Click chemistry oligomerisation of azido-alkyne-functionalised galactose accesses triazole-linked linear oligomers and macrocycles that inhibit Trypanosoma cruzi macrophage invasion

Article abstract of DOI:10.1016/j.tet.2015.04.085

Abstract Reaction of 2-(2-(2-azidoethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-β-d-galactopyranoside (7) under CuAAC conditions gives rise to mixed cyclic and linear triazole-linked oligomers, with individual compounds up to d.p. 5 isolable, along with mixed larger oligomers. The linear compounds resolve en bloc from the cyclic materials by RP HPLC, but are separable by gel permeation chromatography. The triazole-linked oligomers - pseudo-galactooligomers - were demonstrated to be acceptor substrates for the multi-copy cell surface trans-sialidase of the human parasite Trypanosoma cruzi. In addition, these multivalent TcTS ligands were able to block macrophage invasion by T. cruzi.

Full text of DOI:10.1016/j.tet.2015.04.085

Accepted Manuscript
Click chemistry oligomerisation of azido-alkyne-functionalised galactose accesses
triazole-linked linear oligomers and macrocycles that inhibit Trypanosoma cruzi
macrophage invasion
Vanessa L. Campo, Irina M. Ivanova, Ivone Carvalho, Carla D. Lopes, Zumira A.
Carneiro, Gerhard Saalbach, Sergio Schenkman, João Santana da Silva, Sergey A.
Nepogodiev, Robert A. Field
PII:
S0040-4020(15)00595-5
10.1016/j.tet.2015.04.085
TET 26688
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 2 February 2015
Revised Date: 12 April 2015
Accepted Date: 23 April 2015
Please cite this article as: Campo VL, Ivanova IM, Carvalho I, Lopes CD, Carneiro ZA, Saalbach
G, Schenkman S, da Silva JS, Nepogodiev SA, Field RA, Click chemistry oligomerisation of azido-
alkyne-functionalised galactose accesses triazole-linked linear oligomers and macrocycles that inhibit
Trypanosoma cruzi macrophage invasion, Tetrahedron (2015), doi: 10.1016/j.tet.2015.04.085.
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1
ACCEPTED MANUSCRIPT
Graphical Abstract
Click chemistry oligomerisation of azido-alkyne-
functionalized galactose accesses triazole-linked
linear oligomers and macrocycles that inhibit
Trypanosoma cruzi macrophage invasion
Leave this area blank for abstract info.
Vanesa L. Campo,a, ± Irina M. Ivanova,b, ± Ivone Carvalho,a Carla D. Lopes,c Zumira A. Carneiro,c Gerhard Saalbach,b
Sergio Schenkman,d João Santana da Silva,c Sergey A. Nepogodiev,b Robert A. Field b,*

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Tetrahedron
journal homepage: www.elsevier.com
Click chemistry oligomerisation of azido-alkyne-functionalised galactose accesses
triazole-linked linear oligomers and macrocycles that inhibit Trypanosoma cruzi
macrophage invasion
Vanessa L. Campo,a,± Irina M. Ivanova,b,± Ivone Carvalho,a Carla D. Lopes,c Zumira A. Carneiro,c
Gerhard Saalbach,b Sergio Schenkman,d João Santana da Silva,c Sergey A. Nepogodiev,b
Robert A. Field b,*
a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Av. Café S/N, CEP 14040-903, Ribeirão Preto, SP, Brazil
b Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
c Faculdade de Medicina de Ribeirão Preto, Department of Parasitology Microbiology and Immunology, USP, Av. Bandeirantes 3900, CEP 14049-900, Ribeirão
Preto, SP, Brazil
d Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Rua Botucatu 862 8, Andar 04023-062 São Paulo, SP, Brazil
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Reaction of 2-(2-(2-azidoethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-β-D-galactopyranoside (7)
under CuAAC conditions gives rise to mixed cyclic and linear triazole-linked oligomers, with
individual compounds up to d.p. 5 isolable, along with mixed larger oligomers. The linear
compounds resolve en bloc from the cyclic materials by RP HPLC, but are separable by gel
permeation chromatography. The triazole-linked oligomers – pseudo-galactooligomers – were
demonstrated to be acceptor substrates for the multi-copy cell surface trans-sialidase of the
human parasite Trypanosoma cruzi. In addition, these multivalent TcTS ligands were able to
block macrophage invasion by T. cruzi.
Available online
Keywords:
Keyword_1 Click chemistry
Keyword_2 Triazole-linked oligomers
Keyword_3 Pseudo-glycomacrocycles
Keyword_4 Trypanosoma cruzi
Keyword_5 Macrophage invasion
2015 Elsevier Ltd. All rights reserved.
1. Introduction
The blood-borne protozoan parasite Trypanosoma cruzi causes Chagas’ disease, a debilitating and often
lethal condition that afflicts millions of people in South and Central America. There is a desperate need for
new treatments for this disease, with a requirement for the identification of new drug targets and potential
therapeutic agents.1-3 The biology of carbohydrates – glycobiology4 – presents many underexplored targets
and opportunities5-6 for drug discovery.7 Including T. cruzi8 makes extensive use of cell surface mucin
glycoproteins9 and associated enzyme activities, trans-sialidases, in its’ attempt to evade the human
immune response of one hand, and to adhere to and invade host cells on the other.10-11 T. cruzi trans-
sialidase (TcTS) is considered a relevant drug target for Chagas’ disease.12-13 With this in mind, we have
investigated glycopeptide substrates for and inhibitors of TcTS,14-15 including the application of CuAAC
± These authors contributed equally to this study
* Corresponding author: Fax: +44-1603-450018; e-mail: rob.field@jic.ac.uk (R. A. F.)

3
16
20-22
ACCEPTED1M7-19ANUSCRIPT
click chemistry with carbohydrate building blocks
to access libraries of small molecule inhibitors
and multivalent TcTS ligands displayed on calixarene cores.23
There is much current interest in the use of multivalent glycoconjugates as anti-pathogen agents18, 24-25
and click chemistry presents interesting opportunities for the generation of oligomeric triazole-linked
structures.26-28 CuAAC click chemistry has found application in the synthesis of analogues of cyclic
oligosaccharides,27 with medium size macrocycles being prepared in moderate to good yields starting both
from protected29-30 and “free” sugar derivatives.31-32 There are only a few examples of the successful
oligomerisation/polymerisation of such azide-alkyne-functionalised carbohydrates leading to linear
oligomers, including recent proximity-driven click-polymerization of an 4-O-propargyl-β-D-
galactopyranosyl azide in a crystal lattice33-34 and click-polymerisation of an open chain azido-alkyne
derivative of gluconamide.35 We have previously shown that the cyclooligomerisation of azido-alkyne-
functionalised sugars gives rise to relatively rigid 1,6-linked cyclic pseudo-galactooligosaccharides that are
recognised by TcTS.32 In the present study, we wished to access linear oligomers and/or larger
macrocycles, in order to span a greater area that might more adequately map to the distribution of trans-
sialidase on the parasite cell surface, so blocking trans-sialidase-action and associated infection processes36
(Fig. 1). Here we have designed linkers between sugar and azide functionality, in contrast to our previous
work. We opted for ethylene glycol-based linkers to reduce the potential for non-specific interactions with
proteins and membrane components.37 We anticipated that these flexible linkers might also promote
oligomerisation at the expense of cyclisation. Here we report the chemical synthesis of azido-alkyne-
functionalised galactose monomer 7, CuAAC click reactions thereof and characterisation of the resulting
triazole-linked oligomers. Finally, assessment of the interaction of these triazole-linked materials with
TcTS is reported, along with initial assessment of their ability to block macrophage invasion by T. cruzi
parasites.

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Fig. 1. Schematic representation of cell surface-presented Trypanosoma cruzi trans-sialidase and potential for its’ inhibition by 1,2,3-triazole-
linked linear and cyclic pseudo-galactooligomers
2. Results and Discussion
2.1. Synthesis of azido-alkyne galactose-containing monomer
The azido-alkyne-substituted galactose monomer 7 was synthesised in nine steps starting from known 6-
O-propargyl-D-galactopyranose 1,32 as outlined in Scheme 1. Propargyl ether 1 was per-O-benzyolated
and converted into the required hemiacetal 3 using a solution of ammonia in methanol-THF. The resulting
hemiacetal 3 was treated with trichloroacetonitrile and DBU to obtain the corresponding imidate donor 4,
which upon activation with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in the presence of 2-(2-(2-
chloroethoxy)ethoxy)ethanol gave chlorinated β-glycoside 5 in a respectable 85% yield for the
glycosylation step. In the presence of NaN3 and NaI, the chlorinated β-galactoside 5 was converted into the
azido β-galactoside 6 in near quantitative yield.

