Stability of 5(6)-carboxyfluorescein in microwave-assisted synthesis of fluorescein-labelled O-dimannosylated peptides

Article abstract of DOI:10.1055/s-0029-1216820

Methodology for the efficient, automated and microwave-assisted Fmoc solid-phase synthesis of a 5(6)-carboxyfluorescein-labelled Lys(Dde)-Gly-Wang resin that can be further elongated through the lysine N^ε amino group is described. Incorporation

Full text of DOI:10.1055/s-0029-1216820

2210
PAPER
Stability of 5(6)-Carboxyfluorescein in Microwave-Assisted Synthesis of
Fluorescein-Labelled O-Dimannosylated Peptides
S
ynthesis of Fluo
e
rescein-Lab
n
e
lled O-Dim
a
annosylate
t
d Pepti
a
de
s
Kowalczyk,a,c Paul W. R. Harris,a,c Rod P. Dunbar,b,c Margaret A. Brimble*a,c
a
b
c
Department of Chemistry, University of Auckland, 23 Symonds St, Auckland 1142, New Zealand
School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
Fax +64(9)3737422; E-mail: m.brimble@auckland.ac.nz
Received 11 February 2009
compounds that mimic the cluster effect6 are most often
used as antigenic moieties. Some of these constructs were
prepared via mannosylation through the side chain Ne
amino group of a lysine residue.7 There is also evidence
Abstract: Methodology for the efficient, automated and micro-
wave-assisted Fmoc solid-phase synthesis of a 5(6)-carboxyfluores-
cein-labelled Lys(Dde)-Gly-Wang resin that can be further
elongated through the lysine Ne amino group is described. Incorpo-
ration of O-dimannosylated peptides onto this resin using Fmoc-[a- that some receptors are able to recognise less complex
sugar units8 such as terminal single and dimannose units,9
D-Man(OBz)4-(1→6)-a-D-Man(OBz)3a1-]Ser-OH and PEG-[a-D-
Man(OBz)4-(1→6)-a-D-Man(OBz)3a1-]-OH building blocks is
fucose, and N-acetylglucosamine moieties. Given that the
demonstrated. Conditions were optimised to enable the efficient au-
optimal characteristics of sugar spacing for binding and
tomated synthesis of several carboxyfluorescein-labelled dimanno-
uptake, and the mechanism of action of human mannose
sylated peptides. 5(6)-Carboxyfluorescein was shown to be stable to
receptors, are still not fully described8b,10 we wished to de-
the microwave conditions used for glycopeptide synthesis. The
velop synthetic technology to allow optimisation of a suit-
able PRL to target human mannose receptors. Previously,
we focused on the synthesis of monomannosylated
peptides11 incorporated into a polyalanine scaffold that
also contained 5(6)-carboxyfluorescein as a fluorescent
label to facilitate biological screening. Microwave tech-
nology was not used in this preliminary work. This initial
synthetic strategy has now been extended to allow the au-
tomated microwave-enhanced solid-phase synthesis of
5(6)-carboxyfluorescein labelled O-dimannosylated con-
structs under uniform conditions and the results of this
work are reported herein.
methodology described provides a robust, flexible synthetic plat-
form for the preparation of a variety of fluorescently labelled glyco-
peptides (especially O-dimannosylated peptides) for biological
evaluation.
Key words: carbohydrates, glycopeptides, mannosylated amino
acids, glycosylations, solid-phase synthesis
Post-translational glycosylation of proteins has a signifi-
cant influence on a number of biologically important pro-
cesses such as cell growth, cell adhesion, cell
differentiation, and immune defence.1 Abnormal glycosy-
lation of proteins may lead to diseases including cancer
and rheumatoid arthritis.1 Tumour cells and infectious
agents containing unique carbohydrates on their cell sur-
face are recognised by the immune system therefore, syn-
thetic peptides of similar structure can be used as vaccines
to stimulate the immune system. Despite problems associ-
ated with proteolysis and delivery to the immune system,2
peptide-based vaccines have enormous potential due to
their ease of synthesis and purification. Carbohydrate
bearing antigens are recognised by mannose receptors
(MRs) which play an important role in binding antigens,
migration of dendritic cells (DCs) and interaction of DCs
with lymphocytes.3
Synthesis of glycopeptides has been recently comprehen-
sively reviewed by Davis et al.12 There are two general ap-
proaches for the preparation of glycopeptides. The first
strategy requires initial synthesis of the peptide chain fol-
lowed by attachment of the carbohydrate unit. The second
approach involves preparation of a glycosylated amino
acid building block, which is then incorporated into step-
wise on-resin peptide synthesis.13 This latter approach has
found broad utility for the preparation of O-glycosylated
peptides. The limited success realized trying to effect the
direct O-glycosylation of serine or threonine hydroxy
groups on resin14 established that stepwise solid-phase
synthesis of glycopeptides is in fact the method of choice.
Glycopeptide synthesis using a solid support is advanta-
geous over solution-phase chemical methods due to short-
er reaction times, ease of synthesis, the ability to drive
acylation to completion using a large excess of building
blocks, and the potential to automate the process due to its
repetitive nature.13c,15
The work reported herein focuses on developing synthetic
technology to allow optimisation of a suitable pattern rec-
ognition ligand (PRL) to target human mannose receptors
found on human antigen-presenting cells (APCs), for use
in synthetic vaccines. The exact binding requirements for
the mannose receptor are, as yet, unknown, however,
polymannan derivatives,4 mannosylated dendrimers,5 and
Recently, microwave irradiation has gained popularity for
the solid-phase peptide synthesis of difficult sequences.16
Previous reports on the use of microwave-enhanced solid-
phase synthesis of glycopeptides,17 albeit in the absence
of the fluorescent label, established the convenience of us-
SYNTHESIS 2009, No. 13, pp 2210–2222
x
x.
x
x
.
2
0
0
9
Advanced online publication: 14.05.2009
DOI: 10.1055/s-0029-1216820; Art ID: P02709SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of Fluorescein-Labelled O-Dimannosylated Peptides
2211
.
ing microwave irradiation to effect peptide coupling using
sterically demanding residues. Bradley et al.18 have incor-
porated 5(6)-carboxyfluorescein into a simple peptide
chain lacking glycosylated residues at the final stage of
the synthesis using microwave irradiation whilst Katritz-
ky et al.19 have recently described the microwave-assisted
solid-phase peptide synthesis (SPPS) of coumarin-based
fluorescently labelled simple peptides using benzotriazole
derivatives. To date no studies have provided a fast and
reliable method for the preparation of 5(6)-carboxyfluo-
rescein-labelled glycopeptides using microwave irradia-
tion, thus establishing the ability of the fluorescent label
to withstand microwave irradiation over prolonged peri-
ods of SPPS. Herein, we describe a convenient method for
the microwave-assisted synthesis of O-dimannosylated
peptides that also bear a 5(6)-carboxyfluorescein label,
thus providing ready access to a focused library of la-
belled glycopeptides for biological assays.
OBz
OBz
OBz
O
OBz
O
BzO
BzO
BzO
BzO
O
O
OBz
O
OBz
O
BzO
BzO
BzO
BzO
O
O
O
O
OH
O
3
OH
O
N
H
15
O
12
Figure 1 Dimannosylated building blocks 12 and 15
Man(OBz)7]-OH 12 and PEG-[diMan(OBz)7]-OH 15
(Figure 1), that could be readily incorporated into SPPS
was undertaken.
A Fmoc group was used to protect the Na amino group and
the sugar hydroxys could be protected with either benzyl,
acetate, or benzoyl groups. The presence of protecting
groups such as acetate or benzoyl on the hydroxy groups
of the sugar moiety stabilises the glycosidic linkage dur-
ing the final trifluoroacetic acid assisted glycopeptide
cleavage from the resin.24
A carbohydrate moiety can be attached to the peptide
chain via an amino acid residue or a linker via an O- or N-
glycosidic bond. The purpose of the linker is to keep the
sugar and peptide backbone apart thus spacing the newly
incorporated sugar molecules at a distance from the pep-
tide chain thereby minimising unfavourable steric interac-
tions. By employment of a lipophilic or hydrophilic
spacer unit, molecules with the desired physical properties
can be prepared.20 A review on the use of chemical link-
ages to attach sugar moieties has been reported by
Davis.21 Polyethyleneoxy linkers are often used for the in-
troduction of a carbohydrate unit20a,22 with their increased
hydrophilicity improving the desired water solubility of
the final compounds. Moreover, they possess greater flex-
ibility compared to natural oligosaccharides.23 For the
purposes of this study, serine and polyethyleneglycol
were used as mannose carriers.
Different methods for the synthesis of the a(1→6) linked
mannosides are reported in the literature.25 Meldal et al.26
have synthesized benzoyl-protected dimannosylated
building blocks attached to Na-Fmoc-protected serine,
threonine, and hydroxyproline. The carboxy groups were
protected as pentafluorophenyl esters. The preparation of
the a(1→6) benzoyl-protected dimannosylated building
block was achieved via Koenigs–Knörr glycosylation27
using a benzoyl-protected dimannosylated bromide as the
glycosyl donor. Glycosyl halides are unstable
intermediates28 requiring the use of in situ generated do-
nors resulting in lower yields for the glycosylation step.29
Zhu and Kong30 used 6-O-(2,3,4,6-tetra-O-acetyl-a-D-
mannopyranosyl)-2,3,4-tri-O-benzoyl-a-D-mannopyra-
nosyl trichloroacetimidate as the glycosyl donor for the
preparation of a variety of a(1→6)-linked mannose
polysaccharides. Trichloroacetimidates, are stable, easily
prepared crystalline products which are advantageous
over unstable glycosyl halides28 used for Koenigs–Knörr
glycosylation.27 In our case the trichloroacetimidate meth-
od was adopted as this had already proven a reliable meth-
od for our previous preparation11 of the simple Fmoc-
[Man(OBz)4a1-]Ser building block.
A carboxyfluorescein label can be introduced either at the
initial or final stage of solid-phase glycopeptide synthesis
(SPGS) however, previous experience has shown11 that
initial preparation of fluorescently labelled Wang resin
with the fluorescent label attached to the Na amino group
of a lysine residue followed by glycopeptide chain elon-
gation via the Ne amino group of the same lysine residue
is the strategy of choice affording better yields of the de-
sired glycopeptides compared to peptide elongation via
the Na amino group.
In order to develop synthetic technology for the routine
incorporation of glycosylated residues into fluorescently
labelled peptides using solid-phase synthesis, several
5(6)-carboxyfluorescein-labelled O-dimannosylated pep-
tides incorporated into a simple alanine-based peptide
scaffold were prepared. The use of microwave-assisted
SPGS was critical to the successful outcome of this work.
Preparation of Fmoc-Ser[diMan(OBz)7]-OH 12
The synthetic route used for the synthesis of an a(1→6)
linked dimannose unit attached to serine 12 is depicted in
Scheme 1. 2,3,4,6-Tetra-O-benzoyl-a-D-mannopyranosyl
trichloroacetimidate (6)11 was used as a glycosyl donor to-
gether with versatile thioglycosides31 which, depending
on the requirements, served as either a mannosyl acceptor
5 or a mannosyl donor 7.
Preparation of O-Dimannosylated Building Blocks
Initial preparation of the appropriate dimannosylated
building blocks, namely protected Fmoc-Ser[di-
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

2212
R. Kowalczyk et al.
PAPER
.
OR
O
The next step required glycosylation of thioacceptor 5 us-
ing mannosyl trichloroacetimidate 611 as a glycosyl donor
using the Schmidt protocol37 to afford benzoyl-protected
a(1→6)dimannosylated sugar donor 7. Hence, glycosyla-
tion of 5 with benzoyl-protected mannosyl trichloroace-
timidate 611 with trimethylsilyl triflate activation in
dichloromethane afforded the desired dimannosylated
thioglycoside 7 in good yield (88%). The presence of a
participating benzoyl group at C2 in 611 led to formation
OH
OR
OTrt
OH
O
OR
O
a
c
HO
HO
RO
RO
RO
RO
OH
STol
STol
1 R = Ac
2 R = H
3 R = H
4 R = Bz
b
d
e
OBz
OH
f
OBz
O
OBz
O
OBz
BzO
BzO
BzO
BzO
OBz
O
1'
BzO
STol
1
O
7
5
BzO
of the desired O-dimannosylated a-anomer 7. The H
OBz
O
NMR spectrum exhibited two doublets at dH = 5.15 and
dH = 5.76 corresponding to H1¢ and H1, respectively, and
the anomeric configuration of the product was established
by the magnitude of the J1,2 and J1¢,2 coupling constant (1.4
Hz and 1.6 Hz, respectively) and the JC1,H1 and JC1¢,H1¢ het-
erocoupling constants which were 171.0 Hz and 173.8 Hz,
respectively. Tvaroska and Taravel38 reported that larger
1JC1,H1 coupling constants (approximately 170 Hz) are ex-
hibited by a-anomers of pyranoses and oligo- and
O
CCl3
NH
OBz
BzO
BzO
1
6
STol
g
OBz
h
O
HO
FmocHN
BzO
BzO
1'
OAllyl
O
OBz
O
8 O
OBz
BzO
BzO
OBz
O
1
BzO
BzO
j
O
1
polysaccharides while smaller JC1,H1 coupling constants
HO
FmocHN
O
OR
OBz
O
are characteristic of b-anomers (~160 Hz).
FmocHN
OAllyl
Subsequent glycosylation of the Fmoc-Ser-O-Allyl (8)11
acceptor by thioglycoside donor 7 with activation by N-io-
dosuccinimide and silver triflate in dichloromethane af-
forded protected O-dimannosylated serine 11 in moderate
yield (58%).
BzO
BzO
O
8
O
11 R = Allyl
12 R = H
k
OR
9 R = H
10 R = CCl3C(=NH)
i
Scheme 1 Reagents and conditions: (a) 1. Cu(OTf)2, Ac2O, 0 °C to
r.t. 2. BF3·OEt2, TolSH, CH2Cl2, 0 °C to r.t., 24 h, 57% over 2 steps;
(b) 1 M NaOMe, MeOH, r.t., 3 h, 96%; (c) TrtCl, py, DMAP, 0 °C to
r.t., 24 h (crude); (d) BzCl, py, DMAP, 0 °C to r.t., 12 h, 98% (from
crude); (e) TFA–CH2Cl2–i-Pr3SiH (1:94:5), r.t., 1.5 h, 85%; (f) TM-
SOTf, CH2Cl2, –40 °C to r.t., 3 h, 88%; (g) NIS, AgOTf, CH2Cl2, 0
°C, 3 h, 58%; (h) NBS, EtOAc–H2O (1:1), r.t., 24 h, 64%; (i) K2CO3,
CH2Cl2, Cl3CCN, r.t., 12 h, 71%; (j) TMSOTf, CH2Cl2, –40 °C to r.t.,
3 h, 74%; (k) Pd(PPh3)4, PhSiH3, CH2Cl2, Ar, 67%.
A better synthesis of 11 involved preparation of the ben-
zoyl-protected dimannosyl trichloroacetimidate 10 as the
glycosyl donor to which Fmoc-Ser-O-Allyl (8)11 was cou-
pled using Schmidt methodology.37 Dimannosyl trichlo-
roacetimidate 10 was synthesized by anomeric
deprotection of thioglycoside 7 using N-bromosuccinim-
ide in a mixture of ethyl acetate–water to afford dimanno-
side 9 in 64% yield. Subsequent conversion into
trichloroacetimidate 10 in a similar fashion to that used
for monomannosylated unit 611 took place upon treatment
with trichloroacetonitrile and potassium carbonate afford-
ing benzoyl-protected dimannosylated Schmidt donor 10
in good yield (71%). Glycosylation of Fmoc-Ser-O-Allyl
(8)11 using trimethylsilyl triflate (20 mol%) in dichlo-
romethane at –40 °C for three hours afforded Fmoc-[a-D-
Man(OBz)4-(1→6)-a-D-Man(OBz)3a1-]Ser-O-Allyl (11)
in 74% yield. This yield provided a significant improve-
ment compared to glycosylation using thioglycoside do-
nor 7 (58%).
The synthesis started with the preparation of benzoyl-pro-
tected acceptor 5 containing a free 6-OH group that was
successfully executed in six steps. Initial per-O-acetyla-
tion of D-mannose using copper triflate with a stoichio-
metric quantity of acetic anhydride, followed by anomeric
substitution with p-thiocresol under boron trifluoride–di-
ethyl ether complex activation gave thioglycoside 132 in a
one-pot synthesis.33 Alternative strategies for this one-pot
thioglycoside step are also available.34 The next steps re-
quired deacetylation of 132 using catalytic sodium meth-
oxide in methanol (Zemplén conditions)35 followed by
selective formation of a primary trityl ether at C6, and
benzoylation to afford benzoyl-protected thioglycoside 4.
Removal of the trityl protecting group was then achieved
under acidic conditions.36 Initial deprotection using p-tol-
uenesulfonic acid in dichloromethane only afforded the
desired product in low yield with substantial recovery of
starting material. Longer reaction times and an increased
quantity of p-toluenesulfonic acid did not improve the
yield. Gratifyingly, trifluoroacetic acid with triisopropyl-
silane as a carbocation scavenger removed the trityl ether
to afford the desired thioglycoside acceptor 5 in 85%
yield.
The identity of the product 11 was confirmed by the pres-
ence of an [M + H]+ ion in the FAB spectrum (m/z calcd
for C82H70NO22: 1420.4390; found: 1420.4392). In addi-
tion, formation of the glycosylated product was confirmed
by the presence of a signal due to the anomeric proton H1
at dH = 5.20 which was shifted upfield (dH = 6.58 for com-
pound 10 before glycosylation) and a signal at dC = 98.5
due to the anomeric C1 which was shifted downfield
(dC = 94.8 for compound 10 before glycosylation). Finally,
removal of the allyl group using freshly prepared
tetrakis(triphenylphosphine)palladium(0)39 and phenyl-
silane as the allyl group acceptor40 in dichloro-
methane gave the Fmoc-[a-D-Man(OBz)4-(1→6)-a-D-
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

PAPER
Synthesis of Fluorescein-Labelled O-Dimannosylated Peptides
2213
Man(OBz)3a1-]Ser-OH (12) building block in 67% yield could be performed. It was decided to attach the 5(6)-car-
ready for incorporation into a peptide chain.
boxyfluorescein label to the Na amino group of a lysine
with further incorporation of the mannosylated peptide
chain through the side chain Ne amino group.11 The stabil-
ity of the 5(6)-carboxyfluorescein to the microwave con-
ditions was an important issue to be addressed in this
Preparation of PEG-[diMan(OBz)7]-OH 15
In a similar fashion to the method used for the preparation study. Initial preparation of 5(6)-carboxyfluorescein-
of Fmoc-[a-D-Man(OBz)4-(1→6)-a-D-Man(OBz)3a1-]Ser- labelled Wang resin was undertaken. The microwave-
OH (12), the synthesis of PEG-[a-D-Man(OBz)4-(1→6)- assisted automated SPGS was carried out starting from
a-D-Man(OBz)3a1-]-OH (15) was also undertaken commercially available pre-loaded Fmoc-Gly-Wang resin
(Scheme 2).
using a CEM Liberty microwave peptide synthesizer
(Scheme 3).
.
OBz
OBz
O
.
O
Dde
BzO
BzO
FmocHN
1"
Wang
NH
ε
a
O
7
OBz
O
1) deprotect Fmoc (a)
2) Fmoc-Lys(Dde)-OH coupling (b)
O
H
O
α
N
BzO
BzO
HO
Wang
H2N
1'
O
Ot-Bu
O
3) capping (c)
4) repeat step 1
3
O
13
O
1) 5(6)-carboxyfluorescein
coupling (d)
2) remove additional
5(6)-carboxyfluorescein (e)
3) protection of hydroxy groups
of 5(6)-carboxyfluorescein (f)
O
OR
3
14 R = t-Bu
15 R = H
b
HO
Scheme 2 Reagents and conditions: (a) NIS, AgOTf, CH2Cl2, 0 °C,
O
Wang linker
TrtO
77%; (b) TFA, anisole (10:1), r.t., 1.5 h, 80%.
OTrt
O
Dde
NH
ε
O
The synthesis of 15 started with the preparation of ben-
zoyl-protected dimannosylated thiomannoside donor 7
(Scheme 1). Subsequent glycosylation of the tert-butyl-
protected linker 1341 with thioglycoside donor 7 by acti-
vation with N-iodosuccinimide and silver triflate afforded
glycoside 14 in good yield (77%). Thus incorporation of
the PEG linker unit 1341 facilitated the glycosylation step
compared to direct glycosylation of Fmoc-Ser-O-Allyl
(8)11 by dimannosylated thioglycoside donor 7. The struc-
ture of the dimannosylated PEG product 14 was con-
firmed by the presence of the [M + H]+ ion in the FAB
spectrum (m/z calcd for C74H75O23: 1331.4699; found:
O
6
H
α
N
O
Wang
5
N
H
O
O
16
Scheme 3 Reagents and conditions: (a) 20% piperidine–DMF,
MW, 80 °C, 0.5 min plus 3 min; (b) HBTU, DIPEA, DMF, MW,
80 °C, 5 min; (c) 20% Ac2O–DMF, MW, 70 °C, 2 min; (d)
HBTU, DIPEA, DMF, MW, 80 °C, 30 min; (e) 6 cycles of 20%
piperidine–DMF, MW, 80 °C, 0.5 min plus 3 min; (f) TrtCl, DIPEA,
CH2Cl2, r.t., overnight.
The Fmoc protecting group was removed from Fmoc-
Gly-Wang resin using 20% piperidine in N,N-dimethyl-
formamide under microwave conditions followed by in-
corporation of Fmoc-Lys(Dde)-OH [Dde = 1-(4,4-
dimethyl-2,6-dioxocyclohexylidene)ethyl] and subse-
quent Fmoc removal to allow for incorporation of the
5(6)-carboxyfluorescein via the Na amino group of the
lysine. This procedure was based on a conventional SPPS
protocol employed by Brock et al.42 for the preparation of
fluorescently labelled Wang resin using diisopropylcarbo-
diimide (DIC) and 1-hydroxybenzotriazole (HOBt) as
coupling reagent. Bradley et al.18 reported an improved
method for coupling 5(6)-carboxyfluorescein to the side
chain of a lysine residue under microwave conditions,
also using diisopropylcarbodiimide and 1-hydroxybenzo-
triazole as coupling reagent, however in this case the
fluorescent label was introduced at the final stage of the
synthesis. The reaction time was shortened to ten minutes
compared to use of an overnight reaction that was re-
quired when conventional coupling of the label42 was
used. In our case the use of uniform reagents for all cou-
pling steps was preferred hence, N-[(1H-benzotriazol-1-
yl)(dimethylamino)methylene]-N-methylmethanaminium
1
1331.4719). H and 13C NMR spectroscopy also con-
firmed the desired structure of 14 by the characteristic res-
onances for the anomeric proton H1¢ at dH = 5.19 (shifted
upfield compared to the anomeric proton H1 at dH = 5.76
in donor 7) and the anomeric carbon C1¢ at dC = 97.7
(shifted downfield compared to the anomeric carbon C1 at
dC = 86.6 in donor 7). The magnitude of the J1¢,2¢ and J1¢¢,2¢¢
coupling constants (1.3 Hz and 1.5 Hz, respectively) to-
gether with the JC1¢,H1¢ and JC1¢¢,H1¢¢ heterocoupling con-
stants (172.3 Hz and 170.4 Hz, respectively) confirmed
the a-configuration of the product 14.
Deprotection of the tert-butyl ester using trifluoroacetic
acid and anisole afforded the desired dimannosylated
PEG linker 15 building block (80%) ready for further
elaboration into a peptide chain.
Microwave-Assisted Solid-Phase Synthesis of 5(6)-
Carboxyfluorescein-Labelled Wang Resin
With protected glycosylated serine building blocks 12 and
15 in hand, Fmoc solid-phase glycopeptide synthesis
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

