Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides

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Synthesis and Mass Spectral Characterization of Mycobacterial

Phosphatidylinositol and Its Dimannosides

Gregory M. Rankin,^†Benjamin J. Compton,^‡Karen A. Johnston,^‡Colin M. Hayman,^‡Gavin F. Painter,^‡

and David S. Larsen*^,†

^†University of Otago, P.O. Box 56, Dunedin, New Zealand

^‡Carbohydrate Chemistry Team, Industrial Research Limited, P.O. Box 31310, Lower Hutt, New Zealand

S

* Supporting Information

ABSTRACT: A family of naturally occurring mycobacterial

phosphatidylinositol (PI) and its dimannosides (PIM₂,

AcPIM₂, and Ac₂PIM₂) that all possess the predominant

natural 19:0/16:0 phosphatidyl acylation pattern were

prepared to study their mass spectral fragmentations. Among

these, the ﬁrst synthesis of a fully lipidated PIM (i.e.,

(16:0,18:0)(19:0/16:0)-PIM₂) was achieved from

( )-1,2:4,5-diisopropylidene-^D-myo-inositol in 16 steps in 3%

overall yield. A key feature of the strategy was extending the utility of the p-(3,4-dimethoxyphenyl)benzyl protecting group for its

use at the O-3 position of inositol to allow installation of the stearoyl residue at a late stage in the synthesis. Mass spectral studies

were performed on the synthetic PIMs and compared to those reported for natural PIMs identiﬁed from a lipid extract of M.

bovis BCG. These analyses conﬁrm that fragmentation patterns can be used to identify the structures of speciﬁc PIMs from the

cell wall lipid extract.

of synthetic PIM compounds that are present in the

mycobacterial cell wall (i.e., most synthetic preparations of

PIMs do not contain natural acylation patterns). Standard

organic extraction protocols of mycobacteria aﬀord PI, PIM₂,

AcPIM₂, and Ac₂PIM₂as major cell wall components. These

fractions are heterogeneous mixtures with each compound type

consisting of a number of molecules with diﬀerent fatty acid

substituents. Isolation of discrete PIMs is implausible because

of the similarity of these molecules and this complicates

structural elucidation.

INTRODUCTION

■

The waxy cell wall of mycobacteria continues to be of interest

to a number of research groups as it is essential for pathogen

viability inside an infected host.^1,2This makes it a target for

drug design³and also provides a rich source of material that has

a wide range of immune-modulatory activities. Because of this,

we have developed a program aimed at the structural

determination, synthesis, and testing of various cell wall

components from mycobacteria in an endeavor to contribute

to the growing body of evidence that will allow immunological

insight into the mechanism of action for mycobacterial

products. In particular, we have focused on the phosphatidyl-

inositol mannoside (PIM) subclass of molecules because of

their diverse biological activity.⁴Both synthetic and natural

preparations of these molecules have been investigated in

various immunological models. For example, synthetic PIM

glycans display strong adjuvant activity when conjugated to the

carrier protein BSA,⁵and natural PIM extracts can induce a cell-

mediated immune response in a vaccination protocol utilizing

the model antigen OVA.⁶In the latter example, the activity of

the PIM compounds was attributed to their ability to form

liposomes and deliver the vaccine to antigen presenting cells.

PIMs have also been shown to bind various cell surface

receptors including DC-SIGN,⁷CD1d,⁸TLR2/4,^9,10and α₅β₁

integrin.¹¹Along with their adjuvant properties, PIM

compounds have also been shown to block LPS signaling,¹²

reduce allergic airway disease in murine models of asthma,¹³

induce T-cell adhesion to ﬁbronectin,¹¹and modulate T-cell

proliferation.¹⁴⁻¹⁶Although synthetic examples from most PIM

subclasses have been prepared to date, there are few examples

Hsu et al. have undertaken a comprehensive mass spectral

study of a fractionated cell wall extract from Mycobacterium

bovis Bacillus Calmette Guerin to determine the acylation

́

pattern of the major PI and PIM molecules.¹⁷They found that

the most abundant PIMs contain a palmitoyl (16:0) and a (R)-

tuberculostearoyl (19:0) fatty acid attached to the glycerol

moiety of the phosphatidyl group. The fragmentation patterns

of deprotonated molecular ions of these compounds shown by

negative-ion electrospray ionization tandem mass spectrometry

(MS²) indicated that the 16:0 acyl group fragmented more

readily than the 19:0 fatty acid. From this observation, they

proposed that the more abundant PIMs had a 19:0 and a 16:0

fatty acid at the sn-1 and sn-2 glyceryl sites respectively,

exempliﬁed by (19:0/16:0)-PIM₂(1). These observations were

also reported by Gilleron et al.¹⁸in their mass spectral study of

a PIM extract from M. bovis. The hypothesis that the sn-2 acyl

group fragments more readily than that at the sn-1 position was

Received: June 8, 2012

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conﬁrmed by Dyer et al.¹⁹from a mass spectral study of a non-

natural synthetic PIM₂. NMR studies of higher acylated

PIMs^20,21have identiﬁed two additional acylation sites. These

are the primary position of the mannose residue attached at

O-2 of inositol for AcPIMs together with the O-3 position of

inositol for Ac₂PIMs (Figure 1).

data to that of the discrete entity obtained through isolation or,

more commonly, via careful and methodical synthesis.

Although a number of studies have produced synthetic

PIMs,^5,19,23−33only two have reported compounds that contain

the most abundant natural acylation pattern. Speciﬁcally, both

the Seeberger³⁴and Lear³⁵groups have synthesized (16:0-

(19:0/16:0)-PIM₂(2a), and Seeberger et al.³⁴have also

synthesized 16:0-(19:0/16:0)-PIM₆. Unfortunately, no detailed

mass spectral analyses were performed, and therefore, no

comparison with data obtained from natural extracts could be

made. To deﬁnitively ascertain the acylation patterns of fully

lipidated PIMs, we report here the synthesis of a structurally

deﬁned set of these molecules containing the natural 19:0/16:0

acylated phosphatidyl moiety. These encompass the major

components of the PIM₂family proposed to be present in

mycobacterial cell walls. A detailed mass spectral study of these

compounds has substantiated the fragmentation interpretation

of Hsu et al.^17,22Furthermore, inclusion of a fourth acyl group

provides the ﬁrst synthetic example of an Ac₂PIM₂to be

reported and signiﬁcantly extends the available library of

synthetic PIMs that will be tested in biological assays.

Figure 1. Acylation sites of PIM₂species.

Our targets were (19:0/16:0)-PI (4) (Scheme 2), (19:0/

16:0)-PIM₂(1), 16:0-(19:0/16:0)-PIM₂(2a), and an Ac₂PIM₂

(3) (Figure 1). The synthesis of the ﬁrst three targets was by

modiﬁcation of syntheses already developed for other PIM

compounds.^{9,13,15,19,24,25,36,37}However, for Ac₂PIM₂, a diﬀerent

synthetic strategy was required that would allow sequential

glycosylation at O-6 and O-2 of myo-inositol, acylation at O-3,

and phosphatidylation at O-1. In their study, Hsu et al.²²

focused on the fragmentation pattern of an Ac₂PIM₂which they

tentatively assigned the structure as (16:0,18:1)(19:0/16:0)-

PIM₂(3a). We chose to target another analogous abundant

natural Ac₂PIM₂with a structure believed to be (16:0,18:0)-

(19:0/16:0)-PIM₂(3b). This compound (3b) would be

synthetically more readily accessible and would still allow

comparison of the mass spectral data to those from natural

samples.

By analysis of mass spectra (MS, MS², MS³) of a M. bovis

extract, Hsu et al.²²proposed that the most abundant AcPIM₂

species (2) possessed the expected 19:0/16:0 phosphatidyl

acylation pattern and a palmitoyl residue at O-6″, i.e., 16:0-

(19:0/16:0)-PIM₂(2a). They also showed that the major

species from the fully lipidated PIM subgroup (Ac₂PIM₂)

possessed the same phosphatidyl moiety as (19:0/16:0)-PIM₂

(1). By analogy to (16:0-(19:0/16:0)-PIM₂(2a), it was

suggested that the acyl group attached to O-6″ of the major

Ac₂PIM₂subclass was most commonly palmitoyl (16:0). The

acyl residue at O-3 of inositol was predominately oleoyl (18:1),

stearoyl (18:0), or (R)-tuberculostearoyl (19:0). The assign-

ments were made on the assumption that the acyl group

attached to O-6″ fragments more easily than that at O-3.

However, the structural analyses of these fully acylated PIMs

are not deﬁnitive as only limited data pertaining to atom

connectivity can be obtained using mass spectrometry alone.

Thus, the assignments of the acylation pattern at these

positions on the mannose and inositol residues are only

speculative until proven by comparison of the spectroscopic

RESULTS AND DISCUSSION

■

The current study required a phosphatidylation reagent with

tuberculostearoyl (19:0) and palmitoyl (16:0) fatty esters at the

sn-1 and sn-2 positions of the glycerol, respectively. It was

a

Scheme 1. Synthesis of Phosphoramidite 5 and H-Phosphonate 6

a

Reagents and conditions: (a) BnOP(NiPr₂)₂, 1H-tetrazole, CH₂Cl₂, 80%; (b) 8, THF, Py, TEAB buﬀer, quant.

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decided to utilize both phosphoramidite 5 and H-phosphonate

6 as both of these compounds have been used successfully in

previous syntheses of PIM compounds (Scheme 1). The

required diacylglycerol 7 was synthesized from (R)-benzyl

glycidyl ether using the procedure reported by Horst et al.³⁸

Treatment of diacylglycerol 7 with benzyloxybis-

(diisopropylamino)phosphine and 1H-tetrazole aﬀorded phos-

phoramidite 5 in 80% yield. H-Phosphonate 6 was prepared in

quantitative yield by reaction of 7 with chlorophosphite 8. The

data were consistent with those reported by Seeberger et al.³⁴

The ﬁrst target (19:0/16:0)-PI (4) was prepared by

phosphatidylation of known intermediate 9²⁸with H-

phosphonate 6³⁴and then global debenzylation to furnish the

previously unreported compound 4 in 86% yield, which was

shown to be 96% pure by HPLC analysis (Scheme 2).

in multiple gram amounts (Scheme 3). Pseudodisaccharide 10

was mannosylated with trichloroacetimidate donor 11,¹³which

incorporates a methoxyacetate (MAc) group at O-2. This

directing group imparts high α-selectivity giving compound 12

in 85% yield with no β-anomer detected and has the added

advantage that it can be readily removed in the presence of

other ester groups.^12,13Oxymercuration was used to cleave the

enol ether adduct of the Ir(I)-promoted allyl ether isomer-

ization to avoid loss of the acid labile MAc group, and the

resulting alcohol 13 was coupled with H-phosphonate 6

followed by in situ oxidation with iodine. Hydrogenolysis gave

partially protected PIM₂14, and ﬁnally, the methoxyacetate

group was removed with dilute NaOMe in CH₂Cl₂/MeOH to

aﬀord (19:0/16:0)-PIM₂(1) in 78% yield, which was shown to

be 95% pure by HPLC.

The synthesis of AcPIM₂16:0-(19:0/16:0)-PIM₂(2a) began

with the mannosylation of 10 with orthogonally protected

mannosyl phosphate donor 15³⁹(Scheme 4). Promotion of the

reaction with excess TBSOTf resulted in glycosylation, albeit

with partial exchange of the O-6″ silyl protecting group to give

16 as a 2:1 mixture of triisopropyl- and tert-butyldimethyl silyl

ethers in 67% yield. Standard modiﬁcation procedures, namely

a

Scheme 2. Synthesis of (19:0/16:0)-PI (4)

a

́

debenzoylation under Zemplen conditions, followed by

Reagents and conditions: (a) (i) 6, PivCl, Py, (ii) I₂, Py/H₂O, (iii)

Amberlite IRC86 (H⁺) resin; (b) Pd(OH)₂/C, H₂, MeOH, THF, 86%.

benzylation and subsequent removal of the allyl and silyl

ether protecting groups aﬀorded 17 in 77% yield over three

steps. Selective acylation of diol 17 with palmitoyl chloride gave

the AcPIM₂headgroup 18 in 85% yield. The 1H-tetrazole-

mediated coupling of phosphoramidite 5 with 18 followed by in

situ oxidation furnished protected AcPIM₂19 in 88% yield.

Hydrogenolysis of the benzyl groups of 19 gave 16:0-(19:0/

Increasing in complexity, the syntheses of (19:0/16:0)-PIM₂

(1) and 16:0-(19:0/16:0)-PIM₂(2a) were accomplished. We

chose pseudodisaccharide 10^9,28as the starting material for the

synthesis of 1 and 2a as this compound can be easily prepared

a

Scheme 3. Synthesis of PIM₂(1)

a

−

Reagents and conditions: (a) 11, TMSOTf, toluene, 0 °C, 85%; (b) (i) (COD)(MePh₂P)₂Ir⁺PF₆, H₂, THF, (ii) HgO, HgCl₂, (CH₃)₂CO/H₂O,

85%; (c) (i) 6, PivCl, Py, (ii) I₂, Py/H₂O, (iii) Amberlite IRC86 (H⁺) resin; (d) Pd(OH)₂/C, H₂, MeOH, THF, 81%, two steps; (e) (i) 2.5 mM

NaOMe, CH₂Cl₂/MeOH, (ii) Amberlite IRC86 (Na⁺) resin, 78%.

C

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a

Scheme 4. Synthesis of AcPIM₂(2a)

a

Reagents and conditions: (a) 10, TBSOTf, toluene, 0 °C, 67%; (b) NaOMe, CH₂Cl₂/MeOH; (c) BnBr, NaH, DMF; (d) (i)

−

(COD)(MePh₂P)₂Ir⁺PF₆, H₂, THF, (ii) AcCl, MeOH/CH₂Cl₂, 77%, three steps; (e) C₁₅H₃₁COCl, Py/CH₂Cl₂, 85%, (f) (i) 5, 1H-tetrazole,

MeCN, (ii) m-CPBA, CH₂Cl₂, 88%; (g) Pd(OH)₂/C, H₂, MeOH/THF, 90%.

a

Scheme 5. Resolution of myo-Inositol 20

a

Reagents and conditions: (a) 4-bromobenzyl bromide, NaH, THF, 65%; (b) 22, TMSOTf, 4 Å M.S., CH₂Cl₂, −30 °C; (c) NaOMe, MeOH, D-23,

42%, two steps, ^L-24, 41%, two steps; (d) TFA, H₂O, CH₂Cl₂, 0 °C, 2.5 h; (e) BnBr, NaH, DMF, 79%, two steps; (f) TFA, MeOH, CH₂Cl₂, 12 h,

79%; (g) K₃PO₄, Bu₄NBr, Pd(OAc)₂, 3,4-dimethoxyphenylboronic acid, EtOH, 90%; (h) (i) Bu₂SnO, toluene, (ii) CsF, AllBr, DMF, 73%.

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a

16:0)-PIM₂(2a) in 90% yield, which was shown to have a

purity of 98% by HPLC.

Scheme 6. Synthesis of Mannosyl Donors 29 and 30

With the ﬁrst three targets in hand, our attention turned to

the synthesis of (16:0,18:0)(19:0/16:0)-PIM₂(3b) (Scheme

5). Bis-isopropylidene-myo-inositol 20⁴⁰⁻⁴²was chosen as a

convenient starting material as it is readily available and displays

appropriate reactivity (i.e., O-3 is more reactive than O-6). We

planned to use a relay deprotection method at O-3 whereby this

position was protected as a p-bromobenzyl (PBB) ether.^43,44

This group was chosen due to the variety of reactions which

can mediate its selective removal, for example, aromatic

substitution followed by treatment with either a Lewis acid,

protic acid, or oxidant.^43,44

Inositol 21 was prepared by selective alkylation of 20 with p-

bromobenzyl bromide in 65% yield. The mannosylation of 21

with TCA donor 22^45,46and subsequent deacetylation

permitted chromatographic separation of the diastereomeric

mannosides ^D-23 and L-24 in 42% and 41% yields, respectively.

The ^D-myo-inositol conﬁguration of 23 was conﬁrmed by

synthesis from ^D-20, prepared by resolution of ( )-20 via the

corresponding bis-(S)-O-acetylmandeloyl esters.^47,48Selective

acidic hydrolysis of the trans-fused isopropylidene group and

subsequent benzylation of the resulting triol gave 25, which was

then treated with methanolic TFA to aﬀord diol 26. The

incompatibility of the PBB and allyl ether protecting groups in

the Pd-catalyzed amination reaction led to transforming the

PBB ether into a p-(3,4-dimethoxyphenyl)benzyl (DMPBn)

ether via a Suzuki−Miyaura coupling with 3,4-dimethoxy-

phenylboronic acid in excellent yield. Stannylene acetal

promoted alkylation of 27 was used to selectively install the

allyl ether at O-1 to aﬀord 28 in 73% yield.

To avoid the problems of silyl exchange as encountered in

the synthesis of 2a (10 → 16, Scheme 4), mannosylation of 28

with phosphate donor 29, bearing a TBS ether at O-6, was

attempted. Although the reaction aﬀorded a signiﬁcant amount

of the target pseudotrisaccharide, a product resulting from

additional glycosylation at O-6″ was also observed, presumably

by cleavage of the TBS ether and subsequent glycosylation. To

overcome this, a more reactive donor requiring only catalytic

amounts of a promoter was used. The known TCA mannosyl

donor 30⁴⁹was prepared in four steps from tricyclic orthoester

31³⁹via an alternative method (Scheme 6). The Lewis acid

promoted reaction of 31 and allyl alcohol aﬀorded 32 which

was deallylated under standard conditions aﬀording diol 33 in

87% yield. Selective silylation of the primary hydroxyl group

was achieved using TBSOTf to aﬀord 34, which was then

converted to imidate 30.

Mannosylation at O-2 of 28 with TCA donor 30 gave

pseudotrisaccharide 35 in high yield (Scheme 7). Routine

protecting group manipulations, namely debenzoylation to give

36 and benzylation, aﬀorded 37. Mild acidic conditions cleaved

the silyl ether to give 38, and subsequent acylation with

palmitic acid aﬀorded 39 in 79% yield from 35. Attempted

removal of the DMPBn ether of 39 with DDQ, as described for

mono- and disaccharide substrates,⁴³resulted in a complex

mixture of products due to the uncontrolled loss of benzyl

protecting groups. Seeberger et al. had noted that this

protecting group could not be selectively removed from a

protected hexasaccharide in the synthesis of a GPI anchor.⁵⁰

This problematic deprotection was circumvented by hydrolysis

of the DMPBn group using TFA and cation scavenger 40⁵¹to

cleanly aﬀord 41 in 74% yield. Acylation of alcohol 41 with

a

Reagents and conditions: (a) HOP(O)(OBu)₂, CH₂Cl₂; (b) TBSCl,

DMAP, Py, CH₂Cl₂, 73%, two steps; (c) AllOH, BF₃·Et₂O, CH₂Cl₂,

−

99%; (d) (i) (COD)(MePh₂P)₂Ir⁺PF₆, H₂, THF, (ii) AcCl, MeOH/

CH₂Cl₂, 85%; (e) TBSOTf, sym-collidine, CH₂Cl₂, 79%; (f) Cl₃CCN,

DBU, CH₂Cl₂, 83%.

stearic acid and deallylation of 42 gave the Ac₂PIM₂headgroup

43.

