WO2002088667A2 - Oligomerisation enantioselective d'acides carboxyliques alpha-hydroxy et d'acides alpha-amino - Google Patents

Oligomerisation enantioselective d'acides carboxyliques alpha-hydroxy et d'acides alpha-amino Download PDF

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Publication number
WO2002088667A2
WO2002088667A2 PCT/US2002/013708 US0213708W WO02088667A2 WO 2002088667 A2 WO2002088667 A2 WO 2002088667A2 US 0213708 W US0213708 W US 0213708W WO 02088667 A2 WO02088667 A2 WO 02088667A2
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set forth
amino acid
oligomer
oligomers
enantiomer
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PCT/US2002/013708
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English (en)
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WO2002088667A3 (fr
Inventor
Stephen J. Lorbert
Charles S. Schasteen
Paul K. S. Nam
Daniel Forciniti
Mathur P. Rajesh
Shubhender Kapila
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Novus International, Inc.
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Priority to AU2002259100A priority Critical patent/AU2002259100A1/en
Publication of WO2002088667A2 publication Critical patent/WO2002088667A2/fr
Publication of WO2002088667A3 publication Critical patent/WO2002088667A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2/00—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs

Definitions

  • the present invention relates to a process for the enantioselective preparation of oligomers consisting of ⁇ -amino acid isomers and co-oligomers consisting of ⁇ -hydroxy carboxylic acid isomers and ⁇ -amino acid isomers.
  • the present invention also relates to compositions containing such oligomers and co-oligomers and methods of use thereof.
  • oligomer which is protected from degradation in the rumen of a ruminant
  • provision of such an oligomer which provides nutritional or pharmacological benefit to the animal
  • provision of a process for the preparation of such oligomers is the provision of an oligomer which is protected from degradation in the rumen of a ruminant
  • a further object of the present invention is the provision of a co-oligomer and oligomer that provides nutritional or pharmacological benefit to animals, and the provision of a process for the preparation of such co-oligomers and oligomers.
  • a further object of the invention is the provision of a co-oligomeric or oligomeric coating for vitamins, minerals, or nutrients.
  • Another object of the present invention is the provision of a method to purify enantiomeric mixtures of ⁇ -hydroxy carboxylic acids, ⁇ -amino acids, or combinations thereof.
  • the present invention is directed to a process for the preparation of an oligomer consisting of ⁇ -amino acid isomers.
  • the process comprises forming a reaction mixture containing (i) an enzyme and (ii) an an enantiomeric mixture of ⁇ -amino acid, or derivative thereof.
  • An oligomer is formed that incorporates one enantiomer of the enantiomeric mixture of the ⁇ -amino acid or derivative thereof in preference to the other enantiomer.
  • the present invention is further directed to a composition
  • a composition comprising a residue of an ⁇ -hydroxy carboxylic acid bonded to a peptide by an amide or an ester linkage, said peptide comprising two or more ⁇ -amino acid residues, each of said ⁇ -amino acids being independently selected from the group consisting of ⁇ -amino acids.
  • ⁇ -amino acid residues in the peptide are of identical chirality.
  • the present invention is further directed to an oligomer of the formula CA-(AA) n - wherein CA is the residue of an ⁇ -hydroxy carboxylic acid, each AA is the residue of an ⁇ - amino acid or derivative thereof wherein greater than one-half of the AA residues are derived from the group consisting of ⁇ -amino acids or derivatives thereof having the same chiral configuration, and n is at least 2.
  • the present invention is also directed to a process for providing an animal with a food ration.
  • the process comprises providing an oligomer or a co-oligomer prepared from a mixture containing an enzyme, an ⁇ -amino acid, and optionally, an ⁇ -hydroxy carboxylic acid or derivative thereof.
  • the feed ration is administered to the animal by oral administration, eye spray, placement in ear, placement in nasal cavity, and bucchal administration, sublingual administration, rectal administration or injection.
  • the present invention is further directed to an orally administered dietary supplement comprising a vitamin, mineral, or nutrient that is coated with an oligomeric coating.
  • the coating comprises a residue of an ⁇ -hydroxy carboxylic acid bonded to a peptide by an amide linkage.
  • the peptide comprises two or more independent ⁇ -amino acids independently selected from the group consisting of ⁇ -amino acids.
  • the present invention is further directed to a process for providing an animal with a dietary supplement comprising a vitamin, mineral, or nutrient.
  • the process comprises coating the vitamin, mineral or nutrient with a composition to form a dietary supplement and administering the dietary supplement to the animal.
  • the composition comprises a residue of an ⁇ -hydroxy carboxylic acid bonded to a peptide by an amide linkage and the peptide comprises two or more independent ⁇ -amino acids independently selected from the group consisting of ⁇ -amino acids.
  • the present invention is further directed to a process for purifying an enantiomeric mixture of ⁇ -amino acid or derivative thereof.
  • the process comprises forming a reaction mixture comprising (i) an enzyme, (ii) an enantiomeric mixture of ⁇ -amino acid or a derivative thereof, and (iii) an ⁇ -hydroxy carboxylic acid or a derivative thereof.
  • a peptide reaction product is formed from the reaction mixture comprising (i) an oligomer or co- oligomer from the combination which incorporates one of the members of the enantiomeric mixture of ⁇ -amino acid or derivative thereof in preference to a second enantiomer of the enantiomeric mixture, and (ii) unreacted second enantiomer.
  • the oligomer or co-oligomer and unreacted second enantiomer are then separated from the reaction product and each other.
  • the present invention is also directed to a process for purifying an ⁇ -hydroxy carboxylic acid enantiomer or derivative thereof in an enantiomeric mixture.
  • the process comprises forming a reaction mixture comprising (i)an enzyme, (ii) an enantiomeric mixture of an ⁇ -hydroxy carboxylic acid and (iii) an ⁇ -amino acid or a derivative thereof.
  • a reaction product is formed from the reaction mixture comprising (i) a co-oligomer which preferentially incorporates a first enantiomer over a second enantiomer of the enantiomeric mixture, and (ii)unreacted second enantiomer.
  • the co-oligomer and unreacted second enantiomer are then separated from the reaction product and each other.
  • Fig. 1 is aMALDI-TOF graph of methionine oligomers and co-oligomers from a papain catalyzed synthesis.
  • Fig. 2 is a MALDI-TOF graph of HMB-methionine co-oligomers from a papain catalyzed synthesis.
  • Fig. 3 is a HPLC graph of methionine sulfone oligomers.
  • Fig. 4 is a HPLC graph of HMB-methionine sulfone co-oligomers after a incubation period of 10 minutes.
  • Fig. 5 is a HPLC graph of HMB-methionine sulfone co-oligomers after a incubation period of 24 hours.
  • Fig. 6 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of lysine oligomers synthesized in a reverse micellar system.
  • Fig. 7 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of HMB-lysine co-oligomers synthesized in a reverse micellar system.
  • Fig. 8 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of HMB-lysine co-oligomers synthesized in a 2-phase system.
  • Fig. 9 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of lysine oligomers synthesized in a 2-phase system.
  • Fig. 10 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of lysine oligomers synthesized in a 3-phase system.
  • Fig. 11 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of HMB-lysine co-oligomers synthesized in a 3-phase system.
  • Fig. 12 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of lysine oligomers synthesized in a reduced volume 2-phase system.
  • Fig. 13 is a ion-pair liquid chromatography and MALDI-TOF mass spectrometry graph of HMB-lysine co-oligomers synthesized in a reduced volume 2-phase system.
  • Fig. 14A is a chromatogram of persulfonated methionine oligomers using a UN absorption detector.
  • Fig. 14B is a positive ion total ion chromatogram of persulfonated methionine oligomers.
  • Fig. 15A is a chromatogram of persulfonated HMB-methionine co-oligomers using a UN absorption detector.
  • Fig. 15B is a positive ion total ion chromatogram of persulfonated HMB-methionine co-oligomers.
  • Fig. 16 is a positive ion ESI spectra of (Met) 3 sulfone peak eluting at 5.27 minutes.
  • Fig. 17 is a positive ion ESI spectra of (Met) 4 sulfone peak eluting at 7.70 minutes.
  • Fig. 18 is a positive ion ESI spectra of (Met) 5 sulfone peak eluting at 9.47 minutes.
  • Fig. 19 is a positive ion ESI spectra of (Met) 6 sulfone peak eluting at 11.09 minutes.
  • Fig. 20A is a positive ion ESI spectra of (Met) 7 sulfone peak eluting at 12.7 minutes.
  • Fig. 20B is a positive ion ESI spectra of (Met) 8 sulfone peak eluting at 14.26 minutes.
  • Fig. 20C is a positive ion ESI spectra of (Met) 9 sulfone peak eluting at 15.60 minutes.
  • Fig. 21 A is a chromatogram of persulfonated methionine oligomers using a UN absorption detector.
  • Fig. 21B is a total ion chromatogram ESI-negative ion of persulfonated methionine oligomers.
  • Fig. 22 A is a chromatogram of persulfonated HMB-methionine co-oligomers using a UN absorption detector.
  • Fig. 22B is a total ion chromatogram ESI-negative ion of persulfonated HMB- methionine co-oligomers.
  • Fig. 23 is a negative ion ESI spectra of HMB-(Met) 5 sulfone peak eluting at 11.57 minutes.
  • Fig. 24 is a negative ion ESI spectra of HMB-(Met) 6 sulfone peak eluting at 13.86 minutes.
  • Fig. 25 is a negative ion ESI spectra of HMB-(Met) 7 sulfone peak eluting at 15.31 minutes.
  • Fig 26 is a bar graph of the relative distribution of (Met) n wherein n is the number of methionine residues in the methionine oligomers.
  • Fig 27 is a bar graph of the relative distribution of HMB-(Met) n wherein n is the number of methionine residues in the HMB-methionine co-oligomers.
  • Fig. 28 A is a positive ion ESI-MS spectra of HMB-methionine co-oligomers synthesized with HMB methyl ester and methionine ethyl ester.
  • Fig. 28B is a negative ion ESI-MS spectra HMB-methionine co-oligomers synthesized with HMB methyl ester and methionine ethyl ester.
  • Fig. 29 is a parent ion SSI-MS spectra HMB-methionine co-oligomers synthesized with HMB methyl ester and methionine ethyl ester.
  • Fig. 30 is a daughter ion spectrum of (Met) 6 -ethyl ester.
  • Fig. 31 A is a positive ion ESI-MS spectra of tyrosine (Tyr)n oligomers wherein n is the number of tyrosine residues in the oligomers.
  • Fig. 3 IB is a negative ion ESI-MS spectra of tyrosine (Tyr)n oligomers wherein n is the number of tyrosine residues in the oligomers.
  • Fig. 32 A is a positive ion spectra of HMB-tyrosine co-oligomers.
  • Fig. 32B is a negative ion spectra of HMB-tyrosine co-oligomers.
  • Fig. 33 A is a positive ion ESI-MS spectra of leucine oligomers.
  • Fig. 33B is a negative ion ESI-MS spectra of leucine oligomers.
  • Fig. 34A is a positive ion ESI-MS spectra of HMB-leucine co-oligomers.
  • Fig. 34B is a negative ion ESI-MS spectra of HMB-leucine co-oligomers.
  • Fig. 35A is a positive ion ESI-MS spectra of HMB-phenylanaline co-oligomers.
  • Fig. 35B is a negative ion ESI-MS spectra of HMB-phenylanaline co-oligomers.
  • Fig. 36 is a graph of the effect of Aqueous : Non- Aqueous ratios on (Lys) n oligomer yield wherein n is the number of lysine residues in the oligomers in a two-phase system.
  • Fig. 37 is a bar graph of the effect of volumetric ratios on the degree of (Lys) n oligomer yield in a two-phase reaction system wherein n is the number of lysine residues in the oligomers.
  • Fig. 38 is a graph of the effect of additive concentrations on (Lys) n oligomer yield wherein n is the number of lysine residues in the oligomers.
  • Fig. 39 is a bar graph of the effect of additive concentrations on the degree of (Lys) n oligomerization wherein n is the number of lysine residues in the oligomers.
  • Fig. 40 is a graph of the effect of substrate concentrations on (Lys) n oligomer yield wherein n is the number of lysine residues in the oligomers.
  • Fig. 41 is a bar graph of the distribution of lysine oligomers formed in reaction mixtures with varied substrate concentrations.
  • Fig 42 is a graph of the effect of incubation time on total lysine oligomer yield.
  • Fig. 43 is a bar graph of the distribution of lysine oligomers formed after different incubation time periods.
  • Fig 44 is a graph of the effect of aqueous to non-aqueous solvent phase ratios on total lysine oligomer yield in a three-phase system.
  • Fig. 45 is a bar graph of the distribution of lysine oligomers formed in reaction mixtures at various aqueous to non-aqueous solvent ratios.
  • Fig. 46 is a graph of the effect of additive concentrations on the total lysine oligomers yield.
  • Fig. 47 is a bar graph of the distribution of lysine oligomers formed with varied additive concentrations in a 2-phase system.
  • Fig. 48 is a graph of the total lysine oligomer yield formed after different incubation time periods in a 3-phase system.
  • Fig. 49 is a bar graph of the distribution of lysine oligomers formed after a one hour incubation period in a 3-phase system.
  • Fig. 50 is a bar graph of the distribution of lysine oligomers formed after a 24 hour incubation period in a 3-phase system.
  • Fig. 51 is a chromatogram of an enantiomeric mixture of methionine ethyl ester using a UN absorption diode array detector (DAD).
  • DAD absorption diode array detector
  • Fig. 52 is a chromatogram of a enantiomeric mixture of methionine ethyl ester and HMB-ethyl ester using a UN absorption diode array detector (DAD).
  • DAD absorption diode array detector
  • Fig. 53 is a chromatogram of an oligomer and co-oligomer hydrolyzate illustrating the presence of only the L-methionine enantiomer using a UN absorption diode array detector (DAD).
  • DAD absorption diode array detector
  • Fig. 54 is a chromatogram of an oligomer and co-oligomer hydrolyzate illustrating the presence of only the L-HMB enantiomer using a UN absorption diode array detector (DAD).
  • DAD absorption diode array detector
  • Fig. 55 A is a positive ion ESI-MS spectra of the lactic acid - methionine oligomers prepared in Example 15.
  • Fig. 55B is a negative ion ESI-MS spectra of the lactic acid - methionine oligomers prepared in Example 15.
  • Fig. 56A is a positive ion ESI-MS spectra of the lactic acid - tyrosine oligomers prepared in Example 15.
  • Fig. 56B is a negative ion ESI-MS spectra of the lactic acid - tyrosine oligomers prepared in Example 15.
  • Fig. 57A is a positive ion ESI-MS spectra of the lactic acid - leucine oligomers prepared in Example 15.
  • Fig. 57B is a negative ion ESI-MS spectra of the lactic acid - leucine oligomers prepared in Example 15.
  • Fig. 58 A is a positive ion ESI-MS spectra of the lactic acid - tryptophan oligomers prepared in Example 15.
  • Fig. 58B is a negative ion ESI-MS spectra of the lactic acid - tryptophan oligomers prepared in Example 15.
  • Fig. 59A is a positive ion ESI-MS spectra of the lactic acid - phenylalanine oligomers prepared in Example 15.
  • Fig. 59B is a negative ion ESI-MS spectra of the lactic acid - phenylalanine oligomers prepared in Example 15.
  • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS hi accordance with the present invention, it has been discovered that oligomers and co-oligomers of ⁇ -hydroxy carboxylic acids and ⁇ -amino acids maybe prepared in an enzymatically catalyzed reaction.
  • the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid oligomers enzymatically synthesized by the process of the present invention may possess altered properties from those of the ⁇ -amino acid monomers.
  • the ⁇ -hydroxy carboxylic acid/ ⁇ - amino acid oligomers unlike proteins, peptides, or amino acid monomers, are not recognized in the rumen by ruminal microorganisms. As a result, the ruminal microorganisms do not break down the oligomers and the oligomers are available for absorption by the ruminant.
  • solubility properties of many of the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid oligomers of the present invention are also different from their monomeric counterpart.
  • ⁇ -amino acid monomers such as methionine
  • methionine are soluble in water
  • the ⁇ - hydroxy carboxylic acid/ ⁇ -amino acid oligomers and ⁇ -amino acid oligomers formed from methionine monomers are insoluble. This alteration advantageously permits the oligomers to be introduced in aqueous environments without being dissolved in the solution.
  • the oligomers of the present invention comprise the residue of an ⁇ - hydroxy carboxylic acid bonded to the residue of an ⁇ -amino acid by an amide or an ester linkage.
  • the oligomers correspond to the general formula CA-(AA) n wherein CA comprises the residue of an ⁇ -hydroxy carboxylic acid, (AA) n is an oligomeric segment comprising the residue of one or more independent ⁇ -amino acids, n is at least 1 and CA is bonded to (AA) n by an amide or an ester linkage, hi accordance with a preferred embodiment, the ⁇ -hydroxy carboxylic acid is bonded to the residue of an ⁇ -amino acid with an amide bond to effectively create an ⁇ -amino acid oligomer that is "end-capped" by an ⁇ - hydroxy carboxylic acid residue.
  • an ⁇ -amino acid oligomer is formed that corresponds to the formula (AA) n wherein each AA is the residue of one or more independent ⁇ -amino acids, n is at least 2 and the amino acid residues are bonded to each other by an amide linkage or an ester.
  • (AA) n when n is greater than 1, (AA) n may comprise more than one independent ⁇ -amino acid residue. Stated another way, (AA) n comprises a peptide comprising two or more independent ⁇ -amino acids.
  • the composition of the oligomer may be advantageously tailored for specific applications.
  • the oligomer may be designed to meet the essential amino acid requirements of a particular animal by incorporating two or more different amino acid residues (e.g., methionine and lysine residues) into an oligomer.
  • Such an example is an oligomer comprising lysine and methionine residues in a 3:1 ratio, which would meet the essential amino acid requirements of a ruminant.
  • n is less than 20. In some embodiments, n ranges from about 1 to about 10, more typically from about 2 to about 8 and, in some embodiments, from about 3 to about 5. For example, in oligomers comprising methionine residues, n typically ranges from about 4 to about 12, with an average of about 6 to about 8.
  • the oligomers of the present invention may be obtained (and used) as a dimer, trimer, tetramer, pentamer, hexamer, septamer, octamer, nonamer, decamer, etc. in which a residue of the ⁇ -hydroxy carboxylic acid is linked to a residue of an ⁇ -amino acid via an amide or ester linkage.
  • an oligomeric segment may be obtained which is chemically or enzymatically linked to another moiety, for example, through the ⁇ -hydroxy group of the ⁇ - hydroxy carboxylic acid residue, the carboxy terminus of the ⁇ -amino acid residue (for oligomers comprising an amide linkage between the ⁇ -hydroxy carboxylic acid residue and the ⁇ -amino acid residue) or the amino terminus of the ⁇ -amino acid residue (for oligomers comprising an ester linkage between the ⁇ -hydroxy carboxylic acid residue and the ⁇ -amino acid residue).
  • the oligomer or oligomeric segment corresponds to the structure:
  • R 1 is hydrogen, hydrocarbyl or substituted hydrocarbyl
  • R 2 is hydrogen, hydrocarbyl, substituted hydrocarbyl, or a hydroxy protecting group
  • R 3 is hydrogen, hydrocarbyl or substituted hydrocarbyl
  • each AA is the residue of an ⁇ -amino acid selected from the group consisting of ⁇ - amino acids independently of any other ⁇ -amino acid residue
  • n is at least 1.
  • the oligomer or oligomeric segments of the present invention may comprise the residue of any ⁇ -hydroxy carboxylic acid.
  • Preferred ⁇ -hydroxy carboxylic acids correspond to the general structure R 1 R 3 C(OR 2 )COOH wherein R 1 is hydrogen, hydrocarbyl or substituted hydrocarbyl; R 2 is hydrogen, a hydroxy protecting group, hydrocarbyl, or substituted hydrocarbyl; and R 3 is hydrogen, hydrocarbyl or substituted hydrocarbyl, preferably hydrogen.
  • the ⁇ -hydroxy carboxylic residue may be the residue of any of the following naturally occurring ⁇ -hydroxy carboxylic acids (with R 1 for such acid being given in brackets): lactic acid [-CH 3 ], mandelic acid [-C 6 H 5 ], malic acid [-CH 2 COOH], and tartaric acid [-CH(OH)COOH].
  • the ⁇ -hydroxy carboxylic acid residue may be the residue of an ⁇ -hydroxy acid analog of a naturally occurring ⁇ -amino acid, more preferably the residue of the ⁇ -hydroxy analog of an essential ⁇ -amino acid, and still more preferably the residue of the ⁇ -hydroxy analog of methionine, i.e., 2-hydroxy-4- (methylthio)butyric acid.
  • the ⁇ -hydroxy carboxylic acid residue may comprise the residue of an ⁇ - hydroxy carboxylic acid having the D- configuration, the L- configuration, or a racemic or other mixture of the D- and L- isomers. In some embodiments, however, it is generally preferred that the ⁇ -hydroxy carboxylic acid residue comprise the residue of an ⁇ -hydroxy carboxylic acid having the L- configuration.
  • the ⁇ -hydroxy carboxylic acid residue incorporated into the oligomer may comprise the residue of more than one ⁇ -hydroxy carboxylic acid.
  • the residue may comprise a homo-oligomer containing one or more ⁇ -hydroxy carboxylic acid monomers or a hetero-oligomer containing two or more independent ⁇ - hydroxy carboxylic acid monomers.
  • the oligomers of the present invention may comprise the residue of any ⁇ - amino acid.
