Synthesis of 310-helix-inducing constrained analogues of L-proline

Article abstract of DOI:10.1021/jo0401422

A bicyclic indolizidinone carboxylic acid and a tricyclic constrained analogue of L-proline were synthesized and evaluated for their ability to induce helix formation as L-Ala tetrapeptides. Variable-temperature NMR, DMSO titration, CD spectra, and X-ray structure analyses, in conjunction with molecular modeling, confirmed the existence of 3₁₀-helical motifs with di- and tetrapeptides of L-Ala.

Full text of DOI:10.1021/jo0401422

Syn th esis of 310-Helix-In d u cin g Con str a in ed An a logu es of
L-P r olin e
Stephen Hanessian,* Gianluca Papeo, Kamal Fettis, Eric Therrien, and Minh Tan Phan Viet
Department of Chemistry, Universite´ de Montre´al, C. P. 6128, Succ. Centre-Ville,
Montre´al, P.Q., Canada H3C 3J 7
stephen.hanessian@umontreal.ca
Received February 25, 2004
A bicyclic indolizidinone carboxylic acid and a tricyclic constrained analogue of L-proline were
synthesized and evaluated for their ability to induce helix formation as L-Ala tetrapeptides. Variable-
temperature NMR, DMSO titration, CD spectra, and X-ray structure analyses, in conjunction with
molecular modeling, confirmed the existence of 310-helical motifs with di- and tetrapeptides of L-Ala.
In tr od u ction
Studying the underlying principles of protein folding
is crucial to our fundamental understanding of life
processes. The pathogenesis of many diseases which
originate as a result of misfolding of proteins may be
viewed in a different context if a better knowledge of the
relationship between structure and function was avail-
able.1
The ordered structural domains of proteins consist of
R-helices and â-sheets.2 In particular, R-helical motifs
play a crucial role in a number of biological processes.3
R-Helices can adopt interesting three-dimensional struc-
tures, as for example, in the four-helix bundle4 and the
coiled-coil.5 A better understanding of the formation of
R-helices could, in theory, be achieved by using low
molecular weight molecules as models.6a However, this
simulation is difficult because the small free energy
difference necessary to stabilize the folded state over the
unfolded one is difficult to achieve in practice. In addition,
the nucleation of the helix is entropically disfavored
because it requires the first four amino acids to adopt a
helical conformation without being initially stabilized by
hydrogen bonds with subsequent residues in the helix.7,8
Many approaches have been developed to date to
induce folding in short peptides.9 One of the most viable
F IGURE 1. Helix-inducing nucleating motifs.
methods6a,b,10 is to propagate a desired conformation of a
peptide chain from an ordered region starting with a
conformationally restricted template (Figure 1). The
entropy required to fold a linear peptide may thus be
lowered relative to the existing conformational energy
of the unfolded state. These templates normally direct
the nucleation process by using hydrogen bonding or a
mixture of hydrogen bonding and hydrophobic interac-
tions. While L-Pro has a negative effect on helix stability
when incorporated in an internal position of the peptide
chain,2,11 it is frequently found at the N-terminus of
naturally occurring peptides.12 In this latter case, L-Pro
does not interfere with the hydrogen bonding network
of the backbone. Another advantage is that the φ value
for L-Pro (∼60°) is close to the theoretical backbone φ
value expected for a 310 or R-helical conformation.13 The
idea of using simple L-Pro derivatives as potential R-helix
nucleator templates for short peptides has already been
exploited. Studies concerning the peptide Piv-L-Pro-L-Pro-
(1) (a) Baltzer, L.; Nilsson, H.; Nilsson, J . Chem. Rev. 2001, 101,
3153. (b) Maitra, S.; Nowick, J . S. In The amide linkage: structural
significance in chemistry, biochemistry and material science; Greenberg,
A., Breneman, C. M., Liebman, J . F., Eds.; Wiley: New York, 2000;
pp 495-518. (c) DeGrado, W. F.; Summa, C. M.; Pavone, V.; Nastri,
F.; Lombardi, A. Annu. Rev. Biochem. 1999, 68, 779.
(2) (a) Creighton, T. E. Proteins: Structures and molecular properties;
Freeman and Co.: New York, 1984. (b) Richardson, J . S.; Richardson,
D. C. Principles and Patterns of Protein Conformation. In Prediction
of Protein Structure and the Principle of Protein Conformation;
Fasman, G. D., Ed.; Plenum: New York, 1989; pp 1-99.
(3) Andrews, M. J . I.; Tabor, A. B. Tetrahedron 1999, 55, 11711.
(4) Mott, H. R.; Campbell, I. D. Curr. Opin. Struct. Biol. 1995, 5,
114.
(9) For a recent and exhaustive review, see: Schneider, J . P.; Kelly,
J . W. Chem. Rev. 1995, 95, 2169. See also: Muller, K.; Obrecht, D.;
Knierzinger, A.; Stankovic, C.; Spiegler, C.; Bannwarth, W.; Trzeciak,
A.; Engelrt, G.; Labhardt, A. M.; Schoenholzer, P. Perspect. Med. Chem.
1993, 513.
(10) Austin, R. E.; Maplestone, R. A.; Sefler, A. M.; Liu, K.;
Hruzewicz, W. N.; Liu, C. W.; Cho, H. S.; Wemmer, D. E.; Bartlett, P.
A. J . Am. Chem. Soc. 1997, 119, 6461.
(5) Lupas, A. Trends Biol. Sci. 1996, 21, 375.
(6) (a) Kemp, D. S.; Curran, T. P.; Davis, W. M.; Boyd, J . G.;
Muendel, C. J . Org. Chem. 1991, 56, 6672. (b) Kemp, D. S.; Curran,
T. P.; Boyd, J . G.; Allen, T. J . J . Org. Chem. 1991, 56, 6683.
(7) Zimm, B. H.; Bragg, J . K. J . Phys. Chem. 1959, 31, 526.
(8) Lifson, S.; Roig, A. J . Phys. Chem. 1961, 34, 1963.
(11) von Heijne, G. J . Mol. Biol. 1997, 218, 499.
(12) Richardson, J . S.; Richardson, D. C. Science 1988, 240, 1648.
(13) Venkatraman, J .; Shankaramma, S. C.; Balaram, P. Chem. Rev.
2001, 101, 3131.
10.1021/jo0401422 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/25/2004
J . Org. Chem. 2004, 69, 4891-4899
4891

Hanessian et al.
SCHEME 1 a
F IGURE 2. Constrained analogues of L-Pro.
NHMe 1 in organic solvents14 suggested that 310-helix
formation is elicited by the presence of contiguous Pro
residues. However, Xaa-L-Pro-L-Pro-OH derivatives that
can be linked to the N-terminus of the desired peptide,
while attractive because of the potential proper orienta-
tion of three amide carbonyls with the correct pitch and
spacing for helix-type structures, are usually not suf-
ficiently constrained to enforce stable and consistent
helical folding. In fact, energetically similar cis/trans
isomers in the Xaa-L-Pro as well as L-Pro-L-Pro amide
bonds may create multiple conformers. Moreover, L-Pro-
L-Pro is known to be a poor turn-constraining unit,
because of the small energy difference between turn-like
and extended structures.15 Kemp and co-workers16devised
a helical template based on a conformationally restricted
analogue of the Ac-L-Pro-L-Pro sequence 2 (Figure 1). A
stable helix formation in aqueous media for peptides
5-11 residues in length was observed. This template also
allowed independent evaluation of helix propensities for
the Ala residue.6 Bartlett and co-workers10 have studied
the semirigid template 3 in inducing helical conforma-
tions of appended hexapeptides using CD and NMR.
We recently reported the synthesis of constrained
analogues of L-proline, represented by the tricyclic motif
5 (Figure 2).17 The synthesis of a precursor to the
indolizinone 6, which encompasses a constrained bicyclic
L-proline, was also recently communicated.18 In this
paper, we describe the synthesis of the prolyl L-TcaP
(tricyclic constrained proline) 4, and the indolizidinone
scaffold 6 with the objective to study their ability to
induce L-Ala oligopeptides to adopt helical conformations.
