The synthesis and immune stimulating action of mannose-capped lysine-based dendrimers

Article abstract of DOI:10.1016/j.tet.2009.02.018

The syntheses of monodisperse lysine dendrimers, capped with two to sixty-four mono-, di- and tri-α-d-mannopyranosyl residues are described. These syntheses used reactive N-hydroxysuccinimide esters to ensure complete reaction of dendrimer amines with the

Full text of DOI:10.1016/j.tet.2009.02.018

Tetrahedron 65 (2009) 2939–2950
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
The synthesis and immune stimulating action of mannose-capped lysine-based
dendrimers
Ben W. Greatrex a, Samuel J. Brodie a, Richard H. Furneaux a, Sarah M. Hook b, Warren T. McBurney b,
,
Gavin F. Paintera, Thomas Rades b, Phillip M. Rendle a
*
a Industrial Research Limited, PO Box 31-310, Lower Hutt 5040, New Zealand
b School of Pharmacy, University of Otago, PO Box 913, Dunedin 9054, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
The syntheses of monodisperse lysine dendrimers, capped with two to sixty-four mono-, di- and tri-a-D-
Received 23 October 2008
Received in revised form 16 January 2009
Accepted 5 February 2009
Available online 12 February 2009
mannopyranosyl residues are described. These syntheses used reactive N-hydroxysuccinimide esters to
ensure complete reaction of dendrimer amines with the mannosylating reagents. The purities of the
dendrimers were established by RP-HPLC and MALDI-TOF mass spectrometry and were found to be
excellent. The relative ability of these glycodendrimers to induce dendritic cell maturation was mea-
sured, however, no significant trends were observed.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
these multivalent molecules. The choice of method used to attach
the capping units to the dendrimers is crucial to obtain homoge-
Glycodendrimers are hyper-branched, well defined compounds
that are recognized as ideal structures to exploit of the cluster
glycoside effect and so influence cellular communication events.1,2
The ability to construct dendrimers from a range of starting ma-
terials allows the fine-tuning of the size and shape of the dendrimer
and the spacing of residues displayed on the outer shell. The use of
neous products. The coupling must be highly efficient as slight
deficiencies lead to heterogeneity in the resulting products, which
is magnified for higher generations and separation of these impu-
rities is impractical.
In this paper we report the synthesis of monodisperse mannose
and 1-thiomannose mono- and oligosaccharide-capped lysine
dendrimers developed from a benzhydrylamine core. We in-
vestigated the ability of our novel mannodendrimers to activate
a specific type of immune cell, dendritic cells (DCs), by measuring
the expression of activation markers MHC class II and CD86 using
flow cytometry.
L-lysine as a monomer for dendrimer synthesis is attractive for
pharmaceutical targets as amide bond cleavage within the den-
drimers yields the naturally occurring amino acid, thereby reducing
the risk of metabolic toxicity. The L-lysine-based dendrimers were
first described by Denkewalter et al. and can be synthesized in
a stepwise divergent manner, which conserves the chirality of the
a
-stereocentre of the amino acid.3
2. Results and discussion
The specific targeting of mannose-capped dendrimers to man-
nose receptors, highly expressed in cells of the immune system, has
the potential to provide drug/antigen delivery systems for vacci-
nation or treatment of diseases localized in macrophages and other
antigen-presenting cells.4 Due to the potential of this chemical
2.1. Synthesis of mannose-capped dendrimers
Six generations of lysine-based dendrimers G0 to G5 (Fig. 1)
containing two to sixty-four ‘valence’ amines, respectively, pro-
tected as Boc carbamates, were synthesized as previously de-
scribed.8 Capping the dendrimers required both amine-reactive
mannosyl derivatives and a non-mannose hydroxy compound.
The latter could be used to cap the dendrimers and provide
controls with comparative physical properties for the DC assays.
Mannosyl capping reagents included 1-thiomannose analogues,
which are known for their greater stability to glycosidase action
and their abilities to similarly bind to mannose specific re-
ceptors.9 Mannobiosyl and mannotriosyl derived capping re-
agents were also prepared. The active esters 2, 7, 11, 14, 19 and 23
class, the conjugation of
a-D-mannopyranose units to dendritic
supports has been performed by a number of groups.1,5,6 Usually,
the sugar is functionalized through the anomeric position by use,
for example, of glycosyl isothiocyanates or N-hydroxysuccinimide
(NHS) esters, which can react with amino groups located on the
exterior of the dendrimer. Alternatively, it is possible to adopt thiol-
radical addition and cycloaddition7 strategies for the synthesis of
* Corresponding author. Tel.: þ64 4 931 3167; fax: þ64 4 931 3055.
E-mail address: p.rendle@irl.cri.nz (P.M. Rendle).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.02.018

2940
B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
OAc
OAc
OAc
OAc
O
O
O
AcO
H
N
AcO
a)
AcO
b)
AcO
OAc
O
Lys =
O
OAc
O
Lys
Lys
Lys
Lys
O
Lys
Lys
AcO
HN
AcO
AcO
Lys
Lys
AcO
O
OAc
Lys
Lys
Lys
15
16
Lys
Lys
Lys
Lys
Lys
HN
Lys
CCl3
OAc
Lys
Lys
Lys
Lys
Lys
Lys
OAc
OAc
O
Lys
OAc
O
AcO
AcO
AcO
AcO
Lys
Lys
OAc
O
O
OAc
O
O
AcO
AcO
OAc
AcO
AcO
Lys
Lys
Lys
Lys
Lys
O
Lys
OAc
O
O
Lys
Lys
Lys
Lys
Lys
Lys
AcO
R
5
O
AcO
Lys
Ph2CHNH
17 R = OMe
18 R = OH
19 R = NHS
O
c)
O
AcO
Lys
Lys
AcO
O
d)
O
20
OMe
5
Lys
Lys
Lys
Lys
Lys
Lys
G0
Lys
Lys
Lys
Lys
Lys
Lys
Scheme 2. (a) (i) NH2NH2$AcOH, DMF; (ii) CCl3CN, DBU; (b) (i) TMSOTf, CH2Cl2,
HO(CH2)5CO2Me, (ii) Pyridine, Ac2O; (c) (i) NaOMe/MeOH; (ii) NaOH, H2O; (iii) Pyri-
dine, Ac2O; (d) DCC, N-hydroxysuccinimide, EtOAc.
Lys
Lys
Lys
Lys
G1
Lys
Lys
Lys
Lys
Lys
Lys
gave the free acid 6 from which the required active ester 7 was
derived. For the synthesis of the 1-thiomannosides 11 and 14,
a one-pot procedure described by Ibatullin et al. was adapted.11
Thus, peracetylated mannose 3 was allowed to react with thiourea
in the presence of boron trifluoride etherate to give an intermediate
isothiouronium salt, which was alkylated with the appropriate al-
kyl halide 8 or 9 in the presence of triethylamine, the latter halide
being derived from 6-bromohexanoic acid. From acids 10 and 13,
the latter derived by zinc/acetic acid reduction of 12, the required
acylating reagents 11 and 14 were obtained by use of DCC and N-
hydroxysuccinimide in ethyl acetate. The mannosides 7 and 11 with
the glycolate-derived linker were too reactive for purification on
silica gel and were instead partially purified by removing solids by
filtration. Reactive ester 14, with the longer alkyl linker, could be
purified by flash silica column chromatography.
G2
G3
G4
G5
Figure 1. Structure of lysine dendrimers G0–G5 with benzhydrylamine at the core.
were the key building blocks and their syntheses are outlined in
Schemes 1–3.
Glycolic acid-derived capping reagent 2 was prepared as pre-
viously described (Scheme 1).10 Reaction of peracetylated
a-D-
mannose (3) with benzyl glycolate (4) in the presence of boron
trifluoride etherate gave the mannoside 5, which on hydrogenolysis
Roche et al. determined that the mannose disaccharide,
Man-(1/6)- -Man, was taken up most efficiently by the man-
nose receptor, which is one endocytic receptor expressed by DCs.12
In light of this, the -Man-(1/6)- -Man capping reagent 19
was prepared (Scheme 2). Peracetylated (1/6)-mannobiose 15
a-D-
a-D
O
O
a) b)
HO
AcO
OH
NHS
a
-D
a-D
1
2
a
was converted to the acetylated glycosyl trichloroacetimidate 16,
which was treated with methyl 6-hydroxyhexanoate and TMSOTf
to give the glycoside 17. This perester was saponified to give the
heptahydroxy free acid, which, when acetylated, gave heptaacetate
18 from which the required active ester 19, was obtained after
purification by flash chromatography. Although this multi-step
process gave access to the target compound, a complication arose
during the glycosylation of methyl 6-hydroxyhexanoate (1.5 equiv)
with imidate 16 (1 mol equiv). In addition to the desired glyco-
sidation reaction, transfer of the acetyl group from O-2 of the donor
to the acceptor was observed. This 2-deacetylated hexa-O-acetyl-
mannobiosyl glycoside also participated as a glycosylation acceptor
and gave tetrasaccharide glycoside 20 as a by-product (34%). Such
OAc
OAc
OAc
O
OAc
O
OBn
AcO
AcO
c)
AcO
AcO
HO
OH
+
O
O
O
OAc
R
3
4
5 R = OBn
6 R = OH
7 R = NHS
d)
b)
O
I
OAc
OAc
O
8
e)
AcO
AcO
or
+
3
O
O
S
R
n
Br
OCH2CCl3
5
OH
O
OAc
O
AcO
AcO
AcO
a)
10 n = 1, R = OH
HO
HO
b)
9
11 n = 1, R = NHS
12 n = 5, R = OCH2CCl3
13 n = 5, R = OH
HO
OAc
O
OH
O
O
O
f)
O
HO
AcO
b)
O
O
O
O
14 n = 5, R = NHS
O N
HO
HO
AcO
AcO
O
O
NHS
=
OH
R
5
5
O
O
OAc
O
OH
OH
OAc
21
22 R = OH
23 R = NHS
b)
Scheme 1. (a) AcCl; (b) DCC, N-hydroxysuccinimide, EtOAc; (c) BF3$OEt2, CH2Cl2; (d)
H2, 10% Pd/C solvent; (e) BF3$OEt2, (NH2)2CS, CH3CN, 3 h reflux then NEt3 and 8 or 9,
0
ꢀC; (f) Zn, AcOH.
Scheme 3. (a) Pyridine, Ac2O; (b) N-hydroxysuccinimide, DCC, EtOAc.

B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
2941
competitive reactions during glycosylations are not unknown.13 In
the present case this problem was ameliorated by repeating the
reaction with excess of the acceptor and by acetylating the mixture
when the reaction was complete. This both re-esterified the C-2
hydroxyl group of the donor and facilitated the chromatographic
removal of the excess methyl 6-hydroxyhexanoate as its acetate. By
this means the yield of glycoside 17 from imidate 16 was increased
from 45% to 76%.
resulting poly-amine salts could be stored for short periods. The
coupling of the active esters 2, 7, 11, 14, 19 and 23 to these poly-
amine salts G0–G5 proceeded in good yield when facilitated by
Hunig’s base (Table 1) to give capped dendrimers 24a–40a. To in-
crease the likelihood of complete capping, DMF was used as solvent
to ensure homogeneity and the reactions were left overnight.
Precipitation of the products from the DMF using water or diethyl
ether, filtration and purification by flash chromatography afforded
material of good purity as indicated by RP-HPLC. Under-capped
material having shorter retention times was clearly visible, and its
disappearance was an excellent indication of reaction progress. The
extent of mannosylation of each product 27a–40a was readily
determined from the ratios of the integrals of the 1H NMR signals of
the mannose anomeric resonances to those of the benzhydryl
aromatic signals. Both of these signals were cleanly resolved from
other resonances. Structure was also confirmed by electro-spray
mass spectroscopy (ESMS). For larger generation protected glyco-
dendrimers, ionization by ESMS was weak, however, MALDI-TOF
MS of these products gave more intense molecular ions and
therefore became the method of choice. It was difficult to achieve
complete capping using ‘short’ linker active esters (7 and 11 where
Z¼1) in even small dendrimers (n¼4 and 8). Increasing the length
of the linker (to Z¼5) solved this problem and increased the yield of
In previous studies involving the mannose binding proteins
Con A and the Fimbrial lectin of E. coli
a-D-Man-(1/6)[a-D-Man-
(1/3)] -Man was shown to be an avid ligand, which possessed
a-D
a multivalent mode of binding.6 We have previously described
a synthesis of compound 2114 and wished to examine the prop-
erties of a mannodendrimer displaying this subunit in our DC
assay. Thus, acetylation of the mannotrioside acid 21 using pyri-
dine/acetic anhydride followed by aqueous workup afforded the
deca-acetylated acid 22, which, when allowed to react with N-
hydroxysuccinimide in the presence of DCC, afforded ester 23
after isolation by flash chromatography (Scheme 3).
With a series of amine-reactive esters in hand, capping of the
lysine dendrimers could proceed. The Boc protected dendrimers
were deprotected using either HCl/MeOH or TFA/DCM, the latter
yielding cleaner products for higher generation material. The
Table 1
Capping of lysine dendrimers with mannose derived active esters
G0-G5
NHBoc
n
O
O
AcO
HO
G1,G2
N
G1,G2
N
H
HCl/MeOH
(method A)
or
H n
n
24a-26a
24b-26b
NaOMe/MeOH (method C)
or
TFA/CH2Cl2
(method B)
NH3/MeOH/H2O (method D)
NHS ester
DMF / DIPEA
Ac4R1O
OAc
O
R1O
OH
O
AcO
HO
Ac4R2O
R2O
O
O
G0-G5
NH2.HX
Y
Y
G0-G5
N
N
G0-G5
z
n
n
z
H
n
H
27b-40b
27a-40a
n
De-Boc method
NHS ester
Per-OAc
De-Ac method
Yielda (%)
Product
Y
Z
R1
R2
Purityb
MS m/z
Calcd
450.20
752.32
806.25
Speciesc
Observed
2
A
A
A
2
7
11
24a
27a
28a
C
C
C
35
37
74
24b
27b
28b
d
O
S
d
1
1
d
H
H
d
H
H
dd
dd
450.20
752.32
806.25
[MþNa]þ
[MþH]þ
[MþNa]þ
>99
4
A
A
A
B
B
A
2
7
11
14
19
23
25a
29a
30a
31a
32a
33a
C
C
C
C
C
C
49
27
41
60
62
53
25b
29b
30b
31b
32b
33b
d
O
S
S
O
O
d
1
1
5
5
d
d
H
H
H
H
99
800.41
724.81
1534.5
1758.7
2343.0
2991.2
800.41
724.89
1534.3
1758.7
2343.2
2991.3
[MþH]þ
H
H
H
Man
Man
dd
>99
93
[Mþ2H]þþ
[MþNa]þ
[MþNa]þ
[MþNa]þ
[MþNa]þ
99
98
5
Man
8
A
A
B
B
B
2
26a
34a
35a
36a
36a
D
C
C
C
D
29
20
63
72
72
26b
34b
35b
36b
36b
d
S
S
O
O
d
1
5
5
5
d
d
H
H
H
H
dd
>99
99
90
97
772.91
2991.0
3439.5
4608.1
772.91
2991.1
3439.3
4606.1
[Mþ2H]þþ
[MþNa]þ
[MþNa]þ
[MþNa]þ
11
14
19
19
H
H
Man
Man
16
B
B
14
19
37a
38a
C
D
63
45
37b
38b
S
O
5
5
H
Man
H
H
90
>97
6806.4
9186
6805.3
9143
[MþNa]þ
[MþNa]þ
32
64
B
B
14
14
39a
40a
C
C
61
97
39b
40b
S
S
5
5
H
H
H
H
de
de
13,525
26,991
13,558
26,811
[MþNa]þ
[MþNa]þ
a
b
c
Isolated combined yield for the capping and deprotection reactions.
Purity determined by analytical RP-HPLC at 220 nm and results do not take into account differences in extinction coefficients.
Molecular species observed in the mass spectrum.
Purity not determined by HPLC although samples pure by NMR.
Purity could not be unambiguously established by HPLC due to peak broadening.
d
e

