Synthesis of 3'O-(2-cyanoethyl)-2'-deoxythymidine-5'-phosphate as a model compound for evaluation of Cyanoethyl Cleavage

Article abstract of DOI:10.1135/cccc2008183

An essential and challenging task during the development of our sequencing-by-synthesis (SBS) technique is the evaluation of efficient cyanoethyl (CE) cleavage conditions. For this purpose 3'-O-(2-cyanoethyl)-2'- deoxythymidine-5'-phosphate as a model compound as well as a short DNA oligomer bearing the CE function as terminal group were synthesized and used for various deprotection experiments. As it is already known for 2'-O-CE-protected RNA oligonucleotides, the CE function can be cleaved with tetrabutylammonium fluoride (TBAF) in THF. Indeed, by using 3'-O-(2-cyanoethyl)-2'-deoxythymidine- 5'-phosphate as a simple model compound for cleavage tests, we found out that the 3'-O-CE function is quantitatively cleaved with 1 m TBAF in THF. However, the CE group is also cleaved by other small bases like hydroxy groups under alkaline conditions. The CE cleavage with TBAF in THF gives the fastest and quantitative removal of the CE group under mild conditions for our sequencing-by-synthesis (SBS) application. The efficient removal of the 3'-CE group is crucial for the proof-of-principle of our SBS approach using dye-labeled 3'-CE-blocked dNTPs, which is currently under investigation. Herein we describe the application of 3'-O-(2-cyanoethyl)-2'-deoxythymidine-5'- phosphate as model compound for the development of reversible terminators for the SBS technique. Furthermore we suggest that nucleoside phosphates bearing any removable 3'-modification might be suitable model compounds for cleavage studies in a heterogeneous environment comparable to an oligonucleotide/aprotic solvent system.

Full text of DOI:10.1135/cccc2008183

Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
515
SYNTHESIS OF 3′-O-(2-CYANOETHYL)-2′-DEOXYTHYMIDINE-
5′-PHOSPHATE AS A MODEL COMPOUND FOR EVALUATION
OF CYANOETHYL CLEAVAGE
b
a2
Angelika C. KELLERa1,*, Saulius SERVA , Diana C. KNAPP
,
c
a3
Marek KWIATKOWSKI and Joachim W. ENGELS
a
Institut für Organische Chemie und Chemische Biologie, J. W. Goethe Universität
Frankfurt am Main, Max-von-Laue Str. 7, 60438 Frankfurt am Main, Germany;
e-mail: keller@chemie.uni-frankfurt.de, knapp@chemie.uni-frankfurt.de,
1
2
3
joachim.engels@chemie.uni-frankfurt.de
b
c
Fermentas UAB, V. Graiciuno 8, 02241 Vilnius, Lithuania; e-mail: sserva@fermentas.lt
Oligovation AB, Dag Hammarskjölds väg 32B, Uppsala Science Park, 751 83 Uppsala, Sweden;
e-mail: marek.kwiatkowski@oligovation.se
Received Septem ber 8, 2008
Accepted Decem ber 11, 2008
Publish ed on lin e March 18, 2009
An essen tial an d ch allen gin g task durin g th e developm en t of our sequen cin g-by-syn th esis
(SBS) tech n ique is th e evaluation of efficien t cyan oeth yl (CE) cleavage con dition s. For th is
purpose 3′-O-(2-cyan oeth yl)-2′-deoxyth ym idin e-5′-ph osph ate as a m odel com poun d as well
as a sh ort DNA oligom er bearin g th e CE fun ction as term in al group were syn th esized an d
used for various deprotection experim en ts. As it is already kn own for 2′-O-CE-protected RNA
oligon ucleotides, th e CE fun ction can be cleaved with tetrabutylam m on ium fluoride (TBAF)
in THF. In deed, by usin g 3′-O-(2-cyan oeth yl)-2′-deoxyth ym idin e-5′-ph osph ate as a sim ple
m odel com poun d for cleavage tests, we foun d out th at th e 3′-O-CE fun ction is quan tita-
tively cleaved with 1 M TBAF in THF. However, th e CE group is also cleaved by oth er sm all
bases like h ydroxy groups un der alkalin e con dition s. Th e CE cleavage with TBAF in THF
gives th e fastest an d quan titative rem oval of th e CE group un der m ild con dition s for our
sequen cin g-by-syn th esis (SBS) application . Th e efficien t rem oval of th e 3′-CE group is crucial
for th e proof-of-prin ciple of our SBS approach usin g dye-labeled 3′-CE-blocked dNTPs, wh ich
is curren tly un der in vestigation . Herein we describe th e application of 3′-O-(2-cyan oeth yl)-
2′-deoxyth ym idin e-5′-ph osph ate as m odel com poun d for th e developm en t of reversible ter-
m in ators for th e SBS tech n ique. Furth erm ore we suggest th at n ucleoside ph osph ates bearin g
an y rem ovable 3′-m odification m igh t be suitable m odel com poun ds for cleavage studies in a
h eterogen eous en viron m en t com parable to an oligon ucleotide/ aprotic solven t system .
Keyw ord s: DNA; Sequen cin g-by-syn th esis; Polym erase in corporation ; Ph osph orylation s;
Cyan oeth yl protectin g group; 3′-O-Modified n ucleotides; Cleavage reaction s; Oligo-
n ucleotides; Ph osph oram idites; Nucleoside triph osph ates.
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 4, pp. 515–534
© 2009 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc2008183

516
Keller, Serva, Knapp, Kwiatkowski, Engels:
A n ew approach for h igh -th rough put sequen cin g is th e array-based
sequen cin g-by-syn th esis (SBS) tech n ique1. Th is tech n ique does n ot require
gel electroph oresis an d affords a direct read-out of th e base sequen ce via
fluorescen t sign al detection . Th e ch allen gin g part of developin g th is
sequen cin g m eth od is th e evolution of a h igh ly toleran t polym erase th at
in corporates reversible term in ators in to a DNA-tem plate; an oth er require-
m en t is th e syn th esis of four reversible term in ators2. We foun d out th at
certain polym erases accept an d in corporate cyan oeth yl (CE)-bearin g
n ucleotides like 3′-O-(2-cyan oeth yl)-2′-deoxyth ym idin e-5′-triph osph ate
(3′-O-CE-dTTP); th erefore, th is 3′-m odified n ucleotide is a good can didate
for a poten tial reversible term in ator. Besides th e dem an ded term in atin g
properties, th e 3′-O-CE group also h as to be quan titatively rem ovable to
en able th e n ext in corporation step. For th e evaluation of th e m ildest
deprotection con dition s, a sim ple m odel com poun d 3′-O-(2-cyan oeth yl)-
2′-deoxyth ym idin e-5′-ph osph ate was syn th esized first an d used for several
deprotection experim en ts. Due to its h igh polarity an d solubility in water,
th e m on oph osph ate sim ulates well th e beh avior of sm all oligodeoxy-
n ucleotides (ODNs). It also form s a h eterogen eous m ixture in aprotic or-
gan ic solven ts, wh ich leads to differen t deprotection kin etics as in solution .
Based on th e results from th e m on om er, we applied th e optim ized de-
blockin g con dition s to a sh ort DNA oligom er (8m er) bearin g th e CE fun c-
tion on th e 3′-term in us.
CHEMISTRY
We establish ed a sim ple syn th etic procedure for th e preparation of th e
m odel com poun d. Th e syn th esis starts with a on e-flask tran sien t protection
of both h ydroxy groups in th e pen tose m oiety of 2′-deoxyth ym idin e
(Sch em e 1). Followin g th e tran sien t-protection procedure kn own from ref.3,
2.5 equiv. ch lorotrim eth ylsilan e is added to 1 in pyridin e to protect both
OH groups. After 30 m in th e m ixture is treated with 1.1 equiv. ben zoyl
ch loride for selective ben zoylation of N3.
