TITLE OF THE INVENTION
STABILIZED PEPTIDE SWEETENING AGENTS
BACKGROUND OF THE INVENTION Field of the Invention;
The present invention is related to peptide sweeteners for use in foods and beverages. The invention is more specifically related to low caloric, non-toxic, edible synthetic dipeptide sweeteners.
Description of the Background;
Much research effort has been directed within the last 15 years in an effort to achieve a low caloric synthetic sweetener composition which is stable against acid hydrolysis, has high thermal stability, is stable against intestinal enzymes such as chymotrypsin, and yet is generally recognized as safe for consumption by humans.
Although many substances have been proposed and/or synthesized for use as artificial sweetening agents toreplace sugar, most have not been successful. Either they suffer the disadvantage of a bitter aftertaste, they exhibit toxic side effects due to their inherent chemical structure when metabolized in the body of the
consumer, or they are unstable in storage and losetheir sweetness before use.
In 1969, it was reported in Journal of The American Chemical Society, Vol. 91, pages 2684-2691 that lower alkyl esters (especially the methyl ester) of L-aspartyl-L-phenylalanine had good sweetening properties, being approximately 100-200 times sweeter than sucrose. The methyl ester is commonly called Aspartame. However, this product has a serious disadvantage in that it is unstable in the presence of acids and heat, thereby losing its sweetness as it forms by-product diketopiperazines having an unpleasant taste.
In an effort to overcome these difficulties, Chibata et al. in United States Patent 3,971,822, granted in 1976, reported certain novel ester derivatives of N-aspartyl-aminoalkanol, some of which were said to be equal in sweetness to the esters of Laspartyl-L-phenylalanine earlier reported except that the compounds described in this patent were said to be heat stable. However, these compounds never became commercially accepted, apparently because of a poor taste.
In 1979, Lipinski et al. disclosed in United States Patent 4,158,068 a non-peptide sweetener called commercially "Acetosulfame-K." This compound is
currently in use in the United Kingdom but is reported to have also an aftertaste problem. A far greater problem in the United States is that the molecule is entirely new and has therefore no prior history of safe ingestion by consumers. As a practical matter this newness requires a long and difficult proceeding before the Food and Drug Administration before this product can be placed in commercial distribution.
In 1983, Grant E. Dubois described in his United States Patent 4,381,402 still other sweeteners, glycoside steriosides, which are essentially extracts from the leaf of a plant which grows in Paraguay and is quite sweet tasting. These sweet tasting glycosides however suffer from a problem of aftertaste and are not believed by principal users to be the answer to the problem of stability.
In March 1984, Joseph Tsau and James Young obtained United States Patent No. 4,439,460 on sulfate and sulfonate salts of Aspartame and related dipeptides. These salts are disclosed as being heat stable up to 170ºC and therefore suitable for use as synthetic sweeteners when dry blended with maltodextrins for baking cakes and pies.
Also of note in the prior art is a broad disclosure by Brennan and Hendricks reported in United States Patent 4,399,163 in August 1983. This patent
discloses thousands of compounds which are said to be sufficiently stable to be used for baking and cooking at elevated temperatures in the ranges of 300-350ºF. However, all of these compounds have untested and untried configurations, and the metabolic fate of the materials are hence uncertain as to side effects on the user.
Up to the time of the present discovery, therefore, none of the proposed artificial sweeteners had sufficient sweetness and stability to satisfactorily replace sugar in low-calorie foods and beverages without residual problems.
SUMMARY OF THE INVENTION
Accordingly, it is an object of this invention to provide an artificial sweetener having sufficient sweetness and stability to more satisfactorily replace sugar in low-calorie foods and beverages than artificial sweeteners previously available.
It is a particular object of this invention to provide artificial sweeteners having stability against heat, acid, and enzyme degradation, thereby providing sweeteners resistant to long-term storage. Such stabilized sweeteners, being resistant to enzymatic degradation and acid hydrolysis, also resist the production of undesirable by-products after
consumption, thereby lessening the possibility of undesirable and possibly unsafe reactions that might occur. These and other objects of the invention as will hereinafter become more readily apparent have been accomplished by providing a dipeptide sweetener, comprising a compound of the formula:
wherein
X is H, Li, Na, or K; n is 0, 1, or 2; m is 1, 2, 3, or 4;
R is (1) OR1 wherein R1 is a C1 C7 alkyl group; a C2-C7 alkenyl or alkynyl group; or said alkyl, alkenyl, or alkynyl group substituted with a C1-C4 alkoxyl group, a hydroxyl group, or a halogen atom with the proviso that no substitution occurs on C1 of R1; (2) N(R2)2 wherein each R2 independently represents H, an alkyl group containing at least 4 carbon atoms, or a 4-, 5- or 6-membered heterocyclic group containing one sulfur, oxygen, or nitrogen atom in the heterocyclic ring; or (3) R3 wherein R3 is R1 or -CH2R1; and
R' is H, halogen, or phenyl; or a pharmaceutically acceptable salt thereof.
