Synthesis of highly enantioenriched hydroxy- and dihydroxy-fatty esters: substrate precursors for cytochrome P450BioI

Tetrahedron: Asymmetry 22 (2011) 1709–1719

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Synthesis of highly enantioenriched hydroxy- and dihydroxy-fatty esters: substrate precursors for cytochrome P450BioI Arti A. Singh, Siti N. A. Zulkifli, Michaela Meyns, Patricia Y. Hayes ⇑, James J. De Voss ⇑ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane 4072, Australia

a r t i c l e

i n f o

Article history: Received 12 August 2011 Accepted 25 August 2011 Available online 1 November 2011

a b s t r a c t A series of highly enantioenriched hydroxy- and dihydroxy-fatty esters were required as part of our ongoing investigation into cytochrome P450BioI. This mediates the biosynthesis of pimelic acid via C–C bond cleavage of long chain fatty acids within Bacillus subtilis. Herein we report the synthesis of various stereoisomers of methyl 7-hydroxytetradecanoate, methyl 8-hydroxytetradecanoate, and methyl 7,8dihydroxytetradecanoate in highly enantioenriched form, using a combination of asymmetric synthesis and a preparative enantioselective HPLC is reported. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Cytochromes P450 (P450s or CYPs) are a superfamily of oxidative haemoproteins that catalyse a wide variety of oxidative transformations,1 including hydroxylation at unactivated carbon centres2,3 and oxidative C–C bond cleavage reactions.4–8 P450BioI (CYP107H1) from Bacillus subtilis is the first prokaryotic P450 known to catalyse an oxidative C–C bond cleavage.9 It mediates the hydroxylation and C–C cleavage of long chain fatty acids to produce pimelic acid (heptanedioic acid), an important intermediate in the biosynthesis of biotin (Vitamin H).5,9 To investigate the C–C bond cleavage catalysed by P450BioI, a series of hydroxy- and dihydroxytetradecanoic acids 1–3 (Fig. 1) were synthesised and incubated with a catalytically active P450BioI system.5 The enzyme catalysed the oxidation of 7-hydroxytetradecanoic acid 1, but not that of 8-hydroxytetradecanoic acid 2, resulting in pimelic acid production, while processing of 7,8threo-dihydroxytetradecanoic acid threo-3 resulted in a greater production of pimelic acid compared to erythro-3. Probing the stereoselectivity of these transformations required the synthesis of enantioenriched samples of both 1 and threo-3. P450BioI processed alcohol (S)-1 and the corresponding diol (7R,8R)-3 preferentially to pimelic acid over the corresponding enantiomers, but low selectivity was observed, with (R)-1 and (7S,8S)-3 still being good substrates. It was suggested that this lack of selectivity was because the free fatty acids were not the natural substrates for P450BioI. Acyl carrier protein-bound fatty acids (acyl-ACP) have also been isolated in complex with P450BioI when it was heterologously expressed.9 Enzymic oxidation of such complexes led to the spe⇑ Corresponding authors. Tel.: +61 7 3365 3825; fax: + 61 7 3365 4299 (J.J.D.V.). E-mail addresses: [email protected] (P.Y. Hayes), [email protected] (J.J. De Voss). 0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetasy.2011.08.027

cific production of pimelic acid,9,10 whereas the oxidation of free fatty acids primarily produced a range of subterminally (x-1 to x-5) hydroxylated fatty acids.11 This strongly suggested that acyl-ACPs rather than free fatty acids themselves are the natural substrates for P450BioI. Recently-determined X-ray crystal structures of a number of P450BioI-acyl-ACP complexes support this hypothesis and show that the fatty acid chain is oriented in a U shape within the enzyme’s active site, with C-7 and C-8 positioned closest to the haem iron.10,12 These structures also indicate that both the pro-S and pro-R hydrogen atoms at C-7 and C-8 are well positioned for oxygen insertion,10,12 suggesting that there may still be low stereoselectivity observed between the enantiomers of ACP-bound substrates, as previously observed with free hydroxyand dihydroxyacid substrates.5 We wish to explore the stereoselectivity of the C–C bond cleavage catalysed by P450BioI but with ACP-bound hydroxy- and dihydroxy-fatty acids as substrates. Thus, we require highly enantioenriched samples of methyl 7-hydroxytetradecanoate 4, methyl 8-hydroxytetradecanoate 5, 7,8-threo-dihydroxytetradecanoate threo-6 and 7,8-erythro-dihydroxytetradecanoate erythro6. We had previously synthesised enantioenriched samples of 4 and threo-6 but now sought more efficient routes to larger quantities of all of the stereoisomers of 4–6. These would be required for the production of acyl-ACPs and subsequent biochemical and enzyme co-crystallisation studies. Herein we report the synthesis of the enantiomers of the hydroxyesters 4 and 5 utilising preparative enantioselective HPLC, and the enantioselective synthesis of threo dihydroxyesters (7S,8S)-6 and (7R,8R)-6 by a shorter and less demanding route than that reported previously.5 Additionally, an analogous synthesis of the enantiomers of erythro-6 [(7R,8S)-6 and (7S,8R)-6] is detailed, which utilises (S)-methoxyphenylacetic acid (MPA) ester derivatisation to facilitate both enantiomer separation and the determination of the absolute configuration.

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O

OH

RO 1 R = H ,4 R = CH3

O RO OH 2 R = H, 5 R = CH3

O

OH (R)

RO

(R)

OH (7R,8R)-3 R = H, (7R,8R)-6 R = CH3 threo O

OH (S)

RO

reduced with NaBH4 to the racemic hydroxyester rac-7 (83% yield). A practical resolution of 7 by enantioselective HPLC employing a wide range of columns and conditions proved unsuccessful, however, enantiomeric separation on a preparative scale (300 mg) was achieved via enantioselective preparative HPLC (Chiralpak OJ-H column) after derivatisation of rac-7 to its benzoate rac-12. This process yielded (R)-12 and (S)-12 in 33% and 35%, respectively (Scheme 1). The absolute configurations and enantiomeric excesses (ee) of the enantiomers of 12 were determined by enantioselective HPLC comparison with synthetic, enantioenriched (R)-12 (66% ee). Standard (R)-12 was synthesised by asymmetric reduction13 of the acetylenic ketone moiety of ketoester 11 using (9R)CBS-borane to afford the corresponding (R)-configured acetylenic hydroxyester (R)-7 (60% yield), which was then converted to the corresponding benzoate. Enantiomeric excesses of 96% and 88% were determined for the HPLC separated (R)-12 and (S)-12, respectively (Fig. 2). Removal of the benzoate groups from both (R)-12 and (S)-12 using sodium hydroxide in methanol, followed by catalytic hydrogenation (H2, Pd/BaSO4, hexane), afforded the target, saturated hydroxyesters (R)-5 and (S)-5 in good yields.

(S)

OH (7S,8S)-3 R = H, (7S,8S)-6 R = CH3 O

OH (S)

RO

(R)

OH (7R,8S)-3 R = H, (7R,8S)-6 R = CH3 erythro O

OH (R)

RO

(S)

OH (7S,8R)-3 R = H, (7S,8R)-6 R = CH3 Figure 1. Synthesised hydroxy- and dihydroxy-fatty esters and their acid analogues.

2. Results and discussion 2.1. Synthesis of hydroxy-fatty esters: (R)- and (S)-methyl 8hydroxytetradecanoates (R)-5 and (S)-5, and (R)- and (S)-methyl 7-hydroxytetradecanoates (R)-4 and (S)-4 Since we wished to access both enantiomers of the hydroxyfatty esters 4 and 5, a synthetic route that employed resolution at a late stage was attractive. A number of methods to produce these structurally simple molecules can be envisioned but we wished to employ propargylic alcohols such as 7 (Scheme 1) as intermediates. Such molecules can be generated in the enantioenriched form via CBS-borane reduction of the corresponding ynones in a reaction that proceeds with predictable enantioselectivity.13 Thus, standards would be available for assigning the absolute configuration of the two enantiomeric series produced via resolution. Additionally, our previous work had shown that the enantiomers of compounds such as 7 were amenable to analysis via enantioselective chromatography.5 The synthesis of (R)-5 and (S)-5 began with the addition of the anion of THP-protected hept-6-yn-1-ol14 8 (generated by reaction with n-BuLi) to heptanal, which afforded the mono-protected diol rac-9 in 87% yield (Scheme 1). Standard transformations then afforded ketoester 11 in 73% yield over 3 steps, which was subsequently

Figure 2. Enantioselective HPLC traces of rac-12, (R)-12 (96% ee) and (S)-12 (88% ee) following preparative separation, and synthetically (R)-enriched standard (R)-12 (66% ee). Chiralpak OJ-H column, 2% isopropanol in hexane, flow rate 0.5 mL min1, UV detector 254 nm, retention times: (R)-12 (+)-enantiomer, 15.6 min, (S)-12 ()enantiomer, 16.8 min.

A strategy analogous to that employed for the synthesis of (R)-5 and (S)-5 was used to access the enantiomers of methyl 7-hydroxytetradecanoate 4. The key intermediate, methyl 7-hydroxytetradec-5-ynoate 13, was available starting from octanal and THP protected hex-5-yn-1-ol as reported previously.5 As with methyl 8-hydroxytetradec-5-ynoate 7, all attempts to separate the enantiomers of 13 by preparative enantioselective HPLC were unsuccessful, hence rac-13 was converted to the corresponding benzoate rac-14 (Scheme 2). Racemic 14 was subjected to prepara-

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O THPO

a

3

RO

c

3

O

O

OH

O

rac-7

OH

O

O f

O

3

3

O

+

(R)

4

rac-12

4

O

rac-10 R = H

O e

11

rac-9 R = THP

b

3

4

4

8

O

d

3

(S)

3

4

4

OBz

OR

OR

(R)-12 R = Bz

g

(S)-12 R = Bz

g

(R)-7 R = H

(S)-7 R = H

h

h

O

O (R)

O

(S)

3

O

4

3

4

OH

OH

(R)-5

(S)-5

Scheme 1. Synthesis of (R)-5 and (S)-5. Reagents and conditions: (a) (i) n-BuLi, THF, 40 °C, N2; (ii) heptanal, 40 °C to room temperature, N2, 87%; (b) p-TsOH, MeOH, room temperature, 90%; (c) (i) Jones’ reagent, acetone, 0 °C to room temperature;(ii) CH2N2, MeOH, 0 °C to room temperature, 93% over two steps; (d) NaBH4, MeOH, 0 °C, 99%; (e) BzCl, CH2Cl2, pyridine, 65%; (f) Preparative enantioselective HPLC separation (Chiralpak OJ-H column, 1% isopropanol in hexane, flow rate 10 mL min1, UV detector 254 nm), (R)-12 (+)-enantiomer, retention time 18.3 min, 35% yield, 96% ee, (S)-12 ()-enantiomer, retention time 21.1 min, 33% yield, 88% ee; (g) NaOH, MeOH, room temperature, (R)-7 69%, (S)-7 64%; (h) H2, 5% Pd/BaSO4, hexane, room temperature, (R)-5 80%, (S)-5 62%.

