N →π* interactions of amides and thioamides: Implications for protein stability

Article abstract of DOI:10.1021/ja4033583

Carbonyl-carbonyl interactions between adjacent backbone amides have been implicated in the conformational stability of proteins. By combining experimental and computational approaches, we show that relevant amidic carbonyl groups associate through an n→π

Full text of DOI:10.1021/ja4033583

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Communication
n##* Interactions of Amides and Thioamides: Implications for Protein Stability
Robert W. Newberry, Brett VanVeller, Ilia A. Guzei, and Ronald T Raines
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja4033583 • Publication Date (Web): 10 May 2013
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n→π* Interactions of Amides and Thioamides: Implications for
Protein Stability
Robert W. Newberry,† Brett VanVeller,† Ilia A. Guzei,† and Ronald T. Raines*,†,‡
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†Department of Chemistry and ‡Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706,
United States
Supporting Information Placeholder
ABSTRACT: Carbonyl–carbonyl interactions between adjacent
backbone amides have been implicated in the conformational sta-
bility of proteins. By combining experimental and computational
approaches, we show that relevant amidic carbonyl groups associate
through an n→π* donor–acceptor interaction with an energy of at
least 0.27 kcal/mol. The n→π* interaction between two thioam-
ides is 3-fold stronger than between two oxoamides due to in-
creased overlap and reduced energy difference between the donor
Figure 1. (A) Notion of a carbonyl–carbonyl n→π* interaction in
and acceptor orbitals. This result suggests that backbone thioamide
amide 3. (B) Three-dimensional orbital rendering and (C) contour
incorporation could stabilize protein structures. Finally, we demon-
plot showing overlap of n and π* orbitals in the trans exo confor-
strate that intimate carbonyl interactions are described more com-
mation of 3. Images were rendered with NBOView 1.1.
pletely as donor–acceptor orbital interactions rather than dipole–
dipole interactions.
Protein architecture is mediated by a suite of noncovalent inter-
actions within and between polypeptide chains, including the hy-
drophobic effect, hydrogen bonding, Coulombic interactions, and
van der Waals interactions.1 We have put forth an n→π* interac-
tion as an additional means by which peptide bonds themselves
interact.2 In this n→π* interaction, a carbonyl oxygen donates lone
pair (n) electron density into another carbonyl group (Figure 1).
Such donation occurs when the donor and acceptor form a short
contact along the Bürgi–Dunitz trajectory for nucleophilic addi-
tion.3 These interactions have been implicated in many systems,
including small molecules,4 peptides,5 proteins,6 peptoids,7 and
nucleic acids.8
Compound
X
O
S
O
S
O
S
Y
O
O
O
O
S
Z
1
2
3
4
5
6
OMe
OMe
NMe2
NMe2
NMe2
NMe2
S
Figure 2. Compounds used to evaluate n→π* interactions in tor-
sion balance analyses.
We and others have used a torsion balance in a proline model
system to characterize energetic relationships of importance to
peptide and protein structure (Figure 2).2,9,5c,5g,5j,5m,10 Both the cis
and trans isomers of the N-acetyl proline peptide bond are populat-
ed at room temperature and can be distinguished by NMR spec-
troscopy due to their slow interconversion. As the n→π* interac-
tion is only possible in the trans isomer, the ratio of isomers
(Ktrans/cis) reports on the energy of the interaction. All previous stud-
ies employed esters as the n→π* acceptor (1); because esters are
more electrophilic than the amides found in proteins, those studies
overestimated the strength of n→π* interactions at
0.7 kcal/mol.2c,9 Hence, we sought to determine the energy of a
prototypical n→π* interaction between two amides. Primary and
secondary amides can both donate hydrogen bonds to the acetyl
group, obscuring the n→π* interaction in our analysis;11 thus, we
elected to examine the tertiary dimethyl amide (3).
In D2O, the value of Ktrans/cis for amide 3, like that for ester 1, is
greater than unity (Figure 3A). We then employed density func-
tional theory (DFT) calculations at the B3LYP/6-311+G(2d,p)level
of theory with natural bond orbital (NBO) analysis to estimate the
energy of the n→π* interaction.12 We optimized the geometry of 3
in both the Cγ-endo and Cγ-exo puckers of its pyrrolidine ring (Fig-
ure 4) and found the corresponding n→π* energies to be 0.14 and
0.53 kcal/mol, respectively. At room temperature, proline exists
~66% in the endo pucker and ~34% in the exo pucker.2b Based on
this ratio, we estimate the energy of the n→π* interaction in 3 to
be En→π* = 0.27 kcal/mol (Figure 3B). This interaction is weaker
than that in 1, which is consistent with the lower electrophilicity of
the amide acceptor relative to the ester. Importantly, because the
tertiary amide is less electrophilic and more stericially encumbered
than the secondary amides common in proteins, the values we
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Figure 4. (A) Cγ-endo and (B) Cγ-exo pyrrolidine ring pucker of
compounds 1–6. (C) Parameters that denote pyramidalization of
carbonyl groups due to n→π* donation.
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Table 1. Conformational Parameters of Thioamidesa
compound
conformation
d (Å)
θ (deg)
∆ (Å)
Θ (deg)
4
cis, endo
4.6158(14)
66.96(7)
0.0035(14)
0.43(17)
5
6
6
trans, endo
trans, endo
trans, exo
3.2529(12)
3.4248(16)
92.19(4)
96.11(6)
0.0237(8)
0.0243(17)
0.0392(16)
2.61(9)
2.70(19)
4.36(18)
Figure 3. (A) Values of Ktrans/cis for compounds 1–6 in D2O at
25 °C. (B) Energy of n→π* interactions in 1–6 from second-order
perturbation theory. (C) Overlap integrals between the n and π*
orbitals of 3–6. (D) Reciprocal of the energy gap between the n and
π* orbitals of 3–6. Data for esters 1 and 2 are from ref. 9.
3.2433(15) 101.92(7)
aFrom X-ray diffraction analysis of the crystalline compound. Param-
eters are defined in Figure 4.
report here are likely to underestimate the energy of an n→π* inter-
action between most peptide bonds. Thus, we expect that n→π*
interactions in proteins contribute ≥0.27 kcal/mol of stabilization
per interaction.
analysis of crystalline 4–6 to search for this signature of an n→π*
interaction. Thioamide 4 crystallized as its cis isomer and therefore
does not show an n→π* interaction, leaving the acceptor nearly
planar (Table 1). Thioamides 5 and 6 both crystallized as their trans
isomer, with 6 crystallizing in both pyrrolidine ring puckers. In
both 5 and 6, the acceptor carbon is pyramidalized toward the
donor significantly more than in 4, denoting a stronger n→π*
interaction. In addition, pyramidalization of the acceptor in both
conformations of 6 is greater than in 5, which is consistent with
the stronger n→π* interaction in 6. Moreover, the greater pyrami-
dalization of the acceptor in 6-exo than in 6-endo confirms that the
exo ring pucker of proline promotes stronger n→π* interactions.16
Indeed, the pyramidalization in 6-exo is among the largest observed
to date in this proline model system.9,17 These observations are also
consistent with the pyramidalization in crystal structures of thioam-
ide-containing peptides (see: Supporting Information, Figure S1).
Previously, we demonstrated that the substitution of an amide
donor (i.e., 1) with a thioamide (2) increases n→π* donation to an
ester carbonyl (Figure 3A).9,13 This increase arises from sulfur being
a better electron-pair donor than its oxygen congener. This finding
suggested to us that backbone thioamide substitution could en-
hance the n→π* interaction between carbonyl groups and stabilize
the folded structures of proteins. Still, the quality of a thioamide as
an n→π* acceptor has been predicted only computationally.14
Hence, we synthesized 4–6 to evaluate thioamides as both donors
and acceptors of the n→π* interaction for the first time.
Replacing the donor of 3 with the larger thioamide to yield 4 in-
creased the value of Ktrans/cis despite the added steric clash confirm-
ing that sulfur is a stronger n→π* donor than oxygen (Figure 3A).
Interestingly, whereas replacing the acceptor of 3 with a thioamide
to yield 5 reduced Ktrans/cis, replacing the acceptor of 4 with a thio-
amide to yield 6 led to an increase in Ktrans/cis. Our NBO analyses
show that replacing the acceptor with a thioamide reduces orbital
overlap with the donor (Figure 3C), providing a rational basis for
the value of Ktrans/cis for 5 being lower than that for 3.
These data further establish the quantum mechanical nature of
intimate carbonyl–carbonyl interactions. Some have argued that
these interactions are primarily dipolar in nature.18 The dipole
moment of an amide is greater than that of an ester,19 so if a dipo-
lar interaction is dominant, replacing the ester of 1 with the amide
in 3 should cause an increase in Ktrans/cis, which is contrary to ob-
servation (Figure 3A). Moreover, as a thioamide has a still larger
dipole moment than an amide,20 a dipolar origin would predict a
larger value of Ktrans/cis in thioamide 5 than in amide 3. That was
not observed in our experiments. Finally, in a dipolar interaction,
neither of the participating groups has a defined role; rather, they
interact symmetrically. Thus, if intimate carbonyl interactions are
dipolar in nature, then substituting either amide with a thioamide
should have a comparable effect on Ktrans/cis. The conformational
preferences of 4 and 5 suggest otherwise: substituting the n→π*
donor amide with a thioamide increases Ktrans/cis, whereas replacing
the acceptor decreases Ktrans/cis. These data affirm that a dipolar
mechanism is insufficient to describe intimate interactions between
carbonyl groups. Instead, the data are more consistent with an
electronic donor–acceptor effect like the n→π* interaction.
NBO analysis of thioamides 3 and 5 revealed another pertinent
quantum mechanical attribute. The π* orbital of a thioamide is
lower in energy than that of an amide, reducing the energy gap
between donor and acceptor orbitals (Figure 3D). From second-
order perturbation theory, the energy released by the mixing of two
orbitals is proportional to the reciprocal of the energy gap between
those orbitals. Thus, though a thioamide acceptor overlaps less
with an n→π* donor (Figure 3C), a smaller energy gap between the
donor and acceptor can produce more effective orbital mixing and
a stronger interaction overall. The consequences are apparent in
thioamide 6, in which the n→π* interaction is particularly strong
at 0.88 kcal/mol (Figure 3B), demonstrating that pairs of thioam-
ides engage in significantly stronger n→π* interactions than do
pairs of analogous amides.
Individual n→π* interactions between amides are relatively
weak. In abundance, however, they could make a significant con-
tribution to the conformational stability of a protein. We note
another implication as well. Shifts in the equilibrium between α-
helices and β-sheets have been implicated in amyloid fibrillogene-
sis.21 Hydrogen bonding, which is operative in both α-helices and β-
As the n→π* interaction populates the π* orbital of the accep-
tor, it induces pyramidalization of the acceptor toward the donor
(Figure 4C). This distortion is detectable by X-ray diffraction analy-
sis and can provide a signature of n→π* interactions in small mol-
ecules and peptides.4,5k,9,13,15 Hence, we conducted X-ray diffraction
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sheets,22 is unlikely to affect this equilibrium decisively. In contrast,
S. B. Chem. Commun. 2011, 47, 2342-2344. (m) Erdmann, R. S.;
n→π* interactions are common in α-helices but not β-sheets,6b and
thus could play a critical role in the maintenance of protein home-
ostasis. In addition, our finding that the n→π* interaction between
two thioamides is 3-fold stronger than that between two amides
(Figure 3B) encourages efforts to exploit thioamides to enhance
conformational stability in peptides and proteins.23,24 Finally, as
these interactions are not included in conventional force fields, we
argue that accounting for the n→π* interaction could improve the
accuracy of computational investigations of proteins.
Wennemers, H. J. Am. Chem. Soc. 2012, 134, 17117-17124.
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Supporting Information
Synthetic and analytical procedures, and computational data on
compounds 3–6. This material is available free of charge via the
Internet at http://pubs.acs.org.
◼ AUTHOR INFORMATION
Corresponding Author
rtraines@wisc.edu
Notes
The authors declare no competing financial interests.
◼ ACKNOWLEDGMENT
We are grateful to Brian Dolinar (University of Wisconsin–
Madison) for technical assistance and to Amit Chouhdary (Harvard
University) and Grant Krow (Temple University) for contributive
discussions. R.W.N. was supported by NIH Biotechnology Train-
ing Grant T32 GM008349. This work was Supported by grants
R01 AR044276 (NIH) and CHE-1124944 (NSF). High-
performance computing was supported by grant CHE-0840494
(NSF).
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23x7mm (600 x 600 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
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40x16mm (300 x 300 DPI)
ACS Paragon Plus Environment

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Journal of the American Chemical Society
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57x36mm (300 x 300 DPI)
ACS Paragon Plus Environment

Journal of the American Chemical Society
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22x7mm (600 x 600 DPI)
ACS Paragon Plus Environment

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Journal of the American Chemical Society
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35x13mm (600 x 600 DPI)
ACS Paragon Plus Environment