The synthesis of single enantiomers of trans-alkene containing mycolic acids and related sugar esters

Accepted Manuscript

The synthesis of single enantiomers of trans-alkene containing mycolic acids and

related sugar esters

Hanan M. Ali, Gani Koza, Rwoa'a T. Hameed, Richard Rowles, Carys Davies, Juma'a

R. Al Dulayymi, Christopher D. Gwenin, Mark S. Baird

PII:

S0040-4020(16)30884-5

10.1016/j.tet.2016.08.089

TET 28070

DOI:

Reference:

To appear in: Tetrahedron

Received Date: 21 June 2016

Revised Date: 17 August 2016

Accepted Date: 30 August 2016

Please cite this article as: Ali HM, Koza G, Hameed RT, Rowles R, Davies C, Al Dulayymi JR, Gwenin

CD, Baird MS, The synthesis of single enantiomers of trans-alkene containing mycolic acids and related

sugar esters, Tetrahedron (2016), doi: 10.1016/j.tet.2016.08.089.

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ACCEPTED MANUSCRIPT

The synthesis of single enantiomers of trans-alkene

Leave this area blank for abstract info.

containing mycolic acids and related sugar esters

Hanan M. Ali, Gani Koza, Rwoa’a T. Hameed, Richard Rowles, Carys Davies, Juma’a R. Al

Dulayymi, Christopher D. Gwenin and Mark S. Baird,*

School of Chemistry, Bangor University, Gwynedd, LL57 2UW, UK

ACCEPTED MANUSCRIPT

1

The synthesis of single enantiomers of trans-alkene

containing mycolic acids and related sugar esters

Hanan M. Ali, Gani Koza, Rwoa’a T. Hameed, Richard Rowles, Carys Davies, Juma’a R. Al Dulayymi,

Christopher D. Gwenin and Mark S. Baird,*

School of Chemistry, Bangor University, Gwynedd LL 57 2 UW, UK

Abstract— Routes to single enantiomers of hydroxy, methoxy and ketomycolic acids containing an α–methyl-trans-alkene unit as well as

related trehalose, glucose, arabinose and glycerol esters are described. In serodiagnostic assays to detect antibodies to mycobacterial lipids,

the trehalose esters give higher responses to the serum of patients with culture positive tuberculosis than to that of culture negative patients.

Key words: Mycolic acid, TDM, TMM, GMM, trans-

alkene, trehalose monomycolate, trehalose dimycolate

synthetic MAs of different classes matching the structures

of components of natural mixtures are selectively active.¹⁵

In addition, there are a range of generally less abundant

MAs in mycobacteria containing other X and Y groups;

these include molecules containing an α-methyl-trans-

alkene unit.^11,12Keto-mycolic acids 2 with a proximal

trans-alkene substituent and a variety of chain lengths have

been reported (Table 1).^{11, 12,16-18}In these cases, the methyl

group adjacent to the alkene is on the proximal side relative

to the hydroxy-acid. The methyl group in the X position of

keto- and methoxy-MA and at the Y-position in α–methyl-

trans-cyclopropane containing MA, is distal from it.^11,12

1. Introduction

Mycolic acids (MAs) are characteristic components of the

cell envelopes of mycobacteria, often covalently bound to

arabinogalactan as a penta-arabinose tetramycolate.^1-3They

are also seen as trehalose dimycolate (TDM – a potent

signalling agent in the immune system),^4,5trehalose

monomycolate (TMM)⁶and other sugar esters that are not

bound to the cell wall,^7-9or as free mycolic acids.¹⁰MAs

comprise two elements (Figure 1), the β-hydroxy-acid, for

which d is generally 21 or 23, and the meromycolate. The

latter is a long chain usually containing two groups X and

Y.

Table 1. Typical chain lengths of major β-methyl-trans-alkene

containing mycobacterial ketomycolic acids (2)

2

Species

a

17

b

19

c

15

d

23

Ref

11

Mycobacterium tuberculosis,

Mycobacterium bovis, M. bovis

BCG, Mycobacterium microti

Figure 1: Typical mycolic acid structure

M. tuberculosis, M. bovis, M. 17

bovis BCG, M. microti

19

13

23

11

In the most common classes of mycobacterial MA, the two

M. tuberculosis Canetti

Mycobacterium avium

complex (MAC)

17

23

21

11

groups are both cis-cyclopropanes (

α

-MA), or group X is

an –methyl- -methoxy or –methyl-β

α

β

α

-keto group

Mycobacterium marinum

Mycobacterium scrofulaceum

M. aurum

17

15

17

13

17

19

17

21

19

11

19

(methoxy and keto-MA respectively). MAs are generally

present as complex mixtures containing several different

chain lengths of each class, and the detailed composition is

dependent on the mycobacterium.^11,12The presence and

proportion of individual classes of MA is important for

the virulence of diseases such as tuberculosis.^13,14The MAs

are themselves strongly bioactive and indeed individual

--------------------------------------------------------------------

* Corresponding author: e-mail: chs028@bangor.ac.uk.

Fax: +44 1248 370528

M. aurum

A derivative of a hydroxy-MA with a proximal α–methyl-

trans-alkene has also been reported.¹⁶Mycobacterium

smegmatis synthesizes keto-MAs containing about 33% of

a trans-alkene. The same percentage is seen in the

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2

hydroxy-mycolate.²⁰In addition, related methoxy-MA 3

have been reported as in Table 2.¹¹

Table 2. Typical chain lengths of major β-methyl-trans-alkene

containing mycobacterial methoxymycolic acids (3)¹¹

Me

OH

O

OMe

CH₃(CH₂)_a

(CH₂)_c

OH

(CH₂)_dCH₃

(CH₂)_b

3

Scheme 1: (i) LiHMDS, THF (71%); (ii) H₂, Pd/C, EtOAc/EtOH (88%);

(iii) 1-phenyl-1H-tetrazole-5-thiol, K₂CO₃, acetone (86%); (iv) H₂O₂,

(NH₄)₆Mo₇O₂₄.4H₂O, THF/IMS (95%)

Me

Species

a

b

c

d

M. tuberculosis, M. bovis,

M. bovis BCG

M. tuberculosis Canetti

M. microti

17 19 13 23

In order to fix the (R)-stereochemistry of the α-methyl-

45-47

trans-alkene fragment, aldehyde 8,^39,

was chain-

17 19 15 23

17 15 17 21

extended with compound 7 and base, using a modified

Julia–Kocienski reaction,^48-50followed by hydrogenation of

the derived alkenes to give 9. The pivaloate group was

removed and the primary alcohol was converted into

sulfone 11 (Scheme 2) via the bromide 10.

The biosynthesis of these and other MA has been studied

extensively.^21-27Labelling studies show the methyl branch

of trans-MA from M. tuberculosis are exclusively on

carbons derived from the 2-position of acetate, while those

from M. smegmatis, are exclusively derived from the 1-

position.²⁸

Another type of mycolic acid, isolated from M. smegmatis

and Mycobacterium aurum,²⁹Mycobacterium chelonei,³⁰

and Mycobacterium fortuitum,³¹contains an α–methyl-

trans-alkene at the proximal position and a cis-alkene at the

distal position.^32-34In the latter case, the specific rotation of

its methyl ester has been reported to be +1.4 (CHCl₃),³⁵

while that of the acetoxy methyl ester is reported as +3

(CHCl₃), and that of a deacetoxy derivative of a mycolate

containing only an α–methyl-trans-alkene chiral centre

corresponds to a molecular rotation (M_D) of -19.5.³⁵In

addition, the specific rotation of a wax ester containing this

unit has been reported to be +4.3 (CHCl₃).^29,36,37This

allows the contribution to the molecular rotation from the

α–methyl-trans-alkene to be calculated, as the only other

chiral centres in these cases are at the hydroxy-acid

position, the contribution of which to the molecular

rotation is known (+40°). This leads to a value of -25° for

the α–methyl-trans-alkene (for 30% of methyl branched

molecules); this in turn suggests this sub-unit has an (R)-

stereochemistry, based on model compounds.¹⁸

Scheme 2: (i) LiHMDS, THF (86%); (ii) H₂, Pd/C, EtOAc/EtOH (96%);

(iii) KOH, THF, MeOH, H₂O (97%); (iv) N-bromosuccinimide, PPh₃,

NaHCO₃, CH₂Cl₂(92%); (v) 1-phenyl-1H-tetrazole-5-thiol, K₂CO₃,

acetone (95%); (vi) H₂O₂, (NH₄)₆Mo₇O₂₄.4H₂O, THF/IMS (81%)

A modified Julia reaction between the aldehyde 12,^51,66and

sulfone 11 gave a mixture of alkenes which was

hydrogenated. The silyl ether was changed to an acetyl

group as in 13, in order to avoid the presence of two

identical protecting groups at a later stage, and the acetal

group was converted into aldehyde 14 with periodic acid

(Scheme 3).

As part of a study to determine the biological significance

of specific MA structures,^38-44and to confirm regio- and

stereochemistry, we now report the synthesis of hydroxy-

MA, keto-MA and methoxy-MA containing a α–methyl-

trans-alkene unit. Four sugar esters and one glycerol ester

were prepared from one methoxy-MA, to provide the

opportunity for a systematic comparison of their effects on

cytokines and chemokines, as well as their application as

antigens in the serodiagnosis of tuberculosis.

Scheme 3: (i) 11, LiHMDS, THF (86%); (ii) Pd/C, EtOAc (96%); (iii)

HF.pyridine, pyridine (83%); (iv) Ac₂O, pyridine (94%); (v) periodic acid

(80%)

The modified Julia–Kocienski reaction using a 1-phenyl-

1H-tetrazole sulfone and an aldehyde with potassium bis-

(trimethylsilyl)amide in 1,2-dimethoxyethane is known to

lead to an E-alkene with good stereoselectivity, especially

if the sulfone or aldehyde is α-substituted.^52-54Reaction of

the sulfone 15,⁶⁰with aldehyde 14 gave the trans-alkene 16

(Scheme 5). It is essential that no epimerization occurs

adjacent to the aldehyde during this process. There is

2.

Results and discussion

As the first step in the synthesis of alkene-MA 23, 6-

bromohexanal (4) was chain extended to the ester 6 and

then converted into the sulfone 7 (Scheme 1):

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3

considerable precedent for the retention of the chirality.^53,

^56-59Removal of the silyl group gave 17 (R= Ac, R’ = Me)

and hydrolysis of the two esters produced the free hydroxy

acid 17 (R= H, R’ = H). The [α]²¹_Dof this, -2.07 (CHCl₃,

0.743 µmol), corresponding to M_D-26°, is in agreement

with that reported for the hydroxymycolates of M.

smegmatis (M_D-16°, though it must be noted these only

contain ~30% trans-alkene).¹⁸

Scheme 4: (i) LiOH, MeOH, H₂O, THF (72%); (ii) TBDMSCl, imidazole,

DMF; (iii) K₂CO₃, MeOH/H₂O, then KHSO₄, (70%); (iv) PPTS, MeOH,

H₂O, THF (73%); (v) PCC (89%); (vi) HF.pyridine (44%)

In the same way compound 19 was first protected with a

tetrahydropyranyl group to give 24 and then converted into the

keto-MA 25 ([α]²³_D+1.58 (CHCl₃, 0.436 µmol)) (Scheme 5):

Me

OAc

O

OTHP

(CH₂)₁₅

CH₃(CH₂)₁₇

(CH₂)₁₇

OMe

(CH₂)₂₁CH₃

Scheme 4: (i) 14, KHMDS, 1,2-dimethoxyethane (34%); (ii) HF.pyridine,

THF (17 (R= Ac, R’ = Me), 91%); (iii) LiOH, MeOH, THF, H₂O (17 (R=

H, R’ = H), 65%)

24

Me

(i)-(vi)

Me

OH

O

In the same way the enantiomer of 15, compound 18,⁶⁰was

converted into 19 and free hydroxy-MA 20 (Figure 1). The

¹H NMR spectra of each of these in the alkene region was

identical to that reported in the literature,^20,24and to minor

signals in fractions of α-mycolates derived from M.

avium.⁵⁵

CH₃(CH₂)₁₇

(CH₂)₁₇

OH

(CH₂)₁₅

(CH₂)₂₁CH₃

25

Me

Scheme 5: (i) LiOH, MeOH, H₂O, THF (72%); (ii) TBDMSCl, imidazole,

DMF; (iii) K₂CO₃, MeOH/H₂O, then KHSO₄, (70%); (iv) PPTS, MeOH,

H₂O, THF (73%); (v) PCC (89%); (vi) HF.pyridine (44%)

The chain lengths reported for major trans-alkene-

methoxy-MA are somewhat different from the keto-

analogues. In order to prepare the methoxy-MA 3 (a = 17, b

= 19, c = 13, d = 23), aldehyde 8 was treated with 26 and

base, followed by hydrogenation to produce 27, which was

in turn converted into sulfone 28 by standard methods

(Scheme 6):

Figure 1

Oxidation of either 17 (R= Ac, R’ = Me) or 19 led to the

corresponding protected keto-MAs. However, attempted

deprotection of these using LiOH led to epimerization at

the position adjacent to the ketone. In order to avoid this,

the alcohol 17 (R= Ac, R’ = Me) was first protected as

THP-ether 21, followed by hydrolysis of the esters and then

reprotection at the alcohol group as a silyl ether 22.

Removal of the THP-group, oxidation, and then

deprotection now proceeded without epimerization,⁶⁰

leading to the free acid 23 (Scheme 4), matching the

overall structure of the major trans-alkene keto-mycolate

reported for M. marinum.¹¹

Scheme 6: (i) LiHMDS, THF (76 %); (ii) Pd/C,H₂, EtOAc (92 %); (iii)

LiAlH₄, THF (81 %); (iv) diethyl azodicarboxylate, Ph₃P, 1-phenyl-1H-

tetrazole-5-thiol (84 %); (v) (NH₄)₆Mo₇O₂₄.4H₂O, H₂O₂, THF/IMS (84 %).

A further modified Julia reaction on the sulfone 28 and

aldehyde 29,⁴³again followed by hydrogenation, then

reaction with periodic acid led to aldehyde 31 (Scheme 7):

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4

Scheme 9: Formation of S,S-methoxy-MA fragment

Elimination of a proton from 39 would lead to 41, and in a

Scheme 7: (i) LiHMDS, THF (96 %); (ii) Pd/C,H₂(96 %); (iii) periodic

second step to the proximal α–methyl-trans-cyclopropane

acid (87 %).

21-26, 39

unit 42 (Scheme 10).

In this case, it appears that

The S-methoxy-S-methyl substituted aldehyde 32,³⁹was

chain extended to sulfone 34, again using standard

procedures (Scheme 8):

rapid cyclopropanation occurs, as the alkenes 41 are not

commonly reported.

Scheme 8: (i) LiHMDS, THF, (85 %); (ii) Pd/C,H₂, (100 %); (iii) LiAlH₄,

THF (99 %); (iv) diethyl azodicarboxylate, Ph₃P, 1-phenyl-1H-tetrazole-5-

thiol (91%); (v) (NH₄)₆Mo₇O₂₄-4H₂O, H₂O₂(35%), THF/IMS (97 %).

Scheme 10: Formation of trans-cyclopropane

In applying this to the stereochemistry of trans-alkene

mycolates, one possibility is that alkylation of the cis-

alkene 38, again occurs from the bottom face, this time to

the proximal carbon leading formally to 39b; elimination

of a proton would then lead to the (R)-α–methylalkene 43

(Scheme 11). In this case, it appears that cyclopropanation

of the alkene does not occur, as no such regioisomers have

been reported.

A modified Julia reaction between sulfone 34 and aldehyde

31 led, after deprotection, to the final MA, 37.(Scheme 8)

Scheme 8: (i) KHMDS, 1,2-dimethoxyethane (37 %); (ii) HF-pyridene,

THF (96 %); (iii) LiOH, MeOH, H₂O, THF (82 %).

Scheme 11: Proposed stereochemistry of methylation by SAM

The biological activity of mycolic acids is often seen most

strongly in their sugar esters, such as glucose mycolate

(GMM), TDM and TMM.³In natural isolates it is difficult

to identify within the complex mixtures whether particular

sub-classes or chain lengths are selectively responsible for

these properties. To try to understand whether trans-alkene

mycolic acids have specific biological properties, a series

of sugar esters of the methoxy-MA 37 was prepared. The

acid was first reprotected at the β-hydroxy group with

TBDMS to give 44 and then coupled to the protected

trehalose 45:⁶²

These results provide the first synthetic approaches to keto-

, hydroxy- and methoxy-MAs containing a proximal (R)-

α–methyl-trans-alkene unit, and a comparison of molecular

rotations with those in the literature confirms the

stereochemistry of this subunit. It has been proposed that

MA biosynthesis involves alkylation of a cis-alkene by

SAM (S-adenosylmethionine) to provides an intermediate

cation leading to each of the other MA functionalities.⁶¹

Although the actual mechanism may be different,²⁷this

provides a model by which to analyse the stereochemistry

of the various classes of MA. Thus, using 38 as a model

precursor, formal addition of a methyl cation to the distal

carbon of the alkene from the bottom face would produce

cation 39; trapping from the top face could lead eventually

to the known (S,S)-stereochemistry of 40 at the distal

position. (Scheme 9). ^{16, 21}

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5

Scheme 14: (i) CsHCO₃, DMF:THF, 70 %, (ii) Na/NH₃, 47 %.

