1068-1620/01/2706- $25.00 © 2001

MAIK “Nauka

/Interperiodica”0413

Russian Journal of Bioorganic Chemistry, Vol. 27, No. 6, 2001, pp. 413–416. Translated from Bioorganicheskaya Khimiya, Vol. 27, No. 6, 2001, pp. 462–465.Original Russian Text Copyright © 2001 by Malakhov, Korshun, Berlin.

1

Oligodeoxynucleotides containing covalentlyattached fluorescent dyes are widely used in nucleicacid research [1, 2].

2

Thus, fluorescently labeled oligo-nucleotides are employed in the automated sequencingof DNA as well as DNA probes and primers in PCR.They are also used in the study of nucleic acids bymethods based on the alteration of emission of theinteracting fluorophores [3–5]. In this connection, top-ical is the problem of obtaining fluorescent dyes withpredetermined spectral properties and developingmethods of their introduction into oligonucleotides. Forintroducing fluorescent labels, it is most convenient touse standard methods of the automated phosphoramid-ite synthesis of oligonucleotides.

Among promising dyes with interesting fluorescentproperties are some aromatic heterocyclic compounds,for example, benzoxazole derivatives. Such compoundspossess high quantum yields of fluorescence [6, 7] andare used in particular as optic bleachers, organic scintil-lators, and laser dyes [8–10].

Based on 2-[4-phenylethynyl)phenylbenzoxazole(

II

), we developed the synthesis of a novel derivative ofthis fluorophore (

III

) as well as of the solid support (

V

)containing this heterocycle (scheme).

The initial substances in the synthesis were 2-[4-phenylethynyl)phenylbenzoxazole (

II

) [11] and 4-(4-

1

To whom correspondence should be addressed; e-mail:yuber@ibch.ru.

2

Abbreviations: DMAP, 4,4-dimethylaminopyridine; DMTr, 4,4'-dimethoxytrityl; LCAA-CPG, long-chain aminoalkylated con-trolled pore glass; prefix “d” (deoxy) in the oligonucleotide struc-tures is omitted.

iodophenyl)butanediol-1,3 (

I

) [12]. Their couplingunder the Heck–Sonogashira reaction conditions [13](a palladium–copper catalyst in the presence of anorganic base) afforded 4-[4-(2-benzoxazolyl)phenyl-ethynyl)phenylbutanediol-1,3 (

III

) (yield 91%), char-acterized by the

1

H NMR spectrum [

(Py-

d

5

;

δ

): 8.21

(d,2 H,

J

8

Hz,

Ar

H

), 7.80–

7.77 (m, 1 H,

Ar

H

), 7.66

(d, 2H,

J

8

Hz,

Ar

H

), 7.55–7.51

(m, 3 H,

Ar

H

), 7.33

(d, 2 H,

J

8

Hz,

Ar

H

), 7.30–7.25

(m, 2 H,

Ar

H

), 4

.38 (quint, 1H,

J

6.5

Hz,

CH

2

C

H

(OH)CH

2

), 4

.16–4,03 (m, 2 H,

CH

2

C

H

2

OH), 2

.97–2,87 (m, 2 H,

ArC

H

2

CH(OH)),1.96

(quart, 2 H,

J

6

.5 Hz,

CH(OH)C

H

2

CH

2

(OH)

] andmass spectrum [MALDI–TOF,

(

m

/

z

)

+

: found

384.3(

M

+ H)

; calculated

383.45 (C

25

H

21

NO

3

)

]. This com-pound contains a primary and a secondary hydroxylgroup that are connected by a three-carbon chain andmimic the corresponding 5' and 3' hydroxyls of the nat-ural nucleosides. The introduction of the 4,4'-dimethoxytrityl group at the primary hydroxyl gave themonofunctional alcohol (

IV

), which was reacted undertreatment with

N

,

N

'-diisopropylcarbodiimide with the

N

-succinylated polymer support LCAA-CPG [14, 15].The resulting

p

-(2-benzoxazolyl)tolane-containingsupport (

V

) had the anchor fluorophore content of27

µ

mol/g.

