Targeted delivery of fluorescent high-mannose-type oligosaccharide cathepsin inhibitor conjugates

Article abstract of DOI:10.1002/cplu.201500004

Abstract Three fluorescent cathepsin inhibitor glycoconjugates have been designed, synthesized, and evaluated in terms of their cell internalization and cathepsin inhibitory properties. The conjugates are composed of a peptide epoxysuccinate, capable of c

Full text of DOI:10.1002/cplu.201500004

DOI: 10.1002/cplu.201500004
Full Papers
Targeted Delivery of Fluorescent High-Mannose-Type
Oligosaccharide Cathepsin Inhibitor Conjugates
Chung S. Wong, Sascha Hoogendoorn, Gijs A. van der Marel, Herman S. Overkleeft,* and
Jeroen D. C. CodØe*[a]
Three fluorescent cathepsin inhibitor glycoconjugates have
been designed, synthesized, and evaluated in terms of their
cell internalization and cathepsin inhibitory properties. The
conjugates are composed of a peptide epoxysuccinate, capa-
ble of covalent and irreversible binding to cysteine proteases,
coupled to a fluorescent BODIPY dye and functionalized with
a mono-, tri-, or heptamannoside. Mannose-receptor-depen-
dent uptake of the probes in live dendritic cells is shown to
depend on the type of carbohydrate attached. Where uptake
of the monomannoside is poor and mannose-receptor-inde-
pendent, the intracellular labeling of cathepsins by the probes
equipped with a tri- or heptamannoside conjugate appeared
concentration- and mannose-receptor-dependent.
Introduction
The targeted delivery of chemotherapeutics through the inter-
mediacy of cell surface receptors represents an attractive
means to selectively deliver cargo to target tissues or subcellu-
lar compartments. Conceptually different approaches have
been developed over the years to selectively target therapeu-
tics and diagnostics to specific cell types by receptor-mediated
uptake of the deliverables. Various ligands have been used as
a homing device, including antibodies and small synthetic mol-
ecules such as folic acid, peptides, and carbohydrates.[1] Lectins
are carbohydrate-binding receptors, which occur both as mem-
brane-bound and soluble proteins. They play a key role in
a wide variety of cellular recognition and communication pro-
cesses.[2] They are abundantly expressed on dendritic cells
(DCs) and macrophages, cells that have evolved to survey their
surroundings and detect pathogens and danger signals. Many
of the lectins found on these cells are members of the C-type
lectin family and these include the macrophage mannose re-
ceptor (MMR), Dectin-1, Dectin-2, and DC-SIGN.[3] These carbo-
hydrate-binding receptors have been exploited in various anti-
gen-targeting strategies to enable both the efficient uptake of
antigens and simultaneous stimulation of the immune cells.[4]
Peptidases play a key role in the processing of peptides and
peptide antigens and as such play a pivotal role in the com-
plex antigen presentation pathway. To probe the activity of
cathepsins in living DCs we have previously reported the adap-
tation of the broad-spectrum cathepsin inhibitor DCG-04[5] to
obtain targeted activity-based cathepsin probes. DCG-04 was
originally developed by Bogyo and co-workers, in a seminal
paper,[5a] which together with the first paper by Cravatt and
co-workers on serine hydrolase probes,[6] shaped the field of
activity-based protein profiling. Taking the natural product, the
broad-spectrum cysteine protease inhibitor E-64, as a basis,
Bogyo and co-workers appended both a biotin and—in a later
contribution—a set of different fluorophores and showed that
all these structures retain potency and (broad-spectrum) spe-
cificity against numerous mammalian cathepsin cysteine pro-
teases.[5] From these studies, which yielded activity-based
probes currently widely used by the chemical biology com-
munity, it became apparent that cathepsin cysteine proteases
tolerate a wide variety of functional groups appended to the
dipeptide epoxysuccinate core. We capitalized on this by ap-
pending, directly adjacent to a reporter fluorophore, a mannose
cluster to allow for lectin-mediated uptake of the probe (1:
UHG392; see Figure 1).[7,8] Our first-generation activity-based
probe (ABP) 1 contains an artificial mannose cluster built up
from a hexalysine oligopeptide with each lysine side chain
modified to bear a monomannoside residue.[7]
We hypothesized that the nature of the mannose ligand
may influence recognition by the cell surface lectins and con-
sequently the uptake and routing of the conjugates. For exam-
ple, it is known that the prevalent carbohydrate-binding lectins
on DCs, DC-SIGN, and the MR bind oligomannosides better
than monomannosides.[3,4] Glycan microarray studies have re-
vealed that DC-SIGN strongly binds high-mannose-type struc-
tures[9] and available crystal structures of DC-SIGN bound to
natural ligands show that a terminal branched trimannose
structure, featuring a-(1,3) and a-(1,6) mannose branches on
a core mannose residue, fits well in the carbohydrate-binding
[a] C. S. Wong,+ Dr. S. Hoogendoorn,+ Prof. Dr. G. A. van der Marel,
Prof. Dr. H. S. Overkleeft, Dr. J. D. C. CodØe
Leiden Institute of Chemistry
Leiden University, Einsteinweg 55
2333 CC Leiden (The Netherlands)
E-mail: h.s.overkleeft@chem.leidenuniv.nl
jcodee@chem.leidenuniv.nl
[+] C.-S.W. and S.H. contributed equally to this work
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cplu.201500004.
This article is part of the “Early Career Series”. To view the complete
series, visit: http://chempluschem.org/earlycareer.
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Figure 1. Structures of the DCG-04 mannose conjugate 1 previously studied
and the mannosyl DCG-04 probes 2, 3, and 4 reported here.
site of this signaling receptor.[10] The MR also binds oligoman-
nosides and a preference for the same type of branching has
been reported.[11] We therefore designed and synthesized a set
of BODIPY-DCG-04-oligomannose conjugates, bearing oligo-
mannosides that feature natural glycosidic connections and
contain the a-(1!3),a-(1!6)-branched trimannoside structure.
We here describe the assembly of three BODIPY-DCG-04-man-
nose clusters (2, 3, and 4; Figure 1), bearing either a mono-,
tri-, or heptamannoside targeting entity and their efficacy in
the labeling of cathepsins in both cell lysates and live cells.
Scheme 1. Reagents and conditions: a) i. I2, Et3SiH, CH2Cl2, reflux; ii. MeOH,
2,6-lutidine, RT; b) K2CO3, MeOH, RT; c) NaH, BnBr, DMF, 08C to RT; d) DME/
H2O (10:1), pTsOH, 82% over 4 steps; e) Cs2CO3, acetone, (CF3)C(NPh)Cl,
90%; f) 12, DCM, TfOH, activated molecular sieves, À408C to 08C, 84%;
g) NIS, AcOH, DCE/THF (1:1), 94%; h) i. Pd/C, H2, EtOAc/tBuOH/H2O (1:3:4),
ii. Pd/C, H2, H2O; i) Ac2O, pyridine, 08C to RT, 76% over 2 steps; j) propargyl
alcohol, BF3·Et2O, DCM, RT, 61%; k) MeOH, NaOMe, 508C, 68%; l) NIS, DCM/
AcOH (1:1), RT, 40%; m) 14, NIS, TfOH, activated molecular sieves, DCM,
À408C to RT, 62%; n) i. Pd/C, H2, EtOAc/MeOH/H2O (5:4:1); ii. Pd/C, H2,
MeOH/H2O (1:1); o) Ac2O, pyridine, 08C to RT, quantitative yield over 2 steps;
p) H2NNH2·AcOH, DMF, 08C, 79%; q) ClC(=NPh)CF3, Cs2CO3, acetone, quanti-
tative; r) propargyl alcohol, TfOH, DCM, activated molecular sieves, À408C
to 08C, 40%; s) i. NaOMe/MeOH; ii. 0.1m NaOH (aq.), quantitative. DME=1,2-
dimethoxyethane, DMF=N,N-dimethylformamide, DCE=1,2-dichloroethane,
DCM=dichloromethane, Tf=trifluoromethylsulfonyl, NIS=N-iodosuccini-
mide.
Results and Discussion
The three BODIPY-DCG-04 mannose conjugates 2, 3, and 4
were assembled by conjugation of the relevant propargyl man-
nosides (7, 19, and 27; Scheme 1) with azide-functionalized
BODIPY-epoxysuccinate 5 (Scheme 2). a-Propargyl monoman-
noside 7 was synthesized following a literature procedure.[12]
Oligomannosides 19 and 27 were synthesized in a convergent
manner as depicted in Scheme 1. Starting from peracetylated
mannose 6, orthoester 8 was obtained via the intermediate
formation of a mannosyl iodide, as reported by Adinolfi
et al.[13] Intramolecular substitution of the iodide gave orthoest-
er 8, which was deacetylated and subsequently benzylated to
give orthoester 10. Acidic hydrolysis of 10 then yielded hemi-
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with trisaccharide donor 14 was achieved using the NIS/TfOH
promotor couple to yield heptamer 21 as a single product in
62% yield. Hydrogenation of 21 with Pd/C and H2 in
EtOAc/MeOH/H2O (5:4:1) was followed by a second hydro-
genation in MeOH/H2O (1:1) to give the debenzylated hepta-
mer 22, which was directly subjected to global acetylation. At-
tempts to introduce the propargyl moiety onto the peracety-
lated heptamer using BF3·Et2O did not lead to the desired
product and therefore we switched to the use of a more
potent glycosylating agent. To this end, the anomeric acetyl
was chemoselectively deblocked using hydrazine acetate and
the liberated alcohol was converted into the N-phenyl trifluor-
oacetimidate. Glycosylation of propargyl alcohol with donor 25
under mild acid catalysis yielded the peracetylated heptaman-
noside 26 in 40% yield. Deacetylation under standard ZemplØn
conditions led to the partial removal of the acetyl groups, ne-
cessitating an extra saponification step with aqueous 0.1m
NaOH to provide the target heptamer 27.
The BODIPY-DCG-04-mannose conjugates were obtained
through a CuI-catalyzed Huisgen 1,3-dipolar cycloaddition[16] of
azido BODIPY-DCG-04 (5)[7,8] and the propargyl mannosides 7,
19, and 27 (Scheme 2). After HPLC purification the three target
constructs were obtained in 42% (2), 24% (3), and 32% (4)
yield, respectively.
