WO2018094293A1 - A kit for determining the absolute configuration of alcohols using a competing enantioselective conversion approach - Google Patents

Abstract

Provided herein is a kit for the determination of the absolute configuration of alcohols of a competing enantioselective conversion approach.

Description

A KIT FOR DETERMINING THE ABSOLUTE CONFIGURATION OF ALCOHOLS USING A COMPETING ENANTIOSELECTIVE CONVERSION APPROACH CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No.

62/424,380, filed November 18, 2016, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under Grant Nos.

CHE1152449 and CHE1361998, awarded by the National Science Foundation. The

Government has certain rights in the invention.

TECHNICAL FIELD

[0003] Provided herein is a kit for the determination of the absolute configuration of alcohols using a competing enantioselective conversion approach.

BACKGROUND

[0004] Determining the absolute configuration of organic compounds is a challenging problem. There are many potential ways to determine the absolute configuration of organic molecules including X-ray analysis, circular dichroism (CD), the Mosher's advanced method, vibrational circular dichroism (VCD) with computational analysis, and others. Most of these methods are time consuming, labor intensive and not cost effective.

SUMMARY

[0005] Provided herein are methods and kits for determining of the absolute

configuration of secondary alcohols using a competing enantioselective conversion (CEC) approach. The kits presented herein are capable of determining the configuration of secondary alcohols on the micromole to nanomole scale. The absolute configuration of the alcohol can be assigned via qualitative analysis of the relative fast reaction, e.g., by thin-layer chromatography. The kits and methods of the disclosure can utilize as much as a 50-fold reduction in material in direct comparison to other CEC studies. The disclosure provides for multi-use kits, as well as, one-use kits. Product formation from the methods and kits disclosed herein can be determined by using a variety of detecting schemes, including ¹H NMR spectroscopy, and thin-layer chromatography. The methods and kits described herein provide consistent results and exhibit pseudo-first order kinetics in regards to substrates. Therefore, the lower limit of sensitivity for the methods and kits disclosed herein is largely limited by the detection schemes used to detect the ester product from the alcohol substrate.

[0006] In a particular embodiment, the disclosure provides for a competing

enantioselective conversion (CEC) kit used to determine the absolute configuration of a secondary alcohol, comprising, consisting essentially of, or consisting of: a first container comprising a first solution that comprises, consists essentially of, or consists of R- homobenzotetramisole (R-HBTM), an organic acid anhydride, a tertiary amine, and one or more non-protic solvents; and a second container comprising a second solution that consists essentially of, or consists S-homobenzotetramisole (S-HBTM), an organic acid anhydride, a tertiary amine, and one or more non-protic solvents, wherein the first and second containers can be stored for at least six months at temperature of about -20 ºC or lower with minimal to no loss in enantioselective activity, and wherein the absolute configuration of the secondary alcohol can be determined by use of the CEC kit with the secondary alcohol and qualitatively determining the fast reaction via ester product formation. In a further embodiment, the organic acid anhydride of the first solution and/or of the second solution is propionic anhydride. In another embodiment, the tertiary amine of the first solution and/or of the second solution is N,N-diisopropylethylamine. In yet another embodiment, the one or more non-protic solvents of the first solution and/or of the second solution comprises CDCl₃. In a further embodiment, the first and second solution consists essentially of, or consists 0.2 equivalents of S-HBTM or R-HBTM, 15 equivalents of propionic anhydride, and 5 equivalents of N-N-diisopropylethylamine. In yet a further embodiment, the first and second containers are sealed under an inert gas. In another embodiment, the first and second containers are vials, ampoules, tubes, or bottles. In yet another embodiment, the first and second containers are made of a dark material that absorbs ultraviolet radiation. In a further embodiment, the CEC kit further comprises: a third container comprising a protic solvent. In yet a further embodiment, the protic solvent is methanol or methanol-d₄. In a certain embodiment, the CEC kit further comprises: a fourth container comprising a first optically pure secondary alcohol; and optionally a fifth container comprising a second optically pure secondary alcohol, wherein the second optically pure secondary has the opposite rotation to the first optically pure secondary alcohol. In a further embodiment, the first optically pure secondary alcohol and the second optically pure secondary alcohol are different enantiomers of the same compound. In yet a further embodiment, the first optically pure secondary alcohol and second optically pure alcohol are selected from (R)-(+)-1-(2-naphthyl)ethanol and (S)-(í)-1-(2-naphthyl)ethanol. In another embodiment, the CEC kit is manufactured for a single use, such that the first container and second container contain enough solution to determine the absolute configuration of one secondary alcohol. In an alternate embodiment, the CEC kit is manufactured for multiple uses, such that the first container and second container contain enough solution to determine the absolute configuration of multiple secondary alcohols. In yet another alternate embodiment, the CEC kit is manufactured for multiple uses, such that the CEC kit contains multiple containers that comprise the first solution, and multiple containers that comprise the second solution.

[0007] In a particular embodiment, the disclosure further provides for a Competing Enantioselective Conversion (CEC) process for determining the absolute configuration of a secondary alcohol comprising, consisting essentially of, or consists of: providing the CEC kit of any one of the preceding claims; mixing or adding a solution comprising a secondary alcohol with the first solution from the first container; mixing or adding the solution comprising the secondary alcohol, with the second solution from the second container;

incubating the reaction mixtures for 30 min to 2 h, wherein both reaction mixtures are incubated for substantially or the same period of time; stopping the two reactions by adding a protic solvent to the first reaction vessel and the second reaction vessel; analyzing the two reactions by using a qualitative analytical chemical technique(s) to identify the faster-reacting HTBM catalyst; and determining the absolute configuration of the secondary alcohol by using a predictive CEC mnemonic to analyze the results from the analytical chemical technique(s). In a further embodiment, the analytical techniques are thin layer

chromatography (TLC) and/or nuclear magnetic resonance (NMR).

[0008] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the disclosure and, together with the detailed description, serve to explain the principles and implementations of the invention. [0010] Figure 1 presents the R-HBTM and S-HBTM acyl-transfer reagents and a mnemonic for determining the absolute configuration of secondary alcohols using a CEC method disclosed herein.

[0011] Figure 2 presents a graph detailing the conversion of alcohol (R)-(+)-1-(2- Naphthyl)ethanol ((R)-5)) as determined via ¹H NMR spectroscopy and GC-MS analysis. Alcohol (R)-5 was solvated in CDCl₃ and then 100 μL was dispensed into each CDCl₃ CEC kit vial containing 450 μL of a solution of propionic anhydride, N-N-diisopropylethylamine, and HBTM.

[0012] Figure 3 presents the reaction and conditions used in testing substrate-loading solvent compatibility with a CEC kit of the disclosure and alcohol (R)-5. The product yields of (1R)-1-(2-naphthyl)ethyl propionate ((R)-6) from this reaction are presented in Table 8.

[0013] Figure 4 presents results with a kit of the disclosure tested with various benzyl and Į-aryl secondary alcohols. ^(a)TLC images, solvent systems, and stains for each entry are included in the Examples section. The TLC fast reaction was determined by qualitative assessment of reaction conversion. ^(b)Conversion was determined via ¹H NMR spectroscopy. Bolded reaction conversions represent entries where qualitative assessments of the fast reaction by TLC were successful. ^(c)The substrate was loaded in DMSO-d₆ and the kit ran for 1 h. ^(d)The kit ran for 1 h. ^(e)The substrate was loaded in DMSO-d₆ and the kit ran for 10 min.

[0014] Figure 5 presents the structure of Batefenterol, a MABA bronchodilator, which comprises the structure of entry 12 of FIG.4.

[0015] Figure 6 presents the proposed reactivity of entry 12 of FIG.4 when used with a kit of the disclosure.

[0016] Figure 7 shows the results using a kit of the disclosure that was tested with propargylic, allylic, Į-hydroxyester, Į-hydroxyamide, and β-aryl secondary alcohols. ^(a)The TLC fast reaction was determined by qualitative assessment of reaction conversion. TLC images, solvent systems, and stains for each entry are described in the Examples section. ^(b)Conversion was determined via ¹H NMR spectroscopy. Bolded reaction conversions represent entries where qualitative assessments of the fast reaction by TLC were successful. ^(c)The kit ran for 30 min. ^(d)The kit ran for 10 min.

[0017] Figure 8 presents TLC plate images using a kit of the disclosure with (R)-5 (1) the same day of CEC kit preparation and (2) after six months of storage in a freezer. Left TLC lane: R-HBTM; Right TLC lane: S-HBTM. Plates were eluted in 30% ethyl acetate in hexanes. Visualization was achieved by staining with PMA stain. Reaction conversion via ¹H NMR spectroscopic (%): (1) R-HBTM: 15 S-HBTM: 98 (2) R-HBTM: 16 S-HBTM: 96.

[0018] Figure 9 presents embodiments demonstrating how the kits of the disclosure can be used to determine the absolute configuration of a secondary alcohol.

[0019] Figure 10 presents a calibration curve for concentration of (R)-5 relative to GC- MS peak intensity.

[0020] Figure 11 presents a calibration curve for concentration of (R)-6 relative to GC- MS peak intensity.

[0021] Figure 12 presents TLC plate images using the CEC kit conditions for entries 1-6 in Table 4, as further plotted in FIG. 2. The left lane of each TLC plate contains R-HBTM and the right lane contains S-HBTM. Plates were eluted in 30% ethyl acetate in hexanes. Visualization was achieved by UV lamp (bottom row) and staining with PMA stain (top row). Plate numbers correlate to entry numbers in Table 4.

[0022] Figure 13 presents TLC plate images using the CEC kit conditions for entries 1-7 in Table 7. The left lane of each TLC plate contains R-HBTM and the right lane contains S- HBTM. Plates were eluted in 30% ethyl acetate in hexanes. Visualization was achieved by UV lamp (bottom row) and staining with PMA stain (top row). Plate numbers correlate to entry numbers in Table 7.

[0023] Figure 14 provides a comparison of reaction conversion (%) after 30 minutes of (R)-5 to (R)-6 relative to the dielectric constant of the solvent (R)-5 was loaded into to the CEC kit that contained CDCl₃.

[0024] Figure 15 presents TLC images corresponding to each entry tested in FIG.4. Left TLC lane: RHBTM; Right TLC lane: S-HBTM. TLC conditions and stains are listed in Table 3.

[0025] Figure 16 presents TLC images corresponding to each entry tested in FIG.7. Left TLC lane: RHBTM; Right TLC lane: S-HBTM. TLC conditions and stains are listed in Table 3.

[0026] Figure 17 provides various analyses for FIG.4, Entry 12, using a CEC kit of the disclosure.

[0027] Figure 18 shows variation of volume utilized from (R)-5 solution 1, HBTM solutions, and anhydride/base solution 1. The left lane of each TLC plate contains R-HBTM and the right lane contains S-HBTM. Plates were eluted in 30% ethyl acetate in hexanes. Visualization was achieved by staining with PMA stain

[0028] Figure 19 shows variation of equivalents of base utilized from anhydride/base solutions 1, 2 and 3. The left lane of each TLC plate contains R-HBTM and the right lane contains S-HBTM. Plates were eluted in 30% ethyl acetate in hexanes. Visualization was achieved by staining with PMA stain.

[0029] Figure 20 presents ¹H NMR Spectra CEC analysis of Entry 1 from FIG.7.

[0030] Figure 21 presents ¹H NMR Spectra CEC analysis of Entry 2 from FIG.7.

[0031] Figure 22 presents ¹H NMR Spectra CEC analysis of Entry 3 from FIG.7.

[0032] Figure 23 presents ¹H NMR Spectra CEC analysis of Entry 4 from FIG.7.

[0033] Figure 24 presents ¹H NMR Spectra CEC analysis of Entry 5 from FIG.7.

[0034] Figure 25 presents ¹H NMR Spectra CEC analysis of Entry 6 from FIG.7.

[0035] Figure 26 presents ¹H NMR Spectra CEC analysis of Entry 6 from FIG.7 after vacuum removal of solvent and resolvation in CDCl₃.

[0036] Figure 27 depicts the results of initial studies looking at the kinetic resolution of racemic, β-Chiral Primary Alcohols. The reaction conditions were the following: the alcohol substrate (0.1 M), (S)-HBTM (10 mol %), and DIPEA (0.55 equiv.) were combined in CDCl₃, followed by the addition of propionic anhydride (0.55 equiv) at 0 °C. The reaction was quenched after 15 min by the addition of MeOH. ^bThe conversion and selectivity were calculated based upon the ee of the ester and remaining alcohol, which were determined using chiral HPLC analysis.

[0037] Figure 28 demonstrates that the optically pure alcohol 5 was acylated with propionic anhydride and 20 mol % of either (R)- or (S)-HBTM. Reaction progress is plotted as ln[5] vs time, up to 50% conversion. Selectivity factor (s) from the ratio of slopes: (S)- HBTM/(R)-HBTM = í0.0294/í0.0102 = 2.9.

[0038] Figure 29 presents the results of CEC conversion of ȕ-chiral primary alcohols with (R)- and (S)-HBTM Acylation system. Reaction conditions: ^aOptically enriched alcohols (0.015 M) were acylated with propionic anhydride (2 equiv.) in the presence of (R)- or (S)-HBTM (10 mol %) and DIPEA (2 equiv.) in CDCl₃ (400 ^L total volume). After 30 min, the reactions were quenched by the addition of CD₃OD (50 ^L). The reaction was diluted to 600 ^L in CDCl₃, and percent conversion was determined by proton NMR analysis. The results from run to run were reproducible. ^bPercent conversions are the average of two trials ^cCEC reaction run with 0.020 M alcohol and 20 mol % of HBTM. ^dFor compound 16, slightly overlapping peaks introduced small errors in the absolute values of the integrations. The relative conversions were consistent run to run, and no correction was applied. ^eThe enantiomeric excess for all alcohols was ^89%, except for 13 (83% ee) and 21 (84% ee).

[0039] Figure 30A-B provides for (A) Mnemonic for determining the absolute configuration of stereocenters β to a primary alcohol. Aryl groups, certain heteroaromatic and nonaromatic ʌ systems, bromines, and chlorines act as directing groups. (B) Proposed transition state between chiral primary alcohols and acylated (S)-HBTM.

DETAILED DESCRIPTION

[0040] As used herein and in the appended claims, the singular forms "a,” "an,” and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an alcohol" includes a plurality of such alcohol and reference to "the reagent" includes reference to one or more reagents, and so forth.

[0041] 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 to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0042] Also, the use of“or” means“and/or” unless stated otherwise. Similarly, “comprise,”“comprises,”“comprising”“include,”“includes,” and“including” are interchangeable and not intended to be limiting.

[0043] It is to be further understood that where descriptions of various embodiments use the term“comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language“consisting essentially of” or“consisting of.”

