Andrei Szöke
email: andrei.szoke@ach.aphp.fr

Alexandre Méary

Aziz Ferchiou

Anca Trandafir

Marion Leboyer

Franck Schürhoff

Correlations between cognitive performances

Keywords: Schizophrenia, schizotypal, disorganisation, cognition, verbal fluency

Introduction

Important advances have been made in identifying the risk factors and in identifying, describing, and classifying the clinical symptoms of schizophrenia. However, the link between specific risk factors and particular clinical aspects is less well understood. Cognitive deficits may occupy an intermediate position between risk/vulnerability factors and clinical manifestations [START_REF] Heydebrand | Correlates of cognitive deficits in first episode schizophrenia[END_REF]. Thus, studying their correlation with identified risk factors and/or with the clinical dimensions may help to bridge the gap.

A tri-dimensional model for psychotic symptomatology emerged as an empirical finding [START_REF] Liddle | Schizophrenic syndromes, cognitive performance and neurological dysfunction[END_REF] and has since been repeatedly validated [START_REF] Grube | Meta-analysis of symptom factors in schizophrenia[END_REF]. Based on theoretical considerations and empirical findings, specific cognitive deficits have been suggested as explanations for each of the three psychotic dimensions. For example, Frith [START_REF] Frith | The cognitive neuropsychology of schizophrenia[END_REF] hypothesized that the negative dimension is the result of a deficit of willed action, resulting in difficulties in self-initiation and maintenance of action. For the disorganization dimension the proposed deficit consists in difficulties in suppressing inappropriate mental activity [START_REF] Baxter | Neuropsychological deficits associated with schizophrenic syndromes[END_REF] which leads to distractibility and poor inhibition of automatic (inadequate) responses [START_REF] Torres | Symptoms and interference from memory in schizophrenia: evaluation of Frith's model of willed action[END_REF]. Finally, the positive dimension has been linked to deficits in self-monitoring [START_REF] Frith | The cognitive neuropsychology of schizophrenia[END_REF] and, as a consequence, to difficulties in recognising his/her own thoughts and actions.

Studies exploring the correlation between psychotic dimensions and cognitive performances in schizophrenic subjects provided partial support to these hypotheses. Although the negative dimension correlates with most cognitive deficits [START_REF] Rocca | Neuropsychological correlates of reality distortion in schizophrenic patients[END_REF][START_REF] Heydebrand | Correlates of cognitive deficits in first episode schizophrenia[END_REF], impairments on tests that depend on initiation and maintenance of action, such as verbal fluency (VF), were more constantly associated with high scores of negative signs [START_REF] Good | The relationship of neuropsychological test performance with the PANSS in antipsychotic naïve, first-episode psychosis patients[END_REF][START_REF] Howanitz | Verbal fluency and psychiatric symptoms in geriatric schizophrenia[END_REF]. By contrast, most studies using classical neuro-psychological tests did not find associations with the positive dimension [START_REF] Brekke | Cognitive and psychophysiological correlates of positive, negative, and disorganized symptoms in the schizophrenia spectrum[END_REF][START_REF] Gouzoulis-Mayfrank | Semantic hyperpriming in thought-disordered patients with schizophrenia: state or trait?--a longitudinal investigation[END_REF]. However, several studies found an association between the positive dimension (or individual positive symptoms) and source monitoring (SM) deficits [START_REF] Brébion | Positive symptomatology and source monitoring failure in schizophrenia: an analysis of symptom-specific effects[END_REF][START_REF] Brébion | Source monitoring in schizophrenia: characterisation and associations with positive and negative symptomatology[END_REF][START_REF] Franck | Confusion between silent and overt reading in schizophrenia[END_REF] which are explained, at least in part, by difficulties in self-monitoring. Disorganisation was associated with deficits in attention tasks, particularly tasks necessitating sustained attention in the presence of distracters, such as the Stroop Word and Colour test (SWCT) [START_REF] Baxter | Neuropsychological deficits associated with schizophrenic syndromes[END_REF][START_REF] Liddle | Schizophrenic syndromes and frontal lobe performance[END_REF]. A recent meta-analysis [START_REF] Dibben | Is executive impairment associated with schizophrenic syndromes? A meta-analysis[END_REF] of studies reporting correlations between the three psychotic dimensions and commonly used tests of executive function reached similar conclusions. This analysis found that the negative and disorganisation dimensions, but not positive dimension, were significantly associated with impairment on executive tests. Furthermore, in this study VF and SWCT showed some specificity in their correlations to the psychotic dimensions as VF was three times more strongly associated with negative symptoms than with disorganisation, and the SWCT showed a twofold difference in the opposite direction.

Although each of the specific associations mentioned before has been reproduced, there are also several divergent findings in the literature. A possible explanation for the heterogeneity of results is the presence of confounding factors. Among these, medication and the clinical state of the subjects (chronic, acute, in remission etc.) are probably the most important.

To avoid these confounding factors, some authors have studied the correlation between schizotypal traits and cognitive performances in schizotypal subjects [START_REF] Suhr | Factor versus cluster models of schizotypal traits. II: relation to neuropsychological impairment[END_REF], in subjects at risk for schizophrenia [38], and in normal controls [START_REF] Dinn | Positive and negative schizotypy in a student sample: neurocognitive and clinical correlates[END_REF]. Schizotypal traits may be grouped along the same three dimensions as psychotic symptoms (positive, negative and disorganisation), are stable, may be reliably assessed, and are present in subjects that are usually not taking psychotropic medication.

Studies exploring the correlation between the three schizotypy dimensions and neurocognitive deficits are surprisingly rare [START_REF] Kerns | Schizotypy facets, cognitive control, and emotion[END_REF][START_REF] Suhr | Factor versus cluster models of schizotypal traits. II: relation to neuropsychological impairment[END_REF]38] and provide results for only a few neurocognitive measures. Thus, it is unclear if these correlations parallel those seen in schizophrenic subjects.

Most of the studies that investigated correlations between psychotic or schizotypal dimensions and cognitive performance were exploratory in design and used large cognitive batteries. This led to multiple statistical tests and, for this reason, increased the chance of spurious, false positive, findings.

There are currently no data from studies investigating clinical-cognitive correlation, in both schizophrenic and non-psychotic subjects, using the same cognitive battery.

In this study, we tested two hypotheses: 1/ that correlations exist between specific cognitive performances and specific psychotic or schizotypal dimensions and 2/ that correlations in non-psychotic subjects parallel those seen in schizophrenic subjects. To test these hypotheses, we assessed the correlations between cognitive performances and clinical dimensions in schizophrenic subjects and in non-psychotic subjects (first degree relatives of schizophrenic subjects and normal controls).

Methods

Subjects

Patients meeting DSM-IV [START_REF]American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition[END_REF] criteria for schizophrenia or schizoaffective disorder were consecutively recruited. Patients were interviewed by an experienced psychiatrist using the French version of the Diagnostic Interview for Genetic Studies (DIGS) [START_REF] Jijr | Diagnostic interview of genetic studies. Rationale, unique features and training. NIMH Genetics Initiative[END_REF][START_REF] Preisig | Diagnostic interview for genetic studies (DIGS): inter-rater and test-retest reliability of the French version[END_REF] to confirm their diagnosis. Most of the schizophrenic subjects were inpatients at the time of the study.

They were approached for inclusion when their treating psychiatrist considered them able to undergone the study procedures.

First-degree relatives were interviewed with the DIGS to exclude those presenting psychotic disorders.

Healthy controls were blood donors at the "Etablissement Français du Sang" in Créteil.

Controls were included after being interviewed with the DIGS and the FIGS (Family

Interview for Genetic Studies) [START_REF] Maxwell | Family Interview for genetic Studies[END_REF] to confirm the absence of personal or family history of DSM IV axis I or II disorders.

For inclusion in the study, all subjects were required to be normothymic, as evaluated by the Montgomery and Asberg Depression Rating Scale [START_REF] Montgomery | A new depression scale designed to be sensitive to change[END_REF] and the Bech and Rafaelsen mania rating scale [START_REF] Bech | The Bech-Rafaelsen Mania Scale and the Hamilton Depression Scale[END_REF]. Subjects were included only if aged between 18 and 60 years, and if they had no history of neurological disease or current substance abuse.

