NEW PROCESS FOR BIOPHOSPHORYLATING ORGANIC COMPOUNDS

Abstract: Described is a new microbial biophosphorylation process for regiospecifically phosphorylating the hydroxyl group, in a ''phosphate active'' hydroxyl containing organic compound, under biotransformation conditions utilizing the microorganism, Rhizopus oryzae ATCC No. 11145. The phosphorylated compounds produced by the process include e.g., macrolide FK-506 type immunosuppressants which are useful in preventing human host rejection of foreign organ transplants, e.g. bone marrow, liver, kidney, lung and heart transplants. Other hydroxyl containing compounds, applicable in this process include rapamycin, echinocandins, HIV protease inhibitors, simvastatin and zearalenone.

Description: TITLE OF THE INVENTION NEW PROCESS FOR BIOPHOSPHORYLATING ORGANIC COMPOUNDS CROSS REFERENCE TO RELATED APPLICATIONS The present application is a combined continuation-in-part application of SN 594,500 (Case 18238) filed October 9, 1990; SN 594,214 (Case 18240) filed October 9, 1990; SN 595,894 (Case 18208) filed October 11, 1990; SN 691,606 (Case 18261) filed April 26, 1991; SN 691,607 (Case 18382) filed April 26, 1991; SN 701,387 (Case 18340) filed May 16, 1991; and SN 735,963 (Case 18462) filed July 25, 1991. BACKGRQUNP OF THE INVENTION 1. Field of the Invention This invention relates to a new regio- specific biophosphorylation process for producing phosphorylated derivatives of "phosphate active" hydroxyl containing organic compounds utilizing the microorganism Rhizopus oryzae. ATCC No. 11145. The process involves contacting the microorganism and an organic compound containing a free phosphate active hydroxyl group, under biotransformation conditions, which phosphorylate the hydroxyl group. The process involves either resting Rhizopus cells or culturing the microorganism in the presence of the organic compound. Rhizopus oryzae ATCC No. ill45 is known in the art primarily as a hydroxylating agent. It also degrades insect moulting hormones (J.C.S. Chem. Comm. 1974: 656-657, 1974); participates in hydroxylation of steroids (Can, J, Chem. 57:436-440 and 1585-1587, 1979; ibid., 59; 1651-1655, 1981; ibid., 63: 1127- 1131, 1985; H.J. Peppier, ed., Microbial Technology Reinhold, New York, p. 288-297, 1967; U.S. Patent 2,646,370); is involved with transformations of sesquiterpene lactone costunolide (J.C.S, Perkin 1: 3022-3028, 1979); produces 16-hydroxyverrucarin A and B and 3'-hydroxyverrucarin A by transformation of verrucarins A and B (Appl. Environ, Microbiol, 46:480-483, 1983); and produces OH-products of imipramine (J. Pharmaceut. Sci. 70: 151-153, 1981). However, in the above references, there is no description of its ability as a regiospecific hydroxyl phosphorylating agent. Rhizopus is a genus of fungi which commonly occurs on mature fruits, grain and vegetables, as well as soil. Typically they are saprobes and facultative parasites, and form a branched, aseptate mycelium. Rhizopus spp are used commercially, e.g., in the preparation of carboxylic acids or steroids, or in the metabolism of hydrocarbons. Culture or fermentation of Rhizopus arrhizus ATCC 11145 is straightforward, conventional and well known. See, for example, U.S. 4,410,629. Adapta¬ tions, modifications and variations of the protocol are given in the Examples are within the routine skill of the fermentation microbiologist. Regiospecific biophosphorylating agents are desired in the art since standard chemical phosphorylation using, e.g., POCI3 or PCI5, are generally non-regiospecific and do not produce selectively phosphorylated compounds, which can lead to improved water solubility and pharmacokinetics. ATCC PEPQSIT A sample of MF 4974 has been deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville MD 20852. The culture access designation is ATCC No. 11145. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is an ** -H nuclear magnetic resonance (NMR) spectrum taken at 400 MHz of C-32 phosphorylated FR-900520 in CDCI3. Figure 2 exhibits the assigned molecular structure for C-32 phosphorylated FR-900520. Figure 3 is nuclear magnetic resonance (NMR) spectrum taken at 400 MHz of the C-43 methylated phosphate ester of the phosphorylated rapamycin macrolide in MeOH. Figure 4 exhibits the assigned molecular structure for the C-43 phosphorylated rapamycin macrolide. Figure 5 is ^H nuclear magnetic resonance spectrum of echinocandin IIIA. SUMMARY OF THE INVENTION We have discovered a new overall process for selectively biophosphorylating an organic compound containing "phosphate active" hydroxyl groups. The organic compound is suspended in an aqueous medium with a strain of Rhizopus oryzae ATCC No. 11145 for a sufficient time to produce the phosphorylated organic compound. By the term "phosphate active hydroxyl con¬ taining organic compound" as used herein is meant a compound containing a hydroxyl group which can stereo- specifically interact with the Rhizopus microorganism to undergo phosphorylation under biotransformation conditions. A simple test with the organic compound under the biotransformation conditions described herein will determine, without undue experimentation, if the hydroxyl group in the organic molecule is phosphate active. The process involves contacting resting Rhizopus cells in a phosphate buffered medium con¬ taining e.g. glycerol, as a carbon nutrient, or by the fermentation of the microorganism Rhizopus oryzae. ATCC No. 11145 together, in the presence of the hydroxyl containing organic compound, «.g., an FK-506 type macrolide immunosuppressant i.e. FK-520, under submerged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being conducted at a pH of about 7 for a sufficient time, e.g. 24 hours at 27 β C, to selectively biophosphorylate the phosphate active hydroxyl group. Either process can be utilized but preferred is the process using resting cells. It will be recognized by those skilled in the art that phosphorylation by Rhizopus oryzae ATCC 11145 is not limited to that particular strain. Rather, other Rhizopus oryzae strains can also be expected to be capable of acting to perform hydroxyl phosphorylation of hydroxy containing organic compounds. DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS In accordance with this invention there is provided a process for producing a biophosphorylated hydroxyl containing organic compound, wherein said hydroxyl group is phosphate reactive, comprising the step of contacting a strain of Rhizopus oryzae ATCC No. 11145 microorganism together with the hydroxyl containing organic compound in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the biophosphory¬ lated hydroxyl containing organic compound. The present invention involves a biotrans¬ formation process, which involves the contacting of resting cells, or the fermentation of, the micro¬ organism, Rhizopus oryzae. together with an organic compound containing a free hydroxy group, to produce the phosphorylated derivative. The microorganism is currently on deposit with the American Type Culture Collection, 12301 Parklawn Drive in Rockville, Maryland as ATCC No. 11145. The scope of the compounds included within the "phosphate reactive hydroxyl containing organic compounds" includes a C-32 hydroxy-containing macro¬ lide as described in Fujisawa's USP 4,894,366 of the formula: CH 3 0 OCH 3 wherein R is H, C1-C alkyl, R 2 is hydrogen, hydroxy or lower alkanoyloxy, "R- is methyl, ethyl, propyl or allyl, n is an integer of 1 or 2, and the symbol of a line and dotted line is a single bond or a double bond, and a pharmaceutically acceptable basic salt thereof. Specifically included is where the compound FK-506 is where R is methyl, R 2 is hydroxy, R 3 is allyl, n is 2 and the double bond is absent; and the compound FK-520 is where R is methyl, R 2 is hydroxy, R 3 is ethyl, n is 2 and the double bond is absent. Also included are phosphate reactive hydroxyl containing compounds including the following FK-506 type compounds (from USP 4,894,366 to Fujisawa): 7-Allyl-l,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 > 9 ]octacos-18-ene-2,3,10,16-tetraone, l,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- l-methylvinyl]-23,25-dimethoxy-13,19,17,21,27-penta- methyl-11,28-dioxa-4-azatricyclo[22.3.1.0 ~*» 9 ]octacos- 18-ene-2,3,10,16-tetraone, 16-Allyl-l,13-dihydroxy-ll-[-2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-22,24-dimethoxy- 12,18,20,26-tetramethyl-10,27-dioxa-4-azatricyclo- [21.3.1.0 • 8 ]heptacos-17-ene-2,3,9,15-tetraone. Further included within the term "FK-506 type macrolide" as used herein are specifically the compounds disclosed in Fisbn's EP0 0 323 042 Of " the formula: II wherein each vicinal pair of substituents [R ** - and R 2 ], [R 3 and R 4 ], [R 5 and R 6 ] independently: a) represent two vicinal hydrogen atoms, or b) form a second bond between the vicinal carbon atoms to which they are attached; in addition to it significance above, R 2 can represent an C^-C^Q alkyl group; Tt 7 represents H, OH or alkyl, or in conjunction with ! it may represent =0; R- and R 9 independently represent H or OH; R!° represents H; alkyl, wherein said alkyl can be substituted by one or more hydroxyl groups; c l~ c '10 alkenyl, which can be substituted by one or more hydroxyl groups, or C^-C^Q alkyl substituted by =0; X represents 0, (H, OH), (H,H) or -CH 2 0-; Y represents 0, (Ξ, OH), (H,H), N-NR n R 12 or N-OR 13 wherein, RH and R 2 independently represent H, independently represent H or independently represents 0, or it can independently represent (R 2 ^a,H); wherein R 2t ^a independently represents OH, 0-C**_-C 10 alkyl or OCH2OCH2CH2OCH3; n is 1,2, or 3; in addition to their significances above, Y, ~ -~ and R 23 , together with the carbon atoms to which they are attached, can represent a 5- or 6- membered N-, S- 0- containing heterocyclic ring, which is saturated or unsaturated, and which can be substituted by one or more groups selected from alkyl, hydroxyl, c l~^10 alkyl substituted by one or more hydroxyl groups, 0-Cι-Ci Q alkyl, benzyl and -C^Se^g^ ; provided that when X and Y both represent 0; R 9 represents OH; R 14 , R 15 , R i6 , R1 7 , R 18 , R i9 and R 22 each represent methyl; R---& represents OCH3; R 8 and R 23 each represent H; [R 3 and R 4 J and [R 5 and R 6 ] each ^represent a carbon-carbon bond; and pharmaceutically acceptable salts thereof, which includes acid addition salts of any amine groups present. Preferably when R 2 , R 7 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R i7 , R 18 , R 19 , R 20 a, R 22 and R 23 comprise carbon-containing groups, those groups contain up to 10 carbon atoms, more preferably from 1 to 6, e.g., methyl or methoxyl. Also preferred is wherein each of R-*- 4 , R!->, R 16 , R 17 , R 18 , R 19 and R 22 represents methyl. The alkyl groups: R 2 , R 7 , R 11 , R 12 , R i3 , R 14 , R 5 , R 16 , R 17 , RI 8 , R i9 , R 2 0 , R 22 and R 23 can comprise straight chain, branched and cyclic groups. Alkyl groups substituted by =0, include R!° which can represent include ketone and aldehyde groups. Preferably, 10 is allyl (i.e., prop-2-enyl), propyl ethyl or methyl. Preferably, n is 2; R 7 is H or OH; R! and R 2 both represent H; X is preferably 0 or (H, OH); R 20 a represents OH or 0CH 3 ; and when Y, R!° and R 23 together represent a N-,S- or 0- containing heterocyclic ring, preferred is where that ring is five-membered, more preferably a pyrrole or tetrahydrofuran ring. Preferred embodiments as products are C-32 phosphorylated FK-506, o and C-32 phosphorylated FK-520, O CH3O OCH 3 and C-32 phosphorylated FK-523; O CH 3 O OCH 3 and the corresponding C-32 phosphorylated C-31 desmethyl analogs, and also the following specific compounds from EPO 323,042 to Fisons: 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- l-methylvinyl]-18-[(phenylseleno)methyl]-16,26,28- trimethoxy-13,22,24,30-tetramethyl-ll,17,31-trioxa-4- azatetracyclo[25.3.1.0 4 • 9 .0 16 »20 ]hentriacont-21-ene- 2,3,10-trione, 17-Allyl-l,2,14-trihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 - 9 ]octacos-18-ene-3,10,16-trione, 17-Allyl-l,2,14,16-tetrahydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 - 9 ]octacoε-18-ene-3,10-dione, 17-Propyl-l,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 ' 9 ]octacosane-2,3,10,16-tetraone, 17-Propyl-l,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylethyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 ' 9 ]octacos-18-ene-2,3,10,16-tetraone, 17-Propyl-l,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylethyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 ' 9 ]octacosane-2,3,10,16-tetraone, 17-Propyl-l-hydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-l-methylvinyl-23,25-dimethoxy-13,19,21,27- tetramethyl-ll,28-dioxa-4-azatricyclo[22.3.1.0 4 • 9 ]- octacosa-14,18-diene-2,3,10,16-tetraone, 17-Propyl-l-hydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-l-methylvinyl]-23,25-dimethoxy-13,19,21,27- tetramethyl-ll,28-dioxa-4-azatricyclo[22.3.1.0 4 ' 9 ]- octacos]-18-ene-2,3,10,16-tetraone, 17-Allyl-l,14,20-trihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 • 9 ]octacos-18-ene-2,3,10,16-tetraone, 17-(l-Hydroxyproρ-2-enyl)-l,14,20-trihydroxy-12- [2-(4-hydroxy-3-methoxycyclohexyl)-l-methylvinyl- 23,25-dimethoxy-13,19,21,27-tetramethyl-ll,28-dioxa- 4-azatricyclo[22.3.1.0 4• 9 ]octacos-18-ene-2,3,10,16- tetraone, 17-Allyl-l,2-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 > 9 ]octacos]-18-ene-3,10,16-trione, 17-Allyl-l,16-dihydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 ' 9 ]octacosa]-14,18-diene-2,3,10-trione, 17-Allyl-l-hydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 • 9 ]octacos]-18-ene-2,3,10,16-tetraone, 17-(2,3-Dihydroxypropyl)-l,14-dihydroxy-12-[2-(4- hydroxy-3-methoxycyclohexyl)-l-methylvinyl-23,25-di- methoxy-13,19 21,27-tetramethyl- l,28-dioxa-4-azatri- cyclo[22.3.1.O 4 • 9 ]octacos-18-en -2,3,10,16-tetraone, 17-Ethanalyl-l,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 • 9 ]octacoε-18-ene-2,3,10,16-tetraone, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclo- hexyl)-l-methylvinyl]-26,28-dimethoxy-13,22,24,30- tetramethyl-ll,31-dioxa-4,17-diazatetracyclo- [25.3.1.0 • 9 .0 16 •20 ]hentriaconta-16(20),18,21- triene-2,3,10-trione, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- l-methylvinyl]-26,28-dimethoxy-17-(2-hydroxyethyl)- 13,22,24,30-tetramethyl-ll,31-dioxa-4,17-diazatetra- cyclo[25.3.1.0. » 9 .0 16 » 20 ]hentriaconta-16(20),18,21- triene-2,3,10-trione, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- l-methylvinyl]-26,28-dimethoxy-13,22,24,30-tetra- methyl-17-phenylmethyl-ll,31-dioxa-4,17-diazatetra- cyclo-[25.3.1.0 4 - 9 .0 i6 > 20 ]hentriaconta-16(20),18,21- triene-2,3,10-trione, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- l-methylvinyl]-26,28-dimethoxy-13,22,24,30-tetra- methyl-17-phenylmethyl-ll,31-dioxa-4,17-diazatetra- cyclo[25.3.1.0 4 » 9 .θ 6 ' 20 ]hentriaconta-16(20),18,21- triene-2,3,10-trione, 17-Allyl-l,14-dihydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,1 ,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 - 9 ]octacos-18-ene-2,3,10,16-tetraone C16 oxime, 17-Allyl-l,14-dihydroxy-12-[2-(4-hydroxy-3-methoxy- cyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 » 9 ]octacos-18-ene-2,3,10,16-tetraone C16 oxime O-methyl ether, 17-Propyl-l-hydroxy-12-[2-(3,4-dihydroxycyclohexyl)-l- methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl- 11,28-dioxa-4-azatricyclo[22.3.1.0 - 9 ]octacos-18-ene- 2,3,10,16-tetraone, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- l-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetra- methyl-17-(2-oxopropyl)-ll,28-dioxa-4-azatricylo- [22.3.1.0 4 » 9 ]octacos-18-ene-2,3,10,16-tetraone, 17-Allyl-l,14-dihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl)-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-aza-spiro- [tricyclo[22.3.1.0. 4 • ]octacos-18-ene-2,2 » -oxirane,- 3,10,16-trione, 17-Ethanalyl-l,2,14-trihydroxy-12-[2-(4-hydroxy-3- methoxycyclohexyl-l-methylvinyl-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 - 9 ]octacos-18-ene-3,10,16-trione, 1,14-Dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)- l-methylvinyl]-26,28-dimethoxy-18-[(phenylseleno)- methyl-13,22,24,30-tetramethyl-ll,17,31-trioxa-4-aza- tetracyclo[25.3.1.0. 4■ 9 0. 16 • 20 ]hentriaconta-16(20),21- diene-2,3,10-trione, 4-Methyl-[17-Allyl-l,14-dihydroxy-12-[2-(4-hydroxy- 3-methoxycyclohexyl-l-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 ' 9 ]octacos-18-ene-2,3,10-trione-16-ylidene]- hydrazide, or a pharmaceutically acceptable salt of any one thereof, In general, the phosphorylated organic com¬ pound can be produced by either contacting resting cells of Rhizopus. in an aqueous buffered phosphate medium containing a carbon nutrient at ambient temperature, or by culturing (fermenting) the above- described microorganism, Rhizopus oryzae, in the presence of the hydroxy containing organic compound in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.). The aqueous medium is preferably maintained at a pH of about 7 at the initiation and termination (harvest) of the fermen¬ tation process. A higher pH leads to substantial and/or total loss of product. The desired pH may be maintained by the use of a buffer such as morpholino- ethanesulfonic acid (MES), morpholinopropanesulfonic acid (MOPS), and the like, or by choice of nutrient materials which inherently possess buffering properties, such as production media described hereinbelow. The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like. Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like. The preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cotton¬ seed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distiller's solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like. The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and consider¬ able quantities of mineral nutrients, are also suitable for use. When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium -phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like. If necessary, especially when the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added. As one of the starting materials in the process, the FK-520 starting material can be obtained by the fermentation of S. hygroscopicus var. ascomyceticus. ATCC No. 14891, as described in U.S. Patent 3,244,592, and by the fermentation of S. hygroscopicus subsp. yakushimaensis No. 7278, (to produce FR-900520, or "FK-520", and the other FK-506 type macrolides can be obtained by the processes) as described in EPO Publication No. 0184162 to Fujisawa, and PCT WO 89/05304 to Fisons, said above references hereby incorporated by reference for this particular purpose. As to the conditions for the production of phosphorylated organic compound in massive amounts, submerged aerobic cultural conditions are preferred therefor. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism produced in a "slant" and culturing said inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, in which the inoculum is produced, is substantially the same as or different from the medium utilized for the production of the phosphorylated organic compound and is generally autoclaved to sterilize the medium prior to inoculation. The pH of the medium is gener¬ ally adjusted to about 7.0 prior to the autoclaving step by suitable addition of an acid or base, preferably in the form of a buffering solution. Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture. The fermentation is usually conducted at a temperature between about 20 β C and 40 β C, preferably 25-35 β C, for a period of about 10 hours to 24 hours, which may be varied according to fermentation con¬ ditions and scales. Preferably, the production cultures are incubated for about 24 hours at 27 C C on a rotary shaker operating at 220 rpm, wherein the pH of the fermentation medium is maintained at 7.0 to harvest. Preferred culturing/production media for carrying out the fermentation include the following media: Seed and Transformation Medium g___. Glucose 20.0 Soy Flour (Sigma) 5.0 Yeast Extract (Fidco) 5.0 NaCl 5.0 K 2 HP0 4 5.0 Adjust pH to 7.0 The produced phosphorylated organic compound can be recovered from the culture medium by conven¬ tional means which are commonly used for the recovery of other known biologically active substances. The phosphorylated substance produced is found in the cultured mycelium and filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional sol¬ vent, such as methanol and the like, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellu¬ lose, alumina, etc.), crystallization, recrystalliz- ation, and the like. A preferred method is solvent extraction, particularly using methanol. The phosphorylated organic compound obtained according to the resting cell or fermenta¬ tion processes as explained above can be isolated and purified in a conventional manner, for example, extraction, precipitation, fractional crystalliza- tion, recrystallization, chromatography, and the like. Suitable formulations of the material may also include conventional pharmaceutically acceptable biolabile esters of phosphorylated organic compound, formed via the hydroxy groups on the molecule, such as the acetate. The phosphorylated organic compound, and particularly that of FK-520, of the present invention are water soluble possesses pharmacological activity such as i munosuppressive activity, antimicrobial activity, and the like, and therefore are useful for the treatment and prevention of the transplantation rejection of organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc., graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, and the like. The pharmaceutical composition of this invention can be used in the form of a pharmaceu¬ tical preparation, for example, in solid, semisolid or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, supposi¬ tories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisili- cate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semi¬ solid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases. For applying this composition to a human, it is preferable to apply if by parenteral or enteral administration. While the dosage of therapeutically effective amount of the C-32 phosphorylated FK-506, varies from, and also depends upon the age and con¬ dition of each individual patient to be treated, a daily dose (calculated on the basis of a 70 kg man) of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally administered. Rapamvcin It has been further found that a new immunoεuppressant, a phosphorylated rapamycin macrolide, can be obtained via the present process by contacted resting Rhizopus cells in a phosphate buffered medium containing glycerol as a carbon nutrient, or by the fermentation of the microorganism Rhizopus oryzae. ATCC No. 11145 together, in the presence of the macrolide rapamycin, under submerged aerobic conditions in an aqueous carbohydrate medium, containing a nitrogen nutrient, said conditions being conducted at a pH of about 7 for a sufficient time, e.g. 24 hours at 27°C, to selectively C-43 phosphory- late the rapamycin type macrolide. Either process can be utilized but preferred is the process using resting cells. The resultant C-43 phosphorylated macrolide exhibits immunosuppressant activity, similar to rapa¬ mycin i.e., inhibition of T-cell proliferation of mouse T lymphocytes stimulated with the combination of interleukin-2 plus PMA. A positive sample in this assay will inhibit T-cell proliferation, as indicated by reduced tritiated thymidine uptake. Also, in accordance with this invention, there is provided a process for producing an immunosuppressant, identified as a phosphorylated macrolide comprising the step of contacting a strain of a Rhizopus microorganism capable of phosphorylat- ing free hydroxy groups, e.g., Rhizopus oryzae, and specifically Rhizopus orvzae ATCC No. 11145, together with rapamycin macrolide (See U.S. Patent 3,929,992 for its preparartion), in an aqueous medium, containing a carbon nutrient, preferably at a pH below about 8.0, for a sufficient time to produce the C-43 phosphorylated macrolide. Also provided is the unfiltered broth produced by the above process, which exhibits positive inhibition of T-cell activation. Also provided is a new immunosuppressant being a phosphorylated macrolide which exhibits a proton nuclear magnetic resonance spectrum as identified in Figure 3, and a molecular weight of 993 as obtained by (FAB) mass spectrometry. Also provided is a pharmaceutical composition for the treatment of immunoregulatory disorders and diseases containing a therapeutically effective amount of the phosphorylated macrolide in combination with a pharmaceutically acceptable, substantially non-toxic carrier or excipient. In addition, there is provided a method of use for treating a human host to prevent organ transplantation rejection, i.e., heart, kidney, liver, lung, bone marrow, or for treating autoimmune diseases i.e., juvenile diabetes mellitus comprising administering to said host a therapeutically effec¬ tive amount of the phosphorylated macrolide. The present invention involves a biotrans¬ formation process, which involves the contacting of resting cells, or the fermentation of, the micro¬ organism, Rhizopus oryzae. together with a rapamycin macrolide containing a free hydroxy group, to produce the phosphorylated macrolide derivative. The micro¬ organism is currently on deposit with the American Type Culture Collection, 12301 Parklawn Drive in Rockville, Maryland as ATCC No. 11145. By the term "phosphorylated macrolide" as used herein is meant the compound having a proton NMR spectrum as illustrated in Figure 3 (of the methylated derivative), a mass spectrum molecular ion of 993, and an assigned molecular structure of the formula: vfoere R= [HO]2-P- In general, the C-43 phosphorylated macrolide can be produced by either contacting resting cells, of Rhizopus in an aqueous buffered phosphate medium containing a carbon nutrient at ambient temperature, or by culturing (fermenting) the above-described microorganism, Rhizopus oryzae, in the presence of the known rapamycin macrolide, described in U.S. Patent 3,929,992, in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under submerged aerobic conditions (e.g. shaking culture, submerged culture, etc.). The aqueous medium is preferably maintained at a pH of about 7 at the initiation and termination (harvest) of the fermentation process. A higher pH leads to substantial and/ or total loss of product. The desired pH may be maintained by the use of a buffer such as morpholinoethanesulfonic acid (MES), morpholino- propanesulfonic acid (MOPS), and the like, or by choice of nutrient materials which inherently possess buffering properties, such as production media described hereinbelow. The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like. Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like. The preferred sources of nitrogen are yeast extract, meat extract, peptone, gluten meal, cotton¬ seed meal, soybean meal and other vegetable meals (partially or totally defatted), casein hydrolysates, soybean hydrolysates and yeast hydrolysates, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, distiller's solubles, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea, amino acids, and the like. The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and consider¬ able quantities of mineral nutrients, are also suitable for use. When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salts, and the like. If necessary, especially when the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added. The rapamycin starting material can be ob¬ tained by the known fermentation of S. hygroscopicus. NRRL No. 5491, as described in U.S. Patent 3,929,992. As to the conditions for the production of the phosphorylated macrolide in massive amounts, submerged aerobic cultural conditions are preferred therefor. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism produced in a "slant" and culturing said inoculated medium, also called the "seed medium", and then to transfer the cultured vegetative inoculum aseptically to large tanks. The fermentation medium, in which the inoculum is produced, is substantially the same as or different from the medium utilized for the production of the phosphorylated macrolide and is generally autoclaved to sterilize the medium prior to inoculation. The pH of the medium is generally adjusted to about 7.0 prior to the autoclaving step by suitable addition of an acid or base, preferably in the form of a buffering solution. Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture. The fermentation is usually conducted at a temperature between about 20 β C and 40 β C, preferably 25-35 β C, for a period of about 10 hours to 24 hours, which may be varied according to fermentation con¬ ditions and scales. Preferably, the production cultures are incubated for about 24 hours at 27 β C on a rotary shaker operating at 220 rpm, wherein the pH of the fermentation medium is maintained at 7.0 to harvest. Preferred culturing/production media for carrying out the fermentation include the following media: Seed and Transformation Medium g_i__ Dextrose 20.0 Soy Meal (Sigma) 5.0 Yeast Extract (Fidco) 5.0 NaCl 5.0 K 2 HP0 4 5.0 Adjust pH to 7.0 The produced phosphorylated macrolide can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances. The phosphorylated macrolide produced is found in the cultured mycelium and filtrate, and accordingly can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centri- fuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyo- philization, extraction with a conventional solvent, such as methanol and the like, pH adjustment, treat¬ ment with a conventional resin (e.g.anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. acti¬ vated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like. A preferred method is solvent extraction, particularly using methanol. The product phosphorylated macrolide from the fermentation exhibits positive immunosuppressive activity by the "T-cell proliferation assay" and possesses utility on this basis and exhibits the following physical characteristics: 1. White amorphous powder 2. Solubility in methanol 3. Molecular weight of 993, as determined by FAB mass spectroscopy and is consistent with the assigned molecular structure in Figure 3. The phosphorylated macrolide obtained accord¬ ing to the resting cell or fermentation processes as explained above can be isolated and purified in a conventional manner, for example, extraction, precipi- tation, fractional crystallization, recrystallization, chromatography, and the like. Suitable formulations of the material may also include conventional pharmaceutically acceptable biolabile esters of the phosphorylated macrolide, formed via the hydroxy groups on the molecule, such as the acetate. It is to be noted that in the aforementioned fermentation reactions and the post-treatment of the fermentation mixture therein, the tautomeric and conformational isomer(s) of the phosphorylated macrolide, including those due to rearrangement of the hemiketal ring system are also included within the scope of the present invention. The phosphorylated macrolide of the present invention possesses pharmacological activity such as immunosuppressive activity, antimicrobial activity, and the like, and therefore are useful for the treatment and prevention of the transplantation rejection of organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc., graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, and the like. The pharmaceutical composition of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the instant invention compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, supposi¬ tories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisili- cate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semi¬ solid, or liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases. For applying this composition to a human, it is preferable to apply if by parenteral or enteral administration. While the dosage of*therapeutically effective amount of the phosphorylated macrolide, varies from, and also depends upon the age and con¬ dition of -each individual patient to be treated, a daily dose (calculated on the basis of a 70 kg man) of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally administered. Echinocandins The present invention process also is applicable in a process for selectively biophosphorylating a cyclic lipopeptide related to echinocandins and having a peptide skeleton bearing several hydroxy groups wherein in said phosphorylated cyclic lipopeptide, the phosphate group is attached to the hydroxy group of the 4-hydroxyproline component of the lipopeptide. Echinocandins or echinocandin compounds are cyclohexapeptide compounds having a lipophilic side chain and having antifungal properties. Many are natural products but many compounds are semi-synthetic. The natural products are described in the literature by such names as echinocandins, aculeacins, mulundocandin, by number designations or by structure. Many have been known a long time and the structure and properties may be found summarized in the CRC Handbook of Antibiotic Compounds, Vol IV, Part I, pp 355-367, CRC Press, Inc., Boca Raton, Fla. 1980. Still others include a more recently discovered compound such as that described in U.S. Patent 4,931,352. " The present invention is especially directed to a compound having the formula (III): 0 II wherein R is -P(0H) 2 or a cation salt thereof. By "cation salt" is meant a salt of Li, K, Mg, Na, Ca, and (C-^-C^ alkyl)ammonium. 0 II When R is -P(0H)2t the compound may be represented by formula (IIIA), and hereinafter referred to as Compound IIIA. BRIEF DESCRIPTION OF THE DRAWING Fig. 5 is nuclear magnetic resonance 0 II spectrum of Compound III in which R is -P(0H)(0K). MASS SPECTRAL DATA Compound IIIA has a molecular weight of 1144 by FAB-MS (observed (M + Na) + of 1167). NMR DATA -~E NMR Spectra of the compound isolated as a monopotassium salt in CD3OD at 400 MHz is seen in Fig. 5; and -*- C NMR chemical shifts of the same isolate in CD3OD at 100 MHz are as follows: δ c 177.2, 175.9, 174,3, 173.2, 172.9, 172.58, 172.57 f 169.0, 158.4, 133.1, 129.6, 116.2, 77.0, 75.8, 75.2 (doublet, J CP =4.0 Hertz), 74.3, 73.9, 70.6, 70.5, 69.7, 68.3, 62.7, 58.3, 56.3, 56.1, 55.6, 51.2, 47.0, 45.9, 39.4, 38.1, 37.6, 36.7, 35.0, 34.6, 32.9, 31.24, 31.20, 30.8, 30.6, 30.3, 28.1, 27.0, 20.7, 20.2, 19.8, 11.6. On the basis of these and other data, Compound IIIA is believed with considerable certainty to have the structure indicated. Compound IIIA is a white solid soluble in water and polar solvents such as lower alkanols and in dilute alkali metal, magnesium, calcium, and tetra (lower alkyl)ammonium bases. From the bases, salts in which R is a cation salt of phosphate may be obtained. The compound of this invention has similar antibiotic properties as the non-phosphorylated compound and thus would be useful as an antibiotic for the control of parasites, especially Pneumocystis carinii. the causative agent of pneumocystis pneumonia, a particular problem with immune compromised patients, and for the control of fungi. Its antifungal properties are particularly useful against certain strains of yeast, such as Candida albicans and Candida tropicalis- The compound in which R is (0H)£ is conveniently produced by incubating a compound having the formula (Z) (Compound Z) with induced resting cells of Rhizopus arrhizus ATCC 11145 maintained in the Merck Culture Collection as MF 4974. The culture was originally obtained from the American Type Culture Collection at 12301 Parklawn Drive, Rockville, MD 20852. Compound Z maybe produced by cultivating Zalarion arboricola ATCC 74030 in nutrient medium as hereinafter described as well as by methods described in copending applications S.N. 492,025, filed March 12, 1990 and S.N. 492,026, also filed March 12, 1990. The microorganism Rhizopus arrhizus ATCC 11145 is also known as Rhizopus oryzae (J.J. Ellis, 1985, Mycologia 77_: 243-247). The species has also been described under the names Rhizopus nodosus and Rhizopus tritici. The strain MF 4974, ATCC 11145, exhibits all the essential features of E. arrhizus described by M.A.A. Schipper under the name β. orvzae. CBS Studies in Mycology 25.:1-19 (1984). During a recent regrowth of MF4974 ATCC 11145, the following diagnostic characteristics were observed. The strain is apparently heterothallic because zygospore spore formation was not observed. Colonies grow on most standard mycological media, but on cornmeal agar (Difco) are extremely fast-growing, reaching 35 mm in diameter in 36 hours at 20 β C, reaching >90 mm in 36 hours at 37 β C, with sporangiophores completely filling Petri dishes, hyaline at first but soon becoming pale yellowish gray to light gray, brownish gray, finally dark gray. Sporangiophores 200-1000 μm tall, 7.5-19 μm wide, aseptate, straight to curved at base, flared at apex, sometimes furcate, with walls slightly thickened, with minutely granular surface, pale yellowish brown or yellowish gray, arising from rhizoidal hyphae. Rhizoidal hyphae consisting of 5 3-10, thick, often contorted branches. Sporangia globose to subglobose, slightly flattened on underside, 100-230 μm in diameter, opaque, with surface spiny, dark gray to black. Columellae 18-50 μm in diameter, hemispherical 10 to subglobose, smooth, often collapsing, without adhering sporangia remnants. Sporangiospores 5-8 X 2.5-5 μm, subglobose to irregularly elliptical, or angular in sideview, with faint to prominent longitudinal striations, hyaline to pale yellowish 15 brown. Although the invention is discussed hereinbelow principally with respect to a specific strain, not only the strain described above, but varieties and mutants, whether obtained by natural z\ n selection, produced by the action of various mutating agents such as ionizing radiation or chemical agents such as nitrosoguanidine are contemplated within the scope of the present invention. Compound IIIA may be produced by cultivating 25 Rhizopus arrhizus ATCC 11145 in a suitable nutrient medium containing Compound Z under conditions hereinafter described and thereafter recovering from the product medium by extracting the desired product from the fermentation medium with a suitable solvent, 0 concentrating the component containing the desired compound, and then subjecting the concentrated material to chromatographic separation. The cultivation is carried out in a medium containing sources of carbon and nitrogen assimilable by the microorganism. The sources of carbon include