Synthesis of novel coumarin-based fluorescent probes

Article abstract of DOI:10.1135/cccc20070996

We report on synthesis of new fluorescent probes suitable for site-specific incorporation into oligonucleotides. Coumarin derivatives were used as sensitive fluorescent labels and were attached to glycerol unit by two types of linkers as potential building blocks for oligonucleotide synthesis. Spectral characteristics of the functionalized coumarin building blocks were measured.

Full text of DOI:10.1135/cccc20070996

996
Kosiova, Kois:
SYNTHESIS OF NOVEL COUMARIN-BASED FLUORESCENT PROBES
1,
2
Ivana KOSIOVA * and Pavol KOIS
Department of Organic Chemistry, Faculty of Natural Sciences, Comenius University,
Mlynska dolina, Pavilon CH2, SK-84215 Bratislava, Slovak Republic;
1
2
e-mail: kosiova@fns.uniba.sk, kois@fns.uniba.sk
Received March 5, 2007
Accepted Jun e 6, 2007
Dedicated to Professor Štefan Toma on the occasion of his 70th birthday.
We report on syn th esis of n ew fluorescen t probes suitable for site-specific in corporation in to
oligon ucleotides. Coum arin derivatives were used as sen sitive fluorescen t labels an d were
attach ed to glycerol un it by two types of lin kers as poten tial buildin g blocks for oligo-
n ucleotide syn th esis. Spectral ch aracteristics of th e fun ction alized coum arin buildin g blocks
were m easured.
Keyw ord s: Coum arin ; Fluorescen t labelin g; Oligon ucleotides; Fluorescen t spectroscopy;
Click ch em istry; Triazoles; Azides.
Oligodeoxyn ucleotides bearin g reporter groups, e.g. fluorescen t labels, are
useful tools in m olecular biology, m edicin e an d diagn ostics1–10. Various
n ucleosidic as well as n on -n ucleosidic11–22 fluorescen t labeled ph osph or-
am idites h ave been successfully in corporated in to oligom ers in to an y pre-
determ in ed position of a n ucleic acid ch ain .
We report on syn th esis of n ew fluorescen t probes suitable for site-specific
in corporation in to oligon ucleotides. Coum arin s are often used as fluores-
cen t labels for m olecular studies of n ucleic acids an d protein s23–26. For h igh
quan tum yields we used coum arin derivatives 1–5 as sen sitive labels.
7-Hydroxycoum arin derivatives 4 an d 5 are com m ercially available as fluo-
rescen t labels an d coum arin -4-acetic acids 1–3 were syn th esized in our
laboratory by th e Pech m an reaction in th e fram e of n ew coum arin studies.
For furth er application s, fun ction alization of basic coum arin skeleton was
n ecessary. Two lin kers were attach ed to coum arin derivatives. 3-(3-Am in o-
propoxy)propan e-1,2-diol (6) we prepared via a publish ed procedure12 an d
3-azidopropan e-1,2-diol un it (7) was prepared via m eth od com m on in
azidon ucleoside syn th esis27.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 8, pp. 996–1004
© 2007 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20070996

Novel Coumarin-Based Fluorescent Probes
997
We attach ed 3-(3-am in opropoxy)propan e-1,2-diol un it on coum arin -
4-acetic acids 1–3 usin g a m eth od com m on in peptide syn th esis28 (Sch em es
1 an d 2). Th e reaction of coum arin -4-acetic acid with N-h ydroxysuccin -
im ide (NHS) in th e presen ce of dicycloh exylcarbodiim ide (DCC) gave NHS
ester, wh ich was coupled with th e lin ker to give protected product in th e
57–75% after ch rom atograph y. Th e yields were lowered by len gh ty separa-
tion of products from con tam in atin g dicycloh exylurea (DCU). Hydrolysis
of isopropyliden e groups with Dowex WX8 (H+) gave th e desired products
8–10 in 93–96% yields.
