Eskil M E Andersen1, Hsin Wang2, Joshua S H Khoo3, Jose F Cerda4, Ronald L Koder3,5. 1. Department of Biochemistry, The City College of New York, New York, NY, United States of America. 2. Department of Chemistry, The City College of New York, New York, NY, United States of America. 3. Department of Physics, The City College of New York, New York, NY, United States of America. 4. Department of Chemistry, St. Joseph's University, Philadelphia, PA, United States of America. 5. Graduate Programs of Physics, Biology, Chemistry and Biochemistry, The Graduate Center of CUNY, New York, NY, United States of America.
Abstract
Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations. Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR. These Safranines have two-electron midpoint potentials ranging from -272 to -315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties. While this mixture may aid in Safranine O's utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.
Safranine O is widely used in the bioenergetics community as an indicator dye to determine membrane potentials and as an electron transfer mediator in potentiometric titrations. Here we show that two different commercial preparations of Safranine O contain less than sixty percent by weight of the title compound, with the rest primarily consisting of two closely related safranine isomers. All three major isomer components were isolated using reverse phase HPLC and their structures determined using mass spectrometry and two-dimensional NMR. These Safranines have two-electron midpoint potentials ranging from -272 to -315 mV vs. SHE. We have also investigated the absorption and fluorescence spectra of the compounds and found that they display distinct spectral and photophysical properties. While this mixture may aid in Safranine O's utility as a mediator compound, membrane potential measurements must take this range of dye potentials into account.
In order to fully understand biological electron transfer processes and the reactions coupled to them in metabolism, it is necessary to determine the reduction potentials of respiratory and photosynthetic electron transfer cofactors and side chains. These protein properties are typically investigated in solution, necessitating the transport of electrons through the solvent to react with the dissolved protein analyte. Biological redox couples are commonly inert towards electrode surfaces, and the major reason for this is that the redox centers tend to be shielded by the protein structure which prevents close contact with the electrode surface. Oxidation-reduction mediators, which are small organic molecules are therefore required during solution potentiometric measurements to transfer electrons between the electrode and the analyte. Mediators also have the added benefit of creating electrical poise during measurements which helps the experimentalist alter the potential of the solution in a controlled manner.Safranine (also known as Safranine O, Safranin, Safranine T, or basic red is often used as an electron transfer mediator in potentiometric measurements and as an internal colorimetric indicator in reduction potential determinations both in solution [1, 2] and in membranes [3-5]. It is a synthetic phenazine derivative that may be prepared from reacting a 1:1 mixture of o-toluidine and 1.4-diamino-2-methylbenzene followed by nucleophilic addition of aniline [6]. The compound has its origins in the burgeoning dye industry of the 19th century and was first isolated in 1859 [7]. The reduction potential of Safranine O at pH 7.0 was first reported by Prince et al. in 1981 to be −280 mV vs the Standard Hydrogen Electrode (SHE) as determined by polarography [8]. However, based on our findings this value may have been an average of multiple safranine isomers.In addition to being useful as an electron transfer mediator Safranine has received considerable interest because of its photoreduction properties [9-11]. It has been demonstrated to have applications in a number of areas such as solar energy conversion [12, 13], analytical chemistry [14, 15], as a histological stain [16, 17], and in synthetic organic chemistry as an initiator of photopolymerization [18-20]. Many natural phenazine derivatives are biosynthesized by bacteria as well where they play roles in processes such as electron shuttling to terminal acceptors, modification of cellular redox states, regulation of gene expression, and biofilm formation [21]. Finally, we have synthesized a series of safranine analogues for use as artificial electron transfer cofactors in de novo designed enzymes [22, 23].In the scientific literature commercial sources of Safranine O are assumed to consist of a homogeneous compound. We have discovered that they in fact contain a heterogenous mixture of isomeric safranines that are resistant to separation by normal phase chromatography. We have therefore separated and isolated the three major components of a commercial sample of Safranine using C18 reverse phase HPLC. These three components make up more than 90% of the total safranine content in the mixture. We then determined their key physicochemical properties, most importantly their reduction potentials, as well as their ultraviolet and visible absorption and fluorescence properties. Finally, we determined the chemical structures of the components using two-dimensional NMR spectroscopy.