Scheme 2. Assembly of the mannose-BODIPY-DCG-04 conjugates 2, 3, and 4
and structures of cathepsin binding probes 5 (azido-BODIPY-DCG-04), 28
(azido-DCG-04), 29 (green BODIPY(FL)-DCG-04), and 30 (AS44). Reagents and
conditions: a) sodium ascorbate, CuSO4, DMF/H2O (1:1), Man1-BODIPY-DCG-
04 42%, Man3-BODIPY-DCG-04 24%, Man7-BODIPY-DCG-04 32%.
To investigate labeling of cathepsins by activity-based
probes 2, 3, and 4 we first evaluated their activity in cell ly-
sates. To this end, mouse liver lysate was incubated with in-
creasing concentrations of Man1-BODIPY-DCG-04 (2), Man3-
BODIPY-DCG-04 (3), or Man7-BODIPY-DCG-04 (4), after which
the proteins in the lysates were resolved on SDS-PAGE (Fig-
ure 2A). All three mannosyl DCG-04 probes 2, 3, 4 label
cathepsins in a concentration-dependent manner, as is evident
from Figure 2A. A small difference in gel-shift is apparent for
the three constructs and correlates to their molecular weights.
A decrease in binding capacity was observed with increasing
cluster size, suggesting that the steric bulk of the heptaman-
nosyl cluster retards binding and cathepsin inactivation. The
diminished binding efficacy of the larger mannosyl clusters, to-
gether with the difference in the gel-shift of the labeled pro-
teins indicates that mannosidases present in the cell lysate do
not (effectively) trim the probes when bound to the cathepsins
or when unbound in the cell extract. Incubation of immature
mouse dendritic cell (DC) lysate with the probes showed a simi-
lar concentration-dependent binding of cathepsin proteins
(Figure 2B). In line with our previous findings, changing the
pH of the buffer from pH 5.5 (the optimal pH for most cathep-
sin activity)[7] to pH 7 led to abrogation of cathepsin binding,
showing that active enzymes are required for labeling. Next,
a set of competition experiments was performed. The lysates
were pre-incubated with different DCG-04 competitors: azido-
DCG-04 28, green fluorescent probe BODIPY(FL)-DCG-04 29,[7]
and AS44 30[8] (see Scheme 2 for the structures of the compet-
itors), followed by incubation with the probes. As seen in Fig-
ure 2B labeling of the cathepsins with the red mannosyl
BODIPY-DCG-04 conjugates was effectively prevented, leading
to either disappearance of the fluorescent bands in the com-
petition experiment with nonfluorescent azido-DCG-04 and
acetal 11[14] in 82% yield over four steps. Treatment of this
lactol with (N-phenyl)trifluoroacetamidoyl chloride in the pres-
ence of Cs2CO3 afforded (N-phenyl)trifluoroimidate donor 12 in
90% yield. The construction of key trisaccharide 14, which was
used as a precursor for both the propargyl trimannoside 19
and as a building block to construct heptasaccharide 27, was
accomplished by a double glycosylation of diol 13[15] using
donor 12 and a catalytic amount of TfOH. Next, trimer 14 was
converted into the corresponding anomeric acetate 15 using
NIS and AcOH. Removal of all benzyl groups from this trimer
required a two-step sequence. The fully protected trisaccharide
was first treated with Pd/C and H2 in a mixture of EtOAc/
tBuOH/H2O (1:3:4) after which the solvent was replaced with
water for the second reduction event to effect removal of all
benzyl groups. Peracetylation of the crude trimer yielded 17 in
76% yield over the two steps. Propargyl alcohol was then con-
densed with the trimannosyl acetate under the agency of
BF3·Et2O to provide the fully protected trimer 18. Global deace-
tylation under ZemplØn conditions yielded the propargyl tri-
mannoside 19.
The heptasaccharide 21 was assembled using trisaccharide
donor 14 and acceptor 20. The latter building block was ob-
tained by treatment of thiomannoside 13 with NIS and AcOH.
This led to the formation of the a-acetate 20 in 40% yield,
along with its b-anomeric counterpart (37%) and a minor by-
product that was characterized as 1,6-anhydro-2,4-di-O-benzyl-
b-d-mannose (11%). Double condensation of diol acceptor 20
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Figure 3. Uptake and cathepsin binding of the probes in live dendritic cells.
A) DCs were treated with varying concentrations of 2, 3, or 4 (2 h, 378C) or
pre-incubated (1 h, 378C) with azido-DCG-04 (20 mm) or mannan
(3 mgmLÀ1), followed by addition of 2, 3, or 4 (1 mm, 2 h, 378C), washed
with PBS, lysed, and resolved on 12.5% SDS-PAGE. In-gel fluorescence of
BODIPY (Cy3) and total protein stain (CBB) are shown. B) Representative con-
focal microscope images of DCs treated with 1 mm of probes 2, 3, or 4 (left
panels) or with mannan (3 mgmLÀ1, right panels) for 1 h, followed by treat-
ment with the probes. After treatment, cells were washed with PBS, fixed
with 4% formaldehyde, nuclei stained with Draq5, and imaged using the
Cy3 (lex =532 nm) settings for BODIPY (red) and Cy5 (lex =635 nm) settings
for Draq5 as a nuclear stain (blue).
Figure 2. Cathepsin labeling experiments in mouse liver and dendritic cell
lysate. Mouse liver lysate (10 mg total protein, (A)) or immature mouse den-
dritic cell (DC) lysate (8 mg total protein, (B)) was incubated (1 h, 378C) with
an increasing concentration of probe 2, 3, or 4 at pH 5.5 or 1 mm at pH 7. Al-
ternatively, lysates were incubated (1 h, 378C) with azido-DCG-04 (1 or
10 mm), AS44 (10 mm), or BODIPY(FL)-DCG-04 (1 or 10 mm), before treatment
with probe 2, 3, or 4 (1 mm, 1 h, 378C). Proteins were resolved on 12.5%
SDS-PAGE, followed by fluorescence scanning (Cy2 (green): BODIPY(FL),
Cy3 (red): BODIPY(TMR)) and total protein staining with Coomassie brilliant
blue (CBB). M: dual-color protein molecular weight marker.
BODIPY-DCG-04 4 are taken up through the intermediacy of
a carbohydrate binding receptor, where Man1-BODIPY-DCG-04
2 can be internalized (at least in part) through a receptor-inde-
pendent pathway. Receptor-mediated internalization is clearly
more efficient. Although it has previously been reported that
the mannose receptor can bind monomannosides,[3b] in the
case at hand it appears that this is not enough for effective in-
ternalization of the conjugate. With respect to our first-genera-
tion probe (1) it appears that DCG-04 labeling with the triman-
noside probe is equally efficient. We have, however observed
a difference in processing of the probes. Where probe 1 seems
to be processed by mannosidases in living cells (as judged
from the minimal difference in gel-shift for the labeled cate-
psins, indicating only a small shift in molecular size), the cur-
rent probes are more resistant to the endo/lysosomal action of
mannosidases.
AS44, or the appearance of green fluorescent bands in the ex-
periment with the green DCG-04 probe 29.
Next, the probes were tested for uptake and binding of
cathepsins in living DCs (Figure 3). In line with the results ob-
tained with the cell lysates, a concentration-dependent label-
ing pattern was observed (Figure 3A, left panel). The most effi-
cient and selective labeling was achieved with the trimannosyl
probe 3, where the monomannosyl compound 2 showed the
highest background fluorescence. Also in these experiments
the heptamannoside probe 4 labeled the target cathepsins
somewhat less efficiently than its trimannoside counterpart 3.
Competition experiments with the nonfluorescent cell-permea-
ble azido-DCG-04 probe 28 indicated that, also in living DCs,
active cathepsins are labeled by the probes. To test whether
uptake of the probes was carbohydrate receptor mediated the
DCs were incubated with mannan (3 mgmLÀ1) prior to expo-
sure to the probes. In doing so, labeling by the tri- and hepta-
mannosyl probes was effectively blocked, showing that uptake
of these ABPs is receptor-dependent. The receptor-mediated
uptake and labeling was confirmed by confocal microscopy.
Figure 3B shows the clear uptake of Man3-BODIPY-DCG-04 3
and Man7-BODIPY-DCG-04 4 in DCs but little uptake of Man1-
BODIPY-DCG-04 2 (Figure 3B, left panels). Pre-incubation of the
cells with mannan prevented uptake of probes 3 and 4. Com-
bined, the results show that Man3-BODIPY-DCG-04 3 and Man7-
Conclusion
We have reported on the assembly of three fluorescent ca-
thepsin probes functionalized with different mannosides to in-
vestigate the role of these carbohydrate appendages on inhibi-
tion efficacy and internalization efficiency. The size of the man-
nose oligosaccharides proved to influence the amount of in-
hibition, with the largest heptamannoside showing least effec-
tive cathepsin labeling in cell lysates at low inhibitor
concentrations. For effective uptake in live cells it is shown
that the tri- and heptamannoside outcompete the monoman-
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pyranosyl)-1-thio-a-d-mannopyranoside (14): Trifluoro imidate
donor 12 (19.91 g, 30 mmol) and acceptor 13 (4.53 g, 10 mmol)
were dissolved in DCM (200 mL) and stirred over activated molecu-
lar sieves (3 Š) at RT for 30 min. The solution was cooled to À408C
and to the cooled solution was added TfOH (0.18 mL, 2 mmol) and
the reaction mixture was gradually allowed to warm to 08C. At
08C the reaction was quenched with triethylamine and the mixture
was filtered over celite and rinsed with DCM. The organic phase
was washed with H2O and the aqueous phase was extracted with
DCM (4”). The combined organic layers were dried over MgSO4, fil-
tered, and concentrated in vacuo. Purification by column chroma-
tography yielded trimer 14 as a colorless oil (11.7 g, 8.4 mmol, 84%
yield). Spectroscopic data were in accordance with literature.[19]
nosyl probe. The live cell experiments corroborated the more
effective inhibition of the smaller trisaccharide inhibitors over
its larger heptasaccharide counterpart.
Experimental Section
2-O-Acetyl-3,4,6-tri-O-benzyl-d-mannopyranoside (11): To a solu-
tion of peracetylated mannose 6 (114.5 g, 293 mmol) in DCM
(750 mL) was added iodine (104.2 g, 411 mmol) and triethylsilane
(66 mL, 410 mmol). The reaction mixture was heated to reflux.
After 4 h TLC showed complete conversion of the starting material
and mixture was cooled to RT. To the reaction mixture was added
2,6-lutidine (140 mL), MeOH (71 mL) and the reaction mixture was
stirred overnight at RT. The reaction mixture was concentrated in
vacuo, dissolved in EtOAc, washed with water (1”), 10% Na2S2O3
(aq.) (2”), H2O (3”), and brine (2”), dried over Mg2SO4, filtered,
and concentrated in vacuo. The residue was dissolved in MeOH
(500 mL), and to the solution was added K2CO3 (6.6 g, 48 mmol)
and stirred for 4 h at RT. The reaction mixture was concentrated in
vacuo and co-evaporated with toluene (3”). The product was dis-
solved in DMF (1.0 L) and to the solution was added BnBr (158 mL,
1.32 mol). The reaction mixture was cooled to 08C and to the
cooled solution was added NaH (60% mmÀ1) (31.7 g, 1.32 mol) in
small portions over 6 h. The reaction mixture was gradually
warmed to RT and was stirred overnight at RT. The reaction mixture
was cooled to 08C and quenched with MeOH. The solvent was re-
moved in vacuo and the concentrate was dissolved in Et2O,
washed with H2O (4”) and brine (2”), dried over MgSO4, filtered,
and concentrated in vacuo. The crude was dissolved in DME/H2O
(10:1) (1.5 L) and the solution was cooled to 08C. To the cooled so-
lution was added pTsOH (75 mmol, 14.25 g), after 3 h at 08C the re-
action was quenched with sat. NaHCO3 (aq.). Brine was added and
the organic layer was separated. The product was extracted with
DCM (3”) and the combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo. Purification by column
chromatography yielded mannose 11 as a colorless oil (118.4 g,
240 mmol, 82% yield over 4 steps as an a/b mixture 10:1). Spectro-
scopic data were in accordance with literature.[18]
Acetyl
2,4-O-dibenzyl-3-O-(2-O-acetyl-3,4,6-O-tribenzyl-a-d-
mannopyranosyl)-6-O-(2-O-acetyl-3,4,6-O-tribenzyl-a-d-manno-
pyranosyl)-a/b-d-mannopyranoside (15): To a suspension of NIS
(1.25 g, 5.55 mmol) in DCE/THF (1:1) (27 mL) was added acetic acid
(21.2 mL, 370 mmol). To the NIS mixture was added a solution of
trimer 14 (5.17 g, 3.7 mmol) in DCE/THF (1:1) (5 mL) and the reac-
tion mixture was stirred overnight at RT. The reaction mixture was
diluted with EtOAc, washed with 2” 10% Na2S2O3 (aq.) (1”), H2O
(1”), sat. aq. NaHCO3 (3”), H2O (3”), and brine (2”), dried over
MgSO4, filtered, and concentrated in vacuo. Purification by column
chromatography yielded trimer 15 as a colorless oil (4.67 g,
3.5 mmol, 94%). [a]d22 = +50.08 (c=1.0, DCM). FTIR (neat): n˜ =
975.53, 1026.80, 1048.27, 1090.94, 1231.12, 1368.81, 1453.91,
1
1496.72, 1743.42, 2870.34, 3031.12 cmÀ1. H NMR (400 MHz, CDCl3)
(a/b mixture, 1:0.4): d=7.41–7.08 (m, 56H), 6.18 (d, J=2.0 Hz, 1H),
5.59 (s, 0.4H), 5.50 (ddd, J=8.3, 3.3, 1.9 Hz, 2.8H), 5.20 (d, J=
1.8 Hz, 1.4H), 4.96 (d, J=1.9 Hz, 1H), 4.94 (d, J=1.8 Hz, 0.4H), 4.89
(s, 1H), 4.86 (d, J=2.8 Hz, 1.4H), 4.85–4.81 (m, 1.4H), 4.79–4.71 (m,
1.4H), 4.72 (d, J=1.7 Hz, 1H), 4.70 (d, J=2.0 Hz, 0.4H), 4.67 (s,
0.4H), 4.66–4.63 (m, 2.4H), 4.62–4.60 (m, J=1.9 Hz, 2H), 4.57 (d, J=
2.3 Hz, 0.4H), 4.54 (s, 0.4H), 4.51 (s, 0.4H), 4.50–4.38 (m, 10H), 4.12
(dd, J=9.6, 3.1 Hz, 1H), 4.02 (ddd, J=8.1, 5.2, 2.7 Hz, 2H), 4.00–
3.83 (m, 8.4H), 3.84–3.78 (m, 1H), 3.78–3.65 (m, 6H), 3.64 (s, 0.4H),
3.62 (t, J=2.0 Hz, 0.8H), 3.59 (s, 0.4H), 3.57 (s, 0.4H), 3.51 (ddd, J=
8.8, 4.4, 2.0 Hz, 0.4H), 2.35 (s, 1H), 2.15 (s, 3H), 2.14 (s, 1.2H), 2.09
(s, 3H), 2.07 (s, 1.2H), 1.99 (s, 1.2H), 1.95 ppm (s, 3H). 13C NMR
(101 MHz, CDCl3): d=170.3, 170.3, 170.2, 170.1, 169.0, 168.9, 138.6,
138.6, 138.5, 138.3, 138.1, 137.9, 137.8, 137.8, 137.6, 129.1, 128.6,
128.5, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2, 128.1, 127.9,
127.9, 127.9, 127.9, 127.8, 127.7, 127.7, 127.7, 127.6, 127.6, 125.4,
100.1, 99.7, 98.5, 98.3, 92.8, 90.8, 79.7, 78.1, 78.0, 77.7, 76.5, 76.3,
75.3, 75.2, 75.1, 75.0, 74.7, 74.4, 74.3, 74.2, 74.1, 74.1, 74.0, 73.6,
73.5, 73.5, 73.4, 72.4, 72.2, 72.1, 72.0, 72.0, 71.7, 71.6, 71.5, 71.3,
69.2, 69.0, 68.8, 68.7, 68.6, 68.6, 68.4, 68.3, 66.4, 66.3, 21.3, 21.1,
21.0 ppm. HRMS: [M+H]+ calcd for C80H87O19 1351.58361, found
1351.58399.
2-O-Acetyl-3,4,6-tri-O-benzyl-1-O-(N-phenyltrifluoroacetimidoyl)-
a/b-d-mannopyranoside (12): To a solution of mannose 11 (24.6 g,
50 mmol) in acetone (200 mL) was added N-(p-anisyl)-2,2,2-trifluor-
oacetimidoyl chloride (10.4 mL, 68.8 mmol) and the reaction mix-
ture was cooled to 08C. To the cooled solution was added Cs2CO3
(20.7 g, 55 mmol) and the reaction mixture was allowed to warm
to RT. After 6 h the mixture was filtered over celite and the filtrate
was concentrated in vacuo. Purification by column chromatogra-
phy yielded imidate donor 12 as a yellow oil (29.8 g, 44.9 mmol,
90% yield as an 25:1 a/b mixture). [a]d22 +26.48 (c=1.0, DCM).
FTIR: (neat): n˜ =111.48, 1162.48, 1207.57, 1310.77, 1364.72, 1453.91,
1489.32, 1597.42, 1716.21, 1749.15, 2867.10, 3031.71 cmÀ1. Spectro-
scopic data are reported for the major (a) isomer: 1H NMR
(400 MHz, CDCl3, T=328 K): d=7.38–7.01 (m, 18H), 6.79 (d, J=
7.4 Hz, 2H), 6.20 (s, 1H), 5.47 (dd, J=3.2, 2.0 Hz, 1H), 4.87 (d, J=
10.9 Hz, 1H), 4.72 (d, J=11.2 Hz, 1H), 4.64 (d, J=12.0 Hz, 1H), 4.57
(dd, J=11.3 Hz, 2H), 4.51 (d, J=12.0 Hz, 1H), 4.01 (ddd, J=8.3, 3.1,
1.3 Hz, 1H), 3.99–3.89 (m, 2H), 3.78 (dd, J=7.8, 3.7 Hz, 1H), 3.72
(dd, J=11.2, 1.7 Hz, 1H), 2.11 ppm (s, 3H). 13C NMR (100 MHz,
CDCl3): d=169.9, 143.4, 138.5, 138.4, 137.8, 128.9, 128.5, 128.5,
128.3, 128.0, 127.8, 127.8, 127.7, 124.6, 119.6, 77.7, 75.4, 74.5, 74.0,
73.6, 72.4, 68.9, 67.7, 20.9 ppm.
Acetyl 2,4-O-di-acetyl-3-O-(2,3,4,6-O-tetra-acetyl-a-d-mannopyr-
anosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-mannopyranosyl)-a/b-d-
mannopyranoside (17): Trimer 15 (3.38 g, 2.5 mmol) was dissolved
in an EtOAc/tBuOH/H2O (1:3:4) mixture (50 mL) and the solution
was purged with argon. To the solution was added cat. Pd/C (10%)
and the mixture was stirred at RT under H2 (g) atmosphere. After
TLC showed complete conversion to a single spot, the Pd/C re-
moved by filtration through a pad of celite which was rinsed with
MeOH, and the filtrate was concentrated in vacuo. Next, the crude
was taken up in H2O (50 mL) and purged with argon. To the solu-
tion was added cat. Pd/C (10%) and the mixture was stirred at RT
under H2 (g) atmosphere overnight. The mixture was filtered
through a pad of celite which was rinsed with H2O, and the filtrate
Phenyl
2,4-O-dibenzyl-3-O-(2-O-acetyl-3,4,6-O-tribenzyl-a-d-
mannopyranosyl)-6-O-(2-O-acetyl-3,4,6-O-tribenzyl-a-d-manno-
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Full Papers
was concentrated in vacuo. The 1H NMR spectrum of the crude
showed complete removal of aromatic signals and the crude was
co-evaporated with 1,4-dioxane (3”). The crude was dissolved in
pyridine (25 mL) and the solution was cooled to 08C. To the cooled
solution was added acetic anhydride (2.5 mL) and the reaction mix-
ture was gradually allowed to warm to RT. After complete conver-
sion, the reaction mixture was cooled to 08C, quenched with
MeOH, and concentrated in vacuo. The crude product was dis-
solved in EtOAc and washed with 1m HCl (aq.) (1”), sat. NaHCO3
(aq.) (1”), H2O (3”), and brine (2”), dried over MgSO4, filtered, and
concentrated in vacuo. Purification by column chromatography
yielded per-O-acetylated trimer 17 as a colorless oil (1.81 g,
1.9 mmol, 76% yield). [a]d22 = +40.48 (c=1.0, DCM). FTIR (neat):
n˜ =975.27, 1039.52, 1139.46, 1212.30, 1368.79, 1433.73, 1741.84,
MeOH). FTIR (neat): n˜ =981.81, 1042.80, 1131.53, 1363.00, 2490.41,
1
2929.08, 3285.13 cmÀ1. H NMR (400 MHz, MeOD): d=5.06 (s, 1H),
4.91 (d, J=1.8 Hz, 1H), 4.84 (d, J=1.8 Hz, 1H), 4.26 (t, J=2.3 Hz,
2H), 4.04 (dd, J=3.1, 1.8 Hz, 1H), 3.98 (dd, J=3.3, 1.7 Hz, 1H), 3.94
(dd, J=11.1, 5.2 Hz, 1H), 3.89–3.84 (m, 3H), 3.84–3.81 (m, 2H),
3.81–3.78 (m, 2H), 3.78–3.65 (m, 5H), 3.65–3.58 (m, 3H), 2.88 ppm
(t, J=2.5 Hz, 1H). 13C NMR (100 MHz, MeOD): d=104.0, 101.5,
100.2, 80.7, 79.9, 76.2, 74.9, 74.4, 73.8, 72.6, 72.4, 72.1, 72.0, 71.2,
68.7, 68.5, 67.3, 67.1, 62.8, 55.0 ppm. HRMS: [M+H]+ calcd for
C21H35O16 543.19196, found 543.19224.
