During an introductory organic program, carhohydrate chemistry is often presented poorly or not a t all, a situation which might be acceptahle if it were not repeated in higher level courses. It is the purpose of this article to point out to the students a carhohydrate system of current interest in hopes of generating some enthusiasm. In 1891 Villiers isolated a group of unusual non-reducing oligosaccbarides from Bacillus Macerans grown on a medium rich in amylose ( I ) . However, it was not until sometime later that Schardieer (2) accom~lishedthe definitive structural eluridatlon o'f these compiunds, showing them to he cyclic oliaos~ccharidescontaining from six to twelve n-1.4 linked glu>ose units (Fig. 1).
and ord have shown that the glucose residues also maintain this CI(D) conformation in solution (4).although there must certainly be more conformational mobility in solution than in the matrix. In addition it seems hvdroeen hondine also persists in solution. Even in dimethGl szfoxide, a strong competitor for intramolecular hydrogen bonding, the 3-hydroxyl proton nmr signal a t 5.2 6 is unchanged (5). I t is this intramolecular hvdroaen bondine which has been sueeested to account in p a k forihe cone &ape of cycloamylos~s~ Complexes
The most interesting characteristic of these cyclic oligosaccharides is their ability to complex avariety of guest molecules in their cavity. These guest molecules range in size from noble gases to fatty acyl coenzyme A compounds (6), and the stabilitv of the comolexes varies with the size of the euest and 2). If a substrate is too large, i t will simily not fit host into the cavity and therefore willnot bind to the cycloamylose. Conversely, if the substrate is tw small it will pass in and out of the pore with little apparent binding (7). In fact this "best fit" phenomenon is used in separating the polysaccharides from each other (7). Cvclooctaamvlose. for example. . . can be precipitated from aqueous solution with anthracene, while cvcloheptaamvlose and cvclohexaamvlose remain in solution simply because the anthracene will ;ot effectively penetrate their cavities to form the necessary insoluble complex. The fact that the guest molecule was actually contained in the cavity was shown first by X-ray (8).However, that this was the case in solution required further proof. When the guest molecule is aromatic, the host-puest disposition in solution is easy to clarify (9).Both the 3 and 5 methine protons of the glucose units are pointing inside of the cavity, and on complexnrion of an aromatic s h r a t e , these as ohrerved in their nmr spectra. are strongly shielded, indicating that they
me able
Table 1. Molecular Dimeniions of Cvcloamvloses Number of glucose
Cvcloamvlore
residues
cyclonexasmylore 6 7 Cycloheptaamylose ~ ~ ~ I o ~ ~ t a a m y l ~ l e8
Cavity dimensions (A) Diameter Deofh
4.5 -7.0 -8.5
6.7 -7.0 -7.0
Ref. ( 7 )
Figure 2. Front and side view of cyclohexaamylose
The important structural features to notice in these compounds are their toroid shape, their hydrophobic cavity.and surface, and their hvdrophilic faces (Fia. 2). ~ e c a k of e apparent lack of free rotaiion ahout the glycosidic bond which connect8 the glucose units, the cvcloamvloses are not prrfectly cylindrical m.&ecules hut are somewha; cone shaped. The 6-hydroxql face is the narrow side while the 2,3-hydroxyl faceis somewhat wider. Although X-ray studies (3) have not quantitated these differences, they have provided some information about the diameter for several of these oligosaccharides (Table 1). In addition, the X-ray analysis revealed the glucose rings to he in the Cl(D) chair conformation and that the 3-hydroxyl hydrogen is hydrogen bonded to the 2-hydroxyl oxygen of an adjacent ring. Furthermore, both nmr 204 I Journal of Chemical Education
Table 2. Dissociation Constants of Cyclohexaarnylore Complexes Acid anion guest
. ..- .. .Propionate Ikobut~rate Primate Benzoate
Ref. (71
Dissociation constant
Table 3.
Substrate Induced ShiftP (AS)for Cyclohepta-arnylora Protans 191
Benrolc acid +0.04 m - H y d r ~ ~ y b e n z o +0.04 i~ acid pHydroxybenroic +0.04 acld t0.04 Methyl p-hydroxy. Denloate +0.06 Phenol m-t-Butylphenol +0.05 o-t-~utvlohenol +0.04 ..