tive enantioselective HPLC (Chiralpak OJ-H column) to afford enantiomerically pure (R)-(+)-14 (43% yield) and (S)-()-14 (35% yield). Hydroxyesters (R)-()-4 and (S)-(+)-4 were then obtained from (R)-14 and (S)-14, respectively, following benzoate hydrolysis and catalytic hydrogenation. The absolute stereochemistry of the enantiomers of 4 was assigned by comparing their specific rotations with those of the enantioenriched (R)-()-4 (68% ee) and (S)-(+)-4 (74% ee) produced via asymmetric synthesis previously.5

These results also confirmed the configurations of the enantiomers of 14 and were in accordance with those expected from their HPLC elution order: the (R)-enantiomer of both 12 and 14 elutes first. It is worth noting that, despite the fact 4 and 5 are chiral only because of the differences in substituents some six bonds removed from the stereogenic centre, they have small but characteristic specific rotations; the (R)-isomer is the ()-enantiomer in both cases. Additionally, HPLC resolution of a racemate for the production of

OH

OBz

O

O

a 4

O

4

O

3

3

rac-13

rac-14 OR

OR

O

b

O (R)

O

4

(S)

+ O

3

(R)-14 R = Bz

c

(S)-14 R = Bz

c

(R)-13 R = H

(S)-13 R = H

d O O

d OH

3

4

3

(R)

(R)-4

O 4

OH (S)

O 3

4

(S)-4

Scheme 2. Synthesis of (R)-4 and (S)-4. Reagents and conditions: (a) BzCl, CH2Cl2, pyridine, room temperature, 64%; (b) preparative enantioselective HPLC separation (Chiralpak OJ-H column, 2% isopropanol in hexane, flow rate 10 mL min1, UV detector 254 nm), (R)-14 (+)-enantiomer, retention time 14.9 min, 43% yield, >99% ee, (S)-14 ()-enantiomer, retention time 17.4 min, 35% yield, >99% ee; (c) NaOH, MeOH, room temperature; (d) H2, 5% Pd/BaSO4, hexane, room temperature, (R)-4 40%, (S)-4 35% over two steps.

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enantioenriched 4 and 5 compared favourably to an asymmetric synthesis utilising CBS-borane; higher ee’s were achieved (88– 100% vs 68–74%) and the operationally difficult CBS-borane reduction was avoided. 2.2. Synthesis of the threo and erythro enantiomers of methyl 7,8-dihydroxytetradecanoate, (7S,8S)-6, (7R,8R)-6, (7R,8S)-6 and (7S,8R)-6 The previously reported5 synthesis of (7S,8S)-6 and (7R,8R)-6 utilised non-racemic diethyl tartarates as starting materials, with the production of the enantiomers requiring 12 steps for each. We envisioned a much more efficient synthesis by making use of the key alkyne 15. This could be stereoselectively reduced to either an (E)- or (Z)-alkene that, upon osmium catalysed dihydroxylation, would yield the corresponding threo and erythro-diols (threo-6 and erythro-6), respectively. Sharpless asymmetric dihydroxylation (SAD)15 of the achiral alkenes (E)-16 and (Z)-16 would provide the opportunity for enantioselective introduction of chirality. (E)-Alkenes, such as (E)-16, are known to readily undergo SAD reactions to afford vicinal diols with threo stereochemistry and high ee’s, while SAD reactions of (Z)-alkenes [e.g. (Z)-16] provides erythro vicinal diols, albeit with lower ee’s. The key intermediate 15 (Scheme 3) was available in good yield in three steps from commercially available starting materials. Deprotonation of 1-octyne with n-BuLi, and subsequent reaction with THP-protected 6-bromo-1-hexanol16 17 in the presence of HMPA, afforded the desired product 15 in 81% yield. Br HO

a

Br THPO

3

3

17

b

4

THPO

4

3

15 17 Scheme 3. Synthesis of bromide 17 and alkyne 15. Reagents and conditions: (a) 3,4-dihydro-2H-pyran, p-TsOH, CH2Cl2, 0 °C to room temperature, N2, 83%; (b) (i) nBuLi, THF, 40 °C, N2; (ii) HMPA, bromide 17, 40 °C to room temperature, N2, 81%.

Reduction (Li/NH3) of alkyne 15 afforded the (E)-alkene (E)-18 in 83% yield (Scheme 4). Acid catalysed cleavage of the THP moiety, followed by Jones oxidation of alcohol (E)-19 and esterification of acid (E)-20 afforded the methyl ester (E)-16 in 78% yield over three steps. Dihydroxylation of (E)-16 with either AD-mix-a or AD-mix-b under standard SAD conditions provided the desired 7,8-threodihydroxyesters (7S,8S)-6 and (7R,8R)-6 in 81% and 75% yields, respectively. Based upon the Sharpless predictive model,15 the AD-mix-a would provide the enriched (S,S)-enantiomer and ADmix-b the (R,R)-enantiomer. This prediction was confirmed by comparison of the specific rotations of the compounds obtained here with those of (7S,8S)-6 and (7R,8R)-6 synthesised previously from chiral, non-racemic tartrate starting materials.5 Enantioselective HPLC analysis (Chiralpak AD-H column) revealed an ee >99% for both (7S,8S)-6 (from AD-mix-a) and (7R,8R)-6 (from AD-mixb). This route thus provides highly enantioenriched samples of both enantiomers of threo-6 in nine synthetic steps as compared to 24 steps in the synthesis reported previously.5 The synthesis of the erythro-dihydroxyesters (7R,8S)-6 and (7S,8R)-6 similarly utilised alkyne 15, but with catalytic hydrogenation (Lindlar’s catalyst) affording (Z)-18 in quantitative yield (Scheme 5). GC/MS and NMR analysis revealed the presence of a small amount (10%) of the corresponding, chromatographically inseparable (E)-18. This was therefore carried forward through the synthesis for separation at a later stage. The desired (Z)-methyl ester (Z)-16 was obtained in high yield following standard THPdeprotection of (Z)-18 (94% yield), Jones oxidation of alcohol (Z)19 (92%), and esterification of acid (Z)-20 (96%). Dihydroxylation of (E)-alkenes under SAD conditions usually occurs more quickly than that of the corresponding (Z)-alkenes. Thus, oxidation of the mixture of (Z)- and (E)-16 (10:1) at this stage with 0.15 equiv of AD-mix-a (26 h) allowed dihydroxylation of the 10% of (E)-alkene impurity present, while leaving the majority of (Z)-16 unreacted. Purification of the unreacted alkene (Z)-16 by column chromatography provided geometrically pure (Z)-16. Subsequent dihydroxylation on geometrically pure (Z)-16 under standard SAD conditions (AD-mix-a, 210 h) afforded erythro-6 in 97% yield (Scheme 6). Since the oxidation of (Z)-alkenes with the commercially available SAD reagent premixes is known to occur with less enantioselectivity than of the corresponding (E)-alkenes,15 it was not expected that this step would proceed (E)

a 4

THPO

RO

4

3

3

15

(E)-18 R = THP

b

(E)-19 R = H OH

O

(S)

e

O

3

O c

3

(7S,8S)-6 4

O

f d

OH

(E)-20 R = H (E)-16 R = CH3

4

OH

(E)

RO

(S)

(R)

O

3

(R)

4

OH (7R,8R)-6 Scheme 4. Synthesis of (7S,8S)-6 and (7R,8R)-6 from alkyne 15. Reagents and conditions: (a) Li (s)/NH3 (l), t-BuOH/THF (3:5), 78 °C, 83%; (b) p-TsOH, MeOH, room temperature, 95%; (c) Jones’ reagent, acetone, 0 °C to room temperature, 83%; (d) CH2N2, MeOH, 0 °C to room temperature, 99%; (e) AD-mix-a, MeSO2NH2, t-BuOH/H2O (1:1), 0–4 °C, 81%, >99% ee; (f) AD-mix-b, MeSO2NH2, t-BuOH/H2O (1:1), 0–4 °C, 75%, >99% ee (Chiralpak AD-H column, 2% isopropanol in hexane, flow rate 0.5 mL min1, UV detector 215 nm, retention times: (7S,8S)-6 16. 9 min, (7R,8R)-6 16.0 min).

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a 4

THPO

(Z)

RO

3

enantiomers of erythro-6 into their bis-methoxyphenylacetic acid ester derivatives to at once facilitate both analytical and preparative separation, as well as to allow the determination of their absolute configurations (Mosher’s analysis). Thus the dihydroxyester erythro-6 obtained from the dihydroxylation of (Z)-16 was reacted with (S)-methoxyphenylacetic acid to afford the crude diastereomeric bis-(S)-methoxyphenylacetic acid esters erythro-21 (Scheme 6). Enantioselective HPLC analysis (Chiralpak AD-H column), unsurprisingly, revealed a low de of 10%, reflecting a 10% ee for the dihydroxylation of (Z)-16. The diastereomeric bis-(S)-methoxyphenylacetic acid esters were successfully separated by preparative enantioselective HPLC to afford pure (7R,8S)-21 and (7S,8R)-21 in 32% and 42% yields, respectively (>99% de and ee following separation). Base-catalysed methanolysis of the (S)-methoxyphenylacetic acid esters (7R,8S)-21 and (7S,8R)-21 provided the corresponding enantiomerically pure dihydroxy-fatty esters (7R,8S)-6 (65%) and (7S,8R)-6 (58% yield; Scheme 6). The absolute configuration at the 7- and 8-positions of both (7R,8S)-21 and (7S,8R)-21 was determined by NMR spectroscopy. The signals in the 1H NMR spectra of (7R,8S)-21 and (7S,8R)-21 ob-

4

3

15

(Z)-18 R = THP (Z)-19 R = H

b O c

RO

(Z) 3

d

4

(Z)-20 R = H (Z)-16 R = CH3

Scheme 5. Synthesis of (Z)-16 from alkyne 15. Reagents and conditions: (a) H2, Lindlar’s catalyst (5% Pd/CaCO3 poisoned with lead), EtOAc, room temperature, quantitative; (b) p-TsOH, MeOH, room temperature, 94%; (c) Jones reagent, acetone, 0 °C to room temperature, 92%; (d) CH2N2, MeOH, 0 °C to room temperature, 96%.

with high stereoselectivity but it was hoped that preparative enantioselective HPLC would provide the pure enantiomers. However, all attempts to separate the erythro enantiomers (7S,8R)-6 and (7S,8R)-6 were unsuccessful. It was thus decided to convert the

Ph O O

O (Z)

O

3

O

OH (R)

3

a

(S)

b,c

O

O (R)

3

O

OH

(Z)-16 O

O

4

+ O

3

(S)

O

(S)

OH

4

Ph

H

(R)

O

O

O

4

+

O

(S)

3

(S)

O

O

H Ph

(7R,8S)-21

(7S,8R)-21

d O O

O

Ph

OH erythro-6

4

O

O

H

O (R)

(S)

(S) 4

d OH

3

H

(S)

(R)

O

OH (R)

(S) 4

O

3

(S)

OH (7R,8S)-6

4

OH (7S,8R)-6

Scheme 6. Synthesis of (7R,8S)-6 and (7S,8R)-6 from (Z)-16. Reagents and conditions: (a) AD-mix-a, MeSO2NH2, t-BuOH/H2O (1:1), 0–4 °C, 97%; (b) (S)-methoxyphenylacetic acid, DCC, DMAP, CH2Cl2; (c) preparative enantioselective HPLC separation (Chiralpak AD-H column, 10% isopropanol in hexane, flow rate 10 mL min1, UV detector 254 nm), de 10%; (7R,8S)-21 retention time 22.8 min, 32%, (7S,8R)-21 retention time 28.6 min, 42% (both >99% de and ee); (d) NaOH, MeOH, room temperature, (7R,8S)-6 65%, (7S,8R)-6 58%.

Figure 3. Partial 1H NMR spectra (C6D6, 400 MHz) and diagnostics DdRS values for bis-(S)-methoxyphenylacetic acid esters (7R,8S)-21 and (7S,8R)-21. The signals for H-7 and H-8 were assigned from selective 1D TOCSY experiments.

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tained in C6D6 (Fig. 3) were better resolved than the signals in the corresponding spectra obtained in CDCl3; thus the data obtained in C6D6 were used for the stereochemical analysis. Selective 1D TOCSY experiments were used to assign the signals for H-7 and H-8 in both (7R,8S)-21 and (7S,8R)-21. Irradiation of the signal corresponding to the methylene group adjacent to the methyl ester (H2-2) for (7R,8S)-21 revealed that the signal at dH 4.93 ppm corresponded to H-7, whereas H-7 was found to resonate at dH 5.35 ppm for (7S,8R)-21. This was confirmed by irradiation of the signal corresponding to the terminal methyl group (H3-14) for each isomer, which indicated the signals corresponding to H-8: dH 5.39 for (7R,8S)-21 and dH 4.97 for (7S,8R)-21. Diagnostic DdRS values were then calculated17,18 from the 1H chemical shift values observed for these four important signals (Fig. 3). These calculated values were then used to assign the absolute configuration at the 7- and 8-positions of both (7R,8S)-21 and (7S,8R)-21, based upon the model developed by Seco et al.17,18 for determining the absolute configuration of 1,n-diols from the chemical shifts of their bis-methoxyphenylacetic acid ester derivatives. Despite the requirement of chemical derivatisation, subsequent deprotection and preparative enantioselective HPLC separation in this synthesis of the enantiomers of erythro-6, the route has much to recommend it. It provides both (7R,8S)-6 and (7S,8R)-6 enantiomerically pure and in preparatively useful quantities (10 mg each) in only ten synthetic steps. In addition, the use of the bis(S)-methoxyphenylacetic acid derivatives (7R,8R)-21 and (7S,8S)-21 allowed the direct determination of the absolute stereochemistry of each enantiomer.

vanillin and 2.5 mL concentrated H2SO4 dissolved in 250 mL EtOH). H and 13C NMR spectra were recorded on either a Bruker AMX400 or Bruker AV500 spectrometer as indicated. 1H and 13C signals are recorded in parts per million (ppm) on the d scale, with the residual solvent peaks (CDCl3: dH 7.24 and dC 77.0; C6D6: dH 7.15 and dC 128.0) as internal references. GC/MS analyses were performed on a Shimadzu GCMS QP5000 or QP5050 gas chromatograph mass spectrometer, operating at 70 eV, connected to a GC-17A gas chromatograph fitted with a DB-5 column (30 m, internal diameter 0.25 mm, J&W Scientific). Standard GC/MS programme: split mode; column flow 1.0 mL min1; total flow 102.2 mL min1; injector 250 °C; detector 250 °C; oven 100 °C (1.0 min equilibration) held for 2.0 min, ramp 16 °C min1 to 250 °C and held for 24.0 min (total programme time 35.4 min). Melting points were measured on a Buchi Dr. Tottoli apparatus and are uncorrected. High resolution mass spectra were recorded in positive ion ESI mode on a Bruker micrOTOFQ spectrometer. Elemental microanalyses were performed by the Microanalytical Service, School of Chemistry and Molecular Biosciences, at the University of Queensland. Optical rotations were measured at the sodium D line (589 nm) at ambient temperature using a 1 mL quartz cell with a 10 cm path length, using a Jasco P-2000 polarimeter. Analytical and preparative enantioselective HPLC was performed on an Agilent 1200 Series Liquid Chromatograph System equipped with a UV detector and an ALP (Advanced Laser Polarimeter, PDR-Chiral Inc.) using Chiralpak OJ-H or AD-H columns (20  250 mm or 4.6  250 mm, Daicel Chemical Industries, Ltd.) as indicated. 1

4.2. Synthesis of (R)- and (S)-methyl 8-hydroxytetradecanoates (R)-5 and (S)-5 3. Conclusions Using a combination of asymmetric synthesis and preparative enantioselective HPLC, we have successfully synthesised a series of isomeric hydroxy- and dihydroxy-fatty esters with demonstrated high levels of enantioenrichment in preparatively useful amounts. Both 7-hydroxy and 8-hydroxytetradecanoates 1 and 2 have been reported from natural sources; the former from a variety of butterflies19 and the latter from plant leaves.20 However, the stereochemistry of these naturally occurring compounds has not been investigated and the data reported here should help facilitate future work. Indeed, the positive specific rotation observed for both (S)-4 and (S)-5 aligns with a seemingly general trend for the (S)enantiomers of hydroxy-fatty acids. Additionally, all of the compounds reported here will be useful in our future investigations of the stereoselectivity of the C–C bond cleavage process catalysed by P450BioI.

4. Experimental 4.1. General Materials obtained commercially were of reagent grade unless otherwise stated. Anhydrous solvents were dried according to the established procedures and were distilled under vacuum or N2 immediately prior to use. THF was distilled off sodium-benzophenone ketyl. CH2Cl2 and DMSO were dried over and distilled from CaH2. HMPA was dried and stored over CaH2. Moisture or air sensitive experiments were conducted in oven-dried glassware under a N2 or Ar atmosphere. Flash column chromatography was performed on Silica Gel 60 (0.04–0.06 mm, 230–400 mesh, Scharlau or Merck). TLC was performed on Kieselgel 60 F254 plates (Merck, aluminium backed). Compounds were visualised by treatment with PMA (5% phosphomolybdic acid in EtOH), KMnO4 (1.0 g KMnO4, 5.0 g Na2CO3 and 500 mg NaOH dissolved in 100 mL H2O), or vanillin dips (6.0 g

4.2.1. rac-14-(Tetrahydro-2H-pyran-2-yloxy)tetradec-8-yn-7-ol rac-9 A solution of n-BuLi (1.15 M in hexanes, 24.0 mL, 27.6 mmol) was added dropwise to a solution of 2-(hept-6-ynyloxy)tetrahydro-2H-pyran14 8 (4.00 g, 20.4 mmol) in THF (40 mL) stirred at 40 °C under an N2 atmosphere. The solution was stirred at 40 °C for 3 h, then freshly distilled heptanal (5.81 g, 50.8 mmol) was added dropwise. The reaction mixture was stirred at 40 °C for 15 min, then was allowed to warm to room temperature and was stirred for 2 days. Saturated aqueous NH4Cl solution (100 mL) was then added to quench the reaction, and the mixture was extracted with Et2O (5  100 mL). The combined organic extract was washed with brine (100 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 10–50% EtOAc in petroleum spirit 40–60) to afford rac-9 (5.53 g, 17.8 mmol, 87%) as a colourless oil. 1H NMR (400 MHz, CDCl3) d 0.87 (t, J = 6.6 Hz, 3H, H3-14), 1.20–1.88 (m, 22 H), 2.22 (dt, J = 6.9, 2.0 Hz, 2H, H2-5), 3.39 (td, J = 9.6, 6.6 Hz, 1H, H-1), 3.47–3.53 (m, 1H, H-60 ), 3.74 (td, J = 9.6, 6.8 Hz, 1H, H-1), 3.84–3.90 (m, 1H, H60 ), 4.34 (tt, J = 6.5, 1.9 Hz, 1H, H-8), 4.57 (t, J = 3.5 Hz, 1H, H-20 ). 13 C NMR (100 MHz, CDCl3) d 14.1 (C-14), 18.6, 19.7, 22.6, 25.2, 25.5, 28.4, 28.9, 29.2, 29.7, 30.8, 31.7, 38.2, 62.4 (C-60 ), 62.8 (C-8), 67.4 (C-1), 81.5 (C„C), 85.3 (C„C), 98.9 (C-20 ). GC/MS EI m/z (%) 209 (1), 165 (1), 141 (6), 101 (9), 85 (100), 84 (35), 67 (31), 57 (25), 55 (83), 43 (46), 41 (51). Anal. Calcd for C19H34O3: C, 73.50; H, 11.04. Found: C, 73.13; H, 11.29. HRMS ESI calcd for C19H34NaO3 ([M+Na]+): 333.2400. Observed: 333.2389. 4.2.2. rac-Tetradec-6-yne-1,8-diol rac-10 A solution of rac-9 (4.80 g, 15.5 mmol) and p-toluenesulfonic acid (cat., 200 mg) in MeOH (200 mL) was stirred at room temperature for 2 h. Solid NaHCO3 (5.0 g) was added to quench the reaction and the solvent was evaporated under reduced pressure The residue was purified by flash chromatography (silica gel, 5–50%

