Carbohydrate-based templates for synthetic vaccines and drug delivery

Article abstract of DOI:10.1016/S0040-4020(01)00852-3

Methyl tetra-O-allyl, and tetra-O-[2-(tetrahydro-2H-pyranyl)oxy.-3-oxapentyl glucosides, and tetra-O-(cyanoethyl)galactosyl azide were converted into derivatives containing linkers with terminal carboxylic acid functionalities at the anomeric position and

Full text of DOI:10.1016/S0040-4020(01)00852-3

TETRAHEDRON
Pergamon
Tetrahedron 57 '2001) 8733±8742
Carbohydrate-based templates for synthetic vaccines
and drug delivery
Ross P. McGeary,a,b Istvan Jablonkaic,d and Istvan Totha,b,c,p
aSchool of Pharmacy, Steele Building, The University of Queensland, Brisbane Qld 4072, Australia
bAlchemia Pty Ltd, 3 Hi-Tech Court, Brisbane Technology Park, Eight Mile Plains, Brisbane Qld 4113, Australia
cDepartment of Pharmaceutical and Biological Sciences, The School of Pharmacy, University of London, 29-39 Brunswick Sq,
London W1CN 1AX, UK
Â
Institute of Chemistry, Chemical Research Center, Hungarian Academy of Sciences, Pusztaszeri ut 59/67, H-1025 Budapest, Hungary
d
Received 22 May 2001; revised 18 July 2001; accepted 16 August 2001
AbstractÐMethyl tetra-O-allyl, and tetra-O-[2-'tetrahydro-2H-pyranyl)oxy.-3-oxapentyl glucosides, and tetra-O-'cyanoethyl)galactosyl
azide were converted into derivatives containing linkers with terminal carboxylic acid functionalities at the anomeric position and bearing
four arms with phthaloyl- or BOC-protected terminal amino groups. These molecules were suitable for use in solid-phase peptide synthesis
and for the preparation of dendrimers containing multiple copies of peptides. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
needed to elicit an immune response. The advantage of
preparing such a synthetic vaccine using standard solid-
phase peptide synthesis is that carriers and adjuvants
could be included in the constructs.1
Vaccination, the training of the immune system to recognise
foreign microorganisms by exposing the host to killed or
attenuated bacteria or viruses such that when the host
encounters the intact pathogen a strong and rapid immune
response is elicited, has proved to be one of the most
important and cost-effective public health interventions.
Vaccination has led to the world-wide eradication of
smallpox, and the control of such diseases as measles,
rubella, polio and diptheria.
The potential for using dendrimer cores in the preparation of
synthetic vaccines has been recognised. Tam's multiple
antigenic peptide 'MAP) system consists of a core of
branching lysine residues, each terminating in a free
amino group, to which are attached multiple copies of
identical peptide sequences. The advantage of having
multiple copies of identical peptides on the one construct
is that signi®cantly higher antibody titres can be obtained
than for a single peptide sequence when conjugated to a
carrier protein.2
Many problems remain with conventional vaccines, such as
their limited shelf-lives, the need for suitable carriers and
adjuvants, and the potential dangers of using live micro-
organisms. These problems can be addressed by the
development of synthetic vaccines. These contain peptide
sequences corresponding to the antigenic determinant's) of
the invading microorganisms and, under appropriate con-
ditions, the host can raise an immune response to these
synthetic vaccines which offers protection against the
disease. The potential advantages of synthetic vaccines are
that they would not require refrigerated storage 'a great
advantage in developing nations) and they are not prepared
from pathogenic microbes. As synthetic entities, they would
be guaranteed to be of homogeneous composition and, as
such, only very small amounts of these molecules would be
The incorporation of lipidic molecules as adjuvants into
synthetic peptides has been shown to enhance immuno-
genicity of otherwise poorly immunogenic peptides. For
example, Jung and coworkers coupled tripalmitoyl-S-
glyceryl cysteine 'Pam3Cys) to a peptide epitope and
found that the resulting lipopeptide constructs were potent
immunogens with self-adjuvanting properties.3,4 An
advanced system which includes features of both Tam's
and Jung's constructs is the lipid polylysine core 'LCP)
system, which uses lipoaminoacids together with a poly-
lysine core.5 Lipoaminoacids are known to increase peptide
immunogenicity,6 and they also have the potential to be
used for the oral delivery of drugs and peptides.7 In the
LCP system the lipoaminoacids are incorporated as a
lipidic anchor at the C-terminus of the polylysine system,
and can be synthesised by standard solid phase peptide
synthesis.
Keywords: synthetic vaccines; carbohydrates; solid-phase synthesis;
adjuvants/carriers.
Corresponding author. Tel.: 1617-3365-1386; fax: 1617-3365-1688;
e-mail: i.toth@pharmacy.uq.edu.au
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020'01)00852-3

8734
R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
Scheme 1. 'a) allyl bromide, NaH, DMF, 0±508C, 1 h, 67% 'b) i. 9-BBN, THF, D, 6 h; ii. H2O2, NaOH, 08C±rt, overnight, 70% 'c) phthalimide, PPh3, DEAD,
THF, rt, 72 h, 94% 'd) Ac2O, H2SO4, 2208C, 3 days, 78% 'e) TMSN3, SnCl4, CH2Cl2, rt, 1 day, 83% 'f) H2/Pd/C, EtOAc, rt, 2 days, 76% 'g) succinic
anhydride, CH2Cl2, D, overnight, 84%.
The synthesis of carbohydrate-centred glycoclusters and
glycodendrimers has attracted much attention recently8
and, in principle, protecting group manipulation allows for
the attachment of different peptide sequences within the
same construct. This paper describes the synthesis of
glucose- and galactose-based monosaccharides, incorporat-
ing carboxylic acid groups for attachment to solid
supports,9,10 and suitably functionalised with terminal
amino groups for the attachment of multiple copies of
peptide antigen sequences.
[3.3.1]nonane '9-BBN) reagent gave the tetra-O-'3-
hydroxypropyl) derivative 3 in good yield '70%). When
the tetraol 3 was subjected to Mitsunobu conditions
'phthalimide,
diethyl
azodicarboxylate,
triphenyl-
phosphine), the four hydroxyl groups of 3 were converted
to their corresponding phthaloyl-protected amine function-
alities, giving compound 4 in excellent yield '94%).
Acetolysis of the methyl glucoside 4 was accomplished
using a mixture of acetic anhydride and sulfuric acid
'50:1) at 2208C, which afforded predominantly the a-
anomer of the acetate 5 in high yield '78%; a/b<7:1, deter-
mined by integration of the resolved H-1 signals). The
acetate 5 was converted the corresponding azido derivative
6 by treatment with azidotrimethylsilane in the presence of
tin'IV) chloride as catalyst. The azide 6 was obtained as an
anomeric mixture 'a/b<2:1). Catalytic hydrogenation of
the azide 6 provided the anomeric amino derivative 7 with
the same ratio of anomers as it was found for 6. Reaction of
the amine 7 with succinic anhydride yielded the required
carboxylic acid 8, also as an anomeric mixture, but this time
with the ratios of anomers reversed 'b/a<2:1). This fact
suggested that the two anomers of compound 7 were in
dynamic equilibrium, and that the b-anomer reacted faster
with succinic anhydride.
2. Results and discussion
Methyl a-d-glucopyranoside 1 was used as a common start-
ing material for the syntheses of the carbohydrate core
molecules 8 and 16 shown in Schemes 1 and 2, respectively.
