2,6-Disubstituted benzoates as neighboring groups for enhanced diastereoselectivity in β-galactosylation reactions: Synthesis of β-1,3-linked oligogalactosides related to arabinogalactan proteins

Article abstract of DOI:10.1021/jo902100q

(Chemical Equation Presented) Arabinogalactan proteins (AGPs) are plant glycoproteins which contain a β-1,3-linked galactan core. The synthesis of the β-galactopyranose-1,3-β-galactopyranose linkage using various 2-O-acyl-protected glycosyl donors has bee

Full text of DOI:10.1021/jo902100q

pubs.acs.org/joc
2,6-Disubstituted Benzoates As Neighboring Groups for Enhanced
Diastereoselectivity in β-Galactosylation Reactions: Synthesis of
β-1,3-Linked Oligogalactosides Related to Arabinogalactan Proteins
Nathan W. McGill and Spencer J. Williams*
School of Chemistry and Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Parkville, Victoria 3010, Australia
sjwill@unimelb.edu.au
Received September 29, 2009
Arabinogalactan proteins (AGPs) are plant glycoproteins which contain a β-1,3-linked galactan
core. The synthesis of the β-galactopyranose-1,3-β-galactopyranose linkage using various 2-O-acyl-
protected glycosyl donors has been plagued with poor stereoselectivity and side reactions including
orthoester formation and transesterification of the 2-O-acyl group from the donor to the acceptor.
We have investigated the use of 2,6-disubstituted benzoyl groups as bulky neighboring groups on the
glycosyl donor. A 2,4,6-trimethylbenzoyl group was found to be optimal and enabled the formation
of the β-galactopyranose-1,3-β-galactopyranose linkage to disarmed ester-protected acceptors,
suppressing transesterification and reducing orthoester formation while enhancing the β-selectivity
of galactosylation reactions. A series of β-1,3-linked oligogalactosides were prepared and elaborated
to neoglycoconjugates for the study of AGP biosynthesis and AGP binding proteins.
Introduction
water emulsions such as citrus- and cola-based beverages.
AGPs are characterized by a hydroxyproline-rich protein
core, which is often modified at the C-terminus through the
installation of a glycosylphosphatidyl anchor.4 The hydro-
xyproline residues are O-glycosylated with complex glycans
rich in D-arabinofuranose and D-galactopyranose.2 The core
regions of these glycans contain a linear galactan chain,
comprising β-1,3-linked oligogalactosides interrupted with
β-1,6-linkages, as demonstrated by sensitivity to periodate
cleavage.5
Arabinogalactan proteins (AGPs) are plant glycoproteins
with emerging roles in embryogenesis, cellular homeostasis,
and developmental processes.1,2 They are significant articles
of commerce, being the major constituent of gum arabic,
which possesses a wide range of applications in the food and
pharmaceutical industries owing to its activities as an emul-
sion stabilizer, excipient, and demulcent (soothing agent).3
Gum arabic is exulted as the premier emulsifier for oil-in-
*Corresponding author. Tel: þ61 3 8344 2422. Fax: þ61 3 9347 8124.
(1) Seifert, G. J.; Roberts, K. Annu. Rev. Plant Biol. 2007, 58, 137–161.
(2) Showalter, A. M. Cell. Mol. Life Sci. 2001, 58, 1399–1417.
(3) Islam, A. M.; Phillips, G. O.; Sljivo, A.; Snowden, M. J.; Williams, P.
A. Food Hydrocolloids 1997, 11, 493–505.
(4) Gaspar, Y.; Johnson, K. L.; McKenna, J. A.; Bacic, A.; Schultz, C. J.
Plant Mol. Biol. 2001, 47, 161–176.
(5) Tan, L.; Qiu, F.; Lamport, D. T.; Kieliszewski, M. J. J. Biol. Chem.
2004, 279, 13156–13165.
9388 J. Org. Chem. 2009, 74, 9388–9398
Published on Web 11/20/2009
DOI: 10.1021/jo902100q
r
2009 American Chemical Society

McGill and Williams
JOCArticle
SCHEME 1. (a) Mechanism of Glycosylation with Neighboring Group Participation, and Side Reactions Leading to Poor
Stereoselectivity, Orthoester Formation, And Transesterification; (b) Comparison of Dioxolenium Ion Intermediates of
Standard and 2,6-Disubstituted Benzoyl Groups; (c) Protecting Groups Investigated in This Work: Moz = 2,6-Dimethoxybenzoyl;
Mez = 2,4,6-Trimethylbenzoyl
The synthesis of the β-D-galactopyranosyl-1,3-β-D-galac-
topyranose linkage has attracted some attention owing to
its occurrence in the tetrasaccharide core of glycosamino-
glycan proteoglycans,6 the type V core of the ABO blood
group antigens,7 protozoan parasites such as leishmania,8,9
and various plant-based materials including arabino-
galactan10 and AGPs.11-13 However, poor stereoselec-
tivity is commonly observed in many galactosylation
unexpected occurrence of R-galactosides even with a
2-O-benzoyl group on the donor that should be capable
of neighboring group participation.17 Kong and co-
workers have reported the development of galactosyl
donors that give stereoselective R-galactosylation; these
glycosyl donors possess an electron-rich alkyl substituent
at O-3 and a 2-O-benzoyl group.15 Other complicating
processes have been seen with 2-O-acyl galactosyl donors,
including the formation of orthoesters21-23 and transester-
ification of the 2-O-acyl group to the acceptor alcohol.24-26
These processes most likely occur through nucleo-
philic attack of the acceptor alcohol on the dioxolenium
ion carbon of the intermediate formed upon activa-
tion of the glycosyl donor.27 Whitfield and co-workers
have presented computational evidence that orthoester
formation and transesterification are separate processes
but which proceed through a common dioxolenium ion
(Scheme 1a).25,28
reactions.14-20 In a landmark study, Kovac and co-workers
ꢀ
reported the synthesis of a series of β-1,3-linked oligoga-
lactosides, but this work was plagued by the frequent and
(6) Allen, J. G.; Fraser-Reid, B. J. Am. Chem. Soc. 1999, 121, 468–469.
(7) Meloncelli, P. J.; Lowary, T. L. Aust. J. Chem. 2009, 62, 558–574.
(8) McConville, M. J.; Thomas-Oates, J. E.; Ferguson, M. A.; Homans,
S. W. J. Biol. Chem. 1990, 265, 19611–19623.
(9) McConville, M. J.; Turco, S. J.; Ferguson, M. A.; Sacks, D. L. EMBO
J. 1992, 11, 3593–3600.
€
(10) Burgermeister, J.; Paper, D. H.; Vogl, H.; Linhardt, R. J.; Franz, G.
Carbohydr. Res. 2002, 337, 1459–1466.
(11) Chowdhary, M. S.; Navia, J. L.; Anderson, L. Carbohydr. Res. 1986,
150, 173–185.
(12) Ziegler, T.; Adams, B.; Kovac, P.; Glaudemans, C. P. J. J. Carbo-
hydr. Chem. 1990, 9, 135–158.
(13) Wang, L. X.; Sakairi, N.; Kuzohara, H. J. Carbohydr. Chem. 1993,
10, 349–361.
(21) Yang, Z.; Lin, W.; Yu, B. Carbohydr. Res. 2000, 329, 879–884.
(22) Wilstermann, M.; Magnusson, G. Carbohydr. Res. 1995, 272, 1–7.
(23) Seeberger, P. H.; Eckhardt, M.; Gutteridge, C. E.; Danishefsky, S. J.
J. Am. Chem. Soc. 1997, 119, 10064–10072.
ꢀ
(24) Ziegler, T.; Kovac, P.; Glaudemans, C. P. J. Liebigs Ann. Chem.
(14) Yang, F.; Du, Y. Carbohydr. Res. 2002, 337, 485–491.
(15) Chen, L.; Kong, F. Tetrahedron Lett. 2003, 44, 3691–3695.
(16) Jacquinet, J.-C. Carbohydr. Res. 2004, 339, 349–359.
1990, 613–615.
ꢀ
(25) Berces, A.; Whitfield, D. M.; Nukada, T.; do Santos, Z., I.;
ꢀ
(17) Kovac, P.; Taylor, R. B.; Glaudemans, C. P. J. J. Org. Chem. 1985,
50, 5323–5333.
(18) Li, A.; Kong, F. Carbohydr. Res. 2005, 340, 1949–1962.
Obuchowska, A.; Krepinsky, J. J. Can. J. Chem. 2004, 82, 1157–1171.
(26) Murakami, T.; Hirono, R.; Sato, Y.; Furusawa, K. Carbohydr. Res.
2007, 342, 1009–1020.
€
(19) Yang, F.; He, H.; Du, Y.; Lu, M. Carbohydr. Res. 2002, 337, 1165–
1169.
(20) Chen, L.; Kong, F. Carbohydr. Res. 2002, 337, 1373–1380.
(27) Crich, D.; Dai, Z.; Gastaldi, S. J. Org. Chem. 1999, 64, 5224–5229.
ꢀ
(28) Nukada, T.; Berces, A.; Zgierski, M. Z.; Whitfield, D. M. J. Am.
Chem. Soc. 1998, 120, 13291–13295.
J. Org. Chem. Vol. 74, No. 24, 2009 9389

McGill and Williams
JOCArticle
TABLE 1. Effect of Protecting Group on the Glycosylation of
8-Azidooctan-1-ol Using Donors 5, 7, 9, and 10
which is widely acknowledged for its insensitivity to transes-
terification.23,32 These results may be readily rationalized in
terms of activation of the glycosyl donor leading first to an
oxocarbenium ion, which is captured by the neighboring
group to give a dioxolenium ion (Scheme 1a).27 In the normal
course of events, this dioxolenium ion should undergo SN2-
like reaction with a nucleophilic alcohol. However, a reac-
tivity mismatch with an acceptor alcohol may lead to either
nucleophilic attack on the intermediate at the dioxolenium
carbon, leading to transesterification, or reversion to the
oxocarbenium ion, allowing bottom-face attack and forma-
tion of the R-anomer.
entry
donor
yielda (%)
1
2
3
4
5, R = Bz
25b
45b
44c
75
7, R = Piv
9, R = Moz
10, R = Mez
aReactions were performed using donor (1 equiv), acceptor
(1.2 equiv), NIS (1.4 equiv), and TfOH (0.25 equiv) in CH2Cl2 at 0 °C.
bTransesterification product was isolated in 10% yield. cR-Glycoside
was isolated in 6% yield; transesterification product was isolated in 15%
yield.
