The modular synthesis of multivalent functionalised glycodendrons for the detection of lectins including DC-SIGN

Article abstract of DOI:10.1039/c7ra08872h

Glycodendrons are excellent tools for the biological evaluation of lectins. Herein, we present the expedient and efficient synthesis of a versatile second generation dendron scaffold using double exponential growth methodology. The dendron scaffold can be rapidly functionalised, as illustrated by the conjugation of the dendron scaffold to biotin or a fluorescent probe, followed by oxyamine glycan conjugation to the glycan of choice. The use of a fluorescent Lewis^X glycodendron for the detection of the C-type lectin DC-SIGN on macrophages is then demonstrated.

Full text of DOI:10.1039/c7ra08872h

RSC Advances
View Article Online
View Journal | View Issue
PAPER
The modular synthesis of multivalent
functionalised glycodendrons for the detection of
Cite this: RSC Adv., 2017, 7, 45260
lectins including DC-SIGN†
a
a
bc
Bridget L. Stocker*ab
Stefan Munneke, Kristel Kodar, Gavin F. Painter,
ab
and Mattie S. M. Timmer
*
Glycodendrons are excellent tools for the biological evaluation of lectins. Herein, we present the expedient
and efficient synthesis of a versatile second generation dendron scaffold using double exponential growth
methodology. The dendron scaffold can be rapidly functionalised, as illustrated by the conjugation of the
dendron scaffold to biotin or a fluorescent probe, followed by oxyamine glycan conjugation to the
Received 11th August 2017
Accepted 13th September 2017
DOI: 10.1039/c7ra08872h
X
glycan of choice. The use of a fluorescent Lewis glycodendron for the detection of the C-type lectin
rsc.li/rsc-advances
DC-SIGN on macrophages is then demonstrated.
5,6
linker methodology. This oxyamine methodology is not only
highly efficient and allows for the conjugation of naturally
Introduction
Glycoconjugates, such as glycolipids and glycoproteins, are occurring (isolated) glycans, but it also leads to the formation of
essential to all forms of life. These fundamental cellular b-linked GlcNAc residues, which are commonly found conju-
7
constituents are involved in a variety of biological processes, gated to proteins in the form of N-glycans. To this end, we
such as host cell recognition, inammation, cell signalling and proposed that the target glycodendron 1 could be prepared via
1,2
proliferation, with the carbohydrate portion of the glyco- peptide coupling of the carboxy-functionalised dendron 2 to the
conjugate oen interacting with lectins (carbohydrate binding amine-functionalised neoglycoside prepared via conjugation of
proteins) that are expressed on the surface of cells. Lectins, glycan 3 to 3-(methoxyamino)propan-1-amine (4) (Scheme 1).
however, generally have a weak affinity for their carbohydrate The dendron scaffold 2 would in turn contain the molecular
ligands and therefore require multivalent interactions in order probe of choice. The oxyamine ligation methodology has not
3
to induce a biological response. Thus, to study carbohydrate– been used for the conjugation of multiple copies of a glycan to
lectin binding, the glycan epitope is typically conjugated to a molecular scaffold, so we rst envisioned synthesizing a gly-
4
a substrate that allows for multivalent presentation. To this codendron containing GlcNAc as proof-of-concept, before
X
X
end, dendrimers and dendrons have both been used for the extending our methodology to include the glycan Lewis . Lewis
multivalent presentation of glycans. One advantage of the use of is a ligand for the C-type lectin DC-SIGN [also known as Cluster
8
a glycodendron is that its core can be functionalised with of Differentiation 209 (CD209)], which is expressed on the cell
9
–11
reporter groups (e.g., uorescent groups, biotin) or with toxins, wall of several cells types,
which can allow for the detection, isolation or elimination of like’) macrophages expressing higher levels of DC-SIGN
cells that expresses the target lectin.
compared to their classically-activated (‘M1-like’) counter-
with alternatively-activated (‘M2-
12
Given the application of glycodendrons for the study of parts. Moreover, DC-SIGN can be used by the human immu-
carbohydrate–lectin interactions, we became interested in nodeciency virus (HIV), Ebola, hepatitis C, and non-viral
developing a highly efficient synthesis of a dendron scaffold pathogens, such as Mycobacterium tuberculosis, to infect host
Accordingly, DC-SIGN is an attractive target for
choice and with glycans using our recently developed oxyamine glycodendron/glycodendrimer therapeutics.
13,14
that could be readily functionalised with the molecular probe of cells.
15–18
a
School of Chemical and Physical Sciences, Victoria University of Wellington, P.O. Box
6
00, Wellington, New Zealand. E-mail: mattie.timmer@vuw.ac.nz
b
Centre for Biodiscovery, Victoria University of Wellington, P.O. Box 600, Wellington,
New Zealand
c
Ferrier Research Institute, Victoria University of Wellington, P.O. Box 600, Wellington,
New Zealand
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c7ra08872h
Scheme 1 Assembly of glycodendrons using an amine-functionalised
oxyamine linker.
1
45260 | RSC Adv., 2017, 7, 45260–45268
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
Scheme 3 Synthesis of the first generation dendron 8.
Scheme 2 Retrosynthesis of the dendron scaffold.
target dendron scaffold 5 are very efficient (vide infra). More-
over, similar yields have been reported for the tri-O-alkylation of
TRIS using analogous substrates, as illustrated by Dupuy et al.
who reported a 53% yield for the tri-O-alkyl-ation of N,N-
diphenyl-TRIS with tert-butyl bromoacetate using a two-phase
To synthesise the dendron scaffold 2 we proposed a double
19
exponential growth strategy. For the double exponential
growth methodology, a protected rst-generation dendron is
synthesised and then converted to a second-generation den-
dron in a parallel synthesis. This strategy has merit since it
allows for the easy purication of the rst-generation dendron,
minimises the number of steps to assemble larger dendrons,
and therefore facilitates the rapid assembly of second-, third-
24
system, while Newkome et al. prepared a tri-acid functional-
ised dendron core in two-steps via the Michael addition of TRIS
to acrylonitrile followed by nitrile hydrolysis to give the ethyl
25
esters in 54% over two steps. It should also be noted that our
route was readily amenable to synthesis on the gram scale, and
following purication of dendron 6 via silica gel chromatog-
raphy followed by reverse phase (C18 beads) chromatography,
the rst-generation dendron 6 was obtained in excellent purity.
With the rst generation dendron in hand, the synthesis of
the second-generation dendron was then explored using the
double exponential growth approach. Accordingly, the rst
generation dendron 6 was divided into two batches for
conversion into the two reactive substrates (Scheme 4). Depro-
20
and fourth-generation dendrons. Depending on the functional
groups at the periphery of the dendron, a variety of strategies
can then be employed for the conjugation of glycans, including
21
22
copper-catalysed ‘click’ reactions, and reductive amination.
We envisioned that dendron 2 could be obtained from
precursor 5 via Staudinger reduction of the azide in 5 to an
amine and subsequent peptide coupling with a probe equipped
with a carboxylic acid, followed by hydrolysis of the terminal
t-butyl esters groups (Scheme 2). The protected dendron 5 could
in turn be prepared from the rst generation dendron 6 via
double exponential growth methodology. Finally, it was envi-
sioned that dendron 6 could be synthesised in two steps from
the highly versatile branching unit tris(hydroxymethyl)amino-
tection of the tert-butyl esters of 6 via the agency of TFA/CH
2 2
Cl
(1/1, v/v) gave the tri-valent acid 12 in quantitative yield, while
azide reduction of 6, using RANEY® nickel mediated hydroge-
nation, afforded primary amine 13, again in quantitative yield.
0 0
N,N,N ,N -tetramethyl-O-(1H-benzotriazol-1-yl)uronium
Next,
23
methane (TRIS, 7) using amide formation and tri-O-alkylation
with tert-butyl bromoacetate.
