Chain Precursor Research Articles

Application of Elasticity Models with Constraints on Chains and Junctions to Characterization of Randomly Crosslinked Networks

Abstract Characterization of randomly crosslinked networks requires a topological description of imperfect network structure and a molecular-elasticity theory, preferably with a minimum number of adjustable parameters, relating macroscopic stress and strain or swelling ratios to molecular deformation. It is shown that simple relationships exist between the molecular weight between consecutive junctions, the molecular weight of linear precursor chains, the cycle rank, and the number of junctions for networks having no defects other than chain ends. The idealized affine deformation model (in which the constraints on junction fluctuations are infinite) and the idealized phantom-chain model (in which the constraints are absent) are inadequate for network characterization. The more realistic Flory-Erman constrained junction model is applied to the description of cis-polyisoprene cured with dicumyl peroxide. Swelling equilibrium experiments are in agreement with stress-strain measurements. Analysis of these stress-strain data with the constrained-junction and constrained-chain models gives similar results. This validates the Flory hypothesis that it is generally sufficient to concentrate the effects of the constraints on the junctions. Nevertheless, the constrained chain model proposed by Erman and Monnerie has the advantage of depending on one parameter, KG, instead of two, κ and ζ, for the constrained junction model.

Preparative in vitro generation of lacto-series type 1 chain glycolipids catalyzed by β1 → 3-galactosyltransferase from human colonic adenocarcinoma Colo 205 cells

Lacto-series glycolipids, comprising two isomeric types distinguished as type 1 or 2 based upon the linkage of the terminal galactose of the chains, form the basis for a diversity of cell surface antigens expressed on cells. Experimentally, type 2 chain precursors are generally more abundant in tissues for extractive purposes to yield rather large quantities of material compared to the type 1 chain structures. Conditions have been defined for in vitro conversion of terminal Galβ1 → 4GlcNAc linkages of type 2 chain precursors to yield type 1 lacto-series chain based terminal Galβ1 → 3GlcNAc structures in 5- to 10-mg amounts or higher. The terminal galactose of underivatized type 2 chain structures is removed by hydrolysis with jack bean β-galactosidase followed by transfer of galactose in β1 → 3 linkage catalyzed by a β1 → 3-galactosyltransferase from human colonic adenocarcinoma Colo 205 cells which was first depleted of β1 → 4-galactosyl-transferase by chromatography on α-lactalbumin-Sepharose. Scaled-up reaction mixtures provided a final yield of product after isolation of about 90% from the immediate Lc 3Cer precursor in the 5-mg product range. The biosynthetic product was subjected to extensive chemical analysis by 1H NMR and mass spectrometric methods. These results indicated the presence of a high purity terminal Galβ1 → 3-linked product. The amount of material was sufficient for nondestructive characterization by 2-D NMR, with subsequent confirmation of structure by +FAB-MS and methylation analysis by GC-MS. The results indicate an effective means to rapidly generate lacto-series type 1 precursors in vitro as a superior alternative to direct tissue extractive procedures.

Immunoblot analysis of platelet glycoprotein IIb in patients with Glanzmann thrombasthenia in Israel.

Recent studies have indicated that severe ('type I') Glanzmann thrombasthenia is a heterogeneous hereditary disorder caused by quantitative and/or qualitative abnormalities of platelet membrane glycoproteins (GP) IIb and IIIa. Immunoblot analysis of sodium dodecyl sulphate (SDS)-solubilized platelets was carried out on controls and 18 patients (12 Iraqi-Jews, two Iranian Jews and four Arabs) employing three antibodies (one monoclonal and two polyclonal) directed at different sites on GPIIb. Nonreduced control platelet samples contained a major Mr approximately 140k immunoreactive protein that was split into an Mr approximately 120k (alpha) and an Mr approximately 25k (beta) band after reduction with mercaptoethanol. The nonreduced samples from all 18 patients tested had trace amounts of Mr approximately 140k band corresponding to normal GPIIb; the intensity of this band was estimated to be less than 1% of the normal amount. Unlike the control samples, however, this Mr approximately 140k band did not change electrophoretic mobility following reduction. Since GPIIb originates from a single chain precursor molecule of Mr approximately 140k that comprises both the alpha and beta chains, and which does not change mobility with reduction, our data suggest that the platelets of these patients contain small amounts of this precursor.

Histochemical analysis of the chemical structure of blood group-related carbohydrate chains in serous cells of human submandibular glands using lectin staining and glycosidase digestion.

Using lectin staining methods in combination with exo- and endo-glycosidase digestion procedures, we analyzed the chemical structure of different types of blood group-related substances in serous cells of formalin-fixed, paraffin-embedded human submandibular glands. Serous cells produced only H antigen; A and B antigens were not present, and the expression of H antigen is dependent on the secretor status of the tissue donor. Although reactivity with Ulex europaeus agglutinin I (UEA-I) was not markedly reduced by alpha-L-fucosidase digestion, an affinity for peanut agglutinin (PNA) was seen after fucosidase digestion in the cells from secretors. In those from nonsecretors, no PNA reactivity appeared after enzyme digestion. On the other hand, sialidase digestion elicited PNA reactivity in serous cells irrespective of the donor's secretor status. PNA reactivity observed after fucosidase or sialidase digestion was susceptible to endo-alpha-N-acetylgalactosaminidase (endo-GalNAc-dase) digestion. SBA reactivity in UEA-I-negative cells from secretors, or in cells from fetuses and newborn infants, was markedly reduced by beta-galactosidase digestion. After galactosidase digestion, reactivity with Griffonia simplicifolia agglutinin II (GSA-II) appeared in the corresponding cells. This GSA-II reactivity was almost completely eliminated by subsequent beta-N-acetylhexosaminidase digestion. Whereas PNA reactivity in these cells was not reduced by beta-galactosidase treatment, it was significantly diminished by endo-GalNAc-dase digestion. These results suggest that at least two kinds of precursor disaccharides are produced in submandibular serous cells, i.e., SBA-reactive D-galactose-(beta 1-3,4)-N-acetyl-D-glucosamine and PNA-reactive D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine (O-glycosidically linked Type 3 chain or T antigen). Final fucosylation and synthesis of these two types of precursor chain appear to be under the control of the secretor gene.

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis

Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.

The ABO, Lewis and related blood group antigens; a review of structure and biosynthesis.

Numerous studies have shown that the antigenic determinants of the ABO blood group system are closely related in biochemical terms to the antigenic determinants of the Hh, P, Lewis and Ii blood group systems. The blood group antigens of each of these systems are formed by the addition of specific sugars to an oligosaccharide precursor chain which may be bound through sphingosine to fatty acids (glycolipid) or through serine or threonine to a peptide chain (glycoproteins). The direct gene products of each of these blood group systems are the glycosyltransferase enzymes which catalyse the addition of the specific sugar thus conferring the specified blood group activity to the glycolipid or glycoprotein molecule. The antigenic determinants of the ABO and Lewis systems in addition to red cells also exist in the body secretions in soluble form when the relevant genes are expressed in the phenotype. The antigens expressed on both the red cells and in the secretions are determined by the interaction of Hh, Sese, ABO and Lele genes.

Histochemical demonstration of O-glycosidically linked, type 3 based ABH antigens in human pancreas using lectin staining and glycosidase digestion procedures

Histochemical analyses of the chemical structures of sugar sequences with or without blood group specificity were carried out by combined stepwise digestion of tissue sections with exo- and endoglycosidases and subsequent lectin stainings in formalin-fixed, paraffin-embedded human pancreas. In acinar cells from blood group A or AB secretor individuals, sequential digestion with alpha-N-acetylgalactosaminidase and alpha-L-fucosidase imparted reactivity with peanut agglutinin (PNA) in cells reactive with Dolichos biflorus agglutinin as well as those with Ulex europaeus agglutinin I(UEA-I). Simple fucosidase digestion imparted the PNA reactivity only in UEA-I reactive cells. Sequential digestion with alpha-galactosidase and fucosidase likewise liberated the PNA binding sites in Griffonia simplicifolia agglutinin I-B4 reactive cells from blood group B and AB secretors. Sialidase digestion liberated the PNA binding sites not only in acinar cells but also intercalated duct cells, islet cells of Langerhans and endothelial cells. The PNA reactivity obtained by these enzyme digestions was eliminted by endo-alpha-N-acetylgalactosaminidase (endo-GalNAcdase) digestion. Preexisting PNA affinity in acinar cells from non-secretors was also susceptible to endo-GalNAcdase treatment. Following the endo-GalNAcdase digestion, fucosidase or sialidase digestion recovered the PNA reactivity in acinar cells from nonsecretors. These results show that ABH determinants carried on O-glycosidically linked type 3 chain (D-galactose-(beta 1-3)-N-acetyl-D-galactosamine alpha 1-serine or threonine) are secreted in pancreatic acinar cells and suggest that product coded by the secretor gene is required for the complete conversion of type 3 precursor chains into H determinants.

Synthesis and assembly of a functionally active recombinant platelet-derived growth factor AB heterodimer.

A Chinese hamster ovary cell line that stably expresses transfected human platelet-derived growth factor (PDGF) A and B chain precursors was established. All three dimeric combinations of PDGF chains were produced by this cell line; their biosynthesis, assembly, and processing were followed by pulse-chase analyses. PDGF-AA, PDGF-AB, and PDGF-BB were processed to Mr values of about 30,000 and were accumulated in these forms in the medium. In addition, PDGF-BB was further processed to a 24-kDa component, which remained cell-associated. The major secreted component was PDGF-AB, which was purified and shown to have structural and functional characteristics indistinguishable from PDGF-AB purified from human platelets.

Thyroid hormone regulation of alpha-lactalbumin: differential glycosylation and messenger ribonucleic acid synthesis in mouse mammary glands.

Mouse mammary tissue, when cultured in the presence of insulin, corticoids, PRL, and physiological levels of T3, shows increased synthesis and secretion of alpha-lactalbumin. Tissue cultured in the presence of insulin, hydrocortisone, PRL, and T3 synthesizes two distinct forms of alpha-lactalbumin, but secretes only one form. Tissue cultured in the absence of T3 synthesizes and secretes only one form. To address the question of whether these two electrophoretically distinct forms arose by differential glycosylation of the same polypeptide or by synthesis of two different polypeptide precursor chains, mammary tissue was cultured in the presence of insulin, corticoids, and PRL with or without T3, and the mRNA and alpha-lactalbumins were isolated. Northern blot analyses indicated that mammary gland tissue cultured in the presence of T3 contained 2.46 times more alpha-lactalbumin mRNA than tissue cultured only in the presence of insulin, hydrocortisone, and PRL. This enhanced mRNA level was confirmed by in vitro translation experiments where tissue cultured in the presence of insulin, hydrocortisone, PRL, and T3 produced mRNA that resulted in 2.1 times as much radiolabeled alpha-lactalbumin as tissue cultured in the absence of T3. Sodium dodecyl sulfate-polyacrylamide gel analysis of the in vitro translation products revealed only one band, suggesting the presence of only one message. Endoglycosidase digestion of the two forms of alpha-lactalbumin produced in the presence of T3 resolved them into a single band on sodium dodecyl sulfate-polyacrylamide gels. Thus, the electrophoretic differences between the two forms synthesized in the presence of T3 appear to be due to differential N-linked glycosylation of the same polypeptide chain and not to synthesis of two different polypeptide precursor chains.

Heterogeneity in the molecular defect leading to the leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is a recessive autosomal disease characterized by life-threatening recurrent bacterial infections, by defective functions of leukocytes and by deficient membrane expression of leukocyte adhesion glycoproteins. These proteins, LFA-1, Mac-1 and p150,95, are alpha 1/beta 1 heterodimers composed of different alpha chains and of a common beta chain. Patients with the severe phenotype of the disease completely lack the three glycoproteins on cell surface. Previous studies showed a conserved synthesis of the LFA-1 alpha chain precursor in cytosol of patients' cells and an inconstant presence of the beta chain precursor. When present, precursors are in free form and not associated to alpha/beta complexes in the cells of patients with the severe phenotype. The availability of the beta chain cDNA probe allowed us to examine the beta chain gene expression in the lymphoblastoid cell lines of 4 patients. On the basis of the results obtained both at protein and RNA levels we can distinguish 3 types of mutations characterized by (a) barely detectable beta subunit messenger RNA and undetectable beta precursor, (b) decreased level of beta subunit mRNA and undetectable beta precursor and (c) normal beta subunit mRNA level and detectable beta precursor of normal size.

