M Alzaher

N Vannson

O Deguine

M Marx

P Barone

K Strelnikov

Plasticity Brain plasticity and hearing disorders

Keywords:

Permanently changed sensory stimulation can modify functional connectivity patterns in the healthy brain and in pathology. In the pathology case, these adaptive modifications of the brain are referred to as compensation, and the subsequent configurations of functional connectivity are called compensatory plasticity. The variability and extent of auditory deficits due to the impairments in the hearing system determine the related brain reorganization and rehabilitation. In this review, we consider cross-modal and intra-modal brain plasticity related to bilateral and unilateral hearing loss and their restoration using cochlear implantation. Cross-modal brain plasticity may have both beneficial and detrimental effects on hearing disorders. It has a beneficial effect when it serves to improve a patient's adaptation to the visuo-auditory environment. However, the occupation of the auditory cortex by visual functions may be a negative factor for the restoration of hearing with cochlear implants. In what concerns intra-modal plasticity, the loss of interhemispheric asymmetry in asymmetric hearing loss is deleterious for the auditory spatial localization. Research on brain plasticity in hearing disorders can advance our understanding of brain plasticity and improve the rehabilitation of the patients using prognostic, evidencebased approaches from cognitive neuroscience combined with post-rehabilitation objective biomarkers of this plasticity utilizing neuroimaging.

Introduction

Though there are hard-wired configurations for connections between sensory areas and associative cortices, a need for adaptation in a changing environment requires a certain degree of flexibility to modify cortical interactions, at least at the local scale. Therefore, permanently changing sensory stimulation can alter functional connectivity patterns in the healthy brain. As can be expected, these modifications in brain anatomy and function are even more pronounced in the case of sensory dysfunction due to disease or injury [START_REF] Bavelier | Cross-modal plasticity: where and how?[END_REF]. In the pathology case, these adaptive modifications of the brain are referred to as compensation, and the subsequent configurations of functional connectivity are called compensatory plasticity. Inevitably, the deficient input from one sensory modality leads to attempts to compensate for this deficiency based on the remaining sensory modalities, which acquire a novel, often enhanced, mode of functioning. This phenomenon is called cross-modal plasticity, and it aims to supplant the missing sensory input with information from other sensory systems. On the other hand, the term intra-modal plasticity can be applied to the case when partial sensory loss can lead to functional reconfiguration to extract the maximum information from the spared sensory functionality. Naturally, intramodal plasticity can be combined with cross-modal plasticity to obtain the maximal compensation level in the brain.

In case of hearing loss, one can define several types of deafness; they can be conductive (damage in the external, middle ears), sensorineural (inner ear, auditory nerve), and mixed. Each of these types of deafness can be symmetric, affecting equally both ears or asymmetric when the difference of hearing loss between ears is above 15 dB at one frequency. The extreme case of asymmetric hearing loss is unilateral deafness. All types of mild and moderate hearing loss can be rehabilitated by hearing aids, while severe conductive hearing loss is rehabilitated by bone conduction prosthesis and severe sensorineural hearing loss by cochlear implants. A cochlear implant (CI) is a surgically implanted electronic device that provides a sense of sound for profound to total hearing loss patients. This neuroprosthesis bypasses the defunct cochlea by transferring acoustic vibrations of a sound into electric pulses of the electrodes to stimulate the auditory nerve, and subsequently the auditory cortex creating a somewhat synthetic sound perception of the environment [START_REF] Karoui | Searching for the sound of a cochlear implant: evaluation of different vocoder parameters by cochlear implant users with single-sided deafness[END_REF].

The limited number of implanted electrodes (up to 24 compared to more than 3000 internal hair cells) and independent spectral channels lead to the poor spectral resolution aggravated by the interaction between the channels from current spread, which degrades some of the auditory cues used in speech perception [START_REF] Stilp | Acoustic context alters vowel categorization in perception of noise-vocoded speech[END_REF]. However, CI users may experience increased effects of spectral compensation relative to those observed in normal-hearing (NH) listeners [START_REF] Feng | Effects of spectral resolution on spectral contrast effects in cochlear-implant users[END_REF], which may be explained by the long-term brain reorganization in response to the degraded signal provided by the implant. It follows that compensatory brain plasticity is an important factor for the optimal outcomes of deafness rehabilitation with CIs.

The critical question in deafness and its rehabilitation is how clinical scores related to speech and auditory space perception are reflected in brain plasticity mechanisms. We will consider both types of brain plasticity (cross-modal and intra-modal) in this review in relation to symmetric (bilateral) and asymmetric hearing loss and its restoration using cochlear implantation. In the following review, deaf will refer to bilateral deafness; otherwise, it will be specified.

Cross-modal plasticity: Whole-brain level

Visuo-auditory plasticity in deafness

The classical perceptual benefits derived from multisensory interaction facilitate the weaker or masked-by-noise moda-lity's perception. It has been believed for a long time that the acquisition of speech-reading skills is one of the sensory substitution strategies developed by deaf patients, which serves to recover better speech comprehension [START_REF] Grant | Auditory-visual speech recognition by hearing-impaired subjects: consonant recognition, sentence recognition, and auditory-visual integration[END_REF][START_REF] Kaiser | Talker and lexical effects on audiovisual word recognition by adults with cochlear implants[END_REF][START_REF] Summerfield | Lipreading and audio-visual speech perception[END_REF][START_REF] Tyler | Performance over time of adult patients using the Ineraid or nucleus cochlear implant[END_REF]. In a large cohort of deaf patients, our group has shown higher performance in audiovisual conditions compared with auditory-alone conditions [START_REF] Rouger | Evidence that cochlear-implanted deaf patients are better multisensory integrators[END_REF]. Though several studies [START_REF] Rouger | Evidence that cochlear-implanted deaf patients are better multisensory integrators[END_REF][START_REF] Bernstein | Enhanced speechreading in deaf adults: can short-term training/practice close the gap for hearing adults?[END_REF][START_REF] Bernstein | Speech perception without hearing[END_REF][START_REF] Strelnikov | Improvement in speech-reading ability by auditory training: evidence from gender differences in normally hearing, deaf and cochlear implanted subjects[END_REF] indeed provide evidence that deaf people can have higher speechreading performance than NH subjects (Fig. 1), there is some debate in the literature concerning the sources of interindividual variability in speech-reading, and the utility of speech-reading training [START_REF] Summerfield | Lipreading and audio-visual speech perception[END_REF][START_REF] Bernstein | Enhanced speechreading in deaf adults: can short-term training/practice close the gap for hearing adults?[END_REF].

Compensatory cortical plasticity in deafness is inevitably related to imperfect sound perception through the implant and attempts to compensate for this deficient perception via contextual cues, including speech-reading. The cortical network that supports speech-reading in NH subjects [START_REF] Capek | Cortical circuits for silent speechreading in deaf and hearing people[END_REF] shares many areas with auditory speech processing. In addition to the cortical regions involved in visual motion perception, auditory speech areas are also implicated in speech-reading, but their exact contributions are controversial [START_REF] Campbell | The processing of audio-visual speech: empirical and neural bases[END_REF]. While some studies reported an activation by speechreading in the auditory areas of the superior temporal lobe, including the primary auditory cortex [START_REF] Calvert | Activation of auditory cortex during silent lipreading[END_REF][START_REF] Pekkola | Primary auditory cortex activation by visual speech: an fMRI study at 3T[END_REF][START_REF] Que | Language and sensory neural plasticity in the superior temporal cortex of the deaf[END_REF], others found this activation of the primary auditory cortex either variable across subjects [START_REF] Macsweeney | Silent speechreading in the absence of scanner noise: an event-related fMRI study[END_REF] or absent [START_REF] Bernstein | Visual speech perception without primary auditory cortex activation[END_REF]. It was also demonstrated with functional magnetic resonance imaging (fMRI) that congruent visual speech increases activity in the auditory cortex [START_REF] Okada | An fMRI study of audiovisual speech perception reveals multisensory interactions in auditory cortex[END_REF]. Discrepancies among these studies are probably Fig. 1 -Evolution of multisensory performance after cochlear implantation. Percent of correct responses of disyllabic words presentation in silence for patients at different periods after cochlear implant activation for speechreading of words (V), auditory (A) and audiovisual performance (AV) in word perception. Error bars represent standard errors. CI users' speech-reading abilities (blue) remain stable across all post-implantation periods tested. The average auditory performance of implanted patients (green) is at the suboptimal level after 6 months of experience with the implant. The audiovisual performance (red) after 6 months is stable at the maximum level. related to the experimental protocols involving different levels of phonological or lexical processing.

Visuo-auditory plasticity after cochlear implantation

Effect of cochlear implants on auditory perception

Usually, during the first month of experience, implanted patients perceive mostly indistinguishable sounds and noise; the capacity to discriminate and understand speech usually develops after two or three months of experience with the implant. In post-lingual CI patients, speech recovery is optimal during the first year after cochlear implantation [START_REF] Leigh | Evidence-based guidelines for recommending cochlear implantation for postlingually deafened adults[END_REF][START_REF] Ukcisg | Criteria of candidacy for unilateral cochlear implantation in postlingually deafened adults I: theory and measures of effectiveness[END_REF], but there is however an extensive range of variability, and in extreme cases, some CI recipients never develop usable speech and oral language [START_REF] Peterson | Cochlear implants and spoken language processing abilities: review and assessment of the literature[END_REF].

Although performance after cochlear implantation tends to improve with the development of better technologies and surgical techniques, the medical community still faces the problem of a great deal of unexplained individual variability in outcomes after implantation. Several factors are discussed as being associated with patients' final performance [START_REF] Holden | Factors affecting outcomes in cochlear implant recipients implanted with a perimodiolar electrode array located in scala tympani[END_REF]. The most discussed are biographic and audiologic factors such as duration of severe-to-profound hearing loss, age at the definition of this loss, duration of hearing loss, hearing aid use, residual hearing, age at cochlear implantation, preoperative speech recognition and post-operative implant experience. These factors exhibit small positive or negative correlations with long-term implant outcome [START_REF] Lazard | Pre-, Per-and postoperative factors affecting performance of postlinguistically deaf adults using cochlear implants: a new conceptual model over time[END_REF]. Device and surgical factors, such as the brand of the implant, percentage of available electrodes programmed, residual hearing preservation, and electrode position within the cochlea, have also been related to performance. Specifically, electrodes positively correlated with outcomes are suggested to be: (i) located in the scala tympani (ST); (ii) inserted to the intended design depth of the array, i.e., not over-or under-inserted; and (iii) proximal to the modiolar wall. The extent to which biographic, audiologic, and device-related factors interact and individually contribute to speech understanding is not fully understood, especially when speech must be understood in more realistic and complex listening environments, i.e., background noise. Controlling for some factors should allow a more focused examination of others [START_REF] Holden | Factors affecting outcomes in cochlear implant recipients implanted with a perimodiolar electrode array located in scala tympani[END_REF].

Audiovisual integration as a compensatory mechanism after cochlear implantation

Despite the sound provided by the cochlear implant, a progressive increase in speech-reading ability was observed during the first years after implantation [START_REF] Giraud | Cross-modal plasticity underpins language recovery after cochlear implantation[END_REF][START_REF] Giraud | Imaging plasticity in cochlear implant patients[END_REF], which may be a compensation for the poor quality of the sound. However, some studies indicated only mild improvement [START_REF] Bergeson | Development of audiovisual comprehension skills in prelingually deaf children with cochlear implants[END_REF] or no change at all [START_REF] Gray | Patient performance over eighteen months with the Ineraid intracochlear implant[END_REF] in speech-reading capacities after implantation. To address this controversial issue, we performed a longitudinal study of speech-reading performance in a population of about one hundred adults with post-linguistic profound deafness of diverse etiologies and durations, encompassing post-implantation times ranging up to eight years [START_REF] Rouger | Evidence that cochlear-implanted deaf patients are better multisensory integrators[END_REF]. As previously reported [START_REF] Gray | Patient performance over eighteen months with the Ineraid intracochlear implant[END_REF], at the time the CI was switched on, word speech-reading performance in CI patients was much higher (35%) than the performance of NH participants (9%). As these scores were collected while patients had almost no auditory experience with their implant, they mainly reflected word speech-reading abilities in deaf patients without CI, and they were not significantly different from the pre-implantation scores. A somewhat surprising outcome of our study was that CI users' speechreading abilities remained stable across all post-implantation periods tested (i.e., greater than 35% even after several years), even though the patients had reached their maximal auditory performance (Fig. 1). However, the conclusion about the stability of speech-reading performance after cochlear implantation was slightly revised when the gender of subjects was taken into account [START_REF] Strelnikov | Improvement in speech-reading ability by auditory training: evidence from gender differences in normally hearing, deaf and cochlear implanted subjects[END_REF]. The implanted patients' average auditory performance recovered to the suboptimal level (80-90%) after six months experience with the implant, while, remarkably, the audiovisual performance after six months was at about 100% (Fig. 1). Our central hypothesis is that the above-discussed high visual aptitude might induce CI users to improve the multisensory integration mechanisms, which may be higher than in NH subjects. To address this issue, we compared the audiovisual gain of CI patients during a word recognition task to the gain in controls exposed to the same stimuli degraded using a noise-band vocoder paradigm to simulate the degraded sound from the cochlear implants [START_REF] Rouger | Evidence that cochlear-implanted deaf patients are better multisensory integrators[END_REF]. In the vocoder simulation, fine temporal cues within each spectral component are removed while global temporal and spectral acoustic information is preserved. This approach allowed us to make direct comparisons of audiovisual performance at equivalent ranges of non-optimal auditory performance for both controls and CI subjects. The normalization of the bimodal benefit with respect to the auditory performance showed an audiovisual gain in CI patients nearly twice as high as observed in NH participants. Moreover, in another study, CI patients showed significant supra-normal audiovisual integration for linguistic prosodic information compared to hearing controls, as evidenced by CI patients having a visuo-auditory gain more than three times larger than that observed in hearing controls [START_REF] Lasfargues-Delannoy | Supra-normal skills in processing of visuoauditory prosodic information by cochlear-implanted deaf patients[END_REF].

From the clinical point of view, these results suggest that a strong visually and audio-visually based rehabilitation during the first months after implantation would significantly improve and fasten the functional recovery of speech intelligibility.

