Deuterium Isotope Effects in a - F3 Pyranose and in Pyranose-Furanose Interconversions1

Home , Pyranose

JOURNAL OF RESEARCH of the Nationa l Bureau of Standards - A. Physics and Chemistry Vol. 71A, No.2, March- April 1967 . Deuterium Isotope Effects in a - f3 Pyranose and In Pyranose-Furanose Interconversions1

Horace S. Isbell and Clarence W .. lt Wade

Institute for Materials Research, National Bureau of Standards, Washington, D.C. 20234

(Decembe r 15, 1966)

Rates of Illutarotation catalytic coeffi c ient s, a nd isotope e ffects a re re port ed for the mut arotati o ns o(a-D.xylose, a-D·glucose, 'and ,B-D·fruc tose in H 20 and in D20 at 20°C. The isotope effects (kH /k/J) for the muta rotation of ,B-D·fructose (a pyranose·furanose interconvers ion) parallel in striking manne r the isotope e ffect s for the mutarotation of a-D·glucose (an a-,B pyranose anomerization). For each sugar, the isoto pe effect is lowest for the acid·catalyzed reaction, and highest for the water-catalyzed reaction. The parall e li sm of the values o bt ained for the isotope effects unde r various conditions shows that the rat e·dete rmining ste ps in the tw o reactions are s imilar. Presumably, in both instances, the ove~all mutarotation ari ses from concurrent reacti ons o pe rating on differe nt s pecies of the sugar and shoWIng substanti a ll y diffe rent isotope effects. The g radual change in th e isotope effect indicat es that, under the conditions studied, three reactions take place conc urrently. The follo wing isotope effects we re found for the mutarotations at 20 °C: For a-D·g1ucose, kH"o+/kn"o+ = 1.39: kH ,O/ko,o = 3.87; and kB/k~ = 1.83. For ,B-D·fructose, kH ,o +/k'hO + = 1.39; kH,o/kD,o = 3.87; and kB/k~ = 1.92. Mechanisms are presented for the several concurre nt acid· and base·catalyzed mutaro· tation reactio ns.

Key Words: Acid-base catalysis in D2 0, deut erium isotope e ffects, D·fructose, D·glu cose, isotope effects, mechani s m of mutarotation, mutarotat ion , pyranose·furanose int erconver· sions, s ugars in solution.

1. Introduction catalysts. It has been tacitl y assu med that both a-f3 pyranose anomerizations and pyranose-furanose inter The mutarotation process is known to involve the conversions take place through a common, acyclic reversible interconv ersion of several modifications intermediate. However , other mechanis ms are pos of the sugar [1 , 2].2 For mo st sugars, this results sible, especially anome ri zation without ring opening, in an equilibrium that may include two pyranose or a change in ring size by intramolecular ring con forms, two furanose forms, the acyclic form, and traction or expansion. In view of these possibilities, solvated and ionic modifications of these. The muta it seemed desirable to study and compare the two rotation of D-glucose consists almost entirely of an types of mutarotation, in H2 0 and in D2 0. Prior to a-f3 pyranose interconversion (anomerization) . The the present work, others [5, 6, 7, 8] had studied the mutarotations of D-galactose, D-talose, D-ribose, D mutarotation of D-glucose and certain other a-f3 glycero-D-ido-heptose, and certain other sugars give pyranose anomerizations by means of deuterium clear evidence for two types of reaction, namely , a-f3 isotope-effects, and had obtained important infor pyranose anomerization and pyranose-furanose inter· mation on the reaction mechanisms, but no pyranose conversion [3]. The mutarotation of D-fru c tose, furanose interconversion had been so investi gated. however , arises in large meas ure from a pyranose For this reason, we undertook a comparative study of fura nose interconversion [4]. isotope effects in the mutarotation of D-glucose, a The a-f3 pyranose anomeri zations have been investi typical a-f3 pyranose anomerization, and the muta gated thoroughly, but relatively little work has been rotation of D-fructose, a typi cal pyranose-furanose done on pyranose-furanose interconversions. Isbell interconversion. and Pigman [4] showed that the two types of reaction The object of the work was to elucidate the mech differ in their heats of reaction, activation energies, anisms for pyranose-furanose interconv ersions and rates of reaction, and sensitivity to acid and base to show how these reactions differ from a-f3 pyranose

1 Abstracted. in IJarl. from a thesis s ub milled by Cla rence \V. H. Wade 10 Georgetown anomerizations. In the work re ported here, methods Univ ersit y in May 1965. in parti al fulfillme nl or Ihe requiremenl s ror Ih e degree of Doctor or Ph..ilosoph y. were successfully developed for preparing and purify 2 Fi gu res in brackets indicale the li le ral u re rere re nces al the end or Ihis paper. ing the O-de uterated analogs of a-D-glucopyranose,

137 244 - 1420-67- 4 0- H --- B 0 B----H - 0 / ~ HC SLOW HC RAPID "CH ~ , I 0--- HA , !""I 0--- HA :/:"" RAPID SLOW i/ HC HCOH HC I I I + HB + A-

