A glycoprotein can exist as a spectrum of glycosylated forms, where each protein molecule may be associated with an array of oligosaccharide structures. The overall range of glycoforms can have a variety of different biophysical and biochemical properties. As a result of this 'microheterogeneity' there is a need for the synthesis of homogeneously glycosylated proteins for analytical purposes. The synthesis of novel glycoproteins through the selective reaction of glycosyl iodoacetamides with the thiol groups of cysteine has been developed (Figure 10919). Through site-directed mutagenesis, whereby the natural asparagine glycosylation sites can be exchanged for cysteine, it was possible to selectively glycosylated proteins at predetermined sites. This provided a general method for the synthesis of homogeneously glycosylated proteins. We chose the glycoprotein hormone erythropoietin as a model system since the <I>N</I>-glycans at residues 24, 38 and 83 are essential for <I>in vivo</I> biological activity. Using a modified recombinant erythropoietin gene (prepared in Chapter 2) we optimized protein expression, and over-expressed and purified His<SUB>10</SUB>-WThEPO, His<SUB>10</SUB>-Asn24Cys, His<SUB>10</SUB>-Asn38Cys, His<SUB>10</SUB>-Asn83Cys and His<SUB>10</SUB>-Asn38/83 CyshEPO's in yields of 13mgL<SUP>-1</SUP> from <I>E. coli.</I> This allowed us to probe the structure of rhEPO using techniques such as NMR spectroscopy and electrospray MS (Chapter 3). Having access to larger quantities of EPO, we were also able to develop on-line LC-ESI-MS methods, which allowed us to monitor protein glycosylation reactions with glycosyl β-<I>N</I>-iodoacetamides and map the position of the glycosylation site. The synthesis of relevant iodoacetamides was attempted and simple glycosyl iodoacetamides and <SUP>13</SUP>C labeled iodoacetamides were prepared and successfully employed as probes for protein glycosylation.

An investigation into novel protecting group strategies for the prevention of imide formation at the Asp-X segment of a peptide sequence was undertaken. Research centred around the esterification of the aspartyl side chain with an acid labile protecting group which exerted an extreme steric influence on the system. Three protecting group strategies were extensively studied with respect to the aspartyl residue; namely derivatised trityl systems, the 2,2,4,4-tetramethylpent-3-yl (Tmp) system and the 2,4-dimethyl-2,4-bis(4'-methoxyphenyl)pent-3-yl (Dmbp) system. The characteristics of the trityl β-aspartates indicated that they possessed the requirements sought in a successful protecting group, but orthogonal synthesis of such derivatives applicable to automated SPPS proved elusive. The 9-fluorenylmethyloxycarbonyl (Fmoc) amine β-protected aspartyl residue was successfully synthesised with the side chain protected with Tmp and Dmbp groups. The respective β-derivatised aspartyl residues were incorporated into peptide sequences which were known to be susceptible to aspartimide formation, namely the partial sequence of coat protein phage MS2 and ferrodoxin, and tested for their efficacy in preventing rearrangement during Fmoc-based SPPS. The β-aspartyl-protected peptides were subjected to a variety of acidic deprotection protocols and the merits of each were assessed with respect to aspartimide formation.

In recent years, much effort has been directed towards the development of new strategies for the efficient synthesis of libraries of peptidomimetics as potential therapeutic leads. The solid phase inverse synthesis of peptides(N(r)C) has only received a fraction of the attention given to the classical (C(r)N) solid phase assembly of peptides due to oxazolone-induced epimerisation. However, rather than trying to prevent its formation we have demonstrated how the peptide-derived 5(<i>4H</i>)-oxazolone can be used effectively as an ideal precursor to a range of peptidomimetics. (Fig. 9971) The development of a suitable strategy for the solid phase inverse synthesis of peptides is described and investigated as a valuable route into C-terminally modified peptides. This methodology is then applied to the construction of the polymer-supported 5(4<i>H</i>)-oxazolone II and various transformations thereof have been examined, often with solution phase comparisons.

The problems caused by <i>Phytophthora medecaginis; </i>together with the isolation, biological activity and biosynthesis of zwittermicin A (which shows potential as an inhibitor of this disease) are reviewed. Studies towards the synthesis of the aminopolyol antibiotic zwittermicin A I will be described. (Fig. 6438) Chapter 2 outlines the approaches investigated towards the synthesis of the nitrogen-rich fragment II which include: enzymatic separation of racemic 2,3-diaminopropionic acid using a <i>D</i>-amino acid oxidase; and synthetic routes based on a Hofmann rearrangement of <i>N</i>-protected asparagine. Chapter 3 describes studies towards the synthesis of aminopolyol species IV from threonine-derived ketone V and the serine-derived aldehyde VI. Methodology for the “matched” lithium-mediated aldol reaction between these two components was developed, together with directed reductions of the resultant aldol adducts to give either <i>syn </i>or <i>anti </i>diol relationships. The future application of this methodology to the total synthesis of zwittermicin A is discussed in Chapter 4. Chapter 5 presents a preliminary study of high pressure conditions for enantioselective organocatalytic aldol reactions.

No description available.

The increased application of combinatorial synthesis as a valuable tool in the drug discovery process has led to the recent escalation in research into solid phase synthesis techniques. The use of multiple parallel solid phase synthesis has been described as an efficient method of lead compound optimisation. This method is explored for the generation of analogues of a substituted acrylic acid, identified from random screening, in order to increase receptor affinity. The optimised solution phase synthesis of the lead compound <I>via</I> a Horner-Wadsworth-Emmons reaction is reported. The synthetic route is then described for the solid phase synthesis of a simple analogue. Attempts have been made towards the multiple parallel solid phase syntheses of a range of substrates. Alternative methods for facile product purification as alternatives to covalent binding to a polymeric resin have recently been researched. The affinity of tetrabenzo [<I>a, c,g,i</I>]fluorene (Tbf) for charcoal has already been described as a useful method for the purification of peptides and proteins, and this property has been harnessed, for the first time, for application to general organic synthesis. An efficient solution-solid phase synthesis of the quinolone antibacterial, Ciprofloxacin, is described, in which charcoal was used to purify Tbf-bound intermediates in a <I>pseudo</I> solid phase purification step.

The cycloaddition reactions of nitrile oxides with jojoba oil have been investigated. The nitrile oxides were generated <i>in situ</i> by two principal routes: (i) by the base catalysed dehydration of nitromethyl compounds using tolylene-2,4-diisocyanate (the Mukaiyama method), and (ii) by thermolysis of nitro-acetate and -malonate esters (the Shimizu method). In order to faciliate the identification of modified jojoba products and establish optimum conditions for reaction with the unsaturation present in jojoba oil, the cycloaddition reactions of nitrile oxides with selected model alkenes were studied. Terminal alkenes afforded 5-substituted-2-isoxazolines regiospecifically in 21-68% yield, whereas mid-chain alkenes proved to be less reactive and gave a regioisomeric mixture of 4,5- disubstituted isoxazolines (19-69&37 yield). Cycloadditions to unsaturated lipids were also examined; methyl oleate and oleyl acetate both yielded stereospecifically a regioisomeric mixture of <i>cis-</i> isoxazolines when reacted with ethoxycarbonylnitrile oxide. The geometric isomer of methy oleate, methyl elaidate, afforded a mixture of <i>trans</i>-4,5-disubstituted isoxazolines. The ratio of regioisomers in all cases was shown to be 1:1 by the use of high field <SUP>13</SUP>C NMR, which detected small differences in chemical shifts of ring carbons. Proton NMR was also used to characterise lipid isoxazolines, assignment of their spectra being facilitated by comparison with those of model compounds. FAB mass spectrometry enabled the detection of parent ions for these isoxazolines. Application of the Shimizu method to ethyl nitroacetate provided a novel approach to formonitrile oxide; with model alkenes 3-unsubstituted isoxazolines were formed in 19-36% yields. In contrast, ethyl nitroacetate reacted with isocyanate and base to form <i>N</i>-phenyl-α-ethoxycarbonyl-α-nitroacetamide which was itself thermolysed to produce an amide nitrile oxide. Further exploitation of this approach using di-isocyanates lead to the generation of di-nitrile oxides. Cycloaddition reactions of the analogous azomethine oxide, <i>C,N</i>-diphenylnitrone, led to formation of the corresponding ring-saturated isoxazolidine moieties.

There is much interest in the synthesis of unnatural, enantiomerically pure amino acids, including dehydroamino acids and b,b-disubstituted amino acids. Described here is a method of synthesising dehydroamino acids <i>via</i> oxazolones derived from a modified version of the Erlenmeyer reaction. (Fig. 13661A) Rh-DuPHOS and Rh-BPE hydrogenation of these dehydroamino acids led to the synthesis of single products in high d.e. and e.e. Synthesis of such oxazolones on solid phase would provide a method for on-resin amino acid modification, allowing for greater flexibility in the combinatorial synthesis of peptides. Linkage <i>via</i> a silyl linker would allow mild and selective cleavage, while use of an acid functionalised linker would allow inverse (N(r)C) peptide synthesis. Attempts to synthesise I are described, along with a method of oxazolone formation which avoids the use of acid, and is therefore compatible with silyl linkers (Fig. 13661B).

Structure of bicyclic pseudo octapeptides containing a sequence arginine-glycine-aspartic acid (RGD peptides) was investigated by high-resolution NMR spectroscopy. The compounds, synthesized by General Electric Healthcare, are used as contract agents in imaging of cancer. Structures were generated using different protocols in vacuum or explicit solvent. The best results were obtained through the analysis of a series of NOESY spectra acquired using varying mixing times. Full relaxation matrix analysis in explicit solvent was performed on these data yielding a consistent set of structure. The polyethylene glycol moiety and the metal binding site did not show any signs of tertiary structure. The distance restraints for structure calculations were supplemented by dihedral restraints provided by the analysis of vicinal coupling constants. Towards this end, an extensive set of coupling constants related to dihedral angles of amino acids was obtained. Various experiments, including HMBC, HSQC and HETLOC, were evaluated yielding a basic set of experiments suitable for the measurement of coupling constants of peptides with natural abundance of isotopes. The structures of the studied peptides were found to be similar and to adopting a reverse γ-turn centered on the aspartic acid of the RGD motif. Despite the bicycling nature of these peptides the compounds showed signs of flexibility, which was more pronounced in H2O. Side chains of the compounds are flexible as implied from the analysis of the distributions of cl angles. Structures were determined in two solvents, H2O and DMSO. It was found that the water based structures are more extended, while the DMSO based structure are more compact. DMSO structure of one peptide was compared with a published NMR structure of a similar peptide and a good agreement was found for the common RGD loop. The water based structures are similar to a published X-ray structure of a RGD peptide in complex with αvβ3.

2-Aminoadenine or 2,6-diaminopurine (D), is an adenine analogue where three hydrogen bonds are formed in a base pair with thymine, instead of only two hydrogen bonds in the A.T. base pair. Hence the thermodynamic stability of duplexes containing D.T base pairs should be increased relative to those containing A.T. More subtle interactions affect the structural transitions of duplexes containing diaminopurine. Previous attempts at incorporating diaminopurine into DNA during routine oligonucleotide synthesis were hindered due to acid catalysed depurination (dependent on N<SUP>6</SUP>-amino protection) and very poor lability of the N<SUP>2</SUP>-amino protecting group during ammonia deprotection. An improved N<SUP>2</SUP>-amino and N<SUP>6</SUP>-amino protection strategy was developed for the synthesis of the diaminopurine monomer. This monomer was used successfully during routine machine synthesis to make diaminopurine-containing oligonucleotides. Derivatised solid support was synthesised to allow 3'-incorporation of diaminopurine. A novel synthetic route to the free diaminopurine nucleoside was also developed. The ability of nucleic acids to form very stable duplexes has many important applications in diagnostic and therapeutic molecular biology. The hybridisation of DNA probes or PCR primers to their targets may be increased, and hence the specificity; or allow shorter probes/primers to be employed. The same strategy may be applied to antisense therapy, or DNA sequencing primers. In order to predict the physical properties of such duplexes, the thermodynamic stability (UV melting) of duplexes containing diaminopurine was determined, using oligonucleotides with all combinations of nearest neighbours. This data revealed that when diaminopurine replaced adenine, stability of the duplex was increased, with the exception of the TD/AT nearest neighbour interaction. This showed that the additional hydrogen bond does not always increase stability - more complex interactions are responsible for overall duplex stability.