2214
R. Kowalczyk et al.
PAPER
hexafluorophosphate N-oxide (HBTU) was used for intro- and 95% trifluoroacetic acid with triisopropylsilane and
duction of the 5(6)-carboxyfluorescein label. Since the water as scavengers were used to cleave the peptides from
Kaiser test43 performed after ten minutes of microwave ir- resin (20 min cycle). N-[(dimethylamino)-1H-1,2,3-triaz-
radiation revealed incomplete coupling the reaction was olo[4,5-b]pyridin-1-ylmethylene]-N-methylmethanamin-
continued with complete coupling requiring a longer time ium hexafluorophosphate N-oxide/1-hydroxy-7-aza-
of 30 minutes with microwave heating at 80 ° C compared benzotriazole (HATU/HOAt) was used as the coupling re-
to the ten minute cycle at 60 °C reported by Bradley et agent for coupling the mannosylated building block and
al.18 Inefficient 5(6)-carboxyfluorescein acylation was the coupling cycle was carried out for 20 minutes.
also found using a 10-minute cycle18 under diisopropyl-
The free amino group present on the side chain of the Ne-
carbodiimide and 1-hydroxybenzotriazole activation
lysine residue allowed for coupling of a specific number
(manual addition of reagents) and again 30 minutes was
of Fmoc-protected alanine residues (3 or 4) and finally either
required for complete reaction.
Fmoc-[a-D-Man(OBz)4-(1→6)-a-D-Man(OBz)3a1-]Ser-
The discrepancy in the reaction time required to effect OH (12) (compound 18) or PEG-[a-D-Man(OBz)4-
complete coupling of the 5(6)-carboxyfluorescein label (1→6)-a-D-Man(OBz)3a1]-OH (15) (compounds 20 and
might be attributed to the different solid supports used by 22) were introduced into the peptide chain. In order to
Bradley et al.18 and our laboratory. Tentagel44 resin was minimise the use of the dimannosylated constructs 12 and
used during the study by Bradley et al.18 while polystyrene 15, only 1.5 equivalents of the building blocks were used
cross-linked with 1% DVB resin was used during the per coupling step and the dimannosylated serine was pre-
present work. Tentagel44 resin is prepared by grafting activated with HATU/HOAt outside the synthesizer. The
PEG to low (1% to 2%) cross-linked polystyrene. The solution was then added manually to the reaction vessel
swelling properties of Tentagel44 are much improved and subjected to microwave irradiation for 20 minutes (80
compared to polystyrene resin.45
°C). The Kaiser test43 performed after coupling of the gly-
cosylated serine (for compound 18) and glycosylated PEG
linker (for compounds 20 and 22) was negative hence, fi-
nal Fmoc deprotection was undertaken. Subsequent cleav-
age from the resin afforded crude benzoyl-protected
glycopeptides 17, 19, and 21.
Subsequent treatment of the peptidyl-resin with 20% pip-
eridine in N,N-dimethylformamide (6 cycles) ensured
cleavage of additional fluorophore esters.42 Attempts to
effect microwave-assisted introduction of O-trityl-pro-
tecting groups on the resin-bound label using trityl chlo-
ride and N,N-diisopropylethylamine were disappointing. Glycopeptide 17 was purified by reverse-phase HPLC to
Neither prolonged reaction time (CH2Cl2, 40 °C, 15 min) afford the desired product in 8% yield. Pleasingly, re-
compared to the standard coupling cycle (80 °C, 5 min), verse-phase HPLC analysis of the crude glycopeptides 19
nor the use of N,N-dimethylformamide instead of dichlo- and 21 obtained after cleavage from the resin revealed the
romethane and higher temperature (80 °C, 15 min) effect- presence of one major product (ca. 66% and ca. 73%, re-
ed the tritylation. Thus, trityl protection was achieved spectively, based on HPLC analysis of the crude prod-
using standard manual conditions at room temperature ucts). Further analysis by MALDI-TOF spectroscopy
upon treatment with trityl chloride and N,N-diisopropyl- identified the major peaks in the reverse-phase HPLC
ethylamine in dichloromethane overnight to afford fluo- spectra as the desired glycopeptides 19 (m/z calcd:
rescently labelled Wang resin 16 ready for further 2030.6750; found: 2030.6453) and 21 (m/z calcd:
elongation.
2101.7121; found: 2101.9402;) by the presence of their
corresponding [M+] ions.
Finally, deprotection of the benzoate esters on the man-
nose residues of glycopeptide 17 using catalytic sodium
methoxide in methanol and final purification by reverse-
phase HPLC gave the desired glycopeptide 5(6)-carboxy-
fluorescein-{[D-Man(OH)4-(a1→6)-D-Man(OH)3(a1-
O)]Ser-(Ala)4-}Lys-Gly-OH (18) in 73% yield (6% over-
all). The identity of the product was confirmed by the
presence of the [M + Na]+ ion in the MALDI-TOF mass
spectrum (m/z calcd: 1279.4501; found: 1278.9909).
Microwave-Assisted Solid-Phase Synthesis of Glyco-
peptides
Subsequent manual treatment of the peptidyl-resin 16
with 2% hydrazine hydrate effected removal of the Dde-
protecting group in order to extend microwave-enhanced
automated SPPS through the side chain Ne amino group of
the lysine residue (Scheme 4).
Because O-tritylation was undertaken manually it was
also more convenient to perform the subsequent Dde
deprotection step manually. From this point on the synthe-
sis was once again carried out in the CEM Liberty peptide
synthesizer. Thus, HBTU was used as a coupling reagent
for incorporation of the Fmoc-Aaa (5 min cycle) and the
Fmoc group was removed using 20% piperidine solution
in N,N-dimethylformamide (0.5 min then 3 min cycles).
20% Acetic anhydride–N,N-dimethylformamide solution
was used to cap the unreacted amino groups (2 min cycle)
As the crude glycopeptides 19 and 21 were of significant
purity (ca. 66% and ca. 73%, respectively) removal of the
benzoate protecting groups from the protected glycopep-
tides was performed without prior purification. Reverse-
phase HPLC monitoring of the progress of O-debenzoyla-
tion of the crude 19 and 21 indicated almost quantitative
conversion to the desired deprotected products 5(6)-car-
boxyfluorescein-{[D-Man(OH)4-(a1→6)-D-Man(OH)3(a1-
O)]PEG-(Ala)3-}Lys-Gly-OH (20) and 5(6)-carboxyfluo-
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

PAPER
Synthesis of Fluorescein-Labelled O-Dimannosylated Peptides
2215
rescein-{[D-Man(OH)4-(a1→6)-D-Man(OH)3(a1-O)]PEG-
resin allows a more convenient and efficient synthesis of
(Ala)4-}Lys-Gly-OH (22) after three and four hours, re- fluorescently labelled glycopeptides and the fluorescently
spectively.
labelled resin itself. Use of HBTU as a coupling reagent
instead of DIC/HOBt for incorporation of the 5(6)-car-
boxyfluorescein enables the use of uniform reagents in
microwave-enhanced automated process. However, an
additional study needs to be performed to find suitable
conditions for protection of the hydroxy groups on the
fluorescein label under microwave conditions. Related
work by Bradley et al.18 focused on coupling the carboxy-
fluorescein label at the final stage of the synthesis, using
a microwave vessel using DIC and HOBt whereas in the
present work HBTU was used to introduce the label onto
the resin at an early stage. The latter method thus offers
the possibility to readily access a range of glycopeptides
using this pre-labelled resin in SPGS.
Finally reverse-phase HPLC purification afforded pure
glycopeptides 20 and 22 in good yield (66% and 54%, re-
spectively, based on the benzoate removal step). The iden-
tity of the products was confirmed by the presence of the
[M]+ ion in the MALDI-TOF spectrum at m/z 1302.6882
(calcd: 1302.4915) for glycopeptide 20 and by the pres-
ence of the [M + H]+ ion (m/z calcd: 1374.5359; found:
1374.1804), for glycopeptide 22.
In conclusion, the microwave-assisted solid-phase syn-
thesis of 5(6)-carboxyfluorescein-labelled Wang resin
with the label attached to Na amino group of lysine residue
has been optimised and improved. Ready access to this
Dde
H
N
O
NH
ε
NH
ε
n
H
1) deprotect Dde (a)
2) n-times Fmoc-Ala-OH coupling (b), capping (c)
and Fmoc-deprotection (d)
O
H
N
O
α
H
N
α
Wang
Fluoro(Trt)2
N
H
Wang
Fluoro(Trt)2
N
H
O
O
16
n = 3 or
n = 4
1) building block 15 coupling (e)
2) deprotect Fmoc (d)
3) cleave from resin (f)
1) building block 12 coupling (e)
2) deprotect Fmoc (d)
3) cleave from resin (f)
OBz
OBz
O
BzO
BzO
OBz
OBz
O
O
BzO
BzO
OBz
O
BzO
BzO
O
OBz
O
O
BzO
BzO
O
O
H
H
N
N
O
NH
ε
17, 8%
NH
ε
O
H2N
3
4
n
O
O
O
O
H
N
H
N
α
α
OH
OH
Fluoro(Trt)2
Fluoro(Trt)2
N
H
N
H
O
O
19 n = 3; 58% crude, 66% purity
21 n = 4; 62% crude, 73% purity
deprotect Bz (g)
deprotect Bz (n = 4) (i)
deprotect Bz (n = 3) (h)
[α-D-Man(OH)4-(1→6)-α-Man(OH)3α1-O]
H2N-Ser-Ala-Ala-Ala-Ala
Fluoro
[α-D-Man(OH)4-(1→6)-α-D-Man(OH)3α1-O]
PEG-Ala-Ala-Ala-Ala
Lys-Gly-OH
18, 73% (6% overall)
Fluoro
Lys-Gly-OH
22, 54% from crude
[α-D-Man(OH)4-(1→6)-α-D-Man(OH)3α1-O]
O
O
5
PEG-Ala-Ala-Ala
O
6
Fluoro
Lys-Gly-OH
HO
HO
20, 66% from crude
O
O
Wang linker
Fluoro
OH
Scheme 4 Reagents and conditions: (a) 2% NH2NH2·H2O–DMF, r.t., 2 × 3 min; (b) HBTU, DIPEA, DMF, MW, 80 °C, 5 min; (c) 20% Ac2O–
DMF, MW, 70 °C, 2 min; (d) 20% piperidine–DMF, MW, 80 °C, 0.5 min plus 3 min; (e) HATU, HOAt, collidine, DMF, MW, 80 °C, 20 min;
(f) TFA–i-Pr3SiH–H2O, MW, 40 °C, 20 min; (g) NaOMe, MeOH, r.t., pH 12.1, 3 h; (h) NaOMe, MeOH, r.t., pH 11.9, 3 h; (i) NaOMe, MeOH,
r.t., pH 11.9, 4 h.
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