  • Preferred ⁇ -amino acids correspond to the general structure R a R b C(NH 2 )COOH wherein R a is hydrogen, hydrocarbyl, substituted hydrocarbyl or heterocyclo; and R b is hydrogen.
  • the ⁇ -hydroxy amino residue(s) maybe the residue(s) of any of the naturally occurring ⁇ -amino acids, e.g., asparagine, glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, cysteine, methionine, tryptophan, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.
  • ⁇ -amino acids e.g., asparagine, glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, serine, threonine, cysteine, methionine, tryptophan, tyrosine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.
  • the ⁇ -amino acid residue(s) include the residue(s) of one or more essential ⁇ -amino acids, i.e., isoleucine, phenylalanine, leucine, lysine, methiomne, threonine, tryptophan, histidine and valine. Still more preferably, the ⁇ -arriino acid residue(s) include the residue(s) of methiomne and/or lysine.
  • the ⁇ -amino acid residue may comprise the residue of an ⁇ -amino acid having the D- configuration, the L- configuration, or a racemic or other mixture of the D- and L- isomers. hi some embodiments, however, it is generally preferred that the ⁇ -amino acid residue comprise the residue of an ⁇ -amino acid having the L- configuration.
  • the ⁇ -amino acid residue incorporated into the oligomer may comprise the residue of more than one ⁇ -amino acid.
  • the residue may comprise a homo-oligomer containing one or more ⁇ -amino acid monomers or a hetero- oligomer containing two or more independent ⁇ -amino acid monomers.
  • the oligomers of the present invention are enzymatically synthesized in a mixture.
  • the mixture comprises at least one ⁇ -hydroxy carboxylic acid or a derivative thereof, at least one ⁇ -amino acid or a derivative thereof, and an enzyme.
  • the ⁇ -hydroxy carboxylic acid may be present in the mixture as a free acid or as a carboxylic acid derivative, e.g., the corresponding ester, acid halide, amide, anhydride, or ketene.
  • the ⁇ -hydroxy carboxylic acid is preferably present in the mixture in the form of an amide, i.e., whe Y is -NR 6 R 7 and R 6 and R 7 are independently hydrogen or hydrocarbyl, more preferably lower alkyl, still more preferably hydrogen.
  • the mixture may contain more than one ⁇ -hydroxy carboxylic acid species.
  • the mixture may contain the hydroxy analog of methionine (in one or more of its free acid, acid halide, amide, anhydride or ketene forms) and, in addition, one or more other ⁇ -hydroxy carboxylic acids such as lactic acid, mandelic acid, malic acid, or tartaric acid (in one or more of their respective free acid, acid halide, amide, anhydride or ketene forms).
  • the mixture may further contain oligomers (e.g., dimers, trimers, tetramers, pentamer, hexamer, septamer, octamer, nonamer, decamer, etc.) of one or more ⁇ -hydroxy carboxylic acids.
  • oligomers e.g., dimers, trimers, tetramers, pentamer, hexamer, septamer, octamer, nonamer, decamer, etc.
  • the mixture may contain a homo-oligomer formed from HMB or another ⁇ -hydroxy carboxylic acid or a hetero-oligomer of an ⁇ -hydroxy carboxylic acid (e.g., HMB) and at least one other ⁇ -hydroxy carboxylic acid.
  • the ⁇ -amino acids may be present in the mixture as a free acid or as a carboxylic acid derivative, e.g., the corresponding ester, acid halide, amide, anhydride, or ketene.
  • the ⁇ -amino acid is preferably present in the mixture in the form of an amide, i.e., where Y is -NR 6 R 7 and R 6 and R 7 are independently hydrogen or hydrocarbyl, more preferably lower alkyl, still more preferably hydrogen.
  • the mixture may contain more than one ⁇ -amino acid species.
  • the mixture may contain one ⁇ -amino acid (in one or more of its free acid, acid halide, amide, anhydride or ketene forms) and, in addition, one or more other ⁇ -amino acids (in one or more of their respective free acid, acid halide, amide, anhydride or ketene forms).
  • the mixture may contain methiomne (in one or more of its free acid, acid halide, amide, anhydride or ketene forms) and, in addition, one or more other nutritionally important ⁇ -amino acid(s) such as lysine, tryptophan and/or phenylalanine (in one or more of their respective free acid, acid halide, amide, anhydride or ketene forms).
  • methiomne in one or more of its free acid, acid halide, amide, anhydride or ketene forms
  • other nutritionally important ⁇ -amino acid(s) such as lysine, tryptophan and/or phenylalanine
  • the mixture may contain oligomers (e.g., dimers, trimers, tetramers, pentamer, hexamer, septamer, octamer, nonamer, decamer, etc.) of one or more ⁇ -amino acids.
  • the mixture may contain a homo- oligomer formed from methionine, lysine or other ⁇ -amino acid or a hetero-oligomer of an ⁇ - amino acid (e.g., methionine) and at least one other nutritionally important ⁇ -amino acid such as lysine, tryptophan and/or phenylalanine.
  • the reaction mixture further comprises an enzyme.
  • the enzyme may be dissolved in the mixture or, alternatively, it may be adsorbed or otherwise immobilized onto a variety of substrates.
  • the enzyme maybe immobilized onto controlled pore glass, agarose, sepharose, nylon, or polyethylene glycol. Enzymes may also be adsorbed, for example, onto activated charcoal, ion exchange resins, silica, poryacrylamide, collagen, starch, bentonite, ultramembrane filters, cellulose, alumina, titania, and polyvinylchlori.de. hi addition, enzymes may be retained by entrapment, microencapsulation, liposome formation, hollow fiber, inorganic bridge formation, and aggregation.
  • enzymes generally characterized as a protease when included in a reaction mixture along with, for example, an ⁇ -hydroxy carboxylic acid ethyl ester and an ⁇ -amino acid ethyl ester, will cause a peptide reaction product to be formed from the reaction mixture.
  • the peptide reaction product comprises an oligomer comprising ⁇ -amino acids and the ⁇ -hydroxy carboxylic acid bonded together by amide bonds.
  • protease enzymes include serine proteinases (e.g., Trypsin, ⁇ -Chymotrypsin, Elastase, Carboxypeptidase, and Subtilisin), thiol proteinases (e.g., Papain, Ficin, Bromelain, Streptococcal proteinase, Cathepsins, Calpains, Clostripain, and Actinidin), metalloproteinases (e.g., Thermolysin), acid proteinases (e.g., Pepsin, Penicillopepsin, Chymosin, Cathepsin, and Renin), liver esterase (e.g., pig liver esterase), alkaline protease, carbonic anhydrase, nonribosomal peptide synthetase, thrombin, cardosins A or B, or pronase.
  • serine proteinases e.g., Trypsin
  • the reaction mixture containing the lipase enzyme, an ⁇ -hydroxy carboxylic acid, and an ⁇ -amino acid or derivative thereof instead forms a polyester reaction product.
  • Enantioselective lipase enzymes may be obtained from a variety of microorganisms such as Candida cylindracea, Candida lipolytica, Candida antarctica (bacteria) and fungi such as Rhizopus oryzae, Aspergillus niger, and the like.
  • the reaction product will therefore comprise an oligomer wherein the ⁇ -amino acids and the ⁇ -hydroxy carboxylic acid are bonded together by ester bonds.
  • reaction mixture comprises a lipase enzyme and an ester of an ⁇ -hydroxy carboxylic acid or a derivative thereof, an oligomer of ⁇ -hydroxy carboxylic acid will form wherein the monomers are linked together by ester bonds.
  • the mixture contains an enzyme which catalyzes the formation of peptide bonds.
  • Exemplary enzymes include serine proteinases (e.g., Trypsin, ⁇ - Chymotrypsin, Elastase, Carboxypeptidase, and Subtilisin), thiol proteinases (e.g., Papain, Ficin, Bromelain, Streptococcal proteinase, Cathepsins, Calpains, Clostripain, and Actinidin), metalloproteinases (e.g., Thermolysin), acid proteinases (e.g., Pepsin, Penicillopepsin, Chymosin, Cathepsin, and Renin), liver esterase (e.g., pig liver esterase), alkaline protease, carbonic anhydrase, nonribosomal peptide synthetase, thrombin, cardosins A or B, or pronase.
  • serine proteinases e.g., Trypsin,
  • the present invention may be utilized to enzymatically synthesize oligomers, co-oligomers, or segments thereof, consisting of ⁇ -hydroxy carboxylic acid isomers and ⁇ -amino acid isomers or ⁇ -amino acid isomers wherein one enantiomer of the ⁇ -hydroxy carboxylic acids, ⁇ -amino acids, or derivatives thereof is incorporated into the co-oligomer or oligomer in preference to the other enantiomer.
  • peptide or ester reaction products comprising co-oligomers, oligomers or segments thereof can be formed from a reaction mixture comprising an enantiomeric mixture of ⁇ -hydroxy carboxylic acids, ⁇ -amino acids, or derivatives thereof, wherein one enantiomer of the enantiomeric mixture is incorporated into the reaction product in preference to the other enantiomer of the mixture.
  • the oligomers of the present invention are enantioselectively synthesized in a mixture.
  • the mixture comprises at least one at least one ⁇ -amino acid or a derivative thereof as described above, an enantioselective enzyme; and, optionally a ⁇ -hydroxy carboxylic acid or a derivative thereof as described above.
  • the ⁇ -hydroxy carboxylic acids and ⁇ -amino acids may be present in the mixture as enantiomeric mixtures.
  • An enantiomeric mixture contains enantiomeric pairs of the ⁇ - hydroxy carboxylic acids, ⁇ -amino acids, or derivatives thereof.
  • the proportion of each species may vary from a racemic mixture that contains equal proportions of the D- and L- isomer configurations (e.g., 50% of the L-isomer and 50% of the D-isomer), to enantiomeric mixtures wherein one species is proportionally greater than its opposite species (e.g., an enantiomeric mixture containing 70% L-isomer and 30% D-isomer).
  • the reaction mixture contains a racemic mixture of ⁇ -amino acid. In another embodiment, the reaction mixture contains a racemic mixture of ⁇ -hydroxy carboxylic acid, hi still another embodiment, the reaction mixture contains racemic mixtures of both ⁇ -hydroxy carboxylic acid and ⁇ -amino acid.
  • the mixture contains an enzyme which enantioselectively catalyzes the formation of peptide bonds between ⁇ -amino acids having identical chiral configurations (e.g., L-isomers of amino acids).
  • the co-oligomer or oligomer formed from the mixture comprises a residue of an ⁇ -hydroxy carboxylic acid bonded to a peptide by an amide linkage or an ester linkage, wherein the peptide comprises two or more independent ⁇ -amino acid residues having identical chiral configuration.
  • the enzyme further enantioselectively catalyzes the formation of the amide or ester linkage between the ⁇ -hydroxy carboxylic acid residue and the ⁇ -amino acid residue such that the oligomer comprises one ⁇ -hydroxy carboxylic acid enantiomer linked to the ⁇ -amino acid oligomer in preference to another ⁇ -hydroxy carboxylic acid enantiomer.
