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Chemoenzymatic Synthesis of Sialyl Lewisx Glycopeptides.

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were obtained from the resulting solution after i t had been left to stand at room
temperature for two weeks Elemental analysis: Calc. (found). Mn 25.2 (25.4). N
51.4 (51.3). C 22.0 (22.1). H 1 4 (1.4)%.
Received: March 15. 1996 [Z89291E]
German version- A n g e i ~ .Chmi. 1996, 108. 1934-1936
-
-
Keywords: azides complexes with nitrogen hgands layer compounds magnetic properties . manganese compounds
-
[l] H. J. M. de Groot, L. J. de Jongh. R. D. Willet, J. Reedijk. J Appl. P h w 1982,
53,8038; C. Benelli, D. Gatteschi. D. W. Carnegie, R. L. Carlin. J. A m . Chrm.
Soc. 1985. 107, 2560; I. Vasilevesky, N. R. Rose, R. Stenkamp, R D. Willet,
Inorg. Chem. 1991, 30, 4082; G. de Munno, M. Julve, F. LLoret. J. Faus. M.
Verdaguer, A. Caneschi, Angeiv Chum. 1993, 10s. 1122: Angrit.. Chern. Inr. Ed.
Engl. 1993.32, 1046.
121 R. Cortes. L. Lezama. J. L. Pizarro. M. I. Arriortua. X. So1ans.T. Rojo. Angriv.
C h n . 1994. 106, 2520; Angeir. Chem. Inr. Ed. Engl. 1994. 33, 2488.
[3] J. S. Miller. A. J. Epstein, Anger. Chem. 1994, 106. 399; Angen.. Chem. Inr. Ed.
Engl. 1994.33. 385; W. R. Entley, G. S. Girolami, Inorg. CIient. 1994,33, 5165.
[4] D. M. Duggan, D. N. Hendrickson, /not-g. Chem. 1973, 12.2422; J. Commarmond. P. Plumere, J. M. Lehn, Y. Agnus, R. Louis, R. Weiss. 0. Kahn. I.
Morgenstern-Badaraci, J A m . Chrm. Sor. 1982. 104. 6330; P. Chaudhuri. M.
Guttmann. D. Ventur, K . Wieghardt. B. Nuber. J. Weiss. J Chcm. SOL..C h m
Commun. 1985, 1618; T. Rojo, L. Lezama, R Cortes, J. L. Mesa, M. 1. Arriortua, G Villeneuve, J. Magn. Magn. Mur. 1990, 83, 519, R. Cortes. J. I. R. de
Larramendi. L. Lezama, T. Rojo, M. K. Urtiaga, M. I. Arriortua,J. Chem. Soc.
Dalron Trans. 1992. 2723; R. Cortes, M. K. Urtiaga, L. Lezama. J. I . R. de
Larramendi. M. 1. Arriortua, T. ROJO,[hid., 1993, 3685, and references therein.
[5] R. Vicente. A. Escuer, J. Ribas, M. S. El Fallah, X. Solans, M. Font-Bardia.
Iiiorg. Chem 1993, 32, 1920: J. Ribas, M. Monfort, C. Diaz, C. Bastos, X.
Solans. ihid. 1994. 33, 484.
[6] J. Ribas. M. Monfort, C. Diaz. C. Bastos, X. Solans. Inorg. Cl7em. 1993, 32.
3557.
(71 J. Ribas. M. Monfort. R. Costa, X. Solans, Inorg. Chem. 1993. 32, 695.
IS] M. A. Halcrow, J. C. Huffman, G. Christou, Angeir. Cltem. 1995, 107, 971 :
Angew. Chem. I n r . Ed. Engl. 1995, 34, 889.
191 C. G. Pierpont, D. N. Hendrickson, D. M. Duggan, F. Wagner, E. K. Barefield, Inorg. Chem. 1975, f4, 604: A Escuer. R. Vicente. J. Ribas. M. S. El
Fallah. X. Solans, M. Font-Bardia. i b d 1993. 32, 3727; R. Cortes. M. K.
