Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke
Synthesis of new disulfide-based H-phosphonate reagent for solid-phase oligonucleotide functionalization with mercaptopropyl group at one or two termini is described. Using this single reagent, 3'- and 3', 5'-disulfide-linked oligonucleotides were synthesized', disulfide bonds cl...
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| Date: | 1998 |
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Інститут молекулярної біології і генетики НАН України
1998
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| Journal Title: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Cite this: | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke / I.Y. Dubey, D M. Fedoryak // Биополимеры и клетка. — 1998. — Т. 14, № 2. — С. 163-172. — Бібліогр.: 43 назв. — англ. |
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| author | Dubey, I.Y. Fedoryak, D.M. |
| author_facet | Dubey, I.Y. Fedoryak, D.M. |
| citation_txt | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke / I.Y. Dubey, D M. Fedoryak // Биополимеры и клетка. — 1998. — Т. 14, № 2. — С. 163-172. — Бібліогр.: 43 назв. — англ. |
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| description | Synthesis of new disulfide-based H-phosphonate reagent for solid-phase oligonucleotide functionalization with mercaptopropyl group at one or two termini is described. Using this single reagent, 3'- and 3', 5'-disulfide-linked oligonucleotides were synthesized', disulfide bonds cleaved with dithiothreitol and generated thiol groups labelled with iodoacetamidofluorescein. Tis oligonucleotides containing one or two fluorescein residues at 3'- and 5'-ends were prepared in high yields.
Описано синтез нового дисульфідного Н-фосфонатного реагента для твердофазного введення в олігонуклеотиди меркаптопропільних груп по одному чи двох кінцях. З використанням цього реагента було синтезовано З'- та 5'-дисульфідні похідні олігонуклеотидів. Дисульфідні зв'язки розщеплено дитіотреїтолом і тіольні групи, що утворилися, модифіковано йодацетамідофлюоресцеїном. З високим виходом одержано оліго-Т15, які несуть один чи два залишки флюоресиеіну на У- і 5 -кінцях.
Описан синтез нового дисульфидного Н-фосфонатного реагента для твердофазного введения в олигонуклеотиды меркаптопропильных групп по одному или двум концам. С использованием этого реагента синтезированы 3',5'-дисульфидные производные олигонуклеотидов. Дисульфидные связи расщеплены дитиотреитолом и образовавшиеся тиольные группы модифицированы иодацетамидофлюоресцеином. С высоким выходом получены олиго-Т15, несущие один или два остатка флюоресцеина на 3' и 5'-концах.
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ISSN 0233-7657. Биополимеры и клетка. 1998. Т. 14. № 2
Synthesis and use of disulfide-based
H-phosphonate reagent for 3'- and/or 5'-
oligonucleotide labelling via mercaptoalkyl linker
I. Y. Dubey, D. M. Fedoryak
Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine
1 Murmanska str., 253660 Kyiv, Ukraine
Synthesis of new disulfide-based H-phosphonate reagent for solid-phase oligonucleotide functionalization
with mercaptopropyl group at one or two termini is described. Using this single reagent, 3'- and 3',
5'-disulfide-linked oligonucleotides were synthesized', disulfide bonds cleaved with dithiothreitol and
generated thiol groups labelled with iodoacetamidofluorescein. Tis oligonucleotides containing one or two
fluorescein residues at 3'- and 5'-ends were prepared in high yields.
Introduction. The need for methods and reagents to
modify oligonucleotides has increased in an extra
ordinary rate due to new applications of oligo
nucleotide conjugates as potent research tools, diag
nostic and therapeutic agents [ 1 — 5 ] . Oligonucleo
tides labelled with reporter groups, e. g. fluorescent
dyes, are used in non-isotopic detection of nucleic
acids, automated DNA sequencing, studies on nucleic
acid structure and dynamics. A number of molecules,
including intercalators, artificial nucleases, lipophilic
carriers, peptides e t c , has been covalently linked to
oligonucleotides to improve their antisense activity by
increasing cellular uptake, nuclease resistance or
binding affinity.
A wide variety of methods for the preparation of
oligonucleotide conjugates has been discussed in de
tail in comprehensive reviews and books [2, 6—9 ]. In
principle, conjugation is based on the introduction of
appropriate functional group into oligonucleotide, fol
lowed by its specific coupling with another molecule.
Chemical approach to the preparation of labelled
DNA involves incorporation of modified nucleotide or
non-nucleotide reagent during oligonucleotide syn
thesis; once incorporated, these synthons are func-
tionalized post-synthetically, although direct intro
duction of some reporter molecules during solid-phase
synthesis is also possible. The most common labelling
© I. Y. D U B E Y , D . M. FEDORYAK, 1 9 9 8
methods use aminoalkyl linker groups. Another po
pular approach is to employ the highly reactive thiol
function introduced as mercaptoalkyl linker able to
react with thiol-specific groups.
Certainly, the latter strategy seems to be more
promising and flexible, as thiols may be used in
greater variety of reactions than amines. The thiol is
a group capable of selective derivatization in the
presence of oligonucleotide functional groups, i. e.
amines, phosphates and hydroxyls. Two types of thiol
modification are available, to generate stable thiol
ether linkage or easily cleavable disulfide bond.
S-alkylation reaction, that is coupling of highly nuc-
leophilic sulfhydryl group with molecules bearing
electrophilic thiol-specific functions, yields oligonuc
leotide conjugated via thioether bond. Iodo(bro-
mo)acetamides, aziridinyl sulfonamides, y-bromo-a,
/З-unsaturated carbonyls, maleimides and less reactive
acrylates are the functionalities observed to selectively
react with aliphatic mercapto group [10] .
Besides that, thiol group can also form disulfides
with other thiols, usually after activation with 2,2'-
dipyridyl disulfide or related reagent [11—14] . This
approach is especially important for oligonucleotide
coupling with peptides and proteins under mild con
ditions, although conjugation through thioether lin
kage was also used for this purpose [13—18] . An
important feature of disulfide formation is that it is
readily reversed by treatment with mercaptans like
163
D U B E Y I Y., FE D O R YAK D. M.
dithiothreitol. SH-oligonucleotides were reversibly bo
und to thiol-modified solid supports [12, 19] , or to
hydrophobic transport agents like thiocholesterol to
be cleaved within the cell by cellular reducing agents
releasing a free oligonucleotide [20] . S-S bond clea
vage is widely used to generate thiol linkers from
disulfide precursors [11, 13, 2 1 — 2 3 ] . It should be
noted that disulfide formation is a common problem
on storage of thiol oligonucleotides. On the other
hand, circular and looped oligonucleotides were pre
pared by oxidative disulfide bond formation between
mercaptoalkyl tether groups [23, 2 4 ] . An additional
interesting property of mercapto group is its ability to
bond readily to some metals, and thiol-modified
oligonucleotides have been immobilized on metal
electrode surfaces for use in hybridization assays,
biosensors, etc. ( [ 2 5 ] and references therein).
Thus, the unique chemistry of sulfhydryl group
makes thiol oligonucleotides very attractive for diverse
applications, and at the same time the development of
reagents and methods for their preparation and
conjugation is still an important problem. Here we
describe the synthesis and use of H-phosphonate
reagent for solid-phase oligonucleotide functionali
zation with aliphatic thiol groups at both 3 ' - and/or
5'-termini.
Materials and Methods. 4,4'-Dimethoxytrityl
chloride (DMTrCl) , 5 - (iodoacetamido)fluorescein
(IAF) and 1,2,4-triazole (Tri) were purchased from
«Fluka» (Switzerland), pivaloyl chloride (PivCl) and
1,4-dithiothreitol (DTT) from «Мегск» (Germany).
Reagents for gel electrophoresis were from «Sigma»
(USA). Solvents were dried as follows: pyridine was
distilled over ninhydrin and then refluxed over cal
cium hydride for 2 h and distilled; acetonitrile was
distilled over phosphorus pentoxide and calcium hyd
ride. TLC was performed on Kieselgel 6 0 F 2 5 4 plates
(«Мегск») using the following solvent systems: chlo
roform (A), chloroform-methanol 9:1 (B) and iso-
propanol — cone N H 3 — water 7:2:1 (C). To detect
nucleoside derivatives, the plates were sprayed with a
mixture 95 % EtOH — acetic acid — anisaldehyde —
cone H 2 S 0 4 9:0.1:0.5:0.5 (v/v) and heated at 110 °С.
Nucleosides are revealed as blue to black spots [26] .
SH-containing compounds were detected by spraying
TLC plates with 0.5 % ethanol solution of 5,5'-
dithiobis(2-nitrobenzoic acid) («Sigma») as yellow
spots [27 ]. Thiols were also detected by exposing
TLC plates to iodine vapors (white spots on dark
background), although this reagent is less specific
producing similar results with all compounds that
could be oxidized by iodine, e. g. dialkyl sulfides.
Absorbance spectra were recorded on HP 8452
spectrophotometer («Hewlett-Packard», USA). *H
NMR spectra were obtained with Bruker AC-250
spectrometer (250 MHz) in CDC1 3 with TMS as
internal standard. 3 1 P NMR spectra were recorded on
the same device operating at 101 MHz using 85 %
H 3 P 0 4 as external standard. Solid-phase oligonuc
leotide synthesis was performed on Applied Biosys-
tems Model 380B automatic DNA synthesizer by
H-phosphonate method [28 ] using reagents delivered
by instrument supplier. Capping with isopropyl H-
phosphonate has been employed [29 ]. Concentrations
of H-phosphonates, including reagent (5), and piva
loyl chloride (PivCl) as condensing reagent in ace
tonitrile— pyridine (4:1) were 0.03 M and 0.15 M,
respectively. Reverse phase HPLC was performed on
Waters 600E chromatograph using ^Bondapak-Cjg
column (3,9 x 150 mm, «Waters», USA) with a linear
gradient of acetonitrile (0—30 % in 30 min) in 0,1 M
TEAA buffer, pH 7.5 (flow rate 1 ml /min) . Poly-
acrylamide gel electrophoresis (PAGE) was carried
out on standard 20 % denaturing gel. Oligonucleotide
bands were visualized under UV light (254 nm) using
UV-shadowing technique, or at 365 nm for fluorescein
conjugates.
Extinction coefficient e2b0 for T , 5 oligonucleotide
calculated according to [30] was 1.22*10 .
Synthesis of reagent. 3-A c e t y l m e r c a p -
t о p г о p a n о 1-1 (1) and 3-m e r c a p t o p r o -
p a n о 1-1 (2) were prepared according to [31 ].
3 ,3 ' -D i t h i o d i p r o p a n o l (3) was
synthesized by adapting procedure described for the
preparation of diethanoldisulfide [32] . To 2.20 g of
3-mercaptopropanol-l (2, 2.5 mmol) 0.5 eq. of 30 %
hydrogen peroxide was added dropwise with stirring
and ice-cooling. The solution was allowed to stand
overnight at room temperature until thiol had com
pletely disappeared (test with thiol reagent). Water
was removed under reduced pressure. 2.17 g of
colourless oil was obtained (quantitative yield). Purity
of (3) was confirmed by *H NMR: б (ppm) 3.72 (t,
2H, C H 2 0 ) ; 2.78 (t, 2H, CH 2 S) ; 2.40—2.60 (br. s.,
1H, OH); 1.92 (quint, 2H, C H 2 internal).
l - 0 - ( 4 , 4 ' - d i m e t h o x y t r i t y l)-3,3'-d i-
t h i o d i p r o p a n o l (4). 1.82 g of
3,3'-dithiodipropanol (3, 10 mmol) was evaporated
three times with anhydrous pyridine and dissolved in
the same solvent (10 ml). The solution of 1.19 g of
dimethoxytrityl chloride (3.5 mmol) in anhydrous
pyridine (10 ml) was added dropwise with stirring
over 1 h. The mixture was kept at room temperature
for additional hour, poured into 100 ml of 5 %
N a H C 0 3 and extracted with chloroform (3 * 30 ml).
Combined organic layer was washed with 0.25 M
TEAB (pH 7.5, 50 ml) and water, dried over N a 2 S 0 4
and evaporated. After coevaporation with toluene to
164
SYNTHESIS O F D I S U L F I D E - B A S E D H - P H O S P H O N A T E R E A G E N T
remove remaining pyridine, product was isolated by
silica gel chromatography in the gradient 40—80 %
CHCI3 in hexane to give a light yellow oil after
evaporation. Yield 1.20 g (2.48 mmol, 71 % based on
DMTrCl). Rf 0.10 (system A); 0.68 (B). *H NMR: б
(ppm) 7.15—7.45 (m, 9H, Ar); 6.82 (d, 4H, Ar); 3.78
(s, 6H, 2 C H 3 0 ) ; 3.72 (t, 2H, CH 2 OH); 3.15 (t, 2H,
CH 2 ODMTr); 2.70—2.85 (m, 4H, 2CH 2 S); 1.85—
2.05 (m, 4H, 2 C H 2 internal). Ca. 10 % of ditritylated
dipropanoldisulfide were also isolated (Rf 0.61 in
system A).
l - 0 - ( 4 , 4 - d i m e t h o x y t r i t y l )-3,3'-d i-
t h i o d i p r o p a n o І - Г - 0 - Ш - р h o s p h o n a -
t e) (5). Standard procedure for nucleoside phos-
phitylation [28 | was used with some modifications.
1,2,4-TriazoIe (1.86 g, 27 mmol) was evaporated
twice with anhydrous acetonitrile and suspended in
the same solvent (15 ml). N-methylmorpholine
(5.5 ml, 50 mmol) and then phosphorus trichloride
(718 /Л, 8.25 mmol) were added with cooling (5—
10 °С), The mixture was stirred for 30 min at this
temperature. Then solution of monotritylated dithio-
dipropanol (4, 0.80 g, L65 mmol in 10 ml of dry
CH 3 CN) was added dropwise over 20 min with
cooling, and the reaction mixture was stirred for
further 20 min at room temperature. TLC (system B)
showed almost complete conversion of starting mate
rial into product with low mobility. The mixture was
poured into 100 ml of 0.5 M TEAB (pH 7.5) and
10 min later extracted with chloroform ( 3 x 5 0 ml).
Organic extract was washed with 0.25 M TEAB (2 x
x 50 ml) and water (50 ml), dried over sodium sulfate
and evaporated. Product (5) was isolated by flash
silica gel chromatography. Column was washed with
5 % methanol in chloroform and then the desired
H-phosphonate was eluted with 7—8 % MeOH in
chloroform containing 1 % triethylamine. Eluate was
washed with 0.25 M TEAB and water, dried over
N a 2 S 0 4 and evaporated. Product was dried with oil
pump. 0.72 g of viscous pale yellow oil was obtained
(67 %). Rf 0.05 (system B). l H NMR: б (ppm)
7.15—7.45 (m, 9H, Ar); 6.82 (d, 4H, Ar); 6.82 (d,
1H, H-P, J H P 620 Hz); 3.93 (m, 2H, C H 2 0 - P ) ; 3.78
(s, 6H, 2 C H 3 0 ) ; 3.05—3.20 (m, 8H, CH 2 ODMTr +
+ 3CH 2 N (TEAH)); 2.70—2.90 (m, 4H, 2CH 2 S);
1.85—2.10 (m, 4H, 2 C H 2 internal); 1.31 (t, 9H,
ЗСН3 (TEAH)). Purity of reagent was > 95 %,
according to 3 1 P NMR analysis (CDC1 3: 6p 2.95).
Model 5'-functionalization of nucleoside. 33 mg
of reagent 5 (0.05 mmol) and 17 mg of З'-О-
acetylthymidine (0.06 mmol) were evaporated twice
with anhydrous pyridine, dissolved in the same
solvent (600 jul) and treated with pivaloyl chloride
(24/Л, 0.2 mmol). 3 min later TLC (system B)
showed complete conversion of starting H-phospho
nate (Rf 0.05) into trityl-, nucleoside-containing coup
ling product (Rf 0.42) well separated from the slight
excess of starting nucleoside (Rf 0.33). Iodine (25 mg,
0.1 mmol) in 400 //1 of pyridine-water (20:1) was
added, and 10 min later the complete oxidation of
H-phosphonate diester into phosphodiester (trityl-,
nucleoside-positive, Rf 0) was observed. The mixture
was diluted with 0.25 M TEAB (1 ml) and aqueous
solution of N a 2 S 0 3 was added dropwise carefully to
neutralise excess iodine until its colour disappeared.
No disulfide bond cleavage was observed. Further
2 ml of 0.25 M TEAB were added, and the mixture
was extracted with chloroform ( 3 x 1 ml) , extract was
washed with 0.25 M TEAB and water (1 mi each) and
evaporated. Disulfide-functionaiized dT(Ac) contai
ning a little amount of starting nucleoside was dissol
ved in 1 ml of CHCI3-TEA (9:1), and 0.25 mmol of
DTT was added. In 2 hours TLC showed the
complete disappearance of starting trityl-positive ma
terial with Rf 0, to be converted into 2 new products
of S-S bond cleavage: (a) DMTr-negative, nucleoside-
positive, SH-positive, Rf 0 (systems A, B); 0.73 (C),
and (b) DMTr-positive, nucleoside-negative, SH-po
sitive, Rf 0.75 (A); 1.0 (B). Compound (b) was
formed also as the only product upon treatment of
1,1 '-0-bis-dimethoxytrityl-3,3'-dithiodipropanol (see
above) with DTT, it was trityl-positive product of
l-0-dimethoxytrityl-3,3'-dithiodipropanol (4) cleava
ge with DTT, and, finally, the same compound was
formed upon deacylation of AcSCH 2 CH 2 CH 2 ODMTr
with KOH/EtOH. Therefore, compound (b) could be
considered as HSCH 2 CH 2 CH 2 ODMTr, whereas com
pound (a) with low TLC mobility could be 5'-
mercaptopropylphosphate nucleoside derivative (6,
R - - d T ( A c ) ) .
Synthesis of oligonucleotide conjugates. I n t r o
d u c t i o n o f d i s u l f i d e r e a g e n t i n
t o o l i g o n u c l e o t i d e s . The solution of
disulfide H-phosphonate reagent (5) was attached to
the spare port on the synthesizer. For thiol intro
duction at the 3'-terminus, reagent was directly
coupled to thymidine-derivatized solid support (CPG)
with coupling time extended to 5 min (coupling yield
92 % ) . Standard H-phosphonate synthesis of oligo-
T , 5 chain was then performed, with average coupling
yield 98.1 %. After sequence elongation was comp
leted, the support containing З'-disulfide-linked T 1 5
was divided into 2 equal parts. The first one was used
for the preparation of 3'-thiol oligomer, whereas from
the other half of polymer 5',3'-dithiol derivative was
obtained by repeating coupling with reagent (5)
(coupling time 5 min, yield 95 %, as determined by
detritylation of small weighed part of polymer). After
165
D U B E Y I. Y., FEDORYAJK D. M.
standard oxidation (2 % iodine in pyridine-water
98:2, 10 min) oligonucleotides were cleaved from the
support (concentrated ammonia, room temperature
overnight). 5'-DMTr-containing sequences were puri
fied by Oligo-Pak cartridge (Milligen/Biosearch,
USA) following manufacturer's protocol. Oligonuc
leotides were detritylated directly on the cartridge,
eluted and evaporated. (Note that at this step both
oligomers contained additional thymidine residue lin
ked at З'-end through disulfide tether). Normal T , 5
oligonucleotide was also synthesized by standard H-
phosphonate method to be used as control.
D i s u l f i d e b o n d c l e a v a g e .
Disulfide-containing oligonucleotides were quantita
tively reduced before labelling by treatment with
30 mM DTT (0.5 mg per 1 O D 2 6 0 of oligonucleotide)
in 10 mM Tris-HCl buffer (pH 8.0) overnight at
37 °С under argon. Cleavage was monitored by
HPLC. Most of the D T T was removed by extraction
with butanol, and the reduced thiol oligonucleotides
were desalted at PD-10 cartridge («Pharmacia»).
O l i g o n u c l e o t i d e l a b e l l i n g
w i t h f l u o r e s c e i n . 5 O D 2 6 0 of З'-thiol or
3',5'-dithiol oligonucleotide (0.04 mmol) were dissol
ved in 200 ml of 0.1 M sodium carbonate/bicarbonate
buffer (pH 9.0). 20 (for З'-thiol o l igo-T l 5 ) or 30 (for
3',5'-dithiol o l i go -T 1 5 ) eq of 5 - ( i o d o a c e t a m i -
do)fluorescein in 100 /Л of freshly distilled DMF were
added with agitation, and the reaction mixture was
incubated under argon at room temperature overnight
in the dark. Labelling reaction was monitored by
reverse phase HPLC. If necessary, a further 5—10 eq
of IAF could be added. The reaction mixture was
diluted with 500 /Л of water and passed through
PD-10 gel filtration column to remove excess label.
The desired product was eluted with 50 mM TEAB
(pH 7.5) containing 5 % of ethanol, and eluate was
evaporated. In the synthesis of З'-labelled T 1 5 oligo
nucleotide, some non-labelled starting oligomer was
removed efficiently at Oligo-Pak cartridge following
the protocol for DMTr-oligonucleotides isolation, and
3',5'-bis-fluorescein-T 1 5 was purified by reverse phase
H P L C Purity of products was confirmed by PAGE
and H P L C 3'-Fluorescein-T I 5 : yield 78 %. UV/Vis :
A 2 6 0 / A 4 9 4 = 2.03 (calculated ratio 1.96). 3' ,5'-bis-
Fluorescein-T, 5: y i e l d 5 7 %; A 2 6 0 / A 4 9 4
e 1.24
(calculated = 1.15).
Results and Discussion. A number of methods for
the preparation of thiol-modified oligonucleotides has
been described [2, 6 — 9 ] . SH groups can be intro
duced into oligonucleotide using suitably protected
nucleoside mercapto derivatives, e. g. 5'-mercapto-
nucleosides for 5'-derivatization [33] or 5-mercapto-
uridine for site-specific labelling [34] . Cystamine is
widely employed to prepare thiol oligonucleotides, as
it has amino group to react with amine-specific
entities, and cleavable disulfide bond generating thiol
upon treatment with DTT. Cystamine linkers have
been introduced at the internucleotide linkage via
H-phosphonate approach [35 ] , 5'-cystaminyl oligo
nucleotides have been synthesized to yield 5'-thiol
derivatives [13, 16, 2 3 ] , and З'-thiol oligonucleotides
were prepared by cleaving from the special support
with cystamine with subsequent S-S bond cleavage
[23 ]. More common approaches to the introduction of
thiols into 3 ' - or 5'-terminus have been also develo
ped. З'-Thiol oligonucleotides are usually synthesized
on specially designed disulfide-derivatized solid sup
ports [11, 14, 18, 19, 22, 2 3 ] . Preparation of these
supports is rather laborious procedure, although they
are now commercially available. 5'-Thiol oligonuc
leotide functionalization is more simple, and corres
ponding reagents are also available. They are deriva
tives of S-protected mercaptoalcohols, namely S-trityl
mercaptopropanol or -hexanol phosphoramidites [17,
36, 38 ] or H-phosphonates [37, 38 ]. These reagents
can be introduced into oligonucleotides at the last
coupling step of solid-phase synthesis, and then
S-trityl group is removed by A g N 0 3 / D T T . This
approach has been reported to work well with rela
tively short sequences (up to 12-mers), but yields
decreased significantly for longer oligomers, espe
cially with the use of amidite reagents [17, 3 7 ] . Low
yields of thiol oligonucleotides were probably resulted
from the chemical modification of 5'-thiol terminus
during deprotection. So, there are several methods for
oligonucleotide 5'- or З'-end functionalization with
thiol linker groups, but these approaches are usually
not highly efficient, and moreover they are not
applicable for the synthesis of oligonucleotides with
thiols at both termini.
Our intention was to achieve a simple technique
for obtaining 3 ' - and/or 5'-thiol labelled oligonuc
leotides using single reagent. As many methods for
3 ' - and 5'-labelling described above briefly are based
on the cleavage of disulfide precursors, it was clear
that properly designed disulfide-containing functio-
naiizing reagent could be able to label any end of
oligonucleotide. Cystamine has been used for 3' - and
5'-labelling, however we would like to create a reagent
for direct functionalization during solid-phase syn
thesis. We decided that such a reagent has to be
bifunctional, like cystamine, but with hydroxy groups
instead of amines, containing one DMTr-protected
hydroxyl to allow for chain elongation and one
phosphorylating moiety for reagent introduction as
P-component during oligonucleotide synthesis; these
two units should be connected with cleavable disulfide
166
SYNTHESIS O F D L S U L F I D E - B A S E D H - P H O S P H O N A T E R E A G E N T
linker. Dialcohol disulfides were an obvious сіюісе for
our purpose, and reagent resulted from tritylation and
phosphorylation of two hydroxy groups linked via S-S
bond would be introduced at 3 ' - or 5'-end of oligo
nucleotide to obtain 3 ' - or 5'-thiol derivatives after
internal disulfide cleavage. Reagent under design was
based on the same principle as oligonucleotide phos-
phorylating agent derived from the symmetric su-
lfodiethanol, with one hydroxyl being protected with
DMTr group and another phosphorylated; it can be
added at 3 ' - or 5'-end of oligonucleotide, and during
ammonia deprotection central sulfonyl linkage is /?-
eliminated to produce 3 ' - or 5'~phosphate (or both)
[391.
Our first attempts to prepare oligonucleotide
functionalizing reagent based on the easily available
diethanoldisulfide failed: when O-monotritylated in
termediate was phosphorylated with tris(triazo-
lyDphosphinc, starting material disappeared, but sig
nificant cleavage of the product (Rf ca. 0.05 in system
B) was observed during its isolation with formation of
two compounds able to be oxidized with iodine,
probably the result of S-S bond cleavage or another
redox process. This reaction was not further inves
tigated, as it was found in the literature that 2-
mercaptoethyl phosphates are anyway unstable under
basic conditions cleaving ethylmercapto group to yield
free phosphates. Nevertheless, it was demonstrated
that mercaptopropyl and -hexyl phosphates were
completely stable [21, 3 6 ] .
As a result, we have prepared thiol-introducing
H-phosphonate reagent (5) based on 3,3'-dithio-
dipropanol (dipropanoldisulfide). Our first report on
this reagent was presented at the conference in 1993
[40 ], however its synthesis and use still has not been
described in detail. Recently, a similar phospho-
ramidite reagent based on 6,6-dithiodihexanol has
been used to prepare circular oligonucleotides by
oxidizing 5'-, З'-terminal thiol groups [24] ; to our
best knowledge, its synthesis was not yet reported
too.
The preparation of the reagent (5) (Scheme 1)
started from the synthesis of 3,3'-dithiodipropanol
(3). S-acetylmercaptopropanol (1) was obtained by
the addition of thiolacetic acid to allyl alcohol in the
presence of benzoyl peroxide, and deacylated with
10 % NaOH to give 3-mercaptopropanol (2), ac
cording to [31] . Its subsequent oxidation with cal
culated amount of hydrogen peroxide resulted in
quantitative yield of the desired dipropanoldisulfide
(3). The latter was tritylated with DMTrCl in pyri
dine (yield 71 % ) ; this method was found to be more
efficient than previously described tritylation in the
presence of DMAP and TEA (yield 48 %) [14] .
Monotritylated 3,3'-dithiodipropanol (4) has been
previously attached to solid support via succinate
linkage for the preparation of 3'-thiol oligonucleotides
solely [14, 2 3 ] . In our study, this intermediate was
phosphitylated with PTri 3 to produce H-phosphonate
reagent (5) for solid-phase synthesis. No product
cleavage was observed in this case, as was with
diethanoldisulfide derivative. As 3 1 P NMR demon
strated, reagent was completely stable: no additional
signals were observed in the spectrum after one year
storage at - 1 8 °С. So, possible in theory self-
oxidation of monoester P-H bond of reagent (5) by
its S-S group didnot occur. Possible as well sulf-
urization of diester P-H bonds (including intra
molecular reaction of З'-linked disulfide) during so
lid-phase synthesis was not specially studied; how
ever, this process seems to be unlikely to proceed
under normal synthetic conditions, as dialkyl disul
fides are not active sulfurizing agents since their S-S
bond is not activated by electron-acceptor substi-
tuents. This type of side reaction has never been
reported for phosphoramidite oligonucleotide synthe
sis on disulfide-containing supports, although P(III)
species are much more succeptible to sulfurization
with disulfides than tetracoordinated H-phosphona-
tes. Clear HPLC profiles of reaction mixtures from
the syntheses of thiol oligoucieotides and their con
jugates could probably confirm this conclusion to a
certain degree (see below).
To test the efficasy of H-phosphonate reagent for
thiol group introduction into oligonucleotides, model
labelling of nucleoside (З'-O-acetylthymidine) was
first carried out in the solution, according to Scheme
2, a. Nucleoside was phosphitylated with (5) using
pivaloyl chloride as condensing reagent, and hydro-
phosphoryl group of coupling product was oxidized
smoothly with iodine in aqueous pyridine to give
phosphodiester containing disulfide linkage. The lat
ter was cleaved by D T T in the presence of base
(TEA) to result in the formation of nucleoside func-
tionalized with mercapto group (6, J? = -dT(Ac) ) .
This product was not however specially isolated and
studied since model reaction was performed just to
demonstrate that nucleoside could be converted to
thiol-containing product by coupling with H-phos
phonate reagent (5) followed by iodine oxidation and
DTT treatment.
For oligonucleotide functionalization, proposed
reagent can be added as P-component at the first
and/or last coupling cycle of solid-phase synthesis,
according to normal H-phosphonate synthetic protocol
(Scheme 2) . 5'-Thiol oligonucleotides (6) are prepa
red through the addition of reagent (5) at last
H-phosphonate coupling step after the assembly of
167
D U B E Y I V.. FEDORYAK D . M.
Scheme 1
desired oligonucleotide chain was completed. To pre
pare З'-modified oligonucleotides (7), H-phosphonate
reagent has to be introduced at the first addition to
any nucleoside support, followed by the normal
synthesis of target sequence. Post-synthetic reducing
with DTT cleaves S-S bond leaving a thiol group at
5'- or 3-terminus. In such a way, oligonucleotides
labelled at both 3 ' - and 5'-ends could be also easily
prepared.
We have used H-phosphonate (5) to synthesize a
15-mer oligothymidylate containing one or two thiol
groups at 3 ' - and 5'-termini on DNA synthesizer by
conventional H-phosphonate approach. Coupling time
for (5) was increased to 5 min, as is usually
recommended for functionalizing reagents containing
long alkyl chains. In this case, the yields were 92 and
95 % for the first and last coupling step, respectively,
as determined by DMTr cation release, whereas
common nucleoside H-phosphonates were coupled for
2 min with average yield about 98 %. After final
oxidation with aqueous iodine, oligonucleotides were
cleaved from the polymer support with concentrated
NH 4 OH. Ammonolysis was carried out overnight at
room temperature, as only thymidines were present in
the sequence; otherwise common deblocking (e. g. at
55 °С for at least 6 h) should be performed. Disulfide
168
SYNTHESIS OF D I S U L F I D E - B A S E D H - P H O S P H O N A T E R E A G E N T
bond cleavage could be performed simultaneously, by
adding DTT to ammonia solution [19] . In our case,
S-S bonds were kept during oligonucleotide puri
fication to avoid the oxidative dimerization of SH-
functionalized oligonucleotides [11, 12, 3 6 ] . 5 - 0 -
DMTr protecting groups were not removed to simplify
the isolation of product. As HPLC analysis of crude
reaction mixtures showed good chromatographic pat
terns (Fig. 1, я) , no special attempts were made to
purify oligomers by gel electrophoresis or HPLC.
DMTr- and disulfide-containing oligonucleotides were
purified using Oligo-Pak cartridges. Purification is
based there on the principle of reverse phase chroma
tography: the desired oligomer with hydrophobic trityl
group is retained by the support while the failure
sequences without DMTr residue, as well as depro-
tection by-products, are removed from the reaction
mixture by washing to deliver an oligomer of good
purity sufficient for most applications. Detritylation of
oligonucleotides was performed directly on the Oligo-
Pak support after removing side products. All oligo
nucleotides were of good quality after Oligo-Pak
isolation, and no additional purification was carried
out. Then 3 - and 5'-disulfide linkages in purified
oligonucleotides were quantitatively cleaved with DTT
to produce the corresponding thiol derivatives. The
latter should be stored (if necessary) in the presence
of a little amount of DTT (ca. 5—10 mM) to avoid
dimerization. DTT can be removed before conjugation
by butanol or ethylacetate extraction. In principle, it
is not essential to purify 5- th io l oligonucleotides at
this step, as only SH-containing oligomer will react
with labelling reagent and conjugation product could
be easily purified. But in the synthesis of 3'-thiol
oligonucleotides failure sequences also contain 3'-SH
groups, and purification of starting oligomer would be
desirable. Nevertheless, our thiol oligomers were of
good purity, as HPLC showed, and additional puri
fication seemed to be unnecessary. On reverse phase
HPLC, thiol oligonucleotides had somewhat higher
retention time than non-modified T I 5 , but lower than
corresponding disulfide precursors (Fig. 1, b).
To demonstrate the utility of our approach for
oligonucleotide labelling, we have synthesized T I 5
oligonucleotides bearing one or two fluorescein resi
dues at 3 - and both 3 ' - and 5'-ends for the studies
on fluorescence polarization. The coupling step invol
ves reaction of oligonucleotide bearing a nucleophilic
thiol linker(s) with dye reagent containing elect-
rophilic thiol-specific iodoacetamide group (Scheme
3).
Labelling reactions were performed in sodium
carbonate/bicarbonate buffer (pH 9) containing ca.
30 % DMF (where IAF was dissolved). Labelling was
10 20
Retention time, min
30
Fig. 1. Reverse phase HPLC analysis of oligonucleotides: a — crude
reaction mixture containing 5'-DMTr-protected Ti 5 -3'-disulf ide;
b — T i 5 - 3 ' - S H after Oligo-Pak purification of product from a and
disulfide bond cleavage. Arrows indicate the positions of normal T I 5
( / ) and T 1 5 -3 ' -disul f ide (2) on the chromatogram
carried out at room temperature overnight under
argon to avoid possible yield decrease due to oxidative
dimerization of thiol oligonucleotides. As some DTT
is expected to be present in thiol oligonucleotide even
after desalting, the use of sufficient excess of labelling
reagent is recommended. In the presence of 20 eq of
fluorescein reagent, 3'-thiol oligomer was efficiently
labelled with dye during the reported coupling period.
At the same time, in the case of 3 ,5' -dithiol labelling
starting oligomer disappeared too, but about 30 % of
oligonucleotide material remained monolabelled under
these reaction conditions. However, in the presence of
30 eq of IAF almost complete transformation into
bis-labelled product was observed. Excess dye was
removed by gel filtration passing the reaction mix
tures through PD-10 desalting cartridge. Further
purification of conjugates by preparative gel electro
phoresis or HPLC is usually necessary to separate the
conjugation product from unlabelled sequence. In our
case, purification by Oligo-Pak cartridge seemed to be
sufficient, as fluorescein residue, like trityl group, is
enough hydrophobic to retain the conjugate on the
column while impurities are separated. Indeed, this
technique allowed the full separation of some amount
169
D U B E Y I Y , FEDORYAK D. M.
Scheme 3
of non-labelled sequence present in the reaction
mixture from З'-conjugate preparation. However,
Oligo-Pak was not able to separate mono-labelled
oligomer from bis-labelled oligonucleotide product in
the case of 3\5'-bis-fluorescein conjugate preparation.
In this case, preparative HPLC was used to isolate the
desired product.
Oligonucleotide products purified by Oligo-Pak or
reverse phase HPLC were analyzed by HPLC and
PAGE. Fig. 2 showes the HPLC profiles of purified
mono- and bis-labelled oligonucleotides T I 5 -3 ' -Flu
a n d 5 ' -F lu -T ( 5 -3 ' -F lu . 3 ,-Fluorescein-labelled
oligomer was retained longer than non-modified T 1 5 ,
due to hydrophobic character of the dye; additional
Flu residue further increased the retention time.
PAGE analysis showed reasonable purity of oligo
nucleotides. Conjugate bands were easily identified in
the gel by their green fluorescence under long wave
length UV light (365 nm). Substantial mobility shift
was observed for fluorescein-labelled oligonucleotides.
Each dye molecule attached to oligonucleotide decre
ased its mobility in the gel approximately as one
additional nucleotide. Chromatographic and electro-
phoretic behaviour of prepared conjugates is in full
agreement with data on oligonucleotides labelled with
several dye residues by another methods [41—43] .
Fluorescein conjugates were characterized by
UV/Vis spectroscopy. The absorbance spectra of
labelled oligonucleotides at pH 8 are showed in Fig.
3. The spectra of non-modified T 1 5 and IAF are
included to illustrate the relative contribution of the
dye and oligonucleotide to conjugate absorbance.
Absorption pattern of dye residue in the conjugates is
quite similar to that of IAF, with absorbance maxi
mum at 494—495 nm. The extinction coefficient of
fluorescein moiety in the conjugates at 260 nm can be
estimated as ca. 25000 from the spectrum of IAF with
known e 4 9 5 - 75000 [10] . Taking into account dye
extinction at these wavelengths, the observed absor
bance ratios A 4 9 4 / A 2 6 0 for fluorescein-labelled oligo
nucleotides are in good agreement with calculated
values indicating clearly the presence of 1 and 2 dye
residues per oligonucleotide, respectively. It is inte
resting to note that at pH 6 sharp intense peak of
fluorescein absorbance at 494 nm almost disappeared
having been converted into broad band of low inten
sity at 440—500 nm (Am a x = 454 nm), due to the
changes in dye structure; fluorescence at this pH is
also known to be relatively low.
The data presented in this paper demonstrate the
utility of proposed disulfide H-phosphonate reagent
(5) for direct solid-phase oligonucleotide functio-
170
SYNTHESIS O F D 1 S U L F I D E - B A S E D H - P H O S P H O N A T E R E A G E N T
Fig. 3. UV/Vis spectra of oligonucleotides T i 5 - 3 ' - F l u (a) , 5'-Flu-
T I 5 - 3 ' - F l u (b), T 1 5 <c), and IAF dye id) in 25 mM Tris-HCl buffer
(pH 8)
nalization at 3 ' - and/or 5'-end with one or two thiol
groups. It is easily prepared, requires no changes of
common H-phosphonate synthesis protocol, and pro
vides a reliable and flexible way for oligonucleotide
labelling with fluorescent dyes or other molecules at
any terminus.
Acknowledgements. Authors thank G. G. Ra-
rashenkov for the synthesis of 3-mercaptopropanol.
We are also grateful to Dr. S. M. Yarmoluk for
assistance in editing manuscript.
/. Я. Дубей, Д. M. Федоряк
Синтез та використання дисульфідного Н-фосфонатного
реагента для 3 ' - та /або 5'-мічення олігонуклеотидів
через меркаптоалкільний лінкер
Резюме
Описано синтез нового дисульфідного Н-фосфонатного реаген
та для твердофазного введення в олігонуклеотиди меркаптоп-
ропільних груп по одному чи двох кінцях. З використанням
цього реагента було синтезовано З ' - та 5'-дисульфідні похідні
олігонуклеотидів. Дисульфідні зв'язки розщеплено дитіотре-
їтолом і тіольні групи, що утворилися, модифіковано йодаце-
тамідофлюоресцеїном. З високим виходом одержано оліго-Т15,
які несуть один чи два залишки флюоресиеіну на У- і 5 -кінцях.
И. Я. Дубей, Д М. Федоряк
Синтез и использование дисульфидного Н-фосфонатного
реагента для 3 ' - и /или 5'-мечения олигонуклеотидов
через меркаптоалкильный линкер
Резюме
Описан синтез нового дисульфидного Н-фосфонатного реаген
та для твердофазного введения в олигонуклеотиды меркап-
топропильных групп по одному или двум концам. С использо
ванием этого реагента синтезированы 3',5'-дисульфидные про
изводные олигонуклеотидов. Дисульфидные связи расщеплены
дитиотреитолом и образовавшиеся тиольные группы модифи
цированы иодацетамидофлюоресцеином. С высоким выходом
получены олиго-Т15, несущие один или два остатка флюоресце-
ина на. 3' и 5'-концах.
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Received 05.05.97
172
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| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Dubey, I.Y. Fedoryak, D.M. 2019-06-16T07:59:55Z 2019-06-16T07:59:55Z 1998 Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke / I.Y. Dubey, D M. Fedoryak // Биополимеры и клетка. — 1998. — Т. 14, № 2. — С. 163-172. — Бібліогр.: 43 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0004CA https://nasplib.isofts.kiev.ua/handle/123456789/154992 Synthesis of new disulfide-based H-phosphonate reagent for solid-phase oligonucleotide functionalization with mercaptopropyl group at one or two termini is described. Using this single reagent, 3'- and 3', 5'-disulfide-linked oligonucleotides were synthesized', disulfide bonds cleaved with dithiothreitol and generated thiol groups labelled with iodoacetamidofluorescein. Tis oligonucleotides containing one or two fluorescein residues at 3'- and 5'-ends were prepared in high yields. Описано синтез нового дисульфідного Н-фосфонатного реагента для твердофазного введення в олігонуклеотиди меркаптопропільних груп по одному чи двох кінцях. З використанням цього реагента було синтезовано З'- та 5'-дисульфідні похідні олігонуклеотидів. Дисульфідні зв'язки розщеплено дитіотреїтолом і тіольні групи, що утворилися, модифіковано йодацетамідофлюоресцеїном. З високим виходом одержано оліго-Т15, які несуть один чи два залишки флюоресиеіну на У- і 5 -кінцях. Описан синтез нового дисульфидного Н-фосфонатного реагента для твердофазного введения в олигонуклеотиды меркаптопропильных групп по одному или двум концам. С использованием этого реагента синтезированы 3',5'-дисульфидные производные олигонуклеотидов. Дисульфидные связи расщеплены дитиотреитолом и образовавшиеся тиольные группы модифицированы иодацетамидофлюоресцеином. С высоким выходом получены олиго-Т15, несущие один или два остатка флюоресцеина на 3' и 5'-концах. Authors thank G. G. Rarashenkov for the synthesis of 3-mercaptopropanol. We are also grateful to Dr. S. M. Yarmoluk for assistance in editing manuscript. en Інститут молекулярної біології і генетики НАН України Биополимеры и клетка Методы Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke Синтез та використання дисульфідного Н-фосфонатного реагента для 3 ' - та/або 5'-мічення олігонуклеотидів через меркаптоалкільний лінкер Синтез и использование дисульфидного Н-фосфонатного реагента для 3 ' - и/или 5'-мечения олигонуклеотидов через меркаптоалкильный линкер Article published earlier |
| spellingShingle | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke Dubey, I.Y. Fedoryak, D.M. Методы |
| title | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke |
| title_alt | Синтез та використання дисульфідного Н-фосфонатного реагента для 3 ' - та/або 5'-мічення олігонуклеотидів через меркаптоалкільний лінкер Синтез и использование дисульфидного Н-фосфонатного реагента для 3 ' - и/или 5'-мечения олигонуклеотидов через меркаптоалкильный линкер |
| title_full | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke |
| title_fullStr | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke |
| title_full_unstemmed | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke |
| title_short | Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke |
| title_sort | synthesis and use of disulfide-based h-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linke |
| topic | Методы |
| topic_facet | Методы |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/154992 |
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