Synthesis of polyanionic c5-modified 2′-deoxyuridine and 2′-deoxycytidine-5′-triphosphates and their properties as substrates for dna polymerases

Article abstract of DOI:10.3390/molecules26082250

Modified 2^′-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG₆ to PEG₂₄ could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.

Full text of DOI:10.3390/molecules26082250

molecules
Article
Synthesis of Polyanionic C5-Modified 20-Deoxyuridine and
20-Deoxycytidine-50-Triphosphates and Their Properties as
Substrates for DNA Polymerases
Claire Dutson 1, Esther Allen 1 , Mark J. Thompson 1, Joseph H. Hedley 2,†, Heather E. Murton 2,‡
and David M. Williams 1,
*
1
2
Centre for Chemical Biology, Department of Chemistry, Sheffield Institute for Nucleic Acids,
University of Sheffield, Sheffield S3 7HF, UK; claire_dutson@hotmail.com (C.D.);
esther.allen@sheffield.ac.uk (E.A.); drmark64@hotmail.co.uk (M.J.T.)
QuantuMDx Group, Lugano Building, 57 Melbourne Street, Newcastle upon Tyne NE1 2JQ, UK;
Joe_Hedley@hotmail.co.uk (J.H.H.); heathermurton@gmail.com (H.E.M.)
*
†
‡
Correspondence: d.m.williams@sheffield.ac.uk; Tel.: +44-114-222-9502
Present address: Northumbria University, Sutherland Building, Newcastle upon Tyne NE1 8ST, UK.
Present address: Lex Diagnostics, Melbourn Science Park, Melbourn SG8 6EE, UK.
Abstract: Modified 20-deoxyribonucleotide triphosphates (dNTPs) have widespread applications
in both existing and emerging biomolecular technologies. For such applications it is an essential
requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples
of C5-modified dNTPs bearing negatively charged functionality have been described, despite the
fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-
based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides
each of which are labelled with an dianionic reporter group. The reporter group is tethered to the
nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties
of these modified dNTPs with a variety of DNA polymerases have been investigated to study
the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In
general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than
an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from
PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most
able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with
incorporation by Klenow(exo-) generally being very poor.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Dutson, C.; Allen, E.;
Thompson, M.J.; Hedley, J.H.;
Murton, H.E.; Williams, D.M.
Synthesis of Polyanionic C5-Modified
20-Deoxyuridine and 20-Deoxycytidine-
50-Triphosphates and Their Properties
as Substrates for DNA Polymerases.
Molecules 2021, 26, 2250. https://
doi.org/10.3390/molecules26082250
Academic Editor: David R.
W. Hodgson
Keywords: C5-modified dNTPs; polyanionic reporter group; DNA polymerases
Received: 26 March 2021
Accepted: 9 April 2021
Published: 13 April 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Base modified 20-deoxyribonucleoside-50-triphosphates (dNTPs) in which a specific
reporter group, ligand or catalytic moiety is tethered via a linker to the nucleobase allow
the preparation of functionalised nucleic acids that have a wide variety of applications
in biotechnology and chemical biology [1]. Typically dNTPs modified at the C5-position
of pyrimidines or C7-position of 7-deazapurine bases are good substrates for most DNA
polymerases since the modification does not interfere with Watson Crick base pairing. In
addition these modifications are in the major groove whilst most protein-DNA interactions
Copyright:
© 2021 by the authors.
during nucleotide incorporation arise in the minor groove [
2]. Such modified dNTPs can
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
be used in PCR [ ], DNA sequencing [ ] and labelled for use in structural studies [5
1
3
,
4
].
Other applications of modified dNTPs include the directed evolution of novel ligands and
catalysts using SELEX [6,7].
In order to incorporate poorer dNTP substrates, for example with bulkier modi-
fications, a number of groups have evolved existing DNA polymerases to create new
polymerases with altered substrate specificities [4,8–10]. For example, the Marx group
Molecules 2021, 26, 2250. https://doi.org/10.3390/molecules26082250
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2250
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have described [11] the successful incorporation of a dendrimer modified dUTP into an
oligodeoxynucleotide (ODN) using KlenTaq DNA polymerase and a C5-ODN-modified
dUTP using Therminator DNA polymerase [12]. They observed that the rigid alkyne-based
C5-anchor group was sufficient to hold the more flexible dendrimer and ODN side chains
outside of the active sites of the polymerases. Typically, for dNTPs involving C5-modified
pyrimidines, having a rigid linker (typically containing an alkyne or alkene) tethering the
modification to the nucleobase are better substrates for DNA polymerases [13,14].
There are relatively few examples of pyrimidine-modified dNTPs bearing negatively
charged reporter groups in the literature and this is further restricted when examining
dCTP analogues. These types of modified dNTPs would increase the potential for accessing
novel functionalized nucleic acids.
Our interest in these types of nucleotides derives from their potential use in the
diagnostic analysis of nucleic acids using Si-nanowire-based biosensors [15]. In this latter
context nucleic acid hybridization to a Si-nanowire immobilized PNA changes the charge
density and resistance of the nanowire, thereby producing a biosensing signal [16–19].
Monitoring the incorporation of suitably charge-modified dNTPs using such devices might
therefore allow sequence determination. Here we describe the design and synthesis of C5-
pyrimidine modified dNTPs bearing polyanionic side chains and evaluate their substrate
properties for a variety of DNA polymerases.
2. Results and Discussion
There are relatively few examples of C5-modified dUTP analogues with negatively
charged side chains in the literature (Figure 1). Of these, the dUTP analogues
not be incorporated into DNA by polymerases [20]. Compound is a substrate for KOD
XL DNA polymerase [21] and in common with , can be incorporated by PrimeSTAR HS
and Pwo polymerases [22]. The dNTP is a substrate for Taq DNA polymerase [21], whilst
nucleotides of general structure , which have ODNs of differing length tethered to the
base are incorporated into DNA by the engineered polymerase KlenTaq [12]. In the dCTP
1 and 2 could
3
4
5
6
series, the 5-valeric acid-dCTP
7 has been successfully incorporated [23] during primer
extension reactions by Vent (exo-), Pwo and 9◦Nm DNA polymerases in addition to the
Klenow fragment of DNA polymerase I. Successful amplification during PCR was also
demonstrated. The modified dCTP 8, can be incorporated into DNA using KOD XL DNA
polymerase [24]. These studies indicate that the substrate properties of these modified
nucleotides depend upon the type and/or length of the linker between the nucleobase
and charged moiety: Specifically, with a suitably long linker, even bulky substituents can
be tolerated.
Influenced by the findings of previous studies, we designed a series of modified
dNTPs with three features; a rigid C5 anchor attached to the C5 atom of the nucleobase, a
flexible PEG based linker of varying length and a charged reporter moiety (Figure 2).
We chose to use trimesic acid as the anionic reporter group that would be tethered to
the C5 positions of the triphosphates 9 and 10 using polyethyleneglycol (PEG) linkers of
differing lengths. PEG linkers were chosen in order to maintain good water solubility of
the dNTPs. To obtain the desired nucleotides we required PEG linkers functionalized with
amino and carboxylate termini (CA-PEG) that would facilitate coupling to the reporter
group and nucleotides via amide coupling reactions. Thus, commercially available PEG
linkers CA-PEG8 and CA-PEG24 were initially chosen in order to fully explore the effect of
linker length on the substrate properties of the dNTPs. These PEG linkers give approximate
lengths [25] of 2.9 nm (PEG8) to 8.6 nm (PEG24) i.e., distances of around 9 or 25 base pairs
in DNA.
To prepare the desired reporter-linker units we attempted a HATU-mediated amide
coupling between the diester of trimesic acid (11) and commercially available CA-PEG8
(12). However, this formed not only the desired linker-reporter unit 13, but also the
extended product 14 (Scheme 1) that were isolated as colourless oils in 31% and 19% yield,
respectively, following reversed phase HPLC.