Urtidgd, L. Lezdrna, J. L. Pizarro, A. Go%, M. I. Arriortua. T. ROJO,ihid. 1994,
33, 4009.
[lo] J. Ribas, M. Monfort. X. Solans, M. Drillon. Inorg. Chem. 1994. 33, 742;
M. A. S. Goher, F. A. Mautner. Crour. Chein. Acra 1990. 63, 559.
[ l l ] F. A. Mautner. R. Cortes. L. Lezama, T. Rojo, A n p i r . Chem. 1996. IO8, 96,
Angel!. Chem. Int. Ed. Engl. 1996. 35. 78.
[12] L. K. Thompson, S. S. Tandon, M. E. Manuel, Inorg. Chem. 1995, 34. 2356.
[13] R. Cortes. J. L. Pizarro, L. Lezama, M. I. Arriortua, T. Rojo. Inorg. Chem.
1994,33,2697.
(141 G de Munno, M. Julve, F. Nicolo. F.Lloret, J. Faus, R. Ruiz, E. Sinn, Angew.
Chem. 1993.10s. 5 8 8 ; Angeiv. Chem. Inr. Ed. Engl. 1993.32,613; G . de Munno.
R. Ruiz, F. Lloret, J. Faus, R. Sessoli, M Julve. Inorg. Chem. 1995. 34. 408.
[l 51 X-ray structure analysis: Enraf-Nonius CAD-4 diffractometer. Unit cell
parameters were determined from automatic centering of 25 reflections
(12 < 28< 30") and refined by least-squares method. Intensities were collected
with graphite monochromatized Mo,, (0.71069 A) radiation using the o scan
technique. Lorentz polarization and extinction corrections were made. The
structure was solved by direct methods and successive Fourier difference syntheses, using the SHELXL-93 computing program. C,H,N,,Mn, ( M = 436).
monoclinic system. space group P2Ju. a =6.229(1). b =15.104(1).
=
8.923(1) A, /i = 95 72(2)", V = 835.3(2)A3, 2 = 4, pirlcd= 1.734 g ~ m - ii~ =
.
15.4cm-', F(OO0) = 432. The refinements by full matrix least-squares gave
final Rl(F,) = 0 028, n.R2(F:) = 0.1356 and S = 0.78 1161 from 2432 reflections with intensity /22u(1) for 137 variables. All non-hydrogen atoms were
refined anisotropically. The extreme nitrogen atom (N8) of one of the azido
bridges is disordered. A strong pseudo-C symmetry is observed in the compound. Crystallographic data (excluding structure factors) for the structure(s)
reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC-179-81. Copies of the
data can be obtained free ofcharge on application to The Director. CCDC, 12
Union Road, Cambridge CB2 IEZ. UK (fax: Int. code +(1223) 336-033;
e-mail' teched(u chemcrys.cam.ac.uk)
[16] Almost simultaneously to our paper,another papercontaining thesamecrystal
structure was submitted by another research group (see preceeding communication). G. De Munno, M Julve, G. Viau, F. Llore, J. Faus, D Viterbo, Angeir.
Chem. 1996, IOH, 1931; Angeiv. Chem. 1111 Ed. Engl. 1996. 35, 1807.
I171 M. E.Fisher, Am. J. Phys. 1964. 32, 343.
[18] J. H. Van Vleck, Electrical and Magneric Susc~pr~hiliries.
Oxford University
Press, 1965.
