Synthesis of 2'-aminometylmorpholino nucleoside analogues containing the 4'-carboxymethyl linker group

Article abstract of DOI:10.1134/S1068162011060070

A simple and efficient method has been developed for the preparation of 2'-aminomethylmorpholino-4'-carboxymethyl nucleoside analogues and their 2'-N-Boc-modified derivatives as synthons for obtaining oligomers by peptide synthesis methods. Pleiades Publishing, Ltd., 2011.

Full text of DOI:10.1134/S1068162011060070

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 6, pp. 752–757. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © M.F. Kasakin, T.V. Abramova, V.N. Silnikov, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 6, pp. 830–835.
Synthesis of 2'ꢀAminometylmorpholino Nucleoside Analogues
Containing the 4'ꢀCarboxymethyl Linker Group
M. F. Kasakin1, T. V. Abramova, and V. N. Silnikov
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences,
pr. Lavrent’eva 8, 630090 Novosibirsk, Russia
Received: March 25, 2011; in final form, May 30, 2011
Abstract—A simple and efficient method has been developed for the preparation of 2'ꢀaminomethylmorꢀ
pholinoꢀ4'ꢀcarboxymethyl nucleoside analogues and their 2'ꢀNꢀBocꢀmodified derivatives as synthons for
obtaining oligomers by peptide synthesis methods.
Keywords: nucleosides, 2'ꢀaminomethylmorpholino analogues, synthesis, application
DOI: 10.1134/S1068162011060070
1
INTRODUCTION
compounds contain a chiral phosphorus atom and are
a racemic mixture; in addition, their synthesis based
on the reactions of the phosphate groups is inefficient
[4]. To overcome these disadvantages, attempts are
being made to create oligomers that do not contain
phosphorus atoms and chiral centers in the linker
groups between monomer units [10].
The development of methods for the synthesis of
nucleic acids, particularly synthetic oligonucleotides
and their analogues, has had a tremendous impact on
molecular biology. At the same time, improved methꢀ
ods of organic synthesis have made it possible to
obtain nucleic acid analogues with the desired propꢀ
erties. It was possible, in particular, to approach the
design of nucleic acidꢀbased therapeutic agents by
obtaining oligonucleotide methylphosphonate and
phosphorothioate analogues resistant to biodegradaꢀ
tion [1, 2].
The authors of[11] describe the use of 2'ꢀaminomeꢀ
thylꢀ4'ꢀcarboxymethylmorpholine analogues of adeꢀ
nosine and uridine as synthons for the synthesis of modꢀ
ified nucleic acids. 2'ꢀAminomethylmorpholine anaꢀ
logues of nucleosides (Urd, Cyt, Ado, and Guo) were
obtained, and the synthesis of a hexamer containing
oxalate linkers based on the morpholine analogue of
uridine morU6 was described [12]. In this report, to
develop approaches to solidꢀphase synthesis of morꢀ
pholine oligonucleotide analogues containing no chiral
centers in the linker groups between monomers, we
To achieve a noticeable therapeutic effect, it is necꢀ
essary that oligonucleotide analogues possess a numꢀ
ber of properties, namely, resistance to nucleases, high
specificity and strength of binding to complementary
nucleic acids, the ability to penetrate cell membranes,
low toxicity, etc. A wide range of nucleic acid anaꢀ
logues has been created in the last decade [3]. Morꢀ
pholine analogues of oligonucleotides are among the
most promising compounds for creating drugs. Incorꢀ
porating the morpholine ring in place of sugar residue,
these analogues have a high affinity and specificity to
native DNA and RNA, are highly soluble in water, and
easily penetrate the lipid cell membrane [4–6]. They
are the basis for antibacterial [7] and antiviral [8] drugs
and preparations for the correction of genetic disorꢀ
ders [9].
describe the synthesis of 2'ꢀ
NꢀBocꢀprotected 2'ꢀamiꢀ
nomethylꢀ4'ꢀcarboxymethylmorfoline analogues of
uridine, adenosine, cytidine, and guanosine.
RESULTS AND DISCUSSION
Synthesis of Bocꢀprotected 2'ꢀaminomethylmorꢀ
pholine analogues of uridine, adenosine, cytidine, and
guanosine containing the 4'ꢀcarboxymethyl group was
carried out starting from the corresponding ribonucleꢀ
osides containing protected heterocyclic bases
(scheme). The proposed scheme differs methodologiꢀ
cally from that described by Chakhmakhcheva [11]; it
contains three steps fewer (in particular, there is no
stage of dimethoxytritylation of the 5'ꢀhydroxyl group
in the initial nucleosides).