Support (

V

) was employed in standard conditions ofoligonucleotide synthesis to obtain a series of benzox-azolyltolane-labeled oligonucleotide, including thosecontaining, as a second fluorophore, 1-phenylethy-nylpyrene derivative (

VI

), which was introduced usingthe corresponding phosphoramidite reagent (

VII

) [11].The oligonucleotide conjugates synthesized were iso-

LETTERS TO THE EDITOR

Synthesis and Fluorescent Characteristicsof Oligodeoxynucleotides Containing a Novel Fluorescent Label,

p

-(2-Benzoxazolyl)tolane

A. D. Malakhov, V. A. Korshun, and Yu. A. Berlin

1

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,ul. Miklukho-Maklaya 16/10, GSP Moscow, 117997 Russia

Received June 9, 2001; in final form, June 26, 2001

Abstract

—A functional (dihydroxybutyl) derivative of

p

-(2-benzoxazolyl)tolane, a fluorescent label novel forbiopolymers, was synthesized. The functionalized solid support obtained on its basis was employed in the syn-thesis of oligodeoxynucleotides 3-terminally labeled with benzoxazolyltolane (these oligonucleotides also con-tained 1-phenylethynylpyrene residues). This fluorophore within its dihydroxybutyl derivative and the oligonu-cleotides modified with it display an intensive fluorescence characterized by a high Stokes shift. The oligonu-cleotides labeled with this fluorophore are potential probes sensitive to the bioplymer microenvironment.

Key words: modified nucleosides, modified oligodeoxynucleotides, p-(2-benzoxazolyl)tolane, 1-phenylethy-nylpyrene

414

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

Vol. 27

No. 6

2001

MALAKHOV

et al

.

lated by sequential 20% denaturing PAGE and reverse-phase HPLC and characterized by the MALDI–TOFmass spectra (for some of the conjugates the data aregiven in the table). As expected, the modification of thestandard oligonucleotides by the introduction of hydro-phobic substituents considerably increased the reten-tion time values of the conjugates at reverse-phasechromatography.

The resulting conjugates, like the initial butanediolderivatives of the dyes (

III

) and (

VI

), exhibit an inten-sive fluorescence; the spectra, while essentially not dif-fering in shape from those of the initial pseudonucleo-sides, are slightly (approximately by 10 nm) red-shifted(Figs. 1a, 1b).

An interesting feature of the fluorescence spectra ofthe conjugates bearing the

p

-(2-benzoxazolyl)tolane

fluorophore is their large Stokes shift (about 55 nm).This seems to be associated with the fact that this fluo-rophore, harboring electronegative atoms, must possessa considerable dipole moment and, consequently,strongly interact with the polar solvent. Usually, in theelectron-excited states the dipole moment of aromaticcompounds is larger than in the ground state. Therefore,when a fluorophore absorbs a photon, its dipole exertsa perturbing effect on the fluorophore’s environmentand changes. The excitation is followed by the reorga-nization of dipoles of the solvent molecules surround-ing the luminophore, which leads to a long-wave shiftof fluorescence. Thus, this fluorophore can in principlebe used as an indicator of polarity of the sites withinbiopolymers with which this fluorophore is in contact.

We studied the possibility of the nonradiative fluo-rescence energy transfer from

p

-(2-benzox-

HO

OH

I

RO

OH

ON

DMTrO

O

ON

O

NH

O

LCAA-CPG

R1O

OR2

NO

(II) N C N

NHCOCH2CH2CO2H

LCAA-CPG

(III): R = H

(IV): R = DMTrDMTr-Cl/Py

, DMAP

(

I

)

(

V

)

(

VI

): R

1

= R

2

= H;(

VII

): R

1

= DMTr,R

2

= P(OCH

2

CH

2

CN)NPr

2

i

Scheme.

