Elevated conformational rigidity in dipeptides incorporating piperazic acid derivatives

Article abstract of DOI:10.1021/ja9729903

N-Acyl derivatives of piperazic acids display an unusual degree of conformational rigidity. As a consequence, α-N-coupling of a piperazic acid of given configuration (D or L) with an α-aminoacyl unit of opposite configuration (L or D) produces a 'heteroch

Full text of DOI:10.1021/ja9729903

80
J. Am. Chem. Soc. 1998, 120, 80-86
Elevated Conformational Rigidity in Dipeptides Incorporating
Piperazic Acid Derivatives
Ning Xi, Lawrence B. Alemany, and Marco A. Ciufolini*
Contribution from the Department of Chemistry, MS 60, Rice UniVersity, 6100 Main Street,
Houston, Texas 77005-1892
ReceiVed August 25, 1997X
Abstract: N-Acyl derivatives of piperazic acids display an unusual degree of conformational rigidity. As a
consequence, R-N-coupling of a piperazic acid of given configuration (D or L) with an R-aminoacyl unit of
opposite configuration (L or D) produces a “heterochiral” dipeptide that exists in a conformation conducive to
the formation of a peptide turn. Piperazic acids and related compounds may thus be regarded as conformationally
rigid analogues of proline, and because they are readily available in either antipodal form by synthesis, they
should become of great interest in the study of peptide turns.
Introduction
to create a conformationally rigid construct.6 We have found
that 2,3,4,5-tetrahydropyridazine-3-carboxylic acids (“PCAs”),
readily available in either the D or L configuration by synthesis,
behave as conformationally fixed proline equivalents. In
particular, N-2 acylation of PCAs with an R-amino acid of
opposite configuration generates a dipeptide that possesses the
vector properties of a peptide turn. Furthermore, PCAs are good
mimics of proline; therefore, it is likely that PCA dipeptides
may be used as building blocks for turn mimics that more closely
resemble their naturally occurring originals.
There is much current interest in the preparation of peptides
and peptidomimetics with a well-defined conformation.1 These
conformationally constrained peptides are useful to probe issues
of protein folding and to mimic peptide turns. The latter
structural motifs are of special relevance as key features of
â-sheet architectures (hairpin turns)2 and because of their
perceived role in ligand-receptor binding interactions and in
other functional aspects of protein chemistry (“â-turns”, “zinc
fingers”, etc.).3 Turns frequently occur at the site of a proline,
probably because unlike a secondary amide, which is rigidly
held in an s-Z conformation, the flexibility of the tertiary amide
unit of N-acylprolines may accommodate such turns more
readily. Turns are also favored when the proline and the amino
acid contributing its N-acyl group are of opposite configuration.4
However, introduction of a proline within a peptide chain is
insufficient, per se, to induce a turn, because N-acylprolines do
not manifest a sufficiently great preference for that conformer
which promotes turn formation. Indeed, peptide turns must be
further stabilized by a number of intramolecular interactions,
e.g., hydrogen bonds.5 At least 20-30 amino acids seem to be
necessary to stabilize a turn induced by a proline residue and
Results and Discussion
We have recently described dipeptide 1a (Table 1),7 a key
subunit of the antitumor and anti-HIV antibiotics luzopeptins
and quinoxapeptins (“peptin” antibiotics).8 This material,
together with the similar dipeptides 1b (a precursor to luzopeptin
E2) and 1c, all related N-2-acyl derivatives of 4-hydroxy-2,3,4,5-
tetrahydropyridazine-3-carboxylic acid or 2,3,4,5-tetrahydro-
pyridazine-3-carboxylic acid 1d-1l, as well as N-2-acetyl
analogues 2 and 3 exist as a single rotamer about the N-2-acyl
bond within the limits of detection of 1H and 13C NMR
spectroscopy (5.87 T). Compounds 1b-1l, 2, and 3 were
X Abstract published in AdVance ACS Abstracts, December 15, 1997.
(1) Cf. (a) Regan, L.; DeGrado, W. F. Science 1988, 241, 976. (b) Hecht,
M. H.; Richardson, J. S.; Richardson, D. C.; Ogden, R. C. Science 1990,
249, 884. (c) Osterhout, J. J., Jr.; Handel, T.; Na, G.; Arazdordi, T.; Long,
R. C.; Connolly, P. J.; Hoch, J. C.; Johnson, W. C. Jr., Live, D.; DeGrado,
W. F. J. Am. Chem. Soc. 1992, 114, 331.
(2) Cf. Smith, C. K.; Regan, L. Acc. Chem. Res. 1997, 30, 153.
(3) Cf. (a) Creighton, T. E. Proteins: Structure and Molecular Proper-
ties; Freeman: New York, 1983. (b) Rizo, J.; Gierasch, L. Annu. ReV.
Biochem. 1992, 61, 387. (c) Rose, G. D.; Gierasch, L. M.; Smith, J. A.
AdV. Protein Chem. 1985, 37, 1. (d) Ferguson, M. D.; Meara, J. P.;
Nakanishi, H.; Lee, M. S.; Kahn, M. Tetrahedron Lett. 1997, 38, 6961 and
references cited therein.
