Synthesis and hybridization of oligonucleotides modified at amp sites with adenine pyrrolidine phosphonate nucleotides

Article abstract of DOI:10.1135/cccc2009022

Three structurally diverse types of the protected pyrrolidine nucleoside phosphonates were prepared as the monomers for the introduction of pyrrolidine nucleotide units into modified oligonucleotides on the solid phase. Two different chemistries were used

Full text of DOI:10.1135/cccc2009022

Oligonucleotides Modified at AMP Sites
935
SYNTHESIS AND HYBRIDIZATION OF OLIGONUCLEOTIDES
MODIFIED AT AMP SITES WITH ADENINE PYRROLIDINE
PHOSPHONATE NUCLEOTIDES
1,
2
3
4
Dominik REJMAN *, Petr KOČALKA , Radek POHL , Zdeněk TOČÍK and
5,
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: rejman@uochb.cas.cz,
2
3
4
5
kocalka@uochb.cas.cz, pohl@uochb.cas.cz, tocik@uochb.cas.cz, ivan@uochb.cas.cz
Received February 27, 2009
Accepted April 10, 2009
Publish ed on lin e Jun e 1, 2009
Dedicated to Dr. Alfred Bader on the occasion of his 85th birthday.
Th ree structurally diverse types of th e protected pyrrolidin e n ucleoside ph osph on ates were
prepared as th e m on om ers for th e in troduction of pyrrolidin e n ucleotide un its in to m odi-
fied oligon ucleotides on th e solid ph ase. Two differen t ch em istries were used for in corpora-
tion of m odified an d n atural un its: th e ph osph otriester m eth od for th e form er, i.e.,
m on om ers con tain in g N-ph osph on oalkyl an d N-ph osph on oacyl m oieties attach ed to th e
pyrrolidin e rin g n itrogen atom , an d ph osph oram idite ch em istry for th e latter. Sin ce th e syn -
th esized pyrrolidin e n ucleoside ph osph on ic acids are close m im ics of th e 3′-deoxyn ucleoside
5′-ph osph ates, th e in corporation of on e m odified un it in to oligon ucleotides gives rise to on e
2′,5′ in tern ucleotide lin kage. A series of n on am ers con tain in g two or th ree m odified un its,
as well as th e fully m odified aden in e 15-m er, were syn th esized in reverse order, i.e., from
th e 5′ to th e 3′ en d of th e stran d. Th e m easurem en t of th erm al ch aracteristics of th e com -
plexes of m odified n on am ers with th e com plem en tary stran d revealed a destabilizin g effect
of th e in troduced m odification . Th e m odified aden in e h om ooligon ucleotide, was foun d to
form th e m ost stable com plex with oligoth ym idylate of all th e tested m odified oligon ucleo-
tides in term s of ∆Tm per m odification .
Keyw ord s: Oligon ucleotides; Pyrrolidin es; Ph osph on ates; Nucleotides; Nucleosides.
In 1978, th e use of th e so-called an tisen se oligon ucleotides (AONs) com ple-
m en tary to th e term in al sequen ce of RSV (Rous sarcom a Virus) to in h ibit
viral replication in vitro dem on strated1 for th e first tim e th e poten tial of
m eth ods of gen e th erapy. Two m ain m ech an ism s for an tisen se activity
were gradually un covered, alth ough th e problem is very com plex2,3. Th e
AONs bin d by Watson –Crick pairin g to th e target RNAs an d eith er in h ibit
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955
© 2009 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2009022

