The feeding of essential amino acids is warranted under conditions in which there is dietary insufficiency of these amino acids. Such acids are arginine, methionine, lysine, phenylala- nine, tyrosine, valine, leucine, isoleucine, tryptophan, and threonine. Feeding of these amino acids along with nonessential amino acids which include glycine, alanine, serine, cysteine, aspartic acid, asparagines, glutamic, glutamine, tyrosine, proline, hydroxyproline and amino acids that do not occur in protein such as omithine, citruline and beta alanine.

Feeding of these amino acids along with nonessential amino acids could be desirable in tissue level starvations obtained in diabetes and metabolic stress conditions resulting from injury to the body like surgery, infections or burns. Intravenous administration of combinations of es- sential and nonessential amino acids is resorted to under certain conditions of starvation. In this all these situations feeding of compounds of ascorbic acid and amino acids would be beneficial. This is in continuation of our previous invention on the novel synthesis of ascor- bic acid compounds with lysine and its derivatives and/or proline and its derivatives.

Ascorbic acid is a ubiquitous compound essential for the maintenance and preservation of several species. In human beings deprived of ascorbic acid, the deficiency disease scurvy develops which can be life threatening. Ascorbic acid is probably the most effective, efficient and least toxic antioxidant. It is a water soluble, chain-breaking antioxidant, that acts as scav- enger for harmful radicals like superoxide, hydroxyl and singlet oxygen that are produced during normal cellular metabolism. Ascorbic acid is superior to other water soluble and lipid soluble antioxidants. It also protects DNA, enzyme, protein and lipids from oxidative damage and thereby prevents aging, coronary heart diseases, cataract formation, degenerative diseases and cancer. Oxygen radicals have been implicated in initiation and post-initiation stages of carcinogenesis, and in invasion and metastatic processes.

Ascorbic acid has several physiological functions. It is essential for collagen synthesis, pro- teoglygans and various components of extra cellular matrix (ECM). It also helps maintain various enzymes in their reduced forms. Ascorbic acid is involved in the hydroxylation of lysine and proline for which ascorbic acid functions as cofactor. Lysine and proline are prin- cipal components of tendons, ligaments, skin, bone, teeth, cartilage, heart valves, cornea, eye lens and ground substances between cells fibers. Any deficiency of ascorbic results in im- paired collagen formation which leads to tissue weakness and eventually, scurvy. Cellular medicine considers ascorbic acid tile most important natural substances indispensable for maintenance of health at the cellular level. Deficiency of ascorbic acid in humans may lead to various diseases. In addition, ascorbic acid participates in the biosynthesis of camitine and neuroendocrine peptides.

Ascorbic acid has several reactive hydroxy groups that can be used for the synthesis of a number of derivatives. Many substituted compounds at 2-, 3-, 5-and 6-positions have been synthesized. L-ascorbate 2-sulphate is stored in fish and some shrimp. It has ascorbic acid activity for fish such as trout, salmon and catfish. It is 20 times more stable than ascorbic acid. Hence, it has been used in the formulation of feeds. L-ascorbate 2-phosphate is more stable in air than ascorbic acid. This compound is used as source of ascorbic in guinea pigs and rhesus monkeys. L-ascorbyl 6-palmitate, a synthetic lipophilic ascorbic acid derivative, is an effective preservative in foods and pharmaceuticals. In recent years there has been a growing interest in the therapeutic application of L-ascorbic acid and its derivatives as an anti-cancer agent.

The present invention focuses on the synthesis of various compounds of ascorbic acid with essential amino acid, nonessential amino acid and amino acid that do not occur in protein.

These compounds could be used in research medicine, physiology, pharmacology, pharma- ceuticals, nutrition and cosmetic, commercial and industrial application.

The overall objective of this invention is to synthesize ascorbic acid derivatives with essential, nonessential and amino acids that do not occur in protein. The synthesis is carried using L- ascorbic acid and the amino acids. For this purpose, the CH20H of ascorbic acid at 6-position and carboxyl groups of amino acids is utilized.' These new biochemical compounds can provide additional biological effects superior to its individual compounds. Thus, a biochemical synthesis of these compounds in which the amino acids are covalently bound to ascorbic acid is preferable to a simple physical mixture of the amino acids with ascorbic acid. Such unexpected superior biological effects include increased biological stability of these molecules, enhanced absorption by various biological cell compartments and greater biologi- cal efficacy. Such compounds can facilitate and enhance the assimilation of other nutritional components from foods resulting in improved nutritional status of individuals.

