Development of ethynyl-2′-deoxyuridine chemical probes for cell proliferation

Article abstract of DOI:10.1016/j.bmc.2016.07.021

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2′-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.

Full text of DOI:10.1016/j.bmc.2016.07.021

Accepted Manuscript
Development of Ethynyl-2′-deoxyuridine Chemical Probes for Cell Prolifera-
tion
Carrie J. Lovitt, David H. Hilko, Vicky M. Avery, Sally-Ann Poulsen
PII:
S0968-0896(16)30525-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.07.021
BMC 13134
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 May 2016
7 July 2016
11 July 2016
Please cite this article as: Lovitt, C.J., Hilko, D.H., Avery, V.M., Poulsen, S-A., Development of Ethynyl-2′-
deoxyuridine Chemical Probes for Cell Proliferation, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.07.021
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Development of Ethynyl-2-deoxyuridine Chemical Probes for Cell Proliferation
Carrie J. Lovitt,a,# David H. Hilko,b,# Vicky M. Averya and Sally-Ann Poulsenb,*
aDiscovery Biology, Eskitis Institute for Drug Discovery, Griffith University, Don Young
Road, Nathan, Queensland 4111, Australia
bEskitis Institute for Drug Discovery, Griffith University, Don Young Road, Nathan,
Queensland 4111, Australia
*Corresponding author Tel: +61-7-37357825; Fax: +61-7-37356000; E-mail:
s.poulsen@griffith.edu.au.
#Equal first author author
Abbreviations
Nonstandard abbreviations: CuAAC, copper-catalyzed azide-alkyne cycloaddition; EdU, 5-
ethynyl-2´-deoxyuridine; BrdU, 5-bromodeoxyuridine; TK, thymidine kinase; TS,
thymidylate synthase; cLog P, calculated Log P; TBDPSCl, tert-butyldiphenylchlorosilane;
Emax, efficacy at maximum concentrations; FBS, fetal bovine serum; DPBS, Dulbecco’s
phosphate buffered saline

Abstract
A common method of evaluating cellular proliferation is to label DNA with chemical probes.
5-Ethynyl-2´-deoxyuridine (EdU) is a widely utilised chemical probe for labeling DNA, and
upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule
fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and
a reliance on nucleoside active transport mechanisms for entry into cells. Here we have
developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl
ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug
relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were
evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues
incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be
bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.

1. Introduction
Quantitative monitoring of DNA synthesis is used to assess the effect of test compounds on
cancer cell proliferation. Several probes have been generated to evaluate DNA replication in
cells, these probes act through incorporation into DNA during DNA duplication, a method
that directly represents the proliferative potential of cells. Historically, radiolabelled [3H]-
thymidine, was routinely utilised in experiments requiring examination of cell proliferation.
The disadvantages of using this probe include the handling of radioactivity in experimental
procedures and the lengthy methodology involved.1 Next, scientific advances led to the
development of 5-bromodeoxyuridine (BrdU), which permits DNA replication measurements
via microscopy.2 However, there are disadvantages to using BrdU as a proliferative marker,
including cellular toxicity and the denaturing procedures that are required to enable the
detection antibody access to the probe, which can be problematic for the use of additional
stains.3
More recently, 5-ethynyl-2´-deoxyuridine (EdU) was developed as a chemical probe for DNA
replication. Newly synthesised DNA with incorporated EdU is visualised by microscopy
following copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry with a
fluorescent azide.4 The fluorescent azides in use with EdU are small molecules, considerably
smaller than detection antibodies used with BrdU, hence DNA denaturation is not required for
the click chemistry reaction. Thus, limitations associated with the use of BrdU techniques,
including multi-parameter staining, are overcome when using EdU to monitor cell
proliferation.5 Additionally, CuAAC conditions do not compromise cell structure or
antigenicity, and allow multiple fluorescence, high resolution and quantitative microscopy.5
Previous research has successfully utilised EdU to examine cellular proliferation in diverse
fields of study including the nervous system6 and cancer.7 Following cell entry EdU is
converted to EdU-5-monophosphate with thymidine kinase (TK). EdU-5-monophosphate is
an inhibitor of thymidylate synthase (TS) and is responsible for the cytotoxicity associated
with EdU. The use of EdU is thus restricted to experiments that require short exposure times
and/or low probe concentrations.8
The cells uptake of BrdU and EdU is dependent on nucleoside active transport mechanisms
via nucleoside-specific membrane transport carriers (NT). There are seven distinct NT
activities observed in human cells, four have been characterised, and of these, two have broad

selectivity, accepting both purine and pyrimidine nucleosides, one has greater affinity for
pyrimidine nucleosides and one transports only purine nucleosides.9 Human NTs do not
accept phosphorylated compounds, hence once a nucleoside enters a cell via NTs it is
phosphorylated by kinases and then remains trapped intracellularly.9 Additionally, NT
expression profiles are variable across different cancer cell populations9 and this impedes
comparative studies using different cancer cell lines, for example assessing DNA synthesis
with EdU in different cancer cell lines when treated with the same test compound. We
recently reported the ability of 3´ and 3´,5´-acetylated EdU, compounds 1a and 2a,
respectively, to incorporate into nuclear DNA of proliferating mammalian cells.10 Our
rationale was that these compounds, being less polar than EdU, should enter cells by passive
membrane diffusion, independent of nucleoside transporters. Additionally, as the compounds
are simple acyl esters they were expected to be substrates for nonspecific intracellular
carboxylesterase enzymes, with enzymatic hydrolysis of 1a and 2a able to release free EdU
(and acetate). While this earlier study demonstrated that the acetyl group may be used as a
pro-label moiety for EdU, the extent of DNA labeling with 1a and 2a was substantially less
than when using EdU directly or a phosphorylated EdU pro-label.10 In this study we explore
more broadly acylated EdU analogues for their capacity to label DNA in proliferating cells.
Our goal was to identify novel EdU pro-labels that exhibit a balance between good cellular
uptake by passive membrane diffusion and efficient intracellular esterase hydrolysis to release
EdU. Additionally, as EdU pro-labels these compounds may in principle limit the real time
pool of EdU available to TK, and could have the added benefit of reduced cytotoxicity
compared to EdU.
2. Results and Discussion
A chemical probe for bioconjugation must minimally impact on the living cell. Following cell
entry, the probe must be accepted by the cells metabolic enzymes for robust incorporation
into the target biomolecule alongside the canonical counterpart.11 We have developed the
concept of pro-labels to monitor DNA synthesis.10 The pro-label:chemical probe relationship
parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. Just as
a prodrug needs to be modified in vivo to release the bioactive drug, similarly a pro-label is
modified in vivo to release the chemical probe. For a small molecule to have optimal
membrane permeability by passive diffusion, the compounds Log P value is ideally 0 < Log P
< 3. While pro-label compounds 1a and 2a have increased lipophilicity compared to EdU,
their calculated Log P (cLog P) values are still below zero (–1.21 and –0.98, respectively).

These values are indicative of molecules with poor membrane permeability and may account
for the substantially lessened DNA labeling efficiency that was observed with compounds 1a
and 2a compared to EdU in our earlier study.10 Short chain acyl groups that are more
lipophilic than the acetyl group should enhance passive membrane permeability of EdU pro-
labels. Three short chain mono-acylated EdU derivatives 1b-1d and their corresponding bis-
acylated derivatives, compounds 2b-2d, were designed and synthesized (Figure 1). The panel
of EdU pro-labels was designed to provide compounds with an incremental progression of
cLog P values to vary passive membrane permeability (Table 1). Our intention with this
design was to ascertain if there is a ‘sweet spot’ between cellular uptake and esterase
processing, wherein labeling efficiency with an acylated EdU pro-label is comparable to EdU.
EdU, and the acetylated EdU compounds (1a and 1b) were used as control compounds, to
provide a benchmark for comparison. Specifically the short chain acyl groups n-propionyl (1b
and 2b), n-butanoyl (1c and 2c) and n-pentanoyl (1d and 2d) were investigated (Figure 1).
Compound toxicity for EdU and pro-labels 1a-1d and 2a-2d was also investigated.
O
O
N
O
N
NH
NH
NH
N
O
O
O
RO
HO
HO
O
O
O
OR
1a-1d
OR
2a-2d
OH
EdU
R
O
O
O
O
1c, 2c
1d, 2d
1b, 2b
1a, 2a
Figure 1. EdU and acylated EdU pro-label compounds 1a-1d and 2a-2d.
Table 1. Calculated Log P (cLog P) values for EdU and acylated EdU pro-label compounds
1a-1d and 2a-2d.
cLog P a
Compd
EdU
3´ group
5´ group
H
H
–1.44
O
H
–1.21
1a

O
O
O
O
O
O
O
H
H
H
–0.56
–0.14
+0.27
–0.98
+0.32
+1.16
+1.99
1b
1c
1d
2a
2b
2c
2d
O
O
O
O
a Calculated using Instant JChem 5.12.0.
The synthesis of pro-label compounds 1a-1d and 2a-2d was carried out as outlined in Scheme
1.10, 12 To synthesize the mono-acylated compounds 1a-1d the 5 primary hydroxyl group of
5-iodo-2-deoxyuridine (3) was first selectively protected using tert-butyldiphenylchlorosilane
(TBDPSCl) to give intermediate 4. Next, reaction of 4 with trimethylsilylacetylene using
Sonogashira conditions (Et3N, CuI and Pd(PPh3)4 in anhydrous DMF at room temperature)
gave intermediate 5. Acylation at the 3-OH of 5 with the appropriate anhydride in pyridine at
60˚C gave 6a-6d. Finally, the combined removal of the TMS and TBDPS protecting groups
of 6a-6d was achieved using TBAF. The bis-acylated compounds 2a-2d were prepared from
3 following acylation with the appropriate anhydride in pyridine ( 7a-7d), installation of the
TMS protected alkyne ( 8a-8d) and lastly, removal of the TMS protecting groups with
TBAF.