Molecules 2021, 26, 2250
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Figure 1. Examples of base modified dNTPs with negatively charged side chains (ODN = oligodeoxyribonucleotide).
Figure 2. Linker design for C5-modified dUTP and dCTP analogues (left) and structures of precursor C5-propargylamino-
modified nucleotides (9) and (10).
Scheme 1. HATU coupling reaction of diethyl 1,3,5-benzenetricarboxylate (11) with CA-PEG8 forming the desired product
13 (31% yield) and the extended product 14 (19% yield). Reagents and conditions: Dry DMF, HATU, DIPEA, 11, CA-PEG8
◦
(12), 0 C to r.t. over 30 min.
Consequently, in order to improve the yield of 13 we chose an alternative route in
which we converted the acid 11 to its corresponding pentafluorophenyl ester 15. Com-
pound 15 in turn could be reacted cleanly with CA-PEG8 (12) to afford 13 in 61% yield
following RP-HPLC (Scheme 2). CA-PEG24
(16) was conjugated in the same manner to
afford 17 and the analogous PEG4 compound 21 was synthesized from PEG-4 (Scheme 2).

Molecules 2021, 26, 2250
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Scheme 2. Synthesis of linker-reporter units using from diethyl 1,3,5-benzenetricarboxylate penTable 13. (61%), 17 (73%);
◦
(
b
) TsCl, Et3N, r.t., 92%; (
c
) NaN3 in EtOH, 70 C, 79%; (
d
) NaH, THF, BrCH2CO2H, r.t., 88%; (
e) H2, 10% Pd-C in MeOH,
◦
50 C, 30 bar, then 15, DMF, DIPEA, r.t., 25% (two steps).
The reporter-linker units 13
) [13] using TSTU as the coupling agent. The products were then treated with aque-
ous sodium hydroxide solution followed by RP-HPLC to afford the modified dNTPs
22 24 (Figure 3), typically in yields around 60%. Analogous reactions of 13 and 21
, 14, 17 were then conjugated to C5-propargylamino-dUTP
(
9
–
with C5-propargylamino-dCTP (10) [21] gave modified dCTPs 25 and 26, respectively.
Alongside these dNTP analogues we also prepared the dUTP analogue 27. This contains
an O-propargyl-PEG linker. C5-modified dUTPs with an O-propargyl-PEG3 linker are
known to be substrates for KlenTaq DNA polymerase. [26] The linker unit for dUTP
27 was prepared from the O-propargyl-PEG6 compound 28 which was reacted with 1,4-
(dibromomethyl)benzene to give 29 which was then reacted with potassium phthalimide
to give 30. A Sonogashira coupling between compound 30 and 30-O-acetyl-5-iodo-20-
deoxyuridine (31) [27] provided the modified nucleoside 32 (Scheme 3). Phosphorylation
of nucleoside using the Ludwig-Eckstein method [28] followed by treatment with aqueous
methylamine solution provided dUTP 27 in 35% yield.
To compare the effects of the linker on the substrate properties of the modified nu-
cleotides, we first investigated the incorporation of the C5-modified dUTPs in primer
extension reactions using Vent (exo-) DNA polymerase (Figure 4). Initially we chose a
simple template containing the nucleotide sequence AC requiring insertion of dTTP/dUTP
and dGTP. Each reaction contained dATP, dCTP and dGTP together with dTTP or one of
the four dUTP nucleotides (22, 23, 24 and 27). Full length products were observed in each
case. When dTTP or dUTP was absent (i.e., using only dATP, dCTP and dGTP) significant
amounts of n + 1 product were observed (Figure 4, lane 6). In contrast, incorporation of
the modified nucleotides results in a significantly decreased gel mobility of the extended
products that was dependent on the molecular weight of the linker (Figure 4, lanes 2–5).
Furthermore, the nature of the PEG linker (propargylamido (22–24) or O-propargyl (27))
did not appear to affect properties of the modified dUTPs as substrates for Vent (exo-).
In order to investigate the ability of DNA polymerases to extend from the modified
nucleotides following incorporation, we chose a different primer-template requiring the
insertion of the modified nucleotide followed by incorporation of a further 10 natural
dNTPs (Figure 5). In addition, we included the polymerases Bst 2.0, Therminator and Pfu.
In reactions using Vent (exo-) polymerase nucleotide incorporation and extension can be
observed, however in each case there appears to be some misincorporation of the the natural
nucleotides indicated by the presence of faster-mobility full-length product obersved by
PAGE. A similar outcome is observed using Therminator polymerase. However, in each
case the reduced mobility of the full-length products indicates the incorporation of the
modified nucleotides 22
–24 and their subsequent extension albeit with some amounts of
misincorporation of the natural nucleotides. When the polymerases Bst 2.0 and Pfu were

Molecules 2021, 26, 2250
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examined, two product bands were observed by PAGE, indicative of the primer extension
reaction partially stalling after the incorporation of the modified dUTPs. The results also
show that the after incorporation of PEG6-dUTP (27), which contains a propargyl ether in
the linker, further extension by Bst 2.0 was not possible.
Figure 3. Structures of modified dNTPs.

Molecules 2021, 26, 2250
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Scheme 3. Synthesis of compound 33 (precursor to dUTP 26). Reagents and Conditions: (
a
) NaH, DMF, 1,4-dibenzylbromide,
◦
42%; (
b
) potassium phthalimide, DMF, 100 C, 86%; (c) 31, CuI, Pd(PPh3)4, Et3N, DMF, r.t., 38%; ( ) salicyl chlorophosphite,
d
4−
P2O7 , then I2 aq pyridine, then MeNH2, 35%.
Vent (exo-)
PEGn-dUTP
dTTP
Full length
Primer
Figure 4. PAGE gel for the incorporation of PEG modified dUTPs with the use of Vent (exo-) DNA polymerase. All lanes
except 7 contain dATP, dCTP, dGTP and the nucleotide indicated above the gel (from left): Lane 1 = dTTP, dATP, dCTP, dGTP;
Lane 2 = PEG8-dUTP (22), dATP, dCTP, dGTP; Lane 3 = PEG16-dUTP (23), dATP, dCTP, dGTP; Lane 4 = +PEG24-dUTP
(
24), dATP, dCTP, dGTP; Lane 5 = PEG6-dUTP (27), dATP, dCTP, dGTP; Lane 6 = dATP, dCTP, dGTP; Lane 6 = Control,
no dNTPs present. Conditions for all primer reactions; 20 nM primer, 15 nM template, 625 nM dNTPs, Vent (exo-) DNA
◦
polymerase, 40 C, 1 h.

