Protocol for aqueous synthesis of bioactive quaternary ammonium betaine derivatives under blue LED

Summary

Quaternary ammonium compounds exhibit diverse applications as antibiotics, as surfactants, in paper industries, in sewage treatment, and in aquaculture. Here, we present a protocol for synthesizing a library of bioactive quaternary ammonium betaine derivatives under blue LED in water. We describe steps for preparing diazo compounds, synthesizing glycine betaine derivatives, and isolating pure final compounds via precipitation from an aqueous reaction mixture. This protocol promotes a sustainable approach by using water as the reaction medium and room temperature reactions.

For complete details on the use and execution of this protocol, please refer to Rath et al. (2023).¹

Graphical abstract

Highlights

• •Preparation of aryl diazo esters at room temperature
• •Blue LED mediated nitrogen insertion reaction
• •Purification by precipitation from aqueous solution
• •Analysis of the product by NMR and HRMS

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Quaternary ammonium compounds exhibit diverse applications as antibiotics, as surfactants, in paper industries, in sewage treatment, and in aquaculture. Here, we present a protocol for synthesizing a library of bioactive quaternary ammonium betaine derivatives under blue LED in water. We describe steps for preparing diazo compounds, synthesizing glycine betaine derivatives, and isolating pure final compounds via precipitation from an aqueous reaction mixture. This protocol promotes a sustainable approach by using water as the reaction medium and room temperature reactions.

Before you begin

Ubiquitous presence of quaternary ammonium compounds (QAC) as surfactants, antimicrobial agents, dyes and neuromuscular blockers (Figure 1A) makes them an interesting class of molecules.^2,3,4,5,6 They are amphiphiles and subsequently display detergent like mechanisms against various microbes especially against ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species).⁷ Hence, they are used as a broad-spectrum antibiotic (Figure 1B).^8,9 Glycine betaine (GB) derivatives are also used as antimicrobial agents.^10,11 GB- is a natural substance (existing widely among plants), biodegradable, very affordable to source and contains a trimethylalkyl ammonium moiety and a carboxylate functionality (Figure 1C). However, the conventional methods for the synthesis of quaternary ammonium compounds are plagued with disadvantages such as involvement of the acids, bases, metal catalysts, volatile organic compounds and high temperature.^12,13,14 Therefore, the development of a robust synthetic strategy driven by environmental sustainability under mild conditions is highly desirable. In a bid to address this and also inspired by numerous applications of QACs and GB derivatives, the following protocol reports an environmentally compatible synthesis of a novel class of quaternary ammonium betaine derivatives in water under blue LED. The starting materials N-methyl morpholine (NMM) and 1, 4-diazabicyclo[2.2.2]octane (DABCO) are easily available and are reacted with numerous aryl diazo esters in water under blue LED (5 W) to provide the desired compounds in excellent yield.

Figure 1.

Applications of glycine betaine derivatives and our aqueous photolytic synthesis of these molecules

(A–C) QACs and GB derivatives as surfactants, protein denaturation reagent and antimicrobial. Inset scheme: our aqueous photolytic GB synthesis.

Before one begins, the following preparations need to be carried out.

Note: All reagents were purchased from commercial suppliers and used without further purification.

Preparation of the reagents and equipment

A complete list of reagents and equipment can be found in the ‘‘key resources table’’.

• 1.Use flame or oven dried round bottom flasks and 10 mL vial for the synthesis of the aryl diazo ester and the final reaction to prepare Glycine betaine derivatives.
• 2.Use anhydrous solvents such as ‘‘dehydrated solvent system’’ by Finar for aryl diazo synthesis which will be used for the synthesis of Glycine betaine derivatives.