O
N
O
N
TMS
O
N
TMS
O
N
I
NH
NH
NH
NH
(iv)
(ii)
(iii)
O
O
O
O
HO
TBDPSO
TBDPSO
TBDPSO
(i)
O
O
O
O
O
N
OR
1a-1d
OH
OH
OR
6a-6d
I
NH
4
5
O
HO
O
O
TMS
O
N
O
N
R
OH
I
NH
NH
NH
O
O
3
N
O
O
1c, 2c
(ii)
(iv)
1a, 2a
O
RO
RO
RO
(iii)
O
O
O
O
O
1d, 2d
1b, 2b
OR
7a-7d
OR
2a-2d
OR
8a-8d
Reagents and Conditions: (i) imidazole (1.1 equiv.), anhydrous DMF, TBDPSCl (1.15
equiv.), rt, 24 h. (ii) trimethylsilylacetylene (5 equiv.), Et3N (2 equiv.), CuI (0.1 equiv.),
Pd(PPh3)4, (0.05 equiv.), anhydrous DMF, rt, 1-12 h; (iii) acid anhydride R2O (4 equiv. per
OH group), pyridine (20 equiv.), 60 ˚C, 2-8 h. (iv) TBAF (1.2 equiv.), THF, 1 h.
Scheme 1. Synthetic route for preparation of EdU pro-label compounds 1a-1d and 2a-2d.
Pro-label compounds, thymidine derivatives 1a-1d and 2a-2d, were assessed for the ability to
incorporate into newly synthesized nuclear DNA of proliferating MDA-MB-231 and PC3
cancer cells. Cells were treated for 4h with 10, 5 and 1 M of test probe, then fixed and
labeled with Alexa Fluor 488 azide using CuAAC (Figure 2). Cells were counterstained with
Hoechst, a fluorescent DNA stain for all nuclei. Automated quantification of fluorescently
labeled cells provided the percentage of all cells that were labeled with test probe (Figure 2).
Upon application of 10 µM EdU and pro-label analogues, incorporation ranging from 26 ±
4.6% to 37 ± 1.7% was detected in both MDA-MB-231 and PC3 cells labelled with EdU, 1c,
1d, 2c, and 2d (Figure 2). For pro-labels 1a, 1b, 2a, and 2b the observed incorporation levels
were much lower (below 15%) in both cell lines. A similar labeling profile was observed
when the cancer cell lines were exposed to 5 µM of EdU and pro-label compounds, however,
there was reduced labeling with 2c and 2d (17 ± 3.3% and 15 ± 1.2%, respectively) on the
MDA-MB-231 cell line. While the labeling profile for EdU and test compounds in treated
PC3 and MDA-MB-231 cell types was similar upon application of 5 and 10 µM
concentrations, when 1 µM of the compound was applied to cells, differences in cellular DNA
incorporation were observed. EdU demonstrated the highest level of incorporation at 1 µM in

both cell lines (19 ± 2.5% for PC3 cells and 25 ± 1.4% for MDA-MB-231 cells). The labeling
efficacy of the best pro-label compounds, 1c and 1d, was reduced (1c: 11 ± 0.7% and 1d: 12
± 1.7% for PC3 cells and 1c: 5 ± 0.8% and 1d: 10 ± 0.9% for MDA-MB-231 cells). All other
pro-labels gave negligible or non-detectable DNA labeling at 1 µM.
The best performing pro-label compounds for DNA labelling were n-butanoyl mono- and di-
esters (1c and 2c) and n-pentanoyl mono- and di- esters (1d and 2d). The level of DNA
labeling observed with these compounds at 5 or 10 M was very similar to that of EdU. This
is consistent with the cLog P values for these compounds approaching that suited to optimal
membrane permeability by passive diffusion, with cLog P values ranging from -0.14 to +1.99
for the four compounds (Table 1). Compounds 1c, 2c, 1d and 2d did however have notably
less labeling compared to EdU when dosed at 1 M. This likely reflects a delay associated
with esterase processing of the pro-labels to release active EdU, a relationship that is
concealed at the higher test concentrations of 5 and 10 M. A further noticeable structure-
activity relationship is also evident at the lowest test concentration of 1 M with n-butanoyl
and n-pentanoyl derivatives 1c, 2c, 1d and 2d. The mono-esters 1c and 1d labeled more cells
than did the di-esters 2c and 2d. For example, in PC3 cells labeling with 1c and 1d was 11 ±
0.7% and 12 ± 1.7%, respectively, while labeling was <2% for their di-ester counterparts 2c
and 2d. This suggests that the additional processing needed to hydrolyse the ester moieties at
both the 3 and 5 positions 2c and 2d slowed the rate of incorporation into the DNA of
proliferating cells compared to when processing is needed only at the 3 position, as in 1c and
1d. The n-butanoyl moiety was the optimum acyl group in both the mono- and di-ester
derivatives of EdU. For example, labeling with 1c was 34 ± 0.1% and 29 ± 2.3% when dosed
at 10 or 5 M, respectively in PC3 cells (10 µM: 29 ± 2.1%, 5 µM: 27 ± 0.6% for EdU), and
34 ± 2.9% and 32 ± 3.7%, respectively, in MDA-MB-231 cells (10 µM: 37 ± 1.7%, 5 µM: 36
± 1.5% for EdU). The DNA labeling with the control compounds, acetyl esters 1a and 2a was
very low (<1%) at all test concentrations in both cell lines, this indicates that the compounds
have much poorer uptake than EdU, a finding that is consistent with our earlier study.10 The
n-propionyl mono- and di-esters, 1b and 2b, respectively similarly exhibited weak labeling,
however with a marginal improvement when compared to compounds 1a and 2a. The
replacement of acetyl moiety of 1a and 2a with the n-propionyl moiety of 1b and 2b provides
an incremental increase in cLog P (Table 1) to allow this marginal gain, however the n-
propionyl moiety is not sufficiently lipophilic to impart good passive membrane diffusion.

Figure 2. Percentage of (a) PC3 and (b) MDA-MB-231 cancer cells labeled with EdU and
pro-label compounds at concentrations of 10, 5 and 1 µM following 4 h of treatment. Images
of (c) PC3 and (d) MDA-MB-231 cells labeled with pro-label compounds (10 µM). Scale bar
= 50 µm. Data represent ± standard deviation.

The fluorescence intensity range of labeled PC3 and MDA-MB-231 cancer cells was also
examined. The intensity of labeled cancer cells with EdU was compared to those cancer cells
labeled with the best performing pro-label analogues, 1c and 1d. At 5 and 10 µM test probe
concentration, there was similar overall labeling efficiency. Conversely, the fluorescence
intensity distribution profile at 1 µM exhibited variations between EdU and the pro-label
compounds, with the intensity of EdU staining higher in both the PC3 and MDA-MB-231
cancer cell lines than 1c and 1d (Figure 3a, 3b). This observation is indicative of a delayed
labeling with 1c and 1d over EdU, and is consistent with the need to first hydrolyse the acyl
moiety of the pro-label compounds to release EdU, noticeable only at the lowest test
concentration.
Figure 3. Fluorescence intensity values for PC3 (a) and MDA-MB-231 (b) cancer cells
labeled with 10, 5 and 1 µM EdU, 1c and 1d for 4 h. Fluorescence intensity values were
classified as low, medium, high and very high intensity. Data are average values from a
representative dataset.

