Synthesis and Application of Four Fluorescence Labeled Nucleotides Through Disulfide as Reversible Terminators in DNA Sequencing by Synthesis

doi:10.7503/cjcu20140251

Abstract

Abstract:

DNA sequencing is one of the important means of biological research. The development of accurate and high-throughput DNA-sequencing methods has a strong impact on biomedical research. Sequencing by synthesis(SBS) approaches is one of the most important techniques of DNA sequencing. A series of SBS methods have been investigated and implemented. In SBS methods, the principal thing is to synthesize four fluorescently labelled cyclic reversible terminators as the sequencing reagents. These cyclic reversible terminators need to connect the fluorescein through a cleavable linker. In chemical biology, disulfide bridges, which are known to be efficiently and rapidly cleaved by mild reducing agents, have been used in a wide range of applications including drug delivery, functional and structural proteomics, DNA and tumor imaging. The disulfide-based cleavable linker is commonly used due to its rapid cleavage and straightforward synthesis. In this work, starting from 5-iodo-2'-deoxyuridine, 5-iodo-2'-deoxycyridine, 7-deazo-7-iodo-2'-deoxyadenosine and 7-deazo-7-iodo-2'-deoxyguanosine, four kinds of fluorescence labelled necleotides with four different bases through disulfide were synthesized and characterized. The synthesized fluorescence labelled nucleotides can be recognized and accepted by an identified DNA polymerase, incorporated faithfully into DNA strand, followed by cleaved completely by DTT within 1 min. These results demonstrate that the fluorescence labelled nucleotides have potential value as reversible terminators in DNA sequencing by synthesis.

Key words: DNA sequencing by synthesis, Reversible terminator, Cleavable linker, Four fluorescence labelled nucleotide

CLC Number:

O629.11

TrendMD:

TANG Daonian, JIANG Min, LI Xiaowei, KANG Yani, WU Xinyan, GONG Bing, SHAO Zhifeng, ZHAO Xiaodong, SHEN Yumei. Synthesis and Application of Four Fluorescence Labeled Nucleotides Through Disulfide as Reversible Terminators in DNA Sequencing by Synthesis[J]. Chem. J. Chinese Universities, 2014, 35(11): 2346.

