Abstract:
A method for preparing  13 C labeled docosahexaenoic acid (DHA) represented by Formula A: The method comprises the conversion of 2-pentyn-1-ol to  13 C labeled DHA by reaction with propargyl alcohol,  13 C labeled propargyl alcohol and methyl pent-4-ynoate. The various steps involved include tosylation, coupling, bromination, selective hydrogenation and ester hydrolysis to obtain the final product.

Description:
FIELD OF INVENTION 
       [0001]    The present invention relates to methods for the chemical synthesis of fatty acids, and specifically, to methods for the chemical synthesis of  13 C labeled fatty acids such as docosahexaenoic acid. 
       BACKGROUND OF THE INVENTION 
       [0002]    Docosahexaenoic acid (DHA) is an omega-3 unsaturated fatty acid, containing a chain-terminating carboxylic acid group and six cis-double bonds in a 22-carbon straight chain. Its trivial name is cervonic acid, its systematic name is all-cis-docosa-4,7,10,13,16,19-hexa-enoic acid, and its shorthand name is 22:6w3 in the nomenclature of fatty acids. Its chemical structure can be represented as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0003]    DHA is essential for the growth, functional development and healthy maintenance of brain function and is required throughout life from infancy through aging (Horrocks, L. A. and Y. K. Yeo. Pharmacol. Res. 40(3):211-225 (1999)). It is derived from the essential precursor linolenic acid (LNA, 18:3w3). DHA is the main end-product of LNA after successive desaturations and elongations, a metabolic cascade that is assumed to be weak in humans (Burdge G C, Jones A E, Wootton S A (2002) Eicosapentaenoic and docosapentaenoic acids are the principal products of alphalinolenic acid metabolism in young men. Br J Nutr 88:355-363; Brenna J T, Salem N Jr, Sinclair A J, Cunnane S C (2009) Alphalinolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humans. Prostaglandins Leukot Essent Fatty Acids 80:85-91). 
         [0004]    DHA has been attributed to physiological effects such as blood lipid reduction, anticoagulant effect, carcinostatic effect, and improvement in visual functions. DHA was found to inhibit growth of human colon carcinoma cells (Kato T, Hancock R L, Mohammadpour H, McGregor B, Manalo P, Khaiboullina S, Hall M R, Pardini L, Pardini R S (2002). “Influence of omega-3 fatty acids on the growth of human colon carcinoma in nude mice”. Cancer Lett. 187 (1-2): 169-77). Dietary DHA may reduce the risk of heart disease by reducing the level of blood triglycerides in humans. Further, DHA deficiencies are associated with fetal alcohol syndrome, attention deficit hyperactivity disorder, cystic fibrosis, phenylketonuria, unipolar depression, aggressive hostility and adrenoleukodystrophy. In contrast, increased intake of DHA has been shown to be beneficial or have a positive effect in inflammatory disorders (e.g., rheumatoid arthritis), Type II diabetes, hypertension, atherosclerosis, depression, myocardial infarction, thrombosis, some cancers and for prevention of the onset of degenerative disorders such as Alzheimer&#39;s disease (U.S. Pat. No. 7,550,286 B2). 
         [0005]    Due to its various physiological effects, DHA is also administered as a dietary supplement. 
         [0006]    However, the mechanism of action as well as the fate of DHA in the body is still not completely understood. Therefore, it is of interest to study the metabolism of DHA in the body. Also, if DHA is to be administered as a dietary supplement, the fate of the DHA supplement administered needs to be known. 
         [0007]    Thus developing stable metabolic tracers for DHA is needed. To this end,  13 C labeled DHA has been utilized as a metabolic tracer to study the uptake and metabolism of DHA. Further,  13 C labeled DHA was also used to study the placental transfer of DHA from mother to fetus (In vivo investigation of the placental transfer of (13)C-labeled fatty acids in humans. Larquè E, Demmelmair H, Berger B, Hasbargen U and Koletzko B.; J Lipid Res. 44(1):49-55 (2003)). 
         [0008]    Currently known methods of producing  13 C labeled DHA include biosynthetic production. In such methods, micro-organisms capable of producing DHA are cultured on  13 C labeled precursors for DHA such as  13 C glucose,  13 C malonyl CoA (Biosynthetic production of universally (13)C-labeled polyunsaturated fatty acids as reference materials for natural health product research. Le P M, Fraser C, Gardner G, Liang W W, Kralovec J A, Cunnane S C, Windust A J, Anal Bioanal Chem. 389(1):241-9 (2007)). The DHA synthesized is then extracted from such cultures. Another way of studying metabolism of  13 C labeled DHA is by synthesis of phospholipids such as phosphatidyl choline in which  13 C labeled DHA is present at the sn-2 position. This  13 C DHA is released from the phospholipid by phospholipase A2 present in the body. Then the fate of DHA can be followed (Blood compartmental metabolism of docosahexaenoic acid (DHA) in humans after ingestion of a single dose of [(13)C]DHA in phosphatidylcholine. Lemaitre-Delaunay D, Pachiaudi C, Laville M, Pousin J, Armstrong M, Lagarde M., J Lipid Res., 40(10):1867-74 (1999)).  13 C labeled DHA can also similarly be incorporated into triglycerides (Human plasma albumin transports [13C]docosahexaenoic acid in two lipid forms to blood cells. Brossard N, Croset M, Normand S, Pousin J, Lecerf J, Laville M, Tayot J L, Lagarde M. J Lipid Res. 38(8):1571-82. (1997)). However, synthesis of such phospholipids also depends on micro-organisms capable of synthesizing the phospholipid. Thus, the methods known so far are expensive and cumbersome as they involve complex extraction steps. Also, desired product is obtained in low yields. 
       SUMMARY OF THE INVENTION 
       [0009]    There is accordingly a need for new and improved methods for synthesizing  13 C labeled fatty acids, such as but not limited to DHA. The present invention aims to provide such a method. 
         [0010]    In an aspect of the invention, a process is provided for preparing a  13 C labeled fatty acid represented by Formula (i): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0011]    wherein L is —[CH═CH—CH 2 ]—, and n is 0 to 6, preferably 1 to 4, more preferably 3, and the compound comprises at least one  13 C labeled carbon residue. The process comprises: 
         [0000]    (a) converting 2-pentyn-1-ol into a tosylate of Formula (ii), e.g by reaction with tosyl chloride (TsCl): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (b) reacting the compound of Formula (ii) with propargyl alcohol in a coupling reaction, and optionally carrying out one or more additional steps of brominating followed by coupling with propargyl alcohol, to obtain a compound represented by Formula (iii): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein M is —[C≡C—CH 2 ]—, and n is as defined above,
 
(c) carrying out a selective reduction of the compound represented by Formula (iii) to obtain a compound represented by Formula (iv):
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein L and n are as defined above,
 
(d) brominating the compound of Formula (iv) to produce a compound represented by Formula (v):
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein L and n are as defined above,
 
(e) coupling the compound represented by Formula (v) with methyl pent-4-ynoate to obtain a compound represented by Formula (vi):
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (f) carrying out a selective reduction of the compound represented by Formula (vi) to obtain a compound represented by Formula (vii): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and
 
(g) ester-hydrolyzing the compound represented by Formula (vii) to obtain the compound represented by Formula (i),
       wherein the propargyl alcohol used in at least one of the coupling reactions carried out in (b) is labeled with  13 C at C 1 , C 2 , or C 3  of the propargyl alcohol, or a combination thereof.       
 