2216
R. Kowalczyk et al.
PAPER
column (Waters XTerra Prep. C18, 300 mm × 19 mm; 10 mm or Phe-
nomenex Jupiter C4, 250 mm × 10 mm; 5 mm) at a flow rate of 10
mL or 7.5 mL min–1. A linear gradient of 0.1% TFA–H2O (buffer
A) and 0.1% TFA–MeCN (buffer B) was used with detection at 254
nm. Gradient systems were adjusted according to the elution pro-
files and peak profiles obtained from the analytical RP-HPLC chro-
matograms.
The successful synthesis of fluorescently labelled diman-
nosylated peptides 18, 20, and 22 with fluorescent label
attached to the Na amino group of the lysine residue estab-
lished the stability of 5(6)-carboxyfluorescein label to mi-
crowave conditions.
The synthetic protocol developed herein, provides a fast
and effective tool to prepare a diverse library of mannosy-
lated peptides and is a reliable synthetic tool for the con-
structions of glycopeptide ligands for lectin-binding
assays. A significant reduction in reaction time compared
to manual or automated SPPS using conventional meth-
ods has been achieved using automated microwave-assist-
ed SPPS. Employment of a better swelling resin, such as
CLEAR resin46 or ChemMatrix resin47 rather than the
polystyrene-based resin used in the present work may fur-
ther improve this synthetic method.
Synthesis of O-Dimannosylated Building Blocks
4-Methylphenyl 1-Thio-a-D-mannopyranoside (2)
p-Methylphenyl
2,3,4,6-tetra-O-acetyl-1-thio-a-D-mannopyra-
noside32 (1, 5.95 g, 13.1 mmol) was dissolved in MeOH (100 mL)
and 1 M NaOMe in MeOH was added to adjust the pH to 10 (pH
paper). The mixture was stirred for 3 h and a portion of dry ice was
added to neutralise the mixture. After removal of the solvent in vac-
uo, the crude product was purified by flash chromatography (gradi-
ent elution, CH2Cl2–MeOH 9:1 to 7:3) to give 2 (3.62 g, 96%) as a
white amorphous solid; Rf = 0.44 (CH2Cl2–MeOH, 8:2).
[a]D20 +222.5 (c 0.35, MeOH) [Lit.48 +279 (c 0.70, MeOH)].
IR (NaCl): 3428 cm–1 (s br, O–H).
All reagents were purchased as reagent grade and used without fur-
ther purification. Solvents were dried and purified prior to use. Sol-
vents for RP-HPLC were purchased as HPLC grade and used
without further purification. Fmoc-Gly-Wang resin and Fmoc-
Lys(Dde) were purchased from IRIS Biotech GmbH. Fmoc-Ala-
Wang resin, Fmoc-Ala, HOBt, and HBTU were purchased from
Advanced ChemTech. HOAt was purchased from Acros Organics.
HATU and 5(6)-carboxyfluorescein (isomeric mixture) were pur-
chased from Fluka. Analytical TLC was performed using 0.2-mm
plates of Kieselgel F254 (Merck) and compounds were visualised by
UV fluorescence or by staining with 4% H2SO4 in EtOH or ethan-
olic ninhydrin soln (0.3% ninhydrin–EtOH + 1% AcOH), followed
by heating the plate for a few minutes. Flash chromatography was
carried out on Kieselgel F254 S 0.063–0.1-mm (Riedel de Hahn) sil-
ica gel with the solvents indicated. IR spectra were obtained using
a Perkin-Elmer Spectrum One Fourier Transform infrared spectro-
photometer. Optical rotations were determined at the sodium D line
(589 nm), at 20 °C with a Perkin-Elmer 341 polarimeter and are giv-
en in units of 10–1 deg cm2 g–1.
1H NMR (400 MHz, MeOD): d = 2.28 (s, 3 H, CH3-Tol), 3.66–3.82
(m, 4 H, H3, H4, H6), 4.01–4.05 (m, 1 H, H5), 4.06 (dd, J = 2.9, 1.5
Hz, 1 H, H2), 5.34 (d, J = 1.5 Hz, 1 H, H1), 7.08–7.39 (m, 4 H, Ph).
13C NMR (100 MHz, MeOD): d = 21.1 (CH3-Tol), 62.6 (CH2, C6),
68.7 (CH, C3), 73.2 (CH, C4), 74.2 (CH, C2), 76.2 (CH, C5), 90.8
(CH, C1), 130.7 (Cq, C1¢), 130.8 (CH, C3¢, C5¢), 133.5 (CH, C2¢,
C6¢), 138.9 (Cq, C4¢).
NMR data was in agreement with that reported in the literature.48
4-Methylphenyl 6-O-Trityl-1-thio-a-D-mannopyranoside (3)
To an ice cold soln of 2 (0.51 g, 1.77 mmol) in pyridine (7 mL) was
added TrtCl (2.96 g, 10.6 mmol) and a catalytic amount of DMAP.
The mixture was stirred under N2 for 24 h and allowed to warm to
r.t. Ice-cold H2O (5 mL) was added to quench the reaction and the
crude product was extracted with EtOAc (3 × 5 mL), washed with
0.1 M aq HCl (2 × 5 mL) and sat. NaHCO3 (2 × mL), dried
(Na2SO4), and filtered. The solvent was removed in vacuo and the
resulting yellow solid (1.30 g) was used in the subsequent benzoy-
lation step without further purification; Rf = 0.21 (EtOAc–hexane,
1:1).
NMR spectra were recorded as indicated on either a Bruker Avance
DRX300 (1H, 300 MHz, 13C, 75 MHz) or Bruker Avance DRX400
spectrometer (1H, 400 MHz, 13C, 100 MHz) or on a Bruker Avance
DRX600 spectrometer (1H, 600 MHz, 13C, 150 MHz); relative to
TMS (dH 0.00) in CDCl3/TMS or were referenced to the residual
MeOH signal at dH = 3.34 in CD3OD. The 13C values were refer-
enced to the residual CHCl3 signal at dC = 77.0 in CDCl3/TMS or
residual MeOH signal at dC = 49.15 in CD3OD solvent. Assign-
ments were made with the aid of DEPT135, COSY, HSQC, and
HMBC experiments. The ratio of 5-carboxyfluorescein and 6-car-
boxyfluorescein regioisomers was assumed to 60:40 for the assign-
ment of 1H NMR data. LR-MS were recorded on a VG-70SE mass
spectrometer operating at a nominal accelerating voltage of 70 eV
(FAB). HRMS were recorded using a VG-70SE spectrometer at a
nominal resolution of 5000 to 10,000 as appropriate. ESI-MS were
recorded on a Thermo Finnigan Surveyor MSQ Plus spectrometer.
Accurate mass spectra were recorded using Matrix-assisted laser
desorption/ionisation-time of flight mass spectrometry (MALDI-
TOF MS) technique on a Voyager-DE spectrometer with the use of
a-cyano-4-hydroxycinnamic acid (CHCA) as matrix. Analytical
RP-HPLC was performed using Dionex P680 System using an ana-
lytical column (Phenomenex Jupiter C4, 300 Å, 50 mm × 2.0 mm; 5
mm or Phenomenex Gemini C18, 110 Å, 150 mm × 4.6 mm; 5 mm)
at a flow rate of 0.5 mL or 1 mL min–1. A linear gradient of 0.1%
TFA–H2O (buffer A) and 0.1% TFA–MeCN (buffer B) was used
with detection at 210 nm or 254 nm. Gradient systems were adjust-
ed according to the elution profiles. Semi-preparative RP-HPLC
was performed using a Waters 600 System using a semipreparative
4-Methylphenyl 2,3,4-Tri-O-benzoyl-6-O-trityl-1-thio-a-D-
mannopyranoside (4)
To an ice-cold soln of crude 3 (1.30 g, 2.46 mmol) and a catalytic
amount of DMAP in pyridine–CH2Cl2 (1:4, 8 mL) was added BzCl
(1.03 mL, 8.85 mmol) over 10 min. The mixture was stirred over-
night, warmed to r.t., and diluted with EtOAc (10 mL) and H2O (5
mL) was added. The organic layer was washed with 1 M aq HCl (2
× 5 mL), sat. NaHCO3 (2 × 5 mL), H2O (2 × 5 mL), brine (2 × 5 mL),
dried (Na2SO4), and filtered. The solvent was removed in vacuo and
the residue was purified by flash chromatography (EtOAc–hexane,
1:6) to give 4 (1.04 g, 98%) as a white foam; Rf = 0.23 (EtOAc–hex-
ane, 1:2).
[a]D20 –14.8 (c 1.40, CHCl3).
IR (NaCl): 3060 (w), 3032 (w, ArH), 1730 (s, C=O), 1601 (m),
1584 (m, C–C, Ar), 1261 (s, C–O), 762 (s, C–H, Ar), 708 cm–1 (C–
C, Ar).
1H NMR (400 MHz, CDCl3): d = 2.03 (s, 3 H, CH3-Tol), 3.34 (dd,
J = 10.6, 4.6 Hz, 1 H, H6A), 3.43 (dd, J = 10.6, 2.1 Hz, 1 H, H6B),
4.76 (ddd, J = 10.1, 4.6, 2.1 Hz, 1 H, H5), 5.73–5.76 (m, 2 H, H1,
H3), 5.95 (dd, J = 3.2, 1.6 Hz, 1 H, H2), 6.09 (t, J = 10.1 Hz, 1 H,
H4), 7.07–8.14 (m, 34 H, Ph).
13C NMR (100 MHz, CDCl3): d = 21.1 (CH3-Tol), 62.2 (CH2, C6),
67.1 (CH, C4), 70.9 (CH, C3), 71.4 (CH, C5), 72.2 (CH, C2), 86.2
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

PAPER
Synthesis of Fluorescein-Labelled O-Dimannosylated Peptides
2217
(CH, C1), 86.7 (Cq, CPh3), 126.8, 127.6, 127.9, 128.1, 128.3, 128.4,
128.6 (CH, Ph), 129.0, 129.2, 129.4 (Cq, Ph Bz), 129.7 (Cq, C1¢),
129.9, 130.1, 132.4, 133.1, 133.4, 133.5 (CH, Ph), 138.0 (Cq, C4¢),
143.7 (Cq, Ph Trt), 165.1, 165.5 (Cq, Bz).
(d, J = 1.4 Hz, 1 H, H1), 5.85 (dd, J = 3.2, J = 1.6 Hz, 1 H, H2¢),
5.93 (dd, J = 10.1, J = 3.2 Hz, 1 H, H3), 6.02 (dd, J = 10.0, 3.2 Hz,
1 H, H3¢), 6.06 (dd, J = 3.2, 1.4 Hz, 1 H, H2), 6.13 (t, J = 10.0 Hz,
1 H, H4¢), 6.23 (t, J = 10.1 Hz, 1 H, H4), 7.17–8.19 (m, 39 H, Ph).
MS (FAB): m/z (%) = 717 (3), 259 (1), 243 (100), 105 (56) [M +
13C NMR (100 MHz, CDCl3): d = 21.0 (CH3-Tol), 62.4 (CH2, C6¢),
66.5 (CH, C4), 66.8 (CH2, C6), 66.9 (CH, C4¢), 68.8 (CH, C5¢), 70.1
(CH, C2¢, C5), 70.5 (CH, C3, C3¢), 71.9 (CH, C2), 86.6 (d,
J = 171.0 Hz, CH, C1), 98.0 (d, J = 173.8 Hz, CH, C1¢), 128.3,
128.5 (CH, Ph), 128.8 (Cq, C1¢¢), 128.9, 129.0, 129.1, 129.2 (Cq,
Ph), 129.6, 129.7, 129.8, 129.9, 130.1, 132.6, 132.9, 133.0, 133.2,
133.3, 133.4, 133.5 (CH, Ph), 138.4 (Cq, C4¢¢), 165.1, 165.4, 165.5,
165.9 (Cq, Bz).
H]+.
HRMS (FAB): m/z [M + H]+ calcd for C53H45O18S: 841.2835;
found: 841.2833.
4-Methylphenyl 2,3,4-Tri-O-benzoyl-1-thio-a-D-mannopyrano-
side (5)
Compound 4 (1.94 g, 2.30 mmol) was dissolved in a mixture of an-
hyd CH2Cl2 (47 mL) and TFA (0.5 mL) under N2 and then i-Pr3SiH
(2.50 mL, 12.2 mmol) was added. Upon completion of the reaction
which was indicated by the disappearance of the yellow colour (1.5
h), H2O (10 mL) was added and CH2Cl2 was removed in vacuo. The
residue was diluted with EtOAc (30 mL), washed with sat. NaHCO3
(3 × 10 mL), dried (Na2SO4), and filtered. The solvent was removed
in vacuo and the crude product was purified by flash chromatogra-
phy (EtOAc–hexane, 1:3) to give 5 (1.17 g, 85%) as a white foam;
Rf = 0.19 (EtOAc–hexane, 1:3).
MS (FAB): m/z (%) = 1053 (2), 579 (9), 475 (1), 105 (100) [M +
H]+.
HRMS (FAB): m/z [M + H]+ calcd for C68H57O17S: 1177.3317;
found: 1177.3332.
2,3,4-Tri-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-a-D-manno-
pyranosyl)-a-D-mannopyranoside (9)
To a vigorously stirred soln of 7 (0.20 g, 0.17 mmol) in EtOAc–H2O
(1:1, 5 mL) was added NBS (0.08 g, 0.42 mmol) and the mixture
stirred for 24 h. The mixture was diluted with EtOAc (5 mL) and
neutralised with Et3N (pH paper). The organic layer was separated,
washed with brine (3 × 5 mL), and dried (Na2SO4) and the solvent
removed in vacuo. The crude product was purified by flash chroma-
tography (EtOAc–hexane, 2:3) to give 9 (0.12 g, 64%) as a white
foam; Rf = 0.13 (EtOAc–hexane, 1:2).
[a]D20 –18.0 (c 0.97, CHCl3).
IR (NaCl): 3523 (s br, O–H), 2923 (s), 2862 (s, C–H), 1728 (s,
C=O), 1601 (m), 1584 (m, C–C, Ar), 1281 (s), 1262 (s, C–O), 1107
(s, O–C–C), 1093 (s, C–O–C), 709 cm–1 (s, C–C, Ar).
1H NMR (300 MHz, CDCl3): d = 2.33 (s, 3 H, CH3-Tol), 2.54 (br s,
1 H, OH), 3.82 (m, 2 H, H6), 4.60–4.63 (m, 1 H, H5), 5.71 (br s, 1
H, H1), 5.89–5.98 (m, 3 H, H3, H4, H2), 7.14–8.10 (m, 19 H, Ph).
[a]D20 –84.1 (c 0.62, CHCl3).
IR (NaCl): 3444 (s br, O–H), 3064 (w), 3034 (w, C–H, Ar), 1731 (s,
C=O), 1601 (m), 1584 (m, C–C, Ar), 1265 (s, C–O), 1107 (s, O–C–
C), 709 cm–1 (s, C–C, Ar).
1H NMR (300 MHz, CDCl3): d = 3.86 (dd, J = 11.4, 1.8 Hz, 1 H,
H6A), 4.12–4.16 (m, 1 H, H6B), 4.32 (dd, J = 12.2, 3.9 Hz, 1 H,
H6¢A), 4.43–4.49 (m, 1 H, H5¢), 4.61 (dd, J = 12.2, 2.5 Hz, 1 H,
H6¢B), 4.66–4.72 (m, 1 H, H5), 5.18 (d, J = 1.7 Hz, 1 H, H1¢), 5.59
(dd, J = 3.8, 1.5 Hz, 1 H, H1), 5.79–5.84 (m, 2 H, H2¢, H2), 5.99–
6.06 (m, 3 H, H3¢, H3, H4), 6.13 (t, J = 10.0 Hz, 1 H, H4¢), 7.23–
8.17 (m, 35 H, Ph).
13C NMR (75 MHz, CDCl3): d = 21.1 (CH3-Tol), 61.3 (CH2, C6),
67.3 (CH, C4), 70.0 (CH, C2), 71.9 (CH, C3), 72.0 (CH, C5), 89.4
(CH, C1), 128.3, 128.5, 128.6 (CH, Ph), 128.8, 128.9, 129.1 (Cq, Ph
Bz), 129.7 (Cq, C1¢), 129.8, 129.9, 130.0, 132.7, 133.3, 133.6, 133.7
(CH, Ph), 138.5 (Cq, C4¢), 165.3, 165.4, 166.4 (Cq, Bz).
MS (FAB): m/z (%) = 599 ([M + H]+, 2), 475 (13), 355 (3), 233 (5),
105 (100).
HRMS (FAB): m/z [M + H]+ calcd for C34H31O8S: 599.1740; found:
599.1747.
13C NMR (75 MHz, CDCl3): d = 62.5 (CH2, C6¢), 66.7 (CH, C4¢),
67.3 (CH, C4), 67.8 (CH2, C6), 68.9 (CH, C5¢), 69.5 (CH, C5), 69.9
(CH, C3), 70.2 (CH, C3¢), 70.4 (CH, C2¢), 71.1 (CH, C2), 92.5 (d,
J = 175.0 Hz, CH, C1), 98.0 (d, J = 171.7 Hz, CH, C1¢), 128.2,
128.3, 128.4, 128.5, 128.7 (CH, Ph), 129.0, 129.2, 129.4 (Cq, Ph),
129.7, 129.8, 130.0, 133.0, 133.1, 133.4 (CH, Ph), 165.3, 165.5,
165.6, 165.7, 166.2 (Cq, Bz).
4-Methylphenyl 2,3,4-Tri-O-benzoyl-6-O-(2,3,4,6-tetra-O-ben-
zoyl-a-D-mannopyranosyl)-1-thio-a-D-mannopyranoside (7)
2,3,4,6-Tetra-O-benzoyl-a-D-mannopyranosyl
trichloroacet-
imidate11 (6, 0.48 g, 0.64 mmol) and 5 (0.39 g, 0.66 mmol) were
dried together under high vacuum for 8 h prior to the reaction. Ac-
tivated powdered 4Å molecular sieves were added and the mixture
was stirred in anhyd CH2Cl2 (12 mL) under N2 for 30 min. The sus-
pension was cooled to –40 °C and TMSOTf (0.02 mL, 0.13 mmol)
was quickly added. The mixture was stirred for 3 h, Et3N was added
to neutralise the mixture (pH paper) and the temperature of the soln
was slowly raised to r.t. The mixture was then filtered and the fil-
trate washed with sat. NaHCO3 (2 × 5 mL), brine (2 × 5 mL), dried
(Na2SO4), and filtered. The solvent was removed in vacuo and the
crude product was purified by flash chromatography (EtOAc–hex-
ane, 1:3) to give 7 (0.66 g, 88%) as a white foam; Rf = 0.50 (EtOAc–
hexane, 1:1).
MS (FAB): m/z (%) = 1053 ([M – H2O], 1), 579 (9), 475 (1), 231
(5), 105 (100).
HRMS (FAB): m/z [M – OH] calcd for C61H49O17: 1053.2970;
found: 1053.2984.
2,3,4-Tri-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-a-D-manno-
pyranosyl)-a-D-mannopyranosyl Trichloroacetimidate (10)
Compound 9 (2.53 g, 2.36 mmol) was dissolved in anhyd CH2Cl2
(50 mL) under N2 and K2CO3 (0.47 g, 6.49 mmol) was added. The
mixture was stirred for 10 min and trichloroacetonitrile (0.93 mL,
9.33 mmol) was added dropwise. The mixture was vigorously
stirred overnight, filtered, concentrated in vacuo and the crude mix-
ture purified by flash chromatography (EtOAc–hexane, 3:7) to give
10 (2.03 g, 71%) as a white foam; Rf = 0.36 (EtOAc–hexane, 1:2).
[a]D20 –9.4 (c 0.81, CHCl3).
IR (NaCl): 3064 (w), 3033 (w, C–H, Ar), 1729 (s, C=O), 1601 (m),
1584 (m, C–C, Ar), 1263 (s, C–O), 1107 (s, O–C–C), 1095 (s, C–
O–C), 757 (s, C–H, Ar), 709 cm–1 (s, C–C, Ar).
1H NMR (400 MHz, CDCl3): d = 2.23 (s, 3 H, CH3-Tol), 3.80 (dd,
J = 11.00, 1.8 Hz, 1 H, H6A), 4.21 (dd, JAB = 11.0 Hz, J6B,5 = 4.8 Hz,
1 H, H6B), 4.28 (1 H, dd, J = 12.2, 4.1 Hz, 1 H, H6¢A), 4.35 (ddd,
J = 10.0, 4.1, 2.3 Hz, 1 H, H5¢), 4.46 (dd, J = 12.2, 2.3 Hz, 1 H,
H6¢B), 4.95–4.99 (m, 1 H, H5), 5.15 (d, J = 1.6 Hz, 1 H, H1¢), 5.76
[a]D20 –44.8 (c 0.99, CHCl3).
IR (NaCl): 3441 (m), 3336 (m, =NH), 3064 (w), 3033 (w, C–H, Ar),
1730 (s, C=O), 1679 (v, C=N), 1601 (m), 1584 (m, C–C, Ar), 1264
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

