Synthesis of pyrimidine nucleoside and amino acid conjugates

Article abstract of DOI:10.1016/j.tetlet.2020.152598

The synthesis of novel pyrimidine nucleoside bioconjugates with amino acids is presented. The N⁴-amino acid-acylated 2′-deoxycytidine analogues, modified with various amino acids, were synthesized using a three-step synthesis and obtained in moderate overall yields. Novel amino acid-alkylated 2′-deoxycytidine derivatives were obtained during the rearrangement of amino acid-acylated derivatives that occurred during Boc deprotection.

Full text of DOI:10.1016/j.tetlet.2020.152598

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Synthesis of pyrimidine nucleoside and amino acid conjugates
Martyna Koplūnaitė, Kamilė Butkutė, Rolandas Meškys, Daiva Tauraitė
PII:
S0040-4039(20)31097-2
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.152598
TETL 152598
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Tetrahedron Letters
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Revised Date:
Accepted Date:
26 August 2020
14 October 2020
20 October 2020
Please cite this article as: Koplūnaitė, M., Butkutė, K., Meškys, R., Tauraitė, D., Synthesis of pyrimidine
nucleoside and amino acid conjugates, Tetrahedron Letters (2020), doi: https://doi.org/10.1016/j.tetlet.
2020.152598
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Graphical Abstract
Synthesis of pyrimidine nucleoside and amino acid conjugates
Martyna Koplūnaitėa , Kamilė Butkutėa , Rolandas Meškysa and Daiva Tauraitėa,b,*
a
Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life
Sciences Center, Vilnius University, Sauletekio al. 7, LT-10257 Vilnius, Lithuania
b
Department of Chemistry and Bioengineering, Vilnius Gediminas Technical University,
Sauletekio al. 11, LT-10223 Vilnius, Lithuania
* Tel: +370-5-223-4384; Email: daiva.tauraite@bchi.vu.lt
Abstract
The synthesis of novel pyrimidine nucleoside bioconjugates with amino acids is presented. The
N4-amino acid-acylated 2'-deoxycytidine analogues, modified with various amino acids, were
synthesized using a three-step synthesis and obtained in moderate overall yields. Novel amino
acid-alkylated 2'-deoxycytidine derivatives were obtained during the rearrangement of amino
acid-acylated derivatives that occurred during Boc deprotection.
Keywords
Pyrimidine nucleoside; Nucleoside-amino acid conjugates; N4-amino acid-acylated nucleosides;
N4-amino acid-alkylated nucleosides
Introduction

Nucleosides and their analogues are an important family of compounds that possess a
wide range of biological properties. They play an important role in medicinal chemistry, and
selected analogues, such as azidothymidine or gemcitabine, possess antiviral and antineoplastic
properties.1,2 Modified nucleosides are also widely employed as intermediates for the preparation
of nucleoside triphosphates3,4 and functionalized oligonucleotides.5–7 Tremendous efforts have
been made to improve the properties of nucleic acids by either incorporating chemical
modifications in their structure or covalently conjugating reporter groups with biologically
relevant properties, e.g. peptides, carbohydrates, and lipids.8,9 A large variety of modified
oligonucleotides have been synthesized and applied in a diverse range of applications such as
therapeutics or diagnostics.10,11 The development of such hybrid molecules allows the
pharmacokinetic
or
pharmacodynamic
properties
of
the
existing
nucleosides/nucleotides/oligonucleotides to be improved or gives them completely new
properties.
Nucleobase modifications or conjugations with other biomolecules are usually performed
at the C5 position of pyrimidines and the C7 position of 7-deazapurines, since these positions do
not affect complementary base pairing and nucleotides bearing such alterations can be
successfully incorporated into oligonucleotides.12,13 Nucleotides that are modified with amino
acid side chains at these positions have been previously described and employed as substrates for
PCR and primer extension reactions using numerous DNA polymerases.14–17 Less is known
about modifications at other nucleobase positions such as N6 and O6 of purines and N4 and O4 of
pyrimidines, notwithstanding that N6-methyladenosine and N4-acetylcytidine are found in various
RNAs and are involved in the regulation of RNAs activity.18,19 N4-Aminocytidine, originating
from the reaction between hydrazine and cytidine, is known to cause cell mutagenesis by
inducing AT to GC transition.20 Moreover, the synthesis and hybridization of oligonucleotides
containing various N4-acyl-2'-deoxycytidine derivatives have been previously described.21 It is
known from earlier studies that modifications at the N4 position of 2'-deoxycyditine 5'-
triphosphates do not affect hydrogen bonding with guanine and such compounds can be utilized
as substrates by various DNA polymerases.22 In addition, N4-acylcytidines have been
successfully used as tailored substrates for the selection of amidohydrolases in Escherichia coli
uracil auxotrophs23 or the high throughput screening (HTS) of hydrolases based on emulsion
PCR as well as microfluidics.24,25
To expand the existing assortment of N4-modified pyrimidines, we prepared novel 2'-
deoxycytidine N4-amino acid-acylated derivatives and significantly more stable N-(4-(2'-
deoxycytidinyl))amino acid amides – hybrids of nucleoside and amino acids, which could be
further used in HTS systems for novel enzymes or as intermediates for the synthesis of
nucleotides.
Results and Discussion
For the synthesis of nucleoside and amino acid conjugates we modified 2'-deoxycytidine
with glycine, L-alanine, β-alanine, L-leucine, L-isoleucine, L-methionine, L-phenylalanine and
L-tyrosine. The synthesis began by protecting the amino group of selected amino acids using di-
tert-butyl dicarbonate.26 The reactions were carried out at room temperature in a prechilled to 0

