Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn †

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60^c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204 , 1159–1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation. © Munksgaard 1995.     

Resin immobilized synthetic peptides used to characterize phosphorylation and antigenic properties of insulin receptor autophosphorylation domains

To develop a common strategy in peptide design for kinase assay, antibody production and affinity purification, we investigated phosphorylation and antigenic properties of peptides immobilized on an aminated polyacrylic resin (Expansin ?) corresponding to autophosphorylation domains of the insulin receptor tyrosine kinase. Immobilized peptides (1143–1155) and peptide (1314–1330), designated pl 151 and p1322, respectively, were good substrates for the insulin receptor with K_m, of 0.74 and 0.78 mM. By contrast, peptide (952–963), designated p960, was poorly phosphorylated. p1151 showed distinctive behaviour as a substrate, displaying a higher basal phosphorylation, a leftward shift of the insulin dose-response curve (ED₅₀= 0.7 ng mL- ¹ insulin compared to 20 ng mL ¹ for other substrates) and an inhibition by 90% of receptor autophosphorylation (ID₅₀So = 0.5 mM). Similar substrate behaviour was observed with another tyrosine kinase, the pp60^c-src. Antibodies against P1151 and p1322 have comparable reactivity in ELISA, but the antibody against p960 was poor. While purified immunoglobulins (IgG) against both p1151 and p1322 were inhibitors of receptor autophosphorylation and kinase, in immunoprecipitation the IgG against p1151 mainly interacted with the phosphorylated receptor and that against p1322 with non-phosphorylated forms. Functional mapping of the receptor with oligoclonal 1322-antibody revealed inhibition of phosphate transfer to exogenous substrate poly(Glu,Tyr) (4:l) but not towards immobilized p1151. These data provide further support for the distinctive features of endogenous phosphorylation domain 1151. We conclude that immobilized peptides on polyacrylic resin offer a major new potential for use in kinase assays, immunization, immunoabsorbent techniques and purification of well defined oligoclonal antibodies.     

Efficient Fmoc/solid-phase peptide synthesis of O-phosphotyrosyl-containing peptides and their use as phosphatase substrates

A general synthetic method for the efficient preparation of Tyr(P) -containing peptides is described by the use of Fmoc-Tyr(PO₃¹Bu₂) -OH in Fmoc/solid-phase synthesis followed by simultaneous cleavage of the peptide from the resin and peptide deprotection by acidolytic treatment. The applicability of this approach is demonstrated by the synthesis of H-Ser-Ser-Ser-Tyr(P) -Tyr(P) -OH.TFA and the synthesis of the phosphorylated forms of the two physiological peptides, angiotensin II and neurotensin 8–13. In addition, the three phosphorylated peptides were used as substrates in the study of the local specificity determinants of T-cell protein tyrosine phosphatase. In a competition assay using ³²P-radiolabeled [Tyr(P)]⁴-angiotensin II, both un-labeled synthetic [Tyr(P)]⁴-angiotensin II and Ser-Ser-Ser-Tyr(P) -Tyr(P) reduced the release of ³²P and indicated that they efficiently competed as substrates for the phosphatase. Conversely, [Tyr(P)]⁴-neurotensin 8–13 was ineffective as a competitive substrate and indicated that this particular Tyr(P) -containing peptide sequence was not recognized by the enzyme. The marked difference in the recognition of Asp-Arg-Val-Tyr(P) -Ile-His-Pro-Phe and Arg-Arg-Pro-Tyr(P) -Ile-Leu is consistent with the presence of an acidic residue in the -3 position relative to the Tyr(P) residue.     

Common and differential recognition of structural features in synthetic peptides by the catalytic domain and the Src-Homology 2 (SH2) domain of pp60c-src