2942
B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
and 13,851 (base peak). No isotopic distribution patterns were seen
at high molecular weights on the instrument used. Also observed
for 36a was a peak corresponding to loss of a single acetate (6906,
10% base peak height) and a peak at 7078 (10% base peak height,
Mþ114). Altering the laser intensity had no effect on the ratio of
these three peaks. Once deprotected, a sodiated molecular ion was
observed at m/z 4606.6 for 36b, which had an isotope distribution
pattern matching that expected for the molecular formula. Glyco-
sidic bond cleavage giving molecular ions with loss of both one and
two mannose units were observed in the both the mass spectra for
36b and 38b. Since glycosidic bond cleavage was not observed in
the MALDI-TOF mass spectra of 36a and glycosidic bonds are stable
to the deprotection conditions employed, this cleavage is thought
to be due to the acidic matrix (a-cyano-cinnamic acid/dihydroxy-
benzoic acid) used in the MALDI technique. No impurity directly
resulting from the Mþ114 species noted in 36a was observed in
36b. The peak for 38a was broad and two lower m/z broad peaks
were observed that could correspond to singly and doubly under-
capped products, however, no impurities directly resulting from
these under-capped species were observed in 38b.
2.2. Effects of mannose-capped dendrimers on DC activation
Mannodendrimers15
and
other
poly-mannose
neo-
glycoconjugates16,17 can be used in the targeting of molecules to
DCs via the mannose receptor and/or DC-SIGN (dendritic cell-
specific intercellular adhesion molecule-3-grabbing non-integrin).
Mannose-capped dendrimers bind to DC-SIGN with micromolar
affinity, an important receptor involved in DC–T-cell interactions
and HIV virion transport.18 Having at hand a range of mannose-
capped dendrimers with different linkers, ‘valencies’ and number
of sugars per capping group, we investigated their relative ability to
activate DCs derived from the bone marrow of mice (BMDC). This
was established by measuring the expression of activation markers
MHC Class II and CD86 following a two-day incubation period. The
expression of activation markers was determined by flow cytom-
etry and the results expressed as the increase in mean fluorescence
intensity (MFI) over background (i.e., unstimulated DCs), Figure 3.
The mannose-capped dendrimers failed to significantly enhance DC
maturation in vitro relative to LPS making it difficult to elucidate
trends of activity based on structure. There was also little difference
between dendrimers displaying mannose and those ‘control’ mol-
ecules capped with glycolic acid.
Figure 2. MALDI-TOF MS of a [G2] glycodendrimer, acetylated (36a) and deprotected
(36b), and a [G3] glycodendrimer, acetylated (38a) and deprotected (38b).
fully capped dendrimers. This observation is consistent with the
approximation that area on the surface of the dendrimer ‘ball’ per
capping group falls with increasing generation. This effect can be
negated by increasing the radius of the ball by increasing linker
length.
3. Conclusions
Deacetylation of the dendrimers 24a–40a using standard pro-
cedures gave glycolated dendrimers 24b–26b and glycodendrimers
27b–40b. The use of aqueous ammonia in methanol gave improved
yield and purity for larger generation glycodendrimers (for exam-
ple, octavalent 36b). As expected, water solubility of the depro-
tected glycodendrimers increased with generation and number of
sugars per capping group. MALDI-TOF measurements could be used
informatively, as with protected compounds, to analyze product
homogeneity and to confirm structure identity. MALDI-TOF mea-
surements were more useful than NMR for visualizing impurities,
especially on higher generation materials. A broadened peak was
observed for the G5 glycodendrimer 40b, with a m/z range of
25,570–27,990 and an intensity maximum at m/z 26,810. While this
is not unambiguous proof of the structure, the calculated m/z for
glycodendrimer 40b of 26,991 (MþNaþ) falls within the range
observed.
A series of discrete mannose-capped lysine dendrimers have
been described. Variables include: two linker lengths, 1-thio-
mannosides, mono- versus oligo-mannosides and ‘valencies’
ranging from two to sixty-four. The mannodendrimers were com-
pared with glycolated dendrimers and all dendrimers investigated
lacked significant activity compared to LPS in a DC maturation as-
say. Mannose coated dendrimers do not appear to induce DC acti-
vation required for an immune response in vitro in mice. Other uses
for mannose coated dendrimers are being investigated.
4. Experimental
4.1. General methods
Analytical TLC was carried out on pre-coated 0.25 mm thick
Merck 60 F254 silica gel plates and visualization was by thermal
development after dipping in ammonium molybdate and cer-
ium(IV) sulfate in dilute H2SO4. Flash column chromatography was
As representative examples, the MALDI-TOF mass spectra of the
octavalent and hexadecavalent
a-D-Man-(1/6)-a-D-Man capped
dendrimers 36b and 38b and the intermediate acetylated products
36a and 38a, respectively, are presented in Figure 2. When fully
acetylated, the molecules have sodiated molecular ions at m/z 6964
conducted using silica gel 60 (40–60
mm). Analytical RP-HPLC was
conducted on a Waters Symmetry300Ô C18 5
mm, 3.9ꢁ150 mm

B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
2943
Figure 3. Activation of murine bone marrow derived dendritic cells (BMDCs) in response to glycodendrimer constructs. Results are expressed as fold increase in the mean fluo-
rescence index (MFI) of the sample over the background (unstimulated DCs) following incubation with 0.5 nM of glycodendrimer for 48 h. The results represent the meanꢃthe
standard deviation of triplicate experiments.
column eluting with 0.1% trifluoroacetic acid in water/methanol
gradients. Melting points were measured on a Reichert hot-stage
microscope and are uncorrected. Optical rotations were measured
11.9 and 5.1, H-6a), 4.20 (1H, d, J 16.2, OCHHCO2), 4.14 (1H, ddd, J 9.4,
5.1 and 1.8, H-5), 4.05 (1H, dd, J 11.9 and 2.0, H-6b), 2.14 (3H, s,
COCH3), 2.07 (3H, s, COCH3), 2.03 (3H, s, COCH3), 1.99 (3H, s,
COCH3); dC 170.4, 169.6, 169.6, 168.8, 135.0, 128.6, 128.5, 128.3, 97.9
(JC-1,H-1 173.8), 69.1, 68.8, 66.8, 65.8, 64.6, 62.2, 20.7, 20.5, 20.5; m/z
(ESI) 514.1892 ([MþNH4]þ. C23H32O12N requires 514.1919).
using a Perkin Elmer 241 polarimeter and [a]D values are reported
in 10ꢂ1 deg cm2 gꢂ1. Microanalyses were carried out at the Camp-
bell Microanalytical Laboratory, University of Otago, New Zealand.
1H and 13C NMR spectra were recorded at 300 MHz and 75 MHz,
respectively, and run in CDCl3 with TMS as an internal standard
unless otherwise stated; J values are given in hertz. Mass spectra
were recorded on a Applied Biosystems Voyager-DE PRO MALDI-
TOF (MALDI) and externally calibrated or on a Water Micromass Q-
Tof Premier (ESI) mass spectrometer as specified. Dendrimers were
named according to the recommendations set out by Friedhoven
and Vo¨gtle.19
4.3. (N-Succinimidyloxycarbonyl)methyl 2,3,4,6-tetra-O-
acetyl-a-D-mannopyranoside (7)
To a stirred solution of glycoside 5 (1.32 g, 2.66 mmol) in EtOAc
(85 cm3) was added 10% Pd on carbon (10%, 525 mg). The flask was
flushed with hydrogen several times and a hydrogen balloon was
fitted. After 2 h, the reaction mixture was diluted with EtOAc and
filtered through Celite. The solids were washed with EtOAc and the
filtrate and washings were concentrated in vacuo to give carboxy-
4.2. (Benzyloxycarbonyl)methyl 2,3,4,6-tetra-O-acetyl-a-D-
mannopyranoside (5)
methyl tetra-O-acetyl-a-D-mannopyranoside (6) (594 mg, 55%). Rf
0.21 (0.1% AcOH in ethyl acetate); dH 7.99 (1H, br s, CO2H), 5.41–5.33
(2H, m, H-2, H-3), 5.28 (1H, dd, J 9.6 and 9.6, H-4), 4.93 (1H, br s, H-
1), 4.30–4.08 (H-5, m, 5H, H-6a, H-6b, OCH2CO), 2.15 (3H, s, COCH3),
2.09 (3H, s, COCH3), 2.05 (3H, s, COCH3), 1.99 (3H, s, COCH3); dC
174.0, 170.9, 170.2, 169.9, 169.7, 97.7, 69.4, 68.8, 68.8, 66.2, 65.1, 62.5,
20.8, 20.6, 20.6.
To a stirred solution of acid 6 (682 mg, 1.68 mmol) dissolved in
EtOAc (35 cm3), N-hydroxysuccinimide (200 mg, 1.74 mmol) was
added, followed by DCC (360 mg, 1.74 mmol). The reaction mixture
was stirred for 2 h and was filtered. The solids were washed with
EtOAc and the filtrate and washings were concentrated in vacuo.
The residue was taken up in cold CH2Cl2, the solution was filtered
and the filtrate concentrated in vacuo to give the title compound as
To a stirred solution of penta-O-acetyl-a-D-mannopyranose (3)
(1.87 g, 4.78 mmol) and benzyl glycolate (4) (3.12 g, 18.8 mmol) in
CH2Cl2 (30 cm3) at 0 ꢀC under Ar, BF3$Et2O (1.1 cm3, 8.94 mmol)
was added. After 1 h at 0 ꢀC, the ice bath was removed and the
reaction mixture stirred for 72 h, diluted with CH2Cl2 and the or-
ganic layer washed with satd NaHCO3 (aq), brine, dried (MgSO4)
and concentrated in vacuo. Purification of the resulting residue by
flash chromatography (80:20 to 50:50 EtOAc/hexane) afforded
a colourless oil (1.78 g) contaminated with benzyl (2-acetoxy)-
acetate. The crude mixture was taken up in pyridine (3 cm3) and
acetic anhydride (1.5 cm3) was added, the mixture was stirred
overnight and concentrated in vacuo. Purification of the residue by
flash chromatography afforded the pure title compound as a col-
a colourless gum (861 mg, 98%) containing dicyclohexylurea
18
22
ourless oil (1.32 g, 55%). [
a]
þ53.4 (c 0.25, CHCl3); Found: C, 55.8;
(approx. 9% w/w). Rf 0.62 (0.1% AcOH in EtOAc); [
a
]
D
þ48.8 (c 0.25,
D
H, 5.9. C23H28O12 requires C, 55.6; H, 5.7%; dH 7.40–7.31 (5H, m,
C6H5CH2), 5.41–5.24 (3H, m, H-2, H-3, H-4), 5.19 (2H, br s, C6H5CH2),
4.95 (1H, d, J 1.2, H-1), 4.30 (1H, d, J 16.2, OCHHCO2), 4.24 (1H, dd, J
CHCl3); dH 5.38–5.26 (3H, m, H-2, H-3, H-4), 4.97 (d, J 1.3, 1H, H-1),
4.63 (1H, d, J 16.9, OCHHCO), 4.53 (1H, d, J 16.9, OCHHCO), 4.28 (1H,
dd, J 12.2 and 4.7, H-6a), 4.16–4.06 (2H, m, H-5, H-6b), 2.88–2.80

