2009) 4 (22مجلة ابن الهیثم للعلوم الصرفة والتطبیقیة المجلد دراسة تكوین معقد بین حامض الفولیك والفلورین باستخدام مطیافي األشعة تحت الحمراء واألشعة فوق البنفسجیة د كفاء خلف حمود ، ماهر محمود محمد ، عبد اللطیف محمد رؤوف ، سلوان سفیان إبراهیم ، دری عیسى زناد وزارة العلوم والتكنلوجیا ،مركز البحوث الكیمیائیة، بغداد العراق الخالصة للمركبـات الهیدروكربونیـة متعـددة الحلقـات انموذجـاأظهرت دراسة تكون معقد بین حامض الفولیك والفلورین سـم2401االروماتیـة مـن خـالل ظهـور حزمــة أشـعة تحـت الحمـراء جدیـدة فــي -1 فـي حلقــة NH2–C=Nد لجـزء تعـو –للتمـازج فــي نظـام واهــب ) نــم 278، 217( البتیـرین لحـامض الفولیــك ونـشوء قمتــي امتـصاص أشــعة فـوق البنفــسجیة فـي لـیس فقـط عملیـة التحــول مــن ثـم ن تكـون المعقـد غیـر بالــضرورة توزیـع الـشحنة مـع المحـیط وأالنتــائج تؤكـد . π-π مـستقبل .ركبات الهیدروكربونیة متعددة الحلقات االروماتیةالحیوي للحامض بل وسمیة الم IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (4) 2009 Folic Acid With Fluorene: A Complexation Study by UV and FTIR Spectroscopies K. K. Hammud, M. M. Mohamme d, Abdul L. M. Raouf, S. S. Ibrahim, D. E. Znad Ministry of Science and Technology, Centre of Chemistry Research, Baghdad, Iraq Abstract The comp lexation between folic acid and a typ ical p oly aromatic hy drocarbon, fluorene, was investigated usin g FT IR and UV sp ectra. App earance of a new IR b and at 2401cm −1 demonst rates t hat NH2–C=N moiety on p terin ring in fo lic acid is p rotonated when fluorene is introduced. The emergence of two charge transfer bands at 217 nm and 278 nm in UV differ ence sp ectra shows the p resence of π-π comp lexation between fo lic acid and fluorene. These exp eriments confirm t hat fluorene could comb ine with the pterin ring of folic acid through π-π donor– acceptor interaction and induce the p rotonation p rocess in folic acid up on st rengthening electron accep ting ability of pterin ring. The results suggest that comp lexation between fluorene with folic acid necessarily change their char ge distribution and the surroundings. It is inferred that not only biotransformation p rocess of folic acid, but also t he toxicity of p oly aromatic hydrocarbons could be chan ged. Introduction Folic acid is comp osed of p - amino benzoic acid, glutamic acid, and p terin ring (see scheme -1- ). Folic acid, which p lay s a key in one – carbon metabolism, is essential for biosy nthesis of several comp ounds. The p terin ring changed by reductase enzy me to tetrahy drofolate which receives one carbon fragments from donors ( monocarbonic units)such as serine, glycine, and histidine then transfers them to intermediates in the sy nthesis of amino acids, p urines, t hy mine, and p y rimidine found in DNA.[1,2] Poly aromatic hy drocarbons are a class of electron rich aromatic p ollutants with three or more fused benzene rings that are widesp read in natural or artificial forms. So me of them are known t o be muta genic and/or carcinogenic. Transformation and toxicity of Poly aromatic hy drocarbons which were strongly depended on the surroundings was demonst rated.[3-6] The formation of π-π comp lexes between fluorene and some model humic _-acceptor subunits such as O-p henanthroline, py ridine had been invest igated and then exp lained the dependence of the environ mental transformation of p olyaromatic hy drocarbons on the surroundings. [7] Y.Y. He et al [8] reported that Toxicity and transformation p rocess of p olyaromatic hy drocarbons is strongly dep ended on the interaction between p olyaromatic hydrocarbons and the coexisting comp ounds. Complexation between fo lic acid and a typ ical Poly aromatic hy drocarbon, anthracene was invest igated using FT IR and UV sp ectra. App earance of a new IR band at 2362cm −1 demonstrates that NH2–C=N moiety on pterin ring in folic acid is p rotonated when anthracene is introduced. The shift of the characterist ic IR band of the pterin ring and the emergence of two charge transfer bands at 254 nm and 246 nm in UV diff erence sp ectra show the p resence of π-π comp lexation b etween folic acid and anthracen e. These exp eriments confirm that anthracene could combine with the pterin ring of folic acid through π-π donor–accepter interaction and induce the p rotonation p rocess in folic acid up on st rengthening electron accepting ability of pterin ring. IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (4) 2009 The aim of this work is to study the comp lexation between folic acid and another typical p olyaromatic hy drocarbon, fluorene, as a representative comp ound of them by using FT IR and UV sp ectroscop ies. Experime nt M aterials (All materials were from BDH Chemicals Ltd Poole, En gland) 0.01 g fluoren e was dissolved in 100 ml methanol, and the sup ernatant liquid was taken out as fluorene saturated solution. 2.50×10 −4 mol l −1 fo lic acid st ock solution was p repared by dissolving 0.0221 g fo lic acid in 1:1 methanol/water (v:v) or water in 200 ml volumetric f lask. Also HCl, NaOH were used for p H adjustment. Ex perime nt methods [8] FT IR sp ectroscopy : M icroscop ic FT IR-sp ectra of the comp lex formed between folic acid and fluorene as well as the sp ectra of folic acid and fluorene were recorded at FTIR sp ectrop hotometer (IR prestige -21, Sh imadzu, Japan) after packed with p otassium bromide at room temp erature. Solid folic acid and fluorene were used directly to obtain microscopic FT IR sp ectra, while the samples of complex were prepared by mixing the ap p rop riate quantity of folic acid with fluorene. UV absorp tion sp ectroscopy : UV absorp tion sp ectra have often been used to st udy the st ructure of the complex. UV absorp tion sp ectra were recorded as follows: eight of 5.0×10−5 mol l −1 fo lic acid workin g solutions in 1:1 methanol/water (v:v) were resp ectively adjust ed to p H 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 10.0 with HCl and NaOH, which were tagged as group A. Then 50μl fluorene saturated solutions were resp ectively added into each 10ml of the solutions from group A, which were tagged as group B. Fluorene solution of the same concentration without folic acid were used as sp ectra control. The UV absorption sp ectra of the test solutions were recorded usin g a UV–Vis sp ectrop hotometer (UV-1650PC, Shimadzu, Jap an) against a solvent blank (for group A) or fluorene blank (for group B). S cheme-1-: S tructures of folic acid  Results and Discussion Exact information about the functional group s involved in the interaction p rocess would be given by the comp arison of FTIR of folic acid with that of the p roduct after interaction with fluorene. As shown in Fi gur e -1-, the bands for p ure folic acid between 3600 - 3400cm −1 are du e to the hy droxy l (OH) stretching bands of glutamic acid moiety and NH group of pterin ring. The stretching v ibration peak of C=O appears at 1696cm −1 , while the  T he Merck Index ,2000 by Merck & Co Inc., Whitehouse station, NJ, USA N H N N NH2N O H N H N COOH O COOH p - Amino benzoic acid Pt erin ring IBN AL- HAITHAM J . FO R PURE & APPL. SC I VO L.22 (4) 2009 band at 1607cm −1 r elates to the bending mode of NH vibration. The bands between 1511 – 1482 cm −1 are attributed to characterist ic absorption band of the p heny l and pterin ring.[9] When folic acid coexists with fluorene as shown in Fi gur e- 1-, a new absorp tion band appears at 2375cm −1 , and it is ascribed to N + –H st retching vibration band of C= N + H on pterin ring.[10] The appearance of the new band demonst rates that the N atom on pterin ring is p rotonated when fluorene coexists. Because there is no other source of the proton, the change of the IR bands would be caused by the proton transfer from carboxy l at glutamic acid moiety to N atoms at pt erin ring. It is well known that the lone-p air electrons on N atoms at pterin ring are not conjugated with ring π sy stem, so they are cap able to combine with p roton to p roduce p ositive salt. The shift of IR bands at 1511 and 1482cm −1 suggests that p henyl or pterin ring is also involved in the interaction of folic acid with fluorene [11]. Research performed has de monstrated that a p lanar molecular with rich electrons, such as p oly aromatic hy drocarbons, can interact with opp osing sy stem to p roduce comp lexes named as π-π electron donor–accep ter sy stem. [12]. Pt erin ring, a p lanar N-heterocy clic (se e scheme -1-) , would p arallel the p lanar π system of fluorene to p roduce a comp lex by π-π electron donor–accepter interaction through suggested face to face geometry [8]. In consequence, the electron clouds would deflect from fluorene to p terin ring, and then the p roton-accepting ability of N atoms could become st ronger so that the p roton transformation from the carboxy l of glutamic acid to N atoms happens. The interaction between fo lic acid with fluorene is st udied by using the UV sp ectra and UV difference sp ectra as disp lay ed in Figure -2- UV sp ectra of folic acid shows a strong absorb pterin ion band around 283 nm during p H 3.0–8.0 , and it is assigned to the π-π* transition of p terin ring.[13] The dominant form of p terin ring of folic acid is as shown in Scheme -1- dur ing p H 3.0–8.0, and the other acid or basic form could be ignor ed because the p Ka values of the pterin ring are 8.1 and 2.4 [14], which are r esp ectively ascribed to the dissociation of the p rotonated group s of O=C–N and NH2–C=N. This is consistent with the fact that its max of the UV band is remainin g unchan ged during p H 3.0–8.0. Absorption band of fluorene at 289 nm is assi gned to π-π * transition of conju gated π sy stem and it is not interfered by p H. When folic acid and fluoren e coexist, the UV sp ectra against fluorene b lank shows the app earance of two charge transfer bands at 217 and 278 nm during p H 3.0–8.0.T hese two bands are the evidence of the π-π comp lexation between folic acid and fluorene, and they are ascribed to t he dist urbance of the comp lexation on π-π* transition of conjugated π sy stem. The intensities of charge transfer bands should be p rop ortional to the interaction st rength, and the interaction st rength should be p ositive correlation with the p rotonation degr ee of π accepter, so the intensities of charge transfer b ands would be relative to the p rotonation degree of N-atoms on the pterin moiety . The p Ka value 2.4 of the p rotonated group NH2–C=N on p terin ring is much smaller than the inflection. This fact p roves that t he comp lexation between folic acid and fluorene indeed chan ges the char ge distribution of the folic acid, esp ecially the pterin ring. Consequently , the proton accep ting ability of pterin ring increases. Re ferences 1.Lip p incott,s Illustrated Reviews (2005): Biochemistry , Champ e ,P. C.; Harvey , R. A. and Ferrier, D. R. (Editors), Lip p incott Willams & wilkins, USA. P. 372, 2. Birdsall,B.; Feeney , J. S Tendeler,J.B.; Hammond,S.J. and Roberts, G.C.K.(1989), Biochemistry 28:2297–2305. IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (4) 2009 3. Noordman,W.H.; Bruinin g,J.W. ; Wietz es,. D.and Janssen, B. (2000), J. Contam. Hy drol. 44: 119–140. 4. Gensemer, R.W. ; Di xon,D.G.; B.M . Greenberg (1998), Ecotoxicol. Environ. Safety 39:57–64. 5. Chi,F.H. and Amy , G.L. (2004), Chemosp here 55: 515–524. 6. Akkanen, J. ; Pentt inen, S.; Haitzer, M . and Kukkonen ,J.V.K. (2001), Chemosp here 45: 453–462. 7. Wijnja,H. ; Pi gnatello,J.J.and M alekani ,K. (2004), J. Environ. Qual. 33 :265–275. 8. He,Y.Y. ; Wang,X.C. ; Jin b,P.K. and Zhao,Bo Xiaoyuan Fan (2009), Sp ectrochimica Acta Part A 72 :876–879. 9. Zhang,J. ; Rana, S. ; Srivastava,R.S. and M isra, R.D.K. (2008), Acta Biomater 4:40–48. 10. Bellamy L.J. (1980), The Infrared Sp ectra of Comp lex M olecules, Chapman and Hall, London. 11. Szakács,Z. and Nosz ál B. (2006), Electrop horesis 27:3399-3409. 12. M ulliken ,R.S. (1952), J. Am. Chem. Soc. 74: 811–824. 13. Thomas,A.H.; Suلrez,G. ; Cabrerizo,F.M . ; M artino,R. and Capp arelli ,A.L. (2000), J. Phot ochem. Photobiol. A 135:147–154. 14. Albert,A. (1953) Biochem. J. 54 646–654, He,Y. Y. ;Wan g,X.C. ; Jin b, P.K. and Zhao, Bo Xiaoyuan Fan (2009), Sp ectrochimica Acta Part A 72: 876–879. IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (4) 2009 Fig. (1): IR spectra of fluorene and foli c acid before and after interaction with fluorene . 400600800100012001400160018002000240028003200360040004400 1/c m 35 37,5 40 42,5 45 47,5 50 52,5 55 57,5 60 62,5 65 %T flourene + folic acid 400600800100012001400160018002000240028003200360040004400 1/c m 45 47,5 50 52,5 55 57,5 60 62,5 65 67,5 70 72,5 75 77,5 %T 34 12 ,0 8 33 25 ,2 8 31 13 ,1 1 29 24 ,0 9 2 84 3, 07 28 06 ,4 3 23 76 ,3 0 16 95 ,4 3 16 04 ,7 7 13 38 ,6 0 11 05 ,2 1 91 4, 26 76 5, 74 59 4, 08 folic acid Folic acid 400600800100012001400160018002000240028003200360040004400 1/c m 20 25 30 35 40 45 50 55 60 %T flourene FLUORENE FLUORENE+FOLIC ACID 2375cm -1 IBN AL- HAITHAM J. FO R PURE & APPL. SC I VO L.22 (4) 2009 Fig.(2): UV absor ption spectra of fluorene , folic acid, and the ir mixture against fluorene blank respectively 0.01g fluore ne in 100 m l m etha nol Folic a cid a dj usted to pH 2- 10 50μl fluore ne adde d to 10ml (5x10 -5 m ole/l) folic a cid a t pH 2-10 278nm 217 nm