Acetyl 2,4-O-benzyl-a-d-mannopyranoside (20): To a 08C cooled
solution of NIS (0.247 g, 1.10 mmol) in DCM/AcOH (1:1) (20 mL)
was added dropwise a 0.1m solution of thiomannose 13 (10 mL,
1.0 mmol) in DCM. The reaction mixture was stirred at 08C for 1 h
and allowed to warm to RT. After complete consumption of the
starting material the reaction was quenched with 10% Na2S2O3
(aq.) and the product was extracted with EtOAc (4”). The com-
bined organic phases were washed with H2O (2”), sat. NaHCO3
(aq.) (3”), H2O (3”), and brine (2”), dried over MgSO4, filtered, and
concentrated in vacuo. Purification by column chromatography
yielded a-O-acetyl mannose 20 as a colorless amorphous solid
(0.148 g, 0.37 mmol, 40% yield). [a]d22 = +14.68 (c=1.0, DCM). FTIR
(neat): n˜ =1026.60, 1071.89, 1239.98, 1366.52, 1454.28, 1496.96,
2925.08 cmÀ1 1H NMR (300 MHz, CDCl3) (a/b mixture, 1:0.25): d=
.
6.05 (d, J=1.9 Hz, 1H), 5.81 (s, 0.25H), 5.48 (d, J=3.2 Hz, 0.25H),
5.27 (dd, J=8.8, 2.7 Hz, 4.4H), 5.04 (d, J=2.6 Hz, 2H), 5.02 (s,
0.4H), 4.80 (s, 1.2H), 4.35–4.20 (m, 3.4H), 4.19–4.00 (m, 7.2H), 3.90
(dq, J=10.0, 3.1 Hz, 1H), 3.75 (dd, J=11.0, 5.5 Hz, 1H), 3.57 (dd, J=
10.9, 3.2 Hz, 1H), 2.27 (s, 0.8H), 2.25 (s, 2.6H), 2.18 (s, 4H), 2.16 (d,
J=2.1 Hz, 12H), 2.11 (s, 7H), 2.07 (s, 5.5H), 2.05 (s, 3H), 2.00 (s,
4H), 1.99 ppm (s, 4.5H). 13C NMR (75 MHz, CDCl3): d=170.7, 170.6,
170.3, 170.1, 170.0, 170.0, 169.9, 169.8, 169.6, 99.2, 97.6, 90.4, 74.7,
71.5, 69.9, 69.6, 69.5, 69.3, 69.1, 68.5, 68.2, 67.9, 66.9, 65.8, 62.4,
62.3, 60.4, 20.9, 20.9, 20.8, 20.7, 20.7 ppm. HRMS: [M+H]+ calcd
for C40H55O27 967.29252, found 967.29269.
1
1720.48, 2930.75, 3031.24, 3420.07 cmÀ1. H NMR (400 MHz, CDCl3):
d=7.40–7.25 (m, 10H), 6.20 (d, J=1.8 Hz, 1H), 4.90 (d, J=11.1 Hz,
1H), 4.76 (d, J=11.7 Hz, 1H), 4.66 (d, J=11.0 Hz, 1H), 4.59 (d, J=
11.7 Hz, 1H), 3.99 (d, J=7.0 Hz, 1H), 3.83 (dd, J=12.1, 2.6 Hz, 1H),
3.80–3.73 (m, 2H), 3.72–3.65 (m, 2H), 2.52 (s, 1H), 2.32 (s, 1H),
2.05 ppm (s, 3H). 13C NMR (100 MHz, CDCl3): d=169.3, 138.2,
137.25, 128.7, 128.7, 128.3, 128.1, 128.1, 128.0, 91.0, 77.0, 75.6, 75.2,
74.0, 73.1, 71.4, 61.8, 21.0 ppm. HRMS: [M+H]+ calcd for C22H27O7
403.17518, found 403.17527.
Propargyl 2,4-O-diacetyl-3-O-(2,3,4,6-O-tetraacetyl-a-d-manno-
pyranosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-mannopyranosyl)-a-d-
mannopyranoside (18): To a solution of per-O-acetylated trimer 17
(29 mg, 30 mmol) in DCM ( 300 mL) was added a 0.6m propargyl al-
cohol solution (150 mL, 90 mmol) in DCM and a 0.3m BF3·Et2O solu-
tion (150 mL, 45 mmol) in DCM. The mixture was heated to 508C for
6 h after which the reaction mixture was cooled to RT, diluted with
EtOAc, and quenched with sat. NaHCO3 (aq.). EtOAc was added
until the organic phase was transferred to the top phase. The or-
ganic phase was washed with sat. NaHCO3 (2”), H2O (3”), and
brine (2”), dried over MgSO4, filtered, and concentrated in vacuo.
Purification by column chromatography yielded per-O-acetylated
propargyl trimer 18 as a white milky oil (17.6 mg, 18.3 mmol, 61%
yield). [a]d22 = +80.48 (c=1.0, DCM). FTIR (neat): n˜ =978.00,
Acetyl 2,4-O-dibenzyl-3-O-(2,4-O-dibenzyl-3-O-(2-O-acetyl-3,4,6-
O-tribenzyl-a-d-mannopyranosyl)-6-O-(2-O-acetyl-3,4,6-O-triben-
zyl-a-d-mannopyranosyl)-a-d-mannopyranosyl)-6-O-(2,4-O-di-
benzyl-3-O-(2-O-acetyl-3,4,6-O-tribenzyl-a-d-mannopyranosyl)-6-
O-(2-O-acetyl-3,4,6-O-tribenzyl-a-d-mannopyranosyl)-a-d-
mannopyranosyl)-a-d-mannopyranoside (21): Trimer donor 14
(4.20 g, 3.0 mmol) and acceptor 20a (0.402 g, 1.0 mmol) were dis-
solved in DCM (20 mL) and the solution was stirred over activated
molecular sieves (3 Š) at RT for 30 min. NIS (0.74 g, 3.3 mmol) was
added and the solution was stirred at RT. After 15 min the reaction
mixture was cooled to À408C and TfOH (0.3 mmol, 27 mL) was
added to the mixture. The reaction mixture was gradually warmed
to RT and quenched with Et3N. The mixture was filtered over celite
and diluted with DCM. The organic phase was washed with 10%
Na2S2O3 (aq.) and the aqueous phase was extracted with DCM
(5”). The combined organic layers were washed with H2O (1”) and
brine (1”), dried over MgSO4, filtered, and concentrated in vacuo.
Purification by size exclusion chromatography (DCM/MeOH, 1:1)
1038.64, 1136.67, 1214.43, 1368.90, 1433.77, 1741.73, 2926.85 cmÀ1
.
1H NMR (400 MHz, CDCl3): d=5.35–5.17 (m, 7H), 5.04–4.98 (m, 3H),
4.82 (d, J=1.8 Hz, 1H), 4.29–4.25 (m, 3H), 4.24 (s, 1H), 4.21 (dd, J=
9.9, 3.5 Hz, 1H), 4.16 (t, J=2.6 Hz, 1H), 4.13 (d, J=2.4 Hz, 1H), 4.11
(d, J=1.9 Hz, 1H), 4.10–4.06 (m, 1H), 3.88 (ddd, J=9.7, 6.6, 2.5 Hz,
1H), 3.78 (dd, J=10.8, 6.6 Hz, 1H), 3.53 (dd, J=10.8, 2.5 Hz, 1H),
2.50 (t, J=2.4 Hz, 1H), 2.23 (s, 3H), 2.16 (s, 3H), 2.14–2.13 (m, 9H),
2.12 (s, 3H), 2.06 (s, 3H), 2.05 (d, J=1.1 Hz, 3H), 1.99 (s, 3H),
1.98 ppm (s, 3H). 13C NMR (100 MHz, CDCl3): d=170.9, 170.8, 170.6,
170.3, 170.2, 170.2, 170.1, 169.9, 169.74, 99.0, 97.4, 96.0, 78.1, 75.7,
74.1, 70.8, 70.2, 70.1, 69.6, 69.5, 69.2, 68.8, 68.4, 68.4, 67.0, 66.1,
66.1, 62.6, 62.5, 54.8, 21.0, 21.0, 20.9, 20.9, 20.9, 20.8, 20.8 ppm.
