Synthesis of N-substituted morpholine nucleoside derivatives

Article abstract of DOI:10.1080/15257770.2020.1788078

Herein, we report the synthesis of substituted morpholino nucleoside derivatives starting from ribonucleosides. The present protocol shows high functional group tolerance, uses mild reaction conditions, and gives moderate to good yields. This transformati

Full text of DOI:10.1080/15257770.2020.1788078

Nucleosides, Nucleotides & Nucleic Acids
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Synthesis of N-substituted morpholine nucleoside
derivatives
Mikhail D. Nekrasov , Evgeny R. Lukyanenko & Alexander V. Kurkin
To cite this article: Mikhail D. Nekrasov , Evgeny R. Lukyanenko & Alexander V. Kurkin (2020):
Synthesis of N-substituted morpholine nucleoside derivatives, Nucleosides, Nucleotides & Nucleic
Acids, DOI: 10.1080/15257770.2020.1788078
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2020.1788078
Synthesis of N-substituted morpholine nucleoside
derivatives
Mikhail D. Nekrasova,b, Evgeny R. Lukyanenkoa , and Alexander V. Kurkina
b
aLomonosov Moscow State University, Department of Chemistry, Moscow, Russia; Marlin Biotech
LLC, Research Department, Moscow, Russia
ABSTRACT
ARTICLE HISTORY
Received 26 April 2020
Accepted 23 June 2020
Herein, we report the synthesis of substituted morpholino
nucleoside derivatives starting from ribonucleosides. The pre-
sent protocol shows high functional group tolerance, uses
mild reaction conditions, and gives moderate to good yields.
This transformation is based on two sequential pathways: (i)
the oxidation of the ribonucleosides to the corresponding dia-
ldehyde using sodium periodate and (ii) the reductive amina-
tion of the in situ generated dialdehydes with the
hydrochloride salts of various the alkylamines.
KEYWORDS
DNA; PNA; morpholine;
reductive amination;
cycle synthesis
Introduction
DNA mimetics based on a morpholine backbone have been studied and
used as antisense agents for many years. Some morpholino-type nucleic
acid mimetics (those with a morpholinephosphorodiamidate backbone)
have been studied extensively for applications such as gene knock-
down[1–5] and exon skipping.[6] The efficiency of the interaction of mor-
pholino with DNA and RNA in biological systems is based on several
features, such as the low partial charge on the backbone, which
minimises the Columbic repulsion between strands, similar geometry to
DNA strands and high resistance to enzymatic degradation.[7–9] Some
chimeric DNA-morpholino primers have higher precision at the site
because fewer mismatches form with it than with native DNA
primers.[10] This allows their use for single nucleotide polymorphism
detection. Various applications of morpholine-based DNA mimetics
in molecular biology,[11] genetic research,[12] and medicine[6] have also
been reported.
One issue with the preparation of such compounds is the difficulty of
stereoselective morpholine-based monomer synthesis. Traditionally, the
morpholine core has been obtained through an oxidation strategy involving
CONTACT Alexander V. Kurkin
kurkin@direction.chem.msu.ru
Department of Chemistry, Moscow State
University, Leninskie Gory, 1/3, 119991 Moscow, Russian Federation.
ß 2020 Taylor & Francis Group, LLC

2
M. D. NEKRASOV ET AL.
cleavage of the C20-C30 bond in nucleosides because this allows the preser-
vation of the chiral centres at the C10 and C40 positions. Subsequently,
reductive amination is used to form the morpholine cycle. This approach
can be used to convert nucleosides and their 50-silylated derivatives into
secondary[13,14] or tertiary (N-substituted) morpholines. N-substituted mor-
pholines have been synthesised with hydroxyl,[15] alkylamine,[16]
methylcarboxy,[17,18] and various alkyl[19,20] groups. Unfortunately, most of
the above reactions have either low yields or scaling issues and require
further improvement for large-scale monomer synthesis. Our goal in this
study was to find a versatile approach to prepare various N-substituted
morpholino nucleosides starting from protecting group derivatives for
future applications as synthons in morpholine-based monomer synthesis.
With this in mind, nucleosides with exocyclic group protection were
used as starting materials. First, various protocols for the preparation of
morpholines with secondary amino groups were used but found to
be inefficient. These protocols generally use ammonium tetraborates as
nitrogen sources for morpholine cycle formation.[21] Protocols that used
N-substituted amines as substrates did not have high yields (calculated
with respect to the nucleoside) either; thus, alternative reaction conditions
were investigated. Another avenue of research was directed at morpholine-
based peptide nucleic acid (PNA) monomers, so amino acid and hydrazine
derivatives were also investigated as substrates for N-substituted
morpholino nucleoside derivative synthesis.
Results and discussion
The primary goal of our study was to design
a
cycle for
morpholine–nucleoside N-tritylated monomer synthesis. Trial runs with
ammonium biborate proved inconvenient and inefficient, so alternate
pathways were tested (Scheme 1). For this, trityl, diphenylmethyl, benzyl,
and allyl derivatives were synthesised. Direct synthesis of trityl derivatives
failed, probably because of the steric bulk, despite testing the protocol
at weakly acidic and neutral pH. We explored the oxidation of
5-methyluridine (m5U) and 50-O-DMTr-5-methyluridine, subsequent
reductive amination with benzylamine or diphenylmethylamine in aqueous
buffer solution under conditions based on the works of Bell et al.[20] and
Zang et al.[10] (Scheme 1). Reactions with different ratios of dialdehydes
and amines give modest yields of products in all cases.
Our protocol was refined over several iterations until reaching its
current form. Early versions of the protocol involved filtering the iodates
off immediately after dialdehyde formation. This is much desired by
the reaction because to prevent decomposition or overoxidation of the

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
3
Scheme 1. Conventional synthesis of monomer E. Reagents and conditions: (i) DMTr–Cl, NEt3,
Py, rt, Ar; (ii) and (iii) NaIO4, (NH4)2B4O7, CH3OH, rt., NaBH3CN, CH3OH, rt; (ii) and (iv) NaIO4,
PhCH2NH2, rt., NaCNBH3, CH3OH, rt; (v) H2/Pd/C, MeOH; (vi) TrCl, TEA, DMF, rt.
intermediate. In later protocols, the iodates were filtered off with sodium
chloride and other substances precipitating from the solution during aminal
formation (a one-pot strategy - with simultaneous addition of sodium peri-
odate, sodium tetraborate, and amine hydrochloride salts) (Scheme 2).
The most efficient of the previously published reactions was that using
ammonium tetraborate as a nitrogen source. In our study, a commercially
available salt ((NH4)2B4O7 x 10H2O) was replaced with that synthesised in
situ from ammonium chloride and sodium tetraborate. Because these reac-
tions were carried out in alcohols, insoluble sodium chloride formed and
precipitated out of the reaction, thus driving ammonium tetrabo-
rate formation.
Later, amine hydrochloride salts were prepared similarly. Benzylamine
hydrochloride was added as a solid, whereas allylamine hydrochloride (in
later attempts) was added as a 1 M solution in methanol because it is
strongly hygroscopic and hard to store as a solid. After this stage had com-
pleted, the reaction mixture was filtered to remove solid inorganic impur-
ities that could interfere with reduction. Several reduction protocols have
been described in the literature for similar reactions. The most efficient
protocols involve sodium cyanoborohydride as the reducing agent.[10]