(4) One of the earliest examples of the turn-forming ability of N-acyl
proline dipeptides composed of amino acids of opposite configurations
(“heterochiral sequences”) may be found in the structure of gramicidin:
Hull, S. E.; Karlsson, R.; Main, P.; Woolfson, M. M.; Dodson, E. J. Nature
1978, 275, 206. An excellent discussion of the turn-forming ability of
heterochiral sequences appears in ref 3c, p 22 ff.
(5) Alternative techniques used to stabilize turns may include formation
of disulfide bridges (cf. Quinn, T. P.; Tweedy, N. B.; Williams, R. W.;
Richardson, J. S.; Richardson, D. C. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 8747) or metal ligation (cf. Handel, T. M.; Williams, S. A.; DeGrado,
W. F. Science 1993, 261, 879). In extreme cases, a rigid scaffolding
resembling the environment of turns has been used to enforce a desired
conformation: e.g., Kim, K.; Germanas, J. P. J. Org. Chem. 1997, 62, 2847,
2853. Lombart, H. G.; Lubell, W. D. J. Org. Chem. 1996, 61, 9437.
(6) Struthers, M. D.; Cheng, R. P.; Imperiali, B. Science 1996, 271, 342.
(7) Ciufolini, M. A.; Xi, N. J. Org. Chem. 1997, 62, 2320.
(8) Luzopeptins: (a) Konishi, M.; Ohkuma, H.; Sakai, F.; Tsuno, T.;
Koshiyama, H.; Naito, T.; Kawaguchi, H. J. Am. Chem. Soc. 1981, 103,
1241. (b) Arnold, E.; Clardy, J. J. Am. Chem. Soc. 1981, 103, 1243 and
references therein. Quinoxapeptins: (c) Lingham, R. B.; Hsu, A. H. M.;
O’Brien, J. A.; Sigmund, J. M.; Sanchez, M.; Gagliardi, M. M.; Heimbuch,
B. K.; Genilloud, O.; Martin, I.; Diez, M. T.; Hirsch, C. F.; Zink, D. L.;
Liesch, J. M.; Koch, G. E.; Gartner, S. E.; Garrity, G. M.; Tsou, N. N.;
Salituro, G. M. J. Antibiot. 1996, 49, 253. We propose the convenient
descriptor “peptins” to refer to the entire class of these antibiotics.
S0002-7863(97)02990-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/14/1998

Conformational Rigidity in Dipeptides
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 81
Table 1. N-2-Serinyl Derivatives of 2,3,4,5-Tetrahydropyridazine-3-carboxylic Acids (Compounds 1) and N-2-Acetyl Analogues (Compounds
2 and 3) That Exist as Single Rotamers about the Tertiary Amide Structure (1H and 13C NMR Spectroscopy)a
a All compounds were observed in CDCl3 solutions at rt, except 1c, which was observed in CDCl3, CD3CN, (CD3)2CO, and (CD3)2SO solutions.
Different solvents had virtually no effect on the conformation of 1c.
prepared from 4 as shown in Scheme 1.9 The degree of
conformational rigidity observed for 1-3 is unusual, and it is
clearly produced by the PCA residue. Indeed, compounds
similar to 1-3, but displaying a proline or a pipecolinic acid in
place of PCA, exist as mixtures of rotamers about the peptide
bond (compounds 11-13, Table 2).10 Experimental (1H NMR)
and computational11 evidence indicates that 1-3 and 11-13
favor a conformation in which the proline/pipecolinic acid
COOR unit is essentially axial or pseudoaxial, while the N-acyl
carbonyl group points toward the ring atom bearing the COOR
unit and away from the imino linkage. This is described in
Tables 1 and 2 as conformation A, while conformation B,
wherein the N-acyl unit has rotated by approximately 180°, is
disfavored. Molecular mechanics (MM+ force field)11b and
semiempirical (PM3)12 calculations estimate the conformational
energy difference between the two limiting conformers A and
B (∆EAB) of compounds 11-13 to be generally less than 1 kcal/
mol, in line with experiment (rotamer ratio e4:1). In sharp
contrast, the ∆EAB between the two states of several molecules
of type 1-3 may be between about 3.5 (PM3)12 and 5.7 kcal/
mol (MM+), corresponding to a rotamer ratio of 370-15600:1
in favor of A.
Scheme 1.a
(9) Compound 4 was obtained by Schreiber ozonolysis of cyclopentene
to methyl 5-oxopentanoate (Claus, R. E.; Schreiber, S. L. Organic Syntheses;
Wiley: New York, 1990; Collect. Vol. VII, p 168) followed by ketalization,
hydrolysis, and coupling to the Evans auxiliary. Relevant references for
key steps: (step a) Gennari, C.; Colombo, L.; Bertolini, G. J. Am. Chem.
