Todor Deligeorgieva, Iliana Timchevab, Vera Maximovac, Jens-Peter Jacobsend, Karl-Heinz Drexhagee

aUniversity of Sofia, Faculty of Chemistry, 1126 Sofia, BulgariabInstitute of Organic Chemistry with Center of Phytochemistry, Bulgarian Academy of Sciences, 1113 Sofia, BulgariacInstitute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, BulgariadUniversity of Southern Denmark, Odense University, Department of Chemistry, Odense M, DK-5230 DenmarkeUniversity of Siegen, Institute of Physical Chemistry, D-57068 Siegen, Germany

The development in recent years of a number of homo- and heterodimeric asymmetric monomethine cyanine dyes as non-covalently binding nucleic acid fluorescent

labels has a substantial impact on nucleic acid research [1-10]. As a part of our investigations on novel and improved [8] homodimers as nucleic acid probes, we have

synthesized two new homodimeric asymmetric monomethine cyanine dyes N,N,N´,N´-tetramethyl-N,N´-bis{4-[4[(3-methyl-2(3H)-benzoselenazolylidene)methyl]-

quinolinium]prop-1-yl}-1,3-propanediammonium tetraiodide SOSO-1 and N,N,N´,N´-tetramethyl-N,N´-bis{4-[4[(3-(1-pyridiniopropyl)-2(3H)benzothiazolylidene)-

methyl]quinolinium]prop-1-yl}-1,3-propanediammonium hexaiodide TOTO-1-6C.

In this study, we present the synthetic pathway for the preparation of the novel dyes as well as their absorption and fluorescence spectral characteristics.

RESULTS AND DISCUSSION

The dye SOSO-1 5a was synthesized by reacting first 2,3-dimethyl-benzselenazolium methosulfate 1a with 1-(3-iodopropyl)-4-chloroquinol-inium iodide 2 in mildly alkaline media (Scheme 1).

Then N,N,N´,N´-tetramethyl-1,3-propanediamine 4 was bisquaternized with the resulting monomethine cyanine dye 3a (Scheme 2) giving 5a. The same reaction pathway was used for the preparation of the dye TOTO-1-6C 5b. 2-Methyl-3-[(3-pyridinio)propyl]benzothiazolium dibromide 1b [11] was reacted with 1-(3-iodopropyl)-4-chloroquinolinium iodide 2 giving the monomethine cyanine dye 3b (Scheme 1). The preparation of TOTO-1-6C 5b was carried out by bisquaternization of N,N,N´,N´-tetramethyl-1,3-propanediamine 4 with the monomeric monomethine cyanine dye 3b (Scheme 2). The result was a homodimeric asymmetric monomethine cyan-ine dye bearing six positive charges 5b.

The dyes TOTO-1-6C and SOSO-1 do not show fluorescence at the concen-trations used (3x10–6 M (5a) and 1.5x10–6 M (5b)), but become strongly flu-orescent upon excitation at 480 nm after binding to dsDNA (Table 1, Fig.1).

Table 1. Spectral characteristics of the investigated dyes.

Dye Without dsDNA With dsDNAa

abs(nm) (M-1.cm-1) abs(nm) fl(nm) Qf

SOSO-1 495, 520(sh) 110000, (77400) 496, 522 540.4 0.25

TOTO-1-6C 505.5 146700 505.5 534.4 0.35

The concentration of dsDNA is 3g/ml.

Figure 1. Absorption (curve 1) and fluorescence (curve 2) spectra of SOSO-1 in TE buffer in the presence of dsDNA.

Figure 2. Fluorescence spectra of TOTO-1-6C (curve A) and SOSO-1 (curve B) in the presence of dsDNA.