2218
R. Kowalczyk et al.
PAPER
(s, C–O), 1107 (s, O–C–C), 1092 (s, C–O–C), 756 (s, C–H, Ar), 708
cm–1 (C–C, Ar).
HRMS (FAB): m/z [M + H]+ calcd for C82H70NO22: 1420.4390;
found: 1420.4392.
1H NMR (300 MHz, CDCl3): d = 3.87 (dd, J = 11.0, 1.8 Hz, 1 H,
H6A), 4.15 (dd, J = 11.0, 5.3 Hz, 1 H, H6B), 4.31 (dd, J = 12.1, 3.8
Hz, 1 H, H6¢A), 4.41–4.47 (m, 1 H, H5¢), 4.51 (dd, J = 12.1, 2.3 Hz,
1 H, H6¢B), 4.58–4.63 (m, 1 H, H5), 5.14 (d, J = 1.7 Hz, 1 H, H1¢),
5.76 (dd, J = 3.2, 1.7 Hz, 1 H, H2¢), 5.96–6.04 (m, H3¢, 3 H, H3,
H2), 6.12 (t, J = 9.9 Hz, 1 H, H4¢), 6.18 (t, J = 9.9 Hz, 1 H, H4), 6.58
(br s, 1 H, H1), 7.25–8.20 (m, 35 H, Ph), 9.01 (s, 1 H, NH).
13C NMR (75 MHz, CDCl3): d = 62.5 (CH2, C6¢), 66.2 (CH, C4),
66.3 (CH2, C6), 66.7 (CH, C4¢), 68.9 (CH, C5¢), 69.0 (CH, C3), 70.0
(CH, C2, C3¢), 70.3 (CH, C2¢), 72.2 (CH, C5), 90.7 [Cq,
CCl3C(=NH)], 94.8 (d, J = 180.7 Hz, CH, C1), 97.6 (d, J = 175.0
Hz, CH, C1¢), 128.3, 128.4, 128.5, 128.7, 128.8, 128.9 (CH, Ph),
129.2, 129.3 (Cq, Ph), 129.7, 129.8, 129.9, 130.1, 132.9, 133.0,
133.3, 133.4, 133.6 (CH, Ph), 159.8 [Cq, CCl3C(=NH)], 165.1,
165.2, 165.4, 165.5, 166.0 (Cq, Bz).
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4-tri-O-benzoyl-6-O-
(2,3,4,6-tetra-O-benzoyl-a-D-mannopyranosyl)-a-D-mannopyr-
anosyl]-l-serine (12)
Compound 11 (0.73 g, 0.52 mmol) was dissolved in CH2Cl2 (10
mL) and the mixture was degassed under an argon atmosphere.
Pd(PPh3)4 (0.02 g, 20.6 mmol) was added and argon was bubbled
through the yellow soln for 10 min. PhSiH3 (0.51 mL, 4.12 mmol)
was then added and the mixture was stirred under argon for 1.5 h
during which time the colour of the mixture turned black. Upon
completion of the reaction (TLC: EtOAc–hexane, 2:1 + 10%
AcOH), the solvent was removed in vacuo and the crude product
was purified twice by flash chromatography (CH2Cl2–MeOH, 8:2).
The resultant brown solid was lyophilised (t-BuOH) to give 12
(0.48 g, 67%) as a white amorphous powder; Rf = 0.34 (EtOAc–
hexane, 1:1 + 10% AcOH).
MS (ESI): m/z [M + H + Na]+ calcd for C63H51Cl3NNaO18: 1237.21;
found: 1237.43.
[a]D20 –24.2 (c 0.89, CHCl3).
IR (NaCl): 3300–2500 (s br, O–H), 3423 (m), 3350 (m, N–H), 2951
(s), 2873 (s, C–H), 1727 (s, C=O), 1601 (m), 1585 (m, C–C, Ar),
1451 (m, C–O–H), 1263 (s, C–O), 1108 (s, O–C–C), 1095 (s, C–O–
C), 759 (s, C–H, Ar), 709 cm–1 (s, C–C, Ar).
N-(9-Fluorenylmethoxycarbonyl)-O-[2,3,4-tri-O-benzoyl-6-O-
(2,3,4,6-tetra-O-benzoyl-a-D-mannopyranosyl)-a-D-mannopyr-
anosyl]-l-serine Allyl Ester (11)
Compound 10 (0.91 g, 0.75 mmol) and N-(9-fluorenylmethoxycar-
bonyl)-L-serine allyl ester11 (8, 0.28 g, 0.77 mmol) were dried to-
gether under high vacuum for 8 h prior to the reaction. Activated
powdered 4Å molecular sieves were added and the mixture was
stirred in anhyd CH2Cl2 (7 mL) under N2 for 30 min. The suspen-
sion was cooled to –40 °C and TMSOTf (0.03 mL, 0.15 mmol) was
quickly added. The mixture was stirred for 3 h, Et3N was added to
neutralise the mixture (pH paper) and the temperature of the soln
was slowly raised to r.t. The mixture was filtered, the soln was
washed with sat. NaHCO3 (2 × 3 mL), brine (2 × 3 mL), dried
(Na2SO4), and filtered. The solvent was removed in vacuo and the
crude product was purified by flash chromatography (EtOAc–hex-
ane, 1:2 to 1:1) to give 11 (0.79 g, 74%) as a white foam; Rf = 0.14
(EtOAc–hexane, 2:3).
1H NMR (400 MHz, CDCl3): d = 3.82 (m, 1 H, H6A), 4.10–4.21 (m,
2 H, H6B, Serb-HAHB), 4.25–4.31 (m, 3 H, CH Fmoc, H6¢A, Serb-
HAHB), 4.37–4.50 (m, 5 H, H5¢, CH2 Fmoc, H6¢B, H5), 4.79 (m, 1
H, Sera-H), 5.17 (d, J = 1.6 Hz, 1 H, H1¢), 5.19 (br s, 1 H, H1),
5.76–5.78 (m, 1 H, H2), 5.84 (dd, J = 2.9, 1.6 Hz, 1 H, H2¢), 5.95
(dd, J = 10.0, 3.0 Hz, 1 H, H3), 6.05 (dd, J = 10.2, 2.9 Hz, 1 H, H3¢),
6.09–6.10 (m, 2 H, H4, H4¢), 6.41 (d, J = 8.2 Hz, 1 H, NH), 7.20–
8.14 (m, 43 H, Ph).
13C NMR (100 MHz, CDCl3): d = 47.1 (CH, Fmoc), 54.2 (CH,
Sera), 62.4 (CH2, C6¢), 66.5 (CH2, C6), 66.7 (CH, C4¢), 67.4 (CH2,
Fmoc), 68.9 (CH2, Serb), 69.0 (CH, C5¢), 69.9 (CH, C5), 70.1 (CH,
H3, H3¢), 70.2 (CH, C2, C2¢), 97.8 (d, J = 174.0 Hz, CH, C1¢), 98.5
(d, J = 171.0 Hz, CH, C1), 119.8, 125.2, 127.1, 127.6, 128.4, 128.5,
128.6, 128.7, 128.8 (CH, Ph), 129.0, 129.1, 129.2 (Cq, Ph), 129.6,
129.7, 129.8, 130.0, 132.0, 132.1, 132.2, 133.0, 133.1, 133.2, 133.4,
133.5 (CH, Ph), 141.2, 143.8 (Cq, Fmoc), 156.1 (Cq, CONH), 165.2,
165.4, 165.5, 165.6, 166.0 (Cq, Bz), 172.3 (Cq, COOH).
[a]D20 –41.0 (c 1.31, CHCl3).
IR (NaCl): 3427 (m), 3366 (m, N–H), 2952 (s), 289 1 (s, C–H),
1728 (s, C=O), 1601 (m), 1584 (m, C–C, Ar), 1519 (w, N–H), 1263
(s, C–O), 1108 (s, O–C–C), 1095 (s, C–O–C), 759 (s), 742 (s, C–H,
Ar), 709 cm–1 (s, C–C, Ar).
MS (FAB): m/z (%) = 1054 (1), 579 (5), 231 (4), 178 (15), 165 (4),
105 (100).
1H NMR (400 MHz, CDCl3): d = 3.81–3.83 (m, 1 H, H6A), 4.14–
4.18 (m, 2 H, H6B, Serb-HAHB), 4.24–4.36 (m, 7 H, CH Fmoc, H6¢A,
CH2 Fmoc, Serb-HAHB, H5, H5¢), 4.52 (dd, J = 12.2, 2.2 Hz, 1 H,
H6¢B), 4.74–4.87 (m, 3 H, OCH2CH=CH2, Sera-H), 5.17 (br s, 1 H,
H1¢), 5.20 (br s, 1 H, H1), 5.29–5.44 (m, 2 H, OCH2CH=CH2), 5.75
(m, 1 H, H2¢), 5.84 (m, 1 H, H2), 5.90 (dd, J = 10.1, 3.0 Hz, 1 H,
H3¢), 6.00–6.10 (m, 3 H, H4¢, H3, OCH2CH=CH2), 6.13 (t, J = 9.9
Hz, 1 H, H4), 6.30 (d, J = 8.7 Hz, 1 H, NH), 7.19–8.18 (m, 43 H,
Ph).
13C NMR (100 MHz, CDCl3): d = 47.0 (CH, Fmoc), 54.1 (CH,
Sera), 62.5 (CH2, C6¢), 66.4 (CH2, C6), 66.6 (CH, CH2, C4,
OCH2CH=CH2), 66.8 (CH, C4¢), 67.5 (CH2, Fmoc), 68.8 (CH2,
Serb), 69.0 (CH, C5¢), 69.9 (CH, C3¢, C5), 70.0 (CH, C3), 70.2 (CH,
C2¢), 70.3 (CH, C2), 97.6 (d, J = 171.3 Hz, CH, C1¢), 98.5 (d,
J = 173.4 Hz, CH, C1), 119.6 (CH2, OCH2CH=CH2), 119.8, 124.9,
125.2, 127.3, 127.6, 128.2, 128.3, 128.4, 128.5, 128.7 (CH, Ph),
129.0, 129.1, 129.2 (Cq, Ph), 129.6, 129.7, 129.8, 130.0 (CH, Ph),
131.4 (CH, OCH2CH=CH2), 132.9, 133.1, 133.2, 133.4, 133.5 (CH,
Ph), 141.2, 143.8 (Cq, Fmoc), 156.0 (Cq, CONH), 165.2, 165.4,
165.5, 166.0 (Cq, Bz), 169.9 (Cq, COOAllyl).
HRMS (FAB): m/z [M + H]+ calcd for C79H66NO22: 1380.4077;
found: 1380.4065.
tert-Butyl 12-{[2,3,4-Tri-O-benzoyl-6-O-(2,3,4,6-tetra-O-ben-
zoyl-a-D-mannopyranosyl)-a-D-mannopyranosyl)]oxy}-4,7,10-
trioxadodecanoate (14)
Compound 7 (2.00 g, 1.70 mmol) and tert-butyl 12-hydroxy-4,7,10-
trioxadodecanoate41 (13, 0.36 g, 1.31 mmol) were dried together un-
der high vacuum for 8 h prior to the reaction. Activated powdered
4Å molecular sieves were added and the mixture was stirred in an-
hyd CH2Cl2 (7 mL) under N2 for 30 min. The suspension was
cooled to 0 °C, NIS (0.76 g, 3.40 mmol) and AgOTf (0.10 g, 0.39
mmol) were added under a flow of N2. This resulted in a slight pink
mixture that developed continuously during the progress of the re-
action to finally change to yellow indicating that the all starting ma-
terial had been consumed.49 The mixture was neutralised with Et3N
(pH paper), diluted with CH2Cl2 (5 mL), filtered, washed with 10%
aq Na2S2O3 (3 × 5 mL), sat. NaHCO3 (2 × 5 mL), brine (2 × 5 mL),
dried (Na2SO4), filtered and the solvent was removed in vacuo. The
crude product (amber oil) was purified by flash chromatography
(EtOAc–hexane, 2:3) to give 14 (1.74 g, 77%) as a white foam;
Rf = 0.26 (EtOAc–hexane, 1:1).
MS (FAB): m/z (%) = 1053 (2), 579 (8), 231 (6), 178 (20), 105
(100) [M + H]+.
[a]D20 –45.4 (c 0.97, CHCl3).
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