The relative efficiencies of the catalytic domain of the src-family kinase pp60^c-src in phosphorylating four peptide substrates including (i) src-optimal peptide (AEEEIYGEFEAKKKK), (ii) “-YEEI-peptide” (KKTHQEEEEPQYEEIPIYL), (iii) cdc2(6–20) (KVEKIGEGTY GVVYK), (iv) src-autophosphorylation site peptide (ADFGLARLIEDNEY TARG) and the relative efficiencies of its SH2 domain in binding the phosphorylated forms of these peptide substrates were compared. The results show that the src-optimal peptide, “-YEEI-peptide, ” cdc2(6–20) peptide were phosphorylated by the catalytic domain with high efficiency and that the phosphorylated form of all three peptides could bind the SH2 domain of the kinase, confirming the hypothesis proposed by Songyang and co-workers that the catalytic domain of pp60^c-src phosphorylates sites which are recognized by its own SH2 domain (Songyang et al. (1995) Nature 373, 536–539). The four peptides were phosphorylated by the kinase with relative efficiencies in the order of Src-optimal peptide > “-YEEI-peptide” > cdc2(6–20) ? src-autophosphorylation site peptide. However, the Tyr(P)-Src-optimal peptide and [pY]. ¹⁵cdc2(6–20) bound to the SH2 domain of the kinase with an affinity at least an order of magnitude lower than that of the tight-binding peptide, “-pYEEI-peptide.” Thus, our study suggests that the catalytic and SH2 domains of pp60^c-src recognize overlapping but not identical determinants in the local structure around the tyrosine phosphorylation site of the substrate peptides.     

Cyclic peptide substrates of pp60c-src

To study the effects of constrained conformation and amino acid sequence on their kinetic parameters, a series of cyclic peptides were synthesized and each was tested as both a substrate and an inhibitor of pp60^c-src, the product of the src proto-oncogene. The amino acid sequences were derived from Glu-Leu-Pro-Tyr-Ala-Gly and from the autophosphorylation site of pp60^c-src (Ile-Glu-Asp-Asn-Glu-Tyr-Ala-Ala-Arg-Gln-Gly). Linear precursor peptides were synthesized by SPPS on aminomethylated polystyrene resin using the Fmoc-tert-butyl protection scheme with 4-hydroxymethyl-3-methoxyphenoxyacetic acid as the linkage agent. The peptides were cleaved from the support with 1% TFA in dichloromethane with the N-terminal Fmoc and the side-chain protecting groups in place. Removal of the Fmoc group with diethylamine and cyclization with BOP afforded cyclic peptides in 55–78% yield. Side-chain deprotection and further purification gave the final products in 25–48% yields based on their linear precursors. Based on the activities of the linear analogues, cyclization had little effect on the binding (K_i and K_m) and rate of phosphorylation (V_max) of cyclo(Glu-Leu-Pro-Tyr-Ala-Gly) and cyclo(Ile-Glu-Asp-Am-Glu-Tyr-Ala-Ala-Arg-Gln). A series of cyclic decapeptides that contained the dipeptide d -Phe-Pro inserted in various positions in the autophosphorylation sequence showed marked differences in K_i, K_m and V_max. Compared to the well characterized linear substrate Val-5 angiotensin II, the D-Phe-Pro-containing cyclic peptides have higher V_max values but differ little in K_m, with values in the millimolar range.     

Steric constraints in the recognition of peptide substrates for the epidermal growth factor receptor kinase

Epidermal growth factor (EGF) stimulates cellular mitogenesis by binding to and activating its membrane-associated receptor. An important component of signal transduction by the activated receptor is the stimulation of an intrinsic tyrosyl residue-specific protein kinase, which selectively phosphorylates tyrosyl residues in the cytoplasmic tail of the receptor and in other cytoplasmic substrates. A recent study utilizing tyrsub, a new high affinity synthetic peptide substrate for the EGF receptor kinase, provided evidence that in peptide substrate binding, the tyrosyl residue plays the central role in recognition, with residues surrounding the tyrosyl residue contributing to stabilization of docking [Guyer et al. (1994) Arch. Biochem. Biophys. 312 , 573–578], A large body of previous work had identified acidic residues near the site of phosphorylation as most important for binding; therefore, other residues in tyrsub appeared to be promising sites for locating spectroscopic reporter groups. Since tyrsub has neutral residues –4 and + 4 residues from the site of phosphorylation, we prepared two analogs of tyrsub, in each of which one of those residues was substituted with Cys. These cystyrsubs were found to be effectively phosphorylated by EGF receptor prepared from A431 cells, on stimulation with EGF, with high affinities [K^m(app)= 40–50 μM]. Modification of the cystyrsubs with iodoacetamide had no deleterious effect on the ability of the peptide to be phosphorylated by the EGF receptor kinase, while the labeling by 5-iodoacetimidofluorescein completely abolished the productive interaction between the peptide and the EGF receptor. This unexpected failure of the fluorescently labeled peptides to be phosphorylated does, however, provide information on steric limitations to recognition of substrates by the EGF receptor kinase. ©Munksgaard 1996.     