We are interested in synthesizing a series of azidooctyl
β-1,3-oligogalactosides 1-3 as central intermediates in the
preparation of neoglycoconjugates for use as artificial sub-
strates for AGP biosynthetic enzymes and as antigens to
investigate AGP binding proteins. In this work, we have
investigated 2,6-disubstituted benzoates as bulky protecting
and neighboring groups to attempt to overcome poor stereo-
selectivity and transesterification seen in difficult galactosy-
lations (Scheme 1b). Such 2,6-disubstituted benzoates have
been previously investigated by others as neighboring groups
for glycosylation of aliphatic alcohols.25,29,30 This work
represents the first to investigate their use for the formation
of more complex oligosaccharides in a form suitable for
applications as neoglycoconjugates for the study of AGP
biosynthesis and corresponding binding proteins.
These unexpected, but not unprecedented,32 results
prompted us to investigate the use of a 2,6-disubstituted
benzoate as a neighboring group, with the expectation that
the ortho substituents on the benzoyl group should sterically
shield the intermediate dioxolenium ion and prevent trans-
esterification through preventing nucleophilic attack at this
site (Scheme 1b). In addition, the steric bulk of this group
might prevent 1,2-cis-substitution on an intermediate oxo-
nium ion in the event of a reactivity mismatch between
an acceptor alcohol and the derived dioxolenium ion
(Scheme 1a). We chose to investigate 2,6-dimethoxybenzoyl
chloride (MozCl), and owing to its lower cost, 2,4,6-
trimethylbenzoyl chloride (MezCl) was identified as a
pragmatic alternative to 2,6-dimethylbenzoyl chloride
(Scheme 1c). In a glycosylation reaction with 8-azidooctan-
1-ol, 2,6-dimethoxybenzoate 9 gave 11 with poor stereose-
lectivity and also suffered from transesterification (Table 1).
On the other hand, the mesitoate 10 was an effective glycosyl
donor and afforded the β-galactoside 12 in a yield of 75%
with no R-anomer or transesterification products observed.
These results are especially interesting in the context of work
by Whitfield and co-workers, who examined the glycosyla-
tion of a PEG-based primary alcohol acceptor using the
closely related glycosyl donors: pivalate 13, 2,6-dimethox-
ybenzoate 14, and 2,6-dimethylbenzoate 15.25,32 While the
pivalate donor 13 was susceptible to transesterification,
the 2,6-dimethylbenzoate 15 was an adequate glycosyl
donor, although it was prone to orthoester formation.
Conversely, the 2,6-dimethoxybenzoate 14 was pro-
nounced as the optimal donor for glycosylation, although
it underwent transesterification to the acceptor to a limited
extent.
Results and Discussion
Owing to their stability to many of the conditions required
for protecting group manipulation, we employed thioglyco-
sides as glycosyl donors. The 3,4-di-O-isopropylidene galac-
toside 5 was selected as a glycosyl donor as it was anticipated
that the isopropylidene group could be readily removed and
the resultant diol converted to a glycosyl acceptor suitable
for sequential nonreducing-end galactosylations to deliver
the oligogalactosides 1-3. Compound 5 was prepared in two
steps from tetraol 4a31 (see the Supporting Information).
Glycosylation of 8-azidooctan-1-ol using dibenzoate donor
5 afforded the β-galactoside 6 in only 25% yield, accompa-
nied by transesterification product (10%) (Table 1). An
attempt to remedy this poorly performing glycosylation
through the use of a pivalyl-protected donor 7 provided little
improvement; a 45% yield of the β-galactoside 8 was
obtained. The observation of low yields of the desired
β-galactosides, along with amounts of R-anomer and trans-
esterification products, is unusual for a 2-O-pivalyl donor,
(29) Nunomura, S.; Mori, M.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1989,
30, 6713–6716.
(30) Nunomura, S.; Mori, M.; Ito, Y.; Ogawa, T. Tetrahedron Lett. 1989,
30, 5619–5622.
(31) Yde, M.; De Bruyne, K. Carbohydr. Res. 1973, 26, 227–229.
The next phase in our synthesis required the elaboration of
the azidooctyl galactoside 12 to glycosyl acceptor 16. This
ꢀ
(32) Nukada, T.; Berces, A.; Whitfield, D. M. J. Org. Chem. 1999, 64,
9030–9045.
9390 J. Org. Chem. Vol. 74, No. 24, 2009

McGill and Williams
JOCArticle
SCHEME 2
8-azidooctan-1-ol, followed by deacetylation to give 1. Iso-
propylidenation of 1, followed by acetylation afforded 17;
sequential isopropylidene hydrolysis, orthoacetate forma-
tion, and regioselective hydrolysis furnished 18 (Scheme 2).
We also elected to prepare a simpler glycosyl donor, the
tetramesitoate 19 (see the Supporting Information). When
treated with alcohol 18, 19 proved a competent glycosyl
donor, allowing the synthesis of the disaccharides 20a,b in
47% (1:11 R:β). Compound 20b was deprotected using 1 M
LiOH in MeOH/THF/H2O to afford 2.
It was now apparent that the nonreducing-end strategy for
the construction of the target oligosaccharides 1-3, invol-
ving the sequential addition of individual monosaccharide
residues to the nonreducing-end of the growing chain, was
incompatible with the use of a 2-O-mesitoyl group as a
neighboring group, as each subsequent glycosylation would
require the union of a glycosyl donor and acceptor, each
bearing a 2-O-mesitoyl group. We therefore required
a disaccharide donor that could be used to assemble
a trisaccharide in a [2 þ 1] fashion. For this purpose, we
required a galactose acceptor that could be readily converted
to a glycosyl donor after conversion to a disaccharide.
4-Methoxyphenyl alcohol 26 was prepared from galactose
pentaacetate by an eight step route (Scheme 3). This reliable
synthetic procedure requires only two recrystallizations and
no chromatography, and can provide 10 g of alcohol 26 in
just 3 days from galactose pentaacetate.
SCHEME 3
We next sought to compare the performance of the
isopropylidene-protected donor 10 and the tetramesitoate
19. We were mindful of the precedent of Whitfield and co-
workers that the reactivity of related glycosyl donors toward
simple primary alcohol acceptors was optimal when using
relatively high amounts of NIS and TfOH, which also served
to suppress acyl transfer.25 Reaction of 19 and 26 in the
presence of 2.5 equiv of NIS and 1.4 equiv of TfOH provided
the glycoside 27 in only 33% yield (Table 2). Reducing the
amount of TfOH (0.25 equiv) resulted in a sluggish reaction,
which suggested that the reactivities of the donor and
acceptor were mismatched. The reaction conditions were
modified to enhance the reactivity of the donor, while the
amount of NIS was reduced to prevent side reactions.
Reducing the amount of NIS (1.5 equiv) did not affect the
outcome of the reaction, whereas increasing the amount of
TfOH (0.7 equiv) resulted in acceleration of the reaction and
an improvement in yield, presumably owing to an increased
concentration of iodonium triflate in the reaction mixture.
Switching to acid-washed molecular sieves22 resulted in a
was readily achieved by hydrolysis of the isopropylidene
acetal and regioselective 4-O-acetylation via the intermediate
orthoacetate according to the methodology of King and
Allbutt.33,34 However, under a range of conditions, coupling
of mesitoate donor 10 and acceptor 16 was completely
unsuccessful, with no glycoside product being isolated.
Together these results raised some important questions.
Was the failure to glycosylate related to poor reactivity of the
glycosyl donor 10, owing to the presence of the bulky 2-O-
mesitoyl group, or the poor reactivity of the acceptor 16,
which also bears a 2-O-mesitoyl group adjacent to the
hydroxy group?35 More generally, can conditions be
found that 2-O-mesitoyl-protected glycosyl donors can be
used to glycosylate carbohydrate alcohols? Finally, can
mesitoyl groups be utilized as stereodirecting groups to
complete the synthesis of our target β-1,3-linked oligogalac-
tosides 1-3?
The failure of the condensation between 10 and 16 was
attributed to the severe steric congestion arising from the
presence of a 2-O-mesitoyl group on both the donor and
acceptor adjacent to the sites of reaction. As a 2-O-mesitoyl
group was required on the donor as a neighboring group, we
elected to modify the acceptor. Compound 18 was obtained
by ZnCl2-catalyzed26 coupling of galactose pentaacetate and
(33) King, J. F.; Allbutt, A. D. Tetrahedron Lett. 1967, 8, 49–54.
(34) King, J. F.; Allbutt, A. D. Can. J. Chem. 1970, 48, 1754–1769.
(35) An attempted glycosylation of donor 19 and acceptor 16 was also
unsuccessful.
J. Org. Chem. Vol. 74, No. 24, 2009 9391

McGill and Williams
JOCArticle
TABLE 2. Effects of Promotion Conditions, Molecular Sieves, and
Temperature on the Glycosylation of Galactoside 26 with Donor 19
SCHEME 4
NIS
(equiv)
TfOH
(equiv)
mol sieves
˚
(A)
temp
(°C)
yielda
(%)
entry
1
2
3
4
5
6
2.5
2.5
1.5
1.5
1.5
1.5
1.4
4
4
4
4
0
0
0
0
33b
44
47
67
69
80
0.25
0.25
0.7
0.7
0.7
5 (AW)
5 (AW)
0
0 to rt
aReactions were performed in CH2Cl2. b12% of the R-anomer was
isolated.
minor improvement in yield. Finally, running the reaction at
room temperature afforded the disaccharide 27 in 80% yield.
Attempted glycosylation of the alcohol 26 with the donor 10
was completely unsuccessful. Orthoester 28 was the major
product, resulting from nucleophilic attack of the acceptor
on the carbon of the dioxolenium ion intermediate. This
SCHEME 5
ꢀ
result is not without precedent; Berces et al. observed the
formation of an orthoester in attempted glycosylations of a
primary alcohol with the donor 15.25 Nonetheless, this result
is striking, as it demonstrates that the ortho-substituents of
the 2-O-mesitoyl group do not completely sterically block
reaction at the dioxolenium ion carbon, even for a sterically
hindered secondary alcohol. Our attempts to rearrange this
orthoester to the glycoside were fruitless and led only to
recovery of the acceptor 26.
In order to complete the synthesis of the trisaccharide 3, the
4-methoxyphenyl disaccharide 27 needed to be converted into
a glycosyl donor. Deacetylation of 27 proved to be nontrivial.
Under a range of mild conditions (cat. NaOMe/MeOH,
DBU/MeOH, or guanidine/MeOH) the 2-acetate remained
refractory, affording 29. This result is reminiscent of the
difficulty seen in cleaving a hindered acetate in the N3
antigen.36 Application of more forcing conditions (0.5 M
NaOMe in MeOH/THF at 50 °C for 24 h) resulted in cleavage
of the three acetates and incomplete cleavage of the mesitoyl
groups (Scheme 4). The heptamesitoate 30 was obtained by
treatment of the crude product with mesitoyl chloride in
pyridine at 100 °C. Treatment of 30 with thiocresol afforded
the thioglycoside 31 as solely the β-anomer. However, the
yield was somewhat better using a more nucleophilic
alkyl thiol, n-butylthiol, affording the corresponding thiogly-
cosides 32a,b as an anomeric mixture. In this thioglycosyla-
tion reaction, a prolonged reaction time is necessary to
rearrange the intermediate thioorthoesters37,38 to the thiogly-
cosides.
bears seven mesitoyl and three acetyl groups, and complete
deprotection required 5 days. The products were purified by
reversed phase chromatography, which was facilitated by the
lipophilic nature of the azidooctyl aglycon.
