Paul Roux
email: paul.roux@uvsq.fr

Nathan Faivre
email: nathan.faivre@univ-grenoble-alpes.fr

Anne-Sophie Cannavo
email: ascannavo@ch-versailles.fr

Eric Brunet-Gouet

Christine Passerieux
email: cpasserieux@ch-versailles.fr

Exploring Clinical Correlates of Metacognition in Bipolar Disorders Using Moderation Analyses: The Role of Antipsychotics

Keywords: bipolar disorders, metacognition, cognitive complaints, cognition, antipsychotic, impulsivity

The determinants of metacognition are still poorly understood in bipolar disorders (BD). We aimed to examine the clinical determinants of metacognition, defined as the agreement between objective and subjective cognition in individuals with BD. The participants consisted of 281 patients with BD who underwent an extensive neuropsychological battery and clinical evaluation. To assess subjective cognition, participants provided a general rating of their estimated cognitive difficulties. Clinical characteristics of BD were also recorded, along with medication. We studied the potential moderation of the association between cognitive complaints and global objective cognitive performance by several clinical variables with ordinal logistic regressions. Depression and impulsivity were associated with greater cognitive complaints. The only variable that moderated the relationship between objective and subjective cognition in the global model was the prescription of antipsychotics. Patients taking antipsychotics had a poorer association between cognitive complaints and objective neuropsychological performance. This result suggests a role for dopamine in the modulation of metacognitive performance, and calls for the systematic control of antipsychotic medication in future studies documenting metacognitive deficits in severe and persistent mental disorders. Depression and impulsivity should be investigated as potential therapeutic targets for individuals with BD and cognitive complaints, before proposing an extensive neuropsychological evaluation.

Introduction

Metacognition refers to a spectrum of mental activities of which the object is one's own thoughts. In individuals suffering from severe and persistent psychiatric disorders, metacognition is a multi-faceted construct encompassing the recognition that one is ill, the awareness of one's own cognitive style and beliefs, and the awareness of one's own cognitive performance [START_REF] David | Failures of Metacognition and Lack of Insight in Neuropsychiatric Disorders[END_REF]. Lack of insight into the illness is frequent in BD (Bipolar Disorders) [START_REF] Varga | Symptoms and Neurocognition in Bipolar I Patients[END_REF], particularly during the manic phase [START_REF] Camelo | Clinical and Cognitive Correlates of Insight in Bipolar Disorder[END_REF]. Several studies have identified dysfunctional metacognitive beliefs in BD [START_REF] Favaretto | Metacognitions and Repetitive Negative Thinking in Bipolar Disorder and Healthy Controls: A Comparative Study[END_REF], which are associated with a higher level of depression, earlier onset of affective illness [START_REF] Østefjells | An Exploration of Metacognitive Beliefs and Thought Control Strategies in Bipolar Disorder[END_REF], and worse cognitive impairments [START_REF] Van Camp | Metacognitive Functioning in Bipolar Disorder versus Controls and Its Correlations with Neurocognitive Functioning in a Cross-Sectional Design[END_REF]. Cognitive insight in BD is characterized by higher self-reflectiveness, i.e., the capacity to reflect on one's own experiences, which correlates with more severe depression [START_REF] Van Camp | The Longitudinal Course of Cognitive Insight and Mood in Bipolar Disorder[END_REF].

One way to evaluate the awareness of one's cognitive performance is to measure the agreement between objective cognition measured using established neuropsychological tests and subjective cognition assessed using self-reported scales. In this framework, good metacognitive performance implies a close relationship between objective and subjective cognition, whereas metacognitive deficits are reflected by their decorrelation. Several studies have explored the association between objective and subjective cognition but reported inconsistent results. Some reported non-significant or weak (r < 0.3) associations [START_REF] Demant | Assessment of Subjective and Objective Cognitive Function in Bipolar Disorder: Correlations, Predictors and the Relation to Psychosocial Function[END_REF][START_REF] Van Der Werf-Eldering | Is the Lack of Association between Cognitive Complaints and Objective Cognitive Functioning in Patients with Bipolar Disorder Moderated by Depressive Symptoms?[END_REF], thus suggesting poor metacognitive performance in BD. In contrast, other studies reported a more pronounced association [START_REF] Jensen | Optimising Screening for Cognitive Dysfunction in Bipolar Disorder: Validation and Evaluation of Objective and Subjective Tools[END_REF], which appeared to depend on how objective cognition was measured. A previous study indeed reported that the global self-concept of general cognitive ability did not correlate with the general cognitive composite score in BD, whereas self-reported performance just after completing a specific cognitive task correlated with the objective performance [START_REF] Torres | Metacognitive Knowledge and Experience in Recently Diagnosed Patients with Bipolar Disorder[END_REF].

Several attempts have been made to identify the determinants of metacognition in BD. Some proposed computing a meta-sensitivity index as the difference between the individual ranks in objective and subjective cognition, and reported several factors that covaried with this index: mood symptoms, number of hospitalizations, type of BD, socio-occupational difficulties, perceived stress, quality of life, and verbal IQ [START_REF] Miskowiak | Predictors of the Discrepancy between Objective and Subjective Cognition in Bipolar Disorder: A Novel Methodology[END_REF]. Others subtracted the z-scores for objective cognition from the z-scores of subjective cognition. One study using this method found no clinical correlates of metacognition in BD [START_REF] Torres | Metacognitive Knowledge and Experience in Recently Diagnosed Patients with Bipolar Disorder[END_REF], whereas another reported that metacognition was associated with objective cognitive performance [START_REF] Van Camp | Metacognitive Functioning in Bipolar Disorder versus Controls and Its Correlations with Neurocognitive Functioning in a Cross-Sectional Design[END_REF]. However, the computation of a score reflecting a difference in ranks or z-scores between objective and subjective cognition makes it difficult to test whether a significant association between this score and a clinical correlate could be explained by a simpler and more direct relationship between this correlate and objective or subjective cognition.

An alternative strategy is to use moderation analysis, which avoids the limitation of using a difference between scores. One study reported that depressive symptoms did not moderate the association between subjective and objective cognition in euthymic or mildly depressed individuals with BD, thus suggesting a lack of association between depressive symptoms and metacognition in BD [START_REF] Van Der Werf-Eldering | Is the Lack of Association between Cognitive Complaints and Objective Cognitive Functioning in Patients with Bipolar Disorder Moderated by Depressive Symptoms?[END_REF]. On the contrary, a later study reported that the association between subjective and objective cognition was also moderated by depressive symptoms only in individuals with depression and by manic symptoms only in individuals with hypomania/mania [START_REF] Lin | The Associations between Subjective and Objective Cognitive Functioning across Manic or Hypomanic, Depressed, and Euthymic States in Chinese Bipolar Patients[END_REF]. However, these two studies had a relatively limited sample size (n < 150), and investigated only mood symptoms as potential correlates of metacognition in BD, ignoring the potential role of important variables such as medication [START_REF] Clos | Dopamine Is a Double-Edged Sword: Dopaminergic Modulation Enhances Memory Retrieval Performance but Impairs Metacognition[END_REF], psychosocial functioning [START_REF] Miskowiak | Predictors of the Discrepancy between Objective and Subjective Cognition in Bipolar Disorder: A Novel Methodology[END_REF] and childhood trauma [START_REF] Aydin | The Developmental Origins of Metacognitive Deficits in Schizophrenia[END_REF].

Here, we aimed to identify potential clinical correlates of metacognition in a large sample of individuals with BD, beyond mood symptoms, using multiple moderation analysis between cognitive complaints and objective cognition.

Materials and Methods

Study Design and Characteristics of the Recruiting Network

This monocenter, transversal study included patients recruited into the FACE-BD (FondaMental Advanced Centers of Expertise for Bipolar Disorders) cohort from the BD Expert Center of Versailles. The study was pre-registered (NCT04034147). The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human patients were approved by the local ethics committee (Comité de Protection des Personnes Ile de France IX) on 18 January 2010 under French laws for non-interventional studies (observational studies without any risk, constraint, or supplementary or unusual procedure concerning diagnosis, treatment, or monitoring). The board required that all patients be given an informational letter but waived the requirement for written informed consent. However, verbal consent was witnessed and formally recorded. Anonymized data are stored in a national database that was approved by the French body overseeing the safety of computerized databases (Commission Nationale de l'Informatique et des Liberte, DR-2011-069). No incentive was given to patients for their participation.

Participants

General practitioners or psychiatrists referred the patients who were assessed in the Centers of Expertise. The goal of this referral was to improve global care by delivering personalized care plans, after a systematic set of comprehensive assessment tools including a neuropsychological battery. Patients were interviewed by senior psychiatrists, psychologists, or neuropsychologists specialized in bipolar disorders, who were all members of the specialized multidisciplinary teams of the Expert Centres. Senior psychiatrists diagnosed bipolar disorders with the SCID-IV (Structured clinical interview for DSM-IV-TR) [START_REF] First | User's Guide for the Structured Clinical Interview for DSM-IV Axis I Disorders SCID-I: Clinician Version[END_REF]. Outpatients with type 1, type 2, or NOS (Not otherwise specified) BD, who were between 18 and 65 years of age, were eligible for this analysis. Patients who met the following criteria were excluded: history of neurological disorder, dyslexia, dysorthographia, dyscalculia, dysphasia, dyspraxia, substance-related disorders in the previous month (except tobacco use), or electroconvulsive therapy in the past year. No criteria related to the current mood state at inclusion were used to preserve the variability of levels of objective and subjective cognition. However, individuals whose symptom intensity was judged to be incompatible with the one-and-a-half-day evaluation were excluded (for instance, high suicidal risk, agitation, severe distractibility, disability to think or concentrate, or severe indecisiveness). Neuropsychologists screened exclusion criteria related to neurodevelopmental disorders; senior psychiatrists screened exclusion criteria related to neurological and substance-use disorders.

Assessment Tools

The sociodemographic variables collected at inclusion were sex, age, and education level.

Clinical Assessments

The following clinical variables were recorded using the SCID: age at onset of BD, number and type of previous mood episodes, the subtypes of BD, and history of psychotic symptoms. Predominant polarity was determined following previous recommendations [START_REF] Mazzarini | Predominant Polarity and Temperament in Bipolar and Unipolar Affective Disorders[END_REF].

The CGI-S (Clinical Global Impression-Severity) scale assessed illness severity [START_REF] Guy | Clinical global impression scale[END_REF]. We used a yes/no questionnaire for recording patient treatment at the time of evaluation: lithium carbonate, anticonvulsants, antipsychotics, antidepressants, or anxiolytics. Mania was measured using the YMRS (Young Mania Rating Scale) total score [START_REF] Young | A Rating Scale for Mania: Reliability, Validity and Sensitivity[END_REF] and depression using the MADRS (Montgomery Asberg Depression Rating Scale) total score [START_REF] Montgomery | A New Depression Scale Designed to Be Sensitive to Change[END_REF]. The state of anxiety was measured using the total score of the state subscale of the STAI-Y-A (State-Trait Anxiety Inventory, form Y-A) [START_REF] Spielberger | State-Trait Anxiety Inventory and State-Trait Anger Expression Inventory[END_REF]. Impulsivity was assessed using the total score of the BIS-10 (Barratt Impulsiveness Scale version 10) [START_REF] Patton | Factor Structure of the Barratt Impulsiveness Scale[END_REF]. Childhood traumatic events were recorded using the total score of the CTQ (Childhood Trauma Questionnaire) [START_REF] Bernstein | Childhood Trauma Questionnaire: A Retrospective Self-Report Manual [Manual[END_REF]. Domain-based psychosocial functioning was measured using the FAST (Functioning Assessment Short Test) [START_REF] Rosa | Validity and Reliability of the Functioning Assessment Short Test (FAST) in Bipolar Disorder[END_REF]. In this study, the total FAST score was used (higher score meaning poorer functioning). Adherence to medication was measured using the total score of the MARS (Medication Adherence Rating Scale) [START_REF] Thompson | Reliability and Validity of a New Medication Adherence Rating Scale (MARS) for the Psychoses[END_REF]. According to previous reports, all these tools have good psychometric properties (see Supplementary Information S1) for a description of the internal consistency and test-retest reliability for each tool).

