Absorption of an Oxytocin Antagonist (Antocin) and a Vasopressin Analogue (dDAVP) Through a Standardized Skin Erosion in Volunteers

Purpose. Transdermal administration of the peptides [Mpa¹, D-Tyr (Ethyl)², Thr⁴, Orn⁸]-oxytocin (antocin) and [Mpa¹, D-Arg⁸]-vasopressin (dDAVP) was studied in healthy volunteers. Methods. A standardized skin erosion was formed preliminary by suctioning. The peptides were administered in plastic reservoirs through a 5 mm erosion and the absorption was followed for a six-day period with plasma concentration determinations on days 1, 3 and 6 with refilling the reservoirs daily with 15 µm and 10 mM solutions of dDAVP and antocin, respectively. Fourteen healthy non-smoking volunteers divided equally between the sexes, participated in the study. Plasma concentrations were measured using specific radioimmunoassays. Reservoir concentrations and metabolic stability of the peptides were determined using reverse-phase HPLC. Results. Both antocin and dDAVP were absorbed across the skin erosion. The absorption pattern was biphasic with a high initial absorption during days 1 and 2 followed by a lower absorption on days 3 and 6. The absorption on day 1, which was estimated at more than 50% for both peptides during a 24 h period, corresponded to a simultaneous decrease in peptide concentration in the reservoirs. The extent of absorption for antocin on days 3 and 6 was 1/3 to 1/6, respectively, of that observed on day 1. Antocin was minimally degraded in the skin reservoir while dDAVP was intact. However, accumulation of cellular material appeared in the antocin reservoirs. The absorption of antocin was reduced by exposure to intact skin surrounding the skin erosion. No pain was experienced and no scar formation was observed. Conclusions. The observed biphasic absorption may be a consequence of the mild inflammatory response occurring subsequent to eroding the skin. The standardized skin erosion may provide a route for the short-term delivery of otherwise poorly absorbable peptide and protein drugs.     

Differences in Transport Rate of Oxytocin and Vasopressin Analogues Across Proximal and Distal Isolated Segments of the Small Intestine of the Rat

The transmural intestinal passage of some oxytocin and vasopressin analogues (oxytocin, OT; [Mpa¹, D-Arg⁸]vasopressin, dDAVP; [Mpa¹, Tyr (OMe)², carba⁶]oxytocin, carbetocin; [Mpa¹, D-Tyr (OEt)², Thr⁴, Orn⁸]vasotocin, antocin II; [Mpa¹, D-Tyr (OEt)², Thr⁴, desPro⁷Orn⁸Gly⁹NH₂]tocinoic acid-NH(CH₂)₃NH₂, desPOG-antocin II-NH(CH₂)₃NH₂) was studied using isolated proximal and distal segments in the rat. All peptides (measured as peptide-like immunoreactivity) displayed a higher transport rate across distal intestinal segments as determined by radioimmunoassay (RIA). The smallest peptide, des POG-antocin II-NH(CH₂)₃NH₂, was transported at the fastest rate. No correlation of lipophilicity with transport rate was observed. Determination of the amount of peptide remaining in the mucosal media at the end of the incubation period by HPLC did not reveal any visible degradation products. However, the large difference in transport rate between [³H]OT and immuno-reactive OT indicates mucosal metabolism of this peptide. [³H]d-DAVP was distributed in a larger mucosal volume than the extracellular space marker [³H]inulin, indicating tissue uptake, but was too low (<100% of buffer concentration) to make an active transport mechanism likely. The differences in peptide transport rates between proximal and distal intestinal segments are most likely due to a higher distal paracellular permeability despite a decreased absorptive surface area at this region.     

Oxytocin antagonists with changes in the Asn5 position shed light on hormone-oxytocin receptor interactions

Since oxytocin agonists and antagonists have different structure-activity relationships, we have investigated the stereostructural and stereoelectronic requirements of the Asn⁵ residue in oxytocin antagonists by the synthesis of four analogues of the potent, prolonged acting oxytocin antagonist [Pen¹,d -Phe²,Thr⁴,Orn⁸]-oxytocin (I) in which Asn⁵ was replaced respectively with Thr (II), Leu⁵ (III), Asp⁵ (IV) and Tyr⁵ (V). These analogues had pA₂ values in the antioxytocic in vitro rat uterine assay of 7.23 (I), 7.16 (II), 6.67 (III), 7.21 (IV), and 6.76 (IV), respectively. All were also found to be weakly potent in the in vivo anti-vasopressor assay in the rat. These studies demonstrate very different structural and stereoelectronic requirements for oxytocin agonists and antagonists when they interact with the oxytocin uterine receptor.     

