Synthesis, characterization and film formation of modified riboflavin

MaterialsScience and Engineering: C 3 (1995) 273-276

Synthesis, characterization and film formation of modified riboflavin Anant B. Patel, Ratna S. Phadke * Chemical Physics Group, Tata Institute of Funafamental Research, Homi Bhabha Road, Bombay 400 005, India

Abstract Riboflavin (RBF) is not naturally imbued with an amphiphilic character. To impart amphiphilicity the hydroxyl groups of the ribose moiety have been esterified using steak acid, The compound thus modified, 2’,3’,4’,5’-tetrastearoyl riboflavin (SRBF), has been characterized using optical absorption and fluoreslcence spectroscopy, NMR and cyclic voltammetry. Thin filmsof SRBFhave been deposited on IT0 glass using the LB technique. These films exhibit fluorescence properties which are similar to RBF in solution. X-ray diffraction results indicate that the molecules are organized in a highly ordered fashion. Keywords:

Riboflavin; Langmuir-Blodgett film; Isoalloxazine;Synthesis;Characterization

1. Introduction The relentless decrease in the size of solid state electronics components has aroused interest in developing suitable molecular materials [l-3]. This, in turn, has activated research regarding supramolecular assemblies and tailoring of organic/bio molecules to attain desirable properties [ 4,5]. Appropriate interfacing of the molecular microworld to the solid state macroworld is envisaged to lead to the successful fabrication of photochromic and electronic devices, switches, biosensors, etc. [ 61. Flavins are commonly found in most biological tissues. The most important flavins are riboflavin (RBF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The common structural feature of all these molecules is the presence of an isoalloxazine ring. The isoalloxazine ring exists in two stable states: the oxidized and the reduced form. Switching from one state to the other can be achieved chemically as ,well as electrochemically [ 71. RBF does not possess amphiphilic characteristics. To impart amphiphilicity to RBF it is neccessary to modify it by attaching nonpolar groups. We have achieved this by esterification of the hydroxyl groups of the ribose moiety with stearic acid. Characterization of SRBF has been carried out using optical spectroscopy, NMR spectroscopy and cyclic voltammetry. Forty-one layers of SRBF have been deposited on IT0 using the LB deposition technique [ 81. The LB film has been characterized by fluorescence spectroscopy and Xray diffraction techniques.

* Correspondingauthor. 0928-4931/95/$09.50 0 1995Ekvier Science S.A. All rights reserved SSDZO928-4931(95)00083-6

2. Materials and methods

RBF and stearic acid were purchased from Sisco Research Laboratories India. All other ingredients used were of AnalaR grade or better. De-ionised water having a conductivity of 0.5 fi was used. A Bruker AMX 500 spectrometer was used for 13CNMR experiments. Indium-tin oxide (ITO) glass was used as the substrate for the deposition of LB film. A Shimadzu W-Visible Spectrophotometer W-2100 was used for absorption studies and a Shimadzu Spectrofluorometer RF-540 for fluorescence investigations. LB deposition was performed on a KSV-3000 LB deposition system. X-ray diffraction studies were done on a JEOL JDX-8030 X-ray diffractometer.

2.1. Synthesis of SRBF

Esterification of RBF was carried out by the following method. RBF and stearic acid were mixed in the molar ratio 1:4 in the presence of small amounts of concentrated sulphuric acid. The reaction was carried out at 343 K for about 4 h. The reaction mixture was then mixed with ice-cold water and filtered. The residue was washed with sodium bicarbonate solution to remove unreacted stearic acid and RBF. The crude product was recrystalized using ethanol as a solvent to obtain pure SRBF. The purity of SRBF was checked by TLC, using different compositions of ethanol and chloroform as the mobile phase.

214

A.B. Patel, RX Phudke /Muteriuls

El 4’

-COOH

Science arId Engineering:

C 3 (1995) 273-276

C,H20-CO-CH,-(CH,),5-CH3

3

H-C-O-CO-CH2-(CH21,5-CHj I H- q-0-CO-CH2-1 CH,),, -CH3

2’

H-

-CH2,

-CH3

C-0-CO-CH,-(CH,l,,-CH,

-CH,

/

<

Flown

ring

,

Ring

I

Fig. 1. 125.721 MHz broad band proton decoupled 13CNMR spectrum of unpurified SRBF in CDCls at 298 K. Accumulated with relaxation delay 2.5 s, referenced with respect to external TMS. Inset is the molecular structure of SRBF. Assignments of peak positions as indicated [IO].