Materials and methods
Chemicals
Safranine O samples were purchased from Sigma-Aldrich (St. Louis, MO) and Santa Cruz Biotech (Dallas, TX). HPLC grade Acetonitrile was purchased from P212121 (Ann Arbor, MI) and HPLC grade trifluoroacetic acid was purchased from VWR (Radnor, PA). The water used in all purifications and experiments was obtained from a MilliQ system and had a minimum resistivity of 18 MΩ. The mediators used in the potentiometric measurements were methyl viologen (1,1′-Dimethyl-4,4′-bipyridinium dichloride) and pyocyanin (5-Methylphenazin-1-one) also purchased from Sigma-Aldrich. Sodium hydrosulfite and potassium ferricyanide for potentiometric experiments were purchased from VWR.
Chemical purification
Chromatography was performed on a Shimadzu (Kyoto, Japan) HPLC equipped with 2 LP-6AD pumps coupled to an SPD-M20A PDA spectrophotometer. The HPLC column used was a 20x250 mm Proto 300 C18 with 10 μm particle size from Higgins Analytical (Mountain View, CA). Safranine O was dissolved in 4:1 water: acetonitrile. The mixture was separated using an isocratic flow of 27% acetonitrile, 73% water running at a flow-rate of 18 mL/min. After separation, safranine samples were rotary evaporated to remove acetonitrile, and the remaining water was removed by lyophilizing the frozen analytes overnight in 50 mL falcon tubes. During lyophilization, analytes were protected from light by aluminum foil wrappings.
Absorption and fluorescence spectroscopy
The buffer used in all spectroscopy and potentiometry measurements consisted of 50 mM sodium phosphate adjusted to pH 7.0 with phosphoric acid or sodium hydroxide. Absorption spectra were collected on a Hewlett-Packard (Palo Alto, CA) HP 8452A diode array spectrophotometer equipped with OLIS GlobalWorks software for data collection and analysis. Fluorescence spectra were collected on an OLIS (Athens, GA) DM-45 fluorimeter. All Safranines were excited at their wavelength of maximal absorption. Samples were diluted to an absorption of 0.1 AU and degassed with nitrogen in a 1x1 (width x depth) cm quartz cuvette before measurements.
Structural determination
NMR spectra were recorded in perdeuterated DMSO on a Bruker (Billerica, MA) Avance III spectrometer operating at 600 MHz for 1H, 150 MHz for 13C, and 60 MHz for 15N. Assignments were confirmed with 1H-13C HSQC and HMBC, 1H-15N HMBC, and 1H-13C 1, 1-ADEQUATE experiments [24-26]. Amberized NMR tubes were used to protect the samples from photodegradation.
Reduction potential determination
Solution potentiometry [27, 28] was used to determine the redox potentials of the Safranines. Briefly: an airtight cuvette is fitted with a platinum measuring electrode and an Ag/AgCl saturated potassium chloride reference electrode. The reference electrode was standardized using hydroquinone at pH 7 and at 25°C [29]. Potentials were determined in 50 mM sodium phosphate solution pH 7.0 using 5 μM methyl viologen and 5 μM pyocyanin as electron mediators and to establish poise during measurements.Samples were degassed via rubber septum-sealed ports with nitrogen scrubbed of oxygen using a zinc-vanadium bubbler oxygen scrubbing system [30]. Samples were reduced using microliter additions of sodium dithionite in a sodium hydroxide solution (pH>10) and their absorption spectra were determined after each addition. Once the samples had been fully reduced, they were oxidized stepwise using microliter additions of a potassium ferricyanide solution in water while measuring the absorption spectra going in the oxidizing direction to measure the degree of hysteresis. The changes in absorption at the peak reduced wavelength of each safranine was measured and fitted using the Nernst equation (Eq 1) with the number of electrons (n) fixed at 2. In this equation Ecell is the potential of the cell relative to the SHE (), R is the universal gas constant, T is the temperature in Kelvin, F is Faraday’s constant, and [ox] and [red] are the concentrations of oxidized and reduced safranine, respectively.
Results and discussion
Purification
Separation on an analytical C18 HPLC column revealed that the Safranine O mixture is heterogenous and contains a mixture of several components with Safranine type spectra (). Of these compounds I, II and III constitute > 95% of the total area and we therefore decided to investigate the properties of these compounds further. At least 20 mg of each of compound was successfully isolated by RP-HPLC for NMR structure determination.