It was also coupled to the protected arabinose 55. (Scheme

15)

Scheme 15: (i) CsHCO₃, DMF:THF, 71 %, (ii) Na/NH₃, 57 %.

Scheme 12: (i) EDCl, 4-DMAP, CH₂Cl₂, 4 ^oA MS, rt, 6 days; 46 (25%),

47 (50%).

As a final comparison, the glycerol ester (GroMM) 60 was

prepared. (Scheme 16).

The protecting groups were then removed in two steps

(Scheme 13):

Scheme 16: (i) CsHCO₃, DMF:THF, 89 %, (ii) Na/NH₃, 40 %.

Like sugar esters of other synthetic alpha-, keto- and

methoxy-mycolic acids, the sugar esters of trans-alkene

methoxy described above show strong effects on a range of

cytokines and chemokines.⁶³Such sugar esters are also

antigenic to antibodies in serum of patients infected with

active TB.⁶⁴The trans-alkene sugar esters described above

were each evaluated as antigens in ELISA for responses to

antibodies in serum of patients infected with pulmonary

tuberculosis, using modified IgG(Fc) as secondary antibody

to provide a colour response. Twenty samples from a WHO

Specimen Bank were used, 9 culture and smear positive for

TB, 11 smear and culture negative and diagnosed as not

having TB. The samples were all from patients showing

symptoms of suspected TB.⁶⁵The results are presented in

Table 1s (Supplementary Information) and the average

responses are given in Graph 1.

Scheme 13: (i) TBAF, THF, 5 ^oC 1h; (ii) Pyridine, THF, HF-pyridine

complex, 43 ^oC, 17 h, then neutralised with aq. NaHCO₃

The acid 37 was also coupled to protected glucose 52 to

give, after debenzyation, the GMM 54. (Scheme 14)

ACCEPTED MANUSCRIPT

6

Serum samples, provided by the World Health Organisation

from the TDR TB Specimen Bank with the necessary

ethical approval,⁶⁵were all from individuals with

symptoms of suspected tuberculosis.

3.2 (R)-2-[(1R,19R)-1-(tert-Butyldimethylsilanyloxy)-19

(R)-2-[(1R,19R)-1-acetoxy-19-((S)-2,2-dimethyl[1,3]di-

oxolan-4-yl)-eicosyl]tetracosanoic acid methyl ester 13

(i) LiHMDS (8.50 mL, 8.76 mmol, 1.06 M) was added to a

stirred solution of ester 12 (2.62 g, 4.61 mmol)⁶⁶and

tetrazole 11 (2.78 g, 5.07 mmol) in dry THF (50 mL) at –5

^oC under nitrogen. The mixture was allowed to reach r. t.,

stirred for 30 min. Then quenched with sat. aq. NH₄Cl (10

mL) and extracted with EtOAc and petrol (5:1, 3 × 50 mL).

The combined organic layers were washed with water (25

mL), dried, and evaporated; chromatography (petrol/ether,

15:1) gave a colourless oil, methyl (R)-2-[(E/Z)-(1R,19R)-

1-(tert-butyldimethylsilanyloxy)-19-((S)-2,2-

Graph 1: Average responses and standard deviations for 9 serum samples

from pulmonary TB, culture and smear positive patients (TB+) and 11

from culture and smear negative patients (TB0) from the WHO TDR TB

specimen Bank,⁶⁵clinically diagnosed using standard protocols in ELISA

assay using IgG(Fc) secondary antibody

dimethyl[1,3]di-oxolan-4-yleicos-3-enyl]tetracosanote (3.6

g, 86%) as a mixture of isomers in ratio 2.2:1. Palladium

10% on carbon (1.0 g) was added to a stirred solution of the

alkenes (3.45 g, 3.88 mmol) in EtOAc (150 mL).

Hydrogenation was carried out for 1.5 h. The solution was

filtered over a bed of celite and the solvent was evaporated.

Chromatography (petrol/ether, 15:1) gave a colourless oil,

(R)-2-[(1R,19R)-1-(tert-butyl-dimethylsilan-yloxy)-19-((S)-

2,2-dimethyl[1,3]-dioxolan-4-yl)eicosyl]-tetracosanoic acid

methyl ester (3.2 g, 96%), [α]²_D²+3.6 (c 0.94, CHCl₃)

[Found (M+H)⁺: 893.8321, C₅₆H₁₁₃O₅Si requires:

893.8352]; δ_H(500 MHz, CDCl₃): 4.0 (1H, dd, J 6.3, 7.9

Hz), 3.93 – 3.90 (1H, m), 3.87 (1H, br q, J 7.0 Hz), 3.66

(3H, s), 3.61 (1H, br t, J 7.9 Hz), 2.53 (1H, ddd, J 3.8, 7.3,

11.1 Hz), 1.59 – 1.52 (4H, m), 1.41 (3H, s), 1.36 (3H, s),

1.35 – 1.26 (72H, m, v.br), 1.10 – 1.07 (1H, m), 0.97 (3H,

d, J 6.6 Hz), 0.89 (3H, t, J 6.9 Hz), 0.87 (9H, s), 0.05 (3H,

s), 0.03 (3H, s); δ_C(126 MHz, CDCl₃): 175.1, 108.5, 80.4,

76.8, 73.2, 51.6, 51.2, 36.5, 33.7, 32.7, 31.9, 29.9, 29.8,

29.7(v.br), 29.66, 29.62, 29.6, 29.58, 29.5, 29.4, 29.3, 27.8,

27.5, 27.0, 26.6, 25.7, 25.5, 23.7, 22.7, 18.0, 15.6, 14.1, –

4.4, – 4.9; ν_max/cm^-1: 2925, 2854, 1741, 1465, 1368, 1253,

1165, 1068.

(ii) The above ester (3.22 g, 3.61 mmol) was stirred in dry

THF (30 mL) in dry polyethylene vial under nitrogen at 0

°C. Pyridine (1.2 mL) and HF.pyridine complex (5.0 mL)

were added and the mixture was stirred for 18 h at 45 °C.

The mixture was neutralised by slowly pouring it into sat.

aq. NaHCO₃(20 mL). The product was extracted with

petrol/EtOAc (5:1, 3 × 10 mL), and evaporated to give a

white solid. Chromatography (5:1 petrol/EtOAc) gave a

white solid, methyl (R)-2-[(1R,19R)-19-((S)-2,2-dimethyl-

[1,3]dioxolan-4-yl)-1-hydroxyeicosyl]tetracosanoate (2.3 g,

83%), m.p. 67 – 68 ^oC, [α]_D²⁵+14.8 (c 0.970, CHCl₃) [Found

(M+H)⁺: 779.7450, C₅₀H₉₉O₅requires: 779.7487]. This

showed δ_H(500 MHz, CDCl₃): 4.0 (1H, dd, J 6.3, 7.9 Hz),

3.87 (1H, br q, J 6.9 Hz), 3.71 (3H, s), 3.68 – 3.64 (1H, m),

3.60 (1H, br t, J 7.7 Hz), 2.44 (1H, dt, J 5.4, 10.4 Hz), 1.74

– 1.26 (77H, m, v br), 1.41 (3H, s), 1.36 (3H, s), 1.11 –

1.07 (1H, m), 0.96 (3H, d, J 7.0 Hz), 0.88 (3H, t, J 7.0 Hz);

δ_C(126 MHz, CDCl₃): 176.2, 108.5, 80.4, 72.3, 67.8, 51.5,

50.9, 36.5, 35.7, 32.7, 31.9, 29.9, 29.7(v.br), 29.65, 29.62,

29.6, 29.57, 29.55, 29.53, 29.48, 29.4, 29.3, 27.4, 27.0,

Although the standard deviations within this small sample

set are large, these results indicate that the synthetic alkene

containing TDM 49 and TMM 51 may offer potential in the

diagnosis of active TB. It is interesting that, for this series

of compounds, the GMM 54, GroMM 60 and ArMM 57 do

not show significant distinction between pulmonary TB

positive or negative samples. An extended study using a

large serum set is now under way to evaluate these

molecules compared to other synthetic sugar mycolates as

antigens in diagnosis.

3.

Experimental section

3.1 Chemicals were obtained from commercial suppliers

(Sigma, Aldrich, and Alfa Aeser) or prepared from them by

the methods described. Solvents which were required to be

dry, e.g. ether, tetrahydrofuran were dried over sodium wire

and benzophenone under nitrogen, while CH₂Cl₂was dried

over calcium hydride. All reagents and solvents used were

of reagent grade unless otherwise stated. Silica gel (Merck

7736) and silica gel plates used for column chromatography

and thin layer chromatography were obtained from Aldrich;

separated components were detected using variously UV

light, I₂and phosphomolybdic acid solution in IMS

followed by charring. Anhydrous magnesium sulfate was

used to dry organic solutions. Infra-red (IR) spectra were

carried out on a Perkin-Elmer 1600 F.T.I.R. spectrometer

as liquid films or KBr disc (solid). Melting points were

measured using a Gallenkamp melting point apparatus.

NMR spectra were carried out on a Bruker Avance 400 or

500 spectrometer. Specific rotations were recorded in

CHCl₃on a POLAAR 2001 Optical Activity Polarimeter.

Mass spectra were recorded on a Bruker matrix-assisted

laser desorption/ ionisation-time of flight mass

spectrometry (MALDI-TOF MS) values are given plus

sodium to an accuracy of 1 d.p.; accurate mass values were

run on a Bruker LC-MS or on MALDI-TOF MS in Bristol

University.

The detailed procedures for the preparation of compounds 7

– 11 are provided in the Supplementary Information.

ACCEPTED MANUSCRIPT

7

26.6, 25.7, 25.5, 22.7, 15.6, 14.1; ν_max/cm^-1: 3520, 2924,

2854, 1710, 1461, 1377, 1264, 1189, 1164.

(iii) A mixture of acetic anhydride (20 mL) and anhydrous

pyridine (20 mL) was added with stirring to the above ester

(2.24 g, 2.88 mmol) in dry toluene (15 mL), stirred for 20 h

at r.t, then diluted with toluene (20 mL); the solvent was

removed under reduced pressure to give an oil. Chroma-

7.5, 15 Hz), 5.09 (1H, ddd, J 4.1, 7.23, 8.2 Hz), 3.68 (3H,

s), 3.49 (1H, dt, J 3.5, 6.3 Hz), 2.62 (1H, ddd, J 4.4, 7.0,

11.0 Hz), 2.05 (3H, s), 1.99 (2H, q, J 7 Hz), 1.70 – 1.20

(139H, br m including br s at 1.27), 1.08 – 1.00 (1H, m),

0.93 (3H, d, J 7 Hz), 0.89 (6H, t, J 6.7 Hz), 0.88 (9H, s),

0.80 (3H, d, J 7 Hz), 0.04 (3H, s), 0.03 (3H, s); δ_C(126

MHz, CDCl₃): 173.6, 170.3, 136.5, 128.4, 75.9, 74.1, 51.5,

49.6, 37.7, 37.2, 36.7, 33.5, 32.6, 32.5, 31.9, 31.7, 30.0,

29.9, 29.8, 29.7, 29.67, 29.62, 29.6, 29.54, 29.5, 29.43,

29.41, 29.4, 29.1, 28.1, 27.7, 27.5, 27.4, 26.0, 25.9, 25.0,

22.7, 21.0, 20.9, 18.2, 14.4, 14.1, –4.2, –4.4; υ_max: 2923,

2853, 1745, 1464 cm^-1.

tography (petrol/EtOAc, 5:1) gave

a

white solid,

o

compound 13 (2.2 g, 94%), m.p. 37 – 38 C, [α]²_D⁵+15.5 (c

1.03, CHCl₃) [Found (M+Na)⁺: 843.7431, C₅₂H₁₀₀NaO₆

requires: 843.7412]; δ_H(500 MHz, CDCl₃): 5.11 – 5.07

(1H, m), 4.01 (1H, dd, J 6.0, 7.9 Hz), 3.88 (1H, br q, J 6.9

Hz), 3.69 (3H, s), 3.61 (1H, br t, J 7.7 Hz), 2.62 (1H, ddd, J

4.4, 6.9, 10.9 Hz), 2.04 (3H, s), 1.62 – 1.26 (76H, m, v.br),

1.41 (3H, s), 1.36 (3H, s), 1.13 – 1.06 (1H, m), 0.96 (3H, d,

J 7.0 Hz), 0.89 (3H, t, J 7.0 Hz); δ_C(126 MHz, CDCl₃):

173.6, 170.3, 108.5, 80.4, 74.1, 67.8, 51.5, 49.6, 36.5, 32.7,

31.9, 31.7, 29.9, 29.7(v.br), 29.64, 29.61, 29.54, 29.46,

29.42, 29.4, 29.3, 28.1, 27.5, 27.0, 26.6, 25.5, 25.0, 22.7,

21.0, 15.6, 14.1; ν_max/cm^-1: 2924, 2854, 1747, 1465, 1369,

1236, 1163, 1066.

(ii) Compound 16 (150 mg, 0.108 mmol) was dissolved in

dry THF (8 mL) under nitrogen in a dry polyethylene vial,

equipped with a rubber septum, at 0 °C. Pyridine (0.1 mL)

and HF-pyridine complex (0.4 mL) were added and the

mixture stirred for 18 h at 45 °C, then neutralized by slowly

pouring it into sat. aq. NaHCO₃(10 mL) until no more

carbon dioxide was liberated. The product was extracted

with petrol/ether (1:1, 3 × 25 mL), dried and evaporated to

give a white solid. Chromatography (petrol/ether, 10:1)

gave a white solid, methyl (2R,3R,21R,39S,40S,E)-3-

acetoxy-2-docosyl-39-hydroxy-21,40-dimethyloctapenta-

cont-22-en-oate 17 (R = Ac, R’ = Me) (138 mg, 92%), [α]

3.3 (R)-2-((1R,19R)-1-Acetoxy-19-methyl-20-oxoeicosyl)-

tetracosanoic acid methyl ester 14

23

D

–3.59 (c 1.04, CHCl₃); m.p. 36 – 38 °C [MALDI-Found

Periodic acid (1.0 g, 4.4 mmol) was added to the acetal 13

(1.20 g, 1.46 mmol) in dry ether (60 mL) at r.t. under argon

and stirred for 18 h. The mixture was filtered through celite

and washed with ether. The solvent was evaporated;

chromatography eluting with petrol/EtOAc (4:1) gave a

white solid, the title compound 14 (0.87 g, 80%), m.p. 31 –

M+Na⁺: 1290.2; C₈₅H₁₆₆NaO₅requires: 1290.2]; δ_H(500

MHz, CDCl₃): 5.33 (1H, dt, J 6.6, 15.5 Hz), 5.24 (1H, dd, J

7.6, 15.5 Hz), 5.09 (1H, dt, J 3.9, J 8 Hz), 3.69 (3H, s), 3.53

– 3.49 (1H, m), 2.62 (1H, ddd, J 4.4, 7, 10.9 Hz), 2.04 (3H,

s), 1.98 (2H, q, J 7 Hz), 1.63 –1.20 (140H, br m including

br s at 1.27), 1.18 – 1.11 (1H, m), 0.94 (3H, d, J 6.6 Hz),

0.89 (6H, t, J 6.7 Hz), 0.86 (3H, d, J 7 Hz); δ_C(126 MHz,

CDCl₃): 173.6, 170.3, 136.5, 128.4, 75.3, 74.1, 51.5, 49.6,

38.3, 37.2, 36.7, 34.5, 33.5, 32.6, 31.9, 31.7, 30.0, 29.9,

29.8, 29.72, 29.7, 29.63, 29.6, 29.5, 29.45, 29.44, 29.42,

29.4, 29.1, 28.1, 27.7, 27.5, 27.4, 27.3, 26.2, 25.0, 22.7,

21.0, 20.9, 18.2, 14.4, 13.6; υ_max: 3542, 2923, 2843, 1745,

1464 cm^-1.

o

31 C, [α]²_D³+3.2 (c 0.69, CHCl₃) [Found (M+Na)⁺:

771.6834, C₄₈H₉₂NaO₅requires: 771.6837]. This showed δ_H

(500 MHz, CDCl₃): 9.62 (1H, d, J 1.9 Hz), 5.11 – 5.07

(1H, m), 3.68 (3H, s), 2.62 (1H, ddd, J 4.1, 7.0, 10.4 Hz),

2.33 (1H, d sext, J 1.9, 7.0 Hz), 2.03 (3H, s), 1.73 – 1.26

(76H, m, v.br), 1.09 (3H, d, J 7.0 Hz), 0.88 (3H, t, J 7.0

Hz); δ_C: 205.4, 173.7, 170.3, 74.1, 51.5, 49.6, 46.3, 31.9,

31.7, 30.5, 29.7(v.br), 29.62, 29.6, 29.54, 29.5, 29.42, 29.4,

29.3, 28.1, 27.5, 26.9, 25.0, 22.7, 21.0, 14.1, 13.3; ν_max/cm^-

¹: 2922, 2852, 1744, 1467, 1372, 1237, 1167, 1022.

