Peptide Name<\/td>

Tradename<\/td>

Amino Acid Count<\/td>

N-terminal Dipeptide Structure<\/td><\/tr>

Macimorelin<\/td>

Macrilen<\/td>

3<\/td>

Aib-Trp<\/td><\/tr>

Thymopentin<\/a><\/td>

Timunox<\/td>

5<\/td>

Arg-Lys<\/td><\/tr>

Semax<\/a><\/td>

Semax<\/td>

7<\/td>

Met-Glu<\/td><\/tr>

Saralasin<\/a><\/td>

Sarenin<\/td>

8<\/td>

Sar-Arg<\/td><\/tr>

Angiotensin II<\/a><\/td>

Giapreza<\/td>

8<\/td>

Asp-Arg<\/td><\/tr>

Icatibant<\/a><\/td>

Firazyr<\/td>

10<\/td>

D-Arg-Arg<\/td><\/tr>

Alloferon<\/td>

Allokine-alpha<\/td>

13<\/td>

His-Gly<\/td><\/tr>

P-15<\/td>

PepGen P-15<\/td>

15<\/td>

Gly-Thr<\/td><\/tr>

Difelikefalin<\/a><\/td>

Korsuva<\/td>

5<\/td>

Phe-Phe<\/td><\/tr><\/tbody><\/table>

Table 1. Examples of short peptides with a free N\u03b1 group approved by the FDA.<\/figcaption><\/figure>\n\n\n\n

