NOVIOSEGLYCOSIDES
March 20, 1960
1489
[CONTRIBUTION FROM MERCK, SHARP A N D D O I ~ MRESEARCH E LABORATORIES DIVISIONOF MERCK& Co., INC.]
Novobiocin. IX. Noviose Glycosides BY EDWARD WALTON, JOHN 0. RODIN,FREDERICK W. HOLLY,JOHN W. RICHTER, CLIFFORD H. SHUNK AND KARLFOLKERS RECEIVED JULY 10, 1959 From acidic methanolysis of novobiocin, a-methyl 2-O-carbamyl-4-O-methyl-~,~-dimethyl-~-l~op~ranoside as well as the a- and @-methyl3-O-carbamyl-4-O-methy1-5,~-dimethyl-~-lyxoppranosides have been obtained. Alkaline hydrolysis Optical rotational of the carbamyl group has produced the a- and ,%methyl 4-O-methyl-5,5-dimethyl-~-lyxopyranosides. d a t a suggest t h a t novobiocin is an a-glycoside.
Acidic methanolysis of novobiocin (1)1$2 yielded, in addition to a mixture of novobiocic acid and
dilute acid, it reacted with one mole of periodate. This evidence suggested that glycosides I1 and 111 were the a- and 4-anomers of methyl 3-0-carbamylnovioside. The indicated anomeric relationship was confirmed when rotations of samples of glycosides I1 and 111 reached the same equilibrium value following acidic hydrolysis to 3-0-carbarnylnoviose c=o (Fig. 1). As glycoside I11 has the more positive rotation, it is, conventionally, @-methyl 3-0I r(TH2 carbamyl - 4 - 0 - methyl - 5,5 - dimethyl - L cyclonovobiocic acid, 1,3 a methyl glycoside (methyl lyxopyranoside. Glycoside 11, that with the 3-0-carbamylnovioside.ls4 This glycoside ( [a]D negative rotation, is the a-anomer. 2S0) has been shown to be methyl 3-0-carbamyl-4 Having both the a- and P-methyl glycosides of 0 - methyl - 5,5 - dimethyl - L - lyxopyranoside 3 - 0 - carbamyl - 4 - 0 - methyl - 5,5 - dimethyl (1I).lJsKAs the yield of the glycoside I1 was L-lyxopyranoside I1 and 111, it is possible to apply rather low, it was decided to look for other products Hudson's rules of isorotation7 to the question of the of the reaction. After a rather lengthy series of configuration of the glycoside link in novobiocin. From the molecular rotation (- 6250') of a-methyl CHI 3-0-carbamylnovioside (11) and that (+32,500°) of the p-anomer 111, the 2B rotational value is calculated to be +2G,00Oo. As the molecular rotation of novobiocin (-24,000") is negative, the rotational contribution of its glycosidic carbon must be negative. This indicates that the glyco0 I1 I11 vI 0 side link of novobiocin is of the a-L-configuration. On this basis, it is proposed that novobiocin is a - 7 - [4 - hydroxy - 3 - (4 - hydroxy - 3 - [3 C H,, CH, methyl - 2 - butenyl] - benzamido) - 8 - methylcoumaryl] - 3 - 0 - carbamyl - 4 - 0 - methyl - 5,5 dimethyl-L-lyxopyranoside (I). It has been reported's4 that alkaline hydrolysis of a-methyl 3-0-carbamyl-4-0-methyl - 5,5 - diIV V methyl-L-lyxopyranoside (11) removes the 3-0carbamyl group producing a-methyl4-0-methyl-5, fractional crystallizations, two new . glycosidic substances were obtained. One of these glycosides 5-dimethyl-~-lyxopyranoside (IV). Similarly, @( [ a ] -I~ 130') was found to exist in two inter- methyl 4-0-methyl-5,5-dimethyl-~-lyxopyranoside convertible crystalline modifications which had (V)s is obtained from the P-methylglycoside 111. In addition to the a- and @-methyl 3-0-carmelting points of 117-118' and 155-157'. The elemental analyses and functional infrared spectral bamyl - 4 - 0 - methyl - 5,5 - dimethyl - L - lyxobands of this substance indicated that it is isomeric pyranosides (I1 and 111), a third glycoside (VI) with methyl 3-0-carbamylnovioside (11). When was obtained from the acidic methanolysis of the new glycoside (111) was hydrolyzed with novobiocin. The elemental analyses and functional infrared absorption bands of this compound (1) C. H. Shunk, C. H . Stammer, E. A. Kaczka, E. Walton, C. F. were the same as those of the glycosides I1 and Spencer, A. N. Wilson, J. W. Richter, F. W. Holly and K. Folkers, 111. The optical rotation ([a]D-9.9') precluded THISJOURNAL, 78, 1770 (1956). (2) E. A. Kaczka, C. H. Shunk, J. W. Richter, P. J. Wolf, M. M. the possibility of this compound being a polyGasser and K. Folkers, i b i d . , '78, 4125 (1956). morphic forms of the glycosides I1 or 111. Alkaline
-
1
1
(3) J. W. Hinman. H. Hoeksema. E. L. Caron and W. G. Jackson, i b i d . , 7 8 , 1072 (1956). (4) J. W.Hinman, E. L. Caron and H.Hoeksenla. ihid., 79, 3789 (1957). ( 5 ) E. Walton, J. 0. Rodin, C. H . Stammer, F. W. Holly and K . Folkers, i b i d . , 80, 5168 (1958). ( 6 ) J. W. Hinman, E. L. Caron and H. Hoeksema, % b i d . .7 8 , 2018 (1956)
(7) C. S. Hudson, ibid., S1, 66 (1909). (8) This product has also been obtained from &methyl 4-0-methyl5,5-dimethyl-~-lyxopyranoside2,3-cyclic carbonate (ref. 4). These workers have also demonstrated the anomeric relationship of glycosides I V and V. (D) Differences in the infrared spectra (8 to 11 A, region) were not conclusive as only solid state spectra were available.
1400 E.
N'ALTON,
J. 0 . RODIN,F. W. HOLLY,J. W. RICHTER, C. H. SHUNK AND K. FOLKERSVOl. S2
under reduced pressure to a volume of 100 ml. and cooled, giving 6.4 g. of a third solid (C), m.p. 130-185", [ a I z 6 D -22' (c 1 in methanol). The mother liquor from C was concentrated under reduced pressure yielding 50 g. of rcsidual oil. 100" The oil was extracted with 1 1. of boiling ether, and a small amount of insoluble material was removed. One liter of ether was added to the filtrate and a n additional small amount of insoluble material was separated. The ethereal --0 13 solution was concentrated to a volume of 450 ml. and cooled, giving 7.6 g. of white crystalline material ( D ) , m.p. 140190°, [ a I z 6 D -5' (c 1 in methanol). The mother liquor 2 from crop D was concentrated under reduced pressure giving rE 40 g. of residual oil. The oil was dissolved in 100 ml. of boiling chloroforni and cooled giving 30 g. of white crystalline product ( E ) , m . p . 109-116". The mother liquor from crop E was conccn25" trated under reduced pressure t o a volume of 50 ml., and cooled giving 1.3 g. of a second crop ( F ) , 1n.p. 103-153". Concentration of the mother liquor from crop F t o a volume of 25 ml. gave 2 g. of solid ( G ) , m.p. 165-205'. Crops C and D were combined and dissolved in hot ace0" tone. The cooled solution gave 8 g. of product, m.p. 190193', which was combined with crop X giving a total of 50 g. (3lr0) of ~-methyl-3-O-carbamyl-4-O-methyl-5,5-diethyl-25 L-lyxopyranoside (11) .l.&e6 Recrystallization from acetone a purified sample, m.p. 191-192', [ C ~ ] ' ~ D-28' ( c 1.0 0 50 100 150 200 gave in methanol): :"' "_ 2.97. 3.04. 3.16, 5.86 and 6.15 irr, ,. A>:... Time, hours. &: :A 5.80 p . Conibination of crops E and F gave 31.5 g. (20%) of PFig. 1. -Change in rotation with timc at 25' in 3 :V hydromethyl 3-O-carbamyl-4-O-methy1-5,5-dimethyl-~-lyxopychloric acid: 0, a-methyl 2-O-carbamyl-4-O-methyl-5,5- ranoside (111). Recrystallization of a small sample from dimethyl-L-lyxopyranoside (c 1.15); 0 , 0-methyl 3-0-carchloroform gave a product which melted at 117-118". This was recrystallized giving a second crystalline modificabam~l-l-O-inethyl-5,6-dimetl1~l-~-lyxopyranoside (c 2.14). tion of glycoside 111, m.p. 155-157', [ a ] 2 6 ~f130' (c 1.0 in 2.81, 2.89, 3.05, 5.74, 5.84, 6.09 and methanol); ?';A: hydrolysis of compound VI to remove the car- 6.16 p, bamyl substituent gave a good yield of a-methyl Anal. Calcd. for CloHlsSOe (249.26): C, 48.17; H , 1 - 0 - methyl - 5,5 - dimethyl - L - lyxopyranoside 7.68; N, 5.62. Found: C, 47.99; H, 7.50; N,5.85. (IV). When subjected to hydrolysis with dilute \Then the two forms of the 8-methyl glycoside I11 were acid, glyoside VI gave an aldose which failed to mixed, the mixture melted at 155157'. Three recrystallizations of crop B from ether gave areact with periodate which indicates that the 2-O-carbamyl4-O-methyl-5,5-dimethyl-~-ly~0pycarbamyl grouping is in the 2-position. These data methyl ranoside (VI), m.p. 208-21lo, [ a ] " ~ -9.9" (c 1.0 in methlead to the formulation of this third glycoside as anol). a-methyl 2-O-carbamyl-4-O-methyl-5,5-dimethyl-Anal. Calcd. for CloH19S06(249.26): C, 48.17; H, L-lyxopyranoside (VI). This compound has been 7.68; hT,5.62. Found: C, 47.70; H, 7.45; N, 5.34. Solutions of a- and p-methyl 3-0-carbamyl-4-0-methylreportedlo as a hydrolysis product of iso-novobi5,5-dimethyl-L-lyxopyranoside (I1 and 111) in 3 A' hydroocin. It was shown1° that iso-novobiocin is pro- chloric were kept a t room temperature. The rotations duced by a reversible carbamyl migration when of theseacid solutions wcre noted from time to timc, and are innovobiocin is dissolved at I;H 10. It is assumed dicated in Fig. 1. p-Methyl4-O-Methyl-5,5-dimethyl-~-lyxopyranoside (V). that our product is the result of a similar migration occurring during an alkaline stage in the work- --;1 solution of 0.50 g. (2.4 mmoles) of 8-methyl 3-0-carbarn? 1-4-0-methyl-5,5-dimeth~-l-~-lyxopyranoside (111), 2 up of the acidic methanolysis of novobiocin. ml. of 2.5 iv sodium hydroxide and 25 ml. of water was heated on the steam-bath for 1.5 hr. The solution was Experimental cooled, neutralized with carbon dioxide and lyophilized. Acidic Methanolysis of Novobiocin.--A solution of 400 T h e residue was triturated with ether. The ether solution g. (0.65 mole) of novobiocin, 4 1. of methanol and 43 ml. of was concentrated at reduced pressure t o 0.4 g. of residue concentrated hydrochloric acid was refluxed for two hours. which crystallized on standing. The product was recrysWater (5.5 1.) was added and a solid precipitated. The tallized from petroleum ether (b.p. 30-60') giving 0.28 g. mixture was thoroughly cooled and filtered giving 268 g. of ( 7Oy0)of 8-methyl 4-0-methyl-5,5-dimethyl-L-lyxopyranoa mixture of novobiocic and cyclonovobiocic acids, m.p. side, m.p. 62-64'. Two recrystallizations from pFtroleum 145-165'. The filtrate was neutralized with sodium bicar- ether gave an analytical sample, m.p. 66-67.5 , [aI2'D bonate and concentrated to