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This work describes the synthesis of thirteen cyclooctapeptides dicarba-analogues of Somatostatin, containing l- or d-allylglycine (Agl) residues at the termini of the peptide chain, through on resin Ring Closing Metathesis (RCM) of the linear octapeptides. We investigated the influence of the stereochemistry of some strategic amino acids on the propensity to give the cyclic compounds in mild conditions (refluxing DCM). Systematic individual replacement of Phe6,7,11 residues with the corresponding enantiomers, strongly favoured the ring closure by conventional heating. The yield of the cyclic products was strictly correlated to the position of this amino acid on the peptide chain. In particular substitution of Phe6 by Tyr in peptides which did not give the cyclic compounds, allowed the ring formation. The effect of the phenolic −OH function of Tyr side chain on the proximity of the terminal Agl residue was studied by NMR techniques. All the linear precursors gave cyclic somatostatin dicarba-analogues, in good to high yields and in short reaction times, by microwave-assisted RCM, performed with the 2nd generation Grubbs catalyst. The unsaturated dicarba-tether resulted in a mixture of E and Z stereoisomers in a variable ratio, depending on the sequence and the cyclization method. The E isomer was largely the most abundant in all but one the described product.

In vivo somatostatin receptor scintigraphy is a valuable method for the visualization of human endocrine tumours and their metastases. In fact, peptide ligands of somatostatin receptors (sst’s) conjugated with chelating agents are in clinical use. We have recently developed octreotide dicarba-analogues, which show interesting binding profiles at sst’s. In this context, it was mandatory to explore the possibility that our analogues could maintain their activity also upon conjugation with DOTA. In this paper, we report and discuss the synthesis, binding affinity and conformational preferences of three DOTA-conjugated dicarba-analogues of octreotide. Interestingly, two conjugated analogues exhibited nanomolar affinities on sst2 and sst5 somatostatin receptor subtypes.

A limited set of novel octreotide dicarba-analogues with non-native aromatic side chains in positions 7 and/or 10 were synthesized. Their affinity toward the ssts1-5 was determined. Derivative 4 exhibited a pan-somatostatin activity, except sst4, and derivative 8 exhibited high affinity and selectivity toward sst5. Actually, compound 8 has similar sst5 affinity (IC50 4.9 nM) to SRIF-28 and octreotide. Structure-activity relationships suggest that the Z geometry of the double-bond bridge is that preferred by the receptors. The NMR study on the conformations of these compounds in SDS(-d25) micelles solution shows that all these analogues have the pharmacophore beta-turn spanning Xaa7-D-Trp8-Lys9-Yaa10 residues. Notably, the correlation between conformation families and affinity data strongly indicates that the sst5 selectivity is favored by a helical conformation involving the C-terminus triad, while a pan-SRIF mimic activity is based mainly on a conformational equilibrium between extended and folded conformational states.

The synthesis of four biotin derivatives carrying two DOTA moieties for each ligand (BisDOTA set) is reported, for increasing radiation/dose ratio and improving efficiency in the pretargeted avidin-biotin radioimmunotherapy. The biotin-containing scaffold of two BisDOTA was similar to the mono-DOTA derivative previously described. Then the scaffold was elongated by trifunctionalized spacers of different length and conjugated with one of the COOH groups of two DOTA. Two others were prepared starting from a on-resin lysine residue. The lysine alpha-NH2 was bonded to biotin, and then spacers were appended to the epsilon-NH2 and conjugated with two DOTA molecules. One compound contained a p-aminobenzoic acid spacer, which ensured higher head-to-tail distance and increased rigidity of the chain. These last two compounds had a very high ability to bond avidin and were labeled with 90Y at high specific activity. All the compounds were resistant to the action of serum biotinidases.

A series of linear tetrapeptides containing two histidyl residues in position 2 and 4, namely DHGH, DHGdH, KHGH, KHGdH, Ac-DHGH-NH(2), Ac-DHGdH-NH(2), Ac-KHGH-NH(2), and Ac-KHGdH-NH(2), were synthesized and characterised. Their copper(II) binding properties were investigated in depth through a variety of physicochemical methods. Potentiometric titrations were first carried out to establish the stoichiometry and the stability of the resulting copper(II)-peptide complexes. The copper(II) chromophores that are formed in the various cases in dependence of pH were subsequently characterised by extensive spectroscopic analysis (UV-Vis, EPR, CD) in strict correlation with potentiometric data. The effects of the nature of the first amino acid (Lys versus Asp) and of N-terminal amino group protection on copper(II) binding were specifically addressed. On turn, the careful comparison of the copper(II) coordination abilities of the linear peptides with those of their cyclic analogs provided insight into the effects of cyclization on the overall metal binding properties.