4
M. D. NEKRASOV ET AL.
Scheme 2. Modified synthesis (early version) of monomer D. Reagents and conditions: (i)
TBDPSCl, Im, DCM, rt.; (ii) and (iii) NaIO4, CH3OH, Na2B4O7 [ 10H2O, RNH2 x HCl, rt., NaBH3CN,
Et3N, rt., TFA, rt; (iv) 1 M HCl, MeOH, rt.
In one protocol,[12] reduction was performed at neutral pH without buf-
fer for 6 h. This process used with 50-O-dimethoxytritylated nucleosides,
but it did not work well for more polar substrates (yields in range of
20–30% at 1 mmol scale, but lower at larger scales). Another protocol[15]
used mildly acidic conditions for 50-O-silylated substrates. This approach
was not efficient with a non-silylated derivative, and selective silylation was
quite inefficient and required an extra chromatographic purification stage,
so it was deemed unviable. In contrast, reduction under acidic conditions
was faster. In another work,[17] formation of unreduced tertiary morpholine
derivatives and the addition of a reducing agent were carried out under
basic conditions followed by subsequent acidification. This approach
proved to be the most promising method. In our protocol, at the first,
sodium cyanoborohydride was added with trimethylamine, which barely
reacted with aminal at this stage. Next, a 20% solution of trifluoroacetic
acid in methanol was added to lower the pH and increase the speed of the
reduction. Afterwards, the excess of acid was neutralised in mild conditions
to avoid the deprotonation of the substrate or hydrolysis of the protective
groups. This was also attempted with acetic acid based on some earlier pro-
tocols,[19] but acetic acid was not strong enough to counter the effects of
the base and, thus, hence reduction was not efficient.
Finally, the target derivatives were synthesised (Table 1), and the opti-
mised protocol was used for the synthesis of morpholino monomers from
various ribonucleosides and amines (1-4). We attempted to cleavage of the
protective group and obtain activated monomers which need for the syn-
thesis of the morpholine oligomers. Unexpectedly, only complex mixtures
were obtained in the reactions of deprotection monomers. For example, the

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
5
Table 1. Efficiency of the developed method in the synthesis of morpholine-based monomer.
Entry
1
Code
Structure
Yield (%)
95
1a
2
1b
77
3
1c
32
4
1d
57
(continued)

6
M. D. NEKRASOV ET AL.
5
1e
33
6
2a
84
7
2b
64
8
2c
35
9
2d
52
(continued)

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
7
10
2e
25
11
3a
70
12
3b
79
13
3c
34
14
3d
31
(continued)

8
M. D. NEKRASOV ET AL.
15
3e
45
79
60
58
16
17
18
4a
4b
4b
19
4d
31
(continued)

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
9
20
4e
43
General procedure: To a solution of a ribonucleoside (1 mmol) in MeOH (20 mL), sodium periodate (1.02 mmol),
sodium tetraborate decahydrate (2 mmol), and amine hydrochloride (2.3 mmol) were added, and the reaction
mixture was stirred for 2,5 h after which the reaction mixture was filtered. To a solution of the mixture in
MeOH, sodium cyanoborohydride (2.4 mol), and triethylamine (0.33 mL) were added and the mixture was left
to stir for 1 h. Next, 0.4 mL of TFA acid was mixed with 5 mL of methanol and added and the mixture was left
to stir for 3 h.
process removal of benzyl group results in morpholine-nucleobases bond
fission. Allyl removal was also ineffective, we identified two main products
formed in the reaction mixture - target monomer and a symmetric dimer
(on the basis of NMR and mass spectroscopy data).
In some of the mention protocols have used 50-O-silylated derivatives,
which are more soluble in organic solvents and easier to purify. Thus, these
derivatives were used for the synthesis of the activated N-substituted and
N-unsubstituted morpholine cores. Unfortunately, desylilation of N-substi-
tuted morpholine did not work well. 50-O-silylated allyl derivatives under-
went near-quantitative protecting group removal, whereas the benzyl
derivatives, with the exception of the thymine derivatives, was obtained
with low yields, therefore, this pathway to synthesis of activated morpho-
line cores using protective groups, for example, tert-butyldiphenylsilyl
group, cannot be considered as universal. Its useful to note, tert-
butyldiphenylsilyl group (TBDMSi) could be used more routinely because
it allowed for easier purification of the tritylated product.
After the development of the synthetic cycle for the monomers had
completed, research moved to the synthesis of PNA-inspired structures
(Table 1). Two variants were tested: glycine ester synthesis and hydrazine
derivative synthesis. The former could be converted into an amino acid
analogue, whereas the latter could be either reduced to an amine or
deprotected and modified to a hydrazine.
1,1-Diallylhydrazine was chosen for preparation of monomers in which
the nitrogen atom of hydrazine group contained allyl protection group.
This protection group was relative stability towards both oxidative and
reductive conditions that permits to used it in our protocol.
Glycine derivatives were also successfully synthesised (Table 1).
Unfortunately, the yields of this reaction were significantly lower. Similar
literature reactions with glycine have shown better yields,[17] which means
that our approach is not optimal for preparing amino acid derivatives.

