Customizable and Regioselective One-Pot N?H Functionalization of DNA Nucleobases to Create a Library of Nucleobase Derivatives for Biomedical Applications

Article abstract of DOI:10.1002/ejoc.202100786

DNA is one of the most exciting biomolecules in nature for developing supramolecular biofunctional nanoarchitectures owing to the highly specific and selective interactions between complementary Watson-Crick base pairing. Herein, simple and one-pot synthetic procedures have been implemented for producing a library of DNA nucleobase derivatives endowed with reactive functional groups for bioconjugation and cross-linking strategies with other (bio)molecules. Purine and pyrimidine molecules have been regioselectively N?H functionalized either via N-alkylation, N-allylation, N-propargylation or Michael-type reactions and structurally characterized. The influence of the reaction conditions was assessed and discussed. The in vitro biocompatibility of the native and nucleobase derivatives was evaluated by culturing them with human fibroblasts, revealing their cytocompatibility. The library of nucleobase derivatives holds great promise for being coupled to different biomolecules, including biopolymeric materials, lipids, and peptides, thus potentially leading to modular supramolecular nanobiomaterials for biomedicine.

Full text of DOI:10.1002/ejoc.202100786

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Customizable and regioselective one-pot N–H functionalization of
DNA nucleobases to create a library of nucleobase derivatives for
biomedical applications
Authors: Djenisa H. A. Rocha, Carmen M. Machado, Vera Sousa,
Cristiana F. V. Sousa, Vera L. M. Silva, Artur M. S. Silva,
João Borges, and João F. Mano
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202100786
Link to VoR: https://doi.org/10.1002/ejoc.202100786
01/2020

10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
Customizable and regioselective one-pot N–H functionalization of
DNA nucleobases to create a library of nucleobase derivatives for
biomedical applications
Djenisa H. A. Rocha,*[a] Carmen M. Machado,[a] Vera Sousa,[a] Cristiana F. V. Sousa,[a] Vera L. M.
Silva,[b] Artur M. S. Silva,[b] João Borges,*[a] and João F. Mano*[a]
[a]
[b]
Dr. D. H. A. Rocha,* C. M. Machado, V. Sousa, C. F. V. Sousa, Dr. J. Borges,* Prof. Dr. J. F. Mano*
CICECO – Aveiro Institute of Materials
Department of Chemistry
University of Aveiro
Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
E-mail: djenisa@ua.pt (http://compass.web.ua.pt/djenisa-rocha/); joaoborges@ua.pt (http://compass.web.ua.pt/joao-borges/); jmano@ua.pt
(http://compass.web.ua.pt/joao-mano/)
Dr. V. L. M. Silva, Prof. Dr. A. M. S. Silva
LAQV-REQUIMTE
Department of Chemistry
University of Aveiro
Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
Supporting information for this article is given via a link at the end of the document.
Abstract: DNA is one of the most exciting biomolecules in nature for
developing supramolecular biofunctional nanoarchitectures owing to
the highly specific and selective interactions between complementary
Watson-Crick base pairing. Herein, simple and one-pot synthetic
procedures have been implemented for producing a library of DNA
nucleobase derivatives endowed with reactive functional groups for
bioconjugation and cross-linking strategies with other (bio)molecules.
Purine and pyrimidine molecules have been regioselectively N–H
functionalized either via N-alkylation, N-allylation, N-propargylation or
Michael-type reactions and structurally characterized. The influence
of the reaction conditions was assessed and discussed. The in vitro
biocompatibility of the native and nucleobase derivatives was
evaluated by culturing them with human fibroblasts, revealing their
cytocompatibility. The library of nucleobase derivatives holds great
promise for being coupled to different biomolecules, including
biopolymeric materials, lipids, and peptides, thus potentially leading
to modular supramolecular nanobiomaterials for biomedicine.
Figure 1. Purine (A and G) and pyrimidine (T and C) of DNA nucleobases
subjected to N–H chemical functionalization.
Over the last two decades, the interest in the rational design and
development of DNA-based supramolecular multifunctional
nanoarchitectures has notably increased due to recognition of the
DNA as one of the smartest, versatile and powerful natural
building blocks in the biomedical arena.[3-7] However, challenges
associated to the nucleobases themselves, including the
preservation of hydrogen bonding capabilities, the regioselective
introduction of functional groups in high yield, and the poor
solubility in aqueous media and commonly used organic solvents,
remain as the major bottlenecks for modulating the nucleobases’
reactivity, physicochemical and biological properties.[8-13] As such,
increasing efforts have been devoted to improve the solubility of
the nucleobases. For instance, it has been reported that the
addition of appropriate solubilizing groups such as 2-ethylhexyl
chains ensures good solubility to the nucleobases in organic
solvents.[10] The nitrogen atoms in purine and pyrimidine rings
reveal varying degrees of nucleophilicity and, thus, react
differently with several electrophilic compounds, such as
alkylating or acylating agents.[9] The N-alkylation of nucleobases
has been receiving considerable attention since this reaction is a
rapid and direct route to access the acyclic nucleosides frequently
used as chemotherapeutic agents.[9,14-16] To achieve the N-
alkylation of the nucleobases, a wide variety of carbon-
Introduction
Nature provides us with a unique source of biomolecules that are
inherently responsible for supporting and orchestrating the
biological processes that govern life on Earth. Among them, DNA
is unequivocally one of the most powerful biomolecules that has
extended well beyond its classical key role on storing, encoding,
and conveying the genetic information in biological systems
through its basic molecular building blocks – the natural
nucleobases [adenine (A), guanine (G), thymine (T), and cytosine
(C) (Figure 1)].[1,2]
1
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
electrophiles have been used, including alkyl and aryl halides,[9,17]
α,β-unsaturated esters,[18] carbonates,[19] and carboxamide.[20]
However, most of them are known to be toxic and carcinogenic,
which extensively limits their use in the biomedical arena.
Recently, it has been shown that primary alcohols are appropriate
candidates to react with nucleobases, leading to N-alkyl
derivatives in good yield and with low toxicity.[21]
The chemical modification of nucleobases with either terminal
alkyne,[22-24] acrylate,[25-27] allyl,[28,29] acrylonitrile[26,30-32] and thiol[33]
functional groups at the secondary amine has been explored in
the last decades, and in most of the cases, A and T have been at
the limelight. From the chemical functionalization standpoint, the
major bottlenecks associated to these functionalization strategies
include the development of efficient synthetic methodologies for
the successful introduction of reactive terminals at C and G, as
well as simple and direct methods for the regioselective
functionalization of the DNA nucleobases, aiming to preserve
their hydrogen bonding capabilities. As such, there is still plenty
of room for improving the known and developing new and
innovative synthetic approaches for enabling the functionalization
of all DNA nucleobases. The conjugation of the functionalized
nucleobases with either synthetic or natural polymers, lipids
and/or peptides enable the development of complex
supramolecular biofunctional nanomaterials well-suited for a wide
range of biomedical applications.[25,34-39]
Scheme 1. Synthesis of N-alkylated A (1) and (3). i) K2CO3, dry DMF, r.t..
Yields: 53 % (1), 77 % (3).
Initial attempts to react G with 1,5-dibromopentane 6 followed a
synthetic methodology like the one described by Kang et al.[27]
using tetrabutylammonium iodide (TBAI) and C18-crown-6.[43]
However, the reaction was not successful since only unmodified
G was recovered (Table 1, entries 1-2). Therefore, some
modifications were made. In particular, the reactions were carried
out in two steps (Table 1, entries 3-4): firstly, G dissolution and
deprotonation followed by the addition of compound 6. Although
in vestigial quantities, the N-alkylation reaction afforded the
envisioned dialkylated guanine 5 (Scheme 2) solely when the
compound 6 was added dropwise for 30 min at r.t. (Table 1, entry
4). The synthesis of the dialkylated product 5 (Scheme 2) in
vestigial amounts could be explained by the deprotonation of the
acidic N(3) and N(9) protons of G.
Herein we report the one-pot synthesis of a new library of
nucleobase derivatives by regioselective N–H functionalization of
the DNA nucleobases with either alkyl, allyl, propargyl or
propanenitrile functional groups via N-alkylation, N-allylation, N-
propargylation or Michael-type reactions, respectively, aiming to
extend the functionality of the nucleobases towards obtaining
added-value biofunctional materials. Their chemical and
structural characterization was performed by nuclear magnetic
resonance (NMR), mass spectrometry (MS) and high-resolution
mass spectrometry (HR-MS). Furthermore, the in vitro
biocompatibility of the native and nucleobase derivatives was
assessed using primary human dermal fibroblasts (HF), revealing
their non-cytotoxicity and, thus suitability for addressing
biomedical applications.