A new, 14-membered, tetraza cyclic tetrapeptide containing histidine and lysine side-chains, c ( β 3 homoLysdHis β -AlaHis), was designed, synthesized and characterized; its copper(II) binding properties were investigated in dependence of pH by potentiometric and spectroscopic methods. In line with previous studies of similar systems, the progressive involvement of amide nitrogens in copper(II) coordination was evidenced for pH values greater than 6. At physiological pH the dominant species consists of a copper(II) center coordinated by two amide nitrogens, an imidazole nitrogen and a water molecule. In contrast, at pH values higher than 8.7, a copper(II) coordination environment consisting of four amide nitrogens in the equatorial plane and the axial imidazole ligands is formed as clearly indicated by spectroscopic data and theoretical calculations. The behavior of this 14-membered cyclic tetrapeptide is compared to that of its 12-membered cyclic analog, particular attention being paid to the effects of ring size on the respective copper(II) binding abilities.

Head-to-tail histidine containing cyclopeptides can be efficiently synthesised by a three-dimensional orthogonal solid-phase strategy (Fmoc/tBu/allyl) via anchoring the imidazole ring to trityl-resins. Furthermore, Fmoc-His(Trt-®-OA1 can be a useful starting support for the preparation of diketopiperazine combinatorial libraries.

We previously demonstrated that the lipopeptide of the myelin basic protein (MBP) immunodominant epitope in Lewis rat Palm-GpMBP(74-85) (Gp: guinea pig), which induced experimental autoimmune encephalomyelitisin vivo strongly increased the T cell proliferative responsein vitro. We extended this study to the human immunodominant epitope hMBP(83-99), synthesizing different lipophilic peptides bearing a hydrophobic chain linked through an amide or a C-C bond. To this aim, we developed a synthetic pathway for (±)-N-Fmoc-Ahd-OH (Ahd: 2-aminohexadecanoic acid) which was used to synthesize diastereomeric peptides which were successfully separated by reverse-phase high-performance liquid chromatography. MBP-specific T cell lines recognizing the immunodominant epitope hMBP(83-99) have been generated from patients affected by multiple sclerosis. Their proliferative response to the native peptide and to some lipoderivatives has been investigated. In contrast to the animal model, none of the investigated lipopeptides exhibited ‘superagonist’ activity.

A 13-membered ring cyclic tetrapeptide was synthesized by the solid-phase peptide synthesis method, and its copper(II) coordination properties were analyzed by optical spectroscopy, mass spectrometry, and electrochemistry. All collected data strongly support the presence, at alkaline pH, of a stable peptide/copper(III) complex that is formed in solution by atmospheric dioxygen oxidation. On the basis of previous studies on cyclic peptide/copper systems, we suggest that the copper(III) ion is at the center of the ligand's cavity being coordinated to four deprotonated amide nitrogen atoms. This donor set would greatly lower the redox potential for the CuIII/CuII couple, thus allowing easy oxidation of the coordinated copper(II) by atmospheric oxygen.

The tetrapeptide-Cu(II) complex H-( l -His-Gly) 2 -OH/Cu(II), indicated as L-Cu(II), has been investigated, as compared to the Cu(II) inorganic salt CuSO 4 , for its antioxidative and anti-inflammatory properties under a panel of experimental conditions. Both inorganic and organic Cu(II) compounds showed comparable activities in vitro and ex vivo by: (i) protecting, in a dose-dependent manner, rat brain homogenates from Fe(III)/ascorbate- or haemoglobin-induced lipid peroxidation; (ii) inhibiting the superoxide-mediated ferricytochrome c reduction by activated macrophages. CuSO 4 and L-Cu(II) also exhibited similar anti-inflammatory effects in vivo by reducing significantly the extent of carrageenan-induced edema in the rat paw. The activities of the two compounds diverged strikingly only in the xanthine/xanthine oxidase system at low phosphate buffer concentration. L-Cu(II) decreased the rate of NBT reduction by superoxide in a true SOD-like fashion without affecting urate production. Instead, Cu(II) ions caused the rapid xanthine oxidase inactivation thus inhibiting both urate and superoxide production; this effect might be ascribed to the superoxide-mediated generation of the strong oxidant Cu(III) and its interaction with the enzyme. The administration of Cu(II), whether complexed with linear oligopeptides or as an inorganic salt, to animals or tissue extracts, conferred protection against oxidation and ought, conceivably, to interact with endogenous biological molecules and form highly bioavailable complexes which serve, subsequently, as the real scavengers. Moreover, the claimed prominent scavenger activities of Cu(II)-oligopeptide complexes over inorganic copper ions could be realised only in very simple in vitro systems through mechanisms which, although of biochemical interest, are unlikely to be of physiopathological significance.