Synthesis of Neoglycoconjugates. Neoglycoconjugates are
carbohydrates linked to a noncarbohydrate group such as a
protein, fluorescent label, or insoluble polymer support.
Such adducts have become indispensible biochemical tools
owing to the combination of the biological activity of the
carbohydrate with the biological, chemical, or physical
properties of the noncarbohydrate group. Like others inter-
ested in neoglycoconjugates, we have recognized that the
azidooctyl chain provides significant advantages in terms of
(a) its lipophilicity, allowing biphasic partitioning between
water/butanol or purification by reversed-phase chromatog-
raphy, (b) its potential for reduction to a aminooctyl chain
for conjugation chemistry, and (c) its potential for con-
jugation with terminal alkynes using the Cu(I)-catalyzed
azide-alkyne cycloaddition (CuAAC) reaction.39 We were
Glycosylation of the acceptor 18 was attempted using the
previously optimized conditions (Scheme 5). In this case, the
thiobutyl glycosides 32 performed significantly better than
the corresponding thiotolyl glycoside 31. Removal of the
protecting groups from 33 was achieved by saponification
using LiOH in a MeOH/THF/H2O mixture. Compound 33
(36) Kim, H. M.; Kim, I. J.; Danishefsky, S. J. J. Am. Chem. Soc. 2001,
123, 35–48.
(37) Magnusson, G. J. Org. Chem. 1976, 42, 913–914.
(38) Zhang, Z.; Magnusson, G. Carbohydr. Res. 1996, 295, 41–55.
(39) Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952–3015.
9392 J. Org. Chem. Vol. 74, No. 24, 2009

McGill and Williams
JOCArticle
SCHEME 6
interested in the application of the mono-, di-, and trigalacto-
sides 1-3 as substrates for AGP-modifying enzymes such as
glycoside hydrolases40-44 and glycosyltransferases,45,46 and
as antigens for the study of AGP-recognizing proteins.2,41
For the former case, the ability to introduce a dye using the
CuAAC reaction would allow the monitoring of reaction
products by HPLC using sensitive fluorescence detection; in
the latter case, a method for the conjugation of the synthetic
galactosides to proteins was required.
Nitrobenzodiazoles (NBDs) are a useful fluorescent label
owing to their small size and superior water solubility relative
to many commonly used alternatives. Hindsgaul and co-
workers have used neoglycoconjugates with NBD dyes to
streamline the detection of carbohydrate-lectin interac-
tions.47 The alkynyl NBD-dye 34 was prepared in one step
from commercially available 4-chloro-7-nitrobenzofurazan
by nucleophilic aromatic substitution with propargylamine.
This alkyne was coupled with the azidooctyl galactosides 1-3
upon exposure to CuSO4, sodium ascorbate, and the Cu(I)-
stabilizing ligand tris(benzyltriazolylmethyl)amine48,49 to
afford the corresponding conjugates 35-37 (Scheme 6). The
conjugates displayed excellent fluorescent properties with
λex = 470 nm, λem= 540 nm (see the Supporting Information).
Tietze and co-workers introduced the use of squaric acids
as linkers for the conjugation of amine-derivatized carbohy-
drate haptens to proteins.50 Conjugation is realized through
the stepwise reaction of the carbohydrate amine with diethyl
squarate followed by the reaction of the intermediate ethyl
squaramate adduct with the amino residues of a carrier
protein. While a range of other linker methodologies are
available, the squaric acid diester protocol uses commercially
available dialkyl squarates and exhibits good selectivity for
each coupling step, and the chemical stability of the inter-
mediate squaramate ester allows its purification and storage.
One significant limitation for the use of this methodology is
the potent immunogenicity of the squaramide linker,51 which
means that squaramide-linked antigens should not be used
for raising antibodies, but rather for their characterization.
Reduction of the azido groups of 1-3 was achieved
by exposure to triphenylphosphine in 10:1 THF/water
(Scheme 6). The crude products were extracted into water
and coupled with diethyl squarate in pH 8.0 phosphate
(40) Pellerin, P.; Brillouet, J.-M. Carbohydr. Res. 1994, 264, 281–291.
(41) Ichinose, H.; Yoshida, M.; Kotake, T.; Kuno, A.; Igarashi, K.;
Tsumuraya, Y.; Samejima, M.; Hirabayashi, J.; Kobayashi, H.; Kaneko,
S. J. Biol. Chem. 2005, 280, 25820–25829.
(42) Ichinose, H.; Kotake, T.; Tsumuraya, Y.; Kaneko, S. Biosci. Bio-
technol. Biochem. 2006, 70, 2745–2750.
(43) Ichinose, H.; Kuno, A.; Kotake, T.; Yoshida, M.; Sakka, K.;
Hirabayashi, J.; Tsumuraya, Y.; Kaneko, S. Appl. Environ. Microbiol.
2006, 72, 3515–3523.
ꢀ
(44) Tsumuraya, Y.; Mochizuki, N.; Hashimoto, Y.; Kovac, P. J. Biol.
Chem. 1990, 265, 7207–7215.
ꢀ
buffer, according to the excellent procedure of Kovac and
co-workers.52 The products were purified by reversed-phase
chromatography to afford the ethyl squaramate adducts
38-40 in 92-98% yield.
Conclusions
The β-galactopyranose-1,3-β-galactopyranose linkage
presents a special challenge for chemical synthesis owing to
the reactivity mismatch commonly found between requisite
(45) Qu, Y.; Egelund, J.; Gilson, P. R.; Houghton, F.; Gleeson, P. A.;
Schultz, C. J.; Bacic, A. Plant Mol. Biol. 2008, 68, 43–59.
(46) Kato, H.; Takeuchi, Y.; Tsumuraya, Y.; Hashimoto, Y.; Nakano,
ꢀ
H.; Kovac, P. Planta 2003, 217, 271–282.
(47) Dowlut, M.; Hall, D. G.; Hindsgaul, O. J. Org. Chem. 2005, 70,
9809–9813.
(48) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett.
2004, 6, 2853–2855.
€
(50) Tietze, L. F.; Schroter, C.; Gabius, S.; Brinck, U.; Goerlach-Graw,
A.; Gabius, H.-J. Bioconjugate Chem. 1991, 2, 148–153.
(51) Wu, X.; Ling, C. C.; Bundle, D. R. Org. Lett. 2004, 6, 4407–4410.
ꢀ
(52) Hou, S.-J.; Saksena, R.; Kovac, P. Carbohydr. Res. 2008, 343, 196–
210.
(49) Donnelly, P. S.; Zanatta, S. D.; Zammit, S. C.; White, J. M.;
Williams, S. J. Chem. Commun. 2008, 2459–2461.
J. Org. Chem. Vol. 74, No. 24, 2009 9393

McGill and Williams
JOCArticle
donor and acceptor molecules, resulting in side reactions
such as transesterification and orthoester formation. In the
present work, we demonstrate the utility of 2-O-mesitoate
esters as neighboring groups for synthesis of this challen-
ging linkage. The 2-O-mesitoyl group suppressed tran-
sesterification seen for benzoyl- and pivalyl-protected gly-
cosyl donors and afforded excellent β-stereoselectivity
in most cases. Interestingly, the 2-O-mesitoyl group did
not prevent orthoester formation, indicating that steric
hindrance of the central carbon of the intermediate dioxo-
lenium ion does not fully suppress reactivity at this site.
The mesitoyl group requires relatively robust conditions
for its removal (LiOH in MeOH/THF/H2O at 80 °C), which
may prove a limitation for its use in sensitive systems.
In this regard, the investigation of 2,6-dimethyl-4-X-ben-
zoyl groups, where “X” is an electron-withdrawing group,
may prove fruitful in allowing the development of milder
deprotection conditions.25 Using the 2-O-mesitoyl group
we have reported a direct synthesis of a series of β-1,3-
linked oligogalactosides useful for the study of arabinoga-
lactan proteins and demonstrated their elaboration into
fluorescently labeled neoglycoconjugates and activated
forms suitable for synthesis of artificial antigens. On the
basis of these results, the 2-O-mesitoyl group is a convenient
participating group for the synthesis of 1,2-trans-glycosidic
linkages in systems sensitive to transesterification and/
or poor diastereoselectivity and which are sufficiently
robust to withstand the basic conditions required for their
removal.
(34 μL, 0.36 mmol) and pyridine (72 μL, 0.90 mmol) were added
to the solution, which was stirred for 90 min. The reaction was
quenched with aqueous HCl (10 mL, 1 M) and the resultant
mixture extracted with CH2Cl2 (2 ꢀ 10 mL). The organic extract
was washed with 1 M HCl (3 ꢀ 10 mL), satd NaHCO3 (2 ꢀ 10
mL), and brine (10 mL) then dried (MgSO4). The solvent was
removed by evaporation to afford an oil, which was purified by
flash chromatography (EtOAc/petroleum spirits 40%) to yield
17 (40 mg, 51%) as a colorless oil: [R]22D þ19 (c 1.0 in CHCl3);
1H NMR (500 MHz, CDCl3) δ 1.30-1.60 (12 H, m), 1.33-1.56
(6 H, 2 ꢀ s), 2.08-2.09 (6 H, 2 ꢀ s), 3.52 (2 H, t, J 7.0 Hz), 3.63
(1 H, ddd, J 10.0, 7.0, 7.0 Hz), 3.85 (1 H, ddd, J 10.0, 7.0, 7.0 Hz),
3.97 (1 H, ddd, J 2.0, 6.0, 6.0 Hz), 4.15 (1 H, dd, J 7.0, 6.0 Hz),
4.16 (1 H, dd, J 6.0, 2.0 Hz), 4.31 (1 H, d, J 8.0 Hz), 4.33 (2 H, m),
4.95 (1 H, dd, J 8.0, 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ
20.8, 20.9 (2 C), 25.8-29.4 (8 C), 51.4 (1 C), 63.4 (1 C), 69.6, 70.8,
72.9, 73.6, 77.2 (5 C), 100.4 (1 C), 110.7 (1 C) 169.5, 170.8 (2 C);
HRMS (ESIþ) m/z 480.2320 (C21H35N3NaO8 [M þ Na]þ
requires 480.2316).
8-Azidooctyl 2,4,6-Tri-O-acetyl-β-D-galactopyranoside (18).