Degradation of [Mercaptopropionic acid1, D-arginine8] - vasopressin (dDAVP) in Pancreatic Juice and Intestinal Mucosa Homogenate

Abstract: The degradation of the vasopressin analogue dDAVP was studied by reversed phase high-performance liquid chromatography (HPLC) after incubations in pancreatic juice and intestinal mucosa homogenates. dDAVP remained stable in pancreatic juice for a period of 60 min. while the parent hormone arginine vasopressin (AVP) was completely degraded within 5 min. In intestinal mucosa homogenates dDAVP was degraded with half-lives of 9 min. (fast phase) and 161 min. (slow phase), about four times slower than AVP. By amino-acid analysis it was confirmed that the metabolite [Mpa¹, Des-D-Arg⁸-Gly⁹ NH₂]-vasopressin was gradually produced. No other breakdown products were observed. These findings may be of value for the further development of more stable peptide analogues which may be effective upon oral administration.     

Design,Synthesis and Structure–Activity Relationship of New Arginine Vasopressin Analogues Containing Proline Derivatives in Position 2

In this study, we present the synthesis and pharmacological properties of new analogues of arginine vasopressin modified in the N‐terminal part of the molecule with proline derivatives: indoline‐2‐carboxylic acid (Ica) and (2S,4R)‐4‐(naphthalene‐2‐ylmethyl)pyrrolidine‐2‐carboxylic acid. All the peptides were tested for pressor, antidiuretic and in vitro uterotonic activities. We also determined their binding affinity to the human oxytocin receptor. The Ica² substitution resulted in two moderately potent and selective antioxytocic agents: [Mpa¹, Ica², D‐Arg⁸]VP and [Mpa¹,Ica²,Val⁴,D‐Arg⁸]VP (pA₂ = 7.09 and 7.50, respectively). On the other hand, peptides modified with (2S,4R)‐4‐(naphthalene‐2‐ylmethyl)pyrrolidine‐2‐carboxylic acid, apart from their moderate antioxytocic activity, turned out to be weak antagonists of the pressor response to arginine vasopressin. The results of this study provide useful information about the structure–activity relationship of arginine vasopressin analogues and can help to design compounds with desired biological properties.     

The influence of steric interactions on the conformation and biology of oxytocin. Synthesis and analysis of penicillamine6‐oxytocin and penicillamine6‐5‐tert‐butylproline7‐oxytocin analogs

Abstract: Six [Pen⁶]oxytocin analogs were synthesized by substituting penicillamine for cysteine in oxytocin, [Mpa¹]oxytocin, [dPen¹]oxytocin, [5‐t‐BuPro⁷]oxytocin, [Mpa¹, 5‐t‐BuPro⁷]oxytocin and [dPen¹, 5‐t‐BuPro⁷]oxytocin. When tested in the uterotonic test in vitro[Pen⁶]oxytocin, [Pen⁶, 5‐t‐BuPro⁷]oxytocin, [Mpa¹, Pen⁶]oxytocin and [Mpa¹, Pen⁶, 5‐t‐BuPro⁷]oxytocin, all were found to possess both agonistic and antagonistic properties. Their agonistic potency ranged from negligible (0.08 IU/mg) to low (5.85 IU/mg) and their antagonistic potency (pA2) was estimated to range from 6.6 to 7.9. [dPen¹, Pen⁶]Oxytocin and [dPen¹, Pen⁶, 5‐t‐BuPro⁷]oxytocin were found to be pure antagonists with similarly high pA2 values of ≈ 8.2. Replacement of proline by 5‐tert‐butylproline increased binding affinity by a factor of two in [Pen⁶]oxytocin and had no influence on the binding affinity of [Mpa¹, Pen⁶]oxytocin and [dPen¹, Pen⁶]oxytocin. Assignment of the proton signals for prolyl amide cis‐ and trans‐isomers by NMR experiments in water indicated that the Pen⁶?5‐tert‐BuPro⁷ peptide bond cis‐isomer population was augmented relative to the prolyl peptides and measured, respectively, at 20, 35 and 35% in the 5‐tert‐butylproline⁷ analogs of [Pen⁶]oxytocin, [Mpa¹, Pen⁶]oxytocin and [dPen¹, Pen⁶]oxytocin. This augmentation in cis‐isomer population was correlated with a 21‐fold reduction in the agonistic potency and 2‐fold augmentation in antagonistic potency for [Pen⁶, 5‐t‐BuPro⁷]oxytocin relative to [Pen⁶]oxytocin. Augmentation of cis‐isomer population was also correlated to reduced agonist potency without effect on antagonism on conversion of [Mpa¹, Pen⁶]oxytocin to [Mpa¹, Pen⁶, 5‐t‐BuPro⁷]oxytocin. In the potent oxytocin antagonist, [dPen¹, Pen⁶]oxytocin, substitution of 5‐tert‐butylproline for proline augmented the cis‐isomer population without affecting antagonistic potency. The synthesis and evaluation of [Pen⁶]oxytocin and [Pen⁶, 5‐t‐BuPro⁷]oxytocin analogs 1–6 indicated that steric interactions influenced agonist and antagonist activity by modifying peptide conformation. Augmentations in the prolyl cis‐isomer population caused by 5‐tert‐butylproline occurred concurrently with enhanced or maintained antagonistic potency and binding affinity and reduced agonistic potency.     

