New fluorinated peptidomimetics through tandem aza-michael addition to α-trifluoromethyl acrylamide acceptors: Synthesis and conformational study in solid state and solution

Article abstract of DOI:10.1021/jo9001867

A range of partially modified retro (PMR) ψ[NHCH₂] peptide mimetics containing a hydrolytically stable CH₂CH(CF₃)CO unit have been synthesized. The first kind of peptidomimetics is obtained from the highly efficient aza-Michael addition of different amines to α-trifluoromethyl acrylamide acceptors. Subsequent deprotection of the amino group furnishes the key common intermediate for the synthesis of other families of peptidomimetics: dipeptides, tripeptides, peptidomimetics containing a urea moiety, and structures containing two units of a-trifluoromethyl- β²-alanine. Finally, a conformational study of several of the newly synthesized peptidomimetics, performed with the aid of X-ray analysis and NMR techniques, shows a β-turn-like conformation for the structures both in the solid state and in solution.

Full text of DOI:10.1021/jo9001867

New Fluorinated Peptidomimetics through Tandem Aza-Michael
Addition to r-Trifluoromethyl Acrylamide Acceptors: Synthesis and
Conformational Study in Solid State and Solution#
Santos Fustero,*,†,‡ Gema Chiva,‡ Julio Piera,‡ Juan F. Sanz-Cervera,†,‡
Alessandro Volonterio,§ Matteo Zanda,|,$ and Carmen Ramirez de Arellano†,
Departamento de Qu´ımica Orga´nica, UniVersidad de Valencia, 46100 Burjassot, Valencia, Spain,
Laboratorio de Mole´culas Orga´nicas, Centro de InVestigacio´n Pr´ıncipe Felipe, 46013 Valencia, Spain,
Dipartimento di Chimica, Materiali, ed Ingegnieria Chimica “Giulio Natta” Politecnico di Milano, Via
Mancinelli 7, 20131 Milano, Italy, and C.N.R.-Istituto di Chimica del Riconoscimento Molecolare (ICRM)
Via Mancinelli 7, 20131 Milano, Italy
santos.fustero@uV.es
ReceiVed February 1, 2009
A range of partially modified retro (PMR) ψ[NHCH2] peptide mimetics containing a hydrolytically stable
CH2CH(CF3)CO unit have been synthesized. The first kind of peptidomimetics is obtained from the
highly efficient aza-Michael addition of different amines to R-trifluoromethyl acrylamide acceptors.
Subsequent deprotection of the amino group furnishes the key common intermediate for the synthesis of
other families of peptidomimetics: dipeptides, tripeptides, peptidomimetics containing a urea moiety,
and structures containing two units of R-trifluoromethyl-ꢀ2-alanine. Finally, a conformational study of
several of the newly synthesized peptidomimetics, performed with the aid of X-ray analysis and NMR
techniques, shows a ꢀ-turn-like conformation for the structures both in the solid state and in solution.
Introduction
metabolism, reproduction, respiration, and immune defense.1
Nevertheless, the use of peptides in drug development is limited
due to their low metabolic stability in vivo and their poor
bioavailability.2 One strategy to circumvent these drawbacks is
to modify the peptide structure in such a way that the metabolic
stability may be enhanced. Various alterations can be performed
at any point on the peptide chain in order to obtain peptidomi-
metics: amino acids can be deleted, added, or replaced by other
natural or non-natural amino acids; synthetic scaffolds can
substitute for all or part of the peptide backbone; cyclizations
can be performed to increase the rigidity of the molecules, and
finally, peptide bond surrogate units X, usually represented as
ψ(X), can be used instead of the peptidic bond (X instead of
CO-NH).1,3 Another common strategy is the inversion of the
Peptides are essential components of living organisms, acting
as hormones, neurotransmitters, and neuromodulators. As such,
they exert an essential influence on basic functions including
# Dedicated to the memory of Prof. Dr. Herbert Lehmkuhl.
† Universidad de Valencia.
‡ Centro de Investigacio´n Pr´ıncipe Felipe.
§ “Giulio Natta” Politecnico di Milano.
| C.N.R.-Istituto di Chimica del Riconoscimento Molecolare (ICRM).
Corresponding author regarding the X-ray diffraction analysis and discus-
sion. E-mail: carmen.ramirezdearellano@uv.es.
$ Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen
AB25 2ZD, Scotland, UK.
(1) Fletcher, M. D.; Campbell, M. M. Chem. ReV. 1998, 98, 763–795.
(2) (a) Davis, S. S. In PerspectiVes in Medicinal Chemistry; Testa, B., Kyburz,
E., Fuhrer, W., Giger, R., Eds.; Verlag Helvetica Chimica Acta: Basel, 1993;
pp 533-544. (b) Abell, A., Eds. AdVances in Amino Acid Mimetics and
Peptidomimetics; JAI Press, Inc.: Greenwich, 1997. (c) Pauletti, G. M.; Ganwar,
S.; Siahaan, T. J.; Aube, J. AdV. Drug DeliVer. ReV. 1997, 27, 235–256. (d)
Barrett, G. C., Eds. Amino Acid DeriVatiVes; Oxford University Press: Oxford,
1999. (e) Hummel, G.; Reineke, U.; Reimer, U. Mol. BioSyst. 2006, 2, 499–
508.
(3) (a) Olson, G. L.; Bolin, D. R.; Bonner, M. P.; Bo¨ss, M.; Cook, C. M.;
Fry, D. C.; Graves, B. J.; Hatada, M.; Hill, D. E.; Kahn, M.; Madison, V. S.;
Rusiecki, V. K.; Sarabu, R.; Sepinwall, J.; Vincent, G. P.; Voss, M. E. J. Med.
Chem. 1993, 36, 3039–3049. (b) Gante, J. Angew. Chem. 1994, 106, 1780–
1802; Angew. Chem., Int. Ed. Engl. 1994, 33, 1699-1720. (c) Leung, D.;
Abbenante, G.; Fairlie, D. P. J. Med. Chem. 2000, 43, 305–341.
3122 J. Org. Chem. 2009, 74, 3122–3132
10.1021/jo9001867 CCC: $40.75 2009 American Chemical Society
Published on Web 03/23/2009

Fluorinated Peptidomimetics through Tandem Aza-Michael Addition
SCHEME 1. Stereoselective Aza-Michael Addition of
Nitrogen Nucleophiles 1 to N-(r-Trifluoromethyl)acryloyl-
r-amino Esters 2
SCHEME 2. Synthesis of Peptidomimetics Containing
Trifluoroalanine
peptide bonds (NH-CO instead of CO-NH). While inverting
all the bonds generates retropeptides, reversing one or more (but
not all) of the peptide bonds leads to partially modified
retropeptides (PMR)1,4,5 Although retropeptides generally have
a higher resistance to hydrolysis than their peptide analogues,
there are enzymes that easily hydrolyze the retropeptide bond.
One possible approach to avoid proteolytic degradation of both
peptide and retropeptide bonds is to replace them with hydro-
lytically stable groups. Several fluorinated groupings, for
example, are able to substitute the peptide bond and remain
stable in the presence of hydrolytic enzymes.5,6
In previous papers, we have reported on the synthesis of
partially modified retro (PMR) ψ[NHCH2] peptide mimetics
containing a chemically stable and stereodefined CH2CH-
(CF3)CO unit that can be considered to be a substitute for a
trifluoroalanine.7 The key step for the synthesis of such
compounds is a tandem diastereoselective aza-Michael addi-
tion-enolate protonation of nitrogen nucleophiles 1 to N-
trifluorometacryloyl R-amino esters 2 (Scheme 1). The reaction
proceeds with very high diastereoselectivity (up to 42:1), but
is highly dependent on various experimental factors such as the
type of solvent and base used in the reaction as well as the
particular R1 and R2 substituents present. The most important
factor, however, seems to be the presence of carbonyl groups
in both starting materials 1 and 2. It is important to point out
that no reaction is observed unless a trifluoromethyl group is
present in the Michael acceptor 2.
trifluoroalanine unit. We first obtained peptidomimetics 5
through addition of several different amines 4 to the Michael
acceptor 2.8 Subsequent deprotection led to the key common
intermediate 6, which can either give rise to acetylated pepti-
domimetics 7 or react with different substrates to furnish
peptidomimetics 8, which contain a urea moiety, tripeptides 9,
and systems 10, which contain two units of R-trifluoromethyl-
ꢀ2-alanine (Scheme 2).