Molecules 2021, 26, 2250
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Vent (exo-)
Therminator
Full length
Primer
Pfu
Bst 2.0
Full length
Primer
Figure 5. PAGE analysis of the incorporation of PEG modified dUTPs with the use of Vent (exo-), Therminator, Pfu and
Bst 2.0 DNA polymerases. All lanes except “no dNTPs” contain dATP, dCTP, dGTP and the nucleotide indicated above
the gel (from left): Lane 1 = dTTP, dATP, dCTP, dGTP; Lane 2 = PEG8-dUTP (22), dATP, dCTP, dGTP; Lane 3 = PEG16
dUTP (23), dATP, dCTP, dGTP; Lane 4 = PEG24-dUTP (24), dATP, dCTP, dGTP; Lane 5 = PEG6-dUTP, dATP, dCTP, dGTP;
Lane 6 = dATP, dCTP, dGTP; Lane 7 = Control, no dNTPs present. Conditions for all primer reactions; 20 nM primer, 15 nM
-
◦
template, 625 nM dNTPs, 40 C, 1 h.
We then examined incorporation of the dCTP analogues PEG4-dCTP (26) and PEG8-
dCTP (25). Since Vent (exo-) and Therminator had shown incorporation of the modified
dUTPs and subsequent extension of the primer, we chose both of these polymerases to
examine their incorporation together with a further two DNA polymerases, Omni Klentaq
and Klenow (exo-). All polymerases with the exception of Klenow (exo-) successfully incor-
porated the modified dCTPs and subsequently extended the primer (Figure 6). However
misincorporation can still be observed with Therminator and Omni Klentaq. Although
Klenow (exo-) did not successfully incorporate either modified dCTP within the 1 h incuba-
tion period with an extended incubation period of 7 h some incorporation and extension
was seen (data not shown).
In summary, we have described the chemical synthesis of C5-modified dUTP and
dCTP analogues functionalized with anionic reporter group tethered via different length
PEG-based linkers. We have shown that these analogues can be incorporated by a variety
of different polymerases producing full-length products with a significantly decreased
gel mobility. We anticipate that these functionalized nucleotides will have a variety of
applications in biotechnology and will report further on these in due course.

Molecules 2021, 26, 2250
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Omni Klentaq
Therminator
Full length
Primer
Klenow (exo-)
Vent (exo-)
Full length
Primer
Figure 6. PAGE analysis of the incorporation of PEG modified dCTPs with the use of Omni-Klentaq, Therminator, Klenow
(exo-) and Vent (exo-) DNA polymerase. All lanes except “no dNTPs” contain dATP, dGTP, dTTP and the nucleotide
indicated above the gel (from left): Lane 1 = dCTP, dATP, dGTP, dTTP; Lane 2 = PEG8-dCTP (25), dATP, dGTP, dTTP; Lane
3
= PEG4-dCTP (26), dATP, dGTP, dTTP; Lane 4 = dATP, dGTP, dTTP; Lane 5 = Control, no dNTPs present. Conditions for
◦
all primer reactions; 20 nM primer, 15 nM template, 625 nM dNTPs, 40 C, 1 h.
3. Materials and Methods
3.1. General Methods
Dry solvents were obtained from a Grubbs dry solvent apparatus. Column chro-
matography purifications were carried out on silica (60–200 mesh, VWR Chemicals, Hayes,
UK). Thin layer chromatography (TLC) was performed on pre-coated silica gel 60 F254
aluminium backed plates (Merck, London, UK). TLCs were visualised under UV (254 nm).
NMR spectra were recorded on either a Bruker AV250, AV400 or AV500 spectrometer
(as individually stated for all data) (Bruker, Billerica, MA, USA) and chemical shifts are
reported in δ values relative to tetramethylsilane as an external standard. J values are given
in Hz. All 1H, 13C and 31P spectra are available as supplementary materials. Mass spec-
trometry was performed by the University of Sheffield Mass Spectrometry Service using
the method of electrospray ionisation on an LCT Mass Spectrometer (Waters, Millford, MA,
USA) unless otherwise stated. Analytical RP-HPLC was performed on Waters 2695 or 2690
instrumentation using a Gemini C18 5
UK) at a flow rate of 1 mL/min, UV detection was accomplished at 260 nm unless specified
otherwise. Preparative RP-HPLC was performed using a Phenomenex Gemini C18 5
110Å 21.2 250 mm column at a flow rate of 21 mL/min. UV detection was recorded at
µm 4.6
×
250 mm column (Phenomenex, London,
µm
×
260 nm unless specified otherwise. All quoted retention those found by analytical RP-HPLC.
3,5-Diethyl-1,3,5-benzenetricarboxylate (11) was purchased from Sigma-Aldrich (London,
UK) and CA-PEG8 (12) CA-PEG24
MA, USA). Compounds and 10 were synthesized as previously described [13
bis(tri-n-butylammonium) pyrophosphate was also prepared as described [28].
(
16) were purchased from ThermoFisher (Waltham,
9
,
21] and

