Analysis of N-amino-imidazolin-2-one peptide turn mimic 4-position substituent effects on conformation by X-ray crystallography

Article abstract of DOI:10.1002/bip.22327

N-Amino-imidazolin-2-ones, a new class of turn mimics onto which side chains of different amino acids may be added conveniently, have been analyzed by X-ray crystallography. 4-Methyl and 4-benzyl N-amino-imidazolin-2-one tetrapeptide models 2 and 3 were e

Full text of DOI:10.1002/bip.22327

Analysis of N-Amino-Imidazolin-2-One Peptide Turn Mimic
4-Position Substituent Effects on Conformation by X-Ray
Crystallography
Caroline Proulx, William D. Lubell
Dꢀepartement de Chimie, Universitꢀe de Montrꢀeal, C.P. 6128, Succursale Center-Ville, Montrꢀeal, QC H3C 3J7, Canada
Received 28 April 2013; revised 6 June 2013; accepted 7 June 2013
Published online 29 July 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22327
INTRODUCTION
romatic and heterocyclic templates are valuable for
ABSTRACT:
studying relationships between peptide geometry
N-Amino-imidazolin-2-ones, a new class of turn mimics
and activity. They may rigidify dihedral angle
onto which side chains of different amino acids may be
geometry to stabilize turn, sheet or helical conform-
added conveniently, have been analyzed by X-ray crystallog-
A
ers, favor capacity for hydrogen bonds, and enhance
binding pocket occupancy.1 Conformational restrictions by
such templates are typically imposed on the u, w, and x tor-
sion angles of the peptide backbone. For example, a-amino-c-
lactam (Agl, so-called Freidinger-Veber lactam, Figure 1) resi-
dues constrain backbone geometry by covalently linking in a
five-membered ring the a-carbon and amide nitrogen atom of
adjacent amino acids in the peptide. Stabilization of a type II’
b-turn geometry by S-Agl may occur due to restriction of rota-
tional freedom about the w-torsion angle and confinement of
the C-terminal amide to the trans conformation.2 Employment
of Agl residues in peptidomimetics has improved receptor
affinity likely by minimizing the loss in entropy needed to fold
into an active conformer.2
raphy. 4-Methyl and 4-benzyl N-amino-imidazolin-2-one
tetrapeptide models 2 and 3 were examined to study the
influence of the 4-position substituent on turn conformation
and the chi dihedral angle of the neighbouring C-terminal
residue side-chain. The nature of the 4-position substituent
caused substantial effects on the wi1 2 main chain and
C-terminal Phe v1 side-chain dihedral angle values, giving a
preference for b- and g-turn conformers for the methyl- and
benzyl-substituted imidazolin-2-ones, respectively. Confor-
mational analysis in the solid state has provided insight to
guide application of N-amino-imidazolin-2-ones in peptide
Several efforts have focused on introducing and controlling
side-chain orientation (v space) to obtain specific v1 values
among favoured local minima [260ꢀ, gauche (2), 180ꢀ, trans,
and 60ꢀ, gauche (1)], which are separated by relatively low
energy rotational barriers (2–4 kcal/mol, Figure 2).3 The side-
chains of natural L-amino acids in peptides adopt usually one
of the three aforementioned conformations, with the order of
preference being gauche (2) > trans > gauche (1).3a Aromatic
side-chain orientations of Phe, Tyr, and Trp have been
restricted to gauche conformations by cyclic scaffolds, respec-
tively, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic),4
6-hydroxy-2-aminotetretralin-2-carboxylic acid (Hat),5 and
1,2,3,4-tetrahydro-beta-carboline (Tca), which may also induce
turn conformers in peptides (Figure 1).6
C
V
mimicry. 2013 Wiley Periodicals, Inc. Biopolymers (Pept
Sci) 102: 7–15, 2014.
Keywords: peptide mimicry; imidazolin-2-ones; beta
turns; gamma turns
This article was originally published online as an accepted
preprint. The “Published Online” date corresponds to the
preprint version. You can request a copy of the preprint by email-
ing the Biopolymers editorial office at biopolymers@wiley.com
Additional Supporting Information may be found in the online version of this
article.
