2.2. Degree by Which Different Equivalents of Anhydride Are Coupled to Peptides
We selected different equivalents of anhydride for the coupling reaction with the peptide, and the equivalence ratios of the anhydride to the peptide were 10:1, 20:1, 40:1, and 60:1, in which the anhydride was in excess. The reacted system was post-treated and subjected to liquid chromatography to generate a chromatogram, as shown in
Figure 1.
As shown in
Figure 1, the number of peaks is identical for the different equivalence ratios of the liquid chromatograms even though the equivalents of anhydride were different; the area of each peak changes as the anhydride equivalence increases. Each peak was collected and identified by LC-MS. The first peak has a molecular weight of 1105, which is the raw material peak of the peptide. The second peak has a molecular weight of 1221 and is the product peak of a peptide molecule coupled to an anhydride. The molecular weights of the third peak and the fourth peak are 1337, which are generated by a peptide molecule coupled to two anhydride molecules, and only the reaction sites are different. The fifth peak has a molecular weight of 1453 and is generated by a peptide molecule coupled to three anhydride molecules.
Table 1,
Table 2,
Table 3 and
Table 4 show the changes in the peak time and peak area of each peak in the reactions between the peptide and anhydride with different equivalents. The first peak corresponds to the raw polypeptide, and the area of the raw polypeptide peak decreases with increasing anhydride concentration. When the anhydride equivalent was increased to 40 and 60, the areas of the raw peptide peaks varied slightly. The peak area was taken into the standard curve of the peptide, and the conversion rate of the raw material for each equivalence ratio could be obtained, as shown in
Table 5. When the ratio of acid anhydride to peptide reached 60:1, the conversion rate of the raw material no longer increased significantly, and reached equilibrium, with a maximum conversion rate of 87%.
As the amount of anhydride increased, the peak area of the feedstock peptide decreased until an equilibrium was reached. The peak corresponding to the anhydride of the peptide product first increased and then decreased when 40 equivalents of anhydride were added, but the peak area of the peptide product was the highest at this time, indicating that the highest yield of the substance was generated. The peak was observed to increase and then decrease, except for the peptide product coupled to an anhydride. This mainly occurs because the other reaction sites on the product become coupled to more anhydrides when the amount of anhydride continues to increase, and anhydrides are transformed into other coupled products. The peak areas of all other products increased with increasing anhydride equivalents.
2.3. Determination of the Peptide Reaction Site
The sequence of the reactive active sites was progressively determined, and the products were subjected to NMR spectroscopy. For ease of illustration and analysis, the relevant carbon and hydrogen atoms of the histidine in the raw peptide are labeled here, as shown in
Figure 2 below.
Figure 3,
Figure 4,
Figure 5,
Figure 6 and
Figure 7, respectively, show the
1H NMR spectra of raw peptides,
13C NMR spectrum,
1H-
13C HSQC NMR spectrum,
1H-
1H COSY NMR spectrum, and
1H-
13C HMBC NMR spectrum.
In the chemical analysis of the imidazole ring, the hydrogen spectrum (
Figure 3) and the
1H-
13C HSQC spectrum (
Figure 4) provided this paper with the key information to understand its structural composition in a detailed and precise manner. This paper was able to identify the precise data of the proton signals, i.e., δH values of 8.61 (1H, s, H-2) and 7.27 (1H, s, H-4), respectively. Similarly, the carbon signals were also clearly recorded at 134.8 ppm (C-2) and 117.6 ppm (C-4), respectively. These signals reveal the presence of carbon–carbon and hydrogen–hydrogen bonds in the imidazole molecule.
In the
1H-
1H COSY spectra (
Figure 5), a clear correlation signal between H-7 (4.17 ppm) and H-6 (3.23/3.12 ppm) can be observed.
In the detailed analysis of the
1H-
13C HMBC spectra (
Figure 6), the researchers observed a series of distinct and consistent chemical signals. Among these signals, the significant correlation between the H-4 region and the C-2 and C-5 (128.7 ppm) molecules suggests that there may be a specific structural or functional link between them. Further, the H-7 region also exhibited a clear correlation with the neighboring C-5/C-8 (167.7 ppm) region, whereas the H-6 region showed a stable correlation with C-7 (51.8 ppm) as well as the C-8/C-5/C-4 fragment. These multiple correlations confirm the existence of the aforementioned fragments.
