- Email: [email protected]
Alkynylation of carbon nanotube by a peptide bond
Materials Letters 133 (2014) 64–66
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Alkynylation of carbon nanotube by a peptide bond Tong Wu, Yong J. Yuan n Laboratory of Biosensing and MicroMechatronics, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2013 Accepted 21 June 2014 Available online 28 June 2014
Pristine carboxylic multi-walled carbon nanotube (MWCNT) surface was coupled with 4-ethynylaniline by a mild reaction to accomplish alkynyl-modification based on N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a peptide bond, at ambient temperature. Benzyl and alkyl functional groups present were characterized by fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results revealed that the yield of alkyne group immobilized on MWCNT surface was almost 50%. It thus shows the potential of linking suitable substrates for MWCNTs via azide-alkyne Huisgen [2þ 3] cycloaddition. & 2014 Elsevier B.V. All rights reserved.
Keywords: Carbon nanotubes Surfaces Functional Organic
1. Introduction Since their discovery by Iijima [1] in 1991, carbon nanotubes (CNTs) have attracted increasing interest as one of the most fascinating nanoobjects [2–4]. CNTs display unique structural [5], significant mechanical [6,7], thermal [7], optic [8] and electric [9,10] properties which make them promising materials for numerous potential applications. These applications include nanofillers, nanodevices, and nanobiotechnology. However, there are limitations to the construction of nanotube-based functional materials due to extreme difficulties in incorporating highly engineered molecules onto CNT surfaces. The main difficulty is the harsh conditions required for nanotubes functionalization which are detrimental to the functionality of the molecules to be immobilized. This is a critical factor for the success of clickchemistry based reaction [11–13], especially azide-alkyne Huisgen [2 þ3] cycloaddition. The functionalization of nanotubes normally demands the use of an excess of nasty or toxic reagents that are difficult or even impossible to recycle. The conventional method [12,13] is tedious with the reaction being carried out at elevated temperature. As shown on the left hand side of the flow chart part (a) of Fig. 1, the method involves chloridization which requires the use of extremely activated thionyl chloride. This results in acid chlorides being produced on the surface of carboxylic MWCNTs (MWCNT-COOH). The final step involves the reaction between 4-ethynylaniline with active acyl chloride groups in order to obtain the product containing alkyne group according to the Huisgen 1,3-dipolar cycloaddition
n
Corresponding author. Tel.: þ 86 288 760 0980. E-mail address: [email protected] (Y.J. Yuan).
http://dx.doi.org/10.1016/j.matlet.2014.06.130 0167-577X/& 2014 Elsevier B.V. All rights reserved.
reaction. Unfortunately, the conventional method is cumbersome as it requires the process to be carried out initially at 65 1C for 6 h, followed by cooling to 0 1C before the temperature is being raised to 25 1C with stirring for 24 h. Additionally, the reaction has to take place under N2 atmosphere. Therefore, it is necessary to exploit a simple method of CNT surface modification under a mild condition. Here, a new approach of MWCNT modification was proposed by cross-linking carboxyl and amine groups at ambient temperature to form an amide bond, as illustrated on the right hand side of the flow chart part (b) of Fig. 1. In this study we employed water-soluble N-hydroxysuccinimide (NHS) to couple with N-(3-dimethylaminopropyl)-N'ethylcarbodiimide hydrochloride (EDC) as these two reagents are known to have been used to successfully crosslink two proteins by a peptide bond [14–16].
2. Experimental All chemicals were used as received, without further purification. MWNTs-COOH (purity 495%, –COOH content: 3.86 wt%) were purchased from the Timesnano Chengdu Organic Chemicals Co., having an inner diameter of 2–5 nm and outer diameter of 8 nm and an average length of 10–30 μm. N,N-dimethylformamide (DMF), 4-ethynylaniline, EDC and NHS were products of SigmaAldrich. Ultrapure water was obtained from a Spring-R20i and OMNI water purification system and used for solutions preparation and samples washing. Both EDC and NHS aqueous solutions were mixed with pristine MWCNTs to form a dispersed solution under stirring. This was followed by the addition of 4-ethynylaniline in DMF to complete an amine crosslinking reaction with carboxylic acid groups on the
T. Wu, Y.J. Yuan / Materials Letters 133 (2014) 64–66
65
Fig. 1. Flow chart to show comparison of synthetizing MWCNT-alkyne by acyl chloridization reaction (a) and EDC-NHS crosslinking (b).
