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Application of 23Na and 39K in NMR:

23
Na and 39K are both NMR active nuclei. 23Na has an I value of 3/2, a natural abundance of 100% and
relatively high gyromagnetic ratio. 39K, ont he other hand, has an I value of 3/2, a natural abundance of
93,26%, and an exceptionally low gyromagnetic ratio.
23
Na can be measured mainly because of its high natural abundance and high gyromagnetic ratio. It also
has a short magnetic relaxation time, which has been studied in detail in many biological systems. In
contrast, 39K has been the subject of fewer studies published in the past (although its intracellular
concentration is five times that of sodium from a biological point of view), one of the main reasons
being its low measurement sensitivity.
The paper that I found Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens.
Examples of quadrupole ion magnetic relaxation in a crowded protein environment, was written by A.
Stevens, P. Paschalis and T. Scheich and was published in 1992 in the Biophysical Journal Vol. 61, No.
5, on pages 1061-1075.
The purpose of their work was to investigate the kinetic dynamics of 23Na+ and 39K+ in cortical lens
tissues of cattle. To do this, they used single and multiple quantum NMR techniques to characterise the
intralenticular state of the ions. In addition, NMR-visibility studies were used to detect possible
immobilized 23Na+ and 39K+ ions. NMR measurements were performed with General Elecric GN-300
and General Electric GN-500 MHz spectrophotometers.
In the case of lens tissue, maintaining normal function depends in part on the behaviour of the constituent
sodium and potassium ions. Vertebrate lenses provide an excellent system for studying biophysical
phenomena from a biomedical perspective. One of the main reasons for this is the remarkably high
protein content of the lens fibre cells. These proteins typically fall into three broad groups, collectively
known as crystallins. The lens homogenates used for this article were derived from calves aged 1 day to
3 months.
Studies in lens tissue have focused primarily on the distribution of ions. The proximity of the ion to the
polypeptide chain segments was found to determine the relaxation characteristics of the ion (rapid
exchange was assumed for the exchange of the ion from one lattice cell to another). Furthermore, the
NMR data on the amount of "free" mobile ions is consistent with previous findings on the mobility of
sodium and potassium ions in a crowded protein environment.
The results of the study show that a large proportion of intracellular sodium and potassium ions are
"free" and thus able to influence the chemical potential of intralenticular water. The final conclusion of
the paper is that the most likely role of sodium and potassium ions in the lens is to regulate cell volume
by influencing the chemical potential of intralenticular water, as mentioned above.
Briefly, I found one more interesting application of 23Na in an article on the study and treatment of a
joint disease. The paper is Application of sodium triple-quantum coherence NMR spectroscopy for the
study of growth dynamics in cartilage tissue engineering was written by M. Kotecha, S. Ravindran, T.
M. Schmid, A. Vaidyanathan, A. George and R. Magin and was published in 2013 in NMR in
Bimedicine. The use of sodium triple-quantum coherence NMR spectroscopy was investigated to study
tissue growth in three different cartilage tissue-based constructs. The properties of the engineered
cartilage tissues were compared with those of the natural ones. This was easily achieved with 23Na
because articular cartilage contains high amounts of collagen, proteoglycans and glycoproteins. The
proteoglycans contain numerous repeating disaccharides, whose negatively charged carboxylate and
sulphate groups attract sodium ions.

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Experimental session and short discussion of „Facile synthesis of stable selenocystine and
their solution state NMR studies”:
The paper that I found Facile synthesis of stable selenocystine peptides and their solution state NMR
studies was written by R. P. Gokula, K. Patel, S. K. Maurya and H. B. Singh and was published in 2019
in the Organic and Biomolecular Chemistry journal (I didn’t find page numbers, so instead of this I give
the paper’s DOI: 10.1039/C9OB01910C) The article describes a method for synthesizing selenocystine-
containing peptides and their characterization using solution-state nuclear magnetic resonance (NMR)
spectroscopy. The purpose of the work was to develop a simple and reliable approach for synthesizing
selenocystine peptides and to investigate their conformation using NMR spectroscopy.

The experimental session began the use of Fmoc-Sec(Xan)-OH as a precursor to incorporate selenium
in normal peptide sequences, and coupled the other remaining amino acid residues using DIC/HOBt
condition for 6 hours. The synthesized peptides were then purified and they had high purity, as
confirmed by RP-HPLC, and their identities were confirmed by HRMS. The authors also proposed a
mechanism for the formation of -Se-Se- bond from Fmoc-Sec(Xan)-OH, which involves the generation
of a cationic selenium species (selenenium ion) by TFA, followed by the formation of the corresponding
diselenide along with resonance stabilized xanthenyl oxonium ion. The authors did not observe any side
products such as dehydroalanine and acetoxy conjugate of selenopetide (Se-OAc) using their modified
methodology.