The influence of glycosylation on the structure-activity relationship of the tachykinin antagonist MEN 10376 and of its minimal active fragment MEN 10414 was investigated. The antagonist activity was preserved only when β-d-glucosylated Tyr, Ser and Asn were added to the N-terminal position of MEN 10376, while the modification of the side chain of the Tyr2 induced a dramatic decrease in affinity.

Analysis of the 1H–1H NOE effects observed for S-(11,13-dihydroalantolacton-13-yl)-L-cysteinyl-L-alanine methyl ester in (CD3)2SO at 14 T showed that the 7- and 11-protons are in cis positions. The distances between 7-, 8- and 11-protons of the adduct were derived by a semi-quantitative approach based on analysis of NOESY cross peaks and a computed molecular model. The simulated 1H spectrum of the alantolactone moiety is in excellent agreement with the experimental data. The complete assignments of the 13C signals of alantolactone, its peptide adduct and four dipeptide precursors are based on one- and two-dimensional 1H–13C NMR techniques.

The adamantane moiety was introduced in the tachykinin NK2 receptor-selective agonist [β-Ala8]-NKA(4–10) (H-Asp-Ser-Phe-Val-β-Ala-Leu-Met-NH2, MEN 10210) and in different positions of the NK2 receptor antagonist MEN 10376 (H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2) in order to investigate how this substitution affects their biological activity at tachykinin NK1, NK2 and NK3 receptors. 1-Adamantaneacetic acid (1-Ada-CH2COOH) was directly conjugated in the solid phase as the preformed OBt active ester to the N-terminal position of MEN 10210, obtaining MEN 10586 (1-Ada-CH2CO-Asp-Ser-Phe-Val-β-Ala-Leu-Met-NH2). The Pfp ester of adamantaneacetic acid (1) was prepared and used for the acylation of the N-terminal position of MEN 10376, yielding MEN 10606 (1-Ada-CH2CO-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2). Compound 1 was then used to obtain the building block Fmoc-Lys(1-Ada-CH2CO)-OH as a modified amino acid for the synthesis of MEN 10818 [H-Asp-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys(1-Ada-CH2CO)-NH2]. In order to investigate the biological activity of the peptide bearing the adamantane group together with the free N-terminal amino function, we synthesised MEN 10676 [H-Asp(O-2-Ada)-Tyr-d-Trp-Val-d-Trp-d-Trp-Lys-NH2] using Fmoc-Asp(O-2-Ada)-OH, in which 2-adamantanole was the protecting group of the aspartate β-COOH moiety during the peptide synthesis and survived the final peptide cleavage and deprotection carried out under controlled conditions. MEN 10586 showed an agonist activity comparable to that of the parent compound MEN 10210 at NK1 and NK2 receptors of guinea pig ileum, rabbit isolated pulmonary artery and hamster isolated trachea preparations, while it showed a 25-fold higher agonist activity at NK3 receptors of rat isolated portal vein. The three modified antagonist analogs displayed similar or reduced affinity at NK1, NK2 and NK3 receptors as compared to MEN 10376. The drop was particularly evident (>2 log units) at the NK2 receptors of the rabbit isolated pulmonay artery.

Cyclen derivatives, as coordinating ligands, have recovered considerable interest for the development of diagnostic and therapeutic drugs, mimicking the binding site of metalloproteins. We demonstrate that the on-resin reduction of head-to-tail cyclotetrapeptides, anchored to a solid support by the side-chain of a trifunctional amino acid, is an efficient synthetic strategy. The proposed approach leads to the cyclen scaffold still bound to the resin, ready for further decorations.

