Amide III

In 3, the amino functional group is two methylene units removed from the ferrocene nucleus. It appears from the instantaneous and quantitative formation of h from 3 that this feature minimizes steric effects and also enables 3 to undergo the Schotten-Baumann reaction readily without the classical a-metallocenylcarbenium ion effects providing any constraints. The IR spectrum of showed the characteristic N-H stretch at 3320 cm" (s), the amide 1 (carbonyl) stretch at 1625 an - -(s), the amide II (N—H) stretch at 1540 cm (s), and the amide III band at 1310 cm 1(m). In addition, characteristic absorptions of the ferrocenyl group were evident at 1100 and 1000 cm l (indicating an unsubstituted cyclopentadienyl ring) and at 800 cm"l. 

Further evidence for these a-helix ROA band assignments in the extended amide III region comes from the ROA spectrum of poly-L-alanine dissolved in a mixture of chloroform (70%) and dichloracetic acid (30%), known to promote a-helix formation (Fasman, 1987), which shows strong positive ROA bands at 1305 and 1341 cm-1 (unpublished results), and of the cv-helix forming alanine-rich peptide AK21 (sequence Ac-AAKAAAAKAAAAKAAAAKAGY-NHg) in aqueous solution which shows strong positive ROA bands at 1309 and 1344 cm-1 (Blanch et al., 2000). 

IR differences key on frequencies of band maxima and band widths, whereas VCD varies in terms of band shapes associated with those IR transitions, thus being secondarily influenced by frequency and width as well. Two strong bands, the amide I and II, dominate the mid-IR, whereas the amide III is weak and mixed with local CaH deformation modes, spreading its small intensity over a broad region (Diem et al, 1992 Baello et al., 1997 Asher et al., 2001). 

A 4 g 3-indoleacetic acid (I) in ether. I reat with PCi5 as described elsewhere here to get 2.7 g of the chloride (II). Dissolve 2.7 g (II) in 40 ml ethyl acetate and add 2 ml piperidine (or equimolar amount other amine), 3.5 ml N-ethyl-piperidine in 40 ml ethyl acetate. Let stand three hours at room temperature and filter. Wash filtrate with IN HCI, 10% Na carbonate and evaporate in vacuum to get 1 g of the amide (III) (test for activity). Add 1 g (111) in 25 ml ether to 1.4 g lithium aluminum hydride in 50 ml ether stir four hours at room temperature and reflux one-half hour. Cool and carefully add water until no more bubbling. Add 3 ml 20% NaOH and dry. evaporate in vacuum to get 1 g piperidine analog of DMT... 

In addition, for solid samples or peptides in nonaqueous solvents, the amide II (primarily in-plane NH deformation mixed with C—N stretch, -1500-1530 cm-1) and the amide A (NH stretch, -3300 cm-1 but quite broad) bands are also useful added diagnostics of secondary structure 5,15-17 Due to their relatively broader profiles and complicated by their somewhat weaker intensities, the frequency shifts of these two bands with change in secondary structure are less dramatic than for the amide I yet for oriented samples their polarization properties remain useful 18 Additionally, the amide A and amide II bands are highly sensitive to deuteration effects. Thus, they can be diagnostic of the degree of exchange for a peptide and consequently act as a measure of protected or buried residues as compared to those fully exposed to solvent 9,19,20 Amide A measurements are not useful in aqueous solution due to overlap with very intense water transitions, but amide II measurements can usefully be measured under such conditions 5,19,20 The amide III (opposite-phase NH deformation plus C—N stretch combination) is very weak in the IR and is mixed with other local modes, but has nonetheless been the focus of a few protein-based studies 5,21-26 Finally, other amide modes (IV-VII) have been identified at lower frequencies, but have been the subject of relatively few studies in peptides 5-8,18,27,28 ... 

Both amide I and amide III bands are seen in Raman spectra of proteins.30 Lippert et al. devised the following method for estimating the fractions of a-helix, (3 sheet, and random coil conformations in proteins.31 The amide I Raman bands are recorded at 1632 and 1660 cm 1 in DzO (amide I ). The amide III band, which is weak in DzO, is measured at 1240 cm-1 in H20. The intensities of the three bands relative to the intensity of an internal standard (the 1448 cm 1 CH2... 

Other mixed vibration bands known as the amide III, IV and V bands have been identified in various regions of the spectrum but they are of limited diagnostic value. 

Fig. 14.3. (a) Bone section under polarized light, black line outlines where Raman images were acquired. Polarized Raman images of (b) phosphate V2/amide III, (c) phosphate Vi/amide I, and (d) carbonate/phosphate V2 band ratios at the interface between osteon and interstitial bone, (e and f) Three-dimensional view of phosphate Vi/amide I ratio for different polarization directions. Reprinted with permission from [1]... 

In their initial studies, Tfayli et al. [33] acquired spectra from an Episkin model. This model is comprised of human adult keratinocytes which produce stratified epidermis following a 13 h culture period. Raman spectra from this model were compared with normal human skin. Significant differences were noted, particularly in spectral features arising from the 850/830 tyr Fermi doublet (which is sensitive to the H-bonding state of the -OH group [34]) and in the protein amide III region. Usable spectra were acquired to a depth of 15-20 pm. 

Figure 1.5 (a) IR brightfield image of a human skin sample, (b) nucleic acid-amide III absorbance... 

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