Hexapeptides

Cyclic Hexapeptides Containing Acidic Amino Add and Its Derivative 

Cyclic peptides containing glutamic acid or aspartic acid are useful for investigating the effect of the side chains, which can be modified by esterification, on the main-chain conformation. However, few cyclic peptides containing acidic amino acid residues have been synthesized. The present author has synthesized a series of cyclic hexapeptides Cyclo-(Gly-X-Gly)2 where X = Glu(OBzl), Glu(OMe), Glu, Asp (OBzl), and Asp, and investigated their solution conformations using NMR spectroscopy 113). 

The confonnations of Cyclo-(Gly-Glu(OMeH y)2 and Cyclo-(Gly- au-Gly in water were investigated. Observed/values values) suggested for these ( clic hexapeptides the same Cj-symmetric conformation as in DMSO. However, the temperature coefficients implied that in water the internally hydrogen-bonded Qy-NH protons are more solvent-exposed than in DMSO, the latter solvent being less accepting for hydrogen bonding. 

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The smallest sequence possessing most of the neurotensin spectmm of activities and its high potency is the hexapeptide C-terminus (1). [D-Trp ]-Neurotensin acts like a neurotensin antagonist in perfused heart preparations, but acts like a full agonist in guinea pig atria and rat stomach strips (122). 

Figure 6 The topological disconnectivity graph of alanine hexapeptide. (Adapted from Ref. 67.)...

An example of this strategy is the preparation of a synthetic combinatorial library of hexapeptides. The maximum number of sequence combinations for hexapeptides is 20 or 64,000,000. One approach to simplify preparation and screening possibilities for such a library is to specify the first two amino acids in the hexa-... 

This led to the conclusion that these amino acids were essential for the resolution capability and only 6 new libraries of 18 compounds had to be synthesized with these amino acid residues to define the position 3. Surprisingly, the separation abilities of all six libraries were very similar. Therefore, tyrosine was chosen for continuing deconvolution, since it is convenient as its aromatic ring can easily be detected by UV spectrometry. The last step, defining position 5, required the synthesis and testing of 6 individual hexapeptides. 

Fig. 3-3. Comparison of the values of enantiomeric resolution of different DNP-D,L-amino acids at different deconvolution stages of a cyclic hexapeptide sublibrary. Resolution values in a cyclo(Arg-Lys-X-X-X-P-Ala) sublibrary, in the first line, are compared to those obtained in sublibraries with a progressively increasing number of defined positions. All the sublibraries were 30 mM in the running buffer while the completely defined cyclo(Arg-Lys-Tyr-P-Tyr-P-Ala) peptide is used at 10 mM concentration. Conditions cyclopeptide sublibrary in 20 mM sodium phosphate buffer, pH 7.0 capillary, 50 pm i.d., 65 cm total length, 57 cm to the window V = -20 kV, I = 40 electrokinetic injection, -10 kV, 3 s detection at 340 nm. (Reprinted with permission from ref. [75]. Copyright 1998, American Chemical Society.)...

In addition to the development of the powerful chiral additive, this study also demonstrated that the often tedious deconvolution process can be accelerated using HPLC separation. As a result, only 15 libraries had to be synthesized instead of 64 libraries that would be required for the full-scale deconvolution. A somewhat similar approach also involving HPLC fractionations has recently been demonstrated by Griffey for the deconvolution of libraries screened for biological activity [76]. Although demonstrated only for CE, the cyclic hexapeptides might also be useful selectors for the preparation of chiral stationary phases for HPLC. However, this would require the development of non-trivial additional chemistry to appropriately link the peptide to a porous solid support. 

Give the amino acid sequence of hexapeptides that produce the following sets of fragments on partial acid hydrolysis ... 

Structure of GFP and its chromophore. To study the chro-mophore of GFP, a sample of GFP was denatured by heating it at 90°C. It was digested with papain, and then a peptide containing the fluorophore was isolated and purified from the digested mixture. The structural study of the peptide has indicated that the chromophore of GFP is an imidazolone derivative shown below (Shimomura, 1979). This chromophore structure was confirmed later by Cody etal. (1993) in a hexapeptide isolated from GFP. It is intriguing that the structure of the GFP chromophore is a part of the structure of coelenterazine. 

Branchini, B. R., Lusins, J. O., and Zimmer, M. (1997). A molecular mechanics and database analysis of the structural preorganization and activation of the chromophore-containing hexapeptide fragment in green fluorescent protein. /. Biomol. Struct. Dyn. 14 441-448. 

Cody, C. W., et al. (1993). Chemical structure of the hexapeptide chro-mophore of the Aequorea green-fluorescent protein. Biochemistry 32 1212-1218. 

This type of yvn-seleclivc aldol addition has been applied in the synthesis of the unusual L-threonine based amino acid, (2,S, 3/ ,6F)-3-hydroxy-4-methyl-2-methylamino-6-octenoic acid, of cyclosporine104, of the cyclic hexapeptide echinocandin105, and of the antibiotic ionomycin97. 

A repeat of hexapeptides is found to be frequent, whereas the repeat of a nonapep-tide is infrequent. The more frequently a single tripeptide occurs, the more frequent is its presence in such larger repeat hexa-, nona-, and higher peptides. 

Now, it is widely known that proline at the N-terminal position causes problems of steric hindrance by using active ester couplings in the polycondensation step as well as in the synthesis of the tri- or hexapeptides. This is often a stringent restriction also if proline or glycine are intended to be in the C-terminal position. 

The separation on gel beads was more and more refined. Oligomer series of many polyhexapeptides from n = lton = 9 could be completely separated16,20 24) (see Fig. 4). The hexapeptide oligomers could be separated better than the respective tripeptides. 

Fig. 4. Elution of the crude polyhexapeptide (Pro-Ala-Gly-Pro-A]a-Gly) from a Biogel P4 column. (34 X 1400 mm, 200-400 mesh, 0.05 M acetic acid, 50 ml/h, 20 °Q. The upper curve was obtained by the first chromatography, the curves below were obtained by the fifth rechromatrogra-phy. e = absorbance at 230 nm, Ve = elution volume, VD = 310 ml, n — number of hexapeptide units...

Complexity of LR Dynamics in Intact Neutrophils at 37°. At present, our most versatile assay to analyze receptor binding uses fluorescent ligand and a high-affinity antibody to fluorescein which discriminates free from receptor-bound hexapeptide ligand (FLPEP, N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-Fl). The assay has a time resolution of 1... 

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