Nov. 5, 1959
COMMUNfCATIONS TO THE
EDITOR
5837
TABLE I INTERCONVERSION REACTIOM Alkyl group
Yield,
1-Propyl
2-Propyl
1-Butyl
2-Butyl
Derivative
%
Trimethylamine Borane Dihydrobenzoboradiazole Borazole Trimethylamine Borane Dihydrobenzoboradiazole Borazole Trimethylamine Borane Dihydrobenzoboradiazole Borazole Trimethylamine Borane Dihydrobenzoboradiazole Borazole
65 95 88 65 91 79 64 85 91 66 9% 85
bLpb (b.p.1,
C.
-
SH, IiHa+----+
- IiHP+] +
[RBHzKH4C1
2H2
+ 1/3 (-RB=SH)3+
Analyses7
C
H
B
62.64
15.77
9.41
102-103 108 (9 mm) 52.28 62.61 12-1-126 (70/0.5) 52.28 65.14 66-67 57.92 (110/0.6) 65.14 61-62 57.92 (94/0.7)
spectra. The above table gives data concerning the transformations of four representative alkylboroxines. RBHz-
,-
11.70 15.77
N
C
12.18 62.45
15.70 20.32 9 . 4 1 12.18
Found
7 -
H
15.60
B
N
9.50
12.06
52.05 11.51 15.74 20.25 62.54 1 5 . i 9 9.39 12.23
11.70 15.70 20 32 51.95 15.62 8.38 10.86 64.88
11.62 15.60 20 51 15.38 8.23 10.83
12.16 13.04 16.88 57.97 15.62 8 . 3 8 10.86 65.02
12.00 12.94 15.29 8 . 4 1
16 68 10.80
12.16 13.04
1 1 . 9 1 12.90
16.72
16.88 57.90
far toward the direction of glyceraldehyde reduction'; dihydroxyacetone was found to give less than one-fifth the activity obtained with DLglyceraldeyde; and D-glyceraldeyde was found to be more active than the DL-form.
The scope of these reactions are under investigation and will be reported later in greater detail. ROHMAXD HAASCOMPANY REDSTONE ARSEXALRESEARCHDIVISIOS HUNTSVILLE, ALABAMA ?VI. FREDERICK HAWTHORXE RECEIVEDSEPTEMBER 9, 1959
A T P N + SPECIFIC GLYCEROL DEHYDROGENASE
Sir:
FROM LIVER*
The D P N + specific glycerol dehydrogenases of rat and swine liver have been described.l I wish to report here the occurrence of a T P N f specific glycerol dehydrogenase which was found during the process of chromatography of the supernatant fraction of a rat liver sucrose (0.25 M) homogenate. This enzyme may be of general interest since i t was found to oxidize tris-(hydroxymethyl)-aminomethane and other similar compounds used as biochemical buffers. A volume of 20 ml. of supernatant, containing about 200 mg. protein, which had been dialyzed against 5 m M tris phosphate, PH 8.0, was applied to a 1.1 X 30 cm. column of D E d E - ~ e l l u l o s e and , ~ ~ ~initially eluted with about 150 ml. of the same buffer to removed loosely bound proteins. The two glycerol dehydrogenases emerged as separate peaks of activity as shown in Fig. 1. The total activity of the T P N + was about one-eighth that of the D P N + enzyme. The purification of the enzyme during the chromatography was 40-50 fold. The enzyme may be classed as a D-glyceraldehyde hydrogenase : the equilibrium of the reaction lies
*
These abbreviations are used: TPS a n d TPNH, oxidized and reduced forms of triphosphopyridine nucleotide, respectively; D P K oxidized diphosphopyridine nucleotide; DEAE-cellulose, diethylaminoethyl-cellulose. This work was supported by g r a n t C-4110 from t h e National Cancer I n s t i t u t e of t h e National Institutes of Health, United S t a t e s Public Health Service. (1) H . P. Wolf a n d F'. L e u t h a r d t , Helw. Chim. A d a . 36, 1 4 f 3 (1953). ( 2 ) E . A . Peterson a n d H . A . Sober, THISJ O U R K A L , 78 7.51 (1956). ( 3 ) H. A. Sober, I?. 1. C u t t e r , hl. hl. Wycoff a n d fi. A. Petersou, Ibid., 78, 7513 ( 1 U X ) . +
Fig. 1.-Separation of D P S + and T P S + D-glyceraldehyde hydrogenases on DEAE-cellulose. The reaction mixture contained 20 pmole of DL-glyceraldehyde, 4 prnole of triethanolamine pH 7.0, and 0.02 pmole D P S H or T P S H in a volume of 200 microliters. Incubation was 30 min. a t 38". Triangles represent protein (Lowry) ; open circles, enzyme activity with D P S H ; closed circles, with T P S H .
The optimum pH for glyceraldehyde reduction was less than 7.0 with 0.1 hf DL-glyceraldehyde, 0.02 Jf triethanolamine buffer, and 10-4-U T P N H . T.\.hen the reaction was run in the opposite direction, a t 0.1 31 substrate concentration and a t the optimum p H of 9.5 in triethanolamine buffer, glycerol was oxidized, but 2-aniino-2-methyl-?, 3-propanediol was oxidized a t about twice the rate. Tris-(hydroxymethy1)-aminomethane and 2-amino-2-methyl-1-propanol (A411PI)were oxidized at about two-thirds the sate of glycerol, and ethanol was not oxidized a t a measurable rate. The Km's at 38" for glycerol and AlIPz were 0.63 &f and 0.13 114, respectively. The K m AMP2 was for T P N + in the presence of 0.5 1.7 X ;Zf. The K m for D-glyceraldehyde -11. The cniyme was 9570 inwas 6.2 X hibi tcd by 10 -j*l~;-niercuril,cnzoatc.