         [0013]    In one embodiment of the invention, a process is provided for preparing a  13 C labeled DHA represented by Formula A: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where * represents a  13 C labeled carbon residue. 
         [0014]    In this process, 2-pentyn-1-ol of Formula 1: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    is reacted with tosyl chloride (TsCl) to obtain a compound represented by Formula 2: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0015]    In certain non-limiting embodiments, the compound of Formula 2 can be obtained with a yield of 60-68%. 
         [0016]    The compound of Formula 2 is then coupled with propargyl alcohol to produce a compound represented by Formula 3: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0017]    In certain non-limiting embodiments, the compound of Formula 3 can be obtained with a yield of 93-99%. 
         [0018]    The compound of Formula 3 is then reacted with PBr 3  to produce a compound represented by Formula 4: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and the resulting compound is coupled with propargyl alcohol to obtain a compound represented by Formula 5: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0019]    In certain non-limiting embodiments, the compound of Formula 5 is obtained with a yield of 52-62%. 
         [0020]    The compound represented by Formula 5 is reacted with PBr 3  to a produce a compound represented by Formula 6: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and the resulting compound is coupled with propargyl alcohol to obtain a compound represented by Formula 7: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0021]    In certain non-limiting embodiments, the compound of Formula 7 is obtained with a yield of 27-37%. 
         [0022]    The resulting compound of Formula 7 is reacted with PBr 3  to produce a compound represented by Formula 8: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and the resulting compound is coupled with  13 C labeled propargyl alcohol to obtain a compound represented by Formula 9: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where * represents a  13 C labeled carbon residue. 
         [0023]    In certain non-limiting embodiments, the compound of Formula 9 is obtained with a yield of 45-55%. 
         [0024]    Selective reduction of the compound represented by Formula 9 is then carried out to obtain a compound represented by Formula 10: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0025]    In certain non-limiting embodiments, the compound of Formula 10 is obtained with a yield of 63-73%. 
         [0026]    The compound of Formula 10 is then reacted with PBr 3  to produce a compound represented by Formula 11: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0027]    The compound represented by Formula 11 is then reacted with methyl pent-4-ynoate in a coupling reaction to produce a compound represented by Formula 12: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0028]    In certain non-limiting embodiments, the compound of Formula 12 is obtained with a yield of 49-59%. 
         [0029]    Selective reduction of the compound represented by Formula 12 is then carried out to produce a compound represented by Formula 13: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0030]    In certain non-limiting embodiments, the compound of Formula 13 is obtained with a yield of 75-85%. 
         [0031]    Finally, the compound represented by Formula 13 is ester-hydrolyzed to produce the compound of Formula A. In certain non-limiting embodiments, the compound of Formula 1 is obtained with a yield of 82-92%. 
         [0032]    In a preferred, yet non-limiting embodiments of the synthetic process, one or more of the bromination reactions for producing compounds of Formulas 4, 6, 8 and 11 are carried out in presence of pyridine and dichloromethane. The temperature of the bromination reaction is also preferred to be from about 0° C. to about room temperature. 
         [0033]    In yet another preferred embodiment, which is non-limiting, one or more of the coupling reactions for production of the compounds represented by Formulas 5, 7, 9 and 12 are carried out in the presence of CuI, tetrabutylammonium iodide (TBAI) in dry N,N-dimethylformamide (DMF). The temperature of the coupling reaction is also preferred to be from about 0° C. to about room temperature. 
         [0034]    In further non-limiting embodiments, one or more of the hydrogenation reactions for production of the compounds represented by Formulas 10 and 13 are carried out at about room temperature, in an H 2  atmosphere, and using a catalyst such as but not limited to Lindlar&#39;s catalyst. 
         [0035]    In another non-limiting embodiment, LiOH is used for ester hydrolysis of the compound represented by Formula 13, in the presence of THF/H 2 O (3:1), to obtain the compound represented by Formula 1. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0036]    These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein: 
           [0037]      FIG. 1  illustrates the NMR spectra of the  13 C labeled DHA of Formula 1, prepared by an embodiment of a synthetic process of the present invention; 
           [0038]      FIG. 2  illustrates the LC chromatogram of the  13 C labeled DHA of Formula 1, prepared by an embodiment of a synthetic process of the present invention; and 
           [0039]      FIG. 3  illustrates the LC-MS results of the  13 C labeled DHA of Formula (A shows the LC trace, and B shows the MS results), prepared by an embodiment of a synthetic process of the present invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0040]    The present invention provides a useful synthetic process for preparing  13 C labeled fatty acids. The process involves preparing a  13 C labeled fatty acid represented by Formula (i): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein L is —[CH═CH—CH 2 ]—, and n is 0 to 6, preferably 1 to 4, more preferably 3, and the fatty acid comprises at least one  13 C labeled carbon residue. The process comprises:
 
(a) converting 2-pentyn-1-ol into a tosylate of Formula (ii), e.g by reaction with tosyl chloride (TsCl):
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (b) reacting the compound of Formula (ii) with propargyl alcohol in a coupling reaction, and optionally carrying out one or more additional steps of brominating followed by coupling with propargyl alcohol, to obtain a compound represented by Formula (iii): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein M is —[C≡C—CH 2 ]—, and n is as defined above,
 
(c) carrying out a selective reduction of the compound represented by Formula (iii) to obtain a compound represented by Formula (iv):
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein L and n are as defined above,
 
(d) brominating the compound of Formula (iv) to produce a compound represented by Formula (v):
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein L and n are as defined above,
 
(e) coupling the compound represented by Formula (v) with methyl pent-4-ynoate to obtain a compound represented by Formula (vi):
 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    (f) carrying out a selective reduction of the compound represented by Formula (vi) to obtain a compound represented by Formula (vii): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    and
 
(g) ester-hydrolyzing the compound represented by Formula (vii) to obtain the compound represented by Formula (i),
       wherein the propargyl alcohol used in at least one of the coupling reactions carried out in (b) is labeled with  13 C at C 1 , C 2 , or C 3  of the propargyl alcohol, or a combination thereof.       
 