2944
B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
(4H, m, CO(CH2)2CO), 2.14 (3H, s, COCH3), 2.09 (3H, s, COCH3), 2.03
(3H, s, COCH3), 1.98 (3H, s, COCH3); dC 170.5, 169.6, 169.5, 168.4,
164.7, 98.2 (JC-1,H-1 172.9), 69.4, 68.9, 68.6, 65.6, 62.6, 62.0, 25.4,
20.6, 20.5, 20.5; m/z (ESI) 521.25 ([MþNH4]þ. C20H29N2O14 requires
521.16).
(20 cm3) and brine (20 cm3). The organic layer was dried (MgSO4)
and the volatiles were removed. Distillation of the product gave the
title compound as a colourless oil (8.82 g, 89%) with data consistent
with the literature.20 Bp 124 ꢀC, 1.0 mmHg; dH 4.74 (2H, s,
OCH2CCl3), 3.41 (2H, t, J 6.6, CH2Br), 2.49 (2H, t, J 7.4, CH2CO), 1.88
(2H, tt, J 6.9 and 6.7), 1.74 (2H, tt, J 6.3 and 6.3), 1.58–1.48 (2H, m); dC
172.1, 95.3, 74.3, 34.0, 33.6, 32.6, 27.9, 24.2.
4.4. Carboxymethyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-
mannopyranoside (10)
4.7. 5-(2,2,2-Trichloroethoxycarbonyl)pentyl 2,3,4,6-tetra-O-
Penta-O-acetyl-
a
-D-mannopyranose (3) (11.6 g, 30 mmol, azeo-
acetyl-1-thio-a-D-mannopyranoside (12)
tropically dried with toluene 2ꢁ50 cm3) and thiourea (2.49 g,
33 mmol,) were suspended in MeCN (50 cm3, anhydrous grade).
BF3$Et2O (13.2 cm3, 14.8 g, 104 mmol) was added and the mixture
heated under reflux for 2.5 h. The reaction solution was cooled to
0 ꢀC and Et3N (18.5 cm3, 13.5 g, 134 mmol, 4.5 mol equiv) was
added dropwise over 15 min, followed by iodoacetic acid (6.08 g,
33 mmol, 1.1 mol equiv). The reaction mixture was stirred at rt
overnight, and concentrated to a viscous, oily slurry by rotary
evaporation. The residue was partitioned between H2O (350 cm3)
and CH2Cl2 (200 cm3), the organic layer was separated and the
aqueous layer extracted with further CH2Cl2 (4ꢁ100 cm3). The
combined organic extracts were washed with H2O (200 cm3), brine
(200 cm3), dried (MgSO4), filtered and concentrated in vacuo. This
crude product was purified by flash chromatography (98:0.25:2 to
98:0.85:2 CH2Cl2/MeOH/AcOH) to give fractions that were taken to
dryness and then evaporated with toluene (ꢁ2), to give acid 10 as
To a stirred solution of penta-O-acetyl-
a-D-mannopyranose (3)
(9.1 g, 23 mmol) in dry CH3CN (50 cm3), thiourea (1.92 g, 25 mmol)
and BF3$Et2O (11.5 g, 81 mmol) were added. The mixture was
heated under reflux for 3 h then cooled to 0 ꢀC. Et3N (10.5 g,
103 mmol) was slowly added over 15 min followed by 2,2,2-tri-
chloroethyl 6-bromohexanoate (9) (8.2 g, 25.2 mmol). The reaction
mixture was stirred for 16 h at rt and H2O (100 cm3) was added. The
aqueous layer was extracted with CHCl3 (2ꢁ150 cm3) and the
combined organic layers were dried (MgSO4), concentrated in
vacuo and the residue was purified by flash chromatography to give
the title compound (12) as a crystalline colourless solid (4.8 g, 35%).
Mp 77.1–79.4 ꢀC; Rf 0.56 (60:40, hexane/EtOAc); [
22
a
]
þ66.2 (c 1.0,
D
MeOH); dH 5.34–5.24 (4H, m, H-1, H-2, H-3, H-4), 4.75 (2H, s,
CH2CCl3), 4.37–4.29 (2H, m, H-5, H-6a), 4.10 (1H, dd, J 11.6 and 2.0,
H-6b), 2.71–2.53 (2H, m, SCH2), 2.48 (2H, t, J 7.1, CH2CO), 2.16 (3H, s,
CH3CO), 2.10 (3H, s, CH3CO), 2.05 (3H, s, CH3CO),1.99 (3H, s, CH3CO),
1.76–1.62 (4H, m), 1.52–1.43 (2H, m); dC 172.0, 170.8, 170.3, 170.0,
170.0, 95.4, 82.8 (JC-1,H-1 166.95), 42.2, 71.5, 69.8, 69.3, 66.7, 62.8,
34.0, 31.3, 29.3, 28.4, 24.5, 21.2, 21.0, 20.9, 20.9; m/z (ESI) 626.0968
([MþNH4]þ. C22H35Cl3NO11S requires 626.0990).
a pale yellow oil (6.0 g, 48%). Rf 0.57 (94:5:1, CH2Cl2/MeOH/AcOH);
24
[
a]
þ109.5 (c 1.0 in CHCl3); (Found: C, 45.2; H, 5.4; S, 7.3.
D
C16H22O11S requires C, 45.5; H, 5.25; S, 7.6%); dH 5.45 (1H, d, J 1.1, H-
1), 5.39 (1H, dd, J 3.3 and 1.4, H-2), 5.34 (1H, dd, J 10.0 and 9.5, H-4),
5.25 (1H, dd, J 10.0 and 3.4, H-3), 4.37 (1H, ddd, J 9.5, 4.9 and 2.2, H-
5), 4.30 (1H, dd, J 12.2 and 4.9, H-6a), 4.08 (1H, dd, J 12.1 and 2.2, H-
6b), 3.52 (1H, d, J 15.6, SCHHCO2), 3.32 (1H, d, J 15.6, SCHHCO2), 2.17
(3H, s, COCH3), 2.10 (3H, s, COCH3), 2.06 (3H, s, COCH3), 1.99 (3H, s,
COCH3); dC 174.3, 171.3, 170.3, 170.3, 170.1, 82.6 (JC-1,H-1 171.0), 70.6,
69.8, 69.8, 66.5, 62.7, 32.2, 21.2, 21.1, 21.0, 20.9; m/z (ESIꢂ) 421.078
([MꢂH]þ. C16H21O11S: requires 421.080).
4.8. 5-Carboxypentyl 2,3,4,6-tetra-O-acetyl-1-thio-a-D-
mannopyranoside (13)
To a suspension of zinc dust (1.5 g) in acetic acid (10 cm3),
a solution of mannopyranoside 12 (592 mg, 1 mmol) in acetic acid
(5 cm3) was added. The reaction mixture was stirred for 6 h, filtered
and the filtered solids were washed with EtOAc. The filtrate and
washings were concentrated and the residue was purified by flash
4.5. (N-Succinimidyloxycarbonyl)methyl 2,3,4,6-tetra-O-
acetyl-1-thio-a-D-mannopyranoside (11)
chromatography to give the title compound 13 as a crystalline
22
Acid 10 (5.75 g, 14 mmol, azeotropically dried with toluene) was
dissolved in EtOAc (40 cm3, dried over K2CO3) and the solution was
cooled to 0 ꢀC. N-Hydroxysuccinimide (1.57 g, 14 mmol) and then
DCC (2.81 g, 14 mmol) were added and the mixture was stirred at rt
for 1 h. The reaction mixture was filtered, the filtrate taken to
dryness under reduced pressure, dissolved in CH2Cl2 (15 cm3, dried
over K2CO3) and filtered again. The filtrate was concentrated to give
the title compound 11 as a pale yellow foam (6.95 g, 98%); 1H NMR
indicated contamination with 5–10% w/w of dicyclohexylurea. Rf
0.60 (95:5, CH2Cl2/MeOH); dH 5.52 (1H, d, J 1.4, H-1), 5.41–5.33 (2H,
m, H-2, H-4), 5.23 (1H, dd, J 10.0 and 3.5, H-3), 4.41–4.32 (2H, m, H-
5, H-6a), 4.03 (1H, m, H-6b), 3.74 (1H, d, J 15.8, SCHHCO), 3.53 (1H,
d, J 15.8, SCHHCO), 2.85 (4H, s, (CH2)2), 2.17 (3H, s, COCH3), 2.11 (3H,
s, COCH3), 2.06 (3H, s, COCH3), 2.00 (3H, s, COCH3); dC 171.0, 170.2,
170.1, 170.0, 169.1, 165.5, 82.8 (C-1, JC-1,H-1 170.5), 70.3, 70.0, 69.7,
66.3, 62.4, 29.5, 25.9, 21.2, 21.1, 21.1, 21.0; m/z (ESI) 537.1377
([MþNH4]þ. C20H29N2O13S requires 537.1385).
white solid (471 mg, 98%). Mp 90.5–92.7 ꢀC; Rf 0.05 (CHCl3); [
a]
D
þ79.1 (c 1.0, MeOH); dH 8.70 (1H, br s, CO2H), 5.27–5.17 (4H, m, H-1,
H-2, H-3, H-4), 4.40–4.35 (1H, br m, H-5), 4.32 (1H, dd, J 12.0 and
4.8, H-6a), 4.11 (1H, dd, J 12.0 and 1.8, H-6b), 2.73–2.54 (2H, m,
SCH2CH2), 2.37 (2H, t, J 7.5, CH2CO2H), 2.17 (3H, s, COCH3), 2.10 (3H,
s, COCH3), 2.06 (3H, s, COCH3), 2.00 (3H, s, COCH3),1.73–1.60 (4H, m,
SCH2CH2, CH2CH2CO2), 1.51–1.39 (2H, m, CH2CH2CH2); dC 178.3,
170.6, 170.0, 169.8, 169.7, 82.4, 77.4, 77.0, 76.5, 71.1, 69.4, 68.9, 66.3,
62.4, 33.6, 31.0, 28.9, 28.0, 24.0, 20.8, 20.6, 20.5; m/z (ESI) 496.1829
([MþNH4]þ. C20H34NO11S requires 496.1847).
4.9. 5-(N-Succinimidyloxycarbonyl)pentyl 2,3,4,6-tetra-O-
acetyl-1-thio-a-D-mannopyranoside (14)
To a stirred solution of the acid 13 (455 mg, 0.95 mmol) in EtOAc
(3 cm3), N-hydroxysuccinimide (114 mg, 0.99 mmol) followed by
DCC (204 mg, 0.99 mmol) were added. The resulting solution was
stirred for 3 h and filtered through cotton wool. Evaporation of the
solvents and purification by flash chromatography afforded the
reactive ester 14 as a colourless oil, which slowly crystallized upon
4.6. 2,2,2-Trichloroethyl 6-bromohexanoate (9)
A solution of 6-bromohexanoic acid (5.92 g, 30.3 mmol), 2,2,2-
trichloroethanol (6.8 g, 45 mmol) and p-TsOH (250 mg) in benzene
(100 cm3) was heated under reflux using a Dean–Stark trap for 30 h
to remove H2O. Upon cooling, the mixture was diluted with ether
(100 cm3) and washed with satd NaHCO3 (50 cm3, aq) then H2O
standing (439 mg, 81%). Mp 83–84 ꢀC (from CH2Cl2/hexane/ether);
22
Rf 0.50 (30:70, hexane/EtOAc); [
a
]
þ73.7 (c 1.2, CHCl3); Found: C,
D
50.25; H, 5.8; N, 2.4. C24H33O13NS requires C, 50.1; H, 5.8; N, 2.4%; dH
5.33–5.25 (4H, m, H-1, H-2, H-3, H-4), 4.39–4.33 (1H, m, H-5), 4.31
(1H, dd, J 12.0 and 5.4, H-6a), 4.09 (1H, dd, J 12.0 and 3.9, H-6b), 2.83