5
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Scheme 1. Synthetic route to azido-alkyne-containing galactose monomer 7. Reagents and conditions: a) BzCl, Py, 81%; b) NH3, MeOH/THF
(7:3), 64%; c) Cl3CCN, DBU, CH2Cl2, 85%; d) 2-(2-(2-chloroethoxy)ethoxy)ethanol, TMSOTf, CH2Cl2, 85%; e) NaN3, NaI, DMF, 97%; f)
NaOMe, MeOH, 92%.
The presence of azide functionality in 6 was evident from a characteristic signal in the IR spectrum
[2107 cm-1] and the β-configuration followed from the anomeric proton signal (δ 5.75, J1,2=8.0 Hz) in the
1H NMR spectrum. De-O-benzoylation of 6 afforded target azido-alkyne-functionalised galactose
monomer 7 in an overall yield of 25% from 6-O-propargyl galactopyranose 1.38
2.2. Cyclisation and oligomerisation through CuAAC reactions based on azido-alkyne-containing
galactose monomer 7
Once synthesised, the reactivity of azido-alkyne-functionalised galactose monomer 7 was tested under
CuAAC conditions – at 1 M concentration in DMF with CuSO4/Cu turnings either at 110 °C (using
microwave irradiation; Method A) or at ambient temperature (Method B). The progress of both reactions
could conveniently be followed by TLC analysis. The reaction using Method A was complete after 30 min,
compared to 2 days for Method B. TLC analysis showed formation of multiple common products,
consistent with the intended oligomerisation, while different relative spot intensities were evident from the
two methods (Fig 2, TLC lanes A and B). The reaction mixtures from both methods were concentrated
under reduced pressure, redissolved in water and submitted to reverse phase (RP) HPLC purification. This
allowed straightforward separation of cyclic from linear products, as well as resolution of the variously
sized cyclic oligomers from each other (Fig. 2, HPLC traces A and B) resulting in samples of cyclic
monomer 8, cyclic dimer 9, cyclic trimer 10, cyclic tetramer 11, cyclic pentamer 12 and cyclic hexamer 13,
along with mixed linear oligomers which interestingly did not resolve under the reverse phase HPLC
conditions employed to separate the corresponding cyclic materials (Scheme 2). The formation of cyclic
1,4-triazole-linked structures was confirmed by virtue of singlet signals of triazole CH protons at δ 8.10
ppm39 and the absence of propargyl CH signals at δ 2.83 in 1H NMR spectra.

6
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Fig. 2. Reverse phase HPLC and normal phase TLC analyses of cyclic and linear products obtained from 1,3 azido-alkyne cycloaddition
reactions of monomer 7 (1M in DMF). HPLC trace/TLC lane: A , Method A (Cu(I), 110 °C); B, Method B (Cu(I), room temperature); C,
Method C (110 °C); D, Method D (room temperature); TLC lane 7, starting monomer 7; TLC lane L, purified mixed linear oligomer fraction.
O
O
OH
N
OH
HO
O
N
O
HO
OH
O
OH
OH
O
N
O
N
O
O
Method A
Method B
N
O
N
O
O
O
O
O
O
N
O
N
OH
O
HO
Linear oligomers
7
N
N
HO
HO
N
N
N
N
O
N
O
O
N
N
HO
N
O
OH
O
O
O
HO
O
HO
HO
O
OH
O
O
O
OH
8
9
10
A 9.0%
B 6.7%
A 3.0%
B 2.2%
A 1.6%
B 1.0%
O
HO
O
OH
OH
O
O
O
OH
OH
O
HO
O
O
HO
O
N
N
OH
O
N
O
N
O
N
N
OH
OH
O
O
O
O
O
N
OH
O
O
OH
N
HO
N
O
O
O
N
O
O
N
N
OH
O
O
OH
HO
HO
N
N
OH
O
N
O
O
O
N
N
N
N
O
N
O
OH
HO
O
O
O
HO
N
OH
HO
HO
O
N
O
N
O
N
O
N
N
O
O
N
OH
O
O
O
OH
HO
O
O
O
N
N
O
O
HO
O
N
O
N
O
HO
N
OH
N
N
HO
O
O
N
N
N
HO
O
O
N
O
HO
O
O
HO
N
O
N
OH
N
N
O
HO
HO
HO
O
HO
O
O
N
N
N
O
O
HO
OH
O
O
HO
HO
O
O
OH
O
O
O
O
OH
O
11
12
13
B 0.6%
A 1.1%
B 0.8%
A 1.3%
B 0.6%
Scheme 2. Cyclisation and oligomerisation of monomer 7 (1M in DMF), through CuAAC employing Method A (Cu(I), 110 °C) and Method
B. (Cu(I), room temperature). Yields (%) for Method A and Method B.

7
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The linear oligomeric products eluted on reverse phase HPLC as a single broad peak at ca 32 min (Fig.
2, HPLC traces A and B). These compounds were well resolved from each other and from the
corresponding cyclic oligomers on analytical TLC (Fig 2, lane L), linear oligomers 14-18 have slightly
higher Rf values compared to cyclic product of the same molecular size. Monomer 7 was shown to undergo
oligomerisation up to at least a decamer. These analyses alongside isolated yields (see Fig.2, Fig. 3 and
Table S1 in Supplementary Information) also illustrate that the lower reaction temperature (room
temperature vs 110 C) favours formation of linear products over the corresponding cyclic isomers. In
°
contrast to reverse phase HPLC, gel permeation chromatography (GPC) on TSK-HW40S enabled
separation of linear oligomers up to the pentamer (Fig. 3). It should be noted that these linear compounds
contain unreacted azido and alkyne terminal groups capable of further reactions even in the absence of
Cu(I) catalyst. This gave rise to complications during handling and storage due to spontaneous cyclisation
and oligomerisation of purified compounds (data not shown).
The 1,3-dipolar cycloaddition of azido-alkyne galactose monomer 7 generates series of isomeric cyclic
and linear products that have the same molecular formula and hence the same monoisotopic mass.40 This
was confirmed by high resolution MS analyses of individual isolated cyclic compounds 8-13 as well as the
mixture of linear oligomers collected as a single peak in HPLC purification (Fig. 2, HPLC traces A and B;
TLC lane L). Cyclic and linear products from trimer upwards run in MS analyses as multiply charged
species, spectra for which were de-convoluted to obtain monoisotopic masses (Table 1). Cyclic oligomers
1
had distinctive appearances in H NMR spectra: for centrosymmetric macrocyles 8-13 these were
represented by relatively simple spectra of the repeat unit compared to more complex spectra, as be
expected for linear oligomers 14-18.
Table 1 HRMS data of 1,4-triazole-linked cyclic products and linear oligomers.
Calculated
m/z [M+H]+
Monomer 376.1751
Cyclic
Found
Linear
oligomers
7
Found a
compounds m/z [M+H]+
m/z [M+H]+
376.1708
8
376.1701
751.3334
1126.4961
1501.6511
1876.8248
2251.9792
Dimer
751.3357
9
14
15
16
17
18
751.3351
Trimer
1126.4998
1501.6640
1876.8281
2251.9923
10
11
12
13
1126.4985
1501.6631
1876.8273
2251.9897
Tetramer
Pentamer
Hexamer
aData obtained from the analysis of the mixture of linear oligomers obtained as a result of reverse-phase HPLC purification.

