Khawar Hayat

Bello Musa Bodinga

Di Han

Xin Yang

Qingzhu Sun

Lotfi Aleya
email: lotfi.aleya@univ-fcomte.fr

Mohamed M Abdel-Daim

Xiaojun Yang

Xin Yang

Xiaojun Yang

Effects of dietary inclusion of chromium propionate on growth performance, intestinal health, immune response and nutrient transporter gene expression in broilers

Keywords: broilers, chromium, growth performance, organ index, antibody titre, gene expression

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

With the rapid development of the poultry industry, scientists around the globe are now focusing on the role of micronutrients in poultry diets to ensure better production. Although micronutrients are required in minute quantities in the poultry diet, they play an important role in improving birds' health and vigour. In recent decades, the use of Cr in the nutrition of domestic animals has attracted the attention of researchers. Chromium can be present in the environment at numerous oxidation states, with Cr 3+ being the most stable form (NRC, USA, 2005).

Chromium is a micronutrient that is involved in the metabolism of carbohydrates, proteins, lipids and nucleic acids (Khan et al., 1994;[START_REF] Sands | Broilers in heat stress conditions: effects of dietary manganese proteinate or chromium picolinate supplementation[END_REF]. Chromium activates many of the enzymes responsible for protein and nucleic acid synthesis [START_REF] Anderson | Stress effects on chromium nutrition of humans and farm animals[END_REF]. Dietary supplementation with Cr 3+ is recommended by the National Research Council (NRC) Washington, USA for animals suffering from environmental stress (NRC, 1997). However, Cr 3+ supplementation poses a challenge because the dose must be adjusted to provide the actual needed concentration to avoid hepatotoxic, nephrotoxic, oxidative and DNA-damaging effects [START_REF] Deng | Binding of trivalent chromium to serum transferrin is sufficiently rapid to be physiologically relevant[END_REF]Jin et al., 2015). Studies provide indications for the nutritional and physiological role of Cr 3+ as a micronutrient [START_REF] Anderson | Stress effects on chromium nutrition of humans and farm animals[END_REF]. The beneficial effects of Cr 3+ are most obvious in animals that are under environmental, dietary and hormonal stress because Cr 3+ can reduce the harmful effects of stress caused by various environmental factors [START_REF] Lien | Performance, serum characteristics, carcase traits and lipid metabolism of broilers as affected by supplement of chromium picolinate[END_REF][START_REF] Sahin | Chromium supplementation can alleviate negative effects of heat stress on egg production, egg quality and some serum metabolites of laying Japanese quail[END_REF]. Some studies have shown that chromium supplementation of broiler feed positively affects the growth performance and carcass characteristics of birds [START_REF] Debski | Chromium-yeast supplementation of chicken broilers in an industrial farming system[END_REF][START_REF] Toghyani | Performance, carcass traits and hematological parameters of heatstressed broiler chicks in response to dietary levels of chromium picolinate[END_REF][START_REF] Sahin | Chromium picolinate, rather than biotin, alleviates performance and metabolic parameters in heatstressed quail[END_REF][START_REF] Huang | Effects of supplemental chromium source and concentration on growth performance, carcass traits and meat quality of broilers under heat stress conditions[END_REF]. However, other studies reported opposite results [START_REF] Moeini | The effect of different levels of organic and inorganic chromium supplementation on production performance, carcass traits and some blood parameters of broiler chicken under heat stress condition[END_REF][START_REF] Ghazi | Effects of different levels of organic and inorganic chromium on growth performance and immunocompetence of broilers under heat stress[END_REF][START_REF] Kim | Effects of dietary levels of chromium picolinate on growth performance, carcass quality and serum traits in broiler chicks[END_REF].

At present, poultry diseases represent the major threat to the poultry industry. Newcastle disease virus (NDV), a virus that causes a deadly infection, infects many bird species and is highly contagious and fatal to susceptible birds [START_REF] Alexander | Newcastle disease outbreaks in domestic fowl and turkeys in Great Britain during[END_REF]. NDV, which belongs to the genus Avulavirus and the family Paramyxoviridae, is the causative agent of a respiratory and neurological disease (Newcastle disease) that can cause up to 100% morbidity and mortality in infected birds (Alexander, 2003). Newcastle disease is primarily controlled by maintaining a hygienic environment and a good vaccination program. Currently, vaccination against many diseases is helpful in controlling severe sickness and mortality in infected birds [START_REF] Van Boven | Herd immunity to Newcastle disease virus in poultry by vaccination[END_REF]. Recently, lentogenic strains of NDV have been used to induce a protective immune response and studies suggest that different vaccine strategies have specific advantages and disadvantages (Bermudez and Stewart, 2003). Chromium can also increase the immune status of broilers by overwhelming the production of corticosterone, a potent immunosuppressant that interferes with antibody production by decreasing leukocyte populations and interfering with lymphocyte function [START_REF] Sahin | Chromium picolinate, rather than biotin, alleviates performance and metabolic parameters in heatstressed quail[END_REF][START_REF] Lee | Effects of dietary chromium picolinate supplementation on growth performance and immune responses of broilers[END_REF]. To determine the usefulness of chromium addition in producing a good-quality poultry diet that improves birds' performance and resistance to diseases, the present study assessed the effect of dietary Cr 3+ on growth performance, organ index, immune response, histomorphology and nutrient transporter gene expression in broilers.

Materials and methods

Experimental birds and diet

The present study was conducted at the poultry farm of North West Agriculture and Forestry University, Yangling, China. Throughout the experiment, the birds were maintained under constant conditions of temperature, relative humidity, ventilation, light cycle and floor space.

The 756 one-day-old broiler chicks used in this experiment were randomly divided into six experimental groups (A, B, C, D, E and F). Chromium (dry, 0.4%) from chromium propionate is designed to be premixed prior to its addition to complete broiler feed. Premix contain10% chromium (from 0.4% chromium dry) and 90% calcium carbonate (having 35% calcium) delivered Cr @ 0, 0.10, 0.15, 0.20, 0.25 or 0.30 mg/kg in the feed. Each group of 126 chicks was further divided into 7 replicates (18 chicks/replicate). The birds were fed a corn-soybean diet that was formulated to meet all nutritional requirements according to the feeding standard in China (NY/T33-2004) (Table 6).

Experimental records and management

One-day-old birds were weighed, and the average initial body weight of the birds in each group was calculated. The birds were provided with feed and water ad libitum throughout the experiment. The number of dead birds and the temperature and humidity in the birdhouse were recorded on a daily basis. Management requirements and data were recorded regularly. The birds were vaccinated according to routine immunization practice in the region. The broiler house was kept clean and was disinfected on a regular basis.

Clinical observation

During the trial period, the clinical features of the broilers, including their consumption of food and water, were observed daily and bad symptoms (abnormal health conditions or mortality), if any, were noted. All dead birds were dissected, and a detailed post-mortem was conducted.

Growth performance

At 0, 21 and 42 days, the birds were weighed, and the feed intake of each replicate was recorded.

Average daily feed intake (ADFI), average daily weight gain (ADWG), feed conversion ratio (FCR) and mortality were calculated for the periods 0-21 and 0-42 days.

Organ index

Seven birds from each treatment group whose weights were close to the average body weights (birds having minimum difference in body weight to average weight of their respective treatment) of the birds in their respective groups were selected at the age of 21 and 42 days and deprived of feed for 12 h to determine weight loss during the pre-slaughter holding period. The pre-slaughter body weight loss was expressed as g/kg initial body weight of each individual bird.

Later in the study, these birds were slaughtered, and the organ index (relative organ weight of the liver, heart, spleen and thymus) was recorded and expressed as g/kg pre-slaughter live body weight of the individual birds.

Immune response

The birds were tested for serum antibody titres on the 7 th , 14th, 21 st and 28 th days after vaccination against Newcastle disease and on the 28th day after vaccination against avian influenza H5 by HI test according to a standard protocol (OIE, 2012).

Intestinal morphology

At 21 and 42 days of age, seven birds from each treatment group (1 bird/replicate) whose body weights were close to the average body weights of their respective groups were selected. The birds were slaughtered, and the duodenum, jejunum and ileum were removed and cleaned by flushing with pre-cooled saline. Sections (approximately 1 cm in length) were cut from the middle of the duodenum, jejunum and ileum and fixed in 4% paraformaldehyde. The villus height (VH), the crypt depth (CD) and the thickness of the intestinal wall (WT) were measured.

RNA isolation and quantitative real-time PCR

The mRNA expression of SGLT1, GLUT2, rBAT, and CAT1 in intestinal mucosal samples from the duodenum, jejunum and ileum was determined by qRT-PCR. The primers used in these experiments (Table 7) were designed using Premier 5.0 according to sequences obtained from NCBI. Total RNA was isolated using trizol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The quantity and quality of the extracted RNA in each sample were determined using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA). The RNA was stored at -20°C until further use. The RNA was reverse-transcribed using a Takara cDNA transfer kit (Bio-Rad, Hercules, CA, USA). The reaction was conducted in a total volume of 20 μL consisting of 1 μL cDNA, 1 μL forward primer, 1 μL reverse primer, 10 μL SYBR Premix ExTaq and 7 μL nuclease-free water. The qPCR was conducted in triplicate using 96-well microwell plates in a Bio-Rad iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 60°C for 30 s and 72°C for 30 s and a final extension at 72°C for 5 min. The mRNA expression of each gene was calculated using the 2-ΔΔCt method described by [START_REF] Livak | Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[END_REF] after normalizing to βactin.

Statistical analysis

The experimental data were analysed by one-way ANOVA using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). A probability value of P< 0.05 was considered to indicate statistical significance. The notable differences between groups were identified by Duncan's multiple comparisons test.

Results

Growth Performance

Cr 3+ supplementation of the birds' feed resulted in significantly higher ADFI in group B (dietary supplementation with Cr 3+ at 0.10 mg/kg), followed by groups C, A, F, D and E, in that order, at 1-21 days of age. ADWG was significantly higher in the birds in groups B and C (dietary supplementation with Cr 3+ at 0.10 and 0.15 mg/kg, respectively) compared to the other groups at 1-21 and 1-42 days of age, respectively. Moreover, the feed conversion ratio was found to be significantly better in group C (dietary supplementation with Cr 3+ at 0.15 mg/kg) than in any of the other groups, followed by groups D, E, F, B and A, in that order, at 1-21 days of age. At 1-42 days of age, the feed conversion ratio was found to be significantly (P <0.05) better in all chromium-supplemented groups compared to the control. The best FCR was observed in group C, followed by groups B, D, E, F and A (figure 1).

Organ Index

The birds in group B, which were offered a diet supplemented with Cr 3+ at 0.10 mg/kg, showed a significantly increased spleen index at 21 days of age compared with the birds in the control and D, E and F groups (P <0.05). The birds in group F, which received a diet supplemented with Cr 3+ at 0.30 mg/kg, showed a significantly increased thymus index at 21 days of age. At 42 days of age, it was found that the birds in group D, which received a diet supplemented with Cr 3+ at 0.15 mg/kg, had significantly higher heart index compared to the control and to all other treatment groups. Groups C and F, whose feed was supplemented with Cr 3+ at 0.15 mg/kg and 0.30 mg/kg, respectively, showed significantly better thymus index results compared with the control and with groups B and E (Table 1).

a. Immune response

The birds in treatment group C, which were offered a diet supplemented with 0.15 mg/kg Cr 3+ , showed significantly higher antibody titres than the control group and group E on day 7 after immunization against Newcastle disease. At 21 days after immunization, the birds in groups C and D showed significantly higher antibody titres against Newcastle disease than the control group and group E; at 28 days after immunization, they showed significantly higher antibody titres than the control group and group B. The antibody titre against H5 at 21 days after immunization was found to be significantly higher in the birds in group D than in the other treatment groups, although it was not significantly different from the control (Table 2).

b. Intestinal morphology

Figures 234show the intestinal morphology of the animals. The results presented in Tables 345show that the birds in group C, which received a diet supplemented with Cr 3+ at 0.15 mg/kg, had significantly increased crypt depth compared to the control group. Groups C and F showed significantly increased WT in the jejunum compared to all of the other groups at 21 days of age .

In addition, the birds in groups B, C, D and E, which received diets supplemented with Cr 3+ at 0.10, 0.15, 0.20 and 0.25 mg/kg, respectively, showed a significantly reduced VH:CD ratio in the jejunum at 21 days of age (P <0.05).

c. Glucose and amino acid transporter gene expression

Dietary supplementation with Cr 3+ significantly increased the relative expression of the SGLT1 gene in the duodenum of broilers at 21 days of age. The highest expression of the SGLT1 gene was observed in group F (dietary supplementation with 0.30 mg/kg Cr 3+ ), followed by groups D, C, E, B and A. At 42 days of age, CAT1 mRNA gene expression was highest in group D (dietary supplementation with 0.20 mg/kg Cr 3+ ), followed by groups F, A, B, E and A. In the jejunum, CAT1 mRNA expression was significantly higher in group C (dietary supplementation with 0.15 mg/kg Cr 3+ ) than in any of the other groups, while at 42 days of age GLUT2 mRNA expression was significantly higher in all of the chromium-supplemented groups than in the control. However, at 42 days of age, CAT1 mRNA showed significantly higher expression in the jejunum in groups C and F compared to all other groups. In the ileum at 21 days of age, GLUT2 mRNA expression was significantly higher in group C (dietary supplementation with 0.15 mg/kg Cr 3+ ) than in any of the other groups, whereas CAT1 expression was significantly higher in groups B and C compared to all other groups. At 42 days of age, GLUT2 expression in the ileum was highest in group C, followed by groups D, B, F, E and A, and CAT1 expression was significantly higher (P <0.05) in group C compared to all the other groups (figure 5).

Discussion

In the present study, birds were reared under normal conditions without exposure to obvious stress factors. The results indicate that supplementation of the diet with Cr 3+ at various concentrations had significant effects on the growth performance (ADFI, ADWG and FCR) of broilers at different ages. Dietary supplementation with Cr 3+ significantly increased ADFI in the birds in group B at 1-21 days of age. ADWG was significantly higher in the birds in groups B and C than in those in the other groups at 1-21 and 1-42 days of age. Moreover, the feed conversion ratio was found to be significantly better in group C than in any of the other groups at 1-21 days of age. At 1-42 days of age, the feed conversion ratio was significantly better in all chromium-supplemented groups than in the control. Best FCR was obtained in group C. These results are consistent with the results of some previous studies. [START_REF] Toghyani | Performance, carcass traits and hematological parameters of heatstressed broiler chicks in response to dietary levels of chromium picolinate[END_REF] reported that dietary supplementation with CrPic at increased levels (up to 1,500 μg/kg) enhanced body weight and feed intake without any adverse effect on FCR in broilers reared under heat stress. [START_REF] Sahin | Chromium picolinate, rather than biotin, alleviates performance and metabolic parameters in heatstressed quail[END_REF] found a beneficial effect of CrPic on body weight gain in quail. [START_REF] Huang | Effects of supplemental chromium source and concentration on growth performance, carcass traits and meat quality of broilers under heat stress conditions[END_REF] found that supplementation of the diet with chromium improved the dressing percentage of broilers reared under heat stress. [START_REF] Afrin | Response of heat stressed commercial broilers to dietary supplementation of organic chromium[END_REF] found that dietary chromium supplementation significantly increased body weight and feed intake in broilers. [START_REF] Kheiri | Effect of different levels of inorganic chromium on performance and immunity of broiler chicks[END_REF] reported that chromium supplementation of the diet significantly increased body weight in broilers without altering feed intake or FCR. [START_REF] Naghieh | Effect of different sources of supplemental chromium on performance and immune responses of broiler chicks[END_REF] found that the feed intake and body weight of broilers offered a diet supplemented with Cr 3+ were significantly increased. [START_REF] El-Kholy | Dietary supplementation of chromium can alleviate negative impacts of heat stress on performance, carcass yield and some blood hematology and chemistry indices of growing Japanese quail[END_REF] reported that inclusion of chromium in the diet increased the daily body weight gain of broilers reared under heat stress conditions without affecting overall growth performance. However, the results of some other studies are contrary to those of the present study. It was reported that dietary addition of organic and inorganic Cr 3+ at different levels resulted in no difference in weight gain or feed intake in heat-stressed broilers [START_REF] Moeini | The effect of different levels of organic and inorganic chromium supplementation on production performance, carcass traits and some blood parameters of broiler chicken under heat stress condition[END_REF]. In another study, it was found that supplementation of the diet with organic or inorganic Cr 3+ had no effect on feed intake or body weight gain in broilers reared under heat stress conditions. Furthermore, it was observed that chromium supplementation of the diet did not affect any performance parameters (body weight gain, feed intake or feed conversion ratio) in broilers [START_REF] Ghazi | Effects of different levels of organic and inorganic chromium on growth performance and immunocompetence of broilers under heat stress[END_REF]. The differences among the outcomes recorded in various studies could be attributed to various factors such as the use of different forms of chromium, different environmental conditions (i.e., temperature, source of carbohydrate, diet level), the duration of treatment, the treatment dose, the response of the target birds to treatment, and the birds' health status. Variable effects of chromium supplementation of the broiler diet on carcass characteristics have been reported in numerous studies [START_REF] Anderson | Chromium trace element in human and animal nutrition[END_REF]. The results of the current study confirm that variability. Cr 3+ supplementation at 0.10 mg/kg (group B) significantly increased the spleen index. However, Cr 3+ supplementation at 0.30 mg/kg significantly increased the thymus index in 21-day-old broilers. At 42 days of age, the birds in group D showed a significantly increased heart index compared to the other groups.

Furthermore, the birds in groups C and F showed a significantly better thymus index compared to those of control B and E groups. However, the effects of chromium on the liver and the bursa of Fabricius were not significant. Similar variability has been reported in other studies [START_REF] Rao | Effect of dietary supplementation of organic chromium on performance, carcass traits, oxidative parameters, and immune responses in commercial broiler chickens[END_REF][START_REF] Hossain | Growth performance and carcass composition of broilers fed supplemental chromium from chromium yeast[END_REF].

The results of the present study showed that dietary Cr 3+ supplementation boosted antibody titre against Newcastle disease and avian influenza. Treatment group C showed significantly higher antibody titre compared to the control and group E on day 7 post-immunization against Newcastle disease. Birds of groups C and D showed significantly higher antibody titres against Newcastle disease compared to the control and group E on day 21 post-immunization. Furthermore significantly higher titres than the control and group B on day 28 postimmunization. The antibody titre against H5 on day 21 after immunization was found to be significantly higher in the birds of group D compared to all other treatment groups, although it was similar to that of the control. Enhancement of the immune response of birds receiving dietary supplementation with chromium has also been reported by other authors [START_REF] Sahin | Chromium picolinate, rather than biotin, alleviates performance and metabolic parameters in heatstressed quail[END_REF][START_REF] Kheiri | Effect of different levels of inorganic chromium on performance and immunity of broiler chicks[END_REF]. The better immune response of the animals may be due to the antioxidant activity of chromium. This activity may decrease the production of corticosterone, a potent immunosuppressant that decreases leukocyte populations and obstructs lymphocyte function and antibody production [START_REF] Sahin | Chromium picolinate, rather than biotin, alleviates performance and metabolic parameters in heatstressed quail[END_REF][START_REF] Lee | Effects of dietary chromium picolinate supplementation on growth performance and immune responses of broilers[END_REF]. The results of the present study are consistent with those of [START_REF] Cao | Effects of florfenicol and chromium (III) on humoral immune response in chicks[END_REF], who reported improved total antibody titres in broilers fed diets supplemented with chromium. Another reason for the better immune response may be that chromium modulated the immune response through its effects on cytokines. Cytokines are responsible for signalling between cells. Cytokines and their receptors affect the activities of other cells and play a major role in the regulation of the immune system [START_REF] Davison | The immunologists' debt to the chicken[END_REF][START_REF] Callard | Cytokines, chaos, and complexity[END_REF].

It was essential to determine whether the health of the intestinal tract was improved by dietary supplementation with Cr 3+ to promote nutrient utilization. It is recognized that enzymes in the digestive tract play vital roles in the digestion and absorption of nutrients. The birds in group C showed significantly greater crypt depth compared to the control and to the birds in group F at 21 days of age. Groups C and F showed significantly increased WT in the jejunum compared to all other groups at 21 days of age. This may be due to the good health status of the birds and the use of proper management conditions, which can lead to better health of the digestive tract. These results contradict the findings of [START_REF] Makanjuola | Effect of inorganic chromium supplementation on the intestinal mucosa development of heat stressed broilers[END_REF] and [START_REF] Sandikci | The effect of heat stress and the use of Saccharomyces cerevisiae or (and) bacitracin zinc against heat stress on the intestinal mucosa in quails[END_REF], who found a non-significant difference in crypt depth after chromium supplementation of broiler feed. In our study, dietary supplementation with different levels of Cr 3+ probably led to an increase in the relative expression of genes encoding glucose and amino acid transporters in the intestinal tract at different stages of growth, and this is likely to have resulted in better absorption of nutrients and thus better utilization and improved growth performance of the birds.

Conclusions

Chromium has generally been used in the poultry industry to reduce heat stress. In the present study, dietary supplementation with Cr 3+ yielded reasonable results concerning immunity against diseases and intestinal morphology. In addition, Cr 3+ appears to have favourable effects on growth performance, especially on FCR, a finding that was verified by the higher expression of genes encoding nutrient transporters in different parts of the intestine. The concentrations of Cr 3+ (0.10, 0.15, 0.20, 0.25 or 0.30 mg/kg) used in the present study were found to be safe and can be used in the poultry industry to improve broiler health and performance. Supplementation of broiler feed with 0.15 mg/kg Cr 3+ was found to be most effective. Further studies conducted under different conditions are needed to assess the potential beneficial results of Cr 3+ in broiler production.

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. (a-f) Duodenal villus histomorphometry of broilers at 42 days. (A) Control (basal diet

Figure 3 .

 3 Figure 3. (a-f) Jejunal villus histomorphometry of broilers at 42 days. (A) Control (basal diet

Figure 4 .

 4 Figure 4. (a-f) Ileum villus histomorphometry of broilers at 42 days. (A) Control (basal diet

Figure 5 .

 5 Figure 5. (a-f) mRNA expression of specific genes at 21 and 42 days of age in different regions

  Figure 1.

  Figure 2.

Table 1 . Effect of dietary chromium propionate on the organ index of broilers

 1

					Cr levels mg/kg
	Days	Parameters						SEM P Value
			0	0.10	0.15	0.20	0.25	0.30
		liver index	26.85 28.55 27.49 28.07 28.84 26.94 0.264	0.117
		Spleen index	0.79 bc 1.03 a 0.73 c 0.82 bc 0.92 ab 0.93 ab 0.029	0.021
	21	Heart index	5.26	5.74	5.44	5.52	5.68	5.59 0.057	0.213
		Thymus index 4.29 b 4.71 ab 4.71 ab 4.28 b 4.32 b 5.46 a 0.118	0.030
		Bursa index	1.66	1.63	1.78	1.84	2.02	1.82 0.051	0.312
		Liver index	22.48 21.36 22.97 22.15 22.16 24.19 0.386	0.448
		Spleen index	1.41	1.14	1.19	1.25	1.34	1.15 0.039	0.313
	42	Heart index	4.06 b 4.06 b 4.60 a 3.59 c 4.15 b 4.07 b 0.075	0.002
		Thymus index 3.54 b 3.48 b 4.63 a 3.93 ab 3.23 b 4.43 a 0.128	0.002
		Bursa index	1.09	1.16	1.21	1.28	1.36	1.14 0.044	0.566
	a, b, c Mean values within a row with different superscript letters were significantly different (P <
	0.05). Organ index =organ weight g/kg body weight. 0mg/kg: (basal diet, with no chromium propionate
	supplementation; (b) 0.10mg/kg: (basal diet+ 0.10 mg/kg chromium propionate supplementation); (c)
	0.15mg/kg: (basal diet+ 0.15 mg/kg chromium propionate supplementation); (d) 0.20mg/kg: (basal
	diet+ 0.20 mg/kg chromium propionate supplementation); (e) 0.25mg/kg: (basal diet+ 0.25 mg/kg
	chromium propionate supplementation); (b) 0.30mg/kg: (basal diet+ 0.30 mg/kg chromium propionate
	supplementation). Means with different superscripts were significantly different (p < 0.05). Data were
	presented as means ± SE

Table 2 . Effect of dietary chromium propionate on serum antibody titer against Newcastle Disease and Avian Influenza in broilers

 2

					Cr levels (mg/kg)
	Disease	Age							SEM	P Value
			0	0.10	0.15	0.20	0.25	0.30
		Preimmune	6.75	6.75	6.00	6.00	7.50	7.00	1.42	0.867
		7 days after immunization	3.50 c 4.50 abc 5.00 a 4.75 ab 3.67 bc 4.00 abc 0.17	0.032
	Newcastle	14 days after immunization	6.75	6.50	6.25	6.25	5.75	5.75	0.18	0.575
	disease	21 days after immunization	3.50 c 7.00 ab 7.25 a 8.00 a 5.00 bc 7.00 ab 0.397	0.001
		28 days after immunization	7.50 bc 6.25 c	9.25 a 9.00 a 8.00 ab 8.75 ab 0.265	0.001
	H5 AI	21 days	8.50 ab 6.75 c 7.50 bc 8.75 a 7.25 c	7.00 c	0.20	0.002
	a, b, c Mean values within a row with different superscript letters were significantly different (P <
	0.05). 0mg/kg: (basal diet, with no chromium propionate supplementation; (b) 0.10mg/kg: (basal diet+
	0.10 mg/kg chromium propionate supplementation); (c) 0.15mg/kg: (basal diet+ 0.15 mg/kg chromium
	propionate supplementation); (d) 0.20mg/kg: (basal diet+ 0.20 mg/kg chromium propionate
	supplementation); (e) 0.25mg/kg: (basal diet+ 0.25 mg/kg chromium propionate supplementation); (b)
	0.30mg/kg: (basal diet+ 0.30 mg/kg chromium propionate supplementation). Means with different
	superscripts were significantly different (p < 0.05). Data were presented as means ± SE

Table 3 . Effects of dietary chromium propionate on intestinal morphology of duodenum in broilers

 3

	Days Parameters	0	0.10	Cr levels (mg/kg) 0.15 0.20	0.25	0.30	SEM	P Value
	VH (μm) 3282.25 2878.00 3345.34 2965.79 3032.21 3198.09 68.273 0.298
	CD(μm) 161.96	177.83	171.84	193.96	164.38	165.42	4.357	0.287
	21
	WT (μm)	460.33	396.34	435.42	425.17	411.42	414.96	9.297	0.482
	VH/CD	21.04	16.18	19.54	15.48	18.63	19.41	0.676	0.122
	VH (μm) 3561.59 3123.96 3427.94 3645.17 3348.71 3176.62 99.952 0.635
	CD(μm) 25.957	23.73	17.90	29.70	14.44	20.59	4.750	0.475
	42
	WT (μm)	515.21	504.96	526.95	494.55	512.63	476.63 12.099 0.915
	VH/CD	20.89	17.72	22.22	21.35	22.08	17.24	0.800	0.282
	All values were non significant (P < 0.05). 0mg/kg: (basal diet, with no chromium propionate
	supplementation; (b) 0.10mg/kg: (basal diet+ 0.10 mg/kg chromium propionate supplementation); (c)
	0.15mg/kg: (basal diet+ 0.15 mg/kg chromium propionate supplementation); (d) 0.20mg/kg: (basal

diet+ 0.20 mg/kg chromium propionate supplementation); (e) 0.25mg/kg: (basal diet+ 0.25 mg/kg chromium propionate supplementation); (b) 0.30mg/kg: (basal diet+ 0.30 mg/kg chromium propionate supplementation). Means with different superscripts were significantly different (p < 0.05). Data were presented as means ± SE; VH (Villus height), CD (Crypt depth), WT (Wall thickness)

Table 4 . Effect of dietary chromium propionate on intestinal morphology of jejunum in broilers

 4

	Days	Parameters	0	0.10	Cr levels (mg/kg) 0.15 0.20	0.25	0.30	SEM	P Value
		VH (μm)	2591.63 2117.16 2237.79 2111.59 2098.63 2451.00 82.222 0.405
		CD (μm)	159.33 bc 190.67 ab 197.25 a 193.25 ab 176.09 abc 143.25 c 5.843 0.023
	21
		WT (μm)	407.79 b 391.71 b 522.88 a 395.09 b	423.54 b 446.88 ab 13.599 0.029
		VH/CD	16.82 a	11.07 b	11.60 b	11.16 b	11.91 b	17.16 a	0.760 0.014
		VH (μm)	2511.17 2550.21 2144.67 2206.88 2228.13 2126.50 83.058 0.578
		CD (μm)	172.17	178.96	192.38	208.84	184.33	170.75	6.604 0.604
	42
		WT (μm)	481.28	487.25	465.29	494.33	448.87	453.96 13.338 0.919
		VH/CD	15.15	14.54	11.58	11.04	12.19	12.52	0.666 0.478
	a, b, c Mean values within a row with different superscript letters were significantly different (P <
	0.05). 0mg/kg: (basal diet, with no chromium propionate supplementation; (b) 0.10mg/kg: (basal diet+
	0.10 mg/kg chromium propionate supplementation); (c) 0.15mg/kg: (basal diet+ 0.15 mg/kg chromium
	propionate supplementation); (d) 0.20mg/kg: (basal diet+ 0.20 mg/kg chromium propionate

supplementation); (e) 0.25mg/kg: (basal diet+ 0.25 mg/kg chromium propionate supplementation); (b) 0.30mg/kg: (basal diet+ 0.30 mg/kg chromium propionate supplementation). Means with different superscripts were significantly different (p < 0.05). Data were presented as means ± SE; VH (Villus height), CD (Crypt depth), WT (Wall thickness)

Table 5 . Effect of dietary chromium propionate on intestinal morphology of ileum in broilers

 5 All values were non significantly (P < 0.05). 0mg/kg: (basal diet, with no chromium propionate supplementation; (b) 0.10mg/kg: (basal diet+ 0.10 mg/kg chromium propionate supplementation); (c) 0.15mg/kg: (basal diet+ 0.15 mg/kg chromium propionate supplementation); (d) 0.20mg/kg: (basal diet+ 0.20 mg/kg chromium propionate supplementation); (e) 0.25mg/kg: (basal diet+ 0.25 mg/kg chromium propionate supplementation); (b) 0.30mg/kg: (basal diet+ 0.30 mg/kg chromium propionate supplementation). Means with different superscripts were significantly different (p < 0.05). Data were presented as means ± SE; VH (Villus height), CD (Crypt depth), WT (Wall thickness)

	Days	Parameters	0	0.10	Cr levels (mg/kg) 0.15 0.20	0.25	0.30	SEM	P Value
		VH (μm)	1678.17 1424.21 1502.25 1588.79 1599.56 1510.54 58.405 0.880
		CD(μm)	189.75	162.92	183.62	187.04	152.33	183.04	4.545	0.116
	21
		WT (μm)	530.96	490.75	488.71	512.50	469.94	448.17 16.297 0.779
		VH/CD	8.94	8.76	8.15	8.46	10.70	8.26	0.387	0.572
		VH (μm)	1560.50 1776.75 1741.04 1730.17 1832.63 1793.56 61.922 0.945
		CD(μm)	192.92	162.58	178.84	166.06	182.00	166.83	6.096	0.811
	42
		WT (μm)	621.00	483.00	548.71	541.50	534.96	527.94 25.735 0.886
		VH/CD	8.01	10.91	9.76	10.58	10.31	10.87	0.376	0.447

Table 6 . Feed composition offered to broilers during experiment

 6 the premix added in one kg of the diet: Vit. A, 250000 I.U; Vit. D3, 50000 I.U.; Vit. E, 600 I.U; Vit. K3, 40 mg; Vit. B1, 40 mg; Vit. B2, 120mg; B5, 240 mg; Vit.B12, 0.50 mg; Niacin, 1 g; Biotin, 0.65 mg; Folic acid, 25 mg; Zn, 75 mg; Mn, 80 mg; Fe, 80 mg; Cu, 10 mg; I, 0.40 mg; Se, 0.30 mg.

	Ingredients	Starter (%) (1-21days)	Finisher (%) (22-42 days)
	Corn	52.24	55.69
	Soybean meal	32.17	30.1
	Cotton seed meal	2	3
	Corn Gluten 60%	4	4
	Rapeseed meal	3	3
	Calcium	2.5	1.5
	Phosphate (powder)	1.83	0.12
	Lysine sulphate 70%	0.77	1.06
	Salt	0.3	0.36
	DL-methionine	0.28	0.30
	*Premix	0.3	0.3
	Threonine	0.17	0.24
	Zeolite powder	0.17	0.15
	Choline chloride (50%)	0.15	0.1
	Tryptophan	0.12	0.08
	Total	100	100
	Chemical Composition
	Crude Protein % (CP)	21.2	19
	Calcium %	0.96	0.85
	Phosphorous %	0.70	0.58
	Lysine %	1.294	1.272
	Methionine %	0.603	0.651
	Metabolic Energy kcal/kg	2930	3050

*

Table 7 . The sequences of primers used in quantitative real-time PCR Target gene Primer Sequence (5′-3′)

 7

	Target Gene Primer	Sequence	GenBank Accession No.
	Beta-Actin	Forward	ATTGTCCACCGCAAATGCTTC	NM_205518.1
		Reverse	AAATAAAGCCATGCCAATCTCGTC
	SGLT1	Forward	AGCATTTCAGCATGGTGTGTCTTC	XM_415247
		Reverse	GATGCTCCTATCTCAGGGCAGTTC
	GLUT2	Forward	CACACTATGGGCGCATGCT	XM_205209.1
		Reverse	ATTGTCCCTGGAGGTGTTGGTG
	rBAT	Forward	CCCGCCGTTCAACAAGAG	XM_426125
		Reverse	AATTAAATCCATCGACTCCTTTGC
	CAT1	Forward	CAAGAGGAAAACTCCAGTAATTGCA	NC_003424.3
		Reverse	AAGTCGAAGAGGAAGGCCATAA

Acknowledgements

We would like to thank the staff of the Animal Nutrition and Feed Science of Northwest A&F University for their help and support during these experiments. This work was funded by the National Key Research and Development Program of China (2017YFD0500500, 20170502200), the Program for Shaanxi Science & Technology (2017TSCXL-NY-04-04, 2015KTCQ02-19) and the Program for Yangling Agricultural High-tech Industries Demonstration Zone (2018CXY-10).

This research was partially supported by a grant from KEMIN Zhuhai China, Inc.

Conflict of interest

The authors declare no conflicts of interest.


Julien Le Gal

Francesco Tisato
email: tisato@icis.cnr.it

Giuliano Bandoli

Marie Gressier

Joël Jaud

Sandra Michaud

Michèle Dartiguenave

Eric Benoist
email: benoist@chimie.ups-tlse.fr

Synthesis and structural characterization of new oxorhenium and oxotechnetium complexes with XN 2 S-tetradentate semi-rigid ligands (X = O, S, N)

Twelve novel oxo-technetium and oxo-rhenium complexes based on N 2 S 2 -, N 2 SO-or N 3 S-tetradentate semi-rigid ligands have been synthesised and studied herein. By reacting the ligands with a slight excess of suitable [MO] 3+ precursor (ReOCl 3 (PPh 3 ) 2 or [NBu 4 ][ 99g TcOCl 4 ]), the monoanionic complexes of general formula [MO(Ph-XN 2 S)] - could be easily produced in high yield. The complexes have been characterized by means of IR, electrospray mass spectrometry, elemental analysis, NMR and conductimetry. The crystal structures of [PPh 4 ][ReO(Ph-ON 2 S)] 1b and [NBu 4 ][ 99g TcO(Ph-ON 2 S)] 1c have been established. The [MO] 3+ moiety was coordinated via the two deprotonated amide nitrogens, the oxygen and the terminal sulfur atoms in 1b and 1c. In both compounds, the ON 2 S coordination set is in the equatorial plane, and the complexes adopted a distorted square-pyramidal geometry with an axial oxo-group. The chemical and structural identity of the different prototypic complexes (rhenium, 99g Tc complexes and their corresponding 99m Tc radiocomplexes) have been also established by a comparative HPLC study.

Introduction

Technetium and rhenium are known for their useful applications in nuclear medicine. 99m Tc (140 keV c emitter, t 1/2 6.02 h, convenient availability from the 99 Mo/ 99m Tc generator) is widely used for diagnostic imaging, while 186 Re (1.07 MeV b -emitter, t 1/2 90 h) and 188 Re (2.12 MeV b -emitter, t 1/2 17 h) are two of the most attractive isotopes for applications in targeted radionuclide therapy. 1,2 The efforts made in this field in recent years have led to the synthesis of novel, selective and effective chelates. 2,3 Among them, N 2 S 2 , N 2 O 2 or N 3 S frameworks are the most common chelate systems used to bind the [TcO] 3+ and [ReO] 3+ cores. 4 In connection with our interest in the preparation of new substitution-inert technetium and rhenium compounds, we have recently reported the synthesis of a new family of semi-rigid N 2 S 2 , N 2 OS or N 3 S tetradentate ligands, which are characterised by the presence of an aromatic cycle in the framework (Scheme 1). This design (i) enhances the sp 2 character of the nitrogen atom bonded to the metal, (ii) promotes rigidity to the square-pyramidal base which can favour and stabilise the chelate ring by an entropic effect, 5 and (iii) avoids the formation of isomers. Thus, coordination of selected tetraanionic tetradentate ligands (Scheme 1) towards usual rhenium (ReOCl 3 (PPh ON 2 S)] 1b and [NBu 4 ][ 99g TcO(Ph-ON 2 S)] 1c were characterised by single-crystal X-ray analyses, the latter constituting the first example of 99g Tc crystal structure with this kind of semi-rigid ligands.

The reactivity of these ligands with 99m Tc and the stability of the corresponding radiocomplexes, were also investigated. While trianionic tetradentate ligands gave quite stable [ 99m TcO] 3+ complexes with two syn/anti epimers, 6 these tetraanionic tetradentate ligands produced unique and highly stable [ 99m TcO] 3+ species, 7 in accordance to a recent contribution of Papachristou et al. 8 Moreover, our new tetraanionic chelating ligands can be functionalized through the aromatic ring (by amine or acid function) to obtain bifunctional chelating agents (BCAs) for targeting purposes.

The structural identity of the corresponding Re, 99g Tc and 99m Tc compounds was assessed by means of HPLC studies.

Results and discussion

The ligands Four tetradentate semi-rigid ligands (Ph-ON 2 S(Trt), Ph-S(Trt)N 2 S(Trt), Ph-N 3 S(Trt), Ph-N(Me)N 2 S(Trt), general abbreviation, Ph-XN 2 S; Trt = trityl) have been used as chelating agents in the present work. They were synthesised in good yield via multistep reactions as previously described. 7 Ph-XN 2 S ligands act as tetraanionic ligands X -N -N -S -, by coordination via the charged X aromatic function, the two amide nitrogens and the sulfur atoms. Very preliminary studies on radiolabelling of these ligands showed that they form stable 99m Tc complexes 1d-4d at tracer level. 7

Preparation of rhenium(V) complexes

Ligand-exchange reactions of labile ReOCl 3 (PPh 3 ) 2 9 with a slight excess of the relevant tetradentate ligand (1 : 1.3 metal : ligand ratio) in the presence of a deprotonating agent (Scheme 2) Scheme 2 Preparation of the rhenium complexes.

gave [MO(Ph-XN 2 S)] -compounds in high yield. In more detail, reactions of Ph-ON 2 S(Trt) or Ph-S(Trt)N 2 S(Trt) with ReOCl 3 (PPh 3 ) 2 in the presence of sodium acetate in methanol or toluene resulted in oxorhenium complexes 1a or 2a as the sodium salts in 97 and 83% yield, respectively. The cleavage of trityl group was accomplished during the coordination of the ligand to the ReO 3+ core and this is in agreement with the acidic contribution of the metal in the mechanism of sulfur detritylation. 10 By using the above detailed conditions with Ph-N 3 S(Trt) or Ph-N(Me)N 2 S(Trt), only a mixture of several rhenium complexes were obtained. However, use of triethylamine instead of sodium acetate as deprotonating agent determined a quite rapid change of the methanol solution colour from greenishyellow to the final reddish-brown. The coordination took place in 4 h at reflux and led, for each ligand, to a unique oxorhenium complex (3a, 4a) as the triethylammonium salt, in good yield (>85%). The presence of triethylammonium chloride as a persistent contaminant, even after column chromatography purification, suggested the replacement of the countercation with larger PPh 4 + . Metathesis reactions were performed in dichloromethane/methanol solutions at room temperature. Under these conditions, analytically pure complexes 1b-4b were obtained in high yield. Complex 1b gave red crystals suitable for X-ray analysis from dichloromethane-methanol solution. This compound, as well as all other complexes prepared in this study, contains a metal-monooxo core with no additional group coordinated trans to the yl-oxygen. This behaviour is typical of anionic five-coordinated complexes in which the ligand contains good p donor atoms. Rhenium complexation of the trityl protected Ph-ON 2 S(Trt) ligand was also performed in refluxing methanol by direct reduction of tetrabutylammonium perrhenate in the presence of an excess of tin chloride, 13 giving [NBu 4 ][Ph-ON 2 S], 1e.

These oxo-rhenium compounds are soluble in dichloromethane, chloroform, methanol and insoluble in water. They are indefinitely stable in the solid state as well as in organic solvents (for a period of months) as shown by HPLC and NMR. The stability is not affected by the presence of air or moisture.

All the oxo-rhenium complexes were unambiguously identified by conventional analytical and spectroscopic techniques. In particular, IR spectra showed an intense band in the range 948-969 cm -1 attributable to the Re=O stretching vibration, 14 and two strong bands in the range 1618-1667 cm -1 assigned to m C=O of both amides (Table 1). No multiple strong bands were detected in the ranges 1500-1560 and 3200-3400 cm -1 of complexes 1a, 1b, 1e, 2a and 2b, confirming that the amides were deprotonated. [START_REF] Lambert | Introduction to Organic Spectroscopy[END_REF] Conversely, complexes 3a, 4a and 3b exhibited vibrations in the 3200-3400 cm -1 range, due to the presence of the triethylammonium counter-cation and/or of the aromatic amine N-H stretching vibrations, respectively. The IR spectra of complexes 1b-4b exhibited five additional bands of the PPh 4 + moiety. Proton NMR of Re-complexes revealed the loss of both amide and XR (XR = OH, NH 2 or NHMe) hydrogens indicating deprotonation and coordination. In complexes 3a,b and 4a,b the loss of aromatic amine nitrogen hydrogens, which is unusual in amines, may be attributed to the greater acidity imparted to the aniline-like amine hydrogens by conjugation with the aromatic ring. [START_REF] Schultze | [END_REF] Moreover, only a unique signal for hydrogen (3a,b) and the methyl group (4a,b) of the aromatic nitrogen, respectively, was detected indicating sp 2 hybridization for the aromatic amine nitrogen. Methylene protons of the tetradentate ligand framework were found to be diastereotopic (AB pattern) with coupling constants in the range 17-19 Hz. Exo and endo a protons of the glycine residue were detected at 4.54 (H 2a ) and 5.46 ppm (H 2b ) for 1a, downfield shifted compared to the corresponding protons in the uncoordinated ligand (3.76 ppm). Similarly, the a protons close to the thiolate group were deshielded from 3.21 ppm in the uncoordinated ligand to 3.81 (H 1a ) and 4.15 ppm (H 1b ) for 1a (Fig. 1). Similar magnetic inequivalence of CH 2 protons attached to amide donor atoms was observed in MAG 3 Re type complexes. 17 According to the literature, 10 we assigned the downfield signal of both AB patterns to the endo protons (syn to the Re=O group) and the upfield signals to the exo ones (anti to the Re=O group). 18 13 C NMR spectra confirmed coordination of the ligands showing remarkable downfield shifts of the carbon signals nearby the ligating atom set compared to those of the corresponding carbons in the free ligand. For example, the amide carbons were downfield shifted by 20-25 ppm upon coordination, 19 and, analogously, the glycine a carbons moved from 42-45 ppm in the free ligands to 60-62 ppm in the complexes.

Negative-ion ESI-MS spectra of each Re-complex showed the parent peak with the correct isotope distribution pattern consistent with the monomeric anion, without significant fragmentation. Conductivity measurements gave values in methanol consistent with a 1 : 1 electrolyte type.

Synthesis of 99g Tc complexes

99g Tc complexes 1-3c were synthesised by ligand-exchange reactions starting from [NBu 4 ][ 99g TcOCl 4 ] 20 and the relevant ligand in methanol at room temperature. Addition of triethylamine was necessary to isolate pure 3c. In contrast, complex 4c was always collected as a mixture of two species (Scheme 3).

The IR features of 1c-3c were similar to those observed in oxorhenium analogues. The Tc=O stretching vibration appeared consistently at 950 cm -1 , ca. 15-20 cm -1 ipsochromic shifted compared to the vibrations observed in the corresponding oxorhenium compounds. This difference is common in series of isostructural mono-oxo Tc and Re species. 21, 22 Analogously, 1 H and 13 C NMR values do not change significantly on going from oxorhenium to oxotechnetium complexes, indicating structural similarity (Table 1) that was confirmed by the X-ray analysis of complex 1c.

X-Ray crystallography

The refinement as well as selected bond distances and angles involving the atoms of the anions are summarised in Tables 2 and3, respectively. Perspectives drawings of the two complexes are shown in Figs. 2 and3.

Both compounds are monoanionic, the [MO] 3+ core charge being balanced by the four hydrolisable functions of the tetradentate ligand. Each asymmetric unit cell contains only one metal complex, and the absolute configuration around the metal is clockwise. The stereochemical notation to describe this situation is (SP-5-24-C)-1b and (SP-5-24-C)-1c. 23 The acronym means that the complex assumes a square-pyramidal geometry with coordination number 5, 24 is the configuration number according to the CIP (Cahn-Ingold-Prelog) rules, 24 and the symbol C is assigned to that configuration in which the CIP priority numbers of the ligating atoms in the equatorial plane increase proceeding in a clockwise manner when viewing the plane from the ligating atom on the C 4 axis.

Thus, [MO(Ph-ON 2 S)] -complexes exhibit a distorted squarepyramidal geometry with yl-oxygen in the apical position. The square planes defined by the combination of nitrogen, oxygen and sulfur atoms are little distorted, with the two amide nitrogens and the phenolate oxygen in the plane and the sulfur atom lying below the plane by about 0.28 and 0.30 A ˚in 1b and 1c, respectively. Re and Tc lie above the mean plane by 0.67 and

(4)-O(2) 1.225(9) 1.216(5) C(4)-N(2) 1.388(9) 1.375(5) C(5)-N(2) 1.398(8) 1.414(5) C(10)-O(3) 1.347(8) 1.359(4) O(4)-M(1)-N(1) 110.9(3) 109.7(1) O(4)-M(1)-O(3) 109.3(2) 110.8(1) O(4)-M(1)-N(2) 114.1(2) 114.3(1) O(4)-M(1)-S(1) 110.4(2) 110.9(1) O(3)-M(1)-N(2) 78.6(2) 79.2(1) N(2)-M(1)-N(1)
78.4(2) 78.9(1) N( 1 HPLC profiles of Re-, 99g Tc-and 99m Tc-complexes (Fig. 4), obtained by using a combination of UV-vis and radiometric detections, revealed that these three species possessed similar retention times (Table 4), thereby indicating identity of their molecular structures. 7, 28 The minor difference of retention times observed between Tc and Re complexes could be explained by the different polarities of the [Tc=O] 3+ and [Re=O] 3+ group. [START_REF] Hilger | Technetium, Rhenium and Other Metals in Chemistry and Nuclear Medecine 5[END_REF] These results indicate Fig. 4 Comparative reverse-phase HPLC chromatograms: 2b (rhenium complex, UV recording at 270 nm, t r = 2.51 min), 2c ( 99g Tc complex, UV recording at 270 nm, t r = 2.64 min), 2d ( 99m Tc complex, radiometric detection, t r = 2.74 min).

that our semi-rigid ligands form similar 1 : 1 complexes at macroscopic (Re/ 99g Tc) and at tracer level ( 99m Tc).

To conclude, in this paper we have shown that semi-rigid Ph-XN 2 S-type ligands allow stabilization of identical mono-oxo Re and Tc species both in the solid and in the solution states. The incorporation of an aromatic ring fused into the chelate backbone imparts rigidity to a portion of the ligand enhancing the formation and stability of the resulting complex by an entropy effect. The high in vivo stability of the corresponding 99m Tc complexes established before 7 with the structural details and correlations reported here provide support for the potential development of agents as diagnostic ( 99m Tc)/therapeutic ( 188 Re) matched pairs. Incorporation of biologically active fragments into this kind of chelate for the development of in vivo targetspecific radiopharmaceuticals remains our main objective.

Experimental

Safety note CAUTION!: 99g Tc is a weak b-emitter (E b-= 0.292 MeV, t 1/2 = 2.12 × 10 5 years). All manipulations were carried out in laboratories approved for low-level radioactivity using monitored hoods and glove-boxes. When handled in milligram amounts, 99 Tc does not present a serious health hazard because common laboratory glassware provides adequate shielding. Bremsstrahlung is not a significant problem due to the low energy of the b-particles. However, normal radiation safety procedures must be used at all times, especially with solid samples, to prevent contamination and inhalation.

General

All chemicals were of the highest purity commercially available. Solvents were purified by standard methods before use and stored over 0.3 nm molecular sieves. Technetium as [NH 4 ][ 99g TcO 4 ] was obtained from Oak Ridge National Laboratory. Samples were dissolved in water and treated with an excess of aqueous ammonia and H 2 O 2 (30%) at 80 • C prior to use to oxidise residual TcO 2 . Slow evaporation of the solvent with heating at 40 • C afforded [NH 4 ][ 99g TcO 4 ] as a white powder which was converted to the [NBu 4 ][TcOCl 4 ] precursor following a literature method. 20 Rhenium(VII) oxide was purchased from Aldrich Chem. Co. It was converted to ReOCl 3 (PPh 3 ) 2 according to published protocols. 9 The ligands N-( 2

Instrumentation

For rhenium compounds, NMR spectra were recorded on a Bruker AC 200 (50.323 MHz for 13 C and 200.133 MHz for 1 H) or 250 apparatus (62.896 MHz for 13 C and 250.133 MHz for 1 H). Chemical shifts are indicated in d values (ppm) downfield from internal TMS. 1 H NMR and 13 C NMR spectra of each complex were recorded at room temperature in CDCl 3 except for 1a, 2a which were performed in MeOD. Unambiguous assignments, were established by two-dimensional [ 1 H, 1 H] COSY and [ 1 H, 13 C] HMQC and HMBC experiments (Table 1).

IR spectra were recorded as KBr pellets on a Bruker Vector 22 spectrophotometer in the range 4000-400 cm -1 . Negative electrospray mass spectra were obtained on a NERMAG R 10-10 mass spectrometer. Microanalysis was performed by the microanalytical department of the Ecole Nationale Supérieure de Chimie de Toulouse. TLC was performed using precoated Kieselgel 60 plates F 254 (TLC plates, Merck) and was visualised by UV. Column chromatography was carried out using "gravity" silica (SDS silica gel).

For technetium compounds, NMR spectra were collected on a Bruker AC-300 apparatus (75.467 MHz for 13 C and 300.13 MHz for 1 H) using SiMe 4 as internal reference ( 1 H). FT IR spectra were recorded on a Nicolet 510P Fourier transform spectrometer in the range 4000-400 cm -1 in KBr pellets using a Spectra-Tech diffuse-reflectance collector accessory. Mass spectra of selected technetium compounds (ca. 10 -6 M solutions) were recorded on a LCQ instrument (Finnigan, Palo Alto, CA). The complexes have been directly injected by a syringe pump at a flow of 5 lL min -1 . Thin-layer chromatography (TLC) was performed using sheets from Riedel de Haen (Silica gel 60 F 254 ).

HPLC comparison was achieved on a waters 600E gradient chromatography with a Waters Lambda Max UV detector, a SAIP radioactivity detector and an ICS dual integrator for effluent monitoring and a Satisfaction RP18AB column using MeOH-H 2 O-TFA (55 : 45 : 0.1 or 70 : 30 : 0.1) as eluent (flow rate of 1 mL min -1 ). The eluate was monitored by UV absorbance at 270 nm for Re and 99g Tc complexes, or c-ray detection for 99m Tc complexes.

X-Ray crystal structure determination for compounds 1b and 1c

Diffraction measurements for 1b were performed on a Nonius Kappa CCD diffractometer using graphite-monochromated Mo-Ka radiation (k = 0.71073 A ˚) while, for 1c, diffraction data were collected on a Nicolet R3m/V diffractometer (Mo-Ka radiation; k = 0.71073 A ˚). The structures were solved by direct methods for 1b and by heavy-atom methods for 1c. Both were refined with full-matrix least-squares procedure based on F 2 . Structure determination and refinement were performed using the SIR97, [START_REF] Altomare | [END_REF] Maxus 31 and SHELX-97 32 programs. 7350 and 3700 independent reflections with I ≥ 2r(I) were used for refinement of the unit cell for 1b and 1c, respectively. Hydrogens for the disordered chain of the tBu molecule in compound 1c were not located. The most significant details of the crystallography study are reported in Table 2. A collection of selected bond distance and angles are shown in Table 3.

CCDC reference numbers 275918 and 275919. See http://dx.doi.org/10.1039/b508661b for crystallographic data in CIF or other electronic format.

Synthesis of complexes [Na][ReO(Ph-ON 2 S)] 1a and [Na][ReO(Ph-SN 2 S)] 2a.

The synthesis and characterisation of the two complexes 1a and 2a have been reported previously. 7

[PPh 4 ][ReO(Ph-ON 2 S)] 1b. 1a (100.0 mg, 0.22 mmol) and tetraphenylphosphonium chloride (89.2 mg, 0.24 mmol) were dissolved in a mixture of MeOH (15 mL) and CH 2 Cl 2 (15 mL). The solution was stirred for 30 min at room temperature and then allowed to precipitate during 2 days. A red solid was filtered and washed with cold water and dried under vacuum (126.3 mg, yield = 75%).

Found: C, 52. (d, 2 J HH = 18.3 Hz, 1 H, CH 2 N), 5.37 (d, 2 J HH = 18.3 Hz, 1 H, CH 2 N), 6.63 (dt, 3 J HH = 7.6 Hz, 4 J HH = 1.2 Hz, 1 H, CH Ar ), 6.75 (dt, 3 J HH = 7.6 Hz, 4 J HH = 1.5 Hz, 1 H, CH Ar ), 6.99 (dd, 3 J HH = 7.6 Hz, 4 J HH = 1.2 Hz, 1 H, CH Ar ), 7.52 (m, 8 H, CH Ar PPh4 ), 7.69 (m, 8 H, CH Ar PPh4 ), 7.83 (m, 4 H, CH Ar PPh4 ), 8.22 (dd, 3 J HH = 7.6 Hz, 4 J HH = 1.5 Hz, 1 H, CH Ar ); 13 C{ 1 H} NMR (62.5 MHz, CDCl 3 ): d C 40.9 (CH 2 S), 61.8 (CH 2 N), 116.1, 118.7, 119.5, 123.4 (4 CH Ar ), 117.3 (4 C Ar PPh4 ), 131.2, 131.4, 134.9, 135.0, 136.3, 136.4 (20 CH Ar PPh4 ), 141.4, 171.1 (2 C Ar ), 187.5, 193.9 (2 CO); MS (ES -): m/z (%) 437 (60), 439 (100) [M -].

[NBu 4 ][ReO(Ph-ON 2 S)] 1e. To a boiling solution of Ph-ON 2 S(Trt) (148.8 mg, 0.30 mmol) in 40 mL of MeOH was added a solution of tin(II) chloride (143.5 mg, 0.63 mmol, in 1 mL of 0.1 M HCl), followed immediately by a solution of tetrabutylammonium perrhenate (310.6 mg, 0.63 mmol, in 1 mL of MeOH). Refluxing was continued for 5 h, the solution cooled at room temperature and washed with 20 mL of hexane. MeOH was removed and the crude solid was dissolved in CH 2 Cl 2 (20 mL), then washed with water (20 mL). The organic layer was dried with solid Na 2 SO 4 , filtered and concentrated to dryness. The crude material was purified by column chromatography on silica gel (eluent CH 2 Cl 2 -MeOH (95 : 5)) followed by two crystallisations in methanol-ether mixture (3 : 1 v/v). Under these conditions, [Bu 4 N]Cl remained in solution and the complex was obtained as a red-orange powder (105 mg, 52%).

Found: C, 45.75; H, 6.60; N, 5.72. C 27 H 48 N 3 O 4 ReS requires C, 46.53; H, 6.94; N, 6.03%; IR: m max /cm -1 1641 (CO), 968 (ReO); 1 H NMR (250 MHz, CDCl 3 ): d H 0.99 (m, 12 H, CH 3 ), 1.49 (m, 16 H, CH 2 ), 3.07 (m, 8 H, CH 2 ), 3.79 (d, 2 J HH = 17.1 Hz, 1 H, CH 2 S), 4.13 (d, 2 J HH = 17.1 Hz, 1 H, CH 2 S), 4.57 (d, 2 J HH = 18.4 Hz, 1 H, CH 2 N), 5.53 (d, 2 J HH = 18.4 Hz, 1 H, CH 2 N), 6.75 (dt, 3 J HH = 7.8 Hz, 4 J HH = 1.2 Hz, 1 H, CH Ar ), 6.86 (dt, 3 J HH = 7.8 Hz, 4 J HH = 1.5 Hz, 1 H, CH Ar ), 7.05 (dd, 3 J HH = 7.8 Hz, 4 J HH = 1.2 Hz, 1 H, CH Ar ), 8.33 (dd, 3 J HH = 7.8 Hz, 4 J HH = 1.5 Hz, 1 H, CH Ar ); 13 C{ 1 H} NMR (75.5 MHz, CDCl 3 ): d C 13.6 (4 CH 3 ), 19.7, 23.8 (8 CH 2 ), 40.0 (CH 2 S), 58.6 (4 CH 2 ), 61.2 (1 CH 2 N), 115.5, 118.3, 119.3, 123.0 (4 CH Ar ), 140.9, 170.5 (2 C Ar ), 186.9, 193.1 (2 CO); m/z (%) 437 (60), 439 (100) [M -].

[PPh 4 ][ReO(Ph-SN 2 S)] 2b. 2a (105.0 mg, 0.22 mmol) and tetraphenylphosphonium chloride (89.2 mg, 0.24 mmol) were dissolved in a mixture of MeOH (15 mL) and CH 2 Cl 2 (15 mL). After 30 min under stirring, the solvent was removed and the crude was purified by column chromatography on silica gel (eluent: CH 2 Cl 2 -MeOH (95 : 5 then 90 : 10)) to yield the complex 2b as an orange powder (121.0 mg, yield = 73%).

Found 120.6, 122.9, 124.1, 128.1 (4 CH Ar ), 130.6, 130.7, 134.3, 134.4, 135.7, 135.8 (20 CH Ar PPh4 ), 147.3, 152.2 (2 C Ar ), 188.7, 192.2 (2 CO); MS (ES -): m/z (%) 453 (60), 455 (100) [M -].

[Et 3 NH][ReO(Ph-N 3 S)] 3a. To a stirred suspension of Ph-N 3 S(Trt) (144.3 mg, 0.30 mmol) and ReOCl 3 (PPh 3 ) 2 (324.5 mg, 0.39 mmol) in MeOH (40 mL) neat Et 3 N (148.1 lL, 1.20 mmol) was added. The mixture was refluxed for 4 h and then cooled to room temperature. The solvent was removed and the crude purified by column chromatography on silica gel (eluent: CHCl 3 -MeOH (95 : 5 then 90 : 10)) to afford the complex 3a as a red-orange powder (114.2 mg, yield = 89%).

Found (20 mL). The organic layers were dried with MgSO 4 , filtered and dried under vacuum to give a red powder (170.9 mg, yield = 99%).

Found: C, 52. 114.2, 117.6, 118.2, 122.4 (4 CH Ar ), 130.6, 130.8, 134.3, 134.4, 135.7, 135.8 (20 CH Ar PPh4 ), 140.1, 158.3 (2 C Ar ), 187.7, 192.8 (2 CO); MS (ES -): m/z (%) 436 (60), 438 (100) [M -].

[Et 3 NH][ReO(Ph-N(Me)N 2 S)] 4a. Using the same procedure adopted for 3a, Ph-N(Me)N 2 S(Trt) (148.5 mg, 0.30 mmol) gave the complex as a red solid (157.5 mg, yield = 95%) which was contaminated by traces of residual Et 3 NHCl salt.

IR: m max /cm -1 1647, 1622 (CO); 953 (ReO); 1 H NMR (250 MHz, CDCl 3 ): d H 1.25 (t, 9 H, 3 J HH = 7.3 Hz, CH 3 ), 3.01 (q, 3 J HH = 7.3 Hz, 6 H, CH 2 ), 3.83 (d, 2 116.8, 118.0 (4 C Ar PPh4 ), 110.4, 117.4, 117.7, 122.4 (4 CH Ar ), 130.6, 130.8, 134.2, 134.4, 135.8, 135.9 (20 CH (d, 2 J HH = 16.9 Hz, 1 H, CH 2 S), 4.04 (d, 2 J HH = 16.9 Hz, 1 H, CH 2 S), 4.61 (d, 2 J HH = 18.3 Hz, 1 H, CH 2 N), 5.19 (d, 2 J HH = 18.3 Hz, 1 H, CH 2 N), 6.75 (dt, 3 J HH = 7.8 Hz, 4 J HH = 1.1 Hz, 1 H, CH Ar ), 6.89 (dt, 3 J HH = 7.8 Hz, 4 J HH = 1.4 Hz, 1 H, CH Ar ), 7.23 (dd, 3 J HH = 7.8 Hz, 4 J HH = 1.1 Hz, 1 H, CH Ar ), 8.24 (dd, 3 J HH = 7.8 Hz, 4 J HH = 1.4 Hz 1H, CH Ar ); 13 C{ 1 H} NMR (75.5 MHz, CDCl 3 ): d C 13.6 (4 CH 3 ), 19.6 (4 CH 2 ), 23.8 (4 CH 2 ), 36.9 (CH 2 S), 58.6 (4 CH 2 ), 59.9 (CH 2 N), 114. 7, 118.3, 118.6, 122.9 IR: m max /cm -1 1655, 1638 (CO); 945 (TcO); 1 H NMR (300 MHz, CDCl 3 ): d H 0.94 (m, 12 H, CH 3 ), 1.33 (m, 16 H, CH 2 ), 2.86 (m, 8 H, CH 2 ), 3.85 (d, 2 J HH = 16.9 Hz, 1 H, CH 2 S); 4.10 (d, 2 J HH = 16.9 Hz, 1 H, CH 2 S), 4.72 (d, 2 J HH = 18.9 Hz, 1 H, CH 2 N), 4.82 (d, 2 J HH = 18.9 Hz, 1 H, CH 2 N), 6.96 (t, 3 J HH = 7.6 Hz, 1 H, CH Ar ), 7.06 (t, 3 J HH = 7.6 Hz, 1 H, CH Ar ), 7.48 (d, 3 J HH = 7.6 Hz, 1 H, CH Ar ), 8.75 (d, 3 J HH = 7.6 Hz, 1 H, CH Ar ); 13 C{ 1 H} NMR (75.5 MHz, CDCl 3 ): d C 13.6 (4 CH 3 ), 19.6 (4 CH 2 ), 23.7 (4 CH 2 ), 37.3 (CH 2 S), 58.5 (4 CH 2 ), 59.3 (CH 2 N), 120. 6, 122.8, 124.3, 127.0 (15.4 mg, 0.032 mmol) and an excess of triethylamine (22.5 lL, 0.160 mmol). The reaction was stirred for 30 min and the solution turned dark orange. The crude solution was then treated twice with hexane (2 × 3 mL) and then the solvents were removed. The solid was dissolved in CH 2 Cl 2 (5 mL), treated with water (5 mL), and the organic layers were separated, dried and concentrated to dryness. The title compound was obtained as a yellow-orange solid (15 mg, 79%).

1 H NMR (300 MHz, CDCl 3 ): d H 0.95 (m, 12 H, CH 3 ), 1.30 (m, 16 H, CH 2 ), 2.84 (m, 8 H, CH 2 ), 3.72 (d, 2 J HH = 16.7 Hz, 1 H, CH 2 S), 4.07 (d, 2 J HH = 16.7 Hz, 1 H, CH 2 S), 4.58 (d, 2 J HH = 18.6 Hz, 1 H, CH 2 N), 4.93 (d, 2 J HH = 18.6 Hz, 1 H, CH 2 N), 6.65 (t, 3 J HH = 7.2 Hz, 1 H, CH Ar ), 6.80 (m, 2 H, CH Ar ), 8.03 (s, 1 H, NH), 8.28 (d, 3 J HH = 7.2 Hz, 1 H, CH Ar ); 13

  Scheme 1 Ph-XN 2 S Ligands.

Fig. 1

 1 Fig. 1 Proton NMR spectra for complex 1a (MeOD, 25 • C).

  Scheme 3 Synthesis of 99g Tc complexes 1c, 2c, 3c.

Fig. 2

 2 Fig.2An ORTEP drawing of the asymmetric anionic unit [ReO(Ph-ON 2 S)] -of complex 1b with its numbering scheme. Thermal ellipsoids are drawn at the 50% probability level.

Fig. 3

 3 Fig.3Perspective view and atom-labelling scheme of the asymmetric anionic unit [ 99g TcO(Ph-ON 2 S)] -of complex 1c. All non hydrogen atoms are represented by their 50% thermal probability ellipsoids.

a

  Eluent: MeOH-H 2 O-TFA (55 : 45 : 0.1). b Eluent: MeOH-H 2 O-TFA (70 : 30 : 0.1).

  -hydroxyphenyl)-2-[(triphenylmethylthio)methylcarbonylamino]ethanamide, Ph-ON 2 S(Trt); N-[2-triphenylmethylthio)phenyl]-2-[(triphenylmethylthio)methylcarbonylamino]ethanamide, Ph-S(Trt)N 2 S-(Trt);N-(2-aminophenyl)-2-[triphenylmethylthio)methylcarbonylamino]ethanamide, Ph-N 3 S(Trt); N-[2-(N-methylamino)phenyl]-2-[triphenylmethylthio)-methylcarbonylamino]ethanamide, Ph-N(Me)N 2 S(Trt) and the corresponding 99m Tc complexes have been prepared as described previously.7

  (4 CH Ar ), 139.5, 165.2 (2 C Ar ), 182.5, 185.9 (2 CO); MS (ES -) m/z (%) 351 (100) [M -]. [NBu 4 ][TcO(Ph-SN 2 S)] 2c. As for 1c, 25 mg (0.05 mmol) of Ph-S(Trt)N 2 S(Trt) gave the complex as a yellow-brown oil.

  (4 CH Ar ), 140.3, 150.1 (2 C Ar ), 184.0, 186.4 (2 CO); MS (ES -): m/z (%) 367 (100) [M -]. [NBu 4 ][TcO(Ph-N 3 S)] 3c. To a stirred green solution of [NBu 4 ][TcOCl 4 ] (16.0 mg, 0.032 mmol) in MeOH (3 mL) was added Ph-N 3 S(Trt)

11,12

Table 1

 1 Selected 1 H,13 C NMR and IR data of oxorhenium and oxotechnetium complexes 1-4

	NMR

Table 2

 2 Crystal data and structure refinement for 1b and 1c complexes

	Complex	[PPh 4 ][ReO(Ph-ON 2 S)] 1b	[NBu 4 ][ 99g TcO(Ph-ON 2 S)] 1c
	Formula	C 34 H 28 N 2 O 4 PReS	C 26 H 44 N 3 O 4 STc
	M	777.85	592.70
	Crystal system	Orthorhombic	Monoclinic
	Color	Dark red	Brown
	Crystal size/mm	0.21 × 0.14 × 0.64	0.20 × 0.12 × 0.06
	Space group	P2 1 2 1 2 1	P2 1 /n
	a/A ˚9.600(4)	9.114(2)
	b/A ˚14.588(3)	18.383(4)
	c/A ˚21.525(9)	17.360(4)
	b/ •	90	91.32(3)
	V /A ˚3	3015(2)	2907.8(9)
	Z	4	4
	T/K	298	293
	D c /g cm -3	1.714	1.354
	F(000)	1536	1248
	Total reflections	35340	7250
	Independent reflections	9963	7250
	Observed reflections [I > 2r(I)]	6195	3700
	Data/restraints/parameters	6195/0/378	3700/0/315
	Final R indices [I > 2r(I)]	R 1 = 0.0505, wR 2 = 0.0962	R 1 = 0.0454, wR 2 = 0.1064
	R Indices (all data)	R 1 = 0.1070, wR 2 = 0.1080	R 1 = 0.0900, wR 2 = 0.1286
	GOF	0.947	0.881

Table 3

 3

		[PPh 4 ][ReO(Ph-ON 2 S)] 1b	[NBu 4 ][ 99g TcO(Ph-ON 2 S)] 1c
	M(1)-O(4)	1.675(4)	1.653(2)
	M(1)-O(3)	2.000(4)	1.986(2)
	M(1)-N(2)	1.999(5)	1.984(3)
	M(1)-N(1)	1.983(6)	1.960(3)
	M(1)-S(1)	2.276(2)	2.276(1)
	C(1)-S(1)	1.788(9)	1.822(4)
	C(2)-O(1)	1.208(9)	1.219(4)
	C(2)-N(1)	1.356(9)	1.365(5)
	C

Selected bond lengths (A ˚) and angles ( • ) in complexes 1b and 1c

Table 4

 4 HPLC retention times of the 99m Tc, 99g Tc and 185/187 Re complexes

		Metallic complexes
	Ligand	99m TcO	99g TcO	ReO
	Ph-ON 2 S(Trt) a	4.31 (1d)	4.21 (1c)	3.89 (1b)
	Ph-S(Trt)N 2 S(Trt) b	2.74 (2d)	2.64 (2c)	2.51 (2b)
	Ph-N 3 S(Trt) a	3.59 (3d)	3.50 (3c)	3.87 (3b)
	Ph-N(SMe)N 2 S(Trt) b	2.98 (4d)	-	2.87 (4b)

  23; H, 3.63; N, 3.46. C 34 H 28 N 2 O 4 PReS requires C, 52.50; H, 3.63; N, 3.60%; IR: m max /cm -1 1667, 1638 (CO); 969 (ReO); 1 H NMR (250 MHz, CDCl 3 ): d H 3.71 (d, 2 J HH = 16.8 Hz, 1 H, CH 2 S), 4.01 (d, 2 J HH = 16.8 Hz, 1 H, CH 2 S), 4.44

  : C, 50.86; H, 3.48; N, 3.17. C 34 H 28 N 2 O 3 PReS 2 requires C, 51.44; H, 3.55; N, 3.53; IR: m max /cm -1 1665, 1625 (CO); 962 (ReO); 1 H NMR (250 MHz, CDCl 3 ): d H 3.72 (d, 2 J HH = 17.1 Hz, 1 H, CH 2 S), 3.87 (d, 2 J HH = 17.1 Hz, 1 H, CH 2 S), 4.56 (d, 2 J HH = 18.9 Hz, 1 H, CH 2 N), 4.88 (d, 2 J HH = 18.9 Hz, 1 H, CH 2 N), 6.85 (m, 2 H, CH Ar ), 7.49 (m, 9 H, 1 CH Ar + 8 CH Ar PPh4 ), 7.65 (m, 8 H, CH Ar PPh4 ), 7.80 (m, 4 H, CH Ar PPh4 ), 8.75 (m, 1 H, CH Ar ); 13 C{ 1 H} NMR (75.5 MHz, CDCl 3 ): d C 39.3 (CH 2 S), 60.3 (CH 2 N), 116.8, 118.0 (4 C Ar PPh4 ),

PPh 4 ][ReO(Ph-N 3 S)] 3b.

  : C, 35.16; H, 4.52; N, 10.68. C 16 H 25 N 4 O 3 ReS requires C, 35.61; H, 4.67; N, 10.38%; IR: m max /cm -1 1635, 1613 (CO); 956 (ReO); 1 H NMR (250 MHz, CDCl 3 ): d H 1.23 (t, 3 J HH = 7.4 Hz, 9 H, CH 3 ), 3.09 (q, 3 J HH = 7.4 Hz, 6 H, NCH 2 ), 3.81 (d, 2 J HH = 17.2 Hz, 1 H, CH 2 S), 4.02 (d, 2 J HH = 17.2 Hz, 1 H, CH 2 S), 4.55 (d, 2 J HH = 18.7 Hz, 1 H, CH 2 N), 5.12 (d, 2 J HH = 18.7 Hz, 1 H, CH 2 N), 6.62 (m, 1 H, CH Ar ), 6.88 (m, 2 H, CH Ar ), 8.21 (m, 1 H, CH Ar ); 13 C{ 1 H} NMR (75.5 MHz, CDCl 3 ): d C 7.8 (3 CH 3 ), 40.4 (CH 2 S), 46.5 (3 CH 2 ), 60.0 (1 CH 2 N), 114.2, 117.5, 117.8, 123.1 (4 CH Ar ), 140.9, 158.9 (2 C Ar ), 188.1, 194.0 (2 CO); MS (ES -): m/z (%) 436 (60), 438 (100) [M -]. To a stirred solution of 3a (118.8 mg, 0.22 mmol) in 10 mL of MeOH and 10 mL of CH 2 Cl 2 was added tetraphenylphosphonium chloride (71.2 mg, 0.19 mmol). The mixture was stirred for 15 min and then treated with H 2 O (20 mL). The solution was extracted twice with CH 2 Cl 2

	[

4 ][ReO(Ph-N(Me)N 2 S)] 4b.

  This complex was prepared analogously to 2b in 60% yield using CH 2 Cl 2 -MeOH (98 : 2) as eluent for column chromatography purification. Found: C, 52.27; H, 3.89; N, 4.93. C 35 H 31 N 3 O 3 PReS requires C, 53.15; H, 3.95; N, 5.31%; IR: m max /cm -1 1649, 1625 (CO); 954 (ReO); 1 H NMR (250 MHz, CDCl 3 ): 3.79 (d, 2 J HH = 17.1 Hz, 1 H, CH 2 S), 3.97 (d, 2 J HH = 17.1 Hz, 1 H, CH 2 S), 4.14 (s, 3 H, NCH 3 ), 4.39 (d, 2 J HH = 18.6 Hz, 1 H, CH 2 N), 5.01 (d, 2 J HH = 18.6 Hz, 1 H, CH 2 N), 6.53 (m, 1 H, CH Ar ), 6.80 (m, 2 H, CH Ar ), 7.53 (m, 8 H, CH Ar PPh4 ), 7.67 (m, 8 H, CH Ar PPh4 ), 7.82 (m, 4 H, CH Ar PPh4 ), 8.28 (m, 1 H, CH Ar ); 13 C{ 1 H} NMR (75.5 MHz, CDCl 3 ): 40.2 (CH 2 S), 49.8 (CH 3 ), 60.3 (CH 2 N), 110.4 (CH Ar ),

	J HH = 17.1 Hz, 1 H, CH 2 S),
	3.99 (d, 2 J HH = 17.1 Hz, 1 H, CH 2 S), 4.14 (s, 3 H, CH 3 ), 4.49
	(d, 2 J HH = 18.6 Hz, 1 H, CH 2 N), 5.04 (d, 2 J HH = 18.6 Hz, 1 H,
	CH 2 N), 6.66 (m, 1 H, CH Ar ), 6.87 (m, 2 H, CH Ar ), 8.28 (m, 1 H,
	CH Ar ); 13 C{ 1 H} NMR (75.5 MHz, CDCl 3 ): d C 9.1 (3 CH 3 ), 40.8
	(1 CH 2 S), 47.3 (3 CH 2 ), 49.9 (CH 3 ), 60.3 (CH 2 N), 110.9, 117.7,
	118.2, 123.3 (4 CH Ar ), 142.9, 160.0 (2 C Ar ), 187.6, 193.4 (2 CO);
	MS (ES

-): m/z (%) 450 (60), 452 (100) [M -].

[PPh

  Trt) (30 mg, 0.062 mmol) was added. The solution turned orange suddenly. After 1 h stirring at room temperature, the solvent was removed by a gentle stream of N 2 and the crude was purified by column chromatography on silica gel (eluent: CH 2 Cl 2 -MeOH (95 : 5)) to afford a yellow powder(29.4 mg, 80%). IR: m max /cm -1 1654, 1634 (CO); 953 (TcO); 1 H NMR (300 MHz, CDCl 3 ): d H 0.98 (m, 12 H, CH 3 ), 1.36 (m, 16 H, CH 2 ), 2.95 (m, 8 H, CH 2 ), 3.85

	Ar PPh4 ), 143.4, 160.2 (2
	C Ar ), 187.8, 193.2 (2 CO); MS (ES -): m/z (%) 450 (60), 452 (100)
	[M -].
	[NBu 4 ][TcO(Ph-ON 2 S)] 1c. To a stirred green solution of
	[NBu 4 ][TcOCl 4 ] (31 mg, 0.062 mmol) in MeOH (3 mL) Ph-
	ON 2 S(

  C{ 1 H} NMR (75.5 MHz, CDCl 3 ): d C 13.6 (4 CH 3 ), 19.6 (4 CH 2 ), 23.6 (4 CH 2 ), 37.3 (CH 2 S), 58.3 (4 CH 2 ), 59.4 (CH 2 N), 113.6, 117.8, 118.1, 122.8 (4 CH Ar ), 139.9, 153.8 (2 C Ar ), 183.3, 186.2 (2 CO); MS (ES

-): m/z (%) 350 (100) [M -].

Acknowledgements

We are grateful to Prof. R. Chauvin (LCC, Toulouse, France) for helpful discussions.