For this study, the evaluation of EdU and EdU pro-label incorporation was determined
following a 4 h incubation with cells. To assess the toxicity of pro-label compounds on cells
in comparison to EdU, evaluations were undertaken at 4, 24 and 72 h time points on both
cancer cell lines utilised in this study (MDA-MB-231 and PC3) as well as the non-
tumorigenic HEK293 cell line. At the conclusion of each time point, resazurin was applied to
measure the metabolic activity of cells. Parameters of the half maximal inhibitory
concentration (IC50 value) and efficacy at maximum concentrations (Emax value) were
determined. After application of compound for 4 and 24 h time periods, no cytotoxicity was
detected with any compound (Supplementary Material, Figure 1). Following 72 h of EdU or
pro-label exposure, cytotoxicity was observed in PC3 cells and moreso in HEK293 cells, but
not in MDA-MB-231 cells (Table 2). In PC3 cells, a similar toxicity profile was observed for
EdU and the pro-label analogues that incorporated well into DNA (IC50 values: 1c, 9.17 ±
1.464 µM; 2c, 5.91 ± 0.571 µM; 1d, 6.47 ± 0.352 µM; 2d, 7.49 ± 4.426 µM; EdU, 5.70 ±
2.145 µM). The Emax values for the pro-labels were lower than EdU (Emax: 74.35 ± 8.210%).
There were greater differences in toxicity between EdU and the pro-label compounds against
HEK293 cells, with compounds 1c, 2c and 2d more than 10-fold less cytotoxic to HEK293
cells when compared to EdU (IC50 value: 0.06 ± 0.043 µM), however, the activity of 1d (IC50
value: 0.04 ± 0.010 µM) was comparable to that of EdU. The pro-label compounds 1c, 1d, 2c
and 2d displayed similar Emax values to EdU (Emax: 1c, 75.94 ± 5.105%; 2c, 80.62 ± 6.251%;
1d, 83.47 ± 5.374%; 2d, 79.29 ± 7.658%; EdU, 76.96 ± 1.527%), indicating the cellular
sensitivity was comparable at maximal concentrations of EdU and pro-label compounds.
The cell lines utilised in this study, MDA-MB-231 and PC3 cells, have distinct biological
characteristics and greater cytotoxicity may have been detected in the PC3 prostate cancer cell
line due to these distinctions. The HEK293 cell line is a non-tumorigenic cell line, which
perhaps is the reason for the enhanced sensitivity observed with EdU and the pro-labels in
comparison to the cancer cell lines tested. HEK293 cells are routinely utilised in assessing
cytotoxicity of compounds as a model of human tissue for comparison to cancer cells or
microbiological diseases. The decreased sensitivity of HEK293 cells to pro-label compounds
1c, 2c and 2d in comparison to EdU indicates that these compounds were considerably less
toxic than EdU.
Table 2. Cytotoxicity against HEK293, PC3 and MDA-MB-231 cells following incubation
with EdU and pro-label compounds for 72 h.

PC3 Cellsa
Emax (%)
MDA-MB-231 cellsa
HEK293 cellsa
Emax (%)
Compd
IC50(M)
5.70 ± 2.145
NR
Emax (%) IC50(M)
IC50(M)
0.06 ± 0.043
4.27 ± 0.112
5.24 ± 0.003
0.45 ± 0.091
0.04 ± 0.010
NR
74.35 ± 8.210
NRb
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
NR
76.96 ± 1.527
64.33 ± 1.414
65.21 ± 1.697
75.94 ± 5.105
83.47 ± 5.374
NR
EdU
1a
NR
NR
1b
1c
67.72 ± 2.694
64.53 ± 3.316
NR
9.17 ± 1.464
6.47 ± 0.352
NR
1d
2a
NR
NR
63.41 ± 9.977 11.99 ± 2.234
2b
2c
48.31 ± 1.966
46.56 ± 10.218
5.91 ± 0.571
7.49 ± 4.426
80.62 ± 6.251
79.29 ± 7.658
1.13 ± 0.097
0.84 ± 0.139
2d
b
aData represent ± standard deviation. No response (NR) detected at top dose (40 µM),
Emax/IC50 value could not be calculated.
3. Conclusions
Studying cell proliferation via incorporation of chemical probes into DNA allows
visualisation of the population of cells proliferating and subsequent quantification of labeled
cells through image analysis. This approach has the potential to be utilised in a broad range of
applications, including drug discovery for the assessment of test compounds on proliferating
disease specific cells. Non-uniform expression of nucleoside transporters between different
cancer cell lines may lead to non-uniform probe uptake when using EdU, this in turn
diminishes opportunities for quantitative comparison of proliferation between different cancer
cell lines. The novel pro-label analogues described here were designed to enter cells via
passive membrane diffusion rather than using nucleoside transporters. A key advantage of this
design is that it bypasses the hurdle presented by non-uniform nucleoside transporter
expression and so can enable quantitative comparison of proliferation between different
cancer cell lines. Four of the EdU pro-labels, n-butanoyl and n-pentanoyl EdU esters (1c, 2c,
1d and 2d) were shown to incorporate into DNA with a similar profile to EdU upon
application of 5 and 10 µM of compound for 4 h in two distinct cancer cell lines, MDA-MB-

231 (breast cancer) and PC3 (prostate cancer). Furthermore, the pro-labels 1c, 2c and 2d were
less toxic than EdU, which when taken together with the similar incorporation profiles
indicates that they are a viable alternative to EdU when studying cell proliferation. As less
toxic chemical probes for proliferation they may also be more suitable for long-term duration
experiments than EdU. n-Butanoyl and n-pentanoyl monoesters as pro-labels are in the ‘sweet
spot’ of good cellular uptake and esterase processing and low toxicity providing an alternative
to EdU for visualising cell proliferation. This finding may have implications to nucleoside
prodrug development with respect to the effectiveness of acyl esters to support the need for
compound cell entry and ester hydrolysis. As the pro-label:chemical probe relationship
parallels the prodrug:drug relationship it has potential for improving the application of
chemical tools for use in comparative studies.
4. Experimental Section
4.1 Chemical Synthesis. All starting materials and reagents were purchased from commercial
suppliers or synthesised. The synthesis of EdU, pro-labels 1a and 2a and compound 7a was
carried out as reported earlier.8, 10 Reaction progress was monitored by TLC using silica gel-
60 F254 plates with detection by short wave UV fluorescence (
254 nm) and/or staining
with vanillin (5 g of vanillin in a mixture of EtOH:H2O:H2SO4 = 85:10:5). Silica gel flash
1
chromatography was performed using silica gel 60 Å (230-400 mesh). H NMR spectra were
13
1
acquired at 500 MHz and C NMR spectra at 125 MHz at 30 °C. Chemical shifts for H and
13C NMR spectra obtained in DMSO-d6 are reported in ppm relative to residual solvent
1
proton ( = 2.50 ppm) and carbon ( = 39.5 ppm) signals, respectively. Assignments for H
NMR were confirmed by 1H−1H gCOSY, while assignments for 13C NMR were confirmed by
1H−13C HSQC. Multiplicity is indicated as follows: s (singlet); d (doublet); t (triplet); m
(multiplet); dd (doublet of doublet); br (broad). Coupling constants are reported in Hertz (Hz).
Melting points are uncorrected. Mass spectra (LRMS and HRMS) were recorded using
electrospray as the ionization method in positive ion or negative ion modes as stated. All MS
analysis samples were prepared as solutions in acetonitrile. All compounds were analyzed for
purity by HPLC with UV (200 to 400 nm) detection used. Purity of all compounds was ≥
95%. Chemical synthesis procedures and compound characterization data are included in
Supplementary Material.

General Procedure 1 - Acylation
The 2′-deoxyuridine precursor (1 equiv.) was dissolved in anhydrous pyridine (20 equiv.)
under an atmosphere of nitrogen. The corresponding acid anhydride was added (4 equiv. per
hydroxyl group) and the reaction mixture heated to 60 C until complete, as evident by TLC
(vanillin stain/UV  = 254 nm). The reaction was quenched by the addition of ice (25-100
volumes) and EtOAc (5 volumes) followed by vigorous stirring for 24 h. The acylated
product was extracted with EtOAc (3×), the organic fractions combined, washed with
saturated K2CO3 (aq) (3×), 5% HCl (aq) (2×) and brine (1×), dried over MgSO4 and filtered.
The resulting residue was concentrated and dried in vacuo. Most compounds were obtained
analytically pure and used in subsequent steps without further purification unless stated
otherwise.
General Procedure 2 - Sonogashira coupling
The 5-iodo-2′-deoxyuridne precursor (1 equiv.), Pd(PPh3)4 (0.05 equiv.) and CuI (0.1 equiv.)
were dissolved in anhydrous DMF (55 equiv.) under an atmosphere of nitrogen, then Et3N (2
equiv.) added. Next, ethynyltrimethylsilane (5 equiv.) was added and the reaction stirred at rt
until complete, as evident by TLC (vanillin stain/ UV λ = 254 nm). The reaction mixture was
filtered through Celite® and the DMF and Et3N removed under reduced pressure. The crude
product was absorbed onto silica and purified by silica gel flash chromatography.
General Procedure 3 – Removal of silyl protecting groups
To a solution of silyl protected 2′-deoxyuridine precursor (1 equiv.) in anhydrous THF (90
equiv.) under an atmosphere of nitrogen was added TBAF (1.0 M in THF) (1.2 equiv.). The
reaction mixture was stirred at rt until consumption of starting material was evident by TLC
(vanillin stain/UV λ = 254 nm). The crude product was absorbed onto silica and purified by
silica gel flash chromatography.
5′-O-tert-Butyldiphenylsilylether-5-iodo-2′-deoxyuridine (4)13
5-Iodo-2′-deoxyuridine (0.500 g, 1.41 mmol, 1 equiv.) and imidazole (0.096 g, 1.41 mmol,
1.15 equiv.) were combined in anhydrous DMF (3 mL, 35 mmol, 10 equiv.) under an
atmosphere of Ar. The reaction mixture was cooled in an ice bath and tert-butyl-
chlorodiphenylsilane (TBDPSCl) (1.15 equiv.) was added slowly. The reaction was allowed
to warm to rt and stirring continued at rt for 24 h. The crude reaction mixture was absorbed
onto silica and purified using flash chromatography on silica gel (EtOAc/Hexane = 1:1.5 to

