1880
COMMUNICATIONS TO THE EDITOR
We found in these solutions the presence of the following ions : Mo7024-6 ("paramolybdate of Delafontaine") , Mo6020-4(' 'trimolybdate"), Mo6OZ0H --3 ("tetramolybdate"). The paramolybdic ion of Rosenheim (Moe024H-5) does not exist in detectable amount in these solutions. Further details will be published in J . Chim. Phys. or may be found in the author's thesis, "Contribution B l'6tude de 1'6lectrode B quinhydrone: application B la determination des isopolyanions molybdiques." ECOLE SATIONALE SUPERIEURE DE CHIMIEDE STRASBOURG UNIVERSITE DE STRASBOURG STRASBOURG, FRANCE
Vol. 86 CHiOH
HO H L
H, OH NHAc
?OR
D
D
L
D
I,R = NHCHCONHCHCONHCHCONHCHCONHCHCO~H I
I
CHS
J. P. SCHWING
L
(YH2)4
COzH
NHz
I CH3
I
I
(7Hz)~
CH3
D
11,R = NHCHCONHCHCOA I I CHs
RECEIVEDFEBRUARY 24, 1964
(CHz)z
I
L
D
D
CONHCHCONHYHCONHCHCOzH (CH2)4 I CH3 ~ H B I
NHz
Total Syntheses of N". [1-(2. Acetamido3-0-~glucosy1)-D-propionyl-L-alanyl-D- a- and y-glutamyll-~-lysyl-~-alanyl-~-alanine, and Identity of the y-Glutamyl Isomer with the Glycopeptide of a Bacterial Cell Wall Precursor
bodiimide, gave N"-~-BOC-N'-Z-L-L~S-ONP~~ ( I I I ) ,m.p. 83-85', [ a I z 4 D -23.6' (c 2.0, DMF). Condensation of activated ester I11 with H - D - A ~ ~ - D - A ~ ~ - Ogave MBZ~~ N"-t-BOC-N'-Z-L-Lys-D-Ala-D-Ala-ONBZ (IV), m.p. 124-125', [ a I z 4S~9 . 5 ' (c 2.0, DMF). Selective reSir : moval (HCl HOAcl4vl5)of the t-BOC group from Accumulation of uridine nucleotides in a Staphylococtripeptide IV yielded H-N'-Z-L-Lys-D-Ala-D-Ala-oNBz cus aureus was observed' to occur when its growth was * H C 1 . H 2 0(V), m.p. 158-159', [ a ] ' ~ D $37.8' (c 2.8, inhibited by penicillin. On the basis of degradation2r3 DMF) . and enzymatic synthesis4 the principal compound, con~-BOC-(~-OBZ)-D-G~U-OH, obtained as a colorless taining the amino sugar muramic acid [2-amino-3-0viscous oil from y-benzyl D-glutamate, was esteri(D- l-carboxyethyl)-2-deoxy-~-glucose],~~~ was assigned fied with p-nitrophenol to yield t-BOC- ( y-oBZ)-~-Gluthe structure, uridine-6'-pyrophosphoryl-N-acetylmur- ONP (VI), m.p. 120-121', [ a I z 5 D 32.3' (C 2, DMF). amyl - L-alanyl- D -glutamyl- L-lysyl-D-alanyl- D -alanine. Condensation of activated ester VI with tripeptide Further characterization of the nucleotide from peniderivative V in DMF, with addition of one equivalent of cillin-treated cells' and from enzymatic synthesis4d triethylamine, gave N"- [t-BOC-(y-OBV) - ~ - a - G l u ] provided evidence for the Nu-y-glutamyllysyl peptide Nf-Z-~-Lys-~-Ala-~-Aa-ONBZ~0.25H20 (VII), m.p. linkage. The glycopeptide formed by mild acid hy145-147', [ a I z 5 +13.8' ~ (c 2.1, DMF). Removal d r o l y ~ i s of 1 ~the ~ ~uridine ~ nucleotide may then be com(HC1 HOAc) of the t-BOC group from tetrapeptide pletely formulated as 11. derivative VI1 afforded Nu[H-(~-OBZ)-D-(Y-G~~]-N'We wish to record total synthesis of Nu-[ l-(2-acetZ-L-Lys-D-Ala-D-Ala-oNBz. HCl.O.5H20 (VIII), m.p. amido-3-O-~-glucosy~)-~-propiony~-L-alany~-D-aand y123--124'dec., [ ( Y ] ~ ~-9.1' D (c 2, DMF). glutamyl ]-~-lysyl-~-alanyl-~-alanine ( I and 11), and to t-BOC-L-Ala-ONP (IX), m.p. 82-83, [ a ] " ~-60.5' report that the y-glutamyl isomer I1 is identical with (c 2, ethanol), obtained by esterification of L-BOC-Lthe glycopeptide of a bacterial cell wall precursor, as Ala-OH, l 5 was condensed with tetrapeptide derivative shown by two-dimensional paper chromatography. VI11 to yield Na-[~-BOC-L-A~~-(~-OBZ)-D-~-G~U]H - , V : ' - Z - L - L ~ S - O(Na H ~ ~salt) ~ and t-butylazidof or2-L-Lys-D-Ala-D-Ala-ONBZ.0.5H20 (x), m.p. 181matelo in refluxing aqueous t-butyl alcohol gave N"-t182' dec., [ a I z 5+22.5' ~ (c 2, DMF). The latter BOC-N'-Z-L-LYS-OHas a colorless viscous oil which, espentapeptide derivative gave, with HC1 HOAc, N"terified l 1 with p-nitrophenol and N ,N'-dicyclohexylcar[H-L-Ala-(y-OBZ)-D-a-Glu]-N'-Z- L-LYS-D -Ala- D -AlaONBZ.HCl.HzO (XI), m.p. 194-195' dec., [ c Y ] * ~ D (1) ( a ) J . T Park, J . Biol. Chem., 194, 877 (1962); (b) J . T. Park, i b i d . . 194, 885 (1952); (c) J. T. Park, i b i d . , 194, 897 (1952). 19.6'. (2) J . L. Strominger, C o m p f . rend. traw. lab. Carlsbrrg, 81, 181 (1959). Benzyl 2-acetamido-4,6-O-benzylidene-3-0-(~-l-~ar( 3 ) J . T Park and J . L . Strominger, Science, 146, 99 (1957). boxyethyl)-2-deoxy-a-~-glucopyranoside~~ (XII) was (4) (a) E . Ito and J . L. Strominger, J . B i d . Chem., 486, PC 5 (1960); (b) E lto and J . L. Strominger, i b i d . , 487, 2689 (1962); (c) E. Ito and J . L condensed in acetonitrile with pentapeptide X I (with Strominger, i b i d . , 487, 2696 (1962); (d) E . Ito and J. L. Strominger, i b i d . , addition of one equivalent of triethylamine) by means 289, 210 (1964). (5) R . E . Strange and L. H . Kent, Biochem. J . , 71, 333 (1959). of N-ethy1-5-pheny1isoxazo1ium-3'-su1fonate1* to afford
+
+
+
+
(6) Y .Matsushima and J. T . Park, J . Org. Chem., 47, 3581 (1962), and references cited therein. (7) kf. H. Mandelstam and J . L. Strominger, Biochem. Biophys. R e s . C o m m u n . , 6 , 466 (1961). (8) M . Bergmann, L. Zervas, and W. F . Ross, J . Biol. Chem., 111, 245 (1936). (9) The following abbreviations are employed: Ala = alanine, Glu =
glutamic acid, Lys = lysine, I-BOC = t-butoxycarbonyl, BZ = benzyl, S B Z = p-nitrobenzyl, N P = pnitrophenyl, Z = benzyloxycarbonyl, D h l F = h',,\'-dimethylformamide. (10) L . A. Carpino, C . A. Giza, and B . A. Carpino, J . A m . Chem. S o c . , 81, 966 (1959). (11) M . Bodansky and V. du Vigneaud, ibid., 81, 5688 (1959).
(12) Unless otherwise noted, all compounds were obtained as colorless crystals; satisfactory analyses were obtained for these compounds. (13) H . C. Garg, M. C. Khosla, and N. Anand, J . Sci. I n d . Res. (India),
PiB,286
(1962). (14) F . C . McKay and N. F . Albertson, J . A m . Chem. Soc., 79, 4686 (1957). (15) G . W. Anderson and A. C. McGregor, i H d . , 79, 6180 (1957). (16) W. E . Hanby, S. G. Waley, a n d . J . Watson, J . Chem. S o c . , 3239 (1950). (17) H . M. Flowers and R . W. Jeanloz, J . Orp. Chem., 18, 2983 (1963). (18) R . B . Woodward, R . A. Olofson, and H. Mayer, J . A m . Chem. Soc 88, 1010 (1961).
