Identification of a Dipeptide Unknown

(4)
















Veronica Herrera-Caro
CHEM-A402-21
Introduction. 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,
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H-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,
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H-NMR and
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C-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.
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2013 University of Colorado at Boulder, Department of Chemistry and Biochemistry. The information on these pages is
available for academic use without restriction.
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Emission
RAMAN SCAT.

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 PICTURE Chemical Shieft # of C Type of C Assigment
7 29ppm 1 Beta His
12 42 ppm 1 Alpha Gly
9 55 ppm 1 Alpha His
4 117ppm 1 Ring His
- 129ppm - - -
5 133ppm 2 Ring His
2 137ppm 1 Ring His
12 170ppm 1 C=O Gly
9 176ppm 1 C=O His


Chemical Shieft # of Protons Multiplicity Assigment
3.1ppm 1 Doublet Beta C-H His
3.2ppm 1 Doublet Alpha C-H Gly
3.9ppm 1 Quartet C2 - His
4.8ppm 2 Broad Singlet C5 -His
4.6ppm 1 Triplet NHC-H His
7.3ppm 1 Singlet C4 -His
8.5ppm 1 Singlet His 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 Acid Letter Code Rf Value Polar / Non
Polar
(R)
Alanine A 8/12.5 = 0.64 Non Polar
Cystine C 8.5/12.5 = 0.68
Aspartic Acid D 6.6/13 = 0.51 Polar (-)
Glutamic Acid E 7.7/13= 0.59 Polar (-)
Phenylalanine F 11/13= 0.84 Non Polar
Glycine G 6/13 =0.46
Isoleucine I 10.6/13= 0.81 Non Polar
Histidine H 5.8/13= 0.44 Polar (+)
Lysine K 6.2/13= 0.47 Polar (+)
Leucine L 10.9/13= 0.84 Non Polar
Methionine M 9.1/13= 0.70 Non Polar
Proline P 8.5/13= 0.65
Arginine R 6/13= 0.46 Polar (+)
Serine S 6.5/13= 0.50 Polar
Threonine T 7.5/13= 0.58 Polar
Valine V 9.3/12.5= 0.74 Non Polar
Tryptophan W 9.7/12.4= 0.78 Non Polar
Tyrosine Y 9/12.2= 0.73 Non 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= 0.19
Higher 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.4
Supporting Figures.
Figure 8.FDNB Reaction

Figure 9. Ninhydrin Reaction.

Figure 10. Supporting H-C NMR of Suspected Dipeptide