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A new two-step deprotection/cleavage procedure for t-butoxycarbonyl (Boc) based solid phase peptide synthesis is reported. First the protective groups are removed from 4-(oxymethyl)-phenylacetamidomethyl (PAM) resin attached peptide with the weak hard acid, trimethylsilyl bromide-thioanisole/trifluoroacetic acid (TFA). In the second step, the peptide is cleaved from the resin with a stronger hard acid such as trimethylsilyl trifluoromethanesulfonate in TFA or with HF. The method is also shown to deformylate Nin-formyltryptophan moiety efficiently. The usefulness of this procedure for practical solid phase peptide synthesis is demonstrated by comparison with other deprotection methods in the synthesis of urotensin II and human endothelin.

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.

The growth factor receptor-binding protein-Src homology 2 (Grb2-SH2) domain plays an important role in the oncogenic Ras signal transduction pathway, which involves cell proliferation and differentiation. Therefore, the Grb2-SH2 domain has been chosen as our target for development of potential antiproliferative agents. Herein, we report the study of the inhibitory effects of small nonphosphorylated peptide analogs interacting with the Grb2-SH2 domain protein by surface plasmon resonance (SPR) technology. A set of 8 related peptide analogs were synthesized, purified, and characterized. Their inhibitory effects on Grb2-SH2 were evaluated by the SPR technology developed with the BIACORE X instrument. The lead peptide, Fmoc-Glu-Tyr-Aib-Asn-NH2 (Fmoc-E-Y-Aib-N; Fmoc: 9-fluorenylmethyoxycarbonyl; Aib=alpha-amino isobutyric acid) inhibited Grb2-SH2 domain function with an IC50 value of 8.7 microM. A molecular modeling study of the lead peptide indicated that the glutamate in the Fmoc peptide is ideally positioned to form a strong salt bridge to Arg 67 in the Grb2-SH2 domain, using both its backbone carbonyl and its acidic group. Residue Glu 89 in Grb2-SH2 flips inward to fill the binding site and partially replace the phosphate group as a hydrogen-bond acceptor. Results of these studies provide important information for further development of potent nonphosphorylated peptide inhibitors of the Grb2-SH2 domain.

One of the critical intracellular signal transduction pathways involves the binding of the Grb2 SH2 domain to the phosphotyrosine (pTyr) motifs on growth factor receptors, such as epidermal growth factor receptor (EGFR) and erbB2, leading to downstream activation of the oncogenic Ras signaling pathway. Therefore, the Grb2 SH2 domain has been chosen as our target for the development of potential anticancer agents. As a continuation of our earlier work, herein we report the design and synthesis of new peptide analogs, and their inhibitory effect on the Grb2 SH2 domain using surface plasmon resonance (SPR) technology. These novel agents do not contain phosphotyrosine or phosphotyrosine mimics. Binding interactions between these peptides and the Grb2 SH2 domain were measured and analyzed using a BIAcore X instrument, which provides detailed information on the real-time detection of the binding interaction. The results of this study should provide important information for the further development of peptides or peptidomimetics with high affinity for the Grb2 SH2 domain.

A nonphosphorylated disulfide-bridged peptide, cyclo(Cys-Glu1-Leu-Tyr-Glu-Asn-Val-Gly-Met-Tyr9-Cys)-amide (termed G1) has been identified, by phage library, that binds to the Grb2-SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2-SH2 domain association (IC50 of 15.5 microM) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2-SH2 domain in a pTyr-independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side-chains of amino acids Glu1, Tyr3, Glu4, Asn5, and Met8. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2-SH2 domain in a beta-turn-like conformation. Replacement of Tyr3 or Asn5 with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y-X-N consensus sequence similar to that found in natural pTyr-containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu1, i.e. the amino acid N-terminal to Y3, remarkably reduces the binding affinity. The position of the Glu1 side-chain is confirmed to provide a complementary role for pTyr3, as demonstrated by the low micromolar inhibitory activity (IC50 = 1.02 microM) of the nonphosphorylated peptide 11, G1(Gla1), in which Glu1 was replaced by gamma-carboxy-glutamic acid (Gla).

The structures of the cyclic hexapeptide cyclo(-Gly-Tyr-Val-Pro-Met-Leu-) (1) and its phosphotyrosyl (pTyr) derivative cyclo[-Gly-Tyr(PO3H2)-Val-Pro-Met-Leu-] (2), designed as constrained models of a sequence that interacts with the src homology 2 (SH2) region of the p85 subunit of phosphatidylinositol-3-OH kinase (PI-3 kinase), were studied in methanol/water solutions by 500 MHz nmr spectroscopy. Compound 1 was found to exist as a 2:1 mixture of isomers about the Val-Pro bond (trans and cis prolyl) between 292-330 K in 75% CD3O(D,H)/(D,H)2O solutions. A third species of undetermined structure (ca. 5%) was also observed. Compound 2, a model of phosphorylated peptide ligand that binds to the PI-3 kinase SH2 domain, exhibited similar conformational isomerism. When either compound was dissolved in pure solvent [i.e., 100% CD3O(H,D) or (H,D)2O] the ratio of cis to trans isomers was ca 1:1. A battery of one- and two-dimensional nmr experiments at different temperatures and solvent compositions allowed a complete assignment of both the cis and trans forms of 1 and indicated the trans compound to be the major isomer. The spectral properties of the phophorylated derivative 2 paralleled those of 1, indicating like conformations for the two compounds. Analysis of rotating frame Overhauser spectroscopy data, coupling constants, amide proton temperature dependence, and amide proton exchange rates generated a set of constraints that were employed in energy minimization and molecular dynamics calculations using the CHARMM force field. The trans isomer exists with the tyrosine and C-terminal Tyr(+3) (Met) residues at opposite corners of the 18-membered ring separated by a distance of 16-18 A, in contrast with the cis isomer where the side chains of these residues are much closer in space (7-14 A). It was previously shown that the pTyr and the third amino acid C-terminal to this residue are the critical recognition elements for pTyr-peptide binding to the PI-3 kinase SH2 domain. Such cyclic structures may offer appropriate scaffolding for positioning important amino acid side chains of pTyr-containing peptides as a means of increasing their binding affinities to SH2 domains, and in turn provide a conceptual approach toward the design of SH2 domain directed peptidomimetics.

Endothelin converting enzyme (ECE) is essential for generation of the biological effects of endothelin-1 (ET-1) from a precursor, big endothelin-1 (Big ET-1). We synthesized four analogs of human Big ET-1[16–38], substituted with single d-amino acids at P1, P2, P1′and P2′ positions. ECE activity was determined using an ET-1 specific radioimmunoassay system. None of the d-amino acid containing Big ET-1 analogs were apparently cleaved by ECE, however, one of the synthetic peptides, [d-Val22]Big ET-1[16–38], strongly inhibited the ECE activity. Furthermore, when this d-Val22 containing peptide was preadministrated to rat striatum, it was found to inhibit the dopamine release induced by Big ET-1. This result suggests that the d-Val22 containing peptide inhibits the ECE activity in vivo. The d-Val22 containing inhibitor offers hope of developing more potent and highly specific ECE inhibitors of therapeutic significance.

Many biologically important molecules including oncogenes, growth factor receptors, transcription factors, immune cell receptors, protein kinases and cytoskeletal proteins are specifically and reversibly phosphorylated in response to physiological stimuli as well as during differentiation and normal eukaryotic cell cycle progression (1–3). Phosphorylation occurs primarily on the hydroxy 1 group of serine, threonine or tyrosine residues in proteins. While other residues, including histidine, lysine and glutamic acid may be phosphorylated, these phosphoamino acids are chemically less stable and their roles in physiological processes are relatively poorly understood. Protein phosphorylation is accomplished by protein kinases, which catalyze the transfer of phosphate from a nucleotide triphosphate, usually ATP, to the substrate protein. Protein kinases traditionally are grouped by amino acid homology as protein tyrosine kinases or as protein serine/threonine kinases, although a few dual specificity tyrosine/serine/threonine kinases have been described (4,5), and recently, an evolutionarily separate group of serine/threonine kinases related to the phospholipid kinases was discovered (6). Many tyrosine-specific kinases are cell surface receptors or are associated with the plasma membrane as illustrated by the epidermal growth factor, insulin and platelet-derived growth factor receptors (7,8). Serine/threonine kinases more frequently are involved in intracellular signaling and may be linked in cascades with receptor tyrosine kinases to transmit signals from the extracellular environment to the cell nucleus.

We synthesized, separated into enantiomers, and tested for the HIV-1 reverse transcriptase inhibitory activity a group of analogs of dimethyl-1-(1-piperidynyl)cyclobuta[b][1]benzothiophene-2,2a(7bH)-dicarboxylate (NSC-380292). Absolute configurations of the enantiomers were determined based on absolute X-ray structures and analysis of CD spectra. Within pairs of enantiomers the (R,R)-enantiomer was always much more potent HIV-1 reverse transcriptase inhibitor.

N-Nitrosodibutylamine (DBN) and fully deuterated N-nitrosodimethylamine (DMN-d6) have been successfully tritiated using the Wilzbach procedure. Specific activities, after purification and transfer to aqueous solution for biochemical studies, were 180 and 14 mCi/mmole, respectively. 3HN-Nitrosodibutylamine (3H-DBN) was rather sensitive to autoradiolysis, although the unlabelled derivative proved unusually resistant to base-catalyzed exchange with deuterium oxide. No decrease in deuterium content was observed during the preparation of 3HN-nitrosodimethyl-amine-d6 (3H-DMN-d6), demonstrating that tracer quantities of tritium could be incorporated without significantly lowering the deuterium isotopic purity.

Nine previously uncharacterized membrane glycoproteins from normal rat liver have been analyzed by amino acid sequencing from two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after transblotting to Immobilon-P membranes. Three of these components show altered levels of expression in liver tumors. A single electroblotted polyacrylamide gel yielded sufficient quantities of these glycoproteins for amino acid sequencing and the N-terminal structure could be determined for four of them. The remaining five glycoproteins of interest were not sequenceable in this manner, presumably because they had blocked N-termini. Prior to electrophoresis, two enrichment methods were applied to the crude liver membrane preparations: affinity chromatography with concanavalin A to isolate the plasma membrane glycoproteins and then fast protein liquid chromatography on Superose 12 to obtain components having a specific range of molecular weights. These materials were next subjected to 2-D PAGE using pH 4-6 carrier ampholytes in the first dimension and 7.5% sodium dodecyl sulfate gels in the second. The proteins were then electroblotted to Immobilon-P membranes and located by staining with Coomassie Brilliant Blue R-250. Our results demonstrate that N-terminal sequencing (gas-phase) can be achieved on polypeptides obtained from approximately 250 micrograms of total glycoproteins applied to a single 2-D gel.

The one-step synthesis of 14C-methyl(acetoxymethyl)nitrosamine (14C-DMN-OAc) of high specific activity is described. The product was shown to decompose by hydrolysis, as reported previously for the unlabelled material, and by self-radiolysis, a process which occurred even in methylene chloride solution at high specific activities. Both modes of decomposition led to disappearance of label from the solution, at least partly by sorption to the glass storage vessel. Thin-layer chromatography proved less suitable for resolving the decomposition products than radio-gas chromatography. Labelled carbon atoms were found to the extent of only 1% in the formaldehyde produced on esterase hydrolysis of 14C-DMN-OAc, showing that scrambling during the synthesis was not extensive. Storage of aqueous solutions for biological work was best accomplished in frozen aqueous solutions of pH 5.5.

The lanthanide induced chemical shifts and coupling constants have been determined for ring A protons of steroids having a keto, hydroxyl, or acetate functional group at C-3. The assignments were made with the aid of suitably deuterated compounds and should prove useful in steroidal stereochemical and conformational studies.

Zur Umwandlung von (I) in (XI) wird (I) mit Lithiumaluminiumhydrid in Tetrahydrofuran zum Triol (II) reduziert, das Triol acetyliert, das Diacetat (III) mit Phosphoroxychlorid/Pyridin (1 Tag bei Zimmertemp.) zum Gemisch der 313, 18-Diacetoxy-A7,9(11),2U(22)-lanostatrien (IV) (30% Ausbeute) und 313a, 18-Diacetoxy-A7,9(1 1),20(21)-1anostatrien (V) (37%) dehydratisiert, das Gemisch mit Platin zum A790 U-Die? (VI) hydriert, (VI) mit Chromtrioxid/Essigsaure zum A3-7,l 1-Diketon oxidiert und (VII) mit -Zink/Essigsaure zum gesattigten Diketon (VIII) hydriert.