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Towards polypeptides bearing luminescent side-chains as materials for organic light-emitting devices Brunner, Pierre-Louis Marc 2000

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Towards polypeptides bearing luminescent side-chains as materials for organic light-emitting devices by  Pierre-Louis Marc Brunner B. Sc., Bishop's University, 1998  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF SCIENCE  in  THE 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.  THE UNIVERSITY OF BRITISH C O L U M B I A November 2000 © Pierre-Louis Marc Brunner , 2 0 0 0  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 department or by  his or her  representatives.  be granted by the head of  It is understood that copying or  publication of this thesis for financial gain shall not be allowed without my permission.  Department of The University of British Columbia Vancouver, Canada  DE-6  (2/88)  my  written  Abstract  Two different routes, using the Wittig reaction to obtained compound 2.5 are described. The O-benzyl-N-CBZ-L-serine cyclohexyl ammonium salt (2.7b) and the OstilbeneTN-CBZ-L-serine cyclohexyl ammonium salt (2.8b) were then prepared by coupling benzyl bromide or compound 2.5 respectively to iV-CBZ-Z-serine, as precursors to novel polypeptides containing a conjugated chromophore as a side-chain. Compound 2.9 was also obtained as a side-product by elimination of the side-chain in a basic media.  The route toward  these new materials  involved the synthesis  of the  corresponding N C A s of 2.7b and 2.8b. Three different routes using S O C l , Oxalyl 2  chloride or PBr3 as halogenating agents were tried in an attempt to form the 5-membered ring of the N C A s from 2.7a, 2.7b and 2.8b. It was shown that the vinylic bond of the PPV oligomer-like side-chain of 2.8b got chlorinated when SOCI2 was used to form compound 2.11. Attempted syntheses of the polypeptides 2.12a and 2.12b using 4methylmorpholine or diisopropylamine as initiator are also described.  The photoisomerisation of 2.8a and 2.8b were explored. Excitation at 300 nm lead to a 57% conversion of the trans isomer to the cis isomer of the stilbene-like sidechain, where a broader excitation at wavelength greater than 290 nm lead to a 10 % conversion of the trans isomer to the cis isomer of the stilbene-like side-chain.  ii  Table of Content  •  Abstract  ii  •  Table of Content  iii  •  List of Schemes  vii  •  List of Figures  •  List of Abbreviations  •  Acknowledgments  xiii  •  Dedication  xiv  •  Quotation  xv  .  viii ix  C H A P T E R 1: I N T R O D U C T I O N  1  •  1.1 Research Goals  2  •  1.2 Background  4  •  1.2.1 Light-emitting Diodes (LEDs)  ,  ,  4  •  1.2.2 Oligo- and Poly(phenylenevinylene) (PPV)  4  •  1.2.3 Polymer-based Electroluminescent Devices  8  •  1.2.4 Using Polypeptides to Align Organic Materials  9  •  1.2.4.1 Amino Acids  9  •  1.2.4.2 Peptides  10  iii  CHAPTER 2: RESULTS AND DISCUSSION  •  •  •  2.1 Preparation of the Conjugated Side-chains  18  •  2.1.1 Attempted Preparation of 2.2  18  •  2.1.2 Preparation of 2.5  20  •  2.1.2.1 Preparation of 2.5 from /?-Tolunitrile  20  •  2.1.2.2 Preparation of 2.5 fromp-Methyltolualdehyde  22  2.2 Synthesis of Compounds 2.7a, b and 2.8 a, b  24  •  2.2.1 Coupling of Benzyl Bromide and 2.5 with JV-CBZ-Z-serine  24  •  2.2.2 Absorption and Emission spectra of compound 2.7b and 2.8b  29  2.3 Preparation of JV-Carboxyanhydrides 2.10a and 2.10b  30  •  2.3.1 Attempted Preparation of 2.10a  30  •  2.3.2  Attempted  Preparation  of the Z-serine 7Y-carboxyanhydride  Derivative 2.10b •  37  2.4 Polypeptide Synthesis •  40  2.4.1 Polymerization of 2.10a and 2.10b using 4-Methylmorpholine as Initiator  •  40  2.4.2 Polymerization of 2.10a and 2.10b using Diisopropylamine as Initiator  •  •  17  41  2.5 Photoisomerisation of 2.7b and 2.8b  43  •  2.5.1 Photoisomerisation of 2.7b  43  •  2.5.2 Photoisomerisation of 2.8b  46  2.6 Conclusions and Future Considerations  iv  50  CHAPTER 3 : Experimental Section  52  •  3.1 General Experimental Details  53  •  3.2 Starting Marterials  55  •  3.2.1 Preparation of 2.1  55  •  3.2.2 Isomerization of 2.1  55  •  3.2.3 Preparation of a-Bromo-p-tolunitrile (2.3)3*  56  •  3.2.4 Preparation of a-Bromo-p-tolualdehyde (2.4)32  57  •  3.2.5 Preparation of 2.6  57  •  3.3 Conjugated Side-chains  59  •  3.3.1 Attempted Preparation of 2.2  59  •  3.3.2 Preparation of 4-(fraws -2-phenylethenyl)phenylmethyl bromide (2.5) ,  59  •  3.3.2.1 From a-Bromo-p-tolualdehyde (2.4)  59  •  3.3.2.2 From Compound 2.6  60  3.4 iV-CBZ-L-serine Derivatives •  •  •  61  3.4.1 Preparation of TY-CBZ-O-benzyl-I-serine Cyclohexylammonium Salt (2.8a),  61  •  3.4.2 Preparation of TV-CBZ-O-benzyl-JL-serine (2.7a)  62  •  3.4.3 Preparation of 2.8b  63  •  3.4.4 Preparation of 2.7b  64  •  3.4.5 Preparation of TV-CBZ-L-serine Benzyl Ester (2.11)...  65  3.5 N-carboxyanhydrides  66  v  •  3.5.1 Attempted Preparation of O-benzyl-L-serine N-carboxyanhydride (2.10a)  66  •  3.5.1.1 From N-CBZ-O-benzyl-Z-serine (2.7a)  66  •  3.5.1.1.1 Using Thionyl Chloride  66  •  3.5.1.1.2 Using Oxalyl Chloride  66  •  •  3.5.1.2 From JV-CBZ-O-benzyl-L-serine Cyclohexylammonium Salt (2.8a)  67  •  3.5.1.2.1 Using Thionyl Chloride  67  •  3.5.1.2.2 Using Oxalyl Chloride  67  3.5.2 Preparation of 2.10b  68  •  3.5.2.1 From 2.8b  68  •  3.5.2.1.1 Using Thionyl Chloride  68  •  3.5.2.1.2 Using Oxalyl Chloride  68  References  70  vi  List of Schemes  Scheme 1.1:  6  Scheme2.1:  19  Scheme 2.2:  21  Scheme 2.3:  •  22  Scheme 2.4:  23  Scheme 2.5:  24  Scheme 2.6:  28  Scheme 2.7:  31  Scheme 2.8:  32  Scheme 2.9:  •  34  Scheme 2.10:  36  Scheme 2.11:  40  Scheme 2.12:  •.41  Scheme 2.13:  44  Scheme 2.14:  .47  Scheme 2.15:  51  vii  List of Figures  Figure 1.1:  Surface  tethering  of  Polypeptide  a-helices  bearing  pendant  chromophores on the backbone  2  Figure 1.2:  Photoexcitation of a fluorescent polymer  5  Figure 1.3:  Schematic of a two layers O L E D  8  Figure 1.4:  Geometry of the peptide backbone showing the main chain bond angles: cp, v|/ 11  Figure 1.5:  Two peptide strands of an anti-parallel P-sheet. In red are the sidechains (R) carried by the amino acis residues 13  Figure 1.6:  Formation of an a-helix having a chromophore grafted to the backbone by ring-opening metathesis polymerization of the corresponding N C A . . . 15  Figure 2.1:  ' H N M R spectrum (200 M H z , CDC1 ) of 2.7b  Figure 2.2:  ' H N M R Spectrum (400 M H z , CDC1 ) of 2.8b  Figure 2.3:  Absorption and emission spectra of a 1.26 x 10" M solution of  ..27  3  ;  3  27  6  compound 2.7b in CH C1 2  29  2  Figure 2.4:  FTIR spectra of 2.10a and 2.10b  35  Figure 2.5:  ' H N M R simulation of 2.16  38  Figure 2.6:  Comparison of the ' H N M R spectra of 2.7b in acetone-d6. A: Sample before irradiation, B and C: Same sample after 5 and 10 min of irradiation respectively 45  Figure 2.7:  Integrated areas of the selected ' H N M R peaks as a function of time .... 48  Figure 2.8:  Percentage of cis isomer from peak ratios  Figure 2.9:  Comparison of H N M R spectra of 2.8b in CHCI3. A: Sample before irradiation, B to F: Same sample after 10, 15, 20, 25, and 30 min of irradiation 49 !  viii  48  List of Symbols and Abbreviations  Symbol  Description  [a]  specific rotation  A  angstrom  Anal. Calcd.  calculated analysis  Ar  aryl group  br s  broad singlet  bpy  2,2'-bipyridyl  CBZ  carbobenzoxy  1 3  C NMR  carbon Nuclear Magnetic Resonance  COD  1,5-cyclooctadiene  CVD  chemical vapor deposition  °C  degrees Celcius  5  chemical shift  5  +  positive partial charge  5~  negative partial charge  5oop  bending out of plane  d  doublet  D  sodium lamp  dd  doublet of doublets  ix  DIBAL-H  diisobutyl aluminum hydride  DMF  Dimethylformamide  DMSO  dimethylsulfoxide  ECHB  electrons conducting / holes blocking  Et  ethyl  et al  et alii  EL  electroluminescence  eq  equivalent  eV  electronvolt  FTIR  fourier Transform Infrared  g  grams  !  H NMR  proton Nuclear Magnetic Resonance  h  hours  HOMO  highest unoccupied molecular orbital  h  Plank's constant  ITO  indium-tin oxide  IR  infrared  J  coupling constante (Hz)  A. ax  wavelength of maximum absorbance  LED  light-emitting diode  LEDs  light-emitting diodes  LCDs  liquid Crystal Displays  LUMO  lowest unoccupied molecular orbital  m  x  m  multiplet  M  molar  m.p.  melting point  Me  methyl  MeOH  methanol  MHz  mega Hertz  min  minutes  mg  milligrams  mL  milliliters  mmol  millimoles  mol  moles  p  para  pH  potential hydrogen  N  normal  NBS  N-bromosuccinimide  NCA  N-carboxyanhydride  NCAs  A'-carboxyanhydrides  nm  nanometer  Ph  phenyl  ppm  parts per million  PPV  poly(p-phenylenevinylene)  q  quartet  ROMP  ring-opening metathesis polymerization  "  xi  s  singlet  (s)  solid  st  stretch  st sy  symetrical stretch  st as  asymetrical stretch  t  triplet  TBME  f-butylmethyl ether  THF  tetrahydrofuran  UV  utraviolet  Vis  visible  V  frequency (wavenumber)  W  watts  xii  Acknowledgements  First, I would like to thank my supervisor, Dr. Micheal O. Wolf, for its patience and for its presence everytime I needed help, a piece of advice, or simply encouragements to pursue. I must also thank all the past and present members of "The Wolf Pack" in whom I found more then collegues, but good friends. Thank you guys for listening all my stories... I would also like to acknowledge the good work of the supporting staff, especially the N M R technicians, Liane Darge and Marietta Austria, and Peter Borda (elemental analysis). I must not forget the people from the secretary's office, especially Judy and Johann for their kindness and all the nice discussions we had together. I would also like to thanks all the people that helped me at one moment or another during those years, especially Scheffer's group for giving me good advices and letting me use their instruments. Finally, I would like to thank those who supported me and believed in me, especially my parents, who gave me the chance to be where I am today, my brothers for cheering me up, and Karla for her love.  xiii  A la memoire de Nicholai*  xiv  " Qu'importaient les victimes que la machine ecrasait en chemin! N'allait-elle pas quand meme a l'avenir, insoucieuse du sang repandu? Sans conducteur, au milieu des tenebres, en bete aveugle et sourde qu'on aurait lachee parmi la mort, elle roulait, elle roulait, chargee de cette chair a canon, de ces soldats, deja hebetes de fatigue, et ivres, qui chantaient." - Emile Zola  xv  CHAPTER 1  INTRODUCTION  1.1 Research Goals  The goals of the work in this thesis are the preparation of new amino acid derivatives containing a conjugated chromophore as a side chain, as precursors to polypeptides containing these same side-chains. These materials are of great interest because the nanoscale architecture of the polypeptide provides a scaffold for ordering  ^.oI  •?  i  /  1  i ^  *  •%  ^ —CnrornophoreQ »  ^ O  ;•  ,i c  j  *  "  Chromophore  :  .-*  i  <  vo  Lc"*  I  c  ^Chromophore •  binding group L  7  if  iff  Surface  / i f f  Figure 1.1: Surface tethering of polypeptide a-helices bearing pendant chromophores on the backbone^.  2  conjugated chromophores in space. By tethering such polypeptides to surfaces (Figure 1.1), these layers could be used in applications such as electroluminescent devices where the relative orientation of chromophores or lumophores in 3-D space is important for determining the efficiency and performances of these devices.  3  1.2 Background  1.2.1 Light-emitting Diodes (LEDs)  "No one was prepared for the discovery of the plastic sandwich that would emit light when simply connected to a battery."2  In the  1960's, inorganic light-emitting diodes (LEDs) using GaAs-based  semiconductors became widely available. By the mid-1980's initial reports had appeared indicating that fluorescent organic dyes could also be used in LEDs with work by Tang and Van Slyke^, and Saito et alA on materials suitable for use in monochromatic displays. The recent discovery of electroluminescence (EL) in conjugated polymers^, opened the door to another class of materials for LEDs that may directly compete with liquid crystal displays (LCDs) and cathode-ray tubes, particularly for large display devices.  1.2.2 Oligo- and Poly(phenylvinylene) (PPV)  Large band gap polymers have been primarily used as insulators by the electronics industry. However, the low cost, lower density, flexibility, and durability of polymers, compared to inorganic materials, provide the driving force for the search for new smaller band gap conductive and semi-conductive polymers. Conjugated polymers are candidates for these applications, since these materials allow charge transport along  4  the backbones and between chains. For example, poly(phenylenevinylene) or PPV (1.1), is a conjugated polymer where a band gap between the n and n* states of approximately 2.4 eV. Band gaps of between 1.77 eV (red) and 2.95 eV (violet) result in emission in the visible spectrum, so luminescence in PPV occurs with ^  m a x  (emission) = 520 nm by radi-  1.1 ative relaxation to the ground state after excitation either with light or voltage (Figure 1.2).  The energy gap in this polymer can be modified by attaching different substituents  on the polymer backbone, resulting in a luminescence maximum which can be tuned over a wide range of colors.^ P P V is also a good choice of material for application in lightemitting devices, since internal quantum efficiency, which is the number of photons emitted per electron injected, ranges between 0.4 and 20 %. Such organic semiconductor  Figure 1.2: Photoexcitation of a fluorescent polymer^  5  may eventually be used to replace or complement the semiconductors currently used in transistorsP memories, photodiodes,^ or ultimately in ultra-lightweight full color flatpanel displays. 9  PPV is a rigid-rod polymer having tensile strength which allows it to survive the harsh conditions (high temperatures) encountered  during device operation  since  electroluminescence from the polymer is achieved by applying potentials of 5 to 20 Volts, generating an electric field of about 10 Volts/cm across a device^ 5  ( e se  sectionl.2.3). It is completely insoluble in most solvents, but adding substituents to the backbone overcomes this problem2.  ci  tetrahydrothiophene  + CI 1.2  1. N a O H , M e O H / H 0 2  •2. HCI (neutralization) 3. dialysis  CI 180-300 °C n  vacuum, 12 h n  PPV  Scheme 1.1  6  PPV has been synthesized by a variety of routes.  One of the most common  methods involves synthesizing a soluble sulfonium precursor polymerH (Scheme 1.1), which can be deposited as a thin-film onto substrates such as gold or indium-tin oxide (ITO), and thermally converted under vacuum to PPV. P P V and its oligomers can also be synthesized by chemical vapor deposition (CVD),1 ,13 electropolymerization,10 and 2  ring-opening metathesis polymerization (ROMP). 14,15 The Wittig reaction has been used to prepare oligo(phenylenevinylene) from compounds 1.3 and 1.4. The differences in molar mass, molecular weught distributions, the nature and amount of defects and impurities in these polymers and oligomers from the different synthetic routes, affect the electronic properties of the resulting materials and will be reflected in the efficiency and lifetimes of electroluminescent devices fabricated from these materials.  1.3  1.4  Short oligomers of phenylenevinylene containing 2-10 units have also been studied.  Shorter chains result in blue-shifted emission spectra due to the shorter  conjugation length which results in an increase in n - n* band gap.  7  1.2.3 Polymer-based Electroluminescent Devices  Electroluminescent devices based on conjugated polymers are typically prepared as sandwich cells (Figure 1.3), consisting of a layer of polymer sandwiched between two electrodes.  A glass or poly(ethylene terephtalate) (PET) layer is used as a transparent  support in order to allow the emitted light out of the device. ITO is typically used as the anode since it is also transparent, and aluminum, magnesium or calcium, deposited in vacuum, are typically used as the cathode^'^ (Figure 1.3). The cathode is chosen so that its work function favors electron injection to the L U M O of the polymer. A n electrons conducting / holes blocking (ECHB) layer may be added between the cathode and the polymer layer in order to improve the performances of a device, by moving the electrons / holes recombination sites away from the cathode.2  © 5 © © © 1 ©+ t  Cathode E C H B Layer Light-Emitting Layer Anode  Transparent Support  hv  Figure 1.3: Schematic of a two layer O L E D  8  1.2.4 Using Polypeptides to Align Organic Materials  Aligned or ordered materials are of great interest for many practical applications such as memory devices, optical switches and OLEDs. Materials with nanometer-scale architecture could possess unique and superior properties compare to materials which are made without control of nanostructure. For example a highly ordered PPV composite has been recently constructed by filling the channels of a liquid crystal matrix in order to investigate the effects of nanometer-scale engineering on P P V l ? .  It is possible that  ordering the polymer chains can lead to oriented emission, for example alignment of the polymer chains by rubbing was found to be an efficient way to obtain polarized electroluminescence 18.  It has been demonstrated that the introduction of alkoxy side  chains on PPV will interferes with the polymer chain packing and will raise the efficiency of the device by spacing apart the conjugated chains >19. Such spacing could also be 2  achieved by using scaffold such as polypeptide chains.  The proposed approach to  achieve nanoscale control of conjugated, luminescent phenylene vinylene oligomers is to append a polypeptide backbone with such conjugated group.  1.2.4.1 Amino Acids  C0 H 2  I  1.5  9  Naturally occuring amino acids have the general structure, shown in 1.5. Since 1806, when the first natural amino acid (asparagine) was isolated from the asparagus plant by L. N . Vauquelin and P. J. Robiquet, over 150 different natural amino acids have been discovered, differing only in the R group carried on the C . Natural amino acids can a  be obtained by isolation from natural materials or by microbiological methods, as well as chemical synthesis.  For example, glutamic acid and lysine, which are used as food  additives, can be produced on a large scale from simple petroleum derivatives such as acetylene. ^ However, for smaller scale syntheses, commercially available amino acids 2  may be used as starting materials, and their side-chains manipulated to obtain desired materials. It is generally necessary to protect the amine and carboxylic acid functional groups of the chosen amino acid prior to its use as a starting material, and many protected amino acid are commercially available.  For example, in the present work, N-  carbobenzoxy-Z-serine was used in order to prevent side reactions which could occur at the amine group during the formation of the ether side-chain.  1.2.4.2 Peptides  Peptides are composed of two or more amino acid units linked together by amide groups. These peptide bonds are considerably shorter than a conventional single C - N bond due to their partial double bond character, which also results in a planar distribution of atoms and the possibility of both cis and trans conformations being stable. However, the trans configuration is more common than the cis, due to the steric hindrance of the side chains, and will form secondary structures such as a-helices. Anti-parallel P-sheets  10  requiere co (Figure 1.4) to be -180 ° (cis). These types of secondary structures are favoured because of van der Waals interactions, hydrogen bonding, and favourable <p and VJ/ torsion angles (Figure 1.4). The most common helix is composed of 3.6 amino acid residues per turn with a pitch (or translation) of 5.4 A , and hydrogen bond networking which results in 13-membered rings, and thus is termed a 3.6o helix. In such helices, the hydrogen bonds occur between the carbonyl oxygen atom of residue i and the NH group of residue 0'+4).  Helices where the hydrogen bonds form 10-membered rings (3io  helices), are unstable and generally not encountered.  Figure 1.4: Geometry of the peptide backbone showing the main chain bond angles: <p, \|/, and co.2l  11  According to the Zimm-Bragg model, the propensity of a peptide to adopt an helical secondary structure depends on two parameters; the helix initiation constant, a, and the propagation constant s, and is defined as os , where n is the number of amino n  acid residues that constitute the peptide . This implies that the greater the number of 22  residues in the peptide, the more stable the helical structure would be. In fact, peptides formed with fewer than 20 amino acids generally exist only as random coils.  The  parameter cr reflects the probability of a proper alignment of the first three amino acid in an a-helical conformation since hydrogen bonding will not be present prior to the addition of a fourth residue to the growing a-helix. The propagation parameter s reflects the propensity of the added amino acid to adopt the needed a-helical torsion angles 9 and v|/ (Figure 1.4). A n amino acid having a s parameter of less than 1.0 is said to be helixbreaking, but i f s is greater than 1.0, it is said to be helix-making. This last parameter is closely related to the size of the side-chains carried by the amino acids, and the interaction of these side-chains depending on their position around the helix. Bulky sidechains will reduce the range of permitted torsion angles cp and y , and therefore, will lower the s parameter and the propensity for the amino acid to form an a-helix. The same phenomenon occurs with side-chains with strong interactions such as hydrogen bonding between each other.  The a-helicity of a peptide versus a random coil state can be  determined by circular dichroisim (CD). ^ 2  Because a 3.613 a-helix is composed of 3.6 amino acid residues per turn, the sidechains be arranged so that they are staggered by 100° along the axis, and separated in space by 5.4 A because of a structural pitch (or translation) of this amount.  12  These  characteristics make rigid a-helices good candidates for ordering conjugated side-chains in space and can therefore be exploited for constructing nanometer scale structures.  A second structure that peptides can adopt, depending on the permitted torsion angles cp and v|/ for the amino acid residues that are present, is the (3-sheet (Figure 1.5). (3Sheets are formed from P-strands which are helices having only two repeating residues per turn. These P-strands can be aligned in the same direction, forming parallel fJ-sheets, or in the opposite direction forming anti-parallel P-sheets. A s for a-helices, hydrogen bonding between the carbonyl oxygen and the NH group of the P-strands hold the o  structure together. Side-chains pendant to such a structure will be spaced by about 6.8 A .  Figure 1.5: Two peptide strands of an anti-parallel P-sheet. In red are the side-chains (R) carried by the amino acids residues.21  13  One approach to preparing polypeptides is to use A'-carboxyanhydrides (NCAs)  (1.6), a class of compounds which polymerise readily by a living ring-opening mechanism in the presence of various initiators such as amines, alcohols or water (Figure 1.6). Other catalysts, such as the nickel complex Ni(bpy)(COD), have been used in an at-  O  C  5  O 1.6  tempt to get more control of the side reactions. ^ The reactivity at C5 of most N C A s 2  towards even weak nucleophiles makes them difficult to handle without getting decomposition of the ring. The classical synthesis of N C A s involves phosgene, a highly toxic gas, although alternative synthetic routes using less toxic reagents such as thionyl chloride ^, and 2  PBr 26,27 3  are also available. In these methods, an N-alkoxycarbonylamino  acid carrying the desired side-chain reacts with SOCb or PBr to form the acid halide, 3  and the N C A then forms by nucleophilic attack at C5 by the oxygen of the Nalkoxycarbonyl.  Since the initial interest is in synthesizing homopolymers which self-assembled by initiation from a surface, a living polymerisation is desirable(Figure 1.6).  Other  approaches to polypeptides are also possible, but generally would involve tedious coupling and deprotection / protection schemes to build up the polypeptide longer.  14  Figure 1.6: Formation of an a-helix having a chromophore grafted to the backbone by ring-opening polymerization of the corresponding N C A .  H N 2  Whitesell demonstrated that oriented peptides can be used as a scaffold to support functional groups pendant to an a-helix backbone.28 The a-helices have been tethered to a gold surface using a self assembled monolayer of an aminotrithiol "tripod" (1.7) to  15  initiate the polymerisation from alanine NCAs, connecting the a-helices to a gold surface. They obtained a stable directionally oriented peptide layer of 1000 A in thickness which shown stability even after been heated at 180 °C for seven days. They characterized the extremely narrow absorptions for the amide bands by polarized IR.  Amino functionalized silicon wafers have also been used to initiate the polymerization of y-methyl-Z-glutamate 7Y-carboxyanhydride in the melt by heating the sample to just over the melting point ^. Poly(methyl-L-glutamate) (PMLG) have been 2  used as synthetic fibers since the 1950's for the manufacture of synthetic leather. A modified procedure to synthesize PMLG-like  material fibers using poly(amino  acid)urethane copolymers, permits the production of materials that have better physical properties, and that are more suitable for application in the textile industry. In this case, the a-helices are interrupted by polyurethane chain segments which give the structure better elastic recovery and adhesive properties^. Such a strategy could be applied to introduce different desired layers within a L E D by using multiple N C A s to form blockcopolymers.  16  CHAPTER 2  RESULTS AND DISCUSSION  17  2.1 Preparation of the Conjugated Side-chains  Our strategy for ordering conjugated chromophores in space involves the coupling of these chromophores to an a-helix backbone.  It was clear the selected conjugated  chromophore, which bears a functional group that allows coupling to an amino acid had to be first synthesized.  The reaction of primary alkyl halides with alcohols to form  ethers, via nucleophilic substitution, was exploited in order to couple a bromomethyl functionalized conjugated group with JV-CBZ-Z-serine. In this section the synthesis of short, conjugated phenylenevinylene oligomers bearing a bromomethyl  substituent  suitable for such coupling with iV-CBZ-£-serine is described.  2.1.1 Attempted Preparation of 2.2  Compound  2.1  was  prepared  from  commercially  available  p-  xylylenebis(triphenylphosphonium bromide) and /?-tolualdehyde via a Wittig reaction (Scheme 2.1). In this reaction, three different isomers (cis, cis; cis, trans and trans, trans) of 2.1 may form. Since the specie of interest is only the trans, trans isomer, the obtained mixture was converted to all trans by heating to reflux for three days in the presence of a catalytic amount of iodine yielding a highly fluorescent green compound. Unfortunately, the trans, trans-2.1 was not very soluble in most organic solvents, much less so than the mixture of isomers obtained directly from the reaction. The elemental analysis shown that 2.1 was still impure, as a 2.12 % discrepancy is shown for the percentage of carbon present. It was decided to brominate the mixture of isomers using  18  one equivalent of iV-bromosuccinimide (NBS), in an attempt to obtain compound 2.2 (Scheme 2.1). The resulting material was purified by chromatography on silica gel using CH2CI2 as eluant, yielding a green fluorescent powder in 8 % overall yield. The low yield can be attributed to the poor efficiency of the Wittig reaction efficiency to form compounds of this type, as for the yield of the related compound 2.5 formed in 30 % yield via the same reaction. To make 2.2 two Wittig couplings are needed, this reducing the yield of 2.1 to such a low amount. Low yields for other Wittig reactions were also reported by Watakabe et afl 1.  1) 2.1 eq. BuLi DMSO 22°C, 1 6 h 2) 2 e q .  3) I2 (s), toluene, 110°C,3days  benzene, 80 °C, 24 h NBS benzoyl peroxide  2.2  Scheme 2.1  19  2.1.2 Preparation of 2.5  The attempted preparation of compound 2.2 was unsatisfactory, since the product was very insoluble in most organic solvents making it difficult to perform further reactions using 2.2 as a starting material. This is probably due to the rigidity of the rodlike trans isomer, which may need substituents such as methoxy or ether groups to increase its solubility. The extremely low yield of 2.2 also made this synthetic route unappealing for preparing an early intermediate in the synthesis. A n alternative approach to prepare conjugated side-chains bearing a bromomethyl substituent was needed, and the approach by Watakabe et al. to obtain short a\\-trans oligomers of PPV of desired lengths using consecutive Wittig reactions^ 1 was used.  2.1.2.1 Preparation of 2.5 from p-Tolunitrile  Compound 2.5 was prepared using the sequence of reactions shown in Scheme 2.2. a-Bromo-p-tolunitrile (2.3) was synthesised by the bromination of the commercially available p-tolunitrile using N B S and benzoyl peroxide as an initiator. A modified procedure based on the bromination of /?-toluic acid was used.^  2  Excess B r was 2  removed by washing the product with a 5 % solution of hot sodium sulfite, which also removed the succinimide. The nitrile group of a-bromo-/?-tolunitrile (2.3) was reduced to the aldehyde using diisobutylaluminum (DIBAL-H) as reducing agent^ (Scheme 2.2). A modified procedure was used in which the product was not recrystallized as in the literature procedure, but the crude oily material was sublimed to give white crystals,  20  leaving a red powder behind. Over two days the product was obtained in 46 % yield. The sublimation rate can be increased by heating at 50 °C. Compound 2.5 was prepared from the commercially available benzyltriphenyl phosphonium chloride and compound 2.4 following the procedure of Watakabe et al}^  The trans isomer of 2.5 was obtained  in 32 % yield, similar to the yield reported by Watakabe et al for this compound. Despite the relatively low yield, the desired product is obtained pure and without extended workup, simply by filtering the product from the reaction solution.  Br benzoyl peroxide benzene, 80 °C, 24 h  2.3  DIBAL-H chlorobenzene, 0 °C CI  LiOEt  2.5  EtOH,0°C, 1 h  2.4  Scheme 2.2  21  2.1.2.2 Preparation of 2.5 from /j-Methyltolualdehyde  A faster and more efficient way to synthesize compound 2.5 was needed because it is used as a starting material for subsequent reactions and because the preparation of 2.4 is rather slow and tedious. The problems associated with 2.4 were overcome by using  CI" PPh  3  LiOEt EtOH, 0 °C, 1 h 2.6  Br benzoyl peroxide benzene, 80 °C, 24 h  2.5  Scheme 2.3  the approach shown in Scheme 2.3. In this way, the pure trans isomer of compound 2.6 was obtained in 30 % yield. Compound 2.6 was then brominated with N B S , and purified by chromatography on silica gel using a hexahes/chloroform mixture (1:1) as eluant. Recrystallization of the product from methanol yielded the trans isomer of 2.5 in 88 % yield.  22  Scheme 2.4  Using N B S to brominate 2.6 should not lead to bromine addition to the double bond because the concentration of Br2 is low during the reaction.34 However, from close examination of the *H N M R spectrum of the crude product obtained in the second step in Scheme 2.3, it was observed that both the cis isomer of 2.5 and a second compound were also produced in this reaction. The presence of the cis isomer might be explained by the possible isomerization that can occur when bromine is present (Scheme 2.4). The first step in the addition of bromine is an equilibrium between the mono-brominated radical intermediate and the olefin, and therefore, when the concentration of Br2 is low, the reaction favors the olefin. Rotation around the central bond before the elimination of the bromine atom leads to the cis isomer of the olefin. Complete addition of bromine to the olefin also occurs, explaining the presence of a non-fluorescent material (hv lamp excitation) which is left behind on the chromatography column.  23  2.2 Synthesis of compounds 2.7a. b and 2.8 a. b  2.2.1 Coupling of Benzyl Bromide and 2.5 with iV-CBZ-Z,-serine  In order to append the conjugated group to a polypeptide backbone, an amino acid containing the desired pendant group must be synthesized.  TV-CBZ-L-serine was chosen  as a starting material, which is a protected amino acid bearing an hydroxy group on the P carbon suitable for coupling with alkyl halides to form ethers.  /V-CBZ-L-Serine 1) R B r  2) H 0 3  Scheme 2.5  24  +  In order to optimize the conditions for coupling longer conjugated groups to the amino acid precursor, the commercially available benzyl bromide was first coupled to NCBZ-Z-serine.  A modified procedure from  Hruby et al. who  coupled  N-t-  butyloxycarbonyl-Z-serine with benzyl bromide^^ was followed. Here, TV-CBZ-Z-serine  0  2.8 a  2  8  b  was treated with two equivalents of sodium hydride, yielding the dianion. The ether was then formed by adding one equivalent of the appropriate alkyl halide (benzyl bromide or 2.5).  Compounds 2.7a and 2.7b were obtained as oils, and were converted to the  respective cyclohexylammonium salts as white powders in 90% yield, by adding one equivalent  of cyclohexylamine, in order  to  facilitate  their  purification.  The  cyclohexylammonium salts 2.8a and 2.8b (Figure 2.1) could be readily converted back to the respective carboxylic acids by acid workup in cold methanol^ or, as discovered, can also be directly used for the synthesis of the /V-carboxyanhydride serine derivatives (See section 2.3).  25  The H N M R spectra of compounds 2.7b (Figure 2.1) and 2.8b (Figure 2.2) both !  show the characteristic resonance at 8 7.08 for a proton on a trans vinylic bond in conjugation with phenyl groups.  36  By comparison with the -H N M R of c/s-stilbene  36  it  appears that no cis isomer is present. It was observed that the two protons on the P carbon, which are adjacent to the chiral center, are inequivalent. This was also observed for the P-protons of leucine and the P-methyl groups in valine 7. The ' H N M R spectra of 2  compounds 2.8a and 2.8b (Figure 2.2) show that the signals for the two methylene protons on the C B Z group (B) are inequivalent.  In compounds 2.7a and 2.7b these  protons appear as a singlet at 8 5.07, but split into two closely spaced doublets at 8 5.02 in 2.8a and 2.8b. This may be due to association of the bulky tertiary ammonium cation with the carboxylate group which hinders free rotation of the C B Z group. Compound 2.8b was determined to be optically active with a specific rotation of [a] as a 1.71 M solution in chloroform.  2 2 , 5 D  = + 14.8 °  Ffruby et al. obtained the optically active  cyclohexylammonium salt of A -?-butyloxycarbonyl-o-benzyl-L-serine using the same 7  synthetic route and mesured a specific rotation of [a] methanol. 5 3  26  2 5 D  = - 29 ° as a 1.0 M solution in  Ph^H  2.7 b  H ^ P h CH  NH  i.o  7.0  e.o  4Tr*^7r."~-.V  • ,.*„• • ~oTt  Figure 2.1: H N M R spectrum (200 M H z , CDC1 ) of 2.7b l  3  A  C  O * ^ }  Ph^H  rApn  NH  2.8 b  /  B  C  k  n  1  /  J'J  ppm  Figure 2.2: ' H N M R Spectrum (400 M H z , CDC1 ) of 2.8b 3  27  In the synthesis of the amino acid derivatives 2.8a and 2.8b, these compounds were the major products, however a competitive reaction results in side products. From these reactions, compound 2.9 was isolated and characterised by ' H N M R and elemental analysis. Either the excess of NaH or the presence of moisture (generating NaOH) might introduce base, which could lead to the formation of compound 2.9 as shown in Scheme 2.6.  Scheme 2.6  28  2.2.2 Absorption and Emission Spectra of 2.7b and 2.8b  The absorption and emission spectra of a 1.26 x 10" M solution of compound 6  2.7b in CH2CI2 are shown in Figure 2.3. Compound 2.7b exhibits a broad absorption with A-max = 314 nm. When 2.7b is excited at 305 nm, it exhibits a broad emission with Xmax (emission) = 358 nm. These results can be compared with those reported for trans4-aminostilbene, which has X, x = 315 nm, and A,max (emission) = 380 nm.37 ma  compares  well  with  the  absorbance  and  fluorescence  spectra  it also  of p-((p-  ethylphenoxy)methyl)stilbene, prepared by Alquiar et al., where A.max = 310 nm, and A,max (emission) = 358 nm.38  230  255  280  305  330  355  380  405  430  455  480  nm Figure 2.3: Absorption and emission spectra of a 1.26 x 10" M solution of compound 2.7b in C H C 1 6  2  2  29  2.3 Preparation of N-carboxyanhydrides 2.10a and 2.10b  The formation of compounds 2.10a and 2.10b was a crucial step since ringopening of these compounds is expected to yield the desired polypeptides. The high reactivity of N C A s towards even weak nucleophiles make them difficult to handle without decomposition.  2.10a  2.10b  Due to limitations in the amount of the starting amino acid derivatives 2.8a and 2.8b available, the more readily available precursors 2.7a and 2.7b were used as starting materials to make 2.10a, and then the same conditions were used to prepare 2.10b. For safety reasons, the use of phosgene to close the ring was avoided, despite the fact that this reagent is commonly used to convert amino acids to the NCAs.25,27,39  2.3.1 Attempted Preparation of 2.10a  A modified procedure for the synthesis of glycine N C A from JV-CBZ-glycine was used ^ in an attempt to synthesize compound 2.10a from compound 2.7a (Scheme 2.7). 2  A large excess of thionyl chloride was used, and gases (SO2 and HC1) are generated as side products, simplifying the workup and purification of the product. The mechanism of  30  this reaction presumably involves the formation of the acid chloride from 2.7a, followed by a nucleophilic attack at the carbonyl of the C B Z group, and elimination of benzyl chloride as a side product (Scheme 2.8).  O  socu  O  O O  HN-^  Ky  25 °C, 24 h  2.10a OH  T  o  2.7a  oxalyl chloride  ,0  CH CI , DMF 2  2  O  25°C, 16h  b o  2.10a  Scheme 2.7  The /V-carbobenzoxy protecting group was used in this procedure because it is a goog leaving group compared to other possible protecting groups, and this should give the best results in terms of the rate of cyclisation to the N C A s . ? 2  31  Scheme 2.8  After the reaction with thionyl chloride, the resulting oily material was purified by triturating it with diethyl ether and T B M E in order to help it to solidify. Unfortunately, in our hands the product could not be completely purified as determined by ' H N M R spectroscopy.  It is possible that some reaction of the N C A with moisture occurs,  however, only dry solvents, freshly distilled thionyl chloride, and glassware which was dried in an oven for at least 2 hours prior to use, were used. It is difficult to determine i f the conditions used were not suitable for cyclisation or i f the presence of impurities or moisture induced polymerization or decomposition of the N C A after it was formed. Since it is known that N C A s polymerize readily, both in solution and in the solid state,27  32  and should therefore be prepared only a few hours before use,40 all characterization by N M R or FTIR spectroscopy was done immediately after workup. Later experiments on the same samples (3-12 days later) indicated greater amount of unidentified impurities are present, clearly showing that decomposition of the N C A occurs over time.  Because of the difficulties encountered in using compound 2.7a for the synthesis of 2.10a, the same reaction, but using the cyclohexylammonium salt 2.8a as the starting material (Scheme 2.8) was tried. Essentially the same conditions were used as for 2.7a (see above), except that it was necessary to remove both the cyclohexylammonium chloride salt and benzyl chloride formed as side products. Upon addition of T B M E to extract the N C A , the ammonium salt stayed behind as a solid and was easily removed by filtration. Despite the fact that T B M E has a low boiling point (55-56 °C), it was difficult to removed under vacuum as it was trapped in the oily product, and trituration did not give a solid. However, the product was much purer than from the previous attempts using compound 2.7a, as determined by H N M R . The FTIR of compound 2.10a showed l  the characteristic bands of the expected N C A , especially the two bands corresponding to the two carbonyl groups at 1864 cm" and 1795 cm" (Figure 2.4) which were compared 1  1  with the FTIR for the N C A of alanine where two bands are present at 1860 cm' and 1790 1  cm" .27 1  33  PBr CH CI ,25 °C, 16 h 3  2  2  Scheme 2.9  34  Figure 2.4: FTIR spectra of 2.10a and 2.10b  It is interesting to speculate about the mechanism of the reaction between the cyclohexylammonium salt and thionyl chloride. Two mechanisms are proposed: The first is an in situ conversion of the ammonium salt to the carboxylic acid due to the presence of trace acid. It this case, HCI may be present from the reaction of thionyl chloride with trace water. The purity of the product (2.10a) was lower by *H N M R when thionyl chloride was used directly from the supplier. However, a catalytic amount of HCI  35  could also act as an initiator for the conversion of the carboxylate to the carboxylic acid, with more HCI generated during the cyclisation to the N C A .  This may explain why  better results were obtained using the ammonium salt 2.8a instead of the carboxylic acids 2.7a.  A second possible mechanism involves the formation of the acid halide at the carbonyl of the C B Z N-protecting group. Acid halides react with carboxylate salts to form anhydrides (Scheme 2.10).34  m  this mechanism, benzyl alcohol will be produced  as a side-product and may be observed by *H N M R spectroscopy.  Scheme 2.10  36  2.3.2 Attempted Preparation of the X-serine A'-carboxy Anhydride Derivative 2.10b  To prepare 2.10b the same conditions were used as for the conversion of 2.8a to 2.10a, since better results were obtained starting with the cyclohexylammonium salt of the N-CBZ-L-serine derivative (Scheme 2.9).  It was found that, in this reaction,  compound 2.11 was formed as the major product instead of the expected 2.10b as demonstrated by ' H N M R and FTIR where the two characteristic bands corresponding to the two carbonyl groups were present. Under these conditions, it appears that thionyl chloride reacts with the vinylic bond of the conjugated group as indicated by the disappearance of the ' H N M R peak at 8 7.08, and the appearance of a new doublet integrating for two protons at 8 5.15, which are in the spectral region expected for protons on a,a'-dichlorobibenzyl group.41 fraws-Stilbene has been previously chlorinated using either CI2 or tetrabutylammonium iodotetrachloride, to obtain a,a'-dichlorobibenzyl. A n ' H N M R simulation of 2.16 (Figure 2.5), shows clearly that the two a protons appear at 8 5.31.  .  Since undesired 2.11 forms as a side-product when thionyl chloride is used to form the N C A 2.10b, milder conditions were tried in the hope that, with a different reagent, the double bond would not react. Oxalyl chloride was used in an attempt to generate the acid chloride in situ, with subsequent formation of the N C A by cyclisation of 2.8a and 2.8b (Scheme 2.9).  37  CI  5.31  2.16  x  / 89  BO  f>  7o  «»  eo  *.s  SO  41i  40  3S  JO  ?»  70  IS  10  04  OO  Figure 2.5: ' H N M R simulation of 2.16  In these reactions, it was observed that the initially colourless mixtures slowly turn to bright red over the reaction time of 16 h at 25 °C.  This occured with all the starting  materials used (2.7a, 2.8a and 2.8b), indicating that it is not related to the presence of the extended conjugation in 2.8b or 2.10b. When 2.8b was used as starting material a red powder was obtained after workup, the color of which faded to grey when the powder was left in contact with air. In order to separate the product from the ammonium salt,  38  ethyl ether was used, and compound 2.8b was obtained as a yellow solid, the ' H N M R spectrum of which exhibited the characteristic chemical shift for vinylic protons at 5 7.08. By comparing the infrared spectra of 2.10a and 2.10b (Figure 2.6), it can be seen that an extra band appears at 970 cm" in 2.10b, charateristic of the trans vinylic double bond of 1  the stilbene group.  Occasionally, two extra IR bands were detected in the product  mixture at 1712 and 1760 cm" , possibly due to oxalic acid which could be formed from 1  the hydrolysis of oxalyl chloride. Characterization of compound 2.10b by  1 3  C NMR  spectroscopy was not successful since no solvent allowed the preparation of a sufficiently concentrated solution of the compound.  Finally, a third synthetic route was tried to prepare 2.10b from compound 2.8b, using PBr3 in dry CH2CI2.  PBr3 was used since the /V-alkoxycarbonyl amino acyl  bromide, which forms in situ, is known to cyclize more readily than the acid chloride.27 Using a 1:1 molar ratio of 2.8b and PBr3, three different compounds were present as shown by H N M R spectroscopy, including some starting material (2.8b) indicating that !  the reaction did not go to completion after stirring the solution for 15 h at room temperature. It also appears that a carboxylic acid was formed, since a peak at 5 9.79 was also present in the crude product. In this case, it can be speculated that the carboxylic acid is produced first, and the acid bromide forms subsequently from trace HBr present in the solution. If this is the case, it would be consistent with the first mechanism proposed above. Future attempts to synthesise 2.10b in this way should use a large excess of PBr to drive this reaction to completion.  39  3  2.4 Polypeptide Synthesis  2.4.1 Polymerization of 2.10a and 2.10b using 4-Methylmorpholine as Initiator  9 H 9N -{-HN-CH-C-N-CH-Cf *~ J J n T H F , 60 °C, 24 h RO RCT Me-N  O  j_  2.12a, b  2.10a, b  Scheme 2.11  N C A s have been polymerized to polypeptides under many different conditions.  42  The initiator, initiator/monomer ratio, solvent, the reaction time and the temperature all are known to affect the yield from each specific N C A .  4 2  The synthesis of polypeptides  2.12a and 2.12b was attempted from the unpurified N C A precursors 2.10a and 2.10b (Scheme 2.11), using 4-methylmorpholine (1:1) in THF as initiator. These conditions were chosen because they gave the best yield (91 %) for the polymerization of Z,-alanineNCA.42 However, in our case the H N M R and FTIR spectra did not show any evidence !  for the formation of 2.12a and 2.12b. According to Whitesell et al, who prepared polyalanine on a functionalized gold surface,28  40  s u c n  polypeptides exhibit strong  absorptions in the infrared spectrum at 1659 cm' for amide I and 1545 cm" for amide II, 1  1  a shift of about 200-250 cm" from the corresponding N C A s . 1  Characterization of the  material by K N M R spectroscopy is difficult because of the low solubility of such l  polypeptides in many solvents. Kricheldorf has successfully used ' H N M R spectroscopy to demonstrate the formation of poly(glycine), and distinguished the N C A endgroups of the growing chains by the same method since the C -protons were shifted downfield a  compared to the starting NCA.27  2.4.2 Polymerization of 2.10a and 2.10b using Diisopropylamine as Initiator  P R O | V N  ^O  O O 4 - H N — C H - C — N— C H - c 4 H  diisopropylamine T H F , 23 ° C , 1 h  R  0  RO 2.12b  2.10b  Scheme 2.12  The reaction conditions, initiator and initiator/monomer mole ratio to polymerize compound 2.10b  were varied.  Diisopropylamine was used as the initiator (1/10  initiator/monomer mole ratio) in THF at 23 °C and the solution stirred for 1 h (Scheme 2.12). The *H N M R and FTIR spectra again did not show any evidence for the formation of compound 2.12b. It is probable that the lack of purity of the starting material, NCAs  41  2.10a and 2.10b, may be the major reason that formation of the polypeptides was not observed. Katchalski et al. stressed that pure and dry N C A s should be used in these reactions. To obtain purified starting materials, they recrystallized the N C A s several times from dry solvent under nitrogen prior to drying them with P2O5 under vacuum.43  42  2.5 Photoisomerisation of 2.7b and 2.8b  2.5.1 Photoisomerisation of 2.7b  In order to investigate the cis/trans photoisomerisation of 2.7b, the pure trans isomer of compound 2.7b, was dissolved in acetone-d6 (12 mg/mL), the solution degassed, and placed in a sealed NMR tube. Acetone was used as the solvent because 2.7b is poorly soluble in other organic solvents such as benzene or acetonitrile, and chlorinated solvents such as CHCI3 or CH2CI2 are undesirable in photochemical reactions since they are photoreactive . The tube was then irradiated for 5 min under a 450-W 44  Hanova medium-pressure mercury lamp.  In this case, it is possible that the energy  needed to photoisomerize 2.7b is transferred from the triplet excited state of acetone via photosensitization 5,46 A 'H N M R spectrum was collected immediately (Figure 2.6, B, 4  time = 5 min), and by comparison with spectrum A , it is apparent that compound 2.7b is isomerizes to the cis isomer under these conditions, as indicated by the appearance of the characteristic signal of the cis vinylic group at 5 6.63. The singlet at 8 6.63 shows that the two protons of the vinyl group are in similar environments, despite the fact that they are inequivalent. After 5 min, the ratio of the integrated areas of the c/s-vinylic signal at 8 6.63 and the fra/w-vinylic signal at 8 7.08, indicated that the conversion was 9 % (Scheme 2.13). The sample was then irradiated for an additional 5 min, and after a total irradiation time of 10 min, a third  N M R spectra was collected (figure 2.6, C, time =  10 min). From the ratio of peak areas, it is clear that a photostationary state is attained with 10 % of the trans isomer converted to the cis isomer after 10 min.  43  Scheme 2.13  44  Time:  10 min  B  5 min  0 min  Peak #: 8:  4 7.08  3 6.63  Figure 2.6: Comparison of the ' H N M R spectra of 2.7b in acetone-dV A : Sample before irradiation, B and C : Same sample after 5 and 10 min of irradiation respectively.  45  2.5.2 Photoisomerisation of 2.8b  The cis/trans photoisomerization of trans-2.8b was investigated by dissolving a sample of 2.8b in CDCI3 (12 mg/mL), passing it through basic alumina, and finally degassing and sealing the sample under N in an N M R tube. The sample was irradiated 2  in a Rayonet photochemical reactor at 300 nm for 10 min, and then an ' H N M R spectrum was taken immediately (Figure 2.9 B, time = 10 min). By comparison with spectrum A (Figure 2.9) and the ' H N M R of c/s-stilbene, it was apparent that compound 2.8b had 36  photoisomerized from the trans to the cis isomer, as shown by the appearance of the characteristic signal of the cis vinylic group at 5 6.52 in CDCI3 (Figure 2.9, peak # 5). The sample was then irradiated for successive periods of time up to a cumulative time of 30 min. ' H N M R spectra were taken immediately after each irradiation periods (Figure 2.9, A to F). The proton signals for the P h C / / 0 (Figure 2.9, peak # 1,5 4.40 and peak # 2  2, 8 4.46), NH (Figure 2.9, peak # 3, 8 5.86 and peak # 4, 8 5.92), and P h C W C 0 (8 5.06, 2  2  8 5.00) groups shifted downfield by about 0.06 ppm. This difference in chemical shifts was attributed to the presence of the cis isomer. Because of this difference, it was possible to compare the integration of peak # 1 with peak # 2, peak # 3 with peak # 4, and peak # 5 with peak # 6, and this data was plotted over time (Figure 2.7). The shift of the PhC// C02 group (from 8 5.06, to 8 5.00) was not considered because it was 2  impossible to calculate the individual integration of each of the two multiplets overlap due to a lack of resolution. The ratio of the peak integration assigned to the cis isomer over the total integrated area for both trans and cis isomers for each group of peaks, for each successive time period (Figure 2.8) were then plotted. A photostationary state was  46  h v  CHCI,  H  + 3  300 nm 30 min  N ^ >  2.15 57%  Scheme 2.14  achieved in this reaction where 57 % of the trans isomer had isomerized to the cis isomer.  The higher proportion of cis isomer obtained for compound 2.8b (57 %)  compared to the proportion of cis isomer formed for compound 2.7b (10 %) may be attributed to the difference in irradiation bandwidth used to irradiate the compounds. In the case of 2.7b, the irradiation was probably overlapping with the absorption of the cis isomer since a wavelength of > 290 nm was used, converting more cis back to the trans  47  isomer. However, in the case of 2.8b, a narrower bandwidth at around 300 nm probably prevented that to happen since the cis isomer absorbs at higher wavelengths than the trans isomer.  Integration vs Time  20  10  30  40  Time (min)  Figure 2.7: Integrated areas of the selected *H N M R peaks as a function of time.  Percentage of cis isomer formed vs Time 60  -  5  £ 50 co 0 A  n  4  0  7  % cis  —•—1/(1+2)  S) 30 a 1 20  —•-3/(3+4)  o  5/(5+6)  Q> 10  Q.  10  20  30  Time (min)  Figure 2.8: Percentage of cis isomer from peak ratios.  48  40  Time:  30 min  E  25 min  20 min  D  UK  15 min  B  10 min  0 min  Peak #: 8:  6 7.08  5 6.52  43 5.92 5.86  t i  21 4.46 4.40  Figure 2.9: Comparison of H N M R spectra of 2.8b in CHCI3. A: Sample before irradiation, B to F: Same sample after 10, 15, 20,25, and 30 min of irradiation. l  49  2.6 Conclusions and Future Considerations  The exploration of new materials that may be used in electroluminescent devices according to the new approach that is proposed herein, lead to the successful synthesis of the new compounds 2.7b and 2.8b. These amino acids were used for the synthesis of the corresponding NCAs which are expected to serve as precursors for polypeptides containing a conjugated pendant group. Unfortunately, the isolation of pure N C A s 2.10a and 2.10b was unsuccessful, and it is clear that there is a need for a better synthetic route to the N C A s 2.10a and 2.10b. The use of PBr3 to close the ring appears promising, however,  an  alternative  route  which  may  be  worth  bis(trichloromethyl)carbonate to produce phosgene in situ.  considering  is  using  This method has been  explored and successfuly used in peptide synthesis by Falb et al.^  From compound 2.5 it should be straightforward to produce more extended oligomers  by  converting  the  bromomethyl  group  to  the  corresponding  triphenylphosphonium salt and reacting it with either 2.4 or jo-tolualdehyde followed by bromination, thus adding an extra phenylenevinylene unit to the oligomer (Scheme 2.15). It is important to note that solubility problems might be encountered in this approach due to the rigid rod-like nature of the oligomers. Replacing compound 2.4 or p-tolualdehyde by a benzaldehyde derivative containing side-chains such as ethers or esters, should serve to increase the solubility of the oligomers.2  50  pph  3  toluene  2.5 Li EtO" +  EtOH, 0 °C, 1 h 2.4  Scheme 2.15  Should the synthesis of the target polypeptides 2.12a and 2.12b Successful, it will be interesting to examine the behavior of the a-helix in an electric field. It is possible that the spacing between the side-chains may change when an electric field is applied due to the contraction of the helical chain and this could have interesting consequences in devices made from this material.48  51  CHAPTER 3  EXPERIMENTAL SECTION  3.1 General Experimental Details  A l l reactions, unless otherwise indicated, were conducted under a dry N  2  atmosphere using standard Schlenk techniques. Solvents were distilled under N using 2  the appropriate methods: sodium/benzophenone ketyl (THF, hexanes, benzene, diethyl ether), molten sodium (toluene, CH C1 ), NaH (DMF), and D M S O was sequentially dried 2  2  with activated 4 A molecular sieves. Distilled solvents were stored over activated 4A molecular sieves under N . 2  Fine chemicals were obtained either from Aldrich Chemical Co. Ltd., Fisher Scientific, Acros, or Fluka, and used as received.  !  H and C nuclear magnetic resonance (NMR) spectra were recorded in 1 3  CDCI3,  acetone-^ or acetonitrile-rfj at room temperature on Bruker AC-200E, Bruker WH-400, Bruker AV-300 or Bruker AV-400 Fourier transform spectrometers. N M R resonances were referenced to internal C i / C l (5 = 7.24 ppm), C D C O C D / / (5 = 2.04 ppm) or 3  3  2  C D / / C N (5 = 1.93 ppm) for H and CDC1 (8 = 77.0 ppm) for C . !  2  13  3  FT-IR spectra were recorded on a B O M E M MB-Series spectrometer in CH C1 or 2  2  prepared as K B r pellets between two K B r plates. U V - V i s measurments were taken on a HP 8453 diode array spectrophotometer and fluorescence measurements were carried out on a Perkin-Elmer LS-5B fluorometer PE7500-controlled. Melting point determinations were performed on samples placed in capillary tubes using a Gallenkamp apparatus.  53  Flash chromatographic purification was carried out on silica gel 60 (70-230 mesh) which was obtained from B D H . Elemental analyses were performed by Mr. Peter Borda at the University of British Columbia.  54  3.2 Starting Materials  3.2.1 Preparation of 2.1  /?-Xylylenebis(triphenylphosphonium bromide) (13.01 g; 0.01649 mol) was suspended in dry D M S O (150 mL) in a 2-neck round-bottom flask under N . «-BuLi (1.6 2  M in hexanes) (22.3 mL; 0.0357 mol) was added dropwise to the suspension to generate the corresponding ylide. The resulting dark red solution was stirred for 2 h after which time p-tolualdehyde (4.00 mL; 0.0339 mol) was added in one portion. The mixture was stirred overnight at room temperature under N . The resulting orange solid was collected 2  by filtration on a Btichner filter and dried under vacuum on an oil bath (50°C). The solid was then purified by chromatography on silica gel using a hexanes/chloroform mixture (1:1) as eluant. The first fraction was collected and the solvent evaporated in vacuo. The yellow solid was then dried under vacuum (3.79 g; 74 %). *H N M R (200 M H z , CDC1 ) 5 3  7.49 (s, 4 H , Ar) 7.42 (d, J = 8.0, 4 H , Ar), 7.17 (d,J= 8.1, 4 H , Ar), 7.07 (s, 4 H , trans CH=CH), 6.44 (d, J= 7.9, 4 H , cis CH=CH), 2.37 (s, 6 H , CH ). 