Repurposing the Sanger Reagent for Optical Sensing of the Concentration and Enantiomeric Ratio of Chiral Amines, Amino Alcohols, and Amino Acids

F Safia Kariapper; Austin U Wolf; Christian Wolf

doi:10.1002/chir.70062

. 2025 Nov 22;37(12):e70062. doi: 10.1002/chir.70062

Repurposing the Sanger Reagent for Optical Sensing of the Concentration and Enantiomeric Ratio of Chiral Amines, Amino Alcohols, and Amino Acids

F Safia Kariapper ¹, Austin U Wolf ¹, Christian Wolf ^1,^✉

PMCID: PMC12639352 PMID: 41273097

ABSTRACT

The well‐known, readily available Sanger reagent is used for quantitative chiroptical sensing of the amount and enantiomeric composition of amines, amino alcohols, and amino acids. A practical assay that is fast, adaptable to a variety of solvents, and only requires mixing of the sample and probe in the presence of triethylamine prior to the CD and UV measurements is introduced. The analyte tagging generates characteristic CD and UV signals at 405 and 345 nm, respectively, which allow accurate concentration and er analysis without any workup or tedious sample preparation.

Keywords: amino group tagging, chiroptical sensing, circular dichroism spectroscopy, enantiomeric excess analysis, Sanger reagent

Chiroptical sensing of the concentration and enantiomeric composition of amines, amino alcohols, and amino acids labeled with the Sanger reagent is demonstrated. The practical analyte tagging generates characteristic CD and UV signals at 405 and 345 nm, respectively, which allows accurate analysis without workup or tedious sample preparation.

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1. Introduction

The importance of isolation, structure elucidation and in vivo monitoring of natural compounds in the biomedical sciences has been a long‐standing impetus for the development of innovative chemical methods and strategies that accomplish these daunting tasks by selective labeling with small molecules exhibiting favorable chemical reactivity, which enables chemoselective and robust attachment at suitable functional groups, and a distinct spectroscopic signature. To this end, the idea of tagging amino acid residues in peptides and proteins with chromophoric labels has a long history. In fact, it has been eight decades since Sanger used 2,4‐dinitrofluorobenzene, 1, to identify the N‐terminal amino acids in insulin as glycine and phenylalanine in 1945 [1]. Since then, countless small‐molecule labeling reagents have been introduced and used in a large variety of applications. Sanger's seminal work, however, has remained important to date and 1 is still a popular molecular tag or derivatizing agent used in many laboratories [2, 3, 4, 5, 6, 7, 8, 9, 10].

Molecular chirality plays a fundamental role and has multiple implications in the chemical, health, environmental and materials sciences. The analysis of the enantiomeric composition of chiral compounds is often a tedious, time‐consuming process despite considerable advances with chromatographic high‐speed separations [11, 12, 13]. These and other drawbacks have directed increasing attention to other techniques including UV [14, 15], circular dichroism (CD) [16, 17, 18, 19, 20, 21, 22, 23, 24, 25], fluorescence spectroscopy [26, 27, 28], NMR [29, 30, 31, 32, 33, 34, 35], gas‐phase rotational resonance spectroscopy [36], IR thermography [37], mass spectrometry [38, 39, 40, 41], and biochemical methods [42, 43, 44, 45, 46, 47, 48]. Chiroptical sensing has been shown to allow fast determination of the absolute configuration, enantiomeric ratio (er) and total concentration of chiral compounds with minute sample amounts and the assays are generally easy to adapt by any laboratory that is in possession of a CD spectrophotometer. Moreover, CD sensing is amenable to automated multiwell plate readers [49, 50] when high‐throughput screening of hundreds of samples is needed, and significantly reduces solvent waste production in comparison to HPLC methods.

The large majority of chiral molecules are CD‐silent or give weak signals at short wavelengths that can be compromised by optical interferents and preclude accurate quantification efforts. However, impressive methodological advances with complementary chirality sensing concepts that overcome this shortcoming with chromophoric agents designed or optimized to quickly react and either covalently or noncovalently attach to the target molecule have been reported [51]. Although a variety of molecular recognition motifs have been exploited, they all have in common that measurable CD inductions above 300 nm are produced, which are then used with the help of a calibration curve to calculate er values. Assays that operate based on irreversible covalent bond formation are often more robust and less prone to interferences, which is an important consideration, for example, with regard to ease of operation, lab‐to‐lab reproducibility and potential adaptation by others [52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62]. We have previously repurposed amino acid and peptide derivatizing agents for chirality sensing (Figure 1) [63, 64]. In continuation of these efforts, we now wish to report the use of the Sanger reagent for comprehensive sensing of the concentration and enantiomeric composition of amines, amino alcohols, and amino acids by a combination of CD and UV measurements. This is achieved with a practical mix‐and‐measure protocol, and we show that this method is broadly useful and allows quantitative stereochemical analysis with good accuracy.