(iii) Lithium hydroxide monohydrate (17.04 mg, 0.7102

mmol, 30 mol. equiv.) was added to a stirred solution of

ester 17 (R = Ac, R’ = Me) (30.0 mg, 0.0237 mmol) in

THF (3 mL), methanol (0.5 mL) and water (0.5 mL) at RT.

The mixture was stirred at 43 °C for 18 h, then cooled to

RT and acidified with hydrochloric acid (5 %, 2 mL) and

the aqueous layer was extracted with warm petrol/ether

(1:1, 3 × 10 mL). The combined organic extracts were dried

and evaporated; column chromatography (warm

petrol/EtOAc, 5:1) gave a white solid, acid 17 (R = R’ =

3.4 (2R,3R,21R,39S,40S,E)-2-Docosyl-3,39-dihydroxy-

21,40-dimethyloctapentacont-22-enoic acid 17 (R = R’ =

H)

(i) Ester 14 (0.24 g, 0.32 mmol) in dry 1,2-

dimethoxyethane (10 mL) was added to a stirred solution of

sulfone 15 (0.34 g, 0.40 mmol)⁶⁰in dry 1,2-

dimethoxyethane (20 mL) at r.t under nitrogen. The

mixture was cooled to –20 °C and KHMDS (1.04 mL,

0.520 mmol, 0.5 M in toluene) was added, then allowed to

reach r.t and stirred for 1.5 h. Sat. aq. NH₄Cl (25 mL) and

petrol/ ether (1:1, 50 mL) were added. The aqueous layer

was re-extracted with petrol/ether (1:1, 2 × 40 mL) and the

combined organic layers were evaporated. Chromatography

H) (18.6 mg, 65%), [α]²¹–2.1 (CHCl₃, 0.74 µmol)

D

[MALDI-Found M+Na⁺: 1234.3; C₈₂H₁₆₂NaO₄requires:

1234.2]; δ_H(500 MHz, CDCl₃): 5.33 (1H, dt, J 6.6, 15.5

Hz), 5.23 (1H, dd, J 7.6, 15.5 Hz), 3.70 – 3.69 (1H, m),

3.52 – 3.51 (1H, m), 2.45 (1H, br pent, J 4.7 Hz), 2.05 –

2.00 (1H, m), 1.97 (2H, q, J 6.9 Hz), 1.79 – 1.71 (1H, m),

1.66 – 1.59 (2H, m), 1.64 – 1.23 (139H, br m, including br

s at 1.26), 0.94 (3H, d, J 6.6 Hz), 0.89 (6H, t, J 7.0 Hz),

0.86 (3H, d, J 7.3 Hz); δ_C(126 MHz, CDCl₃): 177.3, 136.5,

128.5, 75.4, 72.2, 50.5, 37.3, 36.7, 34.4, 33.4, 32.6, 31.9,

30.0, 29.7, 29.6, 29.52, 29.5, 29.4, 29.1, 27.4, 22.7, 21.0,

16.6, 14.1; ν_max: 3534, 2922, 2854, 1751, 1466 cm^-1.

(petrol/ether, 20:1) gave

a

white solid, methyl

(2R,3R,21R,39S,40S,E)-3-acetoxy-39-((tert-butyl-

dimethylsilyl)oxy)-2-docosyl-21,40-dimethyloctapenta-

cont-22-enoate 16 (0.15 g, 34%), m.p. 25 – 27 °C, [α]_D²³

–

3.18 (c 0.875, CHCl₃) [MALDI-Found M+Na⁺: 1404.2;

C₉₁H₁₈₀NaO₅Si requires: 1404.3] which showed δ_H(500

MHz, CDCl₃): 5.34 (1H, dt, J 6.7, 15 Hz), 5.23 (1H, dd, J

ACCEPTED MANUSCRIPT

8

re-extracted with petrol/EtOAc (3

× 15 mL), and

3.5

(2R,3R,21R,40S,E)-2-Docosyl-3-hydroxy-21,40-

evaporated. The residue was dissolved in THF (6 mL),

methanol (1.0 mL) and water (1.5 mL); to this, potassium

carbonate (300 mg) was added and the mixture stirred at 45

°C for 2 h. The mixture was diluted with petrol/EtOAc

(1:1, 10 mL) and water (1 mL) and acidified with

potassium hydrogen sulfate to pH 6. The aqueous layer was

re-extracted with petrol/EtOAc (5:2, 2 × 10 mL), the

combined organic layers were dried and evaporated.

Chromatography (petrol/EtOAc, 20:1) gave a colourless

oil, (2R,3R,21R,39S, 40S,E)-3-((tert-butyldi-methylsilyl)-

oxy)-2-docosyl-21,40-dimethyl-39-((tetra-hydro-2H-pyran-

2-yl)oxy)octapentacont-22-enoic acid 22 (84 mg, 84%) as a

mixture of diastereisomers [MALDI-Found M+Na⁺:

1433.5; C₉₃H₁₈₄NaO₅Si requires: 1433.5]; δ_H(400 MHz,

CDCl₃): 5.34 (1H, dt, J 6.6, 15.2 Hz), 5.24 (1H, dd, J 7.6,

15.2 Hz), 4.67 (0.5H, br d, J 2.9 Hz), 4.62 (0.5H, br t, J 4.5

Hz ), 3.96 – 3.89 (1H, m), 3.86 – 3.82 (1H, m), 3.50 – 3.42

(2H, m), 2.55 – 2.51 (1H, ddd, J 3.3, 5.6, 12.6 Hz), 2.07 –

2.01 (1H, m), 1.97 (2H, q, J 7.3 Hz), 1.85 – 1.80 (1H, m),

1.75 – 1.1 ( 145H, br m), 0.93 (3H, d, J 6.8 Hz), 0.90 (9H,

s), 0.87 (6H, t, J 6.6 Hz), 0.84 (3H, d, J 7.0 Hz), 0.14 (3H,

s), 0.13 (3H, s); δ_C(126 MHz, CDCl₃): 171.0, 136.4, 128.3,

98.5, 97.8, 81.4, 80.9, 73.5, 62.7, 62.4, 50.7, 37.2, 36.7,

36.4, 35.1, 34.9, 32.6, 32.4, 32.0, 31.9, 31.4, 31.2, 31.17,

30.1, 30.0, 29.82, 29.8, 29.73, 29.7, 29.6, 29.57, 29.52,

29.5, 29.42, 29.4, 29.1, 29.0, 27.7, 27.4, 27.38, 26.3, 25.8,

25.7, 25.2, 22.73, 22.7, 22.6, 21.1, 20.9, 20.4, 19.4, 18.8,

17.8, 14.3, 14.1, 13.6, 11.4, 4.3, –4.9; υ_max: 3425, 2924,

2853, 2362, 1702, 1464 cm^-1.

dimethyl-39-oxooctapentacont-22-enoic acid 23

(i) 2,3-Dihydropyran (0.18 mL, 2.0 mmol) and pyridinium-

p-toluene sulfonate (120 mg, 0.0510 mmol) were added to

a stirred solution of the ester 17 (R = Ac) (130 mg, 0.101

mmol) in dry CH₂Cl₂(1 mL) under nitrogen at r.t. The

mixture was stirred for 1 h, then quenched with sat. aq.

NaHCO₃(3 mL) and extracted with CH₂Cl₂(2 × 15 mL).

The combined organic layers were washed with water (5

mL) and evaporated to give an oil; chromatography

(petrol/EtOAc, 10:1) gave a colourless oil, methyl (2R,

3R,21R,39S,40S,E)-3-acetoxy-2-docosyl-21,40-dimethyl-

39-((tetrahydro-2H-pyran-2-yl)oxy)octapenta-cont-22-en-

oate 21 (128 mg, 92%) as a mixture of diastereoisomers

[MALDI-Found M+Na⁺: 1374.2; C₉₀H₁₇₄NaO₆requires:

1374.3]; δ_H(400 MHz, CDCl₃): 5.33 (1H, dt, J 6.7, 15.2

Hz), 5.24 (1H, dd, J 7.6, 15.2 Hz), 5.11 – 5.07 (1H, m),

4.65 (0.5H, br t, J 2.8 Hz), 4.62 (0.5H, br t, J 2.6 Hz), 3.96

– 3.89 (1H, m), 3.68 (3H, s), 3.50 – 3.44 (2H, m), 2.63 (1H,

ddd, J 4.7, 6.7, 10.8 Hz), 2.1 – 2.0 (4H, including s at

2.03), 1.97 (2H, q, J 6.6 Hz), 1.86 – 1.82 (1H, m), 1.59 –

1.14 (144H, br m including br s at 1.26), 0.94 (3H, d, J 6.7

Hz), 0.89 (6H, t, J 6.6 Hz), 0.84 (3H, d, J 6.7 Hz); δ_C(126

MHz, CDCl₃): 173.7, 170.3, 136.5, 128.4, 98.5, 97.8, 81.4,

80.9, 74.1, 62.7, 62.4, 51.5, 49.6, 37.2, 36.7, 36.4, 35.1,

32.6, 32.5, 32.0, 31.9, 31.7, 31.4, 31.3, 31.2, 29.94, 29.9,

29.8, 29.7, 29.65, 29.6, 29.5, 29.42, 29.4, 29.35, 29.1, 28.1,

27.8, 27.5, 27.4, 27.3, 26.1, 25.7, 25.66, 25.6, 25.0, 22.7,

21.0, 20.9, 20.1, 19.8, 15.2, 14.9, 14.1; υ_max: 2924, 2853m,

2360m, 2341m, 1464 cm^-1.

(iv) Pyridinium-p-toluene sulfonate (PPTS) (128 mg, 0.588

mmol) was added to a stirred solution of the acid 22 (83

mg, 0.058 mmol) in THF (3 mL), and MeOH (0.5 mL) and

the mixture stirred at 40 °C for 6 h, then sat. aq. NaHCO₃

(0.2 mL) was added and the product extracted with

petrol/EtOAc (1:1, 3 × 10 mL). The combined organic

layers were evaporated to give a crude oil; chromatography

(ii) Lithium hydroxide monohydrate (37 mg, 0.88 mmol.)

was added to a stirred solution of ester 21 (120 mg, 0.088

mmol) in THF (8 mL), methanol (1.0 mL) and water (1.5

mL) at r.t. The mixture was stirred at 45 °C for 18 h, then

cooled to r.t., acidified with hydrochloric acid (5 %, 2 mL

pH 6) and extracted with warm petrol/EtOAc (5:2, 3 × 10

mL). The combined organic extracts were evaporated;

chromatography (5:1 petrol/EtOAc) gave a white solid,

(petrol/ EtOAc, 10:1) gave

(2R,3R,21R,39S,40S,E)-3-((tert-butyldimethylsilyl)oxy)-2-

docosyl-39-hydroxy-21, 40-dimethyloctapentacont-22-

a

colourless oil,

(2R,

3R,21R,39S,40S,E)-2-docosyl-3-hydroxy-21,40-

dimethyl-39-((tetrahydro-2H-pyran-2-

enoic acid (76 mg, 97%) [Found (M+Na)⁺: 1348.6;

C₈₈H₁₇₆NaO₄Si requires: 1348.3]; δ_H(400 MHz, CDCl₃):

5.34 (1H, dt, J 6.4, 15.3 Hz), 5.24 (1H, dd, J , 7.4, 15.3

Hz), 3.85 (1H, br, dd, J 6.1, 9.8 Hz), 3.51 (1H, dt, J 4.5, 8.8

Hz), 2.53 (1H, ddd, J 3.8, 5.4, 9.3 Hz), 2.03 (1H, m), 1.97

(2H, q, J 6.6 Hz), 1.73 – 1.62 (1H, m), 1.43 – 1.26 (140H,

br m including, s at 1.26), 0.94 (3H, d, J 6.7 Hz), 0.92 (9H,

s), 0.88 (6H, t, J 6.8 Hz), 0.86 (3H, d, J 6.6 Hz), 0.13 (3H,

s), 0.12 (3H, s); δ_C(126 MHz, CDCl₃): 177.7, 136.5, 128.4,

75.3, 73.7, 50.0, 41.4, 38.2, 37.3, 36.7, 35.8, 34.5, 33.4,

32.6, 31.9, 31.6, 30.1, 30.0, 29.82, 29.8, 29.73, 29.7, 29.64,

29.6, 29.52, 29.5, 29.44, 29.4, 29.1, 29.0, 27.7, 27.4, 27.37,

26.3, 25.8, 25.7, 25.2, 22.72, 22.7, 22.6, 21.1, 20.9, 20.4,

19.4, 18.8, 17.9, 14.3, 14.2, 14.1, 13.6, 11.4, –4.2, –4.9;

υ_max: 3424, 2923, 2852, 1709, 1464 cm^-1.

yl)oxy)octapentacont-22-enoic acid as

a mixture of

diastereoisomers (100 mg, 90%) [MALDI-Found (M+Na)⁺:

1318.1; C₈₇H₁₇₀NaO₅requires: 1318.2]; δ_H(400 MHz,

CDCl₃): 5.33 (1H, dt, J 6.44, 15.4 Hz), 5.25 (1H, dd, J 7.4,

15.4 Hz), 4.67 (0.5H, br t, J 3.1 Hz), 4.44 (0.5H, br t, J 3.9

Hz), 3.97 – 3.89 (1H, m), 3.71 (1H, br q, J 6.7 Hz), 3.55 –

3.43 (2H, m), 2.45 (1H, br dt, J 5.3, 8.6 Hz), 2.05 – 2.03

(1H, br, m), 1.97 (2H, q, J 6.5 Hz), 1.91 – 1.03 (147H, br,

m), 0.93 (3H, d, J 6.7 Hz), 0.88 (6H, t, J 6.3 Hz), 0.85 (3H,

d, J 6.9 Hz); δ_C(126 MHz, CDCl₃): 179.7, 136.4, 128.4,

98.4, 97.7, 81.5, 81.0, 72.1, 62.6, 62.3, 50.8, 37.2, 36.7,

35.5, 32.5, 31.9, 31.2, 29.9, 29.8, 29.7, 29.6, 29.53, 29.5,

29.4, 29.3, 29.1, 27.3, 25.7, 25.6, 22.6, 20.9, 15.1, 14.9,

14.1; υ_max: 3424, 2922, 2852, 2361, 1646 cm^-1.

(iii) Imidazole (510 mg, 0.756 mmol) was added to a stirred

solution of the above acid (98.0 mg, 0.076 mmol) in dry

DMF (1.5 mL) and dry toluene (2.5 mL) at RT followed by

addition of TBDMSCl (0.110 mg, 0.756 mmol) and DMAP

(14.0 mg, 0.115 mmol). The mixture was heated to 70 °C

for 18 h; it was then diluted with petrol/EtOAc (1:1, 15

mL) and sat. aq. NaHCO₃(3 mL). The aqueous layer was

(v) The above acid (75 mg, 0.056 mmol), in CH₂Cl₂(10

mL), was added to a stirred suspension of PCC (36 mg,

0.16 mmol) in CH₂Cl₂(2 mL) at r.t. and stirred for 1 h. The

solvent was evaporated; chromatography (petrol/EtOAc,

5:1) gave a semi solid, (2R,3R,21R,40S, E)-3-((tert-

butyldimethylsilyl)oxy)-2-docosyl-21,40-dimethyl-39-oxo-

ACCEPTED MANUSCRIPT

9

octapentacont-22-enoic acid (61 mg, 82%) [MALDI-

Found (M+Na)⁺: 1346.3; C₈₈H₁₇₄NaO₄Si requires: 1346.3];

δ_H(400 MHz, CDCl₃): 5.34 (1H, dt, J 6.4, 15.2 Hz), 5.24

(1H, dd, J 7.4, 15.3 Hz), 3.86 (1H, br q, J 6.1 Hz), 2.57 –

2.46 (2H, m), 2.41 (2H, dt, J, 1.9, 7.3 Hz), 2.08 – 2.00 (1H,

m), 1.97 (2H, q, J 6.7 Hz), 1.70 – 1.59 (2H, m), 1.60 – 1.49

(4H, m), 1.28 (131H, s), 1.05 (3H, d, J 6.9 Hz), 0.95 (3H, d,

J 6.7 Hz), 0.91(9H, s), 0.88 (6H, t, J 6.6 Hz), 0.12 (3H, s),

0.11 (3H, s); δ_C(126 MHz, CDCl₃): 215.3, 178.0, 136.5,

128.4, 73.7, 50.0, 46.3, 41.2, 37.3, 36.7, 35.8, 33.1, 32.6,

31.9, 29.8, 29.72, 29.7, 29.64, 29.6, 29.58, 29.54, 29.5,

29.48, 29.46, 29.44, 29.4, 29.3, 29.22, 29.2, 27.4, 27.32,

27.3, 25.7, 25.2, 23.7, 22.7, 20.1, 17.9, 16.4, 14.1, –4.2, –

4.9; υ_max: 3419, 2925, 2854, 2360, 2341, 1711, 1464 cm^-1.

