Hans-Jürgen Musiol

Photomodulation of conformational states. Synthesis of cyclic peptides with backbone-azobenzene moieties.

The search for photoresponsive conformational transitions accompanied by changes in physicochemical and biological properties led us to the design of small cyclic peptides containing azobenzene moieties in the backbone. For this purpose, (4‐aminomethyl)phenylazobenzoic acid (H‐AMPB‐OH) and (4‐amino)phenylazobenzoic acid (H‐APB‐OH) were synthesized and used to cyclize a bis‐cysteinyl‐octapeptide giving monocyclic derivatives in which additional conformational restriction could be introduced by conversion to bicyclic structures with a disulphide bridge. While synthesis with H‐AMPB‐OH proceeded smoothly on a chlorotrityl‐resin with Fmoc/tBu chemistry, the poor nucleophilicity of the arylamino group of H‐APB‐OH required special chemistry for satisfactory incorporation into the peptide chain. Additional difficulties were encountered in the reductive cleavage of the S‐tert‐butylthio group from the cysteine residues since concomitant reduction of the azobenzene moiety took place at competing rates. This difficulty was eventually bypassed by using the S‐trityl protection. Side‐chain cyclization of the APB‐peptide proved to be difficult, suggesting that restricted conformational freedom was already present in the monocyclic form, a fact that was fully confirmed by NMR structural analysis. Conversely, the methylene spacer in the AMPB moiety introduced sufficient flexibility for facile and quantitative side‐chain cyclization to the bicyclic form. Both of the monocyclic peptides and both of the bicyclic peptides are photoresponsive molecules which undergo cis/trans isomerization reversibly. Copyright

Synthesis of single- and multiple-stranded cystine-rich peptides

The large abundance of bioactive single‐ and multiple‐stranded cystine‐rich peptides in nature has fostered the development of orthogonal thiol‐protection schemes and of efficient chemistries for regioselective disulfide formation in synthetic replica for decades. In parallel to these entirely synthetic strategies, an increased knowledge of oxidative refolding mechanisms of proteins has been accumulated, and the collective experience with air oxidation of cysteine‐rich peptides into their native disulfide frameworks have largely confirmed Anfinsen′s principle of the self‐assembly of polypeptide chains. In fact, a continuously growing number of cysteine‐rich bioactive peptides from the most diverse sources and with differing cysteine patterns were found to retain the critical sequence‐encoded structural information for correct oxidative folding into the native structures as dominant isomers, although in the biosynthetic pathways the mature peptide forms are mostly generated by posttranslational processing of folded precursors. Such self‐assembly processes can be optimized by opportune manipulation of the experimental conditions or by induction of productive intermediates. But there are also numerous cases where folding and disulfide formation are thermodynamically not coupled and where the application of a defined succession of regioselective cysteine pairings still represents the method of choice to install the desired native or non‐native cystine frameworks. Among our contributions to the state of the art in the synthesis of cystine‐rich peptides, we have mainly addressed the induction of correct oxidative refolding of single‐stranded cysteine‐rich peptides into their native structures by the use of selenocysteine and suitable strategies for disulfide‐mediated assembly of monomers into defined oligomers as mimics of homo‐ and heterotrimeric collagens as a synthetic approach for the development of new biomaterials.

Toward Semisynthetic Lipoproteins by Convergent Strategies Based on Click and Ligation Chemistry

Cell-membrane proteins are anchored to the lipid bilayer by single or multiple insertion of transmembrane helices or by regioselective single or dual lipidation in coand post-translational enzymatic processes, including acylation with fatty acids, prenylation, and rather commonly C-terminal amidation with glycosylphosphatidylinositols (GPI). Procedures for native and neolipidation of peptides have been comprehensively reviewed, and, more recently, even the synthesis of complex GPIs has been reported. However, lipidation of proteins at defined sites and particularly grafting of GPIs or related mimetic structures to the C termini of proteins still represent formidable long-term goals of chemistry and molecular biology. So far, lipoproteins have been obtained by chemical ligation of synthetic lipopeptides with recombinant protein fragments by the highly selective and efficient maleinimide/thiol addition reaction, as shown for the RAS protein, for example, or by total synthesis through orthogonal protection schemes for regioselective lipidation of side-chain amino groups, for example, with 1,2-dipalmitoyl-glycero-3-succinate. In view of the recently developed efficient procedures for the semisynthesis of proteins by native-chemical [6] and expressed-protein ligation, we have performed model studies toward C-terminal lipidation of proteins by exploiting the copper(i)-catalyzed Huisgen’s 1,3-dipolar cycloaddition of terminal alkynes to azides to form a stable triazole product and the transthioesterification between peptide thioesters and N-cysteinyl-lipopeptides followed by intramolecular S!N acyl shift as the synthetic strategy set forth in Scheme 1. Aside from validating the methodology of a combined click and ligation chemistry, incubation of HeLa cells with the micellar solution of the lipopeptide confirmed its fast uptake, as visualized by confocal fluorescence microscopy. It is well established that dual vicinal lipid chains, as present in the di-fattyacyl glycerol moiety of natural GPI anchors are required for an almost irreversible capture of peptides and proteins by lipid bilayers. Correspondingly, to properly mimic the GPI anchor, phosphatidylethanolamine was converted into the corresponding azide 1 by CuSO4-catalyzed diazotransfer with triflyl azide to produce the key intermediate for subsequent application of the click chemistry. The crystalline azide 1 was then used for the 1,3-cycloaddition reaction with the Sprotected model peptide 2, which contained a C-terminal propargylglycine (Pra) residue as suitable reaction partner (Scheme 1). The azide–alkyne cycloaddition was performed with CuI as catalyst in organic solvent, and the lipopeptide derivative 3 was isolated by silica gel chromatography in yields of 70–75 %. Upon removal of the acid-labile S-trityl and NBoc groups from 3 with TFA, the subsequent native chemical ligation of the cysteinyl-lipopeptide with N-dansylor N-rhodamine B-labeled Gly-Pro-Gly-Gly-SPh ester 4 was performed in micellar solutions of 2 % octyl-b-d-glucopyranoside. Ligation was found to proceed smoothly in the presence of tris(2-carboxyethyl)phosphine (TCEP) if excess of the thioesters was carefully avoided to prevent bisacylation as a side reaction. HPLC served to isolate the fluorescence-labeled lipopeptides 5 a,b in yields of 60–70 % as analytically well-characterized compounds, as shown in Figure 1 for compound 5 b. The lipopeptide 6 was obtained in practically quantitative yield by treatment of the C-terminal propargylglycine residue with azide 1 in aqueous–organic media and in the presence of [a] H.-J. Musiol, Dr. S. Dong, Dr. M. Kaiser, Prof. Dr. L. Moroder Max-Planck-Institut f r Biochemie Am Klopferspitz 18, 82152 Martinsried (Germany) Fax. : (+ 49) 89-8578-2847 E-mail : moroder@biochem.mpg.de [b] Dipl.-Phys. R. Bausinger, Dr. A. Zumbusch Institut f r physikalische Chemie Butenandtstraße 5–13, 81377 M nchen (Germany) [c] Dr. U. Bertsch Zentrum f r Neuropathologie und Prionforschung, LMU M nchen Feodor Lynen Straße 23, 81377 M nchen (Germany)

Enantioselective synthesis of S-o-carboranylalanine via methylated bislactim ethers of 2,5-diketopiperazines

Abstract A new synthesis of S - o -carboranylalanine by the Schollkopf-Hartwig procedure is described. The metalated bislactim ether of cyclo-( d -Val-Gly) reacted with propargylbromide to yield with 52 % de the (5 S )-propargyl adduct. Its boronation with 6,9-bis(acetonitrile)-decaborane proceeded in satisfactory yields despite the steric hindrance of the bislactim ether, but surprisingly thermal isomerization was found to occur to some extent at the chiral C-5 atom. Although all attempts to separate the diastereomeric 12:1 mixture failed so far, this synthetic approach represents a significant improvement because of the relatively facile access to S - o -carboranylalanine in high enantiomeric purity.

Synthesis of bivalent inhibitors of eucaryotic proteasomes

Based on the peculiar spatial array of the active sites in the internal chamber of the multicatalytic proteasome, as derived from the X‐ray structure of yeast proteasome, homo‐ and heterobivalent inhibitors were designed and synthesized to exploit the principle of multivalency for enhancing inhibition potency. Peptidic bis‐aldehyde compounds of the octapeptide size were synthesized to address adjacent active sites, whilst a PEG spacer with a statistical length distribution of 19–25 monomers was used to link two identical or different tripeptide aldehydes as binding heads. These bis‐aldehyde compounds were synthesized applying both methods in solution and solid phase peptide synthesis. Bivalent binding was observed only for the PEG‐spaced inhibitors suggesting that binding from the primed side prevents hemiacetal formation with the active site threonine residue. Copyright

Synthesis of ?-(1-azulenyl)-L-alanine as a potential blue-colored fluorescent tryptophan analog and its use in peptide synthesis

Acetyl‐β‐(1‐azulenyl)‐D,L‐alanine has been synthesized in high overall yield by the malonic ester condensation procedure, and the racemate has been enzymatically resolved with acylase I from Aspergillus melleus. The enantiomerically pure L‐amino acid is of interest as a blue‐colored fluorescent tryptophan analog. The bioactivity data of a heptagastrin analog containing it suggests that the planar aromatic azulene moiety may indeed mimic the tryptophan side chain to some extent, and the spectral properties of the azulene moiety makes β‐(1‐azulenyl)‐L‐alanine of potential value as a UV and fluorescence probe in synthetic peptides, and possibly even in proteins if bioincorporation succeeds with chemically misacylated tRNAs. Copyright

Navigation inside a protease: Substrate selection and product exit in the tricorn protease from Thermoplasma acidophilum

The proposed pathway and mechanism of substrate entry and product egress in the hexameric D3 symmetric tricorn protease from Thermoplasma acidophilum were explored by crystallographic studies of ligand complexes and by structure-based mutagenesis. Obstruction of the pore within the 7-bladed beta-propeller (beta7) domain by alkylation or oxidation of an engineered double cysteine mutant strongly decreased enzymatic activities. In line herewith, the crystal structure of the tricorn protease in complex with a trideca-peptide inhibitor modifying the catalytic Ser965 revealed part of the peptide trapped inside the channel of the beta7 domain. The cysteine mutation widening the lumen of the 6-bladed beta-propeller (beta6) domain enhanced catalytic activity, which was restored to normal values after its alkylation. A charge reversal mutant at the putative anchor site of the substrate C terminus, R131E-R132E, drastically reduced the proteolytic activity. The complex crystal structure of a peptide inhibitor with a diketo group at the cleavage site mapped the substrate recognition site and confirmed the role of Arg131-Arg132 as an anchor site. Our results strongly suggest the wider beta7 domain to serve as a selective filter and guide of the substrate to the sequestered active site, while the narrower beta6 domain routes the product to the surface. Moreover, we identified the role of Arg131-Arg132 in anchoring the substrate C terminus.

Aziridine-2-carboxylic acid A reactive amino acid unit for a new class of cysteine proteinase inhibitors

The three‐membered ring of aziridine‐2‐carboxylic acid, which is susceptible to opening by nucleophiles, has been analyzed as a potential useful handle for the design of specific irreversible inhibitors of cysteine proteinases. For this thiol‐reactive amino acid, an imino analogue of proline, a second‐order rate constant of 17.07 M−1 s−1 for inactivation of papain was determined. Thus, the aziridine moiety proved to be remarkably more reactive than activated double bonds, e.g. N‐ethylmaleimide, or halides such as α‐iodopropionic acid or chloroacetic acid. Since it does not alkylate histidine under conditions in which quantitative alkylation occurs with N‐ethyl‐maleimide, it could represent an interesting reactive amino acid unit for the synthesis of a new class of irreversible inhibitors, at least in terms of specificity of the chemical reaction involved in the inactivation process.

CYCLODEXTRINS AS TEMPLATES FOR THE PRESENTATION OF PROTEASE INHIBITORS

Mono(6‐succinylamido‐6‐deoxy)‐β‐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 β‐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 μ‐calpain 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.

Following the energy transfer in and out of a polyproline–peptide†

The intramolecular and intermolecular vibrational energy flow in a polyproline peptide with a total number of nine amino acids in the solvent dimethyl sulfoxide is investigated using time-resolved infrared (IR) spectroscopy. Azobenzene covalently bound to a proline sequence containing nitrophenylalanine as a local sensor for vibrational excess energy serves as a heat source. Information on through-space distances in the polyproline peptides is obtained by independent Förster resonance energy transfer measurements. Photoexcitation of the azobenzene and subsequent internal conversion yield strong vibrational excitation of the molecule acting as a local heat source. The relaxation of excess heat, its transfer along the peptide and to the solvent is monitored by the response of the nitro-group in nitrophenylalanine acting as internal thermometer. After optical excitation, vibrational excess energy is observed via changes in the IR absorption spectra of the peptide. The nitrophenylalanine bands reveal that the vibrational excess energy flows in the peptide over distances of more than 20 Å and arrives delayed by up to 7 ps at the outer positions of the peptide. The vibrational excess energy is transferred to the surrounding solvent on a time scale of 10-20 ps. The experimental observations are analyzed by different heat conduction models. Isotropic heat conduction in three dimensions away from the azobenzene heat source is not able to describe the observations. One-dimensional heat dissipation along the polyproline peptide combined with a slower transversal heat transfer to the solvent surrounding well reproduces the observations.