Background. Clodronate is used in high bone resorption diseases. Its action was defined as "cytotoxic" based on the induced cellular ATP loss, without any experimental verification of reversibility. In the present report the reversibility of clodronate action was tested on cultured human osteoclastic cell cultures. As "in vitro" bioeffects of clodronate are reversible, this compound should not be defined as "cytotoxic".Introduction. Bisphosphonates are pyrophosphate analogs able to inhibit osteoclast-mediated bone resorption widely used in the treatment of diseases with high bone turnover. Several evidences have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R(2)side chain. The nitrogen-containing bisphosphonates act on osteoclasts by preventing protein prenylation, while non-nitrogen-containing bisphosphonates, like clodronate, are metabolized intracellularly to a β-γ-methylene analog of ATP that induces inhibition of the ADP/ATP translocase.Materials and Methods. In order to evaluate clodronate effects on osteoclastic cells and the bioreversibility of its action, we have used a human preosteoclastic (FLG 29.1) cell line and primary cultures of human osteoclast-like (HOC) cells. Functional and differentiative modifications were evaluated with immunocytochemical tartrate-resistant acid phosphatase activity (TRAcP) assay and with rapid quantitative detection of the complex "matrix metalloproteinase 9/tissue inhibitor of metalloproteinase" (MMP9/TIMP1) by RT-PCR analysis based on "TaqMan" technology. The apoptosis phenomenon were detected by DNA ladder analysis and quantified by counting apoptotic cells with Transmission Electron Microscopy (TEM) analysis. Adenosine-5'-[ β - γ -dichloromethylene] triphosphate (AppCCl(2)p) was detected and identified in cell extract by HPLC-ESI-MS-MS Mass Spectrometry. Intracellular ATP modulation in the presence of clodronate was evaluated by luciferin-luciferase assay. The Mann-Whitney "U" test was conducted for statistical analysis.Results. We found that clodronate inhibited both proliferation and differentiative features of cells of the osteoclastic lineage. Furthermore, treatment of both cell types with clodronate caused apoptosis, generation of measurable levels of AppCCl(2)p, and reduction of intracellular ATP levels. Addition of ATP to the culture medium caused an inhibition of the biological actions of clodronate on the human osteoclastic cell lineage.Conclusions. These data indicate that intracellular accumulation of the metabolite AppCCl(2)p is the likely route by which clodronate inhibits osteoclastic function and this effect is reversed by ATP.

The tetrapeptide Ac-Gly-[Cys-Sec]-Gly-NH(2), reproducing the C-terminal motif of the selenoenzyme thioredoxin reductase, was designed and synthesized, and its reactions with a few medicinally relevant gold(i,iii) compounds investigated by ESI-MS. Remarkably, the main reaction products could be unambiguously identified providing valuable insight into the likely mechanisms of enzyme inhibition by gold compounds.

We describe synthesis, conformational studies, and binding to the five somatostatin receptors (sst 1-5) of a few analogues of the cyclic octapeptide octreotide (1), where the disulfide bridge was replaced by a dicarba group. These analogues were prepared by on-resin RCM of linear hepta-peptides containing two allylglycine residues; first- and second-generation Grubbs catalyst efficiencies were compared. The C=C bridge was hydrogenated via two different methods. Binding experiments showed that two analogues had good affinity and high selectivity for the sst5 receptor. Three-dimensional structures of the active analogues were determined by (1)H NMR spectroscopy. Conformation-affinity relationships confirmed the importance of D-Phe(2) orientation for sst2 affinity. Moreover, helical propensities well correlates with the peptide sst5 affinity. The presence of the bulky aromatic side chain of Tyr(Bzl)(10) favored the formation of a 3(10)-helix and enhanced the sst5 selectivity suppressing the sst2 affinity. Finally, a new pharmacophore model for the sst5 was developed.

Spiro[androst-4-en-17 alpha,5'-oxazolidine]-2',3,4'-trione 8a and spiro[androst-4-en-17 alpha,5'-oxazolidine]-2',3,4',11-tetraone 8b, two potentially bioactive spiranes, were prepared from the parent 17-ketones in four steps (64% and 49.5% yield, respectively). The key intermediates were the hydroxyimidates 5a and 5b, which easily underwent cyclization to the corresponding spirooxazolinone 4'-enol ethers when treated with alkylchlorocarbonates. The respective N-amyl derivatives of the spiranes 8a and 8b were obtained with n-pentyl bromide in the presence of KF. A new method for the synthesis of steroid 17 alpha-hydroxy-17-carboxyesters and 17 alpha-hydroxy-17-carboxamides is described. Attempts to synthesize the title compounds from these products were unsuccessful.