3. Results and discussion 3.1. Characterization of SRBF 3.1.1. Solubility and TLC RBF is soluble in water and almost insoluble in nonpolar organic solvents such as chloroform and carbon tetrachloride. SRBF, on the other hand, was found to be marginally soluble in water and readily soluble in chloroform. Thus, solubility considerations indicate that SRBF possesses hydrophobic characteristics. TIC was performed in various compositions of ethanol and chloroform. The most interesting thing to note is that it shows a single sharp spot irrespective of solvent composition. This clearly indicates that the preparation consists of single species of SRBF. 3.1.2. NMR Proton decoupled 13C NMR spectra have been recorded for stearic acid and SRBF dissolved in deuterated chloroform [9,10]. Whereas the carbonyl belonging to stearic acid appears at around 180 ppm, the spectrum of unpurified SRBF shows an additional peak at 174 ppm which is characteristic of carbonyl attached to the ester group (Fig. 1) . This confirms the formation of an ester bond in the compound. 3.1.3. Cyclic voltammetry It is reported that flavins show two redox peaks around - 0.44 V and - 0.76 V respectively. A cyclic voltammetry experiment was performed in a home-built potentiostat having a saturated calomel electrode (SCE) as reference elec-

0

-0.4

-0.8

Voltage/V Fig. 2. Cyclic voltammogram of SRBF: 0.1 M tetrabutylammonium perchlorate in acetonitrile was the supporting electrolyte. Scan rate 50 mV s-‘.

trode, a platinum wire as the working electrode and a platinum mesh as the counter electrode [ 1 l-141. Tetrabutylammonium perchlorate (0.1 M) in acetonitrile was used as a supporting electrolyte. The voltammogram is depicted in Fig. 2.

A.B. Patel, R.S. Phadke / Materials Science and Engineering: C 3 (1995) 273-276

Table 1 Absorption

maxima of RBF and SRBF in different media

RBF SRBF

Solvent

AI (nm)

A2 (nm)

A3 (nm)

Water Ethanol Ethanol Chloroform

267.0 266.5 255.5 264.0

369.5 352.5 342.0 350.0

444.0 445.5 448.0 445.0

T40-

2 Q,

5 iii

2 a

g YL 2

3o \

20IO-

o-

I I.0

0.5

Area Fig. 3. Surface pressure--area

1.5

2.0

per moleculehm

3.2. LBfilm deposition

b

L

z

3

!! s G

20.0

I

0.0

0.0 500.0

600.0

Wavelength

700.0 (nm)

500

I.0

3.1.4. Optical spectroscopy The results of the absorption study of RBF and SRBF are summarized in Table 1. The absorption spectrum of RBF in water shows three absorption bands around 267.0,369.5 and 444.0 nm [ 71. The absorption spectrum of SRBF in chloroform exhibits three bands in the region 250-600 nm which are centered at 264.0, 350.0 and 445.0 nm. The 264.0 and 445.0 nm band positions approximately match with those of RBF. The 369.5 nm band is blue shifted, probably due to the inability of SRBF to form hydrogen bonds and a stable charge transfer complex with chloroform. Whereas in ethanolic solutions RBF exhibits peaks at 266.5,352.5 and 445.5 nm, SRBF has peaks at 262.7,342.0 and 448.0 nm. The large blue shift in 352.5 nm band (about 10 nm) of RBF can be attributed to the tendency of long chain molecules (SRBF) to form aggregates. In general, one can conclude that modification of RBF does not significantly alter the spectral characteristics of the flavin moiety. The fluorescence emission spectra of RBF and SRBF in ethanol have been recorded under identical conditions (excitation and emission slit width 10 nm and temperature 298 K) with excitation at 450.0 nm. Emission peaks in both cases appear at 530.0 and 620.0 nm. Thus, fluorescence study also confirms that modification of RBF by esterifying hydroxyl groups of the ribose moiety does not significantly alter its electronic characteristics.

*

isotherm of SRBF at 294 K.

0

215

I.0

600.0

Wavelength

(nm)

Fig. 4. Fluorescence spectrum with excitation at 450 nm. (a) Riboflavin in aqeous solution, (b) 41 layers of SRBF LB film ‘Y’ type deposition at 25 mN m-‘.

It shows a reduction peak at - 0.34 V and a less intense peak at -0.76 V. In the revers,e cycle an oxidation peak is not observed, probably due to preferential adsorption of oxidized SRBF on to the electrode surface. This indicates that esterification of the compound has not adversely affected the redox characteristics of the flavin ring.

