New phosphoramidite derivatives for the preparation of oligonucleotides containing a hydrazide group in any specified position of the oligonucleotide chain

Article abstract of DOI:10.1007/s11172-006-0175-5

A new versatile method for the preparation of oligonucleotides containing hy drazide groups in any position of the oligonucleotide chain by standard phosphoramidite automated oligonucleotide synthesis is proposed. The method is based on the use of a serie

Full text of DOI:10.1007/s11172-006-0175-5

Russian Chemical Bulletin, International Edition, Vol. 54, No. 11, pp. 2671—2681, November, 2005
2671
New phosphoramidite derivatives for the preparation of oligonucleotides
containing a hydrazide group in any specified position
of the oligonucleotide chain
S. I. Antsypovich,a,bꢀ T. S. Oretskaya,a and G. von Kiedrowskib
аDepartment of Chemistry, M. V. Lomonosov Moscow State University,
Leninskie gory, 119992 Moscow, Russian Federation.
Fax: +7 (495) 939 3181. Eꢀmail: antsypov@rambler.ru
bDepartment of Chemistry, Ruhr University,
150 Universitätsstrasse, Bochum, Germany.
*
Fax: +49 (234) 709 4355
A new versatile method for the preparation of oligonucleotides containing hydrazide groups
in any position of the oligonucleotide chain by standard phosphoramidite automated oligoꢀ
nucleotide synthesis is proposed. The method is based on the use of a series of new modified
components for oligonucleotide synthesis. An original protecting group for the hydrazide group
is proposed. The presence of the hydrazide group in the obtained oligonucleotides and its high
reactivity were demonstrated by the reaction with 4ꢀmethoxybenzaldehyde in solution.
Key words: modified oligonucleotides, hydrazide group, oligonucleotide synthesis,
phosphoramidite derivatives, conjugation.
Synthetic modified oligonucleotides are now widely
used in bioorganic chemistry, molecular biology, and
bioengineering. Of particular interest are oligonucleotides
bearing reactive groups not encountered in natural oligoꢀ
nucleotides, which extends the scope of their applicabilꢀ
ity, as this allows immobilization on various surfaces and
conjugation with molecules of different nature.1—10
poration of such oligonucleotides into extended NA molꢀ
ecules, for covalent binding of complementary and
branched oligonucleotide structures, for example, trisꢀ
oligonucleotides,16—19 and for other purposes.
Methods for preparation of hydrazideꢀcontaining oliꢀ
gonucleotides have not been adequately developed. The
existing methods are not versatile and suffer from some
drawbacks.11—15,20 Oligonucleotides containing the hyꢀ
drazide group at the 5´ꢀ or 3´ꢀend are commercially avaible
from Metabion.13 Their synthesis is based on phosꢀ
phoramidite derivative 1 developed by the Nanogen
Recognomics company.
A topical task of bioorganic chemistry is the developꢀ
ment of efficient methods for the preparation of such
oligonucleotides bearing reactive groups. The hydrazide
group is one of these, which imparts valuable properties to
the oligonucleotide. First of all, this is the possibility of
efficient synthesis of conjugates with molecules and surꢀ
faces containing aldehyde groups to give Nꢀacylhydrꢀ
azones. The reaction requires no activating reagents; it
occurs rapidly under mild conditions and gives products
in high yields.11—15 This method has a number of advanꢀ
tages over the reaction of amines with aldehydes, which
yields Schiff´s bases.11 The latter are less stable than
hydrazones, and additional reduction for the formation of
more stable secondary amines is often required.11,13,14
The application of oligonucleotides containing modiꢀ
fications in the middle of the chain can give conjugates in
which both oligonucleotide ends are free for further modiꢀ
fication, for example, for radioactive labeling, for incorꢀ
However, 3´ꢀmodified oligomers can be obtained only
provided that 5´ꢀOꢀphosphoramidite nucleoside derivaꢀ
tives are used in the automated synthesis. Their cost is
rather high, while the addition efficiency is lower than
that of 3´ꢀOꢀphosphoramidite derivatives. This limits the
applicability of compound 1 for the synthesis of 3´ꢀmodiꢀ
fied oligonucleotides.
The transformation of the ester group into hydrazide
on treatment with hydrazine was used to prepare 3´ꢀmodiꢀ
fied oligonucleotides.12 This approach is rather effective;
however, it cannot claim for being versatile, because the
* Lehrstuhl für Organische Chemie I — Bioorganische Chemie,
Fakultät
für
Chemie,
RuhrꢀUniversität
Bochum,
Universitätsstrasse 150, Gebäude NC 2/170, 44780 Deutschland.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2585—2595, November, 2005.
1066ꢀ5285/05/5411ꢀ2671 © 2005 Springer Science+Business Media, Inc.

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Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005
Antsypovich et al.
postꢀsynthetic procedure based on the hydrazinolysis21 is
accompanied by the formation of large amounts of byꢀ
products due to modification of Nꢀbenzoylcytosine resiꢀ
dues.21 Therefore, hydrazinolysis can be used21—23 only
in combination with special labile protecting groups. For
example, a protecting group more labile than the benzoyl
group is used for the exocyclic amino group of cytidine,
which limits the applicability of the method. In addition,
after removal of the protecting group, the oligonucleotide
may still be unstable against such a strong nucleophile as
hydrazine.24
Recently, we proposed to use phosphoramidite deꢀ
rivative 2 based on bis(2ꢀhydroxyethyl) sulfone for the
introduction of the hydrazide group in the ends of the
oligonucleotide chain.11
This compound allows one to synthesize 5´ꢀ and
3´ꢀmodified oligomers and oligonucleotides containing
hydrazide groups in both ends.11
Note that if the hydrazide group is unprotected, there
is a probability of undesirable formation of an amide
from the hydrazide under the action of ammonia. This
reaction proceeds at a noticeable rate under standard conꢀ
ditions of postꢀsynthetic treatment of oligonucleotides
(25—33% aqueous ammonia at 55 °C)11 both in the case
of unprotected hydrazide and hydrazide that is deꢀ
protected during treatment with ammonia. The eliminaꢀ
tion of bis(2ꢀhydroxyethyl) sulfone takes place in 5 h,
while complete removal of protecting groups from the
oligonucleotide requires 16 h (see Refs 11 and 25). Thus,
the hydrazide function is liberated before the treatment
with ammonia is over. The use of highly labile protection
for exocyclic groups of heterocyclic bases21,26—32 allows
one to remove these groups under mild conditions in
which the hydrazide function remains intact. However,
derivatives with these protecting groups are rather exꢀ
pensive.
Note. CPG is the polymeric support based on controlled pore glass, TBS = ButMe2Si—.

New hydrazideꢀcontaining oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005 2673
This communication describes a new versatile method
for the preparation of deliberately modified hydrazideꢀ
containing oligonucleotides based on a unified proceꢀ
dure, that completely eliminates the formation of byꢀ
products during deprotection. The method implies the
use of an original protecting group for the hydrazide funcꢀ
tion and its selective removal after treatment with ammoꢀ
nia. We developed new hydrazideꢀcontaining phosphorꢀ
amidite derivatives 3 and 4 and a modified polymeric
support 5, which permit the preparation of oligonucleoꢀ
tides containing a hydrazide function at the 5´ꢀend of the
chain, inside the chain, and at the 3´ꢀend.
The protecting group based on
this ester is stable in the presence of
bases if both hydroxy groups are proꢀ
tected. However, it can be cleaved
under mild basic conditions if at least
one of the hydroxy groups is free.
Thus, one of the hydroxy groups should be bound to the
hydrazide function, while the other should be protected;
this will make the overall structure stable during treatꢀ
ment with ammonia.
The tertꢀbutyldimethylsilyl (TBS) group was chosen
as the Oꢀprotecting group in compound 6. This group is
used in the oligoribonucleotide synthesis for the protecꢀ
tion of 2´ꢀhydroxy groups34 and, hence, fully complies
with the above requirements. Indeed, it completely withꢀ
stands the conditions of both the oligonucleotide syntheꢀ
sis and the postꢀsynthetic treatment and can be selecꢀ
tively removed on treatment with tetrabutylammonium
fluoride under mild conditions. A specific feature of the
postꢀsynthetic treatment in the preparation of the modiꢀ
fied oligonucleotide is that the first ammonia treatment of
the immobilized oligonucleotide is carried out with
ethanolic rather than aqueous solutions of ammonia. It
was shown34 that aqueous ammonia may partly remove
the TBS group, which is inadmissible at this stage.
Compound 3 was prepared to be used in the synthesis
of 5´ꢀmodified oligonucleotides. 4ꢀHydroxybutyrohydrꢀ
azide (7) was taken as the starting compound (Scheme 1).
Compound 4 and the polymeric support 5 were synꢀ
thesized starting from 2ꢀaminopropaneꢀ1,3ꢀdiol (11)
(Scheme 2); thus, the distance between two phosphate
Results and Discussion
In the development of new hydrazideꢀcontaining
phosphoramidite derivatives 3 and 4, and modified polyꢀ
meric support 5, the attention was concentrated on the
nature of the protecting group for the hydrazide function.
This group was selected considering the following:
— the protecting group should be completely stable
during the oligonucleotide synthesis with conventionally
protected phosphoramidite derivatives and a standard synꢀ
thetic protocol;3,31,32
— the group should be stable during the standard postꢀ
synthetic treatment with ammonia (16 h at 55 °C);
— the group should be selectively removed under conꢀ
ditions where the hydrazide group remains intact.
Diethyl 2,2ꢀbis(hydroxymethyl)malonate (6) used preꢀ
viously for the synthesis of phosphorylated oligonucleoꢀ
tides served as the key compound.33
Scheme 1
Reagents and conditions: i. N2H4, EtOH; ii. ButMe2SiCl(TBS—Cl), DMAP, Py, CH2Cl2; iii. 4ꢀO2NC6H4OCOCl, DMAP, Py,
CH2Cl2; iv. DMAP, DMF, Py; v. P(OCH2CH2CN)(NPri2)2, Pri2NH•CH2N4, CH2Cl2.

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Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005
Antsypovich et al.
Scheme 2
Reagents and conditions: i. CF3COOEt, Py, CH2Cl2; ii. (MeO)2TrCl, Py, CH2Cl2; iii. NH4OH, EtOH; iv. 4ꢀO2NC6H4OCOCl, Py,
DMAP, CH2Cl2; v. Py, DMAP, CH2Cl2; vi. P(OCH2CH2CN)(NPri2)2, Pri2NH•CH2N4, CH2Cl2; vii. succinic anhydride, DMAP,
Py; viii. Me3SiCl, Py; ix. 2,4,6ꢀPri3C6H2SO2Cl, Nꢀmethylimidazole.
groups existing in natural nucleosides may be retained in
the modified oligonucleotides.5,35—39 The incorporation
of 2ꢀaminopropaneꢀ1,3ꢀdiolꢀbased units in the oligoꢀ
nucleotide results in the formation of a diastereomer mixꢀ
ture; however, for most purposes, this is not crucial.5,35—39
of treatment and about 98% in the case of repeated
treatment.
The sequence of postꢀsynthetic treatments of the modiꢀ
fied oligonucleotides was dictated by their structural feaꢀ
tures. Scheme 3 shows the postꢀsynthetic procedure for a
modified oligonucleotide containing a residue of comꢀ
pound 4. The baseꢀlabile protecting groups were removed
by treatment with a solution of ammonia in anhydrous
ethanol,34 which is a conventional procedure for oligoꢀ
ribonucleotides with standard protecting groups (16 h,
55 °C).40—42
We attempted to select milder conditions for the reꢀ
moval of protecting groups and tested a saturated solution
of ammonia in anhydrous MeOH (60 h, 20 °C or 16 h,
55 °C) and a 2 M solution of ammonia in anhydrous
EtOH (60 h, 20 °C (see Ref. 32) or 16 h, 55 °C). The
standard treatment with 25% aqueous ammonia (16 h,
55 °C) was used as the reference. The reaction mixtures
were analyzed by reversedꢀphase HPLC (see, for example,
Fig. 1, a). It was found that all the conditions mentioned
ensure the quantitative removal of the protecting groups
1
Compounds 3 and 4 were characterized by H and
31P NMR spectroscopy and mass spectrometry.
Using the obtained phosphoramidite derivatives 3
and 4 and the modified polymeric support 5, we syntheꢀ
sized a set of oligodeoxyribonucleotides with 5´ꢀterminal,
intrastrand, and 3´ꢀterminal modifications (Table 1).
The oligonucleotide synthesis was carried out using
the phosphoramidite derivatives of nucleosides with conꢀ
ventional protecting groups according to a standard
phosphoramidite protocol.
At the stage of incorporation of modified units 3 and 4,
the condensation time was increased to 5 min. Initially,
we repeated treatment with derivatives 3 and 4 twice;
however, the incorporation efficiency is rather high
even after a single treatment. The efficiency of the attachꢀ
ment of the modified units was at least 90% in the case

New hydrazideꢀcontaining oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005 2675
Table 1. Hydrazideꢀcontaining oligonucleotides and their nonmodified analogs
Oligonucꢀ
leotide
Primary structure
MALDIꢀTOF mass spectra, [M – H]–
Structure of the units Y1—Y3
(5´→3´)
Found
/
Molecular
formula
Calculated
I
tgg tag ccg cta gat
4607.005
4608.12
4535.971
4537.14
4904.208
4905.67
4833.173
4834.83
4787.104
4788.25
4716.070
4717.48
5084.307
5085.12
5013.271
5014.85
4600.015
4602.16
5262.856a
5265.47a
4519.966
4521.04
4897.217
4898.39
4817.168
4817.96
4593.024
4594.27
5022.340
5023.27
4905.237
4905.98
4718.147
4719.09
С147Н184N57O89P14
С145Н183N56O87P14
С155Н200N60O96P15
С153Н199N59O94P15
С151Н193N59O93P15
С149Н192N58O91P15
С159Н209N62O100P16
С157Н208N61O98P16
С145Н187N58O89P14
С182Н233N58O98P14Sia
С143Н187N54O89P14
С153Н203N61O96P15
С151Н203N57O96P15
С143Н190N59O89P14
С163Н206N60O97P15
С159Н199N59O94P15
С153Н193N58O90P14
II
atc tag cgg cta cca
III
IV
V
tgg tag ccg cta gat Y3
atc tag cgg cta cca Y3
Y1 tgg tag ccg cta gat
Y1 atc tag cgg cta cca
Y1 tgg tag ccg cta gat Y3
Y1 atc tag cgg cta cca Y3
tgg tag ccg cY2a gat
VI
VII
VIII
IX
X
atc tY2g cgg cta cca
XI
Y2 tgg tag ccg cY2a gat
atc tY2g cgg cta cca Y3
tgg Y2ag ccg cY2a gat
tgg tag ccg cta gat Y3A
Y1A tgg tag ccg cta gat
tgg tag ccg cY2Aa gat
XII
XIII
XIVb
XVb
XVIb
a For oligonucleotides IX, the calculated and found masses of the oligonucleotides with 5´ꢀdimethoxytrityl group after treatment with
a concentrated solution of ammonia in anhydrous ethanol are also given.
b Oligonucleotides XIV—XVI are anisaldehyde conjugates of oligonucleotides III, V, and IX, respectively. Y1A, Y2A, Y3A are
anisaldehyde adducts of units Y1, Y2, Y3, respectively.
from nonmodified oligodeoxyribonucleotides. Subseꢀ
quently, modified oligonucleotides were deprotected by
treatment with a saturated solution of ammonia in anhyꢀ
drous ethanol for 17 h at 55 °C (see Scheme 3).
After treatment with ammonia, modified oligonucleoꢀ
tides were isolated by reversedꢀphase HPLC. Their strucꢀ
tures were confirmed by MALDIꢀTOF mass spectrometry.
The spectra contained peaks with m/z corresponding to
the [M – H]– and [M – H – (MeO)2Tr]– ions (see, for
example, Fig. 2, a). The dimethoxytrityl group was reꢀ
moved by treatment with 80% AcOH (the same proceꢀ
dure was applied when using derivative 5). Then oligoꢀ
nucleotides were treated with a solution of Bu4NF in
anhydrous THF, which resulted in elimination of the
tertꢀbutyldimethylsilyl group (see Scheme 3); after that,
hydrazide was liberated under mild alkaline conditions.
To this end, oligonucleotides were treated with 12.5%
aqueous ammonia for 15 min at room temperature (see
Scheme 3). Free oligonucleotides were analyzed and
isolated by reversedꢀphase HPLC. This revealed a major
peak, which always comprised at least 80% (see, for
example, Fig. 1, b). The structures of the target hydrꢀ
azideꢀcontaining oligonucleotides were confirmed by
MALDIꢀTOF mass spectrometry (see Table 1); the specꢀ
tra exhibited peaks corresponding to [M – H]– ions (see,
for example, Fig. 2, b).

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Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005
Antsypovich et al.
Scheme 3
R1, R2 are fragments of protected oligonucleotide; R3, R4 are fragments of the deprotected oligonucleotide; B, B´ are the nucleic base
residues.
Reagents and conditions: 1) NH3/EtOH, 17 h, 55 °C; 2) 80% AcOH, 30 min, 20 °C; 3) Bu4N+F–, THF, 18 h, 20 °C; 4) 12.5% NH4OH,
15 min, 20 °C.
Scheme 4
R1, R2 are oligonucleotide chain fragments.
Oligonucleotides containing a hydrazide function reꢀ
acted with anisaldehyde (Scheme 4). The yield of the
conjugates was as high as 60% (the reaction conditions
were not optimized); they had longer retention times in
the reversedꢀphase HPLC. The adduct of oligonucleoꢀ
tide IX (see Table 1) with anisaldehyde was isolated by
reversedꢀphase HPLC (Fig. 3), its structure being conꢀ
firmed by MALDIꢀTOF mass spectrometry; the specꢀ
trum displayed a peak corresponding to the [M – H]– ion
(see Fig. 2, c).
Thus, the method we proposed for the synthesis of
phosphoramidites 3—5 as the starting compounds for the
preparation of oligodeoxynucleotides provides a facile
route to modified oligonucleotides containing hydrazide
functions in any specified positions of the oligomer, i.e.,
at the 3´ꢀ and 5´ꢀends and within the chain. This is atꢀ
tained using the standard automated phosphoramidite oliꢀ
gonucleotide synthesis. The preparation of oligonucleoꢀ
tides containing several hydrazide functions in the same
chain is also possible.

New hydrazideꢀcontaining oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005 2677
I (rel. units)
A260
5265.47
26.00
100
150
80
60
40
20
0
a
a
100
4963.42
4000
50
0
8000
12000
16000
m/z
I (rel. units)
4602.16
100
5
10
15
20
25
t/min
80
60
40
20
0
A260
b
7.92
60
40
20
0
b
4000
8000
12000
16000
m/z
I (rel. units)
4719.09
100
5
10
15
t/min
80
60
40
20
0
c
Fig. 1. Reversedꢀphase HPLC analysis of the 5´ꢀdimethoxytrityl
groupꢀcontaining hydrazide derivative of oligonucleotide IX isoꢀ
lated from the reaction mixture after treatment of the polymeric
support with a concentrated solution of ammonia in anhydrous
ethanol (a); after deprotection procedure (b). The conditions of
analysis are presented in the Experimental.
Experimental
4000
8000
12000
16000
m/z
Fig. 2. MALDIꢀTOF massꢀspectrometric analysis of oligonucleꢀ
otide IX with a 5´ꢀdimethoxytrityl group after treatment with a
concentrated solution of ammonia in anhydrous ethanol (calcuꢀ
lated: 5262.856, [M – H]–; found: 5265.47) (a); oligonucleotide
IX isolated after completion of the deprotection procedure (calꢀ
culated: 4600.015, [M – H]–; found: 4602.16) (b); conjugate of
oligonucleotide IX with anisaldehyde (calculated: 4718.147,
[M – H]–; found: 4719.09) (c). The conditions of analysis are
presented in the Experimental.
Commercial reagents (Acros Organics, Aldrich, Bayer,
Fluka, Merck, Pharmacia Biotech, and Sigma) were used. All
anhydrous solvents were distilled prior to use. Unless stated
otherwise, all the reactions were carried out with stirring under
dry nitrogen at room temperature. The reaction mixtures
were concentrated, solutions of the residues in CH2Cl2 were
washed with brine (×2), dried with Na2SO4, concentrated,
and chromatographed on a column with SiO2 Kieselgel 60
(230—400 mesh) (Merck) using dichloromethane— ethanol mixꢀ
tures as eluents.
Thinꢀlayer chromatography was performed on Kieselgel
60 F254 plates (Merck) in the following solvent systems: A, diꢀ
chloromethane; B, dichloromethane—ethanol (95 : 5); C, diꢀ
chloromethane—ethanol (9 : 1); D, dichloromethane—ethanol
(85 : 15); and E, dichloromethane—triethylamine (99 : 1).
The optical absorption and UV spectra of oligonucleotide
solutions were recorded on a Varian Cary 1E spectrophotometer
in a quartz cell with an optical path of 1 mm.
derivatives 3 and 4 were measured at 400.131 МHz and the
31P NMR spectra, at 161.992 МHz on a Bruker DRXꢀ400 inꢀ
strument.
Positiveꢀion mass spectra (electrospray ionization, ESI) were
obtained on a Bruker Esquire LC 00153 mass spectrometer.
The negative ion MALDIꢀTOF mass spectra of oligonucleꢀ
otides were recorded on Voyager DEꢀRP (Perseptive Biosystems)
and Bruker Daltonics Autoflex instruments. The samples were
exposed to the radiation of a nitrogen laser with a wavelength of
337 nm. The laser desorptionꢀgenerated ions were energetically
1H NMR spectra were recorded at 200.131 МHz on a Bruker
DPXꢀ200 instrument; the 1H NMR spectra of phosphoramidite

2678
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005
Antsypovich et al.
A260
2ꢀTrifluoroacetamidopropaneꢀ1,3ꢀdiol (12). Anhydrous pyꢀ
ridine (10 mL) was twice added to, and distilled from, aminoꢀ
propaneꢀ1,3ꢀdiol (11) (3.64 g, 40 mmol) and the residue was
dissolved in a mixture of anhydrous pyridine (20 mL) and anꢀ
hydrous CH2Cl2 (20 mL). Ethyl trifluoroacetate (9.52 mL,
80 mmol) was added with stirring. After 24 h, the solvents and
the remaining CF3CO2Et were evaporated. Anhydrous benzene
(20 mL) and then anhydrous CH2Cl2 (20 mL) were added to,
and distilled from, the residue . The resulting compound 12 was
used without further purification. Yield 7.26 g (97.0%), Rf 0.2 (C).
1H NMR (CDCl3), δ: 3.93 (m, 5 H, CH2CHCH2). MS, found:
m/z 187.9 [M + H]+. C5H8F3NO3. Calculated: [М + H] =
188.117.
9.72
8.43
40
30
a
20
10
0
–10
1ꢀOꢀ(4,4´ꢀDimethoxytrityl)ꢀ2ꢀtrifluoroacetamidopropaneꢀ
1,3ꢀdiol (13). A solution of 4,4´ꢀdimethoxytrityl chloride (8.47 g,
25 mmol) in anhydrous CH2Cl2 (100 mL) was slowly added
dropwise with stirring to a solution of compound 12 (7.26 g,
38.8 mmol) in anhydrous pyridine (150 mL). After 1.5 h, the
reaction mixture was concentrated, the residue was dissolved
in CH2Cl2 (200 mL), and the solution was washed with a satuꢀ
rated aqueous solution of NaHCO3, brine, and water. The orꢀ
ganic phase was separated, dried for 2 h with anhydrous sodium
sulfate, and concentrated. The residue was chromatographed on
a column with SiO2 using solvent systems A and B. The yield
was 3.38 g (27.6%), Rf 0.6 (B). 1H NMR (DMSOꢀd6), δ:
3.11 (m, 2 H, CHCH2OH); 3.53 (m, 3 H, TrOCH2CH);
3.79 (s, 6 H, CH3O); 6.93 (d, 4 H, C6H4, J = 8.9 Hz);
7.28 (d, 4 H, C6H4); 7.40 (m, 5 H, C6H5). MS, found:
m/z 488.4 [M + H]+. C26H27F3NO5. Calculated: [М + H]+ =
490.184.
2ꢀAminoꢀ1ꢀOꢀ(4,4´ꢀdimethoxytrityl)propaneꢀ1,3ꢀdiol (14).
A 33% aqueous solution of ammonia (100 mL) was added to a
solution of compound 13 (3.0 g, 6.13 mmol) in ethanol (100 mL).
After 2 h at 55 °C and for 48 h at room temperature, the reaction
mixture was concentrated, and the residue (oil) was dissolved in
CH2Cl2 (100 mL). The solution was washed with a saturated
aqueous solution of NaHCO3 (100 mL) and brine, dried for 2 h
with anhydrous sodium sulfate, concentrated, and chromatoꢀ
graphed on a column with SiO2 with solvent systems A—D as
eluents. Yield 1.55 g (64.3%), Rf 0.2 (C). 1H NMR (DMSOꢀd6),
δ: 2.89 (m, 3 H, CHCH2OH); 3.34 (m, 2 H, TrOCH2); 3.74 (s,
6 H, CH3O); 6.89 (d, 4 H, C6H4, J = 8.7 Hz); 7.25 (d, 4 H,
C6H4); 7.38 (m, 5 H, C6H5). MS, found: m/z 416.2 [M + Na]+.
C24H27NO4. Calculated: [М + Na] = 416.465.
5
10
15
20
25
t/min
A260
9.62
b
30
20
10
8.27
0
–10
5
10
15
20
25
t/min
Fig. 3. Reversedꢀphase HPLC analysis of the reaction mixture
during the synthesis of conjugate of the hydrazideꢀcontaining
oligonucleotide IX with anisaldehyde (a); the isolated conjuꢀ
gate (b). The conditions of analysis are presented in the Experiꢀ
mental.
stabilized during the extraction period of 300 ns and then diꢀ
rected through a linear TOF mass analyzer with a voltage of 25
or 20 kV (for the Daltonics Autoflex instrument). 3ꢀHydroxyꢀ
pyridineꢀ2ꢀcarboxylic acid in a mixture of acetonitrile and water
(1 : 1) was used as the matrix.
Analysis of the reaction mixtures and isolation of the target
modified oligonucleotides were carried out by reversedꢀphase
HPLC on Kontron chromatographs (Kontron Instruments) usꢀ
ing 4×250 mm CC 250/4 Nucleodur 100ꢀ5 C18 ec columns
(MachereyꢀNagel), particle size 7 µm, elution rate 1 mL min–1
(~20 °C). For analysis and isolation of oligonucleotides conꢀ
taining a dimethoxytrityl group, a gradient of acetonitrile
(5—35%) in a 0.1 M aqueous solution of ammonium hydroꢀ
gencarbonate (30 min, 1% per min) was used. In other cases, a
gradient of acetonitrile (5—20%, 0.5% per min, 30 min) was used.
4ꢀHydroxybutyrohydrazide (7). γꢀButyrolactone (15.37 mL,
200 mmol) was dissolved in ethanol (200 mL), and a 35% aqueꢀ
ous solution of hydrazine (18.31 mL, 200 mmol) was added. The
reaction mixture was refluxed for 20 h and cooled, the solvents
were evaporated in vacuo, and the residue was dried in vacuo.
Yield 22.9 g (96.8%). The product was used without further
Diethyl 2ꢀtertꢀbutyl(dimethyl)silyloxymethylꢀ2ꢀhydroxyꢀ
methylmalonate (8). Anhydrous pyridine (20 mL) was twice added
to, and distilled from, diethyl 2,2ꢀbis(hydroxymethyl)malonate
(6) (22.02 g, 100 mmol) and the residue was dissolved in a
mixture of anhydrous pyridine (50 mL) and anhydrous CH2Cl2
(50 mL). This solution was added dropwise to a stirred solution
of tertꢀbutyl(dimethyl)silyl chloride (7.54 g, 50 mmol) and
DMAP (610 mg, 5 mmol) in a mixture of anhydrous pyridine
(50 mL) and anhydrous CH2Cl2 (50 mL), and the mixture was
stirred for 48 h. The usual workup gave compound 8, yield
1
13.20 g (79.0%), Rf 0.5 (A). H NMR (CDCl3), δ: 0.05 (s, 6 H,
(CH3)2Si); 0.85 (s, 9 H, (CH3)3CSi); 1.27 (t, 6 H, CH3CH2, J =
7.2 Hz); 4.06 (s, 2 H, CH2OSi); 4.11 (s, 2 H, CH2OH); 4.22 (q,
4 H, CH3CH2O). MS, found: m/z 335.3 [M + H]+. C15H30O6Si.
Calculated: [М + H] = 335.481.
Diethyl 2ꢀtertꢀbutyl(dimethyl)silyloxymethylꢀ2ꢀ(pꢀnitroꢀ
phenoxycarbonyl)oxymethylmalonate (9). Anhydrous pyridine
1
purification. H NMR (D2O), δ: 1.81 (m, 2 H, CH2CH2OH);
2.27 (t, 2 H, CH2(CH2)2OH, J = 7.6 Hz); 3.59 (t, 2 H, CH2OH,
J = 6.4 Hz). MS, found: m/z 119.2 [M + H]+. C4H10N2O2.
Calculated: [М + H] = 119.134.

New hydrazideꢀcontaining oligonucleotides
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005 2679
(2.67 mL, 33 mmol) and then monohydroxy compound 8
(10.03 g, 30 mmol) were added with stirring to a solution of
pꢀnitrophenyl chloroformate (6.35 g, 31.5 mmol) and DMAP
(366 mg, 10 mmol) in anhydrous CH2Cl2 (200 mL). After 2.5 h,
the precipitate was filtered off and the filtrate was concentrated.
The usual workup gave compound 9, yield 10.95 g (73.0%),
(CH3)2Si); 0.83 (s, 9 H, (CH3)3CSi); 1.23 (t, 6 H, CH3CH2,
J = 7.2 Hz); 2.13 (m, 2 H, CH2CH2OH); 2.40 (t, 2 H,
CH2(CH2)2OCO, J = 7.1 Hz); 4.04 (s, 2 H, CH2OSi); 4.18 (q,
4 H, CH3CH2O); 4.36 (t, 2 H, CH2CH2OCO, J = 6.2 Hz); 4.65
(s, 2 H, CH2OCO); 7.38 (d, 2 H, C6H4, J = 9.4 Hz); 8.27 (d,
2 H, C6H4). MS, found: m/z 644.4 [M + H]+. C27H41N3O13Si.
Calculated: [М + H] = 644.712.
1
Rf 0.7 (A). H NMR (CDCl3), δ: 0.05 (s, 6 H, (CH3)2Si); 0.86
(s, 9 H, (CH3)3CSi); 1.27 (t, 6 H, CH3CH2, J = 7.1 Hz); 4.13 (s,
2 H, CH2OSi); 4.23 (q, 4 H, CH3CH2O); 4.81 (s, 2 H,
CH2OCO); 7.35 (d, 2 H, C6H4, J = 9.2 Hz); 8.27 (d, 2 H,
C6H4). MS, found: m/z 500.3 [M + H]+. C22H33NO10Si. Calcuꢀ
lated: [М + H] = 500.584.
Diethyl 2ꢀtertꢀbutyl(dimethyl)silyloxymethylꢀ2ꢀ[2ꢀ(4ꢀ{Nꢀ
[1ꢀ(4,4´ꢀdimethoxy)trityloxyꢀ3ꢀhydroxypropanꢀ2ꢀyl]carbamoylꢀ
oxy}butyryl)hydrazino]carbonyloxymethylmalonate (16). Comꢀ
pound 15 (2.49 g, 3.86 mmol), anhydrous pyridine (344 µL,
4.25 mmol), and DMAP (47 mg, 0.386 mmol) were added to a
solution of 2ꢀaminoꢀ1ꢀOꢀ(dimethoxytrityl)propaneꢀ1,3ꢀdiol (14)
(1.52 g, 3.86 mmol) in anhydrous CH2Cl2 (25 mL), and the
mixture was stirred for 16 h. The usual workup gave 2.16 g
Diethyl 2ꢀtertꢀbutyl(dimethyl)silyloxymethylꢀ2ꢀ[2ꢀ(4ꢀhydrꢀ
oxybutyryl)hydrazino]carbonyloxymethylmalonate (10). 4ꢀHydrꢀ
oxybutyrohydrazide (7) (2.6 g, 22 mmol) was dissolved in anꢀ
hydrous DMF (100 mL); pꢀnitrophenyl carbonate 9 (10.94 g,
21.9 mmol), anhydrous pyridine (1.618 mL, 20 mmol), and
DMAP (244 mg, 2 mmol) were added. After 16 h, the solvent
was evaporated. After the usual workup of the reaction mixture,
the residue was chromatographed on a column with SiO2 using
solvent systems A—C for elution to give compound 10, yield
1
(62.4%) of compound 16, Rf 0.6 (C). H NMR (DMSOꢀd6), δ:
0.02 (s, 6 H, (CH3)2Si); 0.81 (s, 9 H, (CH3)3CSi); 1.16 (t, 6 H,
CH3CH2, J = 6.9 Hz); 1.78 (m, 2 H, CH2CH2CONHNH); 2.38
(m, 2 H, CH2CONHNH); 2.95 (m, 2 H, CH2OH); 3.48 (t, 2 H,
CH2(CH2)2CONHNH, J = 5.3 Hz); 3.73 (s, 6 H, CH3O); 3.94
(s, 2 H, CH2OSi); 3.95 (m, 2 H, TrOCH2); 4.14 (q, 4 H,
CH3CH2O); 4.40 (s, 2 H, CH2OCONHNH); 4.57 (m, 1 H,
TrOCH2CH); 6.87 (d, 4 H, C6H4, J = 8.8 Hz); 7.23 (d, 4 H,
C6H4); 7.34 (m, 5 H, C6H5). MS, found: m/z 898.7 [M + H]+.
C45H63N3O14Si. Calculated: [М + H] = 899.079.
1
6.34 g (66.3%), Rf 0.5 (C). H NMR (CDCl3), δ: 0.03 (s, 6 H,
(CH3)2Si); 0.84 (s, 9 H, (CH3)3CSi); 1.24 (t, 6 H, CH3CH2,
J = 7.2 Hz); 1.89 (m, 2 H, CH2CH2OH); 2.38 (t, 2 H,
CH2(CH2)2OH, J = 6.8 Hz); 3.70 (t, 2 H, CH2CH2OH, J =
6.1 Hz); 4.05 (s, 2 H, CH2OSi); 4.19 (q, 4 H, CH3CH2O);
4.65 (s, 2 H, CH2OCO). MS, found: m/z 479.3 [M + H]+.
C20H38N2O9Si. Calculated: [М + H] = 479.609.
Diethyl 2ꢀtertꢀbutyl(dimethyl)silyloxymethylꢀ2ꢀ{2ꢀ[4ꢀ(Nꢀ
{1ꢀ(4,4´ꢀdimethoxy)trityloxyꢀ3ꢀ[(diisopropylamino)(2ꢀcyanoꢀ
ethoxy)phosphinoxy]propanꢀ2ꢀyl}carbamoyloxy)butyryl]hydrꢀ
azino}carbonyloxymethylmalonate (4). Diisopropylamine tetrꢀ
azolide (154 mg, 0.9 mmol) was added to compound 16 (674 mg,
0.75 mmol), anhydrous MeCN (10 mL) was twice added to, and
distilled from, the mixture, and the residue was dissolved in
anhydrous CH2Cl2 (15 mL). 2ꢀCyanoethyl bis(N,Nꢀdiisopropyl)
phosphorodiamidite (571 µL, 1.8 mmol) was added with stirꢀ
ring. After 25 min, the reaction mixture was workedꢀup in the
usual way, as described for compound 3 (dried in vacuo for
16 h). The yield of compound 4 was 824 mg (91.0%). Rf 0.75 (B).
The resulting compound 4 was used in the oligonucleotide synꢀ
Diethyl 2ꢀtertꢀbutyl(dimethyl)silyloxymethylꢀ2ꢀ(2ꢀ{4ꢀ[(diisoꢀ
propylamino)(2ꢀcyanoethoxy)phosphinoxy]butyryl}hydrazino)carꢀ
bonyloxymethylmalonate (3). Anhydrous MeCN (5 mL) was twice
added to, and distilled from, compound 10 (263 mg, 0.55 mmol)
and diisopropylamine tetrazolide (113 mg, 0.66 mmol), and
the residue was dissolved in anhydrous CH2Cl2 (10 mL).
2ꢀCyanoethyl bis(N,N´ꢀdiisopropyl) phosphorodiamidite
(420 µL, 1.32 mmol) was added dropwise with stirring. After
30 min, the reaction mixture was washed with cooled brine, the
organic phase was dried for 2 h with anhydrous sodium sulfate
and concentrated, and the residue was dried for 24 h in vacuo.
Cold anhydrous pentane was added, and the mixture was kept at
4 °C for 16 h. The supernatant was decanted, and the residue
was dried in vacuo for 48 h and used in the oligonucleotide
synthesis without further purification. The yield of compound 3
1
thesis without further purification. H NMR (CDCl3), δ: 0.03
(s, 6 H, (CH3)2Si); 0.84 (s, 9 H, (CH3)3CSi); 1.18 (m, 12 H,
((CH3)2CH)2N); 1.26 (t, 6 H, CH3CH2, J = 6.3 Hz); 1.94 (m,
2 H, CH2CH2CONHNH); 2.22 (t, 2 H, CH2CONHNH, J =
6.6 Hz); 2.64 (m, 2 H, CHCH2OP); 2.74 (t, 2 H, CH2CN, J =
6.0 Hz); 3.53 (m, 4 H, CH2CH2CN; CH2(CH2)2CONHNH);
3.69 (m, 2 H, ((CH3)2CH)2N); 3.78 (s, 6 H, CH3O); 4.00 (m,
1 H, TrOCH2CH); 4.05 (s, 2 H, CH2OSi); 4.12 (m, 2 H,
1
was 351 mg (94.0%), Rf 0.7 (C). H NMR (CDCl3), δ: 0.03 (s,
6 H, (CH3)2Si); 0.84 (s, 9 H, (CH3)3CSi); 1.18 (m, 12 H,
((CH3)2CH)2N); 1.26 (t, 6 H, CH3CH2, J = 6.0 Hz); 1.96 (m,
2 H, CH2CH2CH2OP); 2.36 (m, 2 H, CH2(CH2)2OP); 2.75 (t,
2 H, CH2CN, J = 6.0 Hz); 3.53 (m, 2 H, CH2CH2CN); 3.60
(m, 2 H, ((CH3)2CH)2N); 3.79 (m, 2 H, (CH2)2CH2OP); 4.05
(s, 2 H, CH2OSi); 4.18 (q, 4 H, CH3CH2O); 4.65 (s, 2 H,
CH2OCO). 31Р: 147.82. MS, found: m/z 679.1 [M + H]+.
C29H55N4O10PSi. Calculated: [М + H] = 679.827.
TrOCH2); 4.18 (q,
4 H, CH3CH2O); 4.65 (s, 2 H,
CH2OCONHNH); 6.81 (d, 4 H, C6H4, J = 9.0 Hz); 7.26 (d,
4 H, C6H4); 7.30—7.40 (m, 5 H, C6H5). 31Р: 148.46; 148.55.
MS, found: m/z 1120.7 [M + Na]+. C54H80N5O15PSi. Calcuꢀ
lated: [М + Na] = 1121.287.
Immobilization of compound 16 on the polymeric support.
AminopropylꢀCPG (80—120 mesh, pore size 700 Å) with amino
group loading of 44 µmol g–1 (500 mg, 22 µmol based on amino
groups) was suspended in pyridine (3 mL), and succinic anꢀ
hydride (220 mg, 2.2 mmol) and DMAP (10.75 mg, 88 µmol)
were added. The polymeric support was degassed and stirred for
12 h, then the polymer was washed on a filter with pyridine
(5×10 mL), ethanol (5×10 mL), diethyl ether (5×10 mL), and
dried in vacuo. After that, the polymeric support was suspended
in pyridine (2 mL), trimethylchlorosilane (400 µL, 3.13 mmol)
Diethyl
2ꢀtertꢀbutyl(dimethyl)silyloxymethylꢀ2ꢀ{2ꢀ[4ꢀ
(pꢀnitrophenoxycarbonyl)oxybutyryl]hydrazino} carbonyloxyꢀ
methylmalonate (15). Compound 10 (6.44 g, 10 mmol) was disꢀ
solved in anhydrous CH2Cl2 (55 mL). Anhydrous pyridine
(970 µL, 12 mmol), then a solution of 4ꢀO2NC6H4OCOCl
(2.22 g, 11 mmol) in anhydrous CH2Cl2 (20 mL), and DMAP
(122 mg, 1 mmol) were added. After 2 h, the reaction mixture
was workedꢀup in a usual way to give compound 15, yield 4.67 g
(72.5%). Rf 0.7 (B). 1H NMR (CDCl3), δ: 0.02 (s, 6 H,

2680
Russ.Chem.Bull., Int.Ed., Vol. 54, No. 11, November, 2005
Antsypovich et al.
was added, and the suspension was degassed and stirred for 16 h.
The support was separated, washed on the filter with pyridine
(5×10 mL), ethanol (5×10 mL), and diethyl ether (5×10 mL),
and dried in vacuo. Then the polymeric support was suspended
in pyridine (1 mL), and 2,4,6ꢀtriisopropylbenzenesulfonyl chloꢀ
ride (60 mg, 200 µmol) and Nꢀmethylimidazole (46.6 µL,
600 µmol) were added. The mixture was degassed and stirred for
30 min. Then compound 16 (79 mg, 88 µmol) and, after 2 h,
methanol (5 mL) were added, and the mixture was stirred for
16 h. Then the polymeric support was separated, washed on the
filter with pyridine (5×10 mL), ethanol (5×10 mL), and diethyl
ether (5×10 mL), and dried in vacuo.
of the target oligonucleotides were confirmed by MALDIꢀTOF
mass spectrometry (see Table 1).
The reaction of hydrazideꢀcontaining oligonucleotides with
anisaldehyde (general procedure). The desalinated aqueous soluꢀ
tion of the hydrazideꢀcontaining oligonucleotide (content of the
oligonucleotide material 5.0 nmol) was concentrated to dryness
and then dissolved in 0.1 М MES buffer (pH 5.0—5.3) (10 µL).
A 1 M solution of 4ꢀmethoxybenzaldehyde in DMF (4 µL) was
added. After 30 or 60 min at 30 or 37 °C, the solution was diluted
to 500 µL with a 1 mM aqueous solution of ammonium acetate,
and the oligonucleotides were desalted using 1 mM aqueous
ammonium acetate for elution. Then the solution was concenꢀ
trated in vacuo to 400 µL, and the reaction mixtures were anaꢀ
lyzed by reversedꢀphase HPLC. The oligonucleotide and anisꢀ
aldehyde conjugates were isolated by HPLC under the same
conditions. The fractions containing conjugates were collected
and concentrated in vacuo to dryness, and the residue was disꢀ
solved in water (400 µL) and analyzed by HPLC (see, for exꢀ
ample, Fig. 3) and MALDIꢀTOF mass spectrometry (see
Table 1).
The resulting specific loading of the polymeric support by
modified units was determined by spectrophotometry. The modiꢀ
fied polymeric support (1 mg) was treated with a 3% solution of
CF3COOH (50 µL) in 50% aqueous CH3CN. The reaction mixꢀ
ture was centrifuged, the supernatant was withdrawn, and the
polymeric support was washed twice with 50% aqueous CH3CN
(425 µL). The washings were separated from the polymeric supꢀ
port by centrifugation, and combined with the supernatant. The
absorbance of the dimethoxytrityl cation present in the solution
was measured in the visible region at 478 nm. The absorbance
thus found was used to calculate the amount of the dimethoxyꢀ
trityl cation. The specific loading of the polymeric support with
the modified units was taken to be equal to the determined
amount of eliminated dimethoxytrityl cation. The specific loadꢀ
ing of the polymeric support with compound 16 was about
S. I. Antsypovich is grateful to the Alexander von
Humboldt Foundation (Germany) for the research felꢀ
lowship. This work was partially supported by the proꢀ
gram "Universities of Russia" (Project No. 05.02.547).
20 µmol g–1
.
References
Oligonucleotide synthesis (general procedure). Modified oliꢀ
gonucleotides (and their natural analogs) were synthesized on a
Gene Assembler automated DNA synthesizer (Pharmacia
Biotech); the phosphoramidite protocol of the oligonucleotide
synthesis was used. The scale of the synthesis was 1.3 µmol. The
condensation was activated by 4,5ꢀdicyanoimidazole. The conꢀ
centration of the modified phosphoramidite derivatives 3 and 4
was 0.12 mol L–1; the time of the condensation step was inꢀ
creased to 5 min. For the synthesis of 3´ꢀmodified oligonucleoꢀ
tides, polymeric support 5 (65 mg) was used.
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polymer was separated and washed with water (3×500 µL), the
aqueous solutions were combined and concentrated in vacuo
(using a Speed Vac Plus 110A vacuum concentrator (Savant)).
The reaction mixtures were analyzed and the target products
were isolated by HPLC.
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Received April 22, 2005;
in revised form June 27, 2005