We first studied the solution conformation of 4, its methyl
ester, and its L-Ala oligopeptides. While unable to predict
which could be the preferred rotational isomer of the
amide bond between L-proline and the core structure, we
surmized that if properly oriented, the tripeptide Xaa-
L-TcaP-L-Pro derived from 4 could deploy the three
carbonyl groups in the correct pitch and spacing for a
right-handed or a 310-helix. An acetyl group was selected
as a mimic of the first amino acid (Xaa), and the choice
of the methoxy substituent was based on synthetic
precedent and convenience.17 We planned to study L-Ala
oligopeptides as an amide appendage since it is reported
to have a high propensity for helix formation.19 Further-
more, alanine base oligopeptides offer the advantage of
more easily interpretable NMR spectra.
a
Reagents and conditions: (a) (i) Ac2O, K2CO3, CH2Cl2, 0 °C,
(ii) 2 N LiOH, THF/H2O, rt, 86% two steps ;(b) TEMPO, NaOCl,
NaClO2, CH3CN, 35 °C, 98%; (c) L-proline methyl ester hydro-
chloride, PyBOP, DIPEA, CH2Cl2, 0 °C then rt, 82%; (d) 2 N LiOH,
THF/H2O, rt, 91%; (e) H-(L-Ala)n-R, EDC, HOBt, DIPEA, CH2Cl2,
0 °C then rt; (f) H-(L-Ala)4-OtBu, EDC, HOBt, DIPEA, DMF, 0 °C
then rt.
Syn th esis
The previously reported (2S,3aS,8aR)-(6-methoxy-
1,2,3,3a,8,8a-hexahydro-3-aza-cyclopenta[a]inden-2-yl)-
methanol (7)17 was used as the starting material for the
synthesis of Ac-L-TcaP-L-Pro-OH (4) (Scheme 1). N-
Acetylation of the nitrogen was accomplished at 0 °C in
dichloromethane with use of potassium carbonate as the
base. This reaction was also sometimes accompanied by
O-acetylation. Selective hydrolysis of the acetate was
then performed with lithium hydroxide in a mixture of
THF/water at room temperature giving rise, after puri-
fication, to the desired N-acetyl derivative (8) in 86%
yield. Subsequent oxidation of the alcohol to the corre-
sponding acid, using a mixture of NaClO2, commercial
bleach, and catalytic TEMPO,20 afforded the acid Ac-L-
TcaP-OH (9) in quantitative yield. The acid was then
coupled with L-proline methyl ester hydrochloride, using
standard conditions (PyBOP, diisopropylethylamine), to
afford Ac-L-TcaP-L-Pro-OMe (10) in 82% yield, which was
subsequently treated with lithium hydroxide in a mixture
of THF/water at room temperature to afford the desired
Ac-L-TcaP-L-Pro-OH (4) in 91% yield. The L-alanine-based
oligomers were prepared in solution by using conven-
tional chemical manipulation with N-benzyloxycarbonyl
and tert-butyl ester as the orthogonal protective groups.
(14) Venkatachalapathi, Y. V.; Balaram, P. Nature 1979, 281, 83.
(15) Robinson, J . A. Synlett 2000, 4, 429.
(16) Kemp, D. S.; Boyd, J . G.; Muendel, C. C. Nature 1991, 352,
451.
(17) Hanessian, S.; Papeo, G.; Angiolini, M.; Fettis, K.; Beretta, M.;
Munro, A. J . Org. Chem 2003, 68, 7204.
(18) Hanessian, S.; Sailes, H.; Munro, A.; Therrien, E. J . Org. Chem.
2003, 68, 7219.
(19) Marqusee, S.; Robbins, V. H.; Baldwin, R. L. Proc. Natl. Acad.
Sci. U.S.A. 1989, 86, 5286.
(20) Zhao, M.; Li, J .; Mano, E.; Song, Z.; Tschaen, D. M.; Grabowski,
E. J . J .; Reider, P. J . J . Org. Chem. 1999, 64, 2564.
4892 J . Org. Chem., Vol. 69, No. 15, 2004

Analogues of L-Proline
SCHEME 2a
F IGURE 3. Cis/trans acetamide conformational isomers.
reagent as in related cases.21 Subsequent steps involved
functional group manipulations to give the alcohol 17,
followed by oxidation to 18, reduction of the azide, and
N-acetylation to give eventually 6. Peptide coupling with
H-(L-Ala)n-OtBu, using HOBT, gave the di- and tetra-
petides 21 and 22, respectively. We also prepared the
tetrapeptide 23 by the same general procedure as a
control.
Resu lts
Proton NMR spectra of 10 in CDCl3 showed a 3:1 ratio
of two major conformational isomers, as determined by
the ratio of the cleanly resolved pair of doublets at 5.4
and 5.7 ppm, attributed to the C-3a proton of the trans
and cis isomers, respectively (Figure 3). This ratio
changes to 1:1 in D2O, and inverts to 1:1.5 in pyridine-
d5. NOESY experiments in CDCl3 and in pyridine-d5
allowed us to assign the upfield doublet to the trans-
acetamido isomer and the downfield doublet to the cis
isomer. The data are in agreement with literature
precedents showing that the trans-acetamide isomer is
usually predominant in small proline-containing pep-
tides, and that the proportion of the cis isomer increases
in more polar solvents.22,23 The prevalence for the trans
orientation of 10 in solvents of low polarity could be due
to the proximity of the electron-rich phenyl ring and the
a
Reagents and conditions: (a) t-BuLi, CH3I, THF, -78 °C, 76%;
(b) t-BuLi, trisyl azide, THF, -78 °C, 63%; (c) Bu4NF, THF, rt,
81%; (d) TEMPO, NaOCl, NaClO2, CH3CN, 35 °C, 95%; (e) CH2N2,
CH3OH, 100%; (f) (i) H2, Pd/C, CH3OH, (ii) Ac2O, CH3OH, rt, 85%,
2 steps; (g) 2 N LiOH, THF/H2O, rt, 99% crude; (h) H-(L-Ala)n-
OtBu, EDC, HOBt, DIPEA, DMF, 0 °C then rt; (i) H-(L-Ala)4-OtBu,
EDC, HOBt, DIPEA, DMF, 0 °C to rt, 50%.
24
so-called “benzene-oxygen repulsion”.
Ac-L-TcaP-L-Pro-L-Ala-OMe (11) was prepared by cou-
pling template 4 with the commercially available L-Ala
methyl ester hydrochloride (Scheme 1), using a conven-
tional coupling protocol (EDC/ or HOBT, diisopropylethyl-
amine, 67% yield). The same reaction conditions were
employed to obtain Ac-L-TcaP-L-Pro-L-(Ala)2-OtBu (12) in
68% yield. In the case of the longer peptide Ac-L-TcaP-
L-Pro-L-(Ala)4-OtBu (13), the use of DMF instead of
CH2Cl2 afforded the desired product in 44% yield.
The synthesis of the second scaffold 6 follows a recently
published protocol,18 starting with S-pyroglutamic acid
(Scheme 2). Intermediate 14 was transformed to the R-C-
methyl derivative 15 via alkylation of the lithium enolate
with tert-butyllithium as the most suitable base. Refor-
mation of the Li enolate under the same reaction condi-
tions and treatment with trisyl azide21 gave the desired
tertiary azide 16 as the major product. The pseudoaxial
methoxy group at C-8 appears to exert a stereocontrolling
influence in the approach of the electrophilic azidating
To further establish the geometry of the amide bond
between L-TcaP and the L-Pro unit in 10 we relied on
13C chemical shifts as a diagnostic tool. The 13C reso-
nances of â- and γ-carbon atoms of the proline ring are
known to be strongly influenced by the proximity effect
of the carbonyl group of the amide function.25 Comparison
(21) See for example: (a) Hanessian, S.; Therrien, E.; Granberg, K.;
Nilsson, I. Bioorg. Med. Chem. Lett. 2002, 12, 2907. (b) Hanessian, S.;
Wang, W.; Gai, Y. Tetrahedron Lett. 1996, 37, 7477. See also: (c) Evans,
D. A.; Britton, T. C.; Ellman, J . A.; Dorow, R. L. J . Am. Chem. Soc.
1990, 112, 4011. (d) Wasserman, H. H.; Hlasta, D. J . J . Am. Chem.
Soc. 1978, 100, 6780. (e) Kuhlein, K.; J ensen, H. J ustus Liebigs Ann.
Chem. 1974, 369.