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EtOAc in petroleum spirit 40–60) to afford the diol rac-10 (3.16 g, 14.0 mmol, 90%) as a colourless oil. 1H NMR (400 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H, H3-14), 1.22–1.72 (m, 16H), 2.22 (dt, J = 6.9, 2.0 Hz, 2H, H2-5), 3.65 (t, J = 6.5 Hz, 2H, H2-1), 4.33 (tt, J = 6.6, 1.8 Hz, 1H, H-8). 13C NMR (100 MHz, CDCl3) d 14.0 (C-14), 18.6, 22.6, 24.9, 25.2, 28.3, 28.9, 31.7, 32.1, 38.2, 62.7, 62.8, 81.6 (C„C), 85.1 (C„C). GC/MS EI m/z (%) 180 (3), 141 (33), 138 (12), 109 (14), 95 (61), 79 (50), 67 (70), 55 (100), 43 (94), 41 (79). Anal. Calcd for C14H26O2: C, 74.29; H, 11.58. Found: C, 73.88; H, 11.74. HRMS ESI calcd for C14H26NaO2 ([M+Na]+): 249.1825. Observed: 249.1832. 4.2.3. Methyl 8-oxotetradec-6-ynoate 11 To a solution of rac-10 (143 mg, 0.63 mmol) in acetone (6 mL) stirred at 0 °C was added Jones’ reagent (8 M) dropwise until the reaction mixture remained orange in colour. The reaction mixture was allowed to warm to room temperature with stirring over 20 min, then was quenched by the addition of H2O (20 mL). The mixture was extracted with Et2O (4  10 mL) and the combined organic extract was dried over anhydrous MgSO4, filtered and concentrated under reduced pressure. The crude acid was redissolved in methanol (10 mL) and the solution was cooled to 0 °C. An ether CH2N2 solution was added dropwise until a yellowish colour persisted. The excess CH2N2 was evaporated under a stream of N2 and the solvent was removed in vacuo. The residue was purified by flash chromatography (silica gel, 5% Et2O in hexane) to afford 11 (149 mg, 0.59 mmol, 93% over 2 steps) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) d 0.88 (t, J = 6.9 Hz, 3H, H3-14), 1.26–1.37 (m, 8H), 1.61–1.67 (m, 2H), 1.75 (m, 2H), 2.35 (t, J = 7.3 Hz, 2H), 2.39 (t, J = 7.0 Hz, 2H), 2.52 (t, J = 7.3 Hz, 2H), 3.68 (s, 3H, RCO2CH3). 13 C NMR (100 MHz, CDCl3) d 14.0 (C-14), 18.7, 22.4, 24.1, 27.1, 28.6, 29.7, 31.5, 33.4, 45.5 (C-9), 51.6 (RCO2CH3), 81.1 (C„C), 93.1 (C„C), 173.6 (C-1), 188.5 (C-8). GC/MS EI m/z (%) 221 (10), 135 (53), 108 (64), 95 (42), 79 (100), 77 (36), 67 (28), 55 (39), 43 (83), 41 (64). Anal. Calcd for C15H24O3: C, 71.39; H, 9.59. Found: C, 71.20; H, 9.63. HRMS ESI calcd for C15H24NaO3 ([M+Na]+): 275.1618. Observed: 275.1623. 4.2.4. rac-Methyl 8-hydroxytetradec-6-ynoate rac-7 A solution of 11 (149 mg, 0.59 mmol) in MeOH (25 mL) was cooled to 0 °C and then NaBH4 (88 mg, 2.33 mmol) was added portion-wise. The reaction mixture was stirred at 0 °C for 2 h, then aqueous oxalic acid solution (5%, 5 mL) was added. The MeOH was removed by rotary evaporation and then the residue was extracted with CH2Cl2 (4  20 mL). The combined organic extract was washed with saturated aqueous NaHCO3 solution (20 mL) and brine (20 mL). The organic solution was dried over anhydrous MgSO4, filtered and concentrated in vacuo. Purification by flash chromatography (silica gel, 20% EtOAc in hexane) afforded rac-7 (149 mg, 0.59 mmol, 99%) as a colourless oil. 1H NMR (500 MHz, CDCl3) d 0.88 (t, J = 7.1 Hz, 3H, H3-14), 1.23–1.35 (m, 6H), 1.38– 1.46 (m, 2H), 1.54 (m, 2H), 1.59–1.77 (m, 4H), 2.23 (dt, J = 7.0, 1.9 Hz, 2H, H2-5), 2.33 (t, J = 7.5 Hz, 2H, H2-2), 3.67 (s, 3H, RCO2CH3), 4.33 (tt, J = 6.6, 1.9 Hz, 1H, H-8). 13C NMR (125 MHz, CDCl3) d 14.0 (C-14), 18.4, 22.6, 24.1, 25.1, 28.0, 28.9, 31.7, 33.5, 38.2, 51.5 (RCO2CH3), 62.7 (C-8), 81.9 (C„C), 84.6 (C„C), 173.9 (C-1). GC/MS EI m/z (%) 223 (1), 169 (72), 137 (68), 109 (63), 92 (35), 81 (73), 79 (59.), 74 (31), 67 (66), 55 (70), 43 (100), 41 (98). Anal. Calcd for C15H26O3: C, 70.83; H, 10.30. Found: C, 70.68; H, 10.49. HRMS ESI calcd for C15H26NaO3 ([M+Na]+): 277.1774. Observed: 277.1784. 4.2.5. Enantioenriched (R)-Methyl 8-hydroxytetradec-6-ynoate (R)-7 (9R)-CBS-borane (1.0 M in toluene, 0.49 mL, 0.49 mmol) was added to a solution of methyl 8-oxotetradec-6-ynoate 11 (60 mg,

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0.24 mmol) in THF (5 mL) stirred under an N2 atmosphere. After 10 min the solution was cooled to 30 °C and a borane–DMS complex (5 M in Et2O, 53 lL, 0.27 mmol) was added dropwise. After 75 min, the reaction mixture was quenched by the addition of MeOH (1 mL) and was warmed to room temperature and diluted with Et2O (20 mL). The organic solution was washed with a 1:1 solution of 1 M aqueous NaOH and saturated aqueous NaHCO3 (4  20 mL), and then brine (30 mL), and then was dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 20% EtOAc in hexane) to afford enantioenriched (R)-7 (36 mg, 0.14 mmol, 60%) as a colourless oil. ½a25 D ¼ 1:3 (c 0.95, CHCl3). This compound was spectroscopically identical to rac-7. 4.2.6. rac-14-Methoxy-14-oxotetradec-8-yn-7-yl benzoate rac12 Benzoyl chloride (583 lL, 5.0 mmol) was added dropwise to a solution of rac-7 (438 mg, 1.72 mmol) and pyridine (826 lL, 10.2 mmol) in anhydrous CH2Cl2 (30 mL) stirred at 0 °C. The reaction mixture was allowed to warm to room temperature and was stirred for 4 h, then water (5 mL) was added to quench the reaction. The organic and aqueous layers were separated and the aqueous solution was extracted with Et2O (3  20 mL). The combined organic extract was dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (silica gel, 5% EtOAc in hexane) to afford the pure benzoate rac-12 (400 mg, 1.12 mmol, 65%) as a colourless oil. 1H NMR (400 MHz, CDCl3) d 0.87 (t, J = 7.0 Hz, 3H, H3-14), 1.14–1.28 (m, 6H), 1.35–1.47 (m, 4H), 1.57–1.65 (m, 2H), 1.69–1.82 (m, 2H), 2.13 (dt, J = 7.2, 1.9 Hz, 2H, H2-5), 2.41 (t, J = 7.2 Hz, 2H, H22), 3.54 (s, 3H, RCO2CH3), 5.47 (tt, J = 6.5, 1.9 Hz, 1H, H-8), 7.30– 7.35 (m, 2H), 7.42–7.47 (m, 1H), 7.93–7.97 (m, 2H). 13C NMR (100 MHz, CDCl3) d 14.0 (C-14), 18.4, 22.5, 24.1, 25.1, 27.9, 28.8, 31.7, 33.5, 35.2, 51.5 (RCO2CH3), 65.1 (C-8), 78.2 (C„C), 85.5 (C„C), 128.3 (2C), 129.7 (2C), 130.2, 132.9, 165.7 (RCO2Ph), 173.8 (C-1). GC–MS EI m/z (%) 226 (2) 105 (100), 78 (5), 77 (63), 51 (82) 50 (29), 43 (22). Anal. Calcd for C22H30O4: C, 73.71; H, 8.44. Found: C, 73.55; H, 8.73. 4.2.7. Analysis and preparative enantioselective HPLC separation of rac-12 Preparative enantioselective HPLC separation (Chiralpak OJ-H column, 1% isopropanol in hexane, flow rate 10 mL min1, UV detector 254 nm) was performed with 1 mL injections of a 1 mg/ mL solution of rac-12. Retention times: (R)-12 [(+)-enantiomer] 18.3 min, (S)-12 [()-enantiomer] 21.1 min. The enantiomeric purity of each isomer following preparative separation was determined by analytical enantioselective HPLC (Chiralpak OJ-H column, 2% isopropanol in hexane, flow rate 0.5 mL min1, UV detector 254 nm). Retention times: (R)-12 [(+)-enantiomer] 15.0 min, (S)-12 [()-enantiomer] 16.0 min. 4.2.7.1. (+)-(R)-14-Methoxy-14-oxotetradec-8-yn-7-yl benzoate (R)-12. 106 mg, 0.30 mmol, 35%, 96% ee. ½a25 D ¼ þ17:3 (c 0.93, CHCl3). By comparison with the enantioenriched standard (R)-12, the observed retention time of 15.2 min indicated that the major isomer was the (R)-enantiomer. This compound was spectroscopically identical to rac-12. 4.2.7.2. ()-(S)-14-Methoxy-14-oxotetradec-8-yn-7-yl benzoate (S)-12. 100 mg, 0.28 mmol, 33%, 88% ee. ½a25 D ¼ 18:6 (c 0.68, CHCl3). The observed retention time of 16.2 min indicated that the major isomer was the (S)-enantiomer. This compound was spectroscopically identical to rac-12.

