text:
Angélique Igel-Egalon

Nathalie Cheviron

Mickael Hedde

Guillermina Hernandez-Raquet

Christian Mougin

ISTA 14 -Impact of antibiotics from pig slurry on soil microbial communities, including the basidiomycete Trametes versicolor Running head: Impact of antibiotics from pig slurry on micro-organisms

Keywords: veterinary medicine, pig slurry, enzymes, biomarkers, soil micro-organisms

Livestock slurry containing antibiotics is a source of contamination of agricultural soils, with possible effects on soil function and micro-organisms. Extracellular oxido-reductases and hydrolases from the fungus T. versicolor and fungal growth were monitored in liquid cultures in the presence of tetracycline, lincomycine, sulfadiazine and ciprofloxacin for 10 days, in order to assess the suitability of these enzymes as biomarkers. Among the conditions of treatment, statistical analysis demonstrated an increase in manganese-dependent peroxidase after exposure to sulfadiazine at 1 mg/L when compared to the control. Acid phosphatase activity was decreased by lincomycine at 1 or 10 mg/L. Conversely, -glucosidase activity increased in the presence of this antibiotic at 10 mg/L. In Terrestrial Model Ecosystems spiked with contaminated pig slurry, lincomycine at the concentration of 8 or 80 µg/kg dry soil, and ciprofloxacin at 250 ng/kg dry soil decreased the activity of soil dehydrogenase, when compared to a green slurry treatment, over 28day incubations. Laccase activity was similarly decreased in the presence of the highest concentration of antibiotics. We determined bacterial and fungal biomasses using Q-PCR. Bacterial biomass was increased in the presence of lincomycine at 80 µg/kg whatever the time of exposure, and to a lesser extent in the presence of ciprofloxacin at 250 ng/kg, but only at day 28. In contrast, both antibiotics, whatever their concentrations, did not modify fungal biomass in soil. In conclusion, we were unable to demonstrate important effects of antibiotics at concentrations found in the agricultural environment.

INTRODUCTION

Antibiotics, which are used worldwide for the treatment of microbial infections, and thus as growth promoters, are administered in large amounts to livestock. Most of these veterinary medecines are excreted from in faeces, which are often used for the fertilization of agricultural soils (see the reviews by [START_REF] Kemper | Veterinary antibiotics in the aquatic and terrestrial environment[END_REF][START_REF] Sarmah | A global perspective on the use, sales, exposure pathways, occurrence, fate and effects of veterinary antibiotics (Vas) in the environment[END_REF][START_REF] Thiele-Bruhn | Pharmaceutical antibiotic compounsd in soilsa review[END_REF]. Tetracycline, lincomycine, sulfadiazine and enrofloxacin (the later converted to its metabolite ciprofloxacin, [START_REF] Zhou | Excretion of enrofloxacin in pigs an its effect on ecological environment[END_REF] are compounds frequently used in pig rearing. As a consequence, residues of these chemicals have been discovered in slurry-amended or manured soils in concentrations up to 900,000 ng/kg for tetracycline, 8,500 ng/kg for lincomycine, 1,000 ng/kg for sulfadiazine and 52,000 ng/kg for ciprofloxacin [START_REF] Kemper | Veterinary antibiotics in the aquatic and terrestrial environment[END_REF]. Their persistence in soils often varies mainly from several days to several months. Although natural antibiotics are released by micro-organisms in soils, unwanted ecotoxicological effects due to exogenous chemicals, such as alteration of microbial turnover processes in soils and spreading of antimicrobial resistance genes among environmental bacteria, can be expected [START_REF] Kemper | Veterinary antibiotics in the aquatic and terrestrial environment[END_REF]. It is thus important to understand the effects of pharmaceutical antibiotics on soil micro-organisms, particularly concerning lincomycine and ciprofloxacin, whose fates and effects in soils have been poorly studied to date.

One reason for our lack of knowledge is the death of efficient tools to assess the impacts of chemicals on the functional diversity of micro-organisms in soils. It is thus necessary to understand the molecular basis for the enzymatic secretion and to to develop selective and sensitive biomarkers for the ecotoxicity assessment of soils. Fungal liquid cultures allowing high contaminant bioavailability appear suitable for that purpose. The white-rot basidiomycete Trametes versicolor has been studied for many years for its suitability to produce biomarkers for soil ecotoxicity assessment [START_REF] Mougin | Fungal laccases: from structure-activity studies to environmental applications[END_REF]. For example, its laccases have been shown to be induced after exposure to environmental contaminants such as pesticides and polycyclic aromatic compounds [START_REF] Mougin | Enhanced production of laccases by the fungus Trametes versicolor by the addition of xenobiotics[END_REF]. Lebrun et al. in 2010 demonstrated that the activities of several enzymatic systems of this fungus were affected after exposure to copper.

Secondly, the standard tests for toxicity assessment of antibiotics are often conducted under unrealistic conditions. As integrated systems, Terrestrial Model Ecosystems, where natural parameters (moisture, temperature…) can be controlled in order to reduce the variability during the assays, offer more realistic manure application conditions to assess both the environmental fate and effects of chemical contaminants on different soil communities (Ghanem et al., 1996;[START_REF] Fernandez | Ecotoxicological assessment of doxycycline in aged pig manure using multispecies soil systems[END_REF][START_REF] Boleas | Effects of sulfachlorpyridazine in MS 3-arable land: a multispecies soil system for assessing the environmental fate and effects of veterinary medecines[END_REF].

The goals of our programme are to investigate the impact of manure treatment and storage on its antibiotic content, the fate of antibiotics in slurry-amended and manured soils, and the resulting effects of these medicines on the organisms in the topsoil.

In the present study, our specific objectives are 1) to assess the suitability of fungal biomarkers to monitor the effects of antibiotics in liquid cultures and 2) to assess the ecotoxicity of selected antibiotics on the indigenous microflora in soils amended with pig slurry through functional and structural parameters determined in Terrestrial Model Ecosystems. We provide new information concerning the impact of antibiotics on soil micro-organisms in simulated agricultural situations.

MATERIALS AND METHODS

Chemicals and Reagents

Antibiotics and all other high purity chemicals (>99%): e.g. tetracycline (TET), lincomycine (LIN), sulfadiazine (SDZ) and ciprofloxacin (CIP), were purchased from Sigma-Aldrich-Fluka (France).

Analytical grade solvents were obtained from Carlo-Erba (France).

Soil characteristics

The silt loam was collected in the 10-20 cm layer of an experimental site (La Cage, INRA, Versailles, France). It comprises 21.6% sand, 61.1% silt, and 17.3% clay. Its content in organic carbon is 0.98%, and its pHwat was 7.1. Its water holding capacity (WHC) was 15.64 ± 0.21% at pF=2.5 and 6.79 ± 0.21% at pF=4.2. The soil was roughly homogenized, sieved at 2 mm, and used without storage for the preparation of the Terrestrial Model Ecosystems (TMEs).

