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UBC Theses and Dissertations

Studies Towards a Model ϐ-Sheet Causton, Ashley Scott 1994

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STUDIES T O W A R D S A M O D E L (3-SHEET By A S H L E Y SCOTT C A U S T O N B.Sc, The University of Greenwich, 1992 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E O F M A S T E R OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF BRITISH C O L U M B I A July 1994 ©Ashley Scott Causton, 1994 In presenting this thesis in partial fulfilment of the requirements for < an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date 1 ^ O e b W W DE-6 (2/88) 11 Abstract The construction of de novo proteins is a long time goal of many researchers into the protein folding problem. In order to design and construct "tertiary" structure from scratch, an understanding of the forces involved in the lower levels of structure is necessary. Simple models are needed to confidently study the factors involved in stabilizing the secondary and super-secondary structure of proteins. One way in which this can be done is by template assembly. The template limits the degrees of freedom afforded to the attached peptide chains, directing them into pre-determined folding patterns. The long term aim of this project is to model (3-sheets using a cyclotriveratrylene (CTV) molecule as template. The challenge in making this template-assembled synthetic protein is to covalently link six unprotected peptides to the macrocycle. Model studies, using amino acids protected at their C-terminus, were directed towards finding an efficient method that could be used for l inking unprotected peptides. This was achieved by first bromoacetylating the amino terminus of the amino acid models using bromoacetyl bromide in methylene chloride with potassium carbonate as base. These functionalised amino acid models were subsequently coupled to the six hydroxyl moieties on the cyclotriveratrylene template using potassium carbonate as base in degassed dimethylformamide. Thus we have succeeded in coupling six single amino acid esters to a C T V macrocycle in a 90% yield. This represents the first model of a hexa-substituted de novo protein. To successfully nucleate the hydrogen bonding pattern associated with p-sheet formation the template must possess a suitable inter-strand distance. This was investigated by looking for the presence of an internal hydrogen bonding network between the six amino acid Ill strands on the template. This was done using N M R and IR spectroscopy and comparing the results to those obtained for single stranded analogues. A n internal hydrogen bonding network was observed, indicating the suitability of the cyclotriveratrylene macrocycle as a template for modeling (3-sheets. iv Table of Contents Page Abstract i i List of Tables v i List of Figures vi i Abbreviations x Acknowledgments xi CHAPTER 1 INTRODUCTION 1.1 The Protein Folding Problem. . 1 1.2 P ro t e in S t ructure . . . . 2 1.3 Forces Involved in Defining Tertiary Structure 4 1.4 De Novo Protein Design 5 1.5 (3-Sheets 6 1.6 Temp la te Assembly 9 1.7 Pro jec t Goa l s . . 11 1.8 O b j e c t i v e s ... 12 CHAPTER 2 SYNTHESIS 2.1 I n t r o d u c t i o n 13 2.2 Synthesis of the Template 14 2.3 Investigation of l inkers 15 2.4 Reduct ive A m i n a t i o n 18 2.5 A l k y l a t i o n / a c y l a t i o n 21 2.6 Synthesis of Control Compounds for Hydrogen Bonding Study 24 2.7 E x p e r i m e n t a l 25 V CHAPTER 3 HYDROGEN BONDING 3.1 I n t r o d u c t i o n .. 31 3.2 In f ra red Spectroscopy. . . . . . . . . . 33 3.3 N M R Spect roscopy 38 3.4 D i s c u s s i o n . . 44 CHAPTER 4 CONCLUSIONS 4.1 Summary of Results 51 4.2 F u t u r e S tudy . . . 51 References 53 vi List of Tables Table Page 1. Amide proton chemical shifts in CDCI3 solution 39 2. Solvent effect on the amide chemical shift 40 3. Temperature dependence of amide chemical shifts in CDCI3 45 4. Temperature dependent amide chemical shifts for CTV-Phe 45 List of Figures vu Figure Page 1. Schematic diagram of an amino acid and the formation of a biopolymer 3 2. Diagram showing a polypeptide chain where the main-chain units are represented as rigid units forming a plane " 4 3. Schematic illustration of the incremental approach to the design of a four helix bundle 6 4. The inter-strand hydrogen bonds formed by (a) parallel and (b) anti parallel P-sheets 7 5. The mode of P-sheet aggregation 8 6. Template assembly of p-sheets using a single peptide strand. 9 7. Diacylaminopindolidone contains suitable hydrogen bonding moieties for P-sheet nucleation 10 8. Dibenzofuran nucleated P-sheet 11 9. Schematic representation of the C T V template with six P-sheets attached 12 10. Schematic diagram representing the synthesis of the proposed template assembled protein 13 ,11 . Inversion of C T V - O M e 14 12. Synthesis of template , 14 13. Dibenzofuran based amino acid 15 vii i 14. Strategy for making template assembled synthetic proteins based on a single peptide strand using solid phase peptide synthesis. 16 15. Porphyrin based four helix bundle 17 16. Derivitisation of the C-terminus of amino acids as their ethyl esters 18 17. Equilibrium between an aldehyde and amine with an imine and water 19 18. Attempts at Unking peptides to the C T V macrocycle by reductive amination 20 19. Potential side reactions for reductive amination carried out on the C T V macrocycle 21 20. Schematic representation of the alkylation/acylation approach 22 21. Bromoacetylation using bromoacetyl bromide in a mixed acetonitrile/sodium bicarbonate solution , 22 22. Bromoacetylation by brorrioacetic anhydride 23 23. Bromoacetylation using bromoacetyl bromide in methylene chloride with sodium carbonate as base 24 24. Linking six brorrioacetylated amino acid models to the hexahydroxy-CTV macrocycle 24 25. Synthesis of control compounds for hydrogen bonding studies 25 26. Proposed hydrogen bonding network between three peptide strands anchored to a phenyl ring 32 27. Single stranded control compound for hydrogen bonding study 33 28. IR spectrum of the amide region of PhO-Phe 34 ix 29. TR spectrum of the amide region of PhO-Ala 35 30. IR spectrum of the amide region of CTV-Phe 36 31. IR spectrum of the amide region of C T V - A l a 37 32. The C O S Y cross-peaks observed for CTV-Phe in CDCI3 41 33. CTV-Phe proton decoupling experiments 42 34. The N M R signals for non-equivalent protons in CDCI3 become broad in D M S O 43 35. The two possible intra-molecular hydrogen bonds formed by PhO-Phe andPhO-Ala 46 36. Examples of unfavourable hydrogen bonding interactions 47 37. Possible hydrogen bonding network of hexasubstituted C T V involving a 16 membered inter-strand hydrogen bond 49 38. Possible hydrogen bonding network of hexasubstituted C T V involving an 11 membered inter-strand hydrogen bond 50 39. Unsuccessful attempts at making homologues of CTV-Phe 52 X Abbreviations Ala Alanine BaO Barium Oxide CDCI3 Chloroform-dl C H 2 C 1 2 Methylene Chloride (COC l ) 2 Oxalyl Chloride C O S Y Correlated Spectroscopy C T V Cyclotriveratrylene C P K Corey-Pauling-Koltun D B U Diazabicyclo [5.4.0]undec-7-ene D C C Dicycmexylcarbocliimide D C M Methylene chloride D M A N , N-Dimethylacetamide D M F N , N-Dimethylformamide D M S O Dimethyl Sulfoxide D2O Deuterium Oxide IR Infrared K2CO3 Potassium Carbonate M e C N Acetonitrile NaC l Sodium Chloride N a H Sodium Hydride N a H C 0 3 Sodium Bicarbonate N M R Nuclear Magnetic Resonance Phe Phenylalanine Acknowledgments xi I wish to record my sincere appreciation to the following individuals: My research supervisor, Dr. John Sherman, for all his support, guidance and enthusiasm. I would especially like to thank him for stimulating my interest in protein folding. Dr. Janet Fraser and Dr. Bruce Gibb for their input and for doing the lions share of the proof reading. Bob Chapman, Naveen Chopra, Frank Tsai and Adam Mezo for keeping me sane(ish). 1 Chapter 1 Introduction 1.1 The Protein Folding Problem Proteins are essential for life, and have many diverse functions in biological systems. 