Molecules 2021, 26, 2250
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3.2. Chemical Synthesis
3.2.1. (3,5-Diethoxycarbonyl-1-benzenecarbonyl)-CA-PEG8 (13)
CA-PEG8
benzenetricarboxylate (15, 587 mg, 1.35 mmol) were added to a dried flask. Anhydrous
MeOH (10 mL) and dry diisopropylethylamine (200 L, 1.13 mmol) were added and the
(12, 500 mg, 1.13 mmol) and 1,3-diethyl-5-pentafluorophenyl-1,3,5-
µ
reaction stirred at r.t. for 20 h. The crude reaction mixture was analysed by analytical
RP-HPLC (100% A for 25 min; ramp to 95% B over 1 min; hold 5 min; to 100% A over 1 min;
hold 8 min (A = 60% H2O in MeCN + 0.1% TFA, B = MeCN) which showed the product at
14 min retention time. Subsequent purification by preparative RP-HPLC using the same
gradient gave the product (550 mg, 61%). Rf (10% EtOAc in hexane) 0.2; 1H-NMR (400 MHz,
CD3OD) δH 8.76 (s, 1H, phenyl CH), 8.71 (s, 2H, 2
OCH2CH3), 3.87–3.48 (m, 34H, OCH2CH2), 2.56 (t, J = 6.2 Hz, 2H, CH2C = O), 1.45 (t,
J = 7.1 Hz, 6H, 2
OCH2CH3) ppm; 13C-NMR (100.62 MHz, CDCl3) δC 166.0, 165.2, 134.8,
×
phenyl CH), 4.45 (q, J = 7.1 Hz, 4H,
2
×
×
133.1, 132.3, 131.4, 70.5, 70.4, 70.3, 69.7, 66.6, 61.7, 51.7, 50.8, 40.2, 34.8, 14.3 ppm; HR ES-MS
m/z calcd for C32H51NO15 [M + H]+ 690.3337, found 690.3357.
3.2.2. (3,5-Diethoxycarbonyl-1-benzenecarbonyl)-CA-PEG16 (14)
Diethyl 1,3,5-benzenetricarboxylate (11, 526 mg, 1.97 mmol) was dissolved in anhy-
drous DMF (15 mL) under N2, HATU (951 mg, 2.50 mmol) added and the mixture cooled
to 0 ◦C. DIPEA (0.89 mL, 5.2 mmol) was then added and the mixture stirred for 30 min.
CA-PEG8 (12, 1.00 g, 2.27 mmol) was then added and the mixture allowed to warm to
r.t. over 5 h. The mixture was then evaporated to dryness and the residue purified by
prep RP-HPLC using 40% MeCN/water containing 0.1% TFA as the mobile phase. Two
colourless oils were obtained after collection and evaporation: peak A at retention time
12.2 min and peak B with retention time 12.8 min. Peak A was identified as compound 14
(407 mg, 0.37 mmol, 19%) and peak B as compound 13 (422 mg, 0.61 mmol, 31%). Data for
1
13 as described above. Data for 14: H-NMR (400 MHz, D2O) δH 8.21 (s, 2H, 2
×
phenyl
OCH2CH3), 3.65–3.35 (m, 64H,
OCH2CH2), 3.20 (t, J = 6.0 Hz, 2H, CH2CO2H), 2.45 (t, J = 6.2 Hz, 2H, CH2NHCO), 2.35 (t,
J = 6.2 Hz, 2H, CH2N), 1.22 (t, J = 7.1 Hz, 6H, 2
OCH2CH3) ppm; 13C-NMR (100.62 MHz,
CH), 8.19 (s, 1H, phenyl CH), 4.21 (q, J = 7.1 Hz, 4H, 2
×
×
D2O) δC 175.97, 173.96, 167.45, 166.12, 134.41, 132.59, 132.06, 130.74, 69.55, 69.40, 68.77,
66.70, 66.11, 62.73, 39.73, 38.94, 35.91, 34.25, 13.43; HR ES-MS m/z calcd for C51H88N2O24
[M + H]+ 1112.5727, found 1112.5745.
3.2.3. 1,3-Diethyl-5-(pentafluorophenyl)-1,3,5-benzenetricarboxylate (15)
Diethyl 1,3,5-benzenetricarboxylate (11, 1.20 g, 4.50 mmol) was dissolved in anhydrous
DMF (20 mL) under N2 and EDCI (950 mg, 4.95 mmol) added. Pentafluorophenol (910 mg
,
4.95 mmol) was dissolved in anhydrous DMF (5 mL) and added slowly to the reaction
mixture. After stirring for 40 min., the solvent was evaporated and the crude residue
dissolved in acetone (100 mL). To this was added pH 6.2 phosphate buffer (50 mL of a
0.01 M solution). The solution was extracted with EtOAc (3
×
50 mL) and the combined
organic layers dried (MgSO4) and concentrated to give a clear oil. The crude product was
purified by silica column chromatography (eluent 10% EtOAc in hexane) to give a white
solid (1.48 g, 76%). Rf (10% EtOAc in hexane) 0.38; 1H-NMR (250 MHz, CDCl3) δH 9.02
(s, 3H, phenyl CH), 4.49 (q, J = 7 Hz, 4H, 2
×
CH2), 1.47 (t, J = 7 Hz, 6H, 2
×
CH3) ppm;
13C-NMR (100.62 MHz, CDCl3) δC 164.5, 161.3, 142.5, 141.1, 140.0, 139.3, 138.5, 136.7, 136.1,
135.3, 132.2, 128.0, 62.0, 14.3 ppm; HR ES-MS m/z calcd for C19H14O6F5 [M + H]+ 433.0711,
found 433.0729.
3.2.4. (3,5-Diethoxycarbonyl-1-benzenecarbonyl)-CA-PEG24 (17)
CA-PEG24
were added to a dried flask. Anhydrous MeOH (2 mL) and dry diisopropylethylamine
(15 L, 87 mol) were added and the reaction stirred at r.t. for 18 h. The crude reaction
mixture was analysed by analytical RP-HPLC (100% A for 25 min; ramp to 95% B over
(16, 100 mg, 87 µmol) and pentafluorophenyl ester (15) (45 mg, 105 µmol)
µ
µ

Molecules 2021, 26, 2250
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1 min; hold 5 min; to 100% A over 1 min; hold 8 min (A = 60% H2O in MeCN + 0.1%
TFA, B = MeCN), UV detection at 254 nm) which showed the product at 11 min retention
time. Subsequent purification by preparative RP-HPLC using the same gradient gave the
product (88 mg, 73%). Rf (10% EtOAc in hexane) 0.1; 1H-NMR (400 MHz, CD3OD) δH 8.76
(s, 1H, phenyl CH), 8.72 (s, 2H, 2
×
phenyl CH), 4.48 (q, J = 7.1 Hz, 4H, 2
×
OCH2CH3),
3.86–3.49 (m, 98H, OCH2CH2), 2.57 (t, J = 6.2 Hz, 2H, CH2C=O), 1.46 (t, J = 7.1 Hz, 6H,
2
×
OCH2CH3) ppm; 13C-NMR (100.62 MHz, CD3OD) δC 165.0, 132.3, 132.0, 131.4, 70.1,
69.0, 66.4, 61.5, 39.8, 34.4, 13.2 ppm; HR ES-MS m/z calcd for C64H116NO31 [M + H]+
1394.7531, found 1394.7462.
3.2.5. 1-(4-Methylbenzenesulfonyl)-3,6,9-trioxaundecane-11-ol (18)
Triethylamine (54 mL, 0.39 mol) was added to a stirred solution of tetraethylene glycol
◦
(50 g, 0.26 mol) in anhydrous DCM (20 mL). The reaction was cooled to 0 C, freshly
recrystallised 4-toluenesulfonyl chloride (4.9 g, 25.7 mmol) added and the mixture stirred
at r.t. overnight. The solvent was then evaporated to give an oil that was dissolved in DCM
(300 mL) and washed with water (3
acetic acid (0.1 M, 30 mL), water (30 mL), dried (MgSO4) and evaporated to give a clear oil
(8.25 g, 92%). Analytical data corresponded to that described previously [29].
×
20 mL). The organic layer was then washed with
3.2.6. 1-Azido-3,6,9-trioxaundecane-11-ol (19)
Sodium azide (3.85 g, 59.2 mmol) was added portionwise to a stirred solution of
compound 19 (8.25 g, 23.7 mmol) in ethanol (100 mL) over a period of 15 min. Once
homogeneous, the reaction was heated to 70 ◦C. After 20 h. the reaction was cooled to
r.t. and the mixture then evaporated to dryness to give a orange oil that was dissolved in
DCM (100 mL), washed with water (3
evaporated to give a pale yellow oil (4.1 g, 79%); 1H-NMR (250 MHz, CDCl3) δH 3.74–3.55
(m, 14H, 7
CH2), 3.38 (t, J = 5.0 Hz, 2H, CH2), 2.78 (bs, 1H, OH); 13C-NMR (63 MHz,
×
30 mL), and the organic layer dried (MgSO4) and
×
CDCl3) δC 72.48, 70.65, 70.62, 70.55, 70.30, 70.01, 61.65, 50.62; HR ES-MS m/z calcd for
C8H17N3O4 [M + H]+ 220.1292, found 220.1289.
3.2.7. 14-Azido-3,6,9,12-tetraoxatetradecan-1-oic Acid (20)
Sodium hydride (2.46 g, 61.5 mmol) was added portionwise to a stirred solution of
◦
compound 19 in anhydrous THF (60 mL) at 0 C over a period of 30 min. Once in solution,
bromoacetic acid (3.56 g, 25.6 mmol) was added and the reaction stirred overnight at r.t.
The reaction was then quenched with the slow addition of water (5 mL) and stirred for
15 min. The solvent was removed under reduced pressure and the residue dissolved in
DCM (150 mL), washed with HCl (2 M, 50 mL), sat. brine (50 mL) and dried (MgSO4)
before being concentrated to give the title compound as a light orange oil (5.52 g, 88%);
1H-NMR (250 MHz, CDCl3) δH 4.15 (s, 2H, CH2), 3.77–3.70 (m, 2H, CH2), 3.69–3.62 (m,
12H, 6
×
CH2), 3.37 (t, J = 5.0 Hz, 2H, CH2); 13C-NMR (63 MHz, CDCl3) δC 173.18, 71.16,
70.62, 70.56, 70.49, 70.29, 69.96, 68.62, 50.59, 29.65; HR ES-MS m/z calcd for C10H19N3O6
[M + H]+ 278.1347, found 278.1350.
3.2.8. (3,5-Diethoxycarbonyl-1-benzenecarbonyl)-CA-PEG4 (21)
Compound 20 (0.30 g, 1.08 mmol) in MeOH (10 mL) was placed under a H2 atmo-
sphere (30 bar) at 50 ◦C using a ThalesNano H-Cube (Budapest, Hungary). The solution
was then passed over a 10% Pd/C Catcart® at 1 mL/min. The solvent was removed under
reduced pressure to give the title compound as pale yellow oil (0.25 g). The oil was then
dissolved in in anhydrous DMF (10 mL) and dry diisopropylethylamine (170 µL, 1 mmol)
followed by compound 15 (0.52g, 1.2 mmol) and the reaction stirred at r.t. overnight.
Evaporation of the solvent and purification by prep-RP-HPLC gave a clear oil (0.11 g, 25%).
HPLC gradient: 20–70% B over 20 min where A = H2O + 0.1% trifluoroacetic acid and
1
B = MeCN + 0.1% trifluoroacetic acid. Retention time of product = 10.5 min; H-NMR
(250 MHz, CDCl3) δH 8.80 (t, J = 1.6 Hz, 1H, Ar-CH), 8.70 (d, J = 1.6 Hz, 2H, 2
×
Ar-CH),

Molecules 2021, 26, 2250
11 of 16
7.57 (bs, 1H, NH), 4.44 (q, J = 7.1 Hz, 4H, 2
×
CH2), 4.16 (s, 2H, CH2), 3.78–3.59 (m, 16H,
8
×
CH2), 1.44 (t, J = 7.1 Hz, 6H, 2
×
CH3); 13C-NMR (101 MHz, CDCl3) δC 173.05, 165.40,
134.60, 133.39, 132.48, 131.40, 70.79, 70.27, 70.06, 69.67, 68.59, 61.94, 40.37, 14.20; HR ES-MS
m/z calcd for C23H33NO11 [M + H]+ 500.2116, found 500.2132.
3.2.9. [(3,5-Dicarboxy-1-benzenecarbonyl)-CA-PEG8]-5-propargylamido-dUTP (22)
Compound 13 (138 mg, 200
TSTU (60 mg, 200 mol) and DIPEA (70
2 h. stirring at r.t., 5-(3-aminoprop-1-ynyl)-20-deoxyuridine-50-triphosphate (
µ
mol) was dissolved in anhydrous DMF (1.5 mL) and
L, 400 mol) added in quick succession. After
, 100 mol)
µ
µ
µ
9
µ
in sodium borate buffer (1.5 mL of a 0.1 M solution) was added and stirring continued for
24 h. The crude reaction mixture was analysed by analytical RP-HPLC (10% B for 5 min
then 10–100% B over 30 min; (A = 100 mM TEAB, B = 100 mM TEAB/50% MeCN), and
the product (retention time 26 min.) was used without further purification. Aq. NaOH
(4 mL of a 1 M solution) was then added and after stirring at r.t. for 2 h., the mixture
was neutralised by dropwise addition of aqueous acetic acid (1 M). The crude reaction
mixture was analysed by analytical RP-HPLC (5% B for 5 min then 5–100% B over 30 min
(A = 100 mM TEAB, B = 100 mM TEAB/50% MeCN) which showed the product at 16.5 min
retention time. Subsequent purification by preparative RP-HPLC using the same gradient
gave the product 23 (61 µmol, 60%). This was converted to the corresponding Na salt using
Dowex ion exchange resin (Na+ form). 31P NMR (101 MHz, D2O): δP −6.67 (d, J = 20.8 Hz),
−11.40 (d, J = 19.2 Hz), −22.60 (t, J = 20.3 Hz) ppm; ES-MS m/z 1137 [M + H]+.
3.2.10. [(3,5-Dicarboxy-1-benzenecarbonyl)-CA-PEG16]-5-propargylamido-dUTP (23)
Compound 14 (200
mol) and DIPEA (70
5-(3-aminoprop-1-ynyl)-20-deoxyuridine-50-triphosphate (
µ
mol) was dissolved in anhydrous DMF (2 mL) and TSTU (60 mg,
L, 400 mol) added in quick succession. After 2 h. stirring at r.t.,
) (100 mol) in sodium borate
200
µ
µ
µ
9
µ
buffer (2 mL of a 0.1 M solution) was added and the mixture stirred for 24 h. The crude
reaction mixture was analysed by analytical RP-HPLC (10% B for 5 min then 10–100% B
over 30 min; (A = 100 mM TEAB, B = 100 mM TEAB/50% MeCN) and the product (retention
time 30 min.) was used without further purification. Aq NaOH (3.2 mL of a 1 M solution)
was then added and the mixture stirred for 2 h. at r.t. then neutralised by dropwise addition
of acetic acid (1 M). The crude reaction mixture was analysed by analytical RP-HPLC (5%
B for 5 min then 5–100% B over 30 min; (A = 100 mM TEAB, B = 100 mM TEAB/50%
MeCN) which showed the product at 18 min retention time. Subsequent purification by
preparative RP-HPLC using the same gradient gave the product 24 (59
µmol, 59%). This
was converted to the corresponding Na salt using Dowex ion exchange resin (Na+ form).
31P NMR (101 MHz, D2O) δH
−
6.34 (d, J = 20.4 Hz),
−
11.30 (d, J = 19.9 Hz),
−
22.58 (t,
J = 19.4 Hz) ppm; ES-MS m/z 779 [M − 2H]2−
.
3.2.11. [3,5-Dicarboxy-1-benzenecarbonyl)-CA-PEG24]-5-propargylamido-dUTP (24)
Compound 17 (88 mg, 63 mol) was dissolved in anhydrous DMF (1 mL) and TSTU
(19 mg, 63 mol,) and DIPEA (22 L, 126 mol) added in quick succession. After 2 h.
stirring at r.t., 5-(3-aminoprop-1-ynyl)-20-deoxyuridine-50-triphosphate (
, 31.6 mol) in
µ
µ
µ
µ
9
µ
sodium borate buffer (1 mL of a 0.1 M solution) was added and the mixture stirred for
24 h. The crude reaction mixture was analysed by analytical RP-HPLC (10% B for 5 min
then 10–100% B over 30 min; (A = 100 mM TEAB, B = 100 mM TEAB/50% MeCN) and
the product (retention time 32 min) was used without further purification. Aq NaOH
(4 mL of a 1 M solution) was then added and the mixture stirred for 2 h at r.t., then
neutralised by dropwise addition of acetic acid (1 M). The crude reaction mixture was
analysed by analytical RP-HPLC (5% B for 5 min then 5–100% B over 30 min; (A = 100 mM
TEAB, B = 100 mM TEAB / 50% MeCN), which showed the product at 18 min retention
time. Subsequent purification by preparative RP-HPLC using the same gradient gave the
product (60 µmol, 60%). This was converted to the corresponding Na salt using Dowex ion