10
M. D. NEKRASOV ET AL.
The process was modified for the synthesis of monomers with 1,1-
diallylhydrazine fragment, it was made because hydrazines are reducing
agents and react with sodium periodate and iodate, preventing the reac-
tion nucleosides with these reagents. For preventing unforeseen side
reactions, the remaining oxidising agent was filtered off and then
sodium tetraborate tetrahydrate and the hydrochloride hydrazine were
successively added. Another limiting factor in this reaction is low solu-
bility N-protected nucleosides, for example, N4-benzoyl cytidine, in
methanol, which led to a slowdown in the rate of oxidation. The intro-
duction of silyl substituents, as a rule, increased the solubility of the
nucleoside and avoided unnecessary overheating of the reaction (heating
increased the over-oxidation of the dialdehyde) and allowed to increase
of yieldß of target monomers.
The developed approach allowed the synthesis of N-substituted morpho-
lines with reliable yields (up to 95%) and with diverse substituents. The
method proved to be highly efficient for the synthesis of N-alkyl deriva-
tives. This approach to the synthesis of the morpholine cycle preserves the
stereo specificity of the 10- and 50-positions while converting furanose
derivatives to morpholines and avoiding racemisation at the a-positions of
the dialdehyde. All the steps of this synthesis are simple and easily scalable
up to 0.5–1 mol with the use of standard laboratory equipment.
Experimental
Chemistry
All commercially available compounds were purchased and used as
received unless otherwise specified. The solvents tetrahydrofuran (THF)
and dichloromethane (CH2Cl2) were purified by passing through alumina
using solvent purification system. Toluene and acetonitrile (CH3CN) were
distilled from calcium hydride prior to use. Deuterated chloroform (CDCl3)
was stored over 3 Å molecular sieves. Argon gas was purchased from Air
Liquide. All reactions were monitored by thin-layer chromatography (TLC)
carried out on Merck TLC Silica gel plates (60 F254), using UV light for
visualisation and basic aqueous potassium permanganate or phosphomolyb-
1
dic acid stain as a developing agent. H and 13C NMR spectra were
recorded on Bruker Avance 400 instrument with operating frequency of
400 and 100 MHz respectively and calibrated using residual undeuterated
chloroform (dH ¼ 7.28 ppm) and CDCl3 (dC ¼ 77.16 ppm), or undeuterated
DMSO (dH ¼ 2.50 ppm) and DMSO-d6 (dC ¼ 39.51 ppm) as internal refer-
ences. Chemical shifts (d) are reported in ppm, and multiplicities are indi-
cated by s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m
(multiplet), and br (broad). Coupling constants, J, are reported in hertz

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
11
(Hz). All NMR spectra were obtained at room temperature. High resolution
mass spectra (HRMS) were measured on a Thermo Scientific LTQ Orbitrap
instrument using nanoelectrospray ionisation (nano-ESI). The melting
points (m.p.) were not presented as all new substances are of oil or foam
consistency.
General procedure a for N-benzyl morpholino nucleoside synthesis
A ribonucleoside, 5-methyluridine, N2-isobutirylguanosine, N4-benzoylcyti-
dine, or N6-benzoyladenosine (2 mmol), was mixed with sodium periodate
(0.44 g, 2.04 mmol), sodium tetraborate decahydrate (0.80 g, 4 mmol), ben-
zylamine hydrochloride (0.66 g, 4.6 mmol), and 40 mL of methanol with
intense stirring via a stirrer bar. The mixture was stirred for 2.5 h, after
which the reaction mixture was filtered. The reaction flask was then placed
in a water bath, and a stirrer bar, sodium cyanoborohydride (0.30 g,
4.8 mol), and triethylamine (0.66 mL) were added to the reaction mixture,
and the mixture was left to stir for 1 h. Next, 0.8 mL of trifluoroacetic acid
was mixed with 5 mL of methanol and added slowly over 10 min dropwise,
and the mixture was left to stir for 3 h. The reaction was quenched with
potassium carbonate (0.72 g, 5.24 mmol), after which the solution was con-
centrated to 10-mL volume and mixed with 20 mL of water and 90 mL of
dichloromethane. Then, the aqueous layer was extracted twice with dichlor-
omethane–methanol (9:1). The organic layers were combined, dried over
sodium sulphate, filtered, and concentrated under reduced pressure. The
crude product was purified on a silica gel column poisoned with ammonia
in a methanol/dichloromethane system.
General procedure B for N-allyl morpholino nucleoside synthesis
A ribonucleoside, 5-methyluridine, N2-isobutirylguanosine, N4-benzoylcyti-
dine, or N6-benzoyladenosine (30 mmol), was mixed with sodium periodate
(6.52 g, 30.6 mmol), sodium tetraborate decahydrate (13.88 g, 69 mmol), and
600 mL of methanol with intense stirring via a stirrer bar. A stock 1 M
solution of allylamine hydrochloride in methanol (60 mL, 60 mmol) was
added and the mixture was stirred for 2.5 h, after which the reaction mix-
ture was filtered. The reaction flask was placed in a water bath, and a stir-
rer bar, sodium cyanoborohydride (4.16 g, 63 mmol), and triethylamine
(10 mL) were added to the reaction mixture, and the mixture was left to
stir for 1 h. Next, 12 mL of trifluoroacetic acid was mixed dropwise with
24 mL of methanol slowly over 10 min, and the mixture was left to stir for
3 h. The reaction was quenched with 22 mL of 50% potassium carbonate
solution, after which the solution was concentrated to a volume of 100 mL,

12
M. D. NEKRASOV ET AL.
mixed with 100 mL of water, and then mixed with 900 mL of dichlorome-
thane. Then, the aqueous layer was extracted twice with dichloromethane–-
methanol (9:1). The organic layers were combined, dried over sodium
sulphate, filtered, and concentrated under reduced pressure. The crude
product was purified on a silica gel column poisoned with ammonia in a
methanol/dichloromethane system.
General procedure C for N-diphenylmethyl morpholino nucleoside synthesis
A ribonucleoside, 5-methyluridine, N2-isobutirylguanosine, N4-benzoylcyti-
dine, or N6-benzoyladenosine (2 mmol), was mixed with sodium periodate
(0.44 g, 2.04 mmol), sodium tetraborate decahydrate (0.80 g, 4 mmol),
diphenylmethylamine hydrochloride (1.01 g, 4.6 mmol), and 40 mL of
methanol with intense stirring via a stirrer bar. The mixture was stirred for
2.5 h, after which the reaction mixture was filtered. The reaction flask was
placed in a water bath, and a stirrer bar, sodium cyanoborohydride (0.30 g,
4.8 mol), and triethylamine (0.66 mL) were added to the reaction mixture,
and the mixture was left to stir for 1 h. Next, 0.8 mL of trifluoroacetic acid
was mixed with 5 mL of methanol and added slowly over 10 min dropwise,
and the mixture was left to stir for 3 h. The reaction was evaporated dry and
mixed with 10 mL of water and 1.4g of potassium carbonate. Then, the
aqueous layer was extracted twice with dichloromethane, and the organic
layers were combined, dried over sodium sulphate, filtered, and concentrated
under reduced pressure. The resulting oil was dissolved in 10mL tetrahydro-
furan, mixed with 1.00 g of di-tert-butyl-dicarbonate, stirred for 3 h, and then
evaporated dry. The crude product was purified on a silica gel column pois-
oned with ammonia in a methanol/dichloromethane system.
General procedure D for N-glycine morpholino nucleoside synthesis
A ribonucleoside, 5-methyluridine, N2-isobutirylguanosine, N4-benzoylcyti-
dine, or N6-benzoyladenosine (2 mmol), was mixed with sodium periodate
(0.44 g, 2.04 mmol), sodium tetraborate decahydrate (0.92 g, 4.6 mmol), ben-
zyl glycinate hydrochloride (0.81 g, 4 mmol), and 40 mL of methanol with
intense stirring via a stirrer bar. The mixture was stirred for 2.5 h, after
which the reaction mixture was filtered. The reaction flask was placed in a
water bath, and a stirrer bar, sodium cyanoborohydride (0.30 g, 4.8 mol),
and triethylamine (0.66 mL) were added to the reaction mixture, and the
mixture was left to stir for 1 h. Next, 0.8 mL of trifluoroacetic acid was
mixed with 5 mL of methanol and added slowly over 10 min dropwise, and
the mixture was left to stir for 3 h. The reaction was quenched with 5 M
ammonia in methanol, after which the solution was evaporated to oil and

