Regulation of intracellular cardiomyocyte calcium stores by peptides: a new approach to cardiac protection.

The control of cytosolic calcium is a major determinant not only of cardiac function, but also of the capability of myocardial tissue to survive damage. Increase of diastolic calcium leads rapidly to cell injury, and may be induced by a wide range of causes. In this review we describe the major points of calcium control in cardiac myocytes, mainly in mammalian ventricle, focusing on mechanisms of intracellular calcium influx during excitation, voltage gated channels of the sarcolemma and ryanodine receptors of the sarcoplasmic reticulum (SR), and efflux during relaxation, principally the sodium/calcium exchanger in membrane and the SR calcium complex. Mitochondria also depend on calcium concentration while also participating in its control. Moreover, we will outline receptor check points and their roles in physiology and pathology. We will focus on some new aspects of potential protective mechanisms that have been recently described and that involve peptide ligands and that in the case of the Neuregulin1beta/ErbB pathway are already reaching the clinical trial relevance.

Optimizing dissolved air flotation design and saturation.

Dissolved air flotation (DAF) of iron hydroxide precipitates at working pressures lower than 3 atm, using modified flotation units to improve the collection of fragile coagula, was studied. Conventional DAF flotation was studied as a function of saturation pressure in the absence and presence of surfactants in the saturator. Without surfactants, the minimum saturation pressure required for DAF to occur was found to be 3 atm. But, by lowering the air/water surface tension in the saturator, DAF was possible at a saturation pressure of 2 atm. This behavior was found to occur in both batch and pilot DAF operation tests and almost complete recovery of the precipitates was attained. Results are explained in terms of the minimum "energy" which has to be transferred to the liquid phase to form bubbles by a cavity phenomenon. Further, studies were conducted changing equipment design and feed bubbles size distribution (mixing micro and "mid-sized" bubbles). Thus, bubbles entrance position in the collision-adhesion zone ("capture" zone) was compared to bubble entrance position in the water flow inlet below the floating bed. A "mushroom" type diffuser was used for the "capture zone" experiment and better performance was obtained. Results are explained in terms of different mass transfer phenomena in the collection zone and in the separation zone. Finally, results obtained with the use of a column flotation cell working as normal DAF and with a wide bubble size range are presented. Results indicate good performance and some gains in process kinetics with middle size bubbles.

Conformationally constrained analogues of endogenous tripeptide inhibitors of zinc metalloproteinases.

Two diastereomeric furan-2-carbonylamino-3-oxohexahydroindolizino[8,7-b]indole carboxylates, highly constrained analogues of endogenous pyroglutamyl tripeptide inhibitors of snake venom endopeptidases, have been prepared as potential inhibitors of adamalysin II and matrix metalloproteinases. They proved to be inactive against adamalysin II and weak inhibitors of gelatinase A, gelatinase B, stromelysin 1 and human neutrophil collagenase. Evaluation of the mode of binding of the (2R,5S,11bR) isomer in the active site of adamalysin II suggests that the decrease of potency may be due to the reorientation of the acylamino chain in three of the heterocyclic nucleus, to a short contact at the entrance of the S'(1) hydrophobic cleft and to the loss of flexibility of the tetracyclic nucleus in the P'(1), P'(2) region of the inhibitor, which prevents optimal arrangement in the S'(1) specificity subsite.

Two crystal structures of human neutrophil collagenase, one complexed with a primed- and the other with an unprimed-side inhibitor: implications for drug design.

Two crystal structures of human neutrophil collagenase (HNC, MMP-8), one complexed with a primed- and the other with an unprimed-side inhibitor, were determined using synchrotron radiation at 100 K. Both inhibitors contain non-hydroxamate zinc-binding functions. The Pro-Leu-L-Trp(P)(OH)(2) occupies the unprimed region of the active site, furnishes new structural information regarding interaction between the catalytic zinc ion and the phosphonate group, and is the only example of occupation of the S(1) subsite of MMP-8 by the bulky tryptophan side chain. The (R)-2-(biphenyl-4-ylsulfonyl)-1,2,3, 4-tetrahydroisochinolin-3-carboxylic acid, a conformationally constrained D-Tic derivative, accommodates its biphenyl substituent into the deep primary specificity S(1)' subsite, inducing a widening of the entrance to this pocket; this modification of the protein, mainly consisting in a shift of the segment centered at Pro217, is observed for the first time in MMP-8 complexes. Cation-aromatic interactions can stabilize the formation of both complexes, and the beneficial effect of aromatic substituents in proximity of the catalytic zinc ion is discussed. The phosphonate group bound to either a primed- or unprimed-side inhibitor maintains the same relative position with respect to the catalytic zinc ion, suggesting that this binding function can be exploited for the design of combined inhibitors assembled to interact with both primed and unprimed regions of the active cleft.

Serine proteinase inhibition by the active site titrant N alpha-(N, N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester. A comparative study.

Kinetics for the hydrolysis of the chromogenic active-site titrant N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornithine p-nitrophenyl ester (Dmc-azaOrn-ONp) catalysed by bovine beta-trypsin, bovine alpha-thrombin, bovine Factor Xa, human alpha-thrombin, human Factor Xa, human Lys77-plasmin, human urinary kallikrein, Mr 33 000 and Mr 54 000 species of human urokinase, porcine pancreatic beta-kallikrein-A and -B and Ancrod (the coagulating serine proteinase from the Malayan pit viper Agkistrodon rhodostoma venom) have been obtained between pH 6.0 and 8.0, at 21.0 degrees C, and analysed in parallel with those for the enzymatic cleavage of N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). The enzyme kinetics are consistent with the minimum three-step catalytic mechanism of serine proteinases, the rate-limiting step being represented by the deacylation process. Bovine beta-trypsin kinetics are modulated by the acid-base equilibrium of the His57 catalytic residue (pKa approximately 6.9). Dmc-azaOrn-ONp and Dmc-azaLys-ONp bind stoichiometrically to the serine proteinase active site, and allow the reliable determination of the active enzyme concentration between 1.0 x 10-6 M and 3.0 x 10-4 M. The affinity and the reactivity for Dmc-azaOrn-ONp (expressed by Ks and k+2/Ks, respectively) of the serine proteinases considered are much lower than those for Dmc-azaLys-ONp. The very different affinity and reactivity properties for Dmc-azaOrn-ONp and Dmc-azaLys-ONp have been related to the different size of the ornithine/lysine side chains, and to the ensuing different positioning of the active-site titrants upon binding to the enzyme catalytic centre (i.e. to P1-S1 recognition). These data represent the first detailed comparative investigation on the catalytic properties of serine proteinases towards an ornithine derivative (i. e. Dmc-azaOrn-ONp).

Inhibition of adamalysin II and MMPs by phosphonate analogues of snake venom peptides.

Phosphonate analogues of the peptidomimetic N-(Furan-2-yl)carbonyl-Leu-Trp-OH were prepared with the goal of evaluating the effect of phosphonate for carboxylate replacement on binding with snake venom metalloproteinases and MMPs. N-(Furan-2-yl)carbonyl-Leu-L-Trp(P)-(OH)2 showed a 75-fold increase of the inhibiting activity against adamalysin II, a snake venom metalloproteinase structurally related to MMPs and TACE. Both the phosphonate and carboxylate peptidomimetics fit into the active site adopting a retrobinding mode and provide the structural base for a new class of metalloproteinases inhibitors.

Human alpha-thrombin inhibition by the highly selective compounds N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester: a kinetic, thermodynamic and X-ray crystallographic study.

Kinetics, thermodynamics and structural aspects of human alpha-thrombin (thrombin) inhibition by newly synthesized low molecular weight derivatives of alpha-azalysine have been investigated. The thrombin catalyzed hydrolysis of N-ethoxycarbonyl-D-Phe-Pro-alpha-azaLys p-nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) and N-carbobenzoxy-Pro-alpha-azaLys p-nitrophenyl ester (Cbz-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C, and analyzed in parallel with that of N-alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine p-nitrophenyl ester (Dmc-azaLys-ONp). Decarboxylation following the enzymatic hydrolysis of these p-nitrophenyl esters gave the corresponding 1-peptidyl-2(4-aminobutyl) hydrazines (peptidyl-Abh) showing properties of thrombin competitive inhibitors. Therefore, thermodynamics for the reversible binding of D-Phe-Pro-Abh, Cbz-Pro-Abh and Dmc-Abh to thrombin was examined. These results are consistent with the minimum four-step catalytic mechanism for product inhibition of serine proteinases. Eoc-D-Phe-Pro-azaLys-ONp and Eoc-D-Phe-Pro-Abh display a sub-micromolar affinity for thrombin together with a high selectivity versus homologous plasmatic and pancreatic serine proteinases acting on cationic substrates. The three-dimensional structures of the reversible non-covalent thrombin:Eoc-D-Phe-Pro-Abh and thrombin:Cbz-Pro-Abh complexes have been determined by X-ray crystallography at 2.0 A resolution (R-factor = 0.169 and 0.179, respectively), and analyzed in parallel with that of the thrombin:Dmc-azaLys acyl-enzyme adduct. Both Eoc-D-Phe-Pro-Abh and Cbz-Pro-Abh competitive inhibitors are accommodated in the thrombin active center, spanning the region between the aryl binding site and the S1 primary specificity subsite.

2 angstrom X-ray structure of adamalysin II complexed with a peptide phosphonate inhibitor adopting a retro-binding mode.

The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro-binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1'. An adamalysin-based model of tumor necrosis factor-alpha-converting enzyme (TACE) reveals a smaller S1' pocket for this enzyme.

N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p-nitrophenyl ester: a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

The serine proteinase catalyzed hydrolysis of N-ethoxycarbonyl-D-phenylalanyl-L-prolyl-alpha-azalysine p- nitrophenyl ester (Eoc-D-Phe-Pro-azaLys-ONp) was investigated at pH 6.2 and 21.0 degrees C. The results are consistent with the minimum three-step catalytic mechanism. The acylation step is rate limiting for human (Lys 77 species) and porcine plasmin, and for bovine beta-trypsin, the deacylation rate being limiting, on the other hand, for human and bovine alpha-, beta- and gamma-thrombin. Moreover the M(r) 33,000 species of human urokinase and the neuraminidase-treated porcine pancreatic beta-kallikrein-B do not catalyze the hydrolysis of the tripeptide. According to the specificity properties of the serine proteinases considered. Eoc-D-Phe- Pro-azaLys-ONp shows the characteristics of a novel, high selective and optimal chromogenic active site titrant for human and bovine alpha-, beta- and gamma-thrombin.

Separation of airway and tissue properties by transfer respiratory impedance and thoracic gas volume in reversible airway obstruction.

The aim of this study was to establish the ability to estimate separate airway and tissue properties from transfer respiratory impedance (Zrs,tr) data in the presence of airway obstruction. Zrs,tr, thoracic gas volume (TGV) and airway resistance (Raw,pleth) were measured in the presence of obstruction and after use of a bronchodilator (BD) in 13 normal or asymptomatic asthmatic adults and 28 children with symptomatic asthma. An analytical approach was used to solve the equations of a simplified variant of DuBois' model, including airway resistance (Raw*) and inertance (Iaw), tissue compliance (Ct) and resistance (Rt) and pulmonary gas compliance (Cg). The equations of the model could not be reliably solved in four children before BD. Mean Raw,pleth was not different from mean Raw* in adults before (mean +/- SEM) (3.4 +/- 0.5 vs 3.1 +/- 0.3 hPa.s.L-1) or after BD (1.4 +/- 0.2 vs 1.8 +/- 0.2 hPa.s.L-1), or in children after BD (2.9 +/- 0.3 vs 3.2 +/- 0.2 hPa.s.L-1, respectively). In children before BD, Raw* was significantly underestimated compared with Raw,pleth (3.8 +/- 0.4 vs 5.4 +/- 0.6 hPa.s.L-1). Overall, a significant positive correlation was found between the difference [Raw,pleth - Raw*] and Raw,pleth (r = 0.82). In adults, BD induced a decrease in Raw* and Rt, an increase in Ct, and no change in Iaw. In children after BD, there was no significant change in Raw* or Ct, whilst Rt decreased and Iaw increased. Taking Raw,pleth as the gold standard, it is concluded that coherent estimation of parameters of DuBois' model may be obtained from combined Zrs,tr and TGV measurements in normal subjects and moderately obstructed adults, but not in children with significant airway obstruction. This seems to be due to the systematic under-estimation of Raw*.

Thermal artifacts in plethysmographic airway resistance measurements.

Measurements of airway resistance (Raw) by body plethysmography during unconditioned air breathing are implicitly based on the assumption that the warming and humidification of air in the airways are instantaneous. Simulation with a simple model suggests that Raw may be frequency dependent and substantially underestimated at most breathing frequencies if the time constant of gas conditioning (theta) is between 0.01 and 0.3 s. We measured the frequency dependence of the real (Re) and imaginary parts of the relationship between the plethysmographic signal and airway flow from 0.5 to 3 Hz in six healthy subjects in several situations. During breathing of unconditioned air through a heated pneumotachograph, Re increased by 47 +/- 70% between 2 and 3 Hz; the data were consistent with a theta of 0.087 +/- 0.023 s. Additional dead spaces moderately increased theta: 0.105 +/- 0.031 and 0.120 +/- 0.027 s with 50-cm-long polyvinyl chloride and copper tubes, respectively. During breathing of saturated air conditioned at 34, 36, 38, and 40 degrees C, Re exhibited a much smaller positive frequency dependence, most of which was probably due to a flow dependence of Raw. We conclude that unless the inspired gas is conditioned, plethysmographic Raw is likely to be substantially underestimated, particularly when measured during spontaneous breathing.

Fourier analysis versus multiple linear regression to analyse pressure-flow data during artificial ventilation.

Respiratory resistance (Rrs) and elastance (Ers) are commonly measured in artificially-ventilated patients or animals by multiple linear regression of airway opening pressure (Pao) versus flow (V') and volume (V), according to the first order model: Pao = P0 + Ers.V + Rrs.V', where P0 is the static recoil pressure at end-expiration. An alternative way to obtain Rrs and Ers is to derive them from the Fourier coefficients of Pao and V' at the breathing frequency. A potential advantage of the second approach over the first is that it should be insensitive to a zero offset on V' and to the corresponding volume drift. The two methods were assessed comparatively in six tracheotomized, paralysed and artificially ventilated rabbits with and without adding to V' an offset equal to 5% of the mean unsigned flow. The 5% flow offset did not modify the results of Fourier analysis, but increased Rrs and Ers from linear regression by 15.8 +/- 4.6% and 4.55 +/- 0.64%, respectively. Without additional offset, differences between the two methods averaged 30.2 +/- 14.0% for Rrs and 9.3 +/- 6.2% for Ers. The differences almost completely disappeared (2.47 and 0.61%, respectively) when the flow signal was zero-corrected using the assumption that inspired and expired volumes were the same. After induced bronchoconstriction, however, Ers was still slightly larger by linear regression than by Fourier analysis, which may result from nonlinearities and/or frequency dependence of the parameters. We conclude that the regression method requires zero flow correction and that Fourier analysis is an attractive alternative.

Two-frequency analysis of respiratory mechanics in artificially ventilated rabbits.

The frequency dependence of respiratory mechanical properties was studied in 10 paralyzed, artificially ventilated rabbits, by superimposing a single sinusoidal signal with a frequency of 10, 20 or 30 Hz upon the ventilator waveform. The tracheal pressure and flow signals were analyzed both with the usual first order model, which provided total respiratory elastance (Ers) and resistance (Rrs), and by Fourier analysis, which provided respiratory impedance (Zrs) at the breathing frequency (0.85 Hz) and at the superimposed oscillation frequency. The real part of Zrs (Re(Zrs)) decreased by 30% from 0.85 to 10 Hz (P < 0.001), but did not vary significantly from 10 to 30 Hz. This finding is satisfactorily explained by tissue viscoelasticity. Following a histamine aerosol, the frequency dependence of Re(Zrs) changed very little in three out of four rabbits, but increased substantially in the fourth. In that instance, assuming that lung hysteresivity was not markedly modified by histamine, the results suggest inhomogeneous airway obstruction and/or airway wall shunting.

Small-amplitude pressure oscillations do not modify respiratory mechanics in rabbits.

Changes in respiratory mechanics have occasionally been observed during high-frequency ventilation. In this study we investigated whether small pressure oscillations such as those used for respiratory impedance measurements modified total respiratory resistance (Rrs) and total respiratory elastance (Ers). The latter were measured in six paralyzed artificially ventilated rabbits with and without superimposed pressure oscillations at the airway opening. Rrs and Ers were obtained by least square fitting of low-pass filtered tracheal pressure and flow to the usual first-order model. Pressure oscillations of 2-4 hPa peak-to-peak at 10, 20, and 30 Hz applied for periods of 10 min had virtually no effect on Ers (changes ranging from -2.5 to 2.6%) and Rrs (0-8.2%). Analysis of variance did not show a significant difference on the pooled data. Pressure oscillations were also applied every other minute after a histamine aerosol. Ers and Rrs were similarly unchanged. We conclude that the small pressure oscillations used in respiratory impedance measurements do not modify lung mechanical properties and lung response to bronchomotor agents.

Optimal frequency range to analyze respiratory transfer impedance with six-element model.

The aim of this investigation was to assess the optimal frequency range for analyzing respiratory transfer impedance (Ztr) in terms of tissue and airway mechanical properties using the six-element model of DuBois et al. (J. Appl. Physiol. 8:587-594, 1956). Ztr was measured in nine healthy subjects from 2 to 64 Hz by studying the relationship between airway flow and pseudorandom pressure oscillations applied around the chest. The measurements were performed with and without two mechanical loads placed at the mouth: an added inertance of 1.4 Pa.s2.l-1 and an added resistance of 1.65 hPa.s.l-1. The data were corrected for the shunt effect of upper airway walls. The changes in Ztr induced by the loads were very consistent up to 56 Hz with the T-network topology assumed in DuBois's model; the agreement deteriorated at higher frequencies, presumably due to the difficulty of obtaining a homogeneous pressure field around the chest. The fit of the model to the data also worsened sharply at above 56 Hz. In the 2- to 56-Hz frequency range, similar values of the tissue and airway coefficients were obtained with and without the loads. In that frequency range the confidence intervals of the coefficients were better than 10%. We conclude that DuBois's model is valid from 2 to 56 Hz in healthy subjects and allows accurate partitioning of airways and tissue properties. In addition, we present evidence that the upper airway shunt negligibly influences Ztr data and the derived coefficients provided airway flow is measured with a low-impedance pneumotachograph.

Mechanical properties of the upper airway wall in children and their influence on respiratory impedance measurements.

The upper airway wall impedance (Zuaw) may be responsible for a large artifact in the measurement of respiratory system impedance (Zrs) in children. In 17 normal children aged 3.5-13 years Zuaw and Zrs were estimated by varying transrespiratory pressure directly at the mouth (conventional method: Z1) and around the subject's head (head generator method: Z2) from 4 to 32 Hz. Zrs and Zuaw were calculated from Z1 = Zrs.Zuaw/(Zrs+Zuaw) and Z2 = Zrs (1 + Zp/Zuaw), where Zp is the impedance of the pneumotachograph. From the real and imaginary part of Z1, Z2, Zrs, and Zuaw, the corresponding resistance, inertance, compliance and resonant frequency were calculated assuming simple RIC models. No significant difference was found between the mean +/- SE of parameters derived from Zrs (respectively, 6.8 +/- 0.4 cmH2O.L-1.s, 0.034 +/- 0.001 cmH2O.L-1.s2, 10.4 +/- 0.8 m.cmH2O-1, 9.1 +/- 0.3 Hz) and Z2 (6.8 +/- 0.4 cmH2O.L-1.s, 0.038 +/- 0.002 cmH2O.L-1.s2, 10.7 +/- 0.7 ml.cmH2O-1, 8.7 +/- 0.4 Hz). All but the compliance, derived from Z1 were significantly different (P less than 0.01) from those derived from Zrs (5.3 +/- 0.3 cmH2O.L-1.s, 0.008 +/- 0.001 cmH2O.L-1.s2, 11.9 +/- 1.2 ml.cmH2O-1, and 20.3 +/- 1.6 Hz). Respiratory resistance and compliance correlated significantly with height (r = -0.56 and 0.86, respectively), in contrast to upper airway wall resistance (Ruaw) and compliance (Cuaw). Ruaw (8.6 +/- 0.8 cmH2O.L-1.s), Cuaw (1.2 +/- 0.2 m.cmH2O-1), and upper airway wall inertance (0.030 +/- 0.004 cmH2O.L-1.s2) were close to those obtained by direct measurements in adults. The mechanical properties of the upper airway wall are responsible for a significant error in the measurement of Zrs by the conventional method in normal children. Most of the artifact may be corrected for by applying pressure around the child's head.

Within-breath variations of forced oscillation resistance in healthy subjects.

Respiratory resistance (Rrs) was measured by the forced oscillation technique at 10, 20 and 30 Hz in 54 healthy subjects. The sinusoidal pressure oscillations were applied around the head, rather than at the mouth, so as to minimize transmural pressure across extrathoracic airway walls and the corresponding artefact (Peslin et al., J Appl Physiol, 1985, 59, 1790-1795). The flow (V') and volume (V) dependences of Rrs during the respiratory cycle were analysed by least square regression according to: Rrs = K1 + 2.K2.[V']#- K3.V, where K1 and K2 are Rohrer's constants, and where K3 expresses the (negative) volume dependence of Rrs. The analysis was made separately on the inspiratory and expiratory phases. A good fit was usually found between the data and the model, with a root-mean-square error averaging 15% of the mean Rrs at 10 Hz. At all frequencies K2 and K3 were substantially and significantly larger, and K1 slightly lower during expiration than during inspiration. Rrs, K1 and K3 were minimum at 20 Hz, while K2 exhibited a strong positive frequency dependence. The decrease of Rrs from 10 to 20 Hz was entirely explained by the variations of its linear component, and its increase from 20 to 30 Hz was largely due to its flow dependent component. Both the phasic variations and the frequency dependence of the coefficients suggest that the model is purely descriptive and that coefficients K2 and K3 reflect a number of phenomena, including the variations in glottic aperture during the respiratory cycle.

1-Peptidyl-2-haloacetyl hydrazines as active site directed inhibitors of papain and cathepsin B.

Fifteen 1-peptidyl-2-haloacetyl hydrazines, which can be considered halometanes of azapeptides containing Phe in P2 and alpha-aza-Ala or alpha-aza-Gly in P1, were synthesized and tested as models of cysteine-proteases inhibitors. By use of kinetic methods, they proved to irreversibly inactivate papain and cathepsin B via a reversible enzyme-inhibitor intermediate. Second-order rate constants of inactivation in the range 26-23000 M-1s-1 were observed for papain and 2000-39600 M-1s-1 for cathepsin B. KI for the reversible EI adducts ranged from 230 to 0.16 microM for papain and from 11 to 0.37 microM for cathepsin B. Structure of possible reversible EI complex is proposed and used to discuss the effects of structural variation of the inhibitors on the kinetic parameters of inactivation. Title compounds proved to be selective for cysteine-proteases, since no inhibiting activity could be detected toward trypsin, chymotrypsin and porcine pancreatic elastase at 0.1 mM concentration, after 6 h incubation. Relatively low aspecific alkylating properties were also verified in tests using glutathione as the nucleophile.

Synthesis and inhibiting properties toward trypsin like proteases of N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornitine and alpha-azalysine esters.

N alpha-(N,N-dimethylcarbamoyl)-alpha-azaornitine and N alpha-(N,N-dimethylcarbamoyl)-alpha-azalysine phenyl and p-nitrophenyl esters (7-10) were synthesized and tested as inhibitors of trypsin, chymotrypsin and thrombin. The N,N-dimethylcarbamoyl group was chosen to decrease the tendency of acylcarbazates to cyclization into 1,3,4-oxadiazol-2(3H)-ones. Only the p-nitrophenyl alpha-azaornithine derivative 8 was inactivated rapidly by intramolecular acylation of the terminal amino group, rather than by cyclization to oxadiazolone, in aqueous solution at pH 8. The corresponding alpha-azalysine derivative 10 is completely unaffected under the same conditions. Rapid inactivation of thrombin and trypsin only was observed for all alpha-azapeptide esters 7-10 at 0.5 mM inhibitor concentration. No proteolytic activity was restored after 24 h following 2,000 fold dilution of the inhibitor concentration suggesting formation of very stable acylenzymes.