500 550 600 650 7000,0

0,2

0,4

0,6

0,8

1,0

B

A

fluo

resc

ence

inte

nsity

a.u

.

wavelength [nm]

The longest wavelength absorption maximum of SOSO-1 is at 495 nm with a shoul-der at 520 nm. The corresponding molar absorptivities are 110000 and 77400, respec-tively. For TOTO-1-6C this maximum is at 505.5 nm and the molar absorptivity is 146700. The fluorescence maximum of the complex SOSO-1/dsDNA is at 540.4 nm, while that of the TOTO-1-6C/dsDNA complex is at 534.4 nm (Figure 2). The fluores-cence quantum yields are 0.25 and 0.35, respectively. The detection limit of dsDNA is 100 ng/ml with SOSO-1 and 30 ng/ml with TOTO-1-6C. No dependence of the position of the fluorescence maximum on the type of nucleic acid is observed. The complexes are stable for at least 24 hours when kept in sunlight. The dyes are used for agarose gel electrophoresis staining. Clear gels (without fluorescence background) are obtained. The presence of DNA shows up as bright bands of green fluorescence. The dye/DNA complex remains stable during electrophoresis.

1. Haugland, R.P., (1996) Sixth edition, Handbook of fluorescent probes and research chemicals, Molecular Probes, Inc., Eugene, OR.

2. Yue, S., Haugland, R.P., (1995) Dimers of unsymmetrical cyanine dyes containig pyridinium moieties US 5 410 030

3. Yue, S., Johnson, I., R.P., Haugland, (1996) Dimers of unsymmetrical cyanine dyes US 5 582 9774. Rye, H.S, Yue, S., Wemmer, D.E., Quesada, M.A., Haugland, R.P., Maties, R.A., and Glazer,

A.N, (1992) Stable fluorescent complexes of double-stranded DNA with bis-intercalating asym-metric cyanine dyes: properties and applications, Nucleic Acids Res., 20(11), 2803-2811.

5. Benson, S.C., Singh, P., Glazer, A.N., (1993) “Heterodimeric DNA-binding dyes designed for energy transfer: synthesis and spectroscopic properties”, Nucleic Acids Res. 21, 5727-5735.

6. Benson, S.C., Mathies, R.A., Glazer, A.N., (1993) “Heterodimeric DNA-binding dyes designed for energy transfer: stability and applications of DNA complexes”, Nucleic Acids Res., 21, 5720-5726.

7. Benson, S.C., Zheng, Z., Glazer, A.N., (1995) “Fluorescence energy-transfer cyanine hetero-dimers with high affinity for double-stranded DNA. I. Synthesis and spectroscopic properties”, Anal. Biochem. 231, 247-255.

8. Deligeorgiev, T.G., Gadjev, N.I., Timcheva, I.I., Maximova, V.A., Katerinopoulos, H.E., Fukaraki, E., (2000) Synthesis of homodimeric monomethine cyanine dyes as noncovalent nucleic acid labels and their absorption and fluorescence spectral characteristics, Dyes & Pigm., 44(2) 131-136.

9. Timcheva, I., Maximova, Deligeorgiev, T., Gadjev, N., Drexhage, K.H., Petkova, I., (2000) Homodimeric monomethine cyanine dyes as fluorescent probes of biopolymers J. Photochem. Photobiol. B: Biology 58 (2-3) 130-135.

10. Bunkenborg, J., Gadjev, N., Deligeorgiev, T., Jacobsen, J. P., (2000) Concerted intercalation and minor groove recognition of DNA by a homodimeric Thiazole orange dye, Bioconjugate Chem., 11, 861-867.

11. Gadjev, N.I., Deligeorgiev, T.G., Kim, S.H, (1999) Preparation of monomethine cyanine dyes as noncovalent labels for nucleic acids, Dyes & Pigm., 40, 181-186.

The two synthesized novel dyes SOSO-1 and TOTO-1-6C are useful and sen-sitive fluorecent high affinity homodimeric probes for nucleic acid detection in solution and on agarose gel.

CONCLUSION

REFERENCES

Fluorescence Characteristics of Homodimeric Monomethine Cyanine Dyes

SOSO-1 and TOTO-1-6C in the Presence of Nucleic Acids

INTRODUCTION

N

X

R

N I

A

3a X = Se, R = CH3, A = I

3b X = S, R = , A = 2 IN

3 a, b

+ N NCH3

CH3

H3C

H3C2

N

X

R

N N N N

CH3CH3

CH3 CH3 X

N

RA

4

5 a, b

5 a SOSO-1 X = Se, R = CH3, A = 4I

5 b TOTO-1-6C X = S, R = , A = 6I N

SCHEME 2