ꢀ-Turns are, together with R-helices and ꢀ-sheets, the most
important structural elements of peptides and proteins from both
structural and functional points of view. These secondary
structures play an essential role in globular proteins and are
involved in many of the molecular recognition events in
biological systems.9 Therefore, it is important to consider the
conformation adopted by a given peptidomimetic. With this in
mind, we report herein on several X-ray and NMR studies
performed in order to analyze the tendency of these compounds
to adopt turn-like conformations both in the solid state and in
solution.
In the present paper, we extend this methodology to the
synthesis of a class of related peptidomimetics containing a
(4) (a) Goodman, M.; Wilson, C. G.; Chorev, M. J. Am. Chem. Soc. 1977,
99, 8075–8076. (b) Goodman, M.; Chorev, M. Acc. Chem. Res. 1979, 12, 1–7.
(c) Goodman, M.; Chorev, M. Acc. Chem. Res. 1993, 26, 266–273. (d) Goodman,
M.; Chorev, M. Trends Biotechnol. 1995, 13, 438–445.
(5) Volonterio, A.; Bellosta, S.; Bravin, F.; Bellucci, M. C.; Bruch, 940 > e,
L.; Colombo, G.; Malpezzi, L.; Mazzini, S.; Meille, S. V.; Meli, M.; Ramirez
de Arellano, C.; Zanda, M. Chem.sEur. J. 2003, 9, 4510–4522, and references
therein.
(6) See, for example: (a) Bartlett, P. A.; Otake, A. J. Org. Chem. 1995, 60,
3107–3111. (b) Scolnick, L. R.; Clements, A. M.; Liao, J.; Crenshaw, L.;
Hellberg, M.; May, J.; Dean, T. R.; Christianson, D. W. J. Am. Chem. Soc.
1997, 119, 850–851. (c) Wipf, P.; Henninger, T. C.; Geib, S. J. J. Org. Chem.
1998, 63, 6088–6089. (d) Volonterio, A.; Bravo, P.; Zanda, M. Org. Lett. 2000,
2, 1827–1830. (e) Tang, Y.; Ghirlanda, G.; Petka, W. A.; Nakajima, T.; DeGrado,
W. F.; Tirrel, D. Angew. Chem., Int. Ed. 2001, 40, 1494–1496. (f) Zanda, M.
New. J. Chem. 2004, 28, 1401–1411.
Results and Discussion
Addition of Amines 4 to Michael acceptor 2. We began
by investigating the effects of different solvents, bases, and
temperatures on the diastereoselectivity of the process, employ-
ing 2a and benzylamine 4a as model systems. The first
conclusion we reached was that the reaction is quite simple,
efficient, and very fast (30 min) in comparison to other aza-
(7) (a) Sani, M.; Bruche´, L.; Chiva, G.; Fustero, S.; Piera, J.; Volonterio,
A.; Zanda, M. Angew. Chem., Int. Ed. 2003, 42, 2060–2063. (b) Volonterio, A.;
Chiva, G.; Fustero, S.; Piera, J.; Sanchez-Rosello´, M.; Sani, M.; Zanda, M.
Tetrahedron Lett. 2003, 44, 7019–7022. (c) Fustero, S.; Chiva, G.; Piera, J.;
Volonterio, A.; Zanda, M.; Gonza´lez, J.; Mora´n-Ramallal, A. Chem.sEur. J.
2007, 13, 8530–8542. (d) Fustero, S.; Garcia-Sancho, A.; Chiva, G.; Sanz-
Cervera, J. F.; Del Pozo, C.; Acen˜a, J. L. J. Org. Chem. 2006, 71, 3299–3302.
(8) For the preparation of Michael acceptor 2, see ref 7a.
(9) See, for example: (a) Rose, G. D.; Gierasch, L. M.; Smith, J. A. AdV.
Protein Chem. 1985, 37, 1–109. (b) Creighton, T. E. Proteins: Structures and
Molecular Principles, 2nd ed.; Freeman: New York, 1993. (c) Chou, K.-C. Anal.
Biochem. 2000, 286, 1–16. (d) Souers, A. J.; Ellman, J. A. Tetrahedron 2001,
57, 7431–7448.
J. Org. Chem. Vol. 74, No. 8, 2009 3123

Fustero et al.
SCHEME 3. Synthesis of Peptidomimetics 5
TABLE 1. Aza-Michael Reaction of 2 with Aliphatic Amines 4a
pair) (entry 7 vs entry 6, Table 1). Finally, substitution at the
benzyl group of benzylamines (entries 1, 5, and 9, Table 1)
was found to have only a minor effect.
dr
yieldc
entry
2
4
X1 R1
R3
R4 product (R/ꢀ)b (%)
The configuration of the major diastereoisomer 5hr was
determined with the aid of X-ray diffraction analysis while the
configurations of the other major diastereoisomers were assigned
by means of analogy. Suitable monocrystals of 5hr were
obtained through recrystallization from n-hexane; the X-ray
diffraction analysis revealed an S configuration of the new
stereocenter (Figure 1). The crystal consists of two independent
molecules in the asymmetric unit, both presenting a ꢀ-turn-like
secondary structure. The two molecules of the asymmetric unit
are related to each other through hydrogen bonds N-H· · ·OdC
and N-H· · ·F and connected to the molecules of neighboring
asymmetric units by two hydrogen bonds N-H· · ·OdC, thus
generating an infinite zigzag chain.11
1
2a 4a Bn i-Pr Bn
2b 4a t-Bu i-Pr Bn
2a 4b Bn i-Pr allyl
2b 4b t-Bu i-Pr allyl
2a 4c Bn i-Pr p-FC6H4CH2
2a 4d Bn i-Pr (R)-CH(Me)Ph
2a 4e Bn i-Pr (S)-CH(Me)Ph
2b 4e t-Bu i-Pr (S)-CH(Me)Ph
2a 4f Bn i-Pr p-MeOC6H4CH2
2a 4g Bn i-Pr Bn
H
H
H
H
H
H
H
H
H
5a
5b
5c
5d
5e
5f
5g
5h
5i
13:1
8:1
10:1
5:1
14:1
6:1
10:1
8:1
10:1
2:1
2:1
6:1
4:1
87
95
68
70
69
90
75
64
74
80
90
84
80
81
96
2d
3
4
5
6
7
8d
9
10
11
Bn 5j
Bn 5k
H
H
H
H
2b 4g t-Bu i-Pr Bn
12d 2b 4h t-Bu i-Pr (S)-CH(Me)C10H7
5l
13
14
15
2c 4a Bn Bn Bn
2d 4a t-Bu Me Bn
2e 4a t-Bu i-Bu Bn
5m
5n
5o
5:1
7:1
a The reaction was carried out on a 1 mmol scale. Amine 4 (1 equiv)
and DABCO (1 equiv) were added to a solution of the Michael acceptor
2 (1 equiv) in CCl4 (10 mL) and stirred at rt for 30 min. b Determined
with the aid of 19F NMR spectroscopic analysis. c Overall yield of
purified diastereoisomeric mixture. d Results taken from ref 7c.
Michael reactions.10 The best results were obtained when the
reaction was performed at room temperature with CCl4 as a
solvent and 1 equiv of DABCO as a base (87% yield and 13:1
dr).7 To study the scope of the reaction, several different amines
4 were added to Michael acceptors 2 under these optimal
conditions (Scheme 3, Table 1).
FIGURE 1. X-ray structure of 5hr.
Unlike the addition of amino esters 1 to Michael acceptors
2, in which the nature of the X1 group has no significant
influence on the diastereoselectivity of the process,7a in this case,
the influence of the X1 group was quite remarkable, being higher
when X1 ) Bn than when X1 ) t-Bu (entry 1 vs 2, 3 vs 4, and
7 vs 8; Table 1). The R1 side chain of acceptors 2 also had a
marked influence; thus, better stereocontrol was obtained when
a bulkier R1 group was present. This latter trend can be observed
by comparing 5a (R1 ) i-Pr, entry 1, Table 1) with 5m (R1 )
Bn, entry 13, Table 1) or 5b (R1 ) i-Pr, entry 2, Table 1) with
5n (R1 ) Me, entry 14, Table 1) and 5o (R1 ) i-Bu, entry 15,
Table 1). With regard to the effect of the amine structure on
the reaction, higher selectivity was obtained with primary amines
than with secondary ones; in fact, use of the latter resulted in a
dramatic drop in the diastereoselectivity as can be seen, for
example, in the comparison between 5a vs 5j (entries 1 and
10, Table 1) or between 5b vs 5k (entries 2 and 11, Table 1).