Durin g th e aqueous workup, both trim eth ylsilyl protectin g groups are
quan titatively rem oved with trifluoroacetic acid an d th e N3-protected
n ucleoside 2 is obtain ed in an excellen t yield of 83% after purification . Th e
in troduction of th e trityl group at th e 5′-en d usin g stan dard con dition s4
leads to N3-ben zoyl-2′-deoxy-5′-O-(4-m eth oxytrityl)-th ym idin e 3 in 78%
yield. Th en th e protected n ucleoside 3 un dergoes a Mich ael addition re-
action for th e in troduction of th e CE fun ction at th e 3′-en d accordin g
to Sekin e et al.5, wh ich gives th e desired n ucleoside 4 in 70% yield. Th e
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
517
SCHEME 1
SCHEME 2
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518
Keller, Serva, Knapp, Kwiatkowski, Engels:
trityl protectin g group of com poun d 4 is rem oved quan titatively usin g
p-toluen esulfon ic acid. After aqueous workup an d purification by flash col-
um n ch rom atograph y, n ucleoside 5 is obtain ed. Th is com poun d is dis-
solved in m eth an ol an d treated with an excess of con cen trated aqueous
am m on ia (Sch em e 2). Stirrin g at room tem perature un der th ese con dition s
gives 96% of N3-deprotected com poun d 6 with out an y side products. Th e
CE fun ction is stable durin g both deprotection steps as lon g as th e cleavage
of th e ben zoyl group in am m on ia is n ot carried out at elevated tem pera-
tures.
Mon oph osph ate 7 is prepared by a variation of Yosh ikawa’s m eth od6
usin g ph osph orus oxych loride in trim eth yl ph osph ate accordin g to
El-Tayeb’s protocol7. Th e workup procedure of com poun d 7 is tim e-
dem an din g due to product purification by preparative RP-HPLC. Th e 5′-
h ydroxy group is ph osph orylated selectively in a m oderate yield of 52%
after purification . Spectral an alysis by 31P NMR sh ows th e sin glet sign al of
th e desired com poun d. All sign als of th e sugar m oiety an d base could be
1
assign ed by H NMR. Mon oph osph ate 7 is precipitated as sodium salt after
purification by RP-HPLC to give a colorless crystallin e product with defin ed
coun terion s. Th e triph osph ate used for th e in corporation tests, 3′-O-
(2-cyan oeth yl)th ym idin e-5′-O-triph osph ate 8 (see Sch em e 2) was prepared
accordin g to th e protocol of Ludwig an d Eckstein 8. However, th e purifica-
tion of triph osph ate 8 appeared to be tim e-dem an din g, too: Crude separa-
tion from correspon din g m on o- an d diph osph ate was ach ieved by ion -
exch an ge ch rom atograph y (FPLC), furth er purification of th e triph osph ate-
con tain in g fraction was don e by RP-HPLC. Triph osph ate 8 was obtain ed in
12% yield on ly, but in h igh purity as it is n eeded for th e polym erase accep-
tan ce tests. As a referen ce substan ce for th e deprotected product, 2′-deoxy-
th ym idin e-5′-ph osph ate 10 was prepared in th e sam e way as com poun d 7
(Sch em e 3). It is accessible from 2′-deoxyth ym idin e in a m oderate yield of
45% after purification by preparative RP-HPLC with subsequen t precipita-
SCHEME 3
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
519
tion as sodium salt. For th e in corporation in to th e DNA 8m er used for
th e CE deprotection test, 3′-O-(2-cyan oeth yl)-5′-[(2-cyan oeth yl)-(N,N-diiso-
propyl)]-2′-deoxy-ph osph oram idite-th ym idin e h ad to be prepared. Th ere-
fore com poun d 6 was con verted in to its correspon din g ph osph oram idite 9
in 85% yield, purified on a sh ort silica gel colum n un der argon pressure an d
subjected to in verse ODN syn th esis of th e DNA 8m er with 3′-CE-term in us.
Polymerase Incorporation of 3′-O-(2-Cyanoethyl)thymidine-5′-O-triphosphate
Triph osph ate 8 was foun d to be efficien tly in corporated in to DNA sub-
strate, dem on stratin g good term in atin g properties. Figure 1 illustrates ac-
ceptan ce of 8 by a previously selected polym erase1b. Th e DNA prim er
com peten t for m ultiple in corporation s of dTTP was exten ded on ly by a sin -
gle residue durin g polym erization reaction em ployin g triph osph ate 8 in a
tim e-course m ode (lan es 2–4). In parallel, th e resultin g products were ch al-
len ged by addition of dTTP an d th e reaction s were allowed to proceed fur-
th er (lan es 5–7). Th e product of prim er exten sion by 8 (m arked as +1) was
foun d to be resistan t to furth er exten sion (see arrows in th e righ t part of
Fig. 1), wh ile th e un reacted prim er was exten ded furth er (top part of Fig. 1,
lan es 5–7). Th is observation suggests th at th e in corporated m odified
n ucleotide serves as an efficien t polym erization term in ator.
FIG. 1
En zym atic in corporation of 3′-O-(2-cyan oeth yl)th ym idin e-5′-O-triph osph ate in to DNA sub-
strate an d its term in atin g properties
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520
Cleavage Experiments
Keller, Serva, Knapp, Kwiatkowski, Engels:
Th e syn th esized m on oph osph ates 7 an d 10 were used as referen ce m aterials
for th e followin g cleavage experim en ts. Th e ch rom atogram of both th e
startin g m aterial (m odel com poun d) an d th e deprotected m on oph osph ate
are sh own in Fig. 2, wh ile th e com m ercially available ph osph ate 10 was
coin jected for calibration of th e m eth od (see ch rom atogram in th e back-
groun d of Fig. 2).
Figure 2 sh ows th at th e m odel com poun d after CE cleavage, wh ich is
assum ed to be 2′-deoxyth ym idin e-5′-ph osph ate, is well separable from th e
startin g m aterial 7 by th e RP-HPLC m eth od we h ave developed. Th e
deprotected n ucleotide elutes sign ifican tly (at 7.78 m in ) earlier from th e
colum n th an th e CE-protected n ucleotide (deprotection tim e 10.88 m in ).
Th is separation m eth od was also used for th e an alysis of th e cleavage exper-
im en t probes. All cleavage experim en ts don e with th e m odel com poun d
(except experim en t No. 2, Table I) were carried out un der dry con dition s in
argon atm osph ere. Each cleavage test was perform ed by on e stan dard pro-
cedure described in th e Experim en tal.
Th e results of th e first CE cleavage experim en ts usin g th e m odel com -
poun d 3′-O-(2-cyan oeth yl)-2′-deoxyth ym idin e-5′-ph osph ate are sh own in
Table I. At th e begin n in g, we tried to fin d an appropriate cleavin g agen t
efficien t en ough for quan titative CE rem oval (see Table I).
Th e tem perature for th e first four experim en ts listed in Table I was kept
con stan t at 60 °C. We assum e th e CE cleavage followin g a β-elim in ation
FIG. 2
RP-HPLC ch rom atogram of m odel com poun d 7 an d deprotected m on oph osph ate 10
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
521
TABLE I
Cyan oeth yl cleavage tests with variation of cleavin g agen t, reaction tem perature, solven ts
an d am oun t of cleavin g agen t
Reagen t, m ole %
solven t
Tem perature
°C
Cleavage tim e
m in
Experim en t
1
2
3
TEA 3 HF (10), aceton itrile
KOH (40), water
60
60
60
n o cleavage
ca. 30
dicycloh exan o-18-crown -6 (0.1),
potassium fluoride (2), THF
n o cleavage
4
5
1 M TBAF (40), THF
60
25
37
60
66
60
60
60
60
40
40
6
1 M TBAF (40), THF
>60
60
6
6
1 M TBAF (80), THF
7
1 M TBAF (40), THF
8
1 M TBAF (80), THF
3
9
1 M TBAF (5), THF
>20
10
1.5
1
10
11
12
13
14
1 M TBAF (20), THF
1 M TBAF (40), THF/DMF 1:1
1 M TBAF (40), THF/DMSO 1:1
1 M TBAF (40), THF/DMF 1:1
1 M TBAF (40), THF/DMSO 1:1
12
3
FIG. 3
Stacked RP-HPLC ch rom atogram s illustratin g th e OH-in duced 3′-O-CE cleavage of m on o-
ph osph ate 7
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522
Keller, Serva, Knapp, Kwiatkowski, Engels:
m ech an ism ; so on e m igh t con sider rem oval of th e CE group by sm all bases
like h ydroxy groups. Experim en t No. 2 afforded th e assum ed results: h ere,
20 m g of th e m odel com poun d was dissolved in water an d treated with
2 m l of 1 M aqueous KOH at 60 °C. In con trast to th e oth er th ree experi-
m en ts, a h om ogen eous solution was form ed due to com plete dilution of
th e n ucleotide in water. Th e sam ples taken out every m in ute were
quen ch ed in 0.5 M aqueous acetic acid by n eutralization , filtered th rough
syrin ge-filter an d subjected to an alytical RP-HPLC with autosam pler (th e
sam e con dition s as described for th e stan dard procedure). Th e stacked
ch rom atogram s of experim en t No. 2 are sh own in Fig. 3, illustratin g th e
tim e-depen den t CE cleavage. Th e peaks on th e righ t-h an d side in th e
stacked ch rom atogram s (Fig. 3) decrease due to con sum ption of m odel
com poun d 7 (reten tion tim e 10.88 m in ), th e peaks on th e left-h an d side
in crease due to th e form ation of 2′-deoxyth ym idin e-5′-ph osph ate 10 as th e
m ain product (reten tion tim e 7.78 m in ).
Th e depen den ce of th e CE rem oval on alkalin e con dition s was also con -
firm ed by experim en t No. 1 in Table I. Th e reagen t trieth ylam m on ium
trih ydrofluoride in THF is sligh tly acidic (pH 5), wh ich preven ts th e CE re-
m oval. Figure 4 sh ows th e stacked RP-HPLC ch rom atogram s from experi-
m en t No. 1. Not even after 75 m in reaction tim e at 60 °C, cleavage was
observed, on ly th e peak with th e reten tion tim e 10.88 m in rem ain ed.
Experim en t No. 3 (Table I) was a sin gle attem pt to rem ove th e CE fun ction
with potassium fluoride an d dicycloh exan o-18-crown -6 eth er in THF as
FIG. 4
Stacked RP-HPLC ch rom atogram s of cyan oeth yl cleavage experim en t un der acid con dition s
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
523
ph ase tran sfer catalyst. No cleavage was observed due to th e in solubility of
both th e n ucleotide an d fluoride reagen t in THF, wh ich m ean s th at th e re-
action m ixture rem ain ed h eterogen eous th rough th e wh ole experim en t
with out activation by th e crown eth er.
Fin ally we tried to use th e 1 M TBAF/THF stan dard solution as cleavin g
agen t. At 60 °C an d with a forty-fold excess of TBAF (pH 13), th e startin g
m aterial was quan titatively deprotected with in 6 m in . Th erefore, we in ves-
tigated furth er optim ization of CE cleavage with 1 M TBAF/THF solution as
cleavin g agen t.
In th e secon d stage of cleavage experim en ts, both reaction tem perature
an d th e am oun t of cleavin g agen t were varied (see Table I). Th e n ext four
experim en ts (Nos 5, 6, 7 an d 8 in Table I) sh owed clearly a tem perature-
depen den t reaction rate. Con siderin g experim en ts Nos 1 an d 3, deprotect-
ion of th e m odel com poun d 7 occurred ten tim es faster at 60 °C th an at
25 °C, alth ough 25 °C (or 37 °C) as reaction tem perature was preferable due
to biological com patibility. At first sigh t, experim en t No. 8 sh ows th e best
an d fastest CE cleavage with in 3 m in . Un fortun ately, THF boils at 66 °C,
wh ich m akes th is reaction tem perature in applicable to DNA-ch ip arrays.
In th e th ird stage, we varied th e am oun t of cleavin g agen t keepin g th e re-
action tem perature con stan t (see experim en ts Nos 7, 9 an d 10 in Table I).
Regardin g th e m odel com poun d, a m in im um of 40 equiv. TBAF are n eeded
for quan titative cleavage. Lowerin g th e am oun t of fluoride from 40 to
5 equiv., th e cleavage tim e was sign ifican tly prolon ged.
For en h an cin g th e cleavage rate by lowerin g th e reaction tem perature,
we refin ed our cleavage m odel by em ployin g co-solven ts. Th e con dition s
for th e CE cleavage of th e m odel com poun d with co-solven ts are listed in
Table I (experim en ts Nos 11, 12, 13 an d 14). As we prefer th e reaction tem -
perature 40 °C, we optim ized th e cleavage rate by addition of DMF or
DMSO in th e ratio DMSO/THF or DMF/THF of 1:1 (v/v). By com parin g th e
co-solven ts regardin g th e solubility en h an cem en t of th e m odel com poun d,
DMSO sh owed better results. By treatin g 3′-O-(2-cyan oeth yl)-2′-deoxy-
th ym idin e-5′-ph osph ate with 40 equiv. TBAF in DMSO/THF 1:1 (experi-
m en t No. 14) at 40 °C, th e cleavage was four tim es faster th an in DMF/THF
1:1 (experim en t No. 13). In creasin g th e tem perature from 40 to 60 °C
im proved th e cleavage rate, as seen from experim en ts Nos 11 an d 12. Th e
stacked ch rom atogram s of both experim en ts are sh own in Figs 5 an d 6.
Th e ch rom atogram s in Fig. 5 correspon d to experim en t No. 13, th e on es
in Fig. 6 to experim en t No. 14.
In both Figs 5 an d 6 th e ch rom atogram in th e fron t sh ows th e con sum p-
tion of m odel com poun d 7 after 1 m in , th e ch rom atogram in th e back-
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 4, pp. 515–534

524
Keller, Serva, Knapp, Kwiatkowski, Engels:
groun d sh ows th e con sum ption after 3 m in . In experim en t No. 14,
m on oph osph ate 7 is com pletely deprotected after 3 m in . In con trast, th e
CE cleavage was n ot com plete after 3 m in in case of experim en t No. 13.
FIG. 5
Stacked RP-HPLC ch rom atogram s for Experim en t No. 13 (THF/DMF 1:1)
FIG. 6
Stacked RP-HPLC ch rom atogram s for Experim en t No. 14 (THF/DMSO 1:1)
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
525
Cyanoethyl Cleavage on an Oligomer
For th e evaluation of th e CE cleavage on an oligodeoxyn ucleotide (ODN),
we selected th e sequen ce of th e term in al region of th e tem plate oligom er
con sistin g of 8 n ucleotides. First we syn th esized th e correspon din g DNA
8m er with out CE fun ction as a referen ce oligom er. After purification on
an ion exch an ge HPLC an d desaltin g, th e ODN could be obtain ed in h igh
purity as it is sh own in th e MALDI-MS spectrum below (Fig. 7).
