Further probing of the substrate specificities and inhibition of enzymes involved at an early stage of glycosylphosphatidylinositol (GPI) biosynthesis

Carbohydrate Research 337 (2002) 2049–2059

www.elsevier.com/locate/carres

Further probing of the substrate speciﬁcities and inhibition of

enzymes involved at an early stage of glycosylphosphatidylinositol

(GPI) biosynthesis^ꢀ

Arthur Crossman Jr.,^aMichael J. Paterson,^aMichael A.J. Ferguson,^bTerry K. Smith,^b

John S. Brimacombe^a,*

^aSchool of Life Sciences (Chemistry), Carnelley Building, Uni6ersity of Dundee, Dundee DD1 4HN, UK

^bSchool of Life Sciences (Biochemistry), Wellcome Trust Building, Uni6ersity of Dundee, Dundee DD1 5EH, UK

Received 5 April 2002; accepted 26 May 2002

Dedicated to Professor Derek Horton on the occasion of his 70th birthday

Abstract

1-

osyl)-myo-inositol 1-(octadecyl phosphate) (18), 1-

decanoyl-sn-glycerol 3-phosphate) (24), 1- -6-O-(2-amino-2-deoxy-a-

decanoyl-sn-glycerol 3-phosphate) (30) and the corresponding 2-amino-2-deoxy-a-

prepared and tested in cell-free assays as substrate analogues/inhibitors of a-(14)-

D

-6-O-(2-Amino-2-deoxy-a-

D

-glucopyranosyl)-1-O-hexadecyl-myo-inositol (14), 1-

-6-O-(2-amino-2-deoxy-b- -glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexa-

-mannopyranosyl)-myo-inositol 1-(1,2-di-O-hexa-

-galactopyranosyl analogue 36 have been

-mannosyltransferases that are active early

D

-6-O-(2-amino-2-deoxy-a-

D-glucopyran-

D

on in the glycosylphosphatidylinositol (GPI) biosynthetic pathways of Trypanosoma brucei and HeLa (human) cells. The

corresponding N-acetyl derivatives of these compounds were similarly tested as candidate substrate analogues/inhibitors of the

N-deacetylases present in both systems. Following on from an early study, 1-

methyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate) (44) was prepared and tested as an inhibitor of the

-mannosyltransferase. A brief summary of the biological evaluation of the various analogues is provided.

L

-6-O-(2-amino-2-deoxy-a-D-glucopyranosyl)-2-O-

trypanosomal a-(14)-

D

Keywords: Glycosylphosphatidylinositol (GPI) membrane anchors; Substrate analogues of 1-

inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate); GPI biosynthesis in Trypanosoma brucei and HeLa (human) cell-free systems

D

-6-O-(2-amino-2-deoxy-a-D-glucopyranosyl)-myo-

1. Introduction

the bloodstream form of the protozoan parasite Try-

panosoma brucei.²This work is predicated on the belief

that disruption of GPI-anchor biosynthesis would seri-

ously impair the parasite’s ability to survive in a mam-

malian host. The truncated glycosylphosphatidyl-

Over the past decade or so we have used various

analogues of 1-

ranosyl)-myo-inositol

glycerol 3-phosphate) (1) and its N-deacetylated form 2

to probe the substrate speciﬁcities or inhibition of two

enzymes that exert their activities early on in the glyco-

sylphosphatidylinositol (GPI) biosynthetic pathway of

D

-(2-acetamido-2-deoxy-a-

D-glucopy-

1-(1,2-di-O-hexadecanoyl-sn-

inositol anchors 1 and 2,³hereafter denoted by a-

GlcpNAc-PI and a- -GlcpN-PI, respectively, differ

D-

D

from early intermediates of the GPI biosynthetic path-

way only in acylation of positions-1 and -2 of the

sn-glycerol moiety by hexadecanoyl (palmitoyl) rather

than by octadecanoyl (stearoyl) at position-1 and

other long-chain fatty acids at position-2.⁴Since

this exchange has no discernible effect on the ability

of the glycosylphosphatidylinositols 1 and 2 to act as

substrates for their respective enzymes (see be-

^ꢀParasite glycoconjugates, Part 13. For Part 12, see Ref. 1.

* Corresponding author. Tel.: +44-1382-344329; fax: +

44-1382-345517

E-mail address: j.s.brimacombe@dundee.ac.uk (J.S. Brima-

combe).

PII: S0008-6215(02)00187-8

2050

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

mammalian enzyme could not act on any of these

analogues at a detectable rate. Equally signiﬁcant is the

low), they function as ideal surrogates for the natural

substrates.^5,6

discovery that the analogue a- -GlcpNCONH₂-PI (5)