B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
2945
(4H, s, CO(CH2)2CO), 2.72–2.54 (2H, m, SCH2), 2.61 (2H, t, J 6.9,
CH2CO2N), 2.16 (3H, s, COCH3), 2.09 (3H, s, COCH3), 2.09 (3H, s,
COCH3), 1.98 (3H, s, COCH3), 1.79 (2H, tt, J 7.2 and 7.5), 1.68 (2H, tt, J
7.2 and 7.2), 1.57–1.46 (2H, m, CH2CH2CH2); dC 170.9, 170.3, 170.1,
170.1, 169.4, 82.8, 71.5, 69.8, 69.3, 66.7, 62.8, 31.2, 31.1, 29.1, 28.1,
25.9, 24.4, 21.2, 21.0, 21.0, 20.9; m/z (ESI) 593.1981 ([MþNH4]þ.
C24H37O13N2S requires 593.2010).
give the title glycoside 17 as a colourless gum (1.46 g, 71%). Rf 0.40
22
(30:70, hexane/EtOAc); [
a
]
þ58.7 (c 1.16, CHCl3); dH 5.38–5.21
D
(6H, m, H-2, H-3, H-4, H-20, H-30, H-40), 4.87 (1H, d, J 1.3, H-1 or H-
10), 4.78 (1H, d, J 1.4, H-1 or H-10), 4.26 (1H, dd, J 12.1 and 5.3, H-6a0),
4.14 (1H, dd, J 12.1 and 2.1, H-6b0), 4.11–4.05 (1H, m, H-50), 3.95 (1H,
ddd, J 9.5, 5.7 and 2.4, H-5), 3.78 (1H, dd, J 10.9 and 5.8, H-6a), 3.73
(1H, ddd, J 9.5, 6.5 and 6.5, OCHHCH2), 3.68 (3H, s, CO2CH3), 3.57
(1H, dd, J 10.9 and 2.2, H-6b), 3.46 (1H, ddd, J 9.5, 6.5 and 6.5,
OCHHCH2), 2.34 (2H, t, J 7.5), 2.17 (3H, s), 2.16 (3H, s, COCH3), 2.12
(3H, s, COCH3), 2.07 (3H, s, COCH3), 2.05 (3H, s, COCH3), 2.00 (3H, s,
COCH3), 1.99 (3H, s, COCH3), 1.71–1.61 (4H, m, CH2CH2CH2), 1.47–
1.37 (2H, m, CH2CH2CH2); dC 174.0, 170.5, 170.1, 169.8, 169.8, 169.7,
169.5, 97.4 (JC-1,H-1 171), 97.2 (JC-1,H-1 171), 69.6, 69.4, 69.2, 69.2, 68.9,
68.6, 68.0, 66.6, 66.6, 66.0, 62.4, 51.4, 33.8, 28.9, 25.6, 24.6, 20.7,
20.6; m/z (ESI) 787.2639 ([MþNa]þ. C33H48NaO20 requires
787.2637).
4.10. 2,3,4,6-Tetra-O-acetyl-
(1/6)-2,3,4-tri-O-acetyl-
trichloroacetimidate (16)
a
-
D
-mannopyranosyl-
a-D-mannopyranosyl
To a stirred solution peracetylated
a
(1/6)-mannobiose 1521
(6.0 g, 8.8 mmol) in DMF (15 cm3), hydrazine acetate (978 mg,
10.6 mmol) was added. The reaction mixture was warmed at 50 ꢀC
for 30 min and an additional portion of hydrazine acetate (100 mg)
was added. After a further 30 min, the reaction mixture was cooled
to rt and H2O (100 cm3) was added. The aqueous mixture was
extracted with EtOAc (150 cm3) and the organic extracts were
washed with NaHCO3 (2ꢁ50 cm3, aq) and then NaCl solution
(10 cm3, aq). The combined aqueous washings were extracted with
EtOAc (20 cm3) and the combined organic extracts were dried
4.12. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-
mannopyranosyl-(1/6)-[2,3,4,6-tetra-O-acetyl-
mannopyranosyl-(1/6)-2,3,4-tri-O-acetyl-
a-D-
a-D-
a-D-
mannopyranosyl-(1/2)]-3,4-di-O-acetyl-a-D-
mannopyranoside (20)
(MgSO4) and concentrated to give tetra-O-acetyl-
a-D-mannopyr-
anosyl-(1/6)-2,3,4-tri-O-acetyl- -mannopyranose as a col-
a
,
b
-
D
The tetrasaccharide by-product was also isolated from the first
ourless foam (5.49 g, 98% corrected for DMF and EtOAc content). dH
(major anomer) 5.44 (1H, dd, J 10.1 and 3.3), 5.37 (1H, dd, J 9.7 and
3.4), 5.32–5.19 (5H, m), 4.86 (1H, d, J 1.3), 4.28–4.04 (4H, m), 3.96
(1H, br s), 3.78 (1H, dd, J 11.7 and 6.3, H-6a), 3.61 (1H, dd, J 11.7 and
2.6, H-6b), 2.16 (3H, s, COCH3), 2.16 (3H, s, COCH3), 2.11 (3H, s,
COCH3), 2.07 (3H, s, COCH3), 2.06 (3H, s, COCH3), 2.00 (3H, s,
COCH3), 1.99 (3H, s, COCH3). This 1-hydroxy compound (5.49 g,
8.66 mmol) was dissolved in CH2Cl2 (14 cm3) and tri-
chloroacetonitrile (1.52 g, 10.5 mmol) in DBU (133 mg, 0.88 mmol)
was added. The reaction mixture was allowed to stir at rt for 30 min
and, without evaporation, was applied to a column of silica gel and
purified by flash chromatography to give, after removal of the
attempt at the above reaction. Rf 0.17 (30:70, hexane/ethyl acetate);
22
[a
]
þ59.6 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3)
d 5.44–5.24
D
(m, 9H), 5.23 (dd, J 3.0 and 1.8, 1H), 5.16 (dd, J 9.6 and 9.6, 1H), 4.95
(d, J 1.7, 1H), 4.87 (d, J 1.2, 1H), 4.84 (d, J 1.5, 1H), 4.81 (d, J 1.5, 1H),
4.30 (dd, J 8.3 and 5.0, 1H), 4.27 (dd, J 8.6 and 5.2, 1H), 4.18–4.00 (m,
7H), 3.93 (ddd, J 9.7, 6.6 and 2.4, 1H), 3.85–3.69 (m, 3H), 3.67 (s, 3H),
3.60 (dd, J¼11.6 and 2.2,1H), 3.56 (dd, J¼10.8 and 2.6,1H), 3.51–3.42
(m, 1H), 2.37–2.29 (m, 2H), 2.17 (s, 3), 2.15 (s, 3H), 2.15 (s, 3H), 2.13
(s, 3H), 2.10 (s, 3H), 2.10 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 2.06 (s, 3H),
2.05 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H), 1.97 (s, 3H), 1.73–1.59 (m, 4H),
1.47–1.35 (m, 2H); 13C NMR (75 MHz, CDCl3)
d 173.9, 170.6, 170.5,
170.2, 170.0, 169.9, 169.8, 169.7, 169.6, 169.4, 98.8, 98.3, 98.0, 97.3,
70.5, 70.0, 69.5, 69.2, 69.0, 68.9, 68.6, 67.9, 67.4, 67.3, 66.3, 66.1, 65.9,
62.4, 62.2, 51.4, 33.8, 29.0, 25.6, 24.6, 20.8, 20.6; m/z (MALDI-TOF)
1363.50 ([MþNa]þ. C57H80Na1O36 requires 1363.43).
solvent, the
76%). Rf 0.53 (40:60, hexane/EtOAc); [
a-trichloroacetimidate 16 as a colourless foam (5.10 g,
22
a
]
D
þ57.7 (c 1.0, CHCl3); dH
8.82 (1H, s, C(NH)CCl3), 6.23 (1H, d, J 1.8, H-1), 5.47 (1H, dd, J 2.6 and
2.0, H-2), 5.44–5.34 (2H, m), 5.31–5.23 (3H, m), 4.82 (1H, d, J 1.3, H-
10), 4.28 (1H, dd, J 12.5 and 5.2, H-6a0), 4.17–4.06 (3H, m, H-5, H-50,
H-6b0), 3.78 (1H, dd, J 11.0 and 5.4, H-6a), 3.63 (1H, dd, J 11.0 and 2.4,
H-6b), 2.20 (3H, s, COCH3), 2.14 (3H, s, COCH3), 2.10 (3H, s, COCH3),
2.08 (3H, s, COCH3), 2.04 (3H, s, COCH3), 2.01 (3H, s, COCH3), 1.97
(3H, s, COCH3); dC 170.6, 179.9, 169.8, 169.6, 169.6, 159.7, 97.5 (JC-1,H-1
173.9), 94.3 (JC-1,H-1 180.2), 90.5, 71.9, 69.4, 68.9, 68.8, 68.5, 67.9,
66.3, 66.0, 65.8, 62.3, 20.8, 20.7, 20.7, 20.6; m/z (ESI) 802.0889
([MþNa]þ. C28H36NaO18 requires 802.0896).
4.13. 5-Carboxypentyl 2,3,4,6-tetra-O-acetyl-
a
-D
-
mannopyranosyl-(1/6)-2,3,4-tri-O-acetyl-a-D
-
mannopyranoside (18)
Mannopyranoside 17 (850 mg, 1.11 mmol) was dissolved in dry
MeOH (10 cm3) and 30% sodium methoxide in MeOH (seven drops)
was added. The reaction mixture was stirred at rt for 30 min and
then concentrated to 1 cm3 under reduced pressure. The solution
was diluted with H2O (10 cm3) and NaOH (10 cm3, 2 M) was added.
The mixture was stirred overnight and neutralized with Amberjet
1200H resin. After filtration, the filtrate was concentrated in vacuo
to give a colourless foam, which was dissolved in pyridine (5 cm3).
Acetic anhydride (3.96 g, 38.8 mmol) was added and the solution
was left overnight. The volatiles were removed under high vacuum
and the residue taken up in CHCl3 (50 cm3) and washed with HCl
(2ꢁ30 cm3, 1.0 M). The acidic washings were extracted with CHCl3
(50 cm3), the extracts were dried (MgSO4), filtered and concen-
4.11. 5-(Methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-a-D-
mannopyranosyl-(1/6)-2,3,4-tri-O-acetyl-a-D-
mannopyranoside (17)
To
a stirred solution of trichloroacetimidate 16 (2.12 g,
2.71 mmol) and methyl 6-hydroxyhexanoate (790 mg, 5.4 mmol) in
CH2Cl2 (21 cm3), activated 4 Å molecular sieves (2.5 g) were added.
The reaction mixture was stirred at rt for 45 min and then cooled to
ꢂ40 ꢀC and TMSOTf (71 mg, 0.32 mmol) was added. After 15 min,
the cold bath was removed and the mixture was allowed to warm
to rt. Stirring was continued for 1 h, CH2Cl2 (25 cm3) was added, the
reaction was quenched with NEt3 and the mixture filtered through
Celite. The solids were washed with CH2Cl2 and the filtrate and
washings were taken to dryness and the residue was taken up in
pyridine (5 cm3). Acetic anhydride (5 cm3) was added and the so-
lution was left for 16 h. The volatiles were removed under high
vacuum and the residue was purified by flash chromatography to
trated in vacuo to give the title compound 18 as a colourless foam
22
(810 mg, 97%). Rf 0.30 (hexane/EtOAc/AcOH, 40:60:1); [
a]
þ58.9
D
(c 1.20, CHCl3); dH 5.38–5.19 (6H, m), 4.86 (1H, d, J 1.4), 4.78 (1H, d, J
1.4), 4.26 (1H, dd, J 12.3 and 5.1), 4.15 (1H, dd, J 12.2 and 2.2), 4.09
(1H, ddd, J 9.0, 5.1 and 2.0), 3.96 (1H, ddd, J 9.0, 5.8 and 2.2), 3.82–
3.69 (2H, m), 3.56 (1H, dd, J 10.8 and 2.4), 3.52–3.44 (1H, m), 2.39
(2H, t, J 7.1), 2.17 (3H, s), 2.17 (3H, s), 2.12 (3H, s), 2.07 (3H, s), 2.05
(3H, s), 2.01 (3H, s), 1.99 (3H, s), 1.78–1.62 (4H, m), 1.52–1.40
(2H, m); dC 178.0, 170.6, 170.1, 170.0, 169.9, 169.8, 169.7, 169.7, 97.4,

2946
B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
97.2, 69.6, 69.4, 69.2, 69.2, 68.9, 68.6, 67.9, 66.4, 66.4, 66.0, 62.4,
33.6, 28.8, 25.4, 24.3, 20.8, 20.8, 20.6, 20.6, 20.6, 20.6, 20.5; m/z (ESI)
749.2547 ([MꢂH]ꢂ. C32H45O20 requires 749.2510).
(ꢁ3). The combined organic extracts were concentrated in vacuo,
dried and purified by flash silica chromatography (CH2Cl2/MeOH,
24:1) to give acetylated intermediate 24a (59 mg); 55 mg of this
material was dissolved in MeOH (10 cm3) and the pH was adjusted
to 12 with NaOMe in MeOH (30%). After 2 h, silica was added, the
mixture concentrated in vacuo and purified by flash silica chro-
4.14. 5-(N-Succinimidyloxycarbonyl)pentyl 2,3,4,6-tetra-O-
acetyl-
a-D-mannopyranosyl-(1/6)-2,3,4-tri-O-acetyl-a-D-
mannopyranoside (19)
matography (CH2Cl2/MeOH, 9:1) to give the title compound as
20
a white amorphous solid (39 mg, 84%). [
a
]
D
ꢂ7.3 (c 0.52, MeOH); dH
To a stirred solution of acid 18 (810 mg, 1.07 mmol) in EtOAc
(8 cm3), N-hydroxysuccinimide (147 mg, 1.28 mmol) followed by
DCC (263 mg, 1.28 mmol) were added. The reaction mixture was
stirred for 16 h, filtered to remove precipitated urea and the filtrate
was taken to dryness under reduced pressure. Purification of the
residue by flash chromatography (hexane/EtOAc, 30:70) afforded
title compound 19 as a colourless foam (622 mg, 69%). Rf 0.34
(30:70, hexane/ethyl acetate); dH 5.38–5.20 (6H, m), 4.86 (1H, d, J
1.4), 4.78 (1H, d, J 1.4), 4.26 (1H, dd, J 11.9 and 5.2), 4.16 (1H, dd, J
11.9 and 2.2), 4.09 (1H, ddd, J 9.6, 5.3 and 2.1), 3.96 (1H, ddd, J 9.4,
6.0 and 2.3), 3.82–3.70 (2H, m), 3.57 (1H, dd, J 10.7 and 2.3), 3.52–
3.44 (1H, m), 2.84 (4H, br s), 2.65 (2H, t, J 7.2), 2.16 (3H, s), 2.16 (3H,
s), 2.12 (3H, s), 2.07 (3H, s), 2.05 (3H, s), 2.01 (3H, s), 1.99 (3H, s), 1.81
(2H, pent, J 7.0), 1.74–1.63 (2H, m), 1.59–1.47 (2H, m); dC 170.5, 170.1,
169.8, 169.6, 169.1, 168.4, 97.3, 97.2, 69.6, 69.4, 69.2, 68.8, 68.5, 67.7,
66.6, 66.5, 66.0, 62.4, 30.7, 28.6, 25.5, 25.2, 24.2, 20.7, 20.6, 20.5; m/z
(ESI) 870.2633 ([MþNa]þ. C36H45NNaO22 requires 870.2644).
(CDCl3/CD3OH, 4:6) 7.36–7.22 (10H, m, Ph), 6.19 (1H, s, CHPh), 4.54
(1H, dd, J 5.9 and 7.8, NHCHCO), 4.02 (2H, s, CH2OH), 3.97 (2H, s,
CH2OH), 3.20 (2H, dt, J 1.8 and 6.9),1.88–1.66 (2H, m),1.58–1.49 (2H,
m), 1.40–1.29 (2H, m); dC (CDCl3/CD3OH, 4:6) 175.3, 175.2, 173.3,
143.1, 142.9, 130.2, 130.1, 129.2, 129.1, 129.1, 129.0, 63.2 (CH2OH),
58.7 (CHPh2), 54.2 (NHCHCO), 40.1, 34.0, 30.5, 24.3; m/z (ESI)
450.2000 ([MþNa]þ. C23H29N3NaO5 requires 450.2005).
4.17. 1-Benzhydryl-
glycolyl4-cascadane (25b)
L L :
-lysinamide(N,N0):{ -lysinyl(N,N0)}G2x1
To dendrimer [G1]–[HCl]4 (127 mg, 0.178 mmol) and diisopro-
pylethylamine (404
L, 2.31 mmol) in anhydrous DMF (2 cm3),
m
a solution of the reactive ester 2 (169 mg, 0.783 mmol) in anhy-
drous DMF (1 cm3) was added. After 16 h at rt, the solution was
concentrated in vacuo, taken up in water and extracted with CHCl3
(ꢁ3). The combined organic extracts were concentrated in vacuo,
dried and purified by flash silica chromatography (CHCl3/MeOH,
19:1) to give acetylated intermediate 25a (90 mg). MeOH (1 cm3)
and CH2Cl2 (1 cm3) were added to half of this material and the pH
was adjusted to 12 with NaOMe in MeOH (30%). After 1.5 h, the
solution was neutralized with solid CO2, concentrated in vacuo and
purified by flash silica chromatography (CH3CN/H2O/concd NH4OH,
4.15. 5-(N-Succinimidyloxycarbonyl)pentyl 2,4-di-O-acetyl-
3,6-di-O-(2,3,4,6-tetra-O-acetyl-a-D-mannopyranosyl)-a-D-
mannopyranoside (23)
A solution of 5-carboxypentyl (
a
-
D
-mannopyranosyl)-(1/3)-
[(
a
-
D
-mannopyranosyl)-(1/6)]- -mannopyranoside
a
-
D
(21)14
6:1:1) to give the title compound as a white amorphous solid
21
(300 mg, 0.48 mmol) was dissolved in pyridine (5 cm3) and acetic
anhydride (5 cm3) was added. The solution was stirred for 16 h and
the solvents were removed under reduced pressure. The residue
was dissolved in CHCl3 (30 cm3) and washed with HCl (2ꢁ15 cm3,
M) and then H2O (15 cm3). The organic layer was dried and the
volatiles were removed under reduced pressure to give crude 5-
(35 mg, 49%). >99% pure (HPLC, 220 nm); [
a
]
ꢂ15.6 (c 0.7, MeOH);
D
dH (D2O) 7.31–7.19 (10H, m, Ph), 6.02 (1H, s, CHPh), 4.36 (1H, t, J 7.2,
NHCHCO), 4.29 (1H, t, J 7.1, NHCHCO), 4.21 (1H, t, J 7.1, NHCHCO),
4.05 (2H, s, CH2OH), 4.04 (2H, s, CH2OH), 3.99 (2H, s, CH2OH), 3.97
(2H, s, CH2OH), 3.18–3.03 (6H, m), 1.79–1.59 (6H, br m), 1.50–1.17
(12H, br m); dC (D2O) 175.0, 174.8, 174.0, 173.9, 173.1, 141.2, 141.0,
129.3, 129.2, 128.3, 128.1, 127.9, 127.5, 61.3 (CH2OH), 57.8 (CHPh2),
54.3 (NHCHCO), 53.9 (NHCHCO), 53.5 (NHCHCO), 39.4, 38.9, 31.4,
31.0, 28.4, 28.2, 22.8, 22.7; m/z (ESI) 800.4193 ([MþH]þ.
C39H58N7O11 requires 800.4189).
carboxypentyl 2,4-di-O-acetyl-3,6-di-O-(tetra-O-acetyl-
nopyranosyl)- -mannopyranoside (22) (466 mg, 93%). To
stirred solution of this material (215 mg, 0.20 mmol) and
a-D-man-
a-D
a
N-hydroxysuccinimide (36 mg, 0.31 mmol) in EtOAc (3 cm3), a so-
lution of DCC (64 mg, 0.31 mmol) in EtOAc (0.5 cm3) was added.
The reaction mixture was stirred for 16 h and then filtered through
cotton wool. The volatiles were removed under reduced pressure
and the product was purified by flash chromatography to give the
4.18. 1-Benzhydryl-
glycolyl8-cascadane (26b)
L L :
-lysinamide(N,N0):{ -lysinyl(N,N0)}G2x1,,4Gx2
title compound 23 as a foam upon drying under high vacuum
Trifluoroacetic acid (2 cm3) was added to [G2]–[Boc]8 (122 mg,
0.065 mmol) in CH2Cl2 (2 cm3) at 0 ꢀC and the resulting solution
was kept for 16 h at rt then dried in vacuo. The residue was azeo-
tropically dried by the addition and evaporation of CH2Cl2 several
times. To the freshly prepared [G2]–[TFA]8 in anhydrous DMF
22
(218 mg, 93%). Rf 0.50 (EtOAc/hexane, 90:10); [
a]
þ36.2 (c 0.51,
D
CHCl3); dH 5.34–5.12 (7H, m), 5.05–5.00 (2H, m), 4.82 (1H, d, J 1.6),
4.79 (1H, d, J 1.5), 4.33–4.02 (7H, m), 3.90–3.70 (3H, m), 3.54–3.42
(2H, m), 2.83 (4H, br s), 2.63 (2H, t, J 7.2), 2.20 (3H, s), 2.14 (3H, s),
2.13 (3H, s), 2.13 (3H, s), 2.10 (3H, s), 2.05 (3H, s), 2.20 (6H, s), 1.98
(3H, s), 1.97 (3H, s), 1.78 (2H, tt, J 7.5 and 7.2), 1.71–1.59 (2H, m),
1.57–1.45 (2H, m); dC 170.9, 170.8, 170.5, 170.3, 170.1, 170.0, 169.5,
168.8, 99.2, 97.5, 75.2, 71.4, 70.3, 69.8, 69.2, 68.9, 68.7, 68.0, 67.0,
66.4, 66.3, 62.7, 31.2, 29.1, 25.9, 25.6, 24.6, 21.2, 21.0, 21.0; m/z (ESI)
1153.3876 ([MþNH4]þ. C48H69O30N2 requires 1153.3929).
(2 cm3) were added EtiPr2N (362
mL, 2.08 mmol) and reactive ester
2 (223 mg, 1.04 mmol). The reaction mixture became viscous
overnight and further anhydrous DMF (2 cm3) was added. After
a further 24 h, the acetylated intermediate 26a was precipitated by
the addition of water, washed with CHCl3 and collected by filtra-
tion. The precipitate was suspended in MeOH (2 cm3) and water
(1 cm3), and NH4OH (1 cm3, concd) was added. After 30 min stir-
ring the precipitate had dissolved and after 4 h the solution was
concentrated in vacuo and the residue purified by flash silica gel
4.16. 1-Benzhydryl-
(24b)
L
-lysinamide(N,N0):glycolyl2-cascadane
chromatography (CHCl3/MeOH/concd NH4OH/H2O, 60:35:5:3) to
18
To dendrimer [G0]–[HCl]2 (105 mg, 0.273 mmol) and diisopro-
pylethylamine (340
L, 1.92 mmol) in anhydrous DMF (5 cm3),
a solution of the reactive ester 2 (160 mg, 0.744 mmol) in anhy-
drous DMF (5 cm3) was added. After 2 h at rt, the solution was
concentrated in vacuo, taken up in water and extracted with ether
give the capped dendrimer 26b as a white solid (29 mg, 29%). [
a
]
D
m
ꢂ32.7 (c 0.5, H2O); dH (D2O) 7.37–7.24 (10H, m, Ph), 6.07 (1H, s,
CHPh), 4.66–4.14 (7H, m, NHCHCO), 4.09 (8H, s, CH2OH), 4.01 (8H, s,
CH2OH), 3.20–3.03 (14H, m), 1.77–1.63 (14H, br m), 1.51–1.39 (14H,
br m), 1.39–1.15 (14H, br m); dC (D2O) 175.0, 174.9, 174.1, 173.9, 173.1,