         [0042]    In one non-limiting embodiment of the invention, a process is provided for preparing DHA, for example as represented below by Formula A: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where * represents a  13 C labeled carbon residue. 
         [0043]    This synthetic route can, in certain preferred embodiments, yield high purity of  13 C fatty acids, such as DHA, and at reduced cost as compared to other methods through the use of generally abundant and inexpensive reagents. The process also has the advantage that, in certain embodiments, no downstream processing is required. 
         [0044]    It will be appreciated by those skilled in the art that each of the embodiments of the invention described herein may be utilized individually or combined in one or more manners different than the ones disclosed above for the production of  13 C labeled fatty acids, including DHA. In addition, those skilled in the art will be able to select a suitable temperature in view of the reaction conditions being used, in further embodiments of the invention encompassed herein. 
         [0045]    The literature referred to herein establishes knowledge that is available to those with skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. All references cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually incorporated by reference. 
         [0046]    Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. In the case of inconsistencies, the present disclosure, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. 
         [0047]    The term “about” is used herein to mean approximately, in the region of, roughly, or around. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. The term “comprises” is used herein to mean “includes, but is not limited to.” 
         [0048]    The following abbreviations are used throughout the specification: 
       CuI: Copper Iodide 
     DHA: Docosahexanoic Acid 
     DCM: Dichloromethane 
     DMF: Dimethylformamide 
     EtOAc: Ethyl Acetate 
     HCl: Hydrochloric Acid 
     K 2 CO 3 : Potassium Carbonate 
     KOH: Potassium Hydroxide 
     MeOH: Methanol 
     NaHCO 3 : Sodium Carbonate 
     Na 2 SO 4 : Sodium Sulphate 
     PBr3: Phosphorus Tribromide 
     Py: Pyrimidine 
     TBAI: Tetrabutylammonium Iodide 
     THF: Tetrahydrofuran 
     TsCl: Tosyl Chloride 
       [0049]    In one embodiment of the invention, a 13-step chemical synthetic process for preparing  13 C DHA of Formula A is provided. The synthetic process is depicted below in Scheme A. 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
         [0050]    In this synthetic process, 2-Pentyn-1-ol of Formula 1 is used as a starting material, wherein the alcohol group in 2-Pentyn-1-ol is converted to tosyl as represented by Formula 2, using TSCl/KOH. The resulting compound of Formula 2 is coupled with propargyl alcohol using CuI/K 2 CO 3 /TBAI to obtain a compound represented by Formula 3 in good yield. The compound of Formula 3 is coupled with propargyl alcohol, via a bromide represented by Formula 4 to obtain a compound represented by Formula 5. The compound of Formula 5 is coupled with propargyl alcohol, via a bromide represented by Formula 6 to obtain a compound represented by Formula 7. The compound represented by Formula 7 is further coupled with a  13 C labeled propargyl alcohol via the bromide represented by Formula 8 to obtain a compound represented by Formula 9. The resulting compound of Formula 9 is selectively reduced, e.g. using Lindlar&#39;s catalyst, to produce a compound represented by Formula 10 which is then coupled with methyl pent-4-ynoate via a bromide represented by Formula 11 to obtain a compound represented by Formula 12. The compound represented by Formula 12 is selectively reduced, e.g. using a Lindlar&#39;s catalyst, to produce a compound represented by Formula 13, which is ester hydrolyzed, e.g. using LiOH, to produce the  13 C-labeled DHA of Formula A. 
         [0051]    In yet another embodiment of the invention, an alternate, 12-step chemical synthetic process for preparing  13 C DHA of Formula A is provided. The synthetic process is depicted below in Scheme B. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0052]    In the alternate synthetic process, 2-Pentyn-1-ol of Formula 1 is used as a starting material, wherein the alcohol group in 2-Pentyn-1-ol is converted to tosyl as represented by Formula 2, using TSCl/KOH. The resulting compound of Formula 2 is coupled with propargyl alcohol using CuI/K 2 CO 3 /TBAI to obtain a compound represented by Formula 3 in good yield. The compound of Formula 3 obtained is coupled with propargyl alcohol, via a bromide represented by Formula 4 to obtain a compound represented by Formula 5. The compound represented by Formula 5 is coupled with propargyl alcohol, via a bromide represented by Formula 6 to obtain a compound represented by Formula 7. The compound represented by Formula 7 is further coupled with a  13 C labeled propargyl alcohol via the bromide represented by Formula 8 to obtain a compound represented by Formula 9. The resulting compound of Formula 9 is then coupled with methyl pent-4-ynoate via a bromide represented by Formula 14 to obtain a compound represented by Formula 15. The compound represented by Formula 15 is selectively reduced using a Lindlar&#39;s catalyst to produce a compound represented by Formula 13, which is ester hydrolyzed, e.g. using LiOH, to produce the  13 C-labeled DHA of Formula A. 
       EXAMPLES 
       [0053]    The following provides examples of certain preferred embodiments of the synthetic process described herein for producing the  13 C labeled DHA of Formula A. The process is depicted below in Scheme C. 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
       Example 1 
     Synthesis of  13 C DHA Using 13-Step Chemical Synthetic Process 
       [0054]    In the 13-step chemical synthetic process for preparing  13 C DHA of Formula A, 2-Pentyn-1-ol of Formula 1 and tosyl chloride are used as the starting materials. Each of the steps in the chemical synthetic process are described in detail below. 
       Preparation of Compound of Formula 2 (Pent-2-ynyl 4-methylbenzenesulfonate) 
       [0055]    In the first step of the synthetic process, 2-pentyn-1-ol of Formula 1 is converted to the tosyl compound represented by Formula 2 using tosyl chloride in the presence of KOH. The yield of the compound ranges from 60-68%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0056]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 1: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 Moles 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 2-Pentyn- 
                 60 
                 g 
                 84.12 
                 0.71 
                 1 
               
               
                   
                 1-ol 
               
               
                 2. 
                 Tosyl 
                 142.9 
                 g 
                 190.65 
                 0.75 
                 1.06 
               
               
                   
                 Chloride 
               
               
                   
                 (TsCl) 
               
               
                 3. 
                 KOH 
                 79.9 
                 g 
                 56.11 
                 1.42 
                 2 
               
             
          
           
               
                 4. 
                 THF 
                 420 
                 mL 
                 72.11 
                 — 
                 7 
                 vol. 
               