Results
The synthesis of the rst generation dendron commenced with
azide substitution of bromoacetic acid (8) to give azide deriva-
tive 9, which in turn was converted into the activated ester 10 via
a DCC-mediated coupling with N-hydroxysuccinimide (Scheme
3). Conjugation of 10 with excess TRIS (7) then gave triol 11 in
80% yield over three steps. With the triol in hand, alkylation
using NaH and tert-butyl bromoacetate in DMF then gave the
rst generation dendron 6. Here it was determined that the

rapid addition of NaH to a mixture of triol 11 and tert-butyl
bromoacetate in DMF : toluene (1 : 1) at room temperature
minimised the formation of partially alkylated intermediates
and afforded dendron 6 in 47% yield. While this alkylation yield
Scheme 4 Synthesis of second generation dendron 5 using a double
may appear modest, the remaining steps for the synthesis of our exponential growth coupling strategy.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 45260–45268 | 45261

View Article Online
RSC Advances
Paper
X
hexauorophosphate (HBTU)-mediated coupling of tri-acid 12
To synthesise the Lewis glycodendron, a similar reaction
with amine 13 was undertaken to give the second-generation route to that employed for the biotinylation of the dendrimer
dendron 5 in 81% yield and in >95% purity following silica core was undertaken. Accordingly, the azide-group in the tert-
gel chromatography followed by reverse phase (C18 beads) butyl ester-functionalised dendron 5 was rst reduced to the
chromatography. Again, these reactions could be performed on corresponding amine and then coupled to uorescein iso-
the gram scale.
thiocyanate (FITC) to yield uorescein derivative 18 in good
To demonstrate the versatility of our synthetic methodology, yield (71%, 2 steps, Scheme 6). Treatment of 18 with TFA
27
we then sought to functionalise the dendron scaffold 5 with however, resulted in the Edman degradation products 19 and
either biotin or a uorescent group. First, proof-of-concept was 20. To prevent this reaction, the core dendron 5 was rst con-
established via the synthesis of a second-generation biotin- verted into the corresponding amine via Staudinger reduction
functionalised dendron scaffold containing GlcNAc (Scheme and the tert-butyl esters were then removed via treatment with
5
). To this end, the azide in dendron 5 was converted into an TFA to give the amine-functionalised carboxy dendron 21 in
amide via RANEY® nickel hydrogenation and the crude product excellent yield (99%, 2 steps). Next, conjugation with FITC
coupled to D-biotin using an HBTU-mediated peptide ligation to under basic conditions (NEt in DMF) gave the target uores-
give the biotinylated dendron 14 in 95% yield over two steps cent dendron 22, also in very good (74%) yield. The uorescent
Scheme 5). Removal of the tert-butyl esters using TFA in CH Cl dendron was initially obtained as the triethylamine salt aer
3
(
2
2
then occurred smoothly to afford carboxy dendron 15 in excel- size exclusion chromatography, however, as this salt hampered
+
lent yield, which was subsequently conjugated to oxyamine ligation to the neoglycoconjugates, ion exchange (Dowex-H )
glycoside 16, itself prepared in 81% yield via the conjugation of was performed to obtain the dendron as the nona-valent acid.
X
X
GlcNAc to 3-(methoxyamino)propanyl-amine hydrochloride 4
For the assembly of the target Lewis glycodendron, Lewis
5
28
according to previously published procedures. To facilitate the (23) was condensed with amine-functionalised oxyamine linker
10
conjugation, excess oxyamine-functionalised glycan 16 (2 equiv.
4
(Scheme 7). When the reaction was performed at room
per carboxyl group) was used and the reaction mixture was temperature, only a small amount of glycoconjugate was
ꢀ
stirred at room temperature overnight. Dialysis (cellulose ester observed, however, heating the reaction at 40 C for 36 hours saw
dialysis membrane, 500 Da molecular weight cut-off) for 48 complete conversion to the desired oxyamine-linked N-glycan.
hours with a 0.1 M Na HPO buffer at pH 7.5 to prevent oxy- Purication by size exclusion chromatography (BioGel P2) then
2
4
26
amine linker hydrolysis, followed by lyophilisation and reverse allowed for the isolation of the target oxyamine-linked glycan as
phase chromatography (C ) then gave the biotinlyated glyco- the ammonium formate salt. As the presence of this salt could
8
dendron 17 in very good yield (72%).
lead to the formation of formamide by-products in the subse-
quent peptide coupling reaction, the oxyamine-linked glycan was
treated with ion exchange resin (Dowex OH ) to give the free
ꢁ
amine 24 in excellent yield (88%). Next, conjugation with the
FITC-labelled second-generation dendron 22 was performed
using an HBTU-mediated peptide coupling reaction. Here, the
order of addition of reagents was found to be important with the

uorescent dendron 22 being dissolved in freshly distilled DMF
X
and then added to the Lewis glycan 23 followed by the addition
of HBTU and Et N, as the glycan itself had poor solubility in DMF
3
alone, though completely dissolved aer the addition of HBTU
Scheme 5 Functionalisation of the second-generation dendron with
biotin and GlcNAc.
Scheme 6 Synthesis of fluorescent dendron.
45262 | RSC Adv., 2017, 7, 45260–45268
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
could be used to detect DC-SIGN on the human macrophage cell
line THP-1. To this end, human THP-1 cells were converted to
the M2-like phenotype via treatment with phorbol-12-myristate-
29
1
3-acetate (PMA) and interleukin (IL)-4, and the presence of
DC-SIGN on the cells conrmed through use of an anti-DC-SIGN
antibody (Fig. 1A). Glycodendron 25 was then made up in
ꢁ1
a stock-solution of 0.66 mg mL (0.1 mM) and assessed for its
ability to detect DC-SIGN on the THP-1 + PMA + IL-4 cells, with
the unglycosylated dendron 22 being used as a negative control
(Fig. 1B). As illustrated, both the anti-DC-SIGN antibody and the
glycodendron 25 bound DC-SIGN in a concentration dependent
manner (Fig. 1A and B). The unglycosylated dendron 22 did not
show binding to the macrophages at low mM concentrations,
with only the highest concentration tested (100 mM) giving
a slight increase in mean uorescence due to non-specic
X
Scheme 7 Synthesis of the Lewis functionalized glycodendron via
X
conjugation of Lewis to the fluorescein-derived second-generation binding. Next, co-staining of the glycodendron with the anti-
dendron using the bi-functional oxyamine linker.
DC-SIGN antibody and 25 was conducted (Fig. 1C and D).
Here, it is important to note that the co-staining with the anti-
body did not interfere with the glycan-mediated binding.
and Et N. The reaction mixture was then stirred at room
3
temperature overnight, upon which analysis by HRMS indicated
Conclusions
that the desired nona-valent glycodendron was formed (m/z for
4+
285 476 36
[C H N O
168S] calcd: 1781.7393, obsd: 1781.7380). Puri- In conclusion, we have presented the design and synthesis of
cation of the glycodendron using size exclusion chromatography a dendron scaffold that can be readily functionalised at both its
Sephadex CM C-25, 0.1 M aq. NH HCO ) then allowed for the core and periphery. The synthesis of the second-generation
isolation of the uorescent Lewis -functionalised glycodendron dendron scaffold 5 was achieved in six linear steps and in an
5 in 51% yield.
excellent overall yield of 30%. Moreover, the ease of function-
To demonstrate the potential of glycodendron 25 as alisation of the dendron core was demonstrated via its rapid
(
4
2
X
2
a chemical tool, we then assessed whether the glycodendron and efficient conversion to both biotinylated and uorescently
labelled derivatives. The use of oxyamine ligation methodology
to efficiently conjugate multiple glycans to a dendron scaffold
was presented via the conjugation of GlcNAc to the biotinylated
X
dendron scaffold and the conjugation of Lewis to a uo-
rescently labelled dendron scaffold, whereby the uorescently-
X
labelled Lewis glycodendron was used to detect DC-SIGN on
macrophages. Given the efficiency and versatility of our proce-
dures, we envision undertaking future carbohydrate–lectin
binding studies employing glycodendron 25 and related glyco-
dendrons in due course.
