Water-stable helical structure of tertiary amides of bicyclic β-amino acid bearing 7-azabicyclo[2.2.1]heptane. Full control of amide cis-trans equilibrium by bridgehead substitution

Article abstract of DOI:10.1021/ja1017877

Helical structures of oligomers of non-natural β-amino acids are significantly stabilized by intramolecular hydrogen bonding between main-chain amide moieties in many cases, but the structures are generally susceptible to the environment; that is, helices may unfold in protic solvents such as water. For the generation of non-hydrogen-bonded ordered structures of amides (tertiary amides in most cases), control of cis-trans isomerization is crucial, even though there is only a small sterical difference with respect to cis and trans orientations. We have established methods for synthesis of conformationally constrained β-proline mimics, that is, bridgehead-substituted 7-azabicyclo[2.2.1]heptane-2-endo-carboxylic acids. Our crystallographic, 1D- and 2D-NMR, and CD spectroscopic studies in solution revealed that a bridgehead methoxymethyl substituent completely biased the cis-trans equilibrium to the cis-amide structure along the main chain, and helical structures based on the cis-amide linkage were generated independently of the number of residues, from the minimalist dimer through the tetramer, hexamer, and up to the octamer, and irrespective of the solvent (e.g., water, alcohol, halogenated solvents, and cyclohexane). Generality of the control of the amide equilibrium by bridgehead substitution was also examined.

Full text of DOI:10.1021/ja1017877

Published on Web 09/30/2010
Water-Stable Helical Structure of Tertiary Amides of Bicyclic
ꢀ-Amino Acid Bearing 7-Azabicyclo[2.2.1]heptane. Full Control
of Amide Cis-Trans Equilibrium by Bridgehead Substitution
Masahiro Hosoya,† Yuko Otani,*,† Masatoshi Kawahata,‡ Kentaro Yamaguchi,‡ and
Tomohiko Ohwada*,†
Graduate School of Pharmaceutical Sciences, The UniVersity of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan, and Department of Pharmaceutical Sciences at Kagawa Campus,
Tokushima Bunri UniVersity, 1314-1 Shido, Sanuki, Kagawa 769-2193, Japan
Received March 3, 2010; E-mail: otani@mol.f.u-tokyo.ac.jp; ohwada@mol.f.u-tokyo.ac.jp
Abstract: Helical structures of oligomers of non-natural ꢀ-amino acids are significantly stabilized by
intramolecular hydrogen bonding between main-chain amide moieties in many cases, but the structures
are generally susceptible to the environment; that is, helices may unfold in protic solvents such as water.
For the generation of non-hydrogen-bonded ordered structures of amides (tertiary amides in most cases),
control of cis-trans isomerization is crucial, even though there is only a small sterical difference with respect
to cis and trans orientations. We have established methods for synthesis of conformationally constrained
ꢀ-proline mimics, that is, bridgehead-substituted 7-azabicyclo[2.2.1]heptane-2-endo-carboxylic acids. Our
crystallographic, 1D- and 2D-NMR, and CD spectroscopic studies in solution revealed that a bridgehead
methoxymethyl substituent completely biased the cis-trans equilibrium to the cis-amide structure along
the main chain, and helical structures based on the cis-amide linkage were generated independently of
the number of residues, from the minimalist dimer through the tetramer, hexamer, and up to the octamer,
and irrespective of the solvent (e.g., water, alcohol, halogenated solvents, and cyclohexane). Generality of
the control of the amide equilibrium by bridgehead substitution was also examined.
Introduction
Among peptides composed of non-natural amino acids,
ꢀ-peptides have been extensively studied due to their ability to
form ordered structures (especially helices), their structural
resemblance to natural R-peptides, and their enhanced stability
to protease digestion.1 In many cases, helical structures of
oligomers of non-natural ꢀ-amino acids are significantly stabi-
lized by intramolecular hydrogen bonding between main-chain
amide moieties,2 but such structures are generally susceptible
to the environment; that is, helices are often unfolded (desta-
bilized) in protic solvents such as water.3 Thus, development
of ꢀ-peptides with robust organized structure without utilizing
hydrogen bonding is an important goal in the area of peptide
engineering and medicinal chemistry.1b,4
Figure 1. The cis-trans isomerization of amides.
Control of cis-trans (E-Z) isomerization of the amide
linkage is crucial for the robustness of ordered structures.
Nevertheless, tailoring of cis-trans isomerization of non-
hydrogen-bonding amides, that is, tertiary amides in most cases,
is intrinsically difficult due to the small sterical difference with
respect to cis and trans orientations (Figure 1).5 This is
significantly different from the situation in the case of secondary
amides, which show strong preference for trans-conformation
along the main chain, together with hydrogen-bond formation.
Changing the equilibrium of cis-trans isomerization of tertiary
amides in the case of R-amino acids, particularly R-proline and
its mimetics, has been accomplished sterically (by introduction
† The University of Tokyo.
‡ Tokushima Bunri University.
(1) (a) Seebach, D.; Albert, M.; Arvidsson, P. I.; Rueping, M.; Schreiber,
J. V. Chimia 2001, 55, 345–353. (b) Hamuro, Y.; Schneider, J. P.;
DeGrado, W. F. J. Am. Chem. Soc. 1999, 121, 12200–12201. (c) Porter,
E. A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gellman, S. H. Nature
2000, 404, 565.