Binding of calcium to synthetic peptides containing γ-carboxyglutamic acid

The Ca²⁺ binding properties of various γ-carboxyglutamic acid (gla)-containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca²⁺-selective electrode and titration calorimetric techniques. The shortest peptides, FL(gla)(gla)LR, DF(gla)(gla)AK, and the oxidized form of the cyclic hexapeptide CI(gla)(gla)IC, each of which contains one pair of gla residues, have a weak affinity for Ca²⁺, with some peptides probably involved in intermolecular bridging of the Ca²⁺. The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g-atom of Ca²⁺ interacts with 2 mol of peptide (n= 0.5) with a K_d value of 1.6 mM. A second g-atom of Ca²⁺ interacts with 2 mol of this same peptide (n= 0.5) and is characterized by a K_d of 8.8 mM. A longer peptide containing this same sequence, viz. L(gla)R(gla)CI(gla)(gla)IC, possesses two binding sites (n= 2.0)for Ca²⁺ of K_d=16.1 mM, as well as a tighter site (n= 1), Of K_d= 0.4 mM. An increase in stoichiometry of tight binding sites as the peptide is elongated is observed from binding data obtained on a 38-residue peptide that possesses all nine of the gla-residues of protein C in their proper sequence positions. The strongest Ca²⁺ binding sites (n= 3–4) possess an average K_d of 0.4 mM, followed by another class of sites (n= 5–10, average K_d= 1.5–3.0 mM). The affinity and stoichiometry of these stronger sites mimic those observed for binding of Ca²⁺ to the gla region of prothrombin fragment 1. By selective [¹³C]-labeling of the essential gla 16 residue of the 38-mer peptide, we demonstrate that this particular gla residue participates as a donor for a high-affinity Ca²⁺ site. These similarities in binding properties between the synthetic peptide containing the entire gla domain and the gla domain as it exists in proteins and protein fragments indicate suitably designed peptides of this type may constitute appropriate models for investigation of the binding of Ca²⁺ to intact gla-containing proteins.     

Tetrapeptide tyrosine kinase inhibitors

A convenient enantioselective synthesis of p-hydroxymethyl-L-phenylalaninc was developed which produces a 4/1 ratio of L/D enantiomers resulting from a chiral phase-transfer-catalyzed alkylation. This amino acid was coupled into the p561ck tyrosine kinase substrate Ac-Leu-Pro-Tyr-Ala-NHCH₃ as a replacement for Tyr and can subsequently be elaborated into a variety of potential tyrosine kinase inhibitor designs of general structure Ac-Leu-Pro-AA-Ala-NHCH₃, wherein AA is an unnatural amino acid. The contaminating D enan-tiomer was readily removed after coupling to L-Ala-NHCH₃ of this sequence. The utility of the p-hydroxymethyl functionality in an efficient divergent synthetic strategy leading to various inhibitor designs is illustrated with the synthesis of Ac-Leu-Pro-AA-Ala-NHCH3, wherein AA is p-(R,S-hydroxyphosphonomethyl)-L-phenylalanine.     

Analgesic,receptor binding,and peripheral opioid activities of synthetic dermorphin-dynorphin hybrid peptides

The effects of substituting the enkephalin moiety of dynorphin with the dermorphin sequence were studied on the receptor preference, analgesic, and peripheral opioid potencies by using synthetic dermorphin-dynorphin hybrid peptides as the probe. Replacement of the enkephalin moiety of dynorphin with the dermorphin or dermorphin^1–5 sequence caused a remarkable increase in analgesic potency, and a 3-6 fold increase in potency of binding against [³H]-dihydromorphine. The potency of receptor binding against [³H]-EKC was also increased by incorporation of the whole dermorphin sequence into the dynorphin molecule. In the presence of NaCl (100mM), the effect of enhancing binding against [³H]-EKC due to dermorphin substitution disappeared, suggesting the contribution of opioid μreceptor. Peripheral opioid activities assayed by various smooth muscle preparations showed that dermorphin incorporation caused a decrease in the potency of inhibition of the contractions of the guinea pig ileum and the rabbit vas deferens, no change in potency on the mouse vas deferens, and a marked increase in the inhibition of the rat vas deferens. Among the peripheral opioid activities only that assayed with the rat vas deferens appears to correlate approximately with the analgesic and the receptor binding activities. Judging from the relative potencies obtained from all assays, it is evident that the N-terminal dermorphin moiety, but not the C-terminal dynorphin fragment, dominates the opioid activity and receptor preference of the hybrid peptide.     