(vi) The above acid (10 mg, 0.0075 mmol) was dissolved

in dry THF (4 mL) under nitrogen in a dry polyethylene

vial equipped with a rubber septum at 0 °C. Pyridine (0.1

mL) and HF-pyridine complex (0.32 mL, 0.022 mmol.)

were added and the mixture stirred for 18h at 45 °C then

neutralised by slowly pouring into sat. aq. NaHCO₃(3 mL)

until no more carbon dioxide was liberated. The product

was extracted with petrol/ether (1:1, 3 × 10 mL), and

67.4, 60.4, 50.5, 37.3, 36.7, 35.6, 34.5, 32.6, 31.9, 30.9,

30.3, 30.1, 30.0, 29.9, 29.7, 29.6, 29.5, 29.42, 29.4, 29.3,

29.12, 29.1, 28.9, 28.1, 27.3, 26.1, 25.8, 22.7, 22.6, 22.3,

21.1, 21.0, 20.4, 19.4, 14.2, 14.11, 14.06; υ_max: 3424, 2922,

2852, 2361, 1646 cm^-1.

(ii) Imidazole (22.7 mg, 0.333 mmol, 10 mol. equiv.) was

added to a stirred solution of the above acid (45 mg, 0.033

mmol) in dry DMF (0.2 mL) at r. t. followed by addition of

TBDMSCl (50.0 mg, 0.333 mmol, 10 mol. equiv.) and

DMAP (4.01 mg, 0.0333 mmol). The mixture was heated

to 70 °C for 18 h, then diluted with petrol/EtOAc (1:1, 15

mL) and sat. aq. NaHCO₃(3 mL). The aqueous layer was

re-extracted with petrol/EtOAc (3 × 15 mL), and the

solvent evaporated. The residue was dissolved in THF (5

mL), methanol (0.5 mL) and water (0.5 mL); to this,

potassium carbonate (100 mg) was added and the mixture

stirred at 45 °C for 6 h, then diluted with petrol/EtOAc

(1:1, 10 mL) and water (1 mL) and acidified with

potassium hydrogen sulfate to pH 2. The aqueous layer was

re-extracted with petrol/EtOAc (1:1, 2 × 10 mL), and the

combined organic layers dried and the solvent evaporated.

Chromatography (petrol/EtOAc, 20:1) gave a colourless

evaporated to give

a

white solid. Chromatography

oil,

(2R,3R,21R,39R,

40R,E)-3-((tert-butyl-

(petrol/EtOAc, 5:1) gave a white solid, compound 23 (9.0

mg, 98%) [MALDI-Found (M+Na)⁺: 1232.2; C₈₂H₁₆₀O₄Na

requires: 1232.2]; δ_H(500 MHz, CDCl₃): 5.32 (1H, dt, J

6.7, 15.5 Hz), 5.24 (1H, dd, J 7.6, 15.5 Hz), 3.73 (1H, m),

2.50 – 2.45 (2H, m), 2.42 (2H, t, J 7.0 Hz ), 2.02 (1H, m),

1.96 (2H, q, J 6.8 Hz), 1.74 (2H, m), 1.65 – 1.51 (10H, m),

1.26 (126H, br m), 1.05 (3H, d, J 7.0 Hz), 0.95 (3H, d, J 7.0

Hz), 0.89 (6H, t, J 6.7 Hz); δ_C: 215.5, 177.9, 136.5, 128.4,

72.2, 50.6, 46.4, 41.2, 37.3, 36.7, 35.6, 33.1, 32.6, 31.9,

29.8, 29.7, 29.64, 29.6, 29.54, 29.51, 29.5, 29.43, 29.4,

29.3, 29.1, 28.9, 27.4, 27.3, 25.7, 23.7, 22.7, 22.6, 21.0,

19.4, 16.4, 14.1; υ_max: 3420, 3019, 2926, 2855, 1521, 1420,

1215 cm^-1.

dimethylsilyl)oxy)-2-docosyl-21,40-dimethyl-39-((tetra-

hydro-2H-pyran-2-yl)oxy)octapenta-cont-22-enoic acid (34

mg, 70%), [ꢀ]^ꢂ_ꢁ^ꢃ+1.58 (c 0.436 µmol, CHCl₃) [MALDI-

Found (M+Na)⁺: 1432.3; C₉₃H₁₈₄NaO₅Si requires: 1432.3];

δ_H(500 MHz, CDCl₃): 5.34 (1H, dt, J 6.6, 15.1 Hz), 5.24

(1H, dd, J 7.6, 15.1 Hz), 4.67 (0.5H, br t, J 3.0 Hz), 4.44

(0.5H, br t, J 3.8 Hz), 3.97 – 3.89 (1H, m), 3.71 (1H, br q, J

6.7 Hz), 3.57 – 3.42 (2H, m), 2.55 – 2.51 (1H, m), 2.05 –

2.03 (1H, m), 1.97 (2H, q, J 7.0 Hz), 1.88 – 1.82 (1H, m),

1.70 – 1. 31 (145H, br m including br s at 1.26), 0.94 (3H,

d, J 7.0 Hz), 0.93 (9H, s), 0.89 (6H, t, J 6.6 Hz), 0.84 (3H,

d, J 7.0 Hz), 0.15 (3H, s), 0.14 (3H, s); δ_C(126 MHz,

CDCl₃): 177.4, 136.6, 128.3, 98.5, 75.3, 73.7, 67.4, 49.9,

41.7, 39.7, 37.6, 36.7, 35.8, 34.8, 33.3, 32.6, 31.9, 31.6,

30.1, 30.0, 29.82, 29.8, 29.73, 29.7, 29.61, 29.6, 29.52,

29.5, 29.42, 29.4, 29.1, 29.0, 27.7, 27.41, 27.4, 26.3, 25.8,

25.7, 25.2, 22.73, 22.69, 22.6, 21.1, 20.9, 20.4, 19.4, 18.8,

17.8, 14.3, 14.1, 13.6, 11.4, –4.3, –4.9; υ_max: 3425, 2924,

2853, 2362, 1702, 1464 cm^-1.

(iii) PPTS (29.6 mg, 0.118 mmol) was added to a stirred

solution of the above acid (17.3 mg, 0.0118 mmol) in THF

(1 mL), methanol (0.1 mL) and water (0.1 mL) and the

mixture refluxed for 2.5 h followed by stirring at 47 °C for

24 h, then sat. aq. NaHCO₃(0.2 mL) was added and the

product extracted with petrol/EtOAc (1:1, 3 × 5 mL). The

combined organic layers were evaporated to give an oil;

chromatography eluting with petrol/EtOAc (10:1) gave a

colourless oil, (2R,3R,21R,39R,40R,E)-3-((tert-butyldi-

methylsilyl)-oxy)-2-docosyl-39-hydroxy-21,40-dimethyl-

octapentacont-22-enoic acid (12 mg, 73%), [ꢀ]^ꢂ_ꢁ^ꢃ+1.46 (c

1.43 µmol, CHCl₃) [MALDI-Found (M+Na)⁺: 1348.5;

C₈₈H₁₇₆NaO₄Si requires: 1348.3]; δ_H(500 MHz, CDCl₃):

5.31 (1H, dt, J 6.6, 15.3 Hz), 5.24 (1H, dd, J 7.6, 15.3 Hz),

3.83 (1H, dist. pent, J 2.3 Hz), 3.52 – 3.49 (1H, m), 2.55 –

2.51 (1H, m), 2.05 – 2.03 (1H, m), 1.97 (2H, q, J 6.6 Hz),

1.63 – 1.22 (141H, br m including br s at 1.26), 0.97 (3H,

d, J 6.7 Hz), 0.93 (9H, s), 0.89 (3H, d, J 7.3 Hz), 0.88 (6H,

t, J 6.6 Hz), 0.15 (3H, s), 0.14 (3H, s); δ_C(126 MHz,

CDCl₃): 177.7, 136.5, 128.4, 75.3, 73.7, 50.0, 41.4, 38.2,

3.6 (2R,3R,21R,40R,E)-2-Docosyl-3-hydroxy-21,40-

dimethyl-39-oxooctapentacont-22-enoic acid 25

(i) Lithium hydroxide monohydrate (52.8 mg, 1.55 mmol,

30 mol.equiv.) was added to a stirred solution of ester 24

(70.0 mg, 0.0520 mmol) (see Supplementary Information)

in THF (5 mL), methanol (0.5 mL) and water (0.5 mL) at

RT. The mixture was stirred at 43 °C for 18 h, then cooled

to RT and acidified with hydrochloric acid (5%, 2 mL) and

the aqueous layer extracted with warm petrol/ether (1:1, 3 ×

10 mL). The combined organic extracts were dried and

evaporated; chromatography eluting with petrol/EtOAc

(5:2) gave a semi solid, (2R,3R,21R, 39R,40R,E)-2-

docosyl-3-hydroxy-21,40-dimethyl-39-((tetrahydro-2H-

pyran-2-yl)oxy)octapenta-cont-22-enoic acid (46.0 mg,

72%), [α]²²_D+1.75 (c 1.19 µmol, CHCl₃) [MALDI-Found

(M+Na)⁺: 1318.1; C₈₇H₁₇₀NaO₅requires: 1318.3]; which

showed δ_H(500 MHz, CDCl₃): 5.33 (1H, dt, J 6.7, 15.2

Hz), 5.24 (1H, dd, J 7.6, 15.2 Hz), 4.67 (0.5H, br t, J 3.1

Hz), 4.44 (0.5H, br t, J 3.8 Hz), 3.97 – 3.89 (1H, m), 3.71

(1H, br q, J 6.7 Hz), 3.55 – 3.43 (2H, m), 2.49 – 2.42 (1H,

m), 2.05 – 2.03 (1H, m), 1.97 (2H, q, J 7.3 Hz), 1.88 – 1.82

(1H, m), 1.63 – 1.21 (146H, br m including br s at 1.26),

0.94 (3H, d, J 6.7 Hz), 0.89 (6H, t, J 6.6 Hz), 0.84 (3H, d, J

6.7 Hz); δ_C(126 MHz, CDCl₃): 177.5, 145.5, 136.5, 72.2,

ACCEPTED MANUSCRIPT

10

37.3, 36.7, 35.8, 34.5, 33.4, 32.6, 31.9, 31.6, 30.1, 30.0,

29.82, 29.8, 29.73, 29.7, 29.64, 29.6, 29.52, 29.5, 29.44,

29.4, 29.1, 29.0, 27.7, 27.4, 27.37, 26.3, 25.8, 25.7, 25.2,

22.72, 22.7, 22.6, 21.1, 20.9, 20.4, 19.4, 18.8, 17.9, 14.3,

14.2, 14.1, 13.6, 11.4, –4.2, –4.9; υ_max: 3424, 2923, 2852,

1709, 1464 cm^-1.

(iv) The above acid (18.1 mg, 0.0131 mmol), in CH₂Cl₂(1

mL) was added to a stirred suspension of PCC (8.47 mg,

0.0393 mmol, 3 mol. equiv.) in CH₂Cl₂(2 mL) at RT. The

mixture was stirred for 1 h. The solvent was evaporated;

chromatography (petrol/EtOAc, 5:1) gave a white solid,

aqueous layer extracted with warm petrol/ether (1:1, 3 × 10

mL). The combined organic extracts were evaporated;

chromatography (petrol/EtOAc, 5:1) gave a white solid,

(2R,3R,21R,39R,40R,E)-2-docosyl-3,39-dihydroxy-21,40-

dimethyloctapentacont-22-enoic acid (20, R = R’ = H) (8.6

mg, 59%), [α]²⁰+1.67 (c 1.29 µmol, CHCl₃,) [Found

D

(M+Na)⁺: 1234.5; C₈₂H₁₆₂NaO₄requires: 1234.2]; δ_H(500

MHz, CDCl₃): 5.33 (1H, dt, J 6.6, 15.5 Hz), 5.23 (1H, dd, J

7.6, 15.5 Hz), 3.70 – 3.69 (1H, m), 3.52 – 3.51 (1H, m),

2.45 (1H, br pent, J 4.7 Hz), 2.05 – 2.00 (1H, m), 1.97 (2H,

q, J 6.9 Hz), 1.79 – 1.71 (1H, m), 1.66 – 1.59 (2H, m), 1.64

– 1.23 (139H, br m, including br s at 1.26), 0.94 (3H, d, J

6.6 Hz), 0.89 (6H, t, J 7.0 Hz), 0.86 (3H, d, J 7.3 Hz); δ_C

(126 MHz, CDCl₃): 177.3, 136.5, 128.5, 75.4, 72.2, 50.5,

37.3, 36.7, 34.4, 33.4, 32.6, 31.9, 30.0, 29.7, 29.6, 29.52,

29.5, 29.4, 29.1, 27.4, 22.7, 21.0, 16.6, 14.1; υ_max: 3534,

2922, 2854, 1751, 1466 cm^-1.

(2R,

3R,21R,40R,E)-3-((tert-butyldimethylsilyl)oxy)-2-

docosyl-21,40-dimethyl-39-oxooctapentacont-22-enoic

acid (16.0 mg, 89%), [ꢀ]^ꢂ_ꢁ^ꢃ+3.70 (c 0.457 µmol, CHCl₃)

[MALDI-Found

(M+Na)⁺:

1346.4;

C₈₈H₁₇₄NaO₄Si

requires: 1346.3]; δ_H(500 MHz, CDCl₃): 5.33 (1H, dt, J

6.0, 15.3 Hz), 5.24 (1H, dd, J 7.6, 15.3 Hz), 3.85 (1H, m),

2.56 – 2.52 (1H, m), 2.50 (1H, br q, J 6.9 Hz), 2.41 (2H, dt,

J, 1.9, 7.3 Hz), 2.05 – 2.00 (1H, m), 1.97 (2H, q, J 6.6 Hz),

1.75 – 1.68 (2H, m), 1.64 – 1.21 (135H, br m including br s

at 1.26), 1.05 (3H, d, J 6.6 Hz), 0.95 (3H, d, J 7.0 Hz), 0.93

(9H, s), 0.89 (6H, t, J 6.6 Hz), 0.15 (3H, s), 0.14 (3H, s); δ_C

(126 MHz, CDCl₃): 215.3, 178.0, 136.5, 128.4, 73.7, 50.0,

46.3, 41.2, 37.3, 36.7, 35.8, 33.1, 32.6, 31.9, 29.8, 29.72,

29.7, 29.64, 29.61, 29.6, 29.54, 29.5, 29.48, 29.46, 29.44,

29.4, 29.3, 29.23, 29.2, 27.4, 27.32, 27.3, 25.7, 25.2, 23.7,

22.7, 20.1, 17.9, 16.4, 14.1, –4.2, – 4.9; v_max: 3419, 2925,

2854, 2360, 2341, 1711, 1464 cm^-1.

(v) A dry polyethylene vial equipped with a rubber septum,

was charged with the above acid (15 mg, 0.011 mmol) in

dry THF (1 mL) under nitrogen at 0 °C. Pyridine (0.1 mL)

and HF-pyridine complex (0.05 mL, 0.03263 mmol, 3 mol.

equiv.) were added and the mixture stirred for 18h at 43 °C,

then neutralised by slowly pouring it into sat. aq. NaHCO₃

(3 mL) until no more carbon dioxide was liberated. The

product was extracted with petrol/ether (1:1, 3 × 10 mL),

and evaporated to give a solid. Chromatography (petrol/

EtOAc, 5:1) gave a white solid, (2R,3R,21R,40R,E)-2-

docosyl-3-hydroxy-21,40-dimethyl-39-oxooctapentacont-

22-enoic acid 25 (6.0 mg, 44%), [ꢀ]^ꢂ_ꢁ^ꢃ+2.90 (c 0.471 µmol,

CHCl₃) [MALDI-Found (M+Na)⁺: 1232.3; C₈₂H₁₆₀NaO₄

requires: 1233.1]; δ_H(500 MHz, CDCl₃): 5.32 (1H, td, J

6.7, 15.5 Hz), 5.24 (1H, dd, J 7.6, 15.5 Hz), 3.72 (1H, m),

2.50 – 2.45 (2H, m), 2.42 (2H, dt, J 1.6, 7.0 Hz), 2.02 (1H,

m), 1.97 (2H, q, J 7.0 Hz), 1.65 – 1.17 (138H, br m,

including br s at 1.26), 1.05 (3H, d, J 7.0 Hz), 0.95 (3H, d,

J 7.0 Hz), 0.89 (6H, t, J 7.0 Hz); δ_C(126 MHz, CDCl₃):

215.5, 177.9, 136.5, 128.4, 72.2, 50.6, 46.4, 41.2, 37.3,

36.7, 35.6, 33.1, 32.6, 31.9, 29.8, 29.7, 29.64, 29.6, 29.54,

29.51, 29.5, 29.43, 29.4, 29.3, 29.1, 28.9, 27.4, 27.3, 25.7,

23.7, 22.7, 22.6, 21.0, 19.4, 16.4, 14.1; υ_max: 3420, 3019,

2926, 2855, 1521, 1420, 1215 cm^-1.

