Sequences with multiple modifications can be difficult to synthesize and purify, depending on the type and position of the modification. For instance, sequences requiring phosphorylation can be subject to side reactions with subsequently added amino acids, if the amino acids to be modified are close to the C-terminal end. To avoid side reactions during synthesis and improve separation efficiency during purification, GenScript has developed methods using mixed column resins for enhanced separation, as well as highly efficient coupling reagents for increased synthesis efficiency. The methods greatly reduce turnaround time and increase peptide yield.
There are two kinds of phosphopeptides: mono-phosphorylated and multi-phosphorylated. Multi-phosphorylated peptides can be very difficult to synthesize due to their high acidity and ability to reduce the rate of activation during Fmoc solid phase peptide synthesis. GenScript has developed two strategies for reliable phosphopeptide synthesis.
The first is direct step-wise solid phase synthesis of phosphopeptides using mono-benzylated phosphoamino acids and Fmoc chemistry. Two steps in this strategy can be tricky: the coupling of bzl-pAA and the coupling of the next AA. To complete these steps, GenScript applies microwave technology to couple bzl-pAA. This strategy makes phosphopeptide synthesis as simple as conventional solid phase synthesis.
GenScript also employs a global phosphorylation method, which involves the postsynthetic phosphorylation of unprotected hydroxyl groups on a solid support. After synthesis, all of the residues requiring phosphorylation can be phosphorylated in liquid phase in one step. This method has the advantage of avoiding the difficulties of synthesizing bzl-pAA directly.
Sequence: Thr- ... ... ... -pTyr- ... ... ... -pTyr-pTyr- ... ... ... -Lys
Sequence: XXX…XX{PSer}XX…XX{PSer}XXXX Length: 30 AA; M.W.: 3.4 kDa Purity: ˃85%
Sequence: XX…{PSER}XX…XX{PSER}X…X{PTHR}XX Length: 47 AA; M.W.: 5.6 kDa Purity: ˃90%
Sequence: XX…{PTYR}X…X{PTYR}…{PTYR}…XX Length: 25 AA; M.W.: 3.3 kDa Purity: ˃85%
Sequence: XXXX{pSER}X{pSER}XX…XX{pSER}XXXXX Length: 23 AA; M.W.: 2.6 kDa Purity: ˃90%
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The synthesis of peptides containing multiple disulfide bonds can be challenging. This is due to the fact that multiple disulfide bonds can form randomly within any peptide that contains multiple cysteines. Few companies around the world offer multi-disulfide bond peptide synthesis. GenScript, however, has developed two methods to synthesize peptides containing multiple disulfide bonds.
The first of these methods is a stepwise oxidation method utilizing different protecting groups to protect each cysteine pair during the Fmoc SPPS synthesis process. Different deprotecting conditions are used to deprotect each protecting group. These are carefully selected based on the specific needs of the client's project. With subsequent stepwise oxidizations of the disulfide bonds during the liquid phase, we can accurately synthesize peptides with more than two disulfide bonds with great accuracy.
The second method requires single-step oxidation of all of the cysteines of the peptide in liquid phase, to form a peptide containing native multiple disulfide bonds. Zymohydrolysis is subsequently employed to orient the position of the disulfide bonds.
Sequence: H - Cys - Asn - Cys - ... ... ... - Cys - ... ... ... - Cys - ... ... ... - NH2 Disulfide bridge: 1 -11, 3- 15
Sequence: Disulfide bridge: 16-15, 8-13 Length: 18 AA; M.W.: 2.2 kDa Purity: >98%
Sequence: Disulfide bridge: 1-6, 2-10, 5-13) Length: 14 AA; M.W.: 1.5 kDa Purity: >90%
Mono-, di-, and tri-methylated peptides representing histone tails are useful for studying protein-protein interactions between histones and the enzymes that modify them, or between histones and the enzymes that bind to their modified structures. Like multi-phosphorylated peptides, multi-methylated peptides can be difficult to synthesize and purify. Below view examples of multi-methlyated sequences that GenScript has synthesized. In addition, GenScript can combine methylation with any other modification such as acetylation or fluorescent labeling.