8
O
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O
O
HO
HO
O
O
N
OH
N
N
O
HO
O
O
O
O
HO
O
O
O
HO
HO
OH
N3
O
14
O
N
OH
N
N
O
O
O
HO
O
O
O
N
HO
O
O
HO
HO
N
OH
O
N
O
O
N
O
O
HO
OH
N
O
N
O
N3
15
O
HO
O
O
HO
OH
O
O
N
HO
O
O
O
HO
HO
N
OH
N
O
O
O
O
O
N
HO
OH
O
N
N
O
HO
N
O
O
O
N
OH
HO
N
O
O
O
O
O
HO
N
HO
O
O
N
OH
O
N3
O
O
16
HO
HO
HO
N
O
O
O
O
OH
HO
O
N
O
OH
O
N
N
HO
N
O
OH
O
O
N
HO
N
O
O
O
O
HO
O
N
HO
O
N
N
O
OH
HO
N
O
N
O
O
OH
HO
N
O
O
O
O
HO
O
N
HO
O
O
N3
OH
N
HO
17
N
O
OH
O
O
HO
O
N
O
HO
N
OH
N
O
O
O
HO
O
O
N
HO
OH
N
N
O
O
O
HO
O
O
N3
18
HO
OH
Fig. 3. Linear oligomerisation products from the reaction of monomer 7 under CuAAC conditions identified by HRMS. Compounds 14-18
were obtained in a combined yield of 26 % (Method A) and 36 % (Method B).
2.3. Uncatalysed 1,3-dipolar cycloaddition of azido-alkyne-containing galactose monomer 7
Given the noted spontaneous but slow 1,3-dipolar cycloaddition of monomer 7 on standing at room
temperature (vide supra), the reactivity of 7 at 1 M concentration in DMF at 110 °C (under microwave
irradiation; Method C) and at room temperature (Method D) were compared (Scheme 3). Reaction using
Method C was complete after 30 min, while Method D gave approximately 10% conversion of monomer 7
after two weeks. TLC analysis showed even more complex multiple product mixtures than the Cu(I)-
catalysed reaction (Fig. 2, TLC lanes C and D) due to the expected formation of mixed isomeric 1,4- and
1,5-linked triazoles.
The reaction mixture from Method C was concentrated under reduced pressure, redissolved in water and
submitted to the reverse phase HPLC purification. This procedure again allowed separation of cyclic from
linear products, as well as resolution of 1,4- and 1,5-linked isomeric cyclic triazole monomer structures 8
and 19 from each other (Fig. 2, HPLC Trace C). Cyclic oligomers eluted on RP HPLC as mixtures of
poorly resolved regioisomers, with various combinations of 1,4- and 1,5-linked triazole rings incorporated
into macrocycle structures. The ring sizes of mixed 1,4/1,5-linked cyclic dimers (C2), mixed 1,4/1,5-linked

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cyclic trimers (C3), mixed 1,4/1,5-linked cyclic tetramers (C4), mixed 1,4/1,5-linked cyclic pentamers
(C5) and mixed 1,4/1,5-linked cyclic hexamer (C6) were established by mass spectrometry.
The 1,5-linked cyclic monomer 19 was fully characterised by NMR spectroscopy and HRMS. The
formation of the 1,5-linked triazole unit was confirmed by observing the triazole CH singlet signal at δ 7.71
and the absence of a propargyl CH signal at δ 2.83 in the 1H NMR spectra. The 1,4/1,5-linked mixed linear
products were submitted to GPC purification on TSK-HW40S in water, which enabled separation of mixed
linear products up to a tetramer where linear 1,5-linked triazole dimer 20 and linear 1,4-linked dimer 14,
isolable as single compounds, were characterised by NMR spectroscopy and mass spectrometry.
Scheme 3. Spontaneous cyclisation and oligomerisation of monomer 7 leading to a mixture of compounds incorporating both 1,4-linked and
1,5-linked 1,2,3-triazole residues.
The linear structures of dimers 14 and 20 were confirmed by NMR spectroscopy, in particular by
1
observation of a methylene signal of the intact propargyl group at δ ~4.18 in the H NMR spectra. In
addition, DTT reduction of azido group in 20 produced amino-terminated compound which was detected
by MS analysis showing an [M+H]+ peak at m/z 725.28, compared to unreduced precursor with an [M+H]+
1
peak at m/z 751.33. The triazole linkage type in 14 and 20 was also evident from the H NMR spectra,
which showed diagnostic proton resonances of 1,4-linked triazoles at δ 8.04 for 14 and of 1,5-linked
triazoles at δ 7.80 for 20.39
2.4. Cyclic triazole-linked oligomers as acceptor substrates for Trypanosoma cruzi trans-sialidase
(TcTS)
Cyclic compounds 8-10 and 19 were tested for their ability to act as substrates for TcTS,21, 41 with fetuin
serving as a donor substrate for O-3 sialylation of the galactose residues in the triazole-linked macrocycles.
Reactions were carried out over the course of 9 days, with addition of further fetuin and enzyme after 5
days, and addition of fetuin again after 7 days. The reaction mixtures were monitored by TLC and in all

10
cases formation of new compounds was observed, which were identified by HRMS. Thus, reaction of 1,4
ACCEPTED MANUSCRIPT
triazole-linked cyclic trimer 10 resulted in formation of mono-, di- and tri-sialylated products 21, 22 and
23, respectively (Scheme 4). Related reactivity was observed for 1,4-triazole-linked cyclic dimer 9 which
gave two sialylated products, mono-sialylated and di-sialylated derivatives. The 1,4-triazole-linked cyclic
monomer 8 and 1,5-triazole-linked cyclic monomer 19 gave mono-sialylated products as expected (see
Supporting Information). It is therefore evident that cyclic triazole-linked pseudo-galactooligomers are
recognised by, and can act as acceptor substrates for, TcTS.
10
TcTS
Fetuin
HO
HOOC
O
HO
OH
HO
HOO C
O
HO
OH
HO
HO
O
O
O
O
O
O
AcHN
AcHN
O
OH
O
N
N
N
OH
OH
OH
N
N
O
O
N
O
HO
HO
HO
OH
OH
OH
O
O
O
N
O
O
N
O
N
O
O
O
O
OH
O
O
O
O
HO
NHAc
OH
O
O
OH
OH
O
O
HO
O
HO
O
OH
HO
N
N
N
N
N
N
HOOC
N
N
N
O
O
O
N
N
N
N
N
N
N
N
N
HO
HO
HO
HOOC
O
HOOC
O
O
O
HOOC
O
O
OH
HO
OH
HO
HO
HO
OH
HO
HO
HO
O
HO
O
HO
O
O
O
O
O
O
O
AcHN
AcHN
O
O
AcHN
O
O
O
O
OH
OH
OH
21
22
23
Calcd m/z 1415.5806 [M-H]-
Obsd m/z 1415.5994 [M-H]-
Calcd m/z 1706.6760 [M-H]-
Obsd m/z 1706.7021 [M-H]-
Calcd m/z 1997.7714 [M-H]-
Obsd m/z 1997.7947 [M-H]-
Scheme 4. Proposed sialylated products structures 21, 22 and 23 of enzymatic transformation of compound 10. Reagents and conditions: 3
mM fetuin, 1 mM compound 10, T. cruzi trans-sialidase in 100 mM, pH 7.5 phosphate buffer, 28 °C, 9 days.
2.5. Preliminary biological evaluation of triazole-linked oligomers
As the trans-sialidase is an important enzyme associated with host cell invasion by T. cruzi,11, 36 we
evaluated the biological potential of our triazole-linked oligomers as blockers of parasite mammalian cell
entry. The trypomastigote form of the parasite invades mammalian cells, where it differentiates into
amastigote forms that replicate and subsequently de-differentiate to tryposmatigotes. The latter then exit
the cell in search of other cells to infect. Our assays were therefore two-fold: assessment of the number of
trypomastigotes present in the medium, or assessment of the number of amastigotes inside macrophages.
Parasites were mixed with bovine macrophages and triazole-linked oligomers, with or without a pre-
incubation of the parasite and oligomers; where a pre-incubation was employed, free triazole-linked
oligomers were removed by washing before adding the parasites to macrophages. The parasites were left to
invade the macrophages and after 6 days, the trypomastigote form found in the medium was removed and
counted. As shown in Fig. 4 A ( ), the incubation of parasite with triazole-linked oligomers substantially
inhibited the invasion of macrophage by parasites (>90% for trimer-hexamer). To rule out a direct impact