Sensing based on dynamic covalent chemistry with repurposed ninhydrin versus irreversible bond formation with the Sanger reagent.

2. Materials and Methods

2.1. General Information

All chemicals used were purchased and used without purification. NMR spectra were obtained at 400 MHz (¹H NMR) and 100 MHz (¹³C NMR) in deuterated acetonitrile. Chemical shifts are reported in ppm relative to the solvent signal. During initial screening, CD spectra were collected with a data pitch of 0.5 nm, a bandwidth of 1 nm, in a continuous scanning mode with a scanning speed of 500 nm/min and a response of 1 s, using a quartz cuvette (1‐cm path length). The data were baseline corrected and smoothed using a binomial equation. For quantitative determinations of the enantiomeric composition of amine samples, CD measurements were taken as an average of 3 runs each with a scanning speed of 500 nm/min. UV spectra were also collected in triplicate and averaged.

2.2. 2,4‐Dinitro‐N‐(1‐Phenylethyl)aniline, 3

Probe 1 (30.7 mg, 0.165 mmol), amine 2 (20.0 mg, 0.165 mmol), and triethylamine (33.4 mg, 0.33 mmol) were combined in acetonitrile (1.0 mL). The mixture was stirred for 90 min. Compound 3 was obtained as a yellow oil in 99% yield (44.2 mg, 0.163 mmol) after purification by flash chromatography using a mixture of 10% ethyl acetate in hexanes as the mobile phase. ¹H NMR (400 MHz, CD₃CN) δ 8.95 (d, J = 2.7 Hz, 1H), 8.79 (s, 1H), 8.09 (dd, J = 9.6, 2.7 Hz, 1H), 7.46–7.41 (m, 2H), 7.41–7.34 (m, 2H), 7.30 (m, 1H), 6.87 (d, J = 9.6 Hz, 1H) 4.92 (m, 1H), 1.65 (d, J = 6.7 Hz, 3H). ¹³C NMR (100 MHz, CD₃CN) δ 147.58, 142.81, 129.82, 128.99, 127.70, 125.88, 123.59, 115.59, 53.39, 23.54. HRMS (ESI‐TOF) m/z: [M + H]⁺ calcd for [C₁₄H₁₄N₃O₄] 288.0979, found 288.0976.

3. Results and Discussion

At the onset of this study, we decided to test the suitability of the Sanger reagent: 1 for quantitative CD sensing using (R)‐1‐phenylethylamine and 2 as the test analyte. As expected, the chiral amine tagging reaction occurred smoothly at room temperature in the presence of triethylamine, and we were delighted to observe that (R)‐3 gives strong CD inductions with a maximum at approximately 400 nm. Similar negative Cotton effects were obtained in acetonitrile, chloroform, dimethyl sulfoxide, methanol, and tetrahydrofuran (Figure 2). Interestingly, the strongest amplitudes and almost identical CD curves were measured in chloroform, methanol, and tetrahydrofuran while slightly weaker red‐shifted signals were observed in acetonitrile and DMSO.

(a) Reaction of the Sanger reagent 1 with (R)‐2 in the presence of triethylamine forming the dinitroaniline product 3. (b) CD spectra of (R)‐3 obtained in various solvents. The CD measurements were performed at 0.25 mM concentration.

This sensing assay is quite practical and based on a straightforward mix‐and‐measure protocol with a wide solvent selection available. This proved highly advantageous when we continued with the screening of other substrates. We applied probe 1 to various amines, amino alcohols, and amino acids using acetonitrile or methanol as solvent to assure homogeneous reaction and CD sensing conditions. In addition to the sensing of 2, 11 other amines and two diamines, 4–16, were successfully tested and representative CD inductions obtained with 1‐(2‐pyridyl)ethylamine, 8, and 2‐methylpyrrolidine, 14, are shown in Figure 3. Chiroptical sensing of purely aliphatic amino alcohols and structures displaying aryl rings was also possible as shown for (1R,2S)‐2‐amino‐1,2‐diphenylethan‐1‐ol, 19. Finally, we found that the amino acids 21–26 and glutathione, 24, are viable substrates, which is shown for cysteine. Importantly, there is no workup required and the crude reaction mixtures are directly transferred into a cuvette and subjected to the CD analysis. The operational simplicity and continuous workflow would greatly facilitate fully automated sample processing and high‐throughput screening applications if desired.

