Color test for terminal prolyl residues in the solid-phase synthesis of peptides

Kaiser, E. T.; Bossinger, C.D.; Colescott, Robert L.; Olsen, Don B.

doi:10.1016/s0003-2670(01)93726-2

Cited by 71 publications

(43 citation statements)

References 7 publications

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“…Our earlier data showed that replacement of Pro-16 and Thr-17 by their D-isomer in peptide comprising the epitope ( 16 PTPT-GTQ 22 ) had no significant effect on the epitope recognition (15). Therefore, 11 peptides with the sequence of 15 TPTPT-GTQTPT 25 were produced. These compounds contain the PT-GTQ epitope, but the native C-and N-terminal flanking regions were systematically substituted by one to three D-amino acids ( Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…Fmoc groups were removed by 20% piperidine in DMF, or 2% piperidine and 2% diazabicyclo [5.4.0]undec-7-ene in DMF, respectively. The success of the coupling and deprotection steps was monitored by ninhydrin test (24) and͞or isatine assay (25). After the completion of the synthesis, the peptides were cleaved from the resin with trifluoroacetic acid containing 5% water.…”

Section: Methodsmentioning

confidence: 99%

“…[K 12 VTPTPTPTGTQTPT 25 -OH]-BSA conjugate as target antigen at 2 g͞ml in PBS (0.1 mol͞dm 3 , pH 7.3) containing 0.02% NaN 3 was added to 96-well Enzyplate Microtest plates (well capacity, 250 l; Propilén Kft, Pécs, Hungary) at 100 l per well. Plates were incubated overnight at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Partial d -amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide

Tugyi

Uray

Iván

et al. 2005

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

232

179

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The stability of an immunogen against enzymatic degradation is considered an important factor for the design of synthetic vaccines. For our studies, we have selected an epitope from the tandemrepeat unit of the high-molecular-weight MUC2 mucin glycoprotein, which can be underglycosylated in case of colon cancer. In this study, we prepared a MUC2 peptide containing the PTGTQ epitope of a MUC2 protein backbone-specific mAb 996 and its derivatives. In these peptides, the N-and C-terminal flanking regions were systematically substituted by up to three D-amino acids. Peptides prepared by solid-phase synthesis were tested for their mAb 996 binding in competitive ELISA experiments, and their stability was studied in serum and lysosomal preparation. Our data show that the epitope function of peptide 15 TPTPTGTQTPT 25 is retained even in the presence of two D-amino acid residues at its N-terminal flanking region and up to three at its C-terminal flanking region (tpTPTGTQtpt). Also, this partly D peptide shows high resistance against proteolytic degradation in diluted human serum and in lysosomal preparation. These findings suggest that, by appropriate combination of structural modifications (namely, D-amino acid substitution) in the flanks of an Ab epitope, it is feasible to construct a synthetic antigen with preserved recognition properties and high stability against enzymatic degradation. Peptides tPTPTGTQTpt and tpTPTGTQTpt derived from this study can be used for immunization experiments and as potential components of synthetic vaccines for tumor therapy.human serum ͉ rat liver lysosome preparation T he design of appropriate immunogen and its delivery, among other factors (e.g., schedule and route of immunization), are essential for the development of an effective peptide-based subunit vaccine. A major problem limiting the use of peptides is their instability, which is mainly due to the rapid degradation in vivo by proteases. There are different approached for protecting biologically active peptides from enzymatic decomposition, such as alteration of the amide bond (1), cyclization, conjugation to carrier molecule (2), and incorporation of nonproteinogenic amino acids such as ␤-Ala (3) or D-amino acids (4, 5).Sela and Zisman (as reviewed in ref. 6) clearly demonstrated that the presence of D-amino acid residues in linear and multichain polypeptide antigens could improve their stability to proteolysis and alter the biological half-life but also influence B or T cell immunrecognition. The D-amino acids were incorporated into epitope peptides mainly to investigate their effect on peptide-specific B cell and͞or T cell immunogenicity and͞or to analyze the fine specificity and cross-reactivity of Abs͞T cells induced by the all-L, all-D peptides (7). Correlation between the characteristics of immune response and extended biological half-life, as well as their stability to proteolysis, were described (8-10). However, most of the available literature reports on peptides in which all L-amino acid residues were replaced by their...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Partial d -amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide

Tugyi

Uray

Iván

et al. 2005

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

232

179

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“…Sulfated tyrosine was used for the synthesis of EpI and Tyr(tBu) was used for the synthesis of the unsulfated peptide ([Tyr 15 ]EpI). Peptide resin samples (ϳ5 mg) were removed after each coupling step for determination of residual free ␣-amino groups by the quantitative ninhydrin test (20), except for couplings to proline where a coupling efficiency of Ͼ99.5% was assumed where the result of a modification of the Isatin test (21,22) was satisfactory. Double couplings were used to achieve coupling efficiencies of Ͼ99.5%.…”

Section: Peptide Synthesismentioning

confidence: 99%

α-Conotoxin EpI, a Novel Sulfated Peptide from Conus episcopatusThat Selectively Targets Neuronal Nicotinic Acetylcholine Receptors

Loughnan

Bond

Atkins

et al. 1998

Journal of Biological Chemistry

109

100

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We have isolated and characterized ␣-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus. The peptide was classified as an ␣-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has homology to sequences of previously described ␣-conotoxins, particularly PnIA, PnIB, and ImI. However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry. Native EpI was shown to coelute with synthetic EpI. The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides.

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“…In this procedure, the ninhydrine test was performed to check for comple removal of Boc groups, while the isatin test was performed for terminal prolyl residue of Pro peptides [16]. The modification ratios (mmol g …”

Section: Solid-phase Synthesis Of the Cspsmentioning

confidence: 99%

Investigation of Novel Peptide Chiral Selectors Prepared by Solid-Phase Synthesis with a tert-Butoxycarbonyl Amino Acid

Ohyama

Oyamada

Kishikawa

et al. 2009

Chroma

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A new class of chiral stationary phases (CSP) with peptide chiral selectors was prepared by solid-phase synthesis with a tert-butoxycarbonyl-L-amino acid on silica. The type of amino acid that is favorable for this class of CSP is discussed. Using the CSP with the phenylalanine peptide selector, the effect of peptide length on the enantioselectivity was investigated in normal-phase mode. The applicability of the CSP with a phenylalanine peptide to chiral ligand-exchange chromatography was also examined.

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Color test for terminal prolyl residues in the solid-phase synthesis of peptides

Cited by 71 publications

References 7 publications

Partial d -amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide

Partial d -amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide

α-Conotoxin EpI, a Novel Sulfated Peptide from Conus episcopatusThat Selectively Targets Neuronal Nicotinic Acetylcholine Receptors

Investigation of Novel Peptide Chiral Selectors Prepared by Solid-Phase Synthesis with a tert-Butoxycarbonyl Amino Acid

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