D

One of the enzymes of interest is a N-deacetylase that

is a suicide substrate inhibitor of both trypanosomal

N-deacetylates a-

and HeLa (human) cell-free systems⁷to form a-

GlcpN-PI (2). The latter compound is then acted upon

by the other enzyme of interest, an a-(14)- -manno-

syltransferase (MT-1), which transfers an a- -Manp

residue from dolichol phosphate -mannose to form

a- -Manp-(14)-a- -GlcpN-PI (3). In the trypanoso-

D-GlcpNAc-PI (1) in both T. brucei

and human N-deacetylases (Fig. 1).¹¹

The apparent difference in the substrate speciﬁcities

of trypanosomal and HeLa (human) MT-1 enzymes,

D-

D

requiring a-

PI, respectively, as substrates, has been investigated^12,13

using cell-free systems and a series of synthetic a-

D-GlcpN-PI (2) and a-D-GlcpN-2-O-acyl-

D

D-

D

GlcpN-2-O-alkyl-PIs.^14,15Of these, a-

D-GlcpN-2-O-

mal system, this step is followed by the addition of a

fatty acyl group (most often hexadecanoyl) to 2-OH of

methyl-PI (6, R³=CH₃) was shown to be a good

substrate for trypanosomal MT-1, but neither a sub-

strate for nor an inhibitor of HeLa cell MT-1.¹²More-

the

mammalian system wherein inositol 2-acylation occurs

before the ﬁrst a-

-Manp residue is attached.⁸

D-myo-inositol residue, thus differing from the

over, a-

-GlcpN-2-O-octyl-PI (6, R³=C₈H₁₇) and the

D

2-O-hexadecyl compound 6 (R³=C₁₆H₃₃) turned out

to be parasite-speciﬁc GPI pathway inhibitors with

different modes of action: the latter inhibits MT-1

whereas the former inhibits the succeeding inositol

acyltransferase.¹³

Extensive probing of the substrate speciﬁcities/inhibi-

tion of these enzymes has elicited the following infor-

mation. The fatty acid esters assist in the presentation

of the a- -GlcpNAc-PI substrate 1 to the trypanosomal

D

N-deacetylase but are not essential for substrate recog-

nition.⁵Another study⁹examined the substrate speciﬁc-

ities of T. brucei and HeLa N-deacetylases with respect

to the size of the N-acyl (R) group that can be cleaved

The 3-deoxy analogues of the substrates 1 and 2,

denoted by a-

by either the trypanosomal N-deacetylase or MT-1,

whereas the 6%-OH group, as in a-

-6dGlcpNAc-PI,¹⁶is

D

-3dGlcpN(Ac)-PI,¹⁶are not recognised

D

from a series of a-

concluded that the trypanosomal and HeLa enzymes

are active on a- -GlcpNR-PI substrates where R is

D-GlcpNR-PI substrates. It was

dispensable in both the trypanosomal and HeLa N-

deacetylase systems.¹¹The 4%-OH group too is not

essential for substrate recognition by the trypanosomal

N-deacetylase, although methylation of this group re-

duces substrate turnover.¹¹Because the 4%-OH is no

D

acetyl or propionyl but are much less active on sub-

strates where R is butyryl, isobutyryl, pentanoyl or

hexanoyl. A further study¹⁰examined the abilities of

trypanosomal and HeLa N-deacetylases to act on sub-

longer present in a-

D

-4dGlcpN-PI,¹⁶it cannot function

as a substrate for trypanosomal MT-1, which transfers

strates with alkyl substituents at 2-OH of the

inositol residue and their speciﬁcities towards the

conﬁguration ( or ) of the myo-inositol residue. The

D-myo-

an a-D-Manp residue to this position. But it might and,

indeed, does act as an inhibitor.¹⁷

D

L

In continuing to identify essential and nonessential

structural features recognised by the N-deacetylase and

MT-1 enzymes, we have fashioned certain analogues of

the substrates 1 and 2 in order to answer speciﬁc

questions. The phosphate-free analogue 14, for exam-

ple, would indicate whether or not a charged phospho-

ric diester unit is an essential feature recognised by one

or both enzymic activities. A charged phosphoric di-

ester unit is restored in the analogue 18, but a long-

chain alkyl group is introduced instead of the more

complex and synthetically less-accessible 1,2-di-O-hexa-

decanoyl-sn-glycerol in the hope that the simpler ana-

logue 18 might act as a substrate. Three of the ana-

logues, 24, 30, and 36, have a different stereochemistry

at positions-1, -2 and -4, respectively, from those of the

mammalian enzyme is more fastidious than the try-

panosomal enzyme on both counts. Thus, whereas the

trypanosomal enzyme turned over a-

methyl-PI (4, R³=CH₃), a-

-GlcpNAc-2-O-octyl-PI

(4, R³=C₈H₁₇), and a-

-GlcpNAc-[L]-PI, albeit at

lower rates than that for a- -GlcpNAc-PI (1), the

D-GlcpNAc-2-O-

D

natural a-D-glucosaminyl component of GPIs and

should indicate the essentialness of the stereochemistry

at these positions. Equally, though less likely, they

might act to inhibit the enzymes. The diastereoisomeric

analogue 44, containing 1-

was prepared in order to ascertain whether the 2-OH

group of the previously tested a- -GlcpN-[L]-PI en-

gaged in hydrogen bonding in the active site of try-

L-2-O-methyl-myo-inositol,

D

Fig. 1. Some previously prepared analogues of a-

PI (1) and a- -GlcpN-PI (2).

D-GlcpNAc-

D

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

2051

lytic debenzylation, with concomitant reduction of the

2-azido group, then produced the 1-O-hexadecyl ana-

logue 14 (Fig. 2).

The phosphoric diester 18 was accessible from the

known pseudodisaccharide 16³by means of the H-

phosphonate route.²¹Thus, condensation of the H-

phosphonate salt 15 (prepared from 1-octadecanol)

with the pseudodisaccharide 16 furnished a mixture of

diastereoisomeric phosphonic diesters that was con-

verted into the phosphoric diester 17 on oxidation in

situ with iodine in wet pyridine.²¹The ﬁnal transforma-

tion (1718) was accomplished by hydrogenolysis over

20% Pd(OH)₂on carbon.

The synthesis of the other analogues followed a

similar and familiar approach.^3,14,15,22In this, a suitable

glycosyl donor is coupled with the 6-OH group of an

appropriately protected myo-inositol derivative (usually

20³), whereafter the 1-OH group of the coupled product

is exposed for further coupling with the H-phosphonate

22.^14,23Thereafter in situ oxidation²¹provides the re-

lated phosphoric diester, which is transformed into the

fully deprotected analogue on hydrogenolysis.

A synthesis of the unnatural b-linked analogue b-

D-

GlcpN-PI (24) emerged from solvent-assisted

a

coupling²⁴between the trichloroacetimidate 19²⁵and

the glycosyl acceptor 20³in propionitrile at −80 °C

with TMSOTf as the promoter. The formation of an

increased proportion of the b-linked pseudodisaccha-

ride 21 (J_1%,2%9.0 Hz) in the presence of the non-partici-

pating 2-azido group presumably results from an S_N2

displacement²⁶on an a-nitrilium ion intermediate.²⁷

Note too that demethoxybenzylation also occurs at

some stage during the coupling process, thereby freeing

the 1-OH group for subsequent coupling with the H-

phosphonate 22.^14,23Although the 30% yield of the

b-coupled product 21 is rather low and not optimised,

this approach was appealing in the short term insofar

as both the glycosyl donor and acceptor were in hand.

Progression by the H-phosphonate route²¹provided the

fully protected compound 23, which on hydrogenolysis

Fig. 2. Synthesis of 1-

ranosyl)-1-O-hexadecyl-myo-inositol (14) and of triethylam-

monium 1- -6-O-(2-amino-2-deoxy-a- -glucopyranosyl)-

myo-inositol 1-(octadecyl phosphate) (18).

D

-6-O-(2-amino-2-deoxy-a-D-glucopy-

D

panosomal MT-1. This might then explain the some-

what unexpected ﬁnding that this unnatural analogue is

a selective inhibitor of trypanosomal MT-1.¹⁸

2. Results and discussion

A

synthesis of 1-

glucopyranosyl)-1-O-hexadecyl-myo-inositol (14) was

accomplished straightforwardly from the known

D

-6-O-(2-amino-2-deoxy-a-D-

D-

gave b-

Similar approaches to the one described above were

used to prepare a- -ManpN-PI (30) and a- -GalpN-PI

(36), based on couplings between the glycosyl ﬂuorides

26 or 32 and the

-myo-inositol derivative 20,³respec-

D-GlcpN-PI (24) (Fig. 3).

myo-inositol derivative 7,³which on conventional ben-

zylation gave the fully protected compound 8. The

latter compound, unlike previously,³was obtained in

crystalline form and was fully characterised. Removal

of the 1-O-methoxybenzyl group from the compound 8

with triﬂuoroacetic acid (TFA) in CH₂Cl₂provided the

alcohol 9, which on etheriﬁcation with hexadecyl bro-

mide in DMF in the presence of NaH gave the com-

pound 10. A two-step removal of the 6-O-allyl group,

via acidic hydrolysis of the vinylic ether,¹⁹then fur-

nished the glycosyl acceptor 11, which was coupled

with the glycosyl ﬂuoride 12³in diethyl ether in the

presence of zirconocene dichloride and silver

perchlorate²⁰to give the a-linked pseudodisaccharide 13

(J_1%,2%3.5 Hz) after radial-band chromatography. Cata-

D

tively, promoted by zirconocene dichloride–silver per-

chlorate.²⁰The starting glycosyl ﬂuorides 26 and 32

were obtained on treatment of the hemiacetal 25²⁸and

the phenyl selenogalactopyranoside 31,²⁸respectively,

with DAST. In these cases, treatment of the coupled

products with TFA was necessary to effect demethoxy-

benzylation (2728; 3334). The closing sequences

282930 and 343536 were then conducted

without incident (Figs. 4 and 5).

The

L-myo-inositol acceptor 39 needed for the prepa-

ration of a-

D-GlcpN-[L]-2-O-methyl-PI (44) was ob-

2052

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

tained by a conventional methylation of the alcohol 37³

(38), followed by deallylation of the methylated

product (39). Coupling between the glycosyl ﬂuoride

12 and the acceptor 39 in the customary manner²⁰gave

Fig. 3. Synthesis of b-D-GlcpN-PI (24).

Fig. 5. Synthesis of a-D-GalpN-PI (36).

the a-linked pseudodisaccharide 40 in ꢀ64% yield.

Thereafter, the sequence of reactions from the coupled

product 40 to the deprotected analogue 44 was executed

along the lines previously described. In this instance,

the sodium salt 43 (readily prepared from the TEA salt

42) was used for the ﬁnal hydrogenolysis (Fig. 6).

