An Artificial Antiparallel β-Sheet Containing a New Peptidomimetic Template

Full text of DOI:10.1021/jo971431b

7906
J . Org. Chem. 1997, 62, 7906-7907
Ta ble 1. 1H NMR Ch em ica l Sh ifts of th e NH P r oton s of
An Ar tificia l An tip a r a llel â-Sh eet
Con ta in in g a New P ep tid om im etic
Tem p la te
2, a n d 7-9a
Ha
Hb
Hc
Hd
He
Hf
2
7
8
9
9.93
6.31
10.97
10.85
10.45
8.78
4.82
8.29
5.79
Eric M. Smith, Darren L. Holmes, A. J . Shaka,† and
J ames S. Nowick*
4.43
6.37
5.82
6.71
6.00
Department of Chemistry, University of California, Irvine,
Irvine, California 92697-2025
a
Spectra were recorded at 295 K in 1.0 mM CDCl3 solution.
Received August 1, 1997X
During the past few years, we have been developing
compounds that mimic the structures and hydrogen-
bonding patterns of protein â-sheets (artificial â-sheets).1
In 1996, we reported studies of artificial â-sheet 1.2 This
compound comprises a 5-amino-2-methoxybenzamide
template that is linked to a phenylalanylleucine dipeptide
by a hydrogen-bonded 1,2-diaminoethane diurea turn
unit. The 5-amino-2-methoxybenzamide template forms
only one hydrogen bond to the dipeptide and is too short
to hydrogen bond to the carbonyl group of the leucine
residue.3 Here, we report artificial â-sheet 2, which
contains a 5-amino-2-methoxybenzoic hydrazide template
that is of sufficient length to form two hydrogen bonds
to the attached dipeptide.
(Table 1).7 In contrast, protons Hb and Hd exhibit little
(0.12 and 0.39 ppm) downfield shifting. Proton Hf of 2
is shifted upfield by 0.92 ppm relative to that of dipeptide
8, because 8 adopts a â-turn conformation.2,8 In this
conformation, the methylamide NH group is hydrogen
bonded to the urea carbonyl group and is shifted down-
field. For this reason, monopeptide 9 serves as a better
control for Hf.
Artificial â-sheet 2 was synthesized efficiently and in
high yield from diamine 34 (eq 1). Reaction of diamine 3
with isocyanate 45 afforded urea 5. Treatment of urea 5
with phenylalanylleucine methyl ester isocyanate6 gener-
ated diurea 6. Aminolysis with methylamine converted
the methyl ester group of 6 to a methylamide group,
affording artificial â-sheet 2 in 81% overall yield. Arti-
ficial â-sheet 2 was also prepared by solid-phase synthesis
on Merrifield resin in 60% overall yield.5
1H NMR spectroscopic studies establish that 2 is
intramolecularly hydrogen bonded and that it adopts an
antiparallel â-sheet conformation in CDCl3 solution. 1H
NMR chemical shift studies indicate that the appropriate
NH groups are hydrogen bonded. Thus, Ha, Hc, and He
in 2 are shifted downfield by 3.62, 1.67, and 1.92 ppm
relative to the corresponding protons in controls 7 and 8
1H NMR nuclear Overhauser effect (NOE) studies
provide compelling evidence that 2 adopts a â-sheet
conformation. Because the molecular weight of 2 is
moderately high (784), its NOEs are small. For this
reason, we performed these studies in the rotating frame,
using the transverse-ROESY (Tr-ROESY) method.9
A
* To whom correspondence regarding synthetic and structural
studies should be addressed. E-mail address: jsnowick@uci.edu.
† To whom correspondence regarding experimental NMR procedures
should be addressed. E-mail address: ajshaka@uci.edu.
(1) Nowick, J . S.; Smith, E. M.; Pairish, M. Chem. Soc. Rev. 1996,
25, 401.
(2) Nowick, J . S.; Holmes, D. L.; Mackin, G.; Noronha, G.; Shaka,
A. J .; Smith, E. M. J . Am. Chem. Soc. 1996, 118, 2764.
(3) We have also reported a dimeric version of the 5-amino-2-
methoxybenzamide template that is longer than a dipeptide and forms
three hydrogen bonds to a tripeptide: Nowick, J . S.; Pairish, M.; Lee,
I. Q.; Holmes, D. L.; Ziller, J . W. J . Am. Chem. Soc. 1997, 119, 5413.
(4) Nowick, J . S.; Abdi, M.; Bellamo, K. A.; Love, J . A.; Martinez, E.
J .; Noronha, G.; Smith, E. M.; Ziller, J . W. J . Am. Chem. Soc. 1995,
117, 89.
one-dimensional spectrum was first recorded, and 32
distinct resonances were identified and designated 1-32
in order of increasing chemical shift.10 The resonances
were then assigned by a combination of one-dimensional
and two-dimensional (PFG COSY and Tr-ROESY) meth-
ods. These assignments are shown in Figure 1. Separate
(7) These chemical shift studies were performed at 1.0 mM. At this
concentration, negligible self-association occurs, and the observed
chemical shifts are reflective of the unassociated compounds.