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4.2.8. Enantioenriched (R)-14-Methoxy-14-oxotetradec-8-yn-7yl benzoate (R)-12 Following a procedure analogous to that for the synthesis of rac12, enantioenriched (R)-12 (15 mg, 48 lmol, 30%) was synthesised from enantioenriched (R)-7 (35 mg, 138 lmol) and was obtained as a colourless oil. 66% ee (Chiralpak OJ-H column, 2% isopropanol in hexane, flow rate 0.5 mL min1, UV detector 254 nm; retention times: major isomer (R)-12 15.6 min, minor isomer (S)-12 16.8 min). ½a25 D ¼ þ12:5 (c 1.20, CHCl3). This compound was spectroscopically identical to rac-12. 4.2.9. ()-(R)-Methyl 8-hydroxytetradec-6-ynoate (R)-7 Powdered solid NaOH (80 mg, 2.0 mmol) was added to a solution of (R)-12 (106 mg, 0.30 mmol; obtained from resolution of rac-12) in MeOH (10 mL), followed by a drop of water. The solution was allowed to stir at room temperature overnight, and then water was added (20 mL) and the mixture was extracted with EtOAc (2  20 mL). After drying over anhydrous MgSO4, the combined organic extract was filtered and then concentrated in vacuo. The crude product was purified by flash chromatography to afford (R)-7 (52 mg, 0.20 mmol, 69%) as a colourless oil. ½a25 D ¼ 1:7 (c 0.46, CHCl3). This compound was spectroscopically identical to rac-7. 4.2.10. (+)-(S)-Methyl 8-hydroxytetradec-6-ynoate (S)-7 Following a procedure analogous to that for the synthesis of (R)7, the enantiomeric (S)-7 (48 mg, 0.19 mmol, 64%) was synthesised from (S)-7 (105 mg, 0.29 mmol) and was obtained as a colourless oil. ½a25 D ¼ þ1:9 (c 0.90, CHCl3). This compound was spectroscopically identical to rac-7. 4.2.11. (R)-Methyl 8-hydroxytetradecanoate (R)-5 A solution of (R)-7 (52 mg, 0.20 mmol) in hexane (20 mL) was stirred under an H2 atmosphere for 1 h in the presence of a catalytic amount of 5% Pd/BaSO4. After filtration, concentration and purification by flash chromatography (silica gel, 25% EtOAc in hexane), (R)-5 (42 mg, 0.16 mmol, 80%) was obtained as a white solid 1 (mp 29–30 °C). ½a25 D ¼ 1:2 (c 0.68, CHCl3). H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.8 Hz, 3H, H3-14), 1.23–1.46 (m, 19H), 1.56– 1.64 (m, 2H), 2.28 (t, J = 7.5 Hz, 2H, H2-2), 3.52–3.60 (m, 1H, H-8), 3.64 (s, 3H, RCO2CH3). 13C NMR (100 MHz, CDCl3) d 14.1 (C14), 22.6, 24.8, 25.4, 25.6, 29.1, 29.3, 29.4, 31.8, 34.0, 37.4, 37.5, 51.5 (RCO2CH3), 71.9 (C-8), 174.3 (C-1). GC/MS EI m/z (%) 173 (5), 141 (24), 101 (19), 95 (18), 87 (36), 74 (11), 69 (18), 67 (10), 59 (19), 57 (24), 56 (9), 55 (81), 45 (12), 44 (13), 43 (97), 41 (100). Anal. Calcd for C15H30O3: C, 69.72; H, 11.70. Found: C, 70.01; H, 11.88. 4.2.12. (S)-Methyl 8-hydroxytetradecanoate (S)-5 Following a procedure analogous to that for the synthesis of (R)5, the enantiomeric (S)-5 (30 mg, 0.12 mmol, 62%) was synthesised from (S)-7 (48 mg, 0.19 mmol) and was obtained as a white solid (mp 29–30 °C). ½a25 D ¼ þ1:8 (c 0.80, CHCl3). This compound was spectroscopically identical to (R)-5. 4.3. Synthesis of (R)-and (S)-methyl 7-hydroxytetradecanoates (R)-4 and (S)-4 4.3.1. rac-1-Methoxy-1-oxotetradec-5-yn-7-yl benzoate rac-14 Following a procedure analogous to that for the synthesis of benzoate rac-12, benzoate rac-14 (300 mg, 0.84 mmol, 64%) was synthesised from methyl 7-hydroxytetradec-5-ynoate5 rac-13 (330 mg, 1.31 mmol) and was obtained as a colourless oil. 1H NMR (400 MHz, CDCl3) d 0.85 (t, J = 7.0 Hz, 3H, H3-14), 1.35– 1.22 (m, 7H), 1.51–1.44 (m, 2H), 1.89–1.77 (m, 4H), 2.27 (dt,

J = 7.1, 2.1 Hz, 2H, H2-5), 2.41 (t, J = 7.7 Hz, 2H, H2-2), 3.65 (s, 3H, RCO2CH3), 5.56 (tt, J = 6.6, 1.9 Hz, 2H, H-7), 7.44–7.40 (m, 2H), 7.56–7.52 (m, 1H), 8.05–8.02 (m, 2H). 13C NMR (100 MHz, CDCl3) d 14.1 (C-14), 18.2, 22.6, 23.7, 29.1 (2C), 31.7, 32.7, 35.1, 51.5 (RCO2CH3), 65.0 (C-8), 78.7 (C„C), 84.8 (C„C), 128.3 (2C), 129.7 (2C), 130.2, 133.0, 165.6 (RCO2Ph), 173.5 (C-1). GC/MS EI m/z (%) 271 (3), 221 (1), 194 (1), 174 (2), 105 (100), 91 (8), 77 (30), 67 (7), 55 (19), 43 (26), 41 (32). Anal. Calcd for C22H30O4: C, 73.71; H, 8.44. Found: C, 73.88; H, 8.73. HRMS ESI calcd for C22H30NaO4 ([M+Na]+): 381.2042. Observed: 381.2036.

4.3.2. Analysis and preparative enantioselective HPLC separation of rac-14 Preparative enantioselective HPLC separation (Chiralpak OJ-H column, 2% isopropanol in hexane, flow rate 10 mL min1, UV detector 254 nm) was performed with 1 mL injection of a 5 mg/mL solution of rac-14. Retention times: (R)-14 [(+)-enantiomer] 14.9 min, (S)-14 [()-enantiomer] 17.4 min. Both enantiomers were obtained enantiomerically pure (>99% ee) after preparative HPLC separation, as determined by analytical enantioselective HPLC (Chiralpak OJ-H column, 2% isopropanol in hexane, flow rate 0.5 mL min1, UV detector 254 nm). Retention times: (R)-14 [(+)-enantiomer] 13.6 min, (S)-14 [()-enantiomer] 16.9 min.

4.3.2.1. (+)-(R)-1-Methoxy-1-oxotetradec-5-yn-7-yl benzoate (R)-14. 128 mg, 0.36 mmol, 43%, >99% ee. ½a25 D ¼ þ20:4 (c 1.20, CHCl3). This compound was spectroscopically identical to rac-14.

4.3.2.2. ()-(S)-1-Methoxy-1-oxotetradec-5-yn-7-yl benzoate (S)-14. 106 mg, 0.30 mmol, 35%, >99% ee. ½a25 D ¼ 21:7 (c 1.10, CHCl3). This compound was spectroscopically identical to rac-14.

4.3.3. (S)-Methyl 7-hydroxytetradecanoate (S)-4 Powdered solid NaOH (80 mg, 2.0 mmol) was added to a solution of (S)-14 (106 mg, 0.30 mmol) in MeOH (10 mL), followed by a drop of water. The solution was allowed to stir at room temperature overnight, and then water was added (20 mL) and the mixture was extracted with EtOAc (2  20 mL). After drying over anhydrous MgSO4, the combined organic extract was filtered and then concentrated in vacuo to give the crude hydroxyester (S)-13. A solution of crude (S)-13 in hexane (2 mL) was stirred under an H2 atmosphere in the presence of a catalytic amount of 5% Pd/BaSO4 for 1 h. After filtration, concentration and purification by flash chromatography (silica gel, 25% EtOAc in hexane), (S)-4 was obtained (27 mg, 0.10 mmol, 35% over 2 steps) as a white solid (mp 28–30 °C, lit.5 29.5–30.5 °C). ½a25 D ¼ þ0:9 (c 2.20, CHCl3). For enantioenriched (S)-4 (74% ee) synthesised previously,5 ½a25 D ¼ þ0:8 (c 1.20, CHCl3). This compound was spectroscopically identical to the enantioenriched (S)-4 reported previously.5 4.3.4. (R)-Methyl 7-hydroxytetradecanoate (R)-4 Following a procedure analogous to that described for the synthesis of (S)-4, the enantiomeric (R)-4 (37 mg, 0.14 mmol, 40% over 2 steps) was synthesised from (R)-14 (128 mg, 0.36 mmol) and was obtained as a white solid (mp 30–32 °C, lit.5 29.5–30.5 °C). ½a25 D ¼ 1:6 (c 0.95, CHCl3). For enantioenriched (R)-4 (68% ee) synthesised previously,5 ½a25 D ¼ 1:2 (c 1.20, CHCl3). This compound was spectroscopically identical to the enantioenriched (R)-4 reported previously.5