B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
2947
22
141.2, 141.0, 129.3, 129.2, 128.3, 128.2, 127.9, 127.6, 61.4, 57.8, 54.4,
54.2, 53.8, 53.7, 53.5, 39.4, 39.0, 31.4, 31.0, 28.4, 28.2, 22.8; m/z (ESI)
772.9117 ([Mþ2H]þ. C71H115N15O23 requires 772.9140).
solid (65 mg, 99%). >99% pure (HPLC, 220 nm); [
a]
þ188.8 (c 0.5,
D
MeOH); dH (D2O) 7.05–6.97 (10H, m), 5.96 (1H, s), 5.27 (1H, s), 5.25
(1H, s), 4.43 (1H, br t), 4.01–3.97 (2H, m), 3.88–3.66 (10H, m), 3.32–
3.15 (4H, m), 3.00 (2H, br s), 1.66 (2H, br m), 1.34 (2H, br m), 1.21
(2H, br m); dC (D2O) 172.8, 171.8, 141.5, 141.1, 129.0, 127.8, 127.7, 85.3
(Man C-1), 73.9, 71.8, 71.6, 67.3, 61.2, 57.5, 54.3, 39.9, 34.2, 33.8, 31.8,
28.4, 23.0; m/z (ESI) 806.2570 ([MþNa]þ. C35H49N3NaO13S2 requires
806.2599).
4.19. 1-Benzhydryl-
mannopyranosyloxy)ethyl2-cascadane (27b)
L a-D-
-lysinamide(N,N0):1-oxo-2-(
To a stirred solution of [G0]–[HCl]2 (46 mg, 0.12 mmol) in DMF
(5 cm3), reactive ester 7 (148 mg, 0.29 mmol) and then diisopro-
pylamine (0.15 cm3, 0.11 g, 0.85 mmol) were added. The reaction
mixture was stirred at rt for 16 h and then diluted with H2O. The
aqueous mixture was extracted with ether (ꢁ2) and the combined
organic extracts were washed with H2O, dried and concentrated in
vacuo. Purification of the residue by flash chromatography (CHCl3/
4.21. 1-Benzhydryl-
oxo-2-( -mannopyranosyloxy)ethyl4-cascadane (29b)
L L
-lysinamide(N,N0):{ -lysinyl(N,N0)}2Gx1:1-
a-D
To a stirred solution of [G1]–[HCl]4 (228 mg, 0.31 mmol) in DMF
(12 cm3), reactive ester (861 mg, 1.71 mmol) and iPr2NH
7
MeOH, 99:1 to 95:5) gave the acetylated intermediate 27a as
(0.79 cm3, 4.46 mmol) were added. The solution, under Ar, was
stirred at rt for 72 h and diluted with H2O. The aqueous mixture
was extracted with ether (ꢁ3) and the combined organic extracts
were washed with H2O (ꢁ2), dried (MgSO4) and dried in vacuo.
Purification of the residue by flash chromatography (CHCl3/MeOH,
22
a colourless foam (59 mg, 45%). [
a]
þ36.7 (c 0.98, CHCl3); dH 7.43
D
(1H, d, J 7.8), 7.37–7.19 (10H, m), 7.10 (1H, d, J 8.1), 6.58 (1H, dd, J 5.8
and 5.8), 6.25 (1H, d, J 8.2), 5.38–5.23 (6H, m), 4.88 (1H, d, J 1.2), 4.85
(1H, br s), 4.54 (1H, ddd, J 7.9, 7.5 and 6.2), 4.32–4.23 (2H, m), 4.19–
3.93 (8H, m), 3.41–3.28 (1H, m), 3.23–3.12 (1H, m), 2.15 (3H, s), 2.14
(3H, s), 2.08 (3H, s), 2.08 (3H, s), 2.04 (3H, s), 2.01 (3H, s), 1.99 (3H,
s), 1.94 (3H, s), 1.96–1.87 (1H, m), 1.83–1.69 (1H, m), 1.63–1.51 (2H,
m), 1.45–1.28 (2H, m); m/z (ESI) 1088.4047 ([MþH]þ. C51H66O23N3
99:1 to 95:5) gave acetylated intermediate 29a as a colourless foam
20
(243 mg, 36%). [
a
]
þ37.1 (c 1.8, CHCl3); dH 7.74 (1H, d, J 8.2), 7.36–
D
7.16 (12H, m), 7.09 (1H, d, J 7.5), 6.91 (1H, dd, J 5.7 and 5.7), 6.78 (1H,
dd, J 5.8 and 5.8), 6.69 (1H, dd, J 6.0 and 6.0), 6.17 (1H, d, J 8.1), 5.40–
5.23 (16H, m), 4.91 (1H, d, J 1.0), 4.87 (1H, d, J 0.9), 4.85 (1H, br s),
4.81 (1H, d, J 1.0), 4.53–4.33 (3H, m), 4.32–4.21 (4H, m), 4.18–3.92
(15H, m), 3.82 (1H, d, J 15.2), 3.34–3.19 (5H, m), 3.09–2.89 (1H, m),
2.15 (3H, s), 2.15 (6H, br s), 2.14 (3H, s), 2.08 (6H, br s), 2.08 (3H, s),
2.07 (3H, s), 2.04 (3H, s), 2.04 (3H, s), 2.03 (3H, s), 2.02 (3H, s), 2.00
(3H, s), 1.99 (3H, s), 1.99 (3H, s), 1.97 (3H, s), 1.93–1.62 (6H, m), 1.62–
1.42 (6H, m), 1.42–1.25 (6H, m); dC 171.6, 171.2, 170.6, 170.5, 170.0,
169.4, 169.8, 169.8, 169.5, 169.5, 169.4, 168.4, 168.2, 168.0, 167.9,
141.4, 141.3, 128.5, 127.3, 97.8, 97.7, 97.5, 69.0, 68.8, 66.7, 66.6, 65.7,
65.7, 62.1, 56.8, 53.3, 53.0, 52.9, 52.7, 38.6, 38.4, 38.3, 31.7, 31.3, 31.3,
29.0, 28.7, 28.4, 22.8, 22.6, 22.2, 20.7, 29.6, 20.5; m/z (ESI) 1060.9053
([Mþ2H]2þ. C95H131N7O47 requires 1060.9032). The solid 29a
requires 1088.4081). The foam 27a (44 mg, 404 mmol) was taken up
in MeOH (5.5 cm3) and sodium methoxide in MeOH (two drops,
30%) was added. The reaction was stirred at rt for 72 h and the
reaction mixture was adsorbed onto flash silica gel and purified by
flash chromatography to give title compound 27b as a colourless
22
glass (25 mg, 82%). Rf 0.21 (CH3CN/H2O/concd NH4OH, 6:1:1); [a]
D
þ40.6 (c 0.18, H2O); dH (D2O) 7.44–7.23 (10H, m), 6.07 (1H, s), 4.84
(1H, br s), 4.80 (1H, br s), 4.41 (1H, dd, J 7.0 and 7.0), 4.22–4.08 (3H,
m), 4.03–3.95 (3H, m), 3.86–3.77 (4H, m), 3.74–3.51 (6H, m), 3.20–
3.07 (2H, m), 1.87–1.66 (2H, m), 1.53–1.40 (2H, m), 1.33–1.18 (2H,
m); dC (75 MHz, D2O) 173.2, 171.7, 141.1, 140.9, 129.3, 129.2, 128.3,
128.1, 127.8, 127.6, 100.4, 73.6, 70.8, 70.1, 66.9, 66.0, 61.2, 57.8, 53.9,
39.1, 31.4, 28.3, 22.6; m/z (ESI) 752.3209 ([MþH]þ. C35H50N3O15
requires 752.3236).
(224 mg, 106 m
mol) was taken up in MeOH (10 cm3) and NaOMe in
MeOH (two drops, 30%) was added. The mixture was stirred at rt for
16 h and neutralized with Amberlite IRC-50, filtered and concen-
trated in vacuo. The product was taken up in a small volume of H2O,
filtered through cotton wool and concentrated in vacuo to give the
4.20. 1-Benzhydryl-
mannopyranosylthio)ethyl2-cascadane (28b)
L a-D-
-lysinamide(N,N0):1-oxo-2-(
title compound 29b as a colourless gum (113 mg, 74%). Rf 0.18
22
To a stirred solution of [G0]–[Boc]2 (71 mg, 0.14 mmol) in MeOH
(3 cm3), HCl (1.5 cm3, concd) was added. After 30 min at 40 ꢀC, the
resulting solution was concentrated in vacuo, and evaporated with
H2O (ꢁ2) and then with MeCN (ꢁ2). Anhydrous DMF (5 cm3) was
(MeCN/H2O/concd NH4OH, 3:1:1); [
a]
þ24.0 (c 0.27, H2O); dH
D
(D2O) 7.41–7.20 (10H, m), 6.05 (1H, s), 4.86–4.82 (4H, m), 4.38–4.28
(2H, m), 4.25–3.95 (13H, m), 3.89–3.51 (20H, m), 3.20–3.00 (6H, m),
1.88–1.57 (6H, m), 1.52–1.14 (12H, m); dC (D2O) 173.8, 173.7, 173.1,
171.8, 171.7, 141.1, 140.9, 129.3, 129.2, 128.3, 128.0, 127.5, 100.4, 73.6,
70.8, 70.1, 67.0, 66.2, 66.0, 61.2, 57.8, 54.3, 53.9, 53.6, 39.4, 39.1, 31.4,
added to the resulting white solid followed by Et3N (135 mL,
0.97 mmol) and the reactive ester 11 (158 mg, 0.30 mmol) in DMF
(3 cm3). After 2.5 h at rt, the reaction mixture was diluted with H2O
(40 cm3), stirred for 30 min, filtered and the precipitate was puri-
fied by flash chromatography (CHCl3/MeOH, 98:2) to give acety-
lated intermediate 28a (116 mg, 75%). dH 7.44 (1H, d, J 8.1), 7.37–7.17
(10H, m), 7.13 (1H, d, J 8.1), 6.73 (1H, dd, J 6.0 and 6.0), 6.18 (1H, d, J
8.1), 5.40 (1H, br d, J 1.2), 5.37–5.27 (5H, m), 5.23 (1H, dd, J 1.0 and
3.0), 5.23 (1H, dd, J 1.0 and 3.0), 4.49 (1H, ddd, J 7.9, 7.9 and 5.8),
4.36–4.23 (4H, m), 4.16–4.02 (2H, m), 3.47–3.03 (6H, m), 2.14 (3H,
s), 2.13 (3H, s), 2.07 (3H, s), 2.06 (3H, s), 2.05 (3H, s), 2.04 (3H, s),1.99
(3H, s), 1.98 (3H, s), 1.90–1.78 (1H, m), 1.78–1.60 (1H, m), 1.61–1.41
(2H, m), 1.41–1.20 (2H, m); dC 171.1, 171.0, 170.9, 170.4, 170.3, 170.2,
170.0, 168.7, 168.0, 141.8, 141.5, 129.1, 129.0, 127.9, 127.8, 82.6, 82.4,
77.6, 71.1, 70.9, 70.0, 69.7, 66.5, 66.4, 62.7, 57.4, 53.5, 39.8, 34.2, 33.7,
32.4, 29.2, 23.0, 21.2, 21.1, 21.0, 20.9. Acetylated intermediate 28a
(94 mg, 0.084 mmol) was dissolved in MeOH (3 cm3), and the pH of
the solution was adjusted to 12 with NaOMe in MeOH. (30%) After
30 min, the solution was neutralized with solid CO2, concentrated
in vacuo and purified by flash silica chromatography (CH3CN/H2O/
concd NH4OH, 6:1:1) to give dendrimer 28b as a white amorphous
30.9, 28.4, 28.3, 28.1, 22.8, 22.6; m/z (ESI) 724.894 ([Mþ2H]2þ
.
C63H99N7O31 requires 724.819).
4.22. 1-Benzhydryl-
oxo-2-( -mannopyranosylthio)ethyl4-cascadane (30b)
L L
-lysinamide(N,N0):{ -lysinyl(N,N0)}2Gx1:1-
a-D
To a stirred solution of [G1]–[HCl]4 (90 mg, 0.126 mmol) in an-
hydrous DMF (2 cm3), Et3N (229
L, 0.97 mmol) was added. After
m
10 min, a suspension formed to which was added a solution of
reactive ester 11 (320 mg, 0.55 mmol) in DMF (2 cm3) in two por-
tions with 2 h between the additions. After being stirred overnight
at rt, the reaction mixture was diluted with H2O (25 cm3), which
caused a white precipitate to form. The stirring was resumed for
30 min and the precipitate was collected by vacuum filtration and
washed with H2O to give the acetylated intermediate 30a (208 mg,
76%); dH 7.73 (1H, d, J 7.6), 7.57 (1H, d, J 8.0), 7.49 (1H, d, J 6.0), 7.38–
7.11 (11H, m), 7.00 (1H, br dd, J 5.2 and 5.2), 6.90–6.77 (2H, m), 6.12
(1H, d, J 8.0), 5.44–5.11 (16H, m), 4.58 (1H, ddd, J 7.3, 7.0 and 7.0),
4.41–4.17 (10H, m), 4.15–4.01 (4H, m), 3.60–3.00 (14H, m),

