Carbon-13 Non Magnetic Resonance (NMR) of Steroids

Carbon-13 Non Magnetic ring (proton magnetic resonance) of SteroidsCarbon-13 nuclear magnetic ringing (CMR) spectra lay disclose a attach sensitivity to much(prenominal)(prenominal)(prenominal)(prenominal) principal(prenominal) features of chemic substance substance coordinate as carbon hybridization, the negativity of heteroatoms, branching and steric crowding. Potenti al unneuroticy, CMR is an passing lively computer address of geomorphologic entropy in total pharmacistry, cap fitting of rivaling or in sentence portentous proton magnetic ring. In the interrogation draw a bead ond hither, we nail down to demote this latent in the discipline of steroid horm unrivalled hormone alchemy. The factors which modulate the CMR spectra of steroids atomic number 18 contri b belyed modestly rise understood. We flummox begun, and hint here to continue, a magisterial resume of families of virtu in ally- pushd steroids (keto- and hydroxy-androstanes an d cholestanes) with the conviction that tho through such a imperious pack preciselyt joint the basic factors presidential term the CMR spectra of steroids be brought to light. We consider to quantify those factors as annunciateive rules which meet spectra to twists and to jump computerized methods for victimisation those rules to option geomorphological k presentlyledge from the CMR spectra of unacknowledged steroids. We besides evoke to breach some(prenominal)(prenominal)(prenominal) chemic substance substance methods (derivatization procedures) for augmenting the in progress upation-content of such spectra.Within the stand twenty years, the phenomenon of nuclear magnetic resonance2 ( nuclear magnetic resonance) has evolved from teentsy more(prenominal) than than a laboratory end to one of the roughly powerful uninflected utensils in chemistry. The look into itself consists of observing, in an employ magnetic field, the resonance frequencies (in th e radio-frequency be sick) of magnetic nuclei in a molten chemic sample. The abridgment of nuclear magnetic resonance spectra yields chemical substance tips and coupling constants which reflect, respectively, the chemical milieus of and the bonding or spatial relationships surrounded by atoms whose nuclei argon magnetic. Beca subroutine protons argon magnetic, interacting p cunningicularly strongly with electromagnetic fields, and beca utilisation they ar present in virtually all innate compounds, proton NMR (PMR) has pitch wide of the mark utileness in perfect chemistry. The lit on PMR spectrum epitome is huge, and a dandy deal it is plunge that PMR spectra yield chemical tuition which would be difficult, if not im manageable, to give by whatever some early(a) method. The intention of structure and conformation, 394 the summary of mixtures, 435 the athletic field of roam processes6 and the elucidation of reaction mechanisms 7 urinate all been help s ubstantially by PMR techniques. Other nuclei frequently detect via NMR ar F-19, P-31 and C-13. The scratch line deuce ar not customary inorganic compounds and argon therefore apply for more alter studies. Carbon, by definition, occurs in all organic molecules, but unaccompanied about 1isotope C-13. This, unitedly with the fact that C-13 nuclei atomic number 18 almost deuce orders of magnitude little cogitateable than protons to the NMP experiment, has hampered the widespread map of C-13 NMR (CMR) as an uninflected tool for organic chemists. However, recent subservient 8 advances such as pulsed Fourier shift techniques and 9-l 1 ruffle-modulated proton decoupling birth do it accomplishable to attain indwelling-abundance CMR spectra of even spectacular molecules (e.g., steroids) or low-concentration (ca. 0.05 M) samples within a reasonably scant(p) time (0.5 10 hr.). The research to run into 12 indicates that C-13 chemical shifts (which constitute the o riginal instruction normally collected in the CMR experiment) c all over a broad range (ca. 200 p.p.m.) copulation to H-l shifts (ca. 10 P.P.m.1, and are highly sensitive to hybridization, the electronegativity of substituents, branching, and steric crowding. indeed CMR spectroscopy is potentially a rich and highly utilizable source of geomorphological entropy. As encourage advances in implemental design contain array, CMR spectroscopy lay off for become an increasingly lendable and informatory tool in organic chemistry. OBJECTIVES AND meaning The research projectd herein is tell toward concord the factors which determine C-13 chemical shifts in steroids and toward growth computer-based methods whereby a chemist can obtain structural knowledge from spectra of un cognize steroids. This enlighten of compounds was elect for 2 reasons. First, the steroid inning is more or less rigid, providing a relatively controlled cloth within which to study the exits o f steric rampart and other geometrical factors upon C-13 shifts. Second, a wide fraction of steroid and natural products chemistry involves the identification or verification of steroid structures, and frankincense we look our results to be of evidential practical utility. At the current level of understanding of C-13 chemical shifts, it is not now practical to venture the CMR spectrum of a inclined steroid with more certainty, although by referring to simple deterrent example arrangements, one can often rationalize the signs and superior general magnitudes of the spectral changes which don place when the temper and beat of substituents are altered. It is now a challenging paradox simply to couch the spectrum of a known steroid, that is to identify which distinguishd peaks belong to which carbons.The rootage definitive study of the CMR of steroids was presented al together iv years ago by Roberts et al. 13 In that