This compound contains suitable protection at the anomeric
centre that can be transformed to the required carboxylic
functionality at a later stage of synthesis. The synthesis of
`octopus' glycosides has recently been described via per-
allylated glycosides. Allylation of glucoside 1 was initially
carried out using allyl chloride in aqueous sodium
hydroxide in the presence of a phase transfer catalyst, as
described by Dubber and Lindhorst.11 However, the
required 2,3,4,6-tetra-O-allyl derivative 2 was obtained in
very low yield '16%). The yield of the tetraol 2 could be
increased signi®cantly by instead employing allyl bromide
and sodium hydride in DMF at 508C. By this method the
per-allylated derivative 2 was obtained in 67% yield.
Regioselective hydroboration of 2 using 9-borabicyclo-
To modify the physicochemical properties of the templates
molecules, the introduction
of diethyleneglycol
'XCH2CH2OCH2CH2O) moieties appeared to be a feasible
substitution on the monosaccharide core 'Scheme 2).
However, many attempts to obtain such tetra-substituted
derivatives of the tetraol 1 using electrophiles such as

R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
8735
Scheme 2. 'a) NaH, DMF, 0±508C, overnight, 19% 'b) p-TsOH, CHCl3/MeOH, 558C, 2 h, 69% 'c) phthalimide, PPh3, DEAD, THF, rt, 72 h, 86% 'd) Ac2O,
H2SO4, 2208C, 3 days, 78% 'e) TMSN3, SnCl4, CH2Cl2, rt, 1 day, 88% 'f) H2/Pd/C, EtOAc, rt, 2 days, 76% 'g) succinic anhydride, CH2Cl2, D, overnight,
84%.
N3CH2CH2OCH2CH2OTos, ClCH2CH2OCH2CH2OTHP,
ClCH2CH2OCH2CH2Cl and DdeNHCH2CH2OCH2CH2O-
Tos, either in the presence of phase transfer catalysts, or
using strong base 'sodium hydride) at room temperature,
failed. This may have been due either to insuf®cient
reactivity of these electrophiles, or the tendency for these
halides to undergo elimination rather than substitution, lead-
ing to terminal enol ethers.12 Nevertheless, by employing
Scheme 3. 'a) acrylonitrile, DBU, MeCN, rt, overnight, 64% 'b) i. H2/Pd/C, THF, rt, 2 h; ii. adipic acid monobenzyl ester, HBTU, DIPEA, THF, rt, overnight,
55% 'c) i. NaBH4, CoCl2´6H2O, BOC2O, MeOH, 08C±rt, 4 h; ii. LiOH, MeOH/H2O, rt, overnight, 46%.

8736
R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
Scheme 4. 'a) Gly-Wang Resin, HBTU, HOBt, DIPEA, DMF, rt, 2£1 h 'b) i. NH2NH2´H2O, EtOH, 608C, 24 h; ii. TFA/TIPS/H2O, 2 h.
two equivalents of ICH2CH2OCH2CH2OTHP '9) p er
hydroxyl groupof the tetraol 1, and excess sodium hydride
in DMF at 508C, the tetra-alkylated derivative 10 was
obtained, albeit in low yield '19%). Insuf®ciently alkylated
side products were also isolated which, by mass
spectrometry, were shown to be mixtures of the trialkylated
derivatives '17%), and the dialkylated derivatives '21%) of
the starting material 1. Removal of the tetrahydropyranyl
protecting groups of the polyether 10 was accomplished
using 4-toluenesulfonic acid in chloroform±ethanol at
558C to give the tetrahydroxy intermediate 11 in moderate
yield '69%). The tetraol 11 was converted to the tetra-
phthalimide 12, using the same Mitsunobu conditions
employed previously to convert compound 3 to compound
4. Replacement of the methoxyl group at the anomeric posi-
tion of compound 12 with an acetoxyl group, using sulfuric
acid in acetic anhydride, provided the acetate 13, mainly as
the a-anomer 'a/b<7:1, with overall yield 78%), from
which the azido derivative 14 was prepared as anomeric
mixture 'a/b<2:1) by treating compound 13 with azido-
trimethylsilane in the presence of tin'IV) chloride. Hydro-
genation of the azido groupof compound 14 provided the
amino derivative 15, which was re¯uxed with succinic
anhydride in dichloromethane to yield the protected tetra-
amine 16, containing a carboxylic acid functional groupfor
attachment to a solid support.
To reduce the number of steps required to prepare these
template molecules, the synthetic route outlined in Scheme
3 was devised. The crystalline b-galactosyl azide13 17 was
cyanoethylated using acrylonitrile and 1,8-diazabicyclo-
[5.4.0]undec-7-ene 'DBU) in acetonitrile, giving the tetra-
nitrile 18 in 64% yield. It was essential to use a non-aqueous
solvent, as attempts to employ the conditions described by
Bazin and coworkers for cyanoethylating carbohydrates
'acrylonitrile in aqueous sodium hydroxide)14 gave low
yields, and large amounts of the side-product bis'cyano-
ethyl) ether, which proved to be inseparable from the tetra-
nitrile 18. Selective reduction of the azido groupin 18 was
achieved using catalytic hydrogenation. The intermediate
anomeric amine was not isolated, but was coupled directly
with adipic acid, monobenzyl ester, using the peptide
coupling agent HBTU, to give the ester 19, as the pure
b-anomer, and in 55% yield for the two steps. The four
nitrile fuctional groups of 19 were reduced to the corre-
sponding amines using sodium borohydride and cobalt'II)
chloride in methanol.15 By performing this reduction in the
presence of di-tert-butyl dicarbonate 'BOC2O), the transient
amino groups were converted to their corresponding BOC-
protected derivatives in situ. Final treatment of the reaction
mixture with aqueous lithium hydroxide saponi®ed the
ester groupto give the free acid 20, in 46% yield for the
two steps.
Scheme 5. 'a) i. 2-aminododecanoyl-MBHA resin, HBTU, DIPEA, DMF, rt, 30 min; ii. TFA, rt, 2£1 min 'b) i. AcOH, HBTU, DIPEA, DMF, rt, 30 min; ii.
HF-cleavage.

R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
8737
The phthalimido-protected compounds 8 and 16 are suitable
substrates for solid-phase peptide synthesis using either
BOC- or FMOC-chemistries. They were coupled to
preloaded glycine-Wang resin by employing HBTU/
HOBt/DIPEA coupling reagents in DMF 'Scheme 4). The
phthaloyl protecting groups on the resin-bound sugar
derivatives 21 and 22 were removed by treatment with
hydrazine in ethanol at 608C, yielding the corresponding
resin-bound tetraamines. The success of the protecting
groupremoval was con®rmed by a quantitative ninhydrin
analysis on the resin-bound products, and by mass
spectrometry of the products 23 and 24, obtained by
cleavage from the Wang resin with tri¯uoroacetic acid/tri-
isopropylsilane/water.
