 37.3 mmol) was added DMAP (cat) and trimethylamine (1.04 mL, 7.44 mmol) at room temperature. The reaction mixture was stirred at 60 ^°C for 3 h. After completion of the reaction (TLC monitored), the reaction mixture was cooled to room temperature, quenched with water and extracted with CH2CI2 (3 x 10 mL). The combined organic layer was separated, dried over (Na2S0₄) and concentrated under reduced pressure. The residue was dissolved in MeOH (10 mL) and acidified with 1% aq HC1 (3 mL) was added drop wise. The reaction mixture was stirred for 4 h and basified with 2 M KOH to get the pH to 4-5. The solvent was evaporated under reduced pressure to afford the compound 66 (700 mg, 23%) as a white solid. ¾ NMR (400 MHz, CDCh): d 5.25- 4.80 (m, 10H), 4.51 (d, J= 7.6Hz, 1H), 4.43 (dd, 7=12.4, 4.4 Hz, 1H), 4.18 (dd, 7=12.4, 6.0 Hz, 1H), 4.12-3.98 (m, 4H), 3.94-3.81 (m, 2H), 3.73-3.63 (m, 2H), 3.55-3.48 (m, 1 H), 2.23-1.98 (m, 32H), 1.98-1.72 (m, 5H), 1.68-1.43 (m, 11H), 1.23 (s, 3H), 1.16-1.09 (m, 1H), 1.02 (s, 3H), 0.99- 0.95 (m, 1H).

To a solution of 66 (650 mg, 0.53 mmol) and 64 and 64a (580 mg, 0.63 mmol) in CH2CI2 (20 mL), H2O (4 mL), were added K2CO3 (293 mg, 2.12 mmol), TBAB (17.10 mg, 0.05 mmol) at room temperature and stirred in sealed tube at 60 ^°C for 16 h. After completion of the reaction (TLC monitored), the reaction mixture was quenched with water (20 mL) and extracted the compound with CH2CI2 (3 x 10 mL). The organic layer was dried over (Na2S0₄) and concentrated under reduced pressure. The crude compound was purified by silica gel chromatography (70-80 % ethyl acetate in hexanes) to afford 67 and 67a (410 mg, 37%) as a white solid. ESI MS: m/z 2076.75 [M+l8]⁺

To a stirred solution of 67 and 67a (970 mg, 0.47 mmol) in MeOH (5 mL) NaOMe (2.54 mg, 0.04 mmol) was added at 0^°C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (TLC monitored), the reaction mixture was concentrated under reduced pressure. The crude compound was purified by prep. HPLC (Method A, Example 4) to afford CC-00278 (46 mg) and CC-00432 (11 mg) as a white puffy solid. UPLC MS: m/z 1259.71 [M-l] ¹HNMR (400 MHz, CD3OD) (CC-00432): d 5.43(d, 7 = 8.4 Hz, 1H), 5.16 (brs, 1H), 5.02-4.95 (m, 1H), 4.87-4.80 (m,3H), 4.77-4.62 (m, 5H), 4.30- 4.26 (m, 1H), 4.00 (t, 7 = 8.8 Hz, 1H), 3.92-3.67 (m, 6H), 3.60-3.23 (m, 18H), 3.19-3.13 (m, 5H), 2.90 (t, 7= 8.8 Hz, 1H), 2.15-1.65 (m, 8H), 1.60-1.42 (m, 3H), 1.41-1.27 (m, 3H), 1.23-1.17 (m, 2H), 1.14 (s, 3H), 1.05-0.98 (m, 2H), 0.89-0.85 (s, 3H), 0.81-0.72 (m, 2H). HPLC 95.27% (AUC), (Method B), Rt = 14.29 min.UPLC MS: m/z 1260.08 [M-l]

A 5 mg of sample of CC-00432 was dissolved in methanol-d₄ and a suite of 1D and 2D homonuclear and heteronuclear experiments was acquired on the sample utilizing a Bruker Avance III HD spectrometer operating at a nominal proton frequency of 500.13 MHz. Chemical shifts were referenced to the residual methanol signal, 3.31 ppm, in the proton spectrum and 49.0 ppm in the carbon spectrum.