Scheme 2. Synthesis of N-dialkylated G (5). i) NaH (60% mineral oil), dry DMF,
r.t., traces (5).
Some successful examples of T N-alkylations have been reported
in the literature.[27,40,44] Following similar conditions to those
reported in the literature, although using different alkylating
agents, namely 1,2-dibromoethane 2 and 1,3-dibromopropane 4,
different reaction conditions were tested such as the use of phase
transfer catalysts (PTC), solvents [water, dichloromethane,
anhydrous DMF, dimethyl sulfoxide (DMSO), and tetrahydrofuran
(THF)], bases (K2CO3, NaOH and NaH), as well as distinct
heating methods [conventional, microwave (MW) and ohmic
heating]. However, in most cases no reaction occurred or the
expected N-alkylated T was not obtained. Several alkylation
conditions attempted without (Table 1, entry 5) or with phase-
transfer agent (Table 1, entry 6) did not afford the desired
alkylated T. The N-alkylation of T using K2CO3 in anhydrous
DMSO, followed by the addition of 1,2-dibromopropane 4 at r.t.,
afforded 7 with 5% yield (Table 1, entry 7) (Scheme 3). We
hypothesize that the addition of PTC such as TBAI,
tetrabutylammonium bromide (TBAB), tetrabutylammonium
acetate (TBAA), tetrabutylammonium hydroxide (TBAOH) and
crown ether could offer a simple and efficient solution to
counteract the low solubility of the nucleobases in common
organic solvents and could facilitate the N-alkylation. The
Results and Discussion
Functionalization of nucleobases via N-alkylation reaction
The synthesis of N-alkylated A followed previously reported
synthetic procedures.[16,33,40] Briefly, A was treated with potassium
carbonate (K2CO3) in anhydrous N,N-dimethylformamide (DMF),
followed by the addition of 1,2-dibromoethane 2 at room
temperature (r.t.) to obtain 9-(2-bromoethyl)-9H-purin-6-amine 1
in moderate yield (53%).
A was also treated with 1,3-
dibromopropane 4 to generate the 9-(3-bromopropyl)-9H-purin-6-
amine 3 in good yield (77%) (Scheme 1), following a similar
synthetic procedure.
The alkylation of G has revealed to be much more challenging. In
fact, it has been scarcely investigated by the organic chemistry
community due to its very low solubility in common organic
solvents. Moreover, G oxidizes more readily than A. Its high
melting point of 350 °C reflects the intermolecular hydrogen
bonding between the oxo and amino groups in the crystal
structure, being responsible for its insolubility in water.[41,42]
2
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
attempts to couple 1,5-dibromopentane 6 to T at r.t. using the
and the mixture was heated at 65 °C for more 24 h (Table 1, entry
combinations K2CO3/TBAB and NaOH/TBAB were not successful. 14).
However, the increase in the reaction time, temperature and
exchange of TBAB by TBAI led to 1-(5-bromopentyl)-5-
methylpyrimidine-2,4(1H,3H)-dione 8 in low yields (15-20%), as
well as to the recovery of the starting material T (Table 1, entries
8-9).
The use of the conditions described by Zerdan et al.[8] and
different phase-transfer catalysts such as TBAA, TBAOH in 40%
H2O or MeOH and C18-crown-6[43] did not afford the N-alkylated
C derivatives (Table 1, entries 10, 11) or led to the formation of 9
in very low yield (Table 1, entries 12-13).
Scheme 4. Synthesis of N-alkylated C (9). I) K2CO3, TBAI, MeOH, r.t. to 65 ºC.
Yield: 50 % (9).
Scheme 3. Synthesis of N-alkylated T (7) and (8). i) K2CO3, TBAI, dry DMF, r.t.,
ii) K2CO3, DMSO, r.t. Yields: 5 % (7), 20 % (8).
The low yields obtained for T alkylation reactions can be
explained by the challenging purification process and its poor
solubility in common organic solvents. Therefore, the N-alkylation
of T was attempted following other methods described in the
literature. One of them, consisted in the one-pot alkylation using
primary alcohols such as 3-bromopropanol and 6-
bromohexanol.[9] However, none of the methods afforded the
envisioned products. In this regard, it has been postulated that the
two secondary amines present in the T structure contribute to the
Table 1. Screening of the reaction conditions for the synthesis of N-alkylated
nucleobases
Catalyst
(equiv)
Yield
(%)
Entry
Nucleobase
Solvent
T
Base
r.t., 65 h
then reflux,
24 h
TBAI
(0.1)
1[a]
2
G
G
G
MeOH
MeOH
DMF
K2CO3
K2CO3
NaH[c]
N.R.[b]
N.R.[b]
N.R.[b]
C18-
crown-6
reflux
r.t., 72 h
then
50 °C, 48 h
r.t., 72 h
then
50 °C, 48 h
r.t., 24 h
then 80°C,
24 h
low regioselectivity observed in several
T
N-alkylation
3[a]
-
-
-
reactions.[45,46] Thus, the N-protection of one secondary amine
was attempted aiming to reduce the reactive sites of the molecule.
The 3-(2-bromoethyl)-5-methylpyrimidine-2,4(1H,3H)-dione 12
was obtained in three steps: N-protection with tert-butoxycarbonyl
(Boc) protecting group, followed by N-alkylation with 1,2-
dibromoethane 2 and finally the N-deprotection, as illustrated in
the Scheme 5. The low overall yield gathered in the synthesis of
compound 12 can be explained by the fact that the reactions were
incomplete, being necessary to purify the obtained compound in
each step by chromatography. The NMR spectra allowed to
confirm the introduction of the Boc group at structure 10 and the
presence of 2-bromoethyl at product 11.
4[a][d]
G
T
DMF
DMF
NaH[c]
K2CO3
Trace
N.R.[b]
5
TBAB
(0.06)
6
7
T
T
T
T
C
DMF
DMSO
DMF
r.t., 25 h
r.t., 24 h
K2CO3
K2CO3
K2CO3
K2CO3
-
N.R.[b]
5
-
TBAI
(0.08)
TBAI
8
40 °C, 48 h
r.t. 30 h
15
9
DMF
20
(0.06)
r.t.,
overnight
10
TBAA
DMF
N.R.[b]
TBAOH
in 40%
H2O
11
C
DMF
r.t., 24 h
-
N.R.[b]
TBAOH
in
MeOH
TBAI
(0,08)
12
13
C
C
C
DMF
MeOH
MeOH
r.t., 24 h
r.t, 24 h
-
-
10
12
50
r.t., 65 h
then
65 °C, 24 h
TBAI
(0.1)
14[a]
K2CO3
[a] Reaction was carried on in two steps. [b] No reaction. [c] NaH in 60% wt
mineral oil. [d] Addition of 1,5-dibromopentane 6 (1.1) during 30 min. at r.t.
The synthesis of C derivative 9 was performed following the
method described by Kang et al.[27] with some modifications.
Briefly, the compound C was treated with K2CO3 in MeOH in the
presence of TBAI as catalyst, followed by the addition of 1,5-
Scheme 5. Synthesis of 3-(2-bromoethyl)-5-methylpyrimidine-2,4(1H,3H)-dione
(12). i) Boc2O, cat. DMAP, MeCN, r.t., 3 h, ii) NaH, dry DMF, 0 ºC to r.t., 30 min,
iii) K2CO3, MeOH, r.t., 24 h. Yields: 57 % (10), 14 % (11), 63 % (12).
dibromopentane
6
to
generate
the
4-amino-1-(5-
bromopentyl)pyrimidin-2(1H)-one 9 in 50% yield (Scheme 4).
Initially the reaction mixture (C, K2CO3/TBAI and 1,5-
dibromopentane 6) was stirred at r.t. for 65 h. Then, the thin-layer
chromatography (TLC) showed the presence of C in the reaction
medium so, K2CO3/TBAI and 1,5-dibromopentane 6 were added
Functionalization of nucleobases via Michael addition
reaction
The Michael-type addition reaction has been described in the
literature[26,30-32] and involves the addition of the Michael acceptor-
3
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
acrylonitrile to a Michael donor A, T and C, usually in the presence
of a polar solvent and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
or triethylamine (TEA) as deprotonating agent. In this work, the
functionalization of all DNA nucleobases with the acrylonitrile
group followed the reaction conditions described by Graßl et al.[47]
with slight modifications, leading to the nucleobase derivatives 13,
14, 15 and 16 in 12-35% yields (Table 2).
