Propensity for local folding induced by the urea fragment in short-chain oligomers

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Propensity for local folding induced by the urea fragment in short-chain

oligomers†

Lucile Fischer,^aClaude Didierjean,^bFranck Jolibois,^cVincent Semetey,‡^aJose Manuel Lozano,^d

Jean-Paul Briand,^aMichel Marraud,^eRomuald Poteau^cand Gilles Guichard*^a

Received 21st January 2008, Accepted 28th March 2008

First published as an Advance Article on the web 15th May 2008

DOI: 10.1039/b801139g

Examination of local folding and H-bonding patterns in model compounds can be extremely

informative to gain insight into the propensity of longer-chain oligomers to adopt speciﬁc folding

patterns (i.e. foldamers) based on remote interactions. Using a combination of experimental techniques

(i.e. X-ray diffraction, FT-IR absorption and NMR spectroscopy) and theoretical calculations at the

density functional theory (DFT) level, we have examined the local folding patterns induced by the urea

fragment in short-chain aza analogues of b- and c-amino acid derivatives. We found that the urea-turn,

a robust C₈conformation based on 1←3 H-bond interaction, is largely populated in model

ureidopeptides (I–IV) obtained by replacing the a-carbon of a b-amino acid by a nitrogen. This

H-bonding scheme is likely to compete with remote H-bond interactions, thus preventing the formation

of secondary structures based on remote intrastrand interactions in longer oligomers. In related

oligomers obtained by the addition of a methylene in the main chain (V–VIII), nearest-neighbour

H-bonded interactions are unfavourable i.e. the corresponding C₉folding pattern is hardly populated.

In this series, folding based on remote intrastrand interactions becomes possible for longer oligomers.

We present spectroscopic evidence that tetraurea VIII is likely to be the smallest unit capable of

reproducing the H-bonded motif found in 2.5-helical N,N^ꢀ-linked oligoureas.

and thus represents an interesting surrogate. We are interested in

determining the structure of urea-based oligomers belonging to

Introduction

Non-natural oligoamides built from homologated amino acid

the b- and c-peptide lineages, namely compounds of type A and B

(Fig. 1).^12,13

residues (e.g. b- and c-peptides) are the quintessential pep-

tidomimetic foldamers.^1–7Substituting heteroatoms for the carbon

atoms in the backbone of x-amino acid constituents of aliphatic

oligoamides to generate non-amide linkages (e.g. N-oxyamide,^8,9

hydrazide,^10,11urea^12,13) can dramatically alter the pattern of

intrastrand H-bond interactions, and thus represent a promising

strategy to design new foldamers.⁵In particular, the urea group

shares a number of interesting features with the amide linkage,

i.e. rigidity, planarity, polarity, and hydrogen bonding capacity,

^aCNRS, Institut de Biologie Mole´culaire et Cellulaire, Immunologie et

Chimie The´rapeutiques, 15, rue Descartes, F-67000, Strasbourg, France.

E-mail: g.guichard@ibmc.u-strasbg.fr

^bLCM3B, UMR-CNRS 7036, Groupe Biocristallographie, Universite´ Henri

Poincare´, BP 239, 54506, Vandœuvre, France

^cUniversite´ de Toulouse; INSA, UPS, CNRS; LPCNO, 135 avenue de

Rangueil, F-31077, Toulouse, France

^dFundacion Instituto de Immunologia de Colombia (FIDIC), and Universi-

dad Nacional de Colombia, Bogota´, Colombia

^eLCPM, UMR CNRS-INPL 7568, ENSIC-INPL, BP 451, F-54001,

Nancy, France

Fig. 1 a) The 1←3 H-bonding scheme in b-peptide isosteres: ureidopep-

tides of type A, a-aminoxy acid C, N^a-substituted hydrazinoacetic acid D

derivatives; b) the 2.5-helical fold of N,N^ꢀ-linked oligoureas of type B.

† Electronic supplementary information (ESI) available: Inﬂuence of

DMSO-d₆content in CDCl₃–DMSO-d₆mixtures on the NH proton

resonances for II.3, II.4, V.1a and VI.1. NOESY correlations for III.1

in CDCl₃; inﬂuence of the concentration and the solvent on the N–

H stretching absorption in compound V series; formulae of model

compounds I.M and V.M used for DFT calculations; calculated energetics,

backbone torsion angles and hydrogen bond parameters for I.M, I.3a,

V.M, and V.2a; crystal structure data. See DOI: 10.1039/b801139g

Previous work suggests that estimation of nearest-neighbour

interactions in model compounds can be extremely useful to

discriminate between backbones and identify those that may

preferentially adopt compact folding patterns with long range

order. For example, the original demonstration by Gellman and

‡ Present address: Laboratoire Physico-Chimie Curie, CNRS UMR 168,

Institut Curie, Paris, France.

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Table 1 Urea–peptide derivatives I–IV containing a gem-diamino residue

Compound

Code

R¹

R

R^ꢀ

R²

R^ꢀ2

I.1a

I.1b

I.1^ꢀꢀb

I.1c

I.2a

I.2b

I.2c

I.3a

I.3b

I.3c

I.4a

I.4b

I.4c

I.5^a

tBuO

tBu

iBu

H

Me

iPr

Me

iPr

Me

iPr

Me

iPr

Me

H

iBu

H

iBu

H

Me

H

Me

H

Me

H

Me

H

tBuO

Me

iBu

Bn

CH₂OBn

(CH₂)₃

Bn

iPr

H

I.6^b

Me

H

II.1a

tBuO

iBu

—

Me

H

II.1^ꢀa

II.1b

II.1^ꢀc

II.2

tBu

iBu

—

Me

iPr

Me

H

Me

H

tBuO

tBu

tBuO

CH(Bn)CO₂Me

II.3

II.4

BnO

Me

H

Me

iPr

H

III.1

III.2

tBuO

H

iBu

H

—

IV

—

^aRef. 16a. ^bRef. 16b.

coworkers that intramolecular H-bonding between neighbouring

amide groups is not a favourable process in simple diamides

derived from b-alanine led to the proposal that folding based on

remote intrastrand interactions would be favoured in oligomers

composed of b-amino acids.¹⁴

such as a-aminoxy acid C and N^a-substituted hydrazinoacetic acid

D derivatives (i.e. the N–O⁸and hydrazino^10,11(or N–N) turns,

respectively) (Fig. 1a).¹⁵

In contrast, ureido monomers of type B possessing an extra

carbon in their backbone are not expected to favor intramolecular

hydrogen bonding between adjacent residues. This is supported

by the ﬁnding that homochiral N,N^ꢀ-disubstituted oligoureas

of type B form robust helical structures stabilized by remote

H-bond interactions closing 12 and 14-membered pseudorings

(Fig. 1b).¹²To further delineate the propensity for local folding

induced by the urea fragment in compounds of type A and B,

we have now undertaken a detailed conformational investigation

of short-chain ureido peptidomimetics I–VIII (see Tables 1 and

2) using a combination of experimental techniques (i.e. X-

ray diffraction, FT-IR absorption and NMR spectroscopy) and

theoretical calculations using density functional theory (DFT).

Preliminary results from our laboratories suggest that sub-

stituting a nitrogen for the a-carbon in b-amino acid deriva-

tives, to give ureido compounds of type A, makes nearest-

neighbour interactions more favourable than in b-amino acids

counterparts.^13aIn non-polar solvents, type

A

N-acyl-N^ꢀ-

carbamoyl-gem-diaminoalkyl derivatives have been found to

populate a C₈conformation with a 1←3 H-bonding pattern,

reminiscent of the C₇a-peptide c-turn. A particular feature of

this folding pattern (i.e. the urea-turn) is that the urea adopts a

characteristic cis,trans (E,Z) geometry.^13aIt is noteworthy that very

similar turn conformations have been observed in isosteric systems

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Table 2 Urea–peptide derivatives V–VIII having two sp³carbons in the main chain

Compound

Code

R¹

R

R²

R^ꢀ2

V.1a

V.1b

V.1c

V.2a

V.2c

tBuO

Bn

Me

iPr

Me

H

Me

H

Me

CH(Me)OBn

VI.1

VI.2

tBu

Bn

CH(Me)OBn

Bn

Me

H

VII

Bn

Me

H

VIII

Results and discussion

Synthesis of urea-based compounds

The urea-containing peptides and oligoureas I–VIII (Tables 1 and

2) are divided in two families depending on the number of sp³

carbons between the nitrogen atoms in consecutive urea or amide

groups.

Compounds of type A (I–IV, containing a 1,1-diamino

alkyl residue) were synthesized as previously described^13b

by coupling succinimidyl {1-{[(alkyloxy)carbonyl]amino}-1-X-

methyl}carbamates or succinimidyl [1-(acylamino)-1-X-methyl]

carbamates (derived from N-protected a-amino acid and dipep-

tides, respectively) to simple amines or a-amino acid esters.