Compound 17 (180 mg, 0.393 mmol) was dissolved in chloro-
form (10 mL) containing 90% aqueous TFA (2 mL) and was
stirred at rt for 1 h. Evaporation of the solvent gave a residue
which was dissolved in toluene (10 mL) containing p-TsOH (17
mg, 0.090 mmol). Triethyl orthoacetate (270 μL, 1.49 mmol) was
added to the solution, which was stirred at rt for 2 h. The
reaction was neutralized (Et3N) and evaporated to give a solid
residue, which was dissolved in 80% aqueous acetic acid (10 mL)
and stirred at 40 °C for 15 min. Evaporation of the solvent gave a
white residue, which was recrystallized from EtOAc/petroleum
spirits to afford 18 (120 mg, 72% three steps) as fine white
needles: mp 89 °C; [R]20D -10 (c 1.0 in CHCl3); 1H NMR (500
MHz, CDCl3) δ 1.32-1.62 (12 H, m), 2.07-2.19 (9 H, 3 ꢀ s),
3.27 (2 H, t, J 7.0 Hz), 3.48 (1 H, ddd, J 10.0, 7.0, 7.0 Hz), 3.84
(1 H, ddd, J 1.0, 7.0, 7.0 Hz), 3.85 (1 H, dd, J 10.0, 3.5 Hz), 3.89
(1 H, ddd, J 10.0, 7.0, 7.0 Hz), 4.15 (1 H, dd, J 7.0, 11.5 Hz), 4.18
(1 H, dd, J 7.0, 11.5 Hz), 4.42 (1 H, d, J 8.0 Hz), 4.96 (1 H, dd,
J 8.0, 10.0 Hz), 5.33 (1 H, dd, J 3.5, 1.0 Hz); 13C NMR (125
MHz, CDCl3) δ 20.7-20.9 (3 C), 25.7-29.4 (6 C), 51.4 (1 C),
61.8 (1 C), 69.7, 70.1, 70.8, 71.5, 72.8 (5 C), 101.0 (1 C),
170.5-171.1 (3 C); HRMS (ESIþ) m/z 482.2114 (C20H33N3-
NaO9 [M þ Na]þ requires 482.2120). Anal. Calcd for C20H33-
N3O9: C, 52.28; H, 7.24. Found: C, 52.31; H, 7.21.
Experimental Section
General experimental details have been provided pre-
viously.53
8-Azidooctyl 3,4-O-isopropylidene-2,6-di-O-mesitoyl-β-D-ga-
lactopyranoside (12). TfOH (13 μL, 0.16 mmol) was added to a
mixture of donor 10 (0.49 g, 0.62 mmol), 8-azidooctan-1-ol (0.13
˚
g, 0.75 mmol), NIS (0.19 g, 0.87 mmol), and 4 A mol sieves in
CH2Cl2 at 0 °C and stirred for 5 min. The reaction was quenched
with 0.50 M sodium thiosulfate (10 mL), and the organic phase
was washed with satd aq NaHCO3 (2 ꢀ 25 mL) and brine (2 ꢀ 25
mL) and dried (MgSO4). The solvent was evaporated to give an
oil, which was subjected to flash chromatography (5% EtOAc/
toluene) to give 12 (0.31 g, 75%) as a colorless oil: [R]23D þ16 (c
0.8 in CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.26-1.62 (12 H,
m), 1.37-1.65 (6 H, 2 ꢀ s), 2.29-2.33 (18 H, 4 ꢀ s), 3.25 (2 H, t,
J 7.5 Hz), 3.41 (1 H, ddd, J 10.0, J 7.0, J 7.0 Hz), 3.87 (1 H, ddd,
J 10.0, 7.0, 7.0 Hz), 4.14 (1 H, ddd, J 2.0, 5.0, 7.0 Hz, H5),
4.23-4.27 (2 H, m), 4.40 (1 H, d, J 8.0 Hz, H1), 4.60 (1 H, dd,
J 7.0, 11.5 Hz), 4.65 (1 H, dd, J 5.0, 11.5 Hz), 5.29 (1 H, dd, J 8.0,
7.0 Hz), 6.86-6.87 (4 H, m); 13C NMR (125 MHz, CDCl3) δ
19.9-29.8 (14 C), 51.7 (1 C), 64.3 (1 C), 69.8, 71.2, 73.3, 74.1,
77.1 (5 C), 100.5 (1 C), 111.1 (1 C), 128.5-139.8 (12 C), 168.8,
170.0 (2 C); HRMS (ESIþ) m/z 688.3569 (C37H51N3NaO8
[M þ Na]þ requires 688.3568).
8-Azidooctyl β-D-Galactopyranoside (1). ZnCl2 (83.9 mg, 6.14
mmol) was added to a mixture of β-D-galactose pentaacetate
(2.00 g, 5.12 mmol) and 8-azidooctan-1-ol (1.05 g, 6.14 mmol) in
anhydrous toluene (70 mL) at rt. The suspension was warmed
to 60 °C and stirred for 3 days. The reaction was quenched
with satd aq NaHCO3 (5 mL). The organic phase was washed
with satd aq NaHCO3 (2 ꢀ 20 mL) and brine (10 mL) and dried
(MgSO4). The solvent was evaporated to afford a residue,
which was dissolved in MeOH (20 mL) containing catalytic
NaOMe. The solution was stirred for 15 min, neutralized with
Amberlite IR-120 resin (Hþ form), and concentrated to give
a pale yellow oil, which was purified by flash chromato-
graphy (50% EtOAc/petroleum spirits) to yield the tetraol 1
(1.01 g, 59%) over two steps: [R]22 -12 (c 1.0 in CHCl3);
D
1H NMR (500 MHz, MeOH-d4) δ 1.36-1.65 (12 H, m), 3.92
(2 H, t, J 6.5 Hz), 3.46 (1 H, dd, J 9.5, 3.5 Hz), 3.50 (1 H, ddd,
J 1.0, 6.5, 6.5 Hz), 3.51 (1 H, dd, J 7.5, 9.5 Hz), 3.55 (1 H, ddd,
J 9.5, 7.0, 7.0 Hz), 3.72 (1 H, dd, J 6.5, 11.0 Hz), 3.75 (1 H, dd,
J 6.5, 11.0 Hz), 3.84 (1 H, dd, J 3.5, 1.0 Hz), 3.90 (1 H, ddd, J 9.5,
6.5, 6.5 Hz), 4.21 (1 H, d, J 7.5 Hz); 13C NMR (125 MHz,
MeOH-d4) δ 27.0-30.8 (6 C), 52.5 (1 C), 62.5 (1 C), 70.4, 70.8,
72.6, 75.1, 76.6 (5 C), 105.0 (1 C); HRMS (ESIþ) m/z 356.1795
(C14H27N3NaO6 [M þ Na]þ requires 356.1792). Anal. Calcd for
C14H27N3O6: C, 50.44; H, 8.16; N, 12.60. Found: C, 50.50; H,
8.25; N, 12.52.
8-Azidooctyl 2,6-Di-O-acetyl-3,4-O-isopropylidene-β-D-ga-
lactopyranoside (17). A solution of 12 (60 mg, 0.090 mmol)
and methanolic LiOH (1 mL, 1 M) in 90% aqueous THF (2 mL)
was stirred at 60 °C for 5 d. Amberlite IR-120 resin (Hþ form)
was added to neutralize the solution. Filtration and evaporation
of the solvent from the filtrate gave a residue that was dis-
solved in CH2Cl2 (10 mL) and chilled to 0 °C. Acetic anhydride
(53) Dinev, Z.; Gannon, C. T.; Egan, C.; Watt, J. A.; McConville, M. J.;
Williams, S. J. Org. Biomol. Chem. 2007, 5, 952–959.
9394 J. Org. Chem. Vol. 74, No. 24, 2009

McGill and Williams
JOCArticle
8-Azidooctyl 2,3,4,6-Tetra-O-mesitoyl-r-D-galactopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-β-D-galactopyranoside (20a) and 8-Azi-
dooctyl 2,3,4,6-tetra-O-mesitoyl-β-D-galactopyranosyl-(1f3)-
2,4,6-tri-O-acetyl-β-D-galactopyranoside (20b). TfOH (121 μL,
1.37 mmol) was added to a mixture of donor 19 (2.05 g, 2.35
mmol), acceptor 18 (0.901 g, 1.96 mmol), NIS (0.658 g, 2.94
solvent from the filtrate gave a pale yellow residue, which was
recrystallized from n-propanol/petroleum spirits to afford tetra-
ol 22 (1.58 g, 84%) as a white powder: mp 162 °C (lit.55 mp
160-161 °C); [R]23 -39 (c 1 in CHCl3) [lit.55 [R]23 -41.8
D
D
(c 0.28 in CHCl3)]; 1H NMR (500 MHz, CD3OD) δ 3.59 (1 H,
dd, J 9.5, 3.0 Hz), 3.66 (1 H, dd, J 6.0, 6.0 Hz), 3.76 (3 H, s),
3.77-3.83 (3 H, m), 3.92 (1 H, d, J 3.0 Hz), 4.75 (1 H, d, J 8.0
Hz), 6.84-7.09 (4 H, m); 13C NMR (125 MHz, CD3OD) δ 56.2
(1 C), 62.6, 70.3, 72.5, 75.0, 77.0 (5 C), 104.2 (1 C), 115.6-156.7
(6 C); HRMS (ESIþ) m/z 309.0948 (C13H18Na O7 [M þ Na]þ
requires 309.0945).
˚
mmol), and freshly activated 5 A AW molecular sieves in
CH2Cl2 (15 mL) at 0 °C. The mixture was allowed to warm to
rt and was stirred for 4 h. The reaction was quenched by the
addition of 0.50 M sodium thiosulfate (10 mL). The organic
phase was washed with satd aq NaHCO3 (2 ꢀ 25 mL) and brine
(2 ꢀ 25 mL) and dried (MgSO4). The solvent was evaporated to
give an oil, which was subjected to flash chromatography (20%
EtOAc/petroleum spirits). First to elute was the R-linked galac-
toside 20a (105 mg, 4%) as a colorless oil: [R]20D þ84 (c 1.0 in
CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.27-1.60 (12 H),
2.03-2.37 (45 H), 3.28 (2 H, t, J 7.0 Hz), 3.33-3.36 (2 H, m),
3.80 (1 H, ddd, J 10.0, 7.0, 7.0 Hz), 3.91 (1 H, dd, J 10.0, 3.0 Hz),
3.92 (1 H, d, J 8.0 Hz), 3.98 (1 H, dd, J 6.0, 12.0 Hz), 4.07 (1 H,
dd, J 6.0, 12.0 Hz), 4.44-4.51 (1 H, m), 4.58 (1 H, dd, J 7.0, 11.0
Hz), 5.23 (1 H, dd, J 8.0, 10.0 Hz), 5.31 (1 H, dd, J 3.5, 10.0 Hz),
5.43 (1 H, d, J 3.0 Hz), 5.67 (1 H, dd, J 10.0, 3.5 Hz), 5.77 (1 H, d,
J 3.5 Hz), 5.98 (1 H, d, J 3.5 Hz), 6.69-6.89 (8 H, m); 13C NMR
(125 MHz, CDCl3) δ 20.0-29.7 (21 C), 51.5 (1 C), 61.5 (1 C),
63.4-71.0 (10 C), 91.2, 101.6 (2 C), 127.6-140.5 (24 C),
167.7-170.3 (7 C); HRMS (ESIþ) m/z 1228.5564 (C66H83-
N3NaO18 [M þ Na]þ requires 1228.5564). Next to elute was
4-Methoxyphenyl 3,4-O-Isopropylidene-β-D-galactopyranoside
(23). A solution of tetraol 22 (2.0 g, 7.0 mmol) and p-TsOH
(0.13 g, 0.70 mmol) in 2,2-DMP (22 mL) was stirred at rt for 3 h.
The mixture was quenched with Et3N, and the solvent was
evaporated to give an off-white foam. The foam was dissolved
in MeOH (25 mL) containing PPTS (0.18 g, 0.70 mmol) and was
stirred at rt for 10 min. The reaction was quenched (Et3N) and the
solvent evaporated to give white crystals, which were recrystal-
lized from EtOAc/petroleum spirits to yield 23 (1.69 g, 74%) as
white needles: mp 120-121 °C; [R]27D -24 (c 1.2 in CHCl3); 1H
NMR (500 MHz, CD3OD) δ 1.35,1.52 (6 H, 2 ꢀ s), 3.65 (1 H, dd,
J 8.0, 8.0 Hz), 3.75 (3 H, s), 3.78 (1 H, dd, J 4.5, 11.5 Hz), 3.81
(1 H, dd, J 5.5, 11.5 Hz), 3.98 (1 H, ddd, J 2.0, 4.5, 5.5 Hz), 4.11
(1 H, dd, J 8.0, 6.0 Hz), 4.25 (1 H, dd, J 6.0, 2.0 Hz), 4.72 (1 H, d,
J 8.0 Hz), 6.83-7.05 (4 H, m); 13C NMR (125 MHz, CD3OD) δ
26.8, 28.6 (2 C), 55.2 (1 C), 62.6, 74.4, 75.2, 75.4, 81.0 (5 C), 103.2
(1 C), 111.3 (1 C), 115.7-156.9 (6 C); HRMS (ESIþ) m/z
349.1257 (C16H22NaO7 [M þ Na]þ requires 349.1258). Anal.
Calcd for C16H22O7: C, 58.89; H, 6.79. Found: C, 58.85; H, 6.69.
4-Methoxyphenyl 2,6-Di-O-acetyl-3,4-O-isopropylidene-β-
D-galactopyranoside (24). Acetic anhydride (0.87 mL, 9.2 mmol)
was added to a solution of 23 (1.00 g, 3.10 mmol) and DMAP
(112 mg, 0.917 mmol) in pyridine (5 mL) at 0 °C. The mixture
was stirred under N2 for 1 h. The mixture was diluted with
CH2Cl2 (50 mL) and washed with aq HCl (1 M, 25 mL), satd aq
NaHCO3 (2 ꢀ 50 mL), and brine (50 mL). The organic phase
was dried (MgSO4) and concentrated under reduced pressure to
afford an off-white amorphous solid, which was recrystallized
from EtOH/H2O to yield 24 (1.61 g, 84%) as colorless needles:
mp 87-88 °C; [R]23D þ27 (c 1.3 in CHCl3); 1H NMR (500 MHz,
CDCl3) δ 1.36, 1.60 (6 H, 2 ꢀ s), 2.09-2.13 (6 H, 2 ꢀ s), 3.77
(3 H, s), 4.09 (1 H, ddd, J 2.0, 5.0, 5.5 Hz), 4.22 (1 H, dd, J 6.0, 2.0
Hz), 4.26 (1 H, dd, J 7.0, 6.0 Hz), 4.37-4.44 (2 H, m), 4.78 (1 H,
the β-linked galactoside 20b (1.01 g, 43%) as a colorless oil:
1
[R]19 þ32 (c 0.50 in CHCl3); H NMR (500 MHz, CDCl3) δ
D
1.27-1.60 (12 H), 2.05-2.35 (45 H), 3.25 (2 H, t, J 7.0 Hz), 3.37
(1 H, ddd, J 10.0, 7.0, 7.0 Hz), 3.74 (1 H, dd, J 6.5, 6.5 Hz), 3.83 (1
H, ddd, J 10.0, 7.0, 7.0 Hz), 3.99 (1 H, dd, J 10.0, 3.5 Hz), 4.04 (1
H, dd, J 6.5, 12.0 Hz), 4.12 (1 H, dd, J 6.5, 12.0 Hz), 4.22 (1 H, dd,
J 6.0, 6.0 Hz), 4.27 (1 H, d, J 8.0 Hz), 4.47 (1 H, dd, J 6.0, 12.0
Hz), 4.56 (1 H, dd, J 6.0, 12.0 Hz), 5.00 (1 H, d, J 8.0 Hz), 5.17 (1
H, dd, J 8.0, 10.0 Hz), 5.42 (1 H, d, J 3.5 Hz), 5.44 (1 H, dd, J 8.0,
10.0 Hz), 5.63 (1 H, dd, J 10.0, 3.0 Hz), 5.96 (1 H, d, J 3.0 Hz),
6.74-6.89 (8 H, m), 6.77-6.89 (4 H, m); 13C NMR (125 MHz,
CDCl3) δ 20.0-29.3 (21 C), 51.4 (1 C), 62.1-75.5 (11 C), 101.4,
101.6 (2 C), 128.0-140.3 (24 C), 167.1-170.4 (7 C); HRMS
(ESIþ) m/z 1228.5564 (C66H83N3NaO18 [M þ Na]þ requires
1228.5595).
4-Methoxyphenyl 2,3,4,6-Tetra-O-acetyl-β-D-galactopyrano-
side (21). BF3 Et2O (1.95 mL, 15.4 mmol) was added to a stirred
3
solution of p-methoxyphenol (1.91 g, 15.4 mmol) and D-galac-
tose pentaacetate (5.00 g, 12.8 mmol) in CH2Cl2 (100 mL) at
0 °C. The reaction was warmed to rt and stirred for 2 h. The
mixture was quenched with HCl (1 M, 50 mL), and the organic
phase was washed with satd aq NaHCO3 (2 ꢀ 50 mL) and brine
(20 mL). The organic phase was dried (MgSO4) and evaporated
to give a pale yellow residue. The residue was recrystallized from
MeOH to afford tetraacetate 21 (4.87 g, 84%) as colorless
crystals: mp 106-108 °C (lit.54 mp 104 °C); [R]23D þ4 (c 1.0 in
CHCl3); 1H NMR (400 MHz, CDCl3) δ 2.00-2.17 (12 H, 4 ꢀ s),
3.76 (3 H, s), 4.00 (1 H, m), 4.15 (1 H, dd, J 6.4, 11.2 Hz), 4.22 (1
H, dd, J 7.2, 11.2 Hz), 4.91 (1 H, d, J 8.0 Hz), 5.08 (1 H, dd,
J 10.4, 3.6 Hz), 5.42-5.46 (2 H, m), 6.79-6.95 (4 H, m); 13C
NMR (100 MHz, CDCl3) δ 20.5-20.7 (4 C), 55.6 (1 C), 61.2,
66.9, 68.7, 70.8, 70.9 (5 C), 100.8 (1 C), 114.5-155.7 (6 C),
169.3-170.3 (4 C); HRMS (ESIþ) m/z 477.1373 (C21H26NaO11
[M þ Na]þ requires 477.1367).
d, J 8.0 Hz), 5.21 (1 H, dd, J 8.0, 7.0), 6.78-6.97 (4 H, m); 13
C
NMR (125 MHz, CDCl3) δ 20.8-20.9 (2 C), 26.3, 27.5 (2 C),
55.6 (1 C), 63.3, 71.0, 72.5, 73.3, 76.9 (5 C), 99.9 (1 C), 111.0
(1 C), 114.4-155.5 (6 C), 169.5, 170.7 (2 C); HRMS (ESIþ) m/z
433.1468 (C20H26NaO9 [M þ Na]þ requires 433.1469). Anal.
Calcd for C20H26O9: C, 58.53; H, 6.39. Found: C, 58.54; H, 6.32.
4-Methoxyphenyl 2,6-Di-O-acetyl-β-D-galactopyranoside (25).
Compound 24 (2.10 g, 5.08 mmol) was dissolved in chloroform
(40 mL) containing 90% aqueous TFA (3 mL) and was stirred ar
rt for 1 h. Evaporation of the solvent gave a white residue, which
was recrystallized from EtOAc/petroleum spirits to yield diol
25 (1.71 g, 90%) as white needles: mp 112-113 °C; [R]27 þ8
D
(c 1.1 in CHCl3); 1H NMR (400 MHz, CDCl3) δ 2.08-2.16 (6 H,
2 ꢀ s), 3.73 (1 H, dd, J 9.6, 3.2 Hz), 3.75 (1 H, m), 3.77 (3 H, s),
3.97 (1 H, d, J 3.2 Hz), 4.34 (1 H, dd, J 6.8, 11.6 Hz), 4.39 (1 H,
dd, J 6.0, 11.6 Hz), 4.81 (1 H, d, J 8.0 Hz), 5.19 (1 H, dd, J 8.0,
9.6 Hz), 6.78-6.98 (4 H, m); 13C NMR (100 MHz, CDCl3)
δ 20.8, 21.0 (2 C), 55.6 (1 C), 62.6, 68.6, 72.3, 72.5, 73.0
(5 C), 100.4 (1 C), 114.5-155.6 (6 C), 171.1, 171.5 (2 C); HRMS
(ESIþ) m/z 393.1152 (C17H22NaO9 [M þ Na]þ requires
393.1156). Anal. Calcd for C17H22O9: C, 55.13; H, 5.99. Found:
C, 55.09; H, 5.94.
4-Methoxyphenyl β-D-Galactopyranoside (22). A small por-
tion of sodium metal was added to a solution of tetraacetate 21
(3.00 g, 6.60 mmol) in MeOH (100 mL), and the mixture was
stirred at rt for 1 h. Amberlite IR-120 resin (Hþ form) was added
to neutralize the solution. Filtration and evaporation of the
(54) Robertson, A. J. Chem. Soc. 1929, 1820–1823.
(55) Murakata, C.; Ogawa, T. Carbohydr. Res. 1992, 235, 95–114.
J. Org. Chem. Vol. 74, No. 24, 2009 9395

McGill and Williams
JOCArticle
4-Methoxyphenyl 2,4,6-Tri-O-acetyl-β-D-galactopyranoside (26).
Diol 25 (2.00 g, 5.40 mmol) was dissolved in toluene (20 mL)
containing p-TsOH (300 mg, 1.62 μmol). Triethyl orthoacetate
(4.9 mL, 27 mmol) was added, and the mixture was stirred at rt
for 1 h. The reaction was quenched (Et3N), and the solvent
evaporated to give a yellow oil, which was dissolved in 80%
aqueous acetic acid (20 mL) and stirred at 40 °C for 30 min.
Evaporation of the solvent gave a residue, which was recrystal-
lized from EtOAc/petroleum spirits to yield 26 (1.89 g, 85%) as
J 6.0, 12.0 Hz), 4.95 (1 H, d, J 8.0 Hz), 4.97 (1 H, d,
J 8.0 Hz), 5.46 (1 H, dd, J 10.5, 3.5 Hz), 5.60-5.69 (2 H, m),
5.92 (1 H, d, J 3.5 Hz), 5.94 (1 H, d, J 3.5 Hz), 6.56-6.79
(4 H, m), 6.51-6.84 (14 H, m); 13C NMR (125 MHz, CDCl3)
δ 19.8-21.2 (21 C), 55.5 (1 C), 62.8-77.4 (10 C), 99.9-
102.4 (2 C), 114.3-140.3 (48 C), 166.8-169.4 (7 C); HRMS
(ESIþ) m/z 1493.6604 (C89H98NaO19 [M þ Na]þ requires
1493.6595).
4-Methylphenyl 2,3,4,6-Tetra-O-mesitoyl-β-D-galactopyranosyl-
(1f3)-2,4,6-tri-O-mesitoyl-1-thio-β-D-galactopyranoside (31).
fine colorless needles: mp 134-135 °C; [R]22 þ2 (c 0.9 in
D
1
CHCl3); H NMR (500 MHz, CDCl3) δ 2.06-2.20 (9 H, 3 ꢀ
BF3 Et2O (8.40 μL, 0.068 mmol) was added dropwise to a
3
s), 2.70 (1 H, d, J 6.5 Hz), 3.77 (3 H, s), 3.91 (1 H, ddd, J 6.5, 10.0,
3.5 Hz), 3.94 (1 H, ddd, J 1.0, 6.5, 6.5 Hz), 4.18-4.19 (2 H, m,
H6,6), 4.87 (1 H, d, J 8.0 Hz), 5.22 (1 H, dd, J 8.0, 10.0 Hz), 5.37
(1 H, dd, J 1.0, 3.5 Hz), 6.80-6.98 (4 H, m); 13C NMR (125
MHz, CDCl3) δ 20.7-20.9 (3 C), 55.6 (1 C), 61.9, 69.5, 71.2,
71.4, 72.6 (5 C), 100.5 (1 C), 114.5-155.7 (6 C), 170.4-171.1 (3
C); HRMS (ESIþ) m/z 435.1260 (C19H24NaO10 [M þ Na]þ
requires 435.1262). Anal. Calcd for C19H24O10: C, 55.34; H,
5.87. Found: C, 55.64; H, 5.88.
solution of 30 (100 mg, 0.068 mmol) and thiocresol (17 mg,
0.14 mmol) in dry (CH2Cl)2 (2 mL) at rt. The reaction was
heated to 60 °C and stirred overnight. The mixture was cooled
and the reaction quenched by addition of satd aq NaHCO3
(5 mL). The organic phase was washed with satd aq NaHCO3
(2 ꢀ 5 mL) and brine (1 ꢀ 5 mL) and dried (MgSO4). Evapora-
tion of the solvent gave an oil, which was purified by flash
chromatography (15% EtOAc/pet. spirits) to yield 31 (71 mg,
1
71%) as a colorless foam: [R]20 þ36 (c 0.85 in CHCl3); H
D
4-Methoxyphenyl 2,3,4,6-Tetra-O-mesitoyl-β-D-galactopyranosyl-
(1f3)-2,4,6-tri-O-acetyl-β-D-galactopyranoside (27). TfOH was
added to a mixture of donor 19 (1.20 g, 1.40 mmol), acceptor 26
(0.470 g, 1.10 mmol), NIS (0.370 g, 1.65 mmol), and freshly
NMR (500 MHz, CDCl3) δ 2.07-2.34 (66 H), 3.92 (1 H, dd,
J 3.0, 9.0 Hz), 4.15 (1 H, dd, J 10.0, 3.5 Hz), 4.18 (1 H, dd, J 6.0,
6.0 Hz), 4.39-4.55 (4 H, m), 4.62 (1 H, d, J 10.0 Hz), 4.92 (1 H, d,
J 8.0 Hz), 5.42 (1 H, dd, J 10.0, 10.0 Hz), 5.44 (1 H, dd, J 10.5, 3.0
Hz), 5.64 (1 H, dd, J 8.0, 10.5 Hz), 5.91 (2 H, m), 6.50-7.27 (18
H, m); 13C NMR (125 MHz, CDCl3) δ 20.1-21.6 (22 C),
63.0-79.3 (10 C), 87.1 (1 C), 102.6 (1 C), 127.7-140.5 (48 C),
166.8-169.7 (7 C); HRMS (ESIþ) m/z 1493.6429 (C89H98
NaO17S1) [M þ Na]þ requires 1493.6417).
˚
activated 5 A AW molecular sieves in CH2Cl2 (15 mL) at 0 °C.
The mixture was allowed to warm to rt and was stirred for 4 h.
The reaction was quenched by the addition of 0.50 M sodium
thiosulfate (10 mL). The organic phase was washed with satd aq
NaHCO3 (2 ꢀ 25 mL) and brine (2 ꢀ 25 mL) and dried
(MgSO4). The solvent was evaporated to give a foam which
was recrystallized from EtOH to afford disaccharide 27 (1.30 g,
n-Butyl 2,3,4,6-Tetra-O-mesitoyl-β-D-galactopyranosyl-(1f3)-
2,4,6-tri-O-mesitoyl-1-thio-r-D-galactopyranoside (32a) and n-Bu-
tyl 2,3,4,6-Tetra-O-mesitoyl-β-D-galactopyranosyl-(1f3)-2,4,6-
80%) as white crystals: mp 126-127 °C; [R]23 þ47 (c 1.0 in
D
CHCl3); 1H NMR (500 MHz, CDCl3) δ 2.05-2.34 (45 H), 3.76
(3 H), 3.83 (1 H, dd, J 6.0, 7.0 Hz), 4.04 (1 H, dd, J 10.0, 3.5 Hz),
4.07 (1 H, dd, J 6.0, 11.5 Hz), 4.14 (1 H, dd, J 7.0, 11.5 Hz), 4.23
(1 H, dd, J 6.0, 6.0 Hz), 4.60 (1 H, dd, J 6.0, 11.5 Hz), 4.64 (1 H,
dd, J 6.0, 11.5 Hz), 4.70 (1 H, d, J 8.0 Hz), 5.00 (1 H, d, J 8.0 Hz),
5.42 (1 H, dd, J 8.0, 10.0 Hz), 5.46 (1 H, d, J 3.5 Hz), 5.48 (1 H,
dd, J 8.0, 10.0 Hz), 5.63 (1 H, dd, J 10.0, 3.0 Hz), 5.95 (1 H, d,
J 3.0 Hz), 6.74-6.80 (8 H, m), 6.77-6.89 (4 H, m); 13C NMR
(125 MHz, CDCl3) δ 20.0-21.3 (15 C), 55.6 (1 C), 62.2-75.7 (10
C), 101.0, 101.6 (2 C), 114.4-140.4 (30 C), 167.1-170.4 (7 C);
HRMS (ESIþ) m/z 1181.4729 (C65H74NaO19 [M þ Na]þ re-
quires 1181.4717). Anal. Calcd for C65H74O19: C, 67.34; H, 6.34.
Found: C, 67.21; H, 6.35.
tri-O-mesitoyl-1-thio-β-D-galactopyranoside (32b). BF3 Et2O
3
(84.0 μL, 0.679 mmol) was added dropwise to a solution of
30 (1.00 g, 0.679 mmol) and n-butylmercaptan (180 mg,
2.00 mmol) in dry (CH2Cl)2 (20 mL) at rt. The reaction was
heated to 60 °C and stirred overnight. The mixture was cooled
and the reaction quenched by addition of satd aq NaHCO3
(20 mL). The organic phase was washed with satd aq NaHCO3
(2 ꢀ 10 mL) and brine (1 ꢀ 10 mL) and dried (MgSO4). Eva-
poration of the solvent gave an oil, which was purified by flash
chromatography (50% EtOAc/peroleum spirits), a 1:1 R/β
mixture of 32a and 32b (781 mg, 80%) as a colorless foam.
The anomers may be separated by flash chromatography (15%
EtOAc/petroleum spirits). The R-linked disaccharide 32a eluted
4-Methoxyphenyl 2,3,4,6-Tetra-O-mesitoyl-β-D-galactopyra-
nosyl-(1f3)-2,4,6-tri-O-mesitoyl-β-D-galactopyranoside (30).
NaOMe in MeOH (0.50 M, 10 mL) was added to a solution of
27 (1.0 g, 0.87 mmol) in MeOH/THF (2:1, 18 mL), and the
mixture was stirred overnight at 50 °C. The solution was
neutralized with Amberlite IR-120 resin (Hþ form), and the
solvent was removed by evaporation to afford an oil. Mesitoyl
chloride (3.20 g, 17.5 mmol), DMAP (107 mg, 0.870 mmol), and
pyridine (10 mL) were added to the oil, and the mixture was
warmed to 100 °C and stirred for 2 d. The mixture was diluted
with H2O (25 mL) and extracted into CH2Cl2 (50 mL).