Pig slurry sampling and characterisation

The pig slurry, referred to here as green slurry, was from the quarantine livestock building of the IFIP (French Pig Institute) at Romillé (Brittany, France). The animals were cross-bred from Large White and Landrace and weighed 40-100 kg (fattening pigs). They were fed an unchanging cereal formula consisting of wheat (44%), barley (30%), soybeans (18%), molasses (2%) and amino acids and minerals (6%). They did not receive antibiotics.

The pig slurry resulting from about three months of manure accumulation from 20 pigs was homogenized in the pit (about 25 m 3 ) using a submersible pump for about 20 minutes. Then 20 litres of slurry were collected and stored at 4°C for one day until use.

The slurry pH (7.5), dry matter content (26.2 +/-0.1 g/kg), Chemical Oxygen Demand (36 +/-2 gO2/L), Total Kjeldahl Nitrogen (3.05 +/-0.03 g/kg) and Total Ammonia-N (2.3 +/-0.1 g/kg) were determined according to standard methods [START_REF] Apha | Standard Method for the examination of Water and Wastewater, 18[END_REF]. All parameters were analysed in triplicate. These data are in agreement with previous reports on pig slurry characterisation in French husbandry [START_REF] Burton | Manure Management, Treatment Strategies for Sustainable Agriculture[END_REF].

Micro-organism and culture conditions

Trametes versicolor ATCC 32745 was grown on the culture medium described by [START_REF] Abadulla | Decolorization and Detoxification of Textile Dyes with a Laccase from Trametes hirsute[END_REF], containing glucose (10 g/L) and NH4CL (2.5 g/L) as carbon and nitrogen sources. The medium also contained 0.5 g/L MgSO4 7H2O, 5 g/L yeast extract, 0.1 g/L CaCl2 and 0.5 g/L KCl.

FeSO4, ZnSO4, MnSO4 and CuSO4 were added to achieve the final concentration of 0.01 g/L. A mycelial mat on agar plug (10 mm diam.) was inoculated into 10 mL of the culture medium in each 150 mL Erlenmeyer flask. Cultivation was carried out statically in the dark at 25°C for 10 days.

Solutions of antibiotics in water (TET, LIN), diméthylformamide (SDZ) and dimethylsulfoxide (CIP) were added to the cultures to give 0.1, 1 and 10 mg/L (100 µL solution per Erlenmeyer flask).

Biomass and enzymatic determinations were achieved at the beginning of the incubations, then after 3, 5, 7 and 10 days. Each experiment was performed in triplicate.

Set up of Terrestrial Model Ecosystems

Three days before the beginning of the experiments, each Terrestrial Model Ecosystem (TME) was filled with 1 kg dry soil moistened to 50% of its WHC. The soil was then amended with 25 mL pig slurry to simulate agronomic conditions (equivalent to 30 T dry manure/ha). Slurry was spiked or not with antibiotics. CIP was added to the final concentrations of 1 and 10 µg/L in the slurry, 25 and 250 ng/kg in dry soil. LIN was applied in final concentrations of 320 and 3200 µg/L in slurry, equivalent to 8 and 80 µg/kg in dry soil. After mixing, the amended soil was set in the TMEs, a litter-bag (7 x 7 cm, 2 g dry wheat straw) was inserted at mid-height of the soil, and water was added to achieve 100% WHC. TMEs were incubated in the dark at 18 ± 1°C. Biomass and enzyme activities were measured at the beginning of incubation, then after 7, 14 and 28 days. After 28 days, the bags were removed, washed, dried and the straw was weighted. Each experiment was performed in triplicate.

Enzyme activity measurements

Laccase (LAC; E.C. 1.10.3.2) production was assessed in liquid cultures of T. versicolor by measurement of enzymatic oxidation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) at 420 nm ( = 3.6 x 10 4 cm -1 M -1 ) according to [START_REF] Wolfenden | Radical cation as reference chromogens in kinetic studies of one electron transfer reactions[END_REF]. The reaction mixture contained 20 µL extracellular fluid and 980 µL 1 mM ABTS in 0.1 M KH2PO4/citric acid buffer (pH 3.0) at 30°C. The buffer solution was saturated with air by bubbling prior to the experiment. Manganese-dependent peroxidase (MNP; E.C. 1.11.1.14) activity was monitored by the oxidation of 20 µL 5 mM 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one at 334 nm, 200 µL extracellular fluid, 20 µL 5 mM MnSO4, 20 μL 16mM hydrogen peroxide in 740 µL 0.1 M KH2PO4/citric acid buffer (pH 5.0), as described by [START_REF] Paszczynski | Comparison of ligninase-I and peroxidase-M2 from the white-rot fungus Phanerochaete chrysosporium[END_REF]. One unit of enzyme activity is defined as the amount of enzyme that oxidises one µmol ABTS per min.

In TMEs, we used a protocol adapted from [START_REF] Floch | ABTS assay of phenol oxidase activity in soil[END_REF] to determine laccase activity. The reaction mixture contained 1mL soil solution (obtained from 1 g soil in 6 mL KH2PO4/citric acid buffer pH 3.0), and 600 μl of a 50 mM guaiacol solution. After incubation at 37 °C for 20 min, the mixture was centrifuged at 12000 rpm at 4 °C for 2 min. The formation of tetrameric guaiacol in the supernatant was measured at 470 nm.

Measurement of dehydrogenase activity (DHA; E.C. 1.1.1.1), indicating the level of global metabolic process in the soil, was adapted from [START_REF] Schaefer | L'activité déshydrogénasique comme mesure de l'activité biologique globale des sols[END_REF]. 1 mL distilled water and 250 µL aqueous solution of 1,3,5-triphenyltetrazolium chloride (TTC) at 40 g/L were mixed with 1 g fresh soil in 30-mL centrifuge tubes. Controls were prepared without TTC. Incubation was performed at 37°C for 16 hours. After that period, 5 mL ethanol was added to the mixture which was shaken for 5 min in the dark. The medium was incubated at 37°C for 2 hours, and shaked after one hour.

Finally, TTC was added to the control and all the mixtures were centrifuged 5 min at 6000 g. The absorbance of the supernatant (red colour in case of formazan formed) was measured at 485 nm.

Results were expressed in mU per g dry soil.

Acid phosphatase (ACP; E.C. 3.1.3.2, -glucosidase (GLU; E.C. 3.2.1.21) activities were measured according to a method adapted from [START_REF] Dick | Soil enzyme activities as integrative indicators of soil health[END_REF]. 16 g soil were shaken in 100 mL distilled water.

Then, 125 µL of soil suspension were incubated with 25 µL 0.05 M substrate (4-nitrophenyl phosphate for the phosphatase or 4-nitrophenyl ß-D-galactopyranoside for the glucosidase) at 37°C

for one hour in multiwell plates. For each soil sample, a control without substrate was performed.

