Colocalized

CGRP has a wide distribution in the nervous system (19) and was the first peptide to be localized to motoneurons (124). It is also found in primary sensory neurons where it is colocalized with substance P (125). CGRP is derived from a precursor stmcturaHy related to the calcitonin precursor. The latter precursor produces two products, calcitonin itself and katacalcin, while the CGRP precursor produces one copy of CGRP (123). Like other peptides, CGRP is cleaved from its precursor by tryptic breakdown between double basic amino acid residues. 

Neurotensin. Neurotensia [39379-15-2] (NT),j )-Glu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Try-Ile-Leu-OH, is a tridecapeptide that is cleaved from the ribosomaHy synthesized precursor, proneurotensia. NT is distributed through the peripheral and central nervous systems as well as ia certain other cell types (3,67). NT is colocalized with catecholamines ia some neurons. 

Two-dimensional C//correlations such as C//COSY or HC HMQC and HSQC provide the Jqh connectivities, and thereby apply only to those C atoms which are linked to H and not to non-protonated C atoms. Modifications of these techniques, also applicable to quaternary C atoms, are those which are adjusted to the smaller Jqh and Jqh couplings (2-25 Hz, Tables 2.8 and 2.9) Experiments that probe these couplings include the CH COLOC (correlation via long range couplings) with carbon-13 detection (Fig. 2.16) and HC HMBC (heteronuclear multiple bond coherence) with the much more sensitive proton detection (Fig. 2.17)... 

Figure 2.16. CH COLOC experiment of a-pinene [ CD3)2CO, 10% v/v, 25 °C, 50 MHz for C, 200 MHz for H, 256 scans and experiments], (a) stacked piot of the section between 5c = 20.9 and 47.2 C) and Sh = 0.85 and 2.34 ( H) (b) contour piot of (a). One-dimensionai C and H NMR spectra for this section are shown aligned with the abscissa and ordinate of the contour piot. Jch correiation signals which are aiready known from the CH COSY study (Fig. 2.14) and have not been suppressed, are indicated by circies...

These two-dimensional CH shift correlations indicate CH relationships through two and more bonds (predominantly Jch and Jch connectivities) in addition to more or less suppressed Jch relationships which are in any case established from the CH COSY contour diagram. Format and analysis of the CH COLOC or HMBC plots correspond to those of a C//COSY or HSQC experiment, as is shown for a-pinene (1) in Figs. 2.14 - 2.17. 

Figure 2.17. HC HMBC experiment of a-pinene [ CDCb, 5 % v/v, 25 °C, 125 MHz for C, 500 MHz for H, 16 scans, 256 experiments, contour plot]. This experiment gives the same information as Fig. 2.16 within 24 min instead of 8 hrs required for the CH-COLOC in Fig. 2.16 due to higher sensitivity because of proton detection and stronger magnetic field. Deviations of proton shifts from those in Fig. 2.16 arise from the change of the solvent. The methylene protons collapsing in Fig. 2.16 at Sh =2.19 (200 MHz) display in this experiment an AB system with Sa = 2.17 and Sb = 2.21 (500 MHz)...

CH or HC COSY (HMQC) CH bonds CH COLOC or HC HMBC. Jch and Jch relationships between carbon and protons... 

What compound CigHigO can be identified from the CH COSY and CH COLOC plots a with the //NMR speetra shown above and from the C NMR speetra b ... 

Conditions CDCI3, 25 °C, 200 MHz ( //), 50 MHz ( C). (a) CH COSY (shaded contours) and CH COLOC diagrams (unshaded contours) in one diagram with enlarged section (b), and with expanded methoxy quartets (c) (d) sections of C NMR spectra, each with //broadband decoupled spectrum below and NOE enhanced coupled spectrum (gated decoupling) above. 

Knowing the substitution pattern of both benzene rings A and B, one can deduce the molecular structure from the CH connectivities of the CH COSY and CH COLOC plots. The interpretation of both experiments leads firstly to the correlation Table 41.1. 

Further interpretation of the CH COSY / CH COLOC plots allows additional assignments to be made for fragments B and C of aflatoxin B,. 

Since fragment A was clearly assigned with the help of HH coupling constants, all of the C atoms not included in A, which, according to the CH COLOC plot, are two or three bonds apart from the equivalent protons at Sh = 6.72 (Table 43.1), belong to the benzene ring B. 

Two- and three-bond CH eonneetivities of the methyl protons deteeted in the CH COLOC diagram identify the partial structures E and F. Only one bond from 5c = 72.6 to 5c = 44.5 could not be directly identified because of the absenee of the eorresponding eross signal (<5// =1.17 with 5c = 44.5). Nevertheless, partial strueture E is reasonable, assuming the six-membered ring to be retained during metabolism. 

Table 45.1. Partial structures from the CH COSY and CH COLOC plots (the protons are given in Italic numerals, C atoms separated by a single bond are given in bold numerals and C atoms separated by two or three bonds...

The given structure A is confirmed by interpretation of the CH COSY and CH COLOC diagrams. All of the essential bonds of the deealin stmcture are derived from the correlation signals of the methyl protons. In this, the DEPT subspeetra differentiate between the tetrahedral C atoms which... 

The assignment of the umbelliferone residue in A likewise follows from interpretation of the Jqh and relationships in the CH COSY and CH COLOC plots following Table 48.1. The C signals at 5c = 112.9 and 113.1 ean be distinguished with the help of the eoupled NMR spee-... 

Starting from the five CC double bonds, three rings and a 3-methylfuran structural fragment, analysis of the CH COSY and CH COLOC diagrams leads to Table 49.1 and the identification of fragments B-J. 

At this stage of the interpretation, the CH correlations across two or tliree bonds CH COLOC plot) provide more detailed information. The H shifts given in the CH COLOC diagram, showing correlation maxima with the C atoms at a distance of two to tliree bonds from a particular proton, lead to the recognition of eight additional structural fragments B-I (Table 50.2). 

Table 50.2. Partial structures from the CH COLOC plot. Each partial structure B-I is deduced from the two- or three-bond couplings Jew for the H atoms of B-I (with italic values)...

The CH COLOC diagram shows correlation signals for the methyl protons which are particularly clear (Table 51.3). Interpretation of these completes the assignments shown in formula D by reference to those CH multiplicities which have already been established (Table 51.1). 

Table 51.3. Interpretation of the CH COLOC diagram (methyl connectivities) using the CH multiplets derived...

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