J. Biochem., 73, 763-770 (1973) Calorimetric Studies on the Mutarotation of D-Galactose and D-Mannose* Katsutada TAKAHASHI** and Sozaburo ONO Laboratory of BiophysicalChemistry, College of Agriculture, University of Osaka Prefecture, Sakai Receivedfor publication, September 11, 1972 1. Calorimetric measurements were made on the heat change accompanying the mutarotation on D-galactose and D-mannose to evaluate quantitatively the anomeric stability of the two sugars in aqueous solution. 2. It was found that in D-galactose the ƒÀ-anomer is 1,300•}50 J mol-1*** energetically more stable than the ƒ¿-anomer, while in D-mannose the a-anomer is 1,900•}80 J mol-1 more stable than the ƒÀ-anomer at 25•Ž. 3. From stereochemical considerations regarding D-mannose, D-galactose, D-glucose, and D-xylose, it was assumed that the hydroxyl group on C2 in the pyranose ring plays a major role in determining the preferred form of the anomeric pairs. 4. By combining the data with those reported for the isomerization of eq-D-glucose to eq-D-mannose, the energies required for the conversion of a hydroxyl group on C2 of a-D-glucose and ƒÀ-D-glucose in chair-1 form from equatorial to axial were esti- mated to be 7,950 J mol-1 and 10,880 J mol-1, respectively. Conformational stability of pyranose rings has for each a-ƒÀ anomer pair the predominant been considered by Reeves (2) on the basis component in an equilibrium mixture can be of substituent effects. He concluded that any deduced from weighing the above instability substituent other than hydrogen oriented per- factors. In this connection, Sundararajan pendicular to the pyranose ring, introduces and Rao (3) made a potential energy calcu- an instability into the conformation, and that lation for various hexo-, and pentopyranoses in gaseous molecular models using the Kitay- * Presented in part at the 2nd Japanese Calorimetry gorodsky functions ( 4 ). Conference, Tokyo, November 1966 and in part at However, it seems that changes in the the 20th Annual Meeting of the Chemical Society relative position of substituents in pyranose of Japan, Tokyo, April 1967. ** To whom corre- rings have not yet been quantitatively related spondence should be sent. *** In accordance with the decision of IUPAC (International Union of Pure and to the stability of anomers in solution. Applied Chemistry) in 1969 concerning the use of One of the most common experimental symbols, terminology and units ( 1 ), all the thermo- approaches to this problem is obviously the dynamic quantities are given in Joule units, J, in investigation of differences in the ther- this article. For conversion from Joules to calories, modynamic quantities of the anomers. the coefficient 4.184 J cal-1 may be used. For some sugars it is possible to determine Vol.73, No.4, 1973 763 764 K. TAKAHASHI and S. ONO, the thermodynamic quantities ; while free ed polarimetrically by using the following energy difference between ƒ¿- and ƒÀ-anomers values for optical rotation in water at 20•Ž in solution may be calculated from the con- (8, 9); centration ratio of a to ƒÀ in the equilibrium α一D-galactose 【α】D=十144 mixture, enthalpy change can be obtained β一D-galactose [α]D=十52 most accurately by direct calorimetric meas- α-D-mannose [α]D=十30 urement of the heat changes during the β一D-mannose [α]D=-17 mutarotation process.* Direct calorimetric measurements of mu- It was found that the composition of the tarotation have been made on D-glucose by D-mannose sample was 83% a-anomer and 17 Sturtevant (5) and on D-glucose, D-xylose, % ƒÀ-anomer, * while that of D-galactose sample: cellobiose, lactose, and maltose by Kabayama was 100% pure a-anomer, both within the. et al. (6). limits of analysis. In the present study, measurements were Apparatus and Procedure-The calori- calorimetrically attempted on the mutarota- meter used was essentially of a constant-tem- tion process of D-galactose and D-mannose to perature environment type (isoperibol type) obtain further information about the stability with a thermistor as the temperature sensor. of pyranose rings in aqueous solution. The structure of calorimeter is shown in Fig. 1. A Dewar vessel (D) , about 50 cm3 in vol- EXPERIMENTAL ume, provided with a bakelite lid(L) having Materials-Both the sugars studied were a thermistor(T), a heater(H), and a stirrer(S), of the purest grade, purchased from Wako served as a reaction cell. Pure Chemicals, Osaka (D-galactose, mp The thermistor(T) (CS 503, TOA Electro- 167•Ž) and from Shimakyu Pure Chemicals, nics Ltd., Tokyo) with B-values of 3,200 •‹K. Osaka (D-mannose, mp 132•Ž). They were and resistance of 52.41 kƒ¶,at 25•Ž was con- used after vacuum drying at 80•Ž for three nected to a certified DC-Wheatstone bridge: days. The anomer compositions were examin- placed in an air-thermostat. The potential. derived from the bridge was amplified and . continuously recorded. The sensitivity was. 28.65 mV•EK-1 at 25•Ž. As the temperature change is small (several hundredths of a de- gree), a linear relationship between the tem- perature rise and the bridge potential has been assumed. The heater (H) made from platinum wire: having a resistance of 24.509 ƒ¶ at 25•Ž was used for calibration, i.e., determination of the heat capacity. The heater was also used for compensation of the large endothermic heat of sample dissolution, which should be dis- tinguished from the heat change due to mutarotation. Fig.1. Calorimeter structure. * Because of technical reason in doing calorimetry , a material which shows the highest dissolution rate * In some cases it is also possible to deduce the at the mixing with buffer solution was chosen out enthalpy values from the temperature dependence of several specimens. In the case of D-mannose of the equilibrium constant by using the van't Hoff study, however, the sample chosen was unfortunately relation. Such data, that are less reliable, are given not in a pure anomer form but was a mixture of in the review of Isbell and Pigman ( 7 ). α-and β-anomers. J. Biochem. CALORIMETRY ON MUTAROTATION OF SUGARS 765 The stirrer(S) was a glass propeller con- tion was durated for 5 min* and the rotation nected to a synchronous motor. Its rotation rate was then reduced to 300 rpm. The bridge speed could be adjusted in two steps; 300 and output voltage was recorded for 40 min and 2,000 rpm. The higher rate was employed the temperature-time curve, which was due only for sample dissolution at the initiation to mutarotation alone, was analyzed by apply- of the calorimetric run. ing the kinetic equation, including the mu- An accurately weighed sample (2 to 5 g) tarotation rate constant described later. was placed in the Dewar vessel(D). The buffer Mutarotation Rate Constants-There seems solution (20 cm3, 0.025 M acetate buffer, pH to be some evidence that the mutarotation of 4.5) was held in a cylindrical buffer holder certain sugars does not follow simple first- (B) which was suspended from the lid(L) by order kinetics (10). Isbell and Pigman (11) a control shaft(C). The holder had a thin reported in their study on sugar mutarota- rubber film(R) at its bottom which was broken tions made in 1937 that both D-galactose and by a needle tip(N) when the holder was raised D-mannose show a complex mutarotation in up via the control shaft(C). Because of the water. However, in the present study we tensile force of the rubber film, the bottom have carefully performed measurements on of the cylindrical holder(B) was fully opened these two sugars in buffered solutions of at the instant of film breakage so that the various pH values using an advanced recording buffer solution was allowed to contact quickly polarimeter, and we found that their mutarota- the whole of the sugar. tions obey a first-order law over a pH range Since the rate of mutarotation was de- from 3 to 7 and show the minimum rate at pendent upon pH, measurements were per- pH 4.5. formed with a buffered solution at pH 4.5, The polarimeter used was a Yanagimoto where the mutarotation obeyed first-order ORD-185 spectropolarimeter with a quartz kinetics with the minimum rate constant. cuvette of 1 cm optical path. The change in After the whole assembly was put in place, optical rotation with time was recorded at the the calorimeter was submerged in a water fixed wavelength of 370 nm and was analyzed thermostat at 25•}0.001•Ž and left for three by the Guggenheim method (12), which is to four hours. commonly used for the analysis of reaction When thermal equilibrium had been kinetics. From a Guggenheim plot of the reached, as judged from the recorder chart, the optical rotation curve, the rate constants of rotation rate of the stirrer was raised to 2,000 mutarotation in buffered solution at pH 4.5 rpm and electrical heating was started by were obtained as 0.03205 min-1 and 0.06496 sending a known amount of electric current min-1 for D-galactose and D-mannose, respec- (273 mA). Five seconds later the control shaft tively, at 25•Ž. (C) was raised to break the rubber film for sample dissolution. THEORY Electrical heating to compensate for the endothermic dissolution heat was durated for Kinetic Analysis of the Temperature-time 12 to 23 sec. The compensation was control- Curve-If one defines g(t) as the observed tem- led almost empirically by watching the am- perature change at time t in the calorimeter, plifier scale in such a manner that the bridge the true temperature change of a thermal output voltage finally attained nearly zero.