A general method for the synthesis of thioester resin linkers for use in the solid phase synthesis of peptide-α-thioacids

Canne, Lynne E.; Walker, Sharon M.; Kent, Stephen B. H.

doi:10.1016/0040-4039(95)00037-d

Cited by 81 publications

(33 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…To obtain a C-terminal thioacid group on Gly 58 of peptide 1-58 of sPLA 2 , peptide synthesis was started on a 0.15-mmol scale on aminomethyl resin to which a preformed HF-labile Glythioester linker was coupled (15). The usual procedure for His side-chain deprotection (DNP removal) by 2-mercaptoethanol before HF treatment of the peptide resin could not be used because of the vulnerability of the thioester-resin linker.…”

Section: Resultsmentioning

confidence: 99%

Total chemical synthesis of enzymatically active human type II secretory phospholipase A ₂

Hackeng

Mounier

Bon

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Human group II secretory phospholipase A 2 (sPLA 2 ) is an enzyme found in the ␣ granules of platelets and at inf lammatory sites. Although its physiological function is unclear, sPLA 2 can inhibit blood coagulation reactions independent of its lipolytic action. To study the molecular basis of PLA 2 activities, we developed a total chemical synthesis of sPLA 2 by chemical ligation of large unprotected peptides. The synthetic segments PLA 2 -(1-58)-␣ COSCH 2 COOH and PLA 2 -(59-124) were prepared by stepwise solid-phase peptide synthesis and ligated to yield a peptide bond between Gly 58 and Cys 59 . The 124-residue polypeptide product (mass: 13,920 ؎ 2 Da) was folded to yield one major product (mass: 13,905 ؎ 1 Da), the loss of 15 ؎ 3 Da ref lecting the formation of seven disulfide bonds. Circular dichroism studies of synthetic sPLA 2 showed ␣-helix, ␤-structure, and random coil contents consistent with those found in the crystal structure of sPLA 2 . Synthetic sPLA 2 had k cat and K m values identical to those of recombinant sPLA 2 for hydrolysis of 1,2-bis(heptanoylthio)-phosphatidylcholine. Synthetic sPLA 2 , like recombinant sPLA 2 , inhibited thrombin generation from prothrombinase complex (factors Xa, V, II, Ca 2؉ , and phospholipids). In the absence of phospholipids, both synthetic and recombinant sPLA 2 inhibited by 70% prothrombin activation by factors Xa, Va, and Ca 2؉ . Thus, synthetic sPLA 2 is a phospholipidindependent anticoagulant like recombinant or natural sPLA 2 . This study demonstrates that chemical synthesis of sPLA 2 yields a fully active native-like enzyme and offers a straightforward tool to provide sPLA 2 analogs for structureactivity studies of anticoagulant, lipolytic, or inf lammatory activities.

show abstract

Section: Resultsmentioning

confidence: 99%

Total chemical synthesis of enzymatically active human type II secretory phospholipase A ₂

Hackeng

Mounier

Bon

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…C-terminal segments were prepared on -OCH2Pam resins (ABI, Foster City, CA). N-terminal segments were prepared on ␣-thiocarboxylate resin (16). Standard HF cleavage protocols were employed following N-terminal Boc removal and drying of the * The costs of publication of this article were defrayed in part by the payment of page charges.…”

Section: Methodsmentioning

confidence: 99%

Chemically Synthesized SDF-1α Analogue, N33A, Is a Potent Chemotactic Agent for CXCR4/Fusin/LESTR-expressing Human Leukocytes

Ueda

Siani

Gong

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

Stromal cell-derived factor (SDF) 1 is a potent chemoattractant for leukocytes through activation of the receptor CXCR4/Fusin/LESTR, which is a fusion co-factor for the entry of T lymphocytotropic human immunodeficiency virus type 1 (HIV-1). This CXCR4-mediated HIV-1 fusion can be inhibited by SDF-1. Because of its importance in the study of immunity and AIDS, large scale production of SDF-1 is desirable. In addition to recombinant technology, chemical synthesis provides means by which biologically active proteins can be produced not only in large quantity but also with a variety of designed modifications. In this study, we investigated the binding and function of an SDF-1␣ analogue, N33A, synthesized by a newly developed native chemical ligation approach. Radioiodinated N33A showed high affinity binding to human monocytes, T lymphocytes, as well as neutrophils, and competed equally well with native recombinant SDF-1␣ for binding sites on leukocytes. N33A also showed equally potent chemoattractant activity as native recombinant SDF-1␣ for human leukocytes. Further study with CXCR4/Fusin/LESTR transfected HEK 293 cells showed that N33A binds and induces directional migration of these cells in vitro. These results demonstrate that the chemically synthesized SDF-1␣ analogue, N33A, which can be produced rapidly in large quantity, possesses the same capacity as native SDF-1␣ to activate CXCR4-expressing cells and will provide a valuable agent for research on the host immune response and AIDS.

show abstract

“…One limitation to this approach has been the efficient synthesis of C-terminal thioester peptides. Traditionally, these peptides have been obtained in two steps through selective alkylation of thioacid peptides (4) generated from optimized SPPS (10) by using an HF-labile thioacid linker (11,12). However, the need for an individual-solution synthesis of a tert-butoxycarbonyl (Boc)-amino-thioacid linker for each C-terminal amino acid desired has limited the general application of this approach (12).…”

mentioning

confidence: 99%

“…Furthermore, deprotection and cleavage of the polypeptide chain from the resin by anhydrous HF yields the corresponding C-terminal thioacid (COSH) that requires alkylation and purification to obtain the activated thioester (COSR). As a result, only a limited set of ligation pairs has been examined by using Ala (4), Leu (13), or Gly (4, 7) as the C-terminal thioester residue from individual syntheses of amino acid thioacid linkers (12). Other approaches can be used to generate C-terminal thioester peptides directly (5,14,15), for example Asn, but also suffer from the requirement for synthesizing an amino acid-thioester linker of each individual amino acid.…”

mentioning

confidence: 99%

“…Traditionally, these peptides have been obtained in two steps through selective alkylation of thioacid peptides (4) generated from optimized SPPS (10) by using an HF-labile thioacid linker (11,12). However, the need for an individual-solution synthesis of a tert-butoxycarbonyl (Boc)-amino-thioacid linker for each C-terminal amino acid desired has limited the general application of this approach (12). Furthermore, deprotection and cleavage of the polypeptide chain from the resin by anhydrous HF yields the corresponding C-terminal thioacid (COSH) that requires alkylation and purification to obtain the activated thioester (COSR).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology

Hackeng

Griffin

Dawson

1999

Proc. Natl. Acad. Sci. U.S.A.

658

674

View full text Add to dashboard Cite

show abstract

A general method for the synthesis of thioester resin linkers for use in the solid phase synthesis of peptide-α-thioacids

Cited by 81 publications

References 10 publications

Total chemical synthesis of enzymatically active human type II secretory phospholipase A ₂

Total chemical synthesis of enzymatically active human type II secretory phospholipase A ₂

Chemically Synthesized SDF-1α Analogue, N33A, Is a Potent Chemotactic Agent for CXCR4/Fusin/LESTR-expressing Human Leukocytes

Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology

Contact Info

Product

Resources

About

A general method for the synthesis of thioester resin linkers for use in the solid phase synthesis of peptide-α-thioacids

Cited by 81 publications

References 10 publications

Total chemical synthesis of enzymatically active human type II secretory phospholipase A 2

Total chemical synthesis of enzymatically active human type II secretory phospholipase A 2

Chemically Synthesized SDF-1α Analogue, N33A, Is a Potent Chemotactic Agent for CXCR4/Fusin/LESTR-expressing Human Leukocytes

Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology

Contact Info

Product

Resources

About

Total chemical synthesis of enzymatically active human type II secretory phospholipase A ₂

Total chemical synthesis of enzymatically active human type II secretory phospholipase A ₂