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4.4. Synthesis of the threo enantiomers of methyl 7,8dihydroxytetradecanoate, (7S,8S)-6 and (7R,8R)-6 4.4.1. 2-(Tetradec-7-yn-1-yloxy)tetrahydro-2H-pyran 15 n-BuLi (1.42 M in hexanes, 6.0 mL, 8.5 mmol) was added dropwise to a solution of 1-octyne (96%, 1.60 mL, 10.4 mmol) in anhydrous THF (12 mL) stirred at 40 °C under a N2 atmosphere. The solution was stirred at 40 °C for 2.5 h, then HMPA (2.0 mL) was added, followed 15 min later by dropwise addition of 2-(6-bromohexyloxy)-tetrahydro-2H-pyran16 17 (2.45 g, 9.2 mmol). The reaction mixture was allowed to warm to room temperature and was stirred for 24 h. Saturated aqueous NH4Cl solution (20 mL) was added to quench the reaction and the mixture was extracted with petroleum spirit 40–60 (6  25 mL). The combined organic extract was washed with aqueous LiCl solution (4 M, 6  25 mL) and brine (4  25 mL), and then was dried over anhydrous MgSO4 and filtered. The solvent was removed by rotary evaporation and the crude product was purified by flash column chromatography (silica gel, 0.5–2% Et2O in petroleum spirit 40–60) to afford the alkyne 15 (2.04 g, 6.9 mmol, 81%) as a colourless oil. 1H NMR (400 MHz, CDCl3) d 0.87 (t, J = 7.0 Hz, 3H, H3-14), 1.18–1.88 (m, 22H), 2.12 (m, 4H, H2-6 & H2-9), 3.37 (td, J = 9.6, 6.6 Hz, 1H, H-1), 3.48 (m, 1H, H-60 ), 3.71 (td, J = 9.6, 6.9 Hz, 1H, H-1), 3.85 (m, 1H, H-60 ), 4.55 (m, 1H, H-20 ). 13C NMR (100 MHz, CDCl3) d 14.0 (C14), 18.7, 18.8, 19.7, 22.6, 25.5, 25.8, 28.5, 28.7, 29.11, 29.13, 29.7, 30.8, 31.4, 62.3 (C-60 ), 67.6 (C-1), 80.1 (C„C), 80.3 (C„C), 98.8 (C-20 ). GC/MS EI m/z (%) 294 (0.04, M+), 223 (1), 221 (1), 209 (1), 195 (1), 137 (1), 123 (1), 121 (2), 109 (4), 101 (7), 95 (10), 85 (71), 81 (19), 71 (2), 67 (41), 57 (14), 55 (55), 43 (47), 41 (100). Anal. Calcd for C19H34O2: C, 77.50; H, 11.64. Found: C, 77.34; H, 11.91. HRMS ESI calcd for C19H34NaO2 ([M+Na]+): 317.2457. Observed: 317.2437. 4.4.2. (E)-2-(Tetradec-7-en-1-yloxy)tetrahydro-2H-pyran (E)-18 Lithium metal (150 mg, 21.6 mmol) was added to liquid NH3 (50 mL) stirred at 78 °C until the solution remained blue in colour. t-BuOH (3 mL) was added, followed by dropwise addition of a solution of alkyne 15 (595 mg, 2.02 mmol) in anhydrous THF (5 mL). The reaction mixture was stirred at 78 °C for 4 h, then was allowed to slowly warm to room temperature while stirring overnight, during which time the NH3 evaporated. Saturated aqueous NH4Cl solution (50 mL) was added to quench the reaction, and the mixture was extracted with Et2O (6  30 mL). The combined organic extract was washed with brine (2  30 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, 0.5% Et2O in petroleum spirit 40–60) to afford (E)-18 (495 mg, 1.67 mmol, 83%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.9 Hz, 3H, H3-14), 1.17–1.40 (m, 14H), 1.43–1.63 (m, 6H), 1.69 (m, 1H), 1.80 (m, 1H), 1.95 (m, 4H, H2-6 & H2-9), 3.36 (td, J = 9.6, 6.7 Hz, 1H, H-1), 3.48 (m, 1H, H-60 ), 3.71 (td, J = 9.6, 6.9 Hz, 1H, H-1), 3.85 (m, 1H, H-60 ), 4.55 (m, 1H, H-20 ), 5.36 (m, 2H, H-7 & H-8). 13C NMR (100 MHz, CDCl3) d 14.1 (C-14), 19.7, 22.6, 25.5, 26.1, 28.8, 29.0, 29.57, 29.61, 29.7, 30.8, 31.7, 32.5, 32.6, 62.3 (C-60 ), 67.7 (C-1), 98.8 (C-20 ), 130.2 (RHC@CHR), 130.5 (RHC@CHR). GC/MS EI m/z (%) 296 (0.1, M+), 223 (1), 205 (1), 194 (1), 138 (1), 124 (1), 111 (1), 109 (4), 101 (2), 96 (9), 85 (100), 82 (13), 71 (2), 69 (17), 67 (23), 57 (10), 55 (28), 43 (14), 41 (27). Anal. Calcd for C19H36O2: C, 76.97; H, 12.24. Found: C, 76.96; H, 12.08. HRMS ESI calcd for C19H36NaO2 ([M+Na]+): 319.2613. Observed: 319.2597. 4.4.3. (E)-Tetradec-7-en-1-ol (E)-19 Following a procedure analogous to that described for the synthesis of rac-10 from rac-9, THP-ether (E)-18 (495 mg, 1.67 mmol) was deprotected to afford alcohol (E)-19 (336 mg, 1.58 mmol, 95%)

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as a pale yellow oil. 1H NMR (500 MHz, CDCl3) d 0.86 (t, J = 7.0 Hz, 3H, H3-14), 1.15–1.38 (m, 15H), 1.55 (m, 2H), 1.95 (m, 4H, H2-6 & H2-9), 3.62 (t, J = 6.7 Hz, 2H, H2-1), 5.36 (m, 2H, H-7 & H-8). 13C NMR (125 MHz, CDCl3) d 14.1 (C-14), 22.6, 25.6, 28.8, 28.9, 29.55, 29.60, 31.7, 32.5, 32.6, 32.8, 63.1 (C-1), 130.1 (RHC@CHR), 130.5 (RHC@CHR). GC/MS EI m/z (%) 195 (0.1, M+OH), 194 (1, M+H2O), 138 (1), 123 (1), 111 (1), 109 (6), 95 (14), 85 (1), 81 (25), 71 (3), 67 (44), 57 (13), 55 (56), 45 (3), 43 (39), 41 (100). Anal. Calcd for C14H28O: C, 79.18; H, 13.29. Found: C, 78.92; H, 13.44. 4.4.4. (E)-Tetradec-7-enoic acid (E)-20 To a solution of alcohol (E)-19 (220 mg, 1.04 mmol) in acetone (25 mL) stirred at 0 °C was added Jones’ reagent (8 N) dropwise until the reaction mixture remained orange in colour. The reaction mixture was allowed to warm to room temperature with stirring over 20 min, then was quenched by the addition of H2O (50 mL). The mixture was extracted with Et2O (5  25 mL) and the combined organic extract was washed with brine (3  25 mL) and then was extracted with aqueous NaOH solution (5%, 5  25 mL). The combined basic aqueous extract was cooled to 0 °C and concentrated aqueous HCl (32%, 25 mL) was added dropwise until the solution was strongly acidic. The acidic aqueous solution was extracted with Et2O (5  25 mL), and the combined organic extract was washed with brine (3  25 mL), dried over anhydrous MgSO4 and filtered. The solvent was removed in vacuo to afford the acid (E)-20 (195 mg, 0.86 mmol, 83%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.9 Hz, 3H, H3-14), 1.17–1.40 (m, 12H), 1.62 (m, 2H), 1.95 (m, 4H, H2-6 & H2-9), 2.33 (t, J = 7.5 Hz, 2H, H2-2), 5.36 (m, 2H, H-7 & H-8). 13C NMR (100 MHz, CDCl3) d 14.1 (C-14), 22.6, 24.5, 28.5, 28.8, 29.2, 29.6, 31.7, 32.3, 32.6, 33.8, 129.9 (RHC@CHR), 130.8 (RHC@CHR), 179.2 (C-1). GC/MS EI m/z (%) 226 (0.2, M+), 164 (1), 151 (1), 137 (1), 125 (1), 123 (2), 115 (1), 111 (2), 110 (3), 101 (2), 95 (6), 91 (1), 87 (1), 85 (2), 83 (9), 79 (3), 73 (7), 71 (4), 69 (19), 67 (17), 60 (9), 59 (1), 57 (10), 55 (64), 45 (17), 43 (52), 41 (100). HRMS ESI calcd for C14H26NaO2 ([M+Na]+): 249.1830. Observed: 249.1825. 4.4.5. (E)-Methyl tetradec-7-enoate (E)-16 Ethereal CH2N2 was added to a solution of acid (E)-20 (190 mg, 0.84 mmol) in MeOH (20 mL) stirred at 0 °C until the solution remained yellow in colour. The reaction mixture was stirred at 0 °C for 30 min, then was allowed to warm to room temperature and was stirred for a further 30 min. Excess CH2N2 was evaporated under a stream of N2 and the solvent was removed in vacuo to afford the methyl ester (E)-16 (200 mg, 0.83 mmol, 99%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.9 Hz, 3H, H3-14), 1.17– 1.40 (m, 12H), 1.60 (m, 2H), 1.95 (m, 4H, H2-6 & H2-9), 2.28 (t, J = 7.6 Hz, 2H, H2-2), 3.64 (s, 3H, RCO2CH3) 5.36 (m, 2H, H-7 & H8). 13C NMR (100 MHz, CDCl3) d 14.1 (C-14), 22.6, 24.8, 28.6, 28.8, 29.2, 29.6, 31.7, 32.3, 32.6, 34.1, 51.4 (RCO2CH3), 129.9 (RHC@CHR), 130.7 (RHC@CHR), 174.3 (C-1). GC/MS EI m/z (%) 240 (0.6, M+), 209 (2), 208 (3), 190 (0.4), 179 (1), 166 (3), 151 (1), 141 (1), 137 (2), 128 (1), 125 (1), 123 (4), 115 (1), 111 (3), 110 (5), 101 (2), 96 (12), 91 (2), 87 (14), 85 (3), 84 (15), 81 (11), 74 (27), 73 (4), 71 (3), 67 (22), 59 (19), 57 (8), 55 (68), 43 (54), 41 (100). Anal. Calcd for C15H28O2: C, 74.95; H, 11.74. Found: C, 74.99; H, 11.80. HRMS ESI calcd for C15H28NaO2 ([M+Na]+): 263.1987. Observed: 263.1982. 4.4.6. (7S,8S)-Methyl 7,8-dihydroxytetradecanoate (7S,8S)-6 To a solution of t-BuOH and H2O (1:1, 2 mL) stirred at 0 °C was added AD-mix-a (Aldrich, 337 mg) and methanesulfonamide (98%, 25 mg, 0.25 mmol). The mixture was stirred for 15 min, then a solution of (E)-16 (54 mg, 0.23 mmol) in t-BuOH (2 mL) was added. H2O (2 mL) was added and the reaction mixture was stirred at 4 °C for 67 h. The reaction mixture was then re-cooled to 0 °C and solid

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Na2SO3 (44 mg, 0.35 mmol) was added. The mixture was allowed to warm to room temperature with stirring over 30 min and was then extracted with EtOAc (5  10 mL). The combined organic extract was washed with brine (10 mL), dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, 50% EtOAc in petroleum spirit 40–60) to afford (7S,8S)-6 (50 mg, 0.18 mmol, 81%) as a white solid (mp 35–36 °C, lit.5 mp 35–36 °C). >99% ee (Chiralpak AD-H column, 2% isopropanol in hexane, flow rate 0.5 mL min1, UV detector 215 nm, retention time (7S,8S)-6 16.9 min. ½a25 D ¼ 20:3 (c 0.57, MeOH). 1H NMR (400 MHz, CDCl3) d 0.87 (t, J = 6.8 Hz, 3H, H3-14), 1.20–1.54 (m, 16H), 1.63 (m, 2H), 1.91 (d, J = 4.2 Hz, 1H, ROH), 1.99 (d, J = 4.2 Hz, 1H, ROH), 2.30 (t, J = 7.5 Hz, 2H, H2-2), 3.38 (m, 2H, H-7 & H-8), 3.65 (s, 3H, RCO2CH3). 13 C NMR (100 MHz, CDCl3) d 14.1 (C-14), 22.6, 24.8, 25.3, 25.6, 29.1, 29.3, 31.8, 33.4, 33.6, 34.0, 51.5 (RCO2CH3), 74.4, 74.5, 174.2 (C-1). GC/MS EI m/z (%) 159 (10), 142 (5), 129 (1), 127 (25), 115 (2), 110 (17), 101 (2), 97 (6), 87 (25), 85 (3), 81 (29), 74 (20), 73 (2), 71 (4), 69 (17), 59 (20), 57 (33), 55 (92), 43 (97), 41 (100). Anal. Calcd for C15H30O4: C, 65.66; H, 11.02. Found: C, 65.89; H, 10.87. HRMS ESI calcd for C15H30NaO4 ([M+Na]+): 297.2042. Observed: 297.2029.