Experimental
General procedures
Prior to use, THF was distilled from sodium and benzophenone,
CH Cl was distilled from P O , DMF was distilled from BaO,
2 2 2 5
Et N was distilled from KOH. All other reagents were used as
3
5
received. 3-(Methoxyamino)propanyl-amine hydrochloride 4,
5
X
27
oxyamine-linked GlcNAc 16, and Lewis (23) were synthesised
as previously described. All solvents were removed by evapora-
tion under reduced pressure. Reactions were monitored by TLC-
analysis on Macherey-Nagel silica gel coated plastic sheets (0.20
Fig. 1 The expression of DC-SIGN on THP-1 + PMA + IL-4 cells
measured with (A) anti-DC-SIGN antibody and (B) unglycosylated
X
dendron 21 (negative control) and the fluorescent Lewis glycoden-
dron 27. (C) The co-staining with anti-DC-SIGN antibody does not
affect glycodendron binding to THP-1 + PMA + IL-4 cells, single mm, with uorescent indicator UV254) with detection by UV-
staining with 100 mM glycodendron (green), co-staining of the anti-
DC-SIGN antibody with 100 mM glycodendron (red); (D) the expression
of DC-SIGN visualised with glycodendron and anti-DC-SIGN antibody
on THP-1 + PMA + IL-4 cells, using single staining with the anti-DC-
SIGN antibody (blue), co-staining of the antibody with 100 mM gly-
codendron (red) and unstained cells (purple).
absorption (short wave UV – 254 nm; long wave UV – 366 nm),
by dipping in 10% H SO in EtOH followed by charring at
2
4
ꢀ
ꢂ150 C, by dipping in I in silica, or by dipping into a solution
2
ꢀ
of ninhydrin in EtOH followed by charring at ꢂ150 C. Column
chromatography was performed on Pure Science silica gel (40-
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 45260–45268 | 45263

View Article Online
RSC Advances
Paper
6
3 micron). AccuBOND II ODS-C18 (Agilent) was used for reverse the reaction mixture was stirred at r.t. for 4 h. The reaction mixture
phase chromatography. Infrared spectra were recorded as thin was quenched by the addition of ice water (20 mL) and was then
lms using a Bruker Tensor 27 FTIR spectrometer equipped extracted with Et O (2 ꢃ 100 mL). The organic layers were dried

2
with an Attenuated Total Reectance (ATR) sampling accessory with MgSO , ltered and concentrated in vacuo. Purication by
4
ꢁ
1
and are reported in wave numbers (cm ). Nuclear magnetic silica gel column chromatography (PE : EtOAc, 85/15 / 75/25, v/v)
ꢀ
resonance spectra were recorded at 20 C in D
2
O, CD
3
OD, and reverse phase column chromatography (C18, H
2
O/MeOH, 50/
CDCl
00 MHz or Varian VNMRS operating at 600 MHz. Chemical 0.85 mmol, 47%). R
shis are given in ppm (d) relative to solvent residues. NMR 2979, 2935, 2107, 1745, 1684, 1536, 1473, 1428, 1393, 1368, 1229,
3
, or pyridine-d
5
using either a Varian INOVA operating at 50 / 20/80, v/v) yielded tri-alkylated 6 as a white foam (464 mg,
5
f
¼ 0.44 (PE/EtOAc, 70/30, v/v); IR (lm) 3316,
ꢁ
1 1
peak assignments were made using COSY, HSQC and HMBC 1158, 1119, 1037, 971, 915, 845, 731, 647 cm ; H NMR (500 MHz,
experiments.
CDCl ) d 7.86 (s, 1H, NH), 3.95 (s, 6H, 3 ꢃ CH C]O), 3.92 (s, 6H, 3
3
2
1
3
N-Hydroxysuccinimidyl azidoacetate (10). To a solution of ꢃ CH –O), 3.85 (s, 2H, CH –N ), 1.45 (s, 27H, CH tBu); C NMR
2
2
3
3
bromoacetic acid 8 (15.1 g, 0.11 mol) in water (30 mL), NaN
(
3
(125 MHz, CDCl
3
) d 170.2 (3 ꢃ C]O ester), 167.5 (1 ꢃ C]O
tBu), 70.1 (3 ꢃ CH –O), 68.9 (3 ꢃ CH C]O),
tris), 52.9 (CH –N tBu); HRMS(ESI) m/z
), 28.2 (9 ꢃ CH
10] : 547.2974, obsd: 547.2981.
N-Azidoacetyl-1,1,1-tris(carboxymethyloxymethyl)amino-
in vacuo, to give azide 9 as a colourless oil (10.7 g, 0.11 mmol, methane (12). To a solution of dendron 6 (508 mg, 0.93
9%). The crude reaction product was used in the esterication mmol) in CH Cl (5 mL), freshly distilled triuoroacetic acid
30.0 g, 0.43 mmol) was added and the reaction mixture was amide), 82.0 (3 ꢃ C
stirred at r.t. for 3 h. The reaction mixture was diluted with 6 M 60.1 (C
aq. HCl (50 mL) and extracted with Et
O (2 ꢃ 100 mL). The calcd for [C24
organic layers were dried with MgSO , ltered and concentrated
q
2
2
q
2
+
3
3
2
43 4
H N O
4
9
2
2
reaction without further purication. To a solution of crude 2- (5 mL) was added and the reaction mixture was stirred at r.t.
azido acetic acid 9 (9.70 g, 96.0 mmol) in dry CH
N-hydroxysuccinamide (13.2 g, 115 mmol) and N,N -dicyclo- and coevaporated with CH
hexylcarbodiimide (17.8 mL, 115 mmol) were added at 0 C and puried by reverse phase column chromatography (C18
2
Cl
2
(100 mL), for 2 h. The reaction mixture was then concentrated in vacuo
0
2
Cl
2
(10 mL). The residue was
ꢀ
,
the reaction mixture was stirred at r.t. for 18 h. The reaction
mixture was ltered and concentrated in vacuo. To the crude ourless oil. R
residue, EtOAc (150 mL) was added and the solids were removed 5/2/2/1, v/v/v/v); IR (lm) 3418, 2930, 2114, 1727, 1661, 1547,
H
2
O/MeOH, 100/0 / 70/30, v/v) yielded acid 12 as a col-
f
¼ 0.20 (CH Cl /EtOH/MeOH/NH (aq. 33%),
2
2
3
ꢁ1 1
by ltration and the mixture was concentrated in vacuo. The 1427, 1249, 1121 cm ; H NMR (500 MHz, D O) d 4.20 (s,
2
residue was puried by silica gel column chromatography (PE/ 6H, 3 ꢃ CH C]O), 3.98 (s, 6H, 3 ꢃ CH –O), 3.90 (s, 2H,
2
2
1
3
EtOAc, 75/25 / 25/75, v/v) and crystallised from CH
2
Cl
2
/PE to CH
2
–N
3
); C NMR (125 MHz, CDCl
3
) d 174.3 (3 ꢃ C]O),
yield title compound 10 as a white solid (17.9 g, 90.2 mmol, 170.2 (1 ꢃ C]O amide), 69.6 (3 ꢃ CH
2
–O), 68.1 (3 ꢃ CH
2
C]
ꢀ
9
2
1
4%). R
f
¼ 0.25 (PE/EtOAc, 1/1, v/v); mp 110–115 C; IR (lm) O), 59.9 (C
q 2 3
tris), 52.0 (CH –N ); HRMS(ESI) m/z calcd for
+
995, 2935, 2110, 1818, 1781, 1727, 1428, 1416, 1369, 1278, [C12
199, 1087 cm ; H NMR (500 MHz, CDCl ) d 4.24 (s, 2H, CH –
3 2
H
19
N
4
O
10Na] : 401.0915, obsd: 401.0920.
N-Glycyl-1,1,1-tris(tert-butyloxycarbonylmethyloxymethyl)-
ꢁ
1 1
1
3
N ), 2.88 (s, 4H, 2 ꢃ CH C]O); C NMR (125 MHz, CDCl3) aminomethane (13). To a solution of dendron 6 (220 mg,
3
2
d 168.6 (2 ꢃ N–C]O), 164.3 (O–C]O), 48.1 (CH –N ), 25.7 (2 ꢃ 0.40 mmol) in ethanol (1 mL) and THF (1 mL), activated
2
3
aCH ).
