Vangelis Agouridas
email: vangelis.agouridas@ibl.cnrs.fr

Ouafâa El Mahdi

Oleg Melnyk
email: oleg.melnyk@ibl.cnrs.fr

Protein chemical synthesis in medicinal chemistry

Although the majority of proteins used for biomedical research are produced using living systems such as bacteria, biological means for producing proteins can be advantageously complemented by protein semisynthesis or total chemical synthesis. The latter approach is particularly useful when the proteins to be produced are toxic for the expression system or show unusual features that cannot be easily programmed in living organisms. The aim of this perspective article is to provide a wide overview of the use of protein chemical synthesis in medicinal chemistry with a special focus on the production of side-chain or backbone-modified proteins.

INTRODUCTION

The discovery of powerful methods such as solid phase peptide synthesis (SPPS) has accelerated the elucidation of the mode of action of biologically active peptides and the study of their structure-function relationships. As such, the SPPS has fueled the development of peptide therapeutics as well as facilitated their GMP compliant large-scale production. More than a hundred peptides are currently under development in various therapeutic areas that include metabolic diseases, oncology, and cardiovascular diseases. [START_REF] Lau | Therapeutic Peptides: Historical Perspectives, Current Development Trends, and Future Directions[END_REF] Until the 1990s, peptide synthesis methods were limited to the production of polypeptides having less than 40-50 amino acids (AA), and the chemical synthesis of small proteins required considerable efforts. Since then, the domain has witnessed substantial conceptual and technical advances with the advent of chemoselective amide bond forming reactions, also referred to as native ligation reactions. 2- 4 By enabling the coupling of unprotected peptide segments in aqueous media, these methods have facilitated the production of large and highly modified proteins by semi or total chemical synthesis. Such synthetic studies are frequently integrated as part of research programs towards the understanding of protein function in relation to therapeutic interventions based on small molecules (Figure 1). Also, endogenous proteins or proteins derived from other natural sources (animals and plants) constitute a large reservoir of inspiration for the development of therapeutic proteins. In recent years, such approaches have been the focus of numerous studies and are now greatly supported by the richness of the protein chemist's toolbox. The number of proteins prepared by semi or total chemical synthesis has been expanding rapidly since 2010, showing that the developed chemistries fill a gap and respond to important needs in chemical biology research (Figure 2a). This corpus of methods enabled the production of proteins with a mean size of 90 AA obtained through total chemical synthesis (Figure 2b) and nearly 200 AA through semi-synthesis (Figure 2c). reference and the information on how the protein was produced using the PCS-db ID interface (PCS-ID).

In this perspective, we provide a broad overview of the use of protein chemical synthesis for applications in medicinal chemistry with a special focus on its main asset, which is the production of side-chain or backbone-modified proteins (Figure 3). The first section presents the main strategies used for accessing proteins by semi or total synthesis. For the non-specialist, identifying the best synthetic route to a protein target is not an easy task. Hopefully, newly developed decision-support tools, which are available in the form of programs or databases, can provide useful assistance for the design of a synthetic plan. One of these is the Protein Chemical Synthesis database (http://pcs-db.fr), a web-based tool, which inventories biologically relevant peptides and proteins synthesized using chemoselective amide bond forming reactions (> 1000 entries). [START_REF] Agouridas | A Statistical View of Protein Chemical Synthesis Using NCL and Extended Methodologies[END_REF] The content of the PCS database can be interrogated thanks to various search modules, each proposing a large diversity of descriptors for the synthetic design (PCS-db) or for the type of modifications harbored by the proteins (as in the freshly released PCS post-translational modifications module, PCS-PTM). As such, the PCS database and its web-based tools can be considered as a helping-hand for any chemist willing to integrate the synthesis of tailored peptides or proteins in their research work. Browsing the PCS database using the protein identification numbers in the Figures provides a useful complement to the reading of this

Perspective by giving the key information on how the cited proteins were produced (PCS-ID).

The second section gives a description of the main modifications that have been effected by protein chemists and that are relevant for the medicinal chemist. In particular, this section will cover the production of proteins harboring post-translational modifications (PTMs) and their use for deciphering protein function. The third section will discuss the use of protein chemical synthesis as a tool for deciphering biological mechanisms, for target identification and for accessing potential therapeutic proteins.

Figure 3. Scope and content of the Perspective.

DISCUSSION

The semi or total chemical synthesis of proteins Conceptually, the peptide segment coupling methods used for modern protein synthesis differ from classical amide coupling techniques based on the enthalpic activation of carboxylic acids by involving first a chemoselective capture step, which brings the N and C-terminal functionalities close in space (Figure 4a). During this step, the peptide segments get covalently

Post-translational modifications

(or mimics thereof)

The Protein Chemist Toolbox linked by a non peptidic bond. The connecting native peptide bond is formed subsequently after a spontaneous intramolecular rearrangement. The most popular set of techniques used thus far for protein synthesis is undoubtedly native chemical ligation (NCL 2 ) and associated methods. 5- 7 In this case, the reacting partners, a C-terminal peptide thioester and an N-terminal cysteinyl peptide, combine through a reversible thiol-thioester exchange process in water at neutral pH.

The peptide bond is formed from the thioester-linked intermediate by a thermodynamically favorable intramolecular acyl migration reaction from sulfur to nitrogen. Since the introduction of NCL in 1994, the scope of peptide bond formation through thiol-thioester capture was considerably extended by the development of various thioester or selenoester surrogates, thio or seleno amino acids and the application of mild dechalcogenation techniques. [START_REF] Agouridas | A Statistical View of Protein Chemical Synthesis Using NCL and Extended Methodologies[END_REF][START_REF] Agouridas | Native Chemical Ligation and Extended Methods. Mechanisms, Catalysis, Scope and Limitations[END_REF][START_REF] Conibear | Native Chemical Ligation in Protein Synthesis and Semi-Synthesis[END_REF] In particular, the desulfurization of Cys is frequently used for the preparation of peptide bonds to alanine (Ala, Figure 4b). kind that was introduced in 1994. b) The dechalcogenation of thio or seleno amino acids, which extends the principle of NCL to the formation of virtually any type of junction, is illustrated with the dechalcogenation of cysteine into alanine.

Living systems can produce extremely large polypeptides, but show significant limitations or require sophisticated techniques when it comes to going beyond the 20 programmable canonical amino acids. In contrast, a strong asset of protein semi or total synthesis is the use of tailored synthetic peptide segments as building blocks for the assembly of the protein, because these peptide segments can incorporate virtually any type of amino acid (e.g., D-amino acids), modification, label or prosthetic group. Functional groups may also be introduced for further in vitro or in vivo chemical manipulations or for the production of a variety of protein scaffolds such as cyclic or branched proteins. The diversity of modifications attainable through chemical synthesis facilitates the tuning of the functions and properties of therapeutic protein candidates, or the use of proteins as tools for biological investigations through proteomic or imaging studies for example.

A schematic example of protein chemical synthesis from synthetic peptide segments is provided in Figure 5a. In this case, the peptide chemist can freely decide the number, type and sites of modification. The size of the proteins attainable by this kind of approach is in the range 50-450

AAs, but is usually less than 200 AA as the number of ligations to perform above this size can be limiting. The largest proteins prepared so far by chemical synthesis (~45-50 kDa, see PCSdb # in Figure 2b) can be considered as masterpieces. The assembly of four peptide segments as shown in Figure 5a gives access to proteins having an average of 150 amino acid residues, one limiting factor being the size of the peptide segments produced by conventional SPPS (~40-50 AA). The semi synthesis of a protein from two peptide modules is shown in Figure 5b. In this example, one reacting partner is produced recombinantly in a living organism such as bacteria and the other by chemical synthesis. [START_REF] Conibear | Native Chemical Ligation in Protein Synthesis and Semi-Synthesis[END_REF] In this case, the site of modification is restricted to the N or C terminal parts of the target protein, the recombinantly produced segment usually being much larger than the one accessed by chemical synthesis. Although this can restrict the application of semi-synthesis to the production of modified proteins, the approach has proven particularly powerful in several instances. Typically, the semi-synthesis of histone proteins harboring various PTMs on their N-terminal tail using expressed protein ligation (EPL [START_REF] Muir | Expressed Protein Ligation: A General Method for Protein Engineering[END_REF] ), an adaptation of NCL to the use of peptide thioesters of biological origin, has significantly advanced the study of the histone code (for a recent review on the synthesis of modified histones see ref [START_REF] Nakatsu | Toolbox for Chemically Synthesized Histone Proteins[END_REF] ). The assembly approaches can also include an enzymatic step, thereby extending the scope of targets attainable through semi or total synthesis (Figure 6). The enzymatic step can be used for further modifying a peptide segment before its inclusion in a protein assembly (Figure 6a). Such an approach is particularly advantageous when the target sequence features multiple sites that can be modified by the same enzyme. [START_REF] Pan | Chemical Synthesis of Structurally Defined Phosphorylated Ubiquitins Suggests Impaired Parkin Activation by Phosphorylated Ubiquitins with a Non-Phosphorylated Distal Unit[END_REF] Indeed, applying the enzymatic step after assembly might yield a heterogeneous product, while such a risk does not exist if the enzymatic modification is performed on a peptide segment that features a single modification site.

Chemoenzymatic methods can also be used for modifying or extending a chemical group introduced during the preparation of one of the peptide segments and utilized as a primer (Figure 6b). [START_REF] Hojo | Chemoenzymatic Synthesis of Hydrophobic Glycoprotein: Synthesis of Saposin C Carrying Complex-Type Carbohydrate[END_REF] The approach has proven particularly powerful in the context of glycoprotein synthesis. [START_REF] Li | Chemoenzymatic Methods for the Synthesis of Glycoproteins[END_REF] Figure 6. Some examples of chemoenzymatic approaches to protein semi or total synthesis. a) A peptide segment is modified prior to its use for protein assembly. b) A chemical group is introduced on one of the starting peptide segments (e.g., a monosaccharide) to enable postassembly enzymatic manipulations (e.g., coupling of a complex oligosaccharide).