3.8 (R)-12-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)tridecyl

pivaloate 27

LiHMDS (87.70 mL, 92.96 mmol, 1.06 M) was added to a

stirred solution of 2,2-dimethylpropionic acid 9-(2-phenyl-

2H-pentazol-1-sulfonyl)nonyl ester 26 (27.03 g, 61.97

mmol)⁴³and (R)-3-((S)-aldehyde 8 (8.20 g, 47.6 mmol)^{39, 45-}

47

o

in dry THF (200 mL) at –10 C under nitrogen, then

allowed to reach r.t. and stirred for 2 h. Petrol/ether (1:1,

100 mL) and sat. aq. NH₄Cl (100 mL) were added, the

organic layer was separated and the aq. layer was extracted

with petrol/ether (1:1, 2 × 100 mL). The combined organic

layers were evaporated. Chromatography (petrol/ether,

50:1) gave a colourless oil, (E/Z)(R)-12-((S)-2,2-dimethyl-

ꢄ

ꢅ

1,3-dioxolan-4-yl)tridec-9-en-1-yl pivaloate 14 g, 76%

in ratio (2.5:1). Palladium 10 % on carbon (1 g) was stirred

with the above alkenes (14 g, 36 mmol) in IMS (200 mL)

under hydrogen for 2 h. The solution was filtered on a bed

of celite and evaporated to give a colourless oil of the title

compound 27 (13 g, 92%),[ꢀ]^ꢂ_ꢁ^ꢆ+16.1 (c 1.14, CHCl₃)

[Found [M

–

CH₃]⁺: 369.2989, C₂₂H₄₁O₄requires:

369.2989]; δ_H(400 MHz, CDCl₃): 4.05 (2H, t, J 6.6 Hz),

4.0 (1H, dd, J 6.3, 7.9 Hz), 3.87 (1H, br q, J 6.9 Hz), 3.60

(1H, br t, J 7.9 Hz), 1.62 (2H, quintet, J 6.6 Hz,), 1.40 (3H,

s), 1.35 (3H, s), 1.32 – 1.26 (18H, m), 1.18 (9H, s), 1.1 –

1.05 (1H, m), 0.96 (3H, d, J 6.6 Hz). δ_C(126 MHz, CDCl₃)

: 178.6, 108.5, 80.4, 67.8, 64.5, 38.7, 36.5, 32.7, 29.9, 29.7,

29.6, 29.59, 29.54, 29.50, 29.2, 28.6, 27.2, 27.0, 26.6, 25.9,

25.5, 15.6; υ_max/cm^-1: 2926, 2855, 1731, 1463, 1368, 1284,

1158.

3.9 5-(((R)-12-((S)-2,2-Dimethyl-1,3-dioxolan-4-yl)tri-

decyl)sulfonyl)-1-phenyl-1H-tetrazole 28

(i) A solution of (R)-12-((S)-2,2-dimethyl-1,3-dioxolan-4-

yl)tridecyl pivaloate 27 (12.7 g, 33.1 mmol) in dry THF

(100 mL) was added slowly to a stirred suspension of

o

3.7 (2R,3R,21R,39R,40R,E)-2-Docosyl-3,39-dihydroxy-

21,40-dimethyloctapentacont-22-enoic acid 20 (R = R’ =

H)

Lithium hydroxide monohydrate (12.0 mg, 0.355 mmol, 30

mol. equiv.) was added with stirring to ester 19 (15.0 mg,

0.0118 mmol) (see Supplementary Information) in THF (2

mL), methanol (0.2 mL) and water (0.2 mL) at RT. The

mixture was stirred at 43 °C for 18 h, cooled to RT, then

acidified with hydrochloric acid (5 %, 2 mL) and the

LiAlH₄(1.88 g, 49.6 mmol) at –20 C under nitrogen. The

mixture was heated under reflux for 1h, then quenched

with sat. aq. Na₂SO₄(10 mL) at –10 ^oC, stirred until a white

precipitate formed, then MgSO₄(20 g) was added. The

precipitate was filtered through celite and washed with

THF (2 × 50 mL). Column chromatography (5:1 petrol/

ether) gave a colourless oil, (R)-12-((S)-2,2-dimethyl-1,3-

dioxolan-4-yl)-tridecan-1-ol (8.0 g, 81%), [ꢀ]^ꢂ_ꢁ^ꢃ+19.3 (c

0.95, CHCl₃) [Found [M–CH₃]⁺: 285.2443, C₁₇H₃₃O₃

ACCEPTED MANUSCRIPT

11

o

29 (3.27 g, 5.49 mmol)⁴³in dry THF (50 mL) at –10 C

under nitrogen. The mixture was allowed to reach r. t.,

stirred for 2 h, then quenched with sat. aq. NH₄Cl (100 mL)

and diluted with petrol/ether (100 mL). The organic phase

was separated and the aqueous layer was extracted with

petrol/ether (1:1, 2 × 100 mL). The combined organic

layers were dried and tevaporated. Chromatography (15:1

petrol/ether) gave a colourless oil, methyl (E/Z)-(2R)-2-

((1R,15R)-1-((tert-butyldimethylsilyl)oxy)-15-(2,2-di-

methyl-1,3-dioxolan-4-yl)hexadec-3-en-1-yl)hexacosan-

requires: 285.2425]; δ_H(400 MHz, CDCl₃): 3.84 (1H, dd, J

6.2, 7.7 Hz), 3.77 (1H, br q, J 7.0 Hz), 3.50 (1H, br t, J 7.7

Hz), 3.41 (2H, t, J 6.6 Hz,), 1.52 – 1.47 (1H, m), 1.45 (3H,

s), 1.43 – 1.40 (1H, m), 1.36 (3H, s), 1.34 –1.15 (20H, m),

1.01 (3H, d, J 6.9 Hz); δ_C(126 MHz, CDCl₃): 108.7, 80.6,

68.2, 62.7, 37.0, 33.3, 33.2, 30.4, 30.2, 30.18, 30.15, 30.14,

30.12, 30.0, 27.4, 27.0, 26.3, 25.9, 15.9; υ_max/cm^-1: 3395,

2926, 2853, 1466, 1377, 1266, 1205, 1155, 1058.

(ii) Diethyl azodicarboxylate (6.1, 35 mmol) in dry THF (2

mL) was added to a stirred solution of 1-phenyl-1H-

tetrazole-5-thiol (6.20 g, 35.0 mmol), Ph₃P (9.12 g, 35.0

mmol) and (R)-12-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)tri-

decan-1-ol (8.00 g, 27.0 mmol) in dry THF (50 mL) at 0

^oC. The mixture was vigorously stirred at r. t. for 16 h. The

solvent was evaporated and the residue was diluted with

petrol/EtOAc (10:1), refluxed for 0.5 h then filtered,

washed with petrol/EtOAc and the filtrate evaporated.

Chromatography (petrol/ether, 5:1) gave a white solid, 5-

(((R)-12-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)tridecyl)thio)-

1-phenyl-1H-tetrazole (10.0 g, 84%), [ꢀ]^ꢂ_ꢁ^ꢂ+13.6 (c 0.88,

CHCl₃) [Found [M–CH₃]⁺: 445.2635, C₂₄H₃₇N₄O₂S

requires: 445.2637]; δ_H(500 MHz, CDCl₃): 7.60 – 7.52

(5H, m), 4.0 (1H, dd, J 6.3, 7.9 Hz), 3.87 (1H, br q, J 7.1

Hz), 3.60 (1H, br t, J 7.6 Hz), 3.39 (2H, t, J 7.4 Hz,), 1.82

(2H, quintet, J 7.3 Hz), 1.58 – 1.53 (1H, m), 1.46 – 1.41

(2H, m), 1.40 (3H, s), 1.35 (3H, s), 1.33 – 1.25 (15H, m),

1.11 – 1.04 (1H, m), 0.96 (3H, d, J 6.6 Hz); δ_C(126 MHz,

CDCl₃) : 154.5, 133.8, 130.0, 129.7, 123.8, 108.4, 80.4,

67.8, 36.5, 33.3, 32.7, 29.8, 29.6, 29.59, 29.5, 29.1, 29.0,

28.6, 27.0, 26.6, 25.5, 15.6; ν_max/cm^-1: 2925, 2854, 1598,

1501, 1464, 1380, 1245, 1161, 1065.

ꢄ

ꢅ

oate 4.5 g, 96% as a 2.5: 1 mixture. Palladium (10% on

carbon, 1.0 g) was added to the above alkenes (4.5 g, 5.21

mmol) in EtOAc (150 mL) under hydrogen and stirred for

1.5 h. The solution was filtered over celite and evaporated.

Chromatography (petrol/ether, 15:1) gave a colourless oil,

the title compound 30 (4.3 g, 96%), [α] ²_D²+3.6 (c 0.94,

CHCl₃) [Found [M+Na]⁺: 887.7857, C₅₄H₁₀₈NaO₅Si

requires: 887.7863]; δ_H: 4.0 (1H, dd, J 6.3, 7.9 Hz), 3.93 –

3.90 (1H, m), 3.87 (1H, br q, J 7.0 Hz), 3.66 (3H, s), 3.61

(1H, br t, J 7.9 Hz), 2.53 (1H, ddd, J 3.8, 7.3, 11.1 Hz),

1.59 –1.52 (4H, m), 1.41 (3H, s), 1.36 (3H, s), 1.35-1.26

(68H, m, v. br), 1.10 – 1.07 (1H, m), 0.97 (3H, d, J 6.6 Hz),

0.89 (3H, t, J 6.9 Hz), 0.87 (9H, s), 0.05 (3H, s), 0.03 (3H,

s); δ_C(126 MHz, CDCl₃): 175.1, 108.5, 80.4, 73.2, 76.8,

51.6, 51.2, 36.5, 33.7, 32.7, 31.9, 29.9, 29.8, 29.7, 29.66,

29.62, 29.60, 29.58, 29.5, 29.4, 29.3, 27.8, 27.5, 27.0, 26.6,

25.7, 25.5, 23.7, 22.7, 18.0, 15.6, 14.1, –4.4, –4.9; ν_max/cm^-

¹: 2925, 2854, 1741, 1465, 1368, 1253, 1165, 1068.

3.11 Methyl (R)-2-((1R,15R)-1-((tert-butyldimethylsilyl)-

oxy)-15-methyl-16-oxohexadecyl)hexacosanoate 31

(iii) A solution of ammonium molybdate (VI) tetrahydrate

(13 g, 10 mmol) in 35 % H₂O₂(40 mL) was cooled in an

ice bath and added to the above tetrazole (10.3 g, 22.0

Periodic acid (0.40 g, 1.7 mmol) was added to a stirred

solution of ester 30 (0.50 g, 0.58 mmol) in dry ether (35

mL) at r.t. under nitrogen and stirred 18 h. The mixture was

filtered through celite and washed with ether (20 mL). The

solvent was evaporated and the product was purified by

column chromatography eluting with petrol/EtOAc (5:1) to

give a white solid, the title compound 31 (0.4 g, 87%)

[Found [M–^tBu]⁺: 735.6679; C₄₆H₉₁O₄Si requires:

735.6687]; δ_H(500 MHz, CDCl₃): 9.77 (1H, t, J 1.9 Hz),

5.11 – 5.07 (1H, m), 3.92 – 3.89 (1H, m), 3.66 (3H, s), 2.52

(1H, ddd, J 3.8, 7.0, 11.0 Hz), 2.42 (2H, dt, J 1.9, 7.6 Hz),

1.63 (2H, pent, J 6.3 Hz), 1.55 – 1.22 (68H, m), 1.09 (3H,

d, J 7.0 Hz), 0.88 (3H, t, J 7 Hz), 0.87 (9H, s), 0.05 (3H, s),

0.027 (3H, s); δ_C(126 MHz, CDCl3): 202.8, 175.1, 73.2,

51.6, 51.2, 43.9, 33.7, 31.9, 29.8, 29.7, 29.65, 29.6, 29.5,

29.4, 29.35, 29.3, 29.2, 27.9, 27.5, 25.8, 23.8, 22.7, 22.1,

18.0,14.1, –4.4, –4.9; ν_max/cm^-1: 2924, 2853, 1738, 1464.

o

mmol) in THF (100 mL) and IMS (250 mL) at 10 C and

stirred at r.t for 2 h. A further solution of ammonium

molybdate (6.9 g, 5.6 mmol) in 35 % H₂O₂(20 mL) was

added and the mixture was stirred at r.t. for 18 h. The

mixture was poured into water (1.2 L) and extracted with

CH₂Cl₂(1 × 200 mL, 3 × 30 mL). The combined organic

phases were washed with water (500 mL), dried and

evaporated. Chromatography (petrol/ether, 5:1) gave a

white solid, the title compound 28 (9.2 g, 84%), [ꢀ]_ꢁ^ꢂꢇ

+12.6 (c 1.01, CHCl₃) [Found [M – CH₃]⁺: 477.2523,

C₂₄H₃₇N₄O₄S requires: 477.2531]; δ_H(500 MHz, CDCl₃):

7.71 – 7.69 (2H, m), 7.65 – 7.59 (3H, m), 4.00 (1H, dd, J

6.3, 7.9 Hz), 3.87 (1H, br q, J 7.0 Hz), 3.75 – 3.72 (2H, m),

3.60 (1H, br t, J 7.9 Hz), 1.99 – 1.92 (2H, m), 1.53 – 1.47

(2H, m), 1.41 (3H, s), 1.35 (3H, s), 1.32 – 1.26 (16H, m),

1.11 – 1.04 (1H, m), 0.96 (3H, d, J 6.6 Hz); δ_C(126 MHz,

CDCl₃): 153.5, 133.0, 131.4, 129.7, 125.0, 108.5, 80.4,

67.8, 56.0, 36.5, 33.7, 29.8, 29.6, 29.58, 29.5, 29.4, 29.2,

28.9, 28.1, 27.0, 26.6, 25.5, 21.9, 15.6; υ_max/cm^-1: 3069,

2917, 2854, 1594, 1498, 1473, 1356, 1259, 1209, 1150,

1064.

3.12 (21S,22S)-21-Methoxy-22-methyltetracontyl

pivaloate 33

LiHMDS (1.1 mL, 1.0 mmol, 1.06 M) was added dropwise

to a stirred solution of (8S, 9S)-8-methoxy-9-methyl-

heptacosanal 32 (0.34 g, 0.77 mmol)³⁹and 2,2-dimethyl-

propionic acid 13-(1-phenyl-1H-tetrazole-5-sulfonyl)-

tridecyl ester 7 (0.42 g, 0.93 mmol) in dry THF (15 mL )

o

3.10 Methyl (R)-2-((1R,15R)-1-((tert-butyldimethylsilyl)-

oxy)-15-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)hexadecyl)-

hexacosanoate 30

LiHMDS (9.3 mL, 9.9 mmol, 1.06 M) was added to a

stirred solution of sulfone 28 (3.23 g, 6.57 mmol) and ester

under nitrogen at –15 C. The solution was allowed to

reach r.t. and stirred for 2 h. Work up as in 3.10 and

chromatography (petrol/EtOAc, 20:1) gave a colourless oil,

(21S,22S,E)-21-methoxy-22-methyltetracont-13-en-1-yl

pivaloate (0.46 g, 85%) in ratio 2.6:1. Palladium 10% on

ACCEPTED MANUSCRIPT

12

carbon (0.5 g) was added to a stirred solution of the alkene

(0.46 g, 0.65 mmol) in IMS (15 mL) and THF (5 mL)

under hydrogen. Hydrogenation was carried out for 1 h.

The mixture was filtered over celite and the solvent was

evaporated. Chromatography eluting with petrol/EtOAc

(20:1) to give a colourless oil, of the title compound 33

(0.46g, 100 %), [ꢀ]_ꢁ^ꢂꢂ–6.5 (c 1.50, CHCl₃) [Found

[M+Na]⁺: 729.7102, C₄₇H₉₄NaO₃requires: 729.7100]; δ_H:

(500 MHz, CDCl₃): 4.05 (2H, t, J 6.7 Hz), 3.34 (3H, s),

2.97 – 2.94 (1H, m), 1.62 (2H, pent, J 6.7 Hz), 1.46 – 1.22

(70H, m), 1.20 (9H, s), 1.12 – 1.06 (1H, m), 0.88 (3H, t, J

6.7 Hz), 0.85 (3H, d, J 7.0 Hz); δ_C(126 MHz, CDCl₃):

178.5, 85.4, 64.4, 57.7, 38.7, 35.3, 32.4, 31.9, 30.8, 30.5,

30.0, 29.9, 29.7, 29.69, 29.66, 29.6, 29.5, 29.4, 29.2, 28.6,

27.6, 27.2, 26.2, 25.9, 22.7, 14.8, 14.1; ν_max/cm^-1: 2923,

1731,1154.

was added m-chloroperbenzoic acid (0.41 g, 2.35 mmol) at

r.t. and stirred for 24 h. The mixture was quenched with

10% NaOH solution (25 mL) and vigorously stirred for 2 h.

