Studies on the Stability of Simple Derivatives

Transcription

1 Studies on the Stability of Simple Deivatives of Sialic Acid* Fom the Cell Ch.(Jmisty Laboatoy, Depatment of Biochemisty, College of Physicians and Sugeons, Columbia Uniusity, New Yok 32, New Yok (Received fo publication, May 8, 1963) The sinlic acids, whose chemisty and metabolism have been eviewed epeatedly in the ecent past (l-5), ae impotant constituents of many biological heteopolymes. It is quite clea that the multiple types of linkage in which a nonulosaminic acid can engage may influence the outcome of eactions designed to demonstate its pesence, cstimatc its quantity, o detemine its modes of attachment, in diffeent polymes. Even the peteatment of the polyme, in the couse of its puification, may not bc without effect on the attachment of sialic acid to one o the othe of the constitunts though its vaious functional gouljs. This poblem of stability was bought to ou attention in the couse of studies in this laboatoy on the mucolipids of bain (6-13). l he liteatue is not abundant in infomation on this point. It is geneally ecognized that the fee sialic acid is unstable towads acid and especially towads alkali, and that it is eleased eadily, when linked as a glycoside, by vey mild acid teatment and even by autohydolysis. A qualitative study of the stability of sialic acids and thei methyl estes at diffeent ph values has been published (14), and occasional mention of I-aious obsevations on this subject will be found in many pnpcs dealing with othe aspects of the chemisty of sialic acid. -1 detailed investigation, spcc*ifying the exact conditions unde whih the este o the glycositlic link of sialic acid is uptued, howeve, has not come to ou attention. The pesent study descibes tspeimmts that may seve as a fist step in this diection. Some of the findings have been mentioned vey biefly in a peliminay fom (15). l hc stnbility of sialic acid and of thee of its simplest functional deivatives was investigated unde a vaiety of conditions, mainly by means of caloimetic techniques. The thee deiativcs epesent models of the thee pincipal ways in which sinlic acid (I) ma-y be linked to anothe molecule: (a) though an este bond (sialic acid methyl este, II); (b) though a glycosidic bond (methosysialic acid, III); and (c) though a combination of these links (methosysialic acid methyl este, IV). The stuctues of the fou coml)omds unde investigation, in thei cuently accepted configuations (16), ae shown in Fig. 1. The majoity of th expeiments wee caied out with a * These studies wee suppoted by a eseach gant fom the Sational Institutes of Health, United States Public Health Sevice. t This epot is fom a dissetation submitted by John D. Kakas in patial fulfillment of the equiements fo the degee of Docto of Philosophy in the Faculty of Pue Science, Columbia LVniesity. Duing pat of the t.ime, J. D. K. held a fellowship fom the Geek State Fellowship Foundation. pepaation of sialic acid isolated fom ox seum poteins and composed of 37 y0 N-ncetyl- and 63% N-glycolylneuaminic acid. In addition, a few oienting studies wee pefomed with a specimen of N-acctylneuaminic acid secued fom human seum poteins. Expeiments with sialic acid fom sheep seum ae omittd fo the sake of bevity, as the esults only duplicated those pesented hee. ESPERIMENT.4L Analytica. PROCEUURE Methods The diect Ehlich eaction was caied out as descibed in the liteatue (17), but the volumes wee educed so that a final eaction mixtue of 3 ml esulted. The pocedue fo the thiobubituate eaction has been descibed befoe (18), as has been that fo the esocinol eaction (19) ; in the latte instance, heating at 100 was applied. Fo the hydoxamic acid eaction, a modification developed fo suga estes (20) was followed; the eaction time was educed to 30 minutes, as peliminay expeiments showed that with the methyl cstcs of sialic acid and methosysialic acid the maximal nbsobances wee attained within a few minutes. The oxidation expimc~nts with peiodate wee ljefomed in unbuffeed aqueous 0.01 M solutions of SnIOh at oom tempeatue in the dak. In samples emovcad afte vaious intevals, the emaining peiodate was detemined, afte dilution with an equal volume of phosphate buffe (12 g of Na2HPOI. 12H20 and 20 ml of N HtSOl in 100 ml), ph 6.5, by the addition of 1 dop of a concentated I<1 solution and titation of the libeated iodine with a 0.01 JI solution of sodium thiosulfate in the pesence of stach. Gilmont micobuettes sved fo the emoval of all samples and fo the titations. The melting points, epoted without coection, wee deteminctl on an elccticnlly h&cd stage (Fishe-Johns). Mateials Siulic i4cids (I)-Pepaations wee isolated fom seum poteins of man, sheep, and ox by hydolysis and ion exchange chomatogal)hy on Dowe; 50 and Dowcs l-x8, essentially as descibed in the liteatue (21). The c,ude substsnccs wee ecystallized thee times fom wate-methanol-ethe. The melting point (with decomposition) of all thee pepaations was (uncoected). The atios of A-acetyl- to W-glycolyhleuuminic acid, as found by micoestimation of glycolic acid cm, wee: bovin, 37:63; ovine, 87.5: 12.5; in human sialic 949

2 950 Sialic Acid Deivatives Vol. 239,?\ o. 4 COOH COOCH3 %-OH &OH H&H H-&H 0 H&OH 0 H-&OH AcHN%-H A&N-&H I- 6-H L- &ti HI&-OH H-&OH H%-OH H-A-OH %H~OH 6~~0~?OOH C-OCH3 H-6-H 0 H-&OH AcHN-L-H I I It III m COOCHJ FIG. 1. Stuctues of sialic acid (I), sialic acid methyl este (II), methoxysialic acid (III), and methoxysialic acid methyl este (ZV). AC = acyl. &OCHs H-6-H 0 H-&OH AcHN-k-H 6-H I- 6-H H-k-OH H-b-OH H-&OH H-k-OH LH~OH 6~~0~ acid, the eading fo glycolic acid was essentially negative (less than 2%). These findings ae in emakable ageement with the liteatue (compae p. 33 of a ecent monogaph (3)). The sialic acid fom ox seum seved fo the pepaation of the este and methoxy deivatives investigated in this study. N-Acetylneuaminic acid: N-Glycolylneuaminic CnH1gNO9 (309.3) Calculated: C 42.7, H 6.19, N 4.53 acid: GIHISNOIO (325.3) Calculated: C 40.6, H. 5.89, N 4.31 Calculated fo ox seum sialic acid: C 41.4, H G.00, N 4.39 Found (bovine) : C 40.80, H 6.17, N (Dumas) 4.69 Found (human) :* C 42.62, H 5.99, N (Dumas) 4.28 &a& Acid,%ZethyZ Este (II)-This deivative was pepaed by heating, unde eflux fo 30 minutes, sialic acid in dy methanol in the pesence of a small quantity of Dowex 50 which had peviously been twice efluxed with dy methanol fo 1 hou. The filtate fom the esin then was passed though a column of Dowex I-fomate in ode to emove sialic acid that had not eacted. The solvent was emoved, and the esidue was died ove Pz05 in a vacuum. The este, ecystallized twice fom methanol-ethe, melted with decomposition at (uncoected). The N-acetyl to N-glycolyl atio was 35:65. N-Acetylneuaminic acid methyl este: C,ZH2,NOg (323.3) Calculated : N 4.33 N-Glycolylneuaminic acid methyl este: CzHNO,n (339.3) Calculated : N 4.13 Found : N (I)umas) 3.94 Methoxysialic Acid Methyl Este (IV)-This compound was pepaed accoding to Blis et al. (23) by heating sialic acid, unde a eflux, with thee changes of dy methanol in the pesence of Dowex 50 fo a total of 15 hous. The poduct, ecystallized twice fom methanol-ethe-petoleum ethe, melted at 195 (uncoected). The N-acetyl to N-glycolyl atio was 1 These analyses wee pefomed by Mico-Tech Laboatoies, Skokie, Illinois. 2 These analyses wee pefomed by Schwazkopf Micoanalytical Laboatoy, Woodside, New Yok. 36 : 64. Methosy-N-acetylncuaminic acid methyl este: C,oH?xNOy (337.3) Calculated: C 46.3, H 6.87, N 4.15 Methoxy-N-glycolylneuaminic acid methyl este: Calculated: C 44.2, H 6.5G, N 3.97 Found :I C 45.0, H 6.75, N (Dumas) 4.11 XethoxysiaZic Acid (III)-This deivative was pepaed fom methoxysialic acid methyl este by mild alkaline hydolysis. An aqueous solution of the este (IV) was maintained at ph 10 and oom tempeatue fo 4 hous by means of an automatic titato (Radiomete, Copenhagen), when 1 mola equivalent of NaOH had been consumed and the hydoxamic acid eaction fo este had become negligible. The solution was passed though a column of Dowex 50 (H+ fom) and evapoated in the fozen state in a vacuum. Attempts at cystallization having failed, a semicystalline powde was obtained by pecipitation with ethe fom aqueous methanol. This poduct gave almost no colo in the thiobabituate eaction; howeve, afte hydolysis in citate buffe, ph 3, at 100 fo 45 minutes, the absobance was the same as that shown by fee sialic acid. No hydoxamic acid eaction was obseved. The extent of the esocinol test was the same as that affoded by fee sialic acid. The N-acetyl to N-glycolyl atio was 37:63. Methoxy-Nacetylneuaminic acid: C?H2N09 (323.3) Calculated: C 44.6, H 6.55, N 4.33 Methoxy-N-glycolylneuaminic acid : C,tHz,NOIo (339.3) Calculated: C 42.5, H 6.24, N 4.13 Found :3 C 43.5, H 6.32, N 3.80 RESULTS AND DISCUSSION Colo Reactions The mola absobances of the sialic acids (human, sheep, ox) and of the thee deivatives of sialic acid fom OS seum unde investigation in the diect Ehlich, esocinol, and thiobabituate eactions, pefomed by the techniques mentioned befoe, ae pesented in Table I. The thiobabituate eaction is by fa the most sensitive eaction fo sialic acids, as long as the hydoxyl on the anomeic cabon is unsubstituted. Its usefulness fo glycosidically bound sialic acid is limited, howeve, because of the dependence on an effective hydolysis. As the esults of the expeiments descibed below indicate, the conditions fo such a hydolysis must be chosen caefully, with the application of coection factos fo destuction, accoding to the type of binding of sialic acid in the paticula compound-infomation not always easily secued. The esocinol eaction is quite sensitive and vey epoducible. Ou esults also indicate that, in contast to the findings with the diect Ehlich eaction, the absobance is independent of the pesence of substituents on the cabosgl and 3 These analyses wee pefomed by Analytica Copoation, New Yok.

3 Apil 1964 J. D. Kakas and E. Chagaff 951 cabonyl cabons of sialic acid. The intefeence by many substances, and especially by galactose, howeve, endes its use athe difficult in the case of natual mateials. In the diect Ehlich eaction, on the othe hand, which appeas to be the most specific eaction in the case of comples mistues (compae IL 60 of (3)), the extent of absobance was found to depend on the substitution of the hemiacetalic hydosyl. Thus, wheeas sinlic acid methyl e.+ exhibited the same absobance as the fee sialic acid, the mola absobances of methosysialic acid and its methyl este wee 85 and 8O/,, espectively, of the absobance of the fee acid. The esults of a time study of the colo development in the diect Ehlich eaction ae illustated in Fig. 2. As can be seen, the poduction of colo inceases linealy with time, with slopes almost identical fo the fou compounds, afte a chaacteistic initial peiod of etadation. The linea ise in extinction continues much beyond the Ijeiod shown in Fig. 2. In this eaction, cyclization to yield a pyolic compound has been postulated as the fist event (3, 14, 24, 25). The diffeences obseved in the shapes of the cuves in Fig. 2 do not contadict such a mechanism. The fee abonyl equied fo the cyclization is pesent in sialic acid and its methyl este, but must fist be poduced in methosysialic acid and methosysialic acid methyl este by the emoval of the glycosidic methosyl, an event occuing moe easily in methosysialic acid than in its methyl este, as will be shown below. The cuves in Fig. 2 ae constucted in tems of the optical density. If the mola absobances ecoded in this expeiment afte a teatment of 30 minutes, the time equied by the standad technique (17), wee computed, they would be found to be ai)peciably lowe than those listed in Table I; moeove, the diffeences in colo development between the fee cabonyl compounds, sialic acid and its methyl este, and the glycosides, methoxysialic acid and its methyl este, ae moe ponounced. These discepancies ae obviously due to the diffeent espeimental aangement equied by the time study: the size and TABLE I Mola absobances of sialic acids and dekvatives in vaious colo eactions Sialic acid souce and deivative A-Acyl Mola absobances (X 10-a) substituents ead at specified wave lengths Acetyl Human seum Sheep seum Ox seum. 37 Sialic acid methyl este 35 Methoxysialic acid. 37 Methoxysialic acid methyl este. 36 Methoxysialic acid, afte hydolysist. GI - co yl Diect Ehlich ITI@ ii-l Aveage deviat,ion * These eadings ae, in view of the aveage deviation, not significant. t At ph 3 and 100 fo 45 minutes RCSOTcinol -I- 580 m/i Thiobabituate 549 m&l (2.2)* 10.9 (2.8)* IO Fo. 2. IXect Ehlich eaction: colo development by sialic acid (fom ox seum) and its deivatives. A solution of 10 p~noles of the compound in 00 ml of N HCl containing 0.83$; of p-dimethylaninobenzaldehgde was heated in a boiling wate bath. At the times indicated, 3-ml samples wee withdawn, cooled in ice wate, and bought to oom tempeatue, and thei absobance was detemined spectophotometically at 565 mp. A, siali acid; A, sialic acid methyl este; 0, methosgsialic acid; l, methoxysialic acid methyl este. I I I 1, I I I I I I IO ihk-& HOURS FIG. 3. Oxidation of sialic acid (fom ox seum) and deivatives by sodium metapeiodate. To a solution of 20 Mmoles of the compound in 10 ml of wate, the same volume of a 0.01 M aqueous solution of NaIO, was added. Equal samples (1 ml) wee emoved at diffeent intevals and mixed with 0.3 ml of phosphate buffe, ph 6.5, and 1 dop of a concentated KI solution. Five minutes late, the libeated iodine was titated with 0.01 M Na&;zOz solu- tion in the pesence of stach. A, sialic acid; A, sialic acid methyl este; 0, methoxysialic acid; l, methoxysialic acid methyl este. type of the vessels and the heating bath used, the volumes of the assay mixtue, the ate of tempeatue equilibation, etc., all of which ae not without infuence on the colo value. Peiodate Oxidation Uptake of Oxidant-The peiodate osidation of sialic acid (10, 23, 26) and of methosgsialic acid methyl este (23) has been investigated befoe. We show, in Fig. 3, a einvestigation of these compounds togethe with sialic acid methyl este and methospsialic acid unde unifom conditions. An initial veq apid uptake of 2 moles of the osidant pe mole of substance is obseved with all compounds. In the case of the glycosiditally substituted deivatives, methosgsialic aid and methosysialic acid methyl este, the cuves level off at this point, with

