N-Pyrrolidine-based α/β-peptides incorporating ABOC, a constrained bicyclic β-amino acid, for asymmetric aldol reaction catalysis

Article abstract of DOI:10.1016/j.tet.2016.02.027

A series of N-pyrrolidine-based α,β-peptide catalysts incorporating a constrained 2-aminobicyclo[2.2.2]octane carboxylic acid (ABOC) residue were synthesized and evaluated in the asymmetric aldol reaction from acetone and some p-substituted benzaldehydes. Their catalytic properties were shown to be highly dependent on the amino acid sequences and on the absolute configuration of the ABOC residue that played a determinant role. Among the peptides tested, the heterochiral tripeptide H-Pro-(R)-ABOC-Asp-OCH₃ 13, that adopts a turn conformation in the solid state, proved to be the most efficient catalyst affording β-hydroxy ketones in high yields and good enantioselectivities (up to 87%).

Full text of DOI:10.1016/j.tet.2016.02.027

Accepted Manuscript
N-Pyrrolidine-based α/β-peptides incorporating ABOC, a constrained bicyclic β-amino
acid, for asymmetric aldol reaction catalysis
Pierre Milbeo, Kelly Maurent, Laure Moulat, Aurélien Lebrun, Claude Didierjean,
Emmanuel Aubert, Jean Martinez, Monique Calmès
PII:
S0040-4020(16)30090-4
10.1016/j.tet.2016.02.027
TET 27497
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 7 December 2015
Accepted Date: 9 February 2016
Please cite this article as: Milbeo P, Maurent K, Moulat L, Lebrun A, Didierjean C, Aubert E, Martinez J,
Calmès M, N-Pyrrolidine-based α/β-peptides incorporating ABOC, a constrained bicyclic β-amino acid,
for asymmetric aldol reaction catalysis, Tetrahedron (2016), doi: 10.1016/j.tet.2016.02.027.
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Graphical abstract
Leave this area blank for abstract info.
N-Pyrrolidine-based α/β-peptides incorporating
ABOC, a constrained bicyclic β-amino acid, for
asymmetric aldol reaction catalysis
Pierre Milbeoa, Kelly Maurenta, Laure Moulata, Aurélien Lebruna, Claude Didierjeanb,c, Emmanuel Aubertb,c,
Jean Martineza, Monique Calmèsa*
IBMM UMR 5247 CNRS-Université Montpellier-ENSCM Bâtiment Chimie (17), Faculté des Sciences Place Eugène
Bataillon 34095 Montpellier cedex 5, France ; Université de Lorraine, UMR 7063 CRM2, Vandoeuvre-lès-Nancy,
a
b
France. c CNRS, UMR 7063, Vandoeuvre-lès-Nancy, France.

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N-Pyrrolidine-based α/β-peptides incorporating ABOC, a constrained bicyclic
β-amino acid, for asymmetric aldol reaction catalysis
Pierre Milbeoa, Kelly Maurenta, Laure Moulata, Aurélien Lebruna, Claude Didierjeanb,c, Emmanuel
Aubertb,c, Jean Martineza, Monique Calmèsa*
a Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CNRS-Université Montpellier-ENSCM Bâtiment Chimie (17), Faculté des Sciences
Place Eugène Bataillon 34095 Montpellier cedex 5, France. E-mail: monique.calmes@univ-montp2.fr
b Université de Lorraine, UMR 7063 CRM2, Vandoeuvre-lès-Nancy, France. c CNRS, UMR 7063, Vandoeuvre-lès-Nancy, France.
Abstract A series of N-pyrrolidine-based α,β-peptide catalysts incorporating a constrained 2-aminobicyclo[2.2.2]octane carboxylic acid
(ABOC) residue were synthesized and evaluated in the asymmetric aldol reaction from acetone and some p-substituted benzaldehydes. Their
catalytic properties were shown to be highly dependent on the amino acid sequences and on the absolute configuration of the ABOC residue
that played a determinant role. Among the peptides tested, the heterochiral tripeptide H-Pro-(R)-ABOC-Asp-OCH3 13, that adopts a turn
conformation in the solid state, proved to be the most efficient catalyst affording β-hydroxy ketones in high yields and good
enantioselectivities (up to 87%).
Keywords Short α/β-peptide - Bicyclic β-amino acid - Organocatalysis - Asymmetric aldol reaction
1. Introduction
The development of organocatalysts, which are metal-free organic catalysts, has shown a great potential in the last decade
for various asymmetric transformations such as aldol, Mannich and Michael reactions.1 Asymmetric aldol reaction provides
chiral β-hydroxy carbonyl units often included in complex natural or synthetic compounds and constitutes one of the most
important and useful carbon-carbon bond-forming reaction.2 Among the various organocatalysts, bifunctional catalysts bearing
both free secondary amine and carboxylic acid functions that are involved in the activation and in the control of the geometrical
position of the reagents, are particularly relevant.3. In particular, proline, its derivatives as well as short N-terminal proline-
containing peptides frequently promote efficient the carbon-carbon bond formation via enamine activation in the aldol
condensation.4 It can be noted that these peptides generally possess greater reactivity than single amino acids, which results in
shorter reaction times and reduced amount of catalyst while providing an excellent stereocontrol. An important characteristic of
peptide-based catalyst concern their secondary structure. Short peptides lead to efficient catalysts when both the amino and
carboxylic acid functions are correctly positioned, creating an appropriate chiral environment in the transition state. This can
result from a privileged turn structure adopted by the peptide allowing the two functions to be in close proximity.5
In our program dedicated to the synthesis and application of new enantiopure β-amino acids, we recently demonstrated the
ability of the highly constrained bicyclic β-amino acid, named ABOC, to promote a stable reverse turn when located in the
central position of α,β-hybrid tripeptides,6 as well as its effectiveness to favor helical folding in both oligoureas and α,β-hybrid
peptides.7 Based on these results and on the studies more particularly developed by Wennemers’s5a, 5d,e and Reiser’s8 groups
showing the high efficiency of pyrrolidine-based α- and α,β-peptide catalysts respectively, we designed and synthesized short
α,β-peptides incorporating proline and ABOC (Figure 1) to study and compare their catalytic activity in the aldol reaction.
In this context, di- and tripeptides (Figure 1) with a N-terminal proline residue were synthesized and studied for their
catalytic effect to promote the aldol reaction via enamine activation.4a,b The ABOC residue, located at the C-terminal position in
dipeptides or in the central position of tripeptides, was used for its ability to favor a suitable secondary structure either by
restricting the conformational freedom or by inducing the formation of a reverse turn. In order to verify the importance of the
absolute configuration of the ABOC residue, both the study of homo- and heterochiral sequences have been investigated.
Therefore, peptides containing either the (R)- or the (S)-ABOC residue were prepared. Moreover, to study the structural
requirements of the C-terminal residue bearing the carboxylic acid function in tripeptides, we prepared a range of compounds
containing an achiral (Gly), aromatic (Phe), functionalized (Asp), or sterically hindered (Aib) C-terminal amino acid. In some
sequences, we also investigated the effect of a C-terminal amide function using in particular the N-acylsulfonamide group
considering the high acidity of its proton that may replace the carboxylic proton in the catalytic cycle.9

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Fig. 1 Chemical formula of di- and tripeptides evaluated in this study.
2. Results and discussion
Four dipeptides and eleven tripeptides (Fig. 1) with the general formula H-Pro-[(R) or (S)]-ABOC-X and H-Pro-[(R) or (S)]-
ABOC-Xaa-X respectively were prepared in solution following Boc standard peptide strategy (Schemes 1 and 2). Xaa was either a
proteogenic or a non-proteogenic α-amino acid and X either an hydroxyl, methoxy or a secondary amino group. The Boc-[(R) and
(S)]-ABOC-OH residues were obtained according to our previously reported procedure.10 For the synthesis of peptides 5-8, 10, 11,
14 and 15, the benzyl ester (OBn) was used as protection of the C-terminal carboxylic acid function and for the carboxylic acid side
chain of aspartic acid. Thereby, as this orthogonal Boc-amine protecting group was removed by hydrogenolysis together with the Z
protection used for the proline residue, the peptide catalysts were obtained directly free of salt at the end of the synthesis. Starting
from Boc-Asp(OBn)-OCH3 a similar peptide strategy afforded tripeptides 12 and 13 with only one free carboxylic acid function on
the C-terminal aspartic acid side chain. In the case of peptides 3, 4 and 9, the C-terminal amide function was introduced at the
beginning of the synthesis using the suitable N-Boc-protected ABOC or α-amino acid derivative. Finally, the methyl ester
protection of the ABOC residue was used for the synthesis of peptides 1 and 2, due to the effectiveness of its synthesis with respect
to that of the corresponding benzyl ester.
Scheme 1 Dipeptide syntheses: a: TMSCl, MeOH; b: i) Z-Pro-OH, HBTU, DIEA; ii) LiOH, H2O; c: NH2R, EDCI, DMAP; d: i)
TFA, CH2Cl2; ii) Z-Pro-OH, HBTU, DIEA; e: H2/Pd/C/MeOH. X = NHiPr or NHTs.