COMMUNICATIONS TO THE EDITOR
May 5 , 1964
1881
L-lysyl-D-alanyl-D-alanine (XX) (obtainedz1 by the the ~-a-glutamylisomer of the fully protected N-acetylaction of venom phosphodiesterase and alkaline phosmuramyl pentapeptide, Nu-[ 1-(2-acetarnido- 1-0-benzyl phatase on uridine-5'-pyrophosphoryl-N-acetylrnur4,6 - 0 -benzylidene- 2 - deoxy- 3 - 0 - D -glucopyranosyl)-DD - alanpropionyl-L-Ala-(-~OBZ)-Da-Glu l - N ' - z - ~ - L y s - ~ - A l a - ~ amyl-~-alanyl-~-glutamyl-~~-C-~-lysyl-~-alanylinezz) were cochromatographed on paper with isobuAla-ONB2.Hz0 (XIII), m.p. 211-214' dec., [ a I z 5 D tyric acid : 0.1M ammonium hydroxide (5 : 3) (solvent A) $58.5' (c 1, DMF). Hydrogenolysis (Hz/Pd black/ used for the first dimension and pyridine :water (4 : 1) 10% Pd-C/85yo HOAc) of X I I I , and purification (solvent B) for the second. The a-glutamyl isomer I of the resulting product by means of column chromawas more mobile than the labeled glycopeptide with the tography on Celite diatomaceous earth, yielded with ratio of mobilities of CY = 0.83 in solvent A and 0.97 in 4.5 holdback volumes (H.B.V.) of butanol-acetic solvent B. acid-water (6 : 1:4) the D-a-glutamyl isomer I of the NNu-(L-Alanyl-D-7-glutamyl)-L-lysyl-D-alanyl-D - alanacetylmurarnyl pentapeptide as a colorless hygroscopic ine (obtained by hydrogenolysis of fully protected penamorphous solid which sinters a t 105', m.p. 145-148' tapeptide XVII) was cochromatographed on paper with dec., [ a I z 5 D $33.6" (c 1.3, water). Anal. Calcd. for ~-alanyl-~-glutamyl-~~C-~-lysyl-~-alanyl-~-alanine (obC81H53N7015.2.5HzO: C, 46.1; H, 7.23; N, 12.1. tainedzl by the action of acetylmuramyl-L-alanine Found: C, 46.1; H, 7.18; N, 12.2. amidasez3on the I4C-labeled N-acetylmuramyl pentaa-Benzyl D-glUtamate, l9 t-butylazidoformate, and peptide XX), and was clearly differentiated from N"sodium carbonate in refluxing aqueous dioxane gave t(L-alanyl-D-a-glutamyl)-L-lysyl-D-alanyl-D-alanine (obBOC-D-G~u-OBZas a colorless oil which was esterified with p-nitrophenol to yield t-BOC-(r-ONP)-D-Glu-OBZ tained by hydrogenolysis of the corresponding protected pentapeptide). The ratio of mobilities of y / a was 0.77 (XIV), m.p. 101-102°, [ a I z 5 D $22.2' (C 2.5, DMF). in solvent A and 0.60 in solvent B. Activated ester XIV was condensed with tripeptide derivative V to yield N"- [~-BOC-(CY-OBZ)-D-~-GIU]-Acknowledgment.-We wish to thank Drs. J. S. N'-Z-L-Oys-D-Ala-D-Aa-ONBz (xv), m.p. 174-175', Anderson and J. L. StromingerzOfor paper chromato[ a I z 5 D $18.7' (c 2.5, DMF). Removal of the t-BOC graphic comparisons of their enzymatically synthesized group from XV (HCI HOAc) afforded N"-[H-(acompounds with I, 11, and the corresponding pentapepO B Z ) - D - ~ G ~ ~ ] - N ' - Z - L-Ala - L ~-~D--AlaD ONBZ. HCl . tides, Mr. L. Brancone and staff for microanalyses, Mr. 0.5Hz0 @VI), m.p. 141-142' dec., [ a I z 5 D +5.5' (c 2, W. Fulmor and staff for optical rotation measurements, DMF) . Mr. J. W. Marsico for paper chromatography, and Mr. Condensation of activated alanine ester I X with tetC. Pidacks for column chromatography. rapeptide ester XVI yielded N a -[t-BOC-L-Ala-(CYJ. L. Strominger,20private communication. O B Z ) - D - ~ - G ~ U ] - N ~ - Z - L - L ~ ~ - D(XVII), - A ~ ~ - D -(21) A ~J.P~. S.M-Anderson N B Zand .O Meadow, J. S. Anderson, and J. L. Strominger, Biochem. Bio(22) p k y s . Res. Commun., 14, 382 (1964). m.p. 187-188' dec., [ a I z 5 D + l l . O ' (c 2, DMF). Re(23) J. M . Ghuysen and J. L. Strominger, Biochemistry, 3, 1110 (1963) moval (HCl HOAc) of the t-BOC group from XVII afforded Nu[H-Ala-(CY-OBZ) - D - ~ - G ~ ] - N ' - Z - L - L ~ S - ORGANIC DCHEMICAL RESEARCH SECTION A. E. LANZILOTTI LEDERLE LABORATORIES DIVISION E. BENZ Ala-D-Ala-ONBZ.HCl.Hz0 (XVIII), m.p. 153-154' AMERICAN CYANAMID COMPANY L. GOLDMAN dec., / a I z 3 D $24.2' (c 2, DMF). PEARLRIVER,NEWYORK The base from pentapeptide salt XVIII was conRECEIVED MARCH2, 1964 densed with protected muramic acid XI1 by means of N ethyl-5-phenylisoxazolium-3'-sulfonate in acetonitrile to yield the ~-7-glutamylisomer of the fully protected muramyl pentapeptide, Nu- [ 1-(2-acetamido-l-O-benzyl C13 Hyperfine Splittings in the 4,6-O-benzylidene- 2 - deoxy - 3 - 0 - D - glucopyranosyl) - D7,7,8,8-TetracyanoquinodimethaneAnion Radical propionyl-L-A~~-(~-OBZ)-D-~-GI~]-N'-Z-L - Lys- D -AlaSir : D-Ala-ONBZ (XIX), m.p. 215-218' dec., [ c Y ] ~ $46.9' ~D (c 1, acetic acid). Hydrogenolysis of X I X and chromaWe have measured the CI3 hyperfine splittings of the tography as for XI11 afforded (with 7.9 H.B.V.) the D-7- 7,7,8,8-tetracyanoquinodimethane (TCNQ) anion radiglutamyl isomer I1 of the N-acetylmuramyl pentapepcal in which C13 was substituted in positions 1 (4) and tide as a colorless, hygroscopic, amorphous solid, m.p. 9 (11, 13, 15). Our results (Table I) show (1) that the 148-150' dec., [ a I z 5 D $14.0' (c 0.9, water). A n a l . Calcd. forC31H63N7016.Hz0: C, 47.6; H, 7.09; N, 12.5. Found: C, 47.5; H, 7.25; N, 12.5. Two-dimensional paper chromatography was employed to compare isomers I and I1 with enzymatically synthesized glycopeptide (kindly carried out by AnderTCNQ son and StromingerzO). 20a A single radioactive and ninhydrin-positive spot was obtained when 7-glutamyl isomer I1 and N-acetylrnuramyl-~-alanyl-~-glutamyl-~4CCl8 splittings calculated from simple Hiickel-LCAO or McLachlanl theory and the 0-r parameters of (19) H. Sachs and E. Brand, J . Am. Ckem. Sac., 7 6 , 4610 (1953). Fraenkel, et al.,2t3are not in very good agreement with (20) Department of Pharmacology, University of Wisconsin Medical School, Madison, Wis. experiment and (2) that previous assignments4 based
+
+
(2Oa) N O T E ADDED I N Pnooa.--identity of the acetylmuramyl pentapeptide and the pentapeptide from enzymatically prepared nucleotide with the (1) A. D . McLachlan, Mol. P h y s . , 3, 233 (1960). D-y-glutamyl isomer I1 and Na-(L-aIanyl-~-~-glutamyl)-~.lysyl-~-alanyl-~-(2) M.Karplus and G. K . Fraenkel, J . Ckem. P k y s . , 36, 1312 (1961) alanine was also established by means of a paper electrophoresis on What(3) P. H . Rieger and G . K . Fraenkel, i b i d . , 5 7 , 2795 (1963). man 3 M M paper in 0.18 M pyridine acetate buffer of pH 4 . 1 at a potential (4) P . H . H. Fischer and C . A . McDowell, J . A m . Ckem. Soc., 86, 2fi94 gradient of 16 valts/cm. a t ' 0 for 5 hr. (1963)