               
                 5. 
                 Ethyl 
                 600 
                 mL 
                 88.11 
                 — 
                 10 
                 vol. 
               
               
                   
                 Acetate 
               
               
                 6. 
                 Water 
                 2 × 100 
                 mL 
                 18 
                 — 
                 2 × 1.67 
                 vol. 
               
               
                 7. 
                 Brine 
                 2 × 50 
                 mL 
                 — 
                 — 
                 2 × 0.83 
                 vol. 
               
             
          
           
               
                 8. 
                 Na2SO4 
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0057]    To a solution of 2-Pentyn-1-ol (60 g, 0.71 mol) in THF (420 mL) cooled to −5° C., tosyl chloride (142.9 g, 0.75 mol) and KOH (79.9 g, 1.42 mol) were added and the reaction mixture was stirred at room temperature for 1 h. After completion of starting material, the reaction mixture was extracted with ethyl acetate (300 mL×2), washed with water (100 mL×2), brine (50 mL×2) and dried over Na 2 SO 4 . The combined organic extracts were evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, 20% EtOAc-hexane) to furnish pent-2-ynyl 4-methylbenzenesulfonate (110 g, 64%) as a light red liquid. 
       Preparation of Compound of Formula 3 (Octa-2,5-diyn-1-ol:) 
       [0058]    The compound of Formula 2 obtained as described above is then coupled with propargyl alcohol in the presence of CuI, K 2 CO 3  and TBAI to produce the compound represented by Formula 3. The yield of the compound ranges from 93-99%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0059]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 2: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 Moles 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 60 
                 g 
                 84.5 
                 0.71 
                 1 
               
               
                   
                 represented 
               
               
                   
                 by Formula 
               
               
                   
                 2 
               
               
                 2. 
                 Propargyl 
                 15.52 
                 g 
                 56.06 
                 0.27 
                 0.38 
               
               
                   
                 alcohol 
               
               
                 3. 
                 Potassium 
                 47.8 
                 g 
                 138.2 
                 0.34 
                 0.48 
               
               
                   
                 Carbonate 
               
               
                 4. 
                 CuI 
                 43.9 
                 g 
                 190.45 
                 0.23 
                 0.32 
               
               
                 5. 
                 TBAI 
                 85.30 
                 g 
                 369.37 
                 0.23 
                 0.32 
               
             
          
           
               
                 6. 
                 DMF 
                 440 
                 mL 
                 73.09 
                 — 
                 7.33 
                 vol. 
               
               
                 7. 
                 Ethyl 
                 2 × 300 
                 mL 
                 88.11 
                 — 
                 2 × 5 
                 vol. 
               
               
                   
                 acetate 
               
               
                 8. 
                 Cold water 
                 2 × 200 
                 mL 
                 18 
                 — 
                 2 × 3.33 
                 vol. 
               
               
                 9. 
                 Brine 
                 2 × 100 
                 mL 
                 — 
                 — 
                 2 × 1.67 
                 vol. 
               
             
          
           
               
                 10. 
                 Na 2 SO 4 , 
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
                 anhydrous 
               
               
                   
               
             
          
         
       
     
         [0060]    To a stirred solution of potassium carbonate (47.8 g, 0.34 mol), CuI (43.9 g, 0.23 mol), and TBAI (85.30 g, 0.23 mol) in DMF (440 mL) cooled to 0° C., propargyl alcohol (15.52 g, 0.27 mol) was added portion wise at room temperature followed by compound represented by Formula 2 (55 g, 0.23 mol) and the reaction mixture was stirred at room temperature for 16 h. After completion of starting materials, the reaction mixture was cooled to 0° C. and diluted with cold water, ethyl acetate (300 mL×2), filtered through celite bed and washed with ethyl acetate. The combined organic extracts were washed with cold water (200 mL×2), brine (100 mL×2) and dried over anhydrous Na 2 SO 4 . Solvent was evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, 20% EtOAc in hexane) to furnish octa-2,5-diyn-1-ol (55 g, 98%) as a light red liquid. 
       Preparation of a Compound of Formula 4 (1-bromoocta-2,5-diyne) 
       [0061]    The compound of Formula 3 obtained as described above is then brominated with PBr 3  to produce the compound represented by Formula 4. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0062]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 3: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 Moles 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 55 
                 g 
                 122.22 
                 0.45 
                 1 
               
               
                   
                 represented 
               
               
                   
                 by Formula 
               
               
                   
                 3 
               
               
                 2. 
                 PBr 3   
                 17.13 
                 mL 
                 270.69 
                 0.18 
                 0.4 
               
             
          
           
               
                 3. 
                 Diethylether 
                 550 
                 mL 
                 74.12 
                   
                 10 
                 vol. 
               
             
          
           
               
                 4. 
                 Pyridine 
                 3.6 
                 mL 
                 79.1 
                 0.04 
                 0.009 
               
             
          
           
               
                 5. 
                 Ethyl 
                 2 × 200 
                 mL 
                 88.11 
                 — 
                 2 × 3.63 
                 vol. 
               
               
                   
                 acetate 
               
               
                 6. 
                 Cold water 
                 100 
                 mL 
                 18 
                 — 
                 1.82 
                 vol. 
               
               
                 7. 
                 Brine 
                 100 
                 mL 
                 — 
                 — 
                 1.82 
                 vol. 
               