At the end of the incubation, 25 µL of CaCl2 (0.5 M) were added in order to stabilize humic acid, and 100 µL Tris 0,1 M pH12 were used to stop the reaction. The amount of p-nitrophenol (PNP) formed was obtained by the measurement of the supernatant absorbance at 405 nm. Results were expressed in mU per g dry soil.

Biomass determination

Biomass was determined from soil genomic DNA. Total genomic DNA was extracted from 0.5g of frozen soil using the Fast DNA kit for soil (MP Biomedical) and suspended in 100 µL of sterile deionised water. rDNA was then amplified in 20 µL final qPCR mixture containing the following total amounts: 10 ng of total soil DNA, 25 pmoles of each primer, 10 µL of iq-SYBR®Green super Mix (Biorad). For bacterial 16S-rDNA, the forward primer used was S-BCT 5'-CAG GCC TAA CAC ATG CAA GTC-3' (primer 63f from [START_REF] Marchesi | Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA[END_REF] and the reversed primer was AS-BCT1 5'-CTG CTG CCT CCC GTA GG-3' (primer 341F from [START_REF] Muyzer | Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA[END_REF]. For fungal 18S-rDNA, the forward primer was S-FUN1 5'-GGA AAC TCA CCA GGT CCA GA-3' (primer nu-SSU-1196 from [START_REF] Borneman | PCR Primers that Amplify Fungal rRNA Genes from Environmental Samples[END_REF] and the reversed primer was AS-FUN1 5'-ATT GCA ATG CYC TAT CCC CA-3' (primer nu-SSU-1536 from [START_REF] Borneman | PCR Primers that Amplify Fungal rRNA Genes from Environmental Samples[END_REF]. All cycles were programmed on Chom4 (Biorad) to perform 35 cycles consisting of: 95°C for 40s, 64°C for 45s and 72°C for 30s with bacterial rDNA and 95°C for 20s, 62°C for 30s and 72°C for 30s with fungal rDNA. Each amplification was followed by a final extension step of 10 minutes at 72°C.

Data analysis

Prior to statistical analyses, Shapiro and Bartlett tests were used to verify the normality and the homoscedasticity of the data, respectively. If both hypotheses were not refuted, ANOVAs and Tukey significant differences test were performed. In all other cases, equivalent non-parametric tests were preferred (Kruskall-Wallis and Kolmogorov-Smirnov tests, respectively).

For liquid culture results, two-way ANOVA was used to test the effect of "exposure time" and "antibiotic concentration" factors on fungi biomass and enzyme activities. Exposure times were 0, 3, 5, 7 and 10 days. The concentration levels were 0 (i.e. the corresponding control), 0.1, 1 and 10 mg/L for all antibiotics. When ANOVA was significant, Tukey HSD tests for mean comparison were realized. Similar analyses were made for TMEs with "exposure time" (0, 7, 14 and 28 days) and the "antibiotic concentration" as the independent variables. The spread concentrations were 0 (control), 8 and 80 µg/kg dry soil for lincomycine and 0 (control), 25 and 250 ng/kg dry soil for ciprofloxacine. All tests were realized with R software (R core development team, 2009) at the rejection level α = 0.05.

RESULTS AND DISCUSSION

Impact of antibiotics on T. versicolor cultured in liquid conditions

The effects of incubation time and antibiotic concentration on T. versicolor cultured in liquid conditions have been assessed on fungal growth and activities of extracellular oxido-reductases (laccase, LAC; Mn-dependant peroxidase, MNP) and hydrolases (-glucosidase, GLU; acid phosphatase, ACP). Levels of medium spiking (0.1, 1 and 10 mg/L) were chosen to cover a range of values previously reported to modify fungal protein activity. Considering overall effects, most of the variance in data was explained by exposure time (Table 1). Bell-shape curves were observed, with maxima noticed after 5 days for fungal biomass, and after 7 days for extracellular hydrolases.

In contrast, LAC activity presented two peaks, after 3 and 10 days of exposure, whereas MNP activity increased during the course of incubation with maxima noticed after 10 days. These results are consistent with the previous study of [START_REF] Lebrun | Development phase-depending response of extracellular enzymatic system to copper stress in Trametes versicolor[END_REF].

On the other hand, few statistical differences appeared when regarding the effect of antibiotic concentration on the enzymatic activities used as biomarkers. TET and CIP from 0.1 to 10 mg/L did not exhibit any impact on the selected activities in our experimental conditions. It remains noteworthy that the well known inducer xylidine [START_REF] Kollmann | Oligomeric compounds formed from 2,5-xylidine (2,5-dimethylaniline) are potent inducers of fungal laccases[END_REF], used as a positive control in the present experiments, led to a 13-fold increase of LAC after 3 days of fungal exposure. SDZ presented an effect only on MNP, with higher activities in 1 mg/L concentration treatment than in other cases (Figure 1). LIN significantly influenced both ACP and GLU. The activity of ACP was decreased when the antibiotic was brought at 1 or 10 mg/L (e.g. -34% at the peak of activity, p < 0.05)(Figure 2A). Conversely, GLU activity increased in the presence of LIN at 10 mg/L when compared to the control (+10%, p < 0.05)(Figure 2B).

Our knowledge of the possible effects of antibiotics on filamentous environmental fungi is scarce.

Because of their molecular mode of action, it was unlikely that the antibiotics in the present study exert direct effects on T. versicolor. Nevertheless, [START_REF] Dhawan | Effect of antibiotics on growth and laccase production from Cyathus bulleri and Pycnoporus cinnabarinus[END_REF] demonstrated that TET at 200 mg/L inhibited fungal growth and increased laccase production from Cyathus bulleri and Pycnoporus cinnabarinus. Because environmental contaminants (PAHs, pesticides, [START_REF] Mougin | Fungal laccases: from structure-activity studies to environmental applications[END_REF]xylidine, Kollmann et al., 2005) modify the secretion of enzymes from T. versicolor, they were intended to be used as potential biomarkers for ecotoxicity assessment. Our results show that fungal oxido-reductases and hydrolases do not behave as relevant biomarkers of exposure to antibiotics. That poor sensitivity could be due to the ability of white rot basidiomycetes to secrete extracellular lignin-degrading enzymes efficiently degrading antibiotics, as reported for TET [START_REF] Wen | Degradation of tetracycline and oxytetracycline by crude lignin peroxidase prepared from Phanerochaete chrysosporium -a white rot fungus[END_REF].

Impact of antibiotics on soil functional diversity in Terrestrial Model Ecosystems

LIN and CIP are two antibiotics widely quantified in French slurry or manure samples (our programme, but also data reported by [START_REF] Kuchta | Fate of lincomycin in snowmelt runoff from manure-amended pasture[END_REF][START_REF] Zhou | Excretion of enrofloxacin in pigs an its effect on ecological environment[END_REF]. Our knowledge of the effects of these chemicals on soils organisms is scarce. We observed a slight effect of LIN on the ACP and GLU activities of T. versicolor. CIP was recently described to affect bacterial communities in soils [START_REF] Zhou | Excretion of enrofloxacin in pigs an its effect on ecological environment[END_REF]. Here we studied their effects in TMEs filled with a silt loamy soil supplemented by green or spiked slurry samples, each applied at 30T/ha. Final concentrations used in our study, of agricultural relevance, were 8 and 80 µg/kg dry soil for LIN, 25 and 250 ng/kg dry soil for CIP. Modification of soil functional biodiversity was assessed by enzyme activity measurements (LAC, DHA, GLU, ACP).

