PRODUCTS OF HYDROLYSIS AND ALCOHOLYSIS OF ~IETHYLATED DISACCHARIDES 38 1
March, 1945
[CONTRIBUTION FROM THE
DEPARTMENT OF CHEMISTRY,
STATE UNIVERSITY OF I O W A ]
The Separation and Identification of the Products of Hydrolysis and Alcoholysis of Methylated Disaccharides BY GEORGEH. COLEMAN, DONALD E. REES,' ROBERTL. SUNDBERG AND CHESTER M. MCCLOSKEY Structures of disaccharides have frequently been determined by hydrolysis of the completely methylated compounds and separation of the hydrolytic products by partition between suitable solvents. In the case of certain 6-linked disirup has saccharides the 2,3,4-trimethyl-~-glucose been identified by conversion to the methyl @-Dglucoside. The yields of crystalline product are usually low. In the present work it has been shown that the point of linkage between the units of a disaccharide can be determined by the preparation and separation of the azoyl (p-phenylazobenzoyl) derivatives of the products obtained by hydrolysis or methyl alcoholysis of fully methylated' disaccharides. Two methods based on this procedure have been developed.
r---l
-
CH3OCH
HCOCHI I CHJOLH
I
~
1
I]
HCO---I I i H C O i
I
I I
H c1
CHIOCH
CHsOH
HCOCH, I
HCO-
I
CH*OCH,
CHeOCHa
Methyl heptamethyl-8-cellobioside
I I
HCO-
HCO CHiOCHa
AzCl
I
CHiOCHa Methyl 2,3,4,6-tetramethyl-D-glucoside
CsHsNs CHSOCH
I
HCOCH, I CH~OCH I O
I
Method 1.-The first method is based upon methyl alcoholysis followed by azoylation as illustrated in the appended equations. The methyl monoazoyltrimethylglycoside is separated from the methyl tetramethylglycoside by difference in water solubility, and identified by comparison with synthetic products. Quantitative isolation of the methyl tetramethylglycoside was not carried out in method A. Method 11.-The second method involves aqueous hydrolysis and azoylation as illustrated. Separation of the tetramethylglycosyl azoate from the azoyltrimethylglycosyl azoate is accomplished by chromatographic adsorption on silicic acid. Separations of mixtures of this type have been reported2 in which the pure a- or @-modifications were used. Since a- and p-mixtures of each derivative are obtained by these methods, separations were first tried with known synthetic azoates prepared under standardized equilibrium conditions. Separation of 1,2,3,4-tetramethyl-~-glucosyl azoate from 4-azoyl-2,3,6-triniethyl-~-glucosyl azoate occurred readily. A mixture of 4azoyl-2,3,6-trimethy~-~-g~ucosyl azoate and 6azoy~-2,3,4-trimethy~-~-g~ucosy~ azoate required a longer development period before separation occurred. Very good yields of azoates are obtained by both 'methods and the large azoyl group increases the molecular weight of the products to such an extent that only a small amount of methylated disaccharide is necessary. Synthetic azoates were prepared corresponding to the products which would result from hydrolysis or methyl alcoholysis of four types of interglucose linkage. These azoates were prepared under standardized conditions producing an equilibrium mixture of a- and @-modifications which could be identified by specific rotation3 and analysis for per cent. a ~ o y l . There ~ was fortunately considerable difference in the specific rotations of the compounds involved in each method. These values and the results of analysis for per cent. azoyl are given in Table I. Both methods were tested on five fully methylated disaccharides. The specific rotations of the separated products were in reasonable agreement with the values of synthetic derivatives. Results of method I applied to the five methylated disaccharides are given in Table 11. (2) Mertzweiller, Carney and Farley, THISJOURNAL, 66, 2367 Myrbaek and T a m m , Svensk. K e r n . T i d . , 63, 441-447
I
CHiOCHo
(1943); (1Y41).
(3) T h e specific rotations of all azoates were taken in U. S. P.
at 25' (c = 0.6 approx.). The light Methyl 4-azoyl-2,3,6-trimethyl-~-glucoside chloroform ciiry-cadmium light with a Wratten F filter. (1) Research Fellow of the Cnrn Prodiicts Rrfinine: Cn.
