Journal of Research of the National Bureau of Standards Vol. 46 No. 2, February 1951 Research Paper 2186 Mechanisms for the Mutarotation and Hydrolysis of the Glycosylamines and the Mutarotation of the

Horace S. Isbell and Harriet L. Frush

A study has been made of t he kinetics of t he mutarot ation and hydrolysis reactions of L-arabinosylamine, and a m echanism has been devised to accoun t for the striking sensit ivity of t he glycosylamines to hydrolysis in a limited pH range. The concepts presented seem applicable for the interpretation of the reactions of other compounds of t he aldehyde ammonia type.

1. Introduction

In the development of methods for the preparation of the amides of uronic acids [1] 1 it became necessary to determine conditions whereby the amino group II. of a l-amino-uronic amide could be hydrolyzed without alteration of the amide group. It was L-Arabinosylamine found that the rate of hydrolysis of the amino group of 1-aminomannuronic amide (I ) is extraordinarily OAc OAc H sensitive to the acidity of the reaction medium. Thus, when the compound is mixed quickly with R 2 c - 6 - 6 --6 - C H.NH.Ac one equivalent of a strong acid, hydrolysis requires I ~ 11 I I a period of many hours, but when the acid is added ~O OAc drop wise to the compound in solution, hydrolysis is III. complete in 15 min or less. Further study showed that 1-aminomannuronic amide is surprisin gly T et raacetyl-L- a rabinosylamine stable both to strong acid and to alkali, and that OR OR H hydrolysis can be effected rapidly only in the pH I I I range 4 to 7. B ecause of the presence of the hy­ drolyzable amide group in 1-aminomannuronic H ' P--~ -~;;--rNHA' amide, the study of the hydrolysis was directed to th e l-aminosugars, more properly designated gly­ '---~ O IV. cosylamines. Exploratory experimen ts with glucosylamine, N-Acety l-L- arabinosylamine galactosylamine, and arabinosylamine showed that the glycosylamines in gen eral possess the peculiar II. Structure and Chemical Properties of sensitivity to hydrolysis in a limited pH range that L-Arabinosylamine had been noted for 1-aminomannuronic amide [2]. In this paper, the mutarotation and hydrolysis of L-arabinosylamine (II) was prepared by treating L-arabinosylamine arc consider ed in detail. M echa­ L- in with ammonia essentially by nisms for the mutarotation of the sugars, comparable the method of de Bruyn and Van Leent [3] . Acetyla­ to those for the mutarotation of the glycosylamines, tion of the compound with acetic anhydride and correlate the properties of the two groups of sub­ pyridine yielded a new tetraacetate (III). Catalytic stances and account for the over-all rate of mutaro­ deacetylation of this substance with barium methy­ tation of both the glycosylamines and the sugars as late in methanol gave a crystalline N -acetyl-L­ a function of pH. arabinosylamine. 2 The latter substance was found to react quickly with 2 moles of sodium periodate, as required for an N -acetylpentosylamine having the H H OH OH structure IV. Since acetylation and de­ I I I I o = c-c - c --c - c - c H· N H , acetylation were carried out by mild reactions that ordinarily cause no change in ring structure, the hH, ~ ~ II I . original L-arabinosylamine and its tetraacetate arc tentatively classified as pYTanoses. I· In a slow titration of L-arabinosylamine, one l-Aminomannuro nic amide equivalent of acid was required for neutralization ' T his co mpo un d is analogous to the N-acctyl·D-glueosylamine prepared by 1 Figures in brackets ind icate the li terature references at tbe ena of tbis paper. Brigl and Keppler [4] and shown by N iemann and lIays [5] to be a pyranose. 132 8 were treated wi th acids and bases in solutians ~ b ufl'cred at vari ous pH values , and the ensuing 7 reactions were followed by op tical rotation. Al­ ~ r------~ though the mu taTotatian and hydrolysis reactions of 6 L-arabinosylamine take place simultaneo usly, they r\ can be studi ed separately because their rates at a given pH value ditrer widely, and because the change 4 \ in accompanying each of the two reactions is large. For study, the change in optical 3 u..., rotation was co nsidered to. consist of two periads: a (--"'0 2 short period b eginning at zero time and character­ a 0..1 0.. 2 0..3 0..4 0.5 0..6 0..7 0..8 0..9 1. 0. 1.1 1.2 1.3 iz ed by a decrease in dextrorotation ; and a long EQUIVA LENTS HCL / MaLE L - ARABINaSYL AM INE period begilming when the initial change was com ­ plete, characterized by an increase in dextrorotation 1"1 0 URE 1. Titration of L-arabinosylamine with acid. and extending until the optical rotation reached a value corresponding to complete hydrolysis (+ 10 5.2°). (fi g. 1). Separatian of crystalline L-arabinase in T he rate af the mutarotation reaction "vas obtained nearly quantitative yield from the mixture after from the data for the first period and the rate af the titr atian established the fact that tIlls treatment wi th hydrolysis reaction from the data for the second dilu te acid had caused hydrolysis af the glycosyla­ period . Suitable data. were ob tained for the mu taro­ mine and formation of the free . tation reaction in alkaline solutions, in 'which the ' Vhen dissalved in water, L-arabinasylam ine under­ hydrolysis is extremely slow, and for the hy drolysis goes spon taneous changes that give rise to a decrease reaction in weakly alkaline or acid solutions, in which in optical ratati on and then to an increase. The the mutarotation reaction is almost instan taneous. changes can be ascribed principally to a mutarata­ Satisfactory rate constan ts for the mutarotation of tion rcaction that establishes equilibrium between L-arabinosylamine were ob tained in the pH r ange 7.8 the various modifications of the glycosylam ine, and to 12 by application of the customary formula 4 for hydralysis of the amino group.3 The relative rates a first-order reaction to the data of table l. Ta ble 2 of the two rcactians vary wi th cxperimen tal concE­ gives the mutarotation constants that were obtained tions (fig . 2). Thus, when L-arabinosylamine is for a series of experimen ts used to evaluate the disf;olve d in strong acid, the op tical rotati on drops catalytic effects af the oxo nium, hydroxyl, and am­ almost at once to abou t + 69°, and then incr eases monium . The dotted curve of figure 3 represen ts slowl y over a periaJ of several weeks (curve I). these mu tarotatian constants corrected for the cata­ H owevcl", if it is di ssolvcd in weak ac id , fo r example lytic effect of the ammonium . The res ul ts in water saturated with carbon dioxide, the op tical clearly show that the mutarotation of L-arabino­ rotation drops immediately to about + 70° and then sylamine is strongly catalyzed by acids bu t not ap­ increascs rapidly to + 105° (curvc II). In a weakly preciably by bases b The mutarotation differs from alkaline solution prepared by dissolving th e sub­ stance in carbon dioxide-free watcr, the specific rota Lion decreases in the co urse of a few hours from + 86 ° to. a m inimum, and thcn slowly riscs (c urve + 110. III). In strong alkali there is almast no changc 10.5 (curve IV). V 1I 100 Thcse qualiLative abservations m.ay be summa­ z rized as fo llows: Q 9 5 1/ ~ Q 0 9 0 a: TIl 0 / 1\ 1 utarotation LL 85 Solution H ydrolys is reaction G reaction w a. 8 0. I ~ .r.. "'-0 '" ~ ill Strongly acid _____ Vcry rapid ______Slow. 7 5 _____ do ______I ' Vcak lyacid ______Rapid, but mcas- 70. U1·a ble. ' Vcaklyalkaline ___ Rapid, b ut meas- SlolI'. 6 5 L m·able. o 4 8 12 16 20. 24 28 32 36 40 4 4 48 52 56 Strongly alkalin c __ Slow ______Do. TIME , HOURS

F I r; URE 2. l'o1utw'otation oI L-ambionsylamine.

To ob tain quantitative data as to the effect of the I , III 2.5 N H CI; IT, in C 0 2-saturated water; In, in CO,-free water; IV, in 0.01 oxanium ion concentration, samples of L-arabinosyl- NNaOH . •1 'he firs t-ordor formu la is applicable if th e concen tration s of the acid and base 3 Thc process appears to be complicated Wldet· som e conditions by the formation catalysts arc held conslan t. of tile diglycosylamine, and possibly by other reaclions tbat depend on the :I HOWC\'Cf , unpublished work has shown that the mutarotation of a-D-galacto presence of an intermediate imine cation . See pa ge 142. sylallline is weakly but defin itely catalyzed by bases.

133 the mutarotations of the sugars in that the latter are TABLE 1. M.111arolalion and hydl'ol ysis measw'e menis- Con. catalyzed by bases more strongly than by acids. The 0.5 g of L-arabinosylamine dissol ved in suffi.cient acid, base, or buffer to give a reason for this difference will be considered in the volume of 25 ml. Solve n ts are listed opposite experiment n umbers. next section. Mutarotation co n· Hydrolysis rate Time pH stant,a constant,s khydrol (minutes) [al~ 1 Ttl-Tm 1 T I1-TCIl TABLE 1. N[ utarolalion and hydrolysis measurements k m =-- log --- = -- log-­ l z-tl Tt2-Tm h-ll Ttz-T CIl

0.5 g of 1.-arabinosylamine dissolved in sufficient acid, basp, or huffer to give a Experiment 7. I volume of 1 JvI KH, PO.+1 volume of I N NaOH. pH 10.0 volume of 25 ml. Solvents are listed oppos ite experiment numhc".

2. 7 +82. 4 ------Mutarotation coo­ HydrolysIs rate 4.3 81.5 0.019 ------.------Time stant,s constant, a kbydl'ol 4 l O. . 02 1 [al ~ pH I 6.0 80. 0 ------(minutes) 1 Tt,-Tm 1 T tl- T oo 12.8 76. 4 . 026 ------= -- log -- 17. I 74. 7 . 025 j km=t2_t , IOg rt2_rm tz-ll Tt2- f oo ------60.0 70.3 ------b (69. 0) ------Experimen t 1. om N NaOH Avg .. 0.023 210.0 + 72.2 10 _.- --.------+86. 4 I. 750 81. 6 _.- -- ._--. _------0. 00009 32 86.1 0. 00034 3.570 84.6 . 00006 nO 86.0 .00040 ------.------4, 560 85. I ------. 00005 120 85 1 . 00031 d (105. 2) 10. 4 ------.--- _. 270 82. 7 12. 1 .00040 ------. - - --- _. -- 1. 250 77.6 Avg .. 0. 00007 b (69.0)

0.00036 Experiment 8. 1 volume of 1 N Na,CO,+1 vo lume of 1 N NaHCO" pH 9.4

Experiment 2. 0.1 NHCl+NH , to pH 10.5 2. 0 +82.2 3.8 79.2 0. 060 4.6 78.0 . 063 1. 7 +85. 0 5.5 77.0 9.4 . 061 1.1 84.2 0.001 7 10.1 73.6 . 055 60 82.2 . 001·1 30.0 70.9 12.1 79.6 10.5 . 001 5 b (69.0) 300 75.5 . 0013 1, 290 70. 3 Avg_. 0. 060 b (69.0) 420 + 74.5 Avg .. 0.0015 1,380 78. l 0. 000056 2,800 82. 6 . 000056 4, 320 84.3 9.5 . 000043 d (105.2) Experiment 3. 2.5 N HCl+NH, to pH 10.5 0.000051 3.3 + 7S.7 4.9 77.2 0.025 Experimen t 9.1 volume of lNNa,CO,+10 volumes oflNNaHCO" pH 8.6 10. 2 73. I . 026 20. I 67. 7 .027 30.2 65.3 10.5 .026 1.6 +79.0 120.0 , (61.9) 2.4 77.2 0.108 5.5 72.9 8.6 . 105 Avg.. 0. 026 6.6 72.0 . 105 12. 1 70. 4 b (69. 0) --.------E xperiment 4. 0.1 N HCl+NH 3 to pH 8.0 Avg .. 0. 106 ISO. 0 +72.7 S. 7 2. 2 +82.2 1.140 77. 8 0.000077 6. 1 79.2 0. 024 4.320 88.4 . 000069 12. 1 76.0 8.5 . 023 8. 640 93.1 . 000051 21. 1 73.2 . 019 d (l05. 2) 145 66.9 Avg .. 0. 000066 Avg.. 0. 022

Experiment 10. 10 volumes of I lv{ KH,PO,+7 volumes of 1 N N aOll E xperiment ,1 . INHCl+NH , to pH 8.0 diluted 10 20 volumes, pH 7.0

1.6 +S1. 5 1.4 + 71. 3 6.5 75.6 0. 057 6.0 78. 1 0.023 8.7 74 . 0 8. 2 . 056 10.5 81. 5 . 018 17.0 70.7 . 056 20.4 87.6 . 016 30.2 69. 0 1500. 103.1 7.2 A vg .. 0. 056 Avg .. 0. 019

Experiment 11. 2 volumes of 1 lVI KH,PO,+1 volume oft N NaOH Experiment 6. 1 N HCl+ NH, to pH 7.5 diluted to 4 volumes, pH 6.6

1.9 + 78. 2 1.3 + 73.9 3.8 74.5 0. 116 6.6 g'I. O 0.033 4. S 73. 2 7.8 . 116 10.0 87.4 . 029 6. 9 71. 3 . IlS 19.9 93.6 . 024 21. 8 68.9 110.0 104 .3 6.8 Avg.. 0. 11 7 Avg .. 0. 029

134 TABLE j . NItitarotation and hydrolysis measmemenis- Co ll . TABLE]. NIutarotation and hydrolysis measw·ements- Con. 0.5 g of r,·ara binosylaminc dissol ved in sufficient acid, base , or buffer to gh -o :\ 0.5 g of J.-arai)inosylaminc dissoln'd in sufficient aCid, bflSC , or bufror to give a volume of 25 ml. Solvents are listed opposite experiment numbers. volu me of 25 m!. Solvents arc li sted opposite ex periment numbers.

M utarotation con· Hydrolysi' rate Mutarotation con­ H ydrolysis rate Time stant,a constant,a h y d ro l sta nt, 1t co nst ant,akhydro l [" l~ pH 'nme (min utes) 1 '(11 - T", 1 T t l - r oc ra]~ pH 1 Tt,-T nt I Ttl-Teo km=- log -- (minutes) km= - Iog -­ =-- log-- t2-t , T'2-Tm =l2-11 log r,~-rC'l> t2-l, . T t2-T m t z- l l TI2 -Teo

E xperiment 12. 5 volumes of 1 j\I KH,PO.+1 volume of 1 N N aOH dilu ted to 10 volnmes. pH 5.9 Experiment 19 . 1 1V oxalic acid , p H 0.8

1.4 '+67.1 1.4 + 77.J .>. 0 69.3 6. I 89.5 0.057 480 72. 1 0.000074 9. 6 94.2 . 054 1, 440 77.7 . 000081 11. 3 96. 0 . 054 1,890 80.6 0.8 .000087 60.0 103.9 6. 2 d (105. 2) 0. 055 0.00008 1

E xperiment 13. 10 ,'olullles of I A1 KH, PO.+1 volume of 1 N NaOH Experiment 20. 2.5 NRCI d ilulee! to 20 volu mes, pH .1.5 3.8 '+69.0 30.0 • 69. 9 2. ~ +80. 1 60.0 70.0 5.6 90. 6 0.069 3.300 71. 2 0.0000046 9.7 97. 0 . 058 35, 300 8 l. 2 0.2 .0000047 60. 0 101.2 5. 9 d (105. 2) ------Avg __ 0. 068 A vg _. 0. 0000047

Expf'rimcnt 14. 5 volumes of 1 ]..T ace tic acid +3 volumes of 1 N Na OH • Symbols are d efin ed in section V IT, 5. b Postulated eq u ili brium value. dilulNI to 10 volu mes. pH 4.7 o Sec page 144 . d Postulated final value. e Value not used in calculations .

1. 3 +69. 4 TABLE 2. NIutarotation constants oj rrarabinosylamine at 9. 9 100. 1 5. 0 0. 108 20° C 12. 0 101. G . 105 13. 1 102.5 . 111

17. 9 104. 2 Expe- i­ k ll, co r­ pH [ J[+] [O ll-] [NH : l IllCpt recteci 8. Avg .. 0. 108

L . _____ 12. 1 7. P4 X 10-" 1. 2flX IO-' 0.001 0. 0001 Experiment Hi. 5 voluJl) rs of 1 N ace Li c acid +2 volumes of l N NaOH 2 _____ 10. 5 3. lO X 10- 11 3 16X IO- 1 O. I . 001 5 . 0000 diluled 1010 VO lumes, pH 4. 4 3 ______10.5 3. 16X 10- 11 3. 16X 10-' 2.5 . 02(\ . 001

1. 9 + 70.7 where kH2o, leH , and leOH are constants ch aracteristic 4. 7 80.2 0. 051 9.7 90.1 3.6 . 047 of the particular sugar, and [H+] and [OH- ] represent 14 .5 95.3 .044 150 104.8 3. 7 the con centraticns of the oxonium and hydroxyl ions, Avg __ 0. 047 respectively. :More recently it h as been established that th e reaction is subject to general acid and base Experiment 18. 10 volumes of 5.14 Nacetie acid + 1 volume of 1 NNaOn, catalysis, and consequently the expression has been pH 2.7 extended to include terms accounting for the cata­ lytic eff ect of undissociated acids, anions of weak 1.8 + 66. 5 2. 6 68.0 0. 022 acids and. cations of weak bases [7]. Thus eq 1 is 9. 6 78. 3 .021 replaced by eq 2: 20. 0 87. 6 . 021 105 104. 2 3. 1 le m= kH2o+ lcH[H +]+ A vg __ 0. 021 (2) 135

) /----~------,

in which HA signifies any acid, neutral or mutarotation of tetramethylglucose is negligible in ionic, and B signifies any base, neutral or ionic. either cresol or pyridine, but rapid in a mixture of Although several mechanisms have been advanced the two solvents 6 Lowry [10] advanced a mechanism for the mutarotation reaction, no single mechanism that involves the addition of a proton at one point in accounts for all of the experimental facts. To the sugar molecule and simultaneous elimination of a account for certain results with acid and base proton at another point. The process may be catalysts, including the observation [8] that the represented in the following manner : 7

H OO H H O- O HB HO = O

(H 0 .H 6 ~ 0 + B + H A P ( HO . H 6 ~ O H A P (H O . H 6 )n+H B++ A-. (3) - 6 / -6/ - 6 0H 1 1 1

This mechanism would give rise to a third-order reaction, expecially in amphoteric solvents. C81·ttj,in term in the rate expression [11], but as pointed out experimental facts, however, may be explained more by Swain [12] , this is not detectible because of the satisfactorily by separate acid- and base-catalyzed relative magnitude of the terms, and hence the rate mechanisms, and such mechanisms have come to be constant on the basis of this mechanism can be quite generally accep ted. It seems probable that represented within the experimental error by eq 1. they act concomitantly with Lowry's mechanism, The simplicity of the mechanism makes it most and that the exten t to which each mechanism is a ttractive for interpretation of the mutarotation effective is determined by the experimental conditions.

Acid-catalyzed mechanisms

H O OH H C OH H O= OH+ HO OH H O OH 1"- kJ 1"- k 2 1 k3 1"- k , 1"- R O+ H A ~ R OH A ~ R ~ R OH A ~ R O+ H A ( 4a) k- l 1/ k - 2 1 k-3 1/ k - 4 1/ - 6 / - 0 - O OH A- - 0 - 0 1 1 1 1 1 t!

all open-chain forms

HONH 2 H CNH 2 HO= N Ht H 2 NO H H 2 N OH 1"- k, 1"'- k2 1 k3 1"- k4 1"'- R O+ H A ~ R OH A ~ R ~ R OH A ~ R O+ H A (4b) 1/ k _ , 1/ k -2 1 k- a 1/ k-4 1/ - 0 - 0 - OOHA- - 0 - 0 1 1 1 1 1

a ll op en-cha in forms

- - HO OR OO1"'-H ] OOH1"- 1"'- [ R OH B ~ R O + HB ~ R O+ B- 1/ -6/ -6/ - 0 1 1 1 (5) (,'Base-catalyzed" mechanism of [14])

6 Sim ilarly, Butler, Smith, and Stacey [9] have recently observed tbat the 7 Although the mechanism of eq 2 was represented in a ge neri c fashion with Ir es pect to 1£.'1 and B , in eq 3 and succeeding mechanisms charges have been a · and II-tetraacetates of N -pbenylglueosylamine are extremely stable in dry indicated for clarity. Nevertheless. tbe mechan isms may be regarded as ge neric. pyridine, b ut mutarotate to an equili brium value on addition of a drop of water. 'l'hus, if B in eq 6a is a neutral substance. t hen H B becom es HB+. 136 Base-catalyzed mechanisms

HCOH HCOHB- HC= O BHOCH BO CH I k. k2 I k3 1"'- k4 1"- p 1"- 1"- R O+ B- R 0 P R + HB P R 0 P R O + B- (6a) 1/ k_. 1/ k -2 1 k-3 k _, - c - c - co- -6/ -6/ 1 1 I 1 ~i all open-chain forms

HCNH2 H CNH2 B- HC= NH BH2NC H H 2 NCH 1"- k. 1"'- k 2 I k 3 1"- k , 1"- R O+ B- p R 0 P R + HB P R 0 P R O+ B- (6b) k _ . 1/ k -2 1 k-3 1/ k - 4 1/ -6/ - c - co- - c - c I I 1 I 1 ~i a ll open-chain forms

HCUH 1"'- R O + HB 1/ -- C 1

("Acid-catalyzed" mechan ism of (1 4))

In eq 4 and 6 there are proposed acid- and base­ of a proton, the product establishes equilibrium with catalyzed mechanisms that we believe represent the open-chain form of the sugar. R estoration of rea onable courses for the mu tarotation of the sugars asymmetry on carbon 1 through ring formation, and the glycosylamines. These mechanisms differ facilitated by combination of the conjugate base in cer tain respects from the mechanisms commonly with the carbonyl e tablishes equilibrium of accepted. the cyclie modifications. The glycosylamine, how­ The acid-catalyzed mechanism presented here for ever, does no t reaet to a great extent by a base­ the mutarotation of the sugars Ceq 4a) begins with catalyzed mechanism Ceq 6b) analogo us to that of the addition of an acid catalyst to the ring oxygen. the free sugar because there is less tendency on the This i followed by ruptm-e of the ring tlu'ough ah part of the amino nitrogen to release a proton. electron hift in which a lone electron pail' of the The above mechanisms differ from those generally glycosyl oxygen facilitates release of electrons to the accepted primarily in regard to the species of the ring oxygen. The product quicldy establishes equi­ sugar undergoing reaction. According to H ammett librium with all species of the open-chain modifica­ ([13 , p. 337]) " ... the only reasonable mechanisms tion. Cyclization can then proceed tlll'ough com­ are the following: for the acid catalysis the mobile bination of carbon 1 with the oxygen of either carbon and reversible addition of a proton to the oxy­ 4 or carbon 5. This gives rise to the alpha and beta gen followed by a rate-determining reaction with a furano e and in equilibrium proportions. base . . . for the base catalysis the mobile and The aeid-catalyzed mutarotation of the glycosyl­ reversible removal of a proton, followed by a rate­ amine is formulated in similar manner, as shown determining reaction with an acid." These mecha­ in eq 4b. In this case, a shift of a lone electron pair nisms, originally advanced by Fredenhagen and Bon­ of the amino nitrogen res ults in the formation of the hoeff er [14] and based in part on the work of Bon­ imonium ion -C= NH2+' R eaction of carbon 1 of hoeff er and R eitz [1 5], have been extended to the

1 glycosylamines by Howard, K enner, Lythgoe, and the imonium ion with the oxygen of carbon 4 or Todd [16] . The mechanisms of [1 4] arc outlined in carbon 5 produces the various ring isomers. A eq 5 and 7, in which the steps con tained in brackets mechanism of this type will aecount for the striking have been postulated by us. Comparison shows that sensitivity of the mutarotation of the glycosylamines our acid-catalyzed m echanism of eq 4a resembles the to acid catalysts. base-catalyzed m echanism of eq 5, and that our base­ A base-catalyzed meehanism for the mutarotation catalyzed mechanism of eq 6a resembles the prior of the sugars may be represented as shown in eq 6a. acid-catalyzed m cchanism of eq 7. It will be seen Combination of the base with the hydrogen of the that the two m echanisms classified here under acid­ glycosidi c hydroxyl gives an activated complex that catalysis Ceq 4a and 5), operate on different species decomposes with separation of the conjugate base of the sugar; this is likewise true of the two classified ancl l' up t ul"e of the ring. By the reversible addition under base-catalysis Ceq 6a and 7). The proportions 137 (

I of the species are determined by the hydrogen ion Base-catalyzed system : concen tration and th e base str ength of the c3.l'bohy­ kl drate, and hence the relative importance of the sev­ a+ B +=±XI eralmechanisms in any solution depends on the sub­ k_1 stance and the pH. k2 Presumably all of the m echanisms, in addition to X1+=±X,+ l-ln Lowry's, are applicable to the sugars, with eq 5 and k-2 6a favored in alkaline solution and 4a and 7 in acid k3 solution. It appears that eq 4b would be the main X 2 + HB +=±X 3 course for the primary and secondary and k-3 indeed the only one that is applicable to tertiary k4 amines. The mutarotation of ter tiary glycosyl­ X 3+=±i3 + B amines was observed by Kuhn and Birkhofer [17], who k_. concluded that the reaction involves the formation of a substituted ammonium ion followed by elimina­ In these expressions, HA represents an acid tion of a proton, to give the imonium ion - C= NHi . catalyst, B a base catalyst, a and {3 the modifica­ I tions of the sugar, and Xl, X 2, and X3 reaction intermediates. The r ate constants and the inter­ Although eq 4b involves the same imonium ion, we mediates arc not the same for the two systems. beli eve that it is produced directly from the amine Application of th e Christiansen equations ([13, p. rath er than from the corresponding ammonium ion . 107]) to the acid- and base-catalyzed systems for the It should be pointed out that the m echanism of eq sugars, and combination of r ate constan ts, lead to 4a and 4b requires only an acid catalyst and is at the following expressions, respectively for the rates variance with the curren tly held concept that both of r eaction: an acid and a base catalyst are necessary fo r the mutarotation reaction. As applied to the glycosides, a m echanism analogous to eq 4a will ac­ count adequately for acid-ca talyzed interconversion in nonaqueous solven ts, a reaction for which mecha­ nisms analogous to those of eq 5, 6a, and 7 are inade­ for catalysis by an acid , and quate because the aglycone group does not permit attack by a b ase ca talyst. Apparently the acid- and base-catalyzed reactions of the sugars take place side by side, and the over-all reaction can be regarded as the sum of the par allel for cat alysis by a base. At equilibrium, and competing r eactions, the rates of which depend on the acid and base catalysts present . It is of _ d[a] = o interest to examine the newly postulated mechanisms dt . to ascer tain whether they will account for the effect of acid and b ase catalysts on the rate of mutarota­ Thus k[a] - k'[i3] = O in both cases, and k/le' is fixed tion . For this purpose, the mechanisms of eq 4a by the equilibrium r atio. By adjusting the arbitrary and 6a can be formulated in the following manner: constants kA and kD' the same value in the two equations is assigned to k and likewise to k' , so that (k[a]- k'[{3]) becomes a common factor for all catalysts. The over-all rate then becomes Acid-catalyzed system:

for i acid catalysts and j base catalysts. If the only catalysts are oxonium ion, hydroxyl ion, and 'water eq 8 reduces to

wher e kH 20 includes both acid and base catalysis by the water molecule. This equ ation, when integrated and expressed in terms of optical rotation by Hud­ son's method [18] 8 becomes

k, 8 At the timc tha t Hudson Dres~ nt c d his classical development of the kirret ics of m utarotation , it was though t that the equilibrium of wn s established X 3 +=±i3 + H A between a ring form and an open-cha in form (referred to in his article as lactone k_. a nd hydrate, respectively). 138

l When this reaction is taken into account, it can +In ;:0=::", =(kH 20 + lcu[H +] + leoH[OH -])(Ic + le' ). be shown by application of the mass law that t '" By reducing 10 wmmon logarithms and combining constants, ! lo ro - r",= t g r - 1' t '"

This is Lhe familiar expression that Hudson found to represent the mutarotation of in acid, neutral, and alkaline solutions [6]. This derivation where J{ is the equilibrium constant for eq 13. Ap­ shows that the postulated acid- and base-catalyzed plication of eq 14 to the data of table 2 gives a series reactions arc in accord with the previously known, of simultaneous equations that show that in the pH experimentally determined relationship correlating range where the mutarotation is sufficiently slow the rate constant for the mutarotation of the sugars to be m easurable, the effect of the term J{ [H+] is less wi til the catalysts present. than the experimental error. Consequently, the The Christiansen eq uations may be similarly term can be neglected and eq 12 can be applied to applied to the mllLarotation of the glycosylamines the mutarotation of L-arabinosylamine. (eq 4a and 4b ), and the summation then takes th e From the data of table 2, numerical values were form: obtained for the catalytic coefficient of eq 12. At low concentrations of oxonium and ammonium ion, the quantity leH20 + leoH[OH - ] approaches le ,n , and each term must be less than the lowest observed rate (0.0004 at pH 12) ; hence lc H20 and leOH are less than 4X 10- 4 and 4X 10- 2, respectively. From experi­ The expressIOn calls for general acid and base men ts 2 and 3, lcN U , was found to be 10- 2 . Inasmuch catalysis. It has been found experimentally that the mutarotation ra te is increased by the presence as lcu20 and leou[OH - ] are negligibly small in com­ of ammonium, phosphate, and carbonate buffers. parison with the values of lc", in experiments 4, 5, and However, for systems in which the only catalysts are 6, values of le H were calculated on the a sumptionthat water, oxonium ion, hydroxyl ion, and ammonion ion, le",= lcu[H+]+ 1O - 2[NHt ]. The average value of leH eq 11, when treated in the same manner as eq 8, so obtained is 6.9 X 10 6 . The mutarotation con tant reduees Lo of table 2, cOlTected for the catalytic effect of am­ monium ion, are given in the last column of the table and are shown by the dotted curve of figure 3.9 A comparison of the numerical values of the catalytic coeffi cients for L-arabinosylamine with those for D-glucose is given in table 3. It may be seen that the principal catalyst for the mutarotation of D-glucose is the hydroxyl ion , whereas for the mutarotation of In tbe case of the glycosylamines, there is theo­ L-arabinosylamine it is the oxonium ion. The retically a complicating factor. In the derivation of striking difference arises primarily from the fact that the expression for the rate of mutarotation of the the glycosyl oxygen has a greater tendency than the sugars, it is possible to assume that a direct relation­ nitrogen to release a proton (eq 6a and 6b) and less ship exists between the concentrations of the alpha tendency than the ni trogen to share a lone electron and b ta isomers and the optical Tota tion regardless pair (eq 4a and 4b). of the presence of either more or less acid. This assumption is valid because only a minute part of the sugar exists under any given conditions in other TABLE 3. Catalytic coeffici ents oj mutarotation 1'eactions at than the normal forms. Because of the basic properties 20° C of the amino group, however, part of both the alpha and the beta glycosylamine probably exist in solution L-Arabinosyl- CatAlyst Sy mbol amine o-Olucose' as th~ corresponding substituted ammonium ion. The proportion of the free amine in aqueous solutions H 2O kH20 < 4X IO- ' 2.6X iO- ' is determined by the equilibrium: H + kJ-l 6.9 X IOG J .6XIO- 1 OB - kOR 4X IO- 2 8X I03 NUt kNH, I X IO- 2 1.2XlO- 3

• Data from [19]. (13)

9 In a preliminary publication [ 2~, an expression was given to represent em­ pirically the rSle of mutarotation in experiments I, 7, 8, and 9. No attempt was madA to evaltnte the general acid and base catalySis of tbe buffers used. 139 ·12 I structure CD ), addition can take place by attachment I of a nucleophilic group to the catbon, and a shift of I Q el ec~r.ons. . If the nucleophi.lic group is hydroxyl, I .10 f\ addltIOn Yields an mtcrmedlate aldehyde ammonia I CF). This grouping is known to be unstable and I MUTAROTAT ION gives up ammonia readily. Thus the aldehydic '2 \ I properties of the glycosylamine account for the (!) 0 .08 ~ -' facility with which ammonia is eliminated. Z ~ (j) -1\ Proposed mechanism for the hydrolysis oj glycosyl­ I-z .06 amines : f'! • I (j) I z 0 6 CH· NH2 CH ·NR+ <.) I .04 I w I'" 1 '" 3 \:i I R 0 R 0 0:: I 1/ 1/ J",,"o""s \ - C - C .02 \ 1 1 \\ ( B ) ( C ) 0 k- 2tl(Hto) l/ ~-o---- (H 2 0 ) I, k2 o 2 4 6 8 10 12 nH RC= NRt HC= NH

1 (H 20 )k3 1 FI GURE 3. Rate constants 101' mutarotation and hydrolysis of R f=! R L-arabinosylamine. 1 (H j O)k3 i - COR -COH

1 1 IV. Hydrolysis Reaction (D ) ( F, )

It has already been mentioned that the rate of k _'i1~? H-) hydrolysis of L-arabinosylamine was calculated from the increase in optical rotation that follows the H initial decrease. The rate of hydrolysis obtained 1 from the data of table 1 is shown in figure 3 as a HO- C- NR2 1 function of pH. The results bring out clearly the R striking sensitivity of L-arabinosylamine to hydrolysis 1 within a limited pH range. lO - COR A satisfactory mechanism for this reaction must 1 account for the ease of hydrolysis and the restriction (F ) of the reaction to a limited pH range. It follows from the well-known stability of amines to acid k- 5tl (H 2 0) hydrolysis 11 that the hydrolytic reaction is not (HtoJ I ks characteristic of the C-NHzlinkage but must be due H to the glycosyl structure. It was mentioned in 1 connection with the acid-catalyzed mutarotation - O- C - NH2 1 that the glycosylamine is able to form the imonium R Sugar -C= NHi , ion, by rupture of the ring following the 1 (X) [ -C- OR addition of a proton to the ring oxygen. A mecha­ 1 nism for hydrolysis including this substance as an (G) intermediate is given in the next column.12 With this It will be shown next that the proposed reaction 10 The reaction-rate- pH curve is similar in shape to many of the enzyme activit y-pH curvcs, among which may be cited tbose of urcase [20], /i·glucosidase mechanism will account for the fact that hydrolysis [21], a·am ylase [22], aud cellulase [231. It seems poss iblc tbat the sensit.i vity of certain enzyme systems to a change in pH may also arise from equilibria compa­ is rapid only in a limited pH range. Presumably rable to those cited here. the first step in the hydrolysis is the formation of the II Tbe stability of the C-N linkage of am ines is illustrated by the fa ct that glucosam ine is obtained by treatment of with conccntrated hydrochloric imonium ion CD) by acid catalysis; this is accom­ acid [24] . 12 The glycosylamines are somewhat analogous to the dialkylaminometh yl panied by a side reaction to form the glycosylammo­ alkyl , which have becn studied b y Slewart and Bradley [251. These authors fo und that the com pounds are rapidly h ydrol yzcd by acid and explained nium ion CC). The second step is the addition of a the case of hydrolysis by a mechanism invo lving the formation of an iminium ion hydroxyl ion to D , to yield the aldehyde ammonia (imonium ion) and condensation of this wi th hydroxyl ion . Our mechanism includes analogo us steps. CF ); it is also accompanied by a side reaction forming

140 the imine (E). The last part of the process covel' and denominator by [I-P], and combining constants the elimination of ammonia from the aldehyde am­ Lo obtain monia. It is represented as proceeding through the ionization of F (reaction 5), followed by an acid­ d(A - X ) (A- X) catalyzed rearrangement of the anion (G) to yield the dt K' + K"[H+] + K'''[OH- ] aldehyde form of the sugar, and ultimately the equi­ librium mixture. The ammonia is converted to am­ After integration, evaluation of th e integration con­ monium ion, thus driving the reaction to completion. stant and conversion to common logarithms, the In the derivation of the rate constant as a function of equation becomes the oxonium ion concentration, it is postulated that in the pH range in which hydrolysis occurs, equilibria 1 A 1 for all reactions except 6 are established quickly and T log (A- X) maintained throughout the reaction. It is also as­ sumed that within this range reaction 6 is irreversible. where k bYdrol is the hydrolysis rate constant. Inas­ Inasmuch as equilibrium conditions arc postulated much as the change in optical rotation during the for all r eactions except 6, the rate of hydrolysis is hydrolysis period is l'. measure of the change in represented by (A- X), the expression in terms of optical rotation becomes d(A-A) (15) dt 1 1' t 1 - 1'", 1 t - t log ~= K +K [H+l + K [On--l= lch Ydro ,o where A is the stUn of all components and X is the 2 1 t2 '" 1 2 3 sugar formed by hydrolysis. From mass law it fol­ (16) ows that By use of the data of table 4, nine simultaneous equations corresponding to eq 16 were set up. Com­ [Fl [H20llc 5 = [Gl [I-r+lk _5 bination of these in groups of 4,2, and 3, and solution by the m e thod of averages gave values of 7.9, 6.1 X I 04 [Dl [OH - ]lC4 = [FlL4 and 4.2 X 108 , respectively, for K 1, K 2, and K 3 . Thus,

[El [H+lL3 = [Dl [HzOllc 3 1

[Bl [H+lkz= [Dl [I-I20 lL2 A plot of this expression is shown by Lhe solid curve of [Cl [H 20lL1= [Bl [I-Plk 1 figure 3; the experimental results arc in good agree­ ment with the curve. Thus, the postulated mecha­ The equations may be solved for B , C, D , E, and F nism and the equation developed from kinetic con­ in terms of G, and the values substituted in the ex­ siderations account for the fact that hydrolysis is pression A = B + C + D + E + F + G + X to obtain rapid only in a limited pH range.

(A - X)- [G] ( L5L 4L2 + [H +]L5L 4L2kl + T ABLE 4. Summa,·y oj data Jar the hydrolysis ,'eaetion at 20°C - [OH llc 5k 4k2 [H20][OH lk5lc 4lc zL[

Experim ent pH [H+] [OH -] k· bydrol [H+]L5L4 + k -5L 4k3 + 10_. ______7.2 0.63X IO- 7 10X IO-s 0. 019 [H20 ][OH ]k 5k4 [OH ]k5k 4L 3 lL ______6.8 1. OXI0-7 6.3XIo-S .029 12 ______6.2 0.3XlO-7 l. 6XI0-s .055 13 ______5.9 13X 10 -7 0.8X lO-' . 068 [H+] k - 5 14 ______+ 1). 15 ______5.0 1. OXIO-' lXlO-' . lOS [H20]k5 4.6 2.5Xlo-' 0. 4X10-' . 108 16 ______17 ______4.0 1. OX 10-' lO X 10-" . 008 3.7 2. OX 10- ' 5Xl o-" .047 This, when simplified by combining constants, and 18 ______3. 1 7. 9XIO- ' 1. 3XlO- 1l .021 expressing the results in terms of new constants becomes B kbydrol= l/(tz-tl) log (Ttl-T cx,,)f(T /2- Tcc), w here Ttl is the first measurement iu t he peri od used for evaluatin g the rate or hyd rolysis . (A- X)[OH- F [G] [OH F+ k'[OH ]+k" In the preceding discussion, it was necessary to omit consideration of the indi vidua.l reaction con­ If the derived value for [G] is substituted in eq 15, stants and of catalysis by acids and bases other it becomes than oxonium and hydroA'}'l ions.13 Fmthermore, it was not possible to consider the several ring d(A-X) (A- X)[OH- ]2[H+]k6 isomers of the system separately. In the mechanism dt [OH F + k'[OH ]+ k'" on page 140, intramolecular reaction of carbon 1 13 InasTll uch as equilibrium co nditions prevail during the hydrolysis, general catalysis in the formation or the imonium ion does not a ITeet tbe rate or h ydroly­ which may be simplified by multiplying numerator sis. ~lth ugh it Mcolcrat s th rate of mutarotlltiol1 . 141 of D with the hydroxyl of carbon 4 would give the VI. Role of the Imonium Ion in Other alpha and beta furanosylamines; reaction with the Reactions of Glycosylamines hydro:x-yl of carbon 5 would give the alpha and beta pyranosylamines. Hence Band 0 symbolize It is believed that the imonium ion, considered to all cyclic modifications of the glycosylamine, and the corresponding cation, respectively, and the constants be an intermediate in the hydrolysis reaction, ac­ of reactions 1 and 2 represent composite rates. co~nts not only for the formation of the diglycosyl­ Similarly, F includes both stereomeric modifications am me but for several other reactions. Thus the addi­ of carb

In some cases the mutarotation and hydrolysis VII. Experimental Details appear to be complicated further by a side reaction that takes place during the early part of the reaction 1. L-Arabinosylamine period and varies in importance with the experi­ mental conditions. Evidence of this complicating Dry ammonia was passed slowly, at room temper­ factor may be seen from the data of table 5. It ature, into 100 ml of methanol containing 1 g of will be observed that when an excess of acid is ammonium chloride and 20 g of L-arabinose in suspen­ added slowly to L-arabinosylamine, the optical rota­ sion. Addition of ammonia was discontinued when tion drops to a lower point than when the acid is the sugar had entirely gone into solution. Ether added quickly. The product having the lower rota­ was then ~dded to the point of incipient turbidity, tion appears to be hydrolyzed more rapidly than the and the mIxture was allowed to stand in the refriger­ other, as the optical rotation of the solution rises ator until crystallization of L-arabinosylamine oc­ more rapidly and ultimately exceeds that of the curred (3 weeks). The crystals were collected on a solution containing less of the byproduct. It seems filter, washed with methanol, and air-dried; the probable that the complication is caused by the yield was 15 g. Recrystallization was conducted by formation of a diarabinosylamine. Evidence of this dissolving the crude arabinosylamine in 30 ml of a reaction is also found in experiments 19 and 20 of 1:10 mixture of concentrated ammonia and water. table 1. In these, the initial observed rotation is The solution was filtered and diluted with 60 ml of lower than usual, and the early change in optical methanol saturated with ammonia, and then with rotation is too rapid to be due entirely to the hydroly­ 150 ml of absolute ethanol. The resulting crystals sis of L-arabinosylamine. As the reaction, after the were separated after 2 hours, washed with methanol first few minutes, appears to follow the first-order saturated with ammonia, and dried at room tempera­ course, the calculations of the rate were made with­ ture in a vacuum desiccator containing phosphoric out the use of the early anomalous values. In experi­ anhydride. The recrystallized product weighed ment 18 of table 1, however, although the initial 10.8 g, and melted at 124° O. At a concentration of rotation is likewise low, the entire mutarotation 2 g/100 ml in 002-free water, [al~ = + 86.3° (5 min)· follows the first-order course. The side reaction . 83.8° (60 min) : 78.6° (7 hr) ; 80.5° (54 hr). D~ which under other conditions may become of majo; Bruyn and Van Leent [2], who first prepared the importance, is being studied further. compound, reported mp 124° 0 and [ al~o + 83° (H20, c= 10). Figure 1 shows the titration curve obtained when TABLE 5. Reaction of L-arabinosylamine with acid (evidence 0.1 N was added portionwise to 75 ml of a for a side reaction) HOI soluti?n co?-taining 0.1493 g of L-al'abinosylamine. 0.25 g-of L·arabinosy!amine dissolved in sufficient 2.5 N HC! to give a volume SuffiCIent hme was allowed after each addition of of 25 m! acid for the pH of the solution to reach equilibrium. At the end of the experiment, the optical rotation Optical rota· Optical rota· Time acter begin. tion after rapid tion after drop· corresponded to that of L-arabinose, and after the ning the addi· addition of wise addition ammonium chloride and excess acid were removed tion of acid acid of acid during 2.5 minutes by trea:tment :vith ion-excha:nge resins, 0.14 g of crystallme arabmose was obtamed from the solution. lv[inutes [alll' [a];~ This is a nearly quantitative yield. 3.8 69.0 67.2 7.0 69. 3 68.2 15.0 69.7 69.4 30.0 69.9 70.5 2. Tetraacetyl-L-Arabinosylamine 60. 0 70.0 7l. 0 Change in [alll' 4- mixture of 4 ~ ?f finely powdered ~ - arabinosyl­ in 1 bL __ . ____ _ l.0 3.8 amme, 40 ml of pyn dme and 16 ml of acet.IC anhydride in a flask equipped with a mechanical stirrer, wa~ 142 kept ill an ice bath and stilTed until solution was H A c complete (4 Ins). After 18 hours at 0° C, the solu­ H 2 C- CHO oHc - 6 n tion was poured into a mixture of ice and water, and the product was extracted with chloroform. E vap­ IL--__ O __--l i oration of the chloroform gave a crystalline residue that was recrystallized from hot ethanol. Tbe is characteristic of all N-aeetyl-pentapyranosyla­ yield was 3.65 g. After several recrystallizations, mines having the same configuration for carbon 1 as tetraacetyl-L-arabinosylamine melted at 177° to that of N-acetyl-L-arabinosylamine, 17 ° C; [al~o =+ 9.6° (CHCb, c= 1.6). Analysis: Calculated for C I3H 190 gN: C, 49.21; H , TABLE 6. Oxidation of N -acetyl-L-ambinosylamine with 6.04; N, 4.41; CO·CH 3, 54.26. Found: C, 49.3; sodium metaperiodate H , 6.1; ,4.4; CO·CH3, 52.4 .

Moles peri- R eaction Optical TO- odate per 3. N-Acetyl-L-Arabinosylamine time tat- ion e. mo le amin e

Six grams of tetraacetyl-L-arabinosylamine was Jlfinutes [a]'g 2.5 +19. 6 dissolved in 100 ml of anhydrous methanol, and 10 3. '1 - 6. 9 5. 0 1. 92 ml of 0.1 N barium methylate in methanol was ,\ . 3 -30.9 added. The was removed by distillation 7. 3 - 42.8 under reduced pressure, with exclusion of moisture. 10. 0 1. 99 10. 7 - 47. 4 The residue wa dissolved in water and treat ed 14 . 2 - 49. 0 succe sively with a cation and an anion exchange 30 2. 00 60 - 44 . resin to remove barium salts. The resulting olu­ 120 2. 01 tion, after evaporation, gave 3.6 g of crystalline product. To recrystallize, the material was dis olved • Based on weight of N -Rcctyl-L-arabinosylamine. in 6 ml of hot water , and the solution was treated with a decolorizing carbon. After filtrat,ion, the 5. Mutarotation and Hydrolysis Measurements solution, combined with 2 ml of washings, was diluted with 2 volumes of methanol, and the mixture was The course of the mutarotation and hydroly is allowed to stand for several hours at 0° C. The reactions was followed by measurements of optical crystalline N -acetylarabinosylamine was separated rotation on solutions of L-arabinosylamine containing and washed with methanol. Melting point, 222° to known amounts of acid and bases. In each experi­ 224° C; r a]~ =+ 69.1° (H 20 , c= 4). The constants ment, 0.5 g of crystalline L-arabino ylamine wa were unchanged after further crystallization . placed in a 25-ml volmnetric flask. Sufficient acid, Analysis: Calculated for C7H 130 5 : C, 43.97; H , base, or buffer solution at 20° C was added rapidly to 6.85; N, 7.33 . Found: C, 44.1; H, 6.8; N, 7.4. give a volumo of 25 ml, and measurements were made of' the optical rotation in a 4-dm tube. Time was measured from the moment the solvent was added 4 . Periodate O xidation of N-Acetyl-L to the crystals. At the end of the eA":periment, and in Arabinosylamine some cases at intermediate points, the pH of the reaction mixture was measured. As 1 mole of It was fOlmd that the optical rotation of a olu­ ammonia is liberated during the hydrolysis, it was tion that was 0.1 M with respect t o N -acetyl-L-arab­ not po ible to maintain the hydrogen ion concentra­ inosylamine and 0.25 M with respect to sodium tion constant, and in each case the pH of the solution metaperiodate (N aI04) quicldy changed from a dex­ increased somewhat as the reaction proceeded. For tro to a levo direction and attained a maximum pmpose of comparison, the values obtained at the ( [ a l ~ = -49° ) in 14 minutes. After this time, there end of the experiment were used in the calculations. was a very gradual decrease in levo-rotation. At In solutions more acid than pH 7.8, the initial intervals, 10-ml ali quots of the solution were with­ decrease in optical rotation that characterizes the drawn, and each sample was treated with 10 ml of mutarotation was too rapid to detect. In the pH a saturated solution of sodium bicarbonate, 25 ml range 7.8 to 12, velocity ,constants for the mutarota­ of 0.1 N sodilUn arsenite, and 1 ml of a 20-percent tion wore calculated by the formula solution of potassimn iodide. The excess arsenite was then titrated with a 0.1 N solution of iodine. The values in table 6 show that two moles of period­ ate ,vere consumed per mole of glycosylamine in 10 minutes, and that further consumption of per'iodate was negligible. These results are in harmony with where rtl and r t 2 are the specific rotations at times a pyranosidc structure for N -acetyl-Irarabinosyla­ tl and t2, and rm , the minimwn optical rotation, i mine. The change in optical rotation that takes asswned to be the equilibrium rotation in the place during the oxidation is also given in table 6. absence of hydrolysis. In some experiments the The levorotatory product, presumably minimum rotation was not reached because the

91G7 3- 51- 5 143 reaction either was too slow, or was complicated by were prepared : Tetraacetyl-L-arabinosylamine competitive hydrolysis. In these cases, a value of (C13H190sN), mp 177° to 178 0 C, fa11° = + 89.6° + 69°, the minimum rotation found for uncompli­ (CHCI3 • c= 1.6); N-acetyl-L-arabinosylamine (C7- cated cases, was used for rm. H130 5N), mp 2220 to 224 0 C, [a11° =+69 .1° (H 20 , It is noteworthy that the equilibrium rotation c= 4). The latter substance was found to react obtained in highly alkaline solution in the presence of quicldy with 2 moles of sodium periodate, as required 2.5 N ammonium chloride (experiment 3, table 1), is for an N-acetylpentosylamine having the pyranose appreciably lower than that found at the same pH structure. in the presence of only 0.1 N ammonium chloride (experiment 2). The cause of the difference is not known, but it may be due to the side reaction The authors gratefully acknowledge the assistance mentioned on page 142. of Nancy B . Holt in various phases of the work, and The rate of hydrolysis was calculated from the rise of Rolf A. Paulson, who analyzed the compounds in optical rotation that takes place after the primary reported. They also express their appreciation to mutarotation reaction is complete. In the calcula­ T. D . Stewart of the University of California for tions it is assumed that the second change in rotation k~ndly and helpful criticism of the reaction mecha­ is due solely to the hydrolysis reaction and that the nIsms. optical rotations of both the arabinosylamine and the IX. References product, L-arabinose, remain constant throughout the period chosen for the rate measurement. [1] H . L. Frush and H . S. Isbell, J . Research NBS 41, 609 (1948) RP1943. The values for the hydrolysis rate constant given [2] H. S. Isbell and H . L. Frush, J . Am. Chern. Soc. 72, in table 1 were calculated from the formula 1043 (1950). [3] C. A. Lobry de Bruyn and F . H. Van Leent, Rec. trav. chim. Pays-Bas. 14, 129 (1895). [4] ' P . Brigl and H . Keppler, Z. physiol. Chern. 180, 38 (1929). [5] C. Niemann and J . T. Hays, J . Am. Chern. Soc. 62, 2960 (1940). where r II and r 12 are the rotations at times i l and i 2, [6J C. S. Hudson, J. Am. Chern. Soc. 29, 1571 (1907). respectively, and r oo is the of the [7] J. N. Bronsted and E. A. Guggenheim, J . Am. Chem. Soc. 49, 2554 (1927). fully hydrolyzed material. [8] T . M. Lowry and I. J . Faulkner, J . Chern. Soc. 127, 2883 (1925). VIII. Summary [9] K Butler, F . Smith, and M . Stacey, J . Chern. Soc. 1949, 3371. [10] T. M. Lowry, J. Chern. Soc. 127, 1371 (1925). It has been found that L-arabinosylamine under­ [11] G. E. K . Branch and M. Calvin, Theory of organic goes a mutarotation reaction that is followed or , p. 404 (Prent ice-Hall, Inc., New York, accompanied by hydrolysis of the amino group. The N. Y., 1944). mutarotation takes place at measurable rates [12] C. G. Swain, Abstracts of papers, p. 47P (116th Meeting, Am. Chern. Soc., Atlantic City, N. J ., Sept. 21, 1949). between pH 7.8 and 12, but it is too rapid to observe [13] L. P . Hammett, Physical organic chemistry (McGraw­ in solutions of higher oxonium ion concentrations. Hill Book Co., Inc., New York, N . Y., 1940). Mechanisms are proposed for the mutarotations of [14] H. Fredenhagen and K. F. Bonhoeffer, Z. physik. Chern. the glycosylamines and of the sugars. The mutaro­ [A] 181, 392 (1938) . [15] K F. Bonhoeffer and O. Reitz, Z. physik. Chern. [AI 179, tation of the glycosylamines, in marked contrast to 135 (1937). that of the sugars, is not appreciably catalyzed by [16] G. A. Howard, G. W. Kenner, B. Lythgoe, and A. R. hydroxyl ion, but is strongly catalyzed by oxonium Todd, J. Chern. Soc. 1946, 855. ion. Apparently mutarotation and hydrolysis of the [17] R. Kuhn and L. Birkofer, Bel'. deut. chern. Ges. 71, 1535 (1938). glycosylamines take place through an intermediate [18] C. S. Hudso n, Z. physik. Chern. 44, 487 (1903). imonium ion. [19] T . M. Lowry and G. F . Smith, Rapports sur les hydrates The rate of hydrolysis of L-arabinosylamine is de carbone, p. 116, 10th Conference of t he Inter­ unusually sensitive to the acidity of the medium, is national Union of Chemistr y ( Li e ~e, 1930). [20] S. F . Howell and J . B. Sumner, J . BioI. Chern. 104, 619 greatest at pH 5, and is negligible in both alkaline (1934). and highly acid solutions. A mechanism is proposed [21] K Hill, Bel'. Verhandl. sachs Akad. Wiss. Leipzig, to account for these facts and by application of mass Math. phys. Klasse. 86, 115 (Hl34). See also, W. W. law to the reaction scheme and evaluation of the Pigman and R. M . Goepp, Jr., chem­ istry, p . 490 (Academic Press, New York, N . Y., 1948). constants, the following expressIOn is obtained for [22] E. Ohlsson, Z. physiol. Chem. 189, 17 (1930). the rate of hydrolysis: [23] G. A. Greathouse, T extile Research J . 20, 227 (1950). [24] C. S. Hudson and J. K. Dale, J . Am. Chern. Soc. 38, 1 1431 (1916). khYdrol [25] T . D. Stewart and W. E. Bradley, J . Am. Chern. Soc. 54, 4172 (1932). [26] H. S. Isbell, Ann. Rev. Biochem. 12,206 (1943). To provide information concerning the structure of L-arabinosylamine, the following new compounds WASHINGTON, July 25,1950

144