Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis

Wöhr, Torsten; Wahl, Franck Olivier; Nefzi, Adel; Rohwedder, Barbara; Sato, T.; Sun, Xicheng; Mutter, Manfred

doi:10.1021/ja961509q

Cited by 333 publications

(230 citation statements)

References 38 publications

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“…Fmoc group removal was with a mixture of 2% (v/v) piperidine and 2% (v/v) 1,8-diazabicyclo(5,4,0)undec-7-ene in dimethylformamide. To minimize aspartimide formation, Asp in the scrambled sequence was coupled as the Asp-Ser pseudo-proline (22). When the sequence was complete, the resin was washed with methanol and peroxide-free ether, and dried under nitrogen before the addition of dichloromethane (4 ml) and dimethylpropylene urea (0.5 ml).…”

Section: Methodsmentioning

confidence: 99%

Disruption of the c-JUN-JNK Complex by a Cell-permeable Peptide Containing the c-JUN δ Domain Induces Apoptosis and Affects a Distinct Set of Interleukin-1-induced Inflammatory Genes

Holzberg

Knight

Dittrich–Breiholz

et al. 2003

Journal of Biological Chemistry

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The transcription factor activator protein 1 (AP-1) 1 was one of the first mammalian transcription factors to be identified, but its physiological functions are still being unraveled. AP-1 is involved in cellular proliferation, transformation, survival, cell death, and the immune response (1, 2). AP-1 converts extracellular signals into changes of the expression of target genes, and AP-1 binding sites are found in a large number of genes. AP-1 is not a single protein, but a homo-or heterodimer composed of members of the JUN, FOS, ATF, and other protein families (1, 2). With the exception of cell cycle regulatory genes, because of the structural and regulatory complexity of AP-1, the knowledge of AP-1 target genes mediating AP-1 functions is far from complete (1, 2). c-JUN is one of the best characterized components of AP-1 (3). Genetic evidence suggests that c-JUN is essential for development and proliferation (1-3). Activity of c-JUN is controlled at multiple levels, first by changes in gene transcription, mRNA turnover, and protein stability, second by interaction with other transcription factors, and third by phosphorylation of its NH 2 -terminal transactivation domain (1-6).The phosphorylation sites required for inducible c-JUN activation have been mapped to serines 63 and 67. A family of 10 highly homologous serine/threonine protein kinases derived from three genes by alternative splicing has been identified that specifically phosphorylate these residues in c-JUN and has therefore been named c-JUN NH 2 -terminal protein kinases (JNK) 1-3 (7, 8). So far, no other protein kinases have been identified that phosphorylate the NH 2 terminus of c-JUN (6, 7). Importantly, early work demonstrated that JNK not only phosphorylate c-JUN, but also bind to a region called the ␦ domain, which is located immediately NH 2 -terminal of the c-JUN transactivation domain (6 -10). Binding of JNK to c-JUN is a remarkable example of a high affinity signaling complex (6 -10). Further studies of the complex in intact cells showed that it does not require the JNK catalytic activity or the presence of the phospho-acceptor sites in c-JUN (11). Presumably, the c-JUN-JNK interaction serves two purposes, first it provides the specificity of JNK for c-JUN, and second it helps to increase the local concentration of JNK at gene promoters that bind c-JUN, thereby enhancing c-JUN-mediated transcription (4).Like the c-JUN protein, JNK have been implicated in numerous biological roles in response to growth factors, stress, and inflammatory cytokines, implying that JNK may mediate their gene regulatory effects mainly through c-JUN (12-14). However, for several reasons this is unlikely. First, JNK phosphorylate other transcription factor substrates, such as ATF-2 (15) and ELK-1 (16); second, individual JNK isoforms display different affinities for c-JUN in vitro (8, 9); and third, the phenotypes of c-JUN and JNK knockout mice show no obvious overlapping phenotype (13,17,18). Therefore, one of the most intriguing questions regarding the c-JUN-JNK i...

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Section: Methodsmentioning

confidence: 99%

Disruption of the c-JUN-JNK Complex by a Cell-permeable Peptide Containing the c-JUN δ Domain Induces Apoptosis and Affects a Distinct Set of Interleukin-1-induced Inflammatory Genes

Holzberg

Knight

Dittrich–Breiholz

et al. 2003

Journal of Biological Chemistry

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“…Thus, pseudoproline dipeptides are prepared by reaction of Fmoc-AA-Ser or Fmoc-AA-Thr with 2,2-dimethoxypropane. 261 Most of the other backbone protectors are introduced by reductive amination of the aldehyde of the protecting group with the amine of the corresponding amino acid, followed by either α-amino protection or dipeptide formation. 262,258,259 6.3.…”

Section: Introduction Of the Protecting Groupsmentioning

confidence: 99%

“…Protecting groups removed by acid -Pseudoprolines (ΨPro). The most used are dimethyloxazolidines (Ψ Me,Me pro) because of their major acid lability (removed by TFA within minutes) 261 Pseudoproline derivatives have been extensively applied to the synthesis of difficult peptides. 257,,263 However, they are limited to Ser and Thr.…”

Section: Introduction Of the Protecting Groupsmentioning

confidence: 99%

Amino Acid-Protecting Groups

2009

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“…Acetic anhydride was then used to cap unreacted amino groups. To suppress aggregation, pseudo-proline building blocks (24,25) were used as structure-disrupting agents at different stages of the solid phase synthesis of such a long peptide. Thus, residues 11-12, 39 -40, and 45-46 were introduced as Fmoc-Ala-Ser( …”

mentioning

confidence: 99%

A Cyclic Peptide Mimicking the Third Intracellular Loop of the V2 Vasopressin Receptor Inhibits Signaling through Its Interaction with Receptor Dimer and G Protein

Granier

Terrillon

Pascal

et al. 2004

Journal of Biological Chemistry

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In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5-O-(3-thio)triphosphate. This peptide also affected the specific binding of [ 3 H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V 2 receptor (V 2 -R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V 2 -R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5-O-(3-thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V 2 -R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs.G protein-coupled receptors (GPCRs), 1 a superfamily of seven transmembrane domain proteins, act as molecular switches that are able to transmit signals from extracellular stimuli to G proteins present on the cytoplasmic side of the plasma membrane. These receptors promote ligand dependent GDP/GTP exchange in the G␣ subunit of the heterotrimeric G protein leading to the activation of effectors. It is well established that intracellular loops of these receptors are important structural elements for their coupling to the heterotrimeric G proteins (1). Yet the precise understanding of the mechanisms by which GPCRs regulate G protein activity remains limited because of the general difficulties in characterizing the structure of integral membrane proteins. One approach considered to circumvent these problems is the use of synthetic peptides mimicking receptor cytosolic domains to probe the events responsible for G protein activation. For the rat vasopressin V 1a receptor, a peptide mimicking its second intracellular loop was found to specifically inhibit vasopressin-specific binding (2). In other cases, short peptides mimicking GPCR third intracellular loops were found to act on the coupling between the receptor and the G protein (3-8). Expressed as a minigene, the ␣ 1b -adrenergic i3 loop was shown to inhibit Gq signaling (9). In ...

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Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis

Cited by 333 publications

References 38 publications

Disruption of the c-JUN-JNK Complex by a Cell-permeable Peptide Containing the c-JUN δ Domain Induces Apoptosis and Affects a Distinct Set of Interleukin-1-induced Inflammatory Genes

Disruption of the c-JUN-JNK Complex by a Cell-permeable Peptide Containing the c-JUN δ Domain Induces Apoptosis and Affects a Distinct Set of Interleukin-1-induced Inflammatory Genes

Amino Acid-Protecting Groups

A Cyclic Peptide Mimicking the Third Intracellular Loop of the V2 Vasopressin Receptor Inhibits Signaling through Its Interaction with Receptor Dimer and G Protein

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