SCHEME 1
(i) 1.0 m olar equiv. NHS, 2.0 m olar equiv. DCC, dry dioxan e; (ii) Dowex WX8 (H+), aqueous
m eth an ol
SCHEME 2
(i) 1.0 m olar eq u iv. NHS, 2.0 m olar eq u iv. DCC, d ry d ioxan e; (ii) 1.0 m olar eq u iv. 6; (iii)
Dowex WX8 (H+), aqueous m eth an ol
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998
Kosiova, Kois:
Copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition was applied to
lin kin g protected 3-azidopropan e-1,2-diol (7) to th e coum arin derivatives
with term in al alkyn e fun ction ality 11 an d 12, wh ich we prepared by th e re-
action of coum arin 4 an d 5 with propargyl brom ide29 (Sch em e 3). Huisgen
1,3-dipolar cycloaddition is on e of th e ideal ch em oselective reaction s,
wh ere two un saturated reactan ts fuse togeth er in m ild con dition s, gen erat-
in g n o byproduct30–39. Th e reaction of azides with term in al alkyn es is
regioselective on ly in th e presen ce of copper(I) ion s. A n um ber of copper(I)
sources can be used32,35, m ost often Cu(I) prepared by in situ reduction of
CuSO4·5H2O. As a catalyst we used 0.15 m olar equivalen t of CuSO4·5H2O
an d 0.30 m olar equivalen t of sodium ascorbate in aqueous tert-BuOH an d
1,4-substituted triazole bridged products were obtain ed. Th e con version of
all reaction s was alm ost quan titative an d m in or byproduct 7-h ydroxy-
coum arin (4) an d 7-h ydroxy-4-m eth ylcoum arin (5) were form ed alon g with
desired products. Th e isolated yields of protected products after ch rom ato-
graph y were in th e ran ge 88–90%. Hydrolysis of isopropyliden e group with
Dowex WX8 (H+) gave th e desired products in yield 93% for com poun d 13
an d 96% for com poun d 14.
SCHEME 3
(i) 1.0 m olar equiv. K2CO3, 1.0 m olar equiv. propargyl brom ide, dry aceton e; (ii) 0.15 m olar
equiv. CuSO4·5H2O, 0.30 m olar equiv. sodium ascorbate, tert-BuOH–H2O = 1:1 (v/v); (iii)
Dowex WX8 (H+), aqueous m eth an ol
Spectral ch aracteristics of isolated products are sum m arized in Table I. All
n ewly prepared derivatives display on ly on e peak in th e fluorescen ce spec-
trum in m eth an ol. Th e absorption an d fluorescen ce m axim a of our com -
poun ds are sim ilar to m axim a of coum arin s 1–5. Th e fluorescen ce in ten sity
of th e com poun ds depen ds on th e site of coum arin m odification . We ob-
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 8, pp. 996–1004

Novel Coumarin-Based Fluorescent Probes
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served on ly sligh t ch an ges of fluorescen ce in ten sity (Fig. 1) an d quan tum
yields in th e series of derivatives of coum arin -4-acetic acids (8–10). On th e
con trary, th e m odification of th e coum arin C7-h ydroxy groups (13 an d 14)
caused decreasin g of in ten sity (Fig. 2) an d quan tum yields in com parison
with th at of correspon din g un m odified coum arin s 4 an d 5.
TABLE I
Total yields an d spectral ch aracteristics of fluorescen t probes 8–10, 13 an d 14
ε
Com pda
Yield, %
Φb
λabs, n m
λem s, n m
cm –1 l m ol–1
1
8
–
73
–
326
326
12487
11883
3032
392
396
418
426
417
417
392
387
387
381
0.21
0.24
0.40
0.30
0.10
0.10
0.08
0.02
0.15
0.05
2
323
9
75
–
323
11291
7444
3
318/349
319/350
325
10
4
58
–
8195
14509
11301
15552
20338
13
5
80
–
320
322
14
78
319
a
b
c = 10–4 m ol l–1 in m eth an ol. An th racen e as a stan dard (Φ = 0.27 in eth an ol), excitation
wavelen gth : 322 n m .