Protein synthesis and the role of PTMs

As mentioned before, one hallmark of protein chemical synthesis is its capacity to access proteins that cannot be obtained recombinantly. [START_REF] Liu | Chemical Synthesis of Proteins That Cannot Be Obtained Recombinantly[END_REF] Logically, a significant proportion (~50%) of the proteins prepared by semi or total synthesis are modified, that is are not solely made of the concatenation of the 20 canonical L-amino acid residues. According to the PCS-db, a large fraction of the studied modifications are post-translational modifications (Figure 7a), and include by order of importance glycosylation or mimics thereof, lysine modification by ubiquitin or ubiquitin-like modifiers, phosphorylation, lysine acetylation or methylation (Figure 7b). level. However, proteins harboring the latter modifications paradoxically constitute the majority of achievements, a situation that reflects their increasing importance in chemical biology and medicinal chemistry (Figure 7b). In the following paragraphs, we will review the situation by providing a selection of recent examples of phosphorylated, ubiquitinylated and glycosylated protein variants exploited for studying the role of PTMs.

Protein phosphorylation or dephosphorylation is used by the cell machinery for orchestrating most cellular processes but its malfunction results in the development of diseases such as cancer, neurodegenerative disorders or diabetes. Therefore, the exploration of protein phosphorylation patterns and the identification of the enzymes involved in their regulation are key to the development of many target-based therapeutic strategies. The inability of living systems to produce substantial amounts of phosphoproteins with a full control of the phosphorylation site is, however, regarded as a serious bottleneck for deciphering the role of protein phosphorylation, for understanding signaling networks and for developing novel phosphoprotein-based therapeutics. [START_REF] Oza | Robust Production of Recombinant Phosphoproteins Using Cell-Free Protein Synthesis[END_REF] Of course, the presence of phosphoserine (pSer) or phosphothreonine (pThr) can sometimes be mimicked by introducing negatively charged amino acid residues, i.e. aspartic (Asp) or glutamic (Glu) acids, or by other means that include phosphonate amino acid surrogates. [START_REF] Chen | Synthetic Approaches to Protein Phosphorylation[END_REF] However, the mimicry of pSer/Thr does not always succeed in reproducing the effects of phosphorylation, [START_REF] Paleologou | Phosphorylation at Ser-129 but Not the Phosphomimics S129E/D Inhibits the Fibrillation of -Synuclein[END_REF] and explains why the synthesis of homogeneous phosphoproteins remains a significant goal and the focus of many synthetic efforts.

One typical example of the importance of protein phosphorylation in disease onset and progression is Tau protein phosphorylation in Alzheimer's disease (AD). An abnormal phosphorylation level of the Tau protein is an early and crucial event in the pathogenesis of AD by leading to the formation of insoluble neurofibrillary tangles. Deciphering the role of phosphorylation and other PTMs in the properties and function of the Tau protein is therefore recognized as an important step toward the conception of future therapeutics. [START_REF] Šimić | Tau Protein Hyperphosphorylation and Aggregation in Alzheimer's Disease and Other Tauopathies, and Possible Neuroprotective Strategies[END_REF] A very recent study from Becker's group describes the semisynthesis of Tau variants featuring a pSer or a carboxymethylated lysine residue within the central repeat domain of Tau4 spanning residues 291-321, a domain involved in the binding to tubulin and in Tau aggregation. [START_REF] Ellmer | Single Posttranslational Modifications in the Central Repeat Domains of Tau4 Impact Its Aggregation and Tubulin Binding[END_REF] The phosphorylation of Ser293 or Ser305 residues decreased the propensity of Tau protein to aggregate without changing the binding to tubulin. In contrast, carboxymethylation of lysine 294, an advanced glycation end product modification that cannot be introduced enzymatically or programmed in living cells, altered the binding to tubulin without changing the aggregation behavior of Tau. Another recent report from Haj-Yahya and Lashuel also used a semisynthetic approach for installing a pSer residue in the C-terminal part of the Tau protein and for deciphering the role of this and other PTMs in healthy and diseased states. [START_REF] Haj-Yahya | Protein Semisynthesis Provides Access to Tau Disease-Associated Post-Translational Modifications (PTMs) and Paves the Way to Deciphering the Tau PTM Code in Health and Diseased States[END_REF] Ubiquitinylated proteins [START_REF] Burlina | Auxiliary-Assisted Chemical Ubiquitylation of Nemo and Linear Extension by HOIP[END_REF] and proteins modified by ubiquitin-like modifiers such as SUMO [START_REF] Bouchenna | Total Chemical Synthesis of All SUMO-2/3 Dimer Combinations[END_REF] are another class of modified proteins that are actively pursued by protein chemists due to the difficulty in obtaining them recombinantly. Although the role of ubiquitinylation in protein degradation by the proteasome is well established, there is increasing evidence that reversible ubiquitinylation also regulates cell cycle events through nonproteolytic mechanisms. The reversion of the (poly)ubiquitin signal is carried out by deubiquitylating enzymes (DUBs), [START_REF] Clague | Breaking the Chains: Deubiquitylating Enzyme Specificity Begets Function[END_REF] which constitute potential therapeutic targets, especially for the development of anticancer drugs. [START_REF] Yuan | Inhibition of Ubiquitin-Specific Proteases as a Novel Anticancer Therapeutic Strategy[END_REF] Decoding the ubiquitin signal and characterizing the properties of DUBs is essential for making progress in this area (for reviews see ref [START_REF] Van Tilburg | Synthetic and Semi-Synthetic Strategies to Study Ubiquitin Signaling[END_REF] ) and already benefits from the outstanding advances made in recent years in the synthesis of polyubiquitin chains [START_REF] Tang | Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-Linked Ub Isomer[END_REF][START_REF] Bondalapati | Total Chemical Synthesis of SUMO-2-Lys63-Linked Diubiquitin Hybrid Chains Assisted by Removable Solubilizing Tags[END_REF][START_REF] Qu | A Highly Efficient Synthesis of Polyubiquitin Chains[END_REF][START_REF] Pan | Chemical Protein Synthesis Enabled Mechanistic Studies on the Molecular Recognition of K27-Linked Ubiquitin Chains[END_REF] and ubiquitinylated proteins. [START_REF] Sun | The Journey for the Total Chemical Synthesis of a 53 kDa Protein[END_REF][START_REF] Bondalapati | Expanding the Chemical Toolbox for the Synthesis of Large and Uniquely Modified Proteins[END_REF] The same holds true for small ubiquitin-like modifiers (SUMO), which can change the localization, the stability, or the activity of the target proteins by modulating proteinprotein interactions and by competing or intersecting with other lysine modifications such as ubiquitin. The protein chemical synthesis toolbox now enables access to SUMO conjugates with an atom control of their branching structure. [START_REF] Bouchenna | Total Chemical Synthesis of All SUMO-2/3 Dimer Combinations[END_REF][START_REF] Bondalapati | Total Chemical Synthesis of SUMO-2-Lys63-Linked Diubiquitin Hybrid Chains Assisted by Removable Solubilizing Tags[END_REF][START_REF] Weller | Aromatic Thiol-Mediated Cleavage of N-O Bonds Enables Chemical Ubiquitylation of Folded Proteins Nat[END_REF] Such tools are already used for characterizing the biochemical and conformational properties of SUMO isoforms [START_REF] Bouchenna | The Role of the Conserved SUMO-2/3 Cysteine Residue on Domain Structure Investigated Using Protein Chemical Synthesis[END_REF] and SUMO deconjugating enzymes (SENPs), which have emerged as potential therapeutic targets. [START_REF] Jia | Chemical Tools and Biochemical Assays for SUMO Specific Proteases (SENPs)[END_REF] The power and versatility of protein chemical synthesis for studying protein ubiquitinylation and the cross-talk between ubiquitinylation and other PTMs such as phosphorylation can be appreciated in the light of a recent study. [START_REF] Pan | Chemical Synthesis of Structurally Defined Phosphorylated Ubiquitins Suggests Impaired Parkin Activation by Phosphorylated Ubiquitins with a Non-Phosphorylated Distal Unit[END_REF] Liu and coworkers reported the semisynthesis of non-phosphorylated or phosphorylated variants of Lys6-linked di-ubiquitin (Ub K6 Ub) to investigate the link between Ub phosphorylation by PINK1 and Parkin's E3 ligase activation at the molecular level (Figure 8). tyrosine 57 (H2A Y57p). [START_REF] Jbara | Chemical Synthesis of Phosphorylated Histone H2A at Tyr57 Reveals Insight into the Inhibition Mode of the SAGA Deubiquitinating Module[END_REF] These two proteins were used to reconstitute nucleosomes and test the effect of Y57 phosphorylation on H2B K120 Ub deubiquitinylation by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator complex. Y57 phosphorylation was found to significantly reduce (~30 fold) the rate of deubiquitinylation of H2B by the SAGA a result that provided the first direct proof of a negative regulation of H2B deubiquitinylation by H2A Y57p. These results are consistent with the X-ray crystal structure of the SAGA DUB module bound to ubiquitinylated nucleosomes. [START_REF] Morgan | Structural Basis for Histone H2B Deubiquitination by the SAGA Dub Module[END_REF] Histone ubiquitinylation is a critical epigenetic mechanism regulating chromatin assembly, DNA transcription activation and DNA damage repair. Since its deregulation plays a major role in oncogenesis, histone ubiquitinylation is considered as a potential target for developing anticancer therapeutics. [START_REF] Jeusset | Developing Targeted Therapies That Exploit Aberrant Histone Ubiquitination in Cancer[END_REF] Therefore, identifying the factors regulating histone ubiquitinylation is a significant goal to pursue. Protein semi and total synthesis has already greatly contributed to enlarge our knowledge of the chromatin code and continues to play a major role in this field. [START_REF] Qi | Total Chemical Synthesis of Modified Histones Frontiers in[END_REF][START_REF] Boichenko | Chemical and Biophysical Methods to Explore Dynamic Mechanisms of Chromatin Silencing[END_REF][START_REF] Dann | ISWI Chromatin Remodellers Sense Nucleosome Modifications to Determine Substrate Preference[END_REF] Another PTM essential to the activity of many proteins is glycosylation. Understanding the relationship between the glycosylation profile or position and the properties or function of the glycoprotein is an important step in the development of glycan-based drugs. [START_REF] Valverde | Glycans in Drug Discovery[END_REF] The study of glycoproteins is often challenging due to the difficulty in accessing substantial amounts of homogeneous protein glycoforms. Their semi or total synthesis is also particularly demanding since it requires a double expertise in oligosaccharide and protein production, which is not common. 41 The outstanding efforts towards the production of homogeneous glycosylated erythropoietin (EPO) variants or surrogates are certainly regarded by all protein chemists as great achievements during the last decade. [START_REF] Payne | Total Synthesis of Erythropoietin through the Development and Exploitation of Enabling Synthetic Technologies[END_REF][START_REF] Fernández-Tejada | Total Synthesis of Glycosylated Proteins[END_REF][START_REF] Wang | At Last: Erythropoietin as a Single Glycoform[END_REF] EPO glycoprotein induces the maturation of red blood cells and its glycosylation is critical for achieving full biological activity. Several EPO variants are used in the clinic for treating anemia resulting from various pathological states and in particular kidney dysfunctions. [START_REF] Foley | Emerging Erythropoiesis-Stimulating Agents[END_REF] The development of future erythropoiesis-stimulating drugs heavily depends on the understanding of the relationship between the number of oligosaccharides, their composition and site of attachment and the in vivo bioactivity of EPO, an objective pursued for example by Kajihara and coworkers using synthetic [START_REF] Murakami | Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex-Type Disialyloligosaccharide[END_REF][START_REF] Murakami | Chemical Synthesis of Erythropoietin Glycoforms for Insights into the Relationship between Glycosylation Pattern and Bioactivity[END_REF] or semisynthetic approaches. [START_REF] Hirano | Design and Synthesis of a Homogeneous Erythropoietin Analogue with Two Human Complex-Type Sialyloligosaccharides: Combined Use of Chemical and Bacterial Protein Expression Methods[END_REF] In the previous examples, the glycan moieties were produced and attached to the peptide segments prior to the assembly of the protein, according to the general strategy described in Figure 4a. An example of total chemoenzymatic synthesis of a glycoprotein that corresponds to the approach depicted in Figure 5b Several other PTMs have been worked out by protein chemists such as protein acetylation [START_REF] Ali | Lysine Acetylation Goes Global: From Epigenetics to Metabolism and Therapeutics[END_REF] and methylation [START_REF] Luo | Chemical and Biochemical Perspectives of Protein Lysine Methylation[END_REF] which are frequent in the proteome (Figure 7). Writers, readers and erasers of protein acetylation or methylation marks are the subject of intense research as therapeutic targets, and include histone deacetylases, bromodomains and protein methyltransferases.

Protein synthesis has also been used to tackle less frequent PTMs such protein sulfation, [START_REF] Watson | Rapid Assembly and Profiling of an Anticoagulant Sulfoprotein Library[END_REF][START_REF] Franck | Semisynthesis of an Evasin from Tick Saliva Reveals a Critical Role of Tyrosine Sulfation for Chemokine Binding and Inhibition[END_REF] nitration [START_REF] Burai | Elucidating the Role of Site-Specific Nitration of -Synuclein in the Pathogenesis of Parkinson's Disease via Protein Semisynthesis and Mutagenesis[END_REF] or featuring advanced glycation end products (AGE). 18

Protein synthesis: from target identification to protein therapeutics

One asset of protein chemical synthesis is its capacity to access proteins harboring virtually any type of modification. Figure 9 summarizes the different categories according to which the modified proteins are classified in the PCS database. Besides the PTM-modified proteins discussed in the previous section, the other categories include backbone cyclized peptides and proteins, tagged proteins and proteins made of D-amino acids (D-proteins).

The production of proteins modified by tags is a frequent application of protein synthesis for enabling their detection or their manipulation. For example, Simonneau et al. used sitespecifically biotinylated hepatocyte growth factor (HGF) domains and streptavidin technology to study the importance of multivalency in HGF receptor activation. [START_REF] Simonneau | Semi-Synthesis of a HGF/SF Kringle One (K1) Domain Scaffold Generates a Potent in Vivo Met Receptor Agonist[END_REF] The modification can also be a reactive group designed to trap a binding partner or an enzyme. For example, the modification of Ub or SUMO proteins by tags and electrophilic groups proved particularly successful for detecting the conjugates formed with DUBs in an activity-based manner. [START_REF] Ovaa | Probing Ubiquitin and SUMO Conjugation and Deconjugation[END_REF] One remarkable application of protein synthesis is the production of D-proteins. D-proteins are mirror images of L-proteins and their interest goes from structural studies to peptide or protein drug selection. Quasi-racemic mixtures of D and L-proteins have the capacity to crystallize more easily than D or L-proteins taken alone. This property is exploited in quasi-racemic X-ray crystallography for obtaining high resolution structural data for proteins that are naturally reluctant to crystallize. [START_REF] Kent | Racemic & Quasi-Racemic Protein Crystallography Enabled by Chemical Protein Synthesis[END_REF] In a different application, the production of D-proteins enables the implementation of a technique called mirror-image phage display (MIPD). MIPD uses the Dprotein as bait to select L-peptide or protein binders from a randomized phage library. By symmetry, the D-analogs of the L-peptide binders obtained from the selection process can bind the natural L-protein. Made of D-amino acid residues, such peptide ligands are nonimmunogenic and significantly more stable toward proteolytic enzymes than L-peptides, making them more likely to become future drugs. The power of the approach has recently been illustrated by the design of a D-protein antagonist of natural vascular endothelial growth factor A (VEGF-A). [START_REF] Uppalapati | A Potent D-Protein Antagonist of VEGF-A Is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo[END_REF] As stated at the beginning of this Perspective article, natural product based screening is a powerful means for small molecule drug discovery [START_REF] Harvey | The Re-Emergence of Natural Products for Drug Discovery in the Genomics Era[END_REF] but also for identifying and developing peptide or protein therapeutics. After the initial screening and identification step, developing a synthetic access to the hits is often needed in order to be independent of the natural source, to provide substantial quantities of compounds of interest and to give access to analogs for drug optimization. The natural protein pool is a growing source of potential protein drugs thanks to the constantly increasing performance of modern protein synthesis. The research on cyclotides is particularly representative of the contribution of modern protein synthesis to the natural product-based protein drug discovery approach. [START_REF] De | Cyclotides: From Structure to Function[END_REF] Cyclotides are backbone cyclized disulfide bond-stabilized small proteins produced by various plants. They have a wide range of applications in medicine by exploiting their natural bioactivity or for their capacity to acquire new functions by grafting an epitope from a bioactive peptide or protein onto the cyclotide scaffold. An analog of cyclotide kalata B1 called T20K has reached clinical trials for its immunomodulatory properties and its potential for the treatment of multiple sclerosis. [START_REF] Gründemann | An Immunomodulatory Cyclotide on Its Way to the Clinic[END_REF][START_REF] Thell | Oral Activity of a Nature-Derived Cyclic Peptide for the Treatment of Multiple Sclerosis[END_REF] Defensins, another class of cyclic peptides, have attracted widespread attention for their appealing potential as antimicrobials, antivirals and immunomodulators. In a program aiming at improving the inhibitory activity of RTD-1 against Anthrax metalloprotease, Camarero and coworkers achieved its full positional amino acid scanning using a chemical approach based on the combination of cyclative NCL and oxidative folding. [START_REF] Li | Full Sequence Amino Acid Scanning of Θ-Defensin RTD-1 Yields a Potent Anthrax Lethal Factor Protease Inhibitor[END_REF] To give another example from the PTM category, very recent reports from Payne's laboratory discuss the potential of tick-derived sulfoproteins produced by chemical synthesis for the design of thrombin inhibitors having anticoagulant activities, [START_REF] Watson | Rapid Assembly and Profiling of an Anticoagulant Sulfoprotein Library[END_REF] or for the development of chemokine inhibitors having promising antiinflammatory properties. [START_REF] Franck | Semisynthesis of an Evasin from Tick Saliva Reveals a Critical Role of Tyrosine Sulfation for Chemokine Binding and Inhibition[END_REF] Figure 9. Modified proteins produced by semi or total synthesis are classified into five distinct categories. Note the overlap between certain categories, especially between PTM and tagged protein categories (30 proteins in common).

CONCLUSION AND PERSPECTIVE COMMENT

The growth of the field of protein therapeutics in the future will be sustained by the development of novel synthesis techniques that are more respectful of the environment. Classical SPPS methods are expensive, use toxic solvents (e.g. N,N-dimethylformamide) and reagents (e.g. uronium coupling agents) [START_REF] Mcknelly | Anaphylaxis Induced by Peptide Coupling Agents: Lessons Learned from Repeated Exposure to HATU, HBTU, and HCTU[END_REF] and generate a large amount of waste. Hopefully, SPPS is already experiencing a smooth transition to greener chemistries, [START_REF] Al Musaimi | Greening Fmoc/tBu Solid-Phase Peptide Synthesis[END_REF] which include the use of water as the solvent for the concatenation of amino acids. [START_REF] Knauer | Sustainable Peptide Synthesis Enabled by a Transient Protecting Group[END_REF] Post-translationally modified proteins (333) Tagged proteins (63) ᴅ-proteins Another trend, which is critical to the discovery of small protein therapeutics, is the development of fast methods for the production of synthetic protein libraries. The development of fast synthesis techniques will enable full benefit from the high throughput methods that are available for screening small molecules. Recent studies show spectacular advances in synthesis time by adapting Fmoc SPPS protocols to automated flow reactors. [START_REF] Hartrampf | Synthesis of Proteins by Automated Flow Chemistry[END_REF] The system enabled the synthesis of a functional 164 AA protein, sortase A, in a few hours. Although the equipment needed to achieve such syntheses will require some time to become popular and affordable, in the future the protein chemist will probably see a dramatic reduction of the synthesis time needed for the SPPS of small proteins or of the peptide building blocks utilized for protein assembly. We can predict that the protein assembly step itself will experience the same technological revolution with the recent development of microfluidic systems able to perform an NCL reaction in a few minutes, [START_REF] Chisholm | Native Chemical Ligation-Photodesulfurization in Flow[END_REF][START_REF] Ollivier | Accelerated Microfluidic Native Chemical Ligation at Difficult Amino Acids toward Cyclic Peptides[END_REF] and eventually a post-ligation treatment such as desulfurization. [START_REF] Chisholm | Native Chemical Ligation-Photodesulfurization in Flow[END_REF] Stimulated by the asset of solid phase chemistry for automation, developments for assembling proteins on the solid phase are also actively pursued. [START_REF] Agouridas | Strategies and Open Questions in Solid-Phase Protein Chemical Synthesis[END_REF][START_REF] Ollivier | A Simple and Traceless Solid Phase Method Simplifies the Assembly of Large Peptides and the Access to Challenging Proteins[END_REF] Complementary to the advances based on solid phase or microfluidic technologies, the field of protein chemical synthesis by peptide segment assembly will benefit from the discovery of novel ligation chemistries enabling the acceleration of protein assembly and the extension of the type of junctions that can be produced chemoselectively in water. [START_REF] Dunkelmann | Amide-Forming Chemical Ligation via O-Acyl Hydroxamic Acids[END_REF] Last but not least and due to their intrinsic nature, peptides and proteins will continue to raise solubility issues [START_REF] Li | Removable Backbone Modification Method for the Chemical Synthesis of Membrane Proteins[END_REF] or side-reactions [START_REF] Neumann | Prevention of Aspartimide Formation During Peptide Synthesis Using Cyanosulfurylides as Carboxylic Acid-Protecting Groups[END_REF]

Figure 1 .