Sequence and characteristics of the conjugates synthesized

Structure (5' 3')* Retention time, min**

Mol. mass

found(MALDI-TOF)*** calculated

TGT-ATT-ATT-TTA-TTT-TTT-TAT-CC

X

25.1 7415.2 (

M

+ Li) 7409.92

TGT-ATT-ATT-TTA-TTT-

Y

TT-TAT-CC

X

29.1 7564.8 (

M

+ Li) 7559.17

TGT-ATT-AT

Y

-TTA-TTT-TTT-TAT-CC

X

27.8 7566.7 (

M

+ Li) 7559.17

*

X

,

p

-(2-benzoxazolyl)tolane residue;

Y

, 1-phenylethynylpyrene residue. ** HPLC was performed on a C-18 reversed-phase SOTA column (4

×

250 mm); linear gradient (5 50%) of MeCN in 0.1 MNH

4

OAc within 60 min; a Beckman 165 instrument (United States).*** Voyager-DE BioSpectrometry Workstation (PerSeptive Biosystems; time-of-flight mass spectrometer with matrix-assisted laser

desorption/ionization (MALDI-TOF).

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

SYNTHESIS AND FLUORESCENT CHARACTERISTICS 415

azolyl)tolane onto 1-phenylethynylpyrene. In the exci-tation spectra of oligonucleotides containing thesedyes, along with emission at 460 nm, which corre-sponds to the fluorescence maximum of phenylethy-nylpyrene, there is a distinct maximum at 346 nm, char-acteristic of p-(2-benzoxazolyl)tolane (Fig. 1c). Thisindicates that phenylethynylpyrene can be excitedthrough a nonradiative energy transfer from p-(2-ben-zoxalyl)tolane. At the same time, because of the largeStokes shift of p-(2-benzoxazolyl)tolane, the fluores-cence spectra of these two fluorophores strongly over-lap. Indeed, upon excitation of the system with a lightwhose wavelength corresponds to the p-(2-benzoxa-zolyl)tolane absorption maximum (λex 335 nm), partialexcitation of phenylethynylpyrene is also observed.Thus, the maximum characteristic of p-(2-benzox-azolyl)tolane is smoothed and a wide maximumappears, characteristic of phenylethynylpyrene(Fig. 1d). It was also found (our unpublished data) thatthe presence of two p-(2-benzoxazolyl)tolane residuesat the neighboring units within an oligonucleotide (sim-ilarly to the analogous disposition of two phenylethy-nylpyrene residues [16]) can lead to the excimer forma-

tion. This property of the fluorophore described can beused in constructing sensitive DNA probes.

ACKNOWLEDGMENTS

We are grateful to I.I. Mikhalev and I.V. Grechishni-kova for measuring fluorescence spectra. This workwas supported by the Russian Foundation for BasicResearch, projects nos. 00-03-3271, 01-03-06124, and00-15-97947 (support of leading scientific schools). 1HNMR spectra were measured at the NMR SpectrometryComplex supported by the Russian Ministry of Scienceand Technology (registration number 96-03-08).

REFERENCES

1. Lakowicz, J.P., Principles of Fluorescence Spectroscopy,New York: Kluwer, Academic/Plenum, 1999.

2. Haugland, R.P., Handbook of Fluorescent Probes andResearch Chemicals, Eugene, OR: Molecular Probes,1996.

3. Masuko, M., Ohuchi, S., Sode, K., Ohtani, H., and Shi-madzu, A., Nucleic Acids Res., 2000, vol. 28, p. 34.

1 2

2

3

3 4

4

300 350 400 450 500

120

80

40

0

(‡) (b)

(c) (d)

nm

300 350 400 450 500

1 2500

250

0

1

2

300 350 4000

2

100

50

1 2

400

1

2

2

360 440 480

300

200

100

0nm

Relative intensity

Relative intensity

Fig. 1. Normalized spectra of the fluorescent pseudonucleosides and labeled oligonucleotides: (a) excitation spectra of (1) p-(2-ben-zoxazolyl)tolane diol (III) (λem 400 nm) and (2) 1-phenylethynylpyrene diol (VI) (λem 440 nm), fluorescence spectra of (3) diol