The invention is also related to compositions containing compounds of the invention, such compositions also being referred to as dipeptide sweeteners or as sweetener compositions, depending on the context of the term. These compositions can also contain a stabilizing amount of an edible, food-grade stabilizing agent, which is typically a hydrocolloidal stabilizer, such as a polysaccharide gum. Compositions of the invention are particularly useful in preparing goods that will be baked as they can withstand baking temperatures and therefore make available readily produced dietetic baked goods.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention has two principle aspects;
(1) dipeptide compounds which themselves are sweet and are stabilized against acid and enzymatic hydrolysis by the presence of a cycloalkyl ring in the amino acid adjacent to the ester functionality and
(2) compositions containing these dipeptides that are further stabilized by the presence of an additional stabilizing agent.
Compounds of the invention are those having the formula:
wherein
X is H, Li, Na, or K; n is 0, 1, or 2; m is 1, 2 , 3, or 4;
R is (1) OR1 wherein R1 is a C1-C7 alkyl group; a C2-C7 alkenyl or alkynyl group; or said alkyl, alkenyl, or alkynyl group substituted with a C1-C4 alkoxyl group, a hydroxyl group, or a halogen atom with the proviso that no substitution occurs on C1 of R1; (2) N(R2)2 wherein each R2 independently represents H, an alkyl group containing at least 4 carbon atoms, or a 4-, 5-, or 6-membered heterocyclic group containing one sulfur, oxygen, or nitrogen atom in the heterocyclic ring; or (3) R3 wherein R3 is R1 or -CH2R1; and
R' is H, halogen, or phenyl; or a pharmaceutically acceptable acid-addition salt thereof.
These compounds are related in structure to Aspartame and its related compounds but differ in several significant ways, particularly in the use of a cyclic amino acid analog of either alanine or phenylalanine. This cyclic amino acid analog aids in the stabilization of compounds of the invention to both acid and enzymatic hydrolysis by a mechanism that is unknown in detail but that is believed to be related to
the presence of the cycloalkyl ring in the alpha position which hinder acid and enzymatic hydrolysis, perhaps by steric hinderance during transition state formation. Furthermore these compounds are stable to heat at normal baking temperatures for reasons that remain unknown.
Compounds falling within the scope of the present invention and equivalents thereof can readily be recognized by their sweetness. In order to provide a low-calorie sweetener, compounds having sweetness at least 50 times that of sucrose are preferred, with a sweetness at least 100 times that of sucrose being more preferred.
Preferred compounds of the invention are those in which X is H, Na, or K; n is 0 or 1; m is 1 or 2; and R is as defined above. Even more preferred are compounds in which n and m are both 1. Within these groupings, compounds in which R' is H are particularly preferred. When R' is not H, compounds in which the stereo-chemical configuration at the carbon to which R' is attached is the S configuration are preferred. The non-cyclic, di-basic amino acid residue represented by the left portion of the formula as shown is preferably from an L-amino acid in all compounds of the invention.
Although Aspartame contains a phenylalanine residue, which is related to compounds in which R'
represents a phenyl group, compounds in which R' represents a hydrogen are preferred since these compounds are analogs of alanine, a less toxic amino acid, and are also not chiral, which simplifies synthesis. When R' is H and m is 1, the cyclic amino acid residue is a residue of cyclopropylalanine (1-aminocyclopropane carboxylic acid), a known constituent of apples and other fruit. When R' is halogen, fluorine, chlorine, bromine, and iodine are preferred halogens.
Compounds in which R represents an ester group are generally preferred over compounds in which R represents an amide group, although these latter compounds can also be sweet. It has been determined through experimental work in the inventors laboratory that the compound in which X is H; n is 1; m is 1; R' is hydrogen and R is NHCH2CH2CH3 is tasteless. Such amides also appear to be tasteless in the Aspartame series. However, as in the present invention, U.S.