Figures/Tables 4

References 28

[1]	Hyman E. D., Anal. Biochem., 1988, 174, 423—436
[2]	Ronaghi M., Karamohamed S., Pettersson B., Uhlén M., NyrénP., Anal. Biochem., 1996, 242, 84—89
[3]	Li Z. M., Bai X. P., Ruparel H., Kim S., Turro N. J., Ju J. Y., Proc. Natl. Acad. Sci., 2003, 100, 414—419
[4]	Seo T. S., Bai X. P., Ruparel H., Li Z. M., Turro N. J., Ju J. Y., Proc. Natl. Acad. Sci., 2004, 101, 5488—5493
[5]	Seo T. S., Bai X. P., Kim D. H., Meng Q. L., Shi S. D., Ruparel H., Li Z. M., Turro N. J., Ju J. Y., Proc. Natl. Acad. Sci., 2005, 102, 5926—5931
[6]	Meng Q. L., Kim D. H., Bai X. P., Bi L. R., Turro N. J., Ju J. Y., J. Org. Chem., 2006, 71, 3248—3252
[7]	Bi L. R., Kim D. H., Ju J. Y., Meng Q. L., Turro N. J., Bai X. P., J. Am. Chem. Soc., 2006, 128, 2542—2543
[8]	Ju J. Y., Kim D. H., Bi L. R., Meng Q. L., Bai X. P., Li Z. M., Li X. X., Marma M. S., Shi S. D., Wu J., Edwards J. R., Romu A., Turro N. J., Proc. Natl. Acad. Sci., 2006, 103, 19635—19640
[9]	Ju J. Y., Kim D. H., Bi L. R., Meng Q. L., Li X. X., Four-color DNA Sequencing by Synthesis Using Cleavable Fluorescent Nucleotide Reversible Terminators, US 7883869,2011-02-08
[10]	Knapp D. C., Onofrio J. D., Engels J. W., Bioconjugate Chem., 2010, 21, 1043—1055
[11]	Balasubramanian S., Chem. Commun., 2011, 47, 7281—7286
[12]	Turcatti G., Romieu A., Fedurco M., Tairi A. P., Nucleic Acids Res., 2008, 36(4), e25-1—e25-13
[13]	Baddiley J., Michelson A.M., Todd A. R., J. Chem. Soc., 1949, 582—586
[14]	Michelson A.M., Todd A. R.,J. Chem. Soc., 1949, 2487—2490
[15]	Gillerman I., Fischer B., Nucleosides, Nucleotides & Nucleic Acids,2010, 29(3), 245—256
[16]	Ludwig J., Acta Biochim. Biophy. Acad. Hung., 1981, 16, 131—133
[17]	Kevin B., Dan C., Chem. Rev., 2000, 100, 2047—2059
[18]	Zlatev I., Lavergne T., Debart F., Vasseur J. J., Manoharan M., Morvan F., Org. Lett., 2010, 12, 2190—2193
[19]	Warnecke S., Meier C., J. Org. Chem., 2009, 74, 3024—3030
[20]	Wu W., Freel Meyers C. L., Borch R. F., Org. Lett., 2004, 6, 2257—2260
[21]	Dixit V. M., Poulter C. D., Tetrahedron Lett., 1984, 25, 4055—4058
[22]	Davisson V. J., Davis D. R., Dixit V. M., Poulter C. D., J. Org. Chem., 1987, 52, 1794—1801
[23]	Caton W. J., Smith M., Carrasco N., Huang Z., Org. Lett., 2011, 13(16), 4156—4159
[24]	Caton-Williams J., Fiaz B., Hoxhaj R., Smith M., Huang Z., Science China Chemistry,2011, 55(1), 80—89
[25]	Knapp D. C., Serva S., D’Onofrio J., Keller A., Lubys A., Kurg A., Remm M., Engels J. W., Chem. Eur. J., 2011, 17, 2903—2915
[26]	Hashmi A. S. K., Häffner T., Yang W. B., Pankajakshan S., Schäfer S., Schultes L., Rominger F., Frey W., Chem. Eur. J., 2012, 18, 10480—10486
[27]	Borsenberger V., Kukwikila M., Howorka S., Org. Biomol. Chem., 2009, 7, 3826—3835
[28]	Baumann S., Schoof S., Bolten M., Haering C., Takagi M., Shin-ya K., Arndt H. D., J. Am. Chem. Soc., 2010, 132, 6973—6981

Compd.	Appearance	ESI-HRMS, m/z	Yield(%)	Purity(%, HPLC)
1a	Red solid	1095.1483[M-H]^-	18	90
1b	Yellow solid	1071.0604[M-H]^-	35	98
		991.0912[M-H₂PO₃]^-
1c	Blue solid	1169.3229[M-H]^-	18	100
1d	Brown solid	1422.2546[M-H]^-	43	93
2	White liquid		67
4a	Pale yellow solid		61
4b	Pale yellow solid		58
4c	Pale white solid		92
4d	White solid		39
5a	White solid	522.0070[M+H]⁺	34	100
5b	White solid	519.0059[M-H]^-	34	98
5c	White solid	542.0222[M-H]^-	30	100
5d	White solid	558.0179[M-H]^-	27	98
6a	White solid	716.0039[M-H]^-	15
6b	White solid	716.0039[M-H]^-	11
6c	White solid	739.0201[M-H]^-	35
6d	White solid	755.0166[M-H]^-	33
7a	Red solid	490.1780[M+H]⁺	95
7b	Yellow solid	465.0565[M-H]^-	80
7c	Blue solid	542.3200[M]	25
7d	Red solid	777.2483[M-H]^-	26
9	White solid		21
10	Yellow solid		86