4 952 Sialic Acid Deivatives Vol. 239, 1-o. 4 I I,, I I I! I I Id IO I I I I FIG. 4. Alkaline hydolysis of the este link of sialic acid methyl este (A) and methoxysialic acid methyl este (B). Solutions of 40 pmoles of compound in 10 ml of COz-fee wate wee used to detemine the equied equivalents of alkali, added in the fom of 0.01 N NaOH, in ode to maintain the indicated ph. An automatic titato (Radiomete, Copenhagen) was employed. o@li;t- COOCH3 &OH H-+-H H-k-OH H-C-OH bh20h II A$~~ = OOCH3 q o H-G-H $OOCH3 H-F-OH H-C-OH H-q-0 H H-q-0 H CH20H CH20H II A Fo. 5. Tautomeic stuctues of sialic acid methyl este vey little additional uptake obsevable in the couse of 24 hous. Sialic acid and sialic acid methyl este, on the othe hand, continue to consume peiodate, although at a much slowe ate, 1 additional mola equivalent being taken up in the couse of 20 hous. The initial apid consumption of 2 moles of oxidant is obviously due to scission between cabon atoms 8 and 9 and cabon atoms 7 and 8 (compae Fig. 1). The uptake of a thid mole by the two compounds possessing a fee hemiacetalic hydoxyl (sialic acid and its methyl este) points to the opening of the pyanose ing as a equisite fo the second phase of the oxidation, which pesumably concens the uptue between cabon atoms 6 and 7. This assumption would explain the slowe ate at which the thid equivalent of oxidant is consumed, since the opening of the ing would now become the ate-limiting step. A slight degee of oveoxidation beyond 3 equivalents is indicated by the cuves fo sialic acid and sialic acid methyl este (Fig. 3), which pesumably ae attibutable to a slow hydoxylation (27) of the methylenic cabon 3. Colo Reactions of Oxidation Poducts-Attempts to isolate the aldehydes expected to esult fom the action of peiodate on the fou compounds studied hee have until now not been success- ful. The eactivity of the cude oxidation poducts in the colo eactions nomally applied to sialic acid and its deivatives could, howeve, be examined. The compounds wee subjected to the action of NaI04 unde the conditions outlined above. and the excess of the oxidant was educed by means of ethylene glycol to iodate, which was pecipitated by the addition of the equied quantity of baium acetate. The filtates wee assayed diectly. Because of the sensitivity of colo tests to admixtues, contol solutions wee pepaed in which the peiodate eagent was, with the omission of the substate, put though the opeations mentioned hee (eduction, pecipitation, etc.); the esulting solution then seved as the solvent fo the unteated sialic acid deivative, whose absobance was compaed with that of the coesponding oxidation poduct. The following obsevations wee made on the oxidation poducts. (a) The absobance in the thiobabituate eaction n-as not alteed. This was to be espected, as peiodate oxidation actually is the fist step in this eaction. (b) The absobance of sialic acid in the esocinol eaction was almost doubled, with a shift of the absoption maximum fom 580 to 600 mp. (c) The absobance in the diect Ehlich eaction deceased by about 657, fo methoxysialic acid and its methyl este and by about 80% fo sialic acid and its methyl este. Alkaline Hydolysis of Este Link The este link of sialic acid is known to be cleaved unde vey mild alkaline conditions (21, 23). In the couse of the pesent studies, the hydolysis, at constant ph, of the este bond in sialic acid methyl este was compaed with that of methoxysialic acid methyl este. Aqueous solutions of the estes wee kept at a constant ph by means of an automatic titato. The equivalents of alkali consumed in these espeiments ae plotted against time in Fig. 4. A paallel set of espeiments, in which the gadual disappeaance of the este was followed at constant ph by means of the hydoxamic acid eaction, confimed that the alkali had actually been consumed by the saponification pocess. As shown in Fig. 4, the este link of sialic acid methyl este is boken at oom tempeatue and ph 8 within 20 minute*. wheeas methoxysialic acid methyl este equies a highe l)h and a much longe time fo complete saponification. The following eaction ate constants wee found fo the conditions specified in Fig. 4: sialic acid methyl este, lc = 2.7 x lop3 se0 ; methoxysialic acid methyl este, k = 3.1 x lo- set?. The substitution of the neighboing hemiacetalic hydoxpl thus appeas to ende the este link moe esistant to alkali. Although steic hindance could be invoked, it is moe likely that anothe facto is of impotance. In contast to fee sialic acid, the methyl este has been epoted to exhibit, in weakly alkaline solution, a tansitoy absoption in the ultaviolet egion (maximum at 265 mm) which has been attibuted (14) to the enol fom of the open chain stuctue (11 B in Fig. 5), in analogy to obsevations on ethyl acetoacetate (28). In the open stuctue, sialic acid methyl este would be expected to behave as an LYketo acid este, wheeas methoxysialic acid methyl este, owing to the stabilization of the pyanose ing (1Z in Fig. 5) by the substitution of the hemiacetalic hydoxyl, would be expected to show the eactivity of an c-methoxy acid. Estes of a-keto acids ae known to be saponified by alkali at ates much highe than those exhibited by thei aliphatic countepats o by the substituted a-hydoxy acids: the ate of alkaline hydolysis of