These novel compounds have applications in a variety of areas including but not limited to nutrition, medicine, and pharmacology.

Figure 1 shows the structure of various essential amino acids.

Figure 2 shows the structure of various nonessential amino acids.

Figure 3 shows the structure of various amino acids that do not occur in proteins.

Figure 4 shows the scheme for the synthesis of ascorbic acid derivatives with essential amino acids. A typical example is that of ascorbyl 6-phenyalanine.

Figure 5 shows the scheme for the synthesis of ascorbic acid derivatives with nonessential amino acids. A typical example is that of ascorbyl 6-glycine.

The present invention provides the synthesis of biochemical compounds where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein.

The present invention provides the synthesis of a biochemical compound where one essential amino acid is bond to ascorbic acid in C-6 position, The present invention provides the synthesis of a biochemical compound where one essential amino acid is bond to ascorbic acid in C-2 position.

The present invention provides the synthesis of a biochemical compound where one essential amino acid is bond to ascorbic acid in C-6 and C-2 positions.

The present invention provides the synthesis of a biochemical compound where one non es- sential amino acid is bond to ascorbic acid in C-6 position.

The present invention provides the synthesis of a biochemical compound where one nones- sential amino acid is bond to ascorbic acid in C-2 position.

The present invention provides the synthesis of a biochemical compound where on nonessen- tial amino acid is bond to ascorbic acid in C-6 and C-2 positions.

The present invention provides the synthesis of a biochemical compound where an amino acid that do not occur in protein is bond to ascorbic acid at C-6 position. The present invention provides the synthesis of a biochemical compound where an amino acid that do not occur in protein is bond to C-2 position.

The present invention provides the synthesis of a biochemical compound where an amino acid that do not occur in protein is bond to C-6 and C-2 position.

The present invention provides the synthesis of biochemical compound where two or more essential amino acid are bond to ascorbic acid at C-6 position.

The present invention provides the synthesis of a biochemical compound where two or more essential amino acids arc bond to ascorbic acid at C-2 position.

The present invention provides the synthesis of a biochemical compound where two or more essential amino acids are bond to ascorbic acid at C-6 and C-2 positions.

The present invention provides the synthesis of a biochemical compound where two or more nonessential amino acid are bond ascorbic acid at C-6 position.

The present invention provides the synthesis of a biochemical compound where two or more nonessential amino acids are bond to ascorbic acid at C-2 position.

The present invention provides the synthesis of a biochemical compound where two or more nonessential amino acids are to ascorbic acid at C-6 and C-2 position.

The present invention provides the synthesis of biochemical compound where two or more amino acids that do not occur in protein are bond to ascorbic acid at C-6 position.

The present invention provides the synthesis of a biochemical compound where two or more amino acids that do not occur in protein are bond to ascorbic acid at C-2 position.

The present invention provides the synthesis of a biochemical compound where two or more amino acids that do not occur in protein are bond to ascorbic acid at C-6 and C-2 positions.

The present invention provides the synthesis of a biochemical compound where one or more essential amino acids are bond to ascorbic acid at C-6 position and one or more. nonessential amino acids are bond to 2-position. The present invention provides the synthesis of a biochemical compound where one or more essential amino acids are bond to ascorbic acid at C-6 position and one or more amino acids that do not occur in protein arc bond to C-2 position.

The present invention provides the synthesis of a biochemical compound where one or more nonessential amino acids are bond to ascorbic acid at C-6 position and one or more essential amino acids are bond to C-2 position.

The present invention provides the synthesis of a biochemical compound where one or more nonessential amino acids are bond to ascorbic acid at C-6 position and one or more amino acids that do not occur in protein are bond to C-2 position.

The present invention provides the synthesis of a biochemical compound where one or more amino acids that do not occur in protein are bond to ascorbic acid at C-6 position and one or more essential amino acids are bond to C-2 position.

The present invention provides the synthesis of a biochemical compound where one or more amino acids that do not occur in protein are bond to ascorbic acid at C-6 position and one or more nonessential amino acids are bond to C-2 position.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein. to prevent oxidation.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein to prevent and/or retard aging of organic and inorganic ma- terials.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein to be used in preventive and therapeutic medicine.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein to stabilize connective tissue and prevent the degradation of extracellular matrix.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein to be used in the identification, development and use of pharmaceutical compounds and their preparations.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein to be used in foods, beverages, and nutritional supplements.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that to prevent do not occur in protein to be used in chemical synthesis process.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein to be used in the industry process.