Key resources table

REAGENT or RESOURCE	SOURCE (Figure 2)	IDENTIFIER
Chemicals, peptides, and recombinant proteins
N-methyl morpholine 99%	Sigma-Aldrich	CAS: 109-02-4
N-methyl piperidine >99%	TCI	CAS: 626-67-5
DABCO >99%	Sigma-Aldrich	CAS: 280-57-9
Acetonitrile 99.9%	Finar	75-05-8
Ethyl acetate 99%	Finar	141-78-6
Methyl(4-chlorophenyl)acetate >98%	TCI	CAS: 52449-43-1
Methyl 3-bromophenylacetate >98%	TCI	CAS: 150529-73-0
Methyl phenylacetate >99%	TCI	CAS: 101-41-7
Methyl 4-tert-butylphenylacetate >98%	Sigma-Aldrich	CAS: 3549-23-3
Methyl(4-bromophenyl)acetate >99%	TCI	CAS: 41841-16-1
Methyl(4-fluorophenyl)acetate 98%	GLR	CAS: 34837-84-8
Methyl(4-methoxyphenyl)acetate 97%	GLR	CAS: 23786-14-3
Methyl(4-methylphenyl)acetate >99%	TCI	CAS: 23786-13-2
Methyl (3-chlorophenyl)acetate 99%	GLR	CAS: 53088-68-9
Methyl (3-fluorophenyl)acetate 98%	GLR	CAS: 64123-77-9
Methyl (3-trifluoromethylphenyl)acetate 98%	TCI	CAS: 62451-84-7
Methyl (3-methoxyphenyl)acetate 97%	Alfa Aesar	CAS: 18927-05-4
Methyl(4-iodophenyl)acetate 98%	Alfa Aesar	CAS: 63349-52-0
Methyl 2-(o-tolyl)acetate 98%	TCI	CAS: 40851-62-5
Methyl(2-bromophenyl)acetate 98%	Alfa Aesar	CAS: 57486-69-8
Methyl (2-methoxyphenyl)acetate 98%	GLR	CAS: 27798-60-3
Methyl(3,4-dimethylphenyl)acetate 98%	TCI	CAS: 57486-71-2
Methyl 2-(thiophen-3-yl)acetate 98%	Alfa Aesar	CAS: 58414-52-1
DBU >98%	Spectrochem	CAS: 6674-22-2
p-ABSA 97%	Sigma-Aldrich	CAS: 2158-14-7
Software and algorithms
ChemDraw Professional 18.0	PerkinElmer	https://www.perkinelmer.com/category/chemdraw
Other
NMR	Bruker	N/A
HRMS	Agilent 6540 accurate-mass Q-TOF LC/MS	N/A
Magnetic stirrer	IKA	C-MAG HS7
Silica gel 100–200 mesh	Finar	63231-67-4
Round-bottom flasks (50 mL)	Borosil	Product code-4380C12
10 mL reaction vials	Borosil	N/A
Disposable syringe	Dispovan	N/A
Disposable needle	Dispovan	N/A
Spatula	Borosil	Product code- LACS8888008
NMR tubes	Norell	Model no-507HP
Separating funnel	Borosil	Product code- 6403017
Rotary evaporator	IKA	Model no-RV-10

Figure 2.

Labware and reagents used for synthesis

Step-by-step method details

Part 1: Synthesis of aryl diazo esters

Timing: 4 h

In this step, the synthesis of aryl diazo acetates was accomplished within 4 h as shown in the scheme below and Scheme 1. Troubleshooting 1.

• 1.To a 50 mL of oven dried round bottom flask equipped with magnetic stir bar, dissolve Aryl acetate (5 mmol, 1 equiv) in acetonitrile (10 mL) and DBU (1,8-diazabicyclo[5.4.0] undec-7-ene) (0.9 mL, 1.2 equivalents, 6 mmol).
• 2.Stir for 10 min.
• 3.Then add p-ABSA (4-acetamidobenzenesulfonyl azide) (1.44 g, 1.2 equivalents, 6 mmol) to the reaction mixture and again stir for 4 h in the dark at r.t as shown in Figure 3.

Scheme 1.

Synthesis of the aryl diazo acetates

Figure 3.

Synthesis and purification of aryl diazo esters

Part 2: Purification of aryl diazo esters

Timing: 2 h

In this step, the pure aryl diazo acetate is obtained within 2 h through extraction and column chromatography Figure 3.

• 4.After completion of the reaction, remove acetonitrile by using rotavapor (100-50 mbar at 35°C, ∼ 15 min).
• 5.Dilute the reaction mixture with ethyl acetate (25 mL) and transfer it to a 125 mL separating funnel.
• 6.Add 10 mL distilled water and brine solution (5 mL) to it.
• 7.Shake the separatory funnel vigorously and allow the aqueous phase and organic phase to separate properly.
• 8.Collect both organic phase and aqueous phase separately in two 50 mL conical flask.
• 9.Pour the aqueous phase back into the separatory funnel and add 10 mL of ethyl acetate for extraction.
• 10.Again, shake the separatory funnel vigorously and allow the aqueous phase and organic phase to separate and transfer them to their corresponding flask.
• 11.Repeat the process 2–3 times.
• 12.Add sodium sulfate to the organic phase, shake the flask slowly and filter the solution in a 100 mL round bottom flask.
• 13.Remove the solvent by using rotavapor (250 mbar at 35°C, ∼ 15 min) to afford the dried crude reaction mixture.
• 14.Add 2 mL dichloromethane to the crude and add 70 mg of 100–200 mesh silica to it. Carefully evaporate the solvent by rotavapor (600 mbar at 35°C, ∼ 10 min) to prepare the slurry.
• 15.Purify crude product by column chromatography (100–200 mesh silica) using 5% ethyl acetate in hexane. Troubleshooting 2.
• 16.Monitor the fraction by TLC. The product has an R_f = 0.8 in 10% ethyl acetate in hexane.
• 17.Collect the combined fractions of pure product and concentrate by using rotavapor under reduced pressure to afford pure aryl diazo ester. Troubleshooting 3.

Note: Prolonged exposure of Aryl diazo esters to light might degrade it. Hence the reaction as well as the purification should be performed in dark.

Part 3: Preparation of photoreactor

Timing: 2 h

Photoreactors are devices used to facilitate photochemical reactions by providing a controlled environment for the interaction of light and chemicals. Instead of purchasing high cost photoreactor one can easily set up his/her own in lab by assembling locally available tools as we did in our lab as follows. If you have higher quality of photoreactor, then you can skip this step.

• 18.Tools needed: 5 W 12 V blue LED strips with suitable adapter (AC-DC), one transparent container (15 mm diameter, 7 mm height) and aluminum foil as shown in Figure 4.
• 19.Attach Blue LED strips at the outside bottom of the transparent container ensuring that the light can fully illuminate the reaction mixture.
• 20.Create a reflective surface around the transparent container using aluminum foil or any other reflective material. This will help redirect the light towards the reaction mixture, maximizing light exposure.
• 21.To carry out a reaction, place the whole light set up on a magnetic stirrer and place a vial of 25/10/20 mL inside the light chamber just above the light source.
• 22.Turn on the LED light source and start the stirring mechanism if applicable.
• 23.Observe the reaction and record any changes or progress over time.

Note: Here, all blue light batch reactions were carried out under air as specified in Photochemical Reactor- Aldrich Micro Photochemical Reactor, blue LED lights (ALDKIT001-1EA). LED light is an IP68 double density 12 V DC waterproof blue light with a spectral range of 435–445 nm with a wall plug power supply of 500 mA with 5–6 W. The irradiation vessel material is borosilicate glass. The distance of the irradiation vessel from the light source is 2 cm.

Figure 4.

Overview of the photoreactor set up

Part 4: Synthesis of glycine betaine derivatives 2–4

Timing: 10 h

In this step, the synthesis of Glycine betaine derivatives are accomplished within 10 h as shown in the scheme below and Scheme 2.

• 24.To a 5 mL of oven dried reaction vial equipped with magnetic stir bar, add aryl diazo acetate (0.39 mmol, 1 equiv) and NMM (N-Methyl Morpholine)/NMP (N-Methyl Piperidine)/DABCO (0.39 mmol, 1 equiv) in water (1 mL). Troubleshooting 4.
• 25.Irradiate the reaction mixture under blue LED for 10 h (Figure 5). The completion of reaction can be understood by discoloration of the reaction mixture and precipitation of the product. Troubleshooting 5.

Note: One should wear light protecting goggles to prevent excessive exposure of blue light.

Scheme 2.

Synthesis of the glycine betaine derivatives from 1

Figure 5.

Blue LED reaction set up

Part 5: Purification of compounds 2–4

Timing: 1 h

• 26.The desired compounds are precipitated from the reaction mixture as shown in Figure 5.
• 27.Evaporate the solvent under by using rotavapor (100-10 mbar at 50°C).
• 28.Add 2 mL ethyl acetate to the reaction mixture.
• 29.The desired compound being insoluble in ethyl acetate got precipitated.
• 30.Filter and wash the precipitates with ethyl acetate 8 to 9 times to obtain the pure compound 2–4.
• 31.Dry it using rotavapor (250-0 mbar at 45°C) for 30 min.
• 32.Characterize the compounds 2–4 by NMR spectroscopy (¹H and ¹³C NMR) by dissolving it in CD₃OD or CDCl₃.

Note: Quaternary ammonium betaine derivatives are highly stable compounds and can be stored at 30°C–35°C and 1013.25 mbar for several weeks.

Expected outcomes

By following the protocol mentioned in part 2, methyl 2-(4-bromophenyl)-2-diazoacetate (1a) is obtained by using methyl 2-(4-bromophenyl)-2-diazoacetate (5 mmol, 1.1 gm), DBU (6 mmol, 913.4 mg) and P-ABSA (6 mmol, 1.44 gm) in 10 mL of acetonitrile in 1.2 gm, 98% yield as yellow solid.

By following the protocol mentioned in part-4, 2a was synthesized by using 1a (99.4 mg, 0.39 mmol) and NMM (39.45 mg, 0.39 mmol) in 95.2 mg, 78% yield as white solid, 3a was synthesized by using 1d (80.41 mg, 0.39 mmol) and DABCO (43.74 mg, 0.39 mmol) in 102.38 mg, 95% yield as white viscous oil and 4a was synthesized by using 1b (99.4 mg, 0.39 mmol) and NMP (38.67 mg, 0.39 mmol) in 72.7 mg, 80% yield as white solid.

Note: Refer to the original paper for substrate scope.¹

Quantification and statistical analysis

Methyl 2-(4-bromophenyl)-2-diazoacetate, (1a):

¹H NMR (400 MHz, CDCl₃) δH 7.49 (d, J = 8 Hz, 2H), 7.36 (d, J = 8 Hz, 2H), 3.86 (s, 3H) ppm. ¹³C NMR (100 MHz, CDCl₃) δ 165.3, 132.1, 125.4, 124.8, 119.4, 52.2 ppm.

2-(4-bromophenyl)-2-(4-methylmorpholino-4-ium)acetate,(2a):

¹H NMR (400 MHz, CD3OD) δH 7.68 (d, J = 8 Hz, 2H), 7.60 (d, J = 8 Hz, 2H), 4.98 (s, 1H), 4.13–4.05 (m, 1H), 4.05–3.93 (m, 4H), 3.92–3.85 (m, 1H), 3.78–3.62 (m, 2H), 3.39 (s, 3H) ppm.

¹³C NMR (100 MHz, CD3OD) δH 169.4, 135.4, 133.3,129.6, 126.1, 81.7, 61.7, 61.4, 60.0, 59.5, 42.4 ppm. HRMS (ESI-TOF) m/z:[M + H]⁺ calcd for C₁₃H₁₇BrNO₃ 314.0386, found 314.0380.

Melting point: 168–170°C.

2-(1,4-diazabicyclo[2.2.2]octan-1-ium-1-yl)-2-(4-methoxyphenyl)acetate, (3a):

¹H NMR (400 MHz, CD₃OD) δ_H 7.55(d, J = 8 Hz, 2H), 7.03 (d, J = 8 Hz, 2H), 4.70 (s, 1H), 3.88–3.78 (m, 6H), 3.55–3.47 (m, 3H), 3.14 (t, J = 8 Hz, 6H) ppm.

¹³C NMR (100 MHz, CD₃OD) δ_C 170.4, 162.7, 134.8, 122.0, 115.4, 80.9, 55.9, 51.5,47.1, 46.2 ppm.

HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₅H₂₁N₂ O₃ 277.1547, found 277.1546.

2-(1-methylpiperidin-1-ium-1-yl)-2-phenylacetate, (4a):

¹H NMR (400 MHz, CD₃OD) δ_H 7.70–7.68 (m, 2H), 7.54–7.46 (m, 3H), 4.94 (s, 1H), 3.89–3.83 (m, 1H), 3.66–3.61 (m, 1H), 3.42–3.34 (m, 2H), 3.25 (s, 3H), 2.00–1.93 (m, 4H), 1.81–1.74 (m, 1H), 1.63–1.58 (m, 1H).¹³C NMR (100 MHz, CD₃OD) δ_C 170.3, 133.5, 131.5, 130.8, 129.9, 80.5, 61.0, 60.8, 43.9, 22.3, 21.2, 20.9. HRMS (ESI-TOF) m/z: [M + H]⁺ calcd for C₁₄H₂₀N O₂ 234.1489, found 234.1488.

Melting point: 180–182°C.

Note: Refer to the original paper for spectral images.¹

Limitations

This protocol is limited to cyclic tertiary amines only.

Troubleshooting

Problem 1

Part 1 (step-by-step method details): All steps involve toxic and volatile organic compounds that can harm human body.

Potential solution

All reactions should be performed inside the fume hood only and personal protection equipment e.g., lab coat, gloves, safety goggles should be used.

Problem 2

Step 15 (step-by-step method details): Silica dusts are harmful to lungs.

Potential solution

One should use mask while packing column.

Problem 3

Step 17 (step-by-step method details): Aryl diazo esters might degrade at room temperature.

Potential solution

It should be kept in freezer at 7–10°C temperature and can be stored up to 3–4 months.

Problem 4

Step 24 (step-by-step method details): N-methyl morpholine has very pungent smell.

Potential solution

Once should use double mask to cover nose and mouth.

Problem 5

Step 25 (step-by-step method details): Excessive and prolonged exposure to blue LED light can be Harmful to eyes.

Potential solution

The photoreactor should be placed in a confine place to prevent light leak. The fume hood glass can be covered with plastic black light proof bag or the operator can wear sun glasses while setting up the reaction.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Prof. Subhabrata Sen (subhabrata.sen@snu.edu.in).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Ms. Suchismita Rath (sr657@snu.edu.in).

Materials availability

All other data supporting the findings of this study are available within the article or from the lead contact upon reasonable request.

Data and code availability

All data reported in this paper will be shared by the lead contact upon reasonable request.

Any additional information required to reanalyze the data reported in this paper can be obtained from the lead contact upon request.