Th e m odified DNA 8m er con sistin g of th e sam e sequen ce like th e
un m odified ODN was th en syn th esized bearin g th e CE fun ction on th e 3′-
term in us. Th e resultin g oligom er was purified in th e sam e m an n er like
th e un m odified referen ce m aterial givin g th e pure DNA 8m er ready for
th e cleavage experim en t (Fig. 8).
Th e m odified DNA 8m er was treated with a h igh excess of TBAF in
THF/DMF 2:3 (v/v) at 45 °C for 15 m in (see Experim en tal for details).
Figure 9 sh ows th e resultin g MALDI-MS spectrum after purification an d de-
saltin g. Th e m ass of th e obtain ed peak is iden tical with th e on e of th e un -
m odified referen ce ODN wh ich proves th e efficien t CE cleavage em ployin g
TBAF in THF/DMF.
FIG. 7
MALDI-MS spectrum of th e un m odified DNA 8m er
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526
Keller, Serva, Knapp, Kwiatkowski, Engels:
FIG. 8
MALDI-MS spectrum of th e m odified DNA 8m er with 3′-CE-term in us
FIG. 9
MALDI-MS spectrum of th e m odified DNA 8m er after cyan oeth yl cleavage
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
527
DISCUSSION
TBAF was used by San eyosh i et al.9 as a deprotection agen t for 2′-O-CE-
oligon ucleotides in RNA syn th esis. It can also be used for rem oval of CE
groups in DNA n ucleotides like our m odel com poun d 3′-O-(2-cyan oeth yl)-
2′-deoxyth ym idin e-5′-ph osph ate. Th e CE fun ction is cleaved in an alkalin e
en viron m en t with stron g bases like fluoride; th e cleavage rate is sign ifi-
can tly im proved at elevated tem peratures, for exam ple at 60 °C. Furth er-
m ore th e cleavage efficien cy depen ds also on th e solven t an d TBAF
am oun t. As a result of deprotection tests, th e cleavage in th e m odel com -
poun d 7 is en h an ced by addition of polar solven ts like DMF or DMSO to
1 M TBAF/THF stan dard solution . Th ese cleavage con dition s were also
applied to a sh ort oligom er with 3′-CE-term in us leadin g to quan titative
rem oval of th e CE fun ction alth ough a sign ifican tly h igh er am oun t of
fluoride is n eeded th an for th e m on oph osph ate. We are n ow at th e stage
of applyin g th e optim ized CE cleavage con dition s to dye labeled 3′-O-CE-
blocked (oligo)n ucleotides th at are used for our SBS approach wh ich is
curren tly un der in vestigation .
EXPERIMENTAL
Biological Materials an d Meth ods
In corporation of 3′-O-(2-cyan oeth yl)th ym idin e-5′-O-triph osph ate 8 was perform ed in th e
presen ce of 1 µM polym erase, 50 µM 3′-O-CE-dTTP an d 10 n M DNA duplex substrate con sist-
in g of th e prim er radioactively labeled by 33P at 5′-term in us (as in dicated by asterisk):
5′-*TGCAGGCATGCAAGCTTGGCGTA-3′
an d
tem plate
oligon ucleotide:
5′-AAAAAAAAAAAATACGCCAAGCTTGCATGCCTGCA-3′. Polym erization was perform ed in
20 µl of th e reaction m ixture at 37 °C. After th e in dicated tim e (see th e legen d of Fig. 1), an
aliquot of th e reaction m ixture was supplem en ted with dTTP (up to 50 µM fin al con cen tra-
tion ) an d th e reaction was allowed to proceed for addition al 5 m in at th e sam e tem perature.
Th e reaction s were stopped by addin g an equal volum e of a STOP solution (Ferm en tas cata-
logue, item #K1711: CycleReader DNA sequen cin g kit) con tain in g EDTA, products resolved
on 15% 29:1 den aturin g (7 M urea) polyacrylam ide (PAA) gel run at 50 °C, dried on
Wh atm an paper sh eet an d autoradiograph ed usin g Fuji ph osph orim ager screen .
a
Oligon ucleotide Syn th esis
Th e 3′-ph osph oram idites for th e syn th esis (direction 3′ to 5′) of th e un m odified DNA
8m er were purch ased from Ph arm acia Biotech , th e 5′-ph osph oram idites for th e in verse syn -
th esis (direction 5′ to 3′) of th e m odified DNA 8m er from Glen Research . Th e oligo-
deoxyn ucleotides were syn th esized on Expedite Nucleic Acid Syn th esis System usin g 500 Å
CPG colum n s from Applied Biosystem s. Th e sequen ce of th e un m odified referen ce oligom er
is 3′-TATGCGGT-5′, th e sequen ce of th e m odified oligom er is 3′-CE-TATGCGGT-5′ wh ere
CE is cyan oeth yl. Th e deprotection test on th e oligom er was perform ed in a h eatin g block
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 4, pp. 515–534

528
Keller, Serva, Knapp, Kwiatkowski, Engels:
Th erm om ixer com fort from Eppen dorf. OD m easurem en ts were carried out with Hitach i
U-1100 Spectroph otom eter. Th e oligom ers were purified on an ion -exch an ge HPLC con sist-
in g of Dion ex DNA Pac
PA-100 colum n (9 × 250 m m ), Jasco in telligen t UV-vis detector
UV-970, Jasco in telligen t HPLC pum p PU-980, Jasco tern ary gradien t un it LG-980-02, Jasco
3-lin e degasser DG-980-50, an d Jasco in terface LC-NetII/ADC. Th e purified oligom ers were
desalted with PD-10 colum n s (prepacked with Seph adex
G-25 M) from GE Health care,
dried un der reduced pressure an d ch aracterized by MALDI-TOF m ass usin g 6-aza-2-th io-
th ym in e (ATT)/am m on ium citrate m atrix. Th e CE cleavage was perform ed as th e followin g:
Th e m odified 8m er (2.4 OD) was dissolved in 330 µl of DMF, th en treated with 230 µl
(7500 equiv.) of 1 M TBAF in THF an d in cubated with gen tle sh akin g at 45 °C for 15 m in .
Th e reaction was quen ch ed with 1 m l of water, th e m ixture was freeze-dried an d th e residue
purified on an ion -exch an ge HPLC. After desaltin g an d dryin g, th e residual ODN was ch arac-
terized by MALDI spectroscopy.
Ch em ical Syn th esis
NMR spectra (1H an d 13C) of n ucleosides were run in CDCl3 or DMSO-d6 on a Bruker AM 250
spectrom eter operatin g at 250.132 MHz. NMR spectra (1H, 13C, 31P an d two-dim en sion al
spectra) of th e m on oph osph ate, triph osph ate an d ph osph oram idite were run in D2O or
aceton e-d6 on a Bruker Avan ce 400 spectrom eter operatin g at 400.132 MHz (161.984 MHz
for coupled 31P NMR, m easured again st 85% ph osph oric acid as extern al stan dard). Ch em i-
cal sh ifts (δ) are given in ppm down field from tetram eth ylsilan e an d couplin g con stan ts (J)
in Hz. All spectra were m easured at 300 K. Peak pattern s of each sign al are ch aracterized
with th e followin g abbreviation s: s for sin glet, d for doublet, dd for double-doublet, t for
triplet, ψt for pseudo-triplet, q for quartet an d m for m ultiplet. Mass spectra were recorded
on a Fison s MALDI VG tim e-of-fligh t (Tofspec) m ass spectrom eter in positive or n egative
m ode usin g ATT m atrix or on a Fison s Electrospray (ES) VG Platform II m ass spectrom eter.