Phosphorylation of pseudotrisaccharide 43 with phosphor-

amidite 5 was unsuccessful; however, treatment with H-

phosphonate 6 and in situ oxidation furnished protected

Ac₂PIM₂44 as the triethylammonium salt in 87% yield. Cation

exchange and global deprotection under hydrogenolytic

conditions aﬀorded Ac₂PIM₂3b as the sodium salt in high

purity (96% by HPLC). A well-resolved H NMR spectrum of

3b was obtained in the mixed solvent system CDCl₃/CD₃OD/

D₂O (70:40:6) at 30 °C.

With all four synthetic targets in hand, we set about

comparing their corresponding fragmentation patterns by

negative ion ESI-MSⁿboth to each other and to those reported

from natural sources. All MS²and pseudo-MS³spectra are

provided in the Supporting Information.

At the core of the PIM family is (19:0/16:0)-PI (4), which

shows a deprotonated molecular ion [M − H]⁻at m/z 851.6.

ESI-MS²fragmentation of this parent ion gave two major peaks

at m/z 595.3, attributed to the loss of the sn-2 palmitoyl (16:0)

residue, and at m/z 433.3, resulting from the loss of the inositol

and the sn-2 fatty acid. Related, less intense peaks from the

analogous losses of the sn-1 tuberculostearoyl (19:0) and

inositol moieties were observed at m/z 553.3 and 391.2, which

reaﬃrms that the sn-2 fatty acid fragments more readily than

that at the sn-1 position (Figure 2). Overall, the fragmentation

pattern revealed in the MS²spectrum of our synthetic target

matched that of the natural product reported by Hsu et al.¹⁷

(Figure S1, Supporting Information).

1

(19:0/16:0)-PIM₂(1), with a parent ion [M − H]⁻at m/z

1175.7 was fragmented, giving rise to two daughter ions at m/z

919.4 and 877.4 corresponding to the loss of the sn-2 and sn-1

acyl residues respectively from the phosphatidyl moiety (Figure

S2B, Supporting Information). The dominant ion of the pair at

m/z 919.4 was consistent with the data reported by Hsu et al.¹⁷

for the preferential loss of the sn-2 palmitoyl ester. This MS²

spectrum of 1 also displays signals of m/z 433.3 and 391.2 also

seen in the MS²spectrum of 4 above. These data conﬁrm that

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a

Scheme 7. Synthesis of Ac₂PIM₂(3b)

a

Reagents and conditions: (a) TMSOTf, 4 Å M.S., toluene, 0 °C, 99%; (b) NaOMe, CH₂Cl₂/MeOH, 93%; (c) BnBr, NaH, DMF, 99%; (d) AcCl,

CH₂Cl₂/MeOH, 92%; (e) C₁₅H₃₁COOH, DMAP, DCC, CH₂Cl₂, 93%; (f) 40, 30% TFA, CH₂Cl₂, 74%; (g) C₁₇H₃₅COOH, DMAP, DCC, CH₂Cl₂,

−

84%; (h) (i) (COD)(MePh₂P)₂Ir⁺PF₆, H₂, THF, (ii) HgO, HgCl₂, (CH₃)₂CO/H₂O, 61%; (i) (i) 6, PivCl, Py, (ii) I₂, Py/H₂O, 87%; (j) (i) Dowex

50WX4-400 (Na+) resin, CHCl₃/MeOH, (ii) Pd(OH)₂/C, H₂, MeOH, THF, 99%.

Figure 2. Negative-ion ESI-TOF MS²spectrum of the ion at m/z 851.6 of 19:0/16:0-PI (4).

the natural acylation pattern of the phosphatidyl group of the

more abundant PIMs is as synthesized here (i.e., contains a

19:0 and 16:0 fatty acid at the sn-1 and sn-2 positions

respectively).

Information). This MS²spectrum also showed a product ion at

m/z 803.3 which is attributed to the loss of the diacyl glycerol

unit. Pseudo-MS³data obtained for m/z 803 of 2a compares

well to the equivalent m/z 803 MS³(1413→803) spectrum of

the natural extract obtained by Hsu et al.²²with prominent ions

at m/z 385.0 and 547.1 attributable to the loss of the O-2

The fragmentation of the [M − H]⁻ion at m/z 1413.9 of

16:0-(19:0/16:0)-PIM₂(2a) showed a similar sn-2 fragmenta-

tion bias as observed for 4 and 1 (Figure S3, Supporting

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Figure 3. Negative-ion ESI-TOF MS²spectrum of synthetic Ac₂PIM₂3b (A) and negative ion ESI-TOF MS²spectrum of the ion at m/z 1680.1 (B)

from a lipid extract of M. bovis AN5.

palmitoylated mannosyl pyranose residue and the palmitic acid

residue respectively (Figure S4, Supporting Information).

The MS²and pseudo-MS³spectra of Ac₂PIM₂, (16:0,18:0)-

(19:0/16:0)-PIM₂(3b) ([M-H]⁻, m/z 1680.2), were compared

to those reported by Hsu et al.²²for the ion at m/z 1678.1 from

the natural extract, the structure of which was proposed to be

(16:0,18:1)(19:0/16:0)-PIM₂(3a) (Figure S5, Supporting

Information). The fragmentation pattern of the 16:0 and 19:0

fatty acids on the phosphatidyl moiety in the MS²spectrum of

the [M − H]⁻molecular ion was consistent with those

observed for PI 4, PIM₂1, and AcPIM₂2a. The MS²spectrum

displayed an ion at m/z 1069.6 arising from the loss of the

diacylglycerol moiety, which was critical for the analysis of the

fragmentation behavior of the O-6″ and O-3 fatty acids. The

pseudo-MS³spectrum of the ion at m/z 1069.6 showed a more

intense signal at m/z 813.4 arising from loss of the O-6″

palmitoyl fatty acid compared to the signal at m/z 785.3

attributed to the loss of the O-3 stearoyl fatty acid. Hsu et al.

observed the same pattern of intensities for the loss of the

palmitoyl and oleoyl fatty acids in the MS³spectrum for the

analogous fragment ion at m/z 1067.7 (1678→1067) for 3a

from the natural extract (Figure S6, Supporting Information).

Further analysis of the pseudo-MS³spectrum of the ion at

m/z 1069.6 of 3b shows the fragment ion at m/z 907.5

attributed to the loss of the O-6 mannose moiety. Additional

fragmentation of this ion results in the signals at m/z 651.3 and

669.3, due to the loss of the O-6″ 16:0 acyl residue as an acid

and ketene, respectively, which are much more intense than

those at m/z 623.3 and 641.3 from the analogous loss of the

O-3 18:0 residue. Given that the structure of 3b is deﬁned by

synthesis and the palmitoyl fatty acid is at O-6″ of mannose,

this conﬁrms the proposal of Hsu et al. that the fatty acid on the

mannose moiety fragments more readily than that on the

inositol. Furthermore, this also conﬁrms the acylation pattern

for natural Ac₂PIM₂3a.

To enable direct comparison of the mass spectral data of 3b

with Ac₂PIM₂from a mycobacterial source, we investigated a

lipid extract of M. bovis AN5. The MS²spectrum of the m/z

1680.2 ion from the MS of 3b was compared to that of the

corresponding ion from the bacterial extract (Figure 3). The

fragmentation pattern and relative intensities of the molecular

ions matched, thereby conﬁrming the speciﬁc acylation pattern

of Ac₂PIM₂from the natural extract.

CONCLUSIONS

■

In conclusion, we have reported the ﬁrst syntheses of PI, PIM₂,

and Ac₂PIM₂along with AcPIM₂, all possessing the naturally

predominant 19:0/16:0 acylation pattern on the phosphatidyl

moiety. The synthesis of Ac₂PIM₂3b was enabled by extending

the utility of the DMPBn protecting group methodology

pioneered by Seeberger. Complete characterization was

achieved by a combination of NMR and MS with purity

established by HPLC. Comprehensive mass spectral analyses

show that the glyceryl sn-2 acyl group fragments more readily

G

dx.doi.org/10.1021/jo301189y | J. Org. Chem. XXXX, XXX, XXX−XXX

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25.0, 27.2, 29.2, 29.4, 29.5, 29.57, 29.61, 29.8, 30.07, 30.12, 32.0, 32.9,

34.2, 34.4, 37.2, 43.1, 43.3, 61.6, 61.7, 61.9, 62.6, 65.4, 65.6, 70.1,

127.0, 127.4, 128.3, 173.1, 173.4; ³¹P NMR (202 MHz, CDCl₃) δ

148.7, 148.9; HRMS-ESI [M + H]⁺calcd for C₅₁H₉₅NO₆P 848.6897,

found 848.6912.

than that at the sn-1 position of the phosphatidyl group. For

Ac₂PIM₂3b pseudo-MS³analyses show that the O-6″ acyl

group on the O-2 mannosyl residue fragments more readily

than that at the O-3 position of inositol. This study conﬁrms

that mass spectrometry provides a powerful tool for deﬁnitively

elucidating the acylation patterns of PIMs from natural

mycobacterial extracts.

Triethylammonium 1-O-((R)-10-Methyloctadecanoyl)-2-O-

hexadecanoyl-sn-glycero-3-H-phosphonate (6).³⁴Diacylglycerol

7 (0.21 g, 0.35 mmol) was dried by coevaporation with pyridine (3 × 5

mL) and then dissolved in dry THF/pyridine (10:1, 5.5 mL). This

solution was added dropwise over a period of 30 min to a stirred

solution of salicyl chlorophosphite 8 (0.088 g, 0.44 mmol) in dry THF

(5 mL) under argon at rt. The reaction mixture was stirred for 3 h,

after which time TLC analysis (CHCl₃/CH₃OH/H₂O = 100:15:1)

showed complete conversion of the starting material into a product of

lower R_f. The reaction mixture was quenched with 1 M aqueous

triethylammonium bicarbonate (TEAB) buﬀer (20 mL), stirred for 15

min, and then extracted into CHCl₃(3 × 30 mL). The combined

organic layers were washed with TEAB buﬀer (3 × 20 mL) and dried

(MgSO₄). After ﬁltration, the solvent was removed in vacuo to give the

title compound 6 (0.322 g, quant) as a colorless oil which was used

without further puriﬁcation: ¹H NMR (500 MHz, CDCl₃) δ 0.83 (d, J

= 6.6 Hz, 3H), 0.88 (t, J = 6.9 Hz, 6H), 1.21−1.32 (m, 51H), 1.34 (t, J

= 7.3 Hz, 9H), 1.56−1.63 (m, 4H), 2.27−2.32 (m, 4H), 3.10 (q, J =

7.3, Hz, 6H), 4.03 (dd, J = 5.3, 8.0 Hz, 2H), 4.18 (dd, J = 6.4, 11.9 Hz,

1H), 4.38 (dd, J = 3.7, 11.9 Hz, 1H), 5.21−5.25 (m, 1H), 6.88 (d, ¹J_PH

= 628 Hz, 1H), 12.87 (brs, 1H); ³¹P NMR (202 MHz, CDCl₃) δ 4.34

EXPERIMENTAL SECTION

■

¹H NMR spectra were obtained at 400 or 500 MHz and referenced to

tetramethylsilane (TMS) (0.0 ppm) or the residual solvent peak (¹H

CHCl₃δ 7.26 ppm). ¹³C NMR spectra were recorded at 75 or 125

MHz and referenced either to TMS (0.0 ppm) or internal solvent

(CDCl₃δ 77.0 ppm). ³¹P NMR spectra were recorded at 202 MHz

with H₃PO₄(δ 0.0 ppm) as the external reference. Electrospray

ionization (ESI) mass spectra were recorded on Q-TOF mass

spectrometers. Anhydrous solvents were sourced commercially and

used without further treatment unless stated. Powdered molecular

sieves were ﬂame-dried under vacuum immediately prior to use. Flash

column chromatography was carried out using Davisil LC60 Å 40−63

μm silica gel unless otherwise stated. All ﬂash chromatography solvents

were AR-grade. Petroleum ether used was bp 60−80 °C range. All

compounds were isolated after silica gel column chromatography and

fractions collected were one spot by TLC. Thin-layer chromatography

(TLC) plates were visualized under an UV lamp and/or with a spray

consisting of 5% w/v dodecamolybdophosphoric acid in ethanol with

subsequent heating. HPLC purity of target compounds: 4 (95.6%), 1

(95.1%), 2a (99.0%), and 3b (96.2%).

1

(d, J_PH= 628 Hz); HRMS-ESI [M + H]⁺calcd for C₄₄H₉₁NO₇P

776.6533, found 776.6543; HRMS-ESI [M − Et₃HN]⁻calcd for

C₃₈H₇₄O₇P 673.5172, found 673.5181.

1-O-(1-O-((R)-10-Methyloctadecanoyl)-2-O-hexadecanoyl-

sn-glycero-3-phosphoryl)-^D-myo-inositol (4). Alcohol 9 (0.030 g,

0.047 mmol) and H-phosphonate 6³⁴(0.055 g, 0.071 mmol) were

coevaporated from dry pyridine (3 × 5 mL) then placed under high

vacuum for 30 min. The reagents were dissolved in dry distilled

pyridine (5 mL), pivaloyl chloride (0.033 mL, 0.238 mmol) was added,

and the reaction mixture was stirred for 2 h at rt. Iodine (0.036 g,

0.143 mmol) in pyridine/H₂O (9:1, 5 mL) was added, and the mixture

was stirred for 1 h before being diluted with CHCl₃(20 mL) and

washed with 10% aq Na₂S₂O₃(20 mL). The aqueous phase was re-

extracted with CHCl₃(2 × 20 mL) then EtOAc (20 mL), the

combined organic layers were dried (MgSO₄) and ﬁltered, and the

solvent was removed in vacuo. The residue was dissolved in CH₂Cl₂/

MeOH (1:1, 10 mL), treated with Amberlite IRC86 resin (H⁺form),

and ﬁltered, solvent was removed, and the residue was semipuriﬁed by

column chromatography on silica gel (CHCl₃/MeOH = 1:0 to 9:1).

Pd(OH)₂/C (20%, 34 mg, 0.048 mmol) was added to the residue in

THF/MeOH (2:3, 10 mL), and the reaction mixture was stirred under

an atmosphere of H₂. After 4 h, the reaction was ﬁltered through Celite

and the solvent removed in vacuo. The residue was puriﬁed by column

chromatography on silica gel (CHCl₃/MeOH/H₂O = 70:40:0 to

70:40:6) to aﬀord the title compound 4 (0.035 g, 86%, 95.6% pure by

HPLC) as a white solid: ¹H NMR (500 MHz, CDCl₃/CD₃OD/D₂O =

70:40:6) δ 0.85 (d, J = 6.5 Hz, 3H), 0.88 (t, J = 7.0 Hz, 6H), 1.06−

1.40 (m, 51H), 1.57−1.64 (m, 4H), 2.30−2.36 (m, 4H), 3.26 (t, J =

9.5 Hz, 1H), 3.45 (dd, J = 3.0, 9.5 Hz, 1H), 3.64 (dd, J = 9.5, 9.5 Hz,

1H), 3.76 (t, J = 9.5 Hz, 1H), 3.87−3.93 (m, 1H), 4.00−4.09 (m, 2H),

4.18−4.21 (m, 2H), 4.43 (dd, J = 2.8, 12.2 Hz, 1H), 5.24−28 (m 1H);

¹³C NMR (125 MHz, CDCl₃/CD₃OD/D₂O = 70:40:6) δ 13.8, 19.5,

22.5, 24.78, 24.84, 27.0, 29.0, 29.1, 29.2, 29.5, 29.6, 29.9, 31.8, 32.7,

34.0, 34.2, 37.0, 62.9, 63.7, 70.5, 70.6, 71.1, 71.6, 71.6, 72.4, 74.4, 76.5,

174.0, 174.3; ³¹P NMR (202 MHz, CDCl₃/CD₃OD/D₂O = 70:40:6)

δ 0.4; HRMS-ESI [M − H]⁻calcd for C₄₄H₈₄O₁₃P 851.5650, found

851.5649.

1-O-Allyl-2-O-(2-O-methoxyacetyl-3,4,6-tri-O-benzyl-α-^D-

mannopyranosyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-mannopyra-

nosyl)-3,4,5-tri-O-benzyl-^D-myo-inositol (12). TMSOTf (5 μL,

0.028 mmol) was added to a mixture of alcohol 10^9,28(0.230 g, 0.227

mmol) and trichloroacetimidate 11¹³(0.270 g, 0.405 mmol) in dry

toluene at 0 °C. After 45 min, the reaction was quenched with Et₃N

and ﬁltered through Celite and the solvent removed in vacuo. The

The HPLC analyses of 4 and 1 used system 1; HPLC: column:

Phenomenex Synergi 4 μm Fusion-RP (80 Å, 3.0 × 250 mm).

Detection: charged aerosol detector (ﬁlter = none). Flow rate: 0.5

mL/min. Column temperature: 40 °C. Solvents: A, H₂O; B, water

containing 50 mM NH₄OAc adjusted to pH 5.0; C, methanol.

Gradient program: 0−10 min 10−0% A, 10% B, 80−90%C, 10−28

min hold (0% A, 10% B, 90% C), 28−30 min return to starting

conditions then equilibrate for 10 min prior to next injection.

Chromatograms were corrected for detector nonlinearity. The HPLC

analysis of 2a used system 2; Phenomenex Synergi 4 μm Fusion-RP

(80 Å, 4.6 × 250 mm). Detection: as for system 1. Flow rate: 0.8 mL/

min. Column temperature: 40 °C. Solvents: A 1:1 water/MeOH + 50

mM NH₄OAc adjusted to pH 5.0 prior to dilution; B, MeOH; C, 2-

propanol. Gradient program; 0−20 min 10% A, 60−50% B, 30−40%

C; 20−21 min hold (10% A, 50% B, 40% C); 21−23 min return to

starting conditions then equilibrate for 17 min prior to next injection.

The HPLC analysis of compound 3b used system 3; HPLC: column

Phenomenex Jupiter C4 5 μm (300 Å, 4.6 × 250 mm); detection: as

for system 1. Flow rate: 0.8 mL/min. Column temperature: 40 °C.

Solvents: A, 75:15:10 CHCl₃/MeOH/H₂O containing 20 mM

NH₄OAc and 1%AcOH; solvent B, 1:1 CHCl₃/MeOH containing

20 mM NH₄OAc and 1% AcOH. Gradient program: 0−15 min 100−

30% A, 0−70% B, 15−20 min hold (30% A, 70% B), 20−25 min

return to starting conditions and equilibrate for 10 min prior to next

injection. Chromatograms for systems 2 and 3 were not corrected for

detector nonlinearity.