Cortical plasticity and audiovisual integration in CI patients

Two complementary mechanisms of brain reorganization occur in adult CI deaf patients (see Fig. 2). Firstly, brain reorganization is induced by the complete deafness manifesting as the compensatory shift toward a visual mode of communication (speechreading and/or sign language). In this case, the cortical circuit that supports visual speech perception in CI patients may differ from those described in congenitally deaf and in NH subjects. Secondly, after implantation, the change in speech processing strategies is associated with a functional reorganization, as the communication mode relies on enhanced visio-auditory proficiencies. Auditory recovery is associated with an increasing reactivation of auditory cortical areas [START_REF] Mortensen | Restored speech comprehension linked to activity in left inferior prefrontal and right temporal cortices in postlingual deafness[END_REF] and with increased audiovisual integration observed in CI patients, which is consistent with the cross-modal activations of low-level visual areas during auditory-only speech perception in CI patients [START_REF] Giraud | Cross-modal plasticity underpins language recovery after cochlear implantation[END_REF]. Furthermore, we found that CI patients present a higher activity level in the visual cortex (inferior occipital gyrus) than controls in the resting state with an increment of activity in these areas from the time of implant activation to less than a year after implantation [START_REF] Strelnikov | Does brain activity at rest reflect adaptive strategies? Evidence from speech processing after cochlear implantation[END_REF]. The higher activity in visual areas at rest may correspond to higher capacities for audiovisual integration of speech because it was also accompanied by an increment of activity in the auditory and speech-related cortices. By integrating EEG and functional near-infrared spectroscopy (fNIRS) signals, Chen et al. [START_REF] Chen | Enhanced visual adaptation in cochlear implant users revealed by concurrent EEG-fNIRS[END_REF] found significantly enhanced visual adaptation and lower visual cortex activation in CI users than in controls. That is, responses to repeated visual stimuli decreased more prominently in CI users than in controls. The results may suggest that CI users process visual stimuli more efficiently than controls [START_REF] Doucet | Cross-modal reorganization and speech perception in cochlear implant users[END_REF]. Further, as numerous studies have demonstrated how sensory loss is accompanied by colonization of deprived sensory areas by the remaining modalities [START_REF] Pekkola | Primary auditory cortex activation by visual speech: an fMRI study at 3T[END_REF][START_REF] Finney | Visual stimuli activate auditory cortex in the deaf[END_REF][START_REF] Nishimura | Sign language ''heard'' in the auditory cortex[END_REF][START_REF] Rauschecker | Compensatory plasticity and sensory substitution in the cerebral cortex[END_REF], Rouger et al. [START_REF] Rouger | Evolution of crossmodal reorganization of the voice area in cochlear-implanted deaf patients[END_REF] showed in adult CI patients a robust cross-modal reorganization of the auditory voice area of the right superior temporal sulcus (rSTS). Significant correlation between activity in the rSTS and speech-reading performance in inexperienced CI patients demonstrated that this auditory area is involved in visual information processing. While one can logically think that this visually induced reorganization could have harmful effects on the functional recovery after cochlear implantation, the longitudinal analysis revealed that the level of crossmodal rSTS activation decreases within the first months postimplantation, while patients are recovering auditory speech comprehension. Such a result clearly demonstrates that in adults, cross-modal plasticity can be reversed by rehabilitating the sensory loss, at least for the auditory loss.

In addition, using fNIRS, Chen et al. [START_REF] Chen | Increased cross-modal functional connectivity in cochlear implant users[END_REF] investigated taskinduced intra-modal functional connectivity between hemispheres for both visual and auditory cortices and cross-modal functional connectivity between visual and auditory cortices in post-lingually deaf CI users and age-matched NH controls. Compared to controls, CI users exhibited decreased intramodal functional connectivity between hemispheres and increased cross-modal functional connectivity between visual and left auditory cortices for both visual and auditory stimulus processing. Besides, higher cross-modal connectivity for auditory than visual stimuli was associated with better speech recognition abilities, pointing to a new functional reorganization pattern related to successful hearing restoration with a CI.

Another study with fNIRS [START_REF] Anderson | Adaptive benefit of cross-modal plasticity following cochlear implantation in deaf adults[END_REF] showed that increased crossmodal activation after adult cochlear implantation is associated with increased auditory responsiveness and better speech understanding with a CI, indicating an adaptive benefit of cross-modal plasticity following implantation. Such results are in perfect agreement with our longitudinal study of audiovisual coupling based on independent components Fig. 2 -Oral speech comprehension, speechreading skills and brain reorganization as a result of cochlear implantation (explanatory scheme). While oral comprehension improves after cochlear implantation, the speechreading level remains rather stable. During deafness, voice areas in the temporal cortex are colonized by visual processing. There is further crossmodal reorganization in the temporal cortex after cochlear implantation. The + sign means that there is a positive regression between the areas (the occipital and the posterior temporal ones). STS: superior temporal sulcus, STG: superior temporal gyrus.

analysis applied to the cortical activity during visual and visuo-auditory speech processing [START_REF] Strelnikov | Increased audiovisual integration in cochlear-implanted deaf patients: independent components analysis of longitudinal positron emission tomography data[END_REF]. Patients at both the early and late periods after implantation had a task-related component in the visual cortex. Of importance, the time course of temporal and visual activity was highly coupled only in experienced patients (Fig. 2), suggesting that visuo-auditory synergy is crucial for adjusting cross-modal plasticity, which in turn is necessary to recover speech comprehension.

A correlation analysis was performed using source analysis in electroencephalography (EEG) to test the hypothesis of a relationship between auditory cross-modal activation and audiovisual integration strength [START_REF] Stropahl | Auditory cross-modal reorganization in cochlear implant users indicates audiovisual integration[END_REF]. The CI group showed a significant relationship between cross-modal activation in the left auditory region and the frequency of fusion percepts (i.e., creation of multisensory objects), such that higher crossmodal activation was associated with more fusion in the McGurk illusion. However, a similar correlation was not observed in NH subjects and patients with moderate hearing loss. This observation confirms the reorganization of cortical activity in CI patients specifically for audiovisual integration.

Altogether, this set of brain imaging studies points to the importance of the synergy that occurs in both the visual and auditory areas and confirms that such audio-visual synergy participates in the progressive recovery of auditory speech comprehension.

Predictive role of visual activity for the auditory speech restoration

Several studies in congenitally deaf children have revealed that the visual cross-modal reorganization of the auditory cortex is deleterious for auditory speech recovery after cochlear implantation [START_REF] Giraud | Predicting cochlear implant outcome from brain organisation in the deaf[END_REF][START_REF] Lee | Cortical activity at rest predicts cochlear implantation outcome[END_REF]. This claim was under debate in adult-acquired deafness as the cross-modal reorganization was shown to regress with the recovery by a cochlear implant. A possible approach to test this hypothesis is to analyze what regions of brain activity shortly after implantation are correlated with the level of auditory recovery several months later in post-lingually deaf adults, i.e. what regions are predictive of the success or failure to develop speech perception using the cochlear implant. Having adopted this approach to analyze brain activity induced by a speechprocessing task, we found strong positive correlations in areas outside the auditory cortex [START_REF] Strelnikov | Visual activity predicts auditory recovery from deafness after adult cochlear implantation[END_REF]. Moreover, these correlations were task-independent; they were present during visual, audiovisual speech, and at rest. The highest positive correlations independently of the task were found in the occipital cortex involved in low visual processing and the posteriortemporal cortex known for audio-visual integration. This finding reinforces the suggested role of visuo-auditory synergy in the rehabilitation of auditory speech comprehension. However, in this study, a deleterious impact of cross-modal reorganization in associative auditory areas was also found at the level of the right middle superior temporal gyrus/superior temporal sulcus (STG/STS) in post-lingual adults, which was similar to the one previously demonstrated in prelingually deaf children [START_REF] Lee | Cortical activity at rest predicts cochlear implantation outcome[END_REF]. In our study, the right STG/STS cortical locus closely matched the STS region reported in the literature as voice-sensitive cortical regions belonging to the temporal voice areas network [START_REF] Belin | Voice-selective areas in human auditory cortex[END_REF][START_REF] Kriegstein | Distinct functional substrates along the right superior temporal sulcus for the processing of voices[END_REF] considered as being predomi-nantly an auditory area. Furthermore, the peaks for all the experimental conditions (visual, audiovisual, and rest) coincided (within a small error due to smoothing) with the subpeak, which was activated during lip-reading in adult CI users [START_REF] Rouger | Evolution of crossmodal reorganization of the voice area in cochlear-implanted deaf patients[END_REF] or by sign language in prelingually deaf patients [START_REF] Sadato | Age-dependent plasticity in the superior temporal sulcus in deaf humans: a functional MRI study[END_REF]. Consequently, it is now firmly demonstrated that the capacity for reversal of cross-modal reorganization of the STG/STS region is crucial to the ability of CI users to recover speech comprehension.

Anderson et al. [START_REF] Anderson | Plasticity in bilateral superior temporal cortex: effects of deafness and cochlear implantation on auditory and visual speech processing[END_REF] suggested that plasticity could be differently expressed on each hemisphere and may impact CI outcome differently. The authors maintain that speechreading may guard against maladaptive plasticity in left phonological areas, and left hemisphere audio-visual interactions may facilitate auditory recovery with a CI. However, the right hemisphere cross-modal plasticity is associated with poor CI outcomes. The confirming observations are that CI patients have poor results for prosody perception, primarily located in the right hemisphere [START_REF] Marx | Speech prosody perception in cochlear implant users with and without residual hearing[END_REF], and for emotional face recognition, which also happens mainly in the right hemisphere [START_REF] Ambert-Dahan | Emotional recognition of dynamic facial expressions before and after cochlear implantation in adults with progressive deafness[END_REF].

All these considerations taken into account, the predictive link between visual activity early post-implantation and the efficiency for auditory speech perception in experienced CI patients, as demonstrated by neuroimaging, provides evidence that visuo-auditory synergy is crucial for cross-modal plasticity and it fosters speech-comprehension recovery in adult cochlear-implanted deaf patients.

Intra-modal plasticity: within the auditory system

Basics of binaural hearing

Binaural hearing is defined as hearing with two healthy ears [START_REF] Moore | Anatomy and physiology of binaural hearing[END_REF], a remarkable process responsible for sound localization in mammals and for speech recognition in noise for humans. This process relies on the perfect integration of two auditory cues within the horizontal plane: the interaural time and level differences (ITD and ILD) and the spectral cues (from the shape of the pinna) in the vertical plane [START_REF] Grothe | Mechanisms of sound localization in mammals[END_REF]. The ITD is the essential auditory cue for the lower frequencies (< 1500 Hz), whereas the ILD is critical to the higher frequencies (> 3000 Hz). In between 1500 and 3000 Hz, binaural hearing relies on both cues. The ILD and ITD are embedded within three neurophysiological binaural hearing mechanisms: the central binaural summation and squelch effects [START_REF] Avan | Importance of binaural hearing[END_REF] and the passive headshadow effect. The head-shadow effect is simply the presence of the head within the auditory field that favors the signal-tonoise ratio (SNR) up to 10-15 dB of the incoming signals for the closest to the source of the sound ear. The central summation effect increases the SNR by summing up (or combining) the incoming signal arriving simultaneously in both ears. This effect increases the perception of the sound by around 3 dB [START_REF] Heil | Towards a unifying basis of auditory thresholds: binaural summation[END_REF]. The squelch effect occurs when the noise and the signals arrive from two different locations (for instance, the noise from the left and the signal from the right). Again, the brain will favor the SNR by decreasing or ''squelching'' the noise and increasing the signal. This binaural effect can increase the SNR up to 12 dB [START_REF] Avan | Importance of binaural hearing[END_REF].

Unilateral hearing loss: behavioral consequences

A disruption of binaural hearing caused by a unilateral hearing loss (UHL, i.e., one healthy ear and one impaired ear) leads to a misperception of ITD and ILD [START_REF] Bronkhorst | Binaural speech intelligibility in noise for hearing-impaired listeners[END_REF], thus affecting all three binaural auditory mechanisms [START_REF] Vannson | Quality of life and auditory performance in adults with asymmetric hearing loss[END_REF]. Therefore, subjects, either children or adults, experiencing asymmetrical hearing loss present deleterious behavioral consequences [START_REF] Vannson | Quality of life and auditory performance in adults with asymmetric hearing loss[END_REF][START_REF] Bess | Auditory performance of children with unilateral sensorineural hearing loss[END_REF][START_REF] Slattery | Monaural sound localization: acute versus chronic unilateral impairment[END_REF]. For example, in children, profound unilateral hearing loss stirs developmental [START_REF] Ead | Pilot study of cognition in children with unilateral hearing loss[END_REF] and language [START_REF] Bess | Auditory performance of children with unilateral sensorineural hearing loss[END_REF] delays inducing lower school performances (higher grade failure up to 33% [START_REF] Bess | Auditory performance of children with unilateral sensorineural hearing loss[END_REF][START_REF] Lieu | Longitudinal study of children with unilateral hearing loss[END_REF], thus requiring intervention as early as possible to avoid future handicaps in their daily life [START_REF] Lee | Consistent and chronic cochlear implant use partially reverses cortical effects of single sided deafness in children[END_REF][START_REF] Vila | Asymmetric and unilateral hearing loss in children[END_REF]. In UHL adults compared to NH peers, consequences are presented as degraded localization and speech recognition-in-noise performances [START_REF] Arndt | Comparison of pseudobinaural hearing to real binaural hearing rehabilitation after cochlear implantation in patients with unilateral deafness and tinnitus[END_REF][START_REF] Vannson | Speech-in-noise perception in unilateral hearing loss: Relation to pure-tone thresholds and brainstem plasticity[END_REF] and a lower global quality of life [START_REF] Vannson | Quality of life and auditory performance in adults with asymmetric hearing loss[END_REF]. It's worth mentioning that the higher the hearing loss asymmetry, the lower the behavioral performances. Furthermore, these lower performances are also correlated with lower global quality-of-life scores [START_REF] Vannson | Quality of life and auditory performance in adults with asymmetric hearing loss[END_REF], confirming that disruption of binaural hearing is a handicap for most UHL adults in their social life.

Neural processing of spatial auditory information and relationship with behavior in UHL Slattery and Middlebrooks [START_REF] Slattery | Monaural sound localization: acute versus chronic unilateral impairment[END_REF] investigated spatial adaptation in patients after UHL. Their study showed that despite the detrimental consequence of hearing asymmetry on auditory spatial performances, some UHL presented normal localization performances compared to monaural controls and NH subjects. The investigation of the relationship between behavioral performances after hearing loss and demographic factors of hearing-impaired patients is essential to further understand the origin of neural adaptation after hearing loss.

Therefore, one approach to understanding the source of behavioral variability is to seek typical profiles in the UHL population by applying cluster analysis using K-means that allows defining homogeneous subgroups of UHL. A cohort of adult patients suffering from UHL with variable hearing loss levels ranging from 36 dB to total deafness was recruited and evaluated for behavioral performances that involve binaural integration, including localization errors (root mean square errors), speech-in-noise performances (speech reception thresholds), and quality-of-life questionnaire (Speech, Spatial and Qualities of Hearing Scale). These behavioral scores were included in a cluster analysis fitted in an unsupervised learning model as defining factors.

As Fig. 3 shows [START_REF] Alzaher | Spatial auditory plasticity reflected by mismatch negativity in unilateral hearing loss patients[END_REF], the cluster analysis separated the UHL cohort into three sub-groups: UHL 1 to 3. UHL1 (n = 9) patients had a ''normal'' localization profile when compared to NH listeners (148 vs. 198). This profile is an example of efficient compensatory strategies underlying neural adaptation after hearing loss. In normal cases, introducing an earplug of 40 dB attenuation dramatically reduces spatial localization abilities due to the alteration of binaural integration and, by default, reducing the perception of ILD and ITD in the horizontal plane. However, UHL1 subjects, who had hearing loss greater than the monaural plug (60 dB), presented normal localization behavior. Thus, as Fig. 3 shows, even UHL2 with very high pure-tone audiometry thresholds (98 dB) were as good as monaural controls. UHL 1 and 2 had a ''good'' spatial resolution compared to monaural controls. Based on their performances, we suggest that those patients adapted to monaural deafness, possibly through ''alternative'' compensatory strategies. Until now, only anecdotal results have been reported on the possibility of recovery of spatial hearing after UHL, and these results highlight that mechanisms such as head movements, monaural cues, and the reliance on visual input could contribute to the effective sound integration: Fig. 3 -A

Head movements

Sound localization in natural auditory scenes requires active interaction with the environment that includes moving the head to explore space and reduce ambiguities by modifying binaural cues relative to the sound, particularly modifying spectral shape cues and the influence of shadow effect on the sound perception of the sound wave.

Monaural spectral shape cues

Animals and humans can benefit from spectral cues to perceive sound location. Indeed, the pinna's physical aperture that interferes in amplifying and attenuating some frequencies entering the ear could help disambiguate sound source locations [START_REF] Batteau | The role of the pinna in human localization[END_REF][START_REF] Hofman | Relearning sound localization with new ears[END_REF]. Different studies trying to manipulate pinna shapes to investigate their influence on perceiving auditory space have tested the importance of spectral cues' implication [START_REF] Schnupp | On hearing with more than one ear: lessons from evolution[END_REF].

Multisensory integration and the importance of visual inputs

Visual inputs are important in guiding and orienting the head towards the source location. Head movements are usually initiated after eye movements. In addition, coupling of auditory and visual spatial cues is very efficient for the occurrence of adaptive cross-modal brain plasticity after hearing loss [START_REF] Bavelier | Cross-modal plasticity: where and how?[END_REF] (see the Section 2 on cross-modal plasticity). The target location is encoded in auditory and visual systems in different manners. However, to correctly identify and detect stimulus sources spatially, both modalities must provide sufficiently informative inputs about both the sound and its visual representation. For example, by reducing visual gain in participants wearing half-lenses to compress visual spatial space by half, it has been shown that the reduction of visual spatial gain induced in three days a similar decrease in spatial auditory localization [START_REF] Zwiers | Plasticity in human sound localization induced by compressed spatial vision[END_REF], meaning that the auditory space was re-calibrated to the spatially scaled visual space probably through brain adaptive mechanisms. These findings suggest that the compressed visual field was responsible for brain plasticity and adaptive changes in sound localization.

All the above-enumerated factors constitute the natural integration and interpretation of the sound and its elements (frequency, intensity, location) in its natural ecological environment, including the visual context [START_REF] Valzolgher | The impact of a visual spatial frame on real sound-source localization in virtual reality[END_REF]. The alteration of one of these factors, such as the binaural cues in the case of UHL, is probably compensated by one of the ''alternatives'' customarily used in the natural listening activity. The reliance on these compensatory strategies would further shape the nature of neural adaptation and cortical activation in response to auditory inputs. More importantly, the issue that needs to be addressed here is how these ''compensatory'' mechanisms influence cortical reorganization and different processes underlying cortical reorganization. To answer this question, further work is needed to explore in detail the correlation between behavioral and neural representations of spatial auditory perception in hearing-impaired patients.

Functional relationship between behavioral and neural adaptation

As already explained in the previous section, the multisensory nature of auditory perception provides the listener with various mechanisms capable of accurately decoding oral speech and auditory space. It has been hypothesized that the alteration of one of these mechanisms, such as binaural cues in UHL, can be compensated by one or more mechanism(s), including monaural cues, head movements, and visual dominance. Unfortunately, few studies investigated the nature of behavioral adaptation after hearing loss and its impact on cortical reorganization.

In our case (Fig. 3), UHL1 had a ''normal'' spatial resolution compared to monaural controls that showed a reduction in sound location. This adaptation can be investigated on a neural level using different neural markers. For example, Cai et al. [START_REF] Cai | Auditory spatial discrimination and the mismatch negativity response in hearing-impaired individuals[END_REF] used mismatch negativity as a neural marker that reflected spatial detection in UHL. They associated the reduction of mismatch negativity amplitude in patients who presented high spatial scores with a decrease in the recruitment of cortical neurons and a broader distribution of neural reactivity over the auditory cortex. Similarly, patients with hearing loss present a broader distribution of the P2 component of late auditory evoked potentials [START_REF] Campbell | Compensatory changes in cortical resource allocation in adults with hearing loss[END_REF] confirming that different activation patterns occur after hearing loss and that these activation patterns can further manifest on the behavioral level. Although few studies depicted the correlation between behavioral and neural adaptation in UHL, we can conclude, based on previous literature, that there is a possible behavioral and neural adaptation due to functional changes on different levels of the auditory system. Despite the ample evidence regarding this adaptation to monaural situations, the underlying cortical reorganization is not fully understood.

Brain plasticity after unilateral hearing loss

Unilateral hearing loss offers us an excellent model of intramodal plasticity as one ear only is impaired. Indeed, UHL induces a brain reorganization that affects cortical sound treatment [START_REF] Scheffler | Auditory cortical responses in hearing subjects and unilateral deaf patients as detected by functional r e v u e n e u r o l o g i q u e x x x ( 2 0 2 1 ) x x x -x x x NEUROL-2518[END_REF]. In NH subjects, stimulation of one ear (right or left) induces relatively higher auditory activity in the contralateral hemisphere. For example, the sound coming from the right ear will cross the midline to reach the left auditory cortex within the left hemisphere (Fig. 4). In contrast, in UHL patients, stimulation of the better (right or left) ear leads to a cortical sound treatment that activates both hemispheres almost symmetrically (Fig. 4) [START_REF] Scheffler | Auditory cortical responses in hearing subjects and unilateral deaf patients as detected by functional r e v u e n e u r o l o g i q u e x x x ( 2 0 2 1 ) x x x -x x x NEUROL-2518[END_REF]. The onset of brain reorganization in adults after unilateral hearing loss appears (or is detectable) after five weeks [START_REF] Suzuki | Cortical representation of hearing restoration in patients with sudden deafness[END_REF], and it seems to reach its maxima (in terms of voxel activation in fMRI) after one year [START_REF] Bilecen | Cortical reorganization after acute unilateral hearing loss traced by fMRI[END_REF]. This slow dynamic in cortical reorganization could be explained by physiological and cytoarchitectonic mechanisms described as a loss of contralateral inhibition and a reinforcement of the number of fiber connections along the healthy ear auditory pathway [START_REF] Bilecen | Cortical reorganization after acute unilateral hearing loss traced by fMRI[END_REF].

To deepen cytoarchitectonic aspects of brain reorganization depicted earlier by Scheffler [START_REF] Scheffler | Auditory cortical responses in hearing subjects and unilateral deaf patients as detected by functional r e v u e n e u r o l o g i q u e x x x ( 2 0 2 1 ) x x x -x x x NEUROL-2518[END_REF] at the whole-brain level, an fMRI study among UHL adults [START_REF] Vannson | Evidence of a functional reorganization in the auditory dorsal stream following unilateral hearing loss[END_REF] focused on the primary (Te 1.0, Te 1.1, and Te 1.2) and secondary (Te 3.0) auditory cortices. In this study, the contralateral dominance was shown to be preserved in the primary auditory cortex for both ears (the better and the poorer). On the contrary, after stimulation of the right better ear, a more symmetrical pattern of cortical sound processing was observed in the non-primary auditory cortices (Fig. 4). A more precise computation of the laterality of the cortical activation is provided by the weighted lateralization index (WLI in [START_REF] Vannson | Evidence of a functional reorganization in the auditory dorsal stream following unilateral hearing loss[END_REF]). The WLI offers a direct comparison of the strength of activation through a single ear in the cortical areas of both hemispheres. The lateralization in NH subjects is reported as being contralateral, meaning that the auditory pathways only weakly activate the auditory cortex ipsilateral to the stimulated ear. The reduced ipsilateral activation is due to local neuronal interactions and the various excitatory/inhibitory nature of ipsilateral inputs that reach the auditory cortex (see [START_REF] Gutschalk | Stimulus dependence of contralateral dominance in human auditory cortex[END_REF]). In unilateral deafness, at the level of the non-primary auditory cortex, patients present a reversed ipsilateral dominance. Indeed, the WLI of the normal ear is shifted toward the ipsilateral auditory cortex suggesting a reorganization of the neuronal interactions in the auditory cortex of the same side. Crucially, the shift toward an ipsilateral aural dominance in UHL patients is directly related to a deficit in sound localization performances (Rho = 0.8; P < 0.01). This last result suggests that the cortical plasticity resulting from the hearing loss is deleterious to spatial hearing abilities. Furthermore, a regression analysis (Fig. 5) at the whole-brain level was computed to investigate the cortical network whose activity accounts for sound localization performances. This regression analysis revealed the weaker involvement of the areas located in the auditory dorsal stream in UHL patients compared with controls. The auditory dorsal stream is primarily involved in processing spatial attributes of the sounds in opposition to the auditory ventral stream involved in the identification of incoming sounds [START_REF] Rauschecker | Maps and streams in the auditory cortex: nonhuman primates illuminate human speech processing[END_REF] (Fig. 5-top panel). Fig. 5 -Regression analysis and auditory streams. In the top panel, A1 refers to the primary auditory cortex. The directions of the auditory dorsal and ventral streams are depicted. In the bottom panel the primary auditory cortex is outlined in white. Blue areas are results of the regression analysis and show brain regions linked to auditory performances (the dorsal stream in the posterior superior temporal gyrus), which are less implicated in unilaterally deaf patients compared with normal-hearing controls.

Fig. 4 -fMRI cortical activation after a sound stimulation at the whole brain level and WLI index. For all panels, NH and UHL are Normal Hearing controls and Unilateral Hearing Loss respectively. For upper panels (fMRI images), L and R depict the Left and Right ear/hemisphere. The arrows represent the side of sound stimulation. For the lower panels (bar graphs), the labels ''Binaural'', ''Right ear'' and ''Left ear'' on the horizontal axis depict the side of the sound stimulation. The vertical axis represents the WLI (Weighted Laterality Index), where positive values indicate the left hemisphere dominance and negative values correspond to the right hemisphere dominance. In normal hearing controls, activity has contralateral shift for each ear, while in UHL activity is ipsilateral to the good right ear. N.S stands for non-significant with respect to zero. Asterisks stand for significant with respect to zero at P < 0.05. Error bars represent standard deviations. NPAC: non-primary auditory cortex. r e v u e n e u r o l o g i q u e x x x ( 2 0 2 1 ) x x x -x x x Thus, our analysis emphasized that UHL degrades the auditory dorsal stream, disabling UHL patients to localize sound in space. After UHL, these biological markers of the brain reorganization reflect the handicap encountered by UHL patients in their daily lives. Overall, these objective neuroimaging measures are in favor of binaural hearing rehabilitation.

Cortical recovery of unilateral hearing loss

Different binaural hearing rehabilitation techniques exist in both children and adults and depend on the degree of UHL. In the most severe cases of single-sided deafness (SSD), a cochlear implant provides the highest quality of life and higher behavioral performances, such as localization and speech recognition in noise Cortical activity recovery after SSD rehabilitation was shown for the moment only with EEG within a pediatric SSD population [START_REF] Lee | Consistent and chronic cochlear implant use partially reverses cortical effects of single sided deafness in children[END_REF][START_REF] Polonenko | Cortical organization restored by cochlear implantation in young children with single sided deafness[END_REF]. These two studies provide some evidence that CI devices could partially restore cortical sound treatment asymmetry approaching NH listeners. However, the onset of SSD (early or late) might be crucial for brain plasticity [START_REF] Lee | Consistent and chronic cochlear implant use partially reverses cortical effects of single sided deafness in children[END_REF]. In early-onset SSD children (0-5 years of age), sound stimulation through CI creates an asymmetrical cortical sound representation in the contralateral hemisphere whereas in late-onset SSD children (10-16 years of age) even with the same implant duration as the earlyonset group (one year on average), sound representation is more symmetrical in both hemispheres probably because children still strongly rely on the better ear.

In addition, within an adult SSD population, our recent positron emission tomography (PET) study [START_REF] Karoui | Restoring cortical processing for spatial hearing after cochlear implantation in AHL: a PET scan H2O15 study[END_REF] demonstrates that a CI reverses the ipsilateral dominance for better ear stimulation, making auditory activity shift toward the contralateral side. Moreover, the level of this restoration of auditory asymmetry is highly correlated with spatial localization scores highlighting our previous hypothesis that restoring binaural hearing in SSD patients is needed.

Conclusions

Cross-modal brain plasticity may have both beneficial and detrimental effects on hearing disorders. It has a beneficial effect when it serves to improve a patient's adaptation to the visuo-auditory environment. However, the invasion of the auditory cortex by visual functions may be deleterious for the restoration of hearing with CIs. In what concerns intra-modal plasticity, the loss of interhemispheric asymmetry in asymmetric hearing loss is deleterious for auditory spatial localization. However, the causal effects underlying correlations of the loss of asymmetry with spatial localization scores remain unclear. An important question is whether the loss of symmetry in the auditory cortex presents an independent deleterious factor or reflects the loss of the auditory function by the deficient ear. Research on brain plasticity in hearing disorders can advance our understanding of brain plasticity, it can improve the rehabilitation of patients using prognostic, evidence-based approaches from cognitive neuroscience combined with post-treatment objective biomarkers of brain plasticity employing neuroimaging.

Perspectives

Given that each neuroimaging technique has its advantages and limitations, it is necessity to compare the sensitivity and specificity of different neuroimaging modalities to establish the most reliable prognostic approach. An outstanding question with significant clinical importance is whether brain activity measured with 18 FDG PET can predict hearing restoration after cochlear implantation. For clinical purposes, it would be important to compare the prognostic values of 18 FDG PET and morphometric MRI. The emerging neuroimaging method fNIRS, a portable and inexpensive device with a high temporal resolution, can have a high potential to estimate brain plasticity. In addition to studying the global levels of auditory activity, one can assess changes in its spatial activity patterns [START_REF] Sadoun | Stimulus-specific information is represented as local activity patterns across the brain[END_REF] due to deafness and its rehabilitation methods. Concerning EEG, there is a significant perspective for studies of automatic brain reaction reflected by mismatch negativity as an objective and relatively simple way to assess the capacity of the brain to discriminate auditory features at different stages of deafness and post-implantation. All this work provides crucial information about the impact of brain plasticity on the recovery of sensory function through a neuroprosthesis (CI) and provides theoretical insights about the intra-and cross-modal adaptive mechanisms. It indicates the possibility of an early predictive diagnosis of cochlear implantation outcomes based on brain imaging techniques.

  Fig. 3 -A. Division of patients with unilateral hearing loss according to K-mean clustering. The x axis represents the dimension 1 (Dim1) of the cluster analysis, the y axis represents the dimension 2 (Dim2). Dim 1 and Dim 2 are the representative dimensions of Speech Reception Thresholds (SRT) and Root Mean Square errors (RMS) respectively. Dim 1 have the major contribution on the cluster division (50,3%). B. The y axis represents the RMS errors in degree, the x axis represents the normal hearing (NH) groups and the unilateral hearing loss (UHL) groups. NH-bin, NH-mon stand for Normal Hearing binaural (two ears) and monaural (one ear plugged) respectively. Groups of UHL depicted here are UHL1: hearing loss of 60 dB; UHL 2: hearing loss of 98 dB and UHL3: hearing loss of 100 dB. Error bars represent standard deviations.

r e v u e n e u r o l o g i q u e x x x ( 2 0 2 1 ) x x x -x x x

Please cite this article in press as: Alzaher M, et al. Brain plasticity and hearing disorders. Revue neurologique (2021), https://doi.org/10.1016/ j.neurol.2021.09.004

NEUROL-2518; No. of Pages 12 Please cite this article in press as: Alzaher M, et al. Brain plasticity and hearing disorders. Revue neurologique (2021), https://doi.org/10.1016/ j.neurol.2021.09.004

Funding

This research is funded by Agir pour l'audition (APA-RD2015-6B) and ANR AgeHear ( ANR-20-CE28-0016-01). r e f e r e n c e s

Disclosure of interest

The authors declare that they have no competing interest.