HRMS: [M+H]+ calcd for C41H55O26 963.29761, found 963.29723.
yielded benzylated heptamer 21 as
a colorless oil (1.84 g,
0.62 mmol, 62% yield). [a]d22 = +43.6 (c=1.0, DCM). FTIR (neat):
n˜ =977.68, 1026.77, 1051.75, 1232.44, 1368.31, 1453.93, 1496.62,
Propargyl 3-O-(a-d-mannopyranosyl)-6-O-(a-d-mannopyranosyl)-
1742.78, 2868.56, 3032.00 cmÀ1 1H NMR (400 MHz, CDCl3): d=
.
a-d-mannopyranoside (19): To
a
solution of per-O-acetylated
7.36–7.07 (m, 90H), 6.17 (d, J=1.8 Hz, 1H), 5.54–5.46 (m, 4H), 5.21
(s, 3H), 4.97 (d, J=1.8 Hz, 1H), 4.93 (s, 1H), 4.90 (s, 1H), 4.84 (dt,
J=10.5, 5.2 Hz, 5H), 4.75–4.69 (m, 3H), 4.64 (d, J=3.8 Hz, 1H),
4.63–4.46 (m, 12H), 4.46–4.35 (m, 14H), 4.34 (d, J=2.2 Hz, 2H),
4.24 (d, J=12.1 Hz, 1H), 4.15 (ddd, J=18.1, 9.2, 3.0 Hz, 2H), 4.05
(dd, J=9.2, 3.2 Hz, 2H), 3.95 (d, J=8.9 Hz, 2H), 3.93–3.82 (m, 10H),
3.83–3.71 (m, 3H), 3.71–3.46 (m, 15H), 3.39 (dd, J=10.8, 5.9 Hz,
2H), 2.12 (s, 6H), 2.06 (s, 3H), 2.04 (s, 3H), 2.02 ppm (s, 3H).
propargyl trimer 18 (18.3 mg, 17.6 mmol) in MeOH (370 mL) was
added a 5 mm NaOMe solution (370 mL, m1.83 mmol) in MeOH.
After complete conversion the reaction was quenched with Amber-
lite IR-120 H+ (pHꢀ7). The solids were filtered and the filtrate was
concentrated in vacuo. Purification by column chromatography fol-
lowed by lyophilization yielded propargyl trimer 19 as a white
powder (6.2 mg, 11.3 mmol, 61% yield). [a]d22 = +114.08 (c=1.0,
ChemPlusChem 2015, 80, 928 – 937
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Full Papers
13C NMR (100 MHz, CDCl3): d=170.3, 170.2, 170.1, 170.1, 169.0,
138.8, 138.7, 138.6, 138.5, 138.3, 138.3, 138.2, 138.2, 138.2, 138.0,
138.0, 137.9, 137.9, 137.9, 137.8, 137.2, 128.7, 128.6, 128.6, 128.5,
128.4, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.0,
128.0, 127.9, 127.9, 127.8, 127.8, 127.8, 127.8, 127.7, 127.7, 127.6,
127.6, 127.6, 127.5, 127.5, 127.4, 127.4, 127.2, 127.1, 99.7, 98.5, 98.3,
97.2, 90.7, 78.3, 78.2, 78.0, 77.5, 77.2, 76.8, 76.4, 75.2, 75.1, 74.9,
74.9, 74.6, 74.5, 74.2, 74.1, 74.1, 73.8, 73.45, 73.4, 73.4, 72.3, 72.1,
72.0, 72.0, 71.9, 71.8, 71.8, 71.6, 71.6, 71.3, 71.0, 68.9, 68.8, 68.8,
68.6, 68.3, 68.2, 66.3, 21.2, 21.2, 21.1, 21.1, 21.1 ppm. MALDI: [M+
H]+ calcd for C178H191O41 2985.28889, found 2985.28898
(0.498 g, 0.235 mmol) in DMF (2.8 mL) was added hydrazine ace-
tate (23.3 mg, 0.259 mmol). The reaction mixture was stirred at 08C
for 1 h and 30 min at RT. After TLC showed complete conversion
the reaction was quenched with acetone and the reaction mixture
was concentrated in vacuo. The crude was dissolved in Et2O and
the organic phase was washed with brine (3”), dried over MgSO4,
filtered, and concentrated in vacuo. Purification by column chro-
matography yielded 1-OH-acetylated heptamer 24 as a white
amorphous solid (0.384 g, 0.185 mmol, 79% yield). FTIR (neat): n˜ =
978.58, 1038.62, 1081.66, 1137.06, 1215.09, 1369.26, 1433.38,
1
1741.35, 2926.28 cmÀ1. H NMR (400 MHz, CDCl3): d=5.37 (dd, J=
3.4, 1.6 Hz, 1H), 5.33–5.24 (m, 8H), 5.21 (dd, J=10.4, 3.2 Hz, 2H),
5.17 (d, J=3.0 Hz, 2H), 5.11 (t, J=10.1 Hz, 1H), 5.07–5.02 (m, 3H),
4.99 (dd, J=4.8, 1.8 Hz, 2H), 4.95 (s, 1H), 4.86–4.82 (m, 3H), 4.76
(d, J=5.0 Hz, 1H), 4.37–4.22 (m, 4H), 4.18 (dd, J=9.8, 3.4 Hz, 1H),
4.15–3.96 (m, 12H), 3.94 (ddd, J=9.7, 5.0, 2.2 Hz, 1H), 3.86 (ddd,
J=10.2, 6.0, 2.5 Hz, 1H), 3.76 (tt, J=7.6, 2.7 Hz, 3H), 3.55 (dd, J=
11.4, 3.2 Hz, 1H), 3.51 (dd, J=11.4, 3.2 Hz, 1H), 3.47 (d, J=9.5 Hz,
1H), 2.21 (s, 3H), 2.18 (s, 3H), 2.17–2.13 (m, 24H), 2.13–2.11 (m,
12H), 2.06 (s, 3H), 2.05–2.03 (m, 9H), 2.00–1.97 ppm (m, 12H).
13C NMR (100 MHz, CDCl3): d=171.1, 170.9, 170.8, 170.8, 170.7,
170.5, 170.5, 170.5, 170.4, 170.3, 170.2, 170.2, 170.1, 170.1, 167.0,
169.9, 169.9, 169.8, 169.7, 169.7, 99.5, 99.2, 99.0, 97.9, 97.6, 97.4,
91.4, 77.5, 77.2, 76.8, 75.3, 75.0, 74.5, 72.7, 70.9, 70.8, 70.1, 70.0,
69.9, 69.7, 69.6, 69.6, 69.5, 69.4, 69.3, 69.2, 68.6, 68.5, 68.5, 68.4,
68.2, 68.1, 67.7, 67.3, 66.8, 66.7, 66.3, 66.0, 65.7, 62.4, 62.3, 62.2,
62.1, 21.2, 21.1, 21.0, 21.0, 20.9, 20.8, 20.8, 20.7 ppm. HRMS: [M+
H]+ calcd for C86H117O58 2077.62003, found 2077.62039.
Acetyl 2,4-O-diacetyl-3-O-(2,4-O-diacetyl-3-O-(2,3,4,6-O-tetraace-
tyl-a-d-mannopyranosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-manno-
pyranosyl)-a-d-mannopyranosyl)-6-O-(2,4-O-diacetyl-3-O-
(2,3,4,6-O-tetraacetyl-a-d-mannopyranosyl)-6-O-(2,3,4,6-O-tetra-
acetyl-a-d-mannopyranosyl)-a-d-mannopyranosyl)-a-d-manno-
pyranoside (23): Benzylated heptamer 21 (896 mg, 0.30 mmol)
was dissolved in EtOAc/MeOH/H2O (5:4:1) (6 mL) and the solution
was purged with argon. To the solution was added cat. Pd/C (10%)
and the mixture was stirred overnight at RT under H2 (g) atmos-
phere. Pd/C was removed by filtration through a pad of celite,
which was rinsed with methanol, and the filtrate was concentrated
1
in vacuo. The H NMR spectrum of the crude showed the presence
of aromatic signals. The crude was taken up in MeOH/H2O (1:1)
(6 mL) and purged with argon. To the solution was added cat. Pd/
C (10%) and the mixture was stirred at RT under H2 (g) atmosphere
overnight. Pd/C was removed by filtration through a pad of celite,
which was rinsed with methanol, and the filtrate was concentrated
in vacuo. The debenzylated intermediate was co-evaporated with
pyridine (3”) and dissolved in pyridine (10 mL), and the solution
was cooled to 08C. To the cooled solution was added Ac2O (1 mL)
dropwise and the reaction mixture was allowed to warm to RT.
After complete conversion of the starting material the mixture was
cooled to 08C and the reaction was quenched with MeOH. The re-
action mixture was concentrated in vacuo and the crude was dis-
solved in EtOAc. The organic phase was washed with 1m HCl (aq.)
(1”), sat. NaHCO3 (aq.) (2”), H2O (3”) and brine (3”), dried over
MgSO4, filtered, and concentrated in vacuo. Purification by column
chromatography yielded peracetylated heptamer 23 as a colorless
amorphous solid (635 mg, quantitative yield). FTIR (neat): n˜ =
976.88, 1038.56, 1084.01, 1138.17, 1213.21, 1369.06, 1432.60,
2,4-O-Diacetyl-3-O-(2,4-O-diacetyl-3-O-(2,3,4,6-O-tetraacetyl-a-d-
mannopyranosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-mannopyrano-
syl)-a-d-mannopyranosyl)-6-O-(2,4-O-diacetyl-3-O-(2,3,4,6-O-
tetraacetyl-a-d-mannopyranosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-
mannopyranosyl)-a-d-mannopyranosyl)-1-O-(N-phenyltrifluoro-
acetimidoyl)-a/b-d-mannopyranoside (25): To a solution of 1-OH-
acetylated heptamer 24 (56.9 mg, 27.4 mmol) in acetone (274 mL)
was added a 0.15m N-phenyl trifluoroacetimidoyl chloride solution
(274 mL, 41.1 mmol) in acetone. To the reaction mixture was added
Cs2O3 (15.5 mg, 41.1 mmol) and the mixture was stirred at RT until
TLC showed complete conversion of the starting material. The re-
action mixture was concentrated in vacuo and directly purified
without further workup. Purification by column chromatography
yielded heptamer imidate donor 25 as a slightly yellow oil
(61.6 mg, 27.3 mmol, quantitative yield). FTIR (neat): n˜ =1040.72,
1084.61, 1138.28, 1215.66, 1370.23, 1435.52, 1674.33, 1743.57,
1742.41, 2935.07 cmÀ1 1H NMR (400 MHz, CDCl3): d=5.98 (d, J=
.
