Fluorescence Quenching and Enhancement by H.doc

Fluorescence Quenching and Enhancement by H-bonding Interactions in Some Nitrogen Containing FluorophoresCHANDAN TAMULY, NILOTPAL BAROOAH, MOITREE LASKAR, RUPAM J. SARMA and JUBARAJ B. BARUAH*Department of Chemistry, Indian Institute of Technology, Guwahati 781 039, India(Received 26 October 2005, 9 March 2006; Accepted 10 March 2006)一些含氮荧光团由于氢键相互作用而产生的荧光猝灭和增强The benzodebenzo4,5imidazo2,1-aisoquinolin-7-one (1) shows solvent dependent fluorescence emission.The compound shows quenching of fluorescence emission on interaction with polyhydroxy compounds such as 1,3-dihydroxybenzene, 1,4-dihydroxybenzene. The result on fluorescence quenching is attributed to the hydrogen bonding interactions. Similar observation of fluorescence quenching by protic solvent is observed with isoindolo2,1-a perimidin-12-one (2). In the case of 2-(2-amino-phenyl)-isoindole-1,3-dione (3) enhancement of fluorescence emission in methanol solution is observed over benzene solution. This is explained on the basis ofintermolecular hydrogen bonding. The crystal structure of adduct of 1 with 1,3-dihydroxybenzene, 1,4-dihydroxybenzene respectively and 3 are reported.Keywords: Fluorescence; Quenching; H-bonding; Solvatochromicity苯并二苯并 4,5 咪唑 2,1 - 异喹啉 - 7 -酮表明溶剂依赖荧光发射。这个化合物表明在与多羟基化合物反应的荧光发射的猝灭如1,3 -苯二酚， 1,4苯二酚，结果表明荧光猝灭是由于氢键相互作用。类似的观察荧光猝灭是由质子溶剂观察isoindolo2,1-a perimidin-12-one (2).如2-(2-amino-phenyl)-isoindole-1,3-dione (3)在甲醇溶液中加强荧光发射是在苯溶液观察的。这个解释是建立在分子间氢键的基础上。1分别与1,3 -苯二酚，1,4苯二酚的加成化合物和3的晶体结构是有报道的。INTRODUCTIONThere is a definite need for understanding enhancement or quenching of fluorescence emission of a fluorophore by externally added substrate/s 1,2.Extensive literature is available on the fluorescence properties of naphthalimide derivatives 39.Several such studies are devoted to identify on and off fluorescence states in these derivatives 1013.However, there are limited studies on understandingfixed geometry responsible for quenching orenhancement of fluorescence emission. This is predominantly because of the fact that such studiesare generally performed in solution. In solution themolecules are relatively loosely bound, and thismakes it difficult to explicitly demonstrate all theweak interactions such as pp or hydrogen bondinginteractions 14. On the other hand in solid state thedifficulty is to identify an organized state that wouldserve as a model. Recently, it is pointed out that insolution pp interaction between aromatic ringsleads to enhancing of electroniccommunicationbetween the two subsystems and such effect isreflected in fluorescence emission of the interactingsystem 15,16. Moreover, the naphthalimide derivatives have biological importance and in analogous molecules it was reported that pp interactions play a decisive role in fluorescence emission 16. We felt that if suitable motifs are identified and their properties in solid state are studied, it will add real value in determining motifs responsible for fluorescence quenching or enhancement. Thus, we have studied the fluorescence properties of a few substrates (17; Fig. 1) with an aim to understand the role of hydrogen bonding in fluorescence emission in these compounds.引言：对于通过在外表上添加底物来理解增强和猝灭荧光团的荧光发射是有明确的需要1,2。有大量的文献是关于萘衍生物的荧光性39。一些此类研究都致力于识别这些衍生物的荧光或非荧光集团。但是，有限的研究都认为荧光的增强和猝灭都是由于固定的空间结构。这主要是因为这一事实，即这类研究通常表现在溶液中。在溶液中的分子受着相对松散的约束，因此，很难明确地表明所有弱相互作用等如或氢键 。另一方面在固态物质中很难一个典型的组织集团。最近，有人指出，在溶液中芳香环之间的相互作用导致两个子系统之间电子效应增强并且这种效应反映了荧光发射的相互作用系统 15,16。此外，萘衍生物具有生物重要性和类似分子性说明它对荧光发射发挥决定性的作用 16 。我们觉得如果合适的主题被确定并且固态性质被研究，这将增加我们决定课题对于荧光淬火或加强真正价值。