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
13
purified on a silica gel column poisoned with ammonia in a methanol/
dichloromethane system.
General procedure E for N,N-diallyl hydrazine morpholino nucleoside
synthesis
A ribonucleoside, 5-methyluridine, 50-tert-butyldiphenylsilyloxy-N4-benzoyl-
cytidine, N2-isobutirylguanosine, or N6-benzoyladenosine (2 mmol), was
dissolved in 40 mL of methanol and mixed with sodium periodate (0.44 g,
2.04 mmol) dissolved in 2 mL of water. The mixture was stirred for 30 min,
after which the reaction was deemed complete, and the mixture was filtered
to remove the inorganic precipitate. Then, 0.66 mL of triethylamine,
sodium tetraborate decahydrate (0.80 g, 4 mmol), and 1,1-diallylhydrazine
dihydrochloride (0.85 g, 4.6 mmol) were added. The mixture was stirred for
2 h, after which sodium cyanoborohydride (0.28 g, 4.4 mol) was added to the
reaction mixture and left to stir for 5 min. Next, 0.8mL of trifluoroacetic
acid was mixed with 5 mL of methanol and added slowly over 10 min drop-
wise, and the mixture was left to stir for 3 h. The reaction was then evapo-
rated dry and mixed with 10mL of water and 1.4 g of potassium carbonate.
Then, the aqueous layer was extracted twice with dichloromethane, and the
organic layers were combined, dried over sodium sulphate, filtered, and con-
centrated under reduced pressure. The resulting oil was purified on a silica
gel column poisoned with ammonia in a methanol/dichloromethane system.
1-((2s,6R)-4-Benzyl-6-(hydroxymethyl)morpholin-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (1a)
It follows general procedure A.
White foam, yield 95% (1.86 g, 5.61 mmol). TLC Rf ¼ 0.3 (CH2Cl2/CH3OH
20:1). Spectral data matched that previously reported for compound 1a:[20]
1H NMR (CDCl3, 400 MHz), d (ppm): 1.89 (3H, s, CH3), 2.03 (2H, d,
J¼ 10.7 Hz), 2.77 (1H, d, J¼ 10.7 Hz), 2.97 (1H, d, J¼ 10.7 Hz), 3.52-3.62 (2H,
m, PhCH2), 3.62-3.74 (2H, m), 3.90-4.04 (1H, m), 5.83-5.95 (1H, dd, J¼ 10.5,
1.4 Hz), 7.26 (1H, s, 6-CH), 7.27-7.38 (5H, m, Ph), 9.56 (1H, br s, NH). 13C
NMR (CDCl3, 100 MHz) d (ppm): 12.4 (CH3), 52.7, 55.9, 62.4 (PhCH2), 63.3,
77.0, 79.5, 110.7, 127.5, 128.4, 129.0 & 135.7 (Ph-C), 136.5, 149.9, 163.9.
1-((2r,6S)-4-Allyl-6-(hydroxymethyl)morpholin-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (1 b)
It follows general procedure B.
White foam, yield 77% (6.49 g, 23.09 mmol). TLC Rf ¼ 0.3 (CH2Cl2/
1
CH3OH 20:1). H NMR (CDCl3, 400 MHz), d (ppm): 1.90 (3H, s), 2.00

14
M. D. NEKRASOV ET AL.
(2H, t, J ¼ 10.8 Hz), 2.62-2.92 (2H, m), 2.96-3.14 (3H, m), 3.64-3.76 (2H,
m), 3.94-4.01 (1H, m), 5.15-5.25 (2H, m), 5.76-5.91 (2H, m), 7.24 (1H, s),
9.48 (1H, br s.). 13C NMR (CDCl3, 100 MHz) d (ppm): 12.5, 52.9, 55.7,
61.1, 63.4, 77.0 (in solvent peak), 79.6, 110.8, 119.1, 133.6, 135.7, 149.9,
163.8. HRþMS (ESI): m/z observed 282.1449, calculated for
C13H20N3O4 282.1448.
1-((2r,6S)-4-Benzhydryl-6-(hydroxymethyl)morpholin-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (1c)
It follows general procedure C.
White foam, yield 32% (0.26 g, 0.64 mmol). TLC Rf ¼ 0.1 (CH2Cl2/
1
CH3OH 20:1). H NMR (CDCl3, 400 MHz), d (ppm): 1.88 (3H, s), 1.93
(2H, td, J1 ¼ 10.8 Hz, J2 ¼ 5.3 Hz), 2.29 (1H, br s.), 2.81 (1H, d,
J ¼ 11.6 Hz), 2.97 (1H, d, J ¼ 11.1 Hz), 3.55-3.70 (2H, m), 3.97-4.06 (1H,
m), 4.32 (1H, s), 5.96 (1H, dd, J1 ¼ 9.9 Hz, J2 ¼ 2.4 Hz), 7.15-7.35 (7H, m),
7.37-7.43 (4H, m), 8.87 (1H, br s.). 13C NMR (CDCl3, 100 MHz) d (ppm):
12.4, 51.9, 54.8, 63.5, 75.9, 77.1, 79.7, 110.8, 127.4, 127.5, 127.8 (4 C),
128.76 (2 C), 128.83 (2 C), 135.6, 140.9, 141.2, 14þ9.8, 163.5. HRMS (ESI):
m/z observed 408.1918, calculated for C23H26N3O4 408.1918.
Benzyl 2-((2S,6R)-2-(hydroxymethyl)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-
1(2H)-yl)morpholino)acetate (1d)
It follows general procedure D.
Bluish-white foam, yield 57% (0.44 g, 1.13 mmol). TLC Rf ¼ 0.3 (CH2Cl2/
1
CH3OH 10:1). H NMR (CDCl3, 400 MHz), d (ppm): 1.92 (3H, s), 2.42
(1H, br s.), 2.49 (1H, t, J ¼ 11.0 Hz), 2.55 (1H, t, J ¼ 10.4 Hz), 2.81 (1H, d,
J ¼ 10.6 Hz), 2.98 (1H, d, J ¼ 10.6 Hz), 3.42 (2H, d, J ¼ 3.7 Hz), 3.64-3.75
(2H, m), 3.95-4.03 (1H, m), 5.15 (2H, s), 5.87 (1H, dd, J1 ¼ 9.8 Hz,
J2 ¼ 2.6 Hz), 7.23 (1H, d, J ¼ 1.2 Hz), 7.31-7.41 (5H, m), 9.04 (1H, br s.).
13C NMR (CDCl3, 100 MHz) d (ppm): 12.5, 51.8, 54.7, 57.9, 63.3, 66.5,
77.2, 79.5, 110.9, 128.4 (2 C), 128.5, 128.6 (2 C), 135.3, 135.6, 149.9, 163.7,
169.7. HRþMS (ESI): m/z observed 390.1662, calculated for
C19H24N3O6 390.1660.
1-((2r,6S)-4-(diallylamino)-6-(hydroxymethyl)morpholin-2-yl)-5-methylpyrimid-
ine-2,4(1H,3H)-dione (1e)
It follows general procedure E.
Colourless oil, yield 33% (0.22 g, 0.66 mmol). TLC Rf ¼ 0.3 (CH2Cl2/
1
CH3OH 20:1). H NMR (CDCl3, 400 MHz), d (ppm): 1.91 (3H, s), 2.48
(2H, td, J1 ¼ 10.3 Hz, J2 ¼ 3.9 Hz), 2.61 (1H, br s.), 2.91 (1H, d, J ¼ 10.5 Hz),
3.12 (1H, d, J ¼ 10.1 Hz), 3.20 (4H, d, J ¼ 6.0 Hz), 3.65-3.76 (2H, m), 3.83-

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
15
3.91 (1H, m), 5.08 (2H, dd, J1 ¼ 10.2 Hz, J2 ¼ 1.4 Hz), 5.15 (2H, dd,
J1 ¼ 17.2 Hz, J2 ¼ 1.5 Hz), 5.75-5.91 (3H, m), 7.23 (1H, d, J ¼ 1.1 Hz), 9.21
(1H, br s.). 13C NMR (CDCl3, 100 MHz) d (ppm): 12.5, 48.7, 52.4, 53.3
(2 C), 63.4, 76.6, 79.6, 110.8, 116.8 (2 C), 135.7, 136.0 (2 C), 149.8, 163.7.
þ
HRMS (ESI): m/z observed 337.1872, calculated for C16H25N4O4 337.1870.
N-(9-((2R,6S)-4-Benzyl-6-(hydroxymethyl)morpholin-2-yl)-9H-purin-6-yl)benza-
mide (2a)
It follows general procedure A.
Greenish-white foam, yield 84% (0.75 g, 1.5 mmol). TLC Rf ¼ 0.7
1
(CH2Cl2/CH3OH 10:1). H NMR (DMSO-d6, 400 MHz), d (ppm): 2.05
(1H, t, J ¼ 10.9 Hz), 2.80-2.92 (2H, m), 3.01 (1H, q, J ¼ 7.5 Hz), 3.12 (1H, d,
J ¼ 10.2 Hz), 3.40-3.53 (1H, m), 3.65 (2H, q, J ¼ 11.6 Hz), 3.82-3.91 (1H,
m), 4.82 (1H, br s), 5.97 (1H, dd, J ¼ 7.8 Hz, 2.0 Hz), 7.24-7.30 (1H, m),
7.32-7.48 (4H, m), 7.55 (2H, t, J ¼ 7.6 Hz), 7.64 (1H, t, J ¼ 7.3 Hz), 8.05
(2H, d, J ¼ 7.2 Hz), 8.67 (1H, s), 8.76 (1H, s), 9.5-12.0 (1H, br s.). 13C
NMR (DMSO-d6, 100 MHz) d (ppm): 45.8, 55.2, 61.6, 62.0, 76.9, 79.3,
125.3, 127.2, 128.3 (2 C), 128.5 (5 C), 129.0 (2 C), 132.5, 133.3, 137.3, 142.8,
150.4, 151.8, (signal of CO lost because of brþoadening). HRMS (ESI): m/z
observed 445.1979, calculated for C24H25N6O3 445.1983.
N-(9-((2R,6S)-4-Allyl-6-(hydroxymethyl)morpholin-2-yl)-9H-purin-6-yl)benza-
mide (2 b)
It follows general procedure B.
White foam, yield 64% (7.56 g, 19.18 mmol). TLC Rf ¼ 0.48 (CH2Cl2/CH3OH
1
10:1). H NMR (CDCl3, 400 MHz), d (ppm): 2.44 (1H, t, J¼ 10.3 Hz), 2.82 (1H,
d, J¼ 11.1 Hz), 3.11 (2H, d, J¼ 6.4 Hz), 3.28 (1H, d, J¼ 10.8 Hz), 3.57-3.80 (2H,
m), 3.98-4.13 (2H, m), 5.17-5.28 (2H, m), 5.30 (1H, s), 5.77-5.90 (1H, m), 6.02
(1H, d, J¼ 8.7 Hz), 7.47-7.57 (2H, m), 7.57-7.65 (1H, m), 8.07 (2H, d,
13
J¼ 7.3 Hz), 8.12 (1H, s), 8.82 (1H, s), 9.76 (1H, s). C NMR (CDCl3, 100 MHz)
d (ppm): 52.7, 56.6, 61.0, 63.0, 77.2, 80.3, 119.1, 122.1, 128.1 (2 C), 128.6 (2 C),
132.6, 133.6, 133.8, 140.40, 149.8, 152.9, 165.1. HRMS (ESI): m/z observed
þ
395.1830, calculated for C20H23N6O3 395.1826.
N-(9-((2R,6S)-4-Benzhydryl-6-(hydroxymethyl)morpholin-2-yl)-9H-purin-6-yl)ben-
zamide (2c)
It follows general procedure C.
White foam, yield 35% (0.34 g, 0.72 mmol). TLC Rf ¼ 0.4 (CH2Cl2/
CH3OH 20:1). 1H NMR (CDCl3, 400 MHz), d (ppm): 2.11 (1H, t,
J ¼ 11.1 Hz) , 2.44 (1H, t, J ¼ 10.6 Hz), 2.85 (1H, d, J ¼ 11.1 Hz), 3.10-3.20
(2H, m), 3.54-3.77 (2H, m), 4.04-4.13 (1H, m), 4.40 (1H, s), 6.10 (1H, d,