Ureido compounds of type B (V–VIII containing a 1,2-

diamino alkyl residue) were prepared from succinimidyl {2-

{[(tert-butoxy)carbonyl]amino}-2-X-ethyl}carbamates as previ-

ously reported.¹⁷Compounds IV,^13bVII and VIII were obtained

by repetitive urea formation with appropriate succinimidyl carba-

mates.

Comparative spectroscopic study of ureido compounds of type A

and type B

The relative propensity for local folding induced by the urea

fragment in model ureas with a 1,1-diaminoalkyl residue (I, type A)

and a 1,2-diaminoalkyl residue (V and VI, type B) was studied in

solution using a combination of FT-IR and NMR spectroscopies.

The IR data (Table 3) of compounds I with R^ꢀ2= H are not

signiﬁcantly sensitive to dilution below 10 mM in CH₂Cl₂and

2 mM in CCl₄, indicating a similar behaviour to analogous peptide

models.^18,19

The broad absorption in the 3300–3400 cm⁻¹region, which is

assigned to H-bonded NHs, shifts to low frequencies when R²=

Me is changed into iPr, and disappears for R^ꢀ2= Me (Fig. 2a,

Table 3). Therefore it may be assigned to the H-bonded N²H

=

Fig. 2 Superimposition of a) NH stretch and b) C O stretch region

FT-IR data for 2 mM compounds I.3a, I.3b and I.3c in CH₂Cl₂.

vibrator.¹⁹On the other hand, the stretching absorption of the

CO¹carbonyl is shifted from about 1695 cm⁻¹for R^ꢀ2= H to

about 1715 cm⁻¹for R^ꢀ2= Me (Fig. 2b, Table 3). We may then

conclude that part of molecules I with R^ꢀ2= H are folded by an

N²H-to-CO¹intramolecular H-bond closing an 8-membered ring

(Fig. 3a).

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=

Table 3 N–H and C O stretching frequencies for the urea–peptide models I and V

1

2

N²–H + N^ꢀ2–H

Free

N²–H

Free

C

O

C

O

=

Cmpd

Solvent

Bonded

Free

Bonded

Free

I.1a

I.1b

I.1^ꢀꢀb

I.3a

I.3b

I.2a

CH₂Cl₂

CCl₄

CH₂Cl₂

CCl₄

CH₂Cl₂

3453^sh

3431

3350

1718^sh

1695

1676

1667

————3430————

————3437————

3339^m,br

3286 ^m,br

3363^w,br

3344^w,br

3352^m,br

3346^m,br

3336^m,br

3346^m,br

—

1662^br

3453

3431

n.v.

1698

1692

1693

1694

1702

—

1679

1669

1677

1689

1667

1682

1657

————3431————

3431

3462/3439

————3431————

3462/3439

3445

3450^sh

—

1718^sh

1714^sh

1713

I.2b

—

I.2c

I.4a

I.4b

I.4c

—

3429

3451^sh

3312^br

3292^br

—

1696^sh

1693

—————1673————––

—————1670————––

—

————3431————

3455

—

1656

V.1a

CH₂Cl₂

CCl₄

CH₂Cl₂

CCl₄

3432

3442

3436

3472/3432

3480/3442

3452

3459

3448^sh

—

3366^w,br

3356 ^br

3367^br

—

1703

1707

1704

1707

n.v.

1677

1690

1682

1648

1658

V.1b

V.1c

—

compounds I.1a–I.3a) (see Fig. 4 (heavy line), Fig. 6 (left panel)

and Fig. 7 (left panel)).

The trans,trans conformation of the urea fragment is not

compatible with the above N²H–CO¹H-bond, and a cis,trans

conformation is geometrically required. This point was conﬁrmed

by NOESY experiments in solution in CDCl₃(Fig. 5).

Molecule I.1a in CDCl₃actually exhibits a C^aH/N²H NOE

correlation, denoting a short interproton distance typical of

the cis,trans conformation, but no N²H/N^ꢀ2H NOE correlation

that would denote the trans,trans conformation (Fig. 5, lower

panel). For molecules I.1a and I.1c, we veriﬁed that the cis,trans

conformation of the urea fragment is not strictly dictated by

the occurrence of the N²H–CO¹intramolecular H-bond. The

cis,trans conformation is effectively retained by I.1a in DMSO,

where solvation breaks this H-bond. In I.1c (R²= R^ꢀ2= Me),

where the single resonance for both methyl groups indicates a

rapid rotation of the CO²–N²Me₂bond, the Me/C^aH (in CDCl₃)

and Me/N^ꢀ2H (in DMSO-d₆) NOE correlations denote a solvent-

induced transition from the cis (CDCl₃) to the trans (DMSO-d₆)

conformation of the N^ꢀ2–CO²bond (Fig. 5, upper part).

Fig. 3 Schematic representation of (a) the C₈c-like folded structure

induced by the urea–peptide motif in I.1a, and (b) the minor folded C₉

structure induced by the urea–peptide motif in V.1a, showing the cis,trans

conformation of the N,N^ꢀ-disubstituted urea fragment.

The N¹H and N^ꢀ2H bonds are free from any intramolecular

interaction, as demonstrated by NMR DMSO-d₆titration exper-

iments in CDCl_3.By progressive addition of DMSO-d₆in CDCl₃,

their proton resonances actually experience a higher shift (Dd :

1.6–2.1 ppm for N¹H and 0.83–1.22 ppm for N^ꢀ2H in compounds

I.1a–I.3a) to low ﬁelds than N²H (Dd : −0.22 to 0.42 ppm in

Fig. 4 Inﬂuence of DMSO-d₆content in CDCl₃–DMSO-d₆mixtures on the NH proton resonances for I.2a (solid line) and V.1a (dotted line).

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Fig. 5 NOESY correlations demonstrating the cis,trans conformation of the N,N^ꢀ-disubstituted urea fragment for I.1a in CDCl₃and DMSO-d₆(bottom)

and the transition from the cis (CDCl₃) to the trans (DMSO-d₆) conformation for the urea N^ꢀ2–CO²bond in I.1c (top).

Qualitative analyses of IR data reveal a similar behavior between

compounds I and V (Table 3), but the intensity of the broad

contribution in the 3300–3400 cm⁻¹region is much weaker in

CH₂Cl₂for the latter, indicating a smaller percentage of folded

molecules (see Fig. S1†).

In CCl₄, the intensity of the low frequency component is highly

sensitive to the concentration down to 0.2 mM, which denotes

a great tendency to molecular aggregation (see Fig. S1†). The

residual absorption at 0.04 mM reﬂects the existence of a minor

percentage of folded molecules with an N²H–CO¹intramolecular

H-bond closing an 9-membered ring (Fig. 3b).

N²H–CO¹H-bond implies that the cis–trans conformation of the

urea fragment is populated (albeit to a low extent) in solution.

The molecular ﬂexibility of the urea models deriving from an

1,2-diaminoalkyl residue is corroborated by the magnetically

equivalent main-chain CH₂protons in compound V series. In

addition, the larger chemical shifts experienced by N²H in the

compounds V series compared to compound I series upon

progressive addition of DMSO-d₆in CDCl₃indicates a higher

solvent accessibility (see Fig. 4, right panel). The NH accessibility

for diurea VI.1 in CDCl₃–DMSO-d₆mixtures revealed that N²H is

a little less accessible in VI.1 than in V.1a (see Fig. S2†), suggesting

that the percentage of the N²H–CO¹H-bond is a little higher,

probably being related to the higher basicity of the urea carbonyl

in VI.1 compared with the urethane carbonyl in V.1a.

Because of the small concentration (0.2 mM) required to have

non-aggregated molecules V in CCl₄, NOESY experiments could

not be carried out in this case. However, the existence of the

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Fig. 6 Inﬂuence of DMSO-d₆content in CDCl₃–DMSO-d₆mixtures on the NH proton resonances for I.1a (left), II.1^ꢀa (middle) and II.1a (right).

that proline does not prevent the occurrence of the urea-turn.

It is noteworthy that the high and low solvent accessibility for

the N²H proton contributions in II.1a depends on the cis and

trans conformation of the Boc–Pro bond, respectively (Fig. 6, right

panel). Combined with the persistence of a broad NH absorption

at 3306 cm⁻¹for II.1^ꢀc, this observation indicates that N²H in

II.1^ꢀa is partly engaged in a 1←3 interaction with CO¹to give

Fig. 7 Schematic representation of the C₈urea-turn conformation

overlapping c- and urea-turns (Fig. 7).

associated with the c-folded Pro residue in II.1^ꢀa.