Induction of a state of tolerance (hyposensitization) in alantolactone-sensitized guinea pigs was attempted by subcutaneous injections of S -(dihydroalantolacton-10-yl)- l -cysteinyl- l -alanine methyl ester 7 , N -acetyl- l -cysteinyl- l -alanine methyl ester 11 or N -acetyl- l -cysteine. Compound 7 was prepared by addition of N -benzyloxycarbonyl- l -cysteinyl- l -alanine methyl ester 5 to alantolactone, followed by the removal of the Z-group. Dipeptide 11 was obtained from N -acetyl- S -ethylcarbamoyl- l -cysteinyl- l -alanine methyl ester. Treatment of dipeptide 5 with HBr-AcOH mixture afforded mainly the S -acetyl derivative 10 , from which dipeptide 11 was also obtained. Biological assays showed that the alantolactone-adduct 7 or N -acetyl- l -cysteine did not significantly modify the positive skin response to alantolactone in alantolactone-sensitive guinea pigs. In marked contrast, dipeptide 11 significantly decreased the skin reaction to alantolactone tested at either 0.25 or 0.08 μg. Control animals did not show skin responses to alantolactone neither after treatment with dipeptides 7,11 or N -acetyl- l -cysteine. The data suggest that dipeptide 11 is an efficient and non-toxic tolerogen in the case of guinea pigs sensitized to alantolactone.

Three novel cyclic tetrapeptides, containing either l- or d-histidine residues and either Lys or Asp side chains, namely c(HGd-HK) (1), cHGHD (2) and c(HGd-HD) (3), were designed, synthesized, characterized and tested as potential copper(II) ligands. Their pH dependent copper(II) binding properties were analysed in depth by a number of potentiometric and spectroscopic determinations. A rather exhaustive description of the species existing in solution has emerged for each copper(II)/oligopeptide system; solution structures for the individual species are proposed. The specific role of the various side chains in the overall metal coordination process is discussed in comparison with the case of Cu(II)-c(HGHK), previously reported. Data obtained in this study highlight the strong impact of the d-His residue on the metal binding abilities of these cyclic peptides. Remarkably, the cyclic tetrapeptides containing two l-His residues are able to form, at physiologically relevant pH values, a characteristic chromophore where the mononuclear copper(II) centre is simultaneously coordinated by two imidazole nitrogens and two amidic nitrogens of the tetrazadodecane ring. This latter type of copper(II) chromophore has been carefully modelled by computational methods. The potentialities of the applied experimental strategy are stressed.

Cyclotetrapeptides are constrained cyclic peptides whose synthesis is considered a difficult task. A methodology based on on-resin head-to-tail cyclization by anchoring the side chain of a trifunctional amino acid was investigated. A series of model cyclotetrapeptides containing the RGD sequence cyclo(Xaa-Arg-Gly-Asp) (Xaa = Ala, Phe, Phg, D-Ala, D-Phe, D-Phg) was synthesized with no cyclodimerization by-products. An evaluation and optimization study of all of the parameters directly involved in the ring closure was performed.

The complexes between copper(II) and four synthetic tetrapeptides bearing a single histidine residue within the sequence (AcHGGG, AcGHGG, AcGGHG and AcGGGH, respectively), have been investigated by potentiometric and spectroscopic methods (UV-Vis, circular dichroism and electron paramagnetic resonance). Potentiometric studies in the pH range 4-12 allowed identification and quantitative determination of the species present in solution for each copper-peptide complex. In all cases, upon raising pH, copper(II) coordination starts from the imidazole nitrogen of the His; afterwards three deprotonated amide nitrogens are progressively involved in copper coordination, except in the case of AcGHGG. Based on the potentiometric and spectroscopic results, detailed molecular structures are proposed for the dominant copper(II) tetrapeptide species existing in solution, either at neutral or alkaline pH. The structural consequences of the presence and of the location of a unique histidine residue within the tetrameric sequence are specifically analyzed. Results are discussed in relation to the modeling of copper(II) binding sites in proteins, particular emphasis being devoted to the copper complexes of the prion protein.

Stoichiometry, stability constants and solution structures of the copper(II) complexes of the N -acetylated tetrapeptide HisGlyHisGly were determined in aqueous solution in the pH range 2–11. The potentiometric and spectroscopic data (UV–Vis, CD, EPR and Raman scattering) show that acetylation of the amino terminal group induces drastic changes in the coordination properties of AcHGHG compared to HGHG. The N 3 atoms of the histidine side chains are the first anchoring sites of the copper(II) ion. At pH 4.7 and 5.6 both the imidazole rings cooperate in the formation of a 2N equatorial set, while, at higher pH values, 3N and 4N complexes are formed through the coordination of peptide N − atoms. The log β values of the copper complexes of AcHGHG are by far lower than those of the corresponding species in the parent Cu II –HGHG system.

We report for the first time the immunoadjuvant effects of lipoconjugation of peptide antigens in an in vitro system by using CD4+ T-cells. The lipopeptides obtained by conjugating a palmitoyl moiety at the N(alpha)-terminal of Gln(74) or at the epsilon-NH(2) of Lys(75) of GpMBP(74-85) induced increased T-cell responsiveness compared to the native nonlipidated peptide. On the other hand, lipoderivatives of GpMBP(82-98) did not increase the T-cell response, demonstrating that the superagonist inducing effect of lipoconjugation is epitope-specific. Digestion of the two native peptides with cathepsin D and L, both implicated in antigen processing, and with a complete lysosomal fraction of a EBV-transformed B cell line shows that GpMBP(74-85) is resistant to cellular proteases, while GpMBP(82-98) is easily digested by these enzymes. These results suggest that the first prerequisite for increasing the T-cell response by lipoconjugation is the stability of the native peptide to peptidases, providing an important insight into the understanding of the immunoadjuvant effect of lipoderivative antigens.

The complex formation between copper(II) and the tetrapeptide l -histidyl-glycyl- l -histidyl-glycine (HL) has been studied in aqueous solution in the pH range 2–10.5, by potentiometric and spectroscopic methods (visible, CD, EPR, 1 H NMR and Raman scattering). Between pH 3 and 6 the species [CuHL] 2+ and [CuH −1 L] have been detected. The former complex co-ordinates through two nitrogen and two oxygen atoms in the equatorial plane while, for the latter, the spectroscopic data (particularly Raman spectra) suggests an unusual structure with the two imidazole rings and two peptide NH in the plane. The species at pH 7, [CuH −2 L] − , is involved in the co-ordination by the amino group, the His 1 imidazole and two peptide nitrogens, while, for the complex [CuH −3 L] 2− , which seems to be predominant in alkaline medium, a third deprotonated backbone NH, replaces the imidazole nucleus.

The two histidyl-glycyl containing peptides, H(L-His-Gly)2-OH and its methyl ester (H(L-His-Gly)2-OCH3, have been structurally characterized by liquid secondary-ion mass spectrometry. Both high-internal-energy ion fragmentations produced in the source and metastable decompositions occurring in the first field-free region have been studied. The mass spectra show the presence of y-, a- and b-type ions, a1 being the most abundant fragment ion. The metastable decompositions are dominated by the loss of a water molecule and by y-type ions. The interactions of the two peptides with alkali metal ions (Li, Na, K) have been evaluated both by normal mass spectrometry and by tandem mass spectrometry to obtain selected daughter-ion spectra. The occurrence of mono-, bi- and trimetallated species has been detected in the gas phase. While, in the case of the protonated species, y-type ions are predominant, in the presence of an alkali metal ion (Cat), they show lower abundances (Cat = Li) or are absent (Cat = Na, K) both in the mass spectra produced in the source and in metastable decompositions. In most of these cases, a very intense low-internal-energy ion, which is represented by [a3 + Cat - H]+ and which can be produced by interaction of the metal with a deprotonated amide nitrogen, is recorded. This mechanism should be favored by the ?anchoring? effect exerted by the imidazole ring of the histidine which promotes interaction with metals. Other metastable decompositions yield abundant [b3 + Cat - H]+ ions or involve the loss of the side-chain of the histidine. The formation of [b3 + Cat +OH]+ ions, observed only in the case of the free acid peptide, is due to the interaction of the metal ion with the C-terminus carboxyl group. Bi- and trimetallated species have also been detected in the gas phase and characterized.