A monolayer of SRBF was formed at the air-water interface by spreading 200 ml of chloroform solution of SRBF (1.5 mg ml-‘) and allowing it to stabilize. The film was compressed at the rate of 1 cm* min- ‘. The surface pressuremolecular area isotherm is depicted in Fig. 3. The isotherm initially shows a gradual rise corresponding to a highly compressible gaseous state. When the pressure becomes greater than 2 mN m- ’ the slope of the curve starts increasing rapidly, corresponding to the liquid phase. At 25 mN m-’ the curve becomes abruptly parallel to the surface pressure axis (small compressibility) which indicates the beginning of the “solid” phase. The stability of the monolayer at a surface pressure 25 mN m -’ was checked and was found to be excellent for a period of 10 min and longer. Forty-one layers of SRBF were deposited on an indium-tin oxide (ITO) coated glass surface. The transfer ratio was found to be close to unity. 3.3. Characterization of LBfilm The fluorescence spectrum of LB film with excitation at 450.0 nm is shown in Fig. 4. The spectrum consists of an intense peak around 528.0 nm and a broad less intense peak at 630.0 nm. One notices that the intense peak has characteristics similar to those of RBF in water. This indicates that emission characteristics of the flavin moiety does not change by organizing it in a well defined and ordered fashion. The

A.B. Patel. R.S. Phadke /Materials Science and Engineering: C 3 (1995) 273-276

276

characteristics similar to the native molecule. SRBF can be used to prepare stable LB films.

600

Acknowledgements

0

I 3.0

8.0

13.0

18.0

23.0

The authors would like to thank Dr. A. Dhanabalan of the Chemistry Department, IIT Bombay, for technical support. The facilities provided by the High Field NMR National Facility supported by DST, DBT, CSIR and TIER, Government of India, located at TIFR, Bombay are gratefully acknowledged.

2 8 (deg.1 Fig. 5. X-ray diffraction of 41 layers of SRBF LB film on IT0 surface. Cu Kcur\ = 0.154 nm.

appearance of the broad peak in curve (b) may be attributed to intermolecular interactions of fluorophores in the solid phase which results from the juxtapositioning of fluorophores belonging to the consecutive monolayers. X-ray diffraction is diagnostic of the uniformity of the organization of LB films [ 151. Fig. 5 depicts the X-ray diffraction pattern of LB film of SRBF. One can see several well defined regularly spaced intense peaks for 28 ranging from 4” to 20”, demonstrating the periodic structure of the film. The peak intensity alternates and decreases with 28. Weakening of higher order peaks can be attributed to well organized assembly of the hydrocarbon chains which is a characteristic feature of X-ray diffraction patterns of LB films. The observed spacing of the lattice repeat distance, 5.36 f 0.14 nm, corresponds to approximately twice the chain length of SRBF long axis. This indicates that the repeat unit is a bilayer. This, in turn, confirms the ‘Y’ type deposition. Thus we can conclude that esterification of RBF has resulted in an amphiphilic molecule which exhibits spectral

References [ 11 F.L. Carter (ed.), Molecular Electronics Devices II, Marcel Dekker, New York, 1987. [2] G.J. Ashwell, Molecular Electronics, John Wiley, New York, 1992. [3] F.T. Hong (ed.), Molecukrr Electronics: Biosensors and Biocomputers, Plenum, New York, 1989. [4] H. Kuhn, Phys. Rev. A, 34 (1986) 3409. [5] J.M. Lehn, Angew. Chem. Int. Ed., 27 (1988) 89. [6] F.T. Hong (ed.). Molecular electronics, science and technology for the future, IEEE Eng. Med. Biol., Sot. Msg., Special issue on molecular electronics., 1994. [7] J. Koziol, Methods Enzymol., 18B (1971) 257. [8] G.G. Roberts (ed.), Langmuir-Blodgett Films, Plenum, New York, 1990. [91 E. Breitmaier, G. Jung and W. Voelter, Angew. Chem. Int. E&L Engl., IO (1971) 679. [ 101 E. Breitmaier and W. Voeiter, Eur. J. Biochem., 31 (1972) 234. [ill V.R.BhagatandK.S.V.Santhanam,J.Sci.Ind.Res.,30(1971)235. [121 F. Sheller, G. Giiard, B. Neumann, H. Kuhn and W. Ostrowski, Bioelectrochem. Bioenergy, 6 (1979) 117. [ 131 B. Janike and P.J. Elving, J. C/rem. Rev., 68 (1968) 312. [141 R.M. Lanniello, T.J. Lindsay and A.M. Yacynych, Anal. Chem., 54 (1982) 1098. [ 151 L. Vittarorio, X-ray diffraction of lipid water system, in D. Chapman (ed.), Biological Membranes, Academic Press, 1969, p. 71.