The research ethics board of Salpêtrière Hospital reviewed and approved the study. The study was described in detail to the subjects, who then gave written informed consent for participation.

Clinical assessment

The symptoms in schizophrenic subjects were assessed using the Signs and Symptoms of Psychotic Illness (SSPI) scale [START_REF] Houenou | Psychometric properties of the French version of the Signs and Symptoms of Psychotic Illness (SSPI) scale[END_REF][START_REF] Liddle | Signs and Symptoms of Psychotic Illness (SSPI): a rating scale[END_REF]. As noted by Grube et al. [START_REF] Grube | Meta-analysis of symptom factors in schizophrenia[END_REF], some symptoms tend to be associated with different dimensions across various studies because their polyfactorial nature and/or differences in subject characteristics across studies. Using symptoms that are inconsistently associated with one dimension for calculating the score for that dimension may lessen its validity. Thus, in this study we used only the core symptoms of each dimension, i.e. signs and symptoms that loaded similarly in the two validation studies (see table 1) [START_REF] Houenou | Psychometric properties of the French version of the Signs and Symptoms of Psychotic Illness (SSPI) scale[END_REF][START_REF] Liddle | Signs and Symptoms of Psychotic Illness (SSPI): a rating scale[END_REF].

The three schizotypy dimensions were derived in healthy subjects (controls and relatives) based on the answers to the Schizotypal Personality Questionnaire -SPQ [START_REF] Dumas | Validation de la version française du questionnaire de personnalité schizotypique de Raine -approche catégorielle et dimensionnelle des traits de personnalité schizotypique dans une population d'étudiants[END_REF][START_REF] Raine | The SPQ: a scale for the assessment of schizotypal personality based on DSM III R criteria[END_REF]. As for the SSPI, we calculated the three schizotypal dimensions using only the traits that loaded on similar dimensions in the two validation studies (table 1)

Insert Table 1.

Cognitive assessment

We tested our hypotheses using a battery of three tests: a Source Monitoring Test (SMT), the SWCT and a test of Semantic (category) VF.

The SMT was specifically designed for this study. We defined four semantic categories that are finite and are everyday knowledge: temperate climate fruits, Olympic sports, European countries and numbers from 50 to 70. Each possible example from these categories was printed on a unique card. At the first stage of the test, twelve items were selected from each category. Two were generated by the subjects after the category was announced, two were read aloud by the subject, two more were read silently by the subject, two were read aloud by the examiner and four were added from the remaining cards ("new" items). The recognition stage took place after the subject underwent the two other tests (SWCT and VF). Only at this stage was the subject informed that the aim of the test was to remember the source of the items with which he/she was presented. The 48 selected cards were presented to the subject in random order and he/she had to decide if this was his/her example, one of the examples he/she read silently or aloud, an example read by the examiner or a new item. The variables derived from this test were: total number of correct responses (CR), number of false alarms (FA = new items that were falsely designated as previously presented), self-discrimination index (SDI = number of times the subject correctly identified himself as the source of the item) and external bias (EB = number of items erroneously attributed to the examiner).

For the VF, the subjects were instructed to provide as many names of animals as possible in a 2-minute period. The production of items in VF tasks results from two processes [START_REF] Troyer | Clustering and switching as two components of verbal fluency: evidence from younger and older healthy adults[END_REF]: clustering, i.e. production of related items (e.g. sheep, cow, goat, pig -farm animals) and switching between those clusters (e.g. switching from farm animals to pets). We used the total number of items produced (excluding errors), the number of switches (Swi) and number of clustered items (Clust) calculated according to the procedures described by Troyer et al. [START_REF] Troyer | Clustering and switching as two components of verbal fluency: evidence from younger and older healthy adults[END_REF].

The SWCT consisted of three trials of 45 seconds each, in which the subject successively read aloud the names of colours printed in black, names the colour with which strings of "X"s are printed and finally names the colour with which the name of a different colour is printed (e.g. red printed in blue -correct answer blue). Two variables were generated using the number of items from each trial: the number of items in the last trial (IntTot) and an interference index (IntIdx), reflecting the extra time needed per item in the last trial compared with the second trial.

Statistical analysis

We compared demographic characteristics (age, sex distribution, education level) of the three groups using an ANOVA (for continuous variables) and the Chi2 test (for categorical variables).

We compared the cognitive performances of the three groups using the SAS mixed procedure. This procedure, based on a general linear mixed model, enabled us to adjust comparisons for demographic characteristics and also for non-independence of observations in subjects from the same family.

Our main analysis (testing our hypotheses of specific and similar clinical-cognitive correlation) involved two steps. In the first step, we selected the clinical dimensions that had a statistically significant influence on the cognitive measures using a stepwise backward regression. This is a classical automatic procedure for selecting the best subset of predictor variables -for a detailed presentation see [START_REF] Armitage | Statistical methods in medical research[END_REF]. For this step of the procedure all demographic and clinical variables were entered as potential explanatory variables. Only the significant variables, at a threshold of 0.1, were kept in the final model. The stepwise backward regression was applied in schizophrenic and in non-psychotic (relatives and controls) groups separately. For the non-psychotic group, "family" was entered as a random effect variable.

Analyzing the two non-psychotic groups together limited the number of statistical tests (and thus the risk of false positive findings) and augmented the sample size (thus the statistical power). We used this procedure, as we did not expected different correlations within the two non-psychotic groups.

Then, we calculated partial correlations between clinical and cognitive variables for all the significant associations identified in step I. If needed, the correlation sign was changed so that negative correlations always reflected poorer cognitive performance in more symptomatic subjects.

When significant correlations were observed in the combined group, we also calculated the correlations in each non-psychotic group.

All statistical analyses were generated using SAS software, Version 9 of the SAS System for Windows (Copyright © 2002-2003 by SAS Institute Inc., Cary, NC, USA).

Results

A total of 133 subjects (54 schizophrenic subjects, 37 of their first degree relatives and 42 controls) were included in this study. Their demographic and clinical characteristics are summarized in Table 2. There were significant differences between groups for all demographic variables, mainly due to the schizophrenic group being younger, less educated and with a larger proportion of male subjects.

Insert Table 2

The results of cognitive testing in the three groups are presented in Table 3.

Insert Table 3

In schizophrenic subjects, 4 cognitive variables (CR from the SMT, the two variables from the SWCT and Swi from the VF) showed significant association with clinical dimensions. Partial correlations showed that for all these variables better results were associated with less negative symptoms. The total number of items in the last trial (interference condition) of the SWCT was also negatively correlated with the disorganisation dimension.

Only three cognitive variables were significantly related to clinical dimensions in nonpsychotic subjects and all these variables were derived from the VF test. The total number of items and Clust were both positively correlated with the disorganisation dimension (i.e. subjects with greater disorganisation scores had better results). In contrast, Swi was negatively correlated with the negative dimension.

All significant clinical-cognitive correlations are presented in Table 4.

When correlations were calculated separately in controls and relatives the results (not shown) remained essentially the same. Correlations were in the same direction as those in the pooled samples, and their values were similar to those in the combined group (between 0.26 and 0.46).

Because some authors calculated the raw (un-partialled) correlations between the cognitive variables and clinical dimensions we also calculated them to allow comparisons. Using this procedure we obtained similar results.

In non-psychotic subjects the same three correlations (involving variables from the VF) reached statistical significance (all three correlations greater than .25). All other correlations were less than .25 and did not approach significance (P>.1).

In schizophrenic subjects only correlations between the negative dimension and the variables from the Stroop test and number of switches in the VF test remained greater than .25 and reached statistical significance. All other correlations were less than .25 and did not approach significance (P>.1). (Detailed results available on request)

Insert Table 4

Discussion

Our first hypothesis was that specific correlations exist between cognitive variables and clinical dimensions. In schizophrenic patients, our results lend partial support to this hypothesis as significant negative correlations were observed between the total number of items in the SCWT and disorganisation, and also between one of the variables reflecting VF performance (Swi) and the negative dimension.