+0.05 +0.19 +0.09
+0.04
+0.16 +O.11
+O.O3 +0.04
c0.19
+0.04 +0.04
+0.14
+0.04
+0.21
r0.06
+0.03
+0.14
+0.03
+0.21
+0.05
+0.08
+0.09
. .. +0.03
+0.26
+0.17
+0.02
+0.03
+0.04 +0.04
+0.20 +0.21
+0.20
+0.13 +O.11
Ref. (91 a Determined from chemical shifts measured a t 100 M H z relative to Me,Si as external reference in 0 , O solution. b Accuracy t 0.02 P P ~ .
are in the magnetic field of the aromatic pi electron cloud (Table 3). In addition to nmr, fluorescence enhancement studies have also provided strongevidence for cavity penetration in solution (6). For example, when a dye which fluoresces, l-anilino-8-napthalenes"1fonate (ANSI, moves from an aqueous to a hydrophobic environment in complexing with apomyoglobin (a protein), the fluorescence of A N S isenhanced (10). It follows that if ANS moves out of water into the cycloamylose cavity, a hydrophobic environment, its fluorescence should be enhanced. Furthermore, since cyclohexamylose with an internal diameter of 4.5 .&canonlv accommodate the aniline residue of ANS, it should give a smaller enhancement than cvcloheotaamvlose which can accommodate at least oart of the naithalene portion of the molecule. These pred:lctions were verified (Fie. 3). The emission soectra eiven here are not corrected for'thi wavelength-dependent sensitivity of the photomultiplier (RCA 1P21).
F~gure3 Flwrescencs Specrmm of 10.' MANS m 0 1 Mphosphate buffer, FU 6 8 The spscnum of 10'' MANS solutlon m 0 1 Mphosphals buffer. pH 6 8, and 8 X M )-cyclodextrln. ,s (dent cel with me spectrum ol ANS at a Pdextrin concenhation of M. Ref. (70).
Thermodynamics and Klnetlcs of Complexation
Cycloamylose-induced suhstrate spectral changes of the type seen above have been used to determine a variety of thermodynamic and kinetic parameters for the complexation process. For example, by obsening changes in the ultraviolet spectra of an aromatic substrate as a function of cycloamylose concentration, it is pmihle to determine the binding constant for the complex (6),as well as the rates of formation and dissociation of the comolex. An excellent example of this treatment is found in the p nitro~henolate-cvclohexaamvlose comdex. The ultraviolet spec&m of the aromatic anion changes on addition of the polysaccharide, and two isosbestic points appear indicating a 1:l complex is formed (Fig. 4). The perturbation introduced in the ultraviolet spectrum of the aromatic anion hy formation of this complex can be represented by the term Abs defined by the following expression (11). AAbs = Abs
Figure 4. Spechum of pnitrophenol anion at varying a-cyclodextrin concentrations: solvent, phosphate buffer: pH 11.0 (I = 0.5);20'. The cyclodextrin concentrations(MI are 2.5 X lo-" lo@. and read hom A to 8. The cancentration of pnitrophenol is 5 X M.
- e A - e3S
A and S are the initial concentrations of the cycloamylose and suhstrate. and t, and t. are their resnective extinction coefficients. The eq&hriconcentration of the complex is given by C = AAbslAr where A < = r, - c -
.
and the formation constant Kf is given by Kf = CI(A - C ) ( S - C)
which transforms to C = AS/(A + S
+ llKf)
I
4
6
A
1b.10-3
& . c ~[MI Figwe 5. Determination of Me equilibrium constant of the nitrophenol-azyclDdextrin complex acwrdlng to Hildebrand and Bsmtsi. Reference (6).
