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 J. S. Anderson, and J. L. Strominger, Biochem. Bio(22) .O Meadow, pkys. 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 MARCH 2, 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 3MM 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. at ' 0 for 5 hr. (1963)
COMMUNICATIONS TO THE EDITOR
1882
on the above theories and the hyperfine splittings arising from CI3in natural abundance are in error. TABLE I Li+TCNQ- SPLITTING CONSTANTS' Positionb or splitting constant
-Calculated,c oerstedHiickel McLachlan
ANI( AH CL3(l) CL3(2) CI3(7) CI3(9)
-3 11 +0.48
+5 23 - 7 17
.-5 02 +O 79 +8.49 -8 44
--Exptl Ref. 4
1 02 1.44 4.40 0.62 7.18 6.38
, oersted-Cia subsd
1 , 0 0 9 ( f O . 005) 1.415(+0.004) 1.52(+0.04)
7 0 6 ( 1 0 04)
g = 2.00263 f 0.00005, M in tetrahydrofuran. See See ref. 3 and 4. T C N Q structure for position numbering. g value and hyperfine splittings obtained by comparison in a hl solution in T H F of lithium tetradual cavity with a cyanoethylenide whose g = 2.00270 f 0.00005 and . 4 ~= a
1.574.
We have observed in the unlabeled TCNQ- spectrum two sets of low intensity lines. One set arises from C13 a t position 9. The other with one-half the signal intensity of that arising from position 9 has a splitting of 4.6 oersted. If the latter is due to CI3 splitting, it must arise from the methylene carbons ( ; . e . , 7 , 8). We have been unable to observe any other resonances in the unlabeled TCNQ- spectrum. Table I shows that the C13 splitting constants for positions 1 and 9 differ from those previously assigned4 and from those calculated from theory. The difference is even more pronounced if the value of 4.6 oersted can be associated with the C13 splitting by position 7. One concludes that either (perhaps both) the current theory of C13 hyperfine splitting or spindensity calculation is inadequate and requires revision. The two C13 enriched TCNQ anion radicals were synthesized as follows. For TCNQ enriched by CI3 in position 1 (4) (Scheme I ) , ethyl P-iodopropionate was treated with potassium cyanide-C13 to give Pethoxycarbonyl propionitrile-l-C13 (I). Treatment of
Vol. 86
ing from combustion of the labeled TCNQ gave a value for C13/C12corresponding to -5% C13 in each of positions 1 and 4, in excellent agreement with the expected value as well as with the relative e.s.r. line amplitudes. TCNQ enriched by CI3 in position 9 (11, 13, 15) was prepared by the reaction of potassium cyanide-CI3 with TCNQ to give K+TCNQ- 9(11, 13, 15)-C13 which was then oxidized to the correspondingly labeled TCNQ.' Mass spectral analysis again gave a value for C13/C12corresponding to ~ 5 C13 7 in ~each of the positions, in good agreement with relative e.s.r. line amplitudes. (7) This method of labeling is analogous t o t h a t used in t h e preparation [O. W. Webster, W Mahler, and
of cyano-labeled tetracyanoethylene-C" K E . Benson, ibid , 84, 3678 (1962)l.
CONTRIBUTION No. 946 CENTRAL RESEARCH DEPARTMENT E. I . D U PONTDE NEMOURS A N D COMPANY STATION EXPERIMENTAL WILMISGTON, DELAWARE
M. T. JONES W. R. HERTLER
RECEIVED FEBRUARY 1. 1964
The Direct Synthesis of Amine Alanes
Sir: In previous papers we have reported the use of the Al-HZ reducing system for the synthesis of NaAIH41 and amine boranes3 Now we wish to report the first successful direct synthesis of an amine alane, triethylenediamine alane, by reaction of aluminum and hydrogen in the presence of triethylenediamine at moderate temperature and pressure. AlH8
t
In a typical experiment 30 g. of triethylenediamine was dissolved in 100 ml. of tetrahydrofuran. To this SYNTHESIS OF TCNQ-l(4)-Cl3 solution was added approximately 6 g. of activated * aluminum powder. The resulting mixture was heated for 6 hr. a t 70' and 5000 p s i . hydrogen pressure. A light gray solid was isolated by filtration and analyzed for aluminum, hydrogen, and nitrogen. The NC. ,CN nitrogen analysis was performed by potentiometric titration of the solution obtained on hydrolysis of the product, after removal of the Al(OH)3. Anal. Calcd. for CeHl2NZ.AlH3:Al, 19.0; N, 19.7; H , 21.2mmoles/ g. Found: Al, 21.8;N, 19.1; H, 22 4 mmoles/g. Deuterolysis of the product showed that 92.4% of the evolved gas was D H and 7.6Yo was Dz, indicating I with ethanolic hydrogen chloride gave diethyl suca low concentration of unreacted aluminum. The prodcinate-l-C13 which was treated with sodium ethoxide uct is thermally stable to >200° and reacts violently in ether to give 2,5-bis(ethoxycarbonyl)-1,4-cyclo- with water. I t is insoluble in the common organic hexanedione-C13 which was hydrolytically decarboxsolvents tested and for this reason no molecular weight ylated to 1,4-cyclohexanedione-1(4)-C13. The C13determination has been made. However, it is speculabeled cyclohexanedione was converted to TCNQlated that the product is not monomeric, because of the 1(4)-C13 and LiTCNQ-1(4)-C13 by the usual prodifunctional nature of the amine. The ready formation c e d u r e ~ . ~Mass ,~ spectral analysis of C 0 2 result(1) E . C. Ashby (to Ethyl Corp.), French Patent 1,235,680 ( M a y 30, SCHEMEI
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(5) D . S . Acker and W. R . Hertler, J . A m . Chcm. Soc., 84, 3370 (1962). (6) L. R . Melby, R . J. Harder, W. R. Hertler, W . Mahler, R . E. Benson, and W. E . Mochel, ibid , 84, 3374 (1962).
1960) ( 2 ) E.C.Ashby, Chem. I n d . (London), 208 (1862). (3) E . C Ashby and W. E. Foster, J . A m . Chcm. Soc., 84, 3407 (1962).