Patent 4,399,163 discloses that compounds of the formula Asp-D-Ser-NHR in which R represents an alkyl group or a heterocyclic group containing one sulfur atom in the heterocyclic ring with the alkyl or heterocyclic group containing at least four carbon atoms, are sweet. Accordingly, compounds of the invention having amides with similar bulky substituents
are likewise believed to be suitable as dipeptide amide sweeteners. Preferred dipeptide sweeteners of the invention are those of which R2 is -CHR3R4 in which R3 and R4 independently represent alkyl groups containing 2-5 carbon atoms or R3 and R4 together represent (1) a divalent alkyl group wherein -CHR3R4 represents a cycloalkyl group or (2) a divalent alkyl group containing a sulfur atom between the terminals of the divalent alkyl groups wherein -CHR3R4 represents a heterocyclic group. Particularly preferred are those compounds in which R3 and R4 independently represent propyl, isopropyl, or cyclopropyl groups as well as those compounds in which -CHR3R4 represents a 5-or 6membered cycloalkyl group or a 4-membered heterocyclic ring containing one sulfur atom in the ring. The sulfur in the heterocyclic ring can be either a divalent sulfur atom or an oxidized form of sulfur such as a sulfone or sulfoxide group.
When R represents the preferred OR1 group, R1 can represent a C1-C7 alkyl group; a C2-C7 alkenyl or alkynyl group; or said alkyl, alkenyl or alkynyl group substituted with a C1-C4 alkoxyl group, a hydroxyl group, or a halogen atom with the proviso that no substitution occurs on C1 of R1. Here and throughout this application, the word "alkyl" represents both cyclic and acyclic alkyl groups. Thus, an alkyl group
containing 5 carbons can be either a cyclopentyl group, an n-pentyl group, a 2-pentyl group, a 3-methyl cyclobutyl group, a cyclobutylmethyl group, a 1,1dimethylpropyl group, or any other 5-carbon-containing alkyl group. Since the various combinations of organizing the atoms within these small alkyl groups are well known to those skilled in the art and are readily available either in the form of alcohols or inthe form of precursors of alcohols that can be used to make esters of the invention, all such alkyl groups are individually contemplated as if each such compound were individually named in this application. For example, one skilled in the art would readily recognize that a cyclopropylmethyl group falls within the scope of this definition of R and that this invention contemplates such esters as if they had been individually named, even if the cyclopropylmethyl group had not been individually written out. Particularly preferred alkyl groups are those containing 2-5 carbon atoms, with those containing 3 or 4 carbon atoms being particularly preferred. Examples of specific alkyl groups (for those whose experience in organic chemistry is limited) are ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, cyclopropylmethyl, methylcyclopropyl (substituted at the 1 or 2 position), n-pentyl, iso-pentyl,
1-methylbutyl, cyclopentyl, cyclobutylmethyl, methylcyclobutyl (the methyl being at the 1, 2, or 3 position), and cyclopentyl groups.
The ester substituent may also contain a C-C double bond, a C-C triple bond, or an electronegative substituent, with the proviso that the electronegative substituent is not on the carbon attached to the oxygen of the ester (i.e., not on C1). The alkenyl and alkynyl groups can contain up to 7 carbons. Particularly preferred groups are the propargyl and allyl groups.
Electronegative substituents may be present on either the alkyl, alkenyl, or alkynyl groups, although electronegative substituents are preferably present on alkyl groups rather than alkenyl or alkynyl groups. Preferred electronegative substituents are C1-C4 alkoxyl groups, hydroxyl groups, and halogen atoms. Fluorine is a preferred halogen atom with chlorine and bromine being less preferred in that order. Examples of substituted esters include 2,2,2-trifluoroethyl, 2- methoxyethyl, 2,3-dihydroxypropyl, bis(hydroxymethyl)methyl, 2-hydroxyethyl, and 2,3- bis(methoxy)propyl. Preferred are esters in which R1 is -CH2R5 wherein R5 is methyl, ethyl, or propyl substituted with a C1-C4 alkoxyl group, a hydroxyl group, or a halogen.
