Fmoc-Pro-Pro-OH

General procedure: A mixture of Boc-l-phenyl alanine-ASUDester (Table 1, entry 6) (3.0g, 6.7mmol), l-phenyl alanine methyl ester hydrochloride (1.46g, 6.7mmol), and triethylamine (1.71g, 16.9mmol) in dry THF (15mL) was stirred overnight at room temperature (15h). After completion of the reaction, the reaction mixture was filtered (remove TEA·HCl salt) and washed with THF (5mL). The filtrate was concentrated under vacuum to obtain an oily crude. The oily crude was dissolved in ethyl acetate, washed with 1.0percent NaHCOThe active substance Lixisenatide is a polypeptide amide composed of 44 amino acids; acetate functions as counterion. In the one-letter code, the amino acid sequence of Lixisenatide is as follows: H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-l-E-W-L-K-N-G-G-P-S-S- G-A-P-P-S-K-K-K-K-K-K-N H2 The peptide chain was constructed by means of linear solid-phase synthesis, starting from the C-terminus, Lys-44. The method of synthesis is Fmoc solid-phase peptide synthesis, in which a Rink amide resin was used in order to obtain a peptide amide. The reactions were carried out in DMF at room temperature. Between the reactions, washing was carried out repeatedly, mostly with DMF, with one of the middle washing steps being carried out with isopropanol. The synthesis of Lixisenatide on the polymeric support can be broken down into the following steps: ' Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin ' Capping of the un reacted amino group ' Cleavage of the temporary protecting group Fmoc ' Coupling of the further Fmoc-amino acids or Fmoc-dipeptides ' Capping of the un reacted amino group ' Final Fmoc cleavage ' Cleavage of Lixisenatide from the resin and simultaneous removal of the side chain protecting groups The synthesis cycle is illustrated in Figure 1. 1.1 Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin Before the synthesis began, the Rink amide resin was swollen in DMF. The swelling was carried out for 2-15 h. Subsequently, the temporary protecting group Fmoc was cleaved from the Rink amide resin using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. The coupling of the first Fmoc-amino acid, Fmoc-Lys(Boc)-OFI, was carried out in an excess of 2.4 eq, in order to load the resin. HOBt hydrate, HBTU and DIPEA served as coupling reagents. The coupling time was 60-120 min. In order to completely load the Rink resin with Fmoc-Lys(Boc)-OFI, a further loading was carried out with the coupling reagents FIOBt hydrate and DIC. The coupling time was 6-18 h. The mixture was stirred while step 1.1 was carried out. The capping was subsequently carried out. 1.2 Capping of the unreacted amino group The consequence of incomplete loading of the resin is that as yet unreacted amino groups are found on the resin. These were inactivated, and hence made unavailable for further coupling, by adding a mixture of acetic anhydride/DIPEA/DMF (10:5:85). The capping mixture remained on the resin for 20 minutes while stirring. The remaining free amino group is acylated. Subsequently, the resin was washed repeatedly with DMF and once with isopropanol. A capping method according to the invention at least at 5 positions of a Lixisenatide synthesis is described in examples 4 and 5. 1.3. Cleavage of the temporary protecting group Fmoc The temporary protecting group Fmoc was cleaved using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. 1.4 Coupling of the further Fmoc-amino acids or Fmoc-dipeptides The next Fmoc-amino acid was coupled to the deprotected amino group on the resin. The coupling was carried out in DMF at different equivalents. The coupling times were between 2 h and 18 h. HOBt/DIC, and also HBTU/DIPEA, were used as coupling reagents. The following derivatives were used as Fmoc-amino acids: ' Fmoc-Lys(Boc)-OFI ' Fmoc-Ser(tBu)-OFI ' Fmoc-Pro-OFI ' Fmoc-Ala-OH x FI2O ' Fmoc-Gly-OH ' Fmoc-Asn(Trt)-OH ' Fmoc-Leu-OH ' Fmoc-Trp(Boc)-OH ' Fmoc-Glu(OtBu)-OFI x H2O ' Fmoc-lle-OFI ' Fmoc-Phe-OFI ' Fmoc-Arg(Pbf)-OFI ' Fmoc-Val-OH ' Fmoc-Met-OH ' Fmoc-Gln(Trt)-OFI ' Fmoc-Asp(OtBu)-OH ' Fmoc-Thr(tBu)-OH ' Fmoc-His(Trt)-OH Alternatively, it was also possible to use Fmoc-dipeptides (method according to the invention): · The active substance Lixisenatide is a polypeptide amide composed of 44 amino acids; acetate functions as counterion. In the one-letter code, the amino acid sequence of Lixisenatide is as follows: H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-l-E-W-L-K-N-G-G-P-S-S- G-A-P-P-S-K-K-K-K-K-K-N H2 The peptide chain was constructed by means of linear solid-phase synthesis, starting from the C-terminus, Lys-44. The method of synthesis is Fmoc solid-phase peptide synthesis, in which a Rink amide resin was used in order to obtain a peptide amide. The reactions were carried out in DMF at room temperature. Between the reactions, washing was carried out repeatedly, mostly with DMF, with one of the middle washing steps being carried out with isopropanol. The synthesis of Lixisenatide on the polymeric support can be broken down into the following steps: ' Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin ' Capping of the un reacted amino group ' Cleavage of the temporary protecting group Fmoc ' Coupling of the further Fmoc-amino acids or Fmoc-dipeptides ' Capping of the un reacted amino group ' Final Fmoc cleavage ' Cleavage of Lixisenatide from the resin and simultaneous removal of the side chain protecting groups The synthesis cycle is illustrated in Figure 1. 1.1 Coupling of the first Fmoc-amino acid (Fmoc-Lys(Boc)-OH) to Rink resin Before the synthesis began, the Rink amide resin was swollen in DMF. The swelling was carried out for 2-15 h. Subsequently, the temporary protecting group Fmoc was cleaved from the Rink amide resin using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. The coupling of the first Fmoc-amino acid, Fmoc-Lys(Boc)-OFI, was carried out in an excess of 2.4 eq, in order to load the resin. HOBt hydrate, HBTU and DIPEA served as coupling reagents. The coupling time was 60-120 min. In order to completely load the Rink resin with Fmoc-Lys(Boc)-OFI, a further loading was carried out with the coupling reagents FIOBt hydrate and DIC. The coupling time was 6-18 h. The mixture was stirred while step 1.1 was carried out. The capping was subsequently carried out. 1.2 Capping of the unreacted amino group The consequence of incomplete loading of the resin is that as yet unreacted amino groups are found on the resin. These were inactivated, and hence made unavailable for further coupling, by adding a mixture of acetic anhydride/DIPEA/DMF (10:5:85). The capping mixture remained on the resin for 20 minutes while stirring. The remaining free amino group is acylated. Subsequently, the resin was washed repeatedly with DMF and once with isopropanol. A capping method according to the invention at least at 5 positions of a Lixisenatide synthesis is described in examples 4 and 5. 1.3. Cleavage of the temporary protecting group Fmoc The temporary protecting group Fmoc was cleaved using 25% piperidine in DMF. This cleavage was undertaken twice; cleavage time of 5 minutes and 20 minutes. Following the Fmoc cleavage, the resin was washed repeatedly with DMF and once with isopropanol. 1.4 Coupling of the further Fmoc-amino acids or Fmoc-dipeptides The next Fmoc-amino acid was coupled to the deprotected amino group on the resin. The coupling was carried out in DMF at different equivalents. The coupling times were between 2 h and 18 h. HOBt/DIC, and also HBTU/DIPEA, were used as coupling reagents. The following derivatives were used as Fmoc-amino acids: ' Fmoc-Lys(Boc)-OFI ' Fmoc-Ser(tBu)-OFI ' Fmoc-Pro-OFI ' Fmoc-Ala-OH x FI2O ' Fmoc-Gly-OH ' Fmoc-Asn(Trt)-OH ' Fmoc-Leu-OH ' Fmoc-Trp(Boc)-OH ' Fmoc-Glu(OtBu)-OFI x H2O ' Fmoc-lle-OFI ' Fmoc-Phe-OFI ' Fmoc-Arg(Pbf)-OFI ' Fmoc-Val-OH ' Fmoc-Met-OH ' Fmoc-Gln(Trt)-OFI ' Fmoc-Asp(OtBu)-OH ' Fmoc-Thr(tBu)-OH ' Fmoc-His(Trt)-OH Alternatively, it was also possible to use Fmoc-dipeptides (method according to the invention): · General procedure: A mixture of Boc-l-phenyl alanine-ASUDester (Table 1, entry 6) (3.0g, 6.7mmol), l-phenyl alanine methyl ester hydrochloride (1.46g, 6.7mmol), and triethylamine (1.71g, 16.9mmol) in dry THF (15mL) was stirred overnight at room temperature (15h). After completion of the reaction, the reaction mixture was filtered (remove TEA·HCl salt) and washed with THF (5mL). The filtrate was concentrated under vacuum to obtain an oily crude. The oily crude was dissolved in ethyl acetate, washed with 1.0% NaHCO3 solution and water. The ethyl acetate layer was dried over Na2SO4 and distilled under vacuum to give an oily crude product.

Molecular Weight:434.5
XLogP3:3.4
Hydrogen Bond Donor Count:1
Hydrogen Bond Acceptor Count:5
Rotatable Bond Count:5
Exact Mass:434.18417193
Monoisotopic Mass:434.18417193
Topological Polar Surface Area:87.2
Heavy Atom Count:32
Complexity:720
Defined Atom Stereocenter Count:2
Covalently-Bonded Unit Count:1
Compound Is Canonicalized:Yes