FIG. 1
Fluorescen t in ten sities of 5,7-dim eth oxycoum arin -4-acetic acid (2) an d its derivative 9,
fun ction alization at carboxyl group causes sligh t ch an ges in fluorescen ce; ᭺ 2, ᭢ 9
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Kosiova, Kois:
FIG. 2
Fluorescen t in ten sities of 7-h ydroxy-4-m eth ylcoum arin (5) an d its derivative 14,
fun ction alization at h ydroxyl group causes large decreasin g of fluorescen ce; ٗ 5, ★ 14
CONCLUSION
Novel coum arin -based fluorescen t probes were prepared by th e reaction of
coum arin -4-acetic acid esters an d 7-(propargyloxy)coum arin s with appro-
priate lin ker. Th e products were obtain ed in good yields an d th eir spectral
ch aracteristics were determ in ed. Work is in progress to exten d th is flexible
approach to th e preparation of differen t types of labeled oligon ucleotides.
EXPERIMENTAL
Solven ts were distilled an d dried by th e establish ed m eth ods40. Meltin g poin ts were m ea-
sured on a Koffler h ot stage an d are un corrected. NMR spectra were recorded in CDCl3 an d
DMSO-d6 with a Varian VX Un ity spectrom eter (300 MHz for 1H an d 75 MHz for 13C).
Ch em ical sh ifts are referen ced to Me4Si (1H) or to th e residual solven t sign al (13C) an d are
given in ppm (δ-scale), couplin g con stan ts (J) are given in Hz. All m easurem en ts were run at
room tem perature. Th e 1H an d 13C assign m en ts were based on 1H-1H COSY, 13C-1H HSQC
an d 13C-1H HMBC experim en ts. Elem en tal an alysis was perform ed on an an alyzer m odel
1106 (Carlo Erba Strum en tacion e). In frared spectra were recorded on a Bruker IFS 55 Equi-
n ox FTIR in KBr, waven um bers are given in cm –1. Absorption spectra were recorded usin g a
UV-VIS spectroph otom eter Agilen 8453, cuvette len gth 1 cm . Fluorescen ce spectra were re-
corded usin g Hitach i F-2000. TLC were perform ed on precoated plates of silica gel 60 F254
(Merck) with ch loroform –m eth an ol (9:1) as eluen t. Th e crude products were purified by
colum n ch rom atograph y on silica gel with ch loroform –m eth an ol (9:1) as eluen t.
7-Hydroxy-2H-chrom en-2-one (7-hydroxycoum arin) and 7-hydroxy-4-m ethyl-2H-chrom en-2-one
(7-h ydroxy-4-m eth ylcoum arin ) were obtain ed from Aldrich , 2-(2-oxo-2H-ch rom en -4-yl)acetic
acids were prepared as described in th e literature41
.
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Gen eral Procedure A
To a solution of 2-(2-oxo-2H-ch rom en -4-yl)acetic acid 1–3 an d NHS (1.0 m olar equiv.) in dry
dioxan e, DCC (2.0 m olar equiv.) in dry dioxan e was added. Th e resultan t m ixture was
stirred at room tem perature for 4 h . Th en lin ker 6 (1.0 m olar equiv.) was added an d th e
m ixture was stirred at room tem perature for 5 h . Th e DCU byproduct was filtered off, th e
solven t was rem oved un der reduced pressure an d th e crude product was purified by colum n
ch rom atograph y.
Th e protected products were dissolved in aqueous m eth an ol an d Dowex WX8 (H+) was
added. Th e reaction m ixture was stirred at room tem perature un til startin g m aterial was con -
sum ed (TLC). Dowex WX8 (H+) was filtered off, th e solven t was rem oved un der reduced
pressure an d th e crude product was purified by crystallisation from eth yl acetate–h exan e
m ixture.