The organic phase was washed with aq HCl (1 M, 4 ꢀ50 mL),
satd aq NaHCO3 (2 ꢀ50 mL), and brine (25 mL) and dried
(MgSO4). Evaporation of the solvent gave a residue, which was
purified by flash chromatography (10-15% EtOAc/petroleum
spirits) to afford the protected disaccharide 30 (1.10 g, 86% two
steps) as an off-white foam. An analytical sample was crystal-
as a colorless foam: [R]19 þ93 (c 1.0 in CHCl3); 1H NMR
D
(500 MHz, CDCl3) δ 1.13-1.46 (7 H, (CH2)2CH3), 2.01-2.37
(63 H), 2.37-2.49 (2 H, m), 4.23 (1 H, dd, J 6.5, 6.5 Hz), 4.37
(1 H, dd, J 10.0, 3.0 Hz), 4.45 (4 H, m), 4.73 (1 H, dd, J 4.0, 8.0
Hz), 5.03 (1 H, d, J 8.0 Hz), 5.43 (1 H, dd, J 5.5, 10.0 Hz), 5.46 (1
H, dd, J 11.0, 3.0 Hz), 5.55 (1 H, dd, J 8.0, 11.0 Hz), 5.92-5.95 (2
H, m), 5.92 (1 H, d, J 5.5 Hz), 6.50-6.85 (14 H, m); 13C NMR
(125 MHz, CDCl3) δ 13.6-31.5 (3 C), 20.1-22.1 (22 C),
63.0-74.8 (10 C), 82.3 (1 C), 102.5 (1 C), 127.9-140.3 (42 C),
166.7-169.7 (7 C); HRMS (ESIþ) m/z 1459.6575 (C86H100
-
NaO17S1 [M þ Na]þ requires 1459.6573). The β-linked disac-
charide 32b eluted as a colorless foam: [R]19 þ36 (c 1.0 in
D
CHCl3); 1H NMR (500 MHz, CDCl3) δ 0.83-1.20 (7 H),
2.08-2.39 (63 H), 2.68 (2 H, m), 3.99 (1 H, dd, J 5.0, 8.0 Hz),
4.16 (1 H, dd, J 10.0, 3.5 Hz), 4.23 (1 H, dd, J 6.5, 6.5 Hz), 4.42 (1
H, d, J 10.0 Hz), 4.49-4.60 (4 H, m), 4.97 (1 H, d, J 8.0 Hz), 5.49
(1 H, dd, J 11.0, 3.5 Hz), 5.53 (1 H, dd, J 10.0, 10.0 Hz), 5.70 (1 H,
dd, J 8.0, 11.0 Hz), 5.96-5.98 (2 H, 2 ꢀ d, J 3.5, 3.5 Hz),
6.53-6.89 (14 H, m); 13C NMR (125 MHz, CDCl3) δ 13.4-31.4
(3 C), 19.8-21.8 (22 C), 62.7 (1 C), 64.2-79.0 (10 C), 83.9 (1 C),
102.3 (1 C), 127.3-140.2 (42 C), 166.4-169.4 (7 C); HRMS
(ESIþ) m/z 1459.6577 (C86H100NaO17S1) [M þ Na]þ requires
1459.6573).
lized from EtOH affording white cubic crystals: mp 176-177 °C;
D
1
[R]20 þ32 (c 1.0 in CHCl3); H NMR (500 MHz, CDCl3) δ
2.04-2.35 (63 H), 3.69 (3 H, s), 4.00 (1 H, dd, J 3.5, 9.0 Hz), 4.21
(1 H, dd, J 6.0, 6.0 Hz), 4.26 (1 H, dd, J 10.0, 3.5 Hz), 4.44-4.53
(1 H, dd, J 3.5, 12.0 Hz), 4.44-4.53 (1 H, dd, J 9.0, 12.0 Hz),
4.48-4.58 (1 H, dd, J 6.0, 12.0 Hz), 4.48-4.58 (1 H, dd,
9396 J. Org. Chem. Vol. 74, No. 24, 2009

McGill and Williams
JOCArticle
8-Azidooctyl 2,3,4,6-Tetra-O-mesitoyl-β-D-galactopyranosyl-
(1f3)-2,4,6-tri-O-mesitoyl-β-D-galactopyranosyl-(1f3)-2,4,6-
tri-O-acetyl-1-β-D-galactopyranoside (33). (i) TfOH (8.1 μL,
0.091 mmol) was added to a mixture of 31 (190 mg, 0.130 mmol),
acceptor 18 (75 mg, 0.16 mmol), NIS (43.9 mg, 0.195 mmol), and
in EtOH (5 mL) was stirred at rt for 30 min. The solvent was
evaporated, and the residue was purified by flash chromatog-
raphy (30% EtOAc/pet. spirits) to afford 34 (108 mg, 50%) as an
orange residue: mp 145-147 °C; 1H NMR (500 MHz, CDCl3) δ
2.37 (1 H, t, J 2.0 Hz), 3.08 (1 H, s), 4.21 (2 H, d, J 2.5 Hz),
6.28-8.48 (2 H, m); 13C NMR (125 MHz, CDCl3) δ 33.5 (1 C),
74.3 (1 C), 78.8 (1 C); 101.1-145.6 (6 C); HRMS (ESIþ) m/z
241.0332 (C9H6N4NaO3 [M þ Na]þ requires 241.0332). Anal.
Calcd for C9H4N4O3: C, 49.55; H, 2.77; N, 25.68. Found: C,
49.47; H, 2.91; N, 25.70.
˚
freshly activated 5 A AW molecular sieves in CH2Cl2 (5 mL) at
0 °C. The mixture was allowed to warm to rt and was stirred for
2 h. The reaction was quenched by the addition of 0.50 M
sodium thiosulfate (10 mL). The organic phase was washed with
satd aq NaHCO3 (2 ꢀ 25 mL) and brine (2 ꢀ 25 mL) and dried
(MgSO4). The solvent was evaporated to give an oil, which was
subjected to flash chromatography (20% EtOAc/petroleum
spirits) to yield trisaccharide 33 (28 mg, 12%) as an amorphous
solid: [R]20D þ32 (c 1.0 in CHCl3); 1H NMR (500 MHz, CDCl3)
δ 1.30-1.60 (12 H, m), 1.98-2.36 (72 H), 3.20 (2 H, t, J 7.0), 3.31
(1 H, ddd, J 10.0, 7.0, 7.0 Hz), 3.60 (1 H, dd, J 6.5, 6.5 Hz), 3.78
(1 H, ddd, J 10.0, 7.0, 7.0 Hz), 3.82 (1 H, dd, J 10.0, 3.5 Hz), 3.95
(1 H, dd, J 3.0, 6.5 Hz), 3.99 (1 H, dd, J 6.5, 11.5 Hz), 4.03 (1 H,
dd, J 6.5, 11.5 Hz), 4.16 (1 H, d, J 8.0 Hz), 4.18 (1 H, dd, J 10.0,
3.5 Hz), 4.23 (1 H, dd, J 6.5, 6.5 Hz), 4.38-4.59 (4 H, m),
4.80-4.95 (2 H, 2 ꢀ d, J 8.0, 8.0 Hz), 5.02-5.06 (2 H, 2 ꢀ d,
J 10.0, 10.0 Hz), 5.33 (1 H, d, J 3.5 Hz), 5.52 (1 H, dd, J 11.0, 3.5
Hz), 5.62 (1 H, dd, J 8.0, 11.0 Hz), 5.89-5.92 (2 H, 2 ꢀ d, J 3.5,
3.5 Hz), 6.43-6.86 (14 H); 13C NMR (125 MHz, CDCl3) δ
19.8-29.7 (30 C), 51.4 (1 C), 62.2 (1 C), 62.7-78.2 (15 C),
101.1-102.8 (3 C), 127.0-140.3 (42 C), 166.4-170.4 (10 C);
HRMS (ESIþ) m/z 1828.8318 (C102H123N3NaO26 [M þ Na]þ
requires 1828.8287). (ii) According to the procedure outlined
above, a 1:1 anomeric mixture of 32a,b (950 mg, 0.658 mmol)
was condensed with acceptor 18 (280 mg, 0.601 mmol). After
purification by flash chromatography (15% EtOAc/petroleum
spirits), trisaccharide 33 was isolated as an amorphous solid
(308 mg, 28%).
4-[(7-Nitrobenzofurazan-4-yl)aminomethyl]-triazol-1-yloctyl
β-D-Galactopyranoside (35). A solution of azide 1 (19.7 mg, 59.1
μmol), alkyne 34 (15.4 mg, 69.9 μmol), CuSO4 (0.37 mg, 2.3
μmol), sodium ascorbate (2.3 mg, 18 μmol), and TBTA (1.6 mg,
2.3 μmol) in THF/H2O (1:1, 5 mL) was stirred at rt for 1 h. The
mixture was evaporated to dryness to give a residue, which was
purified by flash chromatography (17:2:1 EtOAc/MeOH/H2O)
to afford 35 (30 mg, 92%) as an orange residue: [R]20 -13 (c
D
0.50 in DMSO); 1H NMR (500 MHz, DMSO-d6) δ 1.16-1.80
(12 H, m), 3.29-3.40 (5 H, m), 3.42-3.54 (2 H, m), 3.62 (1 H, m),
3.87 (1 H, ddd, J 9.5, 6.5, 6.5 Hz), 4.29 (1 H, d, J 8.0 Hz), 4.32
(2 H, m), 6.49-8.51 (2 H, m), 8.10 (1 H, s); 13C NMR (125 MHz,
DMSO-d6) δ 25.8-30.1 (6 C), 49.8 (2 C), 60.8 (1 C), 68.6-75.5
(5 C), 100.3-145.0 (6 C), 103.4 (1 C), 123.7, 143.0 (2 C); HRMS
(ESIþ) m/z 574.2232 (C23H33N7NaO9 [M þ Na]þ requires
574.2232).
4-[(7-Nitrobenzofurazan-4-yl)aminomethyl]triazol-1-yloctyl
β-D-Galactopyranosyl-(1f3)-β-D-galactopyranoside (36). A so-
lution of azide 2 (84.0 mg, 170 μmol), alkyne 34 (44.0 mg, 202
μmol), CuSO4 (1.1 mg, 6.7 μmol), sodium ascorbate (6.7 mg, 34
μmol), and TBTA (4.3 mg, 34 μmol) in THF/H2O (1:1, 10 mL)
was stirred at rt for 1 h. The mixture was evaporated, and the
residue was purified by reversed-phase chromatography
(0-40% MeOH/H2O) to afford 36 (113 mg, 94%) as an orange
8-Azidooctyl β-D-Galactopyranosyl-(1f3)-β-D-galactopyra-
noside (2). A solution of disaccharide 20b (32 mg, 0.027 mmol)
in a 1:1:0.1 mixture of 1 M LiOH in MeOH, THF, and H2O
(2 mL) was heated at 80 °C for 5 d. The reaction was neutralized
by addition of Amberlite IR-120 resin (Hþ form), and the
solvent was evaporated to afford an oil, which was dissolved
in H2O (2 mL) and purified by reversed-phase chromatography
residue: [R]20 -7 (c 0.50 in DMSO); 1H NMR (500 MHz,
D
MeOH-d4) δ 1.31-1.89 (12 H, m), 3.46-3.77 (13 H, m), 3.82 (1
H, d, J 3.0 Hz), 3.87 (1 H, ddd, J 9.5, 6.5, 6.5 Hz), 4.12 (1 H, dd,
J 3.0, 0.5 Hz), 4.26 (1 H, d, J 8.0 Hz), 4.39 (2 H, t, J 7.0 Hz), 4.49
(1 H, d, J 8.0 Hz), 6.46-8.53 (2H, m), 8.02 (1 H, s); 13C NMR
(125 MHz, DMSO-d6) δ 25.8-30.1 (6 C), 49.8 (2 C), 60.8 (1 C),
67.9-84.1 (10 C), 100.4-145.5 (6 C), 103.2, 105.7 (2 C), 123.8
(1 C), 143.0 (1 C); HRMS (ESIþ) m/z 736.2754 (C29H43N7-
NaO14 [M þ Na]þ requires 736.2760).