However, no significant correlation was found between the positive dimension and performance with the SMT. Our results contrast those from several other studies that found significant correlation between the SMT deficits and the positive dimension [START_REF] Brébion | Source monitoring in schizophrenia: characterisation and associations with positive and negative symptomatology[END_REF] or individual positive symptoms [START_REF] Brébion | Positive symptomatology and source monitoring failure in schizophrenia: an analysis of symptom-specific effects[END_REF][START_REF] Keefe | Source monitoring deficits for self-generated stimuli in schizophrenia: multinomial modelling of data from three sources[END_REF]. However there are also reports of results similar to those obtained in the present study. Vinogradov et al. [START_REF] Vinogradov | Clinical and neurocognitve aspects of source monitoring errors in schizophrenia[END_REF] found that SMT errors were not associated with positive symptoms and were only weakly related to negative symptoms. Important differences in the characteristics of the SMT used (e.g. time between generation and recognition phases, difficulty of the task, etc.) and the clinical state of subjects included (chronic or acute, stable or not etc.) make comparison between studies difficult [START_REF] Ditman | A source-monitoring account of auditory verbal hallucinations in patients with schizophrenia[END_REF]. However, most previous studies did not investigate correlations with the negative dimension. Furthermore, with the exception of the Vinogradov et al. study [START_REF] Troyer | Clustering and switching as two components of verbal fluency: evidence from younger and older healthy adults[END_REF], most studies investigating the negative dimension were based on a dichotomous (positive-negative) model for schizophrenic symptomatology, thus differing from the trichotomous model used in this study.

In contrast to our hypothesis of particular clinical-cognitive correlation, the negative dimension appears to be linked to a more general cognitive impairment showing negative correlations with performances in all three tests. This last finding (the fact that general cognitive deficit was associated with the negative dimension) is consistent with results from several previous studies [START_REF] Harvey | Negative symptoms and cognitive deficits: what is the nature of their relationship?[END_REF]. One possible explanation for this pattern of results (significant correlations with the negative dimension that contrast with the paucity or absence of correlations with the positive and disorganised dimensions) is that the negative dimension is more stable than the other two. For clinical dimensions that show marked temporal variation, the SSPI (which is scored based on the previous week's symptoms) reflects neither the clinical state at the time of testing nor a more general, trait-like characteristic that could, for example, be derived from life-time assessments. The choice of the time frame is clearly of little importance in the case of more stable clinical dimensions (like the negative dimension).

Various populations of subjects (for example patients with chronic and stable positive and disorganised symptoms) should be studied to overcome this potential limitation. Another option, which was used in this study, is to study healthy subjects in which schizotypal traits (i.e. stable measures) are used for the clinical assessment.

Results in the group of non-psychotic subjects were not consistent with our initial hypothesis.

In particular, no correlation was found between measurements from the SMT and SCWT and the clinical dimensions.

To our knowledge this is the first study to explore, in non-psychotic subjects, the correlation between schizotypal dimensions and a source monitoring performances. A possible explanation for our negative findings is the low mean scores and limited variation in the schizotypal dimensions in our sample. Further studies, in populations showing greater variations in scores of schizotypal dimensions, in particular of positive dimension (e.g. subjects with schizotypal personality disorder), are needed to further explore the presence of such correlations.

In the sample of non-psychotic subjects, the correlation between the number of switches in the VF test and the negative dimension of schizotypy was the only result consistent with our initial hypothesis.

An unexpected finding was the positive correlation between disorganisation and both the

Clust and the number of total items in VF, i.e. better performances in subjects with greater disorganisation scores. In our study, the "disorganisation dimension" in the SPQ equated to the "odd speech" trait, which is mainly derived from items that are used to assess poor speech organisation. Several previous studies have suggested that speech or thought disorder (TD) in schizophrenic [START_REF] Gouzoulis-Mayfrank | Semantic hyperpriming in thought-disordered patients with schizophrenia: state or trait?--a longitudinal investigation[END_REF] or normal subjects [START_REF] Moritz | Increased automatic spreading activation in healthy subjects with elevated scores in a scale assessing schizophrenic language disturbances[END_REF] result from an excessive automatic spreading of activation in semantic networks. Our results suggest a similar hypothesis, in which excessive automatic (thus rapid) associations promote large clusters and a greater number of items in the VF but at the same time lessen the logical organisation of speech. Furthermore, functional neuroimaging studies suggest that specific abnormalities are present in the temporal regions of schizophrenic patients with TD [START_REF] Kircher | Reversed lateralization of temporal activation during speech production in thought disordered patients with schizophrenia[END_REF]; the temporal region is also the region implicated in the clustering (but not in the switching) process of VF. This also suggests that thought/speech abnormalities and the clustering process are linked.

We also hypothesised that similar correlations exist in the two populations studied.

Only one correlation, between the negative dimension and the number of switches in VF was similar in the two populations. This correlation was consistent with our initial hypotheses.

However, as discussed previously, in the sample of schizophrenic subjects, the negative dimension was associated with poor results in all tests, leading to significant, negative correlations with almost half of the cognitive measures used in this study. Thus, unless these findings are reproduced in future studies, the possibility that in our study similar correlations were observed by chance cannot be excluded.

The disorganisation dimension showed different correlations in the two samples. Several differences in the clinical measures used in the two groups may explain these differences. One difference already mentioned is that the SSPI explores the clinical state, whereas the SPQ explores stable traits. Another difference is that the SSPI is based on clinician's assessment although the SPQ is based on self-report. Probably, a more important difference is that impaired attention, an item that usually enters into the composition of disorganisation, is not explored by the SPQ. This may explain why the SCWT measurements did not correlate with the SPQ dimension of disorganisation. Thus, to explore whether similar correlations exist with schizotypal traits and with psychotic symptoms, greater effort must be devoted to developing parallel assessments (i.e. containing similar items) for schizophrenic and schizotypal symptoms.

Conclusion

Our study lent partial support to our initial hypothesis of specific correlations between schizophrenic dimensions and cognitive performances but did not support the existence of similar correlations in schizophrenic and non-psychotic subjects. Before our initial hypotheses can be rejected (or validated), several limitations of the present study must be addressed in further research.

The association between Clust and measures of poorer speech organisation in non-psychotic subjects was not initially hypothesised. Thus, although this result is in line with prior findings linking thought disorder and excessive automatic spreading of activation in semantic networks, it needs also to be validated in further studies.

[ 38 ]

 38 Vollema MG, Postma B. Neurocognitive correlates of schizotypy in first degree relatives of schizophrenia patients. Schizophr Bull 2002;28(3):367-77.

  s) on which the item loaded in the English version/ Dimension(s) on which the item loaded in the French version (Pos = Positive Dimension, Neg = Negative Dimension, Dis = Disorganisation, Exc = Psychomotor Excitation, Dep = Depressive Dimension)

  differences [F (2) = 12.01, P < 0.0001] b significant differences[Chi2 (2) = 6.31, P = 0.004] c significant differences [Chi2 (2) = 26.86, P < 0.0001] d Max = total number of items for the dimension = maximum score;

Table 1 .

 1 The structure of the clinical dimensions used in our study

		SSPI	SPQ
		Hallucinations	Unusual perceptual experiences
	Positive	Delusions	Magical thinking
			Ideas of reference
		Flat affect	Constricted affect
	Negative	Poverty of speech	Social anxiety
		Underactivity	No close friends
		Thought form disorder	Odd speech
	Disorganisation
		Impaired attention
	Excluded items (items	Peculiar behaviour (Exc/ Dis) a	Odd behaviour (Dis/ Pos) a
	that load on different	Inappropriate affect (Dis/ Pos) a	Paranoid ideation (Pos & Neg/ Pos) a
	dimensions in the

Table 2

 2 Clinical and demographical variables in the three groups of subjects

	Schizophrenic	Relatives	Normal Controls
	subjects

Table 3

 3 Results of the cognitive assessment in the three groups =< 0,005 (significance threshold after Bonferoni correction for 10 comparaisons)

			Schizophrenic	Relatives	Normal	F(2)
			Subjects		Controls	(P)
	Source	Total Correct	29.04 (6.81) b, d 31.68 (4.52) 32.71 (5.30) 7.36 (0.003)
	Monitoring	False Alarms	3.06 (4.10) c	2 .03 (1.98)	2.83 (2.87) 3.90 (0.03)
	[Mean (SD)]	Self Discrimination	6.13 (1.81) a	6.92 (1.14)	7.02 (1.00) 2.61 (0.09)
		External Bias	4.26 (3.70)	4.11 (3.21)	4.90 (3.88) 1.66 (0.21)
	Stroop test	Total items	34.56 (9.20) b, d 41.76 (8.57) a	47.67	21.73
	[Mean (SD)]				(10.05)	(<0.0001)
		Interference Score	0.67 (0.50) b, c	0.47 (0.19)	0.43 (0.18) 5.55 (0.02)
	Verbal	Total items	24.39 (8.58) b 29.24 (8.65) a	33.90	5.08 (0.01)
	Fluency				(10.77)
	[Mean (SD)]	Number of switches	11.28 (4.11)	12.32 (4.45) 12.31 (3.56) 0.07 (0.92)
		Number of clustered	15.02 (7.38) a 18.14 (7.51) a 22.40 (9.88) 4.47 (0.23)
		items
		compared to controls;
		c P < 0,05 compared to relatives;

a P < 0,05 compared to controls; b P d P =< 0,005 compared to relatives;

Table 4

 4 Significant partial correlations [Pearson's r (P)] between clinical and cognitive

	variables

Acknowledgements

This research was supported by grants from INSERM and "Délégation à la Recherche Clinique de l'AP-HP" (PHRC AOM 98152). A.S. and A.M. received grants from the "Fondation pour la Recherche Médicale". We thank M.J. Pereira-Gomes and E. Abadie for technical assistance.


Ralph-Olivier Moussodia

Samir Acherar

Andrea Bordessa

Régis Vanderesse

Brigitte Jamart-Grégoire
email: brigitte.jamart@ensic.inpl-nancy.fr

An expedient and short synthesis of chiral -hydrazinoesters

Keywords: Pseudopeptide, -Hydrazinoester, Hydrazinopeptide, 1:1 [/-N  -hydrazino]mers, S N 2, hydrazinoturn

Different -hydrazinoesters with high optical purity have been obtained in large scale via a S N 2 protocol. A coupling reaction with a natural aminoacid leads to the corresponding dimers which have been oligomerized in order to obtain the 1:1 [/-N  -hydrazino]mer series.. Conformational studies show that these mixed oligomers are self-organized in solution via a succession of -turn and hydrazinoturn whatever the absolute configuration of the chiral carbons.

Introduction

For several years now, important efforts have been devoted to improve the pharmacological properties of natural peptides by a structure modification of the peptide bond CONH group. 1 Among the numerous possibilities, the replacement of one (or more) peptidic bond(s) by one (or more) hydrazidic bond(s) leads to hydrazinopeptides. 2 Hydrazinopeptides represent a valuable class of mimetic replacements for the natural peptides backbone often leading to analogous of bioactive peptides which preserve biological activity 3 and enhanced proteolytic stability. 4 Furthermore, this additional backbone nitrogen might behave as a hydrogen bond acceptor able to stabilize secondary structure in oligomers. 4,5 In 2003, Lelais and Seebach succeeded in building oligomers of optically pure -hydrazinoacids 4 units.

Unfortunately, due to poor resonance dispersion and fast exchange between the NH signals, the NMR structural analyses performed on these kinds of oligomers didn't yield definitive conclusion on the conformation. However, X-Ray diffraction of the starting -hydrazinoacid (compound 4a) showed that the N  -atom adopts a pyramidal conformation with an (S)-configuration and participates in the stabilization of a hydrazinoturn.

More recent works have shown that mixed oligomers alternating a natural amino acid and an amino acid analogs were also able to fold in solution leading to the formation of a new kind of foldamers. 6 In this paper, we describe the synthesis and the conformational analysis of two series of 1:1 [/-N  -hydrazino]mers alternating natural -amino acid units and -hydrazino acid ones.

In our preliminary work, 7 we demonstrated that bis-nitrogen containing amino acid analogs such as N  -Me, N  -Boc-protected -hydrazinoesters derivatives 3a could be easily synthesized in large scale with high optical purity via a S N 2 reaction between tertbutyl-2-methylhydrazinecarboxylate 1a and the triflate derivative of (S)-hydroxyesters 2 (Scheme 1). In this paper, we decided to extend this protocol in order to obtain new -hydrazinoesters 3 bearing an alkyl or a benzyl group (Bn, i-Bu and i-Pr) on the N  atom, and to study the impact of their presence on the yield of the S N 2 reaction (Scheme 1). In a second part, we will describe the oligomerization of these new building blocks leading to a series of 1:1 [/-N  -hydrazino]mers. The conformational analysis of homo (R,R) and hetero (S,R) series were investigated in order to assess the impact of the chirality on the conformation.

Results and Discussion

Synthesis of -hydrazinoesters 3 and 1:1 [/-N  -hydrazino]mers

-hydrazinoesters 3 were prepared by using the same protocol as described in a previous work. 7 The results summarized in Table 1, indicate that the S N 2 reaction also gives good yields (58-87%) when R corresponds to Me, Bn and i-Bu groups. -hydrazinoesters 3aa to 3dc can be obtained on a multigram scale in only one reaction.

Conversely, we showed that this reaction was very sensitive to the steric hindrance of 2.

As a result, compounds 3ad to 3bd (R = i-Pr) were obtained with very poor yields and no reaction occurs between 1d and 2d. As a consequence, compounds 3ad and 3bd cannot be isolated in a pure form but are mixed with 2d even after purification by flash chromatography.

On the contrary the comparison of the yields obtained for the synthesis of compounds 3aa to 3da, 3bb to 3db and 3ac to 3dc shows that the steric hindrance of R' has little influence on the yield of the reaction. Compounds 3ba and 3bc were then involved in an oligomerization process in order to obtain the hetero (S,R) and homo (R,R) 1:1 [/-N  -hydrazino]mer series (Scheme

2).

Heterochiral compounds 6a-b, 9a-b, 11a-b and 13a-b were obtained in very good yields ranging from 77 to 96% by an iterative sequence of deprotection and coupling reactions as described in Scheme 2. The saponification of the ester group of 3ba and 3bc led to corresponding free C-terminal compounds 4a and 4b respectively. The use of thionyl chloride in methanol 8 led simultaneously to the deprotection of hydrazine group and to the esterification of carboxylic to give compounds 5a and 5b in quantitative yields directly.

Various conditions (EDC, HOBt, HATU, HBTU…) were used in order to perform the coupling reaction between 5a and Boc-L-Leu-OH and between 5b and Boc-L-Ala-OH. The best results were obtained when using HATU and NMM which generate dimers 6a and 6b with excellent yields. The same protocol was also used for the synthesis of tetramers 9a-b, hexamers 11a-b and octamers 13a-b.

When applied to 5a and Boc-D-Leu-OH, these conditions led to the corresponding homochiral compounds 6'a and 9'a. Compounds 6a, 9a, 6'a and 9'a can easily be transformed into corresponding C-terminal amide compounds 14a, 15a, 14'a, 15'a which will be used for the conformational analysis.

It is interesting to notice that both 13

(R) (R) (R) (R) (R) (R) 8a (R = Me, R 1 = i-Bu), 100% N H N OMe O O BocHN i-Bu Bn Me N H N OMe O O TFA.H 2 N i-Bu Bn Me (R) (R) N H N OH O O BocHN i-Bu Bn Me (R) N H N OMe O O H N i-Bu Bn Me Boc (R) (S) N H N NH 2 O O BocHN i-Bu Bn Me N H N OMe O O TFA.H 2 N R 1 Bn R N H N OMe O O BocHN R 1 Bn R (S) (S) (S) N H N OMe O O H N R 1 Bn R Boc N H N OH O O BocHN R 1 Bn R (S) (S)
(R) N H N NH 2 O O BocHN i-Bu Bn Me NH 4 OH MeOH (R) (R) Bn BocHN N OH O R MeOH 4a (R = Me), 98% (R) Bn HCl.H 2 N N OMe O R 5a (R = Me), 99% (R) Bn BocHN N OMe O R 3ba (R = Me) 14a 6a (R = Me, R 1 = i-Bu), 96% NH4OH MeOH NH 4 OH MeOH NH 4 OH MeOH (R) 13a (R = Me, R 1 = i-Bu), 77% 7a (R = Me, R1 = i-Bu), 96% N H N OMe O O H N R 1 Bn R Boc (S) 9a (R = Me, R 1 = i-Bu), 87% 11a (R = Me, R 1 = i-Bu), 83% SOCl 2 (2.1 equiv.) NaOH 1N (2.0 equiv.) MeOH / H 2 O (2:1) 3bc (R = i-Bu) 4b (R = i-Bu), 96% 5b (R = i-Bu), 99% 6b (R = i-Bu, R 1 = Me), 94% 7b (R = i-Bu, R1 = Me), 95% TFA, CH 2 Cl 2 8b (R = i-Bu, R 1 = Me), 100% 9b (R = i-Bu, R 1 = Me), 90%
HATU (1.0 equiv.) NMM (4.0 equiv.) -NaOH 1N (2.0 equiv.) (94-96%)

Couplages (77-83%)

...

Deprotections

-TFA (100%) IR and NMR experiments were carried out in order to determine the ability of these oligomers to fold in solution. Then we compared the conformation of homo (R,R) and hetero (S,R) dimers 14a and 14'a and of tetramers 15a and 15'a.

11b (R = i-Bu, R 1 = Me), 82% n n = 3 13b (R = i-Bu, R 1 = Me), 79% n =

Conformational analysis of homo and hetero oligomer series

Conformational analysis of homo and hetero dimers 14a and 14'a

Le Grel et al. 9 demonstrated that chemical shifts difference , calculated from the 1 H NMR signals of the two C-terminal amidic protons, can give information on the structuration of aza- 3 and amidoxy derivatives (Figure 1). 9,10 We decided to use this parameter to obtain information on the conformation of 14a and 14'a. -The signals of the two amidic NH protons respectively at  = 7.98 ppm and  = 5.24 ppm allowed us to calculate a  = 2.74 ppm for hetero dimer 14a.

-Similarly, we assigned the two amidic NH protons of homo dimer 14'a, at  = 7.99 ppm and  = 5.40 ppm respectively which accounts for a  = 2.59 ppm.

Theses values were in the same order as those obtained by P. Le Grel in the aza- 3 series which is known to fold via an eight-membered H-bonded ring (hydrazinoturn).

To confirm this result, we decided to study the effect of varying ratios of solvent in the mixture of CDCl 3 and DMSO-d 6 on the chemical shift of the NH protons of compounds 14a and 14'a (Figure 2a). Solvents with hydrogen bond acceptor atoms, such as DMSO-d 6 , are able to form intermolecular H-bonds producing a downfield displacement for free acidic protons which is less significant when protons are involved in an intramolecular H-bond. -We observed that the chemical shift of both NHc and NHd (amidic and NHBoc) of hetero dimer 14a shifted downfield when the volume of DMSO-d 6 increased (DMSO-d -CDCl3 = + 1.82 ppm and + 2.26 ppm respectively) confirming that the NH protons were not involved in an intramolecular H-bond (free) (Figure 2a).

-A downfield shift was also observed for NHb. However, the difference between DMSO-d and CDCl3 was lower (DMSO-d -CDCl3 = + 1.58 ppm) and suggested that the hydrazidic proton was free or was involved in a weak intramolecular Hbond. If we take into account the results obtained by Le Grel and al. for the aza- 3 -oligomers (Figure 3), this weak H-bond may well be the result of the presence of a -turn. 5b,11

-Finally, and more interestingly, a very low variation of the chemical shift was observed for the NHa amidic proton when switching from CDCl 3 to DMSO-d 6

(DMSO-d -CDCl3 = -0.15 ppm) confirming the presence of a strong H-bond.

This result is in agreement with the proposed hydrazinoturn structuration (bifurcated H-bond, Figure 3).

It is important to note that same results were obtained for both hetero and homo dimers 14a and 14'a. This showed that the structuration was not dependent on the chirality of the -carbon and that both series fold via a succession of seven-(-turns)

and eight-membered H-bonded rings (hydrazinoturns).

Conformational analysis of homo and hetero tetramers 15a and 15'a

1 H-NMR signals of tetramers 15a and 15'a were fully assigned using a combination of 2D-COSY and 2D-ROESY experiments (see Figures S5-S8 in Supporting Information). As observed in the dimer series, the variation of the chemical shift of all NH protons (Figure 2b) was quite similar for homo tetramer 15'a and hetero tetramer 15a which suggested an identical self-structuration for both series.

-The chemical shifts of the two amidic NH protons of hetero tetramer 15a, were respectively at  = 8.12 ppm and  = 5.37 ppm, given a  = 2.75 ppm).

-Similarly, a  = 2.58 ppm was calculated for the homo tetramer 15'a ( = 7.84

Conclusion

Various -hydrazinoesters 3aa to 3dc building blocks can be obtained with high optical purity using a general protocol. Starting from 3ba and 3bc, we were able to obtain the synthesis of two 1:1 [/-N  -hydrazino]mer series. Conformational studies by 1 H NMR show that 1:1 [/-N  -hydrazino] mers are self-organized in solution via a succession of -turn and hydrazinoturn whatever the absolute configuration of the chiral carbons.

Experimental

General

Unless otherwise stated, all chemicals were purchased as the highest purity commercially available and were used without further purification. Reactions were monitored by thin-layer chromatography (TLC) using aluminium-backed silica gel plates (Macherey-Nagel ALUGRAM ® SIL G/UV254). TLC spots were viewed under ultraviolet light and by heating the plate after treatment with a staining solution of phosphomolybdic acid. Product purifications were performed using Geduran 60 H Silica Gel (63-200 mesh). Dichloromethane was distilled from phosphorous pentoxide under argon atmosphere and others reagent grade solvents were used as received. 1 H and 13 C NMR spectra were recorded on a Bruker Advance 300 spectrometer.

Multiplicities are reported as follow: s = singlet, d = doublet, q = quartet, sept = septuplet, m = multiplet, br = broad, Ar. = aromatic and coupling constants (J) are given in Hertz (Hz). IR spectra were recorded on a Brucker Tensor 27. Melting points (mp) were recorded on a Kofler hot-stage apparatus and were uncorrected. Electron spray ionization mass spectra (ESI-MS) were recorded on a Brucker MicroTof-Q HR spectrometer in the "Service commun de Spectrométrie de Masse", Faculté des Sciences et Techniques, Vandoeuvre-lès-Nancy, France. Optical rotations were determined on an Anton Paar MCP 300 polarimeter in 5 cm cells. Tert-butyl-hydrazinecarboxylate derivatives 1a-d and (S)--hydroxyesters 2a-d were prepared according to ref [12] and [13] respectively.

Representative procedure for the preparation of -hydrazinoesters 3

A solution of (S)--hydroxyester 2 (1.0 equiv., 30 mmol) and 2,6-lutidine (2.3 equiv., 69 mmol) in CH 2 Cl 2 (40 mL) at 0°C, under an atmosphere of nitrogen, was treated with triflic anhydride (1.1 equiv., 33 mmol). After 15 min, a solution of BocNHNHR' 1 (1.0 equiv., 30 mmol) in CH 2 Cl 2 (30 mL) was added dropwise for 30 min and the mixture was stirred for 4-6 h at 0°C until completion (monitored by TLC). After evaporation of the solvent, Et 2 O (50 mL) was added and 2,6-lutidinium triflate was precipitated by storing the reaction mixture overnight in the refrigerator. The 2,6-lutidinium triflate was filtered and the filtrate was diluted in Et 2 O (100 mL) and washed with water (20 mL), dried over MgSO 4 , filtered, and evaporated in vacuo. The resulting crude material was purified and characterized as reported below. 3362, 2982, 2857, 2908, 2873, 2843, 1740, 1734, 1710, 1690, 1678, 1659, 1562, 1510, 1483, 1469, 1462, 1452, 1412, 1389, 1380, 1368, 1238, 1200, 1160, 1124 3371, 2982, 2944, 2908, 2881, 2843, 1745, 1728, 1711, 1692, 1683, 1498, 1490, 1486, 1471, 1460, 1452, 1391, 1383, 1366, 1332, 1275, 1246, 1198, 1161, 1128 Leu, 2 γCH Leu), 3.47 (q, 1H, J = 6.9 Hz, αCH Ala),

(R)-(-)-N α -methyl-N β -Boc-(D)-hydrazinoalanine methyl ester or Boc-h(Me

(R)-(-)-N α -isobutyl-N β -Boc-(D)-hydrazinoalanine methyl ester or Boc-h(i-Bu

(R)-(-)-N α -isopropyl-N β -Boc-(D)-hydrazinolalanine methyl ester or Boc-h(i-Pr

Representative procedure for the amidation reaction with NH 4 OH/MeOH

Ammonium hydroxide (10 mL, 25% aq.) was added to a stirred solution of the appropriate oligomer ester (0.6 mmol) in MeOH (10 mL). The reaction flask was sealed with a rubber septum and stirred vigorously at room temperature for 2 days. The reaction mixture was then concentrated, neutralized with HCl 1N until pH=7, extracted with AcOEt (3 x 20 mL) and evaporated in vacuo. The resulting crude material was purified and characterized as reported below.

Boc-L-Leu--h(Bn)D-Ala-NH 2 , 14a

aScheme 1 .

 1 Scheme 1. Synthesis of -hydrazinoesters derivatives 3.

Scheme 2 .

 2 Scheme 2. Synthesis of hetero (S,R) and homo (R,R) 1:1 [/-N  -hydrazino]mers.

Figure 1 .

 1 Figure 1. Comparison of  values between hydrazidic and amidoxy links by 1 H NMR (300 MHz, CDCl 3 /10 mM).

Figure 2 .

 2 Figure 2. Effects of varying ratios of solvent in the mixture of CDCl 3 and DMSO-d 6 on the chemical shift of the NH protons of a) hetero dimer 14a and (homo dimer 14'a) and of b) hetero tetramer 15a (and homo tetramer 15'a) in 1 H NMR (300 MHz, 10 mM).

Figure 3 .

 3 Figure 3. Postulated structuration of 14a (and 14'a) in CDCl 3 solution.

(Figure 4 .

 4 Figure 4. Postulated structuration of 15a (and 15'a) in CDCl 3 solution.

  )D-Ala-OMe, 3aa. Purified by flash chromatography (Petroleum ether/AcOEt = 50:50), Yield: 87%, yellow oil, [] D 25 = -9.1 (c=1.00, MeOH); IR (ATR)  max /cm -

4. 2 . 2 .

 22 (R)-(-)-N α -benzyl-N β -Boc-(D)-hydrazinoalanine methyl ester or Boc-h(Bn)D-Ala-OMe, 3ba. Purified by flash chromatography (Petroleum ether/AcOEt = 80:20), Yield: 83%, yellow oil, [] D 25 = -9.2 (c=1.00, MeOH); IR (ATR)  max /cm -

  )D-Ala-OMe, 3ca. Purified by flash chromatography (Petroleum ether/AcOEt = 70:30), Yield: 80%, yellow oil, [] D 25 = -20.9 (c=1.00, MeOH); IR (ATR)  max /cm -1 :

  ;1 H NMR (300 MHz, CDCl 3 ) δ 0.9 (d, 3H, J = 6.5 Hz, CH 3 , N α -iBu), 0.97 (d, 3H, J = 6.5 Hz, CH 3 ,N α -iBu), 1.37 (d, 3H, J = 7.2 Hz, βCH 3 Ala), 1.44 (s, 9H, Boc), 1.62-1.81 (m, 1H, CH, N α -iBu), 2.20-2.45 (m, 1H, CH 2 , N α -iBu), 2.55-2.77 (m, 1H, CH 2 , N α -iBu),3.48-3.65 (m, 1H, αCH Ala), 3.71 (s, 3H, O-CH 3 ), 6.33 (br s, 1H, NH); 13 C NMR (75 MHz, CDCl 3 ) δ 16.7 (βCH 3 Ala), 21.7 (2 CH 3 , N α -iBu), 27.2 (CH, N α -iBu), 28.9 (3 CH 3 , Boc), 52.0 (O-CH 3 ), 63.4 (αCH Ala); 65.3 (CH 2 , N α -iBu), 80.0 (C, Boc), 156.3 (C=O, Boc), 175.2 (C=O, ester); HRMS (ESI) calculated for C 13 H 26 N 2 O 4 [M+Na] + m/z 297.1791, found 297.1785.

  )D-Ala-OMe, 3da. Purified by flash chromatography (Petroleum ether/AcOEt = 70:30), Yield: 62%, yellow oil, [] D 25 = -28.2 (c=1.00, MeOH); IR (ATR)  max /cm -1 :

  ; 1 H NMR (300 MHz, CDCl 3 ) δ 1.08 (d, 6H, J = 6.2 Hz, CH 3 , N α -iPr), 1.37 (d, 3H, J = 7.1 Hz, βCH 3 Ala), 1.44 (s, 9H, Boc), 3.03 (sept, 1H, J = 6.2 Hz, CH, N α -iPr), 3.70 (s, 3H, O-CH 3 ), 3.82 (q, 1H, J = 7.1 Hz, αCH Ala), 6.47 (br s, 1H, NH); 13 C NMR (75 MHz, CDCl 3 ) δ 17.1 (βCH 3 Ala), 20.9 (CH 3 , N α -iPr), 21.1 (CH 3 , N α -iPr), 28.9 (3 CH 3 , Boc), 52.1 (O-CH 3 ), 56.0 (CH, N α -iPr), 58.7 (αCH Ala), 79.9 (C, Boc), 157.0 (C=O, Boc), 175.9 (C=O, ester); HRMS (ESI) calculated for C 12 H 24 N 2 O 4 [M+Na] + m/z 283.1634, found 283.1628. 4.2.5. (R)-(-)-N α -methyl-N β -Boc-(D)-hydrazinophenylalanine methyl ester or Boc--h(Me)D-Phe-OMe, 3ab. Purified by flash chromatography (Petroleum ether/AcOEt = 70:30), Yield: 84%, yellow oil, [] D 25 = -37.0 (c=1.00, MeOH); IR (ATR)  max /cm -

4. 2 . 6 .

 26 (R)-(-)-N α -benzyl-N β -Boc-(D)-hydrazinophenylalanine methyl ester or Boc--h(Bn)D-Phe-OMe, 3bb. Purified by flash chromatography (Petroleum ether/AcOEt = 80:20), Yield: 82%, yellow oil, [] D 25 = -5.2 (c=1.00, MeOH); IR (ATR)  max /cm -

4. 6 . 4 . 6 . 1 .

 6461 Representative procedure for the coupling reaction with HATU/NMMThe appropriate trifluoroacetate or hydrochloride salt (1.0 equiv., 9 mmol) was dissolved in CH 2 Cl 2 (10 mL) and cooled to 0°C. This solution was treated successively with NMM (4.0 equiv., 36 mmol), a solution of the Boc-protected fragment (1.0 equiv., 9 mmol) in CH 2 Cl 2 (5 mL), and a solution of HATU (1.0 equiv., 9 mmol) in a minimum amount of DMF. The mixture was allowed to warm to room temperature and then stirred until TLC indicated complete reaction. Subsequent addition of CH 2 Cl 2 was followed by washing with HCl 1N (1 x 25 mL), saturated aqueous NaHCO 3 solution (1x 25 mL), and saturated aqueous NaCl solution (2 x 25 mL). The organic layer was dried over MgSO 4 and concentrated under reduced pressure. The resulting crude material was purified and characterized as reported below. Boc-L-Leu--h(Bn)D-Ala-OMe dimer, 6a. Purified by flash chromatography (Petroleum ether/AcOEt = 70:30), Yield: 96%, white powder: mp = 112 °C; IR (ATR) max /cm -

Table 1 .

 1 Synthesis of -hydrazinoesters 3 performed on a 30 mmol scale.

		O
	HO	(S)
		OMe
		R

2 O (1.1 equiv.), 2,6-lutidine (2.3 equiv.), 0°C, 15 min 2-BocNHNHR' 1 (1.0 equiv.), 0°C, 4 hr, CH 2 Cl 2 (i): Yields calculated after purification by flash chromatography, (ii): Yields determined by 1 H NMR integration relative peaks after purification by flash chromatography, (iii): No reaction after 3 days.

  N α -Me), 3.57-3.64 (q, 1H, J = 7.2 Hz, αCH Ala), 3.72 (s, 3H, O-CH 3 ) 6.52 (br s, 1H, NH);13 C NMR (75 MHz, CDCl 3 ) δ 16.6 (βCH 3 Ala), 28.8 (3 CH 3 , Boc), 44.0 (N α -Me), 52.0 (O-CH 3 ), 63.1 (αCH Ala), 80.2 (C, Boc), 155.9 (C=O, Boc), 174.6 (C=O, ester); HRMS (ESI) calculated for C 10 H 20 N 2 O 4 [M+Na] + m/z 255.1321, found 255.1315.

	1 :
	3362, 3005, 2982, 2955, 2890, 2843, 2799, 1746, 1728, 1689, 1658, 1641, 1630, 1564,
	1553, 1513, 1483, 1462, 1450, 1414, 1393, 1368, 1242, 1210, 1163, 1130, 1111; 1 H
	NMR (300 MHz, CDCl 3 ) δ 1.37 (d, 3H, J = 7.2 Hz, βCH 3 Ala), 1.45 (s, 9H, Boc), 2.67
	(s, 3H,

  CDCl 3 ) δ 1.28 (s, 9H, Boc), 1.31(d, 3H, J = 7.2 Hz, βCH 3 Ala), 3.56 (q, 1H, J = 7.2 Hz, αCH Ala), 3.66 (s, 3H, O-CH 3 ) 3.82-4.01 (m, 2H, CH 2 , N α -Bn), 6.48 (br s, 1H, NH), 7.13-7.36 (m, 5H, Ar.);13 C NMR (75 MHz, CDCl 3 ) δ 16.9 (βCH 3 Ala), 28.9 (3 CH 3 , Boc), 52.2 (O-CH 3 ), 60.8 (αCH Ala), 61.3 (CH 2 , N α -Bn), 80.3 (C, Boc), 128.1 (CH, Ar.), 128.9 (2 CH, Ar.), 130.0 (2 CH, Ar.), 137.6 (C, Ar.), 156.1 (C=O, Boc), 175.2 (C=O, ester); HRMS (ESI) calculated for C 16 H 24 N 2 O 4 [M+Na] + m/z 331.1634,

	1 :
	3370, 3091, 3064, 3032, 2984, 2955, 2907, 2846, 1737, 1728, 1709, 1659, 1641, 1630,
	1564, 1494, 1486, 1462, 1456, 1435, 1424, 1393, 1367, 1237, 1213, 1159; 1 H NMR
	(300 MHz, found 331.1628.

  Yield: 95%, white amorphous solid; 1 H NMR (300 MHz, CDCl 3 ) δ 0.73 (d, 3H, J = 6.0 Hz, δCH 3 Leu), 0.85 (d, 3H, J = 6.0 Hz, H NMR (300 MHz, CDCl 3 ) δ 0.79 (d, 3H, J = 6.3 Hz, δCH 3 Leu), 0.83 (d, 6H, J = 6.3 Hz, 2 δCH 3 Leu), 0.87 (d, 3H, J = 6.3 Hz, δCH 3 Leu), 1.27 (d, 3H, J = 6.9 Hz, βCH 3 Ala), 1.37 (d, 3H, J = 6.9 Hz, βCH 3 Ala), 1.45 (s, 9H, Boc), 1.49-1.68 (m, 6H, 2 βCH 2

	δCH 3 Leu), 1.25 (d, 3H, J = 7.2 Hz, βCH 3 Ala), 1.40 (s, 9H, Boc), 1.40-1.52 (m, 1H,
	βCH 2 Leu), 1.60-1.71 (m, 1H, βCH 2 Leu), 1.96-2.06 (m, 1H, γCH Leu), 3.48-3.57 (m,
	1H, αCH Leu), 3.97-4.16 (m, 2H, CH 2 , N α -Bn), 4.31-4.47 (m, 1H, αCH Ala), 5.69 (d,
	1H, J = 7.2 Hz, NHBoc), 7.22-7.43 (m, 5H, Ar.), 8.39 (br s, 1H, NH hydrazidic), 8.62
	(s, 1H, OH). The crude product was used in a next step without further purification.
	4.3.5. Boc-(L-Leu--h(Bn)D-Ala) 2 -OH, 10a. Yield: 94%, white amorphous solid;

1 : 3362, 3091, 3065, 3031, 2980, 2958, 2907, 2842, 1741, 1726, 1707, 1661, 1626, 1605, 1587, 1496, 1482, 1454, 1389, 1367, 1241, 1217, 1157; 1 H NMR

4.3.4. Boc-L-Ala--h(Bn)D-Leu-OH, 7b.

1

Representative procedure for the preparation of compounds 5a and 5b

  , 5H, 2 CH 2 , N α -Bn, αCH Leu), 4.08-4.21 (m, 1H, αCH Leu), 5.31 (br s, 1H,

	4.4. To a solution of Boc-acid 4a or 4b (1.0 equiv., 22 mmol) in MeOH (50 mL) was
	added SOCl 2 (2.1 equiv., 46.2 mmol) dropwise at 0°C and the mixture was stirred for 48
	h at room temperature until completion (monitored by TLC). Evaporation of MeOH in
	vacuo gives the corresponding chlorhydrate ester 5a or 5b as a white amorphous solid
	in 99% yield. Compounds 5a and 5b were engaged immediately in the next step.
	4.5. Representative procedure for the N-terminal deprotection with TFA
	Boc-ester compound (1.0 equiv., 9 mmol) was treated with a mixture of CH 2 Cl 2 /
	TFA (9 mL / 6 mL) and stirred for 3 h at room temperature. Excess of TFA was co-
	evaporated with MeOH and Et 2 O until to obtain the corresponding trifluoroacetate salt
	compound as a white amorphous solid in quantitative yield. Compounds 8a, 8b, 12a,
	12b and 8'a were engaged immediately in the next step.
	NHBoc), 7.22-7.42 (m, 10H, Ar.), 7.82 (s, 1H, NH), 8.23 (s, 1H, NH), 8.52 (br s, 1H,
	NH). The crude product was used in a next step without further purification.
	4.3.6. Boc-(L-Ala--h(Bn)D-Leu) 2 -OH, 10b. Yield: 96%, white amorphous solid;
	1 H NMR of the major form (300 MHz, CDCl 3 ) δ 0.72-1.15 (m, 18H, 4 δCH 3 Leu, 2
	δCH 3 Leu,), 1.24-1.99 (m, 6H, 2 βCH 2 Leu, 2 γCH Leu), 1.39 (s, 9H, Boc), 3.42-3.63
	(m, 2H, 2 αCH Leu), 3.67-3.81 (m, 4H, 2 CH 2 , N α -Bn), 3.88-4.23 (m, 2H, 2 αCH Ala),
	4.94 (br s, J = 5.7 Hz, NHBoc), 7.15-7.51 (m, 10H, Ar.), 8.02 (br s, 1H, NH), 8.12 (s,
	1H, NH), 8.56 (s, 1H, NH), 9.17 (br s, 1H, OH). The crude product was used in a next
	step without further purification.
	4.3.7. Boc-D-Leu--h(Bn)D-Ala-OH, 7'a. Yield: 82%, white amorphous solid; 1 H
	NMR (300 MHz, CDCl 3 ) δ 0.84 (d, 3H, J = 6.0 Hz, δCH 3 Leu), 0.86 (d, 3H, J = 6.0 Hz,
	δCH 3 Leu), 1.20-1.50 (m, 6H, βCH 3 Ala, βCH 2 Leu, γCH Leu), 1.43 (s, 9H, Boc), 3.65-
	3.80 (m, 1H, αCH Ala), 4.06 (s, 2H, CH 2 , N α -Bn), 4.10-4.25 (m, 1H, αCH Leu), 4.93
	(m, 1H, NHBoc), 7.20-7.50 (m, 5H, Ar.), 8.14 (br s, 1H, NH hydrazidic). The crude
	product was used in a next step without further purification.

  Hz, 2 δCH 3 Leu),1.38 (d, 3H, J = 7.2 Hz, βCH 3 Ala), 1.43 (s, 9H, Boc), 1.46-1.79 (m, 3H, βCH2 Leu, γCH Leu), 3.63-3.78 (m, 1H, αCH Ala), 3.73 (s, 3H, OCH 3 ), 3.88-4.09 (m, 3H, CH 2 , N α -Bn, αCH Leu), 4.77 (br d, J = 6.0 Hz, NHBoc), 7.23-7.43 (m, 5H, Ar.), 7.98 (s, 1H, NMR of the major form (300 MHz, CDCl 3 ) δ 0.81 (d, 3H, J = 6.6 Hz, δCH 3 Leu), 0.83 (d, 3H, J = 6.6 Hz, δCH 3 Leu), 0.85 (d, 3H, J = 6.6 Hz, δCH 3 Leu), 0.90 (d, 3H, J = 6.6 Hz, δCH 3 Leu), 1.29 (d, 3H, J = 7.2 Hz, βCH 3 Ala), (βCH 2 Leu), 48.3 (αCH Ala), 52.3 (O-CH 3 ), 61.0 (CH 2 , N α -Bn), 61.3 (αCH Leu), 80.4 (C, Boc), 128.3 (CH, Ar.), 128.9 (2 CH, Ar.), 129.9 (2 CH, Ar.), 137.1 (C, Ar.), 156.2 (C=O, Boc), 172. (C=O, hydrazid), 175.1 (C=O, ester); HRMS (ESI) calculated for C 22 H 35 N 3 O 5 [M+H] + m/z 422.2655, found 422.2649. Purified by flash chromatography (Petroleum ether/AcOEt = 20:80), Yield: 75%, white powder: mp = 143 °C; IR (ATR)  max /cm -1 : 3440, 3348, 3311, 3234, 3089, 3065, 2961, 2937, 2872, 2855, 1740, 1728, 1697, 1660, 1655, 1548, 1551, 1520, 1498, 1470, 1457, 1438, 1391, 1369, 1318, 1293, 1259, 1162; 1 H NMR of the major form (300 MHz, CDCl 3 ) δ 0.67-0.91 (m, 12H, 4 δCH 3 Leu), 1.13-1.49 (m, 12H, 2 βCH 3 Ala, 2 βCH 2 Leu, 2 γCH Leu), 1.40 (s, 9H, Boc), 3.43-3.62 (m, 1H, αCH Ala), 3.65-3.90 (m, 3H, αCH Ala, CH 2 , N α -Bn), 3.74 (s, 3H, OCH 3 ), 3.90-4.11 (m, 3H, CH 2 , N α -Bn, αCH Leu), 4.12-4.27 (m, 1H, αCH Leu), 4.82 (m, 1H, NHBoc), 7.08-7.47 (m, 10H Ar.), 7.57 (br s, 1H, NH), 7.75 (br s, 1H, NH), 8.30 (br s, 1H, NH); 13 C NMR (75 MHz, CDCl 3 ) δ 17.2 (βCH 3 Ala), 17.6 (βCH 3 Ala), 22.4 (γCH Leu), 23.0 (γCH Leu), 23.3 (δCH 3 Leu), 23.4 (δCH 3 Leu), 24.8 (δCH 3 Leu), 25.4 (δCH 3 Leu), 29.0 (3 CH 3 , Boc), 40.6 (βCH 2 Leu), 40.7 (βCH 2 Leu), 52.3 (αCH Ala), 52.4 (αCH Ala), 52.8 (O-CH 3 ), 60.9 (CH 2 , N α -Bn), 61.1 (αCH Leu), 61.1 (CH 2 , N α -Bn), 64.7 (αCH Leu), 80.6 (C, Boc), 128.2 (CH, Ar.), 128.3 (CH, Ar.), 128.8 (2 CH, Ar.), 128.9 (2 CH, Ar.), 129.8 (2 CH, Ar.), 130.1 (2 CH, Ar.), 137.1 (C, Ar.), 137.3 (C, Ar.), 156.6 (C=O, Boc), 172.4 (3 C(NH)=O), 175.4 (C=O, ester); HRMS (ESI) calculated for C 38 H 58 N 6 O 7 [M+H] + m/z 711.4445, found 711.4440.

	1 : 3322, 3217, 3072, 3037, 2983, 2958, 2938, 2873, 2853, 1754, 1738, 1707, 1681, 1671, 1612, 1558, 1523, 1501, 1457, 1439, 1392, 1368, 1319, 1297, 1271, 1247, (δCH 3 Leu), 25.4 (δCH 3 Leu), 29.0 (3 CH 3 , Boc), 41.9 (βCH 2 Leu), 48.2 (αCH Ala), 52.3 (O-CH 3 ), 60.7 (αCH Leu), 60.8 (CH 2 , N α -Bn), 80.6 (C, Boc), 128.3 (CH, Ar.), 129.0 (2 CH, Ar.), 130.0 (2 CH, Ar.), 137.0 (C, Ar.), 156.0 (C=O, Boc), 171.8 (C=O, hydrazid), 175.3 (C=O, ester); HRMS (ESI) calculated for C 22 H 35 N 3 O 5 [M+H] + m/z 422.2655, found 422.2649. 4.6.2. Boc-L-Ala--h(Bn)D-Leu-OMe dimer, 6b. Purified by flash chromatography (Petroleum ether/AcOEt = 80:20), Yield: 94%, white powder: mp = 68 °C; IR (ATR)  max /cm -1 : 3338, 3280, 3068, 3031, 2977, 2963, 2939, 2909, 2869, 1704, 1688, 1509, 1500, 1456, 1394, 1371, 1289, 1249, 1205, 1165; Leu), 3.45-3.54 (m, 1H, αCH Leu), 3.72 (s, 3H, OCH 3 ), 3.85-4.12 (m, 3H, CH 2 , N α -Bn, αCH Ala), 4.98 (br s, NHBoc), 7.23-7.45 (m, 5H, Ar.), 8.08 (br s, 1H, NH hydrazidic); 13 C NMR (75 MHz, CDCl 3 ) δ 19.5 (βCH 3 Ala), 21.9 (γCH Leu), 23.7 (δCH 3 Leu), 24.7 (δCH 3 Leu), 29.0 (3 CH 3 , Boc), 39.9 (βCH 2 Leu), 49.9 (αCH Ala), 52.2 (O-CH 3 ), 61.2 (CH 2 , N α -Bn), 62.5 (αCH Leu), 80.4 (C, Boc), 128.3 (CH, Ar.), 128.9 (2 CH, Ar.), 130.0 (2 CH, Ar.), 137.0 (C, Ar.), 155.8 (C=O, Boc), 171.5 (C=O, hydrazid), 175.5 (C=O, ester); HRMS (ESI) calculated for C 22 H 35 N 3 O 5 [M+Na] + m/z 444.2475, found 444.2469. 4.6.3. Boc-(L-Leu--h(Bn)D-Ala) 2 -OMe tetramer, 9a. Purified by flash chromatography (Petroleum ether/AcOEt = 20:80), Yield: 87%, white powder: mp = 125 °C; IR (ATR)  max /cm -1 : 3352, 3247, 3066, 3033, 2958, 2940, 2871, 1740, 1729, 1723, 1711, 1691, 1680, 1674, 1657, 1561, 1513, 1499, 1467, 1457, 1388, 1368, 1321, 1198, 1172, 1125; NH hydrazidic); 13 C NMR (75 MHz, CDCl 3 ) δ 17.0 (βCH 3 Ala), 22.3 (γCH Leu), 23.4 1293, 1253, 1214, 1168, 1125; 1 H

1

H NMR (300 MHz, CDCl 3 ) δ 0.86 (d, 6H, J = 6.6 1 H NMR (300 MHz, CDCl 3 ) δ 0.72 (d, 3H, J = 6.0 Hz, δCH 3 Leu), 0.86 (d, 3H, J = 6.0 Hz, δCH 3 Leu), 1.24 (d, 3H, J = 7.2 Hz, βCH 3 Ala), 1.44 (s, 9H, Boc), 1.60-1.73 (m, 2H, βCH 2 Leu), 1.88-2.03 (m, 1H, γCH 4.6.10. Boc-(D-Leu--h(Bn)D-Ala) 2 -OMe tetramer, 9'a.

Acknowlegments

This work was supported by the "Ministère de l'Enseignement Supérieur et de la Recherche" with a Doctoral fellowship for R.-O. Moussodia, the "Conseil Régional de Lorraine" and the "Institut National Polytechnique de Lorraine" with a Postdoctoral fellowship for Dr A. Bordessa.