The organic layer was separated and the aqueous layer was

extracted with CH₂Cl₂(2 × 50 mL). The combined organic

layers were washed with water, dried and evaporated.

Chromatography (petroleum/ether, 10:1) gave a white

o

solid, sulfone 34 (0.81 g, 97%), mp.: 44 – 46 C; [ꢀ]_ꢁ^ꢂꢂ

–

6.19 (c 1.39, CHCl₃) [MALDI–Found: [M+Na]⁺: 837.3,

C₄₉H₉₀N₄NaO₃S requires: 837.6]; δ_H(500 MHz, CDCl3):

7.73 – 7.71 (2H, m), 7.65 – 7.61 (3H, m), 3.75 (2H,

distorted t, J 7.9 Hz), 3.36 (3H, s), 2.98 – 2.96 (1H, m),

1.97 (2H, br pent, J 7.5 Hz), 1.68 –1.61 (2H, m), 1.52 (2H,

br pent, J 7.5 Hz), 1.45 – 1.22 (66H, m), 1.15 – 1.07 (1H,

m), 0.90 (3H, t, J 6.6 Hz), 0.87 (3H, d, J 6.9 Hz); δ_C(126

MHz, CDCl3): 153.5, 133.1, 131.5, 129.7, 125.1, 85.5,

57.7, 56.0, 35.3, 32.4, 31.9, 30.5, 30.0, 29.9, 29.7, 29.65,

29.6, 29.5, 29.4, 29.2, 28.9, 28.2, 27.6, 26.2, 22.7, 21.9,

14.9, 14.1; ν_max: 2924, 2849, 1343, 1157, 1096 cm^-1.

3.13 5-(((21S,22S)-21-Methoxy-22-methyltetracontyl)-

sulfonyl)-1-phenyl-1H-tetrazole 34

(i) A solution of pivaloate 33 (0.75 g, 1.06 mmol) in HPLC

grad THF (15 mL) was added to a stirred solution of LAH

(0.2 g, 1.6 mmol) in THF (35 mL) at –20 C under argon.

3.14 (2R,3R,17R,39S,40S,E)-3-Hydroxy-39-methoxy-17,

40-dimethyl-2-tetracosyloctapentacont-18-enoic acid 37

(i) Ester 31 (0.30 g, 0.38 mmol) in dry 1,2-

dimethoxyethane (60 mL) was added to stirred solution of

sulfone 34 (0.34 g, 0.42 mmol) in dry 1,2-dimethoxyethane

(25 mL) under nitrogen at RT. The mixture was cooled to –

o

The mixture was allowed to warm and refluxed for 1h, then

o

sat. aq. sodium sulfate was added at –20 C until a white

precipitate had formed and THF (20 mL) was added. The

mixture was stirred at r.t. for 30 min, filtered through celite

and evaporated. Chromatography (petrol/EtOAc, 10:1 then

o

20 C and potassium bis (trimethylsilyl)amide (1.08 mL,

5:1) gave a white solid, (21S,22S)-21-methoxy-22-methyl-

0.42 mmol, 0.5 M in toluene) was added and the mixture

allowed to reach r.t. and then stirred for 1.5 h. Sat. aq.

NH₄Cl (30 mL) and petrol /ether (1:1, 55 mL) were added.

The aqueous layer was extracted with petrol/ether (1:1, 2 ×

45 mL). The combined organic layers were evaporated.

Chromatography (petrol/ether, 18:1) gave a semi solid,

o

tetracontan-1-ol (0.65 g, 99 %), mp. 46 – 48 C, [ꢀ]_ꢁ^ꢂꢂ

–

8.21 (c 1.21, CHCl₃) [Found [M–H]⁺: 621.6545, C₄₂H₈₅O₂

requires: 621.6549]; δ_H: (500 MHz, CDCl₃): 3.66 (2H, t, J

6.6 Hz), 3.36 (3H, s), 2.99 – 2.96 (1H, m), 1.67 – 1.64 (1H,

m), 1.59 (2H, pent, J 6.6 Hz), 1.49 (1H, br s), 1.48 – 1.22

(69H, m), 1.14 – 1.07 (1H, m), 0.90 (3H, t, J 7.0 Hz), 0.87

(3H, d, J 6.7 Hz); δ_C(126 MHz, CDCl₃): 85.5, 63.1, 57.7,

35.3, 32.8, 32.4, 31.9, 30.5, 30.0, 29.9, 29.7, 29.68, 29.63,

29.5, 29.4, 27.6, 26.2, 25.7, 22.7, 14.9, 14.1; ν_max/cm^-1:

3373, 2921, 1098,1076.

methyl

(2R,3R,17R,

39S,40S,E)-3-((tert-

butyldimethylsilyl)oxy)-39-methoxy-17,40-dimethyl-2-

tetracosyloctapentacont-18-enoate 35 (0.18 g, 37%)

[MALDI–Found [M+Na]⁺: 1404.3; C₉₂H₁₈₄NaO₄Si

requires: 1404.3]; δ_H: 5.33 (1H, dt, J 6.36, 15.0 Hz), 5.24

(1H, dd, J 7.4, 15.0 Hz), 3.9 (1H, td, J 4.3, 6.8 Hz), 3.65

(3H, s), 3.35 (3H, s), 2.98 – 2.94 (1H, m), 2.53 (1H, ddd, J

3.7, 7.0, 10.8 Hz), 2.05 – 2.0 (1H, m), 1.97 (2H, q, J 6.9

Hz), 1.68 – 1.26 (142H, m, v.br), 1.08 – 1.01 (1H, m), 0.94

(3H, d, J 7.0 Hz), 0.90 (6H, t, J 6.7 Hz), 0.87 (9H, s), 0.85

(3H, d, J 7.0 Hz), 0.52 (3H, s), 0.03 (3H, s); δ_C: (101 MHz,

CDCl₃): 175.2, 136.5, 128.4, 85.4, 73.2, 57.7, 51.6, 51.2,

37.3, 36.7, 35.3, 33.7, 32.6, 32.4, 31.9, 30.5, 30.0, 29.9,

29.84, 29.8, 29.7, 29.66, 29.6, 29.5, 29.45, 29.4, 29.1, 27.8,

27.6, 27.5, 27.4, 26.2, 25.8, 23.7, 22.7, 20.9, 14.9, 14.1, –

4.4, –4.9; v_max/cm^-1: 2924, 2854, 1741, 1465, 1377, 1254,

1099.

(ii) DEAD (0.27 g, 1.6 mmol) in dry THF (2 mL) was

added to a stirred solution of 1-phenyl-1H-tetrazole-5-thiol

(0.28 g, 1.6 mmol), Ph₃P (0.41 g, 1.6 mmol) and the above

o

alcohol (0.75 g, 1.2 mmol) in dry THF (25 mL) at 0 C,

The mixture was vigorously stirred and refluxed for 2.5 h,

then 5:1 petroleum/EtOAc (100 mL) was added. The

mixture filtered through celite and the solvent was

evaporated. Chroma-tography (petrol/ether, 5:1) gave a

white

solid,

5-(((21S,

22S)-21-methoxy-22-

methyltetracontyl)thio)-1-methyl-1H-tetrazole (0.80 g,

91%), [ꢀ]^ꢂ_ꢁ^ꢂ–6.4 (c 1.2, CHCl₃) [MALDI–Found (M+Na)⁺:

805.3, C₄₉H₉₀NaN₄OS requires: 805.6]; δ_H: (500 MHz,

CDCl₃): 7.65 – 7.61 (5H, m), 3.66 (2H, t, J 6.3 Hz), 3.36

(3H, s), 2.98 – 2.96 (1H, m), 1.97 (2H, br pent, J 7.5 Hz),

1.68 – 1.61 (2H, m), 1.52 (2H, br pent, J 7.5 Hz), 1.45 –

1.22 (66H, m), 1.15 – 1.07 (1H, m), 0.90 (3H, t, J 6.6 Hz),

0.87 (3H, d, J 6.9 Hz); δ_C: (126 MHz, CDCl₃): 153.5,

133.1, 131.5, 129.7, 125.1, 85.5, 57.7, 56.0, 35.3, 32.4,

31.9, 30.5, 30.0, 29.97, 29.7, 29.65, 29.6, 29.5, 29.4, 29.2,

28.9, 28.2, 27.6, 26.2, 22.7, 21.9, 14.9, 14.1; ν_max/cm^-1:

(ii) The ester 35 (0.70 mg, 0.05 mmol) was dissolved in dry

THF (10 mL) in a dry polyethylene vial under nitrogen at

r.t. and stirred. Pyridine (0.07 mL) and HF–pyridine

complex (0.5 mL) were added; the mixture was stirred for

17 h. at 42 °C, neutralized with sat. aq. NaHCO₃, then

extracted with petrol/EtOAc (1:5, 3 × 50 mL). The

combined organic layers were washed with brine, dried and

evaporated; chromatography (petrol/EtOAc, 20:1) gave a

white solid, methyl (2R,3R,17R,39S, 40S,E)-3-hydroxy-39-

methoxy-17,40-dimethyl-2-tetracosyloctapentacont-18-en-

3072, 2917, 2853, 1343, 1096

.

(iii) To a stirred solution of the above tetrazole (0.80 g, 1.0

mmol) and NaHCO₃(0.34, 4.1 mmol) in CH₂Cl₂(50 mL)

o

oate 36 (0.62 g, 96%), m.p.: 34 – 36 C [MALDI–Found

ACCEPTED MANUSCRIPT

13

[M+Na]⁺: 1290.3; C₈₆H₁₇₀NaO₄requires: 1290.2]

; δ_H: 5.33

m), 1.1 – 1.0 (1H, m), 0.95 (3H, s), 0.94 (9H, s), 0.89 (6H,

t, J 6.7 Hz), 0.85 (3H, d, J 6.9 Hz), 0.16 (3H, s), 0.14 (3H,

s); δ_C(101 MHz, CDCl₃): 174.9, 136.5, 128.4, 85.5, 73.7,

57.7, 50.0, 37.3, 36.7, 35.8, 35.3, 32.6, 32.4, 31.9, 30.5,

30.1, 30.0, 29.9, 29.8, 29.7, 29.63, 29.6, 29.54, 29.5, 29.4,

29.36, 29.1, 27.6, 27.4, 27.36, 26.2, 25.9, 25.7, 25.5, 25.1,

22.7, 20.9, 17.9, 14.9, 14.1, –4.3, –4.9; ν_max: 2922, 2852,

1709, 1467, 1362, 1253, 1180, 1101, 836, cm^-1.

(1H, dt, J 6.4, 15.0 Hz), 5.24 (1H, dd, J 7.5, 15.0 Hz), 3.72

(3H, s), 3.68 – 3.63 (1H, m), 3.35 (3H, s), 2.98 – 2.93 (1H,

m), 2.46 – 2.41 (1H, td, J 5.4, 9.1 Hz), 2.05 – 2.0 (1H, m),

1.97 (2H, br q, J 7 Hz), 1.76 – 1.68 (1H, m), 1.65 – 1.5

(12H, m), 1.48 – 1.2 (128H, m), 1.15 – 1.05 (1H, m), 0.95

(3H, d, J 6.7 Hz), 0.89 (6 H, t, J 7.1 Hz), 0.86 (3H, d, J 6.8

Hz), 1.96 (2H, br q, J 7 Hz); δ_C(101 MHz, CDCl₃): 176.2,

136.5, 128.4, 85.4, 72.3, 57.7, 51.5, 50.9, 37.2, 36.7, 35.7,

35.3, 32.6, 32.4, 31.9, 30.5, 30.0, 29.9, 29.8, 29.7, 29.6,

29.56, 29.5, 29.4, 29.36, 29.1, 27.6, 27.4, 27.36, 26.2, 25.7,

22.7, 20.9, 14.9, 14.1; ν_max/cm^-1: 3431, 2917, 2850, 1739,

1466, 1099.

3.16 6,6’-Bis-O-(2R,3R,17R,39S,40S,E)-3-((tert-butyldi-

methylsilyl)oxy)-39-methoxy-17,40-dimethyl-2-tetra-

cosylocta-pentacont-18-enoic-2,3,4,2’,3’,4’-hexakis-O-

(trimethylsilyl)-α,α’-trehalose 46 and 6-O-(2R,3R,17R,

39S,40S,E)-3-((tert-butyldimethylsilyl)oxy)-39-methoxy-

17,40-dimethyl-2-tetracosylocta-pentacont-18-enoic-2,3,

4,2’,3’,4’-hexakis-O-(trimethylsilyl)-α,α’-trehalose 47

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimidehydro-

chloride (EDCI) (0.044 g, 0.230 mmol) and DMAP (0.028

g, 0.23 mmol) were added to a stirred solution of acid 44

(0.090 g, 0.065 mmol), protected α,α᾽-trehalose 45 (0.022

g, 0.028 mmol)⁶⁷and powdered 4 Ǻ molecular sieve in dry

CH₂Cl₂(1 mL) at r. t. under nitrogen. The mixture was

stirred for 6 days, diluted with CH₂Cl₂(3 mL) and silica (5

g), then the solvent was evaporated. Chromatography

(petroleum/EtOAc, 20:1) gave a first fraction, the TDM 46

(0.228 g, 25%), [ꢀ]^ꢂ_ꢁ^ꢂ+19 (c 1.2, CHCl₃) [MALDI–Found

[M+Na]⁺: 3496.4; C₂₁₂H₄₃₀NaO₁₇Si₈requires: 3496.0]; δ_H

(400 MHz, CDCl₃): 5.35 (2H, dt, J 6.4, 15.3 Hz), 5.25 (2H,

br dd, J 7.4, 15.3 Hz), 4.85 (2H, br d, J 3.0 Hz), 4.36 (2H,

br d, J 10.8 Hz), 4.01 (2H, br tt, J 3.1, 10.8 Hz), 3.94 (2H,

br pent, J 5.1 Hz), 3.9 (2H, br t, J 8.8 Hz), 3.52 (2H, br t, J

9.0 Hz), 3.38 (2H, dd, J 2.9, 9.3 Hz), 3.34 (6H, s), 2.96

(2H, br pent, J 4.2 Hz), 2.57 – 2.53 (2H, m), 2.03 (2H, br

pent, J 6.5 Hz), 1.96 (4H, br q, J 6.7 Hz), 1.7 – 1.2 (292H,

m), 1.13 – 1.07 (2H, m), 0.9 (6H, d, J 6.7 Hz), 0.89 (12H, t,

J 6.6 Hz), 0.88 (18H, s), 0.16 (18H, s), 0.15 (18H, s), 0.14

(18H, s), 0.06 (12H, s); δ_C(101 MHz, CDCl₃): 173.8,

136.5, 128.4, 94.8, 85.4, 73.5, 73.4, 72.8, 71.8, 70.7, 62.4,

57.7, 51.9, 37.3, 36.7, 35.3, 32.6, 32.4, 31.9, 30.5, 30.0,

29.95, 29.9, 29.7, 29.66, 29.6, 29.5, 29.4, 29.2, 27.6, 27.4,

26.2, 25.8, 22.7, 20.9, 18.0, 14.9, 14.1, 1.1, 0.9, 0.2, –4.5, –

4.7; ν_max: 2924, 2853, 1743, 1466, 1377, 1251, 1163, 1100

cm^-1. The second fraction was TMM 47 (0.072 g, 50%),

[ꢀ]^ꢂ_ꢁ^ꢂ+32 (c 1.1, CHCl₃) [MALDI–Found [M+Na]⁺:

2146.7; C₁₂₁H₂₅₀NaO₁₄Si₇requires: 2146.7]; δ_H(500 MHz,

CDCl₃): 5.34 (1H, dt, J 6.4, 15.3 Hz), 5.24 (1H, dd, J 7.4,

15.3 Hz), 4.91 (1H, d, J 3.0 Hz), 4.84 (1H, d, J 3.0 Hz),

4.35 (1H, dd, J 2.0, 11.8 Hz), 4.08 (1H, dd, J 4.1, 11.8 Hz),

4.0 (1H, td, J 2.2, 9.5 Hz), 3.96 – 3.92 (2H, m), 3.89 (1H,

dd, J 5.2, 8.9 Hz), 3.84 (1H, td, J 3.0, 9.4 Hz), 3.73 – 3.64

(2H, m), 3.5 (1H, dd, J 3.7, 9.0 Hz), 3.49 (1H, dd, J 3.7, 9.0

Hz), 3.47 (1H, dd, J 3.7, 9.0 Hz), 3.43 (1H, dd, J 3.0, 9.3

Hz), 3.4 (1H, dd, J 3.0, 9.3 Hz), 3.35 (3H, s), 2.96 (1H, br

pent, J 4.2 Hz), 2.56 (1H, ddd, J 3.36, 5.4, 9.7 Hz), 2.1 –

2.0 (1H, m), 1.96 (2H, br q, J 6.7 Hz), 1.72 (1H, br t, J 5.2,

6.9 Hz), 1.67 – 1.57 (3H, m), 1.5 – 1.15 (138H, m), 1.3 –

1.07 (1H, m), 0.94 (3H, d, J 6.7 Hz), 0.89 (6H, t, J 6.6 Hz),

0.87 (9H, s), 0.85 (3H, d, J 7.0 Hz), 0.17 (9H, s), 0.16 (9H,

s), 0.15 (9H, s), 0.148 (18H, s), 0.12 (9H, s), 0.06 (3H, s),

0.055 (3H, s); δ_C(101 MHz, CDCl₃): 174.1, 136.5, 128.4,

94.5, 94.4, 85.4, 73.4, 73.3, 72.9, 72.8, 72.7, 72.0, 71.4,

70.7, 62.5, 61.7, 57.7, 51.8, 37.3, 36.7, 35.3, 33.4, 32.6,

(iii) Lithium hydroxide monohydrate (0.31 g, 7.3 mmol)

was added with stirring to ester 36 (0.62 g, 0.49 mmol) in

THF (15 mL), methanol (1.3 mL) and water (1.5 mL) at r.