if AS >> C2. Combining the two expressions for C generates the linear equation
+
+
K diss + (A S) ASlAAbs = Ac AG
'
If (A S ) is plotted against (ASIAAbs)this provides a slope of l/Ar and an intercept of KDIAcfrom which the dissociation constant can be determined simply by dividing the intercept by the slope (Fig. 5). Furthermore, by measuring the KD (dissociation constant) at a number of different temperatures, Volume 54, Number 4, April 1977 1 205
the enthalpy (AH) and thus the entropy (AS) of complexation can be determined (7) (Table 4). The cvcloam~lose-induced&awe in the ultraviolet spectra of p-nit~ophen&late has also been b e d to determine the rates at which the complex is formed and dissociated (6). This was accomplished with temperature jump kinetics. The technique involves heatinn an aqueous sample of the complex up several degrees over a period &a few mi&oseconds and obse&ng the time the system requirea to relax to equilibrium at the new temperat&e. For a 11complex the reciprocal of the relaxation time, T, the time required for the perturbed system to come back to equilibrium, is given by
where k, is the reformation rate of the perturbed system, kd, the dissociation rate, CN and C,. CD the concentrations of free p-nitrophenolate and free cyclohexaamylose, respectively. Holding the cycloamylose in excess and plotting 1/T against C, .CD gives a slope of k, and an intercept of, kd (Fig. 6) (6). Table 5 indicates some of the results of a number of these types of experiments. I t is clear that the formation and dissociation rate constants span a broad range. The driving forces for these complexations are probably hydrophobic in nature although this is still not certain (11). lnsoection of Table 4 reveals that. 'unlike classical hvdrophobic interactions characterized by a favorable entropy of association, enthalpy seems to be the major driving force. It has been suggested that the water molecules in the cavity are
unlike the bulk solvent in that they cannot have a full complement of hydrogen bonds and are thus "enthalpy rich." Expulsion then, of this "enthalpy rich" water on complexations would be the driving force for the process (7). Enzyme Active SIIe Mode* The most interesting application of the cycloamyloses has been their use as enzyme active site models. Here they participate in more than just com~lexformation; covalent bonds are-actually being broken and formed (12). The hydrolysis of substituted phenyl esters (Fig. 7) in base smoithly, the rate conitants being lin& with the appropriate Hammet sigma values. However, in the presence of cycloamyloses, the hydrolysis rates of substituted phenyl esters (13)are accelerated, Table 6, and the linear relationship between rate constanb and sigma values (7) completely disappears (Fig. 8).
.~~~~~ ~
~
Table 4. Thermodynamic Parameters for the Formation of Cyclohaxaamylor. Complexes at 25% Substrate
A P
A.@
(kcallmole)
Ikcal/mole)
AP( e . 4
4 . 2 -7.2 -1 t 1 4 . 6 t 0.7 -2.5 t 0.7
-2.8 -8.7
-3.4 -3.7 -3.1 -3.1 -3.7
-5.7 f 1.3 - 6 . 6 f 0.4
-8.6 f 3.8 --9.8 f 1.2
-3.4
-5.2
p-Nitrophenol pNltro~hsnolateion rn-chlorophenyl acetate m-Ethylphenyl acetate 3,4.5-TrimethylphenyI acetate Bsnzoylacstic add p-~ethylben~oylacatic acid m-~hlorobenloylacetl~ acid
-3.4 4 . 6
2
4.0
f 1.1
Tabla 5.
No,
2.6
R-N-N
0-
R-N-N
0-
b All values refer to 1 4 ' ~ .
* 3 f 3 f 3
* 3.3
Rates of Inclusion of
K,.Mb
Substrat&
HO
8
-3
x
lo-'
Guem into Cycloamylols Cavity kR,M-' s-' 24
x lo'
kD,< I b 210'
so1v.nt (phosphate buffer)
I = 0.5. pH 3.5
Cydoamy~osesa s Catalysts
I
I
1
I
I
l
l
1
I
Table l
l
6. M a x i m a l R a t e Constants and Dissociation Constants of Cycloamylore-Phenyl Acetate Complexes
I
Ref. (13)
Table 7.
T h e Relationship between Metalpara Specificity and t h e Size of t h e Cvcloamvlosa Cavitv
Acetate
Cvclohexaamylase
kobs/kun
Cycloheptsamvlose
Cycloocraarnylosa
Hommett Sigma F i p 8. W p h of the iq)arimm of Uw acceItnatlon of the rateof phenol mleese due 10 0.01 M~ycloamyloseagainst the Hammen substiherd constant, c:(0). cyclosmytose: (0).cycloheptaamylose (VanEnen et al.. 1967a)Reference
(7).
Ref. (7)
droxvl mouos. a more favorable ~ositionfor reaction. thus incr;as;ng k; i ~ i9).~ . This is a so-called "steric steerine" rate enhancement which holds for any set of para and meta s;bstituted esters. However, the metahara rate ratios decrease with the lareer cvcloamvloses (11)&ply because there is less steric steering p o s s i ~ e (Table 7). Acetate is more slowly generated than phenolate (3).This fact, at first puzzling, ran he understood on the basis that the generated must now be acyl cy~loam~loseintermediate cleaved by hydroxide, a hydrolysis which would not be expected to at an un"sual rate. In fact, the cycloarnyluse acyl intermediates have been isolated in several cases (14). Summary The cycloamyloses provide an example of how several spectroscopic techniques combined can precisely define the structure of macroscopic complexes in both a matrix and in solution. In addition, they provide an example of how soectroscopic changes occ"rringduring complex~tioncan be ised to measure both the thermodynamic and kinetic oarameters for such processes. Finally, the cycloamyloses serve as excellent models for enzyme active site studies exhibiting substantial hydrolytic catalysis, substrate specificity, and competitive inhibitions. Clearly then, they represent carbohydrate systems which can be exciting and valuable to study. Literature Cited
-
Figure 9. A comparison of how c d b and metannrophenyl seemtea sii In h e cycl~amylosescavity.
The only observable trend remaining is that meta substituted phenyl esters are hydrolyzed significantly faster than their ortho or para analogs. Furthermore, the meta to para rate ratio decreases in going from cyclohexa to cycloheptaarnylwe, and in all cases the phenolate anion appears in solution much faster than acetate anion. Finally, the rate enhancements were markedly decreased in the presence of guests which were known to bind in the cavity. A mechanistic picture which is consistent with all of these observations is indicated below (7)
where S = substrate, c = cycloamylose, Se = inclusion complex, P = product, and un = uncatalyzed. Clearly anything which binds with the cycloamylose, thereby lowering the concentration of the Sc complex, decreases the overall rate; thus the competitive inhibition phenomenon is understandable. Once the complex has formed, the hydroxyl groups of the host are in a position to react with the carbonyl of the ester, thus releasing the phenolate into solution. When the phenyl ester is meta, this suhstituent forces the acyl group closer to one side of the cycloamylose and therefore closer to the hy-
I41 Beychuk, s., and Ksbat, E. A,,Rimhemixrry. 4,2565 11%Sl. IS) Caru,B..Reggisni. M., Tslrahsdnm. 24.803 (1968). lfil Cramer,F..Sacnycr. W.,andSpatz, H., J. Amer. Chem. Soc., 89.1,(1%71. 171 Criffltha,D. W..sndBencher,Mynm L.."Advanceain CaMlysis,"Vul.23 E1ey.D. 0.. lEdilwx Pine. Herman. and Weis. Paul 8.1, Academic Press. New Ycrk. 1973. 181 Hyhl. A,, Rundle. R. E..and Williams, D. E., J. Amsr Cksm. Sm., 87,2779 (196SI. 19) Demare0.P. V..andThskkar,A. L.. C k e m Commun., 2 (19701. I101 Brand,L..and Withult,B., Msthadr Eruymol., 11,776 (19671. (111 Jencks. W. P., "Catalysiri inChemilvand Enzymolm1.I Mffiraw-HiIlBa* Company.
."....
NOW V,wb
(121 Cramer,F..sndOismhe, W.,Chem. Rcr.. 92.37811959l. (13) Van Etten.R. L..Sehastian,d. F.. Clowes. G.A..and 6ender.H. L.. J. Amer Chrm. Soe.. 89.:3242 l19fi71. 1141 Van Etten.R. L.. Cbms,G.A.. Sebaation. J. F..andBender.H. L..J Amer. Chrm. Soc. 89,3253 (19671.
Volume 54. Number 4. April 1977 1 207