(22) (a) Madison, V.; Schellman, J . Biopolymers 1970, 9, 511. (b)
Higashijima, T.; Tasumi, M.; Miyazawa, T. Biopolymers 1977, 16, 1259.
(23) Gallo, E. A.; Gellman, S. H. J . Am. Chem. Soc. 1993, 115, 9774.
(24) Duan, G.; Smith, V. H., J r.; Weaver, D. F. J . Phys. Chem. A
2000, 104, 4521.
(25) (a) Kessler, H. Angew. Chem., Int. Ed. Engl. 1982, 21, 512 and
reference cited therein. (b) Venkatachalapathi, Y. V.; Venkataram
Prasad, B. V.; Balaram, P. Biochemistry 1982, 21, 5502. (c) Bean, J .
W.; Kopple, K. D.; Peishoff, C. E.; J . Am. Chem. Soc. 1992, 114, 5328.
J . Org. Chem, Vol. 69, No. 15, 2004 4893

Hanessian et al.
TABLE 1. 13C NMR Ch em ica l Sh ift of th e Câ a n d Cγ of
th e P r olin e for Com p ou n d s 4 a n d 10 (CDCl3)
compd
Câ
Cγ
R ) Me (10)
29.2 ppm (trans)a
29.0 ppm (cis)a
28.2 ppm (trans)a
28.7 ppm (cis)a
25.3 ppm (trans)a
25.4 ppm (cis)a
R ) H (4)
25.4 ppm (trans)a
a
Indicated isomer corresponds to acetamido cis/trans orienta-
tion.
between the resonances of the â- and γ-carbon atoms in
CDCl3 led us to conclude that a trans orientation of the
proline amide is preferred in 10, regardless of the
geometry of the acetamido group on the L-TcaP unit as
indicated in the perspective drawings (Figure 3). These
results were also valid in pyridine-d5 (data not shown).
The lack of NOE between the two R-protons in L-TcaP
and L-Pro in CDCl3 further confirmed the trans orienta-
tion of the inter-Pro bond.
One- and two-dimensional NMR experiments in CDCl3
of the acid 4 showed a trans/cis ratio of 7:1 for the
acetamide bond. 13C resonances of â- and γ-carbons of
the major isomer in CDCl3 conform to the reported range
for a trans-amide bond26 (Table 1). The increase in the
ratio of the trans vs cis isomer in the acid 4 compared to
the ester 10 can be explained on the basis of an increased
difficulty in aligning three carbonyl dipoles. In fact, the
7:1 trans-cis ratio observed in CDCl3 for 4 drops to 1:1
in D2O showing a strong solvent dependence.
L-Tca P L-Ala Oligop ep tid es. A qualitative assess-
ment of the presence of intramolecular H-bonding in
oligomeric peptides 11-13 was done by analyzing the
amide N-H stretching in the region of 3200-3500 cm-1
by FT-IR27 at a concentration of 1 mM in CDCl3. An
enhancement of intramolecular H-bonding was observed
at 3320-3340 cm-1 compared to that of the solvent-
F IGURE 4. 1H NMR (600 MHz) spectra in CD2Cl2 of com-
pounds 11, 12, and 13 (A, B, and C, respectively). The shoulder
at 5.49 ppm is a CH2Cl2 satellite peak.
isomer. The appearance of a second trans-isomer peak
at 5.50 ppm corresponds to a minor component in 4 and
10, which is amplified in 11 after attachment of the first
Ala unit. This may be related to a H-bond resulting in
an enthalpic advantage over the two other isomers. To
gain some insight, we performed titration experiments30
with increasing amounts of DMSO-d6 (0-15% v/v) and
temperature dependence of the NH chemical shifts31 in
the range 210-290 K in CD2Cl2.29 The presence of one
intramolecularly H-bonded NH was obvious, which cor-
responded to the trans-acetamido isomer. A γ-turn H-
bonded array was ruled out since it is known to have little
or no enthalpic advantage over the non-H-bonded state.32
exposed free N-H amide at 3412-3418 cm-1 27
. Analysis
of Cbz-L-(Ala)4-OtBu used as a control at the same
concentration in CDCl3 showed only a small hydrogen
bond stretching band, probably due to an irregular
γ-turn-like structure.28,29
Ac-L-TcaP-L-Pro-L-Ala-OMe (11) was analyzed by one-
and two-dimensional NMR at a concentration of 5.8 mM
(Figure 4A). Three conformers can be seen in a ratio of
1:1.6:1.4, as measured from the ratio of the clearly
resolved doublets of C-3a at 5.66, 5.50, and 5.47 ppm, as
well as by the ratios of the NH resonances. NOESY
experiments in the same solvent confirm that the doublet
peaks at 5.50 and 5.47 pm correspond to the major trans
(30) (a) Karle, I. L.; Pramanik, A.; Banerjee, A.; Bhattacharjya, S.;
Balaram, P. J . Am. Chem. Soc. 1997, 119, 9087. (b) J ain, R. M.;
Rajashankar, K. R.; Ramakumar, S.; Chauhan, V. S. J . Am. Chem.
Soc. 1997, 119, 3205.
(26) Siemion, I. Z.; Wieland, T.; Pook, K. H. Angew. Chem., Int. Ed.
Engl. 1975, 14, 702.
(27) (a) Aaron, H. S. Top. Stereochem. 1980, 11, 1. (b) Haque, T. S.;
Little, J . C.; Gellman, S. H. J . Am. Chem. Soc. 1994, 116, 4105. (c)
Maxfield, F. R.; Leach, S. J .; Stimson, E. R.; Powers, S. P.; Scheraga,
H. A. Biopolymers 1979, 18, 2507.
(31) (a) Gellman, S. H.; Dado, G. P.; Liang, G.-B.; Adams, B. R. J .
Am. Chem. Soc. 1991, 113, 1164. (b) Liang, G.-B.; Dado, G. P.; Gellman,
S. H. J . Am. Chem. Soc. 1991, 113, 3994. (c) Diaz, H.; Espina, J . R.;
Kelly, J . W. J . Am. Chem. Soc. 1992, 114, 8316. (d) Tsang, K. Y.; Diaz,
H.; Graciani, N.; Kelly, J . W. J . Am. Chem. Soc. 1994, 116, 3988.
(32) (a) Liang, G.-B.; Riro, C. J .; Gellman, S. H. Biopolymers 1992,
32, 293. (b) Liang, G.-B.; Riro, C. J .; Gellman, S. H. J . Am. Chem. Soc.
1992, 114, 4440.
(28) Goodman, M.; Saltman, R. P. Biopolymers 1981, 20, 1929.
(29) See Supporting Information.
4894 J . Org. Chem., Vol. 69, No. 15, 2004

Analogues of L-Proline
F IGURE 5. Lowest energy structures obtained by restrained
conformational calculations from 2D NOESY data.
NMR studies on Ac-L-TcaP-L-Pro-L-(Ala)2-OtBu (12) in
CD2Cl2 at a concentration of 8.9 mM showed three
conformers in a ratio of 1:3.4:1.8, as measured from the
C-3a doublets at 5.65, 5.54, and 5.47 ppm (Figure 4B).
As expected, COSY experiments showed that the two
downfield doublets attributed to H-bonded NH groups
belong to two different alanine units corresponding to the
major trans-acetamido isomer. NOE contacts from 2D
NOESY data allowed us to obtain a lowest energy
structure using restrained conformational calculation
(HyperChem) (Figure 5A). As depicted, the structure
suggests a bifurcated H-bonding motif involving the
trans-acetamido carbonyl group.
F IGURE 6. (A) DMSO titration experiment for compound 22;
(B) temperature variation experiment for compound 22; and
(C) % DMSO variation experiment for compound 23.
tions and temperature-dependent NH shifts were difficult
to quantitate due to overlapping signals.
In d olizid in on e L-Ala Oligop ep tid es. We had an-
ticipated that the L-Ala oligopeptides 21 and 22 attached
to the bicyclic scaffold would adopt a helical folding
shape, starting with one or more â-turn-type H-bonding
arrays, with the axially disposed N-acetyl carbonyl and
the first L-Ala amide. The prospects for such folding and
structural organization were substantiated by NMR
experiments. Thus, titration of 22 in CDCl3 with increas-
ing amounts of DMSO-d6 clearly showed the prevalence
of four NH signals that were unaffected up to 10% v/v of
DMSO (Figure 6 A).
The only NH chemical shift change was due to the free
N-Ac group. This behavior was corroborated by temper-
ature variation studies for 22, where the same four N-H
signals were not influenced in the range 223-293 K
(Figure 6B). As a control, the DMSO titration of the azido
tetrapeptide 23 showed a similar pattern compared to
the N-acetyl analogue 22, except for the absence of a
second â-turn H bond and distinctly different chemical
shifts. The CD spectra of 21-23 in CDCl3 or MeOH
showed a similar pattern as for 11-13 with bands at ca.
208 and 222 nm characteristic of a 310-helix.29 2D NOESY
experiments with 22 corroborated the proposed helical
NMR studies on Ac-L-TcaP-L-Pro-L-(Ala)4-OtBu (13) in
CDCl3 at a concentration of 5.6 mM showed the presence
of three conformers in a ratio of 1:8.5:6.5 at 5.66, 5.56,
and 5.48 ppm (Figure 4C). COSY experiments showed
that the four doublets of the NH groups belong to four
different alanine units, corresponding to the major trans-
acetamido isomer. NOE contacts and 2D NOESY data
with restrained conformational calculation (HyperChem)
generated a motif encompassing an intramolecular H-
bonding network (Figure 5B). The bifurcated H-bonded
unit observed for 12 is now complemented by further
contacts leading to a 310-helical folding in 13. CD spec-
tra29 for 12 and 13 in MeOH at a concentration of 1.9 ×
10-4 and 1.4 × 10-4 M, respectively, showed bands at 205
and 222 nm indicating a propensity for an 310-helix,32
rather than an R-helix. Unfortunately, DMSO-d6 titra-
J . Org. Chem, Vol. 69, No. 15, 2004 4895

Hanessian et al.
measurement, suggesting a 310-type folding, as well.
Goodman and Saltman28 have already demonstrated the
importance of a nucleating template based on the obser-
vation that linear homooligopeptides showed unfolded or
irregular γ-turn-like structures in a variety of solvents.
Studies are ongoing in our laboratory to uncover other
types of core structures capable of conferring stable
secondary structures to appended polypeptides.
Exp er im en ta l Section
The synthesis of compounds 8, 9, and 10 is given in the
Supporting Information.29
Ac-L-Tca P -L-P r o-OH (4). To a stirred solution of Ac-L-
TcaP-L-Pro-OMe (10; 65 mg, 0.168 mmol) in 1.3 mL of THF,
at room temperature, was added a 2 N aqueous solution of
LiOH (0.17 mL, 0.336 mmol) dropwise and the biphasic system
was stirred at room temperature for 3 h. The solvent was
evaporated; the resulting mixture was diluted with water (5
mL) and then washed with diethyl ether (2 × 5 mL). The
organic layers were discarded while the aqueous one was
acidified to pH 3-4 with 1 N HCl, then extracted with
dichloromethane (3 × 10 mL) and dichloromethane/methanol
9:1 (3 × 10 mL). The combined organic phases were dried (Na2-
SO4) and evaporated to dryness to afford a white solid (57 mg,
91%): mp 134-136 °C, 153-154 °C dec; [R]D -209.0 (c 0.5,
CHCl3); 1H NMR (CDCl3, 400 MHz) for a mixture approxi-
mately 1:7 of major rotamers δ 7.68, 7.26 (0.12 H, d, J ) 8.1
Hz; 0.88 H, d, J ) 5.5 Hz), 6.81-6.72 (2 H, m), 5.75, 5.46 (0.12
H, d, J ) 7.0 Hz; 0.88 H, d, J ) 7.0 Hz), 4.68 (1 H, d, 5.8 Hz),
4.58, 4.52 (0.88 H, d, J ) 9.0 Hz; 0.12 H, d, J ) 9.0 Hz), 3.81,
3.78 (2.64 H, s; 0.36 H, s), 3.76-3.68 (1.76 H, m), 3.54-3.47
(2.24 H, m), 3.12-3.07 (1 H, dd, J ) 16.4, 6.5 Hz), 2.71-2.67
(1 H, d, J ) 16.4 Hz), 2.42 (3.52 H, m), 2.33-2.29 (0.12 H, m),
2.14-2.06 (2.12 H, m), 2.05 (1.24 H, m), 1.87-1.78 (1 H, m);
13C NMR (CDCl3, 75 MHz) δ (174.0), 173.7, 173.0, (171.1),
170.7, 160.5, (160.2), 143.6, (142.7), (136.4), 134.2, (128.6),
125.7, 113.6, 111.5, (110.5), 68.2, (67.9), 59.9, (59.6), 59.5, 55.8,
(55.7), 47.6, 42.9, (39.6), 35.2, 33.9, (28.7), 28.2, 25.4, 22.9,
(22.8); MS (EI+) m/e 372 (M)+; HRMS (EI+) calcd for
F IGUR E 7. Top (A): Ortep diagram representation of 21.
Bottom (B): Modeled tetrapeptide 22.
structure based on the NH correlations. We were grati-
fied to obtain X-ray quality crystals of the dipeptide 21.29
As anticipated from the titration experiments we ob-
served two characteristic intramolecular â-turn-type H
bonds linking the lactam carbonyl and acetamide carbo-
nyl oxygen atoms of the bicyclic scaffolds to NH amide
groups of Ni and Ni + 1 Ala units (Figure 7A). On the
basis of the H-bonding pattern seen in the X-ray struc-
ture of 21, the titration and temperature variation
experiments, as well as the CD spectra characteristics,
and 2D NOESY data,29 we propose that the tetrapeptide
22 adopts a 310-helical shape as depicted in Figure 7B.
C
20H24N2O5 372.16852, found 372.16870.
Ac-L-Tca P -L-P r o-L-Ala -OMe (11). To a chilled solution of
L-alanine methyl ester hydrochloride (3 mg, 0.0156 mmol) in
2 mL of dichloromethane was added Ac-L-TcaP-L-Pro-OH (4;
5 mg, 0.013 mmol), HOBt (3 mg, 0.02 mmol), 1-[3-(dimethy-
lamino)propyl]-3-ethylcarbodiimide hydrochloride (4 mg, 0.02
mmol), and diisopropylethylamine (3 µL, 2.5 equiv). The
reaction mixture was stirred for 1 h at 0 °C and 48 h at room
temperature. Water (2 mL) was then added to the reaction
mixture and the phases were separated. The organic layer was
washed with saturated NaHCO3 aqueous solution (2 × 2 mL)
and brine (1 × 2 mL), dried (Na2SO4), and evaporated to
dryness. The resulting solid was purified via silica gel flash
column chromatography (AcOEt/MeOH, 85:15). Compound 11
was obtained as a white solid (4 mg, 67%): [R]D -158 (c 0.1,
CHCl3); 1H NMR (CD2Cl2, 600 MHz) for a mixture of ap-
proximately 1:1.4:1.6 of major rotamers δ 8.53-8.52 (0.4 H,
d, J ) 7.8 Hz), 7.62-7.61 (0.25 H, d, J ) 9.2 Hz), 7.32-7.31
(0.35 H, d, J ) 8.1 Hz), 7.30-7.29 (0.4 H, d, J ) 8.1 Hz), 7.16-
7.15 (0.35 H, d, J ) 6.5 Hz), 6.95-6.94 (0.25 H, d, J ) 6.0
Hz), 6.83-6.81 (1.2 H, m), 6.75-6.74 (0.8 H, m), 5.67-5.65
(0.25 H, d, J ) 7.1 Hz), 5.51-5.50 (0.4 H, d, J ) 7.1 Hz), 5.48-
5.47 (0.35 H, d, J ) 7.1 Hz), 4.61-4.60 (1 H, d, J ) 9.1 Hz),
4.57-4.54 (0.6 H, m), 4.53-4.48 (0.5 H, m), 4.45-4.40 (0.35
H, quint, J ) 7.3 Hz), 4.32-4.29 (0.15 H, m), 4.25-4.24 (0.4
H, d, J ) 8.1 Hz), 3.82 (2.2 H, s), 3.80 (0.8 H, s), 3.76 (1.5 H,
s), 3.74-3.71 (0.5 H, m), 3.59-3.50 (1.4 H, m), 3.55 (0.6 H,
m), 3.42-3.37 (0.8 H, m), 3.13-3.03 (1.2 H, m), 2.73-2.69 (1
H, m), 2.53-2.50 (0.5 H, dd, J ) 12.4, 6.3 Hz), 2.40 (1 H, s),
2.38 (1.25 H, s), 2.32-2.23 (1.25 H, m), 2.19-1.87 (2.4 H, m),
1.94 (1 H, s), 1.86-1.80 (1.2 H, m), 1.77-1.69 (1 H, m), 1.68-
Con clu sion
We have described the synthesis of two novel con-
strained amino acid core structures that induce the
formation of 310-helices from appended tetraalanine
peptides in organic solvents. The first motif, consisting
of Ac-L-TcaP-L-Pro tetrapeptide, exists in different con-
formations around the Pro-Pro amide bond with respect
to the N-acetyl. The trans-acetamide conformation is
favored in nonpolar solvents, which corroborates the
observed bifurcated intramolecular H bond of two NHs
on different Ala residues with the acetamide carbonyl.
The second motif utilized an indolizidinone as a
potential nucleator for a di- and tetraalanine peptidic
appendage. A 310-helical arrangement was observed by
X-ray crystallography of the dipeptide 21. Titration and
variable-temperature experiments revealed that only one
NH (NHAc) was affected under the NMR conditions of
4896 J . Org. Chem., Vol. 69, No. 15, 2004

Analogues of L-Proline
1.62 (0.4 H, m), 1.50-1.48 (1.2 H, d, J ) 7.3 Hz), 1.44-1.42
(0.75 H, d, 7.1 Hz), 1.41-1.40 (1.05 H, d, J ) 7.1 Hz); 13C NMR
(pyridine-d5, 125 MHz) δ 174.5, 173.2, 171.3, 170.9, 160.9,
(144.5), 143.2, 137.7, 130.4, (127.7), 116.0, (115.9), (113.2),
112.3, (69.6), 69.5, (67.7), 63.2, 61.8, (61.4), (56.9), 56.8, 53.2,
49.5, (49.4), (48.3), 48.2, (43.6), 40.4, 36.8, 36.3, (35.9), (34.5),
30.4, (30.0), 26.3, (25.9), (23.4), 23.2, 17.7, (16.3); MS (EI+)
m/e 458 (M+H)+; HRMS (EI+) calcd for C24H32N3O6 (M + H)
458.22911, found 458.22770.
6.86-6.76 (2.94H, m), 5.66 (0.06H, d, J ) 7.7 Hz), 5.56 (0.53H,
d, J ) 7.1 Hz), 5.49-5.47 (0.41H, d, J ) 6.9 Hz), 4.66-4.64
(0.06H, d, J ) 9.8 Hz), 4.61-4.59 (0.06H, d, J ) 8.7 Hz), 4.57-
4.55 (0.12H, m), 4.51-4.47 (0.41H, br s), 4.45-4.43 (0.06H,
m), 4.40 (0.41H, m), 4.39 (0.41H, m), 4.35-4.33 (0.53H, d, J )
9.0 Hz), 4.30-4.26 (1.94H, m), 4.24-4.21 (0.53H, m), 4.16-
4.12 (1.47H, m), 3.82 (1.23H, s), 3.81 (1.59H, s), 3.80 (0.18H,
s), 3.74-3.71 (1H, m), 3.61-3.57 (1H, m), 3.49-3.44 (0.59H,
m), 3.43-3.38 (0.41H, m), 3.18-3.14 (0.41H, dd, J ) 16.4, 6.6
Hz), 3.14-3.10 (0.59H, dd, J ) 16.5, 6.9 Hz), 2.81-2.78 (0.41H,
d, J ) 16.4 Hz), 2.75-2.71 (0.06H, d, J ) 16.5 Hz), 2.73-2.70
(0.53H, d, J ) 16.5 Hz), 2.46 (1.59H, s), 2.44 (1.23H, s), 2.36-
2.12 (2H, m), 2.07-2.03 (1H, m), 2.02-1.95 (1H, m), 1.93
(0.18H, s), 1.88-1.81 (2H, m), 1.56-1.54 (1.77H, d, J ) 7.3
Hz), 1.48-1.46 (1.23H, m), 1.48 (3.69H, s), 1.47 (4.77H, s),
1.45-1.43 (1.23H, d, J ) 7.4 Hz), 1.41-1.39 (1.23H, d, J )
8.1 Hz), 1.39-1.38 (1.77H, d, J ) 7.4 Hz), 1.30 (0.54H, s), 1.23-
1.21 (1.77H, d, J ) 7.3 Hz), 1.20 (1.77H, d, J ) 7.3 Hz), 1.20-
1.19 (1.23H, m); 13C NMR (pyridine-d5, 125 MHz) δ 173.5,
173.0, 172.9, 172.7, 171.6, 171.0, 170.8, 160.6, 143.3, 137.3,
128.8, 114.1, (111.5), 110.5, 81.1, (68.1), 69.7, 61.7, 60.8, (55.6),
55.5, 50.0, 49.7, 49.5, 49.1, (47.5), 47.4, 36.1, 35.7, (30.0), 29.7,
28.1, 25.5, (23.1), 22.9, 19.0, 18.9, 18.8, 18.6, 17.7; MS (FAB+)
m/e 735 (M + Na)+, 713 (M + H)+, 657 (M - (CH3)2CdCH2);
Ac-L-Tca P -L-P r o-L-Ala -L-Ala -OtBu (12). To a chilled solu-
tion of L-alanyl-L-alanine tert-butyl ester (13 mg, 0.06 mmol)
in 5 mL of dichloromethane was added Ac-L-TcaP-L-Pro-OH
(4; 19 mg, 0.05 mmol), HOBt (11 mg, 0.08 mmol), 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (15
mg, 0.08 mmol), and diisopropylethylamine (23 µL, 0.13 mmol).
The reaction mixture was stirred for 1 h at 0 °C and 40 h at
room temperature. Water (5 mL) was then added to the
reaction mixture and the phases were separated. The organic
layer was washed with saturated NaHCO3 aqueous solution
(2 × 5 mL) and brine (1 × 5 mL), dried (Na2SO4), and
evaporated to dryness. The resulting solid was purified via
silica gel flash column chromatography (AcOEt/MeOH, 85:15).
Compound 12 was obtained as a light-yellow solid (19 mg,
1
68%): [R]D -110 (c 0.2, CHCl3); H NMR (CD2Cl2, 600 MHz)
for a mixture approximately 1:1.8:3.4 of major rotamers δ
7.82-7.77 (0.55H, d, J ) 7.3 Hz), 7.63-7.60 (0.16H, d, J )
8.8 Hz), 7.33-7.30 (0.29H, d, J ) 8.0 Hz), 7.30-7.27 (0.55H,
d, J ) 8.0 Hz), 7.26-7.22 (0.29H, d, J ) 7.5 Hz), 7.14-7.10
(0.55H, d, J ) 8.0 Hz), 7.03-7.00 (0.16H, d, J ) 7.1 Hz), 6.84-
6.74 (2H, m), 6.70-6.66 (0.29H, d, J ) 6.6 Hz), 6.57-6.54
(0.16H, d, J ) 7.1 Hz), 5.67-5.64 (0.16H, d, J ) 7.5 Hz), 5.55-
5.53 (0.55H, d, J ) 7.5 Hz), 5.48-5.45 (0.29H, d, J ) 6.6 Hz),
4.69-4.65 (0.55H, d, J ) 8.8 Hz), 4.62-4.59 (0.16H, dd, J )
8.4, 3.5 Hz), 4.59-4.57 (0.29H, d, J ) 9.6 Hz), 4.57-4.54
(0.29H, d, J ) 8.3 Hz), 4.42-4.27 (2.71H, m), 3.82 (0.87H, s),
3.80 (1.65H, s), 3.79 (0.48H, s), 3.76-3.72 (0.16H, m), 3.60-
3.56 (1.68H, m), 3.55-3.35 (1.16H, m), 3.14-3.08 (1H, dd, J
) 16.6, 7.5 Hz), 2.75-2.69 (1H, dd, J ) 16.6, 6.6 Hz), 2.52-
2.47 (0.58H, m), 2.44-2.42 (1.65H, s), 2.40-2.38 (0.87H, s),
2.34-2.27 (0.32H, m), 2.17-2.10 (1.1H, m), 2.09-2.00 (2.29H,
m), 1.98-1.90 (1.19H, m + s), 1.89-1.80 (0.84H, m), 1.80-
1.73 (0.16H, m), 1.49 (1.44H, s), 1.48-1.46 (2.52H, d, J ) 7.4
Hz), 1.42-1.40 (0.48H, d, J ) 7.2 Hz), 1.40-1.38 (0.48H, d, J
) 7.2 Hz), 1.31 (2.61H, s), 1.25 (4.95H, s), 1.23-1.21 (2.52H,
d, J ) 7.3 Hz); 13C NMR (pyridine-d5, 125 MHz) δ 173.3, 172.8,
172.4, 171.6, 170.3, (170.1), 160.6, 143.2, 137.4, 128.8, (126.1),
114.1, 110.5, 81.1, (68.2), 68.0, 61.8, 60.7, (60.5), (60.1), (55.5),
55.4, 49.5, (47.4), 47.3, (42.8), 39.8, 36.2, 35.7, (34.0), 30.1, 29.6,
(29.3), 28.0, 25.5, (25.3), (23.1), 22.8, (18.9), 18.8, 17.8; MS
(FAB+) m/e 593 (M + Na)+, 571 (M + H)+; HRMS (FAB+)
calcd for C30H42N4O7Na 593.29512, found 593.29260.
HRMS (FAB+) calcd for
C36H53N6O9 713.38740, found
713.38600.
(3S,8S,9S)-3-(ter t-Bu t yld ip h en ylsila n yloxym et h yl)-8-
m eth oxy-6-m eth ylh exa h yd r oin d olizin -5-on e (15). To a
stirred solution of lactam 1418 (1.18 g, 2.694 mmol) in 10.8 mL
of THF at -78°C was added dropwise a 1.5 M solution of
t-BuLi (1.98 mL, 2.239 mmol) in THF. The mixture was stirred
for 30 min at this temperature then methyl iodide (0.24 mL,
3.853 mmol) was added. The reaction mixture was stirred for
30 min at -78 °C, and then the reaction was neutralized by
adding slowly a saturated solution of NaHCO3. The mixture
was extracted with ethyl acetate and the combined organic
phases were dried over sodium sulfate, filtered, and evaporated
to dryness. The residue was purified via flash column chro-
matography on silica gel (30% AcOEt /hexanes) to give the
mixture of diastereoisomers as a yellow oil (921 mg, 76%). A
sample of the major isomer (R-Me) was separated via chro-
matography for spectroscopic data: 1H NMR (CDCl3, 400 MHz)
δ 7.65-7.58 (4H, m), 7.43-7.34 (6H, m), 4.23-4.20 (1H, m),
4.12-4.08 (1H, dd, J ) 10.0, 4.0 Hz), 3.76-3.72 (1H, m), 3.70-
3.66 (1H, dd, J ) 10.0, 2.2 Hz), 3.56 (1H, m), 3.34 (3H, s), 2.47-
2.32 (2H, m), 2.05-1.96 (3H, m), 1.85 (1H, m), 1.50-1.42 (1H,
t, J ) 13.1 Hz), 1.30-1.27 (3H, d, J ) 7.0 Hz), 1.04 (9H, s);
13C NMR (CDCl3, 100 MHz) δ 172.0, 135.9, 134.1, 133.9, 130.0,
129.9, 128.1, 128.0, 72.8, 64.6, 64.2, 58.4, 56.7, 33.2, 31.4, 27.3,
26.9, 26.8, 25.1, 19.8, 18.4; MS (FAB) m/e 452 (M + H)+; HRMS
(EI+) calcd for C27H38NO3Si 452.26209, found 452.26169.
Ac-Tca P -L-P r o-L-Ala -L-Ala -L-Ala -L-Ala -OtBu (13). To a
chilled solution of L-alanyl-L-alanyl-L-alanyl-L-alanine tert-
butyl ester (15 mg, 0.04 mmol) in 2 mL of dry DMF was added
Ac-L-TcaP-L-Pro-OH (4; 17 mg, 0.045 mmol), HOBt (10 mg,
0.075 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (15 mg, 0.075 mmol), and diisopropylethylamine
(22 µL, 0.125 mmol). The reaction mixture was stirred for 1 h
at 0 °C and 48 h at room temperature. DMF was evaporated
under high vacuum and the orange residue was dissolved in
dichloromethane (50 mL). The solution was washed with water
(1 × 20 mL), saturated NaHCO3 aqueous solution (2 × 20 mL),
and brine (1 × 20 mL), dried (Na2SO4), and evaporated to
dryness. The resulting orange solid was purified via silica gel
flash column chromatography (AcOEt/MeOH, 80:20). Com-
pound 13 was obtained as a light yellow solid (16 mg, 44%):
[R]D -90 (c 0.2, CHCl3); 1H NMR (CD2Cl2, 600 MHz) for a
mixture of approximately 1:6.5:8.5 of major rotamers δ 7.62-
7.60 (0.06H, d, J ) 8.1 Hz), 7.62-7.60 (0.06H, d, J ) 8.8 Hz),
7.56-7.54 (0.53H, d, J ) 4.5 Hz), 7.40 (0.41H, br s), 7.30-
7.29 (0.41H, d, J ) 8.1 Hz), 7.26-7.25 (0.53H, d, J ) 5.4 Hz),
7.21-7.19 (0.53H, d, J ) 8.3 Hz), 7.12-7.10 (0.41H, d, J )
8.0 Hz), 7.03 (0.18H, m), 6.97-6.96 (0.94H, d, J ) 7.0 Hz),
(3S,6S,8S,9S)-6-Azid o-3-(ter t-bu tyld ip h en ylsila n yloxy-
m et h yl)-8-m et h oxy-6-m et h ylh exa h yd r oin d olizin -5-on e
(16). To a stirred solution of lactam 15 (920 mg, 2.035 mmol)
(mixture of diastereoisomers) in 8.2 mL of THF at -78 °C was
added dropwise a 1.5 M solution of t-BuLi (1.49 mL, 2.239
mmol) in THF. The mixture was stirred for 1 h at this
temperature then a solution of trisyl azide (756 mg, 2.442
mmol) in THF was added. The reaction mixture was stirred
for 3 h at -78 °C and neutralized with a saturated solution of
NaHCO3; the mixture was then extracted with ethyl acetate
and the combined organic phases were dried over sodium
sulfate, filtered, and evaporated to dryness. The mixture of
diastereoisomers was purified via flash column chromatogra-
phy on silica gel (20% AcOEt /hexanes) to give the major
isomer 16 as a yellow oil (628 mg, 63%): [R]D -120 (c 0.6,
CHCl3); FT-IR (ν, cm-1) 2957, 2103, 1645, 1428, 1109; 1H NMR
(CDCl3, 400 MHz) δ 7.67-7.63 (4H, m), 7.43-7.39 (6H, m),
4.25-4.19 (2H, m), 3.85-3.82 (1H, m), 3.66-3.63 (1H, dd, J
) 9.7, 1.6 Hz), 3.55-3.53 (1H, m), 3.36 (3H, s), 2.39-2.35 (1H,
dd, J ) 14.7, 3.8 Hz), 2.09-2.01 (3H, m), 1.94-1.92 (1H, m),
1.79-1.75 (1H, dd, J ) 14.7, 2.1 Hz), 1.69 (3H, s), 1.06 (9H,
J . Org. Chem, Vol. 69, No. 15, 2004 4897

Hanessian et al.
s); 13C NMR (CDCl3, 100 MHz) δ 168.4, 135.9, 133.9, 133.6,
130.1, 130.0, 128.2, 128.1, 73.8, 64.2, 63.5, 60.6, 59.2, 57.3, 37.3,
27.2, 27.0, 25.2, 25.1, 19.7; MS (EI) m/e 492 (M)+, 435; HRMS
(EI+) calcd for C27H36N4O3Si 492.25567, found 492.25560.
(2 mL) and hydrogenated (1 atm) with Pd/C 10% (10 mg, 10%
mol) for 16 h at room temperature. After removal of the
catalyst via filtration through Celite, the solvent was removed
in vacuo to give 25 mg (quantitative) of amine as a yellow oil.
To a solution of the amine (18 mg, 0.07 mmol) in methanol
(500 µL) was added acetic anhydride (17 µL, 0.18 mmol). The
reaction mixture was stirred at room temperature for 16 h,
the solvents were removed under reduced pressure, and the
residue was purified via flash column chromatography on silica
gel (AcOEt/MeOH 8:2) to give the desired compound 20 as a
(3S,6S,8S,9S)-6-Azid o-3-h yd r oxym et h yl-8-m et h oxy-6-
m eth ylh exa h yd r oin d olizin -5-on e (17). To a stirred solution
of protected alcohol 16 (50 mg, 0.101 mmol) in THF was added
a 1 M solution of tetrabutylammonium fluoride in THF (203
µL, 0.203 mmol) dropwise. The reaction mixture was subse-
quently stirred at room temperature for 16 h. The mixture was
concentrated under reduced pressure and was loaded directly
onto a column of silica gel for purification (AcOEt/hexanes,
1:1). The desired compound 17 was obtained as a yellow oil
(21 mg, 81%): [R]D -199.8 (c 1.0, CHCl3); 1H NMR (CDCl3,
400 MHz) δ 4.75 (1H, br s), 4.12-4.17 (1H, quad, J ) 8.0 Hz),
3.69-3.72 (2H, m), 3.55-3.61 (2H, m), 3.36 (3H, s), 2.35-2.39
(1H, dd, J ) 14.8, 3.6 Hz), 2.07-2.17 (1H, m), 1.97-2.04 (1H,
m), 1.79-1.85 (1H, m), 1.73-1.77 (1H, dd, J ) 14.8, 2.0 Hz),
1.66 (3H, s), 1.39-1.48 (1H, m); 13C NMR (CDCl3, 100 MHz) δ
171.4, 73.5, 67.6, 63.5, 63.0, 60.9, 57.3, 37.1, 26.8, 26.4, 24.9;
MS (MAB) m/e 254 (M)+; HRMS (EI+) calcd for C11H18N4O3
254.13789, found 234.13597.
(3S,6S,8S,9S)-6-Azido-8-m eth oxy-6-m eth yl-5-oxo-octah y-
d r oin d olizin e-3-ca r boxylic Acid (18). To a stirred solution
of 17 (20 mg, 0.079 mmol) in acetonitrile (0.4 mL) was added
a sodium phosphate buffer (0.3 mL of a 0.67 M aqueous
solution of NaH2PO4‚H2O buffered at pH 6.8 with 2 N aqueous
sodium hydroxide) and the mixture was vigorously stirred at
room temperature for 5 min; TEMPO (1.0 mg, 0.005 mmol), a
solution of sodium chlorite in water (18 mg dissolved in 0.1
mL of water, 0.158 mmol), and a commercial bleach solution,
previously diluted with water (2 µL of a 5.25% solution diluted
in 0.4 mL of water, 0.025 mmol), were subsequently added.
The resulting red-brown solution was stirred and heated at
35 °C for 24 h, cooled to room temperature, and diluted with
water (2.0 mL) and the pH was adjusted to pH 8 by adding 5
drops of 2 N aqueous sodium hydroxide. The solution was then
cooled to 0 °C and quenched with aqueous sodium sulfite
solution (64 mg dissolved in 1.21 mL of water), stirred for 30
min at 0 °C, then extracted with ether (2 × 10 mL). The
organic phases were discarded, while the aqueous one was
acidified (pH 2-3) with 1 N HCl then extracted with ether (2
× 10 mL) and then with CH2Cl2 (5 × 10 mL). The combined
organic phases were dried over sodium sulfate, filtered, and
evaporated to dryness. The resulting crude acid was obtained
as a white foam and was used in the next step without any
further purification (20 mg, 95%): [R]D -241 (c 1.0, MeOH);
1H NMR (CDCl3, 400 MHz) δ 4.57-4.60 (1H, t, J ) 8.4 Hz),
3.80-3.82 (1H, m), 3.58-3.61 (1H, m), 3.37 (3H, s), 2.36-2.40
(1H, dd, J ) 14.8, 3.8 Hz), 2.27-2.33 (2H, m), 2.03-2.13 (1H,
m), 1.98-2.01 (1H, m), 1.78-1.82 (1H, dd, J ) 14.8, 2.4 Hz),
1.69 (3H, s); 13C NMR (CDCl3, 75 MHz) δ 176.0, 168.8, 73.7,
62.8, 60.5, 60.7, 57.2, 37.2, 27.8, 27.0, 25.3; MS (MAB) m/e 268
(M)+; HRMS (EI+) calcd for C11H16N4O4 268.11715, found
268.11722.
1
yellow oil (17 mg, 85%): [R]D -148.7 (c 1.0, MeOH); H NMR
(CDCl3, 400 MHz) δ 6.20 (1H, s), 4.39-4.43 (1H, t, J ) 8.1
Hz), 4.06-4.10 (1H, m), 3.73 (3H, s), 3.58-3.59 (1H, d, J )
2.8 Hz), 3.37 (3H, s), 2.61-2.66 (1H, dd, J ) 14.7, 3.3 Hz),
2.40-2.44 (1H, dd, J ) 14.7, 1.7 Hz), 2.30-2.33 (1H, m), 2.12-
2.17 (1H, s), 1.93 (3H, s), 1.87-1.93 (2H, m), 1.54 (3H, s); 13
C
NMR (CDCl3, 100 MHz) δ 173.0, 171.0, 170.2, 74.1, 62.5, 59.5,
57.0, 55.5, 52.6, 35.6, 27.9, 27.3, 27.0, 23.9; MS (FAB+) m/e
299 (M)+; HRMS (FAB+) calcd for C14H23N2O5 299.16069,
found 299.16108.
(3S,6S,8S,9S)-6-Acetylam in o-8-m eth oxy-6-m eth yl-5-oxo-
oct a h yd r oin d olizin e-3-ca r b oxylic Acid (6). To a stirred
solution of methyl ester 20 (17 mg, 0.057 mmol) in 500 µL of
THF was added dropwise a 2 N aqueous solution of LiOH (60
µL, 0.114 mmol) and the biphasic system was stirred at room
temperature for 24 h. The solvent was evaporated and the
resulting mixture was diluted with water (5 mL) then washed
with ethyl acetate (2 × 5 mL). The organic layers were
discarded while the aqueous one was acidified to pH 2 with 2
N HCl. The solution was then concentrated to dryness and
the residue was used in the next step without any further
purification.
L-Bca P -L-Ala -L-Ala -OtBu (21). To a chilled solution of
H2N-L-Ala-L-Ala-OtBu hydrochloride (9 mg, 0.042 mmol) in 500
µL of DMF was added 6 (10 mg, 0.035 mmol), HOBt (7 mg,
0.053 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (10 mg, 0.053 mmol), and diisopropylethylamine
(18 µL, 0.106 mmol). The reaction mixture was stirred for 1 h
at 0 °C and 48 h at room temperature. The solvent was
removed under reduced pressure and the residue was dissolved
in dichloromethane (2 mL). Water (2 mL) was then added and
the phases were separated. The organic layer was washed with
saturated NaHCO3 aqueous solution (2 × 2 mL) and brine (1
× 2 mL), dried (Na2SO4), and evaporated to dryness. The
resulting solid was purified via silica gel flash column chro-
matography (AcOEt to AcOEt/MeOH, 4:1). The desired com-
pound 21 was obtained as a white solid (7 mg, 41%): [R]D
-102.5 (c 0.2, MeOH); 1H NMR (CDCl3, 400 MHz) δ 7.50-
7.52 (1H, d, J ) 7.4 Hz), 7.46-7.48 (1H, d, J ) 8.9 Hz), 6.18
(1H, s), 4.48-4.53 (1H, quint, J ) 7.4 Hz), 4.36-4.47 (1H,
quint, J ) 8.9 Hz), 4.31-4.35 (1H, quint, J ) 7.3 Hz), 3.99-
4.04 (1H, m), 3.61 (1H, d, J ) 3.2 Hz), 3.38 (3H, s), 2.49-2.56
(1H, dt, J ) 12.6, 7.7 Hz), 2.33-2.37 (1H, dd, J ) 14.2, 2.1
Hz), 2.27-2.31 (1H, dd, J ) 14.2, 3.4 Hz), 2.10-2.16 (1H, m),
1.91 (3H, s), 1.80-1.95 (3H, m), 1.51 (3H, s), 1.44 (9H, s), 1.42
(3H, d, J ) 6.1 Hz), 1.35 (3H, d, J ) 7.2 Hz); 13C NMR (CDCl3,
75 MHz) δ 172.9, 172.0, 171.3, 171.0, 81.6, 73.9, 63.2, 61.9,
57.3, 54.9, 49.2, 48.9, 36.4, 28.7, 28.3, 27.3, 27.0, 23.5, 17.8,
17.4; MS (FAB+) m/e 483 (M + H)+; HRMS (FAB+) calcd for
(3S,6S,8S,9S)-6-Azido-8-m eth oxy-6-m eth yl-5-oxo-octah y-
d r oin d olizin e-3-ca r boxylic Acid Meth yl Ester (19). To a
stirred solution of 18 (20 mg, 0.074 mmol) in methanol was
added diazomethane dropwise until the reaction was complete.
The reaction mixture was subsequently quenched with acetic
acid and the mixture was concentrated under reduced pressure
to give the desired compound as a yellow foam (21 mg, 100%):
[R]D -198.0 (c 1.0, MeOH); 1H NMR (CDCl3, 400 MHz) δ 4.47-
4.50 (1H, t, J ) 8.2 Hz), 3.91-3.93 (1H, m), 3.75 (3H, s), 3.58
(1H, d, J ) 2.8 Hz), 3.36 (3H, s), 2.31-2.37 (2H, m), 2.07-
2.16 (1H, m), 1.82-1.95 (3H, m), 1.64 (3H, s); 13C NMR (CDCl3,
100 MHz) δ 172.8, 168.8, 73.7, 62.6, 60.5, 59.1, 57.2, 52.8, 37.5,
28.1, 27.2, 25.1; MS (MAB) m/e 282 (M)+; HRMS (EI+) calcd
for C12H18N4O4 282.13280, found 282.13342.
C
23H39N4O7 483.28187, found 483.28038.
L-Bca P -L-Ala -L-Ala -L-Ala -L-Ala -OtBu (22). To a chilled
solution of H2N-L-Ala-L-Ala-L-Ala-L-Ala-OtBu hydrochloride (16
mg, 0.042 mmol) in 500 µL of DMF was added 6 (10 mg, 0.035
mmol), HOBt (7 mg, 0.053 mmol), 1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimide hydrochloride (10 mg, 0.053 mmol), and
diisopropylethylamine (18 µL, 0.106 mmol). The reaction mix-
ture was stirred for 1 h at 0 °C and 48 h at room temperature.
The solvent was removed under reduced pressure and the
residue was dissolved in dichloromethane (2 mL). Water (2
mL) was then added and the phases were separated. The
organic layer was washed with saturated NaHCO3 aqueous
solution (2 × 2 mL) and brine (1 × 2 mL), dried (Na2SO4), and
(3S,6S,8S,9S)-6-Acetylam in o-8-m eth oxy-6-m eth yl-5-oxo-
octah ydr oin dolizin e-3-car boxylic Acid Meth yl Ester (20).
Compound 19 (20 mg, 0.071 mmol) was dissolved in methanol
4898 J . Org. Chem., Vol. 69, No. 15, 2004

Analogues of L-Proline
evaporated to dryness. The resulting solid was purified via
silica gel flash column chromatography (AcOEt/MeOH, 4:1).
The desired compound 22 was obtained as a yellow foam (7
mg, 32%): [R]D -65.0 (c 0.7, MeOH); 1H NMR (CDCl3, 400
MHz) δ 7.66 (1H, d, J ) 7.1 Hz), 7.62 (1H, d, J ) 6.7 Hz), 7.25
(1H, s), 7.11 (1H, d, J ) 7.7 Hz), 7.03 (1H, d, J ) 7.2 Hz),
4.41-4.43 (1H, quint, J ) 7.7 Hz), 4.30-4.35 (2H, m), 4.27-
4.30 (1H, t, J ) 8.2 Hz), 4.16-4.18 (1H, quint, J ) 7.2 Hz),
4.05-4.07 (1H, m), 3.63 (1H, br s), 3.37 (3H, s), 2.50-2.54 (1H,
m), 2.28-2.36 (2H, m), 2.14-2.16 (1H, m), 1.96 (3H, s), 1.94-
1.98 (1H, m), 1.82-1.84 (1H, m), 1.50 (3H, s), 1.44 (3H, d, J )
7.4 Hz), 1.43 (9H, s), 1.42 (3H, d, J ) 8.0 Hz), 1.40 (3H, d, J
) 7.3 Hz), 1.38 (3H, d, J ) 7.2 Hz); 13C NMR (CDCl3, 100 MHz)
δ 174.0, 173.4, 172.8, 172.1, 171.8, 171.7, 170.8, 81.2, 73.1, 63.0,
61.8, 56.7, 54.2, 50.9, 49.7, 49.0, 48.8, 36.1, 28.4, 27.8, 26.7,
26.3, 22.8, 17.3, 17.1, 16.6, 16.1; MS (FAB+) m/e 647 (M +
Na)+, 625 (M + H)+; HRMS (FAB+) calcd for C29H49N6O9
625.35610, found 625.35623.
× 2 mL), dried (Na2SO4), and evaporated to dryness. The
resulting solid was purified via silica gel flash column chro-
matography (AcOEt/MeOH, 9:1). The desired compound 23
was obtained as a yellow foam (11 mg, 50%): [R]D -155 (c 0.25,
MeOH); 1H NMR (CDCl3, 400 MHz) δ 7.46 (1H, d, J ) 6.6 Hz),
7.04 (1H, d, J ) 8.2 Hz), 7.00 (1H, d, J ) 7.3 Hz), 6.54 (1H, d,
J ) 5.2 Hz), 4.54-4.50 (1H, quint, J ) 7.5 Hz), 4.39-4.31 (3H,
m), 4.25-4.16 (1H, dq, J ) 8.1, 7.0 Hz), 4.00-3.93 (1H, m),
3.61-3.60 (1H, m), 3.37 (3H, s), 2.47-2.42 (2H, m), 2.22-2.07
(1H, m), 2.05-1.92 (2H, m), 1.87-1.83 (1H, dd, J ) 15.1, 2.1
Hz), 1.66 (3H, s), 1.49 (3H, d, J ) 7.0 Hz), 1.46 (9H, s), 1.44
(3H, d, J ) 7.5 Hz), 1.43 (3H, d, J ) 7.0 Hz), 1.39 (3H, d, J )
7.5 Hz); 13C NMR (CDCl3, 100 MHz) δ 173.1, 172.7, 172.4,
172.2, 172.1, 171.5, 81.8, 73.4, 63.9, 62.5, 60.6, 57.3, 51.3, 50.6,
49.3, 49.2, 37.7, 28.3, 28.1. 27.2, 24.7, 18.2, 17.7, 17.6, 17.2;
MS (FAB+) m/e 609 (M + H)+; HRMS (FAB+) calcd for
C
27H45N8O8 609.33603, found 609.33549.
Azid e-L-Bca P -L-Ala -L-Ala -L-Ala -L-Ala -OtBu (23). To a
chilled solution of H2N-L-Ala-L-Ala-L-Ala-L-Ala-OtBu hydro-
chloride (17 mg, 0.045 mmol) in 500 µL of DMF was added 18
(10 mg, 0.037 mmol), HOBt (8 mg, 0.056 mmol), 1-[3-(di-
methylamino)propyl]-3-ethylcarbodiimide hydrochloride (11
mg, 0.056 mmol), and diisopropylethylamine (26 µL, 0.149
mmol). The reaction mixture was stirred for 1 h at 0 °C and
48 h at room temperature. The solvent was removed under
reduced pressure and the residue was dissolved in dichlo-
romethane (2 mL). Water (2 mL) was then added and the
phases were separated. The organic layer was washed with
saturated NaHCO3 aqueous solution (2 × 2 mL) and brine (1
Ack n ow led gm en t. We thank NSERC for financial
assistance, Pharmacia (Nerviano, Italy) for a sabbatical
leave to G.P., and Dr. Michel Simard for the X-ray
crystal structure analysis.
Su p p or tin g In for m a tion Ava ila ble: Selected experimen-
tal procedures, 1H, 13C NMR, NOESY, CD, and FT-IR spectra,
and DMSO-d6 and variable-temperature titrations. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
J O0401422
J . Org. Chem, Vol. 69, No. 15, 2004 4899