Objective and Subjective Cognition

•

Objective cognition: the battery of cognitive tests

The standardized test battery included 11 tests, amongst which five were subtests from the WAIS (Wechsler Adult Intelligence Scale) version III [START_REF] Wechsler | WAIS-III, Wechsler Adult Intelligence Scale: Administration and Scoring Manual[END_REF] or version IV [START_REF] Wechsler | WAIS-IV: Wechsler Adult Intelligence Scale[END_REF]. Participants were included between June 2009 and December 2018. The French version of the WAIS-IV became available during this period, and we decided to start to use this version in May 2012.

The rationale behind this decision was to use the most recent normative data available, as they gave a more accurate picture of the distance in cognitive performances between the recruited participants and the norm. The battery evaluated six domains:

-Processing speed: Digit symbol coding (WAIS-III) or coding (WAIS-IV), WAIS symbol search, and TMT (Trail-Making Test) [START_REF] Reitan | Validity of the Trail Making Test as an Indicator of Organic Brain Damage[END_REF] [START_REF] Golden | A Manual for the Clinical and Experimental Use of the Stroop Color and Word Test[END_REF], semantic and phonemic verbal fluency [START_REF] Lezak | Neuropsychological Assessment[END_REF] and TMT part B -Verbal and perceptual reasoning: WAIS vocabulary and matrices Raw scores were transformed to demographically corrected standardized z-scores based on normative data [START_REF] Conners | Continuous Performance Test II[END_REF][START_REF] Golden | A Manual for the Clinical and Experimental Use of the Stroop Color and Word Test[END_REF][START_REF] Godefroy | The GREFEX battery: Normative data[END_REF][START_REF] Poitrenaud | French Adaptation of the California Verbal Learning Test[END_REF]. Higher scores reflected better performance. We computed a mean score for each cognitive domain. Then we computed a global score for objective cognition by averaging the cognitive domain scores. WAIS-III and WAIS-IV standardized scores were analysed together, as this method has been used in several cohort studies [START_REF] Ehrminger | Longitudinal Relationships between Cognition and Functioning over 2 Years in Euthymic Patients with Bipolar Disorder: A Cross-Lagged Panel Model Approach with the FACE-BD Cohort[END_REF][START_REF] Roux | Minimum Clinically Important Differences for the Functioning Assessment Short Test and a Battery of Neuropsychological Tests in Bipolar Disorders: Results from the FACE-BD Cohort[END_REF][START_REF] Roux | Psychiatric Disability as Mediator of the Neurocognition-Functioning Link in Schizophrenia Spectrum Disorders: SEM Analysis Using the Evaluation of Cognitive Processes Involved in Disability in Schizophrenia (ECPDS) Scale[END_REF][START_REF] Roux | Screening for Cognitive Deficits with the Evaluation of Cognitive Processes Involved in Disability in Schizophrenia Scale[END_REF][START_REF] Brunet-Gouet | Outcome Prediction with a Social Cognitive Battery: A Multicenter Longitudinal Study[END_REF].

•

Subjective cognition: cognitive complaints Cognitive complaints were assessed with item 10 of the QIDS-SR16 (Quick Inventory of Depressive Symptomatology-Self-Report): "During the past seven days, there has been no change in my usual capacity to concentrate or make decisions" (scored 0); "I occasionally feel indecisive or find that my attention wanders" (scored 1); "Most of the time, I struggle to focus my attention or to make decisions" (scored 2); "I cannot concentrate well enough to read or cannot make even minor decisions" (scored 3). Previous studies have reported evidence of validity and reliability of this single-item assessment. Its test-retest reliability over six months is good (Cronbach alpha of 0.74) [START_REF] Gili | Psychometric Properties of Spanish Version of QIDS-SR16 in Depressive Patients[END_REF]. Concurrent validity for this item has been investigated against a similar item rated by a clinician within the clinical-rated form of QIDS, and it was found to be satisfactory [START_REF] Bernstein | Self-Report Versions of the Quick Inventory of Depressive Symptomatology in a Public Sector Sample[END_REF]. Finally, we checked the concurrent validity of this item within our sample. We ran a correlation analysis between item 10 of QIDS and the cognitive functioning factor of FAST, which assesses the subjective level of cognitive functioning perceived by the clinician. We found a moderate-to-strong correlation (Pearson correlation coefficient of 0.48, t = 9.148, df = 273, p-value < 0.001), thus suggesting a good concurrent validity of the single-item assessment of cognitive complaints used in this study.

Statistical Analyses

First, missing data were estimated using multivariate imputations by chained equations (50 imputations, mice package of R). The fraction of missing information (fmi) and the proportion of total variance due to missingness (λ) are reported in the results.

Metacognition was quantified as the strength of the association between cognitive complaints and the average cognitive performance on the neuropsychological battery: a negative and significant association between cognitive complaints and objective cognition was interpreted as good metacognition, whereas a lack of an association or a positive association was interpreted as impaired metacognition. This association was operationalized through an ordinal logistic regression with cognitive complaints as the dependent variable and objective cognition as the independent variable. We first ran successive moderation analyses with cognitive complaints as the dependent variable and two independent variables (one clinical moderator belonging to the variables listed above and objective cognition) by declaring their main effects and their interaction in the model. Assuming that metacognition reflects the strength of the association between objective cognition and subjective complaints, a variable was interpreted as a potential moderator of metacognition if its interaction with objective cognition was significant.

We then ran a multiple ordinal logistic regression with several independent variables to check whether the potential clinical moderator identified in the simple logistic regressions remained significant while simultaneously accounting for all the effects. Beyond the main effect of objective cognition, the independent variables were included in this multiple model if:

the level of significance of their interaction with objective cognition was p < 0.25. This threshold is usual for selecting variables for multiple regression [START_REF] Mickey | The Impact of Confounder Selection Criteria on Effect Estimation[END_REF][START_REF] Hosmer | Applied Logistic Regression[END_REF]. In this case, both the interaction term and the main effect of the clinical covariate was included in the model the level of significance of their interaction with objective cognition was p ≥ 0.25 and the level of significance of their main effect was p < 0.25. In this case, only the main effect of the clinical covariate was included in the model

Results

Characteristics of Participants

We included 281 participants. Socio-demographic, clinical, and functional characteristics of the sample are presented in Table 1. The participants consisted mostly of women with type 2 BD and indeterminate polarity of mood episodes. Most participants were not in an active characterized mood episode during the evaluation: only 13.9% were currently undergoing a major depressive episode. None were manic and only 1.4% were hypomanic. The average severity of illness was between moderate and marked (4.5 ± 0.9).

The MADRS correlated above |r| > 0.5 with the end of the last characterized episode <3 months, the presence of a current major depressive episode, and the state subscale of STAI-YA. These last three variables were thus discarded from the following analyses to avoid multicollinearity issues in the multiple regression. Due to the small sample size, the NOS diagnosis (n = 30) was combined with that of type 2 BD (its nearest neighbor, n = 146) and manic polarity (n = 22) with the indeterminate class (its nearest neighbor, n = 86) of predominant mood polarity.

The neuropsychological results are presented in Table 2. WAIS-III was proposed to 36 participants, and WAIS-IV to 245 participants. The worst performance in objective cognition was found for attention (-0.4 ± 0.7) and the best for reasoning (0.6 ± 0.7).

For cognitive complaints, the distribution of answers to item 10 of the QIDS was the following (see Supplementary Figure S2): 31.8% for "No change in usual capacity to concentrate and decide"; 34.3% for "Occasionally feels indecisive or notes that attention often wanders"; 20.9% for "Most of the time struggles to focus attention or make decisions"; and 13.0% for "Cannot concentrate well enough to read or cannot make even minor decisions". The mean cognitive complaints score was 1.2 (sd:1, n = 277), suggesting occasional subjective cognitive difficulties on average in this sample.

Imputation diagnostic is reported in Supplementary Information S3. Data were considered missing at random, as no systematic relationship between domains of missingness and participants' socio-demographic characteristic were found.

Moderation Analyses

The bivariable ordinal logistic regression of cognitive complaints on objective cognition was not significant (OR = 0.70 (0.44-1.1), t(257.5) = -1.6, p = 0.113, λ = 0.051, fmi = 0.058). Overall, the study sample showed a non-significant association between objective and subjective cognition.

Trivariable Ordinal Logistic Regressions

The results of the ordinal logistic regressions of cognitive complaints with objective cognition and several successive clinical moderators as independent variables are presented in Table 3 for the interaction between objective cognition and clinical moderators, and Supplementary Tables S4 andS5 for the main effect of clinical moderators and objective cognition respectively. Table 3. Results for the trivariable ordinal logistic regressions with cognitive complaints as the dependent variable and objective cognition, several successive clinical moderators, and the interaction between objective cognition and the clinical moderators as independent variables. This table reports only the interaction effect between objective cognition and the clinical moderator (the main effect of objective cognition and clinical moderator are reported in Supplementary Tables S4 andS5).

Interaction between the Mean Cognitive Performance and a Clinical Moderator

OR (95% CI) Significant interactions with objective cognitive performance were found for predominant mood polarity and antipsychotic medication (see Table 3). The distribution of objective cognition according to the level of cognitive complaints and polarity in the observed dataset are reported in Supplementary Figure S6. The association between objective cognition and cognitive complaints was significant for depressive polarity (OR = 0.33 (0.13-0.80), t(66.6) = -2.5, p = 0.015, λ = 0.224, fmi = 0.246), but not indeterminate/manic polarity (OR = 1.02 (0.56-1.87), t(132.6) = 0.1, p = 0.947, λ = 0.179, fmi = 0.192). The distribution of objective cognition according to the level of cognitive complaints and the prescription of antipsychotics in the observed dataset are presented in Figure 1. The association between objective cognition and cognitive complaints was significant for patients not taking antipsychotics (OR = 0.48 (0.28-0.78), t(215.8) = -2.9, p = 0.004, λ = 0.048, fmi = 0.057), but not for those taking antipsychotics (OR = 2.82 (0.73-10.8), t(36.5) = 1.6, p = 0.127, λ = 0.086, fmi = 0.132).

The clinical moderators that were significantly associated with more cognitive complaints in the absence of a significant interaction with objective cognition were a type 2/NOS BD (OR = 1.75 (1.11-2.76), t(269.6) = 2.4, p = 0.017), a lack of history of psychosis (OR = 1.80 (1.01-3.22), t(177.1) = -2, p = 0.047), greater illness severity measured based on the CGI-S (OR = 1.84 (1.38-2.44), t(267.9) = 4.2, p < 0.001), more severe depressive symptoms measured based on the MADRS (OR = 1.14 (1.1-1.17), t(265.6) = 8.4, p < 0.001), anxiolytic (OR = 2.12 (1.22-3.67), t(270.6) = 2.7, p = 0.008), a lifetime substance use disorder (OR = 1.7 (1.02-2.83), t(259.4) = 2, p = 0.043), greater impulsivity measured based on the BIS (OR = 1.08 (1.06-1.1), t(268.4) = 6.7, p < 0.001), an history of trauma assessed based on the CTQ (OR = 1.04 (1.02-1.05), t(261.2) = 4.7, p < 0.001), poorer functioning measured based on the FAST (OR = 1.08 (1.06-1.1), t(269.5) = 8.1, p < 0.001) and poorer medication adherence measured based on the MARS (OR = 0.87 (0.78-0.96), t(234.2) = -2.7, p = 0.008, see Supplementary Table S4).

Multiple Ordinal Logistic Regression

The multiple ordinal logistic regression included cognitive complaints as the dependent variable and the following independent variables: predominant mood polarity, CGI-S, lithium carbonate, antipsychotic, anxiolytic and FAST (their main effect and their interaction with objective cognition), and the main effect of objective cognition, educational level, type of BD, history of psychosis, MADRS, any lifetime substance use disorder, BIS, CTQ, MARS and type of WAIS (see Table 4). The only interaction with objective cognition that remained significant in the multiple moderation analysis was found for antipsychotics (OR = 6 (1.13-31.7), t(216.9) = 2.1, p = 0.035). The variables which were significantly associated with increased cognitive complaints were MADRS (OR = 1.1 (1.06-1.14), t(240.5) = 5.2, p < 0.001) and BIS (OR = 1.07 (1.04-1.1), t(243.5) = 4.4, p < 0.001).

Discussion

We aimed to identify clinical correlates of metacognition in BD using moderation analysis between cognitive complaints and objective cognition.

First, this study confirmed the weak correlation between objective performance on a battery of neuropsychological tests and subjective perception of cognitive functioning reported in previous studies [START_REF] Demant | Assessment of Subjective and Objective Cognitive Function in Bipolar Disorder: Correlations, Predictors and the Relation to Psychosocial Function[END_REF]. Among all investigated clinical variables, only the predominant mood polarity and the prescription of antipsychotics influenced the strength of the association between cognitive complaints and objective cognition. In the absence of covariates, the association between objective cognition and cognitive complaints was more robust for the depressive polarity than for the indeterminate/manic polarity. A negative impact of manic episodes on objective cognition has been reported previously [START_REF] Bourne | Neuropsychological Testing of Cognitive Impairment in Euthymic Bipolar Disorder: An Individual Patient Data Meta-Analysis[END_REF], which may progressively lead to an impairment of metacognition. An alternative interpretation is that preserved metacognition facilitates the emergence of depression, whereas impaired metacognition may lead to disinhibition and mania. However, the moderating effect of polarity did not resist the introduction of covariates in the model.

The main factor influencing the strength of the association between cognitive complaints and objective cognition was the prescription of antipsychotics, for which the moderating effect remained significant in the multiple analysis. This result is compatible with the hypothesis that confidence in cognitive performance is modulated by dopamine [START_REF] Lou | Dopaminergic Stimulation Enhances Confidence and Accuracy in Seeing Rapidly Presented Words[END_REF]. More specifically, our results suggest that dopamine antagonists, such as antipsychotics, may decrease metacognitive accuracy. A previous study reported that dopamine administration increased metacognition in healthy participants, in parallel with increased amplitudes of MEG oscillations in the medial prefrontal cortex [START_REF] Joensson | Making Sense: Dopamine Activates Conscious Self-Monitoring through Medial Prefrontal Cortex[END_REF]. Another study recently demonstrated that the administration of haloperidol, a dopamine antagonist, impaired metamemory in healthy individuals in parallel with aberrant fMRI activity in frontostriatal circuits [START_REF] Clos | Dopamine Is a Double-Edged Sword: Dopaminergic Modulation Enhances Memory Retrieval Performance but Impairs Metacognition[END_REF]. Of note, this effect may depend on the antipsychotic investigated, as it was not replicated with amisulpride [START_REF] Hauser | Noradrenaline Blockade Specifically Enhances Metacognitive Performance[END_REF]. This is the first study to report an association between antipsychotic medication and metacognition in BD. However, it is not possible to conclude a causal link of antipsychotics on metacognitive impairments, as we did not investigate the dose effect of antipsychotics on metacognition in this study. Longitudinal studies are needed to clarify the effect of antipsychotics on metacognition in BD and should account for the specific psychopharmacological properties of a particular antipsychotic, along with the daily dosage and serum level, duration of exposure, and therapeutic response. Despite these limitations, our result strongly encourages controlling for antipsychotic medication in studies investigating metacognition in severe and persistent psychiatric disorders.

Our results do not support a moderating effect of mood symptoms on the relationship between objective and subjective cognition in BD. This result is consistent with those of previous reports showing a lack of association between mood symptoms and metacognition in euthymic [START_REF] Van Der Werf-Eldering | Is the Lack of Association between Cognitive Complaints and Objective Cognitive Functioning in Patients with Bipolar Disorder Moderated by Depressive Symptoms?[END_REF][START_REF] Torres | Metacognitive Knowledge and Experience in Recently Diagnosed Patients with Bipolar Disorder[END_REF] and mildly depressed [START_REF] Van Camp | Metacognitive Functioning in Bipolar Disorder versus Controls and Its Correlations with Neurocognitive Functioning in a Cross-Sectional Design[END_REF] individuals with BD. In contrast, mood symptoms were associated with a weaker relationship between cognitive complaints and objective cognitive performance in acutely ill individuals with BD [START_REF] Lin | The Associations between Subjective and Objective Cognitive Functioning across Manic or Hypomanic, Depressed, and Euthymic States in Chinese Bipolar Patients[END_REF]. This apparent discrepancy between studies can be explained by heterogeneity in the level of mood symptoms, which were low in our sample. Our results did not replicate the findings that hospitalizations and more significant socio-occupational difficulties correlate with metacognition, which were associated with disproportionately more subjective complaints than objective impairment in a previous report involving remitted individuals with BD [START_REF] Miskowiak | Predictors of the Discrepancy between Objective and Subjective Cognition in Bipolar Disorder: A Novel Methodology[END_REF].

The participants' profile might have influenced some of our results, which may not generalize to all individuals with BD. First, there was a majority of BD 2 (52%) compared with BD 1 (37%). We did not find in the present study a significant association between the type of BD and metacognition. However, a previous study has reported that BD type II was associated with having more subjective complaints than objective impairment [START_REF] Miskowiak | Predictors of the Discrepancy between Objective and Subjective Cognition in Bipolar Disorder: A Novel Methodology[END_REF]. Thus, prevalence of BD type II in this sample might have biased recruiting toward individuals who underestimated their cognitive performance. Second, patients had very low manic symptoms. The majority of participants were euthymic or slightly depressed. Again, this might have biased the recruitment toward excluding individuals who overestimated their cognitive performance and lacked insight.

The multiple regression analysis also identified two robust correlates of cognitive complaints: depressive symptoms and impulsivity. Greater depressive symptoms have been consistently identified as important determinants of cognitive complaints in BD [START_REF] Demant | Assessment of Subjective and Objective Cognitive Function in Bipolar Disorder: Correlations, Predictors and the Relation to Psychosocial Function[END_REF][START_REF] Van Der Werf-Eldering | Is the Lack of Association between Cognitive Complaints and Objective Cognitive Functioning in Patients with Bipolar Disorder Moderated by Depressive Symptoms?[END_REF][START_REF] Lin | The Associations between Subjective and Objective Cognitive Functioning across Manic or Hypomanic, Depressed, and Euthymic States in Chinese Bipolar Patients[END_REF] along with less strong manic symptoms [START_REF] Lin | The Associations between Subjective and Objective Cognitive Functioning across Manic or Hypomanic, Depressed, and Euthymic States in Chinese Bipolar Patients[END_REF]. The association between impulsivity and cognitive complaints may be explained by symptoms of attention deficit hyperactivity disorder [START_REF] Pierre | Detection of Adult Attention Deficit Hyperactivity Disorder with Cognitive Complaint: Experience of a French Memory Center[END_REF], which were not investigated in our sample. Our results are consistent with those of a previous study reporting a lack of greater cognitive complaints by individuals with BD taking lithium or antipsychotic medication [START_REF] Jensen | Optimising Screening for Cognitive Dysfunction in Bipolar Disorder: Validation and Evaluation of Objective and Subjective Tools[END_REF]. Our results did not replicate the findings that a higher number of episodes, especially the number of mixed episodes, longer duration of the illness and onset of the illness at an earlier age [START_REF] Martínez-Arán | Do Cognitive Complaints in Euthymic Bipolar Patients Reflect Objective Cognitive Impairment?[END_REF], and impairment in psychosocial functioning [START_REF] Ott | Relation between Functional and Cognitive Impairments in Remitted Patients with Bipolar Disorder and Suggestions for Trials Targeting Cognition: An Exploratory Study[END_REF] are associated with more subjective complaints in BD.

The most important limitation was related to the assessment of subjective cognition, which was based only on one question extracted from a scale not specifically designated to measure cognitive complaints. Further studies should be conducted to replicate our results using a scale validated in BD, such as the Cognitive Complaints in Bipolar Disorder Rating Assessment [START_REF] Rosa | Validity and Reliability of a Rating Scale on Subjective Cognitive Deficits in Bipolar Disorder (COBRA)[END_REF]. The measure of objective cognition used in this study was able to identify performance above the norm, whereas the assessment of subjective cognition could not measure better self-reported performance, as cognitive complaints have a low boundary corresponding to normal performance. Moreover, the cognitive complaints question used in this study evaluated subjective cognitive performance relative to the usual capacity of the patient, whereas the measure of objective cognition referred to the distance to a norm of healthy subjects. Others have proposed using a different question to assess subjective cognition in order to resolve these two issues: "compared to healthy individuals of your age, your cognitive skills (concentration, memory, problem-solving, . . . ) are profoundly below average, well below average, below average, average, above average, well above average, or superior" [START_REF] Van Camp | Metacognitive Functioning in Bipolar Disorder versus Controls and Its Correlations with Neurocognitive Functioning in a Cross-Sectional Design[END_REF][START_REF] Torres | Metacognitive Knowledge and Experience in Recently Diagnosed Patients with Bipolar Disorder[END_REF]. As a consequence, the way we measured subjective cognition in this study may have led to an underestimation of the strength of the association with objective cognition. Another important limitation was the investigation of global cognition only, without exploring the correlates of metacognition within each specific cognitive domain. This may be problematic, as metacognition varies by the domain of impairment, with patients being particularly unaware of attention and processing speed problems [START_REF] Miskowiak | Predictors of the Discrepancy between Objective and Subjective Cognition in Bipolar Disorder: A Novel Methodology[END_REF]. Further studies investigating metacognition in BD may benefit from less biased measures such as the ratio between meta-d' and d [START_REF] Maniscalco | A Signal Detection Theoretic Approach for Estimating Metacognitive Sensitivity from Confidence Ratings[END_REF]. The participants included in the present study were heterogeneous regarding their mood symptoms, mixing participants with a characterized depressive episode and euthymic participants. Further studies should control mood symptoms by recruiting separated subgroups with euthymic, hypomanic and depressed participants with equal sample size. Finally, grouping antipsychotics in just one class was an important limitation of the current study. Some antipsychotics are known to have a potential direct procognitive effect, such as lurasidone [START_REF] Yatham | Lurasidone versus Treatment as Usual for Cognitive Impairment in Euthymic Patients with Bipolar I Disorder: A Randomised, Open-Label, Pilot Study[END_REF], and an indirect procognitive effect through an antidepressant action (ex: quetiapine, lurasidone).