N-Acetylated derivatives of the foregoing analogues,

required for biological studies with the N-deacetylases,

were prepared by standard procedures.⁶

Details of the results of enzymic studies with the

above analogues are or will be reported elsewhere. In

brief, the phosphate-free analogue 14 and its N-acetyl

derivative are neither substrates for nor inhibitors of

GPI biosynthesis in either trypanosomal or HeLa cell-

free systems, indicating the importance of a charged

phosphodiester unit for substrate recognition. This re-

turns on restoration of the phosphodiester linkage, as

in a- -GlcpN-IP-C18 (18), which is a substrate for

D

both trypanosomal and (after 2-O-acylation in situ)

HeLa MT-1 enzymes.¹⁷This lipid-modiﬁed analogue of

Fig. 4. Synthesis of a-

D

-ManpN-PI (30).

a-D-GlcpN-PI (2) is a perfectly good substrate in the

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

2053

trypanosomal system but is a poorer substrate in the

3. Experimental

HeLa system, as is its N-acetyl derivative for the HeLa

N-deacetylase.¹¹The unnatural b-linked analogue 24

and its N-acetyl derivative are substrates for trypanoso-

mal MT-1¹⁷and N-deacetylase,¹¹respectively, but are

not recognised as substrates/inhibitors of the corre-

sponding HeLa enzymes.^11,17Thus is revealed a funda-

mental difference between the two systems. This

General methods.—¹H NMR and COSY spectra

were recorded on either a Bruker AM 300 MHz or AC

500 MHz spectrometer using deuteriochloroform as the

solvent and tetramethylsilane as the internal standard,

unless otherwise indicated. ³¹P NMR spectra used 85%

phosphoric acid in D₂O as the external standard. Opti-

cal rotations were measured using a Perkin–Elmer 141

or 343 polarimeter. Electrospray mass spectra (ESMS)

were recorded with a VG Quattro system (VG Biotech,

UK). Melting points were determined on a Reichert

hot-plate apparatus and are uncorrected. TLC was

performed on Kieselgel 60 F₂₅₄(E. Merck) with detec-

tion under UV light or by charring with 3:17:1 H₂SO₄–

water–ethanol. Radial-band chromatography (RBC)

was performed using a Chromatotron (model 7924T,

TC Research, UK) with Adsorbosil Plus-P (6–15 mm)

(Alltech) as the adsorbent. Light petroleum refers to the

fraction having a boiling point 60–80 °C. All anhy-

drous solvents were purchased from Aldrich Chemical

Co. Ltd.

contrasts with the behaviour of a-

D

-ManpN-PI (30),

a- -GalpN-PI (36) and their N-acetyl derivatives

D

which, perhaps not unexpectedly, are neither substrates

for nor inhibitors of any of the enzymes of interest.^11,17

Whereas the unnatural a-

D-GlcpN-[L]-PI is a selective

inhibitor of trypanosomal MT-1, 2-O-methylation of

the

L

-myo-inositol residue (44) abolishes any

inhibition.¹⁸

1-D-6-O-Allyl-2,3,4,5-tetra-O-benzyl-1-O-(4-meth-

oxybenzyl)-myo-inositol (8).—To a stirred and cooled

(0 °C) solution of the alcohol 7³(0.5 g, 0.82 mmol) in

DMF (20 mL) under argon was added NaH (84 mg, 3.5

mmol) and the solution was stirred for 15 min before

benzyl bromide (0.16 mL, 1.31 mmol) was added drop-

wise. The reaction mixture was stirred at rt for 2 h,

quenched with MeOH and diluted with Et₂O (50 mL).

The ethereal solution was washed with water (3×25

mL), dried (MgSO₄) and concentrated under reduced

pressure. RBC (2:1 cyclohexane–Et₂O) of the residue

yielded the fully protected compound 8 (0.29 g, 54%);

1

mp 78–79 °C; [h]_D²⁵−2° (c 1.0, CHCl₃); H NMR: l

7.42–6.84 (24 H, C₆H₄, 4×Ph), 6.04–5.96 (m, 1 H,

CH₂CHꢀCH₂), 5.27–5.11 (m, 2 H, CH₂CHꢀCH₂),

4.92–4.51 (10 H, 5×CH₂Ar), 4.40–4.34 (m, 2 H,

CH₂CHꢀCH₂), 4.04 (t, 1 H, H-6), 3.98 (t, 1 H, H-2),

3.93 (t, 1 H, H-4), 3.82 (s, 3 H, OCH₃), 3.42 (t, 1 H,

J_4,5=J_5,69.2 Hz, H-5), 3.33 (dd, 1 H, J_1,22.2, J_1,69.2

Hz, H-1), 3.25 (dd, 1 H, J_2,32.2, J_3,49.2 Hz, H-3); Anal.

Calcd for C₄₅H₄₈O₇: C, 77.12; H, 6.90. Found: C, 77.06;

H, 6.90.

1-D-6-O-Allyl-2,3,4,5-tetra-O-benzyl-myo-inositol

(9).—A solution of the methoxybenzyl derivative 8 (83

mg, 0.12 mmol) in CH₂Cl₂(10 mL) containing TFA (92

mL, 1.2 mmol) was set aside at rt for 1 h, whereafter it

was neutralised with Et₃N, washed successively with

water and brine, dried (Na₂SO₄) and concentrated un-

der reduced pressure. RBC (20:1 CH₂Cl₂–Et₂O) of the

residue gave the alcohol 9 (52 mg, 75%), mp 67–68 °C;

1

[h]²_D⁵−10° (c 1.2, CHCl₃); H NMR; l 7.50–7.20 (20

H, 4×Ph), 6.00–5.90 (m, 1 H, CH₂CHꢀCH₂), 5.30–

5.13 (m, 2 H, CH₂CHꢀCH₂), 5.10–4.60 (8 H, 4×

CH₂Ph), 4.40–4.20 (m, 2 H, CH₂CHꢀCH₂), 4.04 (t, 1

Fig. 6. Synthesis of a-D-GlcpN-[L]-2-O-methyl-PI (44).

2054

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

,

H, J_1,2=J_2,32.4 Hz, H-2), 4.02 (t, 1 H, J_1,6=J_5,69.6

Hz, H-6), 3.67 (t, 1 H, J_3,4=J_4,59.5 Hz, H-4), 3.50–

3.40 (3 H, H-1, 3, 5), 2.31 (d, 1 H, J_1,OH6.5 Hz, OH);

Anal. Calcd for C₃₇H₄₀O₆: C, 76.53; H, 6.94. Found: C,

76.42; H, 6.96.

conocene dichloride (242 mg, 0.83 mmol) and 4 A

molecular sieves (500 mg). After stirring of the mixture

at rt for 15 min under argon, it was cooled to 0 °C and

pre-dried silver perchlorate (169 mg, 0.75 mmol) and

1,1,3,3-tetramethylurea (21.5 mL, 180 mmol) were

added. Stirring of the mixture under argon at 0 °C was

continued in the dark overnight, whereafter it was

percolated through a short column of silica gel (further

elution with Et₂O) and the eluent was concentrated

under reduced pressure. RBC (1:10 EtOAc–hexane) of

the residue gave the a-linked pseudodisaccharide 13

1-D-6-O-Allyl-2,3,4,5-tetra-O-benzyl-1-O-hexadecyl-

myo-inositol (10).—To a stirred solution of the alcohol

9 (533 mg, 0.92 mmol) in DMF (15 mL) at 0 °C were

added NaH (88 mg, 3.7 mmol) and hexadecyl bromide

(550 mL, 1.8 mmol). The reaction mixture was stirred at

rt overnight, whereafter MeOH was added to destroy

the excess of NaH and the resulting solution was ex-

tracted with Et₂O (3×25 mL). The ethereal extracts

were combined and washed successively with water (25

mL) and brine (25 mL), dried (Na₂SO₄), and concen-

trated under reduced pressure. RBC (1:5 Et₂O–cyclo-

hexane) of the residue gave the hexadecyl derivative 10

(653 mg, 88%) as an amorphous solid; [h]²_D⁵−4° (c 1.2,

(100 mg, 55%); [h]_D²⁵+34° (c 1.0, CHCl₃); H NMR: l

7.40–6.90 (35 H, 7×Ph), 5.68 (d, 1 H, J_1%,2%3.5 Hz,

H-1%), 5.00–4.20 (14 H, 7×CH₂Ph), 4.15 (t, 1 H,

1

J

_3,4=J_4,59.8 Hz, H-4), 4.05 (t, 1 H, J_1,6=J_5,69.8 Hz,

H-6), 4.00 (t, 1 H, J_1,2=J_2,32.0 Hz, H-2), 3.95 (m, 1 H,

H-5%), 3.85 (t, 1 H, J_2%,3%=J_3%,4%9.0 Hz, H-3%), 3.65 (t, 1

H, J_3%,4%=J_4%,5%9.0 Hz, H-4%), 3.40–3.35 (4 H, OCH₂,

H-1, 5), 3.25 (2 H, H-2%, 3), 3.20 (dd, 1 H, J_5%,6%a1.8,

J_6%a,6%b11.0 Hz, H-6%a), 3.10 (dd, 1 H, J_5%,6%b2.0 Hz,

H-6%b), 1.52 (m, 2 H, OCH₂CH₂), ꢀ1.25 (26 H,

[CH₂]₁₃), 0.80 (t, 3 H, J 7.0 Hz, CH₂CH₃); ESMS(+):

m/z 1239.0 [M+NH₄]⁺.