  • a reaction mixture containing papain, an enantiomeric mixture of methionine ethyl ester isomers and an enantiomeric mixture of HMB ethyl ester isomers will form oligomers consisting of L-HMB linked to one or more L-methionine residues.
  • the enantiospecificity of the enzyme is dependent upon the ⁇ -amino acid and ⁇ -hydroxy carboxylic acid being oligomerized.
  • exemplary enantioselective enzymes include thiol proteinases (e.g., Papain, Bromelain, Cathepsin s, Cathepsin b, and Cathepsin c) and serine proteinases (e.g., some forms of Subtilisin).
  • the enantioselective enzyme enzymatically links the carboxy terminus of the ⁇ -hydroxy carboxylic acid to the amino terminus of the ⁇ -amino acid.
  • either of the ⁇ -hydroxy carboxylic acid residue or the ⁇ - amino acid residues may comprise an oligomer (i.e., a dimer, trimer, etc.) as described above and still be enantioselectively incorporated into the oligomers of the present invention.
  • the enantioselective enzyme in the reaction mixture is suitable for incorporating oligomers comprising the L-enantiomer, the enzyme will catalyze the oligomerization of any ⁇ -hydroxy carboxylic acid residue or ⁇ -amino acid residue having an L- configuration.
  • the enantioselective enzyme may be dissolved in the mixture or, alternatively, it may be adsorbed or otherwise immobilized onto a variety of substrates.
  • the enzyme may be immobilized onto controlled pore glass, agarose, sepharose, nylon, or polyethylene glycol.
  • Enantioselective enzymes may also be adsorbed, for example, onto activated charcoal, ion exchange resins, silica, polyacrylamide, collagen, starch, bentonite, ultramembrane filters, cellulose, alumina, titania, and polyvinylchloride.
  • enzymes may be retained by entrapment, microencapsulation, liposome formation, hollow fiber, inorganic bridge formation, and aggregation.
  • the oligomer formed from the enantioselective oligomerization comprises a composition comprising a residue of an ⁇ -hydroxy carboxylic acid bonded to a peptide by an amide or an ester linkage, wherein the peptide comprises two or more ⁇ -amino acid residues and each of the ⁇ -amino acids of the peptide are independently selected from the group consisting of ⁇ -amino acids.
  • more than 50% of the ⁇ -amino acid residues in the peptide are of identical chirality and, more preferably, essentially all of the ⁇ - amino acid residues in the peptide are of identical chirality.
  • the oligomer or oligomeric segment formed by an enantioselective enzyme co ⁇ esponds to the structure:
  • R 1 is hydrogen, hydrocarbyl or substituted hydrocarbyl
  • R 2 is hydrogen, hydrocarbyl, substituted hydrocarbyl, or a hydroxy protecting group
  • R 3 is hydrogen, hydrocarbyl or substituted hydrocarbyl, each AA is the residue of an ⁇ -amino acid or derivative thereof wherein greater than one-half of the AA residues are derived from ⁇ -amino acids or derivatives thereof having the same chiral configuration, and n is at least 2.
  • a mixture of papain, an enantiomeric mixture of HMB ethyl ester, and an enantiomeric mixture of D, L-methionine ethyl ester in a reaction mixture has been found to fo ⁇ n homo-oligomers of L-methionine and hetero-oligomers of L-HMB/L-methionine wherein the L-HMB is linked through its carboxy terminus to the amino terminus of the methionine oligomer.
  • the homo-oligomers and hetero-oligomers may be separated out of the solution by precipitation, filtration, selective extraction, column chromatography, lyophilization, and evaporation techniques.
  • an oligomer comprising about nine methionine amino acids will precipitate out of solution and may be easily filtered or centrifuged away from the reacting mixture containing the free hydroxy acids and ⁇ -amino acids.
  • Soluble methionine oligomers comprised of lower numbers of methionine residues can be separated from the free amino and hydroxy acids using membrane filtration.
  • the remaining reaction mixture contains papain, a significant amount of monomers of D-HMB ethyl ester and D-methionine ethyl ester (e.g., about 95%o of the total amount of HMB ethyl ester and methionine ethyl ester isomers), and a small amount of L-HMB ethyl ester and L-methionine ethyl ester.
  • Papain may be removed from the solution by size exclusion chromatography or similar separation technique known in the art.
  • the remaining monomers of D-HMB ethyl ester and D-methionine ethyl ester maybe removed from solution by rotary evaporation for purification or transformed to their respective L-isomer form through base racemization and recycled.
  • AA is the residue of an ⁇ -amino acid comprising two or more independent ⁇ -amino acids wherein greater than one- half of the AA residues are derived from ⁇ -amino acids or derivatives thereof having the same chiral configuration, n is at least 2, and the amino acid residues are bonded to each other by an amide linkage. Typically, n will be less than 20.
  • n will range from about 2 to about 10, more typically from about 2 to about 8 and, in some embodiments, from about 3 to about 5.
  • hi methionine oligomers for example, n typically will range from about 4 to 12, with an average of 6 to 8.
  • the enzymatic reaction is carried out in a single phase, aqueous solution under conditions typically employed in enzyme catalyzed reactions for the preparation of oligomers and co-oligomers of ⁇ -amino acids.
  • Such systems are typically used in enzymatic biochemical reaction. See, e.g., Lehninger, Nelson, and Cox, Principles of Biochemistry, 1993, Worth Publisher, NY, NY.
  • the enzymatic reaction is carried out in a two-phase system comprising an aqueous phase and an organic phase.
  • the organic phase comprises an organic solvent selected from the group consisting of alkanes, alkenes, aryls and suitable derivatives thereof. See, e.g., Olmsted and Williams, Chemistry the Molecular Science, 1994, Mosby Publisher, St. Louis, MO.
  • the enzymatic reaction is carried out in a reverse micelle system.
  • a reverse micelle system Such a system comprises a continuous organic phase, a dispersed aqueous phase, and a surfactant to obtain and stabilize micelle phase.
  • the organic phase comprises an organic solvent selected from the group consisting of alkyl, aryl, and suitable derivatives thereof, and the surfactant is selected from the group consisting of ionic or non-ionic surfactants.
  • organic solvent selected from the group consisting of alkyl, aryl, and suitable derivatives thereof
  • surfactant is selected from the group consisting of ionic or non-ionic surfactants.
  • reverse micelle systems are typically used for biotechnological reactions. See, e.g., Vicente, Aires-Barros, and Empis, J. Chem. Tech. Biotechnol 1994, 60, 291.
  • the enzymatic reaction is carried out in a three-phase system comprising an aqueous phase, a first organic phase and a second organic phase with the two organic phases being immiscible.
  • the first organic phase comprises an organic solvent selected from the group consisting of hydrocarbon solvents and the second organic phase comprises an organic solvent selected from the group consisting of halogenated hydrocarbon, perhalogenated hydrocarbon, and halogenated hydrocarbyl solvents.
  • Such three phase systems are routinely used for chemical and biochemical reactions.
  • the reaction may be carried out over a relatively wide range of temperatures, e.g., about 4 °C to about 50 °C, typically about 35 to about 40 °C.
  • the pH of the aqueous phase is typically about 5.5 to about 9.
  • the ratio of the water phase to the organic phase may range from 100:0 to 0.1 :99.9 parts by weight, respectively.
  • Reaction time may varying from minutes to hours (e.g., from about 10 minutes to about 24 hours or more) depending on the desired yield and the synthesis may be achieved both with and without physical agitation of the reaction mixture.
  • oligomers and co-oligomers can be separated from the reaction mixtures through precipitation, filtration, selective extraction, column chromatography, lyophilization, and evaporation techniques. Often, the oligomeric and co-oligomeric products are precipitates which may be easily filtered or centrifuged away from the peptide and ester reaction product mixture containing free hydroxy acids and unreacted ⁇ -amino acids. For example, soluble oligomer and co-oligomeric products can be separated from reaction product mixture using membrane filtration. Alternatively, free amino acids and ⁇ -hydroxy acids may be removed from the product mixture using ion exchange or other applicable chromatographic technique. The selection of separation procedure is dependent on the desired oligomers and co-oligomers.
  • the reaction mixture after the reaction is formed will contain the enzyme and a greater proportion of the non-selected enantiomers of ⁇ -hydroxy carboxylic acid and ⁇ -amino acid than the non-selected enantiomers.
  • the enzyme may be removed from solution by filtration and recycled, thereby leaving a solution primarily containing monomers of the non-selected enantiomers.
  • the non-selected enantiomers may be separated from the solution by rotary evaporation or by other methods known in the art. After separation, the non-selected enantiomers may be transformed into the monomeric form of the selected isomer through base racemization and recycled.
  • the non-selected enantiomers may be transformed into the monomeric form of the selected isomer through base racemization and recycled.
  • the mixture will primarily be comprised of papain, D-methionine, and D-HMB.
  • Papain may be simply filtered from the reaction mixture by size exclusion chromatography leaving a solution primarily containing D-methionine (e.g., approximately 95% or more of the remaining racemic mixture of methiomne) which may be isolated by rotary evaporation.
  • D-methionine e.g., approximately 95% or more of the remaining racemic mixture of methiomne
  • the process of the present invention can be used to recover the selected enantiomer from the separated oligomer.
  • the recovered oligomer may be hydrolyzed with acid to separate the first, selected enantiomer or derivative thereof from other hydrolyzates.
  • the separated enantiomer may then be racemized and recycled for further use.
  • Biological systems such as ruminants, poultry, swine, and aquatic animals readily absorb and utilize the L-isomers of amino acids but are unable to utilize the corresponding D- isomer without first transforming the D-isomer into the L-isomer through an oxidation followed by transamination enzymatic reactions.
  • feed supplements of L-isomer oligomers and co-oligomers are advantageous as they can be utilized by the animal with minimal expenditure of energy, which ultimately improves the growth rate of the animals.
  • the enantioselective oligomerization and co-oligomerization of specific enantiomers of ⁇ -hydroxy carboxylic acids, ⁇ -amino acids, or derivatives thereof results in the purification of the species in the original enantiomeric mixtures.
  • the resulting oligomers and co-oligomers formed are therefore a more pure form of L-enantiomers.
  • the L-enantiomers may then be isolated through addition of acid to the oligomers and co-oligomers thereby hydrolyzing them into the L-enantiomers with which they are comprised.
  • the L-isomers may be further isolated from each other through chromatographic or other separation means known in the art.
  • the reaction mixture contains an increasingly greater proportion of the non- selected enantiomer species, for example, the D-enantiomers.
  • the remaining unreacted enantiomers such as D- ⁇ -hydroxy carboxylic acids, D- ⁇ -amino acids, or derivatives thereof, may be recovered from the reaction mixture by rotary evaporation or other means.