It is noteworthy that the same trend i.e. c-turn nucleation, albeit

to a lower extent, was observed when alanine was substituted

for proline (see Fig. S3†). The chemical shift variation observed

for N²H in II.3 when increasing the DMSO concentration from

Spectroscopic studies of type A amide/urea hybrids II–IV

An additional series of compounds of type A (II–IV, Table 1) have

1.25% to 100% is larger than when a prolyl residue was present

(0.92 ppm versus 0.37 ppm) but is signiﬁcantly lower than

that of a fully accessible amide NH (ca. 1.28 ppm for N³H

in compound III series; vide infra). Substituting D-Ala for L-

Ala in II.3 (i.e. heterochiral versus homochiral sequences) has

no signiﬁcant effect on the chemical shift variation of N²H

(Dd = 0.94 ppm).

been prepared to evaluate the inﬂuence of i) the position of the

urea moiety in the peptide chain (central in III versus terminal in

II), and ii) the number of urea moieties (IV) on folding propensity

and C₈urea-turn formation.

When proline precedes the gem-diamino residue, the NH

stretching for II.1^ꢀa exhibits a strong, broad absorption at

3327 cm⁻¹which considerably decreases for II.1^ꢀc (R^ꢀ2= Me).

The low solvent accessibility for the N³H proton in II.1^ꢀa is quite

similar to that for N²H in I.1a (Fig. 6).

The incorporation of the urea group between two peptide bonds

in III.1 and III.2 does not affect signiﬁcantly the urea spectroscopic

data. For example, the N²H proton resonance is the less sensitive

to solvation while those for N¹H and N³H experience a large shift

to low ﬁelds (Fig. 8).

Substitution of Boc for Piv in II.1a allows the cis–trans

equilibrium around the Boc–Pro bond, but has no inﬂuence on

the N³H solvent accessibility (Fig. 6). All these data indicate

Fig. 8 Inﬂuence of DMSO-d₆content in CDCl₃–DMSO-d₆mixtures on the NH proton resonances for I.3a (left), III.1 (middle) and III.2 (right).

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Moreover, the proﬁles of NH resonances for III.1 and III.2

are practically superimposed, indicating that the chirality of the

sequence (homochiral versus heterochiral) has no inﬂuence on

the conformational properties induced at a short distance by

the urea group. The N²H/C^aH NOE correlation again conﬁrms

the cis,trans conformation of the urea group (See Fig. S4†).

This NOE pattern is conserved in longer hybrid oligomers

made of alternating amide and urea bonds such as tetramer IV

(Fig. 9).

thermodynamic as well as to kinetic data. cis,trans-DMU actually

lies at D_rG^◦=1.3 kcal mol⁻¹above its trans,trans- counterpart,

=

indicating that rotation about C( O)–N bonds in ureas leads

to a less unstable isomer. It should be noted that, according to

theoretical calculations, some alkyl-substituted urea derivatives

are expected to be more stable in their cis conformation.²⁰In

addition, while the lowest²¹free energy of isomerisation of NMA

lies 19.6 kcal mol⁻¹above the trans isomer, the transition state

which connects trans,trans and cis,trans isomers of DMU lies only

9.1 kcal mol⁻¹above the former geometry. This value is similar to

those obtained by previous experimental²²or theoretical²⁰studies

on ureas. In other words, rotation about an urea bond is less

demanding than rotation about a peptide bond and cis,trans urea

bonds should be signiﬁcantly more populated than cis peptide

bonds in oligomers.

Preliminary studies on model compounds with R¹= R =

R²= Me ((1R)-N-[1-(3-methylureido)ethyl]-acetamide and (1S)-

N-[1-methyl-2-(3-methylureido)ethyl]acetamide (See Fig. S5†),

hereafter called I.M and V.M) have provided a ﬁrst overview on

the structures and energies of different folding patterns. In order

to check the role of substituents and side-chains on the stability

scale found for the models, we have also addressed two of the

urea-containing derivatives studied in the experimental part of

this work, namely I.3a and V.2a. All structures considered in this

theoretical work are shown in Figs. 10 and 11 for compounds I

and V, respectively.

The nomenclature ﬁrst refers to unfolded patterns (u), or to the

n-membered (Cn) H-bonded ring, followed by a capital letter when

several geometrical patterns lead to the same ring size. Eight- and

nine-membered rings are only observed in the case of urea-turns

in I and V respectively (C8–I and C9–V types). Besides this folding

pattern, for topological reasons hybrid amide–urea models such

as I can only exhibit an unfolded structure (u–I) or 6-membered

rings. Similarly, compounds V can be unfolded or may exhibit

7-membered pseudocycles. The stability of the conformations

envisaged in this work is mainly ruled by three factors : i) the

energy associated with the backbone, ii) the directionality and

bond length of hydrogen bonds, iii) the energy cost associated

to trans→cis inversion of urea bonds. Considering the intrinsic

penalty of 2.5 kcal mol⁻¹generated by trans→cis isomerisation of

peptide bonds, we only considered peptide fragments in their trans

conformation.

Fig. 9 NOESY correlations demonstrating the cis,trans conformation of

N,N^ꢀ-disubstituted urea fragments for IV in DMSO-d₆.

Theoretical calculations

Urea models containing a gem-diamino residue (type A). All

the results are collected in Table S1†. Relative Gibbs free energies

(D_rG^◦) differ from DE by less than 1 kcal mol⁻¹, thus showing

that entropic effects do not play a signiﬁcant role in the energies.

Except for C6C–I.M, we systematically found two conformations

associated with a given folding pattern. They differ by the torsion

angles φ and w, and in most cases by the hydrogen bond

parameters, i.e. the angle between N–H and H · · · O (h) and the

distance between H and O (r_hb). In the case of the urea-turn, it is

interesting to notice that according to φ and w, structures of type

C8–I and C8^ꢀ–I are reminiscent of the C₇a-peptide classic and

inverse c-turns. None of the geometry parameters not signiﬁcantly

differ between compounds I.M and I.3a.

To gain additional information on interactions between nearest

neighbours in ureido compounds of type A and B, we have

performed theoretical investigations using density functional

theory (DFT) on model compounds I and V.

Before starting the exploration of potential energy surfaces of

ureidopeptides, a brief comparison of the energy properties of N-

methyl acetamide (NMA) and N,N^ꢀ-dimethylurea (DMU), which

are parent representatives of peptide and urea plaques, may be

instructive. The lower thermodynamic stability of cis-NMA with

respect to trans-NMA (by 2.6 kcal mol⁻¹at the present level of

calculation), restrictions in conformational space, non-covalent

stabilization of trans amide bonds, and the so-called steric clash

between successive side-chains in all-cis peptides, are invoked for

explaining the extreme scarcity of cis peptide bonds in natural

proteins. Rotation about urea bonds is less unfavourable, owing to

The model compound in its unfolded geometry (u–I.M) is not

a minimum on the potential energy surface, whereas we have

found two stable geometries for the “real” system I.3a. Such a

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Fig. 10 Molecular conformations for compound I.M and selected conformation for compound I.3a. Calculated Gibbs free energies (D_rG^◦, in kcal mol⁻¹

)

are also given for I.M (plain text) and similar conformations for I.3a (italic). Hydrogen bonds are systematically depicted, even though values for h and

r_hbindicate that they are very weak for C6A^ꢀ–I, C6B^ꢀ–I, C6C–I, C6D^ꢀ–I. See the ESI† for additional details.

structure may thus exist in the gas phase, but it is signiﬁcantly

less stable than the urea-turn C8^ꢀ–I.3a by 3.3 kcal mol⁻¹. The

most important result is that one of the two structures which

exhibits a C₈conformation is the most stable one among all

the isomers considered in this work. This is already true for the

model compound I.M, and more marked for I.3a, since the next

structures in the energy scale (C6A^ꢀ–I.3a and C6B^ꢀ–I.3a, with trans

peptide and urea bonds) lie ca. 2.5 kcal mol⁻¹above. We have also

undertaken the study of two conformations which exhibit cis,trans

and trans,cis bonds in NH–CO–NH (C6C–I and C6D–I). These

structures are relatively high in energy.

In summary, it is noteworthy that the stability of the urea-turn

is important enough to ensure that compound I.3a preferentially

adopts this conformation. The relative stability of C8^ꢀ–I.M

suggests that this preference for the urea-turn motif should resist

to substitution.

This interaction is allowed by the ﬂexibility introduced by the

additional methylene group. Although this structure could be

favoured under speciﬁc conditions (solvent or substituents), it was

not the lowest-energy conﬁguration in the gas phase. The two

most stable conformations are C7A–V and C7B–V, for both the

model and real systems. The directionality of the hydrogen bond

(h) together with the r_hbdistance are in the range of relatively

strong hydrogen bonds. It is also the case for the urea-turn

C9–V, which is probably unstabilized owing to the backbone.