RANEY® nickel (120 mg) was added and hydrogen gas was
2
N-Azidoacetyl-1,1,1-tris(hydroxymethyl)aminomethane (11). then bubbled through the reaction mixture at r.t. for 18 h. The
To a solution of succinimidyl ester 12 (8.83 g, 44.6 mmol) in reaction mixture was ltered, washed with sat. NaCl (aq.)
freshly distilled DMF (100 mL), TrisRIS(hydroxymethyl)amino- (50 mL) containing 1 M NaOH (1 mL), extracted with CH
methane 7 (26.9 g, 222 mmol) and Et
added and the reaction mixture was stirred at r.t. for 18 h. The ltered and concentrated in vacuo. The residue was puried
reaction mixture was concentrated in vacuo, then co-evaporated by silica gel column chromatography (EtOAc/MeOH, 100/0 /
2 2
Cl
3
N (0.6 mL, 4.5 mmol) were (2 ꢃ 50 mL) and the combined organic layers were dried,
with H O to remove traces of DMF. The residue was puried by 85/15, v/v) yielded amine 6 as a colourless oil (207 mg,
2
silica gel column chromatography (dry-loading, PE : EtOAc, 50/ 0.40 mmol, 99%). R ¼ 0.09 (EtOAc/MeOH, 90/10, v/v); IR
f
5
0 / 0/100, v/v) yielded the title compound 11 as a white foam (lm) 3336, 2978, 3933, 1745, 1674, 1520, 1368, 1300, 1229,
ꢁ
1 1
(7.84 g, 38.4 mmol, 86%). R
f
¼ 0.50 (CH
2
Cl
2
/EtOH/MeOH/NH
3
1159, 1121, 1044, 847 cm ; H NMR (500 MHz, CDCl
C]O), 3.88 (s, 6H, 3 ꢃ CH
), 2.07 (bs, 2H, NH
) 1.42 (s, 27H, 9 ꢃ
tBu); C NMR (125 MHz, CDCl
) d 173.2 (1 ꢃ C]O
3
) d 7.82
(
2
1
aq. 33%), 5/2/2/1, v/v/v/v); IR (lm) 3359, 2979, 2946, 2890, (s, 1H, NH), 3.92 (s, 6H, 3 ꢃ CH
2
2
–
113, 1739, 1650, 1540, 1454, 1367, 1282, 1229, 1217, O), 3.30 (s, 2H, CH
2
–NH
2
2
ꢁ
1
1
13
053 cm ; H NMR (500 MHz, D
2
O) d 4.00 (s, 2H, CH
2
–N
3
), 3.76 CH
3
3
1
3
(
s, 6H, 3 ꢃ CH –O); C NMR (125 MHz, D O) d 170.4 (C]O amide), 170.3 (3 ꢃ C]O ester), 81.8 (3 ꢃ C tBu), 70.2 (3 ꢃ
2
2
q
amide), 62.1 (C tris) 60.1 (3 ꢃ CH –O), 51.9 (CH –N ); CH –O), 69.0 (3 ꢃ CH C]O), 59.5 (C tris), 45.6 (CH –NH ),
q
2
2
3
2
2
q
2
2
+
HRMS(ESI) m/z calcd for [C H N O ] : 205.0931, obsd: 28.2 (9
ꢃ
CH
tBu); HRMS(ESI) m/z calcd for
10Na] : 543.2888, obsd: 543.2891.
N-Azidoacetyl-1,1,1-tris(1,1,1-tris[tert-butyloxycarbonylmethyl-
6
13
4
4
3
+
205.0930.
45 2
[C24H N O
N-Azidoacetyl-1,1,1-tris(tert-butyloxycarbonylmethyloxymethyl)
aminomethane (6). To a solution of triol 11 (366 mg, 1.79 mmol) oxymethyl]methylamidocarbonylmethylamidocarbonylmethylox-
in DMF (9 mL) and toluene (9 mL), tert-butyl bromoacetate (1.06 ymethyl)aminomethane (5). First generation tri-acid 12 (118 mg,
mL, 7.16 mmol) and NaH (286 mg, 7.16 mmol) were added and 0.31 mmol) and primary amine 13 (728 mg, 1.40 mmol) were co-
45264 | RSC Adv., 2017, 7, 45260–45268
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
evaporated with DMF (2 ꢃ 5 mL). To the mixture DMF (3.1 mL), (t, 6H, JCH ,NH ¼ 4.3 Hz, 3 ꢃ CH
2
N), 3.92 (s, 18H, 9 ꢃ CH
2
O),
2
HBTU (590 mg, 1.56 mmol) and NEt (0.65 mL, 4.67 mmol) were 3.88–3.80 (m, 26H, 9 ꢃ CH C]O, 3 ꢃ CH C]O, CH N), 3.08
3
2
2
2
added and the reaction mixture was stirred at r.t. for 14 h. The (dt, 1H, J ¼ 4.8 Hz, J ¼ 7.0 Hz, H5-biotin), 2.85 (dd, 1H, J
5
,6
4,5
7,8a
reaction mixture was diluted with Et O (50 mL) and washed with ¼ 5.3 Hz, J
¼ 13.0 Hz, H8a-biotin), 2.69 (d, 1H, J
¼
2
8a,8b
8a,8b
0
.1 M HCl (aq.) (2 ꢃ 50 mL). The combined water layers were 12.8 Hz, H8b-biotin), 2.28–2.16 (m, 2H, CH
2
-1-biotin), 1.71–1.50
extracted with Et
were dried with MgSO
residue was puried by silica gel column chromatography (PE/ (C]O dendron), 170.1 (9 ꢃ C]O), 170.0 (3 ꢃ C]O), 169.0 (3 ꢃ
EtOAc, 75/25 / 0/100, v/v; followed by EtOAc/MeOH 100/0 / C]O), 163.9 (C]O urea biotin), 81.9 (9 ꢃ C tBu), 70.6 (3 ꢃ
5/5, v/v) and reverse phase column chromatography (C , H O/ CH C]O), 70.4 (3 ꢃ CH –O), 70.0 (9 ꢃ CH –O), 68.8 (9 ꢃ
2
O (2 ꢃ 50 mL) and the combined organic layers (m, 6H, CH
2
-2, CH
2
-3, CH
2
-4-biotin), 1.41 (s, 81H, 27 ꢃ CH
3
1
3
4
, ltered and concentrated in vacuo. The tBu); C NMR (125 MHz, CDCl
3
) d 174.4 (C]O biotin), 170.6
q
9
1
8
2
2
2
2
MeOH, 50/50 / 0/100, v/v) yielded second generation dendron CH C]O), 61.8 (C6-biotin), 60.1 (C7-biotin), 59.9 (3 ꢃ C tris),
2
q
5
as a colourless oil (475 mg, 0.25 mmol, 81%). R ¼ 0.48 (EtOAc/ 59.5 (1 ꢃ C tris), 55.5 (C5-biotin), 43.7 (1 ꢃ CH –N), 42.2 (3 ꢃ
f
q
2
MeOH, 95/5, v/v); IR (lm) 3316, 3970, 2934, 2107, 1745, 1670, CH
2
–N), 40.3 (C8-biotin), 35.2 (C1-biotin), 28.2 (C2-biotin), 28.0
ꢁ
1
1
1526, 1368, 1231, 1159, 1120, 917, 846, 731 cm
;
H NMR (27 ꢃ CH ), 27.9 (C4-biotin), 25.3 (C3-biotin); HRMS(ESI) m/z
3
2
+
(500 MHz, D
2
O) d 7.62 (s, 3H, 3 ꢃ NH-TRIS), 7.44 (s, 1H, 1 ꢃ NH- calcd for [C94
H
162
N
10
O
39S] : 1043.5355, obsd: 1043.5351.