As highlighted in Table 2, the optimal condition for the synthesis
of the compounds 13, 15 and 16 involves the addition of TBAI to
facilitate the nucleobases solubility.
A was reacted with
acrylonitrile 17 in the presence of TBAI under microwave
irradiation for 60 min (Table 2, entry 1) but the expected
compound 13 was not isolated. However, after conventional
heating at 70 ºC for a longer reaction time, 13 was obtained in
12% yield (Table 2, entry 2). Following similar conditions, the
reaction of acrylonitrile 17 with G did not provide the compound
14 at all or it was obtained in vestigial amounts (Table 2, entries
3, 4 and 5). For the introduction of the propanenitrile group at the
secondary amine of G, the reaction was performed in slightly
basic conditions with the addition of NaOH (2 M) for better
solubility, thus being possible the synthesis of 3-(2-amino-6-oxo-
1,6-dihydro-9H-purin-9-yl)propanenitrile 14 in modest yield (30%)
(Table 2, entry 6). Even using the solution of NaOH (2 M) as base,
it was not possible to improve the reaction yield of 14 (Scheme 6).
Scheme 7. Synthesis of N-propanenitrile nucleobases (15-16) by Michael-
addition reaction: (i) TBAI, MeOH, 70 °C. Yields: 33 % (15), 35 % (16).
Table 2. Screening of the reaction conditions for the synthesis of N-
propanenitrile nucleobases by Michael addition reaction.
Entry
1[a]
Nucleobase
A
Solvent
T
Yield (%)
MW
(60 °C, 60
min.)
MeOH
-
a
]
2[
A
G
G
MeOH
MeOH
MeOH
70 °C, 65 h
55 °C, 89 h
70 °C, 65 h
12
-
3
4
traces
MW
(100 °C, 30
min.)
a
]
5[
G
MeOH
traces
60 °C, 48.5
h
a , b
] [
]
6[
G
T
MeOH
MeOH
30
33
a
]
7[
70 °C, 72 h
MW
(60 °C, 60
min.)
8[a]
T
MeOH
40
Scheme 6. Synthesis of N-propanenitrile nucleobases (13-14) by Michael-
addition reaction. i) TBAI, MeOH, 70 ºC, ii) TBAI, MeOH, NaOH (2M), 60 ºC,
Yields: 12 % (13), 30 % (14)
9
C
C
MeOH
MeOH
55 °C, 89 h
70 °C, 65 h
20
35
a
10[
]
Still, using similar conditions, T and C were successfully modified
in modest yield (Table 2, entries 8 and 11). The Michael addition
of T and C were screened using conventional (Table 2, entries 7,
9-10) and MW heating (Table 2, entries 8-9), affording the desired
products 15-16 in 20-42% yields. However, using MW irradiation,
15 and 16 were obtained in 40% and 42% yields (Table 2, entries
8 and 11), respectively. The synthesis of the compound 16 was
obtained in low yield (Table 2, entry 9) without TBAI at 55 ºC
(Scheme 7). We hypothesize that the low to modest yields of the
Michael addition reactions could be assigned to the weak
nucleophilic nature of the nucleobases, their general difficult
manipulation/purification and absence of base in the reaction
medium. Nevertheless, the desired compounds 13-16 were
obtained without the use of DBU or TEA.
MW
(60 °C, 60
min.)
11[a]
C
MeOH
42
[a] Catalytic amount of TBAI was used as phase-transfer catalyst. [b] NaOH 2
M (40 drops) was used as base.
Functionalization of nucleobases via N-allylation and N-
propargylation reactions
The regioselective synthesis of N-allyl and N-propargyl A and T,
using commercially available allyl bromide 22 and propargyl
bromide 27, have been described in the literature.[23,24,28,48-49] In
general, the products were obtained in low-to-high yields (25-
89%) typically using conventional heating overnight or for more
than one day. The N-allylation of T was also described involving
two reaction steps, first heating at 65 ºC for 30 minutes followed
by MW irradiation for 2 minutes.[29]
In this work, the treatment of A, T, C and G with allyl bromide 22,
in basic conditions under MW, led to the desired N-allylic
pyrimidine and purine derivatives 18-21 in low to moderate yields
4
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
(16-43%, Table 3) in a shorter reaction time. Moreover, the
introduction of the propargyl terminal in A, T and C was also done
in basic conditions in modest to high yields 25-61% (Table 4) in
reaction times lower than those described in the literature.
Changing the reaction conditions of N-allylation by resorting to
classical heating did not result in the desired products. It is
interesting to note that the reaction conditions for the synthesis of
each nucleobase derivative changed slightly. For instance, in the
case of A (Table 3, entries 1-2) there was the need to reduce the
amount of allyl bromide 22 (from 3.5 to 1 equiv) to obtain the
desired product 18. Moreover, the increase in the MW irradiation
time did not improve the reaction yield. Initial attempts to couple
allyl bromide 22 with G (Table 3, entry 3) were not successful
(only the starting material was recovered). The 9-allyl-2-amino-
1,9-dihydro-6H-purin-6-one 19 (Table 3, entry 4) was obtained in
low yield (26%) using MW in the first reaction step that consisted
in the deprotonation of G followed by stirring at r.t. during the
second step (Scheme 8).
significantly under MW conditions and a fast and environmentally
friendly method was developed.
Scheme 9. Synthesis of N-allyl nucleobases (20-21). i) H2O/NaOH (2M), MW
(40 °C, 15 min). ii) H2O/NaOH (2M), MW (40 °C, 40 min). Yields: 20 % (20),
43% (21).
The N-propargylated nucleobases 23-26 were obtained following
the procedures described elsewhere,[23,24,48-50] with some
modifications. The A, T and C were initially treated with NaH in
dry DMF, followed by the addition of propargyl bromide 27 at r.t.
to generate the products in modest to high yield (25-63%) (Table
4). The compound 23 was obtained in a modest yield (25-30%;
Table 4, entries 1-2), probably due to the inherent loss of
compound during the purification process by recrystallization. The
analysis of the reaction conditions (Table 4, entries 2-3) screened
for the synthesis of T derivative 24 showed that a decrease of the
amount of base and of the reaction time after the addition of
propargyl bromide 27 led to the formation of the desired
compound as main product in moderate yield (41%).
Nevertheless, the N(1) and N(3) bis-adduct 25 was also isolated
as a reaction product in 30% yield (Scheme 10).
Scheme 8. Synthesis of N-allyl nucleobases (18-19): i) H2O/NaOH (2M), MW
(40 °C, 15 min); ii) H2O/NaOH (2M), MW (40 °C, 5 min), then r.t. for 3 h. Yields:
16 % (18), 26 % (19).
Table 3. Screening of the reaction conditions for the synthesis of N-allyl
nucleobases.
Entry
1[a]
Nucleobase
T
Yield (%)
Traces
16
MW (40 °C, 5 min.), then addition
A
A
G
G
T
of 22 40°C, 35 min.
MW (40 °C, 5 min.), then addition
2[a]
of 22 40°C, 10 min.
MW (40 °C, 5 min), then addition
3[a]
-
of 22 40°C, 30 min.
MW (40 °C, 5 min.), then addition
of 22 stirring under r.t. for 3h
4[a]
26
MW (40 °C, 5 min.), then addition
5[a]
Traces
20
of 22 40°C, 35 min.
MW (40 °C, 5 min.), then addition
6[a]
7[a]
T
of 22 40°C, 10 min.
MW (40 °C, 5 min.), then addition
C
C
Traces
43
of 22 40°C, 15 min.
MW (40 °C, 5 min.), then addition
8[a]
of 22 40°C, 35 min.
Scheme 10. Synthesis of N-propargyl nucleobases (23-26). i) dry DMF, NaH
60 % mineral oil, r.t. Yields: 25 % (23), 41 % (24), 63 % (26).
[a] Mixture of H2O/NaOH 2 M was used as solvent.
The allylation of T followed the same reaction conditions used for
A allylation (Table 3, entries 5-6). As for C, the reaction conditions
(Table 3, entries 7-8) allowed its chemical functionalization in 43%
yield using 3.5 equiv of allyl bromide 22 and longer irradiation time
(Scheme 9). This chemical modification was improved
The propargylation of C was carried out using K2CO3 in MeOH,
leading to the compound 26 in low yield (20%; Table 4, entry 4).