(4) Coleman and MrClnskry.
T H I S JOTTRNAL.
soiirce was a mer-
6 6 , 1588 (1948)
r - - 1
I
I IICOII I
I
:ICoCI I I CII,OCI-I lICOCI&
d o
---J
I
CN,OCHs 2,X,1,0-?'ctrainetliylD glucoie
CEIIOCI~ I O
CI130CH
I
IICOCHa I
azoate
azoate
TABLE I SYNIIIETIC Azoi\riis [ 1 1 ]%18 l l e t h y l 3-azoyl-R.4,G-trimethyl-o-glucoside I I U " l!(i" Xlrthyl 3-nzoyl-2.~.6-trimethyl-o-glucoside 32 28 h l e t h y l 4-azoyl-2,3.tj-trimetliyl-o-glucoside - g .- 2 h l e t h y l 6-azoyl-2,3,4-trimeth>-l-i)-gluco.irie ,;I 30 2.3 ~ , 6 - T e t r a m e t h y l - u - ~ l u c n s yl a z o a t e 35 32 33 2,3.4,(j-Tetramethyl-o-~~~l~ctosyl a z o a t e 63 2-Azoyl-3,4,6-triniethyl-~plucosyl a z o a t e 80 3-Amyl-2.4.6-trimethyl-uglucosyl a z o a t e 98 107 9,5 4-Azoyl-2,3,6-trimt?thyl 1)alucosyl a z o a t e - 1 1 --r, 6-Azoyl-2,3,4-trimethyl-r~glucosyl a z o a t e -38 -29 -36
T A B L E 11
[email protected] ROTATIONS OF AZOATES FORMED AFTBK ? r f ~ l ' l i Y I ~A 1 ~ c o i ~ i ~ i . v sOP r s LfLIETfIYLATED DISACCHARIDES Azoate [rr]%,8
hlethyl hIethpl Methyl hlethyl Xlethyl
heptametl1yl-8-cellohioside heptanirtbyl-8-lactoside heptamethylmaltoside heptamethyl-8-gentiobioside heptainethyl-8-melibioside
-4'
-3O
-8 -4 -6
53 41) 46
Synthetir product [n19h6,.tr
-60 - (i -6 52
52
Two specific rotatioiis for one compound represent rotations of equilibriuin mixtrrres obtained in duplicate experiments. a
65.2
65.53
Two or more specific rotations listed for one compound represent rotatioils of equilibrium mixtures obtained in separate experiments. 0
In method B the upper band formed by chromatographic separation of the azoates was always found to be the nionoazoate. The rotation of this band indicated that there was a little contamination from tlie lower diazoate band. IVith cellobiose there was incomplete separation on the first column. The upper band was therefore srj'arated on another column. With methyl
heptamcthyl-fl-lactoside and methyl heptamethylinaltoside a peculiarity was observed. By the chromatographic separation of the azoate mixture L: band was obtained which was less strongly adsorbed than either the monoazoates or diazoates. After separation of this band which passed down the column more rapidly, the upper band which consisted of the monoazoates and diazoates was separated on another column. Values corresponding to the synthetic products were obtained. The identity of the band which first separated has not been determined. It constituted about 25y0 of the original azoate mixtures and had a specific Table 111 shows the rerotation of about -'40°. sults obtained by method B applied to methylated disaccharides. Comparable values for corresponding synthetic products are also given. iVhen nzoyl chloride dissolved in pyridine is
?rfarch, 19.15
PRODUCTS OF
HYDROLYSIS AND ALCOHOLYSIS OF
TABLE 111 CHROMATOGRAPHIC SEPARATIONS OF AZOYLATED HYDROLYZATES (METHOD 11) Synthetic product [al’kas
Weight, g.
lal%a8
1. Methyl heptamethyl-8-gentiobioside Upper band 0 . 173s 22 36 0,2523 -31 -38 Lower hand 2. Methyl heptamcthyl-0-nielibioside Uppcr band 0 1319 42 0.1980 - 41 Lower hand
03
-38
3. Methyl heptaniethyl-8-cellohioside 0.2587 18 36 0.1076 - 3 - 1
Upper band Lower band
Uppcr band separated on another column 28 30 Upper band 0.1718 Lower ‘band 0.0811 3 - 1 4. Methyl heptaniethyl-8-lactoside Upper band 0.1812 19 03 Lower band 0.1031 -38 - 1 Upper band separated on another coluinii 0,0475 GO 63 Upper band Lower band 0.0789 - 2 - 1
5. Methyl heptamethylmaltoside 0.3284 22 Upper baud 40 Lower band 0.1310
36 - 1
-
Uppcr b m d separated on another column 31 3G Upper band 0.1215 Lower band 0.1936 7 - 1
added to a large excess of water azoic anhydride is formed in relatively high yield.4 If slightly more than the theoretical amount of water required for hydrolysis of the anhydride is added to the pyridine solution of azoyl chloride and the mixture allowed to stand for fifteen minutes, only a small amount of anhydride is obtained. A postulated mechanism for the formation of azoic anhydride is given in the following equations
X
=
S
~
&
-
Azoyl chloride
r
C
lN +
i
--f
Pyridine 0
0
3%3
If only a limited amount of water is added the anhydride which forms remains in solution and is soon hydrolyzed. When the pyridine solution is poured into a large excess of water the anhydride is formed and is immediately precipitated due to its insolulility in water. The small amount of azoic anhydride present in the crude synthetic azoates was removed by chromatographic adsorption on silicic acid. The anhydride passed down the column quite rapidly. The synthetic azoates corresponding to the products formed by method I were separated by chromatographic adsorption on silicic acid into fractions having markedly different specific rotations, presumbaly the a- and /%forms. The specific rotations of these fractions and their relative weights are given in Table IV. In order to purify the fractions further, each was recrystallized twice from ligroin. The final melting points, specific rotations, crystal form, and color are given in the last columns of Table IV. In certain cases it was necessary to prepare additional material for this recrystallization. It should be noted that this series represents crystalline derivatives of all eight methyl trimethyl-~-glucopyranosides. The two samples of methyl Cj-azoyl-3,X,4-trimethyl-~glucoside obtained by the application of method I to inethyl heptaniethyl-0-gentiobioside and methyl heptamethyl- P-melibioside wcre each separated chroinatogrnphically on silicic acid. The fractions obtained in each case corresponded closely in specific rotation to those obtained from the synthetic methyl G-azoy1-3,3,4-triethyl-Dglucoside. The sample of methyl 4-azoyl-2,3,0triniethyl-D-glucoside obtained by the application of method I to methyl hep tatnethyl-0-cellobioside was separated chroiiiatographically into the a aiid p modifications. These also corresponded closely in specific rotation to the fractions obtained froin the synthetic methyl 4-azoyl-2,,3,Ci-triniethylD-glucoside. The products thus obtained froin the three disaccharides were recrystallized froin ligroin and found to be identical with the correspoiitiiiig rxrystallized synthetic products.
r
0
~
RfETIiYLATED DISACCHARIDES
l+
0
r
0
1-
r
0 1 Azoic anhydride
TABLE11' CHROMATOGRAPHIC SEPARATIOXS OF SYNTIIETIC AZOATESINTO r_
Upper band (8) Lower hand ( a )
0.2496 0.4:130
ITpperband ( a ) Lower harid (p!
0.1
Upper band ( R ) Lower band fp,
(!.9132 11.4183
i
-.
01- AND &MODIFICATIONS Recryst,rllized product------11. p . , "C. Crystal f o r m Color
~~~~
...
[ ( V I'~BW jm]%w Methyl 2-azoyl-3,1,t~,-trimethyl-~-glucoside Large plates 43 ' t 113.5--11-1 Thick irregular masses 1:33 1;I.'< !)IJ 8 ! l l . j
-,
Pink Deep red
Ifethyl 3-azoyl-2,-l.,G-triniet~yl-D-glucosid~ ,> 1 6:: st'. 7--3:j.2 Flat needles, clusters -4 -1:; IlO--llO 5 l'lntcs
Red ortinge Orange
l i e t hyl 4-azoyl-2,3,~j-tri~nethyl-r~-glucoside 27 25 8 0 , ii--O( I Long slender iieedles -63 -- r:; !!?. S -!I;j, !i" Microscopic
Lustrous red Light pink
I.0
lletliyl ! j - ; i z o y l - 2 , 3 , i - l r i ~ ~ ~ c t h y l - D - ~ l ~ i c o ~ i ~ l e -. C'pper band ( a ) 0,2143 r3 :E) SO, 2-80, 7 Sinall plates Light pink 0.U831 12 Light pink Lower h i d ( 3 ) :~i the ntciting point of methyl 4-azoyl-2,3,6a 12reudenbcrg aiid Plaxikeiihorn, B u . , 73, 1 ~ ~ ~ - i ' , : ~ , ~ - t l ~ i l l l ~ t h y l - ~ - D - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ rri;itcthpl-li-D-gluco.iiil~aitd 122" a$ t h c iiirltiiig
Recrystallization of 01 and $ equilibrium mix- filtrate evaporated t o dryness uiider reduced pressure. I n to remove t h e last traces of methanol, toluene or tures of these azoates did riot prove t o be satis- order ligroin w i s adtied, mixed thoroughly with the residue aud iactory alone for the separation of the CY and ,3 evaporated. forins. However, after the chromatographic s e p Azoylation' and Separation.-The resulting sirup was rations the fractions thus obtained could lie jluri- t i i ~ ~ o l v e tinl ;5 nil. of dry pyridine and 3.0 g. of azoyl cliloride added. 'l'lie renction mixture was allowed to fied readily by recrystallizatim. staiitl at about 3 1 ' for tiveiity-four hours with occa~iorial In Tables 11,- and V the a- c)r 3-confiquration sliakiti:. Two niilliliters of water was added to the azoylatioii niisturc. After allout fifteen niinutes the mixwas assigned to the purified products or1 the b x i s of the specific rotation. If the rotation of the coin- t u r e bec;iiiic liotnogcneous aitrl was treated with an excess po\vtlcrcd ,sudiuni I,i. l l c t h v l d r o h t ) l y ~ at -iiiitc
f , J I I I I ( l f.0
I>('< ' l J l l l l l l C ' t ?
t i i ~ ~ t l t y lr r i o n o a z r ~ ~ l r r i i i i ~ ~ t l t ~ ~ ~ ~ -01)taitied ~ - g l i i c ~by ~~i~l~~~ iiierliod 1 \vas i , L i t - r i u l o i i t o i l -.ilici t u 'l'hc reactioii nii\turc \\a,.kclit a t tlivii ciiolctl and iiciitraliml n i t h 'i'hc, s o l i ( l iiiat~~ri:iI \ w s iiltercti iroiii t h e tiiixturv arid : l i t ,
Method I1 :"I.
ihI ,' T I I I L J,,I K S . ~ I , , 66, 1Xi.3
, I!J44).
c1~3)c-c(;;;,~,
CH.3
M C S
( 2 ) P r e s e n t addrris. fIerciilra I ' o x < i e r Cr>nipauy. \\'ilniington, Iklawnrr 1 3 ) dn P u n t I'elinm i n C h e m i s t r y . 15-12 1 ! + 4 3 ( 4 ) 'The enul furni of 9-forrnylfluorene is st;rl,le [Wislicrnua and \\.akimttIier, l j t r . , 42, 7 8 3 [ I i, LVi.liceni~. a n i i lliitrs. I ? ; I 9 (1910)] a n d , ii t h e aIdehy,ie i, r I ~ t > ~ w l
Tile first of these, %mesityl- l-phenyl-l-propeii1-01 (111), differs structurally from the known
t o this tirnc t h e r e l . ~ t i \ eteritlenc~ei of v i i i y l a l c o h o l s t u niLe h . i r c r i o L bcen nie.i.iircri iicctir:iti.l>.. V n u i i ii hizh caii be t e d , u i r l ch ir:bc.tt?rirv