1

CHCl₃); H NMR: l 7.50–7.20 (20 H, 4×Ph), 6.05–

5.95 (m, 1 H, CH₂CHꢀCH₂), 5.30–5.10 (m, 2 H,

CH₂CHꢀCH₂), 4.95–4.60 (8 H, 4×CH₂Ph), 4.40–4.35

(m, 2 H, CH₂CHꢀCH₂), 4.00 (2 H, H-2, 4), 3.85 (t, 1 H,

H-6), 3.55–3.45 (m, 2 H, OCH₂), 3.40 (t, 1 H, J_4,5=J_5,6

9.3 Hz, H-5), 3.35 (dd, 1 H, J_2,32.3, J_3,49.9 Hz, H-3),

3.15 (dd, 1 H, J_1,22.2, J_1,69.8 Hz, H-1), 1.56 (m, 2 H,

OCH₂CH₂), ꢀ1.25 (26 H, [CH₂]₁₃), 0.89 (t, 3 H, J 7.1

Hz, CH₂CH₃).

1-D-6-O-(2-Amino-2-deoxy-h-D-glucopyranosyl)-1-

O-hexadecyl-myo-inositol (14).—A solution of the pro-

tected compound 13 (88 mg, 0.072 mmol) in 1:2 THF–

MeOH (9 mL) containing 20% Pd(OH)₂on carbon (100

mg) was stirred under 3 atm of hydrogen for 2 h. It was

then percolated through a short column of Chelex 100

on a bed of Celite (further elution with 1:2 THF–

MeOH) and the eluent was concentrated under reduced

pressure to give the hexadecyl derivative 14 (40 mg,

98%); [h]²_D⁵+17° (c 0.4, 10:10:3 CHCl₃–MeOH–wa-

1-D-2,3,4,5-Tetra-O-benzyl-1-O-hexadecyl-myo-inos-

itol (11).—A solution of the allyl derivative 10 (491 mg,

0.61 mmol) in anhyd Me₂SO (20 mL) containing potas-

sium tert-butoxide (595 mg, 5.3 mmol) was heated and

stirred at 100 °C for 1 h, cooled and then poured into

ice water (100 mL). The resulting aqueous solution was

extracted with EtOAc (3×25 mL), and the organic

extracts were combined and washed successively with

water and brine, dried (Na₂SO₄), and concentrated

under reduced pressure. The resulting propenyl deriva-

tive in 1 M 1:9 HCl–acetone (20 mL) was boiled under

reﬂux for 10 min, whereafter the solvents were removed

under reduced pressure. A solution of the residue in

Et₂O was percolated through a short column of silica

gel and the eluent concentrated under reduced pressure.

RBC (1:5 Et₂O–cyclohexane) of the residue gave the

deallylated compound 11 (380 mg, 81%); [h]²_D⁵−5° (c

1.2, CHCl₃); ¹H NMR: l 7.50–7.20 (20 H, 4×Ph),

5.00–4.60 (8 H, 4×CH₂Ph), 4.15–4.03 (3 H, H-2, 4,

6), 3.50–3.30 (4 H, OCH₂, H-3, 5), 3.02 (dd, 1 H, J_1,2

2.1, J_1,69.8 Hz, H-1), 2.55 (s, 1 H, 6-OH), 1.57 (m, 2 H,

OCH₂CH₂), ꢀ1.28 (26 H, [CH₂]₁₃), 0.87 (t, 3 H, J 7.0

Hz, CH₂CH₃).

1

ter); H NMR (10:10:3 CDCl₃–CD₃OD–D₂O): l 5.39

(d, 1 H, J_1%,2%3.0 Hz, H-1%), 4.26 (bs, 1 H, H-2), 3.97 (m,

1 H, H-5%), 3.87 (t, 1 H, J_2%,3%=J_3%,4%9.7 Hz, H-3%), 3.81

(2 H, H-4, 6%a), 3.74 (dd, 1 H, J_5%,6%b4.0, J_6%a,6%b12.1 Hz,

H-6%b), 3.65–3.30 (7 H, OCH₂, H-1, 3, 4%, 5, 6), 3.22

(dd, 1 H, H-2%), 1.63 (m, 2 H, OCH₂CH₂), ꢀ1.27 (26

H, [CH₂]₁₃), 0.88 (t, 3 H, J 7.0 Hz, CH₂CH₃); ESMS(+

): m/z 566.6 [M+H]⁺.

Triethylammonium octadecyl hydrogenphosphonate

(15).—1-Octadecanol (200 mg, 0.74 mmol) was dried

by evaporation of pyridine therefrom and was after-

wards dissolved in dry 10:1 THF–pyridine (11 mL).

This solution was added dropwise over 30 min to a

stirred solution of salicylchlorophosphite (180 mg, 0.89

mmol) in dry THF (5 mL) under argon at rt. After 2 h,

TLC (19:1 CHCl₃–MeOH) indicated complete conver-

sion of the starting material into a product of lower

mobility (R_f0.30). The reaction mixture was quenched

1-D-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-h-D-

glucopyranosyl)-2,3,4,5-tetra-O-benzyl-1-O-hexadecyl-

myo-inositol (13).—After drying overnight over P₂O₅in

a vacuum desiccator, the glycosyl donor 12³(100 mg,

0.21 mmol) and the acceptor 11 (115 mg, 0.15 mmol)

were dissolved in anhyd Et₂O (15 mL) containing zir-

with 1 M triethylammonium hydrogen carbonate

(TEAB) buffer solution (10 mL) and the resulting

aqueous solution was stirred for 30 min. Chloroform

(30 mL) was then added and the organic layer was

separated and washed with 1 M TEAB buffer solution

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

2055

(2×10 mL), dried (MgSO₄), and concentrated under

reduced pressure. The crude hydrogenphosphonate

TEA salt 15 (300 mg, 93%) was used in the next

experiment without further puriﬁcation; ³¹P NMR: l_p

2.00 (with heteronuclear decoupling), J_PH635 Hz;

ESMS(−): m/z 333.4 [M−NEt₃−H]⁻.

H-3, 4%), 3.00 (t, 1 H, H-5), 2.85 (dd, 1 H, H-2%), 2.80

(m, 6 H, 3×CH₂CH₃), 1.25 (m, 2 H, OCH₂CH₂),

ꢀ0.95 (39 H, 3×CH₂CH₃, [CH₂]₁₅), 0.56 (t, 3 H, J 7.1

Hz, CH₂CH₃); ³¹P NMR (10:10:3 CDCl₃–CD₃OD–

D₂O): l_p1.80 (with heteronuclear decoupling);

ESMS(−): m/z 672.4 [M−NEt₃−H]⁻.

Triethylammonium 1-

D-6-O-(2-azido-3,4,6-tri-O-ben-

1-D-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-i-D-

zyl-2-deoxy-h- -glucopyranosyl)-2,3,4,5-tetra-O-ben-

D

glucopyranosyl) - 2,3,4,5 - tetra - O - benzyl - myo - inositol

(21).—To a stirred solution of the donor 19²⁵(117 mg,

0.19 mmol) and the acceptor 20³(139 mg, 0.21 mmol)

in dry propionitrile (10 mL) at −80 °C was added

TMSOTf (34.5 mL, 0.19 mmol). Stirring of the reaction

mixture was continued at −80 °C for 2 h, whereafter it

was diluted with satd aq NaHCO₃(25 mL) and ex-

tracted with Et₂O (3×15 mL). The ethereal extract was

washed with brine (25 mL), dried (MgSO₄), and the

solvent removed under reduced pressure. RBC (elution

ﬁrst with hexane and then with 4:1 hexane–EtOAc) of

the residue gave the alcohol 21 (57 mg, 30%); mp

157–159 °C; [h]_D²⁵−9° (c 1.1, CHCl₃); ¹H NMR: l

7.50–7.00 (35 H, 7×Ph), 5.00–4.75 (10 H, 5×

CH₂Ph), 4.67–4.60 (ABq, 2 H, J_AB12.2 Hz, CH₂Ph),

4.56–4.43 (d+ABq, 3 H, J_1%,2%9.0, J_AB12.2 Hz, H-1%,

CH₂Ph), 4.15–4.03 (3 H, H-2, 4, 6), 3.71–3.63 (2 H,

H-4%, 5%), 3.61–3.45 (6 H, H-1, 2%, 3, 5, 6%a, OH), 3.42 (2

H, H-3%,6%b); Anal. Calcd for C₆₁H₆₃N₃O₁₀: C, 73.40; H,

6.36; N, 4.21. Found: C, 73.10; H, 6.55; N, 4.20.

