Selenium-derivatized oligonucleotides

Article abstract of DOI:10.1080/10426500801898242

Selenium-labelled macromolecules can be effectively analyzed by X-ray crystallography due to the phenomenon of multi-wavelength anomalous dispersion (MAD). The originally developed nucleoside 2-selena-1,3,2-oxathiaphospholane derivatives allow for synthes

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Phosphorus, Sulfur, and Silicon
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Selenium-Derivatized
Oligonucleotides
a
a
a
Wojciech J. Stec , Anna Maciaszek & Piotr Guga
a
Centre of Molecular and Macromolecular Studies,
Polish Academy of Sciences, Department of
Bioorganic Chemistry , Łódź, Poland
Published online: 29 Oct 2008.
To cite this article: Wojciech J. Stec , Anna Maciaszek & Piotr Guga (2008) Selenium-
Derivatized Oligonucleotides, Phosphorus, Sulfur, and Silicon and the Related
Elements, 183:4, 897-907, DOI: 10.1080/10426500801898242
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Phosphorus, Sulfur, and Silicon, 183:897–907, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500801898242
Selenium-Derivatized Oligonucleotides
Wojciech J. Stec, Anna Maciaszek, and Piotr Guga
Centre of Molecular and Macromolecular Studies, Polish Academy
of Sciences, Department of Bioorganic Chemistry, Lꢀ o´ d z´ , Poland
Selenium-labelled macromolecules can be effectively analyzed by X-ray crystallog-
raphy due to the phenomenon of multi-wavelength anomalous dispersion (MAD).
The originally developed nucleoside 2-selena-1,3,2-oxathiaphospholane derivatives
allow for synthesis of fully modified, stereodefined oligomers (up to approximately
15-mers) with any combination of internucleotide phosphoroselenoate linkages of
RP or SP absolute configuration as well as unmodified phosphate bonds, which, in
general, cannot be achieved using enzymatic methods.
Keywords Oxathiaphospholane method; phosphoroselenoate; PSe-diasteoisomer; PSe-
oligonucleotide
INTRODUCTION
In our long-lasting research projects, selenium has been always
1
present in an “intimate pair” with phosphorus. Phosphane selenides,
2
phosphoroselenonoates, and isomeric phosphoroselenoloates, phos-
phoroselenocyanidates,3 and phosphoroisoselenocyanates were con-
sidered as excellent model compounds for studies on the mechanisms
of nucleophilic substitution at phosphorus atom, molecular rearrange-
ments, chemical transformations, and structural assignments. Direct
selenium-phosphorus bond expanded the array of P-chiral organophos-
phates making them excellent model compounds for stereochemical
studies on nucleophilic and metal-ion assisted substitution at P-atom,
as well as intermediates for stereocontrolled conversions to other impor-
4
5
tant classes of compounds, e.g., dinucleoside methanephosphonates.
Selected examples are shown below to demonstrate these early
endeavors.
Financial support by Polish national sources: KBN, grant 3 T09A 059 28 to W.J.S.,
MNiI, grant 3T09A 072 26 to P.G. and CMMS PAS statutory funds, is gratefully acknowl-
edged.
Address correspondence to Wojciech J. Stec, Centre of Molecular and Macromolecular
Studies, Polish Academy of Sciences, Department of Bioorganic Chemistry, Sienkiewicza
1
12, 90-363 Lꢀ o´ d z´ , Poland. E-mail: wjstec@bio.cbmm.lodz.pl
897

8
98
W. J. Stec et al.
Trisubstituted phosphane selenides were obtained in enantiomeri-
cally pure form and were found to be configurationally stable; under
treatment with LiAlH4 they were reduced to parent phosphanes in a
stereocontrolled manner (Scheme 1).1
SCHEME 1
Since natural selenium contains ca. 7% of NMR active 77Se isotope
1
(
spin number = /2), its derivatives are useful for NMR studies. Nuclear
3
1
77
spin-spin coupling constants between directly bonded P and Se in
several classes of organophosphorus compounds (with selenium linked
to a phosphorus atom through a single or double bond) were measured,
2
and this type of information has been used for structural assignments.
Numerous experiments revealed that for diastereomeric cyclic phos-
phoroselenoate compounds with an exocyclic selenium atom in either
axial or equatorial positions (Scheme 2) the corresponding coupling con-
1
1
stants JPSe are distinctly different and the absolute value of JPSe (ax)
1
6
is always lower than JPSe (eq).
Enantiomerically and diastereomerically pure phosphoroselenoates,
as well as phosphorothioates and isotopically labelled phosphates,
became much more easily accessible after implementation of
Wadsworth-Emmons-Horner Reaction for PN→PX conversion in start-
7
ing phosphoranilidates, as well as in their phosphorothioate- and phos-
phoroselenoate congeners (Scheme 3).