TRIS), 7.24 (t, 3H, JNH,CH ¼ 5.0 Hz, 3 ꢃ NH gly), 4.06 (s, 6H, 3 ꢃ
N-(2-D-Biotinylamidoacetyl)-1,1,1-tris(1,1,1-tris[carboxymethyl-
2
CH
2
C]O), 4.01 (d, 6H, JCH ,NH ¼ 4.5 Hz, 3 ꢃ CH
2
–N), 3.98 (s, 2H, oxymethyl]methylamidocarbonylmethylamidocarbonyl-methyloxy-
2
CH –N ), 3.95 (s, 18H, 9 ꢃ CH C]O), 3.93 (s, 6H, 3 ꢃ CH –O), methyl)aminomethane (15). To a solution of second-generation
2
3
2
2
1
3
3
.89 (s, 18H, 9 ꢃ CH –O), 1.45 (s, 54H, 18 ꢃ CH t-Bu); C NMR dendron 14 (12.0 mg, 5.8 mmol) in CH Cl (1 mL), freshly
2
3
2
2
(
125 MHz, CDCl ) d 170.3 (9 ꢃ C]O ester), 169.6 (3 ꢃ C]O distilled triuoroacetic acid (1 mL) was added and the reaction
3
amide), 168.5 (3 ꢃ C]O amide), 168.2 (1 ꢃ C]O amide), 82.0 mixture was stirred at r.t. for 3 h. The crude reaction mixture was
tBu), 70.7 (3 ꢃ CH –O), 70.6 (3 ꢃ CH C]O), 70.2 (9 ꢃ CH concentrated in vacuo and coevaporated with water (10 ꢃ 1 mL).
O), 68.9 (9 ꢃ CH C]O), 60.0 (3 ꢃ C tris), 59.8 (1 ꢃ C tris) 52.5 Purication by reverse phase column chromatography (C18, H O/
CH –N –NH), 28.3 (27 ꢃ CH tBu); HRMS(ESI) MeOH, 100/0 / 70/30, v/v) yielded nona-acid 15 (8.8 mg,
), 42.2 (3 ꢃ CH
m/z calcd for [C84 37] : 943.4920, obsd: 943.4929. 5.6 mmol, 97%). R
¼ 0.1 (EtOAc/MeOH, 80/20, v/v); IR (lm) 3360,
N-(2-D-Biotinylamido-acetyl)-1,1,1-tris(1,1,1-tris[tert-butyl-oxy- 3005, 2990, 2978, 2948, 1721, 1656, 1552, 1472, 1431, 1343, 1245,
(C
q
2
2
2
–
2
q
q
2
(
2
3
2
3
2
+
146
H N
10
O
f
ꢁ
1 1
carbonylmethyloxymethyl]methylamidocarbonyl-methylamido- 1200, 1123, 1032, 1018, 979 cm ; H NMR (500 MHz, D O) d 4.57
2
carbonylmethyloxymethyl)aminomethane (14). To a solution of (dd, 1H, J7,8a ¼ 5.1 Hz, J ¼ 7.5 Hz, H7-biotin), 4.39 (dd, 1H, J
¼
6,7
5,6
second generation dendron 5 (139 mg, 73.7 mmol) in ethanol 4.6, J6,7 ¼ 7.5 Hz, H6-biotin), 4.18 (s, 18H, 9 ꢃ CH
1 mL) and THF (1 mL), activated RANEY® nickel (100 mg) was 3 ꢃ CH CO), 3.95 (s, 6H, 3 ꢃ CH NH), 3.88 (s, 18H, 9 ꢃ CH
added and hydrogen gas was bubbled through the reaction 3.86 (s, 6H, 3 ꢃ CH O), 3.28 (dt, 1H, J5,6 ¼ 5.3 Hz, J4,5 ¼ 8.0 Hz, H5-
mixture at r.t. for 20 h. The reaction mixture was ltered, biotin), 2.96 (dd, 1H, J7,8a ¼ 5.0 Hz, J8a,8b ¼ 13.0 Hz, H8a-biotin),
washed with sat. aq. NaCl (50 mL) containing 1 M NaOH (1 mL), 2.74 (d, 1H, J8a,8b ¼ 13.0 Hz, H8b-biotin), 2.30 (t, 2H, J1,2
extracted with CH Cl (2 ꢃ 50 mL) and concentrated in vacuo. 7.2 Hz, CH -1-biotin), 1.74–1.50 (m, 4H, CH -4-biotin, CH -2-
2
CO), 4.12 (s, 6H,
(
2
2
2
O),
2
¼
2
2
2
2
2
1
3
The resulting oil was then passed through a silica gel plug biotin), 1.39 (p, 2H, J2,3 ¼ J ¼ 7.4 Hz, CH -3-biotin); C NMR (125
3,4
2
(
EtOAc/MeOH, 100/0 / 85/15, v/v) to yield crude amine as MHz, CDCl ) d 77.1 (C]O biotin), 174.2 (9 ꢃ C]O), 172.8 (3 ꢃ
3
a colourless oil, which was then used without further purica- C]O), 171.3 (1 ꢃ C]O), 170.7 (3 ꢃ C]O), 165.2 (C]O urea
2
+
tion. HRMS(ESI) m/z calcd for [C84
30.4960. To the crude second generation dendron amine in 68.0 (9 ꢃ CH
DMF (0.73 mL), D-biotin (27.0 mg, 110 mmol), HBTU (55.9 mg, tris), 59.6 (1 ꢃ C
47 mmol) and NEt (25 mL) were added and the reaction CH –N), 39.6 (C8-biotin), 35.1 (C1-biotin), 27.9 (C2-biotin), 27.6
mixture was stirred at r.t. for 18 h. The reaction was diluted with (C4-biotin), 25.0 (C3-biotin); HRMS(ESI) m/z calcd for
H
148
N
8
O
37] : 930.4967, obsd: biotin), 70.0 (3 ꢃ CH
C]O), 62.0 (C6-biotin), 60.2 (C7-biotin), 59.8 (3 ꢃ C
tris), 55.2 (C5-biotin), 43.0 (1 ꢃ CH –N), 42.4 (3 ꢃ
2
C]O), 69.8 (3 ꢃ CH
2
–O), 69.7 (9 ꢃ CH
2
–O),
9
2
q
q
2
1
3
2
2+
Et O (50 mL) and washed with sat. aq. NaCl (2 ꢃ 50 mL). The [C H N O S] : 791.2538, obsd: 791.2536.
2
58 90 10 39
combined water layers were extracted with Et O (2 ꢃ 50 mL)
N-(2-D-Biotinylamido-acetyl)-1,1,1-tris(1,1,1-tris[3-(N-(2-acet-
amido-2-deoxy-b-D-glucopyranosyl)-O-methyl-hydroxylamine)
2
and the combined organic layers were dried with MgSO
4
,

ltered and concentrated in vacuo. Purication by column propylamidocarbonylmethyloxymethyl]methyl-amidocarbonyl-
chromatography (CH Cl /MeOH, 100/0 / 90/10, v/v) and methylamidocarbonylmethyloxy-methyl)aminomethane (17).
reverse phase column chromatography (C18, H O/MeOH, 50/50 Second-generation dendron nona-acid 15 (1.5 mg, 0.95 mmol)
0/100, v/v) yielded second-generation dendron 14 as a col- and GlcNAc-amine 16 (5.3 mg, 17.1 mmol) were co-evaporated
2
2
2
/
ourless oil (146 mg, 70.1 mmol, 95%). R ¼ 0.50 (CH Cl /MeOH, with freshly distilled DMF (2 ꢃ 2 mL). To the mixture DMF
f
2
2
9
1
0/10, v/v); IR (lm) 3309, 3064, 2979, 2933, 1744, 1668, 1527, (0.2 mL) was added and the reaction mixture was half
368, 1230, 1158, 1118, 1035, 846, 733 cm ; H NMR (500 MHz, concentrated under reduced pressure to remove traces of
ꢁ
1 1
CDCl
3
) d 7.66 (s, 3H, 3 ꢃ NH-TRIS), 7.64 (t, 1H, JCH ,NH ¼ 5.7 Hz, Et
2
NH. To the reaction mixture, HBTU (13.7 mg, 36 mmol) and
2
NH-gly), 7.57 (s, 1H, NH-TRIS), 7.54 (t, 3H, JCH ,NH ¼ 5.1 Hz, 3 ꢃ distilled NEt
3
(25 mL) were added and the reaction mixture was
2
NH-gly), 6.30 (s, 1H, NH biotin), 5.65 (s, 1H, NH biotin), 4.45 stirred at r.t. for 14 h. The reaction mixture was diluted with
(
¼
dd, 1H, J7,8a ¼ 5.3 Hz, J6,7 ¼ 7.0 Hz, H7-biotin), 4.27 (dd, 1H, J5,6 water (2 mL) and puried using a dialysis in a Na
2 4
HPO solu-
ꢁ
1
5.1 Hz, J6,7 ¼ 7.0 Hz, H6-biotin), 4.00 (s, 6H, 3 ꢃ CH
2
O), 3.97 tion (1 g L ), to remove the conjugation byproducts. The water
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 45260–45268 | 45265

View Article Online
RSC Advances
Paper
was replaced twice a day for 4 days. The dendron was then 20, CH
2
Cl
2
/MeOH, 50/50, v/v) and concentrated in vacuo to
puried using reverse phase column chromatography (C , H O/ give the trimethylamine salt of dendron 22. The dendron was
8
2
MeOH, 100/0 / 70/30, v/v) to give glycodendron 17 as a col- co-evaporated with H O and puried using ion exchange
2
1
+
ourless oil (2.8 mg, 0.68 mmol, 72%). H NMR (600 MHz, D O) chromatography (Dowex H , elute with H O) to give dendron 22
2
2
d 4.58 (dd, 1H, J7,8a ¼ 5.1 Hz, J6,7 ¼ 7.7 Hz, H7-biotin), 4.40 (dd, (22.3 mg, 12.8 mmol, 74%) as a yellow oil. R
f
¼ 0.05 (tBuOH/
O, 4/1/1, v/v/v); IR (lm) 3463, 3366, 3074, 3015, 2970,
C]O dendron), 2946, 1738, 1656, 1548, 1436, 1366, 1229, 1216, 1206, 1119,
1
9
4
H, J5,6 ¼ 4.7 Hz, J6,7 ¼ 7.7 Hz, H6-biotin), 4.32 (d, 9H, J1,2
.7 Hz, 9 ꢃ H-1 GlcNAc), 4.12 (s, 6H, 3 ꢃ CH
.07 (s, 18H, 9 ꢃ CH C]O dendron), 3.96 (s, 6H, 3 ꢃ CH
N dendron, 9 ꢃ H-2 H-3), 8.13–7.98 (m, NH), 7.94 (s, 3 ꢃ NH), 7.88 (s, 1 ꢃ NH), 7.84
GlcNAc, 9 ꢃ H-6a GlcNAc, 9 ꢃ CH -dendron), 3.72 (dd, 9H, (d, 1H, J ¼ 8.8 Hz, H-5), 7.17 (d, 1H, J ¼ 8.8 Hz, H-6), 6.78–
¼
AcOH/H
2
2
ꢁ
1 1
2
2 3
–N 1051, 1032, 897 cm ; H NMR (500 MHz, CD OD) d 8.22 (s, 1H,
dendron), 3.92–3.83 (m, 37H, 1 ꢃ CH
2
2
5,6
5,6
J
¼ 5.5 Hz, J
¼ 12.4 Hz, 9 ꢃ H-6b GlcNAc), 3.51 (dd, 9H, 6.63 (m, 4H, 2 ꢃ H-13, 2 ꢃ H-10), 6.58 (d, 2H, J
¼ 8.2 Hz, 2 ꢃ
5
,6a
6a,6b
9,10
J2,3 ¼ 8.8 Hz, J ¼ 9.8 Hz, 9 ꢃ H-3 GlcNAc), 3.48 (s, 27H, 9 ꢃ H-11), 4.35 (s, 2H, 1 ꢃ CH NH), 4.20–4.02 (bs, 24H, 3 ꢃ CH C]
3,4
2
2
NOCH
3
), 3.43–3.28 (m, 28H, 9 ꢃ CH
2
a-3 linker, 9 ꢃ H-4 O, 9 ꢃ CH
2
C]O), 4.02–3.92 (m, 12H, 3 ꢃ CH
2
O, 3 ꢃ CH
2
NH),
1
3
GlcNAc, 9 ꢃ H-5 GlcNAc, H5-biotin), 3.24 (dt, 9H, J2,3
¼
3.92–3.86 (m, 18H, 9 ꢃ CH
2 3
O); C NMR (125 MHz, CD OD)
7
9
.2 Hz, J3a,3b ¼ 13.4 Hz, 9 ꢃ CH
ꢃ CH
2
b-3 linker), 3.01–2.87 (m, 19H, d 183.5 (C]S), 174.5 (9 ꢃ C]O), 173.0 (3 ꢃ C]O), 172.0 (1 ꢃ
2
-1 linker, H8a-biotin), 2.75 (d, 1H, J8a,8b ¼ 12.9 Hz, C]O glyc), 171.4 (3 ꢃ C]O glyc), 171.2 (C]O C-1), 161.5, 154.2
-1-biotin), 2.03 (s, (C-12, C14), 149.7 (C-7), 142.2 (C-2/4/8), 132.1 (C-5), 130.4 (C-
7H, 9 ꢃ N–Ac), 1.88–1.78 (m, 1H, H4a-biotin), 1.77–1.69 (m, 13), 130.3 (C-2/4/8), 128.9 (C-2/4/8), 125.8 (C-6), 120.3 (C-3),
H8b-biotin), 2.30 (t, 2H, J1,2 ¼ 7.3 Hz, CH
2
2
1
8H, 9 ꢃ CH -2 linker), 1.69–1.58 (m, 1H, H4a-biotin) 1.58–1.50 113.8 (C-11), 111.5 (C-9), 103.6 (C-10), 71.4 (3 ꢃ CH –O), 71.0
2
2
(
m, 2H, CH -2-biotin), 1.44–1.36 (m, 2H, CH -3-biotin); (9 ꢃ CH O), 69.3 (3 ꢃ CH C]O), 69.2 (9 ꢃ CH C]O), 61.3 (3 ꢃ
2
2
2
2
2
3
+
HRMS(ESI) m/z calcd for [C166
395.6648.
H
298
N
37
O
84S] : 1395.6641, obsd:
C
q
tris), 61.2 (1 ꢃ C
q
tris), 48.9 (1 ꢃ CH
2
NH), 43.5 (3 ꢃ CH
2
NH);
2
+
1
87 9
HRMS(ESI) m/z calcd for [C69H N O42S] : 872.7329, obsd:
N-Glycyl-1,1,1-tris(1,1,1-tris[carboxy-methyloxymethyl]methyl- 872.7335.
amidocarbonylmethylamidocarbonylmethyloxymethyl)amino- N-(2-Acetamido-2-deoxy-3-O-(a-L-fuco-pyranosyl)-4-O-(b-D-
methane triuoroacetic acid (21). To a solution dendron 5 galactopyranosyl)-b-D-glucopyranosyl)-N-(3-aminopropyl)-O-
X
(
60.5 mg, 31.8 mmol) in MeOH/H O/CH Cl (3 mL, 3/1/1, v/v/v), methylhydroxylamine (24). To a solution of Lewis 23
2 2 2
triphenylphosphine (25.0 mg, 95.4 mmol) was added and the (5.6 mg, 10.6 mmol), in a AcOH/NH OAc buffer (0.5 mL, 2 M,
4
reaction mixture was stirred at r.t. for 18 h. The crude reaction freshly prepared, pH 4.5), 3-(methoxyamino)propanyl-amine
mixture was concentrated in vacuo and co-evaporated with hydrochloride 4 (15.9 mg, 113.2 mmol) was added and the
ꢀ
CH
2
Cl
2
(2 ꢃ 10 mL). The residue was dissolved in CH
2
Cl
2
(3 reaction mixture was stirred at 40 C for 35 h. The crude
mL), TFA (3 mL) was added and the reaction mixture was mixture was directly loaded onto a size exclusion column
stirred at r.t. for 3 h. The solvent was evaporated by an argon (Bio-Gel P-2, 1200 ꢃ 18 mm) and eluted with 0.1 M aq.