Chromatogram showing the separated peaks of the commercial Safranine O mixture.
The mixture was separated on a Higgins Proto 300 4.6x250 mm column. The chromatographic method is isocratic and consists of 27% acetonitrile/73% water with 0.1% trifluoroacetic acid at a flowrate of 2 mL/min. The chromatographic trace was made by plotting the absorption at 520 nm vs. time. The peaks are numbered in order of elution and correspond to the scheme in Table 1.
Table 1
Safranine O components and their properties.
Compound
Compound
Redox potential [mV vs. SHE]
Peak wavelengths [nm]
Extinction coefficient [M-1cm-1]
Emission λmax[nm]
Relative maximal emission
2,8-diamino-3,9-dimethyl-10-phenylphenazin-10-ium
I
−315 ± 2
206
19,376
none detected
N/A
278
27,833
524
19,107
2,8-diamino-3.7-dimethyl-10-phenylphenazin-10-ium
II
−272 ± 2
206
20,644
581
3.06
250
22,115
276
29,372
520
29,800
2,8-diamino-3,6-dimethyl-10-phenylphenazin-10-ium
III
−276 ± 5
210
13,437
594
1
250
12,743
278
20,579
516
19,209
As shown in Fig 2 and Table 1; safranine I exhibits absorption peaks at 206, 278 and 524 nm in the experimental buffer. Contrary to II and III, I is not fluorescent. II shows absorption peaks at 206, 250, 276 and 520 nm and has an emission maximum at 581 nm. III evinces absorption maxima at 210, 250, 278 and 516 nm with an emission maximum at 594 nm. Not only does III absorb at a shorter wavelength (516 nm), but it also emits at a longer wavelength (594 nm). This implies that there must be a greater level of vibrational relaxation in this molecule as compared to II. In addition, the quantum yield for II is greater, and for the same absorption of 0.1 AU, II is more than 3 times as fluorescent as III.
Fig 2
Absorption and fluorescence spectra of three safranine isomers.
The absorption extinction coefficient is shown in black, while fluorescence is shown in red. I) The absorbance spectra of safranine I (this compound is not fluorescent). II) The absorption and fluorescence spectra of safranine II. III) The absorption and fluorescence spectra of safranine III. Fluorescence spectra were obtained by exciting the Safranine at their absorption maxima around 520 nm. The absorption at the wavelength of excitation was set to 0.1 AU at the long wavelength absorption maximum for both emission spectra.
Absorption and fluorescence spectra of three safranine isomers.
The absorption extinction coefficient is shown in black, while fluorescence is shown in red. I) The absorbance spectra of safranine I (this compound is not fluorescent). II) The absorption and fluorescence spectra of safranine II. III) The absorption and fluorescence spectra of safranine III. Fluorescence spectra were obtained by exciting the Safranine at their absorption maxima around 520 nm. The absorption at the wavelength of excitation was set to 0.1 AU at the long wavelength absorption maximum for both emission spectra.All three safranines have identical masses as determined using mass spectrometry. The major HPLC component, II, displays 1H and 13C-spectra characteristic of a molecule with C2 symmetry (Fig 3), and can be readily identified as authentic safranine O. The assignments of all resonances of II are straightforward with the use of two-dimensional HSQC [31] and HMBC [32] (13C as well as 15N) spectroscopies.
Fig 3
One-dimensional 1H (left) and 13C (right) spectra and resonance assignments, exclusive of the methyl region, in DMSO-d6 of safranines I-III.