o

t. The mixture was stirred at 43 C for 18 h. The mixture

was cooled to r.t. and acidified with HCl (5%, 2 mL) and

the aqueous layer was extracted with warm petroleum/ether

(1:1, 3 × 50 mL). The combined organic layers were dried

and evaporated; chromatography (warm petroleum/EtOAc,

7:2) gave a white solid, compound 37 (0.50, 82%), m.p.: 43

–

45 ^oC [MALDI–Found [M+Na]⁺: 1276.2861;

C₈₅H₁₆₈NaO₄requires: 1276.2835]; δ_H(500 MHz, CDCl3):

5.33 (1H, dt, J 6.4, 15.2 Hz), 5.24 (1H, dd, J 7.4, 15.2 Hz),

3.73 – 3.70 (1H, m), 3.35 (3H, s), 2.98 – 2.96 (1H, m),

2.49 – 2.44 (1H, m), 2.0 –1.97 (1H, m), 1.96 (2H, br q, J

6.7 Hz), 1.77 – 1.70 (1H, m), 1.67 – 1.57 (2H, m), 1.55 –

1.15 (141 H, m), 1.14 – 1.05 (1H, m), 0.94 (3H, d, J 6.7

Hz), 0.89 (6H, t, J 6.5 Hz), 0.85 (3H, d, J 6.9 Hz); δ_C(101

MHz, CDCl₃): 179.5, 136.5, 128.4, 85.6, 72.1, 57.7, 50.7,

37.3, 36.7, 35.6, 35.3, 32.6, 32.4, 31.9, 30.5, 29.9, 29.7,

29.5, 29.4, 29.36, 29.1, 27.6, 27.4, 22.4, 21.0, 14.9, 14.1;

ν

_max/cm^-1: 3470, 2917, 2854, 1726, 1689, 1471, 1392, 1202,

1099, 968, 887, 839.

3.15 (2R,3R,19R,41S,42S,E)-3-((tert-Butyldimethylsilyl)-

oxy)-41-methoxy-19,42-dimethyl-2-tetracosylhexacont-

20-enoic acid 44

Imidazole (0.125 g, 1.80 mmol) was added to a stirred

solution of acid 37 (0.23 g, 0.18 mmol) in dry DMF (1.5

mL) and dry toluene (2.5 mL) at r. t. followed by the

addition of TBDMSCl (0.27 g, 1.8 mmol) and DMAP (10

mg). The mixture was stirred at 70 ^oC for 24 h. The solvent

was evaporated under reduced pressure and the residue was

diluted with EtOAc (50 mL) and water (20 mL). The

organic layer was separated and the aqueous layer was

extracted with EtOAc (2 × 30 mL). The combined organic

layers were washed with water, dried and evaporated to

give a colourless oil. The residue was dissolved in THF (8

mL) and n-Bu₄NOH (0.9 mL, 4%) and stirred for 10 min. at

r. t. then diluted with water (10 mL) and extracted with

EtOAc (2 × 20 mL), dried and evaporated to give a residue;

column chromatography (petroleum/EtOAc, 20:1) gave a

colourless oil, the title compound 44 (0.25 g, 96%)

[MALDI–Found [M+Na]⁺: 1390.3; C₉₁H₁₈₂NaO₄Si

requires: 1390.3]; δ_H(500 MHz, CDCl₃): 5.33 (1H, dt, J

6.4, 15.0 Hz), 5.25 (1H, dd, J 7.5, 15.0 Hz), 3.86 – 3.80

(1H, m), 3.35 (3H, s), 2.99 – 2.94 (1H, m), 2.57 – 2.51

(1H, m), 2.07 – 2.01 (1H, m), 1.96 (2H, br q, J 6.8 Hz),

1.73 – 1.68 (1H, m), 1.65 –1.54 (4H, m), 1.5 – 1.2 (138H,

ACCEPTED MANUSCRIPT

14

32.3, 31.9, 30.5, 30.0, 29.9, 29.8, 29.7, 29.66, 29.5, 29.4,

29.2, 28.1, 27.6, 27.4, 26.4, 26.2, 25.8, 24.8, 22.7, 20.9,

18.0, 14.9, 14.1, 1.1, 1.0, 0.9, 0.85, 0.2, 0.04, –4.5, –4.7;

ν_max: 3608, 2925, 2854, 1743, 1466, 1251, 1100, 1077, 844

cm^-1.

3.18

6-O-(2R,3R,17R,

39S,40S,E)-3-Hydroxy-39-

methoxy -17,40-dimethyl-2-tetracosylocta-pentacont-18-

enoic-α,α’-trehalose 51

(i) n-Bu₄NF (0.16 mL, 0.16 mmol, 1M) was added with

stirring to monoester 47 (0.058 g, 0.027 mmol) in dry THF

(2 mL) at 5 C under nitrogen, stirred at r.t. for 1 h, then

o

worked up and purified as before to give 6-O-(2R,3R,17R,

39S,40S,E)-3-((tert-butyldimethylsilyl)oxy)-39-methoxy-

17,40-dimethyl-2-tetracosyloctapentacont-18-enoic-α,α’-

trehalose 50 (0.030, 65%) as a colourless oil, [ꢀ]^ꢂ_ꢁ^ꢂ+39.5

(c 0.75, CHCl₃) [MALDI–Found [M+Na]⁺: 1714.2;

C₁₀₃H₂₀₂NaO₁₄Si requires: 1714.4]; δ_H(500 MHz, CDCl₃+

few drop of CD₃OD): 5.28 (1H, dt, J 6.5, 15.5 Hz), 5.19

(1H, br dd, J 7.6, 15.5 Hz), 5.05 (2H, br t, J 3.5 Hz), 4.3

(1H, dd, J 4.6, 12.3 Hz), 4.23 (1H, dd, J 2.3, 12.3 Hz), 3.9

(1H, br d, J 8.3 Hz), 3.87 – 3.84 (1H, m), 3.81 – 3.71 (4H,

m), 3.65 (1H, dd, J 5.2, 11.6 Hz), 3.47 (1H, dd, J 3.9, 5.9

Hz), 3.46 (1H, dd, J 5.9, 9.7 Hz), 3.3 (3H, s), 2.93 – 2.91

(1H, m), 2.6 – 2.48 (1H, m), 1.99 – 1.95 (1H, m), 1.91 (2H,

br q, J 7.0 Hz), 1.6 – 1.55 (1H, m), 1.45 – 1.15 ( 150H, m),

1.07 – 1.03 (1H, m), 0.89 (3H, d, J 6.7 Hz), 0.83 (6H, t, J

7.0 Hz), 0.81 (9H, s), 0.8 (3H, d, J 7.0 Hz), –0.003 (3H, s),

–0.024 (3H, s); δ_C(101 MHz, CDCl₃+ few drops of

CD₃OD): 174.9, 136.4, 128.3, 93.5, 93.4, 85.5, 73.2, 72.2,

71.7, 70.7, 70.3, 70.0, 62.7, 62.0, 58.7, 57.6, 52.1, 51.7,

50.1, 37.2, 36.6, 35.2, 32.5, 32.3, 31.8, 30.4, 29.8, 29.8,

29.7, 29.7, 29.6, 29.5, 29.4, 29.2, 29.0, 27.4, 27.3, 26.0,

25.7, 25.1, 23.8, 22.6, 20.8, 20.0, 19.6, 17.9, 14.7, 14.0,

13.4, 13.38, –4.6, –5.0; υ_max: 3903, 3368, 2918, 2850, 1733,

1468, 1378, 1253, 1148, 1102, 1078, 1051, 992, 941, 836,

775, 721 cm^-1.

(ii) Compound 50 (0.025 g, 0.015 mmol) and pyridine

(0.05 mL) in dry THF (3 mL) in a dry polyethylene vial

equipped with a rubber septum was stirred at r. t. under

argon, and hydrogen fluoride-pyridine complex (~70% HF,

0.25 mL) was added. The mixture was stirred at 43 ^oC for

17 h, then worked up and purified as above to give the title

compound 51 (0.015, 65%), [ꢀ]^ꢂ_ꢁ^ꢂ+34 (c 0.52, CHCl₃)

[MALDI–Found [M+Na]⁺: 1600.3879; C₉₇H₁₈₈NaO₁₄

requires: 1600.3897]; δ_H(400 MHz, CDCl₃): 5.26 (1H, dt, J

6.6, 15.2 Hz), 5.17 (1H, br dd, J 7.4, 15.2 Hz), 5.03 (1H, d,

J 3.2 Hz), 4.98 (1H, d, J 3.3 Hz), 4.61 (1H, br d, J 11 Hz),

4.18 (1H, br t, J 7.9 Hz), 3.95 (1H, dd, J 7.8, 12.0 Hz), 3.87

– 3.82 (1H, m), 3.79 – 3.71 (2H, m), 3.62 –3.55 (2H, m),

3.5 (1H, dd, J 3.2, 9.8 Hz), 3.44 (1H, dd, J 3.3, 9.8 Hz),

3.32 (8H, br s), 3.28 (3H, s), 3.23 (1H, br t, J 9.5 Hz), 3.18

(1H, br t, J 9.6 Hz), 2.93 – 2.89 (1H, m), 2.38 –2.33 (1H,

m), 1.99 – 1.94 (1H, m), 1.9 (2H, br q, J 6.8 Hz), 1.57 –

1.10 (143H, m), 1.06 – 0.98 (1H, m), 0.87 (3H, d, J 6.7

Hz), 0.81 (6H, t, J 6.4 Hz), 0.78 (3H, d, J 7.1 Hz); δ_C(101

MHz, CDCl₃): 175.4, 136.3, 128.3, 94.6, 85.5, 72.6, 72.4,

72.3, 71.5, 71.2, 71.1, 70.8, 69.9, 64.2, 62.3, 62.1, 57.5,

52.3, 37.1, 36.5, 35.2, 34.6, 32.4, 32.2, 31.8, 30.3, 29.8,

29.7, 29.7, 29.5, 29.4, 29.3, 29.2, 29.0, 27.3, 27.2, 27.1,

25.9, 25.1, 22.5, 20.7, 14.6, 13.9; υ_max: 3364, 2918, 2850,

1728, 1467, 1148, 992 cm^-1.

3.17

6,6’-Bis-O-(2R,3R,17R,39S,40S,E)-3-hydroxy-39-

methoxy-17,40-dimethyl-2-tetracosylocta-pentacont-18-

enoic-α,α’-trehalose 49

(i) n-Bu₄NF (0.1 mL, 0.1 mmol, 1 M) was added to a

stirred solution of diester 46 (0.06 g, 0.017 mmol) in dry

THF (5 mL) at 5 C under nitrogen, allowed to reach r.t.,

o

stirred for 1 h, then evaporated under reduced pressure.

Chroma-tography (CHCl₃/MeOH, 10:1) gave 6,6’-bis-O-

(2R,3R,17R, 39S,40S,E)-3-((tert-butyldimethylsilyl)oxy)-

39-methoxy-17,40-dimethyl-2-tetracosyloctapentacont-18-

enoic-α,α’-trehalose 48 (0.035 g, 67%), [ꢀ]^ꢂ_ꢁ^ꢂ+19.5 (c

0.50, CHCl₃) [MALDI–Found [M+Na]⁺: 3063.9;

C₁₉₄H₃₈₂NaO₁₇Si₂requires: 3063.8]; δ_H(500 MHz, CDCl₃+

few drops of CD₃OD): 5.27 (2H, dt, J 6.5, 15.3 Hz), 5.18 (

2H, dd, J 7.4, 15.3 Hz), 5.04 (2H, d, J 3.4 Hz), 4.3 (2H, br

dd, J 3.8, 12.0 Hz), 4.2 (2H, br d, J 10.84 Hz), 3.91 (2H, br

d, J 9.4 Hz), 3.85 (2H, br q, J 5.0 Hz), 3.76 (2H, br t, J 9.3

Hz), 3.44 (2H, dd, J 3.4, 9.6 Hz), 3.32 (2H, br m), 3.3 (6H,

s), 2.94 – 2.91 (2H, m), 2.53 – 2.48 (2H, m), 1.97 (2H, br

pent, J 6.3 Hz), 1.91 (2H, br q, J 6.8 Hz), 1.57 – 1.1 (292H,

m), 1.07 – 1.0 (2H, m), 0.88 (6H, d, J 6.7 Hz), 0.84 (12H, t,

J 6.3 Hz), 0.81 (18H, s), 0.8 (6H, d, J 7.3 Hz), –0.003 (6H,

s), –0.023 (6H, s); δ_C(101 MHz, CDCl₃+ CD₃OD): 175.2,

136.4, 128.3, 93.5, 85.5, 73.2, 73.1, 71.6, 70.2, 69.9, 62.9,

57.6, 51.59, 37.1, 36.57, 35.2, 33.5, 32.5, 32.3, 31.8, 30.4,

29.83, 29.8, 29.7, 29.63, 29.6, 29.57, 29.4, 29.2, 29.0, 27.7,

27.4, 27.3, 26.9, 26.0, 25.7, 25.6, 24.1, 22.6, 20.8, 17.8,

14.7, 13.9, –4.6, –5.0; ν_max: 3400, 2924, 2853, 1742, 1464,

1100 cm^-1.

(ii) A dry polyethylene vial equipped with a rubber septum

was charged with 48 (0.022 g, 0.007 mmol) and pyridine

(0.05 mL) in dry THF (1.3 mL) and stirred at r. t. under

argon. To it was added hydrogen fluoride-pyridine complex

(~70% HF, 0.2 mL). The mixture was stirred at 43 ^oC for

17 h, then poured slowly into sat. aq. NaHCO₃until no

more CO₂was liberated. The product was extracted with

CHCl₃(3 × 50 mL); the combined organic layers were

evaporated to give

a

residue. Chromatography

(CHCl₃/MeOH, 10:1) gave compound 49 (0.015, 75%),

[ꢀ]^ꢂ_ꢁ^ꢂ+18 (c 1.0, CHCl₃) [Found [M+Na]⁺: 283.6751;

C₁₈₂H₃₅₄NaO₁₇requires: 2835.6728]; δ_H(500 MHz, CDCl₃

+ few drops of CD₃OD): 5.3 (2H, dt, J 6.2, 15.6 Hz), 5.21

(2H, dd, J 7.2, 15.6 Hz), 5.0 (2H, br d, J 3.4 Hz), 4.68 (2H,

br d, J 11.9 Hz), 4.23 (2H, br t, J 9.4 Hz), 4.0

3.74 (2H, br t, J 9.3 Hz), 3.67 3.62 (2H, m), 3.5 (2H, dd, J 2.8,

9.6 Hz), 3.32 (6H, s), 3.21 (2H, t, J 9.3 Hz), 2.96 2.93 (2H, m),

2.4 (2H, m), 1.97 2.02 (2H, m), 1.93 (4H, br q, J 6.7 Hz), 1.65

1.01 (294H, m), 0.91 (6H, d, J 6.7 Hz), 0.86 (12H, t , J 6.7 Hz),

0.82 (6H, d, J 7 Hz); δ_C(101 MHz, CDCl₃+ few drops of

CD₃OD): 175.2, 136.2, 128.2, 94.0, 85.5, 72.8, 72.3, 71.3, 70.6,

69.8, 63.5, 57.4, 52.4, 37.0, 36.5, 35.1, 34.6, 32.3, 32.1, 31.7,

30.2, 29.7, 29.6, 29.6, 29.4, 29.3, 29.3, 29.2, 29.1, 29.0, 28.9,

27.2, 27.1, 27.1, 25.8, 25.1, 22.4, 20.6, 14.5, 13.8; υ_max3584,

3395, 2923, 2853, 1732, 1464, 1376 cm^-1.

– 3.94 (2H, m),

–

3.19

methoxy -17,40-dimethyl-2-tetracosyloctapentacont-18-

enoate-α/β- -glucopyranoside 54

6-O-(2R,3R,17R,39S,40S,

E)-3-Hydroxy-39-

D

(i) The acid 37 (0.054 g, 0.043 mmol) was dissolved in a

mixture of THF and DMF (THF-DMF, 5:1, 1.5 mL) at r. t.