The present invention provides a method of use of a biochemical compound where ascorbate molecules are covalently bound to essential amino acids, nonessential amino acids and amino acids that do not occur in protein to be used in the industry producing cosmetic products.

Synthesis of the above described biochemical compounds were carried out by using standard procedures.

6-Deoxybromcascorbate is synthesized by reacting L-ascorbic acid with hydrogen bromide in acetic acid following the procedure of Block, Lundt and Pedersenj K. Block, 1. Lundt and C.

Pederson. Carbohydrate Research. 68: 313 (1979)) 6-Deoxyamino L-ascorbate is synthesized according to the method of Suskovic (B. Suskovic.

Croat Chem Acta. 62: 537 (1989)) C, H and N analysis are performed on all the compounds synthesized and their melting points are determined. NMR, IR, UV and GC/MS are used to establish the structures of the com- pounds. Purity of the compounds are judged by TLC or HTLC chromatography.

Examples of these standard procedures are described as follows: Example 1 Synthesis of L-ascorbyl-6-lvsine The standard procedure of condensation of an alcohol with an acid is used. The procedure of Cousins et al R. C. Cousins, P. A. Seib, R. C. Hoseney, C. W. Deyoe, Y. T. Lianc, and D. W.

Lillard. , J Am Chem Soc: 54,308 (1977) is detailed as follow.

In brief, ascorbic acid (8 mmoles) is added to a solution of lysine (10 mmoles) in about 20 ml of sulfuric acid. After being stirred for about 2 hours at room temperature, the reaction mix- ture is allowed to stay at room temperature overnight. It is then poured over crushed ice.

Exacted twice with ether and washed with water and dried over sodium sulfate. Ether is re- moved. The product is crystallized with ethanol and dried in vacuum.

Example 2 Synthesis of L-ascorbvl-6-proline The procedure is used to synthesized ascorbyl 6-proline. Ascorbic acid (8 mmoles) is added to a solution of proline (10 mmoles) in about 20 ml of sulfuric acid and stirred for about 2 hours at room temperature and allowed to stay overnight. The reaction mixture is poured over crushed ice, extracted twice with ether and washed with water. Dried over sodium sulfate, ether is removed and the product is crystallized from ethanol, dried in vacuum.

Example 3 Synthesis of Ascorbyl-2-lysine and ascorbyl 2-proline Ascorbyl-2-lysine and ascorbyl-2-proline are also synthesized. To prepare the derivatives at 2-position the hydroxy groups at 5-and 6-positions have to be first protected. Jack and Jones (K. G. A. Jackson and J. K. N. Jones, Can J Chem. 47: 2498 (1969) ) procedure is adopted to prepare 5,6-0-isopropylidene ascorbic acid. 5,6-0-isopropylidene ascorbic acid (5 mmoles) in dry pyridine and acetone is added to lysyl chloride (7 mmoles) or prolyl chloride- (7 mmoles) and the products are worked up according to Cousins et al. and crystallized from ethanol.

In additional, 2,6-di-substituted derivatives of lysine or proline are synthesized by reacting L- ascorbic acid either with excess of lysyl chloride or prolyl chloride in dry pyridine as de- scribed above.

Furthermore, di-substituted derivatives of L-ascorbic acid with different groups at 2-and 6- positions are synthesized by reacting with respective chlorides by combination of techniques as described above.

Example 4 Synthesis of 6-Deoxyascorbate lysine 6-Deoxybromo ascorbate (8 mmoles) is reacted with lysine (10 mmoles) in dry pyridine to give the desired product. The-amino group of lysine is protected so that the-group is available for reaction. The reaction is carried out overnight. Pyridine is removed under re- duce pressure, poured in ice, extracted with ether, washed with water, dried over sodium sul- fate. Ether is removed and crystallized from ethanol.

The other combination of amino acid with ascorbic acid at 2-and 2,6-positions are synthe- sized by the combination of methods as described above.

6-Deoxyascorbate proline is synthesized by the exact procedure described above for 6- deoxyascorbate lysine. 6-Deoxybromo ascorbate (8 rnmoles) is reacted with proline (10 mmoles) in dry pyridine to give 6-deoxyascorbate proline. The product is crystallized form ethanol, dried in vacuum.