The ¾ and ¹³C assignments are provided in Table 26:

Table 26: ³H and ¹³C NMR assignments of CC-00432

Sugar Position ³H Chemical Shift ¹³C Chemical Shift

1 1.89 (m), 0.87 (m) 41.1

2 2.15 (m), 1.45 (m) 20.2

3 2.08 (d), 1.08 (m) 39.1

4 45.1

5 1.08 (d) 58.3

6 1.97 (m), 1.85 (m) 24.4

7 1.58 (m), 1.42 (m) 43.2

8 41.8 9 0.96 (d) 55.3

10 40.6

11 1.77 (m), 1.62 (m) 20.6

12 2.18 (m), 1.48 (m) 39.0

13 89.5

14 2.23 (d), 1.57 (d) 44.0

15 2.10 47.1

16 152.6

17 5.25 (s), 4.81 (s) 105.4

18 1.24 (s) 28.5

19 178.7

20 0.97 (s) 17.2

Glc l 1 5.53 (d), ³JHH=8.3 HZ 95.3

2 4.00 (t) 76.7

3 4.38 (t) 87.8

4 3.45 (t) 69.9

5 3.45 (m) 78.3

6 3.81,3.67 (m) 62.2

Glc II r 4.79 (d), ³JHH=7.9 HZ 96.0

2 3.48 (t) 80.9

3’ 4.10 (f) 87.6 4’ 3.40 (t) 70.6

5’ 3.27 (m) 77.9

6 3.79 (m), 3.66 62.8

Glc III 1 4.75 (d), ³JHH=7.7 Hz 104.8

2 3.35 (t) 75.8

3 3.33 (t) 78.3

4 3.01 (t) 72.9

5 3.29 (m) 78.7

6 3.85, 3.57 63.9

Glc IV 1 4.78 (d), ³JHH=8.0 Hz 104.0

2 3.28 (t)

3

4 3.28 (t) 71.6

5 3.54 (m) 77.9

6 3.93, 3.62 62.8

Glc V 1 5.09 (d), ³JHH=7.9 HZ 103.8

2 3.37 (t) 75.9

3

4 3.25 (t) 73.2

5 3.33 (m) 75.8

6 3.94, 3.64 63.9

Xyl I 1 4.82 (d), ³JHH=7.9 Hz 104.7

2 3.28 (t) 75.3

3 3.28 77.8

4 3.54 71.0

5 3.91, 3.42 67.2

It was determined that CC-00432 is [(l3-[(2-0-P-D-glucopyranosyl-3-0-P-D- glucopyranosyl-P-D-glucopyranosyl)oxy] c///-kaur- 16-en- 19-oic acid-[(2-0-P-D- glucopyranosyl-3-0-P-D-xylopyranosyl-P-D-glucopyranosyl) ester]. The molecule contains six sugar units, five glucose and one xylose units. Each sugar unit exists as the b-anomer based upon a large ³JHH coupling constant (> 7 Hz) for the respective anomeric protons.

EXAMPLE 13: SENSORY ANALYSIS

The following samples were evaluated by 5 panelists trained in sweetener evaluation:

Samples were evaluated using a single sip protocol as follows:

• Samples were served at approximately 4°C

• Panelists were instructed to take 1 sip of the sample, hold in mouth for 5 seconds, expectorate, and rate the given attributes

• A 5 minute break was placed between each sample and panelists were instructed to cleanse their palates with at least 1 bite of unsalted cracker and 2 sips of filtered water

• Due to limited sample quantity panelists were given 7 mL aliquots of each sample

• Samples were randomized within each session for each panelist

• All samples were presented in replicate in each session

Samples evaluated for (1-10 scale):

• Sweet taste intensity: maximum level of sweetness in mouth during 5 seconds

• Bitter taste intensity: maximum level of bitterness in mouth during 5 seconds

• Overall maximum sweet intensity: maximum sweet intensity experienced from the time the sip is taken up to 1 minute

• Overall maximum bitter intensity: maximum sweet intensity experienced from the time the sip is taken up to 1 minute

• Sweet linger intensity: sweet intensity 1 minute after tasting the sample Bitter aftertaste intensity: bitter intensity 1 minute after tasting the sample

An ANOVA, with Sample as fixed effects and Panelist and interaction as random effects was used to determine significance between the samples for each attribute at the 95% Confidence Level, two-tailed. Fishers LSD was used to determine significant differences between mean scores.

Results:

Table 1 : Means table for CC-00392 and CC-00393 compared to Reb M 95% at 400 ppm in water at 4°C

Figure 1 shows a comparison of each attribute of CC-00392 and CC-00393 compared to Reb M. No significant differences were found for the attributes tested at 95% Confidence Level (CL).