11
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of the triazole-linked compounds on the macrophages, pre-incubation of parasites with triazoles, plus
washing to remove excess triazole, was assessed. While the impact on parasite invasion was more modest
than when the triazole was present throughout (Fig. 4 A ), this is to be expected given the dramatic
reduction in concentration of the inhibitor in these experiments. Gratifyingly, inhibition of macrophage
invasion was still pronounced.
Fig. 4. Inhibition of T. cruzi invasion of bovine macrophages in the presence of 1,4/1,5-triazole-linked cyclic dimers (C2), trimer (C3),
tetramer (C4), pentamer (C5), hexamer (C6), heptamer (C7) and a series of mixed 1,4/1,5-triazole-linked linear oligomers. A: Parasites were
applied to macrophages without ( ) or with ( ) pre-incubation with triazoles and following 6 days incubation the numbers of trypomastigotes in
the medium was quantified as % reduction in the number of parasite released from macrophages (% inhibition). B: Parasites and triazoles were
applied to macrophages and the number of amastigotes present inside the macrophages was quantified as an average number of parasite per
macrophage (T. cruzi/Macrophage).
To complement the above assays, we also assessed differentiated amastigote-form T. cruzi numbers
inside infected macrophages, and the impact of triazole-linked oligomers on these numbers. In keeping
with the trypomastigote results (Fig. 4 A), triazole-linked oligomers resulted in a reduction in the number of
parasites found inside macrophages (Fig. 4 B; Fig. 5), with a general trend towards larger structures giving
greater effect.
Taken together, these data confirm the potential of triazole-linked trans-sialidase ligands to block
macrophage invasion by T. cruzi parasites.
Fig. 5. Representative images illustrating amastigote-form parasite numbers present inside macrophages infected with T. cruzi in (A) the
absence and (B) the presence of 250 µM mixed length 1,4/1,5 triazole-linked linear oligomers. Arrows indicate cells infected with T. cruzi.

12
3. Conclusions
ACCEPTED MANUSCRIPT
In this study we have investigated the CuAAC-mediated oligomerisation of an azido-alkyne-
functionalised galactose building block 7 for the development of novel oligomeric, triazole-linked
compounds with potential for impact against the human parasite Trypanosoma cruzi. Despite our initial
expectation that the installation of flexible ethylene-glycol-based linker might favour polymerisation of
monomer over cyclisation, mixed populations of linear and cyclic triazole-linked oligomers of d.p. 1 to >10
were evident. The cyclic compounds were separable from each other and from the corresponding linear
materials by RP HPLC, whilst separation of linear materials up to d.p. 5 could be achieved by gel
permeation chromatography.
The overall yield of triazole-linked products obtained from
polymerisation/cyclooligomerisation of monomer 7 was around 54%, with a higher proportion of linear
products (36%) compared to cyclic products (18%) formed in CuAAC reactions conducted at room
temperature.
Judicious choice of the sites of galactose modification in monomer 7 (the 1- and 6-positions) results in
oligomeric triazole-linked materials that present the 2-, 3- and 4-hydroxyl groups of the sugar units that are
required for binding to T. cruzi trans-sialidase.41 This was borne out by in vitro biotransformations, which
established that triazole-linked 1,6-substituted pseudo-galactoligomers are indeed recognised as acceptor
substrates by T. cruzi trans-sialidase. Given the high copy number presentation of trans-sialidase on the
surface of T. cruzi, multivalent ligands for the enzyme might block the surface of the parasite and inhibit
TcTS-mediated host cell invasion. Encouragingly, preliminary assessment of linear and cyclic triazole-
linked pseudo-galactooligomers clearly establishes their ability to block T. cruzi invasion of macrophages,
with distinct advantage demonstrated by pre-incubation of triazole-linked oligomers with parasite prior to
exposure to macrophages. While this phenomenon would be difficult to reproduce medicinally, it suggests
that there is scope for optimisation of ligand presentation. The versatile nature of CuAAC-based
oligomerisation approach offers significant scope in this regard.
4. Experimental section
Chemicals were purchased as reagent grade and used without further purification. Ammonia solution
(7N NH3 in methanol) and methanolic sodium methoxide (0.5M NaOMe in methanol) were purchased from
Sigma Aldrich. All moisture-sensitive reactions were performed under a dry nitrogen atmosphere using
oven-dried glassware. Anhydrous solvents were purchased from Sigma Aldrich and dichloromethane was
freshly distilled from CaH2 prior to use. Microwave-assisted reactions were carried out in Biotage Initiator
microwave. Reactions were monitored by thin-layer chromatography (TLC) on aluminium-backed, pre-
coated silica gel plates (Silica Gel 60 F254, E. Merck) with the indicated eluents. Compounds were
visualised under UV light (λ 254 nm) and by dipping in ethanol-sulfuric acid (95:5, v/v) followed by

13
heating. Flash chromatography was performed on a Biotage Isolera MPLC system using pre-packed
ACCEPTED MANUSCRIPT
silica gel cartridges. HPLC was carried out using a Dionex system equipped with a Corona Charged
Aerosol (CAD) detector. Gel permeation chromatography was performed on a TSK-HW40S column (1.6 ×
80 cm) eluted with water at 0.4 mL/min flow rate. Optical rotations were measured at 20 °C in 1 mL cell in
the stated solvent using a Perkin-Elmer 341 polarimeter equipped with a sodium lamp. Nuclear magnetic
resonance spectra were recorded on a Bruker Avance III 400 NMR or Bruker Avance 600 spectrometer at
298 K. Chemical shifts (δ) are reported in parts per million (ppm) with respect to internal tetramethylsilane
(TMS) in CDCl3 and residual HOD signal in D2O. NMR signal assignments were made with the aid of
COSY and HSQC experiments. High resolution ESI MS data were obtained using a Waters Synapt G2
mass spectrometer; data for multiply charged species were deconvoluted using the Mass X3 program.
Infra-red spectra were obtained on a Perkin-Elmer FTIR Spectrum BX instrument equipped with MIRacle
single reflection horizontal accessory.
4.1. 2,3,4-Tri-O-benzoyl-6-O-(prop-2-ynyl)-1-O-trichloroacetimidoyl-α-D-galactopyranose (4)
Benzoyl chloride (25 mL, 215 mmol) was added dropwise to a stirred solution of 6-O-propargyl
galactose (1)32 (9.7 g, 43 mmol) in pyridine (95 mL) at 0 °C, the mixture was stirred for 1 h at room
temperature and ice-cold water (100 mL) was carefully added. The product was extracted with CH2Cl2 (3 x
125 mL), the organic extracts were combined and washed with ice-cold 1M HCl (3 x 100 mL), saturated
aqueous NaHCO3 solution (4 x 100 mL), dried (MgSO4), filtered and concentrated under reduced pressure.
The obtained residue was purified by flash column chromatography (toluene/EtOAc, 7:3) to give
compound 2 (21.9 g, 81%, mixture of α/β-anomers) as a colourless solid. Benzoate 2 (6.6 g, 104 mmol)
was dissolved in anhydrous THF (70 mL), 7 N NH3 in methanol (23 mL) was added, the solution was
stirred for 26 h at room temperature and the solvent was removed under reduced pressure. The residue was
purified by flash column chromatography (hexane/EtOAc, 7:3) to give hemiacetal 3 (3.5 g, 64%).
Compound 3 (2.8 g, 5.2 mmol) and trichloroacetonitrile (1.9 g, 13.0 mmol) were dissolved in anhydrous
CH2Cl2 (10 mL), the solution was cooled to 0 °C, stirred for 2 h and concentrated under reduced pressure.
The residue was purified by flash column chromatography (hexane/EtOAc, 7:3) to afford
trichloroacetimidate 4 (2.9 g, 99 %) as a colourless syrup. Rf 0.55 (hexane/EtOAc, 7:3); [α]D + 1.2 (c 0.6,
CHCl3); δH (400 MHz, CDCl3) 8.61 (1H, s, NH), 8.09–8.07 (2H, m, Ph), 7.96–7.93 (2H, m, Ph), 7.81–7.79
(2H, m, Ph), 7.63 (1H, m, Ph), 7.52–7.24 (8H, m, Ph), 6.88 (1H, d, J1,2=3.6 Hz, H-1), 6.08 (1H, dd, J3,4=3.4
Hz, J4,5=1.3 Hz, H-4), 6.04 (1H, dd, J3,4=3.4 Hz, J2,3=10.6 Hz, H-3), 5.92 (1H, dd, J1,2=3.6 Hz, J2,3=10.6
Hz, H-2), 4.68 (1H, m, H-5), 4.12 (2H, m, CH2C≡CH), 3.75 (2H, d, J=6.2 Hz, H-6a, H-6b), 2.24 (1H, t,
J=2.3 Hz, CH2C≡CH); δC (100.6 MHz, CDCl3) 165.7-165.4, 133.6-133.2, 129.9-128.3, 93.9, 75.3, 70.3,
68.6, 68.1, 67.5, 67.1, 58.7; HRMS (ESI) m/z calcd for C32H26Cl3NO9Na+ ([M+Na]+): 696.0571, found:
696.0565.

