Anna Maria

PhD Cédric Rentier

Prof Solange Lavielle

Prof Claudio Toniolo

Prof Thierry

Dr Olivier Monasson

Dr Elisa Peroni

Mlle Matthaia Ieronimaki

Dr Mr Nicolas Pasternak

Dr Lotfi Tak-Tak

Andrea Bordessa

Mme Mr Simon Gonzalez

Jocelyne Gougerot

Dr Francesca Nuti

Dr Giulia Pacini

Dr Giada Rossi

Dr Feliciana Real-Fernandez

Dr Enrico Koenig

Dr Mario Chelli

Dr Giuseppina Sabatino

Dr Fabio Rizzolo

Dr Dott Francis Ciolli

Dr Chiara Testa

Dr Caterina Tiberi

Margherita Di Pisa

Dott Ssa

Simona Pascarella

Dr Dott Raffaelo Paolini

Alessandro Pratesi

Dott.ssa Martina Trabucco

Martina Crulli

Grazie

Prof Annunziata Lapolla

Piero Traldi

Marco Roverso

Prof Selmi Carlo

PhD Cédric Rentier

Prof Alfonso Carotenuto

Paola Migliorini

I risultati hanno dimostrato

As a project that occupied four years of my life, it would have happened much differently (or not happen at all) without a few people. In the next paragraphs, I will express my gratitude and thoughts to a few of these people, in the same way I worked these last few years: in three

Cédric Rentier, PhD thesis xviii General introduction -1. Autoimmunity: introduction to the immune system Cédric Rentier, PhD thesis 1

General introduction 1. Autoimmunity: introduction to the immune system

Immunity is one of the most wonderful examples of complex machineries merging together chemistry and biology to serve a very simple, yet complex purpose: protecting the organism against any form of external agent.

In the following sections, this thesis will outline the basis of immunity, in order to give a comprehensive overview of notions such as autoimmunity, antigens and antibodies.

1.1. Innate immune system and adaptive immune system Protection against external aggressions, i.e. pathogens, such as bacteria or viruses can be seen as a multilayer system.

The first layer is purely physical. In the case of human beings, the easiest example is the skin, but gastrointestinal system and respiratory airways are protected as well.

The second layer, which intervenes in the case of breaching of the first barrier, is the innate immune system, a general and immediate (but non persistent) response of the organism against pathogens. This system is present in almost all living organisms. Cells of the innate immune response are called leukocytes (or also white blood cells) and can be of a diverse nature: phagocytes (macrophages, neutrophils, monocytes, dendritic cells) absorb pathogens to destroy them, releasing inflammatory mediators, e.g. cytokines, which stimulate other responses from the organism against the potential threat; or natural killer cells, which induce apoptosis in infected cells. The complement system, which is a biochemical cascade of reactions between specific plasma proteins that "complement" the immune system in cleaning, tagging or destroying pathogens, is also a part of the innate immune system.

The third layer, much more specific, is the adaptive immune system. Unlike the innate immune system, it is found exclusively in vertebrates and is highly specific to a given pathogen. Adaptive immunity can create immunological memory after an initial response to a specific pathogen, leading to an enhanced response to subsequent encounters with that pathogen.

The adaptive response mainly involves the clonal expansion of a subtype of leukocytes, namely B and T lymphocytes, which are produced by stem cells in the bone marrow.

T lymphocytes (also called T cells) are characterized by the presence at their surface of T cell receptors, while B cells express B cell receptors. The main difference between T cells and B cells lies in their recognition of foreign objects. B cells can recognize linear or conformational epitopes on their own, while T cells need the assistance of antigen presenting cells. Their mode of action is also different: T cells will either enhance the immune response by cytokines production (T helper cells, also known as CD4 + T cells), or destroy infected cells (cytotoxic T cells, also known as CD8 + T cells); while B cells either produce antibodies that will bind to antigens, or survive in the immune system for long periods of time (up to years) in order to quicken the reaction of the immune system in case of exposure to the same antigen.

Discriminating self and non-self: antigens and antibodies

2.1. Discrimination between "self" and "non-self" Discrimination between self-and non-self recognition is absolutely fundamental for the adaptive immune system. This is partly accomplished by the major histocompatibility complex (MHC), a molecule expressed at the surface of the cells, that can be either class I or class II.

MHC class I molecules are expressed by nearly all nucleated cells in the organism and comprise peptides of 8-9 amino acids in length presented to CD8 + T cells to alert possible intracellular infections (this is pictured in Figure 1 below). If an anomaly is detected in presentation by MHC, death of the defective cell is programmed by apoptosis. proteolytic cleavage of the antigens. The epitope-MHC complex is presented to CD4 + T cells that then activate the B cells that will then start to produce specific antibodies.

B cells can also recognize antigens by themselves, without the assistance of MHC molecules, as the B cell receptors expressed are antigen-specific.

Binding of a B cell with its cognate antigen, if a CD4 + T cell gives an additional signal, leads to the production of specific antibodies.

Thus, self-and non-self recognition is strongly dependent on presentation of epitopes to T lymphocytes by MHC molecules (either class I or class II, each one eliciting a different mechanism), or direct recognition by B lymphocytes.

Fortunately, the immune system is able to distinguish between the self and therefore peptides presented in this way to MHC do not trigger any immune response under normal circumstances. This is called "immune tolerance".

Antigens and antibodies

Strictly speaking, an antigen (Ag) can be defined as a substance that can bind to the receptors of the adaptive immune response (T and B cell receptors).

However, three different kinds of antigens are recognized:

-exogenous antigens: these Ags have entered the body from the outside, after breaching through physical barriers and innate immune system.

-endogenous antigens: these Ags are generated inside the cells, as by-products of normal cellular metabolism, or after a cell infection by viruses or bacteria.

-autoantigens: these Ags consist of normal peptides or proteins, components of the organism that are not recognized anymore as self by the immune system because of a malfunction of the "immune tolerance" system.

An immunogen is an Ag that elicits an immune response from the adaptive immune system. If an immunogen is always an Ag, the contrary is not true.

The best example that can be given are haptens, a family of low molecular weight antigenic compounds that are not immunogenic, unless they are bound to a carrier molecule.

Antibodies (Abs), also termed immunoglobulins (Igs), are molecules composed of two heavy (~55 kDa) identical polypeptides chains and two identical light (~25 kDa) polypeptide chains, held together by interchain disulfide bonds.

These large proteins (~150 kDa), produced by plasma cells (differentiated B lymphocytes) are Y-shaped and are used by the organism to selectively identify and neutralize Ags 1,2 .

The general structure of an Ab is represented below (Figure 2). Sugars can be linked to some of their amino acid residues, thus Igs are glycoproteins. The "arms" of the antibody are termed the Fab region (for Fragment antigenbinding), whilst the "base" is called Fc region (Fragment crystallizable). The Fab region is involved in Ag binding (and thus important for specific Ag recognition), while the Fc region is more involved in interactions with surfacecell receptors of the immune system: for example, they allow recognition and ingestion of pathogens by phagocytic cells; or they can interact with proteins activating the complement system.

The connection between Fab and Fc regions is rich in Proline, Threonine, and Serine and is known as the hinge. In mammals, there are five different types of Abs, termed isotypes: IgG, IgM, IgA, IgE, and IgD. They differ mainly in their biological properties, and each isotype contains a different heavy chain. This is summarized in the Table 1. IgG is the most important isotype among the five, providing the majority of antibody-based immunity against exogenous Ags. Found in blood and extracellular fluids in monomeric form, it is the most abundant (~75%) of serum Igs in humans. It is involved in the secondary immune response, produced in the later stages of the immune process, but lasting longer in the organism. Characterized by its ability to easily cross cell membranes, it is the only Ab able to cross the placenta to passively immunize the fetus.

IgM is the largest isotype, usually found in a pentameric form (~975 kDa), forming a "star" of Y-shaped Abs. They are found in blood and form the very first response to a pathogenic invasion, being the primary isotype produced by B cells. Although they are effective at binding Ags because of their higher valency, they cannot cross cell membranes.

IgA isotype is principally found in mucosal areas as a dimer (gastrointestinal, genitourinary, and respiratory tracts), but also in tears, saliva and mucus. It plays a critical role by eliminating pathogens in these zones (mainly by agglutination), thus avoiding pathogenic colonization.

IgE antibodies are present in mucous membranes, lungs, and skin. They have a function in immune response towards allergens and are also believed to protect against certain parasitic worms. It is the least common isotype of Ab in the organism and comes as a monomer.

IgD is another monomeric antibody (with IgG and IgE) found in very small amounts in blood serum. They are mainly expressed on B cell surfaces as antigen receptors and may play a role in eliminating the B-lymphocytes, which generate self-reactive autoantibodies.

Antigen-antibody interactions: epitopes and paratopes

Antigen-antibody interaction is performed through an epitope-paratope interaction, which can be seen as a key-lock system. The epitope is the part recognized by the Ab and usually comprises 6-7 amino acids; the paratope is the binding part of the Ab that selectively recognizes the epitope. The paratope is located at the end of the Fab region, and usually is the same for both arms of an antibody.

These interactions are weak, non-specific and of a complex nature 3,4 . This means binding between an antibody and an antigen is reversible and for this reason interactions between antibodies and antigens are to be considered as relative rather than absolute. The formation and dissociation of antigenantibody complexes follows the basic thermodynamic principles of any reversible biomolecular interaction.

Epitopes can be divided in two categories:

-linear epitopes: these epitopes are composed of a given amino acid sequence.

Also termed continuous epitopes, they are accessible for interaction with antibodies if they are exposed, e.g. at the surface of an antigen or in a conformationally extended region of a folded protein antigen.

-conformational epitopes: also called discontinuous epitopes, these need a particular conformation for the formation of the epitope. It often comes from the protein folding, which allows distant amino acids in the sequence to be spatially close one to each other.

Because most antigens are highly complex, they present numerous epitopes that are recognized by a large number of lymphocytes. Each lymphocyte is activated to proliferate and differentiate into plasma cells, and the resulting antibody response is polyclonal. In contrast, monoclonal antibodies are antibodies produced by a single B lymphocyte clone.

Autoimmune diseases: from self-to non-self recognition

Under normal circumstances, the self-and non-self are well differentiated by the organism and the innate and adaptive immune systems. A balance between immune response and regulatory mechanisms of the immune system allows the whole mechanism to be kept under control.

An autoimmune disease (AID) is characterized by loss of the tolerance to the self and an autoreactivity against self-Ags. A crucial feature of an autoimmune disease is that tissue injury is caused by the immunologic reaction of the organism to its own tissues, and the production of antibodies against the organisms own tissues (autoantibodies, autoAbs). It may lead to destruction of one or more types of body tissues, abnormal growth of an organ, and changes in organ functions. These attacks of the immune system can be directed against a particular type of cells, or instead can be more systemic and widespread.

A disease is considered as an AID if it is in line with Witebsky's postulates 5,6 :

direct evidence from transfer of pathogenic antibodies or pathogenic T cells;

indirect evidence based on reproduction of the autoimmune disease in experimental animals; and circumstantial evidence from clinical clues.

They affect about 8% of the total world population 7 , with 3 patients out of 4 being women 8 , which generally have a higher risk of AIDs development (although exceptions exist) 9 . Direct consequence of these diseases is the high social impact they have on the patients.

AIDs are always complex diseases, with mechanisms that might not always be completely understood. Currently, it is accepted that AIDs are multifactorial diseases: genetics, xenobiotic contaminants, bacterial or viral infections have been proved to be important aetiological agents.

Three pathways for immune tolerance breakdown are outlined in the following sections: by loss of the immune system downregulation (T-reg cells), by crossreactivity of a self protein with microbial specific Abs (molecular mimicry), and by chemical modification of self proteins (aberrant post-translational modifications).

Loss of T regulatory cells effect

T regulatory cells (formerly known as T suppressor cells) play a crucial role in the control of this balance, by being responsible for the elimination of Cédric Rentier, PhD thesis 10 autoreactive T cells. They downregulate or suppress the reactivity of the CD4 + and CD8 + cells towards self-components of the organism.

If, for any reason, the balance between effector T cells (helpers and cytotoxics) and regulatory T cells is disrupted, then the immune system downregulation is lost and an immune process can be initiated. This is a possible cause for Systemic Lupus Erythematosus and rheumatoid arthritis, as reviewed by Chavele et al. 10 and pictured below (Figure 3).

Molecular mimicry

Due to the wide diversity of molecular structures, it is possible to have exogenous pathogens (in particular microbial molecules) that have similar structure and/or conformation to mammalian surface glycoproteins and/or glycolipids, as shown in the Figure 4. 10 Chavele, K.M., Ehrenstein, M.R. FEBS Lett. 585, 3603-3610 (2011).

Cédric Rentier, PhD thesis 11

A possible outcome is the cross-reactivity of the self molecule and the exogenous Ag, leading to activation of autoreactive lymphocytes. This is termed molecular mimicry.

Molecular mimicry has been reported in type I diabetes mellitus (T1DM), RA, and multiple sclerosis (MS) 11 . The most commonly identified triggering agents are pathogens having carbohydrate sequences (considered the antigens) in common with the peripheral nervous tissue.

Post-translational modifications of proteins

Post-translational modifications (PTMs) are chemical modifications of proteins, after their synthesis or during their life cycle. It is estimated that between 50 and 90% of proteins are post-translationally modified in vivo 13 .

PTMs are diverse and abundant, and most proteins undergo proteolytic processing and/or the attachment (or removal) of functional groups to control protein folding, localization, activity, lifespan, and interactions with other molecules. Common examples of PTMs include glycosylation, 11 Oldstone, M.B.A. Cell 50, 819-820 (1987). 12 Oldstone, M.B.A. The FASEB J. 12, 1255-1265 (1998). 13 Doyle, H.A., Mamula, M.J. Trends Immunol. 22, 443-9 (2001).

phosphorylation, ubiquitinylation, acylation, methylation, and amino acid conversions (Figure 5).

Most PTMs are the result of controlled enzymatic action, but modifications may also be introduced as a consequence of aging or exposure of proteins to chemically reactive environments. When PTMs are introduced in a noncontolled manner, they are referred as aberrant PTMs. Such alterations in amino acid structures or sequences can generate neoepitopes from self-proteins, and thus result in the loss of immune recognition 15 , initiating immune system self-reactivity, ultimately leading to AIDs 16,17 .

Cédric Rentier, PhD thesis 13

Autoimmune diseases diagnosis and biomarkers

AIDs are a particular class of diseases, with high social impact on patients' lives, and complex mechanisms. Diagnostic methods exist, but since every AID is different, there is not one single diagnostic method for every AID.

Moreover, cures for immune diseases, when they are available, are extremely expensive and have huge secondary effects on the patients (e.g. immunosuppressant treatments).

Thus, it appears crucial to have at disposal methods that allow an early diagnosis of AIDs. Most of these methods are based on the detection either of the inflammatory markers that are usually associated with the immune response; or on auto-Abs produced by the immune system.

There are several methods for the detection of Ag-Ab complexes (Table 2). Each method has different sensitivities and ideally a test should be characterized by a good sensitivity, low-invasivity, low-cost, and ease of execution. Unfortunately, to date, in most cases the diagnostic and prognostic methods used for AID do not meet these criteria.

Some of these are based on the tendency of antibody-antigen complexes to come out of solution in what is called a "precipitation reaction". Some are based on the ability of antibodies to stick cells together, called an "agglutination reaction". The third category comprises the immunoassays, including:

-Radio immunoassay: this uses radio-labeled Ags (e.g. 125 I attached to Tyr) to measure Ag-Ab interactions 18 . Sensitive and precise, this technique however requires specialized equipment, precautions and licensing, since radioactive substances are used.

-Immunofluorescence: this technique uses Abs labeled with fluorescent dyes that will emit light at a particular wavelength when excited. It can be direct immunofluorescence, if the label is attached directly to the Ab of interest, but these are costly and the number of fluorophores that can be linked is limited.

Indirect immunofluorescence uses a labeled secondary Ab that can recognize the primary Ab (more precisely, they are specific to the heavy chain of Igs considered). It is currently the golden standard for AIDs diagnostics, although it can suffer from cross-reactivity or photobleaching (loss of fluorescence during excitation). In addition, this procedure is invasive as it requires tissue sections or isolated cells.

-Flow cytometry: this is a laser-based, biophysical technology that allows simultaneous multiparametric analysis of the physical and chemical characteristics of up to thousands of particles per second by suspending cells in a stream of fluid and passing them through an electronic detection apparatus.

-Enzyme-Linked ImmunoSorbent Assay (ELISA): Described for the first time in 1971 by Engvall et al. 19 ELISA is based on the primary immunologcal concept of Ag binding to specific Ab. The greatest advantage of this technique lies in the immobilization of one of the reactants to a solid surface: this feature makes ELISA easy to design and to perform, and enables the separation of bound from non-bound material during the assay, facilitating the measuring of specific analytes within a crude preparation. Similar to immunofluorescence, ELISA can be direct or indirect.

Biomarkers and chemical reverse approach

A biomarker is defined 20 as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention."

Specific autoAbs produced during the course of AIDs can serve as biomarkers.

This makes auto-Abs a valuable tool in the diagnosis of AIDs 21,22 , especially when auto-Abs are produced long before the apparition of first symptoms of the disease 23,24,25 ; or when seric concentration of auto-Abs can be correlated with the course of the disease (emission/remission, efficiency of a treatment, etc.).

Unfortunately, in AIDs only very low specific Abs were demonstrated to be detectable in serum, possibly because proteins used in the assays cannot 19 Engvall, E., Perlmann P. Immunochemistry 8, 871-874 (1971). 20 Biomarkers Definition Working Group, Clin Pharmacol Ther. 69, 89-95 (2001). 21 Leslie, D., Lipsky, P., Notkins, A.L. J. Clin. Invest. 108, 1417-1422 (2001). 22 Alcaro, M.C., Lolli, F., Migliorini, P., Chelli, M., Rovero, P., Papini, A.M. Chem. Today 25, 14-16 (2007). 23 Bizzaro, N. Autoimmun. Rev. 6, 325-333 (2007). 24 Bizzaro, N. Clin. Rev. Allergy Immunol. 34, 326-331 (2008). 25 Hu, Z.D., Deng, A.M. Clin. Chim. Acta. 437, 14-18 (2014).

Cédric Rentier, PhD thesis 16 reproduce the real Ag. Moreover, an entire protein can contain more than one epitope, thus leading to reactivity towards multiple Abs.

These observations account, at least in part, for the limited success attained in the discovery of biomarkers for AIDs using proteomic analysis and/or protein microarrays.

The diagnostics and prognostics of AIDs can be done using specific auto-Abs as biomarkers. It was observed that native or recombinant proteins are difficult to obtain (isolation, synthesis) with the correct sequences or the correct post-or co-translational modifications; and the tridimensional structure (folding). This makes such proteins inconvenient tools for simple and reproducible AIDs diagnostics.

Synthetic peptides mimicking autoantigens

Therefore, only the relevant part of the Ag is synthesized by classical means (solid-and liquid-phase synthesis), derived from native protein sequences and reproducing specific epitopes, or mimicking immunogenic modifications. This approach could be even more effective and specific than native proteins in antibody detection.

Its principal advantage is the complete control of the synthesized molecule:

either on the sequence or on the insertion of the correct PTM at the relevant position 26 . The folding issue can also be partly avoided: synthetic peptides, through sequence optimization, can adopt specific conformations (α-helical, βturns, etc.). The most difficult aspect remains the identification of the immunogenic part, i.e. to decide which sequence should be synthesized, which modification should be introduced. Usually, the construction of the correct peptide is based on the molecular mechanism of and the analysis of circulating Abs for each disease.

This novel approach was developed a few years ago at the Laboratory of Peptide and Protein Chemistry and Biology of the University of Florence (PeptLab@UniFi) and called the "Chemical Reverse Approach" (CRA) 27 .

"Reverse" because the screening of the synthetic antigenic probe is guided by autoantibodies circulating in autoimmune disease patients' sera. "Chemical" because autoantibody recognition drives selection and optimisation of the "chemical" structure from defined peptide collections. The whole process is represented in Figure 6. The upper part of the figure (blue and purple) represents the immune-mediated process in AIDs: an aberrant PTM together with a loss of the immune tolerance triggers the immune response. In particular the creation of auto-Abs directed against the neo-Ag. The lower part illustrates the CRA. Synthetic peptides are designed, trying to reproduce immunogenic parts of neo-Ags (continuous/discontinuous epitopes, PTM involved). These synthetic peptides are used to detect possible specific auto-Abs, biomarkers of the AID.

Cédric Rentier, PhD thesis 18

Synthetic peptides in SP-ELISA

The most simple way to evaluate synthetic peptides as molecular probes for AIDs auto-Abs is by ELISA.

The general ELISA procedure (indirect ELISA, shown in Figure 7) involves the immobilization of the Ag on a solid phase (coating), usually on a 96-well microtiter plate, and the consequent blocking of any un-coated areas of the well with a solution of non-reactive protein (e.g. fetal bovine serum, FBS). Then the given biological sample is applied, allowing the binding of the coated Ag to a specific Ab, which is subsequently detected bound by a secondary enzymecoupled Ab. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product.

Secondary Abs are commonly labeled with alkaline phosphatase or horseradish peroxidase, and a large selection of substrates is available. The choice depends upon the required assay sensitivity and the instrumentation available for signaldetection. -easy to set up and can be routinely performed -a large number of sera can be tested in a short period of time -do not need any special instrumentation or reagents -relatively low cost -high potential of automation

The association of this technique with CRA introduces synthetic peptides as a source of stable, reproducible and well-defined antigens.

These concerns are relevant to the set-up of diagnostic assays directed towards clinicians and medical staff. A successful development would provide low-cost ready-to-use systems, with clear and interpretable results and no false positives that do not require training in immunochemistry nor immunology.

Chemical reverse approach and Multiple Sclerosis

As a proof-of-concept, CRA led to the design of a synthetic glycopeptide, the CSF114(Glc), as an antigenic probe for specific auto-Abs of Multiple Sclerosis (MS) in certain subtypes of patients.

A synthetic N-glycosylated peptide analogue of the immunodominant epitope of human myelin oligodentdrocyte glycoprotein, the [Asn 31 (Glc)]hMOG (30-50) was able to recognize auto-Abs in MS patients' sera, whereas its nonglycosylated analogue was inactive despite similar conformations; leading to the conclusion that positive results were related to the N-linked sugar moiety 28,29,30,31 . Further tests using irrelevant sequences but including Nglycosylated building blocks allowed highlighting of the role of the sugar moiety as a putative minimal epitope. 28 Mazzucco, S., Mata, S., Vergelli, M., Fioresi, R., Nardi, E., Mazzanti, B., Chelli, M., Lolli, F., Ginanneschi, M., Pinto, F., Massacesi, L., Papini, A.M. Bioorganic Med. Chem. Lett. 9, 167-172 (1999). 29 Carotenuto, A., D'Ursi, A.M., Nardi, E., Papini, A.M., Rovero, P. J. Med. Chem. 44, 2378-2381 (2001). 30 Lolli, F., Mulinacci, B., Carotenuto, A., Bonetti, B., Sabatino, G., Mazzanti, B., D'Ursi, A.M., Novellino, E., Pazzagli, M., Lovato, L., Alcaro, M.C., Peroni, E., Pozo-Carrero, M.C., Nuti, F., Battistini, L., Borsellino, G., Chelli, M., Rovero, P., Papini, A.M. Proc. Natl. Acad. Sci. U. S. A. 102, 10273-10278 (2005). 31 Lolli, F., Mazzanti, B., Pazzagli, M., Peroni, E., Alcaro, M.C., Sabatino, G., Lanzillo, R., Brescia Morra, V., Santoro, L ., Gasperini, C., Galgani, S., Milco D'Elios, M., Zipoli, V., Sotgiu, S., Pugliatti, M., Rovero, P., Chelli, M., Papini, A.M. J. Neuroimmunol. 167, 131-137 (2005).

Cédric Rentier, PhD thesis 20 In particular, the CSF114(Glc), a 21-mer designed synthetic peptide, gave the best results. It was shown to adopt a type I' β-turn conformation in solution, and to expose its modification at the top of the hairpin (represented Figure 8) cirrhosis and, ultimately, the need for a liver transplantation 34 .

The diagnosis can be established when two of the following criteria are met:

-biochemical evidence of cholestasis based on alkaline phosphatase measurement -presence of serum anti-mitochondrial Abs (AMA) of the IgG class by indirect immunofluorescence -liver biopsy providing histological evidence of non-suppurative destructive cholangitis and destruction of interlobular bile ducts 35 It may happen that two criteria are not sufficient (e.g. when measurement of alkaline phosphatase is at limit within acceptable range), therefore the biopsy is needed. This makes PBC a disease with a rather invasive method of diagnosis, and high social impact on patient's lives.

Even if factors leading to PBC remain poorly understood, the disease is considered a model AID 36,37 because of high specificity bile duct damage. It is 34 Hohenester, S., Oude-Elferink, R.P.J., Beuers, U. Semin. Immunopathol. 31, 283-307 (2009). 35 Lindor, K.D., Gershwin, M.E., Poupon, R., Kaplan, M., Bergasa, N.V., Heathcote, E.J. a unique and critical feature of PBC and suggests the occurrence of an intense autoimmune response directed against biliary epithelial cells.

Furthermore, PBC has a serological hallmark signature. This is represented by anti-mitochondrial antibodies (AMA), highly disease-specific auto-Abs found in 90-95% of patients but in less than 1% of healthy controls 38 .

The aetiology of PBC is multifactorial. It was widely hypothesized that the disease is triggered by environmental exposure in association with a genetic background allowing autoimmunity development 39 40,[START_REF] Matsugo | ITE Letters on Batteries[END_REF][START_REF] Brufani | Alpha lipoic acid derivatives and their use in drug preparation[END_REF] . This illustrates how PBC could be due to exposure to bacterial Ags. 38 that M2 is made up of multiple subunits of the 2-oxo-acid dehydrogenase complex (2-OADC) 45,46 , which is located in the inner mitochondrial membrane and is responsible of the oxidative decarboxylation of keto-acid substrates 47 .

It is important to underline that AMA auto-Ags share a substantial homology, having a common highly-conserved N-terminal domain containing a lipoylated Lys residue (Figure 9) 37 . In particular, the targets of AMA are represented by:

-dihydrolipoamide acetyltransferase (E2 component of the pyruvate dehydrogenase complex, PDC-E2), which is the most frequently detected -dehydrogenase dihydrolipoyl transacylase (E2 subunit of the branched-chain PDH is formed by three subunits: E1 (thiamine diphosphate-dependent dicarboxylase; PDC-E1), E2 (dihydrolipoyl acetyltransferase; PDC-E2) and E3

(dihydrolipoyl dehydrogenase; PDC-E3) working together as described in Lipoyl moiety is covalently bound to proteins and serves as a carrier of various reaction intermediates between the different sites of the enzymes. Perham described these as "swinging arms" 48 because of the high flexibility of the carbon chain of lipoyl-Lys.

Identification of the immunogenic region of PDC-E2

Although it is widely demonstrated that PDC-E2 is the main target of AMA, the definition of its immunodominant epitope is still controversial, as demonstrated by the numerous studies performed over the last 25 years.

Authors (year) Description of Ag tested Lipoylation required for Ab recognition?

Gershiwn et al. (1987) 38 Fused polypeptide (obtained by screening of rat liver cDNA library) expressed in E.coli. 45 Synthetic peptides of rPDC-E2 (mapping of the fused polypeptide obtained by Gershwin 38 ).

Lipoylation status was not taken into account during the Ag characterization. Three points can be further discussed: the sequence, the lipoylation and the conformation.

Immunogenicity of the sequence

From Table 3, it is clear that the situation is quite complicated. This is partly because of the great diversity of Ags used in the studies detailed above:

variable origin of Ags tested (human, rat, porcine, bacterial), whole proteins or sub-domains, conjugated or not. Their preparation also differs (synthetic peptides, recombinant, molecular biology).

In a nutshell, several AA sequences have been described in the various studies regarding auto-Ags in PBC. However, the most widely described -and accepted -is the inner lipoylated domain: PDC-E2(167-186) fragment, although there are some studies that are contradictory with these results.

We can cite the work of Braun et al. 56 that recently questioned the identification of the main immunogenic region. In this work, 33 synthetic overlapping peptides covering the entire length of PDC-E2 (see Figure 10)

were tested by SP-ELISA. The best results (up to 74% of the PBC sera reacted) came from two peptides within the catalytic domain (AA 407-431 and 475-499) and the peptide 101-125 in the first hinge region. The most successful results did not, however, come from peptides containing the immunodominant epitopes (inner and outer lipoyl domains).

Immunogenicity of the lipoylation

The identification of the main immunogenic region with the inner lipoyl domain of PDC-E2 prompted the investigation on the role of lipoic acid cofactor. Braun et al. 56 also evaluated the function of lipoic acid by synthesizing the immunodominant peptide PDC-E2(167-184) in both unlipoylated and lipoylated form. Either peptide was coupled with ovalbumin (OVA). Compared with the unconjugated analogues, the OVA conjugates exhibited stronger reactivity. However, most sera showed high reactivity also with OVA alone, disclosing a huge limitation of this study. After subtracting the reactivity to OVA alone from the OVA-coupled peptides, 44% of PBC sera had IgG response to the unlipoylated peptide (OVA-167-184) and 22% to the lipoylated peptide (OVA-167-184-LA). IgMs were found in 57% and 31%, respectively. Therefore, the higher Ab response was detected by the unlipoylated peptide.

These results are in contradiction with others studies, such as Tuaillon et al. 52 or Quinn et al 53 . who showed the necessity of lipoylation for Ab recognition.

Another aspect that has not been considered until now is the chirality of the lipoylation. Since the C-6 carbon is chiral, it can be found as its (R)-(+)enantiomer, its (S)-(-)-enantiomer, or as racemic mixture. However, only the (R)-enantiomer is endogenously synthesized in vivo once linked to proteins 57,58 . 57 barrel containing two antiparallel β-sheets. Each of these sheets contain three major strands and one minor strand (Figure 12) 60 .

However, conformation of peptides is highly dependent on their environment and physico-chemical parameters: e.g. pH, solvent, temperature, concentration, purity of the sample.

In the case of a variety of tested Ags (as those reported in Table 3), conformation can depend on the antigenic source (synthetic peptide or recombinant protein), or the conditions of the experiment.

Thus, it is important to have reliable information regarding the tridimensional structure of the Ags tested as molecular probes for Abs detection. 59 Dardel, F., Laue, E.D., Perham, R.N. Eur. J. Biochem. 209, 203-209 (1991). 60 Howard, M.J., Fuller C., Broadhurst R.W., Perham, R.N., Tang J.G., Quinn, J., Diamond, A.G., Yeaman, S.J. Gastroenterology 115, 139-146 (1998).

General overview of lipoylation chemistry in vivo and in vitro

Lipoylation plays an essential role in the mitochondria energy cycle, and is possibly crucial in the etiology of PBC.

Attachment of lipoic acid to a molecule is called lipoylation. In vivo, the lipoylation occurs on the side chain of a lysine residue in the form of the amino acid itself to give the so-called "lipoyl-Lys", or in a protein to provide the "lipoylated protein" (Figure 3). Lipoylation is a post-translational modification with a strong structural requirement for Lysine 61 ; however, various amino acids are tolerated at the neighboring residues 62 . This contrasts to Nglycosylation which requires typically a clear consensus sequence 63 . Lipoic acid was first isolated and characterized by Reed et al. 64 in 1951. After a 300,000-fold purification, approximately 10 tons of insoluble "liver residue" 64 Reed L.J., DeBusk B.G., Gunsalus I.C., Hornberger C.S. Jr. Science 114, 93-94 (1951). 65 Reed L.J. J. Biol. Chem. 276, 38329-38336 (2001). 66 Bullock M.W., Brockman J.A. Jr., Patterson E.L., Pierce J.V., Von Saltza M.H., Sanders F., Stoksad E.L.R. J. Am. Chem. Soc. 76, 1828-1832 (1954).

Enantiomerically pure lipoic acid was subsequently prepared by resolution of the racemate by formation of diastereomeric salts using respectively (R)-and (S)-α-methylbenzylamine 67 .

Lipoylation in vivo

Although lipoic acid was extensively studied in the past 50 years 68 , the mechanism by which it is biosynthesized and attached to protein was only recently elucidated 69 . In vivo, lipoic acid exists in very small proportion as free acid. It primarily resides as a cofactor bound to the ε-amino group of specific Lys on lipoyl-accepting protein domain. A large part of the studies on lipoic acid biosynthesis were performed using Escherichia coli (E. coli), enabling the identification of two different pathways, which depend on different sources of substrate. The first pathway is characterized by the use of exogenous lipoic acid, which is activated with adenosine triphosphate to form lipoyl-adenosine monophosphate by lipoate-protein ligase (LplA) 70 . The same enzyme then transfers the molecule to the side chain of a Lys residue on the protein with release of adenosine monophosphate (Figure 17) 71 . LplA is also able to use octanoic acid as substrate, but with reduced efficiency. The second pathway involves endogenous lipoic acid synthesized from fatty acids. In this case, a lipoyl (octanoyl)-transferase called LipB transfers both lipoyl or octanoyl groups conjugated to an acyl carrier protein (ACP) to lipoyl accepting domains 73 . As octanoyl-ACP represents a lipoyl-ACP precursor, the use of this compound to obtain a lipoylated protein requires an additional stepthe introduction of sulfur atoms catalyzed by the N-octanoyltransferase LipA 74,75 (Figure 18). 72 Booker, S.J. Chem. Biol. 11, 10-12 (2004). 73 Zhao X., Miller J.R., Jiang Y., Marletta M.A., Cronan J.E. Chem. Biol. 10, 1293-1302 (2003). 74 Cicchillo R.M., Booker S.J. J. Am. Chem. Soc. 127, 2860-2861 (2005). 75 Miller J.R., Busby R.W., Jordan S.W., Cheek J., Henshaw

.2. Lipoylation in vitro

As far as preparation of lipoylated peptides or proteins goes, two different strategies exist:

-the convergent strategy, which is the direct attachment of a LA to the side chain of the target -the building-block strategy, dealing with the insertion of a carefully designed amino acid in the peptide sequence through classical peptide synthesis.