FIGURE l. Concerted mechanism of Lowry [10]. a-o-xylopyranose, and ,B-o-fructopyranose. Accurate vent isotope-effects. Ordinarily, kinetic isotope-effects rates of mutarotation were determined for the 0- are greater than unity, whereas solvent isotope-effects hydrogenated sugars in H 20 and for the O-deuterated are less than unity. Hence, the overall isotope-effect, sugars in D20 in the presence of certain acid and base kH/kD' may have a value smaller than, equal to, or catalysts. From these data, catalytic constants and greater than unity. Kinetic isotope-effects depend on deuterium isotope-effects were calculated. Mech differences, in the H2 0 and D20 systems, in the anisms were formulated for the mutarotation reactions, energy involved in the alteration of bonds in the tran and these are discussed in relation to isotope effects. sition state; in general, the stronger the altered bond, Numerous workers have sought to explain how the greater the kinetic isotope-effect [18, 19, 20]. mutarotation reactions take place. Lowry and co Usually, the heavier isotope produces the lower reac workers [9] studied the mutarotation of tetra-O-methyl tion rate; hence, isotope effects greater than unity are o-glucopyranose in solvents of low dielectric constant termed "normal," and those less than unity are termed and found that the reaction is slow in dry pyridine, or "inverse_" A large, normal isotope-effect indicates in dry cresol, but is fast in a mixture of the two sol that a proton is transferred in the rate-determining vents, or in either solvent when moist. From these step, with rupture or formation of a bond. observations, Lowry [10] concluded that, for muta Solvent isotope-effects have their origin in differ rotation to occur, both an acid and a base must be ences, in the behavior of the reactants, that are due to present. He then proposed that the mutarotation of differences in the isotopic solvents; usually, they are o-glucopyranose involves a ternary reaction of the inverse effects. The ion product of D20 at 20°C is sugar, an acid, and a base, as shown in figure L The 10-15.05, whereas that of H20 is 10-14. 17 [21]. Thus, a rate-determining step was assumed to be the opening water molecule has a greater tendency to form an of the ring by the simultaneous attack of a base H:tO ion than a D20 molecule has to form a D:tO ion. catalyst and an acid catalyst to form the acyclic Because of the difference in the base strengths of aldehyde. the solvents, a sugar molecule is able to compete more The mechanism involves simultaneous transfer effectively with the D20 molecule for the deuteron of a proton from the acid to the sugar and of a different than with the H 20 molecule for the proton. This proton from the sugar to the base, with a rearrangement situation leads to a higher concentration of the con of the valence electrons. In aqueous solutions, jugate acid of the sugar in D2 0 than in H20 [22]_ The water molecules provide both the acid and the base same situation exists in certain other reaction systems. functions. The work of Swain and Brown [11] on the Examples are (a) the acid hydrolysis of sucrose [23]; mutarotation of tetra-O-methyl-o-glucopyranose in the (b) the base-catalyzed enolization of acetone [24]; (c) presence of the bifunctional catalyst, 2-pyridinol, and the acid-catalyzed decomposition of ethyl diazoacetate other evidence [12] support the concerted mechanism. [25]; and (d) the acid-catalyzed anomerization of acetyl However, there is considerable evidence for other ated sugars [26]. These reactions are subject to reaction-paths, especially for a two-step mechanism specific hydrogen (deuterium) ion catalysis, and the [5, 13, 14, 15, 16, 17]. Regardless of whether proton isotope effect, kH/kD' ranges from 0.39 to 0.69_ All of transfer is by a concerted or a consecutive (two-step) these reactions arise from solvent isotope'effects; mechanism, the overall process for the mutarotation they involve a rapid proton (deuteron) transfer from requires that the sugar molecule accept a proton from the catalyst to the substrate, followed by a rate an acid catalyst and release another proton to a base determining step with no proton transfer. Thus, these catalyst. The concerted and two-step mechanisms isotope effects arise from differences in the ·.concen differ primarily in the mode of proton transfer. Deu tration of the conjugate acid, and not from a rate terium isotope-effects, discussed in the next section. determining cleavage reaction. provide a means for testing various reaction Reactions showing kinetic isotope-effects differ mechanisms_ from those just cited in that they are more rapid in H20 than in D2 0 . Examples are (a) the decomposition 2. Discussion of Isotope Effects of nitramide [27]; (b) the mutarotation of sugars [5, 6, 7, 8]; (c) the bromination of 3-methyl-2,4-pentanedione In H 20/D20 systems, overall isotope-effects arise [28]; and (d) the hydrolysis of acetamide by strong from a combination of kinetic isotope-effects and sol- acids [29]. The latter reactions are subject to general 138 acid-catalysis_ In e ach instance, the kineti c isotope In the present investigation a striking paralleli sm effect is superimposed on a solvent isotope-effect. has been found in the values of kH2o l ko£], I.-I-I:

Numerous comparative studies have been made of a-o·GLUCOSE the mutarotation of a-D-glucopyranose in H20 and

D20. Pacsu [6] found that the rate of mutarotation Serial So lve nt 'Catalyst Conen. sug

Long and Bigeleisen [8] extended the theory of 14 H,O O.015N sulfuric acid .. 4 1.72 0.0353 Challis and co-workers by relating kH I ko and its mag 14' 0,0 0.015N s ulfuric acid .. 4 1.32 .0151 15 H2O O.OO IN K acid phthala te .. 4 4.40 .0201 nitude to (1) the strength of the catalyst, (2) specific IS' 0,0 O.OOIN K a cid ,)htha late .. 4 4.70 .00552 hydrogen-ion and general acid-base catalysis, and (3) il Similar measurement s in H20 a nd 0 20 a re numbered al ike. but a re distinguished by the exchange equilibrium between protons of the use of asteris ks. /) P P for the DzO solutions equals the p H. mete r reading + 0.4. solvent and protons at the reaction center of the sub ' (k, + k,) = I/t [log (f, - f x) - log (f, - f . JJ . strate. With these relationships mathematically represented, they accounted, on the basis of general acid-catalysis, for differences in the isotope effects for 3. Mutarotation Measurements acids of various strengths. They pointed out that the isotope effects found for catalysis by strong acids (L37) Mutarotation measurements, reported in table 1, and by acetic acid (2.6) are of the expected magnitude, were made for the normal forms and the O-deuterated and that the isotope effe ct found for water catalysis is fotms of a-D-glucopyranose, a-D-xylopyranose, and consistent with a reaction "in which the water molecule f3-D-fru ctopyranose, using H 20 and DzO solutions is acting simultaneously as a proton acceptor and containing acids and bases. The H2 0 and D2 0 solu donor" (i.e., the concerted mechanism). ti ons were designed to be molecularly equivalent, The isotope' effects and other experimental facts and the equivalence of the buffered solvents was com clearl y indicate that the mutarotation of D-glucose, pared by measuring the pH after dilution (to the same an a-f3 pyranose anomerization, takes place by acid composi tion) of the H2 0 solvent with D2 0, and of the catalyzed and base-catalyzed mechanisms, involving D2 0 solvent with H2 0. The mutarotation measure acyclic intermediates. ments were conducted in the conventional manner 139 [33] , and the equilibrium rotations, specifi c catalytic of D-fructose to acid catalysts. In weakly acid solution effects, and isotope effe cts were calculated and (region of minimum rate), the mutarotation ratios of compared. D-fructose to D-glucose are almost the same in H2 0 Comparison of the equilibrium optical rotations and D20 (9.2 and 9.4, respectively). This agreement (see table 2) of the sugars in H20 and in D20 shows shows that the catalytic effects of the H 2 0 molecule that D·glucose is slightly less dextrorotatory in D20 and the D2 0 molecule on the rates of mutarotation than in H20, whereas D·fructose is sli ghtly less levoro are alike. tatory in D2 0 than in H2 0. In strongly acid solution, the mutarotation ratios of D-fructose to D-glucose show large differences for TABLE 2 . Equilibrium rotations of o·glucose and o.Jructose in H2 0 and D2 0, but those of D-xylose to D-glucose do H2 0 and in D20 not. Thus, in 0.015 M sulfuric acid, the mutarotation ratios of D-fructose to D-glucose are 21.4 in H20 and Suga r So lvent gflOO ml °C os' [aJ" 30_8 in D20, whereas those of D-xylose to D-glucose are 3.43 and 3.37, respectively. The high mutarotation D-Glu cose H,O 4 20.0 + 12. 18 + 52.7 0 ,0 4 20.0 + 11.88 + 5 1.4 ratios of D-fructose to D-glucose in the alkaline region H,O 4 3.9 + 12.09 + 52.3 arise from the higher sensitivity of the mutarotation 4 0,0 3.9 + 11.80 + 51. 1 of D-fructose to base catalysts. The last two experi D-Fructose H,O 4 20.0 -21. 305 - 92.4 0,0 4 20.0 - 20.76 - 89.8 ments reported in table 3 were conducted on solutions H,O 4 3.9 - 23.41 - 1Ol.3 tnat differed slightly in alkalinity. This fact may 0,0 4 3.9 - 22.76 - 98.5 account for the seemingly anomalous values for the 11 The solutions were read" in a 2-dm tube with a Bates saccharim t: ter. using: a dichromate fructose/ glucose ratios. filter and either a white light or a sodium light. No significant difference was found in the reading obtained with the two lights. The readings were converted inl o'[a1o on the Because the mutarotations are affected to different assumption that 1 05 = 0.3462 a ngular degree. degrees by acids and bases, evaluation of isotope effects in D20 and H2 0 requires consideration of the specific catalytic effects of the catalysts present. 4. Catalytic Effects in a-f3 Pyranose Anomeri According to Bronsted and Guggenheim, and others zations and Pyranose-Furanose Intercon [34, 35, 36], the mutarotation constant (k t + 1.- 2 ) for a versions sugar in the presence of acid and base catalysts can Isbell and Pigman showed that pyranose-furanose be represented by an equation of the following type: interconversions are much more sensitive than a-f3 pyranose anomerizations to acid and base catalysts, and that the effect on the rate of mutarotation of small where the symbols in brackets represent the concen changes in acidity (or basicity) is greater for D-fructose trations of the catalysts. The term k H20 represents than for D-glucose or D-xylose. Therefore, it is of interest to intercom pare the rates of mutarotation of the catalytic effect of the solvent, water, and the coefficients kHAj' k Bn , etc., represent the specific these sugars in H20 and in D2 0 at various pH (or pD) values (see table 3). catalytic constants of the substances indicated in the The mutarotation ratios of D-xylose to D-glucose at subscripts. In aqueous solutions at various pH values, two widely different pH values (4.40 and 1.72) show the principal catalysts are water molecules, hydronium only a small difference in either H2 0 or D2 0. Thus, ions, hydroxyl ions, and sugar anions (S-), and the changes in the acid and base catalysts appear to affect mutarotation constant is represented by the following the mutarotation constants of D-xylose and D-glucose equation, from which the catalytic constants may be to almost the same degree. On the other hand, the calculated: mutarotation ratios of D-fructose to D-glucose show wide variation in both solvents over the same range of pH. The high values of the ratios in acid solution arise from the greater sensitivity of the mutarotation

TABLE 3. Relative rates oj mutarotation of Oi-o-glucose (C), {3-ojructose (Fru) and Oi-o-xylose (Xyl) at 20°C at The catalytic constants of the deuterium analogs may approximately equal acidities be obtained from a similar equation by substitution of deuterium for hydrogen. In evaluating catalytic effects, values of [H3 0+] and pH (or pO) of solution Ratio of (k l + k1.) Solvent Catalyst [D 30 +] were obtained by measurements with a glass C fru Xyl fru/C XyliC electrode at 20°C, assuming that pD = pH + 0.4_ The values of k H20 and k 020 given in tables 4 and 5 were H,O O.O]SN sulfuric acid ...... -. 1.72 1.72 1.72 21.4 3.43 estimated by plotting the mutarotation constants ob- ~ 0,0 O.01SN sulfuric acid ...... 1.72 1.72 1.72 30.8 3.37 H,O O.OOSN sulfuric acid ...... 2. 10 2.10 . 105. 7 . tained in acid solutions against the hydrogen ion (or I 0,0 O.OOSN sulfuric acid ...... •• .. 2. 11 2.11 22.5 . H,O O.002SN s ulfuric acid ...... 2.38 2.38 . 13.2 . deuterium ion) concentrations. In each instance, a 1),0 O.OO2SN s ulfuric acid ...... 2.40 2.40 . 17.3 . line was obtained whose intercept on the vertical axis H,O 0.001 N K H phthalate .. 4.40 4.40 4.40 9.2 3.17 1),0 O.OOIN K H phthalate ... 05.10 5.10 05.10 9.4 3.39 H,O O.OOO02N sodi um hydroxide .. 7.30 7.305 . 31.7 . 0,0 0.00002N sodi um hydroxide .. 7.80 7.73 . 24.5 . :I Although h- H 0 represents the catalytic ,-frect 01 tile solvent. water, the specific (molar) catalyti c consla~ t for the water molec ule, h-~20' is h- H.O/SS,S,

140 TABLE 4. CataL ytic constants a for rn utarotatio ns in H20 solutions T ABLE 5. CataLytic co nstants a for rnutarotations in D,O solutions ~ W~ ~W~

Serial k B" Serial I.: (. No. Nu.

n-D·G LUCOSE n-D·GLUCOSE

X 2 I 1.72 1.9 10 - I") 0.0103 (0.0063) 0.2 1 ...... 1* 1.72 1.9 X 10 - ' Id) 0.00448 (0.00163) 0 .1 50 ...... 7.9 x lO - c d 2 2. 10 I ) .0103 (0.0063) .20 . 2* 2. 1\ 7.8 X 10 - ' Id) . 00277 (0.00163) .146 . X 10- ' d 3 2. 38 4.2 I ) .0 103 (0.0063) .2 1 ...... 3* 2. 40 4.0 X 10- ' Id) . 00226 (0.00163) .158 .. d 4 4.40 I ) I") .0103 (0.0063) .. 4* 5. \0 Id) 1.1 2 x 10- 10 .00 169 (0.00163) ... 5 7.30 Id) 1.35 X 10 - ' . 0103 (0.0063) .. 1. 1 X 10" 5* 7.60 I") 3.55 x 10 - ' .00 177 (0.00163) .. 3940 6 6.70 Id) 3.39 X 10-' . 0 103 (0.0063) . 0.9 X 10' 6* 7.80 (") 5.62 x 10 - ' . 00 198 (0.00 163 . 6230 7 9.45 (d) 1. 91 X IO- ' . 0103 (0.0063) .. 0.9 X 10' 7* 10.20 (d) 1. 4 1 X 10 - ' .0669 (0.00163) .. 4630 Average (0.0063) 0. 2 1 0.9 X 10' Av e rage (0.00 163 ) 0. 157 4930

(3-D·FR UCTOSE (3- D.FRUCTOSE

X d 8 1.72 1.9 10 - ' I ) 0.220 (0.058 ) 8.5 ...... 8* 1.72 1. 9 X 10 - ' Id) 0 .1 380 (0.015 6.47 . 9 2. 10 7.9 x lO - ' (d) . 124 (0.058 ) 8.4 . 9* 2. 11 7.8 X 10 - ' (") . 0623 (0.015) 6.06 . \0 2.38 4. 2 X 10 - ' Id) .0952 (0.058) 8.9 . 10* 2.40 4.0 X 10- ' (d) .0390 (0.01 5) 6.00 . II 4.40 (<0) (<0 ) ... .0580 (0.058 ) \1 * 5. 10 Id) 1.1 2 X 10 - " .0 153 (0.0 15 . (d) X 12 7.35 1. 5 1 10 - ' .248 (0058) . 1. 3 X 10' 12* 8.62 Id) 3.72 X 10- ' . 3320 (0.0 15 . 8.5 x 10' 13 6.70 (d) 3.39 X 10 - ' . 11 8 (0.058 ) . 1.8 X 10' 13* 7.73 (") 4.79 X 10- ' .0 485 (0. 0 15 .. 7. 0 X 10' Average (0.058 ) 8.6 1. 5 X 10' Ave rage (0.01 5 6. 19 7.8 X 10'

n-D·XYLOSE noD-XYLOSE

14 1.72 1. 92 X 10 -' 0.0353 (0.020 1 0.7 ...... 14* 1. 72 1.9 X 10 - ' Id) 0.0151 (0.0055 0.5 ...... 15 4.40 (d) .020 1 (0.020 1 ...... 15* 5. 10 (It) (d) .0055 (0.0055 Average (0.020 1 0.7 ...... Average (0.0055 0.5 ......

Ii (k l + /'2) = k ll ,o + k 1I ,,0 ,+[H :10 " J+ ku '[OH - }. '(k; + k; )= 1.- ",0 + k " ,0 , [D"O ' I + k; rOD - ]. ".Calcul ated fro m p H. assumi ng tha i the ion product a l 20 °C is 10- 1.. . 11 , "Calcul at ed fro m pD . assumin g Ih at the ion product at 20 °C is 1O - 1 ~. o 5 . ~ kB[OH- ]= ks- IS - ] + kml- IOH - j. /;u is concentration-de pe nde nt , as shown in eqs (2) I < k : [OD - I= k ~ _IS- I + /'"" -IOD - I. and (8). d CO ll c e ntra tion of catalys t considered negli gible. d Concent rati on of catalyst cons ide red negligible. was kH20 or ko,o . The values of kHaO+ and kOao+ were In neutral and alk alj ne solutions, catalysis by hydrogen calculated from the measurements in acid solutioll , (o r deuterium) ions is negli'gible, and values for kB and assuming that k ~ were calculated from the equations

(4) and and

(5)

In the calculati ons, the values for kH ,o and k D20 were The values of kH,o, k020 , kHp + , and ko"o+ found for D-glucose are in fair agreement with values reported derive d as previously described, and those for [OH - ] by Hamill and La Mer [7] and other workers [34, 35, and [OD-] were obtained from pH or pD measure me nts, on the assumption that [H 30 +][OH- ]= 10- 14. 17 , 36]. No similar data have heretofore been reported for the mutarotation ot D-fructose. and that [D 30 +][OD- ] = 10- 15.05 r21]. Evaluation of the catalytic constants " OH- and " s The values of kB and k ~ found by solving eqs (4) is complicated by the fact that the mutarotation reac and (5) are given in tables 4 and 5. The catalytic tio n becomes too rapid for accurate measurement in constants were then used to obtain the isotope effects gi ven in table 6. The results show a striking parallel hydroxyl ion concentrations greater than 10- 6• Sepa rate determination of koH- [OH - ] and k s- [S - ] is ism in the isotope effects for the water-catalyzed, the acid-catalyzed, and the base-catalyzed mutarotations especially difficult because the latter term is over of a-D-glucose and j3-D-fructose_ In each instance, shadowed by the fo rmer. Hence, we re presented the the isotope effect is lowest for the acid- catalyz ed re overall effect of base catalysis in H 20 with hydroxyl acti on and highest fo r the water-catalyzed reacti on. ion concentration [OH] as: The parallelism supports the hypothesis that the two reactions, one an a -j3 pyranose anomeri zati on and the other a pyranose-furanose interconversion, take place by similar mecha ni s ms. Isotope effects had not been reported previously for the mutarotation of D-fructose. As already mentioned , the catalytic constant k B for base catalysts includes the effect of the hydroxyl ion

141 and that of the sugar anion. Necessary data are TABLE 7. Calculated catalytic effects for the sugar anions in the available for the separate evaluation of the two effects mutarotations of a-D-glucose and f3-D-fru ctose in water at 20 °C in H20 but not in D20. Catalytic Conen. [5- 1' Catalytic effect for Percent Seri al No. S ugar mole/l pH lll oJe/l co nstant s ugar of (k, + k,1 TABLE 6. Isotope effects in the mutarotations ofD-glucose, D-fructose; ks- a nio n and D-xylose at 20 °C a ks-[S -]

Sugar k lhO l kp~ kH,o ... /k[}so+- kH /;": 5 o-Gl ucose 0.22 7.30 1O- s.6 1 45. 0.00011 1.7 6 1.00 6.70 10 - 5.~ 45. .00013 2.1 1.00 12.25 0.50 (0) a-o-Clucose 3.87 1.39 1.83 {J-D-Fructose 3.87 1.39 1.92 12 D-Fructose 0.22 7.35 10- 5 .31 330. 0.00162 2.83 a-D-Xylose 3.65 1.40 13 1.00 6.70 10- 5 .30 330. .00166 2.86 1.00 12.00 0.50 (0) a The values are based on the averages for the catalytic constant s given in tables 4 and 5. a Calculated from the pH. the concentration of the sugar, and the acid dissociation COll sta nt of the sugar. II Mutarotation too rapid to be de termined. According to Bronsted [37] the catalytic constant for a base is given by the following expression: A simple calculation shows that the relative impor tance of catalysis by the sugar anion depends on the (6) concentration of the sugar. Thus, if K is the acid dissociation constant of the sugar, and Kw is the ion where G and yare parameters evaluated from measure product of water, then ments with a series of comparable base-catalysts, and KB is the reciprocal of the dissociation constant [S-]=K [HS]=K [HS] [OH-] of the conjugate acid. From the measurements of [H30 +] Kw Bronsted and Guggenheim [36], y has a value of about 0.4 for the base-catalyzed mutarotation of D-glucose. and The values of G for the mutarotation of D-glucose and D-fructose may be estimated from the respective catalytic constants for the water molecule, given in (8) table 4. Thus, (7) Substitution of the appropriate values in eq (8) gives k~zo=GKX, the following expressions:

where k~zO is the specific (molar) catalytic constant for 44.7 X 10- 12 .25 [HS] [OH-] the water molecule (i.e., kH zO /55.5), y is 0.4, and K B, for D-glucose, ksJS-] = 10-14.17 by definition, is the reciprocal of the dissociation con stant of the conjugate acid, H30 +; for this acid, KA is = 3720[HS](OH -]; 55.5. For D-glucose at 20 °e, k~zo is 0.0063/55.5, and G, calculated from eq 7, is 10-3.25. Similarly, for 331 X 10-12.00 [HS] [OH-] and for D-fructose, ksJS-] = 10- . D-fructose at 20 °e, ~ zo is 0.058/55.5, and G is calcu 14 17 lated to be 10- 2.28. These values can now be used to estimate ks- , the = 49,000[HS] [OH-]. catalytic constant for the sugar anion. From the data of Urban and Schaffer [38], we calculated the acid These values may now be used in eq (2) to calcu dissociation constants for D- glucose and for D-fructose kOH - from kB[OH-]. Thus, for D-glucose, in water at 20 °e to be 10- 12.25 and 10-12 .°°, respectively. kB[OH -] = (kOH - + 3720[HS])[OH], Therefore, from eq (6), for D-glucose, or kOH - = kB - 372O[HS]. ks _ = 10-3.25(1012.25)°.4 = 45, For D-fructose, and for D-fructose, kOH - = kB -49,000[HS]. ks- = 10-2.28(101 2. °°)°.4 = 330. At pH values less than 8, [HS] is substantially equal The latter value is based on the assumption that the to the molar concentration of the sugar. It follows parameters of eq (6) are the same fo r the mutarotation from the above equations that the values of kB and k: of D-fructose as those determined for the mutarotation should be dependent on the concentration of the sugar. of D-glucose. The above values were used to obtain However, because of the high catalytic effe ct of base the catalytic effects of the sugar anions gi ven in table catalysts, the errors in measuring kB and k~ in the 7. There are no data on the ionization of sugars in present study were too large to reveal the concentra D20 , and, therefore, calculations similar to those in tion factor. The sugar concentrations for the measure table 7 could not be made for the D20 system. ments in D20 were the same as those for the cor-

