稳定同位素~(13)C标记富勒醇结构的探索
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摘要
富勒醇是具有良好生物相容性的富勒烯衍生物,因其具有低毒、抗肿瘤活性、抗病毒活性、抗氧化活性及光敏性等优势被广泛研究,发现其碳笼表面修饰的含氧基团决定其在生物医学应用中的生物效应.然而,富勒醇的结构比较复杂,尚未能精确描述.本文以稳定同位素~(13)C标记技术,通过富勒烯(C60)胺碱催化-氧化合成~(13)C骨架标记的富勒醇,结合质谱、红外光谱、X光电子能谱(XPS)及~(13)C核磁共振谱(~(13)C NMR)对其测试表征,探索富勒醇的精确结构.结果发现,稳定同位素~(13)C碳笼骨架标记极大地提高了~(13)C NMR信号强度,富勒醇分子结构的~(13)C NMR谱中清晰呈现出乙烯基醚碳、未反应烯碳、羟基化碳的信号,即不仅存在未氧化的碳和单氧化的碳,还存在高氧化的碳,与XPS测试结果吻合.富勒醇的碳笼表面修饰基团以羟基、半缩醛/酮、环氧以及羰基等结构存在.富勒醇复杂表面基团修饰结构的确定对其未来广泛的生物医学应用具有重要意义.
Fullerenes are novel carbon nanomaterials that hold great promise in diverse areas, but the limited solubility make them hardly dispersible in aqueous environment and the applications in hydrophilic systems are restricted, in particular for biomedical areas. Currently, chemical functionalization is the main approach to increase the hydrophilicity of fullerenes, where the soluble fullerene derivatives show lower toxicity and interesting bio-effects. Among these fullerene derivatives, hydroxylated fullerene(fullerenol) has good biocompatibility, antitumor activity, antivirus activity, antioxidation activity and photosensitive activity, thus is widely studied and applied. The structure and functionalization of fullerenol determine the bio-effects in biomedical applications. However, the structure of fullerenol is very complicated and the precise structure of fullerenol has not been achieved yet, such as the hydroxyl group number, the position of functional groups and the chemical forms of surface groups. Herein, we adopted ~(13)C staple isotope labeling to investigate the chemical structure of fullerenol. ~(13)C-skeleton labeled fullerene C_(60) was prepared by arc discharge method. Then, ~(13)C-C_(60) was hydroxylated by tetrabutylammonium hydroxide(TBAH) amine alkali catalysis-oxidation to obtain ~(13)C-fullerenol. The ~(13)C-fullerenol was characterized by TOF-MS, IR, X-ray photoelectron spectroscopy(XPS), NMR to analyze the structure of fullerenol. The results indicated that ~(13)C atoms were labeled on the skeleton of fullerene cage. Hydroxylation did not break the carbon cage, which was reflected by the strong C_(60) peak in the TOF-MS spectrum. The Poisson distribution of mass spectra indicated the isotopic effects and the skeleton labeling by ~(13)C. Based on the TOF-MS, there were about five ~(13)C atoms on each fullerene cage. The higher mass signals were assigned to the oxidized fullerene cage and the lack of very large mass signal suggested the detachment of functional groups during the laser irradiation. The oxygen containing groups were confirmed by IR spectrum. A 3340, 1372 and 1074 cm~(-1) peaks were attributed to be C-O/C-OH groups. XPS analyses indicated the chemical components of fullerenol as C 76%-82%, O 14%-18% and Na 0.6%-4%. The C 1s XPS spectra were divided into three major components, namely 284.56-284.86 eV for pristine carbon, 286.07-286.38 eV for C-O and 288.57-288.82 eV for C=O. Based on these, the chemical formulation of fullerenol was defined as Na_n~+[C_(60)O_x(OH)_y]~(n-), where the oxidation degree of fullerenol was regulated by the alkali concentration. The ~1H NMR spectrum showed signals at δ 1.23, 2.50 and 3.34. Only the weak peak at δ 1.23 was due to hydroxyl groups of fullerenol, the rest signals were due to water and dimethyl sulphoxide(DMSO). Enhanced ~(13)C NMR signals were observed at δ 175, 137 and 75-80, which were assigned to C=C-O, C=C and C-OH. Other weak signals were found at δ 160 for O=C-OH, δ 100 for RO-RCH-OH or RO-R_1CR_2-OH and δ54 for C atoms in epoxy structure. In conculsion, the structure of ~(13)C-fullerenol was explore to reach the formula of Na_n~+[C_(60)O_x(OH)_y]~(n-) and the exact numbers depended on the reaction parameters during the hydroxylation. ~(13)C isotope labeling largely enhanced the ~(13)C-NMR signals and revealed the intact fullerene cage during the hydroxylation according to the TOF-MS. The complicated surface functional groups were relfected by IR, XPS and NMR, where multiple forms were identified as hemiacetal/hemiketal groups, epoxy groups, carboxylic ester, carbonyl groups, and so on. The results would definitely benifit the ongoing exploration of fullerenol bio-effects/bioapplications and structure-activity relationship. The ~(13)C labeled fullerene and fullerenol could be used for the quantification of carbon nanomaterials in biological systems.