zyl-myo-inositol 1-(octadecyl phosphate) (17).—Each of

the compounds 15 (148 mg, 0.34 mmol) and 16³(100

mg, 0.10 mmol) were dried overnight over P₂O₅in a

vacuum desiccator, whereafter anhyd pyridine (5 mL)

was evaporated therefrom. They were then dissolved in

dry pyridine (10 mL), pivaloyl chloride (258 mL, 2.1

mmol) was added and the resulting solution was stirred

under argon at rt for 1 h. A freshly prepared solution of

iodine (173 mg, 0.68 mmol) in 19:1 pyridine–water (10

mL) was then added and stirring of the reaction mix-

ture was continued for 45 min. After the addition of

CHCl₃(30 mL), the organic solution was washed suc-

cessively with 5% aq NaHSO₃(25 mL), water (25 mL),

and 1 M TEAB buffer solution (3×15 mL), dried

(MgSO₄), and concentrated under reduced pressure.

RBC of the residue (elution ﬁrst with CHCl₃and then

with 20:1 CHCl₃–MeOH) afforded the TEA phosphate

derivative 17 (110 mg, 77%); [h]²_D⁵+53° (c 1.3, CHCl₃);

¹H NMR: l 7.50–6.90 (35 H, 7×Ph), 5.87 (d, 1 H,

J_1%,2%3.5 Hz, H-1%), 5.05–4.25 (15 H, H-2 and 7×

CH₂Ph), 4.34 (2 H, H-1, 6), 4.14 (m, 1 H, H-5%), 4.09 (t,

1 H, J_3,4=J_4,59.4 Hz, H-4), 4.02 (t, 1 H, J_2%,3%=J_3%,4%

10.0 Hz, H-3%), 3.97 (t, 2 H, J 6.9 Hz, OCH₂), 3.71 (t,

1 H, J_3%,4%=J_4%,5%10.0 Hz, H-4%), 3.54 (bd, 1 H, J_3,49.4

Hz, H-3), 3.47 (t, 1 H, J_4,5=J_5,69.4 Hz, H-5), 3.38 (m,

2 H, H-6%a,b), 3.20 (dd, 1 H, H-2%), 2.92 (q, 6 H, J 6.0

Hz, 3×CH₂CH₃), 1.60 (m, 2 H, OCH₂CH₂), ꢀ1.25

(30 H, [CH₂]₁₅), 1.18 (t, 9 H, J 7.2 Hz, 3×CH₂CH₃),

0.88 (t, 3 H, J 6.9 Hz, CH₂CH₃); ³¹P NMR: l_p–4.27

(with heteronuclear decoupling); ESMS(−): m/z

1328.3 [M−NEt₃−H]⁻.

Triethylammonium 1-

D-6-O-(2-azido-3,4,6-tri-O-ben-

zyl-2-deoxy-i- -glucopyranosyl)-2,3,4,5-tetra -O-ben-

D

zyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol

3-phosphate) (23).—This compound was obtained from

the alcohol 21 (84.4 mg, 0.085 mmol) and 1,2-di-O-hexa-

decanoyl-sn-glycerol 3-hydrogenphosphonate TEA salt

(22)^14,23(124 mg, 0.17 mmol) essentially as described

for the 1-(octadecyl phosphate) 17. RBC (elution ﬁrst

with CHCl₃and then with 19:1 CHCl₃–MeOH) gave

the TEA salt 23 (67 mg, 46%); [h]²_D⁵−5° (c 1.0,

CHCl₃); ¹H NMR: l 7.50–7.10 (35 H, 7×Ph), 5.30 (m,

1 H, H-2 glycerol), 5.10–4.90 (5 H, H-1%, 2×CH₂Ph),

4.85–4.50 (11 H, H-2, 5×CH₂Ph), 4.45–4.23 (t+m, 3

H, J_1,6=J_5,69.9 Hz, H-6, 1- or 3-CH₂glycerol), 4.15–

4.05 (4 H, H-1, 4, 1- or 3-CH₂glycerol), 3.75–3.50 (5

H, H-3, 4%, 5%, 5, 6%a), 3.37 (dd, 1 H, J_1%,2%8.4, J_2%,3%9.9

Hz, H-2%), 3.28–3.20 (2 H, H-3%, 6%b), 2.88 (6 H, 3×

CH₂CH₃), 2.25 (m, 4 H, 2×COCH₂), 1.50 (m, 4 H,

2×COCH₂CH₂), ꢀ1.20 (57 H, 3×CH₂CH₃, 2×

[CH₂]₁₂), 0.85 (t, 6 H, J 6.7 Hz, 2×CH₂CH₃); ³¹P

NMR: l_p−3.50 (with heteronuclear decoupling);

ESMS(−): m/z 1626.2 [M−NEt₃−H]⁻.

Triethylammonium 1-D-6-O-(2-amino-2-deoxy-h-D-

glucopyranosyl)-myo-inositol 1-(octadecyl phosphate)

(18).—A solution of the TEA salt 17 (60 mg, 0.042

mmol) in 2:2:1 n-propanol–THF–water (10 mL) con-

taining 20% Pd(OH)₂on carbon (50 mg) was stirred

under 3 atm of hydrogen for 1 h before it was perco-

lated through a short column of Chelex 100 on a bed of

Celite (further elution with 1:1 n-propanol–water). The

eluent was concentrated under reduced pressure to give

the deprotected product 18 (30 mg, 94%); [h]²_D⁵+14° (c

1

0.1, 10:10:3 CHCl₃–MeOH–water); H NMR (10:10:3

Triethylammonium 1-D-6-O-(2-amino-2-deoxy-i-D-

CDCl₃–CD₃OD–D₂O): l 5.22 (d, 1 H, J_1%,2%3.3 Hz,

H-1%), 3.82 (dd, 1 H, J_1,22.5, J_1,69.6 Hz, H-1), 3.76 (t,

1 H, J_1,2=J_2,32.5 Hz, H-2), 3.73 (m, 1 H, H-5%), 3.62 (t,

1 H, J_1,6=J_5,69.6 Hz, H-6), 3.58–3.46 (4 H, OCH₂,

H-3%, 6%a), 3.40 (dd, 1 H, J_5%,6%b4.0, J_6%a,6%b12.3 Hz,

H-6%b), 3.33 (t, 1 H, J_3,4=J_4,59.6 Hz, H-4), 3.12 (2 H,

glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-

sn-glycerol 3-phosphate) (24).—The TEA salt 23 (45

mg, 0.026 mmol) in 2:2:1 n-propanol–THF–water (5

mL) containing 20% Pd(OH)₂on carbon (90 mg) was

stirred under 3 atm of hydrogen for 2 h. Work-up as

described for the compound 18 gave b- -GlcpN-PI (24)

D

2056

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

(27 mg, 96%); [h]_D²⁵+7° (c 0.3, 10:10:3 CHCl₃–

MeOH–water); ¹H NMR (10:10:3 CDCl₃–CD₃OD–

D₂O): l 5.15 (m, 1 H, H-2 glycerol), 4.75 (d, 1 H, J_1%,2%

8.3 Hz, H-1%), 4.35–4.05 (3 H, H-2, 1- or 3-CH₂glyc-

erol), 4.00–3.90 (m, 2 H, 1- or 3-CH₂glycerol), 3.77

(dd, 1 H, J_1,21.9, J_1,69.9 Hz, H-1), 3.64 (2 H, H-6, 6%a),

3.54 (2 H, H-4, 6%b), 3.42 (1 H, H-3%), 3.35 (dd, 1 H, J_2,3

2.7, J_3,49.1 Hz, H-3), 3.30–3.20 (8 H, H-4%, 5%, 3×

CH₂CH₃), 3.17 (t, 1 H, J_4,5=J_5,69.4 Hz, H-5), 2.77 (t,

1 H, J_1%,2%=J_2%,3%8.3 Hz, H-2%), 2.25 (m, 4 H, 2×

COCH₂), 1.50 (m, 4 H, 2×COCH₂CH₂), ꢀ1.20 (57

H, 3×CH₂CH₃, 2×[CH₂]₁₂), 0.80 (t, 6 H, J 7.0 Hz,

2×CH₂CH₃); ³¹P NMR (10:10:3 CDCl₃–CD₃OD–

D₂O): l_p0.70 (with heteronuclear decoupling);

ESMS(−): m/z 970.2 [M−NEt₃−H]⁻.

