Patent Publication Number: US-5155029-A

Title: Process for producing a cyclic ether

Description:
PRIOR RELEVANT APPLICATIONS 
     This application is a continuation-in-part of application for U.S. Pat. Ser. No. 399,826 filed on Aug. 28, 1989 now U.S. Pat. No. 4,970,163 issued on Nov. 13, 1990. 
    
    
     BACKGROUND OF THE INVENTION 
     The compound sclareolide having the structure: ##STR3## has been found to be a valuable intermediate in preparing the compound having the structure: ##STR4## an important material for use in perfumery. 
     The compound which is the cyclic ether having the structure: ##STR5## has the been shown to be useful in U.S. Pat. No. 4,798,799 issued Jan. 17, 1989 as an intermediate in the creation of the compound having the structure: ##STR6## The compound having the structure: ##STR7## has been shown to be useful in U.S. Pat. No. 4,798,799 issued Jan. 17, 1989 as an intermediate in the creation of the compound having the structure: ##STR8## and has also been shown to be a useful precursor of the compound having the structure:  at column 8, lines 58-60 of U.S. Pat. No. 4,798,799. 
     Indeed, U.S. Pat. No. 4,798,799 discloses the utilization of a culture containing the microorganism Hyphozyma roseoniger having the identifying characteristics of CBS 214.83 and ATCC 20604 capable of producing the diol having the structure: ##STR9## in a recoverable quantity upon the transformation of compounds including the compound having the structure: ##STR10## (sclareol) as well as the cyclic ether having the structure: ##STR11## 
     Table IV, thereof at column 12, lines 15-28 discloses yield of 96% when carrying out the reaction: ##STR12## under fermentation conditions using ATCC 20624. 
     There is no teaching or suggestion in the prior art of either (a) carrying out the reaction: ##STR13## via microbiological methods whereby the compound having the structure: ##STR14## is formed in relatively high yields or (b) carrying out the reaction: ##STR15## via a microbiological method using the organism Bensingtonia ciliata, ATCC 20919 or (c) carrying out the reaction: ##STR16## via a microbiological method using the organism Cryptococcus laurentii, ATCC 20920. 
     Furthermore, the organisms: 
     Cryptococcus albidus, ATCC 20918; 
     Bensingtonia ciliata, ATCC 20919; 
     Cryptococcus laurentii, ATCC 20920; and 
     Cryptococcus albidus, ATCC 20921. 
     are novel organisms. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a GC-MS spectrum for the starting material of Example III. The peak indicated by reference numeral 21 is the peak for sclareol having the structure: ##STR17## The peak indicated by reference numeral 20 is the peak for the internal standard, the compound having the structure: ##STR18## 
     FIG. 2 is the NMR spectrum for the reaction product of Example II, the compound having the structure: ##STR19## 
     FIG. 3 is the NMR spectrum for the reaction product of Example III having the structure: ##STR20## 
     FIG. 4 is the NMR spectrum for the reaction product of Example IX, the compound having the structure: ##STR21## 
    