 1 Figure 1. Protein semi or total synthesis is sustained by the development of small-molecule

Figure 2

 2 Figure 2. a) Number of proteins produced by semisynthesis or total chemical synthesis by

Figure 4 .

 4 Figure 4. a) The covalent capture concept and the principle of chemoselective peptide bond

Figure 5 .

 5 Figure 5. Principle of the total (a) or semi-synthesis (b) of proteins.

Figure 7 .

 7 Figure 7. A large fraction of the proteins prepared by semi or total synthesis feature a post-

  10 PINK1 and Parkin's E3 ligase are key players in the quality control of mitochondria. Mitochondrial damage induces the activation of Parkin's E3 ubiquitin ligase by PINK1, an event which ultimately results in selective mitochondrial autophagy, also called mitophagy. Abnormal PINK1 and Parkin's E3 ligase functions due to gene mutation are involved in diseases such as juvenile Parkinson's disease. With the knowledge that phosphorylated monomeric ubiquitin can activate wild-type Parkin's E3 ligase (w-Parkin), Liu and coworkers embarked on the production of all four possible non-phosphorylated or phosphorylated Ub K6 Ub variants, to see which form interacts with and activates w-Parkin or phosphorylated Parkin (p-Parkin). The synthetic strategy, which is illustrated in Figure 8 with the synthesis of the diphosphorylated Ub P K6 Ub P dimer, enabled the production of the nonphosphorylated or the monophosphorylated variants as well. The latter could be produced with a full control of the phosphorylation site because enzymatic phosphorylation was performed on each Ub domain separately and before dimer assembly. This mode of modification and assembly of phosphorylated Ub dimers follows the general strategy depicted in Figure 5a. Note that the Lys6 residue is mimicked by an S-(2-aminoethyl)-cysteine residue formed by reacting the glycyl cysteamine derivative aGCA on a Ub domain featuring a dehydroalanine derivative (Dha) at position 6. Because the aGCA derivative provides the C-terminal glycyl residue of the proximal Ub domain, the hydrazide Ub segment used as a thioester surrogate was produced recombinantly with a missing glycine at its C-terminus. Of the four possible variants, only Ub P K6 Ub and Ub P K6 Ub P , which are dimers featuring a phosphorylated distal Ub domain, could activate w-Parkin. In contrast, Ub K6 Ub P equipped with a phosphorylated promixal Ub domain could only activate p-Parkin.

Figure 8 .

 8 Figure 8. Semisynthetic approach to phosphorylated diubiquitins by Liu and coworkers.

  has been reported byHojo et al. with the synthesis of Saposin C (80 AA).[START_REF] Hojo | Chemoenzymatic Synthesis of Hydrophobic Glycoprotein: Synthesis of Saposin C Carrying Complex-Type Carbohydrate[END_REF] Saposin C activates GlcCer-β-glucosidase (GCase) and is involved in the degradation of beta-glucosylceramide (GlcCer) in lyzosomes. Deficiency in saposin C due to mutations causes a rare form of Gaucher's disease. A minimally glycosylated precursor of saposin C bearing an N-acetylglucosamine (GlcNAc) residue at asparagine 21 was produced by ligating two peptide segments using NCL. It was further elaborated into saposin C carrying a complex nonasaccharide by coupling a synthetic octasaccharide to the GlcNAc residue. This saposin C product, as well as other glycoprotein analogs, was used to investigate the role of the sugar moiety on GCase activity.

7 #

 7 B670, 166 AA Levinson 2017 #B748, 201 AA Jbara 2016 #C049, 159 AA Burlina 2019 #C019, 116 AA Shu 2019 #B691, 180 AA Ollivier 2017 #C005, 158 AA Zhang 2019 Biotinylation Fluorophores Non-coded amino acids #C099, 155 AA Xin 2019 #B771, 133 AA Harmand 2017 #B251, 115 AA Qi 2015 #B889, 84 AA Witting 2018 Other (solubility, peptide tag) #C081 Sun 2019 #B672, 352 AA Pech 2017 #B585, 70 AA Rohrbacher 2017 #B274, 61 AA Wang 2015 #B732, 18 AA Li 2017 PEGylation #B687, 207 AA Araman 2017 Lipidation #C040, 50 AA Boross 2019 Advanced Glycation End prod. #B885, 205 AA Matveenko 2018 #C115, 178 AA Ling 2020 #C162, 8 AA Yim 2020

  whose occurrence must be minimized for extending the scope of protein chemical synthesis.*O.M.: e-mail: oleg.melnyk@ibl.cnrs.fr. *V.A.: e-mail: vangelis.agouridas@ibl.cnrs.fr.ORCIDOleg Melnyk: 0000-0002-3863-5613 Vangelis Agouridas: 0000-0003-0911-1527.NotesThe authors declare no competing financial interest.BiographiesVangelis Agouridas obtained his Ph.D. in organic chemistry from the University of Versailles Saint-Quentin (France) in 2006. After a first postdoctoral fellowship in natural product synthesis at Boston University (USA), he returned to France for two additional fellowships in medicinal chemistry at Paris V and Paris XI faculties of pharmacy. In 2010, he was appointed assistant professor at the Ecole Nationale Supérieure de Chimie de Lille (ENSCL, France) and joined the group of Dr. Oleg Melnyk in 2013. His main research activity is focused on developing amide-bond metathesis reactions applied to peptides and proteins. Ouafâa El Mahdi obtained her Ph.D. degree in organic chemistry from the University of Montpellier in 1997. She then joined Oleg Melnyk's team (Lille, France) for two years as postdoctoral research fellow where she developed novel biofunctionalization chemistries for hybrid nanoparticle synthesis. In 2003, she was appointed assistant professor at the University of Sidi Mohamed Ben Abdellah (Fez City, Morocco) and was promoted to full professor in 2017 at the same University. She is interested in chemical ligation methods for the synthesis of peptides and proteins. Oleg Melnyk graduated as an engineer in chemistry in 1989 from the Ecole Nationale de Chimie de Paris (ENSCP, France). He received his Ph.D. degree in 1994 from the University of Paris VI (France). During his thesis work, he developed a semisynthetic route to cortisone. After post-doctoral training from 1994 to 1996 in the group of Prof. A. Tartar (Lille, France) he was recruited by the Centre National de la Recherche Scientifique (CNRS) in Lille. He is now director of research at CNRS and his main research interests are the development of chemical methods for protein synthesis and the study of protein function.

ACKNOWLEDGMENTS

We acknowledge financial support from CNRS, INSERM, CHU, Univ Lille and Institut Pasteur de Lille.

AUTHOR INFORMATION

Corresponding Authors

Protein chemical synthesis in medicinal chemistry


Haidong Li

Romain Duwald

Simon Pascal

Silvia Voci

Céline Besnard

Johann Bosson

Laurent Bouffier

Jérôme Lacour
email: jerome.lacour@unige.ch

Neso Sojic
email: neso.sojic@enscbp.fr

Near-Infrared Electrochemiluminescence in Water through Regioselective Sulfonation of Diaza [4] and [6]helicene Dyes

come

Electrochemiluminescence (ECL) is a special case of chemiluminescence where light emission by the excited state of a luminophore is initiated by an electrochemical reaction. 1,2 The electrogenerated species, which involve classically a luminophore and a sacrificial coreactant species undergo highenergy electron-transfer reactions in solution to give excited states. Nowadays, ECL is an ultrasensitive and powerful analytical method, which is on the market for clinical diagnostic. In water, the most employed ECL system is composed of tris (2,2′-bipyridyl) ruthenium(II) luminophore, noted [Ru(bpy)3] 2+ , and tri-n-propylamine (TPA) coreactant. 1,3 ECL is a very active field of research with a special focus on the development of new ECL-active dyes and original analytical strategies in aqueous media. [4][5][6][7] In that context, ruthenium and iridium complexes remain attractive despite their extremely low luminescence efficiencies and short excited state lifetimes in near-infrared region. [8][9][10][11] On the other hand, organic dyes offer a wide chemical diversity with highly tunable photophysical and electrochemical characteristics. [12][13][14][15] The electronic characterization of a new family of dyes featuring a helical geometry was reported recently. 16,17 The chromophores are configurationally stable cationic helicenes that bear structural analogy to both triarylcarbenium ions and classical carbo-or heterohelicenes. 18 In addition, these dyes feature tunable electronic and optical properties depending on the nature of the bridging heteroatoms as well as the presence of pendant functional groups. 16 Also, they are ECL-active in aprotic solvents such as acetonitrile with high ECL yield. 19 Various regioselective functionalizations of the helicene core can be readily achieved leading to a greater chemical diversity as well as water solubility. 16,17 Recently, we reported self-enhanced multicolor ECL using a water-soluble diaza [4]helicene bearing a tertiary dimethylamino-terminated group. 20 Aside those helicene luminophores, very efficient organic ECL dyes have been designed and reported in organic media. [13][14][15]21 However, a major challenge is to obtain such bright organic ECL emitters operating in physiological conditions (i.e. in aqueous buffer at pH 7.4 where bioassays and cell microscopy are relevant), especially in the near-infrared region 11,22 due in part to strong nonradiative processes as governed by the energy gap law. 23 Indeed, this spectral domain is particularly attractive for Scheme 1 Structure and preparation of water-soluble helicene derivatives. Experimental conditions: (a) 1,3-propanesultone (10 equiv), DIPEA (15 equiv), CH3CN, reflux, 16 h (94% yield), (b) H2SO4 (18 equiv), PPA, 60 °C, 1 h (57% yield), (c) H2SO4 (90 equiv), PPA, 80 °C, 2 h (61% yield), (d) H2SO4 (187 equiv), 20 °C, 48 h (63% yield). DIPEA = diisopropylethylamine, PPA = polyphosphoric acid.

biological ECL applications such as cell and tissue microscopy and sensing since it enables lower background interference and longer penetration depth into biological tissues. 24 Herein, we report the successful functionalization of diaza heli-cenes with either one or two sulfonate groups at different aromatic or alkyl positions affording suitable electrooptical properties for aqueous ECL (Scheme 1). Four new molecules exhibiting either [4] or [6]helicene geometries were characterized by electronic absorption, fluorescence and electrochemistry. All molecules were found to be ECL-active in the presence of TPA in phosphate-buffered saline (PBS). They display an ECL wavelength falling in the red to near-infrared region with a good ECL yield.

To achieve solubility in water, the introduction of polar groups was targeted, and sulfonate groups were selected. Two strategies were thus foreseen (Scheme 1). The sulfonate group(s) could be introduced on a terminus of a pendant aliphatic side chain ([4]-C3-Sulfo). Alternatively, those functional groups could be added to the periphery of the helicene cores via a late-stage functionalization strategy ([4]-monoSulfo, [4]-diSulfo and [6]-diSulfo). In a general fashion, cationic [4]helicenes are prepared in a single step from the corresponding hexamethoxy triaryl carbenium ion through successive nucleophilic aromatic substitutions with primary amines. 25,26 Recently, the procedure was adapted for the synthesis of pH-sensitive [4]quinacridine. 27 The presence of the N-atom is advantageous for a derivatization by nucleophilic substitution. In the present case, alkylation of [4]quinacridine with 1,3-propanesultone afforded [4]-C3-Sulfo in 94% yield and single crystals suitable for diffraction analysis were obtained (see ESI). Although being cationic, [4] and [6]helicenes are electron-rich species. 16,17 In fact, these moieties react with electrophiles and, in strongly acidic reaction mixtures, they provide derivatives functionalized regioselectively at position 2 and/or 12 and 3 and/or 15, of [4] and [6]helicenes, respectively (Scheme 1). 28 Practically, treatment of [4]helicene with H2SO4 in polyphosphoric acid (PPA) at 60 °C for 1 h led to the formation of [4]-monoSulfo in 57% yield. Conducting this reaction at 80 °C for 2 h instead led to the preferential formation of [4]-diSulfo in 61% yield. Finally, stirring [6]helicene in conc. H2SO4 for 48 h at 20 °C led to the clean formation of [6]-diSulfo isolated as the bis(2-ethylhexyl)ammonium salt in 63% yield. 29 The electronic absorption spectra of the newly prepared chromophores were recorded in aqueous 0.1 M PBS buffer (pH 7.4) in the 450-800 nm range (Fig. 1). The four derivatives exhibit absorption in the visible domain with maximum wavelength ranging from 615 nm for [4]-C3-Sulfo and [6]-diSulfo to 648 nm for [4]-diSulfo derivatives (Table 1). The [4]-monoSulfo absorption band is centered on a maximum peaking at 632 nm. As previously described, the functionalization of the side chain of [4]helicene ([4]-C3-Sulfo) and at positions 3 and 15 of [6]helicene ([6]-diSulfo) does not influence the optical properties, which is in stark contrast to the functionalization of [4]helicene at position(s) 2 and/or 12 ([4]-monoSulfo, [4]-diSulfo) that can induce a strong modulation of the electronic properties. 28 The solubility of the derivatives in aqueous medium was further investigated by UV-vis absorption from 10 -3 to 10 -6 M concentration (see ESI). In PBS, the sulfonate groups are deprotonated. As depicted with the case of [4]-C3-Sulfo, the introduction of the sulfonate group on the side chain is not sufficient to allow a perfect solubility in PBS and the extinction coefficient is found to be varying considerably, from 500 to 4700 M -1 cm -1 , going from ca. 10 -3 to 2×10 The photoluminescence properties were also characterized in aqueous solution by recording fluorescence spectra (Fig. 1). The spectra reveal typical large emission bands for the diaza helicenes with a full-width half-maximum value between 75 nm ([6]-diSulfo) and 95 nm ([4]helicene derivatives). The fluorescence maximum wavelength is observed in the red to near-infrared region (661 nm up to 710 nm). The Stokes shift value is at least 46 nm and rises up to 61-62 nm for the more bathochromic emitters ([4]-monoSulfo and [4]-diSulfo. Again the emission properties of [4]-C3-Sulfo and [6]-diSulfo are similar to that of non-functionalized [4] and [6]helicene whereas noticeable bathochromic shifts are observed for [4]-monoSulfo and to a larger extend for [4]-diSulfo. The highest fluorescence quantum yield is measured for compound [6]-diSulfo (ΦFL = 6.8%) whereas the three other dyes exhibit weaker emission ranging from 0.6% to 2.8% (Table 1). The non-sulfonated analogues [4] and [6]helicenes showed a similar photophysical behavior in acetonitrile. Indeed, in organic media, the quantum yield of [6]helicene was ~4 times larger than the one from [4]helicene. 19 The electrochemical properties of the diaza helicenes were investigated by cyclic voltammetry (CV) and/or differential pulse voltammetry (DPV) depending on the intrinsic solubility of each derivative in water. A representative CV recorded for [4]-diSulfo, which is the only compound fully soluble at a concentration of 10 -3 M (vide supra) is provided in Fig. 2. A quasi-reversible reduction is monitored at -0.71 V with a peak separation of about 100 mV. It is assigned to a mono-electronic reduction of the helicene cation core. Considering the stability of the reduced form as evidenced by a ratio of cathodic/anodic -1,5 -1,0 -0,5 0,0 0,5 peak currents of 0.92, it offers the opportunity to explore its cathodic ECL behavior in future works. On the contrary, the oxidation process is not reversible at the time scale of the CV with an irreversible peak at 1.29 V. This anodic behavior in water contrasts from the oxidation of diaza helicenes monitored in an aprotic solvent, which is usually reversible. 19 However, this is not surprising since this oxidation corresponds to the formation of a dication radical core that is likely to be rather unstable in aqueous phase. CV of [6]-diSulfo (Fig. S24) shows a relatively similar electrochemical behavior as [4]-diSulfo. But the irreversible oxidation occurs at less positive potential (vide infra) and the reduction process is less reversible.

The mono-sulfonated derivatives that were not soluble enough to be studied by CV were then characterized by DPV in 0.1 M PBS at a concentration of 2×10 -5 M (Fig. S25-S26). At this concentration, the mono-electronic reduction of [4]-monoSulfo is observed at -0.83 V. On the other hand, the oxidation pointing at 1.17 V is slightly superimposed with water oxidation but still remains clearly visible by comparison to DPV recorded only in the presence of the supporting electrolyte. [4]-C3-Sulfo is the only compound where the sulfonate group was introduced far apart from the helicene cation core, by modification of one of the lateral propyl chain. This diaza [4]helicene is the easiest to oxidize (Eox = 1.13 V) whereas it is the hardest to be reduced (Ered = -0.94 V). By comparison, the introduction of the sulfonate directly on the core like in [4]-monoSulfo results in an easier reduction at -0.83 V. This could be ascribed to the presence of the electron-withdrawing group, which lower the electron density of the helicene core. The carbocation can accept more easily an electron, resulting in a less negative reduction potential. When comparing the [4]-diSulfo with [6]-diSulfo, the latter is easier to oxidize (Eox = 1.13 versus Eox = 1.29 V for [6]-diSulfo and [4]-diSulfo, respectively) meaning that the charge of the dication core radical is more stabilized in the most extended [6]helicene structure. In acetonitrile, the oxidation and reduction reactions of [4] and [6]helicenes showed a reversible behavior with very similar potential values. 19 The ECL capability in water was evaluated in oxidation, which is the main mechanistic pathway in aqueous solution. For that, 0.05 M TPA was employed as a sacrificial coreactant. Typically, the potential is scanned towards positive values until the coreactant and the dye are oxidized (Fig. S27). The oxidation of TPA generates a radical cation (TPA •+ ), which dissociates spontaneously by losing a proton. The corresponding neutral radical (TPA • ) is a strong reducer that is characterized by a rather negative potential Ered ~ -1.7 V vs. Ag/AgCl. 10,30 Fig. S27 shows a first small ECL peak appearing before the oxidation of the helicenes. The corresponding mechanistic pathway can be described by the so-called "revisited route" detailed in ESI (Eq. S1-S5). The predominant ECL peak occurs at the potentials where the dyes are oxidized and involves a very exergonic electron-transfer reaction between a strong oxidant (helicene radical) and a strong reducer (TPA • ) that are formed simultaneously in the vicinity of the electrode (see ESI). The free enthalpy of reaction ΔrG° of the corresponding electrontransfer process can be easily estimated by the difference of redox potentials (Eox(helicene) -Ered(TPA • )), leading to an exergonic situation for all four compounds. The corresponding ΔrG° values span from 2.83 to 2.99 eV (without taking into account the entropic contribution (typically, 0.1 eV) that remains constant and negligible compared to the enthalpic term). In all cases, the enthalpy released by the redox reaction is much higher than the energy corresponding to the wavelength of emission that can be calculated according to the formula (EFL (in eV) = 1239.81 / λFL (in nm). These calculated values of EFL range from 1.75 to 1.88 eV confirming that all four molecules should be indeed ECL-active. The ECL spectra recorded by applying a potential of 1.4 V are gathered in Fig. S28. It is noteworthy that it is the first time that this series of molecules are investigated in aqueous media revealing an intense ECL signal. The maximum value for ECL was compared to fluorescence (Table 1) showing that the same excited state is involved with λECL values very close to λFL ± 5 nm.