             
          
           
               
                 8. 
                 Na 2 SO 4 , 
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
                 anhydrous 
               
               
                   
               
             
          
         
       
     
         [0063]    To a stirred solution of compound 3 (55 g, 0.45 mol) in diethylether (550 mL) cooled to 0° C., pyridine (3.6 mL, 0.04 mol), PBr 3  (17.13 mL, 0.18 mol) were added at 0° C. and the reaction mixture was stirred at room temperature for 16 h. After the completion of starting material, the reaction mixture was cooled to 0° C., diluted with cold water, and extracted with ethyl acetate (200 mL×2). The combined organic extracts were washed with cold water (100 mL×1), brine (100 mL×1), dried over anhydrous Na 2 SO 4  and evaporated under reduced pressure to furnish 1-bromoocta-2,5-diyne (75 g, crude) as a red liquid which was carried to the next step without further purification. 
       Preparation of a Compound of Formula 5 (undeca-2,5,8-triyn-1-ol) 
       [0064]    The compound of Formula 4 obtained as described above is coupled with propargyl alcohol to produce the compound of Formula 5. The yield of the compound ranges from 52-62%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0065]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 4: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 Moles 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 75 
                 g 
                 187.5 
                 0.40 
                 1 
               
               
                   
                 represented 
               
               
                   
                 by Formula 
               
               
                   
                 4 
               
               
                 2. 
                 Propargyl 
                 27.2 
                 g 
                 56.06 
                 0.48 
                 1.2 
               
               
                   
                 alcohol 
               
               
                 3. 
                 Potassium 
                 83 
                 g 
                 138.2 
                 0.60 
                 1.5 
               
               
                   
                 Carbonate 
               
               
                 4. 
                 CuI 
                 77 
                 g 
                 190.45 
                 0.40 
                 1 
               
               
                 5. 
                 TBAI 
                 149.5 
                 g 
                 369.37 
                 0.40 
                 1 
               
             
          
           
               
                 6. 
                 DMF 
                 450 
                 mL 
                 73.09 
                 — 
                 6 
                 vol. 
               
               
                 7. 
                 Ethyl 
                 300 
                 mL 
                 88.11 
                 — 
                 4 
                 vol. 
               
               
                   
                 acetate 
               
               
                 8. 
                 Cold water 
                 2 × 100 
                 mL 
                 18 
                 — 
                 2 × 1.33 
                 vol. 
               
               
                 9. 
                 Brine 
                 100 
                 mL 
                 — 
                 — 
                 1.33 
                 vol. 
               
             
          
           
               
                 10. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0066]    In an exemplary embodiment of this step, to a stirred solution of potassium carbonate (83 g, 0.60 mol), CuI (77 g, 0.40 mol) and TBAI (149.5 g, 0.40 mol) in DMF (450 mL) cooled to 0° C., propargyl alcohol (27.2 g, 0.48 mol) and compound represented by Formula 4 (75 g, 0.40 mol) were sequentially added and stirred at room temperature for 16 h. After the completion of starting materials, the reaction mixture was cooled to 0° C. and diluted with cold water, ethyl acetate (300 mL), filtered through a Celite™ pad using Buchner funnel and washed with ethyl acetate. The filtrate was taken and the organic layers were separated. The combined organic extracts were washed with cold water (100 mL×2), brine solution (100 mL×1), dried over Na 2 SO 4  and evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, 20% EtOAc in hexane) to furnish undeca-2,5,8-triyn-1-ol (37 g, 57%) as a pale yellow liquid. 
       Preparation of a Compound of Formula 6 (1-bromoundeca-2,5,8-triyne) 
       [0067]    The compound of Formula 5 obtained as described above is then brominated with PBr3 to produce the compound of Formula 6. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0068]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 5: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 Moles 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 37 
                 g 
                 160.87 
                 0.23 
                 1 
               
               
                   
                 represented 
               
               
                   
                 by Formula 
               
               
                   
                 5 
               
               
                 2. 
                 PBr 3   
                 0.79 
                 mL 
                 270.69 
                 0.09 
                 0.39 
               
             
          
           
               
                 3. 
                 Diethylether 
                 370 
                 mL 
                 74.12 
                   
                   
                   
               
             
          
           
               
                 4. 
                 Pyridine 
                 1.86 
                 mL 
                 79.1 
                 0.02 
                 0.09 
               
             
          
           
               
                 5. 
                 Ethyl 
                 100 
                 mL 
                 88.11 
                 — 
                 2.7 
                 vol. 
               
               
                   
                 acetate 
               
               
                 6. 
                 Cold Water 
                 2 × 50 
                 mL 
                 18 
                 — 
                 2 × 1.35 
                 vol. 
               
               
                 7. 
                 Brine 
                 50 
                 mL 
                 — 
                 — 
                 1.35 
                 vol. 
               
             
          
           
               
                 8. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0069]    To a stirred solution of the compound represented by Formula 5 (37 g, 0.23 mol) in ether (370 mL) cooled to 0° C., pyridine (1.86 mL, 0.02 mol), PBr 3  (0.79 mL, 0.09 mol) were added at 0° C. and stirred at room temperature for 16 h. After the completion of starting material, the reaction mixture was cooled to 0° C. and diluted with cold water, and extracted with ethyl acetate (100 mL). The combined organic extracts were washed with cold water (50 mL×2), brine solution (50×1), dried over Na 2 SO 4  and evaporated under reduced pressure to furnish 1-bromoundeca-2,5,8-triyne (42 g, crude) as a pale yellow color liquid which was carried to the next step without further purification. 
       Preparation of Compound of Formula 7 (tetradeca-2,5,8,11-tetrayn-1-ol) 
       [0070]    The compound of Formula 6 obtained as described above is coupled with propargyl alcohol to produce the compound of Formula 7. The yield of the compound ranges from 27-37%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0071]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 6: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 Moles 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 42 
                 g 
                 233.33 
                 0.18 
                 1 
               
               
                   
                 represented 
               
               
                   
                 by Formula 
               
               
                   
                 4 
               
               
                 2. 
                 Propargyl 
                 14 
                 g 
                 56.06 
                 0.25 
                 1.39 
               
               
                   
                 alcohol 
               
               
                 3. 
                 Potassium 
                 38 
                 g 
                 138.2 
                 0.27 
                 1.5 
               
               
                   
                 Carbonate 
               
               
                 4. 
                 CuI 
                 35.85 
                 g 
                 190.45 
                 0.18 
                 1 
               
               
                 5. 
                 TBAI 
                 69.5 
                 g 
                 369.37 
                 0.18 
                 1 
               
             
          
           
               
                 6. 
                 DMF 
                 250 
                 mL 
                 73.09 
                   
                 5.95 
                 vol. 
               
               
                 7. 
                 Cold water 
                 200 
                 mL 
                 18 
                   
                 4.76 
                 vol. 
               
               
                 8. 
                 Ethyl 
                 200 
                 mL 
                 88.11 
                   
                 4.76 
                 vol. 
               
               
                   
                 acetate 
               
               
                 9. 
                 Ethyl 
                 2 × 100 
                 mL 
                 88.11 
                   
                 2 × 2.38 
                 vol. 
               
               
                   
                 acetate 
               
               
                 10. 
                 Cold Water 
                 2 × 50 
                 mL 
                 18 
                   
                 2 × 1.19 
                 vol. 
               
               
                 12. 
                 Brine 
                 50 
                 mL 
                 — 
                   
                 1.19 
                 vol. 
               