[0044] Any publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

[0045] In view of the continued rapid development of asymmetric strategies for the synthesis of organic molecules, the assignment of absolute configuration of stereogenic centers is an important task for a chemist. As of yet, there is no“one size fits all” analysis for absolute stereochemistry. Several approaches have been reported to facilitate this process, including the use of chiral derivatization reagents followed by NMR spectroscopy, chiral- shift reagents, the exciton chirality method with electronic circular dichroism, vibrational circular dichroism coupled with density functional theory simulations, specific rotation coupled with the use of Hartree-Fock and density functional theory simulations, X-ray diffraction of single crystals, enantiomeric pairs of molecularly imprinted polymers, and detection of enantiomers of chiral molecules with microwave spectroscopy. The

determination of the absolute stereochemistry of small molecules remains an active challenge for researchers. The method used depends on the functionality surrounding the stereogenic center as well as the availability and properties of substance.

[0046] Provided herein is a competing Enantioselective Conversion (CEC) method for the determination of absolute configuration of stereogenic centers. The enantiomerically enriched compound of interest is reacted in parallel reactions with each enantiomer of a chiral kinetic resolution reagent over a given period of time. The relatively fast (versus slow) reaction of the pair is discovered by analyzing conversion using one of several

characterization methods. The identity of the fast reaction is then compared to an empirical mnemonic in order to assign the absolute configuration of a stereogenic center. The CEC method has been applied to secondary alcohols, oxazolidinones, lactams, thiolactams, and primary amines. Characterization techniques used to determine the reaction conversion include 1H NMR spectroscopy, thin-layer chromatography, and electrospray ionization-mass spectrometry (ESI-MS).

[0047] Alcohols are common in natural product structures and are useful handles for absolute structure determination. The CEC method disclosed herein uses the chiral acyl- transfer reagent homobenzotetramisole for configuring the stereogenic center of secondary alcohols. Propargyl and a-aryl secondary alcohols have previously been analyzed with 1H NMR spectroscopy and thin-layer chromatography on the micromole scale to assess the relative fast and slow reactions with a quantitative or qualitative measure of reaction progress. For the CEC methods disclosed herein, an enantioselective catalyst developed for kineti c resolutions of a particular class of compounds is selected. Two reactions are set up in parallel containing an enantioenriched molecule of interest and either the R or S enantiomer of the enantioselective catalyst with the other necessary reagents to conduct the

transformation. Many enantiopure substrates will react faster with one enantiomer of the catalyst. Once the reaction is stopped, the faster reacting catalyst is identified by measuring the relative conversion in each reaction.

After the fast reaction is determined, the predictive mnemonic for the HBTM system identifies the absolute configuration (see FIG.1). The CEC methods used herein were independently evaluated on secondary alcohols with Fu’s planar-chiral DMAP catalysts.

[0048] It was found that the CEC methods disclose herein had notable advantages in comparison to other chemical derivatization methods, including, but not limited to, the kinetic resolution catalysts of the disclosure can be used to identify the absolute configuration of optically pure molecules containing various functional groups; the CEC method presented herein is much less labor intensive than the Mosher’s derivatization methods; the CEC method presented herein can be used to determine the configuration of secondary alcohols and primary alcohols.

[0049] Provided herein are studies directed to using a CEC based method to determine stereogenic centers using a broad sampling of alcohol substrates, including secondary alcohols (e.g., Į-aryl secondary alcohols, β-aryl secondary alcohols, propargyl secondary alcohols, ʌ-systems, etc.); and primary alcohols (e.g., β-chiral primary alcohols). Initial studies with Į-aryl secondary alcohols indicated that high enantioselectivity can be achieved via to a proposed interaction of the aryl system with the HBTM catalyst in the transition state during the acyl transfer. Additionally, it was postulated that β-aryl alcohols might also provide selectivity and were considered good candidates to expand the substrate scope of the methods disclosed herein. It was rationalized that, based on the reported effectiveness of propargyl secondary alcohols that hybridization at the Į-position to the secondary alcohol would result in selectivity with the HBTM catalyst. Therefore, ʌ-systems such as alkene or carbonyl groups were likely to impart selectivity during the acyl transfer.

[0050] Further provided herein is a competing enantioselective conversion (CEC) kit that allows for determining the absolute configuration of secondary alcohols. In a particular embodiment, the CEC kit disclosed herein comprises one or more containers that comprise one or more solutions (e.g., a R-homobenzotetramisole (R-HBTM) containing solution and/or a S-homobenzotetramisole (S-HBTM) containing solution) that can be used to determine the absolute configuration of one or more primary or secondary alcohols.

[0051] In regards to the containers, the containers should be made of material that is largely chemically inert, such as glass and chemically resistant plastics. Preferably, the container should be sealable. For example, the container may comprise screw threads for attaching screwable caps. Alternatively, the container may be hermetically sealed, e.g., ampoules. Generally, the container should be made of a dark material (e.g., amber glass) that absorbs ultraviolet light. The containers may further comprise labels. The labels can provide information such as the listing the contents in the container, directions for use, directions for storage, and/or hazard warnings. In a particular embodiment, a CEC kit is provided herein which comprises a first container which comprises a first solution which comprises R- HBTM, an organic acid anhydride, a tertiary amine, and one or more non-protic solvents; and a second container which comprises a second solution which comprises S-HBTM, an organic acid anhydride, a tertiary amine, and one or more non-protic solvents. Examples of organic acid anhydrides, include, but are not limited to, acetic anhydride, butyric anhydride, benzoic anhydride, and propionic anhydride. In a further embodiment, the first and second container comprises propionic anhydride. In regards to the tertiary amine, any suitable basic tertiary amine can be used with the solutions described herein. Moreover, in certain instances the tertiary amine may be replaced with a non-tertiary amine base, preferably a sterically hindered base. Examples of tertiary amines or sterically hindered bases that can be used in the solutions disclosed herein, include but are not limited to, pyridine, tributylamine, triethylamine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 2-tert-butyl- 1,1,3,3-tetramethylguanidine, 1,5,7-triazabicyclo(4.4.0)dec-5-ene, 7-methyl-1,5,7- triazabicyclo(4.4.0)dec-5-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 1,1,3,3- tetramethylpiperidine, pempidine, 1,4-diazabicyclo[2.2.2]octan, collidine, and 2-6-lutidine. In regards to non-protic solvents, examples include, but are not limited to, toluene, toluene-d₈, CHCl₃, CDCl₃, THF-d₈, MeCN-d₃, DMF-d₇, CH₂Cl₂, CD₂Cl₂, pyridine-d₅, cyclohexane, cyclohexane-d₁₂, DMSO, DMSO-d₆, dioxane, dioxane-d₈, acetone, and acetone-d_6. In a particular embodiment, the HBTM solutions described herein comprises CDCl₃. In a further embodiment, the HBTM solutions described herein comprise from 0.1 to 2, 0.15 to 1, 0.2 to 0.5 equivalents of S-HBTM or R-HBTM. In another embodiment, the HBTM solutions described herein comprise from 1 to 30, 5 to 20, 10 to 20, or 10 to 15 equivalents of an organic acid anhydride. In yet another embodiment, the HBTM solutions described herein comprise from 1 to 10, 2 to 8, 3 to 7, 4 to 6, or about 5 equivalents of a tertiary base. In a particular embodiment, the HBTM solutions described herein comprise 0.2 equivalents S- HBTM or R-HBTM, 15 equivalents propionic anhydride, and 5 equivalents N-N- diisopropylethylamine. In a further embodiment, the disclosure provides for containers which comprises HBTM solutions as described herein that are sealed under an inert atmosphere so as to prevent the HBTM solutions from coming into contact with air or water vapor. Examples of inert atmospheres include nitrogen gas and argon. In a particular embodiment, containers which comprises the HBTM solutions described herein are sealed under argon.

