2003 WILEY-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim (Germany)Supporting Information for ChemBioChem F 678

Pyrenemethyl ara-uridine-2’-carbamate: a strong interstrand excimer in themajor groove of DNA duplex

Natalia N. Dioubankova, Andrei D. Malakhov, Dmitry A. Stetsenko, Michael J. Gait, Pavel E.Volynsky, Roman G. Efremov, Vladimir A. Korshun*

[*] N.N. Dioubankova, Dr. P.E. Volynsky, Dr. Sc. R.G. Efremov, Dr. V.A. KorshunShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya16/10, 117997 Moscow (Russia)Fax: (+7) 095-3306738E-mail: korshun@mail.ibch.ruDr. A.D. Malakhov, Dr. D.A. Stetsenko, Dr. M.J. GaitMRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH (UK)

General Methods and MaterialsButylamine, 1-pyrenylmethylamine hydrochloride, triethylamine trihydrofluoride, triethylamine, were from

Aldrich; N,N-carbonyldiimidazole (CDI) was from Sigma, 4,4’-dimethoxytritylchloride (DmtCl) was fromAvocado; uracil-1-â-D-arabinofuranoside and bis(N,N-diisopropylamino)-2-cyanoethoxyphosphine were fromFluka; diisopropylammonium tetrazolide was prepared as described [M. H. Caruthers, A. D. Barone, S. L.Beaucage, D. R. Dodds, E. F. Fisher, L. J. McBride, M. Matteucci, Z. Stabinsky, J.-Y. Tang, Meth. Enzymol.1987, 154, 287–313].

DCM (Fisher) was always used freshly distilled over CaH2. Anhydrous pyridine was from Aldrich; THF(BDH) was freshly distilled over powdered LiAlH4 and stored over 4Å molecular sieves under nitrogen. Othersolvents, toluene (BDH), chloroform, ethyl acetate, acetone, acetonitrile, hexane, absolute ethanol (Fisher)and methanol (Fluka) were used as received.

TLC and column chromatography were carried out with MERCK silica gel (Kiesegel 60 F254 and Kieselgel60 0.040–0.063 mm).

Purification of Modified OligonucleotidesAftes synthesis CPG bound oligonucleotides were treated with concentrated ammonium hydroxyde at

55 ºC for 10 hs, evaporated, and precipitated from of 1 M LiClO4 (0.4 mL) by dilution with acetone (1.6 mL).In all cases, the 2’-pyrene-carbamate function was found to be completely stable to the conditions ofoligonucleotide synthesis as well as to final deprotection. Oligonucleotides were isolated usingelectrophoresis in 20% denaturing (7 M urea) PAGE in Tris-borate buffer, pH 8.3, and desalted usingPharmacia NAP-10 column and standard purification procedure.

Reversed-phase HPLC of pyrene-modified oligonucleotides was carried out using a Phenomenex RP-C18(3.90 × 300 mm) and dual-wavelength (215 and 254 nm) UV detection using gradient of acetonitrile (0–5%,5 min, 5–15%, 10 min, 15–40, 30 min, 40–80%, 10 min, 80–0%, 10 min). Appropriate fractions werepooled, evaporated, and precipitated from 1M LiClO4 (0.4 mL) by addition of acetone (1.6 mL). Examples ofreverse-phase HPLC traces are shown on Figure S–1. Oligonucleotide concentrations were determined byabsorbance at 260 nm and the calculated single-strand extinction coefficients based on a nearest neighbormodel [J. D. Puglisi, I. Tinoco, Jr., Meth. Enzymol. 1989, 180, 304–325]. A pyrene’s extinction coefficient is22400 [R. A. Friedel, M. Orchin, Ultraviolet spectra of aromatic compounds, Wiley, New York, 1951].Examples of UV curves of pyrene-modified oligonucleotides are shown on Figure S–2.

The mass of each oligonucleotide was checked by MALDI-TOF mass spectrometry on a PerseptiveBiosystems Voyager DE mass spectrometer in positive ion mode using a 1:1 v/v mixture of 2,6-dihydroxyacetophenone (40 mg/mL in MeOH) and aq. diammonium hydrogen citrate (80 mg/mL) as amatrix premixed just before loading the samples onto a plate (Table S–1). Examples of mass-spectra areshown on Figure S–3 - Figure S–8.