4.4.7. (7R,8R)-Methyl 7,8-dihydroxytetradecanoate (7R,8R)-6 Following a procedure analogous to that described for the synthesis of (7S,8S)-6, (E)-16 (54 mg, 0.23 mmol) was dihydroxylated by the action of AD-mix-b (Aldrich, 339 mg) over 115 h to afford (7R,8R)-6 (46 mg, 0.17 mmol, 75%) as a white solid (mp 35–36 °C, lit.5 mp 35–36 °C). >99% ee (Chiralpak AD-H column, 2% isopropanol in hexane, flow rate 0.5 mL min1, UV detector 215 nm, retention time (7R,8R)-6 16.0 min. ½a25 D ¼ þ21:0 (c 0.51, MeOH). This compound was spectroscopically identical to (7S,8S)-6. Anal. Calcd for C15H30O4: C, 65.66; H, 11.02. Found: C, 65.69; H, 10.99. HRMS ESI calcd for C15H30NaO4 ([M+Na]+): 297.2042. Observed: 297.2033.

4.5. Synthesis of the erythro enantiomers of methyl 7,8dihydroxytetradecanoate, (7S,8R)-6 and (7R,8S)-6 4.5.1. (Z)-2-(Tetradec-7-en-1-yloxy)tetrahydro-2H-pyran (Z)-18 A flask containing a solution of alkyne 15 (502 mg, 1.70 mmol) in EtOAc (20 mL) was evacuated and purged twice with N2 gas, then twice with H2 gas. Lindlar’s catalyst (5% Pd on CaCO3 poisoned with lead, 42 mg) was added and the mixture was stirred under a H2 atmosphere for 30 min at room temperature. The reaction mixture was filtered through a pad of Celite™, which was thoroughly washed with additional EtOAc. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (silica gel, 1–2% EtOAc in petroleum spirit 40–60) to afford (Z)-18 (504 mg, 1.70 mmol, quantitative, 10% (E)-isomer) as a colourless oil. 1H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.9 Hz, 3H, H3-14), 1.17– 1.43 (m, 14H), 1.43–1.63 (m, 6H), 1.69 (m, 1H), 1.80 (m, 1H), 1.99 (m, 4H, H2-6 & H2-9), 3.36 (td, J = 9.6, 6.7 Hz, 1H, H-1), 3.48 (m, 1H, H-60 ), 3.71 (td, J = 9.6, 6.9 Hz, 1H, H-1), 3.85 (m, 1H, H-60 ), 4.55 (m, 1H, H-20 ), 5.32 (m, 2H, H-7 & H-8). 13C NMR (100 MHz, CDCl3) d 14.0 (C-14), 19.7, 22.6, 25.5, 26.1, 27.1, 27.2, 29.0, 29.1, 29.69, 29.72 (2C), 30.8, 31.8, 62.3 (C-60 ), 67.6 (C-1), 98.8 (C-20 ), 129.7 (RHC=CHR), 130.0 (RHC@CHR). GC/MS EI m/z (%) 296 (0.02, M+), 281 (0.03, M+CH3), 138 (1), 123 (1), 111 (1), 109 (3), 101 (3), 95 (8), 85 (86), 81 (16), 71 (3), 69 (19), 67 (34), 57 (18), 55 (68), 43 (47), 41 (100). Anal. Calcd for C19H36O2: C, 76.97; H, 12.24. Found: C, 76.84; H, 12.27. HRMS ESI calcd for C19H36NaO2 ([M+Na]+): 319.2613. Observed: 319.2595.

4.5.2. (Z)-Tetradec-7-en-1-ol (Z)-19 Following a procedure analogous to that described for the synthesis of rac-10 from rac-9, THP-ether (Z)-18 (448 mg, 1.51 mmol) was deprotected to afford alcohol (Z)-19 (301 mg, 1.42 mmol, 94%, 10% (E)-isomer) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.8 Hz, 3H, H3-14), 1.17–1.40 (m, 15H), 1.54 (m, 2H), 2.00 (m, 4H, H2-6 & H2-9), 3.61 (t, J = 6.6 Hz, 2H, H2-1), 5.32 (m, 2H, H-7 & H-8). 13C NMR (100 MHz, CDCl3) d 14.1 (C-14), 22.6, 25.6, 27.1, 27.2, 28.96, 29.05, 29.69, 29.71, 31.8, 32.8, 63.0 (C-1), 129.7 (RHC@CHR), 130.0 (RHC@CHR). GC/MS EI m/z (%) 195 (0.1, M+OH), 194 (1, M+H2O), 138 (1), 123 (1), 111 (1), 109 (5), 95 (13), 85 (1), 81 (25), 71 (3), 67 (45), 57 (13), 55 (57), 45 (3), 43 (38), 41 (100). Anal. Calcd for C14H28O: C, 79.18; H, 13.29. Found: C, 79.18; H, 13.48. 4.5.3. (Z)-Tetradec-7-enoic acid (Z)-20 Following a procedure analogous to that described for the Jones’ oxidation of alcohol (E)-19 to afford acid (E)-20, alcohol (Z)-19 (251 mg, 1.18 mmol) was oxidised to afford acid (Z)-20 (246 mg, 1.09 mmol, 92%, 10% (E)-isomer) as a pale yellow oil. 1 H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.9 Hz, 3H, H3-14), 1.17– 1.40 (m, 12H), 1.62 (m, 2H), 1.98 (m, 4H, H2-6 & H2-9), 2.33 (t, J = 7.5 Hz, 2H, H2-2), 5.33 (m, 2H, H-7 & H-8). 13C NMR (100 MHz, CDCl3) d 14.1 (C-14), 22.6, 24.6, 27.0, 27.2, 28.7, 29.0, 29.3, 29.7, 31.8, 34.0, 129.4 (RHC@CHR), 130.3 (RHC@CHR), 179.8 (C-1). GC/MS EI m/z (%) 226 (0.2, M+), 208 (1), 164 (1), 137 (1), 125 (1), 123 (2), 115 (1), 111 (2), 110 (3), 101 (2), 96 (6), 91 (1), 87 (2), 85 (2), 83 (9), 79 (4), 73 (7), 71 (4), 69 (20), 67 (16), 60 (8), 59 (3), 57 (10), 55 (65), 45 (14), 43 (51), 41 (100). Anal. Calcd for C14H26O2: C, 74.29; H, 11.58. Found: C, 73.89; H, 11.57. HRMS ESI calcd for C14H26NaO2 ([M+Na]+): 249.1830. Observed: 249.1817. 4.5.4. (Z)-Methyl tetradec-7-enoate (Z)-16 Following a procedure analogous to that described for the synthesis of ester (E)-16 from acid (E)-20, acid (Z)-20 (195 mg, 0.86 mmol) was esterified using ethereal diazomethane to afford the methyl ester (Z)-16 (198 mg, 0.82 mmol, 96%, 10% (E)-isomer) as a colourless oil. 1H NMR (400 MHz, CDCl3) d 0.86 (t, J = 6.9 Hz, 3H, H3-14), 1.17–1.40 (m, 12H), 1.61 (m, 2H), 2.00 (m, 4H, H2-6 & H2-9), 2.28 (t, J = 7.6 Hz, 2H, H2-2), 3.65 (s, 3H, RCO2CH3) 5.32 (m, 2H, H-7 & H-8). 13C NMR (100 MHz, CDCl3) d 14.1 (C-14), 22.6, 24.9, 27.0, 27.2, 28.8, 29.0, 29.4, 29.7, 31.8, 34.1, 51.4 (RCO2CH3), 129.4 (RHC@CHR), 130.2 (RHC@CHR), 174.3 (C-1). GC/MS EI m/z (%) 240 (0.2, M+), 209 (1, M+OCH3), 208 (2, M+MeOH), 166 (2), 151 (1), 141 (1), 137 (1), 128 (1), 125 (1), 123 (2), 115 (1), 111 (2), 110 (3), 101 (1), 96 (9), 86 (10), 85 (3), 81 (9), 74 (23), 73 (3), 71 (2), 67 (18), 59 (18), 57 (7), 55 (61), 43 (54), 41 (100). Anal. Calcd for C15H28O2: C, 74.95; H, 11.74. Found: C, 74.67; H, 11.99. HRMS ESI calcd for C15H28NaO2 ([M+Na]+): 263.1987. Observed: 263.1982. 4.5.5. erythro-Methyl 7,8-dihydroxytetradecanoate erythro-6 Following a procedure analogous to that described for the synthesis of (7S,8S)-6 from (E)-16, (Z)-16 (47 mg, 0.20 mmol) was dihydroxylated by the action of AD-mix-a (Aldrich, 300 mg) over 210 h to afford a mixture of (7R,8S)- and (7S,8R)-methyl 7,8dihydroxytetradecanoate erythro-6 (52 mg, 0.19 mmol, 97%) as a 1 white solid (mp 89–90 °C). ½a25 D ¼ þ0:4 (c 0.42, MeOH). H NMR (500 MHz, CDCl3) d 0.87 (t, J = 6.9 Hz, 3H, H3-14), 1.20–1.54 (m, 16H), 1.63 (m, 2H), 1.76 (br s, 1H, ROH), 1.80 (br s, 1H, ROH), 2.30 (t, J = 7.5 Hz, 2H, H2-2), 3.57 (m, 2H, H-7 & H-8), 3.65 (s, 3H, RCO2CH3). 13C NMR (125 MHz, CDCl3) d 14.1 (C-14), 22.6, 24.8, 25.6, 26.0, 29.1, 29.3, 30.9, 31.3, 31.8, 34.0, 51.5 (RCO2CH3), 74.5, 74.7, 174.2 (C-1). GC/MS EI m/z (%) 207 (0.3), 189 (1), 159 (14),