(III) (λex 335 nm) and (4) diol (VI) (λex 320 nm) in MeOH (c 10–6 M); (b) (1) excitation spectrum (registration by emission at λex400 nm) and (2) fluorescence spectrum (excitation at λex 335 nm) of oligonucleotide (5')TGT-ATT-ATT-TTA-TTT-TTT-TAT-CCX

(X stands for p-(2-benzoxazolyl)tolane residue); oligonucleotide concentration (here and henceforth) 5 × 10–7 M in K-phosphatebuffer, pH 7.0; (c) excitation spectra (registration by emission at λex 460 nm) of (1) oligonucleotides (5')TGA-TTT-TAT-TTY-TTT-ATC-CX and (2) (5')ACG-AGG-YAA-GCG-TAA (Y stands for phenylethynylpyrene residue); (d) fluorescence spectra of a mixtureof modified oligonucleotides (5')TGT-ATT-ATT-TTA-TTT-TTT-TAT-CCX and (5')ATT-ATT-TTY-TTT-TTT-TAT (1) at λex 335and (2) 375 nm.

416

RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 27 No. 6 2001

MALAKHOV et al.

4. Kandimalla, E.R. and Agrawal, S., Bioorg. Med. Chem.Lett., 2000, vol. 8, pp. 1911–1916.

5. Zhang, P., Beck, T., and Tan, W., Angew. Chem., Int. Ed.,2001, vol. 40, pp. 402–405.

6. Pushkina, L.N. and Tkachev, V.V., Zh. Prikl. Spektrosk.,1964, vol. 1, pp. 275–279.

7. Reiser, A., Leyshon, L.J., Saunders, D., Mijovic, M.V.,Bright, A., and Bogie, J., J. Am. Chem. Soc., 1972,vol. 94, pp. 2414–2421.

8. Bazyl’, O.K., Gruzinskii, V.V., Danilova, V.I., Kopy-lova, T.N., and Maier, G.V., Opt. Spektrosk., 1980,vol. 48, pp. 262–266.

9. Borisevich, N.A., Gorelenko, A.Ya., Kazak, N.S.,Kalosha, I.I., Morgun, Yu.F., Agashkov, A.V., Tol-kachev, V.A., and Tugbaev, V.A., Zh. Prikl. Spektrosk.,1980, vol. 32, pp. 357–359.

10. Vasil’eva, O.A., Gorelenko, A.Ya., Davydov, S.V.,Kalosha, I.I., and Tolkachev, V.A., Zh. Prikl. Spektrosk.,1987, vol. 46, pp. 642–645.

11. Korshun, V.A., Prokhorenko, I.A., Gontarev, S.V., Sko-robogatyi, M.V., Balakin, K.V., Manasova, E.V., Mala-

khov, A.D., and Berlin, Yu.A., Nucleosides Nucleotides,1997, vol. 16, pp. 1461–1464.

12. Malakhova, E.V., Malakhov, A.D., Kuznitsova, S.V.,Varnavskii, O.P., Kadutskii, A.P., Kozhich, D.T., Kor-shun, V.A., and Berlin, Yu.A., Bioor. Khim., 1998,vol. 24, pp. 688–695.

13. Sonogashira, K., Tohda, Y., and Hagihara, N., Tetrahe-dron Lett., 1975, pp. 4467–4470.

14. Damha, M.J., Giannaris, P.A., and Zabarylo, S.V.,Nucleic Acids Res., 1990, vol. 18, pp. 3813–3821.

15. Caruthers, M.H., Barone, A.D., Beaucage, S.L.,Dodds, D.R., Fisher, E.F., McBride, L.J., Matteucci, M.,Stabinsky, Z., and Tang, J.-Y., Meth. Enzymol., 1987,vol. 154, pp. 287–313.

16. Malakhov, A.D., Synthesis and Spectral Properties ofFluorescent Phenylethynylpyrene and Bis(phenylethy-nyl)anthracene Pseudonucleosides and OligonucleotidesLabeled with Them), Cand. Sci. (Chem.) Dissertation,Moscow: Shemyakin–Ovchinnikov Inst. of BioorganicChemistry, Russian Academy of Sciences, 2000.