(0-40% MeOH/H2O) to yield disaccharide 2 (11 mg, 86%) as
D
1
an amorphous solid: [R]21 þ2 (c 0.8 in H2O); H NMR (500
MHz, MeOH-d4, internal acetone) δ 1.36-1.66 (12 H, m), 3.33
(2 H, t, J 7.0 Hz), 3.61-3.98 (13 H, ddd), 4.21 (1 H, m), 4.46 (1 H,
d, J 8.0 Hz), 4.62 (1 H, d, J 8.0 Hz); 13C NMR (100 MHz,
MeOH-d4, acetone) δ 25.5-29.3 (6 C), 51.8 (1 C), 61.4 (1 C),
61.5-83.1 (10 C), 103.0-104.9 (2 C); HRMS (ESIþ) m/z
518.2320 (C20H37N3NaO11 [M þ Na]þ requires 518.2320). Anal.
Calcd for C20H37N3O11: C, 48.48; H, 7.53; N, 8.48. Found: C,
48.34; H, 7.67; N, 8.59.
4-[(7-Nitrobenzofurazan-4-yl)aminomethyl]triazol-1-yloctyl
β-D-Galactopyranosyl-(1f3)-(β-D-galactopyranosyl)-(1f3)-β-
D-galactopyranoside (37). A solution of azide 3 (5.0 mg, 7.6
μmol), alkyne 34 (1.8 mg, 8.1 μmol), CuSO4 (49 μg, 0.31 μmol),
sodium ascorbate (31 μg, 1.5 μmol), and TBTA (160 μg,
0.31 μmol) in THF/H2O (1:1, 2 mL) was stirred at rt for 1 h.
The mixture was evaporated to dryness to give a residue,
which was purified by reversed-phase chromatography
8-Azidooctyl β-D-Galactopyranosyl-(1f3)-β-D-galactopyranosyl-
(1f3)-β-D-galactopyranoside (3). Trisaccharide 33 (100 mg, 0.0553
mmol) was dissolved in 1:1:0.1 mixture of 1 M LiOH in MeOH,
THF, and H2O (10.5 mL), and the solution was heated at 80 °C for
5 d. The reaction was neutralized with Amberlite IR-120 resin (Hþ
form), and the solvent was evaporated to afford an oil, which was
dissolved in H2O (2 mL) and purified by reversed-phase chroma-
tography (0-40% MeOH/H2O) to yield trisaccharide 3 (26 mg,
72%) as a colorless glass: [R]21D 22 (c 1.0 in H2O); 1H NMR (500
MHz, D2O) δ 1.22-1.52 (12 H, m), 3.19 (2 H, t, J 7.0 Hz),
3.61-3.98 (16 H, m), 4.07 (2 H, m), 4.33-4.55 (3 H, 3 ꢀ d, J1,2
8.0 Hz); 13C NMR (100 MHz, MeOH-d4, acetone) δ 25.4-30.8
(6 C), 51.8 (1 C), 61.4-82.9 (16 C), 102.9, 104.4, 104.8 (3 C); HRMS
(ESIþ) m/z 680.2842 (C26H47N3NaO16 [M þ Na]þ requires
680.2849).
(0-40% MeOH/H2O) to afford 37 (4.4 mg, 65%) as an
D
1
orange residue: [R]19 -15 (c 0.1 in MeOH); H NMR (500
MHz, D2O) δ 1.13-1.85 (12 H, m), 3.51-3.84 (19 H), 3.90, 4.16,
4.19 (3 H, 3 ꢀ d, J 3.2 Hz), 4.37, 4.59, 4.65 (3 H, 3 ꢀ d, J 8.0 Hz),
4.40 (2 H, m), 6.41-8.58 (2H, m), 8.04 (1 H, s); 13C NMR
(200 MHz, D2O) δ 25.1-29.0 (6 C), 50.5 (2 C), 61.1 (1 C),
68.1-82.1 (15 C), 102.3, 103.9, 104.1 (3 C), 123.8 (1 C), 124.1,
138.6 (2 C); HRMS (ESIþ) m/z 898.3290 (C35H53N7NaO19
[M þ Na]þ requires 898.3288). 13C NMR data were acquired
by HSQC spectroscopy.
8-(2-Ethoxycyclobutene-3,4-dione-1-ylamino)octyl β-D-
Galactopyranoside (38). PPh3 (94 mg, 0.36 mmol) was added
to a stirred solution of azide 1 (30 mg, 0.090 mmol) in THF/H2O
(10:1, 5 mL) at 0 °C. The mixture was warmed to rt and stirred
overnight. The mixture was concentrated under reduced pres-
sure to give a residue, which was dissolved in EtOAc (5 mL). The
organic phase was extracted with H2O (5 ꢀ 3 mL), and the
7-Nitro-4-(prop-2-ynylamino)benzofurazan (34). A solution of
4-chloro-7-nitro-benzofurazan (200 mg, 0.992 mmol), propar-
gylamine (71 μL, 1.1 mmol), and pyridine (8 μL, 0.1 mmol)
J. Org. Chem. Vol. 74, No. 24, 2009 9397

McGill and Williams
JOCArticle
aqueous phase was concentrated to dryness to afford the crude
amine. The amine was dissolved in a mixture of EtOH/H2O/0.1
M phosphate buffer (2:1:1, 4 mL, pH 8), and diethyl squarate
(613 μL, 90 μmol) was added. The mixture was stirred for 3 h and
then concentrated to dryness. The residue was purified by
reversed-phase chromatography (0-50% MeOH/H2O) to af-
8-(2-Ethoxycyclobutene-3,4-dion-1-ylamino)octyl β-D-galac-
topyranosyl-(1f3)-(β-D-galactopyranosyl)-(1f3)-β-D-galacto-
pyranoside (40). PPh3 (30 mg, 115 μmol) was added to a stirred
solution of azide 3 (19 mg, 29 μmol) in THF/H2O (5:1, 2.2 mL) at
0 °C. The mixture was warmed to rt, stirred overnight, and then
concentrated under reduced pressure to give a residue, which was
dissolved in EtOAc (3 mL). The organic phase was extracted with
H2O (5 ꢀ 3 mL), and the aqueous phase was concentrated to
dryness to afford the crude amine. The amine was dissolved in a
mixture of EtOH/H2O/0.1 M phosphate buffer (2:1:1, 2 mL,
pH 8), and diethyl squarate (5.5 μL, 37 μmol) was added.
The mixture was stirred for 3 h and then concentrated to dryness.
The residue was purified by reversed-phase chromatography
(0-20% MeOH/H2O) to afford 40 (22 mg, 95%) as a colorless
oil: 1H NMR (500 MHz, D2O) δ 1.37-1.65 (15 H, m), 3.37-3.80
(20 H, m), 4.06-4.07 (2 H, 2 ꢀ d, J 3.0 Hz), 4.31, 4.49, 4.55 (3 H,
3 ꢀ d, J 7.5 Hz), 4.65 (2 H, m); HRMS (ESIþ) m/z 778.3109
(C32H53NNaO19 [M þ Na]þ requires 778.3104). The 13C NMR
spectrum for this compound was poorly resolved. We attribute this
to the presence of tautomeric isomers.
1
ford 38 (36 mg, 92%) as a colorless oil: H NMR (500 MHz,
D2O) δ 1.36-1.65 (15 H, m), 3.40-3.62 (6 H, m), 3.73 (1 H, dd, J
6.0, 11.5 Hz), 3.75 (1 H, dd, J 6.5, 11.0 Hz), 3.83 (1 H, dd, J 3.5,
1.0 Hz), 3.89 (1 H, ddd, J 9.5, 6.5, 6.5 Hz), 4.21 (1 H, d, J 7.0 Hz),
4.74 (2 H, m); HRMS (ESIþ) m/z 454.2048 (C20H33NNaO9 [M
þ Na]þ requires 454.2048). The 13C NMR spectrum for this
compound was poorly resolved. We attribute this to the pre-
sence of tautomeric isomers.
8-(2-Ethoxycyclobutene-3,4-dion-1-ylamino)octyl β-D-galac-
topyranosyl-(1f3)-β-D-galactopyranoside (39). PPh3 (36 mg,
137 μmol) was added to a stirred solution of azide 2 (17 mg,
34 μmol) in THF/H2O (5:1, 3 mL) at 0 °C. The mixture was
warmed to rt and stirred overnight. The mixture was concen-
trated under reduced pressure to give a residue, which was
dissolved in EtOAc (4 mL). The organic phase was extrac-
ted with H2O (5 ꢀ 3 mL), and the aqueous phase was con-
centrated to dryness to afford the crude amine. The amine
was dissolved in a mixture of EtOH/H2O/0.1 M phosphate
buffer (2:1:1, 3 mL, pH 8), and diethyl squarate (6.5 μL,
45 μmol) was added. The mixture was stirred for 3 h and then
concentrated to dryness. The residue was purified by reversed-
phase chromatography (0-20% MeOH/H2O) to afford 39 (18
mg, 92%) as a colorless oil: 1H NMR (500 MHz, D2O) δ
1.37-1.65 (15 H, m), 3.41-3.91 (13 H, m), 3.83 (1 H, dd, J
3.0, 1.0 Hz), 3.91 (1 H, ddd, J 9.5, 6.5, 6.5 Hz), 4.12 (1 H, dd, J
3.0, 1.0 Hz), 4.27 (1 H, d, J 7.5 Hz), 4.50 (1 H, d, 7.5 Hz), 4.76 (2
H, m); HRMS (ESIþ) m/z 616.2578 (C26H43NNaO14 [M þ Na]þ
requires 616.2576). The 13C NMR spectrum for this compound
was poorly resolved. We attribute this to the presence of
tautomeric isomers.
Acknowledgment. We express our appreciation to Mr.
J. Johnson for his assistance. This work was supported by
Biosupplies Australia Ltd. and the Australian Research
Council. N.W.M. gratefully acknowledges financial support
from the Albert Shimmins Memorial Fund. The late
Emeritus Prof. Bruce Stone is acknowledged as a source of
inspiration for this work.
Supporting Information Available: 1H and 13C NMR spec-
tra for 1-3, 4b, 5-19, 20a,b, and 23-40, fluorescence spectra
for 35-37, HPLC chromatograms for 37 and 40, and experi-
mental procedures for compounds 4b, 5-11, 16, 19, 28, and 29.
This material is available free of charge via the Internet at http://
pubs.acs.org.
9398 J. Org. Chem. Vol. 74, No. 24, 2009