Patent Application
20250034094
The present invention generally relates to a process for the preparation of N-retinylidene-N-retinylethanolamine (A2E). In particular, this fast and efficient process enables an eighty-seven-fold reduction in reaction time, from about 48 hours to about 33 minutes, with an accompanying yield improvement from about 49% to about 78%.
This section introduces aspects that may help facilitate a better understanding of the disclosure. Accordingly, these statements are to be read in this light and are not to be understood as admissions about what is or is not prior art.
The retinal pigment epithelium (RPE) is a cell monolayer that separates the retina from choroid circulation and is vital for the phagocytic recycling of photoreceptor waste, nutrient supply, ionic balance maintenance, and many other critical activities required for proper functioning of the retina1. Light detection by photoreceptors starts with the conversion of 11-cis-retinal, the prosthetic group sensitive to light in visual pigments, into its isomer, 11-trans-retinal (a.k.a., all-trans-retinal or ATR). ATR then needs to be replaced by another 11-cis-retinal to regenerate rhodopsin as part of the visual cycle process. All-trans or 11-cis retinaldehydes in photoreceptors tend to spontaneously dimerize into lipid bisretinoids that accumulate as retinal lipofuscin. Failures in ABCA4 function, a protein that flips retinaldehydes to the cytosol where deshydrogenases convert them to non-toxic retinols, exacerbate the formation of lipid bisretinoids2. As a consequence, lipid bisretinoids end up in the lysosomes of RPE cells, where they remain indefinitely because they are not susceptible to degradation by lysosomal enzymes. The accumulation of bisretinoid-rich lipofuscin in the RPE has been shown to induce cell death3 and is believed to be a key culprit in the etiology of conditions such as Stargardt's disease, cone-rod dystrophy, Best's macular dystrophy and potentially age-related macular degeneration (AMD)4.
N-Retinylidene-N-retinylethanolamine (A2E) is the most studied lipid bisretinoid. It forms lipofuscin deposits in the retinal pigment epithelium (RPE), causing vision impairment and blindness in eye conditions, such as Stargardt's disease, cone-rod dystrophy, Best's macular dystrophy and potentially age-related macular degeneration (S. Ben-Shabat et al., 2001). Synthetic A2E is often used for inducing the accumulation of lipofuscins within the lysosomes of RPE cells in culture as an in vitro surrogate of retinal lipofuscin buildup, providing insights into the mechanisms of these eye conditions. Many reports describing the use of synthetic A2E employ material that has been prepared using a one-pot reaction of all-trans-retinal (ATR) and ethanolamine at room temperature for 48 hours (C.A. Parish et al., 1998), and the quality of the product is questionable due to various impurities. There are unmet needs for an efficient, fast, and reliable synthetic process to provide a high-quality material for biomedical researches of eye diseases.
This synthesis was revisited by performing a design of experiments (DoE) and high throughput experimentation (HTE) workflow that was tailored to identify the most productive combination of the variables (temperature, solvent, reagent equivalences) for optimization of A2E yield. The DoE findings revealed that the interaction of ethanolamine with acetic acid and ATR was pivotal for the formation of A2E in high yield, likely indicating that imine formation is a critical step in the reaction. Armed with these results, the method was optimized using a microfluidic reactor system before upscaling those conditions for continuous flow synthesis of A2E. This revised method enabled an eighty-seven-fold reduction in reaction time, from 48 hours to 33 minutes, with an accompanying yield improvement from 49% to 78%. Furthermore, a simple method was developed to evaluate the quality of the A2E produced, using optical spectroscopy and LC-MS characteristics to assure that the biological properties observed with A2E samples are not confounded by the presence of occult impurities.
In an embodiment, described herein is a process for the production of N-retinylidene-N-retinylethanolamine (A2E) substantially free of impurities, from ethanolamine and all-trans-retinal (ATR) in the presence of acetic acid, the process comprising the steps of:
In another embodiment, described herein is a rapid method to access the purity of a sample of A2E, the method comprising the steps of:
The above and other aspects, objects, features, and advantages of the present disclosure will become more apparent and better understood when taken in conjunction with the following description, claims and drawings wherein identical reference numerals have been used, where possible, to designate identical features that are common to the figures.