Conclusions

This exploratory study investigated several clinical correlates of metacognition in BD using moderation analysis. The main factor influencing metacognition was the prescription of antipsychotics. The strength of the relationship between cognitive complaints and objective cognitive performance was weaker for individuals taking an antipsychotic. This result suggests a crucial role of dopamine in the modulation of metacognitive performance. Our study emphasizes the importance of controlling for antipsychotic medication when assessing metacognition in severe and persistent mental disorders, such as schizophrenia and BD, in future studies. This result also suggests not to rely on the sole self-report evaluation of cognitive functioning in patients with BD who are taking antipsychotics, and complete this evaluation with objective measures of cognitive performance. Depressive symptoms and impulsivity were associated with poorer subjective cognition and may be considered as potential therapeutic targets for individuals with BD and cognitive complaints. Our results may also guide future programs of metacognitive training in BD.

Supplementary Materials:

The following are available online at https://www.mdpi.com/article/ 10.3390/jcm10194349/s1, Supplementary Information S1: Test-retest reliability and internal consistency for the clinical scales; Supplementary Figure S2: Histogram of cognitive complaints, Supplementary Information S3: Diagnostic for imputation of missing data; Supplementary Table S4: Results for the trivariable ordinal logistic regressions with cognitive complaints as the dependent variable and objective cognition, several successive clinical moderators, and the interaction between objective cognition and the clinical moderator as independent variables. This table reports only the main effect of clinical moderators (the main effect of objective cognition and the interaction between objective cognition and the clinical moderator are reported in Supplementary Table S5 & Table 3 respectively); Supplementary Table S5: Results for the trivariable ordinal logistic regressions with cognitive complaints as the dependent variable and objective cognition, several successive clinical moderators, and the interaction between objective cognition and the clinical moderators as the independent variables. This table reports only the main effect of objective cognitive performance (the main effect of the clinical moderators and the interaction between objective cognition and the clinical moderators are reported in Supplementary Table S4 & Table 3 respectively); Supplementary

Institutional Review Board Statement:

The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. All procedures involving human patients were approved by the local ethics committee (Comité de Protection des Personnes Ile de France IX) on 18 January 2010 under French laws for non-interventional studies (observational studies without any risk, constraint, or supplementary or unusual procedure concerning diagnosis, treatment, or monitoring).

Informed Consent Statement:

The ethical committee required that all patients be given an informational letter but waived the requirement for written informed consent because it considered this study a non-interventional one. However, verbal consent was witnessed and formally recorded.

JFigure 1 .

 1 Figure 1. Distribution of objective cognition according to the level of cognitive complaints in the observed dataset: (a) in individuals with antipsychotic; (b) in individuals without antipsychotic. The width of the boxes is proportional to the sample size in each level of cognitive complaints.

Figure S6 :

 S6 Distribution of objective cognition according to the level of cognitive complaints in the observed dataset: (a) in individuals with predominant depressive polarity; (b) in individuals with predominant indeterminate/manic polarity. Author Contributions: P.R., N.F., E.B.-G. & C.P. contributed substantially to conception and design of the study. P.R., N.F. & E.B.-G. contributed substantially to the analysis and interpretation of data. P.R., A.-S.C. & C.P. contributed substantially to acquisition of data. All authors drafted the article and revised it critically for important intellectual content. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by a grant from Centre Hospitalier de Versailles (Bourse Registre 2019), by the Fondation FondaMental, Créteil, France, and the Investissements d'Avenir Programs managed by the ANR under references ANR-11-IDEX-0004-02 and ANR-10-COHO-10-01.

Table 1 .

 1 Participants' socio-demographical, clinical, and functional characteristics.

	Variable	Mean or %	SD	n
	Age (years)	40.2	11.1	281
	Sex	42.3 (M)		281
	Educational level (years)	14.3	2.5	281
	Diagnosis: Type 1	37.4		281
	Type 2	52
	NOS 1	10.6
	Total number of mood episodes	8.1	7.5	170
	Predominant polarity: depressive	36.5		170
	Indeterminate	50.6
	Manic	12.9
	Age at onset (years)	23.3	8.2	271
	History of psychosis	19.8		232
	Rapid cycling	7.6		263
	CGI 2 Severity (1-7)	4.5	0.9	279
	Current major depressive episode	13.9		281
	Current hypomanic episode	1.4		281
	Current manic episode	0		281
	MADRS 3 (0-60)	10.4	8.5	281
	YMRS 4 (0-60)	2.2	3.4	281
	STAI-YA 5 (state subscale) (20-80)	43.4	14.5	278
	End of last characterized episode > 3 months	55.6		279
	Antidepressant	22.1		281
	Anticonvulsant	25.3		281
	Lithium Carbonate	13.5		281
	Antipsychotic	16.4		281
	Anxiolytic	19.2		281
	Any lifetime substance use disorder	26		273
	BIS 6 (34-136)	66.9	10.6	279
	CTQ 7 (28-140)	43.7	15.1	277
	FAST 8 total (0-72)	20	13	280
	MARS 9 (0-10)	6.8	2.2	254

Table 2 .

 2 Objective neuropsychological performances expressed in standard deviations from the norm.

		Mean	SD	n
	Verbal Memory	0.41	0.9
	CVLT 1 Immediate recall	0.58	1.26	279
	CVLT 1 Short delay free recall	0.29	1.11	279
	CVLT 1 Long delay free recall	0.37	1.09	278
	CVLT 1 Total recognition	0.38	0.59	278
	Working Memory	-0.14	0.7
	Digit Span	-0.24	0.86	273
	Spatial Span forward	-0.08	0.88	270
	Spatial Span backward	-0.12	0.85	270
	Executive Functioning	-0.15	0.77
	TMT 2 Part B	-0.14	1.18	279
	Stroop color/word condition	0.04	1.02	267
	Phonemic fluency	-0.07	1.05	278
	Semantic fluency	-0.42	0.96	278
	Processing speed	-0.11	0.67
	Coding	-0.2	0.92	271
	Symbol search	0.01	0.88	270
	Stroop word condition	-0.08	0.79	268
	Stroop color condition	-0.45	0.85	267
	TMT 2 Part B	0.18	0.96	280
	Attention	-0.38	0.67
	CPT 3 omission	-0.84	1.23	266
	CPT 3 commission	-0.15	1.06	266
	CPT 3 variability	-0.29	1.1	266
	CPT 3 detectability	-0.23	0.98	266
	Reasoning	0.56	0.71
	Vocabulary	0.83	0.87	251
	Matrices	0.34	0.79	270

1 CVLT: California Verbal Learning Test; 2 TMT: Trail Making Test; 3 CPT: Continuous Performance Test.

Table 4 .

 4 Results for the multiple moderation analysis with ordinal logistic regression, including cognitive complaints as the dependent variable.

	Independant Variable	OR (95% CI) 1	Statistic	p	λ	fmi
	Predominant Polarity (Indeterminate/Manic vs. Depressive)	1.17 (0.6-2.29)	t(116) = 0.5	0.646	0.353	0.364
	CGI Severity	1.37 (0.96-1.96)	t(236.4) = 1.7	0.083	0.055	0.063
	Lithium carbonate	1.42 (0.66-3.06)	t(248.9) = 0.9	0.366	0.019	0.026
	Antipsychotic	1.4 (0.63-3.1)	t(240.5) = 0.8	0.408	0.044	0.052
	Anxiolytic	1.89 (1-3.57)	t(248.7) = 2	0.051	0.019	0.027
	FAST	1.02 (1-1.05)	t(245.8) = 1.9	0.058	0.028	0.036
	Objective cognition	0.05 (0-1.58)	t(213.8) = -1.7	0.09	0.111	0.119
	Educational level	0.97 (0.88-1.08)	t(247.5) = -0.5	0.596	0.023	0.031
	Diagnosis (Type 2/NOS 2 vs. Type 1)	1.41 (0.67-2.98)	t(187.3) = 0.9	0.363	0.17	0.179
	History of psychosis	1.22 (0.49-3.02)	t(121.2) = 0.4	0.664	0.337	0.348
	MADRS 3	1.1 (1.06-1.14)	t(240.5) = 5.2	<0.001	0.044	0.052
	Any lifetime substance use disorder	0.81 (0.45-1.45)	t(231.2) = -0.7	0.48	0.069	0.077
	BIS 4	1.07 (1.04-1.1)	t(243.5) = 4.4	<0.001	0.035	0.043
	CTQ 5	1.02 (1-1.03)	t(245.2) = 1.8	0.067	0.03	0.038
	MARS 6	0.93 (0.82-1.06)	t(196.3) = -1.1	0.264	0.15	0.159
	Type of WAIS 7 (IV vs. III)	0.75 (0.36-1.59)	t(246.1) = -0.7	0.458	0.027	0.035
	Objective cognition: Polarity (Indeterminate/Manic vs. Depressive)	1.42 (0.37-5.47)	t(126.5) = 0.5	0.605	0.321	0.332
	Objective cognition: CGI 8	1.52 (0.73-3.17)	t(228.2) = 1.1	0.265	0.076	0.084
	Objective cognition: Lithium carbonate	0.49 (0.09-2.67)	t(221.4) = -0.8	0.405	0.093	0.101
	Objective cognition: Antipsychotic	6 (1.13-31.7)	t(216.9) = 2.1	0.035	0.104	0.112
	Objective cognition: Anxiolytic	0.52 (0.13-2.13)	t(209.5) = -0.9	0.36	0.121	0.129
	Objective cognition: FAST 9	1.03 (0.98-1.08)	t(224.6) = 1.3	0.199	0.085	0.093

J. Clin. Med. 2021, 10, 4349. https://doi.org/10.3390/jcm10194349 https://www.mdpi.com/journal/jcm

NOS: not otherwise specified;

CGI: Clinical Global Impression scale;

MADRS: Montgomery Asberg Depression Rating Scale

; 4 YMRS: Young Mania Rating

Scale; 5 STAI-YA: State-Trait Anxiety Inventory

YA form; 6 BIS: Barratt

Impulsiveness Scale; 7 CTQ: Childhood

Trauma Questionnaire; 8 FAST: Functioning

Assessment Short Test; 9 MARS: Medication Adherence Rating Scale.

Odds Ratio [95% Confidence Interval];

NOS: not otherwise specified;

MADRS: Montgomery Asberg Depression Rating Scale;

BIS: Barratt Impulsiveness Scale;

CTQ: Childhood Trauma Questionnaire;

MARS: Medication Adherence Rating Scale;

Wechsler Adult Intelligence Scale;

CGI: Clinical Global Impression scale;

FAST: Functioning Assessment Short Test. Significant results (p < 0.005) are in bold.

Acknowledgments:

We are grateful to the Fondation FondaMental, in particular the FACE-BD collaborators and data managers. We also thank the psychologists working at the Versailles Center of Expertise in Bipolar Disorders for gathering the data: Anne-Marie Galliot, Isabelle Grevin, and Géraldine Liaskovski. We thank the Centre Hospitalier de Versailles and William Hempel (Alex Edelman and Associates) for editorial assistance.

Data Availability Statement:

The data presented in this study are available on request from the corresponding author. The data are not publicly available as participants did not give their consent for sharing these data.

Conflicts of Interest:

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


Mylène Richard

Julen Ariztia

Sandrine Lamandé-Langle

Nadia Pellegrini
email: nadia.pellegrini@univ-lorraine.fr

Nadia Pellegrini Moïse

Sugar γ-Amino Acids as Building Blocks for the Synthesis of Cyclic Neoglycopeptides

Keywords: sugar amino acids, cyclopeptides, C-glycosyl lactone, multivalent scaffold

come   L'archive ouverte pluridisciplinaire

as templates for anchoring of a wide range of biomolecules or detection dyes. Anomeric sugar -amino acids efficiently obtained by nucleophilic addition of nitronate anion on sugar olefins are usefull building blocks for the preparation of new cyclic glycopeptides. We report here the synthesis of two types of cyclic systems containing -glyco amino acid and -amino acid units. A first example was prepared to demonstrate the feasibility of the cyclization of these original structures. To explore these systems as template for multivalency, -amino acid and sugar amino-acid moieties bearing additional functions were choosen as building blocks for the second macrocyle. Furthermore, we describe and explain some unexpected results obtained during deprotection of these new cyclic glycopeptides.

Cyclic peptides are naturally occurring substances exhibiting improved properties compared to the linear ones. Indeed, linear peptides usually show low metabolic stability limiting their use as drugs and display conformational flexibility reducing the strength of the ligand-target binding. [1][2][3][4][5][6] Therefore, the development of original cyclopeptide-based therapeutics is currently a growing interest. Some of them like the well-known vancomycin and its derivatives, 6,7 gramicidin, 8,9 colistin, 10 octreotide, 11 and RGD derivatives 12,13 exhibit useful biological properties as antibiotics, anticancer and integrin inhibitors. Cyclic peptides also proved to be useful for the design of functional nanotubes from self-assembly. 14,15 Cyclic peptides or peptidomimetics can be useful in drug design [16][17][18][19][20][START_REF] Fransson | Strategies for conversion of peptides to peptidomimetic drugs, Ed by Vale, Nuno[END_REF] and serve as templates for attaching and assembling a wide variety of ligands. 5,22,23 Such templates allow the facile and versatile attachment of multiple ligands (multivalency) by suitable functions. 5,[23][24][25] One noteworthy example are the Regioselectively Addressable Functionalized Templates (RAFT) developed by Dumy et al. [26][27][28] which have found powerful applications in biology and imaging. 27,29,30 A wide range of multivalent (glyco)cyclopeptides and synthetic glycoclusters based on cyclic peptides have been described for their related applications in glycobiology. 5,25,[31][32][33][34] Designing and synthetizing new types of biocompatible and functionalizable molecular scaffolds for multivalent presentation are great challenges for synthetic chemists. As unusual amino acids with multiple functionalization points, sugar amino acids are good candidates and can be incorporated into peptidic structures. A few years ago, we developed anomeric sugar and -amino acids [35][START_REF] Pellegrini-Moïse | Carbohydrate Chemistry[END_REF][START_REF] Enderlin | [END_REF] and we integrated them into small mixed peptides, 38,39 and cyclic glycopeptides. 38,40 In connection with our research on new carbohydrates-containing peptides, we proposed the construction of original hybrid macrocyclic structures built with sugar -amino acids and amino acids. Furthermore, to explore the scope of these structures as templates for multivalency, both amino-acid moieties were carefully chosen to provide additional anchoring points. We give in this paper a full account of the synthesis of these original cyclic hybrid glycopeptides.

Results and discussion

The nucleophilic addition of a nitronate anion on push-pull anomeric sugar olefins has been previously studied in our group (Scheme 1). 35 Michael adducts 1 were obtained in excellent yield as a mixture of inseparable nitro esters starting from the corresponding erythroglycosylidene (14/86:ratio), the major anomer bearing the nitro group on the less hindered face of the sugar. We have previously demonstrated that both anomers are in equilibrium, likely as a result of opening and closure of the sugar ring. The configuration of the major anomer (methyl carboxylate and isopropylidene on the same face) was established on the basis of 1 H NMR spectroscopic data by NOESY experiments on α/β mixture and X-ray crystallographic analysis. 35 Anomeric mixture of nitro esters 1 was treated with lithium hydroxide in THF/H 2 O to give the corresponding carboxylic acid in very good yield. At this stage, the separation of both anomers could be performed by chromatography and the subsequent steps were carried out on the major isomer. Reduction of the nitro group required medium hydrogen pressure (40 psi) To go further with these macrocyclic structures, we envisaged to modulate their hydrosolubility by removing the sugar hydroxyl protecting groups, namely 4,5isopropylidene. Moreover, these hydroxyls could also be considered as additional functionalization points. The cycloglycopeptide 8 was treated by a mixture of TFA/H 2 O (1/1) at 0°C, commonly used for isopropylidine removal (Scheme 3). Unexpectedly, we only observed the formation of C-glycosyl lactone 21 in quantitative yield, as a result of several concommitant intramolecular amide-to-ester conversions of 8. We postulated that compound 21 results from TFA promoted acetal removal followed by nucleophilic attack of O4 on carboxamide function. Few intramolecular lactonizations of C-glycosyl ester derivatives have already been described in the literature and led to the formation of C-glycosyl bicyclic lactones where the lactone ring is annulated to a furanoid 42 or a pyranoide 43 system. However, to the best of our knowledge, no lactonization of amide derivatives in such saccharidic systems has been described. This prompted us to study this reaction on less elaborate precursors in order to find experimental conditions enabling deprotection while preventing lactonization (Scheme 4). We first envisioned the 4,5-isopropylidene removal on -nitro ester These results unambiguously suggested that the minor -anomer 11 displaying the methyl carboxylate on one side of the sugar ring and the isopropylidene on the other side could be a valuable unit for cyclic hybrid glycopeptides synthesis and efficient isopropylidene deprotection. A similar pathway was thus investigated starting from 11 (see SI, scheme S1).

However, it is important to note that compound 11 was obtained in 15% yield after addition of nitromethane anion (minor anomer). Furthermore, after preliminary intramolecular coupling experiments, the cyclic product could not be isolated. The nature and structure of the linear precursor clearly affected the macrocyclization process. In particular, the anomeric configuration did not seem to induce a favorable folding of this linear precursor or provide the required spatial arrangement between amino and carboxylic acid moieties. Moreover, the steric hindrance around the anomeric aminomethyl moiety (on the same face of isopropylidene and hydroxymethyl chain) could prevent the cyclization reaction.

Conclusion

To sum up this work, we have synthesized new and original cyclic glycopeptides containing two types of amino acids, a -glycoamino acid and an -amino acid. The construction of the linear precursors based on two building blocks was achieved by a multistep sequence. A first macrocycle was obtained as a proof of concept to validate the cyclization capabilities of these glycopeptides. The second structure is more elaborate and has proved challenging to synthetize and purify. Nevertheless, this later presents an extended number of chemical functionalities, with the idea of exploring their scope as template for multivalency. During acid catalyzed removal of isopropylidene of these new cyclic glycopeptides, a C-glycosyl lactone in which the lactone ring is annulated to a furanoid system was unexpectedly and spontaneously formed. Intense investigations are now devoted to functionalization of these cyclopeptides via coupling of the amino or hydroxymethyl moieties to relevant biomolecules and/or dyes for biological applications.

Experimental part

Reagents and general methods

DMF was dried by distillation from calcium hydride. Other solvents and reagents were purchased from commercial sources and used without further purification. TLC analyses were performed using standard procedures on Kieselgel 60 F254 plates (Merck). Compounds were visualized using UV light (254 nm) and 30% methanolic H 2 SO 4 /heat as developing agent.

Column chromatography was performed on silica gel SI 60 (63-200 m) or Lichroprep RP-18 (40-63m) (Merck). FTIR spectra were recorded on a Perkin-Elmer spectrum 1000 on NaCl windows (film) or KBr pellets (cm -1 ). Melting points were determined with a Tottoli apparatus and are uncorrected. Optical rotations were measured on an Anton Paar MC300

polarimeter. 1

Procedure A: General procedure for peptidic coupling with HATU

To a solution of amine (1.1 mmol, 1.1 eq.) and carboxylic acid (1 mmol, 1 eq.) in DMF (10 mL) were added HATU (570 mg, 1.5 mmol, 1.5 eq.) and DIEA (363 µL, 2.1 mmol, 2.1 eq.) at

0°C under argon atmosphere. After stirring at room temperature for 24h, the solvent was evaporated. The residue was dissolved in ethyl acetate (20 mL), washed with HCl 1N (5 mL), a saturated aqueous solution of NaHCO 3 (5 mL) and brine (5 mL). The organic layer was dried over MgSO 4 , filtered and concentrated. The product was purified by column chromatography (silica gel, cyH/EA).

Procedure B: General procedure for saponification with LiOH

To a solution of methyl ester (2 mmol) in THF (15 mL) and water (5 mL) was added LiOH (144 mg, 6 mmol, 3 eq.) and the mixture was stirred until completion of the reaction. THF was removed under reduced pressure, ethyl acetate (20 mL) was added and the pH was adjusted to 2-3 with aqueous 1N HCl. The organic layer was washed with brine and dried over MgSO 4 , filtered and concentrated. When necessary, the crude residue was purified by column chromatography (silica gel, cyH/EA).

Procedure C: General procedure for nitro reduction or Cbz hydrogenolysis

To a solution of protected compound (10 mmol) in MeOH (20 mL) was added Pd/C(10%) (40%w/w). The mixture was hydrogenated under H 2 atmosphere (40 psi) for 24 h. The reaction mixture was then filtered through a pad of celite and concentrated to give the desired product. The crude product was used without further purification.