Correspondence to: William D. Lubell, Dꢀepartement de Chimie, Universitꢀe de
Montrꢀeal, C.P. 6128, Succursale Center-Ville, Montrꢀeal, Quꢀebec H3C 3J7, Canada;
e-mail: william.lubell@umontreal.ca
In the case of c-lactam scaffolds, constrained Ser and Thr
residues may be obtained using a b-hydroxy-a-amino-c-
lactam (Hgl, Figure 1)7 residue in which the b-carbon
Contract grant sponsors: Natural Sciences and Engineering Research Council of Canada
(NSERC), Canadian Institutes of Health Research (CIHR), Boehringer Ingelheim
C
V
2013 Wiley Periodicals, Inc.
PeptideScience Volume 102 / Number 1
7

8
Proulx and Lubell
FIGURE 1 Structures of Agl, Hgl, Nai, Tic, Hat, and Tca residues.
stereochemistry and the pyrrolidinone ring puckering direct using aza-propargylglycinamide residues. Analysis of tetrapep-
the hydroxyl side-chain v1 dihedral angle. For example, in the tide model 1 by X-ray crystallographic and NMR spectroscopic
X-ray crystal structure of N-Fmoc-(2R,3S)-Hgl-D-Leu-OMe, analyses demonstrated that the 4-methyl Nai residue adopted
Cb-exo ring puckering of the pyrrolidinone core furnished a turn conformations (Figure 3).10 In the crystal structure of 1,
v1 dihedral angle value of 142.4ꢀ (a 37.6ꢀ deviation from the two distinct conformers were observed in the solid state: one
ideal trans conformation) with the hydroxyl side-chain having exhibiting a type II’ b-turn and the other an inverse c-turn con-
little consequence on the overall type II b turn geometry con- formation.10 The key difference between the two conformers
ferred by the c-lactam backbone.7 In other strategies, side- was due to rotation about the wi1 2 dihedral angle (Table I).
chain functionality has been added at different positions on
azabicyclo[X.Y.0]alkanone amino acid8 and proline9 scaffolds reduced flexibility of the u and w torsion angles may arrive
to improve binding affinity. from lone pair-lone pair electronic repulsion between the two
In addition to the ring constraints of the Nai residue,
Recently, N-amino-imidazolin-2-one (Nai, Figure 1) resi- adjacent nitrogen atoms and the planarity of the urea. In
dues were introduced as aza-analogs of Agl residues in which model peptide 1, the single chiral center of the D-Phe residue
the a-carbon is replaced by nitrogen.10 An advantage of the was responsible for the observed type II’ b- and inverse c-turn
Nai residue is the ability to add side chain groups at the 4- conformers, which would be expected to be type II b- and nor-
position of the imidazolinone heterocycle by approaches fea- mal c-turn conformers for its mirror image L-Phe
turing 5-exo-dig cyclizations and Sonogashira cross-couplings enantiomer.11
FIGURE
model 1.10
3
4-methyl N-amino-imidazol-2-one tetrapeptide
FIGURE 2 Three low-energy conformations about v1 of an amino
acid residue.
Biopolymers (Peptide Science)

Analysis of N-Amino-Imidazolin-2-Ones
9
Table I u and w Dihedral Angles (in Degrees) From Crystal
Analyses Compared With Ideal Turns and v1 Phenylalanine
Dihedral Angle
dene aza-propargylglycinyl-phenylalanine dipeptide building
blocks created by alkylation of their corresponding benzhydry-
lidene aza-glycinyl-phenylalanine dipeptides with propargyl
bromide, followed by Sonogashira cross-coupling in the case of
3 (Schemes 1 and 2). In the case of the 4-methyl analogue 2,
benzhydrylidene aza-glycinyl-D-phenylalanine N0-iso-propyl
amide 4 was alkylated with propargyl bromide and potassium
tert-butoxide in THF to afford in 71% yield aza-
propargylglycine 5, which underwent NaH-promoted 5-exo-
dig cyclization to give imidazol-2-one 6 in 78% yield.10
Removal of the benzhydrylidene protection was performed
using hydroxylamine hydrochloride in pyridine.10,13–15 With-
out further purification, the resulting N-amino-imidazolin-2-
one was treated with excess 4-methoxybenzoyl chloride (500
mol%) and di-iso-propylethylamine (2000 mol%) to afford
N,N-bis(p-methoxybenzamido)24-methyl-imidazolin-2-one 2
in 70% yield over two steps from the corresponding
benzophenone-protected N-amino imidazol-2-one 6. 4-
Methoxybenzoyl derivatives were synthesized in order to facili-
tate crystallization of tetrapeptide mimics for X-ray analysis,
without reducing the basicity of the carbonyl moiety, which
may participate in a 10-member hydrogen bond between the i
and i 1 3 residues.