5 Apil 1964 J. D. Kakas and E. Chagaff 953 ethyl pyuvate was found to be 18,000 times highe than that of ethyl popionate and 840 times highe than that of methoxyglycolic acid ethyl este (29). Teatment with Acid Fee Sialic Acid-This substance is known to be decabosylated when heated in stong acid (23) but has been consideed, on the basis of the esocinol eaction, to be elatively stable unde less dastic condit,ions (30). A simila appaent stability of sialic acid was obseved in the pesent study when the diect Ehlich eaction was employed (Fig. 6). The same figue, howeve, shows that, unde identical expeimental conditions, the colo values ecoded with the thiobabituate eaction decease consideably in a linea fashion, appoximately one-half of the initial absobance being found at the end of 1 hou. A simila obsevation has been epoted ecently (31). ;\s is also shown in Fig. 6, Wacetylneuaminic acid (fom human seum) gave identical esults; this excludes the possibility that the two diffeent.v-acyl substituents in the sialic acid fom ox seum contibuted in diffeent mannes to the behavio of the latte in the colo eactions. It is obvious that the action, even of weak acid, on sialic acid has complex consequences (14, 32), but the poducts cannot yet be identified conclusively. That some of the degadation poducts eact with both the Ehlich and the esocinol eagents has been pointed out befoe (14). The teatment with acid obviously esults in a modification of the molecule that depesses the fomation of the thiobabituate chomogen, but does not intefee mateially with the othe colo eactions. Dccaboxylation does not seem to play a ole, as shown by the behavio of the degadation poduct towads the peiodatethiobabituate eagent. The teatment of sialic acid with HI04 in a stongly acidic envionment pesumably affods B-fomylpyuvic acid, in analogy to the 2-keto-3-deosy suga acids (33, 34), wheeas pevious decaboxylation would lead to the fomation of malondialdehyde (35). These two aldehydes fom, in the eaction with thiobabituic acid, coloed compounds whose absoption specta can be distinguished though the position of the centes of absoption and the espective mola absobances: the poduct fomed with P-fomylpyuvic acid has a maximum at 549 nm, and that fomed with malondialdehyde, at 532 np; the mola estinction shown by the latte poduct is about twice as high as that of the fist (34). As we have obseved, sialic acid heated in 0.1 N HCl at 100 fo 1 hou (Fig. 6) exhibits no shift in the position of the absoption maximum (549 mp), and this, togethe with the decease in the mola absobance, speaks against the occuence of decaboxylation. The patial lactonization of sialic acid (32) could account fo the lack, o the etadation, of the esponse to peiodic acid and fo the diminution of the colo value in the thiobabituate eaction. Dimeization though the fomation of a glycosidic bond between the cabonyl goup of one molecule and a hydoxyl of anothe could also be invoked. No diect evidence of eithe step, howeve, is available. The cyclization to a pyole stuctue, as discussed befoe, would also block the oxidative fomation of fomylpyuvic acid. Thee is, in fact, some evidence that such an event takes place: we have obseved that afte sialic acid has been heated in 0.1 N HCl at 100 fo 30 minutes, the solution foms a puple colo in the cold when mixed with the p-dimethylaminobenzaldehyde eagent; this is chaacteistic of pyoles, wheeas unteated / I I I I I I I IO FIG. 6. Action of 0.1 N HCl at 100 on sialic acid pepaations fom ox seum (A) and human seum (B). Solutions of 10 pmoles of the compounds in 10 ml of 0.1 N HCI wee heated in a boiling wate bath unde a eflux. Measued samples (1 ml) wee emoved at the indicated times and neutalized with an equal volume of 0.1 N NaOH; 1 ml of the mixtue was used in the diect Ehlich eaction (Cuve 1), 0.1 ml in the thiobabituic acid eaction (Cuve 8)) and 0.2 ml in the esocinol eaction (Cuve 3). sialic acid develops the colo only afte being heated with the eagent fo some time. It is not unlikely, theefoe, that seveal events occu simultaneously: lactonization, pyole fomation, and futhe degadation; the extent to which each contibutes to the final esult, howeve, cannot yet be assessed. Este Link of Sialic and Methoxysialic.lcidsThe effect of the teatment of the methyl estes of these ompounds with 0.1 N HCl at 100 fo 1 hou is illustated in Fig. 7. l-ide these conditions, the este link of sialic acid methyl este is cleaved apidly (Cuve S, Fig. 78); the esults of the diect Ehlich and thiobabituate eactions esemble, as expected, those obtained with sialic acid (Fig. 6). The este link of methoxysialic acid methyl este, on the othe hand, appeas to be much moe stable (Cuve S, Fig. 7B): the hydoxamic acid values obseved in the couse of 1 hou diminish only little. The thiobabituate eaction, negative fo methoxysialic acid methyl este in the beginning of the teatment, inceases with time, owing no doubt to the libeation of the cabonyl goup. Fo the same eason a slight incease in the diect Ehlich values is ecoded (compae Table I). That the absobance values in the thiobabituate test incease moe steeply than the hydoxamate values dop (Fig. 7B) could be taken to indicate that the emoval of the glycosidic methoxyl pecedes that of the este methoxyl. Glycosidic Link of Methoxysialic Acid and Its Methyl Este- The acid lability of the glycosidic link of sialic acid was ecognized vey ealy; the fist isolation of the ompound made use of the

6 954 Sialic Acid Deivatives Vol. 239, No. 4 GO IO I I I, lq0 0-0 $ 0.2-? occus moe apidly, but the fee sialic acid evidently is degaded futhe. The stability of the glycosidic link towads acid is heightened by the substitution of the neighboing caboxyl. No cleavage of methoxysialic acid methyl este is appaent at ph values as low as 3. The compound is hydolyzed slowly in 0.1 N HCl and moe apidly in 0.5 N HCl, but the libeation of sialic acid is ovetaken by its destuction. Hence, the complete hydolysis and quantitative ecovey of the libeated sialic acid appea unattainable if it is doubly linked though its hemiacetalic hydoxy and caboxy goups. The hydogen of the cabosyl plays an impotant ole in the emoval of the glycosidic methoxyl. This intamolecula catalytic effect suggests the fomation of a bond between this hydogen and the glycosidic oxygen, I.--I I IO FIG. 7. Action of 0.1 N HCl at 100 on (A) sialic acid methyl este and (B) methoxysialic acid methyl este. Solutions of 20 pmoles of the compounds in 5 ml of 0.1 N HCl wee heated in a boiling wate bath unde a eflux. Measued samples (0.5 ml) wee emoved at the indicated times and neutalized with an equal volume of 0.1 N NaOH. Of the mixtue, 0.7 ml was used in the hydoxamic acid eaction (Cuve 9); 0.25 ml was diluted to 3 ml, and 2.5 ml of this dilution wee used in the diect Ehlich eaction (Cuve 1)) and 0.2 ml in the thiobabituic acid eaction (Cuve 2). zo.5 s Fo.4 a I I I I 1 1 I 0 IO FIG. 8. Hydolysis of the glycosidic link in methoxysialic acid and its methyl este at 100 and at diffeent ph values. Solutions of 5 moles of the compounds in 25 ml of 0.05 M solutions of citic acid, sodium citate, o mixtues of both, at the indicated ph values, and in 0.1 N and 0.5 N HCl, wee heated in a boiling wate bath unde a eflux. Measued samples (0.2 ml) wee withdawn at the indicated times and mixed immediately with the peiodate solution fo the thiobabituate eaction. 0, methoxysialic acid; l, methoxysialic acid methyl este. autohydolysis of submaxillay mucin (36). The esults of ou study of the stability of substitution of the hemiacetalic hydoxyl at ph values between 2 and 7 and in 0.1 and 0.5 N HCl ae illustated in Fig. 8. Between ph 5 and 7, methosysialic acid was not hydolyzed appeciably; modeate cleavage took place at ph 4. The optimal condition fo the hydolysis of the glycosidic link seems to be at ph 3, since the uptue is completed in a elatively shot time, without the degadation of the libeated sialic acid. At highe degees of acidity, cleavage Such hydogen bonds, esulting in the fomation of a &membeed ing, ae not as stong as those of thei 6-membeed countepats, but ae still capable of influencing the ate of chemical eactions (37). In the pesent case, the emoval of the methosyl, which fist involves the addition of a poton to the methosyl oxygen atom, would be facilitated if the poton aleady wee hyclogenbonded to it-an intenal catalysis that is ljevented in the doubly substituted methosysialic acid methyl este, which consequently equies a highe acidity fo hydolysis. Teatment with Sialidase The enzyme was peljaed fom cultue filtates of 17iixio choleae and used in the puification stage efeed to as Stage 6 in a pevious publication (9). When methosysialic acid o its methyl este, in concentations of 10 o 100 pg pe ml of 0.01 M Tis-acetate buffe, ph 6.6 (4 m&i with espect to Ca++), containing 100 sialidase units (9), was incubated fo 24 hous at 37, no libeation of sialic acid was obseved. I-ide the same conditions of ensymic assay, ox bain mucolipid (8) and sialyllactose4 (38) showed consideable cleavage even afte 1 hou of incubation. The libeation of sialic acid was followed by means of the thiobabituatc test. It is likely that the esistance of methosysialic acid and its methyl este to attack by sialidase, when compaed to the susceptibility of the natually occuing sialic acid glycosidcs, is due to the a&cone athe than to diffeences in the steeochemisty aound cabon 2. Teatment with Glacial ;lcetic -4cid and Pyidine Mucolipids and elated deivatives of sialic acid ae, in the couse of thei pepaation, often exposed to solvents such as pyidine o glacial acetic acid. It was of some inteest to examine the stability of the simple deivatives studied hee to teatment with these solvents. Solutions of the fou compounds in eithe pyidine o acetic acid wee heated at 100. The samples wee tested by the diect Ehlich and thiobabituic acid eactions. The expeimental conditions and esults ae summaized in Figs. 9 and We ae vey gateful to D. Kal Meye fo a sample of this compound.

7 Apil 1964 J..!I. Kalcas and B. C hagaff The teatment with glacial acetic acid esults in the apid degadation of sialic acid (Fig. 9-l), as shown by both the diect Ehlich and the thiobabituate eactions. In the case of methoxysialic acid (Fig. 9C), consideable degadation is shown by the diect Ehlich eaction, while the thiobabituate values fail to indicate an accumulation of fee sialic acid. To what extent the glycosidic bond is boken in this case cannot be assessed, as most of the sialic acid libeated would be futhe degaded. The methyl este of sialic acid (Fig. 9B) is degaded slowly; it will be noticed that thee is an initial, as yet unex- plained, incease of the absobance in the thiobabituate eaption. N-hcetylneuaminic acid has a highe mola absobance than N-glycolylneuaminic acid (18), but a tansamidation, effecting the eplacement of :V-glycolyl by N-acetyl goups, is not likely unde the expeimental conditions. Methosysialic acid methyl este (Fig. 9D) appeas to be athe stable; the small initial incease of absobance in both colo eactions could indicate some cleavage of the glycosidic link. It should be noted that the most sevee degadation is suffeed by the two compounds possessing a fee cabosyl goup, sialic acid and methoxysialic acid. Whethe the degadation is caused by a specific action of the acetic acid o is due in pat to the acidity of sialic acid itself cannot be decided without futhe expeimentation. The teatment with pyidine esults in a apid degadation of sialic acid methyl este (Fig. lob), which was confimed b3 both colo eactions. With sialic acid (Fig. loa) and mcthoxy- ** O.-----o h I I I I I *w c I I I I I I H :, U R S FIG. 10. Teatment with pyidine at 100 of (A) sialic acid, (B) sialic acid methyl este, (C) methosysialic acid, and (D) methoxysialic acid methyl este. Solutions of each compound in pyidine (16 ml; lM) wee heated in a boiling wate bath unde a eflux. Measued samples (2 ml) wee emoved at the times indicated, cooled in ice wate, evapoated immediately unde educed pessue at 35, and died ovenight in a vacuum desiccato ove concentated H?SOl. The esidues wee dissolved in wate, and samples coesponding to 0.45 and nole of the stating compound wee used fo the diect Ehlich (--) and the thiobabituate (- - -) eactions, espectively. FIG. 9. Teatment with glacial acetic acid at 100 of (A) sialic acid, (B) sialic acid methyl este, (c ) methoxysialic acid, and (I)) methoxysialic acid methyl este. A 0.5 n~ solution of sialic acid (80 ml) and 16 ml of 2.5 lllm solntions of sialic acid methyl este, methoxysialic acid, and methoxysiali aid methyl este in glacial acet,ic aid wee heated in a boiling wate bath unde a eflux. Measued samples (10 ml fo siali aid and 2 nt fo the othe deivatives) wee emoved at the indicat,ed times, cooled in ice wate, evapoated immediately unde educed pessue at 35, and died ovenight in a vacuum desiccato ove KOH. The esidues wee dissolved in wate, and samples coesponding to 0.45 and finote of the stating compound wee used fo the diect Ehlich (--) and t,he thiobahitunte (- - -) eactions, espectively. -I sialic acid (Fig. loc), on the othe hand, a disageement between the two colo tests was obseved, simila to that mentioned befoe in the teatment with HCI: absobance in the diect Ehlich eaction emained pactically unchanged wheeas the thiobabituate eaction indicated a maked degadation. I yole fomation, which is moe ponounced unde alkaline condi- tions, could account fo this discepancy, as discussed befoe. Methosysialic acid methyl este (Fig. 1OD) also appeas stable by the diect Ehlich test; the compaison, howeve, of the esults of the hydosami a&l eaction befoe and afte the teatment (3 hous) indicated a dop in the xte content of 507..< mentioned befo, the stability of the glyosidic link to acetic acid o pyidine could not be detemined by teatment of the simple deivatives, owing to futhe degadation. Fo this eason, an oienting expeiment with a l)epaation of OS bain mucolipid may be of inteest; it shows that the petat- ment accoded these co~n~;les and delicate substances may not be without consequence..i specimen of mucolipid fom OS bain puified to Stage III (8) was found, by means of the diect Ehlich eaction with ou l)cpaation of cystalline sialic acid as the standad, to contain 32.4 ;, of sialic acid.5 Potions 6 The pepaation of ox bain mucolipid investigated peviously in detail (8) had a sialic acid content of 2F.O%. Since that time, we have encounteed numeous instances of pepaations of the same degee of puity that contained consideably moe sialic acid. Fo instance, in as yet unpublished studies in collaboation with I)s. 0. W. Gaigan and N. %. Stanace, 10 pepaations wee examined whose sialic aid ontent anged fom 27.3 to 32.4%, with a mean of 30.1% and a standad deviation of 1.4.

8 956 Sialic Acid Deivatives Vol. 239, X0. -I (5 ml each) of 0.6% solutions of the lipid in pyidine and in glacial acetic acid wee kept fo 5 minutes at 100 and fo 20 hous at oom tempeatue, and then evapoated unde educed pessue at 35. The aqueous solutions of the esidues wee subjected to dialysis against unning wate fo 24 hous and against distilled wate at 2 fo the same peiod. The pepaation that had been teated with pyidine was ecoveed by lyophilization in a yield of 80y0 of the stating mateial and contained 25.6c/;, of sialic acid; the poduct teated with glacial acetic acid was similaly ecoveed in a yield of 70yc and contained 20.4y, of sialic acid. An unteated contol specimen, subjected to a paallel dialysis and ecovey pocedue, had a pactically unchanged sialic acid content of 32.2%. Concluding Remaks The heteopolymes occuing in biological mateial ae usually detemined though the quantitative estimation of one o moe of thei chaacteistic monomeic constituents. This equies, among othe things, the availability of pocedues pemitting the complete elease and ecovey of the intact monome. Fo the poteins and the nucleic acids these equiements can, on the whole, be met, but this is less tue of the lipids and the polysacchaides. The inceasing inteest in the biological function of vaious deivatives of sialic acid occuing in natue pompted the pesent study, which deals with the simplest epesentatives of the vaious types of links in which sialic acid is able to engage. Since the investigation of the quantity and the manne in which sialic acid is integated into a polyme will have to make empiical use of eactions that mostly ae not entiely specific, the esults epoted hee may seve to place cetain limitations on the intepetation of such studies. Anothe cautionay esevation which may accue fom the pesent obsevations egads conclusions as to stuctue dawn fom espeiments on the action of peiodic acid on polymes containing sialic acid, such as wee attempted in a pevious study fom this laboatoy (10). The finding that the poducts of the action of peiodate on the sialic acid deivatives studied hee still give some diect Ehlich eaction makes difficult the quantitative intepetation of oxidation expeiments pefomed on an intact polyme. The pesent studies also emphasize the necessity of gaining an insight into the types of links of sialic acid pevailing in the polyme to be investigated befoe the esults of quantitative estimations can be evaluated popely. They may contibute to the choice of suitable means of elease and detemination of sialic acid and of the standads to be used in a given case. The geat lability of the este link in a sialic acid molecule that is not stabilized by glycoside fomation (sialic acid methyl este is completely hydolyzed at ph 8 and 25 within 20 minutes) makes it evident how difficult the isolation of an intact macomolecule may be unde cetain cicumstances. It is not unlikely that many discepancies in the liteatue ae attibutable to this sot of labilit,y having been ovelooked. What this study undelines is that fo esults to be meaningful the appopiate standad must be chosen, the histoy of the sample must be fully known, and the hydolysis method must be in keeping with the type o types of linkage in which sialic acid occus in the polyme. Some of these polymes, in whose stuctue sialic acid paticipates, pobably ae to be counted among the most delicate and labile instances of macomolecules found in natue. SUMMARY The stability of sialic acid (isolated fom ox seum poteins and composed of 63% of N-glycolylneuaminic acid and 375; of N-acetylneuaminic acid), of its methoxy deivative, and of the espective methyl estes of these compounds was studied unde vaious conditions, mainly by means of caloimetic techniques. A few oienting expeiments wee also pefomed with N-acetylneuaminic acid fom human seum poteiny. The mola absobance of sialic acid in the caloimetic testy was dependent on the pesence of substituents on the cabonyl cabon atom. Peiodate oxidation was found to modify, but not to abolish, the eactivity in the colo tests. The este link of sialic acid methyl este was cleaved el apidly at ph 8 and 25 and elatively fast in 0.1 N HCl at loo, but simultaneous substitution of the neighboing hemiacetalic hydosyl, as in methosysialic acid methyl este, endeed the este bond much moe esistant to hydolysis both by acid and by alkali. Teatment with 0.1 N HCl at 100 was found to cause a modification of the molecule of sialic acid, esulting in a loss of eactivity in the thiobabituate, but not in the diect Ehlich, eaction. The hydolysis of the glycosidic link of methoxysialic acid was investigated at vaious ph values; ph 3 was found to be the most suitable fo a apid hydolysis without futhe degadation of the libeated sialic acid. Hee again, the same effect of double substitution was obseved: esteification of the neighboing caboxyl, as in methoxysialic acid methyl este, inceased makedly the esistance of the glycosidic link to acid hydolysis. Sialidase fom Vibio choleae was inactive against the methoxyglycosides of sialic acid and its methyl este. Heating with glacial acetic acid o pyidine caused degadation of sialic acid and, to a lesse extent, of its monosubstituted deivatives, wheeas the doubly substituted methosysialic acid methyl este appeaed athe stable. 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