Thermal Denaturation ExperimentsThe results of thermal denaturation studies are given in Table S–2. Duplexes containing bis-pyrenylated

oligonucleotide ON09 show distinct S-shaped “reverse” melting curve detected at 350 nm. Tm values frommelting experiments at 350 nm are similar to those obtained at 260 nm, except cases when pyrene residueof complementary oligonucleotide in the duplex is interacting with one of pyrenes of ON09 (duplexesON09×ON13 and ON09×ON16). An examples of 260 nm and 350 nm UV melting curves for duplexescontaining one pyrene residue (ON08×ON02) and two pyrene residues in different strands (ON08×ON12)are shown on Fehler! Verweisquelle konnte nicht gefunden werden.s S–9 and S–10, respectively.

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Fluorescence MeasurementsFluorescense spectra of duplexes containing one strand longer than other ON07×ON14, ON07×ON15,

ON07×ON16 are given in Figure S–11. The presence of a four nucleotide dangling tail clearly does notaffect significantly on the shape of fluorescence spectra (cf. spectra of duplexes ON07×ON11,ON07×ON12, ON07×ON13 in Figure 3).

The examples of fluorescence spectra for DNA–DNA and DNA–RNA duplexes containing one pyrene indifferent positions or two pyrene are given in Figures S–12, S–13, and S–14.

Molecular ModelsMolecular models of duplexes ON07×ON11, ON07×ON12, ON07×ON13 (Figures S–15 – S–19) were

assembled using Cochranes molecular models, DNA kit (Aldrich). In case of the duplex ON07×ON13pyrenes are brought together and the complanar pyrene pair has considerable degree of freedom forlocation in the major groove of the DNA (Figures S–15 – S–17). The increased distance between pyrenes induplexes ON07×ON12 and ON07×ON11 is less suitable for excimer formation (Figures S–18 and S–19,respectively).

Table S-1

# Sequence, 5’→3’ MALDI MS Calculated mass, [M+H]+

ON01 CTCCCAGGCTCAAAT 4490.39 4496.00ON02 ATTTGAGCCTGGGAG 4643.75 4643.00ON04 CTCCCAGGC UBCAAAT 4597.35 4598.05ON05 CUBCCCAGGCTCAAAT 4598.44 4598.05ON06 CUBCCCAGGCUBCAAAT 4697.89 4698.15ON07 CTCCCAGGC UPCAAAT 4748.51 4756.21ON08 CUPCCCAGGCTCAAAT 4751.38 4756.21ON09 CUPCCCAGGCUPCAAAT 5009.61 5015.46ON10 CTCCCAGGCTCAAAUPCTGG 6002.29 6007.99ON11 AUPTTGAGCCTGGGAG 4903.53 4907.29ON12 ATUPTGAGCCTGGGAG 4902.88 4907.29ON13 ATTUPGAGCCTGGGAG 4903.37 4907.29ON14 CCAGAUPTTGAGCCTGGGAG 6121.86 6128.07ON15 CCAGATUPTGAGCCTGGGAG 6120.60 6128.07ON16 CCAGATTUPGAGCCTGGGAG 6118.99 6128.07

3

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

20

.0

19

.5

23

.03

2

1

Minutes

Figure S–1. Analytical HPLC of pyrene-modifiedoligonucleotide ON07 (1), ON08 (2), ON09 (3).

250 300 350 4000,0

0,1

0,2

0,3

0,4

0,5

A

λ / nm

3

2

1

3

2

1

Figure S–2. UV spectra of pyrene-modifiedoligonucleotides ON07 (1), ON08 (2), ON09 (3)in water, normalized by pyrene absorbanse.