Type of Turn
ui 1 1
wi 1 1
ui 1 2
wi 1 2
Phe v1
b-II0
Inverse c
60
n/a
2120
n/a
280
270
0
60
n/a
n/a
1a
1b
2
58.9
62.1
88.3
260
291.4
2153.3
2166.1
2177.3
120
269.1
271.7
270.4
80
24.6
65.7
31.2
0
54.6
57.8
67.7
n/a
b-II
3
2174.9
79.2
263.6
2166.7
Considering the global peptide conformation may be influ-
enced by the affect of the 4-position substituent on the C-ter-
minal residue geometry, particularly its v1 dihedral angle, we
have synthesized and performed a conformational analysis of
4-methyl and 4-benzyl N-amino-imidazolin-2-one tetrapep-
tide models 2 and 3 (Figure 4). Employing X-ray crystallogra-
phy to study Nai models 2 and 3 in the solid state, we have
found that the 4-position substituent can have a significant
effect on the turn geometry as well as on the side chain v1
dihedral angle of the C-terminal residue.
In light of the importance of turn conformations for the
function of natural and synthetic peptides,12 the utility of lac-
tam constraints (e.g., Agl and Hgl residues) for exploring
structure-activity relationships, as well as the promising attrib-
utes of the Nai residue, which may be readily modified at its
4-position substituent, the results of this analysis may have
significant impact on the future design of peptidomimetics for
studying biologically active peptides.
4-Benzyl N-amino-imidazolin-2-one model 3 was prepared
from benzhydrylidene aza-glycinyl-L-phenylalanine tert-butyl
ester 7 (Scheme 2). As previously reported, alkylation of 7 with
propargyl bromide, Sonogashira cross-coupling with iodoben-
zene, 5-exo-dig cyclization with NaH, and tert-butyl
ester removal with trifluoroacetic acid gave 4-benzyl N-amino-
imidazolin-2-one dipeptide 12.10 Acid 12 was then coupled to
iso-propylamine by activation as a mixed anhydride using iso-
butyl chloroformate to afford amide 13,10,15 which as described
for 6 above, was treated with hydroxylamine hydrochloride in
pyridine to remove the benzhydrylidene.10,13–15 The resulting
N-amino imidazolinone hydrochloride was subsequently
treated without purification with 4-methoxybenzoyl chloride
(200 mol%) and DIEA (300 mol%) in dichloromethane to
provide N,N-bis(p-methoxybenzamido)24-benzyl-imidazo-
lin-2-one 3 in 56% yield from 13.
RESULTS
Synthesis
The syntheses of 4-methyl and 4-benzyl N-amino-imidazolin-
2-one tetrapeptide models 2 and 3 were performed by routes
featuring NaH-promoted 5-exo-dig cyclizations of benzhydryli-
FIGURE 4 4-Methyl- and 4-benzyl-N-amino-imidazol-2-one tetrapeptide models 2 and 3.
Biopolymers (Peptide Science)

10
Proulx and Lubell
SCHEME 1 Synthesis of N,N-bis(p-methoxybenzamido)24-methyl-imidazolin-2-one 2.
and 3, which were compared to the structures previously
observed for 1,10 in order to examine the influence of the ring
substituent and the additional N-p-methoxybenzoyl group.
Conformational Analysis
Crystallizations and X-ray analyses were employed to evaluate
the conformers of 4-methyl and 4-benzyl imidazolin-2-ones 2
SCHEME 2 Synthesis of N,N-bis-p-methoxybenzamido imidazol-2-one iso-propyl amide 3.
Biopolymers (Peptide Science)

Analysis of N-Amino-Imidazolin-2-Ones
11
FIGURE 5 X-ray structures of N,N-bis-p-methoxybenzamido imidazolin-2-ones 2 (left) and 3
(right).
Crystals were grown by slow diffusion of hexanes into a chlo- from a 4-methyl to a 4-benzyl substituent. On the other hand,
roform solution of 4-methyl Nai 2 and an ethyl acetate solu- the Phe wi1 2 dihedral angle was influenced significantly by
tion of 4-benzyl Nai 3, respectively. X-ray diffraction revealed the imidazolin-2 4-position substituent. As mentioned, two
turn conformations in the solid states for both 2 and 3 conformers were observed in the crystal structure of 4-methyl
(Figure 5, Tables I and II).
N-p-methoxybenzamido imidazolin-2-one 1 and they differed
Note the configuration of the phenylalanine residue, L- and primarily with respect to the wi1 2 dihedral angle values, which
D-Phe in 2 and 3 influenced the N1 configuration of the Nai corresponded, respectively, to type II0 b- and inverse c-turns
residue, which conformed, respectively, to geometry similar to (24.6 and 65.7ꢀ, respectively).
that of the i1 1 residue in ideal type II and II0 b-turns possess-
In the case of 4-methyl N,N-bis-p-methoxybenzamido
ing dihedral angles of similar magnitude, yet opposite sign. imidazolin-2-one 2, the wi1 2 value was situated at 31.2ꢀ, which
The influence of two versus one p-methoxybenzoyl groups on is intermediate, yet 6 30ꢀ within the ideal values for both type
the Nai residue may be inferred by comparing the crystal struc- II0 b- and c-turns. In contrast, the wi1 2 value (263.6ꢀ) for 4-
tures of 4-methyl N,N-bis-p-methoxybenzamido imidazolin-2- benzyl N,N-bis-p-methoxybenzamido imidazolin-2-one 3 cor-
one 2 with the X-ray crystal structure reported previously for 4- responded with a c-turn conformer. The preference for c- ver-
methyl N-p-methoxybenzamido imidazolin-2-one 1.10 A rela- sus b-turn appears to increase with the size of the 4-position
tively minimal effect was observed on the Nai residue in the substituent. Due likely to interactions between the Nai ring
solid-state. The most striking difference (626–29ꢀ) was seen at substituent and the side chain of its C-terminal residue, this
the /i1 1 dihedral angle, which remained in the 60 6 30ꢀ range effect may also be responsible for the significant rotation
of an ideal type II0 b-turn. The influence of the additional p- observed about the Phe v1 torsion angle. In the X-ray crystal
methoxybenzoyl group was less significant on the wi1 1 value structures of 4-methyl Nai analogs 1 and 2, the Phe v1 angle
(611–24ꢀ); however, the tendency for the imidazolone hetero- adopted a gauche conformer (55–68ꢀ). On the other hand, the
cycle to adopt a nearly planar geometry caused this dihedral v1 angle of the 4-benzyl Nai analog 3 adopted a trans con-
angle to deviate by 33–57ꢀ from that of an ideal type II0 b-turn. former. Although this may be due solely to steric interactions,
In sum, the extra p-methoxybenzoyl group caused a greater notable CH-p interactions16 were observed between the aro-
twist about ui1 1 and flattened wi1 1 to a value of nearly 180ꢀ. matic rings of the 4-benzyl Nai analog 3. For example, the dis-
In model peptide 2, the geometry of N1 of the Nai residue tance between the donor carbon atom of the Phe residue and
˚
adopted the R configuration yet was nearly planar, with the sum the centre of the 4-benzyl acceptor ring was 3.8 A. Moreover,
of angles about N1 equal to 359.7ꢀ, compared to 350.9 and the 4-benzyl substituent of the imiazolin-2-one was involved in
357.9ꢀ in the type II0 b- and inverse c-turn conformers of 1, a second edge-to-face interaction with one of the p-methoxy-
respectively.
benzyl groups, albeit with a greater carbon-centroid distance of
˚
The ring substituent of the Nai residue appears to alter the 4.3 A. These aromatic interactions may stabilize the c-turn
conformation about the Phe residue with minor effects on its structure observed in the solid-state of 3.
ui1 2 torsion angle, which remained relatively constant (69–
72ꢀ) on addition of a second p-methoxybenzoyl group to the 3 can be viewed, respectively, as constrained analogs of unnatu-
N-terminal of the peptide, yet rotated about 9ꢀ on shifting ral amino acids norvaline and homohomophenylalanine. The
4-methyl- and 4-benzylsubstituted imidazolin-2-ones 2 and
Biopolymers (Peptide Science)

12
Proulx and Lubell
Table II Crystal Data and Structure Refinement for 2 and 3
2
3
Empirical formula
Formula weight
Temperature
C32 H34 N4 O6
570.63
100 K
C38 H38 N4 O6
646.72
100 K
˚
1.54178 A
˚
1.54178 A
Wavelength
Crystal system
Space group
Unit cell dimensions
Monoclinic
P21/c
Monoclinic
P21
ꢀ
ꢀ
a 5 10.2486(2) A, a 5 90
˚
˚
a 5 10.55739(18) A, a 5 90
b 5 27.092(5) A, b 5 97.226(1)
c 5 10.4745(2) A, c 5 90
˚
2972.13(10) A
ꢀ
ꢀ
˚
˚
b 5 32.3613(7) A, b 5 95.6185(11)
c 5 10.3363(2) A, c 5 90
˚
3411.65(12) A
ꢀ
ꢀ
˚
˚
3
3
Volume
Z
4
4
Density (calculated)
Absorption coefficient
F(000)
1.275 g/cm3
0.729 mm21
1208
1.259 g/cm3
0.698 mm21
1368
Crystal size
Theta range for data collection
Index ranges
0.20 3 0.02 3 0.02 mm
3.26–70.84ꢀ
0.10 3 0.04 3 0.02 mm
2.73–71.23ꢀ
212 ꢁ h ꢁ 12, (33 ꢁ k ꢁ 33, (11 ꢁ ‘ ꢁ 12
73651
211 ꢁ h ꢁ 12, 239 ꢁ k ꢁ 39, 212 ꢁ ‘ ꢁ 12
91047
Reflections collected
Independent reflections
Absorption correction
Max. and min. transmission
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I>2sigma(I)]
R indices (all data)
Absolute structure parameter
Largest diff. peak and hole
5622 [Rint 5 0.054]
Semi-empirical from equivalents
0.9855 and 0.8246
Full-matrix least-squares on F2
5622/34/429
1.128
12811 [Rint 5 0.045]
Semi-empirical from equivalents
0.9861 and 0.8809
Full-matrix least-squares on F2
12811/38/891
1.043
R1 5 0.0575, wR2 5 0.1402
R1 5 0.0651, wR2 5 0.1451
–
R1 5 0.0505, wR2 5 0.1298
R1 5 0.0548, wR2 5 0.1340
0.19(15)i
3
3
˚
˚
0.443 and 20.359 e/A
0.167 and 20.268 e/A
iX-ray crystal structure of imidazolone 3 revealed two compounds. Although the stereochemistry of the Phe residue was uncertain in one of the two
structures, the compounds appear to be mirror images, suggesting that imidazolone 3 crystallized as a racemic mixture.
n-propyl side-chain v1 and v2 chi angles adopt trans geometry substituent of the Nai residue in model peptides 3 and 4 was
with values of 176.2ꢀ and 2174.6 to 2179.2ꢀ, respectively. found to influenced turn conformations by imposing differen-
Alternatively, compound 2 and 3, which align the 4-substituents ces in the wi1 2 dihedral angle. 4-Methyl and 4-benzyl groups
with the nitrogen of the preceding amino acid residue, can be favored type II b- and c-turns, respectively. The 4-position
considered as constrained N-iso-propyl- and N-(2-phenethyl) substituent also had a notable influence on the v1 dihedral
phenylalanine analogs, which are evocative of peptoids.17 We angle of the C-terminal residue. In light of such insight, poten-
are currently investigating the synthesis of 5-substituted N- tial exists to employ different 4-position substituents to sys-
amino imidazol-2-ones to situate the Nai residue side chain in tematically study structure activity relationships. Incorporation
an orientation closer to that of a natural amino acid residue.18
of 4-methyl- and 4-benzyl-N-amino-imidazolinones into bio-
logically active peptides is currently under investigation, and
will be reported in due time.
CONCLUSION
N-amino imidazolin-2-one (Nai) residues combine the cova-
lent constraint of Agl residues with the electronic restriction EXPERIMENTAL SECTION
conferred by aza-amino acids. The Nai residue offers the added
advantage of presenting side-chain functionality at its 4- General
position. 4-Substituted Nai residues may thus be used to study Unless specified, all nonaqueous reactions were run under an
biologically active peptides, because they constrain both back- inert atmosphere; glassware was oven-dried or flame-dried and
bone and side chain geometry. The nature of the 4-position let cool under an inert atmosphere prior to use. Anhydrous
Biopolymers (Peptide Science)

Analysis of N-Amino-Imidazolin-2-Ones
13
solvents (THF, CH2Cl2, CH3CN) were obtained either by filtra- evaporation of the volatiles under reduced pressure afforded
tion through drying columns of a GlassContour system 280 mg of semicarbazide contaminated with benzophenone
(Irvine, CA). Diisopropylamine and N,N-diisopropylethyl oxime by-product. Without further purification, semicarba-
amine were distilled over potassium hydroxide. Analytical zide (92 mg, 0.27 mmol) was dissolved in 2 mL of DCM,
thin-layer chromatography (TLC) was performed on glass- treated with 4-methoxybenzoyl chloride (140 mg, 0.82
backed silica gel plates (Merck 60 F254). Visualization of the mmol) and DIEA (0.36 mL, 2.0 mmol), stirred for 12 h, and
developed chromatogram was performed by UV absorbance or washed with 5% citric acid (3 3 10 mL). The organic phase
staining with ceric ammonium molybdate. Silica gel chroma- was dried over Na2SO4, filtered and evaporated under
tography was performed using 230–400 mesh silica (Silicycle). reduced pressure. The residue was purified by flash chroma-
Melting points were obtained on a Buchi melting point appa- tography on silica gel using a gradient from 20 to 50% ethyl
ratus and are uncorrected. Infrared spectra were taken on a acetate in hexanes. Evaporation of the collected fractions
Perkin Elmer Spectrum One FTIR instrument and are reported afforded bis-benzamide 2 as a light yellow foam (68 mg,
in reciprocal centimetres (cm21). Nuclear magnetic resonance 70% yield), which crystallized from chloroform after slow
spectra (1H, 13C, COSY) were recorded either on a Bruker AV diffusion of vapors from hexanes: mp 77–80ꢀC; Rf 0.17 (1:1
300, AMX 300, AV 400 or AMX 400. Optical rotations were EtOAc: hexanes); [a]D20 8.45 (c 0.85, CHCl3). 1H NMR (400
determined with a Perkin-Elmer 341 polarimeter at 589 or 546 MHz, CDCl3) d 1.12–1.16 (6H, m), 1.58 (3H, s), 3.36 (1H,
nm. Data are reported as follows: [a]k temp, concentration (c dd, J 5 3.6, 13.7 Hz), 3.60 (1H, t, J 5 12.8 Hz), 3.83 (3H, s),
in g/100 mL), and solvent. High resolution mass spectra were 3.86 (3H, s), 3.99–4.07 (1H, m), 4.37 (1H, dd, J 5 3.3, 11.8
obtained by the Centre rꢀegional de spectromꢀetrie de masse de Hz), 5.97 (1H, s), 6.87 (2H, d, J 5 8.6 Hz), 6.93 (2H, d,
l’Universitꢀe de Montrꢀeal.
J 5 8.5 Hz), 7.02–7.14 (5H, m), 7.21 (1H, m), 7.78 (2H, d,
J 5 8.5 Hz), 7.86 (2H, d, J 5 8.5 Hz). 13C NMR (100 MHz,
CDCl3) d 170.7, 170.3, 168.8, 163.8, 152.7, 137.8, 131.9,
131.7, 129.3, 128.8, 127.0, 125.1, 121.0, 114.2, 108.7, 61.7,
55.8, 42.1, 35.1, 30.0, 22.7, 10.8. HRMS m/z 571.2553,
(M 1 H)1 calcd for [C32H35N4O6]1: 571.2551. IR (neat)
2926, 1689, 1601, 1510, 1456, 1239, 1165, 1106, 1024,
842, 750.
Reagents
Propargyl bromide and iodobenzene were purchased from
Aldrich and filtered on a silica plug prior to use. Copper iodide
was purchased from Aldrich and purified by dissolving in a
boiling saturated solution of aqueous NaI, followed by dilution
with water, filtering and washing.19 Potassium tert-butoxide,
sodium hydride, N-methylmorpholine, isobutyl chloroformate,
hydroxylamine hydrochloride, pyridine, Pd(PPh3)2Cl2, 4-
methoxybenzoyl chloride, all were purchased from Aldrich,
Alfa Aesar, or Strem Chemicals and used without further puri-
fication. Fmoc-D- and Fmoc-L-Phe-OtBu were purchased from
GL BiochemTM and used as received.
(20R)21-((Diphenylmethylene)amino)23-(30-Phenyl-
N0-iso-propyl-20-propionamide)24-Benzyl-
Imidazolin-2-One (13)
(20R)21-((Diphenylmethylene)amino)23-(30-phenyl-20-prop-
anoate)24-benzylimidazolin-2-one (12, 164 mg, 0.33 mmol)
was dissolved in 4 mL of THF, cooled to 215ꢀC, treated
sequentially with isobutyl chloroformate (56 mL, 0.43 mmol)
and N-methyl morpholine (47 mL, 0.43 mmol), stirred for 15
min, treated with iso-propylamine (42 mL, 0.50 mmol), and
stirred at 215ꢀC for 1.5 h. The volatiles were removed on a
rotary evaporator under reduced pressure to give a residue,
which was dissolved in EtOAc. The organic phase was washed
with 5% NaHCO3 (3 3 5 mL) and 5% citric acid (3 3 5 mL),
SYNTHETIC EXPERIMENTAL PROCEDURES
AND CHARACTERIZATION DATA
(20S)21,1-Bis(p-Methoxybenzamido)24-Methyl-3-
(30-Phenyl-N0-iso-propyl-20-propionamide)-
Imidazolin-2-One (2)
Benzhydrylidene 6 (260 mg, 0.56 mmol, prepared according dried over MgSO4, filtered, and evaporated under reduced
to Ref. 10) was dissolved in 20 mL of pyridine, treated with pressure. The residue was purified by chromatography on silica
hydroxylamine hydrochloride (154 mg, 2.23 mmol), heated gel using a 10–60% gradient of diethyl ether in petroleum
to 60ꢀC, and stirred for 12 h. The volatiles were evaporated ether. Evaporation of the collected fractions afforded 56 mg
under reduced pressure. The residue was dissolved in DCM (31% yield) of amide 13 as a yellow oil: Rf 0.32 (1:1 EtOA-
20
1
and evaporated to remove residual pyridine. Digestion of the c:hexanes); [a]D 39.7 (c 1.15, CHCl3); H NMR (400 MHz,
residue with EtOAc (2 3 20 mL), decantation from the CDCl3) d 1.13 (3H, d, J5 6.6 Hz), 1.19 (3H, d, J 5 6.5 Hz),
insoluble material (e.g., hydroxylamine hydrochloride), and 3.06 (1H, d, J 5 15.8 Hz), 3.23–3.33 (2H, m), 3.98–4.03 (1H,
Biopolymers (Peptide Science)

14
Proulx and Lubell
m), 4.38 (1H, m), 5.01 (1H, s), 6.74 (2H, m), 7.06–7.45 (16H,
m), 7.65 (2H, d, J 5 7.4 Hz), 7.81 (1H, s).13C NMR (100 MHz,
CDCl3) d 169.3, 166.0, 151.0, 137.7, 137.4, 135.4, 134.8, 131.2,
129.9, 129.4, 129.3, 129.0, 128.9, 128.8, 128.83, 128.7, 128.4,
127.0, 126.9, 123.1, 108.0, 62.3, 41.9, 35.6, 31.2, 22.7, 22.6.
HRMS m/z 543.2755, (M 1 H)1 calcd for [C35H35N4O2]1:
543.2754. IR (neat) 3266, 3061, 2971, 1664, 1548, 1494, 1454,
1444, 1398, 1171, 1135, 748, 695.
REFERENCES
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(20R)21,1-Bis(p-Methoxybenzamido)24-Benzyl-3-
(30-Phenyl-N0-iso-propyl-20-propionamide)-
Imidazolin-2-One (3)
Benzhydrylidene 13 (73 mg, 0.135 mmol) was dissolved in 5
mL of pyridine, treated with hydroxylamine hydrochloride
(37 mg, 0.54 mmol), heated to 60ꢀC, and stirred for 12 h.
The volatiles were evaporated under reduced pressure. The
residue was dissolved in DCM and evaporated to remove
residual pyridine. Digestion of the residue with EtOAc (2 3
5 mL), decantation of insoluble material (e.g., hydroxyla-
mine hydrochloride) and evaporation of the volatiles under
reduced pressure afforded 80 mg of semicarbazide contami-
nated with oxime by-product. Without further purification,
semicarbazide (80 mg, 0.13 mmol) was dissolved in 1 mL of
DCM, treated with 4-methoxybenzoyl chloride (45 mg, 0.26
mmol) and DIEA (68 mL, 0.39 mmol), stirred for 12 h, and
washed with 5% citric acid (3 3 5 mL). The organic phase
was dried over Na2SO4, filtered and evaporated under
reduced pressure. The residue was purified by flash chroma-
tography on silica gel using a gradient from 10 to 25% ethyl
acetate in hexanes. Evaporation of the collected fractions
afforded bis-benzamide 3 as a light yellow foam (47 mg, 56
% yield), which crystallized from ethyl acetate after slow dif-
fusion of vapors from hexanes: Rf 0.24 (1:1 EtOAc: hexanes);
[a]D20 2105 (c 0.72, CHCl3). 1H NMR (400 MHz, CDCl3) d
1.07 (3H, d, J 5 6.8 Hz), 1.14 (3H, d, J 5 6.4 Hz), 3.11 (2H,
s), 3.37–3.40 (1H, m), 3.53 (1H, t, J 5 12.6 Hz), 3.86 (3H, s),
3.90 (3H, s), 3.94–4.00 (1H, m), 4.26–4.28 (1H, m), 5.79
(1H, s), 6.86–6.99 (8H, m), 7.06–7.33 (6H, m), 7.79 (2H, d,
J 5 8.8 Hz), 7.89 (2H, d, J 5 8.4 Hz). 13C NMR (100 MHz,
CDCl3) d 170.8, 170.4, 168.2, 163.9, 163.8, 152.7, 138.2,
135.5, 132.0, 131.7, 129.6, 129.2, 129.0, 128.9, 127.6, 127.1,
125.4, 125.3, 124.6, 114.3, 110.0, 62.0, 55.9, 42.2, 35.0, 31.2,
22.8. HRMS m/z 647.2842, (M 1 H)1 calcd for
[C38H39N4O6]1: 647.2864. IR (neat) 1702, 1602, 1509, 1239,
1165, 1024, 749, 699.
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Analysis of N-Amino-Imidazolin-2-Ones
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