(8) Nowick, J . S.; Smith, E. M.; Noronha, G. J . Org. Chem. 1995,
60, 7386.
(9) (a) Hwang, T. L.; Shaka, A. J . J . Am. Chem. Soc. 1992, 114, 3157.
(b) Hwang, T. L.; Shaka, A. J . J . Magn. Reson. Series B 1993, 102,
155.
(5) Holmes, D. L.; Smith, E. M.; Nowick, J . S. J . Am. Chem. Soc.
1997, 119, 7665.
(6) Nowick, J . S.; Holmes, D. L.; Noronha, G.; Smith, E. M.; Nguyen,
T. M.; Huang, S.-L. J . Org. Chem. 1996, 61, 3929.
S0022-3263(97)01431-X CCC: $14.00 © 1997 American Chemical Society

Communications
J . Org. Chem., Vol. 62, No. 23, 1997 7907
and the isobutyryl methyl groups of the hydrazide (3 and
4). One weak cross-peak between the phenylalanine NH
proton (19) and the proton at the 6-position of the
aromatic ring (29), which is not consistent with the model
shown in Figure 1, may be an artifact resulting from
coupling between 19 and 20.9b ROEs between the upper
urea proton (30) and the 1,2-diaminoethane backbone
protons (13, 15, and 16) provide evidence that the 1,2-
diaminoethane diurea forms a turn structure. The
presence of moderate-strong ROEs between 30 and at
least three of the backbone protons suggests that the 1,2-
diaminoethane diurea turn comprises multiple conforma-
tions (e.g., two diastereomeric anti conformers of the 1,2-
diaminoethane backbone).4,12
Strong ROEs between the R and NH protons of
adjacent residues (20 and 27, 18 and 21), and large (8.4
3
and 9.2 Hz) J HNR coupling constants, provide evidence
for a â-strand conformation in the phenylalanylleucine
peptide strand.13 Weak-moderate ROEs between the
phenylalanine and leucine NH groups (19 and 27), and
between the leucine NH and leucine methylamide NH
groups (27 and 21), suggest that non-â-strand conformers
may also be present.
In conclusion, these studies show that the 5-amino-2-
methoxybenzoic hydrazide template forms a hydrogen-
bonded antiparallel â-sheet with the phenylalanylleucine
dipeptide in artificial â-sheet 2. Artificial â-sheet 2 is
similar in structure to systems developed by Kemp et al.14
and by Michne and Schroeder.15 These systems also
contain templates that mimic peptide â-strands; however,
the templates are tetracyclic and bicyclic aromatic mol-
ecules. The simplicity of the 5-amino-2-methoxybenzoic
hydrazide template renders it an attractive alternative
to these polycyclic aromatic templates.
F igu r e 1. Model of artificial â-sheet 2 illustrating interstrand
ROEs. The model was generated using MacroModel V5.5 with
the AMBER* force field. The starting geometry (before
minimization) was chosen to reflect the preferred (anti)
conformation of the 1,2-diaminoethane diurea backbone.4,12
The starting conformation of the leucine side-chain was chosen
to reflect measured coupling constants and NOEs; that of the
phenylalanine is largely arbitrary.
regions of some resonances (14 and 14′, 22 and 22′) were
identified through the two-dimensional NMR studies.
The Tr-ROESY studies show long-range ROEs between
the upper (peptidomimetic) and lower (peptide) strands
that are indicative of antiparallel â-sheet structure.11
Most notably, the proton at the 6-position of the aromatic
ring (29) exhibits ROEs to the phenylalanine R-proton
(20) and the leucine side-chain (1, 5, and 7). These ROEs
are shown as arrows in Figure 1. Also noteworthy are
ROEs between the leucine methylamide methyl group (9)
Ack n ow led gm en t. This work was supported by the
National Institutes of Health Grant GM-49076 and
National Science Foundation Grants CHE-9553262 and
CHE-9625674. D.L.H. and E.M.S. thank the National
Institute on Aging for support in the form of a training
grant (National Research Service Award AG00096-12).
J .S.N. thanks the following agencies for support in the
form of awards: the Camille and Henry Dreyfus Foun-
dation (Teacher-Scholar Award), the National Science
Foundation (Presidential Faculty Fellowship), and the
Alfred P. Sloan Foundation (Alfred P. Sloan Research
Fellowship).
