A series of new peptidyl (alpha-aminoalkyl)phosphonate diphenyl esters containing the 4-amidinophenyl group were synthesized and tested as irreversible inhibitors for thrombin and other trypsin-like enzymes. These phosphonates irreversibly inhibited several coagulation enzymes and trypsin. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 is the best human thrombin inhibitor in the series with a k(obs)/[I] value of 11,000 M-1 s-1, and it inhibits thrombin more than 5-fold more effectively than the other enzymes tested. Z-(4-AmPhGly)P(OPh)2 is the best inhibitor for plasma kallikrein with a k(obs)/[I] value of 18,000 M-1 s-1. Generally, the (4-AmPhGly)P(OPh)2 derivatives are better inhibitors of thrombin and trypsin than the corresponding (4-AmPhe)P(OPh)2 derivatives which contain an extra CH2 separating the amidinophenyl group from the peptide backbone. The amidino phosphonates did not inhibit acetylcholinesterase and were chemically stable in neutral buffers. In addition, the inhibited trypsin derivative did not regain any enzyme activity after removal of excess inhibitor and incubation in a pH 7.5 buffer for 1 day. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 and D-Phe-Pro-(4-AmPhe)P(OPh)2 prolonged the prothrombin time ca. 2-fold and prolonged the activated partial thromboplastin time ca. 3-4-fold in human plasma at concentrations of 63 and 125 microM, respectively. The novel amidine-containing peptidyl phosphonates reported here are thus effective anticoagulants in vitro, and they may have utility for use in vivo.

A series of dipeptides which contained phosphonate analogs of proline and piperidine-2-carboxylic acid (homoproline) have been synthesized and tested as inhibitors of DPP-IV. The rates of inhibition of DPP-IV by these compounds are moderate, but the inhibitors are quite specific. The best inhibitor in the series is Ala-PipP(OPh-4-Cl)2 (13), which has a k(inact) of 0.353 s-1 and KI of 236 microM. The DPP-IV inhibitors Ala-ProP(OPh)2 (6), Ala-ProP(OPh-4-Cl)2 (12), and Ala-PipP(OPh-4-Cl)2 (13) do not inhibit trypsin, human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), acetylcholinesterase, papain, and cathepsin B. However, compounds 12 and 13 inhibited chymotrypsin slowly. Most of these dipeptides containing a homoproline phosphonate residue (PipP) or a Pro phosphonate residue (ProP) at the P1 site are stable in a pH 7.8 buffer with half-lives of several hours to several days. DPP-IV inhibited by 6, 7 (Ala-PipP(OPh)2), 12, or 13 is quite stable, and no enzyme activity was recovered after removal of excess inhibitor and incubation in buffer for 1 day. Since the phosphonate inhibitors are specific toward DPP-IV and the inhibited enzymes are stable, they should be useful in establishing the biological functions of DPP-IV and may be useful therapeutically in the prevention of the rejection of transplanted tissue.

X-ray structures of trypsin from bovine pancreas inactivated by diphenyl [N-(benzyloxycarbonyl)amino](4-amidinophenyl)methanephosphonate [Z-(4-AmPhGly)P(OPh)2] were determined at 113 and 293 K to 1.8 angstrom resolution and refined to R factors of 0.211 (113 K) and 0. 178 (293 K). The structures reveal a tetrahedral phosphorus covalently bonded to the O gamma of the active site serine. Covalent bond formation is accompanied by the loss of both phenoxy groups. The D-stereoisomer of Z-(4-AmPhGly)P-(OPh)2 is not observed in the complex. The L-stereoisomer of the inhibitor forms contacts with several residues in the trypsin active site. One of the phosphonate oxygens is inserted into the oxyanion hole and forms hydrogen bonds to the amides of Gly193, Asp194, and Ser195. The second phosphonate oxygen forms hydrogen bonds to N epsilon 2 of His 57. The p-amidinophenylglycine moiety binds into the trypsin primary specificity pocket, interacting with Asp189. The amide forms a hydrogen bond to the carbonyl oxygen atom of Ser214. The inhibitor moiety, from the 113 K structure of trypsin inactivated by the reaction product of Z-(4-AmPhGly)P(OPh)2, was docked into human thrombin [Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S. R., & Hofsteenge, J. (1989) EMBO J. 8, 3467-3475] and energy minimized. The inhibitor fits well into the thrombin active site, forming favorable contacts similar to those in the trypsin complex with no bad contacts.

For some years we have used thianthrene cation radical perchlorate (Th'+C104-) for studying the reactions of Th'+ in solution. Th'+C104-was chosen for use because it is easily prepared in the crystalline state and its counter ion is not nucleophilic. However, use of solid Th*+C1O4-can be hazardous. A warning about its use was issued in 1969 after a sample exploded when being transferred from a filter funnel.2 Because of this hazard, Th'+C104-should be (and has been in all of our work) made in small quantities, handled carefully, and used soon after preparation. Because of the potential for explosion, we tried on a number of occasions to prepare other isolable salts. Hexachloro-and hexafluoroantimonates can be made by reaction of T h with antimony pentachloride or pentafluoride. However, we found the use of these salts to be troublesome because of the difficulties of removing antimony compounds from products and, in the case of Th'+SbC16-, because of the presence of nucleophilic chloride ion in solution. The tetrafluoroborate (Th'+BF4-) was an obvious, attractive alternative. Some years ago, attempts were made to prepare solid cation radical tetrafluoroborates by reactions of T h and analogues with nitrosonium tetrafluoroborate (NOBF4). Success was achieved with some cation radicals, for example, those of phenothiazine and 10-methyl-and 10-phenylphenothiazine. However, we were unable to prepare solid Th'+BF;. We were able to prepare solutions of Th'+BF4and to use them successfully soon after preparation, but the solutions were not stable for storage during a day or Therefore, use of Th'+BF4-solutions was discontinued. Th'+BF4-had, in fact, been prepared earlier by disproportionation of Th and its 5-oxide (Tho) in fluoboric acid, but the preparation required the use of dry H F s B F~~ We have now found that solid Th'+BFc can be prepared in good yield and quality by a simple control of the method of reaction between Th and NOBF4. The brown, solid salt can be prepared in large quantities as compared with preparations of Th'+C104-. In order to validate the usefulness of the salt, we used it quantitatively in some reactions that in the past had given excellent results with Th'+C104-, namely, reaction with water, dimethylmercury, and diphenylmer~ury.~ Reaction with water gave equal and quantiative yields (by GC) of Th and T h o (eq 1). Reaction with the mercurials gave the expected 5thianthreniumyl tetrafluoroborates (eq 2) in good yields.(1) On leave from the Institute of Organic and Physical Chemistry,(2) Murata, Y.; Shine, H. J.

The crystal structure of di-n-butyltin pyridine-2-phosphonate-6-carboxylate, [C14H24NO6PSn]2, features centrosymmetric dimers disposed about a central Sn2O2 core. The phosphonate carboxylate dianion is μ2-tetradentate, coordinating one tin atom via one of the phosphonate oxygen atoms, the pyridine nitrogen atom, and one of the carboxylate oxygen atoms; the latter atom also coordinates the second tin atom of the dimer. The remaining positions in the seven-coordinate, distorted pentagonal bipyramidal geometry are occupied by a water molecule and two n-butyl groups that occupy axial positions. The lattice is stabilized by hydrogen-bonding contacts leading to an arrangement of parallel, orthogonally related chains of dimeric units. In methanol solution, the dimer is involved in a dissociation equilibrium that is fast on the NMR time scale.