dryness under reduced pressure +l06" (c 0.7 in water). (the temperature was kept below 40"). The crystalline Anal. Calcd. for CsHlsOs(206.23): C, 52.41; H, 8.79; residue was triturated with six 250-1111. portions of acetone, OCH,, 30.1. Found: C, 52.51; H,8.59; OCH3,31.0. and the acetone solution was treated with Darco G-60 and In a similar manner, alkallne hydrolysis of 16 g. of afiltered. The filtrate was concentrated under reduced presmethyl 3-O-carbamyl-4-O-methyl-5,5-dimethyl-L-lyx~p~sure t o a volume of 500 nil. Tlie solution was kept a t 5' and 42 g. of crystalline solid ( A ) , m.p. 190-193', was ob- ranoside (11) gave 11.5 g. (87%) of a-methyl 4-0;methyl5 , 5 - d i m e t h v l - ~ - l ~ ~ x o p y ~ ~ n (IV),l35 o s i d e m.p. 69-71 , [ a I 2 ' D tained. -50" (c 1 . i 3 inwater). The mother liquor from crop .4 was concentrated under Alkaline Hydrolysis of a-Methyl 2-O-Carbamyl-4-0reduced pressure to a volume of 200 ml. and cooled, yieldmethyl-5,5-dimethyl-~-lyxopyranoside(VI) .-A solution of ing 9 g. of solid in the form of white platelets ( B ) , m.p. 2081.0 P. of VI in 50 ml. of water and 4 ml. of 2.5 N sodium 212'. The mother liquor from B was further concentrated hydroxide was heated on the steam-cone for one hour. T h e solution was neutralized (pH 8) with carbon dioxide (10) J. W, Hinman, E. I,. Caron and H. Hoeksema, THISJOURNAL, and lyophilized. The residue was triturated with four 79, 5321 (1957). portions of ether and the ethereal extract was concentrated ( 1 1 ) T h e authors are indebted t o Mr. R. N. Boos and associates for yielding 0.8 g. of oil. The residue was crystallized from pcthe microanalyses a n d to Dr. N. R. Trenner and Mr. R.W. Walker for troleum ether. The recovery of w-methyl 4-0-rnethyl-5,5the infrared spectra.
h
I
DISSOCIATION O F -4NTIGEN-24NTIBODY
March 20, l9GO TABLE I
Moles 1 0 4 - consumed12 ,--per mole cpd 15 min. 60 min.
--
Compound hydrolyzed
a-Methyl 4-0-methyl-5,5-dimethylL-lyxopyranoside ( I V ) a-Methyl 3-0-carbamyl-4-0-methyl5,5-dimethyl-~-lyxopyranoside(11) 9-Methyl 3-0-carbamyl-4-0-methyl5,Z-dimeth yl-L-lyxopyranoside (111) a-Methyl 2-0-carbamyl-4-0-methyl5,5-di1nethyl-~-Iyxopyranoside (VI)
FROM THE
1491
ml.). T h e hydrolysates were neutralized with equivalent amounts of 1 N sodium hydroxide and aliquots (containing approximately 0.1. mmole of aldose) were oxidized with excess 0.1 M sodium metaperiodate. T h e results are shown in Table I.
1.76
1.84
0.76
0.77
(12) Hinman, Caron and Hoeksema have shown (ref. 4) t h a t acidic hydrolysis of a-methyl 3-0-carbamyl 4-O-methyl-5,5-dimethyl-~lyxopyranoside (11) yields in addition t o 3-0-carbamyl-4-0-methyl5,5-dimethyl-~-lyxosea by-product which they have formulated as the bicyclic compound VII. Formation of such a by-product would account for the low consumption of periodate in t h e oxidations of I1 and
0.68
0.68
111.
0.03
0.03
dimethyl-L-lyxopyranoside ( I V ) , m.p. 69.5-71°, [ a I z 6 D -45' (c 1.5 in water), was 0.65 g . (80%). When this product was mixed with a n authentic sample of IV, the mixture melted a t 69-71'. Periodate Oxidation Studies.-Samples of the following glycosides were hydrolyzed on the steam-bath for a n hour with 0.1 N hydrochloric acid (approximately 0.033 mmole/
[CONTRIBUTION No. 2501
PRECIPITATES
6HNEi
0
\ / $
VI1
6
RAHWAY, N. J.
GATESAND CRELLINLABORATORIES OF CHEXISTRY OF TECHNOLOGY]
THE
CALIFORNIA INSTITUTE OF
The Dissociation of Antigen-Antibody Precipitates in Alkali Chloride Solutions B Y ISIDORE
s. EDELMAN' A N D W I L L I A M P. BRYAN RECEIVED SEPTEMBER 8, 1959
The dissociation of antigen-antibody precipitates in solutions of alkali chlorides was studied by measuring the solubility of specific precipitates of purified bovine serum albumin and rabbit anti-bovine serum albumin in solutions of LiC1, NaCl, KCI and CsCl over the concentration range 0.08 to 1.6 molar. At electrolyte activities below 0.25 the solubility of the precipitate a t a given activity has the order CsCl > KCl > NaCl > LiC1. Above an activity of 0.25 different results are obtained. T h e order of the results below an activity of 0.25 is most readily explained b y the interaction of the hydrated cations with negative sites involved in the antigen-antibody bonds.
Introduction
The evidence currently available indicates that The properties of precipitates of antigen (Ag) treatment of such precipitates with saline solutions and antibody (Ab) have been shown to depend on preferentially extracts antibody.4 ~ 5 Although it has been pointed out that different the pH and the concentration of the inorganic salt solutions in which the Ag-Ab reaction takes place. inorganic salts vary in their ability to reduce the The rate of flocculation and the amount of precip- amount of an Ag-Ab precipitate a t equivalent itate formed in these reactions are influenced by ionic strength, there has been no systematic study the pH and the electrolyte concentration of the of this effect with respect to the members of the alkali medium in a variety of Ag-Ab systems, including metal ions series compared a t equivalent activities. polysaccharide :protein as well as protein :protein A comparison of this kind might disclose different precipitin reactions. 2-8 I n general, these experi- degrees of interaction between the inorganic ions ments show that the amount of precipitate formed and the surfaces of the antigen and antibody molebetween antigens and rabbit antibodies is reduced cules or, alternatively, might indicate that the disa t 5 > PH > 8 and is also reduced by increasing sociative effect of the electrolytes is a non-specific electrolyte concentration up to 2 11.1. Furthermore, function of electrolyte activity. In the former a portion of the Ag-Ab precipitate is rendered case, one might expect that the smaller the ion the closer the distance of approach to the protein surface soluble by resuspension in NaCl soluti0ns.~~~0 (perhaps to the specific combining site of the anti(1) Senior Research Fellow in Chemistry, California Institute of gen or antibody) and the greater the dissociation Technology, on sabbatical leave-of-absence from t h e University of of the precipitate. In the latter case, all of the California School of Medicine, San Francisco 22, California. This univalent :univalent salts would have the same study was performed during t h e tenure of a Special Fellowship from the National Heart Institute of the United States Public Health Service. dissociative effects a t the same activities and tem(2) S. Schmidt, 2 . Immunifdfs/orsch., 67, 197 (1930). peratures. (3) M. Heidelberger, F. E. Kendall and T. Teorell, J . Ezp. M e d . , 63, This paper reports the dissociative effects of the 819 (1936). alkali chlorides (Li, Na, K, Cs) on precipitates ( 4 ) J. R . Marrack and H . F. Hollering, Bril. J . E x p . Palhol., 1 9 , 424 (1938). formed from bovine serum albumin and rabbit ( 5 ) J. Oudin and P. Grahar, An?r. I n s l . Pasfeur, 70, 7 (1944). antiserum a t 3". 448
(6) F. Aladjem and M. Lieberman, J . Imntunol., 69, 117 (1952). (7) J. R. Marrack and R. A. Grant, Bvil. J . E x p . Palhol., 34, 263
(1353). ( 8 ) J. R. Marrack, Immunology, 1 , 251 (1358). (9) W.Heidelberger and F . E. Kendall, J . E x $ . J i e d . , 6 2 , 607 (1335).
(10) D. W. Talmage and P. H . hlaurer, J . Iufeclrous Diseases, 92, 288 (1953). (11) F . Haurowitz, R Sowinski and H F Cheng. THISJ O U R N A L , 79, 1882 (1957).