Table 2: Means table for CC-00404 & CC-00405 compared to Reb M 95% at 400 ppm in water at 4°C

* Attribute was significant at the 95% CL (p < 0.05)

** Attribute was significant at the 90% CL (p < 0.10)

A 3-way ANOVA (Panelist, Sample, Panelist *Sample) was used to compare the sweeteners for each attribute at p < 0.05* and p <0.10**

Within a row, means with a different uppercase letter beside them are significantly different at either p <0.05* or p <0.10**

Compared to Reb M 95%, CC-00405 was not perceived as significantly different for the attributes tested. Compared to Reb M 95%, CC-00404 was significantly lower in: Sweet Intensity in Mouth, Overall Max Sweetness Intensity, and Sweet Linger Intensity at the 95% CL.

Table 3: Means table for CC-00422, CC-00423, & CC-00424 compared to Reb M 95% at 400 ppm in water at 4°C

*Within a column, means with a different uppercase letter beside them are significantly different at p < 0.05

Compared to Reb M 95%, CC-00422 was not perceived as significantly different for the attributes tested. Compared to Reb M 95%, CC-00423 was significantly lower in: Sweet Intensity in Mouth, Overall Max Sweetness Intensity, and Sweet Linger Intensity at 95% CL. Compared to Reb M 95%, CC-00424 was significantly lower in: Sweet Intensity in Mouth, Overall Max Sweetness Intensity and Sweet Linger Intensity at 95% CL. Compared to Reb M 95%, CC-00424 was significantly higher in: Bitter Intensity in Mouth at 95% CL.

Table 4: Means table for CC-00428 compared to Reb M 95% at 400 ppm in water at 4°C

CC-00432 was determined to have 7-8% sucrose equivalence.

EXAMPLE 14: PREPARATION AND CHARACTERIZATION OF CC-00375

Preparation of 2 (IN-AYR-A-38-1):

To a stirred solution of 1 (1.2 g, 3.5 mmol) in pyridine (10 mL), was added AC2O (4.97 mL, 52.5 mmol) and the reaction mixture was stirred at room temperature for 16 h. The mixture was azeotroped with toluene (30 mL ^c 2) followed with CH2CI2 (30 mL ^c 2) and dried under vacuum to obtain compound 2 (1.5 g, 63%, IN-AYR-A-38-1) as a white solid. The crude compound was directly used in the next step. ESI MS: m/z 696 [M+NH₄]⁺; ¾ NMR (300 MHz, CDCh): <76.32 (d, 7= 3.3 Hz, 1H), 5.47 (t, J= 9.6 Hz, 1H), 5.31 (t, 7= 9.6 Hz, 2H), 5.12-5.03 (m, 3H), 4.92 (dd, J= 10.2, 3.3 Hz , 1H), 4.31-4.04 (m, 5H) ,3.95 (dd, J= 10.2, 3.6 Hz, 1H), 2.22 (s, 3H), 2.12 (s, 3H), 2.08 (s, 6H), 2.05 (s, 6H), 2.01 (s, 3H), 1.98 (s, 3H).

Preparation of 3 (IN-AYR-A-39-1):

To a stirred solution of 2 (1.5 g, 2.21 mmol) in CH2CI2 (30 mL) at 0 °C, was added HBr in acetic acid (3 mL) in a drop-wise manner over a period of 30 min. After complete addition, the reaction mixture was warmed to room temperature and stirred for 4 h. After completion of the reaction (by TLC), the reaction mixture was quenched with ice cooled water and extracted with CH2CI2 (3 x 30 mL). The combined organic extract was washed with saturated bicarbonate solution (2 ^c 50 mL), dried (Na2S0₄) and concentrate under reduced pressure to obtain compound 3 (1.4 g, 80%, lot # IN-AYR-A-39-1) as a light yellow solid. The crude product was directly used in the next step. ¾ NMR (300 MHz, CDCh): 6.44 (d, J= 3.6 Hz, 1H), 5.52 (t, J= 9.6 Hz, 1H), 5.38 (t, j= 9.6 Hz, 1H), 5.23 (d, j= 3.6 Hz, 1H), 5.06 (q, j= 9.6 Hz , 2H), 4.79 (dd, j= 10.2, 3.6 Hz, 1H) ,4.39-4.26 (m, 2H), 4.20 (d, J= 3.0 Hz, 2H), 4.14-4.02 (m, 2H), 3.85 (dd, J= 9.6, 3.6 Hz, 1H), 2.12 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.02 (s, 6H).