3000 4000 5000 6000 7000 8000 90000

10000

20000

30000

40000

50000

47

48

.5

Counts

Mass (m/z)

Figure S–3. MALDI TOF spectrum of ON07

3000 4000 5000 6000 7000 8000 90000

10000

20000

30000

40000

50000

60000

70000

80000

47

51

.3

Counts

Mass (m/z)

Figure S–4. MALDI TOF spectrum of ON08

3000 4000 5000 6000 7000 8000 9000

0

10000

20000

30000

40000

50000

60000

70000

5009

.6

Counts

Mass (m/z)

Figure S–5. MALDI TOF spectrum of ON09

3000 4000 5000 6000 7000 8000 90000

10000

20000

30000

40000

50000

60000

4597

.35

Counts

Mass (m/z)

Figure S–6. MALDI TOF spectrum of ON04

3000 4000 5000 6000 7000 8000 90000

10000

20000

30000

40000

50000

60000

4597

.35

Counts

Mass (m/z)

Figure S–7. MALDI TOF spectrum of ON05

3000 4000 5000 6000 7000 8000 90000

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20000

4598

.44

Counts

Mass (m/z)

Figure S–8. MALDI TOF spectrum of ON06

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Table S–2. Thermal stabilities of modified duplexesDuplex Schematic structure Tm, 260 nm Tm, 350 nm

ON07×× ON115'

3' 57.2 n.d.

ON07×× ON125'

3' 56.6 n.d.

ON07×× ON135'

3' 55.3 n.d.

ON07×× ON145'

3' 56.8 n.d.

ON07×× ON155'

3' 55.9 n.d.

ON07×× ON165'

3' 54.6 n.d.

ON08×× ON115'

3' 58.3 n.d.

ON08×× ON125'

3' 57.9 n.d.

ON08×× ON135'

3' 57.0 n.d.

ON08×× ON145'

3' 56.6 n.d.

ON08×× ON155'

3' 56.5 n.d.

ON08×× ON165'

3' 56.3 n.d.

ON09×× ON115'

3' 57.0 57.4

ON09×× ON125'

3' 55.9 56.1

ON09×× ON135'

3' 53.8 59.0

ON09×× ON145'

3' 56.0 56.1

ON09×× ON155'

3' 54.8 55.1

ON09×× ON165'

3' 53.1 60.0

ON10×× ON02 5'3'

58.3 n.d.

ON10×× ON115'

3' 55.7 n.d.

ON10×× ON125'

3' 58.4 n.d.

ON10×× ON135'

3' 59.2 n.d.

ON10×× ON145'

3' 62.1 n.d.

ON10×× ON155'

3' 63.5 n.d.

ON10×× ON165'

3' 63.9 n.d.

ON01×× ON115'

3' 57.7 n.d.

ON01×× ON125'

3' 55.8 n.d.

ON01×× ON135'

3' 57.0 n.d.

ON01×× ON145'

3' 56.6 n.d.

ON01×× ON155'

3' 56.5 n.d.

ON01×× ON165'

3' 56.2 n.d.

5

20 30 40 50 60 70 800,015

0,020

0,025

0,20

0,25

2

1

A

T / oC

Figure S–9. Termal denaturation curves of theduplexes ON08×ON02 in hybridization bufferdetected at 260 nm (1) and 350 nm (2)

20 30 40 50 60 70 80

0,03

0,04

0,05

0,20

0,25

2

1

A

T / o C

Figure S–10. Termal denaturation curves of theduplexes ON08×ON12 in hybridization bufferdetected at 260 nm (1) and 350 nm (2)

350 400 450 500 550 600

0

2

4

6

8

10

12

14

16

18

20

223

2

1

I

�/nm

Figure S–11. Fluorescense spectra of duplexesON07×ON14 (1), ON07×ON15 (2),ON07×ON16 (3).

360 380 400 420 440 460 480 500

0

1

2

3

4

5

6

7

3

2

1

I

�/nm

Figure S–12. Fluorescense spectra monopyrene-labelled oligonucleotide ON07 (1) and its duplexeswith complementary RNA ON03 (2) and DNA(ON02).

360 380 400 420 440 460 480 5000

1

2

3

4

I

3

2

1

�/nm

Figure S–13. Fluorescense spectra monopyrene-labelled oligonucleotide ON08 (1) and its duplexeswith complementary RNA ON03 (2) and DNA(ON02).

350 400 450 500 550 6000

2

4

6

8

10

12

I

3

2

1

�/nm

Figure S–14. Fluorescense spectra bis-pyrene-labelled oligonucleotide ON09 (1) and its duplexeswith complementary RNA ON03 (2) and DNA(ON02).

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Figure S–15. Molecular model of the duplexON07×ON13; general view.

Figure S–16. Molecular model of the duplexON07×ON13; pyrenes in the major groove.

Figure S–17. Molecular model of the duplexON07×ON13; possible hydrogen bondsstabilizing close and complanar location of pyreneresidues in the major groove of DNA duplex. Thereare two additional hydrogen bonds: betweencarbamate NH and phosphate O (Hb1) in the onestrand and between carbamate NH and guanineN7 (Hb2) in the other.

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Figure S–18. Molecular model of the duplexON07×ON12; general view.

Figure S–19. Molecular model of the duplexON07×ON11; general view.

Molecular DynamicsSix molecular dynamics with additional geometrical constraints were run to appreciate the influence of

hydrogen bonds Hb1 and Hb2 on pyrene-pyrene interaction. The same calculation protocol was used in allMD simulations. Then geometry characteristics of pyrene-pyrene interactions were analyzed. Distances andangles were measured in program InsightII; surfaces of contact were calculated in program Connoly [M. L.Connolly, Science 1983, 221, 709–713]

Result of simulations pointed on Figures S–20 – S–28.

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Figure S–20. Distance (Å) between centers of pyrene rings versus MD time for model 1 (duplexON07×ON13). The upper graph corresponds to cis conformation of amide bonds in carbamate linker, andthe lower – to trans conformation. Black line corresponds to MD without any constraints (stabilizinghydrogen bonds), red line correspond MD with Hb1 constraint, and blue line represent MD with both Hb1and Hb2 constraints.

Figure S–21. Distance (Å) between centers of pyrene rings versus MD time for model 2 (duplexON07×ON12). The upper graph corresponds to cis conformation of amide bonds in carbamate linker, andthe lower – to trans conformation. Black line corresponds to MD without any constraints (stabilizinghydrogen bonds), red line correspond MD with Hb1 constraint.

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Figure S–22. Distance (Å) between centers of pyrene rings versus MD time for model 3 (duplexON07×ON11). Black line corresponds to cis conformation of amide bonds in carbamate linker, and red line– to trans conformation.

Figure S–23. Surface overlap (Å2) of pyrene rings versus MD time for model 1 (duplex ON07×ON13). Theupper graph corresponds to cis conformation of amide bonds in carbamate linker, and the lower – to transconformation. Black line corresponds to MD without any constraints (stabilizing hydrogen bonds), red linecorrespond MD with Hb1 constraint, and blue line represent MD with both Hb1 and Hb2 constraints.

10

Figure S–24. Surface overlap (Å2) of pyrene rings versus MD time for model 2 (duplex ON07×ON12). Theupper graph corresponds to cis conformation of amide bonds in carbamate linker, and the lower – to transconformation. Black line corresponds to MD without any constraints (stabilizing hydrogen bonds), red linecorrespond MD with Hb1 constraint.

Figure S–25. Surface overlap (Å2) of pyrene rings versus MD time for model 3 (duplex ON07×ON11).Black line corresponds to cis conformation of amide bonds in carbamate linker, and red line – to transconformation.

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Figure S–26. Angle (degrees) between pyrene rings versus MD time for model 1 (duplex ON07×ON13).The upper graph corresponds to cis conformation of amide bonds in carbamate linker, and the lower – totrans conformation. Black line corresponds to MD without any constraints (stabilizing hydrogen bonds), redline correspond MD with Hb1 constraint, and blue line represent MD with both Hb1 and Hb2 constraints.

12

Figure S–27. Angle (degrees) between pyrene rings versus MD time for model 2 (duplex ON07×ON12).The upper graph corresponds to cis conformation of amide bonds in carbamate linker, and the lower – totrans conformation. Black line corresponds to MD without any constraints (stabilizing hydrogen bonds), redline correspond MD with Hb1 constraint.

Figure S–28. Angle (degrees) between pyrene rings versus MD time for model 3 (duplex ON07×ON11).Black line corresponds to cis conformation of amide bonds in carbamate linker, and red line – to transconformation.

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Figure S–29. Two of possible space filling model of DNA duplex ON07×ON13. Strands are light blue andyellow, phosphate groups are red, and chromophores are blue.