Selenium-Derivatized Oligonucleotides
899
SCHEME 2
SCHEME 3

9
00
W. J. Stec et al.
The abovementioned examples document our long-lasting interest
in stereochemical aspects of phosphoroselenoates, extended also on
8
phosphoroselenoate congeners of oligonucleotides. Although the first
diribonucleotide phosphoroselenoates were synthesized in solution by
9
Ogilvie in 1980, our adaptation of the Caruthers’ and - Beaucage’s solid
phase phosphoramidite approach1 to the synthesis of oligo(nucleoside
0
phosphoroselenoate)s opened an access to this class of valuable con-
8
geners of oligonucleotides. In retrospective analysis, we have to men-
tion that oligo(nucleoside phosphoroselenoate)s have appeared to be
toxic to numerous cell lines;11 thus, endeavors towards stereocontrolled
synthesis of oligo(nucleoside phosphoroselenoate)s (vide infra) were
abandoned. We focused our efforts on the development of stereocon-
trolled synthesis of phosphorothioate analogues of DNA (PS-Oligos, 1,
X = S) (Inset 1).
For many applications, including so called antisense or antigene
strategies, PS-Oligos—the ionic analogues of DNA with enhanced nu-
cleolytic stability—were highly demanded, although their use for in vivo
studies also appeared to be limited by non-specific binding to certain
1
2
13,14
proteins and immune-related side effects.
Importantly, despite of
the fact that PS-Oligos are isoelectronic with natural DNA, the presence
of a sulfur atom affects the properties of internucleotide bonds, mostly
due to different steric requirements of sulfur atom (P S vs. P O bond
length), different affinity towards metal ions (“soft” sulfur vs. “hard”
oxygen), and changes in negative charge distribution in the phospho-
rothioate anion. Therefore, the hydration pattern of PS-Oligos is differ-
ent from that of natural oligonucleotides, obstructing the evaluation of
results on kinetics of “rescue effects” of thiophilic metal ions, and mak-
ing analysis of direct or water-mediated contacts between metal ions
and phosphate groups much more difficult. However, this is a calcu-
lated and accepted toll paid for increased stability and preserved good

Selenium-Derivatized Oligonucleotides
901
hybridization properties of phosphorothioate oligonucleotide probes.
It is important to note that PS-Oligos are P-chiral species and the
shortest such modified DNA fragments, i.e., dinucleotides, consist of
the mixture of two P-diastereomers of either RP or SP absolute con-
figuration. For oligonucleotides consisting of n nucleoside units and
containing n − 1 modified internucleotide bonds, the number of exist-
n−1
ing P-diastereomeric forms N is given by the equation N = 2 . This
means, that for oligonucleotides as long as decamers, the product ob-
8
tained on non-stereocontrolled way (e.g., standard phosphoramidite
9
or H-phosphonate methods) exists as the mixture of 2 = 512 diastere-
omers and the content of given single diastereomer drops below 0.2%
1
5
(
1/512). Short PS-Oligos prepared with a non-stereocontrolled or par-
tially stereoselective mode, can be separated into diastereomeric species
by means of chromatographic techniques,16 but this method cannot be
considered general as the efficiency of separation depends upon the se-
quence of nucleobases and the composition of the buffered eluent. At
the time being, several phosphorothioate analogues of DNA (as ran-
dom mixtures of all possible P-diastereomers) are evaluated in clini-
ꢀ
R
1
7
cal studies (Table 1), while one compound—Vitravene (fomivirsen
sodium)—targeting cytomegalovirus IE-2 gene has been accepted by
FDA for the treatment of CMV -induced retinitis. Properties of anti-
sense probes and discussion of the mechanism of their action can be
found in several excellent reviews.1
8−22
Since virtually all biomolecules the oligonucleotides interact with,
are chiral and exist in single stereochemical forms, each diastereomer
of a P-chiral oligonucleotide may interact with them in a slightly dif-
ferent way. Therefore, stereochemical aspects should be taken into
consideration in a design of P-chiral oligonucleotides to assure their
most favorable biological activity. Similar functional alterations and
stereochemical consequences are seen in another class of compounds,
closely related to phosphorothioates, consisting of phosphoroselenoate
oligonucleotides (PSe-Oligos, 1, X = Se), where one of the oxygen
atoms of internucleotide bond is replaced with selenium—the down-
stairs neighbour of sulfur in the Periodic Table. Solid-phase syntheses of
PSe-Oligos were performed using a phosphoramidite or H-phosphonate
III
11,23−28
approach with “selenation” of the P intermediate.
Although it
has been found that stereorandomal PSe-Oligos have a diminished hy-
bridization capability to complementary DNA and RNA templates, as
1
1
compared with both the unmodified and phosphorothioate oligomers,
they have been evaluated as research tools for studies of interactions
with metal ions. Although the enzymatic synthesis of stereodefined
PSe-Oligo based on the use of DNA polymerase, which, rather un-
expectedly, accepted both SP and RP diastereomers of TTPαSe, has

9
02
W. J. Stec et al.
TABLE I Phosphorothioate Analogues of DNA (As Mixtures of All
1
7
Possible P-Diastereomers) Evaluated in Clinical Studies
Disease or
indication
Clinical
status
Company
Isis
Drug
Target
ISIS 301012 ApoB
High cholesterol
Diabetes
Re-stenosis
Hepatitis C
Drug metabolism
West Nile virus
Solid tumors, blood
cancers
Solid tumors
Renal cell carcinoma, Phase 2
acute myeloid
leukemia
Prostate and kidney Phase1/2
tumors
Phase 2
Phase 2
Phase 2
Phase 1b
Phase 1
Phase 1
Phase 3
Pharmaceuticals ISIS 113715 PTP-1B
AVI BioPharma Resten-NG C-Myc
AVI-4065
AVI-4557
AVI-4020
Hepatitis C virus
Cytochrome P450
West Nile virus
Genta
Lorus
Genasensea Bcl-2
G4460a C-Myb
Phase 1
GTI-2040a R2 subunit of RNR
GTI-2501a R1 subunit of RNR
Therapeutics
Topigen
TPI-ASM8 Chemokine
receptor-3/IL3,5
TPI-1100
Asthma
Phase 2
Pre-IND
Phase 2
Phosphodiesterases
PDE4 and PDE7
COPD
Oncogenex
VIRxSYS
OGX-011a Clusterin
Prostate and breast
cancer, non-small
cell lung cancer
VRX496
HIV
Chronic HIV infection Phase 1
been published,29 stereocontrolled method for chemical synthesis, free
of limitations typical for enzymatic reactions, was highly demanded.
3
0
Such a method has been developed in our Laboratory. It is based on
an oxathiaphospholane approach (Scheme 4, its mechanism is shown
on Scheme 5), originally developed for to the solid phase synthesis of
3
1,32
stereodefined phosphorothioate oligonucleotides
and employs sepa-
ꢀ
ꢀ
rated diastereomerically pure 5 -O-DMT-nucleoside-3 -O-(2-selena-4,4-
pentamethylene-1,3,2-oxathiaphospholane) monomers 2 (X = Se) in-
stead of their 2-thio congeners (2, X = S). A set of deoxyribonucleoside
ꢀ
Bz
Bz
iBu,DPC
monomers 2 (B = Ade , Cyt , Gua
and Thy) was synthesized
as depicted in Scheme 6.
The monomers 2 were obtained with satisfactory yield (45–60%)
3
1
and in their P NMR spectra, the resonances in the range of
9–100 ppm accompanied by satellite doublets resulting from the di-
9
3
1
77
1
rect P- Se spin-spin interactions ( J(P,Se) = 945–954 Hz) were found.
The monomers were then chromatographically separated into pure P-
diastereoisomers. Correlation of chromatographic mobility of fast- and

Selenium-Derivatized Oligonucleotides
903
SCHEME 4
SCHEME 5
slow-2 with the respective RP and SP absolute configuration of resulting
internucleotide phosphoroselenoate bond in d(NPSeT) (N = dA, dC, T or
dG) has been achieved enzymatically using RP-specific snake-venom
phosphodiesterase (svPDE) and SP-specific nuclease P1 (nP1).23 It was
found that for all four nucleobases fast-eluting monomers were pre-
cursors of dinucleotides with RP absolute configuration of phosphorus
ꢀ
Bz
atom. Then, 2-selena monomer fast-2 (B = Ade ) was used for elonga-
ꢀ
tion at the 5 -end of natural oligomer d(AGCGGTCGGC) synthesized

9
04
W. J. Stec et al.
SCHEME 6
by phosphoramidite approach (using the DBU-resistant sarcosinyl-
3
3
ꢀ
succinoyl linker ) to yield 5 -O-DMT-d(APSeAGCGGTCGGC) with RP
absolute configuration of phosphoroselenoate internucleotide bond.
Figure 1 shows a spectrum from MALDI TOF analysis of resulting
ꢀ
FIGURE 1 MALDI TOF spectrum for 5 -O-DMT-d(APSeAGCGGTCGGC) with
P
R absolute configuration of phosphoroselenoate internucleotide bond.

Selenium-Derivatized Oligonucleotides
905
oligomer. In the spectrum, a molecular ion m/z 3746 is observed,
whereas a signal at m/z 3444 corresponds to the compound detritylated
upon action of acidic matrix used in the experiment. This synthetic ap-
proach can be used for synthesis of diastereomerically pure Se-labelled
oligonucleotides for X-ray analysis.
It must be emphasized that the developed method allows for the syn-
thesis of fully modified, stereodefined oligomers (up to approximately
15-mers) with any combination of internucleotide phosphoroselenoate
linkages of RP or SP absolute configuration as well as unmodified
phosphate bonds, which, in general, cannot be achieved using enzy-
matic methods. Obtained PSe-Oligos were used for investigations on
thermodynamic stability of their complexes with DNA and RNA tem-
plates. Such the complexes of stereodefined phosphorothioate DNA ana-
logues have been analyzed thoroughly, and it has been found that, usu-
ally, PS-DNA/DNA and PS-DNA/RNA complexes have lower thermal
3
4
stability as compared to unmodified congeners. However, we have
also found that this rule is not valid for certain homopurine [All-RP-
PS]-DNA oligomers interacting with complementary (in the Watson-
Crick sense) RNA templates. Detailed analysis, including titration and
fluorescence quenching experiments, showed that if the homopurine
sequences of at least six nucleotides are palindromic, parallel triplexes
RNA/PS-DNA/RNA are formed, while only duplexes RNA/DNA are ob-
3
5
served for [PO]-, [Mix-PS]- and [All-SP-PS]-DNA. It was interest-
ing to check whether the same phenomenon could be seen for corre-
sponding PSe-Oligos. For that purpose two stereoregular dodecamers
[
All-RP-PSe]- and [All-SP-PSe]-dA12were synthesized. They were ana-
lyzed by MALDI TOF MS and PAGE, and, then, melting temperatures
ꢀ
of their complexes with complementary 2 -OMe-RNA templates were
measured. Obtained data confirmed the stereodependence of melting
ꢀ
temperatures, and for a series of [PX]-dA12 mixed with 2 -OMe-U12 at
◦
◦
◦
a molar ratio 1:2, melting temperatures of 35 C, 53 C and 58 C were
found for [PO]-, [All-RP-PS]- and [All-RP-PSe]-, respectively, whereas
for [All-SP-PSe]-dA12 Tm of only 28 C was assigned. Undoubtedly, the
◦
highest thermal stability of the triplex [All-RP-PSe]-dA12/2 × U12 (a rel-
evant melting curve has good S-shape indicating high cooperativity of
the transition, Figure 2) was found with surprise.
In our interpretation, this finding reflects astonishing functional-
ity of phosphoroselenoate moieties, where selenium atoms present
in proper spatial orientation confer the oligomers unique hybridiza-
tion and thermodynamic properties. Important characteristics of those
interactions are expected to be obtained from analysis of charge
distribution in phosphoroselenoates moieties. Ongoing project aim-
ing the assignment of the order of P-Se bond in internucleotide

9
06
W. J. Stec et al.
ꢀ
P
FIGURE 2 Melting curve for complex of [All-S -PSe]-dA12 with 2 -OMe-U12
template at pH 7.2.
phosphoroselenoate linkages involves the measurements of coupling
3
1
77
2
constants P- Se, the method recalled at the beginning of this account
and initiated more than 35 years ago.
With conclusions presented in this account, one has to admit that the
3
6
role of selenium in biological systems is of increasing interest. Ana-
logues of proteins and oligonucleotides, with sulfur or oxygen atoms
replaced with selenium, have been evaluated as research tools for
studies of interactions with metal ions, and selenium-labelled macro-
molecules can be effectively analyzed by X-ray crystallography due to
3
7
the phenomenon of multiwavelength anomalous dispersion (MAD).
Although a selenium atom can be introduced into an oligonucleotides
ꢀ
in numerous ways, like via incorporation of 2 -SeMe derivatives of nu-
3
7−40
cleosides (relevant methods are under continuous development),
the internucleotide phosphoroselenoate moiety offers, besides relevance
for X-ray structural studies, other possibilities for non-reductionistic
“in-solution” investigation of structural polymorphism and dynamics
of nucleic acids.
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