glycerol, sugars, sugar alcohols, starches and other carbo¬ hydrates, or carbohydrate deivatives such as dextran, cerelose, as well as complex nutrients such as oat flour, corn meal, millet, corn and the like. The exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount of carbohydrate between 0.5 and 40% by weight of the medium is εatifactory. These carbon sources can be used individually or several such carbon sources may be combined in the same medium. The sources of nitrogen include amino acids such as glycine, arginine, threonine, methionine and the like, ammonium salt, and complex sources such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, soybean meal, casein hydrolysates, yeast extracts, corn steep liquors, distillers solubles, cottonseed meal, meat extract, and the like. The various sources of nitrogen can be used alone or in combination in amounts ranging form 0.2 to 10 percent by weight of the medium. In addition, the medium should contain a phosphate salt. The phosphate salt should be at least about 10 percent by weight of the solid components. It is preferably from about 12 to about 15 percent. A particularly suitable medium is soy-glucose medium of the following composition which may be employed both as a seed medium and a culture medium: Soy-Glucose Medium g/1 Glucose 20.0 Soya meal 5.0 Fidco yeast extract* 5.0 NaCl 5.0 K 2 HP0 4 5.0 Adjust pH to 5 *Fidco yeast extract is a nitrogen source, product of Difco Laboratories, Detroit MI. The fermentation may be carried out by first preparing a seed culture. In preparing a seed culture, spores of Rhizopus arrhizus are obtained from oatmeal agar slants of MF 4974 maintained in the Merck Culture Collection and dispersed in water to obtain a spore suspension containing about 7 x 10 9 spores per milliliter. The spore suspension of MF 4974 is inoculated into a seed flask containing the soy glucose broth and the inoculated suspension incubated on a rotary shaker in the temperature range of from about 15°C to about 30 β C, preferably 25 β to 28 β C. The agitation may be up to 400 rpm but generally about 220 rpm is preferred. The incubation is carried out over a period of at least 24 hours to about two days. When growth is abundant, the mycelia are harvested by filtering through a nylon mesh. For biophosphorylation, the mycelia are suspended in a phosphate buffer containing 3 percent glycerol or some other simple carbon source. Compound Z then is added at a concentration of about 50 μg/ml in dimethylsulfoxide (DMSO). The pH of the production medium is important. The flasks are incubated, pre¬ ferably with shaking at 220 rpm at 27 β C for 24 to 48 hours to produce Compound IIIA. It is critical that it be maintained in the range of about 6.0 to 6.3. After completion of the incubation period the contents of all the flasks are pooled and filtered through a nylon mesh filter. The mycelial cake on the filter is slurried with aqueous methanol and filtered. The procedure is repeated with the filter cake and the filtrate loaded onto a styrene/ divinylbenzene column. The column is then washed with water and the phosphorylated product eluted with 20 percent aqueous acetonitrile and the remaining metabolite and substrate eluted with 70 percent aqueous acetonitrile. After elution, the fractions may be assayed by HPLC. The fractions determined to have the desired product as indicated by a retention time of 12.8 mins. are combined and concentrated under reduced pressure to obtain the product as residue. The salts, i.e., where R is a cationic salt of the phosphate, may be prepared by intimately contacting a base corresponding to the cation in an alcoholic or other polar solvent, then concentrating to initiate crystallization of the salt. Thereafter, the salt is recovered by filtration. One method of preparing salts is to apply an aqueous solution of the acid onto a non-functional- ized resin column. Representative resins include "AMBERCHR0M"-161 (divinylbenzene/polysyyrene resin, obtainable from TosoHaas; trademark name registered by Rohm and Haas), "DIAION" HP-20 and SP-207 (cross- linked styrene-divinylbenzene and brominated εtyrene- divinylbenzene, respectively, products of Mitsubishi Chemical). The column is then washed with aqueous H2PO4 or '(H2P0 )2 where M and M 1 are monovalent and divalent cations respectively, thereby converting the acid to a mono-cation salt form. The column is washed with water to remove excess inorganic phosphate salt. The product M or M' salt is then removed from the column by applying an aqueous eluant having greater than 50 percent organic content. Useful eluants are 80 percent acetonitrile, 80 percent ethanol or 80 percent methanol. The product is isolated by concentration to dryness and/or lyophilization of the eluate. This procedure also may be employed to prepare one salt from another. Alternatively, the acid is dissolved in an aqueous mobile phase containing low amount of organic solvent such as acetonitrile and containing phosphate salt thereby forming a solution of the salt of the acid. The solution is subjected to reduced pressure to remove the acetonitrile, then applied to a C-18 extraction column to retain the salt of the product on the column. The salt of the product then may be removed as above described. As previously noted, the phosphate is a compound which is active against certain yeast fungi such as (_. albicans and £. tropicalis- The activity may be seen in a microbroth dilution assay employing a Yeast Nitrogen Base (Difco) with 17. dextrose (YNBD). In carrying out the assay, Compound IIIA was solubilized in 10 percent dimethyl sulfoxide (DMSO) and diluted to 2560 μg/ml. The compounds were further diluted to 256 μg/ml in YNBD. Then 0.15 ml of the suspension was dispensed to the first row of a 96-well plate (each well containing 0.15 ml of YNDB) resulting in a drug concentration of 128 μg/ml. Two¬ fold dilutions were then made to obtain final drug concentrations ranging from 128 to 0.06 μg/ml. The yeast cultures, maintained on Sabouraud dextrose agar were transferred to YM broth (Difco) and incubated overnight at 35°C with shaking (250 rpm). After incubation, each culture was diluted in sterile water to yield a final concentration of 1-5 x 10 6 colony forming units (CFU)/ml. 96-well microplates were inoculated using a MIC-2000 (Dynatech) which delivers 1.5 μl per well yielding a final inoculum per well of 1.5-7.5 x 10 3 cells. The microplates were incubated at 35°C for 24 hours. The minimum inhibitory concentrations (MICs) were recorded as the lowest concentrations of drug showing no visible growth. After recording the MIC, the plates were shaken to resuspend the cells. Thereafter, 1.5 μl samples from the wells in the 96-well microplate were transferred to a single well tray containing Sabouraud dextrose agar. The inoculated trays were incubated 24 hours at 28 β C and then read. The MFC is defined as the lowest concentration of drug showing no growth or less than 4 colonies per spot. Minimum Fungicidal Concentration Fungus (μ_gial) Strain No. om ound IIIA Candida albicans MY 1055 64 MY 1028 64 Candida fP JC lJS MY 1012 32 10 The foregoing illustrates particular suitability for treating mycotic infections. The compounds of the present invention may be employed in inhibiting or alleviating Pneumocvstis 15 carinii infections. In such use, Compound III/IIIA or a composition containing Compound III/IIIA may be administered in a therapeutically effective or inhibitory amount to subjects infected with or susceptible to being infected with Pneumocystis 2 Δ 0- _____.__.__._ _ The suitability of the compounds of the present invention for therapeutic or anti-infective purposes may be determined in studies on immuno- suppressed rats when Sprague-Dawley rats (weighing 5 approximately 200 grams) are immunosuppressed with dexasone in the drinking water (2.0 mg/L) and maintained on a low -protein diet for five weeks to induce the development of pneumocystis pneumonia from a latent infection. Before drug treatment, two rats 30 are sacrificed to confirm the presence of Pneumocystis carinii pneumonia (PCP). Six rats then are injected twice daily for four days intravenously (I.V.) via the tail vein with Compound III in 0.25 ml of vehicle (distilled water). A vehicle control is also carried out. All animals continue to receive dexasone in the drinking water and low protein diet during the treatment period. At the completion of the treat¬ ment, all animals are sacrificed, the lungs are removed and processed, and the extent of disease determined by microscopic analysis of stained slides. A similar experiment may be carried out in which the rats are injected intraperitoneally (I.P.) twice daily for four days and then sacrificed, the lungs removed and processed, and the extent of disease determined by microscopic analysis of stained slides. The outstanding properties are most effec- tively utilized when the compound is formulated into novel pharmaceutical compositions with a pharmaceu¬ tically acceptable carrier according to conventional pharmaceutical compounding techniques. The novel compositions contain at least a therapeutic antifungal or antipneumocystis amount of the active compound. Generally, the composition contains at least 1% by weight of Compound III. Concentrate compositions suitable for dilutions prior to use may contain 90% or more by weight. The compositions include compositions suitable for rectal, topical, parenteral (including subcutaneous, intramuscular, and intravenous), pulmonary (nasal or buccal inhalation), nasal administration, or insufflation. The compositions may be prepacked by intimately mixing Compound III with the components suitable for the medium desired. When the compound is for antifungal use any method of administration may be used. For treating mycotic infection, oral administration is frequently preferred. When oral administration is to be employed, it may be with a liquid composition or a solid composition. For liquid preparations, the therapeutic agent is preferably formulated with water or aqueous compositions, but if desired, may be formulated with glycols, oils, alcohols, and the like. For solid preparations such as capsules and tablets, solid carriers such as starches, sugars, kaolin, ethyl cellulose, calcium and sodium carbonate, calcium phosphate, kaolin, talc, lactose, generally with lubricant such as calcium stearate, together with binders, disintegrating agents and the like. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage form. It is especially advantageous to formulate the compositions in unit dosage form (as hereinafter defined) for ease of administration and uniformity of dosage. Composition in unit dosage form constitutes an aspect of the present invention. The Compound III is preferably formulated in aqueous therapeutic compositions for intravenous or intraperitoneal injection or aerosol when use against Pneumocystis carinii is contemplated, and may be presented in unit dosage form in ampoules or in multidose containers, if necessary with an added preservative. The compositions may also take such forms as solutions in aqueous vehicles such as 0.85 percent sodium chloride or 5 percent dextrose in water, and may contain formulating agents such as stabilizing and/or dispersing agents. Buffering agents as well as additives such as saline or glucose may be added to make the solutions isotonic. The drug also may be solubilized in alcohol/propylene glycol or polyethylene glycol for drip intravenous administration. Alternatively, the active ingre¬ dients may be in powder form for reconstituting with a suitable vehicle prior to administration. The term "unit dosage form" as used in the specification and claims refer to physically discrete units, each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceu¬ tical carrier. Examples of such unit dosage forms are tablets, capsules, pills, powder packets, wafers, measured units in ampoules or in multidose containers and the like. A unit dosage of the present invention may contain from 100 to 1000 milligrams of one of the compounds. HIV Protease Inhibitors - I The present invention is also concerned with a compound which inhibits the protease encoded by human immunodeficiency virus (HIV). The compound, or pharmaceutically acceptable salt thereof, is of value in the prevention of infection by HIV, the treatment of infection by HIV and the treatment of the resulting acquired immune deficiency syndrome (AIDS). The present invention also relates to pharmaceutical compositions containing the compounds, and to a method of use of the present compounds with or without other agents for the treatment of AIDS &. viral infection by HIV. A retrovirus designated human immunodefi¬ ciency virus (HIV) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is the extensive post-trans- lational processing of precursor polyproteins by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Interruption of this processing appears to prevent the production of normally infectious virus. For example, Crawford, S. __t _ ,. , J. Virol., ___., 899, 1985, demonstrated that genetic deletion mutations of the protease in murine leukemia virus which prevent processing of precursor structural proteins result in non-infectious viral particles. Unprocessed struc¬ tural proteins also have been observed in clones of non-infectious HIV strains isolated from human patients. These results suggest that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV. Nucleotide sequencing of HIV shows the presence of a pol gene in one open reading frame [L. Ratner, _± -Al.. Nature, H , 277(1985)]. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an endo- nuclease and an HIV protease [H. Tol, s__ Al. , EMBO J. 4, 1267 (1985); M. D. Power, ≤i. aL. , Science, 231. 1567 (1986); L. H. Pearl, fit al. , Nature ___!, 351 (1987)]. Applicants demonstrate that the compound of this invention is an inhibitor of HIV protease. The compound of this invention provides a prodrug for the inhibition of HIV protease. A biotransformed compound as herein defined is disclosed. This compound is useful in the inhibition of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or not as a prodrug or as a combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV are also disclosed. This invention is also concerned with the use of a compound given below, or pharmaceutically acceptable salts thereof, in the inhibition of HIV protease, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS). The biotransformed compound is the product of the incubation of Rhizopus arrhizus (ATCC 11145) in the presence of L-702,083, an HIV protease inhibitor. It is defined as follows: or pharmaceutically acceptable salts, hydrates or esters thereof. The pharmaceutically-acceptable salts of the compound of the present invention (in the form of water- or oil-soluble or disperεible products) include the conventional non-toxic salts or the quaternary ammonium salts of this compound, which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalene- sulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-ρropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. Hydrates or esters are also encompassed by the present invention. Such hydrates or esters are those which would readily occur to the skilled artisan, and include, for example, C- j ^alkyl esters. The compound of the present invention is useful in the inhibition of HIV protease, the prevention or treatment of infection by the human immunodeficiency virus (HIV) and the treatment of consequent pathological conditions such as AIDS. Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of states of HIV infection: AIDS, ARC (AIDS related complex), both symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the compound of this invention is useful in treating infection by HIV after suspected past exposure to HIV by e.g., blood transfusion, accidental needle stick, or exposure to patient blood during surgery. For these purposes, the compound of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles. Thus, in accordance with the present inven¬ tion there is further provided a method of treating and a pharmaceutical composition for treating HIV infection and AIDS. The treatment involves admini¬ stering to a patient in need of such treatment a pharmaceutical composition comprising a pharmaceu¬ tical carrier and a therapeutically-effective amount of the compound of the present invention. These pharmaceutical compositions may be in the form of orally-administrable suspensions or tablets; nasal sprays; sterile injectable prepara¬ tions, for example, as sterile injectable aqueous or oleagenous suspensions or suppositories. When administered orally as a suspension, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants known in the art. When administered by nasal aerosol or inhal¬ ation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. The injectable solutions or suspensions may be formulated according to known art, using suit¬ able non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid. When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug. Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are useful in the treatment or prevention of the above-indicated conditions, with oral doses two-to-five times higher. For example, infection by HIV is effectively treated by the administration of from 10 to 50 milligrams of the compound per kilogram of body weight from one to three times per day. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the acti¬ vity of the specific compound employed, the metabolic stability and length of action of that compound, the age of the patient, body weight, general health, sex, diet, mode and time of administration, rate of excre¬ tion, drug combination, the severity of the particular condition, and the host undergoing therapy. The present invention is also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS. For example, the compound of this invention may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of other AIDS antiviralε, immunomodulatorε, anti-infectives, or vaccines. HIV Protease Inhibitor - II The present invention is also further con¬ cerned with another compound which inhibits the protease encoded by human immunodeficiency virus (HIV). The compound, or pharmaceutically acceptable salt thereof, is of value in the prevention of infection by HIV, the treatment of infection by HIV and the treatment of the resulting acquired immune deficiency syndrome (AIDS). The present invention also relates to pharmaceutical compositions containing the compound, and to a method of use of the present compound with or without other agents for the treatment of AIDS & viral infection by HIV. A retrovirus designated human immunodefi- ciency virus (HIV) is the etiological agent of the complex disease that includes progressive destruction of the immune system (acquired immune deficiency syndrome; AIDS) and degeneration of the central and peripheral nervous system. This virus was previously known as LAV, HTLV-III, or ARV. A common feature of retrovirus replication is the extensive post-trans- lational processing of precursor polyproteinε by a virally encoded protease to generate mature viral proteins required for virus assembly and function. Interruption of this processing appears to prevent the production of normally infectious virus. For example, Crawford, S. fit. al. , J. Virol., 52., 899, 1985, demonstrated that genetic deletion mutations of the protease in murine leukemia virus which prevent processing of precursor structural proteins result in non-infectious viral particles. Unprocessed struc¬ tural proteins also have been observed in clones of non-infectious HIV strainε isolated from human patients. These results suggest that inhibition of the HIV protease represents a viable method for the treatment of AIDS and the prevention or treatment of infection by HIV. Nucleotide sequencing of HIV shows the presence of a pol gene in one open reading frame [Ratner, L. e± al. , Nature, ____, 277(1985)]. Amino acid sequence homology provides evidence that the pol sequence encodes reverse transcriptase, an endonuclease and an HIV protease [Toh, H. e_t al- » EMBO J. 4, 1267 (1985); Power, M.D. ___ ___. , Science, 231. 1567 (1986); Pearl, L.H. fit Al.. Nature 221, 351 (1987)]. Applicants demonstrate that the compound of this invention is an inhibitor of HIV protease. The compound of this invention provides a prodrug for the inhibition of HIV protease. A biotransformed compound as herein defined is disclosed. This compound is useful in the inhibi¬ tion of HIV protease, the prevention of infection by HIV, the treatment of infection by HIV and in the treatment of AIDS and/or ARC, either as a compound, pharmaceutically acceptable salt (when appropriate), pharmaceutical composition ingredient, whether or not as a prodrug or as a combination with other antivirals, anti-infectives, immunomodulators, antibiotics or vaccines. Methods of treating AIDS, methods of preventing infection by HIV, and methods of treating infection by HIV are also disclosed. This invention is concerned with the use of a compound given below, or pharmaceutically accept¬ able salts thereof, in the inhibition of HIV protease, the prevention or treatment of infection by HIV and in the treatment of the resulting acquired immune deficiency syndrome (AIDS). The biotransformed compound is the product of the incubation of Rhizopus arrhizus (ATCC 11145) in the presence of L-689,502, a HIV protease inhibitor. It is defined as follows: o II R= -P-OH I OH or pharmaceutically acceptable saltε, hydrates or esters thereof. The pharmaceutically-acceptable saltε of the compound of the preεent invention (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts or "the quaternary ammonium saltε of thiε compound, which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalene- εulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potasεium εalts, alkaline earth metal salts such aε calcium and magnesium εaltε, εaltε with organic baεeε such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfateε, long chain halideε εuch aε decyl, lauryl, myriεtyl and εtearyl chlorideε, bromideε and iodideε, aralkyl halideε like benzyl and phenethyl bromideε and others. Hydrates or esters are also encompassed by the present invention. Such hydrates or esters are those which would readily occur to the skilled artisan, and include, for example, C- j ^alkyl esters. The compound of the present inventions is useful in the inhibition of HIV protease, the prevention or treatment of infection by the human immunodeficiency virus (HIV) and the treatment of conεequent pathological conditions such as AIDS. Treating AIDS or preventing or treating infection by HIV is defined as including, but not limited to, treating a wide range of εtates of HIV infection: AIDS, ARC (AIDS related complex), both symptomatic and asymptomatic, and actual or potential exposure to HIV. For example, the compound of this invention is useful in treating infection by HIV after εuspected past exposure to HIV by e.g., blood transfuεion, accidental needle εtick, or expoεure to patient blood during εurgery. For these purposes, the compound of the present invention may be administered orally, parenterally (including εubcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically-acceptable carriers, adjuvants and vehicles. Thus, in accordance with the present inven¬ tion there is further provided a method of treating and a pharmaceutical composition for treating HIV infection and AIDS. The treatment involves admini¬ stering to a patient in need of such treatment a phar- maceutical composition comprising a pharmaceutical carrier and a therapeutically-effective amount of the compound of the present invention. These pharmaceutical compositionε may be in the form of orally-administrable suεpenεionε or tabletε; naεal εprays; sterile injectable preparations, for example, as sterile injectable aqueouε or oleagenous suεpensions or εuppoεitorieε. When adminiεtered orally as a suspen¬ sion, these compositionε are prepared according to techniques well-known in the art of pharmaceutical formulation and may contain microcrystalline cel¬ lulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweetnerε/flavoring agentε known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluentε and lubricantε known in the art. When adminiεtered by naεal aerosol or inhal¬ ation, these compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservativeε, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or disperεing agents known in the art. The injectable solutions or suspensionε may be formulated according to known art, uεing suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable diεperεing or wetting and εuεpending agentε, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acidε, including oleic acid. When rectally administered in the form of εuppoεitories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidity and/or diεεolve in the rectal cavity to releaεe the drug. Dosage levels of the order of 0.02 to 5.0 or 10.0 grams-per-day are uεeful in the treatment or prevention of the above-indicated conditionε, with oral doses two-to-five times higher. For example, infection by HIV is effectively treated by the administration of from 10 to 50 milligrams of the compound per kilogram of body weight from one to three times per day. It will be understood, however, that the specific doεe level and frequency of doεage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stabi¬ lity and length of action of that compound, the age of the patient, body weight, general health, εex, diet, mode and time of adminiεtration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. The present invention is also directed to combinations of the HIV protease inhibitor compound with one or more agents useful in the treatment of AIDS. For example, the compound of this invention may be effectively administered, whether at periods of pre-exposure and/or post-expoεure, in combination with effective amountε of other AIDS antiviralε, immunomodulators, anti-infectives, or vaccines. Simvastatin Analogs Also included by the process of this invention are two phosphorylated derivatives of simvastatin, whose chemical name is 6(R)-[2-(8' (S)-2",2"-dimethylbutanoyloxy-2'(S),6'(R)-dimethyl- l',2',6',7',8',8'a (R)-hexahydronaphthyl-l' (S)- ethyl]-4(R)-hydroxy-3,4,5,6-tetrahydro-2H-pyran- 2-one. See EPO Publication No. 0033 358 and The Merck Index, Eleventh Edition, for a description of the compound, its synthesis and utility as an anti-hypercholesterolemic agent in interfering with cholesterol biosyntheεiε. Contacting simvastatin with the Rhizopus microorganisim under the conditions described herein results in two phosphorylated derivatives: O II L-706, 526 15 L-706, 527 30 The above two compounds of this invention are useful as antihypercholesterolemic agentε for the treatment of atherosclerosis, hyperlipemia and like diseases in humans. They may be administered orally or parenterally in the form of a capsule, a tablet, an injectable preparation or the like. It is usually desirable to use the oral route. Doses may be varied, depending on the age, severity, body weight and other conditions of human patients but daily dosage for adults is within a range of from about 2 mg to 2000 mg (preferably 10 to 100 mg) given in three or four divided doses. Higher doseε may be favorably applied as required. The compounds of rhis invention alεo have useful anti-fungal activities. For example, they may be used to control strainε of Penicillium εp., Aspergillus ni_ge , Cladosporium εp., Cochlioboluε miyabeanns and Helminthoεporium cynodnotis. For those utilities they are admixed with suitable formulating agents, powders, emulsifying agents or εolventε εuch aε aqueous ethanol and sprayed or dusted on the plants to be protected. The pharmaceutically acceptable salts of this invention include those formed from cations εuch aε εodium, potaεsium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium as well as those salts formed from amines such as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N*-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzyl- phenethylamine, l-p-chlorobenzyl-2-pyrrolidine-l'- yl-methylbenzimidazole, diethylamine, piperazine, and tris(hydroxymethyl)aminomethane. Zearalenone Also included by the procesε of thiε invention is the biotransformation product: alpha zearalenol 6'-phoεphate: cx-zearalenol-6' -phos phate The present invention also relates to the above new compound and an object of the present invention is to provide compounds which exhibit estrogenic activity or aid in increasing the rate of growth in meat-producing animals, e.g. cattle, lamb and swine. The compound can be administered to animals by any εuitable method including oral and parenteral administrations. For example, the compound can be blended with ordinary feed containing nutritional values in an amount sufficient to produce the desired rate of growth and can thus be fed directly to the animals, or the compound can be suspended in a suit¬ able injection suspension medium εuch as peanut oil and injected parenterally. The amount of compound fed to an animal, of course, varies depending upon the animal, desired rate of growth and the like. When the new products are to administered in feeds, an animal feed composition may be prepared con¬ taining the usual nutritionally-balanced quantities of carbohydrates, proteins, vitamins and minerals, together with the compounds of the present inven¬ tion. Some of these usual dietary elements are grains, such as ground grain and grain by-products; animals protein substanceε, εuch as those found in fish meal and meat εcrapε; vegetable proteinε like εoybean oil meal or peanut oil meal; vitaminaceouε materials, e.g. vitamin A and D complex members; and bone meal and limestone to provide mineralε. A type of conventional feed material for uεe with cattle includeε alfalfa hay and ground corn cobε together with supplementary vitaminaceous εubεtanceε iε deεired. The εtarting material racemic (±)-zearalenone, a potent anabolic agent uεeful in the raising of meat-producing animalε, originally waε prepared by fermenting the microorganism, Gibberella zeae (Gordon), on a suitable nutrient medium according to the techniques described in U.S. Patent No. 3,169,019. More recently there haε been described the total chemical εyntheεiε of racemic (±)- zearalenone and the 2,4-dimethyl ether derivative thereof (Taub et al., Chemical Communications, 1967, p. 225). The procesε deεcribed in U.S. Patent No. 3,551,455 and in U.S. Patent No. 3,239,545 affordε new and more direct routeε to the total εyntheεiε of (±)-zearalenoneε. Example IA A spore sand culture containing Gibberella zeae (Gordon) NRRL-2830 was aseptically placed in a sterile tube containing 15 milliliters of Czapek's- Dox solution and a small amount of agar. Thiε medium was then incubated for about 168 hours at approxi¬ mately 25 C C. At the end of the incubation period, the medium was washed with 5 milliliters of sterile deionized water and transferred to a sterile tube containing 45 milliliters of Czapek's-Dox solution. The contents of the tube were then incubated for about 96 hours at about 25 β C after which the material was available for use in inoculation of a fermentation medium. The following example illustrateε the fermentation of the organiεm Gibberella zeae (Gordon) NRRL-2830 to produce zearalenone. Example IIA To a 2 liter flaεk were added 300 gramε of finely divided corn. The flask and its contents were then sterilized and after sterilization 150 milliliters of sterile deionized water were added. To the mixture in the flaεk were then added 45 milliliters of the inoculum prepared by the process of Example IA and the material was thoroughly mixed The mixed material was then incubated for about 20 days at 25 β C in a dark room in a water-saturated atmosphere. The following example illustrateε the recovery of the zearalenone from the fermentation medium. Example IIIA A 300 gram portion of fermented material produced by the method of Example IIA was placed in 500 milliliters of deionized water and εlurried. The εlurry waε then heated for about 15 minuteε at 75 β C, 300 gramε of filter aid were then added and the material waε filtered. The solid filtered material containing the anabolic substance was then air dried, and 333 grams of the dried cake were then extracted with 500 milliliters of ethanol. This procedure was repeated three more times. The ethanol extract was evaporated to drynesε under vacuum to give 6.84 gramε of εolid material. Thiε εolid material waε then diε- εolved in 20 milliliters of chloroform and extracted with 30 milliliters of an aqueous solution containing 5% by weight of sodium carbonate having an adjusted pH of about 11.2. The extraction proceεε waε repeated εeven more timeε. The pH of the sodiumcarbonate extract was then adjusted to 6.2 with hydrochloric acid, to yield an anabolic subεtance-containing precipitate. The precipitate and the aqueouε εodium carbonate extract were then each in turn extracted with 75 milliliters of ethyl ether. This procedure was repeated three more times to yield a light yellow ethereal solution, which was then evaporated to yield 116 milligramε of εolid anabolic εubεtance. This material was then subjected to multiple transfer countercurrent distribution using 100 tubes and a εolvent εyεtem conεiεting of two parts chloroform and two parts carbontetrachloride as the lower phase and four parts methanol and one part water as the upper phase, all parts by volume. The solid material obtained from the multiple transfer countercurrent distribution was zearalenone. The following examples are given for the purpose of illustrating the present invention and should not be construed as being limitations on the scope or spirit of the instant invention. EXAMPLE 1 C-32 Phosphorylated FK-520 Microorganism and Culture Conditions Sporeε of Rhizopuε oryzae MF4974 (ATCC No. 11145) cultivated on an oatmeal agar flask were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmer flask and shaken at 27°C on a rotary shaker at 220 rpm for 24 hours. The εecond εtage flaεk (50 ml in a 250 ml Erlenmeyer flaεk) were inoculated with 2.5 ml of seed culture and incubated on a rotary shaker (220 rpm) at 27 β C for 24 hours. Following incubation, each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM pH 7.0 PO buffer containing 3% glycerol. F -520 was added to achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary shaker (220 rpm) at 27°C for 48 hours. Following incubation, the whole broth was extracted as described in the Isolation/Purification Section below. The whole broth (400 ml) was adjusted to pH 3.5 and extracted three times with methylene chloride (3 x 400 ml). Methylene chloride extracts were combined and evaporated to dryness under reduced presεure at 30 β C. The reεulting oil waε diεsolved in methanol and εubjected to HPLC purification. HPLC was carried out on Whatman Partisil 10 ODS-3, 9.4 mm x 25 cm at 50 β C and monitored at 205 nm. The column was developed at 3 ml/minutes with a linear gradient from 35% acetonitrile in 0.1% phosphoric acid to 80% in 0.1% phosphoric acid in 60 minutes. The compound was collected during repeated injections of the above described extract. The fractions at retention time, 32 minutes, were pooled, adjuεted to pH 3 and evaporated to remove acetonitrile. The compound was further purified using C18 Sep Pak (Water Associates) and methanol-water elution solvent to yield 4.5 mg pf product. Characterization C-32 Phosphorylated F -520 (FK-900520) was characterized by FAB mass spectrometry and FK-520 via NMR spectrometry yielding the proton NMR spectrum of Figure 1, which confirms the asεigned molecular structure in Figure 2. EXAMPLE 2 T-Cell Proliferation Assay 1. Sample Preparation Purified C-32 phosphorylated FK-520, as prepared by HPLC above, was dissolved in absolute ethanol at 10 mg/ml. 2. Assay Spleens from C57B1/6 mice were taken under sterile conditions and gently disεociated in ice-cold RPMI 1640 culture medium (GIBCO, Grand Iεland, N.Y.) εupplemented with 10% heat-inactivated fetal calf εerum (GIBCO). Cellε were pelleted by centrifugation at 1500 rpm for 8 minuteε. Contaminating red cellε were removed by treating the pellet with ammonium chloride lysing buffer (GIBCO) for 2 minuteε at 4 β C. Cold medium waε added and cellε were again centrifuged at 1500 rpm for 8 minutes. T lymphocyteε were then iεolated by separation of the cell suspenεion on nylon wool columnε as follows: Nylon wool columns were prepared by packing approximately 4 grams of washed and dried nylon wool into 20 ml plastic syringeε. The columns were εterilized by autoclav- ing at 250 β F for 30 minuteε. Nylon wool columnε were wetted with warm (37 β C) culture medium and rinsed with the same medium. Washed spleen cellε resuspended in warm medium were slowly applied to the nylon wool. The columns were then incubated in an upright position at 37 β C for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell suspensions were spun aε above. Purified T lymphocytes were resuspended at 2.5 x lO** 5 cells/ml in complete culture medium composed of RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 100 mM glutamine, 1 mM sodium pyruvate, 2 x 10 "* ** M 2-mercaptoethanol and 50 μg/ml gentamycin. Ionomycin was added at 250 ng/ml and PMA at 10 ng/ml. The cell suεpenεion waε immediately diεtributed into 96 well flat-bottom microculture plates (Costar) at 200 μl/well. The control, being the medium without test drug, and various below-indicated dilutions of the above sample of purified C-32 phosphorylated FK-520 to be tested were then added in triplicate wells at 20 μl/well. FK-520 was used as a standard. The culture plates were then incubated at 37"C in a humidified atmosphere of 3% Cθ2- 5% air for 44 hours. The proliferation of T lymphocytes waε assessed by measurement of tritiated thymidine incorporation. After 44 hours of culturing, the cells were pulse-labelled with 2 μCi/well of tritiated thymidine (NEN, Cambridge, MA). After another 4 hours of incubation, cultures were harvested on glass fiber filters using a multiple sample harvester. Radioactivity of filter discε correεponding to individual wellε waε measured by standard liquid scintillation counting methods (Betacounter). Mean counts per minute of replicate wells were calculated and the results expressed as percent inhibition of tritiated thymidine uptake (proliferation) as follows: Mean cpm sample tested % Inhibition = 100 - Mean cpm control medium ■***• • *- ( * ) " The results of % inhibition at various concentrations of C-32 phosphorylared FK-520 (C-32-P F -520) are presented in the following table: __*_____ Inhibition of T-Cell Proliferation bv C-32-P FK-520 - - - - (ng/ml) % Inhibition 97 95 89.6 71.3 28.7 0.0 Notes: 1. Mouse T cell cultures were pulsed with H-thymidine for 4 hours prior to harvesting at 48 hours. 2. Standard FK-520 (4 ng/ml) gave 97% inhibition. 3. The mean IC 50 for C-32-P FK-520 was determined to be: 15.0 + 2.1 ng/ml (17.2 ± 2.4 nM) in 3 independent experiments. 4. Inhibition of T-Cell proliferation was reversed by the addition of 100 units/ml of recombinant human IL-2 at the initiation of culture. EXAMPLE 3 C-32 Phosphorylated FK-506 Fermentation Spores of Rhizopus oryzae MF 4974, cultivated on oatmeal agar, were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmeyer flask and shaken at 25 β C on a rotary shaker at 220 rpm for 24 hours. The second stage flasks (50ml in a 250 ml Erlenmeyer flask) were inoculated with 2.5 ml of seed culture and incubated on a rotary εhaker (220 rpm) at 27 β C for 24 hours. Following incubation, each flask was harvested by centrifugation, washed once with sterile water, and resuspended in equal volume of 100 mM P04 buffer containing 3% glycerol, FK506 was added to achieve a final concentration of 0.2 mg/ l. The charged flasks were incubated on a rotary shaker (220 rpm) at 27 β C for 24 hours. Following incubation, the whole broth was worked up as described below. Adjust pH to 7.0 ISOLATION AND PURIFICATION The whole broth (200 ml) was maintained at pH 6.8 and centrifuged. The mycelial cake was washed with water, then discarded. The clear filtrate and washings were pooled and pasεed thru a Spe-ed octadecyl cartridge (14% carbon load, Applied Separationε) under vaccum. The column was washed with 100 ml of water. Column effluent and wash did not contain microbial transformation product when tested with HPLC. The cartridge was eluted with 200 mL methanol. Methanol was evaporated to drynesε under reduced preεεure at 30 β C. The resulting oil was dissolved in methanol and subjected to HPLC purification. HPLC was carried out on Whatman Magnum 20 Partisil 1- ODS-3 Column (C lg ,22.1 mm ID x 25 cm) at 50 β C and monitored at 205 nm. The column waε developed at 7 mL/min with linear gradient from 35% acetonitrile in 0.1% phosphoric acid to 80% acetonitrile in 0.1% phosphoric acid in 70 minutes. The compound was collected during repeated injections of the above described extract. Fractions of retention time 50 minutes were pooled, adjusted to pH 3.0 and evaporated to remove acetonitrile. The compound was desalted using a C**^ Sep Pak (Water Asεociate) to yield 20 mg of product. CHARACTERIZATION The compound was characterized by MS and confirmed by NMR as the C-32 phosphate ester derivative of F 506. EXAMPLE 4 C-32 Phosphorylated C-31-desmethyl FK-520 The fermentation procedure of Example 3 was carried out substantially identical except that C-31 desmethyl F -520 (available as for example, by the procedure in EPO Publication 0 349 061, publiεhed January 3, 1990) was used in place of FK-506. The isolation/purification procedures were virtually identical to those described in Example 3. The C-32 phosphorylated C-31 desmethyl FK-520 was characterized by mass spectrometry and proton nuclear magnetic resonance in which the obtained spectra was consiεtent with the assigned structure. EXAMPLE 5 Carrying out the above IL-2 aεsay described above in Example 2 for C-32 phosphorylated FK-506 (P-FK-506) and C-32 phosphorylated C-31 desmethyl FK-520 (P-31-desMe FK-520) yielded the following reεults: Inhibition of T cell proliferation by P-FK-506 Concentration % of of P-FK-506 inhibition (mg/ml) 50 98.5 25 97.7 12.5 93.3 6.2 80.3 3.1 42.9 IC 50 = 3.4 ng/mL (3.9nM) Inhibition of T cell proliferation bv P-31-desMe FK-520 Concentration 7« of of P-31-desMe F -520 inhibition (mg/ml) 250 95.2 125 90.3 62.5 79.5 31.2 53.8 X5.6 21-1 IC 50 = 31.3 ng/mL (36.7 nM). EXAMPLE 6 Phosphorylated Rapamvcin Macrolide Microorganism and Culture Conditions Spores of Rhizopus orvzae MF4974 (ATCC No. 11145) cultivated on an oatmeal agar flask were inoculated into 50 ml Soy-Glucose medium in a 250 ml Erlenmer flask and shaken at 27°C on a rotary shaker at 220 rpm for 24 hourε. The second stage flask (500 ml in a 1000 ml Erlenmeyer flask) were inoculated with 25 ml of seed culture and incubated on a rotary shaker (220 rpm) at 27 β C for 24 hours. Following incubation, each flask was harvested by centrifuga¬ tion, washed once with sterile water, and resuspended in equal volume of 100 mM pH 7.0 phosphate buffer con¬ taining 3% glycerol. Rapamycin macrolide was added to achieve a final concentration of 0.2 mg/ml. The flasks were then incubated on a rotary shaker (220 rpm) at 27°C for 24 hours. Following incubation, the whole broth was extracted as described in the Isolation/Purification Section below. Media Soy Glucose Medium g/i Dextrose 20.0 Soy Meal 5.0 Fido yeaεt extract 5.0 NaCl 5.0 K 2 HP0 4 5.0 Adjust pH to 7.0 ISOLATION AND PURIFICATION The whole broth (500 ml) was maintained at pH 6.8 and centrifuged. The mycelial cake was washed with water, then dicarded. The clear filtrate and washingε were pooled and paεεed through a Speed octadecyl cartridge (14% carbon load, Applied Sepa¬ rations) under vacuum. The column was washed with 100 ml of water. Column effluent and wash did not contain microbial transformation product when tested with HPLC. The cartridge was eluted with 200 ml methanol. Methanol was evaporated to dryness under reduced pressure at 30°C. The resulting oil was dissolved in methanol and subjected to HPLC puri¬ fication. HPLC was carried out on Whatman Magnum 9 Partisil 10 ODS-3, 9.8 mm i.d. x 25 cm at 25 β C and monitored at 225 nm. The column was developed at 3 ml/minutes with a linear gradient from 35% to 80% acetonitrile in 0.1% phosphoric acid in 30 minutes. The compound was collected during repeated injections of the above described extract. The fractions of retention time 17.3 minutes, were pooled, adjusted to pH 3 and evaporated to remove acetonitrile. The compound was further purified using C18 Sep Pak (Water Associates) and methanol-water elution solvent to yield 14 mg pf product. Characterization The C-43 phosphorylated macrolide was charac¬ terized by FAB masε εpectrometry and NMR spectrometry as the methyl phosphate ester derivative yielding the proton NMR spectrum of Figure 3, which confirms the assigned molecular structure in Figure 4. Methylation to produce the phosphate ester was neceεεary to minimize the considerable line broad¬ ening which characterized the proton NMR spectrum of the free acid. The key features were the downfield displaced H-42 and H-43 signals (which are H-31 and H-32 in FK-506 nomenclature) at 3.87 ppm and 4.12 ppm, respectively, and the additional fine structure of H-43 resulting from coupling with the phosphorus. The downfield shift of H-43 is a normal consequence of replacing the active hydrogen with an electron withdrawing substituent. H-42 is also displaced downfield by proximity to the new substituent. The obtained spectrum of the phoεphorylated macrolides as the phosphate methyl ester with assignments of H-42, H-43, and the methyl ester peaks is shown in Figure 3, EXAMPLE 7 τ-Cell Proliferation Assay 1. Sample Preparation Purified phosphorylated macrolide, as prepared by HPLC above, was diεεolved in absolute ethanol at 1 mg/ml and serially diluted in culture medium prior to addition to the cultures. 2. Assay The asεay waε carried out aε deεcribed in J. Immunol. Vol. 144, pp.251 (1990) by F. Dumont, et al. Spleens from C57B1/6 mice were taken under sterile conditions and gently disεociated in ice-cold RPMI 1640 culture medium (GIBCO, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO). Cells were pelleted by centrifugation at 1500 rpm for 8 minutes. Contaminating red cells were removed by treating the pellet with ammonium chloride lysing buffer (GIBCO) for 2 minutes at 4 β C. Cold medium was added and cells were again centri¬ fuged at 1500 rpm for 8 minuteε. T lymphocytes were then isolated by separation of the cell εuεpenεion on nylon wool columns as follows: Nylon wool columns were prepared by packing approximately 4 grams of washed and dried nylon wool into 20 ml plastic syringes. The columns were sterilized by autoclav- ing at 250 β F for 30 minutes. Nylon wool columns were wetted with warm (37 β C) culture medium and rinsed with the same medium. Washed spleen cells resuspended in warm medium were slowly applied to the nylon wool. The columns were then incubated in an upright posi¬ tion at 37°C for 1 hour. Non-adherent T lymphocytes were eluted from the columns with warm culture medium and the cell εuεpensions were spun as above. Purified T lymphocytes were resuspended at 2.5 x 10- *1 cells/ml in complete culture medium composed of RPMI 1640 medium with 10% heat-inactivated fetal calf εerum, 100 mM glutamine, 1 mM εodium pyruvate, 2 x 10 " - 5 M 2-mercaptoethanol and 50 μg/ml gentamycin. Human recombinant interleukin-2 (50 μg/ml) and PMA at 10 ng/ml. The cell suεpenεion vaε immediately diεtributed into 96 well flat-bottom microculture plateε (Coεtar) at 200 μl/well. The control, being the medium without teεt drug, and various below- indicated dilutions of the above sample of purified phosphorylated macrolide to be tested were then added in triplicate wells at 20 μl/well. Rapamycin (U.S. Patent 3,929,992) was used as a standard. The culture plates were then incubated at 37 β C in a humi¬ dified atmosphere of 5% Cθ2~95 air for 44 hours. The proliferation of T lymphocytes waε aεεeεεed by measurement of tritiated thymidine incorporation. After 44 hours of culturing, the cells were pulse- labelled with 2 μCi/well of tritiated thymidine (NEN, Cambridge, MA). After another 4 hours of incubation., cultures were harvested on glasε fiber filterε using a multiple sample harvester. Radioactivity of filter discε correεponding to individual wells was measured by standard liquid scintillation counting methods (Betacounter). Mean counts per minute of replicate wells were calculated and the results expressed as percent inhibition of tritiated thymidine uptake (proliferation) aε followε: 'Mean cpm sample tested % Inhibition = 100 - X 100 Mean cpm control medium Proliferation waε assesεed at 48 hours of culture by tritiated thymidine uptake. The results, expressed as IC50 values of three independent experiments, are presented in the following table: Notes: 1. Mouse T cell cultureε were pulsed with 3 H-thymidine for 4 hours prior to harvesting at 48 hours. 2. The mean IC5 Q for the C-43 phosphory- lated rapamycin was determined to be: 7.6 ± 0.2 ng/ml and 0.4 ± 0.1 ng/ml for rapamycin in 3 independent experiments. EXAMPLE 8 SYNTHESIS The following preparation and synthesis follows, in general, U.S. Patent 4,661,473; Evans, B.E. fit al, J. Qrg. Chem.. £, 4615, (1985) and Evans, B.E. ≤±. al- , "A Stereocontrolled Synthesis of Hydroxyethylene Dipeptide Isoεtereε," Proc. Am. Pept. Symp., 1, 743-6(1985), and Luly, J.R. fit al, J. Org. Chem, 52., 1487 (1987). N(2(R)-Hydroxy-l(S)indanyl)-5(S)-((1,1-dimethylethoxy- carbonyl)amino)-4(S)-hydroxy-6-ρhenyl-2(R)-3-(4-(2- (morpholino)ethoxy)phenyl)prop-2-en-l-yl)hexanamide: Step A: Preparation of 4-tert-butyldimethylsilyloxy- henvlprop-2-en-l-vl bromide: To a 1 L round bottomed flaεk with a εtirring bar and an argon inlet waε added 26.25 g (160 mmol) of p-hydroxycinnamic acid, 50.62 g (335 mmol) of tert- butyldimethylεilyl chloride, 32.68 g (480 mmol) of imidazole, and 250 mL of dry DMF. Thiε mixture was stirred at room temperature for 24 hours. The DMF was removed jji vacuo and the residue was partitioned between EtOAc and 10% aqeous citric acid. The layers were separated and the organic phase was washed with water (4x) and brine. Drying (MgS0 4 ), filtration and removal of the solvent in vacuo gave 62.3 g of the biεsilylether-ester. This ester was placed in an oven dried 2 L round bottomed, 3 necked flask with a mechanical stirrer, argon inlet, and addition funnel. Ether (460 mL) was added and the εolution was cooled in an ice bath to 0 C C. To this solution was added a solution of Dibal-H 397 mL of a 1.0 M solution in hexanes), dropwiεe over 1 hour. The mixture waε stirred for 1 hour and the reaction was then quenched by careful addition of 1 L of saturated aqueous sodium potasεium tartrate εolution. This viscous mixture was stirred for 20 hours at room temperature. The mixture was filtered through a celite pad, the filtrate layers were separated and the organic phase was extracted with 2 portions of EtOAc. The combined organic phases were washed with brine, dried (MgS0 4 ), filtered and concentrated in vacuo to give the crude alcohol. This material was chromatographed on 500 g of silica gel using 20% EtOAc in hexanes aε eluant. There waε obtained 34.9 g of 4-tert-butyldimethylεilyloxycinnamyl alcohol as a viscous oil. This material crystallized on cooling to -15 C C. A portion of thiε alcohol (18.5 g, 69.96 mmol) waε placed in a 300 mL round bottomed flask with a stirring bar and 125 mL of dry ether. This εolution was cooled to 0°C and 20.83 g (76.95 mmol) of Br3 was added dropwise with a syringe over 5 minutes. This εolution waε maintained at 0 β C for 20 minutes, diluted with hexanes (IL) and washed with aqueous aHC03- This solution was washed with brine, dried (MgS0 4 ), filtered through a pad of silica gel, and concentrated in vacuo to give 17.53 g of -tert- butyldimethylεilyloxypenylprop-2-en-l-yl bromide aε a colorleεε oil. Step B: Preparation of 5(S)-(l,l-dimethylethoxycar- bonyl)amino-4(S)-(l' ,1'-dimethylethyl-1,1- dimethylεilyloxy)-6-phenyl-2(R)-(4-(l',l'- dimethylεilyloxy)phenylprop-2-en-yl)hex- noic acid: To a 500 mL, 3-necked, oven dried round bottomed flask with an argon inlet, stirring bar, low temperature thermometer, and a jacketed addition fun¬ nel was added 75 mL of dry THF and 15.39 mL (109.78 mmol) of diisopropylamine. This solution was cooled to -20 β C and n-butyllithium (42.84 mL, 107.11 mmol of a 2.5M solution in hexanes) was added slowly. The resulting solution was cooled to -78 β C and a solution of (5S,1'S)-5-((1,l-dimethylethoxycarbonyDamino)- 2-phenylethyl)dihydrofuran-2-(3H)-one (16.36 g, 53.55 mmol) in 75 mL of dry THF was added at such a rate that the temperature of the solution did not riεe above -70 β C (ca. 40 minuteε required for thiε addi¬ tion). The reεulting εolution waε aged at -78 β C for 1 hour and the dropping funnel waε charged with a solution of 4-tert-butyldimethylsilyloxyphenylprop- 2-en-l-yl bromide (17.53 g, 53.55 mmol) in 75 mL of dry THF. The bromide εolution was cooled to -78 β C and was then added to the enolate solution dropwiεe over 45 minuteε, keeping the temperature below -70°C. When the addition was complete the solution waε aged at -78 β C for 1 hour, warmed to -50 β C and quenched with a εolution of NaHS0 4 (33 g) in 250 mL of water. The mixture was diluted with EtOAc and the layers were separated. The organic phase was washed with NaHC03 solution and brine. Drying (MgS0 4 ), filtration, and removal of the solvent is. vacuo provided the crude alkylation product as an oil. This material was chromatographed on 1000 g of silica gel using 20% EtOAc in hexanes aε eluant. There was obtained 23.6 g of pure (5S,3R,1'S)3- (4-(l » ,1'-dimethylethyl-1,1-dimethysilyloxy)phenyl- prop-2-en-l-yl)-5-(l-((l,l-dimethylethoxycarbonyl)- amino)-2-phenylethyl)dihydrofuran-2-(3H)-one aε a colorless foam. This material was dissolved in 325 mL of DME and a solution of LiOΞ (6.99 g, 291.97 mmol) in H 2 0 (325 mL) was added. This solution was stirred at room temperature for 24 hours. The DME was removed in vacuo and the aqueous residue was acidified with 10% aqueous citric acid. Thiε milky εuspension was extracted with several portions of EtOAc and the combined extracts were washed with water and brine, dried (MgS0 4 ), filtered, and concentrated j__ vacuo. To the crude α-hydroxy acid was added imidazole (56.79 g, 834 mmol), tert-butyl- dimethylεilyl chloride (62.87 g, 417 mmol) and dry DMF (325 mL) . Thiε mixture waε εtirred at room temperature for 24 hourε. The mixture waε then treated with methanol (325 mL) for 4 hourε at room temperature. The εolvents were removed in vacuo at 60 β C, 20 torr. The residue was dissolved in IL of EtOAc and washed with 10% aqueouε citric acid, water and brine. Drying (MgS0 4 ), filtration and removal of the εolvent gave 28.53 g of 5(S)-(l,l-dimethylethoxy- carbonylamino)-4(S)-(1' ,1'-dimethylethyl-1,1-dimethyl- silyloxy)-6-phenyl-2(R)-(4-(l » ,1'-dimethylethyl-1,1- dimethylεilyloxy)phenylprop-2-en-l-yl)hexanoic acid aε a colorleεs foam. Step C: Preparation of N-(2(R)-hydroxy-l(S)-indanyl)- 5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)- (1' ,1'-dimethylethyl-1,l-dimethylsilyloxy)-6- phenyl-2(R)-((4-(hydroxyphenyl)prop-2-en-l- vDhexanamide: To a 2L round bottomed flaεk with a εtirring bar and an argon inlet was added 5(S)-(l,l-dimethyl- ethylethoxycarbonylamino)-4(S)-(l » ,l'-dimethylethyl- l,l-dimethylεilyloxy)-6-phenyl-2(R)-(4(l' ,1'-dimethyl¬ ethyl-1,l-dimethylsilyloxy)phenylprop-2-en-l-yl) hexanoic acid (28.53 g, 41.71 mmol) from Step B, 2(R)- hydroxy-l(S)-aminoindane (6.85 g, 45.88 mmol), 3-(N,N- dimethylaminopropyl)ethylcarbodiimide hydrochloride (8.79 g, 45.8 mmol), 1-hydroxybenztriazole hydrate (6.20 g, 45.88 mmol), and dry DMF (300 mL) . When all of the solids had dissolved, triethylamine (12.79 mL, 91.76 mmol) was added and the mixture was stirred at room temperature for 18 hours. The mixture waε partitioned between EtOAc (1500 mL) and 10% aqueouε citric acid (1500 mL). The layers were separated and the organic phase was washed with water (3 x 1000 mL), and brine. Drying (MgS0 4 ), filtration and removal of the solvent in vacuo gave 32 g of a yellow foam. This material waε diεεolved in 500 mL of methanol and LiOH (4.99 g, 208.55 mmol) waε added. Thiε εolution was stirred at room temperature for 1 hour. The solution was acidified with aqueouε citric acid and the methanol was removed in vacuo. The resulting aqueous residue was extracted with EtOAc (IL). The EtOAc extract was dried (MgS0 4 ), filtered, and concentrated in vacuo. , Thiε material waε chromatographed on 1 Kg of silica gel using 8L of 40% EtOAc in hexanes as eluant. There was obtained 20.07 g of N-(2(R)-hydroxy-l(S)-indanyl)-5(S)-(l,l-dimethyl- ethoxycarbonyl)amino-4(S)-(l' ,1'-dimethylethyl-1,1-dim ethylsilyloxy)-6-phenyl-2(R)-((4-hydroxyphenyl)prop-2- en-l-yl)hexanamide as a colorlesε foam. Step D: Preparation of N-(2(R)-hydroxy-l(S)-indanyl)- 5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)- ethox phenylprop-2-en-l-yl)hexanamide: To a IL round bottomed flaεk with a εtirring bar and an argon inlet waε added N-(2(R)-hydroxy-l(S)- indanyl)-5(S)-(l,l-dimethylethoxycarbonyl)-4(S)-(l' ,- 1'-dimethylethyl-1,l-dimethylsilyloxy)-6-phenyl-2(R)- (4-(l' ,1'-dimethylethyl-1,l-dimethylsilyloxy)phenyl- prop-2-en-l-yl)hexanamide (19.49 g, 28.1 mmol) 1,4- dioxane (400 mL), 4-(2-chloroethyl)morpholine (12.61 g, 84.3 mmol), and powdered cesium carbonate (27.5 g, 84.3 mmol). Thiε mixture was heated at 80 β C with vigorous stirring for 3 hours. The cooled reaction mixture was filtered through a celite pad and the 1,4-dioxane was removed in vacuo. To this residue was added a εolution of tetrabutylammonium fluoride in THF (280 