One-dimensional 1H (left) and 13C (right) spectra and resonance assignments, exclusive of the methyl region, in DMSO-d6 of safranines I-III.The other two HPLC peak compounds (I and III) do not have such symmetry properties. We posited initially that IV in Fig 4 might be a good candidate for peak #5 because its 1H-spectrum displays 4 aromatic ring protons that are singlet or weakly-coupled. None of the other NMR spectra are consistent with this picture. An unambiguous clue was given by the 1, 1-ADEQUATE [26] experiment which showed that one of the two NH2-bearing aromatic carbons at ~156 ppm is adjacent to two CH-neighbors (boxed peaks in Fig 5). This is only possible if on one of the side rings the NH2 and CH3 groups are not adjacent to each other. III is thus the only remaining candidate. Indeed with this insight, all NMR data fall into place: 1) the carbon resonances can be uniquely assigned with 1, 1-ADEQUATE and agrees completely with 13C-HMBC; 2) the weak ~2 Hz splitting of H(9) is the result of a coupling with the meta-positioned H(7) whereas the broad feature of H(7) resulted from additional 4-bond J-coupling with the CH3 [H(18)]; 3) the information-rich 15N-HMBC data (Fig 6) can be completely understood with 3-bond, 2-bond, and even a few weak 4-bond J-couplings. With the discovery that one methyl group rearranges itself on the ring in III, I can be readily identified to be structured as in Fig 4. The ortho-positioned H(6) and H(7) displayed a J-coupling of 9.1 Hz with each other. All the other NMR data including 1,1-ADEQUATE are consistent with this model and can be interpreted in a straightforward manner. Interestingly, the phenyl ring protons H(f2), H(f3), H(f4) do not display the expected doublet, triplet, and triplet patterns, respectively, as in the other two compounds. In this case H(f3) and H(f4) are nearly degenerate and strong-interaction of J-couplings result in complicated multiplet patterns.
Fig 4
The three major Safranine structures determined by NMR.
The number annotations in red refer to the atom assignments done during the analysis of the NMR spectra and follow the biochemical numbering system of flavin and flavin-like molecules. The IUPAC names of the compounds: (I) 2,8-diamino-3,9-dimethyl-10-phenylphenazin-10-ium. (II) 2,8-diamino-3,7-dimethyl-10-phenylphenazin-10-ium (III) 2,8-diamino-3,6-dimethyl-10-phenylphenazin-10-ium (IV) 2,7-diamino-3,8-dimethyl-10-phenylphenazin-10-ium–an alternate possible structure of III ruled out on the basis of the 1,1 ADEQUATE spectrum depicted in .
Fig 5
The 1,1 ADEQUATE spectrum of safranine III.
The boxed area at the lower left is unambiguous evidence that the methyl group in III is at position 6.
Fig 6
Natural abundance 15N-HMBC spectrum of safranine III.
At the top is the correlating 1-dimensional 1H spectrum. The weak crosspeaks between H(4)—N(10) and H(7)–N(5) result from 4-bond J-couplings in this highly conjugated system.
The three major Safranine structures determined by NMR.
The number annotations in red refer to the atom assignments done during the analysis of the NMR spectra and follow the biochemical numbering system of flavin and flavin-like molecules. The IUPAC names of the compounds: (I) 2,8-diamino-3,9-dimethyl-10-phenylphenazin-10-ium. (II) 2,8-diamino-3,7-dimethyl-10-phenylphenazin-10-ium (III) 2,8-diamino-3,6-dimethyl-10-phenylphenazin-10-ium (IV) 2,7-diamino-3,8-dimethyl-10-phenylphenazin-10-ium–an alternate possible structure of III ruled out on the basis of the 1,1 ADEQUATE spectrum depicted in .
The 1,1 ADEQUATE spectrum of safranine III.
The boxed area at the lower left is unambiguous evidence that the methyl group in III is at position 6.
Natural abundance 15N-HMBC spectrum of safranine III.
At the top is the correlating 1-dimensional 1H spectrum. The weak crosspeaks between H(4)—N(10) and H(7)–N(5) result from 4-bond J-couplings in this highly conjugated system.Also, there is a 1:1:1 triplet in the region of 7.0–7.4 ppm in 1H-spectra (Fig 3) with a splitting of 50.8 Hz due to J-coupling to the quadrupolar 14N. The 15N-HMBC (Fig 6) on the other hand showed a 1H – 15N splitting of 70 Hz at 23.0 ppm. These are characteristic of NH4+ ion [33].
Fig 7 depicts the potentiometric titration of each purified compound, revealing that safranines II and III have similar reduction potentials (−272 and −276 mV vs SHE respectively), while safranine I has an approximately 40 mV more negative potential, at −315 mV vs. SHE, than II and III.
Fig 7
Potentiometric titration of safranines I-III.
Line drawn is a fit to Eq 1 with the number of electrons fixed at 2. Errors are errors of the fit.
Potentiometric titration of safranines I-III.
Line drawn is a fit to Eq 1 with the number of electrons fixed at 2. Errors are errors of the fit.In general, a methyl group in a conjugated system contributes electron density through an inductive effect that is unaffected by solution pH [34]. The Hammett equation describes the thermodynamic equilibrium effects of substituents on aromatic rings for different kinds of reactions where a reaction center is attached to the aromatic ring [35]. It has been shown that the Hammett equation pertains to reduction potentials as well [36]. It is formulated as where describes the ratio of the equilibrium constant of a reaction; K, vs. a reference reaction; K0. σ is dependent on the position and identity of the substituent and ρ is a reaction specific constant. ρ is positive for reactions where negative charge is built up, or where positive charge disappears which is the case when Safranines are reduced. The Hammett constant, σ, is negative for methyl groups which means that the product of ρ and σ is a negative number. This means that the general effect of methyl substituents that are part of the conjugated aromatic system should be to stabilize the oxidized state of the Safranines, lowering their reduction potentials.I (see Table 1 for the compound numbering scheme) has a methyl substituent with the closest proximity to the positively charged nitrogen of the oxidized Safranine with its two-bond distance as well as being the closest spatially. In II the distance is four bonds, and in III it is three bonds. It is arguable that the methyl groups stabilize the positive charge both through its electron donating “Hammett” effect as well as its electric field effect since the inductive effect is bond-distance dependent as well as through-space dependent (the strength of the through-space effect being inversely related to the dielectric strength of the solvent) [37]. In fact, the redox potential follows the rank order of the bond distance from the methyl group so this may also explain why the redox potential of II is slightly lower than III as well. In previous work we have shown that the N(5) nitrogen chemical shift in both safranine analogues altered at the para position of the N(10) phenyl ring [23] and in the N(5) chemical shift of the related flavin molecule in various hydrogen bonding complexes [38, 39] correlates closely with the molecular reduction potential. In this case there is no such correlation. This may be a result of the fact that the methyl groups whose positions change are directly attached to the central phenazine moiety and thus have a much greater inductive effect on the N(5) electron density.
Conclusions
We have identified the three major components in the commercial Safranine O dye and determined their structures using NMR. In all these compounds the amino groups remain at the same positions (on C2 and C8) on the opposite sides of the fused ring system whereas the methyl groups to seem to scramble on the carbons of the outer rings. The mechanism for methyl scrambling is not yet known. It seems likely that these other safranine isomers are side products in synthesis as opposed to breakdown products—samples in amber-colored NMR tube are stable for months and we do observe any changes for samples stored in clear tubes and exposed to sunlight for a similar period of time either. These compounds can only be isolated by reversed-phase HPLC: silica gel chromatography does not separate these isomers at all, and in our hands the mixture has been resistant to all attempts at crystallization. Furthermore the presence of the commonly used negative counterion trifluoracetate in the mobile phase makes the safranines co-elute even on the C18 column, probably because they act as the counterion of the positively charged dyes. We have further interpreted the results of potentiometric titrations and have argued that the differences in their potential values can be explained by structural induction effects from the position of the methyl substituents on the phenylphenazinium ring.Proportions of these safranin compounds may differ depending on vendor formulation which would cause shifts in the apparent and average midpoint potentials of Safranine O. This could be especially problematic when Safranine O is used as a potential standard, as has been the case in a great number of experimental measurements of mitochondrial membrane potentials using safranine fluorescence and absorbance [40]. On the other hand there are also advantages to using a mixture of safranines with a range of midpoint potentials in potentiometric titrations because they establish poise over a greater range of potentials compared to any one pure compound.(XLSX)Click here for additional data file.(PDF)Click here for additional data file.7 Apr 2022
PONE-D-22-05311
Oxidation-reduction and photophysical properties of isomeric forms of Safranin
PLOS ONE
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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript is clearly written and describes the careful characterization of commercially prepared Safranine O, which is commonly used as a mediator in electrochemistry. Specifically, they note that during synthesis, two distinct molecules are produced with unique spectral and electrochemical properties. Given that these compounds are widely used and the mixture of compounds complicates their application, it is important that this work be published.Experiments are well described and the data is clearly presented. Only one minor issue - a relict from a previous submission:"The reduction potential of Safranine O at pH 7.0 was first reported by Prince et al. *in this journal*in 1981 to be −280 mV vs the Standard Hydrogen Electrode (SHE) as determined by polarography [9]"Reviewer #2: This manuscript by Andersen et al describes an analysis of two commercial preparations of safranin, revealing it to be made up of a number of isomers with varying optical and redox properties. The findings will be of interest to users of safranine as a mediator or dye.The research has been conducted to a high standard and the conclusions drawn seem reasonable. The manuscript is mostly well written and clear, but would benefit from a thorough proof-read.It would have been helpful for the authors to have included page numbers in their manuscript. 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12 May 2022Dear Dr. Niederman,Please find attached a re-revised version of our manuscript “Oxidation-reduction and photophysical properties of isomeric forms of Safranin”, PONE-D-22-05311. We have incorporated changes that address all the comments brought forth by both reviewers, and we believe the paper is greatly improved as a result of these suggestions. We thank the reviewers first for these suggestions, and second for responding so quickly.A detailed list of the reviewer comments and our responses follows:Journal Requirements:(1) Please ensure that your manuscript meets PLOS ONE's style requirements.We have reformatted the manuscript to fit the Journal’s style guide.(2) However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement.We have removed the Acknowledgements section of the text completely. However, we were recently reminded that we should also acknowledge the funding that supported the NMR spectrometers used in the paper. As such, the we have added NIH grants S10OD018509, S10OD016432, and P41GM066354.(3) In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found.The minimal data set, including NMR data and potentiometric titration data, has been uploaded as supplemental information.Reviewer 1:(1) Only one minor issue - a relict from a previous submission: "The reduction potential of Safranine O at pH 7.0 was first reported by Prince et al. *in this journal* in 1981 to be −280 mV vs the Standard Hydrogen Electrode (SHE) as determined by polarography [9]"The offending verbiage has been removed. The sentence now reads: “The reduction potential of Safranine O at pH 7.0 was first reported by Prince et al. in 1981 to be −280 mV vs the Standard Hydrogen Electrode (SHE) as determined by polarography [9].” We thank the reviewer for catching our embarrassing error.Reviewer 2:(1) Page 8, second line from bottom – random use of italics.If we’ve found the correct area, these italics are meant to indicate the atom type being discussed in NMR assignments. This is a common usage in the organic literature.(2) Page 9, line 9 – the authors write “in this journal in 1981”. Does this betray a previous, unsuccessful submission to BBA?The offending verbiage has been removed. The sentence now reads: “The reduction potential of Safranine O at pH 7.0 was first reported by Prince et al. in 1981 to be −280 mV vs the Standard Hydrogen Electrode (SHE) as determined by polarography [9].” We thank the reviewer for catching our embarrassing error.(3) Page 14, Figure legend. Some additional punctuation would be helpful here.A number of missing periods were added. We thank the reviewer for catching this.(4) Page 14, last line and Page 15, first line - what does “Figure 3c and 3d” refer to here?This verbiage was left over from a previous version of the figure. The sentence now reads: “The major HPLC component, II, displays 1H and 13C-spectra characteristic of a molecule with C2 symmetry (Figure 3), and can be readily identified as authentic safranine O.” We thank the reviewer for catching this.Alterations in the manuscript are highlighted in yellow. We thank you for your time and attention, and hope these changes make a convincing argument for publication.Best Regards,Ronald L. Koder, Ph.D.Submitted filename: Response to reviewers.pdfClick here for additional data file.3 Jun 2022Oxidation-reduction and photophysical properties of isomeric forms of SafraninPONE-D-22-05311R1Dear Dr. Koder,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Please note that an error on p.13, line 12 of the revised Ms. was found (should read: why the redox potential of II is slightly lower than III as well.) that can be corrected in the galley proofs.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.Kind regards,Robert A. Niederman, Ph.DAcademic EditorPLOS ONEAdditional Editor Comments (optional):Please note that an error on p.13, line 12 of the revised Ms. was found (should read: why the redox potential of II is slightly lower than III as well.) that can be corrected in the galley proofs.16 Jun 2022PONE-D-22-05311R1Oxidation-reduction and photophysical properties of isomeric forms of SafraninDear Dr. Koder:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.If we can help with anything else, please email us at plosone@plos.org.Thank you for submitting your work to PLOS ONE and supporting open access.Kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Robert A. NiedermanAcademic EditorPLOS ONE
Authors: Jose F Cerda; Ronald L Koder; Bruce R Lichtenstein; Christopher M Moser; Anne-Frances Miller; P Leslie Dutton Journal: Org Biomol Chem Date: 2008-04-28 Impact factor: 3.876
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