The crystal and molecular structures of the title compounds were determined by x-ray diffractometric analysis. Torsion angles and puckering parameters are reported for both compounds. In 1 the 5 alpha-cyano group influences the A-ring conformation. The carbonate ester 3 crystallizes in the monoclinic P2(1) space group with two molecules (I and II) in the asymmetric unit. The D-ring conformation is to some extent different between I and II.

Chemical and spectroscopic data clearly demonstrate that all the title compounds are acetylated only at the exocyclic nitrogen.

The 70 eV electron impact mass spectra of six 2-aminooxazoles are discussed in detail with the aid of exact mass measurements, metastable ion detection in the defocused mode and labelling experiments. The fragmentation is markedly influenced by strong electron donating substituents at the 2-position. Some 2-acetylamino and 2-diacetylamino derivatives have also been considered, which lose ketene from [M]+⋅ giving the corresponding aminooxazole molecular ions. The mass spectral behaviour is peculiar and the most important differences between the fragmentation pathways of these compounds and those reported in the literature for oxazole (alkyl and aryl) derivatives are outlined. The mass spectrum of 4-methyl-5-phenylimidazol-2-one is compared with that of the isomeric 4-methyl-5-phenyl-2-aminooxazole.

The reaction of some 2-aminooxazoles and imidazolidin-2-ones with hydrogen peroxide afforded stable trans-β-hydroxyhydroperoxy- or dihydroperoxyimidazolidin-2-ones according to the reaction conditions. In the case of 3a-hydroxy-7a-hydroperoxyoctahydro-2H-benzimidazolidin-2-one, the trans isomer is forbidden and cis adduct was isolated. A radical pathway to these hydroperoxides is proposed. From 2-amino-4-methyloxazole and hydrogen peroxide 2-amino-2-hydroxy-4-methyloxazolidin-4-yl peroxyacetate was also isolated.

Dimethylphosphate esters of vitamins D2 and D3 and of 7-dehydrocholesterol give satisfactory mass spectra under electron impact using the direct introduction probe, while this method is not suitable for the related phosphorodichloridates, which undergo to thermal decomposition-polymerization processes. Such processes are avoided using the direct electron impact method. Abundant molecular ions or their protonated analogues are obtained by field desorption.

The study of the tautomerism of 2-amino-2-oxazolin-4-one (1) and its methyl derivatives has been completed. The methylation of 1 gave 2-imino-3-methyloxazolidin-4-one (4) and 2-methyl-amino-2-oxazolin-4-one (7). All attempts to isolate O-methyl derivatives failed. The uv, nmr, ir, and Raman spectra led to the definite conclusion, in agreement with the chemical evidence, that the 2-aminooxazolinic form a, if it is theoretically possible, predominates in all compounds investigated. Otherwise the 2-iminooxazolidinic form b predominates. The assignment of the carbonyl stretching is discussed on the basis of ir and Raman data. The nmr spectrum of compound 7 shows anomalous behaviour, most probably due to the existence of a monomer-dimer equilibrium.

The infra-red spectrum of 1,3,4-thiadiazole has been measured between 4000 and 400 cm −1 in the vapour and solid phases and in solution. The analysis of the rotational band envelopes alone is not sufficient for a complete assignment of the fundamental vibrations. Additional informations are however gained from the crystal spectrum in polarized light and from the crystal spectrum at −184°C, and the complete vibrational assignment is presented.

A rare secocarotenoid, semi-β-carotenone, has been isolated from young leaves of Ceratozamia fuscoviridis (a form of C. mexicana Brongn.) and of C. kuesteriana. This is the first time that this carotenoid has been obtained from photosynthetic tissue. New data on the chromatographic behaviour and on the spectroscopic properties of the carotenoid are presented.

The heterocyclic moiety of 17 beta-(2-aminooxazol-4-yl) steroids is sensitive to the oxidizing action of hydrogen peroxide and yields products mainly from the opening of the amino-oxazole ring. Unlike simple 2-aminooxazoles, it does not rearrange to 2-imidazolone and the expected steroidal hydroperoxyimidazolidinones were not detected. Among the substances we isolated, N-(aminocarbonyl)-17 alpha-hydroxy-17-carboxamides (2a) and (3a) undergo spontaneous cyclization, in the reaction conditions, giving steroid-17-spirooxazolidinediones (2d) and (3d). Spirane (2d) was synthesized in high yields from (2a) in strongly alkaline medium.