2 4 2
stream, H O (10 mL) was added, and the phosphine salts were NH HCO . Lyophilisation of the product fractions afforded
removed via ltration, and the resulting residue was concen- neoglycoside 24 (5.7 mg, 88%). R ¼ 0.10 (CH Cl /EtOH/
f
2
2
1
9.5
trated in vacuo to give crude amino acid 21. (45.9 mg, 31.6 MeOH/NH (aq. 35%), 5/2/2/1, v/v/v/v); a
¼ ꢁ9.3 (c ¼ 0.2,
3
D
mmol, 99%). R ¼ 0.05 (tBuOH/AcOH/H O, 4/1/1, v/v/v); IR MeOH); IR (lm) 3341, 2925, 2852, 17 117, 1647, 1586, 1466,
f
2
(
1
(
CH
(
lm) 3344, 3003, 2981, 2950, 2937, 2930, 1718, 1651, 1543, 1451, 1415, 1380, 1350, 1302, 1233, 1193, 1085, 1026, 968,
ꢁ
1
1
ꢁ1
1
473, 1458, 1434, 1241, 1199, 1120, 1032, 952 cm ; H NMR 917 cm
;
H NMR (500 MHz, D
2
O) d 5.12 (d, 1H, J
1
000,2000
¼
0
00
000
500 MHz, D
2
O) d 4.06 (s, 18H, 9 ꢃ CH
2
C]O), 4.00 (s, 6H, 3 ꢃ 3.9 Hz, H-1 ), 4.84 (q, 1H, J
5
000,6000 ¼ 6.7 Hz, H-5 ), 4.49–4.42
0 00 0 0
2
C]O), 3.83 (s, 6H, 3 ꢃ CH
2
N), 3.77 (s, 6H, 3 ꢃ CH
2
O), 3.75 (m, 1H, H-1 , H-1 ), 4.05 (m, 2H, H-2 , H-6a ), 3.93–3.81 (m,
1
3
000
00
0
0
0
s, 18H, 9 ꢃ CH
2
O), 3.71 (s, 2H, 1 ꢃ CH
2
NH
2
); C NMR (125 5H, H-3 , H-4 , H-4 , H-3 , H-6b ), 3.80 (d, 1H, J
3
000,4000 ¼ J
4
000,5000
¼
0
00
00
00
000
MHz, D O) d 174.1 (9 ꢃ C]O CO H), 172.6 (3 ꢃ C]ONH), 2.9 Hz, H-4 ), 3.76 (m, 3H, H-6a , H-6b , H-2 ), 3.64 (dd, 1H,
2
2
0
0
00
00
1
(
70.6 (3 ꢃ C]O), 166.7 (1 ꢃ C]O), 162.8, 162.5, 162.2, 161.9 J300 00 ¼ 3.2 Hz, J 00 00 ¼ 9.9 Hz, H-3 ), 3.60–3.55 (m, 1H, H-5 ),
,4
2 ,3
0
0
0
C]O TFA), 119.4, 117.1, 114.8, 112.5 (CF TFA), 69.83, 69.77 (3 3.54–3.46 (m, 1H, H-5 ), 3.54–3.46 (m, 5H, OCH , H-5 , H-2 ),
3
3
ꢃ CH
2
O, 3 ꢃ CH
2
C]O), 69.6 (9 ꢃ CH
2
O), 67.9 (9 ꢃ CH
2
C]O), 3.11–2.92 (m, 4H, CH
2
-1, CH
2
3
-3), 2.02 (s, 3H, CH Ac), 1.95
0
00 13
5
9.9 (1 ꢃ C
q
tris), 59.7 (3 ꢃ C
q
tris), 42.3 (3 ꢃ CH
2
NH), 40.6 (1 (m, 2H, CH
2
-2), 1.17 (d, 3H, J
5
000,6000 ¼ 6.7 Hz, H-6 ); C NMR
2
+
00
000
ꢃ
CH
2
NH
2
); HRMS(ESI) m/z calcd for [C48
H
76
N
8
O
37] : (125 MHz, D
2
O) 170.5 (C]O), 101.8 (C-1 ), 98.7 (C-1 ), 90.5
0
0
0
00
00
00
0
6
78.2150, obsd: 678.2136.
(C-1 ), 76.9 (C-5 ), 76.1 (C-3 ), 74.8 (C-5 ), 73.2 (C-4 ), 72.4
0
0
0
00
000
000
00
N-[N-([(3 ,6 -Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9 -(9H)- (C-3 ), 71.8 (C-4 ), 70.9 (C-2 ), 69.1 (C-3 ), 68.3 (C-4 ), 67.6
xanthen]-5-yl)amino]-thioxomethyl)glycyl]-1,1,1-tris(1,1,1-tris (C-2 ), 66.7 (C-5 ), 61.4 (C-6 ), 60.9 (OCH ), 59.7 (C-6 ), 52.5
0
00
000
0
3
0
[carboxymethyloxymethyl]methylamido-carbonylmethylamido- (C-2 ), 47.2 (C-1), 37.5 (C-3), 24.5 (C-2), 22.1 (CH Ac), 15.2
3
0
00
+
carbonylmethyloxymethyl)aminomethane (22). To a solution of (C-6 ); HRMS(ESI) m/z calcd for [C24H46N O ] : 616.2923,
3
15
dendron 21 (25.1 mg, 17.3 mmol) in DMF (3 mL), uorescein obsd: 616.2938.
0
0
0
isothiocyanate isomer I (7.4 mg, 19.0 mmol) and Et
3
N (1 mL)
N-[N-([(3 ,6 -Dihydroxy-3-oxospiro[isobenzofuran-1(3H),9 -
were added and the reaction mixture was stirred at r.t. for 18 h. (9H)xanthen]-5-yl)amino]-thioxomethyl)glycyl]-1,1,1-tris(1,1,1-
The crude reaction mixture was concentrated in vacuo. The tris[3-(N-(2-acetamido-2-deoxy-3-O-(a-L-fuco-pyranosyl)-4-O-(b-
residue was puried with size exclusion chromatography (LH- D-galactopyranosyl)-b-D-gluco-pyranosyl)-N-(3-aminopropyl)-O-)-O-
45266 | RSC Adv., 2017, 7, 45260–45268
This journal is © The Royal Society of Chemistry 2017

View Article Online
Paper
RSC Advances
methyl-hydroxylamine)propylamidocarbonylmethyloxymethyl]
methylamidocarbonyl-methylamidocarbonylmethyloxy-methyl)
aminomethane (25). To the oxyamine-functionalised Lewis
trisaccharide 24 (6 mg, 9.8 mmol), a solution of FITC labelled
second generation dendron 22 (0.94 mg, 0.54 mmol) in freshly
distilled and half-concentrated DMF (250 mL) was added, followed
2 R. D. Cummings, Mol. BioSyst., 2009, 5, 1087–1104.
3 (a) M. Mammen, S.-K. Choi and G. M. Whitesides, Angew.
Chem., Int. Ed., 1998, 37, 2754–2794; (b) J. J. Lundquist,
S. D. Debenham and E. J. Toone, J. Org. Chem., 2000, 65,
8245–8250.
4 C. M u¨ ller, G. Despras and T. K. Lindhorst, Chem. Soc. Rev.,
2016, 45, 3275–3302.
X
by the addition of HBTU (5.6 mg, 15 mmol) and Et N (10 mL, 72
3
mmol). The reaction mixture was stirred at r.t. for 18 h. The crude
5 S. Munneke, J. R. Prevost, G. F. Painter, B. L. Stocker and
M. S. M. Timmer, Org. Lett., 2015, 17, 624–627.
mixture was then diluted with H
exclusion chromatography (Sephadex CM C-25, 0.1 M aq.
NH HCO ). Lyophilisation of the product afforded uorescent
glycodendron 25 (1.9 mg, 0.28 mmol, 51%) as a yellow foam. UV-VIS:
2
O (1 mL) and puried by size
6 S. Munneke, E. M. Dangereld, B. L. Stocker and
M. S. M. Timmer, Glycoconjugate J., 2017, 34, 633–642.
7 P. Stanley, H. Schachter and N. Taniguchi, N-Glycans, in
Essentials of Glycobiology, ed. A. Varki, R. D. Cummings
and J. D. Esko, et al., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor (NY), 2nd edn, 2009, ch. 8.
8 T. B. Geijtenbeek, R. Torensma, S. J. van Vliet, G. C. van
Duijnhoven, G. J. Adema, Y. van Kooyk and C. G. Figdor,
Cell, 2000, 100, 575–585.