[0052] In other embodiments, the disclosure further provides a CEC kit which further comprises a container of a protic solvent that can be used to stop the reaction conversion. Examples of protic solvents include, but are not limited to, water, acetic acid, alcohols (e.g., methanol, ethanol, propanol, isopropanol, etc.). In yet another embodiment, the disclosure also provides a CEC kit which further comprises disposable micropipettes, or droppers that can be used to transfer the solutions from the containers described herein. In a preferred embodiment, the disposable micropipettes, or droppers comprise one or more markings to facilitate transferring the correct volume of the HBTM solutions disclosed herein.

[0053] Further provided herein are processes or methods for determining the absolute configuration of enantio-enriched secondary alcohols that utilize a CEC kit described herein. The absolute configuration can then be determined via qualitative analysis of the relative fast reaction by thin-layer chromatography. The methods using the CEC kits described herein utilize 50-fold less material than what is required in similar CEC protocols. Accordingly, in direct contrast to other CEC protocols described in the literature, the CEC kits of the disclosure have greatly improved sensitivities. Moreover, instead of using two or three solutions that have to be prepared fresh each time, the CEC kits described herein combine all the reagents in single ready to use solutions. Furthermore, the solutions of the CEC kits described herein are surprisingly stable and storable for months in a freezer or even at room temperature. One might have expected that the various pathways of decomposition, including acylation reactions, condensations and destruction of the catalyst or decomposition of the tertiary amine would occur to some point based upon the teachings in the art. No such degradations or decompositions were seen with any of the components in CEC kits described herein. Moreover, the CEC kits described herein comprise components that facilitate product analysis by TLC and NMR. For example, in certain embodiments described herein, the HBTM solutions described herein comprise a deuterated nonprotic solvent (e.g., CDCL₃), which allows for the reaction mixture to be used directly for ¹HNMR without having to undergo any solvent exchange or purification steps. Moreover, the disclosure teaches a CEC mnemonic that can be used to quickly determine the absolute configuration of the secondary alcohol based upon the results of the analytical methods. For the purposes of the disclosure "CEC mnemonic" refers to the mnemonic presented in FIG.1, unless provided for otherwise.

[0054] The invention is further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.

EXAMPLES

[0055] General Methods. All reactions were carried out under an atmosphere of air with anhydrous solvents unless otherwise noted. All glassware was oven-dried prior to use.¹H and ¹³C NMR spectra were recorded at 298.0 K at 500 MHz. CDCl₃ was used as an internal reference for ¹H NMR (G = 7.27) and ¹³C NMR (G = 77.16) spectra. Thin-layer

chromatography (TLC) was performed on silica gel plates and visualized using various stains that are detailed for specific compounds. Stains were prepared according to the procedure described in Pirrung, M. C. The Synthetic Organic Chemist’s Companion; John Wiley & Sons, Inc., Hoboken, NJ, USA 2007 pp 171–172.

[0056] Chemicals. All commercially available reagents and solvents were used without further purification unless otherwise noted. Hexanes, ethyl acetate, toluene, methanol, propionic anhydride, N,N-diisopropylethylamine, toluene-d₈, CDCl₃, THF-d₈ , acetone-d₆, MeCN-d₃, DMF-d₇, and DMSO-d₆, and 3 were purchased from various chemical suppliers. (R)-5 was prepared according to the procedure described in Wagner et al. (J. Chem. Educ. 2014, 91, 716–721). Certain compounds were either obtained, while other compounds were synthesized de novo, or prepared by following procedures described in Tan et al. (A. B. Chem. Eur. J.7:1845–1854 (2001)), and Lewis et al., (Synlett 12:1923–1928 (2009)).

[0057] Calibration Curves of (R)-5 and (R)-6 with GC-MS for the data presented in FIG. 2.

Stock Solutions

(R)-5. To a 10 mL volumetric flask was added (R)-5 (5.0 mg, 0.029 mmol). The flask was filled to the line with dichloromethane, thereby generating a solution of (R)-5 (2.9 mM).

(R)-6. To a 10 mL volumetric flask was added (R)-6 (6.6 mg, 0.029 mmol). The flask was filled to the line with dichloromethane, thereby generating a solution of (R)-6 (2.9 mM). Calibration Solutions.

All solutions are summarized in Table 1.

Solution 1. To a GC-MS vial was added stock solutions of (R)-5 (909 ^L) and (R)-6 (91.0 ^L), thereby generating a solution ratio for (R)-5:(R)-6 of 10:1 at 2.6 mM and 0.26 mM.

Solution 2. To a GC-MS vial was added stock solutions of (R)-5 (833 ^L) and (R)-6 (167 ^L), thereby generating a solution ratio for (R)-5:(R)-6 of 5:1 at 2.4 mM and 0.48 mM.

Solution 3. To a GC-MS vial was added stock solutions of (R)-5 (667 ^L) and (R)-6 (333 ^L), thereby generating a solution ratio for (R)-5:(R)-6 of 2:1 at 1.9 mM and 0.97 mM.

Solution 4. To a GC-MS vial was added stock solutions of (R)-5 (500. ^L) and (R)-6 (500. ^L), thereby generating a solution ratio for (R)-5:(R)-6 of 1:1 at 1.5 mM and 1.5 mM.

Solution 5. To a GC-MS vial was added stock solutions of (R)-5 (333 ^L) and (R)-6 (667 ^L), thereby generating a solution ratio for (R)-5:(R)-6 of 1:2 at 0.97 mM and 1.9 mM.

Solution 6. To a GC-MS vial was added stock solutions of (R)-5 (167 ^L) and (R)-6 (833 ^L), thereby generating a solution ratio for (R)-5:(R)-6 of 1:5 at 0.48 mM and 2.4 mM.

Solution 7. To a GC-MS vial was added stock solutions of (R)-5 (91.0 ^L) and (R)-6 (909 ^L), thereby generating a solution ratio for (R)-5:(R)-6 of 1:10 at 0.26 mM and 2.6 mM. Table 1. Mixed ratios of (R)-5 and (R)-6 in solutions used for GC-MS calibration curves.

[0058] Vials of solutions 1-7 were capped and subsequently analyzed via GC-MS with a programmed method that increased the temperature from 50 ºC to 290 ºC at a rate of 5 ºC per min. Solutions 1-7 were compared to pure samples of (R)-5 and (R)-6 at concentrations of 2.9 mM to match retention times.

[0059] Integration of peak intensities in solutions 1-7 was collected and compared to the concentrations of (R)-5 and (R)-6 within each solution to develop calibration curves (Table 2). GC-MS peak intensities were divided by 100,000,000 to simply the resulting graphs for (R)-5 (FIG.10) and (R)-6 (FIG.11). Table 2. GC-MS integration of peak intensities of (R)-5 and (R)-6 for solutions 1-7.

[0060] CEC One-Use Kit Preparation: R-HBTM vials: R-HBTM (9.5 mg, 0.036 mmol) was added to a 10 mL volumetric flask followed by the addition of CDCl₃ (2.0 mL). To the solvated R-HBTM solution was added propionic anhydride (342 μL, 2.67 mmol) and N,N- diisopropylethylamine (155 μL, 0.890 mmol) via micropipette. The flask was filled to the line with CDCl₃, thereby generating a solution of R-HBTM (0.0036 M), propionic anhydride (0.267 M), and N,N-diisopropylethylamine (0.0890 M). The R-HBTM stock solution (450 μL) was added to oven-dried 1 mL amber vials via syringe. The vials were sealed under argon. A 10 mL volumetric batch was used to prepare 21 R-HBTM vials.

S-HBTM vials: S-HBTM (9.5 mg, 0.036 mmol) was added to a 10 mL volumetric flask followed by the addition of CDCl₃ (2.0 mL). To the solvated S-HBTM solution was added propionic anhydride (342 μL, 2.67 mmol) and N,N-diisopropylethylamine (155 μL, 0.890 mmol) via micropipette. The flask was filled to the line with CDCl₃, thereby generating a solution of S-HBTM (0.0036 M), propionic anhydride (0.267 M), and N,N- diisopropylethylamine (0.0890 M). The S-HBTM stock solution (450 μL) was added to oven-dried 1 mL amber vials via syringe. The vials were sealed under argon. A 10 mL volumetric batch was used to prepare 21 S-HBTM vials.

Kit Assembly: One sealed R-HBTM vial and one sealed S-HBTM vial, each containing 450 μL of their respective stock solutions, were placed in a 20 mL scintillation vial and capped under argon. The vial was then sealed with electric tape and stored in a freezer.

[0061] CEC One-Use Kit Procedure (see FIG.4 and FIG.7).

Preparation of Solutions: Substrate. Substrate (0.020 mmol) was solvated in a deuterated solvent (250 μL) in a 1 dram vial. The deuterated solvent was CDCl₃ unless otherwise noted.

One-Use Kit CEC Method: The resulting alcohol solution (100 μL) was dispensed to both the R-HBTM and S-HBTM CEC kit vials via microsyringe with a one-minute gap between additions. A needle was inserted into the CEC kit vial to equalize the pressure before addition of the alcohol solution. The solutions were agitated to ensure homogeneity and were allowed to sit for 30 min (see FIG.4, entries 1-12) or 1 h (see FIG.7, entries 1-12). Methanol-d₄ (50 μL) was added via microsyringe to halt reaction progress. The solution was again agitated to ensure homogeneity.

TLC Analysis: To a TLC plate with 2 lanes was spotted the R-HBTM reaction (4.0 μL), and the S-HBTM reaction (4.0 μL) via micropipette. The plate was run, dried, stained, and heated by oven (160 ºC and ~ 1 min unless otherwise noted), and photographed.

1H NMR Spectroscopic Analysis: The quenched solution was then analyzed by ¹H NMR spectroscopy to assess reaction conversion via measurement of peak integration, traditionally of the proton geminal to the alcohol and ester functional groups on the substrate and product respectively. Table 3. Masses, TLC solvent systems, and TLC plate staining solutions used for compounds in FIG.4 and FIG.7 with the CEC kit protocol

[0062] Testing Reaction Conversion by Varied Initial Concentrations of (R)-5 with the CEC Kit Conditions (FIG.2)

CEC Protocol: Substrate (R)-5 was solvated in CDCl3 in a 1 dram vial. Masses and the volume of CDCl₃ added are included in Table 4. Table 4. CEC Kit substrate loadings analyzed for (R)-5 in FIG.2.

The resulting alcohol solution (100 ^L) was dispensed to both the R-HBTM and S-HBTM CEC kit vials via microsyringe with a one-minute gap between additions. A needle was inserted into the CEC kit vial to equalize the pressure before adding the alcohol solution. The solutions were agitated to ensure homogeneity and allowed to sit for 30 min. Methanol-d4 (50 ^L) was added via microsyringe to stop the reaction. The solution was again agitated to ensure homogeneity.

TLC Analysis [0063] To a TLC plate with 2 lanes was spotted the R-HBTM reaction (4.0 ^L), and the S-HBTM reaction (4.0 ^L) via micropipette. The plate was run (30 % ethyl acetate in hexanes), dried, stained (PMA), heated by oven (160 ºC, ~ 1 min), and photographed. Images are included in FIG.12.

[0064] ¹H NMR Analysis: The quenched solution was then analyzed by ¹H NMR spectroscopy to assess reaction conversion via measurement of peak integration of the proton germinal to the alcohol and ester functional groups on the substrate and product respectively. Spectra from entries 1-6 of FIG.7 are provided in FIG.20 to FIG.26.

[0065] GC-MS Analysis: The R-HBTM and S-HBTM CEC kit solutions were concentrated in vacuo. The concentrated mixture was diluted with methylene chloride (2 mL), washed with saturated aqueous NaHCO₃ (2 × 2 mL) and brine (1 × 2 mL). The organic layer was dried (MgSO₄), filtered, and concentrated in vacuo to afford crude reaction mixtures for each entry. Based on the initial amount of substrate added to the reaction kit, each crude mixture was then diluted with methylene chloride in order to give an assumed additive concentration between (R)-5 and (R)-6 of 2.5 mM. If the total solution was in excess of 1.0 mL, then a portion of the solution (1.0 mL) was transferred to a GC-MS vial and capped under air for subsequent analysis via GC-MS with a programmed method for increasing the temperature from 50 ºC to 290 ºC at a rate of 5 ºC per min. Volumes and the resulting GC-MS integration data are included below. The GC-MS integration data for (R)-5 and (R)-6 was utilized with previously developed calibration curves to calculate

concentrations in each reaction mixture and the reaction conversion for each entry. Data are summarized for R-HBTM and S-HBTM reactions with entries 1-6 in Table 5 and Table 6.

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A

1⁹

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W

2⁷

-⁰

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[0066] Development of a One-Use CEC Kit. The previous section described a refined microscale protocol and an expanded substrate scope. This success led to a new goal:

development of a commercially viable CEC kit. Initially, a set of three solutions (R-HBTM, S-HBTM, and a mixture of propionic anhydride and N-N-diisopropylethylamine) in toluene was proposed. The proposed target market was industrial research and development programs and academic research groups.

[0067] One of the first concerns about the kit was the solvent choice. While toluene was ideal for small volume measurements without significant volume loss due to evaporation, its dielectric constant and resultant poor compatibility with polar organic compounds greatly limited its utility in a medicinal chemistry setting. Another issue raised was the desirability of collecting both qualitative and quantitative data for the analysis. When industrial chemists were asked, the requirement for micromoles of material was not considered a problem. More important was the desire to record quantitative data for reaction conversion. A one-use kit was particularly convenient for the user and was preferred by the medicinal chemists polled.

[0068] The proposal for a commercially viable CEC kit was revised based on this feedback. The substrate loading in each reaction was adjusted to 8 μmol to accommodate ¹H NMR spectroscopic analysis. The total volume of the system was established at 550 μL with a 50 μL methanol quench, providing a slight excess of the volume required for standard NMR spectroscopy tubes. The solvent was switched from toluene to CDCl₃ to accommodate direct NMR analysis and solubility for a broader spectrum of organic compounds.

[0069] For convenience, the substrate would be added in a 100 μL portion. The protocol was optimized to use 20 μmol of substrate (5.0 mg for a substrate with a molecular mass of 250, for example) and 250 μL of CDCl₃ to assure that two equal 100 μL portions could be withdrawn and added to each CEC kit vial.

[0070] The second problem to be resolved was the equivalents and concentration of CEC kit reagents. The optimized microscale protocol for secondary alcohols (0.2 equivalents HBTM, 15 equivalents propionic anhydride, 5 equivalents N-N-diisopropylethylamine) were applied to the new CEC kit parameters (8 μmol substrate and total additive volume of 550 μL) and the reaction conversion of alcohol (R)-5 to propionate ester (R)-6 via the CEC method was studied. The CEC kit represented a 3-fold increase in substrate concentration relative to the microscale protocol, so reactions were halted after 30 min instead of 1 h. Quantitative analysis by ¹H NMR spectroscopy revealed conversions for R-HBTM and S- HBTM of 15% and 98%, in alignment with the predictive mnemonic for assignment of absolute configuration. Quantitative analysis by GC-MS lead to nearly identical reaction conversions when compared to the ¹H NMR spectroscopic data. These concentrations were considered appropriate for the desired substrate loading of 8 μmol at an initial substrate concentration of 14 mM in the CEC kit.

[0071] Based on prior kinetic analysis, the reaction system was expected to be pseudo- first order in substrate. This expectation was tested by varying the substrate loading to give initial concentrations between 1.4 mM and 29 mM while maintaining the same CEC kit concentrations of HBTM, propionic anhydride, and N-N-diisopropylethylamine (see FIG.2). If the hypothesis was correct, similar reaction conversions would be observed for both HBTM reactions over the span of the substrate concentrations. If incorrect, a change in the substrate concentration would result in a change in reaction conversion. After quantitative analysis of reaction conversion for the R-HBTM and S-HBTM reactions by both ¹H NMR spectroscopy and GC-MS, a plot of reaction conversion compared to initial substrate concentration over this 20-fold range revealed no significant change in reaction conversion for both the R-HBTM and S-HBTM reactions. Thus, the reaction was confirmed to be pseudo-first order in the secondary alcohol. These reaction conditions offer a significant advantage to the user compared with previous CEC protocols. The CEC kit will theoretically work with any quantity of substrate less than the recommended 20 μmol as long as the analytical method is capable of measuring the substrate conversion. The microscale protocol is more convenient for most situations with limited sample quantities, but the sensitivity of either procedure is limited only by the analytical method.

[0072] Testing Reaction Conversion by Varied Solvent to Solvate (R)-5 with the CEC Kit in CDCl₃ (FIG.3).

Preparation of Solutions: Substrate. Substrate (R)-5 (3.4 mg, 0.020 mmol) was solvated in a deuterated solvent varying between entries 1-7 of FIG.3 (250 ^L) in a 1 dram vial.

CEC Kit. Prepared as previously described in the Experimental Section.

CEC Protocol. The resulting alcohol solution (100 ^L) was dispensed to both the R- HBTM and S-HBTM CEC kit vials via microsyringe with a one-minute gap between additions. A needle was inserted to the CEC kit vial to equalize the pressure before addition of the alcohol solution. The solutions were agitated to ensure homogeneity and were allowed to sit for 30 min. Methanol-d4 (50 ^L) was added via microsyringe to stop the solution. The solution was agitated to ensure homogeneity. Deuterated solvents tested are included below in Table 7.

TLC Analysis. To a TLC plate, 2 lanes were spotted with the R-HBTM reaction (4.0 ^L), and the S-HBTM reaction (4.0 ^L) via micropipette. The plate was run (30 % ethyl acetate in hexanes), dried, stained (PMA), heated by oven (160 °C, ~ 1 min), and

photographed. Images are included in Figure 13.

1H NMR Analysis. The quenched solution was then analyzed by ¹H NMR

spectroscopy to assess reaction conversion via measurement of peak integration of the proton germinal to the alcohol and ester functional groups on the substrate and product respectively. Table 7. Deuterated solvents used in solvating (R)-5 for use in the CEC kit in CDCl₃

[0073] During testing, another issue raised was the possibility of developing CEC kits in solvents with higher dielectric constants in order to solvate increasingly polar pharmaceutical intermediates that displayed limited or no solubility in CDCl₃. CEC kits in THF-d₈, acetone- d₆, MeCN-d₃, DMF-d₇, and DMSO-d₆ were considered. THF-d₈, MeCN-d₃, and DMF-d₇ were ruled out because of the substantial cost associated with large volumes of these deuterated solvents. Acetone-d₆ and DMSO-d₆ were also ruled out because of the potential for water adsorption and possible side reactions occurring with the solvents in the CEC kit solutions when stored over long periods of time. In recognition of these issues, solvation of polar substrates in one of the aforementioned solvents followed by injection to a one-use CEC kit in CDCl₃ was considered (see FIG.3 and Table 8). Table 8. Testing substrate-loading solvent compatibility with CDCl₃ CEC kit and alcohol (R)- 5.

[0074] A general trend was observed using solvent mixtures created from injection of the substrate in different solvents to the CEC kit in CDCl₃. When loading substrates in toluene- d₈ (entry 1), CDCl₃ (entry 2), THF-d₈ (entry 3), and acetone-d₆ (entry 4), a range of conversion with R-HBTM was observed in 11-18% and a range of conversion with S-HBTM was observed in 93-99%. With DMF-d₇ (entry 6) as the loading solvent, a noticeable decrease in conversion with R-HBTM and S-HBTM of 8% and 83% was observed. The highest dielectric constant tested, DMSO-d₆ (entry 7), the reduced conversion was more pronounced. Overall, with an increase in the dielectric constant of the substrate solvent, there was a general decrease in reaction conversion over the same 30 min time period. However, a clear difference in reaction conversion was observed both qualitatively and quantitatively with the S-HBTM reaction proceeding as the fast reaction. Therefore, for most substrates, the use of a standard CEC kit with CDCl_3, combined with an appropriate substrate solvent, should be satisfactory.

[0075] To confirm the validity of the kit with additional substrates, a series of benzyl and Į-aryl secondary alcohols were examined with both a qualitative analysis of the fast reaction by TLC and a quantitative analysis of reaction conversion by ¹H NMR spectroscopy (see FIG.4). Entries 1-7, previously tested in the microscale protocol, all proceeded effectively with the new CEC kit system. Qualitative and quantitative determinations of the fast reaction as R-HBTM (entries 2, 5, 6) and S-HBTM (entries 1, 3, 4, 7) to give configuration assignments in alignment with the predictive mnemonic were made. With the capability to assess reaction conversion quantitatively, entries 1-4 and 7 all progress with comparable reaction conversion (slow HBTM reaction: 10-26%, fast HBTM reaction: 92-98%) and therefore selectivity in the protocol. Entry 5 achieves a slightly lower reaction conversion for the fast reaction (76%) and for the slow reaction (7%). The fast reaction in sterically hindered entry 6, the intermediate for the enantiomer of crizotinib, proceeds with the lowest conversion of this group of compounds (48%), but is still operating at high selectivity, with the slow reaction proceeding to only 8% conversion.

[0076] Į-Aryl pyridines (entries 8-9) and benzimidizoles (entries 10-11) also proved amenable to quantitative determination of the fast reaction and subsequent alignment with the predictive mnemonic. Entries 8-9 were also capable of qualitative assessment of the fast reaction by TLC. The difference in reaction conversion determined quantitatively by ¹H NMR for entries 10-11 was unable to be qualitatively visualized by TLC despite applying several different staining procedures. Lastly, entry 12 was considered with the CEC kit. Entry 12 is a key intermediate in the synthesis of batefenterol (TD-5959, GSK961081): a multivalent muscarinic antagonist and β₂-agonist (MABA) bronchodilator for treatment of moderate to severe chronic obstructive pulmonary disease (COPD).