Compounds which are ketones instead of esters are also encompassed by this invention when sweet. Such compounds have as the substituent R either R1 or CH2R1. The latter substituent is preferred as the -CH2- replaces -O- in the esters. Preferred ketones are those in which R1 of -CH2R1 is the same as a preferred R1 in an ester described above.
Another grouping of preferred compounds includes those compounds within the scope of the present disclosure but outside the scope originally claimed or specifically identified in U.S. Application Serial Nos. 636,091 and 677,901.
The following are examples of compounds of the invention:
Compounds of the invention can readily be synthesized using known techniques. Typically, the individual amino acids will be synthesized first and subjected to a condensation reaction in order to form the dipeptide. The free-amino-containing amino acid, as indicated by the general formula, is aminomalonic acid, aspartic acid, or glutamic acid. These compounds are readily available commercially. The amino acid containing the cycloalkyl group is available commercially or can be readily synthesized using known techniques. For example, previous applications in the present sequence, such as U.S. Patent Application 523,808, filed August 16, 1983, describe techniques of synthesizing these amino acids. U.S. Patent 4,298,760 also describes a process for preparing 1-aminocyclopropane carboxylic acid which can then be coupled to the second amino acid using standard techniques. Techniques for performing the coupling
reaction are also described in U.S. Patent Application 523,808.
One method of synthesizing any of the desired cycloalkyl amino acids or ketones used in the invention (other than the cyclopropane derivatives, which can be made as described above) is amination of the corresponding alpha-halo acid or ketone. The necessary alpha-halo acids or esters can be prepared by the HellVolhard-Zelinsky halogenation of the unsubstituted acid, all of which are available commerically (e.g., cyclobutane carboxylic acid, cyclopentane carboxylic acid, and cyclohexane carboxylic acid). Other standard reactions for preparing amino acids are the Gabriel phthalimide sythesis, which uses alpha-halo esters instead of alpha-halo acids, and the phthalimidomalonic ester method, which is a combined malonic acid-Gabriel sythesis. Halogenation of ketones is likewise a known process and can be used to prepare the necessary alphahalo ketones from commerically available starting materials.
Compounds in which R' is phenyl or halogen, as well as compounds in which R' is hydrogen, can be prepared from a ketone using a Strecker synthesis. For example, 2-phenylcyclohexanone can be reacted with KCN and ammonium carbonate to produce 2-phenyl- 1-cyanocyclohexylamine which is then hydrolyzed in acid
to produce 1-amino-2-phenylcyclohexane-1-carboxylic acid.
Compounds of the invention as described above are resistant to acid and enzymatic hydrolysis and to self condensation as a result of the presence of the cycloalkyl group. Accordingly, these compounds find use as stabilized dipeptide sweeteners. For examole, Aspartame and a comdound of the invention. were comparison tested for stability to enzymatic hydrolysis by alphachymotrypsin. After 15 minutes at room temperature, Aspartame had been hydrolyzed to give the acid and methanol while the compound of the invention failed to hydrolyze even after 24 hours. Thus, food products containing compounds of the invention have been demonstrated to have a sharp and distinct new property of resistance to peptide degradation by enzymatic hydrolysis and thus should avoid such side effects as behavior modification, hyperactivity, genetic changes, and brain tumors that have been indicated to be possible side effects of Aspartame resulting from metabolic derivatives of Aspartame. The acid resistance likewise is useful in preserving sweetness during storage, particularly in acid foods, such as carbonated beverages.
Compounds of the invention can be used in the same manner in which Aspartame and related compounds are now used. For example, compounds of the invention can be substituted for Aspartame in food and beverage compositions and in other types of sweet comestible products using known techniques. Examples of such compositions are set forth in U.S. Patent Application Serial No. 636,091, filed August 3, 1984.
Although compositions containing the sweeteners of the present invention are stabilized against heat, acid, and enzymes, further stabilization can be achieved against heat by including an ingestible polyhydroxypolyme'r, preferably a hydrocolloidal polysaccharide gum, in a composition containing the dipeptide compound of the invention. The polyhydroxypolymer is not an essential component for producing stability to baking temperatures. However, the extra stability produced by the use of such a material is believed to result from the formation of a polyhydroxypolymer ester of the dipeptide sweetener by a transesterif ication reaction. Hydrocolloidal gums are thus preferred because they are known ingestible polyhydroxy polymers and typically contain catalytic amounts of acidic substances. Since baked goods normally contain starches and other polysaccharides (which are polyhydroxypolymers), transesterification is
a possible reaction in any baked good even in the absence of an additional stabilizer. This proposed mode of action of the stabilizers is currently not known with scientific certainty, although empirical results do indicate increased stability as indicated. Thus, this invention is not limited by such a theoretical consideration.
These concurrent properties of continuous intense sweetness; stability in the face of acids, enzymes and high heat and acceptable food texture; and structural integrity at baking conditions makes these complexes quite valuable. Their principal uses are as low caloric sweeteners for candy formulations, syrups for carbonated beverages, and in non-carbonated beverage mixes,. baked goods compositions, processed vegetables, fruit and meat products, dessert toppings, gelatin foods and similar food products where the combination of properties are essential to a successful product.
Preferred compositions include those in which a hydrocolloidal polysaccharide gum comprises a majority of the composition with the remainder being the compound of the invention, optionally mixed with other sweeteners or with binders, flavoring, colorings, or the like. Gum:peptide ratios are preferred to be in the range of from 100:1 to 2:1, with compositions in the range of 20:1 to 5:1 being preferred with a ratio
of approximately 10:1 being most preferred. Although these two components can be present in the presence of other materials (such as cake mixes and other solid materials described later in more detail), one preferred embodiment of the invention comprises a sugar-like sweetener that can be used as a dry sweetening composition and that consists essentially of the two components, optionally containing a binding or drying agent. In such a dry sweetening agent, the composition will preferably consist essentially of the two components:
A - from about 1 to 33 parts by weight of a stabilized dipeptide of the invention
B - from 99 to 67 parts by weight of a hydrocolloidal polysaccharide gum, especially gum tragacanth, gum acacia, pectin, gum karaya, psyllium seed gum, larch gum, gum gatti, guar gum, locust bean gum, carrageenan, or agar.
Component A of such mixtures consists of the dipeptides sweeteners whose structure and composition has been previously described in this application.
The hydrocolloidal, naturally occurring, polysaccharide gums are known, commercially available
materials and are described in many references such as the ENCYCLOPEDIA OF CHEMICAL TECHNOLOGY (3rd Edition 1983) Vol. 12, pages 57 to 67, published by John Wiley and Sons New York. Although the term gum was formerly used to denote a wide range of compounds, the term as currently used generally and as specifically used in this application refers to industrially useful polysaccharides and their derivatives that hydrate in water to form viscous solutions or dispersions. Such gums are generally classified into two broad classes: natural and modified gums. Natural gums includes gums obtained by microbial fermentation, plant exudates, sea weed extracts, and polysaccharides obtained from the seeds, roots, and other parts of plants. Modified gums are also referred to as semisynthetic gums. These include cellulose and starch derivatives and other industrially produced materials such as modified alginates (propylene glycol and triethylene alginate) and other modified natural gums (low-methyloxypextin, carboxymethyl bean gum, and carboxymethyl guar gum).
Gums are generally classified according to their polysaccharide content, which is in turn classified on the basis of the sugar constituents present in the polysaccharides. For example gum tragacanth is a mixture of acidic polysaccharides containing galacturonic acid, galactose, fucose, xylose and
arabinose. It is an exudate from the Astralaqus tree found in Iran, Syria and Turkey. Solutions are weakly acidic with a pH of 5.0 - 6.0 and a molecular weight range of 10,000 to 250,000. On the other hand gum acacia is a dried exudate obtained from the acacia tree found chiefly in the African Sudan. It has a large molecular weight in the range of 200,000 - 1,160,00 and is stable in a slightly acid pH to neutral range.
Both gums are quite water soluble and exhibit a high propensity for their many free hydroxyl groups to complex with a cycloalkyl dipeptide sweetener of the type described above. What is quite unexpected is the fact that the complex formation does not impede or interfere with the sweetness of this peptide while it does stabilize the material against heat degradation.
Other gums useful in the practice of the present invention include agar (obtained from marine algae belonging to the class Rhodophyceae), algin (a generic description for salts of alginic acid, obtained from the brown sea weed Phaeophyceae), carrageenan (a complex mixture of sulfated polysaccharides extracted from certain genera and species of Rhodophyceae), gum arabic (a dried exudate from species of the acacia tree), gum karaya (also known as sterculia gum, the dried exudate of the Sterculia urens tree), gum ghatti (an exudate from Anogeissus latifolia), guar gum
(derived from the seed of the guar plant), locust bean gum (produced by milling the seeds from the lagomerous evergreen plant Ceratonia siliquia), tamarind gum (obtained from the seed colonels of the tamarind tree), psyllium seed gum (obtained from Plantaqo ovata), quince seed gum, larch gum, pextin (a generic term for a group of polysaccharides consisting principally of methoxylated polygalacturonic acids which are located in the cell walls of all plant tissues), dextrin (produced from sucrose by species of the bacterium Lauconostoc), and xanthan gum (a saccharide produced by the bacterium Xanthomonas champestirs).