The ECL efficiency ΦECL was estimated with respect to [Ru(bpy)3] 2+ in the same experimental conditions (Fig. S27). Significant ΦECL values were found from 5.4% for [4]-diSulfo up to 33.9% for [4]-C3-Sulfo derivative. The lowest ECL yield is obtained for the compound that exhibits the highest oxidation potential but that suffers from a poor fluorescence quantum yield (ΦFL = 0.6%). On the other hand, the brightest ECL emitter is [4]-C3-Sulfo. It illustrates the influence of the position of the sulfonate group(s) on the ECL emission. ECL is brighter when the sulfonate group is located further from the diaza core. In PBS, ΦECL of [4]-C3-Sulfo is larger to that of [6]-diSulfo despite a similar oxidation potential (Eox = 1.13 V). The behavior was similar in acetonitrile where ΦECL value of [4]helicene was 2.3 times greater than [6]helicene using also TPA. 19 But, contrary to the aqueous media, the situation was different since the oxidized radicals of both [4] and [6]helicenes were stable in CH3CN. In PBS, the brightest ECL dye (i.e. [4]-C3-Sulfo) emitting at 671 nm exhibits a lower quantum yield in to [6]-diSulfo, which emits at 661 nm (Table 1). The variations of quantum yield (Table 1) are coherent with the energy gap law, which implies that the rate of nonradiative process increases with the longer emission wavelengths. 23 Considering the energetics of the ECL process, both dyes have the same free enthalpy of reaction with TPA • . Since the lower energy of the excited state is just about 0.03 eV due to a 10 nm red-shift in λECL, this very small difference of exergonicity (0.03 eV) of the reaction generating the excited state cannot explain the observed difference in ΦECL. On the other hand, the stability of the electrogenerated species is a crucial parameter governing the ECL efficiency. 31,32 Therefore, we can hypothesize that this higher ECL efficiency of [4]-C3-Sulfo is related to a better stability of the oxidized form.

Herein, the synthesis of a new series of water-soluble diaza helicenes functionalized with either one or two sulfonate groups is reported. Consequently, these molecules are soluble in aqueous phase. The optical as well as electrochemical behavior was characterized in PBS. In brief, these dyes strongly absorb in the visible region. They emit in the red to nearinfrared region (with maxima between 661 and 710 nm) and the excited state can be reached either by photo-excitation or ECL. These molecules are indeed redox-active and can be oxidized to afford the corresponding radical in water. Although such an oxidation appears to be irreversible in water, the stability of the electrogenerated species is sufficient to undergo redox chemistry with electrogenerated TPA • radical. This classic ECL pathway leads to a strong ECL emission with an ECL efficiency ranging from 5.4% up to 33.9% by comparison with [Ru(bpy)3] 2+ standard. The brightest ECL emitter was found to be the [4]-C3-Sulfo derivative, which gathered a high oxidation potential with a red ECL occurring readily at 671 nm. Finally, the [4]-diSulfo compound exhibits the highest solubility in water together with an ECL emission at 688 nm that is very promising for future applications such as multiplexed bioassays and ECL imaging in biorelevant samples.

Fig. 1

 1 Fig. 1 Normalized absorption (plain lines) and fluorescence (dashed lines) spectra recorded in 0.1 M PBS.

Fig. 2

 2 Fig. 2 CV of 10 -3 M [4]-diSulfo in 0.1 M PBS degassed for 5 minutes. Scan rate 0.1 V s -1 .

Table . 1

 . Photophysical, electrochemical and ECL data recorded in 0.1 M PBS.

		Electroche	Absorption	Fluoresce	ECL
		mistry a			nce
	dYE	Eo	Ered	λ	ε / M -1	λFL /	Φ	λECL	Φ
		x / V	/ V	/ nm	cm -1	nm	FL	/ nm	ECL b
	[4]-C3-	1.	-	6	4700	672	2.	671	3
	Sulfo	13	0.94	15			8%		3.9%
	[4]-	1.	-	6	3900	693	1.	688	2
	monoSulfo	17	0.83	32			3%		1.7%
	[4]-	1.	-	6	4800	710	0.	708	5.
	diSulfo	29	0.71	48			6%		4%
	[6]-	1.	-	6	13500	661	6.	661	1
	diSulfo	13	0.84	15			8%		9.6%
	a Eox (V) and Ered (V) vs. Ag/AgCl refer to the peak potentials measured by DPV or CV for
	mono-and di-sulfonated derivatives, respectively. b The values have been measured
	with respect to [Ru(bpy)3] 2+ using 0.05 M TPA in the same conditions (see ESI).

Acknowledgements

We thank A. Kapturkiewicz for helpful discussions on ECL efficiency. RD, SP, JB and JL thank the University of Geneva and the Swiss National Science Foundation for financial support (SNF 200020-172497 and 200020-184843).

Notes and references

Conflicts of interest

There are no conflicts to declare.


Mustapha Derras
email: mustapha.derras@berger-levrault.com

Laurent Deruelle
email: laurent.deruelle@berger-levrault.com

Nicole Lévy

Francisca Losavio
email: francisca.losavio@ciens.ucv.ve

Berger Levrault

Nicole Levy
email: nicole.levy@cnam.fr

Towards bridging the gap between domain and application design

Keywords: software product line, reference architecture, domain and realization design, application design

published or not. The documents may come

Towards bridging the gap between domain and application design

I. INTRODUCTION

The correspondence between two architectural views [START_REF] Krutchen | Architectural Blueprints-The "4+1" View Model of Software Architecture[END_REF] of a software system has been widely discussed in the literature, however it still remains a problem. A Reference Architecture (RA) for a Software Product Line (SPL) [START_REF] Clements | SPL: practices and patterns, Addison Wesley 3rd edition[END_REF], is built in the Domain Design phase as the main asset of the Domain Engineering (DE) lifecycle; the SPL Engineering (SPLE) Model of [START_REF] Böckle | SPL Engineering: Foundations, Principles, and Techniques[END_REF], now incorporated into the new ISO/IEC 26550 [START_REF]Software and Systems Engineering -Reference Model for Software and Systems PL[END_REF] is followed, see Fig. 1. The main idea of this work is to refine the RA variability model before starting the Application Design, using the Domain Realization phase of DE. The mapping between the architectural logic view and the physical or deployment view will be discussed as a first step, by specifying the technology related to each architectural component; new components may be introduced at this stage. Then the architectural component interfaces will be defined. We will explore if the gap between different architectural abstraction levels, the Domain Design and the Application Design, can be reduced. The Domain Realization will be used as an intermediate abstraction level, to facilitate the configuration step in the Application Engineering (AE) lifecycle. Configuration is unavoidable in SPL during the Application Design phase to produce concrete applications derived from the RA core assets and the variability model; for example, the systems migrating to a cloud structure need to be configured to be used by different clients; the configuration consists in setting the convenient variants. Note that businesses are coping with the challenge of upgrading and optimizing enterprise applications performing configuration using native tools, without modifying the system code, and avoid a customization process that requires code manipulation [START_REF] Dai | Intelligent Information Management[END_REF]. Vendors are providing more options to configure applications for particular needs and industries. But in some cases, configuration options are getting so numerous and layered that they present challenges of their own. Any way, a convenient instance of the RA has to be configured to obtain a concrete software application or product of the SPL family, responding to client requirements. This step is still on the threshold between DE and AE. The problem of relating the abstract components of the SPL RA [START_REF] Clements | SPL: practices and patterns, Addison Wesley 3rd edition[END_REF], [START_REF] Böckle | SPL Engineering: Foundations, Principles, and Techniques[END_REF] with the concrete components of a software product of the SPL family using the right reusable assets, is not completely solved. The reusable assets offered by the actual technology (toolkits, APIs, tools, etc.) used in industrial developments, solve nowadays aspects that had to be carefully programmed before. Some examples are the user interface quick development including the MVC maintainability concern often solved by Angular Js/RESTful, and the portability of the relational database to objects with the Hibernate tools. However, it is claimed that the architecture is still the key to the success of any software project since it is the first design artefact that begins to place requirements into a solution space. The quality attributes of a system, such as performance, modifiability, and availability should be considered by the architecture; if the architecture is not suitable from the beginning for these qualities, it is very difficult to achieve them by some miracle later [START_REF] Northrop | Framework for Software Product Line Practice[END_REF]. The architecture determines the structure and management of the development project as well as the resulting system, since teams are formed, and resources allocated around architectural components. In a previous work [START_REF] Derras | Reference Architecture Design: a practical approach[END_REF], a RA was constructed for the Vacation Request subsystem of the Berger-Levrault SEDIT Human Resources (HR) system [START_REF] Berger-Levrault | Schéma architecture -SEDIT REST -Evolution[END_REF], using a bottom-up strategy with a single product already built by the enterprise. The RA was obtained with a variability model containing mostly nonfunctional components related to the functional components of the core asset; these non-functional components represent the quality properties to be satisfied by each functional component in order to be compliant with functional suitability [START_REF]SQuaRE, Quality model[END_REF]. It is important to notice that these non-functional components were introduced to assume the responsibility to check that the quality property will be actually present in the concrete product. All the non-functional components are variation points that implement different solutions according to the technological evolution. Each choice will be linked to a variant attached to a variation point, realizing the traceability. For example, the non-functional component <<Compliance with law(s)>> will be directly linked to the rules specifying the law(s). Fig. 1. ISO/IEC 26550 SPLE (Software Product Lines Engineering) Reference Model Framework [START_REF]Software and Systems Engineering -Reference Model for Software and Systems PL[END_REF] Recall that the RA or domain architecture determines the structure and the texture of the concrete applications or products of the SPL. According to [START_REF] Böckle | SPL Engineering: Foundations, Principles, and Techniques[END_REF], the structure describes the decomposition that is valid for all applications of the SPL (commonality and variability). The texture is the collection of common rules guiding the design and realization of the parts, and how they are combined to form applications. It defines common ways to deal with variability in domain or application design and realization; it consists of coding rules and general mechanisms such as styles [START_REF] Shaw | Software Architecture. Perspectives of an emerging discipline[END_REF] and design patterns [START_REF] Gamma | Design Patterns: Elements of Reusable OO Software 1st Edition[END_REF] to deal with specific situations/solutions that may occur during design, realization, and coding. The Domain Realization phase is focused on building this architectural texture that will be used to design the concrete applications of the SPL family.

A process was defined in [START_REF] Derras | Reference Architecture Design: a practical approach[END_REF], following the first three phases of the DE lifecycle to obtain the RA, shown in grey in Fig. 1. Our present goal is to reduce the gap between the highlevel abstract RA and the more concrete application design by defining first traceability links between the RA components and the reusable technological assets. In order to do this, the SPLE Domain Realization phase guidelines [START_REF] Böckle | SPL Engineering: Foundations, Principles, and Techniques[END_REF], [START_REF]Software and Systems Engineering -Reference Model for Software and Systems PL[END_REF] will be adapted to deal with a more detailed design concerning the reusable software components and their variants.

The RA represented in UML 2.0 in Fig. 2 is a logic view of the architecture, incorporating elements of the process view, such as the layered domain style used by HR systems. According to [START_REF] Krutchen | Architectural Blueprints-The "4+1" View Model of Software Architecture[END_REF], architectural views can be combined. However, in this UML 2.0 notation, components interfaces are not shown, nor the provide/require in the connections; the components, which are variation points are denoted as stereotypes by << component name >>. Components that are not variation points are considered common components that will be present in all the products of the SPL family. Fig. 2. Reference Architecture for the Vacation Request subsystem of BL SEDIT [START_REF] Derras | Reference Architecture Design: a practical approach[END_REF] This paper is structured as follows besides this introduction: the second section describes the Domain Realization phase: the correspondence between architectural components and the technological tools and the definition of the components interface. Guidelines to perform these activities are provided. The third section discusses some related works and finally the conclusion and perspectives are presented in the fourth section.

II. DOMAIN REALIZATION

A. Generalities

Domain Design is the DE process of SPLE in which the commonality and the variability of the SPL are defined to conform to the RA structure. The input for the domain realization sub-process consists of the RA including the list of reusable software artefacts already present or to be developed. Each component should be planned, designed, and realized for reuse in different contexts supported by the component interface. It is important to note that the result of this phase consists of loosely coupled, configurable components, not of a running application. Domain realization can incorporate configuration mechanisms into the components to realize the variability of the SPL. Traceability links between the artefacts of the reusable platform will facilitate systematic and consistent reuse. Our problem concerns the way to establish consistent traceability links between the RA abstraction level and the design level of the concrete product, saying Application Design and Realization (see Fig. 1). The output of Domain Realization encompasses the design and "implementation" aspects of reusable software components, and their interfaces, for example some kind of configuration files. Components realise variability by providing suitable configuration parameters in their interfaces. Notice that Domain Realization differs from the realization of single systems mainly because the result consists of loosely coupled, configurable components, not of a running application.

The industrial case study of the Vacation Request system will illustrate our approach.

B. Correspondence between architectural components and technical tools

As a first step to provide a rough process for the Domain Realization phase, we will establish the correspondence between architectural components and the technological tools used by the enterprise, which are known reusable and available assets. Notice that technology changes constantly, then all the mentioned tools are potential variants, considering the SPL variability model approach. In our case, each RA architectural component will correspond to a set of modules, which have been already built by Berger-Levrault. For example, two systems implemented in Java are the main modules of the SEDIT Vacation Request subsystem: -the X.Net human resources manager; it allows the configuration of a validation system for a vacation demand to check the hierarchy of the staff responsible of authorizing the request; it corresponds to the RA <<Signature Hierarchy>>; -the e.SEDIT RH to automatize the staff administrative tasks, favouring also collaborative work; it corresponds to the RA component Administrative Tasks [START_REF] Rodríguez | Analyse fonctionnelle de domaine dans le cadre de logiciels de gestion de collectivités territoriales[END_REF]. The architectural style for the RA is event-based, layers and follows a client/server model for communication [START_REF] Shaw | Software Architecture. Perspectives of an emerging discipline[END_REF], used in the HR domain.

The SEDIT tools of the technological platform, i.e., main frameworks, toolkits, APIs and services used in the development stage [START_REF] Berger-Levrault | Schéma architecture -SEDIT REST -Evolution[END_REF] are mostly open source and they are shown in Table 1.

Guidelines:

Input: the RA (in our case, the Vacation Request structure of RA in Fig. 2), the list of reusable software artefacts that are already available or to be developed. In the SEDIT case, the list of technological tools is used.

-For each component and sub-component in a layer, the relation with the corresponding technical tool or with an architectural component present in the subsequent layer, are shown.

-Technological (s) solution (s) is (are) listed for each component. Output: loosely coupled components that can be configured by their interfaces.

All the connections between layers follow the REST system architectural style, where resources are directed only through their URLs, via the http/https protocol. The JAMon system monitors all connections between architectural components, including message passing and RMI (Remote Method Invocation). The technical tools/solutions in Table 1 are used to implement the architectural components as reusable modules at Application design level.

C. Specification of components' interfaces

A new version of the Vacation Request RA presented in Fig. 3 in UML 2.0 is built in this second step. Components and their interfaces are shown in terms of required/provided resources [START_REF] Bell | UML basics -The Component Diagram -UML 2.0 diagrams[END_REF]. The study of the technology used by the enterprise (see Table 1) has provided some hints to update the abstract logic view of the original RA design (see Fig. 2), for example by adding/eliminating components or subcomponents. A new <<Data Access>> component has been located in Process Layer; it will be used to interface the Process and Data Layers, to ensure data portability and persistency/availability. Context is an external system that will provide information to the <<Compliance with Law (s)>> component, through a configuration operation, parameterized by the entity, the staff characteristics, the law text, and a configuration file that captures the specificity of the law's evolution. Notice that one of the main problems found in [START_REF] Böckle | SPL Engineering: Foundations, Principles, and Techniques[END_REF] and [START_REF] Derras | Reference Architecture Design: a practical approach[END_REF], was that the variability in HR systems was due mostly to the frequent law changes and not much to changes in the main functionalities. Moreover, this process is usually performed in a quasi-manual way at a quite high cost.

The Context external system is not connected to the User Interface (UI) Layer since the user that will operate this system does not belong to the Vacation Request set of users (Employee, Supervisor and Administrator); he will operate the Vacation Request configuration externally, updating the law changes. A solution benefiting for example from the Java language features, such as the injection principle and related design patterns [START_REF] Gamma | Design Patterns: Elements of Reusable OO Software 1st Edition[END_REF], to make changes independently from the coded lower levels modules, could be implemented within the Configuration component. It is clear that other configuration solutions could be available, that is why the main component <<Implementation>> of the Context system is a variant. If the Vacation Request system is offered on the cloud, it will be available and used by each client with the appropriate configuration, once the Context system, operated by an external user, has concluded its tasks.

The parameters in the component interfaces could be defined as: entity = {country, region, city, community. …}; staff-id = {id, password, status, …}; access, notify, signapproval, rights-check: Boolean; period: dates for the vacation request or number of days required and period of the year; eval-required: Boolean (push button to activate the evaluation in Process Layer; updated-data: results of administrative tasks related with the vacation request of an employee; law: data structure representing the law text that can be expressed as a set of rules; updated-config: updated configuration file; configfile: reusable configuration file; this information can be retrieved from the Context system expressed and executed in a programming language, for example Java, benefiting from the language features, such as injection principle and design patterns [START_REF] Gamma | Design Patterns: Elements of Reusable OO Software 1st Edition[END_REF], to perform the configuration according to the law changes, independently from code. Input: RA components annotated with reusable technological tools (see Table 1); -For each component, establish the provided/ required resources; new components can be added, and/or existing components can be deleted; specify the corresponding parameters; define the main methods (corresponding to the RA abstract subcomponents, see Fig. 2) used by the component. Output: RA structure expressed in UML 2.0 showing also the RA texture with components' interfaces (see Fig. 3).

Let us note that most of the functional components of the RA are common components; the SEDIT technological platform provides concrete solutions for several variants of the RA variation points which are non-functional components (see Table 1): <<MVC Client-side>> with Angular Js (client-side), <<MVC Server-side>> with Angular Js (server-side), <<Login>> with LDAP, <<Signature hierarchy>> with Spring Security, Kerberos Security, <<Data Access>> with Hibernate, and <<Data Base>> that is actually settled to Oracle. As we have already pointed out, RESTful and JAMon are followed for communications.

The problem remains however with the <<Compliance with Law(s)>> variation point for which some solution has to be provided. In this case the Context external system could be a solution with the Configuration component. The <<Implementation>> will consist of using the Java injection facility and laws will be expressed as a set of rules [START_REF] Douin | La proposition VIP : Variability & Injection Pattern, Internal communication[END_REF]. The design of this system is still an on-going work.

III. RELATED WORKS

Four works are discussed in this section, the first two are dedicated to the SPL development process, and the last two focuses on the HR domain and the migration to the cloud structure, but do not treat the SPL context. In the near future, Berger-Levrault would like to offer a cloud solution for his clients by software product lines for the main functionalities offered by the SEDIT system. Our work will contribute to design a configuration system to treat the problem of laws variation and compliance found in HR systems; as it has been pointed out, these configuration tasks are in general manually solved, requiring much effort and cost.

The paper of Käkölä [START_REF] Käkölä | Standards initiatives for SPL engineering and management within the international organization for standardization[END_REF] discusses the projects and future directions for SPL standardization. The new standard ISO/IEC 26550 [START_REF]Software and Systems Engineering -Reference Model for Software and Systems PL[END_REF], which was an on-going project, is discussed. It presents a reference model for Software Product Line Engineering. To obtain maximum benefits from the SPLE development methodology, businesses need to implement coordinated changes in development methodologies, tools, product architectures, organizational designs, and business models. The SPLE Reference Model involves higher levels of abstraction than the engineering of single systems partly because the platforms require substantial investments, have long life cycles, and have to provide product line architectures and features generally applicable to a wide range of products, services, and markets. Without appropriate abstractions, the platforms with predefined variability cannot be built and managed effectively. On the other hand, standardized methods and tools for developing product lines cannot be easily adopted, tool vendors face difficulties in developing tools to enable SPLE, and universities cannot effectively set up SPLE courses because an internationally accepted curriculum is missing. However, two surveys and workshops are actually organized to derive recommendations for educators to continue improving the state of practice of teaching SPLs, aimed at both individual educators as well as the wider community [START_REF] Acher | Teaching Software Product Lines: A Snapshot of Current Practices and Challenges[END_REF]. The International Organization for Standardization (ISO) has initiated several projects to create a set of international standards for SPLE, such as the ISO/IEC 26550 Reference Model, where guidelines and available tools and techniques are provided. ISO hopes that researchers and practitioners can enrich this initiative.

Our research aims to make a practical use of the SPLE methodology, integrating practices such as the early consideration of quality issues in the RA design, and applying it to an industrial case study.

Guidelines, practices, benefits, and risks to adopt a SPL approach are discussed in the SEI SPL framework by Northup and Clements [START_REF] Northrop | Framework for Software Product Line Practice[END_REF] "… substantial production economies can be achieved when the systems in a SPL are developed from a common set of assets in a prescribed way, in contrast to being developed separately, from scratch … It is exactly these production economies that make the SPL approach attractive". Reuse has evolved from subroutines (1990s) to services (2000s). Most organizations produce families of similar systems, differentiated by features, and a reuse strategy makes sense. Commonalities are exploited differently according to the enterprise domain; hence the study of the domain is essential for the SPL development. Enterprises that have succeeded with product lines vary widely in considering activities such as the nature of their products, the market or mission, business goals, organizational structure, culture and policies, the software process discipline, and the maturity and extent of legacy artefacts. Nevertheless, there is not one correct set of practices for every organization [START_REF]Software and Systems Engineering -Reference Model for Software and Systems PL[END_REF]; activities and practices emerge, having to do with the ability to construct new products from a set of common assets while working under the constraints of organizational contexts, and this problem is still not solved. The recommendation in this work to derive a concrete product, is to follow a production plan, which details how the core assets are to be used to build the product. However, each of the main activities for SPL development, saying, the core asset development (where the RA is built), the product development, and management is individually essential, and they are a blend of technology and business practices. However, the threshold between these activities and how to put them smoothly and correctly together is not discussed in the framework, and it is the problem we are focusing. We are putting into industrial practice an SPL development approach that blends several practices and techniques: a bottom-up architecture-cantered strategy to build the RA [START_REF] Derras | Reference Architecture Design: a practical approach[END_REF], the extraction of functional components from business goals, the use of the properties of the domain architectural styles, the early account of quality requirements as architectural components, as the major responsible of the SPL variation. In our work we use the Domain Realization guidelines of [START_REF] Böckle | SPL Engineering: Foundations, Principles, and Techniques[END_REF], [START_REF]Software and Systems Engineering -Reference Model for Software and Systems PL[END_REF], studying the available technology and developing the component interfaces; our goal is to reduce the gap between the abstract RA and the concrete product derivation, to produce in the near future, a semi-automatic product configuration process.

The paper of Dai, He and Xing [START_REF] Dai | Intelligent Information Management[END_REF] proposes a "6+1" structure as a cloud service platform for Human Resource Management; it does not concern SPL, only the HR domain. HR management includes standard management tasks such as wage, attendance (including leave and vacation demands), and personnel file. A rather classic 6-layered structure is proposed for the platform architecture: 1. UI layer to face clients and to collect data; clients choose the services (e.g. salary, vacation request or recruitment management). 2. Data integrity control layer. 3. Process layer for data processing; it is the platform computation core. 4. Data base logic layer, to control the operations of data in data tables. 5. Data table definition layer, and 6. Data Base layer. Our interest is focused on the "+1" layer. In practice work, HR management business is not always normalized, such as organizational structure adjustment, salary system design and psychological counselling to the employees; the laws are not normalized neither and can be applied differently to each entity. These problems usually require relevant experts to solve, based on their experience, implying huge costs. Meanwhile, many experts have characteristic methods and techniques, they need working platforms to provide services. The cloud service platform can help them build their own sub-working platforms and offers technical support. The "+1" structure will implement characteristic HR management service. The Configuration component in our Context external system for the Vacation Request SEDIT subsystem, could fit into this "+1" layer since it will be used by external experts, usually located in third-party enterprises.

The adoption of the cloud technology for HR systems is the subject of Kumar work [START_REF] Kumar | Cloud Technology and HR Management[END_REF], but it does not concern SPL. The National Institute of Standards and Technology (NIST) defined cloud computing as "a model that provides ubiquitous, convenient, on-demand network access to a shared pool of computing resources like servers, networks, storage, applications and services with minimal management effort and service provider interaction". The most updated version of the cloud technology in the field of HR is the Software as a Service (SaaS) technology. This is the most popular form of the technology. The server, in this case, usually provides the entire software to the user through an application, which does not need to be installed or upgraded because the vendor automatically does this onto the cloud. The user has only to upload and manage information stored in the cloud. Neither upgrading nor updating need to be done by the user. In our case, the whole HR SEDIT system of Berger-Levrault seeks to be offered though the cloud as a SPL in the HR domain. The Vacation Request subsystem is an important functionality that is common to many entities covered by SEDIT. A huge configuration effort must be performed to offer the appropriate configuration to the great number of SEDIT clients. A SaaS configuration service should be included, starting to configure the Vacation Request subsystem to face the law's evolution, being this a major problem of HR systems. Other configuration problems could be solved in this way; but this subject is still an on-going work.

IV. CONCLUSION In a previous work [START_REF] Derras | Reference Architecture Design: a practical approach[END_REF] we have designed a RA for a HR SPL. We have now proceeded to link the high-level RA core asset to the lower level application design by adapting the Domain Realization phase of SPLE [START_REF] Böckle | SPL Engineering: Foundations, Principles, and Techniques[END_REF], [START_REF]Software and Systems Engineering -Reference Model for Software and Systems PL[END_REF]. An industrial case study, the Vacation Request subsystem of the SEDIT system of Berger-Levrault illustrates our approach.

The main result of this preliminary work is a new version of the RA including the specification of the component interfaces. In particular, we studied how the non-functional abstract components that were introduced to take in charge qualities, are realized using technological solutions. A problem that still remains, is to define an external configuration component that will be used to satisfy laws evolution, which is a huge problem in HR systems. The perspectives would be to have the SEDIT HR system on the cloud and an external system to take account of law changes using Java injection and design patterns facilities, without modifying the code [START_REF] Douin | La proposition VIP : Variability & Injection Pattern, Internal communication[END_REF].

Fig. 3 .

 3 Fig. 3. The Vacation Request RA with the interfaces of components Guidelines:

TABLE I

 I

		(Suitability -Correctness)	Informix, MySql;
	Communication Layer
	•	<<Network>>	-LAN, WAN
	•	<<Network Security>>	-http, https;
		(Security -Confidentiality -
		Integrity -Authenticity)
	<<Network Reliability>> (Reliability	-It depends on the stability of the
	-Availability)	connection;
	Context External System • <<Implementation>> o Configuration o Customization Law (s) DB	-Spring -Set of Rules	. CORRESPONDENCE BETWEEN ARCHITECTURAL AND TECHNOLOGICAL COMPONENTS Architectural components Solutions realized technological or architectural with components
				Presentation Layer (UI)	-Angular Js (client-side), Flying
				Saucer, RESTful;
				•	User
				o	Check Access Rights	-<< Access Rights Policy>>;
				•	<<Login>> (Security-	-Angular Js (client-side), Flying
				Authenticity)	Saucer, LDAP;
				•	Supervisor
				-Angular Js (client-side), Flying
				o	Check Signature Rights	Saucer, RESTful, D3JS, KSL;
				o	Take decision	-<<Signature Hierarchy>>;
				-Send Notification; Send
				•	Employee	Response;
				-Angular Js (client-side), Flying
				o	Submit Request	Saucer, RESTful;
				o	Receive Response	-Evaluate Case; Send
				•	Administrator	Notification; Send Response;
				-Angular Js (client-side), Flying
				o	Handle administrative	Saucer, RESTful;
				tasks	-Administrative Tasks;
				<<MVC Client Side>>
				(Maintainability -Modularity)	-Angular Js (client-side), Flying
				Saucer, RESTful;
				<<MVC Server-Side>>
				Process Layer
				•	<<MVC Server-Side>>	-Angular Js (server-side), Spring,
				Log4j, SLF4j; <<MVC (Client-
				•	<<Signature Hierarchy>>	Side)>>; -X.Net, Signature Rights DB,
				(Security -Authenticity)	Spring, Log4j, SLF4j, Spring
				•	Evaluate Case	Security, Kerberos Security,; -Spring, Log4j, SLF4j;
				•	Send Notification	-Spring, Log4j, SLF4j;
				• •	Send Response Administrative Tasks	-Spring, Log4j, SLF4j; -e-SEDIT, Spring, Log4j, SLF4j;
				•	<<Compliance with Employees Rights>>	-Employees Rights DB;
				(Suitability -Appropriateness)
				•	<<Compliance with Law(s)>>	-Law (s) DB;
				(Suitability -Appropriateness)
				•	<<Access Rights Policy>>	-Access Rights DB, Spring, Log4j,
				SLF4j, Spring Security, Kerberos
				Security;
				• <<Data Access -Portability -Persistency/Availability >>	-Hibernate, Hibernate 4GWT, Hibernate JPA,
				Hibernate Envers, CAS;
				Data Layer (Data Base)
				•	<<Data Base>>	-Oracle, SqlServer, PostgreSql,
				o	Administrative DB	Informix, MySql;
				o	Employee Rights DB
				o	Access Rights DB
				o	Signature Rights DB
				•	Data Base Schemas	-Oracle, SqlServer, PostgreSql,
		Funded by Berger-Levrault