16
M. D. NEKRASOV ET AL.
J ¼ 7.9 Hz), 7.14-7.63 (13H, m), 8.02 (2H, d, J ¼ 7.6 Hz), 8.07 (1H, s), 8.78
(1H, s), 9.42 (1H, br s.). 13C NMR (CDCl3, 100 MHz) d (ppm): 51.9, 54.1,
55.4, 63.1, 75.6, 80.0, 127.4, 127.5, 127.8 (4 C), 128.0 (2 C), 128.6 (2 C),
128.75 (2 C), 128.78 (2 C), 132.6, 133.6, 140.7, 140.8, 141.1, 149.6, 151.0,
152.8, 165.0, (one aromatic signal lost because of brþoadening). HRMS
(ESI): m/z observed 521.2298, calculated for C30H29N6O3 521.2296.
Benzyl 2-((2 R,6S)-2-(6-benzamido-9H-purin-9-yl)-6-(hydroxymethyl)morpho-
lino)acetate (2d)
It follows general procedure D.
White foam, yield 52% (0.52 g, 1.03 mmol). TLC Rf ¼ 0.5 (CH2Cl2/
CH3OH 10:1). 1H NMR (CDCl3, 400 MHz), d (ppm): 2.62 (1H, t,
J ¼ 10.9 Hz), 2.83 (1H, d, J ¼ 10.6 Hz), 2.94 (1H, t, J ¼ 10.4 Hz), 3.30 (1H, d,
J ¼ 10.3 Hz), 3.45 (2H, s), 3.59-3.81 (3H, m), 4.02-4.10 (1H, m), 5.17 (2H,
s), 6.05 (1H, dd, J1 ¼ 9.9 Hz, J2 ¼ 2.4 Hz), 7.31-7.40 (5H, m), 7.52 (2H, t,
J ¼ 7.6 Hz)), 7.61 (1H, t, J ¼ 7.3 Hz), 8.06 (d, J ¼ 7.3 Hz, 2H), 8.13 (s, 1H),
8.81 (s, 1H), 9.64 (br s., 1H). 13C NMR (CDCl3, 100 MHz) d (ppm): 52.0,
55.7, 58.1, 62.9, 66.6, 77.2, 80.2, 117.7, 128.1 (2 C), 128.4 (2 C), 128.5, 128.6
(4 C), 132.6, 135.3, 140.4, 149.8, 150.9, 153.0, 16þ6.9, 169.6. HRMS (ESI): m/
z observed 503.2040, calculated for C26H27N6O5 503.2037.
N-(9-((2R,6S)-4-(diallylamino)-6-(hydroxymethyl)morpholin-2-yl)-9H-purin-6-
yl)benzamide (2e)
It follows general procedure E.
Colourless oil, yield 43.3% (0.39 g, 0.86 mmol). TLC Rf ¼ 0.3 (CH2Cl2/
CH3OH 20:1). 1H NMR (CDCl3, 400 MHz), d (ppm): 2.65 (1H, t,
J ¼ 10.6 Hz), 2.86-2.98 (2H, m), 3.15-3.29 (4H, m), 3.42 (1H, d, J ¼ 9.8 Hz),
3.59-3.76 (2H, m), 3.88-3.96 (1H, m), 4.22 (1H, br s.), 5.10 (2H, d,
J ¼ 10.3 Hz), 5.17 (2H, d, J ¼ 17.2 Hz), 5.80-5.97 (3H, m), 7.52 (2H, t,
J ¼ 7.5 Hz), 7.61 (1H, t, J ¼ 7.4 Hz), 8.07 (2H, d, J ¼ 7.3 Hz), 8.12 (1H, s),
8.81 (1H, s), 9.89 (1H, br s.). 13C NMR (CDCl3, 100 MHz) d (ppm): 25.6,
48.5, 53.4 (2 C), 63.1, 76.7, 80.3, 116.8 (2 C), 128.1 (2 C), 128.6 (2 C), 132.6,
133.8, 135.9 (2 C), 140.5, 149.8, 150.9, 153.0, 165.1. HRMS (ESI): m/z
þ
observed 450.2244, calculated for C23H28N7O3 450.2248.
N-(1-((2R,6S)-4-benzyl-6-(hydroxymethyl)morpholin-2-yl)-2-oxo-1,2-dihydropyr-
imi-din-4-yl)benzamide (3a)
It follows general procedure A.
Greenish-white foam, yield 70% (0.62 g, 1.4 mmol). TLC Rf ¼ 0.3
1
(CH2Cl2/CH3OH 20:1). H NMR (CDCl3, 400 MHz), d (ppm): 1.92-2.03
(2H, m), 2.83 (1H, d, J ¼ 10.6 Hz), 3.01 (1H, d, J ¼ 10.6 Hz), 3.42-3.63 (4H,

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
17
m), 3.74-3.84 (1H, m), 4.81 (1H, br s.), 5.71- 5.77 (1H, m), 7.24-7.40 (6H,
m), 7.51 (2H, t, J ¼ 7.7 Hz), 7.62 (1H, t, J ¼ 7.3 Hz), 8.01 (2H, d, J ¼ 8.5 Hz),
8.17 (1H, d, J ¼ 7.8 Hz), 10.83 (1H, br s.). 13C NMR (CDCl3, 100 MHz) d
(ppm): 56.0, 61.5, 62.0, 77.0, 80.6, 96.6, 127.2, 128.3 (2 C), 128.45 (2 C),
128.48 (2 C), 129.0 (2 C), 132.8, 133.1, 137.2, 145.5, 153.7, 163.1. HRMS
þ
(ESI): m/z observed 421.1864, calculated for C23H25N4O4 421.1870.
N-(1-((2R,6S)-4-Allyl-6-(hydroxymethyl)morpholin-2-yl)-2-oxo-1,2-dihydropyrimi-
din-4-yl)benzamide (3 b)
It follows general procedure B.
White foam, yield 79% (8.77 g, 23.7 mmol). TLC Rf ¼ 0.68 (CH2Cl2/
CH3OH 10:1). 1H NMR (CDCl3, 400 MHz), d (ppm): 1.85 (1H, t,
J ¼ 9.6 Hz), 2.03 (1H, t, J ¼ 11.1 Hz), 2.87 (1H, d, J ¼ 11.1 Hz), 3.05 (2H, d,
J ¼ 6.0 Hz), 3.24 (1H, d, J ¼ 10.6 Hz), 3.58-3.81 (3H, m), 3.95-4.07 (1H, m),
5.07-5.23 (2H, m), 5.75-5.88 (1H, m), 5.93 (1H, d, J ¼ 8.4 Hz), 7.43-7.66
(4H, m), 7.89 (2H, d, J ¼ 7.2 Hz), 7.96 (1H, d, J ¼ 7.2 Hz), 9.01 (1H, br s.).
13C NMR (CDCl3, 100 MHz) d (ppm): 29.7, 53.0, 56.7, 61.1, 63.5, 77.3 (in
solvent peak), 81.4, 96.9 (br.), 118.9, 127.6 (2 C), 129.0 (2 C), 133.2, 133.8,
144.8 (br.), 154.4 (br.), 162.3 (br.). HRMS (ESI): m/z observed 371.1718,
þ
calculated for C19H23N4O4 371.1714.
N-(1-((2R,6S)-4-benzhydryl-6-(hydroxymethyl)morpholin-2-yl)-2-oxo-1,2-dihydro-
pyrimidin-4-yl)benzamide (3c)
It follows general procedure C.
White foam, yield 34% (0.36 g, 0.68 mmol). TLC Rf ¼ 1 (Ethyl acetate).
1H NMR (DMSO-d6, 400 MHz), d (ppm): 1.85- 1.95 (2H, m), 2.84 (1H, d,
J ¼ 11.2 Hz), 2.93 (1H, d, J ¼ 10.8 Hz), 3.35- 3.50 (2H, m), 3.82-3.92 (1H,
m), 4.44 (1H, s), 4.79 (1H, t, J ¼ 5.8 Hz), 5.83 (1H, d, J ¼ 7.7 Hz), 7.15-7.25
(2H, m), 7.27-7.37 (5H, m), 7.45 (4H, t, J ¼ 8.6 Hz), 7.49 (2H, t, J ¼ 7.9 Hz),
7.60 (1H, t, J ¼ 7.4 Hz), 7.98 (2H, d, J ¼ 7.5 Hz), 8.13 (1H, d, J ¼ 7.2 Hz),
11.25 (1H, s). 13C NMR (DMSO-d6, 100 MHz) d (ppm): 52.5, 54.9, 62.0,
74.5, 76.8, 80.6, 127.14, 127.17, 127.7 (4 C), 128.4 (6 C), 128.7 (4 C), 132.8,
141.7, 141.9, 145.5 (br. s), 168.6 (br. s), (2 signals lost because of broaden-
ing). HRMS (ESI): m/z observed 497.2185, calculated for
þ
C29H29N4O4 497.2183.
Benzyl 2-((2 R,6S)-2-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-6-(hydroxymethyl)-
morpholino)acetate (3d)
It follows general procedure D.
Colourless oil, yield 31% (0.30 g, 0.63 mmol). TLC Rf ¼ 0.9 (CH2Cl2/
CH3OH 10:1). 1H NMR (CDCl3, 400 MHz), d (ppm): 2.16 (1H, t,

18
M. D. NEKRASOV ET AL.
J ¼ 10.8 Hz), 2.46 (1H, t, J ¼ 10.4 Hz), 2.65 (1H, d, J ¼ 10.4 Hz), 2.72 (1H, d,
J ¼ 9.8 Hz), 3.11-3.21 (1H, m), 3.29 (2H, d, J ¼ 2.5 Hz), 3.40-3.48 (1H, m),
3.50-3.58 (1H, m), 3.77-3.87 (1H, m), 5.15 (2H, s), 5.48-5.56 (1H, m), 5.77
(1H, dd, J1 ¼ 10.0 Hz, J2 ¼ 2.0 Hz), 6.19 (1H, br s.), 7.29-7.46 (8H, m), 7.89
(2H, d, J ¼ 7.2 Hz), 8.38 (1H, d, J ¼ 7.5 Hz). 13C NMR (CDCl3, 100 MHz) d
(ppm): 52.5, 53.4, 57.1, 58.5, 63.2, 66.5, 79.7, 127.5 (2 C), 128.36 (2 C),
128.43 (3 C), 128.6 (2 C), 130.1, 131.8, 133.4, 135.4, 155.2, 167.3, 168.2,
169.7, 186.0. HRMS (ESI): m/z observed 479.2237, calculated for
þ
C25H27N4O6 479.1925.
N-(1-((2R,6S)-6-(((tert-Butyldiphenylsilyl)oxy)methyl)-4-(diallylamino)morpholin-
2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide (3e)
It follows general procedure C.
Colourless oil, yield 43.3% (0.39 g, 0.86 mmol). TLC Rf ¼ 0.3 (CH2Cl2/
1
CH3OH 20:1). H NMR (CDCl3, 400 MHz), d (ppm): 1.08 (9H, s), 2.28
(1H, t, J ¼ 9.7 Hz), 2.54 (1H, t, J ¼ 10.5 Hz), 3.03 (1H, d, J ¼ 10.5 Hz), 3.14-
3.26 (4H, m), 3.42 (1H, d, J ¼ 9.7 Hz), 3.71-3.84 (2H, m), 3.85-3.93 (1H,
m), 5.10 (2H, d, J ¼ 10.2 Hz), 5.17 (2H, d, J ¼ 17.1 Hz), 5.78-5.94 (3H, m),
7.35-7.56 (9H, m), 7.61 (1H, d, J ¼ 7.4 Hz), 7.67 (4H, t, J ¼ 7.6 Hz), 7.77
(1H, d, J ¼ 7.5 Hz), 7.92 (2H, d, J ¼ 7.6 Hz), 8.80 (1H, br s.). 13C NMR
(CDCl3, 100 MHz) d (ppm): 19.2, 26.7 (3 C), 49.8, 53.4, 53.6 (2 C), 64.6,
76.5, 81.4, 116.7 (2 C), 127.5 (2 C), 127.70 þ 127.72 (5 C), 129.0 (2 C), 129.8
(2 C), 133.0, 133.20, 133.24, 135.5 (5 C), 136.2 (2 C), 144.6 (br s.) (2 C),
162.0, (1 aromatic signal lost because of broadening). HRMS (ESI): m/z
observed 664.3320, calculated for C38H46N5O4Siþ 664.3314.
N-(9-((2R,6S)-4-Benzyl-6-(hydroxymethyl)morpholin-2-yl)-6-oxo-6,9-dihydro-1H-
purin-2-yl)isobutyramide (4a)
It follows general procedure A.
Greenish-white foam, yield 79% (0.68 g, 1.36 mmol). TLC Rf ¼0.6
1
(CH2Cl2/CH3OH 10:1). H NMR (DMSO-d6, 400 MHz), d (ppm): 1.10-1.12
(6H, m), 2.02 (1H, t, J ¼ 10.9 Hz), 2.62 (1H, t, J ¼ 10.5 Hz), 2.73-2.87 (4H,
m), 2.96 (1H, d, J ¼ 10.5 Hz), 3.61 (2H, d, J ¼ 4.06 Hz), 3.73-3.78 (1H, m),
4.78-4.83 (1H, m), 5.67 (1H, dd, J1 ¼ 10.2 Hz, J2 ¼ 2.2 Hz), 7.24-7.30 (1H,
m), 7.31-7.37 (5H, m), 8.16 (1H, s), 11.51 (1H, br s). 13C NMR (DMSO-d6,
100 MHz) d (ppm): 18.87, 18.91, 34.8, 45.8, 53.6, 61.6, 61.9, 77.1, 78.8,
127.3, 128.4 (2 C), 129.0 (2 C), 137.36, 137.43 (br s.), 148.1 (br s.), 148.3 (br
s.), 179.7, (2 aromatic signals lost because of broadening). HRMS (ESI): m/
þ
z observed 427.2083, calculated for C21H27N6O4 427.2088.