1 In the early 1950's Sanger proved a protein to be a linear amino acid polymer, constructed from a combination of the 20 different natural amino acids. Furthermore, he determined that the specific sequence of these amino acids was unique to a given protein. 2 Wi th the pioneering work of Anfinsen in the late 1950's, the significance of reversible denaturation / renaturation to a protein's biological properties became clear in terms of its folded state.3 Anfinsen concluded that the primary amino sequence alone contained all the information necessary to define three dimensional structure and thus the function of a protein. 4 Determining how this three dimensional structure arises from its primary sequence has come to be known as the protein folding problem. The three dimensional architecture of these biopolyrners is unique because it has a relatively fixed structure.3 This results from a multitude of non-covalent interactions involving the protein backbone and the various amino acid side chains, which may be polar, non-polar or charged. 5 The ultimate goal for researchers probing the protein folding problem is to understand how three dimensional structure arises in terms of the chemistry and physics of interactions within the biopolymer. 6 The solution to this problem has important implications for the biotechnology industry where there is an ever-growing interest in new catalysts, especially for use at elevated temperature. Thus, the de novo design of proteins is a major goal in this area. 6 2 Recent advances in protein engineering have had a great impact on this research by making larger quantities of desired native and mutant proteins available for study. 6 - 7 This, coupled with advances in X-ray crystallography and protein N M R has made structural determination more expedient, increasing the knowledge base available and fueling interest in this area. The three dimensional architectures of approximately 500 natural proteins are known, revealing some common features.7 There is a small total energy difference between the folded and unfolded state of proteins, which is easy to measure but currently impossible to calculate from first principles. This difference is 5-20 kcal/mol in a protein, for which the sum of all non-covalent interactions in the folded or random states is on the order of 200-400 kcal/mol.7>8 Even i f it were possible to calculate the structure with the lowest free energy, the protein may still adopt the kinetically most accessible architecture along the folding pathway. 3 Therefore prediction of a protein's three dimensional structure remains one of the major unsolved problems in molecular biology. However, the rules concerning how a protein folds are far from understood, and thus the prediction of the tertiary structure from primary sequence is not possible with any reliability. 9 1.2 Protein Structure Before one can understand the three dimensional structure of a protein, an understanding of the lower levels of structure must be developed. The primary structure refers to the specific amino acid sequence of a protein and is dictated genetically or by design. The protein biopolymer is formed by condensation between the carboxyl and amino termini of sequential amino acids (Figure l ) . 6 - 7 These amide bonds have significant double bond character and exists almost exclusively in an s-trans configuration. Therefore the only two bonds around which rotation can readily occur in the backbone are the C - C a bond (psi angle) and the N-Coc bond (phi angle) (Figure 2). Only certain combinations of these two angles occur due to steric interactions between the side chains of these chiral L-amino acid residues 3 and the peptide backbone . 5 - 7 ' 1 0 Repetition of suitable combinations of phi and psi angles results in the formation of recognisable secondary structural features such as oc-helices and |3-sheets.5-7 Secondary structure refers to the local geometry of the polypeptide chain. Studying the factors involved in P-sheet structure is one of the long term goals of this project and therefore w i l l be discussed further in section 1.5. From studies of the known protein structures it has been observed that the secondary structures can pack into simple geometric arrangements, these commonly occurring motifs are known as the super-secondary structure. The overall arrangement of all these lower levels of structure leads to the tertiary structure. 5 ' 7 Figure 1: Schematic diagram of an amino acid and the formation of a biopolymer. H R = side-chain C, H 2 N \ ^ C O O H R L-amino acid O R 2 N-Terminus R-i H C-Terminus Dipeptide O R H O R Polymerisation R H O R H A n amino acid residue Polypeptide 4 F igure 2: Diagram showing a polypeptide chain where the main-chain units are represented as rigid peptide units forming a plane. Bond rotation within the peptide backbone tends to occur at the C a - C ' = 0 bond (psi, \|/) and the N - C a (phi, (()) bond The common themes seen i n the lower levels of structure can occur with different amino acid sequences. 1 1 However, the various amino acids do demonstrate preferences for taking part in the different secondary structures. These intrinsic propensities have been investigated by statistical 1 2 and experimental 1 3 methods, and represent a significant component in then formation of tertiary structure. 1.3 Forces Involved in Defining Tertiary Structure A knowledge of the physical properties of molecules, coupled with the trends that are observed in protein structure has led to an understanding of the dominant forces that occur in proteins. In aqueous solution the folded and unfolded states of a protein result from a balance of forces. Opposing protein folding is the unfavorable entropy associated with restricting the degrees of freedom of the chain. The dominant force for protein folding is the hydrophobic effect,5 i.e., the aversion for water of the non-polar residues which tend to be found at the core of the protein. 1 4 Hydrophilic polar and charged amino acids tend to be found at the surface of the protein. The interior tends to be packed as tightly as possible within steroelectronic considerations, since the presence of any cavities is known to destabilize the folded state of the 5 protein. Within the "non-polar" core that is formed by the hydrophobic residues, a protein's overall architecture can be stabilized by hydrogen bonding within the main-chain (which leads to secondary structure). Non-covalent interactions involving suitably paired polar or charged side-chains can also stabilize the protein's interior. 5 Another example of side-chain interactions that can contribute to protein stability occurs covalently when two cysteine residues that are spatially close in the overall architecture can form a disulfide bond. 7 1.4 De Novo Protein Design Although it is not yet possible to predict the three dimensional structure of a protein from its amino acid sequence alone, the rules of protein folding are sufficiently understood to design protein sequences that can adopt simple folded conformations. One of the most successful example of a de novo designed protein that folds into a monomeric conformation has been demonstrated by the work of DeGrado (Figure 3 ) . 1 5 Isolated peptide strands containing 16 residues with a high propensity for helix formation were seen to aggregate into a four helix bundle at high concentrations, packing along designed hydrophobic faces, while the hydrophilic faces of these amphiphilic peptides were exposed to the aqueous solvent. The stepwise addition of suitable turn residues led to a 74 residue peptide strand that successfully folded into a four helix bundle, a commonly occurring super-secondary structural motif. Thus, using a basic knowledge of protein structure, the feasibility of de novo protein design has been clearly demonstrated. ' A more thorough understanding of the forces involved in the formation of secondary structure is essential for designing the correctly folded protein tertiary structure from its primary amino acid sequence. Current methods for de novo design rely primarily on the correct prediction of secondary structural elements from the primary sequence. 5 They subsequently rely on the correct packing of these secondary structural elements into a stable folded conformation. 6 F igure 3: Schematic illustration of the incremental approach to the design of a four helix bundle. Lines represent unordered peptide strands, cylinders represent a-helixes. Step 1 Tetramer of 4 helices Step 2 Step 3 Monomer 1.5 (3-Sheets Unlike a-helices, which have been studied extensively, 1 6 our knowledge of the forces involved in (3-sheet formation is currently insufficient for the denovo design of well defined 13-sheet structures. In proteins, (3-sheets can be built up from several regions of the polypeptide that may be distant in the primary structure. The constituent P-strands are usually 3-10 residues in length and come together in a parallel or antiparallel fashion by forming hydrogen bonds betweeen the strands (Figure 4). Few simple models exist to study P-sheets, and most Figure 4: The inter-strand hydrogen bonds formed by (a) parallel and (b) antiparallel p-sheets. (a) ^ yr'yr y N - H N - H N - H N - H (b) R - V o . . . . . R - v ; . . . . . R - V o . . , R - V o H - N " H - N ' H - N H - N V-..R ) - . R : V -R V -R o=< ,.o=< ...o=< .....o=< N - H ' " ' N - H " ' N - H " * N - H R _ / R - / R - / R - / > o s >=o...... >=o.....( >=o H - N H - N H - N H - N ^ . . . . R ^ . . . R ^ . . . R ^ - R The side-chains point alternately above and below the plane that is formed by the inter-strand hydrogen bonds N - H ! » 0 = ( ' N - H 0 = ^ ; R - < > - R R " < > - R > = 0 - " H - N > = 0 H - N H - N \ = 0 H - N N = 0 V - R R < V -R R ( 0 = < N - H - 0 = < N - H N - H 0=( N - H ' R - < > - R r - < > = 0 H - N V = 0 ' H - N > = 0 H - N 4"R R " ' X l4"R information on their structure has been gleaned from homopolymers, copolymers and small pept ides. 1 7 A l l these systems aggregate in aqueous solution, demonstrating the difficulty associated with studying isolated P-sheets in this medium. This aggregation can occur by "infinite" hydrogen bonding between the P-strands, or by the "face to face" packing of these P-8 sheets (Figure 5). 5 In order to confidently study the factors involved in the formation of P-sheets controlled monomelic folding is required. F igure 5: The mode of P-sheet aggregation. R 9 1.6 Template Assembly The potentially complex folding patterns of a polypeptide can be overcome by attachment to a template molecule that can direct peptide blocks to predetermined chain topologies. A suitably designed template has interpeptide strand distances that approximate to those found in nature, and works by reducing the degrees of freedom afforded to the peptide, thereby enhancing the potential for intramolecular interactions and facilitating the desired folding pattern. This is especially relevant to P-sheet formation, for which there is a large entropic penalty to be paid upon folding. 5 One example of this technique used a single peptide strand, and more recently a cyclised peptide strand. 1 8 As a method for modelling parallel P-sheets there is some experimental evidence that this general approach has been sucessful (Figure 6 ) . 1 9 . F i gure 6: Template assembly of p-sheets using a single peptide strand. P-sheets are represented as arrows. 1 0 Template assembly was used to model ant iparal le l P-sheets us ing a diacylaminopindolidione based species. This mimics the backbone of two antiparallel P-sheets with functional groups that can undergo favourable hydrogen bonding with attached peptide strands. Incorporation of a suitable turn sequence into these strands led to the formation of monomeric P-sheet structure in certain solvents. Concentrating solely on four hydrophobic residues, recognizable P-sheet propensities were detected in 60% water/DMSO (Figure 7 ) . 2 0 Figure 7: Diacylaminopindolidone contains suitable hydrogen bonding moieties for P-sheet nucleation. Currently the only template that has resulted in a monomeric water soluble p-sheet, is a dibenzofuran molecule onto which are linked two peptide strands (Figure 8 ) . 2 1 This acts as a suitable reverse turn with interstrand distances that approximate to those found in nature for P-1 1 turns. The template also forms a hydrophobic cluster that is thought to be an important factor in the nucleation of antiparallel P-sheets.22 Figure 8: Dibenzofuran nucleated p-sheet. O Rj H O H Rj O H 1.7 Project Goals The long term aim of this project is to model P-sheets using templated assembly. This w i l l be accomplished using a cyclotriveratrylene (CTV) template covalently l inked to six peptide strands (Figure 9). The name "cavitein" has been proposed for these bioconjugate molecules since they are a combination of a cayitand (a host molecule with a convergent binding site) and a protein. The C T V template, which has six phenol functionalities, was chosen for its rigidity, synthetic availability and, more importantly, because the inter-peptide strand distances would approximate those found in nature. Another feature of this template is its enforced cavity, which has been shown to bind hydrophobic molecules in aqueous so lut ion : 2 3 The attached peptide strands w i l l have alternating hydrophobic and hydrophilic residues, such that the hydrophobic residues w i l l point into the core and the hydrophilic residues w i l l remain on the surface. This system is designed to circumvent the aggregation problem associated with modelling isolated P-sheets in aqueous media. The six P-strands wi l l 1 2 self-assemble around the hydrophobic core, leading to controlled "face to face" packing; and interstrand hydrogen bonding wi l l then form a closed surface compound, overcoming the side to side "infinite" hydrogen bonding. Water solubility w i l l be imparted by the solvent exposed hydrophilic residues. F igure 9: Schematic representation of the C T V template with six P-sheets attached. 1.8 Objectives The aims of this project were to: [1] Examine suitable methods for linking the peptides to the macrocyclic template using model amino acids that had been protected at their C-terminus. [2] Investigate the suitability of our template for P-sheet formation in terms of the interstrand distance. This was done by comparing the hexasubstituted C T V compounds to single stranded analogues, and looking for a hydrogen bonding network using IR and N M R spectroscopy. 13 Chapter 2 Synthesis 2.1 Introduction The main aim of this project was to find an efficient method for producing the desired template-assembled synthetic proteins. Synthetically, the greatest challenge in making these macromolecules is to jo in the peptide strands to the template. This requires a linker that can covalently bond them in a controlled and selective manner (Figure 10). This approach is viable because the hexahydroxyl-CTV template 2 4 (hereafter CTV-OH ) has previously been shown to react with six electrophiles, namely alkyl bromides 2 3 and tosylates. 2 5 The covalent linkage of peptides and proteins to organic molecules (and other proteins) has received great attention recently, furnishing us with a few potential methods for linking six peptides. 2 6 F igure 10: Schematic diagram representing the synthesis of the proposed template assembled synthetic protein. C T V Template 6 Linkers 6 Peptides Cavitein 14 2.2 Synthesis of the Template The hexamethoxy-CTV (hereafter C T V - O M e ) macrocycle was first synthesized in 1915 , 2 7 but the correct structure was not proposed for another fifty years. 2 8 The bowl-l ike structure of this macrocycle (Figure 11) is somewhat flexible, with a macrocyclic inversion barrier calculated to be 26.5 kcal/mol. 2 9 It is prepared by the acid catalyzed reaction between 1,2-dimethoxybenzene (veratrole) and formaldehyde. 3 0 Demethylation has been successfully achieved using boron tribromide (Figure 12), forming C T V - O H . 3 0 a Figure 11: Inversion of C T V - O M e . 15 2.3 Investigation of Linkers Many of the strategies for l inking peptides to templates have relied upon techniques derived from solid phase peptide synthesis. One example is encountered with the dibenzofuran molecule that was employed as a reverse turn mimic for the template assembly of P-sheets by Kel ly (described in section 1.6).21 This template has both amine and carboxylic acid moieties (Figure 13), thus allowing it to be incorporated into a standard cycle for peptide synthesis. 3 1 Figure 13: Dibenzofuran based amino acid. Mutter assembled peptide blocks onto a straight chain peptide (described in section 1.6) that was attached to a solid support r e s in . 1 9 Lysine groups were designed into the chain, which were subsequently deprotected and used for the attachment of peptide blocks, thus generating the molecule required for study (Figure 14). One disadvantage of this method was that the polypeptides were "grown" from the template by the addition of one residue at a time. This approach led to difficulties in producing the purified template-assembled synthetic protein. More recently, two methods have been reported in which whole peptide chains have been linked to the template. 3 2 ' 3 3 The only example in which multiple peptide blocks have been directly attached to an organic template molecule was in the synthesis of a helix bundle. 3 4 This was constructed on a porphyrin ring which was derivatised with four alkyl carboxylic acid functionalities. The acid 16 F igure 14: Strategy for making template assembled synthetic proteins based a single peptide strand using solid phase peptide synthesis. Peptide template containing protected lysine residues is constructed on solid support N H 0 N H { ? ] JP | = protecting group Deprotection of lysine groups Coupling of peptide blocks Template Assembled and full deprotection Synthetic Prote in Cleavage from resin 17 Figure 15: Porphyrin based four helix bundle. Ac i d moieties on the porphyrin are activated with N-hydroxysuccinimide and subsequently linked to the N-terminus of a protected peptide. Deprotection of the peptide affords the desired motif, which contained a hydrophobic pocket for binding substrates. Me C O O H i (1) (2) H 2 N-Pept ide (protected) (3) Deprotection L substrate L (ax ia l l igand) 18 moieties were converted to the activated esters which were then reacted with the N-terminus of a suitably protected peptide (Figure 15). Protected peptides are notorious for their low solubility in organic solvents, which may create problems in purification and in the coupling reaction. Therefore, we sought a method that would make use of unprotected peptides. The first goal of this investigation was to find suitable literature methods for covalently l inking proteins to organic molecules. 3 5 The investigation of suitable linker molecules was undertaken using amino acids protected at their C-terminus as the ethyl ester to increase their solubility in organic solvents. The amino acids were derivatised by the standard procedure of stirring the amino acid overnight at room temperature in ethanolic HC1 (Figure 16). 3 6 Figure 16: Derivitisation of the C-terminus of amino acids as their ethyl esters. 2.4 Reductive Amination When mixed together, an aldehyde and an amine are in equilibrium with the resultant dehydration product, the imine (Figure 17). 3 7 It is possible to selectively reduce the imine to the secondary amine in the presence of the free aldehyde, thereby shifting the equilibrium to the r ight . 3 7 This approach to functionalising amino groups in proteins was first reported in 1968 using sodium borohydride as the reducing agent, which also reduced the aldehydes and ketones under investigation at a competing rate. 3 8 Sodium cyanoborohydride 3 9 is now the reagent of choice for reductive amination because it is mild enough not to react with aldehydes R H C l / E t O H R 1 0 0 % R = - C H 2 P h , -Me 19 or ketones. 4 0 Therefore the initial strategy was to functionalise the C T V macrocyclic template with six aldehyde moieties, and react these with the a-amino group of the peptide. F igure 17: Equi l ibr ium between an aldehyde and amine with an inline and water, and the resultant reduction products. H I + R " N H 2 R' ^ O Reduct ion t R' ^ O H H Reduct ion t R' N ' i H .R" The C T V macrocycle was functionalised with six protected aldehyde moieties by reacting C T V - O H with six equivalents of 3-bromopropionaldehyde diethyl ace ta l 4 0 in D M F solvent using sodium hydride as base. Deprotection using trifluroacetic acid 4 1 followed by sodium cyanoborohydride reductive amination with various model amino acids (Figure 18) led to the formation of a tar at each attempt. One of the major side reactions associated with this procedure is that once formed, the secondary amine can undergo further condensation with the aldehyde. This problem is usually overcome by using an excess of the amine. Unfortunately due to the highly organised nature of the hexaaldehyde-CTV, there is a high " local"-concentration of aldehydes with respect to the amine, which may lead to various side reactions 20 (Figure 19). This approach for l inking unprotected peptides to our macrocyclic template system was therefore not pursued any further. F igure 18: Attempts at linking peptides to the C T V macrocycle by reductive amination. EtO EtO 100% 21 F i gure 19: Potential side reactions for reductive amination carried out on the C T V macrocycle. None of these compounds were isolated. 2.5 Alkylation/acylation This approach involved a bifunctional reagent with acylating/alkylating properties. 3 5 The mono-functional bromoalkylation of an enzyme by an organic molecule was first reported in 1971 . 4 2 Since then the nucleophilic side-chains of proteins have been used as a target for labeling with organic molecules 4 3 or for covalently l inking short peptide sequences. 4 4 Thus, acylation of the a-amino group of the model amino acid, by a bi-functional reagent that also contains an alkyl halide moiety produced a suitably functionalised species to undergo 22 nucleophil ic displacement by the hydroxyl groups of the C T V (Figure 20). These functionalised amino acid models can be prepared by several methods, 2 6 and those that were successfully applied to our model system are outlined below. Figure 20: Schematic representation of the alkylation/acylation approach. . 0 B r ^ ^ i L + H 2N-Peptide Y = activated leaving group C T V - O H + B r ^ N H p e p U d e ~ - C T V " 0 ^ N H -Pep , ide o NH-Peptide Bromoacetyl bromide has been used to functionalise short peptide fragments using the mixed solvent of water/acetonitrile and sodium bicarbonate. 4 4 This method was successfully applied to a model amino acid, but as a procedure it was not practical for larger scales because the acid bromide was hydrolysed at a competing rate (Figure 21). Figure 21 : Bromoacetylation using bromoacetyl bromide in a mixed acetonitrile/sodium bicarbonate solution. O C H 2 P h M e C N / H 2 0 H V ^ R , H , N C O , E t NaHCOg « B r 2 0 2 E t C 0 3 Q ^ 7 5 % 23 Another method for bromoacetylation that has been applied directly to peptides during standard peptide synthesis was also investigated. 4 5 Bromoacetic anhydride is generated from bromoacetic acid using dicyclohexylcarbodiimide (DCC) , before addition to the free amino terminus of the model compound (Figure 22). F igure 22: Bromoacetylation by Bromoacetic anhydride. Coupl ing of bromoacetyl bromide to the amino acid model was also achieved in methylene chloride solvent using sodium carbonate as base (Figure 23 ) . 4 6 Using this method the starting materials were easily washed away during work-up, and hydrolysis of the acid bromide was negligible. Thus, this was the method of choice for these model studies. H 4 2 % 24 F igure 23: Bromoacetylation using bromoacetyl bromide in methylene chloride with sodium carbonate as base. H r» M e D C M 1 Br 1 I : B r / V N - C ° 2 E t B r ^ ^ B r H 2 N ^ C 0 2 E t N a 2 C 0 3 H ^ 60% 'c The next step was to attach these functionalised amino acid models to the C T V macrocycle: For this coupling, only a narrow range of solvent/base conditions were successful. The best results were achieved in dimethylformamide (DMF ) using potassium carbonate as base (Figure 24). Degassing of the solvent was found to be essential, since failure to do so led to oxidation of the C T V in competition with the desired alkylation. Overall yields for this coupling were 68 to 90%, which is surprisingly high for the creation of six new bonds to a sterically hindered site! F i gure 24: L ink ing six bromoacetylated amino acid models to the hexahydroxyl-CTV macrocycle. CTV-OH + B ^ V 0 2 * - - o J ^ H , O R D M F ^ R = C H 2 P h (90%) = M e ( 6 8 % ) 2.6 Synthesis of Control Compounds for Hydrogen Bonding Study The initial approach in making these compounds was to link the bromoacylated amino acid models to phenol. Again this coupling only worked within a narrow range of conditions. 25 Al though using dimethylacetamide (DMA)/diazabicyclo[5.4.0]undec-7-ene ( D B U ) or DMA/potassium carbonate were succesful for this coupling, low yields (20-30%) resulted in these control compounds being made by an alternative method. Phenoxyacetic acid was converted to the acid chloride and subsequently reacted with the amine moiety of the model amino acid to give the monomeric product (Figure 25). F igure 25: Synthesis of control compounds for hydrogen bonding studies. O H (COC l ) 2 C H 2 C 1 2 Overall Yields: R = C H 2 P h (83%) = Me (73%) D M F t R H 2 N ^ C 0 2 E t O R N C 0 2 E t H 2.7 Experimental Genera l Comments . Chemicals were reagent grade (Aldrich or B D H ) . Dimethyl formamide was dried over 4A molecular sives and degassed by bubbling dry N2 through it for 20-30 mins. Dimethylacetamide was stirred over B a O then distilled under N2 onto 4A molecular sieves. The * H N M R spectra were run on a Bruker A C - 2 0 0 E or WH-400 spectrometer. Residual lH signals from deuterated solvents were used as the reference. Mass spectra were run on a Kratos Concept IIH32. IR specra were run on an AT I Mattson Genesis 26 Series FTIR spectrometer using N a C l cells of path length 0.516 mm. Samples for IR were solutions in CDCI3, which was subtracted from the spectra. Peaks were referenced to the 1600 c m " 1 peak of polystyrene. Melt ing points were recorded on a Thomas Hoover Unimelt capil lary melt ing point apparatus. S i l i ca gel (230-400 mesh, B D H ) was used for chromatography and silica gel glass-backed analytical plates (0.2 mm, Aldrich) were used for t.l.c. with U V detection and I2 staining where necessary. Compounds are not fully characterised. The fol lowing compounds were prepared by literature methods; 10,15-Dihydro-2,3,7,8,12,13-hexamethoxy-5H-tribenzo[a,d,g]cyclononene (hexamethoxy-c y c l o t r i v e r a t r y l e n e ) , 3 0 10 , 15 -D ihyd ro -2 , 3 , 7 , 8 , 12 ,13 -hexahyd roxy -5H -tribenzo[a,d,g]cyclononene (hexahydroxyl -cyclotr iveratrylene) , 3 0 a E t h y l phenylalaninate hydrochloride salt (phenylalanine ethyl ester) , 3 6 E t h y l alaninate hydrochloride salt (alanine ethyl ester),3 6 and phenoxyacetic ac id . 4 7 Ethyl iV-(2-bromoethanoyl)phenylaIaninate (TV-Bromoacetylphenylalanine ethyl ester), and Ethyl iV-(2-bromoethanoyl)alaninate (iV-bromoacetylalanine ethyl ester). Procedure A . To phenylalanine ethyl ester hydrochloride (0.50 g, 2.6 mmol) in a mixture of acetonitrile (20 mL) and 50% saturated sodium bicarbonate solution (30 mL), at 0°C was added bromoacetyl bromide (1.05 g, 5.2 mmol) dropwise over 5 mins, maintaining the p H between 8-10 by further addition of saturated sodium bicarbonate solution (30 mL) . The reaction mixture was stirred at <5°C for 1 hr before being carefully poured into a mixture of ice (60 g) and concentrated HC1 (15 mL) i n an oversize container. The acidic mixture was extracted with diethyl ether (2 x 75 mL) . The combined organic phases were washed with saturated sodium bicarbonate solution (75 mL) and brine (75 mL) before being dried over magnesium sulfate. Evaporation of solvent in vacuo resulted in a pale yellow oi l . Purification by column chromatography (DCM) led to a white solid, y ie ld = 0.61 g (75%) of N-27 bromoactetylphenylalanine ethyl ester. ! H N M R (200 M H z , CDC1 3 ) ; 8 7.33-7.03 (m, 5 H , Ph), 8 6.81 (d, I H , N-H, J = 7.1), 8 4.77 (dt, IH , N-CH, J = 7.1, 5.6), 8 4.13 (q, 2H , C / / 2 C H 3 , J = 6.4), 8 3.79 (s, 2 H , B r C # 2 ) , 8 3.10 (d, 2 H , CH2Ph, J = 5.6), 8 1.18 (t, 3 H , CH2CH3, J = 6.4). M p 71-2°C. Procedure B. A solution of bromoacetic acid (278 mg, 2 mmol) in (dry) methylene chloride was treated with dicyclohexylcarbodiimide (207 mg, 1 mmol) under dry conditions. This mixture was stirred at room temperature for 30 min, and a precipitate was seen to form. The solution was then filtered, and the solid was washed with a little dry methylene chloride. Dimethylformamide (4 mL) was added to the filtrate and the methylene chloride was then removed in vacuo. This resulted in bromoacetic anhydride as a solution in D M F , which was then added to a mixture Of phenylalanine ethyl ester hydrochloride (115 mg, 0.5 mmol) containing disopropylamine (0.5 mmol) in D M F (3 mL) . This mixture was stirred at room temperature under dry conditions for 2 h. The solvent was removed in vacuo and the residue was partitioned between ethyl acetate (25 mL) and saturated sodium bicarbonate solution (25 mL). The organic phase was washed with brine (25 mL) and dried over MgSC>4. Removal of solvent in vacuo yielded a pale yellow waxy solid. Column chromatography ( D C M ) led to a white so l id , y i e ld ,=• 60 mg (42%) o f N-bromoactetylphenylalanine ethyl ester. Procedure C . Bromoacetyl bromide (2.56 g, 13.8 mmol) was added to a mixture of alanine ethyl ester (1.47 g, 12.5 mmol), sodium carbonate (1.46 g, 13.8 mmol) in methylene chloride (25mL). The reaction mixture was stirred at room temperature under dry conditions for 1 h before being washed with 2 N HC1 solution (20 mL) , water (2 x 20 mL) and dried over M g S 0 4 . Evaporation of the solvent resulted in a waxy colourless solid, yield = 1.79 g (60%) of A/-bromoacetylalanine ethyl ester. * H N M R (200 M H z , CDCI3); 8 7.03 (broad, IH , N-H), 8 4.53 (dq, I H , N-CH, J = 6.9, 6.2), 8 4.22 (q, 2 H , C / / 2 C H 3 , J = 6.4), 8 3.88 (s, 2 H , B r C H 2 ) , 8 1.43 (d, 3 H , C H C / / 3 , J = 6.9), 8 1.26 (s, 2H, C H 2 C # 3 , J = 6.4). M p 61-2 G C. 28 Ethyl Ar -^-phenoxyethanoyOphenylalaninate (PhO-Phe). To a mixture of phenoxyacetic acid (152 mgs, 1 mmol) in dry methylene chloride (5 mL) , was added oxalyl chloride (127 mg, 1 mmol), and 2 drops of D M F . The reaction mixture was stirred under dry conditions at room temperature for 2 h. The solvent was removed in vacuo and the oily green residue added to phenylalanine ethyl ester (193 mg, 1 mmol) in D M F (5 mL) . This mixture was stirred under dry conditions for a further 2 h, before removal of the solvent in vacuo. The reaction mixture was partitioned between diethyl ether (25 mL) and saturated sodium bicarbonate solution (20 mL). The organic layer was washed with 2 N HC1 solution (20 mL) , brine (20 mL) and dried over MgSC>4. Evaporation of solvent resulted in a colourless solid which was recrystallised from diethyl ether / petroleum ether to produce 271 mg (83%) of fine colourless needles. * H N M R (200 M H z , CDC1 3 ) ; 8 7.34-6.84 (m, 11H, Ph, A r O , N-H) , 5 4.93 (dt, I H , N-CH, J = 6.5, 8.2), 5 4.48 (s, 2H , A r O C # 2 ) , 5 4.16 (q, 2 H , C # 2 C H 3 , J = 7.1), 8 3.13 (d, 2H , CH2?h, J = 6.5), 8 1.22 (t, 3H , C H - C H 3 , J = 7.1); IR (CDCI3, cm" 1 ) 3413 (NH), 1730 (ester C=0), 1684 (amide C=0). M p 75-6°C. Ethyl N-(2-phenoxyethanoyl)alaninate (PhO-Ala). Prepared by the method outlined above except using 118 mg (1 mmol) alanine ethyl ester. Purification was by column chromatography (1:1 diethyl ethenpetroleum ether) to produce a 181 mg (72%) of colourless oi l . * H N M R (200 M H z , CDCI3); 8 7.35-6.91 (m, 6H , PhO, N H ) , 8 4.65 (dq, I H , N C H , J = 7.2, 7.0), 8 4.49 (s, 2 H , PhOCt f 2 ) , 8 4.20 (q, 2H , C / / 2 C H 3 , J = 7.1), 8 1.45 (d, 3 H , C H C / / 3 , J = 7.2), 8 1.27 (t, 3H, C H 2 C t f 3 , J = 7.1); IR (CDCI3, cm-' ) 3419 (NH), 1734 (ester C=0), 1675 (amide C=0). 10,15-Dihydro-2,3,8,12,13-hexakis[Ethyl N-(2-oxyethanoyl)phenylalaninate]-5H-tribenzo[a,d,g]cyclononene (CTV-Phe). To a mixture of hexahydroxyl-CTV (13 mg, 3.6 x 10' 5mol) and potasium carbonate (44 mg, 3.2 x 10" 4 mol) in dry, degassed D M F , was added bromoacetylphenylalanine ethyl ester (100 mg, 3.2 x 10 ' 4 mol). The reaction 29 mixture was stirred for 20 h at room temperature in a stoppered flask. The D M F was removed in vacuo. The residue was dissoved in ethyl acetate and filtered through a pad of silica gel with ethyl acetate as eluent producing 56 mg (90%) of a pale yellow oi l . Further purification was achieved by column chromatography (3:1, diethyl ether:acetone) to produce the pale yellow glass. ] H N M R (200 M H z , C D C I 3 ) ; 8 7.58 (d, 3 H , N t f a , J = 7.2), 8 7.25-6.68 (m, 39H, N H H , As, Ph), 8 4.88 (ddd, 3H , NC// a , J = 7.2, 6.8, 6.8), 5 4.75 (ddd, 3H , NCHb, J = 7.6, 6.4, 6.4), 8 4.63 (d, 3H , AnCH axial, J = 15.0), 8 4.42 (d, 3H , A r O C t f 2 , J = 15.2), 8 4.35 (s, 6 H , ATOCH2), 6 4.25 (d, 3 H , ArOC// 2 , J = 15.2), 8 4.10 (q, 12H, C / / 2 C H 3 , J = 6.3), 8 3.50 (d, 3 H , A r 2 C # equatorial, J = 15.0), 8 3.12 (d, A B quartet, 6 H , P h C # 2 a , J = 28.8, 6.8), 8 2.92 (d, A B quartet, 6H , P h C # 2 b , J = 20.8, 6.4), 8 1.16 (t, 18H, C H 2 C # 3 , J = 6.3); M S ( LS IMS , 3-nitrobenzylalcohol) m/e 1766 (M+H+); IR ( C D C I 3 , cm" 1 ) 3410 3370 (NH), 1734 (ester C=0) , 1675 (amide C F O ) , 1266 (ester C-O), 1201 (ether C -O) . 10,15-Dihydro-2,3,8,12,13-hexakis[Ethyl N- (2-oxyethanoyl )alaninate] -5H-tribenzo[a,d,g]cyclononene (CTV-Ala). Same procedure as above except (500 mg, 2.1 mmol) bromoacetyl alanine added to C T V - O H (85 mg, 0.23 mmol), potassium carbonate (289 mg, 2.1 mmol) in 8 m L D M F . Purification by column chromatography (4:1 diethyl ethenacetone) yielded 204 mg (68%) of pale brown viscous oi l . *H N M R (200 M H z , C D C I 3 ) ; 30 6 7.53 (d, 3 H , NH , J = 8.0), 5 7.41 (d, I H , N # , J = 8.0), 8 6.99 (s, 3H , A r H ) , 8 6.88 (s 3 H , ATH), 8 4.77-4.40 (m, 9 H , A r 2 C H axial, NCH), 8 4.29-4.02 (m, 24H , CU3CH2, AxOCH2), 8 3.53 (d, 3H, Ax2CH equatorial, J = 14.8), 8 1.48 (d, 18H, C H C # 3 , J = 6.5), 8 1.27 (t, 18H, C H 2 C H 3 , J = 6.4); M S (LSIMS, 3-nitrobenzylalcohol) m/e 1310 (M+H+); IR ( C D C ^ c r r r 1 ) 3413 (NH), 1734 (ester C=0) , 1675 (amide C=0) , 1210 (ester C-O), 1154 (ether C-O). 31 Chapter 3 Hydrogen Bonding 3.1 Introduction Much of the current knowledge about the hydrogen bonds formed by amides has come from the investigation of small peptides using IR and N M R spectroscopy. These types of studies are often carried out in solvents of low polarity, because high polarity solvents w i l l compete for the hydrogen bonding moieties of the molecule under investigation. Studies of this type on linear peptides have found that the most favourable main-chain hydrogen bonding distances are consistent with those seen in the secondary structure of proteins, namely a -he l i ces 4 8 and reverse turns. 4 9 The prediction of reverse turns from the primary sequence is especially important for de novo protein design. Lifson anchored three peptides to an organic molecule, and provided an example where non-linear peptide strands have been investigated for their interstrand hydrogren bonding potential (Figure 26). Based on IR and N M R spectral data as well as computer modeling, the amide protons were proposed to exist in one of three categories: (1) no hydrogen bonds between the strands, (2) hydrogen bonding between a pair of strands arranged anti-parallel, or (3) forming a clockwise "belt" of hydrogen bonds between all three peptide strands. 5 0 The hydrogen bonding network between the six amino acids on the C T V was studied by TR and N M R spectroscopy. The amide region of the IR spectra gave qualitative information about the different orientations and strengths of the hydrogen bonds. The main information gleaned from the N M R was obtained by comparing the spectra in chlorofornW; (CDCI3) and in dimethyl su l f ox ide -^ (DMSO) . If an intramolecular hydrogen bonding network were present for the test compounds in CDCI3, it would manifest itself by restricting the 32 conformations of the six amino acid strands. The more polar D M S O solvent would disrupt the intramolecular hydrogen bonding network, causing an ensemble of conformations to be observed. In the N M R spectra the chemical shift difference of the amide protons between CDCI3 and D M S O was also related to their ability to form intra-molecular hydrogen bonds. A qualitative estimation of the degree of amide hydrogen bonding was carried out by deuterium exchange and by variable temperature N M R . Sections 3.2 and 3.3 describe the results obtained using IR and N M R respectively, and the implications of these results w i l l be discussed in section 3.4. Figure 26: Proposed hydrogen bonding network between three peptide strands anchored to a phenyl ring. The degree of inter-strand hydrogen bonding increases with the size of the side-chain. • Boc = terf-butyloxycarbonyl R = i -Bu, s-Bu, /-Pr, Me The C T V based molecules studied were linked to either six phenylalanine (hereafter CTV-Phe) or six alanine (hereafter CTV-A la ) derivatives. The results were compared to those obtained for a 33 control molecule: a single stranded analogue of the C T V based compounds consisting of phenol linked to the respective amino acid (Figure 27), which was either a phenylalanine (hereafter PhO-Phe) or an alanine (hereafter PhO-Ala) derivative. F igure 27: Single stranded control compound for hydrogen bonding study. O R N X C ( ) 2 E t phO-Phe R = C H 2 P h H P h O - A l a R = Me 3.2 Infrared Spectroscopy The N - H stretching frequencies for the amides in the IR spectra were surveyed for evidence of hydrogen bonding in CDC I3 . IR spectroscopy has the advantage over N M R that hydrogen bonds arising from different geometries can be distinguished 5 1 because hydrogen bonding between amides is usually in fast exchange on the N M R time scale in C D C I 3 , which yields the weighted average signals for all species present. A completely non-hydrogen bonded N - H stretch observed with N-methyl acetamide has been measured in the gas phase 5 2 to be 3490 cm" 1 . In chloroform this value has typically been found to be around 3437 c n r 1 , indicating some degree of hydrogen bonding with the solvent. 5 1 The stronger the hydrogen bond that the N - H is involved in, the lower the energy required to stretch it, and therefore the lower the stretching frequency 5 3 The IR spectra of the single stranded controls PhO-Phe (Figure 28) and PhO-A l a (Figure 29) exhibited single N - H stretches at 3413 c m - 1 and 3408 c m - 1 respectively, indicating 34 F igure 28: IR spectrum of the amide region of PhO-Phe. Sample run 25 mmol solution C D C I 3 (subtracted from backround), using NaC l cells of 0.516 mm path length. tmp80 MonJun 2715:48:00:661994 \ — 1 1 i 3600 3500 3400 3300 Wavenumbers 35 F igure 29: IR spectrum of the amide region of PhO-Ala . Sample run 25 mmol solution CDCI3 (subtracted from backround), using NaC l cells of 0.516 mm path length. tmp78 MonJun 27 15:40:57:92 1994 / W 7CH % T r a n s m i t t a n c e 60-i 50i 40-i 3600 3500 3400 Wavenumbers 3300 3200 36 F igure 30: IR spectrum of the amide region of CTV-Phe. Sample run 25 mmol solution CDCI3 (subtracted from backround), using N a C l cells of 0.516 mm path length. tmp77 MonJun 27 15:34:58:25 1994 3600 3500 3400 3300 3200 Wavenumbers 37 F igure 31: IR spectrum of the amide region of C T V - A l a . Sample run 25 mmol solution in CDCI3 (subtracted from backround), using NaC l cells of 0.516 mm path length. tmpSI Mon Jun 27 15:53:27:63 1994 3600 . 3500 3400 3300 3200 Wavenumbers 38 fairly weak hydrogen bonding in one geometric orientation. CTV-Phe exhibited two amide stretching frequencies, one sharp at 3410 cm" 1 and the other broad at 3370 c n r 1 (Figure 30). This is indicative of two distinct geometric orientations of the amide protons. The signal at 3410 c m - 1 is similar to that seen in the control, with the newly observed band being consistent with a stronger hydrogen bond. C T V - A l a showed a similar spectra to CTV-Phe , with a weakly hydrogen bonding peak at 3410 c m - 1 , and a very broad peak centered at approximately 3350 cm" 1 (Figure 31). A l l four compounds studied showed concentration independent IR spectra from 1 mmol to 25 mmol. This was the expected result for the CTV-Phe and C T V - A l a which are both designed for concentration independent intramolecular hydrogen bonding. This concentration independence was somewhat unexpected for the controls and was attributed to a weak but stable intramolecular hydrogen bond (which wi l l be discussed further in section 3.4). 3.3 NMR Spectroscopy The equilibrium between the hydrogen bonded and non-hydrogen bonded states for the amide protons was observed to be fast on the N M R time scale, the chemical shifts of these signals being the weighted averages for this equi l ibr ium. 5 4 Typically amide protons come in the range of 5-8.5 p p m 5 4 in CDCI3 depending upon their degree of deshielding. A downfield shift can result from an amide proton involved in a hydrogen bond: electrons wi l l be withdrawn from the proton, therefore deshielding the nucleus and requiring less energy to effect a transition. Other sources of magnetic anisotropy may also effect the chemical shift of the amide proton. The amide proton chemical shifts for all the compounds in CDCI3 are tabulated below (Table 1). The two controls PhO-Phe and PhO-Ala show amide proton shifts of 6.99 ppm and 7.16 ppm respectively, both indicating some degree of hydrogen bonding. CTV-Phe shows two distinct amide chemical shifts at 7.18 ppm and 7.58 ppm in a 1:1 ratio, consistent with two hydrogen bonded amides, one stronger than the other. The amide proton chemical shifts of 39 C T V - A l a were at 7.41 ppm and 7.58 ppm, again exhibiting a 1:1 ratio and consistent with hydrogen bonding. Table 1: Amide proton chemical shifts in CDCI3 solution. C o m p o u n d Chemi ca l Shi f t (ppm) PhO-Phe 6.99* PhO-Ala 7.16 CTV-Phe 7.18*, 7.58 CTV-A la 7.41, 7.58 * Amide proton coincident with aromatic protons, therefore estimated values A l l four compounds were found to exhibit concentration independent N M R in CDCI3 from 1 mmol to 100 mmol, consistent with the IR data and further indicating intramolecular rather than intermolecular hydrogen bonding. The amide A8s obtained by changing the solvent from CDCI3 to the strongly hydrogen bonding D M S O are tabulated below (Table 2). In D M S O the amide protons wi l l be extensively hydrogen bonded with the solvent, therefore a smaller A8 value for changing the solvent from CDCI3 to D M S O would indicate a larger degree of hydrogen bonding present in C D C I 3 . For the two controls PhO-Phe and PhO-A la the A8's (CDCI3 to D M S O ) were 1.44 ppm and 1.31 ppm respectively. The A8's experienced by CTV-Phe were 1.05 ppm and 0.75 ppm, and by C T V - A l a of 0.92 ppm and 0.85 ppm. These results are consistent with stronger intramolecular hydrogen bonding in C D C I 3 of the hexasubstituted C T V analogues than in the single stranded controls. 40 Table 2: Solvent effect on the amide chemical shift. C o m p o u n d CDCI3 D M S O A 8 PhO-Phe 6.99* 8.43 1.44* PhO-Ala 7.16 8.47 1.31 CTV-Phe 7.18*, 7.58 8.23, 8.33 1.05*, 0.75 CTV-A l a 7.41,7.58 8.33, 8.43 0.92, 0.85 * Amide proton coincident with aromatic protons, therefore estimated values The occurrence of a hydrogen bonding network in the CTV-Phe compound is strongly indicated by the presence of sharp non-equivalent protons in CDCI3, while only broad peaks were seen for the same protons in D M S O . The spectrum of CTV-Phe in CDCI3 (Figure 32) shows distinct signals for each of the two strands attached to each C3 symmetric aryl unit of the C T V template. C O S Y N M R spectroscopy exhibited crosspeaks for one amino acid N H -C a / / - C r Y 2 P h strand at 5's 7.58, 4.88 and 3.12 respectively, and for the other NH-CaH-C#2Ph strand at 8's 7.18, 4.75 and 2.92 respectively (Figure 32). Further confirmation of these distinct strands was obtained by decoupling experiments (two of which are shown in Figure 33). The ester groups of both strands did not exhibit any difference in shift values. The CTV-O - C H 2 protons of the methylene linkers are split into two types, one set of signals is the predicted A B quartet, the other set exists as an apparent singlet. This feature is unexpected but does become broad at low temperature, indicating a more restricted species. Changing solvents to the more polar D M S O disrupts the internal hydrogen bonding network, resulting in an ensemble of conformations, which leads to the broadening and convergence of signals for protons (Figure 34). The N M R spectrum of C T V - A l a did not show any noticeable non-equivalence in CDCI3 (except the N - H of the amide protons), the presence of overlapping 41 peaks precluding any firm conclusions. The single stranded controls do not show any significant difference in the shape of their spectra by changing solvent. Figure 32: The COSY NMR cross-peaks observed for CTV-Phe in CDCI3. The two sets of cross-peaks represent non-equivalent NH-C aH-CH2 strands. For a more complete assignment of the spectra see section 2.7. C0 2 Et C0 2 Et P h C H 2 - / P h C H 2 —( b N - H N - H 42 F igure 33: CTV-Phe proton decoupling experiments (only the 5.0-2.7 ppm region of the spectra is shown). Decoupling of the C-Ha signals at (a) 4.85 ppm, or (b) 4.75 ppm, simplify the related C / ^ P h signals at 3.12 ppm and 2.92 ppm respectively, into A B quartets when compared to the original spectra (c). 1 1 1 • 1 f 1 r — , , 1 , — , , r — , , , -, 1 , r S . C 4 . 5 4 - 8 . ~i-S l-Z 43 Figure 33: The N M R signals for non-equivalent protons in CDCI3 become broad in D M S O . (1) A rOC// 2 and N O / CO.Et a b 5J. 0 4.5 PPM PhCH, — / " a N CO :Et PhCH, —( b —f—, i—i—i J—r—1 r 5.0 4.5. P P M (2) Gf7 2Ph CDCI3 D M S O 3 . 0 CDCI3 1 ' I ' 3 .0 D M S O 44 Further evidence for a fairly stable hydrogen bonding network comes from the rates of disappearence of the amide proton N M R signals in CDCI3 upon adding D2O. Over a period of twelve hours the further downfield amide proton for the C T V analogues, i.e., the amide proton with a higher degree of hydrogen bonding, exchanges at a slower rate than the one at higher field. This result supports the presence of a relatively "fixed" conformation with one hydrogen bond being stronger than the other. Temperature dependent N M R was first used as a tool for investigating the conformation of small cyclic peptides in solvents of high polarity, discerning between solvent exposed or internally hydrogen bonded amide protons. 5 5 More recently it has been used to determine the most favourable hydrogen bonding interactions for short peptide sequences in non-polar solvents, giving qualitative data on their strengths, and therefore preferred conformations. 4 8 Temperature dependent N M R studies were carried out in CDCI3, and the A5/AT measured for the amide protons (Table 3 and 4). In chloroform a small A8/AT (e.g. ,-0.0024 ppm/K) indicates no change in the degree of solvent exposure of the proton oyer the temperature range studied, i.e., it remains unaltered in its state of either hydrogen bonding or non-hydrogen bonding. A larger value for A8/AT (e.g., -0.0070 ppm/K) indicates a change in solvent exposure, i.e., from an intramolecular or intermolecular hydrogen bonded proton to a solvent exposed amide proton (or vise versa). The control PhO-Ala exhibited a A8/AT value of -0.0036 ppm/K which compares to the values for C T V - A l a of -0.0032 ppm/K and -0.0040 ppm/K, indicating some degree of change in the conformational equilibria for both these compounds. PhO-Phe essentially showed no temperature dependence in its state of hydrogen bonding with a A8/AT of -0.0020 ppm/K. CTV-Phe demonstrated A8/AT values which averaged -0.0032 ppm/K for the "weaker" hydrogen bonded amide proton and -0.0067 ppm/K for the further downfield "stronger" amide proton, this is consistent with two distinct hydrogen bonding species. The error in these A8/AT measurements could be as high as 25%. 45 Table 3: Temperature dependence of amide chemical shifts in CDCI3 C o m p o u n d 298 K 323 K A8/AT PhO-Phe 6.99* 6.94 - 0.0020 PhO-Ala 7.16 7.07 * - 0.0036 CTV-Phe 7.18*, 7.58 7.12, 7.14 - 0.0024, - 0.0056 CTV-A la 7.41, 7.58 7.33, 7.43 - 0.0032, - 0.0040 * Amide proton coincident with aromatic protons, therefore estimated values Table 4: Temperature dependent amide chemical shifts for CTV-Phe in CDCI3 and A8/AT values (based on shift values of 7.18* and 7.58 ppm at 298 K) Tempera ture (K) Chemica l Shi f t (ppm) A8/AT 323 7.12,7.44 - 0.0024*, - 0.0056 283 7.24, 7.69 - 0.0040*, - 0.0073 263 7.29, 7.83 -0 .0031* , -0.0071 * Amide proton at 298 K coincident with aromatic protons, therefore estimated values 3.4 Discussion The most unexpected result was the intramolecular hydrogen bonding observed in the single stranded controls, PhO-Ala and PhO-Phe. The presence of an intra-molecular hydrogen bond was concluded from the amide N M R chemical shift, IR stretching frequency and from the concentration independent N M R and IR spectra. The presence of intermolecular and non-hydrogen bonding states would almost certainly have been observed over the concentration range studied. The amide proton of these control compounds could be internally hydrogen 46 bonded to either the carbonyl of the ester or to the ether oxygen (Figure 35). However, forming a five membered ring between a carbonyl and the amide within the same amino acid in a peptide chain is known to be unfavourable. 4 8 For amide to carbonyl hydrogen bonds the optimum angle of N - H - - - 0 generally approaches linearity (160° is optimum). The angle C = 0 — H is more flexible, the only requirement being that it is greater than 90°. C P K models reveal that neither of these criteria are met. The hydrogen bonding properties of aryl ethers have not been extensively studied. It is known that of the two lone pairs of the ether oxygen, 2-3 kcal/mol is gained when one pair is in conjugation with the 7t system of the aryl r i n g . 5 6 Hydrogen bonding to the oxygen of an ether tends to lie within in the plane of, the two lone pairs of the sp 3 hybridised oxygen. 5 7 C P K modeling indicates this is a more feasible internal hydrogen bond than that to the carbonyl oxygen. The IR and N M R data are consistent with this being a fairly weak but stable hydrogen bond. F igure 35: The two possible intra-molecular hydrogen bonds formed by PhO-Phe and PhO-Ala . R O R = Me , C H 2 P h The internal hydrogen bonding network for the CTV-Phe analogue is most evident from the occurrence and behavior of non-equivalent protons when comparing the N M R spectra obtained in CDCI3 and D M S O (section 3.3). Evidence from the IR spectra, amide proton exchange and temperature dependent N M R studies indicates the presence of two distinct hydrogen bonds. One is consistent with the intrastrand bond seen in the controls, the-other is 47 F igure 36: Examples of unfavourabe hydrogen bonding interactions., Hydrogen bonding from N - H to C = 0 of the esters can also be excluded because of the unfavourable bond angles that would result. E t 0 2 C E t Q 2 C C 0 2 E t R H - N E t 0 2 C 48 somewhat stronger and is attributed to interstrand hydrogen bonding. C P K modeling can exclude the hydrogen bonding networks of the types shown below for reasons of unfavourable bond rotations, steric interactions and loss of symmetry (Figure 36). Molecular models suggest the most likely hydrogen bonding networks for CTV-Phe (Figure 37 and 38). A l l of these are consistent with the N M R and IR data, in having one inter-strand hydrogen bond and one intra-strand amide to aryl ether bond. Due to the symmetry of the N M R spectra all of these hydrogen bonds must point in the same direction around the C T V base. This indicates some degree of communication between all of the strands, and therefore one of the proposed conformations must dominate. For steric reasons the side-chains may be expected to point outward, leading to an anti-clockwise hydrogen bonding network (when viewed from above). C P K models tentatively indicate that a 16 membered interstrand system (Figure 37) is more consistent with the results than a l l membered (Figure 38), because the paired intra-strand hydrogen bonding can occur in a manner similar to that seen in the controls. s The N M R spectral data for C T V - A l a was inconclusive as to its hydrogen bonding. The very broad amide hydrogen bonding peak seen in the IR indicated that it may be able to adopt a greater number of conformations. 49 F igure 37: Possible hydrogen bonding network of hexasubstituted C T V involving a 16 membered inter-strand hydrogen bond. The hydrogen bond network is shown in a clockwise fashion when viewed from above (with the side-chains pointing out from the bowl). A n anti-clockwise network is also a possibility. intrastrand hydrogen bond E t 0 2 C E t 0 2 C 50 F igure 38: Possible hydrogen bonding network of hexasubstituted C T V involving an 11 membered inter-strand hydrogen bond. The hydrogen bond network is shown in a clockwise fashion when viewed from above (with the side-chains pointing out from the bowl). A n anti-clockwise network is also a possibility. CO^Et 11 membered interstrand R — ^ ^ hydrogen bond N - H ) R qv >- C 0 2 E t O O — ' H intrastrand hydrogen bond 51 Conclusions Chapter 4 4.1 Summary of Results The major goal of the project, i.e., to find a viable approach to a template assembled synthetic protein based on a C T V - O H macrocycle, has been achieved in good yield using a method that wi l l be compatible with linking unprotected peptides. Thus, by treating amino acid models with bromoacetyl bromide, a suitably functionalised species is produced that can be covalently linked to the template. The optimum conditions for this coupling require potassium carbonate as base and degassed dimethylformamide as the solvent. A suitable inter-strand distance for the C T V macrocycle has been confirmed by the observation of an internal hydrogen bonding network. Although the results do not indicate a complete network of hydrogen bonding between the six attached amino acids, the degree of inter-strand communication between the six single residues augers wel l for inter-strand hydrogen bonding with six polypeptides. 4.2 Future Study Further characterization of the hydrogen bonding network of these compounds could be achieved by computer modeling, which in combination with nOe data and C P K modeling may give more information on the conformation of the macrocycle. A n investigation of other amino acids, e.g., valine, leucine, isoleucine, for any relationship between side-chain and conformational preferences, may help to elucidate the direction of the hydrogen bonding network. Addit ion of a second amino acid to the strand, and therefore a second potential hydrogen bond, would yield more information on the inter-strand hydrogen bond nucleating potential of the C T V template. Thus, i f the C T V template does not possess the characteristics 52 for complete inter-strand hydrogen bonding between the residues nearest to the template, their conformational preference should preorganise the second residue sufficiently to facilitate full inter-strand hydrogen bonding between dipeptides. Template assembly works by reducing the degrees of freedom afforded to a peptide strand. If the peptide strands are too restricted, then they may not be able to orientate into favourable folding patterns, whereas a lack of "directing" by the template w i l l defeat its purpose. In the model systems a lack of conformational freedom may have been a reason for the non-optimum hydrogen bonding network. Therefore, the question of "templation verses conformational freedom" needs to be investigated. In our model system this could be done by extending the length of the alkyl chain between the peptide model and the template (Figure 38). The peptide models with different chain lengths have been synthesised, but initial attempts at coupling them to the C T V - O H macrocycle have so far proved unsuccessful. The next stage in this work wi l l be to make the desired template assembled synthetic protein and study its structure. This wi l l be done by addition of six hexapeptide strands to the C T V - O H macrocycle according to the procedures outlined in this thesis. 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