             
          
           
               
                 11. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
               
               
                   
               
             
          
         
       
     
         [0072]    In an exemplary embodiment of this step, to a solution of potassium carbonate (38 g, 0.27 mol), CuI (35.85 g, 0.18 mol) and TBAI (69.5 g, 0.18 mol) in DMF (250 mL) cooled to 0° C., propargyl alcohol (14 g, 0.25 mol) and the compound represented by Formula 6 (42 g, 0.18 mol) were added drop wise for 30 min and stirred for 16 h at room temperature. After the completion of starting material, the reaction mixture was cooled to 0° C. and diluted with cold water (200 mL), ethyl acetate (200 mL), filtered through Celite™ bed using Buchner funnel and washed with ethyl acetate (100 mL×2). The organic layers were separated and the combined organic extracts were washed with cold water (50 mL×2), brine solution (50 mL×1), dried over Na 2 SO 4  and evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, 20% EtOAc in hexane) to furnish tetradeca-2,5,8,11-tetrayn-1-ol (12 g, 32%) as a pale yellow solid. 
       Preparation of a Compound of Formula 8 (1-bromotetradeca-2,5,8,11-tetrayne) 
       [0073]    The Compound of Formula 7 obtained as described above is brominated with PBr 3  to produce the Compound of Formula 8. The yield of the compound ranges from 18-28%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0074]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 7: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 mM 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 7.5 
                 g 
                 198.4 
                 37.8 
                 1 
               
               
                   
                 represented 
               
               
                   
                 by Formula 7 
               
               
                 2. 
                 PBr3 
                 1.44 
                 mL 
                 270.69 
                 15.15 
                 0.4 
               
             
          
           
               
                 3. 
                 Dichloro- 
                 75 
                 mL 
                 84.93 
                   
                 10 
                 vol. 
               
               
                   
                 methane 
               
             
          
           
               
                 4. 
                 Pyridine 
                 0.3 
                 mL 
                 79.1 
                 3.78 
                 0.1 
               
             
          
           
               
                 5. 
                 Dichloro- 
                 2 × 100 
                 mL 
                 84.93 
                 — 
                 2 × 13.33 
                 vol. 
               
               
                   
                 methane 
               
               
                 6. 
                 Water 
                 2 × 25 
                 mL 
                 18 
                 — 
                 2 × 3.33 
                 vol. 
               
               
                 7. 
                 Brine 
                 2 × 25 
                 mL 
                 — 
                 — 
                 2 × 3.33 
                 vol. 
               
             
          
           
               
                 8. 
                 Na2SO4 
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0075]    To a stirred solution of compound represented by Formula 7 (7.5 g, 37.8 mmol) in dry dichloromethane (75 mL), cooled to 0° C., pyridine (0.3 mL, 3.78 mmol) and PBr 3  (1.44 mL, 15.15 mmol) were added at 0° C., then the reaction mixture was stirred at room temperature for 16 h. After the completion of starting material, the reaction mixture was quenched with ice cold water and then extracted with dichloromethane (100 mL×2). The combined organic extracts were washed with water (25 mL×2), brine (25 mL×2), dried over Na 2 SO 4  and evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, 1% EtOAc in hexane) to furnish 1-bromotetradeca-2,5,8,11-tetrayne (2.3 g, 23%) as a yellow color solid. 
       Preparation of a Compound of Formula 9 (heptadeca-2,5,8,11,14-pentayn-1-ol) 
       [0076]    The compound of Formula 8 obtained as described above is coupled with  13 C labeled propargyl alcohol to produce the compound of Formula 9. The yield of the compound ranges from 45-55%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0077]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 8: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 mM 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 1.7 
                 g 
                 280.34 
                 6.53 
                 1 
               
               
                   
                 represented 
               
               
                   
                 by Formula 8 
               
               
                 2. 
                   13 C labeled 
                 0.36 
                 g 
                 56.06 
                 6.42 
                 0.98 
               
               
                   
                 Propargyl 
               
               
                   
                 alcohol 
               
               
                 3. 
                 Potassium 
                 1.35 
                 g 
                 138.2 
                 9.78 
                 1.49 
               
               
                   
                 Carbonate 
               
               
                 4. 
                 CuI 
                 1.24 
                 g 
                 190.45 
                 6.53 
                 1 
               
               
                 5. 
                 TBAI 
                 2.41 
                 g 
                 369.37 
                 6.53 
                 1 
               
             
          
           
               
                 6. 
                 DMF 
                 14 
                 mL 
                 73.09 
                 — 
                 8.23 
                 vol. 
               
               
                 7. 
                 Cold water 
                 10 
                 mL 
                 18 
                 — 
                 5.88 
                 vol. 
               
               
                 8. 
                 Ethyl acetate 
                 2 × 50 
                 mL 
                 88.11 
                 — 
                 2 × 29.41 
                 vol. 
               
               
                 9. 
                 Cold Water 
                 2 × 25 
                 mL 
                 18 
                 — 
                 2 × 14.7 
                 vol. 
               
               
                 10. 
                 Brine 
                 25 
                 mL 
                 — 
                 — 
                 14.7 
                 vol. 
               
             
          
           
               
                 11. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0078]    To a stirred solution of potassium carbonate (1.35 g, 9.78 mmol), CuI (1.24 g, 6.53 mmol) and TBAI (2.41 g, 6.53 mmol) in DMF (14 mL) cooled to 0° C.,  13 C labeled propargyl alcohol (0.36 g, 6.42 mmol) and the compound represented by Formula 8 (1.7 g, 6.53 mmol) were added drop wise and stirred at room temperature for 16 h. After completion of starting materials, the reaction mixture was cooled to 0° C. and diluted with cold water (10 mL), ethyl acetate (50 mL×2), filtered through a Celite™ pad using Buchner funnel and washed with ethyl acetate. The filtrate was taken and the organic layer was separated using a separating funnel. The combined organic extracts were washed with cold water (25 mL×2), brine solution (25 mL×1), dried over Na 2 SO 4  and evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, 16% EtOAc in hexane) to furnish heptadeca-2,5,8,11,14-pentayn-1-ol (750 mg, 50%) as a yellow solid. 
       Preparation of Compound of Formula 10 
       [0079]    The  13 C labeled compound of Formula 9 obtained as described above is selectively reduced with Lindlar&#39;s Catalyst to produce the compound represented by Formula 10. The yield of the compound ranges from 63-73%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0080]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 9: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 mM 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound of 
                 1.4 
                 g 
                 239.31 
                 5.85 
                 1 
               
               
                   
                 Formula 9 
               
               
                 2. 
                 Lindlar&#39;s 
                 1.44 
                 g 
                 — 
                 — 
                 — 
               
               
                   
                 catalyst 
               
             
          
           
               
                 3. 
                 Methanol/ 
                 24 
                 mL 
                 — 
                 — 
                 17.14 
                 vol. 
               