Preparation of 5 (IN-AYR-A-16-1):

To a stirred solution of 4 (3.5 g, 4.35 mmol) in acetic anhydride (6.2 mL, 65.3 mmol) was added DMAP (cat) and Et₃N (1.8 mL, 13.05 mmol) at room temperature and the reaction mixture was stirred at 60 °C for 3 h. After completion of the reaction (by TLC), the reaction mixture was cooled to room temperature, quenched with water and extracted with CH2CI2 (3 x 10 mL). The organic extract was separated, dried (Na2S0₄) and concentrated under reduced pressure. The residue was dissolved in MeOH (10 mL) and acidified with 1% aq HC1 (3 mL). The reaction mixture was stirred for 4 h and basified with 2 M KOH to reach the pH 4-5. The solvent was evaporated under reduced pressure to obtain compound 5 (4 g, 75%, lot # IN-AYR-A-16-1) as a white solid. ¾ NMR (400 MHz, CDCh): <75.25-4.80 (m, 10H), 4.51 (d, J= 7.6Hz, 1H), 4.43 (dd, 7 = 12.4, 4.4 Hz, 1H), 4.18 (dd, 7 = 12.4, 6.0 Hz, 1H), 4.12-3.98 (m, 4H), 3.94-3.81 (m, 2H), 3.73-3.63 (m, 2H), 3.55-3.48 (m, 1 H), 2.23-1.98 (m, 32H), 1.98-1.72 (m, 6H), 1.68-1.50 (m, 3H), 1.50-1.43 (m, 3H), 1.34-1.25 (m, 2H), 1.23 (s, 3H), 1.16-1.09 (m, 1H), 1.02 (s, 3H), 0.99-0.95 (m, 1H), 0.91-0.82 (m, 2H).

Preparation of 6 (IN-AYR-A-40-1):

To a stirred solution of 5 (0.75 g, 0.612 mmol) and 3 (0.52 g, 0.73 mmol) in CH2CI2 (5 mL), H2O (1 mL), were added K2CO3 (0.34 g, 2.45 mmol) and TBAB (0.02 g, 0.06 mmol) at room temperature. The reaction mixture was stirred at 60 °C for 16 h. After completion of the reaction (by TLC), the reaction mixture was quenched with water and extracted with CH2CI2 (3 x 30 mL). The organic extract was separated, dried (Na2S0₄) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (70 - 80 % ethyl acetate in hexanes) to obtain compound 6 (450 mg, 40%, AMRI lot # IN-AYR-A-40-1) as a white solid; ¾ NMR (400 MHz, CD3OD): d 5.82 (d, ,7=8.0 Hz,IH), 5.36 (t, J= 9.6 Hz, 1H), 5.23 -4.8l(m, 16H), 4.78-4.72 (m, 3H), 4.62 (d, 7 =8.0 Hz, 1H), 4.38-3.58 (m, 15H), 2.12-1.85 (m, 55H), 1.82-1.31 (m, 12H), 1.12 (s, 3H), 1.07-0.89 (m, 3H), 0.84 (s, 3H), 0.83-0.72 (m, 1H).

Preparation of CC-00375 (Target-11, IN-AYR-A-43-2):

To a stirred solution of 6 (450mg, 0.244 mmol) in MeOH(3 mL) NaOMe (2 mg, 0.024 mmol) was added at 0 °C and stirred for 5 min. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction ( by TLC), the solvent was removed under reduced pressure. The residue was triturated with n-pentane (2 ^ 5 mL), the solvent was decanted and dried under reduced pressure to obtain crude CC-00375. The residue was purified by preparative HPLC (Method A) to afford CC-00375 (90 mg, 32%, AMRI lot # IN-AYR-A-43-2) as a white fluffy solid; UPLC-MS m/z 1128 [M-H]⁺; ¾ NMR (400 MHz, CDCh): d 5.55 (d, 7 = 8.0 Hz, 1H), 5.15(s, 1H), 5.04 (d, J= 4.0 Hz, 1H), 4.55 (dd, 7=12.4, 7.6 Hz, 2H), 4.02-3.95 (m, 1H), 3.80-3.4l(m, 17H), 3.32-3.23 (m, 10H), 3.19-3.11 (m, 4H), 2.16-1.69 (m, 10H), 1.58-1.30 (m, 6H), 1.17 (s, 3 H), 1.02-0.90 (m, 3H), 0.85 (s, 3H), 0.79-0.73 (m, 1H); HPLC 99.4% (AUC), (Method B), Rt = 27.85 min.