1.9 Hz, 1H), 5.37–5.17 (m, 13H), 5.15 (s, 1H), 5.10–5.04 (m, 2H),
5.01 (s, 2H), 4.99 (s, 2H), 4.93 (s, 1H), 4.88–4.78 (m, 2H), 4.28 (dtd,
J=18.4, 10.1, 8.2, 4.0 Hz, 4H), 4.20–3.86 (m, 13H), 3.85–3.65 (m,
4H), 3.62–3.52 (m, 2H), 3.50 (d, J=2.5 Hz, 1H), 2.27–1.92 ppm (m,
69H). 13C NMR (101 MHz, CDCl3): d=170.7, 170.6, 170.6, 170.6,
170.5, 170.5, 170.4, 170.4, 170.3, 170.2, 170.0, 170.0, 169.9, 169.9,
169.8, 169.8, 169.8, 169.7, 169.7, 169.6, 169.6, 169.6, 169.5, 169.5,
168.4, 168.0, 99.6, 99.2, 99.0, 98.7, 97.4, 97.3, 90.5, 90.5, 77.5, 76.0,
75.6, 75.3, 75.2, 75.0, 74.7, 73.4, 71.4, 70.9, 70.8, 70.7, 70.6, 70.1,
70.0, 69.8, 69.8, 69.6, 69.5, 69.4, 69.4, 69.3, 69.1, 69.0, 69.0, 68.8,
68.7, 68.5, 68.5, 68.4, 67.7, 67.5, 67.5, 67.2, 66.9, 66.3, 66.0, 65.9,
65.8, 65.7, 65.6, 62.3, 62.2, 62.1, 62.0, 20.8, 20.8, 20.8, 20.8, 20.7,
20.7, 20.6, 20.6, 20.6, 20.5 ppm. MALDI: [M+H]+ calcd for
C88H119O59 2119.63059, found 2119.63084.
2854.33, 2924.27 cmÀ1 1H NMR (400 MHz, CDCl3): d=7.28 (t, J=
.
7.5 Hz, 2H), 7.09 (t, J=7.5 Hz, 1H), 6.85 (d, J=7.7 Hz, 2H), 5.40–
5.16 (m, 16H), 5.10–5.05 (m, 2H), 5.04–4.95 (m, 4H), 4.91 (s, 1H),
4.84 (d, J=1.7 Hz, 1H), 4.81 (s, 1H), 4.36–4.22 (m, 4H), 4.22–3.88
(m, 13H), 3.87–3.79 (m, 1H), 3.79–3.70 (m, 3H), 3.60–3.46 (m, 3H),
2.25–2.01 (m, 54H), 2.01–1.95 (m, 9H), 1.92 ppm (s, 3H). 13C NMR
(100 MHz, CDCl3): d=170.8, 170.7, 170.7, 170.7, 170.5, 170.2, 170.1,
170.1, 170.1, 167, 167.0, 169.9, 169.8, 169.8, 169.7, 169.7, 169.5,
143.2, 128.9, 124.7, 119.5, 99.6, 99.4, 99.1, 97.6, 97.6, 75.5, 75.2,
71.9, 70.8, 70.8, 70.0, 69.9, 69.9, 69.8, 69.6, 69.5, 69.5, 69.4, 69.1,
68.7, 68.7, 68.6, 68.6, 67.8, 67.4, 66.8, 66.4, 66.1, 66.0, 65.9, 65.7,
62.5, 62.2, 62.1, 29.8, 21.0, 20.9, 20.9, 20.8, 20.8, 20.7, 20.7,
20.6 ppm.
2,4-O-Diacetyl-3-O-(2,4-O-diacetyl-3-O-(2,3,4,6-O-tetraacetyl-a-d-
mannopyranosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-mannopyrano-
syl)-a-d-mannopyranosyl)-6-O-(2,4-O-diacetyl-3-O-(2,3,4,6-O-
tetraacetyl-a-d-mannopyranosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-
mannopyranosyl)-a-d-mannopyranosyl)-a/b-d-mannopyrano-
side (24): To a 08C cooled solution of peracetylated heptamer 23
Propargyl 2,4-O-diacetyl-3-O-(2,4-O-diacetyl-3-O-(2,3,4,6-O-tetra-
acetyl-a-d-mannopyranosyl)-6-O-(2,3,4,6-O-tetraacetyl-a-d-man-
nopyranosyl)-a-d-mannopyranosyl)-6-O-(2,4-O-diacetyl-3-O-
(2,3,4,6-O-tetraacetyl-a-d-mannopyranosyl)-6-O-(2,3,4,6-O-tetra-
acetyl-a-d-mannopyranosyl)-a-d-mannopyranosyl)-a-d-manno-
ChemPlusChem 2015, 80, 928 – 937
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pyranoside (26): Heptamer imidate donor 25 (43.9 mg, 19.5 mmol)
was dissolved in a 0.274m propargyl alcohol solution (356 mL,
97.5 mmol) in DCM and the mixture was stirred over activated mo-
lecular sieves (3 Š) for 30 min at RT. Then the mixture was cooled
to À408C and to the cooled mixture was added a 0.11m TfOH so-
lution (36 mL, 3.9 mmol) in DCM and the reaction mixture was grad-
ually warmed to 08C. The reaction was quenched with a 0.1m Et3N
solution (0.1 mL) in DCM and the solution was filtered over celite
and concentrated in vacuo. Purification by column chromatogra-
phy yielded peracetylated propargyl mannose heptamer 25 as
a white milky oil (16.5 mg, 7.8 mmol, 40% yield). FTIR (neat): n˜ =
66.6, 66.4, 62.4, 62.2, 56.2 ppm. HRMS: [M+H]+ calcd for C45H75O36
1191.40325, found 1191.40308.
Man1-BODIPY-DCG-04 (2): To a solution of propargyl mannose 7
(1.75 mg, 8 mmol) and BODIPY-DCG-04 (5) (8.6 mg, 7.6 mmol) in
DMF/H2O (1:1) (3 mL) was added 0.1m sodium ascorbate (aq.)
(160 mL, 16 mmol) and 0.1m CuSO4 (aq.) (16 mL, 1.6 mmol). The re-
sulting mixture was stirred for 2 h at room temperature, before
being concentrated and co-evaporated with toluene. Purification
by HPLC-MS (A: 25 mm NH4OAc, B: linear gradient 20!35% aceto-
nitrile (ACN) in 12 min) followed by lyophilization from H2O yielded
Man1-BODIPY-DCG-04 (2) (4.3 mg, 3.2 mmol, 42% yield). 1H NMR
(600 MHz, CDCl3/MeOD): d=8.02 (s, 1H), 7.92 (t, J=5.6 Hz, 1H),
7.86 (d, J=7.5 Hz, 2H), 7.83 (d, J=7.6 Hz, 1H), 7.76 (t, J=5.6 Hz,
1H), 7.40 (s, 1H), 7.06 (d, J=4.1 Hz, 1H), 7.01 (d, J=8.5 Hz, 2H),
6.95 (d, J=8.9 Hz, 2H,), 6.69 (d, J=8.5 Hz, 2H, 2), 6.59 (d, J=
4.1 Hz, 1H), 4.80 (d, J=12.4 Hz, 1H), 4.67–4.60 (m, 3H, CH2), 4.45 (t,
J=7.5 Hz, 1H), 4.40–4.36 (m, 1H), 4.30–4.19 (m, 3H), 4.08 (t, J=
5.8 Hz, 2H), 3.85 (dd, J=11.8, 2.1 Hz, 1H), 3.82–3.77 (m, 1H), 3.74–
3.65 (m, 3H), 3.64–3.55 (m, 3H), 3.15–3.11 (m, 3H), 3.06–3.01 (m,
1H), 2.99–2.93 (m, 1H), 2.86–2.80 (m, 1H), 2.75 (t, J=7.4 Hz, 2H),
2.50 (s, 3H), 2.42 (p, J=6.5 Hz, 2H), 2.33 (t, J=7.3 Hz, 2H), 2.25 (s,
3H), 2.21–2.16 (m, 2H), 1.78–1.69 (m, 1H), 1.62–1.48 (m, 6H), 1.46–
1.41 (m, 2H), 1.41–1.35 (m, 2H), 1.31–1.29 (m, 5H), 1.22–1.15 (m,
2H), 0.92 (d, J=6.4 Hz, 3H), 0.88 ppm (d, J=6.4 Hz, 3H,). 13C NMR
(150 MHz, CDCl3/MeOD): d=177.02, 176.02, 174.68, 173.56, 172.96,
168.66, 168.31, 160.68, 160.59, 157.16, 156.37, 145.22, 141.69,
136.49, 135.67, 131.84, 131.81, 131.78, 131.68, 131.30, 129.27,
128.81, 127.14, 125.60, 124.63, 119.12, 116.19, 115.14, 100.70, 74.84,
72.42, 71.93, 68.54, 65.67, 63.19, 62.90, 60.64, 56.36, 54.34, 54.11,
53.33, 53.14, 48.49, 41.56, 40.15, 40.12, 38.14, 36.86, 36.58, 32.64,
30.93, 29.87, 29.74, 27.32, 26.39, 25.80, 24.17, 23.29, 22.02, 21.29,
14.35, 13.28, 9.62 ppm. HRMS: [M+H]+ calcd for C65H89BF2N11O17
1344.64935, found 1344.65139.
1037.06, 1136.93, 1214.09, 1369.36, 1433.95, 1740.68, 2926.41 cmÀ1
.