Two pathways were developed for the convergent strategy that have been widely used over the last years. A chemical method which is focused on the creation of the amide bond (for a review see Valeur et al. 76 ) between LA and the side chain of a relevant Lys; and a more biological method using the enzymes previously described in the LplA and LipB pathways.

Orthogonal protecting groups were critical for peptide lipoylation by chemical synthesis. For example, Tuaillon et al. 52 Similarly (Figure 21), in Fmoc/tBu strategy Long et al. 77 Amano applied the same lipoyl NHS ester strategy to selectively lipoylate short unprotected peptides of the PDC-E2 sequence (15 amino acids) coupled to an agarose gel 78 . The desired peptide is synthesized incorporating a modified diaminopropionic acid amino acid (N β -Boc-amino-oxyacetyl)-L- (ethylenedioxy)bis(ethylamine)), followed by N-terminal acetylation. The resulting peptide-spacer-linker is cleaved from the resin then coupled to a modified methyl-ketone agarose via an oxime bond. Lipoylation is performed as the last step using the lipoyl NHS ester (Figure 22). This approach has been used as a convenient standard method since then 79,80,81,82 . The drawbacks to peptide acylation include incomplete lipoylation, which is typically verified using the Kaiser test 83,84,85. In addition, the N-terminal residue lipoylation of a Glu-Ala dipeptide was performed using lipoic acid activated with N,N-disuccinimidyl carbonate on >100g scale (Kates et al 86 , Figure 23). The elucidation of in vivo lipoylation mechanisms led to discovery and partial purification of two enzymes from E.Coli (LplA and LplB) that were subsequently employed in vitro for direct lipoylation of proteins 87 . For example, the inner lipoyl domain of human PDC-E2 was successfully obtained Cédric Rentier, PhD thesis 40 using the in vitro lipoylation approach 53 . The ligases were subsequently purified by heparin-agarose chromatography after disruption of bacteria 88 .

Moreover, the octanoyl-Lys substrate of the tetrapeptide Thr-Glu-Lys-Ile was converted to the lipoyl peptide using expressed and purified lipoyl synthase (LipA) from Sufolobus solfataricus 89 (Figure 24). Various strategies were used to prepare suitably protected N ε -lipoyl lysine [Lys(N ε -lipoyl)] analogs for subsequent peptide synthesis. For example, the lysine-copper complex was acylated with racemic lipoyl isobutyl carbonic anhydride 90,91,92 albeit in low yield due to polymerization. Lipoic acid can polymerize by a ring-opening mechanism featuring formation of intermolecular disulfide linkages to produce an insoluble polymer 93 . Lipoic acid was coupled to alkylamines using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide 88 Green D.E., Morris T.W., Green J., Cronan Jr J.E., Guest J.R. Biochem. J. 309, 853-862 (1995). 89 Bryant P., Kriek M., Wood R.J., Roach P.L. Anal. Biochem. 351, 44-49 (2006). 90 Recently, novel lipoylated derivative have been synthesized 96,97 . The amide linkage was replaced by bioisosteric groups, such as 1,2,4-and 1,3,4oxadiazoles, 1,2,3-triazoles, thiazoles or tetrazoles (Figure 26). Such lipoylderivatives may offer interesting alternatives due to their increased stability to proteases. 94 Regarding the disease itself, it is clear from the literature that the situation is complicated and requires clarification. In particular the following points: AA sequence, conformation, and PTMs. Indeed, up to now, the Ags used in the various studies are of a diverse nature, not always well characterized nor defined. This concerns both the primary sequence and the tertiary structure as well as the PTMs introduced.

We propose to take advantage of the CRA, using both organic synthesis and Solid Phase Peptide Synthesis (SPPS) tools to synthesize completely characterized molecular probes, and use these probes in ELISA tests to evaluate their utility as Ags for the detection of AMA.

The first part will deal with the synthesis of Lys(N ε -lipoyl). Both convergent synthesis and building-block approaches were described in the literature, but low yields, inadequate products for liquid-/ solid-phase synthesis or possibly incomplete introduction of lipoylation hampered the production of well-defined lipoylated molecules. Therefore, it was essential to develop adequately protected Lys(Nε-lipoyl) building blocks for SPPS via the Fmoc/tBu strategy.

However, we wanted also to pay attention to the chirality of the lipoyl moiety.

In fact, stereochemistry can play an important role in the Ag-Ab interactions, and to the best of our knowledge, this was never performed on PBC, disregarding the existence of two enantiomers of LA, thus two diastereomers of L-Lys(N ε -lipoyl).

Regarding the peptide synthesis, we selected one peptide from the native protein, and one peptide from a completely designed structure.

As the native sequence, we chose to synthesize the PDC-E2(167-186) peptide.

It is the most widely accepted immunogen involved in PBC.

To assess the role of the AA sequence in Ag-Ab recognition, we also included in our studies the designed type I' β-turn peptide, the CSF114.

Tridimensional structures of peptides were also investigated. Figure 28 shows the hypothetical in-solution structures of both peptides, based on existing data:

CSF114 and its type I' β-turn, while PDC-E2 displaying a type I β-turn around the inner lipoyl domain. This allowed the examination of the exact role of the AA sequence, and a possible condition for lipoylation presence on the Ag.

Results and discussion

Synthesis of the lipoylated lysines

As presented in the introduction, lipoylation of peptides can be performed either by the convergent pathway (lipoylation on peptide, as a final step after the peptide synthesis), or by the building-block strategy (the modified AA is synthesized then introduced as a classic AA during the course of peptide synthesis).

The convergent pathway presents several major disadvantages. First, the necessity of a third degree of orthogonality, which means specific protecting groups for SPPS. Second, the reactivity of reactions directly on peptide is lower because of steric hindrance caused by aggregation and side-chains interactions. Third, side-products resulting from the incomplete and/or incorrect introduction of the chemical modification on the peptide.

In comparison, the building-block (BB) strategy is flexible and highly selective. BB can be prepared in advance, purified, and stored. The shortcoming is the need for the BB to be synthesized, because only a few BBs are sold as commercial products. Regarding the production of Lys(N ε -lipoyl) BB, this is based on the coupling between LA and the side-chain of a Lys, as represented in Figure 29: The synthesized BB should be compatible with Fmoc/tBu SPPS strategy, in order to facilitate its subsequent use in manual and automated peptide synthesis.

Considering that there are two enantiomers of LA, Fmoc-L-Lys as starting However, the coupling reaction described in Figure 29 to yield products shown in Figure 30 requires activation of the LA carboxyl group to form the amide bond.

Two-step reaction: the pentafluorophenylester route

The synthesis takes advantage of commercially available (S)-(-)-α-lipoic acid and (R)-(+)-α-lipoic acid, as highly pure starting materials. Their main drawback, however, is their cost (especially for the non-natural (S)enantiomer).

Chapter One: Primary Biliary Cirrhosis -2. Results and discussion Cédric Rentier, PhD thesis 48

The initially selected synthetic strategy for this building block uses the lipoyl pentafluorophenylester (Pfp esters) as a reactive intermediate to facilitate the coupling to the side chain of the lysine.

This method, developed by Kisfaludy99 , yields the Pfp esters as stable and often crystalline compounds, which makes the Pfp esters route a very suitable coupling strategy for SPPS100 . These compounds are activated by the electronwithdrawing nature of the substituted phenyl ring.

This involves the reaction of pentafluorophenol with LA, as shown in Figure 31. Purification of products synthesized using this method is also convenient, as the by-product obtained is dicyclohexylurea. It is not soluble in dioxane, thus purification is performed through a simple procedure of filtration.

In order to obtain the desired Fmoc-L-Lys(N ε -lipoyl) BBs, the synthesized lipoyl Pfp esters 1, 2 and 3 are coupled to the side chain of a Fmoc-L-Lys.

The reaction is carried out in presence of Fmoc-L-Lys, hydroxybenzotriazole (HOBt) and diisopropylethylamine (DIPEA), as shown in Figure 32: After 24 h of reaction and subsequent purification by Flash Column Chromatograpy (FCC), lipoylated BBs are obtained in poor yields. Despite a very good yield on the formation of the lipoyl Pfp esters, the yield over the two steps of this reaction is very low. This is summarized in Table 5. This might be due to the low solubility of the lipoyl pentafluorophenyl ester intermediates. In fact, these species have been found to be poorly soluble in various solvents (ethyl acetate, acetonitrile, dichloromethane, dioxane, tetrahydrofurane, DMF, water, and 20% acetic acid aqueous solution), possibly because of a polymerization through a ring-opening mechanism 93 .

The building-blocks 5 and 6 have been fully characterized by 1D and 2D

NMR, HPLC-MS and optical rotations. So far, due to the two chiral centers being separated by the long flexible lateral chains acting like a "swinging arm" 48 , only the optical rotation allows differentiation between the two molecules.

Production of Fmoc-L-Lys(N ε -lipoyl) diastereomers was successfully achieved, in order to introduce them into the selected peptide sequences to serve as synthetic probes for PBC diagnostics.

However, the yields were not satisfactory. Considering the cost of the starting material, an alternative solution had to be considered. The problem of photo instability of LA has been known since the 1950s101,102 .

The potential of lipoic acid derivatives to polymerize has not been generally addressed in the synthetic literature, but the effects of solvents on LA degradation and polymerization have been evaluated 103 . In particular, the polymerization problem may arise during concentration and/or recrystallization of lipoyl-containing products. It was found that solvents with readily extractable hydrogen inhibited light induced polymerization of LA and reported that this tendency was minimized in the presence of 2-propanol104 .

This phenomenon is consistent with a mechanism of LA polymerization via the photolytic opening of the dithiolane ring structure resulting in a diradical, followed by propagation through intermolecular disulfide bond formation.

Changing solvents of the reaction for 2-propanol, although possible, cannot guarantee the optimal solubility or reactivity of the reagents. Moreover, the most efficient way to avoid polymerization of the lipoylated intermediates during synthetic and purification processes is to proceed through a one-step reaction.

It is possible to perform an "in-situ" activation of the carboxyl group, prior to its coupling to the side chain of the Lys residue.

The "in-situ" activation involves a short preactivation of the lipoic acid in the presence of a base and a stoichiometric amount of coupling reagent to obtain its activated ester form (depending of the coupling method used). The Fmocprotected Lysine is then added to yield, after one night, the desired lipoylated Lysine in various yields. This is highlighted in Figure 33.

Cédric Rentier, PhD thesis 52 These reactions were all performed on racemic LA for cost reasons. Three coupling reagents were selected for this optimization (Figure 34):

-HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) 105,106 , despite its recent reclassification within the class 1 explosive category 107,108 remains a very effective and widely used coupling reagent.

-OxymaPure (ethyl-2-cyano-2-(hydroxyimino)acetate) 109 was developed recently and is described as a promising alternative to HATU.

-DMTMM-BF 4 (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate) 110 is a triazine-based coupling reagent. It was described as an activator forming more reactive "super-esters". The best results with this method were obtained using HATU as a coupling reagent, with a yield of 60%. Coupling with Oxyma and triazine coupling reagents gave poor yields, with respectively 18% and 37% yield.

Longer coupling times with COOH-unprotected Lysine led, in every case, to the formation of a side product. The observed mass in UPLC-MS spectra (m/z = 907) matches a structure resulting from an undesired coupling of a second Lys residue on the Lys(N ε -lipoyl) BB. The undesired product might be a εbranched dipeptide Lys[N ε -Lys(N ε -lipoyl)], with various proportions as reported in Table 6 for each method. Such a side-product, represented in Figure 35 is difficult to separate from the single-coupling adduct in normal-phase chromatography and requires the adjustment of parameters to obtain longer elution times.

Reverse-phase FCC is possible, and theoretically could allow a good separation of 4 and its side-product. However, the low solubility of the mixture in the solvent system acetonitrile/water does not make this a convenient solution.

To address this limitation, the reaction was carried out in the same conditions as described above, with careful monitoring over time using UPLC. The Lys-Lys side product formation is extremely rapid and its progress is carefully followed, in order to quench the reaction as soon as it is formed. In those conditions, within 3 h, the lipoylated lysine is obtained in a one-pot reaction with 53% yield and less than 4% side-product, thus rendering the purification by normal phase FCC more convenient.

Entry

Coupling conditions Solvent °C Yield lipoylated lysine (4) Yield sideproduct a HATU (1 eq) DIPEA (2 eq) DMF rt 53% < 4% A possible strategy for obtaining the diasteromerically pure building blocks is based on the synthesis of the mixture of diastereomers. This is acquired through the method presented above (e.g. Figure 36), and then by separating the two diastereomers.

This strategy appeared very difficult and time consuming. In fact the This is likely because of the high flexibility of the carbon chains and the distance between the two chiral centers (ca. 15 Å). RP-HPLC co-injections of both diastereomers 5 and 6 lead to a single-peak system, as well as the three building-blocks 4, 5 and 6. We encountered the same problem using normalphase HPLC (eluents DCM/MeOH).

Our failure is supported by a recent paper in which Kates et al. reported the same problem in the case of the synthesis of N α -lipoylated Glu-Ala dipeptide 86 .

In line with Kates et al., we can conclude that diastereomer separation instead of the strategy we propose herein, requires several time consuming steps, i.e., 1) reduction to dihydrolipoamide; 2) derivatization; 3) selective removal of the chiral adduct; 4) reformation of the 1,2-dithiolane ring possibly leading to dithiolane ring-opening polymerization. This is outlined in Figure 37.

Therefore, the final cost (including additional time, reagents, solvents, purifications, waste treatment, etc.) will not be as advantageous as we envisaged.

Cédric Rentier, PhD thesis 56 The two-step reaction, despite a low yield, allowed easier purifications.

Subsequent optimization of the synthesis was performed using the racemic LA.

This enabled higher yields and a faster reaction time (3 hours instead of 16), despite side-products and harder purification.

Separation of the diastereomers did not appear as an interesting strategy.

Peptide synthesis

This project aimed to synthesize modified peptides as molecular probes for Abs of a selected immune-mediated disease, the PBC.

It was essential to select the relevant chemical modifications and sequences to be synthesized and tested, to precisely define the role of each of these parameters on Ag-Ab immune recognition.

It is now accepted that PTMs may affect the immunogenicity of self-protein antigens, triggering an autoimmune response and creating neo-Ags. Therefore, post-translationally modified peptides represent a more valuable tool with respect to isolated or recombinant proteins.

Selection of the peptide sequences

Our laboratory extensively demonstrated that synthetic peptides [i.e., CSF114(Glc)] exposing optimally an aberrant PTM (an N-glucosylation) can detect antibodies in multiple sclerosis. The rationale of this "aberrant modification" is included in the introduction, and the concept of CRA was used to further investigate this hypothesis.

The selection of the synthetic antigenic probes for antibody recognition in PBC was based on the following criteria:

-PDC-E2(167-186), Figure 38 -CSF114, Figure 39: our laboratory previously reported 111,112,113 Thus, we synthesized by Fmoc/tBu SPPS a series of peptides based either on the native immunodominant epitope PDC-E2(167-186) or on the irrelevant βturn structure CSF114 in order to identify the minimal epitope recognized by AMA in PBC patients' sera.

In fact, the minimal epitope involved in Ab recognition could be either the unique modified amino acid (or its stereochemistry), the amino acid sequence 111 Lolli, F., Mulinacci, B., Carotenuto, A., Bonetti, B., Sabatino, G., Mazzanti, B., D'Ursi, A.M., Novellino, E., Pazzagli, M., Lovato, L., Alcaro, M.C., Peroni, E., Pozo-Carrero, M.C., Nuti, F., Battistini, L., Borsellino, G., Chelli, M., Rovero, P., Papini (linear epitope) or the secondary structure (conformational epitope). CSF114 is used as mimic to understand the role of antibody recognition in PBC of the specific amino acid sequence of PDC-E2(167-186) versus the solely β-turn secondary structure.

Chemical modifications introduced in the peptide sequences

We propose to compare the role of both native peptide PDC-E2(167-186) (modified at position 173 where lipoylation is found in vivo on the inner lipoyl domain) and the previously developed synthetic β-turn peptide structure CSF114 (modified at position 7 on the tip of the β-turn). Furthermore, we suggest that the single exposed chemical modification might be sufficient for antibody recognition, independent of the scaffold used.

Each chemical modification was introduced on both sequences to evaluate this hypothesis, and peptides with proteinogenic amino acids were used as controls.

Table 8 summarizes the selected building block that we introduced in our peptides.

In order to evaluate the role of lipoylation in the recognition of the antigen by the serum auto-Abs, the three synthetic Lys(Nε-lipoyl) building blocks were successfully introduced into the selected peptide sequences. In particular BB 4, 5 and 6 were introduced as possible minimal epitopes involved in PBC auto-Abs.

Chapter We extensively demonstrated that aberrant N-glucosylation is involved in some immune-mediated diseases such as multiple sclerosis and Rett Syndrome.

ε-[5-(1,2-dithiolan-3(R)-yl)- 1-oxopentyl]-Lys-OH (5) (R)- Lipoamide-Lys NHFmoc NH O O (R) HO S S (S)-α-[[(9H-fluoren-9- ylmethoxy)carbonyl]amino]- ε-[5-(1,2-dithiolan-3(S)-yl)- 1-oxopentyl]-Lys-OH (6)
Furthermore, there is some evidence that N-glucosylation of proteins might be occurring in mitochondria 114 . It is in these organelles that glucose and its byproducts are ultimately converted into energy for the organism (adenosine triphosphate).

For these reasons we also introduced Asn(Glc) and Asn(GlcNAc) in both sequences.

Preparation of a collection of modified peptides for PBC diagnosis

The previous parts of this work highlighted the peptide sequences and the modified AAs that were selected to be used in the construction of molecular probes directed towards PBC diagnostics.

Peptides were synthesized by SPPS using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using either PLS 4×4 (Advanced ChemTech), Apex 396 (Aapptec) or SyroII (Biotage).

The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.31 mmol/g for PDC-E2(167-186) peptides; 0.7mmol/g for CSF114 peptides).

Modified amino acids described in Table 8, e.g. Asn(Glc) or Asn(GlcNHAc)

were introduced using the protected building-blocks suitable for Fmoc/tBu SPPS, e.g.

Fmoc-L-Asn[β-Glc(OAc) 4 ]-OH and Fmoc-L-Asn[β- GlcNAc(OAc) 3 ]-OH.
Couplings were performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of DIPEA, for 1 hour at room temperature. All the linear peptides were purified by semi-preparative RP-HPLC, as described in General procedure for purification, and characterized by ESI-MS.

Analytical data are reported in Table 9 for CSF analogues and in Table 10 for PDC-E2(167-186) analogues.

Glycosylated peptides were deacetylated following the Deprotection of Glc moieties protocol described in the Experimental part.

The initial synthesis of the PDC-E2(167-186) fragment showed that the strongly hydrophobic sequence is insoluble in water. Analysis and purifications were therefore greatly complicated. Consequently, the use of a short cationic tag of four lysine residues 116 in C-terminus of all our PDC-E2 peptides allowed us to enhance the solubility of these products significantly.

Lipoylated peptides cleavage: stability of the S-S bond

During the synthesis and purification process of our lipoylated peptides, we came across a particular problem of stability of the dithiolane moiety.

Cleavages yielded the peptide as an impure mixture of products, including the desired peptide (m/z 1325.2, [M+2H] 2+ ) and a peptide with a +2 amu (m/z 1326.6, [M+2H] 2+ ). Both had almost identical UPLC retention times.

Cleavages performed using the TFA/TIS/H 2 O cocktail 117 , with the addition of EDT, or with Reagent K 118 did not allow for the acquisition of one single peptide alone (either the dithiolan-or dihydrolipoyl-containing residue). As shown in Figure 40, the crude chromatogram contains many products. 115 We hypothesized that during the cleavage, the dithiolane ring opened to yield the dihydrolipoamide moiety. To confirm this assumption, the fraction containing both peptides was isolated, lyophilized, and diluted with water In order to optimize the cleavage conditions to selectively obtain one or another form (dithiolane or dihydrolipoyl residue), a systematic study using LA in cleavage conditions gave the results described in Table 11. With regards to these results, it appeared difficult to find a selective cleavage strategy. Indeed, the presence of 5% TIS in TFA already yields reduced product after an hour. Presence of EDT yields a reduced product, but also an unidentified degradation product.

Thus, we decided to use the Reagent K as a conventional cleavage method and to manage the lipoylation status afterwards.

In order to obtain the lipoylated peptides with a completely reduced disulfide linkage, we used reduction with DTT119 , producing a cyclic oxidized byproduct, readily removable from the peptide by washing the reaction mixture with cold Et 2 O. This allowed us to directly control the state of the lipoylation of the peptide, as we could either oxidize it completely to the dithiolane, or reduce it to the dithiol (Figure 42). This procedure might prove useful in the biological test phase, to check if autoantibodies preferentially recognize one or the other form. However, care should be exercised with regards to the reduced Cédric Rentier, PhD thesis 66 form since it tends to be unstable and to re-oxidize quite fast whenever conditions are favorable. In Table 12 are given the analytical data for the dihydrolipoamide peptides.

Management of lipoylated peptides S-S bond in ELISA

Solid phase ELISA protocol is characterized by several steps. Each step placed our peptides in particular conditions (pH, °C, etc.) for extended periods of time before the sera of patients were applied:

-coating to the microtiter plate in carbonate buffer pH 9.6 overnight at 4°C

-blocking of the nonspecific reactive positions of the plate using Fetal Bovine

Serum (FBS)

-several washes with aqueous saline solution.

Thus, it was relevant to investigate the behavior of the lipoyl moiety in such conditions. Indeed, "S-S" or "SH SH" might lead to different Ag-Ab recognition, and both should be tested. A good characterization of the S-S bond in ELISA conditions was required to understand which Ag had being used.

The test protocol, described in Experimental part, consisted of dissolving the lipoylated peptide (1mg/ml) in sealed HPLC vials, placed at 4°C, and the execution of an UPLC-MS analysis immediately and after 1h30, 3h, 18h and 24h.

In particular, we examined the changes on the lipoyl moiety when the peptide was placed in coating conditions, using a carbonate buffer pH 9.6. Because of the complex nature of the protein matrix used in FBS buffer, analysis of the peptide in such conditions was not possible by UPLC-MS.

The initial tests were performed on peptide XVII using mercaptoethanol, a common reducing reagent used in molecular biology. We followed the evolution of the lipoyl moiety over time by UPLC-MS. This is pictured on Figure 43.

Results show that H 2 O reoxidizes the DHLA form to the LA form, but in a much slower manner than using the conditions described in Figure 41.

Addition of mercaptoethanol (data not shown in Figure 43) does not change the chromatographic profile significantly. Regarding the use of a carbonate buffer, basic conditions greatly enhance the formation of the dithiolane. This is true even at the very start of the experiment (dithiol/dithiolane ratio at 30:70), meaning that the closure is extremely rapid (oxidation is almost complete after 3 hours). Addition of mercaptoethanol decreases the rate of oxidation. At the start the ratio is roughly 40:60 (dithol/dithiolane), but it slows notably as it progresses.

The complete conversion is reached after 1 night, and not 3 hours like in the previous experiment.

However, a 50 mM concentration of mercaptoethanol is clearly not enough to provide sufficient stability to the dithol compound in an overnight incubation in these conditions. This behavior is likely due to the equilibrium between our peptide and mercaptoethanol, as shown in Figure 44: the reaction is reversible. The test protocol is the same as described above. UPLC-MS analyses were performed immediately and after 2h, 18h and 24h. We followed by UPLC-MS the behavior of a mix of peptides XII and XVIII in carbonate buffer pH 9.6 with 10mM DTT and 10mM TCEP, respectively. Results are shown in Figure 45.

Both DTT 10mM and TCEP 10mM are able to completely and immediately reduce the S-S bond. However, only the TCEP reducing reagent is able to perform an irreversible reaction because of the P=O bond formed (Figure 46). The reduced product is completely stable in basic conditions up to 24 hours.

Reaction with DTT is similar to the mercaptoethanol: reaction is an equilibrium, thus can be reverted, as shown in Figure 47.

In conclusion, the most efficient reducing agent to use in ELISA is the TCEP, as it yields the reduced dihydrolipoamide in an efficient and irreversible way. Absorbance values observed against CSF114 series are lower than the one obtained with the corresponding PDC-E2 analogues.

Importance of the peptide sequence

These findings suggest that the AA residues are required for Ab recognition:

the type I' β-turn irrelevant peptide sequence is not able to detect Abs in PBC patients, and the lipoylation alone is not sufficient.

Cédric Rentier, PhD thesis 76

N-glucosylation relevance in PBC

Peptides I, II, VIII and IX were tested for both IgG and IgM on a cohort of 65 PBC patients using SP-ELISA. This is represented in Figure 53 and Figure 53. These results demonstrate that the higher titers detected in both CSF114 and PDC-E2 series concerns Asn(Glc) peptides (orange bars in the CSF114 series, blue bars in the PDC-E2 series). Asn(GlcNAc) peptides are unable to recognize Abs in PBC patients' sera.

It is important to remember that N-glucosylated PDC-E2 could also mimic a different self-Ag modified with an aberrant N-glucosylation. For this purpose, further investigations on the involvement of N-glucosylation in PBC are required.

Use of modified peptides for PBC diagnosis

Studies on a cohort of PBC patients demonstrated that PDC-E2 sequence is mandatory (either lipoylated or not); lipoylation chirality is not significantly discriminating; and Asn(Glc) might be relevant for detection of Abs in PBC.

Based on the previous results four peptides were selected:

- Indeed, this peptide is able to recognize 63% of PBC patients, although with lower titers compared with the lipoylated peptide [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK, which is able to discriminate 37% of patients.

Interestingly, we obtained 0% positivity percentage for IgM against the unlipoylated PDC-E2 peptide in both rheumatoid arthritis and scleroderma cohorts, while OAID have 23% positivity.

IgG results, on the other hand, appear less appealing: positivity of peptides incorporating unmodified AA (Lys & Asn) or (R)-Lipoamide is lower than the one observed in IgM; and differentiation of the various sera categories using the N-glucosylated analog is poor (titers very high).

Tests of dihydrolipoylated peptides

Peptides incorporating the dithiolane moiety, with the disulfide bond oxidized, were extensively tested. However, the influence of the oxidized/reduced state of the disulfide bond of the lipoyl moiety in Ag-Ab recognition shall be answered.

Methods previously described in this thesis work well only when applied to specific situations: washes with diethyl ether (Figure 42) are not suitable with ELISA conditions/ELISA plates; and TCEP (Figure 46) cannot be applied in every ELISA buffer solution because it would change the pH (TCEP in solution tends to lower the pH).

We decided to optimize the ELISA protocol, and introduce a special washing step just before applying the sera of patients. The general idea is represented in Figure 58: after coating the peptide, a short step of washing the plate with a diluted solution of TCEP is performed before applying Abs.

Cédric Rentier, PhD thesis 86

We previously demonstrated that lipoylated peptides are quickly and efficiently reduced to the dihydrolipoylated moiety by TCEP. For each peptide, we selected 7 sera: 1 control and 6 PBC. ELISA experiments were simultaneously performed in normal and reducing conditions on the same plate. Below are shown the results in IgG (Figure 59) and IgM (Figure 60).

IgM CSF(Glc) and [Lys173]PDC-E2(167-186)-KKKK

Normal TCEP 5mM

Chapter One: Primary Biliary Cirrhosis -2. Results and discussion Cédric Rentier, PhD thesis 88 Overall, based on these preliminary results, it appears that the reduced disulfide bond is less significant in regards to interaction with antibodies. In particular, peptide XIII showed a higher tendency towards the helical structure at low HFA concentrations (5 -10%) as judged by the values of the minimum at 222 nm (Figure 63 and Figure 64). HFA/water solvent mixture is a stabilizing agent, which increases the intrinsic tendency of the amino acid

Chapter One: Primary Biliary Cirrhosis -2. Results and discussion Cédric Rentier, PhD thesis 90 sequence to fold in defined secondary structures. HFA molecules effectively provide a "teflon face" which secludes the peptide in a hydrophobic environment 120 . To further investigate this structural difference, NMR spectra of peptides XI and XIII were acquired in 10% HFA/water v/v solution.

Data obtained indicate that the region around Lys 173 tend to form a turn or a helix for both peptides. However, all parameters indicate that lipoylated peptide XIII has the highest helical content which expands itself to the residue Thr 171 preceding Lys 173 . Structure calculation using the NMR constraints indicated that a likely explanation of the observed difference is a hydrophobic interaction between lipoic acid chain on Lys 173 and Ile 176 side chain in peptide XIII (Figure 65). Existing NMR data previously reported by Howard et al. 60

Conclusion

In this chapter, we aimed to obtain molecular probes to aid in the diagnostic procedure of PBC. We took advantage of the CRA, using both organic synthesis and SPPS tools to synthesize completely characterized molecular probes, and used these probes in ELISA to evaluate their utility as Ags for the detection of AMA.

Up to now, the Ags used in the various studies are of a diverse nature, not always well characterized nor defined. Consequently, the following points were carefully considered: the AA sequence of our Ags, the PTMs introduced in our synthetic Ags, and the conformation adopted in-solution by the modified peptides.

This involved the synthesis of modified peptides, including novel Lys(N εlipoyl) Fmoc/tBu building blocks for SPPS. This permitted the selective introduction of both (R)-and (S)-enantiomers of lipoic acid on our synthetic antigens, and the role of lipoylation stereochemistry in Ab-Ag recognition in PBC was evaluated for the first time.

Other modifications such as N-glucosylation and N-glycosylation (with GlcNAc) were investigated for the hypothetical role of such PTM in autoimmunity in general with a possible role in mitochondrial processes.

Peptides synthesized included the native sequence of PDC-E2(167-186), with a C-terminal Lys-tag to enhance its solubility. We also included in our studies Diabetes diagnosis is based on several criteria shown in Table 13. It is important to note that there is an intermediate state between "normal" and "diabetic", often referred as "pre-diabetic", or impaired glucose tolerance.

Criteria for diabetes diagnosis were reevaluated on a regular basis to take in consideration these populations 124 . It represents a key stage in the natural history of type II diabetes as these people are at a much higher future risk than the general population for developing this condition. The destruction of the β-cells of the pancreas leads to a partial or complete absence of insulin production. The rate of destruction of β-cells is variable, generally quite fast in young patients, and slower in older individuals 126 . In some patients, particularly children and adolescents, one of the first manifestations of the disease is ketoacidosis, because of the absence of insulin, free fatty acids are released from adipose tissues and converted in the liver into ketones (β-hydroxybutyrate and acetoacetate) for energetic purposes. However, these ketones lower the pH of the blood, creating an acidic environment and natural buffering systems (i.e. bicarbonate buffering system) are insufficient to combat this.

Cédric Rentier, PhD thesis 99

In adults, because of the slower β-cell destruction many retain residual β-cell function; this may be sufficient to prevent ketoacidosis for a long time (several years) 127 .

In addition to the criteria for hyperglycaemia described in Table 13, pancreatic auto-Abs have been found in T1D and may have a diagnostic value. There are five types: glutamic acid decarboxylase antibodies, islet cell antibodies, insulin autoantibodies, protein tyrosine phosphatase antibodies, and zinc transporter protein.

However, this method of diagnosis has two drawbacks:

-most commercial laboratories do not have reliably sensitive or specific assays that measure all five auto-Abs -auto-Ab titers may not correlate with the course of the disease and differ among different populations of patients.

Type-2 diabetes

This form of diabetes, that represents about ~90-95% of those with diabetes, previously referred to as non-insulin-dependent diabetes or adult-onset diabetes, encompasses individuals who have insulin resistance and usually have relative (rather than absolute) insulin deficiency (Figure 69). At initial stages of the disease, and often throughout their lifetime, individuals suffering T2D do not need insulin treatment to survive. Although the specific etiologies are not known, autoimmune destruction of β-cells does not occur. Causes are thought to be a combination of genetics and environmental factors.

Cédric Rentier, PhD thesis 100 Most patients with this form of diabetes are obese, and obesity itself causes some degree of insulin resistance. This form of diabetes frequently goes undiagnosed for many years because the hyperglycemia develops gradually and during the earlier stages it is often not severe enough for the patient to notice any of the classic symptoms of diabetes.

Ketoacidosis can also occur in T2D patients with modest hyperglycemia, however, it needs to be triggered in the presence of infection or other stress.

Chapter Two: Diabetes -1. Introduction Cédric Rentier, PhD thesis 101

Post-translational modifications possibly involved in diabetes

The common factor between T1D and T2D is hyperglycaemia. This is defined as an excess of glucose in blood and biological fluids.

The four most common complications of diabetes (macrovascular disease, nephropathy, retinopathy and neuropathy) share numerous mechanisms by which hyperglycaemia can disrupt cell and organ function, with vascular dysfunction also affecting the kidneys, eyes and nervous system 128 .

Excess glucose directly stimulates the activation of the polyol pathway 129 and also the activity of mitochondria 130 and protein kinase C 131 , which results in the production of reactive oxygen species. Hyperglycaemia also leads to the formation of advanced glycation end products, which irrevocably alter the vasculature, leading to vascular stiffening owing to extensive protein crosslinking 132 .

Finally, it has also been demonstrated that over-processing of glucose into hexosamine biosynthetic pathway can cause several manifestations of diabetic complications 133 .

Chemical modifications induced by these pathways are numerous and complex.

However The consensus sequence is not only necessary for the modification, it has also been hypothesized that the sequence allows the adoption of the so-called Asparagine turn. This conformational change allows the activation of the chemically unreactive amide group 138 .

The addition of the N-linked precursor is catalyzed by an oligosaccharyltransferase in the endoplasmic reticulum 139 . After transfer of the oligosaccharide to the protein and proper folding, the oligosaccharide can then be further modified in the Golgi apparatus. This increases the variability according to the final use of the protein as shown in Figure 70. Bacteria can be involved in N-glycosylation as well. Today, many pathways by which prokaryotes can promote glycosylation mechanisms have been reported.

Considering the huge diversity of prokaryotic glycoproteins discovered in recent years, it is clear that glycosylation in these organisms is the norm rather than the exception. The discovery of a general N-glycosylation system in 138 Imperiali, B., Shannon K.L., Unno, M., Rickert, K.W. J. Am. Chem. Soc. 114, 7944-7945 (1992). 139 Silberstein, S., Gilmore, R. FASEB J. 10, 849-858 (1996).

Cédric Rentier, PhD thesis 104

Campylobacter Jejuni is an example that can represent the starting point of progress in understanding prokaryotic glycosylation 140 .

It was also shown a few years ago that Haemophilus influenzae produces a glycosylated adhesion protein termed HMW1 with an unusual N-linked carbohydrate modification 141 .

In contrast to the enzymatic attachment of sugar units to proteins, catalyzed by OST, there is another pathway for addition of sugars to proteins: the glycation reaction.

Glycation of proteins occurs through spontaneous and reversible condensation of a reducing sugar and the free amino group of a protein to form a Schiff base.

This then undergoes a rearrangement into the more stable ketoamine known also as the Amadori product142 . In the case of glucose, the initially formed Schiff base rearranges into the more stable 1-deoxyfructosyl moiety.

Subsequent dehydration, condensation, fragmentation, oxidation, and cyclization reactions lead to the irreversible formation of advanced glycation end products. This is shown in Figure 71.

Even though proteins contain many surface amino groups, only a few are preferentially glycated. This observation was explained when the identification of glycated amino groups in proteins with known three-dimensional structure revealed that glycation preferably occurs at amino groups that are either close to an imidazole moiety or part of a Lysine doublet. Proximity (~5 Å) of an amino group to an imidazole moiety is the strongest predictor of susceptibility to glycation 143 . This site specificity of protein glycation is the consequence of localized acid-base catalysis of the aldimineketoamine tautomerization 144,145 . This makes the glycation phenomenon sequence-dependent.

O-Glycosylation and phosphorylation

Protein glycosylation is structurally divided into O-and N-linked glycosylation. In the former, the first sugar of the oligosaccharide is attached to the hydroxyl group of Ser, Thr, Hyl, or Hyp residues. Uridine-diphosphate-N-acetylglucosamine is the final product of a metabolic pathway of glucose, called hexosamine biosynthetic pathway.

When glucose enters into the cell, a small percentage (~5%) is processed through this pathway (Figure 72). Glucosamine-6-phosphate is generated via an amino-transfer reaction between fructose-6-phosphate and the amino acid glutamine by the action of the rate-limiting enzyme, glucosamine:fructose-6phosphate aminotransferase.

This pathway also intersects with production of acetyl-CoA, the product of glycolysis, to give rise to N-acetylglucosamine.

O-GlcNAc transferase and O-GlcNAcase enzymes respectively introduce or remove O-GlcNAc modification from proteins (Figure 73). However, they do not seem to require consensus sequences on their protein substrates. Rather, their action is likely to be driven by protein-protein interactions 147 .

O-GlcNAc has been shown to be extremely important in cell cycle progression, transcription, intracellular signaling, nutrient sensing, and neuronal plasticity.

An excess of glucose in the organism might be the starting point for an over activation of the hexosamine biosynthetic pathway, thus abnormal levels of proteins modified with O-GlcNAc.

Recent studies demonstrated that the modulation of these cellular pathways and Sites of O-GlcNAcylation may often be directly at or located proximal to the same serine or threonine residues alternatively used by kinases, or the sugar may also occur at sites on a polypeptide distant from phosphoryation sites.

Like O-GlcNAcylation, phosphorylation is directly involved in the regulation of many cellular processes151 .

Thus, dysregulation of O-GlcNAcylation may provoke a change in phosphorylation, as shown in Figure 74. The first enzymatic phosphorylation of proteins was described in 1954 by Kennedy's group 152 . Phosphorylation usually occurs on serine, threonine, tyrosine and histidine residues.

The addition of a phosphate (PO 4

3-

) group to a polar side chain of an amino acid residue can turn a hydrophobic portion of a protein into a polar and extremely hydrophilic site. In this way it can introduce a conformational change in the structure of the protein via interaction with other hydrophobic and hydrophilic residues within the protein.

A phosphate group is given by adenosine triphosphate, yielding a phosphorylated residue and adenosine diphosphate as highlighted in Figure 75.

These changes are catalyzed by two enzymes: kinases and phosphatases.

Objectives of the project

In line with the concept of CRA, we propose the synthesis of several modified peptides for detection of specific auto-Abs in the sera of diabetic patients.

Cédric Rentier, PhD thesis 110

Possible PTMs involved in diabetes have been mentioned in the introduction of this section, and are related closely to products obtained, directly (direct addition of sugar moieties) or indirectly (biochemical pathways triggered by hyperglycaemia). Thus, it is relevant to introduce these PTMs as chemical modifications on synthetic peptides. The ability of such peptides to be used as molecular probes of AID will be assessed by ELISA. Specifically, the Nglycosylation (N-GlcNAc), the O-glycosylation (O-GlcNAc), the glycation (deoxyFru) and the phosphorylation (Phos) will be looked at (Figure 76). Autoantibodies were also found against some mitochondrial proteins, i.e.

153 Allina, J., Hu, B., Sullivan, D.M., Fiel, M.I., Thung, S.N., Bronk, S.F., Huebert, R.C., Van de Water, J., LaRusso, N.F., Gershwin, M.E., Gores, G.J., Odin, J.A. J. Autoimmun. glycerophosphate dehydrogenase, involved in oxidative phosphorylation (step following the glycolysis) to produce adenosine triphosphate 155 .

In addition to the PDC-E2(167-186) sequence, the CSF114 was also used as a synthetic scaffold. Its optimized β-turn conformation could serve to maximize the exposition of PTMs to the external media, in order to assess a hypothetical specificity towards auto-Abs of the chemical modifications introduced.

155 Fabregat, M.E., Gasa, R., Rodriguez, C., Novials, A., Gallart, T., Malaisse, W.J., Gomis, R. Diabetes Res. Clin. Pract. 38, 115-121 (1997).

Cédric Rentier, PhD thesis 112

Results and discussion

Sequence selection

Our laboratory extensively demonstrated that synthetic peptides (i.e., CSF114)

exposing optimally an aberrant PTM can detect antibodies in autoimmune diseases. In particular the rationale of a possible role of "aberrant modifications" in triggering autoimmune conditions is included in the introduction, and we aimed to use the concept of CRA to further investigate this hypothesis.

As discussed in the introduction, the peptide sequences of interest were the CSF114 and the PDC-E2(167-186).

In the case of diabetes (either T1D or T2D), the CSF114 could prove useful exposing at the best an aberrant post-translational modification. Its optimized β-turn conformation maximizes the exposition of the modified amino acid at the top of the hairpin to the external media, thus facilitating the possible epitope-Ab interaction.

The PDC-E2(167-186) was tested as a candidate peptide for Abs detection because of its strong involvement at mitochondrial level in the energetic cycle of cells and glucose metabolism.

Therefore, using Fmoc/tBu SPPS we synthesized a series of peptides based either on the PDC-E2(167-186) or on the designed β-turn structure CSF114 in order to identify a PTM possibly recognized by Abs in diabetes.

Modifications introduced into peptides

The chemical modifications introduced into peptides were also briefly discussed in the Introduction. In the Table 14 A more detailed discussion on lipoylation and N-glucosylation is available in Chapter One (PBC).

N-glycosylation and O-glycosylation were introduced using the corresponding protected N-acetylglucosamines coupled to an Asn and a Ser, respectively. Glycation comes from the completely protected BB Fmoc-L-Lys(Boc)(2,3:4,5di-O-isopropylidene-1-deoxyfructopyranosyl)-OH.

Phosphorylation was inserted on the side chain of a Ser, using a phosphorylated amino acid, protected by a Bzl group.

With the exception of lipoylation and N-glucosylation all these building blocks are commercial and were used in SPPS like the classic AAs. However, due to their cost (more expensive than unmodified AA), they were introduced using less equivalents during the syntheses.

Chapter Two: Diabetes -2. Results and discussion Cédric Rentier, PhD thesis 114

Peptide synthesis

The previous parts of this work highlighted the peptide sequences and the modified AAs that were selected to be used in the construction of molecular probes directed towards diabetes diagnostics.

Syntheses of CSF114 and PDC-E2(167-186)-KKKK analogues containing Ser(PO 3 H 2 ) and Ser(GlcNAc) were undertaken with the assistance of the Master student Martina Crulli, at the PeptLab@UniFi of the University of Florence.

Peptides were synthesized by SPPS using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using either PLS 4×4 (Advanced ChemTech), Apex 396 (Aapptec), LibrtyBlue (CEM) or SyroII (Biotage).

The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.31 mmol/g for PDC-E2(167-186)-KKKK peptides; 0.7mmol/g for CSF114 peptides).

Modified amino acids described in Table 14, e.g. Ser(PO 3 H 2 ) or Asn(GlcNAc)

were introduced using the protected building-blocks suitable for Fmoc/tBu SPPS, e.g. Fmoc-L-Ser(PO 3 Bzl,H)-OH and Fmoc-L-Asn[β-GlcNAc(OAc) 3 ]-OH. Couplings were performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of DIPEA, for 1 hour at room temperature.

Each coupling was monitored by the Kaiser test156 and double couplings were performed if necessary.

All the linear peptides were purified by semi-preparative RP-HPLC, as described in General procedure for purification, and characterized by ESI-MS.

Analytical data of peptides not previously described in Chapter One are reported in Table 15 for CSF analogues and in Glycosylated building blocks were introduced as their peracetylated counterparts (Table 14). Deacetylation of the sugar moieties was performed in basic conditions (pH 11-12) using a fresh sodium methanolate solution. This is a common procedure in carbohydrate chemistry, widely known as Zemplen deacetylation.

Zemplen et al. 157 described the reaction between acetyl groups and sodium methanolate (sodium methanolate in methanol solution), which results in methyl acetate formation (Figure 77). The transesterification usually starts immediately upon methanolate addition and proceeds rapidly. For this reason, the deprotection reaction has to be monitored over time by HPLC-MS. The reaction was stopped when the elimination product appeared; neutralization was performed with HCl conc. and the mixture was immediately lyophilized after solvent evaporation. Increased cleavage time gives higher yields of unprotected peptide. This behavior of the glycated moiety was similar between PDC-E2(167-186) and CSF114 analogues and appeared to be sequence-independent. Thus, cleavage of glycated peptides was routinely performed in 6 hours instead of 3 in a classical protocol.

H 3 C O O R - O-CH 3 H 3 C O O - R O CH 3 MeOH Na + H 3 C O O R O CH 3 -MeO -+ Na H H 3 C O O CH 3 + HO-R

Phosphorylated peptides

Phosphorylated serine is introduced in peptide sequences using a suitably protected AA (represented Figure 81), which was synthesized for the first time Despite the presence of the Bzl group, it was previously reported in the literature 162 that during the course of the peptide synthesis this AA could undergo a β-elimination process similar to O-linked glycosyl moieties 163 (Figure 82). Removal of the Fmoc-protecting group is responsible for the increase in acidity of the α-carbon and subsequent susceptibility to attack by piperidine. 160 Wakamiya, T., Nishida, T., Togashi, R., Saruta, K., Yasuoka, J., Kusumoto, S. Bull. Chem. Soc. Jap. 69, 465-468 (1996). 161 Vorherr T., Bannwarth W. Bioorg. Med. Chem. Lett. This mechanism proceeds via piperidinyl oxidation of the phosphoseryl αcarbon, leading to the generation of a dehydroalanyl residue. Subsequent nucleophilic attack by piperidine on the dehydroalanyl group then leads to 3-

(1-piperidinyl)alanyl formation.
This is a unique feature of mono-benzylphosphorylated Ser as this side reaction is not observed with mono-benzylphosphorylated Thr/Tyr (although both dibenzyl-protected Ser/Thr undergo piperidine-promoted β-elimination164 ). It appears to be promoted by the use of MW-deprotection conditions and reduced with the use of primary amines, such as cyclohexylamine.

However, using classical protocol with 20% piperidine in DMF for Fmocdeprotections (5 min + 15 min at room temperature) did not yield any critical amounts of β-elimination products in our peptide syntheses.

SP-ELISA

The Peptides reported in Table 17 below were tested on 31 type-1 diabetes sera.

Data distribution for IgG (Figure 83) and IgM (Figure 84) are reported hereafter.

Cédric Rentier, PhD thesis 122 17.

From the data reported in Figure 83, it appears that both peptides (IX and II) 17.

containing
In IgM (Figure 84) the highest response is directed towards lipoylated PDC-E2(167-186)-KKKK analogues (peptides XII, XIV, XIII); and, to a lesser extent, to [(S)-Lipoamide-Lys 7 ]CSF114 (peptide VII) and N-linked GlcNAc PDC peptide (peptide X).

Interestingly, lipoylation does not seem a relevant modification when introduced in CSF114 sequence. PDC-E2(167-186)KKKK peptides display overall higher absorbance values compared to the corresponding CSF analogues.

To verify if antibodies detected in patients' sera could have a diagnostic value for type-1 diabetes, a second cohort of 30 NBDs' sera was tested as a control on selected peptides. Data distribution is reported in Figure 85 for IgG and in 17.

In IgG differences between the two populations are not significant. IgG values of [Asn(Glc) 173 ]PDC-E2(167-186)-KKKK (peptide IX) are shown in Figure 57.

The most significant differences between the two populations were obtained for IgM with the following peptides:

- The area under the ROC curve quantifies the overall ability of the assay to discriminate patients and controls. ROC curve areas are typically between 0.5 and 1.0. The smaller the P value (or significance value) is, the higher will be the ability of the assay to distinguish between the two groups, i.e. T1D patients and healthy controls.

From this ROC based analysis, the four peptides resulted to be able to detect specific antibodies in T1D sera according to different cut-off values for the assays. The most interesting peptide was [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (peptide XIII). Considering a diagnostic cut-off value for the assay of 0.881, specific IgM were revealed in the 42% of T1D sera with a specificity of 90% and with a high statistical significance (p value <0.0001). A third cohort of 30 sera of type-2 diabetes was tested on 5 selected peptides and IgM distribution is reported below (Figure 88). Results indicated that peptide 20 significantly discriminates T1D from T2D patients' sera, with a sensitivity of 32.3% and a specificity of 96.5%.

In a nutshell, several points should be highlighted regarding the results of ELISA tests:

-the Ab levels observed are notably higher in IgM than IgG for a majority of the peptides studied in T1D sera.

-for the study of Ab interactions with our synthetic Ags, the PDC-E2(167-186)-KKKK sequence seems more relevant than the designed β-turn structure of CSF114. This may indicate a biological relevance for this mitochondrial epitope.

-based on IgM levels, the peptides selected are the [(R)-Lipoamide- -Lipoyl moiety stereochemistry does not appear to have a particular impact on Ab detection in patients' sera. However, the (R)-enantiomer is the best discriminating antigen because it detects the lowest IgM titer in NBD sera.

-O-linked glycosylation vs N-linked glycosylation: the introduction of Ser(GlcNAc) seems to have greater impact than Asn(GlcNAc) on the PDC-E2 sequence, in particular concerning differentiation between T1D and T2D populations in IgM. This highlights the importance of sugar structure (GlcNAc is better than 1-deoxyfructopyranosyl or Glc moieties) but also on sugarpeptide linkage.

-Phosphorylated PDC-E2 specificity and sensitivity provide evidence for a possible role of this PTM in diabetes, through an aberrant protein phosphorylation triggered by deregulation of O-GlcNAc/Phos equilibrium.

Conclusion

This chapter investigated the role of various aberrant PTMs possibly involved in diabetes, either type-1 or type-2. Hyperglycemia may affect self proteins in several ways and induce aberrant glycosylation, glucosylation, glycation and phosphorylation. Mitochondrial proteins may also be implicated in diabetes because of the high degree of interaction between mitochondria and glucose metabolism / energetic cycle of cells.

We proposed the collection of synthetic peptides including the aforementioned chemical modifications in strategic positions on selected sequences: the CSF114, which is an optimally designed β-turn conformation; and the PDC-E2(167-186) fragment, which is a mitochondrial protein component of the pyruvate dehydrogenase complex.

These peptides were tested in SP-ELISA with a cohort of diabetes sera, either type-1 (immune-mediated form) or type-2 and NBD sera as control, to assess for peptide XXVIII (Figure 87 and Figure 93). On the other hand sensitivity is 36% for peptide XIII, 29% for peptide XXVII and 32% for peptide XXVIII in the case of T1D vs T2D (Figure 94).

This highlights the importance of the native mitochondrial PDC-E2(167-186) protein, as well as the hypothetical involvement of lipoylation, O-glycosylation and phosphorylation in T1D as aberrant PTMs exposed in vivo.

Our study does not provide any efficient synthetic probe for T2D diagnosis.

However, [Ser 173 (GlcNHAc)]PDC-E2(167-186)-KKKK, despite a mediocre differentiation between T2D and NBD populations, presents higher Ab titres.

Thus, it could be used to correlate the clinical status of a patient (insulinresistance) to its Ab titre. Future experiments will focus on establishing a possible correlation between these two parameters.

Perspectives of this project include more studies on this disease, with the synthesis of other peptides including hyperglycaemia-related chemical modifications. Indeed, advanced glycation end products can be of a diverse nature, including molecular cross-links between various residues (i.e.

pentosidine results from the covalent linkage between Lys and Arg). This may provide new insights into the mechanisms of this complex disease. Symptoms and signs vary depending on the location and severity of the plaques. MS is a complex disease that can exist in various clinical subtypes but generally has a relapsing-remitting course, often developing into secondary progressive MS.

Relapsing-remitting MS is characterized by clearly defined disease relapses with full recovery or with sequels and residual deficit upon recovery; in the period between disease relapses patients are stable with lack of disease progression166 .

Secondary progressive MS is defined as a disease that has "an initial relapsingremitting disease course followed by progression with or without occasional relapses, and minor remissions".

Central nervous system inflammation is the primary cause of nervous system damage in MS. The exact factors that initiate inflammation are unknown, but it is generally believed that MS is caused by environmental factors in a Cédric Rentier, PhD thesis 136 genetically susceptible host that trigger a T-cell autoimmune response against the central nervous system 167 .

It has been suggested that the adaptive immune system drives the early stages of MS and the innate immunity drives the progressive stage 168 , although the degree to which the adaptive and the innate immune systems affect each other is uncertain.

The kynurenine pathway

The kynurenine pathway (KP) is a metabolic pathway leading to the production of nicotinamide adenine dinucleotide from the degradation of the essential amino acid Trp 169 .

It is represented in Figure 96 and is characterized by several steps:

-action of indoleamine-2,3-dioxygenase (IDO) or tryptophan- -N-formylkynurenine, by the action of kynurenine formamidase, loses its Nformyl group to give the kynurenine (Kyn). It is the first key metabolite intermediate.

-Kyn can be further processed by enzymes into several metabolites, in particular kynurenic acid (KynA) and 3-hydroxykynurenine (3-OH-Kyn).

-by successive transformations, 3-OH-Kyn ultimately yields quinolinic acid (QuinA), which is a key intermediate in the production of nicotinamide adenine dinucleotide.

Two different enzymes initiate the catabolism of Trp, and both enzymes act as the rate-limiting step for the kynurenine metabolic pathway: TDO and IDO.

Located primarily in the liver, TDO is induced by tryptophan and metabolic steroids, and is highly specific for the substrate tryptophan 170,171,172 .

IDO is found in many tissues, is induced by the pro-inflammatory cytokine interferon-gamma during an immune response 173 and has less substrate specificity.

Until the year 2000, metabolites of the KP were mainly known for their neuroactive properties. Indeed, QuinA is a selective agonist for glutamate receptors of N-methyl-D-aspartate 174 , while KynA is an antagonist of these receptors as well 175 . More importantly in the context of the KP, KynA can attenuate efficiently the excitotoxicity of the QuinA: while QuinA can induce cell toxicity, KynA is generally considered as a neuroprotective compound. However, according to some studies, Kyn and its metabolites could have much broader biological functions. Most intriguingly, Trp and its degradation products have been found to possess remarkable immunomodulatory properties.

Several metabolites such as 3-OH-Kyn and QuinA, when applied exogenously in micromolar concentrations, suppress the proliferation of activated T cells and thus affect or facilitate tolerance to non-harmful antigens 177,178 . B cells and natural killer cells also succumb after being exposed to these metabolites 179 . This is a significant phenomenon because the endogenous formation of Kyn and its downstream metabolites is greatly enhanced in immunocompromised situations due to the activation of IDO by interferon-gamma and other cytokines 180,181 .

If the immune system is stimulated, and cytokines are released by leukocytes, IDO is strongly upregulated, thus converting systemic tryptophan to Nformylkynurenine 182,183 . N-formylkynurenine is rapidly broken down to Kyn in many tissues and the bloodstream. Later, the Kyn will be processed into its metabolites by the KP.

The KP is involved in several neurodegenerative disorders, migraine, pain syndromes and autoimmune diseases such as Huntington's disease and MS. It is worthy noticing that modulation of kynurenine metabolism might be of therapeutic value 184 .

Cédric Rentier, PhD thesis 140

In eye lenses, studies show that kynurenines are prone to deamination and oxidation 185 . The deamination results in formation of α, β-unsaturated ketones that chemically react and modify lens proteins. Such reactions occur mostly at cysteinyl, histidinyl, and lysyl residues 186 . The predominant modifications appear to be Michael adducts, as demonstrated by using Abs specific for Kynmodified proteins 187 .

Deregulations of the Kyn metabolites levels in the organism have been associated with some diseases:

-KynA deregulations are associated with schizophrenia 188 , epilepsy 189 and in several neurological diseases such as MS, amyotrophic lateral sclerosis, chronic brain injury and acquired immune deficiency syndrome-dementia 190 .

-QuinA deregulations have been found in Alzheimer's disease, Parkinson's disease, acquired immune deficiency syndrome-dementia and in amyotrophic lateral sclerosis 190,191,192 In the course of diseases, the balance in KP can be affected and thus result in exacerbation of neurotoxicity of QuinA, or a decrease in neuroprotection by KynA.

In physiological conditions, QuinA is present in nanomolar concentrations and used as a substrate by cells to synthesize nicotinamide adenine dinucleotide.

However, QuinA is predominantly synthesized by infiltrating macrophages and microglia, which associates the neurodegenerative effects of kynurenine pathway activation with neuroinflammation. Thus, during neuroinflammation, QuinA may be produced in excess and kill brain cells.

Cédric Rentier, PhD thesis 141

In particular, in MS, there is some evidence that KP metabolism is misregulated and might be involved in the pathology of the disease, in both aspects of neurotoxicity and immunomodulation. In vivo, it is the first stable metabolite of Trp and is obtained through the KP, and is obtained after catabolitic cleavage of Trp by IDO and TDO, and subsequent deformylation of the N-formylkynurenine (Figure 96).

Kynurenine pathway involvement in Multiple Sclerosis

In vitro, its synthesis was first described in the 50s and consisted of the condensation of diethyl acetamidomalonate sodium salt with o-nitrophenacyl bromide; followed by hydrolysis, decarboxylation and reduction to obtain the racemic kynurenine (Figure 98) 202 . Another pathway explored was the direct ozonolysis of N-acetyl Trp (Figure 99). However, this method suffers from variable yields and various sideproducts which are difficult to separate are obtained In a recent paper, Kleijn et al. 203 tried to apply this strategy on protected L-Trp.

They managed to obtain the desired L-Kyn, however the formation of many over-or under-oxidized by-products did not allow a convenient purification. However, the limitation of this method lies in the complete inactivity of the enzyme when the N α is protected (Boc, Ac) and the free NH 3 + not able to interact anymore with the enzyme. This would probably also hamper the possible use of this strategy for an on-resin Trp indole ring cleavage, although this has not been tested yet.

The convergent method was explored, and most recent works rely on an onresin ozonolysis to selectively cleave the indole ring of Trp to obtain Kyn. This The process, shown in Figure 106, allows a quick and efficient oxidation. The selectivity is good and no other AA is modified, excepted Met residues, that can be oxidized either to sulfoxides or sulfones.

is
Cédric Rentier, PhD thesis 148

Objectives of the project

Literature shows an increased interest in KP, Kyn and its various metabolites, for their role in neurotoxicity, neuroprotection and immunomodulation. While

QuinA is a neurotoxic compound, KynA is neuroprotective, but both are produced from Trp metabolism, and in particular Kyn.

Regarding MS, there is strong evidence of an involvement of KP metabolic products, although their exact role and all the cellular interactions (and their consequences) remain unclear.

In MS patients Trp level may be disregulated, thus leading to an abnormally high level of Kyn in the organism during the course of the disease, modifications in the Trp/Kyn ratio, as well as increased or decreased levels of KynA and QuinA. It might also be possible to modify, through IDO activation,

Trp residues directly on proteins, leading to the production of Kyn-containing aberrantly modified proteins.

Thus, it is not absurd to hypothesize that during MS anti-Kyn auto-Abs may be produced. If such auto-Abs exist, then they can be used as possible biomarkers of MS.

We propose to take advantage of the CRA, using SPPS tools to synthesize completely characterized molecular probes, and use these molecular probes in ELISA to evaluate their utility as synthetic Ags for the detection of anti-Kyn Abs. This project deals with the synthesis and the use of such modified peptides containing Kynurenine.

The selected sequence was the CSF114, which has been developed and extensively studied by our group for its ability to form β-turn structures. A more detailed discussion is available in the first part of the manuscript.

Chapter Three: Multiple Sclerosis -2. Results and discussion Cédric Rentier, PhD thesis 149

After a brief introduction to the Kyn synthetic pathways, we will discuss the synthesis of such kynureninated peptides, the building block selected for SPPS, and also the synthesis and characterization of the peptides.

Results and discussion

Synthesis of Kyn-containing CSF114 analogues

CSF114 and its optimized β-turn conformation maximize the exposition of the modified amino acid at the top of the hairpin to the external media, thus facilitating the possible epitope-Ab interaction. The sequence is represented in The aim of this part of the project was to synthesize Kyn-containing peptides that were tested on MS patients' sera to detect autoAbs directed against Kyn moiety.

The N-glucosylated peptide CSF114(Glc) was extensively studied and shown to recognize specific autoantibodies in a subtype of patients suffering from MS.

In particular it has been validated as clinical tool for the early diagnosis of the disease. It is used as a reference to assess the efficiency of the new Kyncontaining peptides as molecular probes.

[Kyn 7 ]CSF114. The CSF114 sequence was modified on the top of the β-turn (position 7) with Kyn.

[Kyn 18 ]CSF114. The CSF114 sequence was modified at position 18 with Kyn.

Originally position 18 was with a Trp residue, this position was modified to serve as a peptide not exposing optimally the PTM Kyn.

Chapter Three: Multiple Sclerosis -2. Results and discussion Cédric Rentier, PhD thesis 150

The presence of a Met in the sequence of the CSF114 does not allow the use of an on-resin ozonolysis strategy, as described in Figure 106. Moreover, experimental conditions such as the use of ozone and very low temperatures (-78°C) require special equipment and handling.

On the contrary, the use of the BB approach, using a suitable Kyn for Fmoc/tBu SPPS is in line with previous works performed in our laboratory and is overall a safer and more controllable strategy.

Fmoc-L-Kyn-OH (Figure 108) is a commercial AA, ready to use and does not require any particular protecting group. Indeed, the nitrogen located on the aniline ring is strongly deactivated. Peptides were synthesized by SPPS using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using SyroII (Biotage).

The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.31 mmol/g).

Modified AA Kyn was manually introduced using the protected building-block suitable for Fmoc/tBu SPPS, Fmoc-L-Kyn-OH. Couplings were performed with an excess of 3 eq of modified AA, 3 eq of HATU as the activator, and 4 eq of DIPEA, for 1 hour at room temperature.

The last six couplings, after the introduction of AA in position 7, were performed as double couplings. Due to the presence of Kyn, Kaiser test cannot be performed to check couplings. Thus, microcleavages were performed at "strategic points" during peptide synthesis: before coupling a modified AA, and after its coupling. In this way, it was possible to control the synthesis of the peptide and eventually detect any defects in the sequence prior to introducing the modified AA (expensive, thus used cautiously), and to ensure its correct insertion afterwards.

SP-ELISA

The 2 peptides, XXIX and XXX, corresponding to the kynureninated CSF114 analogues were screened on MS patients' sera, to evaluate the role of kynurenine as a possible epitope. The original N-glycosylated CSF114(Glc) was used as a reference for its demonstrated ability to be selectively recognized by Abs in MS patients' sera.

Cédric Rentier, PhD thesis 152

Below are shown the results of indirect SP-ELISA (Figure 109 and Figure 110). Experiments were carried out as described in the Experimental part.

The following sera (Table 19) were used in triplicate with a dilution of 1:100:

Normal Blood Donors In conclusion, our kynurenine-containing peptides are not efficient molecular probes compared to CSF114(Glc) for the detection of autoAbs in MS patients' sera. Ab titres were systematically lower for peptides XIXX and XXX than those observed for the N-glycosylated peptide, in the case of MS sera both for IgG and for IgM.

These preliminary SP-ELISA results highlighted some positive Alzheimer sera for both [Kyn 18 ]CSF114 and CSF114(Glc). Importance of such PTMs and their role in Alzheimer's disease remains to be investigated and should include the application of a more statistically significant number of patients' sera.

Conclusion

The objective of this chapter was to investigate the possible role of kynurenine as a possibly relevant PTM in MS.

Evidence in the literature clearly showed a strong involvement of the kynurenine pathway and its metabolites, i.e. kynurenine, kynurenic acid and quinolinic acid in neurodegenerative diseases, such as MS.

Thus, we synthesized kynurenine-containing peptides. The sequence was selected based on the results previously published in the literature, and takes advantage of the type I' β-turn structure in-solution of the CSF114. It is a 21mer which exposes its modified amino acid at the top of the hairpin, thus maximizing exposition of the side-chain to the external media.

The Kyn modification was introduced as a building-block following the Fmoc/tBu strategy. Other valuable strategies such as on-resin ozonolysis and use of enzymes were considered, but introduction as a building-block suited more the need for a better control of the modification inserted. It was also less demanding in regards to particular procedures or equipments.

The two modified Kyn-containing peptides, [Kyn 7 ]CSF114 and [Kyn 18 ]CSF114 were tested as molecular probes for the detection of autoAbs in MS patients' sera as compared to the reference peptide CSF114(Glc), an Nglycosylated peptide, validated tool for the early diagnostic of MS.

SP-ELISA results confirmed the ability of CSF114(Glc) to be used as probe in the in-vitro study for the detection of specific antibodies in MS patients' sera.

A high IgG antibody level against the Kyn-containing peptides was revealed in only one MS serum. Among the 4 Alzheimer sera tested, 2 gave a positive (IgG) result for the peptide [Kyn 18 ]CSF114 and 1 (IgM) for both Kyncontaining peptides although the significance of these result has yet to be confirmed.

Cédric Rentier, PhD thesis 156

As a future direction for this study, we could envisage using other PTMs inserted in our molecular probes. Indeed, kynurenic acid and quinolinic acid may be more relevant. Nonetheless, their structure is different than kynurenine's, and integration of these chemical modifications into peptides would require optimization. Both compounds are acids so, for example, they might be inserted on the side chain of a Lys.

The significance of the preliminary results obtained using Alzheimer's sera should also be clarified. In order to achieve this, SP-ELISA using a larger cohort of sera should be performed.