1-D-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-h-D-

mannopyranosyl)-2,3,4,5-tetra-O-benzyl-myo-inositol

(28).—Demethoxybenzylation of the compound 27

(227 mg, 0.20 mmol) in CH₂Cl₂(10 mL) containing

TFA (216 mL, 2.8 mmol) was achieved as in the prepa-

ration of the compound 9. RBC (elution ﬁrst with

hexane and then with 3:1 hexane–EtOAc) furnished the

alcohol 28 (123 mg, 62%) as an oil; [h]²_D⁵+30° (c 1.2,

1

CHCl₃); H NMR: l 7.50–7.00 (35 H, 7×Ph), 5.45 (d,

1 H, J_1%,2%1.1 Hz, H-1%), 5.10–4.20 (14 H, 7×CH₂Ph),

4.05–4.00 (3 H, H-2%, 4%, 6), 3.95–3.91 (3 H, H-2, 3%, 5%),

3.88 (t, 1 H, J_3,4=J_4,59.5 Hz, H-4), 3.48–3.41 (2 H,

H-1, 3), 3.37 (dd, 1 H, J_5%,6%a1.9, J_6%a,6%b11.3 Hz, H-6%a),

3.33–3.25 (2 H, H-5, 6%b), 2.25 (d, 1 H, J_1,OH7.0 Hz,

OH); ESMS(+): m/z 1020.6 [M+Na]⁺.

Triethylammonium 1-

D-6-O-(2-azido-3,4,6-tri-O-ben-

2-Azido-3,4,6-tri-O-benzyl-2-deoxy-h-D-mannopyran-

zyl-2-deoxy-h- -mannopyranosyl)-2,3,4,5-tetra-O-ben-

D

osyl ﬂuoride (26).—To a cooled (−30 °C) and stirred

solution of the hemiacetal 25²⁸(158 mg, 0.33 mmol) in

anhyd 1,2-dichloroethane (15 mL) was added DAST

(183 mL, 1.38 mmol). The reaction mixture was allowed

to attain rt over 30 min and it was then partitioned

between ice-water (20 mL) and Et₂O (20 mL). The

aqueous phase was separated and further extracted with

Et₂O (20 mL). The ethereal extracts were combined and

washed with brine (20 mL), dried (MgSO₄), and con-

centrated under reduced pressure. The residue so ob-

tained was percolated through a short silica-gel column

(further elution with Et₂O) and the eluent was concen-

trated under reduced pressure. RBC (elution ﬁrst with

cyclohexane and then with 4:1 cyclohexane–Et₂O) of

the residue gave the a-glycosyl ﬂuoride 26 (106 mg,

zyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol

3-phosphate) (29).—This phosphoric diester was ob-

tained from the H-phosphonate 22^14,23(227 mg, 0.31

mmol) and the alcohol 28 (120 mg, 0.12 mmol) essen-

tially as described for the 1-(octadecyl phosphate) 17.

RBC (elution ﬁrst with CHCl₃and then with 12:1

CHCl₃–MeOH) provided the phosphoric diester as the

TEA salt 29 (145 mg, 70%); [h]²_D⁵+24° (c 1.2, CHCl₃);

¹H NMR: l 7.50–6.90 (35 H, 7×Ph), 5.46 (d, 1 H,

J_1%,2%1.2 Hz, H-1%), 5.16 (m, 1 H, H-2 glycerol), 4.96–

4.47 (14 H, 7×CH₂Ph), 4.42 (2 H, H-2, 2%), 4.30 (m, 2

H, 1- or 3-CH₂glycerol), 4.13 (2 H, H-1, 6), 4.05–3.97

(2 H, H-3%, 4), 3.96–3.82 (4 H, H-4%, 5%, 1- or 3-CH₂

glycerol), 3.46 (dd, 1 H, J_2,31.7, J_3,49.6 Hz, H-3), 3.29

(t, 1 H, J_4,5=J_5,69.0 Hz, H-5), 3.21 (dd, 1 H, J_5%,6%a1.8

Hz, H-6%a), 3.12 (d, 1 H, J_6%a,6%b10.5 Hz, H-6%b), 2.73 (q,

6 H, J 7.0 Hz, 3×CH₂CH₃), 2.15 (m, 4 H, 2×

COCH₂), 1.48 (m, 4 H, 2×COCH₂CH₂), ꢀ1.20 (48

H, 2×[CH₂]₁₂), 1.08 (t, 9 H, J 7.2 Hz, 3×CH₂CH₃),

0.85 (t, 6 H, J 7.0 Hz, 2×CH₂CH₃); ³¹P NMR: l_p

−3.41 (with heteronuclear decoupling): ESMS(−):

m/z 1626.8 [M−NEt₃−H]⁻.

1

67%); [h]²_D⁵+33° (c 1.4, CHCl₃); H NMR: l 7.40–

7.05 (15 H, 3×Ph), 5.55 (dd, 1 H, J_1,21.4, J_1,F50.2 Hz,

H-1), 4.90–4.45 (6 H, 3×CH₂Ph), 4.05–3.95 (3 H,

H-2, 3, 4), 3.88 (m, 1 H, H-5), 3.74 (dd, 1 H, J_5,6a3.9

Hz, H-6a), 3.63 (dd, 1 H, J_5,6b1.9, J_6a,6b11.0 Hz,

H-6b).

1-D-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-h-D-

mannopyranosyl)-2,3,4,5-tetra-O-benzyl-1-O-(4-meth-

oxybenzyl)-myo-inositol (27).—The coupling between

the acceptor 20³(99 mg, 0.15 mmol) and the glycosyl

ﬂuoride 26 (100 mg, 0.21 mmol) was conducted essen-

tially as described for the preparation of the pseu-

dodisaccharide 13. RBC (elution ﬁrst with cyclohexane

and then with 4:1 cyclohexane–Et₂O) afforded the a-

linked pseudodisaccharide 27 (102 mg, 61%) as an oil;

Triethylammonium 1-

mannopyranosyl)-myo-inositol

D

-6-O-(2-amino-2-deoxy-h-

D-

1-(1,2-di-O-hexadec-

anoyl-sn-glycerol 3-phosphate) (30).—A solution of the

TEA salt 29 (105 mg, 0.061 mmol) in 2:2:1 n-

propanol–THF–water (5 mL) containing 20%

Pd(OH)₂on carbon (50 mg) was stirred under 3 atm of

hydrogen for 1 h. Work-up as described for the com-

pound 18 gave a-

D

-ManpN-PI (30) (60 mg, 92%); [h]_D²⁵

1

[h]²_D⁵+13° (c 1.0, CHCl₃); H NMR: l 7.50–6.80 (39

1

+13° (c 0.2, 10:10:3 CHCl₃–MeOH–water); H NMR

(10:10:3 CDCl₃–CD₃OD–D₂O): l 5.03 (s, 1 H, H-1%),

4.98 (m, 1 H, H-2 glycerol), 4.13 (m, 2 H, 1- or 3-CH₂

glycerol), 3.90–3.70 (6 H, H-1, 2, 3%, 5%, 1- or 3-CH₂

glycerol), 3.58 (t, 1 H, J_1,6=J_5,69.5 Hz, H-6), 3.53 (2

H, H-6%a,b), 3.47 (m, 1 H, H-2%), 3.33 (2 H, H-4, 4%),

3.14 (dd, 1 H, J_2,32.6, J_3,49.8 Hz, H-3), 3.04 (m, 1 H,

H-5), 2.89 (q, 6 H, J 7.3 Hz, 3×CH₂CH₃), 2.05 (dt, 4

H, J 7.3 Hz, 2×COCH₂), 1.31 (m, 4 H, 2×

H, C₆H₄, 7×Ph), 5.39 (d, 1 H, J_1%,2%1.1 Hz, H-1%),

5.10–4.20 (16 H, 8×CH₂Ar), 4.19 (t, 1 H, H-6), 4.11

(t, 1 H, H-4), 4.03 (t, 1 H, J_1,2=J_2,32.0 Hz, H-2),

3.96–3.92 (2 H, H-3%, 4%), 3.84 (m, 1 H, H-5%), 3.75 (s, 3

H, ArOCH₃), 3.72 (dd, 1 H, H-2%), 3.37 (dd, 1 H, J_2,3

2.0, J_3,410.0 Hz, H-3), 3.30 (t, 1 H, J_4,5=J_5,610.0 Hz,

H-5), 3.25 (dd, 1 H, J_1,22.0, J_1,610.0 Hz, H-1), 3.19–

3.13 (2 H, H-6%a,b); ESMS(+): m/z 1140.5 [M+Na]⁺.