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
19
N-(9-((2R,6S)-4-allyl-6-(hydroxymethyl)morpholin-2-yl)-6-oxo-6,9-dihydro-1H-
purin-2-yl)isobutyramide (4 b)
It follows general procedure B.
White foam, yield 60% (6.78 g, 18.01 mmol). TLC Rf ¼ 0.3 (CH2Cl2/
1
CH3OH 10:1). H NMR (CDCl3, 400 MHz), d (ppm): 1.19-1.25 (6H, m),
2.09 (1H, t, J ¼ 11.0 Hz), 2.41 (1H, t, J ¼ 10.1 Hz), 2.79-2.92 (2H, m), 2.99-
3.14 (3H, m), 3.62-3.75 (2H, m), 3.94-4.03 (1H, m), 4.20-4.50 (1H, br s.),
5.14-5.25 (2H, m), 5.68 (1H, dd, J1 ¼ 10.0 Hz, J2 ¼ 1.9 Hz), 5.73-5.87 (1H,
m), 7.83 (1H, s), 10.40 (1H, br s.), 12.17 (1H, br s.). 13C NMR (CDCl3,
100 MHz) d (ppm): 18.9, 19.1, 19.7, 36.0, 53.0, 56.2, 61.0, 63.1, 79.4, 119.5
(br s.), 120.2, 133.2 (br s.), 137.0, 147.9, 148.0, 155.7, 179.8. HRMS (ESI):
þ
m/z observed 377.1937, calculated for C17H25N6O4 377.1932.
N-(9-((2R,6S)-4-Benzhydryl-6-(hydroxymethyl)morpholin-2-yl)-6-oxo-6,9-dihydro-
1H-purin-2-yl)isobutyramide (4c)
It follows general procedure C.
White foam, yield 58% (0.65 g, 1.17 mmol). TLC Rf ¼ 0.1 (CH2Cl2/
1
CH3OH 10:1). H NMR (DMSO-d6, 400 MHz), d (ppm): 1.12 (6H, d,
J ¼ 6.8 Hz), 1.93 (1H, t, J ¼ 10.6 Hz), 2.59 (1H, t, J ¼ 10.6 Hz), 2.75-2.91
(3H, m), 3.29-3.37 (1H, m) (partially overlaps with H2O), 3.38-3.47 (1H,
m), 3.78-3.87 (1H, m), 4.46 (1H, s), 4.80 (1H, t, J ¼ 5.8 Hz), 5.71-5.77 (1H,
m), 7.17 (1H, t, J ¼ 7.3 Hz), 7.23 (1H, t, J ¼ 7.3 Hz), 7.29 (2H, t, J ¼ 7.6 Hz),
7.34 (2H, t, J ¼ 7.6 Hz), 7.44 (2H, d, J ¼ 7.2 Hz), 7.48 (2H, d, J ¼ 7.2 Hz),
8.14 (1H, s), 11.70 (1H, s), 12.08 (1H, s). 13C NMR (DMSO-d6, 100 MHz)
d (ppm): 18.87, 18.92, 34.8, 52.7, 54.5, 61.9, 74.7, 76.9, 78.6, 119.7, 127.2
(2 C), 127.6 (2 C), 127.7 (2 C), 128.8 (4 C), 137.4, 141.88, 141.92, 148.1,
148.4, 154.8, 180.2. HRMS (ESI): m/z observed 503.2403, calculated for
þ
C27H31N6O4 503.2401.
Benzyl 2-((2S,6R)-2-(hydroxymethyl)-6-(2-isobutyramido-6-oxo-1H-purin-9(6H)-
yl)morpholino)acetate (4d)
It follows general procedure D.
White foam, yield 31% (0.30 g, 0.62 mmol). TLC Rf ¼ 0.8 (CH2Cl2/
CH3OH 10:1). 1H NMR (CDCl3, 400 MHz), d (ppm): 1.22 (3H, d,
J ¼ 6.8 Hz), 1.26 (3H, d, J ¼ 6.8 Hz), 2.40 (1H, t, J ¼ 10.9 Hz), 2.71 (1H, t,
J ¼ 10.3 Hz), 2.76-2.88 (2H, m), 2.98 (1H, d, J ¼ 9.8 Hz), 3.37 (2H, d,
J ¼ 3.8 Hz), 3.59-3.73 (2H, m), 3.82-3.98 (2H, m), 5.14 (2H, s), 5.60 (1H,
dd, J1 ¼ 9.7 Hz, J2 ¼ 2.1 Hz), 7.29-7.42 (5H, m), 7.79 (1H, s), 10.00 (1H, br
s.), 12.16 (1H, br s.). 13C NMR (CDCl3, 100 MHz) d (ppm): 18.8, 19.1,
36.1, 52.4, 55.7, 58.1, 63.0, 66.6, 77.2, 79.3, 120.3, 128.4 (2 C), 128.5, 128.6

20
M. D. NEKRASOV ET AL.
(2 C), 135.3, 136.9, 147.9, 148.0, 155.8, 170þ.0, 179.6. HRMS (ESI): m/z
observed 485.2144, calculated for C23H29N6O6 485.2143.
N-(9-((2R,6S)-4-(diallylamino)-6-(hydroxymethyl)morpholin-2-yl)-6-oxo-6,9-dihy-
dro-1H-purin-2-yl)isobutyramide (4e)
It follows general procedure C.
Colourless oil, yield 25.5% (0.24 g, 0.55 mmol). TLC Rf ¼ 0.4 (CH2Cl2/
CH3OH 10:1). 1H NMR (CDCl3, 400 MHz), d (ppm): 1.24 (6H, d,
J ¼ 5.7 Hz), 2.72-2.97 (3H, m), 3.10-3.30 (7H, m), 3.65-3.82 (2H, m), 3.91
(1H, br s.), 5.06 (2H, d, J ¼ 10.3 Hz), 5.14 (2H, d, J ¼ 17.2 Hz), 5.57 (1H, d,
J ¼ 8.6 Hz), 5.75-5.90 (2H, m), 7.91 (1H, s), 9.99 (1H, br s.), 12.14 (1H, br
s.). 13C NMR (CDCl3, 100 MHz) d (ppm): 18.9, 19.1, 36.0, 46.4, 48.4, 53.2
(2 C), 63.1, 76.8, 79.7, 116.8 (2 C), 119.9, 135.9 (2 C), 137.4, 147.8, 147.9,
155.8, 179.8. HRMS (ESI): m/z observed 432.2359, calculated for
þ
C20H30N7O4 432.2354.
Conclusions
We have developed a practical synthetic procedure that can be used to syn-
thesise N-substituted morpholine nucleosides containing allyl and benzyl
groups. The developed methodology allows the efficient production of N-
protected morpholine derivatives for future protecting group manipulation.
This method is suitable for the introduction of various protecting groups at
the nitrogen atom that are not sterically hindered (for example, the trityl
group is not suitable). The introduction of an electron-withdrawing car-
bonyl group led to a decrease in yield of over 30%.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Evgeny R. Lukyanenko
Alexander V. Kurkin
http://orcid.org/0000-0003-2197-4384
http://orcid.org/0000-0002-8467-9721
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