Fullerenes are novel carbon nanomaterials that hold great promise in diverse areas, but the limited solubility make them hardly dispersible in aqueous environment and the applications in hydrophilic systems are restricted, in particular for biomedical areas. Currently, chemical functionalization is the main approach to increase the hydrophilicity of fullerenes, where the soluble fullerene derivatives show lower toxicity and interesting bio-effects. Among these fullerene derivatives, hydroxylated fullerene(fullerenol) has good biocompatibility, antitumor activity, antivirus activity, antioxidation activity and photosensitive activity, thus is widely studied and applied. The structure and functionalization of fullerenol determine the bio-effects in biomedical applications. However, the structure of fullerenol is very complicated and the precise structure of fullerenol has not been achieved yet, such as the hydroxyl group number, the position of functional groups and the chemical forms of surface groups. Herein, we adopted ~(13)C staple isotope labeling to investigate the chemical structure of fullerenol. ~(13)C-skeleton labeled fullerene C_(60) was prepared by arc discharge method. Then, ~(13)C-C_(60) was hydroxylated by tetrabutylammonium hydroxide(TBAH) amine alkali catalysis-oxidation to obtain ~(13)C-fullerenol. The ~(13)C-fullerenol was characterized by TOF-MS, IR, X-ray photoelectron spectroscopy(XPS), NMR to analyze the structure of fullerenol. The results indicated that ~(13)C atoms were labeled on the skeleton of fullerene cage. Hydroxylation did not break the carbon cage, which was reflected by the strong C_(60) peak in the TOF-MS spectrum. The Poisson distribution of mass spectra indicated the isotopic effects and the skeleton labeling by ~(13)C. Based on the TOF-MS, there were about five ~(13)C atoms on each fullerene cage. The higher mass signals were assigned to the oxidized fullerene cage and the lack of very large mass signal suggested the detachment of functional groups during the laser irradiation. The oxygen containing groups were confirmed by IR spectrum. A 3340, 1372 and 1074 cm~(-1) peaks were attributed to be C-O/C-OH groups. XPS analyses indicated the chemical components of fullerenol as C 76%-82%, O 14%-18% and Na 0.6%-4%. The C 1s XPS spectra were divided into three major components, namely 284.56-284.86 eV for pristine carbon, 286.07-286.38 eV for C-O and 288.57-288.82 eV for C=O. Based on these, the chemical formulation of fullerenol was defined as Na_n~+[C_(60)O_x(OH)_y]~(n-), where the oxidation degree of fullerenol was regulated by the alkali concentration. The ~1H NMR spectrum showed signals at δ 1.23, 2.50 and 3.34. Only the weak peak at δ 1.23 was due to hydroxyl groups of fullerenol, the rest signals were due to water and dimethyl sulphoxide(DMSO). Enhanced ~(13)C NMR signals were observed at δ 175, 137 and 75-80, which were assigned to C=C-O, C=C and C-OH. Other weak signals were found at δ 160 for O=C-OH, δ 100 for RO-RCH-OH or RO-R_1CR_2-OH and δ54 for C atoms in epoxy structure. In conculsion, the structure of ~(13)C-fullerenol was explore to reach the formula of Na_n~+[C_(60)O_x(OH)_y]~(n-) and the exact numbers depended on the reaction parameters during the hydroxylation. ~(13)C isotope labeling largely enhanced the ~(13)C-NMR signals and revealed the intact fullerene cage during the hydroxylation according to the TOF-MS. The complicated surface functional groups were relfected by IR, XPS and NMR, where multiple forms were identified as hemiacetal/hemiketal groups, epoxy groups, carboxylic ester, carbonyl groups, and so on. The results would definitely benifit the ongoing exploration of fullerenol bio-effects/bioapplications and structure-activity relationship. The ~(13)C labeled fullerene and fullerenol could be used for the quantification of carbon nanomaterials in biological systems.
