July 5 , 1959
SYNTHESIS O F 2-AMINO-2-DEOXY-L-ARhBINOSE [CONTRIBUTION I'KOM THE
DEPARTMENT OF
CHEMISTRY O F
THEOHIO
3477
STATE UNIVERSITY]
Synthesis of 2-Amino-2-deoxy-~-Arabinose(L-Arabinosamine) BY M. L. WOLFROM AND Z. YOSIZAWA~ RECEIVED DECEMBER 19, 1958 Oxidation of ethyl 2-acetan~ido-2-deoxy-l-thio-a-~-ga~actofuranos~de (ethyl 2-acetamido-2-deox~-l-thio-~-g~ycero-P-L nrabino-hexafuranoside) ( I ) with sodium metaperiodate, followed by reduction with sodium borohydride, produced ethy 2-acetamido-2-deoxy-l-thio-p-~-arabinofuranoside (111),which was hydrolyzed with aqueous mercuric chloride solution t 2-acetarnido-2-deoxy-p-~-ara binose (IV)and with hydrochloric acid to 2-amino-3-deoxy-@-~-arabinose hydrochloride ( V ) .
The amino pentoses have come into prominence Elson-Morganll and ninhydrinI2 color tests. The as components of antibiotics2s3 and of possible Dische-Borenfre~nd'~color reaction for amino carcinolytic agents. The first pentosamine, 2- sugars (amino group unsubstituted) showed a amino-2-deoxy-a-D-xylose hydrochloride, dec. 165- maximum absorption a t 490 mw. The assignment of the L-arabinosamine structure 167', [ a ]+a0 ~ + +40' (water), was synthesized by Wolfrom and 4 . n n 0 . ~ 3-Amino-3-deoxy deriv- to the pentosamine follows from the nature of the ative$-? of D-ribose, D-xylose and D-arabinose have reaction and the established configuration of chonbeen synthesized. Three more 2-amino pentoses,* 2-amino-2-deoxy-a-~-ribose hydrochloride, m.p. 142-148' dec., [ a ] -15.6 ~ -+ +6.7' (water), its [ a ] D +14.1 + enantiomorph, m.p. 144-149', - 2.75O (water), and 2-amino-2-deoxy-a-~-~yxose hydrochloride, dec. 148-155', [ a ] +54 ~ -36' (water), have been reported. Also, a 5-amino-5deoxy-1,2-O-isopropy~idene-a-D-xy~ofuranosep toluenesulfonate has been described? H H We wish to report herein the synthesis of 2amino-2-deoxy-P-~-arabinose (' ' L-arabinosamine") , characterized as the hydrochloride V and the NI I H NHAC acetyl derivative IV. The synthesis was carried m out by an extension of the method employed by Wolfrom and Anno? for the synthesis of 2-amino-2- drosamine as ~-amino-2-deoxy-~-ga~actose. l4 Redeoxy-D-xylose. moval of the terminal asymmetric center in the Ethyl 2-acetamido-2-deoxy-l-thio-~-~-galactoa-D-galactoside I results in an L-arabinoside I11 furanoside (ethyl 2-acetamido-2-deoxy-l-thio-~-g~ywith consequent change in anomeric designation cero-P-L-arabino-hexafuranoside)(I) was oxidized from CY-Dto p-L without any change in the order of in the dark a t 0-3' with one mole of sodium meta- groups about C1. periodate. Under these conditions the molecule is Acknowledgment.-Acknowledgment is made cleaved very rapidly a t the C5-CB bond. The oxiA. Thompson in preparing to the assistance of Dr. dation was followed immediately by reduction with sodium borohydride to yield ethyl 2-acetamido-2- this work for publication. (I11), m ,p. 127deoxy- 1-thio-p-L-arabinofuranoside Experimental 129') [ a ] D +172' (water). Hydrolysis of TI1 with Ethyl 2-Acetamido-2-deoxy-~-~-arabinofuranoside (111).aqueous mercuric chloride produced 2-acetamido- Ethyl 2 - acetamido - 2 - deoxy - 1-thio - a-D-galactofuranoside 2-deoxy-P-~-arabinose(IV), m , p . 154-15A0. [ a ] ~(ethyl 2-acetamido-2-deoxy-l-thio-~-g~~~ce~~-~-~-a~ub~n~+147.5 + +94.1° (water). I t gave a positive hexofuranoside) (I)9(1.06 g., 4 millimoles) was dissolved in Morgan-Elsonlo color test for N-acetylamino sugars. 21 mi. of water and tredted with 4.1 millimoles of sodium in 19 ml. of water, a t 0-3", in thedark, for30 Hydrolysis of I11 and IV with hydrochloric acid metaperiodate min. The reaction mixture was passed through a column hydrochlo- (180 X 13 mm., diam.) of Amberlite MB-3l6a.d the column provided 2-amino-2-deoxy-~-~-arabinose was washed with 100 rnl. of water. The combined effluent ride (V), m.p. 133-154' dec., [ a ]+174 ~ + + I 15' washings were concentrated under reduced pressure to 15 (water). The hydrochloride was strongly reducing and ml. T o this solution was added dropwise, with stirring, a to Benedict solution and reacted positively to the solution of 0.15 g. of sodium borohydride in 10 ml. of water,
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(1) Rockefeller Foundation Fellow, 1956-1957; National Science Foundation Research Associate, 1D57--19.58, under Grant NSF-G4404 to The Ohio S t a t e University. ( 2 ) B. R. Baker and R . E. Schaub, THISJOURNAL,?^, 38fi4 (1953). (:O R. R . Baker, R E. Schaub, J . P. Joseph and J. H. Williams, i b i d . , 7 7 . 12 (1955). (4) M. L. Wolfrom and Kimiko Anno, ibzd 75, 1038 (1953). (5) B. R. Baker a n d R. E. Schaub, J . Org. C h e m . , 19,646 (1954). (1;) R . E . Schaub and M. J. Weiss, THISJOURNAL, 80.4683 (1958). (7) B. R . Baker, R . E. Schaub a n d J. H. Williams. ibid., 7 7 , 7 (1955). ( 8 ) M. L. Wolfrom. F. Shafizadeh and R . K. Armstrong, ;bid., 80. 4885 (1958): M. L. Wolfrom, F.Shafizadeh, R . K. Armstrong and T. M. Shen Han, ibid.,81, in press (1959). (9) M L . Wolfrom and Z. Yosizawa, i b i d . , 81. 3474 (1959). 10) W. T. 3. Morgan and I,. A. Elson. Biochenr. J . , 28. 988 (1934).