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145 (1), 142 (8), 129 (1), 127 (37), 115 (1), 110 (24), 101 (2), 97 (4), 87 (29), 85 (2), 81 (39), 74 (21), 73 (2), 71 (4), 69 (16), 59 (17), 57 (39), 55 (99), 43 (100), 41 (87). Anal. Calcd for C15H30O4: C, 65.66; H, 11.02. Found: C, 65.51; H, 11.17. HRMS ESI calcd for C15H30NaO4 ([M+Na]+): 297.2042. Observed: 297.2030. 4.5.6. (7R,8S)- and (7S,8R)-Methyl 7,8-bis((S)-2-methoxy-2phenylacetoxy)tetradecanoates (7R,8S)-21 and (7S,8R)-21 To a solution of dihydroxyester mixture erythro-6 (45 mg, 0.16 mmol) in anhydrous CH2Cl2 (3 mL) under a N2 atmosphere was added DCC (135 mg, 0.65 mmol), DMAP (80 mg, 0.65 mmol) and (S)-methoxyphenylacetic acid (110 mg, 0.66 mmol) and the resulting solution was stirred at room temperature overnight. After filtration of the precipitate and removal of the CH2Cl2 in vacuo, the crude material (45:55 mixture of two diastereomers, >99% ee, 10% de) was subjected to preparative separation by enantioselective HPLC (Chiralpak AD-H column, 10% isopropanol in hexane, flow rate 10 mL min1, UV detector 254 nm, retention times: (7R,8S)21 22.8 min, (7S,8R)-21 28.6 min). Both diastereomers were obtained optically pure (>99% de and ee; 74% combined yield) and their absolute configurations were determined by NMR spectroscopy. (7R,8S)-21: 30 mg, 53 lmol, 32%. Colourless oil. ½a25 D ¼ þ72:2 (c 0.34, CHCl3). 1H NMR (400 MHz, CDCl3) d 0.51–0.73 (m, 2H), 0.79– 0.96 (m, 2H), 0.89 (t, J = 6.8 Hz, 3H, H3-14), 1.02–1.34 (m, 12H), 1.43–1.54 (m, 2H), 2.08 (t, J = 7.6 Hz, 2H, H2-2), 3.42 (s, 3H), 3.43 (s, 3H), 3.68 (s, 3H), 4.46 (s, 1H), 4.75 (dt, J = 10.0, 3.5 Hz, 1H, H7), 4.77 (s, 1H), 5.14 (dt, J = 8.6, 4.2 Hz, 1H, H-8), 7.25–7.33 (m, 5H), 7.36–7.41 (m, 3H), 7.46–7.49 (m, 2H). 13C NMR (100 MHz, CDCl3) d 14.0, 22.5, 24.0, 24.4, 25.3, 27.5, 28.3, 28.9, 30.2, 31.5, 33.7, 51.4 (RCO2CH3), 57.3, 57.5, 74.4, 74.7, 82.1, 82.5, 127.1 (2C), 127.3 (2C), 128.4 (2C), 128.5 (2C), 128.6, 128.7, 136.4, 136.5, 170.1, 170.3, 174.0 (C-1). 1H NMR (400 MHz, C6D6) d 0.64–0.99 (m, 3H), 0.86 (t, J = 7.2 Hz, 3H, H3-14), 1.05–1.47 (m, 15H), 1.96 (t, J = 7.5 Hz, 2H, H2-2), 3.24 (s, 3H), 3.36 (s, 3H), 3.38 (s, 3H), 4.59 (s, 1H), 4.71 (s, 1H), 4.94 (dt, J = 10.3, 3.2 Hz, 1H, H-7), 5.39 (dt, J = 9.8, 3.5 Hz, 1H, H-8), 7.06–7.18 (m, 6H), 7.46 (br d, J = 8.0 Hz, 2H), 7.57 (br d, J = 8.0 Hz, 2H). HRMS ESI calcd for C33H46NaO8 ([M+Na]+): 593.3090. Observed: 593.3092. (7S,8R)-21: 39 mg, 68 lmol, 42%. Colourless oil. ½a25 D ¼ þ49:9 (c 0.70, CHCl3). 1H NMR (400 MHz, CDCl3) d 0.55–0.71 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H, H3-14), 0.85–0.96 (m, 2H), 1.03–1.18 (m, 4H), 1.24– 1.35 (m, 4H), 1.46–1.62 (m, 4H), 2.29 (t, J = 7.6 Hz, 2H, H2-2), 3.42 (s, 3H), 3.43 (s, 3H), 3.69 (s, 3H), 4.48 (s, 1H), 4.76 (dt, J = 9.4, 3.7 Hz, 1H, H-8), 4.78 (s, 1H), 5.13 (dt, J = 8.4, 4.0 Hz, 1H, H-7), 7.27–7.32 (m, 5H), 7.37–7.41 (m, 3H), 7.46–7.49 (m, 2H). 13C NMR (100 MHz, CDCl3) d 14.0 (C-14), 22.3, 24.3, 24.6, 25.0, 27.8, 28.5, 28.7, 30.0, 31.3, 33.8, 51.5 (RCO2CH3), 57.3, 57.5, 74.3, 74.8, 82.1, 82.5, 127.1 (2C), 127.3 (2C), 128.4 (2C), 128.5, 128.6 (2C), 128.7, 136.3, 136.4, 170.2, 170.3, 174.0 (C-1). 1H NMR (400 MHz, C6D6) d 0.75–1.06 (m, 7H), 0.85 (t, J = 7.3 Hz, 3H, H3-14), 1.11– 1.23 (m, 4H), 1.27–1.44 (m, 7H), 2.03 (t, J = 7.4 Hz, 2H, H2-2), 3.25 (s, 3H), 3.36 (s, 3H), 3.38 (s, 3H), 4.61 (s, 1H), 4.71 (s, 1H), 4.97 (dt, J = 10.3, 3.2 Hz, 1H, H-7), 5.35 (dt, J = 9.8, 3.5 Hz, 1H, H8), 7.03–7.18 (m, 6H), 7.48 (br d, J = 8.0 Hz, 2H), 7.57 (br d,

1719

J = 8.0 Hz, 2H). HRMS ESI calcd for C33H46NaO8 ([M+Na]+): 593.3090. Observed: 593.3103. 4.5.7. (7R,8S)-Methyl 7,8-dihydroxytetradecanoate (7R,8S)-6 Powdered solid NaOH (80 mg, 2.0 mmol) was added to a solution of (7R,8S)-21 (29 mg, 51 lmol) in MeOH (10 mL), followed by a drop of water. The solution was allowed to stir at room temperature overnight and then dilute aqueous HCl solution (1%, 15 mL) was added. The mixture was extracted with CH2Cl2 (3  20 mL) and the combined organic extract was washed with brine (20 mL) and water (20 mL). After drying over anhydrous MgSO4, the organic extract was filtered and then concentrated in vacuo. The crude product was purified by flash chromatography (25% EtOAc in hexane) to afford (7R,8S)-6 (9 mg, 33 lmol, 65%) as a white solid (mp 96 °C). ½a25 D ¼ þ1:7 (c 0.40, CHCl3). This compound was spectroscopically identical to the essentially racemic mixture erythro-6. 4.5.8. (7S,8R)-Methyl 7,8-dihydroxytetradecanoate (7S,8R)-6 Following an analogous procedure to that described for the synthesis of (7R,8S)-6 from (7R,8S)-21, the desired compound (7S,8R)6 (9 mg, 33 lmol, 58%) was synthesised from (7S,8R)-21 (32 mg, 56 lmol) and was obtained as a white solid (mp 94 °C). ½a25 D ¼ 2:3 (c 0.24, CHCl3). This compound was spectroscopically identical to the essentially racemic mixture erythro-6. Acknowledgments A.A.S. is grateful for Australian Postgraduate Award scholarship support and S.N.A.Z. is grateful for a University of Queensland Summer Research Scholarship. References 1. Ortiz de Montellano, P. R.; De Voss, J. J. In Cytochrome P450: Structure, Mechanism, and Biochemistry; Ortiz de Montellano, P. R., Ed.; Kluwer Academic/ Plenum Publishers: New York, 2005; pp 183–245. 2. Ortiz de Montellano, P. R. Chem. Rev. 2010, 110, 932–948. 3. Ortiz de Montellano, P. R.; De Voss, J. J. Nat. Prod. Rep. 2002, 19, 477–493. 4. De Voss, J. J.; Cryle, M. J. In Ubiquitous Roles of Cytochrome P450 Proteins: Metal Ions in Life Sciences; Sigel, A., Sigel, H., Sigel, R. K. O., Eds.; John Wiley: Chichester, 2007; pp 397–435. 5. Cryle, M. J.; De Voss, J. J. Chem. Commun. 2004, 86–87. 6. Burstein, S.; Middleditch, B. S.; Gut, M. J. Biol. Chem. 1975, 250, 9028–9037. 7. Mitropoulos, K. A.; Gibbons, G. F.; Reeves, B. E. A. Steroids 1976, 27, 821–829. 8. Matsui, K.; Shibutani, M.; Hase, T.; Kajiwara, T. FEBS Lett. 1996, 394, 21–24. 9. Stok, J. E.; De Voss, J. J. Arch. Biochem. Biophys. 2000, 384, 351–360. 10. Cryle, M. J. Metallomics 2011, 3, 323–326. 11. Cryle, M. J.; Matovic, N. J.; De Voss, J. J. Org. Lett. 2003, 5, 3341–3344. 12. Cryle, M. J.; Schlichting, I. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 15696–15701. 13. Parker, K. A.; Ledeboer, M. W. J. Org. Chem. 1996, 61, 3214–3217. 14. Marino, J. P.; Nguyen, H. N. J. Org. Chem. 2002, 67, 6291–6296. 15. Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem. Rev. 1994, 94, 2483– 2547. 16. Morimoto, Y.; Kitao, S.; Okita, T.; Shoji, T. Org. Lett. 2003, 5, 2611–2614. 17. Seco, J. M.; Martino, M.; Quiñoá, E.; Riguera, R. Org. Lett. 2000, 2, 3261–3264. 18. Seco, J. M.; Quiñoá, E.; Riguera, R. Chem. Rev. 2004, 104, 17–118. 19. Schulz, S.; Francke, W.; Boppre, M. Biol. Chem. Hoppe-Seyler 1988, 369, 633– 638. 20. Tulloch, A.; Hoffman, L. Lipids 1973, 8, 617–622.