The attached drawings are for purposes of illustration and are not necessarily to scale.
While the concepts of the present disclosure are illustrated and described in detail in the description herein, results in the description are to be considered as exemplary and not restrictive in character; it being understood that only the illustrative embodiments are shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected.
As used herein, the following terms and phrases shall have the meanings set forth below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art.
In the present disclosure the term “about” can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range. In the present disclosure the term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more of a stated value or of a stated limit of a range. In the present disclosure, “substantially free of X” indicates that a compound or material substantially free of X has less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, or less than about 0.1% of X in the material. The percentage may be a weight % or a mole % and will be understood based on the context of its use.
In the present disclosure, use of the term “high quality” to describe a compound or material means that the compound or material is substantially free of impurities.
In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading may occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.
The following clauses disclose several non-limiting embodiments of the disclosure.
1. An efficient synthetic process for the synthesis of high quality N-retinylidene-N-retinylethanolamine (A2E) from acetic acid, ethanolamine, and all-trans-retinal (ATR) through optimization of reaction solvents, molar ratios of the starting materials, reaction temperature, reaction time, and chromatographic purification conditions.
2. The efficient synthetic process for the synthesis of high quality A2E according to clause 1, wherein said high quality of A2E has low content of oxidized species, which adversely impact the biological activities of A2E.
3. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said high quality of A2E is measured/characterized according to UV-Vis, NMR and LC-MS data.
4. The efficient synthetic process for the synthesis of high quality of A2E according to clause 3, wherein said high quality of A2E has an UV-Vis spectra having a higher absorbance at about 440 nm than the absorbance at about 331 nm.
5. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said high quality of A2E is qualified by the absorption ratio of 439 nm/339 nm, which detects the A2E contamination with oxidized species or ATR. Only highly purified A2E gave similar concentrations from either 339 and 439 OD values. The 439 nm/339 nm absorption ratio of 1.39 was the highest for pure A2E.
6. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said process is a continuous flow reaction.
7. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said optimized temperature is about 37° C. or lower, preferably at 25° C.
8. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said optimized solvent is methanol or dimethyl sulfoxide (DMSO), preferably DMSO.
9. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said optimized solvent is not ethanol.
10. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said optimized reaction time is achieved by adjusting the flow rate and residence time of the reaction system.
11. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said optimized chromatographic purification condition is a sequential medium pressure liquid chromatography (MPLC) and high pressure liquid chromatograpyy (HPLC) purification process.
12. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said optimized solvent is methanol or DMSO.
13. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said optimized ratios of the starting materials are about 1:10:12 of all-trans-retinal:ethanolamine:acetic acid (ATR:EA:AA).
14. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said process provides a fast reaction with a much-improved yield.
15. The efficient synthetic process for the synthesis of high quality of A2E according to clause 1, wherein said process first provides a mixing point for one equivalent of ATR and about ten equivalents of ethanolamine, then followed by introduction of about twelve equivalents of acetic acid down the stream of continuous reaction flow at about 25° C. using DMSO as the solvent.
16. A product manufactured according to the process of clauses 1-15.
The following clauses recite additional non-limiting embodiments of the disclosure.
17. A process for the production of N-retinylidene-N-retinylethanolamine (A2E) substantially free of impurities, from ethanolamine and all-trans-retinal (ATR) in the presence of acetic acid, the process comprising the steps of:
17b. The process of clause 17 wherein the first solution and second solution are introduced into a mixing chamber prior to being introduced into the continuous flow reactor with the third solution.
18. The process of any one of the preceding clauses wherein each solvent is independently selected from MeOH or DMSO.
19. The process of any one of the preceding clauses wherein each solvent is MeOH or DMSO.
20. The process of any one of the preceding clauses wherein each solvent is DMSO.
21. The process of any one of the preceding clauses wherein the molar ratio of ATR to ethanolamine is 1 to about 10 and the molar ratio of ATR to acetic acid is 1 to about 12.