Molecules 2021, 26, 2250
12 of 16
exchange resin (Na+ form). 31P NMR (101 MHz, D2O) δP −7.70 (d, J = 16.5 Hz),
J = 18.4 Hz), −22.78 (t, J = 19.2 Hz) ppm.
−
11.48 (d,
3.2.12. [3,5-Dicarboxy-1-benzenecarbonyl)-CA-PEG8]-5-propargylamido-dCTP (25)
TSTU (90 mg, 300 mol) and DIPEA (105 L, 600 mol) were added successively
to a solution of compound 13 (207 mg, 300
µ
µ
µ
µ
mol) in anhydrous DMF (2.025 mL). After
0
stirring for 2 h at r.t., a solution of 5-(3-aminoprop-1-ynyl)-2 -deoxycytidine-5 -triphosphate
(
10, 143 µmol) in sodium borate buffer (0.1 M, pH 8.5, 2.25 mL) was added and stirring
continued for 24 h. The mixture was then evaporated. Purification by prep-RP-HPLC
(5–100% B over 30 min, where A = 0.1 M TEAB and B = 50% MeCN/0.1 M TEAB) gave
the desired product (82
22.2 min. After characterisation by ES-MS (Acc. Mass [ESI-]: Calculated for C44H69N5O27P3
[M H] the purified nucleotide was dissolved
H]−: 1192.3393 Observed: 1192.3375 [M
µmol, 57%) that eluted with a retention time of retention time of
−
−
in water (1.5 mL), aq. sodium hydroxide (2 M, 1.5 mL) added and the solution stirred
at r.t. for 2 h. The mixture was then neutralised by dropwise addition of aq. acetic acid
(1 M). Analysis of the reaction by RP-HPLC (5–95% B over 45 min, where A = 0.1 M TEAB
and B = 50% MeCN/0.1 M TEAB) showed the product with a retention time of 13 min.
Purification by prep-RP-HPLC (gradient as above) gave the triphosphate 25 (67
µ
mol,
11.52 (d, J = 20.3 Hz),
H]− 1136.2767,
82%). 31P NMR (101 MHz, D2O) δP −8.25 (d, J = 20.0 Hz),
−
−
22.57 (t, J = 19.9 Hz); HR ES-MS m/z calcd for C40H61N5O27P3 [M
−
found 1136.2736.
3.2.13. [3,5-Dicarboxy-1-benzenecarbonyl)-CA-PEG4]-5-propargylamido-dCTP (26)
TSTU (82 mg, 272 mol) and DIPEA (95 L, 544 mol) were added successively to a
stirred solution of compound 22 (136 mg, 272 mol) at r.t. in anhydrous DMF (2.25 mL)
under N2. After 2 h, a solution of 5-(3-aminoprop-1-ynyl)-20-deoxycytidine-50-triphosph
ate (10, 129 mol) in sodium borate buffer (0.1 M, pH 8.5, 2.25 mL) was added and stirring
µ
µ
µ
µ
µ
continued for 24 h. The mixture was then evaporated. Purification by prep-RP-HPLC
(5–65% B over 30 min where A = 0.1 M TEAB and B = 50% MeCN/0.1 M TEAB) gave the
desired product (96 µmol, 74%) that eluted with a retention time of retention time 26.7 min.
After characterisation by ES-MS (m/z 1002 [M + H]+) the purified nucleotide was dissolved
in water (1.5 mL), aq. sodium hydroxide (2 M, 1.5 mL) added and the solution stirred
at r.t. for 2h. The mixture was then neutralised by dropwise addition of aq. acetic acid
(1 M). Analysis of the reaction by RP-HPLC (5–65% B over 30 min, where A = 0.1 M TEAB
and B = 50% MeCN/0.1 M TEAB) showed the product with a retention time of 10.3 min.
Purification by prep-RP-HPLC (gradient as above) gave the title triphosphate 26 (79
µ
mol,
1
61%). H-NMR (250 MHz, D2O) 31P NMR (101 MHz, D2O) δP −6.58 (d, J = 21.1 Hz),
−
11.42
(d, J = 19.9 Hz),
−
22.72 (t, J = 20.6 Hz) ppm; HR ES-MS m/z calcd for C31H41N5O23P3 [M
− H]− 944.1442, found 944.1405.
3.2.14. [3,5-Dicarboxy-1-benzenecarbonyl)-CA-PEG6]-5-propargylamido-dUTP) (27)
Diethyl 1,3,5-benzenetricarboxylate (15, 5 mg, 18
DMF (1.25 mL) and TSTU (6 mg, 18 mol) and DIPEA (60
succession. After 2 h. stirring at r.t., PEG6-propargyl-dUTP (33, 8.3
µ
mol) was dissolved in anhydrous
L, 36 mol) added in quick
mol) in sodium
µ
µ
µ
µ
borate buffer (1.25 mL of a 0.1 M solution) was added and stirring continued for 20 h. The
crude reaction mixture was analysed by analytical RP-HPLC (10% B for 5 min; ramp to
100% B over 30 min; hold 5 min (A = 100 mM TEAB, B = 100 mM TEAB/50% MeCN),
UV detection at 288 nm) which showed the product at 25 min retention time. Aq. 1M
NaOH solution (2 mL) was added and stirring continued for 2 h at r.t. After this time the
reaction mixture was neutralised by dropwise addition of aq acetic acid (1 M). The crude
reaction mixture was analysed by analytical RP-HPLC (10% B for 5 min then 10–100%
B over 35 min; (A = 100 mM TEAB, B = 100 mM TEAB/50% MeCN), which showed the
product at 19.5 min retention time. Subsequent purification by preparative RP-HPLC using
the same gradient gave the product 28 (3.90 µ
mol, 47%). 31P-NMR (101 MHz, D2O) δP