1:1) to yield the title compound as a white crystalline solid (0.434 g, 50%); mp 184-185 ˚C;
1
Rf = 0.55 (EtOAc). H NMR (500 MHz DMSO-d6) δ = 11.71 (br, 1H), 7.97 (s, 1H), 7.69 –
7.61 (m, 4H), 7.51 – 7.38 (m, 6H), 6.10 (t, J = 6.9 Hz, 1H), 5.29 (d, J = 4.3 Hz, 1H), 4.27 –
4.22 (m, 1H), 3.90 – 3.82 (m, 2H), 3.75 – 3.69 (m, 1H), 2.21 – 2.13 (m, 2H), 1.03 (s, 9H).
LRMS (ESI+): m/z 593 [M+H]+, m/z 615 [M+Na]+.
5′-O-tert-Butyldiphenylsilylether-5-ethynyltrimethylsilane-2′-deoxyuridine (5)
The title compound was synthesized from 4 (0.434 g, 0.732 mmol, 1 equiv.) according to
General Procedure 2. The reaction was complete after 5.5 h. The crude product was purified
by flash chromatography on silica gel (EtOAc/Hexane = 1:4 to 1:1.5) to give the title
compound as a pale yellow crystalline solid (0.405 g, 98%); mp 195-197 ˚C; Rf = 0.8
(EtOAc). 1H NMR (500 MHz DMSO-d6) δ = 11.70 (s, 1H, NH), 7.94 (s, 1H, H6), 7.68 – 7.61
(m, 4H, HAr), 7.53 – 7.38 (m, 6H, HAr), 6.15 – 6.11 (m, 1H, H1′), 5.31 (d, J = 3.6 Hz, 1H,
H3′OH), 4.29 – 4.22 (m, 1H, H3′), 3.86 - 3.76 (m, H4, H5′ or H5′′), 3.71 (dd, J = 4.1, 11.5 Hz, 1H,
13
H5′ or H5′′), 2.26 – 2.11 (m, 2H, H2′,H2′′), 1.02 (s, 9H, C(CH3)3), 0.05 (s, 9H, Si(CH3)3). C
NMR (125 MHz, DMSO-d6) δ = 161.3 (C-4), 149.2 (C-2),143.2 (C-6), 135.1 ( 2× C-Ar),
135.0 (2× C-Ar),132.35 (C-Ar), 132.33 (C-Ar), 129.94 (C-Ar), 129.89 (C-Ar), 127.94 (2× C-
Ar), 127.93 (2× C-Ar), 98.7 (C-5), 97.3 (Si-C≡), 97.2 (≡C-C), 87.0 (C-4′), 84.8 (C-1′), 70.1
(C-3′), 63.8 (C-5′), 40.0 (C-2′), 26.8 (C(CH3)3), 18.7 (C(CH3)3), -0.3 (Si(CH3)3). LRMS
(ESI+): m/z 563 [M+H]+, m/z 585 [M+Na]+.
5′-O-tert-Butyldiphenylsilylether-5-ethynyltrimethylsilane-3′-O-n-propanoyl-2′-
deoxyuridine (6b)
The title compound 6b was synthesized from 5 (0.115g, 0.204 mmol, 1 equiv.) and propanoic
anhydride (0.1 mL, 3.0 mmol, 4 equiv.) according to General Procedure 1. The reaction was
complete after 8 h. The title compound was afforded as a light yellow foam (0.126 g,
1
quantitative); Rf = 0.5 (EtOAc/Hexane = 1:1). H NMR (500 MHz DMSO-d6) δ = 11.74 (s,
1H, NH), 7.95 (s, 1H, H6), 7.76 – 7.55 (m, 4H, 4× Har), 7.54 – 7.35 (m, 6H, 6×HAr), 6.12 (t, J
= 7.10 Hz, 1H, H1′), 5.30 – 5.27 (m, 1H, H3′), 4.09 – 4.06 (m, 1H, H4′), 3.89 (dd, J = 2.9, 11.4
Hz, 1H, H5′ or H5′′), 3.81 (dd, J = 4.1, 11.4 Hz, 1H, H5′ or H5′′), 2.39 – 2.30 (m, 4H, H2′,H2′′,
13
CH2), 1.03 (s, 12H, CH3, Si-C(CH3)3), 0.06 (s, 9H, Si(CH3)3). C NMR (125 MHz, DMSO-
d6) δ = 173.2 (C=O), 161.2 (C-4), 149.2 (C-2), 143.1 (C-6), 135.1 (2× C-Ar), 134.9 (2× C-
Ar), 132.2 (C-Arq), 132.1 (C-Arq), 130.0 (C-Ar), 129.9 (C-Ar), 128.0 (2× C-Ar), 127.9 (2×
C-Ar), 99.0 (C-5), 97.4 (Si-C≡), 97.0 (≡C-C), 84.5 (C-1′), 84.2 (C-2′), 73.5 (C-3′), 63.6 (C-

5′), 37.1 (C-2′), 26.7 (CH2, C(CH3)3) 18.7 (C(CH3)3), 8.7 (CH3), -0.3 (Si(CH3)3). LRMS
(ESI+): m/z 619 [M+H]+, m/z 641 [M+Na]+.
5′-O-tert-Butyldiphenylsilylether-5-ethynyltrimethylsilane-3′-O-n-butanoyl-2′-
deoxyuridine (6c)
The title compound 6c was synthesized from 5 (0.115 g, 0.204 mmol, 1 equiv.) and butanoic
anhydride (0.13 mL, 3.1 mmol, 4 equiv.) according to General Procedure 1. The reaction was
complete after 2 h. The work up was performed twice to give the title compound as a light
yellow foam (0.119 g, 92%); Rf = 0.8 (EtOAc). 1H NMR (500 MHz, DMSO-d6) δ = 11.74 (s,
1H, NH), 7.95 (s, 1H, H6), 7.69 – 7.61 (m, 4H, 4× HAr), 7.51 – 7.40 (m, 6H, 6× HAr), 6.20 –
6.07 (m, 1H, H1′), 5.32 – 5.24 (m, 1H, H3′), 4.09 – 4.05 (m, 1H, H4′), 3.89 (dd, J = 2.9, 11.4
Hz, 1H, H5′ or H5′′), 3.81 (dd, J = 4.1, 11.4 Hz, 1H, H5′ or H5′′), 2.40 – 2.27 (m, 4H, H2′,H2′′,
CH2α), 1.55 (h, J = 7.4 Hz, 2H, CH2β), 1.03 (s, 9H, C(CH3)3), 0.88 (t, J = 7.4 Hz, 3H, CH3),
0.06 (s, 9H, Si(CH3)3)). 13C NMR (125 MHz, DMSO-d6) δ = 172.3 (C=O), 161.1 (C-4), 149.2
(C-2), 143.1 (C-6), 135.0 (2× C-Ar), 134.9 (2× C-Ar), 132.2 (C-Arq), 132.1 (C-Arq), 130.0
(C-Ar), 129.9 (C-Ar), 127.94 (2× C-Ar), 127.90 (2× C-Ar), 99.0 (C-5), 97.4 (Si-C≡), 97.0
(≡C-C), 84.5 (C-1′), 84.2 (C-4′), 73.4 (C-3′), 63.5 (C-5′), 37.0 (C-2′), 35.2 (CH2α), 26.7
(C(CH3)3), 18.7 (C(CH3)3), 17.7 (CH2β), 13.3 (CH3), -0.3 (Si(CH3)3). LRMS (ESI+): m/z 633
[M+H]+.
5′-O-tert-Butyldiphenylsilylether-5-ethynyltrimethylsilane-3′-O-n-pentanoyl-2′-
deoxyuridine (6d)
The title compound was synthesized from 5 (0.115 g, 0.204 mmol, 1 equiv.) and valeric
anhydride (0.24 mL, 3.0 mmol, 4 equiv.) in pyridine (0.25 mL, 3.0 mmol, 15.0 equiv.)
according to General Procedure 1. The reaction was complete after 4 h. The title compound
was afforded as a light yellow foam (0.126 g, 94%); Rf = 0.6 (EtOAc/Hexane = 1:1). 1H NMR
(500 MHz, DMSO-d6) δ = 11.74 (s, 1H, NH), 7.95 (s, 1H, H6), 7.69 – 7.57 (m, 4H, 4× HAr),
7.52 – 7.30 (m, 6H, 6× HAr), 6.12 (t, J = 7.1 Hz, 1H, H1′), 5.30 – 5.26 (m, 1H, 3′), 4.11 – 4.01
(m, 1H, H4′), 3.89 (dd, J = 2.9, 11.4 Hz, 1H, H5′or H5′′), 3.81 (dd, J = 4.1, 11.4 Hz, 1H, H5′or
H5′′), 2.39 – 2.27 (m, 4H, H2′,H2′′,CH2α), 1.51 (p, J = 7.3 Hz, 2H, CH2β), 1.29 (h, J = 7.4 Hz,
13
2H, CH2γ), 1.03 (s, 9H, C(CH3)3), 0.86 (t, J = 7.3 Hz, 3H, CH3), 0.06 (s, 9H, Si(CH3)3). C
NMR (125 MHz, DMSO-d6) δ = 172.4 (C=O), 161.2 (C-4), 149.2 (C-2), 143.1 (C-6), 135.0
(2× C-Ar), 134.9 (2× C-Ar), 132.2 (C-Arq), 132.1 (C-Arq), 130.0 (C-Ar), 129.9 (C-Ar),
127.94 (2× C-Ar), 127.90 (2× C-Ar), 99.0 (C-5), 97.4 (Si-C≡), 97.0 (≡C-C), 84.5 (C-1′), 84.2

