Dr Yajun J Zhao

Prof. Dr Yong Wang

Prof. Dr Xingping P Zhou

Zhigang G Xue

Prof. Dr Xianhong H Wang

Xiaolin L Xie

Prof. Dr Rinaldo Poli

Oxygen Triggered Switchable Polymerization for One-Pot Synthesis of CO2-Based Block Copolymers from Monomer Mixtures

Keywords: Ring-opening copolymerization, organometallic mediated radical polymerization, switch catalysis, block copolymer, polypropylene carbonate

Switchable polymerization affords a unique opportunity to regulate polymer sequence and structure in a one-pot process from mixtures of monomers. Here we report a strategy that uses O2 as an external stimulus to switch the polymerization mechanism from the organometallic mediated radical polymerization (OMRP) of vinyl monomers mediated by (Salen)Co III -R [Salen=N,N'-bis(3,5-di-tertbutylsalicylidene)-1,2-cyclohexanediamine; R = alkyl] to the ringopening copolymerization (ROCOP) of CO2/epoxides. A critical issue is the unprecedented mono oxygen insertion into the Co-C bond, as demonstrated by experimental results and rationalized by DFT calculations, leading to the formation of (Salen)Co III -O-R as an active species to initiate ROCOP. Diblock poly(vinyl acetate)-bpolycarbonate could be obtained from ROCOP of CO2/epoxides with preactivation of (Salen)Co end-capped poly(vinyl acetate). Furthermore, a poly(vinyl acetate)-b-poly(methyl acrylate)-bpolycarbonate triblock copolymer has been successfully synthesized via a (Salen)cobalt-mediated sequential polymerization with an O2triggered switch in a one-pot process.

Introduction

In nature, feedback loops and a variety of trigger-induced effects endow enzymes with necessary temporal and spatial control to ensure highly-ordered biological activities through extremely complex catalytic processes. [START_REF] Traut | Allosteric Regulatory Enzymes[END_REF] Such smart activities have evolved into an intriguing design principle for catalysis systems to simplify the procedures for obtaining a high degree of structural complexities. [2] Specifically, switchable catalysts that display different polymerization activities in response to stimuli provide a unique opportunity to regulate polymer sequence and architecture from mixtures of monomers for fine tuning the material properties at the molecular scale. [3] Typically, modulating the polymerization activity of a known catalyst is achieved by incorporation of stimulus-responsive functional group into the ligand as well as by direct alternation of the oxidation state of the metal center. [4] For example, Diaconescu pioneered a redox switch that allowed the one-pot synthesis of polylactide-b-polycaprolactone. [5] Byers demonstrated that controlling the oxidation state of Fe catalysts by redox or electrochemical triggers enabled the facile discrimination of lactide and epoxide in the ring-opening polymerization (ROP). [6] Long and coworkers have successfully tailored the polyethylene branching via a redox switch. [7] Recently, Williams and Rieger achieved switchable ROCOP of CO2/anhydrides/epoxides and ROP of cyclic esters by controlling the chemistry of metal-chain end-group, demonstrating the possibility to switch the reactivity of a single-site catalyst between different polymerization mechanisms [8] However, the limitation is that ROP and ROCOP share the metal-alkoxide active species. Scheme 1. Mechanistic aspects for ROCOP and OMRP mediated by (Salen)Co complex. a Chain propagation process for cobalt(III) complex mediated ROCOP of CO2/epoxides. b Reversible termination (RT) and degenerative transfer (DT) mechanisms occurring in cobalt(II)/cobalt(III) complex mediated OMRP.

Derived from the ROCOP of CO2/epoxides, CO2-based polycarbonates have been drawing increasing attention owing to their biodegradable and biocompatible properties as well as to the stringent demand for CO2 utilization and sustainable chemistry. [9] Although CO2-based polycarbonates may suffer from poor mechanical/thermal properties and limited functionalities, the living nature of ROCOP in the presence of various metal complexes enables the facile synthesis of CO2-based block copolymers as a possible solution. [10] Current achievements have been mainly focused on the sequential addition of epoxide monomers to afford polycarbonate-b-polycarbonate or on the preparation of polyester-b-polycarbonate by combining the ROCOP of CO2/epoxides and the ROP of cyclic esters. [8] However, the possibility to switch polymerization mechanism gives access to block copolymers with mechanistically incompatible monomers, therefore providing facile access to a wider array of high-value-added nanomaterials with attractive phase separation behavior. [11] To address this, we have recently reported a one-step route to CO2-based block copolymers with tailorable functionalities and compositions by concurrent RAFT polymerization of conjugated vinyl monomers and ROCOP of CO2/epoxides using a bifunctional chain transfer approach. [12] Yet, new methods need to be developed to further expand the structural diversity of CO2-based block copolymers.

Scheme 2. O2-triggerd switch from cobalt mediated OMRP to ROCOP for the synthesis of CO2-based diblock copolymers. Among all the effective catalysts for ROCOP, (Salen)Co III X (X = Cl, OAc etc.) complexes, first employed by Coates, have displayed the most ideal activities, wherein the reversible formation and dissociation of polar Co-O bonds are crucial for the chain propagation process (Scheme 1a). [13] Relative to Co-O bonds, Co-C bonds are less polar and tend to break homolytically, which is key to various enzymatic reactions mediated by vitamin B12. [14] Recently, cobalt complexes have allowed the wellcontrolled organometallic mediated radical polymerization (OMRP) of challenging non-conjugated monomers such as vinyl acetate (VAc), [15] vinyl chloride, [16] N-vinyl amides [17] and vinylidene fluoride (VDF) [18] (Scheme 1b). In particular, the radical polymerization of VAc was shown to be well-controlled also when mediated by the (Salen)Co II system through a degenerative transfer mechanism with AIBN initiation at 60 °C, leading to the generation of (Salen)Co III -PVAc as dormant species. [19] Herein, we report the switch of polymerization mechanism from OMRP to ROCOP using O2 as a trigger and cobalt Salen complexes as mediators (Scheme 2). Key to this protocol is the unprecedented mono oxygen insertion into Co III -C bonds, which generates (Salen)Co III -O-R as an active species for the ROCOP of CO2/epoxides.

Results and Discussion

The first step of the polymer synthesis consisted of the OMRP of VAc mediated by (Salen)Co II , with initiation by AIBN at 60 °C in bulk, following the protocol of Peng et al. [20] The only difference consisted in running the polymerization in an autoclave rather than in regular glassware, in order to facilitate the one-pot switch to the ROCOP step, which requires work under CO2 pressure. This also allowed the reaction to occur under exclusion of light. [21] Various (Salen)Co III -PVAc products were obtained under different conditions (Table 1), confirming the results reported by Peng et al. and also demonstrating the purely thermal nature of this OMRP system. Indeed, comparative experiments conducted in ampules without protection from light displayed faster polymerization rates but inferior control (Table 1, entries 3-6). Moreover, higher AIBN loading significantly accelerated the polymerization but gave products with broader molecular weight distributions. An [AIBN]/[Co II ] ratio of 7/1 could well balance the efficiency and polymerization control (Figures S1-S2). The kinetic plots for the OMRP displayed good linearity and an initiation period of 310 min, which is typical for this system. [20] Also, repeat experiments showed excellent reproducibility. Electrospray ionization (ESI) mass analysis of low molecular weight (Salen)Co III -PVAc (Mn = 2.1 kDa, Đ = 1.05) was performed in methanol, wherein two main series of signals were observed in accordance with [CN(CH3)2C(VAc)n]Na + and [CN(CH3)2C(VAc)n]H + , respectively (Figure . 1a). This suggests that the cyanoisopropyl radical generated from AIBN [ • C(CH3)2CN] predominantly adds to VAc and generates (Salen)Co III -(VAc)n-C(CH3)2CN dormant species (Figure 2). In this respect, this controlling system differs from (TMP)Co II (TMP = tetramesityl porphyrin), which was shown by Wayland et al. to abstract a β-H atom from • C(CH3)2CN and ultimately generate the (TMP)Co III -(VAc)n-H dormant species. [22] Figure 1. Polymer chain end group analysis using ESI spectra. a (Salen)Co III -PVAc (Mn,GPC=2.1 kDa, Đ=1.05). b The acidolysis product of (Salen)Co III -PVAc (Mn,GPC=1.8 kDa, Đ=1.07) in the presence of 3.0 MPa O2 and acetic acid.