  • the D-isomers of ⁇ -hydroxy carboxylic acids, ⁇ -amino acids, or derivatives thereof may then be transformed to their respective L-isomer form through base racemization and reused as reactants in additional oligomerization and co-oligomerization reactions.
  • the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer and ⁇ -amino acid oligomer compositions of the present invention may be provided to animals as an amino acid supplement.
  • the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer and ⁇ -amino acid oligomer compositions may be fed or otherwise administered orally, or sprayed into the eye, ear or nasal cavity of an animal, preferably a ruminant.
  • the compositions may be injected, or administered bucchally (i.e., to the gums), sublingually (i.e., beneath the tongue) or rectally.
  • ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer compositions of the present invention or ⁇ -amino acid oligomer compositions enzymatically formed in the absence of an ⁇ -hydroxy carboxylic acid may also be used to supplement the diets of animals not possessing rumens, such as poultry, swine, and aquatic animals. As with ruminants, these compositions may be fed or otherwise administered orally, bucchally, sublingually, rectally, sprayed into the eye, ear or nasal cavity of an animal, or injected into the animal.
  • compositions may be further used in aquaculture by applying the compositions to an aquatic habitat in a particle size that is able to be ingested by the target animal.
  • the compositions may be used in aquaculture as particles of the ⁇ -amino acid oligomer or ⁇ - hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer itself or as an ingredient in the animal's feed rations. While oligomers and co-oligomers may be utilized in varying sizes, feed mills typically manufacture feed supplements in particle sizes of about 0.25 mm or more for incorporation into feed rations.
  • the feed ration pellets containing the oligomer or co- oligomer ingredient would be sized according to the animal to which it is being fed.
  • fish such as carp and trout
  • the oligomer or co-oligomer compositions may be fed as an ingredient of a feed ration that is applied to the surface of the water.
  • Feed mills typically produce fish feed rations in particle sizes that range between about 4 mm to about 5 mm in diameter.
  • the compositions may be applied to the surface of the water as pure forms of the oligomer or co-oligomer or as ingredients of a feed ration in smaller feed particle sizes.
  • Feed mills typically produce rations for smaller aquatic animals in particles that are at least 1.6 mm in diameter, preferably between about 2 mm to about 3 mm in diameter, more preferably between about 2.2 mm to about 2.4 mm in diameter.
  • the length of the feed ration pellets are typically manufactured to be two to three times the length of the diameter. While feed mills may produce feed rations in particular size ranges, the dimensions of the ration that incorporates the oligomer or co-oligomer composition maybe varied therefrom without diminishing the effectiveness of the oligomer or co-oligomer.
  • the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer or ⁇ -amino acid oligomer compositions may be used as a protective coating for vitamins, minerals, and other nutrient supplements that are ingested by both humans and other animals, for example, ruminants, poultry, swine, and aquatic animals.
  • Vitamins and other nutritional supplements e.g., vitamin A, acetate or palmitate ester, and the like
  • vitamins and other nutritional supplements which are ingested often must be protected against acids and proteolytic enzymes present in the stomach and rumen in order to be available for absorption by the animal in the intestine.
  • the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer or ⁇ -amino acid oligomer compositions provide a superior alternative to animal-based coatings.
  • some oligomer or co-oligomer compositions may be resistant to degradation in the stomach and rumen, as well as insoluble in water, they may be used as vitamins, minerals, and other nutrient coatings.
  • the co-oligomer compositions may coat supplements in order for the vitamins, minerals, and other nutrients to bypass microbial degradation that occurs in the rumen.
  • the co-oligomer compositions are completely degraded wherein the ruminant may absorb the ⁇ -hydroxy carboxylic acids, amino acids, and the previously coated vitamins, minerals, and nutrients. Once absorbed, the ruminant may convert the ⁇ -hydroxy carboxylic acids from the coating to its respective amino acid for utilization by the ruminant. Since the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer and ⁇ -amino acid oligomer coatings are enzymatically synthesized, they do not introduce the risk of infecting the ruminant with a disease that may have been carried by an animal from which a fat or gelatin based coating is derived.
  • gastric acids and enzymes present in the stomach begin to degrade the coating as it passes through the stomach to the intestine. Once in the intestine, the coating is completely degraded, and the previously encapsulated vitamins, minerals, and other nutrients may be absorbed.
  • Some supplements such as vitamin A, are soluble in water. Left uncoated, the soluble supplements would dissolve into the surrounding water and pollute it rather than provide the aquatic animals with the vitamins, minerals, and other nutrient supplements they need.
  • the ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer or ⁇ -amino acid oligomer compositions are also insoluble in water, they also may be beneficially used to coat vitamins, minerals and other nutrient supplements for use in aquaculture.
  • ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer or ⁇ -amino acid oligomer coatings may be achieved by methods known in the art.
  • the oligomer or co-oligomer may be dissolved in a volatile solvent and subsequently spray coated on a fluidized bed of the supplements. As the solvent evaporates, a coating of ⁇ -hydroxy carboxylic acid/ ⁇ -amino acid co-oligomer or ⁇ - amino acid oligomer remains on the supplements which may then be provided to the animal.
  • aquaculture refers to the cultivation of aquatic animals including, but not limited to, freshwater and salt water fish (e.g., carp, trout, catfish, bass, sea bass, cod, salmon, and fish related thereto) and crustaceans (e.g., shrimp, crabs, lobster, freshwater shrimp, and the like).
  • freshwater and salt water fish e.g., carp, trout, catfish, bass, sea bass, cod, salmon, and fish related thereto
  • crustaceans e.g., shrimp, crabs, lobster, freshwater shrimp, and the like.
  • hydrocarbon and “hydrocarbyl” as used herein describe organic compounds or radicals consisting exclusively of the elements carbon and hydrogen.
  • moieties include alkyl, alkenyl, alkynyl, and aryl moieties. These moieties also include alkyl, alkenyl, alkynyl, and aryl moieties substituted with other aliphatic or cyclic hydrocarbon groups, such as alkaryl, alkenaryl and alkynaryl. Preferably, these moieties comprise 1 to 20 carbon atoms.
  • substituted hydrocarbyl moieties described herein are hydrocarbyl moieties which are substituted with at least one atom other than carbon, including moieties in which a carbon chain atom is substituted with a hetero atom such as nitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogen atom.
  • substituents include halogen, heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, protected hydroxy, keto, acyl, acyloxy; nitro, amino, amido, nitro, cyano, and thiol.
  • alkyl groups described herein are preferably lower alkyl containing from one to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include methyl, ethyl, propyl, isopropyl, butyl, hexyl and the like.
  • alkenyl groups described herein are preferably lower alkenyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, hexenyl, and the like.
  • alkynyl groups described herein are preferably lower alkynyl containing from two to six carbon atoms in the principal chain and up to 20 carbon atoms. They may be straight or branched chain and include ethynyl, propynyl, butynyl, isobutynyl, hexynyl, and the like.
  • aryl or “ar” as used herein alone or as part of another group denote optionally substituted homocyclic aromatic groups, preferably monocyclic or bicyclic groups containing from 6 to 12 carbons in the ring portion, such as phenyl, biphenyl, naphthyl, substituted phenyl, substituted biphenyl or substituted naphthyl. Phenyl and substituted phenyl are the more preferred aryl.
  • halogen or halo as used herein alone or as part of another group refer to chlorine, bromine, fluorine, and iodine.
  • heterocyclo or “heterocyclic” as used herein alone or as part of another group denote optionally substituted, fully saturated or unsaturated, monocyclic or bicyclic, aromatic or nonaromatic hydrocarbon groups having at least one heteroatom in at least one ring, and preferably 5 or 6 atoms in each ring.
  • the heterocyclo group preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1 to 4 nitrogen atoms in the ring, and may be bonded to the remainder of the molecule through a carbon or heteroatom.
  • Exemplary heterocyclo include furyl, thienyl, pyridyl and the like.
  • substituents include one or more of the following groups: hydrocarbyl, substituted hydrocarbyl, keto, hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy, alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, and thiol.
  • the acyl moieties described herein contain hydrocarbyl, substituted hydrocarbyl or heterocyclo moieties.
  • hydroxyl protecting group and "hydroxy protecting group” as used herein denote a group capable of protecting a free hydroxyl group ("protected hydroxyl") which, subsequent to the reaction for which protection is employed, may be removed without disturbing the remainder of the molecule.
  • protected hydroxyl a group capable of protecting a free hydroxyl group
  • a variety of protecting groups for the hydroxyl group and the synthesis thereof may be found in "Protective Groups in Organic Synthesis" by T. W. Greene, John Wiley and Sons, 1981, or Fieser & Fieser.
  • Exemplary hydroxyl protecting groups include acetyl (Ac), benzyl (PhCH 2 -), 1-ethoxyethyl (EE), methoxymethyl (MOM), (methoxyethoxy)methyl (MEM), (p-methoxyphenyl)methoxymethyl (MPM), tert- butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBPS), tert-butoxycarbonyl (Boc), tetrahydropyranyl (THP), triphenylmethyl (Trityl, Tr), 2-methoxy-2-methylpropyl, benzyloxycarbonyl (Cbz), trichloroacetyl (OCCCl 3 ), benzyloxymethyl (BOM), tert-butyl (t- Bu), triethylsilyl (TES), trimethylsilyl (TMS), and triisopropylsilyl (TIP S).
  • HMB shall mean the 2-hydroxy analog of methionine, i.e., 2- hydroxy-4-(methylthio)butyric acid.
  • chiral refers to a particular stereoisomer of a molecule.
  • L-methionine refers to a particular stereoisomer of a molecule.
  • L-HMB refers to a particular stereoisomer of a molecule.
  • identical chirality or “identical chiral configuration” refers to the chiral carbon of two or more molecules having the same stereoisomeric configuration.
  • all L-isomers of ⁇ -amino acid have identical chiral configuration.
  • R a R b C(NH 2 )COOH wherein R a is hydrogen, hydrocarbyl, substituted hydrocarbyl or heterocyclo; and R b is hydrogen, the -COOH, -NH2, R a , and R b constituents of L-isomers of ⁇ -amino acid have the same spatial arrangement around the chiral carbon.
  • the two or more L-enantiomers of a specific ⁇ -hydroxy carboxylic acid such as two molecules of L-HMB, will have identical configuration to each other.
  • enantioselective refers to the selection of a specific enantiomer of an enantiomeric mixture and interactions with said enantiomer.
  • an enantioselective enzyme such as papain, selectively catalyzes the linkage of L-methionine ethyl esters to form an oligomer of L-methionine residues.
  • This example demonstrates the enzymatic synthesis of oligomers comprising methionine and co-oligomers comprising HMB-methionine, as well as their characterization using reverse-phase HPLC and matrix assisted laser desorption ionization-time of flight mass spectroscopy (MALDI-TOF MS) analysis.
  • MALDI-TOF MS matrix assisted laser desorption ionization-time of flight mass spectroscopy
  • the oligomerization comprised forming a reaction mixture at a temperature of 37°C consisting of nanopure filtered water (10 mL) containing HMB ethyl ester (0.7 M) and methionine ethyl ester (0.7 M) along with L-cysteine (0.1 M), EDTA (10 mM), sodium citrate (1M) and 1 % papain (by weight of the monomer) at a pH of 5.5.
  • the mixture was allowed to incubate for 24 hours. After 10 minutes, aliquots were removed at regular intervals to monitor the degree of oligomerization and co-oligomerization and the disappearance of the substrate.
  • the experiment was conducted in accordance with reaction conditions for the papain-catalyzed oligomerization of methionine analogs as described by Jost et al. xnHelv. Chim. Ada, 63 (1980) 375-384 (1980).
  • the oligomerization comprised forming a reaction mixture consisting of L-methionine ethyl ester (5 g) and HMB ethyl ester (5 g) dissolved in nanopure water (50 mL) containing sodium bicarbonate buffer (0.1 mole) and L-cysteine (4 mmole). The pH was adjusted to 9 and the solution was made up to 100 mL and incubated for 24 hours at 37°C after adding papain (2 g).
  • oligomers were soluble in dimethyl sulfoxide (DMSO) and tetrahydrofliran (THF) a common mobile phase in GPC for separations.
  • DMSO dimethyl sulfoxide
  • THF tetrahydrofliran
  • Persulfonation of oligomers enhanced the polarity of the oligomers to a point that these could be separated on a C- 18 column with a moderately polar mobile phase (M. Spindler, R. Stadler and H. J. Tanner, J. Agri. Food Chem., 32( 6 ) (1984)1366-1371).
  • the oligomer sulfone residues in the rotary evaporator round bottom flasks were dissolved in a 40:60 acetonitrile / water mixture (5 mL) and filtered through a membrane filter. A 10 ⁇ L aliquot of the solution was injected into a HPLC.
  • the separation of persulfonated oligomers was achieved with a C-18 column using a phosphate buffer - acetonitrile mobile phase. A linear gradient was used to facilitate separations. In this gradient the mobile phase composition was changed from 100% eluant A (phosphate buffer, pH 6.5) to 60%o A and 40% B (20% Acetonitrile) in 20 minutes. The mobile phase flow rate was maintained at 1 mL min " l .
  • the separated oligomers were detected with a UV/NIS diode array detector.
  • the MALDI-TOF spectra of methionine (Met) oligomers are shown in Figure 1.
  • the spectra contain distinct ions which are separated by mass 131.
  • This mass (131) represents the repeating Met moiety (C 5 H 9 ⁇ O S), since the masses of the N and C terminal methionine residues are 132 and 148 respectively. Therefore, a methiomne hexamer (Met) 6 , ( N Met- (Met) 4 -Met c ) + I should appear at m/z 805 and (Met) 7 should appear at m/z 936.
  • the m/z values of the dominant ions did not correspond to this series, instead, one set of dominant ions appeared at m/z 826, 957, 1088, 1219, 1350 and 1481. These ions most likely correspond to ((Met) n + Na + ), where n is an integer between 6 and 11. The second group of ions appeared at m/z 724, 855, 986, 1117, 1248 and 1379. These ions most likely correspond to the series ( N Met-(Met)- Met-O-C 2 H 5 ) + Na + .
  • a third set of ions appeared at m/z 739, 870, 1001 and 1134, these ions most likely correspond to N Met-(Met) n -Met-O-C 2 H 5 + K + .
  • a fourth set of unidentified ions seem to be present at regular intervals in the clusters and which might be assigned the mass values, 842, 973, 1104, 1235 and 1366 co ⁇ esponding to the series N Met-(Met) n -Met c ) + K + . hi all these cases "n" varied between 6 to 11.
  • the apparent absence of these ions does not necessarily mean the absence of HMB- (Met) n co-oligomers in the product mixture.
  • the absence of the ions can be attributed to two factors. The first relates to the low resolving power of the MALDI-TOF MS, which would prevent the resolution of the H + HMB-(Met) n - Met c ions at m/z 806, 937, 1118 from the H + N Met-(Met) n -Met c ions at m/z 805, 936, 1117.
  • the second and more probable cause is the low intensity of the H + HMB- (Met) n - Met 0 ion due to the absence of a good protonation site in HMB- (Met) n co-oligomers.
  • HMB-poly-methionine sulfones The chromatographic separation of HMB-poly-methionine sulfones is shown in Figure 4.
  • This chromatogram contained a number of peaks, which were not present in the poly methionine sulfone chromatogram.
  • the resulting HMB-(Met) n co-oligomers, with the terminal hydroxyl, should be less polar than the corresponding (Met) n oligomers with the terminal amine moiety. Therefore, the HMB containing co-oligomers should elute later than the co ⁇ esponding Met oligomers and this appears to be the case.
  • This example demonstrates four alternative procedures for the enzymatic synthesis of oligomers comprising lysine and co-oligomers comprising HMB-lysine.
  • the experiment was designed to compare three novel synthesis procedures to that of Puigserver et. al. 1 who reported a procedure for papain catalyzed polymerization of lysine.
  • PEG bound Papain system (Puigserver's Method): lOmM of substrate was added to 98 mL of toluene along with 0.8 mL of Diisopropyl amino ethyl and 0.2 mL of mercaptoethanol, followed by 17 mM of PEG 2000 modified Papain. The mixture was allowed to incubate for 24 hours, before being evaporated and redissolved in deionized water and analyzed on a ion-pair liquid chromatography column.
  • Two Phase Toluene Water System : This solvent system was evaluated with varied phase ratios, two of which are described below: a) 10 mM of substrate was added to 98 mL of toluene along with 0.8 mL of Diisopropyl amino ethyl and 0.2 mL of mercaptoethanol, followed by 1 mL of aqueous papain suspension. The mixture was allowed to incubate for 24 hours, before being evaporated and redissolved in DI water and analyzed on a ion-pair liquid chromatography column.
  • Reverse Micellar System 10 mM of substrate was dissolved in 98 mL of a reverse micellar solution containing 150 mM of AOT (3.33 g), 0.8 mL of diisopropyl amino ethyl and 0.2 mL of mercaptoethanol in isooctane. 1 mL of aqueous papain solution was added to the mixture and allowed to incubate for 24 hours, at the end of which the mixture was heated to denature the enzyme and the oligomeric and co-oligomeric products extracted with 1 M NaCl solution. The solution was later analyzed on a ion-pair liquid chromatography column.
  • Three Phase DFP:Octane ater System: To a two phase system comprising of 4.45 mL of DFP and 4.45 mL of octane was added 100 mM of substrate along with 0.08 mL of Diisopropyl amino ethyl and 0.02 mL of mercaptoethanol. The addition of 1 mL of aqueous papain suspension which is insoluble in either of the phases converts this system to a three phase system. The mixture was allowed to incubate for 24 hours, before being evaporated and redissolved in DI water and analyzed on a ion-pair liquid chromatography column
  • HMB-methiomne and HMB-lysine co-polymers were synthesized enzymatically through a papain-catalyzed reaction along with poly-methionine and poly-lysine (as controls) as described in Examples 1 and 2.
  • the biological release of the amino acids from the oligomers was examined using several digestive enzymes including pepsin, trypsin, chymotrypsin, intestinal peptidase and carboxypeptidase.
  • the oligomers were dissolved at lOmg/mL in 0.15 HCl (pH 2.5) or 50 mM KPO 4 (pH 7.5). Samples (0.5 mL) were incubated with 10 units of each enzyme for 2 hours at 37°C.
  • HMB-methionine and HMB-lysine can be hydrolyzed by strong acid and heat.
  • HMB-met is digested only 3.5% by pepsin and not at all by the other proteases.
  • Poly-lysine can be digested by intestinal peptidase (20% in 2 hours at 37° C) but not by other proteases.
  • HMB-lysine is not digested by any of the proteases tested.
  • Met oligomers and HMB-Met co-oligomers produced through papain mediated enzymatic reactions at pH 5.5 and pH 9.0 according to the procedure described in Example 1 were subjected to persulfonation.
  • Persulfone derivatives were separated with the reverse phase liquid chromatography (RPLC).
  • RPLC reverse phase liquid chromatography
  • the separated oligomers and co-oligomers were monitored with a UV absorption spectrophotometeric detector and an electrospray ionization interface (ESI) mass spectrometer.
  • the absorption wavelength was set at 210 nm.
  • the mass spectrometer was operated in positive and negative ion modes.
  • the outputs of the UV absorption detector and the positive ion ESI-MS are shown in Figures 14 and 15.
  • chromatogram of (Met)n persulfones obtained with the UV detector and the positive ion total ion chromatogram (TIC) were similar to the chromatograms obtained from earlier with a earlier experiments using a RPLC-Diode Array Detector (DAD) system. These results had indicated the formation of Met homo-oligopeptides and HMB-Met co-oligomers. The results were supported by data obtained from the matrix assisted laser desorption ionization - mass spectrometry (MALDI-MS) experiments.
  • MALDI-MS matrix assisted laser desorption ionization - mass spectrometry
  • the positive ion TIC of HMB-(Met)n co-oligomers obtained with ESI-MS did not contain extra peaks observed in the LC-UV chromatogram.
  • the spectra of individual peaks in the HMB-Met co-oligomers did not provide any evidence for HMB-(Met)n co-oligomers formation.
  • the confirmation of the HMB-(Met)n was obtained by monitoring negative ions formed through electron attachment to the (Met)n and HMB-(Met)n chains.
  • the TIC of HMB-(Met)n in this case contained extra components (peaks) which corresponded to the extra peaks observed in the LC-UV chromatograms Figures 21 and 22.
  • HMB-(Met)n A few representative spectra for the HMB-(Met)n peaks are shown in Figures 23-25. As expected, the molecular ions for HMB-(Met)n appear at one mass unit higher than the corresponding (Met)n ions, addition, the retention times of HMB-(Met)n peaks are longer than the co ⁇ esponding (Met)n peaks. This is to be expected since the terminal amine group of the (Met)n imparts higher polarity to methionine oligomers than the terminal hydroxyl to the HMB-(Met)n co-oligomers.
  • Both the positive ion and negative ion LC-EShMS data show that the predominant (Met)n are composed of four to ten methionine residues.
  • the negative ion LC-ESI data shows that the predominant HMB-(Met)n co-oligomers contain one HMB residue and four to nine methionine residues.
  • the relative distribution of (Met)n oligomers and HMB- (Met)n co-oligomers is presented in Figures 26 and 27.
  • the positive ion spectra ( Figure 28A) of shows two series of ions that are 28 mass units apart.
  • One series containing ions m/z 674, 805, 936, 1067, 1198 and 1229 co ⁇ espond to ((Met) n + H + ) ions.
  • the other series of ions which occur at m/z 702, 833, 964, 1095, 1226 and 1357 co ⁇ espond to ((Met)n-OET) + H + ions.
