Lumichrome Research Articles

Photophysical properties of lumichrome in aqueous solution tri-block copolymers

We have reported the photophysics of lumichrome (LUM)) in tri-block copolymers micelles of F-127 and P-123, having differences in length of hydrophilic unit. Due to the amphiphilic nature of polymeric micelles, and hydrophilic corona region, we observed the existence of different forms of LUM. We observed an extra emission peak of LUM at ∼ 430 nm and less percentage of existence of the A10 anionic form of LUM in case F-127. From time resolved anisotropy measurement, we have found that the LUM in F-127 micelle faced a more restricted environment than P-123 micelle.

One-step green synthesis of platinum mesoporous nanoparticles by riboflavin for light activated antitumoral therapy

Photodynamic therapy (PDT) has been established as one of the most promising novel cancer therapies with fewer side-effects and enhanced efficacy compared to the currently available conventional treatments. However, its application has been hindered by the limitations that photosensitizers (PS) have. The combination of PS with metallic nanoparticles like platinum nanoparticles (PtNPs), can help to overcome these intrinsic drawbacks. In this work, the combination of PtNPs and the natural photosensitizer riboflavin (RF) is proposed. PtNPs are synthesized using RF (Pt@RF) as reducing and stabilizing agent in a one-step method, obtaining nanoparticles with mesoporous structure for UV triggered PDT. In view of possible future UV irradiation treatments, the degradation products of RF, ribitol (RB) and lumichrome (LC), this last being a photosensitizing byproduct, are also employed for the synthesis of porous PtNPs, obtaining Pt@LC and Pt@RB. When administered in vitro to lung cancer cells, all the samples elicit a strong decrease of cell viability and a decrease of intracellular ATP levels. The antitumoral effect of both Pt@RF and Pt@LC is triggered by UV-A irradiation. This antitumoral activity is caused by the induction of oxidative stress, shown in our study by the decrease in intracellular glutathione and increased expression of antioxidant enzymes.

Photophysical study of lumichrome in 1,4-dioxane-water mixtures

In this work, we have reported the photophysics and dynamics of lumichrome (LUM) in 1,4-dioxane-water solvent mixtures. LUM fluorescence is sensitive to solvent properties such as proton-donating ability (α), proton-accepting ability (β), and viscosity. These properties play a significant role in the conversion of alloxazine-isoalloxazine photo-tautomerization. We have observed an intriguing change in the photophysics of LUM, which varies with the mole fraction of 1,4-dioxane (XD) in the solvent mixture. Different conformers of LUM have been identified in the solvent environment and explained with the help of spectroscopic techniques.

Bacterial phototoxicity of lumichrome photocrosslinked collagen hydrogels

In response to the World Health Organization's (WHO) call to develop alternative antibacterial methods, we investigate endogenous compounds and biopolymers. Lumichrome (LC) is an endogenous, lipophilic degradation product of riboflavin (vitamin B2) that can act as a photosensitizer for antimicrobial applications. LC is also a photocrosslinker of collagen. Crosslinked collagen hydrogels were prepared with two types of cyclodextrins (CDs), namely (2-hydroxypropyl)-β-CD (HPβCD) and -γ-CD (HPγCD), to improve the aqueous solubility of LC. The release of LC from the collagen gels and the phototoxic effect of UVA-irradiated LC against planktonic Staphylococcus (S.) aureus were investigated. Irradiation of LC hydrogels complexed with either CD resulted in similar levels of bacterial death despite the HPγCD gels containing half the LC concentration of the HPβCD gels. This effect may be explained by the higher complexation efficiency and stability of the LC-HPβCD complex, slowing down the LC release. Following treatment, small colony variants of S. aureus were observed. The study is a proof of concept of the release and phototoxic effects of an endogenous photocrosslinker embedded in a biopolymer as a sustainable antibacterial alternative to conventional therapy of, e.g., infected wounds.

Initial Excited State Dynamics of Lumichrome upon Ultraviolet Excitation.

Lumichrome (LC) is the major photodegradation product of biologically important flavin cofactors. Since LC serves as a structural comparison with the flavins; understanding excited states of LC is fundamentally important to establish a connection with photophysics of different flavins, such as lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Herein, we deduce the initial excited state structural dynamics of LC using UV resonance Raman (UVRR) intensity analysis. The UVRR spectra at wavelengths across the 260 nm absorption band of LC were measured and resulting Raman excitation profiles and absorption spectrum were self-consistently simulated using a time-dependent wave packet formalism to extract the initial excited state structural and solvent broadening parameters. These results are compared with those obtained for other flavins following UV excitations. We find that LC undergoes a very distinct instantaneous charge redistribution than flavins, which is attributed to the extended π-conjugation present in flavins but missing in LC. The homogeneous broadening linewidth of LC appears to be lower than that of LF, while the inhomogeneous broadening values are comparable, indicating greater solvent interaction with excited flavin on ultrafast timescale compared with LC, whereas on longer timescale these interactions are almost similar.

Photodynamics of biological active flavin in the presence of zwitterionic surfactants

In the flavin family of photoactive biomolecules, lumichrome (LM) is a very important compound. It contains a tri-cyclic structure with methyl groups at two sides. It formed by the partial decomposition and biodegradation of riboflavin in both acidic as well as in neutral medium. Herein, we have studied the photophysical properties of LM in the presence of two zwitterionic surfactants, namely dodecyldimethyl(3-sulfopropyl) ammonium hydroxide inner salt (DSB), and tetradecyldimethyl(3-sulfopropyl) ammonium hydroxide inner salt (TSB), having the same head group but a different tail part. We have used steady-state absorption, fluorescence emission, and time-resolved fluorescence emission measurements. We observed that in the presence of zwitterionic surfactant aggregates LM shows excitation and emission wavelength dependent emission properties, which demonstrate the structural changes that take place from one form to another prototropic form of LM molecule. The higher rotational relaxation time of LM in the case of DSB compared to TSB demonstrated that LM is facing more rigid environment in DSB micelles compared to TSB micelles.

Selective prototropism of lumichrome in the liposome/graphene oxide interface: A detailed spectroscopic study

The interactions of the biologically active fluorophores like flavins and biomolecules with nano carriers are very much important to produce targeted drug delivery systems. Also studying the interaction of graphene oxide (GO) with liposomes is essential for surface science and which have various applications. Herein, we have reported the interaction of a biologically active flavin molecule lumichrome (LU) with GO, in the absence and presence of zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposome by employing different spectroscopic techniques. These study also shows the prototropic tautomerization of LU, when it interacted with GO in the absence and presence of DMPC liposome. We have found that GO interacted with LU differently in the absence and presence of DMPC liposome. In the absence of DMPC liposome, LU molecules were directly adsorbed on the GO surface involving both edges and basal planes of GO via hydrogen bonding, π-π stacking between aromatic ring of LU and GO, and as a result we observe higher fluorescence quenching of LU. However, in the presence of DMPC liposome, LU molecules were partitioned inside the vesicle and in the presence of GO whole vesicle (in which LU molecules were partitioned) was placed on the edges of GO and here π-π stacking between aromatic ring of LU and GO is not possible, and as a result we observe moderate fluorescence quenching of LU. This study also indicates that zwitterionic DMPC liposome retained its structure when it interacts with GO. We have also found that in LU + GO complex, due to the direct adsorption of LU in GO surface, the prototropic tautomerization of LU is not possible, only the alloxazine form of LU exist. However, in LU + DMPC + GO complex when LU is incorporate inside the vesicle both alloxazine and isoalloxazine form of LU co-exist, indicates the prototropic tautomerization of LU in LU + DMPC + GO complex.

Photoinduced electron transfer in host-guest interactions of lumichrome with p-sulfonatocalix[6]arene

This study explores the complexation and fluorescence quenching of lumichrome (LC), a structural analogue of the biologically important flavin molecules, with a macrocyclic host, p-sulfonatocalix[6]arene (SCX6). It is revealed that the fluorescence quenching involves both static and dynamic interactions and occurs due to photoinduced electron transfer (PET) from the SCX6 host to the excited LC guest. The static quenching constant obtained from the modified Stern-Volmer relationship is in good agreement with the binding affinity of SCX6 with LC. Thermodynamic parameters obtained from the binding isotherms reveal that the SCX6-LC interaction is accompanied by entropy loss, which is consistent with the restriction imposed on the flexibility of the host and guest due to complex formation. Laser flash photolysis studies indicate that PET from SCX6 to LC is followed by proton abstraction by LC from the host or from the medium, to generate the corresponding radical species. The present results help in understanding the fluorescence quenching effect of p-sulfonatocalix[n]arene hosts that is widely exploited in designing fluorescence-based assays. Further, the prolonged lifetimes of the radicals in the SCX6-LC system underlines the notable role of macrocycles in the stabilization of radical intermediates in solution.

Lumichrome tautomerism in alcohol-water mixtures: Effect of carbon chain length and mole fraction of alcohols

Alcohol-water mixtures are important solvent systems, widely used in many applications. In this study, the intriguing water induced tautomerism of the fluorescent chromophore, Lumichrome (LC), has been explored to reveal interesting changes in the solvent environments of alcohol-water mixtures of methanol (MeOH), ethanol (EtOH), n-propanol (PrOH) and tert-butanol (t-BuOH), as a function of the carbon chain length/alkyl group size and concentrations of the alcohols. Since LC remains in the alloxazine form in pure aqueous medium, the appearance of emission from the isoalloxazine tautomer in alcohol-water mixtures provides compelling evidence that addition of alcohol disrupts the H-bonded network structure of bulk water to facilitate the availability of isolated water molecules in the solvent medium, which can form the required LC-water precursor complex for conversion of the alloxazine form to isoalloxazine form. Interestingly, two estimated parameters for LC tautomerism, namely, Rlong, which is the relative emission contribution of the isoalloxazine form in the fluorescence decay traces of LC (and hence a measure of the population of isoalloxazine form in the solution), and τlag, which is the lagtime involved in the evolution of the isoalloxazine emission band in the time-resolved area normalized emission spectra of LC (and hence a measure of the ease with which the LC-water precursor complex is formed), show a non-monotonic dependence on the mole fraction of alcohols (χalcohol) in the mixed solvent systems. The unique variation of Rlong and τlag with χalcohol, indicates the occurrence of three composition zones in the alcohol-water mixtures, which we correlate with characteristic transformations in the microscopic structures of the solvent mixtures. Our results reveal that the ability of the alcohols to disrupt the H-bonded network structure of bulk water increases with increasing size of their hydrophobic alkyl groups, following the trend, t-BuOH > PrOH > EtOH > MeOH.

Dual crosslinked alginate hydrogels by riboflavin as photoinitiator

Photodegradation behavior of riboflavin (RF) as photoinitiator and photosensitizer was investigated. When the visible light was irradiated to RF, the radical activation and decomposition of RF was induced by reactive oxygen species (ROS) that has formed by dissolved oxygen. One of its photoproducts, lumichrome (LC), was a reduced form of RF, which lost its color when photodegraded. Photodegradation mechanisms of RF and its photoproducts by light irradiation were investigated using structural quantitative analysis derived from UV–Vis spectra. It was found that RF provided photocrosslinking reaction to make alginate-based hydrogels. For this investigation, alginate derivative with tyramine moiety was prepared by EDC/NHS chemistry and it used to induce photocrosslinking, which might serve to reinforce the unstable ionic crosslinking in the physiological environment of alginate hydrogel. The structural stability of single-crosslinked (ionically or covalently crosslinked) or dual-crosslinked (ionically and covalently crosslinked) alginate-based hydrogels were compared using rheometer, texture analyzer and SEM.

The effect of albumin in photostabilization of riboflavin: A kinetic study

This investigation involves a study of the photochemical interaction of riboflavin (RF) and bovine serum albumin (BSA) in aqueous solution and the evaluation of this effect on the photostabilization of RF. RF solution (5 × 10–5 M) were irradiated with visible light in the presence of BSA (4.0–6.0 × 10–5 M) at pH 7.4 (0.005 M, phosphate buffer saline) and the apparent first–order rate constants for the photolysis of RF were found to be in the range of 3.92–9.26 × 10–3 min–1. The rate of RF photolysis is inhibited with an increase in the concentration of BSA. The second–order rate constant for the photochemical interaction of RF and BSA at pH 7.4 is 1.22 M–1 min–1. There is a gradual loss of RF fluorescence with an increase in BSA concentration due to the formation of a non–fluorescent complex between the two molecules. The value of Stern-Volmer quenching constant has been determined as 2.673 × 103 M–1. A two-component spectrometric method with correction for irrelevant absorption caused by BSA components has been used to determine RF and its photoproduct, lumichrome (LC), in photolyzed solutions. The mode of photochemical interaction of RF and BSA has been discussed.

Structural transition dynamics of biologically active flavins in alkylglucoside surfactants aggregates.

The photophysics and structural transition dynamics of a bio-active flavin lumichrome (LM) entrapped in two sugars based neutral surfactants were reported. Sugar-based surfactants, which were used for research purpose are potential environmentally friendly, green alternative amphiphilic surface active substance compared to other kinds of common surfactants. Here, two alkyl glucoside surfactants n-octyl-β-D-glucopyranoside (OBG) and n-octyl-β-D-thioglucopyranoside (OBTG) were used. This work is carried out by using steady-state absorption and fluorescence emission spectroscopy along with time-resolved fluorescence emission techniques. Photophysics of LM was modulated several folds in these two sugar-based neutral micelles. LM exhibits excitation and emission wavelength dependent fluorescence properties in these two sugars based neutral micelles. LM confined in the micellar environments exhibited structural transition dynamism, i.e. different kinds of conformers are equilibrated. Herein, different conformers of LM are identified and explained with the help of spectroscopic methods. From the fluorescence anisotropy measurement, it was found that the rotational relaxation time of LM in OBG micelle was more compared to that in OBTG micelle, which indicates that the LM molecule faced much more constrained environment in OBG micellar media.

Investigation of Different Prototropic Forms of Biologically Active Flavin Lumichrome in the Presence of Liposome.

Herein, we reported the photophysical behavior of lumichrome (LC), one of the biologically active flavin molecules, in the presence of small unilamellar vesicle of anionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). With the help of different spectroscopic techniques, we have proposed that anionic DMPC liposome interacts with the cationic form LC in ground state and in excited state and modulate the spectral properties of LC. Photophysical study reveals that different prototropic forms of LC are present in DMPC liposome medium. In the presence of DMPC liposome, fluorescence emission properties of LC vary with change in excitation and emission wavelengths. This indicates switch over between different structural forms of LC. From fluorescence lifetime measurements and fluorescence lifetime imaging (FLIM) study, it was revealed that emission decay profile of LC was fitted biexponentially in the presence of liposome. It suggests that in the presence of liposome, more than one form of LC is present. We have constructed the time-resolved area-normalized emission spectra (TRANES) of LC in the liposome and found one isoemissive point. This confirmed that two emissive species of LC are present in liposome. FLIM study and FE-SEM study give an idea about the structural feature of the complex between LC and liposome.

Simultaneous photoaddition, photoreduction and chemical reduction of riboflavin by sulfur containing dianions: A kinetic study

The present investigation involves the evaluation of the effect of reducing dianions (SO32−, S2O32−, S2O52−) on the kinetics of simultaneous photoaddition, photoreduction and chemical reduction reactions of riboflavin (RF) at pH 6–8. The photoproducts of these reactions have been identified and assayed during the degradation of RF. The final products of the above reactions are cyclodehydroriboflavin (CDRF), lumichrome (LC) and dihydroriboflavin (DHRF), respectively. The first-order rate constants for the loss of RF (kobs) (0.61–3.07 × 10-3 min-1) and formation of photoproducts (0.21–1.61 × 10-3 min-1) in the presence of dianions (0.2–1.0 M), and the second-order rate constants for the photochemical interaction of RF and dianions (1.42–2.01 × 10-3 M-1 min-1) and for the chemical reduction of RF (dark) (2.31–3.25 × 10-3 M-1 min-1) have been determined. The UV and visible spectral and fluorescence characteristics of RF in the presence of dianions have been studied. The fluorescence of RF is quenched by dianions depending upon the extent of their interaction with the molecule. The photoaddition of RF is optimum under neutral pH conditions and is catalyzed by the dianions in the order: S2O52− > SO32− > S2O32−, whereas the chemical reduction is enhanced in the reverse order. The S2O32− dianion is most effective as a reducing agent on the basis of its redox potential which is lower than that of the SO32−dianion. The quantum yields of photodegradation of RF in the presence of dianions (0.018–0.046) have been determined. The mode of photodegradation reactions of RF has been discussed.

Riboflavin as a Bioorganic Solar Fuel: Photoredox Chemistry Rationalized and Accelerated in a Miniaturized Flow Photoreactor

AbstractWith the depletion of fossil‐fuel‐based energies and the corresponding growing environmental concerns, the design of new strategies to store renewable solar energy is highly attractive. Herein, we studied a biomimetic organic solar fuel (Riboflavin – Vitamin B2) to photochemically store solar light into its amphoteric form (leucodeuteroflavin), working as a charge carrier towards a flow fuel cell. We demonstrate that miniaturization of the photoreactor permits significant reduction of the full time solar charge. Nevertheless, due to inherent leucodeuteroflavin (LDH2) degradation into lumichrome (LC), the maximum system charge (at pH=5) is limited to 13 % corresponding to a maximum power density of 0.11 mW cm−2 after 90 min. In comparison with other described photoredox electroactive systems (e. g. polyoxometalates, quinones), organic solar fuels based on flavins appear particularly promising and their chemical structure should be optimized to fit both photostability and economical requirements. In any case, this study enables a clear rationalization and quantification of the mechanism of riboflavin degradation under anaerobic conditions, which has been the topic of many discussions during the last century, and opens the way towards the development of a new generation of low‐cost renewable organic solar fuels.

Photodegradation of formylmethylflavin by side–chain and isoalloxazine ring cleavage in alkaline solution: A kinetic study

The photodegradation of fromylmethylflavin (FMF), a major intermediate product in the photolysis of riboflavin, in the pH range 8.0–12.0 has been studied. FMF undergoes photodegradation by side–chain cleavage to give lumichrome (LC) and lumiflavin (LF) in alkaline solution. It is also degraded by the hydrolysis of isoalloxazine ring to form 1,2–dihydro–1–methyl–2–keto–3–quinoxaline carboxylic acid (KA) and 1,2,3,4–tetrahydro–1–methyl–2,3–dioxoquinoxaline (DQ) and is oxidized to carboxymethylflavin (CMF) in alkaline solution. The photodegradation of FMF follows simultaneous first–order kinetics to form the side–chain, ring cleavage and oxidation products. The apparent first–order rate constants (kobs) for these reactions have been determined and are in the range of 0.15 (pH 8.0) to 4.98 × 102 min–1 (pH 12.0). The rate constants for the formation of the photoproducts (LC, LF, CMF, KA, DQ) have also been determined. The formation of the photoproducts is enhanced with pH due to the sensitivity of flavin triplet to alkaline hydrolysis. FMF and photodegradation products have been determined by a multicomponent spectrofluorimetric method in degraded solutions. It involves the separation of LC and LF by chloroform extraction at pH 2 followed by the choloroform extraction of undegraded FMF at pH 6.5 and their assay at 478 (LC) and 530 nm (LF and FMF), respectively. The aqueous phase is used for the assay of CMF, KA and DQ at 530, 443 and 420 nm, respectively. The mode of photodegradation of FMF has been discussed.

Photolysis of carboxymethylflavin in aqueous and organic solvent: a kinetic study.

This is the first study on the photolysis of carboxymethylflavin (CMF), an intermediate in the photolysis of riboflavin (RF). CMF is photodegraded by removal of side-chain to lumichrome (LC) in acid solution and to LC and lumiflavin (LF) in alkaline solution. It also undergoes alkaline hydrolysis to 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxoquinoxaline (DQ) by cleavage of isoalloxazine ring. CMF degrades to LC in organic solvents. The formation of LC in acid solution and organic solvents takes place by second-order reaction and those of LC, LF, KA and DQ in alkaline solution by first-order reactions. The values of second-order rate constants for the photolysis of CMF at pH 2.0 to 7.0 are in the range of 1.13 to 2.45 M−1 s−1 and those of first-order rate constants (kobs) at pH 8.0–12.0 from 1.53 to 4.18 × 10−4 s−1 and for the formation of photoproducts from 0.37 to 16.6 × 10−5 s−1. The photolysis of CMF is enhanced, with pH, in the alkaline region since the excited state is sensitive to alkaline hydrolysis. The photolysis and fluorescence quantum yields of CMF in aqueous and organic solvents have been reported. CMF and photoproducts have been assayed spectrofluorimetrically. The mode of CMF photolysis is discussed.

Divalent anion catalyzed photodegradation of riboflavin: A kinetic study

A study of the effect of some divalent anions (carbonate, oxalate, phthalate, phosphate) on the kinetics of photodegradation reactions of riboflavin (RF) and its pathways, product composition and fluorescence quenching in the pH range 6.0–8.0 has been conducted. The photodegradation of RF in the presence of 0.2–1.0 M divalent anions on visible irradiation leads to the formation of cyclodehydroriboflavin (CDRF) by photoaddition pathway and lumichrome (LC) by photoreduction pathway as the major end products. These products are formed by simultaneous first–order reactions. The divalent anions deviate the photoreduction pathway of RF in favor of photoaddition pathway to yield CDRF. The extent of this variation depends on the strength of RF–divalent anion complex as indicated by an increase in the loss of RF fluorescence due to complex formation. The first– and second–order rate constants for the photodegradation of RF have been determined and the effect of pH on the rates of these reactions evaluated. The rate of divalent anion catalyzed formation of CDRF is maximum at pH 7 and occurs in the order: carbonate > phosphate > oxalate > phthalate. The catalytic activity of the divalent anions increases with the strength of RF–divalent anion complex resulting in an increase in the rate of formation of CDRF and a decrease in the rate of formation of LC by different pathways.

Observation of Near-Threshold Resonances in the Flavin Chromophore Anions Alloxazine and Lumichrome.

Lumichrome (LC) is the chromophore of the flavin family of photoactive biomolecules, where key biochemical activity involves interplay between redox and photophysical events. Questions remain about the relationship between the redox status of the ground and excited states and demand an improved understanding of the intrinsic photochemistry. Using anion photodissociation spectroscopy, we have measured the intrinsic electronic spectroscopy (564-220 nm) and accompanying photodegradation pathways of the deprotonated anionic form of LC. Experiments were also performed on alloxazine (AL), which is equivalent to LC minus two methyl groups. We observe a resonance state close to 3.8 eV for both anions for the first time, which we tentatively assign to dipole-bound excited states. For AL this state is sufficiently long-lived to facilitate dissociative electron attachment. Our results suggest that the presence of methyl group rotors at key positions along the molecular dipole may reduce the lifetime of the resonance state and hence provide a structural barrier to valence electron capture, and ensuing molecular dissociation.

Multicomponent spectrofluorimetric method for the assay of carboxymethylflavin and its hydrolytic products: kinetic applications.

The simultaneous assay of carboxymethylflavin (CMF), an intermediate in the photolysis of riboflavin, and its hydrolytic side-chain cleavage products, lumichrome (LC) (acid solution) and LC and lumiflavin (LF) as well as isoalloxazine ring cleavage products, 1,2-dihydro-1-methyl-2-keto-3-quinoxaline carboxylic acid (KA) and 1,2,3,4-tetrahydro-1-methyl-2,3-dioxo-quinoxaline (DQ) (alkaline solution) has been carried out by a multicomponent spectrofluorimetric method. The method is based on the adjustment of pH of the degraded solutions to 2.0 and extraction of LC and LF with chloroform. The chloroform extract is evaporated to dryness under reduced pressure, the residue dissolved in pH6.5 citro-phosphate buffer and LC and LF determined at their fluorescence maxima at 478 and 530nm, respectively. The pH of the aqueous phase is re-adjusted to 6.5 and the solution used for the determination of CMF, KA and DQ at the wavelengths of 530, 443 and 420nm, respectively. The proposed method has been validated according to ICH guidelines. The calibration curves for CMF and its hydrolytic products are linear in the concentration range of 0.5-5.0×10-6 M. The mean recovery ranges from 99.0-102.0% with relative standard deviation (RSD) of 0.19-0.99%. The limit of detection (LOD) and the limit of quantification (LOQ) are in the range of 1.17-1.78×10-7 M and 3.55-5.40×10-7 M, respectively. The uniformity of molar balance of CMF and degradation products during hydrolytic reactions indicates the accuracy of the proposed method for the spectrofluorimetric assay of the compounds. It has been applied to study the kinetics of hydrolytic reactions of CMF.