    
     SUMMARY OF THE INVENTION 
     The present invention concerns biologically pure cultures of the microorganisms: 
     Cryptococcus albidus, ATCC 20918; 
     Bensingtonia ciliata, ATCC 20919; 
     Cryptococcus laurentii, ATCC 20920; and 
     Cryptococcus albidus, ATCC 20921. 
     In another embodiment the present invention concerns cultures containing the microorganisms: 
     Cryptococcus albidus, ATCC 20918; 
     Bensingtonia ciliata, ATCC 20919; 
     Cryptococcus laurentii, ATCC 20920; and 
     Cryptococcus albidus, ATCC 20921, 
     said cultures individually capable of producing either the diol having the structure: ##STR22## or the cyclic ether having the structure: ##STR23## or sclareolide having the structure: ##STR24## as follows: Cryptococcus albidus, ATCC 20918 and 
     Cryptococcus albidus, ATCC 20921, 
     capable of producing sclareolide having the structure: ##STR25## from a mixture of sclareol having the structure: ##STR26## and episclareol having the structure: ##STR27## Bensingtonia ciliata, ATCC 20919 capable of producing the diol having the structure: ##STR28## from a mixture of sclareol having the structure: ##STR29## and episclareol having the structure: ##STR30## and Cryptococcus albidus ATCC 20920 capable of producing the cyclic either having the structure: ##STR31## from a mixture of sclareol having the structure: ##STR32## and episclareol having the structure: ##STR33## under aerobic conditions in an aqueous nutrient medium. 
     In still another embodiment the present invention concerns mixtures prepared by cultivating the microorganisms (individually) as follows: 
     Cryptococcus albidus having the identifying characteristics of ATCC 20918; 
     Bensingtonia ciliata having the identifying characteristics of ATCC 20919; 
     Cryptococcus laurentii having the identifying characteristics of ATCC 20920; and 
     Cryptococcus albidus having the identifying characteristics of ATCC 20921 
     under aerobic conditions in an aqueous nutrient medium. 
     A further embodiment of the present invention concerns a process for preparing sclareolide having the structure: ##STR34## which comprises cultivating either the organism Cryptococcus albidus having the identifying characteristics of ATCC 20918 or Cryptococcus albidus having the identifying characteristics of ATCC 20921 under aerobic conditions in an aqueous nutrient medium containing one or more of the compounds having the structures: ##STR35## 
     Still a further embodiment of the present invention concerns a process for preparing the diol having the structure: ##STR36## which comprises cultivating the microorganism Bensingtonia ciliata having the identifying characteristics of ATCC 20919  under aerobic conditions in an aqueous nutrient medium containing one or more of the compounds selected from the group consisting of: 
     (i) sclareol having the structure: ##STR37## (ii) episclareol having the structure: ##STR38## (iii) the acetate having the structure: ##STR39## 
     A further embodiment of the present invention concerns a process for preparing the cyclic ether having the structure: ##STR40## which comprises cultivating the microorganism Cryptococcus laurentii having the identifying characteristics of ATCC 20920 under aerobic conditions in an aqueous nutrient medium containing one or more of the compound&#39;s selected from the group consisting of: 
     (i) Sclareol having the structure: ##STR41## (ii) episclareol having the structure: ##STR42## (iii) the acetate having the structure: ##STR43## 
     The transformation processes involve cultivation of one of the microorganisms: 
     Cryptococcus albidus, ATCC 20918; 
     Bensingtonia ciliata, ATCC 20919; 
     Cryptococcus laurentii, ATCC 20920; or 
     Cryptococcus albidus, ATCC 20921 
     in an aqueous nutrient medium in the presence of one, two or all of the compounds having the structures: ##STR44## Thus, these compounds may be used singularly or as a mixture containing any number of said compounds. 
     Thus, in carrying out the reaction using: 
     Cryptococcus albidus, ATCC 20918 or 
     Cryptococcus albidus, ATCC 20921 
     the following reactions can take place: ##STR45## 
     In carrying out the reaction using Bensingtonia ciliata, ATCC 20919 the following reactions can take place: ##STR46## 
     In carrying out the reaction using Cryptococcus laurentii ATCC 20920, the following reactions can take place: ##STR47## 
     The form in which the microorganisms are used is not critical. They can be used as the culture (suspension), i.e., including the cells and the corresponding nutrient solution, or in the form of cells suspended in a buffer solution. The cells, or an enzyme extract thereof, may be immobilized on a suitable solid support, which may then be used to effect transformations. 
     The suspended culture mixture is prepared by inoculation of a suitable aqueous nutrient medium with the microorganisms. A suitable nutrient medium is one which contains nitrogen sources, inorganic salts, growth factors, the desired substrate(s), and optionally other carbon sources. Some carbon sources suitable for use in the inventive process include, for example, glucose, galactose, L-sorbose, maltose, sucrose, cellosbiose, trehalose, L-arabinose, L-rhamnose, ethanol, glycerol, L-erythrithol, D-mannitol, lactose, melibiose, raffinose, melezitose, starch, D-xylose, D-sorbitol, a methyl-D-glucoside, lactic acid, citric acid and succinic acid. Suitable nitrogen sources include, for example, nitrogen-containing organic substances such as peptone, meat extract, yeast extract, corn steep liquor, casein, urea, amino acids, or nitrogen-containing inorganic compounds such as nitrates, nitrites and inorganic ammonium salts. Suitable inorganic salts include, for example, phosphates of magnesium, potassium, calcium, or sodium. The above mentioned culture medium nutrients may be supplemented with, for example, one or more vitamins of the B group and/or one or more trace minerals such as Fe, Mo, Cu, Mn, and B, as desired. The vitamins or trace minerals are not necessary when a small amount of yeast extract is added to the medium. Addition of an antibiotic, such as chloroamphenicol or chlorotetracycline, may be desirable when bacterial contamination is a problem. The cultivation of the microorganism may be carried out as a stationary culture or as a submerged (e.g., shaking culture, fermentor culture) under aerobic conditions. One may suitably work in the pH range of from about 2.5 to about 9.0, and preferably in the range of from about 3.0 to about 7.5 and most preferably between about 3.0 and 6.5. The pH may be regulated by the addition of inorganic or organic acids, such as hydrochloric acid, acetic acid, and oxalic acid, or by the addition of bases, such as sodium hydroxide, and ammonium hydroxide, or by the addition of a buffer, such as phosphate or phthalate. The incubation temperature should suitably be maintained between about 12° C. and about 33° C., with a range between about 15° C. and about 30° C. being more preferred, and a range between about 18° C. and about 28° C. being most preferred. 
     The process in accordance with this invention may be conveniently carried out by adding one or a mixture of the compounds having the structures: ##STR48## to the nutrient medium at the onset of cultivation, as the sole carbon source. Alternatively, the substrate may be added in combination with another carbon source, such as dextrose, either during cultivation, or when the carbon source is depleted. The only restriction on the concentration of substrate in the culture medium is that of being able to effectively aerate the culture. However, the substrate concentration is preferably in the range of between about 0.1 g/L and about 130 g/L, more preferably in the range of between about 0.5 g/L and about 120 g/L, and most preferably in the range between about 2.5 g/L up to about 100 g/L. The transformation can be suitably carried out under any of the above mentioned conditions. 
     The total transformation time (after initial cultivation period) may vary depending on the composition of the nutrient medium and the substrate concentration. In general, shaking flask cultures require form between about 12 hours and about 264 hours. However, when a fermentor is used the cultivation time may be reduced to about 48 hours or less. 
     The transformation may be carried out using the cells of the microorganism isolated from the culture solution, or with an enzyme extract isolated from the cells in a manner well known to the art. In this case, the transformation can be conveniently carried out in a variety of aqueous nutrient media including, for example, in a buffer solution, in a physiological salt solution, in a fresh nutrient solution, or in water. The isolated cells or enzyme extract may be immobilized on a solid support and the desired transformation effected. Also, transformation of the substrate may be effected by mutants of this organism. Such mutants can be readily obtained by method well known in the art, for example, by exposing the cells to UV or X-rays, or known mutagenic substances, such as, for example, acridine orange. 
     The substrate can be added to the medium as a powder, or a slurry in an emulsifier such as TWEEN® 80 (polyoxyethylenesorbitan mono-oleate), or as a solution in an emulsifier, or as a solution in a hydrophilic solvent such as acetone, methanol, ethanol, ethylene glycol, or dioxan. A surface-active agent, or a dispersion agent can also be added to an aqueous suspension of the substrate, or the substrate can be emulsified using ultrasound. 
     Conventional antifoam agents, such as silicone oils (e.g., UCON), polyalkyleneglycol derivatives, maize oil, or soya oil, can be used to control foaming. 
     The transformation of the substrate can be monitored using standard analytical techniques such as GLC, TLC, HPLC, IR and NMR. If a rapid disappearance of the substrate is observed more substrate can then be added, in order to maximize the transformation capacity of the microorganism. The process is generally terminated when most of the substrate has disappeared from the culture medium. Depending upon the microorganism used, the compound having the structure: ##STR49## or the compound having the structure: ##STR50## 
     or the compound having the structure: ##STR51## may be recovered from the aqueous nutrient medium. 
     The compound having the structure: ##STR52## may be reacted to form the compound having the structure: ##STR53## according to the reaction: ##STR54## The compound having the structure: ##STR55## may be cyclized to the compound having the structure: ##STR56## as stated at lines 52 and 53, at column 8 of U.S. Pat. No. 4,798,799 the specification for which is incorporated by reference herein. The compound having the structure: ##STR57## may also be used as is for its flavor or fragrance value or it may be reduced to the compound having the structure: ##STR58## according to the reaction: ##STR59## 
     Production of the compound having the structure: ##STR60## may take place by (i) first reducing the compound having the structure: ##STR61## to the diol having the structure: ##STR62## or (ii) first reacting the cyclic ether having the structure: ##STR63## via fermentation to produce the diol having the structure: ##STR64## and then rearranging the diol to form the compound having the structure: ##STR65## according to the reaction: ##STR66## or fermenting the compound having the structure: ##STR67## according to the reactions: ##STR68## 
     Preferably, the reduction reaction, to wit: ##STR69## is carried out in the presence of an inert solvent such as toluene using a reducing agent such as VITRIDE®, registered trademark of Eastman Kodak Company of Rochester, N.Y. identifying sodium bis (2-methoxyethoxy) aluminum hydride specifically described in U.S. Pat. No. 3,507,895 the specification for which is incorporated herein by reference. The reaction is preferably carried out at a temperature in the range of from about 70° C. up to about 90° C. for a period of between about one hour and about three hours. 
     The rearrangement reaction, to wit: ##STR70## is preferably carried out in basic media, e.g., aqueous alkali metal hydroxide such as aqueous potassium hydroxide or aqueous sodium hydroxide as more specifically described in Example VIII, infra. 
     Isolation and purification of the compounds having the structures: ##STR71## from the fermentation broths may be achieved by conventional techniques including, filtration or centrifugation, solvent extraction, distillation, crystallization, and the like. 
     The compound having the structure: ##STR72## may be converted to the compound having the structure: ##STR73## by conventional cyclization methods well known in the art as specified at lines 58-68, at column 8 of U.S. Pat. No. 4,798,799 and at column 9, lines 1 and 2 of U.S. Pat. No. 4,798,799. 
     Each of the microorganisms employed in this invention was isolated from a soil sample obtained from various geographical locations. Each of the strains has been deposited with the American Type Culture Collection with the accession numbers as follows: 
     Cryptococcus albidus, ATCC 20918; 
     Bensingtonia ciliata, ATCC 20919; 
     Cryptococcus laurentii, ATCC 20920; and 
     Cryptococcus albidus, ATCC 20921. 
     The organisms Bensingtonia ciliata and Cryptococcus laurentii were also studied by Centralbureau voor Schimmel Cultures (CBS). CBS assigned both of these organisms the name: 
     Lecythophere hoffmannii (van Beijma), W. Gams (synonym Phialophora hoffmannii) 
     because this is a filamentous fungus according to CBS. 
     The organisms Cryptococcus albidus, ATCC 20918 was also studied by Centraalbureau voor Schimmelkultur (CBS). CBS named this culture to be Cryptococcus albidus var. albidus (Saito) Skinner. 
     the organism Cryptococcus albidus, ATCC 20918 is described as follows: 
     Morphology: Growth in liquid medium showed unipolar budding cells. A film stayed on the surface of the liquid while heavy sediment was observed. Growth on solid agar was unicellular with colonies being while to slightly pinkish, very shiny, bright, slimy, round and with smooth borders. No pseudohyphae were formed on corn meal aga. 
     Physiology and Biochemistry: 
     