Benzyl (1-O-((R)-10-Methyloctadecanoyl)-2-O-hexadecano-

yl-sn-glycero)diisopropylphosphoramidite (5). 1H-Tetrazole

was added to a solution of benzyloxybis(diisopropylamino)phosphine

(0.620 g, 1.83 mmol) and diacylglycerol 7 (0.560 g, 0.917 mmol) in

dry CH₂Cl₂(10 mL) and 4 Å sieves under argon at rt. After 2 h, the

reaction mixture was diluted with CH₂Cl₂, washed with saturated

NaHCO₃, dried (MgSO₄), and ﬁltered and the solvent removed in

vacuo. The residue was puriﬁed by column chromatography on silica

gel (Et₃N/EtOAc/petroleum ether = 3:15:80) to aﬀord the title

compound 5 (0.620 g, 80%) as a colorless oil: [α]²_D²+6.34 (c = 1.34,

1

CH₂Cl₂); H NMR (500 MHz, CDCl₃)) δ 0.84 (d, J = 6.5 Hz, 3H),

0.88 (t, J = 7.0 Hz, 6H), 1.05−1.38 (m, 63H), 1.57−1.64 (m, 4H),

2.27−2.31 (m, 4H), 3.59−3.82 (m, 4H), 4.15−4.20 (m, 1H), 4.32−

4.37 (m, 1H), 4.63−4.76 (m, 2H), 5.17−5.21 (m, 1H), 7.31−7.35 (m,

5H); ¹³C NMR (75 MHz, CDCl₃) δ 14.2, 19.8, 22.8, 24.6, 24.7, 24,8,

H

dx.doi.org/10.1021/jo301189y | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

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residue was puriﬁed by column chromatography on silica gel (EtOAc/

petroleum ether = 1:9 to 2:3) to aﬀord the title compound 12 (0.30 g,

85%) as a colorless oil: H NMR (500 MHz, CDCl₃) δ 3.18 (dd, J =

MeOH/H₂O = 70:40:0 to 70:40:6) to aﬀord the title compound 14

(0.032 g, 81%) as a white solid: ¹H NMR (500 MHz, CDCl₃/

CD₃OD/D₂O = 70:40:6) δ 0.81 (d, J = 6.5 Hz, 3H), 0.86 (t, J = 6.5

Hz, 6H), 1.05−1.33 (m, 51H), 1.55−1.58 (m, 4H), 2.28 (t, J = 7.5 Hz,

2H), 2.33 (t, J = 7.5 Hz, 2H), 3.29−2.35 (m, 1H), 3.41 (s, 3H), 3.46

(brd, J = 11 Hz, 1H), 3.55−3.69 (m, 4H), 3.73−3.84 (m, 5H), 3.94−

4.23 (m, 11H), 5.10 (s, 1H), 5.15 (s, 1H), 5.20−5.23 (m, 1H), 5.25 (s,

1H); ¹³C NMR (125 MHz, CDCl₃/CD₃OD/D₂O = 70:40:6) δ 14.4,

20.1, 23.2, 25.4, 25.5, 27.57, 27.61, 29.65, 29.72, 29.3, 29.85, 29.89,

29.94, 30.07, 30.12, 30.15, 30.16, 30.20, 30.23, 30.51, 30.54, 32.4, 33.3,

34.6, 34.7, 37.60, 37.63, 59.5, 61.6, 62.2, 63.5, 64.1, 67.4, 68.0, 69.5,

69.8, 70.7, 71.0, 71.1, 73.27, 73.34, 73.5, 73.8, 73.9, 77.1, 78.9, 79.1,

98.6, 102.1, 171.3, 174.6, 174.9; ³¹P NMR (202 MHz, CDCl₃/

CD₃OD/D₂O = 70:40:6) δ 3.21; HRMS-ESI [M − H]⁻calcd for

C₅₉H₁₀₈O₂₅P 1247.6923, found 1247.6884.

1

2.0, 9.5 Hz, 1H), 3.26−3.40 (m, 5H), 3.42 (s, 3H), 3.53 (dd, J = 3.0,

10.5 Hz, 1H), 3.80−4.21 (m, 14H), 4.21−4.30 (m, 2H), 4.43 (d, J =

10.5 Hz, 1H), 4.47 (d, J = 11.0 Hz, 1H), 4.57−4.94 (m, 18H), 5.11 (d,

J = 10.5 Hz, 1H), 5.16 (s, 1H), 5.23 (d, J = 17.5 Hz, 1H), 5.76 (dddd, J

= 5.0, 5.0, 10.5, 17.5 Hz, 1H), 7.11−7.43 (m, 50H); ¹³C NMR (125

MHz, CDCl₃) δ 59.3, 68.4, 68.5, 69.0, 70.8, 71.4, 71.8, 72.0, 72.1, 72.5,

73.2, 73.4, 74.0, 74.7, 74.9, 75.0, 75.3, 75.7, 75.9, 76.7, 77.3, 77.4, 78.7,

80.0, 80.9, 81.3, 81.5, 98.8 (¹J_C−H= 171 Hz), 99.0 (¹J_C−H= 172 Hz),

117.4, 127.17, 127.20, 127.24, 127.35, 127.37, 127.51, 127.53, 127.54,

127.56, 127.58, 127.7, 127.8, 127.89, 127.90, 127.95, 127.99, 128.1,

128.16, 128.17, 128.23, 128.24, 128.34, 128.37, 128.44, 128.52, 133.9,

137.9, 138.0, 138.1, 138.4, 138.58, 138.61, 138.7, 138.9, 139.1, 169.3;

HRMS-ESI [M + Na]⁺calcd for C₉₄H₁₀₀NaO₁₈1539.6802, found

1539.6793.

Sodium 2,6-Di-O-α-^D-mannopyranosyl-1-O-(1-O-((R)-10-

methyloctadecanoyl)-2-O-hexadecanoyl-sn-glycero-3-phos-

phoryl)-^D-myo-inositol (1). A freshly prepared solution of sodium

methoxide in MeOH (0.5 M, 0.02 mL) was added dropwise to a

stirred solution of 14 (0.015 g, 0.012 mmol) in CH₂Cl₂/MeOH (1:7, 4

mL). After 10 min, the reaction mixture was quenched by the addition

of IRC-86 resin (H⁺form) and ﬁltered. The resulting solution was

treated with IRC-86 resin (Na⁺form) and ﬁltered and the solvent

removed in vacuo. The residue was puriﬁed by column chromatog-

raphy on silica gel (CHCl₃/MeOH/H₂O = 70:40:6) to aﬀord the title

compound 1 (11 mg, 78%, 95.1% pure by HPLC) as a white solid: ¹H

NMR (500 MHz, CDCl₃:CD₃OD:D₂O = 70:40:6) δ 0.75 (d, J = 6.5

Hz, 3H), 0.79 (t, J = 6.5 Hz, 6H), 1.05−1.35 (m, 51H), 1.53−1.58 (m,

4H), 2.23 (t, J = 7.5 Hz, 2H), 2.28 (t, J = 7.5 Hz, 2H), 3.19 (t, J = 9.5

Hz, 1H), 3.33−3.39 (m, 1H), 3.49−3.73 (m, 10H), 3.85−3.96 (m,

7H), 4.09−4.13 (m, 1H), 4.20 (brs, 1H), 4.33 (brs, 1H), 5.00 (s, 1H),

5.02 (s, 1H), 5.17−5.19 (m, 1H); ¹³C NMR (125 MHz, CDCl₃/

CD₃OD/D₂O = 70:40:6) δ 14.5, 20.2, 23.2, 25.5, 25.6, 27.66, 27.70,

29.7, 29.8, 29.9, 29.98, 30.04, 30.2, 30.25, 30.32, 30.60, 30.63, 32.5,

33.4, 34.7, 34.8, 37.69, 37.72, 61.9, 62.1, 63.5, 64.3, 67.7, 70.8, 71.0,

71.1, 71.2, 71.3, 73.4, 73.6, 74.0, 79.1, 79.2, 102.2, 174.8, 175.1; ³¹P

NMR (202 MHz, CDCl₃/CD₃OD/D₂O = 70:40:6) δ 3.09; HRMS-

ESI [M − H]⁻calcd for C₅₆H₁₀₄O₂₃P 1175.6711, found 1175.6676.

1-O-Allyl-2-O-(2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopro-

pylsilyl-α-^D-mannopyranosyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-D-

mannopyranosyl)-3,4,5-tri-O-benzyl-^D-myo-inositol (16).

TBSOTf (95 μL, 0.413 mmol) was added to a mixture of alcohol

10^9,28(0.384 g, 0.379 mmol) and dibutylphosphate 15⁵(0.280 g,

0.344 mmol) in dry toluene at 0 °C. After 45 min, the reaction was

quenched with Et₃N, ﬁltered through Celite and the solvent removed

in vacuo. The residue was puriﬁed by column chromatography on silica

gel (EtOAc/petroleum ether =1:9 to 1:4) to aﬀord a mixture of the

triisopropyl and tert-butyldimethyl silylated pseudotrisaccharides

(approximately 2:1 by ¹H NMR) 16 (0.49 g) as a colorless oil.

From the mixture a sample containing mostly the triisopropylsilyl

2-O-(2-O-Methoxyacetyl-3,4,6-tri-O-benzyl-α-^D-mannopyra-

nosyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-3,4,5-

tri-O-benzyl-^D-myo-inositol (13). (1,5-Cyclooctadiene)bis-

(methyldiphenylphosphine)iridium(I) hexaﬂuorophosphate (0.030 g,

0.040 mmol) was added to a stirred solution of 12 (0.300 g, 0.200

mmol) in dry THF at rt under an atmosphere of argon. The argon

atmosphere was replaced with H₂for ca. 1 min, followed by a gentle

stream of argon being passed over the reaction. After 2 h, the solvent

was removed in vacuo and the residue dissolved in acetone/H₂O (9:1,

40 mL). To the solution were added mercury(II) chloride (0.117 g,

0.403 mmol) and mercury(II) oxide (0.111 g, 0.508 mmol), and the

reaction was stirred under reﬂux for 90 min. Once cooled to rt, the

suspension was ﬁltered through Celite and the solvent removed in

vacuo. The residue was dissolved in CH₂Cl₂(50 mL), washed with

10% aq KI (50 mL), dried (MgSO₄), and ﬁltered and the solvent

evaporated. The residue was puriﬁed by column chromatography on

silica gel (EtOAc/petroleum ether = 1:4 to 1:1) to aﬀord the title

1

compound 13 (0.25 g, 85%) as a white foam: H NMR (500 MHz,

CDCl₃) δ 3.30−3.43 (m, 4H), 3.48 (s, 3H), 3.48−3.51 (m, 1H),

3.56−3.64 (m, 3H), 3.79 (brs, 1H), 3.83−4.03 (m, 6H), 4.11−4.23

(m, 5H), 4.35 (d, J = 12.0 Hz, 1H), 4.39 (d, J = 12.0 Hz, 1H), 4.47−

4.50 (m, 3H), 4.55−4.92 (m, 15H), 5.32 (d, J = 1.5 Hz, 1H), 5.38 (brs,

1H), 5.60 (brs, 1H), 7.17−7.40 (m, 50H); ¹³C NMR (125 MHz,

CDCl₃) δ 59.4, 68.6, 69.2, 69.4, 69.7, 71.4, 71.6, 72.0, 72.20, 72.22,

72.23, 72.6, 73.4, 73.5, 74.2, 74.6, 75.0, 75.1, 75.2, 75.4, 75.7, 76.1,

77.4, 77.7, 78.6, 79.5, 80.1, 80.6, 81.3, 96.1, 99.3, 127.46, 127.56,

127.59, 127.60, 127.65, 127.67, 127.69, 127.72, 127.79, 127.84, 128.04,

128.07, 128.09, 128.34, 128.35, 128.37, 128.45, 128.49, 128.53, 128.54,

138.0, 138.1, 138.25, 138.34, 138.5, 138.6, 138.69, 138.73, 169.7;

HRMS-ESI [M + Na]⁺calcd for C₉₁H₉₆NaO₁₈1499.6489, found

1499.6437.

6-O-α-^D-Mannopyranosyl-2-O-(2-O-methoxyacetyl-α-D-man-

nopyranosyl)-1-O-(1-O-((R)-10-methyloctadecanoyl)-2-O-hexa-

decanoyl-sn-glycero-3-phosphoryl)-^D-myo-inositol (14). Alco-

hol 13 (0.047 g, 0.032 mmol) and H-phosphonate 6³⁴(0.065 g, 0.082

mmol) were coevaporated from dry pyridine (3 × 5 mL) and then

placed under high vacuum for 30 min. The reagents were dissolved in

dry pyridine (5 mL) before the addition of pivaloyl chloride (0.035

mL, 0.254 mmol) and stirred for 2 h at rt. Iodine (0.040 g, 0.159

mmol) in pyridine/H₂O (9:1, 5 mL) was added and the reaction

mixture stirred for 1 h before being diluted with CHCl₃(20 mL) and

washed with 10% aq Na₂S₂O₃(20 mL). The aqueous phase was re-

extracted with CHCl₃(2 × 20 mL) and then EtOAc (20 mL), the

combined organic layers were dried (MgSO₄) and ﬁltered, and the

solvent was removed in vacuo. The residue was dissolved in CH₂Cl₂/

MeOH (1:1, 10 mL), treated with Amberlite IRC86 resin (H⁺form)

and ﬁltered, and solvent removed in vacuo, and the residue was

semipuriﬁed by column chromatography on silica gel (CHCl₃/MeOH

= 1:0 to 9:1). Pd(OH)₂/C (20%, 34 mg, 0.048 mmol) was added to

the residue dissolved in THF/MeOH (2:3, 10 mL), and the reaction

mixture was stirred under an atmosphere of H₂. After 4 h, the reaction

was ﬁltered through Celite and the solvent removed in vacuo. The

residue was puriﬁed by column chromatography on silica gel (CHCl₃/

1

protected material was obtained for NMR and HRMS analysis: H

NMR (500 MHz, CDCl₃) δ 1.05−1.08 (m, 18H), 3.22 (dd, J = 2.0,

10.0 Hz, 1H), 3.32−3.38 (m, 3H), 3.48 (dd, J = 3.0, 11.5 Hz, 1H),

3.67 (d, J = 10.5 Hz, 1H), 3.87−4.07 (m, 8H), 4.11−4.17 (m, 3H),

4.20−4.30 (m, 3H), 4.37 (s, 1H), 4.55 (d, J = 11.0 Hz, 1H), 4.65−4.78

(m, 10H), 4.82−4.85 (m, 2H), 4.89−4.98 (m, 6H), 5.08 (d, J = 13.0

Hz, 1H), 5.24 (d, J = 17.0 Hz, 1H), 5.26 (s, 1H), 5.60 (s, 1H), 5.76 (s,

1H), 5.74−5.81 (m, 1H), 7.16−7.49 (m, 47H), 7.59 (t, J = 6.5, 6.5 Hz,

1H), 8.15 (d, J = 8.0 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 12.2,

18.2, 25.8, 26.2, 62.4, 68.8, 69.4, 70.9, 71.1, 71.8, 72.2, 72.4, 72.6, 72.8,

73.4, 74.1, 74.9, 75.0, 75.3, 75.4, 75.9, 76.0, 77.9, 79.2, 80.2, 81.2, 81.7,

81.9, 99.0, 99.1, 117.7, 127.2−128.5, 130.2, 130.5, 133.0, 134.1, 138.2,

138.3, 138.4, 138.6, 138.9, 139.0, 139.1, 139.2, 139.3, 165.4; HRMS-

ESI [M + Na]⁺calcd for C₁₀₀H₁₁₄NaO₁₇Si 1637.7723, found

1637.7739.

6-O-(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-2-O-

(2,3,4-tri-O-benzyl-α-^D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-

myo-inositol (17). Sodium methoxide in MeOH (6 mL, 30%

solution) was added dropwise to a stirred solution of 16 (0.550 g) in

CH₂Cl₂/MeOH (1:2, 6 mL). After 18 h, the reaction mixture was

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diluted with saturated NH₄Cl (30 mL), the aqueous phase extracted

with CHCl₃(2 × 30 mL), dried (MgSO₄), and ﬁltered, and the solvent

removed in vacuo. The residue was puriﬁed by column chromatog-

raphy on silica gel (EtOAc/petroleum ether = 1:9 to 3:7) to aﬀord the

corresponding mixture of TIPS and TBS ether protected alcohols

(0.455 g) as a colorless oil. NaH (60% dispersion in oil, 38 mg, 0.942

mmol) was added to a stirred solution of the residue (0.475 g) and

benzyl bromide (131 μL, 1.10 mmol) in dry DMF (10 mL) at 0 °C

and the resulting suspension allowed to slowly warm to rt over 18 h.