936
Rejman, Kočalka, Pohl, Točík, Rosenberg:
protein tran slation by steric h in dran ce by blockin g ribosom e processin g, or
activate th e ubiquitous RNase H cleavage of th e RNA part of th e form ed
h eteroduplex. However, ch em ical m odification s of such oligon ucleotides
(ON) were soon foun d n ecessary for th e developm en t of real th erapeutics
an d for in vivo application s, as th e n atural oligon ucleotides are un stable to-
ward cellular en zym es. In addition , it was foun d th at th e m odified oligo-
n ucleotides m ust be also capable of pen etratin g in to cells, sufficien tly
h ybridize with th eir RNA target sequen ces, an d recruit th e RNase H activity
wh ile exertin g n o appreciable side effects4–7. Th e ph osph oroth ioate oligo-
n ucleotides8,9 developed as poten tial th erapeutics of th e first gen eration
were able to m eet several im portan t dem an ds like h igh er cellular uptake,
en h an ced m etabolic stability, an d th e ability to elicit RNase H activity, an d
Vitraven e, a ph osporoth ioate oligon ucleotide, was approved by FDA for
treatm en t of HCMV (Hum an Cytom egalovirus)-in duced retin itis4,5. How-
ever, n on -specific in teraction s of ph osph oroth ioate oligon ucleotides with
protein s an d th e presen ce of a large n um ber of diastereoisom ers in on e
ph osph oroth ioate oligom er due to ch irality of th e ph osph orus atom were
th e drawbacks th at en forced th e syn th esis an d evaluation of a series of
oth er in tern ucleotide lin kage-m odified oligon ucleotides10. Th e process of
assem blin g th e ch im eric (secon d gen eration ) AONs com posed of two or
m ore types of m odified un its started, an d h as sin ce reach ed con siderable
progress wh ereby em ployin g, e.g., m odification s ch em ically as differen t
from each oth er as th e con form ation ally locked n ucleic acids11, peptide
n ucleic acids12, m orph olin o n ucleic acids13, or n on -ch arged m eth ylph os-
ph on ates14, am on g oth er m odification s attem ptin g to exploit th e growin g
kn owledge of th e overall stereoch em istry of th e n ucleic acid ch ain s15
.
Given th e n um ber of very specific biological targets to deal with , an d th e
still in com plete in form ation on various structural an d dyn am ic features of
n ucleic acids, it is n ot surprisin g th at th e existin g m odification s of n atural
n ucleic acid ch ain s can on ly partly satisfy th e n eeds in th e ever expan din g
area of gen e th erapy m eth odologies16. Th erefore, th e ch em ical syn th eses
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Oligonucleotides Modified at AMP Sites
937
con tin ue in order to m eet th e dem an ds for m ore structural varian ts to opti-
m ize th e m en tion ed properties of ONs an d get rid of th e side-effects17.
Th e presen t work describes n ovel oligon ucleotides com bin in g procedures
from two distin ct syn th etic fields: th e preparation of pyrrolidin e-based
m on om eric un its an d th eir con n ection by ph osph on ate lin kages.
On ly a few ONs con tain in g pyrrolidin e rin gs h ave been syn th esized.
Most of th em are based on th e PNA (peptide n ucleic acid)-type
oligon ucleotides usin g am ide m oiety as th e in tern ucleotide lin kage. Th e
m odified ON of type 1 was described by Altm an n 18. A h om opyrim idin e
oligom er con tain in g th is m odification exh ibited a sign ifican t affin ity to
com plem en tary RNA in a sequen ce-specific fash ion , wh ile n o bin din g was
observed to com plem en tary DNA stran d. Pen tam eric th ym idylate ON of
type 2 sh owed a h igh affin ity to th e com plem en tary sin gle-stran ded RNA
an d DNA, wh ilst exh ibitin g kin etic preferen ces for RNA over DNA 19. Th e
m odification 3 was th e result of an idea of con strain in g flexibility an d in -
troducin g positive ch arge in th e PNA backbon e via lin kin g th e α-carbon of
th e glycyl backbon e to th e β-carbon of th e side ch ain on an
(2-am in oeth y)lglycyl PNA 20
. Hetero-oligom eric PNAs con sistin g of
m on om eric buildin g blocks of type 4 sh owed a stron ger bin din g to th e
com plem en tary DNA th an th e origin al PNA described by Jordan 21
.
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938
Rejman, Kočalka, Pohl, Točík, Rosenberg:
Modified ONs in corporatin g trans-4-h ydroxy-N-acetyl-L-prolin ol (trans-
4-HO-L-NAP-NA), or its D an alogue con tain in g aden in e an d th ym in e as
n ucleobases were described by Ceulem an s22. Efim ov described syn th esis
of m odified oligon ucleotides con tain in g 1-acetyl-4-h ydroxypyrrolidin e-
2-ph osph on ic acid backbon e pHypNA with various stereoch em istry. Th ese
ONs exh ibited h igh h ybridization an d discrim in ation ch aracteristics as well
as th e ability to h ybridize to com plem en tary n ucleic acid in low salt con -
cen tration 23
.
As far as th e evaluation of th e role of th e in tern ucleotide backbon e as a
wh ole is con cern ed th e replacem en t of ph osph odiester in tern ucleotide
lin kage with n on isosteric isopolar ph osph on ate m oiety O–P–CH2–O h as
been in th e focus of our in terest24 because of its stability to n ucleolytic
en zym es. Moreover, h ybridization experim en ts with dim ers con tain in g
th is kin d of ph osph ate alteration sh owed th at in troduction of th is m odifi-
cation was n ot likely to block th e form ation of a duplex26,27. A set of
oligon ucleotide-based HIV-I in tegrase in h ibitors con tain in g ph osph on ate
m odification h as been also described28
.
In th e presen t work, we deal with th e com poun ds in wh ich th e stan dard
pen tofuran ose m oiety is replaced with pyrrolidin e rin g. Specifically, th e
4′-carbon of ribose is replaced by n itrogen atom wh ile th e oxygen atom of
ribose is replaced by m eth ylen e groupin g. Th e un derlyin g idea for th is al-
teration was th e assum ption th at th e pyrrolidin e n itrogen atom , upon
proton ation th at was expected to occur at ph ysiological pH, would bear
positive ch arge wh ich in turn sh ould partially n eutralize th e
in tern ucleotide n egative ch arge in th e oligon ucleotide duplex by m ean s of
a decrease in repulsion of th e n egatively ch arged ch ain s. An in crease in sta-
bility of th e duplex would th us be th e result. Moreover, a lower depen den ce
on th e n ature of m etal cation (Na+ or Mg2+) was expected. Ph osph on o-
m eth yl (5), ph osph on oeth yl (6), an d ph osph on oacetyl (7) m oieties were
ch osen as in tern ucleotide lin kages for th e study. Modification 5 an d 6 were
expected to sh ow th e relation between th e in tern ucleotide lin kage len gth
an d duplex stability. Th e idea of in troduction of m odification 7 con tain in g
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Oligonucleotides Modified at AMP Sites
939
am ide m oiety con sisted in th e assum ption th at such less-flexible system
could im prove th e en tropic factor kn own to be im portan t for duplex stabil-
ity. As m en tion ed above, our m odified un its resem ble th e un n atural 2′,5′
lin ked system . Th e oligoribon ucleotides con tain in g 2′,5′-ph osph odiester
lin kages27 sh ow, in con trast to DNA, a h igh selectivity towards com plem en -
tary RNA ch ain s. For th e syn th esis of desired pyrrolidin e oligon ucleotides
with th e m on oester C–P–O lin kage, we ch ose a h igh ly efficien t varian t of
ph osph otriester m eth od em ployin g th e (4-m eth oxy-1-oxido-2-pyridyl)-
m eth yl ph osph oester protectin g group29–32 wh ich also acts as an in tra-
m olecular catalyst of th e con den sation reaction . Th e com bin ation of th e
triester an d stan dard ph osph oram idite m eth ods was used for th e solid
ph ase syn th esis of th e m ixed-backbon e ONs.
RESULTS AND DISCUSSION
Synthesis of Monomers
Pyrrolidin e n ucleoside in term ediate 8 h as been prepared accordin g to th e
described procedure33. Ph osph on ylation of 8 with (i) form aldeh yde an d
diisopropyl ph osph ite, (ii) diisopropyl vin ylph osph on ate, an d (iii) N-
h ydroxysuccin im idyl diisopropylph osph on oacetate led to diisopropyl es-
ters 9, 14, an d 19, respectively (Sch em e 1). Aden in e m oieties of both
ph osph on ates were protected with th e dibutylam in om eth ylen e (DBAM),
an d th e free h ydroxy groups were dim eth oxytritylated usin g dim eth oxy-
trityl ch loride (DMTrCl) in pyridin e. Fully protected diisopropyl ph os-
ph on ates 11, 16, an d 21 were subjected to th e tran sesterification reaction
usin g trim eth ylsilyl brom ide wh ich provided th e protected ph osph on ic
acids 12, 17, an d 22. Th e fin al step con sisted of 2,4,6-triisopropylben zen e-
sulfon yl ch loride (TPSCl) con den sation with (4-m eth oxy-1-N-oxido-
2-pyridyl)m eth an ol (MOPM) followed by h eatin g with 60% aqueous
pyridin e to obtain m on o(4-m eth oxy-1-N-oxido-2-pyridyl)m eth yl (MOP)
ph osph on ates 13, 18, an d 23.
Solid Phase Synthesis of Modified Oligonucleotides
Seven oligon ucleotides con tain in g eith er two (ON1, ON2, ON3), th ree
(ON4, ON5, ON6), or fifteen (ON7) m odified un its were prepared. Th e
oligom ers ON1–ON6 were n atural oligodeoxyn ucleotides con tain in g in -
serts of two or th ree m odified ph osph on ate un its. As already m en tion ed,
a com bin ation of stan dard ph osph oram idite m eth od em ployin g com m on
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940
Rejman, Kočalka, Pohl, Točík, Rosenberg:
SCHEME 1
(i) H2CO, (iPrO)2P(O)H; (ii) di-n-butylform am ide dim eth yl acetal, MeOH; (iii) DMTrCl, pyri-
din e; (iv) TMSBr, lutidin e, aceton itrile; (v) 1. MOPM, TPSCl, pyridin e or MOPM, DCC, pyri-
din e; 2. 60% aq. pyridin e 60 °C; (vi) diisopropyl vin ylph osph on ate, MeOH;
(vii) N-h ydroxysuccin im idyl diisopropyl ph osph on oacetate
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Oligonucleotides Modified at AMP Sites
941
ph osph oram idite m on om ers an d th e triester on e for in corporation of th e
m odified un its was used. Certain difficulties h ad to be dealt with . Th us,
durin g th e ph osph otriester syn th esis of h om ooligon ucleotide ON7 con tain -
in g fifteen m odified un its, we observed, after several con den sation steps,
a stron gly reduced repetitive yield. We con cluded th at m ost probably,
trich loroacetic acid (TCA) used in th e detritylation step form ed a salt with
th e pyrrolidin e m oiety (in fact, a tertiary am in e) wh ich , in turn , could de-
crease th e con den sation yield by con sum in g partly th e activated m on om er
an d/or TPSCl as a con den sin g agen t (form ation of a m ixed an h ydride of
TCA with th e ph osph on ate un it). Th erefore, after detritylation step in th e
elon gation protocol we in cluded a “n eutralization step”, n am ely a sh ort
wash of th e colum n with 20% TEA in aceton itrile to release TCA from its
salt. Th is precaution was able to restore th e expected repetitive yields. Th e
solid ph ase syn th esis of ON7 em ployed th e recen tly developed cyan oeth yl-
type lin ker suitable for th e solid ph ase syn th esis of oligodeoxyn ucleotides
possessin g term in al 3′-ph osph ates/ph osph on ates34.
Th e syn th esized oligon ucleotides were subjected to th ioph en ol/trieth yl-
am in e/dioxan e treatm en t to rem ove MOP protectin g group followed by
con cen trated am m on ia treatm en t to rem ove th e n ucleobase an d
cyan oeth yl ester protectin g groups an d to cleave th e ON from th e solid sup-
port. Crude ONs bearin g 5′-O-dim eth oxytrityl group were purified by RP
HPLC an d th en detritylated on th e sam e colum n .
In sum m arizin g our syn th etic work, th e m on om ers 13, 18, an d 23
for solid ph ase syn th esis of th e m odified ONs were prepared an d success-
fully used as m on om ers for th e syn th esis of two m ixed-backbon e 9-m ers
(Table I) of th e sequen ces 5′-d(GTG ATA TGC)-3′ (ON1, ON2, an d ON3)
an d 5′-d(GCA TAT CAC)-3′ (ON4, ON5, an d ON6) wh ere A represen ts th e
m odified un it. A h om ooligon ucleotide 15-m er of type 5 (ON7) was also
syn th esized usin g m on om er 13. Th e kn owledge acquired with perform ed
ch em ical tran sform ation s, often laborious, can be of a m ore gen eral use in
Aden in e fully m odified pen tadecam er
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942
Rejman, Kočalka, Pohl, Točík, Rosenberg:
th is less explored area of ch em istry, an d our elaboration of th e protocol of
solid-ph ase assem bly of th e m ixed-type oligom ers can also ben efit to future
syn th eses com bin in g ph osph otriester an d ph osph oram idite strategies.
Th e evaluation of th erm al (Tm ) ch aracteristics of th e prepared oligo-
n ucleotides h as revealed th at, in prin ciple, th e prepared pyrrolidin e
oligon ucleotide ch ain s do n ot m eet th e assum ption on poten tial stabiliza-
tion of th e com plex with th e respective oligom eric coun terpart based on
TABLE I
Th erm al stability of duplexes of th e m odified oligon ucleotides with th eir n atural coun ter-
parts (see structures 5–7)
100 m M Na+
10 m M Mg2+
Mon om er/
scaffold
Duplex
Tm
°C
∆Tm /m odif
°C
Tm
°C
∆Tm /m odif
°C
ON8/
ON9
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
–
32.7
–
34.2
–
ON1/
ON9
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
13/5
13/5
23/7
23/7
18/6
18/6
–
21.0
16.1
25.0
–5.8
–5.6
–3.8
–4.9
–
23.1
17.1
27.1
–5.6
–5.7
–3.6
–3.8
–
ON8/
ON4
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
ON2/
ON9
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
ON8/
ON5
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
18.1
22.9
ON3/
ON9
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
a
a
ON8/
ON6
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
a
a
–
–
ON10/
ON11
dA15/dT15
48.0
34.1
28.1
17.2
–
–
–
–
–
–
ON7/
ON11
dA15/dT15
13/5
–
–0.9
–
–
ON8/
ON12
5′-d(GTG ATA TGC)-3′
5′-r(GCA TAT CAC)-3′
–
ON1/
ON12
5′-d(GTG ATA TGC)-3′
5′-r(GCA TAT CAC)-3′
13/5
–5.4
–
ON2/
ON12
5′-d(GTG ATA TGC)-3′
5′-r(GCA TAT CAC)-3′
23/7
18/6
15.2
–6.4
–
–
–
–
–
a
ON3/
ON12
5′-d(GTG ATA TGC)-3′
5′-r(GCA TAT CAC)-3′
a
No duplex form ation (n o sigm oidal profile was observed).
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Oligonucleotides Modified at AMP Sites
943
th e decrease in repulsive forces due to th e presen ce of proton ated
pyrrolidin e rin g. In stead, th e stron gly decreased h ybridization capabilities
were foun d, an d th e data sh ow th at obviously, th e expected effect of th e
pyrrolidin e rin g positive ch arge is offset by effects of m ixed 2′,5′ an d 3′,5′
in tern ucleotide lin kages on oligom er stereoch em istry an d by effects caused
by stereoch em ical arran gem en t of m odified pyrrolidin e un its. Origin ally we
an ticipated a certain con form ation al adaptability of th e N-proton ated
N-ph osph on oalkyl m oiety but our latest results con cern in g also th e con for-
m ation of prolin ol35 an d pyrrolidin e36 n ucleoside ph osph on ic acids sh owed
th at th ese com poun ds existed predom in an tly in a trans arran gem en t of th e
n ucleobase an d N-ph osph on oalkyl m oiety (5′-ph osph ate m im ic) an d n ot in
th e expected an d un am biguously advan tageous cis orien tation like in 5′-n u-
cleotides. In addition , th e fully N-proton ated form was observed with in a
large ran ge of pH values (2–10). Th ese results suggest th at just th e trans ori-
en tation of a n ucleobase an d N-ph osph on oalkyl m oiety togeth er with 2′,5′
lin kage seem to be th e m ain factors con tributin g to duplex destabilization .
Th us, oligom ers of type 5 an d 7 con tain in g eith er two (ON1, ON2) or
th ree (ON4, ON5) m odification s exh ibited sign ifican tly lower m eltin g
poin ts (Tm ) th an n atural on es in th e ran ge from –3.5 to –6 °C per m odifica-
tion . In troduction of th e m odification of type 6 (ON3, ON6) abolish ed th e
duplex form ation com pletely. In con trast to th e type 5 (N-ph osph on o-
m eth yl), th e type 6 (N-ph osph on oeth yl), wh ich is on e m eth ylen e group
lon ger, brin gs to th e system on e m ore degree of freedom . So th e
con tributon of rotation en tropy will be m uch h igh er th an in th e case of
sh orter type 5 an d also in th e case of isosteric but con form ation ally m ore
restricted type 7 (am ide bon d). Th e en tropic factor wh ich relates to th e
stran d pre-organ ization seem s to be m ost im portan t for th e duplex stabil-
ity. Th e results suggest th e n eed to study th e beh avior of an oth er m odifica-
tion com bin in g type 5 an d 7, i.e., th e P–C(O)–N n tern ucleotide lin kage.
In con trast to studied m odified n on am ers, th e prepared h om ooligom er
con sistin g of fifteen m odified un its (scaffold 5) exh ibited a con siderably
lower decrease in th e m eltin g poin t of on ly –0.9 °C per m odification wh ich
suggests th at th e con form ation of th e m odified stran d accom m odates
th e con form ation of th e n atural couterparts or vice versa. Th e study on
pyrrolidin e-based h om ooligom ers an d types of th eir com plexes (duplex,
triplex), orien tation of th e stran ds, etc. is un der way.
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944
Rejman, Kočalka, Pohl, Točík, Rosenberg:
EXPERIMENTAL
Syn th esis of Mon om ers
Un less stated oth erwise, all used solven ts were an h ydrous. Fin al products were lyoph ilized
from dioxan e or ben zen e, an d dried over ph osph orus pen toxide at 50–70 °C an d 13 Pa. TLC
was perform ed on silica gel pre-coated alum in ium plates Fluka or Merck Silica gel/TLC-cards
with a 254 n m fluorescen t in dicator. Th e com poun ds were detected by UV ligh t (254 n m ),
by h eatin g (detection of dim eth oxytrityl group; oran ge color), an d by sprayin g with 1% so-
lution of 4-(4-n itroben zyl)pyridin e in eth an ol followed by h eatin g an d treatm en t with gas-
eous am m on ia (blue color of m on o- an d diesters of ph osph on ic acid). Preparative colum n
ch rom atograph y was carried out on silica gel (40–60 µm ; Fluka) n eutralized with trieth yl-
am in e (1 m l/100 g), an d elution was perform ed at a flow rate of 40 m l/m in . Th e followin g
solven t system s were used for TLC an d preparative ch rom atograph y: toluen e–eth yl acetate
4:1 (T1), 1:1 (T2); toluen e–aceton e 1:1 (T3); ch loroform –eth an ol 9:1 (C1); eth yl acetate–
aceton e–eth an ol–water 4:1:1:1 (H1), 12:2:2:1 (H3). Th e con cen tration s of solven t system s are
given in volum e percen ts (%, v/v). Mass spectra of com poun ds were recorded on a ZAB-EQ
(VG An alytical) in strum en t usin g FAB (ion ization with Xe, acceleratin g voltage 8 kV) with
glycerol an d th ioglycerol as m atrices wh ile for th e oligon ucleotide m olecular ion determ in a-
tion , MALDI-TOF m easurem en t on a Reflex IV (Bruker Dalton ics, Germ an y) in strum en t was
used. NMR spectra were m easured on Varian Un ity 500 in strum en t (1H at 500 MHz, 13C at
125.7 MHz) in deuterated ch loroform or h exadeuteriodim eth yl sulfoxide (DMSO-d6).
Oligon ucleotides were syn th esized on autom ated GENSYN syn th esizer (IOCB Prague) an d
th e Tm values were m easured usin g an UV VIS spectroph otom eter Varian Cary 500.
Diisopropyl [(3R,4R)-3-(Aden in -9-yl)-4-h ydroxypyrrolidin -1-yl]m eth ylph osph on ate (9)
14.5 M aqueous form aldeh yde (1.4 m l, 20 m m ol) was added to th e suspen sion of 8 (0.9 g,
4.087 m m ol) in diisopropyl ph osph ite (3.3 g, 20 m m ol). Th e m ixture was h om ogen ized in
ultrason ic bath an d stirred at 60 °C for 5 h . Th en , 0.02 M sulfuric acid (50 m l) was added
an d th e reaction m ixture was stirred at r.t. for 2 days. Th e solution was applied on to a col-
um n of 40 m l of Dowex 50 (H+). Th e resin was wash ed with 50% aqueous eth an ol (200 m l).
Th e crude title product was obtain ed by elution with 3% am m on ia in eth an ol–water (1:1)
m ixture. Pure com poun d 9 was obtain ed by colum n ch rom atograph y on silica gel usin g lin -
ear gradien t of eth an ol in ch loroform in a 78% yield as a colorless oil (1.27 g, 3.18 m m ol).
HR FAB+: calculated (M + H) 399.190967, foun d 399.190494. 1H NMR (400 MHz, DMSO-d6):
1.24 an d 1.25 (2 × d, 12 H, Jvic = 6.2, (CH3)2CH); 2.56 (dd, 1 H, Jgem = 10.0, J5b,4 = 4.7,
H-5b-pyrr); 2.89 (dd, 1 H, Jgem = 15.0, JH,P = 10.7, CHaHb-P); 2.98 (dd, 1 H, Jgem = 15.0, JH,P
=
12.4, CHaHb-P); 3.09 (d, 2 H, J2,3 = 5.8, H-2-pyrr); 3.35 (dd, 1 H, Jgem = 10.0, J5a,4 = 6.8,
H-5a-pyrr); 4.40 (bm , 1 H, J4,5 = 6.8, 4.7, J4,OH = 4.8, J4,3 = 3.3, H-4-pyrr); 4.60 an d 4.61 (2 ×
dh , 2 × 1 H, JH,P = 7.8, Jvic = 6.2, CH(CH3)2); 4.70 (td, 1 H, J3,2 = 5.8, J3,4 = 3.3, H-3-pyrr);
5.54 (d, 1 H, JOH,4 = 4.8, OH); 7.23 (bs, 2 H, NH2); 8.14 (s, 1 H, H-8); 8.18 (s, 1 H, H-2).
13C NMR (100.6 MHz, DMSO-d6): 24.00 an d 24.08 (d, JC,P = 4, (CH3)2CH); 50.94 (d, JC,P
=
163, CH2P); 58.79 (d, JC,P = 10, CH2-2-pyrr); 61.67 (CH-3-pyrr); 62.69 (d, JC,P = 10,
CH2-5-pyrr); 69.99 (d, JC,P = 6, CH(CH3)2); 75.06 (CH-4-pyrr); 118.87 (C-5); 139.47 (CH-8);
149.52 (C-4); 152.54 (CH-2); 156.19 (C-6). 31P NMR (162 MHz, DMSO-d6): 22.84.
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