FPLC purification of th e triph osph ate was perform ed on a Ph arm acia ion -exch an ge FPLC
system con sistin g of LCC-500 Plus con troller, two P-500 pum ps an d on e sin gle-path UV
Mon itor UV-1. Th e colum n was packed with DEAE-Seph arose gel DEAE Seph adex A 25 from
Ph arm acia. Th e triph osph ate sam ple was loaded on colum n with peristaltic pum p P1, frac-
tion s were collected with a fraction collector RediFrac. Furth er purification of th e tri-
ph osph ate was perform ed on a sem ipreparative colum n LiCh roCart 250-10 RP-18e (5 µm )
from Merck on th e Merck-HPLC system con sistin g of Merck–Hitach i D-6000 in terface,
Merck–Hitach i L-4250 UV-vis-detector, Merck–Hitach i L-6220 in telligen t pum p, Sh im adzu
PF-535 fluorescen ce m on itor an d Merck–Hitach i LC-Organ izer. Th e aqueous fraction s con -
tain in g purified m on oph osph ate an d triph osph ate were lyoph ilized on a Ch rist alph a 2-4
freeze-dryer. HPLC purification of both m on oph osph ates 7 an d 10 after syn th esis was don e
on a preparative colum n Ph en om en ex Jupiter 4 µ Proteo 90 Å (250 × 15 m m ) (at 300 K in
Th erm otech n ic-products Jetstream plus colum n th erm ostat) on a Jasco RP-HPLC system con -
sistin g of Jasco in terface LC-NetII/ADC, Jasco in telligen t HPLC pum p PU-2080 Plus, Jasco in -
telligen t UV/Vis detector UV-2075 Plus, Jasco tern ary gradien t un it LG-2080-02, Jasco
in telligen t sam pler AS-950-10, an d Jasco 3-lin e degasser DG-2080-53. Th e crude m on o-
ph osph ates as well as th e sam ples from th e deprotection tests were filtered with syrin ge fil-
ters Spartan 13/0,45 RC (0.45 µm ) from Wh atm an . Elem en tal an alyses were perform ed on
a CHN-O-Rapid an alyzer from Foss–Heraeus. Th in layer ch rom atograph y was carried out on
Silica Gel 60 F254-coated alum in um sh eets from Merck. Th e separated com poun ds were visu-
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
529
alized by UV-absorption at λ = 254 n m (Cam ag UV lam p). Colum n ch rom atograph y was
perform ed at atm osph eric pressure usin g Silica Gel 60 from Roth , pH values were m easured
with a pH-m eter 761 Calim atic from Kn ick.
CE Cleavage Tests with th e Model Com poun d
Dry 3′-O-(2-cyan oeth yl)-2′-deoxyth ym idin e-5′-ph osph ate 7 (10 or 20 m g, depen din g on th e
solven t volum e) as sodium salt was suspen ded in water-free solven t (except experim en t
No. 2 in Table I) an d h eated up to th e desired reaction tem perature with stirrin g. After keep-
in g th e suspen sion at th at tem perature for a few m in utes, th e cleavin g reagen t was added in
on e portion . Sam ples of 100 or 200 µl each were taken out from th e reaction ch am ber after
selected tim e periods an d quen ch ed with 500 µl of dem in eralized water. Th e probes were
tran sferred after syrin ge filtration in to sm all vials an d subjected to an alytical RP-HPLC with
autosam pler. Both m on oph osph ates 7 an d 10 were ch aracterized by th eir reten tion tim es
after separation on an alytical colum n (Ph en om en ex Jupiter 4 m Proteo) usin g th e follow-
in g eluen ts: eluen t A – 1 M aqueous trieth ylam m on ium acetate (TEAA); eluen t B – water;
eluen t C – aceton itrile (ACN); lin ear gradien t 0–30% buffer C durin g 20 m in . Th ym idin e-
5′-m on oph osph ate disodium salt h ydrate was purch ased from Sigm a–Aldrich an d coin jected
as referen ce m aterial.
N3-Ben zoyl-2′-deoxyth ym idin e (2)
To a solution of 2′-deoxyth ym idin e 1 (6.06 g, 25 m m ol), wh ich was ren dered an h ydrous by
repeated coevaporation with dry pyridin e in 50 m l of absolute pyridin e, eth yldiiso-
propylam in e (21.8 m l, 125 m m ol) an d ch lorotrim eth ylsilan e (8 m l, 62.5 m m ol) were added.
Th e m ixture was stirred un der argon atm osph ere at room tem perature for ca. 30 m in un til
th e startin g m aterial was fully con sum ed. Th en ben zoyl ch loride (4.4 m l, 37.5 m m ol) was
added an d stirrin g con tin ued for an oth er h our. After con sum ption of th e startin g m aterial,
th e reaction m ixture was quen ch ed by addition of 20 g of KH2PO4 an d 100 m l of ice water.
After stirrin g th e m ixture for several m in utes, colorless crystals precipitated wh ich were col-
lected by filtration , wash ed with 200 m l of cold water an d dried un der reduced pressure.
Th e solid was dissolved in 200 m l of m eth ylen e ch loride/m eth an ol 1:1 an d treated with
3.5 m l of trifluoroacetic acid. Th e m ixture was stirred at room tem perature for 30 m in un til
all startin g m aterial was desilylated, th en tran sferred in to a separatory fun n el an d wash ed
on ce with 100 m l of aqueous 5% NaHCO3 solution . Th e aqueous layer was extracted th ree
tim es with 100 m l of m eth ylen e ch loride an d th e com bin ed organ ic layers were dried over
an h ydrous MgSO4. After filtration an d evaporation to dryn ess, th e crude product was puri-
fied by colum n ch rom atograph y usin g m eth ylen e ch loride/m eth an ol (5–10% m eth an ol) as
eluen t to give 7.2 g (83%) of th e pure crystallin e com poun d. 1H NMR (400 MHz, DMSO-d6):
7.97 (dd, 2 H, HA ben zoyl, J = 7.32, 8.42), 7.78 (t, 1 H, HC ben zoyl, J = 7.46), 7.60 (t, 2 H,
HB ben zoyl, JB,C = 7.63), 6.15 (ψt, 1 H, H-1′, J1′,2′ = 6.57), 5.26 (d, 1 H, 3′-OH, J3′,OH = 4.29),
5.11 (t, 1 H, 5′-OH, J5′,OH = 5.07), 4.29 (m , 1 H, H-3′), 3.81 (m , 1 H, H-4′), 3.62 (m , 2 H,
H-5′), 2.24–2.14 (m , 2 H, H-2′), 1.86 (s, 3 H, H-7). 13C NMR (100 MHz, DMSO-d6): 169.6
(C=O ben zoyl), 162.5 (C-4), 149 (C-2), 137.1 (C-6), 135.5 (CC ben zoyl), 131.1 (C-quart
ben zoyl), 130.3 (CA ben zoyl), 129.5 (CB ben zoyl), 109.3 (C-5), 87.6 (C-4′), 84.5 (C-1′), 70.2
(C-3′), 61.1 (C-5′), 39.6 (C-2′, overlapped with DMSO triplet), 12.2 (C-7). MS (ES), m/z: 346.9
(M + H)+.
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530
Keller, Serva, Knapp, Kwiatkowski, Engels:
N3-Ben zoyl-2′-deoxy-5′-O-(4-m eth oxytrityl)th ym idin e (3)
Dry n ucleoside 2 (3.46 g, 10 m m ol) was dissolved in 40 m l of absolute pyridin e un der argon
atm osph ere. 4-Meth oxytrityl ch loride (4.14 g, 13 m m ol) was added in on e portion an d th e
yellow solution was stirred at room tem perature overn igh t. Th e reaction was stopped by ad-
dition of 10 m l of m eth an ol an d stirred for an oth er 30 m in . Th e solven t was evaporated an d
th e yellow oily residue was diluted with 200 m l of m eth ylen e ch loride. Th e organ ic layer
was wash ed th ree tim es with 100 m l of water, dried over an h ydrous MgSO4 an d con cen -
trated. Th e crude product was purified by colum n ch rom atograph y with m eth ylen e ch loride/
m eth an ol (2–5%) to give 4.85 g (78%) of th e pure product as a pale yellow foam . 1H NMR
(400 MHz, CDCl3): 7.93 (m , 2 H, HA ben zoyl), 7.70 (s, 1 H, H6), 7.63 (t, 1 H, HC ben zoyl, J =
7.36), 7.48 (t, 2 H, HB ben zoyl, JB,C = 7.58), 7.44–7.27 (m , 12 H, MMT), 6.87 (d, 2 H, MMT,
J = 8.84), 6.38 (dd, 1 H, H-1′, J1′,2′ = 6.06, 7.33), 5.26 (br d, 1 H, 3′-OH), 4.59 (m , 1 H, H-3′),
4.04 (m , 1 H, H-4′), 3.81 (s, 3 H, MMT-OCH3), 3.45 (m , 2 H, H-5′), 2.39 (m , 2 H, H-2′), 1.43
(s, 3 H, H-7). 13C NMR (100 MHz, CDCl3): 169.2 (C=O ben zoyl), 163.0 (C-4), 159 (C-OCH3),
149.4 (C-2), 143.9 (C-quart MMT), 135.6 (C-6), 135.1 (CC ben zoyl), 134.9 (MMT), 131.1
(C-quart ben zoyl), 130.7 (CA ben zoyl), 130.5 (MMT), 129.3 (CB ben zoyl), 128.5 (MMT),
128.2 (MMT), 127.5 (MMT), 113.5 (MMT), 111.5 (C-5), 87.4 (C-4′), 86.3 (C-quart MMT),
85.1 (C-1′), 72.5 (C-3′), 63.7 (C-5′), 55.4 (OCH3), 41.3 (C-2′), 12 (C-7). MS (ES), m/z: 641.2
(M + H + Na)+. For C37H34N2O7 (618.24) calculated: 71.83% C, 5.54% H, 4.53% N; foun d:
71.81% C, 5.59% H, 4.55% N.
N3-Ben zoyl-3′-O-(2-cyan oeth yl)-2′-deoxy-5′-O-(4-m eth oxytrityl)th ym idin e (4)
In a well-dried Erlen m eyer flask with m agn etic stirrer, com poun d 3 (1.24 g, 2 m m ol) was
dissolved in fresh ly distilled acrylon itrile (2.7 m l, 40 m m ol) an d tert-butyl alcoh ol (5 m l). Af-
ter a few m in utes, cesium carbon ate (652 m g, 2 m m ol) was added an d th e pale yellow sus-
pen sion was vigorously stirred un der argon atm osph ere at room tem perature for about 2.5 h .
Th e in soluble m aterial was rem oved by filtration th rough Celite, th en th e filtrate was con -
cen trated an d purified on a silica gel colum n with m eth ylen e ch loride/m eth an ol (0–1%) to
give 1.23 g (1.84 m m ol, 92%) of th e product as a pale yellow foam . 1H NMR (400 MHz,
CDCl3): 7.93 (dd, 2 H, HA ben zoyl, J = 7.07, JA,B = 8.59), 7.71 (s, 1 H, H-6), 7.64 (t, 1 H,
HC ben zoyl, JB,C = 7.58), 7.49 (t, 2 H, HB ben zoyl, JB,C = 7.58), 7.43–7.26 (m , 12 H, MMT),
6.87 (d, 2 H, MMT, J = 8.84), 6.31 (dd, 1 H, H-1′, J1′,2′ = 5.81, 8.08), 4.24 (m , 1 H, H-3′), 4.15
(q, 1 H, H-4′, J3′,4′,5′ = 2.78), 3.81 (s, 3 H, MMT-OCH3), 3.63 (t, 2 H, H-8, J8,9 = 6.04),
3.54–3.33 (m , 2 H, H-5′), 2.55 (t, 2 H, C-9, J8,9 = 6.04), 2.51–2.28 (m , 2 H, H-2′), 1.51 (s, 3 H,
H-7). 13C NMR (100 MHz, CDCl3): 169.2 (C=O ben zoyl), 162.9 (C-4), 159 (C-OCH3), 149.4
(C-2), 143.8 (C-quart MMT), 135.4 (C-6), 135.1 (CC ben zoyl), 134.8 (MMT), 131.1 (C-quart
ben zoyl), 130.6 (CA ben zoyl), 130.5 (MMT), 129.8 (MMT), 129.3 (CB ben zoyl), 128.5 (MMT),
128.3 (MMT), 127.6 (MMT), 117.5 (C-10), 113.5 (MMT), 111.4 (C-5), 87.5 (C-1′), 85.1 (C-4′),
84.1 (C-quart MMT), 80.5 (C-3′), 64.1 (C-8), 63.7 (C-5′), 55.4 (OCH3), 37.9 (C-2′), 19.1 (C-9), 12 (C-7).
MALDI(+)-MS, m/z: 694.8 (M + Na + H)+. For C40H37N3O7 (671.26) calculated: 71.52% C,
5.55% H, 6.26% N; foun d: 71.24% C, 5.54% H, 6.35% N.
N3-Ben zoyl-3′-O-(2-cyan oeth yl)-2′-deoxyth ym idin e (5)
Com poun d 4 (671 m g, 1 m m ol) was dissolved in a solution of 10% p-toluen esulfon ic acid in
m eth ylen e ch loride/eth an ol 1:1. Th e reaction m ixture was stirred at room tem perature for
Collect. Czech. Chem. Commun. 2009, Vol. 74, No. 4, pp. 515–534

Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
531
2 h un til all th e startin g m aterial was con sum ed. Subsequen tly 100 m l of aqueous saturated
NaHCO3 solution was added an d th e organ ic layer separated. Th e aqueous layer was ex-
tracted th ree tim es with 100 m l of m eth ylen e ch loride, th e com bin ed organ ic fraction s were
dried over an h ydrous MgSO4 an d con cen trated. Th e resultin g colorless oil was purified by
colum n ch rom atograph y with m eth ylen e ch loride/m eth an ol 95:5 (v/v) to give 399 m g
(1 m m ol, 100%) of th e pure product. 1H NMR (250 MHz, DMSO-d6): 8.0–7.56 (m , 6 H, H-6,
H-ben zoyl), 6.11 (t, 1 H, H-1′), 5.20 (t, 1 H, 3′-OH), 4.19 (m , 1 H, H-3′), 3.97 (m , 1 H, H-4′),
3.64 (t, 2 H, H-8, J8,9 = 6.04), 3.63 (m , 2 H, H-5′), 2.78 (t, 2 H, H-9, J8,9 = 6.04), 2.35–2.28
(m , 2 H, H-2′), 1.87 (s, 3 H, H-7). 13C NMR (250 MHz, DMSO-d6): 169.6 (C=O ben zoyl),
162.5 (C-4), 149.0 (C-2), 137.0 (C-6), 135.5 (CC ben zoyl), 131.1 (C-quart ben zoyl), 130.4
(CA ben zoyl), 129.5 (CB ben zoyl), 119.2 (C-10), 109.5 (C-5), 84.9 (C-1′), 84.5 (C-4′), 70.2
(C-3′), 63.5 (C-8), 61.4 (C-5′), 36.5 (C-2′), 18.3 (C-9), 12.2 (C-7). MS (ES), m/z: 399.9 (M +
H)+. For C20H21N3O6 (399.14) calculated: 60.14% C, 5.30% H, 10.52% N; foun d: 60.39% C,
5.47% H, 10.66% N.