Compositions containing a hydrocolloidal gum and dipeptide of the invention can be readily prepared using known techniques. Typically, a dipeptide is dry blended with an appropriate amount of the selected hydrocolloidal gum. Further formulation may be conducted if desired (e.g., suspending agents, binders, flavorings, and the like can be added) but a simple mixture of the two components is sufficient to provide the increased stability described in this application. If desired, a liquid formulation can be prepared by dissolving the two components in water or any other ingestible solvent. In some cases, the composition will not dissolve completely but will instead form a stable suspension or dispersion. Such
materials are usable in that form without further treatment.
In addition to the preferred hydrocolloidal gums, other polyhydroxypolymers useful in the practice of this aspect of the invention include polysaccharides, such as cellulose, starch, amylose, and amylopectin, and artificial polyhydroxy compounds such as polyvinylalcohol. Polyethylene glycol (which has two free hydroxy groups per molecule and has been used as a pharmaceutical carrier for peptides) is within the scope of this aspect of the invention. No particular structure is required of the polyhydroxypolymer other than multiple hydroxy groups and ingestibility. Preferred polymers have an average molecular weight of at least 100,000 daltons, preferably with a minimum molecular weight of 1,000 daltons.
The edible sweeteners of the present invention are particularly useful as stabilized sweeteners for fruit juices, fruit preparations, canned vegetables and fruits, dairy products such as egg products, milk drinks, ice cream, syrups, chocolate syrups and bars, candy, icing and dessert toppings, meat products and especially carbonated and non-carbonated beverages.
A. SAMPLE FORMULATION OF SWEETENER COMPLEX In a suitable mixer of the Banberry type, dry blend 10 parts of the peptide product of Example 1 with 90 parts of a pulverulent dried exudate of the astralagus tree found in Syria commonly referred to as gum tragacanth or gum arabic. Both ingredients are water soluble white crystalline solids. When moistened slightly with water or other aqueous fluids such as whole milk, the mixture will form a pasty complex which is itself water soluble.
This complex is the sweetener ingredient employed as a replacement for sugar in step B which involves the formation of a natural-tas.ting yellow cake which differs from prior cakes in a notable respect - it contains no sucrose.
B. SAMPLE PROCEDURE FOR PREPARING A CAKE MIX WITH THE NEW SYNTHETIC SWEETENER AND BAKING A SWEET YELLOW CAKE
A cake mix recipe, such as a standard yellow cake taken from page 67 of Chapter 4 of the Better Homes and
Gardens Cookbook 1972 printed by Better Homes and
Gardens magazine New York, NY, can be altered to substitute the new sweetener complex for the sugar ingredient of the recipe. The new cake formula hence is as follows:
The margarine is creamed and the synthetic sweetener as a wet paste is added slowly over 10 minutes with constant stirring until light. The two eggs are then added along with the vanilla flavor ingredient. The mixture is then beaten at moderate speed till it is fluffy.
The dry ingredients (sweetener, cake flour, sodium bicarbonate, and salt) are also mixed and sifted. They are then added slowly to the creamed mixture in several equal amounts with intermittent addition of whole milk and beating for 3 minutes after each addition.
Beat the entire mixture as a dough briskly for about 1-2 minutes.
Place the doughy batter into a pie greased and lightly floured 9 x 1½ inch round cake pan and place into an oven pie heated to a bake temperature of 350°F.
Bake the batter for from 30-35 minutes at the 350°F constant temperature to obtain a browned cake. Take out of the oven and cool for about 10 minutes before removing the cake from the pan.
Cool to room temperature and to obtain a tasty sweet cake with no sucrose or calories derived therefrom.
The invention now being generally described, the same will be better understood "by reference to certain. specific examples which are included herein for purposes of illustration only and are not intended to be limiting of the invention or any embodiment thereof unless so specified.