(C-4′), 73.4 (C-3′), 63.5 (C-5′), 37.0 (C-2′), 33.0 (CH2α), 26.7 (CH2β), 26.3 (C(CH3)3), 21.5
(CH2γ), 18.7 (C(CH3)3), 13.5 (CH3), -0.3 (Si(CH3)3). LRMS (ESI+): m/z 670 [M+Na]+
5-Ethynyl-3′-O-n-propanoyl-2′-deoxyuridine (1b)
The title compound was synthesized from 6b (0.097 g, 0.157 mmol, 1 equiv.) according to
General Procedure 3. The reaction was complete after 1.5 h. The crude product was purified
by flash chromatography on silica gel (EtOAc/Hexane = 1:1 to 4:1) to give the title compound
1
as a yellow foam (0.048g, quantitative); Rf = 0.4 (EtOAc). H NMR (500 MHz, DMSO-d6) δ
= 11.65 (s, 1H, NH), 8.28 (s, 1H, H6), 6.14 (t, J = 7.1, 1H, H1′), 5.31 – 5.18 (m, 2H, OH5′, H3′),
4.10 (s, 1H, HC≡), 4.02 (s, 1H, H4′), 3.66 – 3.62 (m, 2H, H5′, H5′′), 2.41 – 2.26 (m, 4H, H2′,H2′′,
CH2), 1.04 (t, J = 7.5Hz, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ = 173.2 (C=O), 161.4
(C-4), 149.4 (C-2), 144.2 (C-6), 98.0 (C-5), 85.1 (C-4′), 84.6 (C-1′), 83.7 (C-8), 76.2 (C-7),
74.4 (C-3′), 61.1 (C-5′), 37.3 (C-2′), 26.7 (CH2), 8.8 (CH3). LRMS (ESI+): m/z 331 [M+Na]+.
HRMS (ESI+): Calcd for C14H16N2NaO6 [M+ Na]+ 331.0901, found 331.0918.
5-Ethynyl-3′-O-n-butanoyl-2′-deoxyuridine (1c)
The title compound synthesized from 6c (0.095 g, 0.149 mmol, 1 equiv.) according to General
Procedure 3. The reaction was complete after 3 h. The crude product was purified by flash
chromatography on silica gel (EtOAc/Hexane = 1:2.33 to 2.33:1) to yield the title compound
1
as a yellow foam (0.039 g, 82%); Rf = 0.4 (EtOAc/Hexane = 4:1). H NMR (500 MHz,
DMSO-d6) δ = 11.65 (s, 1H, NH), 8.28 (s, 1H, H6), 6.16 – 6.09 (m, 1H, H1′), 5.30 – 5.19 (m,
2H, OH5′, H3′), 4.10 (s, 1H, HC≡), 4.05 – 3.98 (m, 1H, H4′), 3.64 (s, 2H, H5′, H5′′), 2.37 – 2.23
13
(m, 4H, CH2α, H2′,H2′′), 1.56 (h, J = 7.3 Hz, 2H, CH2β), 0.89 (t, J = 7.40 Hz, 3H, CH3). C
NMR (125 MHz, DMSO-d6) δ = 172.3 (C=O), 161.4 (C-4), 149.4 (C-2), 144.2 (C-6), 98.0
(C-5), 85.1 (C-4′), 84.6 (C-1′), 83.7 (C-8), 76.2 (C-7), 74.3 (C-3′), 61.1 (C-5′), 37.3 (C-2′),
35.2 (CH2α), 17.8 (CH2β), 13.3 (CH3). LRMS (ESI+): m/z 345 [M+Na]+. HRMS (ESI+): Calcd
for C15H18N2NaO6 [M+ Na]+ 345.1057, found 345.1057.
5-Ethynyl-3′-O-n-pentanoyl-2′-deoxyuridine (1d)
The title compound was synthesized from 6d (0.095 g, 0.149 mmol, 1 equiv.) according to
General Procedure 3. Reaction was complete after 2.25 h. The crude product was purified by
flash chromatography on silica gel (EtOAc/Hexane = 1:2.33 to 2.33:1) to give the title
1
compound as a yellow foam (0.058 g, 90%); Rf = 0.5 (EtOAc/Hexane = 4:1). H NMR (500
MHz, DMSO-d6) δ = 11.65 (s, 1H, NH), 8.28 (s, 1H, H6), 6.18 – 6.09 (m, 1H, H1′), 5.32 –