Compd.	Appearance	ESI-HRMS, m/z	Yield(%)	Purity(%, HPLC)
1a	Red solid	1095.1483[M-H]^-	18	90
1b	Yellow solid	1071.0604[M-H]^-	35	98
		991.0912[M-H₂PO₃]^-
1c	Blue solid	1169.3229[M-H]^-	18	100
1d	Brown solid	1422.2546[M-H]^-	43	93
2	White liquid		67
4a	Pale yellow solid		61
4b	Pale yellow solid		58
4c	Pale white solid		92
4d	White solid		39
5a	White solid	522.0070[M+H]⁺	34	100
5b	White solid	519.0059[M-H]^-	34	98
5c	White solid	542.0222[M-H]^-	30	100
5d	White solid	558.0179[M-H]^-	27	98
6a	White solid	716.0039[M-H]^-	15
6b	White solid	716.0039[M-H]^-	11
6c	White solid	739.0201[M-H]^-	35
6d	White solid	755.0166[M-H]^-	33
7a	Red solid	490.1780[M+H]⁺	95
7b	Yellow solid	465.0565[M-H]^-	80
7c	Blue solid	542.3200[M]	25
7d	Red solid	777.2483[M-H]^-	26
9	White solid		21
10	Yellow solid		86

Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
1a	1.24—1.31(m, 1H), 2.04—2.15(m, 1H), 2.26—2.32(m, 1H), 2.65—2.75(m, 2H), 2.94—2.99(m, 2H), 3.17(t,J=4.8 Hz, 2H), 3.25(d, J=9.6 Hz, 8H), 3.72—3.77(m, 2H), 3.85—3.90(m, 3H), 3.96(s, 2H), 4.10—4.16(m, 2H), 4.49(s, 1H), 5.47(s, 1H), 5.84—5.88(m, 1H), 6.70—6.76(m, 2H), 6.91(d, J=9.6 Hz, 1H), 7.00—7.05(m, 1H), 7.18(d, J=9.6 Hz, 1H), 7.30(d, J=9.6 Hz, 1H), 7.60(d, J=8.0 Hz, 1H), 7.80(s, 1H), 8.18(d, J=7.6 Hz, 1H), 8.29(s, 1H)
1b	2.01—2.14(m, 1H), 2.29—2.39(m, 1H), 2.56—2.70(m, 2H), 2.85—3.01(m, 4H), 3.73—3.82(m, 1H), 3.85—4.25(m, 6H), 4.45(s, 1H), 5.92(t,J=4.0 Hz, 1H), 6.73—6.78(m, 4H), 7.31(dd, J=2.8, 8.8 Hz, 2H), 7.37(s, 1H), 7.62(d, J=8.8 Hz, 1H), 7.79(s, 1H), 7.92(d, J=8.0 Hz, 1H)	-21.22, -11.07, -8.27
2	2.34(t, J=2.8 Hz, 1H), 4.16(dd, J=2.4, 5.2 Hz, 2H), 6.61(s, 1H)
4a	2.11(t, J=5.1 Hz, 2H), 3.56—3.58(m, 2H), 3.78(d, J=4.0 Hz, 1H), 4.21(d, J=5.1 Hz, 3H), 5.08(t, J=5.1 Hz, 1H), 5.23(d, J=4.2 Hz, 1H), 6.09(t, J=6.6 Hz, 1H), 8.18(s, 1H), 10.05(t, J=4.8 Hz, 1H), 11.63(s, 1H)
4b	1.94—2.01(m, 1H), 2.12—2.18(m, 1H), 3.51(s, 1H), 3.55—3.62(m, 2H), 3.79(q,J=3.2, 6.8 Hz, 1H), 4.20(d, J=3.2 Hz, 1H), 4.28(s, 1H), 5.05(t, J=4.8 Hz, 1H), 5.20(d, J=3.6 Hz, 1H), 6.10(t, J=6.4 Hz, 1H), 6.84(s, 1H), 7.81(s, 1H), 8.15(s, 1H), 9.96(s, 1H)