2948
B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
2.17–1.96 (16ꢁ(3H, s)), 1.79–1.00 (18H, m). MeOH (5 cm3) was
added to the crude product (171 mg, 0.078 mmol) and the pH of the
suspension was adjusted to 12 with 30% NaOMe in MeOH. After 2 h,
the reaction was diluted with EtOH, filtered and the precipitate
washed with EtOH (ꢁ3). The combined filtrates were concentrated
in vacuo and purified by flash chromatography (3:1:1, CH3CN/H2O/
and 5.3), 6.20 (1H, d, J 8.0), 5.36–5.18 (24H, m), 4.86–4.84 (4H, br s),
4.77–4.74 (4H, br s), 4.52 (1H, ddd, J 7.2, 7.2 and 7.2), 4.40–4.20 (6H,
m), 4.17–4.04 (8H, m), 3.97–3.88 (4H, m), 3.82–3.66 (8H, m), 3.60–
3.52 (4H, m), 3.48–3.40 (4H, m), 3.25–3.0 (4H, m), 2.40–1.80 (100H,
m), 1.70–1.20 (40H, m); m/z (MALDI-TOF) 3519.20 ([MþNa]þ.
C159H225N7NaO79 requires 3519.37). To a stirred solution of in-
concd NH4OH) to give the capped dendrimer 30b as a white solid
termediate 32a (65 mg, 18.5 m
mol) in MeOH (5 cm3), NaOMe in
21
(64 mg, 54%). >99% pure (HPLC, 220 nm); [
a
]
þ143.6 (c 0.5, H2O);
MeOH (five drops, 30%) was added. The reaction mixture was
stirred for 30 min and sufficient iPrOH was added to precipitate the
product. The white precipitate was collected, washed with a small
amount of iPrOH, dissolved in H2O and lyophilized to give the title
compound 32b as a fluffy white solid (34 mg, 79%). 99% pure (HPLC,
220 nm); dH (D2O) 7.37–7.26 (10H, m), 6.06 (1H, s), 4.84 (4H, br s),
4.78 (4H, br s), 4.34 (1H, dd, J 6.3 and 6.3), 4.19 (1H, dd, J 6.3 and
6.3), 4.12 (1H, dd, J 6.9 and 6.9), 4.02–3.40 (56H, m), 3.16–3.05 (4H,
br s), 3.00 (2H, br t, J 6.2), 2.29–2.10 (8H, m),1.80–1.20 (42H, m); m/z
(MALDI-TOF) 2343.20 ([MþNa]þ. C103H169N7NaO51 requires
2343.07).
D
dH (D2O) 7.30–7.19 (10H, m), 6.01 (1H, s), 5.28 (2H, s), 5.24 (2H, s),
4.37 (1H, t, J 7.1), 4.23 (1H, t, J 7.0), 4.13 (1H, t, J 7.0), 4.01 (4H, br s),
3.85–3.66 (20H, m), 3.92–2.97 (14H, m), 1.83–1.54 (6H, br m), 1.52–
1.11 (12H, br m); dC (D2O) 172.6, 171.8, 170.9, 170.8, 139.9, 139.7,
128.0, 127.1, 126.7, 126.3, 84.0, 72.6, 70.4, 70.3, 66.0, 59.9, 56.5, 53.5,
53.2, 53.0, 38.6, 38.1, 32.9, 32.5, 30.0, 29.8, 27.0, 21.6; m/z (MALDI-
TOF) 1534.36 ([MþNa]þ. C63H97N7NaO27S4 requires 1534.52).
4.23. 1-Benzhydryl-
oxo-6-( -mannopyranosylthio)hexyl4-cascadane (31b)
L L
-lysinamide(N,N0):{ -lysinyl(N,N0)}2Gx1:1-
a-D
To a stirred solution of [G1]–[TFA]4 (24 mg, 0.06 mmol) in DMF
(0.5 cm3), reactive ester 14 (118 mg, 0.20 mmol) was added fol-
lowed by Pr2NH (44 mg, 0.34 mmol). The reaction mixture was
4.25. 1-Benzhydryl-
L
-lysinamide(N,N0):{
-mannopyranosyl)-a-D-
L
-lysinyl(N,N0)}2Gx1:1-
oxo-6-{3,6-di-O-(
a
-D
i
mannopyranosyloxy}hexyl4-cascadane (33b)
stirred for 16 h and diluted with H2O (20 cm3). The precipitated
product was collected by vacuum filtration and washed with H2O,
then purified by flash chromatography (MeOH/CHCl3, 1:99 to 5:95)
to give acetylated intermediate 31a as a white powder solid (62 mg,
76%). dH 7.56–7.45 (3H, m), 7.36–7.17 (10H, m), 7.05 (1H, dd, J 7.7 and
4.2), 6.57 (1H, d, J 8.4), 6.35 (1H, br t, J 5.6), 6.27 (1H, br t, J .54), 6.19
(1H, d, J 8.1), 5.37–5.21 (20H, m), 4.53 (1H, ddd, J 7.5, 7.5 and 7.5),
4.39–4.23 (13H, m), 4.15–4.05 (5H, m), 3.61–3.47 (1H, m), 3.27–3.12
(3H, m), 3.09–2.91 (2H, m), 2.71–2.49 (8H, m), 2.16 (6H, br s), 2.15
(3H, s), 2.13 (3H, s), 2.09 (9H, br s), 2.08 (3H, s), 2.05 (9H, br s), 2.04
(3H, s), 1.99 (6H, br s), 1.98 (3H, s), 1.96 (3H, s), 1.85–1.25 (42H, m);
dC 174.1, 173.4, 173.1, 171.1, 170.9, 170.3, 170.1, 170.0, 141.9, 141.8,
128.9, 128.8, 127.8, 82.8, 71.5, 69.8, 69.3, 66.7, 62.8, 57.3, 53.3, 52.8,
39.4, 39.0, 36.7, 36.5, 31.4, 30.6, 29.7, 29.4, 28.6, 28.5, 25.5, 23.4, 23.1,
21.8, 21.2, 21.1, 21.0, 20.9. Capped dendrimer 31a was dissolved in
MeOH (5 cm3) and CH2Cl2 (2 cm3) and three drops of NaOMe in
MeOH (30%) were added. The mixture was stirred for 30 min and
solid CO2 and H2O (1 cm3) were added to neutralize the reaction
mixture. The MeOH was removed under reduced pressure and the
precipitated solid triturated with cold H2O to remove inorganic
To a stirred solution of [G1]–[HCl]4 (10 mg, 0.014 mmol) in DMF
(1 cm3), reactive ester 23 (100 mg, 0.088 mmol) and EtiPr2N (19 mg,
0.14 mmol) were added. The reaction mixture was stirred for 16 h,
diluted with H2O and the precipitate was collected. The aqueous
filtrate was extracted with CHCl3 (15 cm3) and the organic layer was
dried and concentrated in vacuo. The residue was combined with
the precipitate and triturated with hexane, to give a white amor-
phous solid. Purification by flash chromatography (CHCl3/MeOH,
97:3 to 96:4) gave acetylated intermediate 33a (46 mg, 68%). Rf 0.32
(CHCl3/MeOH, 95:5). This solid was dissolved in dry MeOH (5 cm3)
and NaOMe in MeOH (three drops, 30%) was added. After stirring
i
for 1 h, PrOH (3 cm3) was added to precipitate the deacetylated
product, which was collected and washed with a small volume of
cold isopropanol. The solid was dissolved in H2O and lyophilized to
give 33b as a white solid (24 mg, 56%). >99% pure (HPLC, 220 nm);
22
[a
]
þ54.5 (c 0.079, H2O); dH (D2O) 7.39–7.27 (10H, m), 6.07 (1H, s),
D
5.07 (4H, s), 4.85 (4H, s), 4.77 (4H, s), 4.34 (1H, dd, J 7.2 and 7.2), 4.19
(1H, dd, J 7.1 and 7.1), 4.11 (1H, dd, J 7.1 and 7.1), 4.04–3.60 (76H, m),
3.49 (4H, br s), 3.01 (4H, br s), 3.01 (2H, br t, J 7.0), 2.24–2.51 (8H, m),
2.80–1.20 (42H, m); dC (D2O) 176.9, 129.3, 128.0, 127.5, 79.0, 73.7,
73.1, 71.5, 71.5, 71.0, 70.8, 70.4, 70.1, 68.2, 67.1, 66.1, 65.7, 61.3, 39.3,
36.2, 28.6, 25.4; m/z (MALDI-TOF) 2991.38 ([MþNa]þ.
C127H209N7NaO71 requires 2991.28).
salts and give dendrimer 31b as a white solid (40 mg, 68%). Rf 0.26
22
(6:1:1, iPr/H2O/NH4OH); HPLC 93% pure, 220 nm; [
a]
þ51.6 (c 0.5,
D
MeOH); dH (MeOH-d4) 7.39–7.25 (10H, m), 6.15 (1H, s), 5.24 (4H, s),
4.42 (1H, dd, J 8.3 and 5.9), 4.30 (1H, dd, J 8.0 and 6.1), 4.24 (1H, dd, J
8.3 and 5.9), 3.97–3.68 (24H, m), 3.17–3.12 (6H, m), 2.73–2.56 (8H,
m), 2.26 (4H, t, J 7.0), 2.21 (4H, m), 1.90–1.41 (42H, m); dC (75 MHz,
MeOH-d4) 176.7, 129.8, 128.9, 128.8, 86.6, 75.0, 73.9, 73.3, 68.9, 62.7,
58.5, 55.0, 40.3, 37.2, 36.9, 32.7, 32.0, 30.4, 30.1, 29.4, 26.7, 26.6, 25.4,
24.3; m/z (MALDI-TOF) 1758.776 ([MþNa]þ. C79H129N7NaO27S4 re-
quires 1758.772).
4.26. 1-Benzhydryl-
oxo-2-( -mannopyranosylthio)ethyl8-cascadane (34b)
L L
-lysinamide(N,N0):{ -lysinyl(N,N0)}2Gx1,,4Gx2:1-
a-D
To a stirred solution of [G2]–[Boc]8 (11 mg, 0.0059 mmol) in
MeOH (4 cm3), HCl (2 cm3, concd) was added. After 10 min at 40 ꢀC,
the resulting solution was concentrated in vacuo, evaporated with
H2O (ꢁ2) and then MeCN to give [G2]–[HCl]8. To the resulting solid,
4.24. 1-Benzhydryl-
oxo-6-( -mannopyranosyl-(1/6)-
L L
-lysinamide(N,N0):{ -lysinyl(N,N0)}2Gx1:1-
a
-D
a-
D-
anhydrous DMF (1 cm3) and EtiPr2N (51
mL, 0.293 mmol) were
mannopyranosyloxy)hexyl4-cascadane (32b)
added. After the mixture was stirred for 10 min the HCl salt dis-
solved and reactive ester 11 (24 mg, 0.047 mmol) was added. After
being left to stand overnight at rt, the solution was diluted with
H2O (5 cm3) and stirred for 30 min. The precipitate was collected by
vacuum filtration, washed with H2O and purified by flash silica
chromatography (MeOH/CH2Cl2, 7:93) to give acetylated in-
termediate 34a (5 mg, 20%). dH 7.81 (1H, br d, J 6.0), 7.69 (1H, br d, J
7.8), 7.63–7.46 (5H, m), 7.36–7.16 (11H, m), 7.16–7.07 (3H, m), 7.02–
6.83 (4H, m), 6.15 (1H, d, J 8.1), 5.47–5.13 (32H, m), 4.66–4.56 (1H,
m), 4.55–4.19 (22H, m), 4.18–3.96 (8H, m), 3.53–2.97 (30H, m),
2.14 (24H, br s), 2.08 (24H, br s), 2.04 (24H, br s), 1.97 (24H, br s),
To a stirred solution of [G1]–[TFA]4 (24 mg, 0.024 mmol) in DMF
(0.5 cm3), reactive ester 19 (120 mg, 0.141 mmol) and iPr2NH
(30 mg, 0.24 mmol) were added. The mixture was stirred for 72 h
and the volatiles were removed under high vacuum. Purification of
the residue by flash chromatography (CHCl3/MeOH, 97:3) gave
acetylated intermediate 32a as a colourless gum (66 mg, 79%). Rf
0.40 (CHCl3/MeOH, 95:5); 94% pure (HPLC, 220 nm); dH 7.56 (1H, d,
J 8.6), 7.49–7.44 (2H, m), 7.30–7.20 (10H, m), 7.05 (1H, dd, J 6.3 and
4.6), 6.73 (1H, d, J 8.0), 6.37 (1H, dd, J 5.2 and 5.2), 6.29 (1H, dd, J 5.3

