N → φ* Interaction and n)(φ pauli repulsion are antagonistic for protein stability

Article abstract of DOI:10.1021/ja100931y

In many common protein secondary structures, such as α-, 3 ₁₀, and polyproline II helices, an n → φ* interaction places the adjacent backbone amide carbonyl groups in close proximity to each other. This interaction, which is reminiscent of the Buergi-Dunitz trajectory, involves delocalization of the lone pairs (n) of the oxygen (O _i-1) of a peptide bond over the antibonding orbital (-*) of C_iO_i of the subsequent peptide bond. Such a proximal arrangement of the amide carbonyl groups should be opposed by the Pauli repulsion between the lone pairs (n) of O_i-1 and the bonding orbital (-) of C_iO_i. We explored the conformational effects of this Pauli repulsion by employing common peptidomimetics, wherein the n → -*interaction is attenuated while the Pauli repulsion is retained. Our results indicate that this Pauli repulsion prevents the attainment of such proximal arrangement of the carbonyl groups in the absence of the n → φ* interaction. This finding indicates that the poor mimicry of the amide bond by many peptidomimetics stems from their inability to partake in the n → φ* interaction and emphasizes the quantum-mechanical nature of the interaction between adjacent amide carbonyl groups in proteins.

Full text of DOI:10.1021/ja100931y

Published on Web 04/26/2010
n f π* Interaction and n)(π Pauli Repulsion Are Antagonistic for Protein
Stability
Charles E. Jakobsche,† Amit Choudhary,‡ Scott J. Miller,*,† and Ronald T. Raines*,§
Department of Chemistry, Yale UniVersity, New HaVen, Connecticut 06520, and Graduate Program in Biophysics
and Departments of Biochemistry and Chemistry, UniVersity of Wisconsin, Madison, Wisconsin 53706
Received February 2, 2010; E-mail: scott.miller@yale.edu; rtraines@wisc.edu
The interplay between electronic effects and steric effects
underlies molecular conformation. For example, the common
CdO· · · H-N hydrogen bonds within protein main chains may be
viewed as favored by the delocalization of an oxygen lone pair (n)
into the antibonding orbital (σ*) of the N-H bond but disfavored
by Pauli repulsion1 between n and the N-H bonding orbital (σ).2
Here we report on a second example of this type of dichotomy
within protein main chains.
Figure 1. Definition of equilibrium constant Ktrans/cis, distance d, planar
angle θ, and dihedral angles φ and ψ. X ) O in 1, 2, and 4-6; X ) S
in 3.
In common elements of protein secondary structure, the oxygen
(Oi-1) of a main-chain amide is proximal to the carbon (Ci′) of the
subsequent amide.3 This short contact is promoted by n f π*
electronic delocalization, wherein an oxygen lone pair (n) overlaps
with the Ci′dOi antibonding orbital (π*) of the subsequent peptide
bond.3-5 We suspected that, as in a hydrogen bond, this electronic
effect is antagonized by a steric effect, here arising from Pauli
repulsion between n and the Ci′dOi bonding orbital (π).
To unveil any n)(π Pauli repulsion, we sought a π system that
is isosteric with a carbonyl group but provokes little n f π*