         ______________________________________                                    
                Carbon Assimilation:                                      
                (Growth)                                                  
______________________________________                                    
Carbon Assimilation:                                                      
(Growth)                                                                  
Glucose        +      D-ribose        U                                   
Galactose      +      L-rhamnose      +                                   
L-sorbose      +      D-glucosamine   +                                   
Maltose        +      Ethanol         U                                   
Sucrose        +      Erythritol      -                                   
Cellobiose     +      Glycerol        U                                   
Trehalose      +      Adonitol (Ribitol)                                  
                                      +                                   
Lactose        +      Dulcitol (Galactitol)                               
                                      +                                   
Melibiose      -      D-mannitol      +                                   
Raffinose      +      D-sorbitol (glucitol)                               
                                      +                                   
Melezitose     +      a-methyl-D-glucoside                                
                                      +                                   
Inulin         -      Salicin         +                                   
Soluble Starch -      Inositol        +                                   
D-xylose       -      Lactic acid     -                                   
L-arabinose    -      Citric acid     -                                   
D-arabinose    -                                                          
Succinic acid  +                                                          
Growth at 30° C.                                                   
               +                                                          
Growth at 37° C.                                                   
               -                                                          
Vitamin free growth                                                       
               -                                                          
Splitting of arbutin                                                      
               +                                                          
Nitrogen assimilation:                                                    
NH.sub.4 NO.sub.3                                                         
               +                                                          
KNO.sub.3      +                                                          
NO.sub.2       +                                                          
Ethylamine     +                                                          
Fermentation (Acid from):                                                 
Glucose        -                                                          
Galactose      -                                                          
Maltose        -                                                          
Sucrose        -                                                          
Lactose        -                                                          
Raffinose      -                                                          
Melibiose      -                                                          
Inulin         -                                                          
Cellobiose     -                                                          
Melezitose     -                                                          
Starch         -                                                          
Trehalose      -                                                          
______________________________________                                    
 Note: U = undecided or questionable                                      
 