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Scheme 2 Tripeptide syntheses: a: Boc-[(R) or (S)]-ABOC-OH, HBTU, DIEA; b: BnOH, HBTU, DIEA; c: CH3I/DBU, ∆; d:
NH2Ts, EDCI, DMAP; e: i) TFA, CH2Cl2; ii) Boc-[(R) or (S)]-ABOC-OH, HBTU, DIEA; f: i) TFA, CH2Cl2; ii) Z-Pro-OH,
HBTU, DIEA; g: H2/Pd/C/MeOH; X = OCH3 or NHTs.
In the first attempt we selected the aldol reaction of 4-nitrobenzaldehyde with acetone in DMF at room temperature as a
model to investigate the efficiency of the peptide-based catalysts 1, 2, 5-8, 10, 11, 14 and 15 bearing a free secondary amine
and one or two carboxylic acid functions. The results are reported in table 1.
The first general observation was that the conversion rate and the stereoselectivity were widely dependent on the absolute
configuration of the ABOC as well as, in the case of tripeptide, on the nature of the C-terminal amino acid. Furthermore,
increasing the reaction time was often correlated to an important formation of the side product 17 obtained by an elimination
process.
Table 1 Aldol reaction between nitrobenzaldehyde and acetone catalyzed by short peptides incorporating the constrained β-
Amino acid ABOC.
Entry
catalyst
Time
h
Conversion
16a/17
(%)a
54
95
99
97
69
98
88
81
90
81
-
ee (%)a
16a
12
22
24
34
30
24
78
77
8
8
-
-
78
66
38
Abs. Conf.a
16a
(R)
(R)
(R)
(R)
(S)
(%)a
28
95
91
91
97
98
76
95
77
78
5
1
2
3
4
5
6
7
H-Pro-(S)-ABOC-OH 1
H-Pro-(R)-ABOC-OH 2
96
72
24
24
48
24
24
72
24
72
24
48
48
4
H-Pro-(R)-ABOC-NHiPr 3
H-Pro-(R)-ABOC-NHTs 4
H-Pro-(S)-ABOC-Gly-OH 5
H-Pro-(R)-ABOC-Gly-OH 6
H-Pro-(S)-ABOC-Phe-OH 7
(S)
(S)
(S)
(S)
(S)
-
-
(S)
8
9
H-Pro-(R)-ABOC-Phe-OH 8
H-Pro-(S)-ABOC-Phe-NHTs 9
8
-
10
11
12
H-Pro-(S)-ABOC-Asp-OH 10
H-Pro-(R)-ABOC-Asp-OH 11
H-Pro-(S)-ABOC-Asp-OCH3 12
87
95
96
72
97
92
(S)
(S)
24
13
H-Pro-(R)-ABOC-Asp-OCH3 13
2
99
98
78
(S)
14
15
H-Pro-(S)-ABOC-Aib-OH 14
H-Pro-(R)-ABOC-Aib-OH 15
72
4
94
97
82
99
48
18
(S)
(S)
All reactions were carried out at 25°C using 0.05 mmol of nitrobenzaldehyde and 20 mol% of catalyst, 0.9 ml of a mixture 1/2
of dry acetone and DMF.
a
Conversion, aldolisation/crotonisation ratio, and enantioselectivity of the aldol product were determined by analytical chiral
HPLC analysis on a chiralpak AS-H column, detection at 270 nm. Direct measurement of the aldolisation/crotonisation ratio by
chiral HPLC was valid since the analysis at 270 nm of an equimolar mixture of compounds 16a and 17 led to the same area for
each peak. The absolute configuration of the major enantiomer was assigned by comparison with literature data.11

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The two dipeptides 1 and 2 proved to be globally less efficient catalysts with slower reaction time and stereoselectivity than
tripeptides (Table 1, entries 1 and 2). As a general remark, the reaction time required to achieve the same degree of conversion
was longer when the reaction was catalyzed with homochiral tripeptides rather than with their corresponding heterochiral
sequences (Table 1, entries 5 vs 6; 10 vs 11; 12 vs 13 and 14 vs 15). A specific result was obtained for the two phenylalanine-
containing tripeptides 7 and 8 since they afforded a similar moderate reaction rate with a large difference in the enantiomeric
excess (Table 1, entries 7 and 8). Among the tested tripeptides, the absence of a third chiral center on the sequence led to low or
medium stereoselectivity (Table 1, entries 5 and 6). However, it can be noted that introducing a steric constraint on the C-
terminal amino acid, i.e. the use of the achiral α,α-disubstituted Aib residue known to be a strong turn inducer,12 greatly
accelerated the reaction rate for one of the two diastereoisomers although the stereoselectivity remained weak (Table 1, entries
14 and 15). The best results were obtained with tripeptides including an aspartic acid at the C-terminal position. The two
heterochiral tripeptides 11 and 13 catalyzed efficiently the reaction, which was nearly quantitative after only 2 and 4 hours
respectively whereas 24 and 48 hours were required for their corresponding homochiral diastereoisomers (Table 1, entries 10,
11, 12 and 13). However, the higher stereoselectivity (ee 78%) was obtained when using peptide 13, having only one free
carboxylic acid function located on the aspartic acid side chain (Table 1, entry 13). The presence of a second acidic function on
compound 11 that could also play a role in the transition state, resulted in a lower stereoselectivity (Table 1, entry 11). In
contrast to the homochiral catalyst 12, the homochiral dicarboxylic acid tripeptide 10 afforded a good enantioselectivity (Table
1, entries 10 and 12). However, owing to a low conversion rate, a large amount of the crotonisation product 17 could be
detected when using compound 12. As mentioned above the same was also observed when using peptide 7 making these two
catalysts not completely suitable for the considered reaction. Exchange of the carboxylic acid function of dipeptide 2 by an
amide function increased the reaction rate and slightly improved the enantioselectivity that nevertheless remained modest
(Table 1, entries 3 and 4). The same modification made on tripeptide 7 to yield compound 9 had a deleterious effect since
practically no reaction occurred after 48 hours (Table 1, entry 9).
Thanks to the crystallization of tripeptide 13 obtained by slow evaporation of methanol, a crystal structure (Fig. 2) was
obtained that provided important insight into a preferred conformation of the most efficient catalyst. As previously observed,
the ABOC residue induced a turn,6 allowing the formation of a salt-bridge between the secondary amine and the carboxylic
acid. The position of these two functions in close proximity was consistent with the formation of a favorable aldol condensation
4b,c,h,5a,e,8
transition state to afford chiral β-hydroxy carbonyl compounds with high degree of stereoselectivity.
In the crystal
structure, the presence of a methanol molecule near the salt-bridge was observed and the carboxylic methyl ester pointed away
from the N-terminus. No intramolecular hydrogen bonding between the backbone amino and carbonyl groups was involved in
the stabilization of the turn. However, peptide bonds were rather engaged in intermolecular interactions that stabilized the
crystal packing.
Fig. 2 DRX of tripeptide 13.
Unfortunately, all attempts to crystallize tripeptide 12 have failed, thus limiting our insight on its preferred solid-state
conformation. Therefore the preferred conformations of the homochiral peptide H-Pro-(S)-ABOC-Asp-OCH3 12 and of the
corresponding diastereoisomer H-Pro-(R)-ABOC-Asp-OCH3 13 were investigated by means of molecular dynamics and
Density Functional Theory calculations to define their relative stability as a function of the ABOC absolute configuration.
As an important remark, these calculations indicated that both tripeptides 12 and 13 preferentially adopted a turn
conformation in solution (Fig. 3). The overall most stable conformation obtained for compound 12 (SSSa-12) was 12.8kj.mol-1
lower in energy than the most stable conformation of compound 13 (SRSa-13). Moreover, the calculated conformation
corresponding to the DRX structure (SRSb-13) did not appear to be the most stable conformation of the catalyst 13. In the solid
state the conformation was stabilized by intermolecular interactions (see above), that could explain this difference. For the two
tripeptides, the 10 lowest energy conformations were stabilized by both an ionic interaction between the secondary amine and
the carboxylic acid ([dH+..O-] = 1.612 Å and 1.646 Å for the lowest energy structure of 12 and 13, respectively) and a hydrogen
bond involving the ABOC NH group as donor. However, whereas the hydrogen bond accepting group was the carboxylate
group in compound 13, it was the carbonyl group of the aspartic methyl ester in compound 12 (13: dH..O = 1.965 Å and 12: dH..O
= 2.044 Å). Due to the measured distances it was difficult to explain the energy difference between the two tripeptides 12 and
13. It can be assumed that conformational constraints within the molecules could explain the overall energy stabilization for
tripeptide 12 with respect to tripeptide 13. The calculated “turn structures” of the 10 lowest energy conformations were almost
well superimposed for each compound, although catalyst 13 exhibited a slightly less compact turn with more degrees of