  • Ions in both series are 131 mass units or one methionine residue (C 5 H 9 NOS) apart, hi both series, the value of n lies between 4 - 10.
  • the polymethionine and HMB-polymethionine prepared from Met-ethyl ester and HMB-ethyl ester were also subjected to MS-MS experiments.
  • the freeze-dried precipitates were dissolved in DMSO (2 ⁇ g/ ⁇ l) and the solution was introduced into the mass spectrometer with the sonic spray interface at the rate of 1 mL/hr.
  • the solution was mixed with 1:1 acetonitrile : water mixture containing 0.1% acetic acid.
  • the total fluid volume entering the SSI-MS was maintained at 0.2 mL/min.
  • the parent ion and spectra obtained with the system are shown in Figure 29.
  • Tyr-OMe (equal amounts (wt%) in the case of HMB-OEt and Tyr-OMe) were dissolved in 9.5 mL of 1M citrate buffer. EDTA and L-Cysteine were added . The reaction mixture was adjusted to a pH of 5.5 and 0.5 mL of papain suspension was added to the mixture. The reaction mixture was incubated in a shaker for 24 hours. The enzyme was then denatured by heating the reaction mixture to 80°C for 10 minutes. The reaction mixture was cooled to room temperature.
  • the reaction mixture was filtered to collect the precipitate.
  • the oligomer and co- oligomers in the precipitate were dissolved in DMSO to separate them from the monomers which are relatively insoluble in the solvent.
  • the separated solvent was then evaporated to re-precipitate the oligomer and co-oligomers, which were then washed with water and freeze- dried.
  • the protonated ion at 862 most probably represents the tyrosine oligomers with five residues and an ethyl ester attached to the C-terminal end.
  • the ion at m/z 1025 most likely results from the (Tyr) 6 OEt+H
  • the ion at m/z 997 results from (Tyr) 6 +H + .
  • the presence of oligomers with 5 to 8 Tyr residues is clearly evident, furthermore, the (Tyr) 6 was found to be the most prominent oligomer.
  • the positive ion mass spectrum of HMB-tyrosine co-oligomer is shown in Figure 32A.
  • the dominant ions in this spectrum were the same ions observed in the positive ion spectrum of polytyrosines, Figure 31 A.
  • additional ions appeared at m/z 831, 994 and 1157. These ions appear at a mass difference of 133, suggesting the presence of a HMB residue in the co-oligomer.
  • the peak at m/z 831 most probably represents the co- oligomer with one HMB residue and 4 tyrosine residues with the ethyl ester moiety (HMB- (Tyr) 4 OEt + H "1" ).
  • the residues at m/z 994 and 1157 represent co-oligomers with one HMB residue and 6 and 7 tyrosine residues respectively.
  • the weak intensity of these ions in part relate to lower proton affinity of the hydroxyl group.
  • the papain-catalyzed synthesis of leucine and HMB co-oligomers was performed. Synthesis of leucine oligomers and HMB-leucine co-oligomers was initiated with leucine ethyl ester and HMB ethyl ester as the substrates. The overall synthesis and purification approach was similar to the one used in the case of methionine and HMB-methionine. Reaction rates similar to those obtained with methionine and tyrosine were achieved. Approximate oligomer yield was 58%. The freeze-dried oligomer precipitates were solubilized in DMSO. The solution concentration was brought to approximately 2 ⁇ g/ ⁇ l.
  • the negative ion mass spectrum of the leucine oligomers is shown in Figure 33B.
  • the overall appearance of the spectra is similar to that of the positive ion spectra.
  • the two dominant ion in this spectra co ⁇ espond to (Leu) 6 -OEt +Na and (Leu) 7 -OEt +Na.
  • HMB-Leu co-oligomers The positive and the negative ion spectra of HMB-Leu co-oligomers are shown in Figure 34. As expected, the positive spectra contained all of the dominant ions observed in the positive ion ESI-MS spectra of (Leu) n . However, three additional strong ions at m/z 740, 853 and 866 were also found. These masses co ⁇ espond to sodiated co-oligomers HMB- (Leu) 5 + Na + , HMB-(Leu) 6 + Na + and HMB-(Leu) 7 + Na + respectively. Thus, formation of co-oligomers with one HMB residue with five to seven leucine residues is clearly evident.
  • Papain catalyzed synthesis of phenylalanine and HMB co-oligomers was also conducted. Synthesis of phenylalanine oligomers and HMB- phenylalanine co-oligomers was initiated with phenylalanine ethyl ester and HMB ethyl ester as the substrates. The overall synthesis and purification approach was similar to the one used in the case of methionine and HMB-methionine in Example 1. The oligomerization reaction did not proceed when phenylalanine was the only substrate present in the reaction mixture. The reaction did proceed when HMB-ethyl ester was added to the reaction mixture. Reaction rates similar to those with methionine and tyrosine were achieved.
  • Approximate oligomer yield was 90%.
  • the freeze-dried oligomer precipitates were solubilized in DMSO.
  • the solution concentration was brought to approximately 2 ⁇ g/ ⁇ l.
  • the solution was mixed with 1 : 1 acetonitrile: water mixture containing 0.1% acetic acid.
  • the total fluid volume entering the ESI-MS was maintained at 0.2 mL min. "1 As stated earlier, phenylalanine homo-oligomers were not formed.
  • the ESI-MS results of HMB-PheHMB co-oligomerization reaction are given in Figure 35.
  • the positive ion spectra of HMB-Phe co-oligomers are depicted in Figure 35 A, while the negative ion spectra are depicted in Figure 35B.
  • the positive spectra of the co- oligomers yielded three ion peaks at m/z 790, 937 and 1084.
  • the two-phase reaction system consisted of a small amount of polar phase and a larger amount of an immiscible non-polar phase.
  • the polar phase comprised water, isopropyl amino ethyl and mercaptoethanol. This phase also contained the amino acid ester substrate and papain.
  • parameters such as the volume ratio of the aqueous and the non-aqueous phase, composition of additives, concentrations of the additives, concentrations of the substrates, and the concentration of the enzyme were varied. The effect of these parameters on the degree of oligomerization and yield were monitored.
  • the results of the experiments are summarized below: A.1 Aqueous : Organic Phase Ratio
  • the graph shows that the reaction is nearly complete within the first four hours and only a marginal increase in yield is obtained at longer incubation periods. Analyses of oligomers obtained after different time periods showed that the time periods shorter than 4 hours yield an even distribution of oligomers from (Lys) 2 to (Lyn) 8 , while the longer periods yield higher concentrations of (Lyn) 4 to (Lys) 6 oligomers, Figure 43.
  • the three-phase system consisted of an aqueous phase present between two immiscible non-aqueous phases, one lighter than the aqueous phase and the other heavier than the aqueous phase.
  • the heavier phase comprised decafluoropentane and the lighter phase comprised n-octane.
  • Isopropyl ethyl amine and mercaptoethanol additives were added to the aqueous phase along with the lysine ethyl ester (substrate) and papain (enzyme).
  • the effects of parameters such as the relative volumes of aqueous to non-aqueous phases, the concentration of the additives, the substrate concentration and the enzyme activity on oligomers yield and degree of oligomerization were monitored through a set of experiments.
  • the ratio of the non-aqueous phase volume to aqueous phase volume was varied by changing the volumes of the two organic solvents in equal proportion while holding the aqueous phase volume constant. Substrates, antioxidant additives and enzyme were added to the aqueous phase. The reactants and the enzyme were placed in a sti ⁇ ed reactor and allowed to incubate at 37 °C for 24 hours. The total oligomers yield was determined gravimetrically, while the degree of oligomerization was determined through RPLC analysis. Results of gravimetric determination are represented in Figure 44.
  • the incubation period for the three-phase reaction system was examined through another set of experiments.
  • the experiments were conducted with three phase reaction mixtures consisting of aqueous phase and total organic phases, the aqueous : organic phase ratio was set at 1 :9.
  • the additive concentrations were varied.
  • the incubation periods were varied from 30 minutes to 30 hours. After each time period, total oligomer yield and degree of oligomerization were examined. Results are shown in Figure 48. The results indicate that the reaction reaches completion in approximately six hours and nearly 90 % of the initial substrate mass is converted into the oligomers. Longer incubation periods did not lead to higher yields.
  • Trp-OMe was dissolved in 4.5 mL of 2M citrate buffer along with the EDTA and L- Cysteine. The pH was adjusted to 5 and 0.5 mL of papain suspension was added to the mixture. The mixture was placed in a shaker for three days and incubated. After three days, the enzyme was denatured by heating the broth for a duration of 10 minutes at 80°C.
  • the broth was filtered to collect the precipitate.
  • the broth could be centrifuged to collect the precipitate.
  • the oligomer and co-oligomer precipitate was dissolved in DMF to separate them from the monomers which are relatively insoluble in the solvent.
  • the solvent was evaporated and the precipitate was washed with water, followed by freeze-drying the precipitate to obtain the dry oligomers and co-oligomers.
  • a procedure similar to one used for the synthesis of Met oligomers and HMB-Met co-oligomers of Example 1 was employed for the synthesis and purification of leucine, phenylalanine, and tryptophan oligomers.
  • An amino acid ester e.g., 1 mmole
  • 2M phosphate buffer solution pH 7.5 containing lmM dithiothreithol and 5mM EDTA.
  • a papain suspension e.g., 0.1 mmole was added to the solution. The solution was incubated for two days with continuous shaking, after which the precipitate formed was filtered and washed with water several times to remove the free monomers. The precipitate was dried in vacuo and then subjected to analysis.
  • L-amino acid ethyl ester or D, L-amino acid esters and racemic HMB ethyl ester were dissolved in buffer containing L-cysteine, EDTA and sodium citrate at pH 5.5 as detailed in Tables 5-9.
  • the buffer pH was set at 5.5 and 0.5 mL of papain suspension containing 15 mg of protein was added to the reaction broth. After incubation in a shaker for 24 hours at 35 °C, the enzyme was denatured by heating the broth to 80°C for 10 minutes. The broth with denatured enzyme was then cooled to room temperature.
  • the oligomer and co-oligomer precipitate obtained from the reaction was washed exhaustively with water to remove the monomers and the washed precipitate was then freeze-dried.
  • the freeze-dried oligomer and co-oligomer precipitate was solubilized in DMSO to form a 2 ⁇ g/ ⁇ L solution.
  • HMB co-oligomers were re-precipitated by addition of distilled deionized water.
  • the purified oligomers and co-oligomers were freeze dried.
  • oligomer and co-oligomer were dissolved in appropriate solvents or mixtures solution (DMSO and 1:1 acetonitrile: water mixture) for chemical characterization with liquid chromatography (LC) diode a ⁇ ay detector, LC-electrospray mass spectrometry (ESI-MS) , sonic spray ionization - MS (SSI-MS) and matrix assisted desorption ionization - MS (MALDI-TOF).