Through the investigation have used various 1D (1H, 13C, 77Se) and 2D NMR experiments, including
correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser
effect spectroscopy (NOESY), to determine the conformation of the peptides accurately. The NMR
spectra were analyzed to identify the selenium-proton interactions and to obtain information about the
conformation of the peptides. The authors reported that the NMR studies revealed an unique
conformation of the selenocystine peptides, which was attributed to the presence of selenium atoms in
the peptide backbone. The proton signals in all the peptides were assigned on the basis of peak position,
integration, and coupling constant. The 77Se NMR spectra of all the peptides showed a signal in the
range of 291-310 ppm, indicating the formation of -Se-Se- bond. The authors also studied the stability
of the peptides in different solvents.

The use of solution-state NMR spectroscopy allowed the researchers to gain detailed insights into the
conformation of the selenocystine peptides. The study demonstrated the potential of NMR spectroscopy
in providing structural information about peptides and small molecules, which is critical for
understanding their biological activities.

In conclusion, the authors successfully synthesized stable selenocystine peptides using a modified SPPS
method and characterized them using various techniques such as HRMS, RP-HPLC, and NMR
spectroscopy. The peptides obtained were of high purity and did not require further purification, and
provided high-quality NMR spectra. The authors proposed a mechanism for the formation of -Se-Se-
bond from Fmoc-Sec(Xan)-OH, and did not observe any side products using their modified
methodology. The characterization of the peptides using various NMR spectroscopy techniques
provides valuable insights into the structure of these peptides and their stability in different solvents.
This work could have implications in the synthesis and characterization of other selenocystine-
containing peptides and their potential applications in various fields.

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Copy of the original Experimental part from the manuscript:

In our modification, the reagents used to cleave the resin include
TFA/phenol/water/thioanisole/ethanedithiol28 in place of TFA/triisopropylsilane/water used by
Flemer.19 The reagents used in our protocol eliminate the formation any other byproduct along with the
peptide. The peptides obtained do not require further purification and provide high quality NMR spectra
(vide infra). We also report, for the first time, in-depth characterization of the peptide by 1D NMR (1H,
13C, 77Se) in addition to 2D (COSY, TOCSY) and high-resolution mass spectrometry (HRMS). The
purity of the peptides has been confirmed by RP-HPLC. Further the stability of the peptides in different
solvents has been probed. All the selenocystine tripeptides (1-10) with different acidic, basic and neutral
amino acids were synthesized by SPPS method using Rink amide AM resin as a solid support (Scheme
1, Fig. 1).13 Fmoc-Sec(Xan)-OH was used as a precursor to incorporate selenium in the normal peptide
sequences. The coupling reaction was accomplished under DIC/HOBt condition for 6 hours. The other
remaining amino acid residues were coupled using the same procedure. After the synthesis of the desired
length of the peptide, the resin was treated with the cleaving reagent (TFA with water, phenol,
thioanisole, ethanethiol) for 3 hours to get the final product. The peptide was precipitated after the
cleavage from resin with 10 mL of diethyl ether and kept at -5° C overnight. Finally, it was centrifuged
and lyophilized to get the peptide as a solid powder in 30-35 % yield with respect to the resin. It is worth
noting that the cleavage of resin using Xan as a protecting group is a very effective protocol for the in-
situ generation of -SeH and subsequent formation of -Se-Se- bond.

Fig. 1 Selenocystine peptides 1-10. The purity of these peptides was established using RP-HPLC. In
HPLC, by eluting with gradient of 0-1 % solvent A (acetonitrile containing 0.1% TFA) in solvent B
(water containing 0.1% T FA) over 30 min (flow rate of 1 ml/min), a single peak in HPLC
chromatograms was observed which indicated the high-level purity of the peptides (Fig. 2a). The
molecular ion peaks of the peptides with characteristic selenium isotopic pattern in HRMS confirmed
the identities of selenocystine peptides (Fig. 2b).

Fig. 2 (a) HPLC chromatogram and (b) HRMS of peptide 3. The mechanism for the formation -Se-Se-
bond from Fmoc Sec(Xan)-OH involves generation of a cationic selenium species (selenenium ion) by
TFA. This selenenium ion forms -SeH with ease and subsequently forms the corresponding diselenide
along with resonance stabilized xanthenyl oxonium ion (Fig. 3). To confirm whether the formation of
selenol involved the proton from TFA, attempts were made to record 1H NMR of the product using
deuterated TFA. When deuterated TFA was added to the protected amino acid (in deuterated
DMF/DMSO), an immediate precipitation was observed. Thus, it could not be established, whether the
hydrogen of selenol came from TFA or any other source. On the other hand, it is important to mention
that formation of two side products; dehydroalanine and acetoxy conjugate of selenopetide (Se-OAc),
has been reported during the synthesis of selenocystine peptides.19 We do not observe any such side
product(s) using our modified methodology as evident from the HPLC chromatograms (vide supra) and
NMR spectra of the peptides (vide infra).

Fig. 3 Mechanism for the formation of Se-Se bond from Fmoc Sec(Xan)-OH. All the NMR spectra
along with HRMS of the peptides are given in the supporting information (ESI). Proton signals in all of
these peptides were assigned on the basis of peak position, integration and coupling constant. There are
two main regions in 1H NMR spectrum; (1) chiral region, 3.5-5 ppm and (2) amide region, 7-9 ppm.
The 2D NMR spectra analyses have been used to correlate these two regions and identify all the signals
in a very simple and definite way. In 77Se NMR spectra, all the peptides showed a signal in the range
of 291- 310 ppm revealing the formation of -Se-Se- bond. A representative spectrum of peptide 3 is
discussed in detail. The COSY and TOCSY NMR spectrum of the representative peptide 3 is shown in

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Fig. 4. The three chiral center protons of amino acids appear at 3.64, 4.28 and 4.67 ppm, which are
assigned as H G and F-type protons respectively. These protons (G F -type) showed cross peak
correlation with doublets of secondary amides in COSY spectrum in the range of 8.26- 8.81 ppm
assigned as type B A protons (Fig. 4a). Terminal amine proton (C-type) at 8.04 ppm and two singlets
for primary amides (E D-type) appeared at 7.09 and 7.46 ppm respectively in the proton NMR spectrum.
In the TOCSY NMR spectrum shown in Fig. 4b and all the chemical shifts assigned in the spectra are
given in Table 1. There was only one peak in 77Se NMR spectrum of peptide 3 at 306.77 ppm, which
indicated that no monoselenide containing amino acid fragment was present in the peptide i.e, there is
complete conversion to the Se-Se bond (vide infra). On the other hand, in the 13C NMR there are three
signals in the amide carbonyl region and three signals in the range of 50-60 ppm suggesting the
formation of the tripeptide (Fig. S12).

Fig. 4 (a) 1H-1H COSY, (b) TOCSY NMR spectra of peptide 3 Regarding stability of the peptide in the
solid as well as in solution state, we found that peptides are not stable at room temperature for long
period of time and stable only below -5 °C.29 The stability in the solution was evaluated by 1H NMR
experiments [in DMSO Fig. (5a), D2O (5b) and CDCl3 (5c)] carried out for peptide 3. It was observed
that the peptide was extremely sensitive to CDCl3. However, it achieves remarkable stability when it is
dissolved in D2O and DMSO as validated by the solution state NMR studies.

It is noteworthy that broad signals were observed in the proton NMR spectra when Arginine (Arg) type
of basic amino acid residue was present in the peptide. This kind of broadness of signals may be due to
the presence of hydrogen bonding in peptides (Fig. S27). On the other hand, there was no signal from
the terminal amine and side chain acid group in peptide 8 (Fig. S32) and 9 (S37) indicating the possibility
of the proton to exchange with deuterium. Then, we went on to reduce the Se-Se bond of peptide 3 by
dithiothreitol (DTTred) to demonstrate the formation of -SeH in DMSO. The 77Se NMR peak observed
at 458.55 ppm for Fmoc Sec(Xan)-OH [Fig. 6(a)] shifted to 306.77 ppm in peptide 3 [Fig. 6(b)]. The
attempted reduction of 3 with only DTT in DMSO was unsuccessful and no change in peak position was
observed. Interestingly, when the reduction with DTT was carried out at pH 10, the peak shifted to
upfield region and appeared at -106.67 ppm [Fig. 6(c)].

Fig. 6 77Se NMR spectra (a) Fmoc-Sec(Xan)-OH, (*this study), (b) peptide 3 (c) a 1:4 mixture of 3 and
DTTred in DMSO with NaOH (pH 10). Here, R=Val-Sec-Leu To conclude, the synthesis of
selenocystine tripeptides by using Fmoc-Sec(Xan)-OH as a precursor amino acid is achieved. We have
successfully synthesized ten peptides using this method. The purity and stability are established in HPLC
and NMR studies. Further, we have shown the formation of RSeH by DTTred under basic conditions.
These small selenocystine peptides may play an important role in protein folding pathway, drug scaffold
and affect the enzymatic activity of different selenoproteins.