Catching elusive glycosyl cations in a condensed phase with HF/SbF5 superacid.

Glycosyl cations are universally accepted key ionic intermediates in the mechanism of glycosylation, the reaction that covalently links carbohydrates to other molecules. These ions have remained hypothetical species so far because of their extremely short life in organic media as a consequence of their very high reactivity. Here, we report the use of liquid hydrofluoric acid-antimony pentafluoride (HF/SbF5) superacid to generate and stabilize the glycosyl cations derived from peracetylated 2-deoxy and 2-bromoglucopyranose in a condensed phase. Their persistence in this superacid medium allows their three-dimensional structure to be studied by NMR, aided by complementary computations. Their deuteration further confirms the impact of the structure of the glycosyl cation on the stereochemical outcome of its trapping.

Beta-1,2-linked oligomannosides inhibit Candida albicans binding to murine macrophage.

Interaction of Candida albicans with cells of the macrophage lineage was examined by using heat-killed (HK) and live yeast cells. Laminarin, an analogue of the cell wall beta-glucans, strongly inhibited HK yeasts adherence to J774 cell line but had no effect on live yeast binding. Phosphopeptidomannan (PPM) from Saccharomyces cerevisiae had a limited effect on the binding of both HK and live yeasts but significant inhibition was achieved by the use of C. albicans PPM. The role of beta-1,2-oligomannosides was examined with regard to their exclusive presence within C. albicans PPM. PPM acid labile beta-1,2-oligomannosides or a synthetic beta-1,2-mannotetraose, inhibited yeasts binding in a manner comparable to the original PPM. These latter results were confirmed by using mouse peritoneal macrophages, thus suggesting a general role for beta-1,2-oligomannosides in the adherence of the yeast to the macrophage membrane.

The role of organic synthesis in glycoconjugate research: selected examples.

Chemical synthesis of various oligosaccharides allowed the determination of structural requirements for specific interaction between heparin and anti-thrombin III. The synthesis of a sialylated tetrasaccharide, subunit of the human blood group Cad determinant, is also reported.

N-p-methoxybenzylidene derivatives of 2-amino-2-deoxy-D-glucose as glycosyl donors: a reinvestigation.

6-O-Acetyl-3,4-di-O-benzyl-2-deoxy-2-p-methoxybenzylideneamino-D- glucopyranosyl chloride, 3,4,6-tri-O-acetyl-2-deoxy-2-p-methoxybenzylideneamino-alpha-D-glu copyranosyl bromide, 3,4,6-tri-O-acetyl-2-deoxy-2-p-methoxybenzylideneamino-alpha- and -beta-D- glucopyranosyl trichloroacetimidate, and 3,4,6-tri-O-acetyl-2-deoxy-2-p-nitrobenzylideneamino-alpha-D-gluco pyranosyl bromide have been synthesised, and their behaviour as glycosylation agents with various soluble promoters has been investigated. The results obtained question the accepted non-participating character of the N-p-methoxybenzylideneamino group.

[Synthesis of 2-acetamido-2-deoxy-6-0-(alpha-D-glucopyranosyl)-alpha-D-galactopyranose and its p-aminophenyl-alpha-glycoside]. / Synthèse du 2-acétamido-2-désoxy-6-O-(alpha-D-glucopyranosyl)-alpha-D-galactopyranose et de son p-aminophenyl-alpha-glycoside

Benzyl 2-acetamido-3, 4-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside was condensed with 2, 3, 4-tri-O-benzyl-6-O-p-nitrobenzoyl-alpha-D-glucopyranosyl bromide or with 2, 3, 4-tri-O-benzyl-6-O-p-methoxybenzoyl-alpha-D-glucopyranosyl bromide in benzene at 50 degrees in the presence of pyridine, to give benzyl 2-acetamido-3, 4-di-O-acetyl-2-deoxy-6-O-[2, 3, 4-tri-O-benzyl-6-O-p-nitro(or methoxy)benzoyl]-alpha-D galactopyranoside in excellent yield. The title disaccharide was obtained in crystalline form after deacylation and catalytic hydrogenation. It proved identical with a disaccharide isolated from Salmonella johannesburg 5.58 (40) converted by phage phi 1 (40). In order to bind this disaccharide covalently onto various proteins, p-aminophenyl 2-acetamido-2-deoxy-6-O-(alpha-D-glucopyranosyl)-alpha-D-galactopyranoside has been obtained in an analogous way, starting from p-nitrophenyl 2-acetamido-3, 4-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside.

Streptococcus pneumoniae type XIV polysaccharide: synthesis of a repeating branched tetrasaccharide with dioxa-type spacer-arms.

beta-Glycosides of 2-acetamido-2-deoxy-D-glucopyranose were synthesised, using either 7-methoxycarbonyl-3,6-dioxa-1-heptanol or 8-azido-3,6-dioxa-1-octanol. Selective beta-lactosylation of 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside with hepta-O-acetyl-lactosyl-trichloroacetimidate, followed by beta-galactosylation of the secondary hydroxyl group with O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl)trichloroacetimida te, catalytic hydrogenolysis, and O-deacetylation, gave 7-methoxycarbonyl-3,6-dioxaheptyl 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-6-O-(4-O-beta-D- galactopyranosyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside. Selective beta-lactosylation of 8-azido-3,6-dioxaoctyl 2-acetamido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside with hepta-O-acetyl-lactosyl bromide in the presence of silver triflate, followed by condensation with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide in the presence of silver triflate, catalytic hydrogenolysis, and O-deacetylation, gave 8-azido-3,6-dioxaoctyl 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-6-O-(4-O-beta-D- galactopyranosyl-beta-D-glucopyranosyl)-beta-D-glucopyranoside.

[Synthesis of 2-acetamido-2-deoxy-4-O-alpha-L-fucopyranosyl-alpha-D-glucopyranose]. / Synthèse du 2-acétamido-2-désoxy-4-O-alpha-L-fucopyranosyl-alpha-D-glucopyranose

Condensation of 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide with benzyl 2-acetamido-3,6-di-O-benzyl-alpha-D-glucopyranoside in dichloromethane-N,N-dimethylformamide, in the presence of tetraethylammonium bromide, diisopropylethylamine, and molecular sieve (halide ion-catalyzed reaction), gave benzyl 2-acetamido-3,6-di-O-benzyl-2 deoxy-4-O-(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)-alpha-D-glucopyranoside in crystalline form in 82% yield. Hydrogenolysis of the benzyl groups gave the title disaccharide, in crystalline form in 90% yield, which was characterized by a crystalline peracetylated alpha-D derivative.

Chemical synthesis of the human Pk-antigenic determinant.

Condensation of 1,2,3, 6-tetra-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl) -alpha-D-glucopyranose with 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl chloride in 1,2-dichloroethane in the presence of 2,4,6-trimethylpyridine, silver triflate, and molecular sieve 4 A gave 1,2,3, 6-tetra-O-benzoyl-4-O-[2,3,6-tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzyl-a lpha -D-galactopyranosyl)-beta-D-galactopyranosyl]-alpha-D-glucopyranose. Catalytic hydrogenolysis and debenzoylation then gave 4-O-(4-O-beta-D-galactopyranosyl-beta-D-galactopyranosyl)-D-glucopyranos e, the human blood-group Pk-antigenic determinant. A similar sequence of reaction was performed starting from 1,2,3, 6-tetra-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-beta-D-galactopyranosyl)-beta -D-glucopyranose.

Syntheses of trisaccharide C-D-E and tetrasaccharide B-C-D-E fragments found in orthosomycins.

1,5-Anhydro-3-O-benzyl-2,6-dideoxy-4-O-(3,4-di-O-benzyl-2,6-dideoxy-beta -D- arabino-hexopyranosyl)-D-arabino-hex-1-enitol (17), which corresponds to the B-C fragment of various orthosomycins, was prepared from phenyl 2,3-di-O-benzyl-6-deoxy-4-O-(3,4-di-O-benzyl-2,6-dideoxy- beta-D-arabino-hexopyranosyl)-1-thio-beta-D-glucopyranoside (16) by reductive lithiation. The synthesis of 16 involved a stereoselective coupling of phenyl 2,3-di-O-benzyl-6-deoxy-1-thio-beta-D-glucopyranoside (9) and 1,2-di-O-acetyl-3,4-di-O-benzyl-6-deoxy-beta-D-glucopyranose (14) followed by deoxygenation at C-2'. Glycosylation of methyl 2-O-benzyl-6- deoxy-4-O-methyl-beta-D-galactopyranoside (25) with 3,4,6-tri-O-acetyl-2-deoxy- 2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate, followed by deamination at C-2', led stereospecifically to methyl 2-O-benzyl-6-deoxy-4-O-methyl-3-O-(3,4,6-tri-O-acetyl-2-deoxy-beta-D-ara bino- hexopyranosyl)-beta-D-galactopyranoside (26). The 2-deoxy unit of 26 was then modified by consecutive axial introduction of a C-Me group at position 3', protection of HO-3', and deoxygenation at C-6', in order to obtain methyl 3-O-(3-O-benzoyl-2,6-dideoxy-3-C-methyl-beta-D-arabino-hexopyranosyl)-2- O- benzyl-6-deoxy-4-O-methyl-beta-D-galactopyranoside (39), which corresponds to the D-E fragment of orthosomycins. A glycosyloxyselenation-oxidation-elimination sequence was performed on 39 and either 1,5-anhydro-3,4-di-O-benzyl-2,6- dideoxy-D-arabino-hex-1-enitol (40) or 1,5-anhydro-3-O-benzyl-2,6-dideoxy-4-O-(3,4-di-O-benzyl-2,6- dideoxy-beta-D-arabino-hexopyranosyl)-D-arabino-hex-1-enitol (17) to give the C-D-E tri-and B-C-D-E tetrasaccharide fragments, respectively. Each fragment contained the spiro-ortholactone junction with an (R) configuration at the anomeric carbon atom of the C-unit.

Glycosylation using a one-electron-transfer, homogeneous reagent. Application to an efficient synthesis of the trimannosyl core of N-glycosylproteins.

Double glycosylation of methyl 2,4-di-O-benzyl-beta-D-mannopyranoside with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-1-thio-alpha-D-mannopyranoside using as promoter tris(4-bromophenyl)ammoniumyl hexachloroantimonate, a stable, commercial, and crystalline radical cation, afforded after debenzoylation methyl 2,4-di-O-benzyl-3,6-di-O-(3,4,6-tri-O-benzyl-alpha-D-mannopyranoside in excellent yield. Other mannosyl donors were also investigated.

Chemical synthesis of the desialylated human Cad-anti-genic determinant.

Benzyl 2-azido-2-deoxy-beta-D-galactopyranoside was converted into benzyl 2-azido-4,6-O-benzyl-2-deoxy-beta-D-galactopyranoside via benzylidenation, p-methoxybenzylation, acid hydrolysis, benzylation, and selective oxidation. Condensation of 1,2,3,4,6-penta-O-acetyl-beta-D-galactopyranose with benzyl 2-azido-4,6-di-O-benzyl-2-deoxy-beta-D-galactopyranoside in the presence of trimethylsilyl triflate gave crystalline benzyl 2-azido-4,6-di-O-benzyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D-ga lactopyranosyl)-beta-D-galactopyranoside (76%), which was converted into benzyl 2-azido-4,6-di-O-benzyl-2-deoxy-3-O-(2,6-di-O-benzyl-beta-D-galactopy ranosyl)-beta-D-galactopyranoside and condensed with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-galactopyranosyl bromide in the presence of silver silicate on alumina and molecular sieve 4 A to give 61% of benzyl O-(3,4,6-tri-O-acetyl-2-azido-2-deoxy-beta-D-galactopyranosyl)-(1----4)- O-(2,6-di- O-benzyl-beta-D-galactopyranosyl)-(1----3)-2-azido-4,6-di-O-benzyl-2-deo xy- beta-D-galactopyranoside. Reduction with sodium borohydride followed by N-acetylation, O-deacetylation, and catalytic hydrogenolysis then gave O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1----4)-O-beta-D-gal actopyranosyl-(1----3)-2-acetamido-2-deoxy-D-galactopyranose, the desialylated human Cad-antigenic determinant.

Synthesis of oligosaccharins: a chemical synthesis of propyl O-beta-D-galactopyranosyl-(1----2)-O-alpha-D-xylopyranosyl-(1----6)- O-beta-D-glucopyranosyl-(1----4)-beta-D-glucopyranoside.

Condensation of allyl 3,4-di-O-benzyl-beta-D-xylopyranoside with 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-galactopyranosyl chloride in dichloromethane in the presence of silver triflate gave allyl 2-O-(2-O-acetyl-3,4-6-tri-O-benzyl-beta-D-galactopyranosyl)-3,4-di-O benzyl-beta-D-xylopyranoside (7, 83%). Compound 7 was converted in five steps into 2-O-(2-O-acetyl-3,4-6-tri-O-benzyl-beta-D-xylopyranosyl bromide (13), which was condensed immediately with allyl 2,3,6-tri-O-acetyl-4-O-(2,3,di-O-acetyl-beta-D-glucopyranosyl)-beta-D- glucopyranoside to give crystalline allyl O-(2-O-acetyl-3,4,6-tri-O-benzyl-beta-D-galactopyranosyl)- (1----2)-O-(3,4,-di-O-benzyl-alpha-D-xylopyranosyl)-(1----6)-O-(2,3,-di- O-acetyl-beta-D-glucopyranosyl)-(1----4)-2,3,6-tri-O-acetyl-beta-D-gluco pyranoside (23, 50%). O-Deacylation of 23 followed by catalytic hydrogenolysis gave the title glycoside.

Synthesis of disaccharide fragments of dermatan sulfate.

Condensation of crystalline methyl 2-azido-4,6-O-benzylidene-2-deoxy-beta-D-galactopyranoside with methyl (2,3,4-tri-O-acetyl-alpha-L-idopyranosyl bromide)uronate in dichloromethane, in the presence of silver triflate and molecular sieve, provided 54% of methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-alpha-L-idopyranosyluronate)-beta-D-galactopyranoside . The use of methyl (2,3,4-tri-O-acetyl-alpha-L-idopyranosyl trichloroacetimidate)uronate as glycosyl donor, in the presence of trimethylsilyl triflate, improved the yield to 68%. Regioselective opening of the benzylidene group with sodium cyanoborohydride followed successively by O-sulfation with the sulfur trioxide-trimethylamine complex, saponification, catalytic hydrogenolysis and selective N-acetylation gave the disodium salt of methyl 2-acetamido-2-deoxy-3-O-(alpha-L-idopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside. Condensation of methyl 2-azido-4,6-O-benzylidene-2-deoxy-beta-D-galactopyranoside with methyl (2,3,4-tri-O-acetyl-alpha-D-glucopyranosyl bromide)uronate in dichloromethane, in the presence of silver triflate and molecular sieve, gave methyl 2-azido-4,6-O-benzylidene-2-deoxy-3-O-(methyl 2,3,4-tri-O-acetyl-beta-D-glucopyranosyluronate)-beta-D-galactopryano side in 85% yield. The sequence already described then gave the disodium salt of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside.

[Structure of chromogen I of the Morgan-Elson reaction]. / Structure du chromogène I de la réaction de Morgan-Elson

Alkaline treatment of 2-acetamido-2-deoxy-3,4:5,6-di-O-isopropylidene-aldehydo-D-glucose, or of the analogous D-mannose derivative, gave the same crystalline material, unambiguously identified as 2-acetamido-2,3-dideoxy-alpha-D-erythro-hex-2-enofuranose (3) by n.m.r. and mass spectrometry, and crystallography. Treatment of 3 with methanol in acidic medium gave methyl 2-acetamido-2,3-dideoxy-alpha,beta-D-erythro-hex-2-enofuranoside (11). The identical compound was obtained from Chromogen I under identical conditions.

Binding of heparin to antithrombin III: a chemical proof of the critical role played by a 3-sulfated 2-amino-2-deoxy-D-glucose residue.

Known methyl (prop-1-enyl 2,3-di-O-benzyl-alpha-D-glucopyranosid)uronate was first converted into methyl (prop-1-enyl 2,3-di-O-benzyl-4-O-levulinyl-alpha-D-gluco-pyranosid)uro nat e. Acid hydrolysis, followed by treatment with (bromomethylene)-dimethylammonium bromide, gave methyl (2,3-di-O-benzyl-4-O-levulinyl-alpha-D-glucopyranosyl bromide)uronate. Condensation of this bromide with 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose gave 1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O- levulinyl-beta-D-glucopyranosyluronate)-beta-D-glucopyranose. Acetolysis, followed by selective anomeric O-deacetylation and treatment with (bromomethylene)dimethylammonium bromide then gave 6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-4-O-(methyl 2,3-di-O-benzyl-4-O-levulinyl -beta-D-glucopyranosyluronate)-alpha-D-glucopyranosyl bromide. Condensation of this bromide with benzyl 6-O-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-4- O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-alpha-D- glucopyranoside provided benzyl O-(methyl 2,3-di-O-benzyl-4-O-levulinyl-beta-D- glucopyranosyluronate)-(1----4)-O-(6-O-acetyl-2-azido-3-O-benzyl-2-deoxy - alpha-D-glucopyranosyl)- (1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)- 6-O-acetyl-3-O-benzyl-2-benzyloxycarbonylamino-2-deoxy-alpha-D-glu copyranoside. Removal of the levulinyl group followed by condensation with 6-O-acetyl-2-azido-3,4-di-O -benzyl-2-deoxy-alpha-D-glucopyranosyl bromide provided benzyl O-(6-O-acetyl-2- azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di- O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(6-O-acetyl-2-azido-3- O- benzyl-2- deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-acetyl-3-O-benzyl-alpha-L- idopyranosyluronate)-(1----4)-6-O-acetyl-3-O-benzyl-2-benzyloxycarbon ylamino-2- deoxy-alpha-D-glucopyranoside in 78% yield. O-Deacetylation followed by re-esterification, O-sulfation, catalytic hydrogenolysis, saponification, and N-sulfation gave the non-sodium salt of O-(2-deoxy-6-O-sulfo-2-sulfoamino-alpha-D-glucopyranosyl)-(1----4) -O- (beta-D-glucopyranosyluronic acid)-(1----4)O-(2-deoxy-6-O-sulfo-2-sulfoamino- alpha-D-glucopyranosyl)-(1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)- (1----4)-2-deoxy-6-O-sulfo-2-sulfoamino-D-glucopyranose. This synthetic pentasaccharide neither binds to antithrombin III nor induces anti-factor Xa activity.

Synthetic C-oligosaccharides mimic their natural, analogous immunodeterminants in binding to three monoclonal immunoglobulins.

The binding of three monoclonal antigalactan immunoglobulins, IgAs X24, J539 and X44 to their natural haptens methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside, and the corresponding tri- and tetrasaccharides, was compared to the binding of these immunoglobulins with the comparable C-linked oligosaccharide analogues 1-3. The near identity of affinities of the two sets of oligosaccharides indicated the absence of any hydrogen bond involvement by the intersaccharidic oxygen atoms in the natural immunodeterminants.

Chemical synthesis of the human blood-group P1-antigenic determinant.

Condensation of known benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(2,3,6-tri-O-benzyl-beta-D- galactopyranosyl)-alpha-D-glucopyranoside with 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl chloride in dichloromethane in the presence of 2,4,6-trimethylpyridine, silver triflate, and molecular sieve 4A gave benzyl O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-(1 leads to 4)-O-(2,3,6-tri-O-benzyl-beta-D-galactopyranosyl)-(1 leads to 4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside. Catalytic hydrogenolysis gave crystalline O-alpha-D-galactopyranosyl-(1 leads to 4)-O-beta-D-galactopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-alpha -D-glucopyranose, the human blood-group P1-antigenic determinant. A similar sequence of reactions was performed starting from allyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside, in order to prepare a derivative of this determinant suitable for linkage to carrier molecules.

Synthesis of heparin fragments: a methyl alpha-pentaoside with high affinity for antithrombin III.

The synthesis is described of the methyl alpha-glycoside of the pentasaccharide which represents the sequence in heparin responsible for binding and activation of the anticoagulant protein Antithrombin III. It was obtained in a yield much better than that of the previously synthesised pentasaccharide and exhibited the same biological properties.

Electrosyntheses of disaccharides from phenyl or ethyl 1-thioglycosides.

Constant current electrolyses of the glycosyl donors phenyl and ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside in dry acetonitrile in the presence of various primary and secondary sugar alcohols, performed in an undivided cell, gave beta-linked disaccharide derivatives selectively in good yields. Phenyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-glucopyranoside gave the beta-glucosides exclusively in good to moderate yields.

Conformation of the pentasaccharide corresponding to the binding site of heparin for antithrombin III.

The conformation in solution of the pentasaccharide methyl glycoside (As-G-A*-Is-AM; 1), which represents the binding site of heparin for Antithrombin III, has been investigated using molecular mechanics and 1H-n.m.r. spectroscopy. The pentasaccharide has a rather rigid (As-G-A*) and a more flexible (Is-AM) region. A simplified model of 1, comprising two conformations, corresponding to the 1C4 and the 2S0 forms of the iduronate residue, and modified at the G-A* glycosidic linkage with respect to the energy minimum, reproduces most of the observed 3J values and n.O.e. enhancements. The possible role in the binding to Antithrombin III of a low-energy conformer, not observed in solution, is discussed.