The chemical shift value in the carbonyl or stacked alkene region is typically defined as 150 ppm, but it commonly exceeds 165 ppm. When the chemical shift value surpasses 200 ppm, it usually indicates the presence of aldehydes and ketones. In the analyzed carbon spectral data, eight specific carbonyl carbon signals can be clearly identified in this paper, which are 173.4, 172.3, 172.2, 171.6, 171.4, 171.3, 171.2, and 167.7. These signals indicate precise matches to the corresponding structural units in the raw material.
Based on the information provided, the significant chemical shifts in this polypeptide chain are identified in this paper, as shown in
Figure 8.
After analyzing the NMR spectra of the raw peptide, this paper proceeds to examine the product following the reaction, which is a peptide coupling product linked to a diethylene glycol anhydride product. It was originally analyzed by LC-MS (liquid chromatography–mass spectrometry) that the terminal amino group of the peptide was coupled to a diethylene glycol anhydride molecule. Because this reaction site is the first to initiate the coupling of the peptide, it plays a crucial role in the selectivity of the coupling reaction. In this paper, the structure of the product was further verified through NMR hydrogen spectroscopy.
For ease of analysis, this paper labels the relevant positional behavior of the carbon atoms at the coupling site, as illustrated in
Figure 9. The labeling of the histidine-associated carbon–hydrogen atoms in the coupling product is as described above and shown in
Figure 2.
The hydrogen spectrum (
Figure 10), along with the
1H-
13C HSQC spectrum (
Figure 11), can determine that the diethylene glycol anhydride has reacted with -NH
2. The
1H-
13C HMBC spectra show significant correlation signals between H-3 and C-2/C-4, and H-2 and C-1/C-3. The -NH
2 of the peptide is involved in the reaction, forming the amide active hydrogen proton -NH with a chemical shift of 8.13 ppm. The proton signals in the imidazole ring were δH 8.68 (1H, s, H-2) and 7.22 (1H, s, H-4), while the carbon signals were at 134.1 ppm (C-2) and 116.8 ppm (C-4), respectively.
In the HMBC spectrum (
Figure 12), the active hydrogen proton is observed to have a clear correlation signal with C-1, and the peak splits into a doublet peak, suggesting a linkage to the -CH hypomethyl group in the peptide. In addition, H-4 shows a significant correlation signal with C-2/C-5 (130.2 ppm) [
29].
In the
1H-
1H COSY spectra (
Figure 13), a clear correlation signal between H-7 (4.62 ppm) and H-9 (8.13 ppm) can be observed.
In the detailed analysis of carbon spectra (
Figure 14), a series of distinctive signals for carbonyl carbon were observed in this paper. These signals appear at 10 specific positions: 173.4, 172.4, 172.1, 171.6, 171.5, 171.3, 171.2, 171.1, 170.0, and 169.2. Through an in-depth study and comparison of these signals, it can be confirmed that they match the chemical structures present in the products.
Comparison with the raw material revealed that the amino group at position 9 in the product disappeared and an amide was formed, which appeared as an amide–active hydrogen proton signal at 8.13 ppm and cleaved into a d-peak; these results suggested that the group was linked to the -CH hypromethyl group.
Compared with that of the raw material, the proton signal of the 7-position hypomethyl group changed from 4.17 ppm to 4.62 ppm (a change of 0.45 ppm), suggesting that -NH2 may have reacted by connecting the electron-withdrawing group, resulting in enhanced deshielding and a large chemical shift.
In the HHCOSY spectrum obtained for the product, the -NH at position 9 is significantly correlated with H-7, indicating that -NH2 is involved in the reaction.
In addition to performing NMR spectroscopic analyses of the peptide raw material and the peptide products coupled to an anhydride molecule, we determined the peptide sequence of all coupled peptide products, and the following results were obtained.
The chemical shift values of important structures in peptide products coupled with an anhydride molecule are shown in
Figure 15.
In addition to performing NMR spectroscopic analyses of the peptide raw material and the peptide products coupled to an anhydride molecule, we determined the peptide sequence of all coupled peptide products; the specific results are shown in
Table 6.
Table 6 shows that the first reaction site of the whole peptide is the terminal amino group of the peptide. After the terminal amino group is coupled to an anhydride, the other sites continue to react with the anhydride via a ring-opening reaction. The next site of reaction is the amino group on the lysine side chain of the peptide at the tetra- and hepta-positions, which are almost identical. As the anhydride equivalent increases, all three reaction sites on the peptide couple to the anhydride molecule.