Synthesis of nucleotide-amino acid conjugates designed for photo-CIDNP experiments by a phosphotriester approach

Article abstract of DOI:10.3762/bjoc.9.326

Conjugates of 2'-deoxyguanosine, L-tryptophan and benzophenone designed to study pathways of fast radical reactions by the photo Chemically Induced Dynamic Nuclear Polarization (photo-CIDNP) method were obtained by the phosphotriester block liquid phase synthesis. The phosphotriester approach to the oligonucleotide synthesis was shown to be a versatile and economic strategy for preparing the required amount of high quality samples of nucleotide-amino acid conjugates.

Full text of DOI:10.3762/bjoc.9.326

Synthesis of nucleotide–amino acid conjugates
designed for photo-CIDNP experiments by a
phosphotriester approach
Tatyana V. Abramova*1,2, Olga B. Morozova2,3, Vladimir N. Silnikov1
and Alexandra V. Yurkovskaya2,3
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2013, 9, 2898–2909.
1Institute of Chemical Biology and Fundamental Medicine, SB RAS,
Lavrent’ev Ave, 8, Novosibirsk 630090, Russia, 2Novosibirsk State
University, Pirogova St. 2, Novosibirsk 630090, Russia and
3International Tomography Center, SB RAS, Institutskaya 3a,
Novosibirsk 630090, Russia
doi:10.3762/bjoc.9.326
Received: 23 September 2013
Accepted: 15 November 2013
Published: 18 December 2013
Email:
Associate Editor: D. Spring
Tatyana V. Abramova* - abramova@niboch.nsc.ru
© 2013 Abramova et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
CIDNP; nucleotide–amino acid conjugates; oligonucleotide synthesis;
phosphotriester approach
Abstract
Conjugates of 2’-deoxyguanosine, L-tryptophan and benzophenone designed to study pathways of fast radical reactions by the
photo Chemically Induced Dynamic Nuclear Polarization (photo-CIDNP) method were obtained by the phosphotriester block liquid
phase synthesis. The phosphotriester approach to the oligonucleotide synthesis was shown to be a versatile and economic strategy
for preparing the required amount of high quality samples of nucleotide–amino acid conjugates.
Introduction
Maintaining the integrity of the genome is of paramount bio- endogenous natural and synthetic compounds, which were
logical importance, since the damage of DNA is considered to extensively studied in chemical systems [1]. This “chemical
cause aging and various degenerative diseases. To prevent the way” of the DNA repair efficiently competes with the forma-
pathological DNA damage, cells evolve the DNA repair ma- tion of modified sites, which are targets for the enzymatic
chinery, which restores the chemical information encoded in repair. Since DNA radicals, like all others, are extremely chemi-
genome. In addition to the enzymatic DNA repair system, a new cally active, they are short-lived, and their concentration is very
and quite different repair mechanism, i.e. the non-enzymatic low to be detected by the conventional electron paramagnetic
repair, has been discovered. This non-enzymatic system refers resonance method. An alternative approach has been developed
to the removal of transient products of the DNA damage like for the indirect detection of elusive radicals by utilizing nuclear
short-lived DNA radicals, with a very high reaction rate by spin hyperpolarization, which occurs in transient radical pairs
2898

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
on a microsecond time scale. Nuclear spin hyperpolarization is Guanosine is the most easily oxidized nucleoside [12] in
preserved in diamagnetic products of chemical reactions for photoreactions of which pronounced CIDNP effects could be
several seconds allowing for NMR detection. This approach is detected [13]. Tryptophan was found as one of the most effi-
based on the phenomenon of Chemically Induced Dynamic cient reducing agents for different protonated forms of oxidized
Nuclear Polarization (CIDNP).
guanosine 5′-monophosphate in a wide pH range [14]. The 2,2’-
dipyridyl dye, which is used as a photosensitizer in most of our
Nowadays, the time-resolved variant of CIDNP (TR CIDNP) TR CIDNP experiments, has a disadvantage of a relatively low
becomes a powerful method in the investigation of vitally absorbance at a wavelength of 308 nm at physiological pH. As
important processes with participation of biological macromole- an alternative, in our investigations water soluble carboxylic
cules [2-5]. The possibility of tracking the electron transfer in derivatives of benzophenone (namely, 4-carboxy-, 3-carboxy-
the reactions of damaged DNA bases by NMR using in situ and 3,3’,4,4’-tetracarboxybenzophenone) were used as efficient
photoinitiation and the TR CINDP detection opens a new way photoinitiating agents for electron transfer from nucleotides and
to profound investigation of mechanisms of DNA repair. amino acids [15-18].
Preliminary experiments in this area [6,7] revealed the neces-
sity to study electron transfer processes in detail using a wide Based on the above mentioned reasons, we have chosen
number of specially designed model compound conjugates of 4-benzoylbenzoic acid, 2’-deoxyguanosine, and L-tryptophan
the amino acid, nucleotide, and dye residues, where key partici- [7,9,13-20] as the dye, nucleoside, and amino acid building
pants (dye, amino acid, and nucleoside) have to be drawn blocks, respectively, to construct conjugates 1–8 (Figure 1)
together mimicking the biologically important DNA repair linked in the uniform manner through the phosphate groups.
processes.
Conjugates 1 and 6 (Figure 1) are binary molecules combining
To fulfill the requirements of the photo-induced TR CIDNP the dye and nucleoside residues or the amino acid and nucleo-
experiments, a synthetic strategy for obtaining the model conju- side residues, respectively. Conjugates 2 and 3 include all three
gates should provide a synthetic versatility, easy scaling up, and residues differing in length of the linker between the amino acid
high purity of the title compounds. Although there is a number and nucleoside residues. Since the amino acid–nucleoside
of well-developed methods of the automatic solid phase conjugates linked through the phosphamide bond have insuffi-
supported synthesis (SPSS) of oligonucleotide–peptide conju- cient stability even at neutral pH [21], we used L-tryptophanol
gates [8], this strategy does not ensure the availability of the instead of L-tryptophan in conjugates 3 and 5 which do not
model conjugates for TR NMR photo-CIDNP experiments due have the linker group between the amino acid and nucleoside
to the lack of versatility and difficulties in scaling up the residues to avoid this problem. Phosphodiester linkages in
process. As shown in [9], at least 0.01–0.03 mmol of a com- conjugates 1–8 are stable in a wide pH range.
pound is required for each photo-CIDNP experiment.
Conjugates 4 and 5 containing no guanine nucleobase are
In this report, we have designed conjugates consisting of control compounds for conjugates 2 and 3, respectively. Prelim-
amino acid, nucleotide, and dye residues including linkers of inary results revealed a rapid reduction (with the characteristic
different lengths (Figure 1). Target compounds 1–8 have been time of less than 1 μs) of the guanosyl radical by means of elec-
synthesized by the phosphotriester block liquid phase synthesis tron transfer from the linked tryptophan moiety in conjugate 6.
(LPS).
This efficient reduction was strictly confirmed by comparing
CIDNP kinetics of the photoreaction of conjugate 6 (with 2,2’-
dipyridyl as photosensitizer) and CIDNP kinetics of the
photoreactions of conjugates 7 or 8 under the same conditions.
Results and Discussion
Design of model compounds
Our previous studies revealed a great potential of the TR NMR
Synthesis of model compounds for time
photo-CIDNP technique for the investigation of electron
transfer in oxidized peptides and between oxidized DNA resolved photo-CIDNP NMR experiments
nuclear bases and amino acids and small peptides [10,11]. The It is easy to notice that conjugates 1–8 are designed as a kind of
further development of this work implies the investigation of di- and trimer nucleotide species. So, the key points of its syn-
the electron transfer processes and the detection of elusive radi- thesis are the choice of the strategy (SPSS or LPS) and the
cals in biomolecules using model compounds, where key partic- method of nucleotide condensation. As mentioned in review
ipants of the reaction (dye, amino acid, and nucleoside) are [22], LPS is the preferable strategy for obtaining semi-prepara-
spatially drawn together mimicking the biologically important tive and preparative quantities of short oligonucleotides. This
DNA repair processes.
strategy was successfully used in the synthesis of native oligo-
2899

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
Figure 1: Model compounds for the TR NMR photo-CIDNP experiments: conjugates of 4-benzoylbenzoic acid, 2’-deoxyguanosine, L-tryptophan, and
L-tryptophanol.
deoxynucleotides and phosphorothioate hexa-2’-oligodeoxy- having in hands the key suitably protected 5’-phosphorylated
nucleotides by the phosphoramidite method [23,24]. Moreover, 2’-deoxyguanosine derivative [27], we used the LPS strategy in
LPS was employed for preparing amino acid–nucleotide conju- combination with the phosphotriester approach for the oligonu-
gates [25] and trinucleoside blocks [26] further used in SPSS. cleotide synthesis to obtain target conjugates 1–8. The
The method of nucleotide coupling in LPS presumably depends phosphoramidite condensation in LPS usually leads to the lower
on the availability of parent compounds and is not limited to the yields in these reactions due to insufficient stability of active
use of more active P(III) nucleotide derivatives.
phosphoramidites and the absence of the condensation agent,
which dries the solvent and other reagents in the reaction mix-
Taking into account the need for obtaining semi-preparative ture [28]. Functionalized derivatives of commercially available
amounts of conjugates sufficient for TR NMR CIDNP and 4-benzoylbenzoic acid, L-tryptophan, and L-tryptophanol, were
2900

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
synthesized by the activation of the carboxylic groups of the Target conjugates 1–8 were obtained after the deprotection of
suitably protected samples to obtain their active esters followed fully blocked derivatives 18, 20, 21, 25, 28, 29, 31, and 33. The
by the introduction of the additional linker moiety and phos- pretreatment of these phosphotriester derivatives with TBAF
phorylation, if necessary (Scheme 1), according to the well- under neutral conditions before aqueous ammonia prevented the
known procedures [29-31].
cleavage of the linker containing the oxoethyl fragment because
of β-elimination.
The synthetic routes for obtaining conjugates 1–8 are depicted
in Scheme 2, Scheme 3, and Scheme 4. 5-O-(4,4'-Dimethoxy- It is worthy to notice that the TBAF treatment is essential when
trityl)-1,4-anhydro-2-deoxy-D-ribitol (22) was obtained as the phosphotriester group contains the aromatic residue
described in [32]. The synthetic approach outlined in Scheme 2, (p-chlorophenyl in our case), which is much more stable to
Scheme 3, and Scheme 4 includes the phosphtriester conden- ammonia than the 2-cyanoethyl group usually used in the
sation of specially designed and suitably protected blocks con- phosphoramidite oligodeoxynucleotide synthesis. After the
taining dye 9, nucleotide 17 or its abasic analogues 22 and 32, selective cleavage of the aromatic phosphotriester bond by the
and the amino acid (or its reduced derivative) 11, 13, 15, and F− anion, the oxoethyl fragment is quite stable to β-elimination.
16. A series of these blocks and the standard LPS procedures
provided a versatile route to obtain semi-preparative amounts We succeeded in the mild deprotection of derivative 20 contain-
(100–200 mg) of the model compounds for the TR CIDNP ing the Boc protective group with formic acid but, to our
NMR experiments.
surprise, we failed in this way when the L-tryptophan moiety
Scheme 1: Synthesis of functionalized derivatives of 4-benzoylbenzoic acid (9), L-tryptophan (11, 16) and L-tryptophanol (13). i) N,N’-Dicyclohexyl-
carbodiimide (DCC), N-hydroxysuccinimide (NHS), 1,4-dioxane, then 2(2-aminoethoxy)ethanol); ii) 2(2-aminoethoxy)ethanol), triethylamine (TEA),
1,4-dioxane; iii) p-chlorophenyl dichlorophosphate, 1,2,4-triazole, pyridine (Py); iv) ethyl trifluoroacetate, TEA, MeOH.
2901

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
Scheme 2: Synthesis of conjugates 1–3: i) 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl), 1-methylimidazole (MeIm), Py; ii) tetrabutylammonium
fluoride (TBAF) in Py/H2O, then NH3/H2O; iii) N2H4/H2O in Py/AcOH; iv) TBAF in Py/H2O, then NH3/H2O, then HC(O)OH.
was linked to the 5’-OH group of 2’-dG (conjugate 6). In this protective group for the linker to obtain conjugate 7 because
case, the deprotection was accompanied by the cleavage of the such protection is orthogonal to the protective groups of
guanine base at pH 1–5 in aqueous solutions and under the nucleotide block 17.
action of a TFA/CH2Cl2 mixture. So, we replaced Boc by the
trifluoroacetyl protective group and synthesized the additional After the deprotection, target conjugates 1–8 were purified by
building block 15 to obtain conjugate 6 (Scheme 4).
the anion exchange and reversed phase chromatography (RPC)
at a medium pressure, which provides the high purity of the
Keeping in mind the possibility of a further modification of final products and easy scaling up in laboratory conditions. The
fully protected derivative 31, we chose the acid labile MMTr homogeneity and structures of all key intermediates and final
2902

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
Scheme 3: Synthesis of conjugates 4 and 5: i) TPSCl, MeIm, Py; ii) AcOH/H2O; iii) p-chlorophenyl dichlorophosphate, 1,2,4-triazole, Py; iv) TBAF in
Py/H2O, then HC(O)OH; v) TBAF in Py/H2O, then NH3/H2O.
compounds were confirmed by thin-layer chromatography radical reactions with microsecond time resolution by Chemi-
(TLC), RPC, NMR and mass-spectrometry analysis.
cally Induced Dynamic Nuclear Polarization (CIDNP). Based
on our fruitful experience in the CIDNP investigation of conju-
gates 1, 6, 7, and 8 (will be published soon separately), we are
Conclusion
We designed and synthesized a number of model conjugates to confident that the expansion of this study to other analogous
study the role of electron transfer and the elusive radical forma- model systems is worth doing for elucidation of complex chem-
tion in biologically significant processes. By any standards, the istry, which is developed in nature for maintaining the integrity
use of the hyperpolarization approach is extraordinary to eluci- of the genome encoded in DNA
date the interactions between hidden transient intermediates of
nucleosides and amino acids in conjugates, which mimic bio- Experimental
logically relevant processes. The liquid phase synthesis (LPS) We used L-tryptophan (Fisher Scientific, USA), Boc-NH-L-
in combination with the phosphotriester approach appeared to tryptophan pentachlorophenyl ester (Reanal, Hungary), 2-(2-
meet the requirements for the in situ NMR detection of the aminoethoxy)ethanol (Acros Organics, USA). Other reagents
2903

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
Scheme 4: Synthesis of conjugates 6–8. i) TPSCl, MeIm, Py; ii) TBAF in Py/H2O, then NH3/H2O; iii) TBAF in Py/H2O, then NH3/H2O, then
AcOH/H2O; iv) Ac2O, Py, then AcOH/H2O.
were from Sigma-Aldrich, Inc. (USA). Organic solvents were Kieselgel 55–100 μm (Merck, Germany); RPC, a Porasil C 18
dried and purified by standard procedures. The reaction (55–105 μm, 125 A) (Waters, USA); and anion exchange chro-
mixtures were analyzed on a Milichrom A02 analytical chro- matography, DEAE Sephadex A-25 (Pharmacia, Sweden).
matograph system (Econova, Russia) using a ProntoSIL 125 Eluent composition is given in v/v per cent. NMR spectra were
C18 column (2 × 75 mm) and gradient of buffer B (0.1 M acquired on Bruker AM-400 and AV-300 instruments (Bruker,
TEA–AcOH, pH 7.0, 80% acetonitrile) in buffer A (0.1 M Germany) in appropriate deuterated solvents at 30 °C. Chem-
TEA–AcOH, pH 7.0, water) with UV detection at 250, 260, ical shifts (δ) are reported in ppm relative to the TMS signal. In
280, and 300 nm. TLC was carried out on Kieselgel 60 F254 the case of 31P and 19F, external standards of 85% H3PO4 and
plates (Merck, Germany) in the proper solvent systems (see C6F6, respectively, were used. Coupling constants J are reported
below) and spots were visualized by UV irradiation, ninhydrin in Hertz. Mass spectra were registered in The Center of
(for amine groups) or cysteine/aqueous sulfuric acid (for Cooperative Use “Proteomics”, Russian Academy of Sciences,
nucleosides and tryptophan) solution. Evaporations were on an Autoflex III mass spectrometer (Bruker Daltonics, Inc.)
performed under reduced pressure at 40 °C. The preparative using 2,5-dihydroxybenzoic acid as a matrix (MALDI–TOF) in
silica gel column chromatography was performed using positive or negative mode.
2904

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
Compound 22 was synthesized according to the published The appropriate fractions were pooled and evaporated. After
method [32]. Trifluoroacetamido-NH-tryptophan was obtained drying, 2-N-isobutyry-3’-O-levulinyl-2’-deoxyguanosine
as described in [33]. The syntheses of compounds 9, 10, 12, 15, 5’-monophosphate (6.0 g, 10.0 mmol) was obtained as a glass-
30, and 32 were carried out according to the well-known like residue.
methods [29,30] (see Supporting Information File 1 for details).
2-N-Isobutyryl-3’-O-levulinyl-2’-deoxyguanosine 5’-mono-
Synthesis of 2-N-isobutyryl-3’-O-levulinyl-2’-
phosphate (6.0 g, 10.0 mmol) was dissolved in CH2Cl2
deoxyguanosine 5’-O-(p-chlorophenyl)phos-
phate (17)
(45 mL). Triphenylphosphine (8.0 g, 30 mmol), 2,2′-
dithiodipyridine (6.6 g, 30 mmol), and MeIm (4.8 mL,
60 mmol) were added to the nucleotide solution. After 30 min
of incubation, the solution of p-chlorophenol (6.4 g, 50 mmol)
and TEA (21 mL, 150 mmol) in CH2Cl2 (30 mL) was added,
The synthetic scheme and physicochemical data of intermedi-
ate compounds see Supporting Information File 1.
2’-Deoxyguanosine 5’-monophosphate (5 g, 12.5 mmol, and the reaction mixture was stirred for 3 h. CH2Cl2 was then
disodium salt, 0.1 M solution in 20% aqueous EtOH) was evaporated, the residue was dissolved in 50% aqueous Py
converted into NH4+ form on a DEAE A-25 column (200 mL) (100 mL), The solution was washed with diethyl ether (2 ×
by elution with 1 M NH4HCO3. The desired fractions were 100 mL), and the product was extracted with a CH2Cl2/1-
evaporated; traces of buffer were removed by coevaporation butanol mixture (7/3, 2 × 75 mL). The organic layer was evapo-
with water. The last portion of water was added along with TEA rated, the residue was dissolved in a minimal volume of 30%
(14 mL, 100 mmol). The residue was coevaporated with aceto- aqueous EtOH and loaded on the top of the column containing
nitrile (3 × 25 mL) and Py (2 × 20 mL) and dissolved in Py reversed phase resin equilibrated with water. The target product
(60 mL). Chlorotrimethylsilane (8.2 mL, 6 mmol) was added to was purified by RPC in a linear gradient of acetonitrile in water
the solution under vigorous stirring for 30 min, followed by the (0–50%). The appropriate fractions were pooled and evapo-
addition of isobutyryl chloride (2.7 mL, 26 mmol). After stir- rated. After drying, 2-N-isobutyryl-3’-O-levulinyl-2’-
ring the mixture overnight, it was cooled in an ice bath fol- deoxyguanosine 5’-O-(p-chlorophenyl)phosphate (1/2 TEA salt,
lowed by the addition of water (7 mL) and then (after 10 min) 5.5 g, 7.5 mmol, 60% yield relative to initial 2’-deoxyguano-
25% aqueous ammonia. The cooling bath was removed and the sine 5’-monophosphate) was obtained as a dry glass-like
reaction mixture was stirred for 1 h at room temperature. After yellowish hydroscopic powder. Rf: 0.63 (iPrOH/H2O 4/1);
evaporation, the residue was dissolved in water, and the target 31P NMR (CD3OD) 4.75; 1H NMR (CD3OD) 8.68 (br.s, 1H,
product was purified by RPC in a linear gradient of EtOH in NH-Gua), 8.14 (s, 1H, H8-Gua), 7.16–7.07 (m, 4H, H-pClPh),
water (0–20%). The appropriate fractions were pooled and 6.31 (dd, J = 5.3, 9.3, H1’), 5.48 (m, 1H, H3’), 4.35–4.24 (m,
evaporated. After drying, 2-N-isobutyryl-2’-deoxyguanosine 3H, H-5’,5’’,4’), 3.22 (q, J = 7.3, 3H, CH2-TEA), 2.89–2.83
5’-monophosphate (5.40 g, 12.0 mmol) was obtained as a (m, 3H, H2’, O(O)CCH2), 2.78 (sep, J = 6.8, 1H, CH(CH3)2,
cream-coloured powder. Before the next step (protection of the 2.64–2.57 (m, 2H, CH2C(O)CH3,), 2.47–2.38 (m, 1H, H2’’),
3’-OH group by the levulinyl residue), the nucleotide with the 2.20 (s, 3H, C(O)CH3), 1.32 (t, 6H, CH3-TEA), 1.21 (d,
protected nucleobase was converted to the TEA salt by adding J = 6.8, 3H, CH(CH3)2), 1.19 (d, J = 6.8, 3H, CH(CH3)2);
TEA (5 mL, 35 mmol) to its solution in 50% aqueous Py MALDI–TOFMS (m/z): [M + H]+ calcd for C25H30ClN5O10P,
(50 mL), evaporation of resulting mixture and drying the oil 626. 14; found, 626. 05; [M
+ Na]+ calcd for
residue by coevaporation with acetonitrile (2 × 25 mL) and Py C2 5 H2 9 ClN5 NaO1 0 P, 648. 12; found, 648. 04.
(2 × 20 mL).
General phosphorylation procedure
Levulinic acid (11.6 g, 100 mmol) and DCC (10.3 g, 50 mmol) Compound 10, 12, 15, 23, or 26 (0.2 mmol) and 1,2,4-triazole
was dissolved in diethyl ether (150 mL), and the mixture was (0.08 g, 1.2 mmol) were coevaporated with Py (3 × 1 mL),
stirred for 3 h. After filtration, the diethyl ether was removed dissolved in Py (1 mL), and 4-chlorophenyl dichlorophosphate
from the filtrate by evaporation; the residue was dissolved in Py (0.24 mmol, 0.04 mL) was added to the solution under vigorous
(50 mL), and the solution was transferred to the flask contain- stirring. After 30 min, the reaction was stopped by the addition
ing 2-N-isobutyryl-2’-deoxyguanosine 5’-monophosphate (TEA of several drops of aqueous 5% NaHCO3, and the reaction mix-
salt) and MeIm (4 mL, 50 mmol). The reaction mixture was left ture was evaporated with water to remove Py. The residue was
for 3 h, followed by adding water (50 mL) and stirring the mix- suspended in water, and the target product (compound 11, 13,
ture for 3 h. The reaction mixture was then evaporated several 16, 24, or 27) was purified by RPC in a linear gradient of aceto-
times with water to remove traces of Py. The target product was nitrile in water (0–50%). The appropriate fractions were pooled
purified by RPC in a linear gradient of EtOH in water (0–40%). and evaporated. Typical yield was 90% (1/2 Py salt). See
2905

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
Supporting Information File 1 for physicochemical characteris- [M + H]+ calcd for C28H32N6O10P, 643.19; found, 643.19;
tics of compounds 11, 13, 16, 24 and 27.
[M + Na]+ calcd for C28H31N6NaO10P, 665.17; found, 665.21;
[M + K]+ calcd for C28H31KN6O10P, 681.15; found, 681.20;
[M − H]− calcd for C28H30N6O10P, 641.18; found, 641.62.
Phosphotriester condensation
The coupling reactions afforded fully blocked derivatives 18,
20, 21, 25, 28, 29, 31, and 33 were performed according to [34]. Conjugate 2
After silica gel chromatography, the appropriate fractions were Partly deprotected by TBAF treatment conjugate 20 was
evaporated, and the residue of the fully protected conjugate was dissolved in a minimal amount of 30% aqueous EtOH and
subjected to deprotection. Target conjugates 1–8 were purified placed on the top of the column containing reversed phase resin
and characterized (see below). The DMTr protective group was equilibrated with water. Elution was performed with a linear
removed after the coupling reaction (compounds 23 and 26) gradient of EtOH in water (0–50%) in the presence of 0.05 M
according to [28]. The selective removal of the Lev protective NH4HCO3. The appropriate fractions were pooled and evapo-
group from compound 18 was performed according to [35]. See rated. The residue was treated with concentrated (25%) aqueous
Supporting Information File 1 for physicochemical data of com- ammonia for 48 h at room temperature under stirring, and the
pounds 19, 23, and 26.
mixture was evaporated. The residue was dissolved in formic
acid (1.5 mL). After 3 h, crude conjugate 2 was precipitated by
diethyl ether (15 mL), the tube was frozen at −20 °C, and the
precipitate was collected by centrifugation. Target product 2
Deprotection of fully blocked derivatives and
purification of conjugates 1–8
Compounds 18, 20, 21, 25, 28, 29, 31, or 33 after purification was purified by RPC in a linear gradient of acetonitrile in water
by silica gel chromatography (see above) were mixed with (0–30%) in the presence of 0.05 M NH4HCO3. After drying,
0.33 M solution of TBAF in 50% aqueous Py (pH 7.0, 1.0 mL 0.06 g of conjugate 2 (0.06 mmol, 15% calcd to 19 taken into
per 0.05 g of the fully protected derivative) and stirred at 40 °C the coupling reaction) was obtained. Rf: 0.43 (iPrOH/H2O, 4/1);
overnight. The reaction mixtures were then evaporated several 31P NMR (D2O) 0.82 (s, 1P), −0.10 (s, 1P); 1H NMR (D2O)
times with water to remove Py. The subsequent purification of 7.75 (s, 1H, H8-Gua), 7.71–7.57 (m, 7H, H-Ar), 7.51 (t, J = 7.7,
products by anion exchange chromatography and RPC 2H, H-Ar), 7.40 (d, J = 7.8, 1H, H-Trp), 7.29 (d, J = 7.8, 1H,
depended on the combination of the protective groups and a H-Trp), 7.12 (s, 1H, H-Trp), 7.04 (t, J = 7.8, H-Trp), 6.95 (t,
number of negatively and positively charged residues.
J = 7.8, H-Trp), 5.84 (t, J = 7.0, 1H, H1’), 4.91–4.83 (m, 1H,
H4’), 4.29–4.22 (m, 1H, H3’), 4.10 (t, J = 6.5, 1H,
CH(NH2)CH2), 4.00–3.87 (m, 8H, OCH2CH2OP), 3.87–3.44
Conjugate 1
Partly deprotected by the TBAF treatment conjugate 18 was (m, 10H, NHCH2CH2O, H5’5”), 3.42–3.32 (m, 1H,
treated with concentrated (25%) aqueous ammonia for 48 h at CH(NH2)CH2), 3.24–3.12 (m, 1H, CH(NH2)CH2), 2.56–2.37
room temperature under stirring, and the mixture was evapo- (m, 2H, H2’H2”); MALDI–TOFMS (m/z): [M + H]+ calcd for
rated. The residue was dissolved in 40% aqueous EtOH (10 mL C43H52N9O15P2, 996.30; found, 996.20; [M + Na]+ calcd for
per 0.05 g of the fully protected derivative), and the solution C43H51N9NaO15P2, 1018.29; found, 1018.19.
was applied to a column with DEAE Sephadex A-25 in 40%
aqueous EtOH. Elution was performed with a linear gradient of Conjugate 3
NH4HCO3 (0–0.5 M) in 40% aqueous EtOH. The appropriate Partly deprotected by TBAF treatment conjugate 21 was
fractions were pooled and evaporated. Target product 1 was subjected to RPC as described for partly deprotected by TBAF
then purified by RPC in a linear gradient of acetonitrile in water conjugate 20. The residue was then treated with concentrated
(0–20%) in the presence of 0.05 M NH4HCO3. After drying, (25%) aqueous ammonia for 48 h at room temperature under
0.120 g of conjugate 1 (0.19 mmol, 37% calcd to 9 taken into stirring. After evaporation, target product 3 was purified by
the coupling reaction) was obtained. Rf: 0.53 (iPrOH/H2O, 4/1); RPC as described for conjugate 2. After drying, 0.03 g of conju-
31P NMR (D2O) 1.05 (s); 1H NMR (D2O) 8.01 (s, 1H, gate 3 (0.033 mmol, 33% calcd to 19 taken into the coupling
H8-Gua), 7.91 (dt, J = 8.2, 1.6, 2H, H-Ar), 7.79 (dt, J = 8.2, reaction) was obtained. Rf: 0.40 (iPrOH/H2O, 4/1); 31P NMR
1.6, 2H, H-Ar), 7.76, (dt, J = 7.4, 1.5, 2H, H-Ar), 7.71 (tt, (D2O) 0.76 (s, 1P), −0.66 (s, 1P); 1H NMR (D2O) 7.76 (s, 1H,
J = 7.5, 1.2, 1H, H-Ar), 7.56 (tt, J = 7.4, 1.5, 2H, H-Ar), 6.20 H8-Gua), 7.66–7.38 (m, 10H, H-Ar, H-Trp), 7.19 (s, 1H,
(t, J = 7.1, 1H, H1’), 4.65–4.59 (m, 1H, H4’), 4.08–4.02 (m, H-Trp), 7.18–7.10 (m, 1H, H-Trp), 6.96–6.85 (m, 2H, H-Trp),
1H, H3’), 4.05–4.00 (m, 2H, OCH2CH2OP), 3.98–3.92 (m, 2H, 5.71 (dd, J = 6.1, 8.1, 1H, H1’), 4.79–4.68 (m, 1H, H4’),
OCH2CH2OP), 3.70 (t, J = 5.3, 2H, NHCH2CH2O), 3.68–3.64 4.17–4.10 (m, 1H, H3’), 4.08–4.01 (m, 1H, CH2CH(NH2)CH2),
(m, 2H, H5’5”), 3.59 (t, J = 5.3, 2H, NHCH2CH2O), 2.54–2.43 4.00–3.86 (m, 4H, OCH2CH2OP), 3.86–3.73 (m, 2H,
(m, 1H, H2’), 2.34–2.24 (m, 1H, H2”); MALDI–TOFMS (m/z): CH2CH(NH2)CH2, CH2CH(NH2)CH2), 3.72–3.59 (m, 4H,
2906

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
NHCH2CH2O), 3.58–3.48 (m, 2H, H5’5”), 3.24–3.01 (m, 2H, CH2CH(NH2)CH2), 3.83–3.71 (m, 4H, OCH2CH2OP),
CH2CH(NH2)CH2), 2.33–2.18 (m, 1H, H2’), 2.06–1.93 (m, 1H, 3.70–3.56 (m, 6H, CH2CH(NH2)CH2, CH2CH(NH2)CH2,
H2’’); MALDI–TOFMS (m/z): [M + H]+ calcd for NHCH2CH2O), 3.55–3.43 (m, 2H, CH2CH(NH2)CH2),
C39H45N8O13P2, 895.26; found, 895.65; [M + Na]+ calcd for 3.03–2.95 (m, 2H, H5’5’’), 2.01–1.86 (m, 1H, H2’), 1.85–1.74
C39H44N8NaO13P2, 917.24; found, 917.51; [M + K]+ calcd for (m, 1H, H2’’); MALDI–TOFMS (m/z): [M + H]+ calcd for
C39H44KN8O13P2, 933.21; found, 933.48; [M − H]− calcd for C34H41N3O12P2, 746.22; found, 746.44; [M + Na]+ calcd for
C39H43N8O13P2, 893.24; found, 893.14.
C34H41N3NaO12P2, 768.21; found, 768.43; [M − H]− calcd for
C34H40N3O12P2, 744.21; found, 744.41.
Conjugate 4
Partly deprotected by TBAF treatment conjugate 25 was Conjugate 6
subjected to RPC as described for partly deprotected by TBAF Partly deprotected by TBAF treatment conjugate 29 was
conjugate 20 using gradient of EtOH in water (0–75%). The dissolved in 20% aqueous EtOH (10 mL per 0.05 g of the fully
Boc protective group was removed by formic acid as described protected derivative), and the solution was applied to a column
for conjugate 2. The residue was dissolved in 20% aqueous with DEAE Sephadex A-25 in 20% aqueous EtOH. Elution was
EtOH (10 mL per 0.05 g of fully protected derivative), and the performed with a linear gradient of NH4HCO3 (0–0.5 M) in
solution was applied to a column with DEAE Sephadex A-25 in 20% aqueous EtOH. The appropriate fractions were pooled and
20% aqueous EtOH. Elution was performed with a linear evaporated. Nucleobase deprotection and the subsequent purifi-
gradient of NH4HCO3 (0–1.0 M) in 20% aqueous EtOH. After cation by RPC were performed as described for conjugate 1.
drying, 0.125 g of conjugate 4 (0.15 mmol, 60% calcd to 9 After drying, 0.06 g of conjugate 6 (0.09 mmol, 50% calcd to
taken into the coupling reaction) was obtained. Rf: 0.59 (iPr/ 15 taken into the coupling reaction) was obtained. Rf: 0.38 (iPr/
H2O, 4/1); 31P NMR (D2O) 0.75 (s, 1P), −0.09 (s, 1P); H2O, 4/1); 31P NMR (D2O) 0.85 (s); 1H NMR (D2O) 7.78 (s,
1H NMR (D2O) 7.54 (d, J = 7.8, 2H, H-Ar), 7.48–7.15 (m, 9H, 1H, H8-Gua), 7.36 (d, J = 8.1, 1H, H-Trp), 7.27 (d, J = 8.1, 1H,
H–Ar, H–Trp), 7.06 (s, 1H, H–Trp), 7.00 (t, J = 7.2, 1H, H-Trp), 7.07 (s, 1H, H-Trp), 7.04 (t, J = 7.5, 1H, H-Trp), 6.94
H–Trp), 6.83 (t, J = 7.2, 1H, H–Trp), 4.52–4.42 (m, 1H, H4’), (t, J = 7.5, 1H, H-Trp), 6.01 (t, J = 6.8, 1H, H1’), 4.61–4.55 (m,
4.01 (t, J = 7.1, 1H, CH(NH2)CH2), 3.96–3.89 (m, 1H, H3’), 1H, H4’), 4.18–4.13 (m, 1H, H3’), 4.10 (t, J = 7.0, 1H,
3.88–3.80 (m, 2H, H1’1”), 3.79–3.50 (m, 8H, OCH2CH2OP), CH(NH2)CH2), 4.05–3.94 (m, 2H, CH(NH2)CH2), 3.83–3.69
3.48–3.36 (m, 4H, NHCH2CH2O), 3.33–3.15 (m, 5H, (m, 2H, H5’5’’), 3.44–3.23 (m, 4H, OCH2CH2OP), 3.22–3.04
NHCH2CH2O, CH(NH2)CH2), 3.13–2.98 (m, 2H, H5’5”), (m, 4H, NHCH2CH2O), 2.61–2.51 (m, 1H, H2’), 2.44–2.35
2.96–2.83 (m, 1H, CH(NH2)CH2), 2.04–1.80 (m, 2H, H2’2”); ((m, 1H, H2’’); MALDI–TOFMS (m/z): [M + H]+ calcd for
MALDI–TOFMS (m/z): [M + H]+ calcd for C38H49N4O14P2, C25H34N8O9P, 621.22; found, 621.26; [M + Na]+ calcd for
847.27; found, 847.46; [M + Na]+ calcd for C38H48N4NaO14P2, C25H35N8NaO9P, 643.20; found, 643.24; [M + K]+ calcd for
869.25; found, 869.43; [M + K]+ calcd for C38H48KN4O14P2, C25H34KN8O9P, 659.17; found, 659.20.
885.23; found, 885.42; [M − H]− calcd for C38H47N4O14P2,
845.26; found, 845.60; [M − 2H + Na]+ calcd for Conjugate 7
C38H46N4NaO14P2, 867.24; found, 867.50.
Partly deprotected by TBAF treatment conjugate 31 was
dissolved in minimal amount of EtOH and treated with concen-
trated (25%) aqueous ammonia for 48 h under stirring at room
Conjugate 5
Partly deprotected by TBAF treatment conjugate 28 was temperature. After evaporation, the residue was dissolved in
subjected to RPC as described for partly deprotected by TBAF 40% aqueous EtOH (10 mL per 0.05 g of fully protected deriva-
conjugate 20 using gradient of EtOH in water (0–65%). The tive), and subjected to anion exchange chromatography as
TFA protective group was removed by concentrated (25%) described for conjugate 1. The appropriate fractions were
aqueous ammonia for 3 h. After evaporation, target product 5 pooled and evaporated. The residue was treated with 80%
was purified by anion exchange chromatography as described aqueous acetic acid (5 mL per 0.1 g of the fully protected
for conjugate 4. After drying, 0.09 g of conjugate 5 derivative) for 30 min, diluted tenfold with water and chilled.
(0.125 mmol, 50% calcd to 9 taken into the coupling reaction) The solution was neutralized by addition of concentrated (25%)
was obtained. Rf: 0.67 (iPr/H2O, 4/1); 31P NMR (D2O) 0.73 (s, aqueous ammonia. Target conjugate 7 was purified by RPC as
1P), −0.64 (s, 1P); 1H NMR (D2O) 7.65 (d, J = 8.2, 2H, H-Ar), described for conjugate 1. After drying, 0.015 g of conjugate 7
7.62–7.52 (m, 6H, H-Ar), 7.45 (d, J = 7.8, 1H, H-Trp), 7.43 (t, (0.035 mmol, 25% calcd to 30 taken into the coupling reaction)
J = 7.9, 2H, H-Ar), 7.30 (d, J = 7.9, 1H, H-Trp), 7.13 (s, 1H, was obtained. Rf: 0.1 (EtOH); 31P NMR (D2O) 0.75 (s);
H-Trp), 7.05 (t, J = 7.5, 1H, H-Trp), 6.97 (t, J = 7.5, 1H, 1H NMR (D2O) 8.04 (s, 1H, H8-Gua), 6.29 (t, J = 6.7, 1H,
H-Trp), 4.48–4.41 (m, 1H, H4’), 3.97–3.83 (m, 4H, H3’, 1’1’’, H1’), 4.73–4.68 (m, 1H, H4’), 4.24–4.18 (m, 1H, H3’), 4.01 (t,
2907

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
J = 4.5, 2H, OCH2CH2OP), 3.84–3.78 (m, 2H, H5’5’’), 3.67 (t, Leading Scientists to Russian Educational Institutes” (Grant no.
J = 4.5, 2H, OCH2CH2OP), 3.60–3.46 (m, 2H, NHCH2CH2O), 11.G34.31.0045).
3.19–3.11 (m, 2H, NHCH2CH2O), 2.88–2.77 (m, 1H, H2’),
References
1. Zheng, R.; Shi, Y.; Jia, Z.; Zhao, C.; Zhang, Q.; Tan, X.
2.60–2.49 (m, 1H, H2’’); MALDI–TOFMS (m/z): [M + H]+
calcd for C14H24N6O8P, 435.14; found, 434.99; [M + Na]+
Chem. Soc. Rev. 2010, 39, 2827–2834. doi:10.1039/b924875g
calcd for C14H23N6NaO8P, 457.12; found, 457.00
2. Yurkovskaya, A.; Morozova, O.; Gescheidt, G. Structures and reactivity
of radicals followed by magnetic resonance. In Encyclopedia of
Conjugate 8
Radicals in Chemistry, Biology and Materials; Chatgilialoglu, C.;
Partly deprotected by TBAF treatment conjugate 33 was
subjected to anion exchange chromatography as described for
conjugate 1. The appropriate fractions were pooled and evapo-
rated. The TFA and Ac protective groups were removed by
concentrated (25%) aqueous ammonia treatment for 3 h. After
evaporation, target product 8 was purified by RPC as described
for conjugate 1. After drying, 0.026 g (0.055 mmol, 37% calcd
to 32 taken into the coupling reaction) was obtained. Rf: 0.32
(EtOH); 31P NMR (D2O): 0.85 (s); 1H NMR (D2O) 7.62 (d,
J = 8.0, 1H, H-Trp), 7.52 (d, J = 8., 1H, H-Trp), 7.31 (s, 1H,
H-Trp), 7.26 (t, J = 7.5, 1H, H-Trp), 7.17 (t, J = 7.5, 1H,
H-Trp), 4.30–4.24 (m, 1H, H4’), 4.20 (dd, J = 8.0, 6.2, 1H,
CH(NH2)CH2), 4.02–3.74 (m, 6H, H1’1’’, OCH2CH2OP),
3.65–3.05 (m, 9H, H3’, H5’5’’, NHCH2CH2O, CH(NH2)CH2),
2.17–2.03 (m, 1H, H2’), 1.93–1.82 (m, 1H, H2’);
MALDI–TOFMS (m/z): [M + H]+ calcd for C20H30N2O9P,
472.18; found, 472.02; [M + Na]+ calcd for C20H29N2NaO9P,
494.17; found, 494.02.
Studer, A., Eds.; Wiley and Sons: Chichester, U.K., 2012; Vol. 1,
pp 175–206. doi:10.1002/9781119953678.rad008
3. Hore, P. J.; Broadhurst, R. W. Prog. Nucl. Magn. Reson. Spectrosc.
1993, 25, 345–402. doi:10.1016/0079-6565(93)80002-B
4. Day, I. J.; Maeda, K.; Paisley, H. J.; Mok, K. H.; Hore, P. J.
J. Biomol. NMR 2009, 44, 77–86. doi:10.1007/s10858-009-9322-2
5. Janssen, G. J.; Daviso, E.; van Son, M.; de Groot, H. J. M.; Alia, A.;
Matysik, J. Photosynth. Res. 2010, 104, 275–282.
doi:10.1007/s11120-009-9508-1
6. Morozova, O. B.; Yurkovskaya, A. V. Angew. Chem., Int. Ed. 2010, 49,
7996–7999. doi:10.1002/anie.201003780
7. Morozova, O. B.; Kaptein, R.; Sagdeev, R. Z.; Yurkovskaya, A. V.
Appl. Magn. Reson. 2013, 44, 233–245.
doi:10.1007/s00723-012-0403-0
8. Venkatesan, N.; Kim, B. H. Chem. Rev. 2006, 106, 3712–3761.
doi:10.1021/cr0502448
9. Morozova, O. B.; Kiryutin, A. S.; Sagdeev, R. Z.; Yurkovskaya, A. V.
J. Phys. Chem. B 2007, 111, 7439–7448. doi:10.1021/jp067722i
10.Morozova, O. B.; Kaptein, R.; Yurkovskaya, A. V. J. Phys. Chem. B
2012, 116, 12221–12226. doi:10.1021/jp307956q
11.Morozova, O. B.; Kaptein, R.; Yurkovskaya, A. V. J. Phys. Chem. B
2012, 116, 8058–8063. doi:10.1021/jp301760b
12.Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617–618.
doi:10.1021/ja962255b
Supporting Information
13.Yurkovskaya, A. V.; Snytnikova, O. A.; Morozova, O. B.;
Tsentalovich, Y. P.; Sagdeev, R. Z. Phys. Chem. Chem. Phys. 2003, 5,
3653–3659. doi:10.1039/b305783f
The synthesis and physicochemical characteristics for
compounds 9, 10, 12, 15, 30, and 32, the synthetic scheme
and physicochemical characteristics for intermediates in the
synthesis of compound 17 and the physicochemical
characteristics for compounds 11, 13, 16, 19, 23, 24, 26,
and 27 are provided in the Supporting Information.
14.Morozova, O. B.; Kiryutin, A. S.; Yurkovskaya, A. V. J. Phys. Chem. B
2008, 112, 2747–2754. doi:10.1021/jp0752318
15.Morozova, O. B.; Korchak, S. E.; Sagdeev, R. Z.; Yurkovskaya, A. V.
J. Phys. Chem. A 2005, 109, 10459–10466. doi:10.1021/jp054002n
16.Morozova, O. B.; Yurkovskaya, A. V. J. Phys. Chem. B 2008, 112,
12859–12862. doi:10.1021/jp807149a
Supporting Information File 1
17.Nguyen, T. X.; Kattnig, D.; Mansha, A.; Grampp, G.;
Yurkovskaya, A. V.; Lukzen, N. J. Phys. Chem. A 2012, 116,
10668–10675. doi:10.1021/jp307122h
Syntheses and characteristics for selected compounds.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-9-326-S1.pdf]
18.Saprygina, N. N.; Morozova, O. B.; Kaptein, R.; Yurkovskaya, A. V.;
Sagdeev, R. Z. Dokl. Phys. Chem. 2013, 449, 66–70.
doi:10.1134/S0012501613040027
19.Morozova, O. B.; Hore, P. J.; Sagdeev, R. Z.; Yurkovskaya, A. V.
J. Phys. Chem. B 2005, 109, 21971–21978. doi:10.1021/jp053394v
20.Morozova, O. B.; Saprygina, N. N.; Fedorova, O. S.;
Yurkovskaya, A. V. Appl. Magn. Reson. 2011, 41, 239–250.
doi:10.1007/s00723-011-0252-2
Acknowledgements
This work was supported by the President program “Leading
Scientific Schools” (project nos. NS-64.2012.4 and
2429.2012.3), the Interdisciplinary Integration project no. 60 of
the Presidium of Siberian Branch of Russian Academy of
Sciences, and the grant of the Russian Foundation for Basic
Research (project nos. 12_04_01454_a and 13-03-00437_a).
We also acknowledge support from the Ministry of Education
and Science of the Russian Federation “Measures to Attract
21.Garipova, I. Yu.; Silnikov, V. N. Russ. Chem. Bull. 2002, 51,
1940–1944. doi:10.1023/A:1021329324527
22.Abramova, T. V. Molecules 2013, 18, 1063–1075.
doi:10.3390/molecules18011063
2908

Beilstein J. Org. Chem. 2013, 9, 2898–2909.
23.De Koning, M. C.; Ghisaidoobe, A. B. T.; Duynstee, H. I.;
Ten Kortenaar, P. B. W.; Filippov, D. V.; van der Marel, G. A.
Org. Process Res. Dev. 2006, 10, 1238–1245. doi:10.1021/op060133q
24.Ghisaidoobe, A. B. T.; de Koning, M. C.; Duynstee, H. I.;
Ten Kortenaar, P. B. W.; Overkleeft, H. S.; Filippov, D. V.;
van der Marel, G. A. Tetrahedron Lett. 2008, 49, 3129–3132.
doi:10.1016/j.tetlet.2008.03.025
25.van der Heden van Noort, G. J.; Schein, C. H.; Overkleeft, H. S.;
van der Marel, G. A.; Filippov, D. V. J. Pept. Sci. 2013, 19, 333–336.
doi:10.1002/psc.2508
26.Arunachalam, T. S.; Wichert, C.; Appel, B.; Müller, S.
Org. Biomol. Chem. 2012, 10, 4641–4650. doi:10.1039/c2ob25328c
27.Abramova, T. V. Methodology of obtaining modified
oligodeoxyrobonucleotides for of creation effective tools for molecular
biology investigations. Dr.Sc. Thesis, Institute of Chemical Biology and
Fundamental Medicine, SB RAS, Novosibirsk, Russia, 2012.
28.Abramova, T. V.; Vasileva, S. V.; Koroleva, L. S.; Kasatkina, N. S.;
Silnikov, V. N. Bioorg. Med. Chem. 2008, 16, 9127–9132.
doi:10.1016/j.bmc.2008.09.029
29.Bodanszky, M.; Bednarek, M. A. J. Protein Chem. 1989, 8, 461–469.
doi:10.1007/BF01026430
30.Sinha, N. D.; Striepeke, S. Oligonucleotides with reporter groups
attached to the 5’-terminus. In Oligonucleotides and Analogues: A
Practical Approach; Eckstein, F., Ed.; Oxford University Press: New
York, NY, USA, 1991; pp 185–210.
31.Itakura, K.; Bahl, P.; Katagiri, N.; Michniewicrz, J. J.; Wightman, H.;
Narang, S. A. Can. J. Chem. 1973, 51, 3649–3651.
doi:10.1139/v73-543
32.Eritja, R.; Walker, P. A.; Randall, S. K.; Goodman, M. F.; Kaplan, B. E.
Nucleosides Nucleotides 1987, 6, 803–814.
doi:10.1080/15257778708073426
33.Curphey, T. J. J. Org. Chem. 1979, 44, 2805–2807.
doi:10.1021/jo01329a049
34.Mazzei, M.; Balbi, A.; Grandi, T.; Sottofattori, E.; Garzoglio, R.;
Abramova, T.; Ivanova, E. Farmaco 1993, 48, 1649–1661.
35.van Boom, J. H.; Burgers, P. M. J. Tetrahedron Lett. 1976, 17,
4875–4878. doi:10.1016/S0040-4039(00)78935-0
License and Terms
This is an Open Access article under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
The license is subject to the Beilstein Journal of Organic
Chemistry terms and conditions:
(http://www.beilstein-journals.org/bjoc)
The definitive version of this article is the electronic one
which can be found at:
doi:10.3762/bjoc.9.326
2909