Oligonucleotides Modified at AMP Sites
945
Diisopropyl (3R,4R)-3-{[N6-(Dibutylam in o)m eth ylen eaden in -9-yl]-
4-h ydroxypyrrolidin -1-yl}m eth ylph osph on ate (11)
Com poun d 9 (1.27 g, 3.18 m m ol) was dissolved in m eth an ol (30 m l), an d dibutylform am ide
dim eth yl acetal (0.9 m l, 3.82 m m ol) was added. Th e reaction m ixture was left aside at r.t.
for 2 days an d th en quen ch ed with TEAB (trieth ylam m on ium bicarbon ate) (1 m l). Th e
solven t was evaporated an d th e DBAM derivative 10 was obtain ed in 88% yield (1.51 g,
2.81 m m ol) by colum n ch rom atograph y on silica gel usin g lin ear gradien t of eth an ol in
ch loroform as a colorless oil an d used with out ch aracterization .
Th e obtain ed com poun d was co-evaporated with pyridin e (3 × 40 m l). Th en , 4,4′-di-
m eth oxytrityl ch loride (1.7 g, 5 m m ol) an d silver triflate (1.28 g, 5 m m ol) were added to th e
solution of 10 (1.51 g, 2.81 m m ol) in pyridin e (30 m l). Th e reaction m ixture was stirred at
r.t. overn igh t, quen ch ed with m eth an ol (3 m l), diluted with eth yl acetate (200 m l), an d
wash ed with saturated solution of sodium h ydrogen carbon ate. Organ ic layer was dried over
sodium sulfate, con cen trated, an d applied on to a colum n of silica gel. Th e title product was
obtain ed usin g lin ear gradien t of eth an ol in ch loroform in 77% yield (1.81 g, 2.16 m m ol) as
a yellowish oil. HR-MS: for C46H63N7O6P (M + H)+ calculated 840.45774, foun d 840.45815.
1H NMR (400 MHz, CDCl3): 0.95 an d 0.96 (2 × t, 2 × 3 H, Jvic = 7.3, CH3CH2CH2CH2N);
1.25, 1.27, 1.28 an d 1.29 (4 × d, 4 × 3 H, Jvic = 6.2, (CH3)2CH); 1.32–1.45 (m , 4 H,
CH3CH2CH2CH2N); 1.60–1.71 (m , 4 H, CH3CH2CH2CH2N); 2.24 (dd, 1 H, Jgem = 10.0, J5b,4
5.7, H-5b-pyrr); 2.73 (dd, 1 H, Jgem = 15.0, JH,P = 11.7, CHaHb-P); 2.80 (dd, 1 H, Jgem = 15.0,
JH,P = 12.2, CHaHb-P); 2.84 (dd, 1 H, Jgem = 10.0, J5a,4 = 6.8, H-5a-pyrr); 3.04 (d, 2 H, J2,3
=
=
4.6, H-2-pyrr); 3.40 (t, 2 H, Jvic = 7.3, CH3CH2CH2CH2N); 3.67 an d 3.70 (2 × s, 2 × 3 H,
CH3O); 3.74 (t, 2 H, Jvic = 7.3, CH3CH2CH2CH2N); 4.37 (ddd, 1 H, J4,5 = 6.8, 6.7, J4,3 = 2.5,
H-4-pyrr); 4.67 (dh , 2 H, JH,P = 7.8, Jvic = 6.2, CH(CH3)2); 5.11 (td, 1 H, J3,2 = 4.6, J3,4 = 2.5,
H-3-pyrr); 6.58 an d 6.65 (2
× m , 2 × 2 H, H-m-C6H4-DMTr); 7.07–7.15 (m , 3 H,
H-m,p-C6H5-DMTr); 7.22 an d 7.23 (2 × m , 2 × 2 H, H-o-C6H4-DMTr); 7.37 (m , 2 H,
H-o-C6H5-DMTr); 7.93 (s, 1 H, H-8); 8.56 (s, 1 H, H-2); 9.00 (s, 1 H, CH=N). 13C NMR
(100.6 MHz, CDCl3): 13.68 an d 13.92 (CH3CH2CH2CH2N); 19.75 an d 20.18
(CH3CH2CH2CH2N); 24.04, 24.08 an d 24.11 (d, JC,P = 4, (CH3)2CH); 29.25 an d 31.00
(CH3CH2CH2CH2N); 45.08 (CH3CH2CH2CH2N); 51.26 (d, JC,P
= 165, CH2P); 51.75
(CH3CH2CH2CH2N); 55.05 an d 55.08 (CH3O); 60.26 (d, JC,P = 9, CH2-2-pyrr); 60.63
(CH-3-pyrr); 62.44 (d, JC,P = 11, CH2-5-pyrr); 70.59 an d 70.62 (d, JC,P = 7, CH(CH3)2); 79.15
(CH-4-pyrr); 87.35 (C-DMTr); 112.98 an d 113.05 (CH-m-C6H4-DMTr); 125.38 (C-5); 126.81
(CH-p-C6H5-DMTr); 127.76 (CH-m-C6H5-DMTr); 127.94 (CH-o-C6H5-DMTr); 129.94 an d
130.05 (CH-o-C6H4-DMTr); 135.98 an d 136.00 (C-i-C6H4-DMTr); 140.42 (CH-8); 145.03
(C-i-C6H5-DMTr); 151.25 (C-4); 152.26 (CH-2); 158.18 (CH=N); 158.44 an d 158.80
(C-p-C6H4-DMTr); 159.90 (C-6). 31P NMR (162 MHz, CDCl3): 26.79.
(4-Meth oxy-1-oxido-2-pyridyl)m eth yl (3R,4R)-3-{[N6-(Dibutylam in o)-
m eth ylen eaden in -9-yl]-4-(4,4′-dim eth oxytrityl)oxypyrrolidin -1-yl}m eth ylph osph on ate (13)
Com poun d 11 was co-evaporated with aceton itrile (2 × 30 m l) an d dissolved in th e sam e
solven t (20 m l). Lutidin e (3 m l, 26 m m ol) an d brom otrim eth ylsilan e (1.7 m l, 13 m m ol)
were added un der argon . Th e reaction m ixture was left at r.t. un der argon overn igh t. Aceto-
n itrile was rem oved in vacuo, th e residue was dissolved in th e m ixture of TEAB an d m eth a-
n ol, an d th e volatiles were evaporated. Ph osph on ic acid 12 was obtain ed by colum n
ch rom atograph y on silica gel usin g a lin ear gradien t of H1 in eth yl acetate in 68% yield
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

