Del Bianco

Kung A L Gilliland

' Ursi

Mckee R L Gibbons

PhD Chiara Testa

Le Chavelier

Williams H Y I D Sharpless

K B Fokin

Tetrahedon Lett

INTRODUCTION Bioactive conformations in proteins

Peptides play an important role in many biologically relevant processes and are of outstanding interest in pharmaceutical research. However their use as drugs is limited by their conformational flexibility, instability to proteases, poor oral bioavailability, and pharmacodynamics. A central role in peptide and protein research is the development of rational approaches for the design of peptide and protein ligands, with specific physical, chemical and biological properties. Moreover the determination of the relationship between the conformation and the activity of biologically relevant peptides, e.g. hormones, is of great interest to design potent and very selective agonist/antagonist that could be used in pharmacology.

Information about these interactions improves our understanding of disease conditions and can provide the basis for new therapeutic approaches.

In nature, protein functions, including enzyme activities, are often regulated through conformational change, triggered by ligand-binding or by post-translational modifications at specific sites 1,2 . The conformational state of receptor-protein complexes determines the functional state of a receptor. Peptides are flexible molecules, which tend to adopt myriad of conformations in solution but can assume a single specific conformation only when bound to their receptors. When peptides interact with their receptors, these ligand assume a specific conformation and this folding is crucial for triggering the biological activity.

For example, the interaction of linear and flexible peptides with their macromolecular target, such as G protein-coupled receptors (GPCRs), involves a limited number of closely related conformations that are recognized and after binding either activate or block the biological activity of these targets. The so called bioactive conformation represents only a small subset of the larger ensemble of accessible conformations, which are in a dynamic equilibrium. Identifying the bioactive conformation of the ligand and the structure of the receptor-ligand complex is crucial to design more potent and selective ligands. tein folding.

Two of the basic secondary structural elements that occur locally in protein structures are β-sheets and α-helices. Mimics of these structure can be useful in performing structure/activity relationship studies. In particular, β-turns (Figure 1) 3 are the most common type of non-repetitive structure recognized in proteins and comprise, on average, 25% of the residues 4 . β-turns play an important role in proteins providing a direction change for the polypeptide chain and have been implicated in molecular recognition 5 and in pro

Stabilization of secondary structures

Conformation of peptide fragments is fundamental to express the biological activity in proteins and it is related to the amino acid sequence. Peptides or proteins that do not have the correct conformation do not display their biological role.

The receptor-bound conformation may be poorly populated in solution but this structure may be promoted by incorporating conformational constraints into the peptide. Moreover, in the development of a peptidomimetic the identification of the key residues, and portions of the backbone in the peptide that are responsible for the biological effect, is crucial. A peptidomimetic embodies the conformational and molecular characteristics thought to be important for biological activity of the native sequence. Mimetics might exhibit enhanced potency and might be more selective for various receptor sub-types than their parent sequence, but several generations of variants may need to be prepared before a drug candidate emerges. Characterization of possible relationship between peptide structure and biological activity is often aided by reduction in diversity of the possible conformational states that a linear peptide can adopt by generating cyclic analogues 6 .

The primary approach to design peptide ligands with high potency and specific biological and conformational properties, involves the introduction of conformational constraints. Accessible peptide conformation can be limited through cyclization and further by incorporation of constraints into the cycle. Locking the active conformation in cyclic peptides can give superpotent analogues in matched cases 7 . In addition, conformational constraints provide the basis for receptor selectivity. In fact, different receptors often bind the same flexible substrate in different conformations.

If the conformation stabilized by the constraint closely resembles the structure responsible for the bioactivity, this modification can increase potency and selectivity of the resulting peptide.

Cyclic peptides offer the possibility of conveniently varying both scaffold geometry and R-group functionality. For example, parameters such as ring size can have a dramatic effect on the conformations of cyclic peptides, allowing access to structurally diverse species based on simple modifications in their linear sequences.

Cyclization affects the degrees of freedom of all residues within the ring and thus a macrocycle should adopt a more defined conformation than the equivalent linear sequence (Figure 2). (Kessler H. Angew. Chem. Int. Ed. Engl. 1982, 21, 512).

Biologically active cyclic peptides specifically designed and prepared from the corresponding linear peptides, have been developed and demonstrated to possess several additional attributes including: (i) increased agonist or antagonist potency;

(ii) prolonged biological activity and extended pharmacokinetics; (iii) increased stability to enzymatic degradation; and (iv) increased specificity for a particular receptor 8,[START_REF] Stewart | Hypercalcemia associated with cancer[END_REF][START_REF] Mannstadt | Receptors for PTH and PTHrP: their biological importance and functional properties[END_REF][START_REF] Kluczyk | Microwaveassisted TFA cleavage of peptides from Merrifield resin[END_REF][START_REF] White | Comparative study of conventional and microwave assisted synthesis[END_REF][START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] . The conformation of a peptide can be stabilized by introduction of bridges of various lengths between different parts of the molecule. The bridge can either be local and occurring within a single amino acid residue or being global and linking distant parts of the sequence. Intramolecular side-chain-to-side-chain cyclization is an established approach to achieve stabilization of specific conformations, and has been employed to achieve rigidification that results in restricting the conformational freedom. Therefore a larger conformational constraint can be introduced.

Types of cyclic peptides

Cyclic peptides are polypeptide chains whose amino and carboxyl termini, or two amino acids side chains, are themselves linked together by an amide (CO-NH), disulfide (S-S), carbon-carbon (CH 2 -CH 2 or CH=CH), reduced amide (CH 2 -NH), methylene thioether (CH 2 -S), methylene sulfoxide (CH 2 -SO), methylene ether (CH 2 -O), thioamide (CS-NH), keto methylene (CO-CH 2 ), aza (NH-NR-CO) bond or recently 1,4-[1,2,3]triazolyl bridge (Figure 3) 14,15,16 . In a number of studies, side-chain-to-side-chain cyclization has been performed by lactam bridge formation between N-and C-termini, side-chain and the N-or Cterminus, or two side-chains, to generate highly active peptides characterized by αhelix or β turn structures 17,18,19 . Moreover the introduction of the amide isosteres also results in local and global changes of dipole moments and in the pattern of intramolecular and peptide-receptor hydrogen-bond formation. Thus, incorporation of amide bond isosteres cannot only improve in vivo stability as the mimetic is no longer a substrate for peptidases, but can improve selectivity towards receptor subtypes, can change pharmacological functions and can enhance pharmacokinetic properties.

On the other hand, cyclization in the form of a disulfide bridge between two cysteines, or other thiol-containing amino acids, is the most abundant posttranslational modification resulting in side-chain-to-side-chain cyclization. However, under certain redox potentials, the disulfide bridge will behave as a relatively transient modification yielding either the reduced linear form or generate a variety of intermolecular disulfide containing products.

Moreover, the disulfide bridge can be reducted by specific enzymes such as gluthatione reductase, or by nucleophilic agents. An alternative could be the ring closing metathesis (RCM). This reaction allows the obtainment of cycles, starting from dienic substrates 20 . The substrate scope includes rings of 6 to 20 members. In addressing macrocyclic peptides, a class of tetrapeptide disulfides inspired the synthesis of the carbon-carbon bond analogs. For example, replacement of cysteine residues by allylglycines resulted in the acyclic precursors, which were subjected to RCM to afford the corresponding macrocycles. Ruthenium complexes have been applied to RCM 21 . Moreover the carba replacement (CH 2 -CH 2 ) is non-polar and does not allow the possibility of intramolecular or peptide-receptor hydrogen bonding, while the reduced amide (CH 2 -NH) unit is conformationally different from the amide bond because it does not have any double-bond character.

All of these types of cyclization require orthogonal protection of side chains to afford a peptide cyclization. While side-chain to side-chain cyclization of peptide sequences has been successful in many instances, a number of factors are known to significantly influence the efficiency of the cyclization reaction and the yield of the desired cyclic peptide product.

Therefore cyclizations, which do not require complicated orthogonal protection schemes, are of great interest.

Cyclopeptides containing disulfide bridge: the case of somatostatine

In eukaryotic cells, nature has used disulfide bonds as its major covalent bond for conformational control and stabilization. Disulfide bonds constitute an abundant and important structural element in the folding of proteins and peptides. Disulfide bonds stabilize the properly folded conformation of proteins and/or destabilize denatured conformations by decreasing conformational entropy. While disulphide-containing peptide rings have reduced conformational properties relative to their linear counterparts, many cyclic disulphide-containing peptides still can be flexible. In many proteins and peptides, disulfide bridges are prerequisite for their proper biological function

The somatostatin hormones, or somatotropin release-inhibiting factor (SRIFs) are side-chain to side-chain disulfide-bridged cyclic peptides. The highly potent cyclic peptide somatostatin, SRIF a H-Ala 1 -Gly 2 -c[Cys 3 -Lys 4 -Asn 5 -Phe 6 -Phe 7 -Trp 8 -Lys 9 -Thr 10 -Phe 11 -Thr 12 -Ser 13 -Cys 14 ]-OH (SRIF-14) (Figure 4), was isolated from mammalian hypothalamus and first synthesized by J. Rivier 22 . Later on, it was found that native SRIF occurs in two biologically active forms, SRIF-14 and a 28-residue peptide. The natural hormone is widely distributed in the endocrine and central nervous systems and it has many modulating actions in the body, such as growth hormone (GH) inhibition, and glucagon, insulin, and gastrin release suppression.

A family of five G-protein-coupled somatostatin receptors (sst1-5), located in the cell membrane, mediates the biological effects of SRIF.

It is known that some of these receptors, mainly the sst2 and sst5 subtypes, mediate the antiproliferative effects of SRIF on cellular growth of many sort of tumors. SRIF receptors are strongly expressed in various types of malignant cells, particularly in some neuroendocrine or neuroendocrine-like tumors. A lot of somatostatin analogues radiolabeled, were designed mainly for the targeting of malignant cells with γ-or β-emitting radionuclides, for tumor imaging and for therapy. The clinical use of native SRIF has been precluded by its short half-life in vivo (1-2 min) but the broad spectrum of physiological activity of this hormone has prompted many researchers to investigate the structural requirements necessary to elicit the biological activity. As a consequence, in the past decades, hundreds of peptide and nonpeptide analogues were synthesized and tested for their structureactivity relationships 23 . Among them, Octreotide (trade name Sandostatin ® , provided by Novartis Pharmaceutical), (Figure 5) a cyclic octapeptide SRIF agonist, was synthesized by Sandoz researchers 15 . Octreotide contain the disulfide bridging of the parent peptide and shows high affinity and specificity toward sst2 receptor subtype 24 . It was the first analogue to be used in clinical protocols. However, the administration of octreotide itself as a cell growth inhibitor, was successful only in a few cancer types as acromegaly and metastatic carcinoid diseases 25 . It is used in clinical protocols mainly as a carrier of radionuclides for diagnostic or therapeutic purposes 24,26,27 . Octreotide incorporates D-Trp in place of Trp 8 of SRIF and exhibits the so-called pharmacophore sequence, Phe 7 -D-Trp 8 -Lys 9 -Thr [START_REF] Mannstadt | Receptors for PTH and PTHrP: their biological importance and functional properties[END_REF] . The disulfide tether of octreotide stabilizes a β-turn type II' structure spanning the D-Trp 8 -Lys [START_REF] Stewart | Hypercalcemia associated with cancer[END_REF] residues, supposed to be mandatory for the binding to the ssts 28 . Nevertheless, Octreotide presents side effects and low affinity toward the same receptors, prompting many researchers to look for more stable bridging regions and peptide sequences to produce more selective and long acting analogues. Interestingly, the action mode of sst-related compounds is mediated by a massive endocytosis of the sst-analogue/sstr complex 29 , thus enabling the accumulation of compound within the targeted cell. This property has been thoroughly exploited for imaging cancer cells using radiolabeled analogues of octreotide, in which the cyclopeptide is conjugated to a metal chelating moiety, generally a polynitrogen molecule either linear (e.g. diethylene triamine pentaacetic acid, DTPA) or cyclic (tetraazacyclododecane tetraacetic acid, DOTA) 30 .

On the other hand, it is well-known that the disulfide bridge is prone to be attacked by endogenous reducing enzymes, such as glutathione reductase and thioredoxin reductase, or by nucleophilic and basic agents 31 . Disulfide isomerases, as well as reducing agents and thiols, can affect this covalent bond and can lead to structural rearrangement with a complete loss of activity. For this reason, octreotide and its analogues demonstrated side effects as limited bioavailability and selectivity, as well as chemical instability of the S-S bridge. Furthermore, the labeling of the octreotide derivatives with isotopes such as 99m Tc and 188 Re need a step in reducing medium that can afford ring cleavage, especially at the level of the disulfide linkage. The replacement of this essential structural element by bioisosteric, hydrolytically, and reductively stable substitutes is therefore of great interest.

Several approaches to substitution of the disulfide bridge by more stable covalent connections such as amides, thioleter, diselenides, or carbon-based bridges, have been reported 14,32,33,34,35,36,37 . For example, the effect was studied of the replacement of the disulfide bridge with a dicarba-bond, obtained by RCM reactions, catalyzed by ruthenium, on two allylglycines substituting the Cys 3,14 residues in the linear peptide 20 . Moreover, very recently Meldal and co-workers studied the potential of [1,2,3]triazoles as functional mimetics of multiple naturally occurring disulfide bonds in biologically active peptides 38 . By replacing of cysteines involved in the disulfide bridge of the peptide sequence with unnatural synthetic amino acids able to generate, via copper catalyzed 1,3-dipolar Huisgen reaction, more stable tethers bridging the bioactive site are produced. Moreover, this strategy allows the possibility to investigate the cycle dimension and orientation of triazolyl moiety that can induce the best bioactivity.

(Figure 6).

Triazoles exhibit chemical orthogonality and provide excellent stability against isomerases and proteases 39 , 40 . Moreover, triazoles can be formed in a twocomponent approach, which is comparable to that of disulfide bond formation from two cysteine residues. This strategy allows directional formation of disulfide bond mimetics by the substitution of two or more cysteine residues in a peptide with alkynyl-and azido-functionalized amino acids Cyclization leads to c) the correctly folded hairpin structure or d) incorrectly folded globule-like structure (Holland-Nell K., Meldal M., Angew. Chem. Int. Ed, 2011, 50, 5204-06).

Moreover, when four cysteine residues are involved in disulfide bridging, their replacement with functionalized amino acids able to perform the "click" reaction, can lead to the correct folding in the case of the correct positioning of the reactive groups.

Triazole-containing cyclopeptides

The recently introduced Cu I -catalyzed azido-to-alkyne 1,3-dipolar cycloaddition (CuAAC), or 1,3-dipolar Huisgen's cycloaddition 41,42,43 , as a prototypic "Click chemistry reaction" 44 presents a promising opportunity to develop a new paradigm for intramolecular side-chain-to-side-chain cyclization in peptides. In fact, the 1,4-1,2,3-triazolyl bridge offers interesting mode to generate structural constraint.

The inherent low reactivity of the azide and alkynyl functions toward an expansive range of functional groups and reagents eliminates the need for elaborate protection schemes. In addition, the 1,4-disubstituted 1,2,3-triazolyl bridging moiety is also inert toward a vast variety of synthetic conditions and its proteolytic stability is of great importance for applications in biological and material sciences. Therefore, the 1,2,3-triazolyl bridge offers interesting mode to generate structural constraint.

The 1,2,3-triazole is isosteric to the peptide bond and offers an appealing structural motif in peptidomimetics thanks to its structural and electronic features that are similar to those of a peptide bond. In particular, the 1,4-disubstituted- [1,2,3]-triazole represents a trans-amide peptide bond mimicking moiety 45 , positioning the substituents in positions 1 and 4 at 5.1 Å apart, which is only slightly longer than the distance between two carbons separated by a trans-amide bond (3.9 Å). Triazole moiety is planar and possesses a large dipole moment of ~5 Debye, which bisects the ring plane near atoms N3 and C5, and has the capacity of the N2 and N3 electron lone pairs to serve as hydrogen bond acceptors (Figure 7). However, unlike peptide bonds, triazoles are both proteolytically and metabolically stable. In addition, the azide and alkyne precursors required for their synthesis are relatively inert, making the Huisgen cycloaddition bio-orthogonal. [1,2,3] triazole (Bock V.D., Perciaccante R., Jansen T.P., Hiemstra H., van Maarseveen J.H., Org. Lett., 2006, 8, 919-922). The N-terminal fragments (1-34) of both PTH and PTHrP, regulate calcium in the blood, by interaction with the receptor PTH1-Rc. Moreover they have in common only 8 amino acids in the first 13 residues. Chorev et al. 47,48,49,50 hypothesized that both hormones assume the same conformation when they bind to the receptor. In particular, in biomimetic medium, like H 2 O/TFE (2,2,2-trifluoroethanol), PTH and PTHrP share the N-terminal part, as α-helical structure. Therefore, this particular conformation can be important for their biological activity because interaction with the receptor is higher. Moreover, stabilization of the α-helical structure by lactam ring between Lysine [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] and Aspartic acid 17 (i-i+4) increased antagonist activity 47 .

Starting from the observation that 1,4-disubstituted [1,2,3]triazolyl serves as a rigid linking unit mimicking a trans-amide bond, in previous work we reported the synthesis and conformational analysis of a series of i-to-i+4 side chain-to-side chain 1,4-disubstituted-[1,2,3]triazolyl-bridged cyclo-nonapeptides, analogous of the model i-to-i+4 lactam-bridged cyclo-nonapeptide N α -Ac-PTHrP (11-19)NH 2, that is a truncated version of the α-helical and potent parathyroid hormone receptor 1 (PTHR1) agonist, [Lys [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] (& 1 ),Asp 17 (& 2 )]PTHrP(1-34)NH 2 18,50 , (Scheme 1). In this context, a synthetic strategy was set up to obtain a series of N α -Fmoc-ω-azido-αamino-and N α -Fmoc-ω-ynoic-α-amino acids 51,16 needed for CuAAC.

These previous studies explored the relationship between the size of the bridge containing the 1,2,3-triazolyl moiety, the location of this moiety within the bridge, its orientation relative to the peptide backbone, and the predominant conformations displayed by these heterodetic cyclic peptides 46 .

The CD and NMR conformational analysis allowed to identify the permutations that mimic at the best the cyclo[Lys [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] ,Asp 14 ]PTH (11-19).

Scheme 1. Amino acid sequence of a modified fragment of N α Ac-hPTHrP (11-19)NH 2 (A), lactam cyclopeptide [Ac-Lys-Gly-Lys(&1)-Ser-Ile-Gln-Asp(&2)-Leu-Arg-NH 2 ] (B), i-to-i+4 side-chain to side-chain 1,4-disubstituted [1,2,3]triazolyl-bridged peptide (C).

It was demonstrated that the optimal conformational organization may be related to the right size of the methylen bridge and to the favourable location of triazolyl ring, which supports non-distorting intramolecular interactions and stabilizes ordered regular conformations 52 . The i-to-i+4 side chain-to-side chain 1,4-disubstituted- [1,2,3]triazolyl-bridged cyclo-nonapeptide (Scheme 1C) share conformational properties with its structurally related helical i-to-i+4 lactam-bridged cyclononapeptide (Scheme 1B). Therefore the CuAAC reaction can be used to generate a 1,4-substituted 1,2,3-triazole between side chain azido and alkynyl moieties at the i and i+4 positions of a peptide chain that effectively mimics the use of the analogous lactam bridge.

Optimized synthesis of PTHrP(1-34)NH 2

PTHrP(1-34)NH 2 sequence is: H-Ala 1 -Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp 10 -Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg 20 -Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu 30 -Ile-His-Thr-Ala-NH 2 . Considering the presence of clusters of arginines, sterically hindered and hydrophobic amino acid residues in the 19-28 sequence of PTHrP and the considerable length of the peptide, the synthesis of PTHrP(1-34)NH 2 is quite challenging. Therefore we focused our effort in the optimization of a synthesis protocol for this peptide. In particular, we showed the advantages that the use of microwaves have, in obtaining the best results in terms of yield and purity of the final peptide. The application of microwave irradiation in peptide chemistry has been reported in several publications, most of which describe case studies of successful syntheses of difficult peptides. Microwaves have also been used to monitor the synthesis of the peptide PTHrP(1-34)NH 2 . In fact, during the elongation of the peptide chain, we analyzed by UPLC-ESI-MS intermediate fragments obtained through micro-cleavages assisted by microwaves. This strategy has allowed us, through the characterization of sequences of deletion, to understand what are the critical points of the synthesis that may require the use of microwaves.

Intramolecular i-to-(i+5) side-chain-to-side chain cyclization stabilize the formation of β-turn secondary structures: melanocortin peptides hormones.

Following the previous studies performed on PTH and PTHrP, we decided to apply the triazole strategy to another peptide hormone, involved in a lot of different biological process in human: the melanocortin peptide hormones.

Melanocortins are a family of hormones secreted by pituitary gland and also expressed in the brain and peripheral tissues. Melanocortins include adrenocorticotropin (ACTH) and α-, β-, and γ-melanocyte stimulating hormones (MSHs). These hormones are involved in many biological functions by interacting with five receptors hMC1R-hMC5R. Consequently they are considered a potential target for drugs to treat diseases such as obesity and sexual disfunction. MTII, Ac-Nle 4 -c[Asp 5 -D-Phe 7 -Lys 10 ]αMSH 4-10 -NH 2,53,54,55 , is a potent long acting non-selective super-agonist of MC1R, MC3R, MC4s 56 . This homodetic Asp 5 to Lys 10 side chain-to-side chain bridged lactam stabilizes the pharmacophore containing sequence His 6 -D-Phe 7 -Arg 8 -Trp 9 in a type-II β-turn. The particular importance of melanocortin system, underscores the unmet need for highly selective, pharmacokinetically diverse, and bioavailable agonists and antagonists analogs.

The introduction of [1,2,3]triazole was aimed to stabilize a β-turn conformation replacing the lactam bridge of MTII with an i-to-i+5 side chain-to-side chain cyclization via Cu I -catalyzed azido-to-alkyne 1,3-dipolar cycloaddition (CuAAC) generating 1,4-or 4,1-disubstituted [1,2,3]triazolyl-containing ring structures.

A series of heterodetic MTII related cyclo-heptapeptides, that varied in the size, location and in the orientation of the disubstituted [1,2,3]-triazolyl bridge, was synthesized for a fine-tuning of the best conformation needed for bioactivity.

Studying this modification in the context of the MTII scaffold provides the opportunity to evaluate its potential as a modulator of receptor sub-type selectivity.

Conformational and biological studies were performed on the peptides synthesized to identify the location and direction of the 1,2,3-triazolyl in the bridge that best reproduce the β-turn conformation leading to highly potent and selective MT-II agonists. PTH exerts its actions via a G protein-coupled seven-transmembrane domaincontaining receptor (PTH1Rc and PTH2Rc), [START_REF] Segre | Principles of Bone Biology[END_REF] which is a member of the glucagon/secretin/calcitonin/vasoactive intestinal peptide receptor subfamily 61 .

In analogy to PTH, most of the known biological functions are exerted by the Nterminal PTHrP (1-34) fragment that has 60% sequence similarity to PTH(1-34),

(Figure 9). The hypothesis was made that the N-terminal 1-34 fragments of both hormones share a common bioactive conformation when bound to the receptor and in the past, this sequence was the subject of numerous structure-activity-conformation relationship studies 62,63 . This extensive homology accounts for the ability of PTHrP to bind to and activate classic PTH receptors in bone and kidney and consequently to generate the paraneoplastic effects on calcium and phosphate metabolism that occur in humoral hypercalcemia of malignancy. The amino-terminal peptides of PTHrP share a receptor with PTH, but they also bind to a type of receptor in some tissues that does not bind parathyroid hormone In fact, there are two receptors, PTH1-Rc and PTH2-Rc, which differ in their ligand selectivity. In particular, PTH2-Rc is activated by PTH, but not by PTHrP. Interestingly, the N-terminal fragments (1-34) of both PTH and PTHrP regulate calcium in the blood by interaction with the receptor PTH1-Rc 64,65 present on the surface of cells in target tissues such as bone and kidney.

Moreover, like PTH, some of the effects of PTHrP result from its effects on transepithelial fluxes of calcium, but many of its actions have nothing to do with calcium homeostasis. Most prominently, PTHrP peptides exert significant control over proliferation, differentiation and death of many cell types. They also play a major role in development of several tissues and organs.

Microwave assisted peptide synthesis

Microwave (MW) is a form of electromagnetic energy located between infrared radiation and radiowave with frequencies in the range of 0.3-300 GHz, which corresponds to wavelengths of 1 mm -1 m, but only the frequency of 2.45 GHz, corresponding to a wavelength of 12.24 cm, is utilized by industrial and domestic microwave apparatus, in order to avoid interferences with radar and telecommunication activities operating within this region.

The energy of the microwave photon in this frequency region (0.0016 eV) is too low to break chemical bonds and is also lower than the energy of Brownian motion. It is therefore clear that microwaves cannot induce chemical reactions 66,67,[START_REF] Stuerga | Microwaves in Organic Synthesis[END_REF] . Microwave heating works quite differently than conventional heating that transfers energy to reaction via conduction or convection. Conventional heating is a comparatively slow and inefficient method for transferring energy into the system because it depends on the thermal conductivity of the reaction vessel materials. On the contrary microwave approach is based on temperature increase by dielectric heating, which operates through two mechanisms: dipolar polarization and ionic conduction (Figure 10). Since only the electric field transfers energy to heat a substance, this component of microwave irradiation causes dipoles and ions to align, and as the applied electric field oscillates, the dipole-ion field is forced to realign itself. In both events, since alignment doesn't occur completely, a part of this energy is lost as heat and cause a sudden rise of molecular temperature and, thereby, of the entire reaction mixture.

Thus microwave energy enhances reaction rates and increases yields by limiting side reactions. While some believe that these results can be explained through thermal/kinetic effects, others argue that additional specific non-thermal microwave effects are at work.

Thermal effects

Thermal effects are strictly connected to the different characteristics between microwave and conventional heating. Microwave heating uses the ability of some compounds (liquids or solids) to transform electromagnetic energy into heat. The attitude of a substance to convert electromagnetic radiation into heat depends on its dielectric properties 67 and is calculated by the loss angle, tanδ, which is expressed as tanδ = ε´´/ ε´. The loss factor ε´´ quantifies the efficiency through which the absorbed energy is converted into heat, while the dielectric constant ε´, describes the ability of the molecules to be polarized by an electric field. A reaction medium with a high tanδ value efficiently absorbs electromagnetic radiation and converts it into heat, whereas substances with low tanδ values are not affected by microwaves [START_REF] Kappe | [END_REF] .

Therefore, energy transmission is produced by dielectric losses, which is in contrast to conduction and convection processes observed in conventional heating and the amount of heating is directly dependent on the dielectric properties of the molecules.

These characteristics mean that absorption of the radiation and heating may be performed selectively. Moreover microwave irradiation is rapid and volumetric, with the whole material heated simultaneously, in contrast with conventional heating that is a process starting from the surface of a sample and slowly is transferred inside.

The thermal effects observed under microwave irradiation conditions are a consequence of the inverted heat transfer (overheating) 70,71 , the non-homogeneities of the microwave field within the sample, known as "hot spots" 72 , and the selective absorption of the radiation by polar compounds. These effects can be used efficiently to improve processes, modify selectivities or even to perform reactions that do not occur under conventional conditions.

Specific non-thermal effects

Some authors have also justified the enhancement obtained in microwave applications, by the existence of specific non-thermal microwave effects that essentially result from a direct interaction of the electric field with specific molecules in the reaction medium. These effects can be rationalized considering the Arrhenius law:

k = Ae (-Ea/RT)
It has been affirmed that the presence of an electric field leads to orientation effects of dipolar molecules and consequently changes the pre-exponential factor A or the activation energy (entropy term) in the Arrhenius equation 73,74 . A similar effect should be observed for polar reaction mechanisms, where the polarity is increased going from the ground state to the transition state, thus resulting in an enhancement of reactivity by lowering the activation energy.

Modes

When the microwaves enter a cavity, they are reflected by the walls. The reflections of the waves eventually generate a three dimensional stationary pattern of standing waves within the cavity, called modes. Most early microwave-enhanced synthesis works were done in multi-mode systems. Multi-mode reactors present larger cavities with a microwave field that contains multiple modes of energy of different intensity, so called hot and cold spots. As a consequence, this non uniform microwave irradiation inside the reactor is compensated, if possible, by rotation of samples through the field. While multi-mode strategy is particularly suitable for parallel synthesis, the small individual samples characteristic of drug discovery are problematic in multi-mode systems, due to the hot and cold spots intrinsic in the cavity design. Moreover, the power density in the cavity is low, making it difficult to heat small individual samples. Recently, in order to obtain a well-defined heating pattern for small loads, single-mode cavities with good uniform energy distribution and the ability to couple with small samples more efficiently have become available.

These cavities are better suited for drug discovery work, because the higher power density allows the energy to be more focused. Because of this type of cavity allows only one single mode to be present. Much higher field strengths can be obtained, giving rise to more rapid heating. A suitably designed cavity also prevents the formation of 'hot and cold spots' within the sample, resulting in a uniform heating pattern. This is very important when microwave technology is used in organic chemistry, since the heating pattern for small samples can be well controlled. This allows for higher reproducibility and predictability of results as well as optimization of yields, which are usually more difficult when using a domestic microwave oven.

Synthetic strategies for difficult peptide sequences

Peptide synthesis by solid-phase methods is often hampered by partial unmasking of the N α -protection and the occurrence of incomplete coupling. These problems generally depend on structuration and/or poor solvation of the growing peptide chain and are especially severe in the presence of the so-called "difficult sequences".

Kinetic problems due to intermolecular aggregation or steric hindrance of protecting groups can generate premature termination of the sequence during the different steps of the solid-phase pathway. As a consequence, the desired products are contaminated by a series of structurally and chemically very similar compounds such as diastereoisomers formed after epimerization, or mismatch and incomplete sequences.

Although conventional SPPS remains the principal strategy for peptide synthesis, we were attracted by the possibility to improve peptide reactions, by using microwave energy, decreasing chain aggregation during the syntheses, improving the coupling rates and preventing side reactions in particularly difficult peptide sequences.

The introduction in 1992 of MW irradiation in peptide chemistry stimulated a lot of interest followed by numerous efforts to use it to overcome synthetic difficulties resulting in sluggish reactions, low yields, and complex reaction crude products that were hard to purify 75 .

Several different groups have reported the synthesis of peptides using microwave strategy, dramatically reducing reaction times and improving the yield of final product. During the last decade the growing interest in MW field has been marked by the progress in developing scientific MW equipment to embrace specific research needs 76 . In general, introduction of MW technology led to the shortening of reaction time that resulted in higher purity of crudes and increased yields of the pure final product. The application of MW irradiation in peptide chemistry has been reported in several publications, most of which describe case studies of successful syntheses of difficult peptide sequences 77 .

However, a definitive comparison of conventional RT and MW-assisted-SPPS protocols must be performed on identical instruments. In fact, comparing conventional RT and MW-assisted SPPS carried out on different instruments that apply protocols that differ in the equivalent excess of reagents and their molar ratios, stirring techniques and the nature and number of washing steps, may be misleading.

In addition, the automated synthesizers are generally developed to produce peptides without monitoring of the progress of the synthesis. Therefore by-products caused by side reactions such as aspartimide and diketopiperazine formation, incomplete couplings and deprotections may be detected only after the final cleavage of the deprotected peptide from the resin.

In this part of my PhD we focused the attention on the comparison between the Fmoc/tBu RT-SPPS with the MW-assisted synthesis of the 1-34 N-terminal fragment of PTHrP using the same instrument (Liberty TM , CEM, Matthews, NC, USA), and monitoring the progress of the synthesis by UPLC-ESI-MS of MWassisted mini-cleaved fragments of the growing peptide chain.

Synthesis of PTHrP(1-34)

The sequence of PTHrP(1-34)NH 2 is shown in Figure 11. of sterically hindered (underlined in yellow) and hydrophobic (underlined in red) amino acid residues, represents a synthetic challenge that was the subject of this comparative study. The comparison of conventional RT and MW-assisted-SPPS protocols was performed on identical instruments, Liberty TM automated peptide synthesizer, using the some excess of reagents and molar ratios.

The synthesis was initially carried out following the conventional RT protocol, using the Liberty TM automated peptide synthesizer, excluding MW irradiations. The synthesis was performed on Rink-amide NovaSyn ® TGR resin (0.2 mmol/g, 500 mg), following Fmoc/tBu chemistry. After deprotection of the N-terminal Fmoc group with a 20% solution of piperidine in DMF, Fmoc-protected amino acids (5 equiv), TBTU (5 equiv) and DIEA (10 equiv) in DMF were used in the coupling steps. The RT-SPPS protocol consists of two consecutive deprotection steps of 5 and 10 min employing deprotecting reagent, and 20 min for the coupling steps (Table 1) 78 . In MW-assisted SPPS protocol, the deprotection steps were performed at 75 °C using 35 W for 30 sec for the first one and 60 W for 180 sec for the second one, while the coupling steps were performed at 75 °C using 30 W for 300 sec for all amino acids except Arg and His, for which the protocols reported in Table 1 were applied. Specifically, we have decreased the power of MW irradiation and lowered the temperature in order to avoid side reactions such as δ -lactam formation for Arg and racemization for His 79 .

MW-assisted mini-cleavage of PTHrP(1-34)NH 2 fragments

As modern automated SPPS protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. In fact monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. Although different methods have been developed for monitoring of SPPS, we observed that the use of colorimetric monitoring or continuous-flow UV absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis.

Therefore, we decided to monitor the progress of PTHrP(1-34)NH 2 synthesis by UPLC-ESI-MS analyses of small aliquots of cleaved peptide fragments obtained by MW-assisted mini-cleavages. The application of MW-assisted mini-cleavages of resin-bound peptides has been proposed as a fast, reliable method to monitor SPPS 80 .

Consequently, after specific coupling cycles, suspected to be difficult, we stopped the synthesizer and withdrew a small aliquot for analysis by UPLC-ESI-MS. A small sample of Fmoc-protected peptide-resin beads was removed from the reactor vessel, weighted into a fritted polypropylene tube and treated with a 20% solution of piperidine in DMF (2 × 1 mL) for 5 min. The sample was then washed with DMF (2 × 1 mL) and DCM (3 × 1 mL), and dried under vacuum. Mini-cleavages were performed in a 10 mL glass tube containing 2 mL of cleavage mixture consisting of TFA/TIS/water solution (95:2.5:2.5 v/v/v). Tubes were inserted into the microwave cavity of Discover™ S Class (CEM) and the cleavage was carried out at 45 °C, using 15 W for 15 min assisted by external cooling of the reactor vessel.