30 min allyl bromide '10.4 mL, 120 mmol) was added drop-
wise and the temperature was raised to 508C. When the
reaction was complete 'about 1 h) the reaction ¯ask was
placed in an ice bath and the excess sodium hydride was
quenched by the slow addition of methanol '20 mL). The
mixture was evaporated to dryness, then the residue was
dissolved in CH2Cl2 '200 mL) and washed with water
'3£80 mL). The organic phase was dried 'MgSO4), ®ltered,
and concentrated, then the residual oil was puri®ed by ¯ash
chromatography 'hexane/EtOAc 8:2), yielding the tetra-
ether 2 as a colourless oil '4.77 g, 67%). Rf 0.19 'hexane/
EtOAc 8:2). [a]24 ˆ196.0 'c 1.0, CHCl3). nmax'thin ®lm)
D
3080, 2980, 2910, 2846, 1647, 1460, 1426, 1384, 1349,
1194, 1153, 1083, 1050, 980, 924, 770, 720, 670,
1
560 cm21. MS 'FAB): 377 'M1Na)1, 355 'M1H)1. H
The synthesis of the lipoaminoacid-containing galactoside
26, is shown in Scheme 5. 2-'Butoxycarbonylamino)dode-
canoic acid was attached to MBHA resin then, after
tri¯uoroacetic acid deprotection, the BOC-protected tetra-
amine 20 was coupled to the solid phase. After a second
deprotection and subsequent N-acetylation, the construct
was cleaved from the resin using standard anhydrous hydro-
¯uoric acid/scavenger conditions, giving the galactose-
based core molecule 26, whose identity was con®rmed
by high-resolution mass spectrometry and 1H NMR
spectroscopy.
NMR '500 MHz, CDCl3): d 3.37 's, 3H, CH3), 3.38 'dd,
1H, J2,3ˆ9.0 Hz, H-2), 3.45 'dd, 1H, J3,4ˆ9.2 Hz, H-4), 3.62
'3H, m, H-5, H-6, H-60) 3.68 't, 1H, J3,4ˆ9.2 Hz, H-3),
3.97±4.34 'm, 8H, 4£OCH2CHCH2), 4.74 'd, 1H,
J1,2ˆ3.6 Hz, H-1), 5.10±5.28 'm, 8H, Jgemˆ1.2 Hz,
4£OCH2CHCH2), 5.87±5.93 'm, 4H, Jvicˆ5.6 Hz,
4£OCH2CHCH2). 13C NMR '62.9 Hz, CDCl3): d 55.0
'OCH3), 66.7, 70.0, 72.5, 73.8, 74.2 'allyl-C-1), 77.5,
79.5, 81.5, 98.3 'C-1), 116.2, 116.5, 117.0, 117.4 'allyl-C-
3), 134.7, 134.9, 135.0 'allyl-C-2). Anal. Calcd for
C19H30O6: C, 64.38; H, 8.53. Found: C, 64.21; H, 8.22.
4.1.2. Methyl 2,3,4,6-tetra-O-+3-hydroxypropyl)-a-d-
glucopyranoside +3). 9-BBN '0.5 M solution in THF,
70 mL, 35 mmol) was added to a stirred solution of 2
'1.03 g, 2.9 mmol) in dry THF '25 mL), then the reaction
was heated under re¯ux for 6 h. The excess 9-BBN was
destroyed by the dropwise addition of water '3.0 mL) at
08C. The hydroboration mixture was oxidised by the drop-
wise addition of 3 M NaOH 'aq., 36 mL) and 30% H2O2
'36 mL) at 08C, followed by stirring overnight at room
temperature. The aqueous phase was saturated with
K2CO3 then the THF phase was separated. The aqueous
phase was extracted with THF '2£50 mL), then the
combined THF layers were dried over MgSO4, ®ltered,
and concentrated. The oily residue was puri®ed by column
chromatography '9:1!8:2 CHCl3/MeOH) to yield the
tetraol 3 as a colourless oil '0.86 g, 70%). Rf 0.26 'CHCl3/
MeOH 8:2). nmax'CHCl3) 3630, 2960, 2800, 1650, 1460,
1430, 1380, 1350, 1050, 980, 940, 770, 720, 670,
3. Conclusion
Two glucose-based templates and one galactose-based
template have been prepared, each bearing a carboxylic
acid for attachment to solid supports, and four protected
amines attached to spacers of differing hydrophobicity,
suitable for chain extension via BOC- or FMOC-based
solid phase peptide synthesis. The stability of these
constructs towards the conditions of resin cleavage using
either tri¯uoroacetic acid or hydro¯uoric acid has been
demonstrated, as has their utility for preparing lipoamino-
acid-containing dendrimers on solid supports.
4. Experimental
4.1. General methods
1
560 cm21. MS'FAB): 449 'M1Na)1, 427 'M1H)1. H
NMR '500 MHz, CDCl3):
d
1.77±1.82 'm, 8H,
Optical rotations were determined with AA-10R polari-
meter '1 dm cells, Na-D-line: 589 nm, Optical Activity
Ltd). Mass spectra were run on a Perkin±Elmer API 3000
LC/MS/MS electrospray instrument, or with a VG Analyti-
cal ZAB-SE instrument using fast atom bombardment
'FAB) techniques '20 kV Cs1 ion bombardment) with
2 mL of appropriate matrix '3-nitrobenzyl alcohol or thio-
glycerol) added. 1H NMR spectra were recorded on either a
Bruker AM 500 instrument or a Varian Gemini 300
machine. 13C NMR spectra were run on either a Bruker
AM250 instrument or a Varian Gemini 300 machine. All
reactions were performed under inert gas atmospheres.
4£OCH2CH2CH2OH), 3.24 'dd, 1H, J4,5ˆ9.2 Hz, H-4),
3.28 'dd, 1H, H-2), 3.38 's, 3H, OCH3), 3.48 '1H, t,
J3,4ˆ9.5 Hz, H-3), 3.52±3.74 'm, 20H, 4£OCH2CH2-
CH2OH), 3.80 'm, 1H, H-6), 3.82±3.87 'm, 2H, H-5,
H-60), 4.80 '1H, d, J1,2ˆ3.5 Hz, H-1). Anal. Calcd for
C19H38O10: C, 53.51; H, 9.00. Found: C, 53.60; H, 8.72.
4.1.3. Methyl 2,3,4,6-tetra-O-3-phthalimidopropyl-a-d-
glucopyranoside +4). A solution of diethyl azodicarboxyl-
ate 'DEAD) '0.93 mL, 5.9 mmol) in dry THF '5 mL) was
added dropwise to a solution of the tetraol 3 '0.48 g,
1.13 mmol), phthalimide '0.93 g, 6.30 mmol), and tri-
phenylphosphine '1.57 g, 6.0 mmol) in dry THF '40 mL)
and the reaction was stirred at room temperature for 72 h.
The solvent was evaporated and the residue was dissolved in
CH2Cl2 '50 mL) and was washed with brine, then dried
4.1.1. Methyl 2,3,4,6-tetra-O-allyl-a-d-glucopyranoside
+2). Sodium hydride '60% dispersion in oil, 4.00 g,
100 mmol) was added portionwise to a well stirred solution
of 1 '3.88 g, 20.0 mmol) at 08C in 100 mL dry DMF. After

8738
R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
'MgSO4), ®ltered and concentrated. Puri®cation of the resi-
due by ¯ash chromatography 'EtOAc/hexane 8:2) afforded
the tetraphthalimide 4 '1.00 g, 94%) as a colourless oil. Rf
'500 MHz, CDCl3) 'a-anomer only): d 1.89±1.97 'm, 8H,
4£OCH2CH2CH2N), 3.06±3.15 'm, 2H, H-2, H-4), 3.29 't,
1H, J2,3ˆ9.0 Hz, H-3), 3.44±3.87 'm, 19H, H-5, H-6, H-60,
4£OCH2CH2CH2N), 5.36 '1H, d, J1,2ˆ3.5 Hz, H-1), 7.45±
7.80 'm, 16H, 4£ArH). Integration of the signals of the H-1
protons indicated that the a/b ratio was 2:1. Anal. Calcd for
C50H47O13N7: C, 63.47; H, 4.97. Found: C, 63.41; H, 4.88.
0.28 'EtOAc/hexane 7:3). [a]24 ˆ128.5 'c 1.0, CHCl3).
D
n
max'CHCl3) 3020, 1780, 1716, 1420, 1380, 1216, 1060,
930, 760, 669, 540 cm21. MS 'FAB): 966 'M1H1Na)1,
943 'M1H)1. H NMR '500 MHz, CDCl3): d 1.91±1.98
1
'm, 8H, 4£OCH2CH2CH2N), 3.06±3.11 'm, 2H, H-4,
H-2), 3.29 's, 3H, OCH3), 3.43 't, 1H, J3,4ˆ9.5 Hz, H-3),
3.46±3.63 'm, 8H, 4£OCH2CH2CH2N), 3.65±3.92 'm, 11H,
4£OCH2CH2CH2N, H-5, H-6, H-60), 4.70 'd, 1H,
J1,2ˆ3.5 Hz, H-1), 7.45±7.80 '16H, m, 4£ArH). 13C NMR
4.1.6.
pyranosylamine +7). The azido sugar
2,3,4,6-Tetra-O-3-phthalimidopropyl-d-gluco-
'0.38 g,
6
0.4 mmol) dissolved in EtOAc '10 mL) was hydrogenated
over 10% Pd/C '90 mg) catalyst for 2 days at room tempera-
ture. The catalyst was ®ltered off and washed with EtOAc
'40 mL) and the ®ltrate was evaporated. The residue was
puri®ed by ¯ash chromatography 'EtOAc/Et2O 9:1, contain-
ing 0.5% triethylamine) to give the amine 7 as a colourless
foam '280 mg, 76%). nmax'CHCl3) 3520, 3400, 3020, 1780,
1716, 1380, 1240, 1080, 950, 760, 670, 550 cm21. MS
'62.9 Hz, CDCl3):
d
28.8, 29.3, 29.4, 29.6
'OCH2CH2CH2N), 35.3, 35.7, 35.8 'OCH2CH2CH2N),
54.9 'OCH3), 68.7, 69.2, 69.8, 70.0, 70.6, 71.0, 76.5
'OCH2CH2CH2N, C-5, C-6), 78.24 'C-4), 80.8 'C-2), 81.9
'C-3), 97.7 'C-1), 123.0, 123.1, 131.9, 132.0, 132.2, 132.4,
133.7, 133.8 'ArC), 168.2 'CON). Anal. Calcd for
C51H50O14N4: C, 64.96; H, 5.35. Found: C, 64.68; H, 5.42.
1
'FAB): 951 'M1Na)1, 928 'M)1. H NMR '500 MHz,
CDCl3) 'a-anomer only):
d
1.84±1.99 'm, 8H,
4.1.4. 1-O-Acetyl-2,3,4,6-tetra-O-3-phthalimidopropyl-
d-glucopyranose +5). A solution of the tetraphthalimide 4
'1.00 g, 1.06 mmol) in acetic anhydride '10 mL) was stirred
at 2208C for 10 min. To this stirred solution was added pre-
cooled '08C) Ac2O/H2SO4 '50:1, 5 mL), dropwise during
5 min, then the reaction mixture was left at 2208C for
3 days. The reaction mixture was diluted with CH2Cl2
'100 mL) and was washed successively with saturated
NaHCO3 '50 mL) and water '50 mL). The organic layer
was dried 'MgSO4), ®ltered, and concentrated, then co-
distilled with several portions of toluene. The residue was
puri®ed by ¯ash chromatography 'EtOAc/hexane 7:3) to
yield the acetate 5 as a colourless oil '0.80 g, 78%). Rf
0.19 'EtOAc/hexane 7:3). nmax'CHCl3) 3020, 1780, 1750,
1716, 1380, 1240, 1080, 950, 760, 670, 550 cm21. MS
'FAB): 1104 'M1Cs)1, 994 'M1Na)1. 1H NMR
'500 MHz, CDCl3) 'a-anomer only): d 1.91±1.95 'm, 8H,
4£OCH2CH2CH2N), 2.10 's, 3H, OAc), 3.14±3.19 '2H, m,
H-4, H-2), 3.40 'm, 1H, H-3), 3.45 'm, 1H, H-6), 3.51±3.79
'm, 17H, H-60, 4£OCH2CH2CH2N, 3.82±3.90 'm, 1H, H-5),
6.12 '1H, d, J1,2ˆ3.5 Hz, H-1), 7.45±7.80 'm, 16H,
4£ArH). Integration of the signals of the H-1 protons indi-
cated that the a/b ratio was 7:1. 13C NMR '62.9 Hz, CDCl3)
'a-anomer only): d 21.0 'Ac±C-1) 28.8, 29.2, 29.4, 29.5
'OCH2CH2CH2N), 35.3, 35.6 'OCH2CH2CH2N), 68.5,
69.3, 69.3, 70.9, 71.1, 72.8, 76.5 'OCH2CH2CH2N, C-5,
C-6), 77.5 'C-4), 79.7 'C-2), 81.6 'C-3), 89.6 'C-1), 123.0,
123.1, 131.9, 132.0, 132.2, 132.3, 133.7, 133.7 'ArC), 168.2
'CON). Anal. Calcd for C52H50O15N4: C, 64.32; H, 5.19.
Found: C, 64.41; H, 5.22.
4£OCH2CH2CH2N), 3.01±3.11 'm, 3H, H-4, H-2, H-3),
3.44±3.92 'm, 19H, H-5, H-6, H-60, 4£OCH2CH2CH2N),
4.95 't, 1H, H-1), 7.45±7.80 'm, 16H, 4£ArH). Integration
of the signals of the H-1 protons indicated that the a/b ratio
was 2:1. 13C NMR '62.9 Hz, CDCl3) 'a-anomer only): d
28.7, 28.9, 29.4, 29.6 'OCH2CH2CH2N), 35.4, 35.7
'OCH2CH2CH2N), 69.3, 70.0, 70.2, 70.4, 70.8, 71.0, 75.6
'OCH2CH2CH2N, C-5, C-6), 78.6 'C-4), 84.1 'C-2), 85.9
'C-3), 89.3 'C-1), 123.1, 131.88, 132.4, 133.5, 133.6,
133.7 'ArC), 166.2 'CON). Anal. Calcd for C50H49O13N5:
C, 64.72; H, 5.32. Found: C, 64.41; H, 5.12.
4.1.7. 4-+2,3,4,6-Tetra-O-+3-phthalimidopropyl)-d-gluco-
pyranosylamino)-4-oxobutanoic acid +8). The amino
sugar 7 '140 mg, 0.15 mmol) was re¯uxed with succinic
anhydride '17 mg, 1.7 mmol) in dry CH2Cl2 '5 mL) over-
night. The solvent was evaporated and the residue was puri-
®ed by ¯ash chromatography 'CHCl3/MeOH 93:7) to give
the acid 8 as a colourless foam '130 mg, 84%). Rf 0.33
'CHCl3/MeOH 9:1). [a]24 ˆ124.0 'c 1.0, CHCl3).
D
nmax'KBr) 3460, 3410, 2928, 2874, 1770, 1713, 1650,
1530, 1397, 1371, 1188, 1130, 1098, 1040, 940, 880, 720,
1
531 cm21. MS'FAB): 1051 'M1Na)1, 1029 'M1H)1. H
NMR '500 MHz, CDCl3) 'b-anomer only): d 1.79±1.96 'm,
8H, 4£OCH2CH2CH2N), 2.59 'm, 2H, HNOCCH2), 2.73
'm, 2H, HOOCCH2), 2.96 'm, 1H, H-2), 3.19 'm, 2H,
H-5, H-3), 3.32 'm, 1H, H-6), 3.41 'm, 1H, H-60), 3.46±
3.83 'm, 17H, H-4, 4£OCH2CH2CH2N), 4.86 't, 1H,
J1,2ˆ8.5 Hz, H-1), 7.47 'd, 1H, NH), 7.62±7.80 'm, 16H,
4£ArH). Integration of the signals of the H-1 protons
indicated that the a/b ratio was 1:2. 13C NMR '62.9 Hz,
CDCl3) 'a-anomer only): d 28.8, 29.0, 29.4, 29.6
4.1.5.
2,3,4,6-Tetra-O-3-phthalimidopropyl-d-gluco-
pyranosyl azide +6). A solution of the acetate 5 '0.44 g,
0.45 mmol) in dry CH2Cl2 '20 mL) was stirred with azido-
trimethylsilane '0.15 mL, 1.13 mmol) and tin'IV) chloride
'0.026 mL, 0.23 mmol) for 1 day. The solution was diluted
with CH2Cl2 '20 mL) and washed with 1 M KF solution
'10 mL) then with water '10 mL). The organic extract was
dried 'MgSO4), ®ltered, and concentrated to afford a white
foam '0.36 g, 83%). Rf 0.30 'EtOAc/hexane 7:3).
'OCH2CH2CH2N),
30.7
'NCOCH2)
34.9,
35.6
'OCH2CH2CH2N), 69.0, 69.2, 69.3, 69.6, 70.3, 70.5, 70.8,
76.3 'OCH2CH2CH2N, C-5, C-6), 79.0 'C-4), 78.2, 80.9
'C-2), 81.5 'C-3), 86.3 'C-1), 123.1, 123.2, 123.3, 123.4,
132.0, 132.1, 132.2, 133.8, 133.9, 134.0, 134.1 'ArC),
166.2, 166.37, 166.9 'CON), 173.2 'NHCO). Anal. Calcd
for C54H53O16N5: C, 63.09; H, 5.20; N, 6.81. Found: C,
63.21; H, 5.31, N, 6.64.
[a]24 ˆ151.8 'c 1.0, CHCl3). nmax'CHCl3) 3030, 2120,
D
1780, 1713, 1360, 1240, 1080, 950, 760, 670, 550 cm21
.
4.1.8. 2-[2-+2-Iodoethoxy)ethoxy]tetrahydro-2H-pyran
+9). 2-Chloroethoxyethanol '6.0 g, 48 mmol) was added to
MS'FAB): 977 'M1Na)1, 955 'M11)1. 1H NMR

R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
8739
ice-cold 3,4-dihydro-2H-pyran '15 mL) and the mixture
was stirred for 5 h at room temperature. The excess 3,4-
dihydro-2H-pyran was distilled off under high vacuum.
The pale yellow oily residue '9.8 g; Rf 0.41 hexane/EtOAc
1:1) was added without further puri®cation to a solution of
dry sodium iodide '8.8 g, 58.8 mmol) in dry acetone
'60 mL), then the mixture was heated under re¯ux for
24 h. The precipitated salt was ®ltered off and the solvent
was evaporated. The residue was taken up in CH2Cl2
'60 mL) and the precipitate was again ®ltered off. The
®ltrate was washed with 40 mL of water, then dried
'MgSO4) and concentrated to give the iodide 9 as an oil
'13.5 g, 82%) which was used in the next stepwithout puri-
®cation. Rf 0.30 'hexane/EtOAc 8:2). nmax'thin ®lm) 3510,
2940, 2870, 1464, 1454, 1440, 1384, 1353, 1262, 1201,
Jˆ3.4 Hz, H-1). Anal. Calcd for C23H46O14: C, 50.54; H,
8.48. Found: C, 50.41; H, 8.21.
4.1.11. Methyl 2,3,4,6-tetra-O-+5-phthalimido-3-oxa-
pentyl)-a-d-glucopyranoside +12). A solution of diethyl
azodicarboxylate 'DEAD) '2.69 mL, 17.1 mmol) in dry
THF '5 mL) was added dropwise to a stirred solution of
the tetraol 11 '1.8 g, 3.3 mmol), phthalimide '2.72 g,
18.5 mmol), and triphenylphosphine '4.58 g, 17.5 mmol)
in dry THF '15 mL), then the reaction mixture was stirred
at room temperature for 72 h. The solvent was evaporated
and the residue was dissolved in CH2Cl2 '100 mL) and
washed with brine and dried 'MgSO4), ®ltered, and concen-
trated. Puri®cation of the residue by ¯ash chromatography
'EtOAc/Et2O 9:1) afforded the product 12 as a colourless oil
1078, 1035, 1021, 990, 870, 814, 736, 617, 430 cm21
.
'3.0 g, 86%). [a]24 ˆ160.4 'c 1.0, CHCl3). nmax'CHCl3)
D
1
MS'FAB): 215'M2THP)1. H NMR '500 MHz, CDCl3):
d 1.51±1.83 'm, 6H), 3.37't, 2H), 3.50 'm, 1H), 3.61 'm,
1H), 3.69 'm, 5H), 3.87 'm, 1H), 4.62 'm, 1H, OCHO). 13C
NMR '62.9 Hz, CDCl3): d 19.4, 25.6, 30.5, 50.7, 62.2, 66.7,
70.0, 70.6, 74.5, 99.0.
3020, 1775, 1716, 1460, 1430, 1396, 1216, 1100, 1042,
1
756, 720, 670, 550 cm21. MS'FAB): 1085 'M1Na)1. H
NMR '500 MHz, CDCl3): d 3.16 'm, 2H, H-2, H-5), 3.27
's, 3H, OCH3), 3.42±3.48 'm, 2H, H-6,60), 3.51±3.63 'm,
14H), 3.62±3.77 'm, 10H), 3.80±3.89 'm, 10H), 4.65 '1H,
d, Jˆ3.5 Hz, H-1), 7.44±7.83 '16H, m, 4£ArH). 13C NMR
'62.9 Hz, CDCl3): d 37.3, 37.4 'CH2N), 54.7 'OCH3), 67.5,
67.6, 67.8, 69.5, 69.9, 70.0, 70.2, 70.4, 70.5, 70.8, 72.0,
72.2, 76.5, 77.8, 80.7, 82.1, 98.0, 123.2, 131.9, 132.0,
132.1, 132.2, 133.8, 168.1. Anal. Calcd for C55H58O18N4:
C, 62.14; H, 5.50. Found: C, 62.31; H, 5.32.
4.1.9. Methyl 2,3,4,6-tetra-O-[+5-+tetrahydro-2H-pyra-
nyl)oxy)-3-oxapentyl]-a-d-glucopyranoside +10). Sodium
hydride '60% dispersion in oil, 8.0 g, 200 mmol) was added
to a stirred solution of methyl a-d-glucoside 1 '4.85 g,
25 mmol) in anhydrous DMF '125 mL) at 08C. After stirring
for 30 min, the iodide 9 '60.0 g, 200 mmol) was added drop-
wise at 08C. The cooling bath was removed and the ¯ask
was immersed in an oil bath at 508C. After stirring overnight
the reaction mixture was evaporated, then the residue was
dissolved in CH2Cl2 '300 mL) and washed with water
'2£100 mL). The organic phase was dried 'MgSO4),
®ltered, and concentrated, then the oily residue was puri®ed
by ¯ash chromatography '0!5% MeOH in CHCl3) to afford
the polyether 10 as a yellow oil '4.2 g, 19%). Rf 0.37
'CHCl3/MeOH 95:5). nmax'CHCl3) 3490, 3040, 2870,
1460, 1450, 1384, 1350, 1202, 1078, 1020, 990, 930, 884,
760, 680 cm21. MS'FAB): 906 'M1Na)1. 1H NMR
'500 MHz, CDCl3): d 1.54 'm, 16H, CH2 of THP), 1.68
'm, 4H, CH2 of THP), 1.79 'm, 4H, CH2 of THP), 3.27
'm, 1H, H-2), 3.34 's, 3H, CH3), 3.42±3.85 'm, 4H),
3.51±3.76 'm, 29H), 3.78±3.85 'm, 9H), 3.90±4.07 'm,
3H), 4.59 '4H, s, CH of THP), 4.76 '1H, d, Jˆ3.5 Hz,
H-1). Anal. Calcd for C43H78O18: C, 58.49; H, 8.90.
Found: C, 58.11; H, 8.99.
4.1.12. 1-O-Acetyl-2,3,4,6-tetra-O-+5-phthalimido-3-oxa-
pentyl)-d-glucopyranose +13). A solution of the phthali-
mide 12 '3.5 g, 3.3 mmol) in acetic anhydride '15 mL)
was stirred at 2208C for 10 min. To this solution was
added precooled '08C) Ac2O/H2SO4 '50:1, 7.5 mL) drop-
wise during 5 min, then the reaction mixture was left at
2208C for 3 days. The reaction mixture was diluted with
CH2Cl2 '300 mL) and was washed successively with cold
saturated NaHCO3 '100 mL) and water '2£100 mL). The
organic layer was dried 'MgSO4), ®ltered, and concentrated,
then co-distilled with several portions of toluene. The resi-
due was puri®ed by ¯ash chromatography 'EtOAc/Et2O
85:15) to give the acetate 13 as a colourless oil '2.8 g,
78%). Rf 0.34 'EtOAc/Et2O 9:1). [a]24 ˆ133.0 'c 1.0,
D
CHCl3). nmax'CHCl3) 3020, 1775, 1750, 1716, 1400,
1220, 1100, 1050, 930, 760, 670, 540 cm21. MS'FAB):
1224 'M1Cs)1, 1114 'M1Na)1. 1H NMR '500 MHz,
CDCl3) 'a-anomer only): d 2.02 's, 3H, CH3 of OAc),
3.20±3.28 'm, 2H, H-2, H-5), 3.52±3.75 'm, 24H, CH2
and sugar protons), 3.72±3.91 'm, 12H, CH2 and sugar
protons), 6.14 '1H, d, J1,2ˆ3.5 Hz, H-1), 7.63±7.82 '16H,
m, 4£ArH). Integration of the signals of the H-1 protons
indicated that the a/b ratio was 7:1. 13C NMR '62.9 Hz,
CDCl3) 'a-anomer only): d 21.0, 37.3, 37.4, 67.6, 67.7,
67.8, 67.9, 69.2, 69.9, 70.2, 70.4, 70.6, 70.7, 70.8, 72.3,
72.7, 76.5, 78.8, 81.7, 90.0, 123.2, 132.2, 133.9, 168.6.
Anal. Calcd for C56H58O19N4: C, 61.65; H, 5.36. Found:
C, 61.27; H, 5.23.
4.1.10. Methyl 2,3,4,6-tetra-O-+5-hydroxy-3-oxapentyl)-
a-d-glucopyranoside +11). 4-Toluenesulfonic acid mono-
hydrate '0.23 g, 1.2 mmol) was added to a solution of the
polyether 10 '4.2 g, 4.8 mmol) in CHCl3 '30 mL) and
MeOH '30 mL). The solution was stirred at 558C for 2 h,
then the catalyst was neutralised by the addition of triethyl-
amine '0.2 mL) and the solvent was evaporated. The residue
was puri®ed by ¯ash chromatography 'CHCl3/MeOH 9:1)
to give the tetraol 11 as a colourless oil '1.8 g, 69%). Rf 0.19
'CHCl3/MeOH 85:15). nmax'CHCl3) 3620, 2960, 1460,
1450, 1380, 1360, 1222, 1080, 1020, 990, 940, 872, 750,
680, 550 cm21. MS'FAB): 679 'M1Cs)1, 585 'M1K)1,
4.1.13. 2,3,4,6-Tetra-O-+5-phthalimido-3-oxapentyl)-d-
glucopyranosyl azide +14). A solution of the acetate 13
'1.3 g, 1.2 mmol) in dry CH2Cl2 '30 mL) was stirred with
azidotrimethylsilane '0.4 mL, 3.0 mmol) and tin'IV)
chloride '0.07 mL, 0.6 mmol) for 24 h at room temperature.
The solution was diluted with CH2Cl2 '30 mL) and washed
1
569 'M1Na)1. H NMR '500 MHz, CDCl3): d 3.17 't,
1H, J2,3ˆ9.5 Hz, H-2), 3.26 'dd, 1H, J5,6ˆ6.5 Hz, H-5),
3.31 's, 3H, OCH3), 3.42±3.3.61 'm, 29H), 3.60 'm, 2H),
3.66 'm, 2H), 3.70 'm, 1H), 3.87 'm, 2H), 4.79 'd, 1H,

8740
R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
with 1 M KF solution '30 mL) then with water '50 mL). The
organic phase was dried 'MgSO4), ®ltered, and concentrated
to afford the azide 14 as a colourless foam '1.13 g, 88%). Rf
'50 mL). After stirring vigorously for 3 h a clear, pale
yellow solution had formed and after a further 1 h more
acrylonitrile '6.4 mL) and DBU '0.60 mL) were added.
After stirring overnight the solution was evaporated and
the residue was puri®ed by ¯ash chromatography '50±
75% EtOAc/hexane) to give the tetranitrile 18 as a colour-
0.41 'EtOAc/Et2O 9:1). [a]24 ˆ118.6 'c 1.0, CHCl3).
D
n
max'CHCl3) 3020, 2120, 1775, 1716, 1380, 1220, 1090,
940, 760, 670, 540 cm21. MS'FAB): 1207 'M1Cs)1,
1097 'M1Na)1. H NMR '500 MHz, CDCl3) 'a-anomer
less oil, 2.61 g '64%). Rf 0.69 'EtOAc). [a]24 ˆ219.1 'c
1
D
only): d 3.25 '2H, m, H-2, H-5), 3.47±3.69 '24H, m, CH2
and sugar protons), 3.70±3.88 '12H, m, CH2 and sugar
protons), 5.94 '1H, d, Jˆ3.5 Hz, H-1), 7.54±7.80 '16H,
m, 4£ArH). Integration of the signals of the H-1 protons
indicated that the a/b ratio was 2:1. 13C NMR '62.9 Hz,
CDCl3) 'a-anomer only): d 37.4, 67.9, 68.0, 69.2, 69.5,
70.0, 70.3, 70.5, 70.7, 71.00, 71.4, 72.0, 72.3, 72.5, 76.5,
77.5, 80.8, 81.9, 85.0, 87.8, 91.0, 123.2, 132.2, 133.7, 166.1.
Anal. Calcd for C54H55O17N7: C, 60.39; H, 5.16. Found: C,
60.52; H, 5.01.
1.1, CHCl3). nmax'thin ®lm) 2251, 2118, 1117 cm21. ES-
1
MS: 418 'MH)1, 435 'M1NH4)1, 440 'M1Na) 1. H
NMR '300 MHz, CDCl3)
d
2.59±2.66 'm, 8H,
4£CH2CN), 3.38 'dd, 1H, H-3, Jˆ2.6, 9.6 Hz), 3.45 'dd,
1H, H-2, Jˆ8.0, 9.6 Hz), 3.61±4.26 'm, 12H, 5£CH2O
and 2£CHO), 4.58 'd, 1H, H-1, Jˆ8.0 Hz). 13C NMR
'75 MHz, CDCl3) d 18.9, 19.2, 19.2, 19.4, 65.9, 66.5,
67.3, 67.8, 68.2, 74.5, 74.7, 78.9, 82.4, 90.0, 117.9, 118.0,
118.1, 118.4. HRMS 'TOF) calcd for C18H24N7O5 'M1H)1
418.1839, found 418.1865.
4.1.14. 2,3,4,6-Tetra-O-+5-phthalimido-3-oxapentyl)-d-
glucopyranosylamine +15). The azido sugar 14 '110 mg,
0.102 mmol) dissolved in EtOAc '10 mL) was hydro-
genated using 10% Pd/C catalyst '60 mg) for 2 days at
room temperature. The catalyst was ®ltered off and was
washed with EtOAc '40 mL), then the ®ltrate was evapo-
rated. The residue was puri®ed by ¯ash chromatography
'CHCl3/MeOH 97:3) to give the amine 15 as a colourless
foam '80 mg, 76%). Rf 0.20 'CHCl3/MeOH 95:5).
4.1.17. 6-+2,3,4,6-Tetra-O-+2-cyanoethyl)-b-d-galacto-
pyranosylamino)-6-oxohexanoic acid, benzyl ester +19).
A solution of the tetranitrile azide 18 '1.22 g, 2.92 mmol) in
THF '75 mL) was hydrogenated at atmospheric pressure
over 10% Pd/C '100 mg) for 2 h, at which time TLC
indicated the complete consumption of starting material.
The atmosphere was replaced with argon, and adipic acid,
mono benzyl ester '0.76 g, 3.2 mmol), O-benzotriazol-1-yl-
N,N,N0,N0-tetramethyluronium hexa¯urophosphate 'HBTU)
'1.16 g, 3.07 mmol) and N,N-diisopropylethylamine
'610 mL, 450 mg, 3.5 mmol) were added. After stirring
overnight the mixture was ®ltered through Celitew and the
®lter pad was washed well with methanol. The ®ltrate was
evaporated and the residue was dissolved in EtOAc
'200 mL) and washed with 5% HCl '3£100 mL), saturated
NaHCO3 '2£100 mL) and brine '50 mL) then dried
'MgSO4) and evaporated to a pale yellow syrup. This was
puri®ed by ¯ash chromatography 'EtOAc) to give the
a-anomer benzyl ester 19 as a colourless oil '0.98 g,
55%). Rf 0.39 'EtOAc). nmax'thin ®lm) 2251, 1732, 1670,
1541, 1115 cm21. ES-MS: 610 'M1H) 1, 627 'M1NH4)1,
n
max'CHCl3) 3500, 3410, 3020, 1775, 1720, 1380, 1220,
1090, 950, 770, 670, 540 cm21
.
MS'FAB): 1071
'M1Na)1. H NMR '500 MHz, CDCl3): d 3.12±3.21 'm,
2H, H-2, H-5), 3.47±3.93 'm, 38H), 5.21 'd, 1H,
J1,2ˆ3.5 Hz, H-1), 7.68±7.81 'm, 16H, 4£ArH). 13C NMR
'62.9 Hz, CDCl3): d 37.4, 67.6, 67.8, 69.7, 70.0, 70.2 70.6,
71.7, 71.9, 72.1, 72.2, 75.5, 75.7, 76.5, 77.0, 77.9, 79.6,
81.9, 82.3, 83.9, 85.7, 86.0, 86.4, 89.1, 91.0, 123.2, 132.2,
133.9, 168.1. Anal. Calcd for C54H57O17N5: C, 61.68; H,
5.48. Found: C, 60.31; H, 5.29.
1
4.1.15. 4-[2,3,4,6-Tetra-O-+5-phthalimido-3-oxapentyl)-
d-glucopyranosylamino]-4-oxobutanoic acid +16). The
amino sugar 15 '45 mg, 0.04 mmol) was re¯uxed with
succinic anhydride '5.4 mg, 0.05 mmol) in dry CH2Cl2
'5 mL) overnight. The solvent was evaporated and the resi-
due was puri®ed by chromatography 'CHCl3/MeOH 95:5)
to give the acid 16 as a colourless foam '47 mg, 84%). Rf
0.12 'CHCl3/MeOH 95:5). nmax'KBr) 3470, 3400, 2980,
1778, 1716, 1660, 1530, 1460, 1380, 1240, 1080, 1040,
966, 825, 740, 670, 540, 440 cm21. MS 'FAB): 1171
1
632 'M1Na)1. H NMR '300 MHz, CDCl3) d 1.62±1.68
'm, 4H, CH2CH2), 2.23±2.28 'm, 2H, CH2CON), 2.36 't,
2H, CH2CO2, Jˆ6.9 Hz), 2.51±2.65 'm, 8H, 4£CH2CN),
3.47 'dd, 1H, H6A, Jˆ2.4, 9.4 Hz), 3.57, 'br d, 1H, H6B,
Jˆ9.0 Hz), 3.65±3.98 'm, 12H, 4£CH2O and 4£CHO) 5.04
't, 1H, H-1, Jˆ9.2 Hz), 5.09 's, 2H, CH2OPh), 6.84 'd, 1H,
NH, Jˆ9.3 Hz), 7.33 'br s, 5H, 5£Ar-H). 13C NMR
'75 MHz, CDCl3) d 18.8, 19.2, 19.3, 19.4, 24.2, 24.5,
33.7, 35.9, 65.7, 65.8, 66.2, 67.5, 68.3, 73.4, 74.5, 78.3,
79.0, 83.5, 118.1, 118.2, 118.6, 118.7, 128.0, 128.2, 128.5,
135.9, 173.3, 173.7. HRMS 'TOF) calcd for C31H40N5O8
'M1H)1 610.2875, found 610.2877.
1
'M1Na)1, 1149 'M1H)1. H NMR '500 MHz, CDCl3):
d 2.59 'm, 2H, HNOCCH2), 2.73 'm, 2H, HOOCCH2),
3.10±3.18 'm, 2H, H-2, H-5), 3.44±3.90 'm, 37H), 5.20
'd, 1H, J1,2ˆ3.5 Hz, H-1), 7.67±7.80 'm, 16H, 4£ArH).
13C NMR '62.9 Hz, CDCl3): d 30.8, 31.2, 37.4, 67.6, 67.8,
69.7, 70.0, 70.2 70.6, 71.7, 71.9, 72.1, 72.2, 75.5, 75.7, 76.5,
77.0, 77.9, 79.6, 81.9, 82.3, 83.9, 85.7, 86.0, 86.4, 89.1,
91.0, 123.2, 132.2, 133.9, 168.1. Anal. Calcd for
C58H61O20N5: C, 60.68; H, 5.35. Found: C, 60.41; H, 5.22.
