Solid-phase-supported synthesis of morpholinoglycine oligonucleotide mimics

Article abstract of DOI:10.3762/bjoc.10.115

An efficient solid-phase-supported peptide synthesis (SPPS) of morpholinoglycine oligonucleotide (MorGly) mimics has been developed. The proposed strategy includes a novel specially designed labile linker group containing the oxalyl residue and the 2-aminomethylmorpholino nucleoside analogues as first subunits.

Full text of DOI:10.3762/bjoc.10.115

Solid-phase-supported synthesis of morpholino-
glycine oligonucleotide mimics
Tatyana V. Abramova*1, Sergey S. Belov1,2, Yulia V. Tarasenko1
and Vladimir N. Silnikov1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1151–1158.
1Institute of Chemical Biology and Fundamental Medicine, SB RAS,
Lavrent’ev Ave, 8, Novosibirsk 630090, Russia and 2Novosibirsk
State University, Pirogova St. 2, Novosibirsk 630090, Russia
doi:10.3762/bjoc.10.115
Received: 31 January 2014
Accepted: 23 April 2014
Published: 20 May 2014
Email:
Tatyana V. Abramova* - abramova@niboch.nsc.ru
Associate Editor: S. Flitsch
* Corresponding author
© 2014 Abramova et al; licensee Beilstein-Institut.
License and terms: see end of document.
Keywords:
labile linker; morpholino oligomers; oligonucleotide mimics;
solid-phase-supported peptide synthesis (SPPS)
Abstract
An efficient solid-phase-supported peptide synthesis (SPPS) of morpholinoglycine oligonucleotide (MorGly) mimics has been
developed. The proposed strategy includes a novel specially designed labile linker group containing the oxalyl residue and the
2-aminomethylmorpholino nucleoside analogues as first subunits.
Introduction
The phosphorodiamidate morpholino oligomers (PMO) and methylene caxboxamide [10] groups represent the individual
peptide conjugated PMO (PPMO) are currently promising compounds. It means that their physicochemical properties,
candidates for antisense therapy of a number of infectious and such as thermostability of complementary complexes, do not
hereditary diseases [1-4] despite of some difficulties and limita- depend on the diastereomeric composition of the mixtures,
tions. They also proved to be valuable tools to study funda- which can vary in different preparations. The difference in the
mental problems of gene expression in the course of embryoge- spatial structure, in the extent of base stacking, and in the
nesis [5,6]. A few examples of morpholino oligomers stability of complementary duplexes for individual diastereo-
containing other types of internucleoside bonds were described. mers has been demonstrated for phosphotriester [11] and
An attractive feature of these new morpholino oligonucleotide methylphosphonate [12] oligonucleotide analogues and PMO
analogues is the absence of additional chiral centers. Unlike [13].
commercially available PMO and PPMO, which are mixtures of
diastereomers, oligomers constructed with the use of phos- The degree of protonation at physiological pH and the presence
phoromonoamidate [7], oxalyl diamide [8], amidine [9] and of positively charged centers in oligonucleotide analogues
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Beilstein J. Org. Chem. 2014, 10, 1151–1158.
results in increased water solubility and higher thermal stability
of complementary duplexes of such oligonucleotide mimics
with nucleic acids [14,15]. Some conformationally restricted
protonated PNA or pyrrolidine oligonucleotide mimics exhibit
selective binding with RNA but not with DNA [16,17]. It has
been shown that conjugation of PMO with cationic peptides or
other cationic residues facilitates penetration of PMO into cells
and efficiently prevents the growth of E. coli in vitro and in
vivo [18].
Taking the above considerations into account, methylenecar-
boxamide (glycine) oligomers (MorGly) (Figure 1A), being
protonated at physiological pH, seem to be promising candi-
dates for the development of novel antisense oligonucleotide
mimics.
Figure 2: Dangling residues of MorGly oligomers synthesized by solid
phase supported (A) and liquid phase synthesis (B); the blunt end of
MorGly oligomer (C).
chain. It was shown while studying their tandem complexes that
the impact of cooperative interactions at oligomer junctions on
the thermal stability was higher for modified oligomers than for
native oligodeoxyriboadenylates. This result may indicate that
the contribution of nucleobase stacking to the thermal stability
of complementary complexes is more important for the oligo-
nucleotide mimics than for native oligonucleotides. The substi-
tution of the uracil nucleobases in the MorGly oligomers by
thymines with better stacking properties may solve this
problem. The synthesis of the thymine containing morpholino
monomer 1e was necessary to prove this hypothesis.
Figure 1: Morpholinoglycine oligomers (A) and protected monomers
1a–e (B) for their synthesis.
The promising properties of the MorGly oligomers as potential
antisense agents motivated us to develop the SPPS method for
A convenient synthetic procedure was previously described for the synthesis of these oligonucleotide mimics. We focused our
protected monomers 1a–d (Figure 1B) necessary for the syn- efforts on the development of a new linker group to produce
thesis of the MorGly oligonucleotide mimics [19]. When oligomers without dangling residues (Figure 2C), the synthesis
studying the tandem complementary complexes of MorGly of the corresponding Thy-containing monomers and the
homohexa- and pentamers, we found out that the adenine- improvement of the SPPS protocol for the MorGly oligomers.
containing MorGly oligomers formed more stable complexes
with poly(U) than native oligodeoxyriboadenylates of the same Results and Discussion
length. Moreover, it was revealed that the MorGly oligomers For the synthesis of the thymine-containing monomer 1e we
preferably bind with RNA than with DNA [20]. At the same applied the procedure published for obtaining 1d [19] using
time, the stability of complementary complexes of modified 5-methyluridine as parent compound. Synthetic schemes, pro-
oligomers was shown to depend on the residues dangling the cedures and physicochemical data for intermediates in the syn-
4-end of the MorGly oligomers. In this study, the type of the thesis of monomer 1e are given in Supporting Information
residue dangling the 4-end of the oligomer chain (Figure 2) File 1.
depended on the utilized synthetic method. We found that the
MorGly oligomers containing the residues of type A formed We have previously shown that the morpholino oxalyldiamide
less stable complementary complexes than oligomers containing oligomers were cleaved by aqueous ammonia treatment, which
residues of type B.
resulted in the formation of shortened oligomers with the blunt
end of type C (Figure 2) [8]. So, the oxalyl residue can be used
Another interesting fact demonstrated in the article [20] was the as a labile linker between the solid support and the growing
dependence of the thermal stability of complementary oligomer chain (Figure 3) during SPPS in combination with the
complexes formed by the MorGly oligomers on the hetero- tert-butyloxycarbonyl (Boc)- and acyl-protected morpholino
cyclic base composition (uracil or adenine) of the modified nucleosides.
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Beilstein J. Org. Chem. 2014, 10, 1151–1158.
cedures and physicochemical data for intermediates in the syn-
thesis of monomer 3e.
Our next goal was to attach morpholino monomers 2a,d,e to the
Boc-Gly-PAM resin through the oxalyl linker. We tried
different ways to do this including to change the order in assem-
bling the elements of the whole construction, the direct attach-
ment of the monomer unit to the support without additional
6-aminohexanoic acid and the application of more active bis(2-
cyanoethyl)oxalate instead of dimethyl oxalate. However, only
the route shown in Scheme 2 was successful and gave the
loading of the polymer with the monomers 5a,d,e close to that
of the parent resin. It is interesting that the attachment of the
oxalyl-containing residue to the morpholine nitrogen in
monomers 5a,d,e resulted in the appearance of duplicate signals
of the nucleobases and morpholine protons in the NMR spectra
(see Experimental) similar to morpholinooxalyl nucleosides [8].
Figure 3: Cleavage of MorGly oligonucleotide mimics from solid
support and deprotection of nucleobases by aqueous ammonia treat-
ment: i) NH3/H2O.
After completion of the synthesis, the cleavage of the oligomer
from the solid support and the deprotection of nucleobases can
be performed simultaneously by treatment with aqueous
ammonia as in the solid phase synthesis of native oligonucleo-
tides (ODN) [21]. The oxalyl-mediated attachment of the
growing chain to the solid support is usual in the synthesis of
base sensitive ODN derivatives [22].
Figure 3 indicates the necessity of using monomers 2
(Scheme 1) as first subunits bound to the support through the
oxalyl linker. We synthesized Boc-protected aminomethylmor-
pholino nucleosides 2a,d,e as shown in Scheme 1 starting from
aminomethylmorpholino nucleosides 3a,d,e. The synthesis of
adenine and uracil containing compounds 3a,d was published
earlier [8]. Thymine containing morpholino nucleoside 3e was
obtained similarly to 3d starting from the 5-methyluridine. See
Supporting Information File 1 for the synthetic schemes, pro-
Scheme 2: Loading of the Boc-Gly-PAM resin with the morpholino
nucleosides 5a,d,e: i) Dimethyl oxalate, TEA, MeOH; ii) 4, TEA, Py;
iii) trifluoroacetic acid (TFA)/CH2Cl2/PhSH; iv) coupling with 5a,d,e.
See Table 1 for detailed protocol.
After completion of the loading, the unreacted amino groups
were capped by diethyl pyrocarbonate (DEPC) as described
earlier [23]. The extent of the loading and the rate of the
cleavage of loaded monomers were determined after the
Scheme 1: Synthesis of Boc-protected 2-aminomethylmorpholino
nucleosides: i) di-tert-butyl dicarbonate ((Boc)2O), triethylamine (TEA),
pyridine (Py); ii) AcOH/H2O.
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Beilstein J. Org. Chem. 2014, 10, 1151–1158.
aqueous ammonia/iPrOH treatment of aliquots of the support. allowed us to achieve high yields in the coupling reaction. The
The capacity of loaded supports 6a,d,e was achieved as much as synthesis of MorGly pentamers was performed manually in the
0.61–0.65 mmol/g after 4 h of the coupling reaction between mini spin receiver columns (0.7 mL) according to the improved
monomers 5a,d,e and the Boc-Gly-PAM polymer resin. It was protocol of the SPPS of MorGly oligomers (Table 1). The
found that the cleavage of the monomers from the solid support receiver column was shaken to mix the support and reactants.
in the course of the ammonia treatment was completed within Swelling and capping steps were omitted beginning the second
two days at room temperature (see Supporting Information cycle. After each cycle, a small sample of the support was
File 1). The structure of the monomers obtained after the subjected to aqueous ammonia treatment to analyse resulting
ammonia treatment (Figure 3, n = 0, Base = Ade, Ura, Thy) was oligomers. The homogeneity and the structure of the products
confirmed by thin-layer chromatography (TLC), reversed phase formed after each cycle were confirmed by TLC, RPC and mass
chromatography (RPC), and mass spectrometry (see Supporting spectrometry before and after removal of the Boc protective
Information File 1).
group (see Supporting Information File 1 for details).
The cleavable linker containing the S–S bond has been previ- It is interesting to note that the mobility of the homooligomers
ously used in the synthesis of PMO in the 2→4 direction [23]. in TLC decreased with increasing of their length (Table S1,
This corresponds to the 5’→3’ direction of native oligonucleo- Supporting Information File 1). This fact helped us to estimate
tides. In our case, the MorGly oligomers were synthesized in the comparative length and purity of homooligomers. The yield
the opposite direction (4→2) [20] similar to the standard direc- in each cycle was 96–98%.
tion (3’→5’) of native ODN and peptide nucleic acid (PNA)
synthesis. Probably, the same S–S-containing linker could be After completion of the pentamer synthesis and cleavage of the
used in the synthesis of MorGly oligomers, but it should be oligomer from the support, the Boc protective group was
examined. In our work we propose another approach, which has removed by trifluoroacetic acid (TFA). Target homopentamers
fewer steps and does not require the additional thiol treatment to 7a,d,e (Figure 4) were purified by cation exchange chromatog-
cleave the oligomer from the support.
raphy and RPC.
In our previous work [20], the reaction yields of the monomer Conclusion
attachment to the growing chain have dropped to 50% after the We developed a novel type of the labile linker for the synthesis
two first couplings although the addition of the first and the of MorGly oligomers and improved the SPPS protocol for
second monomer units provided a good yield (95%). The MorGly oligomers providing high yields at each cycle.
change of the solvents in SPPS of MorGly oligomers (see Pentamers 7a,d,e obtained by the proposed strategy will be used
Table 1) similarly to the synthesis protocol for PMO [23] in the further study of the structure and the thermal stability of
Table 1: SPPS protocol for MorGLy oligomers using Boc-Gly-PAM resin (10–30 mg).
Entry Step
Volume
Reagents and solvents
Time
1
2
3
4
5
swelling
0.7 mL
1,3-dimethyl-2-imidazolidinone (DMI)
dichloromethane (DCM)
1 h
washing
2 × 0.5 mL
2 × 0.5 mL
5 × 0.5 mL
2 × 3 min
2 × 10 min
5 × 3 min
2 × 1 min
deprotection
washing
0.4 M PhSH in DCM/TFA, 1:4
DCM, DMI, DCM, DMI, DCM
neutralization 2 × 0.5 mL
1-methyl-2-pyrrolidinone
(NMP)/N,N-diisopropylethylamine (DIPEA), 9:1
6
activation
0.04–0.12 mL
0.2 M 5a,d,e at the first cycle, 1a,d,e at subsequent
cycles; 0.18 M
5–8 min
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
tetrafluoroborate (TBTU); 0.18 M DIPEA; in DMI
7
coupling
0.04 mL of the solution of activated
monomer per 10 mg of the resin
solution of activated monomer obtained at step 6
4 h
8
washing
capping
washing
4 × 0.5 mL
2 × 0.3 mL
2 × 0.5 mL
DMI, DCM, DMI, DCM
DEPC/DCM, 1:9
DCM
4 × 3 min
2 × 5 min
2 × 3 min
9
10
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Beilstein J. Org. Chem. 2014, 10, 1151–1158.
Quantitative analytical RPC were performed on a Milichrom
A02 chromatograph system (Econova, Russia) on a ProntoSIL
125 C18 column (2 × 75 mm) in a gradient of buffer B (0.1 M
TEA–AcOH, pH 7.0, 80% acetonitrile) in buffer A (0.1 M
TEA–AcOH, pH 7.0, water) (0–100 % over 15 min) with an
elution rate of 0.2 mL/min and UV detection at 250, 260, 280,
and 300 nm unless otherwise noted. TLC was carried out on
Kieselgel 60 F254 plates (Merck, Germany) in the proper
solvent systems (see below) and visualized by UV irradiation,
ninhydrin (amine groups) or cystein/aqueous sulfuric acid
(nucleoside and trityl groups). Preparative silica gel column
chromatography, RPC, and cation exchange chromatography
were performed using silica gel (35–70 μm, Acros Organics,
USA), Porasil C 18 (55–105 μm, 125 A) (Waters, USA), and,
Figure 4: MorGly homopentamers containing adenine, uracil, and
thymine nucleobases.
complementary complexes of the MorGly oligomers with native Servacel P-23 (Serva, Germany) or SP Sepharose fast flow (GE
DNA and RNA to elucidate the role of the hydrogen bonds and Healthcare, USA), respectively. The compositions of all liquid
base stacking in their formation and stability. The use of highly mixtures are indicated as v/v percent. All evaporations were
sophisticated constructs containing short modified oligonucleo- performed under reduced pressure. Compounds 1a,d and 3a,d
tides is nowadays a promising strategy in the antisense therapy were synthesized according to the published procedures [8,19].
[24]. Recently, short cationic morpholinoguanidinium
Synthesis of thymine containing monomers
oligomers were shown to penetrate living cells and to facilitate
the inhibition of Gli1 [25].
1e and 3e
{2-[N-(tert-Butyloxycarbonyl)aminomethyl]-6-(thymin-1-
The unique properties of MorGly oligomers, such as the proto- yl)morpholin-4-yl}acetic acid (1e): Thymine containing
nated backbone at physiological pH, stability to nucleases, and monomer 1e was synthesized similarly to uracil analogue 1d
more preferable complex formation with RNA than with DNA, [19] starting from 5-methyluridine (1.29 g, 5 mmol). Yield 1.0 g
make them an attractive alternative in comparison to native (2.50 mmol, 50%). Rf 0.68 (iPrOH/H2O, 4:1); 1H NMR
oligonucleotides and other derivatives or analogues.
(DMSO-d6) 7.53 (s, 1H, H6-Thy), 6.95 (t, J = 5.6 Hz, 1H, Boc-
NH), 5.58 (dd, J = 10.1, 1.8 Hz, 1H, H6), 3.74–3.64 (m, 1H,
H2), 3.11–2.74 (m, 6H, H3, H5, Boc-HNCH2, CH2C(O)OH),
2.30 (app. t, J = 10.3 Hz, 1H, H3), 2.01 (app. t, J = 10.6 Hz, 1H,
Experimental
General
We used 5-methyluridine, O-(benzotriazol-1-yl)-N,N,N’,N’- H5), 1.77 (s, 3H, СН3-Thy), 1.37 (s, 9H, CH3-Boc);
tetramethyluronium tetrafluoroborate, 1-methyl-2-pyrrolidi- MALDI–TOFMS (m/z): [M + H]+ calcd for C17H27N4O7,
none, 1,3-dimethyl-2-imidazolidinone (Sigma-Aldrich, USA); 399.19; found, 399.77; [M + Na]+ calcd for C17H26N4NaO7,
Boc-Gly-PAM resin (substitution 0.76 mmol/g, NovaBiochem, 421.17; found, 421.64. See Supporting Information File 1 for
Germany); sodium azide, glycine (Serva, Germany); sodium the synthetic scheme and physicochemical data of intermediate
periodate, bromotrichloromethane, di-tert-butyl dicarbonate compounds.
(Acros Organics, USA). Receiver columns (REF 740522,
Macherey–Nagel, Germany) were used for SPPS of the MorGly 2-Aminomethyl-4-trityl-6-(thymin-1-yl)morpholine (3e):
oligomers in manual mode. All other reagents and solvents were Thymine containing monomer 3e was synthesized similarly to
from Sigma–Aldrich (USA) and Reachem (Russia). NMR uracil analogue 3d [8] starting from the corresponding
spectra were recorded on a Bruker AV400 spectrometer 2-hydromethyl derivative (2.16 g, 4.24 mmol). Yield 1.05 g,
(Bruker, Germany) in appropriate deuterated solvents at 30 °C. (2.20 mmol, 52%). Rf 0.12 (EtOH/DCM, 1:9); 1H NMR
Chemical shifts (δ) are reported in ppm relative to TMS signals. (CDCl3) 7.50–7.38 (br.s, 6H, o-H-Tr), 7.28 (app.t, J = 7.5 Hz,
Coupling constants J are reported in Hertz. MALDI–TOF mass 6H, m-H-Tr), 7.17 (t, J = 7.2 Hz, 3H, p-H-Tr), 6.98 (q, J = 0.8
spectra were registered on an Autoflex III mass spectrometer Hz, 1H, H6-Thy), 6.15–6.07 (dd, J = 9.8 Hz, 1H, 2.2, H6),
(Bruker Daltonics, Germany) using 2,5-dihydroxybenzoic acid 4.14–4.04 (m, 1H, H2), 3.31 (dt, J = 11.2, 2.3 Hz, 1H, H3), 3.05
as a matrix (MALDI–TOF) in positive or negative mode in The (dt, J = 11.8, 2.1 Hz, 1H, H5), 2.75–2.64 (m, 2H, NH2CH2),
Center of Cooperative Use (“Proteomics”, Russian Academy of 1.81 (d, J = 0.8 Hz, 3H, CH3), 1.39 (dd, J = 11.1, 10.0 Hz, 1H,
Sciences). IR spectra were recorded on a Vector 22 spectrom- H3), 1.35–1.30 (dd, J = 11.6, 10.6 Hz, 1H, H5);
eter (Bruker Optics, Germany) in KBr.
MALDI–TOFMS (m/z): [M – Tr + 2H]+ (the Tr protective
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Beilstein J. Org. Chem. 2014, 10, 1151–1158.
group was removed during the sample preparation) calcd for 327.17; found, 327.02; [M + Na]+ calcd for C14H22N4NaO5,
C10H17N4O3 241.13; found, 241.45. Physicochemical charac- 349.15; found, 349.02.
teristics for intermediate compounds see in Supporting Informa-
tion File 1.
Compound 2e: Rf 0.19 (EtOH/DCM, 1:9), 0.77 (iPrOH/H2O,
4:1); 1H NMR (CDCl3) 7.21 (q, J = 1.1 Hz, 1H, H6-Thy), 5.67
(dd, J = 10.0, 2.6 Hz, 1H, H6), 4.84 (t, J = 5.2 Hz, 1H, Boc-
NHCH2), 3.82–3.74 (m, 1H, H2), 3.44–3.35 (m, 1H, Boc-
NHCH2), 3.13–3.02 (m, 2Н, Boc-NHCH2, H3), 2.91 (dd, J =
General procedure for the synthesis of Boc-
protected 2-aminomethylmorpholino monomers
5a,d,e
2-[N-(tert-Butyloxycarbonyl)aminomethyl]-6-(N6-benzoyl- 12.8, 1.7 Hz, 1H, H5), 2.61 (dd, J = 10.2, 12.2 Hz, 1H, H3),
adenin-9-yl)morpholine (2a), 2-[N-(tert-butyloxy- 2.52 (dd, J = 12.8, 11.3 Hz, 1H, H5), 1.92 (d, J = 1.1 Hz, 3H,
carbonyl)aminomethyl]-6-(uracil-1-yl)morpholine (2d), CH3-Thy), 1.42 (s, 9H, CH3-Boc); MALDI–TOFMS (m/z): [M
2-[N-(tert-butyloxycarbonyl)aminomethyl]-6-(thymin-1- + H]+ calcd 341.18 for C15H25N4O5, found, 340.94; [M + Na]+
yl)morpholine (2e): The morpholino nucleoside 3a,d,e calcd for C15H24N4NaO5, 363.16; found, 362.96.
(0.72 mmol), TEA (0.35 mL, 2.1 mmol), and (Boc)2O (0.19 g,
0.87 mmol) in pyridine (1.5 mL) were stirred for 1 h. The reac- 6-[N-(2-methoxy-2-oxoacetyl)]aminohexanoic acid (4): A
tion mixture was diluted with DCM (30 mL) and washed with mixture of dimethyl oxalate (1.20 g, 10 mmol), 6-aminohexa-
5% aqueous NaHCO3 (2 × 25 mL). The organic layer was evap- noic acid (1.31 g, 10 mmol) and TEA (10 mmol, 1.40 mL) in
orated with toluene several times to remove traces of pyridine. dry MeOH (20 mL) was stirred at room temperature for 24 h.
The residue was dissolved in 80% aqueous acetic acid (AcOH) The reaction mixture was evaporated; the residue was triturated
(5 mL). After 30 min of stirring, the mixture was poured over with diethyl ether (25 mL) and cooled in an ice bath. Diethyl
ice (50 g) and carefully neutralized by adding the calculated ether was decanted, and the residue was dried in vacuum to give
amount of dry NaHCO3 (5.6 g) under vigorous stirring. The the title compound 4 as a partial TEA salt (2.62 g, 8.2 mmol,
aqueous suspension was then washed with diethyl ether colourless semisolid at room temperature). 1H NMR (DMSO-
(50 mL). The aqueous layer was concentrated to 10 mL; the d6) 8.92 (t, J = 5.8 Hz, 1H, NH), 3.76 (s, 3H, OCH3), 3.10 (app.
target product was purified by RPC in the gradient of EtOH in q, J = 6.7 Hz, 2H, NHCH2CH2), 2.65 (q, J = 7.2 Hz, 2H,
water (0–60%). The appropriate fractions were evaporated NCH2CH3), 2.17 (t, J = 7.4 Hz, 2H, CH2CH2C(O)OH),
to give after drying the title compounds (0.36 mmol, yield 1.54–1.38 (m, 4H, NHCH2CH2CH2, CH2CH2CH2C(O)OH),
50%).
1.30–1.18 (m, 2H, CH2CH2CH2CH2CH2), 1.01 (t, J = 7.2 Hz,
3H, NCH2CH3); 13C NMR (DMSO-d6) 175.0, 161.6, 157.1,
Compound 2a Rf 0.14 (EtOH/DCM, 1:9), 0.52 (iPrOH/H2O, 53.1, 45.8, 39.1, 34.2, 28.6, 26.2, 24.6, 10.6.
4:1); 1H NMR (CDCl3) 9.01 (s, 1H, Bz-NH), 8.78 (s, 1H,
H8-Ade), 8.17 (s, 1H, H2-Ade), 8.01 (dt, J = 7.6, 1.2 Hz, 2H, 9-{2-[N-(tert-Butyloxycarbonyl)aminomethyl]-6-(N6-
o-H-Bz), 7.60 (tt, J = 7.5, 1.2 Hz, 1H, p-H-Bz), 7.50 (app. t, J = benzoyladenin-9-yl)morpholin-4-yl}-8,9-dioxo-7-
7.5 Hz, 2H, m-H-Bz), 5.91 (dd, J = 10.1, 2.7 Hz, 1H, H6), 4.85 azanonanoic acid (5a), 9-{2-[N-(tert-butyloxycarbonyl)-
(t, J = 4.6 Hz, 1H, Boc-HNCH2), 3.9–3.86 (m, 1H, H2), aminomethyl]-6-(uracil-1-yl)morpholin-4-yl}-8,9-dioxo-7-
3.47–3.37 (m, 1H, Boc-HNCH2), 3.31 (dd, J = 12.2, 2.4 Hz, azanonanoic acid (5d), 9-{2-[N-(tert-butyloxycarbonyl)-
1H, H3), 3.20–3.11 (m, 1H, Boc-HNCH2), 3.08 (dd, J = 10.5, aminomethyl]-6-(thymin-1-yl)morpholin-4-yl}-8,9-dioxo-7-
12.1 Hz, 1H, H5), 3.00 (dd, J = 12.6, 1.9 Hz, 1H, H3), 2.70 (dd, azanonanoic acid (5e): Boc-protected morpholino nucleosides
J = 12.6, 11.0 Hz, 1H, H5), 1.41 (s, 9H, CH3); 3a,d,e (0.35 mmol), the acid 4 (0.76 g, 3.5 mmol), and TEA
MALDI–TOFMS (m/z): [M + H]+ calcd for C22H28N7O4, (0.42 mL, 3 mmol) in pyridine (3 mL) were heated at 50 °C for
454.22; found, 454.62; [M + Na]+ calcd for C22H27N7NaO4, 48 h. The reaction mixture was then cooled and diluted with
476.20; found, 476.68.
DCM (30 mL). The solution was washed with water (30 mL).
The aqueous layer was extracted with DCM (2 × 30 mL). The
Compound 2d: Rf 0.12 (EtOH/DCM, 1:9), 0.53 (iPrOH/H2O, organic layers were combined and evaporated several times
4:1); 1H NMR (CD3OD) 7.76 (d, J = 8.0 Hz, 1H, H6-Ura), 5.69 with water to remove pyridine. The target product was purified
(d, J = 8.0 Hz, 1H, H5-Ura), 5.67 (dd, J = 10.2, 2.3 Hz, 1H, by RPC in the gradient of MeCN in water (0–50%). The appro-
H6), 3.86–3.77 (m, 1Н, H2), 3.20 (br.d, J = 5.7 Hz, 2H, Boc- priate fractions were evaporated to give after drying the title
NHCH2), 3.04 (dd, J = 12.6, 2.3, 1H, H3), 2.88 (dd, J = 13.0, compounds as a light cream powder. Yield 0.28 mmol, 80%.
2.0 Hz, 1H, H5), 2.64 (dd, J = 12.6, 10.4 Hz, 1H, H3), 2.51 (dd,
J = 13.0, 11.0 Hz, 1H, H5), 1.43 (s, 9H, CH3); Compound 5a: Rf 0.43 (EtOH/DCM, 1:9), 0.73 (iPrOH/H2O,
MALDI–TOFMS (m/z): [M + H]+ calcd for C14H23N4O5, 4:1); 1H NMR (CD3OD) 8.76, 8.75 (2s, 0.5H each, H8-Ade),
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Beilstein J. Org. Chem. 2014, 10, 1151–1158.
8.67, 8.66 (2s, 0.5H each, H2-Ade), 8.10 (br.dt, J = 8.4, 1.2, 2H, Loading of the support with the first monomer
o-H-Bz), 7.68 (br.tt, J = 7.5, 1.2 Hz, 1H, p-H-Bz), 7.59 (br.tt, J Boc-Gly-PAM resin (10–30 mg) was placed in the receiver
= 7.8, 1.3 Hz, 2H, m-H-Bz), 6.09, 6.02 (2dd, J = 10.5, 2.8 Hz, column equipped with a cap at the outlet. Loading of the resin
0.5H each, H6), 4.82–4.78 (br.d, J = 11.8 Hz, 0.5H, H3), was performed according the protocol (Table 1) using the
4.66–4.58 (m, 2.5H, H5, Boc-NHCH2), 4.52 (dt, J = 13.2, 2.2 monomers 5a,d,e for the coupling step. The column was shaken
Hz, 0.5H, H3), 4.21 (br.d, J = 13.2 Hz, 0.5H, H5), 4.05–3.93 to mix resin and reactants. Changing the reactants and solvents
(m, 1H, H2), 3.94 (dd, J = 12.6, 10.6 Hz, 0.5H, H3), 3.58 (dd, J was carried out by centrifugation of the column equipped with a
= 12.2, 10.6 Hz, 0.5H, H5), 3.28–3.23 (m, 2H, NHCH2CH2-), collection tube at 2000 min−1. After drying, a sample of the
2.89 (dd, J = 13.5, 11.4 Hz, 0.5H, H3), 2.32 (t, J = 7.2 Hz, 1H, resin (1–2 mg) was treated with a mixture of iPrOH and
CH2CH2C(O)OH), 2.32–2.28 (m, 0.5H, H5), 2.23 (t, J = 7.2 concentrated (25%) aqueous ammonia (0.1 mL, 1:1) for 48 h at
Hz, 1H, CH2CH2C(O)OH), 1.72–1.55 (m, 4H, NHCH2CH2, room temperature under shaking. The substitution of the
CH2CH2C(O)OH), 1.44, 1.43 (2s, 4.5H each, CH3), 1.43–1.36 supports 6a,d,e was determined after calculating the amount of
(m, 2H, CH2CH2CH2C(O)OH); MALDI–TOFMS (m/z): [M + the cleaved monomer according the data of quantitative analyt-
H]+ calcd for C30H39N8O8, 639.29; found, 639.23; [M + Na]+ ical HPLC using the extinction coefficients ε260
calcd for C30H38N8NaO8, 661.27; found, 661.22; [M + K]+ 15.02 mM−1cm−1 for A, 9.66 for U and 8.56 for T [26] and was
calcd for C30H38KN8O8, 677.24; found, 676.19.
found to be 0.60–0.65 mmol/g. Physicochemical characteristics
of the cleaved monomers are given in Supporting Information
Compound 5d: Rf 0.45 (EtOH/DCM, 1:9), 0.79 (iPrOH/H2O, File 1.
4:1); 1H NMR (CD3OD) 7.83, 7.80 (2d, J = 8.2 Hz, 0.5H each,
H6-Ura), 5.76, 5.74 (2d, J = 8.2 Hz, 0.5H each, H5-Ura), 5.74, Synthesis of MorGly homopentamers 7a,d,e
5.71 (2dd, J = 10.2, 2.9 Hz, 0.5 each, H6), 4.75–4.59 (m, 2H, Pentamers 7a,d,e were synthesized manually in the receiver
Boc-NHCH2), 4.56 (br.d, J = 12.9 Hz, 0.5H, H3), 4.44 (dt, J = columns (see above) on the supports 6a,d,e (10 mg), respective-
13.1, 2.0 Hz, 0.5H, H5), 4.26 (br.d, J = 12.9 Hz, 0.5H, H3), ly, by repeating steps 2–8 of the protocol (Table 1). Monomers
4.10 (dt, J = 13.1, 1.8 Hz, 0.5H, H5), 3.91–3.81 (m, 1H, H2), 1a,d,e were used for the coupling step. Physicochemical charac-
3.33–3.28 (m, 2.5H, H3, NHCH2CH2), 3.14 (dd, J = 13.4, 11.5 teristics of oligomers cleaved from the support after certain
Hz, 0.5H, H5), 2.94 (dd, J = 13.2, 10.4 Hz, 0.5H, H3), 2.76 (dd, cycles are given in Supporting Information File 1. After
J = 13.3, 11.4 Hz, 0.5H, H5), 2.33, 2.31 (2t, J = 7.5 Hz, 2H, completion of the synthesis, the oligomers were cleaved from
CH2CH2C(O)OH), 1.72–1.56 (m, 4H, NHCH2CH2, the support by concentrated (25%) aqueous ammonia/iPrOH
CH2CH2C(O)OH), 1.47, 1.46 (2s, 4.5H each, CH3), 1.47–1.37 (1:1, 1 mL) treatment for 48 h at room temperature under
(m, 2H, CH2 CH2 CH2 C(O)OH); MALDI–TOFMS shaking. The supernatants were evaporated. The Boc-protected
(m/z): [M + H]+ calcd for C22H34N5O9, 512.24; found, 512.07; oligomers were treated with TFA (0.5 mL) for 5 min and evapo-
[M
+ Na]+ calcd for C2 2 H3 3 N5 NaO9 , 534. 22; rated. Crude deprotected oligomers were dissolved in water
found, 534.20; [M + K]+ calcd for C22H33KN5O9, 550.19; containing 0.1% TFA (1 mL) and subjected to cation exchange
found, 550.16.
chromatography (SP Sepharose) in a gradient of NaCl (0–2M)
in aqueous 20% ethanol containing 0.1% TFA. The appropriate
Compound 5e: Rf 0.68 (EtOH/DCM, 1:9), 0.83 (iPrOH/H2O, fractions were evaporated, and the target pentamers were puri-
4:1); 1H NMR (CD3OD) 7.67, 7.64 (2q, J = 1.1 Hz, 0.5 each, fied by RPC in the gradient of MeCN in water (0–40%)
H6-Thy), 5.74, 5.70 (2dd, J = 10.0, 2.7 Hz, 0.5 each, H6), containing 0.1% TFA. The appropriate fractions were concen-
4.75–4.58 (m, 2H, Boc-NHCH2), 4.51 (br.d, J = 12.9 Hz, 0.5H, trated using vacuum centrifugation. After purification,
H3), 4.43 (dt, J = 13.6, 2.0 Hz, 0.5H, H5), 4.21 (br.d, J = 12.8 0.5–1.0 μmol of homopentamers 7a,d,e were obtained. RPC
Hz, 0.5H, H3), 4.09 (dt, J = 13.1, 2.2 Hz, 0.5H, H5), 3.89–3.80 HPLC traces of purified pentamers 7a,d,e are provided in
(m, 1H, H2), 3.32–3.26 (m, 2.5H, H3, NHCH2CH2), 3.15 (dd, J Supporting Information File 1.
= 13.6, 11.4 Hz, 0.5H, H5), 2.99 (dd, J = 13.1, 10.5 Hz 0.5H,
H3), 2.76 (dd, J = 13.1, 11.4 Hz, 0.5H, H5), 2.34, 2.32 (2t, J = Pentamer 7a: MALDI–TOFMS: (m/z): [M + H]+ calcd for
7.5 Hz, 2H, CH2CH2C(O)OH), 1.93, 1.92 (2d, J = 1.1 Hz, 1.5H C58H76N36O9, 1406.66; found, 1406.43.
each, CH3-Thy), 1.71–1.56 (m, 4H, NHCH2CH2,
CH2CH2C(O)OH), 1.47, 1.46 (2s, 4.5H each, CH3-Boc), Pentamer 7d: MALDI–TOFMS (m/z): [M + H]+ calcd for
1.47–1.37 (m, 2H, CH2CH2CH2C(O)OH); MALDI–TOFMS C53H71N20O19, 1291.52; found, 1291.65.
(m/z): [M + Na]+ calcd for C23H35N5NaO9, 548.23; found,
548.27; [M + K]+ calcd for C23H35KN5O9, 564.21; found, Pentamer 7e: MALDI–TOFMS (m/z): [M + H]+ calcd for
564.26
C58H81N20O19, 1361.60; found, 1361.71.
1157

Beilstein J. Org. Chem. 2014, 10, 1151–1158.
12.Thiviyanathan, V.; Vyazovkina, K. V.; Gozansky, E. K.;
Supporting Information
Bichenchova, E.; Abramova, T. V.; Luxon, B. A.; Lebedev, A. V.;
Gorenstein, D. G. Biochemistry 2002, 41, 827–838.
doi:10.1021/bi011551k
Synthetic schemes, procedures and physicochemical data
for intermediates in the synthesis of compounds 1e and 3e;
kinetics of the cleavage of monomers from supports 6a,d;
TLC, RPC HPLC traces and mass spectra data for
oligomers cleaved from the samples of supports 6a,d,e and
for purified pentamers 7a,d,e are provided in the
Supporting Information.
13.Kang, H.; Chou, P.-J.; Johnson, W. C., Jr.; Weller, D.; Huang, S.-B.;
Summerton, J. E. Biopolymers 1992, 32, 1351–1363.
doi:10.1002/bip.360321009
14.Debart, F.; Abes, S.; Deglane, G.; Moulton, H. M.; Clair, P.; Gait, M. J.;
Vasser, J.-J.; Lebleu, B. Curr. Top. Med. Chem. 2007, 7, 727–737.
doi:10.2174/156802607780487704
15.Yeh, J. I.; Shivachev, B.; Rapireddy, S.; Crawford, M. J.; Gil, R. R.;
Du, S.; Madrid, M.; Ly, D. H. J. Am. Chem. Soc. 2010, 132,
10717–10727. doi:10.1021/ja907225d
Supporting Information File 1
16.Kumar, V. A.; Ganesh, K. N. Curr. Top. Med. Chem. 2007, 7, 715–726.
doi:10.2174/156802607780487722
Syntheses and characteristics for selected compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-115-S1.pdf]
17.Worthington, R. J.; Micklefield, J. Chem.–Eur. J. 2011, 17,
14429–14441. doi:10.1002/chem.201101950
18.Mellbye, B. L.; Weller, D. D.; Hassinger, J. N.; Reeves, M. D.;
Lovejoy, C. E.; Iversen, P. L.; Geller, B. L. J. Antimicrob. Chemother.
2010, 65, 98–106. doi:10.1093/jac/dkp392
Acknowledgements
19.Kasakin, M. F.; Abramova, T. V.; Silnikov, V. N. Russ. J. Bioorg. Chem.
2011, 37, 752–757. doi:10.1134/S1068162011060070
20.Abramova, T. V.; Kassakin, M. F.; Tarasenko, Yu. V.; Lomzov, A. A.;
Koval, V. V.; Pyshnyi, D. V.; Silnikov, V. N. Russ. J. Bioorg. Chem.
2012, 38, 400–411. doi:10.1134/S1068162012040024
21.Abramova, T. Molecules 2013, 18, 1063–1075.
doi:10.3390/molecules18011063
The work was supported by the President program “Leading
Scientific Schools” (project no. 1205.2014.4); the grant of the
Russian Foundation for Basic Research (project no. 12-04-
01454-a and 14-04-01018-a); and the Interdisciplinary Integra-
tion project no. 60 of the Presidium of the Siberian Branch of
the Russian Academy of Sciences. We also acknowledge
support from the Ministry of Education and Science of the
Russian Federation, agreement no. 14.В25.31.0028.
22.Alul, R. H.; Singman, C. N.; Zhang, G.; Letsinger, R. L.
Nucleic Acids Res. 1991, 19, 1527–1532. doi:10.1093/nar/19.7.1527
23.Weller, D. D.; Hassinger, J. N.; Cai, B. Z. Oligonucleotide analogs
having cationic intersubunit linkages. U.S. Patent 7,943,762 B2, May
17, 2011.
References
1. Panchal, R. G.; Geller, B. L.; Mellbye, B.; Lane, D.; Iversen, P. L.;
24.Mallikaratchy, P.; Gardner, J.; Nordstrøm, L. U. R.; Veomett, N. J.;
McDevitt, M. R.; Heaney, M. L.; Scheinberg, D. A. Nucleic Acid Ther.
2013, 23, 289–299.
Bavari, S. Nucleic Acid Ther. 2012, 22, 316–322.
2. Leger, A. J.; Mosquea, L. M.; Clayton, N. P.; Wu, I. H.; Weeden, T.;
Nelson, C. A.; Phillips, L.; Roberts, E.; Piepenhagen, P. A.;
Cheng, S. H.; Wentworth, B. M. Nucleic Acid Ther. 2013, 23, 109–117.
3. Watts, J. K.; Corey, D. R. J. Pathol. 2012, 226, 365–379.
doi:10.1002/path.2993
25.Pattanayak, S.; Khatra, H.; Saha, S.; Sinha, S. RSC Adv. 2014, 4,
1951–1954. doi:10.1039/c3ra45257c
26.Cavaluzzi, M. J.; Borer, P. N. Nucleic Acids Res. 2004, 32, e13.
doi:10.1093/nar/gnh015
4. Deleavey, G. F.; Damha, M. J. Chem. Biol. 2012, 19, 937–954.
doi:10.1016/j.chembiol.2012.07.011
5. Bill, B. R.; Petzold, A. M.; Clark, K. J.; Schimmenti, L. A.; Ekker, S. C.
Zebrafish 2009, 6, 69–77. doi:10.1089/zeb.2008.0555
6. Anderson, J. L.; Carten, J. D.; Farber, S. A. In Zebrafish: Cellular and
Developmental Biology, 3rd ed.; Detrich, H. W.; Westerfield, M.;
Zon, L. I., Eds.; Methods in Cell Biology, Vol. 101; Elsevier Academic
Press Inc.: San Diego, CA, USA, 2011; pp 111–141.
7. Zhang, N.; Tan, C.; Cai, P.; Jiang, Y.; Zhang, P.; Zhao, Y.
Tetrahedron Lett. 2008, 49, 3570–3573.
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doi:10.1016/j.tetlet.2008.04.035
8. Abramova, T. V.; Kassakin, M. F.; Lomzov, A. A.; Pyshnyi, D. V.;
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9. Pérez-Rentero, S.; Alguacil, J.; Robles, J. Tetrahedron 2009, 65,
1171–1179. doi:10.1016/j.tet.2008.11.069
The definitive version of this article is the electronic one
which can be found at:
10.Chakhmakhcheva, O.; Andrianov, M.; Buryakova, A.; Choob, M.;
Efimov, V. Nucleosides Nucleotides 1999, 18, 1427–1428.
doi:10.1080/07328319908044742
doi:10.3762/bjoc.10.115
11.Abramova, T. V.; Vorobev, Y. N.; Lebedev, A. V. Bioorg. Khim. 1986,
12, 1335–1347.
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