Subsequently, dry DMF, NaH and C were mixed and stirred at r.t.
Then, the propargyl bromide 27 was added and the reaction
5
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
mixture was stirred for 6.5 h affording 26 in modest to good yield
(34-63%; Table 4, entries 5-6), respectively (Scheme 10).
In summary, the synthesis of N-modified A led to the compounds
1 and 3 in high yield (53-77%) and compounds 13, 18 and 23 in
low yield (12-25%). The lower yield of the later compounds is due
to the required purifications by recrystallization and column
chromatography. For the majority of the reactions with T, the
regioselective modification of N(1) position was achieved in
modest yield (20-33%). However, no product resulting from the
N(3) addition or both N(1) and N(3) additions was isolated in either
the N-alkylation, Michael addition reaction or N-allylation. The
purification process was time-consuming and laborious,
suggesting that eventually other by-products were formed.
However, it was not possible to isolate and quantify them. On the
other hand, when the regioselectivity of the reaction was
controlled using Boc, the overall yield of the compound 12 (45%)
was improved and when it was possible to isolate the bis-adduct
25 the overall yield of the N-propargylation reaction was 71% yield.
The chemical modifications on C gave the desired products 9, 16,
21 and 26 in moderate to high yields (35-63%), thus confirming
that the developed method is regioselective, since the
modification occurred only in the N(1) position. The chemical
modification of G was not successful regarding the alkylation and
propargylation, being necessary the use of 2-amino-6-
Table 4. Screening of the reaction conditions for the synthesis of N-propargyl
nucleobases.
Base
(equiv)
Yield
(%)
Entry
1[a]
2[a]
2[a]
3[a]
4[c]
5[a]
6[a]
7[a]
Nucleobase
T
r.t., 45 min then addition
NaH[b]
(2.0)
A
A
T
25
30
of 27 (1.5) 4 h
r.t., 60 min then addition
NaH[b]
(2.0)
of 27 (1.5) 1.5 h
r.t., 45 min then addition
NaH[b]
(2.0)
Traces
41
of 27 (1.5), 4h
r.t., 45 min then addition
NaH[b]
(1.5)
T
of 27 (1.5), 3 h
K2CO3
(2.0)
C
C
C
G
r.t., 4 days
20
r.t., 60 min then addition
NaH[b]
(2.0)
34
of 27 (1), 5 h
chloropurine 28 as
G surrogates to allow facile N(9)-
propargylation. Indeed, the chemical modification on compound
28 is well-known[8,28,51] due to the difficult chemical manipulation
of G. In this work, the acrylonitrile and allyl moieties were directly
introduced in G, without the need to use G surrogate 28, affording
compounds 14 and 19 in modest yield (26-30%).
r.t., 40 min then addition
NaH[b]
(2.5)
63
of 27 (2), 1.5 h
r.t., 3 hours then
addition of 27 (1), 3 h
NaH[b]
(2.0)
-
r.t., 30 min; 40 °C, 60
min finally addition of 27
(2), 2 h
NaH[b]
(2.5)
8[a]
G
G
-
NMR Spectroscopy
The unequivocal structure elucidation of the nucleobase
derivatives 1, 3, 8, 9, 12, 13-16, 18-21, 23-26 and 30 was
performed by 1D (1H and 13C) and 2D (HSQC, HMBC, NOESY)
NMR (more info on the ESI†). The typical protons in the 1H NMR
spectra of N-alkylated A 1 and 3 (see Figures S1 and S3 on the
ESI†) are the singlets in the aromatic region corresponding to the
resonance of protons 4-NH2, H-2 and H-8 at δH 7.28 ppm, 8.15-
8.43 ppm and 8.18-8.70 ppm, respectively. In the aliphatic region,
it was possible to identify the signals corresponding to the protons
of the bromoethyl and bromopropyl chains, respectively. The
aliphatic protons and carbons appear at δH 2.35–4.57 ppm (δC
20.5-44.6 ppm): as triplets at δH 4.40-4.57 ppm (δC 44.5-44.6
ppm) and 3.95–4.32 ppm (δC 31.6-41.4 ppm) assigned to the
NCH2, BrCH2, and as a quintet at δH 2.35 ppm (δC 20.5 ppm)
attributed to 2’-CH2- of the bromopropyl chain. The 1H NMR
spectrum of compound 8 (see Figure S5 on the ESI†) showcase
the singlet corresponding to the proton resonances of methyl
(CH3) group, at δH 1.93 ppm, the quartet at δH 6.98 ppm
corresponding to proton H-6 with a coupling constant of J = 1.2
Hz due to the coupling with the methyl group 5-CH3, and the broad
singlet at δH 8.45 ppm due to the NH proton resonance. In the
aliphatic region it was possible to see the signals corresponding
to the protons of the alkyl chain. The high frequency chemical shift
of protons NCH2 and BrCH2 at δH 3.40-3.73 ppm is due to the
deshielding effect of the nitrogen and bromo atoms. The 1H NMR
spectrum of compound 12 (see Figure S7 on the ESI†) shows two
triplets in the aliphatic region. Such triplets are assigned to the
methylene groups bonded to N(3) of T that appear at high
chemical shift, δH 4.16 and 4.79 ppm, due to the deshielding effect
of the nitrogen and bromo atoms and the carbonyl groups of C-2
MW (40 °C, 5 min.),
then addition of 27
40°C, 35 min.
NaOH
2M /H2O
(4:1)
9
traces
[a] DMF was used as solvent. [b] NaH in 60% wt mineral oil. [c] MeOH was used
as solvent and TBAI (0.1 equiv) as catalyst.
It is noticeable that the reaction time before and after the addition
of propargyl bromide 27 played a key role in this protocol leading
to an improvement in the yield for almost the double.
Unfortunately, the attempts to generate N-propargyl G using the
reaction conditions tested with A, T and C were not successful
(Table 4, entries 7-8) and attempts to do the propargylation under
MW irradiation and using NaOH 2M as base only provided the
compound 30 in vestigial amounts (Table 4, entry 9). To
circumvent the challenges associated to the manipulation of G, a
protected G derivative was used, namely 4-amino-6-chloropurine
28. The desired compound 30 was obtained using the method
described by Nagapradeep et al.[51] in 65% overall yield (Scheme
11).
Scheme 11. Synthesis of G containing N-propargyl terminal (30). i) K2CO3, dry
DMF, r.t., 48 h, ii) TFA:H2O (3:1), r.t., 48 h. Yields: 50 % (29), 80 % (30).
6
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
and C-4 positions. The 1H NMR spectrum of N-alkylated cytosine
9 (see Figure S9 on the ESI†) presents similar signals of N-
alkylated A and T in the aliphatic region. In the aromatic region,
two typical doublets were denoted at δH 5.83 ppm and 7.80 ppm,
being assigned to protons H-5 and H-6, respectively.
protons H-1’,1’’ of the propargyl chain. The connectivities found in
the 2D NMR spectra allowed the total assignments of all
protonated and quaternary carbon resonances (see Figures S29,
S31, S33 and S35 on the ESI†).
The full characterization of the compounds 13-16 having a
propanenitrile moiety allowed the identification of their main
signals. The H NMR spectra (see Figures S12, S14, S16 and
Cell viability study
1
The in vitro cytotoxicity of the library of N-functionalized
nucleobases (1, 8, 9, 13-16, 18-21, 23, 24, 26, 30) and native
nucleobases (A, T and C) was evaluated using primary human
dermal fibroblast cells (HF) by measuring the changes in the
metabolic activity of the cells through a resazurin-based assay.
The in vitro cytotoxicity of the non-functionalized G could not be
studied due to its poor solubility in both culture media and DMSO.
Due to the presence of active metabolites on the viable cells, they
can convert the blue-nonfluorescent dye (resazurin) to a pink-
fluorescent dye (resorufin), which is proportional to the number of
viable cells over a wide concentration range. We started the
cytotoxicity studies using a 10 nM–100 µM concentration curve
with a dilution factor of 10 (10 nM, 100 nM, 1 µM, 10 µM, 100 µM).
The metabolic activity of HF incubated for 72 h with the
compounds was measured and compared with the one of
compound-free cells (control). All tested compounds exhibited no
significant effects at the studied concentration range. The viability
index presented values higher than 80%, thus demonstrating that
these compounds had no significant effect in the cells’ metabolic
activity. Besides, the obtained results indicated that,
independently of the solution concentration used, no statistically
significant differences in cell viability were observed, denoting the
excellent biocompatibility of the native and functionalized
nucleobase derivatives (Figure 2).