  • LC liquid chromatography
  • ESI-MS LC-electrospray mass spectrometry
  • SSI-MS sonic spray ionization - MS
  • MALDI-TOF matrix assisted desorption ionization - MS
  • a set of experiments was carried out to discern enantioselectivity of papain wherein oligomers and co-oligomers were catalyzed from an amino acid and HMB.
  • the experiments entailed enantioselective determination of the reactants (e.g., methionine and HMB co-oligomerization from enantiomeric mixtures of D, L-methionine and D, L-HMB and separation of supernatant and oligomer and co-oligomer precipitates).
  • the supernatant was filtered to remove suspended matter, and the precipitate was purified through repeated washing and DMSO back-extraction.
  • the purified oligomer and co-oligomer precipitates were hydrolyzed with acid.
  • the Met oligomers and HMB-Met co-oligomers were synthesized through papain mediated enzymatic reactions at pH 5.5.
  • the synthesis involved the following steps:
  • Racemic mixtures of both D, L-Met-OEt and D, L-HMB-OEt were dissolved in 4.5 mL of 2M citrate buffer along with EDTA and L-Cysteine. The pH was set to 5 and 0.5 mL of papain suspension was added to the mixture. The mixture was incubated in a shaker for 3 days.
  • the enzyme e.g., papain
  • the enzyme was denatured by heating the mixture for 10 minutes at 80°C.
  • the mixture was filtered and the precipitate collected. (Alternatively, the mixture may be centrifuged to separate the precipitate from the supernatant).
  • the oligomers and co-oligomers were dissolved in DMF to separate them from the monomers, which are relatively insoluble in DMF.
  • a 25 mg aliquot of purified oligomers and co-oligomers obtained from the racemic Met-OEt and HMB-OEt was placed in 10 mL vials and hydrolyzed with 2 mL of 6.05(N) HCl at 110°C for 24 hours.
  • the clear solutions obtained after hydrolysis were diluted with nanopure water and injected in an LC equipped a diode a ⁇ ay detector (DAD).
  • DAD diode a ⁇ ay detector
  • Enantioselective HPLC analysis was carried out with a proline-Cu based column EC 250/4 Nucleosil Chiral 1 (Macherey-Nagel, hie, Easton, PA).
  • the mobile phase consisted of 0.5 mM cupric sulfate (pentahydrate) solution in nanopure water.
  • Column oven temperature was maintained at 60°C. Separated analytes were monitored with a UV absorbance DAD.
  • This example demonstrates the synthesis of co-oligomers comprising lactic acid (an ⁇ -hydroxy carboxylic acid) with oligomers of the ⁇ -amino acids methionine, leucine, tyrosine, phenylalanine and tryptophan.
  • the experiment consisted of esterifying D,L-Lactic acid with acidified ethyl alcohol by refluxing it at 80°C for 8 firs to produce lactic acid ethyl ester, which was used in each of the oligomerization reactions.
  • the oligomerization consisted of forming a mixture by dissolving each of the amino acid ethyl esters and the lactic acid ethyl ester in various amounts in a pH 5.5 buffer containing, L-cysteine, EDTA and sodium citrate as shown in the tables below. At the end of 24 hours, the mixture was heated to 80°C for 10 mins to denature the enzyme. The supernatant was analyzed on RPLC to determine the yield of the reaction.
  • the precipitate was washed thoroughly with water to obtain a monomer (amino acid and lactic acid) free product.
  • the product was freeze dried and a small part of it was dissolved in DMSO and introduced into the ESI-MS and the mass spectrum obtained was recorded.
  • Lactic acid-methionine oligomers were synthesized using the general procedure described above.
  • the ingredients for the oligomerization reaction mixture consisted of the following:
  • the oligomerization produced an yield of 75%> and the positive ion and negative ion spectra are reproduced in Figs. 55A and 55B respectively.
  • the positive ion spectrum shows the presence of 2 dominant peaks at 834 and 965. These peaks most probably represent the homo-oligomers, N Met-(Met) 4 -Met c -OEt and N Met-(Met) 5 -Met c -OEt respectively which are separated by the repeating methionine residue unit of mass 131.2.
  • the negative ion spectrum shows the presence of a series of peaks each separated by around 131 mass units.
  • One set of peaks appear at 774 and 905 and these most probably represent the deprotonated ions, N LA-(Met) 4 -Met c -OEt and N LA-(Met) 5 -Met c -OEt respectively.
  • Another set of ions appear at 809 and 940 and these mot probably represent the adducts of the above co-oligomers with the chloride ion.
  • Lactic acid-tyrosine oligomers were synthesized using the general procedure described above.
  • the ingredients for the oligomerization reaction mixture consisted of the following:
  • the positive ion and negative ion spectra are provided in Fig. 56A and 56B respectively.
  • the positive ion spectra shows the presence of a evenly spaced sets of two peaks with each peak separated from the other by 28 amu. This mass represents the difference in mass between C-terminal free acid and ethyl ester. Each set of peaks is separated by a mass of 163 units with is the repeating unit of the tyrosine residue.
  • the negative ion spectrum reveals a number of peaks with some of them forming a series separated by 163 units. One set appears at 1096, 1259 and 1422 and these most probably represent the presence of deprotonated co-oligomer ions formed from N LA-Tyr 5 -Tyr c -OEt, N LA-Tyr 6 -Tyr c -OEt and N LA-Tyr 7 -Tyr c -OEt respectively.
  • Lactic acid-leucine oligomers were synthesized using the general procedure described above.
  • the ingredients for the oligomerization reaction mixture consisted of the following:
  • the oligomerization produced a yield of 40% with respect to leucine.
  • the positive and negative ion specta are provided in Figs. 57A and 57B respectively.
  • the positive ion spectrum has a pair of peaks at 726 and 839 and these ions are separated by the repeating residue unit of leucine (113 amu) and they most probably represent the protonated ions of homo-oligomers, N Lleu-Leu 4 -Leu c -OEt and N Leu-Leu 5 -Leu c -OEt respectively.
  • Another ion appears at 698 and this is most probably the protonated ion of the homo-oligomer N Leu-Leu 4 - Leu c -OH.
  • Lactic acid-tryptophan oligomers were synthesized using the general procedure described above.
  • the ingredients for the oligomerization reaction mixture consisted of the following:
  • Lactic acid-phenylalanine oligomers were synthesized using the general procedure described above.
  • the ingredients for the oligomerization reaction mixture consisted of the following:
  • the lactic acid was esterified with iso-propyl alcohol and was used along with the phenylalanine ethyl ester.
  • An oligomerization yield of 80%> was obtained with respect to phenylalanine.
  • the positive and negative ion spectrum is provided in Fig. 59A and 59B respectively.
  • the positive ion spectrum reveals the strong presence of sodiated co-oligomers ions of N LA-(Phe) 3 -Phe c -OEt + Na + and N LA-(Phe) 4 -Phe c -OEt + Na + at masses 730 and 877 respectively. These peaks are separated by 147 units, which is the repeating residue unit of phenylalanine.

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Abstract

L'invention concerne une synthèse et une composition enzymatique d'oligomères et de co-oligomères comprenant des acides carboxyliques α-hydroxy et des acides ou des peptides α-amino. Dans un mode de réalisation préféré, un acide carboxylique α-hydroxy à configuration chirale spécifique est lié par une liaison amide à un acide α-amino spécifique à configuration chirale spécifique, ou est lié par une liaison amide à un peptide constitué de monomères d'acides α-amino présentant des configurations chirales identiques. Des enzymes protéolytiques permettent de catalyser une oligomérisation d'acide carboxylique α-hydroxy et d'acide α-amino. Le degré et la distribution de l'oligomérisation varient en fonction du type et des concentrations des mélanges réactionnels différents utilisés, ainsi que de la longueur de la durée réactionnelle permise. On peut donner les oligomères obtenus à des ruminants en tant que compléments aminoacides biodisponibles résistant à la dégradation dans la panse, et à d'autres animaux tels les porcins, les volailles et les animaux aquatiques.
PCT/US2002/013708 2001-05-02 2002-05-02 Oligomerisation enantioselective d'acides carboxyliques alpha-hydroxy et d'acides alpha-amino WO2002088667A2 (fr)

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DE102008042932A1 (de) 2008-10-17 2010-04-22 Evonik Degussa Gmbh Herstellung und Verwendung von Methionylmethionin als Futtermitteladditiv für Fische und Krustentiere
WO2010112365A1 (fr) 2009-03-31 2010-10-07 Evonik Degussa Gmbh Dipeptides utilisés comme additifs pour l'alimentation animale
WO2011147881A2 (fr) 2010-05-27 2011-12-01 Evonik Degussa Gmbh Dipeptides cycliques utilisés comme additifs d'aliments pour animaux
CN102422984A (zh) * 2011-10-26 2012-04-25 中国海洋大学 一种蛋氨酸寡肽及其在大菱鲆饲料中的应用
CN102429109A (zh) * 2011-10-26 2012-05-02 中国海洋大学 一种蛋氨酸寡肽及其在对虾饲料中的应用
US9655863B2 (en) 2012-07-12 2017-05-23 Novus International, Inc. Matrix and layer compositions for protection of bioactives
WO2017174398A1 (fr) 2016-04-07 2017-10-12 Cysal Gmbh Cyanophycine pour organismes aquatiques à alimentation lente
CN109234343A (zh) * 2018-09-30 2019-01-18 中国海洋大学 一种蛋氨酸寡肽及其制备方法和应用

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DE102008042932A1 (de) 2008-10-17 2010-04-22 Evonik Degussa Gmbh Herstellung und Verwendung von Methionylmethionin als Futtermitteladditiv für Fische und Krustentiere
WO2010112365A1 (fr) 2009-03-31 2010-10-07 Evonik Degussa Gmbh Dipeptides utilisés comme additifs pour l'alimentation animale
DE102009002044A1 (de) 2009-03-31 2010-10-07 Evonik Degussa Gmbh Dipeptide als Futtermitteladditive
WO2011147881A2 (fr) 2010-05-27 2011-12-01 Evonik Degussa Gmbh Dipeptides cycliques utilisés comme additifs d'aliments pour animaux
DE102010029399A1 (de) 2010-05-27 2011-12-01 Evonik Degussa Gmbh Cyclische Dipeptide als Futtermitteladditive
CN102422984A (zh) * 2011-10-26 2012-04-25 中国海洋大学 一种蛋氨酸寡肽及其在大菱鲆饲料中的应用
CN102429109A (zh) * 2011-10-26 2012-05-02 中国海洋大学 一种蛋氨酸寡肽及其在对虾饲料中的应用
US9655863B2 (en) 2012-07-12 2017-05-23 Novus International, Inc. Matrix and layer compositions for protection of bioactives
WO2017174398A1 (fr) 2016-04-07 2017-10-12 Cysal Gmbh Cyanophycine pour organismes aquatiques à alimentation lente
CN109234343A (zh) * 2018-09-30 2019-01-18 中国海洋大学 一种蛋氨酸寡肽及其制备方法和应用

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