The reaction mixture was partioned between Et₂O and H₂O (1:1, 50

mL) and the aqueous phase extracted into Et₂O (2 × 25 mL). The

combined organic layers were washed with saturated NaCl (100 mL),

dried (MgSO₄), and ﬁltered and the solvent removed in vacuo. The

residue was puriﬁed by column chromatography on silica gel (EtOAc/

petroleum ether = 1:9 to 3:7) to aﬀord the corresponding mixture of

TIPS and TBS ether protected intermediate (0.470 g) as a colorless

oil. (1,5-Cyclooctadiene)bis(methyldiphenylphosphine)iridium(I)

hexaﬂuorophosphate (58 mg, 0.069 mmol) was added to a stirred

solution of the residue (0.550 g) in dry THF at rt under an argon

atmosphere. The argon was replaced with H₂for ca. 2 min followed by

a gentle stream of argon being passed over the reaction. After 2 h, the

solvent was removed in vacuo, and the residue was dissolved in

MeOH/CH₂Cl₂(1:4, 20 mL). Acetyl chloride (250 μL) was added,

and the reaction was stirred for 18 h. Solid NaHCO₃was added to

quench the reaction, and then the resulting mixture partitioned

between CHCl₃and H₂O (1:1, 50 mL). The aqueous layer was

extracted with CHCl₃(2 × 30 mL), the combined organic fractions

were dried (MgSO₄) and ﬁltered, and the solvent was removed in

vacuo. The residue was puriﬁed by column chromatography on silica

gel (EtOAc/petroleum ether = 1:9 to 1:1) to aﬀord the title compound

17 (0.45 g, 93%) as a white solid: ¹H NMR (500 MHz, CDCl₃) δ 1.69

(brs, 1H), 3.24−3.29 (m, 2H), 3.40 (brs, 1H), 3.44−3.48 (m, 1H),

3.55−3.61 (m, 4H), 3.76−3.99 (m, 9H), 4.07−4.13 (m, 2H), 4.36−

4.41 (m, 2H), 4.46−4.67 (m, 12H), 4.69−4.76 (m, 3H), 4.81−4.89

(m, 3H), 5.26 (s, 1H), 5.28 (s, 1H), 7.15−7.37 (m, 50H); ¹³C NMR

(125 MHz, CDCl₃) δ 62.3, 69.6, 71.4, 72.0, 72.1, 72.3, 72.6, 73.4, 74.6,

74.86, 74.91, 75.1, 75.2, 75.3, 75.6, 76.6, 78.5, 79.4, 80.0, 80.3, 81.2,

95.7 (¹J_C,H= 168.5 Hz), 99.8 (¹J_C,H= 173.2 Hz), 127.47, 127.53, 127.6,

127.7, 127.8, 127.9, 128.0, 128.1, 128.2, 128.27, 128.31, 128.35,

128.39, 128.43, 128.45; HRMS-ESI [M + Na]⁺calcd for C₈₈H₉₂NaO₁₆

1427.6283, found 1427.6290.

puriﬁed by column chromatography on silica gel (EtOAc/petroleum

ether = 1:4 to 2:3) followed by further puriﬁcation on silica gel

(EtOAc/CHCl₃= 0:1 to 3:97) to aﬀord the title compound 19 (0.090

g, 88%) as a colorless oil: ¹H NMR (500 MHz, CDCl₃) δ 0.83 (d, J =

6.5 Hz, 6H), 0.88 (t, J = 7 Hz, 18H), 1.03−1.39 (m, 150H), 1.45−1.59

(m, 12H), 2.12−2.22 (m, 12H), 3.24−3.33 (m, 5H), 3.36−3.40 (m,

3H), 3.76−3.81 (m, 2H), 3.88−4.20 (m, 35H), 4.43−4.45 (m, 10H),

4.57−4.67 (m, 15H), 4.72−4.84 (m, 8H), 4.88−4.91 (m, 6H), 4.94−

5.07 (m, 5H), 5.11−5.15 (m, 1H), 5.27 (d, J = 1.5 Hz, 1H), 5.32 (d, J

= 1.5 Hz, 1H), 5.48 (d, J = 1.5 Hz, 1H), 5.49 (d, J = 1.5 Hz, 1H),

7.01−7.37 (m, 110H); ¹³C NMR (125 MHz, CDCl₃) δ 14.1, 19.7,

22.7, 24.8, 27.1, 29.16, 29.23, 29.3, 29.4, 29.5, 29.6, 29.7, 30.1, 31.9,

32.8, 33.88, 33.93, 34.0, 34.2, 37.1, 61.4, 61.5, 66.1, 68.7, 69.4, 70.1,

70.2, 70.5, 71.6, 71.7, 71.8, 72.0, 72.3, 72.5, 73.1, 74.0, 74.3, 74.5, 74.7,

74.8, 74.9, 75.1, 75.6, 75.7, 76.2, 78.1, 78.3, 78.8, 80.0, 80.4, 80.9, 81.0,

98.5, 98.6, 99.3, 127.0, 127.2, 127.4, 127.5, 127.7, 127.8, 127.9, 128.0,

128.11, 128.14,128.2, 128.3, 128.4, 128.5, 128.8, 128.87, 128.92, 129.0,

135.1, 137.5, 137.65, 137.74, 138.16, 138.22, 128.3, 138.5, 138.7,

138.9, 139.0, 139.1, 172.5, 172.7, 173.0, 173.5; ³¹P NMR (202 MHz,

CDCl₃) δ −0.16, −0.29; HRMS-ESI [M + Na]⁺calcd for

C₁₄₉H₂₀₁NaO₂₄P 2428.4143, found 2428.4148.

6-O-α-^D-Mannopyranosyl-1-O-(1-O-((R)-10-methyloctadeca-

noyl)-2-O-hexadecanoyl-sn-glycero-3-phosphoryl)-2-O-(6-O-

hexadecanoyl-α-^D-mannopyranosyl)-D-myo-inositol (2a).³⁴Pd-

(OH)₂/C (20%, 37 mg, 0.053 mmol) was added to a solution of 19

(0.085 g, 0.035 mmol) in THF/MeOH (2:3, 10 mL). The reaction

mixture was stirred under an atmosphere of H₂for 4 h at rt and then

ﬁltered through Celite and the solvent removed in vacuo. The residue

was puriﬁed by column chromatography on silica gel (CHCl₃/MeOH/

H₂O = 70:40:1 to 70:40:6) to aﬀord the title compound 2a (0.045 g,

90%, 98% pure by HPLC) as a white solid: [α]²_D²+39.0 (c = 0.12,

CHCl₃/MeOH/H₂O = 70:40:6); ¹H NMR (500 MHz, CDCl₃/

CD₃OD/D₂O = 70:40:6) δ 0.85 (d, J = 6.5 Hz, 3H), 0.89 (t, J = 7.0

Hz, 9H) 1.07−1.43 (m, 75H), 1.56−1.64 (m, 6H), 2.30−2.39 (m,

6H), 3.29 (t, J = 9.0 Hz, 1H), 3.46 (dd, J = 2.5, 10.5 Hz, 1H), 3.57−

3.69 (m, 3H), 3.73−3.83 (m, 5H), 3.96−4.45 (m, 12H), 5.09 (s, 1H),

5.12 (s, 1H), 5.24−5.29 (m, 1H); ¹³C NMR (125 MHz, CDCl₃/

CD₃OD/D₂O = 70:40:6) δ 13.8, 19.5, 22.6, 24.7, 24.8, 24.9, 26.98,

27.03, 29.1, 29.2, 29.3, 29.4, 29.6, 29.9, 31.8, 32.7, 33.9, 34.0, 34.1,

37.0, 61.1, 62.9, 63.4, 63.6, 66.9, 67.1, 70.0, 70.1, 70.3, 70.5, 70.6, 70.7,

72.8, 73.0, 73.3, 76.8, 78.5, 78.7, 101.5, 101.6, 173.9, 174.2, 175.0; ³¹P

NMR (202 MHz, CDCl₃/CD₃OD/D₂O = 70:40:6) δ −0.5; HRMS-

ESI [M − H]⁻calcd for C₇₂H₁₃₄O₂₄P 1413.9003, found 1413.9014.

( )-3-O-(4-Bromobenzyl)-1,2:4,5-di-O-isopropylidene-myo-

inositol (21). To a solution of diol 20 (2.01 g, 7.71 mmol) in dry

THF (100 mL) were added NaH (60% dispersion in oil, 0.383 g,

15.97 mmol) and 4-bromobenzyl bromide (2.15 g, 8.60 mmol), and

the mixture was reﬂuxed overnight under an atmosphere of nitrogen.

The mixture was quenched with H₂O, extracted into EtOAc (3 × 100

mL), and washed with H₂O (150 mL) and saturated NaCl (150 mL).

The organic extract was dried (MgSO₄) and ﬁltered and the solvent

removed in vacuo. The residue was puriﬁed by column chromatog-

raphy on silica gel (EtOAc/petroleum ether = 1:2 to 1:1) to give the

title compound 21 (2.14 g, 65%, R_f= 0.39, EtOAc/petroleum ether 1:1)

and ( )-3,6-di-O-(4-bromobenzyl)-1,2:4,5-di-O-isopropylidene-myo-

inositol (0.709 g, 15%, R_f= 0.88, EtOAc/petroleum ether 1:1), both

as white solids. Data for 21: mp 139−142 °C (EtOAc/PE); ν_max

(ATR-IR) 3474, 2982, 2936, 2884, 1487, 1466, 1403, 1382, 1374,

1344, 1237, 1217, 1161, 1137, 1112, 1083, 1062, 1046 cm⁻¹; ¹H NMR

(500 MHz, CDCl₃) δ 1.35 (s, 3H), 1.45 (s, 3H), 1.48 (s, 3H), 1.55 (s,

3H), 2.40 (d, J = 2.9 Hz, 1H), 3.26 (dd, J = 9.4, 10.6 Hz, 1H), 3.77

(dd, J = 4.3, 10.1 Hz, 1H), 3.89 (dd, J = 6.7, 10.6 Hz, 1H), 3.96 (dd, J

= 4.9, 6.6 Hz, 1H), 4.03 (t, J = 9.7 Hz, 1H), 4.34 (t, J = 4.5 Hz, 1H),

4.75 (d, J = 12.6 Hz, 1H), 4.83 (d, J = 12.6 Hz, 1H), 7.28−7.31 (m,

2H), 7.46−7.49 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 26.0, 27.01,

27.03, 28.3, 71.2, 74.5, 74.7, 76.6, 77.3, 78.4, 81.7, 110.4, 112.6, 121.9,

130.0, 131.6, 136.9; HRMS-ESI [M + Na]⁺calcd for C₁₉H₂₅⁷⁹BrNaO₆

451.0727, found 451.0705.

6-O-(2,3,4,6-Tetra-O-benzyl-α-^D-mannopyranosyl)-2-O-

(2,3,4-tri-O-benzyl-6-O-hexadecanoyl-α-^D-mannopyranosyl)-

3,4,5-tri-O-benzyl-^D-myo-inositol (18).³⁴Palmitoyl chloride (13

μL, 0.043 mmol) was added to a stirred solution of diol 17 (0.050 g,

0.036 mmol) in dry pyridine/CH₂Cl₂(4:1, 2.5 mL) and 4 Å sieves.

The reaction was stirred at 30 °C for 2 h, diluted with CH₂Cl₂(30

mL), washed with 0.5 M HCl (20 mL), dried (MgSO₄), and ﬁltered

and the solvent removed in vacuo. The residue was puriﬁed by column

chromatography on silica gel (EtOAc/petroleum ether = 1:4 to 1:3) to

aﬀord the title compound 18 (0.050 g, 85%) as a colorless oil. ¹H and

¹³C NMR (500 MHz, CDCl₃) data were consistent to those in the

literature.³⁴

1-O-(1-O-((R)-10-Methyloctadecanoyl)-2-O-hexadecanoyl-

sn-glycero-3-benzylphosphoryl)-6-O-(2,3,4,6-tetra-O-benzyl-

α-^D-mannopyranosyl)-2-O-(2,3,4-tri-O-benzyl-6-O-hexadeca-

noyl-α-^D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol

(19). Alcohol 18 (0.07 g, 0.043 mmol) and phosphoramidite 5 (0.150

g, 0.177 mmol) were coevaporated from dry acetonitrile (2 × 30 mL)

and then placed under high vacuum for 30 min. The reagents were

dissolved in dry acetonitrile (15 mL) before the addition of 1H-

tetrazole (13 mg, 0.192 mmol) at 0 °C and stirred for 2 h at rt. Dry

CH₂Cl₂(10 mL) was added, the solution was cooled to −30 °C, and a

dried (MgSO₄) solution of m-CPBA (∼50%, 0.066 g, 0.192 mmol) in

CH₂Cl₂(3 mL) was added to the reaction. After being warmed to rt

over 1 h, the reaction was diluted with Et₂O/H₂O (1:1, 60 mL) and

washed with saturated NaHCO₃(30 mL) and then 10% aq Na₂SO₃

(30 mL). The combined aqueous phases were re-extracted with Et₂O

(2 × 30 mL), the combined organic layers were dried (MgSO₄) and

ﬁltered, and the solvent was removed in vacuo. The crude residue was

Data for ( )-3,6-di-O-(4-bromobenzyl)-1,2:4,5-di-O-isopropyli-

dene-myo-inositol: mp 146−147 °C (EtOAc/PE); ν_max(ATR-IR)

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2985, 2931, 2896, 2880, 1487, 1463, 1402, 1368, 1352, 1339, 1240,

1217, 1159, 1132, 1089, 1067, 1053 cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ 1.34 (s, 3H), 1.41 (s, 3H), 1.45 (s, 3H), 1.47 (s, 3H), 3.33

(dd, J = 9.4, 10.5 Hz, 1H), 3.63 (dd, J = 6.5, 10.6 Hz, 1H), 3.73 (dd, J

= 4.2, 10.1 Hz, 1H), 3.99 (t, J = 9.8 Hz, 1H), 4.06 (dd, J = 5.1, 6.4 Hz,

1H), 4.33 (t, J = 4.6 Hz, 1H), 4.74 (d, J = 13.8 Hz, 3H), 4.82 (d, J =

12.7 Hz, 1H), 7.25−7.30 (m, 4H), 7.43−7.49 (m, 4H); ¹³C NMR

(125 MHz, CDCl₃) δ 26.0, 27.08, 27.12, 28.0, 71.15, 71.29, 74.6, 76.7,

77.1, 78.8, 80.2, 81.1, 110.1, 112.3, 121.4, 121.9, 129.56, 129.99,

131.36, 131.58, 137.0, 137.4; HRMS-ESI [M + Na]⁺calcd for

C₂₆H₃₀⁷⁹Br₂NaO₆619.0301, found 619.0293.

Anal. Calcd for C₄₆H₅₃BrO₁₁: C, 64.11; H, 6.20; Br, 9.27. Found: C,

63.84; H, 6.23; Br, 9.31.

3-O-(4-Bromobenzyl)-1,2:4,5-di-O-isopropylidene-^D-myo-in-

ositol (D-21). To a solution of diol ^D-20^47,48(0.315 g, 1.21 mmol) in

dry THF (60 mL) were added NaH (60% dispersion in oil, 0.087 g,

3.63 mmol) and 4-bromobenzyl bromide (0.333 g, 1.33 mmol), and

the mixture was reﬂuxed overnight under an atmosphere of nitrogen.

The reaction mixture was quenched with H₂O and extracted into

EtOAc (3 × 50 mL) and washed with H₂O (20 mL) and brine (20

mL). The organic extract was dried (MgSO₄) and ﬁltered and the

solvent removed in vacuo. The residue was puriﬁed by column

chromatography on silica gel (EtOAc/petroleum ether = 1:2) to give

the title compound ^D-21 (0.812 g, 67%) as a white solid: [α]²_D⁷−37.0 (c

= 0.95, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 1.36 (s, 3H), 1.45 (s,

3H), 1.48 (s, 3H), 1.55 (s, 3H), 2.40 (d, J = 2.8 Hz, 1H), 3.27 (dd, J =

9.4, 10.4 Hz, 1H), 3.77 (dd, J = 4.3, 10.1 Hz, 1H), 3.88 (dd, J = 6.7,

10.6 Hz, 1H), 3.96 (dd, J = 4.9, 6.6 Hz, 1H), 4.03 (t, J = 9.7 Hz, 1H),

4.35 (t, J = 4.5 Hz, 1H), 4.75 (d, J = 12.6 Hz, 1H), 4.84 (d, J = 12.6

Hz, 1H), 7.28−7.31 (m, 2H), 7.46−7.49 (m, 2H); HRMS-ESI [M +

3-O-(4-Bromobenzyl)-6-O-(3,4,6-tri-O-benzyl-α-^D-manno-

pyranosyl)-1,2:4,5-di-O-isopropylidene-^D-myo-inositol (23)

and 3-O-(4-Bromobenzyl)-6-O-(3,4,6-tri-O-benzyl-α-^D-manno-

pyranosyl)-1,2:4,5-di-O-isopropylidene-^L-myo-inositol (24).

TMSOTf (119 μL, 0.658 mmol) was added to a mixture of alcohol

21 (2.83 g, 6.58 mmol), trichloroacetimidate 22 (5.03 g, 7.90 mmol),

and 4 Å molecular sieves in dry CH₂Cl₂(20 mL) at −30 °C. After

being stirred for 30 min, the reaction was quenched with Et₃N, ﬁltered

through Celite, and evaporated to dryness. The residue was puriﬁed by

column chromatography on silica gel (EtOAc/petroleum ether = 1:9

to 1:2) to give a mixture of diastereoisomers 6-O-(2-O-acetyl-3,4,6-tri-

O-benzyl-α-^D-mannopyranosyl)-3-O-(4-bromobenzyl)-1,2:4,5-di-O-

isopropylidene-^D-myo-inositol and 6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-

α-^D-mannopyranosyl)-3-O-(4-bromobenzyl)-1,2:4,5-di-O-isopropyli-

dene-^L-myo-inositol (5.81 g). Sodium methoxide (prepared from

reacting Na (0.192 g, 8.36 mmol) with MeOH (10 mL)) was added

dropwise to a stirred solution of the diastereoisomers (5.81 g, 6.43

mmol) in MeOH (25 mL) until the pH of the solution remained

greater than 11. The reaction mixture was stirred overnight then

extracted into EtOAc (3 × 100 mL), and the combined organic

extracts were washed with H₂O (100 mL). The solution was dried

(MgSO₄) and ﬁltered, and the solvent was removed in vacuo. The

residue was puriﬁed by column chromatography on silica gel (EtOAc/

petroleum ether = 1:9 to 1:2) to give the title compounds 23 (2.39 g,

42%, two steps, R_f= 0.31, EtOAc/petroleum ether 2:3) and 24 (2.32 g,

41%, two steps, R_f= 0.52, EtOAc/petroleum ether 2:3) both as white

foams. Data for 23: [α]_D²⁹+2.9 (c = 1.06, CHCl₃); ν_max(ATR-IR)

3495, 3063, 3029, 2986, 2932, 1488, 1454, 1373, 1219, 1156, 1116,

1086, 1067, 1045 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.33 (s, 3H),

1.38 (s, 6H), 1.56 (s, 3H), 2.49 (brs, 1H), 3.20 (dd, J = 9.4, 10.7 Hz,

1H), 3.66 (dd, J = 1.9, 10.8 Hz, 1H), 3.70 (dd, J = 4.2, 10.2 Hz, 1H),

3.80 (dd, J = 3.7, 10.8 Hz, 1H), 3.88 (dd, J = 3.3, 9.3 Hz, 1H), 3.92−

4.06 (m, 5H), 4.11 (dd, J = 1.4, 3.0 Hz, 1H), 4.28 (t, J = 4.5 Hz, 1H),

4.51 (d, J = 12.2 Hz, 1H), 4.54 (d, J = 10.6 Hz, 1H), 4.67−4.75 (m,

4H), 4.81, 4.82 (2 × overlapping d, J = 12.7, 10.7 Hz, 2H), 5.33 (d, J =

1.4 Hz, 1H), 7.18−7.20 (m, 2H), 7.25−7.37 (m, 15H), 7.46−7.49 (m,

2H); ¹³C NMR (125 MHz, CDCl₃) δ 26.1, 26.98, 27.16, 28.2, 68.6,

70.7, 71.1, 72.0, 73.4, 74.5, 75.4, 76.6, 76.9, 77.4, 79.9, 80.2, 81.6, 98.9,

110.3, 112.2, 121.8, 127.57, 127.76, 127.79, 127.90, 127.91, 128.22,

128.34, 128.43, 128.57, 130.0, 131.6, 137.0, 138.13, 138.42, 138.43;

HRMS-ESI [M + Na]⁺calcd for C₄₆H₅₃⁷⁹BrNaO₁₁883.2663, found

883.2637. Anal. Calcd for C₄₆H₅₃BrO₁₁: C, 64.11; H, 6.20; Br, 9.27.

Found: C, 63.84; H, 6.27; Br, 9.24.

Na]⁺calcd for C₁₉H₂₅⁷⁹BrNaO₆451.0727, found 451.0722. H NMR

1

and all other analytical data, excluding the optical rotation, were

consistent with ( )-3-O-(4-bromobenzyl)-1,2:4,5-di-O-isopropyli-

dene-myo-inositol 21.

3-O-(4-Bromobenzyl)-6-O-(3,4,6-tri-O-benzyl-α-^D-manno-

pyranosyl)-1,2:4,5-di-O-isopropylidene-^D-myo-inositol (D-23).

TMSOTf (19 μL, 0.103 mmol) was added to a mixture of alcohol

D-21 (0.440 g, 1.03 mmol), trichloroacetimidate 22⁵¹(0.784 g, 1.23

mmol), and 4 Å molecular sieves in dry CH₂Cl₂(15 mL) at −30 °C.