MWCNT surface. The whole process was completed within 6 h at ambient temperature, as compared to at least 27 h required for the conventional method. Finally, the surface-modified MWCNT-COOH was evaluated by fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance spectroscopy (1H NMR) and thermogravimetric analysis (TGA). TGA was carried out by a TGA Q500. All thermograms were scanned at 7 1C/min to 800 1C in N2. FT-IR spectra were obtained from a Nicolet 5700 spectrometer via KBr pellets. 1H NMR spectra were recorded by using a Bruker 400 MHz spectrometer in CDCl3.
3. Results and discussion As evidenced by the FT-IR spectra in Fig. 2, the pristine MWCNT-COOH was modified successfully by 4-ethynylaniline via an amide bond. Apart from alkyl C–H stretching vibration at 2834 and 2913 cm 1, the characteristic absorptions of the amide structure CO NH appeared significantly at 1621, 1681 and 1376 cm 1, due to N–H bending, C¼ O stretching and C–N stretching respectively. It is noteworthy that these experimental FT-IR values compared well with the literature values [17] as respectively displayed in the inset of Fig. 2. As shown in Fig. 2, there are 4 peaks at the wavelengths of 1430, 1506, 1535 and 1577 cm 1 in spectrum (b). Comparing these peaks with literature values [17], these 4 peaks are contributed by C ¼C skeletal of a benzene ring. Their corresponding α and β protons were also detected by NMR analysis of MWCNTs modified by EDC-NHS cross-linking carboxyl and amine groups. As shown in Fig. 3, both α and β protons in solution as observed in spectrum (b) and on MWCNT surface as in spectrum (c) were of
Fig. 2. FT-IR spectra of pristine MWCNT-COOH and modified by EDC-NHS crosslinking. (a) Blank pristine MWCNTs and (b) surface modified MWCNTs.
perfect match. The detailed chemical shifts were listed in the inset of comparison of 1H NMR α and β chemical shifts in Fig. 3. Chemical shifts of 1H (α and β) of a benzene ring towards high field indicate that there was an increase in electron density as 4-ethynylaniline was immobilized onto MWCNTs. Also, there was a decrease in the resonance signal of 1H (α and β) due to a heterogeneous dispersed solution and the presence of less amount of ethynylaniline amide. The quantitative determination of immobilized alkyne by a peptide bond is obtained by using the TGA technique. Fig. 4. depicts the thermograms of pristine MWCNT-COOH and the modified MWCNT-COOH by EDC-NHS crosslinking. TGA showed a weight loss of approximately 12.3% for surface modified MWCNTs and 9.6% for blank pristine MWCNTs at 800 1C. The
66
T. Wu, Y.J. Yuan / Materials Letters 133 (2014) 64–66
Fig. 3. 1H NMR spectra of blank pristine MWCNT-COOH and surface-modified MWCNTs by EDC-NHS crosslinking (400 MHz, CDCl3). (a) Blank pristine MWCNT-COOH, (b) pure 4-ethynylaniline and (c) surface modified MWCNTs.
Acknowledgment
99
Weight(%)
97
This work was funded by the National Natural Science Foundation of China to YJY under General Program Funds 31170954 and 30870664.
95 93
(a)
91
References
89
(b)
87 85
0
100
200
300
400
500
600
700
800
Temperature( ) Fig. 4. TGA Profiles of pristine MWCNT-COOH and modified by EDC-NHS crosslinking. (a) Blank pristine MWCNTs and (b) surface modified MWCNTs.
weight loss observed for surface modified MWCNTs between 280 and 800 1C is due to the destruction of 4-ethynylaniline immobilized by a peptide bond. Based on 3.86 wt% contained MWCNTCOOH used, the conversion rates (yields) of replicative MWCNT alkynylation were calculated from 32 to 50%. Therefore, the amount of alkyne immobilized could be further improved if a higher percentage of contained carboxylic acid was used.
4. Conclusion We have successfully explored the method of crosslinking EDCNHS for the purpose of alkynylating CNTs at a mild condition. This protocol can be exploited to fabricate nanotube-based functional materials which are highly engineered molecules on MWCNT surfaces. It paves the way for cycloaddition by azide-alkyne Huisgen [2þ3] click chemistry, especially for MWCNTs based applications.