Molecules 2021, 26, 2250
13 of 16
−
6.92 (d, J = 18.5 Hz),
−
11.45 (d, J = 19.5 Hz),
−
22.67 (t, J = 18.6 Hz) ppm; HR ES-MS m/z
calcd for C41H53N3O26P3 [M − H]− 1096.2130, found 1096.2080.
3.2.15. 3,6,9,12,15,18-Hexaoxaheneicos-20-yn-1-ol (28)
Hexaethylene glycol (5 g, 17.7 mmol)◦and NaH (60% in oil,1.06 g, 26.6 mmol) were
dissolved in anhydrous DMF (100 mL) at 0 C and stirred for 2 h at r.t. Propargyl bromide
(2.96 mL, 26.6 mmol) was added dropwise and the reaction mixture stirred for 16 h at
r.t. MeOH (10 mL) was then added and the reaction mixture evaporated. Purification
by silica column chromatography (0–10% MeOH in EtOAc) gave a yellow oil (1.73 g,
31%). Rf 0.6 (10% MeOH in EtOAc); 1H-NMR
2H, OCH2C CH), 3.73–3.64 (m, 24H, 6 OCH2CH2), 2.45 (s, 1H, C
δ
(250 MHz, CDCl3) δH 4.22 (d, J = 2.5 Hz
≡
×
≡
CH); 13C-NMR
δ
(101 MHz, CDCl3) δC 72.6, 70.6, 70.4, 70.3, 69.1, 61.8, 58.4 ppm; HR ES-MS m/z calcd for
C15H28O7Na [M + Na]+ 343.1733, found 343.1719.
3.2.16. 1-(4-Bromomethylbenzyl)-1-(1,3,6,9,12,15,18-hexaoxaheneicos-20-yne (29)
Compound 28 (1.73 g, 5.34 mmol) was dissolved in anhydrous DMF (100 mL) at
◦
0 C and NaH (60% in oil, 259 mg, 6.48 mmol) was added. After stirring for 2 h, 1,4-
bis(bromomethyl)benzene (1.71 g, 6.48 mmol) was added gradually. The reaction was
stirred for 16 h at r.t., then the solution was evaporated. Purification by silica column
chromatography (0–1% MeOH in EtOAc) gave the product as an oil (1.13 g, 42%). Rf 0.36
(EtOAc); 1H-NMR (400 MHz, CDCl3) δH 7.43–7.32 (m, 4H, phenyl CH), 4.57 (s, 2H, CH2),
4.51 (s, 2H, CH2), 4.22 (d, J = 2.4 Hz, 2H, OCH2C
≡
CH), 3.78–3.59 (m, 24H, 6
×
OCH2CH2),
2.45 (t, J = 2.4 Hz, 1H, C
128.1, 74.5, 72.8, 70.6, 70.4, 69.6, 69.1, 58.4, 33.4 ppm; HR ES-MS m/z calcd for C23H36O7Br
≡
CH) ppm; 13C-NMR (101 MHz, CDCl3) δC 138.7, 137.1, 129.1,
[M + H]+ 503.1644, found 503.1627.
3.2.17. 1-(4-Phthalimidomethylbenzyl)-1-(1,3,6,9,12,15,18-hexaoxaheneicos-20-yne (30)
Compound 29 (1.31 g, 2.25 mmol) and potassium phthalimide (458 mg, 2.48 mmol,
1.1 eq.) were dissolved in anhydrous DMF (60 mL) and heated to reflux for 16 h. After
cooling to r.t., the solvent was evaporated and the residue dissolved in EtOAc (50 mL) and
washed with saturated brine. The aqueous layer was extracted with EtOAc (3
×
40 mL) and
the organic layers were dried (Na2SO4) and concentrated to give a thick brown oil, which
was purified by silica column chromatography (EtOAc) to give the desired product (1.10 g,
1
86%). TLC: Rf 0.38 (EtOAc); H-NMR (400 MHz, CDCl3) δH 7.88–7.85 (m, 2H, phenyl
CH (phthalimide)), 7.74–7.72 (m, 2H, phenyl CH (phthalimide)), 7.44 (m, 2H, phenyl CH),
7.42 (m, 2H, phenyl CH), 4.86 (s, 2H, CH2), 4.54 (s, 2H, CH2), 4.23 (d, J = 2.4 Hz, 2H,
OCH2C
≡
CH), 3.71–3.65 (m, 24H, 6
×
OCH2CH2), 2.45 (t, J = 2.4 Hz, 1H, C
CH) ppm; 13C-
≡
NMR δC (101 MHz, CDCl3): 134.0, 128.7, 128.1, 123.4, 74.6, 72.9, 70.6, 70.4, 69.4, 69.1, 58.4,
41.4 ppm; HR ES-MS m/z calcd for C31H39NO9Na [M + Na]+ 592.2523, found 592.2544.
0
0
3.2.18. 5-[(4-Phthalimidomethylbenzyl)-PEG6-propargyl)]-3 -O-acetyl-2 -deoxyuridine (32
5-Iodo-30-O-acetyl-20-deoxyuridine (31, 250 mg, 632
mol) [27], copper (I) iodide
24 mg, 126 mol), Pd (PPh3)4 (73 mg, 63 mol) and triethylamine (176 L, 1.26 mmol)
)
µ
(
µ
µ
µ
were dissolved in anhydrous DMF (5 mL). Compound 31 (900 mg, 1.58 mmol) dissolved
in anhydrous DMF (5 mL) and then added portionwise to the mixture over 30 min. After
stirring for 18 h, a 5% aq. solution of disodium EDTA (12 mL) was added and the mixture
stirred until a cloudy green precipitate formed. The solvent was then evaporated and the
residue dissolved in EtOAc. The EtOAc solution was then washed with water (3
dried (Na2SO4) and evaporated. The crude product was purified by silica column chro-
×
30 mL),
matography (0–5% MeOH in DCM) to give a white solid (201 mg, 38%). Rf 0.22 (5% MeOH
in DCM); 1H-NMR
δ (400 MHz, CDCl3) δH 9.00 (br s, 1H, NH), 8.38 (s, 1H, H6), 7.95–7.75
(m, 2H, phenyl CH (phthalimide)), 7.78–7.62 (m, 2H, phenyl CH (phthalimide)), 7.40 (d,
J = 8.1 Hz, 2H, phenyl CH), 7.31 (d, J = 2.2 Hz, 2H, phenyl CH), 6.33 (dd, J = 8.3, 5.8 Hz,
1H, H10), 5.35 (m, 1H, H40), 4.84 (s, 2H, CH2), 4.52 (s, 2H, CH2), 4.37 (s, 2H, OCH2C
CH),
≡