4c	2.12—2.18(m, 1H), 2.41—2.46(m, 1H), 3.45—3.57(m, 2H), 3.78—3.81(m, 1H), 4.28—4.31(m, 3H), 5.06(t, J=5.6 Hz, 1H), 5.25(d, J=4.0 Hz, 1H), 6.43—6.47(m, 1H), 7.74(s, 1H), 8.09(s, 1H), 10.08(t, J=5.2 Hz, 1H)
4d	2.26—2.32(m, 1H), 2.48—2.55(m, 1H), 3.70—3.80(m, 2H), 3.96(dd, J=3.6, 6.8 Hz, 1H), 4.33(s, 2H), 4.47—4.50(m, 1H), 6.38—6.42(m, 1H), 7.25(s, 1H)
5a	2.36—2.47(m, 2H), 4.01(s, 2H), 4.20—4.30(m, 3H), 4.63—4.65(m, 1H), 6.28(t, J=6.0 Hz, 1H), 8.42(s, 1H)	-22.39, -11.35, -9.30
5b	2.27—2.33(m, 1H), 2.44—2.50(m, 1H), 4.05(s, 2H), 4.19—4.31(m, 3H), 4.56—4.59(m, 1H), 6.21(t, J=6.0 Hz, 1H), 8.37(s, 1H)	-22.55, -11.51, -10.31
5c	2.46—2.59(m, 2H), 4.10—4.24(m, 5H), 4.70(s, 1H), 6.49(t, J=6.8 Hz, 1H), 7.78(s, 1H), 8.02(s, 1H)	-21.67, -10.98, -8.50
5d	2.41—2.46(m, 1H), 2.53—2.58(m, 1H), 4.06(s, 2H), 4.11—4.20(m, 3H), 4.73(s, 1H), 6.34(t, J=6.8 Hz, 1H), 7.45(s, 1H)	-22.98, -11.24, -10.59
6a	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6b	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6c	2.46—2.51(m, 1H), 2.59—2.66(m, 1H), 2.74(t, J=6.4 Hz, 2H), 3.15(t, J=6.0 Hz, 2H), 4.18—4.27(m, 5H), 4.71—4.74(m, 1H), 6.52(t, J=6.8 Hz, 1H), 7.00(t, J=5.2 Hz, 1H), 7.55—7.60(m, 3H), 8.05(s, 1H), 8.10(d, J=4.0 Hz, 1H)
6d	2.35—2.40(m, 2H), 2.68(t, J=6.4 Hz, 2H), 3.12(t, J=6.4 Hz, 2H), 4.12—4.21(m, 5H), 4.71(s, 1H), 6.37(t, J=7.6 Hz, 1H), 7.17—7.21(m, 1H), 7.32(s, 1H), 7.77(s, 2H), 8.32(d, J=5.2 Hz, 1H)	-22.52, -11.13, -9.65
7a	1.26—1.31(m, 12H), 2.70(t, J=7.2 Hz, 2H), 3.53(t, J=6.8 Hz, 2H), 6.97(d, J=2.4 Hz, 2H), 7.04(dd, J=2.4, 9.6 Hz, 2H), 7.13(d, J=9.2 Hz, 2H), 7.81(d, J=1.2 Hz, 1H), 8.19(dd, J=1.6, 8.4 Hz, 1H), 8.39(d, J=8.4 Hz, 1H)
7b	2.69(t, J=6.4 Hz, 2H), 3.69(t, J=6.8 Hz, 2H), 6.69—6.72(m, 2H), 6.84(d, J=2.0 Hz, 2H), 6.91(d, J=8.8 Hz, 2H), 7.56(s, 1H), 7.76—7.83(m, 1H), 7.99(t, J=8.4 Hz, 1H)
Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
9	2.49—2.52(m, 2H), 2.58—2.61(m, 2H), 7.26(t, J=7.2 Hz, 3 H), 7.33(t, J=7.2 Hz, 6 H), 7.45(d, J=7.6 Hz, 6 H)
10	2.42(t, J=6.4 Hz, 2H), 3.34(t, J=5.2 Hz, 2H), 6.52(dd, J=2.4, 8.8 Hz, 2H), 6.63—6.65(m, 4 H), 7.12(t, J=7.2 Hz, 3 H), 7.20(t, J=7.6 Hz, 6 H), 7.31(d, J=7.6 Hz, 6 H), 7.45(s, 1 H), 7.76(dd, J=1.6, 8.4 Hz, 1 H), 7.85(d, J=8.4 Hz, 1 H)

Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
1a	1.24—1.31(m, 1H), 2.04—2.15(m, 1H), 2.26—2.32(m, 1H), 2.65—2.75(m, 2H), 2.94—2.99(m, 2H), 3.17(t,J=4.8 Hz, 2H), 3.25(d, J=9.6 Hz, 8H), 3.72—3.77(m, 2H), 3.85—3.90(m, 3H), 3.96(s, 2H), 4.10—4.16(m, 2H), 4.49(s, 1H), 5.47(s, 1H), 5.84—5.88(m, 1H), 6.70—6.76(m, 2H), 6.91(d, J=9.6 Hz, 1H), 7.00—7.05(m, 1H), 7.18(d, J=9.6 Hz, 1H), 7.30(d, J=9.6 Hz, 1H), 7.60(d, J=8.0 Hz, 1H), 7.80(s, 1H), 8.18(d, J=7.6 Hz, 1H), 8.29(s, 1H)
1b	2.01—2.14(m, 1H), 2.29—2.39(m, 1H), 2.56—2.70(m, 2H), 2.85—3.01(m, 4H), 3.73—3.82(m, 1H), 3.85—4.25(m, 6H), 4.45(s, 1H), 5.92(t,J=4.0 Hz, 1H), 6.73—6.78(m, 4H), 7.31(dd, J=2.8, 8.8 Hz, 2H), 7.37(s, 1H), 7.62(d, J=8.8 Hz, 1H), 7.79(s, 1H), 7.92(d, J=8.0 Hz, 1H)	-21.22, -11.07, -8.27
2	2.34(t, J=2.8 Hz, 1H), 4.16(dd, J=2.4, 5.2 Hz, 2H), 6.61(s, 1H)
4a	2.11(t, J=5.1 Hz, 2H), 3.56—3.58(m, 2H), 3.78(d, J=4.0 Hz, 1H), 4.21(d, J=5.1 Hz, 3H), 5.08(t, J=5.1 Hz, 1H), 5.23(d, J=4.2 Hz, 1H), 6.09(t, J=6.6 Hz, 1H), 8.18(s, 1H), 10.05(t, J=4.8 Hz, 1H), 11.63(s, 1H)
4b	1.94—2.01(m, 1H), 2.12—2.18(m, 1H), 3.51(s, 1H), 3.55—3.62(m, 2H), 3.79(q,J=3.2, 6.8 Hz, 1H), 4.20(d, J=3.2 Hz, 1H), 4.28(s, 1H), 5.05(t, J=4.8 Hz, 1H), 5.20(d, J=3.6 Hz, 1H), 6.10(t, J=6.4 Hz, 1H), 6.84(s, 1H), 7.81(s, 1H), 8.15(s, 1H), 9.96(s, 1H)
4c	2.12—2.18(m, 1H), 2.41—2.46(m, 1H), 3.45—3.57(m, 2H), 3.78—3.81(m, 1H), 4.28—4.31(m, 3H), 5.06(t, J=5.6 Hz, 1H), 5.25(d, J=4.0 Hz, 1H), 6.43—6.47(m, 1H), 7.74(s, 1H), 8.09(s, 1H), 10.08(t, J=5.2 Hz, 1H)
4d	2.26—2.32(m, 1H), 2.48—2.55(m, 1H), 3.70—3.80(m, 2H), 3.96(dd, J=3.6, 6.8 Hz, 1H), 4.33(s, 2H), 4.47—4.50(m, 1H), 6.38—6.42(m, 1H), 7.25(s, 1H)
5a	2.36—2.47(m, 2H), 4.01(s, 2H), 4.20—4.30(m, 3H), 4.63—4.65(m, 1H), 6.28(t, J=6.0 Hz, 1H), 8.42(s, 1H)	-22.39, -11.35, -9.30
5b	2.27—2.33(m, 1H), 2.44—2.50(m, 1H), 4.05(s, 2H), 4.19—4.31(m, 3H), 4.56—4.59(m, 1H), 6.21(t, J=6.0 Hz, 1H), 8.37(s, 1H)	-22.55, -11.51, -10.31
5c	2.46—2.59(m, 2H), 4.10—4.24(m, 5H), 4.70(s, 1H), 6.49(t, J=6.8 Hz, 1H), 7.78(s, 1H), 8.02(s, 1H)	-21.67, -10.98, -8.50
5d	2.41—2.46(m, 1H), 2.53—2.58(m, 1H), 4.06(s, 2H), 4.11—4.20(m, 3H), 4.73(s, 1H), 6.34(t, J=6.8 Hz, 1H), 7.45(s, 1H)	-22.98, -11.24, -10.59
6a	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6b	2.18—2.26(m, 1H), 2.36—2.43(m, 1H), 2.66(t, J=6.4 Hz, 2H), 3.08(t, J=6.8 Hz, 2H), 4.08(s, 1H), 4.17(s, 1H), 4.49—4.52(m, 1H), 6.17(t, J=6.4 Hz, 1H), 7.18—7.21(m, 1H), 7.75(d, J=6.0 Hz, 2H), 8.03(s, 1H), 8.29(d, J=4.8 Hz, 1H)
6c	2.46—2.51(m, 1H), 2.59—2.66(m, 1H), 2.74(t, J=6.4 Hz, 2H), 3.15(t, J=6.0 Hz, 2H), 4.18—4.27(m, 5H), 4.71—4.74(m, 1H), 6.52(t, J=6.8 Hz, 1H), 7.00(t, J=5.2 Hz, 1H), 7.55—7.60(m, 3H), 8.05(s, 1H), 8.10(d, J=4.0 Hz, 1H)
6d	2.35—2.40(m, 2H), 2.68(t, J=6.4 Hz, 2H), 3.12(t, J=6.4 Hz, 2H), 4.12—4.21(m, 5H), 4.71(s, 1H), 6.37(t, J=7.6 Hz, 1H), 7.17—7.21(m, 1H), 7.32(s, 1H), 7.77(s, 2H), 8.32(d, J=5.2 Hz, 1H)	-22.52, -11.13, -9.65
7a	1.26—1.31(m, 12H), 2.70(t, J=7.2 Hz, 2H), 3.53(t, J=6.8 Hz, 2H), 6.97(d, J=2.4 Hz, 2H), 7.04(dd, J=2.4, 9.6 Hz, 2H), 7.13(d, J=9.2 Hz, 2H), 7.81(d, J=1.2 Hz, 1H), 8.19(dd, J=1.6, 8.4 Hz, 1H), 8.39(d, J=8.4 Hz, 1H)
7b	2.69(t, J=6.4 Hz, 2H), 3.69(t, J=6.8 Hz, 2H), 6.69—6.72(m, 2H), 6.84(d, J=2.0 Hz, 2H), 6.91(d, J=8.8 Hz, 2H), 7.56(s, 1H), 7.76—7.83(m, 1H), 7.99(t, J=8.4 Hz, 1H)
Compd.	¹H NMR^a, δ	³¹P NMR^b, δ
9	2.49—2.52(m, 2H), 2.58—2.61(m, 2H), 7.26(t, J=7.2 Hz, 3 H), 7.33(t, J=7.2 Hz, 6 H), 7.45(d, J=7.6 Hz, 6 H)
10	2.42(t, J=6.4 Hz, 2H), 3.34(t, J=5.2 Hz, 2H), 6.52(dd, J=2.4, 8.8 Hz, 2H), 6.63—6.65(m, 4 H), 7.12(t, J=7.2 Hz, 3 H), 7.20(t, J=7.6 Hz, 6 H), 7.31(d, J=7.6 Hz, 6 H), 7.45(s, 1 H), 7.76(dd, J=1.6, 8.4 Hz, 1 H), 7.85(d, J=8.4 Hz, 1 H)