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

2057

COCH₂CH₂), 1.13 (t, 9 H, J 7.3 Hz, 3×CH₂CH₃),

ꢀ0.98 (48 H, 2×[CH₂]₁₂), 0.60 (t, 6 H, J 7.1 Hz,

2×CH₂CH₃); ³¹P NMR (10:10:3 CDCl₃–CD₃OD–

D₂O): l_p−1.04 (with heteronuclear decoupling);

ESMS(−): m/z 970.5 [M−NEt₃−H]⁻.

erol 3-phosphate) (35).—This phosphoric diester was

obtained from the pseudodisaccharide derivative 34

(0.068 g, 0.068 mmol) and the H-phosphonate 22^14,23

(0.150 g, 0.2 mmol) essentially as described in the

preparation of the 1-(octadecyl phosphate) 17. RBC

(30:115:1 CHCl₃–MeOH) furnished the phosphoric

diester as the TEA salt 35 (0.079 g, 67%); [h]²_D⁵+46° (c

0.8, CHCl₃); ¹H NMR: l 7.50–7.10 (35 H, 7×Ph),

5.83 (d, 1 H, J_1%,2%3.3 Hz, H-1%), 5.24 (m, 1 H, H-2

glycerol), 5.10–4.58 (15 H, H-2, 7×CH₂Ph), 4.50–4.20

(m, 5 H, 1- or 3-CH₂glycerol, H-1, 5%, 6), 4.18–4.00

(t+m, 3 H, J_3,4=J_4,59.7 Hz, H-4, 1- or 3-CH₂glyc-

erol), 3.78 (dd, J_3%,4%2.4 Hz, H-3%), 3.68–3.60 (2×dd, 2

H, J_1%,2%3.3, J_2%,3%10.8 Hz, H-2%, 4%), 3.57 (dd, J_2,32.0, J_3,4

9.7 Hz, H-3), 3.49 (dd, 1 H, J_5%,6%a5.9, J_6%a,6%b9.3 Hz,

H-6%a), 3.48–3.42 (2 H, J_5,610.2 Hz, H-5, 6%b), 2.95 (m,

6 H, 3×CH₂CH₃), 2.25 (2×t, 4 H, 2×COCH₂CH₂),

1.55 (m, 4 H, 2×COCH₂CH₂), ꢀ1.20 (57 H, 3×

CH₂CH₃, 2×[CH₂]₁₂), 0.90 (m, 6 H, J 7.0 Hz, 2×

2-Azido-3,4,6-tri-O-benzyl-2-deoxy-i-D-galactopyran-

osyl ﬂuoride (32).—To a stirred solution of the

selenogalactopyranoside 31²⁸(0.1 g, 0.16 mmol) in

freshly distilled CH₂Cl₂(10 mL) at −5 °C under argon

was added dropwise DAST (0.034 mL, 0.26 mmol).

Stirring was continued for 1 min, whereafter N-iodo-

succinimide (0.047 g, 0.21 mmol) was added. The re-

sulting mixture was then stirred for 40 min before it

was diluted with CH₂Cl₂(30 mL) and washed with cold

water (30 mL), satd NaHCO₃solution (25 mL) and

brine (25 mL), dried (MgSO₄), and concentrated under

reduced pressure. RBC (12:110:1 hexane–EtOAc) of

the residue, with sacriﬁcial cuts, provided the pure

b-anomer 32 (0.034 g, 44%); [h]²_D⁵−21° (c 1.9, CHCl₃);

1

IR (ﬁlm); w 2100 cm⁻¹(azide); H NMR: l 7.38–7.17

1

CH₂CH₃); ³¹P NMR: l_p−0.27 (with H heteronuclear

(15 H, 3×Ph), 5.08–4.82 (dd+d, 2 H, J_1,27.5, J_1,F

52.5 Hz, H-1, CHaPh), 4.68 (ABq, 2 H, J_AB11.7 Hz,

CH₂Ph), 4.55 (d, 1 H, J 11.5 Hz, CHbPh), 4.45 (ABq,

2 H, J_AB11.7 Hz, CH₂Ph), 4.00–3.88 (2 H, H-2, 4),

3.65–3.55 (3 H, H-5, 6a,b), 3.39 (dd, 1 H, J_2,310.4, J_3,4

1.9 Hz, H-3).

decoupling); ESMS(−): m/z 1626.3 [M−NEt₃−H]⁻.

Triethylammonium 1-

D-6-O-(2-amino-2-deoxy-h-

D-

galactopyranosyl)-myo-inositol

1-(1,2-di-O-hexade-

canoyl-sn-glycerol 3-phosphate) (36).—A solution of

the benzylated compound 35 (79 mg, 0.045 mmol) in

1:1 THF–propanol (4 mL) containing 20% Pd(OH)₂on

carbon (100 mg) was stirred under 3 atm of hydrogen

for 1 h. Work-up as described for the compound 18

1-D-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-h-D-

galactopyranosyl)-2,3,4,5-tetra-O-benzyl-myo-inositol

(34).—The coupling between the b-galactosyl ﬂuoride

gave a- -GalpN-PI (36) (38 mg) in essentially quantita-

D

32 (0.178 g, 0.37 mmol) and the

D-myo-inositol deriva-

tive yield; [h]²_D⁵+25° (c 0.3, 10:10:3 CHCl₃–MeOH–

water); ¹H NMR (10:10:3 CDCl₃–CD₃OD–D₂O): l

5.55 (d, 1 H, J_1%,2%3.7 Hz, H-1%), 5.27 (m, 1 H, H-2

glycerol), 4.42 (dd, 1 H, 1- or 3-CHa glycerol), 4.30 (t,

1 H, J_5%,6%a=J_5%,6%b6.0 Hz, H-5%), 4.23–4.12 (2 H, H-1, 1-

or 3-CHb glycerol), 4.10–3.85 (6 H, H-2, 3%, 4%, 6, 1- or

3-CH₂glycerol), 3.80–3.64 (t+m, 3 H, J_3,4=J_4,59.6,

J_6%a,6%b10.6 Hz, H-4, 6%a,b), 3.45 (2 H, H-2%, 3), 3.35 (m,

1 H, H-5), 3.15 (q, 6 H, J 7.3 Hz, 3×CH₂CH₃), 2.30

(m, 4 H, 2×COCH₂), 1.60 (m, 4 H, 2×COCH₂CH₂),

1.50–1.10 (57 H, 3×CH₂CH₃, 2×[CH₂]₁₂), 0.80 (t, 6

H, J 7.1 Hz, 2×CH₂CH₃); ³¹P NMR (10:10:3 CDCl₃–

tive 20³(0.176 g, 0.27 mmol) in anhyd 1,4-dioxane (15

mL) was carried out essentially as described for the

preparation of the pseudodisaccharide 13. RBC (10:1

4:1 hexane–EtOAc) gave the a-linked pseudodisaccha-

ride 33 (0.154 g, 38%), which was contaminated with a

trace of impurity; ESMS(+): m/z 1140.1 [M+Na]⁺

and m/z 1156.1 [M+K]⁺. This material was used in

the next step without further puriﬁcation.