1H NMR (600 MHz, CDCl3): d=5.35 (dd, J=9.9, 3.5 Hz, 1H), 5.33–
5.32 (m, 1H), 5.29–5.22 (m, 7H), 5.21 (dd, J=10.0, 3.4 Hz, 2H),
5.19–5.16 (m, 1H), 5.07 (dd, J=3.0, 1.9 Hz, 1H), 5.05 (dd, J=3.4,
1.8 Hz, 1H), 5.00–4.97 (m, 3H), 4.97–4.94 (m, 3H), 4.92–4.89 (m,
1H), 4.85 (d, J=1.8 Hz, 1H), 4.84 (d, J=1.9 Hz, 1H), 4.32–4.23 (m,
6H), 4.17 (td, J=10.0, 3.4 Hz, 2H), 4.14–4.09 (m, 2H), 4.08 (s, 1H),
4.07–3.99 (m, 4H), 3.97 (dd, J=9.9, 3.4 Hz, 2H), 3.97–3.88 (m, 3H),
3.87 (ddd, J=10.3, 5.2, 2.7 Hz, 1H), 3.75 (dq, J=10.8, 5.5 Hz, 3H),
3.71 (d, J=5.6 Hz, 1H), 3.56 (dd, J=11.4, 2.2 Hz, 1H), 3.53 (d, J=
2.6 Hz, 1H), 3.52 (d, J=2.6 Hz, 1H), 2.52 (t, J=2.4 Hz, 1H), 2.21 (s,
3H), 2.19 (s, 3H), 2.17 (s, 3H), 2.16–2.13 (m, 21H), 2.12–2.11 (m,
12H), 2.06 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 2.04 (s, 3H), 1.98 (s,
3H), 1.98 (s, 3H), 1.97 ppm (d, J=1.3 Hz, 6H). 13C NMR (150 MHz,
CDCl3): d=171.1, 171.0, 170.9, 170.8, 170.8, 170.8, 170.8, 170.8,
170.6, 170.6, 170.6, 170.5, 170.4, 170.3, 170.3, 170.2, 170.2, 170.2,
170.2, 170.1, 170.1, 170.1, 170.0, 170.0, 170.0, 170.0, 169.9, 169.9,
169.8, 169.8, 169.8, 169.7, 99.4, 99.2, 97.8, 97.7, 97.3, 96.6, 78.8,
75.6, 75.5, 75.3, 75.0, 71.2, 71.0, 70.9, 70.3, 70.0, 70.0, 69.8, 69.6,
69.6, 69.5, 69.5, 69.5, 69.3, 69.2, 68.8, 68.7, 68.6, 68.6, 68.0, 67.8,
67.6, 66.8, 66.8, 66.4, 66.1, 66.1, 66.0, 66.0, 65.8, 62.5, 62.3, 62.2,
55.0, 21.0, 21.0, 21.0, 21.0, 20.9, 20.9, 20.9, 20.9, 20.8, 20.8, 20.8,
20.8 ppm. HRMS: [M+H]+ calcd for C89H119O58 2115.63568, found
2115.63581.
Man3-BODIPY-DCG-04 (3): To a solution of propargyl mannoside
19 (2.4 mg, 4.5 mmol) and BODIPY-DCG-04 (5) (5.1 mg, 4.5 mmol) in
DMF/H2O (1:1) (2 mL) was added 0.1m sodium ascorbate (aq.)
(90 mL, 9 mmol) and 0.1m CuSO4 (aq.) (2.2 mL, 0.22 mmol). The result-
ing mixture was stirred for 1 h at room temperature, before being
concentrated and co-evaporated with toluene. Purification by
HPLC-MS (A: 25 mm NH4OAc, B: linear gradient 20!35% ACN in
12 min) followed by lyophilization from H2O yielded Man3-BODIPY-
Propargyl 3-O-(3-O-(a-d-mannopyranosyl)-6-O-(a-d-mannopyra-
nosyl)-a-d-mannopyranosyl)-6-O-(3-O-(a-d-mannopyranosyl)-6-
O-(a-d-mannopyranosyl)-a-d-mannopyranosyl)-a-d-mannopyra-
noside (27): To a solution of per-O-acetylated propargyl mannose
heptamer 26 (16.5 mg, 7.8 mmol) in MeOH (156 mL) was added
a 125 mm NaOMe (156 mL, 3.9 mmol) solution in MeOH and the re-
action was stirred overnight at RT. TLC-MS analysis showed incom-
plete deacetylation of the starting material and the reaction was
quenched with Amberlite IR-120 H+ (pHꢀ7). The crude was taken
up in H2O (0.5 mL) and a 0.2m NaOH (aq.) (0.5 mL) solution was
added to the solution. The reaction was followed on TLC-MS and
after completion the reaction was quenched with Amberlite IR-120
H+ (pHꢀ7). Solids were filtered and the filtrate was concentrated
in vacuo. The product was lyophilized from H2O without further
purification yielding propargyl mannose heptamer 27 as a white
powder (9.3 mg, 7.8 mmol, quantitative yield). 1H NMR (600 MHz,
D2O): d=5.17 (d, J=1.8 Hz, 1H), 5.14 (d, J=1.8 Hz, 1H), 5.07 (d, J=
1.8 Hz, 1H), 5.02 (d, J=1.8 Hz, 1H), 4.92 (d, J=1.8 Hz, 1H), 4.91 (d,
J=1.8 Hz, 1H), 4.88 (d, J=1.9 Hz, 1H), d 4.36 (s, 2H), 4.26 (dd, J=
3.3, 1.8 Hz, 1H), 4.16 (dd, J=3.3, 1.8 Hz, 1H), 4.11 (dd, J=3.2,
1.7 Hz, 1H), 4.08 (dt, J=3.6, 1.9 Hz, 2H), 4.03 (dd, J=9.6, 3.5 Hz,
1H), 4.01–3.97 (m, 5H), 3.96–3.92 (m, 2H), 3.92–3.87 (m, 11 H),
3.87–3.83 (m, 2H), 3.80–3.72 (m, 8H), 3.70 (ddd, J=12.6, 7.3,
2.1 Hz, 4H), 3.67 (s, 2H), 3.65 ppm (s, 1H). 13C NMR (150 MHz, D2O):
d=103.8, 103.7, 103.5, 100.8, 100.7, 100.6, 100.3, 80.1, 79.7, 79.7,
74.70, 74.6, 74.0, 73.9, 73.1, 72.7, 72.2, 71.9, 71.9, 71.7, 71.4, 71.3,
71.3, 71.2, 70.9, 70.9, 70.8, 68.1, 68.1, 68.0, 67.1, 67.1, 66.8, 66.7,
1
DCG-04 (3) (1.8 mg, 1.1 mmol, 24%) H NMR (600 MHz, MeOD): d=
8.08 (s, 1H), 7.88 (d, J=8.9 Hz, 2H,), 7.43 (s, 1H), 7.07 (d, J=4.0 Hz,
1H), 7.01 (d, J=8.5 Hz, 2H), 6.97 (d, J=8.9 Hz, 2H), 6.69 (d, J=
8.5 Hz, 2H), 6.62 (d, J=4.1 Hz, 1H), 5.05 (s, 1H), 4.85–4.77 (m, 3H),
4.69–4.61 (m, 3H), 4.45 (t, J=7.6 Hz, 1H), 4.38 (dd, J=9.3, 5.7 Hz,
1H), 4.29–4.23 (m, 3H), 4.09 (t, J=5.9 Hz, 2H), 4.05 (d, J=2.5 Hz,
1H), 3.98–3.90 (m, 2H), 3.88–3.54 (m, 17H), 3.16–3.09 (m, 3H),
3.07–2.99 (m, 1H), 2.99–2.93 (m, 1H), 2.86–2.80 (m, 1H), 2.76 (t, J=
7.3 Hz, 2H), 2.51 (s, 3H), 2.42 (p, J=6.5 Hz, 2H), 2.33 (t, J=7.3 Hz,
2H), 2.26 (s, 3H), 2.20–2.14 (m, 2H), 1.77–1.70 (m, 1H), 1.64–1.47
(m, 6H), 1.47–1.41 (m, 2H), 1.41–1.35 (m, 2H), 1.32–1.29 (m, 5H),
1.20–1.15 (m, 2H), 0.92 (d, J=6.4 Hz, 3H), 0.88 ppm (d, J=6.4 Hz,
3H). 13C NMR (150 MHz, MeOD): d=177.14, 176.13, 174.79, 173.69,
173.08, 168.74, 168.43, 165.61, 163.01, 160.81, 160.67, 157.29,
156.48, 150.29, 145.18, 142.19, 136.59, 135.75, 131.90, 131.77,
131.36, 129.35, 128.91, 127.23, 125.71, 124.73, 119.16, 116.24,
115.22, 103.97, 101.33, 100.79, 80.67, 74.96, 74.39, 73.70, 72.66,
72.46, 72.10, 71.27, 68.80, 68.64, 67.48, 67.14, 65.75, 63.23, 62.91,
60.71, 56.47, 54.38, 54.21, 53.42, 53.19, 41.62, 40.20, 38.19, 36.91,
36.63, 32.71, 31.05, 29.95, 29.82, 27.40, 26.48, 25.88, 24.25, 23.30,
ChemPlusChem 2015, 80, 928 – 937
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
22.02, 21.34, 14.35, 9.59. LC/MS analysis (linear gradient 10% !
90% ACN) tR: 6.53 min, ESI-MS (m/z): [M+H]+: 1668.40.
direct labeling experiments, cells were cultured for 2 h (378C; 5%
CO2) in the presence of probes 2, 3, or 4 (0.1, 0.25, 0.5, 0.75 or
1 mm). After incubation, cells were washed with PBS (2”), lysed
(35 mL Invitrogen complete cell extraction buffer), and proteins re-
solved on 12.5% SDS-PAGE, followed by fluorescence scanning
(Cy3 settings) and CBB staining. Image processing was done with
ImageJ, representative gels from at least three independent experi-
ments are shown.
Man7-BDP-DCG-04 (4): To a solution of propargyl mannoside 27
(4 mg, 3.4 mmol) and BODIPY-DCG-04 (5) (3.8 mg, 3.4 mmol) in
DMF/H2O (1:1) (2 mL) was added 0.1m sodium ascorbate (aq.)
(68 mL, 6.8 mmol) and 0.1m CuSO4 (aq.) (6.8 mL, 0.68 mmol). The re-
sulting mixture was stirred for 8 h at room temperature, before
being concentrated and co-evaporated with toluene. Purification
by HPLC-MS (A: 25 mm NH4OAc, B: linear gradient 20!35% ACN
in 12 min) followed by lyophilization from H2O yielded Man7-BDP-
Confocal fluorescence microscopy: Experiments were conducted
on a Leica TCS SPE confocal microscope, using dsRed filter settings
for BODIPY (lex =532 nm) and Cy5 settings for Draq5 (lex =
635 nm). Cells (30–75”104 cells per well) were seeded onto sterile
Labtek II 4- or 8-chamber borosilicate cover glass systems (Fisher
Emergo). Dendritic cells were allowed to attach for 2 h before pre-
incubation with mannan (3 mgmLÀ1) (1 h, 378C, 5% CO2) and sub-
sequent probe incubation (1 mm, 2 h). Cells were then thoroughly
washed (PBS), fixed (4% formaldehyde in PBS), washed again with
PBS, nuclei stained with Draq5 (Thermo Scientific), and imaged. All
experiments were performed at least in duplicate.