Among enzymatic activities, two patterns of response appeared, according to the type of enzyme, which were shared by the two antibiotics. Hydrolase enzymes merely responded to the time of incubation, as shown by the evolution of activities of ACP and GLU in our TMEs. For instance, in the presence of LIN, GLU activity was influenced by the time exposure (F3,32 = 25.7, p<0.001 and F3,32 = 19.1, p<0.001) but not by antibiotic concentration (F3,32 = 2.8 ns and F3,32 = 0.8 ns ) (Figure 3).

In contrast, oxydo-reductases were influenced by antibiotic concentration rather than time of incubation. The activity of DHA was constant during the course of the experiment with green slurry. It was affected by the presence of each of the antibiotics. Spreading LIN-containing slurry decreased the activity of DHA, either at 8 or 80 µg/kg, in comparison with green slurry alone (Figure 4A). For instance, after 7 days of incubation, DHA activity was half lower (-54%) in the LIN-80 µg/kg treatment, than in green slurry one. Regarding CIP effects, only treatment spread at the highest concentrations induced a significant decrease in DHA activity (Figure 4B). For instance, after 7 days, DHA activity in CIP-250 ng/kg treatment was decreased by 45% when compared to green slurry treatment.

LAC activity was similarly decreased in the presence of the highest concentration of antibiotics (data not shown). For example, in the LIN-80 µg/kg treatment, activities after 7 and 14 days were 34% and 23% lower, respectively, than in the control. In the CIP-250 ng/kg treatment, decreases were comparable for the same exposure times (-25% and -40% respectively). Nevertheless, after 28 days no differences were observed between treatments.

Whatever the incubation conditions, 18-24% of the initial amounts of straw were degraded in the litter-bag assays, without a significant effect of the antibiotics (data not schown).

Data concerning the effects of LIN and CIP on soil enzyme activities are lacking. Our results contrast with the study of [START_REF] Zhou | Excretion of enrofloxacin in pigs an its effect on ecological environment[END_REF], who reported no impact of enrofloxacin and its metabolite CIP on populations of soil micro-organisms at concentrations below 0.2 mg/kg. It should be noted that the effects of antibiotics in soils are influenced by their bioavailability for microorganisms.

Impact of antibiotics on soil microbial biomass in Terrestrial Model Ecosystems

In our experiments, bacterial and fungal biomasses were specifically determined using Q-PCR on DNA from soil samples. Bacterial biomass was affected by spreading with antibiotic spiked-slurry.

It was increased in the presence of LIN at 80 µg/kg whatever the time of exposure (Figure 5A), and to a lesser extent in the presence of CIP at 250 ng/kg, but only at day 28 (Figure 5B). In contrast, LIN and CIP, whatever their concentrations, did not modify fungal biomass in soil TMEs. No data are available to date concerning the effects of LIN on microbial communities in agricultural soils.

However, recent laboratory experiments demonstrated contrasted shifts in bacterial diversity according to pH in forest soils [START_REF] Cermak | Bacterial communities of two contrasting soils reacted differently to lincomycine treatment[END_REF]. Only one study [START_REF] Zhou | Excretion of enrofloxacin in pigs an its effect on ecological environment[END_REF] reported an impact of enrofloxacin and its metabolite CIP on the edaphon community, leading to the promotion of bacterial growth utilizing the fluoroquinolones as a nutrient source. Antibiotics (e.g. sulfonamide, SDZ and tetracycline compounds) increased the fungal:bacterial ratio, although in higher concentrations within the soil [START_REF] Hammesfahr | Impact of the antibiotic sulfadiazine and pig manure on the microbial community structure in agricultural soils[END_REF][START_REF] Thiele-Bruhn | Effects of sulphonamide and tetracycline antibiotics on soil microbial activity and microbial biomass[END_REF].

Table 1. Anova results (F values) on the effect of the factors "antibiotic concentration" (0, 0.1, 1 and 10 mg L -1 ), "exposure time" (0, 3, 5, 7 and 10 days) and their interaction on the response of 5 extracellular enzymatic activities of T. versicolor in liquid conditions. TET: tetracycline, LIN: lincomycine, SDZ: sulfadiazine, CIP: ciprofloxacin, ACP: acid phosphatase, GLU: -glucosidase, LIP: lignin peroxydase, MNP: manganese peroxydase, LAC: laccase. * p < 0.05; ** p< 0.01; *** p< 0.001.

Fig 1 .

 1 Fig 1. Effect of sulfadiazine (SDZ) at various concentrations (mg/L) on Mn-dependant peroxidase (MNP) in liquid cultures of T. versicolor. Results are expressed as means ± standard errors.

Figure 4 :

 4 Figure 4: Evolution of dehydrogenase activity (DHA) in TMEs filled with green or slurry spiked with (A) lincomycine (LIN) at two concentrations (µg/kg) or (B) ciprofloxacin (CIP) at two concentrations (ng/kg). Results are expressed as means ± standard errors.

Figure 5 :

 5 Figure 5: Evolution of bacterial biomass in TMEs filled with green or slurry spiked with (A) lincomycine (LIN) at two concentrations (µg/kg) or (B) ciprofloxacin (CIP) at two concentrations (ng/kg). Results are expressed as means ± standard errors.

Acknowledgements: The present study, as a part of the research programme DIPERPHA, was supported by the French ANR programme SEST and by ADEME. We are grateful to Dr. Patrick Dabert (GERE unit, CEMAGREF, Rennes, France) for providing and analysing the samples of green pig slurry. The authors are also indebted to Dr. David Tepfer (PESSAC INRA) for proofreading the manuscript, and to Agathe Brault, Sébastien Breuil and Isabelle Touton for their technical assistance.


Chenghui Shi

Isabelle Correia

Nicolo Tonali

Sandrine Ongeri

Olivier Lequin

Two consecutive aza-amino acids in peptides promote stable β-turn formation in water

come

Two consecutive aza-amino acids in peptides promote stable β-turn formation in water

Introduction

Currently, more than 80 peptides have been approved as drugs for the treatment of various diseases, showing that peptide drugs have gained astounding interest in pharmaceutical industry. [1][2][3] As one type of drug entities, peptides have medium molecular weights between small molecules and antibodies as well as unique pharmacological properties including high affinity, high selectivity and low immunogenicity. However, the lower proteolytic stability, membrane permeability and oral bioavailability of natural peptides still prevent their rapid development in drug discovery despite notable recent advances in peptide delivery technology. 1 Moreover, small peptides are rather flexible and only rarely adopt stable conformations, which may preclude their ability to interact with protein targets or to inhibit protein--protein interactions (PPIs), which involve generally well--defined secondary structures in the hot--spot sequences of interaction. Modified peptides and peptidomimetics represent an alternative to avoid these limitations of peptides. 4 The field of peptide--based foldamers adopting stable conformations able to mimic secondary structures represent also recent promising therapeutic opportunities in particular to target PPIs.

5,6

Azapeptides represent one type of peptidomimetics in which one or more of the residues are substituted by semicarbazide. It has been proven that azapeptides usually show higher selectivity and proteolytic stability than that of parent peptides. [7][8][9] Given the repulsion between adjacent nitrogen lone pairs and the planar structure of the urea moiety, the preferential dihedral angle values of aza--amino acid residue (ɸ, ψ) are close to (±90±30°, 0±30°) or (±90±30°, 180±30°). 10,11 This narrow range of dihedral angle values explains why peptides containing a single aza--amino acid adopt a β--turn structure. 12-17 It has also been demonstrated that this non--extended structure can disrupt the β--hairpin conformation of peptides even containing a D--Pro--Gly template in their sequence. 18 The few reported computational and experimental studies of peptides (mainly di and tripeptides) containing one aza--amino acid residue have shown that this class usually adopts a type II β--turn conformation in solid--state or in organic solvent (CDCl 3 or DMSO). 15,16,19,20 However, only non-classical azapeptides encompassing a N--amidothiourea moiety adopt a type II β--turn structure in water thanks to the enhanced H bond donor ability of the thioureido NH group. 21 To our knowledge, only one statement, made by us, on the more challenging synthesis and conformation study of azapeptides containing two sequential aza--amino acids has been reported so far. 22,23 In this conformational study, we observed that diaza--tripeptide 1 containing two aza-amino acids at its N--terminus (aza/aza/α pseudotripeptide, Fig. 1) is prone to adopt a single type I β--turn or two consecutive type I β-turn conformations in methanol, 23 whereas the corresponding natural tripeptide shows highly disordered conformation. We exploited this property to synthesize the aza/aza/α/aza/aza/α pseudohexapeptide 2 composed of two diaza--tripeptide units (Fig. 1) which was shown to adopt highly folded conformations made of repeated β--turn conformations or even a full--length 3 10 --helix structure. This suggests that the repeat mode aza/aza/α in longer peptide sequences has the propensity to induce helical structuration in peptides. Notably, we found that the insertion of side chains on both aza--amino acids had a beneficial effect to stabilize the turn conformation of the diaza--tripeptides. Indeed, diaza--tripeptide 3 with two azaGly at its N--terminus (Fig. 1) showed higher conformational flexibility by NMR and MM/DFT calculations than diaza--tripeptide 1 bearing azaVal--azaAla, oscillating from a few helical populations to extended structures. On the basis of these findings, we have the aim to use these diaza--peptide units in peptidomimetic foldamers targeting protein--protein interactions involving turn or helical secondary structures. For that purpose, we had to further investigate two new factors: (1) the ability of these units to retain their turn propensity in water, which is an even more challenging solvent than methanol for maintaining intramolecular hydrogen bonding and stable conformations, because solubility in water is mandatory for biological evaluations; and (2) the influence of the nature of the side chain of the aza--amino acid residues on the conformation.

Because of the low aqueous solubility of the diaza--tripeptides 1 and 2 mentioned above, their NMR analysis in water was not possible. Herein we propose to introduce more hydrophilic aza--and natural amino acids in order to improve the aqueous solubility of the target diaza--tripeptides, thereby making possible their NMR analysis in water. Thus, azaLys was used instead of azaVal at the N-terminus and, the natural amino acid Val was replaced by Ser at the C--terminus. The N--Boc protection was replaced by an acetyl group to further decrease the hydrophobic character and better mimic a neighboring amino acid residue. Meanwhile, we used three different aza--amino acids, including azaGly, azaAla and azaLeu as the central residue in diaza--tripeptides 4, 5, and 6 respectively, in order to investigate the influence of the presence or not, and the nature of the side chain on the conformation of diaza--tripeptides.

Fig. 1 Structure of the previously reported aza/aza/α pseudotripeptides and aza/aza/α/aza/aza/α pseudohexapeptide 1--3 23 and of the newly synthesized aza/aza/α pseudotripeptides 4--6. Aza--amino acids are designated with the "a" letter preceding the three--letter code.

Results and discussion

For the synthesis of peptide 4 without side chain on the central aza-amino acid, we used the same procedure described in our previous work (Scheme 1).

22

Compound 7 was activated by 4--nitrophenyl chloroformate to give compound 8 (89% yield), which reacted with hydrazine monohydrate to give compound 9 with a yield of 68%. 9 was activated by phenyl chloroformate to give compound 10 (98% yield), which was coupled with Ser(Bzl)--NH 2 (synthesis described in ESI) to provide compound 11 in a satisfactory yield of 71%. The Boc group of 11 was removed using a solution of HCl in dioxane. The free amine was immediately acetylated using acetic anhydride in the presence of pyridine as base to afford compound 12 in a good yield of 75%. Finally, both Bzl and Cbz groups were simultaneously removed from 12 by catalytic hydrogenation using Pd/C as catalyst to get compound 4 with a yield of 64%.