Moreover, like the aza-Michael addition of R-amino esters to
acceptors 2,7a (S)-amines produced the products with higher
diastereoselectivity (matched pair) than (R)-amines (mismatched
Deprotection of 5b To Afford Key Intermediate 6. The
obvious, most direct way to synthesize the unprotected amine
6 (Scheme 4) is to perform the reaction between Michael
acceptors 2 and ammonia. However, when we tried this
approach, it failed completely, resulting in the formation of a
complex mixture in which products derived from double
addition were identified. We therefore decided to undertake the
selective deprotection of compounds 5. Even though the best
diastereoselectivities were obtained when X1 ) Bn, we chose
5b (X1 ) t-Bu) for the synthesis of intermediate 6 and,
SCHEME 4. Synthesis of Peptidomimetics 6 and 7
(10) R-Substituted acrylamide acceptors are often unreactive as aza-Michael
acceptors. In fact, a substrate similar to compound 2 but with a methyl instead of a
trifluoromethyl group is completely unreactive in the kind of addition that we are
describing in this paper; only starting material is recovered from the reaction.
Therefore, the extraordinary reactivity of adduct 2a is due to the presence of the
electron-withdrawing CF3 group (see ref 7c). A similar behavior is observed in the
case of other aza-Michael reactions with 4-substituted acceptors (see ref 5).
(11) For more details about the packing of the crystal, see the Supporting
Information.
3124 J. Org. Chem. Vol. 74, No. 8, 2009

Fluorinated Peptidomimetics through Tandem Aza-Michael Addition
SCHEME 5. Preparation of Urea-Containing
Peptidomimetics 8
consequently, for the different families of peptidomimetics
7-10. The reason for this choice was that it allowed for the
selective deprotection of the amine moiety while keeping the
carboxylic acid protected. Although this step seemed to be
straightforward at first, it turned out to be a complicated reaction.
Our initial attempts to carry out the hydrogenolysis of 5 with
different catalysts [Pd(OH)2, Pd/C, PtO2, PdCl2], at different
H2 pressures, and with different solvents and reaction times were
thus unsuccessful, resulting in either no reaction or very poor
conversions. Finally, using stoichiometric amounts of Pd(OH)2
in AcOEt under atmospheric pressure of H2, we were able to
obtain the deprotected product 6 in quantitative yields (Scheme
4). We managed to reduce the catalyst loading to 0.6 equiv by
increasing the H2 pressure to 100 psi and still obtain a
quantitative yield of 6; however, further efforts to reduce the
catalyst loading resulted in a dramatic drop in yield and
sometimes in no reaction. Some degree of epimerization (<4%)
for the stereocenter containing the CF3 group was observed.
Acetylated dipeptide 7 was easily prepared by treating 6 with
Ac2O in pyridine (Scheme 4). The major diastereoisomer, 7r,
was easily separated with the aid of column chromatography
and then employed to confirm the configuration. It was also
used in the conformational analyses involving X-ray and NMR
techniques (see Figure 2).
Synthesis of Urea-Substituted Peptidomimetics 8. As stated
above, various moieties have been used to replace the amide
bond in the peptide structure, among them, the urea group.12
For example, ureas have been included in the structure of some
enzymatic inhibitorsD13 and used to induce a reorganization of
the peptide structure through bidimensional turns.14 The syn-
thesis of PMR ψ[NHCH2] ureas that contain an R-trifluoro-
methyl-ꢀ2-alanine 8 can be carried out starting from both
secondary amine 5 and primary amine 6. Indeed, these urea-
containing pseudopeptides were obtained in good to excellent
yields through reaction with the corresponding isocyanate in
dry CH2Cl2 at 0 °C in only 30 min (Scheme 5, Table 2).
We also investigated the scope of the reaction, which proceeds
efficiently with both primary amines 6 (entries 1-4, Table 2)
and secondary amines 5 (entries 5-7, Table 2). A selection of
aromatic (entries 1 and 2, Table 2), linear aliphatic (entries 3,
5 and 6, Table 2), and branched aliphatic (entries 4 and 7, Table
2) isocyanates 11 gave the expected products in high yields. It
is important to note that this method is also suitable for the use
of isocyanates derived from an R-amino ester. In particular, the
reaction between 5b and the isocyanate derived from the tert-
butyl ester of valine gave the pseudotripeptide 8g (entry 7, Table
2) in excellent yield. In this way, the direction of the chain of
the retropeptide can be restored, and at the same time, the
peptide chain can be extended.
TABLE 2. Synthesis of Urea-Peptidomimetics 8a
entry product
X1
R1
R3
R5
yieldb (%)
1
2
3
4
5
6
7
8a
8b
8c
8d
8e
8f
t-Bu i-Pr
t-Bu i-Pr
t-Bu i-Pr
t-Bu i-Pr
t-Bu i-Pr Bn CH2CO2Et
Bn Bn Bn Et
t-Bu i-Pr Bn (S)-CH(i-Pr)CO2t-Bu
H
H
H
H
p-F-C6H4
p-MeO-C6H4
Et
90
60
94
99
85
95
91
(R)-CH(Me)Ph
8g
a When the starting material is 6 (R3 ) H), a mixture of both
diastereoisomers R/ꢀ (28:1) is employed for the reaction, although, only
the major one is shown in Scheme 5. All the ureas obtained as a
diastereoisomeric mixture could be separated by flash chromatography.
b Overall yield of purified diastereoisomeric mixture.
In addition to the synthesis of urea peptidomimetics described
above, a symmetric urea peptidomimetic 8h was prepared
starting with 6 and isocyanate 11. The latter was, in turn,
obtained from 6 upon treatment with triphosgene and NaHCO3.
The symmetric tetrapeptide was obtained in 69% overall yield
in both steps. One noteworthy feature of this synthesis is that
8h has a C2 axis of symmetry (Scheme 6).
Synthesis of Peptidomimetics 9 and 10. The coupling of
N-protected R-amino acids and 6 was carried out in the presence
of TMP, HATU, and HOAt to give tripeptides 9 in good yields
(Scheme 7).15,16 The chosen R-amino acids were phenylglycine
and glycine.
Bis-trifluoromethylated peptidomimetics 10 were obtained in
two steps from intermediate 6. The first step involved the
reaction of 6 with (R-trifluoromethyl)acryloyl chloride 12.17 This
reaction led to a new Michael acceptor 13 in high yield, which
was then reacted with the appropriate nitrogen nucleophiles
(Scheme 8).
Two different representative nitrogen nucleophiles were
chosen for the aza-Michael reaction: an amine (benzylamine)
and an R-amino ester (valine tert-butyl ester hydrochloride).
Thus, the new Michael acceptor 13 was treated with the
corresponding nitrogen nucleophile under the same conditions
described above for the aza-Michael reaction of 2 with amines
(DABCO, CCl4, rt). The expected products were obtained, but
with a dramatic drop in the diastereoselectivity as compared to
the equivalent aza-Michael reactions between the same nucleo-
philes with Michael acceptor 2b (Nu ) NH2CH2Ph: 2:1 vs 8:1;
Nu ) NH2CH[CH(CH3)2]-t-BuCO2: 4:1 vs 38:1) (Scheme 9).
Although this result was somewhat disappointing, it was not
completely unexpected since the main cause for asymmetric
induction in 2b (the stereocenter of the amino acid grouping)
is more distant from the newly formed stereocenter.7c
Secondary Structure of Peptidomimetics Containing a
Trifluoroalanine Unit. Structural studies for a series of
peptidomimetics containing a trifluoroalanine unit were carried
out in order to analyze their backbone secondary structure. The
(12) Castro, J. L.; Ball, R. G.; Broughton, H. B.; Russell, M. G. N.; Rathbone,
Dd.; Watt, A. P.; Baker, R.; Chapman, K. L.; Fletcher, A. E.; Patel, S.; Smith,
A. J.; Marshall, G. R.; Ryecroft, W.; Matassa, V. G. J. Med. Chem. 1996, 39,
842–849. (b) von Geldern, T. W.; Kester, J. A.; Bal, R.; Wu-Wong, J. R.; Chiou,
W.; Dixon, D. B.; Opgenorth, T. J. J. Med. Chem. 1996, 36, 968–981. (c) Burgess,
K.; Linthicum, D. S.; Shin, H. Angew. Chem., Int. Ed. Engl. 1995, 34, 907–909.
(d) Patil, B. S.; Vasanthakumar, G. R.; Babu, V. V. S. J. Org. Chem. 1998, 63,
6088–6089.
(13) Lam, P. Y. S.; Jadhav, P. K.; Eyerman, C. J.; Hodge, C. N.; Ru, Y.;
Bacheler, L. T.; Meek, J. L.; Otto, M. J.; Rayner, M. M.; Wong, Y. N.; Chang,
C.-H.; Weber, P. C.; Jackson, D. A.; Sharpe, T. R.; Erickson-Viitanen, S. Science
1994, 263, 380–384.
(14) (a) Nowick, J. S.; Abdi, M.; Bellamo, K. A.; Love, J. A.; Mart´ınez,
E. J.; Noronha, G.; Smith, E. M.; Ziller, J. W. J. Am. Chem. Soc. 1995, 117,
89–99. (b) Nowick, J. S.; Holmes, D. L.; Macking, G.; Noronha, G.; Shaka,
A. J.; Smith, E. M. J. Am. Chem. Soc. 1996, 118, 1066–1072. (c) Nowick, J. S.;
Mahrus, S.; Smith, E. M.; Ziller, J. W. J. Am. Chem. Soc. 1996, 118, 2764–
2765.
(15) A mixture of both diastereoisomers 6r/ꢀ (96:4) is employed for the
reaction, although only the major one is shown in Scheme 7.
(16) Humphrey, J. M.; Chamberlin, A. R. Chem. ReV. 1997, 97, 2243–2266.
J. Org. Chem. Vol. 74, No. 8, 2009 3125

Fustero et al.
FIGURE 2. X-ray plot of trifluoroalanine-containing peptidomimetic backbone for 5hr, 7r, 8c, and 13 (red oxygen, blue nitrogen, green fluorine,
gray carbon, and white hydrogen atoms).
single-crystal X-ray structure of compounds 5hr, 7r, 8c, and
13 were thus determined.18 The results showed that one (8c,
13), two (5hr), and four (7r) independent molecules are present
in the crystal structure asymmetric unit. The molecular structure
for all the independent molecules of compounds 5hr, 7r, 8c,
and 13 show a ꢀ-turn-like, nine-membered folding pattern,
3126 J. Org. Chem. Vol. 74, No. 8, 2009

Fluorinated Peptidomimetics through Tandem Aza-Michael Addition
SCHEME 6. Synthesis of Symmetric Urea-Containing
Tetrapeptide 8ha
SCHEME 9. Aza-Michael Addition of Nitrogen
Nucleophiles to 13
a A 28:1 diastereomeric mixture for compound 6 was used as starting
material. Compound 8h consisted of a 26:3:3:1 mixture of diastereomers,
only the major of which is shown.
SCHEME 7. Coupling of 6 with N-Protected r-Amino Acids
the peptidic chain. Canonical ꢀ-turns involve a 10-membered
ring with a hydrogen bond between the backbone CO(i) and
the backbone NH(i + 3).20 The simplest and most abundant
ꢀ-turns, those of type I and type II, can be characterized by the
φ and ψ torsion angles of the two residues, i+1 and i+2, in the
middle of the turn (Figure 4a).21 Although ꢀ-turns are mostly
stabilized by hydrogen bonds, it is not clear that such hydrogen
bonds are necessary for the appearance of ꢀ-turns.21b A distance
R
criterion has been established, according to which the CiR-Ci+3
distance must be shorter than 7 Å.22 Therefore, typical
SCHEME 8. Preparation of Michael Acceptor 13
parameters studied for canonical C10 ꢀ-turns are the dihedral
R
angles φi+1, ψi+1, φi+2, and ψi+2 and the CiR-Ci+3 distance.
FIGURE 3. Conformation of Gellman’s and Zanda’s PMR-pep-
tides.
although no intramolecular hydrogen bonding was observed in
the crystal structure (Figure 2).
Several examples of related PMR-peptides, such as triamide
1419 and PMR ψ[NHCH(CF3)]Gly peptides 15,5 with ꢀ-turn-
like, nine-membered conformation have been previously re-
ported. These have all been found to have intramolecular
N-H· · ·OdC hydrogen bonds in the solid state (Figure 3).
Canonical ꢀ-turns are constituted of four amino acid residues
and are characterized by a sudden change in the direction of
(17) For the preparation of (R-trifluoromethyl)acryloyl chloride 12, see: (a)
Hanzawa, Y.; Suzuki, M.; Kobayashi, Y.; Taguchi, T.; Iitaka, Y. J. Org. Chem.
1991, 56, 1718–1725. (b) Yamakazi, T.; Ichige, T.; Takei, S.; Kawashita, S.;
Kitazume, T.; Kubota, T. Org. Lett. 2001, 3, 2915–2918.
(18) For more details about the crystal structure determination, see the
Supporting Information.
(19) (a) Gellman, S. H.; Adams, B. R.; Dado, G. P. J. Am. Chem. Soc. 1990,
112, 460–461. (b) Dado, G. P.; Desper, J. M.; Gellman, S. H. J. Am. Chem.
Soc. 1990, 112, 8630–8632. (c) Gellman, S. H.; Dado, G. P.; Liang, G.-B.;
Adams, B. R. J. Am. Chem. Soc. 1991, 113, 1164–1173. (d) Liang, G.-B.; Dado,
G. P.; Gellman, S. H. J. Am. Chem. Soc. 1991, 113, 3994–3995. (e) Dado, G. P.;
Desper, J. M.; Holmgren, S. K.; Rito, C. J.; Gellman, S. H. J. Am. Chem. Soc.
1992, 114, 4834–4843. (f) Dado, G. P.; Gellman, S. H. J. Am. Chem. Soc. 1993,
115, 4228–4245.
FIGURE 4. (a) C10 ꢀ-turn motif and typical backbone torsion angles
φ (Ci-1-Ni-CR -Ci) and ψ (Ni-CR -Ci-Ni+1) in a simple peptide
i
i
model. (b) C9 ꢀ-turn-like motif and backbone torsion angles φ1
(O2-C1-CR -N3), ψ1 (C1-CR -N3-C4), φ2 (N3-C4-CR -C6), and ψ2
2
2
5
(C4-CR -C6-N7) in trifluoroalanine-containing peptidomimetics.
5
J. Org. Chem. Vol. 74, No. 8, 2009 3127

Fustero et al.
TABLE 3. O(2)· · ·C(8) Distance (Å) and O1 (O2-C1-Cr2-N3), ψ1 (C1-Cr2-N3-C4), O2 (N3-C4-Cr5-C6), and ψ2 (C4-Cr5-C6-N7) Torsion
Angles (deg) for All Independent Molecules of Peptidomimetics 5hr, 7r, 8c, and 13a
torsion angles in residue i + 1
torsion angles in residue i + 2
entry
product
O(2)· · ·C(8) distance
φ1
ψ1
φ2
ψ2
1
2
3
4
5
6
7
8
5hrA
5hrB
7rA
7rB
7rC
7rD
8c
5.7014(26)
5.0325(25)
5.6487(56)
5.5476(62)
5.1404(56)
5.4147(59)
5.2277(30)
5.4156(90)
159.83(15)
-21.43(22)
164.97(41)
168.12(36)
-81.87(44)
165.69(39)
-77.54(22)
146.57(81)
-118.84(18)
-81.73(20)
-113.15(46)
-120.73(44)
-101.74(47)
-131.66(44)
-86.94(23)
-109.88(85)
-114.91(17)
-138.29(16)
-129.74(39)
-124.47(40)
-128.99(40)
-126.11(41)
-128.65(21)
-126.71(65)
58.45(20)
65.34(20)
52.64(45)
45.72(46)
55.91(44)
38.66(50)
74.50(22)
58.47(74)
13
a The O(2)· · ·C(8) distance found for all independent molecules of compounds 5hr, 7r, 8c, and 13 is within the 5.03-5.70 Å range. This is in good
R
agreement with a corresponding CiR · · ·Ci+3 distance being shorter than 7 Å, which is the distance criterion for ꢀ-turns. The ψ1, φ2, and ψ2 torsion
angles determined for all the molecules are in the range of -106 ( 25, -127 ( 12, and 52 ( 13°, respectively. These values are not within the range
defined for type I and type II ꢀ-turns for the φ and ψ torsion angles of the two residues, i+1 and i+2, in the middle of the turn [type I: φi+1 ) -60 (
30°, ψi+1 ) -30 ( 30°, φi+2 ) -90 ( 30°, ψi+2 ) 0 ( 50°; type II: φi+1 ) -60 ( 30°, ψi+1 ) +120 ( 30°, φi+2 ) +80 ( 30°, ψi+2 ) 0 ( 50°].
However, the ꢀ-turn-like folded peptidomimetic torsion angle sequence ψ1 ·φ2 ·ψ2 is similar to a retro-inverso type II ꢀ-turn torsion angle sequence
(-φi+2)·(-ψi+>1)·(-φi+1). The values found for the φ1 torsion angle [157 ( 11° (5hrA, 7rA, 7rB, 7rD), -80 ( 3° (7rC, 8c), and -21.5 ( 0.1
(5hrB)] do not fit in a retro-inverso type II torsion angle sequence. Since the φ1 torsion angle is not involved in the 9-membered, ꢀ-turn-like
conformation present in compounds 5hr, 7r, 8c, and 13, it will therefore not be expected to correspond to the (-Ψi+2) torsion angle present in a C10
ꢀ-turn.
TABLE 4. Hydrogen Bond Lengths (Å) and Angles (deg) for
equilibrium between hydrogen-bonded and non hydrogen-
Trifluoroalanine-Containing Peptidomimetics 5hr, 7r, 8c, and 13
bonded states display a high temperature dependence (∆δ/∆T
g 2.6).24 We thus performed H NMR (CDCl3) experiments
1
compd
X · · ·Y
H· · ·Y
X-H· · ·Y
using a 3 mM solution of 7r at different temperatures. The
notably high ∆δ/∆T value for both Ha and Hb protons (8.39
and 21.60, respectively) indicate that they are in equilibrium
between hydrogen-bonded and non-hydrogen-bonded states
(Figure 6). To determine whether the hydrogen-bonded state
was due to an intra- or intermolecular interaction, H NMR
analysis of 7r under concentrated conditions (300 mM) was
carried out.
5hr
N7A-H7A· · ·F
N7-H7· · ·O1Ai
N7A-H7A· · ·O3D
N7B-H7B· · ·O1C
N7C-H7C· · ·O3B
N7D-H7D· · ·O3A
N7-H7· · ·O3i
3.749(2)
3.152(2)
2.874(5)
3.067(5)
2.928(5)
2.833(5)
2.989(3)
3.124(3)
2.831(7)
2.907(18)
2.288(17)
2.07(2)
2.25(2)
2.13(2)
2.10(3)
2.25(2)
2.58(2)
2.33(6)
168(2)
172(2)
168(5)
171(5)
173(5)
150(5)
147(2)
124(2)
150(9)
7r
1
8c
13
N7-H7· · ·O1i
N7-H7· · ·O2i
a Symmetry operators: 5hr (i: x - 1, y, z), 7r (i: x - 1, y, z; ii: x +
For both Ha and Hb, the chemical shift moved downfield with
the variation being quite remarkable (0.49 ppm and 1.13 ppm,
respectively), a result that indicates that intermolecular hydrogen
bonds are involved. This variation in chemical shifts to
downfield regions for the concentrated samples as compared to
the diluted ones was observed for all the temperature ranges
studied. The ∆δ/∆T value for both Ha and Hb protons was very
high at this concentration (12.50 and 18.75, respectively),
confirming the equilibrium between the two states (Figure 6).
Finally, in order to discard the possibility of intramolecular
interactions and thereby confirm the existence of the intermo-
lecular hydrogen bonds, the chemical shift of protons Ha and
Hb upon the addition of MeOD, a competitive solvent for
hydrogen bond formation, was measured (Figure 7). Amide
protons with intramolecular hydrogen bonds exchange much
more slowly with the solvent than those with intermolecular
hydrogen bonds and thus the chemical shift should not change
significantly in the former case. The considerable increase of
1
1
1, y, z), 8c (i: -x + 1, y + /2, -z), 13 (i: -x + 2, y + /2, -z + 2).
The corresponding torsion angles of the two residues i+1
and i+2 for the analysis of the ꢀ-turn-like nine-membered
folding pattern found for the backbone of compounds 5hr, 7r,
8c, and 13 are φ1 (O2-C1-CR -N3), ψ1 (C1-CR -N3-C4), φ2
2
2
(N3-C4-CR -C6), and ψ2 (C4-CR -C6-N7) (Figure 4b). The
5
5
O(2)· · ·C(8) distance would correspond to the CiR · · ·Ci+3
R
distance if we take into account that in the reported peptido-
mimetics, O(2) corresponds to CiR in a typical peptidic sequence
(Figure 4b). These parameters have all been determined from
single-crystal X-ray diffraction data (Table 3).
In all the crystal structures reported, the N(7)-H moiety is
involved in the 3D intermolecular hydrogen bonding network
rather than in an intramolecular N-H· · ·OdC hydrogen bond
(Table 4). The hydrogen bonding scheme for the four indepen-
dent molecules of compound 7r is shown in Figure 5. This
means that the ꢀ-turn-like conformation found in all the
independent molecular structures of 5hr, 7r, 8c, and 13 is
inherent to the peptidomimetic backbone primary structure and
does not depend on the intramolecular hydrogen bond formation.
Compound 7r was selected to confirm the conformation of
these new peptidomimetics in solution. The temperature de-
pendence of NMR chemical shifts for amide protons that do
not form strong hydrogen bonds with a given solvent, e.g.,
CDCl3, provide valuable information about the formation of
hydrogen bonds.23 Peptide NH protons that either have no
hydrogen bonds or are strongly hydrogen bonded exhibit little
temperature dependence (∆δ/∆T e 2.6) while protons in
(20) For some leading references, see, for example: (a) Sibanda, B. L.;
Thornton, J. M. Nature 1985, 316, 170–174. (b) Wilmot, C. M.; Thornton, J. M.
J. Mol. Biol. 1988, 203, 221–232. (c) Sibanda, B. L.; Thornton, J. M. J. Mol.
Biol. 1993, 229, 428–447. (d) Mattos, C.; Petsko, G. A.; Karplus, M. J. Mol.
Biol. 1994, 238, 733–747. (e) Gunasekaran, K.; Ramakrishnan, C.; Balaram, P.
Protein Eng. 1997, 10, 1131–1141. (f) Gardner, R. R.; Liang, G.-B.; Gellman,
S. H. J. Am. Chem. Soc. 1999, 121, 1806–1816.
(21) See, for example: (a) Rai, R.; Vasudev, P. G.; Ananda, K.; Raghothama,
S.; Shamala, N.; Karle, I. L.; Balaram, P. Chem.sEur. J. 2007, 13, 5917. (b)
Czinki, E.; Csa´sz´ar, A. G.; Perczel, A. Chem.sEur. J. 2003, 9, 1182. (c) Wipf,
P.; Henninger, T. C.; Gelb, S. J. J. Org. Chem. 1998, 63, 6088.
(22) (a) Levitt, M. J. Mol. Biol. 1976, 104, 59. (b) Chou, P. Y.; Fasman,
G. D. J. Mol. Biol. 1993, 229, 428–447.
(23) Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.; Scolastico, C. Eur. J.
Org. Chem. 1999, 389–400.
(24) ∆δ/∆T values are given in ppb/K.
3128 J. Org. Chem. Vol. 74, No. 8, 2009

Fluorinated Peptidomimetics through Tandem Aza-Michael Addition
FIGURE 5. Hydrogen-bonding scheme for the four independent molecules of compound 7r.
FIGURE 6. Temperature dependence of amide protons NHa and NHb of 7r at concentrations of 3 mM and 300 mM.
1
the chemical shift values found in the H NMR experiments
upon the successive addition of MeOD thus implies the absence
of intramolecular hydrogen bonds.
containing an R-trifluoromethyl-ꢀ2-alanine 9, and PMR
ψ[NHCH2] containing two R-trifluoromethyl-ꢀ2-alanine units
10.
Taken together, these results suggest the formation of
intermolecular hydrogen bonds for 7r. These results are in
complete agreement with the X-ray diffraction analysis of the
compound’s crystal structure, in which further evidence of these
intermolecular hydrogen bonds can be found (see Table 4).
The backbone conformation for some selected trifluoroala-
nine-containing peptidomimetics has been analyzed through
single-crystal X-ray diffraction and NMR techniques. It was
observed that trifluoroalanine-containing PMR ψ[NHCH2] pep-
tides provide a preorganized ꢀ-turn-like conformation backbone
even though no intramolecular hydrogen bonding was observed
in either solid state or solution. This type of peptidomimetics
could thus be used to induce ꢀ-turn-like conformations and to
promote ꢀ-hairpin formation.
Conclusion
In this paper, we have described the synthesis of several
families of R-trifluoromethyl-ꢀ2-alanine unit-containing pepti-
domimetics. First, the synthesis of peptidomimetics 5 by means
of a stereoselective aza-Michael addition of amines to the
fluorinated Michael acceptor 2 proceeds very efficiently in
comparison with other similar reactions; in fact, similar
substrates without a trifluoromethyl group do not react at all
under the conditions used here. Hydrogenolysis of N-benzyl-
protected amine 5b gives N-free peptidomimetic 6, which was
then used as an intermediate for the synthesis of other pepti-
domimetics: PMR ψ[NHCH2] urea peptides containing an
R-trifluoromethyl-ꢀ2-alanine 8, PMR ψ[NHCH2] tripeptides
Experimental Section
General Method. The reactions were carried out under nitrogen
atmosphere unless otherwise indicated. The solvents were purified
prior to use: CH2Cl2 and CCl4 were distilled from calcium hydride.
Benzylamine was distilled before use. All reagents were used as
received unless otherwise stated. The reactions were monitored with
the aid of thin-layer chromatography (TLC) on 0.25 mm E. Merk
precoated silica gel plates. Visualization was carried out with UV
light, aqueous ceric ammonium molybdate solution, or potassium
permanganate stain. Flash column chromatography was performed
J. Org. Chem. Vol. 74, No. 8, 2009 3129

Fustero et al.
FIGURE 7. Chemical shift dependence of amide protons NHa and NHb of 7r upon addition of MeOD.
with the indicated solvents on silica gel 60 (particle size 0.040-0.063
mm). Melting points were measured with a “Cambridge Instrument”
apparatus. Optical rotations were measured on a Perkin-Elmer 241
CDCl3) δ ) 0.86 (d, J ) 6.9 Hz, 3H), 0.89 (d, J ) 6.9 Hz, 3H),
1.40 (s, 9H), 2.09-2.20 (m, 1H), 2.64 (br, 2H), 3.07-3.10 (m,
1H), 3.17-3.30 (m, 2H), 4.40 (dd, J1 ) 8.2 Hz; J2 ) 4.5 Hz; 1H),
7.53 ppm (d, J ) 8.2 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) δ )
1
polarimeter. H, 13C, and 19F NMR spectra were recorded on 300
3
MHz Bruker and 400 MHz Bruker Advance spectrometers. Chemi-
cal shifts (δ) are given in ppm. Coupling constants (J) are given in
hertz (Hz). The letters m, s, d, t, and q stand for multiplet, singlet,
doublet, triplet, and quartet, respectively. The letters br indicate
that the signal is broad. High-resolution mass spectra measurements
were carried out on a VGmAutospec instrument (VG Analytical,
Micromass Instruments) by the Universidad de Valencia Mass
Spectrometry Service.
General Procedure for the Synthesis of Peptidomimetics 5.
Amine 4 (0.2 mmol) was added to a solution of Michael acceptor
2 (0.2 mmol) and DABCO (0.2 mmol) in CCl4 (4 mL) at 0 °C.
The reaction mixture was stirred at this temperature until TLC
revealed total consumption of the starting material (30 min). The
solvent was removed under vacuum, yielding a diastereoisomeric
mixture of 5r/5ꢀ. Major diastereoisomer 5r was separated in most
cases by means of flash chromatography (n-hexane/ethyl acetate)
on deactivated silica gel (2% Et3N in n-hexane) and later, when
necessary, washed with n-hexane to yield a white solid.
16.4 (CH3), 17.9 (CH3), 26.9 (CH3), 29.9 (CH), 37.2 (Cq, J(C,F)
) 2.3 Hz), 51.1 (Cq, 2J(C,F) ) 25.3 Hz), 57.0 (CH), 81.3 (C), 123.5
(Cq, 1J(C,F ) 280.5 Hz), 164.6 (Cq, 3J(C,F) ) 2.3 Hz), 170.0 (C);
19F NMR (282.4 MHz, CDCl3) δ ) -66.8 ppm (d, J(H,F) ) 8.2
Hz, 3F); HRMS m/z calcd for C13H23F3N2O3 312.1660, found
312.1619 [M]+.
Synthesis of 7: (2S)-2-[(2S)-2-(Acetylaminomethyl)-3,3,3-trifluo-
ropropanamido]-3-methylbutanoate (7). Acetic anhydride (2.14
mmol, 0.2 mL) and pyridine (2.14 mmol, 0.17 mL) were added to
a solution of 6 (0.54 mmol, 0.169 mg) in CH2Cl2 (12 mL) and
stirred at room temperature for 12 h. The reaction mixture was
quenched with satd aq NaHCO3 (5 mL) and extracted with CH2Cl2
(3 × 5 mL). The combined organic layers were washed with brine
and dried over anhydrous sodium sulfate, and the solvents were
removed under reduced pressure to obtain a white solid, which was
then purified by recrystallization. Yield >99%. Major diastereoi-
somer 7r: mp 202-204 °C; [R]25D ) +101.2 (c ) 1.0 in CHCl3);
1H NMR (300 MHz, CDCl3) δ ) 0.85 (d, J ) 6.9, 3H), 0.89 (d, J
) 6.8, 3H), 1.40 (s, 9H), 1.91 (s, 3H), 2.12-2.20 (m, 1H),
3.34-3.55 (m, 2H), 3.83-3.91 (m, 1H), 4.37 (dd, J1 ) 8.3, J2 )
4.5, 1H), 6.31 (sa, 1H), 6.92 (d, J ) 8.3, 1H); 13C NMR (75.5
MHz, CDCl3) δ ) 17.4 (CH3), 18.9 (CH3), 22.9 (CH3), 27.9 (CH),
30.5 (CH3), 36.3 (Cq, 3JCF ) 3.5), 49.5 (Cq, 2JCF ) 26.4), 58.2 (CH),
(S)-tert-Butyl 2-(S+R)-[2-(allylaminomethyl)-3,3,3-trifluoropro-
panamido]-3-methylbutanoate (5br/5bꢀ). Total yield 95%. Major
diastereoisomer 5br: mp 94-96 °C; [R]25 ) +1.9 (c ) 1.0 in
D
CHCl3); 1H NMR (300 MHz, CDCl3) δ 0.83 (d, J ) 6.9 Hz, 3H),
0.88 (d, J ) 6.7 Hz, 3H), 1.39 (s, 9H), 2.08-2.18 (m, 1H), 2.41
(br, 1H), 2.92-2.98 (m, 1H), 3.14-3.23 (m, 2H), 3.74-3.83 (m,
2H), 4.39 (dd, J1 ) 8.4 Hz; J2 ) 4.3 Hz; 1H), 7.22-7.27 (m, 5H),
7.55 ppm (d, J ) 8.1 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) δ )
1
3
82.1 (C), 124.0 (Cq, JCF ) 266.8), 165.8 (Cq, JCF ) 1.7), 170.9
(C), 171.2 (C); 19F NMR (282.4 MHz, CDCl3) δ ) -67.5 ppm (d,
J(H,F) ) 7.2 Hz, 3F); HRMS m/z calcd for C15H25F3N2O4 ·HCl
355.1844, found 355.1812 [M]+.
3
17.5 (CH3), 18.9 (CH3), 27.9 (CH3), 31.1 (CH), 45.0 (Cq, J(C,F)
2
) 2.8 Hz), 50.5 (Cq, J(C,F) ) 25.2 Hz), 53.9 (CH2), 57.8 (CH),
General Procedure for the Synthesis of Isocyanates 11g and
11h.25 A saturated solution of NaHCO3 (5 mL) and a solution of
triphosgene (0.32 mmol) in CH2Cl2 (1 mL) were added to a solution
of the tert-butyl ester of valine (0.48 mmol) in CH2Cl2 (5 mL) at
0 °C. After the isocyanate was formed (20 min), the organic layer
of the reaction was separated, and the aqueous phase was extracted
with CH2Cl2 (3 × 15 mL). The combined organic layers were dried
over anhydrous sodium sulfate, and the solvents were removed
under reduced pressure to yield the crude product, which was
employed in the next reaction with no further purification.
General Procedure for the Synthesis of Urea-Substituted Pep-
tidomimetics 8. Isocyanate 11 (0.54 mmol) was added to a solution
of 6 (0.54 mmol) in CH2Cl2 (3 mL) under N2 atmosphere. The
reaction mixture was stirred until TLC revealed total consumption
82.1 (C), 124.6 (Cq, 1J(C,F) ) 280.0 Hz), 127.4 (CH), 128.1 (CH),
3
128.5 (CH), 138.7 (C), 165.5 (Cq, J(C,F) ) 2.2 Hz), 170.0 ppm
(C); 19F NMR (282.4 MHz, CDCl3) δ ) -66.7 ppm (d, J(H,F) )
9.2 Hz, 3F); HRMS m/z calcd for C20H29F3N2O5 402.2130, found
402.2218 [M]+.
Synthesis of the Intermediate 6: (S)-tert-Butyl 2-[2-(Aminom-
ethyl)-3,3,3-trifluoropropanamido]-3-methylbutanoate (6). Pearl-
man catalyst Pd(OH)2 (60%) was added to a solution of 5b (0.7
mmol) in AcOEt (30 mL). This mixture was stirred for 24 h under
H2 atmosphere (100 psi) and then was filtered through Celite. The
solvent was removed under vacuum to obtain a diastereoisomeric
mixture 6r/6ꢀ (28:1). Total yield >99%. Data were taken from the
28:1 mixture. Major diastereoisomer 6r: 1H NMR (300 MHz,
3130 J. Org. Chem. Vol. 74, No. 8, 2009

Fluorinated Peptidomimetics through Tandem Aza-Michael Addition
of the starting material (30 min). The solvent was removed under
vacuum to yield a diastereoisomeric mixture of 8r/8ꢀ. Major
diastereoisomer 8r was separated through purification with the aid
of recrystallization or flash chromatography on silica gel as
stationary phase.
2J(C,F) ) 26.4 Hz), 57.9 (CH), 82.5 (C), 121.8 (Cq, 1J(C,F) ) 272.5
Hz), 123.9 (Cq, 1J(C,F) ) 280.5 Hz), 129.0 (Cq, 3J(C,F) ) 5.2 Hz),
133.7 (Cq, 2J(C,F) ) 31.0 Hz), 161.5 (C), 164.7 (Cq, 3J(C,F) ) 2.3
Hz), 170.4 ppm (C); 19F NMR (282.4 MHz, CDCl3) δ ) -64.6 (s,
3F), -67.1 (d, J(H,F) ) 9.3 Hz, 3F); HRMS m/z calcd for
C17H24F6N2O4 435.1718, found 435.1799 [M + 1]+.
General Procedure for the Synthesis of Bis-trifluorometh-
ylated Peptidomimetics 10. To a solution of 13 (0.10 mmol) in
CCl4 (3 mL) under nitrogen atmosphere at 0 °C were added
DABCO (0.10 mmol) and then the nitrogenated nucleophile (0.10
mmol). The reaction mixture was stirred until TLC revealed total
consumption of the starting material (2 h). The solvent was then
removed under vacuum. The crude reaction mixture was subjected
to flash chromatography (n-hexane/ethyl acetate 6:1) to obtain the
product as a diastereomeric mixture (10r/10ꢀ).
(S)-tert-Butyl 2-(S+R)-2-[(3-Ethylureidomethyl)-3,3,3-trifluoro-
propanamido]-3-methylbutanoate (8c). Total yield 94%. Major
diastereoisomer 8cr: mp 204-206 °C; [R]25D ) +53.5 (c ) 1.1 in
1
CHCl3); H NMR (400 MHz, CD3COCD3) δ ) 0.95 (d, J ) 4.6,
3H), 0.97 (d, J ) 4.6, 3H), 1.06 (t, J ) 7.3, 3H), 1.47 (s, 9H),
2.14-2.22 (m, 1H), 3.11-3.19 (m, 2H), 3.42-3.49 (m, 1H),
3.64-3.76 (m, 2H), 4.36 (dd, J1 ) 6.6, J2 ) 5.4, 1H), 5.60 (br,
1H), 5.71 (br, 1H), 8.00 ppm (br, 1H); 13C NMR (100 MHz,
CD3COCD3) δ ) 15.8 (CH3), 17.8 (CH3), 19.3 (CH3), 28.1 (CH3),
3
31.6 (CH), 35.3 (CH2), 37.8 (Cq, J(C,F) ) 1.8 Hz), 50.1 (Cq,
2J(C,F) ) 23.7 Hz), 58.9 (CH), 81.9 (C), 125.9 (Cq, 1J(C,F) ) 277.8
Hz), 166.2 (C), 166.2 (Cq, 3J(C,F) ) 3.0 Hz), 171.3 (C); 19F NMR
(282.4 MHz, CDCl3) δ ) -67.4 ppm (d, J (H,F) ) 8.2 Hz, 3F);
HRMS m/z calcd for C16H28F3N3O4 383.2031, found 383.2082 [M]+.
General Procedure for the Synthesis of Peptidomimetics 9. sym-
Collidine (1.10 mmol), HOAt (0.22 mmol), and HATU (0.22 mmol)
were added to a solution of 6 (0.22 mmol) and the corresponding
N-protected R-amino acid (0.28 mmol) in anhydrous DMF (2.5 mL).
The reaction mixture was stirred overnight, quenched with HCl 1
N (6 mL), and extracted with diethyl ether (3 × 5 mL). The
combined organic layers were washed with water and dried over
anhydrous sodium sulfate, and then the solvents were removed
under reduced pressure to obtain a diastereoisomeric mixture of
9r/9ꢀ. The crude reaction mixture was subjected to flash chroma-
tography (n-hexane/ethyl acetate 6:1) to afford the major diaster-
oisomer 9r.
(S)-tert-Butyl 2-{(S)-2-[(S+R)-2-(benzylaminomethyl)-3,3,3-tri-
fluoropropanamidomethyl]-3,3,3-trifluoropropanamido}-3-meth-
ylbutanoate (10a). The mixture of 10ar/10aꢀ 2:1 was not separable
by FC: total yield 70%; 1H NMR (300 MHz, CDCl3) δ ) 0.80 (d,
J ) 6.8 Hz, 3H), 0.86 (d, J ) 6.9 Hz, 3H), 1.38 (s, 9H), 1.59 (br,
1H), 2.06-2.14 (m, 1H), 2.85-2.90 (m, 1H), 2.95-3.05 (m, 1H),
3.08-3.16 (m, 1H), 3.29-3.38 (m, 1H), 3.46-3.56 (m, 1H),
3.69-3.71 (m, 2H), 3.82-3.89 (m, 1H), 4.36 (dd, J1 ) 8.5 Hz, J2
) 4.6 Hz, 1H), 6.36 (d, J ) 8.5 Hz, 1H), 7.19-7.30 (m, 5H), 7.95
ppm (t, J ) 5.2 Hz, 1H); 13C NMR (75.5 MHz, CDCl3) δ ) 17.4
(CH3), 18.8 (CH3), 27.9 (CH3), 30.9 (CH), 36.1 (Cq, 3J(C,F) ) 2.9
2
2
Hz); 44.6 (CH2); 49.7 (Cq, J(C,F) ) 25.8 Hz); 50.2 (Cq, J(C,F)
) 25.3 Hz), 53.6 (CH2), 58.0 (CH), 82.5 (C), 123.9 (Cq, 1J(C,F) )
280.5 Hz), 124.5 (Cq, 1J(C,F) ) 271.3 Hz), 127.5 (CH), 128.1 (CH),
3
3
128.6 (CH), 138.7 (C), 165.0 (Cq, J(C,F) ) 2.3), 166.3 (Cq, JCF
) 2.3 Hz), 170.9 ppm (C); 19F NMR (282.4 MHz, CDCl3) δ )
-66.7 (d, J(H,F) ) 8.6 Hz, 3F), -67.4 ppm (d, J(H,F) ) 7.8 Hz,
3F); HRMS m/z calcd for C24H33F6N3O4 542.2453, found 542.2471
[M + 1]+.
[6S,10(S+R),13S)-tert-Butyl 13-Isopropyl-2,2-dimethyl-4,7,11-
trioxo-6-phenyl-10-(trifluoromethyl)-3-oxa-5,8,12-triazatetradecan-
14-oate (9b). Total yield 60%. Major diastereoisomer 9br obtained
as a white solid: mp 174-177 °C; [R]25 ) +100.9 (c ) 0.9 in
D
Single-Crystal X-ray Diffraction. Crystals were grown by means
of slow evaporation of n-hexane (5hr), n-hexane/CHCl3 (7r),
acetone (8c), or n-hexane/CH2Cl2 (13) solutions. Crystals suitable
for X-ray diffraction were measured at 100(2) K (13) or 150(2) K
(5hr, 7r, and 8c) on a Nonius-Kappa CCD diffractometer using
graphite-monochromated Mo KR radiation (λ ) 0.71073 Å) and a
ω scan mode. Crystal structures were solved with direct methods,
and all non-hydrogen atoms were refined anisotropically on F2
(program SHELXL-97).26 The hydrogen atoms of the NH groups
were located in a difference Fourier synthesis and refined with
restrained N-H bond lengths. The methyl groups for compounds
5hr, 7r, and 8c were refined as rigid. Other hydrogen atoms were
included using a riding model. For compound 13, the t-Bu group
is disordered over two positions; the disordered atoms were refined
isotropically. The absolute structure could not be determined by
means of anomalous dispersion effects.27 The programs use neutral
atom scattering factors, ∆f′ and ∆f′′, and absorption coefficients
from International Tables for Crystallography.28 Crystal data for
compound 5hr: colorless prism, 0.45 × 0.20 × 0.16 mm size,
orthorhombic, P212121, a ) 9.668(19) Å, b ) 20.336(4) Å, c )
23.189(5) Å, V ) 4559.4(16) Å3, Z ) 8, Dc ) 1.213 g cm-3, 2θmax
) 54.96°, 34939 reflections collected of which 5747 were inde-
pendent (Rint ) 0.045), 547 refined parameters, R1[I > 2σ(I)] )
0.0345, wR2(all data) ) 0.0789. Crystal data for compound 7r:
colorless prism, 0.36 × 0.12 × 0.04 mm size, triclinic, P1, a )
9.5958(19) Å, b ) 12.452(3) Å, c ) 15.882(3) Å, R ) 90.36(3),
ꢀ ) 92.27(3), γ ) 98.29(3)°, V ) 1876.2(7) Å3, Z ) 4, Dc )
1.255 g cm-3, 2θmax ) 55.76°, 8912 reflections collected of which
4747 were independent (Rint ) 0.094), 913 refined parameters, R1[I
> 2σ(I)] ) 0.0648, wR2(all data) ) 0.1325. Crystal data for
1
CHCl3); H NMR (300 MHz, CDCl3) δ ) 0.90 (d, J ) 6.0 Hz,
3H), 0.92 (d, J ) 6.4 Hz, 3H), 1.36 (br, 9H), 1.44 (s, 9H),
2.09-2.22 (m, 1H), 3.14-3.20 (m, 2H), 4.01-4.06 (m, 1H), 4.32
(dd, J1 ) 8.3 Hz, J2 ) 5.5 Hz, 1H), 5.0 (d, J ) 6.2 Hz, 1H), 5.45
(d, J ) 4.9 Hz, 1H), 7.13-7.15 (m, 2H), 7.26-7.34 ppm (m, 5H);
13C NMR (75.5 MHz, CDCl3) δ ) 18.1 (CH3), 19.1 (CH3), 28.0
(CH3), 28.2 (CH3), 29.7 (CH), 37.2 (CH2), 49.8 (Cq, 2J(C,F) ) 27.2
1
Hz), 59.1 (CH), 59.4 (CH), 80.5 (C), 82.7 (C), 123.7 (Cq, J(C,F)
) 280.0 Hz), 127.4 (CH), 129.0 (CH), 129.3 (CH), 136.1 (C), 155.7
(C), 166.7 (C), 171.5 (C), 172.4 ppm (C); 19F NMR (282.4 MHz,
CDCl3) δ ) -67.6 ppm (d, J(H,F) ) 6.1 Hz, 3F); HRMS m/z
calcd for C26H38F3N3O6 546.2790, found 546.2872 [M + 1]+.
Synthesis of Michael Acceptor 13: (S)-tert-Butyl 3-Methyl-2-
(S+R)-3,3,3-trifluoro-2-[(2-(trifluoromethyl)acrylamido)meth-
yl]propanamido butanoate (13). To a solution of 6 (0.72 mmol) in
CH2Cl2 (10 mL) under N2 atmosphere at 0 °C were added sym-
collidine (1.44 mmol) and a solution of (R-trifluoromethyl)acryloyl
chloride 12 (1.44 mmol) in CH2Cl2 (5 mL) followed by a catalytic
amount of DMAP. The reaction mixture was stirred until TLC
revealed total consumption of the starting material (2 h). The
reaction was quenched with NH4Cl (10 mL) and extracted with
CH2Cl2 (3 × 10 mL). The combined organic layers were washed
with HCl 1 M (3 × 10 mL) and dried over anhydrous sodium
sulfate, and the solvent was then removed under reduced pressure
to obtain the crude product. Purification through flash chromatog-
raphy (n-hexane/ethyl acetate 8:1) afforded the pure product 13 as
a white solid: yield 90%; mp 185-189 °C; [R]25D ) +71.4 (c 0.9,
1
CHCl3); H NMR (300 MHz, CDCl3) δ ) 0.82 (d, J ) 6.9 Hz,
3H), 0.87 (d, J ) 6.8 Hz, 3H), 1.39 (s, 9H), 2.08-2.18 (m, 1H),
3.34-3.47 (m, 1H), 3.63-3.72 (m, 1H), 3.84-3.92 (m, 1H), 4.39
(dd, J1 ) 8.7 Hz, J2 ) 4.5 Hz, 1H), 6.17 (q, JHF ) 1.14 Hz, 1H),
6.35 (q, J ) 8.5 Hz, 1H), 6.43 (q, JHF ) 1.5 Hz, 1H), 6.71 ppm
(br, 1H); 13C NMR (75.5 MHz, CDCl3) δ )17.2 (CH3), 18.7 (CH3),
(25) Nowick, J. S.; Holmes, D. L.; Noronha, G.; Smith, E. M.; Nguyen, T. M.;
Huang, S. L. J. Org. Chem. 1996, 61, 3929–3934.
(26) SHELXL-97: Sheldrick, G. M., University of Go¨ttingen: Germany, 1997.
(27) Flack, H. D. Acta Crystallogr. 1983, A39, 876.
3
27.9 (CH3), 30.9 (CH), 36.3 (Cq, J(C,F) ) 2.9 Hz), 49.3 (Cq,
J. Org. Chem. Vol. 74, No. 8, 2009 3131

Fustero et al.
compound 8c: colorless lath, 0.45 × 0.09 × 0.03 mm size,
monoclinic, P21, a ) 10.503(2) Å, b ) 9.1652(18) Å, c ) 10.804(2)
Acknowledgment. We acknowledge the Ministerio of Edu-
cacio´n y Ciencia (CTQ2007-61462 and CTQ2006-01317) and
Centro de Investigacio´n Pr´ıncipe Felipe (CIPF) for financial
support. G.C. thanks the Ministerio of Educacio´n y Ciencia for
a predoctoral fellowship and CIPF for a research assistant
contract, and J.P. thanks the Instituto de Salud Carlos III of
Ministerio de Sanidad (CP07/00314). C.R.d.A. thanks Con-
solider Ingenio 2010 (CSD2007-00006) and Generalitat Valen-
ciana (GV07/087) for financial support.
Å, ꢀ ) 92.64(3)°, V ) 1038(4) Å3, Z ) 2, Dc ) 1.226 gcm-3
,
2θmax ) 52.74°, 7646 reflections collected of which 4230 were
independent (Rint ) 0.059), 253 refined parameters, R1[I > 2σ(I)]
) 0.0440, wR2(all data) ) 0.0904. Crystal data for compound
13: colorless spike, 0.58 × 0.12 × 0.08 mm size, monoclinic, P21,
a ) 10.504(2) Å, b ) 9.4224(19) Å, c ) 11.523(2) Å, ꢀ )
110.52(3)°, V ) 1068.1(4) Å3, Z ) 2, Dc ) 1.351 g cm-3, 2θmax
)
52.74°, 4203 reflections collected of which 2297 were independent
(Rint ) 0.040), 278 refined parameters, R1[I > 2σ(I)] ) 0.0802,
wR2(all data) ) 0.2420.
Supporting Information Available: Characterization data
CCDC-706798 and CCDC-706801 contain 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.
1
and H and 13C NMR spectra for all new compounds. Details
about the packing of the crystal structure of compounds 5hr,
7r, 8c, and 13. This material is available free of charge via the
Internet at http://pubs.acs.org.
(28) International Tables for Crystallography; Kluwer Academic Publishers:
Dordrecht, The Netherlands, 1992; Vol. C, Tables 6.1.1.4 (pp 500-502), 4.2.6.8
(pp 219-222), and 4.2.4.2 (pp 193-199).
JO9001867
3132 J. Org. Chem. Vol. 74, No. 8, 2009