The solubility and properties of a purified ichthyocol in salt solutions of neutral pH

1. Purified citrate-extracted ichthyocol obtained from carp swim bladders has been further characterized with respect to its content of certain amino acids and carbohydrate substances. 2. The degree of solubilization or dispersion of ichthyocol by solutions of certain salts maintained in the range of neutral pH and at a temperature of 0-2 degrees C. has been determined. 3. While a number of salts of monovalent cations had no significant solubilizing effects on ichthyocol, ammonium chloride in a concentration of 1 M did cause solution of the protein. 4. Sodium thiosulfate in a range of concentrations caused the solubilization of ichthyocol but was most effective in an intermediate concentration of 0.25 M. 5. Several salts of divalent cations, in particular the chlorides of calcium, magnesium, and barium, and magnesium thiosulfate in concentrations ranging from 0.3 to 1 M caused the immediate and complete solubilization of the ichthyocol. 6. Solutions of ichthyocol in calcium chloride, magnesium chloride, and sodium thiosulfate buffered or adjusted to pH 7.0, were studied with respect to intrinsic viscosity of the protein, optical rotation, ultracentrifugal sedimentation, and reconstitution into fibers. It was found in each case that the original characteristics of the collagen, as determined previously in acid solution, were maintained when the protein was dissolved in salt solutions of neutral pH. No evidence of denaturation or gelatinization could be found when ichthyocol was solubilized under the stated conditions. 7. Collagen in neutral solution with sodium thiosulfate, calcium chloride, or magnesium chloride was not attacked by trypsin as determined viscometrically at 20.0 degrees C., but was rapidly degraded by a purified bacterial collagenase.     

Morphologies and conformation transition of lentinan in aqueous NaOH solution

Molecular morphologies and conformation transition of lentinan, a beta-(1-->3)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration [s2](1/2), intrinsic viscosity [eta], as well as specific optical rotation at 589 nm [alpha]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix-coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the first time, we confirmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15 degrees C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan.     

Ab initio prediction of optical rotation: comparison of density functional theory and Hartree-Fock methods for three 2,7,8-trioxabicyclo[3.2.1]octanes

We report ab initio calculations of the frequency-dependent electric dipole-magnetic dipole polarizabilities, beta(nu), at the sodium D line frequency and, thence, of the specific rotations, [alpha](D), of 2,7,8-trioxabicyclo[3.2.1]octane, 1, and its 1-methyl derivative, 2, using the Density Functional Theory (DFT) and Hartree-Fock/Self-Consistent Field (HF/SCF) methodologies. Gauge-invariant (including) atomic orbitals (GIAOs) are used to ensure origin-independent [alpha](D) values. Using large basis sets which include diffuse functions DFT [alpha](D) values are in good agreement with experimental values (175.8 degrees and 139.2 degrees for (1S,5R)-1 and -2, respectively); errors are in the range 25-35 degrees. HF/SCF [alpha](D) values, in contrast, are much less accurate; errors are in the range 75-95 degrees. The use of small basis sets which do not include diffuse functions substantially lowers the accuracy of predicted [alpha](D) values, as does the use of the static limit approximation: beta(nu) approximately beta(o). The use of magnetic-field-independent atomic orbitals, FIAOs, instead of GIAOs, leads to origin-dependent, and therefore nonphysical, [alpha](D) values. We also report DFT calculations of [alpha](D) for the 1-phenyl derivative of 1, 3. DFT calculations find two stable conformations, differing in the orientation of the phenyl group, of very similar energy, and separated by low barriers. Values of [alpha](D) predicted using two different algorithms for averaging over phenyl group orientations are in good agreement with experiment. In principle, the absolute configuration (AC) of a chiral molecule can be assigned by comparison of the optical rotation predicted ab initio to the experimental value. Our results demonstrate the critical importance of the choice of ab initio methodology in obtaining reliable optical rotations and, hence, ACs, and show that, at the present time, DFT constitutes the method of choice.     

The characteristics of the intermediates in collagen-fold formation

The kinetics of collagen-fold formation has been analyzed in terms of a series of consecutive reactions, rather than the usual set of parallel reactions. Data on the regain of optical rotation and viscosity for denatured ichthyocol, formaldehyde cross-linked ichthyocol, isolated α1-chains, α2-chains and the β₁₂-component of rat skin collagen have been utilized. The Guggenheim point averaging procedure was used for data analysis for those reaction steps where final equilibrium measurements cannot be made. By considering the data over appropriate time ranges, optical rotation constants characteristic of each sequential intermediate have been determined. Comparisons of the constants for the first nucleated intermediate in the cases of varying degrees of cross-linking and differing pyrrolidine ring contents, show that both hydrogen-bonding interactions and pyrrolidine ring content contribute to the stabilization of the multistranded nucleated fold intermediate. The presence of single chain nucleated intermediate seems highly unlikely.     

A 13C nuclear magnetic resonance and circular dichroism study of the collagen-gelatin transformation in enzyme solubilized collagen.

Natural abundance Fourier transform 13C nuclear magnetic resonance (13C NMR) were obtained for enzyme solubilized collagen at 1 degrees intervals through the transition region. The transition of collagen molecules from the rigid triple helical state to single-stranded, random-coil state is accompanied by a change from broadened carbon resonances unobservable under high-resolution conditions to narrow line spectra. Thus distinction can be made between helical and random-coil states of individual residues. The transition is monophasic, as determined by examination of 14 different carbon resonances, and the entire structure is found to melt cooperatively over a temperature interval of 5 +/- 1 degrees. All the residues seem to be involved in the unfolding process concurrently. The transition was also studied by examining the changes in the circular dichroism spectrum brought about by heating. The experiments corroborated the observation that the transition proceeded cooperatively over a temperature interval of 4 degrees. Enzyme soluble collagen is seen to melt less cooperatively than native collagen. The enthalpy change was determined by assuming an equilibrium between three random coil gelatin chains and tropocollogen molecules. From the enthalpy, the average length of the tripeptide sequences (70-85) involved in the transition can be estimated. The shortening of the cooperative unit could arise as a result of some alteration of the native conformation through proctase treatment.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

The first enantioselective synthesis of the amavadin ligand and its complexation to vanadium

The ligand of the naturally occurring vanadium compound amavadin found in Amanita muscaria, (2S, 2'S)-N-hydroxyimino-2,2'-dipropionic acid (1), was synthesized stereoselectively in two steps with 43% overall yield. After complexation of this ligand to vanadyl acetate, amavadin was isolated in quantitative yield. Due to the chirality at vanadium amavadin consists of a mixture of delta and lambda diastereoisomers. Directly after its synthesis, the delta to lambda ratio of amavadin is 2.27 and it decreases to 0.80 after equilibrium has been reached. During this epimerization the optical rotation for V[(2S,2'S)-N-hydroxyimino-(2,2')-dipropionate]2 (=amavadin) changes from [alpha](D)25 = +36 degrees to +114.0 degrees (c = 0.5, H2O). For V[(2R,2'R)-N-hydroxyimino-(2,2')-dipropionate] the optical rotation changes from [alpha](D)25 = -36 degrees to -113.2 degrees (c = 0.5, H2O).     

Double-helical character of ribonucleic acid from virus-like particles found in Penicillium chrysogenum

1. RNA was isolated from virus-like particles found in Penicillium chrysogenum and resolved into two fractions by gel filtration through agarose columns. 2. Fraction 1 was excluded and had the following properties: 50.9% G+C [AMP 0.246, UMP 0.246, CMP 0.252, GMP 0.255 (mole fraction)]; mol.wt. about 1.2x10(6) daltons; s(20,w) 12.3S and ;melting' temperature about 100 degrees C (solvent 0.15m-sodium chloride-0.015m-sodium citrate pH7.2); optical rotation [alpha](max.) 6000 degrees at 278nm; circular dichroism (epsilon(L)-epsilon(R))(max.)=8.181mol(-1) cm(-1) at 260nm. 3. Properties of fraction 2 include 37.8% G+C [AMP 0.313, UMP 0.312, CMP 0.186, GMP 0.189 (mole fraction)]; mol.wt. about 140000 daltons; s(20,w) 7.3S, T(m) about 85 degrees C (solvent 0.15m-sodium chloride-0.015m-sodium citrate, pH7.2); optical rotation [alpha](max.) 6000 degrees at 278nm; circular dichroism (epsilon(L)-epsilon(R))(max.)=8.241mol(-1) cm(-1) at 260nm. 4. The properties of both fractions were consistent with a double-helical conformation.     

Studies on the activation energy and deuterium isotope effect of human skin collagenase on homologous collagen substrates

The activation energy (EA) and solvent-deuterium kinetic isotope effect (kH/kD) of human skin fibroblast collagenase were studied on the homologous human type I, II, and III collagens in both native and denatured states. Values for EA on human type I and II collagens in solution were 47,000 and 61,000 cal, respectively. The Arrhenius plot for type III collagen, unlike that for the other types, was characterized by a break in EA at approximately 26 degrees C. At temperatures below this point, EA was 42,500 cal; at higher temperatures, EA fell to 29,500 cal. This latter value, intermediate between type I collagen monomers and denatured random gelatin alpha chains, appears to result from a further opening in the already loosened helix of the type III collagen molecule in the region of the 3/4:1/4 collagenase cleavage site. The EA of trypsin on native human type III collagen was also measured and found to be 70,000 cal. This high value calls into question the role of serine proteases in the physiologic degradation of this substrate; a much higher energy expenditure was required for trypsin to cleave type III collagen than for the fibroblast collagenase. Reaction velocity on human collagen types I-III in solution was slowed 15-35% (kH/kD = 1.2-1.5) by the substitution of deuterium for hydrogen in the solvent buffer. This value was far lower than that observed following the aggregation of solution monomers into insoluble fibrils (kH/kD = 9). Denaturation of triple helical monomers into random gelatin alpha chains eliminated any slowing by deuterium, and kH/kD was 1.0 in all cases. Since the same peptide bond hydrolysis accompanies the cleavage of all these forms of the collagen substrate, it would appear that the role of water at the rate-limiting step of collagen degradation may not reside in the hydrolysis of a peptide bond per se, but rather may reflect the difficulty in transporting water molecules to the site of such catalysis, especially following fibril aggregation.     

Isolation and characterization of pepsin-treated type III collagen from calf skin.

Calf skin collagen was solubilized by incubating acid-extracted calf skin with pepsin at pH 2.0 and 25 degrees C, conditions that did not cause degradation of the triple helical region of collagen. Type III collagen was separated from type I collagen by differential salt precipitation at pH 7.5. The isolated type III collagen contained mainly gamma and higher molecular weight components cross-linked by reducible and/or non-reducible bonds. The isolated alpha1 (III) chains had an amino acid composition characteristic of type III collagen. Denatured but unreduced type III collagen, chromatographed on carboxymethyl-cellulose, eluted in the alpha 2 region, while after reduction and alkylation the alpha1 (III) chains eluted between the positions of alpha1 (I) and alpha2. The mid-point melting temperature temperature (tm) of type III collagen (35.1 degrees C) in a citrate buffer at pH 3.7 was somewhat lower than that of type I collagen (35.9 degrees C). Renaturation experiments at 25 degrees C showed that denatured type III collagen molecules with intact intramolecular disulfide bridges (gamma components) reform the triple helical structure of collagen much faster than reduced and carboxymethylated alpha1 (III) chains.     

Production of 2,3-Butylene Glycol from Citrus Wastes. II. The Bacillus polymyxa Fermentation

The conditions required for production of levo 2,3-butylene glycol by Bacillus polymyxa from citrus molasses were studied. Starter cultures required acclimatization to the substrate prior to inoculation of the fermentation medium. Maximal production of butylene glycol with minimal residual sugar was obtained with a medium consisting of molasses, diluted to 20 degrees Brix, and 0.4% urea. Optimal environmental conditions included aeration at 0.11 volumes of air per volume of medium per minute, maintenance of pH at 6.0 to 6.2, a fermentation temperature of 30 C, and a stirring rate of 420 rev/min. The concentration of butylene glycol obtained in the fermentation beer ranged from 2.3 to 4.4%. The optical rotation of the glycol ranged from [alpha](D) (23 degrees ) = -1.01 degrees to -10.45 degrees . The variation in rotation was probably due to the presence of contaminating substances in the distillate.     

On the action of ozone on gelatin

Gelatin, the lower molecular weight derivative of collagen, was treated with ozone and the structural changes were studied with polarimetry, electronic absorption and FT-IR spectroscopy. The resulting ozonized gelatins were studied by thermal analysis (DSC and TGA) in comparison to a reference gelatin. It has been found that at relatively low ozone dose, for instance at ozone/gelatin molar ratios between 0.1 and 0.35 the structural damage introduced by ozone oxidation of gelatin is minimal to negligible and this may facilitate the industrial utilization of ozone in the cold sterilization of gelatin as well as in its bleaching processes required for certain applications. At higher O(3)/gelatin molar ratios the gelatine damage is evident both polarimetrically as suggested by the drop of its specific optical rotation and by spectrophotometrical analyses.     

Odor distinctiveness between enantiomers of linalool: difference in perception and responses elicited by sensory test and forehead surface potential wave measurement

The effects on humans of inhalation of optically active linalools were examined in terms of sensory tests and portable forehead surface electroencephalographic (IBVA-EEG) measurements in order to assess their odor distinctiveness by chiral isomers. (R)-(-)-Linalools with specific rotation of [alpha](D) = -15.1 degrees were isolated by repeated flash column chromatography from lavender oil, while (S)-(+)-linalools with [alpha](D) = +17.4 degrees and (RS)-(+/-)-linalools with [alpha](D) = 0 degrees and content of (R)-form 50.9% and (S)-form 49.1% were obtained from coriander oil and commercial linalool, respectively, by using the same method. With the use of an inhalator, each was administered to subjects both before and after 10 min of work. It was found that administration after work evoked different subjective impressions when compared with that before work depending on the configuration of the isomers and the type of work employed. For instance, inhalation of (R)-(-)-linalool after hearing environmental sounds not only produced a much more favorable impression in the sensory test but was also accompanied by a greater decrease in beta waves after work in comparison with that before work. This is in contrast to the case of mental work, which resulted in a tendency for agitation accompanied by an increase in beta waves. These findings led us to conclude that enantiomeric stereospecificity of linalool evoked different odor perception and responses not only with chiral dependence but also with task dependence. In addition, in comparing these sensory profiling features and IBVA-EEG tendencies between hearing environmental sound and mental work, a tendency was observed for (R)-(-)-linalool to coincide with (RS)-(+/-)-linalool but not with (S)-(+)-linalool.     

Biosynthetic and structural properties of endothelial cell type VIII collagen

A highly unusual endothelial cell collagen (Sage, H., Pritzl, P., and Bornstein, P., (1980) Biochemistry 19, 5747-5755) has been characterized in greater detail. Pulse-chase experiments with bovine aortic endothelial cells revealed two nondisulfide-bonded collagens, of apparent chain Mr = 177,000 and 125,000, with an estimated synthesis and secretion time of 75 min. Stepwise, quantitative processing to stable lower molecular weight forms as described for type I procollagen was not observed. Endothelial collagen was secreted over a temperature range of 24-37 degrees C and, prior to heat denaturation, did not display affinity for a gelatin-binding fragment of fibronectin coupled to Sepharose. The presence of a pepsin-resistant domain (Mr = 50,000) in both the soluble and cell layer-associated forms of this protein was shown by ion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Endothelial collagen was cleaved by vertebrate collagenase into several discrete fragments that differed in molecular weight from the characteristic alpha A and alpha B fragments generated from the interstitial collagens. Nontriple helical domains corresponding to the NH2- and COOH-terminal propeptides of other procollagen types were not found after incubation of endothelial collagen with bacterial collagenase. Additional evidence for the lack of extended noncollagenous sequences was provided by studies with mast cell proteases, which convert native procollagen to collagen but are unreactive toward native interstitial collagens. Endothelial collagen was not cleaved by these enzymes at 37 degrees C, but, as observed for interstitial collagen alpha chains, required prior heating at elevated temperatures for cleavage to occur. In view of this unique set of structural characteristics, and a distribution that is not restricted to the endothelium, we have designated this protein as type VIII collagen.     

Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form

We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35 degrees C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol X mol-1 X h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35 degrees C were: human collagenase, approximately equal to 1500 h-1; microbial (clostridial) collagenase, approximately equal to 100 h-1; and general proteases, 23-52 h-1. In addition it was shown that pronase cleaves type III collagen in solution at 22 degrees C by attacking the same Arg-Gly bond in the alpha 1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8 degrees C increase in denaturation temperature (Tm = 40.2 degrees C in solution; Tm = 47.0 degrees C in fibrils).     

Characterization of a fucoidan from Lessonia vadosa (Phaeophyta) and its anticoagulant and elicitor properties

Blades of Lessonia vadosa (Phaeophyta) were extracted with 2% CaCl(2) solution, affording in 4.4% yield a polysaccharide which contained fucose and sulfate groups in the molar ratio 1.0:1.12. The high negative optical activity value ([alpha](D)(22)=-134.0 degrees ), FT-IR and NMR analysis suggest the presence of a fucoidan. (13)C NMR spectrum of the polysaccharide obtained by solvolytic desulfation of native fucoidan indicated the major presence of 1-->3 linked alpha-l-fucan. Depolymerization of the native fucoidan with H(2)O(2) in the presence of copper(II) acetate gave in 54.8% yield a fraction with 33.7% of sulfate content. The native fucoidan (MW 320,000) showed good anticoagulant activity whereas the radical depolymerized fraction (MW 32,000) presented a weak anticoagulant activity. These polysaccharides showed significant activation of phenylalanine-ammonia lyase (PAL), lipooxygenase (LOX) and glutathione-S-transferase (GST) defence enzyme activities in tobacco plants.     

Differences in thermal stability of frog and rabbit alpha alpha- and alpha beta-tropomyosins determined by optical rotatory dispersion

Frog and rabbit alpha alpha- and alpha beta-tropomyosins were purified, and their thermal stabilities determined by use of optical rotatory dispersion. The tropomyosins were found to be virtually completely helical at 5 degrees C. Regions of different thermal stabilities were seen for all tropomyosins. Rabbit and frog alpha alpha-tropomyosin show very similar thermal properties, with main transitions near 47-49 degrees C. The main transition for frog alpha beta-tropomyosin is at 32 degrees C. The results show that the alpha beta-tropomyosins are less stable than the alpha alpha-forms. Only thermal transitions of the alpha beta-forms appear to be correlated with the body temperatures of the animals.     

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.     

The effect of ultraviolet irradiation on soluble collagen

1. The effect of ultraviolet irradiation on acid-soluble and neutral-salt-soluble calf-skin collagen was studied by chromatography, gel filtration, amino acid analysis and sedimentation of the sub-units, and the reaction kinetics of degradation were obtained from viscosity and optical rotation measurements. 2. It was demonstrated that, whereas the structure of neutral-salt-soluble calf-skin collagen may be represented by the formula (alpha(1))(2)alpha(2), the acid-soluble extract has the formula alpha(1).(alpha(2))(2). The acid-soluble collagen is also unusual in containing a large amount of a component that could be beta(22). 3. Ultraviolet irradiation causes the progressive degradation of the collagen molecule into smaller molecular fragments that subsequently lose their helical nature. The rate constants show that the denaturation of soluble collagens by ultraviolet irradiation is much slower, under the conditions used, than denaturation by heat or enzymes.     

Ab initio calculations for the optical rotations of conformationally flexible molecules: a case study on six-, seven-, and eight-membered fluorinated cycloalkanol esters

Based on the time-dependent density functional response theory, an approach for the prediction of optical rotations of enantiomers of conformationally flexible molecules was developed. The method was applied successfully for the determination of the absolute configuration of trans-2-fluorocycloalkanol acetates with different ring sizes. The largest deviations between experimental and theoretical [alpha](D) values are 10 deg x [dm x (g/cc)](-1) (about 20% error). These theoretical results suggest that the optical rotation in these molecules is dominated by the local (1R;2R) configuration of the two substituents and that different ring and even axial/equatorial orientations play a less important role.