A) Preparative HPLC Method:Gemini@l0pm NX (250 x 4.6 mm, 10 pm); mobile phase,

A= H2O and B= CH3CN; Flow rate: 32 mL/min, Injection volume: 500 pL, Runtime: 40 min, gradient: 10-80%A, 20-90% B (0.0-58 min); UV detection at 210 nm) B) HPLC Method: Phenomenex Hydro RP (250 x 4.6 mm, 5 mm); mobile phase, A= H2O with 0.0284% MBOAc and 0.0116% Acetic acid and B= CH3CN; gradient: 15-70% B (0.0-58 min); UV detection at 210 nm.

A series of NMR experiments including ¾NMR, ¹³C NMR, ¾-¾ COSY, HSQC- DEPT, HMBC, ROESY, and 1D TOCSY were acquired to allow assignment of CC-00375. The sample was prepared by dissolving 3.9 mg in 150 pL of CD30D and NMR data were acquired on a Bruker Avance 500 MHz instrument equipped with a 2.5 mm inverse probe. A summary of the ¾ and ¹³C chemical shifts for the aglycone are found in Table 1. A summary of the 'H and ¹³C chemical shifts for the glycoside at C-19 are found in Table 2. A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-13 are found in Table 3.

Table 1. ¾ and ¹³C NMR (500 and 125 MHz, CD3OD), assignments of the

CC-00375 aglycone.

Resonance obscured by CD3OD, assignment based on HSQC-DEPT data. ¾esonance overlapped with Glcm H-l, assignment based on HSQC- DEPT data.

Table 2. ¾ and ¹³C NMR (500 and 125 MHz, CD3OD), assignments of the

CC-00375 C- 19 glycoside.

^§Four carbon resonances in the range of 78.2-78.3 ppm (78.18, 78.24, 78.26 and 78.30), hence chemical shifts could not be unequivocally assigned.

^Three carbon resonances in the range of 62.6-62.8 ppm (62.57 and 62.79 ppm; two carbons overlapped at 62.79 ppm), hence chemical shifts could not be unequivocally assigned.

Table 3. ¾ and ¹³C NMR (500 and 125 MHz, CD3OD), assignments of the

CC-00375 C-13 glycoside.

§Four carbon resonances in the range of 78.2-78.3 ppm (78.18, 78.24, 78.26 and

78.30), hence chemical shifts could not be unequivocally assigned.

¾lcm H-l and H-17 at 4.86 ppm are partially overlapped.

The ESI-TOF mass spectrum acquired by infusing a sample of CC-00375 showed a [M-H] ion at m/z 1127.4707. The mass of the [M-H] ion was in good agreement with the molecular formula CsoHsoChs (calcd for C50H79O28: 1127.4758, error: -4.5 ppm) expected. The MS data confirmed that CC-00375 has a nominal mass of 1128 Daltons with the molecular formula, C50H80O28. The ion observed at m/z 1225.4410 is most likely due to [M-H+H3PO4] .

The MS/MS spectrum of CC-00375, selecting the [M-H] ion at m/z 1127.0 for

fragmentation, indicated sequential loss of five glucose units at m/z 965.5359, 803.3687, 641.2654, 479.2701 and 317.2029. The data thus indicated the presence of five glucose units in the structure.

It was determined that CC-00375 was l3-[((2-0- -D-glucopyranosyl-3-0- -D- glucopyranosyl)- -D-glucopyranosyl)oxy] c///-kaur- 16-en- l 9-oic acid-[(2-0-a-D-glucopyranosyl)- b-D-glucopyranosyl) ester]

EXAMPLE 15: PREPARATION AND CHARACTERIZATION OF CC-00376

Preparation of 2 (IN-VKT-B-25-2):

To a stirred solution of 1 (2.0 g, 2.10 mmol) in methanol (20 mL) was added NaOH (1.68 g, 42.06 mmol) in one lot at room temperature and stirred at 70 °C for 4 h. After completion of the reaction ( by TLC), the reaction mixture was cooled to room temperature, acidified with 1 N HC1 (P^H 4.0) at 10 °C. The solvent was evaporated at 50 °C to get the crude residue. The crude residue was extracted with n-butanol (3 ^c 50 mL). The combined organic extract was washed with water and separate the organic extract, dried (Na2S0₄), concentrated on reduced pressure. The crude residue was azeotroped with MeOH and concentrated under reduced pressure to afford 2 (1.2 g, 75%, lot # IN-VKT-B-25-2) as a white solid. The crude product was directly used in the next step. ESI MS: m/z 811.3 [M+Na]⁺; ¾ NMK (400 MHz, DMSO-de): S 11.96-11.76 (brs,