5835:
COMMUNICATIONS TO THE EDITOR
Vol. 81
propylcarbodiimide gave crystalline 111, iii 22%, yield, 1n.p. 1fi5-1C,G0. Conipound 111 also was prepared in 25s/i, overall yield by tritylation of natural hniinopenicil( 4 ) B \V. Moore, r e d P I O C18. , 280 (197'3) lanic acid followed by esterification with diazo~VERXSECANCERRESEARCH LABORATOR\ methane, 1n.p. 1&-16(jo, undepressed upon admixLVASHISGTON ~TNIVERSITY SCHOOL OF MEDICIKE BLAKEiv. h l O O R E ture with the corresponding samples made by ST LOUIS10, MISSOURI total synthesis and from penicillin G and havirlg an KECEIVED SEPTEMBER 17, 1959 identical infrared spectrum (KBr) and optical rotation. The niethyl ester of 111was saponified selectively' A GENERAL SYNTHESIS OF THE PENICILLINS to afford (i-tritylamiriopenicillanic acid (II'j as the Sir : crystalline diethylamine salt in 1i This Communication describes both a partial H3rK:i03S;11i.p. l6(i-l6So (dec.), CY% (from penicillin G) and a total general synthesis of ( c 1 in dioxane),; : ;A a t .J.(Xi(vs)u [foulid: C, the penicillins. The key intermediate in the two ( j ! ) . i l ; H,i . 0 0 ; S, 7.9C)j. The salt resrjondetl to routes is ti-aminopenicillanic acid (V) which Tve the quantitative hydroxylamine assay for penicilhave acylated to form both "natural" penicillins lins.' Tritylation; of 1- gave the diethylamine and penicillins not obtainable directly by fer- salt of I V in yield, n1.p. lti&-l(i(io (clec.). mentation.2 Identity of samples of IV prepared from 111 and I n the total synthesis series, removal of the IT,respectively, was established by undepressed phthaloyl group from t-butyl ~-a-4-carboniethoxy- mixed m.p., identical infrared spectra (KI3r) 5 , 5 - dimethyl - a - phthaliniido - 2 - thiazolidine- and rotations. acetate3 as described for the corresponding IJLHC1 isomer produced t-butyl n-a-i-carboiiiethoxy-5,,j- H ? S d S Y dimethyl-a-amino-2-thiazolidineacetate hydrochloCO,R SH--LCO.CH ride (Ia), C1::H25N204SC1,in yield; 1ii.p. 183-1S4" (dec.), c P u 4- 91" (c. 1.2 in methanol) Ia K = - C CH,iS [found: C, 45.65; H, 7.61; X, S.31 I. Cleavage of the t-butyl ester with hydrogen chloride led to a Ci2cSb yield of the dihydrochloride of D-a-4-carboniethoxy - 5,5 - dimethyl - a - amino - 2 - thiazolidineacetic acid (IC),C~Hl3S2O4SCl2; m.p. 04-97" (dec.), CY%~II S2' (c 0.5 in (i -Ir hydrochloric aCidJ [found: C, 33.72, H, 5.SS; N, S.031. Trityl chloride and diethylamine; converted I C into L)0 111 \ cy - 4 - carbomethoxy - 3,5 - dimethyl - a - trityl\ amino-2-thiazolidineacetic acid (11). Treatment of II with N,S'-diisopropylcarbodiiniide in dioxanewater, followed by chromatography over neutral alumina, afforded crystalline aniinopenicillanate (111) in 25 K2OZS; m.p. I(j,j-I(Xjo, a 3 ' D S butyl acetate), ;A:t a t 5.6;3(vs)p [found: C, 1.1 / , c J H os H k.' i l . 2 9 ; H, (i.07; S,5.7X]. r--K - 1 " f I T-SC0,H + By the partial synthesis route, saponification of 0 0 the a-methyl ester grouping of I b (obtained from \'I, ~ieiiicilliiiti penicillin G6) with one equivalent of sodiuiii \', ti-,i,iiiiopenicillatlic acid hydroxide arid tritylation" of this product loriiied Tktritylatioii of 1V with dilute hydrochloric 11 in 2oc,', over-all yield a s a non-crystalline solid, acid gave ;E(;{ of crystalline (j-aminopetiicillanic 45" acid (Vi, ChHl2X2OBS;m.p. 2Oi-2OS" C2sH:jllNy04S;11i.p. Si-SO" (dec.), ~ " I I (dec.!, (c 1.8 in n-butyl acetate) [found: C, W T 4 : H , [natural,' 2os-20no (dec.) 1, undepressed mixed (i.2S; h7, .J.33]. Cyclization of 11 with diiso- 1 i i . p . with natural, C Y " ~ D 2 i 3 ° ( G 1.2 i l l 0 . 1 +V ( I ) J . C. Sheehan, K. R . Henery-l,r~ganand I>. A. J c ~ h u w ~ nT ,H I S hydrochloric acid) ]found: C, 44.43; H, 5..Y : J O U R N A L , 7 5 , Y292 (1!1,53), fuotnote 2 . K , I2.SCil. The infrared spectra (KRr) of ( 2 ) I n a Ciba Funn(latir,n Sympuhiuni Iirld in l . ~ ~ n d l~~ ~ iin g l,a i ~ iirl l, natural and syiithetic V were identical. Synthetic \ l a r c h , 19.33, J C. S. s t a t e d t h a t ". . wc have yrcparr(1 t h i i compound 1' was compared to natural V, ill parallel deter[li amin