因此我们已经研究了一些底物的荧光性质(17; Fig. 1)，目的是了解氢键在这些化合物的荧光发射方面的作用。RESULTS AND DISCUSSIONIt is observed that the position and intensity of fluorescence emission (lex 450 nm) of benzode -benzo4,5imidazo2,1-aisoquinolin-7-one (1) issolvent dependent. The fluorescence properties ofthis compound were reported earlier 17 but theeffect of hydrogen bonding on fluorescence emissionwas not studied. The compound 1 on excitation at450nm in methanol shows emission at 507nmwhereas it shows emission at 485nm in benzene(Fig. 2). When the intensity of fluorescence emissionof samples with identical concentrations of 1 inmethanol is compared with a solution of benzene it is found that there is a considerable amount of reduction in intensity of emission in methanol over a benzene solution. Thus, a protic solvent reduces the intensity of fluorescence emission of 1 and also takes emission towards a higher wavelength. To establish this fact we have prepared two hydrogen bonded adduct of 1, namely 4 with 1,3-dihydroxybenzeneand 5 with 1,4-dihydroxybenzene and determined the crystal structures of these two adducts. The benzode benzo4,5Imidazo2,1-aisoquinolin-7-one(1) forms 2:1 adducts 4 with 1,3-dihydroxybenzeneand 5 with 1,4-dihydroxybenzene respectively (Fig.1). The structure of the adduct 4 has severalinteresting features. In the adduct 4 the H-bondingtakes place between two nitrogen atoms of twomolecules of 1 with the hydroxy groups of onemolecule of 1,3-dihydroxybenzene to form a Ushapedstructure (Fig. 3a). The adduct is formed byweak hydrogen bonding interaction between thenitrogen atom and the hydroxyl groups. For theN HZO interaction the donoracceptor bonddistance and bond angles participating in hydrogenbonding in this adduct are 2.884A and 173.968respectively. The N H and HZO distances are1.958A and 0.93A , respectively. In the crystal latticethese interactions allow two molecules of 1 to appearabove each other as stacks (Fig. 3b). In this orderlyarrangement the hinges are provided by 1,3-dihydroxybenzenes. From the top view it can beseen that the molecular arrangement in the lattice issymmetric to a 908 rotation followed by a translationif each pair is considered to be an object to be rotated.There are two types of p-separations between thearomatic rings in the crystal lattice of the adduct 4.The two separations between the aromatic layers areshown in the packing of the crystal lattice as X and Y(Fig. 3b) are 3.581A and 3.621A . These distances are slightly higher than the limit 3.5A that is conventionally found in molecules having pp interaction14,18,19. Thus, one can say that in this system the pp interaction has lesser significance.Similarly, the adduct 5 formed by benzode benzo4,5imidazo2,1-aisoquinolin-7-one with 1,4-dihyroxybenzenehas the nitrogen atom of the benzode benzo2,1-aisoquinolin-7-one bound to a hydroxylgroup of 1,4-dihydroxybenzene. The other hydroxylgroup of 1,4-dihydroxybenzene holds another nitrogenatomof benzode benzo4,5imidazo2,1-aisoquinolin-7-one. In the N HZO interactions the dDAbond distance and , DZH A angle is 2.884A and75.758, respectively (Fig. 4).结果与讨论观察发现苯并二苯并 4,5 咪唑 2,1 - 异喹啉 - 7 -酮荧光发射(ex=450nm)的位置和强度由溶剂决定。关于这种化合物的荧光性质报道更早 17 但是， 氢键作用的荧光发射没有研究。这种化合物在甲醇450nm处激发说明在507nm处发射，而在苯中则是在485nm（见图2 ） 。当强度的荧光发射相同样品相同浓度的化合物1 ，甲醇与苯相比，在甲醇中有相当数量发射强度降低。因此质子溶剂降低了化合物1的荧光发射强度也将发射的波长增长。为了说明这一事实我们准备了两个化合物1的氢键加合物，即4 ，1,3 -苯二酚和5，1,4 -苯二酚，并测定这两个加合物的晶体结构。苯并二苯并 4,5 咪唑 2,1 - 异喹啉 - 7 -酮的形式2：1加合物4 与1,3 -苯二酚和5与1,4 -苯二酚分别（图1 ）。加合物4的结构有几个有趣的特征。在加合物4中氢键是由1中带氢氧根的两个分子的两个氮原子中形成的，在1,3 -苯二酚，形成U型结构（图3A ）。加合物在氮原子和羟基碱形成弱氢键作用。对于N HO共价键的距离和角度都对加合物氢键形成有影响（分别为2.884A and 173.96）。N H和HO键的距离分别为1.958A 和0.93A 。在晶格中这些相互作用使得1的两分子表现出互相堆砌的样子。在这种有序安排取决于所提供的1,3 - 苯二酚。从上方的看法，可以看到分子安排在格中对称90旋转之后，如果每对转化被认为是一个物体旋转。芳香族环晶格的加合物4间有两种分离的类型。这两个分离的芳香层表现在包装晶格的X和Y （图3B ）是3.581A 和3.621A 。这些距离略高于平常键3.5A 的距离 14,18,19 。因此可以说在这个系统中作用具有较少的意义。同样，所形成的加合物5是由苯并二苯并 4,5 咪唑 2,1 - 异喹啉 - 7 -酮与1,4 - 苯二酚形成的，苯并二苯并 4,5 咪唑 2,1 - 异喹啉 - 7 -酮的氮原子与1,4 - 苯二酚的羟基集团成键。1,4 - 苯二酚的另一个羟基集团与苯并二苯并 4,5 咪唑 2,1 - 异喹啉 - 7 -酮的另一个氮原子相连。在N HO作用中dDA 的距离和D HA的夹角分别为2.884A 和75.758 ，分别为（图4 ） 。The fluorescence emission spectra of the compounds 1, 4 and 5 in solid state were measured by exciting at 450 nm. In the solid state the parent compound 1 on excitation at 450nm has a fluorescence emission at 493nm with a shoulder at 550 nm. Whereas, the adduct 4 has a very feeble fluorescence emission at 530nm (Fig. 5) (lex 450 nm). The adduct 5 also shows a very weak fluorescence emission at 535nm on excitation at 450 nm. From crystallographic study it is evident that there are no pp interactions among the parent fluorophores. So, it may be suggested that by formation of H-bonded adducts, the inter-motif interactions that would have originally been present in the fluorophore are changed. This results in the modification of the energy states, which lowers thefluorescence intensity.We also examined the concentration dependent 1HNMR spectra of the compound 1 in acetonitrile (please refer to supplementary material). No significant changes in the chemical shifts or coupling pattern of the 1HNMR signals on change of concentration except broadening of the signals due to dipolar interactions was observed. This indicates no significant pp interactions among the molecules of 1 in solution also. Alternately,acetonitrile does not change the electronic environment of the molecule 1, and this result also adds to the fact that it does not quench fluorescence of 1 but behaves in a manner similar to that of benzene. The compound isoindolo2,1-a perimidin-12-one (2) is a structural isomer of 1 but has a differentheterocyclic ring due to a difference in the position ofthe nitrogen atoms and carbonyl groups in the ring.The fluorescence emission of 2 on excitation at 540nm is sensitive to solvent. For example, in methanol it shows poor fluorescence emission (Fig. 6)whereas, a solution of 2 in benzene (identicalconcentration) shows emission at 592 nm, which isabout 12-fold higher in intensity than that ofmethanolic solution. The emission of an acetonitrilesolution has an intermediate effect on the intensity ofemission. Thus, the solvent capable of H-bonding such as methanol reduces the intensity of emissiondrastically. To check the possibility of identifying thetype of interactions among the molecules of 2 indifferent solvents the concentration dependent UVvisible spectra were recorded in three differentsolvents namely benzene, methanol and acetonitrile.It was found that the slopes and trends of variation ofconcentration versus absorbance are different foreach solvent. This suggests that the intermolecularsolute solvent interactions causes changes in thespectra and such effects are well documented inthe literature 20. In case of a protic solvent the hydrogen bonded structure changes the originalintermotif interactions and one can see the quenchingof fluorescence due to modification of the ground state. 化合物1 ，4和5固态的荧光发射光谱经测是在450 nm左右处被激发。固态的初始化合物1在450nm激发，在550nm的肩膀493nm荧光发射。鉴于加合物4具有非常虚弱荧光发射在530nm（图5 ）( ex=450nm).加合物5还在535nm显示了非常弱荧光发射在450nm出激发。从晶体学研究很显然，在母体荧光团间并没有作用。因此，可以认为，由形成的H 键加合物，跨序的相互作用，将原来本在荧光的变化。这样的结果使得降低荧光强度的能量集团得到校正。我们还研究了化合物1在乙腈中不同浓度的核磁共振光谱（请参阅补充材料） 。在化学变化中没有重大变化和耦合模式的核磁共振信号由于偶极作用除扩大信号外还改变浓度的说法。这表明在化合物1在溶液中分子间也没有显著的作用，另外，乙腈不会改变分子1电子环境的 ，这一结果也使它不猝灭荧光，但表现的方式类似苯成为事实。 化合物isoindolo 2,1 - perimidin - 12 - 1 （ 2 ）是一个结构性的异构体1 ，但具有不同的杂环由于不同的位置，氮原子和羰基在环内。化合物2的荧光发射激发540nm且对溶剂敏感。例如，在甲醇这表明弱荧光发射（图6 ）而化合物2在苯（相同浓度）显示发射在592nm，这是约12倍的强度比甲醇的溶液。乙腈溶液的发射对发射的强度起媒介作用。因此溶液形成氢键的能力如甲醇大大降低了发射强度。检查的可能性，确定类型之间的相互作用的分子2在不同溶剂中的浓度依赖性紫外可见光谱记录了三种不同的溶剂即苯，甲醇，乙腈。结果发现，每个溶剂斜坡和趋势的变化，浓度与吸光度不同。这表明，分子间溶质溶剂的相互作用造成的光谱变化，而这种影响是有据可查的文献 20 。如果质子溶剂的氢键结构改变了原始的相互作用和看到的荧光猝灭是由于修改的基态。To further strengthen our observations, we studiedanother related molecule 2-(2-aminophenyl)-isoindole-1,3-dione (3) that has a phthalimide ring as wellas an amino group. The position of the amino groupallows it to have conjugation over the two rings. Theprotonation of the amino group of this moleculeaffects the intermolecular hydrogen bonding. Westudied the visible spectroscopic and fluorescenceemission spectra of this compound (3) in differentsolvents. It was found that the compound hasdifferent trends in the concentration dependentprofiles (Figs. 7 and 8) in the UV-spectra in differentabsorption intensity and shifts the absorption to ahigher wavelength. The concentration dependentUV-spectra shows that in methanol the concentrationversus absorbance has a regular growth as expectedfrom Beers Lambert law (Fig. 7, inset) but in the caseof benzene as a solvent there is deviation from thestraight line that would pass through the origin (Fig.8, inset). This may be due to the fact that a proticmedium disrupts the intermolecular hydrogenbonding among the individual molecules whereasbenzene being a non-protic solvent allows intermolecularhydrogen bonding between the moleculesof 2-(2-aminophenyl)-isoindole-1,3-dione. The effectis reflected in the fluorescence emission spectra of thecompound (3) in different solvents (lex 435 nm)also. In methanol the compound shows emission at505 nm, in acetonitrile it emits at 487 nm, whereas itemits at 471nm in benzene (Fig. 9). The observedintensity of emission in different solvents are in the order methanol . acetonitrile . benzene. Thisindicates that the solvent polarity as well ashydrogen bonding may be responsible for such aprocess. Methanol forms hydrogen bonds with the amino group, thus changing the magnitude of intermotif interactions. To distinguish the effect of protonation from H-bonding interactions, we recorded the visible spectra of 3 in the presence ofhydrochloric acid in methanol. The addition of acid to the methanolic solution of the compoundimmediately showed a new absorption peak at456nm with an intensity comparable to the absorption of the parent compound. The effect of addition of acid is also reflected in the fluorescence emission.On excitation of an acidic solution of 3 in methanol at 450 nm it showed altogether a new but very low intensity emission at 572 nm. These results clearly indicates that protonation of the amine quenches the emission in the case of 3. However, the observation on enhancement of intensity of fluorescence by methanol in the parent compound shows that the role of an acid and a protic solvent are totally independent. It may be concluded that hydrogenbondingwith the ZNH2 group with methanol causes disruption of intermolecular hydrogen bonding incompound 3 but does not generate a protic state. The structure of the molecule 3 in solid state hasintermolecular hydrogen bonding between ZNH2groups with the carbonyl group of other molecules.The IR spectra in solid state clearly suggest the presence of intermolecular hydrogen bonding. In theIR spectra of 3 two very sharp absorptions at3462 cm21 and 3385 cm21 due to intermolecular hydrogen bonding are observed. The effect can beexplained by the scheme shown in Fig. 10a.Methanol being a protic solvent cause disruption ofsuch hydrogen bonding and this leads to fluorescenceenhancement. This suggests that the intermolecular hydrogen bonding among 3 is favoured inthe case of benzene. The disruption of H-bondingmakes the nitrogen atom more electron rich to pumpin electron density to the aromatic ring which causesenhancement of intensity. The ability of water toexchange with the NH2 group of 3 is revealed fromthe concentration dependent 1H NMR of thecompound 3. We have recorded the 1H NMR of thecompound 3 in CD3CN at a different concentrationand found that the residual water in the acetonitrilecould be distinguished from NH2 group at lowconcentration (refer to supplementary data). Theposition of the NH2 group was further ascertained byrecording its 1HNMR spectra in CDCl3. At higherconcentrations it was found that the residual waterand the NH2 signals cannot be distinguished as theyappear as a broad signal. This shows that rapidexchange of NH2 protons and water protons takesplace at higher concentration, whereas at lowconcentration the exchange is less. This is obviousas the probability of interaction between two solutecomponents in a dilute solution decreases. Althoughthis result is not conclusive to depict intermolecularhydrogen bonding among the molecules of 3, it doestell us that the compound 3 prefers hydrogen bondformation with water. The fluorescence lifetime ofthe excited state of 3 was determined in methanoland benzene. It has a lifetime of G 45 ns andG 27 ns, respectively. Thus, the effect may bepurely due to the hydrogen-bonding effect where theexcited state is stabilised by hydrogen-bonding asthe methanol has a longer lifetime. It is interesting tonote that Licchelli et al. 21 had observer fluorescencequenching and enhancement of 1,8-naphthlimide-tethered iminopyridine ligands by addingcopper(II) and zinc(II) ions respectively. The resultswere explained in terms of intramolecular excimerspecies, however, a careful look at the structurereveals that there is C O Cu interactions in thecrystal structure whereas the zinc complex is devoidof such interactions. The compound 3 possessesC O HZN interactions which may be comparedto the case of copper complex 21 where the electrondensity of the ring was drawn by this interaction andin our case such an effect is caused by anintermolecular hydrogen bonding.为了进一步加强我们的观测，我们研究了另一个相关的分子2 - （ 2 -氨基苯基） - isoindole - isoindole - 1 ,3 -二酮（ 3 ）具有邻苯二甲酰亚胺环以及作为氨基酸组。氨基酸组的位置允许它有共轭的两个圆环。这种分子氨基酸组的质子化作用影响了分子间氢键的形成。我们研究了化合物（ 3 ）在不同溶剂中的可见光谱和荧光发射光谱的。结果发现，化合物的紫外光谱在不同溶剂中有着不同趋势的浓度依赖性（图7和8 ）。在这种情况下，提高了质子溶剂吸收强度和变化的吸收，以更高的波长。浓度依赖紫外线光谱表明，在甲醇浓度与吸光度有一个定期的增长预期从啤酒的兰伯特法（图7 ，插页） ，但如苯作为溶剂有偏离直线将通过原产地（图8 ，插页）。这可能是由于这样一个事实，即质子中等破坏分子间个人之间的粘合分子，而苯是一个非质子溶剂间氢键使分子之间2 - （ 2 -氨基苯基） - isoindole - 1 ,3 -二酮。这种影响也反映了在荧光发射光谱的化合物（ 3 ）在不同溶剂中( ex=435nm)甲醇复合表明发射在505nm，在乙腈中487nm处发射，而苯在471nm处发射 （图9 ）。所观察到的排放强度在不同溶剂中甲醇乙腈苯。这个表明，溶剂极性以及氢键可导致这样一个进程。甲醇与氨基酸集团形成氢键，从而改变了规模顺序的相互作用。为了区分影响质子的氢键相互作用，我们记录的可见光谱的将3存放于含有盐酸的甲醇。此外该化合物的酸的甲醇溶液立即展示了一个新的吸收峰在456nm的强度可比吸收的母体化合物。加酸的影响也反映在荧光发射。 化合物3在甲醇的酸性溶液中在450nm处激发，这表明一个完全新的，但非常低发射强度在572 nm左右。这些结果清楚地表明，质子化的胺猝灭发射如情况3 。然而，观察增强荧光强度甲醇在母体化合物表明，酸的作用和一个质子溶剂是完全独立。可以得出结论，-NH2与甲醇形成氢键导致了化合物3分子间氢键的破坏，但并不形成质子状态。化合物3分子的固态结构中有-NH2与羰基的其他分子形成的氢键。固体状态的红外光谱显然表明存在分子间氢键。化合物3在红外光谱下在3462cm-1和3385cm