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Org. Synth. 2020, 97, 21-37

DOI: 10.15227/orgsyn.097.0021

Synthesis of 3,6-Bis(dimethylamino)-9H-xanthen-9-one by Stepwise Chemical Redox Cycling

James L. Bachman, Cyprian I. Pavlich, and Eric V. Anslyn*¹

Checked by Jacob E. Dander, Katie A. Spence, and Neil K. Garg

1. Procedure (Note 1)

A. 3,6-Bis(dimethylamino)-9H-xanthen-9-one (1). To a 250-mL round-bottomed flask (24/40 joint) equipped with a 4.0-cm football-shaped Teflon-coated magnetic stirbar is charged pyronin Y (7.40 g, 24.4 mmol, 1.00 equiv) (Note 2) and sodium phosphate tribasic dodecahydrate (18.6 g, 48.8 mmol, 2.00 equiv) (Note 3). To the round-bottomed flask, N-Methyl-2-pyrrolidone (NMP, 150 mL) is added via graduated cylinder and water (5.00 mL, 278 mmol, 11.4 equiv) via syringe (Note 4). The flask is fitted with a rubber septum and temperature probe (Figure 1A). The flask is placed into a preheated silicon oil bath at 110 ºC and the stir rate is set to 650 rpm. The reaction mixture is stirred for 2 h at this temperature, and the conversion can be monitored by TLC (Note 5). After 2 h, the flask is removed from the oil bath and allowed to cool to 23 °C over 15 min. During the course of the first 2 h, the starting material is converted to a 1:1 mixture of product 1 and a non-fluorescent by-product, giving a brown solution (Figure 1B). Iodine (6.20 g, 24.4 mmol, 1.00 equiv) (Note 6) and sodium phosphate (9.30 g, 24.4 mmol, 1.00 equiv) are charged to the flask in single addition. An additional portion of water (2.00 mL, 111 mmol, 4.55 equiv) is added by syringe and the flask is fitted with a rubber septum and placed in a 110 ºC silicon oil bath and stirred overnight. Introduction of iodine to the solution results in a color change to a purple solution (Figure 1C) (Note 7). After stirring for 16 h at 110 ºC following iodine addition, the reaction is complete.

Figure 1. Color change from A (start) to B (2 hours) to C (iodine added)

After cooling to 23 ºC, the crude reaction mixture is filtered through a pad of celite (Note 8) into a 1-L round-bottomed flask (24/40 joint). The celite is washed with methylene chloride (400 mL) (Note 9) followed by methanol (200 mL) (Note 10) until no blue fluorescent product is visible (Figure 2).

Figure 2. Wash celite to remove fluorescent product (A), after washing (B)

The reaction is concentrated on a rotary evaporator (400 mmHg down to 100 mmHg, 40 ºC) for the bulk of the solvent removal. NMP is removed by vacuum distillation while the flask is stirred at 650 rpm (Note 11). The flask is heated to 70 ºC and stirred over 30 min while the pressure is gradually lowered (250 - 50 mmHg). The pressure is subsequently reduced to 1 mmHg and the reaction is stirred and further heated to 85 ºC. Distillation is aided by periodic heating with a heat gun and additional dry ice is packed around the collection flask. The majority of the NMP is removed after 1.5 h and the distillation is halted, affording a dark purple oil. Following the reaction, the product exists in an approximately 17:3 ratio of 1 to the de-methylated side product (Notes 12 and 13).

To convert the side product back to 1, the crude purple oil is dissolved in 150 mL methanol, methylene chloride, and acetic acid (8:1:1, v:v:v) (Note 14) and transferred to a 500-mL round-bottomed flask (24/40 joint) equipped with a 4.0-cm football-shaped Teflon-coated magnetic stir bar. To the flask, 37% formaldehyde solution (0.91 mL, 12 mmol, 0.50 equiv) (Note 15) is added by syringe and the reaction is stirred (650 rpm) at 23 ºC for 1 h. Sodium cyanoborohydride (0.767 g, 12.2 mmol, 0.50 equiv) (Note 16) is charged to the flask and stirred for 3 h, where the reaction is deemed complete by LCMS or TLC (Note 17). The reaction is concentrated under reduced pressure (400 mmHg to 100 mmHg, 40 ºC). To the round-bottomed flask, water (200 mL) and methylene chloride (200 mL) are added. While stirring at 650 rpm, potassium carbonate (15 g) (Note 18) is slowly added to minimize gas evolution, and the final pH is 9-10 (Note 19). The solution is transferred to a 1-L separatory funnel and separated. The aqueous layer is further extracted with methylene chloride (3 x 100 mL). A further 100 mL of methylene chloride is used in rinsing residual product from the glassware used for extraction. The organic extracts are combined and washed with water (2 x 250 mL) and brine (1 x 200 mL, which was further diluted until the layers separated) (Note 20). The organic layer is split into two equal portions and each dried over anhydrous Na₂SO₄ (75 g) (Notes 21 and 22). The sodium sulfate is removed by filtration through a fritted filter (Note 8) into a 1-L round-bottomed flask (24/40 joint) and a portion of methanol (200 mL) is added. To the solution, silica gel (75 g) (Note 23) is added. The solution is concentrated under vacuum (400 mmHg to 50 mmHg, 40 ºC) on a rotary evaporator and then under high-vacuum (Note 24). The silica gel-adsorbed product is then loaded onto a packed chromatography column (Note 25) and purified (Note 26) to give 3.65 g (53%) of 3,6-bis(dimethylamino)-9H-xanthen-9-one (1) (Notes 27, 28, and 29).

2. Notes

1. Prior to performing each reaction, a thorough hazard analysis and risk assessment should be carried out with regard to each chemical substance and experimental operation on the scale planned and in the context of the laboratory where the procedures will be carried out. Guidelines for carrying out risk assessments and for analyzing the hazards associated with chemicals can be found in references such as Chapter 4 of "Prudent Practices in the Laboratory" (The National Academies Press, Washington, D.C., 2011; the full text can be accessed free of charge at https://www.nap.edu/catalog/12654/prudent-practices-in-the-laboratory-handling-and-management-of-chemical. See also "Identifying and Evaluating Hazards in Research Laboratories" (American Chemical Society, 2015) which is available via the associated website "Hazard Assessment in Research Laboratories" at https://www.acs.org/content/acs/en/about/governance/committees/chemicalsafety/hazard-assessment.html. In the case of this procedure, the risk assessment should include (but not necessarily be limited to) an evaluation of the potential hazards associated with pyronin Y, sodium phosphate, iodine, N-Methyl-2-pyrrolidone, methylene chloride, methanol, sodium bicarbonate, ethyl acetate, hexanes, sodium sulfate, silica gel, celite, acetic acid, formaldehyde solution, sodium cyanoborohydride and gas evolution. As well, precaution should be taken when heating compounds to the extent described herein. Additionally, vacuum distillation poses potential risks associated with heating compounds under vacuuming the utmost caution should be taken.

2. Pyronin Y was purchased from Millipore Sigma as the "For microscopy (Bot., Fl., hist)" grade of the compound, this was used as received. Quantitative ¹H-NMR pdf using 1,3,5-trimethoxybenzene (purchased from Acros Organics and purified by vacuum sublimation at 50 ºC) as a standard revealed a purity of 65.7% (¹H NMR sample in 1:1 CD₃OD to DMSO-d₆). This was determined by using 21.9 mg of Pyronin Y and 12.5 mg of 1,3,5-trimethoxybenzene. The bottle of Pyronin Y starting material was used, giving 7.40 g of pure starting material. CD₃OD and DMSO-d₆were purchased from Cambridge Isotope Laboratories and used as received.

3. Sodium phosphate tribasic dodecahydrate (≥98%) was purchased from Millipore Sigma and used as received.

4. N-Methyl-2-pyrrolidone (NMP) (99%) was purchased from VWR chemicals and used as received. Water was sourced from a de-ionized water tap.

5. The reaction was monitored by silica gel TLC (Figure 3) with the mobile phase (2:2:1, v:v:v) ethyl acetate/hexanes/methylene chloride. TLC Silica gel 60 F254 was purchased from Sorbent Technologies, Inc. The Rf value of the product (1) was 0.5, and the Rf of the by-product (2) was 0.9 The by-product spot appears when 2 is exposed to UV-Vis light while on the TLC plate, which reverts it back to pyronin Y. Faint amounts of starting material appear as substantial on the TLC plate.

Figure 3. TLC monitoring at 2 hours, prior to iodine addition

6. Iodine (≥99.99%) was purchased from Millipore Sigma and used as received.

7. Iodine serves as an oxidant to convert the xanthene by-product 2 back to the starting material, pyronin Y, allowing it to participate again in the reaction cycle.

8. Celite 545 filter aid (50 g) was used in a fritted funnel (dimensions: 7 cm diameter and 10 cm height, m porosity). Celite was purchased from Fisher Scientific and used as received.

9. Methylene chloride (certified ACS grade) was purchased from Fisher Scientific and used as received.

10. Methanol (certified ACS grade) was purchased from Fisher Scientific and used as received.

11. The vacuum distillation apparatus (Figure 4) utilizes a high-vacuum (Welch 1405B DuoSeal), and a standard distillation set up including a three-way adapter attached to the round-bottomed flask, a 24/40-jointed condenser, a vacuum adapter and a 500-mL receiving flask (24/40 joint. The crude reaction mixture was distilled from a 500-mL round-bottomed flask. Starting at 60 ºC and 50 mm Hg with 650 rpm stirring, the NMP was rapidly distilled when dry ice was added to the acetone bath around the submerged receiving flask. The temperature was slowly increased to 70 ºC over the course of 2 h until only residual NMP remained, which could be removed by washing in subsequent steps.

Figure 4. NMP evaporation set-up

12. During the course of the reaction, demethylation occurs for 10-20% of the product and the extent of demethylation can be assessed by LCMS (column: ZORBAX Eclipse Plus C18, 95Å, 2.1 x 50 mm, 5 µm, 400 bar pressure limit, Agilent part number: 959746-902; method: 5% MeOH in 95% water to 95% MeOH in 5% water over 12 minutes gradient with a constant 0.1% formic acid background. The ratio of product to de-methylated byproduct was determined by integrating the 280 nm absorbance from the LCMS trace). (Figure 5)

Figure 5. Monitoring demethylation by LCMS

13. Separation of the desired N,N,N'N'-tetramethyl product 1 from the N,N,N'-trimethyl side product by chromatography is challenging, thus reductive amination is used to simplify the purification.

14. Glacial acetic acid was purchased from Fisher Scientific and used as received.

15. Formaldehyde solution (37% wt. with 10-15% methanol as a preservative) was purchased from Fisher Scientific and used as received.

16. Sodium cyanoborohydride reagent grade (95%) was purchased from Millipore Sigma and used as received.

17. The reductive amination was successful to alkylate the trimethyl by-product to form the desired product 1. (Figure 6) Alkylation can be monitored by either LCMS (column: ZORBAX Eclipse Plus C18, 95Å, 2.1 x 50 mm, 5 µm, 400 bar pressure limit, Agilent part number: 959746-902; method: 5% MeOH in 95% water to 95% MeOH in 5% water over 12 min with a constant 0.1% formic acid background. The ratio of product to de-methylated side product was determined by integrating the 280 nm absorbance from the LCMS trace) or silica gel TLC with the mobile phase (2:2:1, v:v:v) ethyl acetate/hexanes/methylene chloride. The demethylated side product and desired product appear when exposed to UV-Vis light while on the TLC plate.

Figure 6. Monitoring reductive amination by LCMS and TLC

18. Potassium carbonate anhydrous (granular powder/ACS certified chemical) was purchased from Fisher Scientific and used as received.

19. Monitoring the pH of the solution was done with hydrion pH (0.0-13.0) paper. A final pH was 9-10 was observed following addition of potassium carbonate.

20. Brine (sat. NaCl) was prepared with sodium chloride (Crystalline/ACS certified) purchased from Fisher Scientific and used as received. Emulsions were apparent with the brine wash and the brine layer was continually increased in volume until layer separation was observed (~300-400 mL in total).

21. Prior to addition of drying reagent, a small amount of acetone (~10 mL) was added the reaction mixture to break up visible emulsions.

22. Sodium sulfate anhydrous (granular/ACS certified) was purchased from Fisher Scientific and used as received.

23. Silica gel premium grade 60 Å was purchased from Sorbent Technologies, Inc.

24. The silica gel-adsorbed product is dried thoroughly to ensure no residual solvent is present that will affect separation. With complete drying, the silica gel is freely flowing and resembles unfunctionalized silica.

25. The column (dimensions 10 cm in diameter, 46 cm in height) is wet-packed with silica gel (450 g) using a solution of hexanes and methylene chloride (4:1, v:v). The silica gel-adsorbed product was loaded directly onto the packed silica.

26. The elution gradient that was used was: hexanes/methylene chloride (8:2) 0.5 L; hexanes/methylene chloride/ethyl acetate (7:2:1) 0.5 L, (6:2:2) 0.5 L, (5:2:3) 1 L, (4:2:4) 1 L, (3:2:5) 2 L, and finally (2.5:2:5.5) 2 L. The product was collected starting at 40% ethyl acetate and ending at 55% ethyl acetate. Due to the fluorescence of the product, it can be tracked during chromatography. Collection in 20-mL test tubes began when the product was at the position in Figure 7A. One full rack (40) of test tubes was collected. Following this, collection was done in Erlenmeyer flasks and a total of 1.5 L was collected before switching back to test tubes, which was done when the product was at the location in Figure 7B. A further 60 20-mL test tubes were used in the collection.

Figure 7. Monitoring product elution by fluorescence

The product was found starting in test tube 25, in all of the Erlenmeyer flasks and out to test tube 95. Thus, tubes 25-95 were collected, in addition the 1.5 L collected from the five Erlenmeyer flasks. (Figure 8) In total, approximately 3 L of fractions were combined and concentrated by rotary evaporation (starting at 400 mmHg and down to 100 mmHg) and then high-vacuum for several hours.

Figure 8. Monitoring chromatography fractions by TLC

27. The following characterization data of the purified 3,6-bis(dimethylamino)-9H-xanthen-9-one 1 was found: brown/orange solid which became more purple over time open to air, ¹H NMR pdf (600 MHz, CDCl₃) δ: 3.05 (s, 12H), 6.42 (s, 2H), 6.65 (d, J = 9.0 Hz, 2H), 8.06 (d, J = 9.0 Hz, 2H). ¹³C NMR pdf (125 MHz, CDCl₃) δ: 40.0, 96.8, 109.1, 111.5, 127.4, 154.5, 158.2, 175.8. IR (powder) 2906, 2808, 2723, 1592, 1530, 1430, 1348, 1330, 1158, 1119, 667 cm^-1. mp 234 - 236 ºC. HRMS calcd (ESI) m/z for C₁₇H₁₈N₂O₂: [M+H]⁺: 283.1441. Found: 283.1447.

28. The purity of 1 was determined to be 98% by quantitative ¹H-NMR pdf using 24.0 mg of 1 and 15.2 mg of 1,3,5-trimethoxybenzene as an internal standard.
The submitter's yields were 72% and 83% (97% and 95% purity, respectively). Checkers obtained 61% yield (97% purity) when the procedure was carried out on half scale. The purity of the pyronin Y bottle in the second run was slightly lower.

Working with Hazardous Chemicals

The procedures in Organic Syntheses are intended for use only by persons with proper training in experimental organic chemistry. All hazardous materials should be handled using the standard procedures for work with chemicals described in references such as "Prudent Practices in the Laboratory" (The National Academies Press, Washington, D.C., 2011; the full text can be accessed free of charge at http://www.nap.edu/catalog.php?record_id=12654). All chemical waste should be disposed of in accordance with local regulations. For general guidelines for the management of chemical waste, see Chapter 8 of Prudent Practices.

In some articles in Organic Syntheses, chemical-specific hazards are highlighted in red "Caution Notes" within a procedure. It is important to recognize that the absence of a caution note does not imply that no significant hazards are associated with the chemicals involved in that procedure. Prior to performing a reaction, a thorough risk assessment should be carried out that includes a review of the potential hazards associated with each chemical and experimental operation on the scale that is planned for the procedure. Guidelines for carrying out a risk assessment and for analyzing the hazards associated with chemicals can be found in Chapter 4 of Prudent Practices.

The procedures described in Organic Syntheses are provided as published and are conducted at one's own risk. Organic Syntheses, Inc., its Editors, and its Board of Directors do not warrant or guarantee the safety of individuals using these procedures and hereby disclaim any liability for any injuries or damages claimed to have resulted from or related in any way to the procedures herein.

3. Discussion

The use of fluorophores in biological imaging and fluorescence-based assays is highly prevalent.² This use has culminated in advancements such as next-generation DNA sequencing³ and more recently peptide sequencing,⁴ as well as countless reports utilizing fluorescent signal for analyte detection.⁵^,⁶^,⁷ The workhorse fluorophores used in many of these studies come from the rhodamine dye family.⁸ First discovered over a century ago in 1887 by Ceresole,⁹ only recently have studies demonstrated substitution of the core rhodamine oxygen with C, Si, P, Ge, and Sn,¹⁰^,¹¹^,¹² which give fluorophores with highly diverse photophysics and emissions well into the IR region. In accessing these compounds, a common intermediate is the corresponding xanthone derivative of the fluorophore scaffold, with the title compound 1 demonstrating the standard rhodamine variant. Significant efforts have been dedicated to the preparation of these xanthones, as substitution at the ketone carbonyl provides a facile method of building complex fluorophores. ¹³^,^14,15^,¹⁶

The title compound 1 is a versatile building block for the synthesis of rhodamine fluorophores. Indeed, fluorophores derived from this compound as well as the C and Si substituted fluorophores have found widespread use in varying fields, and thus improving the synthesis would help to further increase their use. The xanthene scaffold is unique in its ability to be interconverted between different forms of the molecule, with the xanthone appearing to be a thermodynamic sink relative to the xanthene and pyronine forms.

Figure 9. Comparison of various xanthone syntheses

Many reports on xanthone synthesis utilize the oxygen-transfer reagent KMnO₄ to oxidize the xanthene form of the dye^{11, 17} (Figure 9) directly to the ketone. Preceding this, the xanthene molecule is generally accessed through ring-closing or pyronine reduction.

From Pyronin Y, Bertozzi and coworkers demonstrated borohydride reduction followed by permanganate oxidation gave compound 1 in a 21% yield for the two steps.¹³ Similarly, studies out of the Hell and Lavis labs for carbon- and silicon-xanthones utilizes this same xanthene permanganate oxidation.¹¹^,¹⁸ Nagano has developed one method for the direct oxidation of silicon-pyronine to silicon-xanthone, which relies on a sequential three step procedure with KCN, FeCl₃ in acid, followed by reflux in bicarbonate which was shown to proceed in 15-30% yield for various Si-rhodamines^{12, 19} and a 22% yield for the title compound. ²⁰ These methods suffer in part due to the stability of the conjugated pyronine relative to the xanthene. It has been shown that under aerobic conditions, xanthenes oxidize to pyronines and thus cannot participate in the permanganate oxidation.^11,21 As well, the harsh oxidant KMnO₄ may result in product degradation.

The method presented herein takes advantage of the cycle between pyronine and xanthene. With a base-catalyzed addition of water to C9 of the pyronine a dibenzylic alcohol species is generated. A deprotonation of the resulting hydroxyl group results in a transfer of the dibenzylic hydride to another equivalent of pyronine, forming both xanthone and xanthene. In this way, 50% of the pyronine is converted to product in every cycle. Iodine oxidizes the xanthene back to pyronine, which is able to participate in the reaction once more. Due to this cycling between reduced and oxidized forms of the molecule, this methodology can be applied to syntheses that either form the xanthene first and are then treated with iodine or begin from pyronine, as is demonstrated here. In some instances, oxidation of the xanthene with quinones provides a facile route to pyronine.¹⁴ In previous studies, we expanded this methodology to carbo- and siliconpyronines (Table 1) and observed good yields using with this oxidation protocol. We have demonstrated this work with the N,N,N',N'-tetramethyl substitution pattern as it is highly general, but seemingly any alkyl or hydrogen aniline substitution pattern is amenable to this methodology.²²
Table 1. Oxidation of rhodamine variants

References and Notes

Anslyn Laboratory, Department of Chemistry, The University of Texas at Austin, Austin, Tx, 78712, USA. Email: anslyn@austin.utexas.edu. ORCID: 0000-0002-5137-8797. The authors thank Edward M. Marcotte for discussions on this project and acknowledge support from the National Institute of Health (R35 GM122480) to E.M.M. as well as The Welch Regents Chair to E.V.A. (F-0046). Additionally, we thank NSF (1 S10 OD021508-01) for the Bruker AVANCE III 500 NMR.
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Appendix
Chemical Abstracts Nomenclature (Registry Number)

Pyronin Y: Xanthylium, 3,6-bis(dimethylamino)-, chloride (1:1); (92-32-0)

Na₃PO₄: Sodium phosphate tribasic dodecahydrate; (10101-89-0)

I₂: Iodine; (7553-56-2)

: 1-Methyl-2-pyrrolidinone; (872-50-4)

HCHO: Formaldehyde solution (37 wt. % in H₂O); (50-00-0)

NaCNBH₃: Sodium cyanoborohydride; (25895-60-7)

AcOH: Glacial acetic acid; (64-19-7)

	James Bachman was born in Houston, Texas. He obtained a B.S. in Chemistry in 2014 at The University of Texas at Austin where he stayed for his graduate studies under the supervision of Professor Eric V. Anslyn. He is currently a Ph.D. candidate and his dissertation is focused on fluorophore synthesis and peptide-labeling used in single-molecule peptide sequencing. After graduation, James will carry out postdoctoral work at the University of California, Los Angeles with Professor Neil K. Garg.
	Cyprian Pavlich was born in Lancaster, CA and moved to Irving, Texas where he attended and graduated from St. Peter's Academy. He is now an undergraduate research assistant at the University of Texas at Austin under the supervision of Professor Eric V. Anslyn. Cyprian will graduate in spring 2020 with a B.S. in chemistry and is looking forward to continuing his chemical education in a graduate program.
	Eric V. Anslyn received his Ph.D. with Robert Grubbs at Caltech followed by a postdoctoral fellowship with Ronald Breslow at Columbia University. He started his independent career in 1989 at The University of Texas at Austin. He has made his mark studying physical organic chemistry and supramolecular chemistry, including seminal work on differential sensing. He was recently awarded the James Flack Norris Award in physical organic chemistry by the American Chemical Society.
	Jacob E. Dander received his B.S. in Chemistry from Illinois College in Jacksonville, IL, where he performed undergraduate research under Professor Brent Chandler on the total synthesis of the anti-cancer compound xenitorin A. He is currently a Ph.D. Candidate and NSF Graduate Research Fellow in Professor Neil Garg's laboratory at UCLA. His doctoral studies are focused on the development of nickel-catalyzed amide C-N bond activation methodologies and the total synthesis of complex molecules.
	Katie A. Spence received her B.A. degree in Chemistry and Psychology from Williams College in 2018. As an undergraduate, she studied the formation of atmospheric organic aerosols and completed a senior thesis on this topic under the direction of Professor Anthony Carrasquillo. She is currently a second-year graduate student in Professor Neil K. Garg's lab at the University of California, Los Angeles where she develops synthetic methodologies that employ strained intermediates.

Notes

References/EndNotes

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