Synthetic study of peptide aldehydes.

Itô, Akira; Takahashi, R.; Miura, Chieko; Baba, Yoshihiko

doi:10.1248/cpb.23.3106

Cited by 37 publications

(15 citation statements)

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“…Deacetylation generated the ~-cyclodextrin derivative mono-functionalized at the linear, fully flexible 4C-spacer as carboxylic acid. The tripeptide aldehyde HLeu-Leu-Nle-H was prepared as the semicarbazone according to known procedures [12][13][14][15] and was then linked to the cyclodextrin derivative via the PyBOP procedure [18]. Formation of intra-and/or intermolecular ester bonds with the free hydroxyl functions was not observed to occur under these conditions.…”

Section: Resultsmentioning

confidence: 99%

“…jugate Boc-Nle-H was obtained by reduction of the related N-methyl-Nmethoxy carboxamide with lithium aluminum hydride [12] and converted to the stereochemically stable semicarbazone Boc-Nle-Sc [13][14][15]. Upon deprotection of the et-amino group with 25% TFA/DCM the amino acid semicarbazone H-NIe-Sc was extended to the tripeptide Boc-Leu-Leu-Nle-Sc by standard procedures using Boc-Leu-OSu in the acylating steps.…”

Section: Synthesis Of the [3-cyclodextrinll-leucyl-l-leucyl-l-norleucmentioning

confidence: 99%

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Cyclodextrins as templates for the presentation of protease inhibitors

Schaschke¹,

Hj²,

Assfalg-Machleidt³

et al. 1996

FEBS Letters

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Mono(6-succinylamido-6-deoxy)-13-cyclodextrin was synthesized by classical carbohydrate chemistry and used as a template mono-functionalized with the linear, fully flexible 4C-spacer carboxylate for covalent linkage of the calpain inhibitor leucyl-leucyl-norleucinal. Spectroscopic analyses of the conjugate do not support a self-inclusion of part of the hydrophobic peptide tail, but confirm its intra-or intermolecular interaction with the template moiety that leads to full water solubility. The inhibitory potency of the ~-cyclodextrin/peptide aldehyde construct was compared with that of the parent Ac-Leu-Leu-Nle-H against cathepsin B and calpain. Despite the large size of the template the inhibition of cathepsin B was only slightly reduced in full agreement with the X-ray structure of this enzyme which shows full accessibility of the S-subsites. For this enzyme the 4C-spacer is apparently sufficient to guarantee optimal interaction of the peptide tail with the binding cleft. Conversely, for gcalpain a significantly decreased inhibitory potency was obtained with the conjugate suggesting steric interference of the template in the binding process. These results show that the beneficial properties of the cyclodextrin template can be retained in conjugates with bioactive peptides if attention is paid to optimize in each case the size and nature of the spacer for optimal recognition of the grafted biomolecule.

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Section: Resultsmentioning

confidence: 99%

Section: Synthesis Of the [3-cyclodextrinll-leucyl-l-leucyl-l-norleucmentioning

confidence: 99%

Cyclodextrins as templates for the presentation of protease inhibitors

Schaschke¹,

Hj²,

Assfalg-Machleidt³

et al. 1996

FEBS Letters

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“…DnsArgH was prepared by first reaction (Nw-NO2)-L-arginine methyl ester (Sigma) with dansyl chloride by standard techniques to give Na-dansyl-(Nc@-N02)-L-arginine methyl ester, mp 114-116'C. The Ncr-dansyl-(N'@-N02)arginine methyl ester was reduced with diisobutyl aluminum hydride (Aldrich) in anhydrous tetrahydrofuran at -200C, according to the procedure of Ito et al (27), to Na-dansyl-(N@-NO2)-L-argininal. This product was worked-up as a bisulfite salt similar to the procedure of Thompson (28) in the synthesis of peptidylalaninal compounds, and then reacted with semicarbazide HCl in sodium acetate at 80'C to protect the aldehyde function as semicarbazone, yielding Na-dansyl-(N@-N02)-L-arginine semicarbazone, mp 175-1780C.…”

Section: Methodsmentioning

confidence: 99%

“…The Nw-NO2 group was removed by reduction in a Parr Hydrogenator (H2 at 20 psi; 1 psi = 6.89 kPa) in methanol/water/acetic acid (16:2:1) over palladium black, yielding dansyl-L-arginine semicarbazone acetate, mp 143-145°C (crystallized from ethanol/ethyl ether). Finally, the semicarbazone protection was removed in methanol/2 M HCI/formaldehyde, as described for the synthesis of leupeptin (27). The solution was brought to pH 7.0, and the product was extracted with methylene chloride, dried, and concentrated, yielding dansyl-L-argininal-HCl, crystallized from ethanol/ethyl ether, mp 180-185°C.…”

Section: Methodsmentioning

confidence: 99%

Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.

Kozlowski¹,

Wezeman²,

Schultz³

1984

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

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The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteinase inhibitor p-chloromercuribenzoate. Significant evidence exists for the role of proteolytic enzymes in malignant transformation, tumor cell invasion, and tumor cell metastasis (1-3). For example, treatment of normal cells with proteolytic enzymes can lead to the appearance of phenotypic characteristics in the untransformed cells that are similar to those of transformed cells (1, 4-7). In addition, proteolytic enzymes secreted from transformed cells may degrade host tissues into which the tumor cells invade, and may, through the degradation of surrounding tissue, promote tumor cell metastasis (1-3). Particular proteinases are reported to be secreted in larger amounts by transformed cells than their normal counterparts. These identified proteinases secreted by transformed cells include the serinetype proteinase plasminogen activator (1, 2, 8, 9), the lysosomal cysteine-type proteinase cathepsin-B (1, 10), and collagenolytic proteinases (1,3). The existence of a role for proteolytic enzymes in tumorigenesis and metastasis is supported by experiments in which these processes are shown to be inhibited by inhibitors of proteinase enzymes (1, 2).Whereas a large amount of evidence exists for secretion into the extracellular environment of relatively high concentrations of proteinases by tumor cells, recent reports also document the existence of proteinase activity either associated with the outer cell membrane or membrane fractions within transformed cells (1,2,(11)(12)(13)(14)(15)(16)(17)(18)(19). Similar to the types of proteinases reported to be found secreted into extracellular fluid, both a neutral trypsin-like seine proteinase, which may be plasminogen activator (1,2,(11)(12)(13)(14)(15)(16), and a cathepsin-B cysteine proteinase (1, 17) have been reported. A high concentration of cell surface proteinases may provide an intimate mechanism by which the membrane of the transformed cell can both maintain malignant traits within the tumor and promote the degradation of surrounding tissue during tumor cell invasion and metastasis.In this communication we report the development of a general proteinase fluorescent probe, dansyl-L-argininal (DnsArgH), which is capable of detecting both serine and cysteine proteinases with a primary site specificity toward arginine. Thus, both the neutral trypsin-like serine proteinases and the cathepsin-B-like cysteinyl proteinase, previously reported to be associated with transformed cells should be detected with DnsArgH. The fluorescent aldehyde binds to proteinases with a high affinity an...

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“…(6) for the synthesis of enterokinase inhibitors and by Galpin et al (3) for the preparation of chymostatinlike inhibitors. Peptide aldehydes can also be prepared by oxidation of the corresponding alcohols (7), by reduction of the appropriate acids or esters (8) or by enzymatic oxidation of the alcohols with alcohol dehydrogenase (9). Rich et al (10) of pepsin.…”

mentioning

confidence: 99%