Identification of a Dipeptide Unknown (4)

Veronica Herrera-CaroCHEM-A402-21Introduction. The purpose of the experiments performed, was to gain experience in several characterization methods commonly used in both chemistry and biochemistry laboratories. All of the techniques used during this experiment are crucial in numerous areas of biochemistry. The experiment consisted in the identification of an unknown dipeptide through various characterization methods. First, ultraviolet and fluorescence absorption spectroscopy were used to determine the presence of phenylalanine, tryptophan o tyrosine. After confirming the presence or absence of this amino acids, 1H-NMR spectrum at both 60 MHz and 400 MHz where provided to attempt a rough identification of the dipeptides that could then be confirmed using thin layer chromatography.Amino acids are the core skeleton of proteins, which are essential parts of organisms and participate in virtually every process in our cells. In proteins, amino acids are bonded between the amine group of amino acid and the carboxylic acid of another. This chain of amino acids is known as a peptide. Like carbohydrates, a peptide that contains only two amino acids is a dipeptide, three a tripeptide and more then ten polypeptides. Proteins are polypeptides consisting of 20 or more amino acids. This amino acids come primarily form our diet. We ingest them as proteins, and this are broken down into peptides and then into single amino acids that are then bonded again into the needed proteins. This breaking of peptide bonds is carried out through acid catalyzed hydrolysis in the stomach and by proteolytic enzymes further down in our digestive system.

Methods. UV-Vis and Fluorescence. Ultraviolet spectroscopy as we know, is a technique that measures the interaction of molecules with electromagnetic radiation. The energy of the light is used to promote electrons from the ground state to an excited state. A spectrum is obtained when the absorption of light is measured as a function of its frequency or wavelength. Molecules with electrons in delocalized aromatic systems often absorb light in the near-UV (150400nm) or the visible (400800 nm) region. The absorbance of a solute depends linearly on its concentration and therefore absorption spectroscopy is ideally suited for quantitative measure- ments.

The wavelength of absorption and the strength of absorbance of a molecule depend not only on the chemical nature but also on the molecular environment of its chromophores. The peptide groups of the protein main chain absorb light in the far-UV range (180230nm). The aromatic side- chains of Tyr, Trp and Phe also absorb light in this region and, in addition, they absorb in the 240300nm region. Hence, some of us did not have any of this three amino acids, other techniques had to be performed. Fluorescence is one of the three forms of photoluminescence reactions in chemistry that involves the excitation of an electron from its ground state to a higher energy state as shown in Fig 1. After a period of time, the electron decays back to the ground state and energy is released in the form a second photon (fluorescence). Particularly, only the three amino acids previously mentioned are the only ones capable undergoing this phenomenon. Also, it has to be taken into consideration that when performing measurements over water, one finds spectra that include water Raman Figure 1. Photoluminescence reactions.scattering and fluorescence from substances in the water. This is of importance, since if you dont have any of the amino acids that are capable of fluorescing, the Raman scattering may be confused with a signal.Figure 2. Emission Spectra of Water

NMR Specta. Other characterization techniques, such as IR, NMR and Mass-Spec are more specific in the determination of which specific amino acids a peptide might contain. In particular, 1H-NMR and 13C-NMR are very precise in identifying any substance, if one has a pure sample to test. Proton NMR is based on the premise that protons are spin , and only a few isotopes are spin too. 1H, 13C, 15N, and 31P are all NMR active. Nuclei that have a spin and a charge have a magnetic dipole, which means that they are affected by magnetic fields showing up in two possible alignments with field (alpha, low energy) or against the field (beta, high energy). NMR is so to say insensitive to the amount of sample, but is very sensitive to chemical environment. This means, that particular nuclei say any H in X compound may flip (resonate) from the alpha alignment to the beta at a particular frequency (ppm). This is the information that is collected and different nuclei in different environments flip (resonate) at different frequencies, permitting an identification of each one. These nuclei also have specific splitting patterns (energy level affected by energy level of neighbor H), depending on the number of neighbor nuclei that are NMR active. This split so to speak the peaks into doublets, triplets and so on indicating one or more NMR active neighbors. Another helpful characteristic that shows up in NMR spectra is the coupling constant which represent nuclei that are connected through the same atoms. Common amino acids have been studied with NMR spectra, and their signals have been recorderd in tables in order to better identify them in unknown peptides as shown in Fig 3.

Figure 3. NMR of Common amino acids, specifically Histidine

TLC Chromatography. Finally, in order to obtain a specific identification that includes the sequence in which the suspected amino acids that form the unknown peptide are other techniques can be performed. In order to do so, the mixture of amino acids has to be separated and then identified. This can be done using chromatography, and depending on the quality of the separation that wants to be obtained, it can be ion exchange, column or thin layer chromatography.