(4) For examples of hydrogen-bonded ꢀ-peptides with biological activity,
see: (a) Imamura, Y.; Watanabe, N.; Umezawa, N.; Iwatsubo, T.; Kato,
N.; Tomita, T.; Higuchi, T. J. Am. Chem. Soc. 2009, 131, 7353–7359.
(b) Karlsson, A. J.; Pomerantz, W. C.; Weisblum, B.; Gellman, S. H.;
Palecek, S. P. J. Am. Chem. Soc. 2006, 128, 12630–12631. (c) Porter,
E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2002, 124,
7324–7330. (d) Liu, D. H.; DeGrado, W. F. J. Am. Chem. Soc. 2001,
123, 7553–7559.
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Christianson, L. A.; Durell, S. R.; Gellman, S. H. J. Am. Chem. Soc.
2000, 122, 2711–2718. (b) Hete´nyi, A.; Ma´ndity, I. M.; Martinek,
T. A.; To´th, G. K.; Fu¨lo¨p, F. J. Am. Chem. Soc. 2005, 127, 547–553.
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VCH: Weinheim, 2006; and references cited therein.
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14780 J. AM. CHEM. SOC. 2010, 132, 14780–14789
10.1021/ja1017877  2010 American Chemical Society

Bicyclic ꢀ-Amino Acid Bearing 7-Azabicyclo[2.2.1]heptane
A R T I C L E S
Figure 2. (A) 2,2-Disubstituted-pyrrolidine-4-carboxylic acid. (B) ꢀ-Amino acid bearing 7-azabicyclo[2.2.1]heptane and (C) homooligomers of (B) described
in the previous study.11 (D) 1,3-Allylic strain of bicyclic amides.
of alkyl group(s) at the R-position of the nitrogen atom,6,7 etc.)
or stereoelectronically (by use of the gauche effect, which
influences puckering of the pyrrolidine ring, leading to direct
interaction of the amide linkage and the carboxylic functional-
ity,8 etc.). In most cases, the trans-amide structure was favored
over the cis-amide, although 5,5-dimethyl-R-proline6c,d and its
oxazolidine and thiazolidine analogues7 drove the equilibrium
toward the cis-amide. Thus, steric and stereoelectronic factors
can be used to modify the cis-trans amide isomerization to
various extents, but complete control of cis-trans amide
isomerization to obtain a single conformer is very difficult, even
in the case of R-proline derivatives.
homogeneous secondary structures would be obtained, leading
to length-independent induction of ordered structures.
Recently, the synthesis and crystallographic and CD/UV
spectral analyses of ꢀ-amino acid homooligomers based on
bicyclic 7-azabicyclo[2.2.1]heptane have been studied (B and
C, Figure 2).11 The bicyclic skeleton was found to constrain
the torsion angles (φ, θ, and ψ) along the main chain (B).12
This rigid bicyclic system was proposed to drive the formation
of CD-active ordered structures of the long oligomers. Further-
more, the present bicyclic structure and the presence of two
bridgehead hydrogen atoms of the 7-azabicyclo[2.2.1]heptane
amide have a great influence on amide planarity: they promote
nitrogen pyramidalization and twisting of the amide linkage,
while monocyclic amides such as the pyrrolidine amides take
planar structures.13 This nonplanarity stems from angle strain
of the bicyclic structure and 1,3-allylic-type strain between the
amide moiety and the bridgehead hydrogen atoms (D, Figure
2). Therefore, we expected that bridgehead substitution of the
relevant bicylic skeleton could influence cis-trans isomerization
oftheamidegrouptoagreatextent.The7-azabicyclo[2.2.1]heptane
structure is also contained in epibatidine, a biologically active
alkaloid, as the basic skeleton, and thus synthetic access to this
ring system has been well investigated in the past.14-16
In this study, we established synthetic methods leading to
conformationally constrained ꢀ-proline mimics, that is, bridge-
head-substituted 7-azabicyclo[2.2.1]heptane-2-endo-carboxylic
acid (e.g., 3 in Figure 4). We studied the robustness of secondary
structures of the homooligomers and found that bridgehead
substitution completely biased the cis-trans equilibrium to the
cis-amide structure (along the main chain). Helical structures
based on the cis-amide linkage were generated independently
of the number of the residues, from the minimalist dimer through
the tetramer, hexamer, and up to the octamer, and irrespective
of the solvent, including water, alcohol, halogenated solvents,
While control of cis-trans equilibrium of R-proline deriva-
tives has been studied intensively, that of tertiary amide-type
ꢀ-amino acids has been little explored,9 probably because the
N-amide linkage in a ꢀ-amino acid is distal from the intraresidual
carboxylic acid functionality and direct interaction is ineffective.
As a precedent of non-hydrogen-bonding ꢀ-peptides, 2,2-
disubstituted-ꢀ-proline (2,2-disubstituted-pyrrolidine-4-carboxy-
lic acid, A, Figure 2) oligomers have been studied,10 and length-
dependent induction of helical structures has been demonstrated.
If full control of cis-trans isomerization of ꢀ-tertiary amides,
that is, suppression of the contributions of interconverting
rotamers, and that of torsion angles (φ, θ, and ψ) along the
main chain (in A, Figure 2) could be achieved, conformationally
(6) (a) Be´lec, L.; Slaninova, J.; Lubell, W. D. J. Med. Chem. 2000, 43,
1448–1455. (b) Swarbrick, M. E.; Gosselin, F.; Lubell, W. D. J. Org.
Chem. 1999, 64, 1993–2002. (c) Magaard, V. W.; Sanchez, R. M.;
Bean, J. W.; Moore, M. L. Tetrahedron Lett. 1993, 34, 381–384. (d)
An, S. S. A.; Lester, G. C.; Peng, J. L.; Li, Y. J.; Rothwarf, D. M.;
Welker, E.; Thannhauser, T. W.; Zhang, L. S.; Tam, J. P.; Scheraga,
H. A. J. Am. Chem. Soc. 1999, 121, 11558–11566.
(7) For examples of cis-prolyl conformations with pseudoprolines, see:
(a) Wittelsberger, A.; Keller, M.; Scarpellino, L.; Patiny, L.; Acha-
Orbea, H.; Mutter, M. Angew. Chem., Int. Ed. 2000, 39, 1111–1115.
(b) Keller, M.; Sager, C.; Dumy, P.; Schutkowski, M.; Fischer, G. S.;
Mutter, M. J. Am. Chem. Soc. 1998, 120, 2714–2720, and references
therein.
(11) Otani, Y.; Futaki, S.; Kiwada, T.; Sugiura, Y.; Muranaka, A.;
Kobayashi, N.; Uchiyama, M.; Yamaguchi, K.; Ohwada, T. Tetrahe-
dron 2006, 62, 11635–11644.
(12) Ab initio calculations of the cis- and trans-dimers of the bicyclic
ꢀ-amino acid B ((S)-enantiomer) suggested that φ, θ, and ψ converged
to-86°,-162°, and-75°, respectively, while ψ had one additional
minimum at-95° within 0.1 kcal/mol difference.
(13) Otani, Y.; Nagae, O.; Naruse, Y.; Inagaki, S.; Ohno, M.; Yamaguchi,
K.; Yamamoto, G.; Uchiyama, M.; Ohwada, T. J. Am. Chem. Soc.
2003, 125, 15191–15199.
(8) (a) Eberhardt, E. S.; Panasik, N.; Raines, R. T. J. Am. Chem. Soc.
1996, 118, 12261–12266. (b) Bretscher, L. E.; Jenkins, C. L.; Taylor,
K. M.; DeRider, M. L.; Raines, R. T. J. Am. Chem. Soc. 2001, 123,
777–778.
(9) (a) Abele, S.; Vo¨gtli, K.; Seebach, D. HelV. Chim. Acta 1999, 82,
1539–1558. (b) Huck, B. R.; Langenhan, J. M.; Gellman, S. H. Org.
Lett. 1999, 1, 1717–1720. For examples of peptoid oligomers and other
tertiary amide oligomers, see: (c) Fowler, S. A.; Blackwell, H. E. Org.
Biomol. Chem. 2009, 7, 1508–1524. (d) Wu, C. W.; Kirshenbaum,
K.; Sanborn, T. J.; Patch, J. A.; Huang, K.; Dill, K. A.; Zuckermann,
R. N.; Barron, A. E. J. Am. Chem. Soc. 2003, 125, 13525–13530. (e)
Bernardi, F.; Garavelli, M.; Scatizzi, M.; Tomasini, C.; Trigari, V.;
Crisma, M.; Formaggio, F.; Peggion, C.; Toniolo, C. Chem.-Eur. J.
2002, 8, 2516–2525. (f) Crisma, M.; Moretto, A.; Toniolo, C.;
Kaczmarek, K.; Zabrocki, J. Macromolecules 2001, 34, 5048–5052.
(10) (a) Huck, B. R.; Fis, J. D.; Guzei, I. A.; Carlson, H. A.; Gellman,
S. H. J. Am. Chem. Soc. 2003, 125, 9035–9037. (b) Huck, B. R.;
Gellman, S. H. J. Org. Chem. 2005, 70, 3353–3362.
(14) For example: (a) Herna´ndez, A.; Marcos, M.; Rapoport, H. J. Org.
Chem. 1995, 60, 2683–2691. (b) Campbell, J. A.; Rapoport, H. J. Org.
Chem. 1996, 61, 6313–6325. (c) Bai, D.; Xu, R.; Chu, G.; Zhu, X. J.
Org. Chem. 1996, 61, 4600–4606. (d) Singh, S.; Basmadjian, G. P.
Tetrahedron Lett. 1997, 38, 6829–6830. (e) Pandey, G.; Bagul, T. D.;
Sahoo, A. K. J. Org. Chem. 1998, 63, 760–768, refs 15 and 16 and
references therein.
(15) Corey, E. J.; Loh, T.-P.; AchyuthaRao, S.; Daley, D. C.; Sarshar, S.
J. Org. Chem. 1993, 58, 5600–5602.
(16) Fletcher, S. R.; Baker, R.; Chambers, M. S.; Herbert, R. H.; Hobbs,
S. C.; Thomas, S. R.; Verrier, H. S. M.; Watt, A. P.; Ball, R. G. J.
Org. Chem. 1994, 59, 1771–1778.
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A R T I C L E S
Hosoya et al.
to the epibatidine case. The resultant amine (16) was subjected
to benzoylation with benzoyl chloride to give bridgehead-
substituted bicyclic amide 2. The bromoamide 2 was also
converted to the simple N-benzoyl amide 1 via tBuOK-mediated
elimination of HBr, followed by hydrogenation of the resultant
alkene 17.
Evaluation of the Effect of the Bridgehead Methyl Substitu-
ent on Amide Cis-Trans Equilibrium. X-ray crystallographic
analyses showed that benzoyl amides 1 and 2 both take cis-
amide structures; that is, the phenyl group and the bridgehead
H are on the same side with respect to the amide bond (Scheme
1, Figure 5a, and Figure S2; the torsion angle ω(C1-C2-N-C3)
values for 1 and 2 were -32.0° and -41.3°, respectively). The
ω(C1-C2-N-C3) values are close to 0°, consistent with the
cis-conformation of the amides. However, these values appar-
ently deviated from 0° because of nitrogen pyramidalization.
Solution structures of 1 and 2 were also elucidated by 1D-
1H and 2D NMR spectroscopy (Supporting Information). The
1D-1H and 13C NMR spectra in chloroform-d1 and methanol-d4
showed the presence of a single conformer, and NOESY
experiments detected NOE between the phenyl protons of the
benzoyl group and the bridgehead proton in both cases (Figures
5b and Figure S3 (2D NOESY spectrum in CD3OD)). Only a
Figure 3. Energy differences between cis and trans rotamers of the methyl-
substituted dimer at the MP2/6-31G*//B3LYP/6-31G* level.
1
Figure 4. 7-Azabicyclo[2.2.1]heptane derivatives in this study.
cis conformer was detected for compound 1 in the H NMR
spectra in the range of temperature from -20 to 100 °C in
1,1,2,2-tetrachloroethane-d2 (data not shown). These data indi-
cated that both 1 and 2 take a single conformation with the
cis-amide linkage in solution.
and cyclohexane. We also studied the effect of transformation
of the bridgehead substituents to various functionalities.
Results and Discussion
Bridgehead-Substituted Bicyclic ꢀ-Amino Acid Derivatives
and Their Homooligomers. Synthesis of Bridgehead Methoxy-
methyl-Substituted Bicyclic ꢀ-Amino Acid. The above strategy
can be applied to bridgehead-substituted bicyclic ꢀ-amino acid
derivatives. First, we chose an alkoxymethyl group as a
bridgehead substituent because of its synthetic compatibility with
the additional carboxylic acid functionality of the ꢀ-amino acids.
It turned out that this substituent increased the water solubility
of the oligomers, and this feature makes it possible to conduct
various spectroscopic measurements in water. Thus, compounds
3-9 (Figure 4) were chosen as synthetic targets.
For the preparation of the N- and C-protected ꢀ-amino acid
derivative 3 (see Scheme 3), it was necessary to change the
strategy used for the methyl-substituted amides 1 and 2.20 After
several attempts, we decide to utilize the bridgehead ester-
substituted bicyclic alcohol 18 (Scheme 2) as a synthetic
precursor; it was synthesized according to Avenoza et al.21 Next,
the Wittig reaction (leading to 20) was used as a key step in
the introduction of a one-carbon unit at the ꢀ-position, and this
Bridgehead Methyl-Substituted Bicyclic Amide. Molecular
Design and Synthesis. To evaluate the effect of the bridgehead
substituent on the amide cis-trans isomerization, we carried
out quantum chemical calculations for the dimer of bicyclic
ꢀ-amino acid substituted with a methyl group at the bridgehead
(C4) position (Figure 3). The calculations showed that the cis-
conformer is more stable than the trans-conformer by 2.8 kcal/
mol (MP2/6-31G*//B3LYP/6-31G*). This result suggested that
the combination of the present bicyclic skeleton and the
bridgehead substitution would completely tip the equilibrium
toward the cis-amide.
To examine this predicted effect, we synthesized the nitrogen-
amides of 4-methyl-7-azabicyclo[2.2.1]heptane (1 and 2, Figure
4) by means of a modification of the procedure employed in
epibatidine synthesis by Corey et al.15 and Fletcher et al.16
(Scheme 1). First, a methyl group was introduced at the R
position of the carboxylic acid moiety of 10 using lithium
diisopropylamide (LDA) and hexamethylphosphoramide (HMPA),
and then Curtius rearrangement followed by methanol treatment
gave the carbamate (12).17 Because a trifluoroacetyl group is
reported to be the best auxiliary for diastereo-selective bromi-
nation in the next step,18,19 12 was treated with trimethylsilyl
iodide to give the primary amine 13 quantitatively, and 13 was
acylated with trifluoroacetic anhydride to give 14. Bromination
of 14 with 2 equiv of Me3PhNBr3 proceeded stereoselectively
in a manner similar to that of the nonmethylated precursors18
to give a single trans-dibromo compound 15. Its structure was
confirmed by X-ray crystal structure analysis (Figure S1).
Deprotection of the trifluoroacetyl group followed by transan-
nular nucleophilic cyclization both proceeded smoothly under
weakly basic conditions (K2CO3, 35 °C), in a manner similar
(19) Direct trapping of the acyl azide intermediate with trifluoroacetic acid
instead of methanol during the Curtius rearrangement was attempted,
but was unsuccessful with substrate 9 because of steric hindrance of
the methyl group (see below and ref 17).
(17) Pfister, J. R.; Wymann, W. E. Synthesis 1983, 1, 38–40.
(20) Attempts at nucleophilic or electrophilic substitution of the bromo
(18) Kapferer, P.; Vasella, A. HelV. Chim. Acta 2004, 87, 2764–2789.
group of 2 with carboxylic acid equivalents were unsuccessful.
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Bicyclic ꢀ-Amino Acid Bearing 7-Azabicyclo[2.2.1]heptane
A R T I C L E S
Scheme 1. Synthesis of Methyl-Substituted 7-Azabicyclo[2.2.1]heptane Amidea
a THF ) tetrahydrofuran.
Figure 5. (a) X-ray crystallographic structure of 1. (b) Diagnostic NOESY peaks of 1 measured in chloroform-d.
in turn was converted to a carboxylic acid functionality (Scheme
2 and see below).
23 quantitatively by hydroboration with BH3 ·THF (as a mixture
of endo/exo isomers (82:18)), and then 23 was subjected to
Swern oxidation and subsequent Pinnick oxidation to give the
ꢀ-carboxylic acid 25. The ratio of the endo/exo isomers with
Ketone 19 was obtained from alcohol 18 by Swern oxidation,
and then 19 was converted to olefin 20 via the Wittig reaction.
The bridgehead ester was reduced to alcohol 21 by using a
combination of NaBH4 and CaCl2,22 and the alcohol group was
protected with a p-methoxybenzyl (PMB) group to give 22.23
The exocyclic methylene group of 22 was converted to alcohol
(22) Avenoza, A.; Barriobero, J. I.; Busto, J. H.; Cativiela, C.; Peregrina,
J. M. Tetrahedron: Asymmetry 2002, 13, 625–632.
(23) When we tried other protecting groups such as a MOM and a silyl
ether group, the yields of the coupling reactions with camphorsultam
were rather low because of the formation of undefined byproducts. In
the case of benzyl protection, the coupling yield was moderate, but
the subsequent deprotection of the benzyl group by catalytic hydro-
genation proceeded only slowly.
(21) Avenoza, A.; Busto, J. H.; Peregrina, J. M. Org. Lett. 2007, 9, 1235–
1238.
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Hosoya et al.
Scheme 2. Synthesis of Bridgehead-Substituted Bicyclic ꢀ-Amino Acid 25-endoa
a DMSO ) dimethylsulfoxide, TBAI ) tetrabutylammonium iodide, DMF ) N,N-dimethylformamide.
respect to the ꢀ-substituent remained constant during these
oxidation processes. Conversion of the resultant carboxylic acid
25 to methyl ester 26 with (trimethylsilyl)diazomethane enabled
separation of the endo/exo isomers by column chromatography
on silica gel. The isolated endo-ester 26-endo was hydrolyzed
to give the racemic ꢀ-amino acid 25-endo. All the steps
proceeded in high yields, and the present procedure is applicable
at a 10 g scale. The structure of the resultant ꢀ-amino acid was
confirmed by X-ray crystallographic analysis of the methoxy-
methyl-substituted N-benzoyl derivative rac-4a (Figure 6a and
also Scheme 3), which was synthesized in the course of a
preliminary study (see Supporting Information, Scheme S1).
To separate the endo-ester enantiomers, an optically active
camphorsultam group was introduced into the ꢀ-amino acid 25-
endo as a chiral auxiliary to give 27 (Scheme 3). During
optimization to identify suitable conditions for large-scale and
reproducible separation, we found that the enantiomers of the
deprotected hydroxymethyl-substituted derivative 28 could be
separated by silica-gel column chromatography (eluent: n-hex-
ane:Et2O ) 2:1).24 Other protected hydroxymethyl-substituted
derivatives were difficult to separate by column chromatography
on a preparative scale.
The chiral alcohol 28 was converted to the methyl ether 29
with (trimethylsilyl)diazomethane in the presence of aqueous
fluoroboric acid,25 and hydrolysis gave the chiral ꢀ-amino acid
30. The stereochemistry was confirmed by X-ray crystal-
lographic analysis of the homodimer (S)-5 (vide post, see Figure
Figure 6. ORTEP drawings of the crystal structures of (a) rac-4a and (b)
(S)-5.
6b). The values of optical rotation [R]24 of the N-benzoyl-
D
(24) 7 g of 28 can be separated at one time by repeated column
chromatography.
(25) Aoyama, T.; Shioiri, T. Tetrahedron Lett. 1990, 31, 5507–5508.
methyl ester were -34.7° for (R)-4a (c ) 1.56, CHCl3) and
+35.3° for (S)-4a (c ) 1.48, CHCl3), respectively.26
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Bicyclic ꢀ-Amino Acid Bearing 7-Azabicyclo[2.2.1]heptane
A R T I C L E S
Scheme 3. Optical Resolution and Synthesis of Chiral ꢀ-Amino Acidsa
a DDQ ) 2,3-dichloro-5,6-dicyano-p-benzoquinone, TFA ) trifluoroacetic acid.
In the course of homocoupling of the ꢀ-amino acid unit (R)-3
(vide infra), we unexpectedly obtained N-acetyl amide (R)-4b
(Figure 4) during deprotection of the Boc group of (R)-3 in the
presence of HCl in ethyl acetate (acetic acid was present as a
contaminant). The N-acetyl amide (R)-4b was an oily material,
which showed NOE effects between the acetyl methyl protons
and the bridgehead proton in the 2D-NOESY spectrum in
methanol-d4, indicating the presence of cis-amide structure in
solution (Figure S4). Only a single conformer was detected
in methanol-d4, and the spectrum did not change even at 50
°C. These findings show that even the sterically small N-acetyl
group on the nitrogen atom generates the cis-amide structure
in the presence of the bridgehead substituent.
Next, homooligomers, the dimer (R)-5, the trimer (R)-6, the
tetramer (R)-7, the hexamer (R)-8, and the octamer (R)-9 were
synthesized by means of normal solution-phase peptide coupling
procedures using 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide (EDCI) and (4-dimethylamino)pyridine (DMAP) (Scheme
4). The hexamer (R)-8 was synthesized by amide coupling of
the amine of the dimer (R)-5 and the carboxylic acid of the
tetramer (R)-7. The octamer (R)-9 was synthesized by the
coupling of the amine of the hexamer (R)-8 and the carboxylic
acid of the dimer (R)-5. The dimer composed of the (S)-
enantiomer of the ꢀ-amino acid ((S)-5) was synthesized in a
manner similar to that of (R)-5 (see Supporting Information).
Structural Analysis of Monomeric and Oligomeric ꢀ-Amino
Acid. Crystallographic Analyses and NMR Spectroscopy. Single-
conducted (Figure 6). The observed main-chain torsional angles
of these molecules are summarized in Table 1. Both rac-4a
(ω ) -38.2°) and (S)-5 (ω ) -24.8° and 13.4°) take cis-amide
structures. The torsion angle θ was well converged to constant
absolute value, because of the rigid bicyclic skeleton, while the
angles ω and φ vary to some extent depending upon the degree
of nitrogen pyramidalization.27 The free-rotating torsion angle
ψ was -87.5° in the case of (S)-5, which was consistent with
the calculated value of the dimer of the bridgehead-unsubstituted
case (C).12
1D-1H, 1D-13C, and 2D-NMR (COSY, HOHAHA, and
NOESY) analyses were conducted in chloroform-d1 and methanol-
d4 for the dimer (R)-5, the trimer (R)-6, and the tetramer (R)-7
of the unit ꢀ-amino acid (see Supporting Information). Diag-
nostic interresidual NOE effects between the ꢀi proton and the
bridgehead (Ri+1) proton were observed in the 2D-NOESY
spectra (Figure 7 and Supporting Information). All the data
indicated the presence of a single conformer having cis-amide
linkages. The NMR spectra of the trimer (R)-6 were also
measured in various solvents: methylene chloride-d2, methanol-
d4, acetone-d6, and D2O (Figure S5). Only a single conformer
was present in all of these solvents. In the range of the
temperature from 20 to 50 °C, there was no significant change
of the shapes and chemical shifts of the peaks, in either
chloroform-d1 or methanol-d4 (Supporting Information).
The 2D-NOESY NMR spectra of the trimer (R)-6 measured
in D2O showed correlation patterns similar to those observed
in methanol-d4 (Figure S6).
The present results clearly indicated that the short ꢀ-peptide
oligomers ((R)-5, (R)-6, and (R)-7) based on the bridgehead-
substituted bicyclic structure adopted distinct folding structures
and showed highly homogeneous conformations in a range of
solvents from water to chloroform. The combination of the
present bicyclic skeleton and unsymmetrical bridgehead sub-
crystal structure analyses of the racemic N-benzoyl monomer
(rac-4a) and the dimeric ꢀ-amino acid ((S)-5) were successfully
(26) The chiralities of the monomers (R)- and (S)-3 or their homooligomers
were consistent with those of the bridgehead unsubstitued compounds
(ref 11) in terms of the sign of the optical rotations and CD absorptions.
The characteristic deshielding 1H-NMR chemical shift of the bridge-
head (H1) proton of the camphorsultam derivative, (R)-28 (multiplet,
4.77-4.48 ppm), as compared to that of (S)-28 (multiplet, 4.72-4.70
ppm), was also consistent with the case of the corresponding
bridgehead unsubstituted compounds ((R)-camphorsultam amide,
multiplet, 4.71-4.69 ppm; and (S)-camphorsultam amide, multiplet,
4.66-4.64 ppm, respectively (see ref 11)).
(27) The tilt angles of nitrogen pyramidalization of amide bonds for 1, 2,
rac-4a, (S)-5, 45, and 47 are 27.4°, 29.4°, 28.5°, 10.3°, 23.2°, and
40.7°, respectively.
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A R T I C L E S
Hosoya et al.
Scheme 4. Synthesis of the Homooligomers (R)-5, (R)-6, (R)-7, (R)-8, and (R)-9
Table 1. Main-Chain Torsional Angles in the Crystal Structurea
or ꢀ-proline derivatives (A in Figure 2),10 which showed length-
dependent increase of the CD intensities (per residue). Because
a mixture of cis- and trans-conformers was observed in the
NMR spectra of the bridgehead-unsubstituted dimer, it was
suggested that the ratio of cis-amide conformer increased as
the chain length increased in the case of the bridgehead-
unsubstituted oligomers.11 The dimer of the S-enantiomer ((S)-
5) showed a symmetric spectrum with respect to that of (R)-5
in terms of the Cotton effects (Figure S7). Furthermore, the CD
spectrum of the tetramer (R)-7 in water did not change
significantly as the temperature was raised to 80 °C (Figure S8).
Further, the CD spectra of (R)-7 in water in the concentration
range of 25 µM to 1 mM were almost the same (Figure S9a),
and the CD spectra of (R)-9 in water in the concentration range
of 8-32 µM were also almost the same (Figure S9b), which
indicates that no aggregation occurred. The methoxymethyl
groups, which protrude outside the helix (see Figure 9), probably
contributed to the increased solubility in water. We also
measured the CD spectra of the tetramer (R)-7 in other solvents:
methanol, 2,2,2-trifluoroethanol (TFE), and cyclohexane (c-Hex)
(Figure 8b). While (R)-7 in water solution showed about 20%
enhanced CD intensities as compared to the methanol solution,
the CD spectra in all the solvents had similar peak shapes and
intensities, indicating that the structure of (R)-7 in these solvents
is essentially conserved.
compound
residueb
ω (deg)c
φ (deg)
θ (deg)
ψ (deg)
1
1
1
1
1
2
1
1
-32.0
-41.3
-38.2
-24.8
13.4
24.9
-51.8
2d
rac-4ae
(S)-5
157.0
(-80.3)g
-136.5
99.7
162.0
-169.5
-158.4
152.9
(49.1)f
-87.5
(-55.2)f
(65.3)f
45e
47e
167.9
165.9
(38.8)f
a See Figure 6a for the definition of the torsion angles. b Numbering
from the N-terminal. c In the case of compounds 1 and 2, ω means the
torsion angle (C1-C2-N-C3) (Scheme 1). d The values of the enan-
tiomer of (S)-configuration with respect to the C4 atom are shown. e The
values of the (R)-enantiomer are shown. f The torsion angle concerns the
C-terminal ester. g The torsion angle concerns the N-terminal N-Boc
bond.
stitution thus permits unprecedented full control of selectivity
in favor of the cis-amide.
Circular Dichroism. The CD spectra of the dimer (R)-5, the
trimer (R)-6, and the tetramer (R)-7 at 100 µM, the hexamer
(R)-8 at 45 µM, and the octamer (R)-9 at 32 µM in water at 20
°C are shown in Figure 8a. Intensities of the spectra were
normalized in terms of concentration and the number of residues.
The spectra showed strong signals at 190-195 nm and at around
215-223 nm, and, surprisingly, the intensities of the signals
per residue ([θ] (deg cm2 dmol-1 residue-1)) were almost the
same throughout the range of oligomers: [θ]214 ) -2.9 × 104
for (R)-5, [θ]215 ) -2.9 × 104 for (R)-6, [θ]218 ) -2.7 × 104
for (R)-7, [θ]222 ) -2.4 × 104 for (R)-8, and [θ]223 ) -2.4 ×
104 for (R)-9. This is in sharp contrast to the oligomers of
bridgehead-unsubstituted bicyclic amino acid (C, Figure 2)11
These results of the CD spectral studies clearly suggest that
the oligomers 5, 6, 7, 8, and 9 take highly homogeneous and
robust ordered structures, probably cis-helical structures based
on the cis-amide linkage. The calculated CD spectrum of the
dimer (R)-5 at the TD-DFT level (TDDFT/B3LYP/aug-cc-
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Bicyclic ꢀ-Amino Acid Bearing 7-Azabicyclo[2.2.1]heptane
A R T I C L E S
Figure 7. Diagnostic interresidue NOE signals (open circles) of (a) (R)-5, (b) (R)-6, and (c) (R)-7 in CD3OD at 25 °C.
Figure 8. CD spectra of (a) (R)-5, (R)-6, and (R)-7 at 100 µM, (R)-8 at 45 µM, and (R)-9 at 32 µM in water at 20 °C and (b) CD spectra of (R)-7 at 100
µM in methanol, water, TFE, and cyclohexane (c-Hex) at 20 °C. The molar ellipticity [θ] values were normalized in terms of the concentration and the
number of residues.
pVDZ//B3LYP/6-31G*)28 showed that the cis-amide structure
of (R)-5, rather than trans-amide structure, is consistent with
the experimental CD spectra (Figure S10). This and the
aforementioned X-ray and NMR results support the above
interpretation of the CD spectra.
in magnitude to those found in the crystal structures of 4a and
(S)-5 (Table 1). It is anticipated from this model structure that
the bridgehead substituents protrude outside the helix, and they
are expected to be useful for functionalization of the helix
surface.
The computational conformational search of the N-formyl-
(S)-octamer revealed that the cis-helical structure is the lowest-
energy structure (Figure 9), and the torsion angles of the eight
residues (ω ) -23.6° to -16.9°, φ ) -103.3° to -97.5°, θ )
-160.6° to -158.6°, and ψ ) -83.6° to -79.4°) are similar
Transformation of Bridgehead Substituents. Bicyclic ester
derivative 18 could be transformed to compounds substituted
with ethyl (35) and i-propyl (39) groups at the bridgehead
position (Scheme 5, (1) and (2)). The hydroxyl group of 18
was protected with a tert-butyldimethylsilyl (TBS) group, and
then the bridgehead ester of 31 was reduced with NaBH4 and
22
(28) Frisch, M. J.; et al. Gaussian 03; Gaussian, Inc.: Pittsburgh, PA, 2003.
CaCl2 to give 32. The bridgehead hydroxymethyl group of
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A R T I C L E S
Hosoya et al.
Figure 9. Top view (left) and side view (middle) of the energy-minimum structure of the octamer (right) obtained by Monte Carlo conformation search
followed by DFT-optimization (B3LYP/6-31G*). Hydrogen atoms are omitted for clarity.
Scheme 5. Synthesis of Various Bridgehead-Substituted Bicyclic Amine Derivatives
32 was oxidized by Swern oxidation to give aldehyde 33, whose
aldehyde group was converted to alkene via a Wittig reaction
to give 34. The ethyl-substituted bicyclic compound 35 was
obtained by hydrogenation of 34. For the preparation of the
isopropyl-substituted compound, an additional methyl group was
introduced into aldehyde 33 with a methyl Grignard reagent to
give the secondary alcohol 36, which was oxidized to give
ketone 37. While the following Wittig reaction proceeded
slowly, probably due to steric hindrance near the bridgehead
position, the resultant alkene 38 was hydrogenated over PtO2
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Bicyclic ꢀ-Amino Acid Bearing 7-Azabicyclo[2.2.1]heptane
A R T I C L E S
isomerization of the present bicyclic ꢀ-amino acid derivatives
to the cis-amide structure.
Conclusion
Herein, we described the synthesis of ꢀ-amino acids bearing
the 7-azabicyclo[2.2.1]heptane structure substituted with a
functional group at the bridgehead position. We showed that
homooligomers of ꢀ-amino acid bearing a bridgehead meth-
oxymethyl group generated robust secondary structures without
hydrogen bonds. Crystallographic and NMR-NOE analysis
showed that these homooligomers had strong predominance of
the cis-amide structure, and they adopted helix-type folded
structures in the solid state and in solution. Furthermore, the
CD and NMR spectra indicated that these secondary structures
were retained in water solution even in the case of homooli-
gomer as short as the dimer. These results indicate that the
oligomers can be utilized as robust minimalist elements of
ordered secondary structures, both in hydrophobic and in
hydrophilic environments. Furthermore, on the basis of the shape
and sign of the CD spectra, the CD-active structures of the homo-
oligomers of bridghead-unsubstituted 7-azabicyclo[2.2.1]-
heptane-2-endo-carboxylic acid (C in Figure 2)11 can be
concluded to be helix structures involving the cis-amide linkage.
The present robust helical oligomers should therefore be
available for the creation of novel helix-mimicking compounds
with a range of potential biochemical applications.
Figure 10. ORTEP drawing of the crystal structure of bridgehead Br-
substituted N-acetylamide 45.
to give the isopropyl-substituted bicyclic compound 39 in high
yield. These N-Boc-protected alkyl-substituted bicyclic com-
pounds (35 and 39) can be easily converted to ꢀ-amino acid
derivatives by means of the procedure described in this work.
The bromo-substituted compounds 45, 46, and 47 (all
racemic) were synthesized by a modification of the procedure
of Avenoza et al. via bridgehead radical reaction (Scheme 5,
(3)):29 the bridgehead alcohol of 40 was oxidized to the
carboxylic acid 42, and then a mixture of the Barton ester 43
and bromotrichloromethane was irradiated with a 150 W halogen
lamp to give the bromo compound 44 in a good yield, and this
was used for the next step without further purification. Com-
pound 44 can be derived to N-acetamide 45, N-isopropyl amide
1
Acknowledgment. This work was supported by a Grant-in-Aid
for Scientific Research from the Japan Society for the Promotion
of Science. We thank Mr. Hiroaki Kurouchi and Ms. Fumika Karaki
of our group for help in the synthesis of (R)-8 and (R)-9. Some of
the calculations were carried out using Research Center for
Computational Science, Okazaki, Japan. We thank the computa-
tional facility for generous allotments of computer time.
46, and N-benzoyl amide 47 in good yields. H NMR studies,
including the NOE experiments, showed that all of these bromo-
substituted amides (45-47) were present as a single conformer
and took cis-amide conformations (Supporting Information). In
the range of temperature from 20 to 50 °C in methanol-d4, no
1
new peaks appeared in the H NMR spectrum of N-isopropyl
amide 46, excluding apparent equilibrium between the amide
rotamers. The cis-amide structures of the N-acetyl amide 45
and the N-benzoyl amide 47 were also supported by crystal-
lographic analysis (Figure 10 and Figure S11). Therefore, these
bridgehead substituents also strongly bias the amide cis-trans
Supporting Information Available: Supporting scheme and
figures. Experimental section including the details of synthesis,
NMR spectral data, CD and UV spectra, X-ray crystallographic
data, and the details of calculations. This material is available
free of charge via the Internet at http://pubs.acs.org.
(29) Avenoza, A.; Busto, J. H.; Cativiela, C.; Peregrina, J. M. Tetrahedron
2002, 58, 1193–1197.
JA1017877
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