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Peptide Synthesis Case Studies

GenScript's long history of top-quality peptide synthesis has given us an opportunity to examine both common and rare peptide synthesis problems and come up with solutions. Sometimes, we come up with more than one. Below, you will find details of three specific cases exemplifying the competency and innovation of GenScript methods implemented within the FlexPeptideTM peptide synthesis platform. We have found ways to circumvent the problems plaguing the synthesis of multi-cysteine peptides, multi-phosphorylated peptides, and the extremely long peptides.

  • Case Study Ⅰ
  • Case Study Ⅱ
  • Case Study Ⅲ

1. Multi-cystine peptides

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 peptides that contain enough cysteines. Few companies around the world offer multi-cysteine peptide synthesis services for containing two disulphide bonds and none of them can synthesize peptides with more. GenScript, however, has developed two separate ways around this obstacle, making peptide synthesis containing multiple disulphide bonds both possible and affordable.

The first of these is stepwise oxidation 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 disulphide bonds during the liquid phase, we can accurately synthesize peptides with more than two disulfide bonds with great accuracy.

The second method works through folding and oxidization. The key point is to take the peptide at its folding condition in liquid phase and oxidize all the cysteines to form a peptide containing native multiple disulfide bonds in one step and then employ zymohydrolysis to orient the position of disulfide bonds.

Case study: Peptide A
Sequence: H - Cys - Asn - Cys - ... ... ... - Cys - ... ... ... - Cys - ... ... ... - NH2
Disulfide bridge: 1 - 11, 3 - 15
HPLC results:

MS results:

2. Multi-phosphorylated peptides

There are two kinds of phospho-peptides: mono-AA-phosphorylated and multi-AA-phosphorylated. The multi-phosphorylated group can be very difficult to synthesize due to its high acidity and ability to reduce the rate of activation during Fmoc SPPS. 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. This strategy involves two problems: the coupling of bzl-pAA and the coupling of the next AA. To solve them, GenScript applies microwave technology to couple bzl-pAA. This strategy makes phosphopeptide synthesis as simple as general peptide synthesis.

The second is the global phosphorylation method, which involves the postsynthetic phosphorylation of unprotected hydroxyl groups on a solid support. After synthesis, all of the phosphorylated positions in any such peptide can be phosphorylated in liquid phase in one step. This method has the advantage of avoiding the difficulties of synthesizing bzl-pAA directly.

Case study: Peptide B
Sequence: Thr- ... ... ... -pTyr- ... ... ... -pTyr-pTyr- ... ... ... -Lys
HPLC results:

MS results:

3. Extremely long peptides

The FlexPeptideTM system employs GenScrip's proprietary ligation method to achieve high yields even for very long peptides. This is accomplished by first synthesizing several shorter sequences and then ligating them together in high efficiency.

Case study: Peptide C
Sequence: Ala-Glu...-Lys-....-Lys-Lys-....-Glu-Ala-...-Ala-Ala
Length: 149 AA; M.W.: 15971.51
HPLC results:

MS results: