n→π* INTERACTIONS IN THE MOLECULES OF LIFE by Amit

747 Pages·2013·13.6 MB·English

n→π* INTERACTIONS IN THE MOLECULES OF LIFE

by
Amit Choudhary

A Dissertation Submitted in Partial Fulfillment
of the Requirements for the Degree of

Doctor of Philosophy
(Biophysics)

at the
UNIVERSITY OF WISCONSIN–MADISON
2011
© Copyright by Amit Choudhary 2011
All Rights Reserved
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ABSTRACT

n→π* INTERACTION IN THE MOLECULES OF LIFE
Amit Choudhary
Under the supervision of Professor Ronald T. Raines
At the University of Wisconsin–Madison

Noncovalent interactions dictate molecular conformation, reactivity, and function in chemical
and biological systems. An underlying feature of many noncovalent interactions is electron
delocalization. For example, a hydrogen bond involves the delocalization of the lone pair (n) of
the hydrogen-bond acceptor over the antibonding orbital (σ*) of the hydrogen-bond donor. We
discovered another noncovalent interaction, termed the “n→π* interaction”, which involves the
delocalization of the lone pair of the donor group into the antibonding orbital (π*) of a carbonyl
group. This interaction is reminiscent of the Bürgi–Dunitz trajectory for nucleophilic additions to
carbonyl groups. This thesis presents my findings pertaining to this interaction.
Chapter 1 reviews the major findings concerning the n→π* interaction in the last five years.
In chapter 2, I explore the nature of amide carbonyl–carbonyl interactions using a peptidic model
system. The intimacy of the carbonyl groups could arise from a charge–charge or dipole–dipole
interaction, or an n→π* electronic delocalization. By installing isosteric chemical substituents in
a peptidic model system, and using NMR spectroscopy, X–ray diffraction analysis, and ab initio
calculations to analyze the consequences, the intimate interaction between adjacent carbonyl
groups is shown to arise primarily from an n→π* electronic delocalization. These findings lay
foundation for the common signatures of the n→π* interaction.
In many common proteins secondary structures, such as -, 3 , and polyproline II helices, an
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n→π* interaction places the adjacent backbone amide carbonyl groups in close proximity to each
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other. Such a proximal arrangement of the amide carbonyl groups should be opposed by the
Pauli repulsion between the lone pairs (n) of O and the bonding orbital (π) of the carbonyl
i–1
group (C=O). In chapter 3, we explore the conformational effects of this Pauli repulsion by
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employing common peptidomimetics, wherein the n→π* 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→π* interaction. This
finding also indicates that the poor mimicry of the amide bond by many peptidomimetics stems
from their inability to partake in the n→π* interaction.
In chapter 4, we show widespread occurrence of n→π* interactions in proteins. Using
computational analyses and statistical analysis of a large, non-redundant subset of protein
structures determined to high resolution in concert, we find that n→π* interactions are abundant,
and especially prevalent in common secondary structures such as α-, 3 -, and polyproline II
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helices, and twisted β-sheets. I also discuss the evident effects of n→π* interactions on protein
structure and stability.
The n→π* interaction is reminiscent of the Bürgi–Dunitz trajectory for nucleophilic
additions to carbonyl groups and should induce pyramidalization of the acceptor carbonyl. In
chapter 5, we establish this signature of n→π* interactions in α-helices. We use crystal structures
of 14 peptides that contain both α- and β-amino acid residues and that assume a helical structure.
The α-amino acid residues, which adopt the main-chain dihedral angles of an α-helix, display
dramatic pyramidalization, but the β-amino acid residues do not.
In Chapter 6, I review the electronic, structural, and conformational attributes of four such
isosteres—thioamides, esters, alkenes, and fluoroalkenes—in detail. In particular, the ability of
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these isosteres to partake in noncovalent interactions is compared with that of the peptide bond.
Chapter 7 describes how an n→π* interaction was implicated in the origin of life by favorably
orchestrating the reactivity and stability of the prebiotic molecules. In Chapter 8, I describe our
finding pertaining to the existence of an n→π* interaction in a common household drug aspirin. I
also describe the plausible effects of this interaction on the conformational, physical, and
chemical attributes of aspirin.
The chromophore in many fluorescent proteins, including the renowned green fluorescent
protein, contains a highly conjugated imidazolidinone ring. In Chapter 8, I describe the existence
of an n→π* interaction between the carbonyl of a backbone residue and the imidazolidinone
carbonyl. This n→π* electron delocalization prevents structural distortion during chromophore
excitation that can lead to fluorescence quenching. Additionally, I discuss the existence of this
interaction in the on-pathway intermediates leading to the chromophore and its likely role in
chromophore biogenesis.
The partial covalency in an n→π* interaction stems from the delocalization of the electron
pair of a donor group into the antibonding orbital of a carbonyl group. The resulting electron
delocalization can transform an otherwise planar, achiral carbonyl group into a nonplanar, chiral
entity. In chapter 10, I present crystallographic evidence on the ability of the n→π* interaction to
induce chirality. Chapter 11 describes the quantum mechanical origin of the conformational
preferences of the 4-thiaproline and its S-oxides. In chapter 12, I describe our efforts towards
modulating the strength of the n→π* interaction using α-fluoro groups. My observations that
interactions to a carbonyl do not require a dipole are discussed in chapter 13.
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An analysis of high-resolution protein structures in protein databank suggests that these
carbonyl–carbony interactions prefer to occur in pairs. The reluctance of the acceptor carbonyl
(C=O) to engage in more than one n→π* electron delocalization is described in Chapter 14 using
an imidazolidine-based model systems with one acceptor carbonyl and two donor carbonyl
groups. Our result indicates that the electrophilicity of the acceptor carbonyl is reduced when it
engages in an n→π* electron delocalization. This reduction in electrophilicity discourages a
second n→π* electron delocalization to the acceptor carbonyl.
Appendix A describes the installation of ketoproline in collagen and its its utility in allowing
chemoselective bioconjugation. Appendix B describes the use of isosteric probes of an amide
bond to examine the substrate conformational specificity of the enzyme prolyl 4-hydroxylase.
Appendix C lists common small molecules with the signatures of the n→π* interaction. Finally,
appendices D–N describe crystallographic studies on small molecules.
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ACKNOWLEDGEMENTS
The work embodied in this thesis will result in more than fifteen published manuscripts.
However, this would not have been possible without the help of many collegeaus and
collaborators. I will try my best to duly acknowledge everyone who has helped me along the
way. Please pardon me if I miss anyone.
I will start by acknowledging my advisor, Professor Ronald Raines. As an incoming graduate
student, I was profoundly impressed by the diversity of the research in Raines laboratory. So you
can imagine my disappointment when I learned that Ron was not hiring, and my subsequent
elation when Ron told me that he could “make room for me” in his lab. Ron gives a great deal of
independence to pursue our own ideas and truly works hard to make sure that we have enough
resources. He has assembled a diverse work force of smart, yet extremely helpful graduate
students and postdoctoral researchers.
The dynamics in the Raines lab is one of the best on this planet. My fellow lab members have
helped and intellectually challenged me, and made the Raines lab a “home away from home” for
me. Frank Kotch, a former postdoc in our lab, always took the time to show me the ropes. Matt
Shoulders helped me to refine my hypotheses and educated me about everything possible under
the sun concerning the “States”. I have learned a lot from Matt and he is right in describing
himself as my “guru”. Joe Binder and Annie Tam made sure that the lab was open from 7 am till
midnight seven days a week. This motivated me to work hard and it was very comforting to
know that someone was around even at 8 pm on Fridays when the Biochem building wore a
ghostly look. Jeet Kalia regularly participated in our Saturday lab lunches, and I had a number of
very enlightening conversations with him. Ben Caes and Christine Bradford made sure that my
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“a” and “the” articles were in the right place in all of my manuscripts. I have learned a lot about
writing from them. Christine also taught me a lot about the “States” and contributed to my
personal growth. Mike Palte helped me choose colors for my multidimensional plots and
challegened me intellectually. His advice about shaving my mustache off to be popular among
ladies has not borne many fruits, but holds a lot of promise. Mike and Greg Ellis have also gone
over many of my manuscripts and research proposals. Their constructive criticisms have taught
me how to approach and present a biological problem. Eddie Myers, a former postdoc in our lab,
propelled me to be a more disciplined scientist. It was fun working with Katrina Jensen and
Kelly Gorres on a collaborative project described in Appendix B. Nick McGrath taught me how
to run super fast fun columns during the last month of my graduate studies. I wish I had known
about this five years ago! Mike Levine saved a lot of my time by teaching me how to efficiently
use field codes in MSWord documents. Other Raines lab colleagues, including Langdon Martin,
John Lukesh, Ho-Hsuan, and Cindy Chao have contributed to my personal and professional
development in many ways. The vitality displayed by the recent recruits to the Raines lab
including Jim, Rob, Trish, Kristen, and Kevin have made it a great place to work. I am also
thankful to our lab manager Greg who patiently bore my regular pestering for overnight
deliveries of my chemicals.
I am thankful to all of my collaborators. We have a long standing collaboration with Prof.
Grant Krow and his lab. The initial findings on thioamides were simultaneously discovered by
the Krow and Raines laboratories. This provided me an opportunity to interact with Prof. Krow
and Dr. Deepa Gandla, which I enjoyed a lot. We have worked on many projects with Prof.
Krow and I am looking forward to more interactions with him as we work on the manuscripts.
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The work described in Chapter 3 was done in collaboration with Prof. Scott Miller and Dr.
Chuck Jakobsche. We worked with Prof. Dek Woolfson and Dr. Gail Bartlett in our search for
n→π* interaction in proteins. Prof. Sutherland and Dr. Matt Powner provided us with the kinetic
data supporting the role of n→π* interaction in the origin of life.
My committee members and other professors have been a source of knowledge, inspiration
and guidance throughout my PhD. Prof. Helen Blackwell taught us stereoelectronic effects
during my early days of graduate school and later wrote letters for my fellowship applications.
Prof. Sam Gellman critiqued our Protein Science manuscript and has regularly provided many
interesting ideas to test our hypotheses. Prof. Landis and Prof. Mecozzi have also suggested very
interesting ideas. I am also thankful to Prof. Frank Weinhold for going over our computational
methodology used to estimate the strength of n→π* interaction in the different regions of the
Ramachandran map (Chapter 4). It would be ungrateful not to thank Prof. Tom Record. I am
aware that the graduate admissions committee was not very impressed by my graduate
application. If it hadn't been for Prof. Record’s faith and recommendation, I would not have been
admitted to UW-Madison.
I was lucky to have two extremely smart and very hardworking undergraduate students.
Khian Hong worked on the thiaproline project and is now pursuing his graduate studies at
Harvard. Khian contributed not only to my professional growth, but also to my personal growth.
I am extremely grateful that Khian extended his stay in Madison to complete the thiaproline
project. My other undergraduate student, Kim Kamer, worked with me on numerous projects and
has set a new record for the number of published papers as an undergraduate researcher in the
Raines lab. Kim never complained that I was “slave driving” her (which I was) and wound up
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many projects very efficiently. Kim has also joined Harvard’s Chemistry and Chemical Biology
program and I look forward to many more exciting moments with both Khian and Kim.
I have benefitted a lot from the numerous very high quality facilities and facility managers. I
worked with Dr. Charlie Fry on two projects (Chapter 12 and 14). Charlie has always been
available and willing to help with my NMR experiments even if that required him to work till 9
pm on a Friday night. Drs. Monica Ivancic (Chemistry Department), Mark Anderson, and Milo
Westler have patiently helped me on a number of occasions. Dr. Ilia Guzei and Lara Spencer
have guided me through many difficult X-ray structures and made sure that I understood what I
was doing. I was a technical assistant at the Mass Spectrometry facility at the Chemistry
department and learned a lot about MS from Dr. Martha Vestling. Dr. Darrell McCaslin helped
us with CD spectroscopy on our ketoproline containing collagen related peptides and has
inspired me to do “science for the sake of science”. I deeply admire Darrell’s critical and
scholarly approach to science.
I have frequently borrowed ideas and skills that I learned during my undergraduate training. I
would like to thank Prof. Jayant Udgaonkar for exposing me to the area of protein folding and
Profs. K. R. Prasad, S. S. Bari, R. K. Trikha, N. K. Nayar for teaching me the experimental
aspects of synthetic organic chemistry.
My friends have also helped me considerably with my research. Jayashree and Jaganath
taught me computational chemistry and I had several illuminating conversations about protein
folding with Ashok. Anbarasan helped me plan many of my retrosyntheses and my trips to
Bishu’s place in NYC were very refreshing.