Convenient method for the synthesis of phosphoramidite non-nucleotide inserts for preparation of functionalized oligonucleotides

Article abstract of DOI:10.1134/S1068162012060106

A simple approach to the synthesis of amidophosphite synthons of achiral non-nucleotide inserts using 4-(2-(4,4'-dimethoxytrityloxy)ethyl)morpholine-2,3- dione as a backbone of key precursor was suggested. Non-nucleotide synthons were synthesized using this approach that were suitable for synthesis of acridine-containing oligonucleotide derivatives, as well as oligonucleotides with branched carbohydrate-phosphate backbone. Pleiades Publishing, Ltd., 2012.

Full text of DOI:10.1134/S1068162012060106

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2012, Vol. 38, No. 6, pp. 662–666. © Pleiades Publishing, Ltd., 2012.
Original Russian Text © M.S. Kupryushkin, D.V. Pyshnyi, 2012, published in Bioorganicheskaya Khimiya, 2012, Vol. 38, No. 6, pp. 745–750.
LETTER
TO THE EDITOR
A Convenient Method for the Synthesis of Phosphoramidite
NonꢀNucleotide Inserts for Preparation of Functionalized
Oligonucleotides
M. S. Kupryushkin and D. V. Pyshnyi1
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences,
pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia
Received December 19, 2011, in final form, January 20, 2012
Abstract—A simple approach to the synthesis of amidophosphite synthons of achiral nonꢀnucleotide inserts
using 4ꢀ(2ꢀ(4,4'ꢀdimethoxytrityloxy)ethyl)morpholineꢀ2,3ꢀdione as a backbone of key precursor was sugꢀ
gested. Nonꢀnucleotide synthons were synthesized using this approach that were suitable for synthesis of acriꢀ
dineꢀcontaining oligonucleotide derivatives, as well as oligonucleotides with branched carbohydrateꢀphosꢀ
phate backbone.
Keywords: branched DNA, nonꢀnucleotide insert, modified oligonucleotides, fluorescent probes, thermal stability
DOI: 10.1134/S1068162012060106
1
Amidophosphite monomers, including those of a to the final phosphitylation step providing the final
nonꢀnucleotide nature, are widely used for the prepaꢀ product (Scheme 2). The universal building block (
I)
ration of a variety of oligonucleotide derivatives in the is a common reagent for the synthesis of inserts with
context of automatic DNA synthesis. However, the
synthesis of required synthons, such as nonꢀnucleꢀ
otide inserts for the introduction of functional groups
into various positions of the oligonucleotide chain, is a
separate synthetic problem for each case. Hence, the
development of a convenient method for the synthesis
of nonꢀnucleotide inserts carrying different functional
residues is an actual problem.
this method, which allows the production of a series of
various nonꢀnucleotide inserts with structures defined
by the chosen functionalized building block carrying
the required group (groups).
HO
N
O
O
O
N
O
O
O
N
i
ii
(I)
Based on a series of certain synthetic approaches
suggested by various authors [1, 2], a simple method of
synthesis of achiral nonꢀnucleotide inserts for develꢀ
opment of oligonucleotide derivatives using automatic
amidophosphite protocol was proposed. This methods
includes a parallel synthesis of two functionally differꢀ
ent building blocks: an universal building block, 4ꢀ(2ꢀ
(4,4'ꢀdimethoxytrityloxy)ethyl)morpholineꢀ2,3ꢀdione
OH
OH
ODMTr
Reagents and conditions:
) diethyl oxalate, isopropanol, 12 h, 70%. 1H NMR:
3.43 (t, 2 H, >N–CH2–C 2–OH, 5.5 Hz); 3.53 (t, 2 H >N–
2–CH2–OH, 5.5 Hz); 3.71 (t, 2 H, –O–CH2–CH2
N<, 5.1 Hz); 4.49 (t, 2 H, –O–C 2–CH2–N<, 5.1 Hz).
13C NMR
: 45.99, 49.45, 58.25, 65.84, 154.01, 157.77; (ii
DMTrꢀCl, pyridine, 2 h, ( 3.16 (t,
): above 90%. 1H NMR:
2 H, >N–CH2–C 2–ODMTr, 5.3 Hz); 3.59 (t, 2 H, >N–
2–CH2–ODMTr, 5.3 Hz); 3.68 (t, 2 H, –O–CH2–
2–N<, 4.9 Hz); 3.73 (s, 3 H, OMe); 4.49 (t, 2 H, –O–
2–CH2–N<, 4.9 Hz); 6.85–8.66 (m, 13 H, aromatic).
(i
δ
H
J
CH
J
–
(
I
) (Scheme 1), and a functionalized building block,
substituted aliphatic amine (for example compounds
II) and (III)). Combining the two building blocks
J
H
J
δ
)
I
δ
(
H
J
into a unified structure is conducted in the stage prior
CH
CH
CH
J
J
Abbreviations: TBDMS, tretꢀbutyldimethylsilyl; DMTr,
dimethoxytrityl; CEP, 2ꢀcyanoethoxy diisopropylamino phosꢀ
phinyl; Lev, levulinyl; DIPEA, diisopropylethylamine; TEA, triꢀ
ethylamine; DMAP, 4ꢀN,Nꢀdimethylaminopyridine.
Corresponding author: phone: (383)ꢀ363ꢀ5151; eꢀmail: pyshꢀ
nyi@niboch.nsc.ru.
J
Scheme 1. Synthesis of a universal building block (
I)
1
on the basis of substituted morpholineꢀ2,3ꢀdione.
662

A CONVENIENT METHOD FOR THE SYNTHESIS
OH
663
O
O
O
N
OCEP
R2
N
O
R2
N
O
R2
i
(Pr N) POCH CH CN
2
R1
N
R1
N
2
2
2
NH
+
R1
O
O
ODMTr
ODMTr
ODMTr
Scheme 2. General scheme for preparation of nonꢀnucleotide synthon insert
from ‘functionalized’ and ‘universal’ building blocks.
A series of nonꢀnucleotide inserts was synthesized functional groups) as functionalized building blocks
using derivatives of the primary amine (III) (one to demonstrate the possibilities of the method
functional group) and secondary amine (II) (two (Scheme 3).
Cl
OTBDMS
(II)
OCEP
O
H
N
N
OTBDMS
N
N
(IV)
N
H
O
i
ii
HN
N
OMe ODMTr
O
O
O
N
OMe
Cl
OCEP
O
H
iii
i
v
N
N
NH2
LevO
ODMTr
LevO
(V)
O
(III)
ODMTr
Reagents and conditions:
) , DMAP, pyridine, 45
88%. 1H NMR:
–0.25–0.00 (m, 6 H, (C
2.65–2.81 (m, 2 H, –C 2–CN); 3.05–3.25 (m, 2 H, –C
–C
2–CH2–CN); 6.75–8.39 (m, 19 H, aromatic). 31P NMR:
40%; (
) (Pr2iN)2POCH2CH2CN, DIPEA, tetrazole, acetonitrile, 1 h, (
(C 3)CH–, –CH2–C 2–CH2–); 2.1 (s, 3 H, C 3–C(O)–); 2.45 (m, 2H, –CH2–C
4 H, –C 2–CN, CH3–C(O)–C 2–CH2–); 3.16 –3.05 (m, 4 H, –C 2–NH–C(O)–, –C
16 H, >N–C 2–C 2–O–, (CH3)C –, –C 2–CH2–CN, –OC 3); 3.93–4.00 (m, 2 H, –C(O)–O–C
6.85–8.66 (m, 13 H, aromatic). 31P NMR:
147.96, 148.06.
(i
°
C, 12 h, 93%; (ii) (Pr2iN)2POCH2CH2CN, DIPEA, tetrazole, acetonitrile, 1 h, (IV):
δ
H
3)2Si); 0.63–0.68 (m, 9 H, (C
2–O–DMTr); 3.45–4.00 (m, 24 H, –OC
147.74; 147.84; (iii), DMAP, pyridine, 60
): 77%. 1H NMR:
1.1–1.6 (m, 20 H,
2–C(O)–O–); 2.68–2.81 (m,
2–ODMTr); 3.5–3.8 (m,
2–CH2–);
H
3)3CSi); 0.97–1.29 (m, 12 H, (C
H
3)2CH–);
H
H
H
3, –CH
2–C 2–,
H
H
δ
°C, 8 h,
i
v
V
δ
H
H
H
H
H
H
H
H
H
H
H
H
H
H
δ
Scheme 3. Synthesis of biꢀ and monofunctionalized nonꢀnucleotide synthon inserts (IV) and (V), respectively.
A structure of amidophosphite synthon of achiral functionalized amine. The compound (IV) differs
from the analogue synthesized earlier in the shorter
linker between diethanolamine fragments of the insert
backbone.
acridineꢀcontaining nonꢀnucleotide insert that was
suggested previously was synthesized in seven stages
with the total yield of only ~10% [3]. A compound
(
IV) similar to that described in [3] was synthesized in
four stages with a total yield of approximately 60%
with the method of synthesis suggested earlier for
1ꢀ
(2ꢀ(tertꢀbutyldimethylsiloxy)ethyl)ꢀ
2ꢀ(6ꢀchloroꢀ2ꢀ
It was established that inserts with a linker fragment
on the basis of an oxalate residue are significantly
more effective in the production of oligonucleotide
derivatives when compared with analogues based on a
N
N
methoxyacridineꢀ9ꢀyl)ethylene diamine (II) as a glutarate linker [3]. Thus, a monomer (IV) is inserted
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

664
KUPRYUSHKIN, PYSHNYI
Properties of oligoꢀthymidylate derivatives and their DNA complexes
Δ
S
°
,
Δ
H
°
,
m/z M
– H]–)c
([
calc/exp
ΔG3ꢀ 7,
kcal/mol
a
Compound
Tm
,
°
C
Δ
F
, %b
cal/(mol K)
kcal/mol
T6
–117.6
–114.0
–171.2
–135.4
–89.9
–63.6
–
–38.8
–35.4
–58.0
–47.9
–32.3
–23.6
–
–2.4
0.0
6.8
–9.5 d
30.8
35.8
21.6
9.7
T3DÒ3
XT6
X1 T6
–4.9
–5.9
–4.4
–3.8
–
–12.9
–13.5
25.4
24.0
–
2443.4/2443.5
2896.1/2896.9
2443.4/2443.5
2443.4/2443.8
3242.8/3246.5
2443.4/2443.5
2485.5/2484.5
2485.5/2483.7
2
T5XT
T3XT3
(T3)2X2T3
–
T2XT4
–93.3
–113.8
–144.5
–33.3
–39.6
–50.1
–4.4
–4.3
–5.2
21.6
20.5
29.1
18.6
57.5
18.6
T3X*T3
X*T6
D, X, X* are nonꢀnucleotide inserts of the following structure:
D
X
X*
5'
5'
O
O
5'
RO
RO
O
O
N
N
N
N
Cl
O
Cl
O
O
O
O
O
P
NH
NH
O P O
O
O
O
P
N
3'
O
O
N
3'
3'
OMe
OMe
1
2
where in inserts X: R = –TBDMS; X : R = –H; X : R = oligonucleotide fragment (here T );
3
X*: R = –TBDMS.
–4
a Total concentrations of oligonucleotides taken at a stoichiometric ratio is 2 × 10 M.
b
Δ
F
is a relative change in fluorescence at
λ
em
= 500 nm (
λ
ex
= 420 nm ) as a result of hybridization with DNA matrix CA C at 6 C,
°
6
–6
Δ
F(%) = 100%(Fss – Fds)/Fss. Concentration of acridineꢀcontaining nucleotides was 10 M. A 20ꢀfold excess of DNA matrix, CA C,
6
was added. All measurements conducted in a buffer containing 1 M NaCl, 10 mM sodium phosphate (pH 7.3), 0.1 M Na EDTA.
2
c Obtained by V. V. Koval with AutoflexꢀIII MALDI TOF (Bruker Daltonics, Germany) using linear regime of negative ions registration,
N laser (337 nm), matrix, 3ꢀhydroxypicolinic acid.
2
d Thermodynamic parameters of formation of the complex containing insert based on diethylene glycol ether
to the data in [4].
D were calculated according
in a growing chain with an efficiency close to the effiꢀ
ciency of insertion of a standard amidophosphite
monomer, and a nonꢀnucleotide unit obtained with its
help is stable in conditions of postꢀsynthetic deprotecꢀ
tion (tertꢀbutylamine in an aqueous methanol soluꢀ
tion).
A series of linear and branched oligonucleotides
containing the developed nonꢀnucleotide inserts (
and , respectively, Table) in various positions of carꢀ
bohydrateꢀphosphate backbone was synthesized with
an automatic DNA synthesizer ASMꢀ800 (Biosset,
X
Y
Novosibirsk) using the amidophosphites (IV) and (V).
Furthermore, mass spectra and data on the thermodyꢀ
namic stability of duplexes formed with complemenꢀ
tary DNA probes were obtained (table, figure).
In addition, the simplified nonꢀnucleotide insert
(V), lacking the acridine residue derivative obtained
with the help of compound (III), was found to be sufꢀ
ficiently stable in standard conditions of oligonucleꢀ
otide deprotection (concentrated aqueous ammonia).
It can be seen from the Table that the acridine
derivative residue in an insert composition with
oxalate linker
X containing intercalator compensates
Compounds (I), (IV), and (
V
) are characterized
with H NMR (300 MHz, DMSOꢀd6) and 32P NMR
DMSOꢀd6) spectra.
its destabilizing effect caused by the distortion of the
regular structure of the carbohydrateꢀphosphate backꢀ
bone in the duplex composition. The observed melting
1
(
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
No. 6
2012

A CONVENIENT METHOD FOR THE SYNTHESIS
665
5'
5'
71°C
76.5
°
°
C
C
5'
5'
(D)
(Y)
5'
5'
5'
72
5'
(D)
(D)
5'
67°C
69°
C
5'
(Y)
5'
5'
GATCACTCTGGTCTC
CTTTTCCAAATAATC
GAGACCAGAGTGATC
GATTATTTGGAAAAG
5'
5'
5'
5'
D
5'
Y
O
O
O
O
N
O
N
H
O
O
O
O P O
O P O
Structures of DNA duplexes and their melting points. Buffer: 1M NaCl, 0.01 M sodium phosphate (pH 7.3), 0.1 mM Na EDTA,
2
–5
total concentration of oligonucleotide components taken in stoichiometric ratio is 10 M.
point of DNA duplex (T3XT3/СА6С) with insert
however, lower than the melting point of
T3X 3/СА6С complex carrying the similar insert
X
is, the length of combining linker. Acridine residue demꢀ
onstrates a fluorescence decrease in the binding of
modified oligonucleotide with DNA (for example
*T
with the glutarate linker (X*). It is likely that the short,
rigid linker does not provide an optimal interaction
with the intercalating residue with internal base pairs
in the modified duplex composition. The inserts at the
end of duplexes stabilize the double helix by a practiꢀ
cally equal extent independent of the length of the
linker fragment (see table, XT6/СА6С and
X
*
T6) when in the insert with the glutarate linker, and
an increase when in the insert with the oxalate linker
XT6). Hence, even so, insignificant changes of insert
(
structure, such as the structure of the linker fragment
combining different branches of the nonꢀnucleotide
unit, could significantly affect the properties of the
respective oligonucleotide derivatives.
X*T6/СА6С).
A significant effect of the nature of combining the
Two extended branched oligonucleotides (45 nt)
carry three pentadecanucleotide fragments: two idenꢀ
tical 5'ꢀend fragments and one 3'ꢀend were obtained
linker in the insert can be seen with an analysis of the
fluorescence change of oligonucleotide conjugates on
transfer from the singleꢀstranded to the doubleꢀ
using synthon (
capable of duplex formation with a melting point (Tm
significantly lower (Tm = 71 and 67°C) than for an
unmodified duplex of 30 bp length ( m =76.5 ), but
V). It was shown that the oligomers are
stranded state (see F in table). As would be expected,
Δ
)
the formation of a complex of acridineꢀcontaining oliꢀ
gonucleotides with a complementary DNA probe
results in the change of fluorescence of the intercalatꢀ
ing residue. Fluorescence quenching occurs on the
formation of the doubleꢀstranded complex in the case
of internal location of fluorophore residue, indepenꢀ
dent of the combining linker length. Formation of the
duplex with terminal location of acridine derivative is
characterized by different changes of fluorescence
Т
°С
comparable with the melting point of the duplexes of
the same length carrying insert(s) based on the phosꢀ
phodiester of diethylene glycol (D) in the central part
of one or both chains. Hence, the availability of the
second extended singleꢀstranded fragment in the
branching point does not produce a significant destaꢀ
efficiency of the intercalating residue depending on bilizing effect on the DNAꢀduplex.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 38
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2012

666
KUPRYUSHKIN, PYSHNYI
The present work demonstrated that a convenient
REFERENCES
approach to the preparation of amidophosphite synꢀ
thons, allowing the creation of nonꢀnucleotide inserts
of different functionalities with minimal synthetic
steps, was developed.
1. Dioubankova, N.N., Malakhov, A.D., Stetsenko, D.A.,
Korshun, V.A., and Gait, M.J., Org. Lett., 2002, vol. 4,
pp. 4607–4610.
2. Morocho, A.M., Karamyshev, V.N., and Polushin, N.N.,
ACKNOWLEDGMENTS
Bioconjugate Chem., 2004, vol. 15, pp. 569–575.
This work was supported by the Russian Foundaꢀ
tion for Basic Research (project no. 10ꢀ04ꢀ01492a), by
the programs of the Presidium of the Russian Acadꢀ
emy of Sciences “Molecular and cell biology” and
‘BFINN’, and in part by grants from the Siberian
Branch of the Russian Academy of Sciences.
3. Kupryushkin, M.S. and Pyshnyi, D.V., Russ. J. Bioorg.
Chem., 2012, vol. 38, pp. 625–638.
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Pyshnyi, D.V., Dokl. Biochem. Biophys., 2006, vol. 409,
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No. 6
2012