After being stirred for 30 min, the reaction was quenched with Et₃N,

ﬁltered through Celite, and evaporated to dryness. The residue was

puriﬁed by column chromatography on silica gel (EtOAc/petroleum

ether = 1:4 to 1:2) to give pseudodisaccharide 6-O-(2-O-acetyl-3,4,6-

tri-O-benzyl-α-^D-mannopyranosyl)-3-O-(4-bromobenzyl)-1,2:4,5-di-O-

isopropylidene-^D-myo-inositol (0.882 g) as a white foam. Sodium

methoxide (prepared from reacting Na (0.069 g, 1.27 mmol) with

MeOH (5 mL)) was added dropwise to a stirred solution of the

pseudodisaccharide (0.882 g, 0.975 mmol) in MeOH (5 mL) until the

pH of the solution remained greater than 11. The reaction mixture was

then allowed to stir overnight. The reaction mixture was extracted into

EtOAc (3 × 50 mL), and the combined organic extracts were washed

with H₂O (50 mL). The solution was dried (MgSO₄) and ﬁltered, and

the solvent was removed in vacuo. The residue was puriﬁed by column

chromatography on silica gel (EtOAc/petroleum ether = 1:2) to give

the title compound ^D-23 (0.819 g, 80%) as a white foam. ¹H NMR and

all other analytical data were consistent with those of 23, prepared

from ( )-21.

3-O-(4-Bromobenzyl)-4,5-di-O-benzyl-6-O-(2,3,4,6-tetra-O-

benzyl-α-^D-mannopyranosyl)-1,2-di-O-isopropylidene-D-myo-

inositol (25). To a solution of 23 (2.11 g, 2.45 mmol) in CH₂Cl₂(20

mL) were added H₂O (44 μL, 2.45 mmol) and TFA (0.377 mL, 4.89

mmol) at 0 °C. The solution was stirred for 2.5 h, diluted with CH₂Cl₂

(40 mL), and washed with saturated NaHCO₃(20 mL) and saturated

NaCl (20 mL). The organic phase was dried (MgSO₄) and ﬁltered,

and the solvent was evaporated under reduced pressure to give 3-O-(4-

bromobenzyl)-6-O-(3,4,6-tri-O-benzyl-α-^D-mannopyranosyl)-1,2-di-O-

isopropylidene-^D-myo-inositol (2.01 g) as a white foam which was used

without further puriﬁcation: HRMS-ESI [M + Na]⁺calcd for

C₄₃H₄₉BrO₁₁Na 843.2350, found 843.2328. To a solution of crude

triol (2.01 g, 2.45 mmol) in dry DMF (20 mL) were added NaH (60%

dispersion in oil, 0.528 g, 22 mmol) and benzyl bromide (2.61 mL, 22

mmol) at 0 °C, and the reaction mixture was stirred overnight. The

reaction was quenched by careful addition of H₂O (5 mL) and then

extracted into EtOAc (3 × 100 mL). The combined organic layers

were washed with H₂O (100 mL) and saturated NaCl (100 mL), dried

(MgSO₄), and ﬁltered, and the solvent was removed in vacuo. The

residue was puriﬁed by column chromatography on silica gel (EtOAc/

petroleum ether = 1:9 to 1:2) to give the title compound 25 (2.12 g,

79%, two steps) as a white foam: [α]²_D⁹+9.3 (c = 1.00, CHCl₃); ν_max

(ATR-IR) 3062, 3029, 2984, 2865, 1496, 1453, 1362, 1242, 1201,

1089, 1070, 1041, 1026 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 1.35 (s,

Data for 24: [α]³_D⁰+79.5 (c = 1.04, CHCl₃); ν_max(ATR-IR) 3486,

3062, 3029, 2985, 2934, 2896, 1488, 1454, 1403, 1373, 1315, 1219,

1

1156, 1123, 1069, 1047, 1027 cm⁻¹; H NMR (500 MHz, CDCl₃) δ

1.29 (s, 3H), 1.41 (s, 3H), 1.43 (s, 3H), 1.47 (s, 3H), 2.49 (brs, 1H),

3.24 (dd, J = 9.5, 10.5 Hz, 1H), 3.65 (dd, J = 1.9, 10.6 Hz, 1H), 3.69

(dd, J = 4.2, 10.1 Hz, 1H), 3.84 (dd, J = 2.9, 10.6 Hz, 1H), 3.88−3.92

(m, 2H), 3.95−4.02 (m, 3H), 4.06 (dt, J = 2.3, 10.1 Hz, 1H), 4.10 (dd,

J = 1.7, 3.2 Hz, 1H), 4.29 (t, J = 4.5 Hz, 1H), 4.49 (d, J = 12.1 Hz,

1H), 4.51 (d, J = 10.8 Hz, 1H), 4.66−4.75 (m, 4H), 4.82, 4.83 (2 ×

overlapping d, J = 12.7, 10.7 Hz, 2H), 5.33 (d, J = 1.5 Hz, 1H), 7.17−

7.19 (m, 2H), 7.24−7.39 (m, 15H), 7.46−7.49 (m, 2H); ¹³C NMR

(125 MHz, CDCl₃) δ 25.9, 27.08, 27.09, 28.2, 68.4, 68.6, 70.8, 71.1,

71.9, 73.6, 74.2, 74.5, 75.3, 76.5, 76.8, 77.2, 79.0, 79.6, 80.1, 98.1,

110.0, 112.6, 121.8, 127.64, 127.68, 127.77, 127.88, 128.01, 128.07,

128.4, 128.6, 130.0, 131.6, 137.0, 138.12, 138.25, 138.6; HRMS-ESI

[M + Na]⁺calcd for C₄₆H₅₃⁷⁹BrNaO₁₁883.2663, found 883.2646.

K

dx.doi.org/10.1021/jo301189y | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Featured Article

3H), 1.53 (s, 3H), 3.27 (dd, J = 7.8, 9.6 Hz, 1H), 3.51 (dd, J = 1.7,

11.1 Hz, 1H), 3.62−3.65 (m, 2H), 3.80 (dd, J = 1.9, 3.1 Hz, 1H), 3.85

(t, J = 8.0 Hz, 1H), 3.88 (dd, J = 3.2, 9.5 Hz, 1H), 3.95−3.99 (m, 2H),

4.05−4.10 (m, 2H), 4.26 (dd, J = 3.8, 5.8 Hz, 1H), 4.35 (d, J = 12.2

Hz, 1H), 4.51 (d, J = 10.9 Hz, 1H), 4.57 (d, J = 10.9 Hz, 4H), 4.62 (d,

J = 12.2 Hz, 1H), 4.67−4.71 (m, 4H), 4.74 (d, J = 12.5 Hz, 1H), 4.78

(d, J = 12.5 Hz, 1H), 4.89 (d, J = 10.9 Hz, 1H), 5.49 (d, J = 1.7 Hz,

1H), 7.02−7.06 (m, 2H), 7.12−7.16 (m, 3H), 7.20−7.32 (m, 25H),

7.36−7.44 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 25.8, 27.6, 68.9,

71.8, 71.9, 72.3, 72.4, 73.1, 74.49, 74.55, 74.84, 74.90, 75.0, 75.3, 76.8,

77.3, 79.2, 79.6, 80.9, 81.1, 96.3, 110.2, 121.7, 127.3, 127.49, 127.56,

127.71, 127.73, 127.83, 127.87, 127.89, 128.06, 128.17, 128.21, 128.29,

128.30, 128.31, 128.41, 128.44, 129.6, 131.5, 137.3, 137.9, 138.37,

138.45, 138.56, 138.58, 139.0; HRMS-ESI [M + Na]⁺calcd for

C₆₄H₆₇⁷⁹BrNaO₁₁1113.3759, found 1113.3707.

3-O-(4-Bromobenzyl)-4,5-di-O-benzyl-6-O-(2,3,4,6-tetra-O-

benzyl-α-^D-mannopyranosyl)-D-myo-inositol (26). To a solution

of 25 (4.054 g, 3.71 mmol) in CH₂Cl₂(100 mL) were added MeOH

(0.230 mL, 5.57 mmol) and TFA (1.43 mL, 18.56 mmol), and the

reaction mixture was stirred at rt overnight. The reaction mixture was

diluted with CH₂Cl₂(50 mL) and washed with saturated NaHCO₃

(50 mL) and saturated NaCl (50 mL). The organic phase was dried

(MgSO₄) and ﬁltered, and the solvent was evaporated under reduced

pressure. The residue was puriﬁed by column chromatography on

silica gel (EtOAc/petroleum ether = 1:2 to 1:1) to give the title

compound 26 (2.12 g, 79%) as a white foam: [α]³_D¹+24.4 (c = 1.03,

CHCl₃); ν_max(ATR-IR) 3434, 3087, 3062, 3029, 2864, 1592, 1496,

1453, 1360, 1208, 1070, 1048, 1027 cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ 2.55 (brs, 1H), 2.99 (d, J = 8.6 Hz, 1H), 3.28 (t, J = 9.4 Hz,

1H), 3.38 (dd, J = 2.8, 9.5 Hz, 1H), 3.48−3.55 (m, 3H), 3.84−3.86

(m, 1H), 3.88−3.92 (m, 2H), 3.95 (t, J = 9.2 Hz, 1H), 4.03 (t, J = 9.5

Hz, 1H), 4.02−4.07 (m, 2H), 4.35 (d, J = 12.1 Hz, 1H), 4.46 (d, J =

11.1 Hz, 1H), 4.55−4.63 (m, 6H), 4.70−4.79 (m, 5H), 4.84 (d, J =

11.1 Hz, 1H), 5.51 (d, J = 2.0 Hz, 1H), 7.09−7.14 (m, 3H), 7.14−7.19

(m, 3H), 7.22−7.31 (m, 24H), 7.35−7.38 (m, 2H), 7.41−7.44 (m,

2H); ¹³C NMR (125 MHz, CDCl₃) δ 69.2, 70.3, 71.89, 71.92, 71.95,

72.31, 72.33, 73.2, 74.78, 74.81, 74.9, 75.6, 75.8, 78.0, 79.4, 79.8, 80.7,

81.1, 97.0, 121.9, 127.42, 127.45, 127.47, 127.54, 127.64, 127.69,

127.7, 127.8, 127.85, 127.87, 127.88, 128.0, 128.22, 128.25, 128.26,

128.34, 128.37, 128.43, 129.5, 131.7, 136.8, 138.29, 138.31, 138.36,

138.53, 138.55, 138.8; HRMS-ESI [M + Na]⁺calcd for

C₆₁H₆₃⁷⁹BrNaO₁₁1073.3446, found 1073.3400.

7.19 (m, 7H), 7.20−7.30 (m, 23H), 7.37 (d, J = 8.1 Hz, 4H), 7.50−

7.53 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 56.02, 56.05, 69.1,

70.3, 71.9, 72.3, 72.52, 72.55, 73.2, 74.8, 74.9, 75.6, 75.8, 77.8, 79.5,

79.8, 80.7, 81.2, 97.0, 110.5, 111.6, 119.4, 127.0, 127.38, 127.41,

127.43, 127.5, 127.61, 127.63, 127.69, 127.85, 127.88, 127.9, 128.0,

128.20, 128.23, 128.24, 128.32, 128.34, 128.40, 128.44, 133.8, 136.4,

138.31, 138.33, 138.38, 138.5, 138.7, 138.8, 140.8, 148.8, 149.2;

HRMS-ESI [M + Na]⁺calcd for C₆₉H₇₂NaO₁₃1131.4865, found

1131.4854. Anal. Calcd for C₆₉H₇₂O₁₃·H₂O: C, 73.51; H, 6.62. Found:

C, 73.59; H, 6.74.

1-O-Allyl-4,5-di-O-benzyl-3-O-(4-(3,4-dimethoxyphenyl)-

benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-D-

myo-inositol (28). Diol 27 (1.72 g, 1.55 mmol) and dibutyltin oxide

(0.463, 1.86 mmol) were dried together under high vacuum for 30 min

and then ﬂushed with argon. To this was added anhydrous toluene (30

mL), and the reaction mixture was heated at reﬂux for 2 h during

which time the suspension changed to a clear yellow solution. Once

cooled to rt, the solvent was removed in vacuo to give a yellow foam.

Cesium ﬂuoride (0.353 g, 2.34 mmol) was added and the mixture

dried under high vacuum together for 30 min and then ﬂushed with

argon. Anhydrous DMF (20 mL) was added, followed by allyl bromide

(0.201 mL, 2.34 mmol), and the reaction mixture was heated at 50 °C

overnight. After cooling, the reaction mixture was diluted with CH₂Cl₂

(200 mL) and partitioned with H₂O (100 mL). The aqueous layer was

re-extracted with CH₂Cl₂(2 × 50 mL), the combined organic layers

were washed with saturated NaCl (100 mL), dried (MgSO₄), and

ﬁltered, and the solvent was removed in vacuo. The residue was

puriﬁed by column chromatography on silica gel (EtOAc/petroleum

ether = 1:4 to 2:3) to give the title compound 28 (1.30 g, 73%, R_f=

0.71, EtOAc/petroleum ether 1:1) and 2-O-allyl-4,5-di-O-benzyl-3-O-

(4-(3,4-dimethoxyphenyl)benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-

mannopyranosyl)-^D-myo-inositol (0.128 g, 7%, R_f= 0.82, EtOAc/

petroleum ether 1:1), both as white foams. Data for 28: [α]²_D⁶+14.2 (c

= 1.00, CHCl₃); ν_max(ATR-IR) 3488, 3062, 3029, 2865, 1604, 1588,

1526, 1501, 1453, 1399, 1360, 1327, 1250, 1216, 1171, 1050, 1025

cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 2.43 (brs, 1H), 3.22 (dd, J = 2.5,

9.6 Hz, 1H), 3.28 (t, J = 9.5 Hz, 1H), 3.36 (dd, J = 1.6, 11.3 Hz, 1H),

3.40−3.45 (m, 2H), 3.84 (dd, J = 2.0, 2.8 Hz, 1H), 3.88−4.07 (m,

5H), 3.93 (s, 3H), 3.96 (s, 3H), 4.10 (t, J = 9.7 Hz, 1H), 4.15 (t, J =

9.6 Hz, 1H), 4.22 (t, J = 2.6 Hz, 1H), 4.27 (d, J = 12.0 Hz, 1H), 4.48

(d, J = 11.0 Hz, 1H), 4.62 (d, J = 12.1 Hz, 1H), 4.64−4.78 (m, 7H),

4.81 (d, J = 10.6 Hz, 1H), 4.85 (d, J = 11.1 Hz, 2H), 4.93 (d, J = 10.6

Hz, 1H), 5.15 (dq, J = 10.4, 1.3 Hz, 1H), 5.25 (dq, J = 17.2, 1.6 Hz,

1H), 5.53 (d, J = 1.8 Hz, 1H), 5.79 (dddd, J = 5.7, 5.7, 10.2, 17.2 Hz,

1H), 6.96 (d, J = 8.3 Hz, 1H), 7.05−7.17 (m, 7H), 7.17−7.32 (m,

21H), 7.34−7.43 (m, 6H), 7.50−7.54 (m, 2H); ¹³C NMR (125 MHz,

CDCl₃) δ 56.02, 56.05, 66.5, 68.7, 70.7, 71.9, 72.11, 72.16, 72.4, 73.2,

74.9, 75.22, 75.26, 75.8, 76.0, 79.8, 80.0, 80.5, 81.4, 81.5, 98.4, 110.4,

111.6, 117.9, 119.4, 127.0, 127.2, 127.3, 127.37, 127.44, 127.60,

127.61, 127.8, 127.87, 127.91, 128.0, 128.10, 128.14, 128.17, 128.24,

128.3, 128.4, 128.44, 133.8, 134.3, 136.5, 138.2, 138.6, 138.70, 138.71,

138.8, 139.1, 140.7, 148.8, 149.3; HRMS-ESI [M + Na]⁺calcd for

C₇₂H₇₆NaO₁₃1171.5178, found 1171.5082. Anal. Calcd for

C₇₂H₇₆O₁₃.H₂O: C, 74.08; H, 6.73. Found: C, 74.52; H, 6.65.

4,5-Di-O-benzyl-3-O-(4-(3,4-dimethoxyphenyl)benzyl)-6-O-

(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-D-myo-inositol

(27). To an oven-dried Schlenk ﬂask were added diol 26 (0.934 g, 0.89

mmol), 3,4-dimethoxyphenylboronic acid (0.194 g, 1.07 mmol),

tetrabutylammonium bromide (0.029 g, 0.09 mmol), potassium

phosphate (K₃PO₄) (0.565 g, 2.66 mmol), and EtOH (25 mL). The

resulting mixture was subjected to the freeze−pump−thaw cycle three

times to exclude air. Pd(OAc)₂(10 mg, 0.045 mmol) was then added

under a ﬂow of argon, and the reaction mixture was stirred at rt for 3 h.

Once the reaction was complete, the solution was ﬁltered through a

plug of Celite and washed with EtOAc (100 mL). The reaction

mixture was washed with saturated aq NaHCO₃(100 mL), and the

aqueous layer was back-extracted with EtOAc (3 × 50 mL). The

combined organic layers were washed with H₂O (50 mL) and

saturated NaCl (50 mL), dried (MgSO₄), and ﬁltered, and the solvent

was removed in vacuo. The residue was puriﬁed by column

chromatography on silica gel (EtOAc/petroleum ether = 1:2 to 2:3)

to give the title compound 27 (0.881 g, 99%) as a white foam: [α]_D²⁸

+20.7 (c = 1.01, CHCl₃); ν_max(ATR-IR) 3460, 3062, 3029, 2906,

2865, 1604, 1589, 1526, 1502, 1453, 1399, 1360, 1327, 1250, 1217,

Data for 2-O-allyl-4,5-di-O-benzyl-3-O-(4-(3,4-dimethoxyphenyl)-

benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-D-myo-ino-

sitol: [α]³_D¹+23.5 (c = 1.00, CHCl₃); ν_max(ATR-IR) 3506, 3062, 3029,

2927, 2863, 1604, 1589, 1526, 1502, 1453, 1398, 1360, 1327, 1249,

1216, 1170, 1140, 1090, 1049, 1026 cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ 2.44 (d, J = 10.4 Hz, 1H), 3.26 (t, J = 9.3 Hz, 1H), 3.42 (dd,

J = 2.3, 9.9 Hz, 1H), 3.44−3.49 (m, 2H), 3.52 (dd, J = 4.0, 10.9 Hz,

1H), 3.86 (t, J = 2.6 Hz, 1H), 3.88 (m, 1H), 3.89−4.06 (m, 5H), 3.93

(s, 3H), 3.96 (s, 3H), 4.16 (ddt, J = 1.3, 6.2, 12.7 Hz, 1H), 4.30 (d, J =

12.0 Hz, 1H), 4.46 (d, J = 11.0 Hz, 1H), 4.52−4.62 (m, 5H), 4.68−

4.78 (m, 5H), 4.79 (d, J = 10.7 Hz, 1H), 4.86 (d, J = 11.0 Hz, 1H),

4.90 (d, J = 10.7 Hz, 1H), 5.21 (dq, J = 10.5, 1.3 Hz, 1H), 5.28 (dq, J =

17.2, 1.6 Hz, 1H), 5.61 (d, J = 1.4 Hz, 1H), 5.94 (dddd, J = 5.3, 6.1,

10.4, 16.6 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 7.04−7.17 (m, 8H),

7.20−7.30 (m, 22H), 7.36−7.40 (m, 4H), 7.50−7.53 (m, 2H); ¹³C

NMR (125 MHz, CDCl₃) δ 56.03, 56.06, 69.0, 71.75, 71.76, 72.2, 72.9,

1

1171, 1140, 1072, 1049, 1025 cm⁻¹; H NMR (500 MHz, CDCl₃) δ

2.64 (s, 1H), 2.96 (d, J = 6.5 Hz, 1H), 3.29 (t, J = 9.4 Hz, 1H), 3.45

(dd, J = 2.7, 9.5 Hz, 1H), 3.49−3.52 (m, 2H), 3.53 (dd, J = 2.8, 9.6 Hz,

1H), 3.85−3.87 (m, 1H), 3.88−3.99 (m, 3H), 3.93 (s, 3H), 3.95 (s,

3H), 4.01−4.06 (m, 2H), 4.09 (t, J = 2.7 Hz, 1H), 4.34 (d, J = 12.1 Hz,

1H), 4.46 (d, J = 11.1 Hz, 1H), 4.54−4.61 (m, 4H), 4.69−4.76 (m,

5H), 4.79 (d, J = 10.8 Hz, 1H), 4.84 (d, J = 11.1 Hz, 1H), 4.87 (d, J =

10.8 Hz, 1H), 5.54 (d, J = 1.9 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 7.08−

L

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The Journal of Organic Chemistry

Featured Article

73.1, 73.2, 73.9, 74.6, 74.9, 74.87, 75.81, 75.84, 77.9, 78.2, 79.5, 81.1,

81.3, 81.5, 97.3, 110.5, 111.6, 117.38, 119.42, 126.9, 127.31, 127.34,

127.39, 127.47, 127.52, 127.58, 127.7, 127.84, 127.87, 127.9, 128.0,

128.16, 128.20, 128.22, 128.3, 128.4, 133.8, 135.0, 136.7, 138.3,

138.52, 138.53, 138.6, 138.7, 139.0, 140.6, 148.8, 149.3; HRMS-ESI

[M + Na]⁺calcd for C₇₂H₇₆NaO₁₃1171.5178, found 1171.5157.