14
ACCEPTED MANUSCRIPT
4.2. 2-(2-(2-Chloroethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-2,3,4-tri-O-benzoyl-β-D-
galactopyranoside (5)
To a solution of imidate 4 (2.9 g, 5.2 mmol) and 2-(2-(2-chloroethoxy)ethoxy)ethanol (730 mg, 4.3
mmol) in anhydrous CH2Cl2 (50 mL) containing 4 Å mol. sieves TMSOTf (90 µL, 0.5 mmol) was
gradually added at -30 °C, the reaction mixture was allowed to warm to room temperature and stirred for 3
h. The reaction was quenched by addition of Et3N (1.0 mL) and concentrated under reduced pressure. The
residue was purified by flash chromatography (EtOAc/hexane, 7:3) to give compound 5 (2.7 g, 85%) as a
colourless solid. Rf 0.17 (EtOAc/hexane, 7:3); [α]D + 0.9 (c 0.7, CHCl3); IR (neat) 2877, 2360, 1725, 1601,
1584, 1451, 1260, 1094, 750 cm-1; δH (400 MHz, CDCl3) 8.09–8.07 (2H, m, Ph), 7.98–7.96 (2H, m, Ph),
7.79-7.77 (2H, m, Ph), 7.61 (1H, m, Ph), 7.53–7.36 (8H, m, Ph), 5.90 (1H, dd, J3,4=3.5 Hz, J4,5=1.0 Hz, H-
4), 5.75 (1H, dd, J1,2=8.0 Hz; J2,3=10.5 Hz, H–2), 5.56 (1H, dd, J3,4=3.5 Hz; J2,3=10.5 Hz, H-3), 4.90 (1H,
d, J1,2=8.0 Hz, H–1), 4.19–4.12 (3H, m, H–5, CH2C≡CH), 4.11–4.04 (1H, m, GalOCH2), 3.87–3.81 (1H, m,
GalOCH2), 3.76 (2H, m, H-6a, H-6b), 3.69–3.60 (4H, m, CH2), 3.59–3.53 (2H, m, CH2Cl2), 3.51–3.47 (2H,
m, CH2), 3.44–3.38 (2H, m, CH2), 2.30 (1H, t, J=2.3 Hz, CH2C≡CH); δC (100.6 MHz, CDCl3) δ 165.6–
165.2, 133.4–133.2, 130.0–128.3, 101.7, 79.1, 75.0, 72.7, 71.9, 71.3, 70.7, 70.5, 70.4, 69.9, 69.5, 68.5,
67.8, 58.7, 42.7; HR-MS (EI) m/z calcd for C36H37ClO11Na+ ([M+Na]+): 703.1922, found: 703.1917.
4.3. 2-(2-(2-Azidoethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-2,3,4-tri-O-benzoyl-β-D-galactopyranoside
(6)
To a solution of β-galactoside 5 (5.2 g, 7.6 mmol) in DMF (50 mL) NaN3 (2.5 g, 38.0 mmol) and NaI
(1.1 g, 8.0 mmol) were added and the reaction mixture was stirred at 70 ᵒC for 28 h. The reaction mixture
was diluted with water (20 mL) and extracted with CH2Cl2 (3 x 30 mL). The resulting organic extracts
were dried (MgSO4), and concentrated under reduced pressure to give compound 6 (5.0 g, 97%) as a
colourless syrup. Rf 0.17 (hexane/EtOAc, 7:3); [α]D + 0.9 (c 0.7, CHCl3); IR (neat) 2874, 2359, 2107, 1724,
1601, 1584, 1451, 1259, 1094, 752 cm-1; δH (400 MHz, CDCl3) 8.09–8.07 (2H, m, Ph), 7.98–7.96 (2H, m,
Ph), 7.79–7.77 (2H, m, Ph), 7.61 (1H, m, Ph), 7.53–7.36 (8H, m, Ph), 5.90 (1H, dd, J3,4=3.5 Hz, J4,5=1.0
Hz, H-4), 5.75 (1H, dd, J1,2=8.0 Hz; J2,3=10.5 Hz, H-2), 5.56 (1H, dd, J3,4=3.5 Hz; J2,3=10.5 Hz, H-3), 4.90
(1H, d, J1,2=8.0 Hz, H-1), 4.19–4.12 (3H, m, H-5, CH2C≡CH), 4.11–4.04 (1H, m, GalOCH2), 3.87–3.81
(1H, m, GalOCH2), 3.76 (2H, m, H-6a, H-6b), 3.69–3.59 (2H, m, CH2), 3.57–3.52 (2H, m, CH2), 3.52–3.46
(2H, m, CH2), 3.44–3.37 (2H, m, CH2), 3.51–3.29 (2H, m, CH2N3), 2.30 (1H, t, J=2.3 Hz, CH2C≡CH); δC
(100.6 MHz, CDCl3) 165.6–165.2, 133.4–133.2, 130.0–128.3, 101.5, 79.1, 75.0, 72.7, 71.9, 71.3, 70.7,
70.5, 70.4, 69.95, 69.9, 69.5, 68.5, 67.8, 58.7, 50.6; HRMS (ESI) m/z calcd for C36H37N3O11Na+
([M+Na]+): 710.2326, found: 710.2320.

15
ACCEPTED MANUSCRIPT
4.4. 2-(2-(2-Azidoethoxy)ethoxy)ethyl 6-O-(prop-2-ynyl)-β-D-galactopyranoside (7)
A solution of compound 6 (2.4 g, 3.48 mmol) in absolute MeOH (20 mL) was treated with 0.5 M
NaOMe in MeOH (2.7 mL, 1.39 mmol), the solution was kept at room temperature for 1 h, neutralized with
dry ice, concentrated under reduced pressure and purified by flash chromatography (CH2Cl2/MeOH, 9:1) to
give compound 7 (1.2 g, 91%) as a colourless syrup. [α]D -1.6 (c 1.3, MeOH); δH (600 MHz, Methanol-d4)
4.16 (1H, d, J1,2=7.6 Hz, H-1), 4.11 (2H, d, J=2.4 Hz, CH2C≡CH), 3.94–3.84 (1H, m, H-6a), 3.69 (1H, dd,
J3,4=3.4 Hz, , J4,5=1.1 Hz, H-4), 3.67–3.54 (12H, m, H-5, H-6b, CH2), 3.43 (1H, dd, J1,2=7.6 Hz, J2,3=9.7
Hz, H-2), 3.37 (1H, dd, J2,3=9.7 Hz, J3,4=3.4 Hz, H-3), 3.28 (2H, t, J=5.0 Hz, CH2N3), 2.76 (1H, t, J=2.4
Hz, CH2C≡CH); δC (100.6 MHz, D2O) 105.0, 80.6, 76.0, 75.0, 74.7, 72.5, 71.6 (2C), 71.4, 71.1, 70.4, 70.3,
69.7, 59.3, 51.8; HRMS (ESI) m/z calcd for C15H25N3O8Na+ ([M+Na]+): 398.1539, found: 398.1534.
4.5. Cu(I)-catalysed oligomerisation of monomer 7
A solution of azido-alkyne monomer 7 (100 mg, 0.3 mmol) containing Cu turnings (260 mg, 4.1 mmol)
and CuSO4 (11 mg, 0.1 mmol) in DMF (0.27 mL) was placed into a sealed microwave tube. The tube was
either submitted to microwave irradiation at 110 ºC at 50 W magnetron power (Method A) or stirred at
room temperature for 2 days (Method B). The reaction progress was followed by the TLC
(CH3CN/EtOAc/iPrOH/H2O, 85:20:50:50) taking samples at 15 min intervals (Method A) or 24 h intervals
(Method B). After completion the reaction mixtures were separated from Cu turnings and the solvent was
removed by repeated evaporation with toluene under reduced pressure. The residues were re-dissolved in
water and subjected to purification using HPLC on Phenomenex Luna C-18 column (10 × 250 mm). HPLC
method included linear gradient from 0% to 35% in 50 min of MeCN in 0.1% aqueous CF3CO2H at
1mL/min flow rate. Peaks were detected by a CAD and collected as individual compounds or mixtures of
oligomers which were identified using 1H NMR and/or HRMS.
Cyclic monomer 8: HPLC retention time 16.3 min; yield 9.0 mg (9.0%) (Method A), 6.7 mg (6.7%)
(Method B); δH (400 MHz, D2O) 8.15 (1H, s, CH triazole), 4.93 (1H, d, J=13.4 Hz, CH2a-triazole), 4.71-
4.61 (3H, m, CH2b-triazole, CH2N), 4.38 (1H, d, J1,2=7.9 Hz, H-1), 4.02-3.94 (3H, m, OCH2CH2N, H-6a),
3.91 (1H, d, J3,4=3.5 Hz, H-4), 3.90-3.66 (10H, m, CH2, H-5, H-6b), 3.64 (1H, dd, J2,3=9.7 Hz; J3,4=3.5 Hz,
H-3), 3.50 (1H, dd, J1,2=7.9 Hz; J2,3=9.7 Hz, H-2); δC (100.6 MHz, D2O) 143.6, 126.2, 102.2, 74.2, 72.7,
+
70.6, 69.6, 69.4, 69.4, 69.1, 68.9, 68.5, 62.4, 49.7; HR-MS (EI) m/z calcd for C15H26N3O8 [M+H]+:
376.1714, found 376.1710.
Cyclic dimer 9: HPLC retention time 21.3 min; yield 3.0 mg (3.0%) (Method A), 2.2 mg (2.2%) (Method
B); δH (400 MHz, D2O) 8.12 (2H, s, CH triazole), 4.74 (4H, m, CH2-triazole), 4.65 (4H, m, CH2N), 4.38
(2H, d, J1,2=7.9 Hz, H-1), 4.02–3.92 (6H, m, OCH2CH2N, H-6a), 3.91 (2H, d, J3,4=3.6 Hz, H-4), 3.86-3.70
(8H, m, CH2, H-5, H-6b), 3.69–3.57 (14H, m, CH2, H-3), 3.53 (2H, dd, J1,2=7.9 Hz, J2,3=9.9 Hz, H-2); δC

16
ACCEPTED MANUSCRIPT
(100.6 MHz, D2O) 143.7, 125.6, 102.8, 73.5, 72.6, 70.6, 69.6 (2C), 69.4, 68.9, 68.7, 68.6, 63.4, 50.1;
HRMS (ESI) m/z calcd for C30H51N6O16 ([M+H]+): 751.3357, found: 751.3334.
Cyclic trimer 10: HPLC retention time 23.9 min; yield 1.6 mg (1.6%) (Method A), 1.0 mg (1.0%)
(Method B); δH (400 MHz, D2O) 8.10 (3H, s, CH triazole), 4.72 (6H, m, CH2-triazole), 4.63 (6H, m,
CH2N), 4.39 (3H, d, J1,2=7.8 Hz, H-1), 4.00–3.93 (9H, m, OCH2CH2N, H-6a), 3.90 (3H, d, J3,4=3.4 Hz, H-
4), 3.85–3.73 (12H, m, CH2, H-5, H-6b), 3.70–3.58 (21H, m, CH2, H-3), 3.53 (3H, dd, J1,2=7.9 Hz, J2,3=9.9
Hz, H-2); δC (100.6 MHz, D2O) 143.8, 125.5, 102.8, 73.5, 72.6, 70.6, 69.7, 69.6 (2C), 69.4, 68.9, 68.7,
63.4, 50.0; HRMS (ESI) m/z calcd for C45H76N9O24 ([M+H]+): 1126.4998, found: 1126.4961.
Cyclic tetramer 11: HPLC retention time 25.5 min; yield 1.1 mg (1.1%) (Method A), 0.8 mg (0.8%)
(Method B); δH (400 MHz, D2O) 8.10 (4H, s, CH triazole), 4.72 (8H, m, CH2-triazole), 4.63 (8H, m,
CH2N), 4.39 (4H, d, J1,2=7.8 Hz, H-1), 4.00–3.93 (12H, m, OCH2CH2N, H-6a), 3.90 (4H, d, J3,4=3.4 Hz, H-
4), 3.85–3.73 (16H, m, CH2, H-5, H-6b), 3.70–3.58 (28H, m, CH2, H-3), 3.53 (4H, dd, J1,2=7.9 Hz, J2,3=9.9
Hz, H-2); δC (100.6 MHz, D2O) 143.8, 125.5, 102.8, 73.5, 72.6, 70.6, 69.7, 69.6, 69.5, 69.4, 68.9, 68.7,
63.4, 50.0; HRMS (ESI) m/z calcd for C60H101N12O32 ([M+H]+): 1501.664, found: 1501.6511.
Cyclic pentamer 12: HPLC retention time 26.5 min; yield 1.3 mg (1.3%) (Method A), 0.6 mg (0.6%)
(Method B); δH (400 MHz, D2O) 8.10 (5H, s, CH triazole), 4.71 (10H, m, CH2-triazole), 4.63 (10H, m,
CH2N), 4.39 (5H, d, J1,2=7.9 Hz, H-1), 4.01–3.94 (15H, m, OCH2CH2N, H-6a), 3.90 (5H, d, J3,4=3.4 Hz, H-
4), 3.85–3.73 (20H, m, CH2, H-5, H-6b), 3.70–3.58 (35H, m, CH2, H-3), 3.53 (5H, dd, J1,2=7.9 Hz, J2,3=9.9
Hz, H-2); δC (100.6 MHz, D2O) 143.8, 125.5, 102.8, 73.5, 72.6, 70.6, 69.6, 69.6, 69.5, 69.4, 68.9, 68.7(2
C), 63.4, 50.0; HRMS (ESI) m/z calcd for C75H126N15O40 ([M+H]+): 1876.8281, found: 1876.8248.
Cyclic hexamer 13: HPLC retention time 27.2 min; yield 0.5 mg (0.5%) (Method B); δH (400 MHz, D2O)
8.11 (6H, s, CH triazole), 4.72 (12H, m, CH2-triazole), 4.67-4.62 (12H, m, CH2N), 4.39 (6H, d, J1,2=7.8 Hz,
H-1), 4.01–3.96 (18H, m, OCH2CH2N, H-6a), 3.90 (6H, d, J3,4=2.8 Hz, H-4), 3.87–3.71 (24H, m, CH2, H-
5, H-6b), 3.70–3.59 (42H, m, CH2, H-3), 3.53 (6H, dd, J1,2=7.9 Hz, J2,3=9.8 Hz, H-2); δC (100.6 MHz,
D2O) 102.8, 73.4, 72.5, 70.6, 69.5, 69.3, 68.8, 68.7, 63.4, 50.0; HRMS (ESI) m/z calcd for C90H151N18O48
([M+H]+): 2251.9923, found: 2251.9792.
Cyclic oligomers (DP>6): HPLC retention time 28 to 30 min; yield 13.7 mg (13.7%) (Method A), 6.0 mg
(6.0%) (Method B).
Linear oligomers: HPLC retention time 32.0 min; yield 25.8 mg (25.8%) (Method A), 35.7 mg (35.7%)
(Method B); HRMS (ESI) m/z calcd for ([M+H]+): 376.1751; 751.3357; 1126.4998; 1501.6640; 1876.8281;
2251.9923, found: 376.1708; 751.3351; 1126.4985; 1501.6631; 1876.8273; 2251.9897.