946
Rejman, Kočalka, Pohl, Točík, Rosenberg:
(1.4 g, 1.46 m m ol). NMR sign als were too broad to be an alyzed so th is in term ediate was
used with out furth er ch aracterization .
A m ixture of com poun d 12 (0.8 g, 0.83 m m ol) an d (4-m eth oxy-1-N-oxido-2-pyridyl)m eth -
an ol (0.4 g, 2.5 m m ol) was co-evaporated with pyridin e (2 × 10 m l) an d dissolved in th e
sam e solven t (10 m l). TPSCl (1.52 g, 5 m m ol) was added, an d th e reaction m ixture was
stirred at r.t. overn igh t. Pyridin e was rem oved in vacuo, an d th e crude reaction m ixture was
purified on silica gel colum n usin g a lin ear gradien t of H3 in eth yl acetate followed by
lin ear gradien t of H1 in H3. Th e obtain ed m aterial was h eated at 60 °C in 60% aqueous
pyridin e (30 m l) for 5 days. Th e solven ts were rem oved, an d th e obtain ed residue was parti-
tion ed between ch loroform an d TEAB. Organ ic layer was dried over an h ydrous sodium sul-
fate an d con cen trated in vacuo. Th e crude m aterial was co-evaporated with m eth an ol (2 ×
20 m l) an d dissolved in th e sam e solven t (30 m l). Dibutylform am ide dim eth yl acetal
(0.5 m l, 2.12 m m ol) was added, an d th e m ixture was left for 2 days at r.t. 2 M aqueous TEAB
(1 m l) was added an d th e reaction m ixture was con cen trated in vacuo, dissolved in ch loro-
form , an d wash ed with TEAB. Th e product was obtain ed in a 29% yield (0.24 g, 0.24 m m ol)
by reverse ph ase ch rom atograph y usin g lin ear gradien t of m eth an ol in water as a wh ite
am orph ous fluffy solid after lyofilization from water. HR FAB+: calculated (M + H)
893.411525, foun d 893.408434. 1H NMR (500 MHz, CDCl3): 0.95 an d 0.96 (2 × t, 2 × 3 H,
Jvic = 7.4, CH3CH2CH2CH2N); 1.22 (t, 3 H, Jvic = 7.3, CH3CH2N); 1.39 an d 1.65 (2 × m , 2 ×
4 H, CH3CH2CH2CH2N); 2.52 an d 2.88 (2 × bm , 2 H, H-5-pyrr); 2.93–3.07 (m , 6 H, H-2-pyr,
CH2P an d CH3CH2N); 3.40 (t, 2 H, Jvic = 7.3, CH3CH2CH2CH2N); 3.66 an d 3.69 (2 × s, 2 ×
3 H, CH3O-DMtr); 3.70 (t, 2 H, Jvic = 7.3, CH3CH2CH2CH2N); 3.76 (s, 3 H, CH3O-MOP); 4.57
(bm , 1 H, H-4-pyrr); 5.12 (bm , 1 H, H-3-pyrr); 5.14 (bd, 2 H, JH,P = 6.8, CH2-MOP); 6.57 an d
6.63 (2 × m , 2 × 2 H, H-m-C6H4-DMTr); 6.70 (dd, 1 H, J5,6 = 7.2, J5,3 = 3.5, H-5-MOP);
7.05–7.14 (m , 3 H, H-m,p-C6H5-DMTr); 7.15–7.22 (m , 5 H, H-3-MOP an d H-o-C6H4-DMTr);
7.32 (m , 2 H, H-o-C6H5-DMTr); 7.94 (s, 1 H, H-8); 8.06 (d, 1 H, J6,5 = 7.2, H-6-MOP); 8.46 (s,
1 H, H-2); 9.02 (s, 1 H, CH=N). 13C NMR (125.7 MHz, CDCl3): 8.46 (CH3CH2N); 13.70 an d
13.92 (CH3CH2CH2CH2N); 19.77 an d 20.18 (CH3CH2CH2CH2N); 29.24 an d 31.02
(CH3CH2CH2CH2N); 45.07 (CH3CH2CH2CH2N); 45.37 (CH3CH2N); 51.84 (CH3CH2CH2CH2N);
52.06 (d, JC,P
= 150, CH2P); 55.06 an d 55.10 (CH3O-DMTr); 56.15 (CH3O-MOP);
60.82 (CH-3-pyrr); 61.25 (CH2-MOP); 61.80 (CH2-2-pyrr); 62.59 (CH2-5-pyrr); 75.40
(CH-4-pyrr); 87.33 (C-DMTr); 108.83 (CH-3-MOP); 110.65 (CH-5-MOP); 113.00 an d
113.10 (CH-m-C6H4-DMTr); 125.43 (C-5); 126.81 (CH-p-C6H5-DMTr); 127.77 an d 127.9
(CH-o,m-C6H5-DMTr); 129.87 an d 130.02 (CH-o-C6H4-DMTr); 135.87 an d 135.90
(C-i-C6H4-DMTr); 139.66 (CH-6-MOP); 141.03 (CH-8); 144.93 (C-i-C6H5-DMTr); 150.85
(C-2-MOP); 151.18 (C-4); 152.09 (CH-2); 158.42, 158.50 an d 158.58 (CH=N an d
C-p-C6H4-DMTr); 158.78 (C-4-MOP an d C-6).
Diisopropyl 2-[(3R,4R)-3-(Aden in -9-yl)-4-h ydroxypyrrolidin -1-yl]eth ylph osph on ate (14)
Diisopropyl vin ylph osph on ate (1.4 g, 7 m m ol) was added to a suspen sion of com poun d 8
(0.7 g, 3.18 m m ol) in m eth an ol (30 m l). Th e m ixture was refluxed for 5 days, an d th e
solution was applied on to a colum n of Dowex 50 (40 m l). Th e resin was wash ed with
50% aqueous eth an ol (200 m l). Th e crude product was obtain ed by elution with 3% am m o-
n ia in eth an ol–water (1:1) m ixture. Titled com poun d was obtain ed in a 69% yield (0.9 g,
2.19 m m ol) by colum n ch rom atograph y on silica gel usin g lin ear gradien t of eth an ol in
ch loroform as a colorless oil. HR FAB+: calculated (M + H) 413.206617, foun d 413.208356.
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