Quentin Pavic

Sylvain Tranchimand

Loïc Lemiègre

Laurent Legentil
email: laurent.legentil@ensc-rennes.fr

Diversion of a thioglycoligase for the synthesis of 1-O-acyl arabinofuranoses

An arabinofuranosylhydrolase from the GH51 family was transformed into an acyl transferase by mutation of the catalytic acid/base amino acid. The resulting enzyme was able to transfer carboxylic acid onto the anomeric position of arabinose with complete chemo-and stereoselectivity. A wide range of acyl α-l-arabinofuranoses was obtained with yields ranging from 25 to 83 %. Using this method, ibuprofen and N-Boc phenylalanine were successfully transformed into their corresponding acyl conjugates, expanding the scope of the reaction to drugs and amino acids.

Glycosyl esters are a family of carbohydrates with increasing potential as bioactive or industrially relevant compounds. Indeed carbohydrates acylated by aryl carboxylic acids, 1 fatty acids 2 or saponins 3 are known high value water soluble preservatives, surfactants or food additives (Stevioside for example). 4 Such conjugates are naturally present in fruits or vegetables, mainly as 1,2-trans isomers. 5,6 They constitutes also important metabolites as many xenobiotic carboxylic acids undergo phase II conjugation with glucuronic acid to give the corresponding 1-O-acyl β-d-glucuronides that are easily excreted through the bile duct or the kidneys. 7 The lack of stability of glycosyl esters hampers their extractability and their transfer as manufactured fine chemicals is difficult. Different synthetic strategies were therefore implemented to access such useful intermediates starting from either partially protected or unprotected sugar. Most popular methods relied on SN2 nucleophilic substitution of glycosyl halides 8,9 or trichloroacetimidate glycosides, 10 remote activation of methoxypyridyl glycoside, 11 Mitsunobu 12 or carbodiimide-promoted coupling 13 to yield either α-or β-glycosyl esters. Such strategies nevertheless lack either efficiency or selectivity. Alternatively, biotechnological tools were developed to access in particular 1-O-acyl glucuronides. They relied on microsomial preparation of UDP-glucuronyltransferases 14 or the use of lipases 15,16 as catalyst for the transfer of carboxylic acid to nucleotide-or free sugar respectively. Such seducing approaches are however hampered by either the low availability of the substrate or the lack of versatility of the catalyst. Very recently, sucrose phosphorylases have been shown to accept carboxylic acids to provide the corresponding 1-O-acyl α-d-glucoses. 17,18 The reaction proceeds smoothly with benzoic or acetic acid but is limited to sucrose as substrate. If such phosphorylase could serve as an acyl transferase, there is a possibility that less specific hydrolases could perform the same task. Glycosylhydrolases in the presence of a large excess of alcohol are known to perform transglycosylation. 19 However carboxylic acids are poor nucleophiles and the competition with water would be in favor of the latter (Scheme 1). In addition the resulting glycosyl esters are known to be readily hydrolyzed by native glycosylhydrolases. 20 Interestingly, Withers and co-workers have designed an elegant strategy to favour the attack of various nucleophiles by the mutation of the acid/base residue. 21 Such mutation drastically reduces the kinetic of the attack of water allowing competition with other nucleophiles like mercaptan, 22 fluoride 23 and even carboxylic acid. 24 But for the last two examples, the resulting conjugates were only detected and never isolated. In fact, the thioligases have never been diverted for the synthesis of acyl glycoside derivatives. We thus propose herein a novel strategy for the synthesis of glycosyl esters by the use of a thioligase as innovative and original acyl transfer catalyst. Scheme 1 Competition between hydrolysis and acylation of the glycosyl-enzyme intermediate upon action of a glycosylhydrolase.

The drawback associated with the monitoring of glycosyl ester formation is their non-stability due to acyl migration. 1,2-trans Acyl arabinofuranoses are known to be more stable and less prone to such rearrangement. 25 Interestingly Davies and collaborators have reported on a versatile, thermostable hydrolase that efficiently degrades polymers of l-arabinose, the arabinofuranosidase 51 from Rhuminiclostridium thermocellum (CtAraf51). 26 Latter on Ferrières and co-workers used this enzyme as efficient biocatalyst in both self-condensation and transglycosylation of L-arabinofuranose donor. 27,28 Furthermore, CtAraf51 was successfully transformed into the corresponding thioligase by the mutation of the glutamic acid 173 into the alanine. The resulting mutant CtAraf51 E173A was able to recognize a thioimidoyl arabinofuranoside 1 as a donor and transfer the furanose to different thiophenol via a remote activation mechanism. 29 From there, we decided to perform the 1-O-acylation of arabinose from 1 with CtAraf51 E173A as catalyst to provide a wide range of 1-O-acyl arabinofuranoses (Scheme 2).

Scheme 2 Strategy of acylation of L-arabinose by the thioligase CtAraf51 E173A

We tested first our strategy starting from 1 30 and 4-methoxybenzoic acid as the acylating agent (Figure 1). The mutant Araf51 E173A was expressed according to known procedure. 29 The reaction was performed in 50 mM phosphate buffer pH 7 at 25 °C in the presence of an excess of 4-methoxybenzoic acid. In the absence of the catalyst, no reaction occurred. Gratifyingly, when Araf51 E173A was added, TLC analysis showed the formation of a new spot identified after purification as the 1-Omethoxybenzoyl α-L-arabinofuranose 2. The isolated yield reached 52 %. The structure of 2 was unambiguously confirmed by NMR spectroscopy. 1 H and 13 C NMR showed in particular signals at 6.05 ppm and 102.3 ppm respectively, characteristic of an acylated anomeric position. The associated coupling constant reached 1.1 Hz, typical of a 1,2-trans configuration. Such complete diastereoselectivity of the reaction was expected as the selected furanosidase works with retention of configuration. The resulting 1-O-methoxybenzoyl α-L-arabinofuranose 2 was surprisingly very stable and did not undergo acyl migration or hydrolysis in the course of the reaction unlike others acyl glycosides 31 (monitored by 1 H NMR. See ESI). Interestingly, using the native Araf51 as catalyst, sole the arabinose was formed. These preliminary results validated our model and confirm the possibility to implement a new reactivity, the acylation, to a glycosidase after mutation of the acid/base residue. It also confirmed the crucial role of the mutation to prevent the competition of the carboxylic acid acceptor with water. In order to optimize this reaction, different conditions (pH, time, donor/acceptor ratio) were then screened (Table 1). On one hand, at pH below 4.5 (entry 1), the thioimidoyl arabinofuranose 1 was quickly chemically hydrolysed due to proton activation of the benzimidazole. On the other hand, in alkaline conditions, typically the one used for thioligation, no product of acylation could be detected and only the starting thioimidoyl arabinofuranoside 1 and arabinose were found (entry 2). Interestingly, improved isolated yield compared to neutral pH was obtained at acidic pH (entry 3 vs. entry 4). Complete disappearance of 1 was achieved after 24h and the isolated yield reached 78 % (entry 5). It nevertheless dropped to 60 % when using an equimolar equivalent of 4-methoxybenzoic acid (entry 6). In these conditions the kinetic slowed down and a fair amount of hydrolysis occurred. In addition, using 1 H NMR, we confirmed that in neutral and alkaline media, the resulting product 2 was more sensitive to chemical hydrolysis (See ESI). It correlated the larger amount of arabinose found when the reaction occurred at pH above 6 (entry 4 vs. entries 2 and 3). In term of mechanism, the reaction should proceed according to the canonical mechanism described by Withers. 21 The first half-reaction followed a two-steps process i) attack of the nucleophilic residue glutamate 292 and ii) subsequent departure of thiobenzimidazole via a remote activation mechanism 29 (Scheme 3). The second-half of the reaction, that is to say the attack by the carboxylic acid, can proceed according to two different scenarios with either the carboxylic acid or the carboxylate as the nucleophile. Sugimoto et al. demonstrated that the acylation of glucose catalysed by sucrose phosphorylase proceeded best at pH below the pKa of acetic or benzoic acid meaning that the protonated form of the acid probably act as the nucleophile. 17 In our case, the difference in efficiency between pH 6 and pH 7 was not striking and it is probably the carboxylate form that act as the nucleophile. Such form does not need to be activated unlike water. As the mutation of the glutamate residue 173 by an alanine one reduced drastically the activation of water, the carboxylate can compete efficiently to give the corresponding acyl 1,2-trans arabinofuranose as the only product of the reaction (Scheme 3). According to our data, the pH nevertheless influenced greatly the kinetic of the reaction. The Araf51 is known to be stable and active in a large range of pH. 26 It is therefore not the potential degradation of the enzyme that can explain such difference. We suspect that the difference in reactivity is linked to the first half-reaction rather than the second-half one. Acidic media could indeed contribute to accelerate the departure of the thiobenzimidazole aglycon thus increasing the turn-over of the reaction.

Finally, to demonstrate the versatility of the reaction, a large panel of carboxylic acid was screened as acceptor of the acylation reaction (Table 2). ‡ It included aryl and alkyl carboxylic acid as well as amino acids or drugs like ibuprofen. The yields for the furanosylation of aryl carboxylic acid ranged from 18 to 83 % (entries 1 to 5). The lowest yields were found with electron-withdrawing groups on the aromatic ring like bromide, trifluoromethyl or nitro (entries 2 to 4). A follow-up of the reaction between 1 and 4-nitrobenzoic acid was performed and showed the concomitant formation of the corresponding acyl arabinofuranose 20 and arabinose. We then compared the stability of the resulting conjugates at pH 6 in the presence or the absence of Araf51 E173 (See ESI). A strong increase of the kinetic of hydrolysis was found when the enzyme was added. The presence of the electronwithdrawing group must reduce the nucleophilicity of the carboxylic acid and increase the nucleofuge property of the acyl group of the product. Both effects contributed then to increase the competition with water. The same trend was observed using salicylic acid as substrate (See ESI). The strong hydrolysis observed was probably linked to the activation of the ester function by the phenol in ortho position. Interestingly, the alkyl carboxylic acids 2-phenylacetic acid 10 and hexanoic acid 12 were readily converted to the corresponding acyl arabinofuranoses 22 and 23 with yields up to 66 % (entries 6 and 7). For the bulkier pivaloic acid 14, the conversion remained low probably because of the steric hindrance (entry 8). Gratifyingly ibuprofen 11 was transformed into the acyl conjugate 25 with an excellent 83 % yield (entry 9) opening the possibility to transfer the methodology to the synthesis of acyl glucuronides that result from phase II metabolism of drugs. It was worth reporting that no stereoselection occurred when using racemic ibuprofen and a 1:1 ratio of diastereoisomers was obtained (See ESI). Also with 6-hydroxyhexanoic acid 13 no product of O-glycosylation was formed and the corresponding acyl arabinofuranose 26 was obtained in 53 % yield (entry 10). The same chemoselectivity was found using hydroxybenzoic acid 7 (entry 5). Even the 4 -(mercaptomethyl)benzoic acid 8 reacted with 1 without formation of the thiobenzyl arabinoside isomer (entry 11). Thus it showed in our conditions that carboxylic acid was a better nucleophile than the phenol, the alcohol or the aliphatic thiol. Finally, no reaction was detected in the presence of 4-(aminomethyl)benzoic acid 9 (entry 12) or phenylalanine (entry 13). The presence of the ammonium group seemed to be detrimental to the approach of the acceptor in the +1 sub-site. Indeed, when the amine of phenylalanine was protected as a carbamate, the resulting acyl conjugate 30 was obtained with 38 % yield (entry 14). Such result paves the way to the extension of the scope of reaction in the domain of protein glycosylation. To summarize, we report here a novel catalytic pathway for the synthesis of glycosyl esters using a mutated glycosylhydrolase. This methodology has the advantage to use stable reactant and readily available enzyme. It is performed in mild reaction conditions and with a large range of carboxylic acids with good yields and excellent selectivity. This study in particular shows the plasticity of glycosylhydrolases and in particular the arabinofuranosidase towards numerous catalytic activities. In addition to transglycosylation and thioligation, this enzyme is also able to perform acylation of arabinose. Interestingly the reaction could be extended to mimics of L-arabinose, like Dgalactofuranose with potential biological properties. Also the extension of the methodology to thioligases with greater interest like glucuronidase would be a definite advantage for the synthesis of the corresponding acyl glucuronides.

Figure 1

 1 Figure 1 Synthesis of 4-methoxybenzoyl α-l-arabinofuranose 2 catalysed by CtAraf51 E173A

Scheme 3

 3 Scheme 3 Hypothesized mechanism of 1-O-acylation of thioimidoyl arabinofuranoside 1 catalysed by Araf51 E173A.

Table 1

 1 Screening of pH and acceptor amount for the acylation of thioimidoyl arabinofuranoside 1 by 4-methoxybenzoic acid. Conditions: CtAraf51 E173A, 50 mM phosphate buffer, R.T.

	Entry Equivalent	pH Time	Yield of	Ratio
		of acceptor		(h)	2 (%)	Ara/2 a
	1	4	4.5 16	-	1/0
	2	4	8	16	-	1/0 b
	3	4	7	16	52	0.3/1
	4	4	6	16	67	0.1/1
	5	4	6	24	78	0.15/1
	6	1	6	24	60	0.4/1

a determined by NMR. b in this case large amount of 1 remained.

Table 2

 2 Extension to various carboxylic acid acceptors ‡

	Entry Acceptor RCO2H Isolated Yield (%) 1-O-Acyl Araf
	1	3	57	17
	2	4	47	18
	3	5	57	19
	4	6	18 a	20
	5	7	78	21
	6	10	66	22
	7	12	77	23
	8	14	25	24
	9	11	83 a	25
	10	13	53	26
	11	8	100 b	27
	12	9	-	28
	13	15	-	29
	14	16	38	30

a 1:1 ratio of diatereoisomer with (±) ibuprofen b Conversion yield determined by NMR

Acknowledgements

We are grateful to CNRS and the "Ministère de l'enseignement supérieur et de la recherche" for financial supports. We thank the "Centre Regional des Mesures Physiques de l'Ouest (Université de Rennes 1)" for the registration of the mass spectra. This work benefited from the support of the project C-linkAse ANR-15-CE07-0009-01 of the French National Research Agency (ANR).

Conflicts of interest

There are no conflicts to declare Notes and references ‡ General procedure for the enzymatic synthesis of glycosyl esters: A mixture of 4 equivalent of carboxylic acid acceptor in phosphate buffer (20 mL, 50 mM, pH = 6) was prepared and pH was adjusted to 6 with NaOH 2N. The mutant enzyme Araf51 E173A (≈ 5 mg, 0.00025 eq) and 2'-benzimidazolyl-1-thio-α-L-arabinofuranoside 1 (≈ 100 mg, 0.35 mmol, 1 eq) were added. The mixture was stirred at RT for 16 hours. After freeze-drying, the mixture was purified by column chromatography on silica gel to yield the corresponding 1-O-acyl arabinofuranose.