over a period of 5 min., a t room temperature. After stirring for an additional 30 min., the solution was neutralized with N sulfuric acid, passed thrnugh a column (180 X 13mm .) o f Amberlite MB-3,'5 and the column was washed with 100 ml. of water. The effluent and washings were combined, concentrated to a sirup and further dried by repeated evaporation with ethanol under reduced pressure. The matcrial was crystallized from alcohol-ether-petroleum ether; L. A. Elson and W. T. J. Morgan, i b i d . , 27, 1824 (103X) S. Moore and W. H . Stein, J . B i d . C'hent., 176, 367 (15148). Z. Dische and Ellen Borenfreund. i b t l l . , 184, 5 1 7 f l R h O ) Sybil P. James, F. S m i t h , M . Stacey and I,. 1'. Wigains. J . Ckem. S O L , 625 (1946). (15) A monobed ion-exchange resin producrd h y K o h m a t i d kI:i.t., C o . , Resinous Products Division, Philadelphia 5 . l'ciina (11) (12) (13) (14)
3478
COBIMUNIC.4TIONS TO THE
EDITOR
Vol. 81
yield 0.75 g. (80%), m.p. 116-118' (preliminary softening). X-ray powder diffraction datalo: 9.03~11, 7.99s(2), 7 . 0 1 ~ ~ Pure material was obtained after several recrystallizations 4.76s(1), 4.60m, 4.39111, 4.10s(3), 3.53s(2), 3.33vw, 3 . 1 5 ~ . from ethanol-ether-petroleum ether; m.p. 127-129" (pre- 3.03m, 2.82w, 2.66vw, 2.48vw, 2.40vw, 2.23vw, 2 . 1 8 ~ ~ ~ liminary softening), [ o ] ~ ' D $172" (c 0.5, water); X-ray 2.07~~. powder diffraction datai6: 12.8m, 8.29s(3), 6.97w, 6.42w, Anal. Calcd. for CTH~~NOF,:C, 43.98; H , 6.85; N, 4.88s(1), 4.33111. 4.13s(2), 3.93~~1, 3.31vw, 3.19w, 2.85vw, 7.33. Found: C, 44.04; H, 6.85; N, 7.39. 2.26vw, 2.21vw, 2 . 1 4 ~ ~ . Z-Amino-2-deoxy-p-~-arabinose Hydrochloride (V).-An Anal. Calcd. for C & I J ~ O ~ SC, : 45.91; H , 7.28; S , 5.95; S, 13.62. Found: C, 45.94; H , 7.20; N, 5.82; S, amount of 88 mg. of 2-acetamido-2-deoxy-fl-~-arabinose was heated with 2 ml. of 4 N hydrochloric acid in a 13.41. boiling water-bath for 30 min. The hydrolyzate was con2-Acetamido-2-deoxy-j3-~-arabinose(IV).-An aqueous centrated to a sirup and further dried by evaporation w t h solution of 693 mg. of mercuric chloride in 60 ml. of water, methanol-acetone under reduced pressure, after whlch at 50°, was added to a solution of 300 mg. of ethyl 2-acet- treatment the sirup crystallized; yield 78 mg. (S9%), m.p. amido-2-deoxy-l-thio-~-~-arabinofuranose (111) in 15 ml. of 150-154'' dec. Pure material was obtained upon recryswater and allowed t o stand a t room temperature for 5 hr. The tallization from methanol-acetone; m.p. 153-155" dec., white precipitate which formed was removed by filtration. [ L Y ] ~ f174 ~ D (initial, extrapolated) + 115"_(c 0.5, water, T h e filtrate was passed through a column (200 X 18 mm., final); X-ray powder diffraction datal6: i .6iw, 6.20m, diam.) of Amberlite MB-3'6 and the column was washed 5 20m, 4.72s(3), 4.11s(l), 3.83s(2), 3.36vw, 3.15w, 2.88w, with 200 ml. of water. T h e effluent and washings were 2 . 6 7 ~ ~21 ,5Ovw. concentrated t o a sirup which was further dried by evaporaAnal. Calcd. for C I H ~ ~ C ~ SCO, ~32.36; : H, 6.52; N, tion repeatedly with ethanol under reduced pressure. T h e 7.55; C1, 19.10. Found: C, 32.59; H , 6.52; N, 7.46; product crystallized from methanol-acetone-ether; yield 158 mg. (65%), m.p. 151-155' (preliminary softening). C1, 18.84. Pure material was obtained by recrystallization from the The substance was strongly reducing and gave a strong same solvents. The compound was strongly reducing and positive reaction to the Elson-Morgan" and ninhydrin1* exhibited a positive Morgan-ElsoniO test for AT-acetylamino color tests. I n the Dische-Borenfreund test a maximum sugars; m.p. 154-156' (preliminary softening), [ c u ] ~ ~ D mas developed a t X 490 mp.