22. The process of any one of the preceding clauses wherein the TR is from about 3 minutes to about 120 minutes, such as about 3 minutes to 120 minutes or 3 minutes to about 120 minutes.
23. The process of any one of the preceding clauses wherein the TR is about 33 minutes.
24. The process of any one of the preceding clauses wherein the temperature is from about 25° C. to about 50° C., such as about 25° C. to 50° C. or 25° C. to about 50° C.
25. The process of any one of the preceding clauses wherein the temperature is about 25° C.
26. The process of any one of the preceding clauses wherein step e) comprises sequential application of MPLC and preparative HPLC.
27. The process of any one of the preceding clauses yielding A2E which displays a ratio of the U.V. absorbance at 339 nm to the U.V. absorbance at 439 nm of about 1.39 when dissolved in alcohol.
28. The process of any one of the preceding clauses yielding A2E that is substantially free of oxidized A2E, ATR, the enamine of ATR and ethanolamine, and dihydo-A2E.
29. A rapid method to access the purity of a sample of A2E, the method comprising the steps of:
30. The method of clause 29, wherein the A2E is prepared by the process of clause 1 or 2.
One of the most prevalent and most studied of such lipid bisretinoids is A2E, a pyridinium quaternary amine comprised of two retinaldehyde derived moieties. The most cited article on the synthesis of A2E5 uses all-trans-retinal and ethanolamine in a one-step-synthesis (Scheme 1), with one equivalent of acetic acid and ethanolamine in ethanol for 48 hours at room temperature as the best condition for A2E product formation. Scheme 2 shows that the first step in the reaction sequence is the formation of the all-trans-retinal (ATR), ethanolimine, followed by tautomerization to an enamine in a [1,6] proton shift; subsequent addition of a second molecule of ATR produces an iminium ion intermediate. Rearrangement of the iminium ion and subsequent auto-oxidation generates A2E4, 5.
Two aspects of the reaction mechanism are worth noting. In the first step, nucleophilic attack of the aldehyde by ethanolamine will be most favorable under alkaline conditions, since in the presence of acid, the ethanolamine nitrogen will be protonated, thus reducing its nucleophilicity. Conversely, acid conditions favor the second step by protonating the aldehyde oxygen to produce the hydronium leaving group. As the imine formation is an equilibrium, it is necessary to have enough acid to promote the formation of the hydronium ion, but not so much that the nucleophile equilibrium favors full protonation of ethanolamine. Therefore, allowing time for the reaction of all-trans-retinal with ethanolamine prior to the addition of acetic acid and investigation of the equivalence of acetic acid were viewed as crucial parameters to evaluate for optimizing reaction yield.
An additional factor to be considered in the planning of this reaction is the choice of solvent. Jin et al.6 studied the effects of different solvents on the extraction of A2E from eyecups. They found that extraction of A2E with different solvents followed by HPLC analysis revealed that A2E is not stable in THF, CHCl3 or EtOH, but is stable in methanol and DMSO. These findings suggested solvent as another possible parameter to be optimized.
Finally, the role of temperature on reaction efficiency was considered. As the conversion of all-trans-retinal to A2E occurs under physiologic conditions, uncovering whether the A2E yield would increase upon raising the reaction temperature from 22° C. to 37° C. and whether it would be improved at even higher temperatures was investigated.
Given these reaction variables of interest, a design of experiments (DoE) and high throughput experimentation (HTE) strategy was used to improve the synthesis of A2E. DoE is a statistical methodology that aims to identify all major parameters involved in a reaction to reveal how those parameters interact, since reaction parameters are rarely independent of each other7-9. Based on the DoE data obtained, the interplay of reaction parameters can be determined to guide the discovery of optimized conditions.
A key consideration in the DoE approach is the number of experiments to be performed10. For example, for a 23 factorial design, with two variables evaluated at three levels, a total of eight unique experiments are required, not including experimental replicates. The number of experiments required grows exponentially with the number of levels applied to the experiment. To simplify the execution of a large number of experiments and reduce costs, high throughput experimentation (HTE) is a valuable technique that is commonly used for data collection. HTE allows for grouping of common operations so that a series of experiments can be rapidly performed in parallel at microscale. This approach also allows for the automation of procedures, such as liquid handling and data analysis, so that hundreds of experiments can be executed simultaneously, and analyzed using quantitative techniques such LC-MS or semi-quantitatively by DESI-MS11-15. HTE also allows for facile and automated replication of experiments, making it the ideal pairing with the DoE approach, since the labor burden for implementing replicates does not increase considerably when the experiments are executed in a microscale format16, 17.
Due to the light and oxidation sensitivity of A2E, the data inputs from the HTE campaign were applied to continuous flow synthesis to provide better control over these parameters. Flow reactions involve the use of automated reagent delivery systems that are continuously mixed within a tubular reactor and collected downstream with control over residence time, flow rate, reactor temperature, light exposure, and reaction O2 content. The use of continuous flow methods can allow for better control over reaction parameters relative to batch syntheses due to the improved surface area-to-volume ratios in flow reactions that enable more efficient mixing and heat transfer. Microfluidic reactors also offer the advantages of safer handling and use of very small quantities of starting materials during the reaction optimization process. For preparative scales, the small-scale setup can be readily upscaled with the same control over mixing and heat transfer18.
The DESI-MS signal intensities for the A2E product peak (592.45 m/z) for each of the 27 reaction conditions were corrected for background signal, and the measured values were scaled from 0-100% with respect to the highest product peak intensity observed (TABLE 1). Evaluation of the HTE findings from this initial 33 DoE revealed that the best conditions are one equivalent of acetic acid at 37° C. in DMSO.
Utilizing Ellistat software, contour plots were generated to predict reaction gradient profiles (
The results obtained from the HTE and DoE analyses were used to guide an initial screen of continuous flow conditions (TABLE 2). The flow setup (
One indication of A2E purity is its UV-Vis absorption spectrum. When pure, A2E will have two defined bands: one at about 336 nm and a more intense one around 439 nm5, 19, 20. The intensity of the 439 nm band is important, since ATR, the synthetic precursor and potential contaminate in the isolated A2E fraction, contributes more to the sample absorbance at 330 nm than at 439 nm. Neither reaction generated a very pure A2E sample, even though their NMR spectra showed all the expected peaks for this compound. The difference between the UV-Vis spectra obtained for the two experiments is remarkable, with the experiment run at lower temperature and higher acetic acid equivalency showing clearer bands on 333 nm and 432 nm. This observation suggests that both the decrease in temperature and increase in acetic acid equivalency leads to increased reaction yield.
A second DoE was performed with a focus on A2E yield improvement and suppression of byproduct formation. After analyzing the batch experiments that produced A2E in the literature, we observed that there are two different reagent stoichiometries utilized. While Parish et al.5 utilizes a 2.27:1:1 ratio of ATR:ethanolamine:acetic acid, Guan et al.21 utilizes a 1:19:24 ratio of ATR:ethanolamine:acetic acid.
In order to understand the role of each reagent in the synthesis of A2E, another 33factorial DoE was designed, where the equivalence of each reagent was used as the minimum, intermediate and maximum factors to generate a matrix of 27 unique experiments with DMSO as solvent. We also sought to explore the effect of reaction temperatures on A2E yield, so that each set of 27 replicated experiments was performed at 25, 37 and 50° C. (we lowered the highest temperature in the experiments to 50° C. since the first DoE revealed that 100° C. was detrimental to A2E yield). Comparisons between the A2E product ion intensities produced by all 81experiments indicates that the best reaction condition is one equivalent of ATR, 10 equivalents of ethanolamine and 12 equivalents of acetic acid at 25° C. (TABLE 2).
To gain a deeper understanding of the results obtained, a Pareto analysis was generated to detail the importance of each factor and/or combination of factors (
Using the results of the second HTE as a guide, flow syntheses at two different flow rates—1 μL/min (i.e., a residence time, TR, of 3.3 minutes) and 0.1 L/min (TR=33 minutes) were performed. TLC analysis of the products showed that increased residence time produced a more intense spot for the product and less intense spots for the by-products, findings that were confirmed by product isolation after flash column chromatography (TABLE 3). The improved yield when the residence time is increased by an order of magnitude is related to the fact that the one-pot synthesis of A2E is a product of five consecutive reactions over 48 hours, such that a TR=3.3 minutes does not provide enough time for all of these reactions to occur. The maximum residence time possible for the experiment was achieved at 33 minutes due to the size limitations of the 3223 reactor and S1 system chosen for the study.
The flash column chromatography purified A2E produced a UV-Vis spectrum (
LC-MS analysis (
Based on these data, the conditions for preparative HPLC were tailored to produce a clear separation between the compounds in the mixture (ZORBAX ExtendC18 column, 9.4×250 mm, 5 μm, 80 A in a gradient of 85/25 to 95/5 Acetonitrile (ACN)/H2O for 1 hour with a flow rate of 4 mL/min). A2E samples synthesized in flow and purified by this method gave a UV-Vis spectrum with clear peaks at 331 nm and 440 nm (
The flow setup used three syringe pumps to deliver the reagents, with ATR and ethanolamine first engaging in a T-mixer; that mixture then encounters acetic acid in a second T-mixer before flowing this final mixture through a coiled PFA tubing reactor (
Applying the optimized conditions derived from the microscale setup, the reagent equivalencies and residence time were maintained, but three different flow rates of 10, 5 and 2 μL/min were tested. The faster flow rate (10 μL/min) proved to be the most efficient (TABLE 4), a finding that can be attributed to improved mixing with increasing flow rate23.
Pure A2E was obtained by collecting the reaction mixture from the flow reactor and isolating the product by sequential medium pressure liquid chromatography (MPLC) and preparative HPLC. The purity of A2E obtained is corroborated by the UV-Vis spectrum obtained (
1H NMR (CD3OD, 500 MHz): δ 1.07 and 1.08 (6H each, s, C5-(CH3)2 and C5′-(CH3)2); 1.53 (4H, m, C2-H2 and C2′-H2); 1.69 (4H, m, C3-H2 and C3′-H2); 1.75 and 1.77 (3H each, s, Cl-CH3 and Cl′-CH3); 2.07 3H, s, C9-CH3); 2.10 (4H, m, C4-H2 and C4′-H2); 2.18 (3H, s, C13-CH3); 2.20 (3H, s, C9′-CH3); 3.94 (2H, t, CH2—O); 4.56 (2H, t, N—CH2); 6.20 (1H, d, C8-H); 6.27 (lH, C10-H); 6.32 (lH, d, C8′-H), 6.37 (lH, d, C7-H); 6.44 (lH, d, C10′-H); 6.57 (lH, d, C7′-H); 6.63 (lH, d, C12-H); 6.72 (lH, s, C14-H); 6.78 (lH, d, C12′-H); 7.15 (lH, dd, C11-H); 7.89 (lH, d, C13′-H); 7.96 (lH, dd, C14′-H); 8.01 (lH, dd, C11′-H); 8.56 (lH, d, C15′-H). Peaks at 4.89 ppm and 3.34 ppm are HOD and MeOD, respectively.
As a further test of the method, two parameters were changed: residence time and solvent. In a first approach, the flow rate, reagent equivalences, temperature, and solvent were maintained, but the residence time was increased four-fold, from 33 minutes to 120 minutes (TABLE 5). The increased residence time only led to the increased production of by-products, with a major UV-Vis peak at 328 nm, a feature that is highly suggestive of increased of A2-DHP-E content19, 26. In the second case, the flow rate, residence time, reagent equivalences and temperature were maintained, but ethanol instead of DMSO was used. This reaction produced a far greater amount of iso-A2E than A2E (TABLE 6).
In order to develop an approach to rapidly assess and standardize A2E quality, absorption and fluorescence spectra were used. This method was extremely sensitive and easy for detecting differences in sample quality (
The 439 nm/339 nm absorption ratio was the parameter that can most readily detect A2E contamination with oxidized species or ATR. Absorbance data were used to calculate the concentration of the A2E solutions based on published molar extinction coefficients of A2E at 339 and 439 nm24. Only highly purified A2E gave similar concentrations from either 339 or 439 OD value. The 439 nm/339 nm absorption ratio of 1.39 was the highest for pure A2E.