引文
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3 Kraszewski S,Tarek M.Ramseyer C.Uptake and translocation mechanisms of cationic amino derivatives functionalized on pristine C60by lipid membranes:A molecular dynamics simulation study.ACS Nano,2011,11:8571-8578
4 Sijbesma R,Srdanov G,Wudl F,et al.Synthesis of a fullerene derivative for the inhibition of HIV enzymes.J Am Chem Soc,1993,15:6510-6512
5 Chiang L Y,Bhonsle J B,Wang L,et al.Efficient one-flask synthesis of water-soluble[60]fullerenols.Tetrahedron,1996,14:4963-4972
6 Chiang L Y,Swirczewski J W,Hsu C S,et al.Multi-hydroxy additions onto C60 fullerene molecules.J Chem Soc Chem Commun,1992,24:1791-1793
7 Chiang L Y,Upasani R B,Swirczewski J W,et al.Evidence of hemiketals incorporated in the structure of fullerols derived from aqueous acid chemistry.J Am Chem Soc,1993,13:5453-5457
8 Chiang L Y,Lu F J,Lin J T.Free radical scavenging activity of water-soluble fullerenols.J Chem Soc Chem Commun,1995,12:1283-1284
9 Zhu Y S,Sun D Y,Liu G Z,et al.An ESR study on OH-radical scavenging activity of water-soluble fullerenols(in Chinese).Chem JChin Univ,1996,7:1127-1129[祝严师,孙大勇,刘桂珍,等.富勒醇清除OH自由基的ESR研究.高等学校化学学报,1996,7:1127-1129]
10 Chiang L Y,Wang L Y,Swirczewski J W,et al.Efficient synthesis of polyhydroxylated fullerene derivatives via hydrolysis of polycyclosulfated precursors.J Org Chem,1994,14:3960-3968
11 Chiang L Y,Upasani R B,Swirczewski J W.Versatile nitronium chemistry for C60 fullerene functionalization.J Am Chem Soc,1992,26:10154-10157
12 Naim A,Shevlin P B.Reversible addition of hydroxide to the fullerenes.Tetrahedron Lett,1992,47:7097-7100
13 Sun D Y,Liu Z Y,Guo X H,et al.Convenient preparation and properties of C60(OH)x(in Chinese).Chem J Chin Univ,1996,1:19-20[孙大勇,刘子阳,郭兴华,等.C60(OH)x的简便合成及性质.高等学校化学学报,1996,1:19-20]
14 Li T B,Li X H,Huang K X,et al.Synthesis and characterization of hydroxylated fullerene epoxide-An intermediate for forming fullerol.J Central South Univ Technol,1999,1:35-36
15 Li T B,Huang K X,Li X H,et al.Studies on the rapid preparation of fullerols and its formation mechanism(in Chinese).Chem J Chin Univ,1998,6:858-860[李添宝,黄克雄,李新海,等.C60(OH)n的快速制备及其机理研究.高等学校化学学报,1998,6:858-860]
16 Bogdanovi?G,Koji?V,?or?evi?A,et al.Modulating activity of fullerol C60(OH)22 on doxorubicin-induced cytotoxicity.Toxicol In Vitro,2004,5:629-637
17 Li J,Zhang M,Sun B,et al.Separation and purification of fullerenols for improved biocompatibility.Carbon,2012,2:460-469
18 Nel A E,Madler L,Velegol D,et al.Understanding biophysicochemical interactions at the nano-bio interface.Nat Mater,2009,7:543-547
19 Cai W,Piner R D,Stadermann F J,et al.Synthesis and solid-state NMR structural characterization of 13C-labeled graphite oxide.Science,2008,5897:1815-1817
20 Ruan L F,Chang X L,Sun B Y,et al.Preparation and spectra of 13C-enriched fullerene(in Chinese).Chin Sci Bull(Chin Ver),2014,59:905-912[阮龙飞,常雪灵,孙宝云,等.稳定同位素13C标记C60的制备及光谱性质.科学通报,2014,59:905-912]
21 Xing G M,Zhang J,Zhao Y L,et al.Influences of structural properties on stability of fullerenols.J Phys Chem B,2004,31:11473-11479