Chiroptical sensing scope. Top: General workflow and structures of all amines, amino alcohols, and amino acids investigated (only one enantiomer is shown). Bottom: CD inductions measured with the dinitroaniline derivatives of amine (R)‐8, (S)‐14, (S)‐19, and (R)‐24. The CD spectra obtained with Sanger reagent tagged to (R)‐enantiomers are shown in red while the (S)‐enantiomers appear in blue. The CD measurements were performed at 0.25 mM in MeOH.

Although tagging of amino groups with the Sanger reagent is well known, we monitored the reaction by ¹H NMR and UV spectroscopy (Figure 4). In comparison of the NMR spectra of the probe, the test amine and the crude reaction mixture shows that the labeling occurs quantitatively in the presence of stoichiometric amounts of 1 and without any sign of by‐products. We were pleased to find that the formation of 3 does not only give characteristic CD signals but also coincides with a UV induction at 345 nm. The amine and the probe are CD‐ and UV‐silent above 300 nm, which means that the chiroptical signal inductions can be unequivocally assigned to compound 3. It is noteworthy that the UV maximum is reached within 5 min, which demonstrates that the tagging step is very fast and the sample can be subjected to UV and CD analysis without delay. However, this is a robust assay with a wide time window, and we observed no spectral changes after several hours.

¹H NMR and UV analysis of the reaction between probe 1, amine 2, and triethylamine in acetonitrile. Top NMR spectra of (1) Sanger reagent 1; (2) 1‐phenylethylamine 2; (3) crude reaction mixture with 2 equivalents of triethylamine in CD₃CN after 90 min. Please note that the triethylamine signals are cut off; see SI for the full spectrum. Bottom: UV monitoring of the increase in the UV absorption at 345.0 nm due to the formation of 3.

We then investigated the chiroptical response of the probe to the enantiomeric composition and concentration of amine 2. Mixing of 1 and solutions containing 2 in varying enantiomeric excess followed by CD analysis showed a linear correlation between the amplitude at 405.0 nm and the sample ee (Figure 5). This was also the case with the UV maximum at 345.0 nm when the amine concentration was incrementally increased from 0.0 to 50.0 mM. While the linearity of these chiroptical signal inductions is not a requirement for quantitative analysis, it simplifies the mathematical treatment. These results thus set the stage for quantitative sensing experiments, vide infra.

Linear CD and UV response of the Sanger probe to the enantiomeric composition and concentration, respectively, of amine 2. Top: Induced CD signals obtained with 1 and amine 2 at varying %ee (left). Plot of the CD maximum at 407.0 nm versus %ee values (right). The “–” sign indicates that (R)‐(−)‐1‐phenylethylamine was in excess. Middle: UV induction upon formation of the reaction product and the corresponding calibration curve showing the absorption intensity measured at 345.0 nm versus the analyte concentration. Bottom: Selected examples of CD/UV sensing of samples with varying enantiomeric composition and concentration. See SI for details.

A favorable feature of our assay is that the generation of distinguishable diastereomers for quantitative er determination is not required. Unlike chiral agents that are often more expensive and need to be prepared in enantiopure (or highly enantioenriched) form, the use of achiral 1 does not cause undesirable kinetic resolution effects and other systematic errors resulting from the formation of diastereomeric mixtures, which may necessitate the cumbersome use of correction factors. Moreover, the UV inductions are nonenantioselective and thus allow determination of the total concentration of both enantiomers ([R] + [S]) independent of their ratio while the CD signals respond to the chirality of the analyte and can be used for er ([R]/[S]) calculation. With the previously obtained UV and CD calibration curves in hand, we randomly prepared seven samples of 2 with varying concentrations and enantiomeric ratios to thoroughly test the general performance of our sensing assay. We chose to screen samples within a 5.0–50.0 mM concentration range to allow fast and complete amine tagging at room temperature without the risk of formation of aggregates that may cause nonlinear chiroptical responses. These samples were applied in our assay and the measured UV and CD maxima at 345.0 and 407.0 nm, respectively, were analyzed. The sign of the Cotton effects allowed determination of the absolute configuration of the major enantiomer while the chiroptical signal intensities gave the total amine concentration and er values with good accuracy as shown in Table 1. For example, the sensing of Sample 1 containing amine 2 in a 9.6:90.4 (R)/(S) ratio at 13.0 mM gave an er of 6.8 (R):93.2 (S) and a total concentration of 14.1 mM. The analysis of Sample 2, which consisted of 70.0% of the R‐enantiomer and 30.0 of S‐2 ratio at 26.0 mM gave the exact same enantiomeric composition and a total concentration of 25.6 mM (see spectra in Figure 5 and entries 1 and 2 in Table 1). The analysis of the other samples showed similar absolute error margins of less than 5%, which are generally accepted for high‐throughput screening applications.

