Communication pubs.acs.org/jchemeduc
Introducing Students to NMR Methods Using Low-Field 1H NMR Spectroscopy to Determine the Structure and the Identity of Natural Amino Acids Aleksandra Zivkovic,*,† Jan Josef Bandolik,† Alexander Jan Skerhut,† Christina Coesfeld,† Nenad Zivkovic,‡ Miomir Raos,‡ and Holger Stark*,† †
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätstrasse 1, Düsseldorf 40225, Germany ‡ Faculty of Occupational Safety, University of Nis, Carnojevica 10A, 18000 Nis, Serbia S Supporting Information *
ABSTRACT: Nuclear magnetic resonance (NMR) spectroscopy is a widely used analytical technique for molecular structure determination, and is highly valued in the fields of chemistry, biochemistry, and medicinal chemistry. The importance of NMR methods in the European (PhEur) and United States Pharmacopeia (USP) is steadily growing. However, undergraduates often have problems becoming familiar with handling the complex data. We have developed a simple experiment in which undergraduates, who are learning 1H NMR spectroscopy for the first time, investigate natural amino acids, and determine their structure and identity using low-field 1H NMR measurements and simple COSY experiments. These students see and learn the connection between the chemical shift of the αC-proton and the isoelectric point of the amino acid. They engage with the spectroscopic topic by acquiring their own spectra, and processing and interpreting the data. Understanding important natural amino acids and their physicochemical character is highly relevant to all students studying life sciences. KEYWORDS: Analytical Chemistry, Laboratory Instruction, Hands-On Learning/Manipulatives, Problem Solving/Decision Making, Amino Acids, Laboratory Equipment/Apparatus, NMR Spectroscopy, Qualitative Analysis, Second-Year undergraduates
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INTRODUCTION The importance of nuclear magnetic resonance (NMR) spectroscopy for analysis of both small molecules as well as large proteins or nucleic acid chains qualifies it as one of the most important analytical tools in instrumental analysis taught to students. The method generates a high density of information on the molecules. However, for nonexperienced undergraduates, this high density of information is often the source of problems. Students can easily be overwhelmed by the complexity of data obtained from one experiment: chemical shifts, splitting patterns, integrals, line width, roof effect, and so on.1 While sophisticated high-field NMR machines up to 1000 MHz are quite expensive to purchase, operate, and maintain, educators can take advantage of a less costly alternative: using low-field benchtop NMR spectrometers (40−80 MHz), which are more appropriate for the teaching laboratory. We have found the low-field NMR spectrometer to be a useful instrument for improving undergraduates’ understanding of the technique.2,3 Students are encouraged to obtain their own data and perform their own data evaluation to understand the technique. This hands-on experience increases students’ knowledge and motivation compared to theoretical lessons and examples. The first lowfield NMR benchtop spectrometers were introduced in 2011; © 2016 American Chemical Society and Division of Chemical Education, Inc.
since then, more benchtop spectrometers have come on the market. They vary in magnetic field, resolution, size, technical equipment, and costs.4,5 The L-amino acids used in this experiment (see the structures in the student instructions of the Supporting Information) are vitally important structures in life sciences and related areas (protein structure, catalysis, etc.).6 They represent a useful and important source of substances that are homogeneous, yet show enough heterogeneity and variations to be used in a number of structural determination experiments. Students are challenged to combine their physicochemical knowledge on amino acids with their knowledge of NMR spectroscopy to understand the basics of 1H NMR characteristics. The neutral amino acids are present in water solution in a neutral/zwitterion equilibrium. There are two zwitterion forms of acidic and basic amino acids rather than only one zwitterionic form by neutral amino acids (details are shown in the student instructions of the Supporting Information). Autoprotonation/ deprotonation on the side chain changes the protonation extent Received: March 3, 2016 Revised: November 8, 2016 Published: December 15, 2016 115
DOI: 10.1021/acs.jchemed.6b00168 J. Chem. Educ. 2017, 94, 115−120
Journal of Chemical Education
Communication
observe and discuss various physicochemical properties of amino acids. In contrast, the approach described in this paper differs in that we challenge students to determine the unknown structure on their own from the complete pool of all amino acids. After an introductory lecture on NMR theory, the students delve into the task as a first expansion of their knowledge in this field. We want students to develop an understanding of the method and its possibilities with an unknown structure determination without the direct comparison to known 1H NMR spectra. In this experiment we focus on the chemical structure of each amino acid, with its 1D and 2D 1H NMR spectra. Simultaneously we want our students to develop a connection between the physicochemical character of the amino acid and the chemical shift of the αC-proton. 2D 1H NMR spectroscopy is not essential for the identification of an amino acid, and therefore, we use the measurement to introduce for the first time 2D NMR spectroscopy to our students. The main pedagogic goals of the developed experiment are for students to • Interpret the 1H NMR data (both one- and two-dimensional) • Process their own data (here with MNova software) • Understand how chemical character influences a chemical shift • Learn how D2O exchange affects a 1H NMR spectrum • Gain hands-on experience with NMR spectroscopy from preparing the sample, acquiring a spectrum, processing data, and evaluating their results. The first step for the students in the structure determination of an amino acid is to measure the 1H NMR spectrum of an unknown amino acid in D2O. The second step is to determine whether an aliphatic or an aromatic amino acid is present. If students have an aromatic amino acid, their unknown is already narrowed down to four structures. If they have an aliphatic amino acid they need to see if the proton in the αC-position to amino and carboxylic acid is below 3.40 ppm (basic amino acid), between 3.50 and 4.10 ppm (neutral amino acid), or above 4.00 ppm (possible acidic amino acid). A neighboring aromatic structure in the aromatic amino acids is responsible for the low-field position of the αC-proton compared to aliphatic amino acids. An expected increase in the chemical shift (from 3.98 to 4.25 ppm) is observed when comparing the chemical shift of the αC-proton of L-histidine, a basic amino acid with an isoelectric point of 7.47, with L-phenylalanine, a neutral amino acid with an isoelectric point of 5.84. At the beginning of the experiment, students need to become familiar with the basics for the chemical shift and increment calculations7 (Figure 3) because these data are needed for the data evaluation. Introducing increment calculations, even in a quite simplified version (Figure 3), helps students gain an impression of the chemical shifts of different protons. These calculations, though more complex, are used in the programs that predict the chemical shifts (e.g., MNova Software, which our students used). A simplified version of increment calculation does not take into account the protonation status of both the amino and carboxylic groups.