1
DCG-04 (4) (2.5 mg, 1.1 mmol, 32%). H NMR (600 MHz, MeOD): d=
8.28 (s, 1H), 8.09 (d, J=8.1 Hz, 1H), 7.95 (s, 1H), 7.90–7.85 (m, 2H),
7.79 (t, J=5.6 Hz, 1H), 7.42 (s, 1H), 7.07 (d, J=4.1 Hz, 1H), 7.01 (d,
J=8.5 Hz, 2H), 6.98 (d, J=8.9 Hz, 2H), 6.69 (d, J=8.5 Hz, 2H), 6.62
(d, J=4.1 Hz, 1H), 5.11 (s, 1H), 5.07 (s, 1H), 4.99 (s, 1H), 4.81–4.64
(m, 8H), 4.50–4.43 (m, 1H), 4.41–4.36 (m, 1H), 4.31–4.23 (m, 3H),
4.19 (d, J=2.8 Hz, 1H), 4.12–4.07 (m, 4H), 4.03–3.53 (m, 41H),
3.16–3.10 (m, 3H), 3.07–3.02 (m, 1H), 3.00–2.94 (m, 1H), 2.88–2.80
(m, 1H), 2.76 (t, J=7.3 Hz, 2H), 2.51 (s, 3H), 2.44 (q, J=6.4 Hz, 2H),
2.34 (t, J=7.2 Hz, 2H), 2.26 (s, 3H), 2.21–2.17 (m, 2H), 1.78–1.71 (m,
1H), 1.66–1.47 (m, 6H), 1.47–1.41 (m, 2H), 1.41–1.36 (m, 2H), 1.33–
1.28 (m, 5H), 1.23–1.17 (m, 2H), 0.92 (d, J=6.4 Hz, 3H), 0.88 ppm
(d, J=6.4 Hz, 3H). HRMS: [M+H]+ calcd. for C101H149BF2N11O47
2317.96965, found 2317.97256.
Acknowledgements
The Netherlands Organization for Scientific Research (NWO-CW)
and the European Research Council are acknowledged for finan-
cial support.
Cell culture of primary cells: Immature dendritic cells were ob-
tained from the bone marrow of C75BL/6 mice and were a gift
from the Biopharmaceutical Department (Leiden University). The
use of animals was approved by the ethics committee of Leiden
University. Mice were sedated; bone marrow of tibiae and femurs
was flushed out and washed with PBS. Cells were grown in den-
dritic cell selection medium (IMDM containing granulocyte-macro-
phage colony stimulating factor (GM-CSF) (2:1 v/v) containing 8%
FCS, penicillin/streptomycin (100 units/mL), glutamax (2 mm), and
b-mercaptoethanol (20 mm). Cells were selected for 10 days (378C;
5% CO2) and subcultured every 2–3 days before use in the assays.
Keywords: activity-based protein profiling · drug delivery ·
fluorescent probes · mannose receptors · oligomannose
[1] See for some relevant reviews: a) N. Yamazaki, S. Kojima, N. V. Bovin,
A. S. Gabius, H. J. Gabius, Adv. Drug Delivery Rev. 2000, 43, 225–244;
b) J. Sudimack, R. J. Lee, Adv. Drug Delivery Rev. 2000, 41, 147–162;
Z. M. Qian, H. Y. Li, H. Z. Sun, K. Ho, Pharmacol. Rev. 2002, 54, 561–687;
I. Rubio-Aliaga, H. Daniel, Trends Pharmacol. Sci. 2002, 23, 434–440.
[2] W. I. Weis, K. Drickamer, Ann. Rev. Biochem. 1996, 65, 441–473.
[3] a) W. I. Weis, M. E. Taylor, K. Drickamer, Immunol. Res. 1998, 163, 19–34;
b) C. G. Figdor, Y. van Kooyk, G. J. Adema, Nat. Rev. Immunol. 2002, 2,
77–84.
[4] a) Y. Van Kooyk, W. W. J. Unger, C. M. Fehres, H. Kalay, J. J. García-Vallejo,
Mol. Immunol. 2013, 55, 143–154; b) J. M. Irache, H. H. Salman, C.
Gamazo, S. Espuelas, Exp. Opin. Drug Deliv. 2008, 5, 703–724; c) J.
Rauen, C. Kreer, A. Paillard, S. van Duikeren, W. E. Benckhuijsen, M. G.
Camps, A. R. P. M. Valentijn, F. Ossendorp, J. W. Drijfhout, R. Arens, S.
Burgdorf, PLoS One 2014, 9, e103755; d) E. W. Adams, D. M. Ratner, P. H.
Seeberger, N. Hacohen, ChemBioChem 2008, 9, 294–303.
[5] a) D. Greenbaum, K. F. Medzihradszky, A. Burlingame, M. Bogyo, Chem.
Biol. 2000, 7, 569–581; b) D. Greenbaum, A. Baruch, L. Hayrapetian, Z.
Darula, A. Burlingame, K. F. Medzihradszky, M. Bogyo, Mol. Cell. Proteo-
mics 2002, 1, 60–68.
[6] Y. S. Liu, M. P. Patricelli, B. F. Cravatt, Proc. Natl. Acad. Sci. USA 1999, 96,
14694–14699.
[7] U. Hillaert, M. Verdoes, B. I. Florea, A. Saragliadis, K. L. L. Habets, J.
Kuiper, S. van Calenbergh, F. Ossendorp, G. A. van der Marel, C. Driessen,
H. S. Overkleeft, Angew. Chem. Int. Ed. 2009, 48, 1629–1632; Angew.
Chem. 2009, 121, 1657–1660.
[8] a) S. Hoogendoorn, G. H. M. van Puijvelde, J. Kuiper, G. A. van der Marel,
H. S. Overkleeft, Angew. Chem. Int. Ed. 2014, 53, 10975–10978; Angew.
Chem. 2014, 126, 11155–11158; b) S. Hoogendoorn, K. L. Habets, S. Pas-
semard, J. Kuiper, G. A. van der Marel, B. I. Florea, H. S. Overkleeft, Chem.
Commun. 2011, 47, 9363–9365.
Labeling of cathepsins in mouse liver and immature dendritic
cell lysate: Lysates (8–10 mg total protein, determined on a Qubit
2.0 fluorometer, Life Technologies-Invitrogen) in 50 mm sodium cit-
rate pH 5.5 or pH 7 (as indicated), 5 mm DTT, 0.2% CHAPS, and
0.1% Triton X-100 were incubated with the indicated concentra-
tion of probe (total volume: 10 mL) for 1 h at 378C. For competition
experiments, lysates were first incubated with N3-DCG-04 (1 or
10 mm), AS44 (10 mm) or BODIPY(FL)-DCG-04 (1 or 10 mm) for 1 h,
378C, before addition of the probe and incubation was continued
for 1 h. After treatment, 5x Laemli’s sample buffer (including b-
mercaptoethanol) was added and the samples were boiled (1008C,
5 min) and resolved on 12.5% SDS-PAGE. Gels were scanned on
a Typhoon 2000 imager (GE Healthcare) using the Cy2 (lex =
532 nm; lem =526 nm) and Cy3 (lex =532 nm; lem =580 nm) set-
tings. Total protein loading was determined by staining with Coo-
massie brilliant blue and subsequent scanning on a BioRad GS800
calibrated densitometer. Image processing was done with ImageJ,
representative gels from at least three independent experiments
are shown.
Labeling of cathepsins in live immature mouse dendritic cells:
Cells were seeded onto tissue-culture coated 24-well plates
(200.000 cells per well, 250 mL medium) and allowed to attach for
2 h (378C; 5% CO2), before addition of inhibitor or probe to the
medium. Pre-incubations with N3-DCG-04 (20 mm) or mannan
(3 mgmLÀ1) were conducted for 1 h, followed by addition of com-
pound 2, 3, or 4 (1 mm) and incubation was continued for 2 h. For
[9] E. van Liempt, C. M. C. Bank, P. Metha, J. J. García-Vallejo, Z. S. Kawar, R.
Geyer, R. A. Alvarez, R. D. Cummings, Y. van Kooyk, I. van Die, FEBS Lett.
2006, 580, 6123–6131.
[10] Y. Guo, H. Feinberg, E. Conroy, D. A. Mitchell, R. Alvarez, O. Blixt, M. E.
Taylor, W. I. Weis, K. Drickamer, Nat. Struct. Mol. Biol. 2004, 11, 591–598.
ChemPlusChem 2015, 80, 928 – 937
www.chempluschem.org
936
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers
[11] V. KØry, J. J. F. Krepinsky, C. D. Warren, P. Capek, P. D. Stahl, Arch. Bio-
chem. Biophys. 1992, 298, 49–55.
Chem. Int. Ed. 2002, 41, 2596–2599; Angew. Chem. 2002, 114, 2708–
2711.
[12] a) M. Bergeron-Brlek, T. C. Shiao, M. C. Tron, R. Roy, Carbohydr. Res. 2011,
346, 1479; b) R. Daly, G. Vaz, A. M. Davies, M. O. Senge, E. M. Scanlan,
Chem. Eur. J. 2012, 18, 14671–14679.
[17] K. Brix, A. Dunkhorst, K. Mayer, S. Jordans, Biochimie 2008, 90, 194–207.
[18] C. S. Barry, E. J. Cocinero, P. C¸arÅabal, D. P. Gamblin, E. C. Stanca-Kaposta,
´
S. M. Remmert, M. C. Fernµndez-Alonso, S. Rudic, J. P. Simsons, B. G.
[13] M. Adinolfi, A. Iadonisi, A. Ravidà, M. Schiattarella, Tetrahedron Lett.
2003, 44, 7863–7866.
Davis, J. Am. Chem. Soc. 2013, 135, 16895–16903.
[19] S. Serna, J. Etxebarria, N. Ruiz, M. Martin-Lomas, N.-C. Reichardt, Chem.
Eur. J. 2010, 16, 13163–13175.
[14] a) T. G. Mayer, B. Kratzer, R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 1994,
33, 2177–2181; Angew. Chem. 1994, 106, 2289–2293; b) X. Liu, B. L.
Stocker, P. H. Seeberger, J. Am. Chem. Soc. 2006, 128, 3638–3648.
[15] L. J. van den Bos, J. Dinkelaar, H. S. Overkleeft, G. A. van der Marel, J.
Am. Chem. Soc. 2006, 128, 13066–13067.
Received: January 7, 2015
[16] a) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057–
3064; b) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew.
Published online on April 21, 2015
ChemPlusChem 2015, 80, 928 – 937
www.chempluschem.org
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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim