A new linker for solid-phase synthesis of oligonucleotides with terminal phosphate group

Article abstract of DOI:10.1135/cccc20080032

Synthesis of a novel cyanoethyl-type linker suitable for the solid-phase synthesis of oligodeoxynucleotides possessing terminal 3′-phosphate group is described. Since the linker is a 2-substituted 2-cyanoethanol, the release of the synthesized oligonucleotide from the solid support is accomplished by β-elimination in the ammonia deprotection step. 2008 Institute of Organic Chemistry and Biochemistry.

Full text of DOI:10.1135/cccc20080032

32
Pačes, Točík, Rosenberg:
A NEW LINKER FOR SOLID-PHASE SYNTHESIS OF
OLIGONUCLEOTIDES WITH TERMINAL PHOSPHATE GROUP
1
2,
3,
Ondřej PAČES , Zdeněk TOČÍK * and Ivan ROSENBERG *
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, v.v.i.,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: paces@uochb.cas.cz,
2
3
tocik@uochb.cas.cz, ivan@uochb.cas.cz
Received October 30, 2007
Accepted Jan uary 14, 2008
Syn th esis of a n ovel cyan oeth yl-type lin ker suitable for th e solid-ph ase syn th esis of oligo-
deoxyn ucleotides possessin g term in al 3′-ph osph ate group is described. Sin ce th e lin ker is a
2-substituted 2-cyan oeth an ol, th e release of th e syn th esized oligon ucleotide from th e solid
support is accom plish ed by β-elim in ation in th e am m on ia deprotection step.
Keyw ord s: 2-Substituted-2-cyan oeth an ol; β-Elim in ation ; Modified LCAA-CPG; Solid-ph ase
syn th esis; Oligon ucleotide 3′-ph osph ates; Solid support; Deprotection .
A variety of supports for th e solid-ph ase syn th esis of (deoxy)ribon ucleo-
tides h ave been in troduced over past two decades, th e (lon g-ch ain alkyl)-
am in o con trolled-pore glass (LCAA-CPG) playin g a prom in en t role am on g
th em for m ost purposes. However, with in creasin g n um ber of cases in
wh ich oligom ers con tain in g term in al ph osph ate groups were n eeded for
subsequen t en zym atic1,2 or ch em ical3 ligation s as well as for diagn ostic4
purposes, specific approach es were sough t to provide appropriately ad-
justed supports an d protocols.
Th ere are several ch em ically differen t m eth ods for th e solid-ph ase syn -
th esis of th e term in ally ph osph orylated oligon ucleotides wh ich em ploy
various lin kers for th e attach m en t of th e first n ucleotide un it. Th us, Horn
et al.5 described a solid support with th e 2-[(2-h ydroxyeth yl)sulfon yl]-
eth oxy group (Fig. 1, scaffold A). Th e desired 3′-ph osph orylated oligo-
n ucleotide is released from th e support by a β-elim in ation reaction (e.g., by
treatm en t with am m on ia). A ch em ically sim ilar lin ker was also described
later by Markiewicz et al.6.
Th e m eth od usin g an oxim ate-derived support was in troduced by Hän er
et al.7 (Fig. 1, scaffold B). Th e ph osph odiester con jugate of oligon ucleotide
an d oxim ate un dergoes, un der alkalin e con dition s, th e elim in ation reaction
resultin g in th e form ation of term in ally ph osph orylated oligon ucleotide.
Collect. Czech. Chem. Commun. 2008, Vol. 73, No. 1, pp. 32–43
© 2008 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20080032

Solid-Phase Synthesis of Oligonucleotides
33
An oth er strategy for th e preparation of term in ally ph osph orylated oligo-
n ucleotides m ade use of th e oxidation of th e cis-diol system of th e term in al
ribon ucleoside m oiety8–11 with NaIO4, followed by th e β-elim in ation reac-
tion (Fig. 1, scaffold C).
An in terestin g lin ker for th e syn th esis of oligon ucleotides with 3′-term i-
n al ph osph ates an d ph osph oroth ioates was described by Ravikum ar et al.12.
Th eir con cept con sists in th e use of th e 3-(2-h ydroxyph en yl)propan -1-ol
derivative an ch ored to th e solid support via ester bon d with ph en olic h y-
droxyl (Fig. 1, scaffold D). Th e prim ary h ydroxy group serves for th e attach -
m en t of th e first n ucleotide un it. Upon am m on ia treatm en t, th e ph en ol
ester group is cleaved an d th e followin g n ucleoph ilic attack of ph en olic OH
on th e carbon atom bearin g ph osph oester m oiety releases th e 3′-ph osph or-
ylated oligon ucleotide.
Th e use of dieth yl bis(h ydroxym eth yl)m alon ate, an ch ored to th e solid
support via on e of th e h ydroxy groups, was reported by Guzaev et al.13
(Fig. 1, scaffold E). Un der alkalin e con dition s, two elim in ation reaction s
led to fin al release of th e oligon ucleotide from th e solid support.
FIG. 1
Overview of lin kers wh ich em ploy β-elim in ation or related reaction s in th e preparation of ter-
m in ally ph osph orylated oligon ucleotides
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Pačes, Točík, Rosenberg:
In an oth er approach , Letsin ger et al.14 described th e form ation of a ph os-
ph oram idate (P–N) bon d between th e first 3′-n ucleotide un it an d th e am i-
n o group of th e LCAA-CPG (Fig. 2, scaffold F). Th is bon d rem ain s stable
durin g th e wh ole oligon ucleotide syn th esis, in cludin g acid rem oval of th e
dim eth oxytrityl group. On treatm en t with am m on ia, th e ph osph oester pro-
tectin g groups are rem oved, an d th e 3′-ph osph orylated oligon ucleotide at-
tach ed to th e solid support is easily cleaved off with 80% aqueous acetic
acid. Sim ilar m eth od usin g ben zidin e as a ligan d for attach m en t of th e first
n ucleotide un it via th e P–N lin kage was reported by Markiewicz et al.6,
wh ereby th e cleavage of th e P–N lin kage was accom plish ed usin g isopen tyl-
n itrite (Fig. 2, scaffold G).
FIG. 2
Overview of lin kers prepared by differen t ch em istries for use in solid-ph ase syn th esis of term i-
n ally ph osph orylated oligon ucleotides
Collect. Czech. Chem. Commun. 2008, Vol. 73, No. 1, pp. 32–43

Solid-Phase Synthesis of Oligonucleotides
35
Im bach et al.15 developed an altern ative solid support em ployin g a
ph otolabile 2-n itroben zyl-alcoh ol-type lin ker for th e attach m en t of th e first
n ucleotide un it (Fig. 2, scaffold H). Irradiation of th e solid support led to
th e elim in ation reaction un der release of th e attach ed ph osph orylated
oligon ucleotide in to solution . Th is approach seem s to be suitable for th e
syn th esis of base-sen sitive oligon ucleotides.
A un iversal allyl-type lin ker was reported by Zh an g et al.16. Th e allyl ester
group (Fig. 2, scaffold I) was isom erized un der m ild con dition s (Pd(0)
catalysis), an d th e form ed vin yl ph osph ate m oiety was subsequen tly h ydro-
lyzed to release th e oligon ucleotide ph osph ate.
Several auth ors17–19 reported th e use of 2-sulfan yleth an -1-ol attach ed to a
solid support via disulfide bon d (Fig. 2, scaffold J). After syn th esis of th e
oligom er, th e disulfide lin kage was reduced with dith iotreitol un der alka-
lin e con dition s. Th e free 2-sulfan yleth yl ph osph ate un derwen t an in tra-
m olecular n ucleoph ilic attack of th e S-an ion on th e C1 carbon atom an d
released oligon ucleotide ph osph ate an d episulfide.
In th is paper, we report th e syn th esis of a n ovel type of lin ker, 2-substi-
tuted-2-cyan oeth an -1-ol attach ed to th e LCAA-CPG, wh ich is a prom isin g
altern ative to an ch orin g of oligon ucleotides in th e solid-ph ase syn th esis of
term in ally ph osph orylated oligon ucleotides (Fig. 3, scaffold K). Th e release
of th e syn th esized oligon ucleotide from th e solid support is accom plish ed
by β-elim in ation durin g th e am m on ia deprotection step.
FIG. 3
Structure of n ovel lin ker
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Pačes, Točík, Rosenberg:
RESULTS AND DISCUSSION
Preparation of th e key com poun d 9 (Sch em e 1) to be used as th e desired
lin kin g m oiety started from th e kn own m on osilyl derivative of tetra-
20
eth ylen e glycol 2 wh ich was tosylated in pyridin e, an d th e obtain ed tosyl
derivative 3 was treated with sodium salt of eth yl cyan oacetate. Th e n ucleo-
ph ilic displacem en t of th e tosylate group by eth yl cyan oacetate C-an ion
proceeded sm ooth ly to give com poun d 4.
SCHEME 1
Syn th esis of n ovel lin ker of 2-cyan oeth yl type
Wh en th e C-alkylation was perform ed in eth an ol in th e presen ce of so-
dium eth oxide, a low yield of 4 (~15%) was obtain ed. In con trast to th e pre-
vious reaction s, th e use of DBU in DMF to gen erate th e eth yl cyan oacetate
C-an ion followed by th e reaction with 3 provided a m ixture of un iden tified
products. Reduction of th e ester group of com poun d 4 with sodium boro-
h ydride in eth an ol th en proceeded sm ooth ly to yield th e desired h ydroxy
derivative 5 in a good yield (60% based on 2). Th e subsequen t dim eth oxy-
tritylation of 5 yielded com poun d 6. Rem oval of th e silyl protectin g group
of 6 with TBAF in THF un der carefully con trolled con dition s afforded th e
dim eth oxytrityl derivative 7 (60% based on 5). Th e desilylation of 6 was
foun d to proceed very fast with a fresh ly prepared 0.5 M TBAF·3H2O in THF
(ca. 1–2 h ).
Collect. Czech. Chem. Commun. 2008, Vol. 73, No. 1, pp. 32–43

Solid-Phase Synthesis of Oligonucleotides
37
Th is was th e crucial poin t of th e syn th esis sin ce th e prolon ged tim e of
th e desilylation step (e.g., overn igh t) led to β-elim in ation reaction releasin g
acrylon itrile derivative 8 an d dim eth oxytrityl alcoh ol, th e RF of wh ich on
TLC is alm ost iden tical with th at of com poun d 7. Th e desilylated com -
poun d 7 was th en acylated with succin ic an h ydride, an d th e obtain ed
h ydrogen succin ate 9 (94% on spectral ch aracterization ) was an ch ored via
th e am ide bon d to LCAA-CPG by th e curren tly used procedure21. Th e
ach ieved loadin g varied between 40 an d 50 µm ol/g.
Selected oligon ucleotides prepared to provide th e proof of suitability an d
efficien cy of th e design ed solid support by m ean s of furn ish in g 3′-ph os-
ph orylated oligom ers were successfully syn th esized from th e respective
2′-deoxyn ucleoside 3′-ph osph oram idites in con ven tion al way in th e trityl-
off m an n er, as described in Experim en tal. Th e presen ce of th e ph osph oester
group was proved un am biguously by th e en zym atic deph osph orylation
usin g alkalin e ph osph om on oesterase. Th e results were docum en ted by th e
HPLC profiles (Fig. 4) an d th e MALDI-TOF data.
a
b
FIG. 4
HPLC an alyses of: a crude oligon ucleotide 3′-ph osph ates syn th esized on th e cyan oeth yl-CPG
(Lun a C18 5 µm , 4.6 × 150 m m ; 15→40% B/20 m in , A = 10% aceton itrile in 0.1 M TEAA, B =
25% aceton itrile in 0.1 M TEAA, pH 7.5); b th e m ixtures of oligon ucleotide 3′-ph osph ates an d
th eir deph osph orylated products
Th e cyan oeth yl type of ligan d presen ted h ere was already used, in th e
procedure refin em en t an d with out specific data, in our earlier work dealin g
with th e syn th esis of th e 3′-ph osph orylated (n = 0) an d ph osph on ylated
(n = 1) oligon ucleotides (Fig. 5) as HIV in tegrase in h ibitors22, an d with th e
attach m en t of th e protected dA-PMEA ph osph on ate din ucleotide via
in tern ucleotide lin kage followed by syn th esis of th e m odified oligon ucleo-
tide prim ers useful for th e study on eukaryotic DNA polym erases23. Th is
proved com patibility of th e n ovel lin ker use an d th e m eth od of an ch orin g
Collect. Czech. Chem. Commun. 2008, Vol. 73, No. 1, pp. 32–43

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Pačes, Točík, Rosenberg:
for syn th eses with both ph osph ate an d ph osph on ate com pon en ts adds to
th e versatility of th e approach .
FIG. 5
Exam ples of use of n ovel cyan oeth yl-type solid support
CONCLUSION
We succeeded in elaboratin g a syn th etic route to a n ovel lin ker attach ed to
LCAA-CPG for th e preparation of 3′-ph osph orylated oligon ucleotides, uti-
lizable in various con jugation reaction s. Th e n ew lin ker was tested in syn -
th esis of a set of deoxyoligon ucleotides carryin g th e term in al 3′-ph osph ate
group. Th e prepared oligom ers were obtain ed, upon a con ven ien t β-elim i-
n ation release from th e support, in a h igh yield an d purity. Th e syn th esis of
th e lin ker is sim ple, straigh tforward, an d un am biguous an d gives good
yields. Th e subsequen t an ch orin g to th e LCAA-CPG proceeds with a h igh
loadin g efficien cy. Th e oligo(eth ylen e glycol) spacer arm of varyin g len gth s
m ay be used, th us en ablin g th e tun in g of ph ysicoch em ical properties of th e
solid support surface to obtain a m ore con ven ien t en viron m en t for th e con -
den sation . Th e used syn th etic m eth odology offers th e possibility of a sim -
ple th ree-step in troduction of th e 2-cyan oeth yl-type lin ker on to polyeth yl-
en e glycol-grafted polystyren e to obtain even m ore easily th e cyan oeth yl-
type solid support for oligon ucleotides carryin g th e term in al ph osph ate or
ph osph on ate m oieties (study in progress). In sum m ary, th e m eth odology
presen ted h ere provides a tech n ically easy, advan tageous procedure with -
out problem atic steps offerin g an altern ative worth con sideration wh en
search in g for a reliable approach to oligon ucleotides possessin g term in al
ph osph ate/ph osph on ate groups.
Collect. Czech. Chem. Commun. 2008, Vol. 73, No. 1, pp. 32–43

Solid-Phase Synthesis of Oligonucleotides
39
EXPERIMENTAL
Gen eral
Th e solven ts were evaporated at 40 °C an d 2 kPa, an d th e products were dried over ph os-
ph orus pen toxide at room tem perature an d 13 Pa. Th e reaction course was ch ecked usin g
silica gel UV254 TLC sh eets (Merck) wh ereby th e products were detected by UV m on itorin g
an d by sprayin g with 1% eth an olic solution of 4-(4-n itroben zyl)pyridin e followed by gen tle
h eatin g an d subsequen t exposin g th e sh eets to am m on ia vapour (blue colour of tosyl deriva-
tives). For flash colum n ch rom atograph y, a 40–60 µm silica gel (Fluka) was used. Th e TLC
was carried out in th e followin g solven t system s (v/v): toluen e (T), 5% EtOAc–toluen e (T5),
10% eth yl acetate–toluen e (T10), an d 50% eth yl acetate–toluen e (T50). An alytical HPLC
was perform ed on a Lun a C18 colum n (4.6 × 150 m m ; Ph en om en ex) usin g a lin ear gradien t
of m eth an ol an d aceton itrile in 0.1 M TEAA at a flow rate of 1 m l/m in an d UV detection
(260 n m ). Preparative reversed-ph ase ch rom atograph y was carried out on an octadecyl silica
colum n (25 × 250 m m , 20 µm , IOCB Prague); com poun ds were eluted with a lin ear gradien t
of m eth an ol in water at 15 m l/m in . UV spectra were taken on a Cary Bio 100 (Varian ) spec-
troph otom eter. High -resolution FAB m ass spectra were recorded on a ZAB-EQ (VG An alyti-
cal) in strum en t with glycerol an d th ioglycerol as m atrices. MALDI TOF spectra were
perform ed on a Reflex IV (Bruker Dalton ics). NMR spectra (δ, ppm ; J, Hz) were m easured on
a Varian Un ity-500 spectrom eter (1H at 500 MHz; 13C at 125.7 MHz) in DMSO-d6 at 20 °C.
Ch em ical sh ifts were referen ced to solven t sign al (con verted to δ-scale) usin g relation s
δH(DMSO) 2.50 ppm an d δC(DMSO) 39.7 ppm . Proton 2D-COSY spectra were used for th e
structural assign m en t of coupled proton s an d 2D-ROESY spectra for detection of th e NOE
con tacts. 13C NMR ch em ical sh ifts an d couplin g con stan ts J(C,P) were obtain ed from broad-
ban d proton -decoupled spectra usin g APT pulse sequen ce. IR spectra (ν, cm –1) were perform ed
on an IFS-55 (Bruker) in strum en t.
13-[(tert-Butyldiph en ylsilyl)oxy]-2-h ydroxym eth yl-5,8,11-trioxatridecan en itrile (5)
Th e silyl derivative 2 (15 g, 38.6 m m ol)20 was treated with tosyl ch loride (8.60 g, 45 m m ol)
in pyridin e (100 m l) at room tem perature for 16 h . Th e reaction m ixture was cooled in an
ice bath , quen ch ed with water (20 m l) an d, after 10 m in , con cen trated in vacuo. Th e residue
was partition ed between eth yl acetate an d water. Th e organ ic layer was wash ed several tim es
with water, dried over an h ydrous m agn esium sulfate, filtered, an d evaporated to dryn ess.
Th e product was purified by silica gel ch rom atograph y usin g a lin ear gradien t of aceton e in
toluen e (0–10%). Th e obtain ed com poun d 3 (13.34 g, 64%, ligh t yellow oil) was used in th e
n ext step with out ch aracterization .
Sodium h ydride (1.73 g, 43.3 m m ol) was added to a solution of eth yl cyan oacetate (6.6 g,
43.3 m m ol) in dry DMF (150 m l) at 0 °C un der argon atm osph ere. Th en , after 30 m in of
vigorous stirrin g, tosyl derivative 3 in DMF (50 m l) was added dropwise to th e solution , an d
th e reaction m ixture was h eated at 50 °C un der argon for 15 h (TLC in T5). Several drops of
acetic acid were added an d th e m ixture was evaporated. Th e residue was partition ed be-
tween ch loroform an d water, th e organ ic layer was wash ed several tim es with water, dried
over an h ydrous MgSO4, filtered, an d evaporated. Th e product was dissolved in eth an ol
(99%, 50 m l) an d sodium boroh ydride (2.2 g, 43.3 m m ol) was added at 0 °C. After stirrin g
for 30 m in , TLC in T5 in dicated th e absen ce of th e startin g m aterial. Acetic acid (10.4 g,
173.2 m m ol) was slowly added to destroy th e excess of sodium boroh ydride, an d th e m ix-
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40
Pačes, Točík, Rosenberg:
ture was stirred at room tem perature for 30 m in . Th e solven t was evaporated an d th e resi-
due partition ed between ch loroform an d water. Th e organ ic layer was wash ed several tim es
with water, dried over an h ydrous MgSO4, filtered, an d evaporated. Th e product 5 was puri-
fied by silica gel ch rom atograph y usin g a lin ear gradien t of aceton e in toluen e (0–20%).
Yield 3.22 g (60%, ligh t yellow oil). 1H NMR (DMSO-d6): 7.64 m , 4 H an d 7.42 m , 6 H
(arom . H); 5.28 t, 1 H, J = 5.6 (OH); 3.74 dd, 2 H, J = 4.9, 5.3 an d 3.52 m , 14 H (OCH2);
2.86 dddd, 1 H, J = 5.2, 5.6, 6.6 an d 9.2 (CH-C); 1.76 m , 2 H (C-CH2); 0.97 s, 9 H ((CH3)3C).
13C NMR (DMSO-d6): 133.31, 2 C (arom . C); 135.29, 4 C, 129. 96, 2 C an d 128.00, 4 C
(arom . C); 121.65, 1 C (CN); 71.81, 70.20, 70.03, 69.95, 69.95, 69.78, 67.50, 63.44 an d
61.175, 8 C (OCH2); 31.565, 1 C (CH); 28.31, 2 C (CH2); 26.795 an d 18.95, 4 C ((CH3)3C).
FAB HR (glycerol, th ioglycerol, m eth an ol): For C27H39NNaO5Si calculated 508.2495; foun d
508.2448 [(M + Na].
2-[(4,4′-Dim eth oxytrityl)oxy]-13-h ydroxy-5,8,11-trioxatridecan en itrile (7)
Th e silyl derivative 5 (4.9 g, 10.4 m m ol) was treated with 4,4′-dim eth oxytrityl ch loride (4.5 g,
13.11 m m ol) in dry pyridin e (50 m l) at room tem perature overn igh t (TLC in T). Meth an ol
(5 m l) was added an d, after 1 h , th e reaction m ixture was con cen trated to h alf volum e in
vacuo (30 °C bath tem perature). Th e residue was partition ed between ch loroform an d water,
th e organ ic layer was wash ed several tim es with an ice-cold 10% aqueous citric acid to re-
m ove pyridin e an d th en with water, dried over an h ydrous MgSO4, filtered, an d evaporated.
Th e residue was treated with 0.5 M TBAF·3H2O in THF (60 m l) for 1 h . TLC in T10 sh owed
com plete desilylation . Water (20 m l) was added an d th e m ixture was con cen trated in vacuo.
Th e residue was partition ed between ch loroform an d water, th e organ ic layer was wash ed
several tim es with water, dried over an h ydrous Na2SO4, filtered, an d evaporated. Th e prod-
uct 7 was purified by silica gel ch rom atograph y usin g a lin ear gradien t of aceton e in toluen e
(0–30%). Yield 3.22 g (60%, ligh t yellow oil). 1H NMR (CDCl3): 7.45 m , 2 H; 7.34 m , 4 H;
7.30 m , 2 H; 7.22 m , 4 H; 6.84 m , 4 H (arom . H); 3.79 s, 6 H (CH3-O-DMTr); 3.52–3.73 m ,
14 H (H 4, 6, 7, 9, 10, 12, 13); 3.29 dd, 1 H, J = 9.0 an d 5.0 (Ha-CHb-O); 3.21 dd, 1 H, J =
9.0 an d 6.4 (Hb-CHa-O); 3.03 dddd, 1 H, J2,3 = 9.5, 5.7, J2,CH2O = 6.4, 5.0 (H-2); 2.47 t, 1 H,
JOH,13 = 5.9 (OH); 1.9 ddt, 1 H, Jgem = 14.0, J3a,2 = 9.5, J3a,4 = 5.0 (H-3a); 1.86 ddt, 1 H, Jgem
=
14.0, J3b,4 = 8.2, 5.7, J3b,2 = 5.7 (H-3b). 13C NMR (CDCl3): 158.56 (C-p-C6H4-DMTr); 144.34
(C-i-C6H5-DMTr); 135.44 an d 135.48 (C-o-C6H4-DMTr); 129.99 (CH3-o-C6H4-DMTr); 128.03
(C-o-C6H5-DMTr); 127.91 (C-m-C6H5-DMTr); 126.91 (C-p-C6H5-DMTr); 120.84 (CN); 113.20
(C-m-C6H4-DMTr); 86.37 (C-DMTr); 72.45, 70.58, 70.38, 70.30, 67.62, 67.07, 62.66, 61.71
(CH2-13, 12, 10, 9, 8, 6, 4 + CH2O); 55.21 (CH3-O-DMTr); 29.51 (CH-2); 29.38 (CH2-3).
IR (CHCl3): 3596, 3487, 2840, 2246, 1608, 1583, 1509, 1495, 1455, 1443–1447, 1254, 1176,
1153, 1121, 1096, 1083, 1015, 1036, 1003, 915, 830, 700–704. FAB (DS, CH3CN,
(C32H39NO7, 549.27): 594.7 (M + 2 Na – H), 1143.3 (2 M + 2 Na – H). For C32H39NO7 (549.3)
calculated: 67.48% C, 7.15% H, 2.55% N; foun d: 68.23% C, 7.40% H, 2.15% N.
12-Cyan o-13-[(4,4′-dim eth oxytrityl)oxy]-3,6,9-trioxatridecan eh ydrogen succin ate (9)
Th e 4,4′-dim eth oxytrityl derivative 7 (0.4 g, 0.79 m m ol) in pyridin e (5 m l) was treated with
succin ic an h ydride (0.79 g, 7.9 m m ol) in th e presen ce of 4-dim eth ylam in opyridin e (0.36 g,
2.9 m m ol) at room tem perature overn igh t. TLC in T50 sh owed com plete esterification . Th e
reaction was quen ch ed with water (0.5 m l), an d th e solution was con cen trated in vacuo.
Th e residue was partition ed between ch loroform an d water. Th e organ ic layer was wash ed
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Solid-Phase Synthesis of Oligonucleotides
41
several tim es with an ice-cold, 10% aqueous citric acid to rem ove DAP, an d fin ally with wa-
ter. Th e organ ic layer was dried over an h ydrous Na2SO4, filtered, an d evaporated to dryn ess.
Th e product 9 was afforded by ch rom atograph y on a C18 colum n usin g a lin ear gradien t of
m eth an ol in water. Yield 0.45 g (94%, ligh t yellow oil). 1H NMR (DMSO-d6): 7.40 m , 2 H
(H-o-C6H5-DMTr); 7.33 m , 2 H (H-m-C6H5-DMTr); 7.26 m , 4 H (H-o-C6H4-DMTr); 7.24 m ,
1 H (H-p-C6H5-DMTr); 6.91 m , 4 H (H-m-C6H4-DMTr); 4.08 m , 2 H (H-1); 3.74 s, 6 H
(CH3-O-DMTr); 3.38–3.61 m , 12 H (H-2, 4, 5, 7, 8, 10); 3.08, 3.22 m , 2 H (CH2O); 3.05 m ,
1 H (H-12); 2.43, 2.48 m , 4 H (H-2, 3 h ydrogen succin ate); 1.74, 1.83 m , 2 H (H-11). 13C NMR
(CDCl3): 172.41, 173.80 (CO); 158.40 (C-p-C6H4-DMTr); 144.71 (C-i-C6H5-DMTr); 135.30
an d 135.37 (C-i-C6H4-DMTr); 129.82 (C-o-C6H4-DMTr); 128.14 (C-m-C6H5-DMTr); 127.75
(C-o-C6H5-DMTr); 127.05 (C-p-C6H5-DMTr); 121.41 (CN); 113.49 (C-m-C6H4-DMTr); 85.87
(C-DMTr); 69.95, 69.91, 69.81, 69.70, 68.44, 67.29, 66.55, 63.53, 62.51 (CH2-1, 2, 4, 5, 7, 8,
10 + CH2O); 55.25 (CH3-O-DMTr); 29.15 (CH-2); 28.92, 29.02 (C-2, 3 h ydrogen succin ate);
28.68 (CH2-11). IR (CHCl3): 3517, 2840, 2246, 1734, 1608, 1584, 1509, 1495, 1455,
1443–1447, 1254, 1121, 1099, 1083, 1076–1079, 1053, 1036, 1013, 1002, 915, 831, 700–704,
635. ES HR (NOBA, CH3CN): For C36H43NO10 calculated 648.2809; foun d 648.2809 [(M–)].
For C36H43NO10 (649.3) calculated: 66.55% C, 6.67% H, 2.16% N; foun d: 64.67% C, 7.66% H,
2.35% N.
Preparation of 2-Cyan oeth yl-Modified LCAA-CPG
Th e LCAA-CPG 500 A, 80–120 m esh (1 g) was treated with a solution of 3% dich loroacetic
acid in 1,2-dich loroeth an e (20 m l) at room tem perature overn igh t. Th e suspen sion was fil-
tered un der argon , th e CPG support was wash ed successively with 10% trieth ylam in e in
dich lorom eth an e, dich lorom eth an e, an d eth er, an d fin ally dried in vacuo over ph osph orus
pen toxide. Th e support was treated with h ydrogen succin ate of 7 (61 m g, 0.1 m m ol), DAP
(12 m g, 0.1 m m ol), trieth ylam in e (0.28 m l, 2 m m ol), an d 1-[3-(dim eth ylam in o)propyl]-
3-eth ylcarbodiim ide h ydroch loride (382 m g, 2 m m ol) in pyridin e (10 m l) un der gen tle sh ak-
in g at room tem perature for 24 h . Th e m odified CPG was wash ed with pyridin e, an d th e
un reacted am in o groups were acetylated usin g a cappin g m ixture (Table I). Th e loadin g of
th e ligan d to CPG varied between 40 an d 50 µm ol/g (determ in ed spectroph otom etrically as
th e released dim eth oxytrityl cation ).
Solid-Ph ase Syn th esis of Oligon ucleotide 3′-Ph osph ates
Th e oligon ucleotides were syn th esized as trityl-off com poun ds on th e cyan oeth yl-m odified
LCAA-CPG at 0.5 µm ol scale on th e Gen Syn V02 DNA/RNA syn th esizer accordin g to th e
protocol given in Table I.
MALDI-TOF: d (T9Gp ) (C100H131N23O70P10, 3083.477): 3004.419 [M – HPO3 + 2 H],
3084.384 [M
+ H], 3106.336 [M + Na – H], 3198.230 [M + 5 Na + H]; d (T9Cp )
(C99H131N21O70P10, 3043.471): 2965.599 [M – HPO3 + 2 H], 3044.585 [M + H], 3067.545
[M + Na + H], 3159.390 [M + 5 Na + H]; d (T9Ap ) (C100H131N23O69P10, 3067.483): 2989.425
[M – HPO3 + 2 H], 3068.421 [M + H], 3091.367 [M + Na]; d (T9Tp ) (C100H132N20O71P10
,
3058.471): 3059.273 [M + H], 3081.694 [M + Na], 3174.605 [M + 5 Na + H].
Collect. Czech. Chem. Commun. 2008, Vol. 73, No. 1, pp. 32–43

42
Pačes, Točík, Rosenberg:
TABLE I
Oligon ucleotide syn th etic protocol
Cyclus Procedure Reagen t
Tim e, s
180
1.
2.
Detritylation
3% CCl3COOH in 1,2-dich loroeth an e
Ph osph oram idite
couplin g
0.05 M ph osph oram idite in CH3CN (100 µl)
0.50 M tetrazolee in CH3CN (100 µl)
180c
20% acetic an h ydride in CH3CN (150 µl)
DAPa/2,4,6-trim eth ylpyridin e/CH3CN
(6/30/70; w/v/v (150 µl)
3.
Cappin g
60c
4.
5.
Oxidation
1.1 M t-BuOOHb in dich lorom eth an e (300 µl) 60c
Cleavage from CPG 30% aq. am m on ia
an d deprotection
16 h /55 °C
a
b
c
4-Dim eth ylam in opyridin e; tert-butylh ydroperoxide; recyclin g of reagen ts th rough th e
colum n .
En zym atic Deph osph orylation of Oligon ucleotide 3′-Ph osph ates
Th e appropriate oligon ucleotide 3′-ph osph ate (12 n m ol) was treated with alkalin e ph os-
ph atase (4.3 × 10–4 U) in 50 m M Tris pH 10.55 (80 µl) at 35 °C for 45 m in . Th en , an aliquot
(10 µl) of th e m ixture was an alyzed by HPLC on th e reversed-ph ase colum n (Fig. 4a). Th e
co-in jection of th e ph osph orylated an d deph osph orylated oligon ucleotides resulted in two
peaks (Fig. 4b).
Support by Grants No. 203/05/0827 and No. 202/05/0628 (Czech Science Foundation), Centre for
Biomolecules and Complex Molecular Systems (LC512), and ChemGen LC06077 (Ministry of
Education, Youth and Sports of the Czech Republic) under research project Z40550506 is gratefully
acknowledged. The authors are indebted to the staff of the Departments of Mass Spectrometry and
NMR of the Institute for measurements and interpretation of HR MS and NMR spectra.
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