Oligonucleotides Modified at AMP Sites
947
1H NMR (400 MHz, DMSO-d6): 1.234, 1.240, 12.42 an d 1.243 (4 × d, 4 × 3 H, Jvic = 6.2,
(CH3)2CH); 1.92 (m , 2 H, CH2P); 2.43 (dd, 1 H, Jgem = 9.8, J5b,4 = 4.6, H-5b-pyrr); 2.66 (m ,
2 H, CH2N); 2.90 (dd, 1 H, Jgem = 9.6, J2b,3 = 5.1, H-2b-pyrr); 3.00 (dd, 1 H, Jgem = 9.6, J2a,3
=
7.1, H-2a-pyrr); 3.18 (dd, 1 H, Jgem = 9.8, J5a,4 = 7.0, H-5a-pyrr); 4.41 (bm , 1 H, J4,5 = 7.0, 4.6,
J4,OH = 5.0, J4,3 = 3.7, H-4-pyrr); 4.57 an d 4.58 (2 × dh , 2 × 1 H, JH,P = 7.8, Jvic = 6.2,
CH(CH3)2); 4.72 (td, 1 H, J3,2 = 7.1, 5.1, J3,4 = 3.7, H-3-pyrr); 5.52 (d, 1 H, JOH,4 = 5.0, OH);
7.21 (bs, 2 H, NH2); 8.13 (s, 1 H, H-8); 8.24 (s, 1 H, H-2). 13C NMR (100.6 MHz, DMSO-d6):
23.97 an d 23.99 (d, JC,P = 4, (CH3)2CH); 25.60 (d, JC,P = 139, CH2P); 48.95 (CH2N); 57.17
(CH2-2-pyrr); 60.58 (CH2-5-pyrr); 61.60 (CH-3-pyrr); 69.41 (d, JC,P = 6, CH(CH3)2); 75.26
(CH-4-pyrr); 118.88 (C-5); 139.77 (CH-8); 149.56 (C-4); 152.48 (CH-2); 156.18 (C-6).
31P NMR (162 MHz, DMSO-d6): 28.46.
Diisopropyl 2-{(3R,4R)-3-[N6-(Dibutylam in o)m eth ylen eaden in -9-yl]-
4-(4,4′-dim eth oxytrityl)oxypyrrolidin -1-yl}eth ylph osph on ate (16)
Com poun d 14 (0.9 g, 2.19 m m ol) was dissolved in m eth an ol (20 m l), an d dibutylform am ide
dim eth yl acetal (0.72 m l, 3 m m ol) was added. Th e reaction m ixture was left aside at r.t. for
2 days an d th e reaction was quen ch ed with TEAB (1 m l). On evaporation of th e solven t, th e
desired product 15 was obtain ed in a 95% yield (1.15 g, 2.08 m m ol) by colum n ch rom atog-
raph y on silica gel usin g a lin ear gradien t of eth an ol in ch loroform as a yellowish oil, an d
was used with out ch aracterization .
Com poun d 15 was co-evaporated with pyridin e (3 × 40 m l) an d dissolved in th e sam e sol-
ven t (20 m l). Dim eth oxytrityl ch loride (1.4 g, 4 m m ol) an d silver triflate (1 g, 4 m m ol) were
added to th e solution , an d th e reaction m ixture was stirred at r.t. overn igh t. Th e reaction
was quen ch ed with m eth an ol (3 m l), diluted with eth yl acetate (200 m l), an d wash ed with
saturated solution of sodium h ydrogen carbon ate. Organ ic layer was dried over sodium
sulfate, con cen trated, an d th e residue applied on a colum n of silica gel. Th e product was
obtain ed in a 79% yield (1.4 g, 1.64 m m ol) usin g lin ear gradien t of eth an ol in ch loroform
as a yellowish oil. HR FAB+: calculated (M + H) 854.473397, foun d 854.476457. 1H NMR
(500 MHz, CDCl3): 0.95 an d 0.96 (2 × t, 2 × 3 H, Jvic = 7.3, CH3CH2CH2CH2N); 1.27, 1.28
an d 1.29 (3 × d, 12 H, Jvic = 6.2, (CH3)2CH); 1.35–1.49 (m , 4 H, CH3CH2CH2CH2N);
1.61–1.71 (m , 4 H, CH3CH2CH2CH2N); 1.78 (m , 2 H, CH2P); 2.07 (dd, 1 H, Jgem = 10.0,
J5b,4 = 5.8, H-5b-pyrr); 2.55–2.72 (m , 3 H, H-5a-pyrr an d CH2N); 2.79 (dd, 1 H, Jgem = 9.9,
J2b,3 = 2.9, H-2b-pyrr); 2.85 (dd, 1 H, Jgem = 9.9, J2b,3 = 6.6, H-2a-pyrr); 3.40 (t, 2 H, Jvic = 7.3,
CH3CH2CH2CH2N); 3.67 an d 3.70 (2 × s, 2 × 3 H, CH3O); 3.72 (t, 2 H, Jvic = 7.3,
CH3CH2CH2CH2N); 4.38 (ddd, 1 H, J4,5 = 7.1, 5.8, J4,3 = 2.9, H-4-pyrr); 4.66 (dh , 2 H, JH,P
=
8.0, Jvic = 6.2, CH(CH3)2); 5.12 (dt, 1 H, J3,2 = 6.6, 2.9, J3,4 = 2.9, H-3-pyrr); 6.58 an d 6.65
(2 × m , 2 × 2 H, H-m-C6H4-DMTr); 7.07–7.15 (m , 3 H, H-m,p-C6H5-DMTr); 7.21 an d 7.23
(2 × m , 2 × 2 H, H-o-C6H4-DMTr); 7.36 (m , 2 H, H-o-C6H5-DMTr); 7.93 (s, 1 H, H-8); 8.56
(s, 1 H, H-2); 9.02 (s, 1 H, CH=N). 13C NMR (125.7 MHz, CDCl3): 13.66 an d 13.89
(CH3CH2CH2CH2N); 19.78 an d 20.21 (CH3CH2CH2CH2N); 24.00, 24.05 an d 24.06 (d, JC,P
=
4, (CH3)2CH); 26.32 (d, JC,P = 141, CH2P); 29.29 an d 31.08 (CH3CH2CH2CH2N); 45.17
(CH3CH2CH2CH2N); 48.60 (CH2N); 51.80 (CH3CH2CH2CH2N); 55.07 an d 55.11 (CH3O);
58.40 (CH2-2-pyrr); 60.26 (CH2-5-pyrr); 60.53 (CH-3-pyrr); 70.06 an d 70.10 (d, JC,P = 6,
CH(CH3)2); 79.08 (CH-4-pyrr); 87.38 (C-DMTr); 113.04 an d 113.11 (CH-m-C6H4-DMTr);
125.48 (C-5); 126.82 (CH-p-C6H5-DMTr); 127.75 (CH-m-C6H5-DMTr); 128.02 (CH-o-C6H5-DMTr);
129.92 an d 130.02 (CH-o-C6H4-DMTr); 136.05 an d 136.07 (C-i-C6H4-DMTr); 140.52 (CH-8);
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

948
Rejman, Kočalka, Pohl, Točík, Rosenberg:
145.05 (C-i-C6H5-DMTr); 151.38 (C-4); 152.38 (CH-2); 158.25 (CH=N); 158.52 an d 158.60
(C-p-C6H4-DMTr); 159.97 (C-6). 31P NMR (202.3 MHz, CDCl3): 27.50.
(4-Meth oxy-1-oxido-2-pyridyl)m eth yl 2-{(3R,4R)-3-[N6-(Dibutylam in o)-
m eth ylen eaden in -9-yl]-4-(4,4′-dim eth oxytrityl)oxypyrrolidin -1-yl}eth ylph osph on ate (18)
Com poun d 16 (1.4 g, 1.64 m m ol) was co-evaporated with aceton itrile (2 × 30 m l) an d dis-
solved in th e sam e solven t (15 m l). Lutidin e (2.3 m l, 20 m m ol) an d brom otrim eth ylsilan e
(1.3 m l, 10 m m ol) were added un der argon . Th e reaction m ixture was left overn igh t at r.t.
un der argon , aceton itrile was rem oved in vacuo, th e residue was dissolved in a m ixture of
aqueous TEAB an d m eth an ol an d th e solven ts evaporated again . Ph osph on ic acid 17 was ob-
tain ed in a 73% yield (1.17 g, 1.2 m m ol) by colum n ch rom atograph y on silica gel usin g a
lin ear gradien t of H1 in eth yl acetate as a yellowish oil. NMR sign als were too broad to be
an alyzed so th is in term ediate was used with out furth er ch aracterization .
A m ixture of com poun d 17 (0.58 g, 0.6 m m ol) an d (4-m eth oxy-1-oxido-2-pyridyl)m eth a-
n ol (0.3 g, 1.78 m m ol) was co-evaporated with pyridin e (2 × 10 m l) an d dissolved in th e
sam e solven t (10 m l). TPSCl (1.1 g, 3.6 m m ol) was added. Th e reaction m ixture was stirred
at r.t. overn igh t. Pyridin e was rem oved in vacuo, an d th e crude reaction m ixture was puri-
fied on a silica gel colum n usin g lin ear gradien t of H3 in eth yl acetate followed by lin ear
gradien t of H1 in H3. Th e obtain ed m aterial was h eated at 60 °C in 60% aqueous pyridin e
(30 m l) for 5 days, an d th e solven ts were rem oved. Th e obtain ed residue was partition ed be-
tween ch loroform an d TEAB. Organ ic layer was dried over an h ydrous sodium sulfate an d
con cen trated in vacuo. Crude m aterial was co-evaporated with m eth an ol (2 × 20 m l) an d
dissolved in th e sam e solven t (30 m l). Dibutylform am ide dim eth yl acetal (0.5 m l, 2.1 m m ol)
was added, an d th e m ixture was left at r.t. for 2 days. Th en 2 M aqueous TEAB (1 m l) was
added an d th e reaction m ixture was con cen trated in vacuo, dissolved in ch loroform , an d
wash ed with TEAB. Th e product was obtain ed in a 25% yield (0.15 g, 0.15 m m ol) by reverse
ph ase ch rom atograph y usin g a lin ear gradien t of m eth an ol in water as a wh ite am orph ous
fluffy solid after lyofilization from dioxan e. HR FAB+: calculated (M + H) 907.427175, foun d
907.424525. 1H NMR (400 MHz, CDCl3): 0.94 an d 0.96 (2 × t, 2 × 3 H, Jvic = 7.4,
CH3CH2CH2CH2N); 1.27 (t, 3 H, Jvic = 7.3, CH3CH2N); 1.38 an d 1.65 (2 × m , 2 × 4 H,
CH3CH2CH2CH2N); 1.92 (bdt, 2 H, JH,P = 17.7, Jvic = 8.0, CH2P); 2.41 (bm , 1 H, H-5b-pyrr);
2.80 (bdd, 1 H, Jgem = 10.8, J5a,4 = 7.1, H-5a-pyrr); 2.94 (bt, 2 H, Jvic = 8.0, CH2N); 3.03 (q,
2 H, Jvic = 7.3, CH3CH2N); 3.11 an d 3.23 (2 × m , 2 H, H-2-pyrr); 3.40 (t, 2 H, Jvic = 7.3,
CH3CH2CH2CH2N); 3.67 an d 3.70 (2 × s, 2 × 3 H, CH3O-DMtr); 3.71 (t, 2 H, Jvic = 7.3,
CH3CH2CH2CH2N); 3.81 (s, 3 H, CH3O-MOP); 4.57 (bm , 1 H, H-4-pyrr); 5.15 (bm , 1 H,
H-3-pyrr); 5.15 (bd,
2 H, JH,P = 7.3, CH2-MOP); 6.59 an d 6.66 (2 × m , 2 × 2 H,
H-m-C6H4-DMTr); 6.71 (dd, 1 H, J5,6 = 7.3, J5,3 = 3.5, H-5-MOP); 7.08–7.15 (m , 3 H,
H-m,p-C6H5-DMTr); 7.16–7.22 (m , 5 H, H-3-MOP an d H-o-C6H4-DMTr); 7.32 (m , 2 H,
H-o-C6H5-DMTr); 7.92 (s, 1 H, H-8); 8.07 (d, 1 H, J6,5 = 7.3, H-6-MOP); 8.47 (s, 1 H, H-2);
9.02 (s, 1 H, CH=N). 13C NMR (100.6 MHz, CDCl3): 8.46 (CH3CH2N); 13.67 an d 13.89
(CH3CH2CH2CH2N); 19.73 an d 20.15 (CH3CH2CH2CH2N); 25.49 (d, JC,P = 128, CH2P); 29.22
an d 30.98 (CH3CH2CH2CH2N); 45.12 (CH3CH2CH2CH2N); 45.20 (CH3CH2N); 50.82 (CH2N);
51.84 (CH3CH2CH2CH2N); 55.06 an d 55.10 (CH3O-DMTr); 56.20 (CH3O-MOP); 56.52
(CH2-2-pyrr); 59.46 (CH2-5-pyrr); 60.54 (CH-3-pyrr); 60.76 (d, JC,P = 4, CH2-MOP); 77.44
(CH-4-pyrr); 87.48 (C-DMTr); 108.69 (CH-3-MOP); 110.81 (CH-5-MOP); 113.07 an d 113.15
(CH-m-C6H4-DMTr); 125.62 (C-5); 126.92 (CH-p-C6H5-DMTr); 127.82 an d 127.94
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