[0077] Entry 12 presented an interesting challenge because of the secondary amine also present in the molecule. It was envisioned that there would be rapid acylation of the secondary amine, followed by the key esterification of the chiral secondary alcohol (see FIG. 6). After treatment in the CEC kit, loss of starting material was indicated by ¹H NMR spectroscopic analysis and TLC. Isolation of the amide through independent amidation of entry 12 confirmed the formation of the amide first. Then, conversion of the newly formed amide to the propionate ester was measured to assess quantitative reaction conversion. Both qualitative and quantitative methods confirmed a faster reaction with R-HBTM, indicating the stereocenter was (R), which is in accordance with the mnemonic and in alignment with the reported stereochemistry of the compound.

[0078] After testing a series of interesting benzyl and Į-aryl secondary alcohols, it was sought to analyze the utility of the CEC kit with additional substrate classes (see FIG.7). The propargylic alcohol in entry 1 displayed the fastest reactivity observed for the CEC kit of this series, with the fast reaction reaching 99% conversion and the slow reaction at 63% after 30 min. To verify that the rate of reaction was the cause for the unusually high mismatched reaction conversion, the same compound was exposed to the CEC kit conditions again (entry 2). In this second attempt, reaction progress was halted at 10 min. The fast reaction conversion was 93% and the slow reaction dropped to 39%. Next, an allylic alcohol was tested (entry 3). The reaction progressed over 30 min proceeded to a lower reaction conversion compared to previous substrates and therefore was run for 1 h. A noticeable difference in reaction conversion was observed qualitatively and quantitatively, in alignment with the mnemonic for assignment of the conversion of the reactant to a product, if the reactant's alkene is the ʌ-group. Previously tested β-hydroxyesters proceed without selectivity between the HBTM reactions. Therefore, the inclusion of the allylic system appears the primary contributor to the observed reaction conversion difference. After discovering a substrate displaying selectivity with a simple ʌ system directly connected to the stereocenter of study, Į-hydroxyester substrates were considered. Methyl- and ethyl-lactate (entries 4 and 5) displayed a significant difference in reaction rate between the HBTM reactions culminating in a difference in reaction conversion after 10 min in alignment with the mnemonic for assignment of reaction conversion if the carbonyl group is applied in the mnemonic as ʌ. Appending a benzyl group to the ester (entry 6) resulted in a similar outcome, but with a decrease in reaction rate. Additionally, enantiomeric Į-hydroxyamides were studied (entries 7 and 8). These compounds reacted significantly slower, achieving a reaction conversion of 35% for the fast reaction over 1 h. However, the slow reaction in both cases proceeded to far lower conversion, with reaction conversions of 7% and 3%. Again, if the carbonyl is assigned as ʌ, the observed fast reaction aligns with the previously established mnemonic. Finally, four β-aryl secondary alcohols from the microscale studies were also tested (entries 9-12) with the CEC kit over 1 h and concluded with the R-HBTM reaction as the fast reaction according to both qualitative and quantitative analysis. This data also aligns with the conclusions drawn from the microscale studies for all four compounds. The conversion difference between fast and slow reactions was smallest in entry 9, with a β- pyridine group, but still produced a difference of 17% conversion. The studied substrates expand the secondary alcohols applicable to the CEC method with HBTM and the established mnemonic for assignment of absolute configuration.

[0079] One of the final questions about the CEC kit was the stability of the ingredients in CDCl₃ over time. In order to test the stability, a batch of kits was freshly prepared. The CEC kit was tested with (R)-5 the same day. The kits were then stored in a–20 ºC freezer. After six months, a kit from the same batch was removed, warmed to room temperature, and tested with (R)-5 (see FIG.8). Comparison of the TLC plates qualitatively showed identical performance in each reaction lane and the assignment of the fast reaction was clearly made as the S-HBTM reaction. ¹H NMR spectroscopic data also confirmed this conclusion, with nearly identical reaction conversions between the R-HBTM and S-HBTM reactions for plate 1 and plate 2. Therefore, the CEC kit solutions appear stable when stored in a freezer over a six-month period and display no noticeable degradation in performance.

[0080] After vetting the CEC kit system, the influence of substrate alcohol concentration in the microscale procedure was next determined. Between the two processes, there was a three-fold reduction in the concentration of HBTM, anhydride, and base in the microscale protocol. If the reaction shows saturation kinetics with respect to the anhydride, then the pseudo-first order rate constant would be proportional to the HBTM concentration, and thus the rate constant would be about a factor of 3 smaller for the microscale procedure. A serial dilution of alcohol (R)-5 was conducted to produce stock solutions varying between 7.26 mM and 0.907 mM, which resulted in a range of 72 nmol to 9 nmol of substrate per reaction. Because of the scale and the poor detection of (R)-5 at these concentrations via GC-MS, only qualitative data was accessed by TLC. However, the images of plates corresponding to entries 1-4 display comparable reaction conversion for all reaction lanes. This analysis supports the expectation that the microscale protocol displays pseudo-first order kinetic behavior with the alcohol substrate. The limit of detection for effective recognition of the fast reaction of HBTM was 9 nmol per reaction for entry (R)-5 in the PMA stain. However, the theoretical limit for the use of this protocol is based only on the ability to detect the reaction progress. An example of reduced-scale detection was recently illustrated by Poulter and coworkers using the microscale protocol with autoradiography on silica-TLC plates to determine the absolute configuration of hydroxysqualene synthesized in bacterial biosynthesis.

[0081] Initial Studies Using the CEC approach for Kinetic Resolution of Racemic, β- Chiral Primary Alcohols. To determine whether the CEC method could be used with β- chiral primary alcohols, the effectiveness of kinetic resolution of racemic substrates with HBTM was first probed. Because HBTM and related catalysts have been proposed to interact with the substrates through ʌ interactions, alcohols bearing a ʌ-group were tested initially. It was found, that alcohols 1í3 of FIG.27 could be resolved with modest selectivities. Though these selectivities are too low to be of practical synthetic utility, they are more than sufficient to make a stereochemical assignment based upon rate differences using the CEC method. One limitation was apparent as alcohol 4 in FIG.27, with the arene group Ȗ to the alcohol, resulted in almost no selectivity. Other acylation catalysts were investigated (BTM and Cl-PIQ), but they did not provide superior selectivity in comparison to HBTM. While the kinetic resolutions gave superior selectivities at lower temperatures, 0 °C was selected as the standard temperature for CEC investigation due to the reproducibility and convenience inherent to iceíwater baths.

[0082] Determining the order of the reaction with respect to the β-aryl primary alcohol. Acylation reactions that are first order in alcohol are well behaved in the CEC method, and the product conversions can be used to reliably identify the fast-reacting enantiomer. The acylation of enantiopure alcohol 5 of FIG.28 was catalyzed by (R)- or (S)- HBTM in the presence of an excess of propionic anhydride and diisopropylethylamine (DIPEA); conversion was monitored by NMR analysis. The acylation of 5 of FIG.28 was faster with (S)-HBTM and displayed first order behavior with respect to alcohol with either catalyst, validating the assumption that relative conversions reflect relative reaction rates. This rate behavior simplifies the CEC analysis to a one-point conversion analysis. It reduces time and effort needed to identify the faster reacting catalyst and, therefore, the configuration of the stereocenter.

[0083] Establishing a relationship between the absolute configuration of enantioenriched primary alcohols and catalyst selectivity. Most of the enantioenriched alcohols in FIG.29 were prepared by borane reduction of the corresponding acids, which were themselves prepared via enantioselective Į-alkylations. Alcohols 6í8 of FIG.29 all react faster with (S)- HBTM. While methyl-bearing alcohol 6 of FIG.29 shows higher rates of conversion, bulkier alcohols 7 and 8 of FIG.29 show a slightly higher difference in conversion between parallel reactions. Both electron-rich and electron-poor arenes 10 and 11 of FIG.29 are selective with (S)-HBTM. Because most of the alcohols had the same configuration with respect to R¹ and R², enantiomers 5 and 12 of FIG.29 were compared. Both showed approximately equal and opposite conversions with (R)- and (S)-HBTM, serving as positive controls. Previous experience has shown that a sample with lower enantiopurity will reduce the observed difference between the conversions. With the exception of compounds 13 and 21 of FIG.29, all of the samples are ^ 89% ee, so the effect will be negligible. The CEC method also showed selectivity for scopolamine, 14 of FIG.29, demonstrating its utility for natural products containing this functional group array.

[0084] Based upon the results with the β-aryl primary alcohols, alcohols with different groups in place of the arene were investigated. Heteroaromatic alcohols 15 and 16 of FIG. 29 displayed significant differences in conversion, but unfortunately, indole 21 of FIG.29 displayed conversions that were too close to confidently assign the faster reacting catalyst. A brief examination of nonaromatic ʌ-systems revealed that enone 13 of FIG.29 was an effective substrate.

[0085] Interestingly, methyl ester 17 of FIG.29 displayed selectivity opposite what was expected if the ester played the role of a directing group. It was postulated that

intramolecular hydrogen bonding could be altering the conformation of 17 of FIG.29 and thus the selectivity. Curiously, modest but reproducible selectivity was observed for β-halide alcohols 18 and 19 of FIG.29. Lone pairíʌ interactions might account for the selectivity, but further experimental evidence is needed. Phenyl alkyne 22 of FIG.29 showed a slightly higher conversion with the (S)-HBTM catalyst, which was consistent with a directing group effect by the alkyne. The selectivity is modest enough to discourage its application to configuration assignments. Lastly, there was no selectivity for the negative control of alcohol 20 of FIG.29. This outcome is consistent with models for the resolution of secondary alcohols by HBTM, in which a ʌ-directing group is important for resolution.

[0086] Establishing a mnemonic to determine the configuration of ȕ-chiral primary alcohols. As shown in FIG.30, the directing group (ʌ system) drawn to the left of the page and the primary alcohol to the right of the page, if (S)-HBTM is the faster reacting catalyst, than the R group points out of the plane of the page. Conversely, if (R)-HBTM is the faster reacting catalyst, the R group points into the plane of the page. It was postulated that there is a transition state analogous to that proposed by Birman and Houk, in which the alcohol approaches the face of the catalyst unencumbered by the bulky phenyl group and adopts a conformation in which the directing group interacts with the cationic ʌ-system of acylated HBTM (see FIG.30B). An unfavorable steric interaction between the alcohol’s R group and the catalyst imparts the observed stereoselectivity.

[0087] It will be understood that various modifications may be made without departing from the spirit and scope of the invention. Other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS: 1. A competing enantioselective conversion (CEC) kit used to determine the absolute configuration of a secondary alcohol, comprising:

a first container comprising a first solution that comprises R-homobenzotetramisole (R-HBTM), an organic acid anhydride, a tertiary amine, and one or more non-protic solvents; and

a second container comprising a second solution that comprises S- homobenzotetramisole (S-HBTM), an organic acid anhydride, a tertiary amine, and one or more non-protic solvents,

wherein the first and second containers can be stored for at least six months at temperature of about -20 ºC or lower with minimal to no loss in enantioselective activity, and wherein the absolute configuration of the secondary alcohol can be determined by use of the CEC kit with the secondary alcohol and qualitatively determining the fast reaction via ester product formation.

2. The CEC kit of claim 1, wherein the organic acid anhydride of the first solution and/or of the second solution is propionic anhydride.

3. The CEC kit of claim 1 or claim 2, wherein the tertiary amine of the first solution and/or of the second solution is N,N-diisopropylethylamine.

4. The CEC kit of any one of the proceeding claims, wherein the one or more non-protic solvents of the first solution and/or of the second solution comprises CDCl₃.

5. The CEC kit of any one of the preceding claims, wherein the first and second solution comprises 0.2 equivalents of S-HBTM or R-HBTM, 15 equivalents of propionic anhydride, and 5 equivalents of N-N-diisopropylethylamine.

6. The CEC kit of claim 5, wherein the first and second solution consists of 0.2 equivalents of S-HBTM or R-HBTM, 15 equivalents of propionic anhydride, and 5 equivalents of N-N-diisopropylethylamine.

7. The CEC kit of any one of the preceding claims, wherein the first and second containers are sealed under an inert gas.

8. The CEC kit of any one of the preceding claims, wherein the first and second containers are vials, ampoules, tubes, or bottles.

9. The CEC kit of any one of the preceding claims, wherein the first and second containers are made of a dark material that absorbs ultraviolet radiation.

10. The CEC kit of any one of the proceeding claims, wherein the CEC kit further comprises:

a third container comprising a protic solvent.

11. The CEC kit of claim 10, wherein the protic solvent is methanol or methanol-d₄.

12. The CEC kit of any one of the proceeding claims, wherein the CEC kit further comprises:

a fourth container comprising a first optically pure secondary alcohol; and optionally A fifth container comprising a second optically pure secondary alcohol, wherein the second optically pure secondary has the opposite rotation to the first optically pure secondary alcohol.

13. The CEC kit of claim 12, wherein the first optically pure secondary alcohol and the second optically pure secondary alcohol are different enantiomers of the same compound.

14. The CEC kit of claim 13, wherein the first optically pure secondary alcohol and second optically pure alcohol are selected from (R)-(+)-1-(2-naphthyl)ethanol and (S)-(í)-1- (2-naphthyl)ethanol.

15. The CEC kit of any one of the proceeding claims, wherein the CEC kit is manufactured for a single use, such that the first container and second container contain enough solution to determine the absolute configuration of one secondary alcohol.

16. The CEC kit of any one of claims 1 to 14, wherein the CEC kit is manufactured for multiple uses, such that the first container and second container contain enough solution to determine the absolute configuration of multiple secondary alcohols.

17. The CEC kit of any one of claims 1 to 14, wherein the CEC kit is manufactured for multiple uses, such that the CEC kit contains multiple containers that comprise the first solution, and multiple containers that comprise the second solution.

18. A Competing Enantioselective Conversion (CEC) process for determining the absolute configuration of a secondary alcohol comprising:

providing the CEC kit of any one of the preceding claims;

mixing or adding a solution comprising a secondary alcohol with the first solution from the first container;

mixing or adding the solution comprising the secondary alcohol, with the second solution from the second container;

incubating the reaction mixtures for 30 min to 2 h, wherein both reaction mixtures are incubated for substantially or the same period of time;

stopping the two reactions by adding a protic solvent to the first reaction vessel and the second reaction vessel;

analyzing the two reactions by using a qualitative analytical chemical technique(s) to identify the faster-reacting HTBM catalyst; and

determining the absolute configuration of the secondary alcohol by using a predictive CEC mnemonic to analyze the results from the analytical chemical technique(s).

19. The process of claim 18, wherein the analytical techniques are thin layer

chromatography (TLC) and/or nuclear magnetic resonance (NMR).