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Concanavalin A Binds Pyranosides and Their Tetraacetates Stereoselectively.

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Table I . Measured fluorescence lifetimes k - l and electron transfer rates k,,
(room temperature).
Cpd.
k-'
k,, K'l
Zn-1 a
Zn-1 b
2a
2b
Zn-2a
Zn-2 b
Zn-2c
3
Zn-3
Zn-6
253 ps
1.7 ns
500 ps
7.3 ns
275 ps
1.8 ns
1.8 ns
107 ps
25 ps
1.8 ns
3.4x 109
-
1.9 x 10'
-
3.0
109
F = ( 4 7 ~ / . k , n - " ~exp
-
9 2 to9
40.0 x 10'
-
different conformations (dihedral angle) of comparable enand 3.9 x
eV. A value of
ergy vary between 0.2 x
V,,(CS) = 2 . 4 ~
eV is obtained for the conformation
with a dihedral angle of 70" (see above). With this value and
the parameters AGO and ).[l7] one obtains for T = 293 K
according to Equations (1) and (2)["] k,,(CS) = 5.7 x l o l o
and 9.9 x 10" s-' for 2. = 0.62 and 0.9 eV, respectively, in
satisfactory agreement with the experimentally determined
ET rate constant of 4 x 10" s - ' (Table 1).
Summary: For an efficient charge separation in quinones
coupled covalently with porphyrin, the MO coefficient in the
HOMO of the porphyrin at the site of coupling should be as
small as possible and the orientation of the quinone to the
cyclohexane bridge as orthogonal as possible.
Received: January 19, 1990 [Z 3748 IE]
German version. Angew. Chem. 102 (1990) 690.
[l] See, for example, S. Boxer, Biochim. Biophys. Acta 726 (1983) 265; T. A.
Moore, D. Gust, P. Mathis, J.-C. Mialocq, C. Chachaty. R. V. Bensasson,
E. J. Land, D. Doizi, P. A. Liddell, W. R. Lehmann, G A. Nemeth, A. L.
Moore, Nuture (London) 307(1984)630: A. R. Joran, B. A. Leland, G. G.
Geller, J. J. Hopfield, P. B. Dervan. J. Am. Chrm. Soc. 106 (1984) 6090;
J. L. Sessler, M. R. Johnson. Angew. Chem. 99 (1987) 679; Angew Chem.
fnt. Ed. Engl. 26 (1987) 678; M. R. Wasielewski in M . A. Fox, M. Chanon
(Eds.): Photoinduced Electron Transfer, Purr A , Elsevier, Amsterdam 1988,
p. 161; D. Gust, T. A. Moore, Science (Washington D.C.) 244 (1989) 35.
121 G. A. Haggis, L. N Owen, J. Chem. Soc. 1953,404.
[3] N. Kornblum, W. J. Jones, G. J. Anderson, J. Am. Chem. Soc. X I (1959)
4113.
[4] B. Belleau, G. Malek, J Am. Chem. Soc. 90 (1968) 1651
[5] R. Kuhn, I. Hammer, Chem. Ber. 83(1950) 413.
161 N. Jacobsen, K. Torssell, Justus Liehigs Ann. Chem 763 (1972) 135
[7] L. I. Zakharkin, L. P. Sorokina, J. Gen. Chem. U S S R Engl. Pans/. 37
(1967) 525; Zh. Obshch. Khim. 37 (1967) 561.
[a] ENDOR method, see K. Mobius, M. Plato, W. Lubitz. Phys. Rep. 8 7
(1982) 171; H. Kurreck, B. Kirste, W. Lubitz, Angew. Chem. 96 (1984) 171;
Angew. Chem. Int. Ed. Engl. 23 (1984) 173; Electron Nuclear Douhlc Resonance Spectroscopy of Radiculs in Solution, VCH. Weinheim 1988.
[9] H. J. Shine, A. G. Padilla, S.-M. Wu. J. Org. Chem. 44 (1979) 4069.
[lo] C . Heller, H. M. McConnell, J. Chem. Ph-vs. 32 (1960) 1535.
[ll] M . Plato. E.TrGnkle, W Lubitz, F. Lendzian, K . Mobius, Chem. P/iy.$. 107
(1986) 185, M. Plato. W. Lubitz, F. Lendzian, K. Mobius. f s r . J. C i i ~ m2.X
(1988) 109.
[12] In order to avoid complications in the INDO parametrization, Mg was
used instead of Zn in the M O calculations [13].
[I 31 M. Huber. Dissertation, Freie Universitit Berlin 1989.