N-[3-(2,3-Dihydroxypropoxy)propyl]-2-(7-hydroxy-2-oxo-2H-chromen-4-yl)acetamide (8). Wh ite
solid product (yield 280 m g, 0.8 m m ol, 93%), RF 0.1, m .p. 123–130 °C. For C17H21NO7
(351.4) calculated: 58.11% C, 6.02% H, 3.99% N; foun d: 58.32% C, 6.12% H, 4.06% N.
1H NMR (DMSO-d6): 10.59 s, 1 H (HO-7); 8.18 m , 1 H (NH-CO); 7.60 d, 1 H, J5,6 = 8.8 (H-5);
6.79 dd, 1 H, J5,6 = 8.8, J6,8 = 2.5 (H-6); 6.72 d, 1 H, J6,8 = 2.5 (H-8); 6.16 s, 1 H (H-3); 4.63 d,
1 H, JHO,15 = 4.9 (HO-15); 4.49 t, 1 H, JOH,16a = 4.9, JHO,16b = 4.7 (HO-16); 3.63 s, 2 H
(H-9); 3.55 m , 1 H (H-15); 3.35–3.30 m , 2 H (H-16); 3.30–3.21 m , 2 + 1 H (H-13, Ha-14);
3.08–3.19 m , 2 + 1 H (H-11, Hb-14); 1.62 m , 2 H (H-12). 13C NMR (DMSO-d6): 167.54 (10),
161.16 (2), 160.26 (7), 155.00 (4), 151.27 (8a) 126.69 (5), 112.88 (6), 111.49 (3), 111.50 (4a),
102.29 (8), 72.34 (14), 70.49 (15), 68.18 (13), 63.12 (16), 36.19 (11), 29.20 (12). IR (KBr):
3540, 3331, 3300, 3160, 3112, 1719, 1687, 1663, 1624, 1610, 1569, 1519, 1399, 1122, 1106,
1074, 1089, 1055.
N-[3-(2,3-Dihydroxypropoxy)propyl]-2-(5,7-dimethoxy-2-oxo-2H-chromen-4-yl)acetamide (9).
Wh ite solid product (yield 100 m g, 0.3 m m ol, 95%), RF 0.26, m .p. 72–75 °C. For C19H25NO8
(395.4) calculated: 57.71% C, 6.37% H, 3.54% N; foun d: 57.93% C, 6.28% H, 3.36% N.
1H NMR (DMSO-d6): 7.83 t, 1 H (NH-CO); 6.60 d, 1 H (H-5); 6.46 d, 1 H (H-7); 6.06 s, 1 H (H-3);
4.60 d, 1 H, JHO,15 = 4.9 (HO-15); 4.48 t, 1 H, JHO,16a = 5.8, JHO,16b = 5.6 (HO-16); 3.84 s, 3 H
(CH3-7); 3.79 s, 3 H (CH3-5); 3.69 s, 2 H (H-9); 3.58–3.49 m , 1 H (H-15); 3.41–3.20 m , 5 H
(H-13, H-14, H-16); 3.10 m , 2 H (H-11); 1.63 m , 2 H (H-12). 13C NMR (DMSO-d6): 168.38
(10), 162.52 (2), 159.96 (7), 158.21 (5), 156.20 (4), 150.98 (8a), 113.15 (3), 103.67 (4a),
95.28 (8), 93.51 (6), 72.22 (14), 70.35 (15), 63.01 (13), 62.25 (16), 55.81 (CH3-7), 42.83 (9),
35.93 (11), 29.35 (12). IR (KBr): 3417, 3290, 1729, 1700, 1645, 1616, 1607, 1560, 1550,
1496, 1386, 1119, 1068, 1043.