The other combination of amino acid with ascorbic acid at 2-and 2,6-positions are synthe- sized by the combination of method as described above.

Example 6 Synthesis of L-6-deoxyascorbvl lvsine The £-group of lysine is protected (7 mmoles) and is reacted with 6-deoxybromo ascorbate (5 mmoles) in dry pyridine to give the said product. After the reaction is over, the product is worked up as described above and crystallized in ethanol The compounds with 2-substitution are prepared by the method as described above.

Example 7 Synthesis of 6-Deoxyaminoascorbate lysine 6-Deoxyamino ascorbate (5 mmoles) is reacted with lysyl chloride (7 mmoles) in dry pyridine and the reaction product is worked up according to Cousine et al. It is crystallized from etha- nol.

The other combination of amino acids with ascorbic at 2-and 2, 6- positions are prepared as described above.

Example 8 Synthesis of 6-Deoxvaminoascorbate proline 6-Deoxyarnino ascorbate (5 mmoles) is reacted with prolyl chloride (7 mmoles) in dry pyri- dine and worked up by the method as described above and crystallized from ethanol.

The other combination of amino acid with ascorbic acid at 2-and 2, 6- positions are prepared as described above.

6-Deoxyamino ascorbate (5 mmoles) is reacted with prolyl chloride (7 mmoles) in dry pyri- dine and worked up as described above and crystallized from ethanol. Melting point is de- termined and C, H and N analysis are performed. Structure is established by NMR, IR, UV and GC/MS methods.

The other combination of amino acid with ascorbic acid at 2-and 2, 6- positions are prepared as described above.

Experiments have been made with some of the above described compounds: Material s and Method: L-Ascorbic acid, D-isoascorbic acid, L-lysine, L-proline and MTT were purchased from Sig- ma (St. Louis). The structures of ascorbic acid, iso-ascorbic acid, lysine and prolin are shown in Figure. 1 Novozyme 435 (immobilized lipase B from Candida Antarctica), t-amyl alcohol, acetonitrile and actone were purchased from Aldridge (Milwaukee, WI). Thin layer chromatography (TLC) plates coated with silica gel 150 A°, 250uM were from Whatman International Ltd, Maidstone, England.

All other reagents used were of high purity and were obtained either from Sigma or Aldridge company.

Purity of the compounds synthesized were judged by TLC.

General Experimental Conditions: a) Cell Cultutre. Normal human dermal fibroblast (NHDF), human melanoma cells A2058, Hep G2 cells and human breast cancer cells MDA MB 231 were obtained from ATCC.

They were cultured in complete Dulbecco's Modified Eagle Media supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100mg/ml) into 24- well tissue culture plates Costar, Cambridge, MA, and incubated at 37°C in a tissue culture incubator equilibrated with 95% air and 5% CO2. At near confluence the cells were treated with test compounds in triplicate at 0,10 uM, 10 uM, 100 uM, 500 uM and 1000 uM concentrations. b) MTT Assay. Cell viability was assayed by MTT assay. Cells after treatment with test compounds were washed with PBS and O. 5ml of MTT (5mg/ml) in PBS was added to each well. The cultured plates were in cubated at 37°C for an additional 2 hrs. The me- dia was carefully aspirated and lml of DMSO was then added to each well to dissolve blue formazan crystals that formed. Optical density was measured at 590 nm with Bio- Spec 1601, Shimadzu spectrometer. c) Geltinase Zymography. Gelatinase zymography was performed using the the protocol described by Liu et al 1995 and Kubota et al 1991 (Liu, X-H, Rose, D. P Cancer Lett 92,21, 1995; Kubota, S, Fridman, R, Yamada, Y Biochem Biophys Res Comm 176, 129,1991) in 10% pre-cast polyacrylamide gels (Novex, Invitrogen) in presnce of 0. 1% gelatin. Culture media (20 pl) was loaded and SDS-PAGE was performed with tris-glycine SDS buffer. After electrophoresis, the gels were washed with 5% Triton- 100 for 30 minutes. After washing, the gels were incubated for 24 hrs at 37°C in the presence of 50mM Tris-HCl, 5mM CaCl2, 5uM ZnCl2, pH 7.5 and stained with Coo- massie Blue R 0.5% for 30 minutes and destained. Protein standards were run concur- rently and approximate molecular weight were determined. d) Matrigel Invasion Assay. The studies were conducted using Matrigel (Becton Dickin- son) inserts in compatible 24 wells plates. The breast cancer cells MDA MB 231 ( 2x104) suspended in 200 {il of the media supplemented with different dose of test com- pound as dicated by the design of the experiment were seeded on the insert in the well.