[14] R. A. Marcus, N. Sutin, Biochim. Biophys. Acta 8 I t (1985) 265.
[15] See. for example, P. Siders, R. J. Cave, R. A. Marcus. J. Chem. Ph.w 81
(1984) 5613: H. Heitele, M. E. Michel-Beyerle, J. Am. Chrm. Sot. 107
(1985) 8068; B. A. Leland. A. R. Joran, P M. Felker, J. J. Hopfield. A. H.
Zewail, P. B. Dervan, J Phys. Chem. 89 (1985) 5571; G. L. Closs, L. T
Calcaterra, N. J. Green, K . W. Penfield. J. R. Miller, ihid. 90 (1986) 3673;
L. Otha, G. L. Closs, K. Morokuma, N. J. Green. J. Am. Chem. Soc. 108
(1986) 1319; M. R. Wasielewski, M P. Niemczyk in M. Gouteman. P. M.
Rentzepis, K. D. Straub (Eds.): Porphyrins-Exerted States and Dynumre.?,Am. Chem. Soc., Washington, D.C., 1986, p. 154; R. J. Cave, P. Siders,
R. A. Marcus, J. Phys. Chem. YO (1986) 1436: H. Heitele, M. E. MichelBeyerle, P. Finckh, Chem. Phrs. Lett. 134 (1987) 273; A. M. Oliver, D C .
Craig, M. N. Paddon-Row. J. Kroon, J. W. Verhoeven, ihrd. IS0 (1988) 366.
672
VCH Verlugsgesellschaft mbH, 0-6940 Wemheim, 1990
[16] In the case of 3. VDnwas calculated by the MO method RHF-INDO [ l l ]
in analogy lo a concept with which VD,, was recently determined for the
primary ET step in bacterial photosynthesis from the atomic coordinates
of the participating pigments [cf. H. Michel, 0. Epp. J. Deisenhofer, EMBO J. 5 (1986) 24451: M. Plato. K. Mobius, M. E. Michel-Beyerle, M.
Bixon, J. Jortner, J. Am. Chem. Soc. 110 (1988) 7279. Unlike the pigments
of the photosynthesis, D and A in Zn-3 are linked covalently via the
cyclohexanediyl bridge. The influence of the bridge on the electronic structure of D and A was taken into account in the M O calculations.
[17] According to Murcus et al. [14] Equation (2) holds true,
I-(i+ AC0)*/(41-kn7)]
(2)
where i.
i s the total reorganization energy, AGO the free standard reaction
enthalpy, k , the Boltzmann constant, and T the absolute temperature. AGO
values for the charge separation and charge recombination were estimated
from the cyclovoltammetrically determined redox potentials of 211-6 and
5 and the energy of the first singlet excitation state Eoo of zinc tetraphenylporphyrin [E,,, = 2.1 eV, cf. M R. Wdsielewski, M. P. Niemczyk, J Am.
Cheni. Soc. 106 (1984) 50431 to be AGo(CS) = - 0.84, AGo(CR) =
- 1.26 eV. In the estimation of;. = 1, + i.,
[ I ,is the internal reorganization
energy arising from changes in geometry of the reacting porphyrins (P) and
quinones (Q) and j.(,is the external reorganization energy by changes in the
surrounding medium], 2, was neglected, since the changes in geometry
between the IP*Q and P.QQ states caused by photoinduced charge separation can be regarded as small [13,23]; cf. also M. R. Wasielewski, M. P.
Niemczyk, W. A. Svec, E.B. Pewitt, J. Am. Chem. Soc. 1/17. (1985) 1080.
2" was estimated according to the method of Marcus [R. A. Marcus. J.
Chem. Phys. 24 (1956) 9661 using geometric data ( r p = 6, rp = 4, rp.o =
10.6 A) and dielectric data of the solvent CH,CI, (no = 1.42. E = 9.08) to
be i, = 0.62eV [13]. With these AG" and 1. values, Equation (2) gives
F(CS)/F(CR) = 320. This value is regarded as the upper limit, since the
decrease in Fin the inverted region (- AGO > L) is, according to investigations on comparable systems [cf. J. Ulstrup, J. Jortner, J. Chem. Phys. 63
(1975) 43581 significantly smaller than is predicted according to Equation
(2). For comparison with the value of 1 calculated here. an experimentally
= 0.9 eV can be resorted to which was obtained by
determined value of i.
M. R. Wasielewski et al. (see above) from measurements of the transient
absorption for porphyrin-quinones with comparably large center-tocenter separation i-p.o as in the case of Zn-3. With this value of i.,
F(CS)/F(CR) = 4.
[18] T. Murao. 1. Ydmazaki, K. Yoshihara, Appl. Opt. 21 (1982) 2297.
[19] R. Eichberger. F. Willig, W. Storck, Mol. Cryst. Liq. Crysr. 17s (1989) 19.
[20] Excitation was repetitive with laser pulses (6 ps halfwidth, < 10" photons
per pulse) at 590 nm and a repetition frequency of 800 kHz upon time-resolved photon counting and 4 MHz upon measurement with the synchroscan camera. The first method of measurement with 40 ps apparent
laser pulse width was used if the time constants of the decay curves were
greater than 150 ps. If the smallest time constant of the decay curves was
less than 150 ps then the second method of measurement was used with
15-25 ps apparent laser pulse width, depending on the integration time.
[21] J. A Schmidt. A. R. McIntosh,A. C. Weedon. J. R. Bolton. J. S. Connolly,
J. K. Hurley, M. R. Wasielewski. J. Am. Chem. Soc. 110 (1988) 1733.
[22] P. G. Seybold, M. Goutermann, J. Mol. Spectrosc. 31 (1969) 1.
[23] M. D. Archer. V. P. Y. Gadzekpo, J. R. Bolton, J. A. Schmidt, A. Weedon,
J. Chem Soc. Farudq Trans. 2 82 (1986) 2305; J. A. Schmidt. A. Siemiarczuk, A. C. Weedon, J. R. Bolton. J. Am. Chem. Soc. 107(1985)6112; J. A.
Schmidt, J. Y. Lin, 1. R. Bolton, M. D. Archer, V. P. Y Gadzekpo, 1.Chem
Soc. Faraday Truns. t 85 (1989) 1027.
'
Concanavalin A Binds Pyranosides
and Their Tetraacetates Stereoselectively**
By Jiirgen-Hinrich Fuhrhop * and Michael Arlt
The protein concanavalin A (= Con A) stereoselectively
binds carbohydrate head groups that appear as neighboring
hexose units on the surfaces of glycoproteins [I1 or vesicles.[']
[*I Prof. Dr. JLH. Fuhrhop and DipLChem. M. Arlt
Institut fur Organische Chemie der Freien Universitdt
TdkUStrdSSe 3, D-1000 Berlin 3 3
['*I This work was supported by the Deutsche Forschungsgemeinschaft (SFB
312 "Vectorial Membrane Processes"), the Forderungskommission fur
Forschung und wissenschaftlichen Nachwuchs der Freien Universitdt, and
the Fonds der chemischen Industrie.
0570-0833i9010606-0672$03.50 f .2S/0
Angerv. Chem. In[. Ed. Engl. 29 (1990) No. 6
D-Glucopyranose and D-mannopyranose units are agglutinated, whereas D-galactopyranose units are not. Clefts,[31
shallow pockets,[41as well as non~pecific‘~]
or substratedependentu6Ibinding sites have been characterized by X-ray
crystal structure analyses of monosaccharide complexes.
None of the resulting binding models agree with the experimental results described in this paper.
Agglutination experiments with
M aqueous suspensions of the glycolipids 1 a-ld”] and 1 mg of Con A per
mL[*l yielded expected results: D-glUCOSe and D-mannose
la
le
meric spheres in the presence of Con A. At tenfold higher
concentrations of Con A all stereoisomeric vesicles are precipita ted .
None of the binding models of Con A given in the literature accomodate four or eight acetyl groups (assuming the
binding of two carbohydrate head groups) in place of protons. We therefore propose a binding site without a “cleft” or
“pocket” but consisting of planar or slightly convex p-pleated sheets, which constitute 60% of the Con A surface. Such
a binding site would offer a much better explanation for the
Id
lh
lc
lb
If
kl
a - d , R=H
e- h . R=Ac
were bound, D-galactose and L-glucose were not. Con A
therefore differentiates not only between diastereomers but
also between enantiomers. Furthermore, we found that on
sonication the tetraacetates 1e - 1h formed spherical aggregates similar to those obtained from lipids 1 a- 1 d. Electron
micrographs of negatively stained specimens (pH 7; phosphotungstate) showed vesicles (diameter approx. 400 f
100 A) and were identical for both the glucose derivatives 1 a
and 1 e. Light scattering measurements after gel chromatography (Sephadex G100) gave identical results in the dead
volume for 1 a and its tetraacetate 1e. Entrapment experiments with the water-soluble fluorescence dye pyranine,
however, were only successful with la. In the case of the
tetraacetate aggregates, the dye dispersed equally between
the dead volume and the fractions that followed. We assume
that the mechanical stability of the “hydrophobic” tetraacetate membrane does not suffice for remaining impermeable
during gel chromatography. Surface monolayers of la on
water were also more stable (collapse pressure: n, = 50 f
5 mN m-’; molecular area: 39 3 A’) than in the case of
the tetraacetate l e (34 f 3 mN m-’; 54 3 A’ per molecule).