ACCEPTED MANUSCRIPT

15

then warmed gently until all had dissolved. Dry CsHCO₃

(0.072 mg, 0.371 mmol) was added to a stirred solution and

the mixture was left at r. t. for 1 h. then tosylate 52 (0.33 g,

3.20 Methyl 5-O-[(2R,3R,17R,39S,40S, E)-3-hydroxy-39-

methoxy-17,40-dimethyl-2-tetracosyloctapentacont-18-

enoate]-α-D-arabinofuranoside 57

o

0.047 mmol) was added. The mixture was stirred at 70 C

(i) The acid 37 (0.056g, 0.044 mmol) was dissolved in

THF and DMF (5:1, 1.5 mL) at r. t., then warmed gently

until all had dissolved. Dry CsHCO₃(0.061mg,

0.313mmol) was added and stirred at r.t. for 1 h, followed

by the arabinofuranoside 55 (0.033g, 0.066 mmol)⁶⁸in

for 18 h then quenched with water (5 mL). The product was

extracted with CH₂Cl₂(3 × 60 mL) and the combined

organic layers were dried and evaporated. Chromatography

(petroleum/EtOAc, 10:1) gave benzyl 2,3,4-tri-O-benzyl-6-

O-(2R,3R,17R,39S,40S,E)-3-hydroxy-39-methoxy-17,40-

dimethyl-2-tetracosyloctapentacont-18-enoate-β-D-gluco-

pyranoside 53 (0.056, 70%), [ꢀ]^ꢂ_ꢁ^ꢂ+15 (c 1.1, CHCl₃)

[MALDI–Found [M+Na] ⁺: 1798.6; C₁₁₉H₂₀₂NaO₉requires:

1798.5]; δ_H(400 MHz, CDCl₃): 7.4 –7.26 (20H, m), 5.34

(1H, dt, J 6.4, 15.4 Hz), 5.24 (1H, dd, J 7.4, 15.4 Hz), 4.97

(1H, d, J 11.0 Hz), 4.95 (1H, d, J 11.0 Hz), 4.93 (1H, d, J

11.0 Hz), 4.9 (1H, d, J 12 Hz), 4.78 (1H, d, J 11 Hz), 4.71

(1H, d, J 11 Hz), 4.64 (1H, d, J 12 Hz), 4.61 (1H, d, J 11

Hz), 4.54 (1H, br t, J 6.4 Hz), 4.52 (1H, br s), 4.22 (1H, dd,

J 4.6, 11 Hz), 3.67 (2H, br t J 8.7 Hz), 3.54 – 3.47 (3H, m),

3.35 (3H, s), 2.96 (1H, br pent, J 4.2 Hz), 2.5 – 2.46 (2H,

m), 2.06 – 2.01 (1H, m), 1.97 (2H, br q, J 6.7 Hz), 1.8 – 1.7

(1H, m), 1.68 – 1.55 (3H, m), 1.5 – 1.2 (138H, m), 1.15 –

1.05 (1H, m), 0.94 (3H, d, J 6.7 Hz), 0.89 (6H, t, J 7 Hz),

0.85 (3H, d, J 6.7 Hz); δ_C(101 MHz, CDCl₃): 175.2, 138.4,

138.2, 137.7, 137.1, 136.5, 128.5, 128.4, 128.35, 128.1,

128.0, 127.97, 127.9, 127.85, 127.7, 102.3, 85.4, 84.5, 82.3,

77.8, 75.8, 75.1, 74.9, 72.8, 72.3, 71.1, 62.9, 57.7, 51.3,

37.2, 36.7, 35.6, 35.3, 32.6, 32.3, 31.9, 30.5, 30.0, 29.9,

29.8, 29.7, 29.66, 29.6, 29.58, 29.53, 29.5, 29.4, 29.1, 27.6,

27.5, 27.4, 26.2, 25.9, 22.7, 20.9, 14.9, 14.1; υ_max: 3487,

2920, 2851, 1738, 1466, 1072, 727, 696 cm^-1.

(ii) Sodium (~50 mg) was added to liq. ammonia (100 mL)

until the blue colour persisted. The glucopyranoside 53 (48

mg, 0.027 mmol) in 1,4-dioxane (2 mL) was added; the

mixture was stirred for 4 – 5 min, when the blue colour

disappeared, then quenched with sat. aq. NH₄Cl (5 mL) and

ether (30 mL). The ammonia was allowed to evaporate; the

aqueous layer was extracted with ether (2 × 30 mL). The

combined organic layers were evaporated. Chromatography

(CHCl₃/MeOH, 10:1) gave a 6:4 α,β-mixture of compound

54 (18 mg, 47%), [ꢀ]_ꢁ^ꢂꢂ+28 (c 0.50, CHCl₃) [MALDI–

Found [M+Na]⁺: 1438.3385; C₉₁H₁₇₈NaO₉requires:

1438.3363]; δ_H(400 MHz, CDCl₃+ few drops of CD₃OD):

5.28 (1H, dt, J 6.4, 15.2 Hz), 5.18 (1H, br dd, J 7.4, 15.2

Hz), 5.1 (0.6H, d, J 3.8 Hz, H–1α), 4.46 (0.4H, d, J 7.8 Hz,

H–1β), 4.4 (1H, br d, J 11.8 Hz, H–6α,β), 4.24 (1H, br dd, J

5.8, 11.8 Hz, H–6α,β), 3.96 – 3.92 (0.6H, ddd, J 2.2, 5.6,

9.8 Hz, H–5α), 3.67 – 3.58 (1.6H, H–4α + CH-OH MA),

3.48 – 3.44 (0.4H, ddd, J 2.2, 5.6, 9.0 Hz, H–5β), 3.43 –

3.26 (5H, H–2,3α, H–3,4β and s at 3.3 for OCH₃), 3.17

(0.4H, br t, J 8.4 Hz), 2.95 – 2.91 (1H, m), 2.41 – 2.34 (1H,

m), 2.0 – 1.95 (1H, m), 1.9 (2H, br q, J 6.7 Hz), 1.62 – 1.1

(146H, m), 1.07 – 1.0 (2H, m), 0.88 (3H, d, J 6.7 Hz), 0.83

(6H, t, J 6.6 Hz), 0.8 (3H, d, J 7 Hz); δ_Cfor α, β isomers

(400 MHz, CDCl₃+ few drops of CD₃OD): 175.1, 136.4,

128.2, 96.6, 92.2, 85.5, 76.1, 74.5, 73.6, 72.4, 72.2, 70.5,

70.3, 69.2, 63.5, 57.5, 52.7, 52.5, 37.1, 36.6, 35.2, 35.0,

32.4, 32.2, 31.8, 30.4, 29.8, 29.76, 29.7, 29.6, 29.5, 29.41,

29.4, 29.3, 29.2, 29.1, 29.0, 27.4, 27.2, 26.0, 22.5, 20.8,

14.7, 13.9; υ_max: 3400, 2923, 2852, 1720, 1466, 1376, 1100

cm^-1. Starting material (12 mg) was recovered.

o

THF/ DMF (0.5 mL). The mixture was stirred at 70 C for

18 h, quenched with water (3 mL), then extracted with

CH₂Cl₂(3 × 30 mL) and evaporated. Chromatography

(petroleum/ EtOAc, 10:1) gave methyl 2,3-di-O-benzyl-5-

O-[(2R,3R, 17R,39S,40S,E)-3-hydroxy-39-methoxy-17,40-

di-methyl-2-tetracosyloctapentacont-18-enoate]-α-D-

arabinofuranoside 56 (0.049g, 71%) as a colourless thick

oil, [ꢀ]^ꢂ_ꢁ^ꢂ+21 (c 0.51, CHCl₃) [MALDI–Found [M+Na]⁺:

1602.3; C₁₀₅H₁₉₀NaO₈requires: 1602.4]; δ_H(400 MHz,

CDCl₃): 7.4 – 7.28 (10H, m), 5.34 (1H, dt, J 6.3, 15.4 Hz),

5.25 (1H, dd, J 7.4, 15.4 Hz), 4.92 (1H, br s), 4.57 (1H, d, J

12 Hz), 4.56 (1H, d, J 11.8 Hz), 4.5 (1H, d, J 12.0 Hz), 4.48

(1H, d, J 11.8 Hz), 4.33 – 4.26 ( 2H, m), 4.24 – 4.2.0 (1H,

m), 3.99 (1H, d, J 2.1 Hz), 3.84 (1H, dd, J 2.6, 6.5 Hz),

3.65 – 3.62 (1H, m), 3.37 (3H, s), 3.35 (3H, s), 2.98 – 2.94

(1H, m), 2.43 (1H, tt, J 5.5, 9.1 Hz), 2.06 – 2.02 (1H, m),

1.97 (2H, br q, J 6.7 Hz), 1.7 – 1.5 (5H, m), 1.45 – 1.2

(137H, m), 1.11 – 1.04 (2H, m), 0.94 (3H, d, J 6.7 Hz),

0.89 (6H, t, J 6.6 Hz), 0.85 (3H, d, J 6.9 Hz); δ_C(101 MHz,

CDCl₃): 175.0, 137.4, 137.2, 136.4, 128.5, 128.46, 128.4,

128.0, 127.9, 127.86, 107.2, 87.8, 85.4, 83.7, 79.4, 72.4,

72.2, 72.1, 63.5, 57.7, 54.9, 51.5, 37.2, 36.7, 35.5, 35.3,

32.6, 32.3, 31.9, 30.5, 30.0, 29.9, 29.7, 29.6, 29.5, 29.4,

29.35, 29.1, 27.6, 27.4, 27.36, 26.2, 25.8, 22.7, 20.9, 14.9,

14.1; υ_max: 2923, 2853, 1738, 1465, 1100 cm^-1.

(ii) Sodium (50 mg) was added to liq. ammonia (100 mL)

as above. Arabinofuranoside 56 (42 mg, 0.026 mmol) in

1,4-dioxane (2 mL) was added and stirred for 4 – 5 min,

when the blue colour disappeared, then quenched with sat.

aq. NH₄Cl (5 mL) and ether (30 mL). Work up as before

and chromatography (CHCl₃/MeOH, 10:1) gave compound

57 as a thick oil (21 mg, 57%), [ꢀ]^ꢂ_ꢁ^ꢂ+26 (c 0.70, CHCl₃)

[MALDI–Found [M+Na]⁺: 1422.3430; C₉₁H₁₇₈NaO₈

requires: 1422.3414]. This showed δ_H(400 MHz, CDCl₃+

few drops of CD₃OD): 5.28 (1H, dt, J 6.4, 15.2 Hz), 5.2

(1H, dd, J 7.4, 15.2 Hz), 4.78 (1H, br s), 4.33 (1H, dd, J

4.6, 11.7 Hz), 4.28 (1H, dd, J 5, 11.7 Hz), 4.08 (1H, br q, J

4.9 Hz), 3.96 (1H, dd, J 1.3, 2.8 Hz), 3.85 (1H, dd, J 2.9,

5.0 Hz), 3.65 – 3.60 (1H, m), 3.35 (3H, s), 3.3 (3H, s), 2.95

– 2.93 (1H, m), 2.39 (1H, ddd, J 4.8, 7.4, 10.2 Hz), 2.01 –

1.96 (1H, m), 1.91 (2H, br q, J 6.6 Hz), 1.62 – 1.03 (146H,

m), 0.89 (3H, d, J 6.7 Hz), 0.84 (6H, t, J 6.6 Hz), 0.8 (3H,

d, J 7.0 Hz); δ_C(101 MHz, CDCl₃+ few drops of CD₃OD):

175.0, 136.4, 128.3, 108.8, 85.5, 81.9, 81.2, 78.0, 72.4,

63.4, 57.6, 55.0, 52.6, 37.1, 36.6, 35.2, 34.9, 32.4, 32.2,

31.8, 30.4, 29.8, 29.78, 29.7, 29.6, 29.5, 29.48, 29.4, 29.3,

29.2, 20.0, 27.4, 27.3, 27.2, 26.0, 25.3, 22.6, 20.8, 14.7,

14.0; ν_max: 3400, 2925, 2854, 1724, 1466, 1215, 1094 cm^-1.

Starting material (10.2 mg) was recovered.

3.21 (S)-2,3-Dihydroxypropyl (2R,3R,17R,39S,40S,E)-3-

hydroxy-39-methoxy-17,40-dimethyl-2-tetracosylocta-

pentacont-18-enoate 60

ACCEPTED MANUSCRIPT

16

casein/PBS buffer) (50 µl / well) was added and incubated at

25 ºC for 1 h. The plates were washed with 400 µl casein/PBS

buffer 3 times using an automatic washer, and any excess

flicked out onto a paper towel until dry. Secondary antibody

(anti-human IgG (Fc specific) peroxidise conjugated antibody

produced in goat (Aldrich) (diluted to 1:2000 in casein/PBS

buffer) (50 µl / well) was added, and incubated at 25 ºC for 30

minutes. The plates were again washed 3 times with 400 µl

casein/PBS buffer using an automatic washer, and any excess

was again flicked out. OPD substrate (o-phenylenediamine, 1

mg/ml, 50 µl / well) and H₂O₂(0.8 mg/ml) in 0.1 M citrate

buffer) was added, and the plates were incubated for 30

minutes at 25 ºC. The colour reaction was terminated by

adding 2.5 M H₂SO₄(50 µl / well), and the absorbance

measured at 492nm.

(i) CsHCO₃(0.061 g, 0.313 mmol) was added to a solution

of tosylate 58 (0.030 g, 0.067 mmol)⁶⁹and acid 37 (0.056

g, 0.044 mmol) in dry DMF:THF (1:5, 2 mL) at r. t. and

o

stirred at 70 C for two days. The suspension was diluted

with EtOAc (20 mL) and water (10 mL) and the aqueous

layer was extracted with EtOAc (3 × 10 mL). The

combined organic layers were washed with water (10 mL)

and brine (10 mL), dried and evaporated to give a thick oil.

Column chromatography (10:1 hexane/EtOAc) gave (S)-

2,3-bis-(benzyloxy)propyl

(2R,3R,17R,39S,40S,E)-3-

hydroxy-39-methoxy-17,40-dimethyl-2-tetracosylocta-

pentacont-18-enoate 59 (0.060, 89%) [MALDI–Found

[M+Na]⁺: 1530.3; C₁₀₂H₁₈₆NaO₆requires: 1530.4]. This

showed δ_H(400 MHz, CDCl₃): 7.4 – 7.28 (10H, m), 5.34

(1H, dt, J 6.4, 15.3 Hz), 5.25 (1H, dd, J 7.4, 15.3 Hz), 4.7

(1H, d, J 12.0 Hz), 4.65 (1H, d, J 12.0 Hz), 4.6 (2H, br s),

4.44 (1H, dd, J 4, 11.6 Hz), 4.23 (1H, dd, J 5.4, 11.6 Hz),

3.84 (1H, br pent, J 5.4 Hz), 3.64 – 3.57 (3H, m), 3.36 (3H,

s), 2.99 – 2.96 (1H, m), 2.5 (1H, br d, J 5.9 Hz), 2.44 ( 1H,

br td, J 5.4, 9.8 Hz), 2.1–2.03 (1H, m), 1.98 (2H, br q, J 6.7

Hz), 1.73 – 1.2 (142H, br m), 1.15 – 1.07 (1H, m), 0.96

(3H, d, J 6.7 Hz), 0.9 (6H, t, J 6.7 Hz), 0.87 (3H, d, J 6.9

Hz); δ_C(101 MHz, CDCl₃): 175.4, 138.0, 137.9, 136.5,

128.4, 128.35, 127.73, 127.7, 127.6, 85.4, 75.8, 73.5, 72.3,

72.1, 69.6, 63.5, 57.7, 51.4, 37.2, 36.7, 35.5, 35.3, 32.6,

32.4, 31.9, 30.5, 30.0, 29.9, 29.8, 29.7, 29.66, 29.6, 29.5,

29.44, 29.4, 29.1, 27.6, 27.5, 27.4, 26.2, 25.8, 22.7, 20.9,

14.9, 14.1; υ_max: 3474, 2924, 2853, 1735, 1466, 1370, 1100,

968, 908 cm^-1.

Acknowledgements

HMA and RTH wish to thank the Government of Iraq for the

award of PhD studentships. CR thanks the Welsh Government

for support under the A4B programme. We wish to thank Dr

Paul Gates of Bristol University for carrying out accurate mass

MALDI determinations, and Dr A Ramsay and the Special

Programme for Research and Training in Tropical Diseases at

the WHO for access to sera from the TDR TB Specimen Bank.

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Article Doi

The synthesis of single enantiomers of trans-alkene containing mycolic acids and related sugar esters

DOI: 10.1016/j.tet.2016.08.089

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Article abstract of DOI:10.1016/j.tet.2016.08.089

Full text of DOI:10.1016/j.tet.2016.08.089

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