As reported in our previous work, 22 the active carbamate of protected 2--alkyl--hydrazines could not couple with protected 1-alkyl--hydrazines but the active carbamate of protected 1--alkyl-hydrazine is able to react with protected 2--alkyl--hydrazine, so we changed the strategy to synthesize compounds 5 and 6 which have side chains on their central aza--amino acid (Scheme 2). First, the activation of the protected 1--alkyl--hydrazines 13a and 13b using 4--nitrophenyl chloroformate afforded compounds 14a and 14b (76% and 59% yields respectively), which reacted with 7 to give diaza-peptide moieties 15a and 15b with a good yield of 79%. Fmoc groups were cleaved from 15a and 15b using 20% piperidine/DMF to provide 16a and 16b in satisfactory yields (91%). Given the bulkiness of alkyl groups on the nitrogen which must be activated, active carbamates of 16a and 16b were less active to react with Ser(Bzl)--NH 2 . Here, triphosgene was used to activate 16a and 16b to provide more active acyl chloride intermediates, followed by the addition of Ser(Bzl)--NH 2 to get tripeptides 17a and 17b in 46% and 54% yields respectively. Compounds 18a and 18b were then obtained by the replacement of the Boc group by an acetyl group using the same operating protocol as for the synthesis of 12 (75% and 76% yields respectively). Finally, the target diaza-tripeptides 5 and 6 were afforded by the simultaneous cleavage of the Bzl and Cbz groups using catalytic hydrogenation (58% and 59% yields respectively). As expected, the three diaza--tripeptides 4--6 showed good solubility in aqueous solution (greater than 10 mM). We thus investigated their structures in water and compared them when in methanol solvent (Fig. 2), serving as a reference to our previous published work. 23 Different NMR parameters were analysed to assess the hydrogen bonding and folding propensities of diaza-tripeptides, in particular the temperature dependency of the amide proton chemical shift (temperature coefficient Δδ HN /ΔT) and the through--space dipolar 1 H--1 H ROE correlations. Diaza--tripeptide Ac--aLys--aGly--Ser--NH 2 (4) shows evidence of intramolecular hydrogen bond formation in both methanol and water solvents. Indeed, the Ser amide proton chemical shift exhibits a low temperature dependency in comparison with other NH protons, the temperature coefficient being -1.4 ppb/K and -3.4 ppb/K in methanol and water, respectively (Table 1). These coefficients are well below the threshold value of -4.6 ppb/K, which is usually an indicator to consider hydrogen bond engagement. 24 In addition, a ROE correlation is observed in both solvents between the amide proton of Ser and the methyl protons of the N--terminal acetyl group (Fig. 3, S3, S7), supporting the formation of a β--turn folded structure, stabilized by a i, i+3 hydrogen bond between the acetyl CO group and the Ser NH group. NMR structure calculations show that two turn conformations are compatible with the NMR data. Indeed, the diaza--peptide block can adopt either negative or positive ɸ angle values around ± 90°, leading to type I or I' β--turns, respectively (Fig. 4A). Such conformational equilibrium between these two β turn types had been previously described for diaza-tripeptide 1 in methanol, 23 and was found to occur in a slow exchange regime on the NMR time scale. Given that a single set of chemical shifts is observed for diaza--tripeptide 4 in both solvents at room temperature (Fig. 2), it is likely that the absence of side chain in the azaGly residue enables faster rotation around the N-Nα bond, leading to a fast exchange regime on the NMR time scale at room temperature. The appearance of slowly interconverting conformational isomers can only be detected for diaza--tripeptide 4 when cooling down to very low temperatures (228 K) in methanol (Fig. S5).

Additional i, i+2 ROEs were also observed between the Ser amide proton and the side chain methylenic protons (CH 2 γ, δ, ε) of azaLys (Fig. 3). These correlations can be accounted for a compact conformation in which azaLys side chain folds back onto Ser residue. The folding of aLys side chain is further suggested by the observation of a significant chemical shift difference between the two diastereotopic CH 2 ε protons in methanol (0.08 ppm difference), which is usually not the case when a Lys side chain is fully exposed to solvent. Structure calculations indicate that this compact orientation could be stabilized by a hydrogen bond between the ammonium group of azaLys and either the hydroxyl or carbonyl group of Ser (Fig. 4A).

The NMR spectra of diaza--tripeptides Ac--aLys--aAla--Ser--NH 2 (5) and Ac--aLys--aLeu--Ser--NH 2 (6) displayed much higher complexity than diaza--tripeptide 4, owing to the presence of chemical shift heterogeneity. As a matter of fact, different sets of resonances could be observed for the residues of 5 and 6, in water and in methanol (Fig. 2), a situation reminiscent of that observed for diaza-tripeptide 1 in methanol. The NMR spectra of the previously studied diaza--tripeptide 1 in methanol showed two major conformational isomers at room temperature (~45% population) which were ascribed to sign inversion of the ɸ dihedral angle, as aforementioned. Two other minor isomers (~5% population) were ascribed to N--terminal Boc cis/trans isomerization. The situation differs in diaza--tripeptides 5 and 6, in so far as four forms with closer populations could be evidenced. This prompted us to run temperature variation experiments to further investigate the origin of the chemical shift heterogeneity. Experiments in water at low temperature show that the different forms have close chemical shifts. The coalescence of proton signals arises around 298 K for diaza--tripeptide 5 (Fig. S12) and above 308 K for diaza--tripeptide 6 (Fig. S20). Experiments in methanol enabled us to work at much lower temperatures, cooling down to 228 K. At this temperature, proton spectra exhibited much sharper and resolved peaks, providing better discrimination of the 4 forms. The peak integrations on 1D 1 H spectra yielded populations of 42%, 27%, 19% and 12% for diaza--peptide 5 (Fig. S13) and 55%, 22%, 17% and 6% for diaza--peptide 6 (Fig. S21). Exchange cross--peaks were observed on 2D ROESY spectra between the different forms (Fig. S15 and S23), unambiguously proving that the four forms correspond to conformational isomers that interconvert during the mixing time of 2D experiments (250 ms). The chemical shifts of NH protons show linear temperature dependency over the 228-251 K temperature range and the amide protons of Ser residues in the 4 different isomers have the lowest temperature coefficients, between -2.2 and -3.3 ppb/K, supporting their engagement in intramolecular hydrogen bonds (Table 1). The temperature coefficients where much more difficult to measure in water owing to spectral overlap. Partial data could be obtained either after peak coalescence or for isolated HN peaks before coalescence. They indicate that Ser HN proton has the smallest temperature dependency in comparison with other HN protons.

The analysis of ROESY spectra in methanol at low temperature (Fig. S15 andS23) shows diagnostic ROEs of β--turn folding, in particular the characteristic i, i+3 correlation between Ser HN proton and acetyl protons. Strong sequential i, i+1 HN--HN correlations also indicate that the aza--amino acids adopt (ɸ, ψ) angles values around (±90°, 0°). Finally, ROEs are observed between Ser HN proton and aLys methylenic side chain protons, as in diaza-tripeptide 4. All these ROEs are also observed at higher temperature, in both methanol and water (Fig. 3, S11 and S19), where partial coalescence between the different forms occurred. Intriguingly, it was not possible to detect distinctive ROEs between the different forms at low temperature that would provide clues about the nature of the conformational isomerism affecting the different conformers. Importantly, exchange peaks are observed even at the lowest temperature of study in methanol. Therefore, magnetization transfers due to conformational exchange induce some contamination of the through--space dipolar ROE correlations, which may complicate the discrimination of distinctive conformational features. Of note, the sign inversion of ɸ angle between +90 and -90° in the aza--amino acids yields mirror--imaged backbone conformations within the diaza--amino acid unit and consequently gives rise to similar sets of ROEs.

Structure calculations based on NMR restraints show actually two possible turn conformations for diaza--tripeptides 5 and 6 (Figures 4B,4C) corresponding to types I and I', as for diaza-tripeptides 1 23 and 4 (Figure 4A). However, we had previously shown for diaza--tripeptide 1 that a second 10--membered β--turn could be formed if the first turn adopts a type I conformation. This second turn was stabilized by a hydrogen bond involving the C-terminal primary amide group and the carbonyl group of the first aza--amino acid residue. Although this double turn conformation is compatible with the observed ROEs between Ser HN and carboxamide protons, the average temperature coefficient of carboxamide protons in the three diaza--tripeptides 4--6 suggest that this hydrogen bond is not as stable as the acetyl--Ser H--bond.

An unusual N amide ⋯ H--N amide hydrogen bond has been proposed to stabilize the backbone conformation of aza--amino acids with (ɸ, ψ) angles close to (±90±30°, 0±30°). 25,26 In the diaza--tripeptide β-turns, two N amide ⋯ H--N amide hydrogen bonds are formed between consecutive residues (N1⋯ H--N2 and N2⋯ H--N3) which could further contribute to the β--turn stabilization. It should be noted that these atypical hydrogen bonds do not seem to have a strong effect on amide proton temperature coefficients in methanol and water, in comparison with conventional C=O ⋯ H--N hydrogen bonds. This can be shown by comparing the temperature coefficients of HN2 and HN3 in the diaza--tripeptides 1 23 and 4--6 (Table 1).

The particular feature of the studied diaza--tripeptides 5 and 6 is the presence of conformational isomerism that severely complicates NMR analyses, whatever the solvent. We show that the presence and the nature of the aza--amino acid side chain have an effect on the conformational exchange. Diaza--tripeptide 4 displays fast exchange at room temperature while diaza--tripeptides 5 and 6 bearing a side chain on the second aza--amino acid exhibit slow exchange under the same conditions. The bulkiness of the substituent (isobutyl vs methyl) also slows down the kinetics of interconversion, as indicated by differences in coalescence temperatures. One source of postulated conformational isomerism is the slow rotation around the N-Nα bond in substituted aza-amino acids (ɸ = ±90°), yielding 4 possible conformational isomers. Diaza--peptide units having opposite signs of their ɸ angles are not compatible with β--turn formation. As all conformational isomers show H--bonded β--turn features, this implies that the sign inversion of ɸ angle concerns both aza--amino acids simultaneously, yielding only two conformational isomers, i.e. (ɸ 1 ,ɸ 2 ) = (+90°,+90°) or (-90°,-90°). Another possibility of conformational exchange would be E/Z isomerism around amide bonds or Nα--C' bond (ψ angle). Indeed, we had previously suggested in the case of compound 1 that E/Z interconversion of Boc carbamate groups could be a source of conformational isomerism. In the case of N--terminal acetyl groups preceding aza--amino acids, cis--trans isomerism has also been reported for the Ac--azaGly amide bond. 25 Concerning the rotation around the Nα--C' bond, sequential HN/HN ROEs are only compatible with conformations having ψ angles around 0°. Finally, the Nα atom in aza--amino acids has a small pyramidal character, offering the possibility to dynamically orient the side chain in two opposite configurations. Lubell et al. have reported the adaptive chirality of Nα center in aza--amino acids that could be achiral or exhibit either L or D--like chirality. 27 The X--ray structure and DFT analyses of diaza--tripeptide 1 revealed that the pyramidal character of the Nα atom is very weak.

23

Of note, a slow inversion of Nα chiral center has not been observed in peptides incorporating one aza-amino acid unit by NMR analyses. 25,27,28 Nevertheless, it is possible that the situation may differ in peptides incorporating two consecutive aza--amino acids and which are conformationally stabilized.

Conclusion

This work confirms our previous finding that unlike peptides containing exclusively α--amino acids, the conformational restriction conferred by the insertion of two consecutive aza--amino acids in aza/aza/α pseudotripeptides endows them with a strong tendency to adopt 10--membered type I β--turn conformations. The introduction of polar lateral chains on the N--terminus aza--amino acid (azaLys) and on the natural amino acid at the C--terminus (Ser), as well as the replacement of the N--Boc by an acetyl group dramatically increased the solubility in water of compounds 4--6 compared to compound 1. Although the NMR conformational studies of azapeptides 4--6 were hampered by conformational exchange, they reveal striking similarities of the NMR spectra recorded in water and methanol conditions, in terms of HN chemical shifts, HN temperature coefficients, and sets of ROE correlations. Therefore, the conformational space of diaza-tripeptides is largely comparable in both solvents, indicating that stable turn conformations are populated even in the more challenging aqueous solvent. Diaza--tripeptides 4--6 have a strong propensity to adopt a 10--membered β--turn, through a i, i+3 hydrogen bond between the carbonyl group of the acetyl moiety and the amide proton of Ser. Two backbone conformations can be adopted in the diaza--amino acid segment corresponding to types I and I' β--turns. This is of particular interest as the insertion of only one aza--amino acid induces a type II β--turn, that is less abundant in protein structures (19%) than the type I β--turn (46%) 29 and this conformation is stable only in solid--state or in organic solvent but unstable in aqueous solution. 15,16,19,20 However, the nature of the side chain of the aza--amino acid and amino acid residues has an influence on the stability of the double turn conformation. Our previous conformational study 23 of compound 3 containing two azaGly units had shown that the presence of side chains on both aza--amino acids had a beneficial effect in stabilizing the turn conformation of the diaza--tripeptides. This new report on Ac--aLys--aGly--SerCONH 2 4, which has similar β-turn propensity as diaza--tripeptides 5 and 6, indicates that the presence of a single side chain on the first aza--amino acid residue is sufficient to promote turn stabilization.

The presence of conformational isomerism with provision of four conformers observed in the case of diaza--tripeptides 5 and 6 could be explained by the restricted pyramidal inversion of a nitrogen adopting a sp3 hybridization state, that could thus be a source of chirality producing two diastereomers. While this chirality has not been observed in previously reported NMR studies of peptides having one aza--amino acid, 25,27,28 two consecutive aza-amino acids could further constrain and slow down the nitrogen inversion. The presence of polar side chains allowing hydrogen bond between the ammonium group of aLys and either the hydroxyl or carbonyl group of Ser could also further slow down the isomerization.

Overall, this work provides an additional proof that foldamers based on diaza--amino acids units might resolve a major issue in the use of peptides as drugs, by stabilizing turns and promoting helical conformations unlike natural peptides, while having the ability to maintain the selectivity thanks to the lateral chains. The next exciting step will be to investigate the ability of longer peptides containing these diaza--amino acids units to target PPIs involving turn or helical secondary structures 30,31 or to stabilize the helical conformation of intrinsically disordered proteins (IDPs) such as amyloid proteins. represent two of the most challenging drug discovery targets nowadays. Furthermore, our demonstration of the folding propensity in water of peptides incorporating diaza--peptide units suggest the ability of these new peptidomimetic foldamers to be active in biologically compatible media.

Experimental section

General information

Usual solvents were purchased from commercial sources and DCM was dried and distilled over CaH 2 . Thin--layer chromatography (TLC) analyses were performed on silica gel 60 F250 (0.26 mm thickness) plates. The plates were visualized with UV light (λ = 254 nm) or stained by a 4 % solution of phosphomolybdic acid or ninhydrin in EtOH. NMR spectra were recorded on an Ultrafield Bruker AVANCE

NMR conformational studies

NMR experiments conducted over a temperature range from 273 K to 308 K were acquired on a Bruker Avance 500 MHz NMR spectrometer equipped with a cryogenic TCI probe. C chemical shifts were referenced to the methanol solvent signal (residual protonated CHD 2 OH at 3.31 ppm and deuterated 13 CD 3 OH at 49.1 ppm, respectively). We checked that the pseudopeptides 4--6 did not self--assemble in aqueous solution or in methanol in this concentration range, by assessing the absence of concentration dependency on chemical shifts upon a 10 folds dilution for samples in water or a 20 folds dilution for samples in methanol.

NMR structure calculations

Structures were calculated using Amber14 program and ff14SB forcefield, as previously described.

23

Structures were refined using GBSA solvation model. Aza--amino acids parameterization was made with Antechamber, using gaff forcefield atom types and partial charges were calculated with AM1--BCC. 2D ROESY cross--peaks were integrated using NMRFAM--SPARKY. Three classes of distance restraints were defined, corresponding to upper limit distances of 3.0, 3.8 and 5.0 Å.

Scheme 2

 2 Scheme 2 Synthesis of diaza--tripeptide 5 and 6. Reagents and conditions: (a) 4--nitrophenyl chloroformate, pyridine, DCM, r.t, overnight, 76% (14a) and 59% (14b); (b) compound 7, DIPEA, ACN, r.t, 5 h, 79%; (c) 20% piperidine/DMF, r.t, 30 min, 91%; (d) i: triphosgene, DIPEA, DCM, r.t, 20 min; ii: Ser(Bzl)--NH2, DIPEA, DCM, r.t, overnight, 46% (17a) and 54% (17b) for two steps; (e) i: 4 M HCl in dioxane, r.t, 3 h; ii: acetic anhydride, pyridine, DCM, r.t, overnight, 75% (18a) and 76% (18b) for two steps; (f) H2, Pd/C 10%, acetic acid/H2O, r.t, overnight, 58% (5) and 59% (6).

Fig. 2 1D 1 H

 21 Fig.2 1D1 H NMR spectra of diaza--tripeptides 4--6 in water and in methanol, showing the region of HN resonances. Spectra were recorded at 278 K in water and at 228 K in methanol. The peak labelled with an asterisk corresponds to formate impurity. Unlabelled peaks correspond to carboxamide NH2, side chain aLys NH3 + or impurities.

Fig. 3 2D 1 H-- 1 H

 311 Fig. 3 2D1 H--1 H ROESY spectra of diaza--tripeptides 4--6 in water showing the correlations of Ser HN proton with side chain aliphatic protons. Spectra were recorded at 308 K for 4, and 278 K for 5 and 6.

Fig. 4

 4 Fig. 4 NMR structures of diaza--tripeptides 4 (A), 5 (B) and 6 (C). For each peptide, two low--energy NMR structures were selected to represent type I' and type I β--turn conformations. The diagram of ROE correlations is shown on the left. Strong, medium and weak ROE intensities are represented by solid, dashed and dotted lines, respectively.

37

  The synthesis of 13b and Ser(Bzl)--NH 2 are described in ESI. The protocols of synthesis and the characterization of intermediates 11--12, 14a/b--18a/b and of the final diaza-tripeptides 4--6 are detailed in ESI.

13 C

 13 HMBC spectra. Low temperature NMR experiments (225--250 K) were acquired on a Bruker Avance 600 MHz NMR spectrometer equipped with a room temperature TBI probe. NMR data were processed with TopSpin 3.6 and analysed with TopSpin 3.6 or NMRFAM--SPARKY program. For NMR studies in water, about 2.5 mg of compounds 4--6 were dissolved in 0.6 mL of H 2 O/D 2 O (90/10 v/v), pH=5.5. DSS (sodium 4,4--dimethyl--4--silapentane--1--sulfonate, 0.1 mM) was used for chemical shift calibration. For NMR experiments in methanol, 4 to 6 mg of compounds 4--6 were dissolved in 0.6 mL of CD 3 OH.

Table 1

 1 HN proton temperature coefficient ΔδHN/ΔT (ppb/K) for compounds 4--6 in methanol and water. The range of temperature coefficients obtained for the different conformational isomers before signal coalescence is indicated in brackets.

	Compound	Solvant	T (K)	aLys1	aGly/aAla/aLeu2	Ser3	NH2 Z	NH2 E
	4	MeOH H2O	228-251 278-308	-5.8 n.d.	-4.6, -7.9 (α) n.d.	-1.4 -3.4	n.d. n.d.	n.d. n.d.
	5	MeOH H2O	228-251 278-288	[-5.5, -4.8] -7.3	[-4.8, -4.0] -5.9	[-3.3, -2.2] [-4.7, -3.2]	[-7.8, -6.8] -5.5	[-5.8, -4.9] --
	6	MeOH H2O	228-251 278-298	n.d. [-8.8, -7.1]	[-5.0, -4.3] -7.3	[-2.9, -2.2] [-7.4, -4.3]	[-7.4, -6.4] [-6.6, -5.8]	[-5.3, -4.8] [-7.8, -4.0]
	n.d., non--detected proton due to exchange

  CDCl 3 : δ = 7.26 ppm, in CD 3 OD and CD 3 OH: δ = 3.31 ppm; 13 C NMR, CDCl 3 : δ = 77.16 ppm, CD 3 OD and CD 3 OH: δ = 49.00 ppm), and the following abbreviations are used: singlet (s), doublet (d), , mobile phase: ACN/H 2 O + 0.1% TFA (gradient 1--30% in 15 or 20 min). Preparative HPLC were performed on Agilent 1260 Infinity II. Column: Pursuit (C18 10 x 250μm--5μm), mobile phase: ACN/H 2 O + 0.1% formic acid. Compounds 7, 8, 9, 10 and 22 were prepared as reported in our previous literature

	300 (	1 H, 300 MHz,	13 C, 75 MHz) or on a Bruker AVANCE 400 (	1 H, 400
	MHz,	13 C, 100 MHz). Chemical shifts δ are in ppm with the solvent
	resonance as the internal standard (	1 H NMR, residual protiated
	solvent in doublet of doublet (dd), triplet (t), quintuplet (qt), multiplet (m),
	broad multiplet (bm), and broad singlet (brs). Mass spectra were
	obtained using a Bruker Esquire electrospray ionization apparatus.
	HRMS were obtained using a TOF LCT Premier apparatus (Waters)
	with an electrospray ionization source.
	The purity of compounds was determined by HPLC--MS on
	Agilent 1260 Infinity. Column: ATLANTIS T3 column (C18, 2.1 x
	150mm--3μm)22	and 13a was synthesized according to
	published method.

Acknowledgements

The China Scholarship Council (CSC) is thanked for the PhD fellowship of C. Shi. Karine Leblanc (Service d'Analyses-HPLC--Masse BioCIS, Univ. Paris Saclay) is thanked for HPLC and HRMS analyses.

Notes and references

Conflicts of interest

There are no conflicts to declare.


Zakaria Tichout

Rodrigue Rousier

Stéphane Colin

Chambre fluidique portable de mesure transcutanée dynamique pour l'oxymétrie te la capnométrie

and research institutions in France or abroad, or from public or private research centers.