3  3.2.2 Isomerization of 2.1  Compound 2.1 (0.3002 g; 0.9670 mmol) was dissolved in toluene (25 mL) in a round-bottom flask. A catalytic amount of I ( ) was added and the mixture heated to 2 S  reflux for 3 days, after which time a solid precipitated. The solution was cooled and left at 4 °C overnight. The solution was filtered and washed with two portions (5 mL) of cold  55  toluene. The product was isolated as green, shiny crystals (24.3 mg; 0.0783 mmol, 8%). ' H N M R (200 M H z , CDC1 ) 8 7.49 (s, 4 H , Ar), 7.42 (d, J= 8.0, 4 H , Ar), 7.17 (d, J = 3  8.1,4 H , Ar), 7.07 (s, 4 H , trans CH=CH), 2.37 (s, 6 H , CH ). Anal. Calcd. for C H 2 : C, 3  24  2  92.86 %, H , 7.14 %. Found: C, 90.74 %, H , 7.07 %.  3.2.3 Preparation of a-Bromo-p-toIunitrile (2.3)32  p-Tolunitrile (34.91 g; 0.2980 mol) and TY-bromosuccinimide (53.03 g; 0.2980 mol) were dissolved in dry benzene (300 mL) in a round bottom flask (500 mL). Benzoyl peroxide (320.0 mg; 9.105 mmol, 3%) was added, and the mixture was heated to reflux on an oil bath (100 °C) for 24 h. The clear yellow solution was cooled to room temperature and washed twice with a hot 5 % N a S 0 solution (100 mL) to remove B r 3  2  and succinimide. The aqueous solution was washed with benzene and the organic fractions combined. The solvent was removed in vacuo to give a yellow-orange solid, which was recrystallized from methanol to give yellow needles (39.08 g, 0.1993 mol, 67 %). H N M R (200 M H z , CDC1 ) 8 7.59 (d,J= 8.1, 2 H , Ar), 7.45 (d, J= 8.1, 2 H , Ar), !  3  4.40 (s, 2 H , Cift-Br). Anal. Calcd. for C H N B r : C: 49.01 %, H : 3.08 %, N : 7.14 %. 8  6  Found: C: 49.09 %, H : 3.05 %, N : 7.09 %. mp = 107-109 °C  56  3.2.4 Preparation of a-Bromo-p-tolualdehyde (2.4)33  a-bromo-p-tolunitrile (2.3) (8.059 g; 0.04111 mol) was dissolved in chlorobenzene (70 mL) in a round bottom flask (500 mL). The solution was cooled to 0 °C on an ice bath. A 1.0 M solution of D I B A L - H in hexanes (50 mL, 0.050 mol) was added dropwise over a period of 20 minutes at 0 °C. The mixture was stirred for 1 h at 0 °C, diluted with chloroform (130 mL), and acidified with a solution of 3 N HCI (50 mL). The aqueous layer was removed and washed once with chloroform (25 mL). The organic portions were combined and dried over MgSCXt, and the solvents removed in vacuo leaving a yellow oil. The oil was dissolved in a minimum quantity of ethyl ether and transferred to a sublimator. The pure a-bromo-p-tolualdehyde (2.4) was then isolated by sublimation as white crystals (3.769 g, 0.01894 mol, 46 %). U N M R (200 M H z , CDC1 ) l  3  5 9.98 (s, 1 H , CO-H),  7.81 (d, J=  8.1, 2 H , Ar), 7.44 (d, J=  8.1, 2 H , A r ) , 4.42 (s, 2 H ,  C# -Br). 2  3.2.5 Preparation of 2.6  >-Tolualdehyde  (12.02 g; 11.80 mL, 0.1000 mol) and benzyl triphenyl  phosphonium chloride (38.90 g; 0.1000 mol) were dissolved in absolute ethanol (300 mL), and the solution was cooled to 0 °C. A solution of lithium ethoxide was prepared by reacting lithium metal (0.6950 g; 0.1001 mol) in absolute ethanol (200 mL), and added dropwise to the solution containing the aldehyde over a period of 10 min. The solution was then stirred for 1 h, and water (30 mL) was added to assist the precipitation.  57  The resulting white solid was collected by filtration and washed twice with cold hexanes (25 mL), and dried under vacuum (6.22 g, 0.0320 mol, 32 %). *H N M R (200 M H z , CDC1 ) 5 7.58-7.10 (m, 9 H , Ar), 7.08 (s, 2 H , trans CH=CH), 2.38 (s, 3 H , CH ). 3  3  58  3.3 Conjugated Side-chains  3.3.1 Attempted Preparation of 2.2  N-bromosuccinimide (0.1720 g; 0.9664 mmol) was dissolved in warm benzene (30 mL) in a round bottom flask (50 mL). Compound 2.1 (300 mg; 0.966 mmol) was then added and immediately formed a red solution. Benzoyl peroxide (16.0 mg; 0.455 mmol) was added in a catalytic amount (5 %). The mixture was exposed to white light (100 W) and heated to reflux for 24 h on an oil bath (100 °C). The benzene was removed in vacuo from the violet solution on an oil bath (30 °C). The residual solid was washed twice with a boiling solution (5 mL) of 5 % sodium sulphite to remove Br2, succinimide and excess N-bromosuccinimide. The solid was then purified by chromatography on silica gel using methylene chloride as eluant. The first fraction was collected and the solvent removed in vacuo. The product was an impure green powder (35 mg; 0.080 mmol, 8 %).  3.3.2 Preparation of 4-(fra/w-2-phenylethenyl)phenylmethyl bromide (2.5) 3.3.2.1 From a-Bromo-p-tolualdehyde (2.4)  a-Bromo-/?-tolualdehyde  (2.4)  (6.00 g; 0.0302 mol) and benzyltriphenyl-  phosphonium chloride (11.72 g; 0.03000 mol) were dissolved in absolute ethanol (150 mL), and cooled to 0 °C. A solution of lithium ethoxide was prepared by react ing lithium metal (0.2083 g; 0.03001 mol) in absolute ethanol (75 mL). The lithium ethoxide solution was then added dropwise to the previously cooled ethanol solution over a period  59  of four minutes. The solution was stirred for 1 h, and water (10 mL) was added to assist the precipitation. The white powder was collected by filtration on a Biichner filter, washed twice with cold hexanes (25 mL) and dried under vacuum (2.52 g, 9.24 mmol, 31 %). *H N M R (200 M H z , CDC1 ) 5 7.41 (d, J = 7.0, 2 H , Ar), 7.40 (d, J = 7.1, 2 H , Ar), 3  7.38-7.10 (m, 5 H , Ar), 7.06 (s, 2 H , trans CH=CH), 4.42 (s, 2 H , C i / B r ) . 2  3.3.2.2 From Compound 2.6  Compound 2.6 (12.30 g, 0.06330 mol) and N-bromosuccinimide (12.39 g, 0.06963 mol) were dissolved in dry benzene (150 mL) in a round bottom flask (250 mL). Benzoyl peroxide (4.5 %, 100.0 mg; 2.845 mmol) was added, and the mixture heated to reflux on an oil bath (100 °C) for 24 h. The benzene was removed in vacuo at 30 °C, and the yellow residue was then washed twice with a hot N a S 0 solution (5 %, 100 mL) to 3  remove B r and succinimide. The solid was filtered on a Biichner filter, washed twice 2  with boiling water (50 mL) and recrystallized from methanol as a pale yellow solid (15.24 g, 0.05579 mol, 88 %). Purification by chromatography on silica gel using a hexanes/chloroform mixture (1:1) as eluant yielded the trans isomer (95 % pure) (2.5). ' H N M R (200 M H z , CDC1 ) 8 7.42 (d,J= 7.0,2 H , Ar), 7.38 (d, J= 7.1,2 H , Ar) 7.06 (s, 3  2 H , trans CH=CH), 4.40 (s, 2 H , Ci/ Br). Anal. Calcd. for C i H i B r : C: 65.95 %, H : 2  5  4.80 %. Found: C: 66.66 %, H : 4.71 %.  60  3  3.4 7Y-CBZ-Z-serine Derivatives  3.4.1 Preparation of TV-CBZ-O-benzyl-X-serine Cyclohexylammonium Salt (2.8a)  /V-CBZ-L-serine (2.00 g; 8.36 mmol) was dissolved in dry D M F (50 mL) in a round-bottom flask (100 mL). The solution was cooled to 0 °C and sodium hydride (0.441 g; 18.4 mmol) was added in small portions over 2-3 minutes. When no further gas evolved, benzyl bromide (1.57 g; 1.09 mL, 9.20 mmol) was added in one portion. The mixture was then heated to 25-30 °C for 5 h under N . The solvent was then removed in 2  vacuo on an oil bath (40 °C) and the resulting beige oil dissolved in water and extracted twice with ether (20 mL). The water fraction was then acidified to pH 3.5 using a 3 N HC1 solution and extracted five times with ethyl acetate (20 mL). The ethyl acetate portion was washed with water until the washings were neutral, pooled and dried over MgSC>4 ( ). The ethyl acetate was removed in vacuo and leaving a yellow oil. The oil was S  then dissolved in a minimum quantity of hot toluene and set aside to cool. The resulting precipitate was removed by filtration and the solvent removed in vacuo. The resulting residue was dissolved in a minimum quantity of chloroform, and hexanes added until the solution remained cloudy. The solution was heated until the cloudiness disappeared and set aside for cooling. The resulting white solid was dried under vacuum, redissolved in diethyl ether (30 mL), and cyclohexyl amine (0.7033 g, 0.8112 mL, 7.092 mmol) was added in one portion. A white salt precipitated immediately, the solution was sonicated for 2 min, the solid filtered and washed twice with ether (35 mL). The white salt was then recrystallized from ethyl acetate (0.991 g, 2.31 mmol, 28 %). ' H N M R (200 M H z ,  61  CDC1 ) 5 7.28; (m, 10H, Ar), 5.81 (d, J= 7.0,1 H , Nfl), 5.01 (m, 2 H , OfePh), 4.43 (s, 2 3  H , PhC7/ 0), 4.13 (m, 1 H , COCtf) 3.78 (m, 2 H , OGi/ ) 2.64 (m, 1 H) 1.95-0.88 (m, 12 2  2  H,). Anal. Calcd. for C H : N 0 C: 67.27 %, H : 7.53 %, N : 6.54 %. Found: C: 67.04 2 4  3 2  2  5  %, H : 7.57 %, N : 6.54 %. Compound 2.9 was also isolated as a white powder from the toluene portion as a side-product (about 10 % yield). ' H N M R (400 M H z , CDC1 ) 5 8.72 3  (s, 1 H , COO//) 7.40-7.22 (m, 5 H , Ar), 4.97 (d, J,= 14.9, 2 H , CH =C), 4.42 (m, 2 H , 2  PhC7/ 0), 4.12 (m, 1 H , Nfl). Anal. Calcd. for C I I H H N 0 : C: 59.72 %, H : 5.01 %, N : 2  4  6.33 %. Found: C: 58.97 %, H : 4.96 %, N : 6.22 %  3.4.2 Preparation of W-CBZ-O-benzyl-L-serine (2.7a)  N-CBZ-O-benzyl-Z-serine cyclohexylammonium salt (2.8a) (0.5002 g; 1.170 mmol) was dissolved in C H O H (40 mL) in a round-bottom flask (100 mL) and cooled to 3  0 °C. The solution was treated with 2 N HCI (8 mL) and stirred for 1 h. The C H O H 3  was removed in vacuo and water (50 mL) was added to the residue. The suspension was extracted 4 times with ethyl acetate (30 mL). The extracts were pooled together and washed with water until the washes are neutral. The ethyl acetate was removed in vacuo leaving a colorless oil that crystallized slowly under vacuum to give a white solid. The solid was then recrystallized from toluene. (215.0 mg, 0.6528 mmol, 56 %). *H N M R (200 M H z , CDC1 ) 8 7.84 (br s, 1H, COO#), 7.28 (m, 10 H , Ar), 5.65 (d, J = 14.8, 1 H , 3  Nfl), 5.12 (s, 2 H , CH ?h), 4.55 (m, 1 H , COCfl), 4.50 (s, 2 H , P h C i / 0 ) , 3.85 (m, 2 H , 2  2  OCH ). Anal. Calcd. for C18H19NO5: C: 65.64 %, H : 5.81 %, N : 4.25 %. Found: C: 65.71 2  %, H : 5.85 %, N : 4.36 %. m.p. = 97 °C.  62  3.4.3 Preparation of 2.8b  iV~-CBZ-Z-serine (2.329 g; 10.00 mmol) was dissolved in dry D M F (75 mL) in a round-bottom flask (125 mL). The solution was cooled to 0 °C and sodium hydride (0.5290 g; 22.00 mmol) added in small portions over 2-3 min. When no further gas evolved, 4-(rra«s -2-phenylethenyl)phenylmethyl bromide (2.5) (2.732 g; 10.00 mmol) ,  was added and the mixture heated to 25-30 °C for 5 h under N . The solvent was then 2  removed in vacuo on an oil bath (40 °C) and the resulting beige oil dissolved in ethanol (200 mL). The white insoluble solid was filtered on a Buchner filter, and the ethanol removed in vacuo. Water was added (50 mL) and the water suspension extracted with ether (20 mL). The water layer was removed and acidified to pH 3.5 with 2 N HC1. The solution was then extracted with ethyl acetate (80 mL), and the organic portions washed with water until the washing portions were neutral.  The ethyl acetate portions were  pooled together and dried over MgS04( ). The ethyl acetate was removed in vacuo to S  give an oily yellow residue which was dissolved in a minimum quantity of hot toluene and set aside to cool.  The precipitate (N-CBZ-L-serine and 2.9) was removed by  filtration and the solvent removed in vacuo. The resulting residue was dissolved in a minimum of chloroform, and hexanes added until the solution remained cloudy. The solution was then heated until the cloudiness disappeared and set aside for cooling. The resulting white solid was dried under vacuum and redissolved in diethyl ether (200 mL). Cyclohexyl amine (0.130 g; 0.150 mL, 1.31 mmol) was added in one portion. A white salt precipitated immediately and the solution sonicated for 2 min. The solid was then  63  filtered and washed twice with ether (35 mL) (0.631 g, 1.19 mmol, 12.%). [a] D - + 14.8 °. 2 2  H N M R (400 MHz, CDC1 ) 5 7.48-7.21 (m, 14 H , Ar), 7.03 (s, 2 H ,  5  l  3  trans CH=CH), 5.85 (d, J = 14.9, 1 H , N#), 5.02 (m, 2 H , COC// Ph), 4.46 (s, 2 H , 2  PhC/fcO), 4.13 (m, 1 H , COCH) 3.82 (m, 2 H , OCH ) 2.80 (m, 1 H) 1.95-0.88 (m, 12 H). 2  Anal. Calcd. For C 2 H 3 8 : N 0 C: 72.43 %, H : 7.22 %, N : 5.28 %. Found: C: 70.67 %, H : 3  2  5  7.12 %, N : 5.20 %. Compound 2.9 was observed in the crude.  3.4.4 Preparation of 2.7b  Compound 2.8b (0.1850 g; 0.3486 mmol) was dissolved in C H O H (40 mL) in a 3  round bottom flask (100 mL) and cooled to 0 °C. The solution was treated with 2 N HC1 (3 mL) and stirred for 1 h. The C H O H was then removed in vacuo and water (50 mL) 3  added to the residue. The suspension was extracted four times with ethyl acetate (30 mL), the extracts pooled together and washed with water until neutral. The ethyl acetate was removed in vacuo. The resulting residue was dissolved in a minimum amount of chloroform, and hexanes added until the solution remained cloudy. The solution was heated until the cloudiness disappeared and was then set aside for cooling. The resulting solid was filtered and dried under vacuum (176.9 mg; 0.4101 mmol, 85 %). H N M R !  (200 M H z , CDC1 ) 5 7.52-7.08 (m, 14 H , Ar), 7.04 (s, 2 H , trans CH=CH), 5.60 (d, J = 3  14.9, 1 H , Nfl), 5.07 (s, 2 H , COCr/ Ph), 4.50 (m, 1 H , COCH), AAA (s, 2 H , PhCtf 0), 2  3.78 (m, 2 H , OCH ). 2  2  Anal. Calcd. for C H N 0 : C: 71.58 %, H : 5.91 %, N : 3.34 %. 2 5  Found: C: 71.59 %, H : 5.88 %, N : 3.33 %.  64  2 5  5  3.4.5 Preparation of /V-CBZ-I-serine Benzyl Ester (2.11)  Sodium bicarbonate (720 mg; 8.57 mmol) was dissolved in water and  N-CBZ-L-  serine (2.00 g; 8.37 mmol) added in small portions. When no further gas was evolved, the water was removed in vacuo on an oil bath (50 °C). The slightly yellowish salt was dried in a desiccator over NaOH for three days, and was then dissolved in hot D M F (15 mL; 80 °C). Benzyl bromide (5.0 mL; 0.040 mol) was added and the solution was stirred for 20 h at 70 °C. The D M F and excess benzyl bromide were removed in vacuo and the white residue was taken up in ether (50 mL) and water (8 mL). The water layer was discarded and the ether layer washed successively with a saturated solution of sodium bicarbonate (5 mL) and then with water (10 mL). The ether layer was dried over anhydrous sodium sulfate and removed in vacuo leaving a brown oil behind. The residue was then recrystallized by dissolving it in warm CCI4 (12 mL), adding petroleum ether (5 mL, b.p. 35-60 °C) and allowing the solution to cool slowly to room temperature (20 °C). The product precipitated as needles (850 mg; 2.6 mmol; 31 %). H N M R (200 M H z , !  CDCI3) 5 7.45-7.30 (m, 10 H , Ar), 5.72 (d, J = 14.8, 1 H , N#),  5.23 (s, 2 H ,  CO(CH)OCtf Ph), 5.18 (s, 2 H , CO(NH)OC// Ph), 4.52 (m, 1 H , COC#), 4.00 (m, 2 H , 2  2  OCH ). Anal. Calcd. for C H i N 0 : C: 65.64 %, H : 5.81 %, N : 4.25 %. Found: C: 65.68 2  I 8  9  5  %, H : 5.78 % , N : 4.31%.  65  3.5 /y-carboxyanhydrides  3.5.1 Attempted preparation of O-benzyl-L-serine N-carboxyanhydride (2.10a) 3.5.1.1 From TV-CBZ-O-benzyl-Z-serine (2.7a) 3.5.1.1.1 Using Thionyl Chloride  JV-CBZ-O-berizyl-L-serine (2.7a) (0.5010 g; 1.521 mmol) was added to thionyl chloride (10 mL) in a round bottom flask (25 mL). The solution was stirred overnight at 20-25 °C. Excess thionyl chloride and benzyl chloride were removed in vacuo leaving a yellow oil. /-Butylmethyl ether (15 mL) was added and the solution filtered. The solvent was removed in vacuo to give a pale yellow oil, which was then triturated six times with dry ethyl ether. H N M R (200 MHz, CDC1 ) 8 7.40-7.19 (m, 5 H , Ar), 6.32 (s, 1 H , N#), !  3  4.52 (s, 2 H , OCH ?h),  4.38 (m, 1 H , COCH),  2  3.72 (s, 2 H , OC# CH). FTIR (CH C1 ) 2  2  2  1863 cm" (CH-C=0), 1798 cm" (NH-C=0). 1  1  3.5.1.1.2 Using Oxalyl Chloride  TV-CBZ-O-benzyl-Z-serine (2.7a) (100 mg; 0.304 mmol) and D M F (3-5 .%) were dissolved in dry CH C1 (10 mL) in a round bottom flask (25 mL). Oxalyl chloride was 2  2  added (0.18 mL; 2.1 mmol), and the colorless solution stirred overnight at 20-25 °C. After 16 h, the solution was red. The CH C1 , the excess oxalyl chloride and the D M F 2  2  were removed in vacuo leaving a red oil behind which was then triturated six times with dry ethyl ether. *H N M R (400 MHz, CDC1 ) 8 7.40-7.07 (m, 5 H , Ar), 6.50 (s, 1 H , NH), 3  66  4.51 (s, 2 H , OOfcPh), 4.37 (s, 1 H , COCH), 3.74 (s, 2 H , OC# CH). FTIR (CH C1 ) 2  2  2  1864 cm" (CH-C=0), 1795 cm- (NH-C=0). 1  1  3.5.1.2 From A'-CBZ-O-benzyl-X-serine Cyclohexylammonium Salt (2.8a) 3.5.1.2.1 Using Thionyl Chloride  • A -CBZ-0-benzyl-£-serine cyclohexylammonium salt (2.8b) (100 mg; 0.233 /  mmol) was added to thionyl chloride (10 mL) in a round bottom flask (25 mL). The solution was stirred overnight at 20-25 °C. The excess thionyl chloride and benzyl chloride were removed in vacuo to leaving a yellow oil behind. f-Butylmethyl ether (15 mL) was added, and the resulting solid filtered leaving the cyclohexylammonium salt. The solvent was removed in vacuo to give a pale yellow oil, which was then triturated six times with dry ethyl ether. *H N M R (200 M H z , CDC1 ) 8 7.40-7.19 (m, 5 H , Ar), 6.32 (s, 3  1 H , Nfl), 4.52 (s, 2 H , OC# Ph), 4.38 (m, 1 H , COCH), 3.72 (s, 2 H , OCf7 CH). FTIR 2  2  (CH C1 ) 1865 cm" (CH-C=0), 1798 cm' (NH-C=0). 1  2  1  2  3.5.1.2.2 Using Oxalyl Chloride  JV-CBZ-O-benzyl-Z-serine cyclohexylammonium salt (2.8a) (100 mg; 0.233 mmol) and a catalytic amount of D M F (3-5 %) were dissolved in dry C H C 1 (10 mL) in a round 2  2  bottom flask (25 mL). Oxalyl chloride was added (0.17 mL; 2.0 mmol), and the colorless solution stirred overnight at 20-25 °C. After 16 h, the solution was red. The CH C1 , 2  2  excess oxalyl chloride and D M F were removed in vacuo to leaving a red oil behind. The  67  oil was triturated six times with dry ethyl ether. *H N M R (400 M H z , CDC1 ) 8 7.38-7.13 3  (m, 5 H , Ar), 6.17 (s, 1 H , N#), 4.53 (s, 2 H , OCH ?h), 4.40 (s, 1 H , COCH), 3.72 (s, 2 2  H , OCi7 CH), 1.95-1.00 (m, 13 H). FTIR (CH C1 ) 1860 c m (CH-C=0), 1797 cm 1  2  2  -1  2  (NH-C=0).  3.5.2  Preparation of 2.10b  3.5.2.1 From 2.8b 3.5.2.1.1 Using Thionyl Chloride  Compound 2.8b (35.2 mg; 0.0663 mmol) was added to thionyl chloride (10 mL) in a round bottom flask (25 mL). The yellow solution, which became colorless after 10 min., was stirred at 20-25 °C for 24 h. The excess thionyl chloride and benzyl chloride were removed in vacuo leaving a colorless oil behind. ^-Butylmethyl ether (15 mL) was added and the resulting solid filtered leaving the cyclohexylammonium salt. The solvent was removed in vacuo to give a pale yellow oil which was then triturated six times with dry ethyl ether. H N M R (400 M H z , CDC1 ) 8 7.40-7.00 (m, 9 H , Ar), 6.20 (s, 1 H , N#), !  3  5.15 (s, 2 H) 4.50 (s, 2 H , OC# Ph), 4.34 (m, 1 H , COCH), 3.68 (d, 2 H , OC# CH). 2  2  FTIR (CHCL ) 1864 cm" (CH-C=0), 1798 cm" (NH-C=<9). 1  1  2  3.5.2.1.2 Using Oxalyl Chloride  Compound 2.8b (100 mg; 0.188 mmol) and a catalytic amount of D M F (3-5 %) were dissolved in dry CH C1 (10 mL) in a round bottom flask (25 mL). Oxalyl chloride 2  2  68  was added (0.17 mL; 2.0 mmol), and the colorless solution stirred at 20-25 °C for 17 h, after what, the solution was red. The CH2CI2, excess oxalyl chloride and D M F were removed in vacuo leaving a red powder behind. Ethyl ether (10 mL) was then added and the insoluble red powder (14 mg,) filtered. The ether was removed in vacuo leaving a yellow solid (42 mg; 0.13 mmol, 69 %). 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