Tuberculosis: A Deadly Infection

Synthesis of an intermediary compound of mycolic acid motif available within Mycobacteria

Mycolic acids (MAs) are made up of long-chain fatty acids and are present as defensive waxy covering of the mycobacterial cells, the causal agent of the disease (TB) is Mycobacterium tuberculosis (M. tb). The present work is with an objective to prepare an intermediary compound to the repeating structure of MAs via the method of the Julia-Kocienski chemical reaction to interconnect large building blocks.

Tuberculosis is considered to be the most infectious malady that, in 2015, was answerable for the mortality of an expected 1.8 million individuals.1 The causal agent of the disease (TB) is Mycobacterium tuberculosis (M. tb) that are rod structured bacilli that convey the infection airborne and when breathed in, multiply within the lungs. The defensive waxy covering of the mycobacterial cells are made up of long-chain fatty acids called mycolic acids (MAs) 1. 2 The large molecule may differ in their structural orientation due to varied combination of functional moieties that are specialised for the different Mycobacteria sp. and therefore with the aid of structural differentiation the species can be distinguished. 3 The usual structure of the MA incorporates two different parts namely: the mycolic acid motif and the meromycolate chain, demonstrated within Figure 1. Out of all the categories of structures found within the X and Y motifs of the backbone of meromycolate, the 3 major sorts of MAs seen within M. tb are: methoxy-, alpha-, and keto-mycolic acids (Refer Table 1). 2,4 In this regard, author Barry et al. had communicated a condensed summary about mycolic specificities within MAs. Approximately 70% of the predominance is due to Alpha-mycolic acids while the rest such as methoxy and keto- mycolic acids forms the subsequent portion in an identical percentage.

Table 1: frequent combinations of the X and Y motif within the meromycolate chain which specifies the type of mycolic acid within Mycobacteria

Fig 2: (ω-1)-methoxy mycolic acid structure purified from Mycobacteria isolated from environmental samples

It is evident for M. tuberculosis the further methyl group at the α position and the methoxy group demonstrates a S, S constitution.5 MA have been seen to be bonded to cell wall as arabino esters and it can also be purified in the form of trehalose di- (TDM) and mono-mycolates (TMM) from the envelope of the cell or also in the form of hydroxy acid existing in the liberated or free position 1. 6,7. It must be mentioned that free form of MA is secreted in vitro at the time of growth of M. tuberculosis 7. The comparative non allowance of the cell wall offers resistance to the bacterium against antibiotics which are hydrophilic in nature 8,9 . One exception has been observed in terms of resistance for the drug isoniazid that goes within the bacterium and hinders the process of synthesis of MA which hampers the protective factor of the bacterium 10. One of the striking MA purified is (ω-1)-methoxy mycolic acid 2 from the M. tuberculosis found from the river source 11. Varied research studies have been conducted on the biosynthesis of MA and the hereditary material sequencing of the Mycobacteria. With more depth in knowledge about the MA will help to evaluate their functional role in resistance and will pave out the way for comprehending the resistance mechanism of the deadly disease. 2,12–17 Due to the presence of acid group within the structure of MA they remain in the form of esters within the Mycobacteria’s cell wall. Moreover, these ester compounds have been initially formulated 12. The same research study have revealed that the biological functioning of the MA was found to be maximum when existed in the bonded form of esters of trehalose dimycolates (TDMs), trehalose monomycolates (TMMs) and glucose mycolates. Moreover, the MA along with their esters of sugar compounds had stimulated an antigenic reaction against the antibodies present within the patient’s serum, found to be infected with TB. This was observed when a study was conducted with small sample population of TB infected and non – infected samples. 18 Therefore, it is quite interesting to comprehend that these ester compounds could be of immense value in the field of diagnostics for active cases of TB. Within the bacteria, the biosynthesis pathway of the moiety goes through a Claisen-type condensation chemical reaction within which a coupling of the α and β carbons occurs before the process of selective reduction synthesizes the moiety. The other research group have achieved the assemblage of the moieties with the aid of biomimetic methodology and many researchers have also discussed about the organic mode of manufacturing of the whole MA that involved varied combinations and permutations of the distal and proximal located functioning groups available on the backbone of meromycolate chain with expanded rate of success. 12–15,19