49 ABNORMAL SPECIES OF SUCRASE-ISOMALTASE (S-I) ARE ASSOCIATED WITH VARIATIONS OF LUMINAL PANCREATIC ENZYME CONTENT IN RAT INTESTINE

The S-I complex is synthesized as a single chain precursor P. Once it reaches the brush-border surface, P is split by pancreatic proteases into S and I. Recently, we have documented in vitro this cleavage. Among the proteases tested: trypsin (T), elastase (E) and chymotrypsin (C), T was the only one involved in producing normal S-I. But, depending on the protease and its concentration, abnormal forms of S and I were observed. Thus, to document those observations, modifications of pancreatic enzyme balances in intestinal lumen were induced in rats either physiologically (i.e. by diet changes), either as a result of surgical transfert of pancreatic ducts. By electrophoretic analysis of S-I immunoprecipitates: 1) when T was increased over E and C, an abnormal form of I (I'; mw<I) was found S was inchanged; 2) when E was increased over T or C, two species, E1 and E2, larger than normal S and I, were only observed; 3) when T, E and C, all together were hightly increased, I' and a new specie, S (mw<<S) were the only forms characterized. By both S and I enzyme determinations: 1) all complexes, S-I', E1-E2 and S'-I', indicated a significant decrease in the S/I activity ratio; 2) only the S' species showed an increase of the normal S Km value (24.8 v.s 19.1, p 0.001).

Expression of proopiomelanocortin-related peptides in human follicular fluid.

In order to evaluate the expression of the opioid precursor proopiomelanocortin (POMC) in the ovarian follicle, we measured 6 of its main end-products in 23 follicular fluids. We coupled high performance liquid chromatography (HPLC) to specific radioimmunoassays. Seven follicles were immature (diameter less than 9 mm), 10 were obtained from superovulated patients during an in vitro fertilization-embryo transfer program (greater than 22 mm) and six were persistent follicles, collected during the luteal phase [15-31 mm, luteinized unruptured follicles (LUF)]. Follicular fluids were extracted by mean of Sep-pak cartridges and then purified by HPLC with a reverse-phase C-18 column eluted in a linear gradient with acetonitrile/0.01 M hydrochloric acid (from 18:82 to 40:60). Fractions were tested with specific antisera for ACTH (1-39), alpha-MSH, beta-lipotropin (beta-LPH), beta-endorphin (beta-EP) and gamma-endorphin (gamma-EP) immunoreactivities. No presence of beta-LPH, beta-EP and ACTH was confirmed, while gamma-EP, alpha-MSH and des-alpha-MSH were detected for the first time in follicular fluid. In every class of follicles shorter chain peptides predominate over their longer chain precursor. Immature follicles are characterized by the highest amounts of gamma-EP, ACTH, alpha-MSH and des-alpha-MSH if compared to superovulated and LUF. On the contrary, beta-EP amount was highest after superovulation. Apart from this finding, peptide levels in superovulated patients and LUF are similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of blood-group-related carbohydrate antigens by trophoblast subpopulations.

A panel of monoclonal antibodies directed at fucosylated and sialylated carbohydrates on the Type 1 and Type 2 blood group precursor chains was used in an immunohistological study of trophoblast subpopulations. Formalin-fixed paraffin-embedded sections of normal trophoblast throughout pregnancy, hydatidiform moles and gestational choriocarcinoma were examined. Sialyl-Lex was localized in extravillous trophoblast populations in normal and molar pregnancies and choriocarcinoma tumour cells. After neuraminidase treatment, Lex was identified in similar cell populations. Although Lea was identified in syncytial trophoblast cells at the maternofetal interface, no staining was seen for sialyl-Lea. Villous trophoblast did not stain with any of the monoclonal antibodies used. Our results underline the divergent differentiation of trophoblast into villous and invasive extravillous subpopulations. The analogy with tumour cells is discussed.

An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains

It is generally accepted that an aphidicolin-sensitive DNA polymerase elongates the eucaryotic RNA primer (iRNA) into a mature Okazaki piece reaching ca. 200 nucleotides. Yet, as shown here, nascent DNA chains below 40 nucleotides accumulated in simian virus 40 (SV40) DNA replicating in isolated nuclei in the presence of aphidicolin. These products resembled precursors of longer Okazaki pieces synthesized in the absence of aphidicolin (termed here DNA primers) in size distribution, lagging-replication-fork polarity, and content of iRNA. Within the isolated SV40 replicative intermediate, DNA primers could be extended in a reaction catalyzed by the Escherichia coli DNA polymerase I large fragment. This increased their length by an average of 21 deoxyribonucleotide residues, indicating that single-stranded gaps of corresponding length existed 3' to the DNA primers. Incubation with T4 DNA ligase converted most of the extended DNA primers into products resembling long Okazaki pieces. These data led us to propose that the synthesis of an SV40 Okazaki piece could be itself discontinuous and could comprise the following steps: (i) iRNA synthesis by DNA primase, (ii) iRNA extension into a DNA primer by an aphidicolin-resistant activity associated with DNA primase-DNA polymerase alpha, (iii) removal of iRNA moieties between adjacent DNA primers, (iv) "gap filling" between DNA primers by the aphidicolin-sensitive DNA polymerase alpha, and (v) ligation of DNA primer units onto a growing Okazaki piece. Eventually, a mature Okazaki piece is ligated onto a longer nascent DNA chain.

ChemInform Abstract: Construction of the Alkyl Side Chain of 11‐Deoxyprostaglandins Using the Aldol Reaction of Substituted Cyclopentane Carbaldehydes with Heptan‐2‐one.

The aldol reaction of the substituted cyclopentane carbaldehydes 1 with heptan-2-one was demonstrated to offer an effective alternative to the carbonyl olefinations generally used for the build-up of the (E)-3-oxooct-1-enyl side chain of 11-deoxyprostaglandins. In this case dimethyl 2-oxoheptylphosphonate can be substituted advantageously by the cheaper heptan-2-one as side chain precursor.

Keratinocyte Urokinase-Type Plasminogen Activator is Secreted as a Single Chain Precursor

Urokinase-type plasminogen activator (uPA) is produced and secreted by cultured human keratinocytes as a single chain precursor. UPA in keratinocyte conditioned medium is not susceptible to inhibition with diisopropylfluorophosphate (DFP), and it has an apparent molecular weight of 55 kD under both reducing and nonreducing conditions. Cleavage of keratinocyte uPA by plasmin results in the formation of a 96 kD complex comprised of activated uPA and PA inhibitor 2. PA extracted from normal human epidermis is only partially inhibited by DFP, suggesting that precursor uPA is also present in vivo. The synthesis of uPA as a precursor with reduced enzymatic activity as well as decreased affinity for inhibitors is likely to be a mechanism by which normal epidermis regulates plasminogen activation in vivo.

Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells

The insulin receptor is an integral transmembrane glycoprotein comprised of two alpha-(approximately 135 kDa) and two beta-(approximately 95 kDa) subunits, which is synthesized as a single polypeptide chain precursor (alpha beta). The primary sequence of the human insulin receptor (hIR) protein, deduced from the nucleotide sequence of cloned human placental mRNAs, predicts two large domains (929 and 403 residues) on either side of a single membrane spanning domain (23 residues); each of these major domains has a distinct function (insulin binding and protein/tyrosine kinase activity, respectively). To experimentally test this deduced topology, and to explore the potential for independent domain function by the hIR extracellular domain, we have constructed an expression plasmid encoding an hIR deletion mutant which is truncated 8 residues from the beginning of the predicted transmembrane domain (i.e., 921 residues). This domain of the hIR is in fact processed into alpha- and truncated beta-subunits and secreted with high efficiency from transfected CHO cell lines which express this mutant hIR, and the protein accumulates as an (alpha beta)2 dimer in the medium. This molecule is recognized by a battery of 13 monoclonal antibodies to epitopes on the IR extracellular domain, four of which block insulin binding and two of which require the native conformation of the IR for recognition. Further, this domain binds insulin with an apparent dissociation constant comparable to that of the wild-type hIR. However, the secreted dimer displays a linear Scatchard plot, while that of the wild-type membrane-associated hIR is curvilinear.(ABSTRACT TRUNCATED AT 250 WORDS)

Combinatorial calculations of topological defects in tetrafunctional polymer networks

The structure of real polymer networks admits many kinds of topological defects related to the way in which polymer chains are connected to crosslinks. The fractions of crosslink types have been derived from combinatorial considerations concerning the probabilities of various combinations of “structural elements” of crosslinks. 34 fractions of different crosslinks, possible in tetrafunctional systems, have been obtained as explicit functions of concentrations of seven structural elements — free-end chains, single-, double-, triple-, and quadruple-connected chains, loops, and unsaturated (void) functionalities. It was shown that different distributions (and different properties) result from end-to-end linking of precursor chains vs. vulcanization of long chains by bridging along their contours.

Tumor-associated blood group antigen expressions and immunoglobulins associated with tumors.

As outlined in Figures 1 and 2, the biosynthetic pathways for the expression of the A, B and H, and the Lewis determinant carbohydrate sequence structures, as well as sialylated structures, involves both type 1 and type 2 precursor chains (which may be present as glycolipids and N- or O-linked glycoproteins), and many glycosyltransferases. For tumor cells, there appears to be increased expressions of fucosyl- and sialyltransferases yielding such structures as the Le(x), sialyl-Le(a), and many other similar determinants, which are not found on the normal cell progenitor of the tumor. The types of structures expressed on tumor cells is dependent on the particular fucosyl-, sialyl- and other glycosyltransferase genes activated in the transformation and tumor progression events, the availability of the substrates for the glycosyltransferases (both the precursor sequences and the nucleotide-sugar substrates) which is partly dependent on metabolites available to the tumor mass, and on the genotype of the individual regarding particular glycosyltransferases. Both the loss of A, B and/or H blood group antigen expressions of tumor cells and the relative expressions of the Lewis and sialylated-oligosaccharide determinants may be a consequence of the competing biosynthetic pathways and the glycosyltransferases for common substrate sequences, as well as due to the loss of particular glycosyltransferases concomitant with transformation. All of these factors probably account for the variable expressions of the complex of carbohydrate sequence determinants when comparing tumor sections of different individuals as well as the heterogeneity of expression of particular determinants within a single tumor tissue section. As described above, the A, B and/or H determinants, and the precursor sequences, are also expressed to differing extents on epithelial cells depending on the tissue type and cellular location in the tissue. Thus, the differentiation state of the particular epithelial cell also determines the quantity and types of carbohydrate sequences expressed. However, because of the complex nature of the competing biosynthetic pathways for the carbohydrate sequences of glycolipids and glycoproteins, and the relative activations of fucosyl- and sialyltransferases of tumor cells, it would seem that simple deductions as to the state of differentiation of particular tumors with A, B, H and precursor sequence expressions is not warranted.(ABSTRACT TRUNCATED AT 400 WORDS)

Spin delocalisation in heterocyclic captodative radicals1,2

AbstractThe open chain and heterocyclic precursors of 12 captodative radicals 1–4 were prepared in order to perform their ESR spectroscopic evaluation. Each of them contains a ketone as a captor substituent and an amino, a thioether or an ether substituent as a dative group. Spectral comparison shows the influence of geometry since 5‐membered cd‐radicals with thio‐groups are more delocalised than their 6‐membered or open chain analogues. Interestingly, when cyclic conjugation becomes possible because of an additional double bond such as in 4b, no marked difference of spin delocalisation is observed in comparison with dihydroanalogue 2b. As the dative substituent changes from CH3N‐ to S‐ and to O‐ (in 2b‐2d) the delocalisation decreases and the participation of the corresponding carbonyl group increases.