1H), 5.29 (s, 1H), 5.16 (s, 2H), 5.06-4.99 (m, 2H), 4.74 (s, 2H), 4.65 (s, 1H), 4.54 (m, 2H), 4.45 (m, 1H), 4.32-4.30 (m, 2H), 4.08 (m, 1H), 3.98 (t, J= 11.6 Hz, 1H), 3.72 (m, 2H), 3.64-3.58 (m, 2H), 3.38 (m, 1H), 3.40-3.37 (m, 2H), 3.24-3.15 (m, 4H), 3.04 (m, 2H), 2.11-1.95 (m, 4H), 1.81- 1.68 (m, 6H), 1.61-1.58 (d, J= 14.8 Hz, 2H), 1.50-1.46 (m, 2H), 1.36-1.25 (m, 4H), 1.10-1.06 (m, 6H), 1.02-0.98 (m, 1H), 0.91-0.83 (m, 6H).

Preparation of 3 (IN-VKT-B-30-1):

To a stirred solution of 2 (1.0 g, 1.26 mmol), was added AC2O (l .28g, 12.69 mmol) and the reaction mixture was stirred at room temperature for 16 h. The mixture was azeotroped with toluene (30 mL x 2) followed with CH2CI2 (2 x 30 mL) and dried under vacuum to obtain crude residue. The crude residue was purified by silica gel chromatography ( 40-50 % ethyl acetate in hexanes) to afford 3 (270 mg, 18%, lot # IN-VKT-B-30-1) as a pale yellow solid. ESI MS: m/z 1165.3 [M-H]⁺; ¾ NMR (400 MHz, CDCh): d 5.36 (dd, J= 1.2, 4.0 Hz, 1H), 5.31 (s, 1H), 5.29 - 5.24 (m, 2H), 5.13 (brs, 1H), 5.11-5.06 ( m, 2H), 4.91 (brs, 1H), 4.85-4.76 (m, 2H), 4.72 (d, J = 8.0 Hz, 1H), 4.59 (d, J= 8.0 Hz, 1H), 4.46 (dd, 7= 4.0, 12.8 Hz, 1H ), 4.40-4.36 (m, 1H), 4.15- 3.98 (m, 5H), 3.79-3.73 (m, 2H), 3.63-3.57(m, 1H), 2.22 (s, 3H), 2.20-2.13 (m, 2H), 2.10-2.01 (m, 24H), l .95(s, 3H), 1.88-1.73 (m, 5H), 1.65-1.54 (m, 4H), 1.47-1.44 (m, 1H), 1.27-1.21 (m, 4H), 1.16 (d, J= 3.2 Hz, 3H), 1.09-1.04 ( m, 1H), 1.02-0.98 (m, 2H), 0.96 (s, 3H), 0.86-0.83 (m, 2H).

Preparation of 5: (IN-VKT-B-60-1):

To a solution of 3 (200 mg, 0.17 mmol) and 4 (143.6 mg, 0.20 mmol) in CH2CI2 (10 mL), H2O (2 mL), were added K2CO3 (94.68 mg, 0.68 mmol), TBAB (5.52 mg, 0.01 mmol) at room temperature. The reaction mixture was stirred at 60 °C for 16 h. After completion of the reaction, the reaction mixture was quenched with water and extracted with CH2CI2 ( 3 x 30 mL). The organic layer was separated, dried over (Na2S0₄) and concentrated under reduced pressure. The residue was purified by silica gel chromatography (70 - 80 % ethyl acetate in hexanes) to afford compound 5 (110 mg, 43%, lot # IN-VKT-B-60-1) as a white solid; ¾ NMR (400 MHz, CD3OD): d 5.70 (d, J= 7.6 Hz, 1H), 5.39 (s, 1H), 5.33-5.28 (m, 3H), 5.24-5.19 ( m, 4H), 5.01 (t, J= 9.6 Hz, 1H), 4.96 (m, 4H), 4.69-4.62 (m, 3H ), 4.42-4.33 (m, 2H), 4.22-3.97 (m, 9H), 3.94-

3.88 (m, 3H), 3.74-3.66 (m, 2H), 3.55 (t, J= 8.4 Hz, 1H), 2.10 (s, 3H), 2.07-2.05 ( m, 3H), 2.01-

1.89 (m, 39H), 1.87 (m, 3H), 1.83 (m, 4H), 1.80-1.68 (m, 2H), 1.61-1.57 (m, 2H), 1.48-1.35 (m, 3H), 1.19(s, 3H), 1.16-1.12 (m, 5H), 1.06 (d, J= 6.0 Hz, 3H), 1.04 - 0.99 (m, 2H), 0.93 (d, J = 7.6 Hz, 1H), 0.81- 0.77 (m, 5H).