3′-O-(2-Cyan oeth yl)-2′-deoxyth ym idin e (6)
Nucleoside 5 (300 m g, 0.75 m m ol) was dissolved in 10 m l of m eth an ol an d cooled down to
0 °C. NH4OH (3 m l, 27%) was added dropwise an d th e m ixture was stirred at 0 °C for
30 m in , th en at room tem perature for 1.5 h . Afterwards th e reaction m ixture was con cen -
trated an d purified on a silica gel colum n with m eth ylen e ch loride/m eth an ol 95:5 (v/v) to
give 212 m g (0.71 m m ol, 96%) of th e product as colorless crystals. 1H NMR (250 MHz,
DMSO-d6): 11.31 (br s, 1 H, H-3), 7.68 (s, 1 H, H-6), 6.12 (ψt, 1 H, H-1′), 5.12 (m , 1 H,
5′-OH, J5′,OH = 5.38), 4.14 (m , 1 H, H-3′), 3.92 (m , 1 H, H-4′), 3.63 (t, 2 H, H-8, J8,9 = 6.04),
3.58 (m , 2 H, H-5′), 2.78 (t, 2 H, H-9, J8,9 = 6.04), 2.28–2.09 (m , 2 H, H-2′), 1.77 (s, 3 H,
H-7). 13C NMR 250 MHz, DMSO-d6): 163.7 (C-4), 150.5 (C-2), 136.0 (C-6), 119.2 (C-10),
109.6 (C-5), 84.5 (C-1′), 83.7 (C-4′), 79.4 (C-3′), 63.5 (C-8), 61.4 (C-5′), 36.2 (C-2′), 18.3
(C-9), 12.3 (C-7). MS (ES), m/z: 318.0 (M + Na + H)+. For C13H17N3O5 (295.12) calculated:
52.88% C, 5.80% H, 14.23% N; foun d: 53.07% C, 5.91% H, 14.23% N.
3′-O-(2-Cyan oeth yl)-2′-deoxyth ym idin e-5′-ph osph ate (7)
Com poun d 6 (385 m g, 1.3 m m ol) was dissolved in 10 m l of dry trim eth yl ph osph ate an d
cooled down to 0 °C. Th e solution was treated with 1,8-bis(dim eth ylam in o)n aph th alen e
(418 m g, 1.95 m m ol) an d, after stirrin g for 5 m in , ph osph orus oxych loride (0.16 m l,
1.7 m m ol) was added dropwise on coolin g. Th e pale yellow clear solution was stirred at 0 °C
for 3 h , subsequen tly quen ch ed by addition of 20 m l of cold trieth ylam m on ium h ydrogen -
carbon ate buffer (pH 7) an d stirred for 1 h . Th e aqueous layer was extracted five tim es with
100 m l of tert-butyl m eth yl eth er for rem oval of th e proton spon ge an d trim eth yl ph os-
ph ate. Th e aqueous layer was lyoph ilized an d purified by RP-HPLC with 0.1 M trieth yl-
am m on iun acetate buffer/aceton itrile as eluen t. Th e crude product was obtain ed as colorless
glassy oil wh ich was an alyzed by NMR an d m ass spectrom etry th en . For easier h an dlin g an d
storage of th e product, it was precipitated as a sodium salt. Th e resultin g 250 m g
(0.66 m m ol) of m on oph osph ate was dried by freeze-dryin g over th ree days. Th e n ucleotide
was dissolved un der argon atm osph ere at 4 °C in 6.6 m l of absolute m eth an ol to give a
water-free m eth an olic 0.1 M m on oph osph ate solution . A fresh ly prepared an h ydrous sodium
perch lorate solution (m ade from 1.21 g (9.9 m m ol) of sodium perch lorate in 33 m l of abso-
lute aceton e) was added dropwise at 4 °C an d th e sodium salt of th e n ucleoside precipitated
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532
Keller, Serva, Knapp, Kwiatkowski, Engels:
as colorless crystals. Th e crystals were cen trifuged (4 °C, 5 m in , 12 000 rpm ), th e supern atan t
was rem oved an d th e solid dried un der reduced pressure yieldin g 267 m g (52%) of th e prod-
uct as a Na+-salt. 1H NMR (400 MHz, D2O): 7.69 (s, 1 H, H-6), 6.22 (ψt, 1 H, H-1′), 4.31 (m ,
1 H, H-3′), 4.22 (m , 1 H, H-4′), 3.95 (m , 2 H, H-5′), 3.72 (t, 2 H, H-8, J8,9 = 6.04), 2.71 (t,
2 H, H-9, J8,9 = 6.04), 2.39–2.23 (m , 2 H, H-2′), 1.81 (s, 3 H, H-7). 13C NMR (100 MHz, D2O):
166.3 (C-4), 151.5 (C-2), 137.2 (C-6), 119.6 (C-10), 111.6 (C-5), 85.0 (C-1′), 83.5 (C-4′), 80.0
(C-3′), 64.9 (C-8), 63.7 (C-5′), 36.2 (C-2′), 18.3 (C-9), 11.5 (C-7). 31P NMR (161.98 MHz,
D2O): 2.11 (s, P-α). MS (ES), m/z: 374.0 (M – H)–.
3′-O-(2-Cyan oeth yl)-2′-deoxyth ym idin e-5′-triph osph ate (8)
Com poun d 7 (55 m g, 0.19 m m ol) was coevaporated twice with dry pyridin e (3 m l) an d
dried un der vacuum overn igh t. Th e flask was filled with argon an d closed with a septum .
Th e followin g steps were carried out un der sligh tly positive argon pressure. Th e n ucleoside
was dissolved in absolute pyridin e (190 µl) an d an h ydrous dioxan e (570 µl) an d th en treated
with 190 µl (0.19 m m ol) of a fresh ly prepared 1 M solution of 2-ch loro-4H-1,3,2-ben zodioxa-
ph osph orin an -4-on e in an h ydrous dioxan e. After 10 m in , a m ixture of 570 µl (0.28 m m ol)
of 0.5 M solution of bis(tributylam m on ium ) dih ydrogen diph osph ate in an h ydrous DMF
an d 190 µl (0.80 m m ol, 4.3 equiv.) of tributylam in e was quickly added. Th e reaction m ix-
ture was stirred for 10 m in an d subsequen tly treated with 3.8 m l of a 1% iodin e solution in
pyridin e/water 98:2 (v/v). After stirrin g at room tem perature for 15 m in , 5% aqueous
NaHSO3 solution was added un til th e brown reaction m ixture turn ed ligh t yellow due to
iodin e reduction . Th e solution was con cen trated un der reduced pressure, th e residue dis-
solved again in 20 m l of water an d left at room tem perature for 30 m in . Th e m ixture was
treated with 38 m l of con cen trated am m on ia an d stirred at room tem perature for 60 m in .
