Synthesis and Characterization of More Potent Analogues of Hirudin Fragment 1−47 Containing Non-Natural Amino Acids<sup>,</sup>

Filippis, De; Quarzago, Daniela; Vindigni, Alessandro; E, Di Cera; Fontana, Angelo

doi:10.1021/bi980717n

Cited by 37 publications

(65 citation statements)

References 49 publications

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“…R indicates the fully reduced peptide BugArgNal and N the disulfide oxidized species. The correctness of disulfide pairing was established by the peptide mapping strategy previously detailed~De Filippis et al, 1995!. ~De Filippis et al, 1995Filippis et al, , 1998!, the spectral differences observed between the natural fragment 1-47 and its synthetic analogue~s! do not reflect global structural changes, but only their different content in aromatic residues.…”

Section: Resultsmentioning

confidence: 99%

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

et al. 1999

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Abstract:Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potencỹ K d ϭ 0.2-1.0 pM! and selectivity. Hirudin is composed of a compact N-terminal region~residues 1-47, cross-linked by three disulfide bridges! that binds to the active site of thrombin, and a flexible C-terminal tail~residues 48-64! that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant~K d ! of the corresponding thrombin complexes~K d ϭ 30-400 nM!. The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by b-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology~Cys6-Cys14, Cys16-Cys28, Cys22-Cys37!, while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues~De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515!, yielding a molecule that inhibits the fast or slow form of thrombin by 2,670-and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity~K d,fast ϭ 15.4 pM, K d,slow ϭ 220 pM! comparable to that of full-length hirudin~K d,fast ϭ 0.2 pM, K d,slow ϭ 5.5 pM!. Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested~10 mM!.Keywords: anticoagulant peptides; hirudin; noncoded amino acids; peptide synthesis; thrombin Thrombin is a serine protease that plays a pivotal role in maintaining the intricate balance between hemostasis and thrombolysis Davie et al., 1991!, and for this reason it remains the primary target of antithrombotic therapy~Fenton et al., 1998!. Among natural anticoagulants, hirudin is the most potent and specific inhibitor of thrombin~Markwardt, 1994!. Hirudin is a polypeptidẽ ;7,000 Da! isolated from the salivary secretion of the medicinal leech and is composed of a compact N-terminal region~residues 1-47! cross-linked by three disulfide bridges and a flexible negatively charged C-terminal tail~48-64!~Folkers et al. Abbreviations: Standard three-lett...

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Section: Resultsmentioning

confidence: 99%

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

et al. 1999

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“…Chiral phasetransfer catalysts 1 are prepared according to the procedure as described (26). 2 Cl 2 (0.5 M) was added concentrated H 2 SO 4 (catalytic amount), and isobutylene (excess) was introduced at room temperature. After stirring for several hours, the resulting mixture was treated with saturated NaHCO 3 , extracted with ether, and dried over Na 2 SO 4 .…”

Section: Methodsmentioning

confidence: 99%

“…odification of peptides is an essential and flexible synthetic concept for efficient target screening and optimization of lead structures in the application of naturally occurring peptides as pharmaceuticals (1,2). The direct introduction of side chains to a peptide backbone represents an attractive method for the preparation of various unnatural peptides.…”

mentioning

confidence: 99%

Stereoselective terminal functionalization of small peptides for catalytic asymmetric synthesis of unnatural peptides