17
ACCEPTED MANUSCRIPT
4.6. Uncatalysed oligomerisation of monomer 7
A solution of compound 7 (100 mg, 0.3 mmol) was placed into a sealed microwave tube. The reaction
mixture was submitted to microwave irradiation at 110 ºC at 50 W magnetron power (Method C). The
reaction progress was followed by TLC (CH3CN/EtOAc/iPrOH/H2O, 85:20:50:50) taking samples at 15
min intervals (Method C). After 30 min the solvent was removed by repeated evaporation with toluene
under reduced pressure. The residues were redissolved in water and subjected to purification using HPLC
on Phenomenex Luna C-18 column (10 × 250 mm). HPLC method included linear gradient from 0% to
35% in 50 min of MeCN in 0.1% aqueous CF3CO2H. Peaks were detected by a CAD detector and collected
1
as individual compounds or mixtures of oligomers which were identified using H NMR and/or HRMS
(ESI).
Linear dimer 14: HPLC retention time 32.0 min; yield 1.2 mg (1.2%); δH (400 MHz, D2O) 8.02 (s, 1H),
4.60–4.52 (2H, m, CH2-triazole), 4.36–4.27 (2 H, m, H-1, H-1’), 4.23–4.14 (2 H, m, CH2C≡CH), 3.98–3.87
(4H, m, OCH2CH2N), 3.84–3.81 (2H, m, H-4), 3.78–3.52 (26H, m, CH2, H-6, H’, H5, H-3), 3.47–3.41 (4
H, m, H-2, CH2N3); δC (100.6 MHz, D2O) 102.7, 76.9, 73.4, 72.5, 70.5, 68.7, 68.5, 63.3, 58.1, 50.0, 49.9;
Cyclic monomer 19: HPLC retention time 14.9 min; yield 8.7 mg (8.7%); δH (400 MHz, D2O) 7.88 (1H,
s, CH triazole), 4.92–4.83 (2H, m, CH2-triazole), 4.72–4.56 (2H, m, CH2N), 4.42 (1H, d, J1,2=7.9 Hz, H-1),
4.06–4.02 (2H, m, OCH2CH2N), 3.97–3.82 (6H, m, CH2, H-4, H-5,H-6), 3.74–3.57 (7H, m, CH2, H-3),
3.53 (1H, dd, J1,2=7.9 Hz; J2,3=9.9 Hz, H-2); δC (100.6 MHz, D2O) 140.5, 102.2, 74.4, 72.7, 71.1, 70.6,
70.3, 69.7, 69.5, 69.3, 68.8, 61.2, 48.5; HRMS (ESI) m/z Calcd for C15H26N3O8 ([M+H]+): 376.1751,
found: 376.1701.
Linear dimer 20 HPLC retention time 32.0 min; yield 1.5 mg (1.5%); δH (400 MHz, D2O) 7.77 (1H, s,
CH triazole), 4.60–4.54 (2H, m, CH2N), 4.35–4.29 (2 H, m, H-1, H-1’), 4.23–4.14 (2 H, m, CH2C≡CH),
3.97–3.88 (4H, m, OCH2CH2N), 3.84–3.81 (2H, m, H-4),3.78–3.52 (26H, m, CH2, H-6, H-5, H-3), 3.47–
3.41 (4 H, m, H-2, CH2N3); δC (100.6 MHz, D2O) 102.7, 73.1, 72.4, 70.6, 68.7, 68.6, 60.3, 58.1, 50.0, 48.1.
4.7. Trans-sialidase-mediation sialylation of triazole-linked pseudo-galactooligomers
To a solution of fetuin42 as a donor substrate (25 ꢀL, 3 mM in 0.1 mM phosphate buffer pH 7.0) and
triazole-linked acceptor substrates 8-10 and 19 (25 ꢀL, 1 mM in 0.1 mM phosphate buffer pH 7.0) was
added TcTS enzyme (25 ꢀL). The mixture was incubated at 28 °C for 5 days when additional 25 ꢀL
aliquots of TcTS enzyme and fetuin were added; after a further 2 days additional 25 ꢀL aliquot of fetuin
was added. Reaction progress was followed by TLC (CH3CN/EtOAc/iPrOH/H2O, 85:20:50:50). The
protein was precipitated by heating the mixture at 100 °C for 1 min and the denatured enzymes were

18
ACCEPTED MANUSCRIPT
removed by centrifugation (14000 rpm, 5 min). The supernatant was transferred to a new Eppendorf
tube, concentrated by freeze drying and submitted to HR-MS analysis.
4.8. Macrophage invasion assays
Compounds used in preliminary macrophage invasion assays included a series of mixed 1,4/1,5-
triazole-linked cyclic compounds C2, C3, C4, C5, C6, and C7 and a mixture of linear 1,4/1,5-triazole-
linked oligomers generated by Method C (Section 4.6). Trypanosoma cruzi Y strain was cultivated in the
LLC-MK2 host cells, as described elsewhere.43
Preparation of macrophages: Macrophages derived from bone marrow (BMMOs) were obtained as
described previously.44 Briefly, total bone marrow cells were cultured in RPMI 1640 medium (Sigma-
Aldrich), supplemented with 10% Fetal Bovine Serum (Cutilab) and 30% L-929 cell-conditioned media at
37°C under 5% CO2 atmosphere. On the seventh day of culture BMMOs were harvested and plated in 96 or
24 well microplates (at 5 x 105 cells/mL).
Preparation of trypomastigotes: Trypomastigote form Trypanosoma cruzi Y strain (1.5 x 106 cells/mL)
was added into wells containing BMMOs in two different ways: (Treatment 1) trypomastigotes and
triazole-linked compounds at a final concentration of 250 µM were plated at the same time; and (Treatment
2) trypomastigotes and triazole-linked compounds at a final concentration of 250 µM were first pre-
incubated for 1 hour, then the parasites were washed to remove free compound and were plated to the wells
containing BMMOs. In both methods the trypomastigotes were maintained for 2 h in contact with
BMMOs. The parasites that had not invaded macrophages were removed by serial washing after the 2 h
incubation.
Analysis of released trypomastigotes: After six days incubation, free trypomastigotes that had escaped
from macrophages were counted in a Neubauer chamber and results are expressed as a percentage
inhibition with respect to trypomastigotes released from infected cells that had not been treated with
triazole-linked compounds.
Analysis of amastigotes inside BMMOs: Macrophages infected with trypomastigotes as described above
(Treatment 1) were maintained in culture for four days, during which time the parasites differentiated into
amastigote forms. The infected macrophages were fixed with methanol, stained with Giemsa and analysed
using Leica DMI 4000B microscope. The results are expressed as an average number of amastigotes per
macrophage.
Acknowledgments

19
These studies were supported by the UK BBSRC Institute Strategic Programme Grant on
ACCEPTED MANUSCRIPT
Understanding and Exploiting Metabolism (MET) [BB/J004561/1], a DTP PhD studentship (to II) and the
John Innes Foundation. We acknowledge financial support and fellowships from the FAPESP (Fundação de
Amparo à Pesquisa do Estado de São Paulo-Brazil) and CNPq (Conselho Nacional de Desenvolvimento
Científico e Tecnológico). We are grateful to José Carlos Tomaz for conducting MS analyses.
Supplementary data
Supplementary data associated with this article can be found in the online version, at
http://dx.doi.org/10.1016/j.tet.XXXXXXXX. These data include additional details of HRMS, TLC
1
analyses, experimental details of the alternative route to 7, and H and 13C NMR spectra for
cycloligomerisation products.
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(but see reference 28 for examples of pyridyl-containing monomers that do form catenanes).
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ACCEPTED MANUSCRIPT
Supplementary Information
Click chemistry oligomerisation of azido-alkyne-functionalised galactose accesses
triazole-linked linear oligomers and macrocycles that inhibit Trypanosoma cruzi
macrophage invasion
Vanessa L. Campo,a, Irina M. Ivanova,b, Ivone Carvalho,a Carla D. Lopes,c Zumira A. Carneiro,c Gerhard
Saalbach,b Sergio Schenkman,d João Santana da Silva,c Sergey A. Nepogodievb and Robert A. Fieldb,*
a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Av. Café S/N, CEP 14040-903, Ribeirão Preto, SP, Brazil
b Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK
c Faculdade de Medicina de Ribeirão Preto, Department of Parasitology Microbiology and Immunology, USP, Av. Bandeirantes 3900, CEP 14049-900, Ribeirão
Preto, SP, Brazil
d Department of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Rua Botucatu 862 8, Andar 04023-062 São Paulo, SP, Brazil
List of Tables, Schemes and Figures
Yields of 1,4-triazole-linked cyclic and linear products from CuAAC reactions of
monomer 7
2
4
4
Table S1
Table S2
Scheme S1.
HRMS data for products of enzymatic sialylation of triazole-linked cyclic
galactooligomers
Alternative route to compound 7 based on BF3•OEt2-catalysed glycosylation with 1-
acetate 24.
The HRMS spectrum of the series of 1,4-triazole-linked linear products
3
4
Fig. S1
Fig. S2
The TLC analysis of enzymatic transformations of 1,4-triazole-linked cyclic monomer
8, 1,5-triazole-linked cyclic monomer 19, 1,4-triazole-linked cyclic dimer 9 and 1,4
triazole-linked cyclic trimer 10
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
1H NMR and 13C NMR spectra of compound
6
Fig. S3
Fig. S4
Fig. S5
Fig. S6
Fig. S7
Fig. S8
Fig. S9
Fig. S10
Fig. S11
Fig. S12
Fig. S13
Fig. S14
Fig. S15
Fig. S16
4
7
5
8
6
9
7
10
11
12
13
14
15
16
17
18
19
8
9
10
11
12
13
14
19
20
26
1