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freedom especially for the C-terminal methyl ester. This relative largest conformational flexibility of tripeptide 13 could be
correlated to its better efficiency in term of catalysis.
Figure 3 DFT calculated privileged conformations of tripeptide 12 and 13.
An important outcome of this study was the inversion of configuration of the ABOC residue that did not seem to have a
great effect on the spatial orientation of both the acid and amine functions, which are in both cases in close proximity.
Nevertheless, the (R) configuration of ABOC was essential for a good level of enantioselectivity of the reaction. It was
interesting to correlate these data with some previously reported results, showing that the α-tripeptide H-Pro-Pro-Asp-NH2 was
clearly a more efficient catalyst in the aldol reaction than its diastereoisomer H-Pro-(D)-Pro-Asp-NH2.5e Furthermore, using
short α,β-tripeptides, the group of Reiser also highlighted the influence of the configuration of the β-aminocyclopropane
carboxylic acid (β-ACC) incorporated in the central position of two tripeptides H-Pro-(-)-cis-β-ACC-Pro-OH or H-Pro-(+)-cis-
β-ACC-Pro-OH, in the selectivity of the reaction.8 In these two cases, the efficient catalytic properties of peptides were
correlated to the existence of turn-privileged conformations where both the carboxylic acid and amine functions were close to
each other. In our study, considering both the DRX structure of tripeptide 13 and the theoretical structures for the two
diastereoisomeric peptides 12 and 13 that suggest a favorable spatial arrangement of the two functional groups within the two
tripeptides, it appears difficult to rationalize the different results obtained regarding the rate and the stereoselectivity when using
these two catalysts in the aldol reaction. As already been observed in previous investigations5d,e,8 these results pointed out the
complexity in designing new efficient peptide catalysts.
For all the homo- or heterochiral tripeptides of this study, the same direction of stereo-induction leading to the S-enriched
aldol product was observed regardless the absolute configuration of the ABOC residue. This is consistent with previous results
obtained in similar anhydrous conditions when using tripeptides bearing a (S)-proline residue at the N-terminal position.5a,8,13
Likewise, dipeptides partially promote the R absolute configuration as previously observed with (S)-proline alone or N-terminal
(S)-proline containing dipeptide catalysts.3a,14
In an attempt to increase the stereoselectivity of the reaction, several reaction parameters including solvent, temperature and
loading charge, were investigated using the most effective catalyst 13 (Table 2). Among the different solvents tested, DMF and
DMSO proved to be the most suitable for the reaction (Table 2, entries 2 and 6). Neat conditions or other solvents induced
lower enantioselectivity. Introduction of a small amount of water decreased the reaction rate and induced low reverse
enantioselectivity (Table 2, entry 7). Interestingly, by decreasing the catalyst loading to 5 mol% the reaction still occurred,
yielding quantitative yield with the same enantiomeric excess after 4 hours instead of 2 h (Table 2, entry 3). When the reaction
was carried out in DMF at lower temperature (0°C or -20°C) we observed an enhanced stereoselectivity i.e. 81% and 85%
respectively, with an acceptable decrease in the reaction rate (Table 2, entries 8 and 9). At -40°C, a full conversion was
observed after 48h without any positive effect on the stereoselectivity (Table 2, entry 10).

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Table 2 Aldol reaction between nitrobenzaldehyde and acetone catalyzed by H-Pro-(R)-ABOC-Asp-OCH3 13.
Entry
Acetone/
Solvent (v/v)
neat
T °C
Time
h
2
2
4
2
4
2
24
2
Conversiona
16a/17
(%)a
98
98
96
97
96
97
99
ee (%)a
16a
66
78
78
65
68
76
12
Abs. Conf.a
16a
(S)
(%)a
99
99b
99
98
95
95
99
95
99
96
1
2
3
4
5
6
7
8
9
rt
rt
rt
rt
rt
rt
rt
0
-20
-40
DMF (1/2)
DMF (1/2)*
THF (1/2)
DCM (1/2)
DMSO (1/2)
H2O (10/1)
DMF(1/2)
DMF(1/2)
DMF (1/2)
(S)
(S)
(S)
(S)
(S)
(R)
(S)
(S)
99
99
82
81
85
81
8
48
10
(S)
All reactions were carried out using 0.05 mmol of nitrobenzaldehyde and 20 mol% of catalyst, 0.9 ml of a mixture
(1/2) of dry acetone and the selected solvent; *reaction with 5 mol% of catalyst.
a See Table 1 note a.
b 88% isolated yield.
The catalyst H-Pro-(R)-ABOC-Asp-OCH3 13 was then applied to the enantioselective aldol reaction of acetone with some
representative aromatic aldehydes under the optimized conditions. The results obtained are reported in Table 3. All the
reactions proceeded affording the corresponding aldol adducts in good yield and high enantiomeric excess in favor of the (S)
enantiomer (85-87% ee) using both 20 or 5 mol% of tripeptide 13 at -20°C. The reaction time was dependent on the nature of
the substituent of the aromatic aldehyde that was significantly affected in the absence of an electron withdrawing group (Table
3, entry 2). Despite longer reaction times, it was noteworthy that 5 mol% of the effective tripeptide 13 were sufficient, to
catalyze aldol reaction even at -20°C.
Table 3 Aldol reaction between aldehydes and acetone, catalyzed by H-Pro-(R)-ABOC-Asp-OCH3 13.
Entry
Product
16a
R
Catalyst
Time
h
8
24
24
72
8
48
8
Yielda
(%)
88
86
82
78
84
80
85
ee (%)b
Abs. Conf.b
(%)
20
5
20
5
20
5
20
5
1
2
3
4
4-NO2Ph
Ph
85
87
86
87
87
88
86
86
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(S)
16b
16c
4-ClPh
4-BrPh
16d
48
83
All reactions were carried out at -20 °C using 0.2 mmol of nitrobenzaldehyde and 20 or 5 mol% of
catalyst 13, in 3.6 ml of a mixture of dry acetone and DMF (1/2).
a isolated yields.
b See Table 1 note a
3. Conclusion
In conclusion, several α,β-peptide catalysts incorporating a N-terminal proline and the constrained bicyclic β-amino acid
ABOC were synthesized and their catalytic activity in the asymmetric aldol reaction was evaluated. Among the catalyst tested
in this study, that differ either in the C-terminal amino acid or the absolute configuration of the ABOC, the catalyst H-Pro-(R)-
ABOC-Asp-OCH3 proved to be the more efficient, providing the aldol products in both high yield and stereoselectivity. ABOC
appears as an important structuring element of the catalyst and its (R) absolute configuration, not associated with the sense of
the induced stereoselectivity, is crucial for both the catalytic activity and the asymmetric induction. Further studies focusing on
new applications of these α,β-peptide catalysts are now under investigation.
4. Experimental section
4.1. General remarks
All reagents were used as purchased from commercial suppliers without further purification. Solvents were purchased puriss
p.a. from commercial suppliers or dried and purified by conventional methods prior to use. Dry acetone has been employed in