Dibutyl (2-O-Benzoyl-3,4-di-O-benzyl-6-O-tert-butyldime-

thylsilyl-α-^D-mannopyranosyl) Phosphate (29). To a solution of

tricyclic orthoester 31³⁹(0.70 g, 1.57 mmol) and 4 Å molecular sieves

in dry CH₂Cl₂(20 mL) was added dibutyl phosphate (3.10 mL, 15.7

mmol) in one portion. The reaction mixture was stirred at rt

overnight, cooled to 0 °C, and quenched with Et₃N (3 mL). The

solution was warmed to rt and ﬁltered through a plug of Et₃N-

deactivated silica gel and further eluted with CH₂Cl₂(20 mL), and the

solvent was removed in vacuo. The residue was puriﬁed by column

chromatography on silica gel (EtOAc/petroleum ether with 1% Et₃N

= 1:4 to 2:3) to give dibutyl (2-O-benzoyl-3,4-di-O-benzyl-6-O-tert-

butyldimethylsilyl-α-^D-mannopyranosyl) phosphate (0.954 g) as a

colorless oil. tert-Butyldimethylsilyl chloride (TBSCl) (0.572 mL, 3.30

mmol) was added to a stirred solution of dibutyl (2-O-benzoyl-3,4-di-

O-benzyl-6-O-tert-butyldimethylsilyl-α-^D-mannopyranosyl) phosphate

(0.868 g, 1.32 mmol), pyridine (6 mL, 74.2 mmol), and DMAP (0.323

g, 2.64 mmol) in anhydrous CH₂Cl₂(20 mL). The reaction mixture

was allowed to stir at rt overnight. The solvent was removed in vacuo,

and the residue was puriﬁed by column chromatography on silica gel

(EtOAc/petroleum ether with 1% Et₃N = 1:9 to 1:2) to give the title

sulfonate (TBSOTf) (0.317 mL, 1.20 mmol) was added dropwise over

ca. 10 min to a solution of diol 33 (0.506 g, 1.09 mmol) and sym-

collidine (0.435 mL, 3.27 mmol) in dry CH₂Cl₂(25 mL) under an

argon atmosphere at rt. The reaction mixture was stirred for 1 h,

diluted with CH₂Cl₂(50 mL), washed with H₂O (2 × 30 mL), and

dried (MgSO₄). The solution was ﬁltered, and the solvent removed in

vacuo. The residue was puriﬁed by column chromatography on silica

gel (EtOAc/petroleum ether =1:9 to 1:2) to give the title compound

34 (0.498 g, 79%) as a white solid: mp 139−142 °C (EtOAc/PE); ν_max

(ATR-IR) 3363, 3036, 2933, 2899, 2859, 1723, 1605, 1584, 1453,

1352, 1290, 1248, 1125, 1105, 1076, 1059 cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ 0.088 (s, 3H), 0.094 (s, 3H), 0.94 (s, 9H), 2.59 (d, J = 3.7

Hz, 1H), 3.84 (dd, J = 1.6, 11.2 Hz, 1H), 3.94 (ddd, J = 1.4, 3.6, 9.7

Hz, 1H), 4.00 (dd, J = 3.7, 11.2 Hz, 1H), 4.06 (t, J = 9.6 Hz, 1H), 4.15

(dd, J = 3.2, 9.5 Hz, 1H), 4.59 (d, J = 11.4 Hz, 1H), 4.66 (d, J = 10.8

Hz, 1H), 4.79 (d, J = 11.4 Hz, 1H), 4.91 (d, J = 10.8 Hz, 1H), 5.34

(brs, 1H), 5.64 (dd, J = 1.9, 3.2 Hz, 1H), 7.22−7.34 (m, 10H), 7.43−

7.47 (m, 2H), 7.56−7.60 (m, 1H), 8.10−8.12 (m, 2H); ¹³C NMR

(125 MHz, CDCl₃) δ −5.3, −5.1, 18.5, 26.1, 62.4, 69.6, 71.7, 72.7,

74.2, 75.3, 77.8, 92.8, 127.6, 127.7, 128.0, 128.1, 128.3, 128.4, 130.1,

133.2, 138.2, 138.7, 165.9; HRMS-ESI [M + Na]⁺calcd for

C₃₃H₄₂NaO₇Si 601.2592, found 601.2624.

2-O-Benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-α-

D-mannopyranosyl Trichloroacetimidate (30).⁴⁹Mannoside 34

(2.43 g, 4.20 mmol) was dissolved in dry CH₂Cl₂(100 mL) and

cooled to 0 °C under an atmosphere of nitrogen. To the solution were

added trichloroacetonitrile (3.79 mL, 37.8 mmol) and DBU (0.063

mL, 0.420 mmol), and the reaction was stirred for 1 h at this

temperature. The solvent was removed in vacuo, and the residue was

puriﬁed by column chromatography on silica gel (EtOAc/petroleum

ether = 1:9) to give the title compound 30 (2.51 g, 83%) as a colorless

oil: ν_max(ATR-IR) 2952, 2927, 2855, 1727, 1674, 1601, 1587, 1496,

1454, 1361, 1260, 1165, 1148, 1095, 1069, 1047 cm⁻¹; ¹H NMR (500

MHz, CDCl₃) δ 0.10 (s, 3H), 0.11 (s, 3H), 0.95 (s, 9H), 3.86−3.92

(m, 2H), 4.03 (dd, J = 2.9, 11.3 Hz, 1H), 4.14 (dd, J = 3.3, 9.7 Hz,

1H), 4.23 (t, J = 9.6 Hz, 1H), 4.62 (d, J = 11.4 Hz, 1H), 4.69 (d, J =

10.6 Hz, 1H), 4.81 (d, J = 11.4 Hz, 1H), 4.92 (d, J = 10.6 Hz, 1H),

5.73 (dd, J = 2.1, 3.2 Hz, 1H), 6.39 (d, J = 2.0 Hz, 1H), 7.23−7.36 (m,

10H), 7.45−7.49 (m, 2H), 7.58−7.62 (m, 1H), 8.12−8.15 (m, 2H),

8.66 (brs, 1H); ¹³C NMR (125 MHz, CDCl₃) δ −5.3, −5.1, 18.4, 26.0,

61.7, 67.9, 72.0, 73.5, 75.4, 75.6, 77.5, 95.9, 127.8, 128.2, 128.3, 128.4,

128.5, 129.7, 130.2, 133.4, 137.7, 138.5, 160.1, 165.6; HRMS-ESI [M +

Na]⁺calcd for C₃₅H₄₂³⁵Cl₃NNaO₇Si 744.1688, found: 744.1684.

1-O-Allyl-2-O-(2-O-benzoyl-3,4-di-O-benzyl-6-O-tert-butyldi-

methylsilyl-α-^D-mannopyranosyl)-4,5-di-O-benzyl-3-O-(4-(3,4-

dimethoxyphenyl)benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-

mannopyranosyl)-^D-myo-inositol (35). Alcohol 28 (0.417 g, 0.363

mmol) and trichloroacetimidate 30 (0.324 g, 0.448 mmol) were

coevaporated from dry toluene (2 × 20 mL) and dried under high

vacuum for 30 min and then ﬂushed with nitrogen. Anhydrous toluene

(30 mL) was added followed by 4 Å molecular sieves, and the solution

was cooled to 0 °C. TMSOTf (4 μL, 0.018 mmol) was added, and the

reaction mixture was stirred for 1 h at 0 °C. The reaction was

quenched with Et₃N (0.2 mL), ﬁltered through Celite, and washed

with DCM (50 mL) and the solvent evaporated to dryness. The

residue was puriﬁed by column chromatography on silica gel (EtOAc/

petroleum ether = 1:4 to 1:2) to give the title compound 35 (0.360 g,

99%) as a white foam: [α]_D²⁷+26.0 (c = 1.80, CHCl₃); ν_max(ATR-IR)

3062, 3030, 2927, 2856, 1724, 1603, 1587, 1526, 1501, 1453, 1397,

1360, 1266, 1251, 1216, 1170, 1094, 1026 cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ −0.02 (s, 3H), 0.02 (s, 3H), 0.90 (s, 9H), 3.18 (dd, J = 2.0,

9.7 Hz, 1H), 3.29, 3.29 (overlapping dd, t, J = 1.5, 11.2, 9.3 Hz, 2H),

3.34 (dd, J = 2.5, 9.9 Hz, 1H), 3.40 (dd, J = 3.1, 11.3 Hz, 1H), 3.51 (d,

J = 11.3 Hz, 1H), 3.74 (d, J = 10.3 Hz, 1H), 3.83−3.85 (m, 1H), 3.86−

3.97 (m, 4H), 3.92 (s, 3H), 3.94 (s, 3H), 4.02 (dd, J = 5.3, 12.8 Hz,

1H), 4.07−4.12 (m, 4H), 4.16 (t, J = 9.8 Hz, 1H), 4.20 (d, J = 12.0 Hz,

1H), 4.34 (t, J = 2.2 Hz, 1H), 4.48 (d, J = 10.9 Hz, 1H), 4.59−4.70 (m,

8H), 4.75−4.81 (m, 3H), 4.85, 4.85 (2 × overlapping d, J = 10.7, 11.5

Hz, 2H), 4.90 (d, J = 10.8 Hz, 2H), 4.92 (d, J = 10.6 Hz, 1H), 5.03

(dq, J = 10.5, 1.4 Hz, 1H), 5.20 (d, J = 1.8 Hz, 1H), 5.21 (dq, J = 17.2,

1

compound 29 (0.793 g, 78%) as a colorless oil: H NMR (500 MHz,

CDCl₃) δ 0.09 (s, 3H), 0.10 (s, 3H), 0.92−0.96 (m, 15H), 1.37−1.46

(m, 4H), 1.63−1.70 (m, 4H), 3.83 (dd, J = 1.7, 11.4 Hz, 1H), 3.89 (dt,

J = 2.1, 9.7 Hz, 1H), 4.03−4.12 (m, 6H), 4.18 (t, J = 9.7 Hz, 1H), 4.59

(d, J = 11.4 Hz, 1H), 4.68 (d, J = 10.8 Hz, 1H), 4.79 (d, J = 11.4 Hz,

1H), 4.90 (d, J = 10.8 Hz, 1H), 5.66 (t, J = 2.6 Hz, 1H), 5.74 (dd, J =

2.0, 6.4 Hz, 1H), 7.22−7.35 (m, 10H), 7.43−7.47 (m, 2H), 7.57−7.61

(m, 1H), 8.09−8.12 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ −5.4,

−5.1, 13.6, 18.4, 18.7, 26.0, 32.3, 61.6, 67.9, 67.95, 68.0, 68.8, 69.9,

72.0, 73.4, 74.3, 75.4, 96.1, 127.7, 128.0, 128.1, 128.38, 128.43, 128.45,

129.7, 130.1, 133.4, 138.0, 138.6, 165.5; ³¹P NMR (202 MHz, CDCl₃)

δ −3.01; HRMS-ESI [M + Na]⁺calcd for C₄₁H₅₉NaO₁₀PSi 793.3513,

found 793.3505.

2-O-Benzoyl-3,4-di-O-benzyl-α-^D-mannopyranose (33). (1,5-

Cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexaﬂuoro-

phosphate (0.050 g, 0.059 mmol) was added to a stirred solution of

allyl ether 32 (2.40 g, 4.76 mmol) in dry THF (20 mL) at rt under an

atmosphere of argon. The argon atmosphere was replaced with H₂for

ca. 1 min, followed by a gentle stream of argon being passed over the

reaction. The reaction mixture was stirred for 2.5 h, the solvent was

removed in vacuo, and the residue was dissolved in CH₂Cl₂/MeOH

(2:1, 30 mL). Acetyl chloride (0.3 mL, 4.22 mmol) was added to the

solution, and the reaction was stirred overnight. The reaction mixture

was quenched with saturated NaHCO₃(20 mL), and H₂O (50 mL)

was added. The mixture was extracted with CH₂Cl₂(3 × 100 mL), the

combined organic extracts were dried (MgSO₄) and ﬁltered, and the

solvent was removed in vacuo. The residue was puriﬁed by column

chromatography on silica gel (EtOAc/petroleum ether = 1:2 to 1:1) to

give the title compound 33 (1.88 g, 85%) as a white foam: ν_max(ATR-

IR) 3389, 3063, 3031, 2929, 1719, 1601, 1584, 1496, 1452, 1345,

1268, 1210, 1178, 1095, 1070, 1042, 1026 cm⁻¹; ¹H NMR (500 MHz,

CDCl₃) δ 2.33 (brs, 1H), 3.61 (d, J = 3.3 Hz, 1H), 3.77 (dd, J = 4.9,

11.3 Hz, 1H), 3.86 (dd, J = 2.1, 11.8 Hz, 1H), 3.91 (t, J = 9.6 Hz, 1H),

3.99 (ddd, J = 2.7, 4.7, 9.8 Hz, 1H), 4.16 (dd, J = 3.2, 9.2 Hz, 1H), 4.58

(d, J = 11.4 Hz, 1H), 4.64 (d, J = 11.0 Hz, 1H), 4.77 (d, J = 11.4 Hz,

1H), 4.92 (d, J = 11.0 Hz, 1H), 5.28 (dd, J = 1.9, 3.5 Hz, 1H), 5.59

(dd, J = 1.9, 3.1 Hz, 1H), 7.24−7.35 (m, 10H), 7.45−7.48 (m, 2H),

7.56−7.60 (m, 1H), 8.05−8.08 (m, 2H); ¹³C NMR (125 MHz,

CDCl₃) δ 62.4, 69.6, 71.6, 71.9, 74.3, 75.3, 77.7, 92.6, 127.7, 127.9,

128.0, 128.3, 128.4, 128.5, 128.6, 130.0, 133.4, 138.0, 138.1, 165.8;

HRMS-ESI [M + Na]⁺calcd for C₂₇H₂₈NaO₇487.1727, found

487.1712.

2-O-Benzoyl-3,4-di-O-benzyl-6-O-tert-butyldimethylsilyl-α-

D-mannopyranose (34).⁴⁹tert-Butyldimethylsilyl triﬂuoromethane-

M

dx.doi.org/10.1021/jo301189y | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Featured Article

1

1.6 Hz, 1H), 5.55 (d, J = 1.7 Hz, 1H), 5.69−5.77 (m, 2H), 6.94 (d, J =

8.4 Hz, 1H), 7.05−7.06 (m, 1H), 7.09 (dd, J = 2.0, 8.2 Hz, 1H), 7.11−

7.14 (m, 4H), 7.15−7.31 (m, 30H), 7.31−7.36 (m, 7H), 7.39−7.45

(m, 5H), 7.54−7.57 (m, 1H), 8.08−8.11 (m, 2H); ¹³C NMR (125

MHz, CDCl₃) δ −5.4, −5.1, 18.4, 26.1, 56.0, 56.1, 61.9, 68.6, 69.0,

70.8, 71.5, 71.6, 72.06, 72.08, 72.10, 72.2, 72.5, 73.3, 73.9, 74.8, 74.96,

75.02, 75.3, 75.8, 75.9, 77.0, 77.6, 79.1, 80.1, 81.0, 81.5, 81.7, 99.0

(¹J_C−H= 173 Hz), 99.2 (¹J_C−H= 174 Hz), 110.4, 111.5, 117.5, 119.4,

126.8, 127.2, 127.3, 127.40, 127.41, 127.47, 127.59, 127.61, 127.66,

127.67, 127.86, 127.88, 127.95, 127.97, 128.09, 128.11, 128.12, 128.21,

128.22, 128.28, 128.31, 128.39, 128.41, 128.5, 130.1, 130.3, 132.9,

133.86, 133.95, 136.7, 138.1, 138.2, 138.5, 138.7, 138.85, 138.93,

139.1, 139.2, 140.3, 148.7, 149.2, 165.2; HRMS-ESI [M + Na]⁺calcd

for C₁₀₅H₁₁₆NaO₁₉Si 1731.7772, found 1731.7702. Anal. Calcd for

C₁₀₅H₁₁₆O₁₉Si.H₂O: C, 72.98; H, 6.88. Found: C, 72.76; H, 7.15.