Soc. 1986, 108, 6394. Evans, D. A.; Britton, T. C.; Dorow, R. L.; Dellaria,
J. F. J. Am. Chem. Soc. 1986, 108, 6395. Trimble, L. A.; Vederas, J. C. J.
Am. Chem. Soc. 1986, 108, 6397. (Step b) Evans, D. A.; Britton, T. C.;
Dorow, R. L.; Dellaria, J. F. Tetrahedron 1988, 44, 5525. (Steps c and d)
see refs 7 and 10. (Steps g and h) Ishizuka, T.; Kunieda, T. Tetrahedron
Lett. 1987, 28, 4185.
(10) Cf. Ciufolini, M. A.; Xi, N. Tetrahedron Lett. 1995, 36, 6595.
(11) (a) All calculations were carried out with the Hyperchem 4.0
package, available from Hypercube, Inc., Ontario, Canada. (b) No parameters
exist for cyclic hydrazones of the type found in PCA in the MM+ method
implemented in this package. The program will automatically use ap-
proximate parameters in such cases; consequently, the results of MM+
calculations are of qualitative, rather than quantitative, significance. We
relied primarily on PM3 semiempirical methods, because this technique
seems to reproduce experimental conformational population ratios rather
well.
a Reagents and conditions: (a) THF, -78 °C, 65%; (b) MeOH, 0
°C, 80%; (c) MeOH, rt, 93%; (d) sym-collidine, CH2Cl2, 0 °C, 74%;
(e) 90% in H2O, rt, 96%; (f) MeCN, rt, 90%; (g) Et3N, cat. DMAP,
CH2Cl2, 0 °C, 99%; (h) MeOH, 88%; (i) 50% in CH2Cl2; (j) DCC,
70% i-j; (k) TFA, THF, rt, 99%.
(12) Repulsive forces are not as strong in PM3, a reparametrized form
of AM1, as they are in other semiempirical methods (Hyperchem literature
and manuals). Therefore the conformational energy differences calculated
by this method are likely to represent lower-end estimates.
Experimental evidence in support of the calculated confor-
mational preferences of N-acyl-PCAs may be found in work
by Blount.13 Specifically, the X-ray crystal structure of

82 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Xi et al.
Table 2. Rotamer Ratio (1H NMR, 25 °C) for Proline (11, 12) and Pipecolinic Acid (13) Analogs of 1b and 1da
a Rotamer A is always favored over B.
compound 14, a derivative of a diformyl terpenoid, reveals that
the 4-bromobenzoyl group connected to the tetrahydropyridazine
unit exists in a conformation of type A (the carbonyl unit points
away from the imino linkage). This observation is noteworthy,
in that compound 14 is not encumbered by a ring structure that
might perturb the innate conformational preferences of the
Figure 1. Overlay of the PM3-optimized structures of compounds 15
and 16. dPCA may form tighter turns than proline.
N-acyltetrahydropyridazine.14
The energy barrier for rotation about the tertiary amide C-N
bond serves perhaps as a more useful parameter to gauge the
conformational rigidity of N-acyl-PCAs, and since only one
conformer is readily detected by NMR, rotation about the C-N
bond in 1-3 may be either very fast or extremely slow. It seems
unlikely that fast rotation is operative here, since the bond in
question is part of an amide, and indeed, the VTNMR spectrum
of 1c in (CD3)2CO shows no evidence for two rotamers down
to -89 °C. Indeed, little change is observed in the NMR
spectrum at -89 °C relative to room temperature, except for
the expected downfield shifts of NH and OH resonances. This
suggests that the spectral properties of 1c and related structures
are a consequence of hindered rotation. In an effort to determine
the magnitude of the rotational barrier, a VTNMR study of 1c
was conducted at temperatures up to 55 °C in CDCl3, up to 85
°C in CD3CN, up to 93 °C in C6D5CD3, and up to 107 °C in
DMSO-d6. Again, no significant changes other than expected
shifts of NH and OH resonances were apparent in this
temperature range. Similarly, VTNMR studies of 2 up to 120
°C in DMSO-d6 revealed no evidence of thermally induced
conformational motion, suggesting that the activation barrier
for internal rotation of PCA dipeptides is very high.
at 79 °C. At 86 °C or higher, each methyl group appeared as
a doublet, J ) 0.18 ( 0.03 Hz. Between 86 and 93 °C, the
total half-height line width of each methyl resonance was 0.43
Hz. At temperatures between 100 and 121 °C, the total half-
height line width narrowed to 0.40 Hz. Selective irradiation of
the C-3 methine proton at δ 5.07 induced collapse of each pair
of methyl signals into a singlet, thus demonstrating 5JHH between
the methine H and the methyl hydrogens. Such five-bond J
couplings of ca. 0.2 Hz are well documented in very similar
molecular environments.15 We presume that these J couplings
became visible at higher temperatures due to the diminished
viscosity of the DMSO-d6 solvent and the attendant improve-
ment in spectral resolution. Selective irradiation of the C-6
imino proton at δ 6.93 did not cause collapse of either pair of
methyl signals, the only detectable effects being the expected
upfield Bloch-Siegert shifts16 of 0.33 Hz for the ester methyl
group and just 0.20 Hz for the more upfield N-acetyl methyl
group. As expected, irradiation of the C-3 methine proton at δ
5.07 resulted in larger upfield Bloch-Siegert shifts of 0.85 Hz
for the ester Me group and 0.39 Hz for the N-acetyl Me group.
The above results suggest that PCAs might act as confor-
mationally rigid analogues of proline, and as such they might
force peptide turns. Figure 1 shows the overlays of the PM3-
optimized structures of dipeptide 15, a computationally better
The NMR experiments on 2 were carried out at a digital
resolution of 0.0305 Hz. The spectral resolution increased
steadily with increasing temperature. The half-height line width
of the central line of the DMSO-h-d5 quintet steadily narrowed
from 1.30 Hz at room temperature (rt) to 0.40 Hz at >100 °C.
The half-height line width of each of the methyl signals (methyl
ester and acetyl) of 2 narrowed from 0.67 Hz at rt to 0.46 Hz
(15) (a) Davies, D. B.; Abu Khaled, M.; Urry, D. W. J. Chem. Soc.,
Perkin Trans. 2 1977, 1294. (b) Hayamizu, K.; Yamamoto, O. J. Mol.
Spectrosc. 1967, 22, 119. (c) Riggs, N. V.; Verma, S. M. Tetrahedron Lett.
1968, 19, 3767.
(13) Blount, J. F.; Dunlop, R. W.; Erickson, K. L.; Wells, R. J. Aust. J.
Chem. 1982, 35, 5, 145.
(14) We are grateful to one of the reviewers for kindly bringing to our
attention the work cited in refs 13, 17, and 20.
(16) (a) Mersh, J. D.; Sanders, J. K. M. J. Magn. Reson. 1982, 50, 289.
(b) Hosur, R. V.; Ernst, R. R.; Wu¨thrich, K. J. Magn. Reson. 1983, 54,
142. (c) Freeman, R. A Handbook of Nuclear Magnetic Resonance; John
Wiley & Sons: New York, 1988; pp 14-16.

Conformational Rigidity in Dipeptides
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 83
Table 3. Experimental19 and Calculated (PM3) Rotamer Ratios for N-Acyl-2-pyrrolideine 18 and N-Acyl-2-piperideine 19b
a Estimated (25 °C) from an expression of the type ∆∆Hf ≈ ∆G° ) -RT ln(K). b The presence of a π system in the ring is insufficient to induce
significant bias in favor of conformer A.
Table 4. Conformational Energy Differences (PM3) in
tractable congener of molecule 1b, and its proline analogue 16.
N-2-Acyl-PCAs and Analogs
These compounds display the popular D-L amino acid pair that
promotes turn formation.4,6 It is apparent that a great deal of
shape similarity exists between proline and PCA peptides;
furthermore, PCAs seem to favor a tighter turn than proline.
Therefore, it seems quite plausible that N-acyl-PCAs could be
utilized to engineer peptides with enforced turns. Some
evidence in support of this surmise may be gathered from the
work of Bock.14,17 The X-ray crystal structure of oxytocin
antagonist 17 reveals turn motifs at the level of the PCA-type
a Estimated (25 °C) from an expression of the type ∆∆Hf ≈ ∆G° )
-RT ln(K).
Intramolecular hydrogen bonding interactions, or steric ef-
fects, are also unlikely to be primary determinants of the strong
subunits, which interestingly are each part of a heterochiral
sequence and exist in a conformation of type A. A motif similar
to a â-turn, though not a â-turn proper, is also apparent at the
level of the proline. Similar conclusions may be drawn also
from the X-ray crystal structure of luzopeptin A.8b Whereas
the turn feature present in this antibiotic is not produced by the
N-2-(D-serinyl)-PCA amide (the serine N atom carries a quinal-
doyl unit), but it rather occurs in proximity of the depsi bond
between the D-serine and the carboxy group of L-N-methyl-3-
hydroxyvaline,18 it is apparent that the serine N atom and the
PCA amide group are arranged in a conformation that would
promote turn formation. Moreover, the constraints imposed by
the macrocyclic ring cause only slight deviation from the
computed (PM3) global minimum for the serine-PCA subunits.
Proposed Origin of the Conformational Properties of
PCAs. It is unlikely that the presence of a π system in the
PCA ring suffices, per se, to induce the observed bias in favor
of conformer A. This is apparent from the 1H NMR spectra of
enamides 18 and 19: while the π bond does induce a preference
for conformers 18A and 19A over their corresponding B types,
this preference is a modest 1.9:1 and 2.3:1, respectively19 (Table
3). Calculations (PM3) reproduce these experimental values
rather well.
conformational preferences of N-2-acyl-PCAs. All compounds
of type 1-3 display identical conformational behavior, regard-
less of potential intramolecular H bonding interactions (not
possible with 1d, 1f, 1g, 1l, and 2) and regardless of the steric
bulk of the N-2-acyl groups present on the PCA. Furthermore,
PM3 calculations on compound 2, on its N-2-formyl analogue
20, and on structure 21, wherein the ester group is missing
altogether, suggest that conformational energy differences
between the A and B states (∆∆Hf) are large (ca. 3.8 kcal/
mol)12 and practically identical in all three molecules (Table
4). Thus, the steric demand of the N-2-acyl group or the
presence of the ester unit seems to be largely an insignificant
parameter. It is noteworthy, however, that the preference for
conformer A becomes even stronger in PCA amides (cf. 22,
Table 4), which correspond to the form of PCA found in peptin
antibiotics. Conversely, the preference for conformation A is
calculated to be significantly attenuated in PCA N-2-carbamates
(cf. 23).
These computational results suggest that an electrostatic
phenomenon may be at the root of the conformational behavior
of N-acyl-PCAs. We propose that the primary interaction that
determines conformational preferences in such structures is the
electrostatic repulsion between the carbonyl oxygen and the
imino nitrogen atoms in conformers of type B and the attendant
dipolar effects. Further details of PM3 calculations on com-
pound 2 appear in Figure 2. We stress that the following data
probably are useful only in a qualitative, or at most a
semiquantitative, sense.12 It seems that a redistribution of charge
occurs in the molecule in going from conformer 2A to 2B.
(17) Bock, M. G.; DiPardo, R. M.; Williams, P. D.; Pettibone, D. J.;
Clineschmidt, B. V.; Ball, R. G.; Veber, D. F.; Freidiger, R. M. J. Med.
Chem. 1990, 33, 2321.
(18) For a discussion of the ability of depsipeptides to establish turn
motifs, see: Haque, T. S.; Little, J. C.; Gellman, S. H. J. Am. Chem. Soc.
1996, 118, 6975.
(19) Cf. Stille, J. K.; Becker, Y. J. Org. Chem. 1980, 45, 2139.

84 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Xi et al.
Figure 2. Electrostatic repulsion between the carbonyl O and imino N atoms as the proposed origin of the conformational preference in N-2-acyl
derivatives of PCAs (PM3 calculations).
Specifically, the density of negative charge on N-1 is halved in
conformer 2B (the more energetic one) relative to conformer
2A. The negative charge density on O-8 is also slightly less in
2B than it is in 2A, whereas the negative charge density on the
imino and carbonyl carbons, C-6 and C-7, is very similar in
the two conformers. Seemingly, electrostatic repulsion between
the N and O atoms pushes electronic density away from N-1 in
conformer 2B, resulting in polarization of the imino linkage
toward the carbon atom. However, N is more electronegative
than C; therefore, this electronic redistribution is probably
unfavorable. Likewise, bond polarization in the amide carbonyl
is reduced considerably in 2B, signifying that electrostatic
interactions within the molecule must work against the elec-
tronegativity of the O atom. Such effects would be expected
to be less pronounced in carbamate analogue 23, because an O
atom is forced in close proximity to the imino N in either
limiting conformer. This is indeed the case: the energy
difference between conformers A and B is now estimated to be
1.3 kcal/mol. Most of this energy difference may be due to
repolarization of the carbamate carbonyl, but notice that no
charge redistribution occurs within the imino linkage when the
molecule switches between the two conformations.14,20
compound 3, Bock and co-workers14,17 observed that the solution
conformation (NMR) of substances related to 17 differs depend-
ing on whether the PCA units are present as such or in reduced
form. Presumably, the constraints imposed upon these mol-
ecules by a relatively small 18-membered ring suffice to promote
a conformational change upon removal of the important control
element represented by the imino subunit. It is also noteworthy
that biological activity was greatly affected by the precise
oxidation state of the PCA units (cf. 17, X, Y ) H or π bond).
Finally, PCA-like behavior is seen in the interesting proline
analogue 24, a lower ring homologue of 2 (Figure 3).21 In this
case, though, charge redistribution effects are less pronounced
than in 2, probably because atoms N-1 and O-7 are not as close
in conformation 24B as the corresponding N-1 and O-8 are in
conformer 2B. Indeed, the calculated energy difference be-
tween the two conformational states of 24 is reduced to 2.0
kcal/mol.
In summary, N-2-acyl-PCAs display an elevated degree of
conformational rigidity that strongly favors rotamer A (Table
1 and Figure 2). This preference is probably due to electrostatic
repulsion between the ring imino N atom and the carbonyl
oxygen on the PCA N-2-acyl unit. Calculations also suggest
that N-2-acyl PCAs are similar in shape to N-acylprolines. As
a result, it is likely that PCAs may be utilized to replace proline
within peptides whenever and wherever a turn motif is desired.
Further ramifications of these ideas are currently under inves-
tigation, and results in this area will be the subject of future
papers.
A consequence of the above hypotheses is that reduction of
the imino linkage in PCA peptides would be expected to
diminish the conformational bias toward conformers of type A.
Whereas this is not readily apparent from the spectra of our
(20) Results of more sophisticated ab initio calculations concerning charge
distribution and rotational barriers in various carbonyl compounds may be
found in the following: (a) Wiberg, K. B.; Laidig, K. E. J. Am. Chem.
Soc. 1987, 109, 5935. (b) Laidig, K. E.; Cameron, L. M. Can. J. Chem.
1993, 71, 872. These methods may permit calculation of rotational barriers
in PCA peptides; however, we have not yet carried out such studies.
(21) After the submission of this paper the preparation of the remarkable
ring system 24 was described: Mish, M. R.; Guerra, F. M.; Carreira, E. M.
J. Am. Chem. Soc. 1997, 119, 8379.

Conformational Rigidity in Dipeptides
J. Am. Chem. Soc., Vol. 120, No. 1, 1998 85
Figure 3. Summary of computational data for proline analogue 24 (PM3 calculations).
m), 3.70 (3H, s), 3.62 (1H, br), 1.69-1.84 (4H, m), 1.40 (9H, s). 13C:
δ 173.5, 156.2, 103.7, 80.4, 64.8, 62.7, 51.9, 29.6, 28.1, 24.3. IR:
3316, 1732 cm-1. MS (EI): m/z 304 (M+ + H) 248, 232, 204, 57
(100). HRMS (EI): calcd for C13H24N2O6 (M+ + H): 304.1634, obsd
304.1628.
Experimental Section22
Hydrazine 5. A solution of 49 (6.0 g, 18.8 mmol, 1 equiv) in THF
(40 mL) was added slowly to a cold (-78 °C) solution of LDA [22.6
mmol, 1.2 equiv, from i-Pr2NH (4.0 mL, 1.5 equiv) and BuLi in hexane
(2.5 M, 9.0 mL, 22.6 mmol, 1.2 equiv); 1,10-phenanthroline indicator].
The mixture was stirred at -78 °C for 30 min, and then (CbzsNd)2
(11.2 g, 37.6 mmol, 2.0 equiv) in THF (40 mL) was added. After 2
min, the reaction was quenched (glacial AcOH, 5 mL) and partitioned
between CH2Cl2 and pH 7 phosphate buffer. The combined extracts
were washed (saturated aqueous NaHCO3), dried (Na2SO4), and
concentrated. Chromatography of the residue (40% EtOAc/hexane)
yielded 7.5 g (65%) of 5 as a white waxy solid. The NMR spectra of
this compound were broad at rt (slow conformational motion of tertiary
Protected Dipeptide 9. A solution of acid chloride 8 (2.5 g, 13.2
mmol, 1.5 equiv) in CH2Cl2 (15 mL) was slowly added to a cold (0
°C) solution of 7 (2.7 g, 8.8 mmol, 1.0 equiv) and collidine (3.5 mL,
26.4 mmol, 3 equiv) in CH2Cl2 (10 mL) under Ar. The mixture was
stirred at 0 °C for 20 min; then it was directly applied to a short silica
gel column and eluted with EtOAc. The filtrate, containing 9 and
collidine, was concentrated, and the residue was chromatographed (30
f 60% EtOAc/hexane) to yield 3.0 g (74%) of 9 as a waxy solid.
[R]25 ) +17° (0.055 g/mL). 1H: δ 7.50 (1H, br), 5.2-5.3 (2H, m),
D
carbamate). [R]25 ) +35.1° (c ) 0.073 g/mL). 1H (C6D6, 70 °C):
D
4.83 (1H, br t, J ) 3.8), 4.3-4.4 (2H, m), 3.8-4.0 (4H, m), 3.71 (3H,
s), 2.47 (3H, m), 1.8-2.0 (2H, m), 1.6-1.8 (2H, m), 1.46 (9H, s). 13C:
δ 172.1, 171.2, 170.1, 154.9, 152.9, 103.4, 94.9, 82.6, 64.8, 58.2, 52.5,
52.4, 30.0, 27.8, 27.5, 22.8, 22.6. IR (film): 3297, 1792, 1740, 1699
cm-1. MS (EI): m/z 459 (M+), 458, 403, 386, 359, 57 (100). HRMS
(EI): calcd for C19H29N3O10 459.1853, obsd 459.1858.
δ 6.9-7.3 (15H, m), 6.2-6.3 (1H, m), 5.13 (2H, s), 5.16 (1H, d, J )
12.4), 5.06 (1H, d, J ) 12.4), 4.9-5.0 (1H, m), 4.21 (1H, m), 3.3-3.7
(4H, m), 3.06 (1H, dd, J1 ) 2.9, J2 ) 13.67), 2.08-2.46 (6H, m). 13
C
(C6D6, 70 °C): δ 173.7, 157.4, 156.8, 153.3, 137.3, 137.1, 136.4, 130.0,
129.5, 129.4, 129.0, 128.8, 128.5, 128.4, 128.0, 127.7, 127.5, 104.9,
69.5, 69.0, 68.1, 66.9, 65.3, 62.2, 56.1, 41.8, 38.0, 31.4, 24.4. IR: 3316,
1787, 1759, 1721 cm1-1. MS (FAB): m/z 618 (M+ + 1), 91 (100).
HRMS (EI): calcd for C33H35N3O9 617.2373, obsd 617.2373.
Compound 1d. A solution of 9 (2.4 g, 5.2 mmol) in 25 mL of
90% aqueous TFA was stirred at rt for 30 min; then the solvent was
evaporated, and the residue was partitioned between CH2Cl2 and
saturated aqueous NaHCO3 (extra solid NaHCO3 was added to
neutralize all the TFA). The aqueous layer was extracted with more
CH2Cl2; the combined extracts were washed (brine), dried (Na2SO4),
and concentrated to afford 1.6 g (100%) of 1d. Recrystallization from
30% EtOAc/hexanes gave white prisms (1.5 g, 96%), mp ) 152-153
Ester 6. Methanolic MeOMgBr was prepared by dropwise addition
of 8.9 mL of MeMgBr (26.8 mmol of 3 M solution in Et2O, 1.5 equiv)
into cold (0 °C) MeOH (50 mL). A solution of 5 (11.0 g, 17.8 mmol,
1.0 equiv) in CH2Cl2 (50 mL) was slowly added, and the mixture was
stirred at 0 °C for 30 min before being quenched with saturated aqueous
NH4Cl (30 mL). The organic phase was separated, and the aqueous
layer was extracted with CH2Cl2. The combined organic phases were
washed (brine), dried (Na2SO4), and concentrated. Chromatography
of the residue (40% f 70% EtOAc/hexane) retrieved the chiral auxiliary
°C. [R]25 ) +37.1° (0.062 g/mL). 1H: δ 6.92 (1H, m), 5.68 (1H,
D
dd, J1 ) 3.91, J2 ) 9.52), 5.13 (1H, m), 4.56 (1H, t, J ) 9.52), 4.21
(1H, dd, J1 ) 3.91, J2 ) 9.52), 3.71 (3H, s), 2.52 (3H, s), 1.78-2.45
(4H, m). 13C: δ 169.6, 169.3, 168.3, 153.3, 144.0, 65.0, 54.8, 52.8,
51.2, 23.1, 20.4, 18.1. IR (KBr): 3021, 1782, 1748, 1708, 1689, 1641
cm-1. MS (EI): m/z 297 (M+), 255, 237, 231, 83 (100). HRMS (EI):
calcd for C12H15N3O6 297.0961, obsd 297.0962.
and furnished the desired 6 (6.7 g, 80%) as a white waxy solid. [R]25
D
) -7.4° (0.084 g/mL). The NMR spectra of this compound were broad
at rt (slow conformational motion of tertiary carbamate). 1H (50 °C):
δ 7.28 (10H, br), 6.93 (1H, br), 5.14 (2H, s), 5.12 (2H, s), 4.80-4.96
(2H, m), 3.70-3.90 (4H, m), 3.62 (3H, s), 1.70-2.10 (4H, m). 13C
(50 °C): δ 171.4, 156.2, 155.9, 135.7, 135.6, 128.2, 128.0, 127.9, 127.8,
127.5, 103.8, 68.2, 67.4, 65.6, 60.8, 51.9, 30.0, 22.8. IR: 3301, 1740,
1716 cm-1. MS (CI): m/z 473 (M+ + 1), 429, 395, 367 (100). HRMS
(EI): calcd for C24H28N2O8 472.1845, obsd 472.1849.
Compound 1e. The preparation of this compound is fully detailed
in the Supporting Information. Colorless oil. [R]25D ) -9.54° (0.022
g/mL). 1H: δ 6.95 (1H, t, J ) 2.07), 5.60 (1H, br), 5.17 (1H, m), 4.99
(1H, dd, J1 ) 9.3, J2 ) 6.1), 4.72 (1H, t, J ) 9.3), 4.46 (1H, dd, J1 )
9.3, J2 ) 6.1), 3.75 (3H, s), 1.87-2.45 (4H, m). 13C: δ 170.0, 169.4,
158.6, 143.9, 67.0, 53.8, 52.8, 51.2, 20.4, 18.2. IR: 3289, 1754, 1702,
1639 cm-1. MS (EI): m/z 255 (M+), 211, 196, 170, 142, 83 (100).
HRMS (EI): calcd for C10H13N3O5 255.0855, obsd 255.0855.
Monoprotected Hydrazine 7. A mixture of 6 (6.0 g, 12.7 mmol,
1.0 equiv), BOC2O (3.3 g, 15.3 mmol, 1.2 equiv), 10% Pd/C catalyst
(0.6 g, 10 wt % 6), and MeOH (200 mL) was stirred under an H2
atmosphere (balloon) until all the starting material was consumed (TLC,
ca. 12 h). The mixture was filtered (Celite), the filtrate was concen-
trated, and the residue was chromatographed (40% EtOAc/hexane) to
Compound 1f. The preparation of this compound is fully detailed
in the Supporting Information. White crystals, mp ) 166-167 °C.
[R]25 ) +32.3° (0.048 g/mL). 1H: δ 6.87 (1H, br d, J ) 4.1), 5.45
D
yield 7 (3.6 g, 93%) as a light yellow oil. [R]25 ) -15.0° (0.306
D
(1H, dd, J1 ) 9.5, J2 ) 3.9), 5.04 (1H, m), 4.45 (1H, t, J ) 9.5), 4.06
(1H, dd, J1 ) 9.5, J2 ) 3.9), 3.64 (3H, s), 1.77-2.33 (4H, m), 1.37
(9H, s). 13C: δ 169.3, 168.5, 152.0, 148.2, 144.1, 83.3, 64.3, 55.8,
52.4, 51.1, 27.2, 20.1, 18.0. IR (KBr): 3039, 1819, 1746, 1724, 1691,
1634 cm-1. MS (EI): m/z 356 (M+ + 1), 340, 311, 282, 255, 83, 57
(100). HRMS (EI): calcd for C15H21N3O7 355.1379, obsd 355.1378.
g/mL). 1H: δ 6.32 (1H, br), 4.86 (1H, br t, J ) 4.0), 3.76-3.95 (4H,
(22) Melting points (mp) are uncorrected. Unless otherwise stated (a)
1H (250 MHz) and 13C (62.9 MHz) NMR spectra were recorded at 25 °C
in CDCl3, (b) FTIR spectra were obtained from films on NaCl plates, and
(c) optical rotations were measured in CH2Cl2.

86 J. Am. Chem. Soc., Vol. 120, No. 1, 1998
Xi et al.
Compound 1b. The preparation of this compound is fully detailed
of ABX system, br dd, J1 ) 6.0, J2 ) 1.9), 3.77 (3H, s), 3.05 (1H, br,
B part of ABX, J1 ) 13.9 Hz, J2 ) 2.0 Hz), 2.75 (1H, br, ddd, A part
of ABX, J1 ) J2 ) 13.9, J3 ) 3.0), 2.19 (3H, s), 1.8-2.3 (2H, m),
1.3-1.6 (2H, m). 13C (DEPT): δ 52.4, 50.6, 47.1 (CH2), 25.6 (CH2),
21.9 (CH2), 20.7.
in the Supporting Information. Colorless oil. [R]25 ) -45° (0.110
D
g/mL). 1H: δ 6.93 (1H, d, J ) 3.9), 5.76 (1H, d, J ) 6.8), 5.24 (1H,
X part of ABX system, m), 5.12 (1H, m), 3.84 (2H, AB part of ABX
system), 3.69 (3H, s), 2.97 (1H, br), 1.81-2.38 (4H, m), 1.40 (9H, s).
13C: δ 171.1, 169.9, 143.1, 79.7, 64.4, 54.1, 52.6, 51.3, 28.2, 20.3,
18.6. IR: 3434, 1748, 1713, 1691, 1631 cm-1. MS (EI): m/z 330
(M+ + 1), 311, 299, 279, 256, 243, 83 (100). HRMS (EI): calcd for
C14H24N3O6 (M+ + H): 330.1665, obsd 330.1668.
Compound 1c. The preparation of this compound is fully detailed
in the Supporting Information. Pale yellow oil. 1H: δ 9.29 (1H, d, J
) 7.3), 8.29 (2H, br, s), 8.19 (1H, d, J ) 8.5), 7.81 (1H, d, J ) 8.5),
7.75 (1H, t, J ) 8.5), 7.59 (1H, t, J ) 7.5), 7.03 (1H, m), 5.80 (1H, X
part of ABX system, m), 5.28 (1H, br, d, J ) 5.5), 4.18 (1H, dd, B
part of ABX, J ) 11.2, 3.5), 4.04 (1H, dd, A part of ABX, J ) 11.2,
4.9), 3.72 (3H, s), 1.9-2.5 (4H, m). 13C: δ 170.6, 169.9, 154.9, 149.3,
146.4, 143.3, 137.4, 130.1, 129.9, 129.3, 128.0, 127.6, 118.8, 64.7,
54.1, 52.9, 51.5, 20.5, 18.7.
Acknowledgment. We gratefully acknowledge the NIH
(Grant CA-55268), the NSF (Grant CHE 95-26183), and the
Robert A. Welch Foundation (Grant C-1007) for their support
of our research program. M.A.C. is a Fellow of the Alfred P.
Sloan Foundation, 1994-1998.
Supporting Information Available: Hardcopy spectra of
compounds 1b, 1c, 2, and 3, calculated vs experimental energy
differences for compounds 18 and 19, tabulation of calculated
energy differences between conformers A and B of some PCA
derivatives/analogues, and detailed experimental procedures for
the preparation of compounds 1b, 1c, 1f, 1g, 2, and 3 (7 pages).
See any current masthead page for ordering and Internet access
instructions.
Compound 2. The preparation of this compound is fully detailed
in the Supporting Information. Pale yellow oil. 1H: δ 6.83 (1H, m),
5.20 (1H, m), 3.67 (3H, s), 2.30 (3H, s), 1.78-2.31 (4H, m). 13C: δ
172.3, 170.3, 141.4, 52.5, 50.5, 20.9, 20.2, 18.6.
Compound 3. The preparation of this compound is fully detailed
in the Supporting Information. Colorless oil. 1H: δ 5.32 (1H, X part
JA9729903