    
     The organism Bensingtonia ciliata, ATCC 20919 is described as follows: 
     Morphology: On Yeast Maintenance Broth (ATCC medium #200) cells are globose with 1-3 buds per cell. On solid medium cells become filamentous with ballistospores produced. Colonies are tan-salmon in color, flat, dull, with smooth edges. On Corn Meal Agar (ATCC medium #307) true mycelium was noticed after 3 weeks. 
     Physiology: 
     
         ______________________________________                                    
                 Carbon                                                   
                 Assimilation:                                            
______________________________________                                    
Carbon Assimilation:                                                      
Glucose        +       D-ribose       -                                   
Galactose      +       L-rhamnose     +                                   
L-sorbose      +       D-glucosamine  +                                   
Maltose        +       Ethanol        W                                   
Sucrose        +       Erythritol     +                                   
Cellobiose     +       Glycerol       +                                   
Trehalose      +       Adonitol (Ribitol)                                 
                                      W                                   
Lactose        -       Dulcitol (Galactitol)                              
                                      W                                   
Melibiose      +       D-mannitol     W                                   
Raffinose      +       D-sorbitol (glucitol)                              
                                      W                                   
Melezitose     +       a-methyl-D-glucoside                               
                                      W                                   
Inulin         -       Salicin        W                                   
Soluble Starch +       Inositol       -                                   
D-xylose       +       Lactic acid    -                                   
L-arabinose    +       Citric acid    -                                   
D-arabinose    -       Succinic acid  W                                   
Vitamin free growth                                                       
               -                                                          
Nitrogen assimiliation:                                                   
NH.sub.4 NO.sub.3                                                         
               W                                                          
Growth at elevated temp:                                                  
30° C.  W/-                                                        
37° C.  -                                                          
Bensintonia ciliata,                                                      
ATCC 20919 (Cont&#39;d.)                                                      
Taxonomic Description:                                                    
Bensingtonia ciliata                                                      
C.T. Ingold                                                               
Hyphomycete                                                               
(Fungi Imperfecti)                                                        
______________________________________                                    
 Note: W = weak                                                           
 
    
     A ballistosporic fungus (Spores forcibly abjected). 
     The ballistospores are colorless, ovoid, 2×5 c, mostly 8×5 c in liquid culture, pointed at the apex and with a flattened base. 
     The ballistospores germinate with the formation of yeast-like blastospores that upon repeated spore formation result in typical yeast colonies (see photo). 
     Some ballistospores germinate with the formation of short hyphae that produce ballistospores and with repeated spore discharge result in colonies entirely of the hyphae type (see photo). 
     Evaluation from the following media: 
     