PAPER
Synthesis of Fluorescein-Labelled O-Dimannosylated Peptides
2219
IR (NaCl): 2927 (s), 2869 (s, C–H), 1728 (s, C=O), 1601 (m), 1584
(m, C–C, Ar), 1263 (s, C–O), 1108 (s, O–C–C), 710 cm–1 (s, C–C,
Ar).
Solid-Phase Synthesis of Glycopeptides 17–22; General Proce-
dures
Glycopeptides were assembled using SPGS on pre-loaded Fmoc-
Gly-Wang resin with the use of a CEM Liberty microwave peptide
synthesizer using the following conditions: Fmoc removal: 20% pi-
peridine–DMF, 0.5 min plus 3 min cycle at 80 °C; Fmoc-Aaa cou-
pling: Fmoc-Aaa (5.0 equiv), HBTU (4.5 equiv), DIPEA (10
equiv), DMF, 5 min cycle at 80 °C; 5(6)-carboxyfluorescein cou-
pling: 5(6)-carboxyfluorescein (5.0 equiv), HBTU (4.5 equiv), DI-
PEA (10 equiv), DMF, 30 min cycle at 80 °C; O-dimannosylated
building block coupling: O-dimannosylated building block (1.5
equiv), HATU (1.45 equiv), HOAt (1.5 equiv), collidine (4.5
equiv), DMF, 20 min cycle at 80 °C; capping: 20% Ac2O–DMF, 2
min cycle at 70 °C; cleavage: TFA–i-Pr3SiH–H2O (95:2.5:2.5), 20
min cycle at 40 °C; removal of additional ester bound 5(6)-carboxy-
fluorescein: 20% piperidine–DMF, six cycles of 0.5 min plus 3 min
at 80 °C.
1H NMR (400 MHz, CDCl3): d = 1.43 [s, 9 H, (CH3)3C], 2.48–2.51
(m, 2 H, H2), 3.60–3.81 (m, 10 H, H5, H6, H3, H8, H9), 3.83–3.90
(m, 3 H, H11, H12A), 4.05–4.16 (m, 3 H, H12B, H6¢), 4.30 (dd,
J = 12.2, 4.0 Hz, 1 H, H6¢¢A), 4.41 (ddd, J = 10.0, 4.0, 2.4 Hz, 1 H,
H5¢¢), 4.46–4.51 (m, 2 H, H5¢, H6¢¢B), 5.18 (d, J = 1.5 Hz, 1 H, H1¢¢),
5.19 (d, J = 1.3 Hz, 1 H, H1¢), 5.80–5.82 (m, 2 H, H2¢, H2¢¢), 5.97
(dd, J 10.2, 3.4 Hz, 1 H, H3¢), 6.02 (dd, J = 10.0, 3.2 Hz, 1 H, H3¢¢),
6.11 (t, J = 10.2 Hz, 1 H, H4¢), 6.12 (t, J = 10.0 Hz, 1 H, H4¢¢), 7.25–
8.19 (m, 35 H, Ph).
13C NMR (100 MHz, CDCl3): d = 28.0 [CH3, (CH3)3C], 36.2 (CH2,
C2), 62.5 (CH2, C6¢¢), 66.6 (CH2, CH, C3, C4¢¢), 66.8 (CH2, C6¢),
66.9 (CH, C4¢), 67.5 (CH2, C12), 68.8 (CH, C5¢¢), 69.3 (CH, C5¢),
70.0 (CH, C2¢¢), 70.1 (CH2, C11), 70.2 (CH, C3¢, C3¢¢), 70.3 (CH2,
C5), 70.4 (CH, C2¢), 70.5 (CH2, C6), 70.6 (CH2, C8), 70.7 (CH2,
C9), 80.4 [Cq, (CH3)3C], 97.7 (d, J = 172.3 Hz, CH, C1¢), 97.9 (d,
J = 170.4 Hz, CH, C1¢¢), 128.2, 128.4, 128.5 (CH, Ph), 128.9, 129.0,
129.1, 129.2, 129.3 (Cq, Ph), 129.6, 129.7, 129.8, 130.0, 133.0,
133.3, 133.4 (CH, Ph), 165.1, 165.4, 165.5, 165.6, 165.9 (Cq, Bz),
170.9 (Cq, C1).
Glycosylated building blocks were pre-activated before placement
in the synthesizer. Glycosylated building block (1.5 equiv) was dis-
solved in DMF, HOAt (1.5 equiv), and HATU (1.45 equiv) were
then added and the mixture was shaken until dissolved. The soln
was transferred to the reaction vessel, followed by the addition of
collidine (4.5 equiv). The mixture was microwaved for 20 min at 80
°C, and a sample of the resin (1.0 mg) was taken for the Kaiser
test.43
MS (FAB): m/z (%) = 1053 (4), 579 (8), 105 (100).
HRMS (FAB): m/z [M + H]+ calcd for C74H75O23: 1331.4699;
Removal of the Dde protecting group from the Ne amino group of
the lysine residue was performed manually using 2%
NH2NH2·H2O–DMF (2 × 3 min).
found: 1331.4719.
12-{[2,3,4-Tri-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-a-D-
mannopyranosyl)-a-D-mannopyranosyl]oxy}-4,7,10-trioxa-
dodecanoic Acid (15)
5(6)-Carboxyfluorescein(Trt)2-Lys(Dde)-Gly-Wang Resin (16)
Peptidyl-resin 16 was synthesised on a CEM Liberty microwave
peptide synthesizer using Fmoc-Gly-Wang resin (625 mg, 0.80
mmol g–1) and the conditions outlined in the general procedure. Af-
ter removal of the additional ester bound 5(6)-carboxyfluorescein,
the 5(6)-carboxyfluorescein-Lys(Dde)-Gly-Wang resin was trans-
ferred to the fritted glass reaction vessel and dried under a flow of
N2. TrtCl (12 equiv) was then added, followed by the addition of
CH2Cl2 and DIPEA (12 equiv). The mixture was shaken overnight,
filtered, washed with CH2Cl2 (5 ×), dried under a flow of N2 and a
fresh portion of the tritylation reagents was added and the procedure
repeated to yield yellow-coloured 5(6)-carboxyfluorescein(Trt)2-
Lys(Dde)-Gly-Wang resin (16), which was used for further synthe-
sis. A sample of the trityl-protected fluorescein-labelled resin
(25.0 mg) was cleaved from the resin using TFA–i-Pr3SiH–H2O
(95:2.5:2.5) for 2 h, precipitated from cold Et2O, isolated by centri-
fuge and lyophilised from MeCN–H2O + 0.1% TFA to yield 5(6)-
carboxyfluorescein-Lys(Dde)-Gly-OH; HPLC (Phenomenex Jupi-
ter C4 analytical column, 50 mm × 2.0 mm, 0.5 mL min–1, linear
gradient of 1% B to 50% B over 15 min): tR = 12.78 min.
Compound 14 (0.87 g, 0.65 mmol) was dissolved in TFA (15 mL)
then anisole (1.5 mL) was added and the mixture was stirred for 1.5
h. The solvent was removed in vacuo and the crude product was pu-
rified by flash chromatography (EtOAc–hexane 2:1) to give 15
(0.66 g, 80%) as a white foam; Rf = 0.57 (EtOAc–hexane, 1:1 +
10% AcOH).
[a]D20 –49.7 (c 0.65, CHCl3).
IR (NaCl): 3300–2500 (s br, O–H), 2925 (s), 2881(s, C–H), 1728 (s,
C=O), 1601 (m), 1584 (m, C–C, Ar), 1451 (m, C–O–H), 1263 (s, C–
O), 1108 (s, O–C–C), 756 (s, C–H, Ar), 710 cm–1 (s, C–C, Ar).
1H NMR (400 MHz, CDCl3): d = 2.59–2.62 (m, 2 H, H2), 3.62–3.82
(m, 10 H, H5, H6, H3, H8, H9), 3.84–3.91 (m, 3 H, H11, H12A),
4.04–4.16 (m, 3 H, H12B, H6¢), 4.29 (dd, J = 12.2, 4.0 Hz, 1 H,
H6¢¢A), 4.41 (ddd, J = 10.0, 4.0, 2.6 Hz, 1 H, H5¢¢), 4.46–4.52 (m, 2
H, H6¢¢B, H5¢), 5.18 (d, J = 1.5 Hz, 1 H, H1¢¢), 5.21 (d, J = 1.5 Hz, 1
H, H1¢), 5.80–5.82 (m, 2 H, H2¢, H2¢¢), 5.97 (dd, J = 10.1, 3.4 Hz, 1
H, H3¢), 6.02 (dd, J = 10.0, 3.2 Hz, 1 H, H3¢¢), 6.08–6.14 (m, 2 H,
H4¢, H4¢¢), 7.25–8.20 (m, 35 H, Ph).
13C NMR (100 MHz, CDCl3): d = 34.7 (CH2, C2), 62.5 (CH2, C6¢¢),
66.3 (CH2, C3), 66.6 (CH2, CH, C6¢, C4¢¢), 67.0 (CH, C4¢), 67.6
(CH2, C12), 68.9 (CH, C5¢¢), 69.4 (CH, C5¢), 70.1 (CH, C2¢¢), 70.2
(CH2, CH, C11, C3¢¢, C3¢), 70.3 (CH, C2¢), 70.4 (CH2, C5), 70.5
(CH2, C6), 70.6 (CH2, C8), 70.8 (CH2, C9), 97.7 (CH, C1¢), 97.9
(CH, C1¢¢), 128.3, 128.4, 128.5, 128.8 (CH, Ph), 129.0, 129.1, 129.3
(Cq, Ph), 129.7, 129.8, 129.9, 130.0, 130.1, 130.4, 130.5 (CH, Ph),
165.2, 165.5, 165.6, 165.7, 166.0 (Cq, Bz), 171.2 (Cq, C1).
MS (FAB): m/z (%) = 1297 (M + Na+, 1), 1053 (4), 579 (8), 105
(100).
HRMS (FAB): m/z [M + H]+ calcd for C70H67O23: 1275.4073;
found: 1275.4075.
MS (ESI): m/z [M + H]+ calcd for C39H40N3O11: 726.26; found:
726.55.
5(6)-Carboxyfluorescein-{[D-Man(OBz)4-(a1→6)-D-
Man(OBz)3(a1-O)]Ser-(Ala)4-}Lys-Gly-OH (17)
Fmoc-Gly-Wang resin (120 mg, 0.75 mmol g–1) was used for the
synthesis of glycopeptide 17 using a CEM Liberty microwave pep-
tide synthesizer and the conditions outlined in the general methods.
The crude benzoyl-protected product was purified by RP-HPLC
(semi-preparative Waters XTerra Prep. C18 column, flow rate: 10
mL min–1; linear gradient 10% B to 100% B over 35 min) and lyo-
philised to give 17 (15.9 mg, 8%) as a yellow amorphous solid;
HPLC (Phenomenex Gemini C18 analytical column, 150 mm × 4.6
mm, 1 mL min–1, linear gradient of 10% B to 100% B over 35 min):
tR = 29.47 min.
HRMS (MALDI-TOF): m/z [M + Na]+ calcd for C105H100N8NaO32:
2007.6336; found: 2005.7562.
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

2220
R. Kowalczyk et al.
PAPER
NMR data was not recorded.
umn, 50 mm × 2.0 mm, 0.5 mL min–1, 3 min of 1% B followed by
linear gradient of 1% B to 50% B over 15 min): tR = 15.55 min.
5(6)-Carboxyfluorescein-{[D-Man(OH)4-(a1→6)-D-
Man(OH)3(a1-O)]Ser-(Ala)4-}Lys-Gly-OH (18)
1H NMR (600 MHz, MeOD): d = 1.34–1.42 (m, 9 H, 3 × Alab-H3),
1.52–2.02 (m, 6 H, Lysg-H2, Lysd-H2, Lysb-H2), 2.46–2.60 (m, 2 H,
H2), 3.16–3.31 (m, 2 H, Lyse-H2), 3.65–4.07 (m, 28 H, H3, H5, H6,
H8, H9, H11, H12, H2¢, H2¢¢, H3¢, H3¢¢, H4¢, H4¢¢, H5¢, H5¢¢, H6¢,
H6¢¢, Glya-H2), 4.13–4.32 (m, 3 H, 3 × Alaa-H), 4.56–4.59 (m, 0.4
H, Lysa-H 6-CF), 4.68–4.70 (m, 0.6 H, Lysa-H 5-CF), 4.80 (br s, 1
H, H1¢¢), 4.86 (br s, 1 H, H1¢), 6.60–6.76 (m, 6 H, CHFluoro), 7.34–
7.36 (m, 0.6 H, CHFluoro 5-CF), 7.80 (br s, 0.4 H, CHFluoro 6-CF),
8.13–8.14 (m, 0.4 H, CHFluoro 6-CF), 8.23 (d, J = 8.0 Hz, 0.4 H,
CHFluoro 6-CF), 8.29–8.30 (m, 0.6 H, CHFluoro 5-CF), 8.57 (s, 0.6 H,
CHFluoro 5-CF).
13C NMR (150 MHz, MeOD): d = 17.5, 17.6, 17.9, 18.0 (CH3, 3 ×
Alab), 24.1, 24.2 (CH2, Lysg), 29.7, 29.9 (CH2, Lysd), 32.5, 32.6
(CH2, Lysb), 37.2 (CH2, C2), 39.9 (CH2, Lyse), 41.8, 41.9 (CH2,
Glya), 50.9, 51.2, 51.3, 51.7, 51.8 (CH, 3 × Alaa), 55.7 (CH, Lysa),
62.9 (CH2, C6¢¢), 67.5 (CH2, C12), 67.8 (CH2, CH2 PEG), 68.3
(CH2, C6¢), 68.7 (CH, C4¢, C4¢¢), 71.2, 71.3, 71.5, 71.6 (CH2, 5 ×
CH2 PEG), 72.1, 72.2 (CH, C2¢, C2¢¢), 72.7, 72.8 (CH, C3¢, C3¢¢),
73.2, 74.4 (CH, C5¢, C5¢¢), 101.4 (CH, C1¢), 101.9 (CH, C1¢¢), 103.7
(CH, Fluoro Ph), 111.2 (Cq, Fluoro), 114.0, 125.7, 125.9 (CH,
Fluoro Ph), 128.7 (Cq, Fluoro), 130.4, 130.6, 130.8, 135.9 (CH,
Fluoro Ph), 137.5, 140.5, 141.8, 154.4, 162.0 (Cq, Fluoro), 168.5,
168.6, 170.5, 172.8, 172.9, 174.7, 174.8, 174.9, 175.0 (Cq, CONH,
COOH).
Compound 17 (9.5 mg, 4.8 mmol) was dissolved in MeOH (5 mL)
and 1 M NaOMe soln in MeOH was added to adjust the pH to 12.1
(pH meter). When the all starting material had disappeared as
judged by analytical RP-HPLC (3 h), the soln was neutralised with
a portion of dry ice and the solvent was removed in vacuo. The
crude product was purified by RP-HPLC (semi-preparative Phe-
nomenex Jupiter C4 column, flow rate: 7.5 mL min–1, linear gradi-
ent: 1% B to 50% B over 35 min) and lyophilised to give the 18 (4.4
mg, 73%) as a yellow amorphous solid; HPLC (Phenomenex Jupi-
ter C4 analytical column, 50 mm × 2.0 mm, 0.5 mL min–1, 3 min of
1% B followed by linear gradient of 1% B to 50% B over 15 min):
tR = 15.31 min.
1H NMR (600 MHz, MeOD): d = 1.31–1.42 (m, 12 H, 4 × Alab-H3),
1.44–1.69 (m, 4 H, Lysg-H2, Lysd-H2), 1.78–2.04 (m, 2 H, Lysb-
H2), 3.18–3.28 (m, 2 H, Lyse-H2), 3.65–4.08 (m, 15 H, H2, H2¢, H3,
H3¢, H4, H4¢, H5, H5¢, H6, H6¢, Glya-H2, Serb-HAHB), 4.11–4.18
(m, 3 H, Serb-HAHB, Alaa-H, Sera-H), 4.27–4.37 (m, 2 H, 2 ×
Alaa-H), 4.45–5.00 (m, 1 H, Alaa-H), 4.57–4.60 (m, 0.4 H, Lysa-
H 6-CF), 4.69–4.72 (m, 0.6 H, Lysa-H 5-CF), 4.82 (br s, 1 H, H1¢),
4.83 (br s, 1 H, H1), 6.66–6.83 (m, 6 H, CHFluoro), 7.37–7.39 (m, 0.6
H, CHFluoro 5-CF), 7.79 (br s, 0.4 H, CHFluoro 6-CF), 8.17–8.19 (m,
0.4 H, CHFluoro 6-CF), 8.24–8.26 (m, 0.4 H, CHFluoro 6-CF), 8.30–
8.32 (m, 0.6 H, CHFluoro 5-CF), 8.61 (s, 0.6 H, CHFluoro 5-CF).
13C NMR (150 MHz, MeOD): d = 17.8, 17.9, 18.2, 18.3 (CH3, 4 ×
Alab), 24.1, 24.2 (CH2, Lysg), 29.7, 29.9 (CH2, Lysd), 32.5, 32.7
(CH2, Lysb), 39.9, 40.0 (CH2, Lyse), 41.8, 41.9 (CH2, Glya), 50.5,
50.7, 50.8, 50.9 (CH, 4 × Alaa), 54.3 (CH, Sera), 55.6 (CH, Lysa),
63.0 (CH2, C6¢), 67.2 (CH2, Serb), 67.4 (CH2, C6), 68.5, 68.7, 71.4,
72.1, 72.5, 72.6, 73.7, 74.6 (CH, C2, C2¢, C3, C3¢, C4, C4¢, C5,
C5¢),101.3 (CH, C1¢), 102.6 (CH, H1), 103.6 (CH, Fluoro Ph),
114.7 (Cq, Fluoro), 118.1, 126.4, 129.1, 130.7, 131.0, 135.6 (CH,
Fluoro Ph), 137.6, 155.1, 160.7, 161.0 (Cq, Fluoro), 167.4, 168.5,
168.6, 170.1, 170.3, 172.9, 173.0, 174.4, 174.5, 174.7, 174.8, 174.9,
175.1, 175.2 (Cq, CONH, CO2H).
HRMS (MALDI-TOF): m/z [M]+ calcd for C59H78N6O27:
1302.4915; found: 1302.6882.
5(6)-Carboxyfluorescein-{[D-Man(OBz)4-(a1→6)-D-
Man(OBz)3(a1-O)]PEG-(Ala)4-}Lys-Gly-OH (21)
Fmoc-Gly-Wang resin (125 mg, 0.80 mmol g–1) was used for the
synthesis of glycopeptide 21 using a CEM Liberty microwave pep-
tide synthesizer and the conditions outlined in the general methods.
The crude benzoyl-protected glycopeptide 21 [131 mg, 62% yield,
73% purity (RP-HPLC)] was used in the subsequent debenzoylation
step without further purification; HPLC (Phenomenex Jupiter C4
analytical column, 50 mm × 2.0 mm, 0.5 mL min–1, 3 min of 5% B
followed by linear gradient of 5% B to 100% B over 15 min):
tR = 18.75 min.
HRMS (MALDI-TOF): m/z [M + Na]+ calcd for C56H72N8NaO25:
1279.4501; found: 1278.9909.
HRMS (MALDI-TOF): m/z [M]+calcd for C111H111N7O35:
2101.7121; found: 2101.9402.
5(6)-Carboxyfluorescein-{[D-Man(OBz)4-(a1→6)-D-
Man(OBz)3(a1-O)]PEG-(Ala)3-}Lys-Gly-OH (19)
Fmoc-Gly-Wang resin (125 mg, 0.80 mmol g–1) was used for the
synthesis of glycopeptide 19 using a CEM Liberty microwave pep-
tide synthesizer and the conditions outlined in the general methods.
The crude benzoyl-protected glycopeptide 19 (118 mg, 58% yield,
66% purity (RP-HPLC)) was used in the subsequent debenzoylation
step without further purification; HPLC (Phenomenex Jupiter C4
analytical column, 50 mm × 2.0 mm, 0.5 mL min–1, 3 min of 5% B
followed by linear gradient of 5% B to 100% B over 15 min):
tR = 18.75 min.
5(6)-Carboxyfluorescein-{[D-Man(OH)4-(a1→6)-D-
Man(OH)3(a1-O)]PEG-(Ala)4-}Lys-Gly-OH (22)
Crude 21 (32.8 mg, 15.60 mmol) was dissolved in MeOH (15 mL)
and 1 M NaOMe soln in MeOH was added to adjust the pH to 11.9
(pH meter). When the all starting material had disappeared as
judged by analytical RP-HPLC (4 h), the soln was neutralised with
a portion of dry ice and the solvent was removed in vacuo. The
crude product was purified by RP-HPLC (semi-preparative Waters
XTerra Prep. C18 column; flow rate: 10 mL min–1; linear gradient:
1% B to 50% B over 35 min) and lyophilised to give 22 (11.6 mg,
54%) as a yellow amorphous solid; HPLC (Phenomenex Jupiter C4
analytical column, 50 mm × 2.0 mm, 0.5 mL min–1, 3 min of 1% B
followed by linear gradient of 1% B to 50% B over 15 min):
tR = 15.68 min.
HRMS (MALDI-TOF): m/z [M]+ calcd for C108H106N6O34:
2030.6750; found: 2030.6453.
5(6)-Carboxyfluorescein-{[D-Man(OH)4-(a1→6)-D-
Man(OH)3(a1-O)]PEG-(Ala)3-}Lys-Gly-OH (20)
Crude 19 (19.5 mg, 9.61 mmol) was dissolved in MeOH (7 mL) and
1 M NaOMe soln in MeOH was added to adjust the pH to 11.9 (pH
meter). When the all starting material had disappeared as judged by
analytical RP-HPLC (3 h), the soln was neutralised with a portion
of dry ice and the solvent was removed in vacuo. The crude product
was purified by RP-HPLC (semi-preparative Waters XTerra Prep.
1H NMR (600 MHz, MeOD): d = 1.34–1.44 (m, 12 H, 4 × Alab-H3),
1.53–2.02 (m, 6 H, Lysg-H2, Lysd-H2, Lysb-H2), 2.48–2.53 (m, 1 H,
H2A), 2.60–2.64 (m, 1 H, H2B), 3.15–3.26 (m, 2 H, Lyse-H2), 3.63–
3.87 (m, 25 H, H3, H5, H6, H8, H9, H11, H12A, H2¢, H2¢¢, H3¢, H3¢¢,
H4¢, H4¢¢, H5¢, H5¢¢, H6¢, H6¢¢), 3.89–4.07 (m, 3 H, H12B, Glya-H2),
412–4.30 (m, 4 H, 4 × Alaa-H), 4.56–4.58 (m, 0.4 H, Lysa-H 6-CF),
4.67–4.70 (m, 0.6 H, Lysa-H 5-CF), 4.80 (br s, 1 H, H1¢¢), 4.86 (br
s, 1 H, H1¢), 6.61–6.72 (m, 4 H, CHFluoro), 6.77–6.78 (m, 2 H,
C
18 column; flow rate: 10 mL min–1; linear gradient: 1% B to 50%
B over 35 min) and lyophilised to give 20 (8.2 mg, 66%) as a yellow
amorphous solid; HPLC (Phenomenex Jupiter C4 analytical col-
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