Th e solven t was evaporated, th e solid (330 m g) dissolved th en in ca. 12 m l of water, filtered
th rough a 0.2 µm syrin ge filter an d purified in four portion s on a DEAE-Seph arose fast-flow
colum n (1.6 cm diam eter, 15 cm len gth ) at 4 °C (flow rate 4 m l/m in , gradien t 0–500 m l
100% buffer A in 0.05 M TEAB, 500–650 m l 50% buffer A an d 50% buffer B in 1 M TEAB).
Th e product eluted with 0.17 M trieth ylam m on ium h ydrogen carbon ate (TEAB) buffer; 65 m g
of th e obtain ed product was furth er purified by sem ipreparative HPLC. HPLC-purified
28.3 m g of triph osph ate (12%) were isolated after both purification steps. 1H NMR (300 MHz,
D2O): 7.77 (d, 1 H, H-6), 6.34 (dd, 1 H, H-1′, J1′,2′ = 5.8), 4.50 (d, 1 H, H-3′), 4.36 (s, 1 H,
H-4′), 4.21 (m , 2 H, 2 H-5′), 3.85 (t, 2 H, 2 H-8, J8,9 = 6.0), 2.82 (t, 2 H, H-9, J8,9 = 6.0),
2.49–2.35 (m , 2 H, H-2′), 1.93 (d, 3 H, H-7). 31P NMR (161.98 MHz, D2O): –10.9 (s, P-γ),
–11.7 (d, P-α), –23.2 (s, P-β). MALDI(–)-MS, m/z: 533.95 (M – 2 H)–.
3′-O-(2-Cyan oeth yl)-5′-[(2-cyan oeth yl)-(N,N-diisopropyl)]-2′-deoxyph osph oram idite-
th ym idin e (9)
Dried com poun d 6 (236 m g, 0.8 m m ol) was dissolved un der argon in 8 m l of absolute
dich lorom eth an e an d treated with (2-cyan oeth yl)-di(N,N-diisopropyl)ph osph in e (381 µl,
1.2 m m ol) an d 4,5-dicyan oim idazole (110 m g, 0.92 m m ol). Th e m ixture was stirred at room
tem perature for 4 h , th en diluted with 20 m l of dich lorom eth an e an d wash ed on ce with
25 m l of saturated NaHCO3 solution . Th e organ ic layer was separated, dried over Na2SO4,
filtered an d con cen trated. Th e crude product was purified on a sh ort silica gel colum n un der
argon pressure. Th e pure product was obtain ed in 337 m g (85%) yield as an isom eric m ix-
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Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate
533
ture an d stored at –20 °C un der argon . 1H NMR (400 MHz, aceton e-d6): 9.88 (br s, 2 H, NH),
7.69 (s, 1 H, H-6), 7.56 (s, 1 H, H-6), 6.31 (dd, 1 H, H-1′, J1′,2′ = 5.83, 8.38), 6.25 (dd, 1 H,
H-1′, J1′,2′ = 5.60, 8.60), 4.36 (m , 1 H, H-3′), 4.32 (m , 1 H, H-3′), 4.21 (m , 2 H, H-4′), 4.0–3.63
(m , 10 H, H-5′, H-isopropyl ph osph in e, ph osph in e-O-CH2), 3.91 (t, 2 H, 2 H-8, J8,9 = 6.10),
3.81 (t, 2 H, 2 H-8, J8,9 = 6.10), 2.80 (t, 2 H, CH2-CN ph osph in e, J1,2 = 5.81), 2.77 (t, 2 H,
H-9, J8,9 = 6.06), 2.41–2.26 (m , 4 H, H-2′), 1.87 (s, 3 H, H-7), 1.86 (s, 3 H, H-7), 1.28–0.99
(m , 24 H, H-isopropyl ph osph in e). 13C NMR (100 MHz, aceton e-d6): 163.2 (C-4), 150.2 (C-2),
135.4 (C-6), 135.2 (C-6), 118.2 (CN, ph osph in e), 118 (C-10), 110 (C-5), 84.6 (C-1′), 84.3
(C-1′), 83.5 (C-4′), 80.1 (C-3′), 63.8 (C-8), 63.2 (C-8), 61.4 (C-5′), 58.4 (O-CH2, ph osph in e),
42.7 (C-isopropyl, ph osph in e), 36.6 (C-2′), 23.9 (C-isopropyl, ph osph in e), 19.8 (CH2, ph os-
ph in e), 18.0 (C-9), 11.5 (C-7). 31P NMR (75 MHz, aceton e-d6): 148.8 (s, P-α), 148.2 (s, P-α).
ESI(–)-MS, m/z: 494.4 (M – H)–.
2′-Deoxyth ym idin e-5′-ph osph ate (10)
2′-Deoxyth ym idin e 1 (315 m g, 1.3 m m ol) was dissolved in 10 m l of absolute trim eth yl
ph osph ate un der argon atm osph ere an d cooled down to –4 °C. At th at tem perature, ph os-
ph orus oxych loride (0.25 m l, 2.6 m m ol) was added dropwise an d th e reaction m ixture was
stirred at –4 °C for 6 h . Th e reaction was stopped by addition of 20 m l of TEAB buffer (pH 7)
an d stirrin g con tin ued for 30 m in . Th e aqueous layer was extracted five tim es with 100 m l
of tert-butyl m eth yl eth er, lyoph ilized an d purified on RP-HPLC with 0.05
M trieth yl-
am m on ium acetate buffer/aceton itrile as eluen t. Th e crude product was obtain ed as a color-
less glassy oil wh ich could be an alyzed by NMR an d m ass spectrom etry. For facile h an dlin g
an d storage of th e product, it was precipitated as a sodium salt. Th e resultin g 190 m g
(0.59 m m ol) of m on oph osph ate was dried by freeze-dryin g over th ree days. Th e n ucleotide
was dissolved un der argon atm osph ere at 4 °C in 5.9 m l of absolute m eth an ol to give a
water-free m eth an olic 0.1 M m on oph osph ate solution . A fresh ly prepared an h ydrous sodium
perch lorate solution (1.08 g (8.8 m m ol) sodium perch lorate in 29.5 m l of absolute aceton e)
was added dropwise at 4 °C an d th e sodium salt of th e n ucleotide was precipitated. Th e crys-
tals were cen trifuged (5 m in , 4 °C, 12 000 rpm ), th e supern atan t was rem oved an d th e color-
less solid was lyoph ilized to give 203 m g (45%) of th e crystallin e product. 1H NMR (400 MHz,
D2O): 7.58 (s, 1 H, H-6), 6.15 (ψt, 1 H, H-1′), 4.37 (m , 1 H, H-3′), 3.97 (m , 1 H, H-4′), 3.87
(m , 2 H, H-5′), 2.16 (m , 2 H, H-2′), 1.71 (s, 3H, H-7). 13C NMR (100 MHz, D2O): 166.4 (C-4),
151.5 (C-2), 137.3 (C-6), 111.5 (C-5), 85.4 (C-1′), 84.9 (C-4′), 71.0 (C-3′), 64.6 (C-5′), 38.6
(C-2′), 11.5 (C-7). 31P NMR (161.98 MHz, D2O): 0.66 (s, P-α). MALDI(–)-MS, m/z: 373.8 (M –
2 H – Na)–.
This synthetic and biochemical work was financially supported by the EU FP6 CRAFT project
“Array SBS”. The authors are indebted to the staff of the Institute of Organic Chemistry and Chemical
Biology, Hannelore Brill and Ilona Priess for mass spectra measurement, Marianne Christof for
elemental analysis and Dr. J. G. Zimmermann for measurement and interpretation of the 31P NMR
spectra.
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534
Keller, Serva, Knapp, Kwiatkowski, Engels:
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