         ______________________________________                                    
ATCC medium                                                               
______________________________________                                    
#307          Corn Meal Agar (Difco 0386) and                             
              1/2 strength Corn Meal Agar                                 
#200          Yeast Malt Agar (Difco 0712)                                
#331          Neuropora Agar (Difco 0321)                                 
#1245         YEPD                                                        
#324          Malt Extract Agar (Difco 0024)                              
#336          Potato Dextrose Agar                                        
#343          V-8 Juice Agar                                              
______________________________________                                    
 
    
     REFERENCES 
     1. Ingold, C. T. (1986) Bensingtonia ciliata Gen. et. sp. nov., Ballistoporic Fungus. Trans. Br. Mycol. Soc. 86(2): 325-328. 
     2. Ingold. C. T. (1988) Further Observations on Bensingtonia ciliata. Trans. Br. Mycol. Soc. 91(1): 162-166. 
     Cryptococcus laurentii, ATCC 20920 is described as follows: 
     Physiology and Biochemistry: 
     
         ______________________________________                                    
Carbon Assimilation:                                                      
(Growth)                                                                  
Glucose      +        D-ribose      + weak                                
Galactose    +        L-rhamnose    +                                     
L-sorbose    + weak   D-glucosamine V                                     
Maltose      +        Ethanol       +                                     
Sucrose      +        Erythritol    +                                     
Cellobiose   +        Glycerol      +                                     
Trehalose    +        Adonitol (Ribitol)                                  
                                    + weak                                
Lactose      + weak   Dulcitol      V                                     
                      (Galactitol)                                        
Melibiose    +        D-mannitol    +                                     
Raffinose    +        D-sorbitol (glucitol)                               
                                    +                                     
Melezitose   +        a-methyl-D-   +                                     
                      glucoside                                           
Inulin       +        Salicin       +                                     
Soluble Starch                                                            
             +        Inositol      +                                     
D-xylose     +        Lactic acid   +                                     
L-arabinose  +        Citric acid   +                                     
D-arabinose  + weak   Succinic acid +                                     
Nitrogen Assimilation:                                                    
NH.sub.4 NO.sub.3                                                         
             +                                                            
KNO.sub.3    +                                                            
NO.sub.2     +                                                            
Ethylamine   +                                                            
Vitamin free growth                                                       
             +                                                            
Splitting of arbutin                                                      
             +                                                            
Cryptococcus laurentii,                                                   
ATCC 20920 (Cont&#39;d.)                                                      
Fermentation                                                              
(Gas Production):                                                         
Glucose      -                                                            
Galactose    -                                                            
Maltose      -                                                            
Sucrose      -                                                            
Lactose      -                                                            
Raffinose    -                                                            
Melibiose    -                                                            
Inulin       -                                                            
Cellobiose   -                                                            
Melezitose   -                                                            
Starch       -                                                            
Trehalose    -                                                            
______________________________________                                    
 Note: V = variable.                                                      
 
    
     Morphology: Pink slimy colonies; round budding cells, heavy sediment in flask; no Dalmau plate thin hyphae were formed. 
     Reference: C. P. Kurtzman, Mycologia 65; p. 388-395, 1973. 
     Cryptococcus albidus, ATCC 20921 is described as follows: 
     Physiology and Biochemistry: 
     
         ______________________________________                                    
Carbon Assimilation:                                                      
______________________________________                                    
Carbon Assimilation:                                                      
Growth                                                                    
Glucose        +      D-ribose        -                                   
Galactose      +      L-rhamnose      +                                   
L-sorbose      +      D-glucosamine   +                                   
Maltose        +      Ethanol         V                                   
Sucrose        +      Erythritol      -                                   
Cellobiose     +      Glycerol        V                                   
Trehalose      +      Adonitol (Ribitol)                                  
                                      V                                   
Lactose        +      Dulcitol (Galactitol)                               
                                      +                                   
Melibiose      -      D-mannitol      +                                   
Raffinose      +      D-sorbitol (glucitol)                               
                                      +                                   
Melezitose     +      a-methyl-D-glucoside                                
                                      +                                   
Inulin         -      Salicin         +                                   
Soluble Starch +      Inositol        +                                   
D-xylose       +      Lactic acid     -                                   
L-arabinose    +      Citric acid     -                                   
D-arabinose    +      Succinic acid   +                                   
Nitrogen Assimilation:                                                    
NH.sub.4 NO.sub.3                                                         
               +                                                          
KNO.sub.3      +                                                          
NO.sub.2       +                                                          
Ethylamine     +                                                          
Vitamin free growth                                                       
               +                                                          
Splitting of arbutin                                                      
               +                                                          
Cryptococcus albidus,                                                     
ATCC 20921 (Cont&#39;d)                                                       
Fermentation                                                              
(Gas Production):                                                         
Glucose        -                                                          
Galactose      -                                                          
Maltose        -                                                          
Sucrose        -                                                          
Lactose        -                                                          
Raffinose      -                                                          
Melibiose      -                                                          
Inulin         -                                                          
Cellobiose     -                                                          
Melezitose     -                                                          
Starch         -                                                          
Trehalose      -                                                          
______________________________________                                    
 Note: V =  variable.                                                     
 