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the catalytic reactions. The enantiopure compound (S)-1-tert-butyloxycarbonylaminobicyclo[2.2.2]octane-2-carboxylic acid
(Boc-[(R) or (S)]-ABOC-OH) was prepared as previously described.10 Reactions were monitored by TLC performed on silica
gel (60F-254) (detection by irradiation with UV light and/or by treatment with a solution of phosphomolybdic acid) and/or by
HPLC. All intermediate compounds were characterized by LC/MS analysis and used directly in the next synthetic step.
Microwave irradiation was achieved in a Biotage Initiator+ instrument. An automated flash purification system was used for the
flash chromatography on silica gel. Using a Biotage automated flash purification system the TLC compound retention data were
converted into an isocratic elution or linear gradient elution system. Melting points were determined with a Büchi 510 Melting
Point. HPLC analyses were performed with an Agilent model 1220 instrument at 214 nm using: column A, Phenomenex®
Onyx Monolithic HD-C18, 2µ, (50 x 4.6 mm), flow 3 ml/min, H2O (0.1 % TFA)/CH3CN (0.1 % TFA), gradient 0→0 % (30 s),
0→100 % (3 min) and 100 % (1 min); column B, chiralpak AS-H, 5µ, (250 x 4.6 mm), flow 1 ml/min, Hexane/2-propanol
90/10. The ESI mass spectra were recorded with a platform II quadrupole mass spectrometer (Micromass, Manchester, UK)
fitted with an electrospray source. HRMS were recorded in positive mode using NBA (3-nitrobenzylalcohol or GT
(Glycerol/thioglycerol) as matrix. NMR spectra were recorded on Bruker Avance III 600 MHz spectrometer at 298 K. DMSO-
d6 was used as internal reference (1H = 2.50 ppm and 13C = 39.52 ppm). Data are reported as follows: chemical shift (δ),
multiplicity (s = singlet, d = doublet, t = triplet, br = broad, m = multiplet), coupling constants (J/Hz), integration, and
1
1
assignment. The H and 13C assignments were obtained by a combined analysis of H J-coupling correlation spectroscopy (g-
COSY), 1H-13C heteronuclear multiple bond (g-HMBC) and single bond (multiplicity editing g-HSQC15 correlation
experiments.
4.2. Synthesis of catalysts
H-Pro-(S)-ABOC-OH 1 A solution of Boc-(S)-ABOC-OH (150 mg, 0.56 mmol, 1.0 equiv.) and of trimethylsilyl chloride
(TMSCl) (0.31 mL, 2.80 mmol, 5.0 equiv.) in dry MeOH (0.2 M) was stirred at room temperature overnight and then
concentrated under reduced pressure. The expected H-(S)-ABOC-OCH3, HCl was isolated as a white solid by filtration, after
precipitation using diethyl ether. This (S)-amino ester was then added to a solution of the pre-activated benzyloxycarbonyl
proline (Z-Pro-OH) (1.0 equiv.) in DMF [pre-activation using DIEA (6 equiv.) and HBTU (1.0 equiv.)]. The reaction mixture
was activated by microwaves irradiations at 85°C for 30 min (HPLC monitoring, column A). DMF was removed under reduced
pressure and the crude product was diluted with EtOAc, washed with KHSO4 (1 M), NaHCO3 (1 M) and brine. The organic
phase was dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the intermediate protected dipeptide
Z-Pro-(S)-ABOC-OCH3, after automated flash chromatography on silica gel using cyclohexane/EtOAc as eluent. The following
hydrolysis was performed in a THF/H2O mixture (2/1) (0.1 M) at room temperature for 12h using 1.1 equivalents of LiOH.H2O.
Then, THF was removed under reduced pressure and the aqueous phase was acidified with 1N HCl and extracted with EtOAc.
The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the intermediate
compound Z-Pro-(S)-ABOC-OH. The last Z-deprotection step was carried out by using a catalytic amount of Pd/C 10% in
MeOH (0.1 M) under 1 atm of H2. After vigorous stirring for 12 h at room temperature, the reaction mixture was filtered
through celite and concentrated under reduced pressure to give the expected dipeptide H-Pro-(S)-ABOC-OH 1 (97 mg, 0.35
mmol, 65%) as a white powder. Mp 168-169°C; tR (HPLC) column A = 1.81 min; MS (ESI): m/z = 267.2 [(M+H)+]; 1H NMR
(600 MHz, DMSO-d6) δ (ppm): 1.51 (m, 1H, 4-H), 1.53 (m, 4H, 5-H and 8-H), 1.55 (m, 2H, 3’-H), 1.59 (m, 3H, 3-H and 6-H
or 7-H), 1.64 (m, 1H, 2’-H), 1.87 (m, 1H, 2’-H), 2.08 (m, 1H, 3-H), 2.31 (m, 2H, 6-H or 7-H), 2.44 (m, 1H, 2-H), 2.79 (m, 2H,
4’-H), 3.39 (dd, J = 5.7 and 8.4 Hz, 1H, 1’-H), 8.77 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 24.0 (C-4),
26.5 (C-5 and C-8), 26.7 (C-3’), 30.3 (C-6 and C-7), 30.6 (C-3), 30.9 (C-2’), 47.0 (C-4’), 47.4 (C-2), 51.2 (C-1), 61.4 (C-1’),
173.4 (CONH), 177.1 (CO2H); HRMS (ESI) Calcd for C14H23N2O3 (MH+) 267.1709, found 267.1708.
H-Pro-(R)-ABOC-OH 2 was synthetized as described for dipeptide 1 starting from Boc-(R)-ABOC-OH. It was obtained as a
white powder (86 mg, 0.32 mmol, 58%); Mp 206-207°C; tR (HPLC) column A = 1.71 min ; MS (ESI): m/z = 267.2 [(M+H)+];
1H NMR (600 MHz, DMSO-d6) δ (ppm): 1.13 (m, 1H, 3-H), 1.42 (m, 1H, 5-H or 8-H), 1.43 (m, 1H, 6-H or 7-H), 1.47 (m, 1H,
4-H), 1.51 (m, 1H, 6-H or 7-H), 1.52 (m, 2H, 3’-H), 1.55 (m, 1H, 2’-H), 1.58 (m, 1H, 5-H or 8-H), 1.59 (m, 1H, 5-H or 8-H),
1.68 (m, 1H, 5-H or 8-H), 1.89 (m, 1H, 2’-H), 2.10 (dd, J = 5.3 and 10.4 Hz, 1H, 2-H), 2.21 (m, 2H, 4’-H and 6-H or 7-H), 2.25
(m, 1H, 6-H or 7-H), 2.74 (dd, J = 4.9 and 9.5 Hz, 1H, 1’-H), 2.78 (ddd, J = 1.2, 5.3 and 12.8 Hz, 1H, 3-H), 3.62 (ddd, J = 2.0,
6.5 and 9.2 Hz, 1H, 4’-H), 9.32 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 24.1 (C-5 or C-8), 24.2 (C-4), 24.3
(C-6 or C-7), 26.4 (C-5 or C-8), 26.8 (C-3’), 30.2 (C-3), 30.5 (C-6 or C-7), 30.6 (C-2’), 48.9 (C-2), 51.0 (C-1), 53.3 (C-4’), 66.5
(C-1’), 173.6 (CONH), 177.6 (CO2H); HRMS (ESI) Calcd for C14H23N2O3 (MH+) 267.1709, found 267.1707.
H-Pro-(R)-ABOC-NHiPr 3 A solution of Boc-(R)-ABOC-OH (50 mg, 0.19 mmol, 1.0 equiv.), DMAP (5 mg, 0.04 mmol, 0.2
equiv.), EDCI (36 mg, 0.19 mmol, 1.0 equiv.) and isopropylamine (11 mg, 0.19 mmol, 1 equiv.) in DMF (0.1 M) (2 ml) was
stirred 12h at room temperature (HPLC monitoring, column A). DMF was removed under reduced pressure and the residue
diluted with EtOAc and then washed with KHSO4 (1 M), NaHCO3 (1 M) and brine. The organic layer was dried over
anhydrous MgSO4 and concentrated under reduced pressure to afford the crude Boc-(R)-ABOC-NHiPr. The Boc group was
removed using a solution of TFA (1 mL) in CH2Cl2 (1 mL) at room temperature for 30 min. The organic solvent and excess of
TFA were then concentrated under reduced pressure and the residue diluted with DMF (0.1 M) was then added to a solution of
the pre-activated benzyloxycarbonyl proline (Z-Pro-OH) (1.0 equiv.) in DMF [pre-activation using DIEA (6 equiv.) and HBTU
(1.0 equiv.)]. The reaction mixture was activated by microwaves irradiations at 85°C for 30 min (HPLC monitoring, column A).
DMF was removed under reduced pressure and the crude product, diluted with EtOAc, was washed with KHSO4 (1 M),
NaHCO3 (1 M) and brine. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to
afford the intermediate protected dipeptide Z-Pro-(S)-ABOC-NHiPr, after automated flash chromatography on silica gel using