142 responding measurements in H 20 ; hence, the rearrangements of methyl glycosides, presumabl y concentration factor is partially eliminated in the take place by cyclic mechanisms. Thus, Bonner ratios of kB/ k:; given in table 6. [26] found that the rates for the anomerization of penta Table 8 presents values for the catalytic constants O-acetyl-D-glucopyranose and tetra-O-acetyl-n-xylo for the mutarotations of D-glucose and D-fructose in pyranose in 1:1 AcOD-Ac20 with D2S04 catalyst H20. The high sensitivity of the mutarotation of are approximately 1. 7 times those in 1: 1 AcOH D-fructose to hydroxyl ion catalysis is shown by the Ac 20 with H 2S04 catalyst (i.e., kH/ko=0.6, an inverse large value of kOH -, nearly 200 times that for the isotope effect). He explained the result by a solvent mutarotation of D-glucose. The values of kH20 , kH:lO + isotope-effect in the reaction depicted in figure 2. and ks- for D-fructose, 'however, are only approximately Undoubtedly, the reaction takes place by way of a ten times those found for D-glucose. The excep cyclic intermediate, without rupture of the pyranose tionally high sensitivity of the mutarotation of D ring. fructose (and presumably of other pyranose-furanose In striking contrast to this reaction, the mutarota interconversions) to catalysis by hydroxyl ion is anom tion of the unsubstituted sugar is slower in D20 than alous and should be investigated further. in H 20 under all conditions studied. There is no evidence for a mutarotation reaction of a sugar that TABLE 8. Comparison of catalytic constants for the mutarotations proceeds with an inverse isotope-effect. Elimina ofo·glucose and ofructose in H20 at 20°C tion and addition of the anomeric hydroxyl group in a cyclic mechanis m would result in hydroxyl exchange. Seri al Molar Experimentally, oxygen exchange in water labeled . k a b No. Sugar conen. "H lOll-I k ll ~ 1.- 11 ;10 + s- koll - 1.-11 18 of with 0 is much slower than the mutarotation reac suga r tion [39]. Hence, we conclude that the cyclic mech anism is of little importance in the mutarotation of the 5 o -G lucose 0.22 7.30 1.35 x 10- 7 6.3 X 10- ' 0. 21 45. 10.200. 1.1 X 10' free sugar in water. 6 1.00 6.70 3.39 x 10-' 5,300 0.9 x 10' 7 0.22 ' 9.45 1.91 X 10-' 7,200 0.8 XIO' Because a pyranose-furanose interconversion re

Average 0.5 6.3 x I0- · 0.2 1 45. 7,600 0.9 XI0· quires ring contraction , the cyclic mechanism can be excluded in the mutarotation of D-fructose. An 12 o-.Fructose 0.22 7.35 1.51 X 10- 5.8 X 10-' 2.2 330. 1.29 X 10' 1. 3 X 10' intramolecular cleavage reaction analogous to the 13 1.00 6.70 3.39 x 10- 1.74 X 10' rearrangement of methyl 3,6-anhydro-a-D-glucopyra Average 0.6 5.8 x 10-' 2.2 330. 1.5 X 10' 1. 3 x 10' noside to methyl 3,6-anhydro-a-D-glucofuranoside Ratio of constant s fru ctose/glucose 9.2 10.5 7.3 197 [40] can be envisaged [41] for the mutarotation of n-fructopyranose, but this seems unlikely in li ght ' ~ Ca l c ulat e d by mea ns of eq (6). of the similarity in the isotope effects for the muta h Calculated by means of eqs (2) and (8). rotations of D-fructose and D-glucose.

5. Reaction Mechanisms 5.2. Acyclic Mechanisms In the discussion that follows, it is postulated that In the preceding section, it was shown that the iso the mutarotation of free sugars takes place by way of tope effects for the mutarotations of D-glucose and an acyclic intermediate, and that cyclization of the D-fructose in H 2 and in D2 are similar under a 0 0 intermediate gives either a pyranose or a furanose variety of conditions. This result is rather surprising, form of the sugar, depending on the position of ring because the mutarotation of D-fructose involves a closure, change in · the size of the ring, whereas the mutarota When a sugar is dissolved in water, various reac tion of D-glucose involves a change in the configuration tions occur. Hydrogen bonds and ion pairs are formed of the anomeric carbon atom. A change in anomeric between the solvent and polar groups; protons of the configuration could take place either through a cyclic hydroxyl groups are exchanged; ionization takes place or an a cyclic intermediate. with the formation of both cations and anions; and activated complexes are formed with any catalysts 5.1. Cyclic Mechanisms present. The various molecular species exist in Excellent discussions of epimerization and ring dynamic equilibrium and take part in the mutarota isomerization of sugar derivatives have been written tion reactions. The observed mutarotations are, [42, 43, 44]. It was pointed out that anomerization however, dominated by kinetic conditions (not by of acetylated sugarsl in aprotic solvents, and certain true equilibrium conditions). It was mentioned that

/'0 CLH I c::::1 + I OAc H

FIGURE 2. A nomerization of an O-acetylpyranose in a mixture of acetic acid, acetic anhydride,. and sulfuric acid [26 J.

143 oxygen exchange in water labeled with 180 is slow [39] In accordance with the interpretation of Long and in comparison with the mutarotation, and that this Bigeleisen [8], in the transition state, the HA bond is fact precludes a (cyclic) mutarotation mechanism in stronger for weak acids than for strong acids_ Rup volving hydroxyl exchange_ Reversible formation of ture of a strong, isotopic bond in a rate-determining a gem-diol by the carbonyl form of the sugar would step gives a large isotope-effect; this accounts for the also result in oxygen exchange_ The slow exchange fact that the isotope effect is larger weak than for with 180 is in accord with the premise that hydroxyl strong acids_ groups attached to the carbon chain of the sugar react Figure 4 illustrates the general acid-catalyzed muta more readily than water molecules with the nascent rotation reaction with hydronium ion as the catalyst. carbonyl group_ In the reaction mixture, equiJ.ib Cleavage of the hydronium ion and rupture of the rium conditions are established slowly, and this fact ring in the transition state yields the acyclic cation accounts for 180 exchange at elevated temperatures and a molecule of water. The bond joining the proton and during long reaction periods_ to the anomeric oxygen atom must remain intact in In the acid-catalyzed mutarotation the reaction the transition state; otherwise, the reaction would be sequence starts with the rapid, reversible addition the concerted mechanism of figure L The isotope of the acid, HA, to the ring oxygen atom_ This addi effect is small because the bond joining the proton tion is followed by a series of reactions, resulting in an and the water molecule is weak and easily ruptured_ acyclic intermediate which establishes equilibrium Protonation of the ring oxygen atom by strong acids with other acyclic forms_ By recyclization, the acyclic prior to ring cleavage results in specific acid catalysis intermediate yields the ring isomers (or anomers)_ (see fig_ 5)_ The rate of the subsequent reaction de Presumably, the activated complex formed by the pends on the concentration of the cyclic sugar cation addition of HA to the ring oxygen atom can take part formed, which, in turn, depends OIl the concentration in the cleavage reaction directly, or the complex can of the hydronium ion and not on that of any other acid_ ionize prior to cleavage_ The first path gives rise to Thus, the reaction should be subject to specific hy general acid-catalysis (fig_ 3); in this process, rupture dronium ion catalysis_ This reaction differs from of the ring, with release of anion A -, is rate-determining_ general acid-catalysis (see fig'ure 4) in that the H30+

OH OH

~-H -A + HA ~ t::!;;0 ~ ~~ OH OH v H H ~ OH ~ OH s~ +A- C=O+H OH OH ~ t::!I H

~ H~-H-A + HA ~ t::!;;c ~ t::!J COH l l OH ALL ACYCLIC FORMS + HA

H RCO H l l H DH+HA~ RCOH H + A- OH L:- C=O+H H H RCOH RCOH I H ]H!"1

______OH ~ O_\~/O:HA D 0./ H H

FIGURE 3. General acid-catalysis.

144 bond is broken prior to, not in, the rate-determining Weak bases and sugars form complexes which may step_ For this reason, there is no primary isotope react by way of general base-catalysis de pi cted in effect. However, the concentration of the sugar fi gure 7. In this process, cleavage of the proton from cation is higher in D20 than in H20, and this higher the anomeric hydroxyl group and rupture of the ring concentration of the sugar cation in DzO should give occur in the rate-determining step. Coordination of rise to an inverse, solvent isotope-effect, as in the acid the solvent (H 20) with the ring oxygen atom pre hydrolysis of sucrose [23]_ Experimentally, it has sumably facilitates the reaction, but transfer of the not been possible, under any circumstances, to obtain proton to the ring oxygen atom must be subsequent an inverse isotope-effect for the mutarotation reaction to ring cleavage; otherwise, the reaction would be of sugars in aqueous solutions. Hence, we conclude concerted. Presumably, slow cleavage of the proton that specific hydronium-ion catalysis plays, at most, from the anomeric hydroxyl group, with rupture of only a minor part in the overall mutarotation. the ring, is followed by rapid addition of a proton to To account for the very rapid mutarotation of the the nascent anion. The isotope effect is moderately sugars in alkaline solution, a base-catalyzed mecha large because the anomeric 0 - H bond is ruptured ni sm is necessary. The base-catalyzed reaction must in the rate-determining step. involve the anomeric hydroxyl group, because, when Considerable uncertainty exists as to whether the this hydroxyl group is replaced by an amino group or water-catalyzed reaction follows the concerted mech by an alkoxyl group, base-catalyzed anomerization anism of Lowry (fi g. 1) or a two-step process_ In does not occur [17]. both, one molecule of water acts as a general acid A base-catalyzed step, in combination with general catalyst and another acts as a general base-catalyst. acid-catalysis, accounts for the behavior of the s ugars In both mechanis ms, rupture of the complex at the in slightly alkaline solution. Strong bases cause ring oxygen atom yields 'a hydroxyl ion, and transfer of rapid, reversible ionization of the sugar; and the the anomeric proton to a water molecule yields a hy ionization step is follow ed by transfer of a proton from dronium ion. If, as depicted in figure 8, addition and the solvent (or other general acid-catalyst) to the ring elimination of the protons take place simultaneously, oxygen atom, and slow rupture of the ring, as illus the mechanism is concerted. The isotope effect is trated in figure 6. A moderately large, kine ti c isotope high (3.87) because proton transfers occur in the effect arises in H 20 and D20 from alteration of the transition state at the ring oxygen atom and at the relatively strong H - OH bond in the transition state. anomeric hydroxyl group, and both bonds are strong.

SLOW ~ H~O

~ RAPID

FIGURE 4. General acid-catalyzed reaction with hydronwm LO n as catalyst.

SLOW OH ~ + H3~ RAPID C=O c::::I FIGURE 5. Specific acid-catalyzed reaction.

OH ~-HOH SLOW ~ - SUGA R + OH ~ ~ + OH 0- ~ C=O v I

FIGURE 6. Strong-base catalysis.

OH SLOW ~O- SUGAR + B +HB~~ + B kn V---C=O C=O I I

FIGURE 7. Weak-base catalysis.

145 .~O--H- OH SLOW ~OH -----"

~ ~~O- H--OH2 RAPID v------. C = 0 I H

fIGURE 8. Concerted reaction with H20 as catalyst.

H I SLOW O-H------" ? ;;::::: ~ Ij RAPID C=O H-O I I H (WATER COMPLEX)

FIGURE 9. Dimeric H 20 as a bifunctional catalyst.

If transfer of the proton from the anomeric hydroxyl and the rate-controlling step is shifted to the opening group to a water molecule occurs either before or of the ring, with transfer of a proton to the ring oxygen after ring cleavage, the reaction consists of a two-step atom. The size of the isotope effect depends, as in process in which water acts as a general acid-catalyst. general acid-catalysis, on the strength of the bond The isotope effect for this process would be expected joining the conjugate base to the ring oxygen. to be slightly less than that for the concerted mech The mechanisms outlined accommodate the experi anism, because, in this mechanism, proton transfer mental results. All of the reactions proceed concur occurs in the transition state at only one point in the rently, and complex equilibrium states are established. complex, instead of at two points. Figure 9 depicts a possible mechanism in which dimeric water acts as a bifunctional catalyst. The mechanism is spec 6. Experimental Procedures ulative, but seems plausible in view of the high activity of the bifunctional catalysts of Swain and J:!rown [11]. 6.1. Preparation of Sugars Comparison of the results obtained under various conditions reveals that, in passing from highly acid a. a-D-Glucopyranose. D-Glucose (10 g) was dis to slightly acid and to alkaline solutions, there is no solved in 10 ml of water, and the solution was treated abrupt change in the magnitude of the isotope effects. with 0.5 g of decolorizing carbon and filtered. The The isotope effects increase from about 1.4 in highly filtrate and washings were concentrated in a rotary acid solution to about 4 in slightly acid solution, and evaporator at 35 to 40°, to about 15 g of clear solution; then decrease in alkaline solutions. The gradual this was mixed with 20 ml of glacial acetic acid and chan-ge in the isotope effects is in accordance with nucleated with a few crystals of a-D-glucopyranose. the view that, under the conditions studied, three The flask was stoppered and slowly rotated. After reactions take place concurrently. In highly acid three days, the crystalline product was separated by solution, the acid-catalyzed mechanism predominates. filtration and washed successively with 5 ml each of The isotope effect is small for strong acids and large 90 percent aqueous acetic acid, glacial acetic acid, for weak acids, because the rate-determining step and absolute ethyl alcohol. Finally, the crystalline involves transfer of a proton from the catalyst, HA, a-D-glucopyranose was dried for 1 hr over phosphorus to the ring oxygen atom. In the transition state, the pentaoxide in an oven at 55°/1 mm. The product proton is bonded more strongly to A- in weak acids weighed 5.2 g. Measurements of optical rotation and than in strong acids; hence, the isotope effect with of the infrared spectrum showed that the product was the former is greater. In the region of minimum rate, pure a-D-glucopyranose. the solvent molecules (H 20 or D20) are the principal b. {3-D-1'ructopyranose. The following procedure catalysts, and presumably, the reactions take place proved satisfactory for the purification of {3-D-fructo by the two-step mechanism as well as by the con pyranose. D-Fructopyranose (10 g) was dissolved in .certed mechanism of Lowry. In neutral and alkaline 10 ml of water, and the solution was treated with 0.15 solution, the base-catalyzed mechanism predominates. g of decolorizing carbon and filtered. The filtrate In the concerted mechanism, with H20 or D20 as a was concentrated to a thick sirup, which was diluted .catalyst, both proton transfer at the ring oxygen atom with 5 ml of ethanol and again concentrated. The and proton elimination at the anomeric hydroxyl group residue was dissolved in 10 ml of methanol, and the take place in the rate-controlling step. Both of the solution was re-evaporated to a sirup; this procedure bonds are relatively strong; consequently, the isotope was repeated 3 times. Finally, the sirup (aboi.£12 r) effect is large. In the base-catalyzed reaction, the was dissolved in p-dioxane (10 ml), and the clear sugar molecule is principally in the form of the anion, solution was nucleated with {3-D-fructopyranose crys-

146 tals and rotated overnight. The resulting crystals M and 0.0025 M sulfuric acid in deuterium oxide from were separated by filtration, and washed s uccessively the corresponding aqueous acids. with 10 ml each of 85 percent aqueous p-dioxane and After preparation, all of the solutions were trans p-dioxane_ The product was dried for 1 hr over ferred to glass-stoppered bottles and tightly closed_ phosphorous pentaoxide in an oven at 55°/1 mm; The D20 solutions were transferred, in the dry box, weight 5_6 g_ Measurement of optical rotation and to 2S-ml flasks, and were opened only under anhydrous of the infrared spectrum s howed that the s ugar was conditions (so as to prevent dilution of the D20 with pure ,B-D-fructopyranose_ H 20 from the air). c_ ex-D-Xylopyranose_ Commercial D-xylopyranose Proof that the quantity of sulfuric acid was the same (10 g) was dissolved in 6 ml of boiling water, and the in each of the water solutions and in the corresponding hot solution was mixed with 0_5 g of decolorizing carbon deuterium oxide solutions was obtained as follows: Two and filtered_ The filtrate was cooled to room tempera ml of the aqueous acid was mixed with 2 ml of D20; ture, diluted with about 3_5 ml of p-dioxane (to incipient similarly, 2 ml of the deuterated acid was mixed with turbidity), nucleated with crystals of ex-D-xylopyranose, 2 ml of H 20, giving D2 0 - H20 mixtures that should and placed on a rotator. Crystallization began almost contain equal amounts of D20 and H2 0. The pairs immediately, and, after 24 hr, the crystals were sep gave the following pH meter readings: 2.23 and 2.22; arated by filtration and washed successively with 5 ml 2.76 and 2.76; and 2.91 and 2.9l. each of 60 and 85 percent aqueous p-dioxane and b. Potassium Acid Phthalate in D20. Two hundred pure p-dioxane_ The crystals, 7_5 g, were filtered ml of 0_001 M aqueous potassium acid phthalate was and twice recrystallized from water by the same lyophilized; the residue was thrice successively dis procedure_ After the crystals had been air-dried solved in 2 ml of D20 and lyophilized_ The residue (with the exclusion of dust), they were dried for 1 hr was then di ssolved in enough D20 to make 200 ml of over phosphorus pentaoxide in an oven at 50°/1 mm_ solution. (The solution gave a reading of 4.70 on a The optical rotation and the infrared spectrum showed pH meter; the corresponding pD is 5.10.) that the substance was pure ex-D -xylopyranose_ c. Sodium Hydroxide Stock Solution. Approxi d_ O-Deuterated Sugars_ The deuterium analogs mately 8 g of sodium hydroxide was mixed with 8 ml of the hydrogen forms of the s ugars were prepared of H20, and the mixture was allowed to stand until by replacin g the labile hydrogen atoms of each sugar the sodium carbonate had precipitated, leaving a clear with deuterium; this was accomplished by repeated solution. A portion (2 ml) of this carbonate-free solu dissolution of the sugar in D20 followed by freeze tion was transferred to a 2S0-ml volumetric flask and drying_ The crude, deuterated sugars were recrys diluted with enough freshly boiled H 20 to make 250 tallized by dissolution in D20, followed by the addi .ml of solution. The solution was standardized with tion of p-dioxane_ This procedure avoids the use of potassium acid phthalate. large quantities of deuterated solvents, and gives d. Sodium Hydroxide-d in D20 . An aliquot of the high yields of desired products_ The isotopic purity standardized sodium hydroxide stock solution was of the deuterated sugars was established by infrared transferred to a SOO-ml, round-bottomed flas k and absorption measurements which showed negligibly lyophilized. After introduction of dry nitrogen, 2 ml weak bands corresponding to OH groups and strong of D2 0 was added, and the solution was again lyo bands corresponding to OD groups_ philized_ The residue was diluted with D20 under dry In the preparations, commercial deuterium oxide nitrogen to the desired concentration of alkali, and (99_5%) was used without purification_ The infrared the solution was stored in a dry-box protected from absorption spectra showed a lack of appreciable H 20 absorption_ Scrupulous precautions were taken to 6.3. Measurement of Optical Rotation avoid deuterium exchange in the recrystallized, deu terated sugars, and to prevent dilution of the deuterium For each measurement of optical rotation, approxi oxide by atmospheric moisture_ mately 0.4 g of the crystalline sugar was weighed into a dry, 25-ml Erlenmeyer flask, and about 10 ml of the 6_2. Solvents for Mutarotation Measurements solvent to be used was placed in another 2S-ml flask. Both flasks were stoppered and placed in a thermo a. Sulfuric Acid in D20. To prepare 0.015 M sulfuric stated bath at the temperature planned for the ex acid in deuterium oxide, 200 ml of 0.015 M aqueous periment. A 2-dm, glass-jacketed, polarimeter tube sulfuric acid was transferred to a I-liter, round-bot was placed in a Bates, adjustable-sensitivity sac tomed flask and lyophilized; then, the flask was placed charimeter. The temperature was co ntrolled by in a dry box. After removal of moisture from the dry circulating water from the thermostated bath through box by successiv e evacuation and filling of the dry the jacket of the polarimeter tube, and measured by a box with dry nitrogen, 6 ml of D20 was added to the thermometer placed in the line of flow_ The zero flask , and the resulting solution was lyophilized. reading of the instrument was determin ed before the Successive dissolution in 6 ml of deuterium oxide and measurement was started_ After te mperature lyophilization of the solution was carried out twice equilibrium had been attained for the system (1 hr or more. Finally, the sirupy residue was transferred to longer), the solvent was quickly poured into the flask a 200-ml volumetric flask and diluted with sufficient containing the sugar. Time was measured from the D20 to make 200 ml of solution_ moment the solvent was added. The sugar was dis The same procedure was used for preparing 0.005 solved by swirling the partially submerged, stoppered

147 flask, and, upon dissolution of the crystals, the solu [9) T. M. Lowry and E. M. Richards, J. Chern. Soc. 127, 1385 tion was immediately transferred to the polarimeter (1925): T. M. Lowrv and l. J. Faulkner, ibid. 127,2883 (1925). rIOl T. M. Lowry, J. Chern. Soc. 2554 (1<)27). tube_ Optical rotations were read continuqusly dur [ll) C. G, Swain and J, F. Brown, Jr., 1. Am. Chern. Soc. 74,2534, ing the first 30 min for fast reactions and ilt regular 2538 (1952). intervals for slower reactions_ The equilibrium rota [12) E. Pu.rlee, .J. Am . Chern. Soc. 81,263 (1959). tion was read at the end of a 24-hr period for the fast [13) K. J. Pe.dersen, 1. Ph.ys. Chern. 38,581 (1934). [14) C. G. Swain, A. DiMilo, and J. P . .Cordner, J . Am. Chern. Soc. reactions, and after 48 or more hours for the slow 80,5983 (1958). reactions_ All optical rotations recorded in the tables [l5) R. P. Bell and J. C. Clunie, Proc, Roy. Soc. (London) A212, are averages of 10 readings_ 33 (1952); R. P . .Bell and P . J ones, 1. Chern. Soc . .88 : (1953); The rate constant for each buffered sugar solution Y. Pocker, ibid. 1279 (1957). was calculated by use of the conventional formula: [16) H. Fredenhagen and K. F. Bonhoeffer, Z. Ph.ysik. Chern. A181, 392 (1938); L. P .. Hammett, Ph.ysical Organic Chemistry, pp. 221, 337 (McGraw-Hill Book Co. Inc., New York, 1940); S. W. Benson, Foundations of Chemical Kinetics, pp. 559-563 (McGraw· Hill Book Co., New York, 1960). [l7) H. S. Isbell and H. L Frush, J. Res. NBS 46, ;32 (1951) RP2186. [18) S. Glasstone, K. Laidler, and H. Eyring, The Theory of Rate where (kJ + k2 ) is the mutarotation constant for the Processes, pp. 153- 201 (McGraw·Hill Book Co., New York, balanced reaction, rl is the rotation at time tl, r2 is 1941). [19) E. S. Gould, Mechanism and Structure in Organic Chemistry, the rotation at time t2, and roo is the equilibtium rota p. 192 (Henry Holt and Co., New York, 1959). tion, all in "degrees sugar-" [20) L Melander, Isotope Effects on Reaction Rates, pp. 34- 65 After the equilibrium rotation had been taken, the (The Ronald Press Co., Ne w York, 1960). pH of the solution was measured with a glass' electrode [21) A. K. Covington, R. A. Robinson, and R. G. Bates, J. Ph.ys. Chern. 70,3820 (1966). and commercial pH meter- Only the experiments [22) W. F. K. Wynne·Jones, Chern. Rev. 17, ll5 (1935); J. Chern. employing sodium hydroxide solution showed any Ph.ys. 2,381 (1934); Trans. Faraday Soc. 32,1397 (1936). substantial difference in the pH of the original solvent [23) E. A. Moelwyn-Hughes and K. F. Bonhoeffer, Naturwissen and of that of the sugar solution. Values for [H30+] schaften 22, 174 (1934); P . .Gross, H. Steiner, and H. Seuss, and [OH- ] were calculated from the pH arid the ion Trans. Faraday Soc. 32,883 (1936); W. H. Hamill and V. K. La Mer, J. Ch~m. Ph.ys. 4, 294 (1936). product corresponding to pK 14_17 at 20 °e_ Values [24] O. Reitz and J. Kopp, Z. Ph.ysik. Chern. A184, 429 (1939). for pO were obtained from the relationship pD = read [25] P .. Gross, H. Steiner, and F . Kraus, Trans. Faraday Soc. 32, ing on pH meter + 0-4. Values for [030 +] and [00-] 877 (1936); 34,351 (1938); J. D. Roberts, C. M. Regan, and were calculated from the values of pO and the ion l. Allen, 1. Am. Chern. Soc., 74,3679 (1952). product corresponding to pK 15_05 at 20 [26] W. A. Bonner, J. Am. Chem_ Soc. 83,2661 (1961). °e. [27] V. K. La Mer and J. Greenspan, Trans. Faraday Soc. 33, 1266 (1937); S. Liotta and V. K. La Mer, 1. Am. Chern. Soc. 60, 1967 (1938). [29] O. Reitz, Z. Elektrochem. 44, 693 (1938). The authors express their apprecIatIOn to Or. Wil [30] F. A. Long and D. Watson, J. Chern. Soc. 2019 (1958). liam Zorbach of Georgetown University for his interest, [31] W. P . . Jencks and J. Carriuolo, J. Am . Chern. Soc. 82, 675 cooperation, and counsel during the course of the (1960). investigation, and to Or- Harriet L. Frush pf the Na [32] M. 1. Nicolle and F. Weisbuch, Compl. Rend. 240,84 (1955). [33] Ref. 2, p. 446. tional Bureau of Standards for valuable criticism and [34] C. S. Hudson, J. Am. Chern. Soc. 29,1571 (1907). help in the preparation of the manuscript. . [35] T. M. Lowry and G. F. Smith, J. Chern. Soc. 2539 (1927). [36] 1. N. Bronsted and E. A. Guggenheim, 1. Am . Chern. Soc. 49, 2554 (1927). 7. References [37] 1. N. Bronsted, Trans. Faraday Soc. 24, 630 (1928). [38] F. Urban and P .. A. Schaffer, J. BioI. Chern. 94,697 (1931). [II T. M. Lowry and G. F. Smith, Rapports sur les Hydrates de [39] K. Goto and T. Chitani, Bull. Chern. Soc. Japan 16,403 (1941); Carbone, 10th Conference of the International Union of D. Rittenberg and C. Graff, 1. Am. Chern. Soc. 80, 3370 (1958). Chemistry, p. 79 (Liege, 1930), [2) H. S. Isbe ll , in Polarimetry, Saccharimetry and the Sugars, [40] W. N. Haworth, L N. Owen, and F. Smith, J. Chern. Soc. S8 F. 1. Bates, ed. , NBS Circ. 440. U.S. Government Printing (1941). Office, Washington , D. C., 1942, part 3, chapter 29, pp. 439 [41] H. S. Isbell, Ann. Rev. Biochem. 9,65 (1940). to 456. [42] R. U. Lemieux in Molecular Rearrangements, Part 2, pp. [3) H. S. Isbell and W. W. Pigman, J. Res. NBS 18, 141 (1937) 71(}-743, P . . de Mayo, ed. (Interscience Publishers, New RP969. York,1964). [4) H. S. Isbell and W. W. Pigman, J. Res. NBS 20, 773 (1938) [43] E. L Eliel, N. L Allinger, S. J. Angyal, and G. A. Morrison, RPll04. Conformational Analysis, pp. 403-413 (Interscience Pub· [5) C. Challis, F. A. Long, and Y. Po.c ker, 1. Chern. Soc. 4679 (1959). lishers, New York, 1965). [6) F.. Pa.csu, J. Am. Chern. Soc. 55,5066 (1933); 56,745 (1934). [44] F. Shafizadeh, Advan. Carbohydrate Chern. 13, 9 (1958). [7) W. H. Hamill and V. K. La Mer, J. Chern. Ph.ys. 4, 395 (1936). [8) F. A. Long and 1. Bigeleisen, Trans. Faraday Soc. 55, 2077 (1959). (Paper 7lA2-445)

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