    
     Morphology: Yellowish-tan, slimy colonies; round budding cells; heavy sediment in the flask, no pseudomycelium or true mycelium. 
     The following examples serve to illustrate embodiments of the invention as it is now preferred to be practiced but in no way are said Examples meant to limit the scope thereof. Unless otherwise stated weights are in grams, temperatures are in degrees centigrade and pressure is in mm/Hg. 
     EXAMPLE I 
     Effect of pH on Conversion of Sclareol to Sclareolide Using Cryptococcus Albidus (Saito [Skinner Var. Albidus])(ATCC 20918) 
     Reactions ##STR74## 
     The following medium was prepared: 
     
         ______________________________________                                    
       NH.sub.4 NO.sub.3                                                  
                      0.1%                                                
       KH.sub.2 PO.sub.4                                                  
                      0.1%                                                
       MgSO.sub.4.7H.sub.2 O                                              
                      0.05%                                               
       Yeast Extract  0.2%                                                
______________________________________                                    
 
    
     Eleven 500 ml flasks each containing 100 ml of medium and 1 g of sclareol in TWEEN® 80 (ratio of sclareol of TWEEN® 80=2:1). 
     Each flask was inoculated with 5 ml of a 24 hour culture grown on dextrose at 25° C. and 150 rpm. Product and substrate were monitored by TLC against a known standard. 
     The &#34;substrate&#34; is sclareol which is an 80:20 mixture of the compound having the structure: ##STR75## and the compound having the structure: ##STR76## the &#34;Intermediate&#34; is the compound having the structure: ##STR77## The &#34;product&#34; is sclareolide, the compound having the structure: ##STR78## 
     
                       TABLE I                                                     
______________________________________                                    
Flask         DURATION                                                    
No.    pH     24 Hours    48 Hours  72 Hours                              
______________________________________                                    
1      2.5    TP + S + TI P + S + I P + S + TI                            
2      3.0    P + TS + I  P + S + I P                                     
3      3.5    P + TS + TI P + TI    P                                     
4      4.0    P + TS + I  P + TI    P + TI                                
5      4.5    P + TS + I  P + I     P + TI                                
6      5.0    P + TS + I  P + I     P + TI                                
7      7.0    P + S + I   P + I     P + I                                 
8      7.5    P + S + I   P + I     P + I                                 
9      8.0    P + S + I   P + TI + I                                      
                                    P + I                                 
10     8.5    P + S + I   P +  TS + I                                     
                                    P + I                                 
11     9.0    P + S + I   P + TS + I                                      
                                    P + I                                 
______________________________________                                    
 TS: Trace Substrate                                                      
 S: Sustrate                                                              
 TP: Trace Product                                                        
 P: Product                                                               
 I: Intermediate                                                          
 TI: Trace Intermediate                                                   
 
    
     EXAMPLE II 
     Preparation of Diol Intermediate 
     Reactions ##STR79## 
     During screening of ten soil samples from Greenwood Forest, Barnegat Township, N.J., several flasks showed a spot on the TLC corresponding to the compound having the structure: ##STR80## The following medium was prepared: 
     
         ______________________________________                                    
       NH.sub.4 NO.sub.3                                                  
                      0.2%                                                
       KH.sub.2 PO.sub.4                                                  
                      0.1%                                                
       MgSO.sub.4.7H.sub.2 O                                              
                      0.05%                                               
       Yeast Extract  0.2%                                                
       Dextrose       1.0%                                                
______________________________________                                    
 
    
     Into a 500 ml flask was placed 10 ml medium and 1.0 g of a 50:50 mixture of sclareol powder:TWEEN® 80. The flask was inoculated with 400 microliters of isolate of Bensingtonia ciliata, ATCC 20919. After one week at 25° C. and 150 rpm, the resulting product was extracted with 330 ml of ethyl acetate and the extract dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator. The residue was dissolved in hot hexane and ethyl acetate. The resulting extract was permitted to evaporate for a period of 24 hours whereupon were obtained pure crystals (350 mg) of the compound having the structure: ##STR81## were recovered. 
     FIG. 2 is the NMR spectrum of the compound having the structure: ##STR82## 
     EXAMPLE III 
     Preparation of Sclareolide Using Cryptococcus albidus (Saito [Skinner Var. Albidus]), ATCC 20918 
     Reactions ##STR83## 
     
         ______________________________________                                    
MEDIUM         FERMENTER PARAMETERS                                       
______________________________________                                    
NH.sub.4 NO.sub.3                                                         
           0.2%    Temperature:    25° C.                          
KH.sub.2 PO.sub.4                                                         
           0.1%    Aeration:       1.0 l/min.                             
MgSO.sub.4.7H.sub.2 O                                                     
           0.05%   Agitation:      430 rpm                                
Yeast Extract                                                             
           0.2%    pH = 5.8 con-                                          
Antifoam  10.0 g   trolled with 25% NaOH                                  
d-H.sub.2 O                                                               
           8.5 l   Duration:       4 days                                 
______________________________________                                    
 
