Concept of CD, Octant rule and Kroning Kramers transformation

1. Concept of CD, Octant rule and Kroning Kramers transformation Enantiomers that are discriminitaed by biological probes Conformation of coprostane. Stereospecific reactions cis Alkene + anti addition = racemic mixture

7. (-)- Carvone, spearmint odour ; (+)- caraway seed odour Adrenergic agentsEasson–Stedman hypothesis

8. Sex attractant, allures gypsy moth (pheromone) (-)-Aspartame, sweet; (+)- bitter (-)- Menthol,cooling agent; (+)- no Sedative, hypnotic: other form convulsant (+)-Aspargine, sweet; (-)- tasteless

9. (R)- and (S)-limonene have odors of orange and lemon respectively. • Odors of (R)- and (S)-amphetamine also differ. • Differences in the activity of atropine [(dl)-hyoscyamine] and (-)-hyoscyamine and (-)- and (+)-adrenaline. • (+)- and (-)-Nicotine also differs markedly in their physiological action. • The DNA double helix and the protein a-helix, the biopolymers have a right-handed turn. • Enzymes and receptor systems exhibit stereochemical preferences. • (-)-Amosulalol is a nonspecificadrenoceptor antagonist at b- receptor. The (+)-enantiomer is selective and more potent a1- adrenoceptor antagonist.

10. 600 times sweeter and does not get metabolized. Preparation of (L)-Dopafor Treatment of Parkinson’s

13. The structures most likely feasible are : trans- transoid –trans - transoid -trans  (most natural and synthetic steroids have this skeleton, e.g., 5a- dihydrotestosterone) cis-transoid-trans-transoid-trans (some natural steroids have this skeleton,e.g., cholic acids). Conformation of 5b-substituted Coprostane (A/B-cis) cholestane. Conformation of 5a-substituted cholestane

14. Conformation of 5b-substituted coprostane (A/B-cis) cholestane.

15. Brutcher et al 17- β - subtd steroids with C/D trans is best represented by the envelope form where C13 is above the plane of C14, C15, C16 ,& C17 and the 17- β- subtd in a pseudo eq form. The other forms are represented as shown below:

19. Circular dichroism (CD) is the difference in absorption between left and right circularly polarised light by a chiral chromophore. Δε= εl - εr Optical rotation (ORD) and CD contain the same information and are related by Kronig-Kramers Transforms. CD data are more easily interpreted than optical rotation data because it is an absorption rather than dispersion phenomenon. In proteins, CD of the amide chromophore reports on secondary structure (proportions of alpha helix, beta sheet, turns, etc.). Optically pure enantiomers always display opposite. Cotton effect ORD curves of identical magnitude.

20. The enantiomer with more pharmacodynamic activity, eutomer, lower affinity, distomer. • The ratio of affinities, eudismic ratio and its logarithm, eudismic index. • The slope of a plot of eudismic index versus the logarithm of affinity, pA2 or pD2 values (in pharmaco- logy or ki and km values in enzymology) for a homologous series is known as the eudismic affinity quotient (EAQ). • The EAQ is a quantitative measure of the stereoselectivity within a compound series for a particular biological effect, positive slope, indicative of greater difference in activity, agonist or antagonist, between a pair of enantiomers, the greater the specificity exhibited by the eutomer. • A dual action drug, the eutomer may be the distomer in other case.

21. Physics of CD and ORD Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase. A projection of the combined amplitudes perpendicular to the propagation direction thus yields a line.When this light passes through an optically active sample with an absorbance A, the amplitude of the stronger absorbed component will be smaller than that of the less absorbed component. The consequence is that a projection of the resulting amplitude now yields an ellipse instead of the usual line. The occurrence of ellipticity is called Circular Dichroism.Rotation of the polarization plane (or the axes of the dichroic ellipse) by a small angle a occurs when the phases for the 2 circular components become different, which requires a difference in the refractive index n. The change of optical rotation with wavelength is called optical rotary dispersion, ORD.

22. The Cotton effect is the characteristic change in optical rotatory dispersion in the vicinity of an absorption band of a substance. The combination of both( circular birefringence and circular dichroism ) effects in the region in which optically active absorption bands are observed gives raise to a phenomenon called cotton effect.The difference nL- nR (∆n) and eL- eR (∆e) vary with the wave length and can be positive or negetive . • At a given wave length ∆n and ∆e for one enantiomer is equal and opposite for other enantiomer. Positive Cotton effect is where the peak is at a higher wavelength than the trough. Negative Cotton effect is the opposite. Optically pure enantiomers always display opposite Cotton effect of identical magnitude. Zero crossover point between the peak and the trough closely corresponds to the normal UV max.

23. CD is a measurement of how an optically active compound absorbs right- and left-handed circularly polarized light. All optically active compounds exhibit CD in the region of the appropriate absorption band. CD is plotted as l-r vs . For CD, the resulting transmitted radiation is not plane-polarized but elliptically polarized.  is the angle between the initial plane of polarization and the major axis of the ellipse of the resultant transmitted light. A quantity  is defined such that tan  is the ratio of the major and minor axis of the ellipse of the transmitted light. ’ approximates the ellipticity. When expressed in degrees, ’ can be converted to a specific ellipticity [] or a molar ellipticity []. CD is usually plotted as []

24. Chromophores in inherently asymmetrical environments such as helical molecules where chromophore is part of the helix. • Chromophore near a chiral center (common). • Chromophore adjacent to a chiral plane or axis (less common). * C=O has a weak n* transition ca 280 nm, but it can be easily observed by ORD-CD on dilute samples. *Semiempirical rules have been developed to allow conclusions to be drawn about C=O location, ring conformational properties and absolute stereochemistry. *The constitution, conformation or configuration can be determined if two of these three things are known.

25. Determination of optically active substance such as aminoacids, polypeptides, proteins, steroids, antibiotics, terpenes. Stereochemistry of Aliphatic aminoacids : Aliphatic amino acids show a unique cotton effect. α -amino acids of levo configuration show positive effect around 215nm while dextro enantiomers show negetive effect. Stereochemistry of Steroids: In one form the specific rotation increases with decreasing wave length (positive curve) and in the other form the specific rotation decreases with increasing wave length ( negetive curve). Methyl group on carbon 3 lies in the (-) quadrant. All other atoms are symmetrically arrayed about the xz and yz planes, canceling each other out. Predicted Cotton effect is negative, which is what is observed. Example of correlating a Cotton effect sign to an octant model to determine absolute configuration assuming relative stereochemistry is known.

26. Applications of CD in structural biology • Determination of secondary structure of proteins that cannot be crystallised • Investigation of the effect of e.g. drug binding on protein secondary structure • Dynamic processes, e.g. protein folding • Studies of the effects of crystallisation, media on protein structure • Secondary structure of membrane proteins • Study of active site changes • Carbohydrate structure

27. Circular Dichroism • Circular Dichroism (CD) is a type of absorption spectroscopy that can provide information on the structures of many types of biological macromolecules • It measures the difference between the absorption of left and right handed circularly-polarized light by proteins. CD is used for; • Protein structure determination. • Induced structural changes, i.e. pH, heat & solvent. • Protein folding/unfolding. • Ligand binding • Structural aspects of nucleic acids, polysaccharides, peptides, hormones & other small molecules.

28. Optical Rotatory Dispersion(ORD)and Circular Dichroism (CD) • CD are absorption spectroscopy methods similar to ordinary UV-VIS except that they employ polarized light. ORD and CD are also known as chiroptical Techniques. *CD measures the difference between the absorption of left and right handed circularly polarized light. This is measured as a function of wavelength, & the difference is always very small (<<1/10000 of total). After passing through the sample, the L & R beams have different amplitudes & the combination of the two unequal beams gives elliptically polarized light. In ORD the rotation of a beam of linearly polarized light (plane polarized light) is measured as a function of the wavelength. In one sense ORD is very similar to the classical determination of the specific rotation, [α]TD in a polarimeter; the difference being that determination of specific rotation is performed at a single wavelength. * In CD the absorbance of a circularly polarized beam light is measured as a function of the wavelength.*CD measures the differential absorption of chiral light by enantiomers. * Both ORD and CD require that the compound being measured is chiral and “optically active”.

29. If the absorption of the light is negligibly small, then the minor axis of the ellipse will be very small compared to the major axis. The emerging light will be equivalent to plane-polarized light. The orientation of the ellipse corresponds to optical rotation. • Light passing through a chromophore solution may interact with the sample in two main ways, may be refracted or absorbed,is quantitated by the refractive index, n, or by the molar extinction coefficient, ε. Optically active samples have distinct molar extinction coefficients for left (εL) and right (εR) circularly polarized light. εL ≠ εR. The difference in absorbance, A, of the two components, is a measure of Circular Dichroism (CD). i.e., CD = AL – AR.

30. ΔA is the difference between absorbance of LCP and RCP light. ΔA is a function of wavelength, so it must be known. • Molar circular dichroism Applying Beer's lawDA = (eL - eR), Where εL and εR are the molar extinction coefficients for LCP and RCP light, C is the molar concentration l is the path length in centimeters (cm). Then De = eL - eR. If Δε or ΔA or ellipticity, θ, is plotted against wavelength, λ, a CD spectrum may be obtained. The ellipticity is proportional to the difference in absorbance of the two components, AL – AR. Thus, CD is equivalent to ellipticity. In an optically active material, the electric vectors →R E and →L E rotate at different speeds. Since the two circularly polarized components propagate with different speeds, the index of refraction (n) for the two components will be different. This effect is called circular birefringence (CB). The result is a phase shift between the two components, δ, proportional to the refractive index difference, nL– nR. Actually, δ = (2π/λ)/ (nL–nR). When the two components are combined, the phase shift results in a permanent rotation of the long axis (major axis) of the elliptically polarized light. In fact, φ = δ/2 = (π/λ)/(nL–nR).

31. Thus circular birefringence is equivalent to optical rotation. Since the refractive index is always dependent on the wavelength, the angle of rotation is also wavelength dependent. The relationships between CD and ellipticity (θ) and between the circular birefringence and the optical rotation (φ) are given by • φ= 180 l (nL – nR) / λ & θ= 2.303(AL – AR)180/4π = 33.0 (AL – AR) = 33.0 ΔA • Where l is the sample length and θ and φ are measured in degree. For comparison of results from different samples it is necessary to consider molarity. In CD spectroscopy, there is absorption as well as a difference in the index of refraction for the two cpl. The result is both a tilting of the axis of polarization and a loss of ‘planarity’. Chromophores in chiral environments will display a CD signal.

32. Molar ellipticity is circular dichroism corrected for concentration. Molar circular dichroism and molar ellipticity, [θ], are readily interconverted by the equation: [q] = 3298.2De. Elliptical polarized light is composed of unequal contributions of right and left circular polarized light. This relationship is derived by defining the ellipticity of the polarization as: tanq = (b – a)/(b + a). Where ER and EL are the magnitudes of the electric fieldvectors of the right-circularly and left-circularly polarized light, respectively. When ER equals EL (when there is no difference in the absorbance of right- and left-circular polarized light), θ is 0° and the light is linearly polarized. When either ER or EL is equal to zero (when there is complete absorbance of the circular polarized light in one direction), θ is 45° and the light is circularly polarized.

35. Plain” ORD curves obey Fresnel’s equation

41. Classification of Polarization Linear : Two orthogonal plane waves with same phase but possibly different amplitudes. Circular: Two orthogonal plane waves with 90 deg phase shift but same amplitudes. Elliptical: Possibly any degree phase shift with different amplitudes Linearly polarized light: in the form of a plane wave in space. Circularly polarized light:Light is composed of two plane waves of equal amplitude but differing in phase by 90°. Circularly polarized light consists of two perpendicular EM plane waves of equal amplitude and 90° difference in phase. Elliptically polarized light:Two plane waves of differing amplitude, differing in phase by 90°, or other than 90°.

42. Light, electromagnetic radiation, can be considered to behave as two wave motions (electric and magnetic) generated by oscillating orthogonal electric and magnetic dipoles. • Most optical measurements (UV-VIS, ORD, and CD) are only concerned with the electric field; • the orthogonal oscillating magnetic vector is usually ignored. • The electric vector vibrates perpendicularly to the direction of travel of the beam of light. • In ordinary light there are an infinite number of planes perpendicular to the direction of travel • A beam of linearly polarized light is made up of two orthogonal polarization states each of which trace out a helix as a function of time: right circularly polarized light, and left circularly polarized light. • In linearly polarized light one of the planes has been selected by the polarizer. • The right and left directions are determined from the point of the receiver (in radio the point of reference is the transmitter). • The chiroptical phenomena of ORD and CD involves the diastereotopic interactions of enantiomeric materials with these polarization states.

43. Octant Rule • One of the important uses of CD and ORD is to determine the absolute configuration of ketones and aldehydes. • This is accomplished by application of a semi-empirical generalization known as the octant rule. • The rule predicts the sign of the n → π* Cotton effect of a ketone due to the contributions of substituents in each of eight octant sectors surrounding the carbonyl chromophore. • The rule allows one to determine the absolute configuration and also the likely conformation of a molecule from the sign of the Cotton effect.

44. Given below the ORD curves for a-ethylcholestanone and a ketone of similar structure derived from cafestol. Predict the proper configuration for cafestrol. • Steroid 1 has the absolute stereochemistry as shown, displays a positive Cotton effect • This is used to assign the absolute stereochemistry for the rings in the triterpene cafestrol A or A’ and its multistep degradation yields B or B’ which has a negative Cotton effect, therefore A and B must have enantiomeric configurations for the ring substituents.

45. Measurement of how an optically active compoundabsorbs right- and left-handed circularly polarized light.• All optically active compounds exhibit CD in the region of the appropriate absorption band.• CD is plotted as εl-εr vs λ.• For CD, the resulting transmitted radiation is not plane- polarized but elliptically polarized. Optical activity requires both a finite μ and a finite m. The product of these two vectors corresponds to a helical displacement of charge. By analogy with the dipole strength associated with normal absorption, we can define a rotational strength R, that indicates the intensity, or probability, of a CD transition. For most purposes, we can say that R = μm cos θ where θ is the angle between the two dipoles.

46. Two waves reach their peaks and cross the zero line in the same moments. Two waves are added with a phase difference of -90 degrees instead of 90 degreesa 3/4 wavelength difference Front view

48. Any linearly polarized light wave can be obtained as a superposition of a left circularly polarized and a right circularly polarized light wave, whose amplitude is identical. • Plane-polarized waves in an absorbing medium

49. Circularly polarized waves in an absorbing medium

50. Plane-polarized waves in a refracting medium