1-O-Allyl-4,5-di-O-benzyl-2-O-(3,4-di-O-benzyl-6-O-tert-bu-

tyldimethylsilyl-α-^D-mannopyranosyl)-3-O-(4-(3,4-

dimethoxyphenyl)benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-

mannopyranosyl)-^D-myo-inositol (36). Sodium methoxide (pre-

pared from reacting Na (0.27 g, 11.74 mmol) with MeOH (5 mL))

was added dropwise to a stirred solution of 35 (1.62 g, 0.948 mmol) in

CH₂Cl₂/MeOH (1:9, 50 mL) until the pH of the solution remained

greater than 11. The reaction mixture was then allowed to stir

overnight, partitioned with H₂O (50 mL), extracted with CH₂Cl₂(3 ×

50 mL), dried (MgSO₄), and ﬁltered and the solvent removed in

vacuo. The residue was puriﬁed by column chromatography on silica

gel (EtOAc/petroleum ether = 1:4 to 1:2) to give the title compound

36 (1.42 g, 93%) as a white foam: [α]²_D⁷+47.8 (c = 1.57, CHCl₃); ν_max

(ATR-IR) 3488, 3061, 3030, 2926, 2854, 1604, 1588, 1526, 1501,

1453, 1398, 1360, 1250, 1216, 1170, 1091, 1026 cm⁻¹; ¹H NMR (500

MHz, CDCl₃) δ −0.01 (s, 3H), 0.01 (s, 3H), 0.86 (s, 9H), 2.35 (brs,

1H), 3.21 (dd, J = 1.8, 9.7 Hz, 1H), 3.29, 3.31 (overlapping t, dd, J =

9.4, 1.5, 11.2 Hz, 2H), 3.34 (dd, J = 2.5, 9.9 Hz, 1H), 3.42 (dd, J = 3.1,

11.1 Hz, 1H), 3.58 (dd, J = 1.4, 11.4 Hz, 1H), 3.69 (dd, J = 3.7, 11.4

Hz, 1H), 3.83 (dd, J = 1.9, 3.0 Hz, 1H), 3.87−3.95 (m, 5H), 3.94 (s,

3H), 3.95 (s, 3H), 3.96−4.00 (m, 1H), 4.05−4.11 (m, 3H), 4.12−4.18

(m, 2H), 4.22 (d, J = 12.0 Hz, 1H), 4.40 (t, J = 2.1 Hz, 1H), 4.47 (d, J

= 11.0 Hz, 1H), 4.59 (d, J = 12.0 Hz, 1H), 4.62−4.72 (m, 7H), 4.75,

4.76 (2 × overlapping d, J = 12.5, 11.3 Hz, 2H), 4.80, 4.81 (2 ×

overlapping d, J = 10.7, 12.1 Hz, 2H), 4.87, 4.87 (2 × overlapping d, J

= 10.9, 11.0 Hz, 2H), 4.91 (d, J = 10.5 Hz, 1H), 4.95 (d, J = 10.7 Hz,

1H), 5.17 (dq, J = 10.4, 1.4 Hz, 1H), 5.23 (d, J = 1.7 Hz, 1H), 5.26

(dq, J = 17.2, 1.6 Hz, 1H), 5.53 (d, J = 1.6 Hz, 1H), 5.79 (dddd, J =

5.5, 5.5, 10.8, 17.1 Hz, 1H), 6.96 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 2.1

Hz, 1H), 7.08−7.14 (m, 5H), 7.15−7.46 (m, 40H); ¹³C NMR (125

MHz, CDCl₃) δ −5.3, −5.1, 18.4, 26.0, 56.0, 56.1, 62.3, 68.4, 68.7,

69.9, 70.9, 71.89, 71.96, 72.0, 72.11, 72.14, 72.19, 73.2, 74.0, 74.8, 75.0,

75.3, 75.7, 76.05, 76.1, 79.2, 79.5, 80.1, 81.58, 81.6, 81.7, 98.7, 100.0,

110.4, 111.5, 117.8, 119.4, 126.8, 127.27, 127.36, 127.44, 127.45,

127.57, 127.63, 127.85, 127.89, 127.91, 127.93, 127.97, 128.12, 128.14,

128.21, 128.25, 128.27, 128.31, 128.35, 128.38, 128.6, 133.9, 134.0,

136.6, 138.05, 138.11, 138.58, 138.61, 138.7, 138.9, 139.0, 139.1,

140.3, 148.7, 149.2; HRMS-ESI [M + Na]⁺calcd for C₉₈H₁₁₂NaO₁₈Si

1627.7510, found 1627.7554. Anal. Calcd for C₉₈H₁₁₂O₁₈Si.H₂O: C,

72.48; H, 7.08. Found: C, 72.60; H, 7.30.

1216, 1170, 1091, 1049, 1026 cm⁻¹; H NMR (500 MHz, CDCl₃) δ

−0.02 (s, 3H), 0.01 (s, 3H), 0.86 (s, 9H), 3.18 (dd, J = 1.9, 9.7 Hz,

1H), 3.26 (t, J = 9.4 Hz, 1H), 3.28 (dd, J = 1.6, 11.2 Hz, 1H), 3.31 (dd,

J = 2.6, 10.0 Hz, 1H), 3.39 (dd, J = 3.0, 11.1 Hz, 1H), 3.56 (dd, J = 1.4,

11.3 Hz, 1H), 3.70 (dd, J = 3.5, 11.4 Hz, 1H), 3.81−3.95 (m, 6H),

3.92 (s, 3H), 3.94 (s, 3H), 3.95−3.98 (m, 1H), 4.00−4.09 (m, 4H),

4.18 (t, J = 9.8 Hz, 1H), 4.20 (d, J = 11.9 Hz, 1H), 4.37 (t, J = 2.4 Hz,

1H), 4.47 (d, J = 11.0 Hz, 1H), 4.57−4.73 (m, 12H), 4.76 (d, J = 10.9

Hz, 1H), 4.79 (d, J = 12.2 Hz, 1H), 4.85 (d, J = 11.0 Hz, 1H), 4.91,

4.91, 4.92 (3 × overlapping d, J = 10.5, 10.7, 11.0 Hz, 3H), 5.09 (dq, J

= 10.4, 1.2 Hz, 1H), 5.21 (dq, J = 17.1, 1.5 Hz, 1H), 5.23 (d, J = 1.5

Hz, 1H), 5.52 (d, J = 1.8 Hz, 1H), 5.72 (dddd, J = 5.6, 5.6, 10.8, 17.1

Hz, 1H), 6.94 (d, J = 8.3 Hz, 1H), 7.05 (d, J = 2.1 Hz, 1H), 7.07−7.13

(m, 5H), 7.14−7.39 (m, 43H), 7.42 (d, J = 8.1 Hz, 2H); ¹³C NMR

(125 MHz, CDCl₃) δ −5.3, −5.1, 18.4, 26.0, 56.0, 56.1, 62.4, 68.6,

69.8, 71.0, 71.8, 71.9, 72.3, 72.4, 72.5, 72.8, 73.3, 74.4, 74.8, 74.9, 75.0,

75.2, 75.6, 75.7, 76.0, 76.1, 79.06, 79.11, 80.2, 81.4, 81.6, 82.0, 98.3,

98.7, 110.4, 111.5, 117.9, 119.4, 126.8, 127.26, 127.29, 127.34, 127.36,

127.41, 127.46, 127.51, 127.54, 127.60, 127.63, 127.82, 127.84, 127.87,

127.94, 127.97, 128.03, 128.07, 128.12, 128.15, 128.20, 128.23, 128.24,

128.25, 128.27, 128.32, 128.4, 128.5, 133.9, 134.0, 136.7, 138.1, 138.5,

138.56, 138.6, 138.62, 138.8, 138.9, 139.2, 139.3, 140.2, 148.7, 149.2;

HRMS-ESI [M + Na]⁺calcd for C₁₀₅H₁₁₈NaO₁₈Si 1717.7980, found

1717.8070.

1-O-Allyl-4,5-di-O-benzyl-3-O-(4-(3,4-dimethoxyphenyl)-

benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-2-O-

(2,3,4-tri-O-benzyl-α-^D-mannopyranosyl)-D-myo-inositol (38).

Acetyl chloride (0.30 mL, 4.22 mmol) was added to a solution of

37 (0.417 g, 0.246 mmol) in CH₂Cl₂/MeOH (3:7, 20 mL) and the

mixture stirred at rt for 1 h. The reaction mixture was diluted with

CH₂Cl₂(40 mL) and washed with H₂O (40 mL). The aqueous layer

was re-extracted with CH₂Cl₂(2 × 20 mL), the combined organic

layers were washed with saturated NaCl (40 mL), dried (MgSO₄),

ﬁltered, and the solvent was removed in vacuo. The residue was

puriﬁed by column chromatography on silica gel (EtOAc/petroleum

ether = 1:2 to 1:1) to give the title compound 38 (0.226 g, 92%) as a

white foam: [α]²_D¹+39.1 (c = 1.60, CHCl₃); ν_max(ATR-IR) 3483,

3062, 3029, 2925, 2864, 1604, 1588, 1526, 1498, 1453, 1397, 1361,

1

1250, 1216, 1171, 1067, 1025 cm⁻¹; H NMR (500 MHz, CDCl₃) δ

1.84 (brs, 1H), 3.17 (dd, J = 2.0, 9.7 Hz, 1H), 3.25 (t, J = 9.4 Hz, 1H),

3.28 (dd, J = 1.4, 11.0 Hz, 1H), 3.31 (dd, J = 2.5, 10.0 Hz, 1H), 3.38

(dd, J = 3.0, 11.2 Hz, 1H), 3.59−3.63 (m, 2H), 3.80−3.82 (m, 1H),

3.82 (t, J = 9.6 Hz, 1H), 3.85−3.94 (m, 4H), 3.93 (s, 6H), 3.95−4.04

(m, 4H), 4.07 (dt, J = 3.1, 9.6 Hz, 1H), 4.18 (t, J = 9.8 Hz, 1H), 4.19

(d, J = 11.9 Hz, 1H), 4.27 (t, J = 2.2 Hz, 1H), 4.47 (d, J = 11.0 Hz,

1H), 4.57 (d, J = 11.9 Hz, 1H), 4.59−4.80 (m, 13H), 4.85 (d, J = 11.0

Hz, 1H), 4.89−4.95 (m, 3H), 5.12 (dq, J = 10.4, 1.4 Hz, 1H), 5.17 (d,

J = 1.5 Hz, 1H), 5.23 (dq, J = 17.2, 1.6 Hz, 1H), 5.51 (d, J = 1.9 Hz,

1H), 5.74 (dddd, J = 5.4, 5.6, 10.4, 17.1 Hz, 1H), 6.94 (d, J = 8.4 Hz,

1H), 7.06 (d, J = 2.0 Hz, 1H), 7.07−7.12 (m, 5H), 7.14−7.39 (m,

43H), 7.43 (d, J = 8.2 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 56.0,

56.1, 62.2, 68.6, 71.1, 71.3, 71.8, 71.9, 72.1, 72.2, 72.3, 72.4, 72.9, 73.3,

74.5, 74.8, 74.9, 75.0, 75.1, 75.6, 75.7, 75.9, 76.1, 78.8, 78.9, 80.2, 81.4,

81.5, 81.7, 98.7, 99.1, 110.4, 111.5, 117.8, 119.4, 126.8, 127.26, 127.29,

127.37, 127.44, 127.47, 127.49, 127.57, 127.61, 127.65, 127.67, 127.69,

127.83, 127.84, 127.88, 127.93, 128.01, 128.06, 128.11, 128.13, 128.15,

128.17, 128.19, 128.2, 128.3, 128.38, 128.4, 128.5, 133.8, 133.9, 136.4,

138.0, 138.28, 138.30, 138.5, 138.6, 138.68, 138.74, 138.8, 139.1,

140.4, 148.7, 149.2; HRMS-ESI [M + Na]⁺calcd for C₉₉H₁₀₄NaO₁₈

1603.7115, found 1603.7135. Anal. Calcd for C₉₉H₁₀₄O₁₈.H₂O: C,

71.89; H, 6.83. Found: C, 71.57; H, 6.55.

1-O-Allyl-4,5-di-O-benzyl-3-O-(4-(3,4-dimethoxyphenyl)-

benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-2-O-

(2,3,4-tri-O-benzyl-6-O-tert-butyldimethylsilyl-α-^D-mannopyr-

anosyl)-^D-myo-inositol (37). To a solution of alcohol 36 (1.22 g,

0.76 mmol) in anhydrous DMF (30 mL) were added NaH (60%

dispersion in oil, 0.055 g, 2.28 mmol) and benzyl bromide (0.271 mL,

2.28 mmol) at 0 °C, and the reaction mixture was allowed to stir

overnight. The reaction was quenched with H₂O (5 mL) and extracted

into CH₂Cl₂(3 × 100 mL). The combined organic layers were washed

with H₂O (100 mL) and saturated NaCl (100 mL), dried (MgSO₄),

and ﬁltered, and the solvent was removed in vacuo. The residue was

puriﬁed by column chromatography on silica gel (EtOAc/petroleum

ether = 1:9 to 1:2) to give the title compound 37 (0.75 g, 99%) as a

colorless oil: [α]_D²⁷+37.7 (c = 1.16, CHCl₃); ν_max(ATR-IR) 3063,

3030, 2927, 2856, 1604, 1588, 1526, 1500, 1454, 1397, 1360, 1249,

1-O-Allyl-4,5-di-O-benzyl-3-O-(4-(3,4-dimethoxyphenyl)-

benzyl)-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-2-O-

(2,3,4-tri-O-benzyl-6-O-hexadecanoyl-α-^D-mannopyranosyl)-D-

myo-inositol (39). To a solution of alcohol 38 (0.50 g, 0.316 mmol)

in anhydrous CH₂Cl₂(20 mL) were added palmitic acid (0.324 g, 1.26

mmol), DMAP (0.154 g, 1.26 mmol), and DCC (0.261 g, 1.26 mmol).

The reaction mixture was stirred at rt overnight and the solvent

removed in vacuo. The residue was puriﬁed by column chromatog-

raphy on silica gel (EtOAc/petroleum ether = 1:9 to 1:2) to give the

N

dx.doi.org/10.1021/jo301189y | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Featured Article

title compound 39 (0.295 g, 93%) as a colorless oil: [α]²_D⁷+35.0 (c =

1.07, CHCl₃); ν_max(ATR-IR) 3029, 2923, 2853, 1733, 1604, 1587,

1526, 1499, 1454, 1360, 1250, 1216, 1171, 1091, 1054, 1026 cm⁻¹; ¹H

NMR (500 MHz, CDCl₃) δ 0.88 (t, J = 7.0 Hz, 3H), 1.18−1.32 (m,

24H), 1.52−1.58 (m, 2H), 2.24 (t, J = 7.6 Hz, 2H), 3.17 (dd, J = 1.9,

9.7 Hz, 1H), 3.25 (t, J = 9.4 Hz, 1H), 3.27 (dd, J = 1.6, 11.3 Hz, 1H),

3.31 (dd, J = 2.6, 10.0 Hz, 1H), 3.38 (dd, J = 2.9, 11.2 Hz, 1H), 3.77−

3.82 (m, 2H), 3.84−3.92 (m, 4H), 3.93 (s, 3H), 3.94 (s, 3H), 3.95−

4.04 (m, 4H), 4.07 (dd, J = 1.7, 12.0 Hz, 1H), 4.16−4.24 (m, 4H),

4.30 (t, J = 2.1 Hz, 1H), 4.46 (d, J = 11.0 Hz, 1H), 4.51 (d, J = 10.8

Hz, 1H), 4.56, 4.57 (2 × overlapping d, J = 11.6, 11.9 Hz, 2H), 4.61−

4.75 (m, 10H), 4.78 (d, J = 10.8 Hz, 1H), 4.85 (d, J = 11.0 Hz, 1H),

4.89−4.94 (m, 3H), 5.09 (dq, J = 10.5, 1.3 Hz, 1H), 5.21 (dq, J = 17.2,

1.6 Hz, 1H), 5.21 (d, J = 1.2 Hz, 1H), 5.52 (d, J = 1.8 Hz, 1H), 5.71

(dddd, J = 5.5, 5.6, 10.7, 17.0 Hz, 1H), 6.94 (d, J = 8.0 Hz, 1H), 7.05−

7.12 (m, 6H), 7.14−7.38 (m, 43H), 7.43 (d, J = 8.2 Hz, 2H); ¹³C

NMR (125 MHz, CDCl₃) δ 14.2, 22.8, 24.9, 29.27, 29.34, 29.4, 29.6,

29.69, 29.72, 29.75, 29.76, 32.0, 34.3, 56.02, 56.06, 63.1, 68.6, 70.2,

71.2, 71.4, 71.5, 72.0, 72.1, 72.3, 72.4, 72.5, 73.3, 74.1, 74.5, 74.8, 75.0,

75.2, 75.6, 75.7, 75.9, 76.1, 78.5, 78.8, 80.2, 81.4, 81.5, 81.8, 98.7, 98.8,

110.6, 111.5, 117.9, 119.4, 127.0, 127.28, 127.3, 127.38, 127.46,

127.49, 127.51, 127.55, 127.59, 127.63, 127.68, 127.75, 127.78, 127.85,

127.98, 128.0, 128.1, 128.13, 128.16, 128.2, 128.25, 128.3, 128.33,

128.34, 128.36, 128.4, 128.6, 133.9, 134.0, 136.3, 138.0, 138.2, 138.3,

138.5, 138.57, 138.58, 138.76, 138.82, 139.1, 140.6, 148.7, 149.2,

173.7; HRMS-ESI [M + Na]⁺calcd for C₁₁₅H₁₃₄NaO₁₉1841.9412,

found 1841.9352.