               
                   
                 Pyridine (5:1) 
               
             
          
           
               
                 4. 
                 Methanol 
                 — 
                 32 
                 — 
                 — 
               
             
          
           
               
                 5. 
                 Ethyl acetate 
                 2 × 50 
                 mL 
                 88.11 
                 — 
                 2 × 35.71 
                 vol. 
               
               
                 6. 
                 1N HCl 
                 10 
                 mL 
                 36.5 
                 — 
                 7.14 
                 vol. 
               
               
                 7. 
                 Brine 
                 10 
                 mL 
                 — 
                 — 
                 7.14 
                 vol. 
               
             
          
           
               
                 8. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0081]    To a stirred solution of compound represented by Formula 9 (1.4 g, 5.85 mmol) in methanol/pyridine (5:1, 24 mL), Lindlar&#39;s catalyst (1.4 g, w/w) was added. The reaction mixture was stirred under H 2  atmosphere at room temperature for 16 h. After completion of starting material, the reaction mixture was filtered through a Celite™ pad and washed with methanol. The solvent was evaporated under reduced pressure and the crude obtained was extracted with ethyl acetate (50 mL×2), and washed with 1N HCl solution (10 mL×1), brine solution (10 mL×1) and dried over Na 2 SO 4 . The combined organic extracts were evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, 10% EtOAc in hexane) to furnish compound represented by Formula 10 (1.0 g, 68%) as a colorless liquid. 
       Preparation of a Compound of Formula 11 
       [0082]    The compound of Formula 10 obtained as described above is brominated with PBr 3  to produce the compound of Formula 11. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0083]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 10: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 mM 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound of 
                 1.2 
                 g 
                 249.28 
                 4.81 
                 1 
               
               
                   
                 Formula 10 
               
               
                 2. 
                 PBr3 
                 0.52 
                 g 
                 270.69 
                 1.92 
                 0.4 
               
             
          
           
               
                 3. 
                 Dichloromethane 
                 20 
                 mL 
                 84.93 
                 — 
                 16.67 
                 vol. 
               
             
          
           
               
                 4. 
                 Pyridine 
                 0.38 
                 mL 
                 79.1 
                 0.48 
                 0.1 
               
             
          
           
               
                 5. 
                 Cold water 
                 10 
                 mL 
                 18 
                 — 
                 8.33 
                 vol. 
               
               
                 6. 
                 Dichloromethane 
                 2 × 50 
                 mL 
                 84.93 
                 — 
                 41.67 
                 vol. 
               
               
                 7. 
                 Water 
                 15 
                 mL 
                 18 
                 — 
                 12.5 
                 vol. 
               
               
                 8. 
                 Brine 
                 20 
                 mL 
                 — 
                 — 
                 16.67 
                 vol. 
               
             
          
           
               
                 9. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0084]    To a solution of compound represented by Formula 10 (1.2 g, 4.81 mmol) in dry dichloromethane (20 mL) and pyridine (0.038 mL, 0.48 mmol) cooled to 0° C., PBr 3  (0.52 g, 1.92 mmol) was added drop wise and stirred at room temperature for 2 h. After completion of starting material, the reaction mixture was quenched with ice cold water (10 mL×1) and extracted with dichloromethane (50 mL×2). The combined organic extracts were washed with water (15 mL×1), brine (20 mL×1), dried over Na 2 SO 4  and evaporated under reduced pressure to furnish compound represented by Formula 11 (1.2 g, crude) as a yellow liquid which was carried to the next step without further purification. 
       Preparation of Compound of Formula 12 
       [0085]    The compound of Formula 11 obtained as described above was coupled with methyl-pent-4-yonate to produce the compound represented by Formula 12. The yield of the compound ranges from 49-59%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0086]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 11: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 mM 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound of 
                 200 
                 mg 
                 312.5 
                 0.64 
                 1 
               
               
                   
                 Formula 11 
               
               
                 2. 
                 Methyl-pent- 
                 86 
                 mg 
                 111 
                 0.76 
                 1.19 
               
               
                   
                 4-yonate 
               
               
                 3. 
                 Potassium 
                 132 
                 mg 
                 138.2 
                 0.96 
                 1.5 
               
               
                   
                 Carbonate 
               
               
                 4. 
                 CuI 
                 112 
                 mg 
                 190.45 
                 0.64 
                 1 
               
               
                 5. 
                 TBAI 
                 236 
                 mg 
                 369.37 
                 0.64 
                 1 
               
             
          
           
               
                 6. 
                 DMF 
                 10 
                 mL 
                 73.09 
                 — 
                 50 
                 vol. 
               
               
                 7. 
                 Cold Water 
                 10 
                 mL 
                 18 
                 — 
                 50 
                 vol. 
               
               
                 8. 
                 Diethyl ether 
                 2 × 25 
                 mL 
                 74.12 
                 — 
                 2 × 125 
                 vol. 
               
               
                 9. 
                 Water 
                 10 
                 mL 
                 18 
                 — 
                 50 
                 vol. 
               
               
                 10. 
                 Brine 
                 10 
                 mL 
                 — 
                 — 
                 50 
                 vol. 
               
             
          
           
               
                 11. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0087]    To a solution of potassium carbonate (132 mg, 0.96 mmol), CuI (121 mg, 0.64 mmol) and TBAI (236 mg, 0.64 mmol) in dry DMF (10 mL) cooled to 0° C., methyl pent-4-ynoate (86 mg, 0.76 mmol) and the compound represented by Formula 11 (200 mg, 0.64 mmol) in DMF were added and stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with ice cold water (10 mL) and filtered through a Celite™ bed and washed with diethyl ether (25 mL×2), water (10 mL×1), brine solution (10 mL×1) and dried over Na 2 SO 4 . The combined organic extracts were evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, eluted at 2% EtOAc in hexane) to furnish compound represented by Formula 12 (120 mg, 54%) as a colorless liquid. 
       Preparation of a Compound of Formula 13 
       [0088]    The compound of Formula 12 obtained as described above was selectively reduced with Lindlar&#39;s catalyst to produce the compound of Formula 13. The yield of the compound ranges from 75-85%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0089]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 12: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 mM 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound of 
                 500 
                 mg 
                 344.82 
                 1.45 
                 1 
               
               
                   
                 Formula 12 
               
               
                 2. 
                 Lindlar&#39;s 
                 500 
                 mg 
                 — 
                 — 
                 — 
               
               
                   
                 catalyst 
               
             
          
           
               
                 3. 
                 Methanol/ 
                 10 
                 mL 
                 — 
                 — 
                 20 
                 vol. 
               