S18 on the ESI†)) showed two triplets in the aliphatic region
corresponding to protons H-1’ and H-2’ at δH 3.88-4.44 ppm and
2.88-3.17 ppm, respectively, as well as the signals characteristic
of the purine and pyrimidine rings in the aromatic region. The
analysis of the 13C NMR spectrum of each compound was
possible with the aid of the 2D NMR spectra (HSQC and HMBC),
allowing the assignments of all protonated and quaternary carbon
resonances (see Figures S13, S15, S17 and S19 on the ESI†).
The connectivities found in the HMBC spectra for H-1’ played an
important role in the unequivocal assignment of the resonances
of the quaternary carbon C-6 and of C-2 and C-4 (in the case of
modified A and G, respectively) and C-2 (in the case of modified
T and C).
The analysis of the 1H NMR spectra (see Figures S20, S22, S24
and S26 on the ESI†) of compounds 18-21 enabled the
assignment of the chemical shifts of the protons of the allylic
system. The signals of compounds 18, 20 and 21 appear as
double triplet (dt) at δH 4.83 ppm, 4.34 ppm and 4.26 ppm, with
two coupling constants of 1.3-1.6 Hz and 5.4-5.8 Hz assigned to
H-1’, whereas a multiplet at δH 5.66-6.10 ppm was assigned to H-
2’. The protons H-3’ present different splitting pattern due to the
signals overlap of the vinylic system protons. The chemical shift
of H-3’ of the compounds 18 and 21 appears at δH 5.06-5.33 ppm
as two doublets of quartets with cis coupling JH3’_H2’ = 10.3 Hz,
trans coupling JH-3’_H-2’ = 17.2 Hz and the geminal and allyl
couplings JH-3’_H-3’ and JH-3’_H-1’ = 1.6 Hz. The chemical shift of H-3’
of the compounds 19 and 20 appears at δH 5.03-5.26 ppm as two
doublets of doublets with trans coupling JH3’_H2’ = 17.0 Hz, cis
coupling JH3’_H2’ = 10.4 Hz and the geminal and allyl couplings
JH3’_H3’ and JH3’_H1’ = 1.5 Hz. The compound 19 presents a doublet
in the aliphatic region at δH 4.83 ppm and a multiplet in the
aromatic region at δH 5.99-6.12 ppm corresponding to H-1’ and H-
2’.
The connectivities found in the HMBC spectra of compounds 18-
21 allowed the unequivocal assignment of the quaternary carbon
resonances and confirmation of protonated carbons (see Figures
S21, S23, S25, S27 on the ESI†), which were assigned from the
correlations found in their HSQC spectra. The connectivities
found in the HMBC spectra for H-1’ allowed the correct
assignment of the resonances of C-6 (in the case of modified A
and G) and C-2 (in the case of modified T and C).
Figure 2. Cell viability analysis of native and functionalized nucleobases at a
concentration range between 10 nM-100 µM. Data is presented as mean ±
standard deviation of three independent experiments performed in
quadruplicate.
1
The characteristic signals in the aromatic region in the H NMR
spectra of N-propargyl nucleobases 23-24 and 26 (see Figures
S28, S30 and S34 on the ESI†) correspond to protons of the
purine or pyrimidine structures. Moreover, it is also clear the
presence of a triplet at δH 3.34-3.47 ppm and a doublet at δH 4.48-
5.03 ppm, corresponding to the resonances of the protons of the
It is noteworthy that dermal fibroblasts are the main cell types
implicated in the extracellular matrix (ECM) production during the
wound healing process and these preliminary cell viability data
suggest that the clinical use of these compounds might not have
negative effects in the metabolic activity of human fibroblast cells.
This is a very important feature for biomedical applications,
namely for wound tissue regeneration. Moreover, the nucleobase
derivatives can be further functionalized with cell signaling
1
propargyl group. The H NMR spectrum of compound 25 (see
Figure S32 on the ESI†) shows two signals as triplets at δH 3.12
ppm and 3.45 ppm corresponding to protons H-3’’ and H-3’ of the
propargyl chain, respectively, indicating that the modification
occurred in both of the T NH groups. Moreover, one can also
denote a doublet of doublets at δH 4.56 ppm corresponding to the
7
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
commercially available first grade. Melting points were obtained using a
Büchi melting point B-540 apparatus and are uncorrected. Microwave-
assisted reactions were performed in an Ethos MicroSYNTH Labstation
(Milestone Inc) using an infrared sensor to control the temperature. The 1H
and 13C NMR spectra were recorded on a Bruker 300 or 500 [300.13 MHz
moieties, including fibronectin or peptide-mimetic proteins to
prompt cell adhesion, proliferation, and differentiation. As
previously highlighted, the library of functionalized DNA
nucleobase derivatives holds great promise for being coupled to
a wide array of biomolecules, including polysaccharides or
peptides thus potentially leading to innovative high-added value
supramolecular biofunctional nanomaterials for biomedicine.
However, the in vitro biological performance of such innovative
biomaterials should be studied to undoubtedly comment on their
suitability to address biomedical applications.
(1H), 75.47 MHz
( (
13C) or 500.13 MHz (1H), 125.76 MHz 13C)]
spectrometers with tetramethylsilane (TMS) as internal reference.
Unequivocal 1H assignments were made with the aid of 2D COSY (1H /1H),
whereas 13C assignments were made based on 2D HSQC (1H/13C) and
HMBC experiments (delays for one-bond and long-range JC-H couplings of
147 and 7 Hz). The chemical shifts are reported as δ values (ppm) and
coupling constants (J) in Hz. High-mass-resolving ESI-MS were conducted
in
a Q-Exactive® hybrid quadrupole Orbitrap® mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany). The instrument was
operated in positive mode, with a spray voltage at 3.0 kV and interfaced
with a HESI II ion source. The analysis was performed through direct
infusion of the prepared solutions at a flow rate of 10 μL min-1 into the ESI
source, and the operating conditions were as follows: sheath gas
(nitrogen) flow rate 5 (arbitrary units); auxiliary gas (nitrogen) 1 (arbitrary
units); capillary temperature 320 °C, and S-lens rf level 50. Spectra were
analysed using the acquisition software Xcalibur ver. 4.0 (Thermo
Scientific, San Jose, CA, USA).
Conclusion
The DNA nucleobases (A, T, C and G) are important natural
heterocyclic building blocks for the rational design and
development of advanced and modular soft supramolecular
functional nanobiomaterials to be used in the biomedical arena
owing to their unique features. In this work, synthetic procedures
have been developed for the regioselective functionalization of
the secondary amine of the DNA nucleobases with more reactive
functional groups in a simple, one-pot and modular manner
towards the creation of a library of DNA nucleobase derivatives.
The proposed synthetic procedures allowed anchoring distinct
chemical functionalities, including N-terminal alkyl, N-
propanenitrile, N-allyl, and N-propargyl functional groups into the
DNA nucleobases, thus extending their functionalities. The
nucleobase derivatives were regioselectively synthesized, in most
of the cases in a single step, without resorting to the use of
protecting groups and subsequent deprotection, thus avoiding
two time-consuming steps that usually involve the use of toxic or
volatile organic solvents and generate waste. The low cytotoxicity
assigned to the toolbox of N-functionalized nucleobase
derivatives, as well as their intrinsic ability to maintain the high-
fidelity molecular recognition capability and structural
programmability encoded by the complementary Watson-Crick
base pairing open new avenues in the biomedical field. In
particular, the library of nucleobase derivatives holds great
promise as a highly versatile platform to obtain soft biofunctional
materials and complex systems denoting emergent properties
and multifunctionalities. For instance, the toolbox of nucleobase
derivatives can be coupled to and precisely positioning a wide
array of naturally occurring building blocks. Those include
peptides, lipids, and carbohydrates, thus possibly enabling the
A, T, C and G were obtained from Alfa Aesar. 2-Amino-6-chloropurine,
potassium carbonate (K2CO3), tetrabutylammonium iodide (TBAI),
tetrabutylammonium bromide (TBAB), tetrabutylammonium acetate
(TBAA), tetrabutylammonium hydroxide (TBAOH), acrylonitrile, propargyl
bromide were purchased from Aldrich and Merck. N,N-Dimethylformamide
(DMF) was purchased from Aldrich and dried with molecular sieves (4 Å)
prior to use. All other chemicals were used as received without further
purification.