Preparation of CC-00376 (IN-VKT-B-62-3):

To a stirred solution of 5 (110 mg, 0.06 mmol) in MeOH (2 mL) was added NaOMe (0. leq) at 0 °C under inert atmosphere. The reaction mixture was stirred at room temperature for 2 h. After completion of the reaction (by TLC), the solvent was removed under reduced pressure to afford crude residue. The crude residue was triturated with n-pentane (2 x 5 mL), the solvent was decanted and dried under reduced pressure to get the crude residue. The crude residue was purified by preparative HPLC (Method A- same as Example 14) to afford CC-00376 (80 mg, 77%, AMRI lot # IN-VKT-B-62-3) as a white pluffy solid; UPLC-MS : m/z 1111.91 [M⁺-l]_; ¾ NMR (400 MHz, CD₃OD): d 5.10 (d, 7= 8.0 Hz, 1H), 5.34 (s, 1H), 5.05-5.04 (m, 2H), 4.75-4.72 ( m, 3H), 4.52 (d, J= 7.6 Hz, 1H), 4.40 (d, J= 7.6 Hz, 1H), 4.03-3.97 (m, 2H), 3.86-3.24 (m, 25H), 3.17-3.11 (m, 3H), 2.13-2.10 (m, 2H), 2.05 (s, 1H), 1.95-1.68 (m, 6H), 1.57-1.43 (m, 2H), 1.37-1.30 ( m, 2H),l. l6 (s, 3H), 1.11 (d, J= 6.4 Hz, 3H), 1.00-0.95 (m, 2H), 0.90-0.86 (m, 3H), 0.81- 0.72 (m, 1H); HPLC 99.24% (AUC), (Method B- same as Example 14), Rt= 39.52 min

A series of NMR experiments including ¹H NMR, ¹³C NMR, ¾-¾ COSY, HSQC- DEPT, HMBC, ROESY, and 1D TOCSY were acquired to allow assignment of CC-00375. The sample was prepared by dissolving 2.3 mg in 150 pL of pyridine-ds and NMR data were acquired on a Bruker Avance 500 MHz instruments equipped with a 2.5 mm inverse probe and 5 mm broad band probe. and NMR data were acquired on a Bruker Avance 500 MHz instrument equipped with a 2.5 mm inverse probe. A summary of the ¾ and ¹³C chemical shifts for the aglycone are found in Table 1. A summary of the ¾ and ¹³C chemical shifts for the glycoside at C-19 are found in Table 2. A summary of the ¾ and ¹³C chemical shifts for the glycoside at C- 13 are found in Table 3.

Table 1. ¾ and ¹³C NMR (500 and 125 MHz, pyridine-i¾), assignments of the

CC-00376 aglycone.

Table 2. ¾ and ¹³C NMR (500 and 125 MHz, pyridine-i¾), assignments of the CC-00376 C- 19 glycoside.

Table 3. ¾ and ¹³C NMR (500 and 125 MHz, pyridine-i¾), assignments of the

CC-00376 C-13 glycoside.

Assignment based on 1H NMR data acquired at 298K.

^§Two carbon resonances observed at 70.1 ppm are resolved at 70.05 and 70.14 ppm in

¹³C NMR spectrum.

The ESI-TOF mass spectrum acquired by infusing a sample of CC-00376 showed a [M- H] ion at m/z 1111.4786. The mass of the [M-H] ion was in good agreement with the molecular formula C50H80O27 (calcd for C50H79O27 : 1111.4809, error: -2.1 ppm) expected. The MS data confirmed that CC-00376 has a nominal mass of 1112 Daltons with the molecular formula,

C50H80O27. The ions observed at m/z 1147.4485 and 1209.4373 are most likely due to [M-H+Cl]^' and [M-H+H3PO4]^', respectively.