[1] Iijima S. Helical microtubules of graphitic carbon. Nature 1991;354:56–8. [2] Dai H. Carbon nanotubes: synthesis, integration, and properties. Acc Chem Res 2002;35:1035–44. [3] Dai H. Carbon nanotubes: opportunities and challenges. Surf Sci 2002;500:218–41. [4] Terrones M. Carbon nanotubes: synthesis and properties, electronic devices and other emerging applications. Int Mater Rev 2004;49:325–77. [5] Chen J, Hamon MA, Hu H, Chen Y, Rao AM, Eklund PC, et al. Solution properties of single-walled carbon nanotubes. Science 1998;282:95–8. [6] Salvetat JP, Bonard JM, Thomson NH, Kulik AJ, Forro L, Benoit W, et al. Mechanical properties of carbon nanotubes. Appl Phys A 1999;69:255–60. [7] Ruoff RS, Lorents DC. Mechanical and thermal properties of carbon nanotubes. Carbon 1995;33:925–30. [8] Wan XG, Dong JM, Xing DY. Optical properties of carbon nanotubes. Phys Rev B Condens Matter 1998;58:6756–9. [9] Odom TW, Huang JL, Kim P, Lieber CM. Structure and electronic properties of carbon nanotubes. J Phys Chem B 2000;104:2794–809. [10] Moulton SE, Minett AI, Wallace GG. Carbon nanotube based electronic and electrochemical sensors. Sens Lett 2005;3:183–93. [11] Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed 2001;40:200–21. [12] Zhang Y, He H, Gao C. Clickable macroinitiator strategy to build amphiphilic polymer brushes on carbon nanotubes. Macromolecules 2008;41:9581–94. [13] ZhangY HeH, Gao C, Wu J. Covalent layer-by-layer functionalization of multiwalled carbon nanotubes by Click Chemistry. Langmuir 2009;25:5814–24. [14] Timkovich R. Detection of the stable addition of carbodiimide to proteins. Anal Biochem 1977;79:135–43. [15] Staros JM, Wright RW. Swingle DM. Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem 1986;156:220–2. [16] Grabarek Z, Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal Biochem 1990;185:131–5. [17] The Sadtler handbook of infrared spectra [Internet]. Hercules (CA): Bio-Rad Laboratories, Inc., Information Division. C1978-2004 [cited 08.12.13]. Available from: 〈http://www.bio-rad.com/en-us/product/ir-databases〉.
Contents lists available at ScienceDirect
Materials Letters journal homepage: www.elsevier.com/locate/matlet
Alkynylation of carbon nanotube by a peptide bond Tong Wu, Yong J. Yuan n Laboratory of Biosensing and MicroMechatronics, School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu, Sichuan 610031, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 December 2013 Accepted 21 June 2014 Available online 28 June 2014
Pristine carboxylic multi-walled carbon nanotube (MWCNT) surface was coupled with 4-ethynylaniline by a mild reaction to accomplish alkynyl-modification based on N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to form a peptide bond, at ambient temperature. Benzyl and alkyl functional groups present were characterized by fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results revealed that the yield of alkyne group immobilized on MWCNT surface was almost 50%. It thus shows the potential of linking suitable substrates for MWCNTs via azide-alkyne Huisgen [2þ 3] cycloaddition. & 2014 Elsevier B.V. All rights reserved.
Keywords: Carbon nanotubes Surfaces Functional Organic
1. Introduction Since their discovery by Iijima [1] in 1991, carbon nanotubes (CNTs) have attracted increasing interest as one of the most fascinating nanoobjects [2–4]. CNTs display unique structural [5], significant mechanical [6,7], thermal [7], optic [8] and electric [9,10] properties which make them promising materials for numerous potential applications. These applications include nanofillers, nanodevices, and nanobiotechnology. However, there are limitations to the construction of nanotube-based functional materials due to extreme difficulties in incorporating highly engineered molecules onto CNT surfaces. The main difficulty is the harsh conditions required for nanotubes functionalization which are detrimental to the functionality of the molecules to be immobilized. This is a critical factor for the success of clickchemistry based reaction [11–13], especially azide-alkyne Huisgen [2 þ3] cycloaddition. The functionalization of nanotubes normally demands the use of an excess of nasty or toxic reagents that are difficult or even impossible to recycle. The conventional method [12,13] is tedious with the reaction being carried out at elevated temperature. As shown on the left hand side of the flow chart part (a) of Fig. 1, the method involves chloridization which requires the use of extremely activated thionyl chloride. This results in acid chlorides being produced on the surface of carboxylic MWCNTs (MWCNT-COOH). The final step involves the reaction between 4-ethynylaniline with active acyl chloride groups in order to obtain the product containing alkyne group according to the Huisgen 1,3-dipolar cycloaddition
n
Corresponding author. Tel.: þ 86 288 760 0980. E-mail address: [email protected] (Y.J. Yuan).