Substrate phosphorylation capacities of the major tyrosine protein kinase from the human promyelocytic cell line,HL-60

The major tyrosine protein kinase, HPK40, isolated from HL-60, the preparation of which is described elsewhere (Ernould, A.P., Ferry, G., Barret, J.M., Genton, A. and Boutin, J.A., Eur. J. Biochem., 214 , 503–514), was investigated as to its specificity on a number of peptides and proteins. It was found that HPK40 can phosphorylate histones (except histone H4), casein, acid-treated enolase, actin and tubulin but not calmodulin. Phosphorylation specificity of HPK40 was investigated using over a hundred peptidic structures. HPK40 is not related to the ‘src’ family and does not phosphorylate efficiently either the tetrapeptide NEYT derived from the pp60src autophosphorylation domain or the corresponding peptide RRsrc, RRLIEDNEYTARG. VALYDYESR from the SH3 domain of pp60c-src is recognized as a substrate with a high phosphorylation level. DEDYIQD, derived from the phosvitin/casein kinase II, was also highly phosphorylated. In order to determine the minimal recognition sequence of HPK40, the phosphorylation of about 60 dito tetrapeptides was investigated. Some of the tetrapeptides, such as *EEYE and NEYE, were well phosphorylated. Even some tripeptides, such as EYE, DYM, TYS and KYE, were recognized by HPK40, while none of the tested dipeptides was recognized as substrate. Sequences of peptides from DRVYHPF (angiotensin), LEEEEEAYGWMDF (minigastrin) and QEEYSAM (from H-rasl) were examined as substrates. The presence of one or several acidic residues on the N^α-side of tyrosine residue was identified as the only apparently favorable determinant. These results are steps towards the minimum recognition sequence, which in turn will serve as a lead for chemical modifications in view of obtaining a specific, low-molecular-weight, inhibitor of this human tyrosine protein kinase.     

The design,synthesis and activity of pentapeptide pp60c-src inhibitors containing L-phosphotyrosine mimics

Efficient syntheses of 4-(R,S-hydroxyphosphonomethyl)-l -phenylalanine and 4-carboxy-l -phenylalanine within the context of the pentapeptide Ac-Ile-X-Gly-Glu-Phe-NH₂ (wherein X = the unnatural amino acid) illustrate the use of a divergent synthetic strategy from an advanced common peptide intermediate to more readily access peptide-based tyrosine kinase inhibitors. The key intermediate, Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH₂, was synthesized by a facile palladium-catalyzed carbonylation of Ac-Ile-Phe(4-iodo)-Gly-Glu(O-tBu)-Phe-NH₂. Oxidation of Ac-Ile-Phe(4-formyl)-Gly-Glu(O-tBu)-Phe-NH₂ with tetrabutylammonium permanganate or addition of di-t-butylphosphite, both followed by trifluoroacetic acid deprotection, gave the target pentapeptide inhibitors wherein X = 4-carboxy-l -phenylalanine or 4-(R,S-hydroxyphosphonomethyl)-l -phenylalanine, respectively. These two peptides gave somewhat more potent inhibition of the tyrosine kinase pp60^c-src than the corresponding pentapeptide wherein X =l -phenylalanine, demonstrating that appended functionalities at the 4-position are accepted and can enhance binding through added interactions within the catalytic region of the active site.     

Structure–activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin

Abstract: Peptides derived from the third α‐helix of the homeodomain (residues 43–58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N‐ and C‐terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C‐terminal segment ⁵²Arg‐Arg‐Met‐Lys‐Trp‐Lys‐Lys⁵⁸ of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide’s basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.     

Inhibition of binding of phospholipase Cγ1 SH2 domains to phosphorylated epidermal growth factor receptor by phosphorylated peptides

A series of tyrosine-containing peptides 1–12: Asp-Ala-Asp-Glu-Tyr⁹⁹²(PO₃H₂)-Leu-Ile-Pro-Gln-Gln-Gly-OH (1) Asp-Ala-Asp-Glu-Tyr⁹⁹² -Leu-Ile-Pro-Gln-Gln-Gly-OH (2) Phe-Leu-Pro-Val-Pro-Glu-Tyr¹⁰⁶⁸(PO₃H₂)-Ile-Asn-Gln-Ser-Val-OH (3) Phe-Leu-Pro-Val-Pro-Glu-Tyr¹⁰⁶⁸ -Ile-Asn-Gln-Ser-Val-OH (4) Asp-Asn-Pro-Asp-Tyr¹¹⁴⁸JR(PO₃H₂)-Gln-Gln-Asp-Phe-Phe-OH (5) Asp-Asn-Pro-Asp-Tyr¹¹⁴⁸ -Gln-Gln-Asp-Phe-Phe-OH (6) Ala-Glu-Tyr¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (7) Ala-Glu-Tyr¹¹⁷³ -Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (8) Ala-Glu-Tyr¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-OH (9) Ala-Glu-Tyr¹¹⁷³ -Leu-Arg-Val-Ala-OH (10) Tyrl¹¹⁷³(PO₃H₂)-Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (11) Tyr¹¹⁷³ -Leu-Arg-Val-Ala-Pro-Gln-Ser-OH (12) (six pairs with and without the tyrosine phosphorylated) has been synthesized. The peptides were derived from tyrosine autophosphorylation sites in the epidermal growth factor receptor (EGFR): Tyr 992, 1068, 1148 and 1173. Peptide 1, derived from the Tyr 992 site, inhibited binding of a ³⁵S-labelled fusion protein containing both of the SH2 domains from PLCγ1 to the phosphorylated EGFR with an IC₅₀ of 8 μM. All of the phosphorylated peptides except 11 (1, 3, 5, 7 and 9) inhibited this binding to some degree (20–55%) at 10 p μM. The nonphosphorylated peptides were inactive in this assay. The nonphosphorylated peptides 2, 4, 6, 8, 10 and 12 were obtained by standard solid-phase synthetic methodologies using both Boc/benzyl and Fmoc/tert-butyl strategies. The phosphorylated peptides 1, 3, 5, 7, 9 and 11 were similarly obtained using a Fmoc/tert-butyl strategy incorporating unprotected N^x-Fmoc-Tyr, followed by phosphitylation and oxidation of the tyrosine in the resin-bound peptide. In addition, Asp-Ala-Asp-Glu-Phe⁹⁹²(4-CH₂PO₃H₂)-Leu-Ile-Pro-Gln-Gln-Gly-OH (15), an analog of 1 incorporating an enzymatically stable phosphotyrosine mimic, 4-phosphonomethyl-l -phenylalanine, was synthesized and found to be inactive.     

A SYNTHETIC STUDY OF THE EFFECT OF TYROSINE AT POSITION 120 OF RIBONUCLEASE

[Tyr¹²⁰]-RNase 111–124 and [Ala¹²⁰]-RNase 111–124 were synthesized and purified to chromatographic and electrophoretic homogeneity. The peptides were mixed noncovalently with RNase 1–118 that had been prepared by enzymatic degradation of native bovine pancreatic ribonuclease A. The activities of the resulting complexes were compared with that of the corresponding complex containing the natural peptide sequence, [Phe¹²⁰]-RNase 111–124, and with native RNase, using yeast RNA, C > p and U > p as substrates. The Tyr¹²⁰ and Phe¹²⁰ complexes were equally active against RNA and C > p, but the Tyr¹²⁰ complex was twice as active as the Phe¹²⁰ complex against U > p. The complex with alanine at position 120 was only 1% as active toward C > p as the complex containing phenylalanine. The prediction that giraffe RNase, which contains tyrosine at position 120, would be relatively more active toward U > p than C > p compared with bovine RNase was verified.     

达沙替尼的合成工艺研究

目的 优化酪氨酸激酶抑制剂达沙替尼的合成工艺。方法 以2-氯-6-甲基苯胺为起始原料,经酰胺化、环合、取代、取代共4步反应合成达沙替尼。结果与结论 经4步反应合成达沙替尼,其结构经¹H-NMR、MS谱确证。改进后的合成工艺反应条件温和、操作简便,总收率为51.5%。     

Evaluation of New Indole and Bromoindole Derivatives as pp60c‐Src Tyrosine Kinase Inhibitors

A series of N‐benzyl‐indole‐3‐imine‐, amine derivatives and their 5‐bromo congeners were synthesized and their biological activity were evaluated against the pp60^c‐Src tyrosine kinase target. To afford the imine derivatives, aldehydes were reacted with substituted benzylamines and the corresponding amine derivatives were obtained by NaBH₄ reduction of these imines. Except insoluble N‐benzyl‐indole‐3‐imine derivatives, all the derivatives showed some activity against the kinase target. Screening of these compounds for their biological activity revealed that among N‐benzyl‐indole derivatives, those bearing 5‐bromo substitution have the enhanced potency, where the amine derivatives were more active than imines.     