N-[3-(2,3-Dihydroxypropoxy)propyl]-2-(3-oxo-3H-benzo[f]chromen-1-yl)acetamide (10). Wh ite
solid product (yield 252 m g, 0.7 m m ol, 96%), RF 0.28, m .p. 144–148 °C. For C21H23NO6
(385.4) calculated: 65.44% C, 6.02% H, 3.63% N; found: 65.22% C, 6.18% H, 3.95% N. 1H NMR
(DMSO-d6): 8.43 d, 1 H, J10,9 = 8.2 (H-10); 8.25 d, 1 H, J9,10 = 9.1 (H-9); 8.07 d, 1 H, J8,7
=
7.4 (H-8); 7.68–7.57 m , 3 H (H-5, H-6, H-7); 6.58 s, 1 H (H-3); 4.60 d, 1 H, JHO,17 = 5.2
(HO-17); 4.48 t, 1 H, JHO,18a = 5.6, JHO,18b = 5.8 (HO-18); 4.18 s, 2 H (H-11); 3.58–3.49 m , 1 H
(H-17); 3.26–3.18 m , 4 H (H-15, H-16); 3.12 m , 2 H (H-13); 1.58 m , 2 H (H-14). 13C NMR
(DMSO-d6): 167.92 (12), 159.31 (2), 154.25 (4), 151.73 (10a), 133.86 (9), 130.89 (5a), 129.62
(8), 129.17 (8a), 127.91 (6), 125.54 (7), 124.74 (5), 118.19 (10), 117.57 (3), 113.92 (4a),
72.34 (16), 70.48 (17), 68.16 (15), 63.14 (18), 43.83 (11), 36.14 (13), 29.23 (14). IR (KBr):
3045, 3289, 1726, 1703, 1645, 1589, 1552, 1521, 1122, 1065, 1045.
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Gen eral Procedure B
To a solution of coum arin 4 or 5 in dry aceton e, an h ydrous potassium carbon ate (1.0 m olar
equiv.) an d propargyl brom ide (1.0 m olar equiv.) were added. Th e resultin g m ixture was
stirred at 50 °C for 18 h , th en th e m ixture was cooled an d th e solven t was rem oved un der
reduced pressure. Th e residue was treated with 15 m l of water an d extracted with eth yl ace-
tate. Th e com bin ed organ ic ph ases were wash ed with water, dried over an h ydrous sodium
sulfate an d evaporated in vacuum . Th e crude product was purified by crystalisation from an
eth yl acetate–h exan e m ixture.
7-(Prop-2-yn-1-yloxy)-2H-chromen-2-one (11). Ligh t yellow solid product (yield 586 m g,
2.93 mmol, 95%), m.p. 118–120 °C (lit. 119 °C). 1H NMR (DMSO-d6): 8.01 d, 1 H, J3-4 = 9.4 (H-4);
7.66 d, 1 H, J5-6 = 8.6 (H-5); 7.06 d, 1 H, J6-8 = 2.5 (H-8); 7.00 dd, 1 H, J5-6 = 8.6, J6-8 = 2.5
(H-6); 6.33 d, 1 H, J3-4 = 9.4 (H-3); 4.94 d, 2 H, J9-10 = 2.3 (H-9); 3.67 t, 1 H, J9-10 = 2.3
(H-10). 13C NMR (DMSO-d6): 160.19 (2), 160.18 (7), 155.13 (8a), 144.24 (4), 129.53 (5),
112.98 (3), 112.86 (4a), 112.83 (6), 101.78 (8), 78.94 (10), 78.52 (11), 56.11 (9).
4-Methyl-7-(prop-2-yn-1-yloxy)-2H-chromen-2-one (12). Ligh t yellow solid product (yield
565 m g, 2.64 m m ol, 93%), m .p. 130–134 °C (lit. 134 °C). 1H NMR (CDCl3): 7.51 dd, 1 H,
J4-5 = 7.5 (H-4); 6.93 d, 1 H, J5-6 = 2.6 (H-6); 6.91 dd, 1 H, J4-5 = 7.5, J5-6 = 2.6 (H-5); 6.15 d,
1 H, J3-Me = 1.2 (H-3); 4.75 d, 2 H, J9-10 = 2.3 (H-9); 2.56 t, 1 H, J9-10 = 2.5 (H-10); 2.39 d, 3 H,
J3-Me = 1.1 (CH3-4). 13C NMR (CDCl3): 161.34 (2), 160.36 (7), 155.08 (4), 152.40 (8a), 125.62
(5), 114.29 (4a), 112.75 (6), 112.47 (3), 102.17 (8), 76.50 (11), 56.17 (9), 18.70 (CH3-4).
Gen eral Procedure C
To a solution of com poun ds 11 or 12 in tert-BuOH–H2O 1:1 (v/v) CuSO4·5H2O (0.15 m olar
equiv.) an d sodium ascorbate (0.30 m olar equiv.) were added. Th e m ixture was stirred at
room tem perature for 15 m in . Th en lin ker 7 (1.0 m olar equiv.) was added an d th e resultin g
m ixture was stirred at room tem perature un til startin g m aterial 11 or 12 was con sum ed
(TLC). Th en th e reaction m ixture was wash ed with eth yl acetate, th e com bin ed organ ic
ph ases were wash ed with water, dried over an h ydrous sodium sulfate an d evaporated in
vacuum . Th e crude product was purified by colum n ch rom atograph y an d crystallized from
an eth yl acetate–h exan e m ixture.
Th e protected products were dissolved in aqueous m eth an ol an d Dowex WX8 (H+) was
added. Reaction m ixture was stirred at room tem perature un til th e startin g m aterial was
con sum ed (TLC). Dowex WX8 (H+) was filtered off, th e solven t was rem oved un der reduced
pressure an d th e crude product was purified by crystallisation from an eth yl acetate–h exan e
m ixture.
7-{[1-(2,3-Dihydroxypropyl)-1H-1,2,3-triazol-4-yl]methoxy}-2H-chromen-2-one (13). W h ite
solid product (yield 133 m g, 0.4 m m ol, 93%), RF 0.33, m .p. 123–125 °C. For C15H15N3O5
(317.3) calculated: 56.78% C, 4.76% H, 13.24% N; foun d: 56.52% C, 4.90% H, 13.01% N.
1H NMR (DMSO-d6): 8.19 s, 1 H (H-10); 8.01 d, 1 H, J4,3 = 9.6 (H-4); 7.66 d, 1 H, J5,6 = 8.8
(H-5); 7.17 d, 1 H, J8,6 = 2.5 (H-8); 7.05 dd, 1 H, J6,8 = 2.5, J6,5 = 8.5 (H-6); 6.32 d, 1 H, J3,4
9.3 (H-3); 5.26 s, 2 H (H-9); 5.15 d, 1 H, JHO,16 = 5.5 (HO-16); 4.86 t, 1 H, JHO,17a = 5.8,
=
JHO,17b = 5.5 (HO-17); 4.49 dd, 1 H, J15a,15b = 13.9, J16,15a = 3.4 (Ha-15); 4.26 dd, 1 H, J15a,16
=
8.1, J15a,15b = 13.9 (Hb-15); 3.82 m , 1 H (H-16); 3.42–3.30 m , 2 H (H-17). 13C NMR
(DMSO-d6): 161.19 (2), 160.29 (7), 155.32 (8a), 144.32 (4), 141.51 (10), 129.52 (5), 125.82
(14), 112.95 (3), 112.67 (6), 112.56 (4a), 101.53 (8), 70.41 (16), 63.27 (9), 61.67 (17), 52.91
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(15). IR (KBr): 3540, 3416, 3344, 3287, 1741, 1724, 1708, 1697, 1622, 1560, 1509, 1474,
1399, 1107, 1073.
7-{[1-(2,3-Dihydroxypropyl)-1H-1,2,3-triazol-4-yl]methoxy}-4-methyl-2H-chromen-2-one (14).