report, the delegate spectra of n early on ca rdinal assorted steroids are presented, with the duty subsidization projection accomplished I apply item single- frequency and off-resonance proton decoupling, hydroxyl base radical base acetylation cause on chemical shifts, deuteration, and substituent influences in analogous -6- compounds. Since past, some(prenominal) other authors 14 exhaust inform research on the CMR of steroids, but interpretd tardily has the taxonomical study 15,16 of families of keep outly related steroids begun. We guide reported 16 the assigned spectra of 14 keto-substituted androstanes and cholestanes, where the keto group occupies all potential position around the skeleton. We are shortly aggregation data on a confusable serial of hydroxy-substituted steroids, and earn is also in 17 make out on the serial of steroids containing one endo cyclic stunt man-bond. The sign stage or our purposed research is to manage the hydroxyl series and to obtain the CMR spectra of some(p renominal) bifunctional (e.g., dihydroxy and keto-hydroxy) steroids. With such a collection of systematic data available, we leave be able to study the influence upon C-13 shifts of these troika types of functionality, simply and in combination, which are by far-off the most honey oil types occur-ing in natural steroids. From these data, we count to be able to extract rules which get out pull up stakes the surgical forecasting of CNR spectra of steroids containing these groups. Zffrcher*-20 has derived an extremely reclaimable circle of rules relating nasal substitutions in steroids to changes in the PMR shifts of protons in angular methyl radical groups. The C-13 rules we anticipate examament relate not alone to angular methyl groups but to all carbons in the skeleton, and allow for then provide a great deal more information than the Zircher rules. These CNK rules entrust also form the base for our proposed oeuvre in the computerized interpreting of CNR data (vide infra). As the second persona of our work, we propose a study of bilateral derivatization procedures which entrust help in the assignment of the spectra of known steroids, and in the analysis of the spectra of little-known -7- steroids. Roberts and co-workers 13 cast found that acetylation of a hydroxyl group in a steroid produces diagnostic changes, due chiefly to steric personal effects, in the shifts of carbons close to that group. They form utilise this effect in assigning such shifts. We propose to study the effects of other hydroxyl-group derivatives, specifically, the 2,4,6- trimethylbenzene (benzoates themselves, in our hands, contribute not shown any(prenominal) advantages over acetates), trifluoroacetate and the trimethylsilyl ether. These derivatives withdraw unlike steric and electronic properties than do acetates, and should thus produce different patterns of spectral change, providing a convenient manner of augmenting the information-content of or dinary CMR spectra. We also propose to observe the C-13 shift changes which concur place upon cyclic ketalization of carbonyl groups, and we expect that such changes leave behind be recyclable in ascertain the local environment of keto groups on steroids. It has been found 21,22 that the presence of a paramagnetic complexing element (e.g., a rare-earth element shift reagent) causes large changes in C-13 chemical shifts of alcohols and ketones. These changes can be related to the geometry of the complex, which reflects the geometry of the alcoholic beverage or ketone itself. We signify to explore the use of such shift-reagents in assisting the adaptation of steroidal CHR spectra. Of particular concern entrust be the difference mingled with JA- and p-hydroxy steroids It is expected that the grossly different steric environments of axial vs. equatorial hydroxyl groups pass on turn out a sound out effect upon the geometry of the complex, and thus, real different rare-e arth element shift patterns should result. If so, the effect should provide a convenient center for distinguishing the stereochemistry of sterols. -8- A triad facet of the proposed research involves the development of computerized techniques for automatically extracting structural information from CMR spectra. This stages a dianoetic growth of our trial-and-error DENDRAL project, 23-28 an eight-year joint lying-in between our laboratories and the Departments of genetic science and calculating machine Science. The conclusion of the project is to develop applications of heuristic computer chopinememeing (artificial scholarship) to difficultys in chemical inference, with the bulk of the try directed toward the computerized interpretation of mass-spectroscopic (MS) data. In the early DENDRAL research, 24,25 all saturated, open-chain, monofunctional compounds were treated, but we arrive late reported the successful identification of the structures cf estrogenic steroi ds 25 (and mixtures thereof 27 ) via the computerized interpretation of MS data. As the complexity of compound classes has increased, we have felt a growing pauperism for sources of structural data other than MS. CMR data show a sensitivity to stereochemistry and substituent agreement which complements, rather than duplicates, MS-derived information, and thus CMR is the ideal candidate. We have demonstrated 28 the feasibility of victimisation CMR data in automated structure analysis. employ a detailed and accurate tick off of prophetic rules 29 for saturated, acyclic amines, we have produceed a computer syllabus which can reason out the structure of such an amine, showtime from its empirical facial elicition and CMR spectrum. A kindred effort is proposed for the steroids (at least, those containing endocyclic double bonds, carbonyl groups and hydroxyl substituents) in which structural information would be inferred from CMR data apply accurate prophetical rules. This in formation could then be structured with the results obtained from derivatization or excess CMR techniques, 9- and (if necessary) from MS analysis to yield possible structures. Not only would such a system have substantial utility, but it would represent an important advance in the state of the art in both(prenominal) CMR spectroscopy and chemical information-processing. A. CMR Spectra of Steroids We plan to get laid the series of sterols by synthesizing emailprotected, emailprotected, 74-, 9+, lbc-, 14ti-, 16x- and 176- androstanols or cholestanols, whose CMR spectra (pulsed Fourier-transform spectra, obtained at 25 Mhz. utilise noise modulated proton decoupling) go away be record and assigned. We have worked out likely synthetic pathways for the pay off of these using commonly accredited procedures and starting from compounds available in our laboratories. In order to test the extent of additivity relationships and of unhomogeneous interactions of substituents, we shall confusablely combine and record the spectra of twain or trio dozen dihydroxy and keto-hydroxy androstanes and cholestanes. The candidates chosen provide bet upon the results of the analysis of the monofunctional steroids. Using statistical procedures similar to those of Dalling and Grant, 30 and of Lindeman and Adams, 31 we shall attempt to tally s/structural variables with chemical shifts, the terminal being the line of an accurate set of substituent parameters for steroids. In assessing the effects of steric crowding and osseous distortion, we plan to hire a computerized, classical-mechanical warning of the molecular structure, such as the Westheimer-type models recently reviewed by Schleyer. B. Derivatization We propose to analyze the changes in C-13 shifts which take place when the hydroxyl group in several of the above androstanols and cholestanols is derivatized to the 2,4,6-trimethylbenzene, trifluoroacetate and trimethylsilyl ether. We propose similarly to check out the effects of ketalizing (with ethene glycol) several androstenone and cholestanones. We propose to analyze the effects of lanthanide shift reagents (in varying concentrations) upon the CMR spectra of several of the hydroxy- and keto-steroids, with particular accent upon pairs of sterols which differ only in the orientation of the hydroxyl group. These investigations give be directed toward the development of a repertoire of non-destructive, chemical methods for increasing the information available from CMR spectra. C. Computerized interlingual rendition of CMR data there are three phases to our proposed research in this area, all of which will make use of the heuristic platformme techniques authentic in our DENDRAL project. First, we guess to develop a course to assist in the assignment of spectra obtained in arts A and B, using currently available techniques (i.e., using rules for acyclic systems together with analogies from appropriate model systems). The purpose here is twofold On one hand, such a program will energise a time-consuming procedure (in our work, the assignment of spectra requires about as much time as the preparation of samples and the recording of spectra, combined), composition on the other hand, it will provide a setting within which to develop techniques applicable to the more difficult problem of structure identification. Specifically, we will need methods for express ing CMR rules as good computer code, and for decision making whether a good, obvious fit occurs between predicted and observed data. Secondly, we intend to utilize the rules derived in part A, together with derivatization information from B, to economize what is called a think program in the DENDRAL terminology. Such a program is designed to examine the spectrum of an foreign and, referring to a set of heuristics, to attempt to see the presence or absence of specific structural features in the unknown. Whereas the predictive rules allow one to pred ict a spectrum from a given structure, the heuristics represent transformations of the rules which allow one to infer structural information from a given spectrum. The native challenge in constructing the planning program will be the design of heuristics which are as informative as possible, so far which run efficiently. This program will be a useful analytic tool in itself and will be employ in the triplet phase of our proposed research. This ordinal phase will involve conflux the planning program with the existing DENDRAL system, which analyzes MS data for steroids. Modifications will be made to the structure contemporaries program, which can construct all possible sets of acyclic substituents from a given set of atoms and attach those substituents in all possible ways to a given cyclic skeleton. The structure beginning now makes use of IISplanner information, constructing only those steroids which are consistent with it. We shall diversify the algorithm to make use of t he fruit from both the MS and the CMR planners, and shall extend the algorithm to consider questions of stereochemistry, which are currently ignored. We moot that the augmented DENDRAL system will have the capacity to identify, unambiguously, the structures of a wide descriptor of steroids using information from just these two spectroscopic sources. The programs will be scripted in the enounce language, and will thus be matched with the rest of the DENDRAL system. Computer time on the PDP-10 will be provided through the NIH-funded SUMEX forwardness at Stanford, and we indicate no put up in this final cause for computer facilities. Programs developed in our proposed research will be available to the scientific community over the ARPA computer network.