Compound 3

The above described procedure A was applied to 2 (580 mg, 1.59 mmol) and alanine methyl ester hydrochloride to provide 3 (608 mg, 85% yield). Colorless gum. Rf = 0.58 (EA), [α] D = -30.9 (c 0.5 in CHCl 3 ). 1 The above described procedure B was applied to 3 and acid 4 was obtained in quantitative yield. The amine 5 was obtained from 3 in 99 % yield via procedure C. Acid 4 (392 mg, 0.9 mmol, 1 eq.) and amine 5 (313 mg, 0.99 mmol, 1.1 eq.) were coupled according to procedure Compound 6 was deprotected using general procedure C and the corresponding amine (396 mg, 0.66 mmol, 1.1 eq.) was coupled with acid 4 (262 mg, 0.6 mmol, 1 eq.) following procedure A to provide 7 (482 mg, 79% yield). Colorless gum Rf = 0.17 (EA). Compound 7 was deprotected using general procedures B and C. To a solution of the deprotected glyco-amino acid (348 mg, 0.4 mmol, 1 eq.) in CH 2 Cl 2 /DMF (4/1) at 0°C under argon, were added DIEA (346 µL, 2.0 mmol, 5.0 eq.) and HATU (182 mg, 0.48 mmol, 1.2 eq.). After stirring at room temperature for 3 days, the solvents were evaporated and the residue was taken up in ethyl acetate (30 mL). The solution was washed with brine (10 mL),

A

  to give the expected amine subsequently protected with a benzyloxycarbonyl group under standard conditions. The NH-Cbz protected compound 2 thusly prepared was coupled with H-L-ala-OMe giving 3 in 85% yield. At this point, either amine or acid terminal functions of 3 were deprotected by hydrogenolysis (H 2 , Pd/C, MeOH) or by saponification (LiOH, THF/H 2 O) respectively. Building blocks 5 and 4 were then coupled using HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate) as activating agent and led to the tetrapeptide 6 in 78% yield. Compound 7 was obtained in 79% yield by hydrogenolysis of N-benzyloxycarbonyl protecting group and subsequent coupling with 4. Having achieved the synthesis of the linear hexapeptide 7, we next envisioned the cyclization step using classical peptide chemistry. First, saponification of the ester function of 7 with LiOH in THF/MeOH mixture led to the crude carboxylic acid. The N-benzyloxycarbonyl group was then removed by catalytic hydrogenolysis using Pd/C in MeOH. Without purification, the fully deprotected glycopeptide was treated with HATU as coupling agent in CH 2 Cl 2 /DMF mixture for 3 days under high dilution conditions (c = 0.1 mmol/L) to promote macrocyclization over poly(oligo)merization. 41 After purification, the cyclic glycopeptide 8 was obtained in 42% yield. The use of other coupling agent (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, PyBop) did not significantly improve the yield of 8. As expected by the symmetric structure of 8, 1 H and 13 C NMR spectra revealed only one set of signals corresponding to a sugar/alanine unit. Nevertheless, mass spectrometry unambiguously confirmed the formation of expected cyclic structure (see SI, Figure S1, see scheme 1 for atom numbering).

  Scheme 2. a) 1N HCl, MeOH, 0°C, 68%; b) i. NaIO 4 , MeOH; ii. NaBH 4 , MeOH, 0°C, 98%;

Scheme 3 . Deprotection of cyclopeptide 8

 38 Scheme 3. Deprotection of cyclopeptide 8

1 (

 1 [α] D = -35.2° (c 0.6 in CHCl 3 ).1 H NMR (CDCl 3 , 400 MHz): δ = 1.30-1.35 (m, 12H, 4 CH 3 ), 1.37 (d, 3H, J = 7.5 Hz, CH 3 alanine), 1.42 (d, 3H, J = 7.5 Hz, CH 3 alanine), 1.48 (s, 3H, CH 3 ), 1.51 (s, 6H, 2 CH 3 ), 2.59-2.75 (m, 6H, 6 H-2), 3.12 (dd, 1H, J gem = 14.5, J = 5.0 Hz, H-2'), 3.23 (dd, 1H, J gem = 14.5, J = 6.0 Hz, H-2'), 3.30 (dd, 1H, J gem = 14.5, J = 5.5 Hz, H-2'), 3.39 (dd, 1H, J = 6.5 Hz, H-2'), 3.43 (dd, 1H, J = 6.5 Hz, H-2'), 3.51 (dd, 1H, J = 7.0 Hz, H-2'), 3.75 (s, 3H, OCH 3 ), 3.90 (dd, 1H, J gem = 11.0, J = 4.0 Hz, H-6), 3.93-3.99 (m, 5H, 5 H-6), 4.39 (m, 1H, Hα), 4.44-4.55 (m, 4H, 2 H-α, 2 H-4), 4.57 (d, 1H, J 4,5 = 6.0 Hz, H-4), 4.87-4.92 (m, 3H, 3 H-5), 5.08 (d, 1H, J gem = 12.5 Hz, CH 2 -Ph), 5.12 (d, 1H, CH 2 -Ph), 6.06 (t, 1H, J = 6.0 Hz, NHCbz), 6.74 (d, 1H, J = 7.0 Hz, NH), 6.88 (d, 1H, J = 7.0 Hz, NH), 7.07 (d, 1H, J = 7.0 Hz, NH), 7.25 (m, 1H, NH), 7.30-7.39 (m, 5H, 5 H-Ar), 7.46 (t, 1H, J = 6.0 Hz, NH). 13 C NMR (CDCl 3 , 100 MHz): δ = 17.5, 17.7, 18.0 (3 CH 3 alanine), 24.6 (3 CH 3 ), 26.1 (3 CH 3 ), 39.2, 39.7, 39.8, 40.1, 40.7, 41.9 (3 C-2, 3 C-2'), 48.2, 49.2, 49.4 (3 C-α), 52.5 (OCH 3 ), 66.8 (CH 2 -Ph), 71.2, 71.4, 71.6 (3 C-6), 81.2, 81.3, 81.4 (3 C-5), 83.1, 83.3, 83.4 (3 C-4), 85.5, 85.7, 85.8 (3 C-3), 112.4, 112.5 (3 C(CH 3 ) 2 ), 128.0 (3 C-Ar), 128.5 (2 C-Ar), 136.6 (C-Ar), 157.C=O Cbz), 170.5, 170.7, 171.2, 172.9, 173.5 (6 C=O). ESI-HRMS [M+Na] + m/z = found 1041.4649 (calculated for C 48 H 70 N 6 O 18 Na: 1041.4639). IR (NaCl) max /cm -1 3327, 2985, 2938, 1718, 1657, 1529 Cyclopeptide 8

dried over MgSO 4 ,

 4 filtered and concentrated. The product was purified by column chromatography (silica gel, AcOEt/MeOH) to provide 8 (143 mg, 42% yield). White solid. Mp = 180 °C. Rf = 0.33 (CH 2 Cl 2 /MeOH: 9/1). [α] D = -50.5° (c 0.5 in CHCl 3 ). 1 H NMR (CDCl 3 , 250 MHz): δ = 1.36 (s, 3H, CH 3 ), 1.42 (d, 3H, J = 7.0 Hz, CH 3 alanine), 1.53 (s, 3H, CH 3 ), 2.64 (d, 1H, J gem = 14.5 Hz, H-2), 2.79 (d, 1H, H-2), 3.32 (dd, 1H, J gem = 14.5, J 2',NH = 6.0 Hz, H-2'), 3.47 (dd, 1H, J 2',NH = 6.0 Hz, H-2'), 3.98 (d, 1H, J gem = 11.5 Hz, H-6), 4.05 (dd, 1H, J 5,6 = 3.5 Hz, H-6'), 4.48 (m, 1H, H-α), 4.51 (d, 1H, J 4,5 = 6.0 Hz, H-4), 4.94 (dd, 1H, H-5), 6.72 (d, 1H, J = 6.0 Hz, NH ala), 7.40 (t, 1H, NH erythro). 13 C NMR (CDCl 3 , 62.9 MHz): δ = 17.8 (CH 3 alanine), 24.8 (CH 3 ), 26.3 (CH 3 ), 40.7, 41.0 (C-2, C-2'), 49.6 (C-α), 71.7 (C-6), 81.6 (C-5), 83.4 (C-4), 85.9 (C-3), 112.8 (C(CH 3 ) 2 ), 171.

15 and 17 using HATU in DMF led to glycopeptides 18 and then 19 in 69% and 47% yields

	After the validation of this proof of concept, i.e. the successful synthesis of 8 and the
	confirmation of the cyclization of these structures, we continued our work with the synthesis
	of a more functionalized cyclic peptide. This second example is built with-glycoamino acid
	and -amino acid bearing additional reactive groups (Scheme 2). For this purpose, L-lysine
	c with a tert-butyloxycarbonyl as side chain protecting group was selected as -amino acid
	partner. Derivative 9 was chosen as -glycoamino acid partner and modified in order to
	1 display a hydroxymethyl arm. As explained above, the nitro ester 9 was obtained as a mixture
	of anomers (15/85 ratio) by addition of the nitromethane carbanion on the corresponding
	exo-glycal. The selective removal of the 7,8-O-isopropylidene protecting group was achieved
	by treatment with 1N HCl in MeOH. The corresponding diol was obtained as a mixture of
	anomers and separated by column chromatography. 35 We decided to go ahead with the major
	isomer 10 and oxidative cleavage of the diol followed by reduction of the aldehyde
	intermediate led to compound 12 bearing a hydroxymethyl arm. The nitro function acting as
	an amino protecting group was maintained for the first coupling with H-L-Lysine(Boc)-OMe
	and the compound 14 was obtained in 90% yield. We first planned to protect the hydroxyl
	group as a silyl ether derivative but this protection step appeared unnecessary since the
	unprotected hydroxyl was not affecting the peptidic coupling. The nitro group was next
	hydrogenated and the resulting amine 15 was protected with a benzyloxycarbonyl group.
	Methyl ester of 16 could be selectively removed by saponification leading to acid 17 in
	quantitative yield. Coupling of compounds
	Scheme 1. a: LiOH, THF/H 2 O (3/1), 4: quantitative yield; b: 1) H 2 , Pd/C (10%), MeOH, 3
	bars, 80%; 2) TMSCl (2 eq.), DCM, 3) DIEA, CbzCl, 65% three steps; c: H-L-Ala-OMe,
	DIEA, HATU, DMF, 85%; d) H 2 , Pd/C, MeOH, 99%; e) DIEA, HATU, DMF, 78%; f) i. H 2 ,
	Pd/C, MeOH, ii. 4, DIEA, HATU, DMF, 79%; g) i. LiOH, THF/H 2 O, ii. H 2 , Pd/C, MeOH, iii.
	DIEA, HATU, DMF/CH 2 Cl 2 , 42%.

respectively. Cyclic derivative 20 was obtained after amine and acid deprotection followed by activation with HATU and intramolecular coupling in CH 2 Cl 2 /DMF mixture under high dilution conditions (c = 0.1 mmol/L). Retrieval of compound 20 in the crude was tedious and purification inefficient although the expected molecular pic was detected by mass spectrometry ([M+Na] + = 1436.7626, calculated for C 66 H 111 N 9 O 24 Na: 1436.7634). We next turned to PyBop as activating agent in the same high dilution conditions. In this case, the recovery of 20 was easier but also required two purifications by column chromatography on silica gel explaining the low yield of 20 (22%). Even though the cyclization step was tedious, we were pleased to obtain a macrocycle bearing two different reactive groups.

  THF/H 2 O, ii. H 2 , Pd/C, MeOH, , iii. HATU, DIEA, DMF/CH 2 Cl 2 , 0°C, 22%.

4 , MeOH; ii. NaBH 4 , MeOH, 0°C, 98%; c) LiOH, THF/H 2 O, 13 98%, 17 99%; d) HCl.H-L-Lys(Boc)-OMe, DIEA, HATU, DMF, 0°C, 90%; e) H 2 , Pd/C, MeOH, 99%; f) DIEA, CbzCl, DCM, 0°C, 70%; g) HATU, DIEA, DMF, 0°C, 69%; h) i. H 2 , Pd/C, MeOH, ii. 17, HATU, DIEA, DMF, 0°C, 47%; i) i. LiOH,

  We eventually investigated the use of HCl in MeOH. When performed at 50°C, the reaction only gave the lactone 22 in 82% yield in 2 hours (entry 6). TLC monitoring showed that maintaining the temperature at -5° C could led to the expected 4,5-diol 26 without lactonization (compound 26 is more polar than lactone 22 on TLC) but this nitro ester was immediately converted into lactone 22 during evaporation of the solvent, even though the The reaction mixture was maintained at -5°C during 12h and then neutralized with NaHCO3. The formation of the lactone was observed during evaporation of the solvent.

	reaction mixture was neutralized with NaHCO 3 . Despite investigating various experimental
	conditions, it clearly appeared that lactonization of amide 25 during removal of the 4,5-
	isopropylidene moiety could not be prevented. Consequently, removal of the isopropylidene
	group for hydrosolubility modulation or for additional functionalization was not an option in
	this context.
	Table 1. 4,5-isopropylidene removal of 25
	entry	solvent	acid	temperature	reaction time (h)	yield of 22 (%)	recovery of 25 (%)
	1	H2O	TFA	0ºC then rt	7	99	0
	2	H2O	TFA	0ºC	7	0	99
	3	H2O	AcOH	50ºC	48	<10	50
	4	CHCl3/MeOH	acidic resin	40ºC	48	<10	30
	5	CH2Cl2	FeCl3.H2O	40ºC	48	<10	30
	6	MeOH	HCl	50ºC	2	82	0
	7	MeOH	HCl	0ºC then rt	7	80	20
	8	MeOH	HCl	-5ºC	12	80*	20

10 (major isomer with methyl carboxylate and isopropylidene on the same face). As expected, the lactone 22 was obtained by treating 10 with a TFA/H 2 O mixture at 0°C, and no traces of nitro ester 23 were detected. The same reaction performed on -nitro ester 11 (minor anomer) gave the nitro ester 24 in quantitative yield without lactonization reaction since the methyl carboxylate and O4 are not on the same face. Taking into account the formation of lactone 21 resulting from intramolecular esterification of amide functions of 8, we planned to investigate the 4,5-isopropylidene removal on a simple amide like 25, obtained from 10 after saponification of methyl ester and coupling with H-L-ala-OMe (Scheme 4). To find efficient experimental conditions for 4,5-isopropylidene removal leading to compound 26 without concomitant lactonization, the nature of the acid, the solvent, the temperature and the reaction time were studied (Table

1

). Treatment of 25 by a 1/1 TFA/H 2 O mixture led to C-glycosyl lactone 22 in quantitative yield (entry 1) although no reaction was observed while maintaining the temperature at 0°C (entry 2). Similarly, compound 26 was not formed when AcOH (entry 3), acidic resin (Amberlite IR 120, entry 4)

44

or FeCl 3

45

(entry 5) were used. In these conditions, 30 to 50% of starting amide 25 were revovered and lactonization occurred up to *

  C=O), 173.2 (C=O). ESI-HRMS [M+Na] + m/z = found 473.1902 (calculated for C 22 H 30 N 2 O 8 Na: 473.1894). IR (NaCl) max /cm -1 3341, 1725, 1662

	Compound 6
	H NMR (CDCl 3 , 250 MHz): δ = 1.33 (s, 3H, CH 3 ), 1.38 (d, 3H, J =
	7.0 Hz, CH 3 alanine), 1.52 (s, 3H, CH 3 ), 2.61 (d, 1H, J gem = 14.0 Hz, H-2), 2.67 (d, 1H, H-2),
	3.20 (dd, 1H, J gem = 14.5, J 2', NH = 5.5 Hz, H-2'), 3.37 (dd, 1H, J 2', NH = 7.5 Hz, H-2'), 3.72 (s,
	3H, OMe), 3.92 (dd, 1H, J gem = 11.0, J 5,6 = 3.5 Hz, H-6), 4.01 (d, 1H, H-6), 4.46-4.57 (m, 2H,
	H-α, H-4), 4.90 (dd, 1H, J 4,5 = 6.0 Hz, H-5), 5.06 (d, 1H, J gem = 12.0 Hz, CH 2 -Ph), 5.12 (d,
	1H, CH 2 -Ph), 5.66 (t, 1H, N-H), 6.80 (d, 1H, J α, NH = 6.5 Hz, N-H), 7.29-7.40 (m, 5H, H-Ar).
	13 C NMR (CDCl 3 , 62.9 MHz): δ = 17.9 (CH 3 alanine), 24.6, 26.1 (2 C(CH 3 ) 2 )), 40.3 (C-2),

42.1 (C-2'), 48.1 (C-α), 52.4 (OCH 3 ), 66.8 (CH 2 -Ph), 71.4 (C-6), 81.4, 83.0 (C-4, C-5), 85.6 (C-3), 112.6 (C(CH 3 ) 2 ), 128.0 (2 C-Ar), 128.5 (3 C-Ar), 136.4 (C-Ar), 156.8 (C=O Cbz), 170.4 (

  , 32.4 mmol, 2 eq.) under argon. After stirring at room temperature until completion of the reaction, the solvent was removed by half in vacuo. The mixture was diluted with CH 2 Cl 2 (300 mL). The organic layer was washed with water (3 x 100 mL), dried over MgSO 4 , filtered and the solvent was removed under reduced pressure. The crude product was solubilized in MeOH (130 mL) and NaBH 4 (622 mg, 1.51 mmol, 1 eq.) was added at 0°C. After 1 h at room temperature, the solvent was removed under reduced pressure. The residue was diluted in Compound 14 (3.85 g, 7 mmol) was deprotected according to general procedure C to provide amine 15 (3.52 g, 7 mmol, 99%) which was then was then protected with a benzyloxycarbonyl group. The crude was suspended in CH 2 Cl 2 (30 mL) and DIEA (2.18 mL, 12.6 mmol, 1.8 eq.) followed by Cbz-Cl (1.5 mL, 10.5 mmol, 1.5 eq.) were added at 0°C. The 2H, 2 H-6), 4.31 (m, 1H, H-), 4.41-4.48 (m, 2H, H-H-4), 4.69 (d, 1H, J 4,5 = 5.5 Hz, H-4), 4.79-4.88 (m, 4H, 2 H-5, 2 NH Boc), 5.07 (d, 1H, J gem = 13.0 Hz, CH-Ph), 5.16 (d, 1H, CH-Ph), 6.43 (m, 1H, NH sucre), 7.15 (m, 1H, NH lys), 7.24 (m, 1H, NH lys), 7.31-7.41 (m, 6H, 5 H-Ar, NH sucre). 13 C NMR (CDCl 3 , 100 MHz): δ = 22.7, 22.9 (2 CH 2 lys), 24.2, 24.3 (2 CH 3 ), 25.8 (2 CH 3 ), 28.4 (2 C(CH 3 ) 3 ), 29.5, 29.6 (2 CH 2 lys), 30.8, 31.5 (2 CH 2 lys), 39.6, 40.1, 40.7 (2 C-2, C-2', 2 CH 2 lys), 42.9 (C-2'), 52.7 (C-α, OCH 3 ), 53.8 (C-α), 61.4, 61.7 (2 C-7), 66.8 (CH 2 -Ph), 79.2, 79.3 (2 C(CH 3 ) 3 ), 79.9 (2 C-6), 81.2, 81.5 (2 C-5), 83.6, 84.0 (2 C-4), 85.0 (2 C-3), 112.9 (2 C(CH 3 ) 2 ), 128.0 (2 C-Ar), 128.1 (C-Ar), 128.5 (2 C-Ar), 136.5 (C-Ar), 156.2 (2 C=O Boc), 157.3 (C=O Cbz), 171.0, 171.6, 172.9, 173.9 (4 C=O). ESI-HRMS [M+Na] + m/z = found 1131.5654 (calculated for C 53 H 84 N 6 O 19 Na: 1131.5683). IR (NaCl) max /cm -1 3336, 2976, 2933, 2862, 1692, 1655, 1525 Compound 18 was deprotected by catalytic hydrogenation (procedure C) and the corresponding amine (1.32 mg, 1.35 mmol, 1.1 eq.) was coupled with acid 17 (787 mg, 1.23 mmol, 1 eq.) according to procedure A to provide 19 (914 mg, 47% yield). White solid. Mp = 117 °C. Rf = 0.39 (EA/MeOH: 9/1). [α] D = +1.2° (c 0.4 in CHCl 3 ). 1 H NMR (CDCl 3 , 400 MHz): δ = 1.23-1.53 (m, 12H, 6 CH 2 lys), 1.28 (s, 3H, CH 3 ), 1.31 (s, 6H, 2 CH 3 ), 1.45 (s, 27H, 3 C(CH 3 ) 3 boc), 1.50 (s, 9H, 3 CH 3 ), 1.60-1.89 (m, 6H, 3 CH 2 lys), 2.42-2.60 (m, 3H, 3 H-2), 2.75-2.87 (m, 3H, 3 H-2), 3.03-3.19 (m, 7H, 3 CH 2 lys, H-2'), 3.26 (dd, 1H, J gem = 14.0, J 2', NH = 4.0 Hz, H-2'), 3.35 (dd, 1H, J gem = 14.0, J 2', NH = 4.0 Hz, H-2'), 3.46-3.56 (m, 2H, 2 H-2'), 3.65 (m, 1H, H-2'), 3.76 (s, 3H, OCH 3 ), 3.80-3.90 (m, 6H, 6 H-7), 4.13-4.21 (m, 4H, 3 H-6, H-), 4.39-4.55 (m, 5H, 2 H-2 H-4, NH Boc), 4.67 (d, 1H, J 4,5 = 6.0 Hz, H-4), 4.81-4.96 (m, 5H, 3 H-5, 2 NH Boc), 5.07 (d, 1H, J gem = 12.0 Hz, CH-Ph), 5.15 (d, 1H, CH-Ph), 6.47 (m, 1H, NH), 7.12 (m, 1H, NH), 7.31-7.43 (m, 8H, 5 H-Ar, 3 NH), 7.93 (m, 1H, NH). CDCl 3 , 100 MHz): δ = 22.7, 22.8 (3 CH 2 lys), 24.3, 24.4 (3 CH 3 ), 25.8, 25.9 (3 CH 3 ), 28.5 (3 C(CH 3 ) 3 ), 29.4, 29.6, 29.7 (3 CH 2 lys), 30.9, 31.2, 31.7 (3 CH 2 lys), 39.6, 40.2, 40.3, 41.0, 42.7 (3 C-2, 3 C-2', 3 CH 2 lys), 52.5, 52.7 (C-α, OCH 3 ), 53.3 (C-α), 54.3 (C-α), 61.4, 61.7 (3 C-7), 66.8 (CH 2 -Ph), 79.1 (3 C(CH 3 ) 3 ), 79.7, 80.0, 80.3 (3 C-6), 81.3, 81.4, 81.6 (3 C-5), 83.7, 84.0, 84.2, 84.3, 84.9 (3 C-3, 3 C-4), 112.8 (2 C(CH 3 ) 2 ), 112.9 (C(CH 3 ) 2 ), 128.1 (2 C-Ar), 128.5 (3 C-Ar), 136.6 (C-Ar), 156.2 (3 C=O Boc), 157.2 (C=O Cbz), 170.8, 171.4, 171.6, 172.9, 173.2, 173.9 (6 C=O). ESI-HRMS [M+H] + m/z = found 1580.8481 (calculated for C 75 H 122 N 9 O 27 : 1580.8445). IR (NaCl) max /cm -1 3331, 2981, 2933, 2867, 1692, 1655,

	5, 173.2 (2 C=O). ESI-HRMS [M+Na] + m/z = found 875.4012 (calculated for C 39 H 60 N 6 O 15 Na: 875.4009). IR (NaCl) max /cm -1 3312, 3056, 2981, 2933, 2857, 1737, 1657, 1529 Compound 12 To a stirred solution of 10 (5.42 g, 16.2 mmol, 1 eq.) in methanol (340 mL) was added NaIO 4 with water until pH = 7. The organic layer was dried over MgSO 4 , filtrered and evaporated under reduced pressure. The crude product 12 (5.10 g, 98% yield) was used without further purification. Colorless oil. Rf = 0.50 (cH/EA: 1/2). [α] D = -10.4° (c 0.4 in CHCl 3 ). 1 H NMR (CDCl 3 , 250 MHz): δ = 1.31 (s, 3H, CH 3 ), 1.48 (s, 3H, CH 3 ), 2.09 (br s, 1H, OH), 2.90 (d, 1H, J gem = 18.0 Hz, H-2), 3.09 (d, 1H, H-2), 3.73 (s, 3H, OCH 3 ), 3.83-3.90 (m, 2H, H-7, H-7'), 4.11 (m, H, H-6), 4.59 (d, 1H, J gem = 11.5 Hz, H-2'), 4.67 (d, 1H, J 4,5 = 6.0 Hz, H-4), 4.90 (dd, 1H, J 5,6 = 4.0 Hz, H-5), 5.06 (d, 1H, H-2'). 13 C NMR (CDCl 3 , 62.9 MHz): δ = 24.5 5, C-6), 83.3 (C-3), 113.6 (C(CH 3 ) 2 ), 171.1 (C=O). ESI-HRMS [M+Na] + m/z = found 328.1014 (calculated for C 12 H 19 NO 8 Na: 328.1003). IR (NaCl) max /cm -1 3507, 2985, 2943, 1735, 1553 Compound 14 Compound 12 (5.10 g, 15.9 mmol) was saponified by LiOH (general procedure B) to provide 13 (4.78 g, 98% yield). Compound 13 (4.78 g, 15.6 mmol) was coupled with H-L-Lys(Boc)-OMe following procedure A to provide 14 (7.72 g, 90 %). Colorless oil. Rf = 0.21 (cH/EA: 3/7). [α] D = -22.0° (c 0.4 in CHCl 3 ). 1 H NMR (CDCl 3 , 250 MHz): δ = 1.25-1.54 (m, 4H, 2 CH 2 lys), 1.32 (s, 3H, CH 3 ), 1.43 (s, 9H, C(CH 3 ) 3 boc), 1.51 (s, 3H, CH 3 ), 1.66-1.90 (m, 2H, CH 2 lys), 2.77 (d, 1H, J gem = 16.0 Hz, H-2), 2.99-3.12 (m, 3H, H-2, CH 2 lys), 3.75 (s, 3H, OCH 3 ), 3.80-3.92 (m, 2H, 2 H-7), 4.13 (m, 1H, H-6), 4.51-4.64 (m, 3H, H-2', H-, NHBoc), 4.79 (d, 1H, J 4,5 = 6.0 Hz, H-4), 4.92 (dd, 1H, J 5,6 = 4.0 Hz, H-5), 5.09 (d, 1H, J gem = 12.0 Hz, H-2'), 6.49 (d, 1H, J = 8.0 Hz, NH). 13 C NMR (CDCl 3 , 62.9 MHz): δ = 22.4 (CH 2 lys), 24.3 (CH 3 ), 25.8 (CH 3 ), 28.4 (C(CH 3 ) 3 boc), 29.5 (CH 2 lys), 31.9 (CH 2 lys), 38.5 (C-2), 40.4 (CH 2 lys), 52.0, 52.5 (C-α, OCH 3 ), 61.2 (C-7), 76.7 (C-2'), 79.9, 81.5 (C(CH 3 ) 3 boc, C-5, C-6), 83.4 (C-3), 83.9 (C-4), 113.3 (C(CH 3 ) 2 ), 156.2 (C=O boc), 169.4 (C=O), 172.7 (C=O lys). ESI-HRMS [M+Na] + m/z = found 556.2485 (calculated for C 23 H 39 N 3 O 11 Na: 556.2477). IR (NaCl) max /cm -1 3364, 2981, 2933, 2867, 1742, 1690, 1666, 1551, 1529 reaction mixture was allowed to warm to room temperature and stirred overnight. Solvent was removed under vacuum and the residue was dissolved in ethyl acetate (40 mL), washed with 0.5M HCl (20 mL) and brine (20 mL). The organic layer was dried over MgSO 4 , filtered and concentrated. The residue was purified by column chromatography (silica gel, cyH/EA) to provide 16 (3.12 g, 70%). Colorless oil. Rf = 0.08 (cH/EA: 1/1). [α] D = -1.8° (c 0.5 in CHCl 3 ). 1 H NMR (CDCl 3 , 250 MHz): δ = 1.28-1.58 (m, 4H, 2 CH 2 lys), 1.34 (s, 3H, CH 3 ), 1.47 (s, 9H, C(CH 3 ) 3 boc), 1.54 (s, 3H, CH 3 ), 1.66-1.92 (m, 2H, CH 2 lys), 2.38 (br s, 1H, OH), 2.63 (d, 1H, J gem = 14.0 Hz, H-2), 2.72 (d, 1H, H-2), 3.08-3.15 (m, 2H, CH 2 lys), 3.22 (dd, 1H, J gem 3.96 (m, 2H, 2 H-7), 4.15 (m, 1H, H-6), 4.54 (m, 1H, H-), 4.63 (d, 1H, J 4,5 = 5.5 Hz, H-4), (CH 3 ), 28.4 (C(CH 3 ) 3 boc), 29.5 (CH 2 lys), 31.8 (CH 2 lys), 40.1, 40.4 (C-2, CH 2 lys), 42.9 (C-2'), 52.2, 52.5 (C-α, OCH 3 ), 61.5 (C-7), 66.9 (CH 2 -Ph), 79.4, 81.7 (C(CH 3 ) 3 boc, C-5, C-6), 83.7, 84.9 (C-3, C-4), 113.0 (C(CH 3 ) 2 ), 128.0 (2 C-Ar), 128.1 (C-Ar), 128.5 (2 C-Ar), 136.4 (C-Ar), 156.3, 156.9 (C=O Boc, C=O Cbz), 170.9 (C=O), 172.9 (C=O lys). ESI-HRMS [M+Na] + m/z = found 660.3112 (calculated for C 31 H 47 N 3 O 11 Na: 660.3103). IR (NaCl) max /cm -1 3345, 2976, 2933, 2867, 1709, 1662, 1525 Compound 18 Compopund 14 was converted to amine 15 by catalytic hydrogenolysis (procedure C) and acid 17 was obtained by saponification of compound 16 (procedure B). Amine 15 (1.14 g, 2.2 mmol, 1.1 eq.) and acid 17 (1.28 g, 2 mmol, 1 eq.) were coupled according to procedure A to provide 18 (1.53 g, 69%). White foam. Rf = 0.6 (EA/MeOH: 9/1). [α] D = +1.6° (c 0.2 in CHCl 3 ); 1 H NMR (CDCl 3 , 400 MHz): δ = 1.30-1.54 (m, 8H, 4 CH 2 lys), 1.31 (s, 6H, 2 CH 3 ), 1.45 (s, 18H, 2 C(CH 3 ) 3 boc), 1.48 (s, 3H, CH 3 ), 1.51 (s, 3H, CH 3 ), 1.63-1.86 (m, 4H, 2 CH 2 (6.93 gCH 2 Cl 2 (200 mL) and the organic layer was washed with a solution of 1N HCl (70 mL) and (CH 3 ), 25.8 (CH 3 ), 36.3 (C-2), 52.0 (OCH 3 ), 61.0 (C-7), 76.0 (C-2'), 79.7, 81.4, 83.7 (C-4, C-= 15.0, J 2', NH = 6.5 Hz, H-2'), 3.46 (dd, 1H, J 2', NH = 7.5 Hz, H-2'), 3.74 (s, 3H, OCH 3 ), 3.84-Compound 19
	Compound 16

4.70 (t, 1H, J = 5.0 Hz, NH Boc), 4.89 (dd, 1H, J 5,6 = 4.5 Hz, H-5), 5.08 (d, 1H, J gem = 13.0 Hz, CH-Ph), 5.15 (d, 1H, CH-Ph), 5.78 (m, 1H, NH Cbz), 6.98 (d, 1H, J = 7.5 Hz, NH), 7.33-7.41 (m, 5H, 5 H-Ar). 13 C NMR (CDCl 3 , 62.9 MHz): δ = 22.5 (CH 2 lys), 24.3 (CH 3 ), 25.8 lys), 2.50-2.77 (m, 6H, 2 OH, 4 H-2), 3.03-3.18 (m, 5H, 2 CH 2 lys, H-2'), 3.25 (dd, 1H, J gem = 15.0, J 2', NH = 5.5 Hz, H-2'), 3.51 (dd, 1H, J gem = 15.0, J 2', NH = 8.0 Hz, H-2'), 3.58 (dd, 1H, J gem = 14.5, J 2', NH = 7.0 Hz, H-2'), 3.75 (s, 3H, OCH 3 ), 3.82-3.88 (m, 4H, 4 H-7), 4.13-4.18 (m, 13 C NMR (

Acknowledgments

This work was supported by The Région Lorraine and Institut Jean Barriol. The Ministère de l'Enseignement Supérieur et de la Recherche is acknowledged for PhD funding to M.

Richard. We thank P. Lemière and F. Lachaud for technical assistance.

Electronic supplementary information

Copies of 1 H and 13 C NMR spectra, experimental procedures and characterizations of intermediates obtained from minor anomer 11.

Compound 20

Compound 19 was saponified (procedure B) and deprotected by catalytic hydrognolysis (procedure C). To a solution of deprotected glyco-amino acid (573 mg, 0.4 mmol, 1 eq.) in CH 2 Cl 2 /DMF (4/1) at 0°C under argon, were added DIEA (346 µL, 2.0 mmol, 5.0 eq.) and HATU (182 mg, 0.48 mmol, 1.2 eq.). After stirring at room temperature for 3 days, the solvents were evaporated and the residue was taken up in ethyl acetate (30 mL). The solution was washed with brine (10 mL), dried over MgSO 4 , filtered and concentrated. The product was purified by column chromatography (silica gel, AcOEt/MeOH) to provide 20 (123 mg, 22% yield). White foam. Rf = 0.24 (EtOAc/MeOH : 95/5). [α] D = -20.7° (c 0.2 in CHCl 3 ).

Compound 21

To a stirred solution of 8 (42 mg, 0.049 mmol) in H 2 O (0.50 ml) at 0°C was added trifluoroacetic acid (0.50 ml). After completion of the reaction (TLC monitoring), the solvents were removed under reduced pressure and coevaporated with Et 2 O (3x5 mL).