ACCEPTED MANUSCRIPT
cyclohexane/EtOAc as eluent. The last Z-deprotection step was carried out by using a catalytic amount of Pd/C 10% in MeOH
(0.1 M) under 1 atm of H2. After stirring vigorously for 12 h at room temperature, the reaction mixture was filtered through
celite and concentrated under reduced pressure to give the expected dipeptide H-Pro-(R)-ABOC-NHiPr 3 (33 mg, 0.11 mmol,
58%) as a white powder. Mp 105-106 °C; tR (HPLC) column A = 1.66 min; MS (ESI): m/z = 249.1 [(M-NHiPr)+], 308.2
[(M+H)+]; 1H NMR (600 MHz, DMSO-d6) δ (ppm): 1.00 (d, J = 6.5 Hz, 3H, CH3), 1.01 (d, J = 6.5 Hz, 3H, CH3), 1.53 (m, 4H,
5-H, 7-H and 8-H), 1.57 (m, 1H, 4-H), 1.59 (m, 1H, 8-H), 1.64 (m, 1H, 2’-H), 1.66 (m, 1H, 6-H), 1.67 (m, 3H, 3’-H and 3-H),
1.73 (ddt, J = 7.5, 8.9 and 12.5 Hz, 1H, 3-H), 1.89 (ddt, J = 7.5, 8.9 and 12.5 Hz, 1H, 2’-H), 2.08 (td, J = 2.8 and 11.5 Hz, 1H,
6-H), 2.13 (m, 1H, 7-H), 2.67 and 2.83 (2 dt, J = 6.7 and 10.3 Hz, 2H, 4’-H), 3.02 (dd, J = 7.0 and 10.6 Hz, 1H, 2-H), 3.41(dd, J
= 5.2 and 8.9 Hz, 1H, 1’-H), 3.78 (dsept, J = 6.5 and 7.5 Hz, 1H, CHCH3), 7.13 (d, J = 7.5 Hz, 1H, NHiPr), 7.58 (s, 1H,
NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 22.3 (CH3), 23.7 (C-4), 25.5 (C-8), 25.6 (C-3’), 25.7 (C-5), 26.1 (C-7),
29.7 (C-3), 29.9 (C-6), 30.3 (C-2’), 40.2 (CHCH3), 43.8 (C-2), 46.6 (C-4’), 51.3 (C-1), 60.5 (C-1’), 172.2 (CONHiPr), 174.1
(C=OPro); HRMS (ESI) Calcd for C17H30N3O2 (MH+) 308.2338, found 308.2343.
H-Pro-(R)-ABOC-NHTs 4 was synthesized as described for the dipeptide 3 using Toluenesulfonamide instead of
isopropylamine. It was obtained as a white powder (39 mg, 0.09 mmol, 50%); Mp 175-176 °C; tR (HPLC) column A = 2.16
1
min; MS (ESI): m/z = 249.1 [(M-NHTs)+], 420.3[(M+H)+]; H NMR (600 MHz, DMSO-d6) δ (ppm): 1.44 (m, 1H, 8-H), 1.51
(m, 2H, 3-H and 4-H), 1.52 (m, 1H, 8-H), 1.53 (m, 2H, 5-H), 1.62 and 1.75 (2 m, 2H, 7-H), 1.79 (m, 4H, 2’-H, 3’-H and 6-H),
1.90 (m, 1H, 3-H), 2.12 (m, 1H, 6-H), 2.13 (m, 1H, 2’-H), 2.32 (s, 3H, CH3), 2.64 (dd, J = 5.5 and 10.1 Hz, 1H, 2-H), 3.10 and
3.14 (2 dt, J = 6.9 and 11.1 Hz, 2H, 4’-H), 3.85 (dd, J = 6.6 and 8.2 Hz, 1H, 1’-H), 7.20 and 7.65 (2 d, J = 8 Hz, 4H, H-arom),
8.86 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 20.9 (CH3), 23.5 (C-4), 23.9 (C-3’), 25.3 (C-7), 25.5 (C-8),
25.8 (C-5), 28.8 (C-3), 29.4 (C-2’), 29.6 (C-6), 45.6 (C-4’), 46.2 (C-2), 52.2 (C-1), 60.0 (C-1’), 126.8 and 128.2 (C-arom),
140.1 (SO2C-arom), 142.3 (CH3C-arom), 167.2 (C=OPro), 176.0 (CONHTs); HRMS (ESI) Calcd for C21H30N3O4S (MH+)
420.1957, found 420.1956.
General procedure for the synthesis of tripeptides H-Pro-[(R) or (S)]-ABOC-Xaa-OH 5, 6, 7 and 8
To a solution of the pre-activated Boc-[(R) or (S)]-ABOC-OH (100 mg, 0.37 mmol, 1.0 equiv.) in DMF (0.1 M), [pre-
activation using DIEA (0.38 mL, 2.22 mmol, 6.0 equiv.) and HBTU (140 mg, 0.37 mmol, 1.0 equiv.)] was added 1.0 equivalent
of the H-Xaa-OBn salt. The reaction mixture was activated by microwaves irradiations at 85°C for 30 min (HPLC monitoring,
column A). DMF was removed under reduced pressure and the crude product, diluted with EtOAc, was washed with KHSO4 (1
M), NaHCO3 (1 M) and brine. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to
afford the intermediate protected dipeptide Boc-[(R) or (S)]-ABOC-Xaa-OBn. The Boc-deprotection was carried out using a
solution of TFA (1 mL) in CH2Cl2 (1 mL) at room temperature for 30 min. The organic solvent and excess of TFA were then
removed under reduced pressure and the residue diluted with DMF (0.1 M) was then added to a solution of the pre-activated
benzyloxycarbonyl proline (Z-Pro-OH) (1.0 equiv.) in DMF [pre-activation using DIEA (6 equiv.) and HBTU (1.0 equiv.)].
The reaction mixture was activated by microwaves irradiations at 85°C for 30 min (HPLC monitoring, column A). DMF was
removed under reduced pressure and the crude product, diluted with EtOAc, was washed with KHSO4 (1 M), NaHCO3 (1 M)
and brine. The organic layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the
intermediate protected tripeptide Z-Pro-[(R) or (S)]-ABOC-Xaa-OBn, after automated flash chromatography using
cyclohexane/EtOAc as eluent. The last Z-deprotection step was carried out by using a catalytic amount of Pd/C 10% in MeOH
(0.1 M) under 1 atm of H2. After vigorous stirring for 12 h at room temperature, the reaction mixture was filtered through celite
and concentrated under reduced pressure to give the expected tripeptide 5, 6, 7 or 8.
H-Pro-(S)-ABOC-Gly-OH 5 was synthesized according to the general procedure from H-Gly-OBn, pTsOH (125 mg, 0.37
mmol) and Boc-(S)-ABOC-OH. The expected compound was obtained as a white powder (82 mg, 0.25 mmol, 67%). Mp 179
°C; tR (HPLC) column A = 1.77 min; MS (ESI): m/z = 249.1 [(b2)+], 324.2 [(M+H)+]; 1H NMR (600 MHz, DMSO-d6) δ (ppm):
1.31 (m, 1H, 7-H), 1.37 (m, 1H, 6-H), 1.55 (m, 1H, 8-H), 1.57 (m, 2H, 5-H), 1.59 (m, 1H, 4-H), 1.63 (m, 1H, 3-H), 1.66 (m,
1H, 8-H), 1.76 (m, 1H, 2’-H), 1.77 (m, 1H, 3’-H), 1.86 (m, 1H, 3-H), 1.91 (m, 1H, 3’-H), 2.05 (m, 1H, 7-H), 2.11 (m, 1H, 2’-
H), 2.34 (m, 1H, 6-H), 3.19 (dd, J = 2.6 and 17.3 Hz, 1H, CH2CO2H), 3.23 (m, 2H, 4’-H), 3.36 (m, 1H, 2-H), 3.74 (dd, J = 6.4
and 17.3 Hz, 1H, CH2CO2H), 4.09 (dd, J = 5.3 and 8.6 Hz, 1H, 1’-H), 6.85 (dd, J = 2.6 and 6.4 Hz, 1H, NHCH2), 8.14 (s, 1H,
NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 23.4 (C-4), 23.7 (C-3’), 25.4 (C-5 and C-8), 26.6 (C-7), 29.0 (C-3), 29.1
(C-6), 29.7 (C-2’), 43.0 (C-2), 43.4 (CH2CO2H), 45.5 (C-4’), 52.7 (C-1), 58.0 (C-1’), 170.4 (C=OPro), 172.1 (C=OABOC), 172.9
(CO2H); HRMS (ESI) Calcd for C16H26N3O4 (MH+) 324.1923, found 324.1926.
H-Pro-(R)-ABOC-Gly-OH 6 was synthesized according to the general procedure from H-Gly-OBn, pTsOH (125 mg, 0.37
mmol) and Boc-(R)-ABOC-OH. The expected compound was obtained as a white powder (78 mg, 0.23 mmol, 62%). Mp 154-
1
155°C; tR (HPLC) column A = 1.61 min; MS (ESI): m/z = 249.1 [(b2)+], 324.2 [(M+H)+], 647.2 [(2M+H)+]; H NMR (600
MHz, DMSO-d6) δ (ppm): 1.54 - 1.66 (m, 4H, 5-H and 8-H), 1.58 (m, 1H, 4-H), 1.60 (m, 1H, 7-H), 1.68 (m, 1H, 3-H), 1.71 (m,
1H, 2’-H), 1.73 (m, 2H, 3’-H), 1.80 (m, 1H, 3-H), 1.91 and 2.00 (2 m, 2H, 6-H), 2.10 (m, 1H, 7-H), 2.13 (m, 1H, 2’-H), 2.98
(m, 1H, 4’-H), 3.01 (m, 1H, 2-H), 3.06 (m, 1H, 4’-H), 3.32 (dd, J = 4.5 and 17.1 Hz, 1H, CH2CO2H), 3.67 (dd, J = 6.3 and 17.1
Hz, 1H, CH2CO2H), 4.01 (dd, J = 6.9 and 8.1 Hz, 1H, 1’-H), 7.49 (dd, J = 4.5 and 6.3 Hz, 1H, NHCH2), 7.97 (s, 1H, NHABOC);
13C NMR (150 MHz, DMSO-d6) δ (ppm): 23.4 (C-4), 24.0 (C-3’), 25.5 (C-5 and C-8), 26.6 (C-7), 29.4 (C-3), 29.6 (C-2’), 29.9
(C-6), 42.7 (CH2CO2H), 44.1 (C-2), 45.4 (C-4’), 52.8 (C-1), 59.5 (C-1’), 169.9 (C=OPro), 172.3 (C=OABOC), 172.6 (CO2H);
HRMS (ESI) Calcd for C16H26N3O4 (MH+) 324.1923, found 324.1928.

ACCEPTED MANUSCRIPT
H-Pro-(S)-ABOC-Phe-OH 7 was synthesized according to the general procedure from H-Phe-OBn, HCl (108 mg, 0.37 mmol)
and Boc-(S)-ABOC-OH. The expected compound was obtained as a white powder (110 mg, 0.27 mmol, 72%). Mp 174 °C; tR
(HPLC) column A = 2.12 min; MS (ESI): m/z = 249.1 [(b2)+], 414.2 [(M+H)+]; 1H NMR (600 MHz, DMSO-d6) δ (ppm): 1.26
(m, 2H, 6-H and 7-H), 1.57 (m, 2H, 5-H), 1.59 (m, 1H, 4-H), 1.62 (m, 1H, 3-H), 1.66 (m, 2H, 8-H), 1.74 (m, 1H, 2’-H), 1.75
(m, 1H, 3’-H), 1.87 (m, 1H, 3-H), 1.88 (m, 1H, 3’-H), 2.05 (m, 1H, 7-H), 2.14 (m, 1H, 2’-H), 2.36 (m, 1H, 6-H), 2.90 and 2.94
( 2 dd, J = 5.3 and 13.2 Hz, 2H, CH2Ar), 3.08 (ddd, J = 5.4, 6.6 and 11.2 Hz, 1H, 4’-H), 3.12 (dt, J = 7.7 and 11.2 Hz, 1H, 4’-
H), 3.33 (dd, J = 6.3 and 9.7 Hz, 1H, 2-H), 4.12 (dd, J = 4.8 and 8.6 Hz, 1H, 1’-H), 4.18 (td, J = 5.3 and 6.8 Hz, 1H, CHCO2H),
6.68 (d, J = 6.8 Hz, 1H, NHCHCO2H), 7.01 (d, J = 7.3 Hz, 2H, H-ortho), 7.13 (t, J = 7.3 Hz, 1H, H-para), 7.18 (t, J = 7.3 Hz,
2H, H-meta), 8.14 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 23.4 (C-3’ and C-4), 25.4 (C-5 and C-8), 26.8
(C-7), 28.8 (C-6), 28.9 (C-3), 29.7 (C-2’), 37.5 (CH2Ar), 43.1 (C-2), 45.2 (C-4’), 52.7 (C-1), 54.8 (CHCO2H), 57.7 (C-1’),
125.8 (CH-para), 127.6 (CH-meta), 129.4 (CH-ortho), 138.4 (C-arom), 170.2 (C=OPro), 171.5 (C=OABOC), 174.4(CO2H);
HRMS (ESI) Calcd for C23H32N3O4 (MH+) 414.2393, found 414.2397.
H-Pro-(R)-ABOC-Phe-OH 8 was synthesized according to the general procedure from H-Phe-OBn, HCl (108 mg, 0.37 mmol)
and Boc-(R)-ABOC-OH. The expected compound was obtained as a white powder (84 mg, 0.20 mmol, 55%). Mp 146-148°C;
tR (HPLC) column X column A = 1.85 min; MS (ESI): m/z = 249.1 [(b2)+], 414.2 [(M+H)+]; 1H NMR (600 MHz, DMSO-d6) δ
(ppm): 1.47 (m, 1H, 7-H), 1.50 (m, 1H, 6-H), 1.55 (m, 1H, 2’-H), 1.56 (m, 3H, 3’-H and 5-H), 1.59 (m, 1H, 4-H), 1.63 (m, 1H,
3’-H), 1.65 (m, 2H, 8-H), 1.69 and 1.78 (2 m, 2H, 3-H), 1.92 (m, 1H, 2’-H), 2.24 (m, 1H, 7-H), 2.27 (m, 1H, 6-H), 2.92 (m, 1H,
CH2Ar), 2.94 (m, 1H, 4’-H), 2.97 (m, 1H, CH2Ar), 2.99 (m, 1H, 4’-H), 3.23 (m, 1H, 2-H), 3.75 (dd, J = 5.4 and 8.1 Hz, 1H, 1’-
H), 4.18 (m, 1H, CHCO2H), 7.18 (t, J = 7.1 Hz, 1H, H-para), 7.21 (d, J = 7.1 Hz, 2H, H-ortho), 7.26 (t, J = 7.1 Hz, 2H, H-
meta), 7.55 (br s, 1H, NHCHCO2H), 7.79 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 23.6 (C-4), 24.1 (C-3’),
25.5 (C-5 and C-8), 26.6 (C-7), 29.8 (C-2’), 30.2 (C-3 and C-6), 37.2 (CH2Ar), 42.6 (C-2), 46.0 (C-4’), 52.1 (C-1), 54.9
(CHCO2H), 59.4 (C-1’), 126.1 (CH-para), 128.0 (CH-meta), 129.1 (CH-ortho), 138.5 (C-arom), 169.6 (C=OPro), 173.4
(C=OABOC), 173.9 (CO2H); HRMS (ESI) Calcd for C23H32N3O4 (MH+) 414.2393, found 414.2400.
General procedure for the synthesis of the tripeptides H-Pro-[(R) or (S)]-ABOC-Xaa-OH 10, 11, 14 and 15
To a solution of the pre-activated Boc-Xaa-OH (1.0 equiv.) in DMF (0.2M) [pre-activation using DIEA (2 equiv.) and
HBTU (1.0 equiv.)] was added dropwise benzyl alcohol (1.0 equiv.). After stirring at room temperature for 12h (HPLC
monitoring, column A), the DMF was removed under reduced pressure and the crude product, diluted with EtOAc, was washed
with KHSO4 (1 M), NaHCO3 (1 M) and brine. The organic phase was dried over anhydrous MgSO4 and concentrated under
reduced pressure. The Boc-deprotection was carried out using a solution of TFA (1 mL) in CH2Cl2 (1 mL) at room temperature
for 30 min. The organic solvent and excess of TFA were then removed under reduced pressure and the residue diluted with
DMF (0.1 M) was directly added to the pre-activated Boc-[(R) or (S)]-ABOC-OH (1.0 equiv.) in DMF, [pre-activation using
DIEA (6.0 equiv.) and HBTU (1.0 equiv.)]. The reaction mixture was activated by microwaves irradiations at 85°C for 30 min
(HPLC monitoring, column A). DMF was removed under reduced pressure and the crude product, diluted with EtOAc, was
washed with KHSO4 (1 M), NaHCO3 (1 M) and brine. The organic phase was dried over anhydrous MgSO4 and concentrated
under reduced pressure to afford the intermediate protected dipeptide Boc-[(R) or (S)]-ABOC-Xaa-OBn. A new cycle of Boc-
deprotection and coupling reaction using the pre-activated Z-Pro-OH is then directly carried out affording the intermediate
protected tripeptide Z-Pro-[(R) or (S)]-ABOC-Xaa-OBn, after automated flash chromatography using cyclohexane/EtOAc as
eluent. The last Z-deprotection step was carried out by using a catalytic amount of Pd/C 10% in MeOH (0.1 M) under 1 atm of
H2. After vigorous stirring for 12 h at room temperature, the reaction mixture was filtered through celite and concentrated under
reduced pressure to give the expected tripeptide 10, 11, 14 or 15.
H-Pro-(S)-ABOC-Asp-OH 10 was synthesized according to the general procedure from Boc-Asp(OBn)-OH (150 mg, 0.46
mmol) and Boc-(S)-ABOC-OH (125 mg, 0.46 mmol). The expected compound was obtained as a white powder (116 mg, 0.30
mmol, 66%).Mp 186-188 °C; tR (HPLC) column A = 2.31min ; MS (ESI): m/z = 249.1 [(b2)+], 382.0 [(M+H)+], 763.2
[(2M+H)+] ; 1H NMR (600 MHz, DMSO-d6) δ (ppm): 1.32 (m, 1H, 7-H), 1.36 (m, 1H, 6-H), 1.60 (m, 3H, 4-H and 5-H), 1.64
(m, 1H, 3-H), 1.68 (m, 2H, 8-H), 1.72 (m, 1H, 2’-H), 1.85 (m, 2H, 3’-H), 1.90 (m, 1H, 3-H), 1.97 (m, 1H, 7-H), 2.17 (m, 1H,
2’-H), 2.35 (dd, J = 2.9 and 15.6 Hz, 1H, CH2CO2H), 2.38 (m, 1H, 6-H), 2.53 (dd, J = 10 and 15.6 Hz, 1H, CH2CO2H), 3.21
(m, 2H, 4’-H), 3.36 (dd, J =6.6 and 10.5 Hz, 1H, 2-H), 4.10 (dd, J = 6.6 and 8.4 Hz, 1H, 1’-H), 4.21 (ddd, J = 2.9, 6.8 and 10.0
Hz, 1H, CHCO2H), 6.98 (d, J = 6.8 Hz, 1H, NHCHCO2H), 8.08 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm):
23.4 (C-4), 23.6 (C-3’), 25.4 (C-5 and C-8), 26.8 (C-7), 28.9 (C-3), 29.1 (C-6), 29.6 (C-2’), 39.8 (CH2CO2H), 43.0 (C-2), 45.9
(C-4’), 49.2 (CHCO2H), 52.8 (C-1), 59.2 (C-1’), 169.6 (C=OPro), 171.9 (CH2CO2H), 172.2 (C=OABOC), 174.2 (CHCO2H);
HRMS (ESI) Calcd for C18H28N3O6 (MH+) 382.1978, found 382.1983
H-Pro-(R)-ABOC-Asp-OH 11 was synthesized according to the general procedure from Boc-Asp(OBn)-OH (150 mg, 0.46
mmol) and Boc-(R)-ABOC-OH (125 mg, 0.46 mmol). The expected compound was obtained as a white powder (108 mg, 0.28
mmol, 61%). Mp 190 °C (dec); tR (HPLC) column A = 1.60 min; MS (ESI): m/z = 249.1 [(b2)+], 382.2 [(M+H)+]; 1H NMR (600
MHz, DMSO-d6) δ (ppm): 1.46 (m, 1H, 6-H), 1.49 (m, 1H, 7-H), 1.55 - 1.59 (m, 4H, 5-H and 8-H), 1.59 (m, 1H, 4-H), 1.75 (m,
1H, 2’-H), 1.78 (m, 2H, 3-H), 1.81 (m, 2H, 3’-H), 2.11 (m, 1H, 7-H), 2.14 (m, 1H, 2’-H), 2.31 (m, 1H, 6-H), 2.42 (dd, J = 9.4
and 15.4 Hz, 1H, CH2CO2H), 2.47 (dd, J = 3.6 and 15.4 Hz, 1H, CH2CO2H), 3.11 and 3.16 (2 m, 2H, 4’-H), 3.25 (dd, J = 5.7
and 10.6 Hz, 1H, 2-H), 3.96 (dd, J = 6.6 and 8.4 Hz, 1H, 1’-H), 4.07 (ddd, J = 3.6, 5.7 and 9.4 Hz, 1H, CHCO2H), 7.18 (d, J =
5.7 Hz, 1H, NHCHCO2H), 7.93 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 23.4 (C-4), 23.6 (C-3’), 25.4 (C-5
and C-8), 26.7 (C-7), 29.5 (C-6), 29.6 (C-2’ and C-3), 39.3 (CH2CO2H), 43.1 (C-2), 45.7 (C-4’), 49.1 (CHCO2H), 52.7 (C-1),

ACCEPTED MANUSCRIPT
59.4 (C-1’), 168.3 (C=OPro), 172.4 (C=OABOC), 172.5 (CH2CO2H), 173.2 (CHCO2H); HRMS (ESI) Calcd for C18H28N3O6
(MH+) 382.1978, found 382.1983.
H-Pro-(S)-ABOC-Aib-OH 14 was synthesized according to the general procedure from Boc-Aib-OH (75 mg, 0.37 mmol) and
Boc-(S)-ABOC-OH (100 mg, 0.37 mmol). The expected compound was obtained as a white powder (52 mg, 0.15 mmol, 40%).
1
Mp 220 °C (dec); tR (HPLC) column A = 1.71min ; MS (ESI): m/z = 249.1 [(b2)+], 352.2 [(M+H)+]; H NMR (600 MHz,
DMSO-d6) δ (ppm): 1.26 (m, 1H, 6-H), 1.27 (m, 1H, 7-H), 1.35 (s, 6H, CH3), 1.55 (m, 3H, 3-H and 5-H), 1.57 (m, 2H, 4-H and
8-H), 1.65 (m, 1H, 8-H), 1.76 (m, 1H, 3’-H), 1.80 (m, 1H, 2’-H), 1.91 (m, 1H, 7-H), 1.92 (m, 1H, 3-H), 1.98 (m, 1H, 3’-H),
2.14 (m, 1H, 2’-H), 2.41 (m, 1H, 6-H), 3.22 (td, J = 7.7 and 10.6 Hz, 1H, 4’-H), 3.38 (m, 1H, 2-H), 3.42 (ddd, J = 5.7, 6.9 and
10.6 Hz, 1H, 4’-H), 4.17 (dd, J = 4.9 and 8.2 Hz, 1H, 1’-H), 7.39 (s, 1H, NHCCO2H), 8.19 (s, 1H, NHABOC); 13C NMR (150
MHz, DMSO-d6) δ (ppm): 23.5 (C-4), 23.8 (CH3), 24.1 (C-3’), 25.3 (C-5), 25.5 (C-8), 26.9 (C-7), 28.4 (C-3), 29.0 (C-6), 30.1
(C-2’), 42.9 (C-2), 45.7 (C-4’), 52.6 (C-1), 56.9 (CCO2H), 57.4 (C-1’), 170.5 (C=OPro), 171.1 (C=OABOC), 179.7 (CO2H);
HRMS (ESI) Calcd for C18H30N3O4 (MH+) 352.2236, found 352.2240.
H-Pro-(R)-ABOC-Aib-OH 15 was synthesized according to the general procedure from Boc-Aib-OH (75 mg, 0.37 mmol) and
Boc-(S)-ABOC-OH (100 mg, 0.37 mmol). The expected compound was obtained as a white powder (30 mg, 0.09 mmol, 23%).
1
Mp 190 °C (dec); tR (HPLC) column A = 1.62 min; MS (ESI): m/z = 249.1 [(b2)+], 352.2 [(M+H)+]; H NMR (600 MHz,
DMSO-d6) δ (ppm): 1.28 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.51 (m, 1H, 7-H), 1.56 (m, 1H, 4-H), 1.57 (m, 3H, 5-H and 8-H),
1.63 (m, 2H, 3-H and 6-H), 1.64 (m, 1H, 8-H), 1.70 (m, 2H, 3’-H), 1.75 (m, 1H, 2’-H), 1.78 (m, 1H, 3-H), 2.04 (m, 1H, 7-H),
2.07 (m, 1H, 6-H), 2.09 (m, 1H, 2’-H), 2.96 (m, 2H, 4’-H), 3.07 (m, 1H, 2-H), 3.92 (m, 1H, 1’-H), 7.58 (s, 1H, NHCCO2H),
7.91 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 23.5 (C-4), 24.4 (C-3’), 24.5 and 25.3 (CH3), 25.6 (C-5 and C-
8), 26.1 (C-7), 29.4 (C-3), 29.6 (C-6), 30.0 (C-2’), 43.7 (C-2), 45.5 (C-4’), 52.6 (C-1), 56.1 (CCO2H), 59.7 (C-1’), 170.4
(C=OPro), 172.1 (C=OABOC), 177.3 (CO2H); HRMS (ESI) Calcd for C18H30N3O4 (MH+) 352.2236, found 352.2240.
H-Pro-(S)-ABOC-Phe-NHTs 9 A solution of Boc-Phe-OH (100 mg, 0.38 mmol, 1.0 equiv.), DMAP (9 mg, 0.08 mmol, 0.2
equiv.), EDCI (73 mg, 0.38 mmol, 1.0 equiv.) and p-Toluenesulfonamide (65 mg, 0.38 mmol, 1 equiv.) in DMF (0.1 M) (4 ml)
was stirred at room temperature until completion of the reaction (HPLC monitoring, column A). DMF was removed under
reduced pressure and the residue diluted with EtOAc was washed with KHSO4 (1 M), NaHCO3 (1 M) and brine. The organic
layer was dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the crude Boc-Phe-NHiPr. The Boc-
deprotection was carried out using a solution of TFA (1 mL) in CH2Cl2 (1 mL) at room temperature for 30 min. The organic
solvent and excess of TFA were then removed under reduced pressure and the residue diluted with DMF (0.1 M) was then
added to a solution of the pre-activated Boc-(S)-ABOC-OH (1.0 equiv.) in DMF [pre-activation using DIEA (6 equiv.) and
HBTU (1.0 equiv.)]. The reaction mixture was activated by microwaves irradiations at 85°C for 30 min (HPLC monitoring,
column A). DMF was removed under reduced pressure and the crude product, diluted with EtOAc, was washed with KHSO4 (1
M), NaHCO3 (1 M) and brine. The organic phase was dried over anhydrous MgSO4 and concentrated under reduced pressure to
afford the intermediate protected dipeptide Boc-(S)-ABOC-Phe-NHTs. A new cycle of Boc-deprotection and coupling reaction
using the pre-activated Z-Pro-OH is then directly carried out affording after automated flash chromatography using
cyclohexane/EtOAc as eluent the intermediate protected tripeptide Z-Pro-(S)-ABOC-Phe-NHTs. The last Z-deprotection step
was carried out by using a catalytic amount of Pd/C 10% in MeOH (0.1 M) under 1 atm of H2. After stirring vigorously for 12 h
at room temperature, the reaction mixture was filtered through celite and concentrated under reduced pressure to give the
expected tripeptide Z-Pro-(S)-ABOC-Phe-NHTs 9 as a white powder 45 mg, 0.08 mmol, 21%). Mp 186-187 °C; tR (HPLC)
1
column A = 2.47 min ; MS (ESI): m/z = 567.3 [(M+H)+] ; H NMR (600 MHz, DMSO-d6) δ (ppm): 1.26 (m, 1H, 7-H), 1.31
(m, 1H, 6-H), 1.55 (m, 2H, 5-H), 1.57 (m, 1H, 4-H), 1.58 (m, 1H, 2’-H), 1.60 (m, 1H, 3-H), 1.64 (m, 2H, 8-H), 1.76 (m, 1H, 3-
H), 1.79 (m, 2H, 3’-H), 2.10 (m, 1H, 7-H), 2.14 (m, 1H, 2’-H), 2.38 (s, 3H, CH3), 2.46 (m, 1H, 6-H), 2.67 (dd, J = 6.0 and 13.4
Hz, 1H, CH2Ar), 2.82 (dd, J = 5.7 and 13.4 Hz, 1H, CH2Ar), 3.13 (m, 2H, 4’-H), 3.19 (dd, J = 6.5 and 9.5 Hz, 1H, 2-H), 3.77 (t,
J = 8.5 Hz, 1H, 1’-H’), 4.35 (dt, J = 6.0 and 7.8 Hz, 1H, CHCH2Ar), 6.55 (d, J = 7.8 Hz, 1H, NHCHCH2Ar), 6.79 (d, J = 7.2
Hz, 2H, H-orthoPhe), 7.01 (t, J = 7.2 Hz, 2H, H-metaPhe), 7.07 (t, J = 7.2 Hz, 1H, H-paraPhe), 7.26 and 7.61 (2 d, J = 8.2 Hz, 4H,
H-aromTs), 8.01 (s, 1H, NHABOC); 13C NMR (150 MHz, DMSO-d6) δ (ppm): 20.9 (CH3), 23.4 (C-4), 24.3 (C-3’), 25.3 (C-8),
25.4 (C-5), 26.7 (C-7), 28.9 (C-6), 29.0 (C-3), 29.3 (C-2’), 38.6 (CH2Ar), 43.2 (C-2), 45.7 (C-4’), 52.6 (C-1), 55.1 (CHCH2Ar),
59.9 (C-1’), 125.7 (CH-paraPhe), 126.5 (CH-aromTs), 127.5 (CH-metaPhe), 128.4 (CH-aromTs), 129.3 (CH-orthoPhe), 137.4 (C-
aromPhe), 140.3 (CCH3), 141.9 (SO2C-arom), 169.2 (C=OPro), 171.6 (C=OABOC), 176.5 (CONHSO2); HRMS (ESI) Calcd for
C30H39N4O5S (MH+) 567.2641, found 567.2649.
H-Pro-(S)-ABOC-Asp-OCH3 12 A solution of Boc-Asp(OBn)-OH (250 mg, 0.77 mmol, 1 equiv.), DBU (0.15 ml, 0.77 mmol,
1 equiv.) and CH3I (0.25 ml, 3.85 mmol, 5 equiv.) in CH3CN (0.4 M) (2 ml) was refluxed for 4h. CH3CN was removed under
reduced pressure and the residue, diluted with EtOAc, was washed with saturated sodium bicarbonate and brine. The organic
phase was dried over anhydrous MgSO4 and concentrated under reduced pressure to afford the Boc-Asp(OBn)-OCH3. The
Boc-deprotection was carried out using a solution of TFA (1 mL) in CH2Cl2 (1 mL) at room temperature for 30 min. The
organic solvent and excess of TFA were then removed under reduced pressure and the residue diluted with DMF (0.1 M) was
added to a solution of the pre-activated Boc-(S)-ABOC-OH (1.0 equiv.) in DMF [pre-activation using DIEA (6 equiv.) and
HBTU (1.0 equiv.)]. The reaction mixture was activated by microwaves irradiations at 85°C for 30 min (HPLC monitoring,
column A). DMF was removed under reduced pressure and the crude product, diluted with EtOAc, was washed with KHSO4 (1
M), NaHCO3 (1 M) and brine. The organic phase was dried over anhydrous MgSO4 and concentrated under reduced pressure to
afford the intermediate protected dipeptide Boc-[(R) or (S)]-ABOC-Asp(OBn)-OCH3. A new cycle of Boc-deprotection and
reaction coupling using the pre-activated Z-Pro-OH is then directly carried out affording the intermediate protected tripeptide

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Z-Pro-[(R) or (S)]-ABOC-Asp(OBn)-OCH3, after automated flash chromatography using cyclohexane/EtOAc as eluent. The
last Z-deprotection step was carried out by using a catalytic amount of Pd/C 10% in MeOH (0.1 M) under 1 atm of H2. After
vigorous stirring at room temperature for 12 h, the reaction mixture was filtered through celite and concentrated under reduced
pressure to give as a white powder the expected tripeptide H-Pro-(S)-ABOC-Asp-OCH3 12 (141 mg, 0.31 mmol, 46%); Mp 220
°C (dec); tR (HPLC) column A = 1.76 min ; MS (ESI): m/z = 249.1 [(b2)+], 396.2 [(M+H)+] ; 1H NMR (600 MHz, DMSO-d6) δ
(ppm): (presence of two rotamers): rotamer 1 (54%): 1.35 and 1.91 or 1.57 and 1.66 (2 m, 6H, 5-H, 6-H and 8-H), 1.57 (m, 1H,
4-H), 1.64 and 1.88 (2m, 2H, 3’-H), 1.30-2.40 (m, 6H, 2’-H, 3-H and 7-H), 2.36-2.50 (m + dd, J = 6.3 and 15.9 Hz, 2H,
CH2CO2H), 3.14 (m, 2H, 4’-H), 3.18 (m, 1H, 2-H), 3.55 (s, 3H, CH3), 3.96 (t, J = 7.5 Hz, 1H, 1’-H), 4.38 (q, J = 6.9 Hz, 1H,
CHCO2CH3), 7.84 (s, 1H, NHABOC), 7.98 (d, J = 6.9 Hz, 1H, NHCHCO2CH3); rotamer 2 (46%): 1.35 and 1.91 or 1.57 and 1.66
(2 m, 6H, 5-H, 6-H and 8-H), 1.57 (m, 1H, 4-H), 1.64 and 1.88 (2m, 2H, 3’-H), 1.30-2.40 (m, 6H, 2’-H, 3-H and 7-H), 2.27-
2.44 (2 dd, J = 4.6 and 9.2 Hz, 2H, CH2CO2H), 3.14 (m, 2H, 4’-H), 3.46 (m, 1H, 2-H), 3.59 (s, 3H, CH3), 3.96 (t, J = 7.5 Hz,
1H, 1’-H), 4.22 (dt, J = 4.6 and 7.5 Hz, 1H, CHCO2CH3), 7.72 (d, J = 7.5 Hz, 1H, NHCHCO2CH3), 7.16 (s, 1H, NHABOC); 13
C
NMR (150 MHz, DMSO-d6) δ (ppm): (presence of two rotamers): rotamer 1: 23.4 (C-3’), 23.6 (C-4), 25.5 or 26.8 (C-5, C-6
and C-8), 29.0-30.5 (C-2’, C-3 and C-7), 38.2 (CH2CO2H), 43.0 (C-2), 44.9 (C-4’), 49.6 (CHCO2CH3), 51.6 (CH3), 52.6 (C-1),
58.8 (C-1’), 170.7-173.4 (C=OPro, C=OABOC, CO2H and CO2CH3); rotamer 2: 23.4 (C-3’), 23.6 (C-4), 25.5 or 26.8 (C-5, C-6 and
C-8), 29.0-30.5 (C-2’, C-3 and C-7), 38.2 (CH2CO2H), 42.3 (C-2), 44.9 (C-4’), 49.8 (CHCO2CH3), 51.7 (CH3); 52.6 (C-1), 58.8
(C-1’), 170.7-173.4 (C=OPro, C=OABOC, CO2H and CO2CH3); HRMS (ESI) Calcd for C19H30N3O6 (MH+) 396.2135, found
396.2132.
H-Pro-(R)-ABOC-Asp-OCH3 13 was synthesized as described for the tripeptide 12 starting from Boc-(R)-ABOC-OH. White
powder (198 mg, 0.50 mmol, 65%); Mp 182-184 °C ; tR (HPLC) column A = 1.63 min; MS (ESI): m/z = 249.1 [(b2)+], 396.2
1
[(M+H)+], 791.6 [(2M+H)+]; H NMR (600 MHz, DMSO-d6) δ (ppm): 1.50 (m, 1H, 5-H), 1.58 (m, 1H, 4-H), 1.59 (m, 2H, 8-
H), 1.61 (m, 1H, 7-H), 1.65 (m, 1H, 3-H), 1.67 (m, 1H, 2’-H), 1.69 (m, 1H, 5-H), 1.74 (m, 1H, 6-H), 1.76 and 1.80 (2 m, 2H,
3’-H), 1.84 (m, 1H, 3-H), 1.94 (m, 1H, 7-H), 2.06 (m, 1H, 6-H), 2.08 (m, 1H, 2’-H), 2.40 (dd, J = 5.1 and 15.6 Hz, 1H,
CH2CO2H), 2.43 (dd, J = 6.9 and 15.6 Hz, 1H, CH2CO2H), 2.98 (ddd, J = 6.5, 8.4 and 10.7 Hz, 1H, 4’-H), 3.02 (m, 1H, 2-H),
3.07 (ddd, J = 6.2, 7.6 and 10.7 Hz, 1H, 4’-H), 3.59 (s, 3H, CH3), 3.77 (t, J = 8.3 Hz, 1H, 1’-H), 4.47 (dt, J = 5.1 and 6.9 Hz,
1H, CHCO2CH3), 7.43 (s, 1H, NHABOC), 7.70 (d, J = 7.1 Hz, 1H, NHCHCO2CH3); 13C NMR (150 MHz, DMSO-d6) δ (ppm):
23.3 (C-4), 24.0 (C-3’), 25.4 (C-5), 25.6 (C-6 and C-8), 28.4 (C-2’), 29.2 (C-3), 30.8 (C-7), 38.2 (CH2CO2H), 44.3 (C-2), 44.4
(C-4’), 50.0 (CHCO2CH3), 51.7 (CH3), 52.7 (C-1), 60.5 (C-1’), 169.3 (C=OPro), 172.2 (CO2CH3), 172.6 (C=OABOC), 173.0
(CO2H); HRMS (ESI) Calcd for C19H30N3O6 (MH+) 396.2135, found 396.2135.
4.3. Aldol addition procedures
a) General procedure for monitoring the aldol reactions by chiral HPLC A solution of the aldehyde (0.05 mmol) and of
the selected peptide-catalyst (0.0025 or 0.01 mmol, 5 or 20%) either in a mixture of dry acetone and the selected solvent (1/2)
(0.9 ml) or in dry acetone alone (0.9 ml) was stirred at the indicated temperature (Tables 1-2). At intervals, aliquots were
collected and prepared for chiral HPLC analysis. For that, a mixture of EtOAc and water (1/1) (1 ml) was added to the aliquot,
the organic phase was concentrated under reduce pressure and the residue was dissolved in 2-propanol for HPLC analysis.
Conversion, aldolisation/crotonisation ratio and enantioselectivity of all samples were determined by using a Diacel chiralpak
AS-H column with an isocratic mixture of n-hexane and 2-propanol as eluents (Tables 1-3).
b) General procedure for aldol reactions catalyzed by the tripeptide H-Pro-(R)-ABOC-Asp-OCH3 A solution of the
aldehyde (0.2 mmol) and of the tripeptide H-Pro-(R)-ABOC-Asp-OCH3 (0.01 or 0.04 mmol, 5 or 20%) in a mixture of dry
acetone and DMF (1/2) (3.6 ml) was stirred at the -20°C for 8-72 h (Table 3). After completion of the reaction, the reaction
mixture was treated as described above and the crude product was purified by column chromatography on silica gel using
cyclohexane/EtOAc as eluent to afford the corresponding pure product (Table 3). All reaction products had characteristic
spectroscopic data in accordance with literature data.11
4.4. Crystallographic data
X-ray data of 13 were collected at 100K with a Agilent SuperNova-Dual diffractometer using the molybdenum microsource
(MoKα, λ = 0.71073 Å). Diffraction data were processed using CrysAlis RED (Oxford Diffraction, 2012). The structures were
solved by direct methods with SIR200416 and the crystallographic refinements were conducted using SHELXL-2014/7.17
H
atoms were located in difference Fourier maps. Coordinates of N-bonded H atoms were refined freely. The C/O-bonded H
atoms were placed at calculated positions and refined using a riding model. The H-atom Uiso parameters were fixed : at
1.2Ueq(C) for methine C-H, methylene groups and N-H groups and at 1.5 Ueq(C) for methyl groups and the O-H group of the
methanol molecule. CCDC 1435011 contains the supplementary crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif
4.5. DFT Calculations
Conformational searches for tripeptide 12 and 13 were performed using the Replica Exchange Molecular Dynamic method18
with AMBER14 software.19 The proline amino group was considered protonated at the expanse of the carboxylate group. The
AM1-BCC force field was used and for each compound the 500 lowest energy conformations were saved and clustered
according to geometry similarity calculated through RMSD (CPPTRAJ software).20 These representative molecular structures
were then optimized using Density Functional Theory [B97D3 functional including dispersion corrections;21 6-31G(d,p) basis
set] with Gaussian09 software.22 Solvent effects (N,N-Dimethylformamide ) were taken into account by using a polarizable

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continuum model and frequency calculations were performed in order (i) to check that true energy minima were obtained and
(ii) to calculate Gibbs free energies.
Acknowledgments The authors thank the CNRS and the MESR for financial support; the LMP (Laboratoire de Mesure
Physique) Université de Montpellier for NMR facilities, the SCBIM and Université de Lorraine for XRD facilities. This work
was granted access to the HPC resources of TGCC/CINES under the allocation 2015-c2015087449 made by [GENCI].
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ACCEPTED MANUSCRIPT
V.G.; Voth, G.A.; Salvador, P.; Dannenberg, J.J.; Dapprich, S.; Daniels, A.D.; Farkas, O.; Foresman, J.B.; Ortiz, J.V.; Cioslowski, J.; Fox, D.J.
Gaussian, Inc., Wallingford CT, Gaussian 09, Revision D.01, 2013.