Analysis of Sequence Distribution

Sequence distribution studies on several types of rubber by 13C-NMR technique have been reported. Some of the more recent reports include silicone rubbers [28-30], SBR [31], acrylonitrile-butadiene rubber (NBR) [32,33], polyurethane [34,35], polyepichlorohydrin [36], ethylene-norbonene [37] and ethylene-propylene rubber [4, 16, 25, 38-44]. The NMR studies on EPDM have been carried out extensively, because it is one of the important parameters, which control the physical properties of the elastomer. For example, ethylene sequence can influence the crystallisation kinetic and melting behaviour of the rubber [38]. 

Synthetic cis-1,4 polyisoprene has the structural feature similar to PB. As to the NMR analysis of sequence distribution, however, little work has been done after 1978. Detailed assignment may be possible by using modern NMR techniques and magnetic field higher than 100 MHz for 13C-NMR. 

The microstructure of polychloroprene, polymerised at +12 to +70 °C, was analysed using H- and 13C-NMR [32], Signal assignments were made for head-and-tail arrangements of trans-1,4 units, which was the major component of polychloroprene, and for other isomeric units. Polymerisation at high temperature resulted in a slight increase in head-to-head and tail-to-tail linkage of trans-1,4 units as well as the increase in cis-1,4 units. 

The combination of lanthanide shift reagents and 2D NMR techniques (COSY and NOESY) was applied to the H- and 13C-NMR assignments of ethylene-vinyl acetate 

Chemical Shift (8) Sequence Carbon Type Chemical Shift (8) Sequence Carbon Type 

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Carbon 13 NMR has proved to be an excellent technique for analysis of sequence distributions and comonomer contents in ethylene-prq)ylene copolymer. These analyses are particularly straightforward if one of the monomer units is present at a level of 94% or greater because the other monomer will then occur primarily as an isolated unit. 

As a technique for the analysis of sequence distribution in copolymers, high-resolution NMR spectroscopy is particularly useful when the spectral resolution is sufficient to resolve the resonances of the specific sequences. A number of copolymer structural problems can be elucidated by using NMR spectroscopy. The composition of the copolymer can be quantitatively determined. The detection of compositional dyads can be used to determine the distribution of composition, that is, whether the sample is a mixture of homopolymers, a block copolymer, an alternating copolymer, or a random copolymer (see Chapter 1). If resonances are resolved due of the triad sequences of the copolymer, sequence distributions can be determined, and the mechanism of the copolymerization can be tested in terms of Bemoullian, first-order Markov, second-order Markov, or non-Markovian statistics. In rare circumstances, the tactic nature of the copolymer can be determined if distinguishable syndio- and isotactic n-ad resonances are resolved. Such an analysis has been carried out for copolymers of methyl methacrylate-methacrylic acid, for which the a-CHs resonances of all 20 triads have been assigned and have been used to determine the cotacticity of the copolymer [22]. 

Staubli et al. (1991) offer an in depth analysis of the effects of sequence distribution on the Tg of poly(anhydride-co-imide)s and discuss the experimental results with respect to several applicable theoretical models... 

Urinary proteins were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), and a 70-kDa protein was identified as the major component of cat urine (Fig. 4.1 A). Comparative analysis of urinary proteins in several other mammals such as humans, mice, dogs, and cattle did not detect a 70-kDa protein. Therefore, the 70-kDa protein was purified from cat urine and characterized by biochemical methods (Miyazaki, Kamiie, Soeta, Taira and Yamashita 2003). Analysis of tissue distribution indicated that the 70-kDa protein is expressed in the kidney in a tissue-specific manner and secreted from the proximal straight tubular cells of the kidney into the urine (Fig. 4.IB). A full-length cDNA for a 70-kDa protein was cloned from a cat kidney cDNA library. The cDNA clone encoded a polypeptide of 545 amino acid residues. The deduced amino acid sequence shared 47% identity with cat carboxylesterase (CES, EC 3.1.1.1), and contained both the CES family protein motif (EDCLY) and a conserved active site motif (GESAG) associated with... 

Valuable reviews and books of structural analysis of elastomers have been published by several authors [1-6]. Some of these reviews provide excellent explanation on the basic theory of sequence distribution of copolymer and NMR techniques applicable to elastomers. Typical high-resolution 3H- and 13C-NMR spectra of various vulcanisates and raw rubbers are depicted in a book written by Kelm [6]. The assignments and references shown for each rubber are very useful for structural studies of elastomers. In view of recent progress in the hardware and software of NMR, this chapter describes some of the more recent applications of high-resolution NMR to the structural characterisation of elastomers, after a brief description on the fundamental structural features of elastomers. 

Now we move on to consider the analysis of copolymers. There are usually two things we would like to know. First, the composition of the copolymer and, second, some measure of sequence distributions. Again, in the early years, before the advent of commercial NMR instruments, infrared spectroscopy was the most widely used tool. The problem with the technique is that it requires that the spectrum contain bands that can be unambiguously assigned to specific functional groups, as in the (transmission) spectrum of an acrylonitrile/methyl methacrylate copolymer shown in Figure 7-43 (you can tell this is a really old spectrum, not only because it is plotted in transmission, but also because the frequency scale is in microns). 

The potential functions used to evaluate or score a sequence in a structure are usually empirical potentials of mean force estimated from statistical analysis of the distribution of residue-residue distances in the PDB. The potentials are less detailed than full atomic potentials, often treating each residue as a single interaction site. Solvation is the key aspect that these potentials try to capture. Many different potentials have been developed, and new variants continue to be tested. - Most potentials appear to be comparable. 

Preliminary studies towards the development of efficient spectra modelling tools have been pubhshed. These include, for example, the analysis of the distribution of peptide masses generated by in silica digestion tools from protein sequence databases [182, 154] and the influence of a sequence on spectra intensity [143]. 

Structural Analysis.—Two developments in the theory of sequenee statistics have been reported. Bauer has described a numerical solution of sequence distributions for a reaction that modifies the original units, e.g., hydrogenation of a double bond. In the model, it is assumed that the rate constant depends on the structure of the adjacent units, and it is shown how the rate constants can be obtained from the triad proportions as a function of time. Cais et ol. have analysed the distribution... 

Johnston [104, 105] studied the effects of sequence distribution on the 2 of alkyl methacrylate-vinyl chloride and a-methylstyrene-acrylonitrile copolymers by differential scanning calorimetry, differential thermal analysis, and thermomechanical analysis. 

An effective understanding of copolymerization chemistry can only be realized through the analysis of the products of the reactions. There have been many accounts of the applications of n.m.r. spectroscopy to polymers and the evaluation of sequence distributions in copolymers figures highly in some of the reviews. " A computer simulation of the C n.m.r. spectrum has been described. Other techniques which have received recent attention are excimer fluorescence spectroscopy for alternating copolymers, mass spectroscopy for ethylene-propylene oxide copolymers, and pyrolysis g.l.c. > A review of analytical techniques has been made by Fujiwara, Mori, Nishioka, and Takeuchi. In a complementary series of articles infi red and Raman spectroscopy have been reviewed by Tanaka C n.m.r. of branched copolyma by Fujiwara and the particular problems of solid and liquid polymer aiuilysis by Tsuge and Mukoyama, respectively. 

Analysis of tlie global statistics of protein sequences has recently allowed light to be shed on anotlier puzzle, tliat of tlie origin of extant sequences [170]. One proposition is tliat proteins evolved from random amino acid chains, which predict tliat tlieir length distribution is a combination of the exponentially distributed random variable giving tlie intervals between start and stop codons, and tlie probability tliat a given sequence can fold up to fonii a compact... 

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