5.19 (m, 2H, H3′, OH5′), 4.10 (s, 1H, HC≡), 4.03 – 3.99 (m, 1H, H4′), 3.64 (s, 2H, H5′, H5′′),
2.39 – 2.21 (m, 4H, H2′,H2′′, CH2α), 1.52 (m, J = 7.4, Hz, 2H, CH2β), 1.30 (m, J = 7.4 Hz, 2H,
CH2δ), 0.87 (t, J = 7.4 Hz, 3H, CH3). 13C NMR (125 MHz, DMSO-d6) δ 172.5(C=O)
161.5(C-4) 149.4 (C-2), 144.2 (C-6) , 98.0 (C-5) , 85.0 (C-4′) , 84.6 (C-1′) , 83.7 (HC≡), 76.2
(C≡C-C), 74.4 (C-3′), 61.1 (C-5′), 37.3 (C-2′) , 33.1 (CH2α) , 26.4 (CH2β), 21.5 (CH2γ), 13.5
(CH3). LRMS (ESI+): m/z 359 [M+Na]+. HRMS (ESI+): Calcd for C16H20N2NaO6 [M+ Na]+
359.1214, found 359.1222.
5-Iodo-3′,5′-bis-O-n-propanoyl-2′-deoxyuridine (7b)
The title compound 7b was synthesized from 5-iodo-2′-deoxyuridine (0.200 g, 0.564 mmol, 1
equiv.) propanoic anhydride (0.57 mL, 4.51 mmol, 8 equiv.) according to General Procedure
1. The reaction was complete after 1.25 h. The title compound was afforded as a translucent
1
white foam (0.245 g, 93%); Rf = 0.3 (EtOAc/Hexane = 1:1). H NMR (500 MHz, DMSO-d6)
δ 11.74 (br s, 1H, NH), 8.02 (s, 1H, H6), 6.10 (br t, J = 7.1 Hz, 1H, H1′), 5.21 (m, 1H, H3′),
4.34 – 4.24 (d, J = 4.1, 2H, H5′,H5′′), 4.19 (m, 1H, H4′), 2.48 – 2.28 (m, 6H, H2′, H2′′, 2×CH2),
13
1.13 – 1.00 (m, 6H, 2× CH3). C NMR (125 MHz, DMSO-d6) δ 173.3 (C=O), 173.2 (C=O),
160.3 (C-4), 150.0 (C-2), 144.4 (C-6), 84.9 (C-1′) , 81.6 (C-4′), 73.8 (C-3′), 70.0 (C-5), 63.5
(C-5′), 36.2 (C-2′), 26.74 (CH2), 26.68 (CH2), 8.9 (CH3), 8.7 (CH3). LRMS (ESI+): m/z 467
[M+H]+ 489 [M+Na]+.
5-Iodo-3′,5′-bis-O-n-butanoyl-2′-deoxyuridine (7c)
The title compound 7c was synthesized from 5-iodo-2′-deoxyuridine (0.300 g, 0.847 mmol, 1
equiv.) and butanoic anhydride (1.1 mL, 6.7 mmol, 8 equiv.) according to the General
Procedure 1. The reaction was complete after 5 h. The title compound was afforded as very
fine off white crystals (0.418 g, quantitative); mp 122-124 ˚C (EtOAc/Hexane); Rf = 0.4
(EtOAc/Hexane = 1:1). 1H NMR (500 MHz, DMSO-d6) δ = 11.74 (br s, 1H, NH), 8.02 (s, 1H,
H6), 6.10 (dd, J = 6.3, 7.8, 1H, H1′), 5.20 (m, 1H, H3′), 4.32 – 4.23 (m, 2H, H5′, H5′′), 4.20 –
4.16 (m, 1H, H4′), 2.49 – 2.26 (m, 6H, H2′, H2′′, 2× CH2α), 1.63 – 1.51 (m, 4H, 2× CH2β), 0.94
– 0.86 (m, 6H, 2× CH3). 13C NMR (125MHz, DMSO-d6) δ = 172.4 (C=O), 172.3 (C=O),
160.4 (C-4), 150.0 (C-2), 144.3 (C-6), 84.9 (C-1′), 81.5 (C-4′), 73.7 (C-3′), 70.0 (C-5), 63.3
(C-5′), 36.2 (C-2′), 35.24 (CH2α) 35.16 (CH2α), 17.8 (CH2β), 17.7 (CH2β), 13.4 (CH3), 13.3
(CH3). LRMS (ESI+): m/z 495 [M+H]+, m/z 517 [M+Na]+.
5-Iodo-3′,5′-bis-O-n-pentanoyl-2′-deoxyuridine (7d)

The title compound 7d was synthesized from 5-iodo-2′-deoxyuridine (0.300 g, 0.847 mmol, 1
equiv.) and valeric anhydride (1.3 mL, 6.7 mmol, 8 equiv.) according to General Procedure 1.
The reaction was complete after 4.25 h. The crude product was further purified by flash
chromatography on silica gel (EtOAc/Hexane = 1:3) to give the title compound as a white
1
solid (0.346 g, 78%); mp 88-90 ˚C; Rf = 0.6 (EtOAc/Hexane = 1:1). H NMR (500 MHz,
DMSO-d6) δ =11.74 (br, 1H, NH), 8.02 (s, 1H, H6), 6.09 (dd, J = 6.3, 7.8, 1H, H1′), 5.19 (m,
1H, H3′), 4.31 – 4.22 (m, 2H, H5′, H5′′), 4.20 – 4.16 (m, 1H, H4′), 2.48 – 2.27 (m, 6H, H2′, H2′′,
2× CH2α), 1.59 – 1.47 (m, 4H, 2× CH2β), 1.36 – 1.25 (m, 4H, 2× CH2γ), 0.91 – 0.82 (m, 6H,
2× CH3). 13C NMR (125 MHz, DMSO-d6) δ = 172.54 (C=O), 172.45 (C=O), 160.3 (C-4),
150.0 (C-2), 144.3 (C-6), 84.9 (C-1′), 81.5 (C-4′), 73.7 (C-3′), 70.0 (C-5), 63.4 (C-5′), 36.2
(C-2′), 33.1 (CH2α), 33.0 (CH2α), 26.4 (CH2β), 26.3 (CH2β), 21.53(CH2γ), 21.49 (CH2γ), 13.53
(CH3), 13.50 (CH3). LRMS (ESI+): m/z 523 [M+H]+, m/z 545 [M+Na]+.
3′,5′-bis-O-Acetyl-5- ethynyltrimethylsilane -2′-deoxyuridine (8a)
The title compound 8a was synthesized from 5′,3′-bis-O-acetyl-5-iodo-2′-deoxyuridine (0.200
g, 0.464 mmol, 1 equiv.) according to General Procedure 2. The reaction was complete after
12.5 h. The crude product was purified by flash chromatography on silica gel (EtOAc/Hexane
1:2.3 to 1.5:1) to give the title compound as a translucent white foam (0.137 g, 74%); Rf = 0.4
1
(EtOAc/Hexane = 1.5:1). H NMR (500 MHz, DMSO-d6) δ 11.70 (s, 1H, NH), 7.95 (s, 1H,
H6), 6.16 – 6.10 (m, 1H, H1ʹ), 5.22 – 5.16 (m, 1H, H3ʹ), 4.29 – 4.24 (m, 2H, H5ʹ, H5ʹʹ), 4.23 –
4.1 (m, 1H, H4ʹ), 2.54 – 2.46 (m, H2ʹ or H2ʹʹ under solvent residual), 2.38 – 2.28 (m, 1H, H2ʹ or
H2ʹʹ), 2.09 (s, 3H, CH3), 2.06 (s, 3H, CH3), 0.19 (s, 9H, Si(CH3)). LRMS (ESI+): m/z 431
[M+Na]+.
5-Ethynyltrimethylsilane-3′,5′-bis-O-n-propanoyl-2′-deoxyuridine (8b)
The title compound 8b was synthesized from 7b (0.220 g, 0.472 mmol, 1 equiv.) according to
General Procedure 2. The reaction was complete after 2.5 h. The crude product was purified
twice by flash chromatography on silica gel (EtOAc/Hexane = 2:3 and EtOAc/Hexane = 1:4,
respectively) to yield the title compound as a yellow solid (0.175g, 85%); mp 143-145 ˚C; Rf
= 0.65 (EtOAc/Hexane = 4:1). 1H NMR (500 MHz, DMSO-d6) δ = 11.70 (br s, 1H, NH), 7.92
(s, 1H, H6), 6.12 (t, J = 7.0 Hz, 1H, H1′), 5.26 – 5.15 (m, 1H, H3′), 4.29 (d, J = 4.2, 2H, H5′,
H5′′), 4.21 (m, 1H, H4′), 2.49 – 2.29 (m, 6H, H2′, H2′′, 2× CH2), 1.08 – 1.02 (m, 6H, 2× CH3),
13
0.19 (s, 9H, Si(CH3)). C NMR (125 MHz, DMSO-d6) δ = 173.3 (d, 2×C=O), 161.2 (C-4),
149.2 (C-2), 143.9 (C-6), 98.8 (C-5), 97.5 (C≡C-C), 97.3 (TMS-C≡C), 85.0 (C-1′) , 81.7 (C-