EXAMPLE 1 PREPARATION OF PROPYL ESTER OF THE
DIPEPTIDE OF ASPARTIC ACID AND CYCLOPROPYL
ALANINE
Melting points (uncorrected) were taken on a Thomas Hoover capillary melting point apparatus. NMR spectra were recorded on a Varian EM 390 MHz spectrometer. TCL was performed on Whatman precoated silica gel plates with the following solvent systems:
(I) Hexanes-EtOAc (2:1)
(II) Ether-Hexanes (2:1)
(III) Ethanol-CHCl3 (F:95)
(IV) n-BuOH-AcOH-H20 (4:1:5)
(V) nBuOH-AcOH-ρyridine-H2O (4:1:1:2); 0.1% AcOH-nBuOH-pyridine
n-Propyl a-Aminocycloprooane Carboxylate Hydrochloride ( 2 ). To a solution of n-propanol (220 ml) and S0C12 (11 ml), chilled to -10ºC, 1-aminocycloρroρane-1-carboxylic acid (11.47 g, 0.11 mol) was added, and the solution was refluxed for 7 h. The solvent was evaporated to give 2 as an oil, 19.5 g (96%), rf (IV) 0.60; 1H-NMR (CD3OD): 60.97 (t, J = 6 Hz, 3H, methyl), 1.35-1.74 (m, 6H, cyclopropyl H, CH2CH2CH3), 4.15 (t, J = 8 Hz, 2H, OCH2).
N-BOC-B-t-Butyl-L-Aspartyl-n-aminocyclopropane Carboxylic acid n-Propyl Ester ( 3 ). To a solution of N-Boc-aspartic acid-β-t-butyl ester (32.50 g, 0.11 mol) in THF (250 ml), N-methylmorpholine (NMM, 12.35 ml, 0.11 mol) was added and the solution was cooled to -15ºC. Isobutylchloroformate (14.71 ml, 0.11 mol) was added and the reaction mixture was stirred at -15ºC for 10 min. A solution of 2 (18.34 g, 0.10 mol) and NMM (11.23 ml, 0.10 mol) in THF (250 ml) was then added. The reaction mixture was allowed to warm up to room
temperature and was stirred for 3 h. The solvent was evaporated in vacuo, the residue was dissolved in AcOEt 9500 ml), and the solution was washed with 0.5 M citric acid (3x50 ml), brine (2x50 ml), 5% NaHCO3 (3x50 ml), and brine (2x50 ml). After drying over anhyd. MgS04, the solvent was removed in vacuo, and the resulting crude 3 was purified by silica gel (60-200 mesh, Baker) column chromatography (6.0x50 cm). The fractions containing a pure compound (1400-2300 ml) were pooled and the solvent was evaporated. Recrystallization from AcOEt-hexanes afforded 25 g (61%) of pure 3 as white flakes, in two crops; Mp 71-72ºC, Rf (I) o.53, Rf (lI) 0.23, Rf (III) 0.70; 1H-NMR (CDCl3): 60.95 (t, 3H, J 8 Hz, CH3), 1.10-1.25 (m, 2H, cyclopropyl H), 1.48-1.75 (m, 22H, cyclopropyl H, CH2CH2CH3 3xC(CH3)3), 2.65- 2.82 (2H, m, Asp CβH2), 4.00 (t, 2H, OCH2, 4.35-4.55 (m, 1H, Asp CάH), 5.65 (br d, 1H, Asp NH), 7.18 (br s, 1H, Ace NH).
L-Aspartyl-α-aminocyclopropane Carboxylic Acid nPropyl Ester (1). A solution of 3 (24.5 g, 59.1 mmol) in CH2Cl2 (180 ml) was cooled to 0ºC, and TFA (245 ml) was added. The solution was stirred at room temperature for 80 min and evaporated to dryness. The resultant oil was triturated with ether to give a solid which was collected by filtration and washed several
times with ether. The solid salt (4) was carefully dissolved in a 5% NaHCO3 solution and the pH was adjusted to 5 with 5% bicarbonate solution. The precipitated zwitterion 1 was collected by filtration, washed with ice cold water, and dried to give finally 10 g of 1 . Another 4.0 g of dipeptide were recovered from the mother liquor, after the mother liquor was allowed to stand at OºC for a few hours. The crude dipeptide was recrystallized first from water (15 ml) and then from an n-propanol-ether mixture to give 11.0 g (72%) of pure 1 as white needles, mp 168-170ºC (dec.) rf (IV) 0.50, rf(V) 0.54, Rf (VI) 0.68, 1H-NMR (CD3OO): δ 0.95 (t, 3H, CH3), 1.05-1.25 (m, 2H, cyclopropyl H), 1.32-1.80 (m, 4H, cyclopropyl H, CH3CH2O), 2.55-2.78 (m, 2H, Asp CβH2), 4.01 (t, 3H, CH2O and AsP C αH).