Oligonucleotides Modified at AMP Sites
949
(CH-o,m-C6H5-DMTr); 129.82 an d 129.99 (CH-o-C6H4-DMTr); 135.64 (C-i-C6H4-DMTr);
139.73 (CH-6-MOP); 141.14 (CH-8); 144.68 (C-i-C6H5-DMTr); 150.77 (d, JC,P = 7, C-2-MOP);
151.09 (C-4); 152.12 (CH-2); 158.34 (CH=N); 158.48 an d 158.57 (C-p-C6H4-DMTr); 158.74
(C-4-MOP); 159.80 (C-6). 31P NMR (162 MHz, CDCl3): 22.49.
Diisopropyl 2-[(3R,4R)-3-(Aden in -9-yl)-4-h ydroxypyrrolidin -1-yl]-
2-oxoeth ylph osph on ate (19)
A solution of N-h ydroxysuccin im idyl(diisopropoxyph osph oryl)acetate, prepared in situ from
(diisopropoxyphosphoryl)acetatic acid (0.7, 3.1 m m ol), N-hydroxysuccinim ide (0.36 g, 3.1 m m ol)
an d DCC (1.28 g, 6.2 m m ol) in aceton itrile (12 m l), was added to (3R,4S)-3-(aden in -9-yl)-
4-h ydroxypyrrolidin e (8) (0.24 g, 1.1 m m ol). Th e suspen sion was con cen trated in vacuo an d
eth yldiisopropylam in e (0.38 m l, 2.2 m m ol) an d eth an ol (4 m l) were added to th e residue.
Th e suspen sion was stirred at r.t. for 2 days. Th en 25% aqueous am m on ia (100 m l) was
added an d, after 30 m in , th e m ixture was con cen trated an d th e residue co-evaporated with
eth an ol (3 × 50 m l). Th e titled product was obtain ed in 36% yield (170 m g) by colum n
ch rom atograph y on silica gel usin g lin ear gradien t of m eth an ol in ch loroform as a yellowish
oil. HR-MS: for C11H16N1O2 (M + H)+ calculated 427.185882, foun d 427.183416. NMR con -
firm ed a 1:1 m ixture of am ide rotam ers. 1H NMR (400 MHz, DMSO-d6): 1.23–1.28 (8 × d, 8 ×
3 H, Jvic = 6.1, (CH3)2CH); 3.00 (dd, 1 H, Jgem = 14.6, JH,P = 11.1, CHaHbP); 3.04 (dd, 1 H,
Jgem = 14.6, JH,P = 10.9, CHaHbP); 3.10 (t, 1 H, Jgem = JH,P = 14.7, CHaHbP); 3.13 (t, 1 H,
Jgem = JH,P = 14.7, CHaHbP); 3.28 (bdd, 1 H, Jgem = 12.7, JH,P = 1.6, H-5b-pyrr); 3.53 (dd, 1 H,
Jgem = 10.8, J5b,4 = 5.7, H-5b-pyrr); 3.58 (ddd, 1 H, Jgem = 12.7, J5a,4 = 6.0, JH,P = 1.6,
H-5a-pyrr); 3.84 (ddd, 1 H, Jgem = 12.5, J2b,3 = 7.5, JH,P = 1.6, H-2b-pyrr); 3.90 (ddd, 1 H,
Jgem = 12.5, J2b,3 = 6.5, JH,P = 1.6, H-2a-pyrr); 4.07 (dd, 1 H, Jgem = 12.7, J5,4 = 6.3, H-5a-pyrr);
4.16 (dd, 1 H, Jgem = 11.3, J2b,3 = 5.4, H-2b-pyrr); 4.25 (dd, 1 H, Jgem = 11.3, J2b,3 = 7.0,
H-2a-pyrr); 4.61 (m , 1 H, H-4-pyrr); 4.63 (m , 4 H, CH(CH3)2); 4.69 (m , 1 H, H-4-pyrr); 4.80
(ddd, 1 H, J3,2 = 7.5, 6.5, J3,4 = 6.3, H-3-pyrr); 4.85 (dt, 1 H, J3,2 = 7.0, 5.4, J3,4 = 5.4,
H-3-pyrr); 5.80 (d, 1 H, JOH,4 = 4.4, OH); 5.86 (d, 1 H, JOH,4 = 4.9, OH); 7.27 (bs, 4 H, NH2);
8.09 (s, 1 H, H-8); 8.14 an d 8.15 (2 × s, 2 × 1 H, H-2); 8.20 (s, 1 H, H-8). 13C NMR
(100.6 MHz, DMSO-d6): 23.72–24.02 (d, JC,P = 4, (CH3)2CH); 34.94 (d, JC,P = 133, CH2P);
47.62 an d 48.84 (CH2-2-pyr); 51.27 an d 52.20 (CH2-5-pyr); 59.10 an d 60.21 (CH-3-pyr);
70.51–70.70 (d, JC,P = 6, CH(CH3)2); 70.90 an d 71.91 (CH-4-pyrr); 119.23 (C-5); 139.13 an d
139.53 (CH-8); 149.65 an d 149.70 (C-4); 152.53 an d 152.60 (CH-2); 156.27 (C-6); 163.59
an d 163.75 (d, J = 6, C=O). 31P NMR (162 MHz, DMSO-d6): 20.60 an d 20.65.
Diisopropyl 2-{(3R4,R)-3-[N6-(Dibutylam in o)m eth ylen eaden in -9-yl]-
4-(4, 4′-dim eth oxytrityl)oxypyrrolidin -1-yl}-2-oxoeth ylph osph on ate (21)
Com poun d 19 (0.17 g, 0.4 m m ol) was dissolved in m eth an ol (2 m l) an d dibutylform am ide
dim eth yl acetal (0.28 m l, 1.2 m m ol) was added. Th e reaction m ixture was left aside at r.t.
for 5 h an d th en quen ch ed with 2 M TEAB (0.8 m l). Th e m ixture was con cen trated an d co-
evaporated with eth an ol (2 × 10 m l) an d th en with dry pyridin e (2 × 10 m l). To th e residue
in pyridin e (10 m l), DMTrCl (0.34 g, 1 m m ol) an d silver triflate (0.26 g, 1 m m ol) were
added. Th e m ixture was stirred at r.t. for 5 days. Th e reaction was quen ch ed with dry m eth -
an ol (1 m l). Th e suspen sion was filtered th rough a pad of Celite, th e cake was wash ed
several tim es with DCM, an d th e organ ic layer was extracted with aqueous sodium bicarbon -
ate. Th e organ ic layer was con cen trated an d th e title com poun d was obtain ed in 50% yield
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