mL of a 1M solution, 280 mmol) and a stirring bar. This solution waε εtirred at room temperature under argon for 28 hourε. The reaction mixture was poured into 3L of H 2 0, with stirring. The white solid was collected by filtration and dried in vacuo overnight. The crude product was chromatographed on silica gel using 5% MeOH in chloroform as eluant. The chromatographed material was recrystallized from boiling EtOAc-hexanes to give 13 g of analytically pure N-(2(R)-hydroxy-l(S)- indanyl)-5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-(2-(4-morpholino)ethoxy)- phenylprop-2-en-l-yl)hexanamide as a white solid, mp:178-179 β C. EXAMPLE 9 Preparation of N-(2R)-hydroxy-l(S)-indanyl)-5(S)- (l,l-dimethylethyloxycarbonylamino)-4(S)-hydroxy- 2(R)-(4-(2-(4-morpholino)ethoxy)phenyl)prop-2-en-l- yl-6-cvclohexylhexanamide. parent compound L-702.083 Step A: Preparation of (5S,l » S)-5-(l'-((l,l-di- methylethoxycarbonyl)amino)-2'-cyclo- hexylethyl)-4.5-dihydrofuran-2-(3H)-one: A εolution of the (5S,l'S)-5-(l'-((l,l-di- methylethoxycarbonyl)amino)-2'-phenylethyl)-4,5- dihydrofuran-2-(3H)-one waε dissolved in ethyl acetate and rhodium on alumina was added. Thiε mixture was shaken under a hydrogen atmoεphere (50 psi) at 50°C overnight. Filtration and evaporation of the solvent afforded the title compound as a viscous oil, which solidified as a hard glaεε. Step B: Steps B, C and D of Example 1 are repeated except that (5S, l*S)-5-((l,l-dimethylethoxy- carbonyl)amino)-2-phenylethyl)dihydrofuran-2- (3H)-one in Step B is substituted with its cyclohexyl analog, (5S, l'S)-5-((l-dimethyl- ethoxycarbonyl)amino)-2-cyclohexylethyl)- 4,5-dihydrofuran-2-(3H)-one. The title compound, L-702,083, is obtained. EXAMPLE 10 BIOTRANSFORMATION OF L-702.083 Sporeε of Rhizopus arrhizus MF 4974, cultivated on oatmeal agar, were inoculated into 50 ml of seed medium [coπtaining(in grams per liter) dextrose 20.0, soy meal 5.0, Fidco yeaεt extract 5.0, NaCl 5.0, K 2 HP0 4 5.0, pH 7.0 before autoclaving] in a 250 ml 3-baffle Erlenmeyer flaεk. The flask was incubated for 24 hours on a rotary shaker (220 rpm) at 27°C. The 2.5 ml of the seed cultures was used to inoculate 50 ml of the same medium in a 250 ml Erlenmeyer flask and incubated on a rotary shaker (220 rpm) at 27 β C for 24 hours. Following incubation, each flask was harvested by centrifugation, washed once with sterile saline solution, and resuεpended in equal volume of 100 mM phoεphate buffer (pH 7.0) containing 3% glycerol. L-702,083 in DMSO (20 mg/ml) waε then added to achieve a final concentration of 0.1 mg/ml, and cultivation was continued at 27 β C on a rotary shaker at 220 rpm. After 12 hours incubation the whole broth was extracted aε deεcribed in Example 16. EXAMPLE 11 ISOLATION AND PURIFICATION Two εhake flaεks were combined to yield 100 ml of broth. The broth was centrifuged. The mycelial cake was washed with water and discarded. The clear filtrate and the wash were combined and passed thru a Spe-ed octadecyl 14% cartridge (Applied Separations) under vacuum. The column was washed with 100 ml of water and then eluted with 200 ml methanol. Methanol extract was concentrated in vacuo to a slightly oily residue. The residue was taken up in methanol and subjected to preparative HPLC chromatography on a Whatman Magnum 20 Partisil 10 ODS-3,22.1 mm x 25 cm at room temperature at a flow rate of 7 ml/minute. The effluent stream was monitored by U.V. abεorption at 215 nm. Elution was achieved with a linear gradient from 30% acetonitrile in 0.1% phosphoric acid to 80% acetonitrile in 0.17. phosphoric acid in 80 minutes. The fractions at retention time of 35 minuteε were combined, adjuεted to pH 4.0 and evaporated to remove acetonitrile. The compound was then desalted using C-18 Sep Pak (Waters Asεociate) to afford 3 mg of product L-706,579. EXAMPLE 12 NMR CHARACTERIZATION The product of Example 11, L-706,579, waε characterized by NMR as the phosphate ester derivative at C-4 of L-702,083. The key observation was the 0.6 ppm downfield displacement of H*L in the Formula : Compared to its chemical shift in the parent L-702,083, the neighboring methine (m) is slightly displaced downfield and is barely discernible as a εhoulder at the baεe of the morpholine CH 2 0 at 3.74 ppm. Alεo perturbed iε the nearby H κ which now appearε underneath the lower field Hj εignal at 3.10 ppm. Its presence iε revealed only by the additional area of the Hj signal, compared with the Hj double doublet in L-702,083. EXAMPLE 13 ORGANIC SYNTHESIS OF L-706,579, PHOSPHATE ESTER OF L-702,083 A. Acetylation of Indan N-[(2R)-Acetoxy-l(S)-indanyl]-5(S)-(l,l-dimethyl- ethoxycarbonylamino)-4(S)-hydroxy-2(R)-[(4-(2-(4- morpholino)ethoxy)phenyl)prop-2-en-l-yl]-6-cyclo- hexanamide, L-702,083, is prepared by the protocol of Examples 1 and 2, except that 2(R)-hydroxy-l(S)- a inoindan is subεtituted with 2(R)-acetoxy-l(S)- aminoindan in Example 1, Step C. B. L-706.579 The product of εtep A iε reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, R.W. and E . G . Khorana, J. Am. Chem. Soc. 8J1. 3749 (1958). Subsequent treatment with excess ammonia, followed by removal of the acetyl groups under basic conditions, yields L-706,579. C Alternative Synthetic Routes Another phosphorylating agent is dibenzyl phosphorochloridate. An extenεive diεcuεεion of phosphorylation and phosphorylating agents can be found in Y. Mizuno, Studies in Org. Chem, Z__, 171-175 (1986) and references cited therein. EXAMPLE 14 Aεεay for Inhibition of Recombinant HIV Proteaεe Inhibition εtudies were performed on the reaction of the HIV protease expreεεed in Eεcherichia coli with a tritiated peptide εubεtrate [ •**! H]-acetyl- Val-Ser-Gln- Aεn-(beta-napthyl-Ala)-Pro-Ile-Val-Gln- Gly-Arg-Arg-NH 2 (MW 1800). The two arginine reεidueε at the carboxyl terminuε give thiε peptide an overall poεitive charge at acidic pH and enable it to bind to the H + form of DOWEX AG-50W-X8 resin and similar resinε. The HIV protease cleaves between the β-napthyl-Ala and proline reεidueε to yeild a product ( 3 H-acetyl-val-ser-asn-(beta-napthyl-ala) that iε either neutral or εlightly negatively charged and doeε not bind to the cation exchange reεin. It iε therefore poεεible to conveniently εeparate the labelled product from the εubεtrate. Aliquotε of 25 μl containing 6.0-8.0 nM HIV protease in asεay buffer (100 mM εodium acetate, pH 5.5 and 0.1% BSA) are placed in assay tubes. The reaction is initiated by addition of 25 μl aliquots of 4.2 uM tritiated peptide substrate in lOOmM sodium acetate, pH 5.5. After incubation for 60 min at 37 e C, the reaction iε stopped with 100 μl of 5% H 3 P0 4 , then analyεed by application of column chromatography. Reεultε are aε followε The preparation and εyntheεiε follows, in general, U.S. Patent 4,661,473; Evans, B.E. fit al. J. Org. Chem.. 50, 4615, (1985) and Evans, B.E. fit al.. "A Stereocontrolled Synthesis of Hydroxyethylene Dipeptide Isosteres," Proc. Am. Pept. Sy p., 1, 743-6(1985), and Luly, J.R. fit al, J. Org. Chem, 51, 1487 (1987), all herein incorporated by reference. All temperatures are in degrees centigrade, unless indicated otherwise. Preparation of N-(cis-2(R)-hydroxy-l(S)-indanyl)- 5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)-hydroxy- 6-phenyl-2(R)-[(4-(2-(4-morpholinyl)ethoxy)phenyl) methyl " )-hexanamide. L-689.502 Step A: Preparation of N-3(S)-[(l,l-Dimethylethoxy- c_.rbonyl)amino]-2(RS)-hydroxy-4-phenyl-l-tri- methylsilyl butane: To a stirred suspension of magnesium turnings (9.79 g, 403 mmol) in dry diethyl ether (200 mL) under nitrogen was added chloromethyltrimethylsilane (50 mL, 358 mmol). The reaction was initiated by gentle warm¬ ing and then was cooled in an ice bath to maintain gentle reflux. After exotherm was complete the reaction was stirred at room temperature for 1 hour then cooled to -78 β C in a dry ice/acetone bath. To the εolution of the Grignard was added dropwise with stirring a solution of N-2(S)-[(l,l-dimethyl- ethoxycarbonyl)amino]-3-phenyl propionaldehyde (19.3 g, 77.4 mmol) in dry diethyl ether (250 mL) dropwise such that the temperature of the reaction remained below -55°C. The resultant gray suspension was allowed to warm to room temperature where it was stirred for 30 minutes then was quenched by pouring into a mixture of ice (500 g) and 107» citric acid (500 mL). The organic phase was collected and the aqueous phase was extracted with diethyl ether (3 X 300 mL) . The combined organics were washed with 10% citric acid (1 X 300 mL) and brine (1 X 200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to give crude N-3(S)-[(l,l-dimethyl- ethoxycarbonyl)amino]-2(RS)-hydroxy-4-phenyl-l-tri- methylεilyl butane (26.6 g, quantitative crude yield) as a yellow oil. An analytical sample was obtained by low pressure chromatography (silica gel, 230-400 mesh; diethyl ether: hexanes, 30%:70%) followed by recrystallization from heptane, mp = 91-95 β C; elemental analysis. Calcd. for C^g^- j ^^Si (337.53): C = 64.05, H = 9.26, N = 4.15; Found: C = 64.05, H = 9.13, N = 4.22; [a^O = -40.0° Step B: Preparation of 3(S)-Amino-4-phenyl-l-butene. To a stirred solution of the product of Step A (22.8 g, 67.5 mmoL) in dry methylene chloride (400 L) cooled in an ice bath and under nitrogen was added in a fine stream boron trifluoride etherate (43 mL, 345 mmol). The solution was allowed to warm to room temperature where it was stirred for 4 days. Reaction was cooled in an ice bath and quenched by the dropwise addition of 10% sodium hydroxide (400 mL). The organic phase was collected and the aqueous phase was extracted with methylene chloride (2 X 250 mL) . The combined organics were washed with brine (1 X 200 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to give crude 3(S)-amino-4-phenyl-l-butene (14.2 g) as a yellow oil, Step C: Preparation of N-3(S)-[(1,1-Dimethyl- ethp χ ycartOnyl)amino1-4-phgnyl-l-bυt«3ne: A εolution of the product of Step B (14.2 g) and di-tfi-rt-butyl dicarbonate (31.0 g, 142 mmoL) in dry methylene chloride (200 mL) waε εtirred at room temperature for 18 hourε, waεhed with 10% citric acid (3 X 100 mL), water (1 X 100 mL), εat'd. εodium bicarbonate (3 X 125 mL), and brine (1 X 250 mL), dried over anhydrouε magneεium εulfate, filtered and concentrated to yield crude N-3(S)-l[(l,l-dimethyl- ethoxycarbonyl)amino]-4-phenylbutene (34.6 g) aε a yellow oil. Crude product waε purified by low pressure chromatography (silica gel, 230-400 mesh, 10 X 20 cm column; diethylether: hexanes, 20%: 80%) to yield N-3(S)-[(l,l-dimethylethoxylcarbonyl)amino]- 4-phenyl-l-butene (16.3 g, 97.6% yield) as a white solid. An analytical sample was obtained by recrystallization from heptane, mp = 67.5-68.5 C C; elemental analysiε, Calcd. for C 15 H 21 N0 2 (247.34): C = 72.84, H = 8.56, N = 5.66. Found: C = 72.78, H = 8.76, N = 5.64. Step D: Preparation of 1(R)-[1' ethoxycarbonyl^amino-Σ-phenylethylloxirane: To a solution of the product of Step C (9.4 g, 38 mmol) in dry methylene chloride (100 mL) cooled in an ice bath and under nitrogen was added 3-chloroperoxybenzoic acid (technical grade, 80-85%; 41 g, 200 mmol). The mixture was stirred at 0 β C for 18 hours and 25 β C for 23 hourε, then diluted with diethyl ether (300 mL), and poured in ice cold aqeous 10% sodium sulfite (1 L). The organic layer was collected and the aqueous layer waε extracted with diethyl ether (3 X 100 mL). The combined organicε were waεhed with 10% εodium εulfite (3 X 100 mL), satd. sodium bicarbonate (3 X 100 mL), and brine (1 X 100 mL), dried over anhydrous sodium sulfate, filtered and concentrated to give a white solid. Crude product was purified by low presεure chroma¬ tography (εilica gel 230 - 400 meεh, 8 X 15 cm column; ethyl acetate: hexaneε, 25%:75%) to yield 1(R)-[1' (S)-(l,l-dimethylethoxycarbonyl)amino-2- phenylethyl]oxirane (7.0 g, 70% yield) aε a clear oil which cryεtallized upon εtanding. An analytical sample was obtained by recryεtallization from heptane mp = 51.5-52°C; elemental analyεiε, Calcd. for C 15 H 21 N0 2 (263.34): C = 68.42, H = 8.04, N = 5.32. Found: C = 68.22, H = 8.26, N = 5.29; [a]-**-. 20 = 1.34° Step E: Preparation of(5S,l'S)-3-carboethoxy- 5-(l-((l* ,1'-dimethylethoxycarbony1)amino)-2- ρhenylethyl)-dihydrpfuran-2-(3g)-pne- The product from Step D, 9.93 g, was dissolved in 100 mL of absolute ethanol and added to a solution of 2.6 g of sodium and 20.1 mL of diethyl malonate in 170 mL of absolute ethanol. After stirring overnite, the reaction was acidified to pH 4 with 10% citric acid and extracted with 2 X 500 mL of ether. The combined organic extracts were washed 1 X 500 mL H 2 0, 1 X 500 mL sat'd NaHC0 3 , 1 X 500 mL εat'd brine and dried over MgS0 4 . The εolvents were removed and the crude product purified by low pressure chroma¬ tography on silica gel eluting with 50% ether/hexaneε (or EtOAc/hexaneε) . The yield of εemi-solid product was 10.6 g. The later fractions contained 2.5 g of the undesired 5 R isomer as a white solid. Step F: Preparation of (5S,l'S)-3-carboethoxy-3-(4- benzyloxyphenylmethyl)-5-[1-(1,1-dimethyl- ethoxycarbonyl)amino)-2-phenylethyl]dihydro- fυran-2-(3H)-one To a stirred solution of (5S,l'S)-3-carbo- ethoxy-5-[l-((1 » ,1'-dimethylethoxycarbonyl)amino)-7- phenylethyl)-dihydrofuran-2-(3H)-one (product of Step E), 2 g (5.3 mmol) in 25 mL of abεolute ethanol waε added a εolution of 0.13 g of sodium in 2.2 mL of abεolute ethanol followed by 1.30 g (5.5 mmol) of 4-benzyloxybenzyl chloride. The εolution waε heated to 50°C under nitrogen for 1 hour, then cooled in an ice bath and acidified with 20 mL of 10% citric acid and diluted with 200 mL of water. The mixture waε extracted with 3 X 100 mL of ether and the combined ether extracts washed with 50 mL of water, 200 mL of sat'd NaHC0 3 and dried over MgS0 4 . Removal of εolvents under reduced pressure and purification by low pressure chromatography on silica gel, eluting with 40% ether in hexanes gave 1.56 g (51% yield) of a clear colorless glass essentially homogeneous by TLC (50% ether/hexanes). Step G: Preparation of (3R,5S,l'S)-3-(4-benzyloxy- phenylmethyl)-5-(l((l,1-dimethylethoxy- carbonyl)amino)-2-phenylethyl)-dihydrofuran- 2-(3H)-one. The product of Step F, 13.6 g, was disεolved in 250 mL of 1,2-dimethoxyethane, and to it was added 117 mL of 1 M lithium hydroxide at room temperature. After stirring for 12 hourε, the εolventε were removed under reduced preεεure, the reεidue εuspended in 200 mL of 10% citric acid and extracted 3 X 500 mL of diethyl ether. The combined ether extractε were waεhed with 500 mL of brine, dried (MgS0 4 ) and the concentrated to dryneεε. The reεidue waε diεεolved in 250 mL of toluene, heated to reflux for 12 hourε, then concentrated to dryness under reduced presεure. Purification by medium preεεure chromatography over εilica gel eluting with 15% ethyl acetate/hexaneε gave 3.2 g of the 3R-lactone aε a clear foam. Further elution with the εame εolventε gave 6.15 g of the 3S-lactone aε a white εolid. Step H: Preparation of N'-(l,l-dimethylethoxy- carbonyl)-5(S)-amino-4(S)-(l' ,1•-dimethyl¬ ethyl-1,l-dimethylsilyloxy)-6-phenyl-2(R)- (4-benzyloxyphenylmethyl-hexanoic acid. The product of Step G, 0.6 g, was dissolved in 30 mL of a 2:1 mixture of ethylene glycol dimethyl ether/water, and to it was added 5 mL of 1 M lithium hydroxide at room temperature. After stirring for 1 hour, the mixture was partitioned between 200 mL chloroform and 20 mL 10% citric acid. The layers were separated and the aqueous phase extracted with 3 X 20 mL chloroform. The combined organic layers were dried (Na 2 S0 4 ) and the εolvent removed to yield 0.56 g of the crude hydroxy acid. This residue was dissolved in 5 mL of dry DMF and 0.845 g tert-butyl dimethylsilyl chloride and 0.725 g of imidazole were added. After εtirring for 18 hourε, the reaction waε poured into 50 mL of water and extracted with 3 X 20 mL of ethyl acetate. The combined organic extractε were washed with 3 X 20 mL of 10% citric acid, 1 X 20 mL of water, 3 X 10 mL of saturated aqueous solution of Na 2 Cθ3, and 20 mL of brine. After drying (Na 2 S0 4 ), the solvent was removed and the resulting residue disεolved in a mixture of 5 mL of THF, 5 mL of glacial acetic acid, and 2 L of water. The mixture was stirred for 4 hours, then poured into 5v. mL of water and extracted with 3 X 20 mL of ether. The combined ether extractε were waεhed with 2 X 20 mL of water, brine, dried (Na 2 S0 4 ), and the εclvent removed. Purification by medium preεsure chromatography over silica gel, eluting with MeOH/CHCl 3 gave 0.60 g of the product as a white glasεy εolid. Step I: Resolution of l-Amino-2-hydroxyindan From the known racemic l-amino-2- hydroxyindan, the resolution was carried out as described for the 3-amino-l,2-dihydroxyindan in Example 7 below (Steps D and E). The (1S,2R)- l-amino-2-hydroxyindan reεulting from εaponification of the higher R f diastereomer was εhown to have an a D of -58° (c = 1.0, CHC1 3 ). The (1R, 2S)-l-amino- 2-hydroxyindan resulting from εaponification of the lower R f diaεtereomer was found to have an ap of +62° (c = 1.0, CHCI3). Step J: Preparation of N-(2(R)-hydroxy-l(S)-indanyl)- 5(S)-(l,l-dimethylethoxycarbonylamino)-4(S)- hydroxy-6-phenyl-2(R)-(4-benzyloxyphenyl- methyl) hexanamide The product from Step H, 0.12 g, was diεεolved in 2 ml dry DMF and to it waε added 40 mg of l(S)-amino-2(R)-hydroxyindane, (Step I) 25 mg of 1-hydroxybenzotriazole hydrate and 70 mg of dimethyl- 3-(3-dimethyl aminopropyl)carbodiimide hydrochloride. Triethylamine waε added to the εtirred solution until the pH was 8.5 (32 mL). After stirring for concen¬ trated to dryness under reduced presεure, the reεidue waε dissolved in 100 mL of chloroform and worked with 1 X 50 mL of 10% citric acid, 1 X 50 mL H 2 0, 1 X 50 mL sat'd aHC03, dried over MgS0 4 and concentrated to dryness. The residue waε diεεolved in 1 mL of tetrahydrofuran and added to 2 mL of 1 M tetrabutyl- ammonium fluoride in THF. After εtirring overnight at room temperature the reaction mixture waε diluted with 10 mL of 10% citric acid and the white preci- pitate collected by filtration. The product was purified by low presεure chromatography on silica gel eluting with 2% methanol/CH 2 C1 2 to give 85 mg of product which was esεentially homogeneous by TLC (3% methanol/CH 2 C1 2 ). Step K: Preparation of N-(2(R)-hydroxy-l(S)-indanyl)- 5(S)-(l,l-dimethylethoxycarbonylamin *-4(S)- hydroxy-6-phenyl-2(R)-(4- ' hydroxyphenyl- methyl)hexanamide The product of Step J, 85 mg was dissolved in 10 mL of methanol and 10 mL of THF, and to it was added 0.10 g of 10% palladium on carbon. The mixture was stirred under an atmosphere of hydrogen for 48 hours at room temperature, then filtered and concen¬ trated to drynesε. The reεidue waε diεεolved in 10 mL of hot ethanol and 20 mL water waε added. On cooling the white εolid precipitate waε collected and dried under vacuum over P 2 θ5- The yield waε 72 mg (98% yield) of pure product: mp 218-219 β C (efferveεceε, εinters at 215) elemental analysis, Calc'd for C 33 H 40 N 2 0 6 : (560.696): C, 70.69; H, 7.19; N, 5.00; Found: C, 70.62; H, 7.39; N, 4.79. Step L: Preparation of N-(cis-2(R)-hydroxy- l(S)-indanyl)-5(S)-[l,l-dimethylethoxy- carbonylamino)-4(S)-hydroxy-6-phenyl-2(R)- [(4-(2-(4-morpholinyl)ethoxy)phenyl]methyl]- hexanamide A stirred mixture of Step K product, N-(2 (R)-hydroxy-l(S)-indanyl)-5(S)-[l,l-dimethylethoxy- carbonyl)amino]-4(S)-hydroxy-6-phenyl-2(R)-(4-hydroxy- phenylmethyl) hexanamide (0.50 g, 0.9 mmol), anhydrous cesium carbonate (1.0 g, 3 mmol) and N-(2-chloroethyl) morpholine, free base (2.35 g, 17 mmole) in 100 mL of anhydrous dioxane waε heated to 80°C (internal temperature) for 3 hrs. After cooling to room temperature the mixture was diluted with chloroform (50 mL) filtered, concentrated to dryneεε under reduced preεsure, and the residue triturated with 50 ml of anhydrous ether and 10 mL of ethyl acetate. The white solid product was collected and dried under vacuum over P θ5- The yield was 0.54 g (897.) of pure product L-689,502: mp 195-7 β C. elemental analysiε, Calc'd. for C 3 H 51 N 3 0: (673.856): C, 69.52 H, 7.63; N, 6.23; Found: C, 69.19 H, 7.45; N, 6.15. maleate hydrate: mp 112-113°C dec. elemental analyεiε, Calc'd. for C 4 H 4 0 4 .H 2 0: (807.946): C, 63.92 H, 7.11; N, 5.20; Found: C, 64.23 H, 6.94; N, 5.10. EXAMPLE 16 A. Fermentation of L-689.502 A frozen vial (2.0 ml) of Rhizopus arrhizus MF4974 was used to inoculate a 250 ml baffled shake flask containing 50 ml of seed medium A. The seed flask was incubated on a rotary shaker (220 rpm) at 27°C for 24 hourε. A 2.5 ml aliquot of the developed εeed was used to inoculate a 250 ml non-baffled flask containing 50 ml of transformation medium B; L-689,502 in DMSO was added to the fermentation at 0 hour to achieve a final concentration of 0.05 mg/ml. The shake flask contentε were εubsequently incubated at 27 β C on a rotary shaker for 4 days. The resultant whole broth was extracted as described in Section B. The whole broth (400 ml) was extracted three times with 1-butanol (3 x 400 ml). The extracts were combined and concentrated under vacuum to an oily residue. The residue was disεolved in methanol and εubjected to high performance liquid chromatography (HPLC). HPLC waε carried out on Whatman Partiεil 10 0DS-3,9.4 mm x 25 cm at room temperature and monitored at 215 mm. The column waε developed at 3 ml/min with a linear gradient from 0.1% aqueouε H3P0 4 -CH3CN, 80:20, to 0.17. aqueouε H 3 P0 4 -CH 3 CN, 20:80, in 60 minutes. The compound was collected during repeated injections of the above described extract. The fractions at retention time, 19.3 minutes, were pooled, adjusted to pH 6.5 and evaporated to remove acetonitrile. The compound was further purified using a C^g Sep-Pak (Waters Associates) and methanol- water elution solvent to yield 11 mg of product. Anal. Calc'd. for C39H5ø0*LøPN3Na 2 : C, 58.72 H, 6.27; N, 5.27; P, 3.89. Found: C, 59.78 H, 6.58; N, 4.97; P, 4.17. NMR, elemental analysis, and MS showed the εtructure to be L-696, 32 Phosphorus was identified and quantified by complexomatric and gravimetric methods. Phosphoryl¬ ation at C 4 was established by NMR from the 0.5 ppm downfield displacement of the C 4 H relative to its chemical shift in the parent L-689,502. EXAMPLE 17 Enzymatic Treatment of L-696.432 The microbial transformation metabolite L-696,432 was determined to be a phosphate ester of L-689,502, a highly potent HIV protease inhibitor. To determine if the phosphate ester bond can be enzymatically cleaved, treatment with alkaline phosphatase was performed. To 0.5 ml of water containing 0.8 mg of L-696,432 was added 30 microliter of bacterial alkaline phoεphataεe (0.31 unit/microliter), and the reaction mixture was incubated at 37 β C and monitored by HPLC. The reaction was complete after four hours of incubation. The reaction product was purified by HPLC and subjected to FAB MS analysis. [M+H]+ion was observed at m/z 674. The fragmentation ions at m/z 574,556,469 and 425 confirmed the product is L-689,502. The results show that L-696,432 can be enzymatically hydrolyzed back 1:o the parent compound L-689,502. EXAMPLE 18 Assay for Inhibition- of Recombinant HIV Protease Inhibition studies of the reaction of HIV protease expressed in Escherichia coli were carried out with a tritiated peptide εubεtrate, [ 3-acetyl- Val-Ser-Gln-Aεn-(beta-napthyl Ala)-Pro-Ile-Val-Gln- Gly-Arg-Arg-NH (MW=1800). The two arginine residues at the carboxyl terminus give this peptide an overall positive charge at acidic pH and enable it to bind to the H+ form of DOWEX AG-50W-X8 resin and εimilar resins. The HIV protease cleaves between the β-napthyl-Ala and proline residues to yield a product [ • *'H]-acetyl-val-ser-asn-(β-napthyl-ala) that is either neutral or slightly negatively charged and does not bind to the cation exchange resin. It iε therefore poεεible to conveniently separate the labelled product from the substrate. Aliquots of 25 μl containing 6.0-8.0 nM HIV protease in assay buffer (100 mM sodium acetate, pH 5.5 and 0.17. BSA) are placed in assay tubes. The reaction is initiated by addition of 25 μl aliquots of 4.2 μM tritated peptide εubstrate in 100 mM sodium acetate, pH 5.5. After incubation for 60 minutes at 37°C, the reaction is stopped with 100 μl of 5% H 3 P0 4 , then analysed by application of column chromatography. L-696,432 demonstrated 32% inhibition at a concentration of 1 μg/ml in substantially purified form. - Ill - EXAMPLE 19 Organic Synthesis of L-696.432. phosphate ester of L-689.502 Organic Synthesis of L-696.432 A. Acetylation of Indan N-(cis-2(R)-Acetoxy-l(S)indanyl)-5(S)-[l,l- dimethylethoxycarbonylamino)-4(S)-hydroxy-6-phenyl- 2(R)-[(4-(2-(4-morpholinyl)ethoxy)ρhenyl]methyl]- hexanamide is prepared by the protocol of Example 1, except that l(S)-amino-2(R)-acetoxyindane εubεtituteε for l(S)-amino-2(R)-hydroxyindane of step I. B. L-696.432 The product of step A iε reacted with monophenyl phosphorodichloridate according to the principles and practice of Chambers, R.W. and H.G. Khorana, J. Am. Chem. Soc. _\&, 3749 (1958). Sub- εequent treatment with exceεε ammonia, followed by removal of the acetyl and phenyl groupε under baεic conditionε, yields L-696,432. C. Alternative Synthetic Routes Another phosphorylating agent is dibenzyl phosphorochloridate. An extensive diεcuεεion of phoεphorylation and phoεphorylating agents can be found in Y. Mizuno, Studies in Org. Chem, 24. 171-175 (1986) and references cited therein. EXAMPLE 20 A culture of Rhizopus arrhizus MF 4974 from the Merck Culture Collection was inoculated in 50 milliliters of soy-glucoεe medium in a 250 milliliter flask and the flask incubated on a rotary shaker at 220 rpm at 27°C for 24 hours to obtain the first stage seed culture. 2.5 milliliters of this seed culture was inoculated into each of 18 flasks containing 50 milliliters of soy-glucose medium which was also charged with Compound Z to a concentration of 5 μg/ml in 50 microliters of DMS0 and the resulting culture incubated on a rotary shaker at 220 rpm at 27 β C for 18 hours. Following incubation, each flaεk waε harvested by centrifugation, the mycelium washed twice with sterile saline and reεuεpended in pH 7.0 phoεphate buffer containing 1 percent glycerol. Compound Z waε added to a final concentration of 58 μg/ml uεing 100 μl of (DMSO). The flaεkε were incubated on a rotary εhaker at 220 rpm at 27 β C for 48 hourε. At the end of thiε period, the contentε of eighteen flaεks (900 milliliterε) were pooled and the whole broth centrifuged. The mycelial cake waε εlurried with 100 milliliterε of water, the pH adjuεted to 3.5 and the εlurry extracted twice with 100 milliliterε of n-butanol. The εupernatant waε acidified to pH 3.5 and extracted twice with one-half the volume of n-butanol. Each organic extract was asεayed by HPLC. The aεεay condition was as follows: Column: "ZORBAX" (DuPont) C8 Rx 4.5 x 250 mm Mobile Phase: Acetonitrile + lOmM aq. KH P0 4 , gradient 20% to 80% over 20 minutes Temperature: 45 β C Flow rate: 1.5 ml/min. Detection: 210 nm Sample Vol: 50 μl The extracts of the mycelium and the extracts of the supernatant were pooled and evaporated to drynesε at 30 β C at reduced pressure to obtain an oil as residue. The oil was dissolved in the mobile phase of 40/60 acetonitrile/water and further purified using ZORBAX C8 (9.6 mm x 25 cm) semi-preparative column. The column waε developed at 7.05 ml/min. uεing 40 percent aqueouε acetonitrile containing 0.1 percent trifluoroacetic acid (TFA) at 45 β C. Fractions having a retention time of 12.8 minutes were pooled, and the solvent evaporated to obtain 12 mg of Compound III in a yield which calculated to be 24%. The product had the masε εpectral data previouεly εet forth. A portion of the product waε converted to the monopotassium εalt. This was carried out by dissolving the biophosphorylation product in 70 percent aqueouε acetonitrile containing 10 mM H P0 4 (pH 4.5). The mixture waε subjected to reduced pressure to remove the acetonitrile and the aqueous residue loaded onto a water-equilibrated C-18 solid phaεe extraction column. The column was washed with water and then eluted with 70 percent aqueous acetonitrile and the eluate freeze-dried for retention of the salt. EXAMPLE 21 Spores of Rhizopus arrhizus were obtained from oatmeal agar slantε of MF 4974 maintained in the Merck Culture Collection and employed to prepare a spore suspension in water of about 7 x 10^ spores per milliliter for use in the preparation of seed culture. Seed flasks each containing 500 milliliters of soy glucose broth of the composition previously given, were inoculated with 1 milliliter of spore suspension and incubated on a rotary shaker (220 rpm) at 27 β C for 24 hours. Following incubation, the mycelia from each flask were harvested by filtering through a 10 micron nylon meεh and then reεuεpended in an equal volume of a 100 mM pH'6.3 phoεphate buffer containing 3 percent glycerol and added to the broth. Compound Z, waε added to a concentration of about 50 μg/ml in dimethylεulfoxide. The flasks were then incubated on a rotary shaker at 220 rpm at 27 β C for 24 hourε. After completion of the incubation period, the contents of sixteen flaskε totaling 8000 milliliterε were pooled and filtered through a 10 micron nylon meεh. The mycelial cake waε εlurried with 1000 milliliters of 50 percent aqueous methanol and filtered. The mycelial cake waε again extracted with aqueous methanol and the two aqueous methanol filtrates combined and diluted with 2000 milliliters of water. The resulting aqueous solution was applied to a 15 mm x 300 mm column packed to a 220 mm bed height with water equilibrated "DIANION" HP20 resin. The filtrate was pumped in a downflow mode at 15 ml/min. in 2000 ml aliquots. After loading, the column was washed with 500 milliliters of water and the desired phosphorylated product eluted with 500 milliliters of 20 percent aqueous acetonitrile. The remaining substrate and metabolite was eluted with 500 milliliters of 70 percent aqueous acetonitrile. The diluted filtrates from the mycelial extracts were also pumped onto the- column, the column washed and then eluted and the eluate assayed by HPLC. The assay condition was as described in Example 20. HPLC indicated the following mass balance for microbial phosphorylation and "DIAION" HP 20 isolation: Substrate Charged: 402.5 mg Metabolite & Substrate Recovered: 383.8 mg Total Recovery: 95.4% Compound III 234.9. g Bioconverεion yield: 61.2% The eluate waε concentrated under reduced pressure and the reεidue diεsolved in 40 percent aqueouε acetonitrile containing lOmM KH P0 4 to obtain a monopotassium εalt. The aqueouε εolution waε εubjected to reduced preεεure -to remove the acetonitrile and the aqueouε residue loaded onto a water-equilibrated C-18 solid phase extraction column. The column was thereafter washed with water and then the monopotassium salt of Compound III eluted with 70 percent aqueous acetonitrile. The eluant waε freeze dried to obtain the εalt. The NMR spectrum of this salt is that previously detailed. The εalt is converted to the acid by careful acidification. EXAMPLE 22 The following salts are prepared by reacting an appropriate phosphate salt and Compound III in the manner described in Example 21 and concentrating under vacuum. 1000 compressed tablets each containing 500 mg of Compound IIIA are prepared from the following formulation: m The finely powdered ingredients are mixed well and granulated with 107. starch paste. The granulation is dried and compressed into tablets. EXAMPLE 24 1000 hard gelatin capsuleε, each containing 500 mg of mono εodium salt of Compound III are prepared from the following formulation: Compound Grams Compound III, mono sodium salt 500 Starch 750 Dibasic calcium phosphate hydrous 5000 Calcium stearate 2.5 A uniform mixture of the ingredientε iε prepared by blending and uεed to fill two-piece hard gelatin capεuleε. 250 ml of an injectable εolution are prepared by conventional procedureε having the following formulation: Dextroεe 12.5 g Water 250 mL Compound III, mono potaεεium salt 400 mg The ingredientε are blended and thereafter εterilized for uεe. EXAMPLE 25 An aerosol composition may be prepared having the following formulation: Per Canister Compound IIIA 24 mg Lecithin NF Liquid Concentrated 1.2 mg Trichlorofluoromethane, NF 4.026 g Dichlorodifluoromethane, NF 12.15 g Preparation of Starting Materials Compound Z, the εtarting material, waε prepared by inoculating 54 milliliterε of P34-2 medium of the following compoεition per liter: corn εteep liquor, 5 g; D-mannitol 25 g; glucoεe monohydrate, 10 g; "PHARMAMEDIA," (nonhydrolyzed protein, Buckeye Oilseed Products, Memphis, Tenn.) 20 g KH 2 P0 4 , 9 g; FeS0 4 »7H 2 0, 10 mg MnS0 4 «*4H 2 0 10 mg; CuCl 2 »2H 2 0, 0.25 mg; CaCl 2 *»2H 2 0, 1 mg; H3BO3, 0.56 mg: (NH 4 ) 6 Mo 7 0 24 *»H 2 0, 0.19 mg; ZnS0 4 «7H 2 0, 2 mg, with frozen vials of Zalerion arboricola MF5533 ATCC 74030 and the inoculated medium incubated with shaking at 220 rpm at 25 β C for four days. Twenty milliliterε were used ±0 inoculate four 2-liter flasks containing 500 milliliters of P34-2 medium and the inoculated medium incubated at 25 C C for four days at 220 rpm. The flask contents were pooled and used to inoculate into three fermenterε each containing 180 liters of P34-2 medium and 2 ml/L of propylene glycol P-2000 (Dow Chemical) to reduce foaming and the inoculated medium cultivated for six days at 25 β C, an air flow of 90 L/min. a pressure of 0.7 kg/cm--- gauge, and an agitator speed of 200 rpm. A 25 liter sample of the resulting broth was then used to inoculate three fer enters each containing 475 liters of P34-2 medium containing 2 ml/L of P-2000 and cultivated for four days at 25 β C, air flow of 250 L/min, pressure 0.7 kg/cm 2 gauge and 150 rpm. 425 liters of this seed broth was inoculated into each of three production fermenters containing 13,700 liters of TG106 medium of the following composition per liter: D-mannitol, 100 g; NZ-Amine type E (casein hydrolysate, Sheffield Productε, Kraft Inc.) 33 g; Fidco 8005 yeaεt extract (Difco), 10 g; (NH 4 ) 2 S0 4 , 5 g; KH 2 P0 4 , 9 g; P-2000, 2 ml, and the fermenters operated at a temperature of 27 β C, air flow of 2500 literε/minute, a preεεure of 0.7 kg/cm 2 gauge, and an agitator εpeed of 50 rpm. The pH waε allowed to decreaεe from 6.0 to 5.5 and then maintained at 5.5 ± 3. After about 2 1/2 weeks the broth was harvested for product iεolation. The broth from the foregoing cultivation was first extracted with an equal volume of methanol. The methanol-broth was clarified using a liquid-solid separator (centrifuge) to obtain clarified liquid as first extract and solid. The extraction-clarification was repeated. The extracts were combined and the water cont at adjusted to about 50 percent. The resulting solution waε paεεed through a "DIAION" SP-207 adεorption column to adεorb Compound III and the column waεhed with aqueouε methanol. Thereafter Compound III waε recovered with 100 percent methanol. The water content of the methanol containing Compound III was adjusted to 50 percent and the aqueous methanol solution intimately mixed with an equal volume of 1:1 ethyl acetate/hexane and the two liquid phases thereafter separated. The aqueous methanol layer was pasεed through a column of "DIAION" SP-207, the column waεhed with aqueouε methanol, and Compound III eluted with 100 percent methanol. The eluant was vacuum concentrated to a minimum volume and the solvent composition adjusted to about 75:20:5 ethyl acetate/methanol/water. The feed thus prepared was passed through a silica gel column and Compound III eluted with 85:10:5 ethyl acetate/methanol/water. The fractions showing 85 percent or greater area purity by HPLC were combined, vacuum concentrated to remove ethyl acetate and the concentrate adjusted to 50 percent aqueous methanol, the latter pasεed through "DIAION" ΞP-20 in the manner previously described concentrated and Compound III precipitated with acetronitrile and recovered by vaccum filtration and then dried. The starting material also may be prepared by methods described in copending applications Serial Nos. 47/492,025 and 47/492,026. Z. arboricola MF5533 ATCC 74030 iε diεcloεed and claimed in copending application Serial No. 630,457, filed December 19, 1990. Briefly, it may be obtained by (a) inoculating a frozen vegatative mycelium of _ . arboricola ATCC 20957 (diεcloεed and claimed in copending application Serial No. 492,024) into KF seed medium of: corn steep liquor, 5 g/1; tomato; tomato paste 40 g/1, oat flour 10 g/1; glucose 10 g/1; FeS0 4 »7H 2 0 10 mg/1; MnS0 4 *»4H 2 0 10 mg/1; CuCl 2 »2H 2 0, 0.25 mg/1; CaCl 2 * »2H 2 0, 1 mg/1; H3BO3, 0.56 mg/1; (NH 4 ) 6 Mθ7θ 24 »H 2 0, 0.19 mg/1; ZNS0 4 »7H 2 0, 2 mg/1; adding to the medium N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (b) cultivating, thereafter (c) plating a portion of the growth on potato dextroεe agar and (d) incubating for 14 days at 25 β C to obtain sporeε then (e) harvesting the spores, (f) diluting the sporeε with εterile saline (g) plating on potato dextroεe agar (h) incubating for 7 days for colony formation, (i) transferring separate colonies to slants of potato and (j) incubating for 14 days at 25 β C. EXAMPLE 26 Biotransformation Enzymology-Iεolation Of Cell Free Activity Sporeε of Rhizopus oryzae MF 4974, cultivated on oatmeal agar, were inoculated in a Soy-Glucose medium consisting of 20.0 g dextrose, 5.0 g Fidco yeast extract, 5.0 g NaCl, 5.0 g K 2 HP0 4 in a liter of distilled water. The pH of the medium was adjusted to 7.0 before autoclaving. The cultures were incubated at 27 β C for 24 hours on a rotary εhaker at 220 rpm. A freεh Soy-Glucose medium waε inoculated with the 24-hour εeed culture at a ratio of 5% (v/v) and the fermentationε were continued in the manner deεcribed above. The phoεphorylating enzyme acti¬ vity, aε a function of time, was determined. Cell free extracts were obtained by suspend¬ ing the cells in a buffer containing 100 mM phoεphate (pH 7.5) and 2 mM EDTA. The cells to buffer ratio was 2:1. Several procedures were then tried to break the cell membranes and release the cell contents, i.e., a high-pressure French presε, εonication, grinding the frozen cells, and lysozyme treatment. The enzyme reaction was carried out in 0.2 ml mixture containing 0.15 mM FK-520, 5 mM MgCl 2 , 5 μM ATP and various amount of enzyme. The reaction mixture was incubated at different temperatures for 1 hour and terminated by the addition of 0.2 ml methanol. The resulting solution was subjected to HPLC analysis on Whatman Partisil 10 ODS-3 at 55 β C. The column was developed at 1 ml/min with a linear gradient from 45% acetonitrile in 0.1% phosphoric acid to 807. in 0.1% phosphoric acid in 30 minuteε. The retention time of phoεphorylated FK-520 and FK-520 was 16,5 and 20 minutes, respectively. Protein concentration was determined by Pierce BCA protein assay reagent using BSA as standard. In a εearch for phoεphorylating enzyme in cell free extracts of Rhizopus oryzae MF 4974 we have found phosphorylating activity: 1) is highest in the extracts of cells isolated after 24 hours incubation in Soy-Glucose medium; 2) both sonication and macera¬ tion of the cellε under liquid nitrogen can be uεed to break the cell membraneε for liberating the enzyme in εoluble form. The pH optimum iε 7.5; 4) the temperature optimum is 37 β C. 5) The phosphorylation of FK-520 was linear with respect to time and enzyme concentration for one hour and from 0.2 to 2.0 mg protein. The effect of the FK-520 concentration on the rate of phosphorylation have been studied using crude extract. The Km value for the substrate (FK-520) was about 0.5 mM and the Vmax waε 2.In mole/min/mg protein. To determine metal ion require¬ ment the crude enzyme was dialyzed thoroughly against 20 mM Tris-HCl buffer and 2 mM EDTA. The resultε show that the phosphorylating activity requires Mg +2 or Ca +2 as cofactor. EXAMPLE 27 NMR analysis established that the biotrans¬ formation product obtained by incubating 24-deoxy FR-900520 with MF 4974 (Rhizopus arrhizus) is the 32 phosphorylated analog: O I I Contacting 24-deoxy FR 900520 made by the process described in Exampleε 28-29 with the Rhizopus microorganism as per the analogous procedure described for 24-oxy FR 900520, yields the 32-phos- phorylated analog as determined by NMR spectroεcopy. EXAMPLE 28 17-Ethyl-l-hydroxy-12-[2'-(3' ' ,4' '-dihydroxycyclo- hexyl)-l'-methylvinyl]-23,25-dimethoxy-13,19,21,27- tetramethyl-ll,28-dioxa-4-azatricyclo[22.3.1.0 » 9 ]- octacos-14.18-diene-2.3.10.16-tetraone To a stirred solution of 31-desmethyl FK-900,520, 17-ethyl-l,14-dihydroxy-12-[2'-(3" , "- dihydroxycyclohexyl)-!'