1812
$2
VCH VErlugsgesrllscl~ufrmhH, D-69451 Weinherm, 1996
Chemoenzymatic Synthesis of Sialyl LewisX
Glycopeptides""
Gabi Baisch and Reinhold Ohrlein*
Invasive leukocytes play a decisive role in acute and chronic
inflammation.['] They reach the site of inflammation through
a multistep process initiated by "leukocyte rolling".[2, 31 This
"rolling" is caused by the interaction of carbohydrate ligands on
the surface of leukocytes and receptors, the E- and P-selectins,
which are found on the surface of activated endothelial cells.[41
A disruption of this early interaction is considered to be a
promising therapeutic approach.[51The tetrasaccharide sialyl
Lewisx (SLe") and various derivatives have been identified as
ligands for selectins.[61 Because monovalent SLe" shows only
HO
AcHN
OH
OH
NHAc
P
HO
O
H
siaa(2-3)galp(l4)(fuca(1-3))glcNAc = SLex
relatively weak binding to E-selectin (IC5,, = 0.75 mM),17] it is
assumed that the E-selectin- SLe" interaction is enhanced in
vivo through "clustering" (multivalency) by either E-selectin or
SLe"'*] o r both. In some early investigations up to 18 preassembled SLe" moieties were attached with a spacer to bovine serum
albumin in order to evaluate this
The positive
results of assays obtained with these artificial glycoproteins
stimulated the search for methods to synthesize well-defined
oligovalent SLe" conjugates. Thus three chemically synthesized
SLe" moieties were linked to nitromethane tricarbonic acids"
and to cyclopeptide templates.[' 'I More rigid, divalent SLe"
conjugates were obtained chemoenzymatically with trisaccharide templates.[' 21
U p to now there have been no systematic studies concerning
the number of SLe" units necessary and their three-dimensional
arrangements. The following strategy for the synthesis of oligovalent SLexconjugates enables rapid screening of these parameters. We selected oligopeptide chains as the framework on which
to attach the carbohydrate moieties. A proper choice of the
monomeric building blocks renders the backbone more rigid or
more flexible, thus markedly influencing the presentation of the
SLe" moieties. Additionally, the peptide linker can be optimized
by the well-established peptide chemistry.[13. 14]
Complex glycopeptide clusters containing natural hexoses are
most efficiently prepared by enzyme-mediated synthesis. With
the help of glycosyl transferases, glycosylations can be performed with predictable, excellent stereo- and regioselectivity," 51 thereby circumventing the highly demanding chemical
synthesis of the SLe" tetrasaccharide!'
The inefficient and
cumbersome protecting group manipulations inevitable in the
[*] Dr. R. Ohrlein, G. Bdisch
Central Research LdbOrdtOrieS
Schwarzwaldallee 21 1. CH-4002 Basel (Switzerland)
Telefax: Int. code +(61)697-8975
[**I The authors thank Dr. A. Katopodis and Dr. M. Streiff for the biotechnological preparation of n(2-3)sialyl transferase and fucosyl transferase VI.
oS7o-0833~96~3S16-fXlZ
$ I S 00+ ,2510
Angew. Chem. Inr Ed. Engl. 1996, 35, No. 16
COMMUNlCATlONS
H NCH
HN-COOtBu
( 2 ) n b
chemical glycopeptide synthesis are avoided.“ ’I The enzymes
function in aqueous buffer systems without poisonous metal salt
promotors.
The scope of the enzymatic methodology is presently limited
by the availability of transferases[18]and the necessary activated
donor sugars. These have to be synthesized,[”] although they
are commercially available on a small scale. Alternatively some
donor sugars may be produced in situ.12*’
Our synthesis started with the N-acetyl glucosamine building
blocks 2 and 3 (Scheme 1 ) . Deprotection of the amino group of
the glycoside
with morpholine[221provided the amine 2,
which was subsequently N-acetylated, treated with trifluoroacetic acid to remove the tevt-butyl ester, and finally deacetylated to yield the building block 3.