Solid-phase synthesis of (tyrosyl-alanyl-glutamyl)n,by segment condensation

(Tyr-Ala-Glu)_n, n= 1–9, were synthesized by segment condensation using the Fmoc/tert-butyl protection strategy and solid-phase techniques. The C-terminal residue was coupled to the resin and the peptides were built out by adding Fmoc-Glu(O-r-Bu)-Tyr(t-Bu)-Ala-OH units. When the desired lengths were reached the peptides were capped with Fmoc-Tyr(t-Bu)-Ala-OH units. Fmoc-Tyr(t-Bu)-Ala-OH and Fmoc-Glu(O-t-Bu)-Tyr(t-Bu)-Ala-OH were synthesized in aqueous solution by the successive addition of N-hydroxysuccinimide esters of Fmoc-Tyr(t-Bu) and Fnioc-Glu(0-t-Bu) to the growing chain. Neither sequential amino acid addition or segment condensation techniques were successful on polystyrene supports. However, the segment condensations were highly successful on kieselguhr-supported polydimethylacrylamide based resins. (Tyr-Ala-Glu)_n, n= 1–9, were tested as inhibitors of the protein [yosine kinase, pp60C^c-src. Inhibition, as measured by IC₅₀ values, increased with increasing size of the peptide.     

黄色素抑制血管平滑肌细胞增殖与3种蛋白激酶的关系

目的：研究黄色素(SY)对血管平滑肌细胞(VSMC)增殖的作用及机制。方法：以培养的家兔VSMC为材料,用细胞计数和³²P-参入法,观察SY对VSMC的增殖及3种蛋白激酶活性。结果：SY抑制VSMC的增殖,且呈剂量和时间依赖性,1.0 mg.mL^-1作用最强,抑制率为54.6%; SY抑制VSMC的DNA合成,1.0 mg.mL^-1作用2 d,抑制率为80.2%;在一定浓度范围内,随着SY浓度的升高,VSMC中3种蛋白激酶(TPK,PKC及MAPK)活性下降。结论：SY对VSMC的增殖有抑制作用,可能是通过抑制TPK和PKC依赖的MAPK信号传导途径而发挥作用。     

Conformational preferences of oligopeptides rich in α-aminoisobutyric acid. III. Design,synthesis and hydrogen bonding in 310-helices

Two sterically constrained peptides {ⁱBoc-Aib-Aib-Aib-DkNap-Leu-Qx-Ala-Aib-Aib-F1, (Dk⁴Qx⁶[7/9]) and ⁱBoc-Aib-Aib-Aib-DkNap-Leu-Aib-Ala-Aib-Aib-Fl, (Dk⁴7/9)} containing α-aminoisobutyric acid (Aib) and Aib-class amino acids in conjunction with selected mono-α-alkyl amino acids were synthesized by an optimized TBTU/HOBt procedure. The use of Aib-class amino acids (e.g. DkNap and Qx), defined and discussed here, gives rise to the same overwhelmingly 3₁₀-helical backbone conformation as that provided by simpler Aib-rich peptides and homopeptides. The synthetic α,α-dialkylamino acids (DkNap, Qx) are aromatic homologues of the known alicyclic variants of Aib, the Ac₅c and Ac₆c amino acids. Two new organic solubilizing groups for peptides, ⁱBoc and 2-methoxyethylamine, are introduced. The ¹H nuclear magnetic resonance analyses of the Dk⁴s/p[7/9] and Dk⁴Qx⁶[7/9] peptides demonstrate the unambiguous 310s/b-helical hydrogen bonding pattern of these peptides, confirming the design objective of these sequence patterns containing greater than 50% Aib and Aib-class composition. © Munksgaard 1994.     

Identification and characterization of a novel synthetic peptide substrate specific for Src-family protein tyrosine kinases

Using a random combinatorial peptide library method [Wu, J., Ma, Q. N. & Lam, K. S. (1994) Biochemistry 33 , 14825–14833] a novel peptide, YIYGSFK, was identified as a substrate for p60^c-src protein tyrosine kinase. Mass spectrometric analysis showed that tyrosine-3 from the N-terminus was the phosphorylation site. Kinetic studies showed that the K_m of YIYGSFK for p60^c-src was 55 μM, about 6.4-fold lower than a peptide derived from p34^cdc2 [cdc2(6–20), KVEKIGEGTYGVVYK], which had been reported to be a specific and efficient substrate for the Src-family protein tyrosine kinases. Comparison of the specificity of YIYGSFK and cdc2(6–20) as a substrate for various Src-family and non-Src-family protein tyrosine kinases suggests that YIYGSFK is a much more specific and efficient substrate for the Src-family protein tyrosine kinases. © Munksgaard 1995.