˚C mixture of 1 N NaOH, 1,4-dioxane and water (1.1:1:1) and took approximately 3 hours for
completion. The protected amino acids were extracted using ethyl acetate and isolated in 90–
95% yield. The Boc-protected amino acids were then treated with N-hydroxysuccinimide and
dicyclohexylcarbodiimide in ethyl acetate for 24 hours at room temperature to obtain activated
NHS-esters.27 Treatment of the activated amino acid esters with 2'-deoxycytidine in
dimethylformamide for 5 days at room temperature resulted in the formation of N4-acyl-Boc-2'-
deoxycytidine nucleosides 4a–h (Scheme 1). The compounds were purified using column
chromatography (solid phase: silica gel, liquid phase: chloroform-methanol mixture, 100:0 to
85:15) and obtained in an overall yield of 34–85% (Table 1).
Scheme 1. Synthesis of N4-acyl-Boc-2'-deoxycytidine nucleosides.
Table 1. N4-Acyl-Boc-2'-deoxycytidine derivatives produced via Scheme 1.
Entry
Product 4a–h
Yield (%)a,b
1a–h

1
2
3
4
5
6
7
8
53
50
59
65
40
34
85
53
4a
4b
4c
4d
4e
4f
4g
4h
a
Reagents and conditions: 1a–h (6.66 mmol), Boc2O (10 mmol), NaOH (13.32 mmol), 1,4-dioxane (12
mL), water (12 mL), 3 h, 0 ˚C, then neutralization with citric acid and extraction with ethyl acetate; 2a–h,
NHS (7.33 mmol), DCC (7.33 mmol), ethyl acetate (30 mL), 24 h, r. t., then filtration; 3a–h, 2'-
deoxycytidine (6.66 mmol), DMF (8–10 mL), 5 d, r. t.
b Isolated yield

Removal of the N-Boc protecting group using HCl or TFA/CH2Cl2 was unsuccessful and
resulted in N4 amide bond hydrolysis, thereby forming mainly 2'-deoxycytidine as the final
product. Thus, we selected mild and neutral conditions for the Boc deprotection step.28 The
synthesized nucleosides were deprotected using a catalyst-free method by heating at reflux in
water for 45–70 minutes. During the deprotection step the N4-acylated 2'-deoxycytidines (4a–h)
underwent rearrangement to N-(4-(2'-deoxycytidinyl))amino acid amides (5a–h) - significantly
more stable N4-alkylated derivatives bearing a primary amide bond (Scheme 2).
Scheme 2. Observed rearrangement of N4-acyl-Boc-2'-deoxycytidines (4a–h) to N-(4-(2'-
deoxycytidinyl))amino acid amides (5a–h) during the Boc-deprotection step.
Table 2. N-(4-(2'-Deoxycytidinyl))amino acid amides produced via Scheme 2.
Entry
Product 5a–h
Time (min)
Yield (%)a,b
48
1a–h
9
60
5a
10
45
37
5b

11
12
13
14
15
16
45
70
70
70
60
45
57
29
26
51
23
46
5c
5d
5e
5f
5g
5h
a Reagents and conditions: 4a–h (1.0–1.3 mmol), water (10–15 mL), 45–70 min, 100 ˚C.
b Isolated yield
N-(4-(2'-Deoxycytidinyl))amino acid amides 5a–h were purified using reverse phase
chromatography (solid phase: C18 column, liquid phase: water-methanol mixture, 100:0 to
80:20) and isolated in a moderate 23–57% yield. Cleavage of the N4 amide bond was observed
during the deprotection step and 2'-deoxycytidine was formed as an impurity. Hence, around
20% of the starting compound was converted into 2'-deoxycytidine according to HPLC-MS
analysis. As shown in Table 2, the deprotection reaction times varied from 45 to 70 minutes. The
reactions were optimized by observing the formation of products using TLC, and it was
determined that the optimal duration was 45 minutes, since longer reaction times yielded a
higher amount of 2'-deoxycytidine. In general, this did not cause problems with the N-(4-(2'-
deoxycytidinyl))amino acid amide derivatives since 2'-deoxycytidine was easily separated from
the product during the reverse phase chromatography purification step. However, it was
impossible to separate N-(4-(2'-deoxycytidinyl))glycinamide (5a) from 2'-deoxycytidine due to
their similar polarities, and an extra synthesis step had to be performed. N-(4-(2'-
Deoxycytidinyl))glycinamide with remaining impurities of 2'-deoxycytidine was treated with
acetyl-NHS ester in DMF. The obtained N4-acetyl-2'-deoxycytidine was successfully separated
from N-(4-(2'-deoxycytidinyl))glycinamide using reverse phase chromatography.

A mechanism for this reaction was previously proposed by Zhang and co-workers.29 The
rearrangement was observed for all of our synthesized compounds 4a–h, independent of the
amino acid residue, and was confirmed by 1H NMR spectroscopy. A shift of the NH signal was
observed when comparing the Boc-protected N4 acylated and N4 alkylated derivatives after
deprotection. The N4 hydrogen atom in the Boc-protected compounds appeared as a singlet at
around 11 ppm, and no such signal was observed for the deprotected compounds. Since a
rearrangement at the C4 atom of 2'-deoxycytidine occurred, the N4 hydrogen atom no longer had
a carbonyl group nearby and the signal shifted from 11 ppm to around 7.8 ppm and split into a
doublet, triplet or multiplet depending on the amino acid residue. Also, two singlets of the
primary amide NH2 group appeared at around 7.0 and 7.5 ppm. A thorough investigation of the
synthesized compounds by NMR spectroscopy revealed that diastereoisomers are not formed;
therefore it can be concluded that racemization of the amino acid side chains has not occurred.
Conclusion
The synthesized 2'-deoxycytidine and amino acid conjugates are a significant addition to
the existing libraries of modified nucleosides. Since there are numerous amino acids that differ
both in their size and chemical properties, a large number of nucleosides/nucleotides could be
further synthesized and used for various applications including enzyme screening and
antimicrobial therapy or even endow them with new properties.30,31 In addition, applications such
as the formation of therapeutic or catalytic aptamers (e.g. SOMAmers)17,32,33 could be
envisioned.
Acknowledgements
The work was supported by the European Regional Development Fund (project No 01.2.2-LMT-
K-718-03-0082) and the European Social Fund (project No 09.3.3.-LMT-K-712-15-0108) under
grant agreement with the Research Council of Lithuania (LMTLT).

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 Novel pyrimidine nucleoside bioconjugates with amino acids are presented.
 The synthesis of Boc protected N4 amino acid-acylated 2'-deoxycytidine compounds is
reported.
 The rearrangement during Boc deprotection resulted in novel amino acid-alkylated 2'-
deoxycytidine nucleosides.