W h ite solid product (yield 130 m g, 0.4 m m ol, 96%), RF 0.33, m .p. 108–113 °C. For
C
16H17N3O5 (331.3) calculated: 58.00% C, 5.17% H, 12.68% N; foun d: 58.14% C, 5.32% H,
12.47% N. 1H NMR (DMSO-d6): 8.19 s, 1 H (H-14); 7.71 d, 1 H, J5,6 = 8.7 (H-5); 7.17 d, 1 H,
J6,8 = 2.5 (H-8); 7.00 dd, 1 H, J6,5 = 8.7, J6,8 = 2.5 (H-6); 6.22 s, 1 H (H-3); 5.26 s, 2 H (H-9);
5.16 d, 1 H (HO-16); 4.86 t, 1 H (HO-17); 4.52 dd, 1 H, J15a,15b = 13.9, J16,15a = 3.4 (Ha-15);
4.25 dd, 1 H, J15a,16 = 8.1, J15a,15b = 13.9 (Hb-15); 3.80 m , 1 H (H-16); 3.61 m , 2 H (H-17);
2.40 s, 3 H (CH3-4). 13C NMR (DMSO-d6): 161.08 (2), 160.13 (7), 154.66 (8a), 1153.42 (4),
141.53 (10), 126.50 (5), 125.75 (14), 113.32 (3), 112.61 (6), 111.27 (4a), 101.55 (8), 70.38 (16),
63.25 (9), 63.02 (17), 58.86 (15), 18.14 (CH3). IR (KBr): 3447, 3415, 1730, 1713, 1688, 1614,
1562, 1511, 1466, 1393, 1098, 1074.
Support by the Slovak Grant Agency VEGA 1/3559/06, the Science and Technology Assistance
Agency under the contract No. APVV-51-046505 and the Slovak State Programme Project No.
2003SP200280203 is gratefully acknowledged.
REFERENCES
1. Markiewicz W. T., Groger G., Rosch R., Zebrowska A., Markiewicz M., Klotz M., Hinz M.,
Godzina P., Seliger H.: Nucleic Acids Res. 1997, 25, 3672.
2. La Clair J. J.: Angew. Chem., Int. Ed. 1998, 37, 325.
3. Paris P., Langenhan J., Kool E.: Nucleic Acids Res. 1998, 26, 3789.
4. Wojczewski C., Stolze K., Engels J. W.: Synlett 1999, 1667.
5. Okamura Y., Kondo S., Sase I., Suga T., Mise K., Furusawa I., Kawakami S., Watanabe Y.:
Nucleic Acids Res. 2000, 28, e107.
6. Kurata S., Kanagawa T., Yamada K., Torimura M., Yokomaku T., Kamagata Y., Kurane R.:
Nucleic Acids Res. 2001, 29, e34.
7. Molenaar C., Marras S. A., Slats J. C. M., Truffert J.-C., Lemaitre M., Raap A. K., Dirks R. W.,
Tanke H. J.: Nucleic Acids Res. 2001, 29, e89.
8. Yamane A.: Nucleic Acids Res. 2002, 30, e97.
9. Vercoutere W., Akeson M.: Curr. Opin. Chem. Biol. 2002, 6, 816.
10. Wells M.: Curr. Opin. Biotechnol. 2006, 17, 28.
11. Misiura K., Durrant I., Evans M., Gait M.: Nucleic Acids Res. 1990, 18, 4345.
12. Theisen P., McCollum C., Upadhya K., Jacobson K., Vu H. N., Andrus A.: Tetrahedron Lett.
1992, 33, 5033.
13. Reed M. W., Lukhtanov E. A., Gorn V. V., Lucas D. D., Zhou J. H., Pai S. B., Cheng Y.,
Meyer R. B., Jr.: J. Med. Chem. 1995, 38, 4587.
14. Prokhorenko I. A., Korshun V. A., Petrov A. A., Gontarev S. V., Berlin Y. A.: Bioorg. Med.
Chem. Lett. 1995, 5, 2081.
15. a) Neuner P.: Bioorg. Med. Chem. Lett. 1996, 6, 147; b) Fukui K., Iwane K., Shimidzu T.,
Tanaka K.: Tetrahedron Lett. 1996, 37, 4983.