    
     SUBSTRATE PREPARATION 
     500 Grams of sclareol, 250 grams TWEEN® 80 and 1125 grams of water were placed in a blender and mixed for 5 minutes to form an emulsion. 
     Fermenter containing above medium was sterilized at 121° C. for 30 minutes and cooled to 25° C. This fermenter was inoculated with 300 ml of 24 hour grown cells of Cryptococcus albidus (Saito [Skinner var. albidus]), ATCC 20918. 600 Grams of sclareol emulsion were added at time of inoculation and 600 g portions of emulsion were added at 24, 48, and 72 hours. At 96 hours, the contents of the fermenter were filtered through a 400 mesh sieve. The resulting crude solid product was dissolved in IPA, filtered and the solution concentrated to crystallize; 430 g of pure sclareolide were obtained. 
     FIG. 1 is a GC-MS spectrum for the initial reaction mass in this Example III. The peak indicated by reference numeral 21 is the peak for the sclareol which is a mixture (80:20) of the compounds having the structures: ##STR84## The peak indicated by reference numeral 20 is the peak for the internal standard, the compound having the structure: ##STR85## 
     FIG. 3 is the NMR spectrum for the sclareolide produced according to this Example III having the structure: ##STR86## 
     EXAMPLE IV 
     Production of Sclareolide From Sclareol Using Cryptococcus albidus, ATCC 20918 
     Same medium and parameters were used in Example III. The mode of substrate preparation and addition were changed. 
     One hundred sixty grams of sclareol powder and 80 g of TWEEN® 80 were added to the medium prior to sterilization. 
     Additional substrate was prepared by mixing finely ground sclareol (2 parts) and TWEEN® 80 (1 part) to form a paste. Two hundred twenty five gram portions of this paste were added at 24, 48, and 72 hours after inoculation. 
     A total of 655 g of crude sclareolide having a purity of 67.34% was obtained. 
     EXAMPLE V 
     Preparation of Sclareolide From Sclareol Using Cryptococcus albidus, ATCC 20921 
     The same medium and parameters were used as in Example IV. The amount of substrate and agitation were changed. 
     One hundred fifty grams of sclareol and 75 grams of TWEEN® 80 were added to the medium prior to sterilization. 
     Only 241 grams of substrate in paste form were added 24 hours after inoculation. Agitation was started at 430 rpm and later increased to 630 rpm. 
     A total of 491 g crude sclareol having a purity of 44% was obtained, a 94.7% mole/mole conversion. 
     EXAMPLE VI 
     Preparation of Sclareolide From Sclareol, Diol Intermediate and Diol Acetate 
     Reactions ##STR87## The following medium was prepared: 
     
         ______________________________________                                    
       NH.sub.4 NO.sub.3                                                  
                      0.2%                                                
       KH.sub.2 PO.sub.4                                                  
                      0.1%                                                
       MgSO.sub.4.7H.sub.2 O                                              
                      0.05%                                               
       Yeast Extract  0.2%                                                
       Dextrose       0.5%                                                
______________________________________                                    
 
    
     A 10 liter fermenter was used with the following operating conditions: 
     
         ______________________________________                                    
Temperature:      25° C.                                           
pH:               6.0                                                     
Agitation:        430 rpm                                                 
Sterilization:    121° C. for 30 mins.                             
______________________________________                                    
 
    
     The fermenter was inoculated with 100 ml of a 48 hour shake flask culture grown on the same medium at 25° C. and 150 rpm. 
     After 24 hours growth, 200 ml aliquots were removed from the fermenter and centrifuged at 10,000 rpm for 10 minutes in a refrigerated centrifuge. The cells in each tube were washed twice with Butterfield&#39;s buffered phosphate. 
     BUFFER PREPARATION 
     Stock Solution: 
     
         ______________________________________                                    
Monopotassium hydrogen phosphate                                          
                          34.0 g                                          
Distilled water          500.0 ml                                         
______________________________________                                    
 
    
     Adjust to pH 7.2 with about 175 ml 1.0 N sodium hydroxide solution; dilute to one liter and store. 
     Diluent: 
     Dilute 1.25 ml stock solution to 1 liter with distilled water. Prepare dilution blanks in suitable containers. Sterilize at 121° C. for 15 minutes. 
     The cells were then taken up in 100 ml of this buffer. The pH was adjusted to 6 and then the material was transferred to a 500 ml flask. 
     Compounds tested: 
     a. Sclareol paste in TWEEN® 80 (2:1)=Compound 1. 
     b. Acetate, compound having the structure: ##STR88##  mix in TWEEN® 80 (1:1)=Compound 2. c. Diol paste, compound having the structure: ##STR89##  in TWEEN® 80 (1:1)=Compound 3. 
     300 ml Flasks containing 100 ml of buffer and cells (resting cells) were placed in shaker incubator at 25° C. and 150 rpm and samples were analyzed using TLC at 24, 48, and 72 hours against the standard known compound. In the following table: 
     P=Product, sclareolide having the structure: ##STR90## S=Substrate, one of the compounds having the structure: ##STR91## I=Intermediate, compound having the structure: ##STR92## T=Trace. 
     