The crude fragments obtained from the MW-assisted mini-cleavages were analyzed by UPLC-ESI-MS on an ACQUITY™ UPLC system (Waters) coupled with a Micromass ® Q-Tof MICRO™ mass spectrometer (Waters) equipped with an ESI source.

MW-assisted mini-cleavage protocol of 19-34 fragment of

PTHrP(1-34)NH 2 : a case study of multi-arginine containing peptides.

The use of microwave-assisted mini-cleavage protocol to monitor the growing peptide chains was useful to optimize in time the crucial reactions, i.e. multi-arginine containing peptides, with side-chains protected by Pbf group.

The 19-34 fragment of PTHrP(1-34)NH 2 H-Arg 19 -Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala 34 -NH 2 is characterized by a cluster of arginine residues in the region 19-21.

In order to validate the use of microwave irradiation during the mini-cleavage step, we compared the RT and MW-assisted mini-cleavage protocols. A sample of beads carrying Fmoc-protected resin-bound peptide (10 mg) was weighted into a fritted polypropylene tube and treated twice with a 20% solution of piperidine in DMF (1 mL) each time for 5 min. The beads were then washed with DMF (2 × 1 mL) and DCM (3 × 1 mL), dried under vacuum and transferred into a 10 mL glass tube containing 2 mL of TFA/TIS/water cleavage solution (95:2.5:2.5 v/v/v). RT minicleavage reactions was performed by magnetic stirring in 15min as well as MWassisted mini-cleavages. The reaction mixture was then filtered and the crude peptide was precipitated from the cleavage mixture by addition of ice-cold diethyl ether followed by cooling for 5 min at -20 °C. The product was collected by centrifugation and directly subjected to UPLC-ESI-MS analysis. Based on the results obtained by UPLC-ESI-MS reported in (Figure 12A), we can assume that 15 min at RT were not adequate to completely remove the Pbf groups from the arginine side-chain residues.

Charachterization of PTHrP(1-34)NH 2 fragments by UPLC-ESI-MS analysis

Thanks to the analysis of minicleavages intermediate fragments of the PTHrP(1-34)NH 2 by UPLC-ESI-MS ,we were able to detect the presence of the desired peptide as well as of some by-products (Figure 14). In particular, we focused our attention on the PTHrP fragments related to the 19-34 sequence, characterized by clusters of Arg residues and highly hydrophobic residues. 2). It is well known that Arg-containing peptides are difficult to synthesize due to the sterically hindered Pbf group as side-chain protection and the tendency to form δ -lactam leading to low yield couplings 81 . As the length of the resin-bound peptide increases, the related UPLC-ESI-MS analyses become much more complex (Figure 15).

ESI-MS/MS analysis

The characterization of the 12-34 fragment of PTHrP(1-34)NH 2 synthesized by RT-Fmoc/tBu SPPS (Liberty™, CEM) was obtained by deconvolution with the attribution of the deletion sequences, where the fragments desLys [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] /Gln 16 -f (12-34) and desLys [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] /Gln 16 ,Leu 27 -f (12-34) were identified as two isobaric peptide sequences lacking either Lys [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] or Gln 16 residues. Since it is tricky to discriminate among the different combinations of deletion sequences in case of isobaric peptides affecting the SPPS, we suggest to plan rationally stops during the synthesis accurately monitoring the missing amino acid residues.

Thus, we report the UPLC-ESI-MS/MS data of the fragment 16-34 of PTHrP(1-34)NH 2 focusing the attention on the presence of the desGln 16 -f(16-34) consisting of a 40% of deletion sequence The deconvoluted spectrum obtained for the cleaved mixture of this sample resulted in several deletion sequences (Figure 16).

Results

After semi-preparative purification the MW-assisted SPPS of PTHrP(1-34)NH 2 yielded 27 mg of >95% pure peptide, whereas RT-SPPS gave only 18 mg of >95% of pure peptide. This improvement is attributed to the higher purity of the crude cleaved peptide mixture (Figure 17). Although the application of MW-assisted SPPS to the synthesis of PTHrP(1-34)NH 2 led only to a moderate improvement in final yield (6.3% vs 4.4%), it allowed us to obtain a crude product of higher quality (77% vs 35%) and in a shorter time (20 h vs 34 h used for the RT and MW-assisted SPPS strategies). Moreover, we demonstrated the usefulness of the combination of an MW-assisted mini-cleavage protocol and the UPLC-ESI-MS analysis for monitoring the quality of the reaction step. Compared to the ninhydrine colorimetric monitoring, our strategy is faster and the UPLC-ESIMS/ MS analysis is more accurate and more informative.

Advantages of MW irradiation in SPPS

The monitoring of traditional RT-SPPS by MW-assisted mini-cleavages combined with fast, efficient and sensitive UPLC-ESI-MS analysis (15 min/analysis vs 30-45 min/analysis for traditional mini-cleavage) is helpful to identify the presence of difficult coupling steps that result in truncated and deletion sequences.

Moreover we can tune MW-assisted SPPS using modified cycles targeting difficult couplings. In these couplings the first step is carried out at RT (without applying MW power, 20 •C) and for variable duration (25 or 2 min) and the second step is carried out in the presence of MW power (75 or 50 •C) for shorter time intervals (5 or 4 min). In the synthesis of PTHrP(1-34)NH 2 we applied such type of couplings for the three arginine residues (R 19 -R-R 21 ) and for the two histidine residues (H 25 -H 26 ).

Evidently, carrying out the RT and MW-assisted SPPSs on the same instrument allowed unbiased side-by-side comparison and the conclusion that the latter procedure is superior to the RT-SPPS. Although the modification of the coupling steps for Arg and His in the MW-assisted SPPS seems to be empirical in nature, it was guided by strong rational that took into account the distinct capacity of this methodology to overcome putative hydrophobic interactions and aggregation. The impact of these phenomena increases with the progression of the coupling reactions.

We therefore decided to take advantage of the MW radiation only for a short duration after the bulk of the reaction has been already taken place. In this manner, the completion of the coupling reaction was facilitated without causing undesired side reactions. We propose this MW-based protocol as a general strategy for overcoming difficult coupling reactions. Admittedly, applying this strategy to different peptides will require fine tuning that will include the adjustment of parameters such as duration of coupling and recoupling steps as well as the level of MW energy employed in the recoupling step. The typical pathway for hormones signal transduction is endocrine signaling.

However, there are also other modes, i.e., paracrine, autocrine, and neuroendocrine signaling (Figure 18).

Peptide hormones

Peptide hormones exert their endocrine function to control and regulate the activity of certain cells or organs. Like other proteins, peptide hormones are synthesized by amino acids according to an mRNA template, which is itself synthesized by a DNA template inside the nucleus. Peptide hormone precursors (pre-prohormones) are then processed in several stages, typically in the endosplasmic reticulum, including removal of the N-terminal signal sequence and sometimes glycosylation, resulting in prohormones. The prohormones are then packaged into membrane-bound secretory vesicles, which can be secreted by exocytosis in response to specific stimuli. In fact, specific endopeptidases in the cell, cleave the prohormones just before its releasing into the blood stream, generating the mature hormone. Mature peptide hormones then diffuse through the blood to all cells, where they interact with specific receptors on the surface of their target cells. These protein hormones often contain amino acid residues necessary to direct folding of the hormone molecule into its active configuration, but with no function once the hormone folds. In the inactive state, hormones do not have a fixed conformation, but after interaction with their receptor, they assume a specific bioactive conformation that is crucial for the triggering of biological response. The binding of hormones, to hormone-receptors triggers the start of a biochemical signal that can lead to further signal transduction pathways, or triggers activation or inhibition of genes. Steroid hormones, for example, pass through the plasma membrane and, once inside the cell, bind to the nuclear membrane receptors, producing an activated hormone-receptor complex. The activated hormone-receptor complex binds to DNA and activates specific genes, increasing production of proteins. Non-steroid hormones, such as peptide and protein hormones are sufficiently hydro soluble to arrive up to their target cells, in all the organisms by blood circulation. However, they do not penetrate the double plasma membrane, strictly impermeable, but bind to plasma membrane receptors, generating a chemical signal (second messenger) inside the target cell. Peptide hormone receptors are often transmenbrane proteins, such as G-protein-coupled receptors (GPCRs), sensory receptors or ionotropic receptors. These receptors generally function via intracellular second messenger, including cyclic adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate (IP3), and calcium (Ca 2+ )calmodulin system (Figure 21). Second messengers activate the mechanism of "transduction of signals", in which, in addition to the receptors, other molecules take part. A ligand is a molecule able to bind and to form a complex with a biomolecule to serve a biological purpose. Ligands can be small molecules, such as GABA (γaminobutyric acid) having neurotransmitter function, or large ones with molecular weight around ten of thousands daltons like proteins, nucleic acids, polysaccharides or lipids. The tendency or strength of binding is termed affinity. High affinity ligand binding implies that a relatively low concentration of a ligand is adequate to maximally occupy a ligand binding site and trigger a physiological response.

For example it is possible that two different ligands bind to the same receptor binding site, but only one of the agonists can maximally stimulate the receptor and thus can be defined as a "full agonist". An agonist that bind and activate a given receptor, but have only partial efficacy at the receptor relative to a full agonist, is called a "partial agonist". Ligands that bind to a receptor but fail to activate the physiological response are receptor antagonists 82 83 .

The Nervous and endocrine systems

In neurocrine signaling, neurons synthesize neuro-hormones, that are released at the synaptic cleft into a gap between the terminals and the dendrites of the next neuron.

Hypothalamus is a portion of the brain that contains a number of small nuclei with a variety of functions. The hypothalamus is responsible for a lot of metabolic processes and other activities of the autonomic nervous system. One of the most important functions of the hypothalamus is to link the nervous system to the endocrine system via the pituitary gland (hypophysis) (Figure 22). The pituitary gland, often called the "master gland", is located in a small bony cavity at the base of the brain. A stalk links the pituitary to the hypothalamus, which controls release of pituitary hormones. The pituitary gland has two lobes: the anterior and posterior lobes. The posterior pituitary stores and secretes oxytocin and the antidiuretic hormones. Oxytocin is one of the few hormones to create a positive feedback loop. For example, uterine contractions stimulate the release of oxytocin from the posterior pituitary, which, in turn, increases uterine contractions. This positive feedback loop continues throughout labor. The anterior pituitary is glandular and secrete the majority of the hormones that regulate the bulk of the endocrine glands. This includes the thyroid-stimulating hormone (TSH), which regulates the thyroid, the adrenocorticotrophic hormone (ACTH), which regulates the adrenal cortex, the gonadotrophic hormone, which is responsible for regulating the gonads, melanotrophins, prolactin, secreted near the end of pregnancy and prepares the breasts for milk production, and somatotropins (Growth Hormone) (Figure 23). These hormones are released from the anterior pituitary under the influence of the hypothalamus. Hypothalamic hormones are secreted to the anterior lobe by way of a special capillary system, called the hypothalamic-hypophysial portal system (HPA or HTPA axis).

Melanocortin peptide hormones

Melanocortin peptides are endogenous neuropeptides hormones, including adrenocorticotropin (ACTH) and α-, β-, and γ-melanocyte stimulating hormones (α-MSH, β-MSH, γ-MSH). They are involved in many biological pathways, including sexual function [START_REF] Wessells | [END_REF] , energy homeostasis 85,86 , modulation of fever and inflammation, control of food intake, feeding behavior 87,88 , endocrine secretion, pain modulation 89 , thermoregulation, cardiac frequency, blood pressure and skin pigmentation 90,91,92,93 .

Genetic mutations in melanocortin system have been associated with early onset of obesity, adrenal insufficiency, red hair pigmentation, and hyperphagy 94,95,96 . For these reasons, melanocortins peptides are considered leads for drugs to treat diseases such as obesity, pigmentation diseases and sexual dysfunction.

Melanocortin peptides derived from post-translational modification of the proopiomelanocortin prohormone (POMC), a 241 amino acids polypeptide precursor.

The POMC gene, located on chromosome 2p23, is expressed in both the anterior and intermediate lobes of the pituitary gland. In addition to the pituitary POMC expression and peptide processing occur normally in the nervous system and in widespread peripheral tissues. The POMC gene encodes a 285-amino acid polypeptide hormone precursor, the pre-pro-opiomelanocortin (pre-POMC), that undergoes extensive, tissue-specific, post-translational processing via cleavage by subtilisin-like enzymes, known as prohormone convertases 97 .

There are eight potential cleavage sites within the polypeptide precursor, consisting of the sequences, Arg-Lys, Lys-Arg or Lys-Lys. Depending on tissue type, the stimulus and the available convertases, processing may yield as many as ten biologically active peptides involved in diverse cellular functions (Figure 24).

Figure 24. Melanocortin peptides, ACTH and α-, β-, and γ-MSH, derive from post-translational processing of POMC, which is also the precursor for opioid peptides and CLIP (corticotropin-like intermediate lobe peptide) 95 In particular, enzymes responsible for processing of POMC peptides include prohormone convertase 1 and 2 (PC1/2), carboxypeptidase E (CPE), peptidyl αamidating monooxygenase (PAM), N-acetyltrasferase (N-AT), and prolylcarboxypeptidase (PRCP). Data showed that PC1 generated ACTH and βlipotropin (β-LPH), whereas PC2 was required for production of α-MSH and βendorphin 98 . This was the first evidence that tissue-specific processing of POMC could be explained by the relative expression of its convertases.

Thus, in the corticotrophs where PC1 predominates, ACTH and β-LPH, are the final POMC-processing products. In contrast, expression of PC2 in the pituitary gland results in production of α-MSH and β-endorphin. In other tissues, including the hypothalamus, placenta, and epithelium, all cleavage sites may be used, giving rise to peptides with roles in pain and energy homeostasis, melanocyte stimulation, and immune modulation. These include several distinct melanotropins, lipotropins, and endorphins that are contained within the adrenocorticotrophin and beta-lipotropin peptides. ACTH is an important component of the HPA axis and is often produced in response to biological stress. Its principal effect is to increase the production and release of corticosteroids. Therefore it is essential for normal steroidogenesis and the maintenance of normal adrenal weight. ACTH consists of 39 amino acids and in the melanotrophs of the intermediate lobe of the pituitary, is the precursor of α-MSH. α-MSH was found to share the sequence of ACTH (1-13), although α-MSH is acetylated at the N terminus and C-terminally amidated. The primary roles of α-MSH is the regulation of pigmentation. α-MSH is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH 2 , and is one of the first peptide hormones isolated from the pituitary gland. Extensive studies of this hormone have confirmed its role in skin pigmentation as well as in various central nervous system (CNS) [START_REF] Eberle | The Melanotropins[END_REF] . Although adrenal stimulatory effects of ACTH and pigmentary influences of α-MSH have been known for over 50 years, the discovery that melanocortin peptides have multiple effects on the host is much more recent.

β-LPH is a 90-amino acid polypeptide deriving from the carboxy-terminal fragment of POMC. It stimulates melanocytes to produce melanin, and can also be cleaved into smaller peptides such as γ-lipotropin, β-MSH, γ-MSH, α-endorphin, βendorphin, γ-endorphin, and met-enkephalin. β-LPH also performs lipid-mobilizing functions such as lipolysis and steroidogenesis.

G protein-coupled melanocortin receptors: MCRs

Human melanocortin peptide hormones share the common tetrapeptide core [His-Phe-Arg-Trp] 100 101 (Figure 25). All the MCRs intracellularly mediate their effects by activating pathways that are cyclic adenosine monophosphate (cAMP) dependent (Figure 27). All the melanocortin receptors possess unique tissues distributions, and are involved in different physiological effects (Figure 28). The cloning of the human melanocortin receptor genes led to great progress in understanding the biological effects of the melanocortin peptides at the melanocortin receptors 91,103,104 The primary native ligand for MC1R, MC3R, MC4,R and MC5R is α-MSH possibly γ-MSH. These native ligand, presenting the minimum sequence necessary for the activation [His-Phe-Arg-Trp], are rather non-selective for these receptors.

Two melanocortin-receptor antagonists, the agouti (also termed agouti signaling protein) and the agouti-related protein (AgRP), participate in control of melanocortin signaling 113 . Agouti is a competitive antagonist at melanocortin receptors with high affinity at MC1R, although it also shows antagonistic activity for the human MC4R. This receptor antagonist may be important in inflammatory responses. In mice, the agouti gene encodes a paracrine signaling molecule that induces hair follicle melanocytes to synthesize pheomelanin, a yellow pigment, instead of the black or brown pigment, eumelanin. The human agouti is a protein closely homologous to the rodent agouti, but it shows a much wider distribution as it is expressed in adipose tissue, testis, ovary, heart, and, at lower levels, in foreskin, kidney, and liver 114 . where the former decreases the drive for feeding and the latter increases it. Both agouti and AgRP contain cysteine-rich C-terminal b M

Pleiotropic effects of constitutive expression

Development of agonists for MCRs

Melanocortins hormones are involved in a lot of biological processes. For this reason the development of selective, potent and long-acting agonist for MCRs, is of great interest receptor family has promoted considerable research activity over the past decade 117,118,119 . α,β-MSH (Figure 29) are rather non-selective for their receptors in human (hMC1R-hMC5R), and, like other peptide hormones, are biologically unstable, being h MTII is characterized by lactam bridge between residues Asp 5 and Lys 10 stabilizing a type-II β-turn structure, that is critical for its bioactivity. The minimal active sequence [His 6 -D-Phe 7 -Arg 8 -Trp 9 ] 56 , is included in the cyclic portion formed by lactam ring. MT-II, resulted to be 90-100 time more potent than native agonists, but nonselective agonist at the four melanocortin receptors MC1R, MC3R, MC4R, MC5R. MT-II showed during phase I trials side effects including nausea and a "stretching and yawning complex" that correlated with spontaneous penile erections 128 . Therefore the therapeutics for skin disease was interrupted.

Nevertheless, MTII can be considered a lead molecule for the development of new specific and selective analogues, although problems need to be solved. One is that ate or block receptor-signaling pathways that lead only to the esired therapeutic effect. This is of particular importance for the melanocortin ceptors as a potential target for the treatment of skin cancer, food intake, and d approach to achieve stabilization of specific conformations, such as αcking them into receptor synthetic agonists or antagonists may encounter difficulties in reaching their target(s). For example, MC4R is located within the brain and, therefore, ligands must penetrate the blood-brain barrier to exert their effects. This important issue must be addressed before any new MC4R-targeted molecule could be considered for clinical use. The ultimate goal for the development of any new therapeutic agent for melanocortin receptors is to identify a drug that produces the desired effect with minimal side effects. To this end, the concept of directed signaling and functional selectivity has generated significant interest, as a means to develop compounds that can selectively activ d re exocrine disorders.

Stabilization of β-turn conformation in melanocortin like peptide by click chemistry

The biological function of peptides and proteins is defined by their ability to adopt well-defined conformations, that complement those of their corresponding binding partner or receptor. Introduction of 1,2,3-triazole into an amino acid backbone is an estabilishe helices and β-turns, and a recognized strategy to improve resistance toward proteolytic degradation, thus increasing the metabolic stability in vitro and in vivo 39,40 .

Side chain-to-side chain cyclizations represents a strategy to select for a family of bioactive conformations, by reducing the entropy and lo binding conformations. This structural manipulation contributes to increased target specificity, enhanced biological potency, improved pharmacokinetic properties, higher binding affinity and lower metabolic susceptibility.

This approach can provide a convenient and versatile access to cyclic peptidomimetics, by incorporation of the building blocks N α -Fmoc-Xaa(ω-N 3 )-OH and N α -Fmoc-Yaa(ω-yl)-OH and the subsequent solution phase Cu I -catalyzed intramolecular azide-alkyne cycloaddition. In this way is possible to develop a new paradigm for an orthogonal bio-organic and intramolecular side chain-to-side chain ty, is isosteric with the peptide bond and can nction as a surrogate of the classical side chain-to-side chain lactam forming bridge. Moreover is it possible to obtain cycle head to tail, side chain to side chain or head/tail to side chain (Figure 31) 129 . cyclization. In fact, the proteolytic stable side chain-to-side chain connecting 1,4-or 4,1-disubstituted [1,2,3] In this PhD work, the 1,2,3-triazolyl-bridging strategy was applied to stabilize the βturn conformation of melanocortin analogues peptides. In fact, MCRs mediate a plethora of biological functions that include sexual function, feeding behavior, pain modulation, thermoregulation, energy homeostasis, cardiovascular effects, and skin pigmentation, making them potential drug targets for treating pain, food intake and body weight as well as erectile dysfunction. Therefore, development of an expansive toolbox of structural modifications that can be used to fine tune the predominant conformations, to achieve modulation of specificity toward receptor subtypes, physicochemical and pharmacological properties, continues to be of great interest in the development of peptide-based drugs in general, and melanocortinergic drugs in particular. Evidently, the plethora of melanocortins-mediated activities, the multitude of melanocortin receptor subtypes, and the high profile of potential therapeutic targets associated with the melanocortin system, underscores the unmet need for highly selective, pharmacokinetically diverse, and bioavailable agonists and antagonists. Understanding the bioactive conformation of the ligand and the structure of the receptor-ligand complex is crucial to design more potent and MCR-subtype selective ligands. Our studies explored the relationship between the size of bridge containing the 1,2,3-triazolyl moiety, the location of this moiety within the bridge, its orientation relative to the peptide backbone, and the predominant conformations displayed by these heterodetic cyclic peptides (Figure 32). Therefore, we synthesize a series of heterodetic cyclo-heptapeptides, based on the scaffold MT-II, varied in the orientation of the [1,2,3]

Click chemistry

Click chemistry is an entire reaction group in wh joining small units together 44 . A set be useful in this context are defined as follows:

• simple reaction co

• ideally, the process should be insensitive to oxygen and water;

• very high yields; 57 d without using

• be stereospecific (but not necessarily enantioselective);

an efficient "click s of these functional groups towards biological molecules in refluxing luene leading to mixture of the 1,4-and 1,5-regioisomers (Scheme 2), but modified proteins and peptides may not survive in this strong condition.

• only inoffensive by-products that can be remove chromatographic methods; to • high thermodynamic driving force, usually greater than 20 kcal.mol -1 .

One of the most interesting reactions that can enter in the click chemistry definition are the cycloaddition reactions involving heteroatoms, such as Diels-Alder and, especially, 1,3-dipolar cycloaddition, that provide fast access to a variety of five-and six-membered heterocycles. In particular, Huisgen 1,3-dipolar cycloaddition of azides and alkynes 41 can ideally meet all prerequisites for chemistry". The chemistry of alkynyl and azido group is completely orthogonal to the chemistry of all endogenous functional groups in proteins.

Azides and alkynes are crucial functional group for click chemistry. In fact they are easy to install and, being among the most energetic species known, they are also among the least reactive functional groups in organic chemistry. This stability, being purely kinetic in origin, is responsible for the slow nature of the cycloaddition reaction and the inertnes and towards the reaction conditions inside living systems (i.e. aqueous, and mild reducing environments).

Classically the cycloaddition between azides and alkynes is carried out to Scheme 2. [1,2,3]-Triazole formation by [3+2] cycloaddition of an alkyne and an azide.

Moreover, the obtainment of mixture of products is not useful in synthesis of peptidomimetics. Meldal 43 and co-workers and Sharpless 44 and co-workers 58 e Shrarpless 42 ies. A pre-organized catalyst e eventually gives rise to the formation of ditriazoles over statistically expected mixtures, even when a 10-fold excess of azide is used. developed a mild and efficient method to generate 1,4-disubstituted [1,2,3]-triazoles by metal-catalyzed reaction using Cu(I) salts as catalyst even in the presence of H 2 O. This copper catalyzed azide-alkyne cycloaddition (CuAAC), or Huisgen copper catalyzed cycloaddition, represent a new generic application for linking fragments under mild condition, so called bio-orthogonal conditions. The CuAAC was later described by Sharpless as a key example of the click chemistry concept 44 . Meldal and co-workers described the use of Cu(I) salts in the solid phase, whil reported solution phase by in situ reduction of Cu(II) salts. It is also possible to envisage the formation of Cu(I) from the oxidation of copper metallic.

The copper (I) alkyne-azide "click" cycloaddition mediated mechanism follows presumably the pathway outlined in Figure 33. The catalytic cycle shows that a source of Cu(I) is fundamental for the regioselectivity. A dimeric copper species ( 8) may be responsible for the catalytic cycle. This may explain the greatly enhanced reactivity of a second azide in diazide-containing spec th First, the copper(I)-catalyst forms a π-complex with the acetylene ( 6) and subsequently replaces the acidic proton of the acetylene. The azide coordinates to the ition of a base; IPEA and 2,6-lutidine minimize the side-product formation. Therefore, the choice free cycloaddition of phosphoranes and azides for the obtainment, on solid phase, of 1,5-disubstituted [1,2,3]-triazoles containing-peptides, both linear and cyclic 134,135 . copper via negatively charged nitrogen atom ( 8), followed by attack and subsequent formation of a new intermediate (9). This slightly bent allene-like structure then rearranges to form the copper-triazoles species (10). Finally, copper (I) exchange with an acidic proton delivers the triazole (3) and the copper (I) species that can enter the next cycle. However, in aqueous solution the formation of copper species ( 7) is exothermic, a result consistent with experimental findings of a rate acceleration in water. Moreover, the copper coordination lowers the pKa of the alkyne CH, thus making deprotonation in aqueous systems possible without the add D of the condition reaction (solvent, base) is crucial for cycloaddition.

Subsequently, Sharpless and co-workers, set up a Ruthenium catalysed azide alkyne cycloaddition (RuAAC), generating selectively 1,5-disubstituted [1,2,3]-triazoles, but the reaction conditions doesn't respect the criteria for click chemistry 130,131,132,133 .

Nevertheless, this reaction represents an innovate strategy because it allows further control of the conformations of a peptide sequence and provide access to the alternative 1,5-substituted triazoles. More recently, Rademann and co-workers reported a metal-F tr igure 34. CuAAC and RuAAC synthesis of triazoles. Comparison of 1,4-and 1,5-substituted iazoles with trans-and cis-peptide bonds.

Synthetic strategy

In the current study we explored the potential of the i-to-i+5 side chain-to-side chain 1,4-and 4,1-disubstituted [1,2,3]triazolyl-containing bridges to stabilize β-turn conformations. Studying this modification in the context of the MTII scaffold provides the opportunity to evaluate its potential as a modulator of receptor sub-type selectivity. To this end, we designed a series of 1,2,3 In this context, a series of N α -Fmoc-ω-azido-α-amino-and N α -Fmoc-ω-ynoic-αamino acids with different length of the side chain, was synthesized and introduced in position i-i+5 or i+5-i, to replace Asp 5 and Lys 10 residues in the sequence of MTII.

Solution phase intramolecular CuAAC converted linear precursor I'-X' in to the 1,4and 4,1-disubstituted- [1,2,3]triazolyl-containing cyclopeptides, I-X.

Synthesis of non-coded amino acids

The non-coded amino with different lengths of the side chains, were synthesized following the procedure previously described 16,51 (Figure 35). This process utilizes trifluoromethanesulfonyl azide (TfN 3 ) as a "diazo donor" in the Cu(II)-catalyzed conversion of a primary amine, into an azide 136,137,138,139 (Scheme 3).

Scheme 3. TfN 3 in the fer reaction diazotrans

The process is amenable to scale-up, employ commercially available reagents and does not require anhydrous conditions. Moreover, since the transformation does not involve the C α , it is accomplished with a complete retention of the original configuration of the α -amino acid residues. However, this reaction is not without problems. The explosive nature of neat TfN 3 and its relatively poor shelf life necessitate its preparation in solution prior to use. Furthermore, inconsistent yields in the preparation of TfN 3 mean that the solution must either be standardized or used in a liberal excess. The removal of trifluoromethanesulfonamide from polar products has in the past also required specialized workup procedures. Finally, perhaps most significantly, the expense of trifluoromethanesulfonic anhydride, used in the preparation of TfN 3 , prohibits the deployment of this reaction on a large scale. To circumvent these problems a cheap, robust, and safe alternative to TfN 3 is required.

A possible alternative to TfN 3 is represented by imidazole-1-sulfonate-azidehydrochloride. Imidazole-1-sulfonyl azide exhibit very similar reactivity to TfN 3 , miming its ability to act as a diazotransfer reagent, but it also might be less costly to prepare, more stable (hopefully crystalline), and produce more easily removed byproducts. The synthesis of imidazole-1-sulfonate-azide-hydrochloride is easily accomplished by the addition of two mole equivalents of imidazole to chlorosulfonyl azide, preformed in situ by the reaction of equimolar quantities of sodium azide and sulfuryl chloride in acetonitrile. Subsequent aqueous workup and flash chromatography afforded the desired product as a colorless liquid in good yield (72%), (Scheme 4).

Scheme 4. Synthesis of imidazole-1-sulfonyl-azide

To obtain a crystalline diazotransfer reagent, desired for reasons of stability ability and easy purification, we treated the imidazole-1-sulfonyl-azide with HCl in EtOH 140 . We obtained in a one-pot reaction, the imidazole-1-sulfonyl-azide hydrochloride as crystalline solid, in good yield and high purity without the need for chromatography. The imidazole-1-sulfonyl-azide hydrochloride was used as "diazo donor" in a reaction Cu II -catalyzed, starting from the N α -Fmoc amino acids, obtaining the correspondent N α -Fmoc-ω-azido-α-amino acids in good yields and with high purity.

Synthesis of N α -Fmoc-ω-alkynyl-α-amino acids

Inside the series of N α -Fmoc-ω-alkyinyl-α-amino acids, only propargylglycine (n=1, Pra) is commercially available. The higher N α -Fmoc-ω-alkynyl-α-amino acids homologs containing (CH 2 ) n (n=2, 3 and 4), were synthesized by alkylation of a Ni(II) complex of the Schiff base formed between glycine and (S)-2-(Nbenzylprolyl)aminobenzophenone, [Ni II -(S)BPB-Gly] as chiral inducer, with alk-ωynyl bromides respectively 141,142,143,144,145 .

The ω-bromoalkynes used for the alkynilation of the nickel complex were prepared from the corresponding alcohol by treatment with TsCl followed by LiBr, with the exception for 4-bromo-1-butyne, commercially available (Table 3). That is depending by the repulsion between ortho-protons of the benzyl group and equatorial substituents of Cα of glycine 146 . The diastereoisomeric excess can be evaluated by UPLC (Table 4). The separation of the diastereoisomers was successfully achieved by flash column chromatography, FCC. Alkylation of glycine by alkynyl bromides in the presence of the chirality inducer [Ni II -(S)BPB-Gly], p H 8 ,2 h , Peptides I'-X' were synthesized on Rink-amide NovaSyn TGR resin (0.14 mmol/g, 300 mg), on a manual batch synthesizer (PLS 4X4, Advanced ChemTech), following the Fmoc/tBu chemistry. The Fmoc-Rink-amide resin was swelled with DMF (1 mL/100 mg of resin) for 20 min before use. Stepwise peptide assembly was performed by repeating for each added amino acid the standard procedure, using TBTU/HOBt/NMM (2 eq./2 eq./3 eq.) as the coupling system and 2 eq. of the Fmoc protected amino acids, except for N α -Fmoc-Xaa(ω-N 3 )-OH and N α -Fmoc-Yaa(ω-yl)-OH for which 1.5 eq. were used. The coupling was carried out in DMF (1mL/100 mg of resin) for 50 min. The solid phase assembly of the linear precursors I'-X', was performed by incorporation of the N α -Fmoc-ω-azido-α-amino-and N α -Fmoc-ωynoic-α-amino acids in positions i-i+5 and i+5-i, replacing Lys 10 and Asp 5 of MTII, during the stepwise build-up of the resin bond peptide. This strategy was found to be the most convenient approach to introduce the functions essential for the subsequent CuAAC. Moreover, thanks to the relative ease introduction of the azido and alkynyl functions, and their inertness toward a wide range of reaction condition and nucleophiles and electrophiles 44,147 orthogonal protection scheme is not required.

N H N B n O O P h N O N C 6 H 5 N O O N i N i(N O 3 ) 2 •6 H 2 O G ly / M e O H H K O H , iPr N C O O H C O O H H 2 N P h O B n C l N O H 4 0 °C , 6 h 1 ) P C l 5 , a n h y d C H 2 C l 2 3 0 m in , rt 2 )
Each coupling was monitored by Kaiser test 148 and was negative, therefore recouplings were not needed. Once the peptide sequences were completed, the free N-terminal α-amino of the resin-bound peptides was acetylated following the protocol described in Experimental Part. The reaction was monitored by Kaiser test.

All the linear peptides were purified by semi-preparative RP-HPLC, employing a linear gradient 20-to-60% of B in 30min (A -0.1% TFA in H 2 O; B -0.1% TFA in ACN) flow 4 ml/min., and characterized by ESI-MS. Analytical data are reported in Table 3. The click reaction conditions were identical to those reported by us previously 51 . In particular, the complete conversion of the linear precursors I'-X' to the desired cyclic [1,2,3] were further purified by RP-HPLC, using the same conditions reported before, and characterized by ESI-MS (Table 4).

For the yields of the pure cyclopeptides, we did not observe a large difference between the cyclopeptides containing triazolyl ring with alkyl chains of different lengths: from 40% (for the longer alkyl chains) to 55%. fragment. The peptide was purified by RP-HPLC, using the same conditions reported before, and characterized by ESI-MS (Table 5).

Biological activities

The biological studies were performed in collaboration with prof. Carrie Haskell-Luevano, Department of Pharmacodynamics, University of Florida, USA, and Medicinal Chemistry, University of Minnesota, Minneapolis.

The biological activity of all the 1,4-and 4,1-disubstituted- [1,2,3]triazolyl-containing cyclopeptides I ÷ X (Table 7), are compared to the lactam-containing peptide MTII, as well as the linear control precursors I' ÷ X' (Table 6).

The potency of MTII, analogues I ÷ X and I' ÷ X', was pharmacologically evaluated for functional potency (EC 50 ) and efficacy using a reporter gene based bioassay for cAMP in HEK cells stably expressing the murine (mouse) melanocortin receptor subtypes mMC1R, mMC3R, mMC4R, and mMC5R 149,150 . The results show that the cyclic peptides are more potent than their linear precursors, confirming that conformational stabilization in the form of side chain-to-side chain cyclization enhances in vitro potency. Moreover, the clicked cyclic peptides have similar potency compared to the parent lactam-containing MTII analogs, indicating that these types of side chain modifications are a feasible alternative to the lactam types of cyclizations to enhance desired in vivo chemotype biophysical properties. Of interest is the emergence of mMC5R selectivity. While the prototypic MTII is 3-and 5-fold more potent toward mMC1R and mMC4R, respectively, than toward mMC3R and mMC5R (cf. EC 50 = 0.03 and 0.06 nM with 0.18 and 0.19 nM, respectively).