-methylvinyl]-23,25-dimethoxy- 13,19,21,27-tetramethyl-ll,28-dioxa-4-azatricyclo- [22.3.1.0 4 - 9 ]octacos-18-ene-2,3,10,16-tetraone produced aε described in EPO Publication. 0 349 061 (77 mg in 3 ml benzene) was added 6 mg of p_-toluenesulfonic acid and the mixture warmed to 60°C in an oil bath. After 25 minutes, the reaction mixture was cooled to room temperature, neutralized by the addition of a saturated aqueous NSLHCO3 εolution and extracted with ethyl acetate (3 timeε). The combined organics were washed with saturated NaCl solution, dried over Na S0 4 and purified by flash chromatography (207. hexanes in ethyl acetate and 1% MeOH) to yield 40 mg of the title compound. MASS: (FAB) 782 (m + Na). Partial NMR (200 mHz): δ 6.80 (dd, J 1 = 16 Hz, J 2 = 6 Hz), 6.16 (dd, J χ = 16 Hz, J 2 = 1.5 Hz), 4.39 (broad d, J = 14 Hz), 4.26 (broad d, J = 5 Hz), 3.91 (dd, J- = 8.8 Hz, J 2 = 3 Hz). EXAMPLE 29 17-Ethyl-l-hydroxy-12-[2'-(3' » ,4' '-dihydroxycyclo- hexyl)-l'-methylvinyl]-23,25-dimethoxy-13,19,21,27- tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0 4 • 9 j " - octacos-18-ene-2.3.10.16-tetraone To a solution of 17-ethyl-l-hydroxy-12- [2'-(3' ' ,4 » '-dihydroxycyclohexyl)-l'-methylvinyl]- 23,25-dimethoxy-13,19,21,27-tetramethyl-ll,28-dioxa-4- azatricyclo[22.3.1.0 4• 9 ]octacos-14,18-diene-2,3,10,16- tetraone (40 mg in 1.5 ml ethyl acetate) waε added 3 mg of 57. Rh/Carbon catalyst. The reaction flask was fitted with a hydrogen balloon, evacuated and recharged with hydrogen gas (3 timeε). After 45 minuteε, the mixture was filtered over Celite, concentrated and purified by flash chromatography (CH 2 C1 2 : MeOH: Hexane (10:1:2)). to yield 33 mg of the title compound. MASS: (FAB) 768 (m + Li). Partial 1 H NMR (200 mHz): δ 4.55 (broad d, J = 5 Hz), 4.39 (broad d, J = 14 Hz), 3.86 (dd, J χ = 8.8 Hz, J 2 as 3 Hz). EXAMPLE 30 PHOSPHORYLATED ANTI-CHOLESTEROLEMIC ISOLATES L-706.526 AND L-706.527 Contacting simvastatin, whose chemical name is: 6(R)-[2-(8'(S)-2",2"-dimethylbutanoyloxy- 2' (S),6' (R)-dimethyl-l',2',6',7',8',8'a (R)- hexahydronaphthyl-l'(S)-ethyl]-4(R)-hydroxy-3,4,5,6- tetrahydro-2H-pyran-2-one with the Rhizopus. microorganiεm under the analogous conditions described in Example 3 produced two phosphorylated compounds, L-706,546 and L-706,527. The preparation and properties of simvaεtatin are deεcribed in EP Publication No. 0033538 to Merck &. Co., Inc. NMR analysis of the biotransformation products obtained by incubating the above compound simviεtatin with Rhizopus arrhizus confirms their assigned structures as illustrated: L-706, 526 L-706, 527 The compounds exhibit inhibition in the biosynthesis of cholesterol aε alεo deεcribed for simvastatin in EP Publication No. 0 033 538. Key features in the NMR of L-706,526 are the absence of a side chain methyl triplet and the presence of a novel CH3CH-O- moiety with εignalε at 1.10 ppm (CH 3 ) and at 3.93 ppm (CH-0). Irradiating at 1.10 ppm collapsed the methine quintet to a doublet. Since the methine proton in the proposed structure has no vicinal neighbor the appearance as a doublet implies coupling with an unseen hetero atom, i.e., the phosphorus. Phosphorylation at the H-6* site a is sug- geεted by the absence of a typical H-6' signal at 3.69 ppm and the presence of a broad, featurelesε εignal at 4.26 ppm which iε aεεigned to a diεplaced H-6'. Both the magnitude of the diεplacement and the loss of detail are reasonable consequences of phos¬ phorylation. EXAMPLE 31 Phosphorylation of Zearalenone Cheπical structure of -zβaralβnol-6' -phosphate Incubation of zearalenone available from Sigma with resting cells of Rhizopus arrhizus MF 4974 for 24 hours results in two biotransformation products. They have been identified as α-zearalenol and α-zearalenol-6'-phosphate. The fermentation, isolation, purification and enzymatic hydrolysis are described below. FERMENTATION One Ml of a Rhizopus arrhizus (MF 4974) spore suεpenεion, harvested from oatmeal agar, wea incubated into 500 mL Soy-Glucose medium contained in a 2 liter Erlenmeyer flask and shaken a 27°C on a rotary εhaker (220 rpm) for 24 hours. Following incubation, each flask was harvested by filtration washed once with water, then resuspended in 500 mL or mM P0 4 buffer (pH 7.0) containing 4% glycerol. Zearalenone was added to a final concentration of .05 mg/mL, using 0.8 mL DMSO. The charged flasks were incubated on a rotary shaker (220 rpm) at 27°C for 24 hourε. Following incubation, the whole broth was worked up as described below. Media Sov Glucose Medium g___, Dextrose 20.0 Soy Meal 5.0 Fido yeast extract 5.0 NaCl 5.0 K 2 HP0 4 5.0 Adjust pH to 7.0 ISOLATION AND PURIFICATION The whole broth (1000 mL) was diluted with an equal volume of methanol, than centrifuged. The mycelial cake was discarded. The clear filtrate waε applied to a 15mm X 30 cm column packed with HP 20 (Mitsubishi Chemical, 220 mm bed height water equilibrated). The filtrate was pumped onto the column in the downflow mode at 15 mL/min. After loading, the column was washed with 500 mL water. Metabolites or substrates were not detected in the spent broth or wash. The column was eluted with a step gradient of methanol:water. The most polar metabolite eluted between 10% and 30% methanol. The second metabolite and subεtrate eluted in the column waεh (100% methanol). Appropriate fractions were combined and evaporated to drynesε under reduced preεεure at 30 β C to yield yellow oilε that waε εubjected to further purification by preparative HPLC. Preparative HPLC waε carried out on Whatman Magnum 9 Partiεil 10 ODS-3 column (C 18 , 9.8 mm ID x 25cm) at room temperature and monitored at 237 nm. The column waε developed at 4 mL/min with linear gradient from 207. acetonitrile in 0.1%phosphoric acid to 80% acetonitrile in 0.1% phosphoric acid in 40 minutes. The compounds were collected during repeated injections of the above described extract. Fractions of retention times 17.5 and 20.1 minutes were collected, pooled, diluted with three volumns of water and desalted using a Speed C^g column (Applied Separations) to yield 7 mg of 6-phosphate ester and 5 mg of α-zearalenol, respectively. ENZYMATIC HYDROLYSIS To prove that the 6'α-hydroxyl group is phosphorylated, enzymatic hydrolysis was examined using various εulfataεeε and alkaline phoεphataεeε obtained from Sigma. In εummary, a εpecific amount of enzyme was added to one mL of lOOmM phosphate buffer containing -50 ug/mL of zearalenol phoεphate eεter. The buffer εolution waε incubated at 37 β C for 4 hourε. After incubation, 300 uL waε removed and analyεed by HPLC, using a Whatman Partisil 10 0DS-3 column (C^g 10 urn, 4.6 mm ID x 25 cm) at room temperature and monitored at 237 nm. The column was developed at 1 mL/min with a linear gradient gradient from 20% acetonitrile in 0.1% phosphoric acid to 80% acetonitrile in 0.17. phosphoric acid in 20 minutes. Under these conditions, α-zearalenol-6'-phosphate ester and α-zearalenol have retention times of 13.93 and 18.17 minutes, respectively. The reεultε of enzymatic hydrolyεiε are εummarized below: ENZYME pi UNITS ACTIVITY P-4653 Alk. Phosphatase 7.0 1.0 1007. α-OH @ lhr P-5521 Alk. Phosphatase 7.0 100.0 1007. α-OH @ lhr The compounds were identified by MS and NMR and confirmed by enzymatic hydrolysiε aε α-zearalenol-6'-phoεphate and α-zearalenol. Alεo, the preεence of phosphorus was confirmed by an ICP elemental analysis.

Claims: WHAT IS CLAIMED IS: 1. A process for producing a biophosphory¬ lated hydroxyl containing organic compound, wherein said hydroxyl group is phosphate reactive, comprising the step of contacting a strain of Rhizopus oryzae microorganism together with the hydroxy containing organic compound in an aqueous medium containing a carbon nutrient at ambient temperature for a suffi¬ cient time to produce the biophosphorylated hydroxyl containing organic compound. 2. The process of Claim 1 wherein said microorganism comprises resting cells of Rhizopus oryzae in a phosphate buffer containing glycerol as a carbon nutrient. 3. The process of Claim 1 wherein said Rhizopus oryzae is cultured with said hydroxyl containing organic compound under submerged aerobic fermentation conditions in an aqueous carbohydrate medium containing a nitrogen nutrient at a pH below about 8.0 at ambient temperature. 4. The procesε of claim 1 wherein said Rhizopus oryzae strain is ATCC No. 11145. 5. A process for producing an immunos- εuppressant, identified as a C-32 phosphorylated derivative of an FK-506 type macrolide comprising the step of contacting a strain of a Rhizopus oryzae microorganism together with a FK-506 type macrolide, containing a free C-32 hydroxy group, in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the C-32 phosphorylated FK-506 type macrolide immunosup¬ pressant. 6. A compound being the C-32 phosphorylated derivative of an FK-506 type macrolide. 7. The compound of Claim 1 of the formula: CH 3 0 OCH 3 wherein: R is H, C* j _-C 4 alkyl, R 1 is H 2 P0 4 , R 2 is hydrogen, hydroxy or lower alkanoyloxy, R 3 is methyl, ethyl, propyl or allyl, n is an integer of 1 or 2, and the symbol of a line and dotted line is a single bond or a double bond, and a pharmaceutically acceptable salt thereof. 8. The compound of Claim 7 being the C-32 phsophorylated derivative of FK-506, FK-520, FK-523, FK-525, C-31 desmethyl FK-506, C-31 desmethyl FK-520, 9. The compound of Claim 7 being: O CH 3 0 OCH 3 10. The compound of Claim 7 being: 0 CH 3 0 OCH 3 11. The compound of Claim 7 being; O CH 3 0 OCH 3 12. The compound of Claim 7 being: O HO CH.,0 OCH, 13. The compound of Claim 7 being: CH 3 0 OCH 3 14. The compound of Claim 7 being: O II CH 3 O OCH 3 15. The compound of Claim 7 being: O CH 3 O OCH 3 16. A pharmaceutical composition for the treatment of immunoregulatory disorders or diseases comprising a pharmaceutical carrier and a therapeutically effective amount of compound of Claim 7. 17. A pharmaceutical composition for the topical treatment of inflammatory and hyperproliferative skin diseases and or cutaneous manifestations of immunologically-mediated illnesεes compriεing a pharmaceutical carrier and a therapeutically effective amount of compound of Claim 18. A method for the treatment of immunoregulatory disorders or diseases comprising the administration to a mammalian species in need of such treatment an effective of a compound of Claim 7. 19. A process for producing an immunosuppressant, identified as a phosphorylated rapamycin-type macrolide comprising the step of contacting a strain of a Rhizopus oryzae microorganism together with rapamy -n macrolide in an aqueous medium containing a carbon nutrient at ambient temperature for a sufficient time to produce the C-43 phosphorylated rapamycin macrolide immunosuppresεant. 20. The process of Claim 19 wherein said microorganiεm are reεting cellε of Rhizopuε oryzae in a phoεphate buffer containing glycerol aε a carbon nutrient. 21. The proceεε of Claim 19 wherein the resulting aqueous medium containing said phosphoryl¬ ated macrolide exhibitε poεitive inhibition of T-Cell activation. 22. The process of Claim 19 wherein said Rhizopus oryzae is cultured with said rapamycin macrolide under submerged aerobic fermentation conditions in an aqueous carbohydrate medium con¬ taining a nitrogen nutrient at a pH below about 8.0 at ambient temperature. 23. A phosphorylated macrolide of the structural formula: O II • where R= [HO]2-P- 24. A phosphorylated macrolide exhibiting a characteristic proton NMR εpectrum aε illuεtrated in Figure 3 and exhibiting poεitive inhibition of T-cell proliferation, reverεible by recombinant human IL-2, in a T-cell proliferation aεεay. 25. A phosphorylated macrolide having the structural formula as illustrated in Figure 4. 26. A method for producing a phosphorylated cyclic lipopeptide compound having the formula 0 Ii wherein R is -P(0H) which comprises cultivating Rhizopus arrhizus ATCC 11145 in a nutrient medium containing a compound having the formula: and a phosphate εalt, said medium maintained in a pH range of from about 6.0 to 6.3. 27. A method according to Claim 26 in which the phosphate salt is in an amount of at least 10 percent by weight of the solid components. 28. A method according to Claim 26 in which the medium of the following composition in grams/liter: glucose 20.0; soya meal 5.0; Fidco yeast extract 5.0: εodium chloride 5.0; and potaεsium hydrogen phosphate 5.0; at pH 5. 29. A method according to Claim 26 in which the cultivation is carried out in the temperature range of from about 15 β C to about 30 β C with agitation in the range of 220 rpm to 400 rpm for a period of from 24 hours to about two days. 30. A method for selectively phosphorylating a compound having the formula at the 4-hydroxyproline hydroxyl which comprises cultivating Rhizopus arrhizus ATCC 11145 in a soy glucose medium containing said compound in an amount of 40-60 μg/ml, wherein said soy glucose medium is of the following composition in grams/liter: glucose 20.0; soya meal 5.0; Fidco yeast extract 5.0; sodium chloride 5.0; and potassium hydrogen phosphate 5.0; at pH 5. 31. A phosphorylated cyclic lipopeptide compound obtained by biophosphorylating with Rhizopus arrhizus ATCC 11145, a cyclic lipopeptide related to echinocandins and having a peptide skeleton bearing several hydroxy groups wherein in said phosphorylated cyclic lipopeptide, the phosphate group is attached to the hydroxy group of the 4-hydroxyproline component of the lipopeptide. 32. A phosphorylated cyclic lipopeptide compound having the formula: 0 ιι wherein R iε -P(0H) 2 or a cation εalt thereof , 33. A compound according to Claim 32 wherein 0 II R is -P(0H) 2 , 34. A compound according to Claim 32 wherein 0 ii R iε -P(0H)(0K) 35. A compound according to Claim 32 wherein 0 R is -P(0H)(0Na). 36. A composition comprising the compound of Claim 32 in admixture with a pharmaceutically acceptable carrier. 37. A method for controlling the growth of fungi comprising administering an antifungally effective amount of the compound of Claim 32. 38. A method for the treatment of or for the prevention of Pneumocystiε carinii infectionε in mammalε which compriεeε adminiεtering to mammals an anti-infective or therapeutic amount of the compound of Claim 32. 39. A compound of the Formula: or pharmaceutically acceptable salt, hydrate, or ester thereof. 40. A method of preparing the compound of Claim 39, comprising the steps of (a) providing a quantity of N-(2(R)-hydroxy- l(S)-indanyl)-5(S)-(l,l-dimethylethoxy- carbonylamino)-4(S)-hydroxy-2(R)-[(4-(2- (4-morpholino)ethoxy)phenyl)prop-2-en-l- yl]-6-cyclohexyl-hexanamide; (2) incubating the compound of step (a) in a microbial culture of Rhizopus arrhizus MF4974; and (3) isolating the compound of Claim 39. 41. A pharmaceutical compoεition uεeful for inhibiting HIV proteaεe, compriεing an effective amount of a compound of Claim 39, and a pharmaceutically acceptable carrier. 42. A pharamceutical composition useful for preventing or treating infection of HIV or for treating AIDS or ARC, comprising an effective amount of a compound of Claim 39, and a pharmaceutically acceptable carrier. 43. A method of inhibiting HIV protease, comprising administering to a suitable mammal in need of such treatment an effective amount of a compound of Claim 39. 44. A method of preventing infection of HIV, or of treating infection by HIV or of treating AIDS or ARC, comprising adminiεtering to a εuitable mammal in need of εuch treatment an effective amount of a compound of Claim 39. 45. A compound of the Formula: O II R= -P—OH, I OH or pharmaceutically acceptable salt, hydrate, or ester thereof. 46. A method of preparing the compound of Claim 45, comprising the steps of (1) providing a quantity of N-(2(R)-hydroxy- l(S)-indanyl)-5(S)-(l,l-dimethylethoxy- carbonylamino)-4(S)-hydroxy-6-phenyl- 2(R)-[(4-(2-(4-morpholinyl)ethoxy)- phenyl)methyl]-hexanamide; (2) incubation the compound of εtep (1) in a microbial culture of Rhizopus arrhizυε MF4974; and (3) iεolating the compound of Claim 45. 47. A pharmaceutical compoεition uεeful for inhibiting HIV protease, comprising an effective amount of a compound of Claim 45, and a pharmaceutically acceptable carrier. 48. A pharamceutical composition useful for preventing or treating infection of HIV or for treating AIDS or ARC, comprising an effective amount of a compound of Claim 45, and a pharmaceutically acceptable carrier. 49. A method of inhibiting HIV protease, comprising administering to a mammal an effective amount of a compound of Claim 45. 50. A method of preventing infection of HIV, or of treating infection by HIV or of treating AIDS or ARC, comprising administering to a mammal an effective amount of a compound of Claim 45. 51. A phosphorylated simvaεtatin derivative of the following εtructure: -70S, 526 or L-706, 527 52. A phosphorylated zearalenol compound of the following structure:

Inventors: ARISON BYRON H, CHEN SHIEH-SHUNG TOM, DUMONT FRANCIS, HSU ANNJIA T, MATHRE DAVID J, PETUCH BRIAN R, REAMER ROBERT A, SO LYDIA T, WHITE RAYMOND F, WU JANE T, ARISON, BYRON, H., CHEN, SHIEH-SHUNG, TOM, DUMONT, FRANCIS, HSU, ANNJIA, T., MATHRE, DAVID, J., PETUCH, BRIAN, R., REAMER, ROBERT, A., SO, LYDIA, T., WHITE, RAYMOND, F., WU, JANE, T.
Applicants: MERCK & CO INC, MERCK & CO., INC.
Application year: 1991
Main classification: C12P9
IPC codes: A61K31, A61K38, A61P37, C07F9, C07H19, C07K7, C12P9, C12R1