interaction. We suspected that alkenyl groups, which lack the
polarity of carbonyl groups, could have this attribute. To enable
quantitative comparisons, we chose the AcProOMe (1) model
system,6 in which n is directed toward π* in the trans conformation
but not in the cis conformation (Figure 1). The value of Ktrans/cis
reports on the differential stability of the trans and cis conformations
and can be measured by using NMR spectroscopy. We suspected
that replacing the ester of 1 with an isosteric fluoroalkene7 would
attenuate the n f π* interaction. Hence, we synthesized and
analyzed 1 and its fluoroalkenyl isostere, 2.
We performed geometry optimizations, frequency calculations, and
NBO analyses at the B3LYP/6-311+G(2d,p) level of theory on
eight conformations of 1 and 2 (see Tables S1 and S2 in the
Supporting Information). We estimated the stabilization afforded
by n f π* electronic delocalization by using second-order
perturbation theory, as implemented with NBO 5.0. In accord with
our expectation, we found that fluoroalkene isostere 2 does not
partake in an appreciable n f π* interaction (Table 1). The π*
orbital of the carbonyl group in 1 is oriented properly for extensive
n f π* overlap, but that of the fluoroalkenyl group in 2 is not
(Figure 2). Additionally, the energy difference between the n and
π* orbitals of 2 (33.2 kcal/mol) is ∼10-fold greater than that of 1
(3.5 kcal/mol). While the π* orbital of the carbonyl is located
primarily on the single carbonyl carbon, the π* of the fluoroalkene
isostere is distributed evenly between the two alkenyl carbons.
Moreover, the distance between the donor oxygen (Oi-1) and
acceptor carbon (Ci′) is short in all low-energy conformations of 1
but long in 2 (Table S1). Finally, Oi-1 in the low-energy conforma-
tions of 1 is along the Bu¨rgi-Dunitz trajectory9 (θ ≈ 100°), but
Oi-1 of 2 is off of that trajectory (θ ≈ 125°) (Table S1).
The conformational differences between 1 and 2 are evident in
their computational energy landscapes (Figure 3A,B). As the value
of d decreases, the interpenetration of the van der Waals surfaces
of the donor and acceptor groups increases. That endows 1 but not
2 with conformational stability. In 1, the n)(π Pauli repulsion is
offset by a strong n f π* interaction; in 2, the n f π* interaction
We found evidence that unfavorable Pauli repulsion can indeed
antagonize a favorable n f π* interaction. Replacing the carbonyl
acceptor with a fluoroalkene switches the conformational preference
of the amide bond from trans to cis (Table 1). We resorted to hybrid
density functional theory and Natural Bond Orbital (NBO)8 analyses
to reveal the basis for this dramatic shift in conformational preference.
† Department of Chemistry, Yale University.
‡ Graduate Program in Biophysics, University of Wisconsin.
§ Departments of Biochemistry and Chemistry, University of Wisconsin.
Figure 2. Overlaps between n and the π* and π orbitals of 1 and 2 in
their optimized geometries.
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10.1021/ja100931y  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 6651–6653 6651

C O M M U N I C A T I O N S
Table 1. Conformational Properties of Compounds 1-6
a
compound
Ktrans/cis
chemical shift of C′i (ppm)
d (Å)b
θ (deg)b
φ (deg)b
ψ (deg)b
n f π* (kcal/mol)b
1
2
3
4
5
6
3.7 : 1.0
1.0 : 1.7
1.0 : 2.2
1.0 : 2.9
1.4 : 1.0
1.0 : 4.0
ND
156
ND
133
ND
105
3.08
3.28
3.59
3.32
-
99.5
124.9
126.3
126.4
-
-71.12
-82.81
-84.42
-84.02
-78.89
-80.43
152.67
117.01
120.92
116.56
167.16
142.03
0.40
0.01
0.05
0.02
-
3.25
104.1
0.03
b
a Measured in CDCl3 at 25 °C. Computed in the optimized conformation (trans amide bond; Cγ-endo pyrrolidine ring pucker).
If n)(π Pauli repulsion destabilizes the trans conformation of 2,
then its amplification should further reduce the population of that
conformation. Some of us had shown previously that the sulfur of
a thioamide is a better n f π* donor than is the oxygen of an
amide.4e However, because sulfur is larger than oxygen and CdS
bonds are longer than CdO bonds, sulfur should engender greater
n)(π Pauli repulsion. To search for that manifestation, we replaced
the donor oxygen (Oi-1) in amide 2 with sulfur. We found the value
of Ktrans/cis for thioamide 3 to be less than that for amide 2 (Table
1). An origin in increased n)(π Pauli repulsion is supported by NSA
(Table S1).
Likewise, we reasoned that attenuating any n)(π Pauli repulsion
should stabilize the trans conformation. We suspected that a
comparison of alkene 4 with alkane 5, which lacks the acceptor π
orbital, would allow us to test our reasoning. Again, we found
evidence for n)(π Pauli repulsion, as the value of Ktrans/cis for alkane
5 is greater than that for alkene 4 (Table 1).
Compound 4 offers another opportunity to probe for n)(π Pauli
repulsion. The pendant fluoro group that is present in 2 but absent
in 4 polarizes the π orbital, reducing the electron density on the
acceptor carbon (C′i). The net effect is to diminish n)(π Pauli
repulsion, as evidenced by a larger value of Ktrans/cis for 2 than 4
(Table 1). Accordingly, we reasoned that polarizing the π bond in
the opposite direction could increase the electron density on the
acceptor carbon, thereby increasing any n)(π Pauli repulsion.
Indeed, the value of Ktrans/cis for 6 is less than those for 2 and 4.
The correlation between the value of Ktrans/cis for compounds 2, 4,
and 6 and the 13C NMR chemical shift of each acceptor carbon
(Table 1), which reports on its electron density, provides additional
validation for our conclusions.
Some of us have argued4e that intimate carbonyl-carbonyl
interactions, which are ubiquitous in many protein secondary
structures,3 involve n f π* interactions and cannot be interpreted
in terms of classical electrostatic models, such as dipole-dipole11
or charge-charge interactions.12 The results herein support this
argument. First, if the interaction between adjacent carbonyl groups
were manifested as a classical dipole-dipole interaction, replacing
the CdO group with a C(sp2)-F group would not elicit a reversal
in the conformational preference from trans to cis. Second, the value
of Ktrans/cis for 3 is less than that for 2, despite the dipole moment
of CdS being greater than that of CdO.13 Third, the φ and ψ
dihedral angles and the conformational energy landscapes of 2 and
4 (the latter of which lacks a dipole) are similar to each other, yet
distinct from those of 1 (Table 1; Figure 3C).
Figure 3. Conformational energy landscapes of (A) 1, (B) 2, and (C) 4.
Figure 4. Overlap of the σ (CR -H) and σ* (C′i-F) orbitals of 2 in its
i
optimized geometry.
The Oi-1 · · · C′idOi distance is especially small in R-helices.3
These short contacts position distal CdO and H-N groups in
the main chain to form the canonical i f i + 4 hydrogen bond
(Figure 5). Our data indicate that n)(π Pauli repulsion deters
such short contacts and would, unless counteracted by an n f
π* interaction, impair R-helix formation. Indeed, others have
shown that replacing a single amide bond with an alkene or a
fluoroalkene isostere severely disrupts R-helical structure.14
Moreover, we put forth n)(π Pauli repulsion as the basis for the
does not overcome that repulsion. Natural Steric Analysis (NSA)
supports the existence of the antagonistic Pauli repulsion in low-
energy conformations (Table S1).
Fluoroalkene 2 lacks a favorable n f π* interaction despite
restricted rotation of its CR -C′i bond (ψ in Figure 1). The anti
i
rotamer is stabilized by a hyperconjugative interaction between the
CR-H bonding orbital (σ) and the C′i-F antibonding orbital (σ*)
(Figure 4).10 This rotamer gives rise to a larger value of JH,F for
3
the trans conformation (16 Hz) than the cis conformation (8 Hz).
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Acknowledgment. We are grateful to W. L. Jorgensen and
M. D. Shoulders for contributive discussions. This work was
supported by NIH Grants R01 GM068649 (S.J.M.) and R01
AR044276 (R.T.R.).
Supporting Information Available: Synthesis and analysis proce-
dures and computational data. This material is available free of charge
via the Internet at http://pubs.acs.org.
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