4.1.18. 6-+2,3,4,6-Tetra-O-+3-tert-butoxycarbonylamino-
propyl)-b-d-galactopyranosylamino)-6-oxohexanoic acid
+20). Sodium borohydride '2.26 g, 59.7 mmol) was added
cautiously and portionwise to a stirred, chilled '08C) solu-
tion of the tetranitrile 19 '910 mg, 1.49 mmol), CoCl2´6H2O
'2.84 g, 11.9 mmol) and di-tert-butyl dicarbonate '1.63 g,
7.48 mmol) in MeOH '40 mL). The ice-bath was removed
and stirring was continued for 4 h. Water '10 mL) was
added, then the pH was raised to 13 by the addition of
LiOH´H2O. After stirring overnight the MeOH was evapo-
rated and the residue was diluted with water '100 mL) and
4.1.16. 2,3,4,6-Tetra-O-+cyanoethyl)-b-d-galactopyrano-
syl azide +18). Acrylonitrile '6.4 mL, 5.2 g, 97 mmol) was
added to a stirred suspension of the galactosyl azide13 17
'2.00 g, 9.75 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-
ene 'DBU) '0.60 mL, 0.60 g, 3.9 mmol) in acetonitrile

R. P. McGeary et al. / Tetrahedron 57 <2001) 8733±8742
8741
®ltered through a pad of Celitew. The ®ltrate was washed
with Et2O '20 mL), then CHCl3 '200 mL) was added and the
pH of the aqueous phase was lowered to 2 with 5% HCl. The
layers were separated and the organic phase was evaporated.
The residue was dissolved in a little 10% MeOH in CHCl3
and ®ltered through a plug of silica. Evaporation of the
®ltrate gave the product 20 as a colourless foam, 640 mg
The resin was washed with 10% N,N-diisopropylamine in
DMF by shaking the resin for 2£1 min. The resin was
thoroughly washed with DMF, then the resin was shaken
for 10 min with 2-'tert-butoxycarbonylamino)dodecanoic
acid '364 mg, 1.16 mmol, 4.0 equiv.), HBTU '2.4 mL of a
0.5 M solution in DMF) and DIPEA '255 mL, 189 mg,
1.46 mmol), resulting in a negative ninhydrin test. After
rinsing the resin well with DMF, the BOC-groupwas
removed by treatment with neat TFA '2£1 min), then the
resin was again washed well with DMF. Compound 20
'352 mg, 0.577 mmol) was coupled to the resin using the
same protocol. The four BOC groups were removed by
treatment with neat TFA '2£1 min), and the resin was
washed well with DMF, then MeOH and ®nally CH2Cl2,
then dried under vacuum to give 570 mg of loaded resin
'25).
'46%). Rf 0.54 '5% MeOH in EtOAc). [a]24 ˆ17.0 'c 1.0,
D
CHCl3). nmax'thin ®lm) 3348, 1693, 1521 cm21. ES-MS:
1
959 'M1Na)1, 937 'MH)1, 837 'MH2BOC)1. H NMR
'300 MHz) d 1.40 'br s, 36H, 4£CMe3), 1.50±1.80 'm, 12H,
6£CH2), 2.25±2.35 'm, 4H, 2£CH2CO), 3.00±3.75 'm,
22H, 4£CH2N, 5£CH2O and 4£CHO), 4.67 'br s, 1H,
H-1), 4.90±5.10 'm, 5H, 5£NH). 13C NMR '75 MHz,
CDCl3) d 24.1, 24.2, 24.5, 28.3, 29.5, 29.6, 29.7, 30.1,
33.5, 33.6, 35.7, 35.8, 37.4, 37.8, 37.9, 38.5, 64.7, 68.2,
68.4, 69.2, 69.9, 70.2, 70.4, 73.7, 73.8, 78.8, 78.9, 79.2,
156.0, 156.1, 156.4, 156.6, 173.9, 176.6. HRMS 'TOF)
calcd for C44H82N5O16 'M1H)1 936.5757, found 936.5742.
4.3.1. Synthesis of compound 26. The loaded resin 25
'285 mg) was swelled in DMF for 30 min, then coupled
with acetic acid using the protocol described above. The
resin was washed successively with DMF, MeOH and
CH2Cl2, dried under vacuum, then cleaved using anhydrous
hydro¯uoric acid with 10% cresol as scavenger at 08C for
1 h. The product was precipitated with diethyl ether, then
lyophilised from water/acetonitrile to give the tetraaceta-
mide 26 as a white powder '37 mg, 28%). ES-MS: 901
4.2. Protocol for loading compounds 8 and 16 onto Wang
resin
FMOC-Gly-Wang resin '100 mg, 0.069 mmol) was swollen
in DMF in a solid phase peptide synthesis reaction vessel for
3 h. The FMOC protecting group was removed with 20%
piperidine in DMF '10 mL) by shaking the resin twice
'2£10 min). The resin was thoroughly washed with DMF,
then 1.5 molar eqvivalents '0.10 mmol) of either 8 or 16
was added together with 2.0 equiv. of HBTU '52 mg,
0.138 mmol), hydroxybenzotriazole '21 mg, 0.138 mmol)
and N,N-diisopropylethylamine '48 mL, 0.28 mmol) in the
minimum amount of DMF. The vessel was shaken for 1 h,
then the process was repeated with the same amount of
either compound 8 or 16, and the coupling reagents, result-
ing in a negative ninhydrin test. The resin was then washed
with DMF several times, giving either compound 21 or 22,
respectively.
'M1H)1, 802, 451 'M12H)21 1H NMR '300 MHz,
.
'CD3)2SO/CDCl3) d 0.67 't, 3H, CH3, Jˆ6.8 Hz), 1.04 'br
s, 16H, 7£CH2), 1.43±1.67 'br m, 14H, 7£CH2), 1.75 'br s,
12H, 4£Ac), 2.04 'br s, 4H, 2£CH2CO), 2.75±4.12 'm,
22H, Ha, 4£CH2N, 3£OCH and 5£OCH2), 6.12 'br s, 1H,
H-1), 6.95±7.74 'm, 8H, 6£NH and NH2). HRMS 'TOF)
calcd for C44H82N7O12 'M1H)1 900.6022, found 900.6014.
Acknowledgements
The authors thank Mr M. Cocksedge, Dr M. Zloh, Mr W.
Baldeo and Mr Alun Jones for their assistance in obtaining
NMR and mass spectra. This work was supported by
Alchemia and The Wellcome Trust.
4.2.1. Protocol for phthaloyl deprotection of 21 and 22,
and for cleaving 23 and 24from the solid support. Resin-
bound glucosides 21 and 22 were treated with hydrazine
hydrate '67 mL, 1.38 mmol) in ethanol '5 mL) at 608C for
1 day. To check the ef®ciency of the deprotection, a sample
of the resin was thoroughly washed with DMF, then with
CH2Cl2/MeOH, and ®nally with CH2Cl2. The resin was
dried and the glucosides 23 and 24 were cleaved from the
resin by stirring the resin with TFA/water/triisopropylsilane
'95:2.5:2.5) for 2 h. The resin was ®ltered off and washed
with TFA. In both cases the solvent was evaporated and the
residue was lyophilised from water/acetonitrile, then puri-
®ed by ¯ash chromatography 'CHCl3/MeOH 9:1). Analyti-
cal samples were obtained by trituration with diethyl ether.
MS'FAB); 23: 587 'M1Na)1, 565 'M1H)1; 24:707
'M1Na)1, 685 'M1H)1. Anal. Calcd for C24H48O9N6
'23): C, 51.05; H, 8.56; N, 14.88. Found: C, 50.78; H,
8.22, N; 14.50. Calcd for C28H56O13N6 '24): C, 49.11; H,
8.24; N, 12.27. Found: C, 49.41; H, 8.07; N, 12.36.
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