*t13 2-Amino-2-deoxy-P-~-ara+147.5' (initial, extrapolated) ---f 4-94" (c 1, water, final); binose hydrochloride was obtained in the same way by the hydrolysis of ethyl 2-acetaniido-2-deoxp-l-thio-j3-~-ara(16) Interplanar spacing, .&., C u K a radiation. Relative intensity, binofuranoside with 4 N hydrochloric acid. The constants estimated visually: s, strong; m, medium; w, weak; v, very. First were identical with those reported above.
(IT)
three strongest lines are numbered, strongest ( l ) , repeated numbers indicate approximate equality.
COLUMBUS 10, OHIO
COMMUNICATIONS T O T H E EDITOR A STEREOSPECIFIC DIHYDROLIPOIC DEHYDROGENASE FROM
SPINACIA OLE R A CEA
Sir: The oxidation of a-keto acids takes place in a number of integrated steps of which the last two are mediated by dihydrolipoic transacetylase and dihydrolipoic dehydrogenase.' The purification and properties of the dihydrolipoic dehydrogenase from animals and microorganisms have been de~ c r i b e d .Diaphorase ~ ~ ~ ~ ~ ~ was ~ ~also ~ found to have powerful dihydrolipoic dehydrogenase activity.' Recently the involvement of flavoprotein in the pyruvate oxidase complex of E. colis and of diaphorase in a-ketoglutarate oxidase system of pig hearts have been demonstrated. In contrast to dihydrolipoic transacetylase, dihydrolipoic dehydrogenase does not exhibit optical specificity. A stereospecific dihydrolipoic dehydrogenase p6
(1) L. J. Reed. "Advances in Enzymology," Interscience Publishers Inc.. New York, S . Y., 18, 319 (1957). (2) L. P. Hager and I. C. Gunsalus, T H I S J O U R N A L75, . 5787 (1953). (3) I. C . Gunsalus, "The Mechanism of Enzyme Action," Johns Hopkins Press, Baltimore, Maryland, 1954, p. 545. (4) D. R.Sanadi and R . L. Searls, B i o c h i m . B i o p h y s . Acta, 2 4 , 220 (1057). (5) D. R . Sanadi, M. Langley and R . L. Searls. J . Biol. C h e m . . 234, 178 (1959). (6) D. S. Goldman, Biockim. Biophys. A d a , 32, 80 (1959). (7) V. Massey, ibid., 3 0 , 205 (1958). (8) M. Koike and L. J. Reed, THISJOURNAL, 81, 505 (1050). (9) V. Massey, Biochim. Biophys. Acta, 32, 286 (1950).
which has been purified 3040-fold from the acetone powder of Spinacia oleracea is DPN-linked and displays maximum activity a t pH 8.0. The rate of reaction with dihydrolipoamide is faster than that with dihydrolipoic acid (Table I), as observed by Sanadi and S e a r l ~ . ~ The enzyme is active with (-)-dihydrolipoic acid (Table I). The slight activity obsen-ed with (+)-dihydrolipoic acid may be due to the contamination with the other isomer. TABLE I Substrate
f & ) Dihydrolipoic acid ( -)-Dihydrolipoic acid" ( +)-Dihydrolipoic acid" ( rt )-Dihydrolipoamide" Cysteine Rediiced glutathione
D P S H formed in 3 min. (uM.)
0.044 0.033 0,005 0.084
0 0
Prepared from the oxidized compounds by the sodium borohydride reduction method of n'agner, et
The incubation was carried out a t 25' in 1.5 ml. containing 70 phl.TRIS (pH 8.0), 0.5 M M . DPN, 2 p M . of substrate and 30 pg. of dihydrolipoic dehydrogenase. Though the reaction is freely reversible with lipoamide it has not been possible to demonstrate (10) A . F. Wagner, E . Walton, G. E. Boxer, M . P. Pruss, F. W. Holly and K. Folkers, THIS J O U R N A L 78, , 5079 (1956).