THE SOLUTION CONFORMATION OF THE FERRICHROMES.

Ferrichrome, ferricrocin and ferrichrysin are ferric cyclohexapeptides whose general primary composition is represented by Res³-Res²-Gly¹-Orn³-Orn²-Orn¹, where the Res^2,3 sites are occupied by glycyl or l -seryl residues and Orn^1,2,3 stands for δ-N-acetyl-δ-N-hydroxy-l -ornithyl. The 220 MHz proton magnetic resonance spectra of deferriferricrocin and deferriferrichrysin in aqueous and in deuterodimethyl sulfoxide solutions are reported and discussed in terms of the molecular conformations. In (CD₃)₂SO the chemical shift temperature dependences of the amide proton resonances suggest two-fold symmetric structures containing two intramolecular hydrogen bonds. Such hydrogen-bonding, and other features of the data, are consistent with antiparallel β-pleated sheet structures, as in the Schwyzer model for cyclohexapeptides. In terms of sites paired by the hydrogen bonds, however, the β-fold differs. In water a random conformation is suggested for these peptides.     

Influence of L-naphthylalanine in position 3 of AVP and its analogues on their pharmacological properties

We describe the synthesis and pharmacological properties of two series of analogues: one which consists of three peptides having L-1-naphthylalanine in position 3 and the second composed of analogues substituted in position 3 with L-2-naphthylalanine. All peptides were tested in bioassays for pressor and antidiuretic activities. We also checked the uterotonic activity in vitro. We observed that the activity of counterparts in both series is, in two cases, strikingly different. One of. the new analogues, [(L-2-Nal)³, (D-Arg)⁸]VP is among the most potent antagonists of the vasopressor response to AVP. Moreover, it is the first potent V₁ antagonist devoid of antiuterotonic activity. This analogue was designed without modification of position 1, which was previously thought to be essential for substantial pressor antagonism. Two other peptides, [Mpa¹, (L-2-Nal)³, (o-Arg)⁸]VP and [Mpa¹, (L-1-Nal)³, D-Arg)⁸]VP, are highly potent V₂ agonists. The second analogue is highly selective. With the exception of [(L-2-Nal)³]AVP, which showed weak antioxytocic activity, (L-Nal)³ modification resulted in the almost complete removal of interaction of our analogues with oxytocic receptors in vitro. Our results suggest that position 3 in AVP and its analogues is important not only for binding and recognition as previously thought, but also for pressor, antidiuretic and uterotonic activities. We also assume that the hindering effect caused by bulky naphthyl moiety has a significant impact on the bioactive conformations of molecules which contain Nal residue, and can thus influence their interaction with V₁, V₂ and oxytocic receptors. © Munksgaard 1997.     

Evaluation of Thr6-bradykinin purified from Polybia occidentalis wasp venom in the choline uptake of mammal cortices

Context: Thr⁶-bradykinin is a peptide found in the venom of social and solitary wasps. This kinin, along with other bradykinin-like peptides, is known to cause irreversible paralysis in insects by presynaptic blockade of cholinergic transmission. However, this activity has never been tested in mammals.

Objective: As such, the objective of this study was to evaluate the effect of Thr⁶-bradykinin on the cholinergic system of rats.

Materials and methods: The peptide was isolated from the venom of the Neotropical social wasp Polybia occidentalis Olivier (Vespidae). After correct identification and quantification by ESI-MS and MS/MS, the peptide was tested in [¹⁴C]-choline uptake using rat cortical synaptosomes. Each uptake assay was accompanied by lactic acid dehydrogenase (LDH) activity measurement to evaluate synaptosome integrity in the presence of six increasing concentrations of BK or Thr⁶-BK (0.039, 0.156, 0.625, 2.500, 10.000 and 40.000?μM).

Results: Data revealed that neither BK nor Thr⁶-BK at any of the six concentrations tested (from 0.039 to 40.000?μM) affected [¹⁴C]-choline uptake in synaptosomes. Moreover, there was no increase in LDH in the supernatants, indicating that BK and Thr⁶-BK did not disrupt the synaptosomes.

Discussion and conclusion: In contrast to previous reports for the insect central nervous system (CNS), Thr⁶-BK had no effect on mammalian cholinergic transmission. Nevertheless, this selectivity for the insect CNS, combined with its irreversible mode of action may be relevant to the discovery of new sources of insecticides and could contribute to understanding the role of kinins in the mammalian CNS.     

A new neurohypophysial peptide,seritocin ([Ser5,Ile8]-oxytocin), identified in a dryness-resistant African toad,Bufo regularis

From the pituitary neurointermediate lobe of the African toad Bufo regularis, vasotocin, hydrin 2 (vasotocinyl-Gly) and a mesotocin-like peptide have been isolated by HPLC and characterized by mass spectrometry, amino acid sequence and chromatographic coelution with synthetic peptides. The mesotocin-like peptide has been identified as [Ser⁵,Ile⁸]-oxytocin in place of mesotocin ([Ile⁸]-oxytocin) found in all other amphibians investigated to date. The name seritocin is suggested. The molecule is virtually devoid of oxytocic activity on rat uterus in contrast to mesotocin. On the other hand, the molar ratio of hydrin 2 to vasotocin in the pituitary reaches 2, whereas it is about 1 in toads and frogs from temperate regions. B. regularis is an anuran species able to withstand a hot and dry season by burrowing. The possible relationship between occurrence of seritocin and Actaptation to arid environment remains to be demonstrated. © Munksgaard 1995.     

Cyclic analogues of Thr6-bradykinin,N8-Lys-bradykinin and endo-Lys8a-vespulakinin 1

Syntheses are described of the endo-Lys^8a-vespulakinin 1 and of cyclo-Thr⁶- and cyclo-N^ε-Lys-bradykinin. The linear peptides covering the entire sequences of endo-Lys^8a-VSK-1 and Thr⁶-BK, and the decapeptide containing all residues constituting Lys-BK, with a Arg-Lys peptide bond involving the ε-amino function of lysine, were prepared by the solid-phase procedure based on Fmoc chemistry. Cyclization was carried out by the diphenylphosphorazide method. The amino-terminal octapeptide sequence of vespulakinin 1, Fmoc-Thr(tBu)-Ala-Thr(tBu)-Thr(tBu)-Arg(Pmc)-Arg(Pmc)-Arg(Pmc)-Gly-OH, and its N^α-Boc-[(Gal β)Thr³, (Gal β)Thr⁴]-analogue, were used to prepare N^α-(1–8 VSK 1)-cyclo-N^ε-kallidin and N^α-[(Gal β)Thr³, (Gal β)Thr⁴, 1–8 VSK 1]-cyclo-N^ε-kallidin. Peptides and glycopeptides were characterized by amino-acid analysis, optical rotation, analytical HPLC and FAB-MS. Consistent with previous findings, preliminary pharmacological experiments on smooth muscle preparations showed that the cyclic, or partially cyclic, analogues were significatively less potent than the linear ones. © Munksgaard 1995.     

RELATION BETWEEN STRUCTURE AND FUNCTION IN SOME PARTIALLY SYNTHETIC RIBONUCLEASES S‘. II.*

The S-protein activating ability of [Orn¹⁰,Glu¹¹]-, [Orn¹⁰,Leu¹¹]- and [Orn¹⁰,Lys¹¹]-S- peptide was tested, before and after guanidination, by exploring their capacity to regenerate ribonuclease activity when recombined with S-protein. The kinetic parameters K_m and k₂ of the resulting modified enzymes were also calculated using C > p and CpC as the substrates. In agreement with previous observations the results obtained indicate that the carboxamide function in position 11 is not essential for biological activity, but contributes by optimizing the RNase A catalytic activity.     