                       TABLE II                                                    
______________________________________                                    
Flask               Duration                                              
No.    Substrate    24 Hours   48 Hours                                   
                                      72 Hours                            
______________________________________                                    
1      1 g of Compound                                                    
                    P + S + I  P + I  P + I                               
       (1) added                                                          
2      1 g of Compound                                                    
                    P + TS + I P + I  P + TI                              
       (2) added                                                          
3      1 g of Compound                                                    
                    TP + S     P + S  P + TS                              
       (3) added                                                          
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     EXAMPLE VII 
     Formation of Diol Intermediate From Sclareol Using Bensingtonia ciliata, ATCC 20919 
     An experiment using the same procedure as Example II was carried out with the following specifications: 
     
         ______________________________________                                    
Organism: Bensingtonia celiata, ATCC 20919 (IFF-8268C)                    
MEDIUM          FERMENTER PARAMETERS                                      
______________________________________                                    
NH.sub.4 NO.sub.3                                                         
            40.0 g  Temperature:    25° C.                         
Yeast Extract                                                             
            40.0 g  Aeration:       2 l/min.                              
KH.sub.2 PO.sub.4                                                         
            20.0 g  Agitation:      300 rpm                               
MgSO.sub.4.7H.sub.2 O                                                     
            10.0 g  pH = 6.0 con-                                         
Sclareol   160.0 g  trolled with 25% NaOH                                 
TWEEN ® 80                                                            
            80.0 g  Duration:       15 days                               
d-H.sub.2 O                                                               
            19.0 l                                                        
______________________________________                                    
 
    
     Fermenter containing above medium was sterilized at 121° C. for 30 minutes and cooled at 25° C. This fermenter was inoculated with 1 liter of 48 hour grown culture of Bensingtonia ciliata, ATCC 20919. After 15 days of incubation, substrate was converted to product. The contents of the fermenter were filtered through a 400 mesh sieve. The resulting crude solid product was air dried. Total of 101.7 g product was obtained. 
     EXAMPLE VIII 
     Preparation of Decahydro-3A, 6,6,9A-Tetramethyl Naphtho[2-1-Furan 
     Reactions ##STR93## 
     Into a 3 neck 1 liter reaction vessel equipped with mechanical stirrer, thermometer and additional funnel and reflux condenser is added 124 ml VITRIDE® registered trademark of Eastman Kodak Company defining sodium bis (2-methoxyethoxy) aluminum hydride. 
     To the VITRIDE® is added with stirring 500 ml toluene. 50 Grams of sclareolide having the structure: ##STR94## prepared according to the procedure of Example VI is dissolved in 120 ml toluene. The sclareol/toluene solution is then added via addition funnel dropwise over a period of 0.5 hours to the reaction mass and the temperature of the reaction mass is permitted to rise to 55° C. 
     The reaction mass is then heated to 80° C. for a period of two hours. 
     The reaction mass is then worked up by adding thereto 600 ml of 5% aqueous sodium hydroxide slowly while cooling with ice. 
     The toluene layer is removed and washed with saturated aqueous sodium chloride followed by methyl alcohol and a sodium bicarbonate saturated solution (equal volumes). 
     The toluene layer is then concentrated yielding a solid weighing 43.34 grams (86% yield). 
     The resulting solid has the structure: ##STR95## 
     1.5 Grams of the resulting compound is admixed with a solution of 12.0 grams of potassium hydroxide in 30 ml water. The resulting mixture is placed in a flask equipped with heater, stirrer and reflux condenser and refluxed at 80° C. for a period of three hours. At the end of the three hour period, the resulting product is fractionally distilled at a vapor temperature of 162°-164° C. and a vacuum of 15 mm/Hg. yielding substantially pure compound having the structure: ##STR96## 
     EXAMPLE IX 
     Preparation of Cyclic Ether 
     Reactions ##STR97## 
     The following medium was prepared: 
     
         ______________________________________                                    
       NH.sub.4 NO.sub.3                                                  
                      0.2%                                                
       KH.sub.2 PO.sub.4                                                  
                      0.1%                                                
       MgSO.sub.4.7H.sub.2 O                                              
                      0.05%                                               
       Yeast Extract  0.2%                                                
       Dextrose       1.0%                                                
______________________________________                                    
 
    
     Into a 500 ml flask was placed 100 ml medium and 1.0 g of a 50:50 mixture of sclareol powder:TWEEN200  80. The flask was inoculated with 400 microliters of isolate of Cryptococcus laurentii, ATCC 20920. After one week at 25° C. and 150 rpm (pH=6), the resulting product was extracted with 330 ml ethyl acetate and the extract dried over anhydrous sodium sulfate. The solvent was removed on a rotary evaporator. 
     The residue was dissolved in hot hexane and ethyl acetate. The resulting extract was permitted to evaporate for a period of 24 hours whereupon an oil was collected having the structure: ##STR98## 
     FIG. 4 is the NMR spectrum of the compound having the structure: ##STR99##