This particular method involves the synthesis of the intermediary compounds 3, via three stages with the production of two subunits: sulfone 13 (Scheme 1) and the aldehyde 23 (Scheme 2). The particular two subunits is then coupled with the aid of Julia-Kocienski olefination which forms 3 (Scheme 3). The intermediate moiety 3 incorporates the mycolic motif, the unit of α-alkyl β-hydroxy acid 13 which usually consist of the R, R- configuration along with the subunit meromycolate 23 which is coupled collectively to form a chain of carbons of length “c” (Refer Figure 1). 19 After the production of intermediate 3 it can be utilised for the continued production of full compound (ω-1)-methoxy mycolic acid 2. Production of structurally specified MA might be used for the study involving biological functioning. This can be used to study the respective impact of certain permutations of mycolic functioning groups. For the production of sulfone 13, the creation initiated with the aid of an oxirane 4 (Scheme 1) that was manufactured using a pre tested procedures. 20 Regioselectivity was shown by the Oxirane 4which was opened with the aid of Grignard reagent to acquire the chiral alcohol 5. As the alkene group was attached, the shield of alcohol with ester was found to be a necessity to make easy the following oxidative cleavage of the subunit alkene to a carboxylic acid 6.

Fig: 3 incomplete meromycolate chain and the motif which is the matter of this production

The process of transesterification happened thereafter to guard the alcohol along with the reactive acid for the rest of the manufacturing process resulting in the formation of methyl ester 7.

Scheme 1: (a) vinylmagnesium bromide, CuI, THF, -78 °C, 1.25 h, 74%; (b) Ac2O, anhydrous pyridine, rt, 18 h, 92%, (c) OsO4, Oxone ®, N2, DMF, rt, 4 h, 70%; (d) conc. H2SO4, MeOH, 80 °C, 3.5 h, 47%; (e) diisopropylamine, MeLi, allyl iodide, HMPA, -78 °C, 2 h, 58%; (f) imidazole, TBDMS-Cl, DMF, 45 °C, 18 h, 57%; (g) 2,6-lutidine, OsO4, NaIO4, 1,4-dioxane-water (3:1), rt, 2.5 h, 73%; (h) 5-(icosane-1-sulfonyl)-1-phenyl-1H-tetrazole, LiHMDS, 2 h, 73%; (i) H2, Pd(OH)2/C THF/IMS (7:3), 2 h, 95%; (j) 1-phenyl-1H-tetrazole-5- thiol, DEAD, triphenyl phosphine, THF, 3 h, 94%; (k) m-CPBA, NaHCO3, DCM, rt, 24 h, 82%.

For guaranteeing the stereocontrol, while the production of the α-alkyl chain, the procedural steps of the Fráter-Seebach alkylation was used, by keeping at the top position of molecule the chelating lithium ion. This phenomenon helped in the diastereoselectively and eventually helped to accomplish the R, R-constitution of the moiety in the alkene 8. 21,22 For the shielding of alcohol, a silyl ether compound was made used of and following this reaction the oxidative cleavage was employed that generated the aldehyde 9 with a modest yield quantity. Thereafter, a coupling reaction of the compound aldehyde having the C20-unit was accomplished via the tailored procedural protocol of JuliaKocienski olefination which generated the alkene 10 as a mixed product of the E and Z isomers. When the compound 10 was subjected to hydrogenation reaction with the aid of palladium hydroxide Pd(OH)2, it resulted in the breakage of benzyl ether groups along with the reduction of alkene, that generated the α-aliphatic chain along with the production of alcohol 11. Then after the replacement of the hydroxyl group with a thiol resulted in the formation of the thioether 12. Ultimately an oxidative procedure was utilised for the yield of 13 having an overall percentage of 4.5% for the production of sulfone 13. 23 Moreover, the expansion of the meromycolate chain necessitates minor subunits to be produced that would extend the chain in regular increasing steps. It is a well known protocol where the commercially accessible compound D-mannitol 14 was given protection twice to form the compound di-acetal 15 (Scheme 2). It was observed that via an oxidative cleavage of the compound diol in 15 with the aid of chemical named sodium metaperiodate resulted in the formation of intermediary compounds named glyceraldehyde acetonide which extensively chemically reacted with methyl 2- (diisopropoxyphosphoryl) acetate 16 along with aqueous form of potassium carbonate to eventually form (E)-α,β-unsaturated ester 17. The particular chemical reaction occurred without isolation step and is referred as a HornerWadsworth-Emmons reaction. The addition of the moiety methyl by Michael resulted in the formation of ester 18. 24 The yield of ester was completed via a three step process with overall quantity of 27.8%. This particular reaction was continued by coupling reaction with sulfone 13 (Scheme 3) for the production of the subunit aldehyde 23, but the reaction was hindered due to the limitations of time.

Scheme 2: Proposed route to aldehyde 23. (a) acetone, anhydrous ZnCl2, rt, 18 h, 49 %; (b) NaIO4, H2O, 5 % NaHCO3, methyl 2-(diisopropoxyphosphoryl)acetate 16, , 6M K2CO3, rt, 20 h, 89 %; (c) MeLi, ether (dry), N2, -78 °C, 63 %. Further steps were not completed, but are as follows: (d) LiAlH4, THF (dry), 1 h; (e) PCC, DCM, reflux, 30 min.; (f) LiHMDS, THF (dry), rt, 2 h; (g) H2, Pd(OH)2/C, rt 2 h; (h) PCC, DCM, reflux, 30 min.

Scheme 3: (a) LiHMDS, 2 h; (b) H2, Pd/C, 18 h.

Conclusion:

The significance of the R, R-configuration within the subunit of α-alkyl-βhydroxy carboxylic acid was found to be of immense during the production of the motif as there is a necessity because the structure of MA are known to generate antigenic reactions. With the work demonstrated here within this paper, the next part of the work would be the synthesis of compound C12-aldehyde 23 followed by the coupling reaction with sulfone 13 (represented within Scheme 3). The motif coupling with the subunits of varied lengths and functioning capacity would generate an array of compounds to be produced. The reaction demonstrated by the varied composition of MA has the capacity to negatively affect the identification of antibodies against anti-mycolic acid within the serum of TB infected patients.25 With this foundation, esterification of MA for the formation of compounds such as TMMs and TDMs enables to form huge databases with an objective to assess the biological functioning of compounds of Mycobacteria. Research based investigation in this field has demonstrated that TDM shows a cytokine reaction which is quite identical to the response generated by the multidrug resistant TB strains during infection.26,27

References:

World Health Organisation, WHO | Global tuberculosis report 2016, World Health Organization, 2016.

C. E. Barry, R. E. Lee, K. Mdluli, A. E. Sampson, B. G. Schroeder, R. A. Slayden and Y. Yuan, Prog. Lipid Res., 1998, 37, 143–179.

M. Watanabe, Y. Aoyagi, H. Mitome, T. Fujita, H. Naoki, M. Ridell and D. E. Minnikin, Microbiology, 2002, 148, 1881– 1902.

M. S. Glickman, S. M. Cahill and W. R. Jacobs, J. Biol. Chem., 2001, 276, 2228–33.

E. Dubnau, M. A. Laneelle, S. Soares, A. Benichou, T. Vaz, D. Prome, J. C. Prome, M. Daffe, A. Quemard, M.-A. Lanéelle, S. Soares, A. Bénichou, T. Vaz, D. Promé, J.-C. Promé, M. Daffé and A. Quémard, Mol. Microbiol., 1997, 23, 313–322.

D. E. Minnikin, L. Kremer, L. G. Dover and G. S. Besra, Chem. Biol., 2002, 9, 545–553.

K. Takayama, H. K. Schnoes, E. L. Armstrong and R. W. Boyle, J. Lipid Res., 1975, 16, 308–17.

M. Luquin, J. Roussel, F. Lopez‐Calahorra, G. Lanelle, V. Ausina and M. Lanelle, Eur. J. Biochem., 1990, 192, 753–759.

M. Ali, G. Koza, R. T. Hameed, R. Rowles, C. Davies, J. R. Al Dulayymi, C. D. Gwenin and M. S. Baird, Tetrahedron, 2016, 72, 7143–7158.

G. Koza, M. Muzael, R. R. Schubert-Rowles, C. Theunissen, J. R. Al Dulayymi and M. S. Baird, Tetrahedron, 2013, 69, 6285– 6296.

D. Z. Al Kremawi, J. R. Al Dulayymi and M. S. Baird, Tetrahedron, 2014, 70, 7322–7335.

J. R. Al Dulayymi, M. S. Baird, E. Roberts, M. Deysel and J. Verschoor, Tetrahedron, 2007, 63, 2571–2592.

S. T. Cole, R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, A. Krogh, J. McLean, S. Moule, L. Murphy, K. Oliver, J. Osborne, M. A. Quail, M.-A. Rajandream, J. Rogers, S. Rutter, K. Seeger, J. Skelton, R. Squares, S. Squares, J. E. Sulston, K. Taylor, S. Whitehead and B. G. Barrell, Nature, 1998, 393, 537–544.