The media on the insert and the well contained the same supplements. The plates with the insert were then returned to the incubator for 18-20 hrs. After incubation, the media from the wells were withdrawn. The cells on the upper surface of the insert were gently scrubbed away with cotton swab. The cell that had penetrated the matrigel membrane and had migrated on the lower surface of the matrigel were stained with hemacolor stain (EM Sciences) and were visually counted under the microsacope.

EXAMPLE 1 Ascorbyl 6-lysine, Figure 2: The standard procedure of condensation of an alcohol with an acid using solid-phase system in the presence of small amount of organic solvent (t-amyl alcohol) catalyzed by immobilized lipase B from Candida Antarctica was adopted. This procedure has used by Yan, Bornscheu- ier and Schmid for the fatty acid esters of ascorbic acid (Y. Yan, U. T. Bornscheuer and R. D.

Schmid, Biotechnology Letters 21,1051, 1999).

Ascorbic acid (Immole, 176 mg), Lysine (1.5 mmole, 219 mg), Novozyme 435 (200 mg) and t-amyl alcohol (20ml) were stirred at 40°C in nitrogen atmosphere. Heating and stirring continued for 12 hrs. The reaction mixture was then filtered under suction, washed with 10 ml of t-amyl alcohol and 10ml of distilled water. Both t-amyl alcohol and water were removed under reduce pressure. The reside was washed several times with methanol and acetone. The solid thus obtained was crystallized from water and methanol to give ascorbyl 6-lysine. The yield was about 50% (161 mg).

The purity of the product was judged by TLC using a solvent system consisting of acetonitri- le, acetone, water and acetic acid (70: 10: 15: 5) according to Roomi and Tsao (M. W. Roomi and C. S. Tsao, J Agri Fd Chem: 46,1406, 1998) and visulized by exposing to iodine in a chamber. It showed a dark spot of ascorbyl 6-lysine and two faint corresponding to ascorbic acid and lysine EXAMPLE 2 Iso Ascorbyl 6-lysine, Figure 4: Iso ascorbyl 6-lysine was prepared in a similar manner to ascorbyl 6-lysine using iso-ascorbic acid (lmmole, 176 mg), lysine (1.5 mmole, 219 mg), Novozyme 435 (200 mg) in t-amyl alcohol (20 ml). The residue on crystalisation from water and methanol gave 170 mg of iso- ascorbyl 6-lysine (52% yield).

On TLC it showed a dark spot for iso-ascorbyl 6-lysine and two minor spots corresponding to iso-ascorbic acid and lysine.

EXAMPLE 3 Ascorbyl 6-proline, Figure 3: Ascorby 6-proline was synthesized by a similar procedure described for ascobyl 6-lysine.

Ascorbic acid (lmmole, 176 mg), proline (1. 5mmole, 172 mg), Novozyme 435 (200mg) and t-amyl alcohol (20 ml) were stirred at 40°C in nitrogen atmosphere. Heating and stirring we- re continued for 12 hrs. The reaction mixture was filtered under suction and washed with t- amyl alcohol (10 ml) and water (10 ml). Water and t-amyl alcohol were removerd under reduce pressure. The residue was washed several times with methanol and acetone. The reac- tion product was crystallized from water and methanol to give 150 mg of ascorbyl 6-proline (50 % yield).

On TLC it showed a dark spot of ascorby 6-proline and two faint spots corresponding to ascorbic acid and proline.

EXAMPLE 4 Iso-ascorbyl 6-proline, Figure 5 : Iso-ascorbyl 6-proline was synthesized exactly by a similar procedure as described for ascor- byl 6-proline replacing iso-ascorbic acid for ascorbic acid. The reaction product was similarly crystallized from water and methanol. The yield was about 45-50%.

On TLC it showed a dark spot for iso-ascorbyl 6-proline and two faint spots corresponding to iso-ascorbic acid and porline.

The present invention is by no means restricted to these specific embodiments and not to be limited in scope by specific examples described herein. Various modifications of the present invention to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the present invention.