Agglutination experiments with Con A showed that the
vesicles made of the tetraacetates 1e - 1h ( 2 99.8 Y purity)
were precipitated with the same binding constants and velocities as the free carbohydrates la-ld (Fig. 1). This means
that four acetate groups on the vesicle surfaces are bound
stereoselectively by Con A in the same way as four hydroxy
groups. Minute traces of non-acetylated carbohydrate head
groups ( < 0.1 YO)cannot possibly cause the observed precipitation, since a tenfold diluted vesicular solution shows no
precipitate whatsoever under the given reaction conditions.
CICLLIUII
rrllcrogIaprl>0 1 L W I 1 A-aggluLlniiLeu V e S l C l e S >now a
flattening- of the membranes at the contact areas. We therefore
that the
A-covered membranes make direct contact, without a protein link between VesicIe surfaces.
Micrographs of unbound vesicles show exclusively monoA n p w . Chem. In1 Ed. E n d . 29 11990) No. 6
0 VCH
observed stereoselective binding of smooth vesicle surfaces,
since there is no obvious driving force for drawing a carbohydrate pair out of the vesicle surface and into a cleft on the
Con A surface. Furthermore, the low solubility of hydrophobic oligomethylene chains in the region of hydrophilic
head groups would strongly oppose such movement, and in
polymerized vesicles, which are also stereoselectively recognized by Con A,[’] such movement would be almost impossible.
D-
glucose (OAcl
0.L
0.3
t
A 0.2
0.1
0
U
f lminl
-
L”
Fig. 1. Time-dependent absorption A at I = 360 nm for the four diastereomeric and enantiomer,c glycolipids 1 a- 1 d (“OH-) and their tetraacetates ~ . o A ~ - )
in aqueous suspensions after addition of
M Con A.
Vc.rlugs~esell.~chaft
mbH. 0-6940 Weinheim,1990
0570-Ocl3319Oj0606-0673$03.50+ .25!0
673
We therefore propose an essentially planar binding site,
whose stereospecifity is presumably caused by the arrangement of six dipoles.
The binding energy between Con A and a glycoprotein is
known to be 23 kJ mol-'.['lThis energy can be attributed to
the dipole-dipole interactions of these six dipole pairs at an
average distance of 0.45 nm.[lolSuch a distance leaves ample
room for the proposed acetyl groups (Fig. 2). There is even
Synergic Destabilization
by Geminal Ester Groups**
By Sergej Verevkin, Barbara Dogan, Hans-Dieter Beckhaus
and Christoph Riichardt *
Dedicated to Professor Roy Huisgen on the occasion
of his 70th birthday
Stabilization by synergic interaction of geminal donor
substituents on a saturated carbon atom, e.g. alkoxy groups
in acetals, and its consequences, have long been known qualitatively as the "anomeric effect"."' This anomeric stabilization and its dependence on structure was recently determined
quantitatively by us.['] In contrast, geminal nitro groups on
Results of
a saturated C atom lead to de~tabilization.[~"'
ab-initio calculations and other theoretical ~ t u d i e s , f ~ ~ - "
point to analogous effects in the case of other acceptor
groups. We have now examined the synthetically important
malonates 1a-c and the methanetricarboxylates 2 in order
to establish whether geminal ester groups are also synergically destabilizing.
Despite the universal use of malonates, to our knowledge
no measurements of their standard enthalpies of formation
have thus far been reported.[4] In the case of 1a-c, 2, and the
reference compounds ethyl 2-methylbutyrate 3 and ethyl pi-
0 0
o-Glu
Fig. 2. Model of the stereoselective binding of o-glucose with and without
acetyl groups at a smooth surface of Con A with a planar binding site distribution (hatched surface is tilted through 90").
H,
enough space for an additional hydration sphere between
both surfaces, which would explain the observed similarities
of binding constants and rates. The carbonyl dipoles of the
acetyl groups obviously do not play an important role in the
binding process. Formal attempts to bind the wrong enantiomers or diastereomers in the model yield at least two distances which are 20 % larger. This is in agreement with the
observed decrease of binding energies and equilibrium constants.
Received: November 24, 1989 [Z 3650 IE]
German version: Angew. Ckem. 102 (1990) 699
CAS Registry numbers:
l a , 101009-69-2: 1 b, 127062-38-8; Ic, 127062-39-9; I d , 127062-40-2; l e ,
101009-70-5; 1 f, 126979-69-9; 1 g, 126979-70-2; l b , 126979-71-3; Con A,
11028-71-0.
[l] L. Bhattacharyya, C. F. Brewer, Biochem. Biopkys. Res. Commun. 137
(1986) 670-674.
[2] H. Bader, K Dorn, B. Hupfer, H. Ringsdorf. Adv. Polym. Sci.64 (1985)
I.
[3] J. P. Carver. A. E. Mackenzie, K. D. Hardman, Btopolymers 24 (1985)
49-63.
[4] J. W. Becker, G. N . Reeke, B. A. Cunningham, G. M. Edelman, Nature
(London) 259 (1976) 406-409.
[5] I. J. Goldstein, C. E. Hayers, Adv. Carbohydr. Ckem. 35 (1978) 128-316.
[6] 2. Derewenda, J. Yariv, J. R Helliwell, A. J. Kalb, E. J. Dodson, M. 2.
Papiz, T. Wan, J. Campbell, EMBO J. 8 (1989) 2189-2193.
[7] J. H. Fuhrhop, H. H. David, J. Mathieu, U. Liman, H. J. Wmter, E.
Boekema, J. Am. Chem. SOC.108 (1986) 1785-1791.
[S] Con A, Grade IV, Sigma, salt free; exact conditions are given in R. D.
Brown 111. C. F. Brewer. S. H. Koenig, Biochemistry 16(1977) 3883-3896.
1 mg of Con A per mL gave total stereoselectivity; with 25 mg Con A per
mL nonselective precipitations of all vesicles were observed.
[9] T. G. I. Ling, B. Mattiason in T. C. Bsg-Hansen, E. van Driessche (Eds):
Leftins. Voi. 2, de Gruyter. Berlin 1982, p. 563-571.
[lo] Derived from the point dipole model:
674
0 VCH
Verlagsgeselisckafr mbH, 0-6940 Wetnhezm, 1990
,CO,Et
H,
EtO,C/'\CO,Et
,CO,Et
Me/"Et
Me,
3
2
,CO,Et
Me /'\Me
4
valate 4 they were now obtained from their combustion
enthalpies AH:( 1) and vaporization enthalpies
(Table 1.).
Table 1 . Combustion enthalpies AH,O(l), vaporization enthalpies AH", and
standard enthalpies of formation AH: of'la-c and 2-4 [kcal mol-l] [a].
~
l a - 552.38 f 0.15
l b - 708.38 f 0.13
I C - 863.08 ? 0.10
2 -1187.73 f 0.20
3 -1002.62
0.33
4
- 1001.08 f 0.25
14.78 f 0.19
13.81 i 0.17
13.31 k 0.19
18.93k 0.17
10.59 f 0.07
9.86 0.03 [4]
-191.13 t 0.15
-197.50 f 0.13
-205.17 f 0.10
-299.30 f 0.20
-133.94 f 0.33
- 135.48 ? 0.25
-176.35 f 0.24
-183-69 i 0.21
-191.86 f 0.19
-280.37 k 0.26
-123.35 & 0.34[b]
- 125.62 0.26 [c]
[a] Standard deviation of the mean value from 5-8 measurements in each case.
[b] Literature value [4]: - 124 86 f 2.02. [c] Literature value [4]: - 128.10 f
2.00.
For the determination of the extra stabilization or destabilization the method based on group increments, as developed
for anomerically stabilized acetalsJZ1was used, and not that
['I
Prof. Dr. C. Ruchardt, Dr. B. Dogan, Dr. H.-D. Beckhaus
Institut fur Organische Chemie und Biochemie der Universitat
Albertstrasse 21, D-7800 Freiburg (FRG)
Dr. S. Verevkin
Kuibyshew Politechnisches Institut, Kuibyshew (UdSSR)
[**I Geminal Snbstitution Effects, Part 3. This work was supported by the
Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, S . V thanks Prof. Alexandr M . Roznov (Kuibyschew) for special support and the DAAD (Deutscher Akademischer Austauschdienst) for an
exchange grant.-Part 2: [2].
0510-0833190j0606-0674 $03 50+.25/0
Angew Chem. Int Ed Engl 29 (1990) No. 6
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