Analogs III, IV, and VII display higher mMC1R-mediated activities as compared with MTII.

In summary, our study supports our idea that the i-to-i+5 side chain-to-side chain bridged [1,2,3]triazolyl containing-cyclopeptides can successfully replace the classical β-turn stabilizing i-to-i+5 side chain-to-side chain bridged lactam containing cyclopeptides and generate equipotent analogs. This enhancement of their in vitro potency relative to the linear precursors suggests that the imposed structural rigidification achieves the conformational stabilization of biological relevant conformation. In the future it could be interesting to fine-tune this modification to improve receptor-subtype selectivity.

Conformational analysis

NMR conformational studies of cyclopeptides I, IV, V were performed in collaboration with prof. Anna Maria D'Ursi, Dr. Mario Scrima and Manuela Grimaldi, of University of Salerno.

In solution, most of the peptides assume multiple flexible conformations.

Determination of the dominant conformers and evaluation of their populations is the aim of peptide conformational studies, in which theoretical and experimental methods play complementary roles 151 . Cyclic peptides typically assume multiple conformations; these conformations are rather flexible, with torsional angles of the backbone (φ, ψ) as well as of the side chain groups (χ i ) fluctuating within large intervals (Figure 38).

In addition, coupling constants between NH and C α H can give information about the average values of the peptide backbone torsional ϕ angles. that is fundamental for the interactions with the receptor.

NMR analysis

NMR spectra of MTII analogues peptides were acquired in DMSO-d6. To exclude potential aggregation, we recorded the 1D proton spectra of the cyclopeptides at a concentration range spanning 1-0.1 mM. At a peptide concentration of 0.1 mM, the peptides did not display any noticeable effects of aggregation. Chemical shift assignments of the proton spectra were achieved via the standard systematic application of DQF-COSY 152 , TOCSY 153 , and NOESY 154 experiments, using the SPARKY 155 software package according to the procedure of Wüthrich 156 .

Analysis of NOESY spectra evidences a high number of well resolved cross-peaks suggestive of significant presence of ordered conformers. According to the observation of a double signal pattern in the NOESY spectrum, two different bar diagrams (Figure 39A and Figure 39B) are reported for analog I, there of the most abundant evidences a higher number of medium range NOEs, whereas the less abundant evidences less intense and less numerous NOE effects.

Interestingly the less abundant conformational population is characterized by the presence along the whole sequence, of a systematic αβ(i, i+3)NOE correlations.

NOESY spectra of cyclopeptides II and X, shows a drastic reduction of diagnostic cross peaks indicating a minor presence of ordered conformers.

Conclusion

A collection of N α -Fmoc protected unnatural amino acids bearing on the side chain azide or alkynyl functions was synthesized and introduced by SPPS in the MTII peptide sequences to afford by Cu(I) catalyzed Huisgen reaction, a new collection of cyclopeptides containing the triazolyl moiety.

The side-chain-to-side chain cyclization of linear peptides generated via click chemistry lead to cyclopeptides containing the triazolyl moiety linked to the αcarbon of the amino acids by alkyl chains of different lengths.

A conformational study of these pure cyclopeptides was performed to understand, by comparison to the parent lactam model, the structural analogies fundamental for the receptor recognition. As preliminary results, we can assume that cyclopeptides containing residues involved in the intramolecular cyclization, bearing the longer side chain, assume more canonical structure than those containing residues with a shorter side chain.

The NMR conformational study of cyclopeptides analogs containing triazolyl moiety that could mimic at the best the orientation of side chains and the secondary structure of the lactam MTII cyclopeptide, are in progress at the University of Salerno.

PART B 3

Impact of endocrine disruptor chemicals on human health

Endocrine disorders are commonly related to improper functioning of the endocrine system, for example malfunctions in pancreas, pituitary, thyroid and adrenal glands.

The hormone imbalance (or endocrine disease/condition) can affect human health inducing a lot of conditions such as diabetes mellitus, thyroid disease, and obesity. A number of factors are believed to cause endocrine disorders. Genetic factors, seems to play a relevant role in the etiology of endocrine diseases. For example mutations at the level of thyroid can determin congenital hypothyroidism, while mutations of pituitary glands can induce a lot of disease, because pituitary gland secretes a series of hormones that regulate different biological function.

Nevertheless, in many cases, the exact cause of a particular endocrine disorder is not known. In the last years, it took importance the hypothesis that also environmental or nutritional factors could be involved in etiology of endocrine disorders. In fact, the increased rate of metabolic and neurodevelopmental diseases seems to correlate with severe changes in the chemical environment, affected by new industrial and agricultural procedures over the past 40 years. Environmental chemicals with hormone-like activity can disrupt programming of endocrine signaling pathways during development, and result in adverse effects, some of which may not be apparent until much later in life. Recent studies linked the exposure to environmental endocrine disrupting chemicals during development with obesity, diabetes and neurodevelopmental disorders. These particular diseases are quickly becoming significant public health problems and are fast reaching epidemic proportions worldwide. Therefore, understand the role of endocrine disrupting chemicals, such as phthalates, in the etiology of different pathologies is crucial 157 . The detection in human fluid, such as urine, of metabolites of specific compounds, supposed to be endocrine disruptors, could be a valid method to explore the potential causes for endocrine disorders. Phthalates are used in a variety of consumer products, therefore they are considered ubiquitous contaminants. In particular di-(2-ethylhexyl) phthalate (DEHP) was the most used plasticizer for PVC in a lot of products and in particular in medical devices. Very recently, phthalates were suspected to be correlated to two relevant disorders of childhood: early obesity and Autism Spectrum Disordes (ASDs). In fact, children are known to be exposed to DEHP twice as high as compared to adult subjects. In particular, fetuses and neonates are highly sensitive to physiologically active agents because they are exposed during critical periods of human development. Furthermore, in the case of intensive therapy procedures, neonates can have higher exposure to phthalates and to their toxic monoesters supposed to be endocrine disruptors.

Therefore, in this part of PhD, we focused on the investigation of the presence of urinary concentrations of DEHP metabolites by HPLC-ESI-MS, in two populations of obese and autistic patients in comparison with healthy controls.

Phthalates

Polyvinylchloride (PVC) is one of the most widely produced plastic in the world.

PVC is used extensively for a lot of different purposes, ranging from a lining for landfill waste disposal sites to a food wrapper for foods. One of the key attributes of PVC that led to its widespread use, is its stability and flexibility, which is achieved by the incorporation of plasticizers. A plasticizer is a substance which when added to a material, usually a polymer, makes it flexible, resilient and easier to handle. There are more than 300 different types of plasticizers described. The most commonly plasticizers used in PVC are phthalates.

Phthalates, the diesters of phthalic acid, are a family of industrial compounds with a common chemical structure (Figure 44). They are widely used, since their introduction in 1930s, as plasticizers to soften PVC, in solvent, and as additives in many consumer products such as vinyl flooring, wall coverings, food containers, cosmetics 158,159 . End-applications include adhesives and glues, electronics, agricultural adjuvants, building materials, personal-care products, medical devices, detergents and surfactants, packaging, children's toys, modeling clay, waxes, paints, printing inks and coatings, pharmaceuticals, food products, and textiles. MEHP subsequently undergo several biotransformation in the liver and it is metabolized to produce a large number of oxidative metabolites 164,165 (Figure 46).

Oxidative metabolism of MEHP starts with hydroxylation of the alkyl chain at various positions and the formation of primary (ω-oxidation) and secondary (ω-1 oxidation) alcohols. These hydroxylated products can undergo further oxidative reactions to the respective ketones and carboxylic acids. After that, the carboxylated alkyl chain can be subject to α-or β-oxidation to yield shorter carboxylated alkyl chains. In female offspring in utero and lactational exposure to high dose of DEHP results in delay of the time of puberty onset and ovarian polycystic 174 .

PPAR-mediated activity of phthalates

A privileged mechanism for EDs interference with metabolic pathways is their direct Recently signal transduction pathway of α-PPAR was also correlated to progression of neurodegenerative and psychiatric diseases. In fact, the activation of α-PPAR, by phthalates interferes with cellular proliferation and lipid metabolism, also in the brain 177 . Moreover, the peroxisome proliferation, mediated by PPAR, leads to oxidative stress and generation of electrophilic free radicals. A link between oxidative brain damage and neurodevelopmental disorders, such as Autism in children, was observed in the lasts years 178 , 179 . In 2009, South Korean scientists reported findings of a statistically-significant correlation between urine phthalate concentrations in children and symptoms of attention-deficit/hyperactivity disorder (ADHD) 180 . Although more research is needed in order to conclusively determine the relationship between phthalate and ADHD, the article suggests that consumers should be aware of its potential effects on behavior and neurological disorders.

Alteration of Thyroid Hormone Levels

Exposure to DEHP in fetuses and infants

Exposure of the developing fetus or infant to endocrine-disrupting chemicals is of particular concern 187 . Infants are believed to be the most sensitive population, as they are exposed since early in life to several different sources, including breast milk, infant formula, baby food, indoor air, and by dermal and oral exposure via indoor dust containing DEHP. The developing fetus and neonate are particularly sensitive to perturbation by endocrine disrupting chemicals because they are exposed during critical periods of human development 188,189,190 . EDs may also be transferred from mother to developing fetus crossing the placenta, or as neonates incorporating them through breastfeeding 191,192 . Moreover, the placenta does not completely protect the unborn fetus from its external environment and the organism undergoes periods of extremely rapid cell division and differentiation thus resulting in cells that can differentiate abnormally and pass altered programming to subsequent generations. In many cases, the damage that occurs prenatally is irreversible, while adult exposure is usually reversible. The developing organism lacks the protective mechanisms that are available to the adult such as DNA repair mechanisms, a fully competent immune system, detoxifying enzymes, liver metabolism, and has an immature blood/ brain barrier 193 . Also, the developing fetus and neonate have increased rates of lipase activity and/or decreased rates of glucuronidation activity as compared to adults, which in some cases may make them more vulnerable to chemical toxicity, resulting in a possible bioaccumulation of DEHP metabolites in their body.

Exposure to environmental chemicals during development can interfere with complex differentiating endocrine signaling pathways and cause adverse consequences later in life. In general, neonates receive higher doses, in terms of body / weight, of DEHP than the general population and their daily dose to DEHP may increase up to 20 folds the tolerable daily intake.

Moreover, in cases of intensive therapy procedures, neonates can have higher exposure to phthalates and to their toxic monoesters supposed to be EDs. Medical devices and tubing may contain 20-40% DEHP by weight, which easily leach out of tubing when heated (as with warm saline/blood).

The combination of prenatal and postnatal exposures may exacerbate the reproductive hazard. Therefore a concern was raised about potential health effects of DEHP. Specifically, EDs are known to cause learning disabilities, severe attention deficit disorder, cognitive and brain development problems, deformations of the body (including limbs); sexual development problems, feminizing of males or masculine effects on females, metabolic diseases etc. In fact, any system in the body controlled by hormones, can be derailed by hormone disruptors. These disruptions can cause different diseases such as cancerous tumors, birth defects, and other developmental disorders.

Detection of DEHP metabolites in urine

Aim of this part of PhD study was to evaluate the levels of the primary and All the products were purified by Flash Column Chromatography, and were obtained in good yield, with chemical purity >98%. DEHP metabolites were characterized by 1 H, [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] C NMR and RP-UPLC (Table 8). Gradient 50 to 100 % of CH 3 CN in 5 min, flow 0,6 ml/min.

Quantitative analysis

Before analysis all the materials used during the entire process were pre-screened by HPLC-ESI-MS to verify the absence of external contamination. In particular, all the solvents (e.g., acetonitrile, water, buffers, etc.), laboratory equipments (polypropylene vials containing urines, solvent bottles, SPE cartridge, teflon cappedglass bottles, pipettes), and instrumentation used during the entire validated, sensitive, and selective HPLC-ESI-MS analytical process, were verified to be DEHPmetabolites free. To this end, we applied the same HPLC-ESI-MS method reported for phthalates detection in urines, to solutions that have been contained in the equipments used. No DEHP-metabolites could be detected in any of the above mentioned material even after two week storage. The internal standards were prepared in ACN and were used as reported in literature 195,196 .

Calibration curves

The unequivocally characterized synthetic metabolites, MEHP, 6-OH-MEHP, 5-cx-MEPP, 5-oxo-MEHP, 5-OH-MEHP, were used as pure analytical standards (>98%)

for quantitative determination in human urines. The standard calibration curves were constructed using working standards solutions of DEHP metabolites, ranging from 0.00025 to 2.5μg/mL. A linear regression analysis was carried out by plotting the peak area (y) against the concentration (x) for each analyte. The linearity of the calibration curve was expressed by the correlation coefficients (r ^2). The lower limit of detection (LLOD) is defined as the lowest concentration of analyte in a sample that can be detected, but not necessarily quantified, under the stated experimental conditions. It can be calculated from the standard deviation (SD) of the response and the slope associated with the calibration curve 197 , according to the equation:

The

Instrumental and chromatographic conditions

The HPLC-ESI-MS analysis was carried out using an high-performance liquid chromatography tandem mass spectrometry RP-HPLC-ESI-MS (Waters, Alliance 2695, Waters, Micromass ZQ) equipped with phenyl column (Betasil, 5 μm, 50 mm x 3 mm, Keystone Scientific, Bellefonte, PA) and with a Waters 2996 Photodiode Array Detector. An aliquot of each sample (20 μL) was injected into the apparatus.

All standards were injected three times in the same day. Curves with correlation coefficients (R 2 ) greater than 0.998 were generated (MEHP, 5-OH-MEHP, 5-oxo-MEHP 0.999; 6-OH-MEHP, 5-cx-MEHP 0.998). The chromatographic separations of metabolites were resolved using a linear gradient from 3% to 60 % B in 10 min (solvent system A: 0.1% acetic acid in water; B: 0.1% acetic acid in ACN). The flow rate was 0.6 mL/min. The column temperature was 32 °C. A guard column (XBridge Tm Phenyl 3.5μm, 3.0x20 mm) was used to prevent column degradation.

Column eluates were monitored at 215, 230 and 254 nm. The mass specific detection was achieved using a Waters, Micromass ZQ Electrospray ionization (ESI) in positive ion mode. The product ion with higher signal intensity was selected for the quantitative analysis for each of the four phthalates. The optimal MS parameters were as follows: the source and desolvation temperature were 120 °C and 400 °C respectively; the capillary voltage was 3.24 kV; cone voltage 30 kV, nitrogen gas was used as desolvation gas and as cone gas as well; the cone gas and the desolvation flow was 60 L/h and 800 L/h respectively; The collision gas was argon with a flow of 0.60 ml/min. Data were acquired and processed using MassLynx™ software (Waters). The analytical methodology adapted for measuring MEHP and secondary oxidative metabolites in urine was already described in the literature 196,198 199 .

One water reagent blank and one quality-control (QC) sample, were processed through the entire process along with unknown urine sample, and were included in each batch of samples to monitor for contamination. The QC sample was spiked with pooled urine and DEHP metabolites standards in known concentration (200 ng/mL).

When urine analysis resulted in values of metabolites concentration exceeding from linear range of the analytical method, we subjected a new aliquot of the same sample to the entire process (deconjucation and SPE) and we analysed again.

A known standard was also injected into the entire sample set in order to confirm chromatographic resolution. The identification of the peak was confirmed by matching retention times of the unknown sample, with the standard.

Sample pre-treatment

First morning urine specimens were collected. All the samples were subjected to two steps of Solid Phase Extraction (SPE) before RP-HPLC-ESI-MS analysis, in order to remove components from biological matrix that could interfere with the analysis. For SPE treatment of urine, we prepared the following buffers: buffer ammonium acetate 1 M pH 6.5; acid buffer pH 2.0 by preparing a solution of NaH 2 PO 4 (0.14 M) and 1%

of 85% H 3 PO 4 ; basic buffer was prepared by adding concentrated ammonium hydroxide (1 mL 30% NH 3 solution) to a 50:50 acetonitrile/water (200 mL). All buffers were stored in sealed bottles at room temperature: basic buffer was discarded after one week, acid buffer after one month.

Human urines (1 mL) were defrosted, sonicated, mixed, and dispensed in glass tubes.

Then buffer ammonium acetate (250 μL, pH 6.5) was added. Incubation with βglucoronidase (5μL, 200units/mL, Roche Biochemical, Mannheim, Germany) was performed at 37 °C for 90 min, resulting in quantitative glucuronide hydrolysis of phthalates metabolites. Escherichia coli K12 β-glucuronidase has excellent glucuronidase activity and no measurable lipase activity on phthalate diesters 195,196. After deconjugation, samples were treated with two steps of solid phase extraction (SPE), in order to remove contamination of biological matrix (Figure 49). A first cartridge was used to retain hydrophobic compounds while the phthalate metabolites were eluted. A second cartridge was helpful to remove residual salts. Analytes were finally eluted with acetonitrile and ethyl acetate, concentrated, re-suspended in water and transferred into vials. All the samples were analysed by RP-HPLC-ESI-MS. 9. These data demonstrate that the solid phase extraction protocol provides the almost quantitative recovery of all the analytes from urine sample. Moreover, this method exhibits a quite good repeatability (%RSD less than 7%). The data were corrected for creatinine. All specimens displayed urinary creatinine in the children reference value range, dependent on age and lean body mass (0.5-4.0 mg/Kg/24 h). For creatinine measurement, we used a Beckman Synchron AS/ASTRA clinical analyzer (Beckman Instruments, Inc., Brea, CA). We used value (micrograms per liter)/creatinine (grams per liter) for dilution correction in the analyses.

Relationships between urinary concentrations of DEHP metabolites, obesity and insulin sensitivity in obese children

Obesity

Obesity is a medical condition characterized by a pathological excess of body fat, that it may have an adverse effect on health, leading to reduced life expectancy and/or increased health problems. Obesity is a complex disease, affecting virtually all ages, races, sexes, and socioeconomic groups, with serious social and psychological repercussions. Further, obesity and overweight are major contributors to the global burden of chronic diseases and are associated with Type 2 diabetes, hyperinsulinemia, insulin resistance, coronary heart disease, high blood pressure, stroke, gout, liver disease, asthma and pulmonary problems, gall bladder disease, kidney disease, reproductive problems, osteoarthritis, and some forms of cancer. Frequently, many of these illnesses are being reported in children who are overweight or obese, whereas in the past these diseases were only seen in older individuals.

Body mass index (BMI), a measurement which compares weight and height, defines people as overweight (pre-obese) if their BMI is between 25 and 30 kg/m 2 , and obese when it is greater than 30 kg/m 2 .

Obesity is a leading preventable cause of death worldwide, with increasing prevalence in adults and children, and authorities view it as one of the most serious public health problems of the 21 st century. In fact, over the last 2 to 3 decades, the prevalence of obesity has risen dramatically in wealthy industrialized countries, and also in poorer underdeveloped nations where it often coexists with under-nutrition 200 .

In the United States, the Center for Disease Control (CDC) reported in 2008 that obesity has reached epidemic proportions with more than 60% of U.S. adults being either obese or overweight 201 . Similar statistics have been reported for many

European countries, the Middle East, Australia, and China.

The reasons for this sharp increase in overweight/obesity are not well understood but factors such as diets, genetics/epigenetics, higher maternal age, use of certain pharmaceuticals, pollution, and the exposition to endocrine disruptors have all been proposed as playing a role.

Causes

102

Obesity is most likely caused by a complicated interaction between genetic, behavioral, and environmental factors. However, the most common causes are thought to be overeating high calorie fatty diets combined with a sedentary lifestyle, which is imposed on a background of genetic predisposition for the disease.

Although much interest has centered on these factors, the exact etiology of obesity is unknown. Some studies report a link between some persistent organic pollutants, such as phthalates, bisphenol A, heavy metals and polychlorobiphenyl, and increased body weight and diabetes 202 . In a cross-sectional study, concentrations of urinary phthalates metabolites were associated with increased waist circumference and insulin resistance in adult males in the United States 176 . This epidemiological study suggest that environmental exposures to various endocrine disrupting chemicals, plays a role in overweight /obesity and in the complications associated with these diseases, such as diabetes. Recently, the adipocytes were proposed as a new target of EDs 203 . Environmental chemicals, such as DEHP, have a disrupting activity on normal adipocyte development as well as on homeostatic control of adipogenesis and early energy balance. These chemicals have been termed "obesogens" based on the idea that they desregulate the lipid metabolism and adipogenesis, promoting obesity.

Transgeneration effects may also be seen following exposure to endocrine disrupting chemicals during development, not only to the exposed individual but also to subsequent generations. This would imply that the mechanisms of transmission occur through the germ line and they may involve genetic and/or epigenetic events. In the case of epigenetic changes, effects are not due to a genetic impairment but to a modification of factors that regulate gene expression, such as DNA methylation and histone acetylation 204 .

Mechanism of action

The exact mechanisms that may induce phthalates to cause obesity are not known, but it may involve a great variety of pathways.

For example, EDs exert their effects altering thyroid function metabolism and/or energy homeostasis. However, the most commonly proposed mechanism involves direct binding to α/ γ PPAR. EDs could also act indirectly inhibiting enzymatic activity of P450 family members, CYP19 and CYP3A1, which convert testosterone to estradiol, or by activating expression of the P450 enzymes, thus altering hormone levels. Finally, another mechanism that has been proposed is altering the neuronal synapse formation, 205 which could affect release of brain-produced substances that bind to nuclear receptors and may affect energy regulation. An additional complexity is that these proposed mechanisms may be interacting with each other. The altered signal or tissue may involve adipocytes, brain, liver, stomach, pancreas, etc.

Identifying the mechanisms whereby EDs influence weight homeostasis and energy balance is an important area of future research.

Detection of DEHP metabolites in obese children

Data suggest that endocrine-disrupting chemicals, as phthalates, found in a variety of household products, may influence mechanisms related to obesity. In vitro studies have shown that these chemicals may interact with PPARs and play a key role in the differentiation of adipocytes and deposition of triglycerides in adipose tissue.

Therefore, in this study we focused on the evaluation of a possible association between urinary phthalate metabolites and age, gender, pubertal development, age at onset of obesity, BMI, waist circumference, indexes of insulin sensitivity, and metabolic syndrome in obese children.

For the study, 42 obese patients and 39 healthy controls were recruited from the staff of "Centro Turbe dell'Accrescimento della Clinica Pediatrica" Azienda Ospedaliero-Universitaria di Parma. Urine was collected early in the morning. All the subject enrolled for the study were classified according to sex, age, BMI, chronological age onset of obesity, birth weight, etc. The auxological parameters were reported in Table 10. The metabolic syndrome diagnosis was performed on the base of the Global international diabetes federation criteria 209 .

Urine concentrations of DEHP metabolites, MEHP, 6-OH-MEHP, 5-OH-MEHP, 5oxo-MEHP, 5-cx-MEHP, were measured in triplicate using the RP-HPLC-ESI-MS method described above. In urine from obese children we found detectable levels of MEHP, 5-OH-MEHP, 6-OH-MEHP, 5-oxo-MEHP and 5-cx-MEHP in 77%, 85%, 90%, 99%, and 85% of patients respectively. In urine from healthy controls we found detectable levels of MEHP, 5-OH-MEHP, 6-OH-MEHP, 5-oxo-MEHP and 5-cx-MEHP in 90%, 74%, 64%, 63%, and 51% respectively. Significant concentrations of 5-OH-MEHP and 5-oxo-MEHP were detected in obese patients, compared with healthy controls (Table 12). The data were analyzed applying an innovative statistical methods based on Artificial

Neural Networks (ANNs) and principal component analysis and connectivity map 210,211 . This new mapping method, aimed to understand the natural processes and recreate those processes using automatic models, is particularly useful to understand the relationships between variables, especially in non-linear relationships.

The method is based on an artificial adaptive system able to define the strength of the associations of each variable with all the others in any dataset, the Auto Contractive Map (AutoCM). After the training phase, the weights matrix of the AutoCM represents the warped landscape of the dataset. We apply a simple filter to the weights matrix of AutoCM system to show the map of the main connections between the variables. Applying AutoCM we are able to obtain a subsequent semantic map of actual relationship between variables.

Results

A negative correlation of MEHP with the age at onset of obesity was shown (r: -0,4; p: 0,03; Figure 50A). A negative correlation was also found between MEHP and AUCI (r: -0,35; p: 0,04; Figure 50B), and between MEHP and AUCG (r: -0,33; p: 0,05; Figure 50C). On the contrary a positive correlation was observed between MEHP and WBISI (r: 0,33; p: 0,05; Figure 50D). 5-oxo-MEHP instead was significantly correlated with the age at onset of obesity (R:+0.51). The connectivity map showed a direct relationship of 5-oxo-MEHP with BMI SDS (Figure 51). MEHP showed a direct relationship with the insulinogenic index, and normal insulin sensitivity. The sum of the metabolites showed a direct relationship with the height SDS (Figure 51) suggesting that height may be related with the ability to metabolize phthalates. This latter was directly related with 5-oxo-MEHP.

Interestingly, 5-OH-MEHP was directly connected with the HOMA index and abnormal insulin sensitivity.

In conclusion, the results supported the hypothesis that the higher metabolites in urine, the earlier the onset of obesity, and suggested that some DEHP metabolites are capable of influencing insulin sensitivity. However, the less phthalates were metabolized (higher MEHP urine concentrations) the later was the onset of obesity. It remains, thus, to be elucidated whether exposure to phthalates per se is actually the risk factor or if the ability of the body to metabolize phthalates is actually the key point. This could explain also the positive relationship of MEHP with WBISI. ASDs are able to live and work independently as adults.

Etiology of ASDs

ASDs etiology is unknown, but it is believed to result from disruption of normal neurobiological mechanisms primarily in the prenatal period 214 . Moreover, it is widely recognized that ASD might have a genetic component 215 . Nevertheless no more than 5% of all autism cases appear to be due primarily to a genetic disorder. In particular, on a total of about 2,000 autism cases in the last 10 years, about 1% can be attributed to X-fragile syndrome, about 2% to gene microdeletions, about 1% to tuberous sclerosis, 1.5/1000 (0.15%) to phenylketonuria (PKU) or other geneticmetabolic inborn errors of metabolism, about 3/1000 (0.3%) to Down syndrome with autistic features, that all summed up is about a 4.45% fraction of all autism cases observed in a relatively large series. On the other hand, it is difficult to truly ascertain the sub-prevalence of Rett syndrome among the ASDs. However it is well known that the world frequency is of one Rett syndrome case in 8,000 to 10,000 females, that is about 1 case in 16,000 to 20,000 in the general population.

A dramatic increase in frequency of children with ASDs has been reported during the last 10 years. Nevertheless, it is difficult to determine how much of this increase may be due to actual increase in the incidence or to increased awareness and diagnosis.

This data suggest that environmental causes, such as exposure to chemicals such as heavy metals, pesticides, phthalates or childhood vaccines, etc., could play a significant role in the etiology of ASDs.

Consequently, the focus of autism research is shifting from purely genetic influences to multifactorial diseases in which complex set of genes should be associated to relevant environmental factors 216,217 . Moreover, recent studies describe a number of gene-environment interactions in which, exposure to a pre-or post-natal environmental pathogen, causes a behavioral disorder only if an exposed individual carries a specific genetic predisposition 218 .

Prenatal exposure to stressful events was found to have significant effects on postnatal behavior and was also associated with increased risk of ASD.

In fact, prenatal stress can reduce uterine and placental circulation, inducing fetal hypoxia. This could stimulate the release of maternal stress hormones that can cross the placenta and alter the development of the HPA axis. Stress can also disrupt the normal patterns of prenatal exposure to sex hormones that program typical sex differences in brain structure and function, which tend to be atypical in children with

ASDs. ASDs are devastating and there is, with rare exception, no established method for its prevention. Consequently is urgently needed to identify potential environmental factors that can contribute to ASDs. Identification of environmental factors that can be avoided, prevented, or ameliorated by programs of primary prevention is therefore especially important.

As concerns arise about exposure to phthalates during critical periods of human development, assessing phthalate exposure using biological matrix may become desirable to investigate the exposure of potentially vulnerable population such as children. ± 5] were randomly chosen from outpatients who had no pathological symptoms.

Subjects' population

A
Urines from children with ASDs and HCs were collected in polypropylene specimen cups, shared in aliquots (1.0 mL) and frozen at -20 °C until analysis. Field blanks consisted in purified water collected in polypropylene tubes and frozen at -20 °C.

The concentration of DEHP metabolites in urine from ASDs children and HCs were determinate by the RP-HPLC-ESI-MS method described above.

First morning urine specimens were collected. All specimens displayed urinary creatinine in the children reference value range, dependent on age and lean body mass (0.5-4.0 mg/Kg/24 h).

Statistical analysis

All

Results

Among the four metabolites of DEHP measured in urines of ASDs, we detected urinary concentration with geometric mean for MEHP (55 μg/L), 5-OH-MEHP (180 μg/L), 6-OHMEHP (17 μg/L), 5-oxo-MEHP (96 μg/L). On the contrary, in urine from healthy controls we found the following values for MEHP (28 μg/L), 5-OH-MEHP (4 μg/L), 6-OH-MEHP (19 μg/L), 5-oxo-MEHP (4 μg/L).

We found detectable levels of MEHP, 5-OH-MEHP, and 5-oxo-MEHP in 79.2%, 52.1%, and 46.0% of ASDs patients, respectively (Table 13). Interestingly, 5-OH-MEHP was the major metabolite in terms of urine concentrations, followed by 5-oxo-MEHP and MEHP. Urinary excretion of 5-oxo-MEHP (p = 0.005), 5-OH-MEHP (p=0.0224), and MEHP (p=0.0312) was significantly higher in autistic patients compared to gender-and age-matched healthy controls (Table 14). On the contrary, 6-OH-MEHP (p = 0.9305) was not able to discriminate ASDs. High levels of 5-OH-MEHP were detected in 26.6% of healthy controls. A significant positive correlation between 5-OH-MEHP and 5-oxo-MEHP could be observed only in autistic children (rs = 0.668, p < 0.0001), but not in the control population (rs = 0.125, p = 0.5565). The oxidised form of the metabolites pathway, corresponding to 5-oxo-MEHP had a specificity of 91.1% in identifying autistic children (Figure 52). The horizontal lines indicate the selected cut-off values (at mean ± 3SD of the control healthy group) and is graphically represented as a horizontal line.

The efficiency of urinary secondary metabolites in discriminating ASDs patients from healthy controls was evaluated using ROC curve analyses (Table 13, Table 15).

Finally, we analyzed the correlations between total Childhood Autism Rating Scale (CARS) score and levels of the different metabolites. Total CARS scores were (means ± SD) 44 ± 7.4 (range: 31-60).

A positive correlation between CARS scores and urinary MEHP levels was observed (rho = 0.429, p = 0.0033), whereas no significant relationships with the levels of the other examined metabolites were found (total CARS vs. 5-OH-MEHP: rho=0.120, p=0.4298; total CARS vs. 5-oxo-MEHP: rho=0.127, p=0.3931; total CARS vs. 6-OH-MEHP: rho=0.0085, p=0.9529).

Moreover, comparing ASDs with n=10 patients with Rett syndrome (a genetic form of autism due in up to 95% of cases to mutation of a gene. i.e., MeCP2), urinary phthalate metabolites appear to be, once again, significantly elevated in ASDs patients (Table 15). Likewise in ASDs, MEHP was found to be the minor urinary metabolite compared to the corresponding oxidized metabolites as in some adult metabolic diseases 219,220 . It is possible to hypothesize that, in the case of ASDs, a high MEHP urinary excretion does not correspond to external contamination, but rather MEHP could likely act as an endocrine disrupter.

From our data it is possible to speculate that phthalates metabolites as endocrine disrupters may play an important and previously not recognized role either in the neurotransmitter system and/or in neurodevelopment possibly increasing the risk of ASDs. In any case, our findings indicate for the first time that specific DEHP metabolites are statistically significantly increased in ASDs. It is intriguing that urinary levels of the oxidized form of the DEHP metabolites (5-oxo-MEHP) In conclusion, our findings generate the idea that prenatal plus postnatal phthalate exposure may have synergistic and cumulative actions affecting brain development, thus possibly contributing to the ASDs phenotype. While continued exploration of the potential causes for neurodevelopmental disorders is important for the health of the population as well as in reducing the economical burden associated with these devastating conditions, our data suggest the importance of a prenatal / postnatal screening for urinary DEHP metabolites in the population at high risk for ASDs.

EXPERIMENTAL PART

Materials and methods

The chemicals were purchased from Sigma-Aldrich and were used without further purification. The ω-alkynyl-alcohols, were purchased from Alfa Aesar; protected 5 Solid Phase Peptide Synthesis (SPPS).

General procedure for in batch SPPS on a manual synthesizer

Peptides were synthesized on a manual batch synthesizer (PLS 4×4, Advanced ChemTech) using a Teflon reactor (10 mL), following the Fmoc/tBu SPPS procedure. The syntheses were performed on Rink-amide NovaSyn TGR resin (0.14 mmol/g, 300 mg). The resin was swelled with DMF (1 mL/100 mg of resin) for 20 min before use.

Stepwise peptide assembly was performed by repeating for each added amino acid the following deprotection-coupling cycle:

• Swelling: DMF (1 mL/100 mg of resin) for 5 min;

• Fmoc-deprotection: resin is washed twice with 20% piperidine in DMF ( 1mL/100 mg of resin), one wash for 5 min followed by another wash for 20 min); • Resin-washings: DMF (3×5 min);

• Coupling: scale employing TBTU/HOBt/NMM (2 eq./2 eq./3 eq.) as the coupling system and 2 eq. of the Fmoc protected amino acids, except for N α -Fmoc-Xaa(ω-N 3 )-OH and N α -Fmoc-Yaa(ω-yl)-OH for which 1.5 eq. were used.

The coupling was carried out in DMF (1mL/100 mg of resin) for 50 min.

• Resin-washings: DMF (3×5 min) and DCM (1×5 min).

• Each coupling was checked by Kaiser Test 148 : In our case all tests were negative therefore it was not necessary to repeat the coupling reaction.

Kaiser test procedure: to a small amount of peptide-resin placed in a test tube, three drops for each of the following solutions were added: ninhydrine (5 g) in ethanol (100 mL); phenol (80 g) in ethanol (20 mL); KCN (2 mL of 1 mM aqueous solution) in pyridine (98 mL). The tube is heated at 100 °C for 5 min. A positive test (resin beads and solution appears strongly blue-violet) states the presence of at least 5% free amino groups.

