Synthesis of a Novel Organic Molecule for DNA Cleavage†

doi:10.7503/cjcu20130565

Abstract

Abstract:

The artificial nucleic acid cleaving agents have attracted extensive attention due to their potential applications in molecular biological technology and drug development. Metal-free cleaving reagents have been considered safer for their hydrolytic pathway of cleaving the P—O bond of phosphodiester in nucleic acids and have shown clinical potential. It is known that guanidinium is the arginine residue and the key functionality at the active site in staphylococcal nuclease(SNase) for DNA hydrolysis. Some small organic molecules as guanidinium derivatives have been used to cleave phosphodiester for DNA hydrolysis. We report here, according to active groups synergetic catalysis principle, the design and synthsis of a novel phosphodiester receptor 1-(N-guanidinoethyl)-4-(N-hydroxyethyl)-piperazidine hydrochloride(4) and the preliminary studies of its DNA cleavage activity. In the compound, the guanidinium group serves to recognize, bind, and electrophilically activate the anionic phosphodiester through hydrogen bonding and electrostatic interaction. The hydroxyl group works as a nucleophilic group in the transphosphorylation reaction, which is expected to be highly efficient because of the proximity effect. A “couple hardness with softness” piperazidine is designed to connect these two groups. Compound 4 was synthesized via a three-step reaction(nucleophilic substitution, hydrazinolysis and guanylation). Its structure was confirmed by ¹H NMR, ¹³C NMR and LC-MS. The influence of pH on the cleaving of pUC 19 DNA was studied by sugar gel electrophoresis. The mechanism of cleaving DNA by the compound was proved through the free radicals quenches experiments. The way of DNA cleavage was discussed through density functional theory and theoretical investigation by Gaussian. The results indicate that with pH value of 7.2 is the optimal pH for DNA cleavage in the presence of compound 4, the phosphodiester bond of DNA would be cleaved by compound 4 via a transphosphorylation pathway through oxidation-reduction reaction. Thus, this compound may be useful as artificial nucleic acid cleaving agent and the study may be usefully applied to achieve a more effective DNA cleavage for optimizing the structure and the distance of functional group to synergistic catalytic cleavage of the phosphodiester bond. In conclusion, design and synthesis of a novel phosphodiester receptor compound 4 containing guanidinoethyl and hydroxyethyl side arms was achieved successfully. We propose to introduce more such compounds as cleaving agents of nucleic acids to be widely investigated and found to be quite efficient.

Key words: Piperazidine, Phosphodiester bond, Guanidinium group, Spacer

CLC Number:

O623.626

TrendMD:

AN Dong, ZHAO Xiaohui, ZHOU Mi, YE Zhiwen. Synthesis of a Novel Organic Molecule for DNA Cleavage^†[J]. Chem. J. Chinese Universities, 2014, 35(2): 275.

Figures/Tables 5

Scheme 1 Synthetic routes of compound 4

Fig.1 pH-dependence profile for DNA cleavage promoted by compound 4(A) Agarose gel(1%) of pUC 19 DNA(0.05 mmol/L bp) incubated for 15 h at 37 ℃ with 0.042 mmol/L compound 4 in different pH buffers. Lanes 1—10: DNA control, pH=6.5, 6.75, 7.0, 7.25, 7.75, 8.0, 8.25, 8.5 and 9.0, respectively; (B) pH dependent profile for DNA cleavage promoted incubated for 24 h by 0.042 mmol/L compound 4 in different pH buffers(50 mmol/L) at 37℃

Fig.2 Radical scavengers inhibition reactions for DNA cleaved by compound 4(A) Histogram representing of pUC19 plasmid DNA(0.05 mmol/L bp) cleaved by compound 4(0.042 mmol/L) in the presence of standard radical scavengers incubated for 24 h at 37 ℃ in pH=7.2 buffer(50 mmol/L Tris-HCl); a. DNA control; b. no inhibitor; c. NaN3; d. KI; e. t-buolf; f. DMSO. (B) agarose gel of radical scavengers inhibition reactions. Lanes 1—6, DNA control, DNA+4, DNA+ 4+NaN3(10 mmol/L), DNA+4+KI(10 mmol/L), DNA+4+t-BuOH(1 mmol/L) and DNA+4+DMSO(1 mmol/L), respectively.

Fig.3 Coformation of midbody for BDNPP cleavage promoted by compound 4Optimized geometry of the intermediate Ⅲ. The hydrogen bond is represented as dashed line.

Scheme 2 Mechanism for DNA cleaved by compound 4(A) Mechanism for BDNPP cleavage promoted by compound 4; (B) plausible mechanism for DNA cleavage promoted by compound 4.