In opposition to compounds of type I, the two conformations

which exhibit cis,trans and trans,cis bonds in NH–CO–NH (C7C–

V and C7D–V) have low energy. Nevertheless, it is unlikely that

the former could be observed since, according to D_rG^◦, it lies at

least 3 kcal mol⁻¹above the lowest-energy structures C7A–V and

C7B–V.

In summary, in the case of ureido compounds with an additional

methyl group in the backbone, C₉turn conformations are not

expected to be observed. The effect of side-chains or substituents in

V.2a does not improve the stability of the urea-turn with respect to

its counterpart in the model compound V.M. In addition, several

folded structures, as well as the unfolded conformation, compete

in a narrow energy range and are more likely populated than the

urea-turn.

Urea models with an additional methylene group (type B). In

this case, we have only found a single conﬁguration for each folding

pattern. All the results are given in Table S2†. In both the model

system V.M and the real system V.2a, the urea-turn does not

appear viable, being less stable than the extended conformation u–

V and all other folded structures. Interestingly, we have found

an additional structure (//–V) in which the peptide and urea

plaques are almost parallel. This structure is stabilized by a dipole–

dipole interaction (↓↑) between the two chemical fragments. As

a matter of fact, our calculations on NMA and DMU show that

these compounds have similar dipole moments (ca. 3.6 Debye).

Crystal structures

Whereas spectroscopic and theoretical studies above indicate that

nearest-neighbour hydrogen interaction are favoured in ureido

compounds of type A, the situation turned out to be different in

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None of the crystal structures displays an intramolecular

hydrogen bond. Except rac-I.4b and II.1^ꢀa, where proline logically

induces a kink, the molecules assume more-or-less extended

conformations, probably favoured by intermolecular interactions

in the crystal (Fig. 12).

The main torsion angles for the urea-containg unit²³are listed

in Table 4.

In all cases, the NH–CO–NH urea fragment is nearly sym-

metrical and trans,trans planar.²⁴One notes that, similarly to L-

peptides, the φ angle is negative (−133^◦to −89^◦). The pseudo

“w” angle generally adopts a positive value (84^◦to 137^◦), except

for V.1a where the negative value is associated with a skewed

conformation of the 1,2-diamino ethane fragment. In all the cases

except II.2, where the iBu substituent is trans to the nitrogen (v¹=

−173^◦), the iBu or Bn side substituent retains the same gauche-

orientation (v¹= −71^◦to −58^◦). The iPr substituent in I.6 assumes

a

b

the classical disposition, with C–H and side-chain C–H bonds

in the trans conformation.

All compounds form hydrogen-bonded ladder structures

(Fig. 12). Successive molecules in the ladder are either parallel

related by a translation for I.5, V.1a, V.1c and VI.2 or antiparallel

related by a 2₁screw axis for rac-I.1b, I.3c, I.6, II.1^ꢀa and II.2. Com-

pound rac-I.4b is different because adjacent molecules present

the same chirality and are crystallographically independent. The

˚

intermolecular spacing (4.67–5.24 A) is similar to that found for

symmetrically disubstituted ureas.²⁵The NH–CO–NH urea motif

is involved in more-or-less complex contacts, with both urea NHs

being engaged in an H-bond with i) the same amide carbonyl in

I.6, II.1^ꢀa and II.2, ii) the urethane carbonyl in I.1b, or iii) the urea

carbonyl in I.4b, I.5, V.1a and VI.2. The N · · · O distances (2.84–

26

˚

3.58 A) may be above the limiting value for a classical H-bond.

Fig. 11 Molecular conformations for compound V.M. Calculated Gibbs

free energies (D_rG^◦, in kcal mol⁻¹) are also given for V.M (plain text) and

similar conformations for V.2a (italic). See the ESI† for additional details.

˚

The closest double contacts (2.91 A and 2.97 A) are observed for

˚

VI.2, and the loosest ones (3.51 A and 3.58 A) for V.1a. In one

case (II.1^ꢀa), a urea NH is engaged in an H-bond network with

three carbonyls, but the N · · · O distances are rather large (3.46,

˚

3.48 and 3.50 A). One also notes that the NHs of contiguous urea

and/or amide groups in the same molecule may be oriented in the

same (I.1b, I.5, I.6, II.1^ꢀa and II.2) or opposite (V.1a and VI.2)

directions.

the solid state. We obtained crystals suitable for X-ray diffraction

for eight of the urea-containing derivatives (rac-I.1b, I.3c, rac-I.4b,

II.1^ꢀa, II2, V.1a, V.1c, VI.2, see Table S3†).

Table 4 Main torsional angles (^◦) of the urea-containing unit^a

Compound^b

x_1,1

171.4(2)

x_1,2

179.1(2)

φ

m

w

x_2,1

x_2,2

172.6(2)

v¹

v²

I.1b

I.3c

I.4b

I.5^c

−122.8(2)

−115.4(7)

−76.3(2)

−111

—

−92.3(2)

129.8(6)

137.0(2) −173.4(2)

169.3(2)

178.6(6)

−58.1(3)

−63.1(3)/173.2(2)

−177.8(6)

−172.6(6)

−177.9(7)/15(1)

−179.9(2)

−174

−67.0(8)

149(1)

173.1(2)

3.5(3)

30.6(2)

−39.6(2)

—

174

176

178.8(3)

176.2(5)

93

105

−166

−60

−89/91

I.6^d

−113

179

174

−58/180

−58.6(4)

−171.7(6)

—

II.1^ꢀa

II.2

−125.5(3)

−122.5(6)

83.7(4) −168.1(3)

−178.9(3)

−65.3(4)/171.4(3)

118.5(5)

172.1(5)

−179.3(6)

69.8(8)/−166.1(6)

V.1a

V.1c

VI.2

176.8(6)

177.7(2)

−179.4(5)

−167.0(5)

−169.1(2)

177.3(4)

−123(1)

58(1)

−145(2)

112.9(3)

105.4(5)

−173(2)

175.4(2)

175.7(4)

−173(2)

−64.9(9)

−62.6(3)

−85.7(8)/94(1)

−133.0(2) 171.8(2)

175.4(3)/−22.4(4)

−87.7(2)/90.8(3)

−89.2(5) 177.1(4)

−179.3(5)

−71.1(5) −100.5(7)/82.8(7)

^aRef. 23. ^bThe angles are reported for the molecule having the same absolute chirality as the starting L-amino acid. ^cRef. 16a. ^dRef. 16b.

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Fig. 12 Molecular conformations and intermolecular interactions in the crystal structures of compounds I.1b, I.3c, I.4b, II.1^ꢀa, II.2, V.1a, V.1c, and

VI.2 (see Table S3 in ESI for crystal data and structure reﬁnement and Table 4 for torsional angles of the urea-containing unit). Intermolecular NH · · · O

˚

distances in A are indicated.

From unfavourable local folding to remote intrastrand interactions:

¹H NMR studies of short oligomers of type D

helical conformation, we have investigated derivatives containing

three (VII) and four (VIII) urea motifs.

Due to their low solubility in chloroform, compounds VII and

VIII with three and four urea motifs were examined in DMSO-d₆

and in CDCl₃–30% DMSO-d₆and compared to diurea VI.2 (see

According to spectroscopic data collected on monomeric com-

pounds V and VI and theoretical studies, intramolecular hydrogen

bonding between nearest neighbours is not a favourable process

for compounds of type B. This is consistent with previous ﬁnding

from our laboratories showing that longer oligomers of type B

(heptaurea and nonaurea) adopt a helical fold stabilized by remote

intrastrand interactions.^12a,cThis 2.5-helical structure is stabilized

by a double H-bonding scheme where each carbonyl is H-bonded

to both NHs of the urea two residues ahead (See Fig. S6†).

Accordingly, one would expect a triurea to be the smallest

molecule capable of reproducing a turn of the aforementioned

helix. To investigate the minimum length required to nucleate a

1

Tables S4–S12†). Qualitative examination of H NMR spectra

revealed a number of interesting features. The NH regions of

diurea VI.2 and triurea VII remain poorly dispersed in both

pure DMSO and CDCl₃–30% DMSO-d₆. In contrast, the urea

NH signals of tetraurea VIII in CDCl₃–30% DMSO-d₆are very

well dispersed. The NH chemical shifts with DMSO solvation

were generally smaller for VIII than for VI.2. In addition, the

non-equivalence of the main-chain CH₂protons spectacularly in-

creased from diurea/triurea to tetraurea. In CDCl₃–30% DMSO-

d₆, the chemical shift difference changes from 0.2 ppm for VI.2 to

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0.88–1.29 ppm for VIII, thus supporting the existence of a rigid

structure for tetraurea VIII.

dichotomy was noticed earlier by Yang and coworkers in the a-

and b-aminoxy peptide series.^8,9bWhereas in non-polar solvents

short aminoxypeptides adopt helical conformations consisting of

successive N–O turns, extended parallel and antiparallel sheet-

like structures stabilized by intermolecular H-bonds have been

observed in the solid state.