PAPER
Synthesis of Fluorescein-Labelled O-Dimannosylated Peptides
2221
CHFluoro), 7.36 (d, J = 8.0 Hz, 0.6 H, CHFluoro 5-CF), 7.80 (s, 0.4 H,
CHFluoro 6-CF), 8.14 (m, 0.4 H, CHFluoro 6-CF), 8.23 (d, J = 8.0 Hz,
0.4 H, CHFluoro 6-CF), 8.30 (m, 0.6 H, CHFluoro 5-CF), 8.58 (s, 0.6 H,
CHFluoro 5-CF).
(13) (a) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149.
(b) Lavielle, S.; Ling, N. C.; Guillemin, R. C. Carbohydr.
Res. 1981, 89, 221. (c) Haase, C.; Seitz, O. In Glycopeptides
and Glycoproteins: Synthesis, Structure, and Application,
Vol. 267; Wittmann, V., Ed.; Springer: Berlin, 2007, 1.
(14) (a) Hollósi, M.; Kollát, E.; Laczkó, I.; Medzihradszky, K. F.;
Thurin, J.; Otvös, L. Jr. Tetrahedron Lett. 1991, 32, 1531.
(b) Paulsen, H.; Schleyer, A.; Mathieux, N.; Meldal, M.;
Bock, K. J. Chem. Soc., Perkin Trans. 1 1997, 281.
(c) Andrews, D. M.; Seale, P. W. Int. J. Pept. Protein Res.
1993, 42, 165.
(15) Kent, S. B. H. Ann. Rev. Biochem. 1988, 57, 957.
(16) (a) Yu, H. M.; Chen, S. T.; Wang, K. T. J. Org. Chem. 1992,
57, 4781. (b) Erdelyi, M.; Gogoll, A. Synthesis 2002, 1592.
(c) Campiglia, P.; Gomez-Monterrey, I.; Longobardo, L.;
Lama, T.; Novellino, E.; Grieco, P. Tetrahedron Lett. 2004,
45, 1453. (d) Murray, J. K.; Gellman, S. H. Org. Lett. 2005,
7, 1517. (e) Murray, J. K.; Farooqi, B.; Sadowsky, J. D.;
Scalf, M.; Freund, W. A.; Smith, L. M.; Chen, J.; Gellman,
S. H. J. Am. Chem. Soc. 2005, 127, 13271.
13C NMR (150 MHz, MeOD): d = 17.5, 17.6, 17.7, 18.0 (CH3, 4 ×
Alab), 24.2, 24.3 (CH2, Lysg), 29.8, 29.9 (CH2, Lysd), 32.6, 32.7
(CH2, Lysb), 37.2 (CH2, C2), 40.0, 40.1 (CH2, Lyse), 41.9, 42.0
(CH2, Glya), 51.1, 51.4, 51.5, 51.8, 51.9, 52.0, 52.2 (CH, 4 × Alaa),
55.8, 55.9 (CH, Lysa), 63.0 (CH2, C6¢¢), 67.6 (CH2, C12), 67.9
(CH2, CH2 PEG), 68.3, 68.4 (CH2, C6¢), 68.7, 68.8 (CH, C4¢, C4¢¢),
71.5, 71.6, 71.7 (CH2, 5 × CH2 PEG), 72.2, 72.3 (CH, C2¢, C2¢¢),
72.8, 72.9 (CH, C3¢, C3¢¢), 73.2, 74.5 (CH, C5¢, C5¢¢), 101.5 (CH,
C1¢), 102.0 (CH, C1¢¢), 103.8 (CH Fluoro Ph), 111.5 (Cq, Fluoro),
114.4, 125.2, 126.1, 126.8 (CH, Fluoro Ph), 128.9 (Cq, Fluoro),
130.6, 130.8, 131.0, 135.9 (CH, Fluoro Ph), 137.7, 141.9, 154.6,
162.0, 162.1, 162.3 (Cq, Fluoro), 168.6, 168.7, 170.4, 170.5, 172.9,
174.9, 175.0, 175.1, 175.2, 176.0, 176.1, 176.3, 176.4 (Cq, CONH,
COOH).
HRMS (MALDI-TOF): m/z [M + H]+ calcd for C62H85N7O28:
1374.5359; found: 1374.1804.
(17) (a) Matsushita, T.; Hinou, H.; Fumoto, M.; Kurogochi, M.;
Fujitani, N.; Shimizu, H.; Nishimura, S. I. J. Org. Chem.
2006, 71, 3051. (b) Matsushita, T.; Hinou, H.; Kurogochi,
M.; Shimizu, H.; Nishimura, S. I. Org. Lett. 2005, 7, 877.
(18) Fara, M. A.; Diaz-Mochon, J. J.; Bradley, M. Tetrahedron
Lett. 2006, 47, 1011.
Acknowledgment
We thank the Maurice Wilkins Centre for Molecular Biodiscovery
for the award of a Doctoral Scholarship to R.K.
(19) Katritzky, A. R.; Yoshioka, M.; Narindoshvili, T.; Chung,
A.; Johnson, J. V. Org. Biomol. Chem. 2008, 6, 4582.
(20) (a) Roy, R.; Saha, U. K. Chem. Commun. 1996, 201.
(b) Boumrah, D.; Campbell, M. M.; Fenner, S.; Kinsman, R.
G. Tetrahedron 1997, 53, 6977.
(21) Davis, B. G. J. Chem. Soc., Perkin Trans. 1 1999, 3215.
(22) (a) Boumrah, D.; Campbell, M. M.; Fenner, S.; Kinsman, R.
G. Tetrahedron Lett. 1991, 32, 7735. (b) Boullanger, P.;
Sancho-Camborieux, M. R.; Bouchu, M. N.; Marron-
Brignone, L.; Morelis, R. M.; Coulet, P. R. Chem. Phys.
Lipids 1997, 90, 63. (c) Bhattacharya, S.; Dileep, P. V.
Tetrahedron Lett. 1999, 40, 8167.
(23) Schmidt, M.; Dobner, B.; Nuhn, P. Eur. J. Org. Chem. 2002,
669.
(24) Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1988,
27, 1697.
(25) Barresi, F.; Hindsgaul, O. J. Carbohydr. Chem. 1995, 14,
1043.
(26) Franzyk, H.; Meldal, M.; Paulsen, H.; Bock, K. J. Chem.
Soc., Perkin Trans. 1 1995, 2883.
(27) Koenigs, W.; Knörr, E. Chem. Ber. 1901, 34, 957.
(28) Lemieux, R. U.; Hendriks, K. B.; Stick, R. V.; James, K.
J. Am. Chem. Soc. 1975, 97, 4056.
(29) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212.
(30) Zhu, Y.; Kong, F. Carbohydr. Res. 2001, 332, 1.
(31) Garegg, P. J. Adv. Carbohydr. Chem. Biochem. 1997, 52,
179.
(32) Zhang, Z.; Ollmann, I. R.; Ye, X. S.; Wischnat, R.; Baasov,
T.; Wong, C. H. J. Am. Chem. Soc. 1999, 121, 734.
(33) Tai, C. A.; Kulkarni, S. S.; Hung, S. C. J. Org. Chem. 2003,
68, 8719.
(34) (a) Mukhopadhyay, B.; Kartha, K. P. R.; Russell, D. A.;
Field, R. A. J. Org. Chem. 2004, 69, 7758. (b) Agnihotri,
G.; Tiwari, P.; Misra, A. K. Carbohydr. Res. 2005, 340,
1393. (c) Dasgupta, S.; Rajput, V. K.; Roy, B.;
Mukhopadhyay, B. J. Carbohydr. Chem. 2007, 26, 91.
(35) Zemplén, G.; Pascu, E. Chem. Ber. 1929, 62, 1613.
(36) Kocieꢀski, P. Protecting Groups, 3rd ed.; Georg Thieme
Verlag: Stuttgart, 2005.
References
(1) Dwek, R. A. Chem. Rev. 1996, 96, 683.
(2) Powell, M. F.; Grey, H.; Gaeta, F.; Sette, A.; Colon, S.
J. Pharm. Sci. 1992, 81, 731.
(3) Figdor, C. G.; van Kooyk, Y.; Adema, G. J. Nat. Rev.
Immunol. 2002, 2, 77.
(4) Sheng, K. C.; Pouniotis, D. S.; Wright, M. D.; Tang, C. K.;
Lazoura, E.; Pietersz, G. A.; Apostolopoulos, V.
Immunology 2006, 118, 372.
(5) Sheng, K. C.; Kalkanidis, M.; Pouniotis, D. S.; Esparon, S.;
Tang, C. K.; Apostolopoulos, V.; Pietersz, A. Eur. J.
Immunol. 2008, 38, 424.
(6) (a) Drickamer, K. Nat. Struct. Biol. 1995, 2, 437.
(b) Crocker, P. R.; Feizi, T. Curr. Opin. Struct. Biol. 1996,
6, 679. (c) Frison, N.; Taylor, M. E.; Soilleux, E.; Bousser,
R.; Mayer, M. T.; Monsigny, M.; Drickamer, K.; Roche, A.
C. J. Biol. Chem. 2003, 278, 23922.
(7) (a) Biessen, E. A. L.; Noorman, F.; van Teijlingen, M. E.;
Kuiper, J.; Barrett-Bergshoeff, M.; Bijsterbosch, M. K.;
Rijken, D. C.; van Berkel, T. J. C. J. Biol. Chem. 1996, 271,
28024. (b) Angyalosi, G.; Grandjean, C.; Lamirand, M.;
Auriault, C.; Gras-Masse, H.; Melnyk, O. Bioorg. Med.
Chem. Lett. 2002, 12, 2723.
(8) (a) Hamdaoui, B.; Dewynter, G.; Capony, F.; Montero, J. L.;
Toiron, C.; Hnach, M.; Rochefort, H. Bull. Soc. Chim. Fr.
1994, 131, 854. (b) Free, P. Org. Biomol. Chem. 2006, 4,
1817.
(9) Geijtenbeek, T. B. H.; van Vliet, S. J.; Engering, A.; ’t Hart,
B. A.; van Kooyk, Y. Annu. Rev. Immunol. 2004, 22, 33.
(10) Willment, J. A.; Brown, G. D. Trends Microbiol. 2008, 16,
27.
(11) Brimble, M. A.; Kowalczyk, R.; Harris, P. W. R.; Dunbar, P.
R.; Muir, V. J. Org. Biomol. Chem. 2008, 6, 112.
(12) Gamblin, D. P.; Scanlan, E. M.; Davis, B. G. Chem. Rev.
2009, 109, 131.
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York

2222
R. Kowalczyk et al.
PAPER
(44) Rapp, W. In Combinatorial Peptide and Nonpeptide
(37) (a) Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl.
1980, 19, 731. (b) Schmidt, R. R.; Grundler, G. Angew.
Chem., Int. Ed. Engl. 1982, 21, 781.
Libraries: A Handbook; Jung, G., Ed.; VCH: Weinheim,
1996, 425.
(38) Tvaroska, I.; Taravel, F. R. Adv. Carbohydr. Chem.
Biochem. 1995, 51, 15.
(45) Delgado, M.; Janda, K. D. Curr. Org. Chem. 2002, 6, 1031.
(46) Kempe, M.; Barany, G. J. Am. Chem. Soc. 1996, 118, 7083.
(47) Camperi, S. A.; Marani, M. M.; Iannucci, N. B.; Cote, S.;
Albericio, F.; Cascone, O. Tetrahedron Lett. 2005, 46, 1561.
(48) Watt, J. A.; Williams, S. J. Org. Biomol. Chem. 2005, 3,
1982.
(39) Coulson, D. R. Inorg. Synth. 1972, 13, 121.
(40) Dessolin, M.; Guillerez, M. G.; Thieriet, N.; Guibé, F.;
Loffet, A. Tetrahedron Lett. 1995, 36, 5741.
(41) Seitz, O.; Kunz, H. J. Org. Chem. 1997, 62, 813.
(42) Fischer, R.; Mader, O.; Jung, G.; Brock, R. Bioconjugate
Chem. 2003, 14, 653.
(49) Saksena, R.; Zhang, J.; Kováč, P. J. Carbohydr. Chem. 2002,
21, 453.
(43) Kaiser, E.; Colescot, R. I.; Bossinge, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595.
Synthesis 2009, No. 13, 2210–2222 © Thieme Stuttgart · New York