               
                   
                 Pyridine (4:1) 
               
               
                 4. 
                 Methanol 
                 20 
                 mL 
                 32 
                 — 
                 40 
                 vol. 
               
               
                 5. 
                 Ethyl acetate 
                 2 × 30 
                 mL 
                 88.11 
                 — 
                 2 × 60 
                 vol. 
               
               
                 6. 
                 1N HCl 
                 10 
                 mL 
                 36.5 
                 — 
                 20 
                 vol. 
               
               
                 7. 
                 Brine 
                 15 
                 mL 
                 — 
                 — 
                 30 
                 vol. 
               
             
          
           
               
                 8. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0090]    To a solution of compound represented by Formula 12 (500 mg, 1.45 mmol) in dry methanol/pyridine (10 mL, 4:1), Lindlar&#39;s catalyst (500 mg, w/w) was added. The reaction mixture was stir under H 2  atmosphere at room temperature for 16 h. Additionally, Lindlar&#39;s catalyst (250 mg) was added two times at 4 h interval and reaction mixture was stirred under H 2  atmosphere. The reaction mixture was filtered through a Celite™ pad, washed with methanol (20 mL) and evaporated under reduced pressure. The crude obtained was extracted with ethyl acetate (30 mL×2), washed with 1N HCl solution (10 mL×1), brine solution (15 mL×1) and dried over Na 2 SO 4 . The combined organic layer was evaporated under reduced pressure to furnish compound represented by Formula 13 (400 mg, 80%) as a pale yellow liquid. 
       Preparation of  13 C Labeled DHA as Represented by Formula A 
       [0091]    In the last step of the 13-step synthetic process,  13 C labeled DHA of Formula A is obtained by ester hydrolysis of the compound represented by Formula 13 in the presence of lithium hydroxide. The yield of the compound ranges from 82-92%. The reaction scheme involved in this process is as follows: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0092]    In an exemplary embodiment, the raw materials used for this step are illustrated in Table 13: 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
               
                   
               
               
                 S. 
                 Name of the 
                   
                 M. 
                   
                   
               
               
                 No. 
                 Material 
                 Qty. 
                 Wt. 
                 mM 
                 Mole Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1. 
                 Compound 
                 180 
                 mg 
                 346.15 
                  0.52 
                 1 
               
               
                   
                 of Formula 
               
               
                   
                 13 
               
               
                 2. 
                 Lithium 
                 109 
                 mg 
                 23.95 
                 2.6 
                 5 
               
               
                   
                 Hydroxide 
               
             
          
           
               
                 2. 
                 THF/H 2 O 
                 6 
                 mL 
                 — 
                 — 
                 33.33 
                 vol. 
               
               
                   
                 (3:1) 
               
               
                 3. 
                 Ethyl 
                 2 × 30 
                 mL 
                 88.11 
                 — 
                 2 × 166.67 
                 vol. 
               
               
                   
                 acetate 
               
               
                 4. 
                 Water 
                 10 
                 mL 
                 18 
                 — 
                 55.55 
                 vol. 
               
               
                 5. 
                 Brine 
                 10 
                 mL 
                 — 
                 — 
                 55.55 
                 vol. 
               
             
          
           
               
                 6. 
                 Na 2 SO 4   
                 As needed 
                 142.04 
                 — 
                 — 
               
               
                   
               
             
          
         
       
     
         [0093]    To a solution of compound represented by Formula 13 (180 mg, 0.52 mmol) in THF/H 2 O (6 mL, 3:1 ratio), lithium hydroxide (109 mg, 2.6 mmol) was added and stirred at room temperature for 16 h. After completion of starting material, the reaction mixture was quenched with aqueous citric acid solution; pH was adjusted to 4 and extracted with ethyl acetate (30 ml×2). The combined organic extracts were washed with water (10 mL×1), brine solution (10 mL×1) and dried over Na 2 SO 4 . The combined organic extracts were evaporated under reduced pressure to obtain the crude product which was purified by column chromatography (100-200 mesh silica gel, the product eluted at 15% EtOAc in hexane) to furnish the compound represented by Formula A ( 13 C DHA) (150 mg, 87%) as a pale yellow liquid. 
         [0094]    The identity of the Compound of Formula A produced by the synthetic process described above was ascertained by NMR spectroscopy. The NMR spectra obtained is presented in  FIG. 1 . 
         [0095]    Purity of the sample obtained was determined by LC (See  FIG. 2 ) and identity was further characterized by LC-MS (See  FIGS. 3A and 3B ). The purity of the sample was found to be 90%. 
       Example 2 
     Synthesis of  13 C DHA by 12-Step Chemical Synthetic Process 
       [0096]    An exemplary embodiment of the 12-step chemical synthesis process for preparing  13 C DHA is shown in Scheme D: 
         [0000]    
       
                 
         
             
             
         
       
       
                 
         
             
             
         
       
     
         [0097]    In this alternate 12-step synthesis strategy, the steps leading to the formation of the compound represented by Formula 9 are similar to those described in Example 1. The compound of Formula 9 thus produced is reacted with PBr 3  in the presence of Py and DCM to produce the compound represented by Formula 14. The compound of Formula 14 is coupled with methyl-pent-4-yonate in the presence of CuI, K 2 CO 3  and TBAI in DMF to produce the compound of Formula 15. The compound of Formula 15 is then selectively hydrogenated in a H 2  atmosphere using a catalyst, e.g. Lindlar&#39;s catalyst, in the presence of quinoline and MeOH. The reaction is carried out at about room temperature. The selective reduction of the compound represented by Formula 15 results in the production of the compound represented by Formula 13. In the last step of the alternate 12-step synthetic process,  13 C DHA of Formula A is obtained by ester hydrolysis of the compound represented by Formula 13 in the presence of lithium hydroxide, and in the presence of THF/H 2 O. 
         [0098]    It will be apparent to a person having skill in the art that all the common steps of this alternate 12-step strategy can be carried out under similar conditions as those described in Example 1 above. 
         [0099]    The preferred embodiments of the invention described above are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific details relating to the reagents and reaction conditions disclosed herein are not to be interpreted as limiting, but merely as an example. 
         [0100]    It will also be apparent to a person skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.