Influence of serine in position i on conformation and dynamics of reverse turns

NMR spectroscopy has been employed for the conformational analysis of the cyclic hexapeptide cycle(-d -Pro¹-Ala²-Ser³(Bzl)-Trp⁴-Orn⁵(Z)-Tyr⁶-) with and without protecting groups on Ser³ and Orn⁵. This peptide sequence was derived from the active loop sequence of the α-amylase inhibitor Tendamistat (HOE 467). The aim was to investigate the role of serine in position i of a standard β-turn on the conformation and stabilization of this turn. Based on distance and torsion constraints from 2D NMR spectroscopic measurements in DMSO-d₆ solution, structure refinement was accomplished by restrained molecular dynamics (MD) simulations in vacuo and in DMSO. The analysis of both structures in solution reveals a considerable effect of the unprotected serine sidechain on the adjacent β-turn conformation. While in the protected peptide with Ser³(Bzl) a βII-turn is observed between Trp⁴ and Orn⁵, the deprotected compound reveals a βI-turn in this region. The βI-turn is stabilized by a backbone-sidechain hydrogen bond from Orn⁵N_αH to Ser³O_γ. Comparisons with other NMR-derived solution structures of cyclic model peptides and in some protein structures from literature reveal a general structural motif in the stabilization of βI-turns by serine in the i position through backbone-sidechain interactions. © Munksgaard 1995.     

Quantitative comparison between oxytocin and four related neurohypophysial peptides on the human uterus in situ

Quantitative assays of four synthetic peptides related to oxytocin were carried out on the post partum human uterus in vivo by means of external tocography. The following activities (U/mg, means and standard errors) were found: isoleucine⁸-oxytocin 365±70, asparagine⁴-oxytocin 150±40, desamino¹-oxytocin 1,030±300, serine⁴-isoleucine⁸-oxytocin 335±95. None of the conventional bioassay procedures proved to be fully reliable for predicting the oxytocic activity in man of peptides related to oxytocin. If the tocographically recorded effects of equipotent doses of the compounds are compared with those of oxytocin no qualitative differences can be observed.     

Theoretical conformational analysis of six arginine vasopressin analogs with the l‐naphthylalanine in position 3

Abstract: Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with l ‐2‐Nal in position 3 causes moderate antiduretic activity, whereas [Mpa¹, (l ‐1‐Nal)³, (d ‐Arg)⁸] VP and [Mpa¹, (l ‐2‐Nal)³, (d ‐Arg)⁸] VP are potent and selective V₂ agonists. Moreover [(l ‐2‐Nal)³, (d ‐Arg)⁸] VP is among the most potent and selective antagonists of V_1a receptors. In this study we carried out conformational calculations on [(l ‐1‐Nal)³] AVP, [(l ‐2‐Nal)³] AVP, [(l ‐1‐Nal)³, (d ‐Arg)⁸] VP, [(l ‐2‐Nal)³, (d ‐Arg)⁸] VP, [Mpa¹, (l ‐1‐Nal)³, (d ‐Arg)⁸] VP, [Mpa¹, (l ‐2‐Nal)³, (d ‐Arg)⁸] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low‐energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest‐energy conformations in the presence and absence of water had β‐turns at residues Phe³‐Gln⁴ and Gln⁴‐Asn⁵ and Gln⁴‐Asn⁵, respectively. The conformation at the Gln⁴‐Asn⁵ turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).     

SYNTHESIS OF ANALOGS OF THE N-TERMINAL EICOSAPEPTIDE SEQUENCE OF RIBONUCLEASE A

For a better understanding of the role played by glut amine 11 either in the catalytic function of ribonuclease A or in the S-peptide/S-protein association process the following S-peptide analogs have been synthesized: [Orn¹⁰,Leu¹¹]- and [Orn¹⁰, Lys¹¹]- S peptide. The S-protein activating ability of the two synthetic S-peptide analogs was tested, before and after guanidination, by exploring their capacity to generate ribonuclease activity, against RNA as substrate, when recombined with S-protein.     

Analogues of arginine vasopressin and its agonist and antagonist modified in the N‐terminal part of the molecule with l‐β‐homophenylalanine

Abstract: In continuation of our efforts to elucidate the role of positions 2 and 3 in arginine vasopressin (AVP) and its analogues, we designed and synthesized peptides modified in these positions with l ‐β‐homophenylalanine (β‐Hph). Two of them had just this single modification, the next two peptides are analogues of the V₂ agonist, namely [3‐mercaptopropionic acid (Mpa)¹]AVP (dAVP). The last two compounds were designed by substitution of positions 2 or 3 of a potent V_1a antagonist, [1‐mercaptocyclohexaneacetic acid (Cpa)¹]AVP, with β‐Hph. All the peptides were tested for their pressor and antidiuretic and uterotonic in vitro activities in the rat. All the activities tested have been found to be significantly decreased. Three analogues, i.e. [Mpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph²]AVP, [Cpa¹,β‐Hph³]AVP, turned out to be uterotonic antagonists with pA₂ = 6.3 ± 0.2, 6.3 ± 0.1, 6.0 ± 0.3 respectively. The last one exhibited antipressor properties also (pA₂ = 6.4 ± 0.1).     

Design,Synthesis, and Biological Evaluation of Novel Peptide Gly3‐MC62 Analogues as Potential Antidiabetic Agents

Two series of conformationally constrained analogues from Gly ³ ‐ MC 62 were designed by scanning the residues Lys¹, Thr², Met⁴, Lys⁵, Met^7, and Ala⁸ with an i‐(i + 2) lactam bridge consisting of a Glutamic acid–xaa–lysine (Glu–Xaa–Lys) scaffold and a diproline fragment. They were synthesized and evaluated for their antihyperglycemic effects. Through screening in normal and mice with diabetes mellitus, peptides II ‐5 , III ‐3 , III ‐4 , and III ‐5 showed significant improvement in antihyperglycemic and antioxidative activities compared with Gly ³ ‐ MC 62 , especially the compound III ‐4 . The primary mechanism of the compounds ( II ‐5 , III ‐3 , III ‐4 , and III ‐5 ) underlying this effect is the islet β‐cells against oxidative damage induced by STZ, and III ‐4 ‐treated mice showed considerable improvement in the preservation of beta cells in the pancreatic islets of DM mice. These data suggested that III ‐4 could be candidate for the future treatment of diabetes mellitus.     

STUDIES ON RIBONUCLEASE S: THE ROLE OF LYSINE-7 FOR ACTIVATION OF S-PROTEIN*

In order to describe further the role of the invariant amino acid residue lysine-7 in the RNase S' system, potential catalytic activities of des-Lys⁷-[Orn¹⁰]-, [Ala⁷, Orn¹⁰]-, [Lys⁴, Ala⁷, Orn^1.0]-S-peptide and corresponding guanidinated derivatives were studied. New information was obtained about S-protein binding capacities of the analogs relative to that of unmodified S-peptide, and about conformation of the corresponding RNase S' analogs by studies on competitive inhibition, difference spectroscopy, difference circular dichroism and thermal transition. Peptides modified in position 7 were no weaker in strength in peptide-protein noncovalent interaction in the presence of substrate. In the absence of substrate the variously modified S-peptide analogs were differentiated in their S-protein affinity. Conformational changes in the geometry of the active site of the reconstituted enzymes, if present, must be smaller than the detection limits of the techniques used. There is strong implication that the change in catalytic efficiency has to be attributed to an alteration or to the position of the charge and that lysine-7 in the natural enzyme is involved in stabilizing the transition state intermediates during decomposition of the substrate. Further substantiation was provided by substrate inhibition studies as well as 3-CMP binding measurements, since 3-CMP binding affinity of RNase S' modified in position 7 was found to be significantly lower than that of RNase S' itself.     

Peptide conformations. 33. Conformational analysis of cyclic enkephalin analogs of the type Tyr-cyclo-(-N ω -Xxx-Gly-Phe-Leu-)*

Four conformationally restricted cyclic enkephalin analogs of the type Tyr-cyclo(-Nω -Xxx-Gly-Phe-Leu-) with Xxx = l -Orn, d -Orn, l -Lys and d -Lys have been synthesized by conventional methods and the conformation of the Bocprotected, the deprotected as well as the des Tyr¹ analogs analysed by proton and nitrogen n.m.r. spectroscopy. The assignments, of the resonances were performed by two-dimensional homo- and heteronuclear correlated spectroscopy in the normal (COSY, SECSY) as well as a modified (for the detection of small couplings) version. NOE difference spectroscopy was used to distinguish the amino acid residues with aliphatic side chains. The n.m.r. parameters suggest a rather rigid conformation of the ornithine analogs with two internal NH protons whereas the lysine peptides appear to be more flexible. The structure of the Orn²-analogs can be described by a Gly³-CO HN-Leu⁵-γⁱ-turn and a hydrogen bond Orn²-CO HN -Orn². The d -lysine compound seems to include a β-turn (Gly³-CO HN-d -Lys²). The relation of the different conformational properties of the four cyclic peptides and their biological activities are briefly discussed.