Synthetic procedures
9-(2-bromoethyl)-9H-purin-6-amine 1 and 9-(3-bromopropyl)-9H-purin-6-
amine 3 were synthesized following a procedure described by Srivastava
et al.[40] and were obtained in good yields [1, (900 mg) 53%; 3, (980 mg)
77%].
The structural characterization of 9-(2-bromoethyl)-9H-purin-6-amine 1, is
already reported in the literature[40] and therefore it will not be presented
herein.
9-(3-bromopropyl)-9H-purin-6-amine (3), white solid, m.p. 353-354 °C. 1H
NMR (300.13 MHz, DMSO-d6): δ = 2.35 (quint, 2H, H-2’, J 5.6 Hz); 4.32 (t,
2H, H-3’, J 5.6 Hz); 4.70 (t, 2H, H-1’, J 5.6 Hz); 8.43 (s, 1H, H-2); 8.70 (s,
1H, H-8) ppm. 13C NMR (75.47 MHz, DMSO-d6): δ = 20.5 (C-2’); 41.4 (C-
3’); 44.5 (C-1’); 116.8 (C-4); 138.3 (C-5); 141.3 (C-8); 145.8 (C-2); 156.4
(C-6) ppm. MS (ESI+) m/z (%): 176.1 [((M-HBr)+H)+, 100]. HRMS (ESI+)
m/z calcd for C8H10N5 ((M-HBr)+H)+, 176.0931; found: 176.0933.
bottom-up
assembly
of
sophisticated
and
modular
supramolecular nanoarchitectures, including adaptive and
bioinstructive nanobiomaterials, smart systems, and high-
toughness hydrogels that could recreate the structural and
functional features of the native ECM of tissues and organs, and
be potentially used in controlled drug/therapeutics delivery,
biosensing, bioimaging, and tissue engineering strategies.
3-(2-Bromoethyl)-5-methylpyrimidine-2,4(1H,3H)-dione
12
was
synthesized following the procedure described by Jacobsen et al.[45] and
was obtained in good yield (700 mg) 63%. 1H NMR (300.13 MHz, DMSO-
d6): δ = 1.86 (d, 3H, 5-CH3, J 1.1 Hz); 4.16 (t, 2H, H-1’, J 8.5 Hz); 4.68 (t,
2H, H-2’, J 8.5 Hz); 7.58 (q, 1H, H-6, J 1.1 Hz) ppm. 13C NMR (75.47 MHz,
DMSO-d6): δ = 12.2 (5-CH3); 42.4 (C-1’); 66.7 (C-2’); 115.3 (C-5); 150.8
(C-6); 158.8 (C-4); 160.1 (C-2) ppm. MS (ESI+) m/z (%): 153.1 [(M+H)+,
100].
Experimental Section
Synthesis of 1-(5-bromopentyl)-5-methylpyrimidine-2,4(1H,3H)-dione (8)
Materials and methods
A mixture of T (146 mg, 1.16 mmol), TBAI (25 mg, 0.069 mmol), K2CO3
(160 mg, 1.16 mmol) in dry DMF (8 mL) was stirred at r.t. for 30 min and
then 1,5-dibromopentane 6 (266 µL, 1.97 mmol) was added. After 30 h of
reaction, at r.t. under nitrogen atmosphere, the unreacted thymine was
filtered off, the solvent was evaporated, and the crude purified by column
All reagents were commercially available and used without further
purification. Column chromatography was performed on silica gel 60,
Merck 0.032–0.063 mm and 0.063–0.200 mm, aluminum oxide 90, Alox N,
neutral and preparative TLC on silica gel 60 GF254 from Merck, and the
spots were detected with UV light (254 and 365 nm). The solvents were
8
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
chromatography using aluminium oxide grade III and a mixture of
dichloromethane: hexane (6:4) as eluent. White solid, (54 mg) 20%. 1H
NMR (300.13 MHz, DMSO-d6): δ = 1.45-1.55 (m, 2H, H-4’); 1.67-1.77 (m,
2H, H-2’); 1.82-1.91 (m, 2H, H-3’); 1.93 (s, 3H, 5-CH3); 3.42 (t, 2H, H-5’, J
6.6 Hz); 3.71 (t, 2H, H-1’, J 6.6 Hz); 6.98 (q, 1H, H-6, J 1.2 Hz); 8.45 (br s,
1H, 3-NH) ppm. 13C NMR (75.47 MHz, DMSO-d6): δ = 12.6 (5-CH3); 24.9
(C-2’); 28.2 (C-3’); 32.0 (C-4’); 33.3 (C-5’); 48.3 (C-1’); 110.7 (C-5); 110.3
(C-6); 150.7 (C-2); 163.9 (C-4) ppm.
A solution of G (500 mg, 3.31 mmol) in MeOH (10 mL) was stirred at 60 °C
for 30 min, then TBAI (catalytic amount), acrylonitrile (1.54 mL, 23.2 mmol)
and 40 drops of NaOH 2M were added. The reaction mixture was heated
at 60 °C and stirred for more 48 h. The solvent was evaporated under
vacuum and the crude was recrystalised in a mixture of hot H2O/ethanol
(20:80). White solid, mp > 410 °C, (202 mg) 30%. 1H NMR (500.13 MHz,
DMSO-d6): δ = 3.17 (t, 2H, H-2’, J 6.3 Hz); 4.43 (t, 2H, H-1’, J 6.3 Hz); 6.50
(br s, 2H,2-NH2); 7.93 (s, 1H, H-8) ppm. 13C NMR (125.76 MHz, DMSO-
d6): δ = 19.5 (C-2’); 42.0 (C-1’); 107.9 (C-5); 118.4 (C-3’); 142.9 (C-8);
155.0 (C-2); 157.0 (C-4); 160.6 (C-6) ppm. MS (ESI+) m/z (%): 205.1
[(M+H)+, 100]. HRMS (ESI+) m/z calcd for C8H9ON6 (M+H)+, 205.0832;
found: 205.0836.
Synthesis of 4-amino-1-(5-bromopentyl)pyrimidin-2(1H)-one (9)
A mixture of C (162 mg, 1.46 mmol), TBAI (54 mg, 0.146 mmol), K2CO3
(403 mg, 2.92 mmol) in MeOH (10 ml) was stirred at r.t. for 40 min and
then 1,5-dibromopentane 6 (335 µL, 2.48 mmol) was added. After 65 h of
reaction, at r.t. under nitrogen atmosphere, the starting material was
observed by TLC. At that time, K2CO3 (202 mg, 1.46 mmol) and 1,5-
dibromopentane (335 µL, 2.48 mmol) were added and the mixture was
heated at 65 °C for more 24 hours. After this period, methanol was
evaporated under vacuum and the crude was purified by column
chromatography on alumina grade III, initially using a mixture of 5% of
methanol in dichloromethane and finally 20% of methanol in
dichloromethane as eluent. Yellow solid, mp 262-263 °C, (180 mg) 50%.
1H NMR (500.13 MHz, DMSO-d6): δ = 1.33-1.65 (m, 6H, H-2’,3’,4’); 3.54
(t, 2H, H-5’, J 7.0 Hz); 3.69 (t, 2H, H-1’, J 7.0 Hz); 5.82 (d, 1H, H-5, J 7.2
Hz); 7.56 and 8.21 (2 br s, 2H, 2-NH2); 7.79 (d, 1H, H-6, J 7.2 Hz) ppm.
13C NMR (125.76 MHz, DMSO-d6): δ = 24.4 (C-4’); 27.7 (C-2’); 31.8 (C-
3’); 35.0 (C-1’); 48.4 (C-5’); 93.1 (C-5); 146.6 (C-6); 153.7 (C-4); 164.9 (C-
2) ppm. MS (ESI+) m/z (%): 260.1 [(M + H)+, 15]. HRMS (ESI+) m/z calcd
for C9H14N3OBr (M)+, 260.0393; found: 260.0391.
General procedure for the allylation reaction of A, T and C
A suspension of the appropriate nucleobase A, T or C (0.9 mmol) in H2O:
NaOH 2M (1:1) was irradiated for 5 min at 40 °C (300 W), then allylbromide
(0.9 mmol, 1.35 mmol or 3.15 mmol for respectively A, T and C) was added
and the mixture was irradiated for more 10 min, for A and T, and 35 min
for C, at 40 °C, respectively. After removal of the solvent and excess of
allyl bromide, 9-allyl-9H-purin-6-amine (18) and 1-allyl-5-methylpyrimidine-
2,4(1H,3H)-dione (20) were purified by flash chromatography using ethyl
acetate and CH2Cl2: MeOH (98:2), respectively.[28] 1-Allyl-4-
aminopyrimidin-2(1H)-one (21) was filtered off, the solvent evaporated,
and the crude was purified by TLC using a mixture of CH2Cl2: methanol:
ammonia solution (8: 1.6: 0.4).