A solution of the pseudodisaccharide 33 (0.104 g,

0.092 mmol) in anhyd CH₂Cl₂(15 mL) containing TFA

(0.1 mL, 1.3 mmol) was kept at rt for 2 h before it was

diluted with CHCl₃(50 mL), neutralised with Et₃N,

washed with water (50 mL) and brine (50 mL), dried

(MgSO₄), and concentrated under reduced pressure.

RBC (6:15:1 hexane–EtOAc) gave the alcohol 34

1

CD₃OD–D₂O): l_p1.06 (with H heteronuclear decou-

pling); ESMS(−): m/z 970.5 [M−NEt₃−H]⁻.

1-L-6-O-Allyl-3,4,5-tri-O-benzyl-1-O-(4-methoxyben-

1

(0.068 g, 73%); [h]_D²⁵+37° (c 0.7, CHCl₃); H NMR: l

zyl)-2-O-methyl-myo-inositol (38).—This compound

was prepared from the alcohol 37³as described for the

7.60–6.80 (35 H, 7×Ph), 5.38 (d, 1 H, J_1%,2%3.5 Hz,

H-1%), 5.01–4.58 (13 H, 6×CH₂Ph, CHaPh), 4.45 (d, 1

H, J 11.2 Hz, CHbPh), 4.18–4.03 (3 H, H-4, 4%, 5%),

4.03–3.90 (3 H, H-2, 2%, 6), 3.85 (1 H, H-3%), 3.60 (m, 1

H, H-1), 3.49–3.35 (3 H, H-3, 5, 6%a), 3.25 (dd, 1 H,

J_5%,6%b5.7, J_6%a,6%b8.7 Hz, H-6%b); ESMS(+): m/z 1020.2

[M+Na]⁺.

D

-enantiomer.¹⁴It had mp 88–89 °C, lit. (

D

-enan-

tiomer)¹⁴mp 88–90 °C; [h]_D²⁵+10° (c 1.2, CHCl₃), lit.

-enantiomer)¹⁴[h]_D−11° (c 1.0, CHCl₃).

1- -3,4,5-Tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-

(D

L

methyl-myo-inositol (39).—This compound was pre-

pared from the allylated derivative 38 as described for

the

tiomer)¹⁴mp 43–45 °C; [h]_D²⁵+20° (c 1.1, CHCl₃), lit.

-enantiomer)¹⁴[h]_D−24° (c 1.0, CHCl₃).

D D-enan-

-enantiomer.¹⁴It had mp 43–45 °C, lit. (

Triethylammonium 1-

zyl - 2 - deoxy - h - - galactopyranosyl) - 2,3,4,5 - tetra - O-

benzyl-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glyc-

D-6-O-(2-azido-3,4,6-tri-O-ben-

D

(D

2058

A. Crossman, Jr. et al. / Carbohydrate Research 337 (2002) 2049–2059

1-

glucopyranosyl)-3,4,5-tri-O-benzyl-2-O-methyl-myo-

inositol (41).—The coupling between the -myo-inositol

L

-6-O-(2-Azido-3,4,6-tri-O-benzyl-2-deoxy-h-

D

-

decoupling); ESMS(−): m/z 1550.1 [M−Et₃N−H]⁻.

Sodium 1- -6-O-(2-amino-2-deoxy-h- -glucopyran-

osyl)-2-O-methyl-myo-inositol 1-(1,2-di-O-hexade-

L

D

L

derivative 39 (0.189 g, 0.323 mmol) and the glycosyl

ﬂuoride 12³(0.216 g, 0.453 mmol) was carried out

essentially as described for the preparation of the pseu-

dodisaccharide 13. RBC (10:14:1 hexane–EtOAc)

afforded the a-linked pseudodisaccharide 40 (0.214 g,

ꢀ64%) admixed with an inseparable impurity; ¹H

NMR: l 7.60–6.60 (34 H, C₆H₄, 6×Ph), 5.61 (d, 1 H,

J_1%,2%3.1 Hz, H-1%), 5.15–4.35 (14 H, 7×CH₂Ar), 4.28

(m, 1 H, H-5%), 4.18 (t, 1 H, J_3,4=J_4,59.5 Hz, H-4), 3.99

(t, 1 H, J_1,6=J_5,69.5 Hz, H-6), 3.93 (t, 1 H, J_2%,3%=J_3%,4%

9.5 Hz, H-3%), 3.73 (bs, 1 H, H-2), 3.71–3.68 (m, 1 H,

H-4%), 3.65 (s, 3 H, ArOCH₃), 3.60 (s, 3 H, OCH₃), 3.55

(t, 1 H, J_4,5=J_5,69.5 Hz, H-5), 3.40–3.30 (3 H, H-1,

6%a,b), 3.28–3.20 (2 H, H-2%, 3); ESMS(+): m/z 1063.9

[M+Na]⁺. This slightly impure material was used in

the next step.

canoyl-sn-glycerol 3-phosphate) (44).—A solution of

the protected sodium salt 43 (52 mg, 0.031 mmol;

prepared from the TEA salt 42) in 4:4:1 THF–

propanol–water (9 mL) containing 20% Pd(OH)₂on

carbon (130 mg) was stirred under 3 atm of hydrogen

for 1.5 h. Work-up as described for the compound 18

gave a- -GlcpN-[L]-2-O-methyl-PI (44) (32 mg) in es-

D

sentially quantitative yield; [h]²_D³+2° (c 0.3, 10:10:3

CHCl₃–MeOH–water); ¹H NMR (10:10:3 CDCl₃–

CD₃OD–D₂O): l 5.35 (d, 1 H, J_1%,2%3.6 Hz, H-1%), 3.74

(s, 3 H, OCH₃), 3.30 (t, 1 H, J_3%,4%=J_4%,5%9.7 Hz, H-4%),

2.21 (m, 4 H, 2×COCH₂), 1.58 (m, 4 H, 2×

COCH₂CH₂), ꢀ1.30 (48 H, 2×[CH₂]₁₂), 0.77 (6 H,

2×CH₂CH₃); ³¹P NMR (10:10:3 CDCl₃–CD₃OD–

D₂O): l_p3.70 (with ¹H heteronuclear decoupling);

ESMS(−): m/z 984.3 [M−Na]⁻.

The pseudodisaccharide 40 (0.188 g, 0.18 mmol) in

anhyd CH₂Cl₂(10 mL) was treated with TFA (0.19

mL, 2.5 mmol) at rt for 2 h and the reaction mixture

was then worked-up as described for the compound 9.

RBC (6:12:1 cyclohexane–Et₂O) furnished the alco-

Acknowledgements

1

hol 41 (0.157 g, 72%); [h]²_D⁵+39° (c 1.0, CHCl₃); H

This work was supported by a programme grant

from the Wellcome Trust. One of the authors (M.J.P.)

is indebted to the BBSRC for ﬁnancial support. We

also thank Dr C.N. Borissow for some preliminary

experiments.

NMR: l 7.45–7.10 (30 H, 6×Ph), 5.39 (d, 1 H, J_1%,2%

3.9 Hz, H-1%), 5.20–4.61 (8 H, 4×CH₂Ph), 4.60–4.40

(5 H, 2×CH₂Ph, H-5%), 4.02–3.92 (2 H, H-3%, 4), 3.88

(t, 1 H, J_1,6=J_5,69.4 Hz, H-6), 3.71 (t, 1 H, J_1,2=J_2,3

2.5 Hz, H-2), 3.66 (dd, 1 H, J_5%,6%a1.6, J_6%a,6%b10.4 Hz,

H-6%a), 3.61–3.45 (3 H, H-4%, 5, 6%b), 3.56 (s, 3 H,

OCH₃), 3.44–3.38 (2 H, H-1, 3), 3.36 (dd, 1 H, J_2%,3%

10.3 Hz, H-2%), 2.90 (d, 1 H, J_1,OH9.5 Hz, OH);

ESMS(+): m/z 944.0 [M+Na]⁺.

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Article Doi

Further probing of the substrate specificities and inhibition of enzymes involved at an early stage of glycosylphosphatidylinositol (GPI) biosynthesis

DOI: 10.1016/S0008-6215(02)00187-8

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Article abstract of DOI:10.1016/S0008-6215(02)00187-8

Full text of DOI:10.1016/S0008-6215(02)00187-8

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