16. Yamana K., Takei M., Nakano H.: Tetrahedron Lett. 1997, 38, 6051.
17. Su S.-H., Iyer R. S., Aggarwal S. K., Kalra K. L.: Bioorg. Med. Chem. Lett. 1997, 7, 1639.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 8, pp. 996–1004

1004
Kosiova, Kois:
18. Korshun V. A., Prokhorenko I. A., Gontarev S. V., Skorobogatyi M. V., Balakin K. V.,
Manasova E. V., Malakhov A. D., Berlin Y. A.: Nucleosides Nucleotides 1997, 16, 1461.
19. Korshun V. A., Balakin K. V., Proskurina T. S., Mikhalev II, Malakhov A. D., Berlin Y. A.:
Nucleosides Nucleotides 1999, 18, 2661.
20. Putnam W. C., Bashkin J. K.: Chem. Commun. 2000, 767.
21. Adamczyk M., Chan C. M., Fino J. R., Mattingly P. G.: J. Org. Chem. 2000, 65, 596.
22. Dioubankova N. N., Malakhov A. D., Stetsenko D. A., Korshun V. A., Gait M. J.: Org. Lett.
2002, 4, 4607.
23. The Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 10th Web ed.;
www.probes.invitrogen.com/handbook.
24. Brauns E. B., Madaras M. L., Coleman R. S., Murphy C. J., Berg M. A.: J. Am. Chem. Soc.
1999, 121, 11644.
25. Andreatta D., Sen S., Pérez Lustres J. L., Kovalenko S. A., Ernsting N. P., Murphy C. J.,
Coleman R. S., Berg M. A.: J. Am. Chem. Soc. 2006, 128, 6885.
26. Mitsui T., Nakano H., Yamana K.: Tetrahedron Lett. 2000, 41, 2605.
27. Ciuffreda P., Loseto A., Santaniello E.: Tetrahedron 2002, 58, 5767.
28. Masjost B., Ballmer P., Borroni E., Zurcher G., Winkler F. K., Jakob-Roetne R., Diederich F.:
Chem. Eur. J. 2000, 6, 971.
29. Luo F. T., Ko S. L., Liu L. J., Chen H.: Heterocycles 2000, 53, 2055.
30. Tornoe C. W., Christensen C., Meldal M.: J. Org. Chem. 2002, 67, 3057.
31. Rostovtsev V. V., Green L. G., Fokin V. V., Sharpless K. B.: Angew. Chem., Int. Ed. 2002,
41, 2596.
32. Rodionov V. O., Fokin V. V., Finn M. G.: Angew. Chem., Int. Ed. 2005, 44, 2210.
33. Perez-Balderas F., Ortega-Munoz M., Morales-Sanfrutos J., Hernandez-Mateo F.,
Calvo-Flores F. G., Calvo-Asin J. A., Isac-Garcia J., Santoyo-Gonzalez F.: Org. Lett. 2003,
5, 1951.
34. Chan T. R., Hilgraf R., Sharpless K. B., Fokin V. V.: Org. Lett. 2004, 6, 2853.
35. Meng J. C., Fokin V. V., Finn M. G.: Tetrahedron Lett. 2005, 46, 4543.
36. Kolb H. C., Finn M. G., Sharpless K. B.: Angew. Chem., Int. Ed. 2001, 40, 2004.
37. Kolb H. C., Sharpless K. B.: Drug Discovery Today 2003, 8, 1128.
38. Ramachary D. B., Barbas III, C. F.: Chem. Eur. J. 2004, 10, 5323.
39. Manetsch R., Krasinski A., Radic Z., Raushel J., Taylor P., Sharpless K. B., Kolb H. C.:
J. Am. Chem. Soc. 2004, 126, 12809.
40. Armarego W. L. F., Chai C.: Purification of Laboratory Chemicals, 5th ed. Butterwort-
Heinemann, an imprint of Elsevier Science, Amsterdam 2003.
41. Charitos C., Kokotos G., Tzougraki C.: J. Heterocycl. Chem. 2001, 38, 153.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 8, pp. 996–1004