TABLE 1.

Chiroptical sensing of the concentration and er of seven samples of 2.

Sample number	Sample composition			Sensing results
Sample number	Abs. config. ^a	Conc. (mM)	er	Abs. config. ^a	Conc. (mM)	er
1	S	13.0	9.6:90.4	S	14.1	6.8:93.2
2	R	26.0	70.0:30.0	R	25.6	70.0:30.0
3	R	47.0	73.0:27.0	R	48.6	77.4:22.6
4	S	39.0	44.0:56.0	S	40.6	41.4:58.6
5	S	10.0	40.0:60.0	S	10.8	40.3:59.7
6	R	7.0	82.1:17.9	R	8.2	86.3:13.7
7	S	21.0	35.0:65.0	S	22.1	35.0:65.0

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^{^a}

The absolute configuration of the major enantiomer was determined by comparison of the ICD sign with a reference. See SI for details.

4. Conclusions

In summary, we have shown that the well‐known Sanger reagent can be used for quantitative chiroptical sensing of the concentration and enantiomeric composition of amines, amino alcohols, and amino acids. The assay is operationally simple, fast, can be performed in a variety of solvents without any precautions, and only requires mixing of the sample and probe in the presence of a base prior to the CD and UV measurements. The combined UV/CD analysis gives concentration and er values with good accuracy and the practical assay workflow is expected to be easily adaptable to automated experimentation and parallel sample screening platforms.

Supporting information

Figure S1: Sensing of (R)‐(+)‐1‐phenylethylamine with the Sanger reagent in the presence of triethylamine in acetonitrile.

Figure S2: Plot of the CD spectra with the reaction run in different solvents at 0.25 mM concentration.

Figure S3: Plot of the UV amplitudes at 345.0 nm versus time.

Figure S4. 1H NMR (400 MHz) Spectrum of 1 in CD3CN.

Figure S5. 1H NMR (400 MHz) Spectrum of 2 in CD3CN.

Figure S6: 1H NMR (400 MHz) Spectrum of the crude reaction mixture in CD3CN.

Figure S7. 1H NMR (400 MHz) Spectrum of the isolated product 3 in CD3CN.

Figure S8. 13C NMR (100 MHz) Spectrum of the isolated product 3 in CD3CN.

Figure S9: Chiral compounds used in this study (only one enantiomer is shown).

Figure S10. CD spectrum obtained by applying the Sanger probe to (R)‐2 (red) and to (S)‐2 (blue). CD measurements were performed at 0.25 mM in ACN.

Figure S11. CD spectrum obtained by applying the Sanger probe to (S)‐4. CD measurements were performed at 0.25 mM in MeOH.

Figure S12. CD spectrum obtained by applying the Sanger probe to (R)‐5. CD measurements were performed at 0.25 mM in MeOH.

Figure S13. CD spectrum obtained by applying the Sanger probe to (R)‐5. CD measurements were performed at 0.25 mM in ACN.

Figure S14. CD spectrum obtained by applying the Sanger probe to (S)‐6. CD measurements were performed at 0.25 mM in ACN.

Figure S15. CD spectrum obtained by applying the Sanger probe to (R)‐7. CD measurements were performed at 0.25 mM in MeOH.

Figure S16. CD spectrum obtained by applying the Sanger probe to (R)‐8. CD measurements were performed at 0.25 mM in MeOH.

Figure S17. CD spectrum obtained by applying the Sanger probe to (S)‐9. CD measurements were performed at 0.25 mM in MeOH.

Figure S18. CD spectrum obtained by applying the Sanger probe to (R)‐10. CD measurements were performed at 0.25 mM in MeOH.

Figure S19. CD spectrum obtained by applying the Sanger probe to (S)‐11. CD measurements were performed at 0.25 mM in MeOH.

Figure S20. CD spectrum obtained by applying the Sanger probe to (R)‐12. CD measurements were performed at 0.25 mM in MeOH.

Figure S21. CD spectrum obtained by applying the Sanger probe to (R)‐13. CD measurements were performed at 0.25 mM in ACN.

Figure S22. CD spectrum obtained by applying the Sanger probe to (S)‐14. CD measurements were performed at 0.25 mM in MeOH.

Figure S23. CD spectrum obtained by applying the Sanger probe to (1R,2R)‐15. CD measurements were performed at 0.25 mM in ACN.

Figure S24. CD spectrum obtained by applying the Sanger probe to (1S,2S)‐16. CD measurements were performed at 0.25 mM in ACN.

Figure S25. CD spectrum obtained by applying the Sanger probe to (R)‐17. CD measurements were performed at 0.25 mM in MeOH.

Figure S26. CD spectrum obtained by applying the Sanger probe to (1R,2S)‐18. CD measurements were performed at 0.25 mM in MeOH.

Figure S27. CD spectrum obtained by applying the Sanger probe to (1R,2S)‐19. CD measurements were performed at 0.25 mM in MeOH and DMSO.

Figure S28. CD spectrum obtained by applying the Sanger probe to (R)‐20. CD measurements were performed at 0.25 mM in MeOH.

Figure S29. CD spectrum obtained by applying the Sanger probe to (S)‐21. CD measurements were performed at 0.25 mM in MeOH.

Figure S30. CD spectrum obtained by applying the Sanger probe to (S)‐22. CD measurements were performed at 0.25 mM in MeOH.

Figure S31. CD spectrum obtained by applying the Sanger probe to (S)‐23. CD measurements were performed at 0.25 mM in MeOH.

Figure S32. CD spectrum obtained by applying the Sanger probe to (R)‐24. CD measurements were performed at 0.25 mM in MeOH.

Figure S33. CD spectrum obtained by applying the Sanger probe to (S)‐25. CD measurements were performed at 0.25 mM in MeOH.

Figure S34. CD spectrum obtained by applying the Sanger probe (2 equivalents of the probe and Et3N) to (S)‐25. CD measurements were performed at 0.25 mM in MeOH.

Figure S35. CD spectrum obtained by applying the Sanger probe to (S)‐26. CD measurements were performed at 0.25 mM in MeOH.

Figure S36. CD spectrum obtained by applying the Sanger probe (2 equivalents of the probe and Et3N) to (S,S)‐27. CD measurements were performed at 0.25 mM in MeOH.

Figure S37. CD response of 1 to amine 2. CD measurements were taken at 0.34 mM.

Figure S38: Plot of the induced CD amplitudes at 407.0 nm versus sample %ee.

Figure S39. UV response of 1 to amine 2. UV measurements were taken at 0.5 mM (Sanger reagent concentration.

Figure S40: Plot of the induced UV amplitudes at 345.0 nm versus sample %ee.

Table S1: Chiroptical sensing of the concentration and er of seven unknown samples.

Figure S41: CD spectrum (left) and UV spectrum (right) of sample 1.

Figure S42: CD spectrum (left) and UV spectrum (right) of sample 2.

Figure S43: CD spectrum (left) and UV spectrum (right) of sample 3.

Figure S44: CD spectrum (left) and UV spectrum (right) of sample 4.

Figure S45: CD spectrum (left) and UV spectrum (right) of sample 5.

Figure S46: CD spectrum (left) and UV spectrum (right) of sample 6.

Figure S47: CD spectrum (left) and UV spectrum (right) of sample 7.

CHIR-37-e70062-s001.pdf^{(1.7MB, pdf)}

Acknowledgments

We gratefully acknowledge financial support from the NSF (CHE‐2246747). Open Access funding enabled and organized by Georgetown University Lauinger 2025.

Kariapper F. S., Wolf A., and Wolf C., “Repurposing the Sanger Reagent for Optical Sensing of the Concentration and Enantiomeric Ratio of Chiral Amines, Amino Alcohols, and Amino Acids,” Chirality 37, no. 12 (2025): e70062, 10.1002/chir.70062.

Funding: This work was supported by the NSF (CHE‐2246747).

Data Availability Statement

The data that supports the findings of this study are available in the supplementary material of this article.

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