Interestingly, experimental NMR and IR data on (amide/urea)

hybrids II support the formation of overlapping c-turns and urea-

turns (see Fig. 7). The comparison with type III urea/amide

hybrids suggest that c-turn nucleation could be favoured by

the presence of a subsequent urea-turn. This conformational

preference of oligo(amide/urea) hybrids in non-polar solvents

parallels that observed in peptides composed of alternating a-

aminoxy acids and a-amino acids.²⁷

The present ¹H-NMR data obtained for VIII in CDCl₃–

30% DMSO-d₆strongly suggest that the minimum number of

urea units required to initiate folding in N,N^ꢀ-linked oligoureas

is four. In particular, the large³J(N²H,^bCH) values (9.7 Hz), the

large chemical-shift differences (Dd) and strong differentiation of

vicinal coupling constants for diastereotopic protons of the central

3

residue (Dd = 1.29 ppm; J(N^ꢀ3H,^aCH) = 3.2 Hz and 9.6 Hz

for upﬁeld and downﬁeld ^aCH, respectively), as well as the i/i+2

NOE correlations (Fig. 13) observed in VIII, are all spectroscopic

features previously associated with helical oligoureas.

Examination of local folding and H-bonding patterns in model

compounds can be extremely informative to gain insight into the

propensity of longer-chain oligomers to adopt speciﬁc folding

patterns based on remote interactions. Estimation of H-bonding

between nearest-neighbour amide groups in simple b-alanine

and c-amino butyric acid derivatives was used by Gellman and

coworkers as a criterion to estimate the relative propensity of b-

and c-peptide backbones to adopt compact and speciﬁc folding

patterns.¹⁴By analogy, the 1←3 H-bonds that occur in model

ureido compounds of type C and related oligo(urea/amide)

hybrids is likely to compete with long-range order H-bonds,

thus preventing the formation of secondary structures based on

remote intrastrand interactions in longer oligomers. In contrast,

the addition of a methylene in the main chain (e.g. type B residues)

noticeably decreases the stability of the folded structure. Although

the H-bond closing the 9-membered pseudocycle (Fig. 3b) is

clearly visible in CCl₄, it is hardly populated in a slightly more polar

solvent such as CH₂Cl₂. Theoretical calculations conﬁrmed this

trend and identiﬁed a number of alternative conformations that

are more likely to be populated. Folding propensity does not in-

crease signiﬁcantly in corresponding diurea and triurea oligomers.

However, the presence of four consecutive urea fragments in this

series results in the appearance of a more rigid and folded structure,

which probably corresponds to the 2.5-helical turn found in helical

hepta- and nonaurea oligomers,¹²and which is reminiscent of the

14-helical structure of c⁴-peptides.²⁸

Fig. 13 Representative inter-residue NOE connectivities observed for

tetraurea VIII in CDCl₃–DMSO-d₆(70 : 30) at 298K. s = strong, m =

medium, and w = weak. These NOE connectivities are consistent with

VIII being the smallest unit capable of populating a 2.5-helical fold.

Conclusion

All the above spectroscopic data indicate that the N,N^ꢀ-

disubstituted urea is a ﬂexible fragment. In the solid state,

where it is systematically engaged in intermolecular H-bonds, the

Experimental

=

trans,trans conformer placing the N–H and C O bonds in an

General

anti orientation is observed in all cases. In solution, however, the

urea motif recovers a conformational freedom, and the CO–N

bond may assume the cis and trans conformations. In the case of

ureido compounds of type A, it essentially populates the cis,trans

conformation and gives rise to a short-distance interaction, with

the preceding peptide carbonyl in a folded structure originally

being term a urea-turn (Fig. 3a).^13aTheoretical investigations

using density functional theory (DFT) are in good agreement with

experimental data. The H-bond closing an 8-membered ring in the

urea-turn is not essential to the cis–trans conformation of the urea

fragment.

Amino acid derivatives were purchased from NeoMPS or Nov-

abiochem. THF was freshly distilled from sodium/benzophenone

˚

under Ar. Toluene was distilled from P₂O₅and stored over 4 A

molecular sieves. The reactions were carried out under an excess

pressure of Ar. HPLC analysis was performed on a Nucleosil C₁₈

column (5 lM, 3.9 mm × 150 mm by using a linear gradient of

A (0.1% TFA in H₂O) and B (0.08% TFA in MeCN) at a ﬂow

rate of 1.2 mL min⁻¹with UV detection at 214 nm. ¹H NMR and

¹³C NMR spectra were recorded using Bruker Avance Apparatus

DPX-300, ARX-300 and DRX-600. Chemical shifts (d) are given

in ppm, and J values are given in Hz. Optical rotations were

obtained using a Perkin–Elmer polarimeter, with [a]_Dvalues being

given in 10⁻¹deg cm²g⁻¹.

The striking difference between the conformations observed

in solution and those existing in the solid state is not un-

precedented in the ﬁeld of peptidomimetic foldamers. A similar

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IR spectra were obtained in the Fourier transform mode

on a Bruker IFS-25 apparatus. Matrix-assisted laser desorp-

tion ionization–time-of-ﬂight (MALDI-TOF) mass analysis was

performed on a linear MALDI-TOF instrument (Flex Control

generated Xmass Data, 2000 Bruker Datoniks), using a-cyano-

4-hydroxycinnamic acid as the matrix. Mass analyses in ESI

(electrospray ionisation) mode were acquired on a LCQ Advantage

MA MSn LCMS instrument (ThermoFischer Scientiﬁc).

7.99 (1H, d, J 7.5, NHCOCH); d_C(50 MHz, DMSO-d₆) 22.2

(CH₃) 22.3 (CH₃), 23.0 (CH₃), 24.2 (CH), 26.0 (CH₃), 27.8 (CH₃),

30.9 (CH₂), 43.7 (CH₂), 46.4 (CH₂), 55.3 (CH), 59.5 (CH), 78.3

(C), 153.4 (C), 157.3 (C), 171.7 (C); HRMS (ESI) m/z calcd for

C₁₇H₃₃N₄O₄[M + H]⁺: 357.2496, found 357.2467.

Boc-Pro-gLeu-CO-Phe-OMe (II.2). Boc-Pro-gLeu-COOSu

(500 mg, 1.135 mmol) was reacted with HCl·H-Phe-OMe (269 mg,

1.248 mmol) and DIEA (395 lL, 2.27 mmol) according to the

general procedure to yield II.2 (530 mg, 92%): a white solid,

General procedure for the preparation of ureas

◦

mp 168 C. [a]²_D⁵−17.7 (c 1.1 in DMF); HPLC t_R= 10.46 min

To a stirred solution of the amine (1.1 equiv.) in 10 mL of

MeCN or DMF were successively added succinimidyl carbamate

(usually ca. 1 mmol) and Hunig’s base (1.2 equiv.). After 10–

30 min, the mixture was diluted with saturated NaHCO₃and

extracted with EtOAc. The organic layer was washed with 1 N

KHSO₄, brine, saturated NaHCO₃and brine, dried (Na₂SO₄),

and evaporated. Flash chromatography and/or recrystallization

afforded pure ureido peptidomimetics.

(linear gradient, 30–100% B, 20 min); d_H(200 MHz, DMSO-

d₆) 0.69–0.88 (6H, m, CH₃), 1.21–1.54 (12H, s, C(CH₃)₃

+

CH₂CH(CH₃)₂), 1.58–1.81 (3H, m, CH₂CH₂CH₂CH), 1.85–2.08

(3H, m, CH₂CH₂CH₂CH), 2.75–2.97 (2H, m, CH₂Ph), 3.15–3.41

(2H, m, NCH₂CH₂), 3.53 (3H, s, OCH₃), 3.85–4.01 (1H, m,

NHCHCO), 4.25–4.44 (1H, m, NHCHCO), 5.02–5.24 (1H, m,

NHCHNH), 6.36 (1H, br d, J 8.5, NHCONH), 6.45 (1H, br d, J

8.5, NHCONH), 8.97 (1H, br d, J 7.7, NHCOCH); d_C(50 MHz,

DMSO-d₆) 22.3 (CH₃) 22.9 (CH₂), 27.9 (CH₃), 30.9 (CH₂), 37.8

(CH₂), 43.7 (CH₂), 46.4 (CH₂), 51.5 (CH), 51.5 (CH₃), 53.7 (CH),

55.2 (CH), 78.3 (C), 126.4 (CH), 128.1 (CH), 129.0 (CH), 136.9

(C), 153.3 (C), 155.9 (C), 171.6 (C), 172.6 (C); HRMS m/z calcd

for C₂₆H₄₁N₄O₆[M + H]⁺: 505.3021, found 505.2730.

Analytical data for representative compounds I–VII

Boc-gLeu-CONHiPr (I.1b). Boc-gLeu-COOSu (1.716 g,

5.00 mmol) was reacted with isopropylamine (511 lL, 6.00 mmol)

according to the general procedure to yield I.1b (1.153 g, 80%):

white solid, mp 123 ^◦C. [a]_D²⁵−13.7 (c 1.0 in DMF); d_H(200 MHz,

DMSO-d₆) 0.80 (6H, d, J 6.1, CH₂CH(CH₃)₂) 0.99 (6H, dd,

Cbz-Ala-gLeu-CONHiPr

(II.3). Cbz-Ala-gLeu-COOSu

(2.65 g, 5.91 mmol) was reacted with isopropylamine (1.51 mL,

J 6.5, 3.3, NHCH(CH₃)₂), 1.33–1.54 (10 H, m, C(CH₃)₃

+

17.7 mmol) according to the general procedure to yield II.3 (2.10 g,

◦

91%): white solid, mp 170 C. [a]²_D⁰+1.5 (c 0.5 in DMF); HPLC

CH₂CH(CH₃)₂), 3.63 (1H, h, J 6.6, NHCH(CH₃)₂), 4.84–5.09 (1H,

m, NHCHNH), 5.73–5.98 (2H, br m, NHCONHCH(CH₃)₂), 7.05

(1H, br d, J 6.9, NHCOC(CH₃)₃); d_C(50 MHz, DMSO-d₆) 22.2

(CH₃), 23.1 (CH₃), 24.3 (CH), 25.1 (CH₂), 28.1 (CH₃), 40.7 (CH),

56.3 (CH), 77.6 (C), 154.7 (C), 156.1 (C); MALDI-TOF m/z 310.5

[M + Na]⁺, 326.7 [M + K]⁺.

t_R= 8.20 min (linear gradient, 30–100% B, 20 min); d_H(300 MHz,

DMSO-d₆) 0.84 (6H, d, J 5.9, CH₂CH(CH₃)₂) 0.98–1.02 (6H, m,

NHCH(CH₃)₂), 1.17 (3H, d, J 7.1, CbzNHCHCH₃), 1.37-1.59

(3H, m, CH₂CH), 3.58–3.69 (1H, m, NHCH(CH₃)₂), 3.97 (1H,

p, J 7.1, NHCHCO), 5.01 (1H, d, J 2.4, CH₂Ph), 5.14–5.24 (1H,

m, NHCHNH), 5.94 (1H, d, J 7.5, NHiPr), 6.07 (1H, d, J 8.5,

NHCHNHCONH), 7.29–7.36 (5H, m, arom. H), 7.39 (1H, d,

J 7.8, CbzNH), 8.10 (1H, d, J 7.8, CHCONH); d_C(75 MHz,

DMSO-d₆) 23.4 (CH) 27.4 (CH₃), 27.6 (CH₃), 28.3 (CH₃), 28.4

(CH₃), 29.4 (CH), 46.0 (CH), 48.9 (CH₂), 55.2 (CH), 60.4 (CH),

70.5 (CH₂), 132.8 (CH), 132.9 (CH), 133.0 (CH), 133.5 (CH),

142.2 (C), 160.8 (C), 161.4 (C), 177.2 (C); HRMS (ESI) m/z calcd

for C_2OH₃₂LiN₄O₄[M + Li]⁺: 399.2579, found 399.2572.

Boc-gPhe-CONHiPr (I.2b). Boc-gPhe-COOSu (100 mg,

0.26 mmol) was reacted with isopropylamine (68 ll, 0.79 mmol)

according to the general procedure to yield I.2b (74 mg, 87%):

◦

white solid, mp 156 C. [a]²_D⁵−11.0 (c 1.1 in DMF); HPLC t_R

=

12.81 min (linear gradient, 0–100% B, 20 min); d_H(200 MHz,

DMSO-d₆) 1.00 (3H, d, J 6.8, CH₃), 1.03 (3H, d, J 6.7, CH₃),

1.36 (9H, s, C(CH₃)₃), 2.88 (2H, br d, J 6.8, CH₂Ph), 3.65 (1H,

m, CH(CH₃)₂), 5.14 (1H, m, NHCHNH), 5.95 (1H, br d, J 7.3,

CONHCH), 6.09 (1H, br d, J 8.0, CONHCH), 7.16–7.32 (6H, m,

arom. H + NHCO₂C(CH₃)₃); d_C(50 Mhz, DMSO-d₆) 23.1 (CH₃),

28.1 (CH₃), 40.7 (CH), 40.9 (CH₂), 59.2 (CH), 77.7 (C), 126.0

(CH), 127.9 (CH), 129.1 (CH), 137.9 (C), 154.5 (C), 156.0 (C);

MALDI-TOF m/z 344.9 [M + Na]⁺, 360.6 [M + K]⁺.

Cbz-D-Ala-gLeu-CONHiPr (II.4). Cbz-Ala-D-gLeu-COOSu

(2.00 g, 4.46 mmol) was reacted with isopropylamine (1.14 mL,

13.38 mmol) according to the general procedure to yield II.4

(1.32 g, 77%): white solid, mp 105 ^◦C. [a]_D²⁰−4.4 (c 0.5 in DMSO);

HPLC t_R= 8.22 min (linear gradient, 30–100% B, 20 min);

d_H(300 MHz, DMSO-d₆) 0.84 (6H, d, J 3.3, CH₂CH(CH₃)₂) 0.98–

1.02 (6H, m, NHCH(CH₃)₂), 1.17 (3H, d, J 7.1, CbzNHCHCH₃),

1.36–1.57 (3H, m, CH₂CH), 3.58–3.69 (1H, m, NHCH(CH₃)₂),

3.99 (1H, q, J 7.0, NHCHCO), 5.01 (1H, d, J 2.4, CH₂Ph), 5.15–

5.24 (1H, m, NHCHNH), 5.99 (1H, d, J 6.8, NHiPr), 6.06 (1H, d,

J 8.2, NHCHNHCONH), 7.28–7.37 (5H, m, arom. H), 7.35 (1H,

d, J 7.4, CbzNH), 8.13 (1H, d, J 7.5, CHCONH); d_C(75 MHz,

DMSO-d₆) 18.8 (CH) 22.5 (CH₃), 22.9 (CH₃), 23.5 (CH₃), 23.7

(CH₃), 24.7 (CH), 41.3 (CH), 44.2 (CH₂), 50.5 (CH), 55.6 (CH),

65.8 (CH₂), 128.2 (CH), 128.8 (CH), 137.5 (C), 156.0 (C), 156.6

(C), 172.6 (C); HRMS (ESI) m/z calcd for C_2OH₃₂N₄NaO₄[M +

Na]⁺: 415.2316, found 415.2377.

Boc-Pro-gLeu-CONHMe

(II.1a). Boc-Pro-gLeu-COOSu

(500 mg, 1.135 mmol) was reacted with HCl·NHMe (92 mg,

1.362 mmol) and DIEA (395 lL, 2.27 mmol) according to the

general procedure to yield II.1a (320 mg, 79%): a white solid,

◦

mp 184 C. [a]²_D⁵−26.8 (c 1.0 in DMF); HPLC t_R= 5.93 min

(linear gradient, 30–100% B, 20 min); d_H(200 MHz, DMSO-d₆,

signals of rotamers in italics) 0.83 (6H, d, J 5.8, CH(CH₃)₂),

1.30/1.37 (10H, s, C(CH₃)₃+ CH(CH₃)₂), 1.40–1.58 (2H, m,

CH₂CH(CH₃)₂), 1.64–1.85 (4H, m, CH₂CH₂CH₂CH), 2.50 (3H,

d, J 4.6, NHCH₃), 3.16–3.43 (2H, m, NCH₂CH₂), 3.88–4.06 (1H,

m, NCHCO), 5.19 (1H, br. t, J 7.9, NHCHNH), 5.97 (1H, br.

d, J 4.4, NHCONHCH₃), 6.15 (1H, d, J 8.7, NHCONHCH₃),

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Boc-gSer(Bn)-CO-Leu-NHMe (III.1). White solid, mp

186 ^◦C. HPLC t_R= 8.95 min (linear gradient, 30–100% B,

20 min); d_H(300 MHz, DMSO-d₆) 0.83 (6H, d, J 6.5, CH(CH₃)₂)

0.85 (6H, d, J 6.5, CH(CH₃)₂), 1.37 (9H, s, tBuOCO), 1.41–1.23

(2H, m, CH₂CH(CH₃)₂), 1.58–1.45 (1H, m, CH₂CH(CH₃)₂), 2.56

(3H, d, J 4.4, NHCH₃), 3.43–3.40 (2H, m, CH₂OBn), 4.14–4.06

(1H, m, NCHCO), 4.48 (2H, s, OCH₂Ph), 5.28–5.18 (1H, m,

NHCHNH), 6.25 (1H, d, J 8.6, NH), 6.37 (1H, d, J 8.6, NH),

7.17 (1H, d, J 7.8, tBuOCONH), 7.33–7.24 (5H, m, Ph), 7.90

(1H, q, J 4.8, NHCH₃); d_C(75 MHz, DMSO-d₆), 22.4 (CH) 23.4

(CH₃), 24.7 (CH₃), 25.9 (CH₃), 28.6 (CH), 42.8 (CH₂), 51.8 (CH),

57.4 (CH), 71.4 (CH₂), 72.3 (CH₂), 78.4 (C), 127.8 (CH), 127.9

(CH), 128.6 (CH), 138.8 (C), 155.1 (C), 156.7 (C), 173.7 (C);

HRMS (ESI) m/z calcd for C₂₂H₃₆LiN₄O₅[M + Li]⁺: 443.2841,

found 443.2824.

DIEA (160 lL, 0.91 mmol) according to the general procedure

to yield VI.1 (220 mg, 92%): white solid, mp 156 ^◦C. [a]_D²⁰+3.4 (c

1.0 in DMF); HPLC t_R= 7.43 min (linear gradient, 30–100% B,

20 min); d_H(400 MHz, DMSO-d₆) 1.07 (3H, d, J 6.2, CHCH₃),

1.20 (9H, s, C(CH₃)₃), 2.53 (3H, d, J 4.7, NHCH₃), 2.92–2.97

(1H, m, NHCH₂), 3.14–3.20 (1H, m, NHCH₂), 3.53–3.61 (1H,

m, NHCH), 3.58–3.67 (1H, m, CHOBn), 4.41 (1H, d, J 11.5,

OCH₂Ph), 4.55 (1H, d, J 11.6, OCH₂Ph), 5.50 (1H, d, J 9.1,

NHCH), 5.81 (2H, t, J 5.3, NHCONHCH₃), 5.93 (1H, s, tBuNH),

7.26–7.38 (5H, m, Ph); d_C(100 MHz, DMSO-d₆), 16.0 (CH₃) 26.4

(CH₃), 29.3 (3 CH₃), 41.5 (CH₂), 49.0 (C), 53.1 (CH), 70.2 (CH₂),

74.1 (CH), 127.3 (CH), 127.6 (CH), 128.1 (CH), 138.9 (C), 157.7

(C), 158.7 (C); HRMS (ESI) m/z calcd for C₁₈H₃₀LiN₄O₃[M +

Li]⁺: 357.2473, found 357.2431.

1-[2-(3-Benzylureido)-3-phenylpropyl]-3-methylurea

(VI.2).

Boc-gSer(Bn)-CO-D-Leu-NHMe (III.2). Boc-gSer(Bn)-COO-

Su (1.15 g, 2.82 mmol) was reacted with CF₃COOH·H-D-Leu-

NHMe (802 mg, 3.10 mmol) and DIEA (725 lL, 4.23 mmol) ac-

cording to the _◦general procedure to yield III.2 (900 mg, 73%): white

solid, mp 169 C. [a]²_D⁰−6.5 (c 1.0 in DMF); HPLC t_R= 8.90 min

(linear gradient, 30–100% B, 20 min); d_H(300 MHz, DMSO-d₆)

0.85 (6H, d, J 6.5, CH(CH₃)₂) 0.87 (6H, d, J 6.5, CH(CH₃)₂),

1.37 (9H, s, tBuOCO), 1.41–1.24 (2H, m, CH₂CH(CH₃)₂), 1.61–

1.48 (1H, m, CH₂CH(CH₃)₂), 2.55 (3H, d, J 4.6, NHCH₃), 3.46–

3.36 (2H, m, CH₂OBn), 4.12–4.04 (1H, m, NCHCO), 4.48 (2H, s,

OCH₂Ph), 5.26–5.16 (1H, m, NHCHNH), 6.23 (1H, d, J 7.8, NH),

6.34 (1H, d, J 8.6, NH), 7.13 (1H, br, tBuOCONH), 7.36-7.24 (5H,

m, Ph), 7.86 (1H, q, J 4.6, NHCH₃); d_C(75 MHz, DMSO-d₆) 22.5

(CH) 23.4 (CH₃), 24.6 (CH₃), 25.9 (CH₃), 28.6 (CH), 42.8 (CH₂),

51.8 (CH), 57.5 (CH), 71.4 (CH₂), 72.3 (CH₂), 78.4 (C), 127.8

(CH), 127.9 (CH), 128.6 (CH), 138.7 (C), 155.0 (C), 156.7 (C),

173.7 (C); HRMS (ESI) m/z calcd for C₂₂H₃₆LiN₄O₅[M + Li]⁺:

443.2841, found 443.2830.

Compound V.1a (66 mg, 0.32 mmol) was treated with TFA

(2 mL) at 0 ^◦C for 30 minutes. Concentration under reduced

pressure and co-evaporation with cyclohexane left a residue

which was dried under high vacuum. The resulting TFA salt was

treated with benzyl isocyanate (36 lL, 0.32 mmol) and DIEA

(64 lL, 0.58 mmol) according to the gene_◦ral procedure to yield

VI.2 (77 mg, quant.): white solid, mp 197 C. [a]²_D⁰−0.140 (c 0.5

in CH₂Cl₂–MeOH 1 : 3); HPLC t_R= 5.72 min (linear gradient,

1

30–100% B, 20 min); H NMR: See Tables S4–S6†; d_C(75 MHz,

DMSO-d₆) 31.6 (CH₃) 43.7 (CH₂), 48.0 (CH₂), 48.6 (CH₂), 56.8

(CH), 131.1 (CH), 131.7 (CH), 132.1 (CH), 133.3 (CH), 133.4

(CH), 134.4 (CH), 144.1 (C), 146.1 (C), 163.0 (C), 164.1 (C);

HRMS (ESI) m/z calcd for C₁₉H₂₄LiN₄O₂[M + Li]⁺: 347.2054,

found 347.2047.

Triurea VII. Compound V.1a (32 mg, 0.10 mmol) was treated

with TFA (2 mL) at 0 ^◦C for 30 minutes. Concentration under

reduced pressure and co-evaporation with cyclohexane left a

residue which was dried under high vacuum. The resulting

TFA salt was treated with (S)-succinimidyl-[1-benzyl-2-(tert-

butoxycarbonylamino)ethyl]carbamate¹⁷(30 mg, 0.10 mmol) and

NMM (34 lL, 0.31 mmol) according to the general procedure to

yield the corresponding diurea (40 mg, quant.). Treatment with

TFA (2 mL) at 0 ^◦C for 30 minutes followed by concentration

under reduced pressure and co-evaporation with cyclohexane left

a residue which was dried under high vacuum. The resulting TFA

salt was treated with benzyl isocyanate (17 lL, 0.31 mmol) and

DIEA (36 lL, 0.20 mmol) according to the general procedure

to yield the triurea VII (40 mg, 97%): white solid, mp 195 ^◦C.

[a]²_D⁰+0.046 (c 0.5 in CH₂Cl₂–MeOH 1 : 3); HPLC t_R= 8.72 min

[[1-Benzyloxymethyl-2-(3-methylureido)ethyl]carbamic acid tert-

butyl

ester

(V.2a). (S)-Succinimidyl-[(1-benzyloxymethyl-

(400 mg,

2-(tert-butoxycarbonylamino)ethyl]carbamate¹⁷

0.92 mmol) was reacted with methylamine hydrochloride

(155 mg, 2.30 mmol) and DIEA (552 lL, 3.22 mmol) according

to the general procedure to yield V.2a (340 mg, 98%): colourless

oil. [a]²_D⁰+3.9 (c 1.0, DMF); HPLC t_R= 8.58 min (linear gradient,

30–100% B, 20 min); d_H(400 MHz, DMSO-d₆) 1.07 (3H, d,

J 6.0, CHCH₃), 1.38 (9H, s, C(CH₃)₃), 2.53 (3H, d, J 4.5,

NHCH₃), 2.96–2.03 (1H, m, NHCHCH₂), 3.19–3.26 (1H, m,

NHCHCH₂), 3.52–3.59 (2H, m, NHCHCHOBn), 4.45 (1H, d, J

11.8, OCH₂Ph), 4.53 (1H, d, J 11.8, OCH₂Ph), 5.76 (1H, t, J 5.8,

NH), 5.82–5.84 (1H, m, NH), 6.37 (1H, d, J 8.8, NH), 7.25–7.35

(5H, m Ph); d_C(100 MHz, DMSO-d₆) 15.9 (CH₃) 26.7 (CH₃), 28.6

(3 CH₃), 40.7 (CH₂), 54.8 (CH), 70.3 (CH₂), 74.8 (CH), 78.0 (C),

127.6 (C), 127.8 (C), 128.5 (C), 139.3 (C), 156.0 (C), 159.1 (C);

HRMS (ESI) m/z calcd for C₁₈H₂₉LiN₃O₄[M + Li]⁺: 358.2313,

found 358.2303.

1

(linear gradient, 30–100% B, 20 min); H NMR: See Tables S7–

S9†; d_C(75 MHz, DMSO-d₆) 31.6 (CH₃), 43.6 (CH₂), 43.7 (CH₂),

48.0 (CH₂), 48.5 (CH₂), 48.6 (CH₂), 56.5 (CH), 56.6 (CH), 131.1

(CH), 131.7 (CH), 132.0 (CH), 133.3 (CH), 134.4 (CH), 144.1

(2C), 146.0 (C), 163.1 (C), 163.2 (C), 164.1 (C); HRMS (ESI) m/z

calcd for C₂₉H₃₆LiN₆O₃[M + Li]⁺: 523.3004, found 523.2969.

Tetraurea VIII. Compound V.1a (37 mg, 0.18 mmol) was

◦

1-[3-Benzyloxy-2-(3-tert-butylureido)butyl]-3-methylurea (VI.1).

Compound V.2a (240 mg, 0.69 mmol) was treated with TFA

(2 mL) at 0 ^◦C for 30 minutes. Concentration under reduced

pressure and co-evaporation with cyclohexane left a residue

which was dried under high vacuum. The resulting TFA salt

was treated with tert-butyl isocyanate (91 lL, 0.87 mmol) and

treated with TFA (2 mL) at 0 C for 30 minutes. Concentration

under reduced pressure, co-evaporation with cyclohexane left

a residue which was dried under high vacuum. The resulting

TFA salt was treated with (S)-succinimidyl-[1-benzyl-2-(tert-

butoxycarbonylamino)ethyl]carbamate¹⁷(52 mg, 0.18 mmol) and

NMM (59 lL, 0.54 mmol) according to the general procedure to

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yield the corresponding diurea (70 mg, quant.). Deprotection and

coupling steps were repeated to yield the corresponding triurea

(97 mg, 80%). This triurea (40 mg, 0.06 mmol) was treated with

TFA (2 mL) for 30 minutes then concentrated under reduced

pressure. Co-evaporation with cyclohexane left a residue which

was dried under high vacuum. The resulting TFA salt was treated

with benzyl isocyanate (8 lL, 0.06 mmol) and DIEA (21 lL,

0.12 mmol) according to the general procedure to yield the

tetraurea VIII (37 mg, 88%). HPLC t_R= 10.66 min (linear

against F²was carried out on all non-hydrogen atoms using

SHELXL97.³²Hydrogen atoms were included by using a riding

model (SHELXL97). The crystallographic data and the ﬁgures

were prepared using WinGX³³and Pymol,³⁴respectively.

Pertinent crystallographic data are listed in Table S3. Crystallo-

graphic data can be found in the ESI.§

Theoretical calculations

All density functional theory (DFT) calculations were performed

with the Gaussian03 suite of programs,³⁵with the hybrid func-

tional B3LYP. Although DFT approaches cannot be used to

study systems whose intermolecular interactions are dominated

by dispersion,³⁶this method is reliable enough for the study of

hydrogen bonds of the type N–H · · · O. Geometry optimisations

were achieved in the gas phase without symmetry constraints by

using the 6-31G(d,p) basis set. We have checked in some cases that

this basis set yields results in close agreement with larger basis sets.

For example, C6A–I.M lies 3.1 kcal mol⁻¹above C8–I.M in the 6-

31G(d,p) basis set and is barely lowered in the 6-311++G(d,p) basis

set, since it is found at D_rG = 2.9 kcal mol⁻¹. Moreover, geometry

parameters, and among them the hydrogen bond feature, are not

signiﬁcantly modiﬁed. Several structural hypothesis were used as

starting points. In addition, relaxed potential energy surface scans

have been performed in order to probe the existence of alternative

minima by varying w, φ or m. Calculation of vibrational frequencies

was systematically done in order to characterize the nature of

stationary points. Paths were traced from transition states to the

corresponding minima using the Intrinsic Reaction Coordinate

method.³⁷Gibbs free energies G^◦were calculated by means of the

harmonic frequencies, i.e. by a straightforward application of the

statistical thermodynamic equations.³⁸

1

gradient, 30–100% B, 20 min); H NMR: See Tables S10–S12†;

d_C(125 MHz, DMSO-d₆) 28.6 (CH₃), 40.6 (CH₂), 40.7 (CH₂),

40.9 (CH₂), 45.0 (CH₂), 45.2 (CH₂), 45.3 (CH₂), 46.0 (CH₂), 53.0

(CH), 53.4 (CH), 128.0 (CH), 128.1 (CH), 128.6 (CH), 128.8

(CH), 128.9 (CH), 129.2 (CH), 130.2 (CH), 130.3 (CH), 130.4

(CH), 131.3 (CH), 141.0 (C), 141.1 (C), 142.9 (C), 143.1 (C), 160.3

(C), 160.5 (C), 160.6 (C), 161.1 (C); HRMS (ESI) m/z calcd for

C₃₉H₄₈N₈NaO₄[M + Na]⁺: 715.3691, found 715.3650.

Structural analyses in solution

¹H-NMR spectra were obtained with TMS as the internal

reference. Concentrations used were 5 mM in CDCl₃and DMSO-

d₆. Proton resonances were assigned by COSY and NOESY exper-

iments. The solvent sensitivity of the urea and amide NH protons,

which is related to their free or hydrogen-bonded character, was

investigated by considering the inﬂuence of DMSO-d₆content in

CDCl₃–DMSO-d₆mixtures. The resonance of a solvent-exposed

(free) proton is rapidly shifted to low ﬁelds whereas that of a hy-

drogen bonded (solvent-protected) proton is only weakly affected

by DMSO-d₆NH-solvation.^13a,29,30IR spectra were obtained in

the Fourier transform mode in order to investigate the NH (3200–

3500 cm⁻¹) and CO (1580–1720 cm⁻¹) stretching frequencies in

CCl₄, CH₂Cl₂and DMSO. The concentration used depended

both on the solvent and the nature of the compound. The IR

spectra of compounds I–III with one 1,1-diaminoalkyl residue

remained unchanged in DMSO whatever the concentration, but

the concentration must be below 10 mM in CH₂Cl₂, and even

2 mM for compounds I in CCl₄, to exclude molecular aggregation.

The same holds true for compounds V with one 1,2-diaminoalkyl

residue in CH₂Cl₂, but the concentration must be reduced below

0.2 mM in CCl₄. In CH₂Cl₂and CCl₄, a free secondary amide

or urea group exhibits an NH absorption at 3400–3450 cm⁻¹and

a CO absorption at 1650–1700 cm⁻¹. The above frequencies are

reduced by 50–200 cm⁻¹and 10–20 cm⁻¹, respectively, when the

NH and CO groups are hydrogen-bonded.^13a,30The contributions

of the residual water in the solvent, if any, were eliminated by

correction in the 3500–3600 cm⁻¹region, where the peptide does

not absorb.

Acknowledgements

This research was supported in part by the Centre National de

la Recherche Scientiﬁque (CNRS), the Agence Nationale pour

la Recherche (grant number NT05_4_42848), Re´gion Alsace,

ImmuPharma France, and the Colombian–French agreement

ECOS-Nord (project number C05S05). The authors thank Roland

Graff and Lionel Allouche for their assistance with 2D NMR

measurements. Access to NMR facilities of the Service Commun

de Biophysicochimie des Interactions Mole´culaires, Universite´

de Nancy, is acknowledged. We thank the CALcul en MIdi-

Pyre´ne´es (CALMIP) and the Centre Informatique National

de l’Enseignement Supe´rieur (CINES) for allocating computer

resources.

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Propensity for local folding induced by the urea fragment in short-chain oligomers

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