1-O-Allyl-4,5-di-O-benzyl-6-O-(2,3,4,6-tetra-O-benzyl-α-^D-

mannopyranosyl)-2-O-(2,3,4-tri-O-benzyl-6-O-hexadecanoyl-

α-^D-mannopyranosyl)-D-myo-inositol (41). Inositol compound 39

(0.260 g, 0.143 mmol) and scavenger 40⁵¹(0.194 g, 1.43 mmol) were

dissolved in CH₂Cl₂(10.5 mL). To the solution was added TFA (4.5

mL, 30% TFA solution in relation to reaction solvent), and the

reaction was stirred at rt for 1 h. The reaction was quenched with aq

NaHCO₃(20 mL) and extracted into CH₂Cl₂(3 × 30 mL). The

combined organic layers were washed with H₂O (20 mL), saturated

NaCl (20 mL), dried (MgSO₄), and ﬁltered, and the solvent was

removed in vacuo. The residue was puriﬁed by column chromatog-

raphy on silica gel (EtOAc/petroleum ether = 1:4 to 1:2) to give the

title compound 41 (0.168 g, 74%) as a colorless oil: [α]³_D⁰+17.7 (c =

1.19, CHCl₃); ν_max(ATR-IR) 3479, 3063, 3030, 2922, 2852, 1734,

1604, 1496, 1453, 1360, 1250, 1208, 1091, 1055, 1027 cm⁻¹; ¹H NMR

(500 MHz, CDCl₃) δ 0.88 (t, J = 6.9 Hz, 3H), 1.17−1.33 (m, 24H),

1.52−1.60 (m, 2H), 2.10 (d, J = 3.0 Hz, 1H), 2.29 (t, J = 6.7 Hz, 2H),

3.23 (dd, J = 2.2, 9.7 Hz, 1H), 3.25 (t, J = 9.4 Hz, 1H), 3.33 (dd, J =

1.5, 11.1 Hz, 1H), 3.36−3.41 (m, 2H), 3.57 (t, J = 9.6 Hz, 1H), 3.80

(dd, J = 3.0, 9.1 Hz, 1H), 3.82−3.93 (m, 4H), 3.94−4.02 (m, 5H),

4.12 (t, J = 2.4 Hz, 1H), 4.17 (t, J = 9.7 Hz, 1H), 4.19−4.23 (m, 2H),

4.29 (dd, J = 4.8, 12.0 Hz, 1H), 4.46 (d, J = 11.0 Hz, 1H), 4.54 (d, J =

10.6 Hz, 1H), 4.58 (d, J = 11.9 Hz, 2H), 4.64−4.75 (m, 9H), 4.81 (d, J

= 11.1 Hz, 1H), 4.85, 4.86 (2 × overlapping d, J = 11.0, 10.7 Hz, 2H),

4.93 (d, J = 10.6 Hz, 1H), 5.03 (d, J = 1.7 Hz, 1H), 5.10 (dq, J = 10.4,

1.2 Hz, 1H), 5.22 (dq, J = 17.2, 1.5 Hz, 1H), 5.54 (d, J = 1.6 Hz, 1H),

5.70 (dddd, J = 5.5, 5.5, 10.8, 17.0 Hz, 1H), 7.07−7.39 (m, 45H); ¹³C

NMR (125 MHz, CDCl₃) δ 14.2, 22.8, 24.9, 29.1, 29.3, 29.36, 29.43,

29.6, 29.69, 29.73, 29.74, 29.76, 29.77, 32.0, 34.3, 63.4, 68.7, 70.6,

70.92, 70.94, 71.7, 72.0, 72.3, 72.4, 72.7, 73.3, 74.4, 74.7, 74.8, 74.9,

75.27, 75.3, 75.5, 75.6, 75.99, 76.02, 78.9, 80.2, 81.2, 81.7, 81.8, 98.7,

99.6, 117.8, 127.27, 127.31, 127.4, 127.49, 127.51, 127.53, 127.62,

127.66, 127.81, 127.82, 127.84, 127.86, 128.01, 128.09, 128.13, 128.18,

128.20, 128.25, 128.26, 128.34, 128.37, 128.39, 128.5, 128.6, 128.8,

134.0, 138.0, 138.1, 138.2, 138.3, 138.4, 138.6, 138.7, 138.8, 139.1,

173.8; HRMS-ESI [M + Na]⁺calcd for C₁₀₀H₁₂₀NaO₁₇1615.8418,

found 1615.8481.

reaction mixture was stirred at rt overnight and the solvent removed in

vacuo. The residue was puriﬁed by column chromatography on silica

gel (EtOAc/petroleum ether = 1:9 to 1:6) to give the title compound

42 (0.049 g, 84%) as a colorless oil: [α]²_D⁸+26.9 (c = 1.15, CHCl₃);

ν_max(ATR-IR) 3063, 3030, 2922, 2852, 1737, 1605, 1496, 1454, 1360,

1207, 1093, 1048, 1027 cm⁻¹; ¹H NMR (500 MHz, CDCl₃) δ 0.88 (t,

J = 7.0 Hz, 6H), 1.13−1.32 (m, 52H), 1.41−1.49 (m, 2H), 1.56−1.63

(m, 2H), 1.99 (dt, J = 7.6, 16.4 Hz, 1H), 2.12 (dt, J = 7.7, 16.4 Hz,

1H), 2.28 (t, J = 7.2 Hz, 2H), 3.30−3.35 (m, 3H), 3.43 (dd, J = 3.1,

11.2 Hz, 1H), 3.79 (dd, J = 9.3, 10.4 Hz, 1H), 3.83 (dd, J = 2.0, 2.9 Hz,

1H), 3.84−3.90 (m, 3H), 3.90−3.96 (m, 3H), 3.97−4.05 (m, 3H),

4.15 (dd, J = 1.9, 11.8 Hz, 1H), 4.17−4.23 (m, 3H), 4.36 (dd, J = 4.3,

11.8 Hz, 1H), 4.48 (d, J = 11.0 Hz, 1H), 4.52−4.76 (m, 13H), 4.79

(dd, J = 2.7, 10.5 Hz, 1H), 4.83 (d, J = 10.5 Hz, 1H), 4.87 (d, J = 11.0

Hz, 1H), 4.96 (d, J = 10.7 Hz, 1H), 5.11 (dq, J = 10.5, 1.3 Hz, 1H),

5.19 (d, J = 1.8 Hz, 1H), 5.22 (dq, J = 17.3, 1.6 Hz, 1H), 5.54 (d, J =

1.8 Hz, 1H), 5.70 (dddd, J = 5.6, 5.6, 10.6, 17.1 Hz, 1H), 7.03−7.39

(m, 45H); ¹³C NMR (125 MHz, CDCl₃) δ 14.2, 22.8, 24.7, 24.9, 29.2,

29.3, 29.38, 29.41, 29.44, 29.5, 29.6, 29.71, 29.73, 29.74, 29.75, 29.77,

29.78, 29.79, 29.80, 32.0, 34.1, 34.2, 63.1, 68.7, 70.5, 71.1, 71.4, 71.5,

71.6, 72.0, 72.3, 72.5, 72.6, 73.3, 74.1, 74.2, 74.8, 75.0, 75.4, 75.49,

75.52, 76.3, 78.7, 79.6, 80.1, 81.5, 81.6, 98.2, 98.7, 118.2, 127.29,

127.32, 127.4, 127.53, 127.54, 127.57, 127.65, 127.69, 127.81, 127.83,

127.86, 128.14, 128.18, 128.21, 128.23, 128.25, 128.27, 128.36, 128.4,

128.41, 128.5, 128.6, 133.7, 137.8, 138.07, 138.14, 138.18, 138.4,

138.6, 138.7, 138.8, 139.1, 173.1, 173.7; HRMS-ESI [M + Na]⁺calcd

for C₁₁₈H₁₅₄NaO₁₈1882.1027, found 1882.0917.

4,5-Di-O-benzyl-3-O-octadecanoyl-6-O-(2,3,4,6-tetra-O-ben-

zyl-α-^D-mannopyranosyl)-2-O-(2,3,4-tri-O-benzyl-6-O-hexade-

canoyl-α-^D-mannopyranosyl)-D-myo-inositol (43). (1,5-

Cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hexaﬂuoro-

phosphate (6 mg, 7 μmol) was added to a stirred solution of allyl

ether 42 (85 mg, 0.046 mmol) in dry THF (5 mL) at rt under an

atmosphere of argon. The argon atmosphere was replaced with H₂for

ca. 1 min, followed by a gentle stream of argon being passed over the

reaction. The reaction mixture was stirred for 2 h, the solvent removed

in vacuo, and the residue dissolved in acetone/H₂O (9:1, 10 mL). To

the solution were added mercury(II) chloride (0.025 g, 0.091 mmol)

and mercury(II) oxide (0.024 g, 0.110 mmol), and the resulting

solution was then heated at 100 °C for 3 h. Once cooled to rt, the

reaction mixture was ﬁltered through Celite and washed with CH₂Cl₂

(50 mL) and the solvent removed in vacuo. The residue was dissolved

in CH₂Cl₂(30 mL), washed with 5% aq KI (10 mL), dried (MgSO₄),

and ﬁltered and the solvent evaporated. The residue was puriﬁed by

column chromatography on silica gel (EtOAc/petroleum ether = 1:9

to 1:4) to give the title compound 43 (0.051 g, 61%) as a colorless oil:

[α]³_D¹+32.6 (c = 1.54, CHCl₃); ν_max(ATR-IR) 3470, 3063, 3030, 2922,

2852, 1737, 1605, 1496, 1454, 1360, 1207, 1092, 1048, 1027 cm⁻¹; ¹H

NMR (500 MHz, CDCl₃) δ 0.88 (t, J = 6.6 Hz, 6H), 1.14−1.35 (m,

52H), 1.45−1.52 (m, 2H), 1.55−1.63 (m, 2H), 2.06 (dt, J = 7.6, 16.2

Hz, 1H), 2.17 (dt, J = 7.6, 16.2 Hz, 1H), 2.30 (t, J = 7.6 Hz, 2H), 3.32

(t, J = 9.2 Hz, 1H), 3.46 (dd, J = 7.1, 10.0 Hz, 1H), 3.58 (d, J = 4.9 Hz,

1H), 3.62 (dd, J = 1.8, 10.0 Hz, 1H), 3.72−3.87 (m, 7H), 3.89−3.97

(m, 3H), 4.04 (t, J = 2.5 Hz, 1H), 4.11−4.14 (m, 1H), 4.16 (dd, J =

1.7, 11.7 Hz, 1H), 4.34 (d, J = 10.7 Hz, 1H), 4.38 (d, J = 11.6 Hz, 1H),

4.40 (dd, J = 3.8, 11.6 Hz, 1H), 4.46−4.65 (m, 12H), 4.70 (d, J = 11.7

Hz, 1H), 4.72 (d, J = 12.9 Hz, 1H), 4.78 (dd, J = 2.7, 10.6 Hz, 1H),

4.83 (d, J = 11.3 Hz, 1H), 4.94 (d, J = 10.7 Hz, 1H), 5.22 (d, J = 2.3

Hz, 1H), 5.32 (d, J = 1.7 Hz, 1H), 7.16−7.39 (m, 45H); ¹³C NMR

(125 MHz, CDCl₃) δ 14.2, 22.7, 24.8, 25.0, 29.25, 29.32, 29.39, 29.4,

29.5, 29.6, 29.7, 29.78, 29.8, 32.0, 34.21, 34.25, 63.1, 69.6, 70.3, 70.9,

71.48, 71.51, 71.8, 72.1, 72.2, 72.7, 73.5, 73.9, 74.3, 74.6, 75.0, 75.2,

75.3, 75.5, 76.8, 78.6, 79.4, 80.2, 80.4, 95.7, 98.7, 127.57, 127.60,

127.65, 127.72, 127.74, 127.8, 127.86, 127.99, 128.01, 128.07, 128.11,

128.19, 128.28, 128.32, 128.37, 128.40, 128.42, 128.44, 128.46, 128.5,

128.6, 137.62, 138.0, 138.16, 138.17, 138.27, 138.33, 138.42, 138.43,

138.45, 173.0, 173.8; HRMS-ESI [M + Na]⁺calcd for C₁₁₅H₁₅₀NaO₁₈

1842.0714, found 1842.0754.

1-O-Allyl-4,5-di-O-benzyl-3-O-octadecanoyl-6-O-(2,3,4,6-

tetra-O-benzyl-α-^D-mannopyranosyl)-2-O-(2,3,4-tri-O-benzyl-

6-O-hexadecanoyl-α-^D-mannopyranosyl)-D-myo-inositol (42).

To a solution of alcohol 41 (0.092 g, 0.058 mmol) in anhydrous

CH₂Cl₂(2 mL) were added stearic acid (0.066 g, 0.231 mmol),

DMAP (0.028 g, 0.231 mmol), and DCC (0.048 g, 0.231 mmol). The

Triethylammonium 4,5-Di-O-benzyl-3-O-octadecanoyl-6-O-

(2,3,4,6-tetra-O-benzyl-α-^D-mannopyranosyl)-2-O-(2,3,4-tri-O-

O

dx.doi.org/10.1021/jo301189y | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry

Featured Article

benzyl-6-O-hexadecanoyl-α-D-mannopyranosyl)-1-O-(1-O-((R)-

10-methyloctadecanoyl)-2-O-hexadecanoyl-sn-glycero-3-

phosphoryl)-^D-myo-inositol (44). Alcohol 43 (0.060 g, 0.033

mmol) and H-phosphonate 6 (0.128 g, 0.165 mmol) were

coevaporated with pyridine (3 × 5 mL) and then dried under high

vacuum for 30 min. The residue was dissolved in dry distilled pyridine

(4 mL) at rt under argon. Pivaloyl chloride (0.057 mL, 0.461 mmol)

was added in one portion, and the reaction mixture was stirred at rt for

3 h. A freshly prepared solution of iodine (0.084 g, 0.330 mmol) in

pyridine/H₂O (9:1, 10 mL) was added, and the mixture was stirred for

1 h. The solution was diluted with CHCl₃(20 mL), stirred for 15 min,

then washed with 10% aq Na₂S₂O₃solution (30 mL). The aqueous

layer was re-extracted with CHCl₃(2 × 20 mL), and the combined

organic layers were washed with TEAB buﬀer (1 M, 3 × 20 mL). The

solution was dried (MgSO₄) and ﬁltered and the solvent removed in

vacuo. The residue was puriﬁed by column chromatography on silica

gel (CH₂Cl₂/Et₃N/MeOH = 99:1:0 to 98:1:1) to give the title

compound 44 (0.074 g, 87%) as a colorless oil: [α]¹_D⁷+9.5 (c = 1.15,

CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 0.83 (d, J = 6.6 Hz, 3H), 0.88

(t, J = 7.0 Hz, 12H), 1.03−1.33 (m, 112H), 1.40−1.59 (m, 8H), 1.97

(dt, J = 7.6, 16.3 Hz, 1H), 2.09 (dt, J = 7.5, 16.2 Hz, 1H), 2.18 (t, J =

7.5 Hz, 4H), 2.25 (t, J = 7.6 Hz, 2H), 2.87−2.94 (m, 6H), 3.34−3.46

(m, 3H), 3.77 (t, J = 9.9 Hz, 1H), 3.85 (dd, J = 3.0, 9.4 Hz, 1H), 3.88−

3.92 (m, 1H), 3.94−4.36 (m, 15H), 4.39−4.88 (m, 18H), 4.96 (d, J =

10.8 Hz, 1H), 5.14−5.19 (m, 1H), 5.50 (s, 1H), 5.71 (s, 1H), 7.01−

7.47 (m, 45H), 12.17 (brs, 1H); ¹³C NMR (125 MHz, CDCl₃) δ 8.5,

14.2, 19.8, 22.8, 24.78, 24.89, 24.94, 24.97, 27.2, 29.19, 29.24, 29.35,

29.44, 29.55, 29.64, 29.67, 29.74, 29.79, 30.1, 32.0, 32.9, 34.0, 34.1,

34.21, 34.23, 37.2, 45.5, 52.8, 62.4, 63.0, 64.5, 64.55, 69.0, 70.35, 70.39,

70.6, 71.0, 71.58, 71.66, 71.69, 71.9, 72.0, 72.4, 73.2, 73.4, 74.0, 74.7,

74.8, 75.2, 75.3, 75.48, 75.54, 76.4, 76.5, 78.4, 79.5, 81.5, 98.2, 98.4,

127.1, 127.2, 127.3, 127.51, 127.58, 127.68, 127.7, 127.8, 127.9, 128.1,

128.16, 128.19, 128.26, 128.37, 128.41, 128.6, 137.9, 138.1, 138.31,

138.33, 138.6, 138.75, 138.8, 139.24, 139.25, 172.8, 173.1, 173.4,

173.7; ³¹P NMR (202 MHz, CDCl₃) δ −1.20; HRMS-ESI [M −

Et₃HN]⁻calcd for C₁₅₃H₂₂₂O₂₅P 2490.5838, found 2490.5854.

ASSOCIATED CONTENT

■

S

* Supporting Information

Full experimental procedures and characterizations of prepared

1

compounds and copies of H and ¹³C NMR spectra for all

novel compounds. Mass spectra and HPLC chromatograms for

compounds 4, 1, 2a, and 3b. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: dlarsen@chemistry.otago.ac.nz.

ACKNOWLEDGMENTS

■

We thank the IRL Charitable Trust and the University of Otago

for ﬁnancial assistance for G. M. Rankin and also Prof Fong-Fu

Hsu for helpful discussion on the mass spectrometry of PIMs.

This research was supported by the New Zealand Ministry of

Science and Innovation (C08X0808).

REFERENCES

■

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Sodium 3-O-Octadecanoyl-6-O-α-^D-mannopyranosyl-2-O-(6-

O-hexadecanoyl-α-^D-mannopyranosyl)-1-O-(1-O-((R)-10-meth-

yloctadecanoyl)-2-O-hexadecanoyl-sn-glycero-3-phosphoryl)-

D-myo-inositol (3b). A solution of triethylammonium salt 44 (0.017

g, 6.55 μmol) in CHCl₃/MeOH (1:1, 10 mL) was stirred in the

presence of Dowex 50WX4-400 (Na⁺) resin for 3 h. The resin was

ﬁltered oﬀ, and the solvent was removed in vacuo. The residue was

puriﬁed by column chromatography on silica gel (MeOH/CH₂Cl₂=

1:99 to 3:97). The residue was dissolved in THF/MeOH (2:3, 5 mL),

Pd(OH)₂/C (20%, 20 mg) was added, and the reaction mixture was

stirred under an atmosphere of H₂for 2 h at rt. The solution was then

ﬁltered through Celite and washed with CHCl₃/MeOH/H₂O

(70:40:6, 3 × 30 mL) and the solvents removed in vacuo. The

residue was puriﬁed by column chromatography on silica gel (CHCl₃/

MeOH/H₂O = 70:40:0 to 70:40:2) to give the title compound 3b

1

(0.011 g, 99%, 96% pure by HPLC) as a white solid: H NMR (500

MHz, CDCl₃/CD₃OD/D₂O = 70:40:6) δ 0.80 (d, J = 6.6 Hz, 3H),

0.84 (t, J = 6.9 Hz, 12H), 1.18−1.31 (m, 103H), 1.51−1.62 (m, 8H),

2.24−2.42 (m, 8H), 3.32 (t, J = 9.3 Hz, 1H), 3.63 (t, J = 9.7 Hz, 2H),

3.69−3.80 (m, 6H), 3.91−4.01 (m, 5H), 4.07−4.27 (m, 5H), 4.32 (dd,

J = 5.0, 11.9 Hz, 1H), 4.37 (dd, J = 2.7, 12.1 Hz, 1H), 4.70 (dd, J = 2.3,

10.4 Hz, 1H), 5.05 (s, 1H), 5.07 (s, 1H), 5.17−5.22 (m, 1H); ¹³C

NMR (125 MHz, CDCl₃:CD₃OD:D₂O = 70:40:6) δ 13.4, 19.1, 22.2,

24.35, 24.36, 24.4, 24.5, 26.6, 26.7, 28.72, 28.77, 28.83, 28.86, 28.87,

28.87, 28.88, 28.9, 29.0, 29.04, 29.11, 29.14, 29.17, 29.18, 29.19, 29.23,

29.25, 29.26, 29.28, 29.29, 29.5, 29.6, 31.4, 31.5, 32.3, 33.5, 33.6, 33.65,

33.66, 36.6, 36.7, 60.7, 62.4, 63.11, 63.14, 63.4, 66.5, 66.8, 69.7, 70.0,

70.1, 70.18, 70.24, 70.4, 70.6, 71.3, 72.5, 72.8, 75.8, 76.27, 76.33, 78.3,

101.2, 101.3, 173.2, 173.4, 173.7, 174.2; ³¹P NMR (202 MHz, CDCl₃/

CD₃OD/D₂O = 70:40:6) δ −0.57; HRMS-ESI [M + Na]⁺calcd for

C₉₀H₁₆₈Na₂O₂₅P 1726.1402, found 1726.1412; HRMS-ESI [M −

Na]⁻calcd for C₉₀H₁₆₈O₂₅P 1680.1618, found 1680.1652.

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Synthesis and mass spectral characterization of mycobacterial phosphatidylinositol and its dimannosides

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