4s59
Dec. 5. 1961
C O M M U N I CAT1ONS T O T H E E D I T O R ON THE MUCOPEPTIDE FRACTION OF STREPTOCOCCAL CELL WALLS
treated with aqueous acetic anhydride and then with dilute alkali. This treatment was designed Sir : to N-acetylate all non-acylated hexosamine conWhen whole or trypsin-treated walls of Group stituents, and to saponify any 0-acyl groups A hemolytic streptococci are extracted with hot present, since 0-acylation is known to decrease formamide,1 the group-specific C-polysaccharide susceptibility to lysozyme.6 is solubilized. The insoluble residue consists of It should be noted that C-polysaccharide prepaparticles retaining the size and shape of the original rations obtained as stated, exhibit an absorption cell walls as shown by electron micrography.2 band in the ester-carbonyl region of the infrared The composition of the formamide-insoluble cell spectrum ; this band disappears on gentle treatment wall residue2 reflects quite closely the mole ratios with alkali. By means of the hydroxamate method, reported for cell walls exposed to trypsing The the acyl groups were identified as formyl groups. principal amino acids of the cell wall residue were The cell wall residue (after formamide treatment) found to be lysine, glutamic acid, and alanine; in was shown to contain saponifiable formyl and, addition, glucosamine and muramic acid were to a lesser extent, actyl groups, whereas the present. The mole ratios of the amino acids, original cell wall preparation did not give rise to in the order listed, approached the values 1: 1:3. formhydroxamic or acethydroxamic acid, nor did I n their recent paper,2 McCarty and Krause also an ester-carbonyl band appear in the infrared described enzymatic solubilization of cell wall resi- spectrum of the trypsin-treated cell walls. Evidue preparations. dently, the extraction procedure introduces saponiWe have analyzed cell wall residue preparations fiable formyl groups; the acetyl esters may result resulting from hot formamide extraction of trypsin- from an acetyl migration. treated walls of Streptococcus pyogenes, Group A, The lysozyme lysate contained only small Type 14, Strain S 23 by means of hydrolysis and amounts of material that reacted with ninhydrin, quantitative paper chromatography. The results the Morgan-Elson reagent, or silver hydroxide. appear in Table I ; amino acids other than those The material was fractionated according to its listed were absent or present in trace amounts only. solubility in 66% and S5% alcohol; the most soluble The values found gravitate toward a gross compo- portion was richest (29%) in hexosamine, the sition 1:1:4: 1 : 1 (lys, glut, ala, glucosamine, least soluble one, poorest (lo’%). The most soluble muramic acid). fraction was chromatographed on Whatman #3TABLE I M M paper with 1-butanol-acetic acid-water 4: 1:5 . COMPOSITION OF THE INSOLUBLE RESIDUEFROM STREPTO-Most of the material remained immobile a t the site COCCAL CELL WALLS AFTER TRYPSIN TREATMENT AND of application, and only two ninhydrin-negative, EXTRACTION WITH FORMAMIDE reducing (Ag) zones were detected. -Mole ratiosnThe more abundant one of these contained about Lysine 1 . 0 1 0.16 (9) 5% of the total hexosamine solubilized from the Glutamic acid 1.00 (9) cell wall residue. The material appeared homoAlanaine 4 . 0 3 =k 0.45 (9) geneous on re-chromatography in 1-butanol-acetic Glucosamine 1.05 f 0.07 (5) acid-pyridine-water and on electrophoresis on Muramic acid 0.99 0.20 (8) paper in 0.05 M borate a t pH 10, 800 v., for two Two different cell wall residue preparations were hours; in the electrical field, i t migrated slowly a t ,analyzed; the figures in parentheses indicate the total number of “spots” of the particular substance that were cut out about six-tenths the rate shown by N-acetylglucosand evaluated; the values given are averages, together with amine. At pH 3.5 or 10.0 (2 A’ acetic acid and t h e average of the highest negative arid positive deviations 0.1 M carbonate, respectively), the fragment did from the mean. not migrate. The composition was determined The cell wall residue is solubilized rapidly prior to and after electrophoresis on glass fiber sheets, by hot 4 N hydrochloric acid, but appreciable quan- with the results appearing in Table 11. tities of small, ninhydrin-reactive fragments were TABLE I1 not seen until heating had been continued for 2-3 COMPOSITION OF LYSOZYME-RELEASED FRAGMENT hours, when a rather complex mixture resulted. Molar ration Enzymatic solubilization was attempted with Alanine 1.0 (6) Maxted’s enzymeJ4 phage-associated lysin,5 and Glucosamine 0.94 f 0.07 (6) lysozyme. Maximum dissolution of hexosamine0.77 =k 0.06 (6) Muramic acid containing material (85% of the total hexosamine a See, mutat. wutand., note a in Table I. present) was achieved by the action of lysozyme The lysozyme-released fragment was treated on a cell wall residue preparation that had been with beta-N-acetylglucosaminidase~ from hog epi(1) A. T. Fuller, Brit. J. Exg21. Pathol., 19, 130 (1938). didymis with the production of two reducing (2) M. McCarty and R. M. Krause, J. E x p . Med., 114, 127 (1961).
*
*
(3) J. A. Hayashi and S. S. Barkulis, J. Bad., 77, 177 (1959). ( 4 ) W. R. Maxted, Lancet, 2,255 (1948). A commercial preparation from Consolidated Laboratories, Inc., Chicago, was used. ( 5 ) R. X I . Krause, J. E x g . Med., 108, 803 (1958).
(6) W. Brumfitt, Brit. J. E r g . Pnthol., 40, 441 (1959). (7) J. Findlay and G. A. Levvy, Bzochem. J., 77, 170 (1960). W e welcome this opportunity t o thank Dr. Levvy for his generous gift of a sample of his enzyme preparatiou
4860
ComtuNIcArIoNs
entities (Ag), separable by chromatography in Partridge mixture and by electrophoresis in borate, pH 10. One of the products migrated like Nacetylglucosamine and reacted likewise in the Morgan-Elson test. The other product was again subjected to hydrolysis and analysis and was found to contain only alanine and muramic acid. IVhen the chromatographically immobile portion of the lysozyme lysate was subjected to brief, mild treatment with acid, there appeared several small reducing fragments, among them glucosaniine and X-acetylglucosamine, as well as an additional reducing and ninhydrin-negative entity. Hydrolysis of this material liberated glucosamine and alanine in the ratio of approximately 2 : 1, We cannot, a t this time, decide what the detailed structures of the fragments are, and which, if any, of the conceivable structures may occur in the native cell wall. However, we feel that the observations presented give Eew knowledge about the association, with one another, of streptococcal cell wall constituents. * C I B A Fellow RESEARCH DBPARTBIENT C I B A PHARMACEUlICAL PRODUCTS, I N C .
SUMMIT, &-EW
€1
L.n
HEI'MAXY
TO TIIE
Vol. 83
EDITOR
then is added and the reaction mixture heated for several hours a t 150-175°. The hydrogen fluoride by-product from the carbonyl fluoride reaction serves as catalyst for the reaction. For phenol and nz- and p-nitrophenol, the yield of the ether is 60 to 80% over-all for the two steps. The reaction is general for substituted phenols including hydroquinone and resorcinol, provided that the substituents or the aromatic ring do not react with hydrogen fluoride or sulfur tetrafluoride. The ionization constants of the trifluoromethoxyand trifluoromethylthio-anilizium ions and benzoic acids7vahave been determined by standard literature and are reported in Table I. The calculated u-parameters are given in Table I1 and compared with those of several other substituents. The inductive and resonance contributions of the groups can be evaluated from UT and aa-parameters calculated according to Taft and Lewis.12 From these results i t can be seen that the OCF3 group is very much like C1, in that it withdraws electrons inductively but supplies them by resonance. Over-all it. is a slightly stronger deactivating group than the
ZELEZYICK*
TABLE
J. A. MANNIELLO RECEIVED NOVEMBER 2, 1961
JERSEY
r
IONIZATION CONSTANTS Ionization constants (-log h) (25') of XCaHiCCkH in XCaI-IA"Ia 60% ethanol in water +
ARYL FLUOROALKYL ETHERS AND SULFIDES : EVIDENCE FOR SULFUR d-ORBITAL INTERACTION
Szr:
Substituent X
m-OCFr
p-OCF, m-SCF, P-SCFa
5.14 5.19 5.13 4.98
3.25 3.82 3.30 2.78
We wish to report a new general method for the synthesis of aryl perfluoroalkyl ethers, the resill ts of quantitative measurements demonstrating that The SCF3 group has a urn value similar to that fluoroalkoxy groups may be considered as halo- of the OCF3 group, btit unexpectedly has a more gen-like and that sulfur d-orbital interaction is positive up value with an added positive increment significant when sulfur is bonded to the strongly of 0.13 for the up from ionization of anilium ions electron-withdrawing fluoroalkyl group. over the benzoic acids. This result is indicative Aryl trifluoromethyl ethers have teen synthe- of a $-R group. Recently Beishline14 reanalyzed sized by the reaction of hydrogen fluoride or anti- the ionization constant data for the SCI-13,SCOCH, mony fluorides with aryl trichloromethyl ethers' and SCN groups on the basis of Taft's U R paramwhich were prepared by chlorination of the ani( 7 ) T h e aryl trifluoromethy! sultiiies have been prepared b y Auorid e soles or phenyl esters of chlorothiocarbonic acid. replacement in aryl trichloromethyl sulfides [see L. M. Yagupolsky It now has been found that the reaction of sulfur and M. S. Marenets. J . Gen. Chcm. IJSSR (English Trans.), 82, 2273 tetrafluoride* (hydrogen fluoride catalyst) v i t h aryl (19RZ)I. T h e reaction of aryl Grignard reagents with trifluoromethylsulfenyl chloride is a n improved route to this class of sulfide and miil fluorocarbonatesS and perfluoroalkyl esters' pro- be described in a future publication (see W. A. Sheppard, Abstracts of vides a general, direct synthesis of aryl perfluoro- American Chemical Society Meeting, Chicago, Illinois, September. alkyl ethers.6 For the preparation of aryl tri- 19G1, p 7 - M .
o It
HF
ArOCX + SF, + ArOCF2X (where X = F or R f ) fluoromethyl ethers, a convenient procedure is to react the phenols with carbonyl fluorideaa6in a Hastelloy autoclave a t 100'. Without isolation of the fluorocarbonate, the sulfur tetrafluoride ( 5 ) 1,. hf. Yagupolsky and (1) (a) British Patent 763,527 (ID%)" V. I. Troitskaya. J . Cdn. Chem., USSR (English Trans.), 47, 618 (1957). (c) N. N. Iarovenko and A. S. Vasileva. J . Gex. Chcnt., USSR (English Trans.), 28, 2539 (lass). (2) (a) C. W. Tilllock, F. S. Fawcett, W. C. Smith and D. 13. CoEman, J . A m . Chcm. 5m..82, 589 (1960). (h) W.R. Iiasek, W.C. Smith and V. A. Engelhardt, ibid., 82, 543 (1960). (3) €1.J. Emeleusand J. F. Wood, J . Chcm. Soc., 2183 (1948). (4) R. F. Clark a n d J. H. Simons, J . A m . Chem. Sor., '76, 6305 (1953); hi. Green, Chem. a n d Ind.,435 (1901). (5) Ail cew compounds have been characterized by analysis and spectral properties. (6) hl. W. Farlow, E. 11. M a n and C. W.Tullock, Inorgnnic Synthrses. 6 , 155 (1960).
(8) T h e aryl perRuoroa!kyl ethcrs a n d sultides have been shown t o Lave stability comparable t o henzotrifluoride a n d t h e perfluoroalkoxy ond pcrfluoronlkylthio groups 6re inert to normal chemical transforniations involving t h e aromatic residue. T h u s , t h r anilines and benzdic acids were prepared by catalytic reduction of the nitro derivatives and oxidation of t h e tolyl compoiindu. Both t h e trifluorornethoxy and trifluoromethylthio7 groups have been shown to be 0.p-directing t o electiopllilic aromatic substitution. (9) A. Bryson, J. Am. Chcm. Soc., 81,4858 (1960). (10) J. D. Roberts, R. L. Webb a n d E. A. McElhill, ibid., 7 2 , 408 (1950). (11) A preliminary communication of p K a measurements on t h e above benzoic acids was recently reported b y L. M . Yagupolsky and I,. M. Yagupolskaya, Proc. Acad. S r i . (English Trans. from Dokladr A k a d . Nnuk S S S R ) , 134, 1207 (1960)) a n d values are essentially in agreement with those reported iu Table 1. The only discussion of these results was the romrnent t.hnt t h e S C B I and OCFa groups mere like halogens. (12) R. W. T a l l . Jr., and I. C. I.ewiu. J . A m . Che7n. Soc., 81, 5343 (13t~~l.
(13) T h e designation "super-halogrn" has been suggested t o describe t!iis behavior of t h e per0uoroalkoxy groups. (14) R. R.Reishline, J . Org. Ckrrn., 46,2533 (lg6l).