In this particular experiment, the identity of the amino acids in a mixture can be assessed using cellulose thin-layer (TLC) chromatography. A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer of a solid adsorbent (in this case cellulose). A small amount of the sample is spotted near the bottom of this plate. The TLC plate is then placed in a shallow pool of a solvent in a developing chamber so that only the very bottom of the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary action. As the solvent moves past the spot that was applied, equilibrium is established for each component of the mixture between the molecules of that component, which are adsorbed on the solid, and the molecules, which are in solution. In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others. When the solvent has reached the top of the plate, the plate is removed from the developing chamber, dried, and the separated components of the mixture are visualized. If the compounds are colored, visualization is straightforward. The common amino acids can be relatively well separated on the cellulose plate. The amino acids can be visualized by derivatization with ninhydrin, and the Rf values of the mixture compared to the Rf values of known amino acids run on the same separation plate for identification. [footnoteRef:1] [1: 2013 University of Colorado at Boulder, Department of Chemistry and Biochemistry. The information on these pages is available for academic use without restriction.]

Results.pH/UV-Vis/Fluorescence. The pH of the final peptide solution was 8, which does not tell us anything because the peptide was dissolved in water, and the pH could result form the substituent in the amino acid or, to the NH or COOH of the amino acid. If a pH of 5 would have resulted instead, it could indicate the presence of aspartamic acid or glutamic acid, given their acidic nature.First, a fluorescence spectra of the least diluted solution (4a) was taken and compared to that of a solution that only contains water, and as shown in Fig 4, no difference was observed. The only signal present was that of the Raman scattering. Figure 4. Fluorescence Spectra of (a)Water/ (b)compound.

For further characterization, a Uv-Vis Spectra of the same solution was taken, and because of the poor information obtained a spectra of the second diluted solution (4b) was also taken. The wavelength of maximum absorption of the peptide was between 210 and 213, which indicates the presence of a His ring.

NMR.

ON PICTUREChemical Shieft# of CType of CAssigment

729ppm1BetaHis

1242 ppm1AlphaGly

955 ppm1AlphaHis

4117ppm1RingHis

-129ppm---

5133ppm2RingHis

2137ppm1RingHis

12170ppm1C=OGly

9176ppm1C=OHis

Chemical Shieft# of ProtonsMultiplicityAssigment

3.1ppm1DoubletBeta C-H His

3.2ppm1DoubletAlpha C-H Gly

3.9ppm1QuartetC2 - His

4.8ppm2Broad SingletC5 -His

4.6ppm1TripletNHC-H His

7.3ppm1SingletC4 -His

8.5ppm1SingletHis N-H

TLC. After hydrolyzing and labeling the dipeptide, both silica and cellulose thin layer chromatography was performed. In order to have a proper comparison, cellulose TLC was taken of the standard solutions of all common amino acids. After calculating RF values of both the standards, and the unknown (labeled and unlabeled) confirmation of the identity and sequence of the unknowns was attempted. It was observed that non-polar amino acids resulted in higher RF values than polar amino acids. In

Figure 5. Cellulose Thin Layer Chromatography of the Standards

Amino AcidLetter CodeRf ValuePolar / Non Polar(R)

Alanine A8/12.5 = 0.64Non Polar

CystineC8.5/12.5 = 0.68

Aspartic AcidD6.6/13 = 0.51Polar (-)

Glutamic AcidE7.7/13= 0.59Polar (-)

PhenylalanineF11/13= 0.84Non Polar

GlycineG6/13 =0.46

IsoleucineI10.6/13= 0.81Non Polar

HistidineH5.8/13= 0.44Polar (+)

LysineK6.2/13= 0.47Polar (+)

LeucineL10.9/13= 0.84Non Polar

MethionineM9.1/13= 0.70Non Polar

ProlineP8.5/13= 0.65

ArginineR6/13= 0.46Polar (+)

SerineS6.5/13= 0.50Polar

ThreonineT7.5/13= 0.58Polar

ValineV9.3/12.5= 0.74Non Polar

TryptophanW9.7/12.4= 0.78Non Polar

TyrosineY9/12.2= 0.73Non Polar

Figure 6. Silica TLC Plate of Unlabeled and Labeled Unknown Dipeptides.

The RF values of both the unlabeled and labeled amino acids where as follows,Lower Spot0.18/ 0.2 HIS= 0.19Higher Spot0.45/0.71/ GLY0.41 *The spot that disappeared in the labeled solution was the higher spot from the unlabeled solution suggesting that Glycine Is in the N-Terminal

Conclusions.After carefully going through all the data collected, I came to the conclusion that the amino acids present in the unknown dipeptide no. 4 where histidine and glycine, being glycine in the N-terminus, as proven by the result of the cellulose and silica TLC of the labeled solution. The resulting dipeptide is shown in Fig 7. To support this, simulated NMR spectra were obtained and matched the spectra provided by the instructor, proving the identity of the unknown. Furthermore, the UV-Vis results coincide with the expected wavelength of maxima absorption (211) . The fluorescence spectra also supports the result obtained, because neither of the suspected amino acids are capable of giving signal.

Figure 7. Computer generated structure of Dipeptide Unknown no.4Supporting Figures.Figure 8.FDNB Reaction

Figure 9. Ninhydrin Reaction.

Figure 10. Supporting H-C NMR of Suspected Dipeptide