950
Rejman, Kočalka, Pohl, Točík, Rosenberg:
(173 m g) by ch rom atograph y on silica gel usin g lin ear gradien t of m eth an ol in ch loroform
as a yellowish foam . HR-MS: for C11H16N1O2 (M + H)+ calculated 868.455814, foun d
868.452661. NMR spectra con firm ed a 1:1 m ixture of am ide rotam ers. 1H NMR (400 MHz,
DMSO-d6): 0.93 an d 0.94 (2 × t, 2 × 6 H, Jvic = 7.2, CH3CH2CH2CH2N); 1.17–1.26 (8 × d,
24 H, Jvic = 6.2, (CH3)2CH); 1.32 an d 1.61 (2 × m , 2 × 8 H, CH3CH2CH2CH2N); 2.58–2.76
an d 2.82–2.98 (2 × m , 2 × 2 H, CH2P); 3.28–3.50 (m , 6 H, H-5-pyrr an d CH3CH2CH2CH2N);
3.58–3.75 (m , 6 H, H-5-pyrr an d CH3CH2CH2CH2N); 3.65 an d 3.70 (2 × s, 12 H, CH3O); 3.87
(m , 2 H, H-2-pyrr); 4.06 (dd, 1 H, Jgem = 10.6, J2b,3 = 9.1, H-2b-pyrr); 4.23 (dd, 1 H, Jgem
=
10.6, J2a,3 = 8.3, H-2a-pyrr); 4.50–4.62 (m , 4 H, CH(CH3)2); 4.69 an d 4.72 (2 × dt, 2 × 1 H,
J4,3 = 7.5, J4,5 = 6.6, H-4-pyrr); 5.25 an d 5.32 (2 × ddd, 2 × 1 H, J3,2 = 9.1, 8.3, J3,4 = 7.5,
H-3-pyrr); 6.61 an d 6.75 (2 × m , 2 × 4 H, H-m-C6H4-DMTr); 6.97 an d 6.98 (2 × m , 4 H,
H-o-C6H4-DMTr); 7.05–7.25 (m , 14 H, H-o,m,p-C6H5-DMTr an d H-o-C6H4-DMTr); 8.32, 8.34,
8.36 an d 8.38 (4 × s, 4 × 1 H, H-2 an d H-8); 8.99 (s, 2 H, CH=N). 13C NMR (100.6 MHz,
DMSO-d6): 13.67 an d 13.85 (CH3CH2CH2CH2N); 19.27 an d 19.75 (CH3CH2CH2CH2N);
23.64–23.86 (d, JC,P = 4, (CH3)2CH); 28.85 an d 30.63 (CH3CH2CH2CH2N); 34.27 an d 34.60
(d, JC,P = 133, CH2P); 44.58 an d 51.08 (CH3CH2CH2CH2N); 45.49 an d 47.47 (CH2-2-pyrr);
49.63 an d 50.83 (CH2-5-pyrr); 54.99 an d 55.11 (CH3O); 58.44 an d 59.21 (CH-3-pyrr);
70.37–70.47 (d, JC,P = 7, CH(CH3)2); 73.28 an d 73.96 (CH-4-pyrr); 86.31 an d 86.37
(C-DMTr); 113.15 an d 113.31 (CH-m-C6H4-DMTr); 126.12 an d 126.21 (C-5); 126.83 126.88,
127.49, 127.60 an d 127.84 (CH-o,m,p-C6H5-DMTr); 129.45 an d 129.92 (CH-o-C6H4-DMTr);
135.36, 135.42, 135.44 an d 135.61 (C-i-C6H4-DMTr); 142.15 an d 142.41 (CH-8); 145.08 an d
145.11 (C-i-C6H5-DMTr); 151.65 an d 151.71 (C-4); 151.77 (CH-2); 158.01 (CH=N); 158.17,
158.24, 158.41 an d 158.49 (C-p-C6H4-DMTr); 159.60 an d 159.63 (C-6); 163.11 an d 163.18
(d, JC,P = 6, CO).
(4-Methoxy-1-oxido-2-pyridyl)m ethyl 2-{(3R,4R)-3-[N6-(Dibutylam ino)m ethyleneadenin-9-yl]-
4-(4,4′-dim eth oxytrityl)oxypyrrolidin -1-yl}-2-oxoeth ylph osph on ate (23)
Com poun d 21 was co-evaporated with aceton itrile (2 × 10 m l) an d dissolved in th e sam e
solven t (3 m l). Un der argon atm osph ere, lutidin e (0.23 m l, 2 m m ol) an d brom otrim eth yl-
silan e (0.13 m l, 1 m m ol) were added. Th e reaction m ixture was left at r.t. un der argon atm o-
sph ere overn igh t an d th en con cen trated in vacuo. To th e residue, 0.1 M TEAB (6 m l) an d
ch loroform (20 m l) were added. Th e organ ic layer was separated, dried over an h ydrous
sodium sulfate, filtered an d on evaporation of solven ts, th e com poun d 22 was obtain ed in
80% yield (0.13 g) by colum n ch rom atograph y on silica gel usin g a lin ear gradien t of H1 in
eth yl acetate as a yellowish foam . NMR sign als were too broad to be an alyzed so th is in ter-
m ediate was used with out furth er ch aracterization .
A m ixture of com poun ds 22 (0.13 g, 0.16 m m ol) an d (4-m eth oxy-1-oxido-4-pyridyl)m eth -
an ol (0.47 g, 0.3 m m ol) was co-evaporated with pyridin e (2 × 10 m l) an d dissolved in th e
sam e solven t (3 m l). Th en DCC (0.1 g, 0.5 m m ol) was added. Th e m ixture was left to stan d
aside at r.t. After 4 days, water (2 m l) was added an d th e m ixture was h eated at 60 °C over-
n igh t. Th en con cen trated, partition ed between ch loroform an d 0.1 M aqueous TEAB, dried
over an h ydrous sodium sulfate. Th e titled com poun d was obtain ed in 39% yield (63 m g) by
colum n ch rom atograph y on silica gel usin g lin ear gradien t of H1 in H3 folowed by reverse
ph ase ch rom atograph y usin g lin ear gradien t of m eth an ol in water as a yellowish am orph ous
fluffy solid (after lyofilization from dioxan e). HR-MS: for C11H16N1O2 (M + H – Et3N)+ calcu-
lated 921.406440, foun d 921.414407. NMR spectra con firm ed a 1:1 m ixture of am ide
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

Oligonucleotides Modified at AMP Sites
951
rotam ers: 1H NMR (400 MHz, CDCl3): 0.92–1.20 (m , 12 H, CH3CH2CH2CH2N); 1.24 (t, 18 H,
CH3CH2N); 1.27–1.47, 1.55–1.74 an d 1.92 (3 × m , 16 H, CH3CH2CH2CH2N); 2.95–3.22 (m ,
4 H, CH2P); 3.25–3.54 (m , 22 H, H-5-pyrr, CH3CH2CH2CH2N an d CH3CH2N); 3.60–3.70 (m ,
2 H, H-5-pyrr); 3.71, 3.72, 3.73 an d 3.74 (4 × s, 12 H, CH3O-DMTr); 3.856 an d 3.860 (2 × s,
6 H, CH3O-MOP); 3.95 (dd, 1 H, Jgem = 12.2, J2b,3 = 5.2, H-2b-pyrr); 4.03 (dd, 1 H, Jgem
=
12.2, J2a,3 = 5.2, H-2a-pyrr); 4.34 (dd, 1 H, Jgem = 10.7, J2b,3 = 7.6, H-2b-pyrr); 4.50 (dd, 1 H,
Jgem = 10.6, J2a,3 = 8.2, H-2a-pyrr); 4.63 an d 4.73 (2 × q, 2 × 1 H, J4,3 = J4,5 = 6.4, H-4-pyrr);
5.05 (ddd, 1 H, J3,2 = 7.2, 5.2, J3,4 = 6.4, H-3-pyrr); 5.16 (ddd, 1 H, J3,2 = 8.2, 7.6, J3,4 = 6.4,
H-3-pyrr); 5.21 (d,
4 H, JH,P = 7.4, CH2P-MOP); 5.57–6.75 (m , 10 H, H-5-MOP an d
H-m-C6H4-DMTr); 7.08–7.32 (m , 18 H, H-o-C6H4-DMTr an d H-C6H5-DMTr); 7.39 an d 7.41
(2 × d, 2 × 1 H, J3,5 = 3.5, H-3-MOP); 7.97 an d 8.10 (2 × s, 2 × 1 H, CH-8); 8.02 an d 8.04
(2 × d, 2 × 1 H, J6,5 = 7.2, H-6-MOP); 8.39 an d 8.44 (2 × s, 2 × 1 H, CH-2); 9.01 an d 9.02
(2 × s, 2 × 1 H, CH=N), 31P NMR (162 MHz, CDCl3): 14.45 an d 14.60.
Syn th esis of Oligon ucleotides
All used solven ts were an h ydrous. 1-H-tetrazole was resublim ed at reduced pressure. Th e
startin g ph osph on ate m on om ers were lyofilized from dioxan e as TEA salts. Before usin g in
th e syn th esizer, th ey were dissolved in pyridin e an d dried over m olecular sieves for 8 h . Th e
HPLC an alysis of oligon ucleotides was carried out on a Nucleosil 100-5 C18 HD (4.6 ×
150 m m ) colum n with liquid ch rom atograph LC5000 (In gos, Czech Republic) usin g gradien t
of aceton itrile in 0.1 M TEAA. Preparative HPLC of oligon ucleotides was perform ed usin g th e
sam e system with differen t colum n s, as follows: Dyn am ax C18 (Rain in , USA) colum n was
used for purification s of oligon ucleotides bearin g term in al dim eth oxytrityl group. Free
oligon ucleotides were purified on Ph en om en ex C18 (Ph en om en ex, USA) (10 × 250 m m ) col-
um n . Th e detritylation took place on Dyn am ax PureDNA (Rain in , USA) (10 × 50 m m ) col-
um n . All oligon ucleotide solution s were evaporated in a cen trifuge evaporator (Labcon co,
USA). Th erm al ch aracteristic of th e prepared oligon ucleotides were m easured on Cary 100
(Varian , Australia) UV/VIS spectroph otom eter.
Th e solid ph ase syn th esis of oligon ucleotides was carried out on GENESYN (IOCB R&D)
syn th esizer usin g th e “trityl on ” m eth od, on th e scale of 0.5 µm ol (~20 m g of solid support
was used for each syn th esis). Table II sh ows th e syn th esis protocols for oligon ucleotides
ON1–ON6. Oligon ucleotide ON7 was syn th esized by ph osph otriester m eth od. Th e average
yield of th e con den sation step was in th e ran ge of 97–98% wh ile th e overall oligon ucleotide
yield reach ed 66–74%.
Th e (4-m eth oxy-1-oxido-2-pyridyl)m eth yl protectin g group of ph osph on ate m oieties of
th e oligon ucleotides boun d on solid support was rem oved by treatm en t with th ioph en ol–
trieth ylam in e–dioxan e (1:1:2 v/v/v) at r.t. for 48 h . Th e CPG (con trolled pore glass) support
con tain in g th e oligon ucleotide was wash ed with aceton itrile an d dried in th e stream of
argon . Th e oligon ucleotide-bearin g support placed in 37% aqueous am m on ia (2 m l) was
th en kept at 55 °C for 16 h to rem ove rem ain in g protectin g groups an d to cleave th e
oligon ucleotide from th e support. Th e solution of free oligon ucleotide bearin g term in al
dim eth oxytrityl group was evaporated after addition of 2 M aqueous TEAB (200 µl) to avoid
cleavage of DMTr group. Th e oligon ucleotide was dissolved in 0.4 M TEAA (1 m l) an d ap-
plied on Dyn am ax C18; 300 A; 10 × 250 m m colum n . Th e ch rom atograph y was carried out
usin g lin ear gradien t from 0.1 M TEAA in 10% aceton itrile to 0.1 M TEAA in 25% aceton itrile
durin g 20 m in , an d th en to 0.1 M TEAA in 50% aceton itrile over addition al 30 m in . Th e pu-
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

952
Rejman, Kočalka, Pohl, Točík, Rosenberg:
TABLE II
Protocol for solid ph ase syn th esis of ph osph on ate an alogues of oligon ucleotides ON1–ON6
Step
Process
Solven t/Reagen t
Volum e, m l
3
Tim e, s
1
2
Wash in g
Cappin g
CH3CN
30
60
(i) Ac2O–collidin e–CH3CN (2:3:6) 0.2
(ii) 6% DMAP in CH3CN
CH3CN
0.2
3
Wash in g
3
30
Start of ph osph otriester elon gation cycle
4
5
6
7
Wash in g
DCE
3
20
Detritylation
Wash in g
Con den sation b
3% TCA in DCE
pyridin e
6
70
3
60
(j) 0.05 M P-com pon en t
(jj) 0.1 M TPSCl
pyridin e
0.2
300–1800
8
Wash in g
1
1
20
10
CH3CN
9
Cappin g
Wash in g
as step 2
10
CH3CN
3
30
En d of ph osph otriester elon gation cycle
Start of ph osph oram idite elon gation cycle
11
12
13
14
Wash in g
DCE
3
20
Detritylation
Wash in g
Con den sation c
3% TCA in DCE
CH3CN
0.1 M am idite
0.5 M tetrazole
CH3CN
0.01 M iodin e in
CH3CN–collidin e–water (65:6:30)
CH3CN
6
70
3
30
0.075
0.090
1
140
15
16
Wash in g
10
40
Oxidation
0.6
17
18
19
Wash in g
Cappin g
Wash in g
1
10
30
As step 2
CH3CN
3
En d of ph osph oram idite elon gation cycle
Postsyn th etic procedures
23
24
Dryin g with argon
Rem oval of MOP
group
th ioph en ol–Et3N–dioxan e (1:1:2)
1
48 h
16 h
25
26
Wash in g
CH3CN
37% aqueous NH3, 55 °C
30
2
Cleavage from
support, an d fin al
deprotection
a
b
Altern atin g pulses of 20-µl segm en ts of (i) an d (ii). Man ual in jection of fresh ly prepared
c
solution of th e appropriate ph osph on ate an d TPSCl in pyridin e. Altern atin g pulses of 15-µl
segm en ts of (j) an d (jj) reagen ts (tetrazole solution first).
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

Oligonucleotides Modified at AMP Sites
953
rified oligon ucleotide was evaporated, dissolved in 0.4 M TEAA (1 m l) an d applied on
Dyn am ax C18; 300 A; 10 × 50 m m colum n wh ere detritylation took place accordin g to th e
protocol sh own in Table III.
TABLE III
Protocol for detritylation on Dyn am ax C18 PureDNA colum n
Flow rate Tim e
Step Process
m l m in –1
m in
1
(i) Colum n wash in g with m eth an ol
(ii) Equilibration 0.1 M TEAA
9.4
5
2
3
4
5
6
Sam ple in jection (1 m l, 0.94–4.7 OD260
Wash in g 0.1 M TEAA
)
–
2
Detritylation 0.5% TFA
2
Wash in g 0.1 M TEAA
4
Elution with lin ear gradien t 0→100% B in A
A = 0.1 M TEAA
3
15
B = 0.1 M TEAA in 50% aceton itrile
Th e oligon ucleotide was dissolved in 0.4 M TEAA (1 m l) an d applied on Dyn am ax C18;
300 A; 10 × 250 m m colum n . Th e ch rom atograph y was carried out usin g a lin ear gradien t
from 0.1 M TEAA in 10% aceton itrile to 0.1 M TEAA in 25% aceton itrile at a flow rate of
3 m l m in –1. Th e exact con dition s depen ded on tem perature. Rem oval of salts on NAP-10
Ph arm acia colum n filled with Seph adex G-25 in 1% aqueous am m on ia was th e last step. Th e
pure oligon ucleotide was th en lyoph ilized an d stored at –20 °C. Structures of th e prepared
ONs were proved by MALDI-TOF m ass spectrom etry (Table IV).
Measurem en ts of Tm Values
Tm values of duplexes of th e prepared ONs with com plem en tary oligodeoxyribon ucleotide
were m easured in 50 m M Tris-HCl (pH 7.2); 10 m M Mg2+ (or 100 m M Na+), an d 1 m M EDTA
at overall oligon ucleotide con cen tration of 4 µM.
An ON solution was h eated to 60 °C, cooled to room tem perature, an d tran sferred to th e
m easurin g cuvette placed in th erm ostated cell. Th e cuvette with th e sam ple was h eated at a
tem perature gradien t of 5 °C m in –1 to 60 °C in th e spectroph otom eter, an d it was con tin ued
at th is tem perature for 5 m in . Th e tem perature was th en left to decrease to 10 °C at a tem -
perature gradien t of 1 °C m in –1 an d th e absorban ce was m easured. Th e absorban ce was also
m easured at th e reverse direction of tem perature from 10 to 60 °C (1 °C m in –1).
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

954
Rejman, Kočalka, Pohl, Točík, Rosenberg:
TABLE IV
MALDI-TOF determ in ation of ON1–ON7 m olecule size
Oligon ucleotide Sequen ce
Calcd
Foun d
Mon om er/
(MALDI-TOF) A scaffold
ON1
ON2
ON3
ON4
ON5
ON6
ON7
5′-d(GTG ATA TGC)-3′
2718.544
2774.533
2746.575
2630.564
2714.549
2672.611
4459.191
2719.995
2775.791
2747.651
2631.737
2715.448
2673.806
13/5
23/7
18/6
13/5
23/7
18/6
13/5
5′-d(GTG ATA TGC)-3′
5′-d(GTG ATA TGC)-3′
5′-d(GCA TAT CAC)-3′
5′-d(GCA TAT CAC)-3′
5′-d(GCA TAT CAC)-3′
dA15
4460.07
(4461.05)
Support by grants Nos 2B06065, LC06077, and LC06061 (Ministry of Education, Youth and
Sports of the Czech Republic) and KAN200520801 (Academy of Sciences of the Czech Republic),
under research project Z40550506 is gratefully acknowledged. Authors are indebted to the staff of the
Department of Mass Spectroscopy for measurements of HR-MS and MALDI-TOF spectra.
REFERENCES
1. Zamecnik P. C., Stephenson M. L.: Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 280.
2. Crooke S. T.: Biochim. Biophys. Acta 1999, 1489, 31.
3. Stein C. A.: Nat. Med. 1995, 1, 1119.
4. Pirollo K. F., Rait A. R., Sleer L. S., Chang E. H.: Pharmacol. Ther. 2003, 99, 55.
5. Kurreck J.: Eur. J. Biochem. 2003, 270, 1628.
6. Tamm I., Dorken B., Hartmann G.: Lancet 2001, 358, 489.
7. Agrawal S.: Biochim. Biophys. Acta 1999, 1489, 53.
8. Furer P., Billeci T. M., Donati A., Kojima B., Karwowski B., Sierzchala A., Stec W., James
T. L.: J. Mol. Biol. 1994, 269, 1609.
9. Eckstein F.: Antisense Nucleic Acid Drug Dev. 2000, 10, 117.
10. Freier S. M., Altmann K. H.: Nucleic Acids Res. 1997, 25, 4429.
11. Orum H., Wengel J.: Curr. Opin. Mol. Ther. 2001, 3, 239.
12. Hyrup B., Nielsen P. E.: Bioorg. Med. Chem. 1996, 4, 5.
13. Iversen P. L.: Curr. Opin. Mol. Ther. 2001, 3, 235.
14. Miller P. S.: Nat. Biotechnol. 1991, 9, 358.
15. Zamaratski E., Pradeepkumar P. I., Chattopadhyaya J.: J. Biochem. Biophys. Methods 2001,
48, 189.
16. Van Aerschot A.: Antiviral Res. 2006, 71, 307.
17. Verma I. M., Veitzman M. D.: Annu. Rev. Biochem. 2005, 74, 711.
18. Altmann K. H., Hüsken D., Cuenoud B., García-Echeverría C.: Bioorg. Med. Chem. Lett.
2000, 10, 929.
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955

Oligonucleotides Modified at AMP Sites
955
19. Hickman D. T., King P. M., Cooper M. A., Slater J. M., Micklefield J.: Chem. Commun.
2000, 2251.
20. Kumar V., Pallan P. S., Ganesh M., Ganesh K. N.: Org. Lett. 2001, 3, 1269.
21. Jordan S., Schwemler C., Kosch W., Kretschmer A., Stropp U., Schwenner E., Mielke B.:
Bioorg. Med. Chem. Lett. 1997, 7, 681.
22. Ceulemans G., Van Aerschot A., Rozenski J., Herdewijn P.: Chem. Eur. J. 1997, 3, 1997.
23. a) Efimov V. A., Chakhmakheva O. G.: Collect. Czech. Chem. Commun. 2006, 71, 929;
b) Efimov V. A., Klykov V. N., Chakhmakheva O. G.: Nucleosides Nucleotides 2007, 26,
1595.
24. Rosenberg I. in: Frontiers in Nucleosides and Nucleic Acids (R. F. Schinazi and D. C. Liotta,
Eds), p. 519. IHL Press, Tucker (GA) 2004.
25. Hanuš J., Barvík I., Ruszová-Chmelová K., Štěpánek J., Turpin P. Y., Bok J., Rosenberg I.,
Petrová-Endová M.: Nucleic Acids Res. 2001, 24, 5182.
26. Rejman D., Snášel J., Liboska R., Točík Z., Pačes O., Králíková S., Rinnová M., Koiš P.,
Rosenberg I.: Nucleosides Nucleotides 2001, 20, 819.
27. Giannaris P. A., Damha M. J.: Nucleic Acids Res. 1993, 21, 4742.
28. Snášel J., Rejman D., Liboska R., Točík Z., Ruml T., Rosenberg I., Pichová I.: Eur. J.
Biochem. 2001, 268, 980.
29. Efimov V. A., Reverdatto S. V., Chakhmakhcheva O. G.: Nucleic Acids Res. 1982, 10,
6675.
30. a) Efimov V. A., Buryakova A. A., Reverdatto S. V., Chakhmakhcheva O. G., Ovchinnikov
Yu. A.: Nucleic Acids Res. 1983, 11, 8369; b) Zarytova V. F., Knorre O. G.: Nucleic Acids
Res. 1984, 12, 2091.
31. Efimov V. A., Chakhmakhcheva O. G., Polushin N. N., Dubey I. Y.: Nucleic Acid Symp.
Ser. 1987, 18, 213.
32. Szabó T., Kers A., Stawinski J.: Nucleic Acids Res. 1995, 23, 893.
33. Rejman D., Kočalka P., Buděšínský M., Rosenberg I.: Tetrahedron 2007, 63, 1243.
34. Pačes O., Točík Z., Rosenberg I.: Collect. Czech. Chem. Commun. 2008, 73, 32.
35. Vaněk V., Buděšínský M., Rinnová M., Rosenberg I.: Tetrahedron 2009, 65, 862.
36. Rejman D., Kočalka P., Pohl R., Rosenberg I.: Tetrahedron 2009, 65, 3673.
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 6, pp. 935–955