References 28

[1]	Armitage B., Chem. Rev., 1998, 98, 1171—1200
[2]	Liu C., Wang M., Zhang T., Sun H., Coord. Chem. Rev., 2004, 248, 147—168
[3]	Gaglione M., Milano G., Chambery A., Moggio L., Romanelli A., Messere A., Mol. Biosyst., 2011, 7(8), 2490—2499
[4]	Patel M. N., Gandhi D. S., Parmar P. A., Nucleos. Nucleot. Nucl., 2012, 31(6), 445—460
[5]	Shao Y., Zhang J., Tu C., Dai C., Xu Q., Guo Z., J. Inorg. Biochem., 2005, 99, 1490—1496
[6]	Zhou C. Q., Lin Y. L., Chen J. X., Chen W. H., Chem. Biodivers., 2012, 9(6), 1125—1132
[7]	Ding Y., Xu J., Pan Z. Q., Zhou H., Lou X., Inorg. Chem. Commun., 2012, 22, 40—43
[8]	Yousef T. A., Bedier R. A., J. Mol. Struct., 2013, 13(1035), 307—317
[9]	Scheffer U., Strick A., Ludwig V., Peter S., Kalden E., Göbel M. W., J. Am. Chem. Soc., 2005, 127, 2211—2217
[10]	Gnaccarini C., Peter S., Scheffer U., Vonhoff S., Klussmann S., Göbel M. W., J. Am. Chem. Soc., 2006, 128, 8063—8067
[11]	Tjioe L., Meininger A., Joshi T., Spiccia L., Graham B., Inorg. Chem., 2011, 50(10), 4327—4339
[12]	Tjioe L., Joshi T., Forsyth C. M., Moubaraki B., Murray K. S., Brugger J., Graham B., Spiccia L., Inorg. Chem., 2012, 51(2), 939—953
[13]	Yue Y., Li J., Zhang J., Zhang Z. W., Lin H. H., Chen S. Y., Yu X. Q., Chem. Biodivers., 2009, 6(12), 2236—2243
[14]	Liu Q., Zhang J., Wang M. Q., Zhang D. W., Lu Q. S., Huang Y., Lin H. H., Yu X. Q., Eur. J. Med. Chem., 2010, 45(11), 5302—5308
[15]	Ma Y., Chen X., Sun M., Wan R., Amino Acids, 2008, 35(2), 251—256
[16]	Huo F. J., Yin C. X., Yang P., Bioorg. Med. Chem. Lett., 2007, 17, 932—936
[17]	Huo F. J., Yin C. X., Yang P., Chem. Res. Chinese Universities, 2007, 28(5), 894—896
[18]	Muche M.S., Göbel M. W., Angew. Chem. Int. Ed. Engl., 1996, 35, 2126—2129
[19]	Ullrich S., Nazir Z., Buesing A., Scheffer U., Wirth D., Bats J. W., Duerner Gerd., Goebel M. W., Chem. Bio. Chem., 2011, 12(8), 1223—1229
[20]	Yu L. L., Ran Y., Bai X. X., Li A. R., Zhu Y. Y., Qin Y., Qu L. B., Chem. J. Chinese Universities, 2012, 33(12), 2681—2687
	(于岚岚, 冉瑜, 白希希, 李爱荣, 朱艳艳, 覃韵, 屈凌波.高等学校化学学报,2012, 33(12), 2681—2687)
[21]	Sheng X., Lu X. M., Zhang J. J., Chen Y. T., Lu G. Y., Shao Y., Liu F., Xu Q., J. Org. Chem., 2007, 72, 1799—1802
[22]	Shao Y., Sheng X., Li Y., Jia Z. L., Zhang J. J., Liu F., Lu G. Y., Bioconjugate Chem., 2008, 19, 1840—1848
[23]	Shao Y., Ding Y., Jia Z. L., Lu X. M., Ke Z. H., Xu W. H., Lu G. Y., Bioorg. Med. Chem., 2009, 17(13), 4274—4279
[24]	Piatek A. M., Gray M., Anslyn E. V., J. Am. Chem. Soc., 2009, 131(12), 4551
[25]	Salvio R., Cacciapaglia R., Mandolini L., J. Org. Chem., 2011, 76(13), 5438—5443
[26]	Awad A. M., Collazo M. J., Carpio K., Flores C., Bruice T. C.,Tetrahedron. Lett., 2012, 53(29), 3792—3794
[27]	Geoffrey D., Coxon B. L., Harvey J. M., Mark H. M., Mahmoud A., J. Med. Chem., 2009, 52(11), 3457—3463
[28]	Sun X. Q., An D., Shao Y., Acta. Cryst., 2011, E67, O2832