2
Tetrahedron
ACCEPTED MANUSCRIPT
Table S1 Yields of 1,4-triazole-linked cyclic and linear products from CuAAC reactions of monomer 7
Reaction products
Method A
Yield (%)
Method B
Yield (%)
8
9.0
3.0
1.6
1.1
1.3
6.7
2.2
1.0
0.8
0.6
0.5
6.0
35.7
9
10
11
12
13
Cyclic high mol. weight compounds
Mixture of linear compounds
13.7
25.8
14 [M+H]+
751.3351
100
15 [M+H]+
1126.4985
16 [M+H]+
1501.6631
17 [M+H]+
1876.8273
18 [M+H]+
2251.9897
8 [M+H]+
376.1708
1465.4302
1840.6017
2590.9238
2552.9568
1090.2657
2047.7090
1259.4308
964.4451
919.3895
2627.1487
733.3273
589.2822
0
mass
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
Fig. S1 The HRMS spectrum of the series of 1,4-triazole-linked linear products obtained from CuAAC oligomerisation of monomer 7.
2

ACCEPTED MANUSCRIPT
Fig. S2 The TLC analysis of enzymatic transformations of 1,4-triazole-linked cyclic monomer 8, 1,5-triazole-linked cyclic monomer 19, 1,4-
triazole-linked cyclic dimer 9 and 1,4 triazole-linked cyclic trimer 10 into mono-, di- and tri-sialylated cyclic compounds in the presence of
TcTS and a fetuin donor. Sialylated products were identified on TLC on the basis of the time course of enzymatic reactions. Negative
controls (lanes C1, C2, C3 and C4) were carried out in parallel with a donor and an acceptor without the enzyme. The progress of reactions
was monitored by analysing samples after 1 day, 7 days and 9 days. TLC was eluted with CH3CN/EtOAc/iPrOH/H2O (85/25/50/50).
Table S2 HRMS data for products of enzymatic sialylation of triazole-linked cyclic galactooligomers
Starting
material
Mono-sialylated
Di-sialylated
Tri-sialylated
Calcd m/z
Found m/z
Calcd m/z
Found m/z
Calcd m/z
Found m/z
Monomer 8
Monomer 19
Dimer 9
665.2523 ([M-H]-)
665.2523 ([M-H]-)
665.2519 ([M-H]-)
665.2519 ([M-H]-)
1040.4164 ([M-H]-) 1040.4237 ([M-H]-)
1331.5118 ([M-H]-) 13315272 ([M-H]-)
Trimer 10
1415.5806 ([M-H]-) 1415.5994 ([M-H]-) (21) 1706.6760 ([M-H]-) 1706.7021 ([M-H]-) (22) 1997.7714 ([M-H]-) 1997.7947([M-H]-) (23)
.
BF3 Et2O
O
O
O
NaOMe,
MeOH
AcO
AcO
HO
HO
O
CH2Cl2
O
O
OAc
HO
O
AcO
+
AcO
O
O
O
O
O
N3
O
N3
O
N3
AcO
32%
AcO
75%
HO
7
24
25
26
Scheme S1. Alternative route to compound 7 based on BF3•OEt2-catalysed glycosylation with 1-acetate 24.
3

4
Tetrahedron
ACCEPTED MANUSCRIPT
1. Experimental Section
1.1. 2-(2-(2-Azidoethoxy)ethoxy)ethanol (25)
A mixture of 2-(2-(2-chloroethoxy)ethoxy)ethanol (1g, 5.9 mmol) and NaN3 (5.75 g, 88.5 mmol) in DMF
(20 mL) was stirred for 9 h at room temperature. The solvent was removed under reduced pressure, the
residue was resuspended in CH2Cl2 and filtered through Celite. The product 25 was obtained as an yellow
oil, yield 1.03 g, (100%): FTIR (KBr) ν : 3417; 2869; 2104; 1666; 1066 cm-1; δH (CDCl3 , 400 MHz)
max
3.76-3.60 (10H, m, CH2O), 3.41 (2 H, t, J 5.0 Hz, CH2 N3).
1.2. 2-(2-(2-Azidoethoxy)ethoxy)ethyl) 2,3,4-tetra-O-acetyl-6-O-(prop-2-ynyl)-β-D-galactopyranoside
(26)
To a solution of acetate 24 (200 mg, 0.52 mmol) and alcohol 25 (108 mg, 0.62 mmol) in CH2Cl2 (10 mL)
BF3•Et2O (0.095 mL, 0.77 mmol) was added. The reaction mixture was stirred for 20 h at room temperature
and then 6 h at 52 ºC. The resulting solution was diluted with CH2Cl2, washed with satd NaHCO3 solution
and brine, dried (MgSO4), concentrated and purified by flash chromatography (EtOAc/Hex 1:1) to give
compound 26 (83 mg, 32%). [α]D25 + 18.2 (c 1.0 CHCl3);. δH (CDCl3 , 400 MHz) 5.42 (1H, d, J3,4=3.4 Hz,
H-4), 5.20 (1H, dd, J1,2=8.0 Hz; J2,3=10.5 Hz, H-2), 5.02 (1H, dd, J3,4=3.4 Hz; J2,3=10,5 Hz, H-3), 4.56 (1H,
d, J1,2=8.1 Hz, H-1), 4.13 (2H, dd, J=2.4 Hz, J=6.1 Hz, CH
J5,6a=6.3 Hz, J6,6b=12.4 Hz, H-6a), 3.76 (1H, dd, J5,6b=6,3 Hz, J6a,6b=12.2 Hz, H-6b), 3.70-3.60 (10H, m,
CH2), 3.4 (2H, t, J=5.2 Hz, CH2N3), 2.42 (1H, s, CH C≡CH), 2.4–1.9 (9H, 3s, Ac); δC (CDCl3, 100 MHz)
170.2, 170.1, 169.5 0 (COCH3); 101.4 (C-1); 79.1 (CH C≡CH), 74.9 (CH C≡CH); 72.0 (C-5); 71.0 (C-3);
70.75, 70.7, 70.4, 70.0 (OCH2), 69.1 (C-2), 69.0 (C-6), 67.7 (CH2), 67.5 (C-4), 58.6 (CH
2
C≡CH), 3.97 (1H, m, H-5), 3.85 (1H, dd,
2
2
2
2
C≡CH), 50.7
+
11Na ([M+Na] ): 524.1851,
(CH2N3), 20.8, 20.7, , 20.6 (COCH3); HRMS (ESI): m/z calcd for C21
H31
N3
O
found: 524.1841
4

ACCEPTED MANUSCRIPT
BzO
O
O
BzO
BzO
O
CCl3
4
NH
1
Fig. S3 H NMR and 13C NMR spectra of compound 4.
5

6
Tetrahedron
ACCEPTED MANUSCRIPT
BzO
O
O
O
O
BzO
O
Cl
OBz
5
Fig. S4 1H NMR and 13C NMR spectra of compound 5.
6

ACCEPTED MANUSCRIPT
BzO
O
O
O
O
BzO
O
N3
OBz
6
Fig. S5 1H NMR and 13C NMR spectra of compound 6.
7

8
Tetrahedron
ACCEPTED MANUSCRIPT
HO
O
O
O
O
HO
O
N3
OH
7
Fig. S6 1H NMR and 13C NMR spectra of compound 7.
8

N
ACCEPTED MANUSCRIPT
N
O
N
O
O
O
HO
HO
O
OH
8
Fig. S7 1H NMR and 13C NMR spectra of compound 8.
9