22 Yang S T,Wang H F,Guo L,et al.Interaction of fullerenol with lysozyme investigated by experimental and computational approaches.Nanotechnology,2008,39:395101
23 Husebo L O,Sitharaman B,Furukawa K,et al.Fullerenols revisited as stable radical anions.J Am Chem Soc,2004,38:12055-12064
24 Ruan L F.Preparation and property analysis of stable isotope 13C labeling of fullerenes and their derivatives(in Chinese).Master Dissertation.Wuhan:Hubei University of Traditional Chinese Medicine,2014[阮龙飞.稳定同位素13C标记富勒烯及其衍生物的制备和性质分析.硕士学位论文.武汉:湖北中医药大学,2014]
25 Wang C L,Ruan L F,Chang X L,et al.The isotopic effects of 13C-labeled large carbon cage(C70)fullerenes and their formation process.RSC Adv,2015,94:76949-76956
26 Wang Z Z,Chang X L,Lu Z H,et al.A precision structural model for fullerenols.Chem Sci,2014,8:2940-2948
27 Andreeva D V,Ratnikova O V,Melenevskaya E Y,et al.The regioselectivity of fullerenols C60(OH)x determined by high-resolution solid-state 13C and 1H NMR analysis.Int J Polym Anal Charact,2007,2:105-113
28 Yang B X.The development of an novel nucleus targeting nanomaterial C60(OH)24-NLC-E for the radiation protection(in Chinese).Master Dissertation.Suzhou:Soochow University,2012[杨百霞.细胞核靶向富勒醇固体脂质纳米粒的辐射防护作用.硕士学位论文.苏州:苏州大学,2012]
29 Zhang X M,Deng B,Li J Y,et al.Preparation of fullerenol from fullerene by use of UV light oxidation(in Chinese).J Radiat Res Radiat Process,2010,1:1-4[张晓敏,邓波,李景烨,等.紫外光氧化法制备水溶性富勒醇.辐射研究与辐射工艺学报,2010,1:1-4]
2 Kratschmer W,Lamb L D,Fostiropoulos K,et al.Solid C60:A new form of carbon.Nature,1990,6291:354-358
3 Kraszewski S,Tarek M.Ramseyer C.Uptake and translocation mechanisms of cationic amino derivatives functionalized on pristine C60by lipid membranes:A molecular dynamics simulation study.ACS Nano,2011,11:8571-8578
4 Sijbesma R,Srdanov G,Wudl F,et al.Synthesis of a fullerene derivative for the inhibition of HIV enzymes.J Am Chem Soc,1993,15:6510-6512
5 Chiang L Y,Bhonsle J B,Wang L,et al.Efficient one-flask synthesis of water-soluble[60]fullerenols.Tetrahedron,1996,14:4963-4972
6 Chiang L Y,Swirczewski J W,Hsu C S,et al.Multi-hydroxy additions onto C60 fullerene molecules.J Chem Soc Chem Commun,1992,24:1791-1793
7 Chiang L Y,Upasani R B,Swirczewski J W,et al.Evidence of hemiketals incorporated in the structure of fullerols derived from aqueous acid chemistry.J Am Chem Soc,1993,13:5453-5457
8 Chiang L Y,Lu F J,Lin J T.Free radical scavenging activity of water-soluble fullerenols.J Chem Soc Chem Commun,1995,12:1283-1284
9 Zhu Y S,Sun D Y,Liu G Z,et al.An ESR study on OH-radical scavenging activity of water-soluble fullerenols(in Chinese).Chem JChin Univ,1996,7:1127-1129[祝严师,孙大勇,刘桂珍,等.富勒醇清除OH自由基的ESR研究.高等学校化学学报,1996,7:1127-1129]
10 Chiang L Y,Wang L Y,Swirczewski J W,et al.Efficient synthesis of polyhydroxylated fullerene derivatives via hydrolysis of polycyclosulfated precursors.J Org Chem,1994,14:3960-3968
11 Chiang L Y,Upasani R B,Swirczewski J W.Versatile nitronium chemistry for C60 fullerene functionalization.J Am Chem Soc,1992,26:10154-10157
12 Naim A,Shevlin P B.Reversible addition of hydroxide to the fullerenes.Tetrahedron Lett,1992,47:7097-7100
13 Sun D Y,Liu Z Y,Guo X H,et al.Convenient preparation and properties of C60(OH)x(in Chinese).Chem J Chin Univ,1996,1:19-20[孙大勇,刘子阳,郭兴华,等.C60(OH)x的简便合成及性质.高等学校化学学报,1996,1:19-20]
14 Li T B,Li X H,Huang K X,et al.Synthesis and characterization of hydroxylated fullerene epoxide-An intermediate for forming fullerol.J Central South Univ Technol,1999,1:35-36
15 Li T B,Huang K X,Li X H,et al.Studies on the rapid preparation of fullerols and its formation mechanism(in Chinese).Chem J Chin Univ,1998,6:858-860[李添宝,黄克雄,李新海,等.C60(OH)n的快速制备及其机理研究.高等学校化学学报,1998,6:858-860]
16 Bogdanovi?G,Koji?V,?or?evi?A,et al.Modulating activity of fullerol C60(OH)22 on doxorubicin-induced cytotoxicity.Toxicol In Vitro,2004,5:629-637
17 Li J,Zhang M,Sun B,et al.Separation and purification of fullerenols for improved biocompatibility.Carbon,2012,2:460-469
18 Nel A E,Madler L,Velegol D,et al.Understanding biophysicochemical interactions at the nano-bio interface.Nat Mater,2009,7:543-547
19 Cai W,Piner R D,Stadermann F J,et al.Synthesis and solid-state NMR structural characterization of 13C-labeled graphite oxide.Science,2008,5897:1815-1817
20 Ruan L F,Chang X L,Sun B Y,et al.Preparation and spectra of 13C-enriched fullerene(in Chinese).Chin Sci Bull(Chin Ver),2014,59:905-912[阮龙飞,常雪灵,孙宝云,等.稳定同位素13C标记C60的制备及光谱性质.科学通报,2014,59:905-912]
21 Xing G M,Zhang J,Zhao Y L,et al.Influences of structural properties on stability of fullerenols.J Phys Chem B,2004,31:11473-11479
22 Yang S T,Wang H F,Guo L,et al.Interaction of fullerenol with lysozyme investigated by experimental and computational approaches.Nanotechnology,2008,39:395101
23 Husebo L O,Sitharaman B,Furukawa K,et al.Fullerenols revisited as stable radical anions.J Am Chem Soc,2004,38:12055-12064
24 Ruan L F.Preparation and property analysis of stable isotope 13C labeling of fullerenes and their derivatives(in Chinese).Master Dissertation.Wuhan:Hubei University of Traditional Chinese Medicine,2014[阮龙飞.稳定同位素13C标记富勒烯及其衍生物的制备和性质分析.硕士学位论文.武汉:湖北中医药大学,2014]
25 Wang C L,Ruan L F,Chang X L,et al.The isotopic effects of 13C-labeled large carbon cage(C70)fullerenes and their formation process.RSC Adv,2015,94:76949-76956
26 Wang Z Z,Chang X L,Lu Z H,et al.A precision structural model for fullerenols.Chem Sci,2014,8:2940-2948
27 Andreeva D V,Ratnikova O V,Melenevskaya E Y,et al.The regioselectivity of fullerenols C60(OH)x determined by high-resolution solid-state 13C and 1H NMR analysis.Int J Polym Anal Charact,2007,2:105-113
28 Yang B X.The development of an novel nucleus targeting nanomaterial C60(OH)24-NLC-E for the radiation protection(in Chinese).Master Dissertation.Suzhou:Soochow University,2012[杨百霞.细胞核靶向富勒醇固体脂质纳米粒的辐射防护作用.硕士学位论文.苏州:苏州大学,2012]
29 Zhang X M,Deng B,Li J Y,et al.Preparation of fullerenol from fullerene by use of UV light oxidation(in Chinese).J Radiat Res Radiat Process,2010,1:1-4[张晓敏,邓波,李景烨,等.紫外光氧化法制备水溶性富勒醇.辐射研究与辐射工艺学报,2010,1:1-4]