Bhatt, V. Molle, G. S. Besra, W. R. Jacobs and L. Kremer, Mol. Microbiol., 2007, 64, 1442–1454.

M. O. Mohammed, M. S. Baird, J. R. Al Dulayymi, A. Jones and C. D. Gwenin, Tetrahedron, 2016, 72, 2849–2857.

C. H. S. Driver, M. O. Balogun, G. Toschi, J. A. Verschoor, M. S. Baird and L. A. Pilcher, Tetrahedron Lett., 2010, 51, 1185– 1186.

J. A. Frick, J. B. Klassen, A. Bathe, J. M. Abramson and H. Rapoport, Synthesis (Stuttg)., 1992, 1992, 621–623.

G. Fráter, Tetrahedron Lett., 1981, 22, 425–428.

F. L. Ndlandla, V. Ejoh, A. C. Stoltz, B. Naicker, A. D. Cromarty, S. van Wyngaardt, M. Khati, L. S. Rotherham, Y. Lemmer, J. Niebuhr, C. R. Baumeister, J. R. Al Dulayymi, H. Swai, M. S. Baird and J. A. Verschoor, J. Immunol. Methods, 2016, 435, 50–59.

D. Hayashi, T. Takii, N. Fujiwara, Y. Fujita, I. Yano, S. Yamamoto, M. Kondo, E. Yasuda, E. Inagaki, K. Kanai, A. Fujiwara, A. Kawarazaki, T. Chiba and K. Onozaki, FEMS Immunol. Med. Microbiol., 2009, 56, 116–128.

Plante, O. J., Buchwald, S. L., & Seeberger, P. H., Journal of the American Chemical Society, 2000, 122(29), 7148-7149.

Agoston, K., Streicher, H., & Fuegedi, P., Tetrahedron: Asymmetry, 2016, 27(16), 707-728.

Luo, X., Ma, X., Lebreux, F., Markó, I. E., & Lam, K., Chemical Communications, 2018, 54(71), 9969-9972.

Reiter, D., Frisch, P., Szilvási, T., & Inoue, S., Journal of the American Chemical Society, 2019, 141(42), 16991-16996.

März, M., Chudoba, J., Kohout, M., & Cibulka, R., Organic & Biomolecular Chemistry, 2017, 15(9), 1970-1975.

Longwitz, L., & Werner, T. (2019). The Mitsunobu reaction, reimagined. Science, 365(6456), 866-867.

Lewis, D.E. and Olson, T.W., An Explanation of a-Cyanocinnamate Esters as a Replacement for DEAD in the Mitsunobu Reaction, 2017.

Dobbs, A. P., & McGregor-Johnson, C., Tetrahedron letters, 2002, 43(15), 2807-2810.

Appendix

General Information:

The chemicals of high quality purity grade were procured form the commercial manufacturers (Sigma-Aldrich and Alfa Aesar) and the solvents were acquired from the Fisher Scientific. The dried condition of the solvents tetrahydrofuran and ether were prepared using the sodium wire and the chemical benzophenone under nitrogen and in case of solvent dichloromethane, it was desiccated using calcium hydride. All the chemicals used for the experiments were purchased of reagent grade unless anything else was mentioned within the protocol. The catalyst used for the hydrogenation reaction, palladium (II) hydroxide was procured from Alfa Aesar. The chemical reactions were escalated utilising magnetic stirrers. Mildly calcined Celite® Filter Cel was procured from the supplier Sigma-Aldrich. For the purpose of column chromatography silica of 60Å of pore size 40-63 micron were acquired from supplier Fluorochem and the eluent was mentioned for each step. Silica gel of 60 Å on glass plates F224 acquired from Merck-Millipore was utilised for the thin layer chromatography. The purified compounds were identified using different UV radiation, along with chemicals for instances solution of potassium permanganate, I2 and solution of phosphomolybdic acid were used for IMS and then charring was continued. The organic solvents were dried using the chemical anhydrous magnesium sulphate. Bruker Alpha FTIR spectrometer were utilised for Infra red (IR) spectrum utilising fluid films and the values were mentioned to the bordering integer. Bruker UltrashieldTM 400 Plus were utilised for conducting the NMR spectra along with Sample Xpress. Bellingham & Stanley ADP 440 polarimeter were utilised for the measurement of particular rotations using orgainic solvent chloroform. The run of the NMR samples were conducted using solvent CDCl3 unless anything specific was mentioned. Parts per million (ppm) were used to report the chemical drifts and it is considered to be relative to the chloroform-d used internally with values (7.26 ppm for 1H NMR, 77.16 ppm for 13C NMR). The following abbreviations were utilised for the signal coupling reactions such as: br., broad, d, doublet; t, triplet; q, quartet; p, pentuplet; m, multiplet.

Synthesis of Alkene 5: (S)-1-(Benzyloxy)hex-5-en-3-ol:

3.02 g and 15.9 mmol of copper iodide was liquefied in the dry state of 300mL of THF in the condition of room temperature under nitrogen and it was chilled to -75 °C. 155.0 mL of 155.0 mmol Vinyl magnesium bromide was mixed with 1.0 M in THF within the temperature range of -75 °C to -50 °C and the mixture was mixed using stirrer for about half an hour in between the temperature -50 °C to -40 °C. The combination was chilled to -75 °C and then a solution of (R)-2-(2- (benzyloxy) ethyl) oxirane of weight of 9.03 g and strength 50.7 mmol in the dried state of THF was mixed within the range of temperature -75 °C to -40 °C. The chemical reaction was mixed with stirrer for about one hour in between the temperature range of -40 °C to -30 °C and then again for 15 minutes at temperature -20 °C. 400 mL of aqueous solution of saturated ammonium chloride was mixed and extraction was carried out with 900 mL of ethyl acetate. The mixture of the layers of organic solvents was cleansed with water, and then magnesium sulphate was used for drying. Then it was filtered and the evaporation of the solvent was carried out to obtain the crude product. Then purification was done using the column chromatography and the elution was carried out with the ratio of solvents petrol/ethyl acetate of (2:1) and the product of 7.8 g (74%) (S)-1- (benzyloxy) hex-5-en-3-ol as an oil with colour orange-yellow was obtained with [α] 20D = -8.8° (c 0.68, CHCl3); δH (CDCl3, 400 MHz): 7.38 – 7.26 (5 H, m), 5.84 (1 H, ddt, J 17.1, 10.0, 7.1 Hz), 5.11 (1 H, d, J 17.1 Hz), 5.10 (1 H, d, J 10.0 Hz), 4.53 (2 H, s), 3.92 – 3.84 (1 H, m), 3.72 (1 H, dt, J 9.4, 5.4 Hz), 3.65 (1 H, ddd, J 9.3, 6.7, 5.8 Hz), 2.25 (1 H, t, J 6.7 Hz), 1.77 (2 H, m); δC (CDCl3, 101 MHz): 138.1 (-), 135.0 (+), 128.6 (+), 127.9 (+), 127.8 (+), 117.7 (-), 73.5 (-), 70.5 (+), 69.1 (-), 42.1 (-), 36.0 (-); νmax (neat): 3425, 3067, 3032, 2918, 1863, 1641, 1496, 1458, 1362, 1204, 1099 cm-1.

Synthesis of Ester 5.5: (S)-1-(Benzyloxy) hex-5-en-3-yl acetate:

21.0 mL, of strength 222.6 mmol of acetic anhydride was added to 21.0 mL, of strength 259.6 mmol, anhydrous pyridine and then this mixture was further added to a mixed solution of 7.58 g and strength of 36.7 mmol (S)-1-(benzyloxy) hex-5-en-3-ol in the condition of room temperature. The above mixture was mixed for the next 18 hours and it was further diluted using 30mL of toluene. Then the solvent was furthered evaporated to obtain the crude content which was in turn purified by column chromatography and the elution was carried out with solvent petrol / diethyl ether in the ratio of 6:1. 8.4 g (91%) of pallid yellow coloured (S)-1-(benzyloxy) hex-5-en- 3-yl acetate was obtained with values of [α]22D = +48.9° (c 0.65, CHCl3); δH (CDCl3, 400 MHz): 7.37 – 7.27 (5 H, m), 5.76 (1 H, ddt, J 17.8, 10.8, 7.1 Hz) 5.07 (1 H, d, 17.8 Hz), 5.07 (1 H, d, J 10.8 Hz), 5.05 (1 H, s), 4.49 (1 H, d, J 12.0 Hz), 4.46 (1 H, d, J 12.0 Hz), 3.55 – 3.44 (2 H, m), 2.40 – 2.27 (2 H, m), 1.99 (3 H, s), 1.94 – 1.80 (2 H, m); δC (CDCl3, 101 MHz): 170.8 (-), 138.4 (-), 133.7 (+), 128.5 (+), 127.9 (+), 127.7 (+), 118.0 (-), 73.2 (-), 70.9 (+), 66.7 (-), 39.0 (-), 33.9 (-), 21.3 (+); νmax (neat): 3066, 3031, 2922, 2861, 1737, 1643, 1496, 1372, 1241, 1100, 1026 cm-1.

