(17)J. D. Winefordner and M. Tin, Anal. Chim. Acta, 32, 64 (1965). (18)C. Thiery, J. Capette, J. Meunier, and F. Leterrier, J. Chim. fhys. Physicochim. Biol., 66, 134 (1969). (19)“British Pharmacopaeia.” 1973. (20)R. J. Lukasiewicz, P. A. Rozynes. L. B. Sanders, and J. D. Winefordner, Anal. Chem., 44,237 (1972). (21) P. Turano and W. J. Turner, J. Chrornatogr.,75, 277 (1973).

(22)L. A. Gifford, J. N. Miller, D.T. Burns, and J. W. Bridges, J. Chromatogr., 103, 15 (1975).

RECEIVEDfor review November 7, 1974. Accepted April 28, 1975. We thank the Medical Research Council for the award of Project Grants in support of this work.

Determination of Anomeric Configuration of D-Ribofuranosyl Nucleosides from Nuclear Magnetic Resonance Spectra by a Pattern Recognition Technique Jure Zupan, Joie Kobe, and Dugan Had% Chemical Institute Boris Kidriz and Faculty of Natural Sciences, University of Ljubljana. Hajdrihova 19, 6 1000 Ljubljana, Yugoslavia

The difficulty of obtaining an unambiguous answer as to the anomeric configuration of ribosides prompted us to apply the pattern recognition method to this problem. The recently developed criterion ( I ) based on the differences of the NMR chemical shifts of the two methyl groups of the 2,2-dimethyl dioxolane ring has certainly facilitated decisions in the nucleosides series. We felt that the extension of this problem to the riboside series in general, independently of the anisotropic influence of the anomeric substituents to the methyl groups, might be successful if the following parameters were used: the chemical shifts of the l’,2’, and 3’ ribose protons and the corresponding 3 J H H vicinal coupling constants 3J1,2,and 3 5 2 , 3 , (Table I). It might be argued that the parameters used are not independent of the ribose ring puckering and the possible conformational state (syn-anti). Therefore, the chemical shifts within the limits given in Table I were chosen and the values of the coupling constants cover the almost complete number of predicted 3 5 H H for various compositions of conformational equilibrium mixtures of N and S conformers (2, 3 ) . To the best of our knowledge, the chemical shifts limits should be satisfied by the requirements, because neither the difference A6 due to the anisotropic effect of the base and/or substituents nor the effect of the electronegativity of the substituents fall out of our frame. Even the dependence of the constants on the nature of the substituents in nucleosides as well as theoretical predictions do not prevent this application (4).A pattern recognition technique was recently applied to various kinds of structural problems based on different data collections, e.g., mass (5, 6),IR (7-9), NMR (10-12) spectra, search for possible anticancer drugs ( I 3 ) , etc. Table I shows the organization of our data base which

consists of 552 computer simulated spectra, representing R, or Rg patterns. The simulation was carried out using the LAOCN3 computer program for the NMR spectra simulation, developed by A. A. Bothner-By and S. M. Castellano. All 552 simulated NMR spectra were digitalized using digital resolution R of 1 Hz and then transformed using the formula_ suggested by Kowalski and Reilly (IO) into the vector A (0, R, 2R, 3 R , . . .):

and F ( f ) is the digitalized NMR spectrogram as a function of frequency f. The summation over f was cut oftat 750 Hz. The problem was to defin? a decision vector W in such a way that a dot product (W-A) will give positive or negative values for A representing R, or Rb configuration, respectively. About 100 vectors, randomly chosen out of the set of 552 vectors A, were selected as the training set. The training begag with an arbitrary vector W of the same dimension as 4. In order to, improve \rtT, each incorrect answer (giving (W-A) < 0 for A represzntins R, or vice versa) causes a feedback of the form (5):W = W cA

+

so that the new vector W gives a correct classification. The “learning“ process was stopped at such \rtT that all training vectors were classified correctly. After the training proce-

Table I. Representative Chemical Shifts and Coupling Constant Intervals for R,, and R,j Patterns R, Interval, Hz

61

62 63 512 52 3

1702

650-700 450-600 550-600 4-5 6-7 All together

Step, Hz

R0 Interval, Hz

No. of cases

25 3 25 7 25 3 1 2 1 2 3.7.3.2-2 = 252

ANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975

51 62 63 J12 J2 3

600-700 525-625 550-650 0-1 4-5 A l l together

Step, Hz

No. of cases

25 5 25 5 50 3 1 2 1 2 5.5.3.2’2 = 3 0 0

Table 11. Dependence of the Prediction Ability on the Dimension of the Decision Vectora Dimension of the decision vector No. of training vectors N o , of test vector Wrong answers Prediction ability, %

4

11

21

51

101

151

201

251

110

100

106

100

105

107

107

106

442

452

446

452

447

445

445

446

104 76.4

0 100

0 100

Dimension of the decision vector is n

12 97.2

12 97.3

5 98.9

14 96.8

7 98.4

+ 1taking n from Equation 1.

dure was completed the obtained decision vector W was used to classify the remainder of vectors and 75-100% correct answers (depending on the dimension of vector W) were obtained. The results are collected in Table 11. The number of iterations required for training did not exceed 151 in any case. Although the prediction ability is very good, the fact that both anomeric configurations R, and Ra are represented with only 252 and 300 spectra may not be overlooked. For more accurate or finer grade coverage of the peak regions in the NMR spectra of R, or Rp patterns, many more vectors should be used. For example, by simulating NMR spectra of 4 shifts and coupling constants with only 5 different values of each item, we get about 10 million patterns. Furthermore, the prediction ability was not tested as yet on real spectra. However, this work is in progress and will allow the final verification of the practical value of the method. Apart from this shortcoming, we believe that the method itself is highly selective for problems of this kind and it could be of great help in tracing the anomeric configuration of ribofuranosides, especially in the nucleoside se-

ries, irrespective of the type of the blocking groups used during the reaction. LITERATURE CITED (1)J. L. Imbach, J. L. Barascut, E. L. Karn. and C. T. Tapiero, Tetrahedron Led., 129 (1974). (2) J. D. Stevens and H. G. Fletcher, J. Org. Chem., 33, 1799 (1968). (3)C. Altona and M. Sandaralingarn, J. Am. Chem. SOC.,95, 2338 (1973). 40, 144 (1973). (4)C. Giessner-Pattre and E. Pullman, J. Theor. Bo/., (5) T. L. lsenhour and P. C. Jurs, Anal. Chem., 43 (lo),20 A (1971). (6)J. E. Justice and T. L. isenhour. Anal. Chem., 46, 223 (1974). (7)E. R. Kowalski, P. C. Jurs, and T. L. Isenhour, Anal. Chem., 41, 1945 (1969). (8)R. W. Lindell and P. C. Jurs, Appl. Spectrosc., 27, 371 (1973). (9)D.R. Preuss and P. C. Jurs, Anal. Chem., 46, 726 (1974). (10)B. R. Kowalski and C. A. Reilly, J. Phys. Chem., 75, 1402 (1971). (11)R. E. Carhart and C. Djerassi. J. Chem. SOC., Perkin Trans. //, 1753 (1973). (12)C. L. Wilkins, R. C. Williams, T. R. Brunner, and P. J. McCambie. J. Am. Chem. Soc., 96, 4182 (1974). (13)E. R. Kowalski and C. F. Bender, J. Am. Chem. SOC.,96,916 (1974).

RECEIVEDfor review October 21, 1974. Accepted February 18, 1975. Financial support of the Boris KidriE Fund is gratefully acknowledged.

Simplified Method of Calibrating Thermometric Nuclear Magnetic Resonance Standards M. L. Kaplan, F. A. Bovey, and H. N. Cheng Bell Laboratories, Murray Hill, N.J. 07974

One of the more difficult aspects of performing reliable rate measurements by nuclear magnetic resonance techniques is the determination of true sample temperatures. Various methods, including the placement of a thermocouple in the probe or in the temperature regulating gas flow, suffer from the inherent defect that recalibration is often necessary, especially when probe geometry or gas flow rates are changed (1 ). Perhaps the most generally used mode of temperature measurement depends on a secondary standard with a temperature-dependent chemical shift covering a wide temperature range. These criteria are met by the hydroxyl proton in methanol for temperatures below ambient, and by the hydroxyl protons in ethylene glycol for high temperature work. Although, in principle, accurate measurements can be made with these compounds, the standardization of chemical shift as a function of temperature has relied upon some thermocouple method, such as those mentioned above.

Various authors have reported the calibration of these secondary standards. For example, when a methanol sample was calibrated, the thermocouple was placed in a capillary inside a nonspinning sample tube also containing a capillary of methanol ( 2 ) .Large vertical temperature gradients were observed which made thermocouple placement critical. Accuracy was also significantly limited by broadening of the spectral lines due to introduction of the thermocouple. More sophisticated apparatus, which included a specially designed static thermistor probe, permitted the sample tube to be spun during temperature measurements ( 3 ) .The same author has reported on measurements with a spinning thermistor ( 4 ) but states that the static thermistor is more accurate ( 5 ) . Still others have utilized a melting point method for calibrating a t 100 MHz, both ethylene glycol and methanol standards supplied by the manufacturer of their spectromANALYTICAL CHEMISTRY, VOL. 47, NO. 9, AUGUST 1975

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