(10) 1H NMR of 2 (25 mM in CDCl3, 298 K): 0.66 (d, J ) 6.4 Hz, 3
H, 1), 0.79 (d, J ) 6.3 Hz, 3 H, 2), 1.24 (d, J ) 6.9 Hz, 3 H, 3), 1.27 (d,
J ) 6.9 Hz, 3 H, 4), 1.39 (ddd, J ) 13.3, 9.3, 5.4 Hz, 1 H, 5), 1.48 (ddd,
J ) 13.8, 8.0, 6.2 Hz, 1 H, 6), 1.56-1.48 (m, 1 H, 7), 2.68-2.65 (m, 2
H, 8), 2.68 (d, J ) 4.8 Hz, 3 H, 9), 2.81 (septet, J ) 6.9 Hz, 1 H, 10),
2.85 (dd ABX pattern, J AB ) 13.8 Hz, J AX ) 8.7 Hz, 1 H, 11), 3.00 (dd
ABX pattern, J AB ) 13.8 Hz, J BX ) 7.5 Hz, 1 H, 12), 3.48-3.41 (m, 1
H, 13), 3.56-3.45 (m, 2 H, 14 and 14′), 3.69-3.61 (m, 2 H, 15), 3.92-
3.86 (m, 1 H, 16), 4.05 (s, 3 H, 17), 4.44 (td, J ) 9.2, 5.7 Hz, 1 H, 18),
4.82 (d, J ) 8.4 Hz, 1 H, 19), 4.94 (q, J ) 8.2 Hz, 1 H, 20), 5.80 (br q,
J ) 4.7 Hz, 1 H, 21), 6.96-6.94 (m, 2 H, 22), 6.96 (d, J ) 9.0 Hz, 1 H,
22′), 7.15 (appar d, J ) 7.0 Hz, 2 H, 23), 7.29-7.23 (m, 3 H, 24), 7.36
(appar t, J ) 7.2 Hz, 1 H, 25), 7.40 (appar t, J ) 7.1 Hz, 2 H, 26), 8.28
(d, J ) 9.2 Hz, 1 H, 27), 8.39 (dd, J ) 9.0, 2.8 Hz, 1 H, 28), 8.62 (d, J
) 2.8 Hz, 1 H, 29), 9.92 (s, 1 H, 30), 10.44 (d, J ) 7.0 Hz, 1 H, 31),
10.97 (d, J ) 7.5 Hz, 1 H, 32).
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and one- and two-dimensional 1H NMR spectra of
artificial â-sheet 2 (24 pages).
(11)
1H NMR Tr-ROESY cross-peaks for 2 (25 mM in CDCl3, 298
K). (Cross-peaks in the F1 dimension are tabulated for each resonance
in the F2 dimension.) 1: 5 (m), 6 (s), 7 (s), 18 (m), 27 (w), 29 (w). 2: 5
(m), 6 (m), 7 (s), 18 (s), 27 (w). 3: 9 (m), 10 (s). 4: 9 (m), 10 (s). 5: 1 (s),
2 (m), 6 (s), 7 (m), 18 (m), 27 (m), 29 (w). 6: 1 (s), 2 (s), 5 (s), 18 (m),
27 (m). 7: 1 (s), 2 (s), 5 (m), 18 (m), 27 (m), 29 (w). 8: 14 (s), 14’(s). 9:
3 (m), 4 (m), 21 (s). 10: 3 (s), 4 (s), 31 (m). 11: 12 (s), 19 (s), 20 (s), 23
(m). 12: 11 (s), 19 (m), 20 (s), 23 (m). 13: 15 (s), 30 (m). 14: 8 (s), 14’
(s). 14’: 8 (s), 14 (s). 15: 13 (s), 16 (s), 22 (m), 30 (s). 16: 15 (s), 22 (w),
30 (m). 17: 22’ (s). 18: 1 (m), 2 (s), 5 (m), 6 (m), 7 (m), 21 (s), 27 (m).
19: 11 (s), 12 (m), 22 (m), 23 (m), 27 (w), 29 (w). 20: 11 (s), 12 (s), 23
(s), 27 (s), 29 (m). 21: 9 (s), 18 (s), 27 (w). 22: 19 (s), 26 (s). 22’: 17 (s),
28 (s). 23: 11 (s), 12 (s), 19 (m), 20 (s), 24 (s). 24: 23 (s). 26: 22 (s). 27:
5 (m), 6 (m), 7 (m), 18 (m), 19 (m), 20 (s), 21 (w). 28: 22’ (s), 30 (w). 29:
1 (w), 5 (w), 7 (w), 20 (s), 30 (s). 30: 13 (m), 15 (s), 16 (m), 28 (w), 29
(s). 31: 10 (m). 32: 17 (m).
J O971431B
(12) Nowick, J . S.; Mahrus, S.; Smith, E. M.; Ziller, J . W. J . Am.
Chem. Soc. 1996, 118, 1066.
(13) Dyson, H. J .; Wright, P. E. Annu. Rev. Biophys. Biophys. Chem.
1991, 20, 519.
(14) (a) Kemp, D. S.; Bowen, B. R. Tetrahedron Lett. 1988, 29, 5077.
(b) Kemp, D. S.; Bowen, B. R. Tetrahedron Lett. 1988, 29, 5081. (c)
Kemp, D. S.; Bowen, B. R.; Muendel, C. C. J . Org. Chem. 1990, 55,
4650.
(15) Michne, W. F.; Schroeder, J . D. Int. J . Peptide Protein Res. 1996,
47, 2.