Synthesis of (R)-5-((31-methoxydotriacont-15-en-1-yl)sulfonyl)-1-phenyl-1H-tetrazole.

The 15-hydroxypentadecanoic acid methyl ester (2) was prepared from ω-pentadecalactone (1) by reaction with sodium methoxide in methanol at reflux temperature.

The 1H NMR spectrum of (2) showed a singlet at δ 3.66 OCH3 and a triplet at δ 3.63 (J 6.6 Hz) (CH2OH) together with a triplet at δ 2.3 with coupling constant 7.5 Hz for CH2 next to the carbonyl group. The 13C NMR spectrum showed a signal at δ 174.4 for the carbonyl carbon at δ 63.1 for the carbon next to the hydroxyl group (CH2OH) and a signal at δ 51.4 (OCH3) . In addition to these there were other carbon signals for the straight carbon chain assigned as follows δ The IR spectrum showed a broad band at 3298 cm-1 for the OH stretch and the broad peak at 1742 cm-1 for the C=O stretch.

The protection of the alcohol functional group

The use of protecting groups in organic chemistry reaction has been referred to as a a necessary evil. It’s use increases the number of steps in the synthesis by creating two additional steps which does not lead to the formation of the product. This also has an implication on the cost of synthesis in that it increases the cost, but its necessary because it masks reactive functional groups, through steps involving reagents which can react with the group, thus preventing the formation of side products which can complicate the synthesis. It also avails the protected functional group when it is needed to react. This is achieved through selective deprotonation reaction. Ethers are used as protecting groups for alcohols due their inert nature 1. The only potential reaction ethers undergo is hydrolysis under strongly acidic conditions to form alcohols. This makes them suitable protecting groups because a protecting group is supposed to be inert to the reaction conditions applied in the subsequent steps of the reaction before it is deprotected 2. The most commonly used ethers are tetrahydropyranyl and methoxymethyl ethers3. 3, 4- dihydro- 2H-pyran undergoes an addition reaction across the double bond to form methyl 15-((tetrahydro-2H-pyran-2-yl)oxy)pentadecanoate. The reaction is catalysed with pyridinium_ p-toulenesulfonate (PPTS), a weakly acidic catalyst to prevent the possibility of hydrolysis of the ester functional group. The 13CNMR spectrum of (3) shows an oxymethine peak (O-CH) at δC 98.8 (C-1’) and an oxy-methylene peak (O-CH2) at δC 67.7 (C-5’), for the tetrahydro- 2H- pyran carbons and the corresponding proton chemical shifts, from 1HNMR spectrum were, δH 4.58 (t) ( J= 3.2 Hz, 1H) for the methine proton (H-1’) and δH 3.87 (dt) 1H, δH 3.72 (dt) IH for the two protons at (C-5’). The two protons are non-chemically equivalent, because the they are in a ring. This in addition with the proton and carbon-13 signals identical to those of compound (2) confirmed the attachment of the 3,4- dihyro-2H- pyran ring onto the alcohol end of the Methyl-15-hydroxypentadecanoate to form methyl 15-((tetrahydro-2H-pyran-2-yl)oxy)pentadecanoate. Unlike in the spectrum of (2) the two protons at H-15 are now not chemically equivalent due to the electronic environment created by the attachment of the tetrahydropyran ring. The rest of the carbon-13 and proton signals were assigned as shown in the table.

13CNMR and 1HNMR assignments of methyl 15-((tetrahydro-2H-pyran-2-yl)oxy)pentadecanoate

Reduction of the ester functional group to a primary alcohol

Lithium aluminium hydride is used for the reduction in this case because it’s a strongest among the hydride reducing agents, since it is a strong reducing agent and it can reduce the less reactive ester functional group to a primary alcohol. The greater hydride character is because the Al-H bond is more polarised than other hydride reducing agents. Two equivalents of the reducing agent is used in this reaction. The aldehyde is first converted into an aldehyde which is then subsequently reduced. NMR spectroscopy was used to confirm the success of the reaction.