To switch from the VAc OMRP to the CO2/epoxide ROCOP, a selective transformation of (Salen)Co III -PVAc into (Salen)Co III -O-PVAc is needed. Although the ROCOP process catalyzed by an alkoxide derivative of cobalt(III) appears unprecedented, metal alkoxide complexes (L-M-OR) are generally regarded as the active species when the reaction is initiated by related L-M-X complexes (X = halides or carboxylic groups), [13b, 23] which are preferred precatalysts given their lower tendency to decompose by hydrolysis. During the initiation periods, nucleophilic attack by X facilitates the ring-opening of an epoxide and results in the L-M-OR active species. Therefore, we have investigated the reaction of the (Salen)Co III -PVAc product of the (Salen)Co II -mediated VAc polymerization (Mn = 16.1 kDa, Đ = 1.09) with O2 under different conditions. An initial study with O2 bubbling at atmospheric pressure in the presence of acetic acid (HOAc) yielded a colorless polymer by precipitation in hexane, confirmed to be PVAc by 1 H NMR spectroscopy (Figure S4). The FT-IR investigation of this polymer revealed a broad absorption band around 3400 cm -1 , indicative of the substitution of the cobalt chain-end by an OH group (Figure S5). This conclusion was confirmed by the ESI-MS spectrum (Figure 1b), which exhibited two main series of signals in accordance with [CN(CH3)2C(VAc)nOH]Na + and [CN(CH3)2C(VAc)nOH]H + . This result suggests the predominance of oxidation over acidolysis, because the latter should yield PVAc-H. On the basis of the known aptitude of (Salen)Co III -OR to be readily hydrolyzed by protonic reagents to yield ROH, [24] we propose that the observed PVAc-OH results from the acidolysis of a (Salen)Co III -O-PVAc intermediate by HOAc, with the concomitant formation of (Salen)Co III -OAc (Figures S6-S7). Moreover, the GPC curve of the polymer exhibited a bimodal distribution with a main component at 14.9 kDa and a minor one at 31.2 kDa (Figure 3a). Given the monomodal distribution and low Đ of the original (Salen)Co III -PVAc, we reasoned that a side process involving some kind of irreversible radical coupling might have occurred. A comparative reaction, carried out in the autoclave under 3.0 MPa O2, yielded a chemically similar polymer, except that the GPC curve displayed this time a monomodal distribution (Đ = 1.15) (Figure 3b). The radical nature of this reaction is indicated by an experiment run in the presence of TEMPO [2,2,6,6tetramethylpiperidine-1-oxyl], leading only to the generation of (Salen)Co II (Figure S10) and TEMPO-capped PVAc (Figure S11). Thus, the reaction appears to take place by initial Co III -C bond cleavage to produce (Salen)Co II and PVAc• intermediates. Furthermore, an isotopic labeling experiment was conducted by reaction of (Salen)Co III -PVAc with 18 O2 in the presence of HOAc.

In this case, the hydrolysis product was confirmed as PVAc- 18 OH according to the ESI-MS spectrum (Figure S12), suggesting that the single O inserted into the Co-C bonds comes from O2 instead of other reagents.

On the basis of the above results, the two following points are worthy of note: (i) the unexpected insertion of a single O atom between Co and the polymer chain upon oxidation with O2; (ii) the increase of selectivity, relative to the chain-chain coupling side products, upon increasing the O2 pressure. In previous investigations of alkylcobalt(III) oxidation, using a variety of ligands such as porphyrins, [25] dimethylglyoximates, [26] and other substituted glyoximates, [27] alkylperoxocobalt(III) derivatives were generated via a radical mechanism that consists of three steps (Scheme 3): homolytic cleavage Eq. ( 1), dioxygen addition to the alkyl radical to generate a peroxyl radical Eq. ( 2) and radical recombination Eq. (3). A few of these alkylperoxo derivatives, [25b, 27b] as well as others made by other methods, [28] have been In order to throw light onto the above two points, a DFT investigation was carried out using the short alkyl group -CH(Me)OCOMe as model for the PVAc chain (see the computational details in the SI). The results are shown in Figure 4 with the changes of each individual steps shown as ΔG° at 60 °C in kcal/mol. Energy profiles for the entire transformation at 25 and 60 °C are available in the SI. The starting point of the investigation is the structure of (Salen)Co III -CH(Me)OCOMe and the sequence of Eq ( 1), ( 2) and ( 3) in Scheme 3. The starting complex is diamagnetic with a square pyramidal geometry, as experimentally found in several (Salen)Co III X derivatives, e.g. with X = Cl, Br or I, [29] O-3,5-C6H3F2, [30] COOMe, [19b] C(=CH2)CH=CH2, [31] and CONHR (R = CH(Me)Cy, CH2C≡CH, CH2-2-C4H3O). [32] Chelation by the acetate carbonyl group, to yield a 6-coordinated isomer is endoergic, contrary to what was found for the bis(acetylacetonate) analogue, (acac)2Co III -CH(Me)-OCOMe. [33] This is probably related to the cost of rearranging the Salen ligand to a non-planar coordination environment. The homolytic Co III -C bond dissociation is 26.2 kcal/mol at 25 °C and 24.2 kcal/mol at 60 °C. Processes resulting in bond cleavage without spin state change (the antiparallel combination of the two S = ½ products yields an overall S = 0 system) have no electronic barrier for the reverse recombination process and may only have a small steric barrier related to the need to reorganize the metal coordination sphere. This was computationally verified for other similar metalalkyl systems. [34] Therefore, the thermodynamic bond strength can be considered a reasonable lower limit approximation of the kinetic activation barrier. This bond strength is much greater than that of (acac)2Co-CH(Me)OCOMe (recalculated as 9.2 kcal/mol at 25 °C at the same level of theory), in agreement with a more difficult polymerization by reversible termination for the (Salen)Co system. Using the Eyring equation and the bond strength at 60 °C as an approximation of the activation barrier yields a rate of homolytic bond cleavage of 3.4 h -1 and a half-life of 0.2 h (12 min). The O2 addition to the alkyl radical to generate • OOCH(Me)OCOMe (Figure 4 Eq. ( 2)) is exoergic as expected and can be considered quantitative in the presence of a high O2 concentration. The O2 addition to (Salen)Co II , on the other hand, is slightly endoergic, whether the adduct is calculated as a doublet or as a quartet. Trapping of the resulting alkylperoxyl radical by (Salen)Co II (Figure 4 Eq. ( 3)) is also exoergic, but only by 10.8 kcal/mol (9.0 at 60 °C). This O-based radical trapping is another example of bond formation by antiparallel combination of two S = ½ products with no electronic barrier. Thus, the formation of the (Salen)Co III -OOCH(Me)-OCOMe derivative is not an irreversible process under the experimental conditions used for the oxidation. Note that an alternative process for the alkyl radicals generated by Eq (1) is dimerization (Figure 4 Eq. ( 4)), which is a model for PVAc • termination by the combination. This process is calculated as thermodynamically very favorable (irreversible), more than the O2 addition. Hence, this process may occur at low [O2], whereas Eq. ( 2) prevails over Eq. (4) at high [O2]. Dimerization by Eq. ( 4) (Figure S12) may explain at least in part the double molar mass distribution observed in the GPC trace of Figure 3a. Since the alkylperoxyl radical is not irreversibly trapped by Eq. ( 3), it accumulates in the medium. At a certain point, reaction 5, which is thermodynamically quite favorable, becomes possible. Its rate law is k5 4) may form quantitatively before all • CH(Me)OCOMe is consumed by O2 in reaction 2. Note, however, that reaction 2 has a slight degree of reversibility at 60 °C (ΔG = -20.6 kcal/mol) and can therefore serve as a form of moderating equilibrium for the active • CH(Me)-OCOMe radical. In turn, the peroxide generated by Eq. ( 5) can homolytically break in Eq. ( 6) to generate two alkoxyl radicals,

• OCH(Me)OCOMe, with a relatively low energy cost (ΔG = 7.4 kcal/mol at 60 °C). The latter can now be trapped either by an additional • CH(Me)OCOMe radical (Figure 4 Eq. ( 7)), to yield an ether product, MeCOOCH(Me)-O-CH(Me)-OCOMe, in a very favorable (irreversible) step, or by (Salen)Co II to yield the observed (Salen)Co III -O-CH(Me)-OCOMe by Eq. ( 8). Eq. ( 7) may also be responsible for the generation of the double molar mass polymer at low O2 pressure.

It is also possible to envisage an alternative pathway leading to the final (Salen)Co III -O-CH(Me)OCOMe product, which involves a homolytic O-O bond cleavage for the intermediate alkylperoxo complex (reaction 9). This generates the (Salen)Co III -O • and • OCH(Me)OCOMe radicals, and the process is completed by reactions 10 and 8 as indicated in Figure 4. Indeed, there is precedent for the O-O bond cleavage in alkylperoxo-cobalt(III) compounds. [28b, 35] The calculations suggest that the metal complex is a Co III complex with an oxyl radical, rather than a Co IV =O complex: the system has lower energy as S = 3/2 with two unpaired electrons on Co and one on the O atom (spin densities of 1.893 and 0.910, respectively). There is also an excited S = ½ state at 8.9 kcal/mol higher in G that can also be described as an oxyl radical (spin densities of 0.161 on Co and 0.816 on O). The O-O bond cleavage of Eq. ( 9) is quite facile at 60 °C (ΔG° = 15.4 kcal/mol) and is followed by thermodynamically favorable processes. However, the competition between Eq. ( 5) and ( 3), which is much in favor of the former at 60 °C, leads us to prefer the scheme involving the sequence of reactions 1-2-5-6-8. In fact, there is also a third possible pathway, which involves a bimolecular reaction between two PVAc-OO • radicals to produce two PVAc-O • radicals and O2. This is equivalent to the sequence of Eq. ( 5) and ( 6) and would still be followed by trapping of the product PVAc-O • radical by the (salen)Co II complex (Figure 4 reaction 8). However, the second order rate law and especially the much lower calculated exergonicity of this reaction (ΔG = -18.70 kcal/mol) vs. trapping by PVAc • (Figure 4 reaction 5) makes us believe that the latter will be much faster (by application of Hammond's postulate) and prevail for the transformation of 2 PVAc • + O2 to 2 PVAc-O • .

The most important contribution of the computational study is to show that the putative alkylperoxo complex, which is not observed in this work but is amply documented in the literature for other ligand systems, is thermally fragile and not thermodynamically stable at high temperatures. It evolves to the more stable alkoxo derivative in an environment where additional alkyl radicals are present. In all the previous work dealing with alkylperoxo derivatives of Co III , these complexes were generated by either ligand exchange using hydroperoxides, ROOH (no O atom acceptors were present to produce alkoxo derivatives) or by autoxidation of alkylcobalt(III) derivatives as in the present case, but the processes were carried out at low temperatures where trapping by Eq. ( 3) becomes favorable and allows completions of the primary alkyl radical oxidation by Eq. ( 2).

Finally, the (Salen)Co III -O-PVAc product obtained by oxidation, in the absence of acetic acid, of (Salen)Co III -PVAc was tested as the initiator for the ROCOP of CO2/propylene oxide, targeting the synthesis of PVAc-b-PPC diblock copolymers. The results are collected in Table 2. When (Salen)Co III -PVAc (Mn,GPC = 10.5 kDa, Table 1, entry 8) and 0.5 equivalent of 7-methyl-1,5,7triazabicyclo[4.4.0]dec-5-ene (MTBD) (cocatalyst for ROCOP), dissolved in propylene oxide (PO), were placed under 3.0 MPa of CO2 and allowed to stir at room temperature for 48 h, no poly(propylene carbonate) (PPC) was detected by [START_REF] Traut | Allosteric Regulatory Enzymes[END_REF] H NMR (Table 2, entry 1). Also, no polymerization was observed when, either AIBN (Table 2, entry 2) or TEMPO (Table 2, entry 3) was deliberately added to the (salen)Co III -PVAc macroinitiator before the O2-triggered activation phase. It is important to note that the (Salen)Co III -PVAc used as macroinitiator was isolated by precipitation before the O2-triggered switch and subsequent ROCOP, thus eliminating any residual AIBN. When a switch was directly attempted starting from a one-pot mixture of (Salen)Co II /VAc/PO/AIBN/MTBD, the reaction only produced PVAc and cyclic propylene carbonate (PC) according to the 1 H NMR spectrum of the reaction mixture (Figure S 34). Obviously, the presence of these reagents somehow blocks the transformation of (salen)Co III -PVAc to (salen)Co III -O-PVAc. However, a comparative reaction with (Salen)Co III -PVAc preactivated by 3.0 MPa O2 led to a slight pressure drop (Table 2, entry 4). The formation of PPC with a perfectly alternating structure could be confirmed by 1 H NMR spectrum of the reaction mixture, while the GPC curve exhibited a monomodal distribution with a low Đ (Figure S15). The 1 H DOSY NMR spectrum gave a single diffusion coefficient for all the PVAc and PPC resonances, whereas two diffusion coefficients were observed for a PVAc/PPC blend (Figures S16-17). We then evaluated the generality of the ROCOP step using other epoxides including cyclohexene oxide (CHO) and styrene oxide (SO) (Table 2, entries 6-7). In all cases, block copolymers with a completely alternating structure for the polycarbonate segments were obtained. It is notable that all the produced polymers displayed low Đ. Typically, salen catalysts have been shown to be highly active for ROCOP of epoxides/CO2, especially in the presence of active co-catalysts, like MTBD. [36] However, significant decreases of catalytic activity were observed in our experiments (TOF ranging from 2 to 10 h -1 ), which is attributed to the use of a macroinitiatior. [37] Synthesis of a PVAc-b-PMA-b-PPC triblock copolymer.

(Salen)Co III -PVAc proved to be a viable macroinitiator for thermally induced chain extension with methyl acrylate (MA) in the absence of additional radical source (RT pathway), yielding a (salen)Co III -capped PVAc-b-PMA copolymer with increased molecular weight and narrow molecular wright distribution (Fig. 5b). In fact, this polymerization can be carried out in the presence of propylene oxide (but absence of CO2) and only MA was incorporated in the polymer. Starting from the mixture of (Salen)Co III -PVAc (Mn,GPC = 5.3 kDa, Table S1, entry 2), MA and PO (Figure 5a), the first step of MA polymerization by OMRP gave (salen)Co III -PMA-b-PVAc. Subsequent O2 activation and pressurization with CO2 allowed the chain extension by ROCOP with incorporation of CO2/PO, while the residual MA from the previous step was no longer incorporated. This constitutes the synthesis of a PPC-b-PMA-b-PVAc triblock copolymer with monomodal and narrow molar mass distribution in a one-pot procedure (Figure 5b) and the 1 H DOSY NMR spectrum gave a single diffusion coefficient for all the PMA, PVAc and PPC resonances, whereas three diffusion coefficients were observed for a PMA/PVAc/PPC blend (Figures S32-33). This result demonstrates that an efficient one-pot switch from OMRP to ROCOP also occurs from a (salen)Co III -capped PMA chain.

Conclusion

In summary, the first example of a switch from an organometallic mediated radical polymerization of vinyl monomers to ringopening copolymerization of CO2/epoxides has been described, using a (Salen)Co system as a common moderating agent/catalyst and O2 as a trigger for the mechanistic switch. Mechanistic aspects concerning the activation of the OMRP dormant species (Salen)Co III -R by O2 have been reasonably proposed, as confirmed by experimental results and supported by DFT calculations. The key to the switch lies in an unprecedented single oxygen atom insertion into Co-C bonds, resulting in a ROCOP-active (Salen)Co III -O-R species. Temporal control over the polymerization processes has been demonstrated, allowing for the facile synthesis of well-defined CO2-based block copolymers in a one-pot procedure.

Experimental Section

Typical Procedure for OMRP of VAc in autoclave. In an argon filled glove box, (Salen)Co II (260 mg, 0.432 mmol), AIBN (124 mg, 0.756 mmol) and VAc (2 mL, 216 mmol) were charged into a pre-dried 25 mL autoclave equipped with a magnetic stir bar. The autoclave was then taken out of the glove box and allowed to stirred at 60 o C for periods of time before it was cooled down to room temperature. A small aliquot of the polymerization mixture was taken out for 1 H NMR spectroscopy. The remained crude mixture was diluted with CH2Cl2 and precipitated in hexane to yield a dark green powder.

Typical Procedure for ROCOP of CO2/epoxide with pre-activation of (Salen)Co III -PVAc by O2. In an argon filled glove box, (Salen)Co III -PVAc (525 mg, 0.05 mmol, Mn,GPC=10.5 kDa, Đ=1.07), MTBD (3.8 mg, 0.025 mmol) and PO (3.5 mL) were charged into a pre-dried 10 mL autoclave equipped with a magnetic stir bar. The autoclave was taken out of the glove box, pressurized with 3.0 MPa O2 and allowed to stir at 60 o C for 12 h. After O2 was slowly released, the autoclave was pressured with 3.0 MPa CO2 and stirred at room temperature for 24 h. A small aliquot of the copolymerization mixture was taken out for 1 H NMR spectroscopy and the remained crude mixture was precipitated in cold methanol to yield a white powder.

Procedure for O2 triggered switch from OMRP to ROCOP. In an argon filled glove box, (Salen)Co III -PVAc (530 mg, 0.100 mmol, Mn,GPC=5.3 kDa, Đ=1.09), MTBD (7.6 mg, 0.05mmol), MA (1 mL) and PO (3.5 mL) were charged into a pre-dried 10 mL autoclave equipped with a magnetic stir bar. The autoclave was taken out of the glove box and allowed to stir at 60 o C for 10 h. After a sample was withdrawn for NMR spectra and GPC analysis, the autoclave was pressurized with 3.0 MPa O2. The reaction mixture was further stirred at 60 o C for 12 h to activate the chain end before it was cooled down to room temperature. After O2 was slowly released, the autoclave was pressurized with 3.0 MPa CO2 and stirred at room temperature for 8 h. At the end of the reaction, a small aliquot of mixture was taken out for 1 H NMR spectroscopy and the remained crude mixture was precipitated in cold methanol to yield a white powder.

Figure 2 .

 2 Figure 2. Plausible pathway for PVAc with cyanoisopropyl chain end group. Reactions of the cyanoisopropyl radical with (Salen)Co II and VAc.

Figure 3 .

 3 Figure 3. GPC curves of (Salen)Co III -PVAc (Mn,GPC = 16.1 kDa, Đ = 1.14) (black) and of the corresponding oxidation/acidolysis product (red). a Comparison of (Salen)Co III -PVA and the acidolysis product obtained with O2 bubbling at atmospheric pressure. b Comparison of (Salen)Co III -PVA and the acidolysis product obtained under a high O2 pressure (3.0 MPa).

  Scheme 3. Mechanistic aspects referring to the formation of LCo III -OOR via dioxygen insertion into Co-C bonds.

Figure 4 .

 4 Figure 4. Mechanism for the oxidation of (Salen)Co III -CH(Me)OCOMe to (Salen)Co III -OCH(Me)-OCOMe under O2. The bold blue numbers represent individual elementary steps. The bold black numbers are calculated standard Gibbs energy changes (ΔG°) at 60 °C in kcal/mol. In the geometry-optimized structures shown for starting and ending complexes, only the donor atoms of the Salen ligand are shown for clarity.

Figure 5 .

 5 Figure 5. Synthesis of triblock copolymer. a Procedures for O2-triggered switchable polymerization from mixture of (Salen)Co III -PVAc macro-initiator, MA and PO under condition: [(Salen)Co III -PVAc]/[MA]/[PO] =1/220/500, OMRP of MA within 10 h at 60 o C, conv.=30%; O2-Activation within 12 h at 60 o C under 3.0 MPa O2, ROCOP of PO/CO2 within 8 h at 25 o C under 3.0 MPa CO2, conv.=7.5%. b GPC traces of the (Salen)Co III -PVAc macro-initiator, Mn=5.5 kDa, Đ=1.09; (Salen)Co III -PMA-b-PVAc, Mn=11.2 kDa, Đ=1.13; PPC-b-PMA-b-PVAc, Mn=15.0 kDa, Đ=1.18.

Table 1 .

 1 Polymerization of VAc mediated by (Salen)Co II in bulk under different conditions. Reaction conditions: 60 o C in 5 mL VAc with [Co]/[VAc]=1/500. b Conversion values detected by 1 H NMR. c Determined by gel permeation chromatography (GPC) with polystyrene as standard. d Number-average molar mass calculated from 1 H NMR spectra. e Comparative reactions carried out in ampule, which was exposed to natural light. f Conducted in 20 mL VAc with [Co]/[VAc]=1/500.

	c

a Entry [AIBN]/[Co] Time (min) Conv. b (%) b Mn,GPC (kDa) c Mn,NMR (kDa) d Đ a

Table 2 .

 2 ROCOP of CO2/epoxides mediated by (Salen)Co III -PVAc with pre-activation by O2. a Reaction conditions: (Salen)Co III -PVAc (from Table 1, entry 8) = 0.05 mmol, PO=3.5 mL, [Co]/[MTBD]/[epoxide] = 1/0.5/1000; pre-activated by 3.0 MPa O2 at 60 o C for 12 h before the ROCOP was conducted under 3.0 MPa CO2 at 30 o C. b Comparative experiment by (Salen)Co III -PVAc without pre-activation. Comparative experiment in the presence of AIBN, [Co]/[AIBN]=1/2. d Comparative experiment in the presence of TEMPO, [Co]/[TEMPO]=1/2. D etermined by 1 H NMR spectra. f PPC = poly(propylene carbonate). g PC = propylene carbonate. h Turnover frequency (TOF) = moles of product (PPC) per mole of catalyst per hour. i Number-average molar mass calculated from the 1 H NMR spectra. j Determined by GPC in THF, calibrated by polystyrene standards. k (Salen)Co III -PVAc (from Table 1, entry 7) = 0.1 mmol, PO=3.5 mL, [Co]/[MTBD]/[epoxide] = 1/0.5/500.

	Entry	Epoxide	(Salen)Co III -PVAc Mn/kDa	Time/h	PPC yield/% e,f	PC yield/% e,g	TOF/h -1 h	Mn,GPC/kDa i	Mn,NMR/kDa j	Đ f
	1 b	PO	10.5	48	--	2	0	--	--	--
	2 c	PO	10.5	48	--	6	0	--	--	--
	3 d	PO	10.5	48	--	5	0	--	--	--
	4	PO	10.5	24	10	2	5	19.5	20.4	1.11
	5 k	PO	5.6	12	10	1	9	9.4	10.6	1.09
	6 k	CHO	5.6	24	12	1	3	12.5	14.2	1.16
	7 k	SO	5.6	24	10	1	2	12.2	13.8	1.15

a c

Acknowledgements

YW thanks the Financial support from National Key R&D Plan (No.2016YFB0302400) and NSFC (No.21604027) of China. RP thanks the CNRS (Centre National de la Recherche Scientifique) for support and the HPC resources of IDRIS under the allocation 2016-086343 made by GENCI (Grand Equipement National de Calcul Intensif) and of the CICT (Centre Interuniversitaire de Calcul de Toulouse), project CALMIP.

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RESEARCH ARTICLE

Yajun Zhao, [a] Yong Wang,* [a] Xingping Zhou, [a] Zhigang Xue, [a] Xianhong Wang, [b] Xiaolin Xie [a] and Rinaldo Poli [c] Page No. -Page No.

Oxygen Triggered Switchable Polymerization for One-Pot Synthesis of CO2-Based Block Copolymers from Monomer Mixtures


Jérémy Vanhelst

Florian Vidal

Dominique Turck

Elodie Drumez

Djamal Djeddi

Eve Devouge

Claire Spyckerelle

Serge Ganga Zandzou

Céline Legrand

Dr Laurent Michaud

Laurent Béghin

Frédéric Gottrand

Stéphanie Coopman

Delphine Ley

Physical activity is associated with improved bone health in children with inflammatory bowel disease

Keywords: Bone Mineral Density (BMD), Crohn's Disease Inflammatory Bowel Disease (IBD), Physical Activity Lifestyle habits, bone mineral density, pediatrics, inflammation

Background & Aims: Bone health is an important concern in patients with inflammatory bowel disease (IBD). Low bone mineral density (BMD) is a powerful predictor of fracture risk in IBD patients. Physical activity (PA) plays an important role in bone health. However, PA data for children and adolescents with IBD are scarce. The primary aim is to evaluate the relationship between PA and BMD in children with IBD. The secondary aim was to assess the relationship between PA and quality of life.

Methods: Eighty-four IBD pediatric patients (45 boys) aged 14.3 ± 2.7 years were included (disease activity: (i) remission, n=62; (ii) mild, n=18; (iii) severe disease, n=1). BMD was measured using dual-energy X-ray absorptiometry and expressed as age-and sex-based Zscores. Each patient wore a triaxial accelerometer for seven consecutive days for objective PA quantification. Quality of life was assessed using the PedsQL TM and energy intake was assessed prospectively for three days using a dietary diary.

Results: BMD Z-score was -0.96 ± 1.11. Only five patients (6%) fulfilled the recommendation of 60 min of daily moderate-to-vigorous PA (MVPA). The proportion of children with osteopenia and osteoporosis was 51% and 4%, respectively. After adjustment for confounders (pubertal status and body mass index), total PA and time in MVPA were positively associated with BMD (regression coefficient per one standard deviation increase in PA parameters=0.26; P<0.05). There was no association between time spent in MVPA and total PA, and total quality of life score.

Conclusions: PA likely is associated with improved bone health in IBD children. Intervention studies investigating a causal relationship between PA and BMD in pediatric patients with IBD are warranted.

INTRODUCTION

Inflammatory bowel diseases (IBDs), including Crohn's disease (CD), ulcerative colitis (UC) and IBD-unclassified (IBD-U), are characterised by chronic inflammation of the gastrointestinal tract. Over the past 50 years, the incidence of IBD has increased globally, with the highest increase in industrialised countries [START_REF] Molodecky | Increasing incidence and prevalence of the inflammatory bowel diseases with time, based on systematic review[END_REF]. During the last 25 years, the incidence of IBD has increased dramatically among teenagers in northern France, with an increase of 126% and 156% for CD and UC, respectively [START_REF] Ghione | Dramatic increase in incidence of ulcerative colitis and Crohn's disease (1988-2011): A populationbased study of French adolescents[END_REF].

Bone mass acquisition during childhood and adolescence is a major determinant of skeletal health later in life [START_REF] Faulkner | Bone mineral accrual from 8 to 30 years of age: An estimation of peak bone mass[END_REF]. Bone health is an important concern in patients with IBD. The prevalence of low bone mineral density (BMD) in children and adolescents with IBD ranges from 8% to 65% [START_REF] Ahmed | Bone mineral assessment by dual energy X-ray absorptiometry in children with inflammatory bowel disease: evaluation by age or bone area[END_REF][START_REF] Schmidt | Longitudinal assessment of bone mineral density in children and adolescents with inflammatory bowel disease[END_REF][START_REF] Walther | Osteoporosis in pediatric patients suffering from chronic inflammatory bowel disease with and without steroid treatment[END_REF]. Bone mineralisation abnormalities in paediatric IBD are associated with the use of corticosteroids, disease activity, low body weight, young age at onset, pubertal delay, vitamin D deficiency, low calcium intake and intestinal malabsorption [START_REF] Schüle | Prediction of low bone mineral density in patients with inflammatory bowel diseases[END_REF][START_REF] Habtezion | Risk factors for low bone density in Crohn's disease[END_REF][START_REF] Mccarthy | Seasonality of vitamin D status and bone turnover in patients with Crohn's disease[END_REF]. Low BMD is a powerful predictor of fracture risk in IBD patients [START_REF] Card | Hip fractures in patients with inflammatory bowel disease and their relationship to corticosteroid use: a population based cohort study[END_REF].

Physical activity (PA), widely recognised as an important health determinant, plays an important role in growth and development. Increased participation in moderate-to-vigorous PA (MVPA) has major health benefits, including a lower risk of cardiovascular and pulmonary disease, musculoskeletal disorder, psychiatric, neurological and metabolic disease as well as cancer [START_REF] Pedersen | Exercise as medicine -evidence for prescribing exercise as therapy in 26 different chronic diseases[END_REF]. According to international PA recommendations, 60 min of MVPA daily are needed to positively impact child and adolescent health [START_REF] Cale | Exercise recommendations for young people: An update[END_REF]. In contrast, a sedentary lifestyle is associated with adverse health consequences and increased morbidity and mortality in adulthood [START_REF] Saunders | Screen Time and Health Indicators Among Children and Youth: Current Evidence, Limitations and Future Directions[END_REF]. However, PA data for children and adolescents with IBD are scarce.

Thus, the primary aim of the study was to evaluate the relationship between PA and bone health in IBD paediatric patients. The secondary aim was to assess the relationship between PA and quality of life.

SUBJECTS AND METHODS

Study design

From October 2014 to March 2017, consecutive paediatric patients with IBD who were followed in hospitals in northern France were invited to participate in this prospective study. Inclusion criteria were: (i) age 6-18 years; (ii) informed consent signed by the patient and his/her parents; (iii) IBD diagnosis (CD, UC, IBD-U) at least six months prior; and (iv) not currently participating in another biomedical study. Exclusion criteria were: (i) acute or chronic disease (other than IBD) associated with a decrease in PA; and (ii) any recent event (≤15 days) that could affect PA. Eighty-four patients were included.

Before participating, the study aims and objectives were carefully explained to, and written informed consent obtained from, each patient and his/her parents. The study was approved by the Research Ethics Committee of the University of Lille (Comité de Protection des Personnes, Nord Ouest IV, Lille, France). All procedures were performed according to the ethical standards of the Helsinki Declaration of 1975, as revised in 2008, and European Good Clinical Practice.

Measurements

Physical activity

PA was assessed using accelerometry, an objective method for use with youth [START_REF] Vanhelst | Awareness of wearing an accelerometer does not affect physical activity in youth[END_REF]. The triaxial accelerometer was the ActiGraph ® Monitor (Model GT3X; ActiGraph, Pensacola, CA, USA), with dimensions 46 × 33 × 15 mm and weight 19 g. The accelerometer measures acceleration in three spatial dimensions according to vertical (x), antero-posterior (y) and medio-lateral (z) vectors. The vector magnitude (VM) was calculated as: VM = √(x 2 +y 2 +z 2 ).

The epoch length was set at 1 sec. A computer was used to initialise and synchronise the accelerometer, which was calibrated based on patient age, height and weight. The GT3x accelerometer has been validated for measuring PA against oxygen consumption and heart rate [START_REF] Romanzini | Calibration of ActiGraph GT3X, Actical and RT3 accelerometers in adolescents[END_REF]. The inter-instrument reliability for this device is better for moderate and vigorous activity than for sedentary activity [START_REF] Santos-Lozano | Technical variability of the GT3X accelerometer[END_REF]. Consistent with consensus recommendations for assessing PA in youth, patients who did not report at least three days with a minimum of 10 hours of PA per day were excluded from analyses [START_REF] Rich | Quality control methods in accelerometer data processing: defining minimum wear time[END_REF]. PA activity levels were classified as: sedentary (0-180 counts.15sec -1 ); light (181-757 counts.15sec -1 ); moderate (758-1112 counts.15sec -1 ); and vigorous (>1112 counts.15sec -1 ) [START_REF] Romanzini | Calibration of ActiGraph GT3X, Actical and RT3 accelerometers in adolescents[END_REF].

Patients wore the accelerometer on their lower back, beneath their clothing, using an elastic belt with adjustable buckle. They were asked to follow their normal daily routine and instructed to remove the device during contact sports, water-based activities (e.g., swimming, showering, bathing) and overnight. The accelerometer was used to record activity over seven consecutive days during free-living conditions. In addition, patients and parents were instructed to keep a PA diary while the patient wore the accelerometer.

Bone mineral density and body composition

BMD and body composition (fat mass and fat free mass) were measured with dual-energy Xray absorptiometry (DEXA; Hologic Corp., Discovery Type, Software 12.6, Bedford, MA, USA). Patients were scanned in the supine position at high resolution. BMD, fat mass and fat free mass were determined based on whole body scan analyses.

Because BMD measurements are age-and sex-specific, standardised Z-scores were calculated by subtracting the age-and sex-specific mean and dividing by the standard deviation.

According to World Health Organisation recommendations, normal BMD T-scores are ≥-1 standard deviation (SD), osteopenia T-scores <-1 to >-2.5 SD and osteoporosis T-scores ≤-2.5 [START_REF]Prevention and management of osteoporosis : report of a WHO scientific group[END_REF].

Quality of life

The IMPACT III was not used herein because it has not been validated among French youth. provides a total score and two subscale dimensions: (i) physical health (physical functioning scale score); and (ii) psychosocial health (emotional, social and school functioning scale scores). For children 8 to 18 years, each question is scored using a 5-point Likert scale ranging from 0 (never) to 4 (almost/always). Appropriate items were reverse scored and total scores were linearly transformed to a 0-100 scale where: 0 = 100; 1 = 75; 2 = 50; 3 = 25; and 4 = 0. For children 5 to 7 years, each question is scored using a 3-point Likert scale ranging from 0 (not at all), 2 (sometimes) and 4 (a lot). Higher scores indicate a better quality of life.

Based on scoring recommendations, patients replied to at least 50% of the questions.

Patients were asked to complete the age-appropriate PedsQL TM forms and rate the degree to which each item had been a problem for them during the past month, on a 5-point Likert scale for children 8 to 18 years and a 3-point Likert scale for children 5 to 7 years. Total score and subscale dimensions are expressed as percentages of the maximum points possible.

Energy intake

Energy intake was assessed using a prospective dietary diary for three consecutive days, including one weekend day. Patients were interviewed about the types of foods consumed during the three 24-hour periods. For the greatest precision possible, patients could complete the dietary diary with their parents. The same dietitian reviewed all dietary diaries. Patients were assisted by an instruction manual for food codification, including validated photographs of more than 250 foods represented in three different portion sizes. Foods were presented in three sizes permitting, with intermediate and extreme positions, seven choices of the amount [START_REF] Moullec | Validation du manuel photos utilisé pour l'enquête alimentaire de l'étude SU.VI.MAX[END_REF]. All quantities were then calculated using KIDMENU ® software (SHS, Paris, France) to compute energy intake using food composition tables from the French Food Safety Agency [START_REF] Afsaa | French Food Composition Data base. REGAL 2007. Informatic Center for Quality of foods[END_REF].

Clinical assessment

Each patient underwent a detailed medical examination. Pubertal status was assessed by direct observation according to Tanner and Whitehouse [START_REF] Tanner | Clinical longitudinal standards for height, weight, height velocity, weight velocity, and stages of puberty[END_REF]. Body mass was measured to the nearest 0.1 kg using an electronic scale (Seca, Hamburg, Germany) after removal of shoes and heavy outer garments. Height was measured to the nearest 0.1 cm using a stadiometer (Seca, Hamburg, Germany). Body mass index (BMI) was calculated as weight (kg) divided by height² (m²). Patients had a plain radiograph of the left hand; wrist and bone age were expressed in years using the Greulich and Pyle atlas [START_REF] Greulich | Radiographic atlas of skeletal development of the hand and wrist[END_REF]. In addition, 25(OH)vitamin D, haematocrit, C-reactive protein, albumin and erythrocyte sedimentation rate were measured to assess inflammation and calculate disease activity. The Pediatric CD Activity Index (PCDAI) was used for patients with CD and the Pediatric Ulcerative Colitis Activity Index (PUCAI) for patients with UC [START_REF] Hyams | Development and validation of a pediatric Crohn's disease activity index[END_REF][START_REF] Turner | Development, validation, and evaluation of a pediatric ulcerative colitis activity index: a prospective multicenter study[END_REF]. The PCDAI comprises three domains: (i) history (one-week recall of abdominal pain, stools, patient functioning, general well-being); (ii) laboratory results

Sample size calculation and statistical analysis

The study objective was to evaluate the association between PA and BMD in children with IBD. Classification of correlation coefficients was based on Cohen's rule, where <0.3 is weak, 0.3-0.6 moderate and >0.6 high [START_REF] Cohen | Statistical power analysis for the Behavioral sciences[END_REF]. Sample size calculation was based on: H 0 : r = 0 (no correlation between parameters) vs. H 1 : r = 0.3 (average correlation between parameters). For alpha = 5% and power = 80%, N = 84 participants were needed.

Data are presented as counts (percentages) for categorical variables and mean±SD or median

RESULTS

Sample characteristics are presented in Table 1. Mean BMD Z-score (±SD) was -0.96±1.11.

The computer analysis of the accelerometer data showed that 82 participants wore the accelerometer during at least three days with a minimum of 10 hours of PA per day. Two participants were excluded from the analysis because of monitoring failure. Only five patients (6%) fulfilled the recommendation of 60 min of MVPA daily. The prevalence of osteopenia and osteoporosis was 51% and 4%, respectively. Drug therapy is also depicted in Table 1.

Bivariate analyses assessing the relationships between BMD-Z score and potential confounding factors are shown in Table 2. BMI and pubertal status (Tanner grade) were positively associated with BMD Z-score (p<0.05 for both associations).

As shown in Table 3, BMD was not significantly related to any of the PA parameters in bivariate analyses. However, after adjustment for confounding factors, total PA and time spent in MVPA were positively associated during the weekdays with BMD (p=0.041 and p=0.046, respectively).

The PedsQL TM total score (±SD) was 81.6±13.4. Associations of quality of life score and potential confounding factors are presented in Table 4. A positive significant association was found between pubertal status and quality of life (r=0.23).

As shown in Table 5, no significant association was found between time spent in MVPA/total PA and quality of life in multivariable linear regression models adjusted for pubertal status.

DISCUSSION

Osteoporosis is a common skeletal disease and is associated with high morbidity and mortality in adulthood [START_REF] Wehren | Hip fracture: risk factors and outcomes[END_REF]. Even if our study shows that mean BMD Z-score of IBD paediatric patients was within normal range, it is of note that osteopenia was found in half of the patients while osteoporosis was rare. BMD-Z score and quality of life score did not differ significantly according to disease severity. Only a non-significant higher quality of life score in children in remission was found compared to children with mild or severe disease (mean: 83 ± 13 vs. 76 ± 15; p=0.058). In relation to our primary objective, the present study shows a positive association between BMD and both amount of PA and time spent in MVPA. This result is consistent with those reported for healthy children [START_REF] Rodriguez | Extracurricular physical activity participation modifies the association between high TV watching and low bone mass[END_REF][START_REF] Tønnesen | Physical exercise associated with improved BMD independently of sex and vitamin D levels in young adults[END_REF]. Vicente-Rodriguez et al. observed a negative association between sedentary lifestyle and bone health in healthy adolescents. More than three hours daily of sedentary behaviour (e.g., watching TV) was associated with increased risk for low bone mineral content in males [START_REF] Rodriguez | Extracurricular physical activity participation modifies the association between high TV watching and low bone mass[END_REF]. In healthy young adults, PA is associated with improved BMD independent of gender and vitamin D status [START_REF] Tønnesen | Physical exercise associated with improved BMD independently of sex and vitamin D levels in young adults[END_REF]. To our knowledge, the only study assessing bone health and PA in IBD has been performed in adult patients [START_REF] Robinson | Effect of a low-impact exercise program on bone mineral density in Crohn's disease: a randomized controlled trial[END_REF]. Patients were randomised to a control group or a low-impact exercise program with increasing intensity across a 12-month period [START_REF] Robinson | Effect of a low-impact exercise program on bone mineral density in Crohn's disease: a randomized controlled trial[END_REF]. In patients who were able to fully comply with the exercise program, the only significant BMD gain was observed at the greater trochanter (+4.7%); these findings were independent of changes in potential confounding variables (e.g., steroid dose, weight, diet).

IBD have a well-known negative impact on health-related quality of life (HRQoL) [START_REF] Moradkhani | Health-related quality of life in inflammatory bowel disease: psychosocial, clinical, socioeconomic, and demographic predictors[END_REF]. Ng et al studied PA as non-pharmacological approach in the management of IBD for improving or maintaining HRQoL [START_REF] Ng | Low-intensity exercise improves quality of life in patients with Crohn's disease[END_REF]. Another study showed that adult patients engaged higher volumes of MVPA above 150 min/week and walking, particularly above 60 min/week, improved their quality of life [START_REF] Taylor | Associations between physical activity, resilience, and quality of life in people with inflammatory bowel disease[END_REF]. Similar results have been found in people with other chronic diseases or in healthy people [START_REF] Nathan | Exercise in individuals with inflammatory bowel disease[END_REF][START_REF] Abell | Physical activity and health related quality of life among people with arthritis[END_REF][START_REF] Anonyke | Physical activity and health related quality of life[END_REF]. Results from our study do not concur with previous research.

According to youth PA guidelines, children and adolescents should accumulate a minimum of 60 min of MVPA daily through transportation, physical education, sports, free play and planned exercise for positive health outcomes [START_REF] Cale | Exercise recommendations for young people: An update[END_REF]. A very low proportion of our study sample followed this recommendation. These data are alarming in relation to studies of healthy children [START_REF] Riddoch | Physical activity levels and patterns of 9-and 15-yr-old European children[END_REF][START_REF] Ruiz | Objectively measured physical activity and sedentary time in European adolescents: the HELENA study[END_REF]. In 2004, among 2185 children and adolescents assessed with accelerometry, 90% of boys and 80% of girls achieved health-related PA recommendations [START_REF] Riddoch | Physical activity levels and patterns of 9-and 15-yr-old European children[END_REF]. In a population of 2200 European adolescents, 57% of boys and 28% of girls followed the PA recommendations, based on accelerometer [START_REF] Ruiz | Objectively measured physical activity and sedentary time in European adolescents: the HELENA study[END_REF]. Wekstetter et al. compared PA levels between IBD paediatric patients and healthy controls [START_REF] Werkstetter | Lean body mass, physical activity and quality of life in paediatric patients with inflammatory bowel disease and in healthy controls[END_REF]. The differences in PA duration and number of steps between groups did not reach statistical significance; however, female patients had a significantly shorter duration of PA (in total and moderate level). In our study, between patients in remission and patients with mild or severe disease activity, no difference was found in total PA (158.1 vs 176.4 counts.min -1 , p = 0.40) and time spent in MVPA (44.8 vs 42.9 min.day -1 , p = 0.47).

It has been hypothetized that PA could be associated with an increased risk for exacerbation of inflammation [START_REF] Ploeger | Exercise and inflammation in pediatric Crohn's disease[END_REF]. Ploeger et al examined inflammatory cells and cytokines in response to acute bouts of moderate intensity continuous exercise and high intensity intermittent exercise in youth with CD and in healthy matched controls [START_REF] Ploeger | Exercise and inflammation in pediatric Crohn's disease[END_REF]. In CD patients, both types of exercise increased immune cells. Moderate intensity exercise induced a significantly greater increase in leukocytes, neutrophils, lymphocytes, monocytes, IL-6 and IL-17, compared with high intensity exercise. TNF-α did not change significantly with either exercise. These preliminary results suggest that moderate PA of short duration is not associated with a deleterious effect on inflammatory parameters. However, further studies are needed to assess the effects of regular exercise on inflammation in IBD pediatric patients.

The current study has both strengths and limitations. Importantly, strong objective methods were used to assess both PA (using accelerometry) and bone health (using DEXA).

Additionally, confounding factors were accounted for in the statistical analyses. The study limitations include its observational design, which means that associations cannot be interpreted as causal. Moreover, the sample size did not allow to study the impact of gender on PA, quality of life and BMD-Z score, and to test the heterogeneity in PA and BMD score according to disease severity. Regarding the multiple testing issue, we could not exclude false positive findings. Moreover, we could not exclude bias in estimates due to missing data. In addition, the thresholds used to classify PA may have affected the results, as previously described [START_REF] Vanhelst | Impact of the choice of threshold on physical activity patterns in free living conditions among adolescents measured using a uniaxial accelerometer: the HELENA study[END_REF]. Variations in weather conditions (e.g., wind, rain, sunshine) during PA assessments were not recorded but may also have affected our results. Another limitation of our study is the lack of information on the type of PA. Animal data showed that mechanical stress (i.e., mechanical loading) on bone can enhance bone mass [START_REF] Whewheeler | Effects of running on the torsional strength, morphometry, and bone mass of the rat skeleton[END_REF][START_REF] Yinglyingling | The effects of repetitive physiologic loading on bone turnover and mechanical properties in adult female and male rats[END_REF][START_REF] Vavan Der Wiel | Additional weight-bearing during exercise is more important than duration of exercise for anabolic stimulus of bone: a study of running exercise in female rats[END_REF][START_REF] Notomi | Effects of tower climbing exercise on bone mass, strength, and turnover in growing rats[END_REF]. Results in healthy adolescents concur with animal studies. PA involving weight bearing (e.g walking, running) is more beneficial to bone mass as compared to PA with minimal weight bearing (e.g cycling, swimming) [START_REF] González-Agüero | Bone Structure and Geometric Properties at the Radius and Tibia in Adolescent Endurance-Trained Cyclists[END_REF][START_REF] Gomez-Bruton | The Effect of Swimming During Childhood and Adolescence on Bone Mineral Density: A Systematic Review and Meta-Analysis[END_REF]. Another limitation of the current study is that the small sample size did not allow to compare CD patients and UC patients.

In summary, PA was found to be positively associated with BMD in paediatric patients with IBD. Intervention studies investigating a causal relationship between PA and BMD are thus warranted in order to determine whether IBD paediatric patients could benefit from PA promotion programs. In addition, these studies should aim at determining the most adequate PA, including type, intensity, duration and frequency.

Legends

  Instead, quality of life was assessed with the Pediatric Quality of Life Inventory (PedsQL TM ) (Version 4.0), which is divided into age groups 5-7 years, 8-12 years and 13-18 years. Each age group version has 23 questions comprising four dimensions: (i) physical functioning; (ii) emotional functioning; (iii) social functioning; and (iv) school functioning. The PedsQL TM

  (haematocrit, erythrocyte sedimentation rate, albumin); and (iii) physical examination (weight, height, abdomen, perirectal disease, extra-intestinal manifestations). The PUCAI comprises six domains: (i) abdominal pain; (ii) rectal bleeding; (iii) stool consistency; (iv) number of stools per 24 hours; (v) nocturnal stools; and (vi) activity level. Remission was defined as scores <10 and mild activity as scores 10-27.5 on the PCDAI and 10-34 on the PUCAI. Values >37.5 on the PCDAI or >34 on the PUCAI indicated moderate to severe activity. Drug therapy, including 5-ASA, corticosteroids, immunomodulators (azathioprine, methotrexate) and anti-TNF therapy (infliximab, adalimumab) was recorded.

[

  range] for quantitative variables. Normality of distribution was assessed visually using histograms and by using the Shapiro-Wilk test. Associations of PA parameters with outcomes (BMD Z-score and quality of life) were analysed using linear regression models with and without adjustment for confounding factors. For each outcome, confounding factors were selected according to bivariate analyses (p<0.10). Associations of each outcome with potential confounding factors were assessed using Student's t-test for categorical confounding factors and Pearson (or Spearman's rank) correlation coefficients for quantitative (or ordinal) confounding factors. All statistical tests were done at the two-tailed α level of 0.05. Data were analysed using SAS software version 9.4 (SAS Institute Inc., Cary, NC, USA).

Table 1 .

 1 Characteristics of the study patients

Table 2 .

 2 Associations between BMD wb Z-score and potential confounding factors

Table 3 .

 3 Associations between BMD wb Z-score and physical activity

Table 4 .

 4 Associations between quality of life and potential confounding factors

Table 5 .

 5 Associations between quality of life and physical activity Abbreviations: IBD= Inflammatory bowel diseases ; IQR=interquartile range; BMDwb= Bone Mineral Density of whole body, MVPA= Moderate to Vigorous Physical Activity; PA=physical activity; SD=standard deviation.

Table 3 .

 3 Associations between BMD wb Z-score and physical activity Data analysis was performed on 76 patients without missing data on BMD wb Z-score and physical activity parameters. Abbrevations: BMD wb = Bone Mineral Density of whole body; CI= confidence interval; MVPA= moderate to vigorous physical activity β indicate regression coefficient per one standard deviation increase in PA parameters. R² indicates the squared semi-partial correlation coefficient. * Adjusted for between-group difference in potential confounding factors at p<0.10 in bivariates analyses (body mass index and Tanner grade).

	Unadjusted		Adjusted*
	β (95%CI)	P	R² (%)	β (95%CI)	P	R² (%)
	All days
	MVPA 0.11 (-0.16 to 0.38)	0.41	0.9	0.19 (-0.05 to 0.44)	0.13	2.5
	Sedentary 0.18 (-0.08 to 0.44)	0.16	2.6	0.15 (-0.10 to 0.40)	0.25	1.5
	Counts 0.05 (-0.22 to 0.32)	0.72	0.2	0.15 (-0.10 to 0.41)	0.24	1.5
	Week days
	MVPA 0.20 (-0.07 to 0.47)	0.14	3.0	0.26 (0.01 to 0.51)	0.041	4.6
	Sedentary 0.20 (-0.06 to 0.46)	0.14	3.0	0.18 (-0.08 to 0.44)	0.19	1.9
	Counts 0.16 (-0.11 to 0.43)	0.23	1.9	0.26 (0.005 to 0.52) 0.046	4.3
	Week end days
	MVPA 0.05 (-0.21 to 0.30)	0.71	0.2	0.15 (-0.08 to 0.39)	0.21	1.7
	Sedentary 0.16 (-0.09 to 0.42)	0.21	2.1	0.09 (-0.14 to 0.33)	0.42	0.7
	Counts -0.04 (-0.30 to 0.21) 0.74	0.2	0.07 (-0.17 to 0.31)	0.57	3.6

Table 4 .

 4 Associations between quality of life and potential confounding factors Values are mean ± standard deviation for categorical variables and correlation coefficient for continuous variables.

	Values	P

Table 5 .

 5 Associations between quality of life and physical activityData analysis was performed on 82 patients without missing data on PedsQL TM total and physical activity parameters (calculated for all days). Abbrevations: CI= confidence interval; MVPA= moderate to vigorous physical activity. Dimension I: physical health comprising of the physical functioning scale score Dimension II: psychosocial health comprising of the emotional, social and school functioning scales score β indicates regression coefficient per one standard deviation increase in PA parameters. * Adjusted for between-group difference in potential confounding factors at p<0.10 in bivariates analyses (Tanner Grade and Intensity of disease)

	Unadjusted		Adjusted*
	β (95%CI)	P	R²	β (95%CI)	P	R²
	Total score
	MVPA -2.09 (-5.04 to 0.85)	0.16	0.025	-1.47 (-4.61 to 1.67)	0.35	0.011
	Counts -2.71 (-5.63 to 0.21)	0.069	0.041	-2.01 (-5.24 to 1.24)	0.22	0.019
	Dimension I
	MVPA -2.75 (-5.72 to 0.22)	0.069	0.041	-1.73 (-4.83 to 1.38)	0.27	0.015
	Counts -2.98 (-5.94 to -0.03)	0.048	0.048	-1.64 (-4.86 to 1.59)	0.31	0.013
	Dimension II
	MVPA -1.75 (-5.05 to 1.55)	0.30	0.014	-1.34 (-4.87 to 2.20)	0.45	0.007
	Counts -2.56 (-5.83 to 0.71)	0.12	0.029	-2.20 (-5.85 to 1.45)	0.23	0.019

Acknowledgements

The authors thank the participants for taking part in the study.

Funding sources

This work was supported by Lille University Hospital Programme (Fonds hospitalier d'aide à l'émergence et à la structuration des activités et équipes de recherche) (Grant number U5235B012P003). The content of this paper reflects only the authors' views, and the Lille University Hospital is not liable for any use that may be made of the information contained therein.

Conflict of interest

The authors do not have any competing interests.


Daniel L Coward

Benjamin R M Lake

Rinaldo Poli

Michael P Shaver

Radically Initiated Group Transfer Polymerization of Methacrylates by Titanium Amino-Phenolate Complexes

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excellent control. Advantageously, a conventional azo initiator is used as activator, yielding a method which is considerably more robust than present titanium-based alternatives. Amine-phenol ligand precursors were synthesized via facile Mannich-type condensation reactions in high yields (Scheme S1). Ligands were selected to explore the effect of both electronic and steric properties, and coordination geometry, on the polymerization. Complexation of the ligand precursors was straightforwardly achieved using one of two methods (Scheme S2) to yield a family of known (1, 7) and novel [START_REF] Bochmann | Cationic Group-4 Metallocene Complexes and Their Role in Polymerization Catalysis -the Chemistry of Well-Defined Ziegler Catalysts[END_REF][START_REF] Brintzinger | Stereospecific Olefin Polymerization with Chiral Metallocene Catalysts[END_REF][START_REF] Chen | Cocatalysts for Metal-Catalyzed Olefin Polymerization: Activators, Activation Processes, and Structure-Activity Relationships[END_REF][START_REF] Coates | Precise Control of Polyolefin Stereochemistry Using Single-Site Metal Catalysts[END_REF][START_REF] Alt | Effect of the Nature of Metallocene Complexes of Group IV Metals on Their Performance in Catalytic Ethylene and Propylene Polymerization[END_REF] titanium(III) amino-phenolate complexes (Figure 1). Full characterization of all complexes, including several molecular structures, is detailed in the Supporting Information. Scheme 1. Titanium-mediated methods of styrene and methyl methacrylate (MMA) polymerizations. All complexes were screened for their efficacy as mediators in the polymerization of styrene and MMA, with titanium(III) amino-bis(phenolate) (ABP) complexes 1-4 the top performers for rapid, controlled polymerization of MMA under the reaction conditions outlined in Scheme 2. 2,2'-Azobis(4-methoxy-2,4-dimethylvaleronitrile (V-70), which was used as polymerization initiator, is very similar to the ubiquitous AIBN, but permits lower reaction temperatures as the half-life time (at 80 °C) of V-70 is 1.9 mins compared to 73 mins for AIBN.

Polymerizations were fast, achieving high conversions with good control over the molecular weight of the polymer (Table S1 and Figure S1). For example, complex 1 reached 88% conversion within 1 hour, with Mn,th = 8800, Mn = 37800 and Đ = 1.13. This initially seems to be a well-controlled radical polymerization. However, it was surprising to observe that the tacticity of the resultant poly(methyl methacrylate) (PMMA) was significantly dependent on the titanium(III) complex used (Table 1). In a typical free-radical polymerization the mm:rm:rr ratio (a measure of the isotactic:heterotactic:syndiotactic character of a polymer) is ca. 3:33:64. PMMA synthesized using both chlorosubstituted 3 and methyl-substituted 2 possesses a slight isotactic bias compared to a free-radical polymerization. The bulky t-butyl-substituted 1 displayed an even more pronounced isotactic bias. In a typical metal-mediated controlled radical polymerization, one would expect the tacticity of the resultant polymer to be very similar to that of a free-radical polymerization, since monomer addition to a radical chain end occurs away from the metal center and thus is not influenced by the ligand framework. The fact that such a noticeable isotactic bias is present when complexes 1-4 are used as mediators suggested that the initial presumptions about the mechanistic pathway may be wrong, even though a standard radical polymerization protocol had been employed. A non-radical pathway was further supported through the addition of a radical scavenger such as dihydroanthracene (DHA, Table S2). In a free-radical MMA polymerization the addition of dihydroanthracene results in a lower monomer conversion, as radical chain ends irreversibly terminate by reacting with the scavenger. However, the use of DHA in a polymerization mediated by 1 showed negligible change in monomer conversion, which suggests that there are no radical chain ends for the scavenger to react with.

A variety of possible mechanisms were explored to better understand how the complexes control the polymerization of MMA. A combination of experimental and computational techniques were used. DFT studies using a simplified amino-phenolate ligand framework (see Supporting Information) strongly suggest that the polymerization mechanism is not ionic, with a ΔG in excess of +31 kcal/mol to form the ion pair. A typical coordination-insertion polymerization, where the monomer coordinates to the titanium via the alkene bond, was also found to be unlikely, due to the large steric bulk of the ligand framework preventing the monomer from being sufficiently close to the metal center for coordination to occur in this way.

From the kinetic and computational data, we propose that the polymerization mechanism is bimetallic, and involves both a titanium(IV)

and titanium(III) species (Scheme 3). This mechanism has previously been termed group transfer polymerization (GTP), and primarily uses organosilicon mediators. 38,39 To the best of our knowledge, this mechanism has not previously been observed with titanium. We propose that during the initiation phase of the polymerization, a titanium(IV) enolate species is formed, most likely after a few radical monomer additions and this would explain the imperfect PMMA dispersity. DFT studies (Scheme S3) show that this enolate is considerably more energetically favorable than other titanium(IV) species that could feasibly form, such as Ti-R0 (R0 = primary radical of V-70) and Ti-MMAn-R0 (with a Ti-C bond) adducts. This suggests that the proposed mechanism is energetically preferred to an organometallic-mediated radical polymerization (OMRP) mechanism, which would require the formation of a stable but homolytically-reversible Ti-C bond. The computational data suggests that this bond strength is insufficient for OMRP.

The polymerization proceeds via a reaction between this Ti(IV)-enolate species and a Ti(III)-MMA adduct, with the MMA coordinating Experimental data also supports a bimetallic GTP mechanism. With the proposed mechanism, one would expect that varying the coordinating nature of the polymerization solvent would have a significant effect on the conversion (Table S3 and Figure S2). With toluene, a noncoordinating solvent, polymerizations were fast and reached high conversions. This rate decreased when using the more-coordinating solvent 1,4-dioxane and decreased further using THF. This is most clearly evidenced with complex 1, with conversion decreasing from 93% (entry S6) to 12% (entry S12) upon the use of a more-coordinating solvent. This suggests that a vacant coordination site is required on the metal for productive polymerization.

via
Comparing the kinetics between free-radical and 1-mediated MMA polymerization further supports this theory (Figure S3). One would expect a controlled radical polymerization to be slower than the free-radical equivalent. However, using 1 as mediator resulted in a faster polymerization than without, which suggests that the mediator both controls and catalyzes the polymerization.

Furthermore, in a typical free radical polymerization one would expect the polymerization rate to increase with increased initiator concentration (as shown in Figure S3). However, the opposite trend is observed when mediated by 1, with the polymerization rate decreasing as the concentration of the initiator was increased (Figure S4). Molecular weights decreased with increased initiator concentration, which is as expected. These results strongly support the proposed bimetallic mechanism. Increasing the initiator concentration increases the concentration of Ti(IV)-enolate, from which the chains grow. Therefore, the concentration of Ti(IV)-enolate dictates the molecular weight. However, increasing Ti(IV) concentration results in a decreased Ti(III) concentration. As both are required in the bimetallic mechanism, the slow polymerization rate with high V-70 concentration can be attributed to the low Ti(III) concentration. If the concentration of Ti(IV)-enolate controls the molecular weight and the relative concentration of Ti(IV) to Ti(III) controls the polymerization rate, it would be expected that adding additional Ti(III) complex 1 midway through the polymerization would result in a faster rate with no new chains being formed. This is clearly observed in Figure 2 (and Figure S5), where different concentrations of 1 were added after 20 minutes. The polymerization rate was faster with higher concentrations of added 1, whilst no new chains were formed. This is further strong evidence for the proposed bimetallic polymerization mechanism.

DFT calculations were performed to understand this bimetallic mechanism in greater detail, and to determine whether the proposed mechanism is theoretically feasible. It is energetically favorable to form both the TI(IV)-enolate (Scheme S4, ΔG = -34.46 kcal/mol) and the Ti(III)-MMA adduct (Scheme S4, ΔG = -16.48 kcal/mol). There is a very small energy barrier to overcome to bring the two species into close proximity, and subsequently an accessible transition state for the chain propagation step with a ΔG ‡ of +21.86 kcal/mol. The overall reaction has a ΔG of -5.19 kcal/mol, which is as expected for a typical polymerization.

DFT additionally supports a monometallic GTP mechanism (Scheme S5), also starting from a Ti(IV)-enolate, with the necessary vacant coordination site for monomer activation arising from dissociation of the pendant amine donor arm on the ligand framework. However, the chain propagation transition state of this pathway yields a ΔG ‡ of +24.61 kcal/mol, which is significantly higher than for the bimetallic pathway. This suggests that the bimetallic mechanism is preferred.

The presence of both mechanisms has been proved experimentally (Figure S6). Whilst attempts to isolate and characterize a Ti(IV)-enolate analogue of 1 were unsuccessful, it was possible to generate in situ a macroinitiator comprising approximately 30 monomer units. In the presence of this macroinitiator and excess monomer, but the absence of 1, chains grew slowly. This slow polymerization illustrates the presence of the monometallic mechanism, as there is no Ti(III) present. Upon addition of 1, the polymerization rate increased by an approximate factor of nine, demonstrating that the bimetallic pathway is significantly more favored.

Complexes 5-7 (Scheme S2) have a substantially more rigid and bulky ligand framework than complexes 1-4, in which the flexible nitrogen donor arm is replaced by either a bulkier amine donor [START_REF] Coates | Precise Control of Polyolefin Stereochemistry Using Single-Site Metal Catalysts[END_REF] or a further phenolate donor (6 and 7). It is expected that these complexes would be incompatible with the proposed mechanisms, since the rigid and bulky ligand framework would make the processes of formation of the Ti(IV)-enolate and dissociation of a donor arm energetically unfavorable. As a result, polymerization would proceed via an uncontrolled mechanism. This is consistent with the polymerization results (Table S1), where dispersities were considerably larger for complexes 5-7 than for complexes 1-4.

We can conclude that for effective polymerization control, there are some constraints over the ligand framework. A strongly donating or overly bulky framework prohibits the proposed group transfer mechanism, with ligand flexibility promoting formation of the required Ti(IV) species. On the other hand, steric bulk on the phenolate has the positive effect of increasing isotactic bias in the resultant PMMA. This tunability allows for the precise synthesis of PMMA with control over both molecular weight and tacticity.

Complexes 1 and 2 were also used as mediators in the polymerization of other methacrylates (Table S4). Control over the polymerization of n-butyl methacrylate ( n BuMA) was highly dependent on the ligand framework of the metal. Use of 1 as mediator led to a well-controlled polymerization (Figure S7) with a narrow dispersity (Đ = 1.14). However, use of 2 as mediator led to a poorly-controlled polymerization with a much broader dispersity (Đ = 2.12). No polymer was produced during the attempted polymerization of t-butyl methacrylate ( t BuMA) using either 1 or 2. It is believed that the need for an additional two t-butyl-containing molecules to interact with the metal center is too energetically unfavorable for polymerization.

In this contribution, we have demonstrated the exceptional control that titanium(III) amino-phenolate complexes have over the polymerization of MMA. Furthermore, the polymerization can be activated using a conventional azo initiator instead of typical expensive and pyrophoric alternatives. The polymerization is suggested to proceed via a unique group transfer mechanism, with a primary bimetallic and secondary monometallic mode of operation. The presence of both mechanistic pathways has been demonstrated experimentally and computationally.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website. Detailed experimental synthetic procedures, characterization data, polymerization and kinetic data, mechanistic schemes and computational data. (PDF)

Figure 1 .

 1 Figure 1. Titanium complexes 1-7.

Scheme 2 .

 2 Scheme 2. Representative MMA polymerization.

  MMA]:[Ti(III)]:[V-70] = 100:1.0:1.0, MMA:toluene = 1:1 (v/v). 80 °C. a 1 hour. b 2.5 hours.

Scheme 3 .

 3 Scheme 3. Proposed bimetallic group transfer polymerization mechanism.

Figure 2 .

 2 Figure 2. Addition of extra 1 during MMA polymerization.

Table 1 . Polymerization screening of MMA with complexes 1-4. Polym. No. Complex PMMA Tacticity mm:rm:rr 1 a

 1

		-	3:33:64
	2 b	1	15:47:38
	3 b	2	6:39:55
	4 a	3	6:39:55

ACKNOWLEDGMENT

M.P.S., D.L.C., and B.R.M.L. gratefully acknowledge support from the EPSRC (EP/M000842/1 and EP/L015536/1) and the Universities of Edinburgh and Manchester for financial support. We thank Dr. Gary Nichol for collection of the X-ray diffraction data.

AUTHOR INFORMATION