The MS/MS spectrum of CC-00376, selecting the [M-H] ion at m/z 1111.5 for

fragmentation, indicated sequential loss of three glucose units at m/z 949.4459, 787.3648 and 625.3024 followed by loss of one rhamnose unit at m/z 479.2807 and loss of one glucose unit at m/z 317.2115. Alternative fragmentation pathway is also observed in the spectrum. For example, the ion at m/z 641.3145 would correspond to loss of one rhamnose from m/z 787.3648 followed by sequential loss of glucose units at m/z 479.2807 and 317.2115. The data thus indicated the presence of four glucose units and one rhamnose unit in the structure.

It was determined that CC-00376 was l3-[((2-0-a-L-rhamnopyranosyl-3-0- -D- glucopyranosyl)- -D-glucopyranosyl)oxy] _<3///-kaur- 16-en- l 9-oic acid-[(2-0-a-D- glucopyranosyl)- -D-glucopyranosyl) ester] . EXAMPLE 16: SENSORY EVALUATION OF CC-00375

The following samples were evaluated by seven panelists trained in sweetener evaluation:

Samples were evaluated using a single sip protocol as described above in Example 13. Data analysis was also conducted as described in Example 13.

Results:

Means table for CC-00375 compared to Reb M 95% at 400 ppm in water at 4 °C

*A 3-way ANOVA (Panelist, Sample, Panelist *Sample) was used to compare the sweeteners for each attribute at p < 0.05

*Within a row, means with a different uppercase letter beside them are significantly different at p < 0.05

EXAMPLE 17: SENSORY ANALYSIS OF CC-00376 The following samples were evaluated by six panelists trained in sweetener evaluation:

Claims

CLAIMS We claim:

1. An isolated and purified diterpene glycoside selected from the following:

2. A composition comprising at least one isolated and purified diterpene glycoside of claim 1

3. The composition of claim 2, wherein the composition is selected from a sweetener

composition, a sweetness enhancing composition and a flavor enhancing composition.

4. The composition of claim 2, further comprising at least one compound selected from a sweetener, additive and functional ingredient.

5. A consumable comprising at least one diterpene glycoside of claim 1.

6. The consumable of claim 5, wherein the consumable is a beverage or beverage product.

7. The consumable of claim 6, wherein the beverage is selected from the group consisting of enhanced sparkling beverages, cola, lemon-lime flavored sparkling beverage, orange flavored sparkling beverage, grape flavored sparkling beverage, strawberry flavored sparkling beverage, pineapple flavored sparkling beverage, ginger-ale, soft drinks, root beer, frozen carbonated beverage, fruit juice, fruit-flavored juice, juice drinks, nectars, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, enhanced water drinks, enhanced water with vitamins, near water drinks, coconut water, tea type drinks, coffee, cocoa drink, beverage containing milk components, beverages containing cereal extracts and smoothies.

8. The consumable of claim 7, wherein the beverage is an enhanced water beverage.

9. The consumable of claim 6, wherein the beverage is a low- or zero-calorie beverage.

10. The consumable of claim 6, wherein the pH of the beverage is from about 2.5 to about 4.

11. The consumable of claim 6, wherein the beverage comprises from about 1 to about 1,000 ppm of the at least one diterpene glycoside.

12. The consumable of claim 11, wherein the at least one diterpene glycoside is selected from the group consisting of CC-00375, CC-00376, CC-00392, CC-00393, CC-00404, CC- 00405, CC-00422, CC-00423 and CC-00428

13. The consumable of claim 12, wherein the beverage further comprises one or more of the following: water, natural cola flavor, a colorant comprising caramel, an acidulant and caffeine.

14. A method of preparing a target diterpene glycoside comprising

(a) protecting the hydroxyl groups of a starting diterpene glycoside having the following structure:

wherein R¹ and R² are each independently selected from hydrogen, monosaccharide and oligosaccharide, to provide a protected diterpene glycoside having the following formula:

wherein R³ and R⁴ are each independently selected from hydrogen, monosaccharide and oligosaccharide, and when R³ or R⁴ is a monosaccharide or oligosaccharide, the hydroxyl groups present thereon are also protected; (b) coupling the protected diterpene glycoside with a functionalized sugar having the following formula:

wherein R⁹, R¹⁰, R¹¹ and R¹² are each independently selected from hydrogen,

monosaccharide and oligosaccharide, and when R⁹, R¹⁰, R^{1 1} or R¹² is a monosaccharide or oligosaccharide, the hydroxyl groups present thereon are also protected,

to provide a protected, coupled diterpene glycoside of the following formula: