Tryptophan Amide Research Articles

Синтез и анализ зависимости анксиолитического эффекта от структуры лейцилтриптофановых лигандов 18 кДа транслокаторного белка

Ранее в НИИ фармакологии имени В. В. Закусова с использованием оригинальной стратегии создания фармакологически активных дипептидов на основе структур непептидных лекарственных средств получены дипептидные лиганды 18 кДа транслокаторного белка (TSPO), амид N-карбобензокси-L-триптофанил-L-изолейцина (ГД-23) и амид N-фенилпропионил-L-триптофанил-L-лейцина (ГД-102), проявившие анксиолитическую активность в стандартных тестах. В настоящей работе получены аналоги дипептида ГД-102 с обратной последовательностью аминокислотных остатков: метиламид N-фенилпропионил-L-лейцил-L-триптофана (ГД-140), N-фенилпропионил-L-лейцил-L-триптофан (ГД-139), амид N-фенилацетил-L-лейцил-L-триптофана (ГД-141), метиловый эфир N-фенилпропионил-L-лейцил-L-триптофана (ГД-138), амид N-фенилпропионил-L-лейцил-L-триптофана (ГД-136). Исследование в тесте «Открытое поле» (ОП) на мышах при внутрибрюшинном введении показало, что вновь синтезированные дипептиды уступают по активности ГД-102 (0,01 – 0,1 мг/кг). Дипептид ГД-140 проявлял анксиолитическую активность в дозах 0,1, 0,5, 1 мг/кг; ГД-139 в дозах 0,5 и 5 мг/кг; ГД-141 в дозах 1 и 5 мг/кг. Дипептид ГД-138 в дозе 0,1 мг/кг снижал двигательную активность животных в ОП. ГД-136 не проявил активность в интервале доз 0,1 – 5 мг/кг. Молекулярный докинг продемонстрировал возможность размещения изученных соединений в зоне связывания лиганда PK11195 с TSPO (PDB ID: 2MGY). При этом для ГД-136, ГД-139, ГД-140 и ГД-141 выявлено наличие pi-стэкинга фенильного фрагмента молекулы с Trp107 рецептора. У ГД-140 и ГД-102 выявлен еще и pi-стэкинг с Trp95. У ГД-138 pi-стэкинг отсутствовал как с Trp107, так и с Trp95. Сделано заключение о том, что ключевым взаимодействием, влияющим на проявление анксиолитической активности, является pi-стэкинг дипептидного лиганда с Trp95 рецептора, а последовательность Trp-Leu для дипептидных лигандов TSPO предпочтительнее последовательности Leu-Trp.

Characterization of Excipient Effects on Reversible Self-Association, Backbone Flexibility, and Solution Properties of an IgG1 Monoclonal Antibody at High Concentrations: Part 2

In this work, we continue to examine excipient effects on the reversible self-association (RSA) of 2 different IgG1 monoclonal antibodies (mAb-J and mAb-C). We characterize the RSA behavior of mAb-C which, similar to mAb-J (see Part 1), undergoes concentration-dependent RSA, but by a different molecular mechanism. Five additives that affect protein hydrophobic interactions to varying extents including a chaotropic salt (guanidine hydrochloride), a hydrophobic salt (trimethylphenylammonium iodide), an aromatic amino acid derivative (tryptophan amide hydrochloride), a kosmotropic salt (sodium sulfate, Na2SO4), and a less polar solvent (ethanol) were evaluated to determine their effects on the solution properties, molecular properties, and RSA of mAb-C at various protein concentrations. Four of the 5 additives examined demonstrated favorable effects on the pharmaceutical properties of high concentration mAb-C solutions (i.e., lower viscosity and weakened protein-protein interactions, PPIs) with a ranking order of guanidine hydrochloride > trimethylphenylammonium iodide > tryptophan amide hydrochloride > ethanol as measured by various biophysical techniques. Conversely, addition of Na2SO4 resulted in less desirable solution properties and enhanced PPIs. The effect of these 5 additives on mAb-C backbone dynamics were evaluated by hydrogen exchange-mass spectrometry (at high vs. low protein concentrations) to better understand their effects on the molecular sites of RSA in mAb-C.

Identification of cholecystokinin tetrapeptide amide metabolites in liver microsomes of human, Rhesus Monkey, Sprague-Dawley rat and CD1 mouse using ultra-high performance liquid chromatography coupled to high resolution mass spectrometer

Endogenous cholecystokinin tetrapeptide (CCK-4, Trp-Met-Asp-Phe-NH2) is a fragment derived from a larger peptide hormone, cholecystokinin (or gastrin). As a panicogenic agent, CCK-4 is commonly used in clinic settings to induce panic attacks for the study of new anxiolytic drugs. However, few studies on CCK-4 metabolism have been published to date. In the present study, we investigate the metabolism of CCK-4 in liver microsomes of human (HLM), Rhesus Monkey (RMLM), Sprague-Dawley rat (RLM) and CD1 mouse (MLM) using ultra-high performance liquid chromatography coupled to a high resolution mass spetrometer. Ten metabolites, inlcuding tryptophan (M1), tryptophan amide (M2), hydroxy metabolites (M3-M5), truncated peptides (M6-M9), and CCK-4 acid (M10), were identified and 8 of them were reported for the first time. The metabolic pattern of CCK-4 in HLM was distinctly different from these in RMLM, RLM, and MLM. M2 and M9 were the major metabolites in HLM and accounted for 19.8% and 13.4% of initial CCK-4, respectively. In contrast, M2 was the major metabolite in RMLM and accounted for 41.4%, whereas M6 was the major metabolite in RLM and account for 39.1%. Three major metabolites M2, M7 and M8 in MLM accounted for 22.6%, 17.9% and 17.8% of initial CCK-4, respectively. Chemical inhibition experiment showed that aminopeptidase and/or endopeptidase hydrolysis were the major metabolic pathways in human to generate these metabolites. We further showed that cytochrome P450 were also involved in the metabolism of CCK-4 via hydroxylation, but to a less extend. These findings provide valuable information for the metabolic processes of CCK-4 among various species and an important reference basis for its safety evaluation and rational clinical application.

Catalytic resolution of dl-tryptophan amides using the resting cells of Flavobacterium aquatile ZJB-09211 in a two-phase system

The catalytic activity of Flavobacterium aquatile ZJB-09211 towards the kinetic resolution of DL-tryptophan amides was significantly enhanced by ethyl acetate. A maximum enzyme activity of 5118.62 U/g was obtained under the optimized conditions consisting of a mixture of ethyl acetate and Tris–HCl buffer (30:70). In a scale-up reaction, the tryptophan amide concentration was improved to 200 mM, with 49.85% ( e.e. > 99.95%) of the substrate being converted to l -tryptophan. The addition of an organic solvent to the process therefore provided an effective approach for improving the activity of the amidase that could be applied to other amidase-catalyzed bioprocesses. • Ethyl acetate enhanced the activity of amidase and facilitated product separation. • Space-time yield of the bioprocess was improved in this two-phase reaction system. • This methodology may play a pivot role in large scale production of L-tryptophan.

Novel Synthetic Route to Fluorinated β‐Carbolines by One‐Pot Reaction.

Abstract This paper describes a new strategy for the synthesis of 1‐trifluoromethyl‐ 9H‐pyrido[3,4‐b]indole 2 by one‐pot reaction of tryptophan methyl ester hydrochloride or tryptophan amide 1 and trifluoroacetic acid (TFA) in 60–78% yield via a possible intramolecular cyclization. The structures of 2a and 2e were confirmed by X‐ray crystallographic analysis.

The dynamics of conformational isomerization in flexible biomolecules. I. Hole-filling spectroscopy of N-acetyl tryptophan methyl amide and N-acetyl tryptophan amide.

The conformational isomerization dynamics of N-acetyl tryptophan methyl amide (NATMA) and N-acetyl tryptophan amide (NATA) have been studied using the methods of IR-UV hole-filling spectroscopy (HFS) and IR-induced population transfer spectroscopy (IR-PTS), which were developed for this purpose. Single conformations of these molecules were selectively excited in well-defined NH stretch fundamentals. This excess energy was used to drive conformational isomerization. By carrying out the infrared excitation early in a supersonic expansion, the excited molecules were recooled into their zero-point levels, partially refilling the hole created in the ground state population of one of the conformers, and creating gains in population in other conformers. These changes in population were detected using laser-induced fluorescence downstream in the expansion. In HFS, the IR wavelength is fixed and the UV laser tuned in order to determine where the population went following selective infrared excitation. In IR-PTS, the UV is fixed to monitor the population of a given conformation, and the IR is tuned to record the IR-induced changes in the population of the monitored conformer. Besides demonstrating the capability of the experiment to change the downstream conformational population distribution, the IR-PTS scans were used to extract two quantitative results: (i) The fractional populations of the conformers in the absence of the infrared, and (ii) the isomerization quantum yields for each of the six unique amide NH stretch fundamentals (three conformers each with two amide groups). The method for obtaining quantum yields is described in detail. In both NATMA and NATA, the quantum yields show modest conformational specificity, but only a hint of vibrational mode specificity. The prospects for the hole-filling technique for providing insight into energy flow in large molecules are discussed, leaving a more detailed theoretical modeling to the adjoining paper [Evans et al. J. Chem. Phys. 120, 148 (2004)].

A catalytic antibody programmed for torsional activation of amide bond hydrolysis.

Amidase antibody 312d6, obtained against the sulfonamide hapten 4 a that mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1 a-3 a by a factor of 10(3) at pH 8. The mechanisms of both the uncatalyzed and antibody-catalyzed reactions were studied. Between pH 8 and 12 the uncatalyzed hydrolysis of N-toluoylindoles 1 a and 3 a shows a simple first-order dependence on [OH(-)], while hydrolysis of 3 a is zeroth-order in [OH(-)] below pH 8. The pH profile for hydrolysis of the corresponding tryptophan amide 2 a is more complex due to the dissociation of the zwitterion into an anion with pK(a) 9.74; hydrolysis of the zwitterionic and the anionic form of 2 a both show simple first-order dependence on [OH(-)]. Absence of (18)O exchange between H(2) (18)O/(18)OH(-) and the substrate, a normal SKIE for both 1 a (k(H)/k(D)=1.12) and 3 a (k(H)/k(D)=1.24) and the value of the Hammett constant rho for hydrolysis of p-substituted amides 3 a-e are consistent with an ester-like mechanism in which formation of the tetrahedral intermediate is rate-determining and the amine departs as anion. The 312d6-catalyzed hydrolysis of 3 a was studied between pH 7.5 and 9, and its independence of pH in this range indicates that water is the reacting nucleophile. Hydrolysis of 3 a is only partially inhibited by the sulfonamide hapten, and this indicates that non-specific catalysis by the protein accompanies the specific process. Only the nonspecific process is observed in the hydrolysis of amides 3 with para substituents other than methyl. Binding studies on the corresponding series of p-substituted sulfonamides 5 a-e confirm the high specificity of antibody 312d6 for p-methyl substituted substrates.

Hydride stretch infrared spectra in the excited electronic states of indole and its derivatives: Direct evidence for the 1πσ* state

The hydride stretch infrared spectra of indole, indole-H2O, 3-methyl indole, 3-methyl indole-H2O, the main conformer of tryptamine (TRA), two conformers of N-acetyl tryptophan amide (NATA), and three conformers of N-acetyl tryptophan methyl amide (NATMA), have been recorded in the electronically excited singlet states using excited-state fluorescence-dip infrared spectroscopy. NATA and NATMA are methyl-capped dipeptides of tryptophan that have conformational flexibility and exhibit sensitivity in their electronic spectra to the conformation of the dipeptide backbone. In the indole monomer, the indole NH stretch fundamental at the S1 origin is shifted from its ground-state value (3525 cm−1) to 3478 cm−1. The corresponding band in the indole-H2O complex appears at 3387 cm−1, shifted by a similar amount from its ground-state position (3436 cm−1). Higher vibronic levels within 1500 cm−1 of the S1 origin, which have been identified previously [B. J. Fender et al., Chem. Phys. Lett. 239, 31 (1995)] as being 1Lb or 1La in character, all show similar excited state indole NH stretch absorptions. The corresponding spectra in 3-methyl indole, 3-methyl indole-H2O, TRA, and in the C5 conformers of NATA and NATMA all are missing the indole NH stretch absorption. In its place, a broad background absorption appears, spread over the entire 2800-3800 cm−1 region. In these molecules, other CH stretch or amide NH stretch absorptions remain sharp, appearing in their expected frequency ranges. Finally, the C7 conformations of NATA and NATMA, which possess an intramolecular hydrogen bond in the dipeptide backbone, have all infrared transitions washed out, replaced by a stronger broad background absorption. The entire data set can be explained by the presence of an excited 1πσ* state which is dissociative along the indole NH stretch coordinate, as recently predicted by Sobolewski and Domcke [Chem. Phys. Lett. 315, 293 (1999)]. In the weak coupling case (indole, indole-H2O), the gap between the 1πσ* state and the S1 origin is too large to be reached by infrared excitation. The selective loss of the indole NH stretch in the intermediate coupling case reflects the strong coupling of the 1Lb state NH stretch (v=1) level to the 1πσ* state, which is dissociative along the NH stretch coordinate. In the NATA and NATMA C7 conformers, an inversion of ordering of the electronic states occurs, pushing the 1La state below the 1Lb origin, and strengthening the coupling of all hydride stretch vibrational levels to the 1πσ* dissociative continuum. These results highlight the important influence of the conformation of the polypeptide backbone on the photophysics of tryptophan in polypeptides.

The infrared and ultraviolet spectra of single conformations of methyl-capped dipeptides: N-acetyl tryptophan amide and N-acetyl tryptophan methyl amide

A combination of methods, including laser-induced fluorescence excitation, fluorescence-dip infrared (FDIR) spectroscopy, and UV-UV hole-burning spectroscopy, have been used to study the infrared and ultraviolet spectra of single conformations of two methyl-capped dipeptides: N-acetyl tryptophan amide (NATA) and N-acetyl tryptophan methyl amide (NATMA). Density functional theory calculations predict that all low-energy conformers of NATA and NATMA belong to one of two conformational families: C5, with its extended dipeptide backbone, or C7eq, in which the dipeptide backbone forms a seven-membered ring joined by a H bond between the ψ-amide NH and the φ-amide carbonyl groups. In NATA (NATMA), the LIF spectrum has contributions from two (three) conformers. FDIR spectroscopy has been used to record infrared spectra of the individual conformers over the 2800–3600 cm−1 region, free from interference from one another. The NH stretch region provides unequivocal evidence that one of the conformers of NATA is C5, while the other is C7eq. Similarly, in NATMA, there are two C5 conformers, and one C7eq structure. Several pieces of evidence are used to assign spectra to particular C5 and C7eq conformers. NATA(A) and NATMA(B) are both assigned as C5(AP) structures, NATA(B) and NATMA(C) are assigned as C7eq (ΦP), and NATMA(A) is assigned as C5(AΦ). In both molecules, the C5 structures have sharp vibronic spectra, while the C7eq conformers are characterized by a dense, highly congested spectrum involving long progressions that extend several hundred wave numbers to the red of the C5 S1–S0 origins. N-acetyl tryptophan ethyl ester (NATE), which can only form C5 conformers, shows only sharp transitions in its LIF spectrum due to four C5 conformers, with no evidence for the broad absorption due to C7eq. This provides direct experimental evidence for the importance of the peptide backbone conformation in controlling the spectroscopic and photophysical properties of tryptophan.

Second Derivative Fluorescence Spectra of Indole Compounds

The fluorescence emission spectrum of N-acetyl tryptophan amide (NATA) in 20 mM K-phosphate buffer, pH 7.5, with excitation at 295 nm, when subjected to second derivatization, showed two troughs at 340 1.0 nm (A) and 358.5 1.0 nm (B). Linear dependence of derivative intensities at A and B was observed with increasing NATA concentration between 0-30 nM but the intensity ratio (B/A), termed R, was found to be invariant at 0.70 0.05. R remained unaffected with variation of the pH (4-10), temperature (15-70 degrees C), salt concentration (0-2 M NaCl), and excitation wavelength between 280-300 nm. A 50-fold molar excess of N-acetyl tyrosine over 10 nM NATA and inclusion of a quencher like 0.8 M acrylamide, 0.4 M potassium iodide or trichloroethanol had no effect on R. It was, however, linearly dependent on the polarity of the solvent-in 1,4-dioxane it became 0.07 0.05. Derivative spectra of tryptophans of proteins largely resembled that of NATA. Low R values of between 0.02-0.34 were observed for proteins under native conditions, which is consistent with the general buried character of tryptophan residues. R increased to 0.6-0.9 after unfolding with denaturants or extensive proteolysis and decreased to close to the original value after refolding. The equilibrium unfolding transitions of proteins expressed as R largely resembled the transitions measured using other physical parameters. R appears to be a more sensitive index for monitoring the hydrophobic environment of tryptophans in protein compared to parameters like emission maxima or intensity of underivatised spectra.

Pressure Effects on Tryptophan and Its Derivatives

The high pressure effects on fluorescence of free tryptophan (Trp) and its derivatives, N-acetyl-tryptophan (AT), N-acetyl-tryptophanamide (NATA), tryptophanamide (TA), and tryptophan, containing 6-polypeptides in aqueous solution, were investigated in a pressure range from 0.1 to 650 MPa. It was found by analyzing the center of spectral mass in the wavelength range from 300 to 450 nm that high pressure shifted the fluorescence spectra of all these species to red direction: 421 cm−1 for Trp, 305 cm−1 for AT, 310 cm−1 for NATA, 265 cm−1 for TA, and 220 cm−1 for single tryptophan containing 6-polypeptides. All the fluorescence efficiencies (i.e., quantum yield) of the compounds were reduced with pressure except free tryptophan where its fluorescence efficiency was enhanced with pressure. Glycerol, ethanol, and pH obviously influenced the pressure effects on their fluorescence characteristics. Since the tryptophan fluorescence is usually used as a probe for protein structural investigation, these findings suggested that the intrinsic pressure effect on tryptophan (or its derivatives) must be taken in consideration to explain the phenomenon observed in high pressure study on biomolecules when using the usual fluorospectroscopic approaches. In the present investigation, the mechanisms involved for pressure effects on tryptophan and its derivatives were explored and discussed.

Regional Distribution and Immunocytological Localization of Red Pigment Concentrating Hormone in the Crayfish Eyestalk

A polyclonal antibody was raised against synthetic tyrosinated crustacean red pigment concentrating hormone (RPCH-Tyr) with the sequence Tyr-Leu-Asn-Phe-Ser-Pro-Gly- Trp-NH 2 with a tryptophan amide at the carboxyl terminal end. Its specificity was tested in comparison with peptides of similar structure. It appears to recognize the three to five residues near the carboxyl terminal. Native RPCH in the crayfish eyestalk was determined by two methods: (a) immunoenzymatic assay (ELISA) using the aforementioned antibody; and (b) bioassay on segments of isolated crayfish tegumentary epithelium. The unitary content in whole eyestalks was 5.5 ± 1.0 nmol for samples ( n = 18) taken at night. The regional distribution of RPCH content in the eyestalk was determined. The greatest proportion (40%) was found in the sinus gland, and the lowest in the retina plus lamina ganglionaris (6%). The medulla interna, medulla externa, and medulla terminalis contained similar proportions (about 16% each). The highest specific content was in the sinus gland (65.0 vs 24.4 pmol/μg protein for the whole eyestalk). Immunopositive neurons were identified in the various regions of the eyestalk. In 22 preparations, an average of 7 cells were identified in the ventromedial rim of the medulla terminalis, sending axons to the sinus gland, after branching in the neuropil of the medulla terminalis. Dorsally, 2 cells were identified in the medulla interna and 4 large cells and 11 small cells were located in the medulla externa in close proximity to the lamina ganglionaris: none of these cells appeared to project to the sinus gland. Profuse immunopositive fibers were found in the lamina ganglionaris projecting distally toward the base of the retina. Immunopositive axons were also found in the optic nerve.

Peptide Synthesis Catalyzed by an Antibody Containing a Binding Site for Variable Amino Acids

Monoclonal antibodies, induced with a phosphonate diester hapten, catalyzed the coupling of p-nitrophenyl esters of N-acetyl valine, leucine, and phenylalanine with tryptophan amide to form the corresponding dipeptides. All possible stereoisomeric combinations of the ester and amide substrates were coupled at comparable rates. The antibodies did not catalyze the hydrolysis of the dipeptide product nor hydrolysis or racemization of the activated esters. The yields of the dipeptides ranged from 44 to 94 percent. The antibodies were capable of multiple turnovers at rates that exceeded the rate of spontaneous ester hydrolysis. This achievement suggests routes toward creating a small number of antibody catalysts for polypeptide syntheses.

Rat Cathepsin H-Catalyzed Transacylation: Comparisons of the Mechanism and the Specificity with Papain-Superfamily Proteases1

We found that rat cathepsin H showed strong transacylation activity under physiological conditions. It is a feature of cathepsin H to utilize amino acid amides not only as acyl-acceptors but also as acyl-donors in the reaction. The pH-dependence of the transacylation activity was distinct from those of other papain-superfamily proteases. The alkaline limb (pKapp = 7.5) could be regarded as the pKa of the alpha-amino group of the acyl-donor, which was also involved in the original amino-peptidase activity. The acidic limb (pKapp = 5.8) was suggested to be involved in the deacylation step, where amino acid amide attacked the acyl-intermediate as a nucleophile in place of water in the hydrolysis. Although the N alpha-deprotonated acyl-acceptor, which is supposed to govern the nucleophilic attack, has a small population in the acidic pH range (above pH5), the transacylation was detectable even at the acidic pH-range because of the high S1'-site binding ability and suitable nucleophilicity of the acyl-acceptor. In the transacylation between various amino acid amides, the S1 and S1' site appeared to prefer hydrophobic residues without and regardless of a branch at beta-carbon, respectively. From these results and the sequence homology in the papain superfamily, we concluded that the reaction was governed by the acyl-donor having a protonated amino group, the acyl-acceptor having a deprotonated amino group and the remarkable hydrophobic character (especially favoring tryptophan amide) of the S1' site, presumably reflecting the good conservation of Trp177 in papain-superfamily proteases.

Cleavage kinetics and anchor linked intermediates in solid phase peptide amide synthesis

Kinetics and cleavage conditions of peptide amide synthesis were studied using the anchor molecules 5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid (4-ADPV-OH) and 5-(2'-aminomethyl-3'-5'-dimethoxyphenoxy) valeric acid (2-ADPV-OH). Unexpectedly the anchor amide alanyl-4-ADPV-NH2 was isolated and characterized as an intermediate during the cleavage with trifluoroacetic acid (TFA) of alanyl-4-ADPV-alanyl-aminomethyl-polystyrene to yield the alanine amide. As a matter of fact the NH--CH alpha bond of the alanyl spacer has to be cleaved to form this intermediate. Using TFA-dichloromethane (1:9) alanyl-4-ADPV-NH2 was obtained as a cleavage product in 50% yield within 60 min, whereas the isomeric alanyl-2-ADPV-NH2 was formed more slowly under these mild conditions. At high TFA concentration no difference between the 2- and 4-ADPV anchor was observed in the rate of formation of the free alanine amide. The presence of tryptophan amide in the cleavage mixture resulted in an anchor alkylated tryptophan amide, which remains stable in acidic solution but disappears rapidly in the presence of the resin. A low TFA/high TFA cleavage procedure is recommended for peptide amid synthesis applying the ADPV anchor.

Substituent effects on the electronic spectroscopy of tryptophan derivatives in jet expansions

Electronic excitation spectra of seven tryptophan derivatives entrained in a supersonic expansion have been recorded using both resonantly enhanced two-photon ionization and laser induced fluorescence. Two derivatives, tryptophan amide and tryptophan methyl amide, were found to have substantial low frequency vibrational progressions in their excitation spectra, yet in both compounds this behavior was apparent in only one conformer. Other derivatives did not display as much vibronic activity. Conformers which had vibrational progressions were found to emit in a broad band far to the red of excitation. All other conformers were found to fluoresce most strongly in resonance with excitation. The presence of low frequency vibrational activity and red shifted fluorescence correlates well with the ability of the derivative to form an intramolecular hydrogen bond between the amine and the carboxylic acid. Backbone conformers that contain an intramolecular hydrogen bond are expected to have large dipole moments, which may strongly perturb the electronic structure of the indole chromophore.

Pronase-catalysed hydrolysis of amino acid amides

Pronase from Streptomyces griseus exhibits a high activity and enantioselectivity in the hydrolysis of three amino acid amides: leucine amide, phenylalanine amide and tryptophan amide. The influence of pH, temperature and metal ions on pronase (EC 3.4.24.4) amidase activity and enantioselectivity has been studied.

Reaction of [formula omitted] amide with d-xylose or d-glucose in acidic solution

The reaction of N α- acetyl- dl)- tryptophan amide (a model for proteinbound tryptophan) with either d- xylose or d- glucose in acidic solution at 75 or 100°C gave the respective N α- acetyl-1-(β- d- glycopyranosyl)- dl- tryptophan amides as the main products in 10–20% yield. These results indicate that the indole nitrogen of protein-bound tryptophan may react with reducing carbohydrates during acid hydrolysis of proteins or, to a lesser extent, during food processing in acidic conditions.

Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones.

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl-lysine chloromethyl ketone, and tosylamide-phenylethyl-chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero-receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha-fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.

Experimental and calculated conformational characteristics of the bicyclic heptapeptide phalloidin

Several conformations generated from approximate potential energy calculations are presented for the bicyclic heptapeptide phalloidin which are consistent with the conformation-dependent information obtained from proton nuclear magnetic resonance measurements performed on phalloidin in dimethylsulfoxide solution. In each conformation, the cysteine amide proton is intramolecularly hydrogen bonded, the tryptophan amide is internally buried and the methyl group of the alanine residue preceding tryptophan is shielded by the tryptophan ring. Thus, phalloidin appears to be a relatively rigid molecule in solution.