http://dx.doi.org/10.1016/j.matlet.2014.06.130 0167-577X/& 2014 Elsevier B.V. All rights reserved.
reaction. Unfortunately, the conventional method is cumbersome as it requires the process to be carried out initially at 65 1C for 6 h, followed by cooling to 0 1C before the temperature is being raised to 25 1C with stirring for 24 h. Additionally, the reaction has to take place under N2 atmosphere. Therefore, it is necessary to exploit a simple method of CNT surface modification under a mild condition. Here, a new approach of MWCNT modification was proposed by cross-linking carboxyl and amine groups at ambient temperature to form an amide bond, as illustrated on the right hand side of the flow chart part (b) of Fig. 1. In this study we employed water-soluble N-hydroxysuccinimide (NHS) to couple with N-(3-dimethylaminopropyl)-N'ethylcarbodiimide hydrochloride (EDC) as these two reagents are known to have been used to successfully crosslink two proteins by a peptide bond [14–16].
2. Experimental All chemicals were used as received, without further purification. MWNTs-COOH (purity 495%, –COOH content: 3.86 wt%) were purchased from the Timesnano Chengdu Organic Chemicals Co., having an inner diameter of 2–5 nm and outer diameter of 8 nm and an average length of 10–30 μm. N,N-dimethylformamide (DMF), 4-ethynylaniline, EDC and NHS were products of SigmaAldrich. Ultrapure water was obtained from a Spring-R20i and OMNI water purification system and used for solutions preparation and samples washing. Both EDC and NHS aqueous solutions were mixed with pristine MWCNTs to form a dispersed solution under stirring. This was followed by the addition of 4-ethynylaniline in DMF to complete an amine crosslinking reaction with carboxylic acid groups on the
T. Wu, Y.J. Yuan / Materials Letters 133 (2014) 64–66
65
Fig. 1. Flow chart to show comparison of synthetizing MWCNT-alkyne by acyl chloridization reaction (a) and EDC-NHS crosslinking (b).
MWCNT surface. The whole process was completed within 6 h at ambient temperature, as compared to at least 27 h required for the conventional method. Finally, the surface-modified MWCNT-COOH was evaluated by fourier transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance spectroscopy (1H NMR) and thermogravimetric analysis (TGA). TGA was carried out by a TGA Q500. All thermograms were scanned at 7 1C/min to 800 1C in N2. FT-IR spectra were obtained from a Nicolet 5700 spectrometer via KBr pellets. 1H NMR spectra were recorded by using a Bruker 400 MHz spectrometer in CDCl3.
3. Results and discussion As evidenced by the FT-IR spectra in Fig. 2, the pristine MWCNT-COOH was modified successfully by 4-ethynylaniline via an amide bond. Apart from alkyl C–H stretching vibration at 2834 and 2913 cm 1, the characteristic absorptions of the amide structure CO NH appeared significantly at 1621, 1681 and 1376 cm 1, due to N–H bending, C¼ O stretching and C–N stretching respectively. It is noteworthy that these experimental FT-IR values compared well with the literature values [17] as respectively displayed in the inset of Fig. 2. As shown in Fig. 2, there are 4 peaks at the wavelengths of 1430, 1506, 1535 and 1577 cm 1 in spectrum (b). Comparing these peaks with literature values [17], these 4 peaks are contributed by C ¼C skeletal of a benzene ring. Their corresponding α and β protons were also detected by NMR analysis of MWCNTs modified by EDC-NHS cross-linking carboxyl and amine groups. As shown in Fig. 3, both α and β protons in solution as observed in spectrum (b) and on MWCNT surface as in spectrum (c) were of
Fig. 2. FT-IR spectra of pristine MWCNT-COOH and modified by EDC-NHS crosslinking. (a) Blank pristine MWCNTs and (b) surface modified MWCNTs.
perfect match. The detailed chemical shifts were listed in the inset of comparison of 1H NMR α and β chemical shifts in Fig. 3. Chemical shifts of 1H (α and β) of a benzene ring towards high field indicate that there was an increase in electron density as 4-ethynylaniline was immobilized onto MWCNTs. Also, there was a decrease in the resonance signal of 1H (α and β) due to a heterogeneous dispersed solution and the presence of less amount of ethynylaniline amide. The quantitative determination of immobilized alkyne by a peptide bond is obtained by using the TGA technique. Fig. 4. depicts the thermograms of pristine MWCNT-COOH and the modified MWCNT-COOH by EDC-NHS crosslinking. TGA showed a weight loss of approximately 12.3% for surface modified MWCNTs and 9.6% for blank pristine MWCNTs at 800 1C. The
66
T. Wu, Y.J. Yuan / Materials Letters 133 (2014) 64–66
Fig. 3. 1H NMR spectra of blank pristine MWCNT-COOH and surface-modified MWCNTs by EDC-NHS crosslinking (400 MHz, CDCl3). (a) Blank pristine MWCNT-COOH, (b) pure 4-ethynylaniline and (c) surface modified MWCNTs.
Acknowledgment
99
Weight(%)
97
This work was funded by the National Natural Science Foundation of China to YJY under General Program Funds 31170954 and 30870664.
95 93
(a)
91
References
89
(b)
87 85
0
100
200
300
400
500
600
700
800
Temperature( ) Fig. 4. TGA Profiles of pristine MWCNT-COOH and modified by EDC-NHS crosslinking. (a) Blank pristine MWCNTs and (b) surface modified MWCNTs.
weight loss observed for surface modified MWCNTs between 280 and 800 1C is due to the destruction of 4-ethynylaniline immobilized by a peptide bond. Based on 3.86 wt% contained MWCNTCOOH used, the conversion rates (yields) of replicative MWCNT alkynylation were calculated from 32 to 50%. Therefore, the amount of alkyne immobilized could be further improved if a higher percentage of contained carboxylic acid was used.
4. Conclusion We have successfully explored the method of crosslinking EDCNHS for the purpose of alkynylating CNTs at a mild condition. This protocol can be exploited to fabricate nanotube-based functional materials which are highly engineered molecules on MWCNT surfaces. It paves the way for cycloaddition by azide-alkyne Huisgen [2þ3] click chemistry, especially for MWCNTs based applications.
[1] Iijima S. Helical microtubules of graphitic carbon. Nature 1991;354:56–8. [2] Dai H. Carbon nanotubes: synthesis, integration, and properties. Acc Chem Res 2002;35:1035–44. [3] Dai H. Carbon nanotubes: opportunities and challenges. Surf Sci 2002;500:218–41. [4] Terrones M. Carbon nanotubes: synthesis and properties, electronic devices and other emerging applications. Int Mater Rev 2004;49:325–77. [5] Chen J, Hamon MA, Hu H, Chen Y, Rao AM, Eklund PC, et al. Solution properties of single-walled carbon nanotubes. Science 1998;282:95–8. [6] Salvetat JP, Bonard JM, Thomson NH, Kulik AJ, Forro L, Benoit W, et al. Mechanical properties of carbon nanotubes. Appl Phys A 1999;69:255–60. [7] Ruoff RS, Lorents DC. Mechanical and thermal properties of carbon nanotubes. Carbon 1995;33:925–30. [8] Wan XG, Dong JM, Xing DY. Optical properties of carbon nanotubes. Phys Rev B Condens Matter 1998;58:6756–9. [9] Odom TW, Huang JL, Kim P, Lieber CM. Structure and electronic properties of carbon nanotubes. J Phys Chem B 2000;104:2794–809. [10] Moulton SE, Minett AI, Wallace GG. Carbon nanotube based electronic and electrochemical sensors. Sens Lett 2005;3:183–93. [11] Kolb HC, Finn MG, Sharpless KB. Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed 2001;40:200–21. [12] Zhang Y, He H, Gao C. Clickable macroinitiator strategy to build amphiphilic polymer brushes on carbon nanotubes. Macromolecules 2008;41:9581–94. [13] ZhangY HeH, Gao C, Wu J. Covalent layer-by-layer functionalization of multiwalled carbon nanotubes by Click Chemistry. Langmuir 2009;25:5814–24. [14] Timkovich R. Detection of the stable addition of carbodiimide to proteins. Anal Biochem 1977;79:135–43. [15] Staros JM, Wright RW. Swingle DM. Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem 1986;156:220–2. [16] Grabarek Z, Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal Biochem 1990;185:131–5. [17] The Sadtler handbook of infrared spectra [Internet]. Hercules (CA): Bio-Rad Laboratories, Inc., Information Division. C1978-2004 [cited 08.12.13]. Available from: 〈http://www.bio-rad.com/en-us/product/ir-databases〉.