Based on conflicting literature reports about the synthesis methodology for A2E and the probable mechanism for A2E formation, two 23 DoE were created, that determined the best reaction conditions for the one-step synthesis of A2E. DMSO, as reported by Jin et al.6, reduces the extent of A2E degradation as it is being formed. The ratio of acetic acid to ethanolamine also proved to be a significant factor for improving reaction yield. The reaction also proved to be sensitive to high temperatures, with more efficient reactions occurring at 25° C.
After identifying the preferred reaction conditions, A2E was synthesized on small and large scales using continuous flow reactors. This modification reduced the reaction time from 48 hours to 33 minutes of residence time leading to a greatly improved A2E production. It was also discovered that increased reaction time and EtOH as solvent lead to greater byproduct formation. The optimized condition for large production of A2E was achieved with a flow rate of 10 μL/min and a residence time of 33 minutes, utilizing DMSO as a solvent at 25° C. with an equivalence ratio of 1:10:12 of ATR:EA:AA. These conditions improved the reaction yield from 49%5 to 78%.
Finally, different purification methods were investigated in order to obtain A2E in the highest possible purity. The results suggest that a sequential MPLC and HPLC purification process generates highly pure A2E according to UV-Vis, NMR and LC-MS data. It was discovered that the sequential MPLC+HPLC purification sequence is crucial for the correct biological response of the samples, because, surprisingly, the presence of even a small amount of oxidized species appears to result in variable biological performance.
AlamarBlue® was from Invitrogen. All other reagents were purchased from Sigma-Aldrich and used without further purification.
NMR spectra were collected using a Bruker AV-III-500-HD NMR spectrometer in CD3OD and the chemical shifts reported versus TMS.
High-throughput experiments and Desorption Electrospray Ionization-Mass Spectrometry (DESI-MS) were performed using a previously published method14. In brief, a
Biomek i7 liquid handling robot was used to prepare the reactions and a LTQ XL (Thermo Scientific) fitted with a DESI 2D stage (Prosolia Inc.) were used to analyze the reaction outcomes. After planning the experiment, the DoE matrix was transferred to a spreadsheet to be inputted on the Xcalibur software (version 3.0) for future use in the DESI-MS. Then, the reagents were transferred into 96-well heating blocks with the desired amounts (in pre-made solutions with the desired solvent). The heating blocks were set to the correct temperatures and, after 48 hours (and cooling of the heating blocks to room temperature), the solutions were transferred to 384-well plates using the i7 robot. The DESI plates were pinned with the i7 robot, and the DESI plate was analyzed with a linear ion trap mass spectrometer fitted with a DESI imaging source. The average ion counts for each combination in the matrix were replicated from three (HTE 2) to eight times (HTE 1) with their respective blanks. The measured ion counts were averaged and normalized against the ion counts measured for the blank regions of the plate. The yields were calculated from the normalized average ion counts and the collected information was inputted into Ellistat software for statistical analysis.
A2E was synthesized using continuous flow methodology in a Labtrix S1 system (Chemtrix BV, Echt, The Netherlands). Three 1 mL stock solutions of 99% DMSO containing ATR (0.3 mol/L, 85 mg), ethanolamine (0.3 mol/L, 18 mg or 3 mol/L, 183 mg) and acetic acid (0.3 mol/L, 18 mg or 3 mol/L, 180 mg or 3.6 mol/L, 216 mg) were prepared, and the solutions were purged with Ar prior to being loaded to three 1 mL Hamilton syringes (Reno, NV), respectively. ATR and ethanolamine were added via a T junction into the same port of a staggered oriented ridge Chemtrix 3223 reactor chip (10 μL), in which acetic acid was also added into a second port. The syringes and chip were connected by FEP tubing (0.8 mm o.d.×0.25 mm i.d., Dolomite Microfluidics). The respective flow rates are reported in TABLES 5 and 6.
The reaction product solutions were extracted with ACN and washed five times with hexane and 1 M NaOAc. The ACN layer was dried under high vacuum. The resulting red solid was purified by silica gel column chromatography using a step elution with 98:2:0.01 CH2Cl2 (DCM)/MeOH/TFA, 90:10:0.01 DCM/MeOH/TFA and 2:1:0.01 DCM/MeOH/TFA, in sequence. The product fractions were further purified via semipreparative HPLC with a ZORBAX ExtendC18 column, 9.4×250 mm, 5 μm, 80 A in a gradient of 85/25 to 95/5 ACN/H2O for 1 hour with a flow rate of 4 mL/min. The product fractions were flash frozen and lyophilized. The resulting yields are reported in TABLES 5 and 6.
Three 5 mL stock solutions of ATR (0.3 mol/L, 426 mg), ethanolamine (3 mol/L, 916 mg) and acetic acid (3.6 mol/L, 1081 mg) were prepared in 99% DMSO, and the solutions purged with Ar prior to loading into three 25 mL Hamilton syringes, respectively. The syringes were mounted onto two Harvard syringe pumps and connected by FEP tubing ( 1/16×0.010 ft, IDEX) to the flow system according to
After the reactions were complete, the product solutions were extracted with ACN and washed three times with 1 M NaOAc. The ACN layer was dried under high vacuum. The resulting red solid was purified by MPLC normal phase chromatography in gradient mode for 40 minutes, starting with 98:2:0.01 DCM/MeOH/TFA, until 50% MeOH with a flow rate of 15 mL/min. The product fractions were further purified via preparative HPLC with a Waters Prep C18 XBridge column, 30×100 mm, 10 μm, 80 A in a gradient of 85/25 to 95/5 ACN/H2O for 35 minutes with a flow rate of 40 mL/min. The product fractions were dried under high pressure. The resulting yields are reported in TABLE 4.
ARPE19 cells from ATCC were plated at 80% confluency in 96-well plates and pre-treated for 1 hour with inhibitors (33 μM Necrostatin 7 (Cayman Chemicals): 2 mM N-acetyl-cysteine (NAC) (Sigma): 50 μg/ml phoroglucinol (Sigma)), after which the medium was supplemented with A2E/ATRD or vehicle (control) and cells were incubated in serum-free OptiMEM (Invitrogen) medium for an additional 23 hours at 37° C. To assess viability, 20 μL of 10× AlamarBlue® (Invitrogen) were added per well, and cells were incubated for an additional hour before reading the fluorescence in SpectraMax M5e (Molecular Devices, CA, USA) using 555 nm excitation/585 nm emission.
A2E quality was assessed by diluting the A2E lot into alcohol. Absorbance and fluorescence spectra were determined in 96-well plates with black walls and clear bottoms. Absorbance was measured between 300 and 500 nm and fluorescence between 500 and 700 nm exciting with 410 nm, using a Spectramax M5e.
LC-MS analysis was performed on a Quantum TSQ Discovery mass spectrometer (ThermoScientific) equipped with ThermoScientific autosampler, ThermoScientific mass spectrometry pump and ThermoScientific ESI detector. Fifteen microliters of sample solution were loaded onto the column and eluted isocratically (mobile phase containing 95% acetonitrile, 5% water, and 0.1% TFA). The column used was an Agilent Eclipse XDB-C18 (4.8×150 mm) with a flow rate of 1 mL/min. The mass spectrometer was operated in positive ion mode with spray voltage at 5000 V and capillary at 350° C. The Q1 quadrupole scanned from m/z 50 to 1000.
Those skilled in the art will recognize that numerous modifications can be made to the specific implementations described above. The implementations should not be limited to the particular limitations described. Other implementations may be possible.
It is intended that that the scope of the present methods and compositions be defined by the following claims. However, this disclosure may be practiced otherwise than is specifically explained and illustrated without departing from its spirit or scope. It should be understood by those skilled in the art that various alternatives to the embodiments described herein may be employed in practicing the claims without departing from the spirit and scope as defined in the following claims.
This patent application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 63/281,785, filed on 22 Nov. 2021, the contents of which are hereby incorporated by reference in their entirety into this disclosure.
This invention was made with government support under R01EY027422, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/050689 | 11/22/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63281785 | Nov 2021 | US |