of the amino function or deprotonation extent of the carboxylic acid function of the amino acids. These changes influence the chemical shift of the αC-proton. When using water as a solvent, the exact position of this equilibrium depends on the isoelectric point of the amino acid. At the isoelectric point, the zwitterion form(s) is/are more dominant. Because the protonation/deprotonation reactions are on a faster time-scale than NMR measurements, one measures only an average structure and not two or more states in an equilibrium. Previous measurements of the amino acids in acidic and basic solutions showed that the chemical shift of the αC-proton dramatically changes (from ∼3.65 to 4.35 ppm) due to the different protonation/deprotonation extent of the amino and carboxylic groups (Figure 1).7
Figure 1. Neutral/zwitterion equilibrium of amino acids in acidic or basic solution.
As the amino group is the direct substituent on the αC-atom (the same carbon, where the proton whose chemical shift we discuss is located) and the hydroxyl function of the carboxylic acid is the substituent of the carbonyl carbon, it is expected that the protonation/deprotonation of the amino group will have a greater influence on the chemical shift of the αC-proton than the protonation/deprotonation of the carboxylic acid (with its resonance forms). The larger influence of the basicity of the side chain of amino acids than its acidity is easily explained because of the larger distance of the αC-proton to the OH substituent than to the NH2 substituent. Therefore, the chemical shift of the αC-atom of the basic amino acids will be closer to the chemical shift value of 3.60 ppm. For the neutral amino acids, expected values are about 4.00 ppm, and for acidic amino acids a value is slightly higher than 4.00 ppm. The preparation measurements confirmed our educated guess (all measurements shown in this paper were done by students). For illustration we show in Figure 2 the 1H NMR measurements of L-aspartic acid and L-asparagine. Even though the structures are very similar, the chemical shift of the αC-proton of L-aspartic acid was found at 4.31 ppm, whereas the same proton of the L-asparagine was found at 4.05 ppm. In the instructor guidelines of the Supporting Information we show the list of all natural amino acids, their isoelectric points, and the exact chemical shift of the αC-proton Scientific contributions important for teaching NMR techniques are increasingly available. Some of them discuss new theoretical approaches for teaching;8,9 most introduce new lab experiments to address the difficulties that students have in spectra interpretation.9−17 A related experimental setup focuses on dipeptide structure determination after measurement of each of the known two amino acids.18 However, this lab experiment was not designed to
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EXPERIMENTAL SECTION
Materials and Instrumentation
A variety of low-field instruments are now on the market and are suitable for performing this experiment. We have used a Magritek 116
DOI: 10.1021/acs.jchemed.6b00168 J. Chem. Educ. 2017, 94, 115−120
Journal of Chemical Education
Communication
Figure 2. 1H NMR spectrum of L-aspartic acid (∼20 mg/mL in D2O), above, and L-asparagine (∼20 mg/mL in D2O), below.
We recommend using deuterium oxide (heavy water) because of the good-to-moderate solubility of all amino acids and suppression of fast exchanging protons. Prelab Exercise
For successful experiments, it is important that students are familiar with the basic theoretical background of 1H NMR spectroscopy and the amino acids and their structures. They should draw all the structures, be able to use increment calculations to determine the theoretical chemical shifts, and discuss multiplicity on the signals in 1H NMR spectroscopy. Suggested questions may be found in the Supporting Information. Procedure
The procedure is simple and can be performed by inexperienced lab students. We group students in pairs, have them obtain from the instructor four unknown samples of natural L-amino acids, and ask them to determine the unknown structures using 1H NMR and 2D COSY measurements. Within a lab period of 4 h, students should make the measurements, generate the data, and determine the structure. The 1H NMR measurements should be done in deuterium oxide; more details are provided in the instructions for students in the Supporting Information. Students
Figure 3. Basics of the increment calculation for amino acids.
Spinsolve NMR Benchtop (42.5 MHz) (Aachen, Germany) with these resolution parameters: 50% line width