4
2
1
(
7
(
6
H
2
O), lAbs,max ¼ 495 nm; H NMR (600 MHz, D
.88–7.85 (m, 1H), 7.73–7.70 (m, 1H), 7.66–7.64 (m, 1H), 7.38–7.22
m, 4H), 6.85–6.58 (m, 5H), 5.12 (d, 9H, J ¼ 3.9 Hz), 4.84 (q, 9H, J ¼
.7 Hz), 4.49–4.42 (m, 18H), 4.41–4.35 (m, 18H), 4.20–3.55 (m,
71H), 3.54–3.46 (m, 45H), 3.40–3.16 (m, 27H), 3.03–2.88 (m, 18H),
2
O) d 8.05 (bs, 1H),
1
2
.08–1.99 (m, 27H), 1.82–1.65 (m, 18H), 1.17 (d, 27H); HRMS(ESI)
4+
m/z calcd for [C285
476 36
H N O168S] : 1781.7393, obsd: 1781.7380.
9 E. J. Soilleux, L. S. Morris, G. Leslie, J. Chehimi, Q. Luo,
E. Levroney, J. Trowsdale, L. J. Montaner, R. W. Doms,
D. Weissman, N. Coleman and B. Lee, J. Leukocyte Biol.,
THP-1 derived macrophage differentiation
2
002, 71, 445–457.
The THP-1 acute monocytic lymphoma cell line was cultured in
RPMI 1640 medium supplemented with 10% Fetal Calf Serum
1
1
0 W. K. Lai, P. J. Sun, J. Zhang, A. Jennings, P. F. Lalor,
S. Hubscher, J. A. McKeating and D. H. Adams, Am. J.
Pathol., 2006, 169, 200–208.
1 L. Tailleux, N. Pham-Thi, A. Bergeron-Lafaurie,
J.-L. Herrmann, P. Charles, O. Schwartz, P. Scheinmann,
P. H. Lagrange, J. de Blic, A. Tazi, B. Gicquel and
O. Neyrolles, PLoS Med., 2005, 2, e381.
2 A. Dom ´ı nguez-Soto, E. Sierra-Filardi, A. Puig-Kr ¨o ger,
B. P ´e rez-Maceda, F. G ´o mez-Aguado, M. T. Corcuera,
P. S ´a nchez-Mateos and A. L. Corb ´ı , J. Immunol., 2011, 186,
5
(
FCS), 1% Glutamax and 1% Penstrep, and seeded 2.5 ꢃ 10
cells per mL. Differentiation of the THP-1 cells was induced via
the treatment with PMA (50 ng mL , 48 h.) followed by addi-
tion of IL-4 (20 ng mL , 24 h) to obtian ‘M2-like’ Mfs according
to the procedures of Puig-Kr ¨o ger et al. For subsequent anal-
ysis, the differentiated cells were detached from the tissue
culture plates by incubating the cells in PBS on ice.
ꢁ1
ꢁ
1
29
1
Flow cytometry
2
192–2200.
ꢀ
The macrophages were stained by incubating for 1 hour at 4 C 13 B. J. Appelmelk, I. van Die, S. J. van Vliet,
with either FITC-labelled glycodendron 25, negative control
C. M. J. E. Vandenbroucke-Grauls, T. B. H. Geijtenbeek
(non-glycosylated dendron 22) or anti-DC-SIGN antibody,
and Y. van Kooyk, J. Immunol., 2003, 170, 1635–1639.
washed twice with PBS, and DC-SIGN expression measured by 14 Y. van Kooyk and T. B. H. Geijtenbeek, Nat. Rev. Immunol.,
X

ow cytometry. The uorescent Lewis glycodendron and the
2003, 3, 697–709.
negative control were used in various concentrations (10 , 10 , 15 J. J. Garcia-Vallejo, N. Koning, M. Ambrosini, H. Kalay,
ꢁ1
1
2
3
4
5
1
0 , 10 , 10 and 10 nM, diluted in 10% FCS), where the human
anti-DC-SIGN antibody (phycoerythrin-labelled) positive control
was diluted by a factor 2 (1/50 / 1/1600, v/v).
I. Vuist, R. Sarrami-Forooshani, T. B. H. Geijtenbeek and
Y. van Kooyk, Int. Immunol., 2013, 25, 221–233.
16 R. Ribeiro-Viana, M. S ´a nchez-Navarro, J. Luczkowiak,
J. R. Koeppe, R. Delgado, J. Rojo and B. G. Davis, Nat.
Commun., 2012, 3, 1–8.
Conflicts of interest
1
7 F. Lasala, E. Arce, J. R. Otero, J. Rojo and R. Delgado,
Antimicrob. Agents Chemother., 2003, 47, 3970–3972.
8 R. Ribeiro-Viana, J. J. Garc ´ı a-Vallejo, D. Collado, E. P ´e rez-
Inestrosa, K. Bloem, Y. van Kooyk and J. Rojo,
Biomacromolecules, 2012, 13, 3209–3219.
There are no conicts to declare.
1
Acknowledgements
The authors would like to thank the Wellington Medical 19 T. Kawaguchi, K. L. Walker, C. L. Wilkins and J. S. Moore, J.
Research Foundation for nancial support and the Health
Am. Chem. Soc., 1995, 117, 2159–2165.
Research Council of New Zealand (Hercus Fellowship, BLS).
20 M. V. Walter and M. Malkoch, Chem. Soc. Rev., 2012, 41,
4593–4609.
2
1 W. Kowalczyk, A. Mascaraque, M. S ´a nchez-Navarro, J. Rojo
and D. Andreu, Eur. J. Org. Chem., 2012, 4565–4573.
Notes and references
1
S. Weinbaum, J. M. Tarbell and E. R. Damiano, Annu. Rev. 22 A. Nelson and J. F. Stoddart, Carbohydr. Res., 2004, 339,
Biomed. Eng., 2007, 9, 121–167. 2069–2075.
This journal is © The Royal Society of Chemistry 2017
RSC Adv., 2017, 7, 45260–45268 | 45267

View Article Online
RSC Advances
Paper
2
3 For some representative glycodendron syntheses using TRIS 26 S. Munneke, J. C. Hill, M. S. M. Timmer and B. L. Stocker,
as the branching core, see: (a) Y. M. Chabre, C. Contino- Eur. J. Org. Chem., 2017, 3722–3728.
P ´e pin, V. Placide, T. C. Shiao and R. Roy, J. Org. Chem., 27 P. Edman, E. H ¨o gfeldt, L. G. Sill ´e n and P.-O. Kinell, Acta
008, 73, 5602–5605; (b) R. Kapl ´a nek, T. B ˇr ´ı za, M. Havl ´ı k,
Chem. Scand., 1950, 4, 283–293.
P. Mart ´a sek and V. Kr ´a l, J. Fluorine Chem., 2007, 128, 179– 28 S. Munneke, G. Gainsford, G. F. Painter, B. L. Stocker and
83; (c) P. R. Ashton, S. E. Boyd, C. L. Brown, M. S. M. Timmer, Carbohydr. Res., 2015, 414, 1–7.
N. Jayaraman, S. A. Nepogodiev and J. F. Stoddart, Chem.– 29 A. Puig-Kr ¨o ger, D. Serrano-G ´o mez, E. Caparr ´o s,
2
1
Eur. J., 1996, 2, 1115–1128.
4 C. Dupuy, R. Viguier and A. Dupraz, Synth. Commun., 2001,
A. Dom ´ı nguez-Soto, M. Relloso, M. Colmenares,
L. Mart ´ı nez-Mu n˜ oz, N. Longo, N. S ´a nchez-S ´a nchez,
M. Rincon, L. Rivas, P. S ´a nchez-Mateos, E. Fern ´a ndez-Ruiz
and A. L. Corb ´ı , J. Biol. Chem., 2004, 279, 25680–25688.
2
2
3
1, 1307–1313.
5 G. R. Newkome and X. Lin, Macromolecules, 1991, 24, 1443–
444.
1
45268 | RSC Adv., 2017, 7, 45260–45268
This journal is © The Royal Society of Chemistry 2017