Molecules 2021, 26, 2250
14 of 16
4.13 (d, J = 1.7 Hz, 1H, H30), 3.96–3.84 (m, 2H, 50CH2), 3.79–3.50 (m, 24H, 6
×
OCH2CH2),
2.52–2.25 (m. 2H, 20CH2), 2.10 (s, 3H, COOCH3); 13C-NMR (101 MHz, CDCl3) δC 149.1,
144.2, 134.0, 132.1, 128.7, 128.1, 127.9, 123.4, 85.8, 85.7, 75.4, 72.8, 70.5, 70.4, 69.4, 69.1, 62.3,
59.2, 41.3, 38.3, 21.0, 1.0 ppm; Mass Spec: ES+ m/z 838.3 [M + H]+.
3.2.19. 5-[(4-Aminomethylbenzyl)-PEG6-propargyl)]-dUTP (33)
5-(Phthalimido-PEG6-propargyl)-30-O-acetyl-20-deoxyuridine (32, 100 mg, 119
was dried in a vacuum dessicator over P2O5 overnight. The nucleoside was then dis-
solved in 1:3 v/v dioxane/pyridine (480 L) in the reaction flask before salicyl chlorophos-
phite (286 L of a 1 M solution in dioxane, 286 mol) was added. After 30 min bis(tri-
n-butylammonium) pyrophosphate (358 L of a 0.5 M solution in DMF, 179 mol) in
tributylamine (86 L, 358 mol) was added in one portion to the reaction flask. After
µmol)
µ
µ
µ
µ
µ
µ
µ
stirring for a further 10 min. iodine (1.2 mL of a 1% solution in 50/50 pyridine/H2O)
was added, and after another 15 min stirring NaHSO3 (0.5 mL of a 5% aqueous solu-
tion) was added. The reaction mixture was then evaporated to a gum before addition of
methylamine (10 mL of a 10% aqueous solution). After 18 h stirring, the methylamine was
removed under reduced pressure. The crude product was purified by MPLC ion-exchange
chromatography (gradient 50 mM–900 mM TEAB, flow rate 8 mL/min, UV detection at
288 nm). Analytical RP-HPLC (10% B for 5 min; ramp to 65% B over 30 min; to 100% B
over 1 min; hold 6 min (A = 100 mM TEAB, B = 100 mM TEAB/50% MeCN), UV detection
at 288 nm) of the product peak from MPLC ion-exchange chromatography showed that
the triphosphate (retention time 27 min). Purification by preparative RP- HPLC using the
same gradient gave the product (42
µ
mol, 35%). 31P-NMR (101 MHz, D2O) δP −9.15 (d,
22.81 (t, J = 19.3 Hz) ppm, HR ES-MS m/z calcd for
J = 19.1 Hz),
−
11.35 (d, J = 19.8 Hz),
−
C32H49N3O21P3 [M − H]− 904.2071, found 904.2032.
3.3. Nucleotide Incorporation Assays
◦
Using either ploymerases HotStarTaq (heated to 95 C for 3 min prior to use), Phusion,
Vent (exo-), Klenow (exo-), Bst 2.0, Therminator (New England Biolabs, Ipswich, MA,
USA), or Omni-KlenTaq DNA polymerase (DNA Technology, St Louis, MO, USA), primer
extension reactions were performed as 20
µL reactions comprising of: 1 U polymerase in
1
×
polymerase specific buffer (provided by DNA Technology, St Louis, MO, USA), 20 nM
0
Cy5-5 -labelled primer and 15 nM template. To investigate the incorporation of a modified
dCTP, a nucleotide mix (modified-dCTP, dATP, dGTP, dTTP) was included alongside the
three controls (Positive control: dATP, dGTP, dTTP, dCTP; Polymerase fidelity control:
dATP, dGTP, dTTP; Negative control: No polymerase). The incorporation of the modified
dUTPs were investigated in an analogous manner (modified dUTP replacing dTTP). All
dNTP mixes were used at a final concentration of 625 nM. In all cases the polymerase was
added lastly to the reaction mixes before the desired incubation period. After incubation
(time and temperature indicated in main text/figure legends), each reaction was stopped
with the addition of 5 µL complementary sequence (100 µM) in a solution of EDTA (250 mM,
pH 8), formamide and Orange G gel loading dye. The reaction mixtures were denatured
by heating at 95 ◦C for 3 min and analysed by electrophoresis on a 17.5% polyacrylamide
gel (PAGE). The gel was imaged by fluorescence visualisation facilitated by the use of the
Cy5-labelled primer.
4. Conclusions
We have described the chemical synthesis and characterisation of four C5-modified
dUTP and two C5-modified dCTP analogues that are functionalised with polyanionic side
chains linked to the nucleosides with PEG linkers of differing lengths. We have shown that
these nucleotides can be incorporated into DNA by a variety of commercially available
DNA polymerases, particularly Vent (exo-) and Omni Klentaq. Klenow (exo-) was less
efficient at incorporation of the dCTP analogues compared to the other polymerases tested.
Successful incorporation of the modified dNTPs is manifested by a reduced electrophoretic

Molecules 2021, 26, 2250
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mobility that is dependent of the length of the PEG linker. Although all modified dNTPs
were successfully incorporated by all DNA polymerases tested the largest modified dNTP,
the PEG24-dUTP analogue, hindered the continued extension with both Pfu and Bst 2.0
DNA polymerases. The alkyne-ether linkage also examined showed good incorporation
with only Bst 2.0 DNA polymerase failing to continue primer extension after its incorpora-
tion. We have therefore demonstrated that these anionically charged PEG modified dNTPs
are substrates for a number of DNA polymerases and therefore might be exploited for a
variety of biotechnological platforms.
Supplementary Materials: The following are available online, 1H-, 13C- and 31P-NMR spectra of
synthesized compounds.
Author Contributions: All authors, were involved in conceptualization of the research and prepa-
ration of the manuscript. C.D., E.A. and M.J.T. undertook chemical syntheses, C.D. and H.E.M.
undertook DNA polymerase assays. D.M.W. was involved in the supervision of C.D. and E.A. and
J.H.H. involved in the supervision of C.D., E.A. and H.E.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by BBSRC, grant number BB/I016244/1 and QuantuMDx Ltd.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds where still available can be requested from the
corresponding authors.
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