Conventional and MW-assisted synthesis of PTHrP(1-34)NH 2

Room temperature and MW-assisted SPPS of PTHrP(1-34)NH 2 were performed using Liberty™ microwave peptide synthesizer (CEM) on Rink-amide NovaSyn ® TGR resin (0.2 mmol/g, 500 mg), which was suspended in a solution of DMF/DCM (1 : 1 v/v) and swelled for 30 min. The reactions were performed in a Teflon vessel and mixed by nitrogen bubbling. Reaction temperatures were measured by an internal fiber optic sensor. The protocol for RT and for MW-assisted SPPS in showed in Table 16. The ongoing of synthesis was monitored by UPLC-ESI-MS of MW-assisted mini-cleavages of intermediate fragments. During the general coupling cycle, the N-terminal Fmoc-protecting group was removed with a solution of 20% piperidine in DMF. Fresh stock solutions of the Fmoc-protected amino acids (5 equiv., 0.2 M) and TBTU (5 equiv. 0.5 M) in DMF, and DIEA (10 equiv. 2M) in NMP were prepared in separated bottles and used as reagents during the SPPS. In particular, the coupling cycles were performed using 2.5 ml of Fmoc-protected amino acids, 1 ml of TBTU and 0.5 ml of DIEA in NMP of stock solutions. Washing steps are performed with DMF and DCM.

RT-SPPS protocol: consisted of two consecutive deprotection steps of 5 and 10 min, respectively, and a 20 min coupling step.

MW-SPPS protocol: consisted of two deprotection steps performed at 75 •C using 35W for 0.5 min for the first one and 60W for 3 min for the second one, whereas the coupling steps were performed at 75 •C, using 30W for 5 min for all amino acids except for Arg and His residues that required specific coupling parameters performed in two steps.

Amino terminal acetylation. General procedure

The free N-terminal α-amino of the resin-bound peptides was acetylated by two consecutive steps using Ac 2 O/NMM in DCM (20 equiv. 1.6 mL of DCM). The first step of 30 min is followed by an additional step with fresh acetylation mixture of 90 min. The reaction was monitored by Kaiser test.

Deprotection, cleavage and purification of free peptide. General procedure

Peptides cleavage from the resin and simultaneous deprotection of the amino-acids side chains were carried out with the cleavage mixture (1 mL/100 mg of resin-bound peptide). For PTHrP(1-34)NH The minicleavages were carried out with 2 ml of TFA/TIS/water solution (95:2.5:2.5 v/v/v) at 45 •C, using 15W for 15 min with external cooling of the reactor vessel.

The reaction mixture was then filtered and the crude peptide was precipitated from the cleavage mixture by addition of ice-cold diethyl ether followed by cooling for 5 min at-20 •C. The product was collected by centrifugation and directly subjected to UPLC ESI-MS analysis.

HPLC analysis of PTHrP(1-34)NH 2

Crude PTHrP(1-34)NH 2 obtained from both the RT and MWassisted SPPSs were analyzed by analytical RP-HPLC (Alliance 2695 HPLC system equipped with a 2996 photodiode array detector, Waters (Milford,MA,USA)) using a JupiterC18 (5μm,250×4.6 mm) column (Phenomenex, Torrance, CA, USA) at 1 ml/min. The solvents used were A (0.1% TFA in H 2 O) and B (0.1% TFA in ACN). Yield 63%. IR(KBr) 2100 cm -1 (N 3 ). 1 H-NMR (600 MHz, D2O) δ = 7.68 (dd, 1, H, J = 1.3, 2.2 Hz, H-4), 8.09 (dd, 1 H, J = 1.6, 2.2 Hz, H-5), 9.53 (dd, 1 H, J = 1.3, 1.6 Hz, H-2); 13 C-NMR (150.9 MHz, D2O) δ = 120.8, 123.4, 138.3.

UPLC-ESI-MS analysis of intermediate fragments of

Diazotransfert on ω-aminic group of N α -Fmoc-amino acids with

Imidazole-1-sulfonyl azide hydrochloride. General procedure

Imidazole-1-sulfonyl azide hydrochloride (1.32 mmol, 1.5 eq) was added to a solution of N α -Fmoc-amino acid (0.88 mmol, 1 eq.), K 2 CO 3 (n+0.

Synthesis of chiral inducer 6.8.1 Synthesis of (S)-N-benzylproline (BP)

A solution of 10 g of (S)-proline (86.8 mmol, 1 eq.) and 18,5

Synthesis of (S)-2-(N-benzylprolyl)aminobenzophenone (BPB)

N-Benzyl-L-proline (4.37 g, 21.4 mmol, 1 eq.)

was added at RT under N 2 to a stirred freshly prepared solution of PCl 5 (8.91 g, 42.8 mmol, 2 eq.) in anhydrous CH 2 Cl 2 (150 mL). After 30 min, cold petroleum ether was added and the acyl chloride precipitated as a yellow oil.

The oil was dissolved in anhydrous CH 2 Cl 2 (80 mL) under N 2 and 2-aminobenzophenone (4.64 g, 23.54 mmol, 1.1 eq) was added in one portion, followed by Et 3 N until pH 8. The mixture was stirred for 2 h at room temperature, then washed with a saturated solution of Na 2 CO 3 and twice with H 2 O. The organic layer was evaporated under reduced pressure. The crude BPB was recrystallized from dried EtOH. Some product was also recovered from the EtOH washings. The material was dried under vacuum over P 2 O 5 to give the chiral inductor BPB.

Yield 48.5%. RP-UPLC: R t 3.44 min (30-90% di B in 5 min 138.54, 138.12, 133.37, 132.55, 132.48, 130.11, 129.12, 128.30, 128.15, 127.05, 125.32, 122.19, 121.52 (18 Ar), 68.25 (C α ), 59.82 (CH 2 Bn), 53.85 (C δ ), 30.98 (C β ), 24.14 (C γ ).

Synthesis of [Gly-Ni-BPB]

A solution of BPB 3 g (7.79 mmol. 1 eq), glycine

1.17 g (15.58 mmol, 2 eq.), and Ni(NO ), 134.53, 133.11, 132.16, 131.66, 129.67, 129.53, 129.28, 129.06, 128.87, 126.19, 125.60, 125.12, 124.21, 120.82 (18 ), 142.27, 133.81, 133.21, 132.12, 131.54, 129.73, 128.94, 128.89, 128.85, 127.55, 127.14, 126.47, 123.66, ), 142.27, 133.67, 133.24, 132.16, 131.54, 129.71, 129.01, 128.89, 128.86, 127.63, 127.07, 126.38, 123.71, 120.71

N O N C 6 H 5 N O O Ni H N O N C 6 H 5 N O O Ni H C≡C-(CH 2 ) 2 -C),

General procedure for click chemistry

Purified linear peptide precursors I'-X' (>95% purity), were subjected to solutionphase intra chain Cu I -catalyzed side chain-to-side-chain azide-alkyne 1,3-dipolar Huisgen cycloaddition (CuAAC). Linear pure peptides 5 mg (4. B 0.1% TFA in ACN).

Lactam bridge formation. General procedure

The fully protected resin-bound peptide was synthesized on a manual batch synthesizer, as described in general procedure. The formation of the lactam bridges between side chain groups of Lys 10 and Asp 5 was performed in solid phase, following an orthogonal protocol of deprotection. In particular Lys 10 was protected in side chain with 1-[(4,4 -dimethyl -2,6 -dioxocyclohex -1-ylidine)ethyl] group (Dde), stable in the condition of elongation of the peptide. After acetilation of the Nterminal, Dde was removed by two consecutive steps, using hydrazine 20% in DMF (1mL), first for 5 min, followed by an additional step with freshly prepared deprotection mixture for 15 min. After deprotection of Lys 10 , the formation of intramolecular lactam bound was performed using TBTU/HOBt/NMM (2.5:2.5:3.5 equiv.) as coupling system in DMF for 40 min.

The reaction of cyclization was monitored by UPLC-ESI-MS analysis of MWassisted mini-cleavage resin bond fragment. Mini-cleavages were performed using Discover TM S-Class single-mode MW reactor as described before.

cAMP based functional bioassay

HEK-293 cells stably expressing the mouse melanocortin receptors were transiently transfected with 4 µg CRE/β-galactosidase reporter gene as described in the literature 149,150 . Briefly, 5,000 to 15,000 post transfection cells were plated into collagen treated 96 well plates (Nunc) and incubated overnight. Forty-eight hours post-transfection the cells were stimulated with 100 µL peptide (10 -6 -10 -12 M) or forskolin (10 -4 M) control in assay medium (DMEM containing 0.1 mg/mL BSA and 0.1 mM isobutylmethylxanthine) for 6 hrs. The assay media was aspirated and 50 µL of lysis buffer (250 mM Tris-HCl pH=8.0 and 0.1% Triton X-100) was added. The plates were stored at -80°C overnight. The plates containing the cell lysates were thawed the following day. Aliquots of 10 µL were taken from each well and transferred to another 96-well plate for relative protein determination. To the cell lysate plates 40 µL phosphate-buffered saline with 0.5% BSA was added to each well. Subsequently, 150 µL substrate buffer (60 mM sodium phosphate, 1 mM MgCl 2 , 10 mM KCl, 5 mM β-mercaptoethanol, 2 mg/mL ONPG) was added to each well and the plates were incubated at 37 °C. The sample absorbance, OD 405 , was measured using a 96 well plate reader (Molecular Devices). The relative protein was determined by adding 200 µL 1:5 dilution Bio Rad G250 protein dye:water to the 10 µL cell lysate sample taken previously, and the OD 595 was measured on a 96 well plate reader (Molecular Devices). Data points were normalized to the relative protein content. EC 50 values represent the mean of three or more independent experiments. EC 50 estimates, and their associated standard errors of the mean, were determined by fitting the data to a nonlinear least-squares analysis using the PRISM program (v4.0, GraphPad Inc.).

NMR conformational analysis

Sample for NMR were prepared by dissolving 1.2 mg of the lactam-and heterodetic [1,2,3]triazolyl-containing cyclo-heptapeptides in 0.5 mL of aqueous phosphate buffer (pH 6.6, 100 mM). Samples were lyophilized and dissolved in a mixture of DMSO/water (0.5 mL, 80:20, v/v). NMR spectra were recorded on a Bruker DRX-600 spectrometer. One-dimensional (1D) NMR spectra were recorded in the Fourier mode with quadrature detection. The water signal was suppressed by a low-power selective irradiation in the homogated mode. DQF-COSY,TOCSY, and NOESY experiments were run in the phase-sensitive mode using quadrature detection in ω1 by time-proportional phase incrementation of the initial pulse. Data block sizes comprised of 2048 addresses in t2 and 512 equidistant t1 values. Before Fourier transformation, the time domain data matrices were multiplied by shifted sin 2 functions in both dimensions. A mixing time of 70 ms was used for the TOCSY experiments. NOESY experiments were run at 300 K with mixing times in the range of 100-250 ms. The qualitative and quantitative analyses of DQF-COSY, TOCSY and NOESY spectra were obtained using the SPARKY interactive program package.

Complete proton resonance assignments were achieved following Wüthrich's procedure. Sequential and medium range Nuclear Overhauser Effects (NOEs)derived distances were used to generate 3D models of the heterodetic [1,2,3]triazolyl-containing cyclo-heptapeptides. The final pdb files were analyzed and validated using PROMOTIF software.

NMR structure calculation

On the basis of sequential and medium range NOE derived distances, 3D models of lactam-and [1,2,3]triazolyl-peptides were generated with a simulated annealing procedure using the DYANA software package 227 . All structures were energy minimized with the SANDER module of the AMBER 5 program 228,229 using for 1000 steps the steepest descent method and for 4000 steps the conjugate gradient method. A non-bonded cut-off of 12 Å and a distance-dependent dielectric term (e = 4* r) were used. The minimization protocol included three steps in which NOE derived distances were used as constraints with a force constant, respectively, of 1000, 100, and 10 kcal/molÅ. The final pdb files were analyzed and validated using PROCHECK software 230 . 5.80 (H, ddt, J 16.9, 10.1,6.9 Hz, CH=). 3.57 (2H, d,J 4.4 Hz, OCH 2 ), 4.96 (H, dm, J 10, 2 Hz, =CH 2 ), 5.03 (H,dm, J 17 Hz, =CH 2 ), 5.38 (H, ddt, J 17, 10.2, 6.6 All reagents were of at least analytical reagent grade. Calibration curves for the quantitative urine analysis were calculated for all the DEHP metabolites plotting peak area average (y) against concentration of standards (x). Stock solutions (100 μg/mL in ACN) of all the metabolites were prepared and stored at -20 °C in Tefloncapped glass bottle until use. Five working solutions (2.5μg/mL; 1.5μg/mL; 1.0μg/mL; 0.25 μg/mL, 0.025μg/mL) for calibration curve plotting, were prepared for all the metabolites by dilution of the stocks in water. These solutions spanned the desired analytical range for each analyte (2.5 to 2500 ng/mL) without exceeding the linear range of the method. Standard solutions were stable 6 months once stored at 4 °C. The chromatographic separations of metabolites were resolved using a linear gradient from 3% to 60 % B in 10 min (solvent system A: 0.1% acetic acid in water; B: 0.1% acetic acid in ACN). The flow rate was 0.6 mL/min. The column temperature was 32 °C. A guard column (XBridgeTm Phenyl 3.5μm, 3.0x20 mm) was used to prevent column degradation. Column eluates were monitored at 215, 230 and 254 nm. The mass specific detection was achieved using a Waters, Micromass ZQ Electrospray ionization (ESI) in positive ion mode. The product ion with higher signal intensity was selected for the quantitative analysis for each of the four phthalates. The optimal MS parameters were as follows: the source and desolvation temperature were 120 °C and 400 °C respectively; the capillary voltage was 3.24 kV; cone voltage 30 kV, nitrogen gas was used as desolvatation gas and as cone gas as well; the cone gas and the desolvatation flow was 60 L/h and 800 L/h respectively;

EXPERIMENTAL PART B

The collision gas was argon with a flow of 0.60 ml/min. Data were acquired and processed using MassLynx™ software (Waters). All standards were injected three times in the same day. Curves with correlation coefficients (R 2 ) greater than 0.998 were generated (MEHP, 5-OH-MEHP, 5-oxo-MEHP 0.999; 6-OH-MEHP 0.998).

Instrument software Empower Software built 1154 generated a result spreadsheet.

Samples pre-treatment

For urines pre-treatment the following buffers were prepared: buffer ammonium acetate 1 M pH 6.5; acid buffer pH 2.0 by preparing a solution of NaH 2 PO 4 (0.14 M) and 1% of 85% H 3 PO 4 ; basic buffer was prepared by adding concentrated ammonium hydroxide (1 mL 30% NH 3 solution) to a 50:50 acetonitrile/water (200 mL). All buffers were stored in sealed bottles at room temperature: basic buffer was discarded after one week, acid buffer after one month.

Sample preparation: solid-phase extraction (SPE)

Human urine (1.00 mL) defrosted, sonicated, mixed, and dispensed in glass tubes.

Samples were subsequently buffered with ammonium acetate (250 μL, pH 6.5).

Incubation with β-glucoronidase (5μL, 200 units/mL) was performed at 37 °C for 90 min, resulting in quantitative glucuronide hydrolysis of phthalates metabolites.

First, an SPE cartridge was equilibrated with 1.0 mL of acetonitrile followed by 2.0 mL of basic buffer. This SPE cartridge was used to retain compounds that are hydrophobic at basic pH while allowing the more acidic analytes to elute. Next, the deconjugated urine samples were diluted with 1.0 mL of basic buffer solution and vortex mixed for 5 s. These samples were then added to an equilibrated SPE cartridge (3 mL/60 mg of Oasis HLB, Waters). The eluate was collected in borosilicate glass tubes. Residual analyte on the first cartridge was eluted by adding a second 1.0 mL of basic buffer to the cartridge. The combined basic buffer eluates were acidified by adding 3.0 mL of acidic buffer and mixed thoroughly. SPE solvent flows were monitored while samples were on the cartridges to maximize sorbentanalyte interaction (<2 mL/min). A second SPE cartridge (3 mL/60 mg of Oasis HLB, Waters) was equilibrated by washing with acetonitrile (1.0 mL) followed by water (1.0 mL) and acidic buffer (2.0 mL). We further purified the acidified sample by adding it to the second SPE cartridge and washing this cartridge with acidic buffer (3.0 mL, Figure 2). We removed residual salts by washing the second cartridge with water (9 mL). The analytes were eluted with acetonitrile (2 mL) followed by ethyl acetate (2 mL). This pooled eluate was evaporated to dryness under a stream of dry nitrogen. The residue was resuspended in water (200 μL), transferred to a glass autosampler vial insert, and analyzed by RP-HPLC-ESI-MS. Prepared samples could be stored at 4 °C for up to two weeks before analysis without degradation; no significant loss of analyte signal was observed until after 10 days of roomtemperature storage. Long-term storage of unextracted urine specimen at -40 °C for six months showed no decrease in phthalate monoester levels.

Daily protocol

In typical sample set were included water samples as blank, processed through the entire process along with unknown urine samples to monitor contamination, and one Peptide engineering relies on synthetic procedures to fold peptides into bioactive structures.

Side chain-to-side chain cyclization is used to stabilize a bioactive conformation and to reduce proteolytic degradation. Among the numerous modes of cyclization, bioisosteric modifications and cyclizations that do not require orthogonal protection schemes, are of great interest. The recently introduced Cu(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen's cycloaddition [1-3] as click chemistry reaction [4] presents a promising opportunity to develop a new paradigm for intramolecular cyclization. In fact, the proteolytic stable 1,4disubstituted [1,2,3]triazolyl bridge surrogates the isosteric peptide bond for a new structural constraint. We report the preparation of Nα-Fmoc-ω-azido-α-amino acids by diazo-transfer of the Nα-protected ω-amino-α-amino acid or a multistep strategy from the Nα-protected ωhydroxy-α-amino acid. The Nα-Fmoc-ω-ynoic-α-amino acids were prepared by alkylation of Ni(II) complexes of the Schiff bases derived from glycine and a chiral inducer with alk-ωynyl bromides [5]. These building blocks were used in solid phase synthesis of series of 8 linear nonapeptides derived from the sequence of PTHrP (11-19) where ω-azido-and ωethynyl-α-amino acids replaced Lys 13 and Asp 17 . Cleavage from the resin and side chain deprotection, followed by intramolecular Cu(I)-catalyzed click reaction, generated a series of i-to-i+4 1,4-disubstituted [1,2,3]triazole-bridged cyclopeptides. [6] The CD and NMR conformational analysis allowed to identify the permutations that mimic at the best the cyclo[Lys13,Asp14]PTH (11-19) [7]. This study lays the ground work to design novel bioactive cyclopeptidomimetics using this novel intramolecular rigidification mode.

Further applications of this strategy [8] will be shown on octreotide analogs conjugated to tetraazamacrocycles to be used in tumour pretargeting as well as on MTII analogs. AM 2,3 ,Testa C 2,3 ,Nuti F 2,3 ,Grossi E. 4 and insulin(AUCI) were calculated from a five-point OGTT. HOMA-IR and QUICKI, were calculated. Urine concentrations of secondary metabolites derived from di (2ethylhexyl)phthalate (PHTALOX) were measured in triplicate using an RP-HPLC method, using as synthetic standards the corresponding univocally characterised MEHP, mono (2ethyl-6-hydroxyhexyl) and the oxidised forms 6-OH-MEHP, 5-OH-MEHP, and 5-oxo-MEHP.

Relationships between urinary concentrations of PHTALOX

Results were analyzed by standard statistical methods and using a special kind of artificial neural network (Auto-CM) obtaining a subsequent semantic map of actual relationship between variables.

A negative correlation of MEHP with the age at onset of obesity was shown (R:-0,36).

MEHP was correlated also with AUCG(R:-0,33), AUCI(R:-0,35) and WBISI(R: +0,33). 5-Oxo-MEHP instead was significantly correlated with the age at onset of obesity(R:+0.51).

The connectivity map showed a direct relationship of 5-oxo-MEHP with BMISDS. The sum of the metabolites showed a direct relationship with the height SDS suggesting that height may be related with the ability to metabolise phthalates. MEHP showed a direct relationship with the insulinogenic index,and normal insulin sensitivity. This latter was directly related with 5-oxo-MEHP. Interestingly, 5-OH-MEHP was directly connected with the HOMA index and abnormal insulin sensitivity.

In conclusion, the results supported the hypothesis that the higher metabolites in urine, the earlier the onset of obesity, and suggested that some PHTALOX metabolites are capable of influencing insulin sensitivity. However, the less phthalates were metabolised (higher MEHP urine concentrations) the later was the onset of obesity. It remains, thus, to be elucidated whether exposure to phthalates per se is actually the risk factor or if the ability of the body to metabolise phthalates is actually the key point. This could explain also the positive relationship of MEHP with WBISI.

Probing the Bioactive Conformation of melanocortin like peptide by

Click Reaction

Chiara Testa, [a,b,c] Francesca Nuti, [a,b] Mario Scrima, [d] Anna Maria D'Ursi, [d] Nadeje

Lubin Germain [c] , Michael Chorev, [e,f] Paolo Rovero, [a,g] Anna Maria Papini, [a,b,c]

[a] Laboratory of Peptide & Protein Chemistry & Biology, Polo Scientifico e Tecnologico, Università di Firenze, I-50019 Sesto Fiorentino (FI), Italy, [b] Department of Chemistry, Università di Firenze, I-50019 Sesto Fiorentino (Fi), Italy , [c] Laboratoire SOSCO -UMR 8123 Université de Cergy-Pontoise Neuville-sur-Oise, 95031 Cergy-Pontoise, France , [d] Department of Pharmaceutical Science, Via Ponte Don Melillo 11C, Salerno, I-84084, Italy [e] Laboratory for Translational Research, Harvard Medical School, One Kendal Square, Building 600, Cambridge, MA 02139, USA, [f] Department of Medicine, Brigham and Women's Hospital, 75 Francis Street,Boston,MA 02115, USA, [g] Department of Pharmaceutical Science, Polo Scientifico e Tecnologico,University of Firenze,Via Ugo Schiff 3, 50019 Sesto Fiorentino (Fi), Italy MT-II is a potent super-agonist of melanocortin receptors, characterized by lactam bridge between residues i and i+5 stabilizing a type-II β-turn structure fundamental for the bioactivity. [1] The minimal active sequence is identified by the following tetrapeptide: His 6 -D-Phe 7 -Arg 8 -Trp 9 [2] .

Side chain-to-side chain cyclizations are of great interest because they are bioisosterics to the lactam bridge and do not require orthogonal protection schemes.

Intramolecular side chain-to-side chain cyclization is an established approach to achieve stabilization of specific conformations and represents a recognized strategy to avoid proteolytic degradation.

In our previous work [3] we designed and studied a new intramolecular side chain-to-side chain [1,2,3]triazolyl modification, based on the well studied i-to-i+4 side-chain to sidechain structure present in parathyroid hormone-related protein (PTHrP).

Therefore we applied this strategy on MT-II sequence, stabilizing the β-turn conformation by the introduction of i-to-i+5 side chain-to-side chain cyclization via formation of a 1,4disubstituted 1,2,3-triazolyl bridge.

In this context we performed the synthesis of different azido and alkynyl amino acids [4,5] , to introduce them as building blocks in the sequence of MT-II. By click reaction we obtained 1,4-disubstituted 1,2,3-triazolyl-bridge corresponding to the ring size of MT-II, characterized by 5 methylenes. Our goal is to explore all the different permutations in terms of location and orientation of the 1,2,3-triazolyl bridge. Conformational studies are in progress to evaluate the best type-II β-turn behavior required for biological activity.

REACTION SCHEME GENERAL OPTIMIZED PROCEDURE

RT and MW-assisted SPPS of parathyroid hormone-related peptide (PTHrP)(1-34)NH 2 were performed using Liberty™ MW peptide synthesizer (CEM) on Rink-amide NovaSyn® TGR resin (0.2 mmol/g, 500 mg). After deprotection of the N-terminal Fmoc group with a 20% solution of piperidine in DMF, Fmoc-protected amino acids (5 equiv), TBTU (5 equiv) and DIEA (10 equiv) in DMF were used in the coupling steps. The RT-SPPS protocol consists of two consecutive deprotection steps of 5 and 10 min employing deprotecting reagent, and 20 min for the coupling steps. In MW-assisted SPPS protocol, the deprotection steps were performed at 75 °C using 35 W for 0.5 min for the first one and 60 W for 3 min for the second one, while the coupling steps were performed at 75 °C using 30 W for 5 min for all amino acids except Arg and His for which the conditions reported in Table 1 were

Scope and Comments

The introduction in 1992 of MW irradiation in peptide chemistry stimulated a host of interest followed by numerous efforts to use it to overcome synthetic difficulties resulting in sluggish reactions, low yields and complex reaction crude products that were hard to purify [1]. During the last decade the growing interest in MW field has been marked by the progress in developing scientific MW equipment to embrace specific research needs [2-5]. In general, introduction of MW technology led to the shortening of reaction time that resulted in higher purity of crudes and increased yields of the pure final product.

The application of MW irradiation in peptide chemistry has been reported in several publications, most of which describe case studies of successful syntheses of difficult peptide sequences [6-8]. However, a definitive comparison of conventional RT and MW-assisted-SPPS protocols must be performed on identical instruments. In fact, comparing conventional RT and MW-assisted SPPS carried out on different instruments that apply protocols that differ in the equivalent excess of reagents and their molar ratios, stirring techniques and the nature and number of washing steps, may be misleading. Moreover, the automated synthesizers are generally developed to produce peptides without monitoring of the progress of the synthesis. Therefore by-products caused by side reactions such as aspartimide and diketopiperazine formation, incomplete couplings and deprotections may be detected only after the final cleavage of the deprotected peptide from the resin.

In this study we compared the Fmoc/tBu RT-SPPS with the MWassisted synthesis of the 1-34 N-terminal fragment of PTHrP using the same instrument (Liberty  , CEM, Matthews, NC, USA), and monitoring the progress of the synthesis by UPLC-ESI-MS of MWassisted mini-cleaved fragments of the growing peptide chain.

PTHrP is an autocrine, paracrine and intracrine regulator of processes such as endochondrial bone formation and epithelial-mesenchymal interactions during the development of mammary glands. In analogy to PTH, most of the known biological functions are exerted by the N-terminal PTHrP(1-34) fragment that has 60% sequence similarity to PTH (1-34). The sequence of PTHrP(1-34)NH 2 is shown in Figure 1. In the past, this sequence was the subject of numerous structure-activity-conformation relationship studies [9,10]. Importantly, the presence of clusters of arginine, of sterically hindered and hydrophobic amino acid residues in the sequence represents a synthetic challenge that was the subject of our comparative study reported herein.

The synthesis was initially carried out following the conventional RT protocol using the Liberty  automated peptide synthesizer excluding MW irradiations. As modern automated SPPS protocols allow the assembly of larger and increasingly complex peptides, a precise control of the coupling reactions is a crucial prerequisite in peptide synthesis. In fact monitoring the progress of synthesis allows the detection of undesirable products caused by side reactions, incomplete couplings or deprotections. Although different methods have been developed for monitoring of SPPS, we observed that the use of colorimetric monitoring or continuous-flow UV absorbance of the reaction column effluent was not informative enough to identify difficult steps in the synthesis. Therefore, we decided to monitor the progress of PTHrP(1-34)NH 2 synthesis by UPLC-ESI-MS analyses of small aliquots of cleaved peptide fragments obtained by MW-assisted mini-cleavages. The application of MW-assisted mini-cleavages of resin-bound peptides has been proposed as a fast, reliable method to monitor SPPS [11]. After specific coupling cycles, suspected to be difficult, we stopped the synthesizer and withdrew a small aliquot for analysis by UPLC-ESI-MS. In particular, we focused our attention on the PTHrP fragments related to the 19-28 sequence, characterized by clusters of Arg residues and highly hydrophobic residues (see Reaction Scheme and Figure 1).

By UPLC-ESI-MS analyses of intermediate fragments of the PTHrP(1-34)NH 2 included in the 19-34 sequence we noticed the presence of the desired peptide as well as of some by-products (Table 1 and Figure 2). The fragmentation patterns of these byproducts in ESI-MS/MS allowed us to confirm the formation of deletion sequences as reported in Supporting Information (Figure S1).

As the length of the resin-bound peptide increases, the related UPLC-ESI-MS analyses become much more complex. We report as an example the characterization of the 12-34 fragment of PTHrP(1-34)NH 2 . The deconvoluted spectrum obtained for the cleaved mixture of this sample resulted in several deletion sequences (Figure 3). The fragments desLys 13 /Gln 16 -f(12-34) and desLys [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] /Gln 16 ,Leu 27 -f (12-34) were identified as two isobaric peptide sequences lacking either Lys [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] or Gln 16 residues.

The UPLC-ESI-MS/MS analyses of the intermediate resin-bound fragments obtained from the RT-SPPS of PTHrP(1-34)NH 2 confirm that it is a difficult sequence for SPPS. The desired peptide was usually present as the major component in the cleavage mixture, but it was accompanied by some deletion peptides mainly lacking Arg, Leu and His residues. It is well known that Arg-containing peptides are difficult to synthesize due to the sterically hindered Pbf group as side-chain protection and the tendency to form γ -lactam leading to low yield couplings [12].

With the above information in hand we sought to improve the synthesis of this difficult sequence of PTHrP(1-34)NH 2 by employing MW-assisted SPPS using Liberty  automated peptide synthesizer with MW irradiations. To address the difficulties observed during the incorporation of Arg and His residues we applied the protocols reported in Table 2. Specifically, we have decreased the power of MW irradiation and lowered the temperature in order to avoid side reactions such as γ -lactam formation for Arg and racemization for His [13].

Indeed, after semi-preparative purification the MW-assisted SPPS of PTHrP(1-34)NH 2 yielded 27 mg of >95% pure peptide, whereas RT-SPPS gave only 18 mg of >95% of pure peptide. This improvement is attributed to the higher purity of the crude cleaved peptide mixture (Table 3 andFigure 4).

On the basis of the results of the analytical RP-HPLC of the crude PTHrP(1-34)NH 2 , we conclude that the use of MW irradiations in SPPS has enhanced the efficiency of crucial coupling cycles improving the final yield and purity of crude peptide and speeding up the remaining coupling cycles. This improvement can be attributed to the prevention of peptide backbone aggregation and acceleration of deprotection and coupling steps.

In summary, although the application of MW-assisted SPPS to the synthesis of PTHrP(1-34)NH 2 led only to a moderate improvement in final yield (6.3% vs 4.4%), it allowed us to obtain a crude product of higher quality (77% vs 35%) and in a shorter time (20 h vs 34 h used for the RT and MW-assisted SPPS strategies). Moreover, we demonstrated the usefulness of the combination of an MW-assisted mini-cleavage protocol and the UPLC-ESI-MS analysis for monitoring the quality of the reaction step (see Supporting Information). Compared to the ninhydrin colorimetric monitoring, our strategy is faster and the UPLC-ESI-MS/MS analysis is more accurate and more informative. We think that application of the strategy presented in this report will help to improve many syntheses of difficult sequences.

Experimental Procedures

RT and MW-assisted SPPS of PTHrP(1-34)NH 2

All Fmoc-protected amino acids and TBTU were purchased from Iris Biotech. (Marktdrewitz, Germany). The following amino acid side-chain-protecting groups were used: OtBu (Asp, Glu), tBu (Ser, Thr), Pbf (Arg), Trt (Gln, His) and Boc (Lys). Rink-amide NovaSyn  TGR resin was purchased from Novabiochem (Laufelfingen, Switzerland). ACN, DCM and diethyl ether from Sigma-Aldrich (St. Louis, MO, USA), DMF and NMP from Scharlau (Barcelona, Spain), DIEA and TFA from Acros Organics (Geel, Belgium) and TIS from Fluka/Aldrich (St. Louis, MO, USA). The synthesis of PTHrP(1-34)NH 2 was carried out using Fmoc/tBu SPPS strategy on a Liberty  automated peptide synthesizer with a single-mode MW reactor (CEM), by RT and MW-assisted strategies. The reactions were performed in a Teflon vessel and mixed by nitrogen bubbling. Reaction temperatures were measured by an internal fiberoptic sensor. The RT-SPPS protocol consisted of two consecutive deprotection steps of 5 and 10 min, respectively, and a 20 min coupling step. In MW-SPPS protocol, the two deprotection steps were performed at 75 • C using 35 W for 0.5 min for the first one and 60 W for 3 min for the second one, whereas the coupling steps were performed at 75 • C, using 30 W for 5 min for all amino acids except for Arg and His residues that required specific coupling parameters performed in two steps (RT followed by MW irradiation, Table 1).

The syntheses were performed on Rink-amide NovaSyn  TGR resin (0.2 mmol/g, 500 mg), which was suspended in a solution of DMF/DCM (1 : 1 v/v) and swelled for 30 min. During the general coupling cycle, the N-terminal Fmoc-protecting group was removed with a solution of 20% piperidine in DMF. Fresh stock solutions of the Fmoc-protected amino acids (0.2 M) and TBTU (0.5 M) in DMF, and of DIEA (2 M) in NMP were prepared in separated bottles and used as reagents during the SPPS. In particular, the coupling cycles were performed using 2.5 ml of Fmoc-protected amino acids, 1 ml of TBTU and 0.5 ml of DIEA in NMP of stock solutions. The fully assembled peptide was cleaved from the resin by treatment with 7 ml of a TFA/TIS/water solution (95 : 2.5 : 2.5 v/v/v) for 3 h at RT. The resin was filtered and the combined filtrates were concentrated under a stream of nitrogen. The crude peptide was precipitated from the cleavage mixture by addition of ice-cold diethyl ether and stored for 30 min at -20 • C. The precipitated product was collected by centrifugation, washed with diethyl ether (3 × 7 ml) and centrifuged. The remaining solid was dried under a stream of nitrogen and lyophilized. The relative amounts of the deletion sequences are calculated in percentage of area under the peak from the total area under the curve obtained from the TIC chromatogram. Fmoc-protected resin-bound peptide (10 mg) was weighted into a fritted polypropylene tube and treated twice with a 20% solution of piperidine in DMF (1 ml) each time for 5 min. The beads were then washed with DMF (2 × 1 ml) and DCM (3 × 1 ml), dried under vacuum and transferred into a 10 ml glass tube containing the cleavage mixture that was placed into the MW cavity. The minicleavages were carried out with 2 ml of TFA/TIS/water solution (95 : 2.5 : 2.5 v/v/v) at 45 • C, using 15 W for 15 min with external cooling of the reactor vessel at the positions shown in the Reaction Scheme. All the parameters, i.e. pressure, temperature and power, involved in the MW-assisted mini-cleavage reactions were monitored as reported in Supporting Information (Figure S2). The reaction mixture was then filtered and the crude peptide was precipitated from the cleavage mixture by addition of ice-cold diethyl ether followed by cooling for 5 min at -20

MW-assisted

UPLC-ESI-MS Analysis of PTHrP(1-34)NH 2 Fragments

UPLC-ESI-MS system consisted of an ACQUITY  UPLC system (Waters) coupled with a Micromass  Q-Tof MICRO  mass spectrometer (Waters) equipped with an ESI source. The chromatographic separation was achieved on a Symmetry 300 C18 column (100 mm × 2.1 mm, ID 3.5 µm, Waters) with the column temperature set at 30 • C. The flow rate was 0.15 ml/min with a linear gradient running from 0 to 30% of B (solvent A: 2% ACN, 0.1% formic acid in H 2 O; solvent B: 2% H 2 O, 0.1% formic acid in ACN) in 7 min, followed by 30-60% B in the next 3 min, then by 95% B for 2 min, and returned to initial condition for 3 min for re-equilibration. The total run time per sample was 15 min. The ESI-MS analysis was carried out in the positive ESI mode, the optimal MS parameters were as follows: capillary voltage 3.2 kV, cone voltage 30 kV, source temperature 120 • C and desolvation temperature 320 • C. Nitrogen was used as desolvation and cone gas with a flow rate of 450 and 40 l/h, respectively. For MS/MS analyses, argon was used as collision gas, and the collision energy was set to 40 eV. Data were acquired and processed using MassLynx  software (Waters).

