UNIVERSITY OF CINCINNATI
Date:______
I, ______, hereby submit this work as part of the requirements for the degree of: in:
It is entitled:
This work and its defense approved by:
Chair: ______
Creation and Investigation of Protein Core Mimetics and DNA Binding Molecules
A dissertation submitted to the
Division of Research and Advance Studies of the University of Cincinnati
in partial fulfillment of the requirements for the degree of
DOCTORATE OF PHILOSOPHY
in the Department of Chemistry of the College of Arts and Sciences
2005
by
Juris Fotins
B. S., University of Latvia, 1999
M. S., University of Cincinnati, 2002
Committee Chair: David B. Smithrud, Ph. D. Abstract
The goal of this research is to design and synthesize small molecules that mimic structural and functional elements of the zinc finger containing proteins. Two fairly independent areas have been explored to determine stable minimal structure and minimal DNA binding motif of zinc fingers.
Mimetic protein cores were created that align a set of L-Phe, D-Phe, or L-Leu residues in a parallel or an antiparallel arrangement in chloroform. Not all cores show a single conformation at room temperature. Stable structures require a synergistic relationship between the H-bonding groups and the residues within the core. The spatial arrangement of the side chains dictates whether a zippered or a crossed pattern of H-bonds is observed for these cores. Variable-temperature 1H NMR experiments were used to determine the strengths of the H-bonds. The existence of H-bonds was verified through FTIR spectroscopic analysis. Large temperature coefficients exist for some protons of aromatic rings that are held in a T-shaped arrangement. A comparison of these temperature coefficients shows that a more stable core is obtained by combining benzenoid and nitrobenzenoid rings as compared to benzenoid rings. Structures were determined using a combination of 2D NMR analysis and molecular modeling. Detailed results of this studies have been published in “Creation and Investigation of Protein - Core Mimetics with Parallel and Antiparallel Aligned
Amino Acids”, Fotins, J., Smithrud, D. B. J. Org. Chem, Vol. 70, No. 11, 2005.
Based on previously studied DNA binding molecules, we have designed second generation mimetic. In addition to a major groove binder already present in the molecule, the mimetic can be derivatized with an intercalator, such as acridine.
ii Functionalization of the aromatic ring provides another attachment point for oligopeptides containing lysine residues, which are known to bind to DNA at the phosphate backbone. Docking experiments performed with HyperChem supported that the overall geometry of the newly designed mimetic is favorable for multiple mode interactions.
iii Acknowledgements
I would first like to thank my research adviser professor David B. Smithrud for his support, guidance and patience that he extended throughout my graduate career. I also want to extend my appreciation to my research committee members: professors George P. Kreishman, Apryll Stalcup and R. Marshall Wilson for many useful discussions and their encouragement.
I would like to thank Dr. Elwood Brooks for his advice and guidance with
NMR spectroscopy.
It is my pleasure to acknowledge my research colleagues: Dr. Shawn Dickess,
Michael Herr, Brian House and Dr. Inese Smukste for numerous discussions that significantly contributed to my academic and experimental knowledge. I would especially like to acknowledge Dr. Jeff Turk for his pioneering work; without his results, it would have been much harder to develop my project.
I would also like to thank Vadim Dvornikov and Sergei Berdnikov for being my best friends for all these years.
I am indebted to my undergraduate adviser Dr. Valerjans Kauss for his contribution to my professional career. Without his knowledge and passion for chemistry, I would probably never have become an organic chemist.
Above all, I am grateful to my parents, Vilma and Sergei, for giving endless support and unconditional love.
iv Table of Contents
Abstract ii Acknowledgements iv Table of Contents v List of Figures vi List of Schemes viii Abbreviations ix
1. Introduction to Protein Mimicry 1
Protein Mimicry 2 Zinc Finger Proteins 5 Protein Core Mimetics 11
2. Synthesis and Physical Properties of Protein-Core Mimetics with Parallel and Antiparallel Aligned Amino Acids 15
Introduction 16
Mimetics with Parallel Aligned Amino Acids 19 Design and Synthesis 19 Experimental Methods 22 Physical Properties 24
Mimetics with Antiparallel Aligned Amino Acids 30 Design and Synthesis 30 Physical Properties 33
Discussion 36 Conclusion 44
3. Design and Synthesis of Small Molecules that Bind DNA 45
Introduction 46 Design and Synthetic Efforts 49
References 56
Experimental Section 68
Spectral Data 93
v List of Figures
Figure 1. Structure diagram of the classical C2H2 zinc-binding motif 6
Figure 2. Overview of the Zif268–DNA complex, showing the side chains that make 7 direct base contacts
Figure 3. Cartoon representation of typical zinc finger protein containing a 9 hydrophobic core, three-stranded structural domain and the DNA binding domain
Figure 4. Schematic representation of parallel β-sheet and antiparallel β-sheet 12
Figure 5. Artificial β–structures that have stable hydrophobic cores around 13 dibenzofuran and dipyridine scaffold
Figure 6. The “molecular torsion balance” has two gently restricted conformational 14 states
Figure 7. The number of aromatic rings held in a T-shaped arrangement determines 16 whether a zippered or a crossed pattern of H-bonds is formed. A nonsynergistic relationship between the core and H-bonding residues, however, results in multiple structures
Figure 8. (A) The original PCM that displays interactions between both aromatic side 17 chains and one of the scaffold s aromatic rings and two isoenergetic H-bonds. (B) The new PCM also contains a scaffold and H-bonding plane, but its internal amines are not methylated
Figure 9. General design of mimetics with parallel aligned amino acids 19
Figure 10. Typical variable temperature 1H NMR spectrum. With an increase in 23 temperature NH proton signals move upfield and aromatic signals move downfield
1 Figure 11. An overlay of the H NMR spectra of (L, D)-Pheparallel and (L, L)-Pheparallel, 25 showing that a correct matching of core with H-bonding residues will lead to a stable structure
Figure 12. Schematic drawings of the compounds showing key NOE cross-peaks 26 (double-headed arrows). Temperature coefficients (standard deviations are less than (0.1) for amide N-H and aromatic Ar-H protons are given. Highlighted in (L, L)- PheNO2parallel are the observed diastereomeric differences
Figure 13. FTIR spectral data from the N-H stretching region of the PCMs and N- 27 Ac-phenylalanine methyl amide. All samples were 3 mM in CHCl3 at 298 K. Each spectrum had its baseline corrected, and the absorbance of CHCl3 was subtracted
Figure 14. Amino acid that induces β-turn to aligned strands of mimetic in 30 antiparallel fashion
vi List of Figures (continued)
Figure 15. Key NOE cross-peaks (double-headed arrows), temperature coefficients 33 for amide N-H and aromatic Ar-H protons, and FTIR spectral data from the N-H stretching region are given for the antiparallel compounds
Figure 16. The diastereomeric sets of the antiparallel compounds, a cartoon depiction 35 of their calculated stable structures highlighting the orientation of the aromatic rings and whether a single conformer is observed
Figure 17. A zippered or a crossed pattern of H-bonds exists between the amino 37 acids of the parallel aligned compounds and the antiparallel aligned compounds
Figure 18. Low-energy structures obtained from molecular modeling studies that are 38 consistent with the observed properties of the compounds
Figure 19. First generation DNA binder containing hydrophobic core, Arg-His 47 recognition strand and fluorescence tag DAE (dansyl group with ethylene diamine linker)
Figure 20. Representative fluorescence quenching assays of mimetic IV binding to 47 mDd (A) and d(A9T9)2 (B) in 0.1 M phosphate buffer pH 7.0 at 25 ºC
Figure 21. Proposed design of the second generation mimetic that will bind DNA 49
vii List of Schemes
Scheme 1 20
Scheme 2 21
Scheme 3 31
Scheme 4 32
Scheme 5 50
Scheme 6 51
Scheme 7 52
Scheme 8 53
Scheme 9 54
Scheme 10 55
viii Abbreviations
Ac2O acetic anhydride AcOH acetic acid Arg arginine Bn benzyl Boc t-butoxycarbonyl BOM benzyloxymethyl CDI N,N’-carbonyldiimidazole DCM dichloromethane DIEA diisopropylethylamine DMF dimethylformamide DMSO dimethyl sulfoxide EtOAc ethyl acetate His histidine HOBt 1-hydroxybenzotriazole NMR nuclear magnetic resonance Phe phenylalanine PCM protein core mimetic PyBOP (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate RT room temperature TC temperature coefficient TFA trifluoroacetic acid TFAA trifluoroacetic acid anhydride THF tetrahydrofuran
ix
Chapter I
Introduction to Protein Mimicry
1 Protein Mimicry
Peptides are short, sequence- and length-specific oligomers composed of
amino acids. These familiar biomolecules are ubiquitous in living cells and assume
myriad roles. Each role assumed by a bioactive peptide will typically correspond to a
unique three-dimensional structure. Protein – protein interactions are crucial events
in most biological processes and are therefore important targets for drug design.
However, short peptides suffer from a number of disadvantages that compromise their
use as drugs. They are conformationally flexible, existing mainly as random
structures in aqueous solution. Peptides are also susceptible to degradation through
peptide bond cleavage by peptidases, have low bioavailability, and exhibit poor
pharmacological profiles due to rapid clearance rates, and poor membrane
permeability.1 Thus, while biomolecular interactions with peptides provide very useful clues for drug design, changes need to be made to create more pharmacologically acceptable drug candidates.
Early efforts to improve disadvantageous peptide nature have focused on the creation of non-natural peptide mimics. Design of these peptidomimetics is based on oligomers that mimic peptide primary structure through the use of amide bond isosteres and/or modification of the native peptide backbone. For example, properly designed azapeptides, oligocarbamates and oligoureas provide resistance toward proteases while retaining most of biological activity of the parent peptide.2 Another
approach of altering properties of peptides is the incorporation of conformationally
constrained non-natural amino acids.3 This provides a tool to control the position of
2 active residues in three-dimensional space and offers valuable information about
active conformations of peptides.
A new focus of peptidomimetic research - imitation of secondary structure
elements of peptides - is required to meet the growing knowledge of peptide folding
and molecular recognition events. Life depends on biological recognition and
binding. Many protein-DNA interactions4a are mediated through α-helical structure.
Many peptide ligand - receptor interactions and antigen-antibody interactions are
mediated through reverse turns.4b Proteases4c and kinases4d,e recognize their substrates through β-strand structures. Quite naturally, protein folding and
recognition processes became “hot” research topics both in academia and in the
pharmaceutical industry.
Molecular recognition events in biological systems depend on a characteristic
three-dimensional pattern of functional groups, which are presented on peptide active
surfaces. Understanding the nature of these processes provides valuable information
for rational design of small molecules that selectively inhibit the harmful protein
recognition events that lead to the onset of various diseases.5
The shape of proteins depends on multiple non-covalent interactions. These interactions define protein substructures or domains that are responsible for biological function. Most proteins contain a tightly packed hydrophobic core, which is covered by more polar amino acids that provide for binding, recognition or simply solubilization.6
Most of protein – protein interactions are mediated by a key local structure in
the protein. The contact area of binding domains is generally large, with 10-60 amino
3 acids participating in the binding event.7 Moreover, each patch of contact residues is presented from peptide segments that are often distant in the primary sequence. It was long thought that mimicking such large and discontinuous binding surfaces with rationally designed small molecules would be impossible. However, the task may be greatly simplified because only a small number of contact side chains appears to be necessary for tight binding.8
The goals of peptide mimicry are to create conformationally and metabolically stable peptidomimetics or nonpeptidic mimetics. To get the highest affinity for a receptor these molecules must be conformationally preorganized or fixed into a shape that is recognized by a receptor, because it reduces the entropy penalty for adopting the receptor-binding shape. Incorporation of essential functional epitopes on rigid minimal structural scaffolds may permit smaller molecules to be generated that bind at protein – protein interfaces.
The goal of this research was to design and synthesize small molecules that mimic structural and functional elements of the zinc finger containing proteins. Two fairly independent areas have been explored to determine stable minimal structure and minimal DNA binding motif of zinc fingers. The dissertation discloses my research efforts towards creating synthetic protein core mimetics and DNA-binding molecules.
4 Zinc Finger Proteins
The zinc finger is one of the major structural motifs involved in eukaryotic
protein – nucleic acid interactions.9 It appears that many proteins that contain zinc
finger domains are involved in DNA binding which in turn affects many aspects of
eukaryotic gene regulation. For example, such fingers occur in proteins induced by
differentiation and growth signals,10a,b in general transcription factors, 10c and in regulatory genes of lower eukaryotic organisms.10d,e An important question in
understanding crucial processes such as the control of gene expression in
differentiation and development is the manner in which DNA-binding protein
domains are able to discriminate between different DNA sequences. The zinc finger
motif has been studied extensively, with a goal of providing some insight into this
phenomenon.
Zinc finger motifs, which were first discovered in transcription factors,
usually contain cysteine and histidine. NMR studies have shown that these zinc
fingers contain an antiparallel β-sheet and an α-helix.11 The two cysteines, which are near the turn in the β-sheet region, and the two histidines, which are in the COOH- terminal portion of the α-helix, coordinate a central zinc ion. The finger forms a
compact globular domain (Figure 1). In addition to the zinc ion, each finger is also
stabilized by a hydrophobic core. This core contains a number of hydrophobic
residues (Phe, Leu, Val, Ile, and Thr), which form hydrophobic patches on both sides
of the finger and shield the zinc binding site from the solvent.
5
Figure 1. Structure diagram of the classical C2H2 zinc-binding motif. The C2H2 motif found among many transcription factors is comprised of a zinc knuckle (orange), a β-turn (red) and a short β-hairpin (purple) at the N-terminus followed by a small loop and an α-helix (blue).11c
Sequence comparisons and mutational analyses have been used to create a
model for the zinc finger – DNA interactions.12 Definitive information was obtained
from the crystal structure data of zinc finger peptide Zif268 – DNA complex.13 The
overall structure of the complex reveals why repeated zinc fingers of Zif268 are such
efficient motifs for protein – DNA recognition; the semicircular structure formed by
the three zinc fingers fits perfectly into the major groove of DNA. The α-helix of
each zinc finger fits directly into the major groove and residues in the NH2 – terminal portion of each α-helix contact the base pairs in the major groove. The Zif268 – zinc finger peptide makes 11 critical hydrogen bonds with the bases. All of these hydrogen bonds involve bases on the G-rich strand of the DNA. The β-sheet is on the back of the helix, away from the base pairs. The two strands of the β-sheet have very different roles in the complex. The first β-strand does not make any contacts with the
DNA, whereas the second β-strand contacts the sugar phosphate backbone along one strand of the DNA (Figure 2).
6
Figure 2. Overview of the Zif268–DNA complex, showing the side chains that make direct base contacts. The peptide is color-coded by finger: finger one is red, finger two is yellow, and finger three is purple. The DNA is shown in dark blue, and the zinc ions in pale blue.13
Simple covalent linkage of multiple zinc finger domains enables the recognition of extended asymmetric sequences of DNA. It is assumed that specific sidechain – sidechain and sidechain – base interactions determine binding specificity of the zinc finger tandems. A number of groups have used site-directed mutagenesis and phage – display selections in an attempt to find a simple set of rules relating the sequence of the zinc – finger protein to its corresponding DNA site.14 Although
7 patterns of typical contacts exist,15 the binding specificity of zinc finger peptides is
not fully understood.16
Regulation of transcription seems to be the most important task performed by the Zif268-like zinc fingers, but recent discoveries also suggest that this class of structures plays an important role in mediating protein - protein interactions.17
The simple mode of DNA binding and the ability to discriminate between closely related DNA sequences both in vivo and in vitro makes the zinc finger topology an ideal template for design of DNA binding proteins for gene expression control. The first example of the potential of this approach was published in 1994. A three – finger protein was constructed to block the expression of an oncogene that was transformed into a mouse cell line. It has also been shown that it is possible to activate a reporter gene by targeting a specific nine base promoter. Thus zinc finger peptides bound to the domains of genes can selectively switch these genes on or off.18
Ultimately, the most important application of zinc finger proteins might involve their use as therapeutics that control gene expression to cure or prevent diseases. In this regard, they might be used to activate or repress a wide range of genes, including ones that cannot be targeted with conventional pharmaceuticals. It is estimated that only 25-50% of the human genes implicated in disease will provide appropriate targets for small – molecule drugs.19 Using transcription factors based on zinc finger design could provide an alternative strategy for controlling genes involved in disease, including those involved in cancer, cardiovascular disease, viral infections, muscular dystrophy, cystic fibrosis, diabetes, glaucoma and chronic pain.20
8 It appears that typical zinc finger proteins can be divided into three fairly independent yet complementary domains that are responsible for unique DNA – binding efficiency. A few of the amino acids compose tightly packed hydrophobic core. The characteristic structural domain (also called ββα motif) is formed by a short β-sheet and an α-helix, which are held together by zinc ion. This structural domain also plays a role in orienting a zinc finger for DNA binding. The functional domain, containing nonconsecutive Arg and His residues that are incorporated in an
α-helix, is responsible for highly efficient binding (Figure 3). This modular composition makes the zinc finger an ideal model for the design of small synthetic molecules that bind DNA.
Figure 3. Cartoon representation of typical zinc finger protein containing a hydrophobic core (blue), three-stranded structural domain (red) and the DNA binding domain (green).
Using the zinc finger as a model, a number of small molecules were synthesized to identify minimal structural requirements necessary for DNA binding.
First, mimetics that emulate hydrophobic protein cores were designed with the aim of assessing the importance of nonbonding interactions of amino acid sidechains for the structural stability of the core. Second, based on previous successes in our research
9 group in creating small DNA-binding molecules, we designed the next generation of mimetics with the aim of improving binding efficiency and exploring DNA binding specificity.
10 Protein Core Mimetics
Creating compounds with protein-like secondary structures remains a
challenging endeavor. Impressive results have been obtained using nonnatural
oligomers that fold into well-defined three-dimensional structures referred to as
foldamers22 and β-turn mimetics23 that align peptide or peptidomimetic chains to form β -sheet structures.
The simplest peptide structural element is the peptide β-strand. The β-strand is a linear or sawtoothed arrangement of amino acids. The amide bonds are almost coplanar and the side chains alternate above and below the plane of the peptide backbone. Isolated β-strands are not common. β-Strands usually exist as hydrogen
bonded pairs, forming β-sheet structures in proteins. Normally considered a random
structure rather than a discrete element of protein secondary structure, the peptide β- strand is now known to be a crucial structural element.21
Combinations of two or more strands to form β-sheets not only act as important scaffolding elements to stabilize protein structure, but also are key recognition motifs that bind to other proteins or DNA. The β-sheet secondary
structure accounts for over 30% of all protein structure. It consists of two or more
paired β-strands arranged in either parallel, antiparallel, or mixed alignments that are
held together through interstrand hydrogen bonds.
Parallel β-sheets contain strands that run in the same direction and are
characterized by a series of 12-membered hydrogen-bonded rings (Figure 4A).
Antiparallel β-sheets contain strands that run in opposite directions and are
11 characterized by an alternating series of 10- and 14-membered hydrogen bonded rings
(Figure 4B). Mixed β-sheets contain mixtures of both patterns.
Figure 4. Schematic representation of (A) parallel β-sheet and (B) antiparallel β-sheet. Amino acid side chains are represented by R. Hydrogen bonds are indicated by dashed lines. Arrows indicate direction from N- to C-terminus.
Results from the literature indicate that incorporation of chain reversal and
intramolecular hydrogen bonding in mimetics is not sufficient to nucleate β–sheet
formation in sequences that are known to adopt β-sheet structure.24 These failures indicate that local conformational propensities are only part of what is required to achieve stable folding.
Successful folding is possible by using mimetics that promote favorable hydrophobic interactions between the β-turn mimetic and the attached peptide strands in addition to promoting chain reversal and intramolecular hydrogen bonding. Kelly showed that structures I and II (Figure 5) adopt a dynamic antiparallel β-sheet structure in aqueous solution. The efficacy of these residues as a β-sheet nucleator appears to result from the hydrophobic cluster conformation, which is formed by 15- membered hydrogen – bonded ring. The hydrophobic core in these oligopeptides is stabilized by the interactions between the aromatic skeleton and the hydrophobic side chains of the α-amino acid residues.
12 R O O R H N N N H H O N O O O H N O O N N N N H H H R R I II
Figure 5. Artificial β – structures that have stable hydrophobic cores around dibenzofuran I25a and dipyridine II25b scaffold.
Mimetics of protein β – structures could provide valuable information about
the nature of protein folding. β-sheet mimetics may also be very useful
pharmaceutical models, since aggregation of some proteins to form insoluble β-sheet structures is thought to be responsible for a number of neurological disorders. For example, there are over 30 diseases that are characterized by amyloid fibrils composed of proteins that have “misfolded” into β-sheets that aggregate into insoluble polymers.26
Aromatic – aromatic and aromatic – aliphatic interactions along with hydrogen bonds, electrostatic and van der Waals interactions play very important roles in providing stabilization energy for the protein native structure. Edge – to – face aromatic interactions have been invoked to explain the stability of certain protein folding motifs. The free energy contribution of the interaction depends on the environment of the aromatic pair. Energy calculations indicate that a typical aromatic
– aromatic interaction of an aromatic pair buried in hydrophobic core has energy of between – 0.6 and – 1.3 kcal/mol.27 Although the total contribution of this interaction
13 to the stability of the protein is small relative to other types of interactions, it is
comparable to the free energy of protein folding.28
While observations and speculations involving these interactions have been reported in many papers in chemistry29a-c and structural biology29d-f, the origin of
these interactions is imperfectly understood. Only a limited number of model
systems have been created that directly examine energies involved in aromatic –
aromatic and aromatic – aliphatic interactions. It has been shown that structure
stabilization of certain peptides is greatly reduced by substituting aromatic amino
acids that appear to be involved in edge – to – face interactions to Ala.30 To the
contrary, “molecular torsion balance” experiment performed by Wilcox (Figure 6)
failed to reveal any significant energetic difference between aryl – aryl and aryl –
alkyl contacts. This suggests that energy contribution of these two interactions to
protein folding is similar.31
Figure 6. The “molecular torsion balance” has two gently restricted conformational states. Folded and unfolded states are separated by a barrier > 18 kcal/mol, and direct observation of the populations of the two states is easily accomplished by NMR spectroscopy at 25 °C.
14
Chapter II
Synthesis and Physical Properties of Protein-Core Mimetics with Parallel
and Antiparallel Aligned Amino Acids.
15 Introduction
Our approach in creating compounds with protein-like secondary structures is
to use an aromatic or aliphatic assembly to stabilize the interactions between amino
acids or peptides. These protein-core mimetics (PCMs) were inspired by Kelly’s
isoquinoline-based β -sheet mimetics.25 In this system, hydrophobic side chains fold
back onto the isoquinoline ring, providing additional stabilization energy for β -sheet
formation. We postulated that a more stabilized core would be obtained by using a
synthetic scaffold to align aromatic rings in a T-shaped arrangement (Figure 7).
Formation of the core would theoretically not require a large entropic penalty for
folding. A T-shaped (or edge to face) arrangement of aromatic rings has been
observed in the X-ray structures of aromatic compounds,25a observed in proteins,33 studied using model systems,34 and investigated through theoretical calculations. 34e, 35
Figure 7. The number of aromatic rings held in a T-shaped arrangement determines whether a zippered or a crossed pattern of H-bonds is formed. A nonsynergistic relationship between the core and H-bonding residues, however, results in multiple structures.
The design of the PCM is based on the small hydrophobic cores (typically
containing Tyr, Phe, and Leu) found in the zinc finger peptides, which are often used
as a model system for protein stability studies. Although small, these cores provide
16 for a substantial amount of structural stability. Imperiali showed that modified
fingers without a metal binding site can give similar structures as the native finger.36
Mutating the highly conserved Phe of zinc fingers to Leu in a model of the Xfin-31 finger peptide37 and in a model finger of the human Y-encoded protein ZFY38 reduces
their stability. Interestingly, in the latter study, the instability is caused by an increase
in the finger’s dynamics.
Figure 8. (A) The original PCM39 that displays interactions between both aromatic side chains and one of the scaffold s aromatic rings and two isoenergetic H-bonds. (B) The new PCM also contains a scaffold and H-bonding plane, but its internal amines are not methylated.
In the initial study of our PCMs,39 the importance of aromatic rings in the formation of core structures was observed. Aromatic rings provide for more structure and H-bonds between Phe residues, when positioned in a T-shaped arrangement, as compared to scaffolds linked to Leu or to a scaffold without an aromatic core. The internal amides of these compounds were methylated (Figure 8A) to ensure that a 7- membered H-bonded ring would not form between one of these amides and a carbonyl group. We were concerned that this H-bonded ring would control structure formation and not the interactions between aromatic or aliphatic groups. In this study, we investigated the properties of PCMs without these Me groups. We wanted to determine whether a set of core residues could work synergistically with H- bonding residues to form a single stable structure. Nitrated compounds were created
17 and investigated to determine whether a change in the electrostatic forces of a core could enhance the strength of aromatic-aromatic interactions. We found that the nature of the side chain and its chirality determine core structures with either a zippered or crossed pattern of H-bonded rings (Figure 7). In some cases, however, strong aromatic-aromatic interactions can work against the H-bonds, resulting in multiple structures. Investigation of these flexible PCMs will lead to a better understanding of the dynamic instability observed in some peptides, such as in the mutated zinc finger peptides.
18 Mimetics with Parallel Aligned Amino Acids
Design and Synthesis
We designed a template that can accommodate multiple amino acids over an
aromatic base as our mimetic protein core (Figure 9). Incorporating different
combinations of L-phenylalanine, D-phenylalanine and L-leucine allowed us to
explore how interactions between aromatic base and sidechain residues of amino
acids influence H-bonding strength between the strands. To test the importance of the
T-stacking phenomenon, additional templates were created in which the electronic
properties of the aromatic system were changed or the aromatic system was removed
completely.
R O O
N CONHCH3 H N CONHCH3 R, R' = (L)-Bn, (D)-Bn H R' or (L)-i-Bu X = H or NO2
X
Figure 9. General design of mimetics with parallel aligned amino acids.
The synthetic route of the target molecules is outlined in Scheme 1.
Anhydride 3 was prepared according to a modified procedure utilizing a Diels-Alder
reaction between indene and maleic anhydride.40 Nitration of 3 was performed under neutral conditions using ammonium nitrate and trifluoroacetic anhydride (TFAA).41
3 or 4 was opened with a single equivalent of an amino acid N-methyl amide to produce a carboxylic acid, which was used without isolation. A second equivalent of
19 an amino acid was covalently linked using benzotriazol-1-yloxytripyrrolidino- phosphonium hexafluorophosphate (PyBOP) as a coupling reagent.42 These conditions produce diamides as major product in very good yields. Other coupling reagents such as CDI, chloroformates and DCC favor formation of undesirable imides. Alternatively, template 3 can be functionalized under the same conditions with commercially available amino acid methyl esters and the resulting diesters can be transformed to methyl amides with CH3NH2 at room temperature. Unfortunately, in the case of 8 and 9, the resulting mixture of diastereomers could not be separated and these core mimetics were not studied. The rest of the compounds were easily purified by flash chromatography.
R O O
O O O N CONHCH3 a O c H N CONHCH3 O + H R' O 1 X 2 X
5 X = H; R = R' = (L)-Bn 3 X = H b 6 X = NO2; R = R' = (L)-Bn 7 X = H; R = (L)-i-Bu 4 X = NO 2 8 X = H; R = (L)-Bn; R' = (D)-Bn 9 X = H; R = (D)-Bn; R' = (L)-Bn
Scheme 1. (a) p-hydroquinone, tetraline, sealed tube 190-200 °C, 5h, 38%; (b) NH4NO3, TFAA, DCM, 2h, RT, 88%; (c) 1 eq amino acid methyl amide, DMF, 12h, RT then DIEA, PyBOP, 1 eq amino acid methyl amide, 4h, RT.
Protein core mimetic 12 was prepared in a similar fashion (Scheme 2). Anhydride 11 was obtained in the Diels-Alder reaction between maleic anhydride and cyclohexadiene-1,3. Further functionalization was performed using the already established PyBOP procedure with phenylalanine N-methyl amide. N-methyl amides
20 were prepared from the commercially available Boc-protected amino acids using the standard coupling protocol with i-butyl chloroformate and methyl amine.
Ph
O O O O (L) O a b N CONHCH3 O + H O N (L) CONHCH3 H O Ph
110 11 12
Scheme 2. (a) toluene, sealed tube 100 °C, 3h, 80%; (b) 1 eq Phe-NHMe·TFA, DIEA, DMF, 12h, RT then DIEA, PyBOP, 1 eq Phe-NHMe·TFA, 4h, RT, 76%.
21 Experimental Methods
Hydrogen-bond strength determination.
Amide hydrogen-bond strength can be determined by measuring the temperature coefficients of the hydrogen-bonded protons (Figure 10). Temperature coefficients are obtained by measuring the chemical shift of the protons with respect to increasing temperature. In chloroform, amide protons freely exposed to the solvent have temperature coefficients near 2.4 ppb/K (either positive or negative); NH’s which remain hydrogen-bonded in the experimental temperature range are smaller than 2.4 ppb/K, and NH’s that undergo a transition from a bound to unbound state in the same temperature range are larger than 2.4 ppb/K. Chloroform was chosen because it should provide an environment that is similar to the hydrophobic interior of a protein. Additionally, a more hydrophobic solvent should reduce the ability of each template to adopt multiple conformations in solution. Independent verification of the formation of a hydrogen bond can be obtained by examining the N-H stretching region in the IR spectrum. A strong absorbance near 3300 cm-1 is evidence of a hydrogen-bonded amide N-H, while amides that do not form a hydrogen-bond appear near 3400 cm-1 and do not absorb as strongly.
22 280K
275K
270K
265K
260K
ppm (t1) 6.50 6.00
Figure 10. Typical variable temperature 1H NMR spectrum. With an increase in temperature NH proton signals move upfield and aromatic signals move downfield.
Structure Determination.
Template structures were investigated using a combination of two-
dimensional COSY,43a DQF-COSY,43b NOESY,43c TOCSY,43d, e and ROESY43f experiments. A search of the conformational space of the templates was performed using both a Monte Carlo method (MMFF94 force field as presented by Spartan Pro) and a manual search procedure (AMBER force field as presented by HyperChem).
Results of these techniques were compared, and global minima were consistent for each template.
23 Physical Properties.
Attaching L-Phe, D-Phe, or L-Leu to an aromatic scaffold gives a series of
protein core mimetics (PCMs) that align the amino acids in a parallel fashion (Figure
8B). Parallel means that both amino acids are attached to the scaffold through their
amino terminus. The physical properties of the compounds were investigated using
1D NMR analysis to determine H-bond stabilities and 2D NMR analysis for structural
information. To verify the existence of H-bonds, the FTIR spectrum of each
compound was examined for a H-bonded N-H stretching band,44 which is generally
observed between 3300 and 3360 cm-1. A free N-H band is observed between 3410
and 3450 cm-1.
The first compound constructed contains one L-Phe and one D-Phe. This
combination of amino acids, when methylated and attached to the original scaffold
(Figure 8A), produces a stable, unique PCM that displays two stable isoenergetic H-
bonds. Both aromatic side chains make contact with the scaffold’s aromatic ring.
The addition of one L-and one D-Phe to the new scaffold gave diastereomers, which
were separated by HPLC. 2D NMR analysis of one diastereomer revealed the
predicted multiple NOE cross-peaks between the scaffold’s aromatic ring and the
aromatic side chains and a cross-peak between the protons of the external Me groups.
The FTIR spectrum of the (L, D)-Pheparallel compound contains absorbance bands that are consistent with H-bonded N-H groups (data not shown). This compound, however, exists as multiple conformers at room temperature (Figure 11). H-bond stability was not determined because multiple conformations exist from room
24 temperature to 240 K.
1 Figure 11. An overlay of the H NMR spectra of (L, D)-Pheparallel and (L, L)-Pheparallel, showing that a correct matching of core with H-bonding residues will lead to a stable structure.
In an attempt to obtain stable and thus useful PCMs, only L-amino acids were
attached to the scaffold. The (L, L)-Pheparallel compound shows a single conformation
on the NMR time scale from 240 to 300 K (300 K spectrum is shown in Figure 11).
Plotting the chemical shifts of the amide protons against the temperature of the
experiments produced straight lines. Temperature coefficients (TCs) derived from
44a,b,f,45 the slope of these lines provide a measure of H-bond stability in CDCl3.
According to the literature, two N-H s of (L, L)-Pheparallel (TC = -1.3 and -2.2 ppb/K,
(Figure 12) would be considered as being either ring-locked (H-bonds in cyclic peptides) or sheltered from the solvent. The other two coefficients of -3.3 and -3.7 ppb/K are in the range found for non-H-bonded N-H s. Supporting evidence for the existence of H-bonds is the observation of an intense H-bonded N-H band in the
FTIR spectrum of (L, L)-Pheparallel (Figure 13). N-Ac-Phe-CONHMe, dissolved in
CHCl3 to give the same concentration of 3 mM, shows only a single non-H-bonded
N-H band.
25
Figure 12. Schematic drawings of the compounds showing key NOE cross-peaks (double- headed arrows). Temperature coefficients (standard deviations are less than (0.1) for amide N-H and aromatic Ar-H protons are given. Highlighted in (L, L)-PheNO2parallel are the observed diastereomeric differences.
Analysis of NOESY and ROESY spectra of (L, L)-Pheparallel revealed key
NOE cross-peaks: two between one aromatic side chain and the scaffold’s aromatic ring and one between the methyl groups of the external amides. The chemical shift of the ortho proton of the scaffold that is correlated to the side chain is shielded (δAr-H =
5.48 ppm) and greatly affected by temperature (TC = 5.7 ppb/K). These results are
consistent with a T-shaped arrangement of aromatic rings. As the solution was
cooled, the population of compounds with a scaffold proton in the shielding cone of
the side chain’s aromatic ring increased. To further demonstrate the importance of
26 the aromatic-aromatic interactions for structure, compound 12 (Figure 12), which does not have an aromatic ring, was constructed and investigated. In chloroform, a single H-bonded ring possibly forms (TC = -1.9 ppb/K), and the other N-H’s are not
H-bonded. Only a few NOE cross-peaks exist in its ROESY and NOESY spectra and none occur between the amino acid protons. Although a single conformer exists, this scaffold does not promote an alignment of the amino acids or extend the H-bonded network.
Figure 13. FTIR spectral data from the N-H stretching region of the PCMs and N-Ac- phenylalanine methyl amide. All samples were 3 mM in CHCl3 at 298 K. Each spectrum had its baseline corrected, and the absorbance of CHCl3 was subtracted.
Although these findings suggest that strong aromatic-aromatic interactions in
(L, L)-Pheparallel contribute to structural stability, another possibility is that the aromatic side chain is in a T-shaped arrangement because of restricted conformational freedom. In this case, stability would be a result of steric constraints. To explore this possibility, the electronic property of the scaffold was changed by the addition of a
27 nitro group. Favorable aromatic-aromatic interactions should increase35b with (L, L)-
PheNO2parallel. The nitro group pulls electron density out of the ring, making its Ar-H
more acidic and attracted to the electron-rich face of a stacked aromatic ring. (L, L)-
PheNO2parallel was isolated and investigated as an approximately 50:50 diastereomeric
mixture. Each diastereomer exists as a single conformer on the NMR time scale from
240 to 300 K. Both external amides are strongly H-bonded (TC = -6.2 and -6.6
ppb/K, Figure 12). One internal amide forms a moderately stable H-bond, and the
other internal amide is ring-locked or shielded (TC = -4.6 and -1.0 ppb/K,
respectively). As seen with the (L, L)-Pheparallel compound, one Ar-H of the scaffold is significantly shielded (δAr-H = 5.62 ppm) at room temperature. The chemical shift of this proton, however, is more temperature-sensitive (TC = 7.1 ppb/K) than the one observed for the (L, L)-Pheparallel compound (TC = 5.7 ppb/K; ∆TC = 1.4 ppb/K).
This larger temperature coefficient suggests that the nitrated benzenoid ring forms a more favorable aromatic-aromatic interaction than the benzenoid ring. Further evidence for the existence of more favorable aromatic-aromatic interactions was the detection of other temperature-sensitive chemical shifts for more than one scaffold
Ar-H (TC = 2.6, 1.4, and 0.5 ppb/K) and NOE cross-peaks between the scaffold’s aromatic ring and both aromatic side chains. Another different feature observed for
(L, L)-PheNO2parallel as compared to (L, L)-Pheparallel is the existence of an NOE cross- peak between each external Me group and the α-proton of the opposite amino acid
(Figure 12).
To further demonstrate the importance of aromatic-aromatic interactions, the properties of the compounds that contain phenylalanine residues are compared to a
28 compound that contains leucine residues. In previous study,39 we found that
aromatic-aromatic interactions provided for a different structure and H-bond stability
of PCMs when compared to aromatic-aliphatic interactions. (L, L)-Leuparallel (Figure
12) shows a single conformer on the NMR time scale from 240 to 300 K. Three strong H-bonds exist (TC = -8.0, -6.4, and -5.1 ppb/K), and the fourth N-H is not H- bonded (TC = -2.9 ppb/K). The properties of (L, L)-Leuparallel are remarkably similar
to the properties observed for (L, L)-PheNO2parallel. Both compounds show NOE cross-peaks between the external Me groups and between each external Me group and the α-C-H of the opposite amino acid. One major difference is that only one side chain of (L, L)-Leuparallel is positioned close to the scaffold’s aromatic ring. There are also differences in the stability of the H-bonded rings. (L, L)-Leuparallel contains a
very stable 7-membered H-bonded ring (TC = -8.0 ppb/K), and a close to zero
temperature coefficient is not observed.
29 Mimetics with Antiparallel Aligned Amino Acids
Design and Synthesis
The same aromatic scaffolding was used to create mimetics with antiparallel aligned amino acids. The β-turn-like element must be introduced in the mimetic to create an antiparallel alignment of strands. Scaffold III was envisioned as the turn inducer (Figure 14). It was functionalized with Phe and Gly to test the effect of possible edge – to – face interactions would have on the stability of newly synthesized core mimetics.
COOH
NH2
III
Figure 14. Amino acid that induces β-turn to aligned strands of mimetic in antiparallel fashion.
The synthetic route for the antiparallel mimetics is outlined in Scheme 3.
Anhydride 3 was obtained in the Diels-Alder reaction of indene with maleic anhydride. It was opened with sodium methoxide prepared in situ from MeOH/NaH.
The resulting acid was converted to the acyl azide 13. The azide is unstable to chromatography conditions and was used without further purification. Heating acyl azide 13 under reflux in a mixture of toluene and methanol afforded methyl carbamate 14 in good yield. Although deprotecting the methyl carbamate is quite problematic, all attempts to convert the isocyanate to free amine or more labile t-butyl
30 carbamate failed completely. Direct hydrolysis of the isocyanate in aqueous solutions yieded ureas. Reaction with t-BuOH to yield Boc protected amine failed as well.
Benzyl alcohol reacted quite efficiently. It was found later in the synthesis that the
Cbz – protected analogs of 16 were virtually inseparable.
Ester 14 was hydrolyzed under standard conditions, activated with CDI and coupled with (L)-phenylalanine. The resulting mixture of diastereomers was separated by radial thin-layer chromatography.
Ph
O O COOCH3 COOR O CON N CONHCH3 3 COOCH H a, b c N 3 e O N COOCH3 H H
14 R=CH3 16 a,b 313d 15 R=H
Scheme 3. (a) NaH, MeOH, RT, 3h; (b) ClCOO-i-Bu, NaN3, TFHaq, RT, 4h, 66% over 2 steps; (c) touene, MeOH, reflux, 5h, 74%; (d) LiOH·H2O, MeOH-H2O 2:1, RT, 3h, 90%; (e) CDI, DCM, then (L)-Phe-NHMe·HCl, RT, overnight, 73% conversion.
A variety of conditions were tested to remove the methyl carbamate functionality (Scheme 4).46 Only the TMSI deprotection protocol yielded amines in useful quantities.47 The resulting amines were coupled with (L)-phenylalanine, (D)- phenylalanine and glycine to provide mimetics 19-24.
31 Ph R O O 1 O
N CONHCH3 N CONHCH3 H H N NHAc a NH2 b 16a H R2
19 R1 = (L) - Bn; R2 = (L) - Bn 17 20 R1 = (L) - Bn; R2 = (D) - Bn 21 R1 = (L) - Bn; R2 = H
Ph R2 H O O N NH2 NHAc b a N CONHCH3 N O CONHCH3 16b H H R1
22 R1 = (L) - Bn; R2 = (L) - Bn 18 23 R1 = (L) - Bn; R2 = (D) - Bn 24 R1 = (L) - Bn; R2 = H
Scheme 4. (a) TMSCl, NaI, CH3CN, reflux, 20h; (b) Ac-Phe-OH of appropriate chirality or Ac-Gly-OH, PyBOP, DIEA, DMF, RT, 5 min, then 17 or 18, RT, 12h.
32 Physical Properties.
PCMs with antiparallel aligned L-Phe or D-Phe were constructed (Figure 15)
to test the ability of core interactions to control structure. Antiparallel means that one
phenylalanine is attached through its carboxylate and the other is attached through its
amine (Scheme 4). The N-H’s are more directly aligned with the carbonyl oxygen
atom of the opposite amino acid, which could have produced more stable H-bonded
rings as compared to the parallel compounds. Separation of diastereomers 16a and
16b led to two diastereomeric sets 1 and 2 (Figure 16). The addition of a L-Phe or D-
Phe to each diastereomer produced four antiparallel aligned PCMs, which will be
referred to as (L, L)1-Pheantiparallel, (L, D)1-Pheantiparallel, (L, L)2-Pheantiparallel, and (L,
D)2-Pheantiparallel.
Figure 15. Key NOE cross-peaks (double-headed arrows), temperature coefficients for amide N-H and aromatic Ar-H protons, and FTIR spectral data from the N-H stretching region are given for the antiparallel compounds.
33 One compound from each diastereomeric set (1 and 2) shows a single
conformation on the NMR time scale from 240 to 300 K. The other two compounds
display multiple conformations. For the structurally stable (L, L)-Pheantiparallel compound (Figure 15), a stable H-bond and a very stable H-bond exist for one external N-H (TC = -6.3 ppb/K) and the internal N-H of the other phenylalanine residue (TC = -7.6 ppb/K), respectively. For the structurally stable (L, D)-Pheantiparallel
compound, both external amides are involved in moderately stable H-bonds (TC = -
4.5 and -4.3 ppb/K) and one internal N-H forms a very stable H-bond (TC = -7.0
ppb/K). For both compounds, the chemical shift of one internal N-H is temperature-
insensitive, which suggests that it was shielded or existed in a ring-locked H-bond.
The FTIR spectra for the antiparallel compounds (Figure 15) are significantly
different than those observed for the parallel compounds. Multiple peaks are
observed for these compounds at a lower frequency than normally found for H-
bonded amides (low-frequency bands have been observed for β-sheet mimetics).44d
The same IR cell was used for all spectra shown in Figures 13 and 15, and the maximum absorbances were within 0.07-0.1 absorbance units. Therefore, the low- frequency bands observed for the antiparallel cores are not an artifact of the IR cell or the experiment. Multiple NOE cross-peaks between both aromatic side chains and the scaffold’s aromatic ring are observed for both compounds. For (L, D)-
Pheantiparallel, a unique cross-peak exists between its α-protons and no cross-peak is
found between its external Me groups. For (L, L)-Pheantiparallel, a NOE cross-peak is observed between the external Me groups. As seen with the parallel compounds, the spatial arrangement of the aromatic side chains of the antiparallel compounds has a
34 pronounced affect on their structures.
Figure 16. The diastereomeric sets of the antiparallel compounds, a cartoon depiction of their calculated stable structures highlighting the orientation of the aromatic rings (side chain rings are given as rectangles), and whether a single conformer is observed.
35 Discussion
The experimental results show that not all PCMs have a single conformation in chloroform. The structurally stable compounds show a different number of core interactions and H-bonding patterns depending on whether they contained phenylalanine residues, leucine residues, or a substituted aromatic ring. To understand the synergistic relationship between the interactions, a Monte Carlo method was used to search the conformational space of the compounds (MMFF94 force field, package procedure of SpartanPro).48 A series of low-energy conformers were obtained that contained a zippered or a crossed pattern of H-bonds (Figures 7 and 17). One or the other pattern is consistent with the observed properties of the
PCMs. For the parallel compounds, the zippered pattern contains 7- and 12- membered H-bonded rings, and the crossed pattern contains one 7-membered and two
10-membered H-bonded rings. For the antiparallel compounds, the zippered pattern contains 8- and 12-membered H-bonded rings, and the crossed pattern contains 8-,
11-, and 12-membered H-bonded rings. For the antiparallel compounds, the length of each H-bonded ring was considered independent of the other rings. Both patterns of
H-bonds require the internal carbonyls to form an up-down pattern.
36
Figure 17. A zippered (dashed double-headed arrow) or a crossed (solid double-headed arrow) pattern of H-bonds exists between the amino acids of the parallel aligned compounds and the antiparallel aligned compounds. The numbers indicate the length of a H-bonded ring. R is a set of L-Phe, D-Phe, or L-Leu and X is H or NO2.
The necessity of this pattern explains the lack of an observed single conformer for (L, D)-Pheparallel (Figure 11). One low-energy conformer observed in the model study shows a zippered pattern of H-bonds. As seen in this structure (Figure 18), one aromatic ring forms a T-shaped arrangement with the scaffold’s aromatic ring and the other aromatic ring is positioned to also interact with the core. For one conformer, we observed NOE cross-peaks between the scaffold aromatic ring and an aromatic side chain and the existence of a shielded scaffold aromatic proton (5.82 ppm, Figure
11), which are consistent with T-shaped aromatic rings. We speculate that the formation of the second T-shaped arrangement gives the observed multiple structures.
According to the molecular modeling results, when the second aromatic side chain forms a T-shaped arrangement with the scaffold’s aromatic ring, its internal carbonyl shifts up and away from the face of this aromatic ring. Both internal carbonyls are now positioned up, and the 7-membered H-bonded ring breaks. Apparently, the stabilizing energies provided by the 7-membered H-bonded ring and the second T- shaped aromatic interaction are similar. Because these two interactions promote different structures, a nonsynergistic relationship exists for structure formation.
37
Figure 18. Low-energy structures obtained from molecular modeling studies that are consistent with the observed properties of the compounds. For (L, D)-Pheparallel, moving the second aromatic side chain to interact with the scaffold’s aromatic ring forces the internal carbonyl to rotate up (indicated by arrows), which breaks the 7-membered H-bonded ring. The R-proton is shown to indicate that the aromatic interactions do not require bond rotation around the amide bond. For (L, L)-PheNO2parallel as compared to (L, L)-Pheparallel, the more favorable aromatic interactions drives the second aromatic side chain to the scaffolds ring, which rotates the amide bond (indicated by arrows), giving the crossed pattern of H-bonds. This phenomenon also occurs for (L, D)2-Pheantiparallel. T indicates a T-shaped arrangement of rings, and H-atoms not involved in H-bonds are removed for clarity.
The position of the amino acid side chains relative to the scaffold’s aromatic ring dictates which pattern of H-bonds forms. This statement is readily evident when comparing the properties of (L, L)-Pheparallel to (L, L)-PheNO2parallel. In the latter compound, both aromatic side chains are positioned close to the scaffold’s aromatic ring. According to the molecular modeling studies, when the second aromatic ring is
38 placed near the scaffold’s ring, the external amide of this amino acid rotates toward
the internal carbonyls (Figure 18). This rotation gives (L, L)-PheNO2parallel a crossed pattern of H-bonds, which is consistent with its three stable H-bonds and the NOE cross-peaks between each external methyl group to the opposite amino acid’s α- proton. The lack of this cross-peak and the existence of an NOE cross-peak between the external methyl groups suggests that the (L, L)-Pheparallel compound forms a
zippered pattern. Although the temperature coefficients of the possible H-bonded N-
H’s observed for (L, L)-Pheparallel are surprisingly close to zero, the observation of a
strong H-bonded N-H stretching band in the FTIR spectrum supports the existence of
a H-bond. Apparently, the 7- and 12-membered H-bonded rings are the preferred
pattern when one set of benzenoid rings interacts. The less stable crossed pattern is
forced to form when both aromatic side chains interact with the scaffold’s aromatic
ring. This result shows that nitrobenzenoid-benzenoid interactions are more
stabilizing than benzenoid-benzenoid interactions. The enhanced stability is most
likely a result of a more stable H-bond49 or a greater π−σ interaction50 and consistent with the results obtained by Sherrill,35b who performed a computational analysis of the interaction energies between substituted aromatic rings. Any possible steric hindrance imposed by the NO2 group would have only lessened the favorable aromatic interaction energies.
Differences in aromatic-aromatic interactions as compared to aromatic- aliphatic interactions are readily apparent when comparing the properties of (L, L)-
Pheparallel and (L, L)-Leuparallel. As predicted, there is a closer association of the
aromatic core residues as evident by the greater number of NOE cross-peaks for (L,
39 L)-Pheparallel as compared to (L, L)-Leuparallel. The patterns of H-bonds are also different. Surprisingly, the structures and properties of (L, L)-Leuparallel are very similar to those of (L, L)-PheNO2parallel. Both compounds display the required three stable H-bonds and the key NOE cross-peaks between each external methyl group to the opposite amino acids α-proton. Does this mean that benzenoid-aliphatic
interactions are more stable than the interactions between benzenoid rings and
equivalent in energy to interactions between nitrobenzenoid-benzenoid rings? The
answer is probably no. Only a single NOE cross-peak is observed between one
isobutyl side chain and the scaffold’s aromatic ring for (L, L)-Leuparallel, which suggests that extensive aliphatic-aromatic interactions do not occur. Most likely, the very stable 7-membered H-bonded ring of (L, L)-Leuparallel forced the crossed pattern of H-bonds. The N-H in this ring has a temperature coefficient (-8.0 ppb/K) that is substantially smaller than the ones observed for the other parallel compounds and even smaller than the ones observed for the antiparallel compounds. Additionally, steric interactions between the side chains most likely kept them from both residing above the H-bonding plane.
To determine whether the observed structures of the PCMs with antiparallel aligned amino acids depend on the spatial arrangement of the side chains, we had to assign the stable diastereomers. One diastereomer contains two L-Phe and the other contains one L-Phe and one D-Phe. According to the synthetic route, all compounds contain a L-Phe that is attached to the scaffold through its amine.
Because these diastereomers were separated before the attachment of the second amino acid, we know that a compound belongs to one of the two sets of
40 diastereomers, which are called set 1 and 2 (Figure 16). For example, one compound
containing two (L)-Phe’s could either be (L, L)1-Pheantiparallel or (L, L)2-Pheantiparallel.
Results obtained from the 2D NMR experiments and molecular modeling studies
were used to assign the conformationally stable antiparallel compounds. Molecular
modeling results show that a stable zippered pattern of H-bonds should be observed
for one diastereomer of the (L, L)1-Pheantiparallel and (L, L)2-Pheantiparallel pair with both
aromatic side chains in contact with the scaffold’s aromatic ring (Figure 18 and
diastereomer 22 of Figure 16). The other compound (diastereomer 19) would have
had both side chains above the H-bonding plane. (L, L)1-Pheantiparallel displays a zippered pattern and NOE cross-peaks between both aromatic side chains and the scaffold’s aromatic ring and thus is assigned as the stable diastereomer 22. The assignment of (L, L)1-Pheantiparallel removes the possibility that (L, D)1-Pheantiparallel is a stable PCM because only one compound of this diastereomic set displays a single conformer. Thus, the other stable diastereomer has to be the other compound that contains one L-Phe and one D-Phe, which we call (L, D)2-Pheantiparallel (diastereomer
20). For this compound, we observe multiple NOE cross-peaks between both
aromatic side chains and the scaffold’s aromatic ring, a cross-peak between the α-
protons, and no cross-peak between the external Me groups. A low-energy structure
consistent with these results was obtained in a molecular modeling study (Figure 18)
by constraining the aromatic rings at a distance observed for T-shaped aromatic
interactions (5 Å).33b This structure nicely shows both external amides being H-
bonded, a close proximity of the α-protons, and far-separated external Me groups.
The modeling results also demonstrates that when the second aromatic ring is
41 positioned near the scaffold s aromatic ring, its external carbonyl rotates back toward
the scaffold, giving the crossed pattern of H-bonds. This phenomenon also occurs for
(L, L)-PheNO2parallel.
One consistent pattern observed for the Phe-containing compounds is that at
least one aromatic side chain has to interact with the scaffold’s aromatic ring to obtain
a single, stable structure. In the cases of (L, L)-PheNO2parallel and (L, D)-Phe2antiparallel,
the second aromatic side chain also makes contact with the scaffold’s aromatic ring,
resulting in a crossed pattern of H-bonds. In the case of (L, L)-Phe1antiparallel, both
aromatic side chains interact with the scaffold’s aromatic ring, but a zippered pattern
is formed. The lack of a single conformer for (L, D)-Pheparallel (Figure 11) demonstrates that a synergistic relationship will not always occur. Another nonsynergistic relationship appears to occur for (L, D)-Phe1antiparallel (diastereomer
23). Its instability was unexpected. A comparison of the cartoon structures of
diastereomer 23 with 20 as drawn in Figure 16 suggests that both compounds should
have had a stable structure with one pair of interacting rings. Molecular modeling
results bolstered that expectation by showing both diastereomers existing in a stable
zippered pattern of H-bonds with one T-shaped, aromatic arrangement. Although
both aromatic side chains of diastereomer 20 interact with the scaffold’s aromatic
ring, a stable structure is formed. Most likely, two sets of T-shaped arrangements of
rings is formed for diastereomer 23 as well. Unlike with diastereomer 20, molecular
modeling results show that favorable H-bonds are disrupted when the second
aromatic side chain is placed near the scaffold’s aromatic ring of diastereomer 23.
One major difference between diastereomers 23 and 20 is the nature of the H-bonding
42 atoms in the 7-membered ring for the amino acid side chain that is drawn close to the scaffold in Figure 16. For diastereomer 20, its carbonyl is involved in the 7- membered ring. For diastereomer 23, its N-H would be involved in the 7-membered ring. Apparently, this subtle difference is enough to disrupt a synergistic relationship between the core and H-bonding residues for diastereomer 23.
43 Conclusion
A series of protein-core mimetics were constructed to investigate the synergistic relationship between an assemblage of aliphatic or aromatic side chains and H-bond forming groups of the amide backbone that is required to produce stable protein cores. Although H-bonds are very stable in chloroform (used to represent the core environment) and could have dominated the PCM properties, interactions between aromatic or aliphatic groups either stabilize the H-bonds, alter the pattern of
H-bonds, or disrupt the H-bonds. A greater number of NOE cross-peaks are observed between core residues for a PCM with a more acidic Ar-H, which is most likely caused by more favorable T-shaped aromatic interactions. Besides the observation of
NOE cross-peaks, experimental evidence for aromatic interactions is obtained through the observation of temperature-sensitive chemical shifts of aromatic protons.
Our PCMs could potentially provide an experimental measure of T-shaped aromatic interaction energies. Aromatic interactions are not required to obtain a stable core.
The structural stability of the Leu-containing compound, however, most likely arose through a very stable H-bond and steric constraints and not through core interactions.
The study of our PCMs demonstrates that subtle differences in the spatial arrangement of the interacting functional groups of amino acids have a great impact on the properties of the mimetics. Considering that protein cores are much more complex than our model systems, it is not surprising that the a priori creation of proteins remains a challenging endeavor.
44
Chapter III
Design and Synthesis of Small Molecules that Bind DNA
45 Introduction
The recent advances in human genome project makes, gene regulation of transcriptional processes a reality. Transcription can be inhibited if a DNA-binding drug alters the interactions between a promoter region and some transcription factors.
It has already been shown that small drug molecules can compete with transcription factors for the same DNA sequences.51 Many antitumor drugs presently in clinical use perform their activity by binding to DNA. Creating genetic medicines that work at transcriptional level provides an important alternative in curing diseases, many of which cannot be targeted with conventional pharmaceuticals.
Proteins that contain zinc fingers are involved in many aspects of regulation of genetic information. Revealing the specificity code of zinc fingers would allow specific targeting of any DNA sequence. Compact structure and fairly simple mode of action of zinc finger’s ββα motif makes it a very attractive model for mimetic design.
We have long been interested in design of small DNA binding molecules based on zinc finger topology. Early efforts in our research group provided very encouraging results.52 Mimetic IV, which contains hydrophobic core and simple
Arg-His dipeptide (Figure 19), showed remarkable affinity toward short DNA sequences. As expected, the core itself or isolated Arg-His dipeptide interact with
DNA only poorly. This indicates the importance of preorganized geometry of the full mimetic in DNA recognition.
46 TFA · H2N NH
NH
O O H N DAE · TFA N N H O O N
NH · TFA IV
Figure 19. First generation DNA binder containing hydrophobic core, Arg-His recognition strand and fluorescence tag DAE (dansyl group with ethylene diamine linker).
Because zinc fingers exhibit general specificity for G-rich regions of DNA,
53 duplexes of a modified Dickerson dodecamer d(CGGGAATTCCCG)2 mDd and
d(AAAAAAAAATTTTTTTTT)2 d(A9T9)2 were chosen as the targets. Fluorescence spectroscopy was used to determine DNA binding affinities of various mimetics.
Fluorescence quenching spectra and association constants of mimetic IV are shown on Figure 20.
mDd d(A9T9)2 4 -1 4 -1 KA (x 10 M ) KA (x 10 M ) IV 10.4 ± 0.4 18.9 ± 1.4 IV + 0.5 M NaCl 7.5 ± 0.1 2.4 ± 0.5
Figure 20. Representative fluorescence quenching assays of mimetic IV binding to mDd (A) and d(A9T9)2 (B) in 0.1 M phosphate buffer pH 7.0 at 25 ºC. Association constants for compound IV determined using modified Stern-Volmer equation.
47 The experimental results show that mimetic IV has KA in the range generally observed for small compounds that bind to the major or minor groove of DNA.
Although the binding affinity of IV towards mDd and d(A9T9)2 appears to be similar
(after adjusting KA with respect to the DNA sequence length), the binding mechanism is different. The fact that association constant drastically diminishes with increase of salt concentration indicates that interaction between IV and d(A9T9)2 has substantial
electrostatic component which is characteristic for phosphate backbone binding
mode.54 Fluorescence and circular dichroism spectroscopy data as well as molecular
modeling suggest that interaction between IV and mDd is consistent with binding to
the major groove of DNA.55
48 Design and Synthetic Efforts
The experimental data discussed in the previous section suggests that
mimetics with a hydrophobic core and Arg-His dipeptide are suitable models for
further design of DNA binding molecules. Association constants of first generation
mimetics are still far from the 109 M-1 association constant typically observed for zinc
finger proteins. Introducing additional peptide strands to the hydrophobic scaffold
will increase possible contact points with DNA, which should result in increased
affinity and specificity.
In addition to the major groove binder already present in the molecule, the
mimetics can be derivatized with an intercalator, such as acridine.56
Functionalization of the aromatic ring provides another attachment point for oligopeptides containing lysine residues, which are known to bind to DNA at the phosphate backbone (Figure 21).57 Docking experiments performed with HyperChem supported our hypothesis that geometry of the newly designed mimetic is favorable for multiple mode interactions with DNA.
Major groove binding
H2N NH
NH Intercalator N O O H H N N N N N H H Hydrophobic O O N O core NH
O NH2 HN NH
O HN n
Phosphate backbone binder
Figure 21. Proposed design of the second generation mimetic that will bind DNA.
49 In the initial approach toward creating a DNA binding mimetic we decided to
functionalize the aromatic ring at the beginning of the synthesis and leave the
attachment of Arg-His-intercalator sequence to a later stage.
Anhydride 3 was nitrated using an established procedure. Nitro anhydride 4
was treated with methanol under acidic condition to afford diester 25 in good yield.
Hydrogenolysis using palladium catalyst provided almost quantitatively aniline 26,
which was coupled with Boc-protected glycine using either CDI or DCC activation
protocol. Hydrolysis smoothly converted diester 27 into diacid 28.
Unfortunately with 28 in hand it was impossible to add any dipeptide or single
amino acid to the carboxylic functionality. Alternatively, we tried to convert 28 into
an anhydride, but all our attempts using Ac2O, (COCl)2, SOCl2 to induce the closure
were fruitless.
O O CO2CH3 O O CO2CH3 abO c O
NO2 3 42NO2 5
CO CH CO H CO2CH3 2 3 2 CO CH CO H CO2CH3 2 3 2 d e
O O NHBoc NHBoc NH N N 26 2 27 H 28 H
Scheme 5. (a) NH4NO3, TFAA, DCM, 2h, RT, 88%; (b) MeOH, cat H2SO4, reflux, 3h, 88%; (c) H2, 10% Pd-C, MeOH-DMF 5:1, 8h, 97%; (d) Boc-Gly-OH, CDI, CHCl3, RT, 2h, then 26, RT, 12h, 85%; (e) LiOH·H2O, MeOH-H2O 3:1, RT, 12h, 88%.
50 The necessary amino acids to build a recognition strand were prepared according to
Schemes 6 and 7. To produce greater flexibility in the synthetic strategy an orthogonal protection was employed. Using a fully protected amino also significantly simplifies purification.
BOM-Cl was prepared according to literature procedure.58 Histidine ester hydrochloride 32 was first converted to N1-Boc derivative which was then converted to N3-Bn histidine 34 upon treatment with BnOTf59 or N3-BOM histidine 35 with benzyloxymethyl
chloride.60
OH a O SMe b O Cl
29 30 31
NH NBoc N c d N N N R
Cl H3N CO2CH3 BocHN CO2CH3 BocHN CO2CH3 32 33 34 R = Bn 35 R = Bom
Scheme 6. (a) 29, Ac2O, AcOH, DMSO, RT, 40h, 56%; (b) SO2Cl2, DCM, RT, 1h, 79%; (c) Boc2O, Et3N, MeOH, RT, overnight, 92%; (d, R=Bn) Tf2O, BnOH, DIEA, DCM, -78°C, 40 min, then 33, 20h, 51%; (d, R=Bom) BOMCl, DCM, RT, 12h, 93%.
Preparation of the fully protected arginine 41 is outlined in Scheme 7. A guanylation reagent61 was first prepared from amidine 36. Hydrogenolysis of Cbz-
ornithine 39 afforded amine 40, which was smoothly alkylated using 38 to give
arginine 41.61b
51 H2N CbzHN CbzHN NH NH NCbz a b N N N N N N
38 36 37 CbzHN NCbz
NHCbz NH2 NH c d
BocHN CO H 2 BocHN CO2H BocHN CO2H 39 40 41
Scheme 7. (a) Cbz-Cl, DIEA, DCM, 0°C to RT, 2h, 91%; (b) NaH, Cbz-OSu, THF, 0°C to RT, 3.5h, 87%; (c) H2, 10% Pd-C, EtOH-DMF 1:1, 5h, 74%; (d) 38, DIEA, DMF, 12h, 45°C, 87%.
Orn-His dipepetides 44 and 45, which were designed for building a
recognition strand, were assembled according to Scheme 8. Appropriately protected
histidine 35 was treated with TFA to give free amine 42, which was smoothly coupled with N-Boc-Orn(Cbz)-OH using the DCC coupling method to give 43. Both variations of Orn-His dipeptide with free N- and C-terminus (44 and 45 respectively) were prepared to enable better synthetic flexibility in assembling the final molecule.
To assemble the imide moiety in the mimetic, either arginine or Orn-His dipeptide could be used to open nitro anhydride 4. Unfortunately, we could not selectively reduce the nitro functionality to aniline, which is required for attaching polylysine.
Conditions62 that are known to reduce nitrobenzenes in the presence of Cbz-protected
amines in our case readily reduced both functionalities.
52 N
N O N N Bom BocHN N CO2CH3 N a N b H Bom Bom
BocHN CO2CH3 H2N CO2CH3 NHCbz 35 42 c d 43
N N
O N O N Bom Bom H2N BocHN N CO2CH3 N CO2H H H
NHCbz NHCbz 44 45
Scheme 8. (a) TFA, DCM, RT, 1h, 91%; (b) N-Boc-Orn(Cbz)-OH, HOBt, DCC, CH3CN, RT, 5h, 64%; (c) TFA, DCM, RT, 1h, quant.; (d) LiOH·H2O, MeOH-H2O 2:1, RT, 12h, 65%.
An alternative approach for construction of the imide is described in Scheme
9. Anhydride 4 was opened with single equivalent of Orn-(Cbz)-CH3 and then
converted to the imide 46 with oxalyl chloride. Hydrogenolysis gave the fully
reduced amine 47. Alkylation of 47 with amidine 38 under various conditions gave
only traces of arginine 48. To avoid possible lactam formation, 47 was hydrolyzed to
give carboxylic acid 49, but again guanylation of this substrate was unsuccessful.
53 CbzHN NCbz
NHCbz NH2 NH
O O O O O N CO2CH3 N CO2CH3 N CO2CH3 a b c O O O O
NO2 NO2 NH2 NH2 4464748 d
CbzHN NCbz
NH2 NH
O O
N CO H N CO H 2 c 2 O O
NH2 NH2 49 50
Scheme 9. (a) Orn(Cbz)-OCH3·HCl, DIEA, DMF, 65°C, 2h, then (COCl)2, DCM, RT, 12h, 71% over two steps; (b) H2 20 psi, 10% Pd-C, 1.5h, 96%; (c) 38, DMF.
Scheme 10 outlines the synthetic effort toward the acridine intercalators.
Chloroacridine 52 was prepared in a single step from N-phenyl-antranilic acid.63
Alkylation of acridine 52 with mono N-Boc-ethylene diamine64 gave very poor yields of 53. Further optimization of conditions is required.
Acridine derivative 54 could be used as an intercalator, which is in fact better suited for fluorescence spectroscopy as a tag. 54 was mono protected as a benzyl carbamate 55.65 Unfortunately, attempts to introduce a linker failed. No desired
product was detected in reaction of 55 with glutaric anhydride 56 or coupling of amino acid 57 using SOCl2.
54 BocHN CO2H
Cl NH NH a b
N N 51 52 53
c
H2N N NH2 H2N N NHCbz 54 55
O
O BocHN CO2H
O 57 56
Scheme 10. (a) POCl3, reflux, 3h, 72%; (b) mono N-Boc-ethylene diamine, DMF; (c) Cbz- Cl, acetone, reflux, 14h, 32%.
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67 Experimental section.
General Experimental. 1H and 13C NMR spectra were acquired using a
Bruker AC250 and a Bruker AMX400 or a Bruker Avance 400 spectrometers operating at 250.13 MHz and 400.14 MHz for proton and 62.90 MHz and 100.62
MHz for carbon nuclei, respectively. Chemical shifts are in ppm and are referenced using an internal TMS standard. The following are the typical parameters used in the
2D NMR studies: NOESY were obtained at 298 K; mixing time of 150 and 300 ms; and FID acquisition time was 0.15 s. Other parameters were: SW = 5800 Hz, 2K data points and 256 or 512 increments each with 16 transients per FID. TOCSY experiments were run with a 300 ms mixing time, L1 =118 and low power pulse of 15 dB. ROESY experiments were obtained at 298 K with a cw pulse power of 30 dB and a mixing time of 400 ms. A QSINE window function was applied in both dimensions with ssb=2 and the data was phased in the usual fashion. Homonuclear proton – proton decoupling experiments were run on the Bruker AC250 operating at
249.99 MHz.
All syntheses were carried out under positive argon pressure, and DMF was freshly distilled under vacuum over Mg2SO4. K2CO3 was powderized and dried under vacuum for 1 h prior the use. Unless otherwise noted, all other solvents and reagents were used as purchased. A MELTEMP (Laboratory Devices) apparatus was used to determine the melting points of the compounds; these values are given uncorrected.
HPLC analysis was performed on a Shimadzu 10A series HPLC using a C18 reversed
68 phase column and water (0.1% TFA)/CH3CN as the eluent. Water for the HPLC assays was purified on a Millipore water purification system.
Calculated Structure Determination. Global minimum structures of the templates were determined through a Monte Carlo search method using the MMFF94 force field (package procedure of Spartan Pro48). Generally, over 1500 structures were created and analyzed for potential energy through a simulated annealing process starting at 5000 K and ending at 300 K. Molecular dynamic simulations were also performed using the AMBER force field as presented by Hyperchem. A series of conformers were generated for each template by heating them to 800 K. At this temperature, the H-bonds were periodically broken and the side chains could swap their positions, i. e., above and away from the base of the template or closer to the template. A total simulation time of 5 ps was used with 1 fs steps. Random conformers were chosen (ca 20) during the simulation process and minimized using the Polak – Ribiere minimization algorithm (RMS gradient of 0.005 kcal/ Åmol).
Most conformers minimized to a single conformer that was over 1 kcal/mol lower in energy than the other minimized structures. The lowest energy conformers obtained for each template matched the ones obtained from the Monte Carlo search.
69 Experimental Procedures
O
O O
Anhydride (3). A solution of maleic anhydride (25 g, 0.25 mol) and p- hydroquinone (10 g, 0.09 mol) in indene/tetraline (45 mL/ 35 mL) was heated at 190-
200 ºC for 5h with constant stirring. The reaction mixture was cooled to 60-70 ºC and poured into benzene (200 mL). Insoluble byproducts were filtered and filtrate concentrated in vacuum. Crude product was filtered and recrystallized from EtOAc
1 to afford 20.3 g (38%) of 3 as white crystals. H NMR (CDCl3, 250 MHz) δ 7.23 (m,
4H), 3.92 (s, 2H), 3.79 (s, 2H), 2.16 (d, J=10 Hz, 1H), 1.95 (d, J=10 Hz, 1H); 13C
NMR (CDCl3, 62.9 MHz) δ 170.7, 141.5, 128.0, 123.0, 52.6, 48.7, 47.2.
O
O O
NO2
Nitro anhydride (4). To a solution of 3 (107 mg, 0.5 mmol) in CH2Cl2 and trifluoroacetic anhydride (2 mL/ 0.5 mL) NH4NO3 was added and reaction mixture
was stirred for 2h at room temperature. After completion as judged by thin–layer
chromatography reaction mixture was washed with H2O (3×20 mL) and combined
organic layers were dried over Na2SO4. After evaporation crude product was
recrystallized from AcOEt to afford 4 (114 mg, 88%) as a tan solid. 1H NMR
70 (CDCl3, 250 MHz) δ 8.16 (d, J=7.5 Hz, 1H), 8.14 (s, 1H), 7.46 (d, J=7.5 Hz, 1H),
4.05 (s, 2H), 3.92 (s, 2H), 2.25 (d, J=10 Hz, 1H), 2.08 (d, J=10 Hz, 1H); 13C NMR
(CDCl3, 62.9 MHz) δ 169.7, 148.9, 147.9, 143.5, 124.0, 123.8, 118.3, 52.9, 48.2,
47.1.
O
O O
Anhydride (11). Maleic anhydride (0.98 g, 10 mmol), cyclohexadiene-1,3
(1.15 mL, 12 mmol) and toluene (2 mL) were placed in a sealed tube and mixture was stirred for 3h at 100 °C. Solvent was evaporated in vacuum, product filtered and
1 recrystallized from CH2Cl2. 9 (1.54 g, 80%) was obtained as colorless needles. H
NMR (CDCl3, 400 MHz) δ 6.31 (m, 2H), 3.20 (m, 2H), 3.16 (s, 2H), 1.62 (d, 2H,
13 J=7.6 Hz,), 1.40 (d, 2H, J=7.6 Hz,); C NMR (CDCl3, 100 MHz) δ 172.8, 132.9,
44.6, 31.5, 22.8.
Parallel templates (5, 6, 7, 12). Leu-NHMe·TFA (73 mg, 0.28 mmol) and
DIEA (49 µL, 0.28 mmol) were added to a solution of 3 in DMF (1.5 mL). Reaction
mixture was stirred at room temperature for 12h. To stirring solution DIEA (49 µL,
0.28 mmol) and PyBOP (146 mg, 0.28 mmol) were added and immediately followed
by Leu-NHMe (0.28 mmol). After stirring for 4h at room temperature solvent was
evaporated, residue dissolved in CH2Cl2, washed with 1M HCl, saturated NaHCO3 solution and brine. After evaporation crude product was purified by flash chromatography.
71 Bn O O
N (L) CONHCH3 H N (L) CONHCH3 H Bn
1 1 5 (111 mg, 82%). H NMR (CDCl3, 400 MHz) δ Phe δ 7.28 (2H, o-Ph), 7.44
(m, 2H, m-Ph), 7.39 (d, 7.4 Hz, 1H, p-Ph), 6.30 (m, 1H, CONHCH3), 5.86 (d, 9.4 Hz,
1H, α-CHNH), 4.67 (m, 1H, α-CH), 3.61 (dd, 13.5 Hz, 4.4 Hz, 1H, α-CHCH2), 2.87
2 (dd, 13.8 Hz, 5.9 Hz, 1H, α-CHCH2), 2.41 (d, 4.9 Hz, 3H, CONHCH3); Phe δ 7.18
(d, 7.4 Hz, 2H, o-Ph), 7.33 (m, 2H, m-Ph), 7.26 (1H, p-Ph), 6.45 (m, 1H,
CONHCH3), 5.90 (d, 7.4 Hz, 1H, α-CHNH), 4.39 (q, 1H, α-CH), 3.16 (dd, 14.3 Hz,
6.9 Hz, 1H, α-CHCH2), 3.09 (dd, 14.3 Hz, 5.9 Hz, 1H, α-CHCH2), 2.63 (d, 4.4 Hz,
6 5 3H, CONHCH3); Scaffold δ 7.01 (m, 1H, C H), 6.94 (d, 7.4 Hz, 1H, C H), 6.76 (m,
1H, C7H), 5.78 (d, 7.4 Hz, 1H, C8H), 3.32 (m, 2H, C2H, C3H), 3.31 (m, 1H, C4H),
1 9 9 13 3.23 (m, 1H, C H), 1.86 (m, 1H, C H), 1.73 (m, 1H, C H); C NMR (CDCl3, 100
MHz) δ 172.1, 171.4, 170.3, 170.0, 145.0, 142.3, 136.9, 136.8, 130.1, 129.1, 129.0,
128.9, 127.4, 127.1, 126.3, 126.2, 124.1, 121.2, 53.9, 52.5, 52.5, 52.0, 51.1, 50.9,
+ 49.2, 48.4, 45.2, 36.9, 36.2, 26.2, 26.1; MS m/z (MNa C33H36N4NaO4) calcd
575.2634, obsd 575.2609.
Bn O O
N (L) CONHCH3 H N (L) CONHCH3 H Bn
NO2
72 1 1 6 (71%). H NMR (CDCl3, 400 MHz) δ Phe δ 7.49 (m, 2H, m-Ph), 7.26 (m,
2H, o-Ph), 7.22-7.28 (m, 1H, p-Ph), 6.16 and 5.94 (m, 1H, CONHCH3), 2.41 (d, 5.9
1 Hz, 1.5H, CONHCH3), 2.40 (d, 5.4 Hz, 1.5H, CONHCH3); Phe δ 4.60 (m, 0.5H, α-
CH), 5.69 (d, 8.7 Hz, 0.5H, α-CHNH), 3.72 (dd, 13.6 Hz, 3.4 Hz, 0.5H, α-CHCH2),
1 2.78 (dd, 13.2 Hz, 5.8 Hz, 0.5H, α-CHCH2); Phe δ 4.54 (m, 0.5H, α-CH), 5.83 (d,
8.7 Hz, 0.5H, α-CHNH), 3.39 (m, 0.5H, α-CHCH2), 2.89 (dd, 14.0 Hz, 6.8 Hz, 0.5H,
2 α-CHCH2); Phe δ 7.38 (m, 2H, m-Ph), 7.22-7.28 (m, 1H, p-Ph), 7.18 (d, 7.3 Hz,
2H, o-Ph), 6.47 (m, 1H, CONHCH3), 4.39 (m, 1H, α-CH), 6.01 (d, 7.8 Hz, 1H, α-
CHNH), 3.11 (dd, 13.1 Hz, 5.8 Hz, 1H, α-CHCH2), 3.07 (dd, 14.0 Hz, 7.8 Hz, 1H, α-
1 CHCH2), 2.64 (d, 4.4 Hz, 3H, CONHCH3); Scaffold δ 7.96 (d, J=8.4 Hz, 0.5H),
7.13 (s, 0.5H), 7.03 (d, J=8.2 Hz, 0.5H); Scaffold2 δ 7.65 (d, J=8.0 Hz, 0.5H), 7.67
13 (s, 0.5H), 5.75 (d, J=7.8 Hz, 0.5H); C NMR (CDCl3, 100 MHz) δ 171.1, 170.1,
170.5, 170.4, 170.2, 169.9, 152.9, 149.7, 147.0, 146.8, 146.6, 144.1, 137.0, 136.8,
136.6 136.2, 130.1 129.5 129.2, 129.1 128.8, 127.7, 127.6, 127.2, 127.1, 124.3, 122.5,
122.0, 121.9, 118.9, 117.0, 54.1, 53.9, 53.0, 52.7, 51.7, 51.4, 51.1, 50.5, 48.7, 48.5,
48.1, 45.9, 45.3, 37.1, 36.8, 36.6, 36.1, 29.7, 26.2, 26.2, 26.0; MS m/z (MNa+
C33H35N5NaO6) calcd 620.2485, obsd 620.2410.
i-Bu O O
N (L) CONHCH3 H N (L) CONHCH3 H i-Bu
1 1 7 (78%). H NMR (CDCl3, 400 MHz) δ Leu δ 6.41 (m, 1H, CONHCH3),
6.00 (d, J=8.1 Hz, 1H, α-CHNH), 4.26 (m, 1H, α-CH), 2.42 (d, J=4.7 Hz, 3H,
73 CONHCH3), 1.92 (m, 1H, α-CHCH2), 1.36 (m, 1H, α-CHCH2), 1.69 (m, 1H,
CH(CH3)2), 0.98 (d, J=6.7 Hz, 3H, CH(CH3)2), 0.94 (d, J=7.1 Hz, 3H, CH(CH3)2);
2 Leu δ 6.60 (m, 1H, CONHCH3), 6.00 (d, J=8.1 Hz, 1H, α-CHNH), 4.19 (m, 1H, α-
CH), 2.65 (d, J=4.7 Hz, 3H, CONHCH3), 1.89 (m, 1H, α-CHCH2), 1.45 (m, 1H, α-
CHCH2), 1.62 (m, 1H, CH(CH3)2), 0.97 (d, J=6.6 Hz, 3H, CH(CH3)2), 0.92 (d, J=6.9
5 6 7 Hz, 3H, CH(CH3)2); Scaffold δ 7.26 (m, 1H, C H), 7.22-7.25 (m, 2H, C H, C H),
7.20 (m, 1H C8H,), 3.57 (m, 2H, C1H, C4H), 3.50 (m, 2H, C2H, C3H), 2.02 (m, 1H,
9 9 13 C H), 1.87 (m, 1H, C H); C NMR (CDCl3, 100 MHz) δ 172.5, 171.7, 171.6, 171.4,
145.5, 143.1, 126.9, 126.4, 124.9, 121.5, 52.2, 51.9, 51.7, 50.9, 49.4, 48.6, 46.1, 40.5,
+ 40.1, 26.2, 26.0, 25.2, 25.1, 23.3, 23.1, 21.5, 21.5; MS m/z (MNa C27H40N4NaO4) calcd 507.2947, obsd 507.2928.
Ph
O O
N (L) CONHCH3 H N CONHCH3 H (L) Ph
1 1 12 (76%). H NMR (CDCl3, 400 MHz) Phe δ 7.30-7.33 (m, 3H, m-Ph, p-
Ph), 7.16-7.20 (m, 2H, o-Ph), 5.94 (d, J=8.1 Hz, 1H, α-CHNH), 4.56 (m, 1H, α-CH),
2 3.40 (m, 1H, α-CHCH2), 3.06 (m, 1H, α-CHCH2); Phe δ 7.33-7.38 (m, 3H, m-Ph, p-Ph), 7.15-7.18 (m, 2H, o-Ph), 5.76 (m, 1H, α-CHNH), 4.58 (m, 1H, α-CH), 3.12-
3 4 3.26 (m, 2H, α-CHCH2); Scaffold δ 6.32 (m,1H, C H), 5.76 (m, 1H, C H), 2.81 (m,
1H, C1H), 2.70 (m, 1H, C2H), 2.52 (m, 1H, C6H), 2.46 (m, 1H, C5H), 1.46 (m, 2H,
7 8 7 8 13 C H, C H), 1.22 (m, 2H, C H, C H); C NMR (CDCl3, 100 MHz) δ 173.9, 173.6,
170.5, 170.4, 137.1, 136.8, 136.3, 129.2, 129.0, 128.9, 128.7, 127.5, 127.3, 126.9,
74 53.9, 53.7, 51.2, 49.6, 46.2, 37.0, 36.7, 33.4, 30.8, 26.4, 26.1, 26.1, 24.8, 24.6; MS
+ m/z (MNa C30H36N4NaO4) calcd 539.2634, obsd 539.2613.
COOCH3 CON3
Acyl azide (13). NaH (164 mg 60% suspension in oil, 4.09 mmol) was added slowly to a cold MeOH (5 mL). The solution was allowed to warm and 3 (833 mg,
3.89 mmol) in dry methanol (2 mL) was added. The resulting mixture stirred at room temperature for 3h, solvent evaporated in vacuum, solid residue dissolved in dry THF
(5 mL) and solution cooled in ice-bath. Iso-butyl chloroformate (0.605 mL, 4.67
o mmol) was added dropwise at 0 C and reaction mixture was stirred for 30 min. NaN3
(1.26 g, 19.45 mmol) dissolved in a minimum amount of water and added to the cooled reaction mixture. After stirring for 4h, solvent was evaporated, dissolved in
CH2Cl2 and washed with water. Crude product was purified by flash chromatography
1 to afford 13 (699 mg, 66%) as colorless oil. H NMR (CDCl3, 250 MHz) δ 7.10-7.27
(m, 4H), 3.76 (s, 3H), 3.70 (m, 1H), 3.65 (m, 2H), 1.96 (d, J=9.3 Hz, 1H), 1.85 (d,
13 J=10 Hz, 1H); C NMR (CDCl3, 62.9 MHz) δ 178.1, 171.2, 143.7, 143.1, 126.2,
-1 123.2, 123.0, 51.3, 49.7, 49.4, 47.9, 47.5; IR (CHCl3) 2137.0, 1728.4, 1726.6 cm .
COOCH3 COOCH N 3 H
Methyl carbamate methyl ester (14). A solution of 13 (543 mg, 2.00 mmol) in toluene (5 mL) and MeOH (1 mL) was refluxed for 5h. After evaporation crude
75 product was purified by flash chromatography to give 14 (410 mg, 74%) as white
1 crystals. H NMR (CDCl3, 250 MHz) δ 7.17-7.26 (m, 4H), 5.13 (d, J=8.5 Hz, 1H),
4.72 (m, 1H), 3.61 (s, 3H), 3.58 (m, 1H), 3.53 (s, 3H), 3.49 (m, 1H), 3.39 (dd, J=9.8
13 Hz, 3.8 Hz, 1H), 1.89 (d, J=9.8 Hz, 1H), 1.78 (d, J=9.5 Hz); C NMR (CDCl3, 62.9
MHz) δ 171.9, 156.5, 144.4, 142.3, 126.5, 126.3, 123.3, 123.0, 52.5, 51.8, 51.2, 48.9,
48.1, 47.3, 46.5.
COOH
COOCH N 3 H
Methyl carbamate carboxylic acid (15). LiOH·H2O (63 mg, 1.49 mmol) was added to a solution of 14 (410 mg, 1.49 mmol) in water/methanol (3 mL/6 mL).
Reaction mixture was stirred for 3h at room temperature, concentrated in vacuum, extracted with CH2Cl2 (1×15 mL), acidified with 5% HCl and extracted with EtOAc
(3×30 mL), dried over Na2SO4 and evaporated. 15 (350 mg, 90%) obtained as white
1 solid. H NMR (CDCl3, 250 MHz) δ 7.18-7.32 (m, 4H), 4.55 (m, 1H), 4.38 (m, 1H),
13 3.83 (s, 1H), 3.64 (s, 3H), 3.44 (s, 1H), 2.15 (s, 1H), 1.99 (s, 1H); C NMR (CDCl3,
62.9 MHz) δ 187.8, 174.0, 158.7, 142.2, 127.6, 126.7, 122.7, 122.0, 56.3, 55.7, 53.0,
47.6, 47.4, 46.8.
Mono Phenylalanine Amides (16a, b). To a solution of 15 (235 mg, 0.9 mmol) in CH2Cl2 (10 mL) was added CDI (146 mg, 0.9 mmol) and after 1h followed by addition of (L)-Phe-NHMe·HCl (194 mg, 0.9 mmol). Reaction mixture was stirred overnight at room temperature, washed with 5% HCl, saturated NaHCO3 solution and brine. After concentration in vacuum mixture of diastereomers was
76 separated by radial thin-layer chromatography (Chromatotron) to give pure 110 mg of
16a, 115 mg of 16b and 52 mg mixture of 16a and 16b.
Ph
O
N CONHCH3 H N COOCH3 H
1 16a. H NMR (CDCl3, 250 MHz) δ 8.26 (d, J=7.5 Hz, 1H), 7.01-7.16 (m,
9H), 6.36 (d, J=4.0 Hz, 1H), 4.65 (q, J=7.0 Hz, 1H), 4.23 (d, J=7.3 Hz, 1H), 3.88 (m,
1H), 3.54 (s, 1H), 3.51 (s, 3H), 3.22 (dd, J=13.8, 6.0 Hz, 1H), 3.12 (s, 1H), 2.89 (dd,
J=13.5, 8.8 Hz, 1H), 2.59 (d, J=4.5 Hz, 3H), 1.80 (s, 1H), 1.65 (d, J=9.8 Hz, 1H), 1.42
13 (d, J=9.5 Hz, 1H); C NMR (CDCl3, 62.9 MHz) δ 173.3, 171.9, 157.3, 147.3, 142.7,
137.6, 129.4, 128.3, 127.2, 126.5, 126.3, 122.7, 121.6, 56.2, 55.0, 52.4, 47.6, 47.4,
46.4, 37.7, 26.1;
Ph O NHCO2CH3
N CONHCH3 H
1 16b. H NMR (CDCl3, 250 MHz) δ 7.36 (d, J=7.8 Hz, 1H), 7.09-7.24 (m,
9H), 6.86 (m, 1H), 4.72 (q, J=6.3 Hz, 1H), 4.35 (s, 2H), 3.52 (s, 3H), 3.46 (s, 1H),
3.30 (s, 1H), 3.19 (dd, J=13.8, 5.5 Hz, 1H), 3.05 (dd, J=14, 8 Hz, 1H), 2.73 (d, J=4.3
Hz, 3H), 2.10 (d, J=9.5 Hz, 1H), 1.82 (d, J=10 Hz, 1H), 1.77 (s, 1H); 13C NMR
(CDCl3, 62.9 MHz) δ 173.4, 171.8, 157.2, 147.6, 142.7, 137.2, 129.2, 128.4, 127.2,
126.6, 126.4, 122.8, 121.4, 56.4, 56.2, 54.7, 52.2, 47.3, 37.5, 26.2.
77 Mono Phenylalanine Amides (17, 18). A mixture of 16a (110 mg, 0.26
mmol), freshly distilled TMS-Cl (67 µL, 0.52 mmol) and NaI (116 mg, 0.78 mmol) in
acetonitrile (5 mL) was refluxed for 20h. Acidified with 10 mL 1M HCl, stirred for
15 min and extracted with CH2Cl2 (3×20 mL). Aqueous phase made basic with
saturated NaHCO3 solution and extracted with CH2Cl2 (3×20 mL). Combined organic layers were washed with Na2SO3 and brine, dried (Na2SO4) and evaporated.
Obtained 17 (32 mg, 34%) was used without further purification.
Ph
O
N CONHCH3 H NH2
1 17. H NMR (CDCl3, 250 MHz) δ 6.97-7.24 (m, 9H), 6.55 (d, J=8.0 Hz, 1H),
6.13 (d, J=3.8 Hz, 1H), 4.60 (q, J=7.5 Hz, 1H), 3.23 (m, 2 H), 2.88-3.07 (m, 3H), 2.60
(d, J=4.8 Hz, 3H), 1.90 (d, J=10.3 Hz, 1H), 1.68 (d, J=8.8 Hz, 1H), 1.38 (m, 1H); 13C
NMR (CDCl3, 62.9 MHz) δ 174.3, 142.7, 136.7, 129.0, 128.2, 126.6, 126.3, 125.6,
123.6, 120.4, 58.0, 56.5, 54.3, 50.4, 47.3, 46.9, 38.1, 25.9.
Ph O NH2
N CONHCH3 H
1 18 (34 mg, 30 %). H NMR (CDCl3, 250 MHz) δ 7.08-7.34 (m, 9H), 6.67 (d,
J=7.5 Hz, 1H), 6.17 (m, 1H), 4.62 (q, J=7.5 Hz, 1H), 3.74 (m, 1H), 3.16 (s, 2H), 3.10
(d, J=7.3 Hz, 2H), 2.70 (d, J=4.5 Hz, 3H), 2.07 (d, J=9.3 Hz, 1H), 1.80 (d, J=8.3 Hz,
78 13 1H), 1.55 (s, 1H); C NMR (CDCl3, 62.9 MHz) δ 174.3, 148.0, 143.1, 137.0, 129.3,
129.2, 128.6, 126.9, 126.6, 126.0, 124.0, 120.6, 57.2, 56.3, 54.9, 48.1, 47.5, 47.3,
38.5, 26.1.
Antiparallel templates (19-24). PyBOP (27 mg, 0.052 mmol) was added to a
mixture of Ac-D-Phe-OH (11 mg, 0.052 mmol) and DIEA (10 µl, 0.052 mmol) in
DMF. Mixture was stirred at room temperature for 5 min and 17 (19 mg, 0.052 mmol) was added. Reaction was allowed to run for another 12h. Solvent was removed in vacuum, residue dissolved in CH2Cl2, washed with 1M HCl, saturated
NaHCO3 solution and brine. After evaporation crude product was purified by flash
chromatography to give 20 (12 mg, 48%).
Ph
O O (L) N CONHCH3 H N NHAc H (L)
Ph
19. See spectral data section for 1H spectrum.
Ph
O O (L) N CONHCH3 H N NHAc H (D)
Ph
1 1 20. H NMR (CDCl3, 400 MHz) δ Phe δ 7.06 (1H, p-Ph), 7.08 (m, 2H, m-
Ph), 6.88 (d, 7.2 Hz, 2H, o-Ph), 5.96 (d, 7.5 Hz, 1H, α-CHNH), 4.38 (m, 1H, α-CH),
2.94 (dd, 13.4 Hz, 6.3 Hz, 1H, α-CHCH2), 2.81 (dd, 12.4 Hz, 8.5 Hz, 1H, α-CHCH2),
1.97 (s, 3H, NHAc); Phe2 δ 7.31 (m, 2H, o-Ph), 7.29 (m, 2H, m-Ph), 7.23 (m, 1H, p-
79 Ph), 8.56 (d, 8.0 Hz, 1H, α-CHNH), 6.05 (m, 1H, CONHCH3), 4.73 (q, 1H, α-CH),
3.42 (dd, 14.0 Hz, 6.1 Hz, 1H, α-CHCH2), 3.05 (dd, 15.4 Hz, 4.5 Hz, 1H, α-CHCH2),
8 5 2.81 (d, 4.8 Hz, 3H, CONHCH3); Scaffold δ 7.18 (m, 1H, C H), 7.14 (m, 1H, C H),
7.11-7.18 (m, 2H, C6H, C7H), 3.95 (m, 1H, C3H), 3.66 (m, 1H, C1H), 3.06 (m, 1H,
4 9 9 2 13 C H), 1.73 (m, 1H, C H), 1.36 (m, 1H, C H), 1.20 (m, 1H, C H); C NMR (CDCl3,
100 MHz) δ 172.9, 171.6, 171.7, 169.7, 147.1, 142.1, 137.9, 135.6, 129.4, 128.7,
128.7, 128.3, 127.6, 127.2, 126.6, 126.3, 122.3, 122.2, 56.5, 55.0, 54.6, 54.2, 47.348,
+ 47.1, 45.0, 38.4, 37.5, 29.7, 29.7, 26.3, 23.2; MS m/z (MH C33H37N4O4) calcd
553.2815, obsd 553.2815.
Ph
O O (L) N CONHCH3 H N NHAc H
21. EDCl (15 mg, 0.077 mmol) was added to a mixture of Ac-Gly-OH (9 mg,
0.077 mmol) and DIEA (13 µl, 0.077 mmol) in H2O (0.5 mL). Mixture was stirred at room temperature for 5 min and 17 (28 mg, 0.077 mmol) in DMF (0.5 mL) was added. Reaction was allowed to run for another 12h. Solvent was removed in vacuum, residue dissolved in CH2Cl2, washed with 1M HCl, saturated NaHCO3 solution and brine. After evaporation crude product was purified by flash chromatography to give 21 (21 mg, 59%). See spectral data section for 1H spectrum.
80 Ph
H (L) O N NHAc
N O CONHCH3 H (L)
Ph
1 1 22 (11 mg, 37%). H NMR (CDCl3, 400 MHz) δ Phe δ 7.04 (1H, p-Ph),
6.95 (t, 2H, m-Ph), 6.74 (d, 7.2 Hz, 2H, o-Ph), 5.98 (d, 7.3 Hz, 1H, α-CHNH), 4.34
(m, 1H, α-CH), 2.90 (dd, 13.5 Hz, 6.2 Hz, 1H, α-CHCH2), 2.71 (dd, 13.5 Hz, 8.8 Hz,
2 1H, α-CHCH2), 1.96 (s, 3H, NHAc); Phe δ 7.22-7.35 (m, 3H, m-Ph, p-Ph), 7.28 (m,
2H, o-Ph), 7.56 (d, 8.0 Hz, 1H, α-CHNH), 6.59 (m, 1H, CONHCH3), 4.73 (q, 1H, α-
CH), 3.25 (dd, 14.2 Hz, 5.9 Hz, 1H, α-CHCH2), 3.12 (dd, 14.0 Hz, 8.3 Hz, 1H, α-
6 7 CHCH2), 2.80 (d, 4.8 Hz, 3H, CONHCH3); Scaffold δ 7.08-7.20 (m, 2H, C H, C H),
7.17 (m, 1H, C8H), 7.14 (m, 1H, C5H), 4.34 (m, 1H, C3H), 3.49 (m, 1H, C4H), 3.21
(m, 1H, C1H), 1.96 (m, 1H, C9H), 1.82 (m, 1H, C9H), 1.12 (m, 1H, C2H); 13C NMR
(CDCl3, 100 MHz) δ 173.2, 171.9, 171.4, 169.7, 147.2, 142.1, 137.4, 135.6, 129.2,
128.7, 128.6, 128.6, 127.5, 127.1, 126.8, 126.3, 122.5, 121.9, 56.0, 54.9, 54.7, 54.3,
+ 47.3, 47.0, 46.7, 38.5, 37.4, 29.7, 26.4, 23.2; MS m/z (MH C33H37N4O4) calcd
553.2815, obsd 553.2820.
Ph
H (D) O N NHAc
N O CONHCH3 H (L)
Ph
23. See spectral data section for 1H spectrum.
81 H O N NHAc
N O CONHCH3 H (L)
Ph
24 (17 mg, 60%). See spectral data section for 1H spectrum.
CO2CH3
CO2CH3
NO2 Nitro anhydride diester (25). Andydride 4 (640 mg, 2.50 mmol) was dissolved in 20 mL of anhydrous MeOH and catalytic amount of sulfuric acid was added. Mixture was heated at reflux for 5h, solvent was evaporated in vacuum and resulting residue was recrystallized from MeOH to give 4 (674 mg, 88%). 1H NMR
(CDCl3, 250 MHz) δ 1.81 (dd, 1H, J=1.4 Hz, J=1.4 Hz), 1.85 (dd, 1H, J=1.4 Hz,
J=1.4 Hz), 1.91 (dd, 1H, J=1.8 Hz, J=1.8 Hz), 1.95 (dd, 1H, J=1.8 Hz, J=1.8 Hz), 3.48
(s, 3H), 3.49 (s, 3H), 3.55 (m, 2H), 3.73 (m, 2H), 7.41 (d, 1H, J=8.0 Hz), 8.06 (dd,
13 1H, J=2.2 Hz, J=8.0 Hz), 8.08 (m, 1H); C NMR (CDCl3, 62.9 MHz) δ 171.2, 151.7,
145.7, 124.1, 118.5, 51.6, 49.6, 47.5, 47.4.
CO2CH3
CO2CH3
NH2 Aniline diester (26). Nitro anhydride diester 25 (1.40g, 4.58 mmol) was
dissolved in the mixture of MeOH-DMF (60 mL, 5:1), 10% Pd-C catalyst (140 mg)
was added and resulting suspension was vigorously stirred in hydrogen atmosphere
for 8h. Solvent was evaporated in vacuum and crystalline residue was triturated with
82 1 diethyl ether to give 26 (1.23 g, 97%). H NMR (CDCl3, 250 MHz) δ 1.67 (dd, 1H,
J=1.4 Hz, J=1.4 Hz), 1.71 (dd, 1H, J=1.4 Hz, J=1.4 Hz), 1.80 (dd, 1H, J=1.8 Hz,
J=1.8 Hz), 1.84 (dd, 1H, J=1.8 Hz, J=1.8 Hz), 3.42 (m, 2H), 3.48 (s, 3H), 3.49 (s,
3H), 3.53 (m, 2H), 6.44 (dd, 1H, J=2.2 Hz, J=7.8 Hz), 6.67 (d, 1H, J=2.2 Hz), 7.01 (d,
1H, J=7.8 Hz).
CO2CH3
CO2CH3
O NHBoc N H
Diester (27). Boc-Gly-OH (175 mg, 1.00 mmol) was dissolved in 20 mL of
chloroform and solid CDI (162 mg, 1.00 mmol) was added at room temperature.
Mixture was stirred for 2h, then aniline 26 (275 mg, 1.00 mmol) was introduced and
resulting solution was stirred for additional 12h. Reaction mixture was successively
washed with 1M HCl, saturated aqueous NaHCO3 solution and brine and dried over
1 Na2SO4. Evoporation of the solvent afforded pure 27 (368 mg, 85%). H NMR
(CDCl3, 250 MHz) δ 1.48 (s, 9H), 1.74 (d, 1H, J=9.2 Hz), 1.87 (d, 1H, J=9.1 Hz),
3.47 (m, 2H), 3.48 (s, 3H), 3.50 (s, 3H), 3.61 (d, 2H, J=8.0 Hz), 3.89 (d, 2H, 6.0 Hz),
5.16 (m, 1H), 7.13 (dd, 1H, J=1.9 Hz, J=7.9 Hz), 7.22 (d, 1H, J=8.0 Hz), 7.53 (d, 1H,
J=1.6 Hz), 7.93 (m, 1H).
CO2H
CO2H
O NHBoc N H
83 Diacid (28). LiOH·H2O (45 mg, 1.05 mmol) was added to a solution of diester 27 (215 mg, 0.50 mmol) in MeOH-H2O (12 mL, 3:1) mixture. Reaction was stirred for additional 12h at room temperature. Mixture was diluted with EtOAc and was extracted with CH2Cl2 (×1), aqueous phase was acidified with 1M HCl and extracted with EtOAc (×3). Combined organic phases were washed with brine and
1 dried over Na2SO4. Evaporation of the solvent afforded pure 28 (179 mg, 88%). H
NMR (5% DMSO-d6-CDCl3, 250 MHz) δ 1.46 (s, 9H), 1.81 (d, 1H, J=9.4 Hz), 1.97
(d, 1H, J=9.5 Hz), 2.80 (m, 1H), 3.65 (m, 3H), 3.88 (m, 2H), 5.75 (m, 1H), 7.16 (m,
2H), 7.50 (m, 1H), 8.86 (s, 1H).
O SMe
Benzyloxy-methyl-(methyl)sulfane (30). Was prepared according to
1 reference 58. 15.56 g (56% starting from benzyl alcohol). H NMR (CDCl3, 250
MHz) δ 2.19 (s, 3H), 4.62 (s, 2H), 4.69 (s, 2H), 7.35 (m, 5H).
O Cl
Benzyloxy-methylchloride (31). Was prepared according to reference 58.
1 11.40 g (79%). H NMR (CDCl3, 250 MHz) δ 4.72 (s, 2H), 5.50 (s, 2H), 7.34 (m,
5H).
NBoc
N
BocHN CO2CH3
α 1 N -Boc-His-(N -Boc)-OCH3 (33). Mixture of His-OCH3·2HCl (484 mg, 2.00 mmol), tryethyl amine (1.115 mL, 8.00 mmol), and Boc2O (1.091 g, 5.00 mmol) in
84 methanol (10 mL) was stirred at room temperature for 12h. Reaction was diluted
with CH2Cl2 and washed with water and then with brine. Dried over Na2SO4.
Evaporation of the solvent afforded 33 (679 mg, 92%). mp 102-105 °C. 1H NMR
(CDCl3, 400 MHz) δ 7.98 (s, 1H), 7.14 (s, 1H), 5.75 (d, J=7.8 Hz, 1H), 4.57 (m, 1H),
13 3.73 (s, 3H), 3.05 (m, 2H), 1.61 (s, 9H), 1.44 (s, 9H); C NMR (CDCl3, 100 MHz) δ
172.1, 155.3, 146.7, 138.5, 136.7, 114.4, 85.4, 79.6, 53.0, 52.1, 30.1, 28.2, 27.7.
N
N Bn BocHN CO2CH3
α 3 N -Boc-His-(N -Bn)-OCH3 (34). A solution of benzyl alcohol (207 µL, 2.00
mmol) and DIEA (348 µL, 2.00 mmol) in DCM (4 mL) was added dropwise to triflic
anhydride (338 µL, 2.00 mmol) in DCM (5 mL) at –78 °C over 10 min time period.
Reaction mixture was stirred for 40 min and solution of 33 (702 mg, 1.90 mmol) in
DCM (4 mL) was added at –78 °C. Solution was allowed to warm up to room
temperature and was stirred for additional 20h. Reaction was diluted with DCM,
quenched with pH 7 buffer, and was vigorously stirred for 30 min. Layers were
separated and organic phase was washed with pH 7 buffer (×1). Extracts were dried
over Na2SO4. Solvent was evaporated and residue was purified by flash
1 chromatography to give 34 (351 mg, 51%). H NMR (CDCl3, 250 MHz) δ 1.40 (s,
9H), 2.95 (m, 2H), 3.70 (s, 3H), 4.46 (m, 1H), 5.08 (d, 2H, J=4.4 Hz), 5.12 (s, 1H),
6.83 (s, 1H), 7.03 (m, 2H), 7.31 (m, 3H), 7.45 (s, 1H).
N• HCl
N BOM BocHN CO CH 2 3
85 α 3 N -Boc-His-(N -Bom)-OCH3 (35). Benzyloxymethyl chloride (31) (674 mg,
4.30 mmol) solution in DCM (5 mL) was added dropwise to Nα-Boc-His-(N1-Boc)-
OCH3 (33) (1.06 g, 2.90 mmol) in DCM (10 mL) at room temperature and stirred for
12h. Solvent was removed in vacuum and residue was triturated with Et2O, filtered
and precipitate was washed on the filter with Et2O (×2). 35 (1.155 g, 93%) was
obtained as a white crystalline powder after drying in vacuum. mp 140-142°C. 1H
NMR (CDCl3, 400 MHz) δ 9.78 (s, 1H), 7.28-7.35 (m, 5H), 7.18 (s, 1H), 5.84 (s, 2H),
5.35 (d, J=5.6 Hz, 1H), 4.70 (m, 2H), 4.57 (m, 1H), 3.75 (s, 3H), 3.33 (dd, J=4.8 Hz,
13 15.7 Hz, 1H), 3.14 (dd, J=7.4 Hz, 15.8 Hz, 1H), 1.41 (s, 9H); C NMR (CDCl3, 100
MHz) δ 170.8, 155.0, 136.2, 135.5, 129.8, 128.5, 128.3, 128.0, 128.0, 127.9, 118.4,
80.5, 76.3, 72.0, 52.8, 52.0, 28.1, 26.3.
CbzHN NH
N N
Mono-Cbz-amidine (37). Was prepared according to reference 61. 1.11 g,
1 91%. mp 102-104 °C; H NMR (CDCl3, 400 MHz) δ 9.04 (m, 1H), 8.45 (s, 1H),
7.70 (m, 1H), 7.68 (s, 1H), 7.25-7.46 (m, 5H), 6.41 (s, 1H), 5.21 (s, 2H); 13C NMR
(CDCl3, 100 MHz) δ 163.8, 155.4, 143.6, 136.3, 128.8, 128.4, 128.2, 128.1, 109.2,
67.5.
CbzHN NCbz
N N
Bis-Cbz-amidine (38). Was prepared according to reference 61. 328 mg,
1 87%. mp 79-81 °C; H NMR (CDCl3, 400 MHz) δ 9.36 (m, 1H), 8.20 (s, 1H), 7.23-
86 13 7.45 (m, 10H), 6.34 (s, 1H), 5.23 (s, 2H), 5.14 (s, 2H); C NMR (CDCl3, 100 MHz)
δ 157.8, 150.4, 142.7, 137.9, 135.5, 134.2, 128.5, 128.3, 128.1, 127.9, 110.0, 68.3,
68.1.
NH2
BocHN CO2H
Boc-Orn-OH (40). Boc-Orn(Cbz)-OH 39 (1.00 g, 4.72 mmol) was dissolved in a mixture of EtOH and DMF (10 ml, 1:1), 10% Pd-C catalyst (144 mg, 5 mol %) was added and reaction mixture was stirred under H2 atmosphere for 5h. DMF and water (40 ml, 1:1) were added to dissolved formed product and catalyst was removed by filtration through celite pad. Solvent was evaporated in vacuum to give 465 mg
(74%) of pure product 40. 1H NMR (DMSO-d6, 250 MHz) δ 1.42 (m, 13H), 2.28 (m,
2H), 3.41 (m, 1H).
CbzHN NCbz
NH
BocHN CO2H
Boc-Arg(Cbz)2-OH (41). Bis-Cbz-amidine 38 in DMF was added dropwise
to a solution of Boc-Orn-OH 40 in DMF at room temperature. Reaction mixture was
stirred overnight at 45°C. Solvent was removed in vacuum and residue was dissolved
in DCM, washed with 1M HCl (×2) and brine. Organic phase was dried over
Na2SO4. After removal of solvent product was purified on silica gel (4%
1 EtOH/DCM) to give 879 mg (87%) of pure 41. H NMR (CDCl3, 250 MHz) δ 1.44
(s, 9H), 1.69 (m, 3H), 1.93 (m, 1H), 3.45 (m, 2H), 4.32 (m, 1H), 5.12 (m, 2H), 5.17
(s, 2H), 7.37 (m, 10H), 8.03 (m, 1H), 8.36 (m, 1H), 11.71 (m, 1H).
87 N
N Bom H2N CO2CH3
His(Bom)-OCH3 (42). Boc-His(Bom)-OCH3 35 (500 mg, 1.17 mmol) was
dissolved in 10 mL of 20% TFA in DCM and reaction mixture was stirred at room
temperature for 1h. Solvent was evaporated in vacuum, residue dissolved in DCM
and washed with saturated NaHCO3 solution. Organic phase was dried over Na2SO4.
1 Removal of solvent gave 42 (307 mg, 91%). H NMR (CDCl3, 250 MHz) δ 2.92 (dd,
1H, J=7.7 Hz, J=15.2 Hz), 3.11 (dd, 1H, J=5.5 Hz, J=15.2 Hz), 3.70 (s, 3H), 3.73 (m,
1H), 4.41 (s, 2H), 5.33 (s, 2H), 6.90 (s, 1H), 7.31 (m, 5H), 7.49 (s, 1H); 13C NMR
(CDCl3, 62.9 MHz) δ 174.9, 138.3, 136.1, 129.1, 128.7, 128.3, 128.0, 73.3, 69.8,
54.2, 52.2, 29.2.
N
O N Bom BocHN N CO2CH3 H
NHCbz
Boc-Orn(Cbz)-His(Bom)-OCH3 (43). Boc-Orn(Cbz)-OH (389 mg, 1.06
mmol), His(Bom)-OCH3 42 (307 mg, 1.06 mmol) and HOBt·H2O (143 mg, 1.06
mmol) were dissolved in 4 mL of CH3CN and solution of DCC (219 mg, 1.06 mmol)
in CH3CN (1mL) was added dropwise. Reaction mixture was stirred at room
temperature for 5h. Formed DCU precipitate was filtered and filtrate was evaporated.
Residue was dissolved in EtOAc, washed with 2M HCl (×2), saturated NaHCO3 solution and brine. After solvent removal crude product was purified on silica gel
1 (5% EtOH/DCM) to give pure 43 (430 mg, 64%). H NMR (CDCl3, 250 MHz) δ
88 1.40 (m, 2H), 1.43 (s, 9H), 1.49 (m, 2H), 1.81 (m, 2H), 3.15 (m, 2H), 3.70 (s, 3H),
4.45 (s, 2H), 4.90 (m, 1H), 5.05 (s, 2H), 5.10 (m, 1H), 5.30 (s, 2H), 6.85 (s, 1H), 7.34
13 (m, 10H), 7.47 (s, 1H); C NMR (CDCl3, 62.9 MHz) δ 172.2, 171.3, 156.7, 155.5,
138.3, 136.5, 135.9, 129.0, 128.5, 128.3, 127.8, 126.6, 113.8, 79.7, 73.1, 69.7, 66.4,
53.3, 52.3, 51.5, 39.8, 29.8, 28.2, 26.3, 25.8.
N
O N Bom H2N N CO2CH3 H
NHCbz
Orn(Cbz)-His(Bom)-OCH3 (44). Boc-Orn(Cbz)-His(Bom)-OCH3 43 (220
mg, 0.34 mmol) was dissolved in 5 mL of 20% TFA in DCM and reaction mixture
was stirred at room temperature for 1h. Solvent was evaporated in vacuum, residue
dissolved in EtOAc and washed with saturated NaHCO3 solution. Organic phase was
1 dried over Na2SO4. Removal of solvent gave 44 (153 mg, 84%). H NMR (CDCl3,
250 MHz) δ 1.44 (m, 2H), 1.66 (m, 2H), 3.13 (m, 2H), 3.16 (m, 2H), 3.72 (s, 3H),
4.43 (s, 2H), 4.88 (m, 1H), 5.08 (s, 2H), 5.31 (m, 1H), 5.39 (s, 1H), 6.86 (s, 1H), 7.34
(m, 10H), 7.46 (s, 1H), 7.88 (d, 1H, J=9.8 Hz).
N
O N Bom BocHN N CO2H H
NHCbz
Boc-Orn(Cbz)-His(Bom)-OH (45). LiOH·H2O (17mg, 0.42 mmol) was added to a solution of Boc-Orn(Cbz)-His(Bom)-OCH3 43 (253 mg, 0.40 mmol) in mixture of MeOH and water (4.5 mL, 2:1). Reaction mixture was stirred overnight at
89 room temperature. Solvent was removed in vacuum and residue dissolved in DCM,
washed with 0.5 M NaOH solution (×2). Aqueous washes were combined and
acidified, extracted with EtOAc (×3). Organic phase was dried over Na2SO4. Solvent
1 was evaporated to give 45 (161 mg, 65%). H NMR (CDCl3, 250 MHz) δ 1.35 (s,
9H), 1.40 (m, 2H), 1.65 (m, 2H), 3.00 (m, 2H), 3.13 (m, 2H), 4.12 (m, 1H), 4.28 (s,
2H), 4.48 (m, 1H), 5.00 (s, 2H), 5.17 (m, 2H), 6.88 (s, 1H), 7.24 (m, 10H), 7.40 (s,
13 1H); C NMR (CDCl3, 62.9 MHz) δ 176.4, 172.7, 156.7, 137.2, 136.7, 136.2, 128.9,
128.3, 128.0, 128.0, 113.9, 79.8, 73.6, 69.9, 66.4, 54.2, 40.4, 33.8, 28.3, 24.8.
NHCbz
O
N CO2CH3 O
NO2
Nitro anhydride (46). Solution of Orn-OCH3·HCl (64 mg, 0.20 mmol), anhydride 4 (52 mg, 0.20 mmol) and DIEA (34 µL, 0.20 mmol) in DMF (1mL) was stirred for 2h at room temperature and then additional 2h at 65°C. Solvent was evaporated and residue was dissolved in DCM (2 mL). Catalytic amount of DMF was added followed by DIEA (34 µL, 0.2 mmol) and oxalyl chloride (18 µL, 0.20 mmol) at room temperature. Reaction mixture was stirred for 12h. Washed with 1M
HCl, NaHCO3 and brine. Organic phase was dried over Na2SO4. After solvent removal residue was purified on silica gel (1% EtOH/DCM) to give 46 (74 mg, 71%).
1 H NMR (CDCl3, 400 MHz) δ 0.71 (m, 1H), 1.05 (m, 2H), 1.77 (m, 1H), 2.07 (d, 1H,
J=9.7 Hz), 2.19 (d, 1H, J=9.6 Hz), 2.91 (m, 2H), 3.52 (s, 1.5H), 3.55 (s, 1.5H), 3.66
90 (m, 2H), 3.93 (m, 2H), 4.16 (m, 1H), 4.85 (m, 1H), 5.09 (s, 2H), 7.36 (m, 5H), 8.01
13 (s, 1H), 8.05 (m, 2H); C NMR (CDCl3, 100 MHz) δ 175.0, 174.9, 174.8, 174.8,
168.3, 168.2, 162.4, 156.2, 156.1, 150.1, 149.9, 147.5, 147.4, 144.4, 144.3, 136.6,
128.6, 128.3, 127.9, 127.9, 123.4, 123.4, 123.2, 123.2, 123.1, 118.1, 118.0, 68.8, 66.4,
53.3, 52.4, 52.4, 52.4, 52.3, 51.7, 51.6, 47.0, 46.9, 46.7, 46.7, 45.9, 45.8, 45.8, 39.9,
36.3, 31.3, 26.1, 26.1, 25.3, 25.2.
NH2
O
N CO2CH3 O
NH2
Aniline (47). Nitro anhydride 46 (85 mg, 0.16 mmol) was dissolved in EtOH
(5 mL), 10% Pd-C catalyst (10 mg) was added and reaction mixture was stirred under
H2 atmosphere (20 psi) for 1.5h. Catalyst was removed by filtration through celite pad. Solvent was evaporated in vacuum and crude product was purified on silica gel
1 to give 56 mg (96%) of pure product 47. H NMR (CDCl3, 400 MHz) δ 0.42 (m,
0.5H), 0.69 (m, 0.5H), 1.12 (m, 1.5H), 1.46 (m, 0.5H), 1.66 (m, 1H), 1.93 (d, 1H,
J=8.9 Hz), 2.04 (m, 1H), 2.50 (m, 0.5H), 2.72 (m, 1.5H), 3.50 (m, 1H), 3.60 (m, 1H),
3.64 (s, 3H), 3.72 (m, 3H), 4.21 (m, 1H), 6.58 (dd, 1H, J=8.5 Hz, J=8.5 Hz), 6.68 (d,
13 1H, J=12.2 Hz), 7.00 (d, 1H, J=7.8 Hz); C NMR (CDCl3, 100 MHz) δ 172.5, 171.7,
171.5, 171.3, 145.5, 143.1, 127.7, 126.8, 126.4, 124.8, 121.4, 113.9, 52.2, 51.9, 51.7,
50.9, 49.3, 48.5, 46.1, 40.5, 40.1, 26.1, 25.9, 25.2, 25.1, 23.2, 23.0, 21.5, 21.4.
91 Cl
N
9-Chloro-acridine (52). Was prepared according to reference 63. 1H NMR
(CDCl3, 250 MHz) δ 7.62 (ddd, 2H, J=1.1 Hz, J=6.6 Hz, J=8.7 Hz), 7.80 (ddd, 2H,
J=1.4 Hz, J=6.6 Hz, J=8.7 Hz), 8.20 (ddd, 1H, J=0.9 Hz, J=1.2 Hz, J=8.8 Hz), 8.42
(m, 1H).
H2N N NHCbz
Mono-N-CBz-2,7-diaminoacridine (55). Cbz-Cl (1.43 mL, 10.00 mmol)
was added at room temperature to a suspension of diaminoacridine hemisulfate 54
(258 mg, 1.00 mmol) in acetone (10 mL) and reaction mixture was refluxed for 14h.
Solvent was evaporated and residue was shaken in 1M NaOH-DCM two-phase
system. Layers were separated and aqueous phase was extracted with DCM (×2), organic extracts were dried over Na2SO4. After solvent removal crude product was
1 purified on silica gel (5% Et3N-5% MeOH-DCM) to give 110 mg (32%) of 55. H
NMR (CDCl3, 400 MHz) δ 8.50 (s, 1H), 7.92 (s, 1H), 7.87 (d, J=9.0 Hz, 1H), 7.78 (d,
J=8.9 Hz, 1H), 7.73 (d, J=8.9 Hz, 1H), 7.35-7.49 (m, 5H), 7.20 (s, 1H), 7.02 (m, 1H),
13 6.99 (dd, J=1.8 Hz, J=8.8 Hz, 1H), 5.28 (s, 2H), 4.24 (s, 2H); C NMR (CDCl3, 100
MHz) δ 153.1, 151.4, 150.0, 148.5, 139.6, 135.9, 135.6, 129.8, 129.5, 128.8, 128.7,
128.5, 122.0, 121.4, 119.5, 118.0, 113.8, 106.2, 67.3.
92