Hirudin is a small (~7 kDa) disulfide-cross-linked polypeptide known as the most potent and specific thrombin inhibitor. We have previously shown that the N-terminal proteolytic fragment 1-47 of hirudin HM2 from Hirudinaria manillensis maintains inhibitory action toward thrombin [Vindigni, A., et al. (1994) Eur. J. Biochem. 226, 323-333]. Here we report the solid-phase chemical synthesis of an analog of fragment 1-47 bearing a Tyr3→Trp exchange (Y3W analog). The crude, reduced peptide was purified by reverse-phase HPLC and subjected to oxidative folding to the disulfide-cross-linked species. The folding process of the Y3W analog was slower than that of the natural fragment 1-47, but nevertheless still occurred almost quantitatively as the natural species. The overall final yield of the synthetic product was ~35%, and its identity and homogeneity was established by a number of analytical techniques, including electrospray mass spectrometry. The unique alignment of the three disulfide bridges of the Y3W analog was established by peptide mapping as Cys6→Cys14, Cys16→Cys28, and Cys22→Cys37 and shown to be identical to that of the natural fragment. The results of far- and near-ultraviolet circular dichroism and fluorescence emission measurements provided evidence that the Y3W analog retains the structural features of the natural species. The thermodynamic quantities (∆GD, ∆Hm, ∆Sm, and ∆CP) characterizing the reversible and cooperative thermal unfolding processes of the Y3W analog (Tm - 60.5 °C) and the natural fragment species (Tm = 62.5 °C) were evaluated. Despite the relatively high Tm values, the stability of both fragment species at 37 °C was only ~10 kJ mol-1, well below the average 50 kJ mol-1 typical of single-domain globular proteins. The synthetic Y3W species was found to be approximately 5-fold more active (KI = 30 ± 5 nM) than the natural fragment 1-47 (KI = 150 ± 20 nM) in inhibiting thrombin. Of interest was that the difference in the free energies of binding to thrombin at 37 °C, ∆∆Gb, between the Y3W analog and natural species (4.2 kJ mol-1) was that expected for the difference in hydrophobicity between the two polypeptides resulting from the Tyr→Trp exchange. The results of this study indicate that solid-phase chemical synthesis represents a convenient and high-yield procedure to prepare analogs of the biologically active, N-terminal core domain of hirudin with improved functional properties.