Highlights and Limitations

This protocol describes two advantages of MW radiation in SPPS. The first one is monitoring of traditional RT-SPPS by MW-assisted mini-cleavages combined with fast, efficient and sensitive UPLC-ESI-MS analysis (15 min/analysis vs 30-45 min/analysis for traditional mini-cleavage). We use this procedure to confirm the presence of difficult coupling steps that result in truncated and deletion sequences. The second advantage is the specifically tuned MW-assisted SPPS that uses modified cycles targeting difficult couplings. In these couplings the first step is carried out at RT (without applying MW power, 20 • C) and for variable duration (25 or 2 min) and the second step is carried out in the presence of MW power (75 or 50 • C) for shorter time intervals (5 or 4 min). In the reported synthesis we applied such type of couplings for the three arginine residues (R 19 -R-R 21 ) and for the two histidine residues (H 25 -H 26 ). Evidently, carrying out the RT and MW-assisted SPPSs on the same instrument allowed unbiased side-by-side comparison and the conclusion that the latter procedure is superior to the RT-SPPS.

Although the modification of the coupling steps for Arg and His in the MW-assisted SPPS seems to be empirical in nature, it was guided by strong rational that took into account the distinct capacity of this methodology to overcome putative hydrophobic interactions and aggregation. The impact of these phenomena increases with the progression of the coupling reactions. We therefore decided to take advantage of the MW radiation only for a short duration after the bulk of the reaction has been already taken place. In this manner, the completion of the coupling reaction was facilitated without causing undesired side reactions. We propose this MW-based protocol as a general strategy for overcoming difficult coupling reactions. Admittedly, applying this strategy to different peptides will require fine tuning that will include the adjustment of parameters such as duration of coupling and recoupling steps as well as the level of MW energy employed in the recoupling step. We are confident that adapting the strategy outlined in this protocol will be advantageous over the 'one generic MW-assisted coupling cycle fits all'.

Introduction

Interaction of linear and flexible peptides with their macromolecular target, such as GPCRs, involves a limited number of closely related conformations that are recognized by, bind to and either activate or block the biological activity of these targets. Hence, the so called bioactive conformation represents only a small subset of the larger ensemble of accessible conformations which are in a dynamic equilibrium. A growing arsenal of structural rigidifications offers means to capture and stabilize the biological conformation of linear peptides in an effort to enhance target specificity, biological potency, binding affinity and metabolic stability [1,2]. Herein we report on the extension of an innovative strategy for stabilization peptide E-turn conformation by side chain-to-side chain cyclization employing a bridge containing a [1,2,3]triazole [5-8].

Results and Discussion

Melanocortin GPCR receptors (MCRs) are involved in many biological pathways, including sexual function, feeding behaviour, energy homeostasis and pigmentation, making them potential targets for drugs to treat diseases such as obesity and sexual dysfunction. Therefore, understanding the bioactive conformation of the ligand and the structure of the receptor-ligand complex is crucial to design more potent and MCR-subtype selective ligands. MT-II is a potent long acting non-selective super-agonist of MCRs, characterized by lactam bridge between residues Asp 5 and Lys 10 stabilizing a type-II E turn structure that is critical for its bioactivity [3]. The minimal active sequence is identified by the tetrapeptide His 6 -D-Phe 7 -Arg 8 -Trp 9 [4], which is included in the cyclic portion formed by lactam ring. In previous work [5] we designed and studied a new intramolecular side chain-to-side chain [1,2,3]triazolyl-bridged modification. This 1,4-disubstituted [1,2,3]triazolyl moiety is bioisosteric to the peptide bond and was recently introduced by us as an D-helix stabilizing rigidification replacing the i-to-i+4 side chain-to-side chain bridging lactam in peptides derived from parathyroid hormone-related protein (PTHrP). In the current study, we applied the [1,2,3]triazolyl-bridging strategy to stabilize a E-turn conformation in MT-II by replacing the lactam bridge with a i-to-i+5 side chain-to-side chain cyclization via Cu I -catalyzed azido-to-alkyne 1,3-dipolar cycloaddition (CuAAC) generating 1,4-disubstituted [1,2,3]triazolyl-containing ring structures. In this context, we have developed synthesis of N D -Fmoc-Zazido-D-amino-and N D -Fmoc-Z-ynoic-D-amino acids (Figure 1) by diazo-transfer of the N D -protected Zamino-D-amino acids [6,7] and by alkylation of a Ni(II) complex of the Schiff base derived from glycine and a chiral inducer with alk-Z-ynyl bromides respectively [8]. Clicked MT-II analogs I-IV presenting different permutations of bridges containing 5 methylenes and a triazolyl moiety were synthesized by a combination of solid phase assembly of the linear peptide precursor I'-IV' and were followed by solution phase CuAAC. The adenylate cyclase activity (EC 50 ) of the linear precursor I'-IV' and cyclic MTII peptide analogs I-IV was evaluated in the murine melanocortin receptors mMC1R, mMC3R, mMC4R and mMC5R (Table 1). Indeed the cyclic peptides are more potent than their linear precursors confirming that conformational stabilization in the form of side chain-to-side chain cyclization enhances in vitro potency. Moreover, the clicked cyclic peptides have similar potency compared to the parent lactam-containing MTII analog. There are indications of emerging selectivity toward receptor subtypes but these need to be further optimized.

In summary, our studies support our contention that the [1,2,3]triazolyl-bridged peptides achieve conformational stabilization that enhances their in vitro potency. In the future we will continue to fine-tune this modification to improve receptor-subtype selectivity. Fig. 2. Schematic representation of all the possible MTII peptide analogs cyclized by [1,2,3]triazolyl-containing bridges that vary in the size of the bridge and the position and direction of the triazolyl moiety within the ring. In the notation tb stands for [1,2,3]triazolyl-bridged, the superscripts m and n define the number of methylenes connecting the triazole moiety to the N-and C-proximal bridge heads and the numbers in the bracket identify the orientation of the triazolyl within the bridge.

Ying, J

Introduction

Parathyroid hormone-related peptide (PTHrP) is an autocrine/paracrine regulator of endochondral bone development and involved in excessive osteoclastic bone resorption associated with humoral hypercalcaemia of malignancy (HHM) [1]. PTHrP is a 139 to 173amino acid protein having the first thirteen N-terminal amino acids in common with parathyroid hormone (PTH). Like PTH, PTHrP releases calcium from bone and increases secretion of calcium and phosphate in the distal tubule [2]. Similar to PTH the calcium metabolism-related activities are located in the first 34 N-terminal residues. Consequently, our synthetic efforts focused on this bioactive sequence that was the subject of numerous structure-activity-conformation relationship studies [3].

Considering the presence of clusters of arginines, sterically hindered and hydrophobic amino acid residues in the 19-28 sequence of PTHrP and the considerable length of the peptide, the synthesis of PTHrP(1-34)NH 2 is quite challenging. We therefore undertook the synthesis comparing the conventional (room temperature) Solid-Phase Peptide Synthesis (SPPS) vs. the MicroWave-assisted SPPS [4].

Results and Discussion

PTHrP(1-34)NH 2 sequence is: H-Ala 1 -Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp 10 -Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg 20 -Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu 30 -Ile-His-Thr-Ala-NH 2 . The peptide was synthesized by SPPS, following Fmoc/tBu strategy, at room temperature and by microwave irradiation, using in both cases Liberty peptide synthesizer (CEM) and the same reagents concentration. The synthesis was monitored by LC-MS using MW-assisted mini-cleavages of the intermediate fragments using Discover single mode microwave system (CEM).

The SPPS was performed using the NovaSyn TGR (500 mg, 0.2 mmol/g), 0.2 M solution of amino acids in DMF (5 equiv.), 0.5 M solution of TBTU in DMF (5 equiv.), 2 M solution of DIPEA in NMP (10 equiv.). Final cleavage of the peptide from the resin and side-chain deprotections were performed with ad hoc cleavage cocktail TFA/water/TIS solution (95:2.5:2.5 v/v/v) for 3 hours at room temperature.

The room temperature-SPPS cycle (Liberty, CEM) consisted of two deprotection steps (5 and 10 min respectively) and 20 min for the coupling steps. The MW-SPPS cycle (Liberty, CEM), the deprotection steps were performed at 75 °C using 35 W for 30 sec for the first one and 60 W for 180 sec for the second one, while the coupling steps were performed at 75 °C, using 30 W for 300 sec.

MW-assisted mini-cleavage cycles (Discover, CEM) were carried out at 45 °C, using 15 W for 15 min with external cooling of the reaction vessel.

In this comparative study of PTHrP(1-34)NH 2 SPPS, using the same instrumentation and the same reagents concentration, MW-assisted strategy gave an increased purity of the crude peptide as compared with the conventional approach. RP-HPLC purification of the crude peptide was evaluated by LC-MS (ACQUITY UPLC ™ System coupled with Micromass ® Quattro micro API Mass Spectrometer, Waters) yielded 27 mg and 18 mg of >95% pure PTHrP(1-34)NH 2 from the MW-assisted synthesis and SPPS at room temperature, respectively (Table 1). In conclusion, microwave technology has improved SPPS of particularly difficult sequences as compared with the conventional room temperature method. This improvement is attributed to prevention of peptide backbone aggregation and acceleration of deprotection and coupling steps.

In general, MW-assisted SPPS is proposed as a reliable strategy to overcome some limitations encountered in conventional SPPS. In our case, although the application of microwaves in SPPS led only to moderate improvement in final yield it allowed us to obtain a crude PTHrP(1-34)NH 2 of higher quality and enabled the development of a fast micro-cleavages as an alternative method for monitoring SPPS.

Therefore, based on our experience, the advantages of MW-assisted SPPS vs RT-SPPS can be summarized as follow: 1) shortening reaction time, 2) shortening micro-cleavage time, 3) crude peptides of higher quality facilitating the subsequent purification steps.

INTRODUCTION

ASDs (autism spectrum disorders), also known as PDDs (pervasive developmental disorders), are a group of complex neurodevelopment disorders, characterized by social impairments, communication difficulties, and restricted, repetitive and stereotyped patterns of behaviour.

A dramatic increase in frequency of ASDs has been reported over the last 20 years (Weintraub, 2011; Kim et al., 2011). Nevertheless, it is difficult to determine how much of this increase may be due to actual increase in the incidence or to increased awareness and diagnosis. The aetiology is unknown, but it is believed to result from disruption of normal neurobiological mechanisms primarily in the prenatal period (Nelson, 1991). Although it is widely recognized that ASDs may 1 To whom correspondence should be addressed (email annamaria.papini@unifi.it). Abbreviations: ASD, autism spectrum disorder; CARS, childhood autism rating scale; CI, confidence interval; DEHP, di- (2-ethylhexyl) have a strong genetic component (Risch et al., 1999; Anney et al., 2011). To the best of our clinical experience, not more than 5% of all autism cases appear to be due primarily to single gene mutations. In particular, syndromic ASD accounts for 15-20% of ASDs, and other complex genetic factors appear to play a major role in non-syndromic forms of autism. World literature evidence seems to indicate that autism has a strong genetic component (10-20%, Geschwind, 2011) which, by itself, does not fully explain the prevalence of the disorder exponentially increasing overthe last two decades. It is quite likely that exposure to potential environmental factors has differential effects depending on genetic background.

According to our observation, the focus of autism research is shifting from purely genetic influences to multifactorial diseases in which complex set of genes should be associated with relevant environmental factors (Larsson et al., 2009; Herbert, 2010). To date, no information is available on the potential role in the pathogenesis of autism for phthalates, ubiquitous contaminants (Griffiths et al., 1985; Bauer and Herrmann, 1997) used as plasticizers, solvents and additives in many consumer products, i.e. vinyl flooring, wall coverings, food containers and cosmetics (Schettler, 2006; Wormuth et al., 2006). In particular, DEHP [di- (2-ethylhexyl) phthalate] represents one of the most commonly used plasticizer in pharmaceutical and medical devices (U.S. FDA 2011). DEHP is primarily metabolized to monoester, further oxidized in v and v-1 positions leading to a pool of secondary metabolites (Albro, 1986; Koch et al., 2003, 2006), and it can be hypothesized that all DEHP metabolites, derived from a hydrolytic/oxidative pathway could, after glucuronidation, be excreted in urine (Figure 1).

Children are known to be exposed to DEHP levels twice as high as those for adults (Hellerstedt et al., 2008; Wittassek et al., 2009). Foetuses and neonates are highly sensitive to physiologically active agents because they are exposed during critical periods of human development (Latini et al., 2003; Kim et al., 2009; Cho et al., 2010; Suzuki et al., 2010). Furthermore, in the case of intensive therapy procedures, neonates can have higher exposure to phthalates and to their toxic monoesters supposed to be EDs (endocrine disruptors) (Silva et al., 2003a, 2003b; Latini et al., 2004). EDs may also be transferred from the mother to the developing foetus across the placenta or to the newborn through breast-feeding (Hines et al., 2009; Latini et al., 2009). For these reasons, the French National Assembly has required the ban of phthalates and parabens (Bill of Law adopted by French National Assembly on 3 May 2011). Moreover, very recently, prenatal phthalate exposure has been reported to decrease child mental/motor development, and increase internalizing behaviour during the preschool years (Whyatt et al., 2011), thus consolidating the concept of an adverse effect of these contaminants on function of the CNS (central nervous system).

The aim of the present study was to evaluate the levels of the primary and secondary metabolites of DEHP in autistic children by using innovative chemical reverse approach (Alcaro et al., 2009; Papini, 2009).

MATERIALS AND METHODS

Subject population

A total of 48 children with ASD (male: 36, female: 12; age at examination: 11.0¡5 years) were recruited from the staff of Children Neuropsychiatric Department, Siena, Italy. All the 48 patients with ASD, diagnosed by DSM IV (Diagnostic and Statistical Manual of mental disorders) and evaluated using ADOS (autism diagnostic observation schedule), ABC (autism behaviour checklist) and CARS (childhood autism rating scale) scores entered the study. Patients with Rett syndrome, Xfragile syndrome, inborn errors of metabolism, 21 trisomy, tuberous sclerosis and gene microdeletions were excluded from the present study. Informed consent from the parents or tutors was obtained, and institutional review board approval was obtained for the study. Mean age at diagnosis was between 30 and 36 months. However, the diagnosis for the examined ASDs population go back to procedures employed several years ago (today, mean age at the diagnosis for infantile autism is about 18-24 months). Forty-five genderand age-comparable HCs (healthy controls) [male: 25, female: 20; age at examination: 12¡5 years] were randomly chosen from outpatients who had no pathological symptoms.

Urine from children with ASD and HCs were collected in polypropylene specimen cups, divided into aliquots (1.0 ml) and frozen at 220 ˚C until analysis. Field blanks consisted in purified water collected in polypropylene tubes and frozen at 220 ˚C.

Determining secondary metabolites in urine

The metabolites measured in this study included: MEHP [mono-(2-ethylhexenyl) 1,2-benzenedicarboxylate] and 6-OH-MEHP [mono- (2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate], 5-oxo-MEHP [mono- (2-ethyl-5-oxohexyl) 1,2-benzenedicarboxylate] and 5-OH-MEHP [mono- (2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate]. All these metabolites were synthesized in the Laboratory of Peptide and Protein Chemistry and Biology (PeptLab) following their previously described procedure (Nuti et al., 2005). The unequivocally characterized synthetic metabolites were used as pure analytical standards (.98% purity) for quantitative determination in urine from ASD children and HCs.

All the solvents (acetonitrile, water, buffers, etc.), labware (polypropylene vials containing urine, solvent bottles, SPE (solid phase extraction) cartridge, Teflon capped-glass bottles, pipettes) and instrumentation used during SPE procedure and the HPLC-ESI-MS (HPLC electrospray ionization MS) analytical process, to detect MEHP and/or secondary metabolites in ASD patients and HCs, were verified to be MEHP-and secondary oxidative metabolites-free. The internal standards were prepared in acetonitrile and were used as reported in the literature (Blount et al., 2000; Kato et al., 2004).

First morning urine specimens were collected. All specimens displayed urinary creatinine in the children reference value range, dependent on age and lean body mass (0.5-4.0 mg/kg per 24 h). For creatinine measurement, we used a Synchron AS/ASTRA clinical analyser (Beckman Instruments). We used values (mg/l) for creatinine and (g/l) for dilution correction in the analyses.

For SPE treatment of urine, we prepared the following buffers: ammonium acetate buffer 1 M, pH 6.5; acid buffer, pH 2.0 by preparing a solution of NaH 2 PO 4 (0.14 M) and 1% of 85% H 3 PO 4 ; basic buffer was prepared by adding concentrated ammonium hydroxide (1 ml of 30% NH 3 solution) to a 50:50 acetonitrile/water (200 ml). All buffers were stored in sealed bottles at room temperature (20 ˚C): basic buffer was discarded after 1 week, acid buffer after 1 month.

Human urines (1 ml) were defrosted, sonicated, mixed and dispensed in glass tubes. Then ammonium acetate buffer (250 ml, pH 6.5) was added. Incubation with b-glucoronidase (5 ml, 200 units/ml, Roche Biochemical) was performed at 37 ˚C for 90 min, resulting in quantitative glucuronide hydrolysis of phthalates and metabolites. Escherichia coli K12 b-glucuronidase has excellent glucuronidase activity and no measurable lipase activity on phthalate diesters (Blount et al., 2000; Kato et al., 2004). After deconjugation, samples were treated with two steps of SPE, using SPE cartridge (3 ml/ 60 mg of Oasis HBL, Waters) to remove any contamination of biological matrix, following the procedure described by Blount et al. (2000). The first cartridge was used to retain hydrophobic compounds while the phthalate metabolites were eluted. The second cartridge was helpful in removing residual salts. Analytes were finally eluted with acetonitrile and ethyl acetate, concentrated, re-suspended in water and transferred into vials. All the samples were analysed by RP-HPLC-ESI-MS (reverse-phase HPLC-ESI-MS). One blank and one QC (quality control) sample were included in each batch of samples. The QC sample was spiked with pooled urine and MEHP and secondary oxidative metabolite standards in known concentration (200 ng/ml). When urine analysis resulted in values for metabolite concentrations exceeding the linear range of the analytical method, we subjected a new aliquot of the same sample to the entire process (deconjugation and SPE) and we analysed again. Before analysis, known concentration samples were treated by SPE and analysed by RP-HPLC-ESI-MS to test SPE efficiency. In our study the efficiency of SPE procedure is in accordance with the literature (Mazzeo et al., 2007). Treated urine samples could be stored at 4 ˚C without degradation. Storage of untreated urines at 240 ˚C for 6 months showed no decrease in phthalate monoester levels.

The analytical methodology adapted for measuring MEHP and secondary oxidative metabolites in urine had already been described in the literature (Blount et al., 2000; Silva et al., 2003a, 2003b; Kato et al., 2005).

In particular, we used an HPLC tandem MS, RP-HPLC-ESI-MS (Waters, Alliance 2695, Waters, Micromass ZQ) equipped with phenyl column (Betasil, 5 mm, 50 mm63 mm, Keystone Scientific) and with a Waters 2996 Photodiode Array Detector. All reagents were of at least analytical reagent grade. The lower LOQs (limits of quantification) were 0.042 mg/l MEHP, 0.048 mg/l 5-OH-MEHP, 0.049 mg/l 5-oxo-MEHP and 0.008 mg/l 6-OH-MEHP. In urine, the LODs (limits of detection) were 0.014 mg/l MEHP, 0.016 mg/l 5-OH-MEHP, 0.016 mg/l 5-oxo-MEHP and 0.002 mg/l 6-OH-MEHP. The chromatographic separations of metabolites were resolved using a linear gradient from 3 to 60% B in 10 min (solvent system A: 0.1% acetic acid in water; B: 0.1% acetic acid in acetonitrile). The flow rate was 0.6 ml/min. The column temperature was 32 ˚C. A guard column (XBridgeTm Phenyl 3.5 mm, 3.0620 mm) was used to prevent column degradation. Column eluates were monitored at 215, 230 and 254 nm. The mass-specific detection was achieved using a Waters, Micromass ZQ ESI in positive ion mode. The product ion with higher signal intensity was selected for the quantitative analysis for each of the four phthalates. The Finally, we analysed the correlations between total CARS (childhood autism rating scale) score and levels of the different metabolites. Total CARS scores were (means¡S.D.) 44¡7. 4 (range: 31-60). A positive correlation between CARS scores and urinary MEHP levels was observed (r50.429, P50.0033), whereas no significant relationships with the levels of the other examined metabolites were found (total CARS versus 5-OH-MEHP: r50.120, P50.4298; total CARS versus 5-oxo-MEHP: r50.127, P50.3931; total CARS versus 6-OH-MEHP: r50.0085, P50.9529).

Moreover, we compared ASDs with n510 patients with Rett syndrome (a genetic form of autism due in up to 95% of cases to mutation of a gene, i.e. MeCP2), urinary phthalate metabolites appear to be, once again, significantly elevated in ASD patients (Table 3). Actual behaviour for most patients with Rett syndrome is not autistic even if all of them do show autistic traits during stage 2 of the disease. Therefore 5-oxo-MEHP urinary excretion appears to be lower in Rett syndrome (P50.0344), whereas 5-OH-MEHP (P50.0601), 6-OH-MEHP (P50.2246) and MEHP (P50.7098) excretion is comparable with that observed in HCs, despite the heavy medication needed in this multi-system genetic disease. These results indicate that medications are likely not the main cause for the increased urinary phthalate excretion evidenced in ASDs, while suggesting the likely intervention of environmental factors in the pathogenesis of this pervasive development disorder.

DISCUSSION

As ASDs are disorders of brain development, any factors that regulate brain development and are known to be altered in autism should be considered as possibly contributing to the phenotype.

Our findings, for the first time, demonstrate an association between phthalates and ASDs. It is interesting to note that, in 87% of the ASD urine samples, we detected MEHP and 5-oxo-MEHP levels higher than the levels recently reported, by Cho et al. (2010). In that study Cho et al. reported a possible relationship between environmental phthalate exposure (external contamination) and the intelligence level of 667 school-age children randomly recruited from nine elementary schools in five South Korean cities. The reported median values were for MEHP (0.055 mg/ml), 5-OH-MEHP (0.18 mg/ml) and 5-oxo-MEHP (0.096 mg/ml) respectively. As spot urines are among the most commonly used samples in clinical toxicology, we showed the data unadjusted for creatinine. In fact, in epidemiological studies it is not practical to collect 24 h urine samples or, when young children are involved, even first morning voids. In particular, spot urines have also been used in a widely promoted paper relating maternal urinary-phthalate metabolite excretion in mothers to psychomotor development in their children at age 3, recently published (Whyatt et al., 2011). Likewise in ASDs, MEHP was found to be the minor urinary metabolite compared with the corresponding oxidized metabolites as in some adult metabolic diseases (Wittassek and Angerer, 2008). It is possible to hypothesize that, in the case of ASDs, a high MEHP urinary excretion does not correspond to external contamination, but rather MEHP could likely act as an ED.

From our data it is possible to speculate that phthalate metabolites as EDs may play an important and previously not recognized role either in the neurotransmitter system and/or in neurodevelopment, possibly increasing the risk of ASDs. In any case, our findings indicate for the first time that specific DEHP metabolites are statistically significantly increased in ASDs. It is intriguing that urinary levels of the most oxidized form of the DEHP metabolites (5-oxo-MEHP) is able to efficiently discriminate ASDs from HCs (Figure 2). Nevertheless, it has already been reported that the most oxidized DEHP metabolites have longer half-life of excretion in urine (Koch et al., 2006). Therefore it is possible that environmental factors and OS (oxidative stress) imbalance may interact in leading to increased ASD risk, although further investigation is needed in dissecting this interaction in vivo.

A link between oxidative brain damage and ASD has been previously reported by several authors (James et al., 2004; Villagonzalo et al., 2010; Sajdel-Sulkowska et al., 2011). Over the last few years, our team has demonstrated that the biological 'duo' hypoxia-OS is a key player in modulating genotype-phenotype expression in Rett syndrome, a wellestablished genetic form of ASD (De Felice et al., 2009; Pecorelli et al., 2011; Signorini et al., 2011).

Moreover, in previous reports, a correlation between OS and ASD has been widely explored by measuring different molecules, possibly coming from oxidative pathway as metabolic biomarkers of OS, in biological fluids (Chauhan et al., 2004; James et al., 2004).

To the best of our knowledge, a strict relationship between OS entity and the in vivo presence of oxidized forms of DEHP has not been reported before. Therefore we could speculate that, in susceptible subjects, DEHP exposure, when finding OS conditions, can lead to in vivo accumulation of toxic metabolites (i.e. the oxidized phthalate metabolites), possibly acting as EDs. Considering that urinary 5-oxo-MEHP reported in our study as increased in ASDs compared with HCs, we can state that our findings are in agreement with an increased OS environment.

Moreover, phthalates are known as EDs, but it is unclear how they could be involved in aetiology of neurodevelopment disorders. Phthalate metabolites activate a-PPARa (peroxisome proliferator-activated receptor a), interfering with cellular proliferation and lipid metabolism. Activation of PPARa by phthalates may alter lipid metabolism in the brain (Clark-Taylor and Clark-Taylor, 2004). Recently the signal transduction pathway of PPARa was correlated with progression of neurodegenerative and psychiatric diseases.

Phthalates also interfere with the TH (thyroid hormone) system by inducing hypothyroidism. Recent studies correlated in utero hypothyroxinaemia to decreased intellectual capacity, mental retardation and ASD (Roman, 2007), thus reinforcing our speculation. Moreover, exposure to DBP (din-butyl phthalate) seems to affect thyroid activity in pregnant women, thus leading to adverse effects on the foetus (Huang et al., 2007).

On the other hand, transient intra-uterine deficits of THs have been shown to result in permanent alterations of cerebral cortex similar to those found in brains of children with autism (Roman, 2007). As a consequence, the current surge of this disease could be related to transient maternal hypothyroxinemia resulting from exposure to anti-thyroid environmental contaminants.

For the first time in this study we correlated different levels of the primary and secondary metabolites with ASDs compared with HC children. These data generate the idea that either current exposure is higher in children with ASD, or alternatively, and more likely, ASD children may differ in their ability to metabolize phthalates.

In conclusion, our findings generate the idea that prenatal plus postnatal phthalate exposure may have synergistic and cumulative actions affecting brain development, thus possibly contributing to the ASD phenotype, according to the general concept generated by Herbert (2010) of 'environmentally vulnerable physiology'. Therefore a prenatal/ postnatal screening for urinary DEHP metabolites in the population at high risk for ASDs could have an important social impact.

Figure 1 .

 1 Figure 1. β-Turn types

Figure 2 .

 2 Figure 2. Differences concerning the interactions ligand-receptor between a linear peptide and cyclic peptide(Kessler H. Angew. Chem. Int. Ed. Engl. 1982, 21, 512).

Figure 3 .

 3 Figure 3. The most common types of cyclopeptides

Figure 4 .

 4 Figure 4. Somatostatin structure

Figure 5 .

 5 Figure 5. Structure of octreotide

Figure 6 .

 6 Figure 6. Mimicking of disulfide bridges by triazoles in tachyplesin-I by the replacement of cysteine residues by alkyno (X) and azido amino acids (Z), which form triazoles upon the copper-catalyzed cycloaddition of the azides and alkynes ("click" reaction): a) tachyplesin-I and b) linear analogues. Cyclization leads to c) the correctly folded hairpin structure or d) incorrectly folded globule-like structure(Holland-Nell K., Meldal M., Angew. Chem. Int. Ed, 2011, 50, 5204-06).

Figure 7 .

 7 Figure 7. Topological similarities of amide and[1,2,3] triazole (Bock V.D., Perciaccante R., JansenT.P., Hiemstra H., van Maarseveen J.H., Org. Lett., 2006, 8, 919-922).

  Intramolecular i-to-(i+4) side-chain-to-side chain cyclization stabilize the formation of helix-like secondary structures: the case of parathyroid hormone and Parathyroid hormone -related protein Previous studies 46 performed in our Laboratory of Peptide & Protein Chemistry & Biology (PeptLab) were focused on the bioactive conformation of parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTHrP is an autocrine, paracrine and intracrine regulator of processes such as endochondrial bone formation and epithelial-mesenchymal interactions during the development of mammary glands.

  Parathyroid hormone (PTH), the most important hormonal regulator of mineral ion homeostasis in mammals, is secreted by the parathyroid glands as a protein containing 84 amino acids. Parathyroid hormone-related protein (PTHrP) was first identified and cloned from malignant tumor cells and tissues from patients with the syndrome of humoral hypercalcemia of malignancy[START_REF] Strewler | [END_REF][START_REF] Moseley | Proc. Natl. Acad. Sci[END_REF] . PTHrP is an autocrine, paracrine and intracrine hormone, produced by most tissues in the body. It regulates different processes such as endochondrial bone formation and epithelialmesenchymal interactions during the development of mammary glands, produced by most tissues in the body. PTH regulates mineral metabolism and bone turnover by activating specific receptors located on osteoblastic and renaltubular cells, while PTHrP which has partial sequence homology to PTH, is associated with the syndrome of humoral hypercalcemia of malignancy[START_REF] Suva | [END_REF] . PTH acts to increase the concentration of calcium in the blood by acting upon PTH receptor in three parts of the body. Increasing calcium concentration in the blood acts (via feedback inhibition) to decrease PTH secretion by the parathyroid glands (Figure8).

Figure 8 .

 8 Figure 8. Process for the activation of PTH.

Figure 9 .

 9 Figure 9. Peptide sequence of PTH(1-34) and PTHrP(1-34).

Figure 10 .

 10 Figure 10. Methods of heating by microwave irradiation.

Figure 11 .

 11 Figure 11. Characteristics of the PTHrP(1-34)NH 2 sequence are a cluster of arginine residues in the positions 19-21, sterically hindered and hydrophobic amino acid sequences

a

  Arg residues were coupled twice.

Figure 12 .

 12 Figure 12. (A-C) TIC chromatograms and ESI-MS analyses to monitor the 19-34 fragment of PTHrP(1-34)NH 2 (RT-Fmoc/tBu SPPS, Liberty™, CEM) after RT mini-cleavage performed at 15 min (A), 30 min (B), 1 h (C).

Figure 12 .

 12 Figure 12. Continued.

Figure 13 .

 13 Figure 13. TIC chromatogram of the 19-34 fragment of PTHrP(1-34)NH 2 (RT-Fmoc/tBu SPPS, Liberty™, CEM) obtained after MW-assisted mini-cleavage procedure performed at 15 min.

Figure 14 .

 14 Figure 14. (A-D) TIC chromatogram of selected crude mixtures of intermediate resin-bound sequences obtained during the synthesis of PTHrP(1-34)NH 2 (Fmoc/tBu RT-SPPS) generated by the MW-assisted mini-cleavages.

Figure 15 .

 15 Figure 15. TIC chromatogram and deconvoluted spectrum of the cleaved mixture of the intermediate resin-bound sequence PTHrP(12-34)NH2 (Fmoc/tBu RT-SPPS) generated by the MW-assisted minicleavages.

Figure 16 .

 16 Figure 16. TIC chromatogram and MS/MS analysis of the fragment 16-34 of PTHrP(1-34)NH 2 (RT-Fmoc/tBu SPPS, Liberty™, CEM) obtained after MW-assisted mini-cleavages monitoring.

Figure 17 .

 17 Figure 17. Analytical RP-HPLC of crude PTHrP(1-34)NH 2 . Fmoc/tBu RT-SPPS (A) and Fmoc/tBu MW-assisted SPPS (B). HPLC: 10-60% B (0.1% TFA in ACN)in A (0.1% TFA in H 2 O) over 20 min

Figure 18 .

 18 Figure 18. Endocrine, paracrine and autocrine secretion

Figure 19 .

 19 Figure 19. Negative feedback in the thyroxine release reflex.

Figure 20 .

 20 Figure 20. The two pathways of hormonal action in cells.

Figure 21 .

 21 Figure 21. Signal Transduction

Figure 22 .

 22 Figure 22. The roles of the hypothalamus and pituitary glands.

Figure 23 .

 23 Figure 23. The role of pituitary gland

Figure 25 .

 25 Figure 25. Amino acid sequence of melanocortin peptides. All the melanocortins share the invariant sequence HFRW

Figure 26 .

 26 Figure 26. The MCRs belong the family of G protein-coupled.

Figure 27 .

 27 Figure 27. Melanocortin-receptor signaling pathway

  of the mouse gene include adult-onset obesity, increased tumor susceptibility, and premature infertility. The AgRP shows a very distinct expression in the central nervous system, as it is expressed in neural cell bodies of posterior hypothalamus in close vicinity to the POMC-expressing neurons. The POMC and AgRP systems may function as physiologically opposing systems, domains that form disulfide ridges leading to similar folded structures 115 . The entire antagonistic activity for CRs resides in the Cys-rich end of the molecule 116 . for medical applications. The physiological significance of the melanocortin easily ydrolyzed by proteases. In addition the Met 4 residue in the α-MSH sequence is easily oxidized leading to a modified ligand with decreased biological activity.

Figure 29 .Figure 30 .

 2930 Figure 29. α-MSH (Ac-Ser 1 -Try 2 -Ser 3 -Met 4 -Glu 5 -His 6 -Phe 7 -Arg 8 -Trp 9 -Gly 10 -Lys 11 -Pro 12 -Val 13 -NH 2 ), and the pharmacophore core [His 6 -Phe 7 -Arg 8 -Trp 9 ] 93

Figure 31 .

 31 Figure 31. Possible cyclisation approaches utilising triazoles

  Figure 32. Ac[Yaa 5 (& 1 ),Xaa 10 (& 2 )]αMSH 4-10 [{& 1 (CH 2 ) n -1,4-[1,2,3]triazolyl-(CH 2 ) m & 2 }] and Ac[Xaa 5 (& 1 ),Yaa 10 (& 2 )]αMSH 4-10 [{& 1 (CH 2 ) m -1,4-[1,2,3] triazolyl-(CH 2 ) m & 2 }] .

Figure 33 .

 33 Figure 33. Proposed mechanism of the click-reaction

  -triazolyl containing cyclo heptapeptides, derived from the model cyclic peptide lactam MTII, Ac-Nle 4 -c[Asp 5 -D-Phe 7 -Lys 10 ]αMSH 4 -10-NH 2 , varied in the size, the location, and the orientation of the [1,2,3]-triazolyl moiety within the bridge.

Figure 35 .

 35 Figure 35. N α -Fmoc-ω-azido-α-amino-and N α -Fmoc-ω-ynoic-α-amino acids

Table 3 . 64 Scheme 5 .Scheme 6 .

 36456 Scheme 5. Synthesis of the chiral inducer [Gly-Ni(II)-(S)BPB]

Table 4 .

 4 5 °C , 20 min yielded the corresponding α-alkynyl-ω-amino acids in good diastereoisomeric excess. Diastereoisomeric excess of the alkynylated complexes. Products of alkylation of the Gly-Ni-'ω-alkynyl-(S)-α-amino acid was obtained by hydrolysis of the resultant alkylated complex in 2M HCl in MeOH. Workup afforded a pale green solid containing the free alkynylated amino acids in the presence of Ni 0 and BPB. This strategy presents the advantage that the chiral auxiliary, BPB, is regenerated in good yield (90%) at the end of the process and can be re-used in a new reaction, although the excess nickel in the waste water is a potential environmental problem. The traces of Ni 0 , which interfere in the following N α -Fmoc-protection reaction, were removed from the free and crude α-alkynylated ω-amino acids, by overnight solid phase extraction of its MeOH/H 2 O (15:20) solution with Na + Chelex resin. Finally, the free ω-alkynyl-α-amino acids were N α -protected with Fmoc employing Fmoc-O-Su, to yield the building blocks in adequate for the Fmoc/tBu solid phase peptide synthesis 16 .

  triazolyl-(CH 2 ) n & 2 }] (I', II', III', IV', IX) Intramolecular Cu I -catalyzed side-chain-to-side-chain azide-alkyne 1,3-dipolar Huisgen cycloaddition (CuAAC), converted the purified (>95% purity) linear precursors I'-X' in to the 1,4-disubstituted-[1,2,3]triazolyl-containing cyclopeptides I-X, presenting different permutations in terms of size, orientation and position of the triazolyl bridges. Depending on the respective position (i-i+5 or i+5-i) of the azido and alkynyl amino acids in the peptide sequence, we generated two classes of cyclopeptides (Figure 36 and Figure 37).

Figure 36 .

 36 Figure 36. Ac[Xaa 5 (& 1 ),Yaa 10 (& 2 )]αMSH(4-10) [{& 1 (CH 2 ) m -1,4-(1,2,3)triazolyl-(CH 2 ) n & 2 }]

2. 6 . 5

 65 Solid Phase Synthesis of lactam bridge-containing cyclopeptide MTII Ac-Nle 4 -c[Asp 5 -D-Phe 7 -Lys 10 ]αMSH 4-10 -NH 2.MTII, Ac-Nle 4 -c[Asp 5 -D-Phe 7 -Lys 10 ]αMSH 4-10 -NH 2 , was synthesized using a Rinkamide NovaSyn TGR resin on a manual batch synthesizer (PLS 4X4, Advanced ChemTech) applying the same Fmoc/tBu solid procedure reported for the MTII linear analogs I'-X'. The formation of the lactam bridges between side chain groups of Lys 10 and Asp 5 was performed in solid phase, following an orthogonal protocol of deprotection. In particular we used Lys 10 protected in side chain with 1-[(4,4dimethyl-2,6-dioxocyclohex -1-ylidine)ethyl] group (Dde), that is stable in the condition of elongation of the peptide. After acetylation of the N-terminus, Dde was removed using hydrazine 20% in DMF (1mL). After deprotection of Lys 10 , the formation of intramolecular lactam bound was performed using TBTU/HOBt/NMM (2.5:2.5:3.5 equiv.) as coupling system, for 40 minutes. The reaction of cyclization was monitored by UPLC-ESI-MS analysis of MW-assisted mini-cleavage resin bond

Figure 38 .

 38 Figure 38. φ and ψ Dihedral angles.

Figure 39

 39 Figure 39 and 40 show the diagnostic sequential and medium-range connectivities observed in the NOESY spectra of cyclopeptides I, V, II, IV and X.

Figure 39 .

 39 Figure 39. Sequential and medium-range NOEs for [1,2,3]triazolyl-containing cyclopeptides MTII-I (A-B) and MTII-V (C). Panels A and B are relative to the NOE connectivities of the most (A) and the less (B) abundant MTII-I conformers. Data were obtained from a 600 MHz NOESY experiments with a mixing time of 200 ms and collected in DMSO-d6 at 300 K.

Figure 40 .

 40 Figure 40. Sequential and medium-range NOEs for [1,2,3]triazolyl-containing cyclopeptides MTII-II (A), MTII-IV (B) and MTII-X (C). Data were obtained from a 600 MHz NOESY experiments with a mixing time of 200 ms and collected in DMSO-d6 at 300 K

Figure 41

 41 Figure41shows an overlap of the 20 best structures of cyclopeptides I and V conformers, calculated by DYANA and then minimized with the SANDER module of the AMBER 5 software package. In all the sets of structures a good overlap is observed in residues within the cyclic regions of the peptides. The structure bundle relative to the cyclopeptide I shows that the conformational preferences of the most populated conformer (Figure41A) are consistent with the presence of type I β-turn including the residues His 6 -Trp 9 , whereas the less populated conformer (Figure41B)show the presence of α-helical structure including all residues. The cluster of NMR structures relative to the cyclopeptide V (Figure41C) shows the presence of type I βturn including the residues His 6 -Trp 9 .

Figure 41 .

 41 Figure 41. NMR structure bundles of MTII-I (A-B) and MTII-V (C) Best 20 calculated structures are shown. Panels A and B show the most (A) and the less (B) abundant MTII-I conformers respectively

Figure 42 .Figure 43 .

 4243 Figure 42. Comparison of MTII-I conformers representative of the most (A) and the less (B) abundant MTII-I conformer populations

Figure 44 .

 44 Figure 44. General chemical structure of phthalates

Figure 45 . 3 . 1 . 1

 45311 Figure 45. DEHP structure.

Figure 46 .

 46 Figure 46. Metabolism of DEHP(Koch et al., Int J Androl, 2006, 29,155-165)

Figure 47 . 3 . 2 3 . 2 . 1

 4732321 Figure 47. Glucuronidation of phthalate monoester

  or indirect activity on nuclear receptors. These receptors regulate the gene expression program according to the signals that they receive in the form of specific ligands, for example hormones or molecules that belong to metabolic pathways as substrates, intermediates or end-products. In particular, peroxisome-proliferator activated receptor (PPAR) has been shown to act as xenobiotic sensor, triggering adaptive transcriptional responses linked to xenobiotic metabolism that may also interfere with their response to endogenous metabolites.PPARs are a group of nuclear receptor proteins that function as transcription factors regulating the expression of genes. Phthalate metabolites, and in particular MEHP, activate three different PPAR isotypes (PPAR α, β/δ, γ), (Figure48) 175 .

Figure 48 .

 48 Figure 48. PPAR -alpha and -gamma pathways.

A

  normal thyroid function is essential for fetal development of the brain, neurons, heart and other organs during critical points of gestation181 . Some environmental toxicants, such as perchlorate, polychlorinated biphenyls (PCBs), inorganic mercury, and phthalates potentially alter human thyroid function during pregnancy by inhibiting iodide transport, thyroglobulin iodination, or competitively inhibiting thyroid hormone binding receptors 182 . Beside being essential for normal brain development, thyroid hormones play an important role in many physiologic system, and alterations in thyroid hormone levels can lead to a myriad of adverse clinical conditions. The prevalence of subclinical hypothyroidism is 2-5% in pregnant women. Maternal hypothyroidism during pregnancy causes preterm birth and low birth weight, and it impairs post-natal mental development in infants183,184 .Phthalates have thyroid-disrupting properties interfering with the thyroid hormone system inducing altered thyroid activity. Although much is still unknown about mechanism and consequences involved with the relationship between environmental exposures and change in thyroid hormone levels, phthalates and other environmental chemicals may bind to thyroid receptors and influence the thyroid hormone signaling. In particular phthalates are associated to a decreases in serum total thyroxine (T4) and triiodothyronine (T3) concentrations, leading to hypothyroidism 185 . Deficiency of thyroid hormones during critical periods of brain development, both in utero and in early postpartum period is a well-recognized cause of brain damage. Recent studies correlated hypothyroxinemia to permanent alteration of the cerebral cortical architecture, decreased intellectual capacity, mental retardation, and neurological diseases. Transient intrauterine deficits of thyroid hormones (THs) have been shown to result in permanent alterations of cerebral cortex similar to those found in brains of children with autism 186 . As a consequence, the current surge of this disease could be related to transient maternal hypothyroxinemia resulting from exposure to antithyroid environmental contaminants.

  secondary metabolites of DEHP in two different populations. To one side we studied the urinary concentration of DEHP metabolites in children obese and/or affected by metabolic syndrome. This study was in collaboration with professor Bernasconi "Centro Turbe dell'Accrescimento, Clinica Pediatrica Azienda Ospedaliero-Universitaria di Parma", and with professor M. Street, Department of Pediatrics University Hospital of Parma. The study was aimed to evaluate a possible correlation between urinary concentrations of phthalates, obesity and insulin sensitivity in obese children. On the other hand, we evaluate the levels of the primary and secondary metabolites of DEHP in autistic children. This work was in collaboration with professor J. Hayek, Child Neuropsychiatry Unit, University Hospital AOUS of Siena, and C. de Felice, Neonatal Intensive Care Unit, University Hospital AOUS of Siena. Urine concentrations of secondary metabolites derived from DEHP were measured in triplicate applying an HPLC-ESI-MS method. The metabolites measured included: MEHP, 6-OHMEHP, 5-oxo-MEHP, 5-OH-MEHP and 5-cx-MEHP. The unequivocally characterized synthetic metabolites were used as pure analytical standards (>98% purity) for quantitative determination in urines from obese or autistic children and healthy controls.

3. 4 Scheme 7 .

 47 Scheme 7. Reagents and conditions: (a) phthalic anhydride, pyridine (Py), under N 2 ; (b) PdCl 2 , quinone in DMF/H 2 O (7:1); (c) 1 M B 2 H 6 in THF, then H 2 O 2 , OH-in THF; (d) CrO 3 , H 2 SO 4 in acetone; (e) NaBH 4 in EtOH.

  lower limit of quantification (LLOQ), similarly defined, quantifies the lowest concentration of analyte that can be determined with acceptable precision and accuracy under the stated experimental conditions. It can be calculated from the standard deviation of the response and the slope associated with the calibration curve, according to the following equation: In urines, LLOQs were 0.042 μg/L [MEHP], 0.048 μg/L [5-OH-MEHP], 0.049 μg/L [5-oxo-MEHP], 0.047 μg/L [5-cx-MEHP], and 0.008 μg/L [6-OH-MEHP]. In urine, LLODs were 0.014 μg/L [MEHP], 0.016 μg/L [5-OH-MEHP], 0.016 μg/L [5-oxo-MEHP], 0.018 μg/L [5-cx-MEHP], and 0.002 μg/L [6-OH-MEHP].

Figure 49 .

 49 Figure 49. SPE procedure

Figure 50 .

 50 Figure 50. A: Relationship between MEHP and Chronological age at onset of obesity; B: Relationship between MEHP and AUCI; C: Relationship between MEHP and AUCG; D: Relationship between MEHP and WBSI

Figure

  Figure 51. Connectivity map

Figure 52 .

 52 Figure 52. Column scattergrams for 5-oxo-MEHP values in ASDs' urine samples (ASDs, n = 48, and Healthy Controls (HC, n = 23)

  amino acids and Rink-amide NovaSyn® TGR resin were purchased from Novabiochem (Laufelfingen,Switzerland). The following amino acid side-chainprotecting groups were used: OtBu (Asp, Glu), tBu (Ser, Thr), Pbf (Arg), Trt (Gln, His) and Boc (Lys). HOBt and TBTU were purchased from Iris Biotech GmbH (Marktredwitz, Germany); Fmoc-L-Arg(Pbf)-OH and Fmoc-L-His(Trt)-OH amino acids were purchased from CBL (Patras, Greece); Fmoc-L-Nleu was purchased from NeoMPS (Strasburgo, France). β-Glucoronidase (Escherichia coli K12) was purchased from Roche Biochemical (Mannheim, Germany). Peptide-synthesis grade N,N-dimethylformamide (DMF), and N-methylpyrrolidone (NMP) for peptide synthesis were purchased from Scharlau (Barcellona, Spain); acetonitrile (ACN) from Carlo Erba; dichloromethane (DCM), trifluoroacetic acid (TFA), piperidine, acetic anhydride (Ac 2 O), N,N-diisopropylethylamine (DIEA), diethyl ether, and N-metilmorpholine (NMM) were purchased from Sigma Aldrich; TIS was purchased from Fluka/Aldrich. The scavengers for cleavage of peptides from resin, 1,2-ethanedithiol (EDT), thioanisole and phenol (PheOH), were purchased from Acros Organics, Jansenn Chimica and Carlo Erba. TLC were carried out precoated on silica gel plates (Merck; 60 Å F254) and spots located with: (a) UV light (254 and 366 nm), (b) ninhydrine (solution in acetone), (c) Cl 2 /toluidine, (d) fluorescamine, (e) I 2 , (f) a basic solution of permanganate [KMnO 4 (3 g), K 2 CO 3 (20 g), and NaOH (0.25 g) in water (300 ml)], (g) 10% H 2 SO 4 in EtOH. Flash Column Chromatography (FCC) was performed on Merck silica gel 60 (230-400 mesh) according to Still et al.

5. 5

 5 MW-assisted mini-cleavage. General procedure MW-assisted mini-cleavages were performed using Discover TM S-Class single-mode MW reactor equipped with Explorer-48 autosampler (CEM). The mixing of the cleavage reaction was accomplished by magnetic stirring and the reaction temperature was monitored at the bottom of the reactor vessels by an IR sensor. A small sample of beads carrying Fmoc-protected resin-bound peptide (10 mg) was weighted into a fritted polypropylene tube and treated twice with a 20% solution of piperidine in DMF (1 ml) each time for 5 min. The beads were then washed with DMF (2 × 1ml) and DCM(3 × 1 ml), dried under vacuum and transferred into a 10 ml glass tube containing the cleavage mixture that was placed into the MW cavity.

1 6 . 2 6 . 3 6 . 4

 1626364 -MS system consisted of an ACQUITY TM UPLC system (Waters) coupled with a Micromass® Q-Tof MICRO TM mass spectrometer (Waters) equipped with an ESI source. The chromatographic separation was achieved on a Symmetry 300 C18 column (100 mm × 2.1 mm, ID 3.5 μm, Waters) with the column temperature set at 30 •C. The flow rate was 0.15 ml/min with a linear gradient running from 0 to 30% of B (solvent A: 2% ACN, 0.1% formic acid in H 2 O; solvent B: 2% H 2 O, 0.1% formic acid in ACN) in 7 min, followed by 30-60% B in the next 3 min, then by 95% B for 2 min, and returned to initial condition for 3 min for re-equilibration. The total run time per sample was 15 min. The ESI-MS analysis was carried out in the positive ESI mode, the optimal MS parameters were as follows: capillary voltage 3.2 kV, cone voltage 30 kV, source temperature 120 •C and desolvation temperature 320 •C. Nitrogen was used as desolvation and cone gas with a flow rate of 450 and 40 l/h, respectively. For MS/MS analyses, argon was used as collision gas, and the collision energy was set to 40 eV. Data were acquired and processed using MassLynx TM software (Waters). Deprotection of Boc-protecting group. General procedure Cleavage of Boc protecting group of N α -Boc-ω-azido-α-amino acid (3.96 mmol), or N α -Fmoc-N ω -Boc-α-amino acid (2.2 mmol), was achieved by treatment with an excess of HCl 2 M (10 ml) at RT for 2 h. The solution was diluted in water and lyophilized. Fmoc protection of the free amino acid. General procedure A solution of (2,4-dioxo-1-pyrrolidinyl)(9H-fluoren-9-ylmethoxy) carbonate (Fmoc-O-Su 4.36 mmol) in dioxane (20 mL) was added dropwise to a stirred solution of the deprotected amino acid (1.85 mmol, 1 eq.) in dioxane (30 mL). A solution of 1M NaOH was subsequently slowly added until pH 8-9 and the reaction mixture was stirred at RT for 2 h and 30 min. The reaction was checked by TLC (CH 2 Cl 2 /MeOH, 9:2, detected by (a) and (b)). Water (12 mL) was added and the solution was acidified with 2M HCl until pH 3. The product was extracted with DCM (3 x 30 ml), dried with anhydrous Na 2 SO 4 , filtered and the solvent removed under vacuum. The crude material was purified by FCC on silica gel (employing a step-gradient of MeOH in DCM, 2-20%) to obtain the corresponding pure N α -Fmoc-ω-azido-α-amino acids or N α -Fmoc-ω-alkynyl-α-amino acids. Diazotransfert on ω-aminic group of N α -Boc or N α -Fmoc-amin acids with triflic anhydride. General procedure A solution of TfN 3 in CH 2 Cl 2 was obtained as follows: Tf 2 O (1.35 ml, 8.13 mmol, 2eq.) was added dropwise to a vigorously stirred mixture of NaN 3 (2.635 g, 40.5 mmol, 10 eq) in H 2 O (6.5 mL) and CH 2 Cl 2 (11 mL) at 0 o C. The resulting mixture was allowed to warm to RT and stirring was continued for 2 h. The water layer was extracted with CH 2 Cl 2 (2 × 4 mL) and the combined organic layers were washed with saturated aqueous Na 2 CO 3(12.5 mL). The resulting solution of TfN 3 in CH 2 Cl 2 was then added dropwise to a solution amino acid (4.06 mmol, 1 eq.), K 2 CO 3 (0.84 g, 6.08 mmol, 1.5 eq.) e CuSO 4 ·5H 2 O (0.01 g, 0.04 mmol) in H 2 O (13 ml) and MeOH (27 ml). The mixture was stirred overnight and the reaction was checked by TLC (iPrOH/AcOEt/H 2 O 6:1:3, detected by (a) and(b), R f 0.81). The organic solvents were evaporated under vacuum. The water layer was acidified to pH 6 with concd.HCl, diluted with 0.25 M of phosphate buffer at pH 6.2 (25 mL), and extracted with CH 2 Cl 2 (4 × 50 mL). The organic layers were washed with brine (25 mL), dried over Na 2 SO 4 , and concentrated under vacuum. The crude colorless oil was purified by flash chromatography using column of RP-18 LiChroprep by solutions of different concentrations of H 2 O/CH 3 CN to afford the desired product. Synthesis of imidazole-1-sulfonyl azide hydrochloride as diazotransfer reagent Sulfuryl chloride (16.1 ml, 200 mmol) was added drop-wise to an ice-cooled suspension of NaN 3 (13.0 g, 200 mmol) in MeCN (200 ml) and the mixture stirred overnight at room temperature. Imidazole (25.9 g, 380 mmol) was added to the ice-cooled mixture and the resulting slurry stirred for 3 h. at room temperature. The mixture was diluted with EtOAc (400 mL), washed with H 2 O (2 × 400 mL) then with saturated aqueous NaHCO 3 (2 × 400 mL), dried over MgSO 4 and filtered. A solution of HCl in EtOH [obtained by the drop-wise addition of AcCl (21.3 mL, 300 mmol) to ice-cooled dry ethanol (75 mL)] was added drop-wise to the filtrate with stirring, the mixture chilled in an ice-bath, filtered and the filter cake washed with EtOAc (3 × 100 mL) to give imidazole-1-sulfonyl azide hydrochloride as colourless needles.

8 = 7 . 6

 876 , 0.01 eq.) in MeOH (30 ml). The mixture is stirred at RT for 10 min. The reaction is checked by TLC (DCM/MeOH 10:1, detected by (a) and(b)). The solution is diluted with H 2 O (30 ml), acidified until pH 2 with HCl 0.1 M, and extracted with DCM (3x50 ml). The combined organic layers were dried with anhydrous Na 2 SO 4 , filtered and the solvent removed under vacuum. The crude product is purified by Flash Column Chromatography (FCC) on silica gel (employing a step-gradient of MeOH in DCM, 2-20%) to obtain the corresponding pure N α -Fmoc-ω-azido-α-amino acids as yellow oils. RP-UPLC: R t 4.28 min (50-100% B in 5 min.). IR (KBr): 2100 cm -1 (N 3 ). ESI-MS: m/z calcd for C 21 H 22 N 4 NaO 4 [M + Na] + : 417.15; found 417.2. [α] D -2.5 (c 1.0, MeOH). 1 H NMR (CDCl 3 , 400 MHz,): δ 7.74 (d, 2H, J 3,4 = J 5,6 = 7.4 Hz, fluorenyl 4-H e 5-H), 7.54 (d, 2H, J 1,2 = J 7,8 = 7.4 Hz, fluorenyl 1-H e 8-H), 7.37 (pseudo t, 2H, fluorenyll 3-H e 6-H), 7.28 (pseudo t, 2H, fluorenyl 2-H e 7-H), 6.19 (s broad, COOH), 5.46 (m, 1H, NH), 4.49-4.33 (m, 3H, CH 2 -O and α-H), 4.18 (t, 1H, J = 6.4 Hz, fluorenyl 9-H), 3.24-3.21 (m, 2H, ε-H 2 ), 1.70-1.42 (m, 6H, 3 × CH 2 ). 13 C NMR (CDCl 3 , 100 MHz): δ 176.97 (COOH), 156.35 (CONH), 143.75, 143.60, e 141.28 (fluorenyl C-4a, C-4b, C-8a, e C-9a), 127.74, 127.06, e 125.01 (fluorenyl C-2-C-7), 120.00 (fluorenyl C-1 and C-8), 67.14 (CH 2 -O), 53.90 (C-α), 51.02 (C-ε), 47.07(fluorenyl C-9), 31.68 (CH 2 ), 28.31 (CH 2 ), 22.55 (CH 2 ). Anal. Calcd for C 21 H 22 N 4 O 4 : C, 63.95; H, 5.62; N, 14.20. Found: RP-UPLC: R t 3.81 min (50-100% di B in 5 min). IR (KBr): 2100 (N 3 ) cm -1 . [α] D -2.3 (c 1.0, MeOH). ESI-MS: m/z calcd. for C 20 H 20 N 4 NaO 4 [M + Na] + 403.14; found 403.3. 1 H NMR (CDCl 3 , 400 MHz,): δ 7.76 (d, 2H, J 3,4 = J 5,6 = 7.6 Hz, fluorenyl 4-H e 5-H), 7.61 (pseudo d, 2H, J 1,2 = J 7,Hz, fluorenyl 1-H e 8-H), 7.40 (pseudo t, 2H, fluorenyl 3-H e 6-H), 7.31 (pseudo t, 2H, fluorenyl 2-H e 7-H), 6.16 (s broad, COOH), 5.34 (m, 1H, NH), 4.45-4.40 (m, 3H, CH 2 -O e α-H), 4.22 (t, 1H, J = 6.6 Hz, fluorenyl 9-H), 3.37-3.30 (m, 2H, δ-H 2 ), 2.01-1.46 (m, 4H, 2 × CH 2 ). 13 C NMR (CDCl 3 , 100 MHz): δ 175.72 (COOH), 156.72 (CONH), 143.75, 143.57, e 141.33 (fluorenyl C-4a, C-4b, C-8a, e C-9a), 127.76, 127.08, e 125.00 (da fluorenyl C-2 a C-7), 120.02 (fluorenyl C-1 e C-8), 67.12 (CH 2 -O), 53.16 (C-α), 50.76 (C-δ), 47.15 (fluorenyl C-9), 29.62 (CH 2 ), 24.81 (CH 2 ). Anal. Calcd for C 20 H 20 N 4 O 4 : C, 63.15; H, 5.30; N, 14.73. Found: C, 63.09; H, 5.25; N, 14.80. 6.5.3 4-Azido-2S-[[(9H-fluoren-9-ylmethoxy) carbonyl]amino] butanoic acid (Fmoc-Abu(γ-N 3 )-OH) (m=2) Yield 89%. RP-UPLC: R t 3.34 min (50-100% B in 5 mini). IR: 2100 cm -1 (N 3 ). ESI-MS: m/z calcd for C 19 H 18 N 4 NaO 4 [M + Na] + 389.12; found 389.4. [α] D -11.5 (c 1.0, MeOH). 1 H NMR (CDCl 3 , 400 MHz,): δ 7.75 (pseudo d, 2H, J = 7.6 Hz, fluorenyl 4-H e 5-H), 7.54 (pseudo d, 2H, J = 7.4 Hz, fluorenyl 1-H e 8-H), 7.39 (pseudo t, 2H, fluorenyl 3-H e 6-H), 7.31 (pseudo t, 2H, fluorenyl 2-H e 7-H), 6.14 (s broad, COOH), 5.63 (m, 1H, NH), 4.53-4.41 (m, 3H, CH 2 -O and α-H), 4.21 (t, 1H, J = 6.8 Hz, fluorenyl 9-H), 3.42-3.39 (m, 2H, γ-H 2 ), 2.19-1.96 (m, 6H, 3 × CH 2 ). 13 C NMR (CDCl 3 , 100 MHz): δ 172.71 (COOH), 156.26 (CONH), 143.53 e 141.29 (fluorenyl C-4a, C-4b, C-8a, e C-9a), 127.76, 127.08, 125.04, 124.99 (fluorenyl C-2, C-7), 120.00 (fluorenyl C-1, C-8), 67.17 (CH 2 -O), 51.70 (C-α), 47.68 (C-γ), 47.09 (fluorenyl C-9), 31.21 (CH 2

6 . 6 6 . 7

 6667 Yield 75 %. RP-UPLC: R t 4.02 min (50-100% B in 5 min). IR: 2100 cm -1 (N 3 ). ESI-MS: m/z calcd. for C 18 H 16 N 4 NaO 44 [M + Na] + 375.12; found 375.4. 1 H NMR (CDCl 3 , 400 MHz,): d 7.70 (pseudo d, 2H, J = 7.4 Hz, fluorenyl 4-H e 5-H), 7.48 (pseudo d, 2H, J = 7.6 Hz, fluorenyl 1-H and 8-H), 7.40 (pseudo t, 2H, fluorenyl 3-H and 6-H), 7.28 (pseudo t, 2H, fluorenyl 2-H e 7-H), 6.3 (s broad, COOH), 5.78 (m, 1H, NH), 4.57-4.45 (m, 3H, CH 2 -O and α-H), 4.32 (t, 1H, J = 6.8 Hz, fluorenyl 9-H), 3.52-3.35 (m, 2H, γ-H2). Anal. Calcd for C 18 H 16 N 4 O 4 : C, 61.36; H, 4.58; N, 15.90. Found: C, 61.46; H, 4.98; N, 15.68. General procedure for the synthesis of p-toluenesulfonate derivatives p-Toluenesulfonyl chloride (61.49 mmol, 1.5 eq.) in pyridine (10 mL) was added to a stirred solution of the ω-alkynyl-alcohol (40.75 mmol, 1 eq.) in pyridine (15 mL), at 0 °C. The reaction was stirred overnight at room temperature and checked by TLC [AcOEt-n-hexane 1:3, detected by (a)]. The reaction mixture was neutralized with 2 M HCl (10 mL/1 mL pyridine), the p-toluensulfonate derivative extracted with CHCl 3 , and the organic layer evaporated under vacuum to afford the product as a pale yellow oil. TLC: (AcOEt/ n-hexane 1:3, detected with (a), Rf 0.6). 1 H-NMR (CDCl 3 , 200 MHz): δ 7.78 e 7.34 (syst. AA'BB', 4H, J = 8.4 Hz, MeC 6 H 4 ), 3.57 (t, 2H, J = 6.2 Hz, 1-H 2 ), 2.45 (s, 3H, MeC 6 TLC: (AcOEt/ n-hexane 1:3, detected with (a), Rf 0.62). 1 H-NMR (CDCl 3 , 200MHz): δ 7.77 e 7.35 (syst. AA'BB', 4H, J = 8.4 Hz,MeC 6 H 4 ), 4.12 (t, 2H, J = 5.8 Hz, 1-H 2 ), 2.43 (s, 3H, MeC 6 H 4 ), 2.32-2.15 (m, 2H, 3-H 2 ), 1.93 (t, 1H, 5-H), 1.91-1.82 (m, 2H, 2-H 2). Synthesis of ω-alkynyl bromo derivatives. General procedure Lithium bromide (17.95 mmol, 1.5 eq.) was added in portions to a stirred solution of the p-toluensulfonate derivative (11.97 mmol, 1 eq.) in acetone (15 mL) at room temperature. The reaction mixture was heated at 40 °C and stirred for 1 h. The reaction was checked by TLC (AcOEt-hexane 1:3; revealed with KMnO 4 and by UV 254 nm). The solvent was evaporated by flushing with N 2 . The residue was treated with n-hexane, filtration of the residue and evaporation of DCM under nitrogen flux, afforded the bromo derivative as a yellow oil. (t, 2H, J = 7.2 Hz, 6-H 2 ), 2.21 (dt, 2H, J = 3.0 Hz, 7.4 Hz, 4-H 2 ), 2.02-1.91 (m, 2H, 3-H 2 ), 1.96 (t, 1H, J = 3.8 Hz, 1-H), 1.71-1.60 (m, 2H, 5-H 2 ) .

  g of KOH (330 mmol, 3.8 eq.) in 70 mL of iPrOH was prepared with stirring at 40°C. As soon as the solution became transparent, slow addition of freshly distilled BnCl (18.3 g, 130 mmol, 1.5 eq.) was added under stirring at the same temperature for 6h. The reaction mixture was neutralized with concentrated aqueous HCl until pH 5-6 (indicator paper), then was added to the reaction mixture CHCl 3 (30 mL) with stirring. The mixture was left overnight, then filtered and the precipitate was washed with CHCl 3 . The CHCl 3 solutions were combined and evaporated, the residue was treated with cold acetone and the precipitate of crude BP filtered and additionally washed with acetone. Some BP was also recovered from the acetone washings. The crude material was dried in air and then over P 2 O 5 . Yield 23.3%. RP-UPLC: R t 1.57 min (10-60% of B in 5 min). ESI-MS: m/z calcd for C 12 H 15 NO 2 [M-H] -. 204.11; 204.15. 1 H NMR (CDCl 3 , 400 MHz): δ 7.44-7,34 (m, 5H, Bn), 6.99 (br s, OH), δ A = 4.33, δ B = 4.14 (syst AB, 2H, J AB =15.3 Hz, CH 2 Bn),3.78 (dd, 1H, J α,β =16 Hz, J α,β' =7.0 Hz, H α ), 3.64 (ddd, 1H, J δ,δ' =9.0 Hz, J δ,γ =19 Hz, J δ,γ' =27 Hz, Hz, H δ ), 2.88-2.81 (m, 1H, H δ' ), 2.33-2.23 (m, 2H, H β , H β' ), 2.00-1.88 (m, 2H, H γ , H γ' ).[START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] C NMR (CDCl 3 , 100 MHz): δ 171.17(COOH), 131.21, 130.39, 129.41, 129.15 (Bn), 67.64 (C α ), 58.04 (CH 2 Bn), 53.45 (C δ ), 29.04 (C β ), 23.09 (C γ ).

  3 ) 2 .6H 2 O 4.53 g di (15.58 mmol, 2 eq.) in dry MeOH (25 ml) was prepared under N 2 Then, a freshly prepared solution of 2.5 M MeONa/MeOH (8 mL, 20 mmol) was added. The resulting mixture was stirred at 55-65°C for 1h (a prolonged heating of the reaction mixture might result to a partial racemization of the BPB moiety), and then neutralized with 10% aqueous citric acid (500 mL). The product was extracted with CH 2 Cl 2 (4 × 40 mL), dried over Na 2 SO 4 , and the solvent removed under vacuum. Yield 95%. TLC: Rf 0.31 (DCM/Aceton 2:1, detected with UV 254 nm). ESI-MS: m/z calcd for C 27 H 25 N 3 NiO 3 [M + H] + : 498,20 ; found 498,3.

6. 10 6 Hz 2 ×

 1062 Hydrolysis of the alkylated complexes. General procedure A solution of the alkylated complex (1.85 mmol) in MeOH (31 ml) was added to warm 2 M HCl (21.8 mL) and the mixture refluxed for 30 min. The solution changes color from deep red to yellow and was detected by TLC (DCM/Me 2 CO 2:1, detected by UV 254 nm). After cooling to RT, 1 M NaOH was added until pH 6 and the solvent removed under vacuum. The yellow solid residue was washed with DCM and dried under vacuum. BPB was quantitatively recovered by extraction with CH 2 Cl 2 . The dried solid product was dissolved in H 2 O/MeOH 4:3 (23 ml) and then gently swirled overnight with Chelex 100 H + resin (7.7 g ), converted from its Na + form by treatment with HNO 3 2.5 M (46 ml) by washing with H 2 O. The mixture was filtered and the resin washed with water, the layers of combined filtrates were evaporated under vacuum, and the residue lyophilized. The crude was purified by FCC (CH2Cl2 to CH 2 Cl 2 -MeOH 10/1) to obtain the pure amino acid as a yellow oil. TLC: Rf 0.5 (DCM/MeOH 10:1). RP-UPLC: R t 4.12 min (50-100% di B in 5 minuti). ESI-MS: m/z calcd for C 23 H 23 NNaO 4 [M + Na] + 400.15; found 400.3. [α] D -3.1 (c 1.0, MeOH). 1 H NMR (CDCl 3 , 400 MHz): δ 7.75 (pseudo d, 2H, J = 7.6 Hz, fluorenyl 4-H and 5-H), 7.59 (pseudo d, 2H, J = 7.CH 2 ). 13 C NMR (CDCl 3 , 100 MHz): δ 176.63 (COOH), 156.17 (CONH), 143.83, 143.67 and 141.29 (fluorenyl C-4a, C-4b, C-8a, and C-9a), 127.71, 127.06, 125.04 (fluorenyl C-2 to C-7), 119.98 (fluorenyl C-83.97 (HC≡C), 68.69 (CH 2 -O), 67.06 (HC≡C), 53.83 (C-α), 47.15 (fluorenyl C-9), 31.73 and 27.81 (C-β and δ), 24.31 (C-γ), 18.15 (C-ε). TLC: Rf 0.47 (DCM/MeOH 9:1). RP-UPLC: R t 3.54 min. (50-100% B in 5 min). ESI-MS: m/z calcd for C 22 H 21 NNaO 4 [M + Na] + 386.14; found 386.2. [α] D -3.0 (c 1.0, MeOH). 1 H NMR (CDCl 3 , 400 MHz): δ 7.73 (d, 2H, J = 7.2 Hz, fluorenyl 4-H and 5-H), 7.57 (d, 2H, J = 7.4 Hz, fluorenyl 1-H and 8-H), 7.39 (pseudo t, 2H, fluorenyl 3-H and 6-H), 7.30 (pseudo t, 2H, fluorenyl 2-H and 7-H), 6.60 (broad s, COOH), 5.51 (m, 1H, NH), 4.43-4.35 (m, 3H, CH 2 -O and α-H), 4.18 (t, 1H, J = 6.6 Hz, fluorenyl 9-H), 2.08-1.99 (m, 3H), 1.94 (t, 1H, J = 2.4 Hz, HC≡C), 1.80-1.75 (m, 1H), 1.58-1.42 (m, 2H, CH 2 ). 13 C NMR (CDCl 3 , 100 MHz): δ 177.06 (COOH), 156.26 (CONH), 143.81, 143.62 and 141.27 (fluorenyl C-4a, C-4b, C-8a, and C-9a), 127.70, 127.06 and 125.05 (fluorenyl C-2 to C-7), 119.96 (fluorenyl C-1 and C-8), 83.49 (HC≡C), 69.11 (CH 2 -O), 67.06 (HC≡C), 53.77 (C-α), 47.11 (fluorenyl C-9), 31.33 (C-β), 24.28 (C-γ), 18.01 (C-δ).

73 (

 73 thoxy) carbonyl]amino]-5-hexynoic acid (n=2) Yield 32%. RP-UPLC: R t 3.37 min. (50-100% B in 5 min.). ESI-MS: m/z calcd for C 21 H 19 NNaO 4 [M + Na] + 372.14; found 372. 1 H NMR (CDCl 3 , 400 MHz): d 7.m, 3H, CH2-O e α-H), 4.21 (t, 1H, J = 6.7 Hz, fluorenyl 9-H), 2.45-2.31 (m, 3H), 2.22-2.10 (m, 1H), 2.00 (t, 1H, J = 2.1 Hz, HC≡C).

  77 µmol) were added to a solution of CuSO 4 .5H 2 O (10 mg, 40 μmol), ascorbic acid (10 mg, 56 μmol) in 5 mL of H 2 O/tBuOH :2/1 v/v. Mixture was stirred at RT overnight and the solution was concentrated and lyophilized. Complete and clean conversion of all linear precursors into the desired 1,2,3-triazolyl-containing cyclopeptides I-X was observed. The reaction of cyclization was monitored by ACQUITY UPLC (Waters Corporation, Milford, Massachusets) coupled to a single quadrupole ESI-MS (Micromass ZQ) using a 2.1 x 50 mm 1.7 μm ACQUITY BEH C18 at 30 °C, with a flow rate of 0.6 mL/min employing a linear gradient of 10% to 60% of B in A in 5 min (A 0.1% TFA in H 2 O;

  solution (0 °C) of lithium diisopropylamide (31.0 mL of a 2 M solution in THF-n-heptane, 62 mM) was added, under nitrogen, hex-5-enoic acid (2.8 g, 24 mM) in anhydrous THF (28 mL). After stirring at room temperature for 30 min and subsequent cooling to 0 °C, iodoethane (5.7 g, 36 mM) was added. The mixture was stirred for 3 h at room temperature and the reaction was then quenched with 3 M HCl (100 mL). After removal of THF, the reaction mixture was extracted with Et 2 O and the combined organic phases were washed with 1 M NaOH. The aqueous basic layer was acidified to pH ~ 1 with conc. HCl and then extracted with Et 2 O. The crude acid isolated after drying over Na 2 SO 4 and removal of the solvent, was used without further purification in the next step. Yield 96%; TLC: (Et 2 O-nhexane 1:1, detected by (e), Rf = 0.47); 1 H NMR (CDCl 3 ) 0.94 (3H, t, J 7.4 Hz, CH 3 ), 1.55-1.81 (4H, m, CH 2 ), 2.12 (2H, m, CH 2 -CH=CH 2 ), 2.38 (H, m, CH), 5.02 (2H, m, =CH 2 ),

  of 2-ethylhex-5-enoic acid (3.25 g, 23 mM) in dry Et 2 O (65 mL) at 0 °C was slowly added LiAlH 4 (2.15 g, 57 mM). After stirring at room temperature for 2 h, the mixture was quenched with H 2 O (200 mL) and concd. HCl was added until the solution became clear. The resulting mixture was extracted with Et 2 O. The combined organic phases were washed with brine and dried over Na 2 SO 4 , followed by column chromatography on silica gel with Et 2 O-petroleum ether 1 :5, then Et 2 Opetroleum ether 1 :2 as eluent to give 1 (1.8 g, 64%); TLC: (Et 2 O-petroleum ether 1:5, detected by (g), Rf = 0.20); IR(KBr): νmax(film)/cm -1 3340 (OH), 1640 (C=C), 1040 (C-O); 1 H NMR (CDCl 3 ) 0.91 (3H, t, J 7.4 Hz, CH 3 ), 1.26-1.60 (6H, m, CH 2 , CH, OH), 2.09 (2H, m, CH 2 -CH=CH 2 ),

  Hz, CH=); 13 C NMR (CDCl 3 ) 11.03 (CH 3 ), 23.25, 29.66, 30.86 (CH 2 ), 39.12 (CH), 67.35 (OCH 2 ), 114.0 (CH 2 =), 139.0 (CH=).

  -5-hexen-1-ol (2.2 g, 17.2 mmol), phthalic anhydride (2.54 g, 17.2 mmol) and pyridine (1.6 mL) were refluxed under nitrogen for 3 h. The mixture was quenched with cold water and extracted with Et 2 O. The organic phase was washed twice with a solution of 10% HCl to remove pyridine. Finally, the mixture was extracted with 0.4 M K 2 CO 3 . The aqueous basic layer was acidified to pH 1 with 1 M HCl and then extracted with Et 2 O. After drying with Na 2 SO 4 and evaporation of the solvent under vacuum, MEHP was obtained as a colourless oil and used without further purification in the next step. Yield 94%. TLC: (Et 2 O/hexane 1:1, detected by (a), Rf = 0.15). ESI-MS: calcd for [M+H]+ 277.3. Found 277.9. 1 H NMR d 0.90 (3H, t, J = 8 Hz, CH 3 ), 1.24-2.14 [7H, m, CH2= CH(CH 2 ) 2 CHCH 2 CH 3 ], 4.25 (2H, d, J = 6 Hz, COOCH 2 ), 4.96 (2H, dd, J = 10, J = 1.8 Hz, CH 2 =), 5.02 (1H, br, OH), 5.76 (1H, m, CH=), 7.61 and 7.81 (4H, 2 m, aromatic CH). 13 C NMR d 10.9 (CH 3 ), 23.6 (CH 3 CH 2 ), 29.9 (CH 2 CH=CH 2 ), 30.9 (CH 2 CH 2 CH= CH 2 ), 38.1 (CH 3 CH 2 CH), 68.0 (COOCH 2 ), 114.5 (CH=CH 2 ), 128.6-133.4 (aromatic C), 138.5 (CH=CH 2 ), 168.2 (COOCH 2 ), 172.3 (COOH).

  in THF (5.5 mL) was added, drop by drop at 0 °C under nitrogen, to a solution of MEHP (1.52 g, 5.5 mmol) in anhydrous THF (50 mL). After stirring at room temperature for 1 h, excess of hydride was destroyed with H 2 O (0.1 mL). After 5 min, 3 N NaOH (0.5 mL) and 30% H 2 O 2 (0.5 mL) were added and the reaction mixture was stirred for 1 h at 50 °C. The mixture was extracted with Et 2 O, washed with brine and dried over Na 2 SO 4 and FeSO 4 . The crude alcohol was isolated after evaporation of the solvent under vacuum, and purified by FCC (CHCl 3 /MeOH/AcOH 90:2:2) to obtain the product as a yellow oil. Yield 89%. TLC: (AcOEt/petroleum ether 1:1, detected by (a) and (f), Rf = 0.3). ESI-MS: calcd for [M+H]+ 295.3. Found 295.4; calcd for [M+Na] + 317.3. Found 317.3. 1 H NMR d 0.90 (3H, t, CH 3 ), 1.44 [9H, m, CH 3 CH 2 CH(CH 2 ) 3 CH 2 OH], 2.92 (1H, br, OH), 3.84 (2H, m, CH 2 OH), 4.23 (2H, m, COOCH 2 ), 7.56 and 7.74 (4H, 2 m, aromatic CH). 13 C NMR d 11.1(CH 3 ), 22.0 (CH 3 CH 2 ), 24.0 [CH 2 (CH 2 ) 2 OH], 29.9 [CH 2 (CH 2 ) 3 OH], 31.9 (CH 2 CH 2 OH), 38.9 (CHCH 2 CH 3 ), 61.9 (CH 2 OH), 67.4 (COOCH 2 ), 128.5-132.9 (aromatic C), 168.6 (COOCH 2 ), 170.1 (COOH).

7. 1 . 5

 15 Mono(2-ethyl-5-oxohexyl) 1,2-benzenedicarboxylate (5-oxo-MEHP) MEHP (2.46 g, 8.9 mmol) was added, drop by drop in 5 min, to a solution of PdCl 2 (16 mg, 0.089 mmol) and p-benzoquinone (1.06 g, 9.8 mmol) in DMF/H 2 O (7:1, 24 mL). After stirring overnight at room temperature, the reaction mixture was quenched with 3 N HCl (60 mL) and extracted with ether. The organic phase was washed with 10% NaOH and brine. The aqueous basic layer was acidified to pH 1 with 3 N HCl and then re-extracted with Et 2 O. The crude product was purified by FCC (CHCl 3 /MeOH/AcOH 90:2:2), and obtained as yellow oil. Yield 41%. TLC: (AcOEt/petroleum ether 2:1, detected by (a), Rf = 0.2). ESI-MS: calcd for [M+H] + 291.3. Found 291.1. IR (KBr) cm -1: 3500-2500 (OH), 2964.5 (CH), 1714.2 (C=O).

  was carried out using an high-performance liquid chromatography tandem mass spectrometry RP-HPLC -ESI-MS (Waters, Alliance 2695, Waters, Micromass ZQ) equipped with phenyl column (Betasil, 5 μm, 50 mm x 3 mm, Keystone Scientific, Bellefonte, PA) and with a Waters 2996 Photodiode Array Detector. An aliquot of each sample (20 μL) was injected into the apparatus.

FSD

  quality control (QC) sample. The QC sample was spiked with pooled urine and DEHP metabolites standards in known concentration (200 ng/mL). When urine analysis resulted in values of metabolites concentration exceeding from linear range of the analytical method, we subjected a new aliquot of the same sample to the entire process (deconjugation and SPE) and we analysed again. To verify the absence of DEHP metabolites contamination, all materials used during the analysis and SPE, were prescreened by RP-HPLC-ESI-MS. Before analysis, known concentration samples were treated by SPE and analysed by HPLC-ESI-MS to test SPE efficiency. : Electrospray Ionization Mass Spectrometry FDA: US Food and drug administration FGIR: fasting glucose/ insulin ratio Fmoc: 9-H-fluoren-9-yl-methoxycarbonyl Fmoc-OSu: N-(9-fluoren-9-yl-methoxycarbonyloxy)-succinimide HOMA-IR: homeostasis model assessment estimate of insulin resistance HPA/HTPA: Hypothalamic-Pituitary-Testicular Axis HPLC: high performance liquid chromatography IARC: International Agency for Cancer Research IGT

  used. The ongoing of synthesis was monitored by UPLC-ESI-MS of MW-assisted mini-cleavages of intermediate fragments. A small sample of Fmoc-protected peptide-resin beads (10 mg) was removed from the reactor vessel, weighted into a fritted polypropylene tube and treated with a 20% solution of piperidine in DMF (2 × 1 ml) for 5 min. The sample was then washed with DMF (2 × 1 ml) and DCM (3 × 1 ml), and dried under vacuum. Mini-cleavages were performed in a 10 ml glass tube containing 2 ml of cleavage mixture consisting of TFA/TIS/water solution (95 : 2.5 : 2.5 v/v/v). Tubes were inserted into the MW cavity of Discover™ S-Class (CEM) and the cleavage was carried out at 45 °C, using 15 W for 15 min assisted by external cooling of the reactor vessel. The crude fragments were analyzed by UPLC-ESI-MS with a Symmetry 300 C18 column (100 mm × 2.1 mm ID, 3.5 µm, Waters) using the following conditions: flow rate 0.15 ml/min with a linear gradient from 0 to 30% of B (solvent A: 2% ACN, 0.1% formic acid in H 2 O; solvent B: 2% H 2 O, 0.1% formic acid in ACN) in 7 min, 30-60% B in the next 3 min, then to 95% B for 2 min, and returned to initial conditions for 3 min for re-equilibration. The total run time per sample was 15 min

Figure 1 .

 1 Figure 1. Characteristics of the PTHrP(1-34)NH 2 sequence are a cluster of arginine residues in the positions 19-21, sterically hindered and hydrophobic amino acid sequences.

Figure 2 .

 2 Figure 2. (A-D) TIC chromatogram of selected crude mixtures of intermediate resin-bound sequences obtained during the synthesis of PTHrP(1-34)NH 2 (Fmoc/tBu RT-SPPS) generated by the MW-assisted mini-cleavages. The related MS/MS spectra are shown in Supporting Information (Figure S1).

  Mini-cleavage of PTHrP(1-34)NH 2 Fragments Both RT and MW-assisted SPPSs were monitored by UPLC-ESI-MS analysis of MW-assisted mini-cleavages of intermediate resinbound fragments using Discover  S-Class single-mode MW reactor equipped with Explorer-48 autosampler (CEM). The mixing of the cleavage reaction was accomplished by magnetic stirring and the reaction temperature was monitored at the bottom of the reactor vessels by an IR sensor. A small sample of beads carrying wileyonlinelibrary.com/journal/jpepsci Copyright c 2011 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2011; 17: 708-714

Figure 3 .

 3 Figure 3. TIC chromatogram and deconvoluted spectrum of the cleaved mixture of the intermediate resin-bound sequence PTHrP(12-34)NH 2 (Fmoc/tBu RT-SPPS) generated by the MW-assisted minicleavages.

  cycle (step 1 and 2) was performed twice refreshing the coupling solution.

Figure 4 .

 4 Figure 4. Analytical RP-HPLC of crude PTHrP(1-34)NH 2 . Fmoc/tBu RT-SPPS (A) and Fmoc/tBu MW-assisted SPPS (B). HPLC: 10-60% B (0.1% TFA in ACN) in A (0.1% TFA in H 2 O) over 20 min.

  phthalate; ED, endocrine disruptor; HC, healthy control; HPLC-ESI-MS, HPLC electrospray ionization MS; MEHP, mono-(2-ethylhexenyl) 1,2-benzenedicarboxylate; 5-OH-MEHP, mono-(2-ethyl-5-hydroxyhexyl) 1,2-benzenedicarboxylate; 6-OH-MEHP, mono-(2-ethyl-6-hydroxyhexyl) 1,2-benzenedicarboxylate; OS, oxidative stress; 5-oxo-MEHP, mono-(2-ethyl-5-oxohexyl) 1,2benzenedicarboxylate; QC, quality control; RP-HPLC-ESI-MS, reverse-phase HPLC-ESI-MS; ROC, receiver operating characteristic; SPE, solid phase extraction; PPARa, peroxisome-proliferator-activated receptor a; TH, thyroid hormone. E 2012 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http:// creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited. RESEARCH ARTICLE ASN NEURO 4(4):art:e00089.doi:10.1042/AN20120015 asnneuro.org / Volume 4 (4) / art:e00089

Figure 1

 1 Figure 1 Metabolism of DEHP (A) Hydrolytic/oxidative pathway leading to MEHP and secondary metabolite formation. (B) Formation of glucuronic conjugates of DEHP metabolites.

Figure 2

 2 Figure 2 Differences between groups: scatterplots for 5-oxo-MEHP values in ASDs urine samples (ASDs, n548; HC, n545) Data are medians and inter-quartile range.

Figure 3

 3 Figure 3 ROC curve analysis (ASD patients versus HC) for DEHP metabolites

Table 1 .

 1 RT and MW-assisted SPPS deprotection and coupling protocols used for the PTHrP(1-34)NH 2 synthesis.

	240	30	50

Table 2 .

 2 RT Fmoc/tBu SPPS: list of PTHrP(1-34)NH 2 fragments produced by MW-assisted minicleavages of intermediate resin-bound peptides. The relative amounts of the deletion sequences are calculated in percentage of area under the peak from the total area under the curve obtained from the TIC chromatogram.

	Analyzed

sequences Calcd mass Intact sequences (m/z) found Deletion sequences (m/z) found Missing aa residues from deletion sequences

						Deletion
						sequences
						(%)
	27-34	865.51	866.39 [M+H] +	753.41 [M+H] +	Leu 27	7
	25-34	1139.62	1140.90 [M+H] +	1003.95 [M+H] + 890.93 [M+H] +	His 25 Leu 27 His 25	2 5
	22-34	1546.84	1547.41 [M+H] +	1434.44 [M+H] +	Leu 27	8
				952.19 [M+2H] 2+	Leu 27	6
	19-34	2015.15 1008.69 [M+2H] 2+	852.72 [M+2H] 2+	Arg 19 Arg 20	39
				774.73 [M+2H] 2+	Arg 19 Arg 20 Arg 21	51
				1322.29 [M+2H] 2+	Leu 27
	12-34	2756.55 1379.24 [M+2H] 2+	1315.26 [M+2H] 2+ 1258.81 [M+2H] 2+	Gln 16 /Lys 13 Leu 27 Gln 16 /Lys
				1065.43 [M+2H] 2+

  [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF]

1.6 RP-HPLC Semi-preparative of PTHrP(1-34)NH 2

	Lyophilized crude peptide was pre-purified by solid-phase extraction with an RP-18
	LiChroprep silica column from Merck (Darmstadt, Germany) using H 2 O/ACN as
	eluents. The purification of the peptide was performed by semi-preparative RP-
	HPLC on a Supelco C18 180 Å (250 × 10 mm, 5 μm) column (Sigma Aldrich, St.
	Louis, MO, USA); eluents: A 0.1% TFA in H 2 O; B 0.1% TFA in ACN: flow
	4ml/min; gradient 30-60% of B in 30min.

2 Intramolecular i-to-(i+5) side-chain-to-side chain cyclization to stabilize β-turn secondary structures: the melanocortin peptides hormones 2.1 The Endocrine System

	The endocrine system is a complex group of glands, including the pituitary gland and
	hypothalamus in the brain, adrenal glands in the kidneys, and thyroid in the neck, as
	well as the pancreas, ovaries and testes. Hormones are chemical messengers, released
	by a cells or a glands, that transfer a signal from one cell (or group of cells) to
	another. Endocrine system, through the secretion of hormones, regulates the control
	of reproduction, metabolism, growth, development, tissue function, mood, and
	energy balance, in response to external stimulations.

Functions Prevalent Tissue Expression Ligand Affinity MCR Subtype or Figure 28. Affinity

  , distribution, and functions of MCR subtypes

	the eumelanin synthetic pathway. The relative affinity of the human MC1R for the discovered by targeted deletion of the receptor 111,112 . Presence of MC5R in B-and T-
	natural melanocortins is α-MSH ≥ ACTH > β-MSH >>γ-MSH 105 . These differences lymphocytes suggests also a function in immune regulation.
	in affinity reproduce the relative potency of the melanocortin peptides in stimulation Functions Prevalent Tissue Expression Ligand Affinity MCR
	Subtype of melanogenesis and explain the lack of melanogenic activity of γ-MSH. Pigmentary effects Melanocytes α-MSH≥ACTH>>γ-MSH MC1R Pigmentary effects Melanocytes α-MSH≥ACTH>>γ-MSH MC1R
	MC1R functions extend well beyond regulation of melanogenesis MC1 receptor is Antipyretic/anti-Immune/inflammatory cells; Antipyretic/anti-Immune/inflammatory cells;
		keratinocytes; keratinocytes;	inflammatory inflammatory
	also expressed at leukocytes, where it mediates the anti-inflammatory Endothelial cells; glial cells Endothelial cells; glial cells
	property 106,107 . ACTH MC2R ACTH MC2R	Adrenal cortex Adrenal cortex	Steroiogenesis Steroiogenesis
	Anti-inflammatory Anti-inflammatory MC2R, also known as ACTH receptor, is selectively activated by Macrophages Macrophages Autonomic functions CNS >γ-MSH=ACTH≥ α-MSH MC3R Autonomic functions CNS >γ-MSH=ACTH≥ α-MSH MC3R
	adrenocorticotropic hormone 108 . MC2R is expressed only in adrenal cortex zona Control of feeding and CNS α-MSH=ACTH>>γ-MSH MC4R Control of feeding and CNS α-MSH=ACTH>>γ-MSH MC4R
	energy homeostasis; energy homeostasis; reticularis and zona fasciculate, where it mediates production and release of steroids Erectile activity Erectile activity
	hormones. Binding of ACTH to its receptor stimulates adenylyl cyclase and induces Regulation of exocrine Exocrine glands, lymphocites α-MSH≥ACTH>γ-MSH MC5R Regulation of exocrine Exocrine glands, lymphocites α-MSH≥ACTH>γ-MSH MC5R
	increases in cell cAMP; thi	s leads to activation of PKA, which promotes expression sections, sections, immunoregolatory immuno regolatory
	of steroidogenic enzymes			functions functions
	The MC3 receptor is identified in many areas of the nervous system and peripheral
	tissues, in brain, placenta and is involved in energy homeostasis, feeding behavior,
	and inflammation. The MC3R is the only MCR activated by γ-MSH with potency
	similar to that of other melanocortins (γ-MSH = ACTH ≥ α-MSH). Recent data
	suggest that MC3R activation mediates protective influences o	f melanocortins in
	myocardial ischemia/ reperfusion-induced arrhythmias in rats 109 .
	Melanocortin MC4 receptor is expressed predominantly in the central nervous
	system, including the cortex, the thalamus, the hypothalamus, the brainstem, and the
	spinal cord. The order of potency for activation of MC4R is α-MSH = ACTH > β-
	MSH >> γ-MSH Distribution of MC4R is consistent with its involvement in
	autonomic and neuroendocrine functions. It regulates both food intake and sexual
	function. MC4R definciency is related	to maturity-onset obesity, hyperphagia,
	hyperinsulinemia, and hyperglycinemia.
	Finally, the MC5 receptor is ubiquitously expressed in peripheral tissues. It occurs in
	the adrenal glands, fat cells, kidney, liver, lung, lymph nodes, bone marrow, thymus,
	. mammary glands, testis, ovary, pituitary testis, uterus, esophagus, stomach,
	MC1 receptor is mainly expressed in cutaneous melanocytes, where plays a key role duodenum, skin, lung, skeletal muscle, and exocrine glands. It plays a role in in determining skin and hair pigmentation. Binding of α-MSH to its MC1R in regulating exocrine gland function 110 , particularly sebaceous gland secretion
	melanocytes starts a signal cascade that activates adenylate cyclase, increases
	intracellular cAMP, and induces activity of tyrosinase, the rate-limiting enzyme in
				49

Table 5 .

 5 Chemical data of linear precursors I'-X'.

	Linear	Rt (*)	MS + (ESI) m/z
	precursor	UPLC	found	calculated
	I'	4.91 min	[M+H] + =1048.61	[M+H] + =1048.24
			[M+2H] 2+ =525.30
	II'	4.74 min	[M+H] + =1048.61	[M+H] + =1048.24
			[M+2H] 2+ =525.30
	III'	4.51 min	[M+H] + =1048.61	[M+H] + =1048.24
			[M+2H] 2+ =525.30
	IV'	4.46 min	[M+H] + =1048.91	[M+H] + =1048.24
			[M+2H] 2+ =525.45
	V'	4.97 min	[M+H] + =1048.61	[M+H] + =1048.24
			[M+2H] 2+ =525.30
	VI'	4.47 min	[M+H] + =1048.71	[M+H] + =1048.24
			[M+2H] 2+ =525.35
	VII'	4.46 min	[M+H] + =1048.61	[M+H] + =1048.24
			[M+2H] 2+ =525.30
	VIII'	4.77 min	[M+H] + =1048.71	[M+H] + =1048.24
			[M+2H] 2+ =525.35
	IX'	4.41 min	[M+H] + =1034.62	[M+H] + =1034.24
			[M+2H] 2+ =518.31
	X'	4.77 min	[M+H] + =1034.91	[M+H] + =1034.24
			[M+2H] 2+ =518.46

(*) solvent system: A 0,1 % TFA in H 2 O; B 0,1% TFA in ACN; gradient 10-60% B in 5 min. Flow 0.6 ml/min

  triazolyl-containing peptide, was achieved after ON incubation at RT in

	tBuOH/H 2 O (1:2 v/v), in the presence of 4.4-fold molar excess of CuSO 4 •5H 2 O and
	ascorbic acid, obtaining Cu(I) by in-situ formation from Cu(II) salts.
	Under these conditions there is no formation of oligomeric products resulting from
	intermolecular click reactions, thus suggesting formation of a Cu I /acetylide/azide
	complex with high preference for intramolecular cyclizations. Moreover following
	this protocol we didn't observe side products formed by dimerizations and
	macrocyclizations, which are often observed in on-resin CuAAC, thus generating

easy to purify crude products. All copper salts were eliminated by solid phase extraction from the crude material by eluting with water, and cyclopeptides were afforded by elution from 10% to 30% of acetonitrile in water. The clicked peptides

Table 6 .

 6 Chemical data of clicked peptides I-X.

	Clicked	Xaa	Yaa	Rt (*)	MS + (ESI) m/z
	peptide			UPLC	found	calculated
	I	n = 1	m = 4	3.66 min	[M+H] + =1048.61	[M+H] + =1048.24
					[M+2H] 2+ =525.30
	II	m = 2	n = 3	3.67 min	[M+H] + =1048.61	[M+H] + =1048.24
					[M+2H] 2+ =525.30
	III	n = 3	m = 2	3.81 min	[M+H] + =1048.61	[M+H] + =1048.24
					[M+2H] 2+ =525.30
	IV	n = 4	m = 1	3.47 min	[M+H] + =1048.91	[M+H] + =1048.24
					[M+2H] 2+ =525.40
	V	m = 4	n = 1	3.68 min	[M+H] + =1048.61	[M+H] + =1048.24
					[M+2H] 2+ =525.30
	VI	m = 3	n = 2	3.62 min	[M+H] + =1048.71	[M+H] + =1048.24
					[M+2H] 2+ =525.31
	VII	m = 2	n = 3	3.99 min	[M+H] + =1048.61	[M+H] + =1048.24
					[M+2H] 2+ =525.30
	VIII	m =1	n = 4	3.79 min	[M+H] + =1048.71	[M+H] + =1048.24
					[M+2H] 2+ =525.31
	IX	n = 2	m = 2	3.51 min	[M+H] + =1034.62	[M+H] + =1034.24
					[M+2H] 2+ =518.71
	X	m = 2	n = 2	3.73 min	[M+H] + =1034.91	[M+H] + =1034.24
					[M+2H] 2+ =518.61

(*) solvent system: A 0,1 % TFA in H 2 O; B 0,1% TFA in ACN; gradient 10-60% B in 5 min. Flow 0.6 ml/min.

Table 7 .

 7 Chemical data of MTII an MTII linear precursor.

	Peptide	Rt (*)UPLC	MS + (ESI)m/z
			found	calculated
	MTII Linear precursor	2.88	[M+H]+=522.11 [M+H]+=1043,55	[M+H]+=1043,22
	MTII	1.79	[M+2H] 2+ =513.83 [M+H] + =1025,71	[M+H] + =1025,32
	(*) solvent system: A 0,1 % TFA in H 2 O; B 0,1% TFA in ACN; gradient 10-60% B in
	5 min. Flow 0.6 ml/min.

Table 8 .

 8 Adenylate cyclase activity (EC50) of linear precursor (I' ÷ X') compared with MTII.

Table 9 .

 9 Adenylate

cyclase activity (EC 50 ) cyclic analogs (I-X) compared with MTII

Table 10 .

 10 characterized of DEHP metabolites by RP-UPLC.

	metabolites	Rt (min)*	[M+H] +
	MEHP	3.54	277.3
	5-oxo-MEHP	2.86	291.3
	5-cx-MEPP	2.81	309.3
	5-OH-MEHP	2.82	295.3
	6-OH-MEHP	2.84	295.3
	*solvent system: A 0,1% TFA in H 2 O; B 0,1% TFA in CH 3 CN.

Table 11 .

 11 Efficiency

		of the solid phase extraction procedure of 5-OH-MEHP, 6-OH-MEHP, MEHP,
	5-cx-MEHP, 5-oxo MEHP from human urine spiked samples.
	concentration	5-OH-MEHP	6-OH-MEHP	MEHP	5-cx-MEHP	5-oxo-MEHP
	added μg/mL	% Recovery	%Recovery	%Recovery	%Recovery	%Recovery
		mean ± RSD	mean ± RSD	mean ± RSD	mean ± RSD	mean ± RSD
	2,50	95.2 ± 4.5	96.6 ± 5.2	96.1 ± 6.2	96.0 ± 5.3	94.9 ±4.3
	1,00	97.4 ± 3.9	97.8 ± 6.4	98.8 ± 6.3	96.8 ± 7.1	97.8 ± 6.6
	0,25	97.6 ± 3.8	98.1 ± 5.8	94.1 ± 6.1	98.1 ± 4.1	97.1 ± 4.8
	n = 3; RSD = relative standard deviation (%).

Table 12 .

 12 Auxological parameters of obese patients and controls

	Parameter	Obese	Controls
		(mean ± SEM)	(mean± SEM)
	Number	42	39
	Males/Females	20/19	23/16
	Prepubertal/Pubertal	9/30	10/29
	Chronological age	12,23 ± 0,45	12,52± 0,63
	Height SDS	0,94 ± 0,17	-0,78± 0,26
	Genetic target SDS	-0,17 ± 0,16
	BMI SDS Cole	3,34 ± 0,06	1,74± 0,21
	Waist circumference	106,8 ± 2,72
	Waist circumference/	0,68 ± 0,01
	height ratio
	Chronological age onset	4,7 ± 0,5	NO
	of obesity
	Birth weight	3,4 ± 0,09	3,09± 0,11

All the patients and healthy controls were subjected to the oral glucose tolerance test (OGTT). Before the test glycemia, insulin, triglycerides, total cholesterol and High-

Table 13 .

 13 Biochemical data, and fasting indices of insulin-resistance and calculated from the OGTT

	Parameter	Obese	Controls
		(mean± SEM)	(mean± SEM)
	Insulin	15,53 ± 2,02	13,03± 1,29
	Glucose	81,97 ± 1,07	81,63± 0,82
	FGIR	7,42 ± 0,67*	16,34±3,36
	HOMA-IR	3,16 ± 0,4 *	2,04±0,26
	QUICKI	0,34 ± 0,01	//
	Insulinogenic Index	0,19 ± 0,03	0,12±0,01
	WBISI	3,57 ± 0,28	//
	AUCG	238,98 ± 5,39	//
	AUCI	228,44 ± 18,58	//

Table 14 .

 14 Concentrations

		of DEHP metabolites in non-insulin resistant and
	insulin-resistant obese patients compared to healthy controls (μg/ml)
	Metabolites	Non insulin-resistant	insulin-resistant Controls
	MEHP	0.08[0.00-0.23]	0.14[0.00-0.36]	0.06[0.13-0.16]
	5-cx-MEHP	0.55[0.07-1.15]	0.45[0.25-1.11]	-
	5-OH-MEHP 0.38[0.17-2.05]	0.73[0.00-1.52]	0.18[0.03-0.69]*
	5-oxo-MEHP 0.32[0.15-0.72]	0.24[0.05-0.55]	0.18[0.26-0.51]*
	6-OH-MEHP 0.67[0.40-1.36]	0.82[0.27-0.88]	0.36[0.00-0.81]
		.* p<0.05 versus all obese subjects

Table 15 .

 15 ROC curve for di(2-ethylhexyl)phthalate secondary metabolites urinary excretion and infantile autism.

	Urinary	Cut-	AUC±SE	95%	P-	Sens	Spec.	+PV	-PV
	phthalates	off		CI	value	(%)	(%)	(%)	(%)
	5-OH-	>.177	.638±	.531-	.0154	52.1	75.6	69.4	59.6
	MEHP		.057	.735
	5-oxo-MEHP	>.142	.666±	.561-	.0028	46.0	91.1	85.2	60.3
			.055	.759
	MEHP	>.01	.631±	.524-	.0233	79.2	44.2	61.3	65.5
			.058	.730
	ALL	>.724	.671±	.568-	.0021	39.2	97.8	95.2	58.7
			.055	.764

AUC: Area under the Curve; SE: Standard error; 95% CI: 95% confidence intervals for the Area under the Curve; Sens.: sensitivity: spec.: specificity; +PV: positive predictive value; -PV: negative predictive value; ALL: total sum of all the examined di(2-ethylhexyl)phthalate secondary metabolites (5-0H-MEHP + 5-oxo-MEHP + 6-OH-MEHP+ MEHP).

Table 16 .

 16 Comparisons of urinary excretion of secondary metabolites for di(2-ethylhexyl)phthalate in autistic patients (N = 48) vs. healthy controls (N = 45) in (μg/mL).