Conclusions

Cédric Rentier, PhD thesis 157

Conclusions

Peptides are unique and versatile compounds with a huge potential in diagnostics associated with ELISA. They can be produced by efficient synthetic strategies as univocally characterized molecules. In the context of immune-mediated diseases, the use of such synthetic peptides mimicking antigens possibly involved in vivo in the formation of autoantibodies can guarantee specificity and selectivity of recognition. This overcomes existing limitations in the case of the use of recombinant proteins.

This Ph.D. work used the concept of "Chemical Reverse Approach", which lies in the conception and synthesis of modified peptides as synthetic Ags to "fish out" specific autoAbs as biomarkers of selected AIDs. The purpose is to obtain ready-to-use diagnostic tests for AIDs of interest.

After a general introduction on the immune system and autoimmunity, this containing peptides although the significance of these results has yet to be confirmed.

Perspectives for this study should rely on the synthesis of peptides containing various kynurenine-analogues, based on the final metabolites of KP, KynA and QuinA, which have a different molecular structure.

Experimental Part

Materials and Methods

The chemicals were purchased from Sigma-Aldrich and used without further purification. Peptide-synthesis grade N,N-dimethylformamide (DMF) was from Scharlau (Barcelona, Spain) or Sigma-Aldrich (France). HPLC-grade CH 3 CN was purchased from Carlo Erba (Italy) or Sigma-Aldrich (France). HATU, TBTU, protected amino acids and resins were obtained from Iris Biotech AG (Marktredwitz, Germany). Activation product containing triazine was obtained from EspiKem S.r.l (Florence, Italy).

The following amino acid side-chain-protecting groups were used: OtBu (Asp, Glu), tBu (Ser, Thr), Pbf (Arg), Trt (Gln, His) and Boc (Lys). Optical rotations were determined using a JASCO P1010 polarimeter. High resolution mass sprecrometry analyses were performed on a JEOL JMS-GC Mate II.

Melting points were obtained on a Büchi apparatus and are uncorrected.

Microwave

General procedure for Solid Phase Peptide Synthesis

Unless otherwise mentioned, SPPS was carried out following the protocols described below.

Washings: DMF (3 × 3 min).

Cédric Rentier, PhD thesis 164

Coupling reaction: Fmoc amino acids (4 eq), TBTU (4 eq) and NMM (5 eq) were dissolved in DMF with a final dilution of 1 mL DMF for 100 mg of resin.

Couplings were performed for 45 min at room temperature.

Washings

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 ( NMR 1 H and 13 C spectra are identical for all three lipoyl pentafluorophenyl esters. To a solution of DMTMM-BF 4 (328 mg, 1 mmol) in DMF (4 ml) cooled down to 0 °C is added, under stirring, (R,S)-α-lipoic acid (206 mg, 1 mmol, 1 eq) and NMM (110 µl, 1 mmol, 1 eq). After 90 min of stirring at 0 °C is added Fmoc-L-Lys-OH (368 mg, 1 mmol, 1 eq). The reaction solution is stirred 2 h more at 0 °C then at room temperature, and checked by TLC (DCM/MeOH 9:1). After one night, the reaction mixture is concentrated under reduced pressure, and the resulting yellow oil is dissolved in DCM (90 ml). Organic phase is washed successively with water (30 ml), 0.5 M KHSO 4 (30 ml), water (30 ml), 0.5 M NaHCO 3 (30 ml), water (30 ml), brine (30 ml), and water (30 ml). Organic phases are pooled together, dried over MgSO4, filtered and concentrated.

Synthesis of

Resulting yellow solid is purified by normal phase FCC using a gradient of DCM/MeOH (95:5 to 90:10). Fractions are checked with TLC (DCM/MeOH 9:1), pooled and evaporated under reduced pressure to yield the desired product as a yellow solid. Fmoc/tBu SPPS, Fmoc-L-Asn[β-Glc(OAc) 4 ]-OH and Fmoc-L-Asn(Ac 3 AcNHβ-Glc)-OH. Couplings are performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of DIPEA, for 1 hour at r.t. Cleavages and purifications are performed as previously reported 30 .

Analytical data are given in Table 9.

Deprotection of the hydroxyl functions of sugar moieties linked to the Nglycosylated peptides is performed as described in the procedure for deprotection of Glc moieties. Purification and characterization of the peptides were performed as described in General procedure for purification and characterization. Gradients for SLCF and semi-preparative HPLC purifications are given Table 22. Solvent systems Analytical data are given in Table 9. Purification and characterization of the peptides were performed as described in General procedure for purification and characterization. Gradients for SLCF and semi-preparative HPLC purifications are given Table 23. Solvent systems used were: A (0.1% TFA in H 2 O) and B (0.1 % TFA in CH 3 CN).

Synthesis of CSF114 lipoylated analogues

Deprotection of the hydroxyl functions of sugar moieties linked to the Nglycosylated peptides is performed as described in the procedure for deprotection of Glc moieties. Analytical data are given in Table 10. The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.31 mmol/g).

Modified amino acids (R,S)-Lipoamide, (R)-Lipoamide or (S)-Lipoamide are introduced by manual batch SPPS with PLS 4×4 using the protected building-blocks suitable for Fmoc/tBu SPPS, 4, 5 or 6. Couplings are performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of DIPEA, for 1 hour at r.t.

Cleavages were performed as described in General procedure for cleavages and micro-cleavages using a mixture of TFA/thioanisole/H 2 O/phenol/EDT (Reagent K, 82.5:5:5:2.5 v:v:v:v:v).

Purification and characterization of the peptides were performed as described in General procedure for purification and characterization. Gradients for SLCF and semi-preparative HPLC purifications are given Table 24 Analytical data are given in Table 10. Lipoyl-lysine chain of peptide XIII was built using the Insight Builder module (Accelrys Software Inc., San Diego). Atomic potentials and charges were assigned using the consistent valence force field (CVFF) 224 . The conformational space of peptide I was sampled through 100 cycles of restrained Simulated Annealing (ε = 1r). In Simulated Annealing, the temperature is altered in time increments from an initial temperature to a final temperature by adjusting the kinetic energy of the structure (by rescaling the velocities of the atoms). The following protocol was applied: the system was heated up to 1500 K over 2000 fs (time step = 1.0 fs); the temperature of 1500 (XXV) are synthesized by SPPS using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using either PLS 4×4 or SyroII.

K
The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.31 mmol/g).

Modified amino acid (1-deoxyfrucropyranosyl)Lys is introduced by manual batch SPPS with PLS 4×4 using the protected building-blocks suitable for Fmoc/tBu SPPS, Fmoc-L-Lys(Boc)(2,3:4,5-di-O-isopropylidene-1deoxyfructopyranosyl)-OH. Couplings are performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of NMM, for 1 hour at r.t.

Cleavages were performed as described in General procedure for cleavages and micro-cleavages using a mixture of TFA/TIS/H 2 O (95:2.5:2.5 v:v:v) for 6 hours at r.t.

Purification and characterization of the peptides were performed as described in General procedure for purification and characterization, but only purified by semi-preparative HPLC (SLCF not performed). Gradients for semi-preparative HPLC purifications are given Table 25 The resin used was a Fmoc-Lys(Boc)-Wang TG (loading 0.24 mmol/g).

Modified amino acids O-glycosylated or phosphorylated are introduced by manual batch SPPS with PLS 4×4 using the protected building-blocks suitable for Fmoc/tBu SPPS, Fmoc-L-Ser[β-GlcNAc(OAc) 3 ]-OH and Fmoc-L-Ser(PO 3 Bzl,H)-OH. Couplings are performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of DIPEA, for 45 min at r.t.

Cleavages were performed as described in General procedure for cleavages and micro-cleavages using a mixture of TFA/TIS/H 2 O (95:2.5:2.5 v:v:v).

Purification and characterization of the peptides were performed as described in General procedure for purification and characterization. Gradients for SLCF and semi-preparative HPLC purifications are given Table 26.

Solvent systems used were: A (0.1% TFA in H using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using SyroII.

The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.31 mmol/g).

Modified amino acid Kynurenine is introduced by manual batch SPPS with PLS 4×4 using the protected building-blocks suitable for Fmoc/tBu SPPS, Fmoc-L-Kyn-OH. Couplings are performed with an excess of 4 eq of modified amino acid, 4 eq of HATU as activator, and 5 eq of NMM, for 1 hour at r.t.

Cleavages were performed as described in General procedure for cleavages and micro-cleavages using a mixture of TFA/TIS/H 2 O/EDT (94:2:2:2 v:v:v:v).

Purification and characterization of the peptides were performed as described in General procedure for purification and characterization, but only purified by semi-preparative HPLC (SLCF not performed). Gradients for semi-preparative HPLC purifications are given Table 27. Solvent systems used were: A (0.1%

TFA in H 2 O) and B (0.1 % TFA in CH 3 CN). Analytical data are given in Table 18.

Introduction

Among the many technological applications of peptide-based materials recently described in the literature, we pioneered the use of new modified peptides as immunological probes in diagnostic devices detecting antibodies circulating in human sera, as biomarkers of autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis. The selection of these peptides has been obtained using the novel concept of 'chemical reverse approach', in which we perform the synthesis of post-translationally modified peptides as mimetics of (neo)antigens in autoimmune diseases [1]. We are currently extending this approach to several other immune-mediated diseases, and thus we need reliable methods to prepare modified amino acid building blocks. Primary Biliary Cirrhosis (PBC) is an autoimmune chronic cholestatic liver disease characterized by destruction of intrahepatic small bile ducts. PBC is usually characterized by the presence of antimitochondrial antibodies (AMA) circulating in blood in 90-95% of patients long before the first clinical symptoms and that appear to be directed against a family of the 2-oxo acid dehydrogenase complexes (2-OADC) located in the inner mitochondrial membrane [2,3]. The pyruvate dehydrogenase-E2 complex (PDC-E2) has been recognized as the immunodominant autoantigen in which the lipoylated moiety seems to be an important component of the antigenic epitope. The role of PDC is to proceed to the oxidative decarboxylation of pyruvates through the transfer of acetyl groups to CoenzymeA (CoA). This is done in particular by the E2 component, which uses a Lys(N ε -dihydrolipoyl) moiety as a swinging arm [4]. Up to now, causes triggering PBC remain unclear, although self reactivity could be induced by xenobiotics responsible of aberrant posttranslational modifications of the lipoyl moiety and/or the lipoyl domain and loss of immune tolerance [5-7].

The immunodominant sequence of the PDC-E2 fragment (167-186) is contained into the inner lipoylated domain of the human pyruvate dehydrogenase complex, as described by Coppel et al. [8] and Van de Water et al. [9]. Nuclear magnetic resonance (NMR) experiments previously reported by Howard et al. on the PDC-E2 protein showed that the structure of the inner lipoyl domain is a type I β-turn [10]. In order to evaluate possibly important parameters in the recognition of the lipoylated antigen by the serum autoantibodies, we investigated the importance of the lipoylation per se by synthesizing native PDC-E2(167-186) peptide, and we compared it with a synthetic structure-based designed peptide, with regards to the minimal epitope that could be either continuous (sequence-related) or discontinuous (conformation-related).

Following this observation, we selected a peptide structure previously developed in our laboratory: CSF114, a 21-mer peptide displaying a type I' β-turn conformation in solution, originally designed to serve as a molecular probe able to selectively detect autoantibodies in multiple sclerosis [11-13]. Its optimized β-turn conformation maximizes the exposure of the modified amino acid at the top of the hairpin to the external media, thus facilitating the possible epitope-antibody interaction.

Two are the possible strategies for the introduction of α-lipoic acid (LA) into peptide sequences: the convergent and the building block approach. After LA synthesis reported in 1954 by Stoksad group [14], Lys(N ε -lipoyl) was reported for the first time a few years later by Nawa et al. [15]. In this case, yields are quite low and the products obtained are not adequate for liquid or solid-phase peptide synthesis (SPPS). In the case of the convergent approach [16,17] possible side products hampered the characterization of the desired modified peptide. Therefore, it was essential to develop adequately protected Lys(N ε -lipoyl) building blocks for SPPS via the Fmoc/tBu strategy.

LA can be introduced in peptide sequences as (R)-enantiomer, (S)-enantiomer, or the (R,S)-racemic mixture, although only the (R)enantiomer is endogenously synthesized in vivo once linked to proteins [18,19]. However, it is accepted that stereochemistry can play an important role in biological recognition. Therefore, in order to investigate the role of chirality of the lipoyl moiety in antigenantibody recognition in PBC we synthesize both native and designed peptide sequences modified with (R)-or (S)-lipoylated lysine residues. To the best of our knowledge, none previously afforded the possible role of aberrant stereochemistry in immune dysregulation.

Therefore, we developed an efficient synthetic protocol to prepare modified peptides bearing both diastereoisomers of the

N ε -(1,2-dithiolane-3-pentanoyl)-N α -Fmoc-L-Lys-OH. Accord- ingly, herein we describe the first synthesis of (S)-α-[[(9H- fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R,S)- yl)-1-oxopentyl]-Lys-OH, (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl] amino]-ε-[5-(1,2-dithiolan-3(R)-yl)-1-oxopentyl]-Lys-OH and (S)- α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3 (
S)-yl)-1-oxopentyl]-Lys-OH, to be used as building blocks ready for SPPS via Fmoc/tBu strategy. We also describe the use of these building blocks in the synthesis of lipoylated peptides, designed to be used as PBC diagnostics.

Materials and Methods

General Comments

Unless otherwise mentioned, all the reagents were purchased from commercial source. 1 H NMR (400 MHz) and 13 C NMR (100 MHz) were measured on a JEOL ECX400 spectrometer. 1 H NMR (200 MHz) and 13 C NMR (50 MHz) were measured on a Varian Gemini spectrometer. Chemical shifts of 1 H and 13 C NMR are expressed in parts per million downfield from tetramethylsilane (TMS, δ = 0) and are referenced to residual undeuterated chloroform (7.26 and 77 ppm, respectively) as internal standard.

Flash column chromatography (FCC) was performed on SDS 60 Å (40-63 μm) silica gel, employing a mixture of the specified solvents as eluent. Normal-phase thin-layer chromatography (TLC) was performed on Macherey-Nagel (Macherey-Nagel GmbH, Düren, Germany) Alugram XtraSil (Macherey-Nagel silica gel 60 Å F254) plates. Silica TLC plates were analyzed under UV light (254 nm) and stained with a 0.2% solution of ninhydrine in ethanol.

Optical rotations were determined using a JASCO (JASCO Analytical Instruments, Tokyo, Japan) P1010 polarimeter. HRMS analyses were performed on a JEOL JMS-GC Mate II. Melting points were obtained on a Büchi apparatus and are uncorrected.

Chromatographic analysis was performed on a Waters (Waters Corporation, Milford, MA, USA) Acquity Ultra-Performance Liquid Chromatography (UPLC) apparatus, which was equipped with a Waters Acquity UPLC BEH C18 column (1.7 μm, 2.1 × 50 mm). Solvent system was comprised of (A) 0.1% TFA in H 2 O and (B) 0.1% TFA in CH 3 CN.

Synthesis of α-lipoic acid-OPfp esters

To a solution of the (R,S)-, (R)-or (S)-α-lipoic acid (500 mg, 2.42 mmol) dissolved in dioxane (13 ml) at room temperature, pentafluorophenol (446 mg, 2.42 mmol, 1 eq) is added under stirring. The mixture is cooled down to 0 °C, then N,N-dicyclohexylcarbodiimide (500 mg, 2.42 mmol, 1 eq) is added. The reaction is stirred overnight at room temperature. After checking with TLC (DCM/MeOH 7:1), the reaction mixture is filtered and the organic phase is concentrated under reduced pressure. NMR 1 H and 13 C spectra are identical for all three lipoyl pentafluorophenyl esters.

Synthesis of (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-

ε-[5-(1,2-dithiolan-3(R,S)-yl)-1-oxopentyl]-Lys-OH (1a,

1b, and 1c) using lipoyl pentafluorophenyl ester route

To a solution of the (R,S)-(1 g, 2.68 mmol), (R)-(924 mg, 2.48 mmol), or (S)-α-lipoic acid-OPfp ester (872 mg, 2.34 mmol) dissolved in CH 3 CN (20 ml), (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-Lys-OH (1 eq), HOBt monohydrate (1 eq) and DIPEA until pH 7-8 are successively added under stirring. The reaction is stirred and followed by TLC (DCM/MeOH 9:1). After 24 h, solvents are evaporated under reduced pressure. The resulting brown oil is dissolved in DCM and the organic phase is successively washed twice with 0.5 M KHSO 4 , then three times with H 2 O. Organic phase is collected, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure.

The resulting yellow oil is purified by normal phase FCC using a gradient of DCM/MeOH (100 : 0 to 90 : 10). Fractions are checked by TLC (DCM/MeOH 9 : 1) and collected, to yield the desired product as a yellow solid. NMR 1 H (400 MHz) and NMR 13 C (100 MHz) are identical to the racemic compound (refer to the succeeding paragraphs).

(S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2- dithiolan-3(R)-yl)-1-oxopentyl]-Lys-OH (1a) TLC (DCM/MeOH 9:1) R f = 0.5 R t (
(S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2- dithiolan-3(R,S)-yl)-1-oxopentyl]-Lys-OH (1c) TLC (DCM/MeOH 9:1) R f = 0.5 R t (

Synthesis of (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-

ε-[5-(1,2-dithiolan-3(R,S)-yl)-1-oxopentyl]-Lys-OH (1c) using HATU/DIPEA
To a solution of the (R,S)-α-lipoic acid (206 mg, 1 mmol) dissolved in DMF (10 ml) at room temperature, HATU (380 mg, 1 mmol, 1 eq) and DIPEA (350 μL, 2 mmol, 2 eq) are added under stirring. After 10 min, (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-Lys-OH (368 mg, 1 mmol, 1 eq) is added.

The reaction is stirred at room temperature, and checked either by TLC (DCM/MeOH 9:1) or by UPLC (50 to 100% B in A over 3 min). After completion, DMF is evaporated under reduced pressure, and the resulting brown oil is diluted in DCM (50 ml). Organic phase is successively washed with 0.5 M KHSO 4 (2 × 30 ml), H 2 O (2 × 30 ml), and brine (2 × 30 ml), then dried over MgSO 4 , filtered and concentrated under reduced pressure. The resulting orange oil is purified by normal phase FCC using a gradient of DCM/MeOH (95:5 to 92:8). Fractions are checked with TLC (DCM/ MeOH 9:1), pooled together and evaporated under reduced pressure to yield the desired product as a yellow solid.

Synthesis of (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]- ε-[5-(1,2-dithiolan-3(R,S)-yl)-1-oxopentyl]-Lys-OH (1c) using Oxyma/DIC
To a solution of the (R,S)-α-lipoic acid (206 mg, 1 mmol) and Oxyma (142 mg, 1 mmol, 1 eq) dissolved in a mixture DMF/DCM 1 : 1 (5 ml) at room temperature under argon, is N,N-diisopropylcarbodiimide (126 mg, 1 mmol, 1 eq) is added under stirring. After 10 min, a suspension of (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-Lys-OH (368 mg, 1 mmol, 1 eq) in DMF/DCM (1 : 1. 5 ml) is added. The reaction is stirred overnight at room temperature and checked by TLC (DCM/MeOH 9:1). The reaction mixture is then concentrated under reduced pressure and the resulting yellow oil is diluted with DCM (100 ml). The organic phase is successively washed with 1 N HCl (2 × 30 ml), 1 N NaHCO 3 (2 × 30 ml), and brine (2 × 30 ml). Organic phases are pooled together, dried over MgSO 4 , filtered, and concentrated. The resulting yellow gelatin is purified by normal phase FCC using a gradient of DCM/MeOH (95 : 5 to 90 : 10). Fractions are checked with TLC (DCM/MeOH 9 : 1), pooled together and evaporated under reduced pressure to yield the desired product as a yellow solid.

Synthesis of (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-

ε-[5-(1,2-dithiolan-3(R,S)-yl)-1-oxopentyl]-Lys-OH (1c) using DMTMM-BF 4
To a solution of DMTMM-BF 4 (328 mg, 1 mmol) in DMF (4 ml) cooled down to 0 °C is added, under stirring, racemic α-lipoic acid (206 mg, 1 mmol, 1 eq) and NMM (110 μl, 1 mmol, 1 eq). After 90 min stirring at 0 °C, (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-Lys-OH (368 mg, 1 mmol, 1 eq) is added. The reaction solution is stirred 2 h more at 0 °C then at room temperature, and checked by TLC (DCM/MeOH 9:1). After one night, the reaction mixture is concentrated under reduced pressure, and the resulting yellow oil is dissolved in DCM (90 ml). The organic phase is washed successively with water (30 ml), 0.5 M KHSO 4 (30 ml), water (30 ml), 0.5 M NaHCO 3 (30 ml), water (30 ml), brine (30 ml), and water (30 ml) dried over MgSO 4 , filtered and concentrated. Resulting yellow solid is purified by normal phase FCC using a gradient of DCM/MeOH (95 : 5 to 90 : 10). Fractions are checked with TLC (DCM/MeOH 9:1), pooled and evaporated under reduced pressure to yield the desired product as a yellow solid.

Peptide Synthesis

General procedure for peptide synthesis CSF114 sequence (TPRVERxGHSVFLAPYGWMVK) and PDC-E2(167-186) sequence (AEIETDxATIGFEVQEEGYL) were synthesized by SPPS, performed by automatic peptide synthesizer Syro II, following the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy.

The peptides were prepared starting from a Fmoc-Lys(Boc)-Wang resin (peptides 2-3c: 0.7 mmol/g; peptides 4-8c: 0.3 mmol/g). Fmoc deprotections (5 min + 15 min) were performed with 20% piperidine in DMF. Fmoc amino acids were used as 0.5 M in DMF solutions. Coupling reactions were performed with 0.5 M TBTU/DMF (4 eq), 0.5 M amino acids/DMF (4 eq) and 1 M DIPEA/NMP (5 eq).

Modified amino acids (2.5 eq) were coupled manually for 1 h, using HATU (2.5 eq) and NMM (3.5 eq) in DMF.

Cleavage from the resin and side-chain deprotection were performed at room temperature for 3 h using the following mixtures (1 ml/100 mg of resin): TFA/TIS/H 2 O (95:2.5:2.5) for peptides 4-7; TFA/TIS/H 2 O/EDT (94:2:2:2) for peptide 2; TFA/EDT/thioanisole/ H 2 O/phenol (82.5:5:2.5:5:5) for peptides 3a-c and 8a-c. The resin was filtered off and the solution was concentrated by flushing N 2 . The peptides were precipitated from cold Et 2 O, centrifuged and lyophilized. Deprotection of the hydroxyl functions of sugar moieties linked to the peptides 5 and 6 was performed adding 0.1 M methanolic NaOMe solution until pH 12 to a solution of the lyophilized peptides in dry MeOH (1 ml/100 mg of resin). After 3 h, the reaction was quenched by adding conc. HCl until pH 7, the solvent was evaporated under vacuum and the residue lyophilized. All peptides were purified by semipreparative Reverse-Phase High-Performance Liquid Chromatography Waters using a Phenomenex (Phenomenex Inc., Torrance, CA, USA) Jupiter column C18 (10 μm, 250 × 10 mm), at 4 ml/min to obtain a purity of at least 95%.

Peptides characterization was performed by UPLC using a Waters ACQUITY UPLC coupled to a single quadrupole ESI-MS (Waters 3100 Mass Detector) using a BEH C18 (1.7 μm 2.1 × 50 mm) column at 35 °C, with a flow rate of 0.6 ml/min. The analytical data are reported in Table 4.

Procedure for disulfide bond reduction

The peptide is dissolved in milliQ water (1 mg/ml) and DLdithiothreitol is added (100 eq). After overnight stirring, the complete reduction is controlled by Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and the peptide is lyophilized. The solid obtained is washed with cold Et 2 O, centrifuged, supernatant is removed. This procedure is repeated once, and then the peptide is lyophilized.

Results and Discussion

In this work, we optimized the synthesis of diastereomerically pure lipoylated lysine derivatives, to be used as building blocks for the preparation of CSF114 and PDC-E2(167-186)-derived peptides by SPPS.

The synthesis of diastereomeric lipoylated lysines 1a and 1b takes advantage of commercially available (S)-(-)-α-lipoic acid and (R)-(+)-α-lipoic acid, as highly pure starting materials, with the main drawback being their cost. The initially selected synthetic strategy for this building block uses the lipoyl pentafluorophenyl ester as a reactive intermediate [20] to facilitate the coupling to the side chain of the lysine. The α-lipoic acid is esterified overnight in the presence of pentafluorophenol and N, N-dicyclohexylcarbodiimide to obtain the lipoyl pentafluorophenyl esters, which are coupled to the side chain of a Fmoc-protected lysine to yield the desired building blocks (Scheme 1, Pathway A).

The yields of the coupling step with Pathway A are quite low, ca 20-25% (Table 1). This might be due to the low solubility of the lipoyl pentafluorophenyl ester intermediates. In fact, these species have been found to be poorly soluble in various solvents (ethyl acetate, acetonitrile, dichloromethane, dioxane, THF, DMF, water, and 20% acetic acid aqueous solution), possibly because of a polymerization of the building block through a ring-opening mechanism [21].

The building blocks 1a and 1b have been fully characterized by 1D and 2D NMR, High-Performance Liquid Chromatography Mass Spectrometry (HPLC-MS) and optical rotations. So far, because of the two chiral centers being separated by the long flexible lateral chains acting like a 'swinging arm' [4], only the optical rotation allows to differentiate the two molecules. Separation and characterization of the lipoylated building blocks remain a challenging issue. Very recently, Kates et al. [22] reporting the synthesis of the N-terminal lipoylated dipeptide Glu-Ala experienced dithiolane ring-opening polymerization as we probably did, and had characterization issues. Therefore, diastereomerically pure building blocks separation from the racemic mixture appeared a rather difficult strategy.

The subsequent optimization steps were performed on the racemic α-lipoic acid for cost reasons. Although Pathway A, using lipoyl pentafluorophenyl ester, yield the desired products, the unsatisfactory yields led us to use an 'in-situ' activation, as shown in Scheme 1-Pathway B, with HATU [23,24], despite its recent reclassification as a class 1 explosive category [25,26], Oxyma [27], and DMTMM-BF 4 [28].

The 'in-situ' activation involves a short preactivation of the lipoic acid in the presence of a base and a stoichiometric amount of coupling reagent to obtain its activated ester form (depending of the coupling method used), then adding the Fmoc-protected lysine to yield, after one night, the desired lipoylated lysine (1c) in various yields. Conditions and results are summarized in the following paragraphs in Table 2, Entries 1-4.

The best results were obtained using HATU as a coupling reagent (Table 2, Entry 1), with a yield of 60%. Coupling with Oxyma and DMTMM-BF 4 gave poor yields (Table 2, Entries 2-4). Longer coupling times with COOH-unprotected lysine led, in every case, to the formation of various amounts of a side product, according Scheme 1. General scheme synthesis of lipoylated lysines. to UPLC-MS spectra an ε-branched dipeptide Lys[N ε -Lys(N ε -lipoyl)] is possibly formed as a side product occurring from an undesired coupling of a second Lys residue on Lys(N ε -lipoyl) (m/z = 907) as reported in Table 2 for each method. Such side product is hardly separable from the single-coupling adduct in normalphase chromatography. Although reverse-phase FCC is possible, the low solubility of the mixture in the solvent system acetonitrile/water does not make this a convenient solution. To address this limitation, the reaction was carried out in the same conditions as described previously for Entry 1, with a careful monitoring over time using UPLC. Lys-Lys side product formation, that is extremely rapid, is carefully monitored, in order to quench the reaction as soon as it does not progress anymore.

In those conditions, within 3 h, the lipoylated lysine is obtained in a one-pot reaction with 53% yield and less than 4% side product (Table 2, Entry 5), thus rendering the purification by normal phase FCC more convenient.

With the choice of the method for the synthesis of lipoylated lysines (low yields but easier purification, Pathway A; or higher yields and faster reaction time, despite side products and harder purification, Pathway B), the building blocks, as reported in Table 3, are ready to be introduced in peptide sequences. Thus, we synthesized a series of peptides based either on the native immunodominant epitope PDC-E2(167-186) or on the irrelevant β-turn structure CSF114 in order to identify the minimal epitope recognized by antimitochondrial antibodies. In fact, the minimal epitope involved in antibody recognition could be either the unique modified amino acid (and its aberrant stereochemistry), the amino acid sequence (linear epitope), or the secondary structure (conformational epitope).

Accordingly, the three synthetic Lys(N ε -lipoyl) building blocks were successfully introduced into the selected peptide sequences. In particular (R)-Lipoamide-Lys, (S)-Lipoamide-Lys, and (R,S)-Lipoamide-Lys were introduced as possible minimal epitopes involved in PBC autoantibodies. Asn(Glc) and Asn(GlcNAc) were also considered as possible aberrant post-translational modifications because of some recent findings concerning N-glycosylation in mitochondria [29], as well as for their involvement in autoimmunity (as we and other authors extensively demonstrated in some immune-mediated diseases).

Peptides incorporating free Lys and Asn residues were synthesized as controls.

The newly synthesized peptides are reported in Table 4. The building blocks reported in Table 3 were introduced in position 7 in the CSF114 sequence and in position 173 in the PDC-E2(167-186) fragment. Synthesis of CSF114 peptides not listed in Table 4 have been already described in previously published papers [11]. Previously obtained results suggested that the strongly hydrophobic (167-186) fragment of PDC-E2 sequence would be difficultly solubilized in water. The use of a short cationic tag of four lysine residues [30] in C-terminus of all our PDC-E2 peptides allowed us to enhance the solubility of these products.

During the synthesis and purification process of our lipoylated peptides, we came across a particular problem of stability of the dithiolane moiety. Cleavage using the TFA/TIS/H 2 O cocktail [31] yielded the peptide as a mixture of the desired product (m/z 1472.7, [M + 2H] 2+ ) and a peptide with a +2 amu (m/z 1473.7, [M + 2H] 2+ ), with UPLC retention times almost identical. We hypothesized that during the cleavage, the dithiolane ring opened to yield the dihydrolipoamide moiety. To confirm this assumption, the fraction containing both peptides was isolated, lyophilized, and diluted with water (1 mg/10 ml) under stirring in an open vessel. Progressive re-oxidation was monitored through UPLC, and completed in 12 h. Cleavages performed with the addition of EDT, or with Reagent K [32], did not allow obtaining one single peptide (either the dithiolan-or dihydrolipoyl-containing residue). In order to obtain the lipoylated peptides with the completely reduced disulfide linkage, we used DTT as reducing agent [33], which produces a cyclic oxidized byproduct, readily removable by washing the reaction mixture with cold Et 2 O. This allowed us to control directly on the peptide, the state of the lipoylation, as we could either oxidize it completely to the dithiolane, or reduce it to the dithiol (Scheme 2). This procedure might prove useful in the biological test phase, to check if autoantibodies preferentially recognize one or another form, although care must be exercised on the reduced form because it tends to be unstable and to re-oxidize quite fast whenever conditions are favorable.

Conclusion

In this paper, the synthesis of a modified amino acid, the Fmocprotected lipoylated lysine, was described. We successfully obtained and characterized the two diastereomeric building blocks compatible with Fmoc/tBu SPPS strategy, designing two synthetic routes to obtain the Lys(N ε -lipoyl) building blocks. The first strategy is based on the use of the lipoyl pentafluorophenyl ester as a reactive intermediate, with low yields but easier purifications, while the second one consists of a one-pot reaction with 'in-situ' activation, completed in a shorter time with moderate yields, but leading to side products slightly more difficult to remove. The lipoylated building blocks thus prepared were successfully introduced in selected peptide sequences by SPPS to obtain a series of peptides derived from CSF114 and PDC-E2(167-186) sequences. These peptides will serve as immunological probes for the detection of autoantibodies in sera of patients suffering from PBC and possibly other selected immune-mediated diseases. The poor solubility of the PDC-E2(167-186) sequence was improved by C-terminal elongation with a cationic sequence comprised of 4 consecutive Lys residues (K4). We successfully overcame the partial reduction of the dithiolane during the peptide cleavage from the resin and deprotection step by developing a rapid and efficient method with which the deprotected mixture of oxidized and reduced peptide is subjected to either treatment with DTT or exposure to air leading to the fully reduced or fully oxidized forms of the final product, respectively. Scheme 2. On-peptide redox handling of the lipoyl moiety.

■ INTRODUCTION

Primary biliary cirrhosis (PBC) is an immune-mediated chronic cholestatic liver disease characterized by the progressive destruction of small interlobular bile ducts, associated with vanishing bile ducts, periportal inflammation, and progressive fibrosis. The disease ultimately leads to cirrhosis and liver failure, thus requiring liver transplantation. 1 PBC is a multifactorial disease, being triggered by environmental exposure in the context of a susceptible genetic background, 2 as well illustrated by the incomplete concordance in monozygotic twins. 3 The diagnosis of PBC relies on the detection of serum antimitochondrial antibodies (AMA) at indirect immunofluorescence (IIF), being found in 95% of cases with high specificity 4 and constituting one of the three diagnostic criteria. 5 AMA are tested using whole rat kidney tissue as the substrate, enabling the detection of a heterogeneous population of autoantibodies directed against a complex set of proteins. Remarkable efforts have been devoted to the definition of the specific molecular targets of these antibodies, recently reviewed by Selmi et al., 6 reaching the largely accepted conclusion that AMA are directed against members of the 2-oxo-acid dehydrogenase complex (2-OADC). 7 The 2-OADC is a multienzyme complex essential in energy metabolism, including several components relevant in the context of PBC: the pyruvate dehydrogenase complex (PDC), the branched chain oxo-acid dehydrogenase complex (BCOADC), and the oxo-glutarate dehydrogenase complex (OGDC), each consisting of three subunits, E1, E2, and E3. Of note, E2 and E3 share the presence of one or more domains in which lysine is attached to lipoic acid, essential for the catalytic functions. In this complex scenario, PDC-E2 is considered the major AMA autoantigen, and the immunodominant epitope is mapped to the inner lipoyl domain, comprising the amino acid residues surrounding the lipoylated lysine residue in position 173, with the lipoamide function considered necessary and/or sufficient for antigen recognition. 6 Indeed, studies based on bacterial lipoyl domains indicate that the presence of the lipoyl moiety is essential for effective recognition by PBC sera, 8,9 while conflicting data were obtained using synthetic peptides. [10][11][12] The first attempt to use synthetic peptides to map PDC-E2 epitopes was reported by Van De Water et al., 10 which synthesized peptides encompassing distinct hydrophilic peaks of the protein according to the classical approach of the hydrophilicity plot. 13 The obtained peptides were tested for their ability to inhibit in a plate assay PBC patients sera binding to PDC-E2 expressed in Escherichia coli. The only active peptide among the six tested was a 20-mer (AEIETDKATIG-FEVQEEGYL) corresponding to PDC-E2 residues 167-184. Even if this peptide was synthesized without lipoylation of the relevant Lys 173 residue, it was able to inhibit, although only at a relatively high concentration, serum binding to PDC-E2, obtained in a lipoylated form in bacterial cells. Conversely, Tuaillon et al. 11 prepared and tested the lipoylated version of synthetic peptide PDC-E2(167-184), showing that it was able to recognize in an immunoenzymatic assay about 50% of AMA positive PBC sera, but only when it was covalently linked to ovalbumin (OVA), while the free lipoylated peptide detected only 7% of positive sera and the corresponding unlipoylated peptide was inactive. More recently, Braun et al. 12 reported similar low levels of reactivity using both lipoylated and unlipoylated free PDC-E2(167-184) peptides (11% and 7% positivity, respectively), while the reactivity of OVA-conjugated peptides was found to be stronger. Interestingly, the unlipoylated conjugated peptide was much more reactive then the lipoylated ones (44% vs 22% IgG and 57% vs 31% IgM positivity, respectively). The importance of this peptide was also underscored by a conformational study, in which the structure of the PDC-E2 inner lipoyl domain expressed in E. coli was analyzed using nuclear magnetic resonance (NMR) spectroscopy, showing that the relevant lipoyl-lysine residue is prominently exposed on the tip of a tight type I β-turn. 14 Because the development of simple synthetic peptide antigens mimicking the main epitopes remains an unmet need, we report herein a serological study based on unequivocally lipoylated synthetic peptide directly tested in solid-phase enzyme-linked immunoassay (SP-ELISA), with the final aim of developing a panel of well-defined immunological probes able to detect specific autoantibodies in PBC patients sera.

■ RESULTS AND DISCUSSION

Peptides Design and Synthesis. We focused our attention on the inner lipoyl domain of PDC-E2, previously described as the primary autoantibody target in sera of patients affected by PBC. We initially developed a robust synthetic strategy to prepare peptides bearing a lipoyl moiety of welldefined chirality on lysine side chain. This method, based on a building block approach, was reported elsewhere. 15 Once the possibility to prepare unequivocally lipoylated peptides was established, we realized with preliminary immunological assays that our target sequence, i.e. [lipoamide-Lys 173 ]PDC-E2(167-186), was too lipophilic to be obtained in large quantity at a sufficient purity level to be efficiently used in immunoenzymatic assays. In fact, previous immunological studies based on this lipoylated peptide were performed using OVA-conjugated peptides. 11,12 Because we believe that conjugations of antigenic peptides to a carrier protein may modify the relevant epitope of the peptide and certainly hampers a correct interpretation of the immunological results, due to the presence of anticarrier antibodies, as observed also in the previously reported studies, 11,12 we decided to add a tetra-lysine solubilizing tag to the C-terminus of our peptides. 15,16 Because, to the best of our knowledge, the role on autoantibody recognition of the lipoyl chain chirality has not been addressed in previous studies, we prepared the two enantiomers of the immunodominant peptide, i.e. [(R)-lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK and [(S)-lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (entries I a n d I I , r e s p e c t i v e l y , i n T a b l e 1 ; sequence A 167 EIETDK 173 ATIGFEVQEEGYL 186 ), as well as the corresponding Lys-tagged unlipoylated peptide, PDC-E2(167-186)-KKKK (III). Moreover, to study the role in autoantibody recognition of the amino acid sequence surrounding the lipoyl moiety and, most importantly, of its conformational features in the native PDC-E2, we designed a peptide in which a lipoylated lysine residue was inserted on an amino acid scaffold that we have previously exploited for its ability to expose a relevant epitope in the context of a SP-ELISA. 17 In fact, we have shown that this peptide, termed CSF114 (sequence TPRVERXGHSV-FLAPYGWMVK), is characterized by a type I β-turn conformation centered on position 7, 18 and therefore we The peptides were prepared by solid-phase peptide synthesis (SPPS), using the two diastereoisomers of the protected lipoylated building block Fmoc-L-Lys(N ε -lipoyl)-OH, as previously described. 15 Briefly, both enantiomers of α-lipoic acid were coupled to the side chain of Fmoc-L-Lys to yield the 1.

Immunological Studies. The ability of peptides I-V to detect specific antibodies in human sera was tested by SP-ELISA using 50 AMA positive sera, 15 AMA negative sera from patients otherwise fulfilling the international criteria, 55 sera from normal blood donors (NBD), 22 sera from patients with rheumatoid arthritis (RA), and 12 sera from patients with scleroderma (SSC). IgG and IgM measurements were performed separately, and the normal range for antibody reactivities were determined using a cutoff value calculated for each antigen as the mean value of the NBD absorbance plus 3 standard deviation.

Role of Lipoic Acid Chiral Center. Peptides I and II were preliminary tested on 30 PBC patients' sera to reveal possible differences in antibody responses (both IgG and IgM) to lipoamide (R) and (S) enantiomers linked to the native PDC-E2(167-186) sequence. The data distribution reported in Figure 1 does not show significant differences between the two enantiomers for both IgG and IgM (unpaired t tests with Welch's correction P > 0.5 and 0.4, respectively), thus allowing us to assume that the lipoamide chirality has no influence on the antibody recognition.

Role of Lipoamide. To test the role of lipoamide moiety on antibody recognition, we subsequently compared the activity of the lipoylated PDC peptide I with those of its unlipoylated counterpart III and of the designed peptide IV, bearing a lipoylated lysine residue exposed on the tip of a β-turn. In light of the immunological equivalence of the two lipoamide enantiomers showed above, we used for this part of the study only the native R enantiomer. Each antigen was tested in parallel on the AMA positive and NBD groups in order to calculate a cutoff value, as described above. We also tested as a control the unlipoylated designed peptide V. The data distribution reported in Figure 2 indicate that, in the case of IgM (panel A), peptides I, III, and IV discriminate between patients and NBD sera because the respective data distributions are significantly different, as evaluated by unpaired t tests with Welch's correction.

In particular, when considering IgM the values above the cutoff are 19/50 (38%) in the case of the native antigen [(R)lipoamide-Lys 173 ]PDC-E2(167-186) (I), 34/50 (68%) for the unlipoylated PDC peptide III, and 10/50 (20%) for the designed lipoylated peptide IV. At variance, the unlipoylated counterpart of IV, i.e., peptide V, is unable to discriminate between patients and controls. In total, 37/50 sera (74%) are positive to at least one peptide. As far as IgG are concerned (Figure 2B), only the two PDC peptides I and III significantly discriminate between patients and controls, showing however a lower sensitivity, as compared to IgM. In fact we observe 20/50 (40%) positive IgG with peptide I, but only 15/50 (30%) with the unlipoylated peptide III.

When looking at the IgM cross-reactivity of the 37 positive sera among different peptides (Venn diagram, Figure 3A), we observe that 16 patients sera recognize both the native lipoylated PDC peptide I and the unlipoylated peptide III, while the latter is able to recognize as much as 18 sera which are not recognized by the native peptide I. In turn, only three sera were recognized by the lipoylated PDC peptide I but not by its unlipoylated counterpart III, thus indicating that only a few IgM antibodies appear to recognize an epitope including the lipoamide moiety. The role of this portion of the antigen can be further analyzed by considering the reactivity of the designed peptide IV, bearing a lipoamide residue on a sequence unrelated to PDC. Interestingly, we observe a large crossreactivity between the two lipoylated peptides (nine sera), but most of these sera (eight) are recognized also by the unlipoylated PDC peptide III, and, most importantly, no sera were specifically recognized by the designed lipoylated peptide IV. Taken together these data confirm the marginal role of lipoamide in IgM antibody recognition because a large majority of sera are detected by the unlipoylated peptide, while only a few are recognized only by the lipoylated PDC peptide and none is recognized exclusively by the unrelated lipoylated peptide.

IgG cross-reactivity analysis (Figure 3B) indicates that 15 out of 20 positive sera are recognized by both lipoylated and unlipoylated PDC peptides I and III. Interestingly, none of the positive sera is recognized exclusively by the unlipoylated peptide, while only five sera appear to react solely with the lipoylated PDC peptide, thus showing to recognize an epitope including the lipoamide moiety.

D

These observations are in line with previously reported results, showing that the synthetic unlipoylated peptide PDC-E2(167-184) is able to inhibit PBC patients sera binding to PDC-E2 10 and to react directly with 57% (IgM) and 44% (IgG) patients sera when conjugated to a carrier protein (OVA). 12 Conformational Analysis. Conformation of peptide I and of the corresponding unlipoylated peptide III was analyzed by circular dichroism (CD) and solution NMR. CD spectra of the two peptides acquired in water solution indicate a random coil conformation for both peptides (Figure S1, Supporting Information). The addition of a structure-stabilizing agent like hexafluoroacetone (HFA) clearly induced a helical conformation in the two peptides (Figure S1). In particular, peptide I showed a higher tendency to the helical structure at low HFA concentrations (5-10%) as judged by the values of the minimum at 222 nm and maximum at 192 nm (Figure 4).

To gain further details about this structural difference, NMR spectra of peptides I and III were acquired in HFA/water 10% v/v solution. Many NMR parameters ( 3 J HN-Hα coupling constants, amide proton temperature coefficients, H α proton chemical shifts, and NOE contacts) indicate that the region from Lys 173 have a tendency to the turn or helical formation in both peptides (Tables S1,S2, and Figures S2,S3, Supporting Information).

The three-bond coupling constant between the intraresidual alpha and amide protons ( 3 J HN-Hα ) is very useful for secondary structure determinations as it can be directly related to the backbone dihedral angle φ. In particular, coupling constant values <6 Hz can be related to right-handed helix (φ ∼ -60°). 19 Residues from Asp 172 to Gln 181 or from Thr 175 to Glu 183 were in this 3 J HN-Hα range for peptides I and III, respectively. Amide protons involved in hydrogen bond usually show temperature coefficients (Δδ/ΔT) more positive than -4.6 ppm/K while amide protons not forming hydrogen bond show temperature coefficients significantly decreased, to around -7.1 ppb/K. 20 In our peptides, a few amide protons show temperature coefficients in agreement with the presence of stable hydrogen bond (Tables S1,S2). Additional evidence for a helix formation is provided by the analysis of the H α resonances, which are strongly dependent on local secondary structure. 21 Upfield shifts for H α relative to random coil values are generally found for residues implicated in a α-helix or in turns and downfield shifts for those in β-sheets. As shown in Figure S2, H α resonances of all residues starting from Thr 171 in both peptides experience upfield shifts of the NMR signals compared to those observed for the same amino acids in random coil state, indicating a tendency to the helix structure of this peptide segment. Also, diagnostic of a helical conformation is the presence of a stretch of NOE connectivities between the amide protons of consecutive residues d NN (i,i+1), supported by the presence of medium-range d αN (i,i+2), d αN (i,i+3), d αβ (i,i+3), and d αN (i,i+4) connectivities. A number of these diagnostic NOEs are observable in the NOESY spectra of peptide I and III (Figure 5 and Figure S3), again in agreement with the helix structure.

Note that all parameters indicate that peptide I has the highest helical content and that helix structure elongates to residue Thr 171 , preceding Lys 173 . In fact, comparing peptides I and III NMR parameters, the first shows: (i) 3 J HN-Hα coupling constants values significantly lower than peptide III starting from Lys 173 (Tables S1,S2), (ii) amide proton temperature coefficients of residues Ile 176 and Gly 177 are more positive than the corresponding in peptide III (Tables S1,S2), (iii) upfield shifts of the α protons higher than peptide III especially along Lys 173 -Ile 176 segment (Figure S2), NOE contacts between alpha proton of residue Asp 172 and amide and beta protons of residues Thr 175 and alpha proton of residue Lys 173 and amide proton of residues Thr 175 diagnostic for a turn/helical conformation around Lys 173 which were absent in peptide III spectra (Figure 5 and Figure S3).

Using the NOE derived data as input (Table S3), structure calculations by restrained simulated annealing gave the conformers of peptide I shown in Figure 6. Peptide I shows a well-defined α-helix structure along residues 171-177, followed by a distorted helix until residue 181 (backbone rmsd of residues 171-181 is 0.60 Å). Calculated ensemble of conformations suggested a likely explanation of the observed stabilization of the helix around Lys 173 in peptide I. In fact, a hydrophobic interaction between lipoic acid chain on Lys 173 and Ile 176 side chain is observed in most of the calculated conformers (Figure 6). Unfortunately, experimental confirmation of this spatial proximity could not be obtained due to signal overlap in the relevant spectral region.

Model of the Lipoylated PDC-E2 Domain. Starting from the NMR structure of the human unlipoylated (apo) PDC-E2 domain reported by Howard et al. (pdb code 1FYC), 14 a model of the lipoylated domain was obtained. The model was also based on the structural information obtained for the homologue PDC-E2 domain of E. coli by Jones et al. 22 because it was studied by NMR also in the lipoylated (holo) form, even if a 3D structure is available only for the apo form (pdb code 1QJO). In both the structures of PDC-E2 domain in the apo form (human and E. coli), the lipoylable lysine residue resides at the tip of a type-I β-hairpin. Upon lipoylation of PDC-E2 domain in E. coli, large changes in 1 H N and 15 N chemical shift were observed for residues from position -2 to +3 around the lipoyl-lysine. Furthermore, the relaxation data suggested that the β-turn containing the lipoyl-lysine becomes less flexible upon lipoylation. 22 Overall, the data suggest a conformational change of the region around the lipoyl-lysine. Taking into account our result on the isolated peptides, which demonstrate a switch to a helix conformation after the lipoylation around Lys 173 , confirmed by the upfield shifts of many 1 H N and 15 N chemical shift observed in the NMR spectra of the PDC-E2 domain in E. coli, 22 a brief segment of α-helix encompassing lipoyl-lysine residue was imposed in building our PDC-E2 model. Then, molecular dynamics (MD) simulations were performed for PDC-E2 model in explicit water. During the MD simulations, the helical region remained stable and the lipoyl chain steadily interacted with Leu 140 and Ile 176 side chains lying in the groove formed by strands 1 and 5 of the protein (Figure 7). The latter observation confirms a hydrophobic driving force for the conformational change. For comparison purpose, the structure of the apo PDC-E2 (pdb code 1FYC) is also reported in Figure 7. Clearly in the last case, Lys 173 side chain is much more exposed to the solvent.

NMR and MD simulations give a possible explanation for the different antigenicity of the lipoylated vs the unlipoylated peptides as well as the holo vs the apo forms of the protein. In fact, a conformational change is observed upon lipoylation, which likely alters or obscures the epitope for antibody binding. Residual antigenicity observed in the lipoylated peptide I can be ascribed to conformational flexibility, which is still high in water solution. Constrained analogues of peptides I and III would be useful probes to verify our hypothesis.

Diagnostic Relevance of the Antigenic Peptide Probes. The current IIF methodology to detect AMA suffers from the technical limitations of this type of assays, including subjectivity of observers, need of trained personnel, and lack of an automated procedure. Thus, several attempts were reported to develop a simple, plate-based immunoenzymatic assay, alternative to the traditional IIF assay, based on the use of recombinant or chimeric proteins. 23,24 To assess the possibility F of using the active peptides characterized in the present study for the development of a simple ELISA, based on a panel of synthetic antigen, to replace the traditional AMA IIF assay, we tested the specificity of the two more sensitive peptide probes, i.e., peptides I and III, using two control groups, rheumatoid arthritis (RA, n = 22) and scleroderma (SSC, n = 12), as well as a group of AMA negative PBC sera (n = 15).

The results reported in Figure 8 indicate that both peptides detect IgM and IgG in significantly higher amounts in PBC than in RA or SSC, thus showing a very good specificity, particularly in the case of IgM. Interestingly, both peptides recognize IgM but not IgG in AMA negative PBC patients and this may result of interest considering the polyclonal increase in serum IgM invariably associated with PBC. In this respect, it is of interest that specific IgM cross-reacting with xenobioticmodified peptides have been previously reported 25 and putative roles have also been proposed over the years for mucosal IgA, with AMA-IgA detected in saliva and urine of patients with PBC. 26

■ CONCLUSIONS

We studied the immunological role of the inner lipoyl domain of PDC-E2 that is considered the AMA major autoantigen, using synthetic peptides bearing well-defined lipoylation on the side chain of the relevant lysine residue which were tested on PBC and control sera. We initially analyzed the role of the lipoyl chain chirality, an aspect not addressed by previous studies, showing that this parameter does not affect autoantibody recognition. Subsequently, we investigated the role of the lipoamide modification of the lysine residue, confirming that indeed both lipoylated and unlipoylated PDC peptides are able to recognize specific autoantibodies in PBC patients sera. When considering in particular IgM, we found that while the native antigen [(R)-lipoamide-Lys 173 ]PDC-E2(167-186) (peptide I) is able to detect 38% of PBC AMA positive patients, its unlipoylated counterpart III recognize 68% patients. Taken together, our data indicate that 74% of PBC patients sera recognize at least one of the two peptides. Interestingly, very few positive sera (3/37) recognized exclusively the lipoylated peptide, thus suggesting that the lipoamide moiety plays a marginal role within the autoreactive epitope, while the surrounding PDC amino acid sequence may be essential, as shown by the lack of immunological activity of the control peptide IV, bearing a lipoylated lysine residue in the framework of an unrelated sequence. These results are supported by a conformational analysis, based on NMR and MD simulations, showing that the lipoyl moiety of PDC-E2 appears to be involved in hydrophobic interactions, which may limit its exposition and thus its contribution to the complex AMA antigenic epitope. A preliminary analysis of the specificity of the two active PDC peptides indicates that these peptides could be part of a panel of synthetic antigens collectively able to mimic in a simple immunoenzymatic assay the complex AMA positivity pattern detected in immunofluorescence. Such a panel of peptides should be used together with classical IIF in the screening for AMA in unselected patients sera to verify their diagnostic potential and their advantage over currently employed assays.

■ EXPERIMENTAL SECTION

Materials and Methods. Automated peptide syntheses were performed on a Biotage Syro II apparatus (Biotage, Uppsala, Sweden). Semipreparative RP-HPLC were performed on a Waters (Waters Corporation, Milford, MA, USA) apparatus using a Phenomenex (Phenomenex Inc., Torrance, CA, USA) Jupiter column C18 (10 μm, 250 mm × 10 mm) at 4 mL/min with solvent system A (0.1% TFA in H2O) and B (0.1% TFA in CH 3 CN). Analytical UPLC were performed using a Waters ACQUITY UPLC coupled to a single quadrupole ESI-MS (Waters 3100 mass detector) using a BEH C18 (1.7 μm 2.1 mm × 50 mm) column at 35 °C, at 0.6 mL/min with solvent system A (0.1% TFA in H 2 O) and B (0.1% TFA in CH 3 CN). Analytical HPLC were performed using a Waters Alliance chromatograph coupled to a single quadrupole ESI-MS (Micromass ZQ) using a Phenomenex Kinetex C18 (2.6 μm 100 mm × 3.0 mm) column working at 0.6 mL/min with solvent system A (0.1% TFA in H 2 O) and B (0.1% TFA in CH 3 CN). SP-ELISA assays were performed on 96-well plates, Nunc Maxisorp (Sigma-Aldrich, Milano, Italy). Washings steps were executed with automatic Hydroflex microplate washer (Tecan Italia, Milano, Italy). Antihuman IgG-alkaline phosphatase conjugates were purchased from Sigma-Aldrich (Milano, Italy), and antihuman IgM-alkaline phosphatase were purchased from Invitrogen (Life Technologies Italia, Monza, Italy). p-Nitrophenyl phosphate was purchased from Fluka (Milano, Italy). Absorbance values were measured on a Sunrise Tecan ELISA plate reader purchased by Tecan (Tecan Italia, Milano, Italy). HFA and 2 Building Block Synthesis. The synthesis of the lipoylated building blocks was reported elsewhere. 15 Briefly, to a solution of (R)-α-lipoic acid or (S)-α-lipoic acid dissolved in DMF at room temperature was added, under stirring, HATU (1 equiv) and DIPEA (2 equiv). After 10 min Fmoc-L-Lys-OH (1 equiv) was added. The reaction was stirred at room temperature and checked by UPLC (50 to 100% B in A over 3 min). After 3 h, DMF was evaporated under reduced pressure and the resulting brown oil was diluted in DCM. Organic phase was successively washed twice with 0.5 M KHSO 4 , H 2 O and brine, then dried over MgSO 4 , filtered, and concentrated under reduced pressure. The resulting orange oil was purified by normal phase FCC. Fractions were checked with, pooled together, and evaporated under reduced pressure to yield the desired product as a yellow solid.

Peptide Synthesis. The synthesis of the peptides was reported elsewhere. 15 Briefly, all the peptides were synthesized by SPPS, performed by automatic peptide synthesizer Syro II, following the fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (tBu) strategy. The peptides were prepared starting from a Fmoc-Lys(Boc)-Wang resin (peptides I-III, 0.3 mmol/g; peptides IV and V, 0.7 mmol/g). Fmoc deprotections (5 min + 15 min) were performed with 20% piperidine in DMF. Fmoc amino acids were used as 0.5 M in DMF solutions. Coupling reactions were performed with 0.5 M TBTU/DMF (4 equiv), 0.5 M amino acids/DMF (4 equiv), and 1 M DIPEA/NMP (5 equiv). Lipoylated building blocks (2.5 equiv) were coupled manually for 1 h, using HATU (2.5 equiv) and NMM (3.5 equiv) in DMF. Cleavage from the resin and side chain deprotection were performed at room temperature for 3 h using the following mixtures (1 mL/100 mg resin): TFA/EDT/thioanisole/H 2 O/phenol (82.5:5:2.5:5:5) for peptides I, II and IV, TFA/TIS/H 2 O/EDT (94:2:2:2) for peptide III, and TFA/TIS/H 2 O (95:2.5:2.5) for peptide V. The resin was filtered off, and the solution was concentrated by flushing N 2 . The peptides were precipitated from cold Et 2 O, centrifuged, and lyophilized. All peptides were purified by semipreparative RP-HPLC to obtain a purity of at least 95%. Gradients were 30-60% B in 30 min for peptides I, II, and III and 25-55% B in 30 min for peptides IV and V. Peptides characterization was performed by analytical UPLC-MS or by analytical HPLC-MS. The analytical data are reported in Table 1.

Immunoassays. After informed consent was obtained, serum samples were stored at -20 °C until use. Sera from 50 AMA positive PBC patients (mean age, 68; range, 47-88 years; 3 males, 47 female) were screened in parallel with both healthy control sera from 55 NBD (mean age, 49; range, 20-72 years), and pathological control sera from 15 AMA negative PBC (mean age, 62; range, 42-84 years; 15 females); 22 RA (mean age, 54; range, 21-79 years; 5 male, 17 females), and 12 SSC (mean age, 63; range, 32-72 years; 1 male, 11 females) using SP-ELISA. In particular, both AMA positive and negative PBC patients were diagnosed according to internationally accepted criteria, 27 while the diagnosis of RA and SSC was based on the ACR criteria. 28,29 From the serological viewpoint, RA patients were ACPA (22/22) and RF (20/22) positive. Further clinical features of PBC patients are summarized in Table 2.

Activated polystyrene 96-well ELISA plates were coated with each peptide, 100 μL/well of 1:100 diluted peptide in pure carbonate buffer 0.05 M (pH 9.6). After overnight incubation at 4 °C, plates were washed three times using washing buffer (0.9% NaCl and 0.05% Tween 20 in Milli-Q water). Nonspecific binding sites were blocked with 100 μL/well of fetal bovine serum (FBS) buffer (10% FBS in washing buffer) at room temperature for 1 h. FBS buffer was removed, and plates were incubated overnight with sera (100 μL/well, diluted 1:100 in FBS buffer) at 4 °C. After three washes, plates were treated with 100 μL/well antihuman IgG or antihuman IgM alkaline phosphatase-conjugated antibody diluted in FBS buffer (1:8000 for antihuman IgG, 1:1200 for antihuman IgM). After 3 h of incubation at room temperature and three washes, 100 μL of substrate buffer (pNPP 1 mg/mL in in alkaline substrate buffer pH 9.8, consisting of 10% diethanolamine and 0.1% MgCl 2 in Milli-Q water) was added to each well. After 15-30 min of incubation at room temperature, the absorbance of each plate was red in a multichannel ELISA reader at 405 nm (ref 620 nm). ELISA plates, coating conditions, reagent dilutions, buffers, and incubation times were tested in preliminary experiments. All buffers were brought to room temperature prior to use and filtered through 0.22 μm filters (except FBS buffer). The antibody levels are expressed as absorbance in arbitrary units at 405 nm.

Circular Dichroism (CD). All CD spectra were recorded using a JASCO J710 spectropolarimeter at 20 °C between λ = 260 and 190 nm (1 mm path, 1 nm bandwidth, four accumulations, and 100 nm min -1 scanning speed). Measurements were performed with peptides in H 2 O (0.100 mM, pH 7.4) or in different mixtures of H 2 O/HFA.

NMR Spectroscopy. The samples for NMR spectroscopy were prepared by dissolving the appropriate amount of peptide in 20 μL of HFA, 160 μL 1 H 2 O, and 20 μL of 2 H 2 O to obtain a concentration of 1 mM for peptides I and III. At this concentration, peptide I showed broad 1 H NMR signals indicating peptide aggregation, hence its concentration was lowered until it showed narrow signal lines typical of a monomeric state (0.2 mM). All 2D NMR spectra of peptide I were acquired at this concentration. NMR spectra were recorded on a Varian Unity INOVA 700 MHz spectrometer equipped with a zgradient 5 mm triple-resonance probe head in a 3 mm tube. The spectra were calibrated relative to TSP (0.00 ppm) as internal standard. One-dimensional (1D) NMR spectra were recorded in the Fourier mode with quadrature detection. 2D DQF-COSY, 30,31 TOCSY, 32 and NOESY 33 spectra were recorded in the phase-sensitive mode using the method from States. 34 Data block sizes were 2048 addresses in t 2 and 512 equidistant t 1 values. A mixing time of 70 ms was used for the TOCSY experiments. NOESY experiments were run with mixing times in the range of 150-300 ms. The water signal was suppressed by gradient echo. 35 The 2D NMR spectra were processed using the NMRPipe package. 36 The qualitative and quantitative analysis of DQF-COSY, TOCSY, and NOESY spectra were obtained using the interactive program package XEASY. 37 The temperature coefficients of the amide proton chemical shifts were calculated from 1D 1 Structure Calculation of Peptide I. The NOE-based distance restraints were obtained from NOESY spectra collected with a 200 ms mixing time. The NOE cross peaks were integrated with the XEASY program and were converted into upper distance bounds using the CALIBA program incorporated into the DYANA program package. 38 NOE derived upper limit constraint used in the calculations are reported in Table S3. NMR derived upper bounds were imposed as semiparabolic penalty functions with force constants of 16 kcal mol -1 Å -2 . A distance maximum force constant of 250 kcal mol -1 Å -2 was used. Lipoyl-lysine chain of peptide I was built using the Insight Builder module (Accelrys Software Inc., San Diego). Atomic potentials and charges were assigned using the consistent valence force field (CVFF). 39 The conformational space of peptide I was sampled through 100 cycles of restrained Simulated Annealing (ε = 1r). In Simulated Annealing, the temperature was altered in time increments from an initial temperature to a final temperature by adjusting the kinetic energy of the structure (by rescaling the velocities of the atoms). The following protocol was applied: the system was heated up to 1500 K over 2000 fs (time step = 1.0 fs); the temperature of 1500 K was applied to the system for 2000 fs (time step = 1.0 fs) with the aim of surmounting torsional barriers; successively, temperature was linearly reduced to 300 K in 1000 fs (time step = 1.0 fs). Resulting conformations were then subjected to restrained Molecular Mechanics (MM) energy minimization within Insight Discover module (ε = 1r) until the maximum RMS derivative was less than 0.001 kcal/Å, using Conjugate Gradient as the minimization algorithm. Finally, conformations were subjected to 1000 steps of unrestrained MM Conjugate Gradient energy minimization. From the produced 100 conformations, 10 structures, whose interprotonic distances best fitted NOE derived distances, were chosen for statistical analysis.

Model of the Lipoylated PDC-E2 Domain. Starting from the NMR structure of the human unlipoylated (apo) PDC-E2 domain (pdb code 1FYC), 14 lipoyl chain was added to Lys 173 using the Insight Builder module (Accelrys Software Inc., San Diego, CA, USA). Then, an in vacuo brief restrained Molecular Dynamics calculation was performed by imposing helical restraints along the protein segment from Thr 171 to Ile 176 (see Results paragraph). Obtained conformation was subjected to 1000 steps of unrestrained MM Conjugate Gradient energy minimization and then soaked in a box of water. The dimensions of the water box were 35 × 65 × 35 Å 3 for a total of 2165 water molecules. The MD simulation was performed using the CVFF force filed with the Insight/Discover program (Accelrys Software Inc., San Diego, CA, USA). First, the initial configuration was minimized using the steepest descent and the conjugate gradient algorithm until an energy gradient of 0.01 kcal mol Å -1 . Then trajectories were created for 2000 ps at 300 K with a time step of 1 fs under periodic boundary conditions. Atomic coordinates were saved every 4 ps. The last configuration was then minimized using the steepest descent and the conjugate gradient algorithm until an energy gradient of 0.01 kcal mol Å -1 . Minimized structure is reported in Figure 7.

■ ASSOCIATED CONTENT

* S Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: The 1,2-dithiolane sulfur atoms at C-6 and C-8 exist in vivo in the oxidized disulfide bond, which may be reduced to form the respective dithiol, dihydrolipoic acid (DHLA, Figure 2). Enantiomerically pure lipoic acid was subsequently prepared by resolution of the racemate by formation of diastereomeric salts using respectively (R)-and (S)--methylbenzylamine [4].

Lipoylation: in vivo pathways and naturally occurring lipoylated proteins

Attachment of lipoic acid to a molecule is called lipoylation. In vivo, the lipoylation occurs on the side chain of a lysine residue in the form of the amino acid itself to give so-called "lipoyl-Lys", or in a protein to provide the "lipoylated protein" (Figure 3). Lipoylation is a post-translational modification with a strong structural requirement for lysine [5]; however, various amino acids are tolerated at the neighboring residues [6], in contrast to N-glycosylation, which requires typically a clear consensus sequence [7]. Although lipoic acid was extensively studied in the past 50 years [8], the mechanism by which it is biosynthesized and attached to protein was only recently elucidated [9]. In vivo, lipoic acid exists in very small proportion as free acid; instead lipoic acid resides primarily as cofactor bound to the amino group of a specific Lys on a lipoyl-accepting protein domain. Many studies on lipoic acid biosynthesis have been performed using Escherichia coli (E. coli), which has enabled identification of two different pathways, depending on different substrate sources. Indeed, the first pathway is characterized by the use of exogenous lipoic acid, which is activated via ATP to form lipoyl-adenosine monophosphate by lipoate-protein ligase (LplA) [10]. The same enzyme then transfers the molecule to the side chain of a Lys residue on the protein with release of AMP (Scheme 2) [11]. LplA is able to use also octanoic acid as substrate, but with reduced efficiency.

Scheme 2 PDC-E2 lipoylation using exogenous lipoic acid by the LplA pathway (adapted from Booker [12])

The second pathway involves endogenous lipoic acid synthesized from fatty acids. In this case, a lipoyl (octanoyl)-transferase called LipB transfers to lipoyl-accepting domains both lipoyl or octanoyl groups conjugated to an acyl carrier protein (ACP) [13]. As octanoyl-ACP represents a lipoyl-ACP precursor, the use of this compound to obtain a lipoylated protein requires an additional step, namely the introduction of sulfur atoms catalyzed by the N-octanoyltransferase LipA [14,15].

(Scheme 3).

Scheme 3 Biosynthesis of lipoic acid from octanoyl-ACP, involving both LipB and LipA (adapted from

Booker)

The three pathways that employ respectively LipA, LipB and LplA can be summarized as pictured in Scheme 3.

Scheme 4 Summary of PDC-E2 lipoylation pathways in vivo (for detailed molecular structures, refer to Schemes 2 and 3).

Among naturally lipoylated proteins, lipoic acid is found as an essential cofactor of several key enzymes involved in oxidative metabolism: i.e., 2-oxoacid dehydrogenase complex (2-OADC), pyruvate dehydrogenase complex (PDC), 2-oxoglutarate dehydrogenase (2-OGDH) and the glycine cleavage system [16, 17, 18, 19]. The lipoyl moiety is covalently bound to these proteins and serves as a carrier of various reaction intermediates between the different enzyme active sites. Perham described these lipoyl-Lys residues as "swinging arms" [20] because of their high flexibility.

Specifically, lipoylation is important for the cellular energetic cycle in mitochondria, because lipoic acid residues are essential cofactors of key enzymes involved in oxidative metabolism.

One of the most relevant lipoylated protein structures is the PDC enzymatic complex, essential for cell activity and energy production. Providing the link between the citric acid cycle and glycolysis, PDC is involved in Primary Biliary Cirrhosis (PBC), an immune-mediated chronic inflammatory liver disease with slow progression, characterized by destruction of small interlobular bile ducts, gradual cholestasis and portal inflammation. The serological hallmark of the pathology is characterized by high disease specific anti-mitochondrial antibodies (AMA), which target primarily the E2 subunit of the PDC (PDC-E2). As the main immunogenic region of PDC-E2 was identified to contain the inner lipoyl domain of the protein, the involvement of lipoic acid as part of the epitope recognized by AMA has been investigated, albeit no final consensus has been attained.

Scheme 3

The PDH reaction mechanism including E1, E2 and E3 subunits. ThDP: thiamine diphosphate. Lip: lipoyl-Lys moiety covalently bound to E2 subunit. CoA: CoenzymeA. FAD: flavin adenine dinucleotide. NAD: nicotine adenine dinucleotide Another example of lipoylated enzymes found in vivo is the glycine cleavage system, which is responsible for glycine decarboxylation. The glycine cleavage system comprises four proteins sharing similarities to the components of 2-OADC, and catalyzing a coordinated set of reactions (Scheme 6): a pyridoxal phosphate-dependent decarboxylase (P protein); a protein with a lipoyl-Lys residue that becomes reductively aminomethylated (H protein); an aminomethyltransferase T protein (requiring tetrahydrofolate as cofactor); and a dihydrolipoyl dehydrogenase (L protein), responsible for the reoxidation of the dihydrolipoyl group.

Scheme 4

The glycine cleavage system, including H, P (pyridoxal phosphate-dependent decarboxylase), T (aminomethyltransferase) and L (dihydrolipoyl dehydrogenase) proteins.

In vitro lipoylation: synthetic strategies to obtain lipoylated peptides

Strategies for the synthesis of lipoylated peptides have involved both the attachment of lipoic acid to the lysine side chain of the target peptide, as well as construction of an N  -lipoyl lysine suitable for introduction into the sequence through peptide synthesis. Both chemical and enzymatic methods were used for peptide lipoylation [21], the latter being accomplished by harnessing enzymes from the LpLA and LipB pathways. Orthogonal protecting groups were critical for peptide lipoylation by chemical synthesis. For example, Tuaillon et al. [22] synthesized the N-terminal acetylated fragment (167-184) of PDC-E2 peptide, lipoylated in position 173 by on-resin selective cleavage of Fmoc protection from the Lys side chain during Boc/Bzl solid-phase synthesis and acylation using the Nhydroxybenzotriazole ester of racemic lipoic acid (lipoyl HOBt ester, Scheme 7).

Scheme 7

On-resin selective deprotection of a Lys(Fmoc) residue in Boc/Bzl strategy, followed by onresin lipoylation using lipoyl HOBt ester.

Similarly, using a Fmoc/tBu strategy, Long et al. [23] removed selectively the side chain protection of Lys(Dde) with 2% hydrazine in DMF for 5 min, and employed lipoyl N-hydroxysuccinimide ester (lipoyl NHS ester) for the acylation of the Lys side chain (Scheme 8). They synthesized several lipoylated peptides of various lengths (7-, 9-, 10-and 12-mer) encompassing the inner lipoylated domain of the PDC-E2 protein.

Scheme 8 On-resin Fmoc/tBu strategy featuring selective deprotection of a Lys(Dde) residue, followed by on-resin lipoylation using lipoyl NHS ester. Amano et al. applied a similar lipoyl NHS ester strategy to selectively lipoylate short unprotected peptides of the PDC-E2 sequence (15 amino acids) coupled to an agarose gel [24]. A specific peptidespacer-linker combination was assembled by attachment of N α -Fmoc-(N β -Boc-amino-oxyacetyl)-Ldiaminopropionic acid (Fmoc-Dpr(Boc-Aoa)) onto Rink amide resin, followed after Fmoc removal, with N-Fmoc-2,2′-(ethylenedioxy)bis(ethylamine) monosuccinamide as a hydrophilic spacer onto which the peptide sequence was elongated and capped by acetylation. The assembled peptidespacer-linker unit was cleaved from the resin, and coupled to a modified methyl-ketone agarose gel via an oxime bond. Lipoylation was performed at the last step using the lipoyl NHS ester (Scheme 9). Scheme 9 Synthesis of agarose-conjugated lipoylated peptides via oxime bond formation and onagarose lipoylation using lipoyl NHS ester (adapted from Amano et al.). This approach has become a convenient standard method [25, 26, 27, 28]. The drawbacks to peptide acylation include incomplete lipoylation, which is typically monitored using the Kaiser test [29,30,31].

In addition, the N-terminal lipoylation of a Glu-Ala dipeptide was performed using lipoic acid activated with N,N-disuccinimidyl carbonate on >100g scale [32] (Scheme 10). The elucidation of in vivo lipoylation mechanisms led to discovery and partial purification of two enzymes from E.Coli (LplA and LplB) that were subsequently employed in vitro for direct lipoylation of proteins [33]. For example, the inner lipoyl domain of human PDC-E2 was successfully obtained using the in vitro lipoylation approach [34]. The ligases were subsequently purified by heparinagarose chromatography [35].

Moreover, the octanoyl-Lys derivative of the tetraeptide Thr-Glu-Lys-Ile was converted to the lipoyl-Lys containing peptide using expressed and purified lipoyl synthase (LipA) from Sufolobus solfataricus (Scheme 11) [36]. Various strategies were used to prepare suitably protected N  -lipoyl lysine [Lys(N  -lipoyl)] for subsequent peptide synthesis. For example, the lysine-copper complex was acylated with racemic lipoyl isobutyl carbonic anhydride [37, 38, 39], albeit in low yield due to polymerization. In fact, lipoic acid can polymerize by a ring-opening mechanism featuring formation of intermolecular disulfide linkages to produce an insoluble polymer [40].Among other examples, Matsugo et al. [41] coupled racemic lipoic acid to the side chain of a Boc-protected Lys to obtain the mixture of diasteromers N lipoyl N  -(Boc)Lys (Scheme 12). Lipoic acid has also been coupled to alkylamines using 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDCI) [42]. Recently, novel lipoylated derivatives were described in which the amide bond has been replaced by bioisosteric groups, such as 1,2,4-and 1,3,4-oxadiazoles, 1,2,3-triazoles, thiazoles or tetrazoles (Scheme 13) [43, 44]. Such methods offer interesting potential for making lipoyl-derivatives of peptides and proteins with increased protease stability To facilitate the introduction of lipoyl moieties into peptide sequences, our laboratory has focused on the synthesis of enantiomerically pure lipoylated Lys derivatives for application as building-blocks in the Fmoc/tBu strategy for solid-phase peptide synthesis [46]. Employing commercially available (S)-(-)-and (R)-(+)--lipoic acids as enantiomerically pure, albeit expensive starting materials, the corresponding lipoyl pentafluorophenyl esters were synthesized from pentafluorophenol and N,Ndicyclohexylcarbodiimide [47]. The lipoyl pentafluorophenyl esters were coupled to the side chain of

Preparation of dihydrolipoylated peptides and proteins

As mentioned, in vivo modification of lipoic acid derivatives to their dihydrolipoyl counterparts occurs during reductive acylation and aminomethylation of the 1,2-dithiolane ring. The mechanism for reductive acylation by PDC has been well studied [48, 49]. Application of the enzyme complex for the synthesis of dihydrolipoylated peptides and proteins is however not practical, because of the selectivity of PDC-E1 towards the lipoylated domain of the E2 component [50].

The first reduction of lipoic acid to dihydrolipoate in vitro has been performed using sodium tetraborohydride [51]. This method has since proven useful in materials chemistry [52 ,53], but may not be compatible with biomolecules, because the relatively strong reducing conditions could damage other functional groups of peptides and proteins. Moreover, the purification steps to remove the byproducts of reduction may be troublesome. More selective agents for reducing disulfide bridges were subsequently employed to make dihydrolipoyl residues, such as dithiothreitol (DTT) [54] and tris(2-carboxyethyl)phosphine (TCEP) [55, 56]. For example, our research group has developed an effective method for selective reduction of the 1,2-dithiolane ring of lipoylated peptides using DTT [46], which leaves other residues untouched (Scheme 17) [72]. Complete reduction is performed after one night of reaction, yielding the corresponding dihydrolipoamide peptide. Subsequent washes with diethylether, in which peptide is not soluble, enables removal of excess of DTT, as well as cyclic byproduct resulting from DTT oxidation. This convenient method has been used to effectively obtain dihydrolipoamide-containing peptides without chromatographic purification.

Scheme 17 Selective 1,2-dithiolane ring reduction of [(R,S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK Photo-induced reduction of lipoic acid derivatives to their dihydrolipoic acid counterparts was used to coat metallic surfaces [57]. In solution, photo-induced reduction should be performed with caution to avoid polymerization [58], which has been be avoided by careful solvent selection. Using TLC monitoring during photo-induced reduction of lipoic acid, protic solvents were found to decrease polymerization, with best results employing propan-2-ol [59]. The -320 mV reduction potential of the lipoic acid -dihydrolipoic acid couple makes handling of the reduced dithiol challenging due to reoxidation back to the dithiolane necessitating inert conditions and in-situ reduction [1].

Uses of lipoic acid and lipoylated molecules

Lipoic acid and dihydrolipoic acid moieties can chelate metals by way of the two sulfur atoms. Metals and their ions were coordinated by lipoic acid in vitro and in vivo. For example, lipoic acid -metal complexes were made with gold [60], mercury [61], copper [62], iron [63] as well as with ions such as Mn 2+ , Pb 2+ , Zn 2+ [64,65] and Cd 2+ [66]. Dihydrolipoic acid was shown to form complexes with heavy metals such as As, Sb and Bi [67]. Formation of metal complexes by lipoyl residues reduces oxidative damage, by diminishing reactive oxygen species (ROS) with potential to damage biological molecules, such as DNA, proteins or lipids. Both lipoic acid and dihydrolipoic acid can scavenge physiologically relevant ROS in vitro [68]. The action of lipoic acid in vivo is less clear, because of its lower concentration in tissues relative to other intracellular antioxidants such as glutathione or vitamin C, albeit lipoic acid may have an important role as an indirect antioxidant regenerating other antioxidant species and chelating metals [69]. A recent study of the relative antioxidant properties of racemic and enantiomerically pure lipoic acid concluded that the natural (R)-enantiomer was more efficient [70].

The therapeutic utility of lipoic acid was examined to treat different diseases: e.g., diabetes mellitus, multiple sclerosis and Alzheimer disease. In diabetes mellitus, lipoic acid administered intravenously was shown to improve impaired endothelial vasodilatation in neuropathy and vascular complications [71]. Multiple year treatment of diabetic neuropathy by intravenous administration of lipoic acid proved to be efficient for reducing significantly symptoms in diabetic patients [72]. Towards a treatment of multiple sclerosis (MS), lipoic acid was shown to be effective in prevention of Experimental Autoimmune Encephalomyelitis (EAE), an animal model of MS [73]. The use of oral supplements of lipoic acid by patients suffering from MS has shown promise in clinical trials [74]. The use of lipoic acid has also shown promise for the treatment of Alzheimer Disease [75, 76]. In particular, the antioxidant properties and metal chelation properties of lipoic acid may be beneficial to Alzheimer paitients, who exhibited increased acetylcholine production.

The chemical properties of the 1,2-dithiolane ring were used to anchor lipoylated peptides and proteins to surfaces. In particular, Self Assembled Monolayers (SAM) were generated by the introduction of a lipoyl residue at the N-terminus or side chains of peptides and proteins to enable interactions with metals and adsorption to create thin layers on metallic surfaces such as gold [77, 78, 79, 80, 81] and silver [82]. Coupling of biomolecules to metallic surfaces is also possible using this technique, as reported for flavin adenine dinucleotide (FAD, Scheme 18) [83].

Scheme 18 Preparation of FAD-coated Au surfaces using lipoyl moiety as an anchor (adapted from Blonder et al.)

Summary

The in vivo and in vitro synthesis of the lipoic acid derivatives have been reviewed with focus on the implication of the heterocyclic 1,2-dithiolane ring in various fields: e.g., medicine as therapeutic agent, materials science as metal surface coating, and nutrition as antioxidant supplement.

Moreover, lipoylated molecules offer potential as Primary Biliary Cirrhosis synthetic antigens for studying autoimmune mechanisms. Lipoylation of peptides and proteins was performed by different pathways in vivo featuring coordinated enzymatic reactions, as well as in vitro by chemical and enzymatic methods. Considering novel methods for effective lipoylation of peptide and protein structures, the relevance and utility of lipoic acid in biology and materials science, a wealth of future applications of this heterocyclic acid may be anticipated.

INTRODUCTION

Met, one of the eight essential amino acids (1), is characterized by a thioether function on the side chain that is particularly sensitive to oxidation. Indeed, low oxidation potential of Met makes it fairly easily oxidized. The biological relevance of Met oxidation is a relatively new topic (2,3). So far, Met was considered as a generic hydrophobic amino acid, interchangeable with others such as Leu or Val.

which is now considered as a biologically relevant residue, protein function, structuration, and protein-protein interaction (4).

oxidants both in vivo and in vitro, producing Met(O). In vivo, Met is oxidized by Reactive Oxygen Species (ROS) such as hydrogen peroxide (H 2 O 2 ) and hydroxyl radicals (HO . ). Oxidized Met is more hydrophilic than the native amino structures can occur and it is believed to be responsible of inactivation, inhibition and activation of some proteins (5).

Because of these core changes in proteins, oxidized forms of that can generate neoepitopes thus involved in triggering autoimmune responses (6). For example, a correlation between Met(O) levels and disease activity has been already demonstrated in Systemic Lupus Erythematosus (SLE) (7,8). Met-oxidation process usually involves a two electron transfer mechanism ( 9), although single-electron transfer mechanism has been also reported (10). Mild oxidizing agents can oxidize Met to the corresponding sulfoxide Met(O) and to Met-sulfone Met(O 2 ) . In biological systems this is an irreversible process (11), but there are some chemicals able to reverse this process (12,13).

One interesting but often disregarded issue is the chirality of Met(O). Considering the natural L-Met, two are the possible diastereoisomers of the oxidized forms, the (R,S)-Met(O) and the (S,S)-Met(O) which can be relevant for molecular recognition, cellular signaling, and most importantly for sulfoxide reduction in vivo (14). Despite numerous efforts, no consensus sequences have N-glycosylation (15). Recent studies by Liang et al. (16) reported that oxidation of Met residues in Methionine Rich Proteins (MRP) was strongly dependent on the protein substrate. Oxidation of CEDRIC RENTIER 1,2,3 , OLIVIER MONASSON 1,2 , FRANCESCA NUTI 1,3 , PAOLO ROVERO 1,4 , GIUSEPPINA SABATINO 1,3 , ANNA MARIA PAPINI For the MW-SPPS using the Liberty instrumentation by CEM (24) we applied the following protocol: microwave cycle was used for two deprotection steps, the first one was for 30 sec (70-75°C, 25W), the second one for 180 sec (70-75°C, 25W). All coupling reactions were performed in 300 sec (70-75°C, 25W).

For the MW-SPPS using the Biotage SyroWave, we applied the following protocol: two deprotection steps at room the second one in 10 min. All coupling reactions were performed in 300 sec (75°C, 20-35W).

syntheses were performed till solvents were visibly removed.

cleavage, cleavage from the resin and side-chain deprotections were performed, for both peptides, at room temperature under atmospheric conditions for 3 h using two different cocktails (1 ml/100 mg resin): TFA/TIS/H 2 O (95:2.5:2.5 v:v:v) and TFA/TIS/H 2 O/EDT (94:2:2:2 v:v:v:v). An optimized cleavage protocol for the in-situ reduction of Met(O) in the tetradecapeptide was obtained using the cocktail TFA/thioanisole/anisole/EDT (90:5:2:3 v:v:v:v) at room temperature under atmospheric conditions in 3 h. 3 eq. of Bu 4 NBr were then added to the mixture in 5 min, for a total cleavage time of 3 h.

RESULTS AND DISCUSSION

tetradecapeptide GHSVFLAPYGWMVK

We report herein the experimental conditions allowing solid-phase oxidation of Met to Met(O) and a convenient tetradecapeptide GHSVFLAPYGWMVK were chosen as model peptides.

sequence yielding a particularly insoluble peptide, due to its very hydrophobic C-terminal fragment. atmosphere leads rapidly to the Met(O)-containing peptide sequence as a soluble material. Accurate UPLC-MS analysis allowed to detect the presence of 60% of Met(O) in the crude material and up to 100% when no reducing reagent was used in the cleavage cocktail (Figure 2). We hypothesize that the oxidized amino acid is the Met residue. This observation is in line with some data already reported in the literature using the same apparatus (25). Techniques such as nuclear magnetic resonance (NMR) or X-ray photoelectron spectroscopy (XPS) (26) are useful to identify oxidized amino acids in peptides. Nevertheless, we reference compound to verify our hypothesis.

2 O 2 up to 5 times more concentrated, although the exact position mass spectrometry (17,18) and SDS-PAGE. This observation highlights the possible role played by the amino acid sequence surrounding Met and/or conformational effects in the oxidation process. These characteristics are relevant not only in vivo, but also during peptide synthesis when Methionine oxidation can occur as a side-reaction. Although chemical strategies are described to reverse the process (19), these are time consuming and unequivocally affect and/or characterization steps can be troublesome. This paper reports a case study of the synthesis of two the well-known peptide involved in Alzheimer's disease and responsible for the aggregation in plaques found in patients' brains with possible subsequent toxicity (20). In this case it has been previously reported that Met oxidation could and plaque formation (21). The second peptide is the irrelevant tetradecapeptide GHSVFLAPYGWMVK, a fragment developed as a synthetic antigenic probe for detecting circulating autoantibodies in Multiple Sclerosis and Rett Syndrome (22,23). This latter was used in the present paper as a model peptide to demonstrate how it is possible to manage Met oxidation, thus controlling in solid-phase the synthetic the oxidized or the reduced form.

EXPERIMENTAL SECTION

as follows: Fmoc: 9-H tBu: tert-butyl; Boc: tert-butoxycarbonyl; DMF: N,Ndimethylformamide; TBTU: O-(benzotriazol-1-yl)-N,N,N',N' diisopropylethylamine; NMP: N-methyl-2-pyrrolidone; 1,2-ethanedithiol; Et 2 O: diethyl ether; Bu: butyl. The tetradecapeptide GHSVFLAPYGWMVK was synthesized following the Fmoc/tBu strategy starting from Fmoc-Lys(Boc)-Wang resin (0.7 mmol/g), with different instrumentations: automatic batch synthesizer (APEX 396, Aapptec, Louisville, KY, USA) equipped with either a 40 or a 8-wells reaction block, manual batch synthesizer (PLS 4×4, Advanced ChemTech, Louisville, KY, USA), automatic microwaveassisted (MW) peptide synthesizers Syro Wave (Biotage, Uppsala, Sweden) and CEM Liberty, (CEM Corporation, (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) was synthesized manually and with the MW-assisted automatic peptide synthesizers by the Fmoc/tBu strategy, starting from a Fmoc-Val-TGA resin (0.13mmol/g). For the conventional SPPS on the automatic and manual batch synthesizer, we used the following protocol: swelling the resin in DMF for 30 min, Fmoc deprotections with 20% piperidine in DMF solution (5 min + 15 min); resin-washings in DMF (3 x 5 min). Couplings were performed in 45 min at room temperature using Fmoc amino acids (4 eq), TBTU (4 eq) and DIPEA (5 eq) Analysis of the peptide fragments obtained after acid couplings revealed that oxidation of Met residue increases after each cycle: no oxidation is observed after introducing the amino acid at position 10 (Phe) and 35% oxidation is observed after introducing the last amino acid (Gly) (data not shown).

Reduction of Met(O)

In vivo, reduction of oxidized Met is performed by two enzymes, i.e., Met(O) reductases, MsrA (37) and MsrB (38), which are respectively selective for (S,S)-Met(O) and (R,S)-Met(O). These enzymes play an essential role in limiting the metabolic consequences of oxidative stress (39). However, enzymes, even though they work stereoselectively, are conditions if used in vitro. In vitro, several methods have been developed for Met(O) reduction. As an example, reducing agents such as DTT (25), or halide reagents (40-42) can be mentioned. Unlike enzymes, these protocols require low-cost reagents and can work in a wide variety of experimental conditions, but may lack enzyme stereoselectivity. In both cases, it has to be taken into account that these operations are time consuming and require troublesome peptide purification with consequent decrease in peptide yield.

protocol compared to the one previously reported (43) based on a convenient one-pot cleavage/reduction of Met(O) in the model tetradecapeptide. Three equivalents of tetrabutylammonium bromide (instead of 30 eq. used in the original paper) were introduced 5 min before the end of the cleavage performed with TFA/ thioanisole/anisole/EDT (90:5:2:3 v:v:v:v) as cleavage mixture. The high solubility of Bu 4 and rapid reduction, without affecting other functional groups in the peptide and yielding the desired product (Figure 4). This procedure does not require any additional substrates, including the most complex biomolecules. This peptides (25,44). performed in solid-phase using an excess of H 2 O 2 in DMF (in 30 min) after coupling Fmoc-Met-OH. UPLC-MS analysis the oxidized amino acid. Both syntheses (MW-assisted and manual) yielded the same product.

Met(O) is responsible of aggregation properties and thus toxicity. Although in some cases in vitro air and therefore water occur in vivo by ROS or Reactive Chlorine Species (RCS), such as hydrogen peroxide (H 2 O 2 ), chloramines, hypochlorous acid (HOCl) or hydroxyl radicals (HO . ) (27-31). In vitro, dimethylsulfoxide (DMSO) (32) chloramine-T and N-chlorosuccinimide (NCS) (33) have proven to be extremely To prevent oxidation, it is therefore recommended to perform peptide synthesis under N 2 atmosphere and the cleavage in the presence of reducing agents as scavengers (i.e. 1-3% of 1,2-ethanedithiol) avoiding the use of peroxidecontaining reagents. In the model tetradecapeptide GHSVFLAPYGWMVK synthesized in this study only Met at position 12 can be oxidized. Indeed, Ser, Trp and His need stronger conditions to be oxidized, such as metal-catalysis, radical initiators or alkaline medium (34,35). The tetradecapeptide synthesis was performed by MW instrumentations, with and without N 2 atmosphere, and manually. MW-assisted synthesis is recognized to drastically reduce reaction time and decrease peptide chain aggregation (24,36). Nevertheless, manual synthesis did not yield, in any conditions, the oxidized peptide.The synthesis performed on a MW-assisted apparatus working under N 2 atmosphere, gave the desired tetradecapeptide without any detectable oxidation. On the contrary, the same synthesis performed in the same conditions without N 2 atmosphere yielded a product showing a double UPLC signal. UPLC-MS analysis showed that one of the peaks corresponded to the oxidized peptide accounting for 30% of the crude (Figure 3). Met(O 2 ) could not be detected. Peptide cleavage performed either in the presence or not of 1,2-ethanedithiol yielded the same crude peptide.

Figure 1 :

 1 Figure 1: Epitope presentation to the T cell receptor by a MHC molecule

Figure 2 :

 2 Figure 2: Y-Shaped antibody structure (including Fc and Fab regions), and typical Fc-linked N-glycans.

Figure 3 :

 3 Figure 3: Loss of the balance between T effectors and T regulatory cells (by Chavele et al.10)

Figure 4 :

 4 Figure 4: Illustration of the molecular mimicry concept (by Oldstone 12 )

Figure 5 :

 5 Figure 5: Common PTMs, including associated chemical structures and most frequently modified amino acids (by Petersen et al.14)

Figure 6 :

 6 Figure 6: Overall representation of the CRA.

Figure 7 :

 7 Figure 7: Indirect ELISA procedure. The blocking step is not shown, and should be between the first and the second step.

Figure 8 :

 8 Figure 8: In-solution conformation of CSF114(Glc).

Figure 9 :

 9 Figure 9: Comparison of the sequences of the lipoyl domains of autoAgs found in PBC. PDC-E2(157-191) fragment is shown.

Figure 10 :

 10 Figure 10: The sequence of PDC-E2. Outer-and inner-lipoylated domains are represented (from Braun et al. 56 ).

Figure 11 .

 11 Figure 11. E1 acts as a catalyzer for both oxidative decarboxylation of pyruvate and reductive acetylation of the lipoyl moiety. E2 transfers this acyl group to the Coenzyme A. E3 regenerates the lipoyl moiety. Comparable mechanisms are found in 2-OADC systems.

3 C 2 Figure 11 :

 3211 Figure 11: The PDH reaction mechanism including E1, E2 and E3 subunits. ThDP: thiamine diphosphate. Lip: lipoyl-Lys moiety covalently bound to E2 subunit. CoA: CoenzymeA. FAD: flavin adenine dinucleotide. NAD: nicotin adenine dinucleotide.

Figure 12 :

 12 Figure 12: Representation of recombinant hPDC-E2 132-208 polypeptide fold (by Howard et al. 60 ) Lipoylation site is indicated together with the type I β-turn (black). The β sheet containing K 173 residue is shown in dark grey; β sheet containing N-and C-terminal residues is shown in light grey.

Figure 13 :

 13 Figure 13: Lipoyl-Lys (top) and representation of a protein-bound lipoic acid, a so-called lipoylated protein (below).

Figure 14 :

 14 Figure 14: The (S)-and (R)-α-lipoic acid.

Figure 15 :

 15 Figure 15: The dihydrolipoic acid (DHLA).

Figure 16 :

 16 Figure 16: Synthesis of racemic lipoic acid by Bullock et al.66

Figure 17 :

 17 Figure 17: PDC-E2 lipoylation using exogenous lipoic acid by the LplA pathway (adapted from Booker 72 )

Figure 18 :Figure 19 Figure 19 :

 181919 Figure 18: Biosynthesis of lipoic acid from octanoyl-ACP, involving both LipB and LipA.

4 Figure 20 :

 420 Figure 20: On-resin selective deprotection of a Lys(Fmoc) residue in Boc/Bzl strategy, followed by on-resin lipoylation using lipoyl HOBt ester.

Figure 21 :

 21 Figure 21:On-resin selective deprotection of a Lys(Dde) residue in Fmoc/tBu strategy, followed by on-resin lipoylation using lipoyl NHS ester.

Figure 22 :

 22 Figure 22: Synthesis of agarose-conjugated lipoylated peptides via oxime bond formation and on-agarose lipoylation using lipoyl NHS ester.

Figure 23 :

 23 Figure 23: Synthesis of a N-terminal lipoylated dipeptide Glu-Ala by Kates et al.86

Figure 24 :

 24 Figure 24: Synthesis of a lipoyl derivative of an octanoylated tetrapeptide (adapted from Bryant et al. 89 )

Figure 25 :

 25 Figure 25: Synthesis of a N ε -lipoyl N α -(Boc)Lys by Matsugo et al. 95

Figure 28 :

 28 Figure 28: Hypothetical in-solution structure of lipoylated CSF114 (left) and structure of the recombinant PDC-E2(132-208) (right).

Figure 29 :

 29 Figure 29: General strategy for the production of a lipoylated BB.

Figure 30 :

 30 Figure 30: The two diastereomers of Fmoc-L-Lys(N ε -lipoyl).

Figure 31 :

 31 Figure 31: Formation of the pentafluorophenylester of LA.

Figure 32 :

 32 Figure 32: Coupling of the lipoyl Pfp esters.

  -α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R,S)-yl)-1-oxopentyl]-Lys-OH (-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(S)-yl)-1-oxopentyl]-Lys-OH (6) 24%

Figure 33 :

 33 Figure 33: BB formation using an « in-situ » activation.

Figure 34 :

 34 Figure 34: Coupling reagents used for « in-situ » activation.

Figure 35 :

 35 Figure 35: Supposed structure of the ε-branched dipeptide Lys[Nε-Lys(Nε-lipoyl)].

Figure 36 :

 36 Figure 36: Optimal conditions for formation of product 4.

  -α-[[(9H-fluoren-9ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R)-yl)-1-oxopentyl]-Lys-OH (5) and (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(S)-yl)-1-oxopentyl]-Lys-OH (6) have extremely similar RP-HPLC retention times (eluents: A: 0.1% TFA in H 2 O, B 0.1% TFA in CH 3 CN).

FmocFigure 37 :

 37 Figure 37: Derivatization strategy for diastereomers separation.

  a detailed structure-activity relationship study allowing the development of a family of βturn structures termed CSF114 exposing optimally an aberrant posttranslational modification able to detect antibodies in another autoimmune disease (multiple sclerosis). Its optimized β-turn conformation maximizes the exposition of the modified amino acid [Asn(Glc)] at the top of the hairpin to the external media, thus facilitating the possible epitope-Ab interaction.

Figure 39 :

 39 Figure 39: The CSF114 sequence. N 7 is highlighted as the modified AA.

(

(

  1mg/10ml) by stirring in an open vessel. Progressive re-oxidation was monitored through HPLC, and completed in 12 h (Figure 41).

Figure 41 :

 41 Figure 41: Progressive reoxidation of a mixture of dithiolane (first peak) and dithiol (second peak) containing sample. Left chromatogram is at t 0 , mid is after 4 hours and right is after 12 hours.

Figure 42 :

 42 Figure 42: Oxidation or reduction of the disulfide bond directly on peptide.

  0.1% TFA in H2O; B 0.1% TFA in CH3CN. a. HPLC: Phenomenex Kinetex C18 2.6 mm 3*100mm; 5 min; 0,6 mL/min. b. UPLC: Waters Acquity UPLC BEH C18 1.7 mm 2.1*50mm; 3 min; 0,6 mL/min.

Figure 43 :

 43 Figure 43: Over-time UPLC chromtograms of lipoylated CSF114 on conditions simulating SP-ELISA. DHLA form R t = 2.18-2.20 min; LA form R t = 2.11-2.13 min.

Figure 44 :

 44 Figure 44: Reduction of dithiolane by mercaptoethanol.

Figure 45 : 3 OFigure 46 :Figure 47 :

 4534647 Figure 45: Over-time UPLC chromtograms of lipoylated PDC-E2-KKKK on conditions simulating SP-ELISA coating. DHLA form R t = 1.74-1.76 min; LA form R t = 1.69-1.71 min.

Figure 48 :

 48 Figure 48: IgG data distribution of PBC sera against [Lys 173 ]PDC-E2(167-186)-KKKK (XI), [(S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIV) and [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII).

Figure 49 :

 49 Figure 49: IgM data distribution of PBC sera against [Lys 173 ]PDC-E2(167-186)-KKKK (XI), [(S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIV) and [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII).

Figure 51 and

 51 Figure 51 and Figure 51 present the results (IgG and IgM) for peptides XI, XIII, XIV compared to peptides IV, VI and VII. Tests were performed for both IgG and IgM on a cohort of 65 PBC patients. (except IgM for peptide XVII which were tested on a smaller cohort of 30 sera).

Figure 50 :

 50 Figure 50: IgG data distribution of PBC sera against [Lys 173 ]PDC-E2(167-186)-KKKK (XI), [Lys 7 ]CSF114 (IV), [(S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIV), [(S)-Lipoamide-Lys 7 ]CSF114 (VII), [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII) and [(R)-Lipoamide-Lys 7 ]CSF114 (VI).

Figure 51 :

 51 Figure 51: IgM data distribution of PBC sera against [Lys 173 ]PDC-E2(167-186)-KKKK (XI), [Lys 7 ]CSF114 (IV), [(S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIV), [(S)-Lipoamide-Lys 7 ]CSF114 (VII), [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII) and [(R)-Lipoamide-Lys 7 ]CSF114 (VI).

Figure 52 :

 52 Figure 52: IgG data distribution of PBC sera against [Asn 173 ]PDC-E2(167-186)-KKKK (VIII), CSF114 (I), [Asn(Glc) 173 ]PDC-E2(167-186)-KKKK (IX) and CSF114(Glc) (II).

Figure 53 :

 53 Figure 53: IgM data distribution of PBC sera against [Asn 173 ]PDC-E2(167-186)-KKKK (VIII), CSF114 (I), [Asn(Glc) 173 ]PDC-E2(167-186)-KKKK (IX) and CSF114(Glc) (II).

Figure 54 :

 54 Figure 54: Comparison between Asn (blue), Asn(Glc) (orange) and Asn(GlcNAc) (gray) moieties on CSF114 for both IgG (top) and IgM (bottom).

Figure 55 :

 55 Figure 55: Comparison between Asn (green), Asn(Glc) (blue) and Asn(GlcNAc) (yellow) moieties on PDC-E2 for both IgG (top) and IgM (bottom).

  [Lys 173 ]PDC-E2(167-186)-KKKK (XI) -[(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII) -[Asn 173 ]PDC-E2(167-186)-KKKK (VIII) -[Asn(Glc) 173 ]PDC-E2(167-186)-KKKK (IX) Disease-specificity of the detected Abs was evaluated comparing the results obtained in PBC patients with NBD cohort using a cutoff value calculated as mean value of NBDs plus three times standard deviation of NBDs. Furthermore, we included 3 different cohorts of disorders as pathological control groups: Rheumatoid Arthritis (RA; n = 22), scleroderma (SSC; n = 10) and other non-hepatic AID (OAID; n = 47). We reported in Figure 56 and Figure 57 the results of SP-ELISA, quantifying the ability of the 4 synthetic Ags selected to detect specific IgG or IgM antibodies in sera of the various cohorts of patients. The dotted line indicates the cut-off, calculated as previously indicated. Comparing PBC data with NBDs through the cut-off value allowed us to identify [Lys 173 ]PDC-E2(167-186)-KKKK as the most relevant antigenic probe for IgM detection. Cédric Rentier, PhD thesis 82

Figure 56 :

 56 Figure 56: IgM data distribution of PBC patients and control cohorts against PDC-E2 peptides. Dotted line indicates the cutoff value.

Figure 57 :

 57 Figure 57: IgG data distribution of PBC patients and control cohorts against PDC-E2 peptides. Dotted line indicates the cutoff value.

Figure 58 :

 58 Figure 58: Schematic ELISA protocol including a reducing step before applying the Abs.

Figure 59 :

 59 Figure 59: Indirect SP-ELISA absorbance values for IgG measured at 405nm for the 2 peptides on selected sera.

Figure 60 :

 60 Figure 60: Indirect SP-ELISA absorbance values for IgM measured at 405nm for the 2 peptides on selected sera. The additional step with TCEP 5mM does not seem to affect the normal course of the results, either in IgG or IgM, as demonstrated by tests using CSF114(Glc) and [Lys 173 ]PDC-E2(167-186)-KKKK as synthetic antigens.

Figure 61 :

 61 Figure 61: Indirect SP-ELISA absorbance values for IgG measured at 405nm for [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII) on selected sera.

Figure 62 :

 62 Figure 62: Indirect SP-ELISA absorbance values for IgM measured at 405nm for [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII) on selected sera. In the case of the [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK, the 1,2dithiolane ring appears to give slightly better results than dihydrolipoyl in a significative number of sera in IgM (64% -7 out of 11 that are above a 0.75 cutoff value).

2. 4 .

 4 Conformational studies The circular dichroism and NMR studies have been performed in collaboration with Prof. Alfonso Carotenuto, from the Department of Pharmacy at the University of Napoli "Federico II". Possible insights into the in-solution favored conformations and exposition of the lipoyl moiety were investigated by circular dichroism and nuclear magnetic resonance (NMR) experiments. These were performed on unlipoylated peptide [Lys 173 ]PDC-E2(167-186)-KKKK (XI) and the lipoylated [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII).Circular dichroism spectra of the two peptides acquired in aqueous solution indicate a random coil conformation for both peptides (Figure63). The addition of a structure stabilizing agent like hexafluoroacetone (HFA) clearly induced a helical formation to different extents in the two peptides.

Figure 63 :

 63 Figure 63: Circular dichroism spectra of peptide XIII (a) and peptide XI (b) in water (blue), HFA/water 5% solution (red), HFA/water 10% solution (black), HFA/water 20% solution (violet), HFA/water 30% solution (dark yellow), HFA/water 40% solution (green), HFA/water 50% solution (cyan).

Figure 64 :

 64 Figure 64: Circular dichroism spectra of peptide XI (blue) and peptide XIII (red) in a 10% HFA/water solution.

Figure 65 :

 65 Figure 65: Superposition of the ten lowest energy conformers of peptide XIII. Structure models were superimposed using the backbone heavy atoms of residues 171-181. Backbone of the lowest energy conformer is shown as a grey ribbon. Heavy atoms of residues 171-181 have different colors (carbon, green; nitrogen, blue; oxygen, red; sulfur, yellow). Hydrogen atoms are hidden for a better view. *K 173 : lipoyl-lysine.

  (ProteinDataBank code 1FYC) allowed the acquisition of a model of the lipoylated domain of the human protein (see Figure10for the sequence, Figure12for the in-solution conformation). Additional data acquired by Jones et al.121 for homologue protein E. coli (ProteinDataBank code 1QJO) were helpful in building the model, because their data include both apo (without the prosthetic group) and holo (with the prosthetic group) forms. In both forms the structures of PDC-E2 domain in the apo form (human and E. coli) the lipoylable Lysine residue resides at the tip of a type-I β-hairpin. Upon lipoylation of PDC-E2 domain in E. coli, large changes in1 H N and 15 N chemical shift were observed for residues from position -2 to +3 around the lipoyl-lysine. Furthermore, the relaxation data suggested that the β-turn containing the lipoyl-lysine becomes less flexible upon lipoylation 121 . Overall, the data suggests a conformational change of the region around the lipoyl-lysine.

Figure 66 :

 66 Figure 66: (A) Ribbon representation of the lipoylated PDC-E2 final pose obtained by molecular dynamics simulations. Only lipoyl-lysine and side chains of residues 140 and 176 are reported for clarity (backbone in gray ribbon, the side chains are color coded as in Figure 65). (B) NMR structure of the apo form of PDC-E2 (pdb code 1FYC, orange ribbon). Only Lys 173 side chain is reported for clarity. Strands numbering of the original work are reported as S1-S8. Structures are showed with the same spatial orientation. *K 173 : lipoyl-lysine. Our results on the synthetic peptides demonstrated a switch to a helix conformation after the lipoylation, confirmed by the up-field shifts of many 1 H N and 15 N chemical shift around Lys 173 . Taking this into account, a brief segment of α-helix encompassing lipoyl-lysine residue was imposed in building our PDC-E2 model. Then, molecular dynamics simulations were performed for PDC-E2 model in explicit water. During the simulations, the helical region remained stable and the lipoyl chain stably interacted with Leu 140and Ile 176 side chain (Figure66). The latter observation confirms a hydrophobic driving force for the conformational change.

Figure 68 :

 68 Figure 68: Pathway of the raising of glucose blood level in type-1 diabetes.

Figure 69 :

 69 Figure 69: Pathway of the raising of glucose blood level in type-2 diabetes.

Figure 70 :

 70 Figure 70: Transfer of an oligosaccharide chain to a peptide by oligosaccharyltransferase, in ER. □ = N-acetylglucosamine; • = mannose; ∆ = glucose.

Figure 71 :

 71 Figure 71: Glycation reaction and Amadori rearrangement leading to glycation adducts.

Figure 72 :

 72 Figure 72: The hexosamine biosynthetic pathway (adapted from Banerjee et al. 147 ).

Figure 73 :

 73 Figure 73: Addition or removal of O-GlcNAc by the respective action of O-GlcNAc transferase and O-GlcNAcase (adapted from Hart et al. 150 ).

Figure 74 :

 74 Figure 74: Balance between O-GlcNAcylation and phosphorylation (adapted from Hart et al. 150 ).

Figure 75 :

 75 Figure 75: Phosphorylation of an OH residue by adenosine triphosphate, catalyzed by a kinase.

Figure 76 :

 76 Figure 76: PTM of interest in diabetes. N-glycosylation (N-GlcNAc), O-glycosylation (O-GlcNAc), 1-deoxyfructosyl (deoxyFru) and phosphorylation (Phos).Because of the demonstrated role of mitochondrial machinery in diabetes, sequence and chemical modification previously discussed in the first part of this thesis will be included in this study; i.e. PDC-E2(167-186) and lipoylation.This modified protein plays a key role in the energetic metabolism of cells by proceeding to the oxidative decarboxylation of pyruvates, and links glycolysis with citric acid cycle.Lipoylation hypothetically could be involved in immune recognition, following an unexpected exposition of mitochondrial auto-Ags generating Abs as a consequence of breaking tolerance and triggering an immune response 153 . This hypothesis is supported by some work in the literature regarding cellular hypersensitivity to mitochondrial antigens in patients with diabetes mellitus 154 .

14 :

 14 below the various modifications introduced are shown including their structures: For lipoylated building-block and lipoylation, refer to Chapter One For N-glucosylated building-block and glucosylation, refer to Chapter One For N-acetylglucosamine building-block and N-glycosylation, refer to Chapter OneTable Chemical modifications introduced in peptide sequences tested for diabetes.

Figure 77 :Figure 78 :

 7778 Figure 77: Zemplen deacetylation reaction mechanism.

2. 3 . 2 . 4 Figure 79 :

 32479 Figure 79: The Fmoc-L-Lys(Boc)(2,3:4,5-di-O-isopropylidene-1-deoxyfructopyranosyl)-OH building block.

Figure 80 :

 80 Figure 80: Crude UPLC chromatograms of [(deoxyFru) 173 Lys]PDC-E2(167-186)-KKKK (XXVIII). Cleavage conditions: TFA/H 2 O/TIS 95:2.5:2.5, r.t., after 3 hours (left) and 5 hours (right).

- 2 .Figure 81 :

 281 Figure 81: The Fmoc-L-Ser(PO 3 Bzl,H)-OH building block.

Figure 82 :

 82 Figure 82: Mechanism of β-elimination and subsequent 3-(1-piperidinyl)alanyl formation.

  SP-ELISA studies have been performed in collaboration with Ms. Martina Crulli from the PeptLab of the University of Florence, Dr. Giada Rossi, and Prof. Paolo Rovero, from PeptLab@UniFi at the Department NeuroFarBa, Section of Pharmaceutical Sciences and Nutraceutics of the University of Florence.

I g G d a ta d is tr ib u tio n T 1 Figure 83 :

 183 Figure 83: IgG data distribution of T1D sera against some peptides reported in Table17.

Ig M d a ta d istr ib u tio n T 1 Figure 84 :

 184 Figure 84: IgM data distribution of T1D sera against some peptides reported in Table17.

Figure 86 Figure 85 :

 8685 Figure 86 for IgM.

IFigure 86 :

 86 Figure 86 : IgM data distribution of NBD sera against some peptides reported in Table17.

Ig M d a ta d istr ib u tio n T 2 Figure 88 :AFigure 89 :Figure 90 :AFigure 91 :Figure 92 :Figure 94 :

 2888990919294 Figure 88: IgM data distribution of T2D sera against some peptides reported in Table17.

Chapter

  Two: Diabetes -3. Conclusion Cédric Rentier, PhD thesis 134 their usefulness as molecular probes for the detection of specific Abs in sera of such patients. Results show that three peptides, namely [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (peptide XIII), [Ser 173 (GlcNHAc)]PDC-E2(167-186)-KKKK (peptide XXVII) and [Ser 173 (PO 3 H 2 )]PDC-E2(167-186)-KKKK (peptide XXVIII) were able to detect IgM Abs in the sera of T1D patients with sensitivity and specificity defined by the ROC curves. At a minimum 90% specificity level, sensitivity (number of patients) of the synthetic probes for T1D vs NBD are of 42% for peptide XIII, 22% for peptide XXVII and 29%

  MS) is a chronic, inflammatory, demyelinating disease of the central nervous system, characterized by the formation of sclerotic plaques in various areas. These plaques are the result of an inflammatory response most likely caused by activation of autoimmune Th-1 cell targeting oligodendrocytes and the myelin sheath together with activated monocytic cells. These highly immunologically active areas subsequently progress to form "scarring" plaques 165 .

Figure 95 :

 95 Figure 95: Immune status and disease course in MS (from Weiner 168 ).

Figure 96 :

 96 Figure 96: The Kynurenine Pathway (figure by Rodgers et al. 176 )

  It has been shown that patients with chronic MS have low Trp concentrations in serum and cerebrospinal fluid suggesting activation of the KP 193,194 . Analysis of human MS ex vivo samples showed that the levels of KynA are decreased in the cerebrospinal fluid of patients with MS 195 . These results are supported by another study showing that the expression of KAT I and II (enzymes responsible for the production of KynA) are decreased in postmortem MS brain sections 196 . However, other studies found the opposite, that there is an increase in the KynA levels in cerebrospinal fluid samples of MS patients. Hartai et al. demonstrated that KAT I and II activity was increased in red blood cells and plasma from MS patients 197 . These contradictory results may be explained by the sample collection time, suggesting that the KP is induced during the active phase of MS leading to increased production of KynA but as the disease progresses the KP profile changes resulting in decreased KynA levels implicating the shift of the KP towards neurotoxicity 198 . 1.4. Introduction of Kynurenine in peptides Introduction of Kyn into peptides can be performed either by the convergent pathway (kynurenination directly on the peptide), or by the building-block strategy (the modified AA is synthesized and introduced as a classic AA during the course of peptide synthesis). The building-block strategy can provide straightforward methods for the introduction of Kyn into peptides. Some methods, with their advantages and drawbacks, are described below. Kynurenine [2-amino-4-(2-aminophenyl)-4-oxobutanoic acid] is a non-natural amino acid. Its structure is represented in Figure 97.

Figure 97 :

 97 Figure 97: Kynurenine.

Figure 98 :

 98 Figure 98: Preparation of racemic Kyn via condensation of diethyl acetamidomalonate with onitrophenacyl bromide (adapted from Kleijn et al. 203 ).

Figure 99 :Figure 100 : 2 Figure 101 :

 991002101 Figure 99: Preparation of Kyn by direct ozonolysis of N-acetyl Trp (adapted from Kleijn et al.203).

Figure 102 : 2 Figure 103 : 3 OFigure 104 :

 10221033104 Figure 102: L-Kyn synthesis by Trp oxidation and the subsequent β-oxindolylalanine intermediate 2 nd oxidation (adapted from Kleijn et al. 203 ).

Figure 105 :

 105 Figure 105: Selective opening of Trp indole ring by Recombinant human IDO enzyme and mild acidolysis by perchloric acid to obtain Kyn.

Figure 106 :

 106 Figure 106: On-resin Trp ozonolysis followed by peptide cleavage to yield the Kyn containing peptide.

Figure 107 :

 107 Figure 107: The CSF114 sequence. N 7 is highlighted as the modified AA.

Figure 108 :

 108 Figure 108: The BB suitable for Fmoc/tBu SPPS, Fmoc-L-Kyn-OH.

  manuscript presents three AIDs: Primary Biliary Cirrhosis, Diabetes and Multiple Sclerosis.The first chapter deals with PBC. We successfully synthesized the two diastereomers of the Fmoc-L-Lys(N ε -lipoyl)-OH to be used as Fmoc/tBu SPPS building blocks. These BBs were introduced in a synthetic fragment of the native mitochondrial protein PDC-E2(167-186) and on the top of an irrelevant β-turn structure, CSF114.Results showed the high importance of the peptide sequence in IgM recognition: the irrelevant β-turn peptide does not show any Ab titre. Chirality of the lipoic acid is not significant. Conformation of the peptide is involved in auto-Ab recognition: introduction of lipoylation shifts the tridimensional structure towards a helix, decreasing affinity of the peptide for auto-Abs. Conclusions Cédric Rentier, PhD thesis 158 The synthesis of conformationally constrained lipoylated analogues of the PDC-E2(167-186) should be performed in order to further investigate these results. The second chapter deals with Diabetes. The main characteristic of this disease is hyperglycaemia. Therefore, attachment of sugars onto proteins appears a relevant PTM, as well as phosphorylation which is closely related to Oglycosylation; and lipoylation for the mitochondrial involvement of glucose in the energy cycle. Various glucosylated/glycosylated/glycated/phosphorylated/lipoylated peptides were synthesized, based on a synthetic fragment of the native mitochondrial protein PDC-E2(167-186) and on top of an irrelevant β-turn structure, CSF114. Results show that three peptides, namely [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK, [Ser 173 (GlcNHAc)]PDC-E2(167-186)-KKKK and [Ser 173 (PO 3 H 2 )]PDC-E2(167-186)-KKKK were able to detect IgM Abs in the sera of T1D patients with sensitivity and specificity over T2D or NBD populations. Further studies on this disease, including the synthesis of other peptides including hyperglycaemia-related chemical modifications (such as crosslinked Advanced glycation end products) may provide new insights in the mechanisms of this complex disease. The third chapter deals with Multiple Sclerosis. The involvement of Kynurenine Pathway in MS was demonstrated in the literature, and the synthesis of kynurenine-containing peptides is a relevant train of research, as Kyn is the first stable metabolite of Trp involved in KP. Kynureninated CSF114 analogues were successfully synthesized and tested on MS and Alzheimer patients' sera. Results showed a high IgG antibody level in only one MS serum. Among the 4 Alzheimer sera tested, 2 gave a positive (IgG) result for the peptide [Kyn 18 ]CSF114 and 1 (IgM) for both Kyn-Cédric Rentier, PhD thesis 159

1 H

 1 NMR (400 MHz) and13 C NMR (100 MHz) were measured on a JEOL (Tokyo, Japan) ECX400 spectrometer. 1H NMR (200 MHz) and 13C NMR (50 MHz) were measured on a Varian (Palo Alto, CA, USA) Gemini spectrometer. Chemical shifts of 1 H and 13 C NMR are expressed in parts per million downfield from tetramethylsilane (TMS, δ = 0) and are referenced to residual undeuterated chloroform (7.26 and 77 ppm, respectively) as internal standard. Coupling constants are reported in Hertz. The multiplicities were marked as s (singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Flash Column chromatography (FCC) was performed on SDS 60Å (40-63 µm) silica gel, employing a mixture of the specified solvent as eluent. Normal-phase thin-layer chromatography (TLC) was performed on Macherey-Nagel Alugram XtraSil (Macherey-Nagel silica gel 60 Å F254) plates. Silica TLC plates were analyzed with UV light (254 nm) and by a 0.2% solution of ninhydrine in ethanol.

2. 1 .

 1 General procedure for in batch SPPS on the manual PLS 4x4 synthesizer Peptides are synthesized on a manual batch synthesizer 213 (PLS 4×4, Advanced ChemTech) using a Teflon reactor (10 or 20 mL), following the Fmoc/tBu SPPS procedure. The resin is placed in the Teflon reactor, equipped with a filter. Mixing is provided by vortex, while filtration is performed connecting the reactor to a vacuum pump. The resin is swollen for 40 min with DMF (1 mL/100 mg of resin) before use. Each amino-acid cycle is characterized by the following four steps: Fmoc-deprotection: Resin is stirred twice (1 × 5 min + 1 × 15 min) with a solution of 20% piperidine in DMF (1 mL/100 mg of resin).

  : DMF (3 × 3 min) and DCM (2 × 3 min). Fmoc-deprotection of the second residue should be performed by a fast protocol to avoid DKP formation (3 × 5 min). After DCM washings each coupling reaction is monitored by Kaiser test. After the last Fmoc-removal the resin is washed with DCM and dried under vacuum. 2.2. General procedure for in batch SPPS on an automatic peptide synthesizer All the reactions are performed in one of the automated peptide synthesizers described in Materials and Methods using a Fmoc/tBu protection scheme. A robotic needle-like arm allows the distribution of reagents predissolved (0.5 M amino acids, coupling reagent and TBTU solutions in DMF; and 1M DIPEA in NMP). The resin is swollen for 40 min. Each amino-acid cycle is characterized by the following four steps: Fmoc-deprotection: Resin is stirred twice (1 × 5 min + 1 × 15 min) with a solution of 20% piperidine in DMF (1 mL/100 mg of resin).Washings: DMF (3 × 2 min).Coupling reaction: Fmoc amino acids (4 eq), TBTU (4 eq) and DIPEA (5 eq) are added as solutions described above. Couplings were performed for 45 min at room temperature. Washings: DMF (3 × 2 min) and DCM (3 × 2 min). Deprotection of the second residue should be performed by a fast protocol to avoid DKP formation (3 × 5 min). Experimental Part -2. General procedure for Solid Phase Peptide Synthesis Cédric Rentier, PhD thesis 165 Below are described the particularities of each synthesizer and differences with this general protocol. SyroII: All the reactions were performed in a Teflon reactor, where mixing is provided by a vortex and filtration is performed by a vacuum pump. After the removal of the last Fmoc-residue and the washes with DCM, resin is dried by pumping air through the resin during 30 sec. Apex 396: All the reactions were performed in a Teflon reactor, where mixing is provided by a vortex and filtration is performed by a N 2 flow. After the removal of the last Fmoc-residue and the washes with DCM, resin is dried under a N 2 flow for 30 min. Liberty Blue: All the reactions were performed in a Teflon vessel, where mixing and filtration are provided by a N 2 flow. Predissolved reagents are 0.2M amino acids, 0.5M DIC and 1M Oxyma solutions in DMF. Fmocdeprotection cycles are respectively of 15 sec (75°C, 155W) and 30 sec (90°C, 30W). Couplings of Arg residues are performed in 1500 sec (25°C, 0W) + 300 sec (75°C, 30W). Couplings of Cys and His are performed in 120 sec (25°C, 0W) + 240 sec (50°C, 35W). Couplings of other residues are performed in 15 sec (90°C, 170W) + 110 sec (90°C, 30W).

, 5 and 6 4. 2 . 1 .

 621 Fmoc-L-Lys(N ε -lipoyl)-OH building blocks 4Using lipoyl pentafluorophenyl ester route To a solution of the (R,S)-(1 g, 2.68 mmol), (R)-(924 mg, 2.48 mmol) or (S)α-lipoic acid-OPfp ester (872 mg, 2.34 mmol) dissolved in CH 3 CN (20 ml) are successively added under stirring Fmoc-L-Lys-OH (1 eq), HOBt monohydrate (1 eq) and DIPEA until pH 7-8. The reaction is stirred and followed by TLC (DCM/MeOH 9:1). After 24h, solvents are evaporated under reduced pressure. The resulting brown oil is dissolved in DCM and the organic phase is successively washed twice with 0.5 M KHSO 4 , then three times with H 2 O. Organic phase is collected, dried over Na 2 SO 4 , filtered and concentrated under reduced pressure. The resulting yellow oil is purified by normal phase FCC using a gradient of DCM/MeOH (100:0 to 90:10). Fractions are checked by TLC (DCM/MeOH 9:1) then collected, to yield the desired product as a yellow solid. (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R,S)yl)-1-oxopentyl]-Lys-OH (4) TLC (DCM/MeOH 9:1) R f = 0.5 R t (UPLC, gradient 40-100% B in A over 3 min) = 2.70 min ESI-MS: m/z calc.

[ 4 , 5 or 6 .

 46 Lys 173 ]CSF114 (IV), [(R,S)-Lipoamide-Lys 173 ]CSF114 (V), [(R)-Lipoamide-Lys 173 ]CSF114 (VI) and [(R,S)-Lipoamide-Lys 173 ]CSF114 (VII) are synthesized by SPPS using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using either PLS 4×4, Apex 396 or SyroII. The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.70 mmol/g).Modified amino acids (R,S)-Lipoamide, (R)-Lipoamide or (S)-Lipoamide are introduced by manual batch SPPS with PLS 4×4 using the protected buildingblocks suitable for Fmoc/tBu SPPS, Couplings are performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of DIPEA, for 1 hour at r.t. Cleavages were performed as described in General procedure for cleavages and micro-cleavages using a mixture of TFA/thioanisole/H 2 O/phenol/EDT (Reagent K, 82.5:5:5:2.5 v:v:v:v:v).

Table 22 :

 22 used were: A (0.1% TFA in H 2 O) and B (0.1 % TFA in CH 3 CN). Sequences, gradients and yields for purification of peptides IV -VII.

4. 3 . 3 .

 33 Synthesis of PDC-E2(167-186) glycosylated analogues [Asn 173 ]PDC-E2(167-186)-KKKK (VIII), [Asn(Glc) 173 ]PDC-E2(167-186)-KKKK (IX) and [Asn(GlcNAc) 173 ]PDC-E2(167-186)-KKKK (X) are synthesized by SPPS using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using either PLS 4×4, Apex 396 or SyroII.The resin used was a Fmoc-Lys(Boc)-Wang (loading 0.31 mmol/g).Modified amino acids, Asn(Glc) or Asn(GlcNAc) are introduced by manual batch SPPS with PLS 4×4 using the protected building-blocks suitable for Fmoc/tBu SPPS, Fmoc-L-Asn[β-Glc(OAc) 4 ]-OH and Fmoc-L-Asn(Ac 3 AcNHβ-Glc)-OH. Couplings are performed with an excess of 2.5 eq of modified amino acid, 2.5 eq of HATU as activator, and 3.5 eq of DIPEA, for 1 hour at r.t. Cleavages were performed as described in General procedure for cleavages and micro-cleavages using a mixture of TFA/TIS/H 2 O (95:2.5:2.5 v:v:v).

4. 3 . 4 .

 34 Synthesis of PDC-E2(167-186) lipoylated analogues [Lys 173 ]PDC-E2(167-186)-KKKK (XI), [(R,S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XII), [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIII) and [(R,S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (XIV) are synthesized by SPPS using Fmoc/tBu strategy, as described in General procedures for Solid Phase Peptide Synthesis, using either PLS 4×4, Apex 396 or SyroII.

4. 3 . 5 . 4 . 7 .

 3547 Procedure for deprotection of Glc moietiesDeprotection of the hydroxyl functions of sugar moieties linked to the Nglycosylated peptides was performed with a 0.1 M methanolic NaOMe solution until pH 11-12 to a solution of the lyophilized peptides in dry MeOH (1 mL/100 mg of resin) under N 2 atmosphere. Reaction is checked by UPLC-MS every hour to follow the deprotection. Once reaction was complete, mixture triple-resonance probe head in a 3 mm tube. The spectra were calibrated relative to trimethylsilyl propanoic acid (TSP, 0.00 ppm) as internal standard.One-dimensional (1D) NMR spectra were recorded in the Fourier mode with quadrature detection. 2D DQF-COSY215,216 , TOCSY 217 , and NOESY 218 spectra were recorded in the phase-sensitive mode using the method from States 219 . Data block sizes were 2048 addresses in t 2 and 512 equidistant t 1 values. A mixing time of 70 ms was used for the TOCSY experiments. NOESY experiments were run with mixing times in the range of 150-300 ms. The water signal was suppressed by gradient echo 220 . The 2D NMR spectra were processed using the NMRPipe package 221 . The qualitative and quantitative analysis of DQF-COSY, TOCSY, and NOESY spectra were obtained using the interactive program package XEASY 222 . The temperature coefficients of the amide proton chemical shifts were calculated from 1D 1 H NMR and 2D TOCSY experiments performed at different temperatures in the range 25-40 °C by means of linear regression. Structure calculation by molecular dynamics The NOE-based distance restraints were obtained from NOESY spectra collected with a 200 ms mixing time. The NOE cross peaks were integrated with the XEASY program and were converted into upper distance bounds using the CALIBA program incorporated into the DYANA program package 223 . A bar diagram reporting diagnostic medium range NOEs used in the calculations is shown in Figure S2. NMR-derived upper bounds were imposed as semiparabolic penalty functions with force constants of 16 Kcal mol -1 Å -2 . A distance maximum force constant of 250 Kcal mol -1 Å -2 was used.

5 .

 5 was applied to the system for 2000 fs (time step = 1.0 fs) with the aim of surmounting torsional barriers; successively, temperature was linearly reduced to 300 K in 1000 fs (time step = 1.0 fs). Resulting conformations were then subjected to restrained Molecular Mechanics (MM) energy minimization within Insight Discover module (ε = 1r) until the maximum RMS derivative was less than 0.001 kcal/Å, using Conjugate Gradient as minimization algorithm. Finally, conformations were subjected to 1000 steps of unrestrained MM Conjugate Gradient energy minimization. From the produced 100 conformations, 10 structures, whose interprotonic distances best fitted NOE derived distances, were chosen for statistical analysis. Cédric Rentier, PhD thesis 182 Experimental part Chapter Two 5.1. Synthesis of glycated peptides [(deoxyFru)Lys 173 ]CSF114 (XXI) and [(deoxyFru)Lys 173 ]PDC-E2(167-186)-KKKK

5. 2 . 1 . 6 .

 216 Deprotection of Glc moieties Deprotection of the hydroxyl functions of sugar moieties linked to the Oglycosylated peptides was performed with a 0.1 M methanolic NaOMe solution until pH 11-12 to a solution of the lyophilized peptides in dry MeOH (1 mL/100 mg of resin) under N 2 atmosphere. Reaction is checked by UPLC-MS every 20 min to follow the deprotection. Once reaction was complete, mixture was quenched by adding conc. HCl until pH 7, the solvent was evaporated under vacuum and the residue lyophilized. Experimental Part -6. Experimental part Chapter Three Cédric Rentier, PhD thesis 185 Experimental part Chapter Three 6.1. Synthesis of CSF114 kynureninated analogues [Kyn 7 ]CSF114 (XXIX) and [Kyn 18 ]CSF114 (XXX) are synthesized by SPPS

1 H

 1 TLC (DCM/MeOH 7:1) R f = 0.85 Electrospray ionisation mass spectrometry (ESI-MS): m/z calc. 372.0 for C 14 H 13 F 5 O 2 S 2 , found 373.1 ([M + H] + ) NMR (CDCl 3 , 200MHz): 3.59 (quin, 1H, dithiolane CH, J = 6.6 Hz), 3.23-3.05 (m, 2 H, dithiolane CH 2 ), 2.68 (t, 2 H, lipoyl CH 2 ), 2.56-2.40 (m, 1 H, dithiolane 7-H a ), 2.00-1.90 (m, 1 H, dithiolane 7-H b ), 1.87-1.68 (m, 4 H, 2 × lipoyl CH 2 ), 1.64-1.58 (m, 2 H, lipoyl CH 2 ).

13 C

 13 NMR (CDCl 3 , 50 MHz): 168.56 (C = O), 143.27 (m-Ph), 141.38 (C-O), 139.80 (p-Ph), 135.08 (o-Ph), 55.84 (dithiolane CH), 39.82 (dithiolane C-7), 38.12 (dithiolane C-8), 34.50, 32.52, 28.09, and 24.14 (4 × lipoyl CH 2 ).

  UPLC, gradient 40-100% B in A over 3 min) = 2.70 min ESI-MS: m/z calc. 556.2 for C 29 H 36 N 2 O 5 S 2 , found 557.4 ([M + H] + ) [α] D = À31°(c = 1.0, CHCl 3 , 22 °C) mp = 89-91 °C NMR 1 H (400 MHz) and NMR 13 C (100 MHz) are identical to the racemic compound (refer to the succeeding paragraphs). (S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2dithiolan-3(S)-yl)-1-oxopentyl]-Lys-OH (1b) TLC (DCM/MeOH 9:1) R f = 0.5 R t (UPLC, gradient 40-100% B in A over 3 min) = 2.70 min ESI-MS: m/z calc. 556.2 for C 29 H 36 N 2 O 5 S 2 , found 557.4 ([M + H] + ) [α] D = +22°(c = 1.0, CHCl 3 , 22 °C) mp = 95-97 °C

1 H

 1 UPLC, gradient 40-100% B in A over 3 min) = 2.70 min ESI-MS: m/z calc. 556.2 for C 29 H 36 N 2 O 5 S 2 , found 557.6 ([M + H] + ) NMR (CDCl 3 , 200 MHz): δ 7.74 (d, 2 H, J 3,4 = J 5,6 = 7.4 Hz, fluorenyl 4-H and 5-H), 7.57 (d, 2 H, J 1,2 = J 7,8 = 7.4 Hz, fluorenyl 1-H and 8-H), 7.42-7.25 (m, 4 H, fluorenyl 2-H, 7-H, 3-H and 6-H), 5.84 (bs, 1 H, NH ε Lys), 5.71 (bs, 1H, NH α Lys), 4.39-4.35 (m, 3 H, fluorenyl CH 2 and CH), 4.23-4.19 (bt, 1

13 C

 13 NMR (CDCl 3 , 50 MHz): δ 174.9 (COOH), 173.93 (CONH), 156.34 (C = O Fmoc), 143.58 and 141.00 (fluorenyl C-4a, C-4b, C-8a and C-9a), 127.65 and 127.03 (fluorenyl C-2, C-7, C-3 and C-6), 124.51 (fluorenyl C-1 and C-8), 119.89 (fluorenyl C-4 and C-5), 67.07 (fluorenyl CH 2 -O), 56.30 (dithiolane CH), 53.63 (Lys CH), 47.11 (fluorenyl C-9), 40.35 (dithiolane C-7), 39.11 (Lys CH 2 ), 38.41 (dithiolane C-8), 36.35 and 34.48 (2 × lipoyl CH 2 ), 31.80 and 29.7 (2 × Lys CH 2 ), 28.80 and 24.55 (2 × lipoyl CH 2 ), 22.29 (Lys CH 2 ).

RENTIER

  ET AL. wileyonlinelibrary.com/journal/jpepsci Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2015; 21: 408-414

Table 1 .

 1 Yields for 1a, 1b, and 1c obtained using the lipoyl pentafluorophenyl ester route (Scheme 1, Pathway A) -α-[[(9H-fluoren-9-ylmethoxy) carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R)yl)-1-oxopentyl]-Lys-OH (1a) (S)-(-)-α-lipoic acid 21% (S)-α-[[(9H-fluoren-9-ylmethoxy) carbonyl]amino]-ε-[5-(1,2-dithiolan-3(S)yl)-1-oxopentyl]-Lys-OH (1b) (R,S)-α-lipoic acid 24% (S)-α-[[(9H-fluoren-9-ylmethoxy) carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R, S)-yl)-1-oxopentyl]-Lys-OH (1c)

7 ] 7 ]

 77 CSF114 (3a) and [(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (8a) ( S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R)-yl)-1-oxopentyl]-Lys-OH (1a) [(S)-Lipoamide-Lys CSF114 (3b) and [(S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (8b) ( S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(S)-yl)-1-oxopentyl]-Lys-OH (1b) [(R,S)-Lipoamide-Lys 7 ]CSF114 (3c) and [(R,S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK (8c) ( S)-α-[[(9H-fluoren-9-ylmethoxy)carbonyl]amino]-ε-[5-(1,2-dithiolan-3(R,S)-yl)-1-oxopentyl]-Lys-OH (1c) [Asn(Glc) 173 ]PDC-E2(167-186)-KKKK (5) F m o c -A s n [ β-Glc(OAc) 4 ]-OH a [Asn(GlcNAc) 173 ]PDC-E2(167-186)-KKKK (6) F m o c -A s n ( β-GlcNAc(Ac) 3 )-OH b a see reference [34]. b Commercially available.RENTIER ET AL. wileyonlinelibrary.com/journal/jpepsci Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2015; 21: 408-414

  inserted a lipoylated Lys residue in that position, which was shown to be exposed on the tip of the β-turn. On the basis of the immunological results, reported below, indicating that the two enantiomers of lipoic acid are equivalent in terms of antibody recognition, we decided to use the natural R enantiomer, preparing the peptide [(R)-lipoamide-Lys 7 ]-CSF114 (IV), as well as its unlipoylated counterpart, [Lys 7 ]CSF114 (V).

Figure 1 .

 1 Figure 1. IgM and IgG data distribution of R and S enantiomers of lipoylated PDC-E2(167-186) on PBC sera.

Figure 2 .

 2 Figure 2. IgM (A) and IgG (B) data distribution of lipoylated and unlipoylated PDC-E2 peptides and designed analogues on sera from the AMA positive PBC and normal blood donor groups. Cut-off and P-values obtained by unpaired t tests with Welch's correction are shown: **** P < 0.0001; *** P 0.0001 to 0.001; ** P 0.001 to 0.01; * P 0.01 to 0.05; NS P > 0.05.

Figure 3 .

 3 Figure 3. Venn diagram of serum cross-reactivity. (A) IgM, (B) IgG.

Figure 5 .

 5 Figure 5. HN-Hα region of the NOESY spectra of peptide I (a) and III (b). Diagnostic cross peaks are labeled.

Figure 6 .

 6 Figure 6. Superposition of the 10 lowest energy conformers of peptide I. Structure models were superimposed using the backbone heavy atoms of residues 171-181. Backbone of the lowest energy conformer is shown as a gray ribbon. Heavy atoms of residues 171-181 have different colors (carbon, green; nitrogen, blue; oxygen, red; sulfur, yellow). Hydrogen atoms are hidden for a better view. *K 173 : lipoyl-lysine.

Figure 7 .

 7 Figure 7. (A) Ribbon representation of the lipoylated PDC-E2 final pose obtained by MD simulations. Only lipoyl-lysine and side chains of residues 140 and 176 are reported for clarity (backbone in gray ribbon, the side chains are color coded as in Figure 6). (B) NMR structure of the apo form of PDC-E2 (pdb code 1FYC, orange ribbon). Only Lys 173 side chain is reported for clarity. Strands numbering of the original work are reported as S1-S8. Structures are showed with the same spatial orientation. *K 173 : lipoyl-lysine.

  H 2 O (99.9%) was obtained from Aldrich (Milwaukee, WI, USA), and [(2,2,3,3-tetradeuterio-3-(trimethylsilanyl)]propionic acid (TSP) was from MSD Isotopes (Montreal, Canada).

Figure 8 .

 8 Figure 8. Data distribution of antibody response to [(R)-lipoamide-Lys173]PDC-E2(167-186)-KKKK (I, (A) IgM, (C) IgG) and [Lys173]PDC-E2(167-186)-KKKK (III, (B) IgM, (D) IgG) in AMA positive (AMA+) and negative (AMA-) PBC patients sera compared with control cohorts. Dotted line indicate the cutoff value. P-Values obtained comparing AMA+ PBC patients sera with control cohorts by nonparametric Mann-Whitney test are shown: **** P < 0.0001; *** P 0.0001 to 0.001; ** P 0.001 to 0.01; * P 0.01 to 0.05; NS P > 0.05.

  H NMR and 2D TOCSY experiments performed at different temperatures in the range 25-40 °C by means of linear regression.

Fig. 1 (Fig. 2 Scheme 1

 121 Fig. 1 (S)-and (R)-Lipoic acid

Fig. 3

 3 Fig. 3 Lipoyl-Lys (top) and representation of a protein-bound lipoic acid, a so-called lipoylated protein (below)

  Scheme 5). The E1 subunit catalyzes both oxidative decarboxylation of pyruvate and reductive acetylation of the lipoyl moiety. The E2 subunit transfers the acetyl group to Coenzyme A. The E3 subunit regenerates the lipoyl moiety. Comparable mechanisms are found in the 2-OGDH and 2-OADC systems.

Scheme 10

 10 Scheme 10 Synthesis of an N-terminal lipoylated dipeptide Glu-Ala byKates et al.

Scheme 11

 11 Scheme 11 Synthesis of a lipoyl derivative of an octanoylated tetrapeptide (adapted fromBryant et al.)

Scheme 12

 12 Scheme 12 Synthesis of a N  -lipoyl N  -(Boc)Lys by Matsugo et al.

Scheme 13

 13 Scheme 13 Synthesis of lipoyl-derivatives replacing the amide bond with bioisosteric groups (adapted fromKoufaki et al.)

  Scheme 15 Lipoylation of Fmoc-Lys by pentafluorophenyl ester strategy

Figure 1 .33

 1 Figure 1. Met, Met(O) and Met(O 2 ).

2 .

 2 The peptides were precipitated in peroxidefree cold Et 2 O, centrifuged and lyophilized. Peptide characterization was performed by Ultra Performance Liquid Chromatography using a Waters ACQUITY UPLC coupled to a single quadrupole ESI-MS (Waters 3100 Mass Detector) using a Waters (Waters Corporation, Milford, MA, USA) BEH C 18 column (1.7 m, 2.1 × 50 mm) at 35 °C, with H 2 O and B 0.1% TFA in CH 3 CN.

2

 2

Figure 2 .

 2 Figure 2. without (bottom) in cleavage mixture. Oxidized peptide R t = 1.58 min and expected peptide R t = 1.83 min. Gradient 25-60% B in A over 3 min.

Figure 3 .

 3 Figure 3. Crude UPLC chromatogram of the tetradecapeptide using TFA/TIS/H 2 O/EDT cleavage method. Oxidized peptide R t = 2.19 min. Expected peptide R t = 2.38 min. Gradient 20-60% B in A over 3 min.

Table 1 :

 1 The five isotypes of Ig in mammals.

Table 2 :

 2 Sensitivity of various immunoassays

Cédric Rentier, PhD thesis 14

  56

Table 3 :

 3 Summary

of the literature regarding Ags in PBC autoimmunity (adapted with permission from G. Pacini, Ph.D. thesis, University of Florence, 2015)

Table 3

 3 summarizes some notable studies, including the description of the Ag used (sequence-and lipoylation-wise) and the conclusions for each of them regarding lipoylation requirements.

  Immunogenicity of the conformationIt may be possible that, independent of the sequence or the lipoylation status, Abs recognize a discontinuous epitope.Study on Bacillus stearothermophylus PDC-E2 lipoyl domain showed that lipoylation does not impose any conformational change on the secondary structure of the protein59 . If the same applies to the human analog, this means that the Ab recognition of the lipoylated form is not a consequence of any conformational change imposed by the lipoate attachment, but directly involves the presence of the lipoate group53 .

Löffelhardt S., Bonaventura C., Locher M., Borbe H.O., Bisswanger H. Biochem. Pharmacol. 50, 637-646 (1995). 58 Raddatz G., Bisswanger H. J. Biotechnol. 58, 89-100 (1997). Cédric Rentier, PhD thesis 29 1.3.3. The resolution of the 3D structure of lipoylated recombinant hPDC-E2 132-208 by Howard et al. revealed that the inner lipoyl domain is characterized by a β-

Table 4 :

 4 Formation of the lipoyl pentafluorophenylesters.

	Cédric Rentier, PhD thesis

Table 5 :

 5 Formation of the Fmoc-L-Lys(N ε -lipoyl) building blocks.

	Chapter One: Primary Biliary Cirrhosis -2. Results and discussion

Table 6 :

 6 Coupling conditions and yields for the one-step formation of product 4.

	105 Carpino, L.A. J. Am. Chem. Soc.115, 4397-4398 (1993).
	106 Albericio, F., Bofill, J.M., El-Faham, A., Kates, S.A. J. Org. Chem. 63, 9678-9683 (1998).
	107 Wehrstedt, K.D., Wandrey, P.A., Heitkamp, D. J. Hazard. Mater. 126, 1-7 (2005).
	108 Malow, M., Wehrstedt, K.D., Neuenfeld, S. Tetrahedron Lett. 48, 1233-1235 (2007).
	109 Subiros-Funosas, R., Prohens, R., Barbas, R., El-Faham, A., Albericio, F. Chem. Eur. J. 15,
	9394-9403 (2009).
	110 Kaminski, Z.J., Kolesinska, B., Kolesinska, J., Sabatino, G., Chelli, M., Rovero, P.,
	Blaszczyk, M., Glowka, M.L., Papini, A.M. J. Am. Chem. Soc. 127, 16912-16920 (2005).

Table 7 :

 7 Optimal coupling conditions for formation of product 4.

	2.1.3. Separation of the diastereomers

  : the immunodominant sequence as originally described by Coppel et al. and Van de Water et al. at the end of the 1980's, and corresponds to the inner lipoylated domain. NMR experiments previously reported by Howard et al. on the PDC-E2 protein showed that the structure of the inner lipoyl domain is on a type I β-turn.

		*
	H	Ala Glu Ile Glu Thr Asp Lys Ala Thr Ile Gly Phe Glu Val Gln Glu Glu Gly Tyr Leu	OH
		Figure 38: The PDC-E2(167-186) fragment. K 173 is highlighted as the modified AA.

Table 8 :

 8 BB introduced in the peptide sequences as PTMs.

Chapter One: Primary Biliary Cirrhosis -2. Results and discussion Cédric Rentier, PhD thesis 61

Table 9 :

 9 Analytical data for CSF114 analogues synthesized.

	Peptide n°	Peptide synthesized	R t (min)	gradient (%B in A)	ESI-MS m/z observed [M+2H] 2+ (calculated [M+H] + )
	VIII	[Asn 173 ]PDC-E2(167-186)-KKKK	2.13 b	10-40	1372.0 (2742.1)
	IX	[Asn(Glc) 173 ] PDC-E2(167-186)-KKKK	2.03 b	10-40	1452.9 (2904.2)
	X	[Asn(GlcNAc) 173 ] PDC-E2(167-186)-KKKK	1.87 b	10-50	1473.3 (2945.3)
	XI	[Lys 173 ]PDC-E2(167-186)-KKKK	2.06 b	10-40	1378.8 (2756.1)
		[(R,S)-Lipoamide-
	XII	Lys 173 ]PDC-E2(167-186)-	1.68 b	20-60	1472.7 (2944.5)
		KKKK
	XIII	[(R)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK	1.68 b	20-60	1472.7 (2944.5)
	XIV	[(S)-Lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK	1.68 b	20-60	1472.7 (2944.5)

Solvents: A 0.1% TFA in H2O; B 0.1% TFA in CH3CN. a. HPLC: Phenomenex Kinetex C18 2.6 mm 3*100mm; 5 min; 0,6 mL/min. b. UPLC: Waters Acquity UPLC BEH C18 1.7 mm 2.1*50mm; 3 min; 0,6 mL/min.

Table 10 :

 10 Analytical data for PDC-E2(167-186) analogues synthesized.

Chapter One: Primary Biliary Cirrhosis -2. Results and discussion Cédric Rentier, PhD thesis 63 Each coupling was monitored by Kaiser test 115 and double couplings were performed if necessary.

Table 11 :

 11 Effects

of cleavage conditions on the dithiolane ring of LA. Red. = reduction; degrad. = degradation.

Table 12 :

 12 Analytical data for dihydrolipoyl-containing peptides.

Chapter One: Primary Biliary Cirrhosis -2. Results and discussion Cédric Rentier, PhD thesis 67

Table 13 :

 13 Criteria

for the diagnosis of diabetes. A1C: Measurement of HbA1C level, based on the attachment of glucose to hemoglobin; FPG: Fasting Plasma Glucose; OGTT: Oral Glucose Tolerance Test. 1.1.1. Type-1 diabetes This form of diabetes, which accounts for only 5-10% of those with diabetes, previously encompassed by the terms insulin-dependent diabetes or juvenileonset diabetes, results from a cellular-mediated autoimmune destruction of the β-cells of the pancreas 125 . Approximately 1.25 million American children and 124 World Health Organization. Definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia (2006). 125 Chiang, J.L., Kirkman, M.S., Laffel, L.M.B., Peters, A.L. Diabetes Care 37, 2034-2054 (2014). Chapter Two: Diabetes -1. Introduction Cédric Rentier, PhD thesis 98 adults have T1D. Immune-mediated diabetes commonly occurs in childhood and adolescence, but it can occur at any age.

Table 16

 16

	for PDC-E2(167-186)-

Solvents: A 0.1% TFA in H2O; B 0.1% TFA in CH3CN. a. HPLC: Phenomenex Kinetex C18 2.6 mm 3*100mm; 5 min; 0,6 mL/min. b. UPLC: Waters Acquity UPLC BEH C18 1.7 mm 2.1*50mm; 3 min; 0,6 mL/min.

Table 15 :

 15 Analytical data of CSF114 analogues.

	Peptide n°	Peptide synthesized	R t (min)	gradient (%B in A)	ESI-MS m/z observed [M+3H] 3+ (calculated [M+H] + )
	XXV	[(deoxyFru)Lys 173 ]PDC-E2(167-186)-KKKK	1.80 b	10-50	973.6 (2918.3)
	XXVI	[Ser 173 ]PDC-E2(167-186)-KKKK	3.22 a	20-70	905.0 (2713.4)
	XXVII	[Ser 173 (GlcNAc)]PDC-E2(167-186)-KKKK	3.13 a	20-70	972.8 (2916.8)
	XXVIII	[Ser 173 (PO 3 H 2 )]PDC-E2(167-186)-KKKK	3.17 a	20-70	931.8 (2793.4)
	Solvents: A 0.1% TFA in H2O; B 0.1% TFA in CH3CN.
	a. HPLC: Phenomenex Kinetex C18 2.6 mm 3*100mm; 5 min; 0,6 mL/min.
	b. UPLC: Waters Acquity UPLC BEH C18 1.7 mm 2.1*50mm; 3 min; 0,6 mL/min.

Table 16 :

 16 Analytical data of PDC-E2(167-186)-KKKK analogues.

	Chapter Two: Diabetes -2. Results and discussion

  5, 2661-2664 (1995). 162 Otvos, L. Jr, Elekes, I., Lee, V.M. Int. J. Pept. Protein Res. 34, 129-133 (1989). 163 Attard, T.J., O'Brien-Simpson, N.M., Reynolds, E.C. Int. J. Pept. Res. Ther. 15, 69-79 (2009).

	Chapter Two: Diabetes -2. Results and discussion
	Cédric Rentier, PhD thesis	120

  204,205 .

	202 Dalgliesh, C.E.J. J. Chem. Soc. 137-141 (1952).
	203 Kleijn, L.H.J., Muskens, F.M., Oppedijk, S.F., de Bruin, G., Martin, N.I. Tetrahedron Lett.
	53, 6430-6432 (2012).

204

Warnell, J.L., Berg, C.P. J. Am. Chem. Soc. 76, 1708-1709 (1954).

205

Brown, R.R., Price, J.M. J. Am. Chem. Soc. 77,

4158-4159 (1955)

.

  All the linear peptides were purified by semi-preparative RP-HPLC, as described in General procedure for purification, and characterized by ESI-MS. Analytical data are reported in Table18.

		Chapter Three: Multiple Sclerosis -2. Results and discussion
	Peptide n°	Peptide synthesized	R t (min)	gradient (%B in A)	ESI-MS m/z observed [M+2H] 2+ (calculated [M+H] + )
	XXIX	[Kyn 7 ]CSF114	1.67 b	20-60	1261.5 (2521.0)
	XXX	[Kyn 18 ]CSF114	1.51 b	20-60	1225.6 (2448.8)
	III	CSF114(Glc)	2.64 a	30-60	1304.5 (2607.1)
	Solvents: A 0.1% TFA in H2O; B 0.1% TFA in CH3CN.
	a. HPLC: Phenomenex Kinetex C18 2.6 mm 3*100mm; 5 min; 0,6 mL/min.
	b. UPLC: Waters Acquity UPLC BEH C18 1.7 mm 2.1*50mm; 3 min; 0,6 mL/min.
	Cédric Rentier, PhD thesis			151

Table 18 :

 18 Analytical data for CSF114 analogues synthesized.

Table 19 :

 19 Sera tested for the SP-ELISA experiments.

	Figure 110: Indirect SP-ELISA absorbance values measured at 405nm for the 3 peptides on
	various sera. Cut-off values: [Kyn 7 ]CSF114 0.56; [Kyn 18 ]CSF114 0.74; CSF114(Glc) 0.89.

To quantify the ability of the kynureninated CSF114 analogues to detect specific IgG or IgM antibodies in MS sera in comparison to NBDs as control sera, cut-off values were calculated separately for each peptide as the mean value of NBDs plus three times standard deviation of NBDs.

The panel of sera includes MS sera, which is the disease of interest; NBDs were used as healthy controls. Alzheimer sera were initially included in the panel test as an MS-unrelated disease (pathological negative control).

Chapter Three: Multiple Sclerosis -2. Results and discussion Cédric Rentier, PhD thesis 153 Figure 109: Indirect SP-ELISA absorbance values measured at 405nm for the 3 peptides on various sera. Cut-off values: [Kyn 7 ]CSF114 0.16; [Kyn 18 ]CSF114 0.12; CSF114(Glc) 0.43.

the NBDs sera. All the 3 coated peptides did not reveal any specific IgM among the tested MS sera. On the other hand, there is one serum that displays a positive IgM titer against the two kynureninated CSF114 analogues: 2P, an Alzheimer patient's serum.

Table 20 :

 20 Micro-cleavages parameters.

	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

Micro-cleavages are a useful tool to monitor stepwise syntheses. They can be performed at room temperature or alternatively microwave assisted to reduce reaction times. Microwave assisted micro-cleavages were performed using CEM Discover TM (CEM Corporation, Matthews, NC) as reported in the Table

Table 21 :

 21 Peptide purification by SLCF. This table is an example of a 20-50% gradient of B in A in 30 min. Compositions may vary depending on the peptide purified.

	All peptides are further purified by semi-preparative RP-HPLC. Methods and
	solvent system used are reported for each peptide. Fractions are checked by
	analytical RP-HPLC ESI-MS or RP-UPLC ESI-MS, homogeneous ones are
	pooled together, CH 3 CN is evaporated under reduced pressure and then
	aqueous phase is lyophilized.
	All the peptides were obtained with a purity ≥ 95% to be used for autoantibody
	detection.

Fractions are checked by analytical RP-HPLC ESI-MS or RP-UPLC ESI-MS, product-containing homogeneous ones are pooled together, CH 3 CN is evaporated under reduced pressure and then aqueous phase is lyophilized.

Fetal Bovine Serum (FBS) Buffer (10% FBS in Washing Buffer). FBS Buffer was removed, and 100 ml/well of diluted sera sample (1:100 in FBS Buffer) was dispensed. Blank wells were included in all the plates, and were obtained using FBS Buffer instead of serum. Plates were incubated at 4°C overnight, and then washed 3 times with Washing Buffer. 100 ml/well of secondary Ab diluted in FBS Buffer (hIgG 1:8000 and hIgM 1:1200) were dispensed, and plates were incubated 3 h at r.t. Plates were washed 3 times with Washing Buffer, then 100 ml/well of Substrate Solution (1mg/ml pnitrophenylphosphate in Substrate Buffer: 1M diethanolamine, 1 mM MgCl 2 , pH 9.8) were dispensed. Plates were incubated for 15-40 min, and then absorbance at 405 nm of each well was read with a spectrophotometer.

Absorbance value for each serum was calculated as (mean absorbance of triplicate) -(mean absorbance of blank triplicate).

4. Experimental part

Chapter One 4.1. Synthesis of α-lipoic acid-OPfp esters (1, 2 and 3) ESI-MS: m/z calc. 372.0 for C 14 H 13 F 5 O 2 S 2 , found 373.1 ([M+H] + ) 1 H NMR (CDCl 3 , 200 MHz): 3.59 (quin, 1 H, dithiolane CH, J = 6.6 Hz), 3.23-3.05 (m, 2 H, dithiolane CH 2 ), 2.68 (t, 2 H, lipoyl CH 2 ), 2.56-2.40 (m, 1 H,

Table 23 :

 23 Sequences, gradients and yields for purification of peptides VIII -X.

	Peptide n°	Sequence	SLCF gradient	Semi-prep HPLC gradient	Yield (purity ≥95%)
	VIII	AEIETDNATIGFEVQEEGYLKKKK	10-40% B in 30 min	20-35% B in 30 min	24 %
	IX	AEIETDN(Glc)ATIGFEVQEEGYLKKKK	10-40% B in 30 min	20-35% B in 30 min	10 %
	X	AEIETDN(GlcNAc)ATIGFEVQEEGYLKKKK	10-40% B in 30 min	15-40% B in 30 min	27 %

Table 24 :

 24 . Solvent systems used were: A (0.1% TFA in H 2 O) and B (0.1 % TFA in CH 3 CN). Sequences, gradients and yields for purification of peptides XI -XIV.

	Peptide n°	Sequence	SLCF gradient	Semi-prep HPLC gradient	Yield (purity ≥95%)
	XI	AEIETDKATIGFEVQEEGYLKKKK	15-50% B in 30 min	30-60% B in 30 min	16 %
	XII	AEIETDK((R,S)Lip)ATIGFEVQEEGYLKKKK	15-50% B in 30 min	30-60% B in 30 min	9 %
	XIII	AEIETDK((R)Lip)ATIGFEVQEEGYLKKKK	15-50% B in 30 min	30-60% B in 30 min	6 %
	XIV	AEIETDK((S)Lip)ATIGFEVQEEGYLKKKK	15-50% B in 30 min	30-60% B in 30 min	7 %

Table 25 :

 25 Sequences, gradients and yields for purification of peptides XXI and XXV Experimental Part -5. Experimental part Chapter Two Cédric Rentier, PhD thesis 183Analytical data are given in Table15and Table16.

	Peptide n°	Sequence	SLCF gradient	Semi-prep HPLC gradient	Yield (purity ≥95%)
	XXI	TPRVERK(deoxyFru)GHSVFLAPYGWMVK	-	25-40% B in 30 min	4 %
	XXV	AEIETDK(deoxyFru)ATIGFEVQEEGYLKKKK	-	20-35% B in 30 min	15 %

. Solvent systems used were: A (0.1% TFA in H 2 O) and B (0.1 % TFA in CH 3 CN).

Table 26 :

 26 2 O) and B (0.1 % TFA in CH 3 CN). Sequences, gradients and yields for purification of peptides XXII, XXIV and XXVI -XXVIIIAnalytical data are given in Table15and Table16.

	Peptide n°	Sequences	SLCF gradient	Semi-prep HPLC gradient	Yield (purity ≥95%)
	XXII	TPRVERSGHSVFLAPYGWMVK	15-60% B in 30 min	25-50% B in 30 min	15 %
	XXIV	TPRVERS(PO 3 H 2 )GHSVFLAPYGWMVK	15-60% B in 30 min	25-50% B in 30 min	17 %
	XXVI	AEIETDSATIGFEVQEEGYLKKKK	15-50% B in 30 min	25-50% B in 30 min	16 %
	XXVII	AEIETDS(GlcNAc)ATIGFEVQEEGYLKKKK	15-50% B in 30 min	30-70% B in 30 min	20 %
	XXVIII	AEIETDS(PO 3 H 2 )ATIGFEVQEEGYLKKKK	15-50% B in 30 min	30-70% B in 30 min	20 %

Table 27 :

 27 Sequences, gradients and yields for purification of peptides XXIX -XXX.

  H, Lys CH α ), 3.58 (quin, 1 H, dithiolane CH, J = 6.6 Hz), 3.17-3.05 (m, 2 H, dithiolane CH 2 ), 2.68 (t, 2 H, lipoyl CH 2 ), 2.55-2.40 (m, 1 H, dithiolane 7-H a ), 2,16 (t, 2 H, Lys CH 2 ), 2.06-1.93 (m, 1 H, dithiolane 7-H b ), 1.85-1.50 (m, 12 H, 3 × lipoyl CH 2 and 3 × Lys CH 2 ).

Table 2 .

 2 Yields for 1c obtained using a one-pot reaction (Scheme 1, Pathway B)

	Entry n°Coupling conditions	Solvent	°C	Reaction time	Yield lipoylated lysine	Yield side product
	1	HATU (1 eq), DIPEA (2 eq)	DMF	rt	overnight	60%	7%
	2	Oxyma (1 eq), DIC (1 eq)	DMF/DCM (1:1)	rt	overnight	18%	7%
	3	DMTMM-BF 4 (1 eq), NMM (1 eq)	CH 3 CN	0 °C for 2 h then rt	overnight	not observed	not observed
	4	DMTMM-BF 4 (1 eq), NMM (1 eq)	DMF	0 °C for 2 h then rt	overnight	37%	15%
	5	HATU (1 eq), DIPEA (2 eq)	DMF	rt	3 h	53%	less than 4%
	rt, room temperature.

Table 3 .

 3 Building blocks used in peptide synthesis

	Peptides

Table 1 .

 1 Analytical Data of Synthesized Peptides Gradient 10-40% B in 3 min. e Gradient 25-45% B in 3 min. f Gradient 30-60% B in 5 min.

	no.	peptide	ESI-MS [M + 2H] 2+ (m/z) found (calcd)	UPLC/HPLC (R t , min)
	I	[(R)-lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK	1472.7 (1472.8)	1.68 a,c
	II	[(S)-lipoamide-Lys 173 ]PDC-E2(167-186)-KKKK	1472.7 (1472.8)	1.68 a,c
	III	[Lys 173 ]PDC-E2(167-186)-KKKK	1378.8 (1378.6)	2.06 a,d
	IV	[(R)-lipoamide-Lys 7 ]CSF114	1324.6 (1324.7)	1.97 a,e
	V	[Lys 7 ]CSF114	1230.5 (1230.4)	2.61 b,f
	a UPLC performed on Acquity system with 3100 Mass Detector (Waters); column, BEH C 18 1.7 μm, 2.1 mm × 50 mm (Waters); eluents, A 0.1% TFA in H 2 O, B 0.1% TFA in CH 3 CN; flow 0.6 mL/min. b HPLC performed on Alliance Chromatography with Micromass ZQ (Waters); column, Kinetex C 18 2.6 μm, 3 mm × 100 mm (Phenomenex); eluents, A 0.1% TFA in H 2 O, B 0.1% TFA in CH 3 CN; flow 0.6 mL/min. c Gradient 20-60%
	B in 3 min.

d

Table 2 .

 2 Main Characteristics of PBC Patients at Time of Serum Collection or Presentation to the Center a Data are counts (%) or means and (range). a Development of major complications of portal hypertension (ascites, esophageal varices, portal-systemic encephalopathy) or hepatocellular carcinoma. b Including serum levels of alkaline phosphatase exceeding 1.5 times the upper normal levels on at least two determinations taken six months apart in the last year.

	AMA positive	AMA negative

a c Pruritus, jaundice, ascites, variceal bleeding, portalsystemic encephalopathy.

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J. Pept. Sci. 2015; 21: 408-414 Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.wileyonlinelibrary.com/journal/jpepsci

DOI: 10.1021/acs.jmedchem.5b00783 J. Med. Chem. XXXX, XXX, XXX-XXX

Acknowledgements

Acknowledgements ANR Chaire d'Excellence PeptKit 2009-2014 (grant n°ANR-09-CEXC-013-01), French-Italian University (Vinci Project grant n°C 2-133) and Ente Cassa di Risparmio di Firenze are gratefully acknowledged for their financial support. Acknowledgements: ANR Chaire d'Excellence PeptKit 2009-2014 (grant n° ANR-09-CEXC-013-01), French-Italian University (Vinci Project grant n° C2-133) and Ente Cassa di Risparmio di Firenze are gratefully acknowledged for their financial support.

This thesis would not have been possible without the financial support of several institutions that I would like to thank here: the French National Research Agency (ANR) for the Chaire d'Excellence 2009-2014 project "Pepkit: development of peptide-based diagnostic kits for autoimmune diseases" (grant n° ANR-09-CEXC-013-01); the French-Italian University for their mobility program (Vinci Project grant n° C2-133) and the Ente Cassa di Risparmio di Firenze for the support to PeptLab@UniFi.

Special highlights to Sofia, my soon-to-be sister-in-law, who kindly accepted

The significance and the sensitivity of the assays based on these two synthetic probes is lower than the one previously observed for the PDC-E2 peptide lipoylated with the (R)-enantiomer.

We also investigated, through the ROC curves reported in Figure 94, if these peptides could be used to differentiate T1D from T2D populations of patients. To a solution of the (R,S)-α-lipoic acid (206 mg, 1 mmol) dissolved in DMF (10 ml) at room temperature is added, under stirring, HATU (380 mg, 1 mmol, 1 eq) and DIPEA (350 µL, 2 mmol, 2 eq). After 10 minutes is added Fmoc-L-Lys-OH (368 mg, 1 mmol, 1 eq).

The

List of publications and communications of C. Rentier

This list is up to date at the present time (August 2015). A selection of manuscripts is available at the end of this thesis as Supplementary Material.

Publications in peer-reviewed books and journals as research papers:

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Supporting Information

Additional supporting information may be found in the online version of this article at publisher's web site.

CONCLUSION

In conclusion, Met oxidation was observed during the tetradecapeptide GHSVFLAPYGWMVK using MW-assisted and operating without N 2 atmosphere. No oxidation of the tetradecapeptide was observed performing the synthesis both manually and on a MW-assisted automated apparatus working under inert atmosphere (N 2 be taken into account to control Met oxidation under atmospheric conditions. Using this as an advantage, mild oxidation of Met-containing peptides can be performed directly on-resin to obtain the two diastereomeric Met(O) containing products, with no methionine-sulfone formation. Moreover, Met oxidation can be successfully handled using an optimized one-pot cleavage-reduction protocol, in line with previous results reported by other authors (25).

In conclusion, this paper reports convenient protocols to selectively deal with Met oxidation state, and to obtain Readers interested in a full list of references are invited to visit our website at www.teknoscienze.com

ACKNOWLEDGMENTS

Chimica oggi -Chemistry today peer-reviewed journal

With increasing amounts of scientific information being put into the public domain, and a growing number of organisations involved in promoting and discussing scientific research, it can be difficult to judge which research claims should be taken seriously. There is a system called peer review that is used by scientists to decide which research results should be published in a scientific journal. The peer review process subjects scientific research papers to independent scrutiny by other qualified scientific experts (peers) before they are made public. Chimica oggi -Chemistry today follows this evaluation process and can be therefore considered a reliable and credited source. The journal is listed moreover in the Thomson Reuters Journal Citation Reports and claims an impact factor for year 2013 of 0.450. The impact factor is a measure of the frequency with which the "average article" in a journal has been cited in a particular year or period. The annual JCR impact factor is a ratio between citations and recent citable items published. Thus, the impact factor of a journal is calculated by dividing the number of current year citations to the source items published in that journal during the previous two years. The impact factor is frequently used as a proxy for the relative importance of a journal within its field, indicating a high standard, quality of research and prestige. At the many trade / technical events we participate, Chimica oggi -Chemistry today is one of the fewest journals with an impact factor value.