The 13CNMR spectrum of compound (4) showed no signal above 150 ppm indicating that the carbonyl carbon had been reduced. In addition to this there was no methoxy carbon and protons as was seen in the spectra of compound (3) above, and there was an additional oxymethylene carbon indicating the presence of a terminal hydroxyl group. This confirmed that the ester had been reduced to form a primary alcohol. The 13CNMR spectrum of compound (4) showed the presence of an oxymethine peak at δC 98.8 and an oxymethylene peak δC 67.7 assignhed to the carbons C-1’ and C-5’ of the tetrahydrofuran moiety, two other oxymethylene carbons δC 63.1 and δC 62.3 assigned to C-1 and C-15 respectively. This was further supported by the presence of a methine proton δH 4.50 (t) 1H, δH 3.87 (dt) 1H and δH 3.72(dt) 1H (H-5’) for the tetrahydro- pyran moiety. δH 3.53 (dt) 1H and δH 3.38 (dt) 1H (H-15) and δH 3.64 (t) 2H (H-1) for the methylene protons of the straight chain. This confirmed the structure of compound (4) to be 15-((tetrahydro-2H-pyran-2-yl)oxy)pentadecan-1-ol. The other assignments are shown in the table below.

13CNMR and 1HNMR assignments of 15-((tetrahydro-2H-pyran-2-yl)oxy)pentadecan-1-ol

Structure of compound (4)

Oxidation of the primary alcohol to an aldehyde

Pyridinium chloro-chromate (PCC) in the solvent dichloromethane (DCM) is used in order to selectively achieve the partial oxidation of the primary alcohol to aldehyde. The use of other chromium based oxidising agents in aqueous solutions leads to the formation of carboxylic acids instead. The reaction is under anhydrous condition to avoid the reaction of the aldehyde formed from reacting with water to form a dihydrate which can further be oxidised to give a carboxylic acid 4.

The 13CNM R spectrum of compound (5) showed a deshielded carbonyl peak at δC 202.4 typical of an aldehyde carbonyl carbon. This was further supported by the presence of a downfield shifted one-proton singlet at δH 9.77. In the 13CNMR spectrum of compound (5) there were only two oxymethylene carbon signals as opposed to three as in the spectra of compound (4), and in addition to the other signals seen in the spectra of compound (4), there was a methylene carbon at δC 43.9 (C-2) which is an sp3 carbon deshielded by the electronic environment of the aldehyde.

13CNMR and 1HNMR assignments of 15-((tetrahydro-2H-pyran-2-yl)oxy)pentadecanal

The formation of alkene through the Wittig reaction

The Wittig reaction is a powerful reaction for the formation of alkenes from carbonyl compounds (aldehydes and ketones) and phosphonium ylides5. The advantage of using the Wittig reaction is that streochemicall outcomes of the reaction can be determined through the choice of the ylide used. Unstabilised phosphorous ylides lead to the formation of Z-alkenes while stabilised phosphorous ylides form E- alkenes. The reaction forms only one double bond exactly where the carbonyl carbon was6. The Wittig reaction also employs mild reaction conditions5. In this reaction an ustabilised phosphorous ylide is used and a Z-alkene is formed. The reaction proceeds via a [2 +2] cycloaddition followed by a cycloreversion.

The 13CNMR spectrum of compound (6) showed the presence of an olefinic carbon signal at δC 129.9. This peak was for the two olefinic carbons because of the presence of symmetry in the molecule the two are chemically equivalent. The peak is also of high intensity indicating it is as a result of more than one carbon nuclei. In addition to these there were; a methoxy carbon δC 55.9 (C-33), two oxymethine peaks δC 98.8 (C-1’) and δC 76.9 (C-31), two oxymethylene peaks at δC 67.7 (C-5’) and δC 62.3 (C-1), a methyl group at δC 19.1 (C-32) and a set of methylene carbon signals. The 1HNMR spectrum of compound (5) showed the presence of signals at δH 5.36 (t) 2H for the chemically equivalent olefinic protons (H-15 and (H-16), δH 4.58 (t) 1H for the methine proton on the tetrahydropyran ring (H-1’), δH 3.88 1H and δH 3.76, δH 3.50 , δH 3.39 (H-), δH 3.28 1H for the methine proton (H-31), δH 3.32 (s) 3H for the methoxy protons (H-33). The presence of both proton and carbon signals for the olefin group indicated the success of the Wittig reaction. Additionally there was no carbonyl carbon signal indicating the aldehyde had reacted.

Deprotection of the alcohol functional group

The tetrahydro-furan moiety which was used in step one to mask the primary alcohol is removed by acid hydrolysis. A weak PPTS is used in this reaction. In the 13CNMR spectrum compound (7) there is only one oxymethine carbon at δC 76.6 as compared to the spectrum of compound (6) where there were 2 oxymethine peaks on belonging to the tetra-hydropyranyl at δC 98.8. In addition to this only one oxymethylene carbon signal was present in the 13CNMR of compound (7) in comparison with compound (6), where they were two. This confirmed the cleavage of the tetrahydropyranyl which was used in step one as a protecting group for the primary alcohol.

The Mitsunobu reaction

This reaction involves the dehydrative coupling of an alcohol which could be a primary, secondary or tertiary alcohol to a pronucleophile (NuH)7. This dehydrative coupling is mediated by a reaction between a triaryl / trialkylphosponium and dialkylazodicarbonyl 8. As the reaction proceeds the azo species is reduced to a derivate of hydrazine and the phosphine is oxidised to phosphine oxide. The reaction generally proceeds with inversion of configuration in almost all cases when a secondary chiral alcohol is used7. Soeme suitable pronucleophiles for the Mitsunobu reaction include; (thio) carboxylc acids, amides, (thio) phenols and sulphonamides. These lead to the formation of C-S, C-N and C-O bonds. Typical Mitsunobu reagents include; diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD) and triphenyl phosphine (PPh3) 9. The DEAD and (PPh3) react to form a betaine with a pKa of 13. In order for this betain to deprotonate an acidic proton from the pro nucleophile, the pronucleophile must have a pKa of 11 or below as dictated by the pKa rule10. If the pKa is above 11, alkylation of the DEAD will occur instead. Solvents commonly used for the reaction include; THF, toluene, diethyl ether, dichloromethane and sometimes even polar such as DMF, ethyl acetate and acetonitrile are also used.

The synthetic scheme

The 1HNMR spectrum of compound (8) showed the presence of five aromatic protons in an ABC spin system, at δH 7.61 (m) 2H assigned to (H-2’ and H-3’), δH 7.56 (m) 2H assigned to (H-4’ and H-6’) and δH 7.54 (m) 1H assigned to H-5’ confirming the presence of a phenyl substituent. This was further confirmed in the 13CNMR and DEPT NMR spectrum which showed 3 sp2 hybridised, methine carbon signals at δC 130.0 assigned to C-2’ and C-3’, δC 129.9 assigned to C-4’ and C-6’ and δC 129.8 assigned to C-5’. The peak at δC 123.9 in the 13CNMR together with the peak at δH 5.35 (dt) 2H confirms the presence of a C=C bond at C-15 and C-16. The 13CNMR spectrum of compound (8) also showed the presence of a methoxy carbon at δC 55.9 (C-33) with the corresponding protons resonating at δH 3.34 (s) 3H (H-33). In the 1HNMR spectrum of compound (8), methyl protons resonating at δH 1.13 (d) 3H (H-32) was evident with the corresponding carbon resonating at δC 19.2. The 13CNMR showed the presence of an oxymethine carbon δC methylene 76.7 assigned to C-31 with the proton attached to this carbon resonating at δH 4.33 (dd) 1H. peaks δC and δC , not seen in the DEPT spectrum indicating they are quartenary carbons assigned to phenyl carbon C-1’ and the tetrazoline carbon C-7’. The carbon chemical shift value of C-1 is substantially reduced since sulphur is less electronegative compared to oxygen. It’s chemical shift value is δC 36.3. Compound (8) is therefore (R)-5-((31-methoxydotriacont-15-en-1-yl)thio)-1-phenyl-1H-tetrazole , confirming the success of the Mitsunobu reaction.

Oxidation of (R,Z)-5-((31-methoxydotriacont-15-en-1-yl)thio)-1-phenyl-1H-tetrazole compound (7) to form (R,Z)-5-((31-methoxydotriacont-15-en-1-yl)sulfonyl)-1-phenyl-1H-tetrazole.

Hydrogen peroxide is the oxidising agent while the molybdenium complex is a catalyst. The 13CNMR spectrum of compound (9) is similar to that of compound (8) with the exception that the chemical shift of C-1, the carbon adjacent to the oxidised sulphur has become ore deshielded with a chemical shift of δC 56.0. Compound (9) is (R)-5-((31-methoxydotriacont-15-en-1-yl)sulfonyl)-1-phenyl-1H-tetrazole.