9-Allyl-9H-purin-6-amine (18). White solid, mp 138-141 °C, (252 mg) 16%.
1H NMR (300.13 MHz, CDCl3): δ = 4.83 (dt, 2H, H-1’, J 1.5 and 5.7 Hz);
5.21 (dq, 1H, H-3’, J 1.6 and 17.1 Hz) 5.33 (dq, 1H, H-3’, J 1.6 and 10.1
Hz); 5.79 (br s, 2H, 6-NH2); 5.98-6.19 (m, 1H, H-2’); 7.82 (s, 1H, H-2); 8.38
(s, 1H, H-8) ppm. 13C NMR (75.47 MHz, CDCl3): δ = 45.8 (C-1’); 119.0 (C-
3’); 119.6 (C-5); 131.8 (C-2’); 140.4 (C-8); 150.0 (C-4); 153.1 (C-2); 155.4
(C-6) ppm. MS (ESI+) m/z (%): 176.1 ([M+H]+, 100]. HRMS (ESI+) m/z
calcd for C8H10N5 (M+H)+, 176.0931; found: 176.0932.
General procedure for the Michael-type reaction of A, T and C
To a solution of the A, T and C (9 mmol) in MeOH (10 ml) was added TBAI
(catalytic amount) and acrylonitrile (3.6 mL, 55 mmol). The mixture was
heated at 70°C under nitrogen atmosphere for 65, 72 and 65 hours,
respectively. The resulting mixture was evaporated and recrystalized in
appropriate mixture of hot ethanol/H2O (80/20).
1-Allyl-5-methylpyrimidine-2,4(1H,3H)-dione (20). White solid, mp 78-
1
80 °C, (300 mg) 20%. H NMR (300.13 MHz, CDCl3): δ = 1.93 (s, 3H, 5-
CH3); 4.34 (dt, 2H, H-1’, J 1.7 and 5.8 Hz); 5.26 (dd, 1H, H-3’, J 1.4 and
17.0 Hz); 5.31 (dd, 1H, H-3’, J 1.4 and 10.4 Hz); 5.81-5.94 (m, 1H, H-2’);
6.98 (q, 1H, H-6, J 1.2 Hz); 9.30 (s, 1H, 3-NH) ppm. 13C NMR (75.47 MHz,
CDCl3): δ = 12.4 (5-CH3); 49.8 (C-1’); 111.0 (C-5); 119.2 (C-3’); 131.8 (C-
2’); 139.7 (C-6); 151.0 (C-2); 164.4 (C-4) ppm. MS (ESI+) m/z (%): 167.1
[(M+H)+, 65]. HRMS (ESI+) m/z calcd for C8H11O2N2 (M+H)+, 167.0811;
found: 167.0815.
3-(6-Amino-9H-purin-9-yl)propanenitrile (13). White solid, mp 256-257 °C,
(203 mg) 12%. 1H NMR (300.13 MHz, DMSO-d6): δ = 3.17 (t, 2H, H-2’, J
6.5 Hz); 4.44 (t, 2H, H-1’, J 6.5 Hz); 7.30 (s, 2H, 6-NH2); 8.17 (s, 1H, H-2);
8.20 (s, 1H, H-8) ppm. 13C NMR (75.47 MHz, DMSO-d6): δ = 18.1 (C-2’);
38.8 (C-1‘); 118.3 (C-3’); 118.7 (C-5); 140.6 (C-8); 149.4 (C-4); 152.6 (C-
2); 156.0 (C-6) ppm. MS (ESI+) m/z (%): 189.1 [(M+H)+, 100]. HRMS (ESI+)
m/z calcd for C8H9N6 (M+H)+, 189.0883; found: 189.0883.
1-Allyl-4-aminopyrimidin-2(1H)-one (21). White solid, mp 240-242 °C, (585
mg) 43%. 1H NMR (300.13 MHz, DMSO-d6): δ = 4.26 (dt, 2H, H-1’, J 1.5
and 5.4 Hz); 5.06 (dq, 1H, H-3’, J 1.5 and 17.2 Hz); 5.17 (dq, 1H, H-3’, J
1.5 and 10.3 Hz); 5.67 (d, 1H, H-5, J 7.2 Hz); 5.83-5.94 (m, 1H, H-2’); 6.99
and 7.11 (2 br s, 1H, 4-NH2); 7.51 (d, 1H, H-6, J 7.2 Hz) ppm. 13C NMR
(75.47 MHz, DMSO-d6): δ = 50.3 (C-1’); 93.6 (C-5); 116.9 (C-3’); 134.1 (C-
2’); 145.8 (C-6); 155.6 (C-4); 165.9 (C-2) ppm. MS (ESI+) m/z (%): 152.1
([M+H]+, 100)]. HRMS (ESI+) m/z calcd for C7H10ON3 (M+H)+, 152.0818;
found: 152.0816.
3-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)yl)propanenitrile
(15).
White solid, mp 196-197 °C, (532 mg) 33%. 1H NMR (300.13 MHz, DMSO-
d6): δ = 1.77 (s, 3H, 5-CH3); 2.90 (t, 2H, H-2’, J 6.5 Hz); 3.91 (t, 2H, H-1’,
J 6.5 Hz); 7.58 (q, 1H, H-6, J 1.1 Hz); 11.4 (s, 1H, 3-NH) ppm. 13C NMR
(75.47 MHz, DMSO-d6): δ = 12.0 (5-CH3); 16.8 (C-1’); 43.0 (C-2’); 108.8
(C-3’); 118.4 (C-5); 141.0 (C-6); 150.7 (C-2); 164.2 (C-4) ppm. MS (ESI+)
m/z (%): 180.1 [(M+H)+ 15]. HRMS (ESI+) m/z calcd for C8H10O2N3 (M+H)+,
180.0768; found: 180.0767.
3-(4-Amino-2-oxopyrimidin-1(2H)-yl)propanenitrile (16). White solid, mp
246-248 °C, (517 mg) 35%. 1H NMR (300.13 MHz, DMSO-d6): δ = 2.88 (t,
2H, H-2’, J 6.5 Hz); 3.88 (t, 2H, H-1’, J 6.5 Hz); 5.69 (d, 1H, H-5, J 7.2 Hz);
7.10 and 7.18 (2 br s, 2H, 4-NH2); 7.62 (d, 1H, H-6, J 7.2 Hz) ppm. 13C
NMR (75.47 MHz, DMSO-d6): δ = 16.8 (C-2’); 44.8 (C-1’); 93.6 (C-5); 118.6
(C-3’); 145.9 (C-6); 155.5 (C-4); 166.2 (C-2) ppm. MS (ESI+) m/z (%):
165.1 [(M+H)+, 80]. HRMS (ESI+) m/z calcd for C7H9ON4 (M+H)+,
165.0771; found: 165.0773.
Synthesis of 9-allyl-2-amino-1,9-dihydro-6H-purin-6-one (19) by allylation
reaction
A solution of G (200 mg, 1.32 mmol) in H2O:NaOH 2M (1:4) was irradiated
for 5 min, at 40 °C (300 W). Then, 3-bromoprop-1-ene 22 (400 µL, 4.63
mmol) was added and the reaction mixture was stirred for 3 h at r.t. and
protected from light. The solvent was evaporated, and the mixture was
purified by TLC using a mixture of CH2Cl2: methanol: ammonia solution (8:
1.4: 0.6). Greyish solid, mp > 410 °C, (66 mg) 26%. 1H NMR (500.13 MHz,
DMSO-d6): δ = 4.83 (d, 2H, H-1’, J 5.5 Hz); 5.03 (dd, 1H, H-3’, J 1.4 and
17.1 Hz); 5.17 (dd, 1H, H-3’, J 1.4 and 10.1 Hz); 5.99-6.10 (m, 1H, H-2’);
Synthesis of 3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)propanenitrile
(14) by Michael-type reaction
6.12 (br s, 2H, 2-NH2), 7.89 (s, 1H, H-8); 10.76 (br s, 1H, 1-NH) ppm. 13
C
9
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10.1002/ejoc.202100786
European Journal of Organic Chemistry
FULL PAPER
NMR (125.76 MHz, DMSO-d6): δ = 48.0 (C-1’); 108.0 (C-5); 117.2 (C-3’);
134.4 (C-2’); 142.9 (C-8); 144.1 (C-2); 149.4 (C-4); 152.8 (C-6) ppm. MS
(ESI+) m/z (%): 192.1 ([M+H]+, 100].
Cell culture. A human primary dermal fibroblast cell line (HF) (ATCC PCS-
201-012TM) was grown in α-MEM culture medium supplemented with 10%
FBS (South America origin, Thermo Fisher Scientific) and 1%
penicillin/streptomycin solution (Thermo Fisher Scientific). Cell cultures
were maintained at 37 °C in a 5% CO2 humidified atmosphere.
General procedure for the propargylation reaction of A, T and C
Cell viability assay. Serial dilutions ranged from 10 nM to 100 µM to cover
a wide scale for the generation of dose–response curves were prepared
from the stock solution (100 mM). In all experiments, the solvent DMSO
(0.1% v/v) alone was used as a negative control. The HF were seeded in
96-well plates (final volume 100 µL) at a density of 3.5 × 103 viable cells
per cm2 and incubated for 24 h. Cells were then exposed to the test
compounds diluted in culture medium (final volume 200 µL) and 48 hours
post transfection, 100 μL of the culture medium was replaced by test
compounds for additional 24 hours. Thereafter, cell viability was assessed
by using fresh medium containing 10% of resazurin-based alamarBlueTM
reagent (100 µL per well (Life Technologies)), according to the
manufacturer’s instructions. Values considered after 3 hours incubation
were within the linear range of the reading and the cell’s viability was
expressed as a percentage of viability of nontreated cells.
To a solution of the appropriate nucleobase A, T or C (2.7 mmol) in dry
DMF (5 mL) was added NaH 60 % mineral oil (5.40, 4.04 and 6.75 mmol
for respectively A, T and C) and the mixture was stirred for 45 min, for A
and T, and 40 min for C, under nitrogen atmosphere. Then propargyl
bromide 27 (4.04, 4.04 and 5.4 mmol for A, T and C, respectively) was
added and the reaction was stirred for more 4 h, 3 h and 1.5 h, respectively.
9-(Prop-2-yn-1-yl)-9H-purin-6-amine (23) was filtered off, the solvent
evaporated and the crude product purified through recrystallization (twice
from MeOH).[23] 5-Methyl-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione
(24) was purified by column chromatography (CH2Cl2; followed by
CH2Cl2:MeOH, 98:2 and 95:5). 4-Amino-1-(prop-2-yn-1-yl)pyrimidin-
2(1H)-one (25) was evaporated and the crude was recrystalized in hot
ethanol.
9-(Prop-2-yn-1-yl)-9H-purin-6-amine (23). White solid, mp 201-203 °C,
(117 mg) 25%. 1H NMR (300.13 MHz, DMSO-d6): δ = 3.47 (t, 1H, H-3’, J
2.5 Hz); 5.03 (d, 2H, H-1’, J 2.5 Hz); 7.29 (br s, 2H, 6-NH2); 8.17 (s, 1H, H-
2); 8.19 (s, 1H, H-8) ppm. 13C NMR (75.47 MHz, DMSO-d6): δ = 32.2 (C-
1’); 75.8 (C-3’); 78.3 (C-2’); 118.5 (C-5); 140.1 (C-8); 149.1 (C-4); 152.7
(C-2); 156.0 (C-6) ppm. MS (ESI+) m/z (%): 174.1 [(M+H]+, 100]. HRMS
(ESI+) m/z calcd for C8H8N5 (M+H)+, 174.0772; found: 174.0774.
Acknowledgements
This work was funded by the Programa Operacional Regional do
Centro – Centro 2020, in the component FEDER, and by national
funds (OE) through FCT/MCTES, in the scope of the project
“SUPRASORT” (PTDC/QUI-OUT/30658/2017, CENTRO-01-
0145-FEDER-030658). This work was also funded by the
European Research Council under the scope of the project
“ATLAS” (ERC-2014-AdG-669858). D. H. A. Rocha, C. M.
Machado and V. Sousa acknowledge the financial support
through the project “SUPRASORT”. C. F. V. Sousa and V. Sousa
acknowledge FCT for their individual PhD grants (2020.04408.BD,
C. F. V. Sousa; 2020.06771.BD, V. Sousa). J. Borges gratefully
acknowledges FCT for his individual Junior Researcher
5-Methyl-1-(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (24). White solid,
mp 157-158 °C, (182 mg) 41%. 1H NMR (300.13 MHz, DMSO-d6): δ = 1.77
(d, 3H, J 0.7 Hz, 5-CH3); 3.40 (t, 1H, H-3’, J 2.5 Hz); 4.47 (d, 2H, H-1’, J
2.5 Hz); 7.57 (q, 1H, H-6, J 1.2 Hz); 11.38 (br s, 1H, 3-NH) ppm. 13C NMR
(75.47 MHz, DMSO-d6): δ = 11.9 (5-CH3); 36.3 (C-1’); 75.7 (C-3’); 78.7 (C-
2’); 109.4 (C-5); 140.1 (C-6); 150.4 (C-2); 164.2 (C-4) ppm. MS (ESI+) m/z
(%): 165.1 ([M+H]+, 100]. HRMS (ESI+) m/z calcd for C8H9O2N2 (M+H)+,
165.0654; found: 165.0659.
5-Methyl-1,3-di(prop-2-yn-1-yl)pyrimidine-2,4(1H,3H)-dione (25). Yellow
1
(CEECIND/03202/2017)
and
Assistant
Researcher
solid, (50 mg) 30%. H NMR (300.13 MHz, DMSO-d6): δ = 1.84 (d, 3H, J
1.2 Hz, 5-CH3); 3.12 (t, 1H, H-3’’, J 2.4 Hz); 3.45 (t, 1H, H-3’, J 2.4 Hz);
4.56 (dd, 4H, H-1’,1’’, J 2.5 and 4.9 Hz); 7.70 (d, 1H, H-6, J 1.2 Hz) ppm.
13C NMR (75.47 MHz, DMSO-d6): δ = 12.5 (5-CH3); 30.1 (C-1’’); 37.6 (C-
1’); 73.1 (C-3’’); 76.1 (C-3’); 78.4 (C-2’); 79.1 (C-2’’); 108.6 (C-5); 139.3 (C-
6); 149.8 (C-2); 164.1 (C-4) ppm.
(2020.00758.CEECIND) contracts. This work was developed
within the scope of the project CICECO – Aveiro Institute of
Materials (UIDB/50011/2020 & UIDP/50011/2020), and LAQV-
REQUIMTE (UIDB/50006/2020) financed by national funds
through the FCT/MCTES.
4-Amino-1-(prop-2-yn-1-yl)pyrimidin-2(1H)-one (26). Brownish solid, mp
224-226 °C, (254 mg) 63%. 1H NMR (300.13 MHz, DMSO-d6): δ = 3.38 (t,
1H, H-3’, J 2.5 Hz); 4.47 (d, 2H, H-1’, J 2.5 Hz); 5.71 (d, 1H, H-5, J 7.2 Hz);
7.08 and 7.17 (2 br s, 2H, 4-NH2); 7.64 (d, 1H, H-6, J 7.2 Hz) ppm. 13C
NMR (75.47 MHz, DMSO-d6): δ = 37.3 (C-1’); 75.3 (C-3’); 79.5 (C-2’); 94.1
(C-5); 144.8 (C-6); 155.2 (C-4); 166.0 (C-2) ppm. MS (ESI+) m/z (%): 150.1
[(M+H)+, 100]. HRMS (ESI+) m/z calcd for C7H8N3 (M+H)+, 150.0662;
found: 150.0662.
Keywords: Biomedicine • Cell culture • Nucleobases • One-pot
synthesis • Regioselectivity
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Entry for the Table of Contents
Regioselective N–H functionalization of DNA nucleobases (A, T, C, G): One-pot synthetic procedures for producing a library of
nucleobase derivatives enlisted with reactive functional groups for bioconjugation and cross-linking reactions with other biomolecules
are reported. The nucleobase derivatives bearing either N-alkyl, N-propanenitrile, N-allyl or N-propargyl terminals are non-cytotoxic,
denoting great promise for biomedical applications.
Institute and/or researcher Twitter usernames: COMPASS Research Group (@COMPASS_RG), CICECO – Aveiro Institute of Materials
(@ciceco_ua), University of Aveiro (@UnivAveiro), Vera Silva (@VeraLMSilva_Org), João Borges (@Joao_F_Borges), João Mano
(@joaofmano)
12
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