B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
2949
1.89–1.19 (42H, m). Intermediate 34a (16 mg, 0.0037) was dissolved
in MeOH (2 cm3) and CH2Cl2 (2 cm3) and the pH of the suspension
was adjusted to 12 by addition of NaOMe in MeOH (30%). After 2 h,
the solution was neutralized with ion exchange resin (Amberlite
IRC 50, Hþ form), concentrated in vacuo and lyophilized to give the
(1H, s), 4.85 (8H, br s), 4.78 (8H, br s), 4.36 (1H, dd, J 6.3 and 6.3),
4.23–4.14 (6H, m), 4.05–3.38 (112H, m), 3.20–3.00 (14H, m),
2.33–2.10 (16H, m), 1.80–1.20 (90H, m); m/z (MALDI-TOF) 4606.6
([MþNa]þ, C199H337N15NaO103 requires 4608.1 (average molecular
mass)).
capped dendrimer 34b as a white solid (11 mg, quant.). >99% pure
20
(HPLC, 220 nm); [
a
]
þ36.2 (c 0.2, H2O); dH (D2O) 7.41–7.11 (10H,
D
4.29. 1-Benzhydryl-
L
-lysinamide(N,N0):
-manno-
m), 6.06 (1H, s), 5.28 (4H, br s), 5.24 (4H, br s), 4.35 (1H, br dd, J 7.0
and 7.0), 4.28–4.08 (6H, m), 4.00 (8H, br s), 3.92–3.60 (40H, m),
3.40–3.00 (30H, m), 1.80–1.60 (14H, m), 1.53–1.37 (14H, m), 1.37–
{
L
-lysinyl(N,N0)}G2x1,,4Gx2,,8Gx3:1-oxo-6-(
a-D
pyranosylthio)hexyl16-cascadane (37b)
1.17
(14H,
m);
m/z
(MALDI-TOF)
2991.1
([MþNa]þ.
To a stirred solution of [G3]–[TFA]16 (200 mg, 0.0509 mmol) in
DMF (5 cm3), reactive ester 14 (702 mg, 1.22 mmol) followed by
EtiPr2N (395 mg, 3.05 mmol) were added. The reaction mixture was
stirred for 16 h and concentrated in vacuo. The residue was dis-
solved in CHCl3/MeOH and filtered through a bed of silica (Rf 0.50,
CHCl3/MeOH, 90:10) to give acetylated intermediate 37a as a white
solid. This solid was dissolved in dry MeOH (6 cm3) and CH2Cl2
(3 cm3) and NaOMe in MeOH (0.10 cm3, 30%) was added to the
solution. The solution was stirred for 30 min after which time the
product precipitated as a white solid, which was collected on a frit
and washed with a little cold MeOH/CH2Cl2 (1:1). The solid was
dissolved in 1:1 MeOH/H2O, the MeOH removed under reduced
C119H193N15NaO55S8 requires 2991.0).
4.27. 1-Benzhydryl-
oxo-6-( -mannopyranosylthio)hexyl8-cascadane (35b)
L L
-lysinamide(N,N0):{ -lysinyl(N,N0)}2Gx1,,4Gx2:1-
a-D
To a stirred solution of [G2]–[TFA]8 (126 mg, 0.06 mmol) dis-
solved in DMF (3 cm3), reactive ester 14 (416 mg, 0.76 mmol)
followed by EtiPr2N (245 mg, 1.90 mmol) were added. The reaction
mixture was stirred for 24 h and diluted with H2O. The pre-
cipitated product was collected by vacuum filtration and purified
by flash chromatography (MeOH/CHCl3, 1:99 to 5:95) to give the
acetylated intermediate 35a as a white solid (233 mg), which was
dissolved in dry MeOH (5 cm3) and CH2Cl2 (3 cm3). NaOMe in
MeOH (three drops, 30%) was added to the solution and the
mixture was stirred for 30 min. Solid CO2 (10 g) and H2O (10 cm3)
were added, and when all the CO2 had vaporized the volatiles
were removed under reduced pressure (high vacuum) to leave the
product, which was purified by flash chromatography. The
resulting capped dendrimer 35b was obtained as a white solid
(137 mg, 63%). Rf 0.20 (iPr/H2O/NH3(aq), 3:1:1); 99% pure (HPLC,
220 nm); dH (CD3OD/D2O, 5:1) 7.37–7.26 (10H, m), 6.16 (1H, s),
5.25 (8H, s), 4.45 (1H, dd, J 5.4 and 8.1), 4.32–4.25 (6H, m), 3.95–
3.69 (48H, m), 3.17–3.09 (14H, m), 2.71–2.56 (16H, m), 2.29 (8H,
br m), 2.20 (8H, t, J 7.5), 1.80–1.28 (90H, m); dC (CD3OD/D2O, 5:1,
resolved peaks only) 177.6, 177.2, 176.9, 130.1, 129.2, 129.0, 86.8,
75.2, 74.1, 73.5, 69.1, 55.3, 40.5, 37.5, 37.1, 33.1, 32.8, 32.3, 30.6,
30.3, 29.6, 26.9, 24.6; m/z (MALDI-TOF) 3439.32 ([MþNa]þ.
C151H257N15NaO55S8 requires 3439.54).
pressure and the product lyophilized to give capped dendrimer 37b
22
(219 mg, 63%). [
a
]
D
þ92.3 (c 0.31, MeOH/H2O,1:1); 90% pure (HPLC,
220 nm); dH (D2O) 7.42–7.23 (10H, m), 6.16 (1H, s), 5.22 (16H, s),
4.50–4.20 (15H, m), 4.06–3.63 (96H, m), 3.13 (30H, br s), 2.60 (32H,
br s), 2.37–2.13 (32H, m), 1.80–1.20 (186H, m); m/z (MALDI-TOF)
6805.3 ([MþNa]þ. C295H513N31NaO111S16 requires 6806.4 (average
molecular mass)).
4.30. 1-Benzhydryl-
L
-lysinamide(N,N0):{
L
-lysinyl(N,N0)}2Gx1,,4Gx2,,8Gx3
:
1-oxo-6-( -mannopyranosyl-(1/6)-a-D-
a-
D
mannopyranosyloxy)hexyl16-cascadane (38b)
To a stirred solution of [G3]–[TFA]16 (34 mg, 8.6
m
mol) in DMSO
(1.0 cm3), reactive ester 19 (176 mg, 0.207 mmol) followed by
EtiPr2N (44 mg, 0.345 mmol) were added. The mixture was stirred
for 96 h and the product was precipitated by the addition of
water. The solid was collected by vacuum filtration and purified
by flash chromatography (CHCl3/MeOH, 100:0 to 70:30) to give
acetylated intermediate 38a as a colourless gum (65 mg, 55%). 88%
pure (HPLC 220 nm); dH 8.37 (1H, s), 8.00–7.48 (10H, m), 6.83–
6.54 (4H, m), 6.51–6.31 (4H, m), 6.21 (1H, d, J 8.4), 5.37–5.26 (80H,
m), 5.21 (16H, br s), 4.88 (16H, br s), 4.78 (16H, br s), 4.64–4.40
(8H, br s), 4.43–4.32 (7H, br s), 4.27 (16H, dd, J 12.3 and 5.3), 4.16–
4.04 (32H, m), 3.93 (16H, br s), 3.83–3.66 (32H, m), 3.63–3.55
(16H, m), 3.50–3.38 (22H, m), 3.24–2.96 (24H, m), 2.32–2.12 (32H,
m), 2.16 (48H, s), 2.16 (48H, s), 2.11 (48H, s), 2.07 (48H, s), 2.06
(48H, s), 2.00 (48H, s), 1.98 (48H, s), 1.72–1.32 (186H, m); m/z
(MALDI-TOF) 13,841 ([MþNa]þ. C615H897N31NaO319 requires
13,852 (average molecular mass)). To this white solid (65 mg,
4.28. 1-Benzhydryl-
oxo-6-( -mannopyranosyl-(1/6)-
pyranosyloxy)hexyl8-cascadane (36b)
L
-lysinamide(N,N0):{
-manno-
L
-lysinyl(N,N0)}2Gx1,,4Gx2:1-
a
-D
a-D
To a stirred solution of [G2]–[TFA]8 (23 mg, 0.011 mmol) dis-
solved in DMF (0.5 cm3), reactive ester 19 (120 mg, 0.141 mmol)
followed by EtiPr2N (30 mg, 0.24 mmol) were added. The mixture
was stirred for 72 h and the volatiles were removed under high
vacuum. Purification of the residue by flash chromatography
(CHCl3/MeOH, 97:3) gave acetylated intermediate 36a as a col-
ourless gum (52 mg, 64%). Rf 0.42 (CHCl3/MeOH, 95:5); 98% pure
(HPLC, 220 nm); dH 8.0–7.73 (3H, m), 7.63–7.43 (3H, m), 7.36–7.14
(12H, m), 7.11–6.92 (3H, m), 6.72–6.56 (2H, m), 6.37–6.24 (2H, m),
6.20 (1H, d, J 8.0), 5.36–5.18 (48H, m), 4.88–4.84 (8H, br s), 4.80–
4.74 (4H, br s), 4.74–4.66 (1H, m), 4.60–4.40 (4H, m), 4.40–4.20
(10H, m), 4.17–4.04 (16H, m), 3.97–3.88 (8H, br s), 3.83–3.64
(16H, m), 3.62–3.52 (8H, m), 3.49–3.40 (10H, m), 3.30–3.28 (12H,
m), 2.30–1.90 (196H, m), 1.70–1.20 (78H, m); m/z (MALDI-TOF)
6964.3 ([MþNa]þ. C311H449N15NaO159 requires 6964.9 (average
molecular mass)). To a stirred solution of intermediate 36a
4.7 m
mol), methanolic ammonia (3 cm3, 7 M) was added. After 3 h
of stirring an oil precipitated from the solution and was dissolve
by the addition of H2O (3 cm3). The mixture was stirred for 16 h
and concentrated in vacuo. The residue was taken up in water
(0.5 cm3) and precipitated by the addition of PrOH. The suspen-
i
sion was centrifuged (4000 rpm, 10 min), the pellet washed with
i
a little PrOH, then taken up in water and lyophilized to give the
capped dendrimer 38b as a white powder (35 mg, 81%). >95%
pure (HPLC, 220 nm); [
22
(44 mg, 7.2
m
mol) in MeOH (5 cm3), NaOMe in MeOH (five drops,
30%) was added. The mixture was stirred for 30 min and PrOH
was added to precipitate the white product, which was collected,
washed with a small amount of PrOH, dissolved in H2O and ly-
ophilized to give capped dendrimer 36b as a white solid (28 mg,
85%). 90% pure (HPLC, 220 nm); dH (D2O) 7.36–7.26 (10H, m), 6.07
a
]
þ35.1 (c 0.7, H2O); dH (D2O) 7.41–7.22
D
i
(10H, m), 6.11 (1H, s), 4.87 (16H, br s), 4.80 (16H, br s), 4.46–4.37
(1H, br m), 4.30–4.10 (14H, m), 4.00–3.62 (208H, m), 3.55–3.43
(16H, m), 3.13 (30H, br s), 2.32–2.14 (32H, br m), 1.84–1.19 (186H,
m); m/z (MALDI-TOF) 9143 ([MþNa]þ. C393H675N31NaO208 requires
9186 (average molecular mass)).
i

2950
B.W. Greatrex et al. / Tetrahedron 65 (2009) 2939–2950
4.31. 1-Benzhydryl-
L
-lysinamide(N,N0):
a positive control. Data was analyzed using CellQuest Pro (BD, USA).
{
L
-lysinyl(N,N0)}2Gx1,,4Gx2,,8Gx3,1,G64x:1-oxo-6-
The fold increase in the mean fluorescence intensity (MFI) of acti-
vation marker expression was determined by dividing the MFI of
each sample by that of the negative control (media only, no
formulation).
(a
-D
-mannopyranosylthio)hexyl32-cascadane (39b)
To a stirred solution of [G4]–[TFA]32 (250 mg, 0.032 mmol) in
DMF (5 cm3), reactive ester 14 (884 mg, 1.22 mmol) followed by
EtiPr2N (330 mg, 2.56 mmol) were added. The reaction mixture was
stirred for 16 h, ether (50 cm3) was added and an oily product
precipitated. Further stirring caused the oil to form a fine white
precipitate, which was collected by vacuum filtration (473 mg,
78%). The solid was dissolved in dry MeOH (19 cm3) and NaOMe in
MeOH (seven drops, 30%) was added to the solution. The reaction
mixture was stirred for 90 min and CH2Cl2 was added to precipitate
the product. The oily precipitate was washed with a little cold
Acknowledgements
The authors gratefully acknowledge the assistance of Prof.
Robin Ferrier in the preparation of this manuscript, Starpharma
(www.starpharma.com) for providing the lysine dendrimer
starting materials and financial support from the Foundation for
Research Science and Technology, New Zealand, Contract
C08X0303.
MeOH, then dissolved in H2O and the product was lyophilized to
22
give the capped dendrimer 39b (263 mg, 61%); [
a]
þ89.8 (c 0.31,
D
References and notes
H2O); dH (D2O) 7.35–7.20 (10H, m), 6.16 (1H, m), 5.26 (32H, s), 4.20–
4.40 (31H, m), 4.0–3.60 (192H, m), 3.14 (62H, br s), 2.80–2.57 (64H,
m), 2.40–2.15 (64H, m), 1.80–1.20 (378H, m); m/z (MALDI-TOF)
13,558 ([MþNa]þ. C583H1025N63NaO223S32 requires 13,525 (average
molecular mass)).
1. Recent glycodendrimer reviews: (a) Chabre, Y. M.; Roy, R. Curr. Top. Med. Chem.
2008, 8, 1237–1285; (b) Niederhafner, P.; Sebestik, J.; Jezek, J. J. Pept. Sci. 2008,
ˇ
ꢀ
ˇ
ꢀ
14, 2–43; (c) Niederhafner, P.; Sebestik, J.; Jezek, J. J. Pept. Sci. 2008, 14, 44–65;
(d) Li, Y.; Cheng, Y.; Xu, T. Curr. Drug Discov. Technol. 2007, 4, 246–254.
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4.32. 1-Benzhydryl-
-lysinyl(N,N0)}G2x1,,4Gx2,,8Gx3,1,G64x,,G325x:1-oxo-6-
-mannopyranosylthio)hexyl64-cascadane (40b)
L
-lysinamide(N,N0):
{
L
(a-D
4. Irache, J. M.; Salman, H. H.; Gamazo, C.; Espuelas, S. Expert Opin. Drug Deliv.
2008, 5, 703–724.
To a stirred solution of [G5]–[TFA]64 (110 mg, 7.07 mmol) in DMF
5. For examples: (a) Woller, E. K.; Cloninger, M. J. Org. Lett. 2002, 4, 7–10; (b)
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(3 cm3), reactive ester 14 (390 mg, 0.687 mmol) followed by EtiPr2N
(148 mg, 1.14 mmol) were added. The reaction mixture was stirred
for 16 h, water (30 cm3) was added and the resulting precipitate
collected by decanting off the aqueous layer. The solid was dried
under vacuum, ether added and the solid collected by vacuum fil-
tration. The solid was dissolved in dry MeOH/CH2Cl2 (6 cm3, 5:1)
and NaOMe in MeOH (30%) added. The reaction mixture was stirred
for 60 min and a fine white precipitate formed. iPrOH (12 cm3) was
added and the precipitate isolated by vacuum filtration and washed
i
with further PrOH. The solid was then dissolved in H2O and the
product was lyophilized to give crude product, which was purified
´
6. Roy, R.; Page, D.; Perez, S. F.; Bencomo, V. V. Glycoconjugate J. 1998, 15, 251–263.
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by RP-HPLC on
a Phenomenex Synergi 4 m Max-RP 80A,
30ꢁ250 mm column eluting with 0.1% trifluoroacetic acid in water/
22
methanol to give capped dendrimer 40b (84 mg, 44%). [
a
]
D
þ88.3
(c 0.6, H2O); dH (D2O) 7.36–7.20 (10H, m), 6.13 (1H, m), 5.27 (64H, s),
4.38–4.17 (63H, m), 4.03–3.64 (384H, m), 3.15 (126H, br s), 2.72–
2.55 (128H, m), 2.35–2.13 (128H, m), 1.86–1.22 (762H, m); m/z
(MALDI-TOF) 26,811 ([MþNa]þ. C1159H2049N127NaO447S64 requires
26,991 (average molecular mass)).
4.33. Generation and activation of bone marrow dendritic
cells (BMDCs)
Murine BMDCs were generated by culturing bone marrow de-
rived stem cells from C57Bl/6J mice in complete Iscove’s Modified
Dulbecco’s Medium (cIMDM; IMDM supplemented with 5% foetal
bovine serum, 1% penicillin/streptomycin, 1% glutamax, and 0.01%
2-mercaptoethanol; from Invitrogen, CA, USA) with 20 ng/mL
recombinant granulocyte/macrophage colony stimulating factor
(clone kindly supplied by Dr G. Buchan, University of Otago) for 6
days at 37 ꢀC, 5% CO2. Cells were seeded at 5ꢁ105 cells/mL in
cIMDM for pulsing with glycodendrimer formulations. Activation of
BMDC was measured by flow cytometric analysis (FACScalibur, BD,
USA). Cells were stained with the BMDC marker CD11c, and the
expression of co-stimulatory activation markers CD86 and MHCII
on CD11cþve cells was investigated 48 h after the addition of 0.5 nM
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´
´
´
21. Szurmai, Z.; Janossy, L.; Szilagyi, Z.; Vekey, K. J. Carbohydr. Chem. 1998, 17,
of the various formulations or 10
mg/mL lipopolysaccharide (LPS) as
417–437.