		ASDs	Healthy Controls	P-value*
	5-OH-MEHP	0.18 (0.037-0.399)	0.04 (0-0.124)	0.0224
	5-oxo-MEHP	0.096 (0.04-0.17)	0.04 (0.015-0.079)	0.005
	6-OH-MEHP	0.017 (0.01-0.034)	0.019 (0-0.043)	0.9305
	MEHP	0.055 (0-0.11)	0.028 (0-0.059)	0.0312
	Values are medians (95% confidence intervals of the median); * Mann-Whitney test for independent
	samples; statistically significant differences are highlighted in bold.

Table 17 .

 17 Comparisons of urinary excretion of secondary oxidative metabolites of DEHP in ASDs with n=10 patients with Rett syndrome in (μg/mL).

	Urinary Phthalate Metabolite	ASDs	Rett syndrome	p-value
	5-oxo-MEHP	0.0997 (0.0397-0.1708)	0 (0 -0.0755)	0.0344
	5-OH-MEHP	0.1740 (0.0311-0.35)	0 (0 -0.0091)	0.0601
	6-OH-MEHP	0.0107 (0-0.0261)	0 (0 -0.0832)	0.2246
	MEHP	0.0287 (0.0159-0.0847)	0 (0 -0.1344)	0.7098
	(data are expressed as medians and 95% confidence intervals; phthalate metabolite concentrations
	are adjusted to creatinine and are calculated per 100 mg of urinary creatinine)
	Therefore, 5-oxo-MEHP urinary excretion appears to be lower in Rett syndrome (p=
	0.0344), whereas 5-OH-MEHP (p=0.0601), 6-OH-MEHP (p= 0.2246), and MEHP

(p= 0.7098) excretion is comparable to that observed in healthy controls, despite the heavy medications needed in this multi-system genetic disease. These results indicate that medications are likely not the main cause for the increased urinary phthalate excretion evidenced in ASDs, while suggesting the likely intervention of environmental factors in the pathogenesis of this pervasive development disorder.

As ASDs are disorders of brain development, any factors which regulate brain development and are known to be altered in autism should be considered as possibly contributing to the phenotype.

It is interesting to note that in 87% of the ASDs urine samples we detected MEHP and 5-oxo-MEHP levels higher than the levels recently published, by

Cho et al. 188

. In that study

Cho et al.

report a possible relationship between environmental phthalate exposure (external contamination) and the intelligence level of 667 School-Age children randomly recruited from nine elementary schools in five South Korean cities. The reported geometric mean was 21.3 μg/L for MEHP, and 18.0 μg/L for 5oxo-MEHP, while in our study we found 55 μg/L and 96 μg/L, respectively.

  are able to efficiently discriminate ASDs from Healthy Controls. Nevertheless, it has already been reported that the most oxidized DEHP metabolites have longer half-life of excretion in urine 164 . Therefore, it is possible that environmental factors and oxidative stress unbalance may interact in leading to increased ASDs risk, although further investigation is needed in dissecting this interaction in vivo. A link between oxidative brain damage and ASDs has been previously reported by several authors221,222,223 . ASDs 226 .Transient intrauterine deficits of thyroid hormones have been shown to result in permanent alterations of cerebral cortex similar to those found in brains of children with autism 226 . As a consequence, the current surge of this disease could be related to transient maternal hypothyroxinemia resulting from exposure to antithyroid environmental contaminants.

	Considering that urinary 5-oxo-MEHP reported in this study as increased in ASDs
	compared to healthy controls, we can state that our findings are in agreement with an
	increased OS environment. Moreover, phthalates are known as EDs but it is unclear
	how they could be involved in etiology of neurodevelopment disorders. As described
	before, phthalate metabolites activate α-PPAR that may alter lipid metabolism in the
	brain. Recently signal transduction pathway of α-PPAR was correlated to
	progression of neurodegenerative and psychiatric diseases. Phthalates also interfere
	with the thyroid hormone system by inducing hypothyroidism. Recent studies
	correlated utero hypothyroxinemia to decreased intellectual capacity, mental
	retardation, and

Over the last years, De Felice et al. has demonstrated that the biological "duo" hypoxia-oxidative stress (OS) is a key player in modulating genotype-phenotype expression in Rett syndrome, a well established genetic form of ASD 224 .

Moreover, in previous reports, a correlation between OS and ASDs has been widely explored by measuring different molecules, possibly coming from oxidative pathway as metabolic biomarkers of OS, in biological fluids

225

. We could speculate that in susceptible subjects, DEHP exposure, when finding OS conditions, can lead to in vivo accumulation of toxic metabolites (i.e., the oxidized phthalates metabolites), possibly acting as EDs.

  1 . Solutions for Kaiser test were prepared as follows:

	The products were characterized by nuclear magnetic resonance (NMR), infrared
	spectra (IR), liquid chromatography-electrospray ionization/ mass spectrometry (LC-
	ESI-MS), and Ultra Performance Liquid Chromatography (UPLC).
	1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, and 200 and 50 MHz
	respectively, on a Varian spectrometer in deuterated solutions and are reported in
	parts per million (ppm), with solvent resonance used as reference.
	Infrared spectra were recorded on a Perkin Elmer mod. BX II FT-IR spectrometer.
	Products were analyzed and characterized by ACQUITY UPLC (Waters Alliance
	2695 Waters Corporation, Milford, Massachusets) coupled to UV diode array Waters
	2996 and to a single quadrupole ESCI-MS (Micromass ZQ) using a C18
	Phenomenex Kinetex (100x3mm, 2.6 µ, C18, 100Å) at 30 °C, with a flow rate of 0.6
	mL/min. The solvent systems used were A (0.1% TFA in H 2 O) and B (0.1% TFA in
	CH Cartridge for urine pretreatment (SPE) were purchased from Waters (Oasis HBL
	3mL/60 mg). For creatinine measurement, a Beckman Synchron AS/ASTRA clinical
	analyzer (Beckman Instruments, Inc., Brea, CA) was used. We used value
	(micrograms per liter)/creatinine (grams per liter) for dilution correction in the
	ninhydrine (5 g) in EtOH (100 ml), Phenol (80 g) in EtOH (20 ml), KCN 1 mM (2 analyses. The HPLC quantitative analyses were carried out using an high-
	ml) in pyridine (98 ml). performance liquid chromatography tandem mass spectrometry RP-HPLC -ESI-MS
	(Waters, Alliance 2695, Waters, Micromass ZQ) equipped with phenyl column

122

[ 1 ] Still, W. C.; Kahn, M.; Mitra, A. J.

Org. Chem. 1978, 43, 2923-2925.

3 CN). Peptides were analyzed by analytical RP-HPLC (Alliance, model 2695 equipped with a diode array detector, Waters) using a Jupiter C18 (5 μm, 250 × 4.6 mm) column (Phenomenex) at 1 mL/min. The solvent systems used were A (0.1% TFA in H 2 O) and B (0.1% TFA in CH 3 CN). Peptides were purified by semipreparative RP-HPLC on a Supelco C18 180 Å (250 × 10 mm, 5 μm) column (Sigma Aldrich, St. Louis, MO, USA), using methods and solvent system as reported. Fractions were analyzed by ACQUITY UPLC (Waters Corporation, Milford, Massachusetts) coupled to a single quadrupole ESI-MS (Micromass ZQ) using a 2.1 x 50 mm 1.7 μm ACQUITY BEH C18 Purification of Nickel complex [Gly-Ni-BPB], of azido and alkynyl amino acids and of DEHP metabolites were performed as reported by flash chromatography. (Betasil, 5 μm, 50 mm x 3 mm, Keystone Scientific, Bellefonte, PA) and with a Waters 2996 Photodiode Array Detector.

Table 18 .

 18 RT and MW-assisted SPPS deprotection and coupling protocols used for the PTHrP(1-34)NH 2 synthesis.

			MW-assisted Synthesis		RT-Syntesis
	Step	Temp(°C) MW Power (W) Time(min) Temp(°C) MW Power (W) Time(min)
	1 st deprotection	75°	35 W	0,5	20	5
	2 nd deprotection	75°	60 W	3	20	10
	Coupling	75°	30 W	5	20	20

  2, it was used TFA/TIS/water solution(95:2.5:2.5

	5.4 General procedure for solid-phase extraction SPE
	•	SPE were performed on RP-C18 LiChroprep columns, with CH 3 CN in H 2 O
		as eluents. Main steps are reported here:
	•	Column washings with MeOH (3 column volumes) and CH 3 CN (3 column
		volumes)
	•	Column conditioning with H 2 O (3 column volumes)
	•	Dissolving the peptide in H 2 O (1 column volume), checking the pH that
		should be neutral.
	•	Adsorbing peptide solution on silica for 3 times
	•	Eluting with H 2 O (3 column volumes).
	•	Eluting with 5%, 10%, 15%, 20% of H 2 O/CH 3 CN (column volume for each
		concentration), and 100% of CH 3 CN.

v/v/v) as cleavage mixture. For MTII analogues (I'-X'), it was used TFA /thioanisole/1,2-ethanedithiol/phenol/H 2 O solution (94:2:2:2:2 v/v/v/v/v). The cleavage was maintained for 3 h with vigorous stirring at RT. Resins were filtrated and washed with TFA. After partial evaporation under nitrogen flux, filtrates were precipitated with cold diethyl ether, separated by centrifugation, dissolved in H 2 Ο and lyophilized with an Edwards apparatus, model Modulyo.

Lyophilized crude peptides were purified by semipreparative RP-HPLC using methods and solvent system as reported. Fractions were analyzed by ACQUITY UPLC (Waters Corporation, Milford, Massachusetts) coupled to a single quadrupole ESI-MS (Micromass ZQ) using a 2.1 x 50 mm 1.7 μm ACQUITY BEH C18. •

Fractions were checked by analytical UPLC-ESIMS, and then lyophilized.

  ). ESI-MS: m/z calcd for C 25 H 24 N 2 O 2 [M + H] + : 385,18; found 385,2. 1 H NMR (CDCl 3 , 400 MHz): δ 11.52 (s, 1H, NH), 8.56 (d, 1H, J= 8.4 Hz, Bn), 7.79-7.36 (m, 9H, Bn), 7.15 (m, 4H, Bn), δ A = 3.92, δ B = 3.59 (syst AB, 2H, J AB = 12.8 Hz, CH 2 Bn), 3.32 (dd, 1H, J α,β = 4.4 Hz, J α,β' =10.0 Hz, H α ), 3.22 (dd, 1H, J δ,δ' = J δ,γ = 6.4 Hz, H δ ), 2.41 (dd, 1H, J β,β' = 8.8 Hz, J β,γ = 16 Hz, H β' ), 2.26 (ddd, 1H, J δδ' = 6.4 Hz, J δγ =12.8 Hz, J δγ' = 22 Hz H δ' ), 1.96 (ddd, 1H, J β,β' = 8.8 Hz, J β,γ = 4.4 Hz, J βγ' = 16.4 Hz, H β ), 1.85-1.76 (m, 2H, H ,γ ,H ,γ' ).

[START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF]

C NMR (CDCl 3 , 100 MHz): δ 198.03 (Ph-CO-Ph), 174.64 (COOH), 139.16,

  CH 2 Bn),3.51-3.31 (m, 2H, H αpro ), 2.63-2.41 (m, 3H, H δpro , H αα'Gly ), 2.13-1.91 (m, 4H, H β'pro , H δ'Pro , H βPro , H ,γPro , H ,γ'Pro ). 13 C-NMR (CDCl3, 50MHz): δ 181.19 (CO Gly ), 177.18 (CO-N-Ph), 171.49 (C=N

1 H-NMR (CDCl 3 , 200MHz): δ 8.27 (d, 1H, J= 8.8 Hz, Bn), 8.07 (d, 1H, J= 7.2 Hz, Bn), 7.51-7.30 (m, 6H, Bn), 7.24-6.69 (m, 6H, Bn), δ A = 4.48, δ B = 3.69 (syst AB, 2H, J AB = 6.2 Hz,

Alkylation of the Gly-Ni-BPB complex with bromoalkynes. General procedure

  To a stirred mixture of Gly-Ni-BPB(1.56 g, 3.14mmol, 1 eq.) in anhydrous DMF (4 ml) were added, under N 2 , finely powdered NaOH(753 mg, 18.84 mmol, 6 eq., pH 12) and bromoalkyne (4.71 mmol, 1.5 eq.). The reaction was checked by TLC (DCM/aceton 2:1, detected by UV 254 nm). After 20 min., the reaction mixture was treated with 0.1 M HCl (100 ml) and the red product extracted with CH 2 Cl 2 (4 × 40 mL), dried over MgSO 4 , and the solvent removed under vacuum. The crude was purified by flash chromatography on silica gel (CH 2 Cl 2 -Me 2 CO 2/1) affording the product as a red amorphous solid.

	Ar),
	69.96 (C αPro ), 63.23 (CH 2 Bn), 61.38 (C αGly ), 57.60 (C δPro ), 30.90 (C βPro ), 23.89
	(C γPro ).
	6.9 6.9.

1 Alkylation of the Gly-Ni-BPB complex with 6-bromohex-1-yne (n=4)

	Yield 59%. TLC: R f 0.63 (DCM/ Me 2 CO 2:1;
	detected by UV 254 nm). ESI-MS: m/z calcd for
	C 33 H 34 N 3 NiO 3 [M + H] + : 578.2; found 578.4. 1 H
	NMR (CDCl 3 , 400 MHz): δ 8.12 (d, 1H, J = 8.4
	Hz, ArH), 8.04 (d, 2H, J = 7.6 Hz, ArH), 7.50-
	7.44 (m, 3H, ArH), 7.33 (t, 2H, J = 7.6 Hz, ArH),
	7.26-7.10 (m, 3H, ArH), 6.92 (d, 1H, J = 7.6 Hz, ArH), 6.66-6.60 (m, 2H, ArH),
	4.42 (d, 1H, J = 12.8 Hz, PhCH 2 ), 3.91 (dd, 1H, J 1 = 3.4 Hz; J 2 = 7.8 Hz, Pro α-H),
	3.57 (d, 1H, J = 12.8 Hz, PhCH 2 ), 3.52-3.43 (m, 3H), 2.79-2.73 (m, 1H), 2.56-2.46
	(m, 1H), 2.36-2.30 (m, 1H), 2.16-2.02 (m, 4H), 1.97-1.88 (m, 1H), 1.91 (t, 1H, J =
	2.4 Hz, C≡CH), 1.76-1.61 (m, 2H), 1.44-1.32 (m, 2H). 13 C NMR (CDCl 3 , 50 MHz):

δ 180.32 (O-C=O), 179.30 (N-C=O), 170.46 (C=N

  18.14 (C≡C-C). 120.69 (18 × Ar), 83.86 (HC≡C), 70.28 (HC≡C), 70,10 (Pro C-α), 68.83 (Bn CH 2 ), 63.13 (C-COO), 56.99 (Pro C-δ), 34.53 (Pro C-β), 30.76 (C≡C-CH 2 -C), 27.93 [C≡C-(CH 2 ) 3 -C], 24.30 (Pro C-γ), 23.67 ( J = 12.6 Hz, PhCH 2 ), 3.86 (dd, 1H, J 1 = 3.4 Hz; J 2 = 8.8

	6.9.2 Alkylation of the Gly-Ni-BPB complex with 5-bromopent-1-yne (n=3)
	Yield 56%. TLC: R f 0.58 (DCM/ Me 2 CO 2:1; detected
	by UV 254 nm). RP-UPLC: R t 3.59 min. (50-100%
	of B in 5 min). ESI-MS: m/z calcd for C 32 H 32 N 3 NiO 3
	[M + H] + : 564.2; found 564.5. 1 H NMR (CDCl 3 , 400
	MHz): δ 8.12 (d, 1H, J = 8.4 Hz, ArH), 8.04 (d, 2H, J
	= 7.6 Hz, ArH), 7.50-7.41 (m, 3H, ArH), 7.33 (t, 2H,
	J = 7.6 Hz, ArH), 7.25-7.10 (m, 3H, ArH), 6.95 (d, 1H, J = 7.6 Hz, ArH), 6.64-6.60
	(m, 2H, ArH), 4.43 (d, 1H,

Hz, Pro α-H),

3.56 (d, 1H, J = 12.6

Hz, PhCH 2 ),

3.52-3.43 (m, 3H)

, 2.79-2.72 (m, 1H), 2.54-2.48 (m, 1H), 2.35-2.20 (m, 1H), 2.20-2.05 (m, 3H), 2.05-1.95 (m, 2H), 1.93 (t, 1H, J = 2.6 Hz, C≡CH), 1.78-1.70 (m, 2H). 13 C NMR (CDCl 3 , 100 MHz): δ 180.37 (O-C=O), 179.15 (N-C=O), 170.54 (C=N

3 Alkylation of the Gly-Ni-BPB complex with 4-bromo-1-butyne (n=2)

	α-H), 3.52 (d, 1H, J = 12.4 Hz, PhCH2), 3.50-3.42 (m, 3H), 2.8-2.7 (m, 1H), 2.48-
	2.23 (m, 2H), 2.20-2.0 (m, 1H), 2.00-1.95 (m, 2H), 1.89 (t, 1H, J = 2.2 Hz, C≡CH),
	1.75-1.67 (m, 2H).
			N N N N	Ni Ni	N O N O		O O	H H
		O O				C 6 H 5 C 6 H 5	(18) (18)
							(18
	× Ar C), 83.47 (HC≡C), 70.27 (HC≡C), 69.99 (Pro C-α), 69.06 (Bn CH 2 ), 63.11 (C-
	COO), 57.05 (Pro C-δ), 34.53 (Pro C-β), 30.75 (C≡C-CH 2 -C), 24.36 [C≡C-(CH 2 ) 2 -
	C], 23.73 (Pro C-γ), 18.15 (C≡C-C).
	6.9.Yield 32%. TLC: R f 0.62 (DCM/Me 2 CO 2:1;
	detected by UV 254 nm). RP-UPLC: R t 2.56 min.
	(50-100% of B in 5 min). ESI-MS: m/z calcd for C 31 H 30 N 3 NiO 3 [M + H] + : 550.2; found 550.19. 1 H	N N N N	Ni Ni	N O N O	O O	H H
	NMR (CDCl 3 , 400 MHz): δ 8.04-7.94 (m, 3H, J =	O O			C 6 H 5 C 6 H 5
	8.4 , ArH), 7.48-7.35 (m, 3H, ArH), 7.3-7.27 (m,

2H, ArH),

7.25-7.10 (m, 3H, ArH), 6.89 (d, 1H, J = 7.2 Hz, ArH), 6.60-5.50 (m, 2H, ArH), 4.39 (d, 1H, J = 12.4 Hz, PhCH2), 3.79 (dd, 1H, J1 = 3.6 Hz; J2 = 8.4 Hz, Pro

  CH 3 CH 2 ), 23.9 (CH 3 CHOH), 24.7 (CH 3 CH 2 ), 27.2 (CH 2 CH 2 CHOH), 35.7 (CHCH 2 CH 3 ), 39.3 (CH 2 CHOH), 66.7 (CHOH), 69.5 (OCH 2 ), 128.8-131.8 (aromatic C), 168.4 (COOCH 2 ), 170.6 (COOH).

	d 11.1 (
	1 H NMR d 0.92 (3H, t, CH 3 ), 1.3 (2H, m, CH 2 CH 3 ), 1.69 (3H,
	m, CHCH 2 CH 2 CO), 2.19 (3H, s, COCH 3 ), 2.52 (2H, m, CH 2 CO), 4.2 (2H, m,
	OCH 2 ), 7.64 and 7.8 (4H, m, aromatic CH). 13 C NMR d 11.1 (CH 3 CH 2 ), 23.7
	(CH 3 CH 2 ), 24.7 (CH 2 CH 2 CO), 30.0 (aliphatic CH), 38.2 (CH 3 CO), 40.6 (CH 2 CO),
	63.5 (OCH 2 ), 128.7-131.5 (aromatic C),

1

  Laboratory of Peptide & Protein Chemistry & Biology, Polo Scientifico e Tecnologico, University of Firenze, 50019 Sesto Fiorentino, Italy; 2 Dipartimento di Chimica "Ugo Schiff", Polo Scientifico e Tecnologico, University of Firenze, via della Lastruccia 13, 50019 Sesto Fiorentino, Italy; 3 Laboratoire de Synthèse Organique Sélective et Chimie bioOrganique EA4505, University of Cergy-Pontoise 4 Dipartimento di Scienze Farmaceutiche, via Ponte Don Melillo 11C, 84084 Salerno, Italy; 5 Dipartimento di Scienze Farmaceutiche, University of Firenze, via Ugo Schiff 3, Polo Scientifico e Tecnologico, 50019 Sesto Fiorentino, Italy 6 Laboratory for Translational Research, Harvard Medical School, One Kendall Square, Building 600, Cambridge, Massachusetts 02139, USA.

, Bernasconi S

  Data suggest that endocrine-disrupting chemicals, as phthalates, found in a variety of household products, may influence mechanisms related to obesity. In vitro studies have shown that these chemicals may interact with PPARs and play a key role in the differentiation of adipocytes and deposition of triglycerides in adipose tissue.The aim was to evaluate possible associations among urinary phthalate secondary metabolites and age, gender, pubertal development, age at onset of obesity, BMI, waist circumference, indexes of insulin sensitivity and metabolic syndrome in obese children.

	1 University Hospital of Parma, Italy, 2 Laboratory of Peptide & Protein Chemistry & Biology,
	University of Florence, Italy and Laboratoire SOSCO -EA4505 Université de Cergy-Pontoise,
	France; 3 Department of Chemistry "Ugo Schiff", University of Florence, Italy, 4 Bracco Foundation,
	Milan, Italy
	We studied 39 subjects (20 M, 19 F, age:12,23 ±0,45 yr, BMI SDS:3,34±0,06).
	Whole body insulin sensitivity index(WBISI), Areas Under the Curve of glucose(AUCG)

Table 1 .

 1 Fmoc/tBu RT-SPPS of PTHrP(1-34)NH 2 : list of fragments produced by MW-assisted mini-cleavages of intermediate resin-bound peptides

	Amount of	deletion	sequences (%)	7	2	5	8		6	39	51	5		55	3	12
		Missing amino acid residues	from deletion sequences	Leu 27	His 25	Leu 27 His 25	Leu 27		Leu 27	Arg 19 Arg 20	Arg 19 Arg 20 Arg 21	Leu 27		Gln 16 /Lys 13	Leu 27 Gln 16 /Lys 13	Gly 12 -Lys-Ser-Ile-Gln-Asp 17
	Deletion sequences	Qtof MS (ESI+)	(m/z) found	753.41 [M + H] +	1003.95 [M + H] +	890.93 [M + H] +	1434.44 [M + H] +		952.19 [M + 2H] 2+ 852.72	[M + 2H] 2+	774.73 [M + 2H] 2+	1322.29 [M + 2H] 2+		1315.26 [M + 2H] 2+	1258.81 [M + 2H] 2+	1065.43 [M + 2H] 2+
	Intact sequences	Qtof MS (ESI+)	(m/z) found	866.39 [M + H] +	1140.90 [M + H] +		1547.41 [M + H] +		1008.69 [M + 2H] 2+			1379.24 [M + 2H] 2+
	Calculated	monoisotopic	mass	865.51	1139.62		1546.84		2015.15			2756.55
			Sequence	H-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-NH 2	H-His-His-Leu-Ile-Ala-Glu-Ile-His-Thr-Ala-NH 2		H-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu-Ile-His-	Thr-Ala-NH 2	H-Arg-Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-	Ala-Glu-Ile-His-Thr-Ala-NH 2		H-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-	Arg-Arg-Phe-Phe-Leu-His-His-Leu-Ile-Ala-Glu-	Ile-His-Thr-Ala-NH 2
	Analyzed	resin-bound	sequences	27-34	25-34		22-34		19-34			12-34

Table 2 .

 2 Fmoc

/tBu RT and MW-assisted SPPS deprotection and coupling protocols used for the synthesis of PTHrP(1-34)NH 2

Table 3 .

 3 Yield of PTHrP(1-34)NH 2 obtained from the RT versus MW-

	assisted SPPS
		Purity of crude	Yield of >95%
	SPPS strategy	peptide (%)	pure peptide
	RT	35	4.4% (18 mg)
	MW-assisted	77	6.3% (27 mg)

  • C. The product was collected by centrifugation and directly subjected to UPLC-ESI-MS analysis (see Supporting Information).

	HPLC Analysis of PTHrP(1-34)NH 2
	Crude PTHrP(1-34)NH 2 obtained from both the RT and MW-
	assisted SPPSs were analyzed by analytical RP-HPLC (Alliance 2695
	HPLC system equipped with a 2996 photodiode array detector,
	Waters (Milford, MA, USA)) using a Jupiter C18 (5 µm, 250×4.6 mm)
	column (Phenomenex, Torrance, CA, USA) at 1 ml/min. The
	solvents used were A (0.1% TFA in H 2 O) and B (0.1% TFA in
	ACN).

RP-HPLC Semi-preparative of PTHrP(1-34)NH

2

Lyophilized crude peptide was prepurified by solid-phase extraction with an RP-18 LiChroprep silica column from Merck wileyonlinelibrary.com/journal/jpepsci Copyright c 2011 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2011; 17: 708-714

Table 1 .

 1 Biological activity (EC 50 ) of the MTII peptide analogs towards the principal murine melanocortin receptors. *Lin stands for linear precursor.

		(tb4 n ,1 m )MTII		(tb1 n ,4 m )MTII
			mMC1R mMC3R mMC4R mMC5R
		Peptide	EC 50	EC 50	EC 50	EC 50
			(nM)	(nM)	(nM)	(nM)
	I II III IV I' II' III'	MTII (tb4 1 ,1 4 )MTII (tb1 4 ,4 1 )MTII (tb4 2 ,1 3 )MTII (tb1 3 ,4 2 )MTII Lin[Nle 5 (G-N 3 ),Pra 10 ]MTII* Lin[Pra 5 ,Nle 10 (G-N 3 )]MTII Lin[hAla 5 (J-N 3 ),Nva 10 (G-yl)]MTII	---0.11 0.51 1.04 0.58 1.25	0.33 3.14 3.70 0.59 2.95 3.67 4.13 8.57	0.04 1.62 0.58 0.15 0.36 0.71 0.43 1.48	0.27 1.93 1.76 0.47 2.30 6.62 3.91 12.4
	IV'	Lin[Nva 5 (G-yl),hAla 10 (J-N 3 )]MTII	2.11	9.90	1.35	11.8

Table 1 .

 1 Room temperature-vs MW-assisted synthetic approach

	SPPS strategy	Purity of crude peptide (%)	% Yield of pure >95% peptide (mg)
	Room temperature	35	4.4 (18 mg)
	MW-assisted	77	6.3 (27 mg)

Table 3

 3 Comparisons of urinary excretion of secondary oxidative metabolites of DEHP in ASDs with n510 patients with Rett syndrome in mg/ml Data are expressed as medians and 95% CI; phthalate metabolite concentrations are adjusted to creatinine and are calculated per 100 mg of urinary creatinine. Statistically significant differences are highlighted in bold.

	Urinary phthalate metabolite ASD Rett	Syndrome	P-value
	5-Oxo-MEHP	0.0997 (0.0397-0.1708)	0 (0-0.0755)	0.0344
	5-OH-MEHP	0.1740 (0.0311-0.35)	0 (0-0.0091)	0.0601
	6-OH-MEHP	0.0107 (0-0.0261)	0 (0-0.0832)	0.2246
	MEHP	0.0287 (0.0159-0.0847)	0 (0-0.1344)	0.7098

Silva M.J., Barr D.B., Reidy J.A., Kato K., Malek N.A., Hodge C.C., et al. Arch Toxicol,

Where 'n' is the number of mols of acid in the system

E 2012 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

Acknowledgements

Chaire d'Excellence ANR 2009-2013 and Fondazione Ente Cassa di Risparmio di Firenze are gratefully acknowledged. Acknowledgments The present work has been supported by the Agence Nationale de la Recherche chaire d'Excellence 2009-2013 (France). Acknowledgments Ente Cassa Risparmio di Firenze and ANR Chaire d'Excellence 2009-2013 PepKit (France) are gratefully acknowledged. February 2012 ACKNOWLEDGEMENT We gratefully acknowledge Dr R. Zannolli (Dipartimento Materno-Infantile, University of Siena, Siena, Italy) for collecting HC urine samples.

FUNDING This work was partly funded by ANR Chaire d'Excellence 2009-2013 (France) and Ente Cassa Risparmio di Firenze. Re ´gion Ile-de-France supported the 'Cotutelle internationale de the `se de doctorat' of C. Testa (Ecole doctorale University of Cergy-Pontoise, France and Dottorato in Scienze Chimiche of University of Florence, Italy).

Mono(5-carboxy-2-ethylpentyl) 1,2-benzenedicarboxylate (5-cx-MEHP)

Jones'reagent (1 M, 2 mL), prepared from CrO 3 (6.7 g) in 96% H 2 SO 4 (6 mL) (2 COOH), 7.8-7.5 (4H, m, aromatic CH). [START_REF] Loffredo | Microwaveassisted solid-phase peptide synthesis at 60 • C: alternative conditions with low enantiomerization[END_REF] C NMR d 10.9 (CH 3 ), and is not a true hormone. It is synthesized in cartilage and in many more tissues than parathyroid hormone, and its secretion is not regulated by serum calcium. It mimics the actions of PTH as a result of its structural homology and its ability to bind and signal via the PTH/PTHrP receptor in bone and kidney [1].

Based on the amino acid sequence, it was deduced that the first 34 amino-terminal amino acids should be sufficient for biological activity. Availability of highly purified parathyroid polypeptide and active synthetic fragments made it possible to develop radioimmunoassay.

Considering the presence of sterically hindered amino acid residues in the sequences, such as Arginine and Phenylalanine between position 19-23, and the length of the peptide, we optimised the synthesis of PTHrP (1-34) The application of microwave irradiation in peptide chemistry has been reported in several publications, most of which describe case studies of successful syntheses of difficult peptides [1,2,3]. Our herein reported study compares the RT with MW-assisted SPPS of the 1-34 Nterminal fragment of parathyroid hormone-related peptide (PTHrP) using the same instrument (Liberty™, CEM), and monitors the synthesis by UPLC-ESI-MS of microwaveassisted mini-cleaved fragments of the growing peptide chain (Figure 1). Identification of some deletion sequences was helpful to recognize critical couplings and helped to guide the introduction of microwave irradiations to these stages.

PTHrP is an autocrine, paracrine and intracrine regulator of processes such as endochondrial bone formation and epithelial-mesenchymal interactions during the development of mammary glands. In the past, this sequence was the subject of numerous structure-activityconformation relationship studies [4,5]. Importantly, the proximity of clusters of arginines, sterically hindered, and hydrophobic amino acids sequences represent a synthetic challenge that was the subject of our comparative study reported herein.

Figure 1. General SPPS scheme.

Supporting information

Supporting information may be found in the online version of this article. optimal MS parameters were as follows: the source and desolvation temperature were 120 and 400 ˚C respectively;

Proceedings of the

the capillary voltage was 3.24 kV; cone voltage 30 kV, nitrogen gas was used as desolvatation gas and as cone gas as well; the cone gas and the desolvatation flow was 60 and 800 l/h respectively; the collision gas was argon with a flow of 0.60 ml/min. Data were acquired and processed using MassLynx TM software (Waters).

Calibration curves for the quantitative urine analysis were calculated for all analytes plotting peak area average (y) against concentration of standards (x). Five standard solutions (linear range: 2.5-2500 ng/ml) for calibration curve plotting, were prepared for all the metabolites. Curves with correlation coefficients (r 2 ) greater than 0.998 were generated (MEHP, 5-OH-MEHP, 5-oxo-MEHP 0.999 and 6-OH-MEHP 0.998).

Statistical analysis

All variables were tested for normal distribution (D'Agostino-Pearson test) and data were presented as means with 95% CI (confidence interval) for normally distributed variables or medians means and with 95% CI for non-normally distributed data. Differences between groups were evaluated using independentsample t test (continuous normally distributed data), Mann-Whitney rank sum test (continuous non-normally distributed data), x2 statistics (categorical variables with minimum number of cases per cell >5) of Fisher's exact test (categorical variables with minimum number of cases per cell ,5). Associations between variables were tested by unvaried regression analysis. The efficiency of urinary phthalates metabolites in discriminating ASD patients from HCs were evaluated using ROC (receiver operating characteristic) curve analyses. All analyses were considered to be statistically significant for P-values ,0.05. Correction for multiple comparisons was made (Bonferroni's correction). The MedCalc version 9.5.2.0 statistical software package (MedCalc Software) was used.

RESULTS

Among the four metabolites (MEHP and secondary oxidative metabolites) measured in urines of ASDs, we could detect urinary concentration with median values for MEHP (0.055 mg/ml), 5-OH-MEHP (0.18 mg/ml), 6-OH-MEHP (0.017 mg/ml) and 5-oxo-MEHP (0.096 mg/ml). By contrast, in urine from HCs we found the following values for MEHP (0.028 mg/ml), 5-OH-MEHP (0.04 mg/ml), 6-OH-MEHP (0.019 mg/ml) and 5oxo-MEHP (0.04 mg/ml). However, the data illustrated in Table 2 are without urinary creatinine correction. Differences between groups by using creatinine-adjusted data were comparable with those employing raw data (data not shown). The statistical significance of variables was independent of effects of urinary creatinine concentration. In fact, it is well known that urine concentration/dilution can affect the results of measurements of urinary metabolites.

We found detectable levels of MEHP, 5-OH-MEHP and 5oxo-MEHP in 79.2, 52.1 and 46.0% of ASD patients respectively (Table 1). Interestingly, 5-OH-MEHP was the major metabolite in terms of urine concentrations, followed by 5-oxo-MEHP and MEHP. Urinary excretion of 5-oxo-MEHP (P50.005), 5-OH-MEHP (P50.0224) and MEHP (P50.0312) was significantly higher in autistic patients compared with gender-and age-comparable HCs (Table 2). By contrast, 6-OH-MEHP (P50.9305) was not able to discriminate ASDs. High levels of 5-OH-MEHP were detected in 26.6% of HCs. A significant positive correlation between 5-OH-MEHP and 5oxo-MEHP could be observed only in autistic children (r s 50.668, P,0.0001), but not in the control population (r s 50.125, P50.5565) (data not shown). The completely oxidized form of the metabolite pathway, corresponding to 5-oxo-MEHP, had a specificity of 91.1% in identifying autistic children (Figure 2).

The efficiency of urinary secondary metabolites in discriminating ASD patients from HCs was evaluated using ROC curve analyses (Figure 3, Tables 1 and2).