4′), 73.7 (C-3′), 63.4 (C-5'), 36.4 (C-2′), 26.71 (CH2), 26.66 (CH2), 8.9 (CH3), 8.8 (CH3), -0.2
(Si(CH3)3). LRMS (ESI+): m/z 459 [M+Na]+.
5-Ethynyltrimethylsilane-3′,5′-bis-O-n-butanoyl-2′-deoxyuridine (8c)
The title compound was synthesized from 7c (0.372 g, 0.753 mmol, 1 equiv.) according to
General Procedure 2. The reaction was complete after 5 h. The crude product was purified by
flash chromatography on silica gel (EtOAc/Hexane 1:2.33) to give the title compound as a
yellow crystalline solid (0.313 g, 84%); mp 121-122 ˚C (EtOAc/Hexane); Rf = 0.2
1
(EtOAc/Hexane = 1:2.33). H NMR (500 MHz, DMSO-d6) δ = 11.70 (br s, 1H, NH), 7.92 (s,
1H, H6), 6.10 (t, J = 7.0 Hz, 1H, H1′), 5.27 – 5.15 (m, 1H, H3′), 4.34 – 4.25 (m, 2H, H5′, H5′′),
4.21 – 4.18 (m, 1H, H4′), 2.48 – 2.28 (m, 6H, H2′, H2′′, 2× CH2α), 1.62 – 1.52 (m, 4H, 2×
CH2β), 0.89 (t, J = 7.4 Hz, 6H, 2× CH3), 0.19 (s, 9H, Si(CH3)3). 13C NMR (125 MHz, DMSO-
d6) δ = 172.35 (C=O), 172.33 (C=O), 161.1 (C-4), 149.2 (C-2), 143.9 (C-6), 98.7 (C-5), 97.4
(≡C-C), 97.3 (TMS-C≡C), 85.0 (C-1′), 81.6 (C-4′), 73.6 (C-3′), 63.3 (C-5′), 36.4 (C-2′), 35.2
(CH2α), 35.1 (CH2α), 17.8 (CH2β), 17.7 (CH2β), 13.4 (CH3), 13.3 (CH3), -0.2 (Si(CH3)3).
LRMS (ESI+): m/z 465 [M+H]+.
5-Ethynyltrimethylsilane-3′,5′-bis-O-n-pentanoyl-2′-deoxyuridine (8d)
The title compound was synthesized from 7d (0.306 g, 0.585 mmol, 1 equiv.) according to
General Procedure 2. The reaction was complete after 7.25 h. The crude product was purified
by flash chromatography on silica gel (EtOAc/Hexane = 1:2.33) to give the title compound as
a yellow waxy solid that darkened with time (0.251 g, 86%); mp 125–126 ˚C; Rf = 0.2
1
(EtOAc/Hexane = 1:4). H NMR (500 MHz, DMSO-d6) δ = 11.70 (s, 1H, NH), 7.92 (s, 1H,
H6), 6.14 – 6.06 (m, 1H, H1′), 5.23 – 5.16 (m, 1H, H3′), 4.34 – 4.23 (m, 2H, H5′, H5′′), 4.22 –
4.17 (m, 1H, H4′), 2.49 – 2.28 (m, 6H, H2′, H2′′, 2× CH2α), 1.57 – 1.48 (m, 4H, 2× CH2β), 1.36
13
– 1.25 (m, 4H, CH2γ), 0.91 – 0.83 (m, 6H, 2× CH3), 0.19 (s, 9H, Si(CH3)3). C NMR (125
MHz, DMSO-d6) δ = 172.49 (C=O), 172.46 (C=O), 161.1 (C-4), 149.2 (C-2), 143.9 (C-6),
98.7 (C-5), 97.5 (≡C-C), 97.3 (TMS-C≡C), 85.0 (C-1′), 81.6 (C-4′), 73.6 (C-3′), 63.3 (C-5′),
36.3 (C-2′), 33.02 (CH2α), 33.00 (CH2α), 26.4 (CH2β), 26.3 (CH2β), 21.54 (CH2γ), 21.49
(CH2γ), 13.5 (2× CH3), -0.2 (Si(CH3)3). LRMS (ESI+): m/z 515 [M+Na]+.
3′,5′-bis-O-Acetyl-5-ethynyl-2′-deoxyuridine (2a)
The title compound 2a was synthesized from 8a (0.137 g, 0.336 mmol, 1 equiv.) according to
General Procedure 3. The reaction was complete after 2 h. The crude product was purified by

flash chromatography on silica gel (EtOAc/Hexane 1:1.5 to 2.33:1) to give the title compound
1
as a yellow foam (0.0783 g, 69%); Rf = 0.45 (EtOAc/Hexane = 4:1). H NMR (500 MHz,
DMSO-d6) δ 11.69 (s, 1H, NH), 8.00 (s, 1H, H6), 6.14 – 6.10 (m, 1H, H1ʹ), 5.21 – 5.16 (m,
1H, H3ʹ), 4.30 – 4.22 (m, 2H, H5′, H5′′), 4.22 – 4.19 (m, 1H, H4ʹ), 4.13 (s, 1H, HC≡), 2.54 –
2.45 (m, H2ʹ or H2ʹʹ under solvent residual), 2.33 (m, 1H, H2ʹ or H2ʹʹ), 2.09 (s, 3H, CH3), 2.06
(s, 3H, CH3),. LRMS (ESI+): m/z 359 [M+Na]+. HRMS (ESI+): Calcd for C15H16N2NaO7 [M+
Na]+ 359.0850, found 359.0879.
5-Ethynyl-3′,5′-bis-O-n-propanoyl-2′-deoxyuridine (2b)
The title compound was synthesized from 8b (0.110 g, 0.252 mmol, 1.0 equiv.) according to
General Procedure 3. The reaction was complete after 3 h. The crude product was purified by
flash chromatography on silica gel (EtOAc/Hexane = 1:1) to yield the title compound as a
dark yellow waxy solid (0.092g, quantitative); mp 144-145 ˚C; Rf = 0.3 (EtOAc/Hexane =
1
1:1). H NMR (500 MHz, DMSO-d6) δ = 11.70 (br s, 1H, NH), 7.97 (s, 1H, H6), 6.11 (t, J =
7.0 Hz, 1H, H1′), 5.21 (m, 1H, H3′), 4.31 – 4.23 (m, 2H, H5′, H5′′), 4.23 – 4.17 (m, 1H, H4′),
4.13 (s, 1H, HC≡), 2.49 – 2.29 (m, H2′, H2′′, 2× CH2), 1.09 – 1.00 (m, 6H, 2× CH3). 13C NMR
(125 MHz, DMSO-d6) δ = 173.3 (C=O), 173.2 (C=O), 161.4 (C-4), 149.3 (C-2), 144.0 (C-6),
98.1(C-5), 85.0 (C-1′), 83.8 (HC≡), 81.6 (C-4′), 76.0 (≡C-C), 73.6 (C-3′), 63.4 (C-5′), 36.3
(C-2′), 26.67 (CH2), 26.65 (CH2), 8.8 (CH3), 8.7 (CH3). LRMS (ESI+): m/z 365 [M+H]+, m/z
387 [M+Na]+. HRMS (ESI+): Calcd for C17H20N2NaO7 387.1163 [M+ Na]+, found 387.1166.
5-Ethynyl-3′,5′-bis-O-n-butanoyl-2′-deoxyuridine (2c)
The title compound was synthesized from 8c (0.280 g, 0.602 mmol, 1 equiv.) according to
General Procedure 3. The reaction was complete after 1.25 h. The crude product was purified
by flash chromatography on silica gel (EtOAc/Hexane = 1:1.8) to give the title compound as a
slightly beige waxy solid (0.224 g, 95%); mp 135-136 ˚C; Rf = 0.35 (EtOAc/Hexane = 1:1).
1H NMR (500 MHz, DMSO-d6) δ = 11.69 (br s, 1H, NH), 7.97 (s, 1H, H6), 6.11 (br t, J = 7.0
Hz, 1H, H1′), 5.24 – 5.18 (m, 1H, H3′), 4.27 (d, 2H, H5′, H5′′), 4.21 – 4.17 (m,, 1H, H4′), 4.13
(s, 1H, HC≡), 2.48 – 2.29 (m, 6H, H2′, H2′′, 2× CH2α), 1.61 – 1.51 (m, 4H, 2× CH2β), 0.95 –
13
0.80 (m, 6H, 2× CH3). C NMR (125 MHz, DMSO-d6) δ = 172.43 (C=O), 172.35 (C=O),
161.4 (C-4), 149.3 (C-2), 144.0 (C-6), 98.1 (C-5), 85.0 (C-1′), 83.8 (HC≡), 81.6 (C-4′), 76.0
(≡C-C), 73.5 (C-3′), 63.3 (C-5′), 36.3 (C-2′), 35.2 (2× CH2α), 17.82 (CH2β), 17.76 (CH2β),
13.4 (CH3), 13.3 (CH3). LRMS (ESI+): m/z 415 [M+Na]+. HRMS (ESI+): Calcd for
C19H24N2NaO7 [M+ Na]+ 415.1476, found 415.1474.

5-Ethynyl -3′,5′-bis-O-n-pentanoyl-2′-deoxyuridine (2d)
The title compound was synthesized from 8d (0.210 g, 0.426 mmol, 1 equiv.) according to
General Procedure 3. The reaction was complete after 2 h. The crude product was purified by
flash chromatography on silica gel (EtOAc/Hexane = 1:1.5) to give the title compound as a
beige waxy solid (0.175 g, 98%); mp 110-111 ˚C; Rf = 0.35 (EtOAc/Hexane = 1:1). 1H NMR
(500 MHz, DMSO-d6) δ = 11.70 (br, 1H, NH), 7.98 (s, 1H, H6), 6.11 (br t, J = 7.0 Hz, 1H),
5.25 – 5.15 (m, 1H, H3′), 4.29 – 4.25 (m, 2H, H5′, H5′′), 4.21 – 4.17 (m, 1H, H4′), 4.13 (s, 1H,
HC≡), 2.49 – 2.28 (m, 6H, H2′, H2′′, 2× CH2α), 1.52 (p, J = 7.4 Hz, 4H, 2× CH2β), 1.37 – 1.23
(m, 4H, 2× CH2γ), 0.93 – 0.82 (m, 6H, 2× CH3). 13C NMR (125 MHz, DMSO-d6) δ = 172.55
(O=C-O5′) 172.45 (O=C-O3′), 161.4 (C-4), 149.3 (C-2), 143.9 (C-6), 98.1 (C-5), 85.0 (C-5),
83.8 (HC≡), 81.6 (C-4′), 75.9 (≡C-C), 73.5 (C-3′), 63.3 (C-5′), 36.3 (C-2′), 33.02 (CH2α)
33.00 (CH2α) 26.4 (CH2β), 26.3 (CH2β), 21.51 (CH2γ) 21.49 (CH2γ), 13.5 (2× CH3). LRMS
(ESI+): m/z 443 [M+Na]+. HRMS (ESI+): Calcd for C21H28N2NaO7 [M+ Na]+ 443.1789,
found 443.1792.
4.2 Cell Culture. The MDA-MB-231 cell line was cultured in DMEM/F12 cell culture media
with the addition of 10% fetal bovine serum (FBS). The PC3 and HEK293 cell lines were
cultured in RPMI cell culture media with the addition of 10% FBS. All cell lines were
incubated in a humidified incubator at 37 °C with 5% carbon dioxide. Cell lines were
obtained from the American Type Culture Collection (ATCC).
4.3 Cytotoxicity Assay. Cells (MDA-MB-231: 2000-5000 cells per well; PC3: 1000-3000
cells per well; HEK293: 1000-3000 cells per well) were seeded into 384-well microplates and
incubated at a humidified incubator at 37 °C with 5% carbon dioxide for approximately 24h.
Following this incubation period, compounds and relevant controls were added to cells. The
experimental controls consisted of 10% DMSO for the positive control, 0.4% DMSO for the
negative control. The cytotoxic drug, puromycin, was utilised as a reference compound to
assess assay sensitivity and reproducibility. All compounds were diluted in a 12-point dose-
response with final concentrations ranging from 0.01 µM to 40 µM. All compound additions
were performed using the Bravo liquid handling platform (Agilent Technologies).
Compounds were incubated as described above for 4, 24 and 72 h time points. Following
these incubation periods, a final concentration of 600 µM Resazurin sodium salt was added to
each well and incubated in as described above for 4 h. Resazurin is a well-established

metabolic indicator dye for the measurement of cellular viability.14 Measurements were taken
on an EnVision™ multilabel plate reader (PerkinElmer) and analysis was performed utilising
Graphpad Prism™ software.
4.4 Incorporation of EdU and Pro-label Compounds into Cancer Cells. Cells (MDA-MB-
231: 2000 cells per well; PC3: 1000 cells per well) were seeded into 384-well CellCarrier
microplates (PerkinElmer) and incubated (37 °C with 5% carbon dioxide) for 72h prior to
application of EdU and pro-label derivatives (final concentration 1 µM, 5 µM or 10 µM) for 4
h in cell culture media. Following the 4 h incubation period, the cell culture media was
removed and 4% paraformaldehyde was applied for 10min. Next, cells were washed 3 times
with Dulbecco’s phosphate buffered saline (DPBS), then blocked and permeabilized with a
solution of 2% bovine serum albumin and 0.5%Triton X-100 for 20 min. Following a DPBS
rinse, cells were incubated with a freshly prepared cocktail of 5 µM of Alexa Fluor 488 azide,
1 mM copper sulfate and 100 mM sodium ascorbate in DPBS for 30 min at room temperature.
The azide cocktail was removed, cells washed 3 times with DPBS and a final concentration of
10 µM Hoechst added to the cells and incubated at room temperature for 30 min Cells were
rinsed with DPBS twice. Two independent experiments were performed, with three
experimental replicates per concentration of pro-label compound or EdU.
Fixed and stained cells were imaged using an Opera™ confocal imaging platform
(PerkinElmer) with 20 lens magnification (more than 3000 objects measured per well).
Columbus™ data management and image analysis software (PerkinElmer) was utilised for
image analysis. The fluorescence intensity for EdU and novel analogue staining was
quantified with positively nuclei staining defined as staining levels detected above
background. For selected pro-label compounds and EdU, fluorescence intensity values were
binned into four categories: low intensity, medium intensity, high intensity and very high
intensity.
Acknowledgements. We thank the Australian Research Council (Grant number FT10100185
to S-AP) for support. The authors would like to thank Dr Todd Shelper for his assistance with
analysis using Columbus.
1
Supplementary Material. Chemical synthesis, compound characterization data and H and
13C NMR spectra for compounds 4, 5, 6b-6d, 1b-1d, 7b-7d, 8a-8d, 2a-2d. Cytotoxicity for

compounds 1a-1d and 2a-2b at 4, 24 and 72 h time points on PC3, MDA-MB-231 and
HEK293 cell lines.
References
1.
Cavanagh, B. L.; Walker, T.; Norazit, A.; Meedeniya, A. C. B. Molecules 2011, 16,
7980.
2.
(a) Gratzner, H. G.; Leif, R. C.; Ingram, D. J.; Castro, A. Exp. Cell Res. 1975, 95, 88;
(b) Gratzner, H. G. Science 1982, 218, 474.
3.
4.
5.
Taupin, P. Brain Res. Rev. 2007, 53, 198.
Salic, A.; Mitchison, T. J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 2415.
Buck, S. B.; Bradford, J.; Gee, K. R.; Agnew, B. J.; Clarke, S. T.; Salic, A.
BioTechniques 2008, 44, 927.
6.
Chehrehasa, F.; Meedeniya, A.; Dwyer, P.; Abrahamsen, G.; Mackay-Sim, A. J.
Neurosci. Methods 2009, 177, 122.
7.
Laurent, J.; Frongia, C.; Cazales, M.; Mondesert, O.; Ducommun, B.; Lobjois, V.
BMC Cancer 2013, 13, 73.
8.
Neef, A. B.; Luedtke, N. W. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 20404.
9.
Galmarini, C. M.; Mackey, J. R.; Dumontet, C. Nature Leukemia 2001, 15, 875.
10.
Huynh, N.; Dickson, C.; Zencak, D.; Hilko, D. H.; Mackay-Sim, A.; Poulsen, S.-A.
Chem. Biol. Drug Des. 2015, 86, 400.
11. (a) Arrowsmith, C. H.; Audia, J. E.; Austin, C.; Baell, J.; Bennett, J.; Blagg, J.;
Bountra, C.; Brennan, P. E.; Brown, P. J.; Bunnage, M. E.; Buser-Doepner, C.; Campbell, R.
M.; Carter, A. J.; Cohen, P.; Copeland, R. A.; Cravatt, B.; Dahlin, J. L.; Dhanak, D.; Edwards,
A. M.; Frye, S. V.; Gray, N.; Grimshaw, C. E.; Hepworth, D.; Howe, T.; Huber, . V. .;
in, .; napp, S.; otz, . D.; ruger, . G.; Lowe, D.; ader, . .; arsden, B.; ueller-
Fahrnow, .; ller, S.; O’Hagan, . C.; Overington, . P.; Owen, D. .; osenberg, S. H.;
Roth, B.; Ross, R.; Schapira, M.; Schreiber, S. L.; Shoichet, B.; Sundström, M.; Superti-
Furga, G.; Taunton, J.; Toledo-Sherman, L.; Walpole, C.; Walters, M. A.; Willson, T. M.;
Workman, P.; Young, R. N.; Zuercher, W. J. Nature Chem. Biol. 2015, 11, 536; (b) Best, M.
D. Biochemistry 2009, 48, 6571.
12.
Li, T.; Guo, L.; Zhang, Y.; Wang, J.; Li, Z.; Lin, L.; Zhang, Z.; Li, L.; Lin, J.; Zhao,
W. Carbohydr. Res. 2011, 346, 1083.

13.
14.
JohnáJennings, L. J. Chem. Soc. Perkin Trans. 1 1992, 2197.
Borra, R. C.; Lotufo, M. A.; Gagioti, S. M.; Barros Fde, M.; Andrade, P. M. Braz Oral
Res 2009, 23, 255.
Legends for Figures and Schemes
Figure 1. EdU and acylated EdU pro-label compounds 1a-1d and 2a-2d.
Figure 2. Percentage of PC3 (a) and MDA-MB-231 (b) cancer cells labeled with EdU and
pro-label compounds at concentrations of 10, 5 and 1µM following 4h of treatment. Images of
PC3 (c) and MDA-MB-231 (d) cells labeled with pro-label compounds (10µM). Scale bar =
50µm. Data represent ± standard deviation.
Figure 3. Fluorescence intensity values for PC3 (a) and MDA-MB-231 (b) cancer cells
labeled with 10, 5 and 1µM EdU, 1c and 1d for 4h. Fluorescence intensity values were
classified as low, medium, high and very high intensity. Data are average values from a
representative dataset.

Graphical Abstract
O
O
N
NH
NH
ester
hydrolysis
N
O
O
RO
O
HO
O
O
OH
OR
O
R =
labeled DNA of
proliferating cells
EdU pro-labels