EXAMPLE 2 TESTING OF THERMAL STABILITY OF COMPLEX
Thermal Stability of 1. 100 mg of dipeptide 1 was mixed with gum arabic (10 g), and a thick, gummy paste was formed by the addition of water (5 ml). This mixture was placed in an oven and heated at 170ºC for 30 min. A sample of pure 1 (50 mg) containing no gum was also heated under the same conditions. A panel of five volunteers taste-tested the gum-dipeptide mixture
before and after baking it. All tasters agreed that no major loss of sweetening power had occurred after heating the mixture. Furthermore, the crisp, pale yellow solid resulting from the baking process possessed an enhanced flavor. On the other hand, total decomposition and loss of sweetness of 1, heated under the same conditions but in absence of gum, occurred. The same experiment was performed using gum Tragacanth in place of gum Arabic, under exactly the same conditions. The outcome of the experiment was the same. The same panel verified that the sweetness and taste of the gum-dipeμtide mixture were retained after the heating process.
EXAMPLE 3 A series of evaluations of the stability and sweetening power of compounds of the invention have been conducted. These include an organoleptic evaluation of the sucrose equivalent sweetening power of various compounds at pH 7, an organoleptic evaluation of stability in baked goods versus Aspartame using a low-calorie cake formulation, an organoleptic evaluation of stability in various buffered solutions versus Aspartame at pH 3, 5, and 7 over three days at 75ºC, and an analytical analysis by high pressure liquid chromatography of the buffer-stored samples.
The compounds tested were all alkyl esters of L-aspartyl-α-aminocycloρroρane carboxylic acid. Compound Dl was the methyl ether, D2 was the ethyl ester, D3 was the n-propyl ester, D4 was the iso-propyl ester, D5 was the n-butyl ester, and D6 was the isobutyl ester.
Sweetness at pH 7
Compounds D1-D6 were tested by a trained organoleptic evaluation panel to determine the sucrose equivalent sweetening power. The sweetening factors set forth in the following table indicate the relative degree of sweetness of the various compounds. A value of 100 indicates that the compound achieved a sweetness equivalent to the indicated sucrose concentration at a concentration 1-100th of the indicated sucrose concentration. In other words, the compound D4 achieved a sweetness equivalent to 5% sucrose at a concentration of 0.05%.
A baking test was conducted to compare the survival of the compounds of the invention in a baking process to the survival of Aspartame. Standard cake batters were prepared using either Aspartame or a compound of the invention. Sweetness levels were adjusted to produce similar sweetness in all batters. The batters were then baked in a small muffin tin and taste tested by a trained organoleptic panel. In each case tested the Aspartame sample was not sweet after baking, but the sample cakes containing compounds of the invention retained, sweetness. This test was qualitative rather than quantitative in determining sweetness after baking. Compounds D1-D6 all retained some sweetness.
Stability in Buffered Solutions A series of evaluations were undertaken to determine the stability of compounds in the invention versus Aspartame in buffered solutions at pH 3, 5, and 7. Samples were stored for three days with daily testing of two types: (1) organoleptic evaluation and (2) analytical analysis to determine structural integrity. Samples tested at pH 3 and 5 were stored at 75ºC (167ºF) while samples tested at pH 7 were either refrigerated or stored at room temperature. The results of stability testing are set forth in Tables 2-
4, which follow. Values in the table show the percent compound remaining, evaluated either by an organoleptic panel (identified as "taste") or by high pressure liquid chromatography analysis and computation (identified as "AC").
In all cases, sweeteners of the invention were significantly more stable than Aspartame under the same storage-conditions.
Publications (patent and otherwise) are listed and discussed throughout this application as evidence of the level of skill of those practiced in the art to which this invention pertains. All such publications, as well as the prior patent applications referred to, are herein individually incorporated by reference in the locations and for the purposes for which they are cited.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the invention as set forth herein.