Characteristics of Peptides Prone to DKP Formation<\/strong><\/h2>\n\n\n\n
\n
N-terminal Secondary Amino Acid (Proline, Sarcosine)<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\n
When Pro or Sar is positioned as the second residue at the N-terminus of a peptide, the cis configuration of the peptide bond between the second and first amino acid residues is strengthened due to the properties of these secondary amino acids. This stimulation leads to DKP formation in peptides with sequences such as Phe-Pro at the N-terminus, as seen in recombinant human growth hormone (rhGH). Substituting Pro with other amino acids significantly inhibits DKP formation. N-alkyl amino acids appear at the first amino acid position in some marketed peptide drugs, such as oritavancin (N-Me-D-Val) and saralasin (Sar-Arg). It is noteworthy that despite the potential high risk of DKP with Xaa-Pro as the peptide N-terminus, approved peptide drugs like nesiritide (32-amino acid disulfide-bonded peptide) and bivalirudin (20-amino acid linear peptide) have N-terminal sequences containing H-Ser-Pro and H-D-Phe-Pro, respectively.<\/p>\n\n\n\n
\n
C\u03b1,\u03b1-Dialkylated Amino Acids<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\n
C\u03b1,\u03b1-dialkylated amino acids synergistically contribute to peptide DKP formation. Although they do not promote cis amide bonds, they impose enhanced conformational restrictions on C\u03b1-C’ and N-C\u03b1 bonds, favoring the helical secondary conformation. This conformational preference brings N\u03b1 closer spatially to the unstable amide carbonyl, promoting DKP formation. While introducing C\u03b1,\u03b1-dialkylated amino acids may enhance susceptibility to DKP formation, some marketed peptide drugs like semaglutide<\/a> (H-His-Aib, GLP-1<\/a> analog, 31 amino acids) and macimorelin (H-Aib-D-Trp) contain Aib (2-aminoisobutyric acid, C\u03b1,\u03b1-dialkylated amino acid) residues near the N-terminus structural domain.<\/p>\n\n\n
\n
$\"Chemical$ <\/a>
Figure 2. Chemical structures of Semaglutide and Macimorelin.<\/figcaption><\/figure><\/div>\n\n\n
\n
N-terminal Glycine (Gly):<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\nThe correlation between DKP formation and N-terminal residues can be influenced by pKa, volume, amino acid configuration, and the conformational stability of DKP molecules. H-Gly-Pro-peptide is a highly sensitive N-terminal sequence that readily forms DKP due to the spatial promotion effect of Gly as the N-terminal residue and the cis configuration driven by Pro. H-Gly-Gly is another sequence highly sensitive to DKP formation. Marketed peptide drug peginesatide has a branched double N-terminal sequence Gly-Gly, but its N-terminal Gly is acetylated. Another marketed peptide drug terlipressin (cyclic peptide with disulfide bonds) has the N-terminal sequence H-Gly-Gly-Gly, and it is found to be susceptible to DKP formation during the formulation process. Peptide sequences with Gly as the N-terminal third residue also undergo DKP formation. Other marketed peptide drugs with free N-terminal Gly residues include <\/a>desmopressin<\/a> (H-Gly-Arg) and PepGen P-15 (H-Gly-Thr).<\/p>\n\n\n
\n
$\"Chemical$ <\/a>
Figure 3. Chemical structures of Peginesatide and Terlipressin.<\/figcaption><\/figure><\/div>\n\n\n
\n
N-terminal Histidine (His)<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\n
Peptides with the sequence H-His-Pro at the N-terminus are highly sensitive to DKP formation, attributed to the catalytic role of the imidazole group in the His side chain. In this structure, the His-Pro peptide bond adopts a cis conformation favorable for DKP, promoting nucleophilic attack by His-N\u03b1 on -Pro-Xaa3- main chain amide. The enhanced nucleophilicity of His-N\u03b1 is facilitated by the deprotonation of His’s basic imidazole group. This His-mediated catalytic process results in the cleavage of the Pro-Xaa3 peptide bond, ultimately forming the cyclic derivative His-Pro-DKP and complementary truncated peptide fragments.<\/p>\n\n\n
\n
$\"Possible$ <\/a>
Figure 4. Possible mechanism of DKP formation in peptides with N-terminal His-Pro.<\/figcaption><\/figure><\/div>\n\n\n
In terms of DKP formation, the H-His-Gly sequence appears more stable than H-His-Pro. Many approved GLP-1\/2 analog peptides, such as <\/a>exenatide<\/a>, dulaglutide<\/a>, lixisenatide<\/a>, and teduglutide<\/a>, feature N-terminal H-His-Gly sequences. Additionally, Alloferon also has an H-His-Gly N-terminal sequence.<\/p>\n\n\n\n
\n
Opposite Configuration of N-terminal Amino Acids<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\n
Peptide sequences composed of alternating L- and D-amino acids significantly affect DKP formation. In this case, the formed DKP molecule is stabilized thermodynamically due to the positioning of the two side chain groups, R1 and R2, on both sides of the DKP molecule plane, favoring DKP formation.<\/p>\n\n\n
\n
$\"DKP$ <\/a>
Figure 5. DKP formed by peptide with N-terminal L-D amino acid.<\/figcaption><\/figure><\/div>\n\n\n
N-terminal dipeptide sequences with opposite configurations of amino acid residues are found in some marketed peptide drugs, such as <\/a>icatibant<\/a> (H-D-Arg-Arg) and <\/a>bivalirudin<\/a> (H-D-Phe-Pro).<\/p>\n\n\n
\n
$\"Chemical$ <\/a>
Figure 6. Chemical structures of Icatibant and Bivalirudin.<\/figcaption><\/figure><\/div>\n\n\n
\n
Disulfide-Bridged Peptides<\/strong><\/li>\n<\/ul>\n\n\n\nDisulfide bonds can effectively suppress DKP formation by limiting the spatial position of the nucleophilic N\u03b1 group. This feature may explain the higher proportion of free N\u03b1 groups in marketed disulfide-bridged peptides, such as octreotide<\/a>, oxytocin<\/a>, and <\/a>lanreotide<\/a>. Compared to N-terminal unacetylated short linear peptides, they are less likely to form DKP.<\/p>\n\n\n\n
\n
N-terminal H-Xaa-Asp Peptides<\/strong><\/strong><\/li>\n<\/ul>\n\n\n\n
If Asp or Asn is located at the second position of the peptide N-terminus, the formation of aspartimide can promote peptide DKP formation. This aspartimide-induced DKP formation has been detected in many studies. Asn\/Asp at the second N-terminal position initially forms aspartimide, and the formed aspartimide peptide intermediate can undergo differential isomerization into the corresponding non-enantiomeric isomer aspartimide or hydrolyze into Asp- or \u03b2-Asp peptides. Additionally, the aspartimide at the second N-terminal position can undergo nucleophilic attack from N\u03b1 and convert into the corresponding cis-DKP compound. Under neutral to alkaline conditions at room temperature or elevated temperatures, cis-DKP can rapidly isomerize into trans-DKP. The sensitivity of aspartimide at the second N-terminal position to DKP is attributed to the increased electrophilicity of the acyl imide part and the easy racemization of the aspartimide intermediate. In this mode, the six-membered ring of DKP is not excluded from the peptide molecules, resulting in the presence of DKP isomers in the peptides.<\/p>\n\n\n
\n
$\"Possible$ <\/a>
Figure 7. Possible mechanism of DKP induced by H-Xaa-Asp.<\/figcaption><\/figure><\/div>\n\n\n
Plecanatide<\/a> is a marketed peptide drug with both linear and Asp as the second N-terminal residue (H-Asn-Asp). Thymosin \u03b11<\/a> has a Ser-Asp N-terminus, but its N\u03b1 group is acetylated.<\/p>\n\n\n\n
Summary<\/strong><\/strong><\/h2>\n\n\n\n
\n
In FDA-approved short linear peptide drugs, the majority of N\u03b1 groups are acetylated and capped.<\/li>\n\n\n\n
N\u03b1 acetylation and capping can have a significant impact on the physicochemical properties of the parent peptide molecule, such as secondary structure, isoelectric point (PI), solubility, etc., particularly for short peptides.<\/li>\n\n\n\n
Long peptide drugs tend to have a relatively higher proportion of free N\u03b1 groups.<\/li>\n\n\n\n
The occurrence of unacetylated N\u03b1 is more common in disulfide-bridged peptides and amide-cyclized peptides.<\/li>\n\n\n\n
Special attention should be given to the possibility of DKP formation for short peptides containing sensitive N-terminal dipeptide sequences.<\/li>\n<\/ul>\n","protected":false},"excerpt":{"rendered":"
The formation of 2,5-diketopiperazine (DKP) with a bicyclic structure is one of the most detrimental side reactions and degradation pathways affecting peptide synthesis. Despite its crucial role as a versatile building block in drug …<\/p>\n","protected":false},"author":1,"featured_media":1007,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":[],"categories":[75],"tags":[133],"_links":{"self":[{"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/posts\/996"}],"collection":[{"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/comments?post=996"}],"version-history":[{"count":4,"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/posts\/996\/revisions"}],"predecessor-version":[{"id":1010,"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/posts\/996\/revisions\/1010"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/media\/1007"}],"wp:attachment":[{"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/media?parent=996"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/categories?post=996"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.creative-peptides.com\/blog\/wp-json\/wp\/v2\/tags?post=996"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}