The most commonly used and commercially availꢀ
able phosphoroamidate morpholine analogues of oliꢀ
gonucleotides (Gene Tools, United States) are not
without certain shortcomings. In particular, these
Abbreviations: (Boc) O, diꢀtertꢀbutyl carbonate; PriꢀOH, isoꢀ
2
propanol; Ibu, isobutiryl; TEA, triethylamine.
Corresponding author: phone: +7 (383) 363ꢀ51ꢀ83; fax:
+7 (383) 363ꢀ51ꢀ82; eꢀmail: Kassakinm@gmail.com.
1
752

SYNTHESIS OF 2'ꢀAMINOMETYLMORPHOLINO NUCLEOSIDE ANALOGUES
753
O
O
O
N
B*/Ura
B*/Ura
B*/Ura
N3
N3
HO
1
2
HO
OH
HO
OH
O
[
Iа–d
]
[
IIа–d]
OH
IIIа–d
3
1O'
B*/Ura
O
[
]
O
B*/Ura
O
HN
O
H2N
6'
5'
2'
3'
4
O
N
N
4'
OH
OH
IVа–d
[Vа–d
]
[
]
Scheme of synthesis of morpholine nucleoside analogues containing the 4'ꢀcarboxymethyl linker. Derivatives of uracyl (a),
6
4
2
N
ꢀbenzoyladenine (b),
N ꢀbenzoylcytosine (c), and N ꢀisobutirylguanine (d). Asterisk (*) designates derivatives with proꢀ
tected heterocyclic bases. 1, NaN , PPh , CBrCl /DMF; 2, NaIO /EtOH + H O, Gly/EtOH, NaBH CN; 3, PPh /pyridine,
3
3
3
4
2
3
3
NH (conc) for compound (IIIa), PPh /pyridine, 1 M NaOH for compounds (IIIc) and (IIId), or Pd/C + H for compound
3
3
2
(
IIIb); 4, (Boc) O, 1 M NaOH/PriOH.
2
The substitution of the hydroxyl group by the azido to avoid repetition of this stage during oligomer synꢀ
group at the 5'ꢀposition of the ribose ring was perꢀ thesis. The yields of compounds (IIIa)–(IIId) after
formed in one stage by the method proposed in [13] chromatographic separation of the reaction mixtures
for 2'ꢀdeoxynucleosides with minor modifications. were 22, 71, 38, and 44%, respectively, relative to the
The content of the target products in the reaction mixꢀ initial nucleosides (Ia)–(Id).
tures in the case of rA*, rG*, and rC* (an asterisk desꢀ
ignates modified heterocyclic bases; see scheme) was
80–85% according to HPLC data. In the case of the
uridine derivative, the yield did not exceed 50%; there
was, however, no need to optimize the reaction condiꢀ
tions due to the low cost of the initial nucleoside. After
chromatographic separation of the reaction mixtures
on silica gel, azides (IIa)–(IIc) were obtained in a
yield of 46, 74, and 78%, respectively. Despite the high
content of the azidoderivative of rG* in the reaction
mixture, it could not be isolated as an individual comꢀ
pound. Therefore, compound (IIc) containing an
impurity of unknown structure (~7%) was used for
further reactions without additional purification.
Two approaches are used to reduce the azido group
in nucleosides: catalytic hydrogenation [16, 17] or
reaction with triphenylphosphine followed by hydrolꢀ
ysis of the intermediate compound [18]. Both methꢀ
ods allow target compounds to be obtained with a simꢀ
ilar yield, and the choice of methods is mainly deterꢀ
mined by the properties of the initial and final
compounds (solubility, stability under the synthesis
conditions, etc.).
The azido group in compound (IIIa) was reduced
by the method described in [18] using hydrolysis of the
intermediate product with concentrated aqueous
ammonia. However, this method was not applicable to
compounds (IIIc) and (IIId) due to the significant
deprotection of heterocyclic bases during synthesis. In
this case, hydrolysis of the intermediate phosphonium
compounds after reaction with triphenylphosphine
was carried out by treatment with 1 M aqueous NaOH.
The reaction occurred at room temperature for 12 h,
with the protection groups of the heterocyclic bases
being unaffected.
The IR spectra of resultant compounds (IIa)–(IId
)
have a characteristic intensive absorption band of the
azido group in the range of 2100 cm–1. Their NMR
spectra contain no proton signal of the 5'ꢀOH group.
The ribose ring is transformed into the morpholine
fragment in three steps without the isolation of interꢀ
mediate products: oxidation of the 2',3'ꢀdiol group of
ribose to dialdehyde, condensation with an amino
The presence of amino and carboxyl groups in the
component, and subsequent reduction [14]. Morphoꢀ molecules significantly impeded the isolation of comꢀ
line derivatives containing the 4'ꢀcarboxyl linker group pounds (IVa)–(IVd) in individual forms. After prelimꢀ
(
III) were synthesized by methods similar to the ones inary purification by ionꢀexchange chromatography,
proposed in [12, 14, 15]. Unlike previous methods these compounds (80–95% of the main product
[12] for the preparation of morpholine derivatives, we according to HPLC data) were used at the last stage
used glycine instead of ammonium tetraborate to close without additional purification. The analytical samꢀ
the morpholine cycle and introduce the 4'ꢀcarboxymꢀ ples were obtained using reverseꢀphase chromatograꢀ
ethyl linker group. The introduction of the linker phy. The IR spectra of the resultant compounds show
group at the monomer synthesis stage made it possible the absence of the band in the region of 2100 cm–1,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
No. 6
2011

754
KASAKIN et al.
which is typical of the azido group. 1H NMR spectrosꢀ Aꢀ25 (Pharmacia, Sweden), Servacel Pꢀ23 (Serva,
copy and electrospray mass spectrometry data were as Germany). Thinꢀlayer chromatography was perꢀ
expected.
The use of catalytic hydrogenation leads to the
desired compound (IVb) with a comparable yield.
The synthesis of 2'ꢀ ꢀBocꢀprotected comꢀ
pounds (Va )–(Vd) was carried out in a water–organic
medium by the method described for amino acids
[19]. The target compounds (Va )–(Vd) were isolated
by reverseꢀphase chromatography with yields of 8.3,
22.0, 8.6, and 13.7%, respectively, relative to initial
compounds (Ia)–(Id). The structure of the obtained
compounds was confirmed by 1H NMR spectroscopy
formed on silica gel plates (Alufolien Kieselgel 60 F254,
Merck, Germany).
5'ꢀAzidouridine (IIa),
N
6ꢀbenzoylꢀ5'ꢀazidoadenosꢀ
N N
4ꢀbenzoylꢀ5'ꢀazidocytidine (IIc), and 2ꢀ
ine (IIb),
N
isobutirylꢀ5'ꢀazidoguanosine (IId). Triphenylphosꢀ
phine (5.76 g, 22 mmol), sodium azide (6.50 g,
100 mmol), and bromotrichloromethane (2.2 ml,
22 mmol) were added to the solution of uridine (Ia
)
(2.44 g, 10 mmol) in DMF (70 ml). The reaction mixꢀ
ture was stirred for 12 h followed by its evaporation.
N6ꢀbenzoyladenosine (Ib) (3.33 g, 8.9 mmol),
N4ꢀbenzoylcytidine (Ic) (3.44 g, 9.9 mmol), and
N2ꢀisobutirylguanosine (Id) (1.32 g, 3.7 mmol) were
and electrospray mass spectrometry; compounds (Vc
)
and (Vd) were additionally characterized by elemental
used for the synthesis of compounds (IIb–IId),
analysis.
respectively. The ratios of the other compounds and
the solvent were the same as in the synthesis of comꢀ
pound (IIa).
Thus, protected 2'ꢀ
thylmorpholine analogues of nucleosides (IVa)–(IVd
NꢀBocꢀaminoꢀ4'ꢀNꢀcarboxymeꢀ
)
were prepared. These can be used as monomer blocks
for the synthesis of morpholine analogues of oligonuꢀ
cleotides by the Bocꢀstrategy of peptide chemistry.
The synthesis and properties of oligonucleotide anaꢀ
logues based on the use of these nucleoside derivatives
will be presented separately.
In the case of compound (IIa), the residue after
evaporation was dissolved in methylene chloride
(150 ml) followed by extraction with water (2 × 200 ml).
After evaporation of the aqueous solution, ethanol
(200 ml) was added to the oil and the precipitate was
filtered. The residual solution of the nonpurified prodꢀ
uct was evaporated, and suspension of silica gel in
methylene chloride was added to the residue. The
resultant suspension was applied to the top of the colꢀ
EXPERIMENTAL
We used triphenylphosphine (Chemapol, Czechoꢀ
slovakia), bromotrichloromethane and sodium perioꢀ
date (AcRos Organics, United States), sodium azide
and glycine (Serva, Germany), sodium cyanoborohyꢀ
dride (Aldrich, United States), uridine, N6ꢀBzꢀadeꢀ
umn with silica gel (3.7 × 12 cm).
In the case of compounds (IIb and IIc), methylene
chloride (150 ml) was added to the residue followed by
extraction with water (2 × 200 ml). The organic soluꢀ
tion was evaporated with a suspension of silica gel to a
minimal volume and applied to a chromatography
column.
nosine, N4ꢀBzꢀcytidine,
N iBuꢀguanosine (Chem
2ꢀ
Genes Corporation, United States). Organic solvents
and other reactants of Russian origin were qualified as
chemically clean and pure for analysis. Solvents were
purified by standard methods.
Analyses of the reaction mixtures and their fracꢀ
tions were carried out on a Milichromꢀ4 chromatoꢀ
In the case of compound (IId), ethanol (200 ml) was
added to the residue and the precipitate was filtered.
After evaporation of the resultant solution, the residue
was mixed with a silica gel suspension and applied to a
graph (Econova, Russia) on a column (
with Nucleosil Cꢀ18 (5 m, MachereyꢀNagel, Gerꢀ
many). 1H and 13C NMR spectra (
, ppm; SSIC, Hz)
2 × 60 mm)
silica gel chromatography column (3.7
× 12 cm). The
µ
product was eluted in a gradient of ethanol in methylene
chloride (0–30%). Fractions with the desired product
were evaporated and dried in vacuo.
δ
were recorded in DMSOꢀd6 on a Bruker AV 300/400
spectrometer (Bruker, Germany) at 300 MHz; mass
spectra were recorded on a ESI MSD XCT Ion Trap
spectrometer (Agilent Technologies, United States);
IR spectra were recorded on a Vector 22 spectrometer
(Bruker, Germany). A UVICORD SD 2158 detector
(280 nm, LKB, Sweden) was used during the column
chromatography. Compounds were centrifuged on an
Eppendorf 5417 microcentrifuge (13 000 rpm, rotor
170 mm, Eppendorf, Germany) and on a Sigma 204
centrifuge (3700 rpm, rotor 250 mm, Sigma, United
States).
Compound (IIa). Yield: 1.24 g, 46%. Rf 0.70
(EtOH–CH2Cl2, 1 : 4). IR (KBr), cm–1: 2103 (
N
≡
N
).
8.0, H6),
8.0, H5), 5.48
J 4.3, OH2'), 4.14
1H NMR 11.37 (1 H, s, NH), 7.69 (1 H, d,
5.77 (1 H, d, 5.2, H1'), 5.67 (1 H, d,
(1 H, d, 6.0, OH3'), 5.29 (1 H, d,
J
J
J
J
(1 H, m, H4'), 3.92 (2 H, m, H2', H3'), 3.60 (2 H, m,
H5', H5'').
Compound (IIb). Yield: 2.62 g, 74%. Rf 0.42
(EtOH–CH2Cl2, 1 : 9). IR (KBr), cm–1: 2103.8
1
(N
≡
N
). H NMR 11.22 (1 H, bs, NH), 8.78 (1 H, s,
Column chromatography was carried out with the H8), 8.72 (1 H, s, H2), 8.08 (2 H, bd,
use of the following sorbents: Preparative Porasil Cꢀ18 Bz), 7.65 (1 H, bt, 7.3, H4, Bz), 7.55 (2 H, apt,
(55–105 m, 125A) (Waters, United States), Kieselgel H3, H5, Bz), 6.07 (1 H, d, 5.4, H1'), 5.67 (1 H, d,
60 (30–70 m, Merck, Germany), DEAEꢀSephadex 6.0, OH3'), 5.45 (1 H, d, 5.2, OH2'), 4.82 (1 H, m,
J
8.0, H2, H6,
J
J
7.5
,
µ
J
µ
J
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
No. 6
2011

SYNTHESIS OF 2'ꢀAMINOMETYLMORPHOLINO NUCLEOSIDE ANALOGUES
755
H2'), 4.24 (1 H, m, H3'), 4.10 (1 H, m, H4'), 3.54– Bz), 7.52 (2 H, apt, J 7.6, H3, H5, Bz), 6.52 (1 H, bd,
3.78 (2 H, m, H5', H5'').
J
⎯
10.0, H6'), 4.58 (1 H, m, H2'), 4.23 (2 H, s,
CH2COOH), 3.38–4.19 (6 H, m, H5', H3',
Compound (IIc). Yield: 2.86 g, 78%. Rf 0.51
(EtOH–CH2Cl2, 1 : 9). IR (KBr), cm–1: 2103.5
N3CH2–).
Compound (IIIc). Yield: 1.21 g, 49%. Rf 0.46
(PriOH–H2O, 4 : 1). IR (KBr), cm–1: 2103.9 (
).
1H NMR (400 MHz) 11.28 (1 H, bs, NH), 8.08 (1 H,
d, 7.5, H6), 8.00 (2 H, bd, 7.7, H2, H6, Bz), 7.60
(1 H, bt, 7.2, H4, Bz), 7.49 (2 H, apt, 7.7, H3, H5,
Bz), 7.35 (1 H, d, 7.5, H5), 5.76 (1 H, dd, 9.6, 2.3,
H6'), 3.99 (1 H, m, H2'), 3.18–3.53 (5 H, m, H3',
(N≡N
). 1H NMR (400 MHz) 11.26 (1 H, s, NH), 8.16
(1 H, d,
7.60 (1 H, bt,
H5, Bz), 7.37 (1 H, d,
H1'), 5.58 (1 H, d, 5.2, OH2'), 5.27 (1 H, d,
J
7.4, H6), 7.98 (2 H, bd,
7.1, H4, Bz), 7.49 (2 H, apt,
7.4, H5), 5.82 (1 H, d,
J
7.4, H2, H6, Bz),
7.7, H3,
3.5
6.0,
N≡N
J
J
J
J
J
,
J
J
J
J
J
OH3'), 4.10 (1 H, aptdd,
H4'), 3.89 (1 H, aptdd,
(2 H, m, H5', H5'').
J
J
9.0, 5.0, H3'), 3.99 (1 H, m,
11.4, 5.8, H2'), 3.54–3.73
J
J
N3CH2–, –CH2COOH), 3.05 (1 H, bd,
2.81 (1 H, bd, 10.4, H3'), 2.34 (1 H, apt,
J
11.4, H5'),
J
J 10.5, H5').
Compound (IId).
(KBr), cm–1: 2102.7 (
(1 H, s, H8), 5.83 (1 H, d,
5.2, H2'), 4.26 (1 H, apt,
4.5, H4'), 3.49–3.55 (2 H, m, H5', H5''), 2.62 (1 H,
spt, 6.9, –CH<), 1.05 (6 H, d, 6.9, –CH3).
Rf 0.49 (EtOH–CH2Cl2, 1 : 4). IR
1
N
≡
N
J
). H NMR (D2O) 8.06
5.2, H1'), 4.69 (1 H, apt,
J 4.9, H3'), 4.08 (1 H, apq,
Compound (IIId). Yield: 0.59 g, 44% after two
steps. Rf 0.55 (PriOH–H2O, 4 : 1). IR (KBr), cm–1:
2105.9 (
(1 H, dd,
(1 H, bd,
3.44–3.76 (4 H, m, H3', N3CH2–), 3.18 (1 H, apt,
J
J
J
N
≡
J
J
N
). 1H NMR (D2O) 8.18 (1 H, s, H8), 6.18
10.1, 2.0, H6'), 4.44 (1 H, m, H2'), 3.90
11.7, H5'), 3.81 (2 H, s, –CH2COOH),
J
J
J
2'ꢀAzidomethylꢀ4'ꢀcarboxymethylꢀ6'ꢀ(uracylꢀ1ꢀyl)
morpholine (IIIa); 2'ꢀazidomethylꢀ4'ꢀcarboxymethylꢀ
11.7, H5'), 2.76 (1 H, spt,
6.9, –CH3).
J 6.9, ꢀCH<), 1.21 (6 H, d,
6'ꢀ(
domethylꢀ4'ꢀcarboxymethylꢀ6'ꢀ(
morpholine (IIIc); 2'ꢀazidomethylꢀ4'ꢀcarboxymethylꢀ
6'ꢀ(
2ꢀisobutirylguanineꢀ9ꢀyl)morpholine (IIId). Ten
N
6ꢀbenzoyladenineꢀ9ꢀyl)morpholine (IIIb); 2'ꢀaziꢀ
N
4ꢀbenzoylcytosineꢀ1ꢀyl)
2'ꢀAminomethylꢀ4'ꢀcarboxymethylꢀ6'ꢀ(uracylꢀ1ꢀyl)
morpholine (IVa); 2'ꢀaminomethylꢀ4'ꢀcarboxymethylꢀ
6'ꢀ(
4ꢀbenzoylcytosineꢀ1ꢀyl)morpholine (IVc); 2'ꢀ
aminomethylꢀ4'ꢀcarboxymethylꢀ6'ꢀ(
2ꢀisobutirylguaꢀ
nineꢀ9ꢀyl)morpholine (IVd). Triphenylphosphine
(0.59 g, 2.2 mmol) was added to the solution of comꢀ
pound (IIIa) (0.69 g, 2.2 mmol) in a small volume of
pyridine (3–5 ml), and the reaction mixture was left
stirring overnight. Compounds (IVc) and (IVd) were
synthesized using compound (IIIc) (1.15 g, 2.8 mmol)
and (IIId) (0.58 g, 1.4 mmol), respectively. The other
reagents and solvents were taken in the same ratio as in
the case of compound (IVa).
N
N
milliliters of an aqueous solution of sodium periodate
(1.07 g, 5 mmol) was added under stirring to comꢀ
pound (IIa) (1,23 g, 4.6 mmol) in 80 ml of ethanol.
After the 20ꢀmin stirring, 30 ml of the aqueous glycine
solution (0.38 g, 5 mmol), which was preꢀadjusted by
TEA (0.6 ml) to pH 9–9.5, was added to the reaction
mixture. The reaction mixture was stirred for 1 h. The
precipitate was removed by filtration. Sodium
cyanoborohydride (0.41 g, 6.5 mmol) was added to the
solution and the mixture was stirred for 2.5 h. The
solution was acidified by conc. HCl to pH 4, stirred for
12 h, and evaporated; the residue was dissolved in
10 ml of 0.1% aqueous TFA containing ethanol. The
product was isolated by reverseꢀphase chromatograꢀ
N
In the case of compound (IVa), conc. NH3 (27%,
3 ml) was added in 12 h to the reaction mixture and left
under stirring for 24 h. In the case of compounds (IVc
)
and (IVd), the reaction mixture was subjected to a twoꢀ
fold dilution with 1 M NaOH and left under stirring
for 24 h.
phy on a column (
ent (0–30%) containing 0.1% TFA.
Compounds (IIIb IIId) were synthesized using
3 × 18 cm) in a waterꢀethanol gradiꢀ
–
compounds (IIb) (1.59 g, 4.0 mmol), (IIc) (2.15 g,
5.8 mmol) and the residue after the synthesis of comꢀ
pound (IId) (~3 mmol), respectively. The other
reagents and solvents were taken in the same ratio as in
the case of compound (IIIa).
The reaction mixture was evaporated several times
with water followed by the addition of water (150 ml)
to the residue, the resultant solution was filtered to
remove the precipitate, and the filtrate was applied to
a column with Servacel Pꢀ23 (2 × 16 cm) in the
Н⊕ form. The product was eluted in a linear gradient
of NH4HCO3 (0–0.2 M) in 20% aqueous ethanol (the
total volume was 1000 ml).
Compound (IIIa). Yield: 0.69 g, 48%. Rf 0.14
(EtOH–CH2Cl2, 1 : 4). IR (KBr), cm–1: 2113.5
1
(
N≡
N
). H NMR 11.47 (1 H, s, NH), 7.60 (1 H, d,
J
8.0, H6), 5.80 (1 H, dd,
dd, 8.0, 1.9, H5), 4.07 (1 H, m, H2'), 3.54 (2 H, s,
CH2COOH), 3.00–3.62 (4 H, m, H5', H3', N3CH2–),
J 10.0, 2.3, H6'), 5.69 (1 H,
Compound (IVa). Yield: 0.27 g, 42.6%. Rf 0.30
(PriOH–H2O–NH3, 7 : 2 : 1). 13C NMR (100.6 MHz,
D2O) 41.72, 53.38, 54.92, 61.44, 73.05, 80.36, 103.16,
142.86, 152.00, 166.86, 177.48. 1H NMR (400 MHz,
J
⎯
2.73 (1 H, dd,
16.0, H5').
J 10.8, 9.0, H3'), 2.55 (1 H, dd, J 11.4,
D2O) 7.77 (1 H, d, J 8.1, H6), 5.83 (2 H, m, H6', H5),
Compound (IIIb). Yield: 1.68 g, 96%. Rf 0.53 4.16 (1 H, m, H2'), 2.93–3.29 (6 H, m, H5',
(PriOH–H2O, 4 : 1). IR (KBr), cm–1: 2114 ( ). 1H H3',
CH2COOH, NH2CH2–), 2.44 (1 H, apt, 10.8
11.2, H5'). Mass spectrum,
N
≡
N
⎯
J
,
NMR (D2O) 8.87 (1 H, s, H8), 8.77 (1 H, s, H2), 7.95 H3'), 2.22 (1 H, apt,
J
(2 H, bd,
J
7.8, H2, H6, Bz), 7.66 (1 H, bt,
J
7.4, H4, m/z 285.0 [
M
+ H]+
.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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2011

756
KASAKIN et al.
Compound (IVc). Yield: 0.87 g, 81%, Rf 0.60 ~2.7 mmol), and compound (IVd) (0.39 g, 1 mmol),
1
(P
D2O) 8.12 (1 H, d,
H6, Bz), 7.58 (1 H, bt,
7.7, H3, H5, Bz), 7.38 (1 H, d,
bd,
H3', H5', NH2CH2–, –CH2COOH), 2.45 (1 H, apt,
10.7, H3'), 2.36 (1 H, apt, 11.4, H5'). Mass specꢀ
trum, m/z 388.0 [
+ H]+
r
iOH–H2O–NH3, 7 : 2 : 1). H NMR (400 MHz, respectively. The other reagents and solvents were taken
J
7.7, H6), 7.78 (2 H, bd,
J
7.7, H2, in the same ratio as in the case of compound (Va ).
J
7.4, H4, Bz), 7.44 (2 H, apt,
Compound (Va ). Yield: 0.12 g, 88%. Rf 0.5
1
(PriOH–H2O, 4 : 1). H NMR (D2O) 7.71 (1 H, d,
J
J
7.7, H5), 5.87 (1 H,
J
9.7, H6'), 4.21 (1 H, m, H2'), 3.02–3.30 (6 H, m,
J
J
⎯
J
J
8.1, H6), 5.78 (1 H, d, J 8.1, H5), 5.71 (1 H, dd,
10.0, 2.2, H6'), 3.87 (1 H, m, H2'), 3.20 (2 H, bs,
NHCH2–), 3.05 (2 H, s, –CH2COOH), 2.98 (1 H, d,
J
J
M
.
10.8, H3'), 2.83 (1 H, d,
10.8, H3'), 2.12 (1 H, apt,
J
11.2, H5'), 2.31 (1 H, apt,
11.2, H5'), 1.97 (9 H, s,
– H]–, 407.2
J
Compound (IVd). Yield: 0.41 g, 74% Rf 0.61
1
(CH3)3С–). Mass spectrum, m/z 383.3 [
+ Na]+
Compound (Vb). Yield: 0.32 g, 59%. Rf 0.5
(PriOH–H2O, 4 : 1). 1H NMR 8.73 (1 H, s, H8), 8.64
(1 H, s, H2), 8.01 (2 H, bd, 7.6, H2, H6, Bz), 7.45–
7.65 (3 H, m, H3, H4, H5, Bz), 6.96 (1 H, t, 5.8 Bocꢀ
NH–), 5.88 (1 H, dd, 9.6, 2.1, H6'), 3.82 (1 H, m,
H2'), 2.96–3.32 (6 H, m, –NHCH2–, CH2COOH),
H3'), 2.87 (1 H, bd, 11.0, H5'), 2.23 (1 H, apt, 11.0
H5'), 1.33 (9 H, s, (CH3)3C–). Mass spectrum,
m/z 510.2 [
– H]–
M
(PriOH–H2O–NH3, 7 : 2 : 1). H NMR (400 MHz,
D2O) 8.00 (1 H, s, H8), 5.78 (1 H, dd, 10.2, 2.1,
H6'), 4.07 (1 H, m, H2'), 2.86–3.14 (6 H, m, H3', H5',
NH2CH2–, –CH2COOH), 2.79 (1 H, apt, 10.7
H3'), 2.60 (1 H, spt, 6.8 CH<), 2.17 (1 H, apt,
11.2, H5'), 1.09 (6 H, d, 6.8, –CH3). Mass specꢀ
trum, m/z 394.2 [
+ H]+, 416.1 [
[M
.
J
J
,
J
J
, ⎯
J
,
J
J
+ Na]+
.
J
M
M
⎯
2'ꢀAminomethylꢀ4'ꢀcarboxymethylꢀ6'ꢀ(
N
6ꢀbenzoyꢀ
J
J
,
ladenineꢀ9ꢀyl)morpholine (IVb). A suspension of the
Pd/C catalyst (0.3 g, 4% Pd) in ethanol (10 ml) was
kept in an H2 atmosphere for 30 min and then added
to the ethanol solution (40 ml) containing compound
M
.
Compound (Vc). Yield: 0.29 g, 22.6%. Rf 0.62
(PriOH : H2O, 4 : 1). 13С NMR (100.6 MHz, D2O)
28.54, 42.58, 53.48, 55.40, 58.40, 78.23, 79.50, 80.96,
96.79, 128.80, 128.80, 133.14, 133.42, 145.91, 154.05,
156.12, 163.46, 167.87, 171.81. 1H NMR (D2O) 8.33
(
IIIb) (2.3 g, 5.3 mmol). The reaction mixture was left
under stirring for 2 days in H2 atmosphere. After the
removal of the catalyst by filtration, water (up to
300 ml) was added to the solution, which was then
(1 H, d,
7.84 (1 H, bt,
H5, Bz), 7.63 (1 H, d,
H6'), 4.28 (1 H, m, H2'), 3.73 (3 H, m, H3',
CH2COOH), 3.24–3.58 (3 H, m, H5', –NHCH2–),
2.80–3.04 (2 H, m, H3', H5'), 1.53 (9 H, s, (CH3)3С–).
Mass spectrum, m/z 486.4 [
– H]–. Exp., %:
J
7.5, H6), 8.06 (2 H, bd,
7.3, H4, Bz), 7.72 (2 H, apt,
7.5, H5), 6.15 (1 H, bd,
J
7.3, H2, H6, Bz),
7.7, H3,
9.4
applied on a column with Servacel Pꢀ23 (2 × 16 cm) in
J
J
the Н⊕ form. The product was eluted in a linear gradiꢀ
ent of NH4HCO3 (0–0.075 M) in 20% aqueous ethaꢀ
J
J
,
nol. Yield: 1.14 g, 52.6%.R ,
f 0.37 (PriOH–H2O–NH3
⎯
1
7 : 2 : 1). H NMR(400 MHz, (D2O) 8.56 (1 H, s,
H8), 8.45 (1 H, s, H2), 7.84 (2 H, bd, 7.7, H2, H6,
Bz), 7.58 (1 H, bt, 7.3, H4, Bz), 7.45 (2 H, apt, 7.5
H3, H5, Bz), 5.96 (1 H, bd, 10.1, H6'), 4.20 (1 H, m,
H2'), 2.89–3.28 (6 H, m, H5', H3', NH2CH2–,
CH2COOH), 2.84 (1 H, apt, 11.5, H3'), 2.24 (1 H,
apt, 11.7, H5'). Mass spectrum, m/z 412.0 [
+ H]+
J
M
J
J
,
C 55.54, H 5.97, N 14.36. Calc. for C23H29N5O7, %:
C 56.67, H 6.00, N 14.37.
Compound (Vd). Yield: 0.20 g, 42%. Rf 0.67
(PriOH–H2O, 4 : 1). 13С NMR (100.6 MHz, D2O)
20.44, 29.84, 37.35, 43.49, 54.68, 55.16, 60.47, 75.25,
79.70, 81.93, 121.22, 149.93, 150.44, 157.90, 158.99,
172.30. 1H NMR (400 MHz, D2O) 8.24 (1 H, s, H8),
J
⎯
J
J
M
.
2'ꢀ( tertꢀbutyloxycarbonylaminomethylꢀ4'ꢀcarꢀ
boxymethylꢀ6'ꢀ(uracylꢀ1ꢀyl)morpholine (Va); 2'ꢀ(
tertꢀbutyloxycarbonylaminomethylꢀ4'ꢀcarboxymethylꢀ
6'ꢀ(
6ꢀbenzoyladenineꢀ9ꢀyl)morpholine (Vb); 2'ꢀ(Nꢀ
tertꢀbutyloxycarbonylaminomethylꢀ4'ꢀcarboxymethylꢀ
6'ꢀ(
4ꢀbenzoylcytosineꢀ1ꢀyl)morpholine (Vc); 2'ꢀ(
tertꢀbutyloxycarbonylaminomethylꢀ4'ꢀcarboxymethylꢀ
6'ꢀ(
2ꢀisobutirylguanineꢀ9ꢀyl)morpholine (Vd). Diꢀ
Nꢀ
Nꢀ
6.02 (1 H, dd,
3.56–3.64 (3 H, m, H3', –CH2COOH), 3.23–3.39
(4 H, m, H3', H5', –NHCH2–), 2.79 (1 H, spt, 6.8
CH<), 2.71 (1 H, apt, 11.4, H5'), 1.40 (9 H, s,
(CH3)3С–), 1.23 (6 H, d, 6.8, –CH3). Mass specꢀ
trum, m/z 492.3 [
– H]–. Exp., %: C 50.60, H 6.16,
J 10.0, 2.0, H6'), 4.14 (1 H, m, H2'),
N
J
,
N
Nꢀ
⎯
J
J
N
M
tertꢀbutyl pyrocarbonate (0.2 ml) was added to the
solution of compound (IVa) (0.10 g, 0.4 mmol) in a
mixture of 1 M NaOH (0.4 ml) and PriOH (0.4 ml).
N 19.37. Calc. for C21H31N7O7, %: C 51.11, H 6.33,
N 19.87.
The mixture was heated to 60
room temperature, followed by extraction with diethyl
ether ( 0.8 ml). The water–alcohol solution was
evaporated, the residue was dissolved in water (5 ml),
and the solution was adjusted by citric acid to pH 3.
The product was isolated by reverseꢀphase chromatogꢀ
°С and stirred for 2 h at
ACKNOWLEDGMENTS
2
×
The work was supported by the Integration project
of the Siberian Branch of the Russian Academy of Sciꢀ
ences, no. 88.
raphy on a column (2 × 23 cm) in a gradient of water in
REFERENCES
ethanol (0–50%). Compounds (Vb)–(Vd) were synꢀ
thesized from compound (IVb) (0.55 g, 1.3 mmol), the
residue containing compound (IVc) (1.69 g,
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