Maruoka

Tayama

Ooi

2004

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

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The asymmetric phase-transfer catalytic alkylation of peptides has been achieved by the use of designed C 2-symmetric chiral quaternary ammonium bromide 1 as catalyst. Excellent stereoselectivities were uniformly observed in the alkylation with a variety of alkyl halides and the efficiency of the transmission of stereochemical information was not affected by the side-chain structure of the preexisting amino acid residues. This method also enables an asymmetric construction of noncoded ␣,␣-dialkyl-␣-amino acid residues at the peptide terminal. Since this chirality can be efficiently transferred to the adjacent amino acid moiety, our approach provides a general procedure not only for the highly stereoselective terminal functionalization of peptides but also for the sequential asymmetric construction of unnatural oligopeptides, which should play a vital role in the peptide-based drug discovery process. M odification of peptides is an essential and flexible synthetic concept for efficient target screening and optimization of lead structures in the application of naturally occurring peptides as pharmaceuticals (1, 2). The direct introduction of side chains to a peptide backbone represents an attractive method for the preparation of various unnatural peptides. Achiral glycine subunit has generally been used for this purpose (3), and glycine enolates (4-14), glycine radicals (15-18), and glycine cation equivalents (19-22) have been exploited as reactive intermediates. However, the control of the stereochemical outcome of these processes in an absolute sense is a difficult task, especially in the modification of linear peptides, and hence development of an efficient and practical approach to establish sufficient stereoselectivity and general applicability has been eagerly awaited (12,23,24). Although the stereoselective phase-transfer alkylation of Schiff base-activated small peptides, which involves chirality transfer between two adjoining amino acid residues, appears to be an attractive method (6-8), it has never been developed to a useful level because of the lack of well designed, effective chiral catalysts. Here we describe our approach to this problem by using our recently developed, optically pure C 2 -symmetric quaternary ammonium salt 1 as catalyst (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). By fine-tuning the catalyst structure, a variety of new side chains can be introduced into the growing peptide terminal with remarkable stereoselectivity, and this newly created chirality can be efficiently transferred to an adjacent amino acid residue, thereby allowing the asymmetric construction of unnatural oligopeptides (36) (Scheme 1). Experimental SectionGeneral. IR spectra were recorded on a Shimazu FT-IR 8200A spectrometer. 1 H NMR spectra were measured on a JEOL JNM-FX400 (400 MHz) spectrometer, and tetramethylsilane was used as internal standard (␦ ϭ 0). Splitting patterns are indicated as s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; sept, septet; m, multiplet; br, broad peak. Optical rotation...

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“…Hirudin analogues were synthesized, allowed to refold and purified as described previously [18,19]. The inhibitory potency of the synthetic analogues was determined by measuring the release of pNA (p-nitroaniline) from FPR (20 µM), as described previously [12,20].…”

Section: Synthesis and Anti-thrombin Activity Of Hirudin 1-47 Analoguesmentioning

confidence: 99%

“…Hirudin analogues were synthesized, purified to homogeneity, and characterized with respect to their chemical identity and functional properties, as described previously [18,19]. The results of the thrombin-binding experiments (Table 1) can be summarized as follows: first, alanine replacement at either position 1 or position 3 reduces affinity almost exclusively for the fast form; secondly, side-chain enlargement at position 3 with bulky and [21].…”

Section: Structural Mapping Of Thrombin Recognition Sites In the Fastmentioning

confidence: 99%

Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

Filippis

Dea

Lucatello

et al. 2005

Biochemical Journal

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In the present work, the effect of Na+ binding on the conformational, stability and molecular recognition properties of thrombin was investigated. The binding of Na+ reduces the CD signal in the far-UV region, while increasing the intensity of the near-UV CD and fluorescence spectra. These spectroscopic changes have been assigned to perturbations in the environment of aromatic residues at the level of the S2 and S3 sites, as a result of global rigidification of the thrombin molecule. Indeed, the Na+-bound form is more stable to urea denaturation than the Na+-free form by approximately 2 kcal/mol (1 cal identical with 4.184 J). Notably, the effects of cation binding on thrombin conformation and stability are specific to Na+ and parallel the affinity order of univalent cations for the enzyme. The Na+-bound form is even more resistant to limited proteolysis by subtilisin, at the level of the 148-loop, which is suggestive of the more rigid conformation this segment assumes in the 'fast' form. Finally, we have used hirudin fragment 1-47 as a molecular probe of the conformation of thrombin recognition sites in the fast and 'slow' form. From the effects of amino acid substitutions on the affinity of fragment 1-47 for the enzyme allosteric forms, we concluded that the specificity sites of thrombin in the Na+-bound form are in a more open and permissible conformation, compared with the more closed structure they assume in the slow form. Taken together, our results indicate that the binding of Na+ to thrombin serves to stabilize the enzyme into a more open and rigid conformation.

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Synthesis and Characterization of More Potent Analogues of Hirudin Fragment 1−47 Containing Non-Natural Amino Acids^,

Cited by 37 publications

References 49 publications

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Stereoselective terminal functionalization of small peptides for catalytic asymmetric synthesis of unnatural peptides

Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

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Synthesis and Characterization of More Potent Analogues of Hirudin Fragment 1−47 Containing Non-Natural Amino Acids,

Cited by 37 publications

References 49 publications

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Incorporation of noncoded amino acids into the N‐terminal domain 1‐47 of hirudin yields a highly potent and selective thrombin inhibitor

Stereoselective terminal functionalization of small peptides for catalytic asymmetric synthesis of unnatural peptides

Effect of Na+ binding on the conformation, stability and molecular recognition properties of thrombin

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About

Synthesis and Characterization of More Potent Analogues of Hirudin Fragment 1−47 Containing Non-Natural Amino Acids^,