5
~HAC
’+
0
-
H2N\/CootBu
F m o c H N k COOtBu
2
hHAc
Ac%xo
,
Scheme 2. a) THF, EDCI. 62-94%; b) 1 % NaOMe in methanol then MeOH,
H2/Pd/C,93-97%;c)DMF,3+DCC/HOBtor3 +BBC,(56% (n =1),47%(2),
54% (3). 80% (4). 67% (5). 84% (6).97% (7));d)valid f o r n = 4-7: CFJOOH,
0°C. (43% (n = 4 ) , 82% ( 5 ) . 53% (6), 55% (7)); e) DMF. 5 +DCC/HOBt
or 5 BBC, 43-53%; EDCI = l-ethyl-3-[3-(dimethylamino)propyljcarbodiim1de
hydrochloride, DCC = dicyclohexylcarbodiimide,HOBt = hydroxybenzotriazole,
BBC = benzotr~azolyloxybis(pyrrolidino)carbonium hexafluorophosphate
AcO
AcO
NHAc
+
7 ( n = 4,5)
6 ( n = 1-7)
3
ZNH(CH,),COOH
NHAc
7
4, n = 1-7
Scheme 1 a ) Morpholine. 77%; b) CH,CI,. Ac,O. py, 9 6 % ; ~CF,COOH,
)
94%;
d) MeOH. MeONa, 76%, Finoc = 9-fluorenylmethyloxycarbonyl. Z = benzyloxycdrbonyl.
Amino acids 4 of various lengths-flexible spacers-were
synthesized according to the Schotten-Baumann procedure
from the corresponding free amino acids and benzyloxycarbony1 chloride in quantitative yields.[’ 31 The building blocks 2
and 4 were linked in the presence EDC11231and subsequently
deacetylated and hydrogenated (Scheme 2). The resulting compounds 5 were connected with building block 3. DCC/HOBt or
BBC proved to be reliable condensing reagents in this case.[241
The trimeric structures 7rZ5]
were finally reached after deblocking of 6 and subsequent condensation of the resulting acid with
5.The versatility of this strategy can be exploited through repetition of steps (d) and (e) of Scheme 2 to generate defined “tetravalent” compounds and structures of even higher “valency”.
Compounds 6 and 7 were treated with uridine S-diphosphogalactose (UDP-ga1)[261and commercially available galactosyl
transferase (EC 2.4.1 .22)[271in a sodium cacodylate buffer
(pH 7.5) in the presence of calf intestine alkaline phosphatase
(CIAP, EC 3.1.3.1) (Scheme 3).[281The latter enzyme destroys
the inhibiting uridine 5’-dipho~phate,’~~]
which is concomittantly formed as a by-product. The completion of the enzymatic
reaction can be checked by thin-layer chromatography or
preferably by MALDI-TOF (matrix-assisted laser desorption
ionization time-of-flight) mass spectrometry of a smaIl sample
of the incubation mixture (see Fig. 1).
The galactosylated intermediates were filtered through Sephadex ((3-25 superfine (Pharmacia)) and characterized by ‘H
and l3C N M R spectroscopy. Subsequently sialic acid was introduced by incubation with cytidine 5’-monophospho-sialic acid
( C M P - S ~ ~ ) ‘ ~and
’ ] a(2-3)sialyl transferase (EC 2.4.99.6)13‘I at
Angrn,. C‘hrm. Inr. Ed. EngI. 1996. 35, No. 16
‘0VCH
NHAc
OH
on
on
OH
OH
Ho
no
8
:N
OH
HO
Wc
OH
HO
HO
Scheme3. a)2.2and3.3equivUDP-gal,gal-tfor8(75%,85‘%,92%,7Oo~,81%,
96%. 64%) and 9, respectively (88%. 75%); b) 2 2 and 3.3 equiv CMP-sia,
~(2-3)sia-tfor 8 (73%. 97%. 73%. 99%. 64%. 77%, 7796) and 9. respectively
(85%,99%);c)2.2and 3.3 equiv. GDP-fuc.fuc-tVI for8(99%. 85%.95%, 77%,
73%. 74%, 67%) and 9, respectively (68’4, 67%)
pH 6.5 in the presence CIAP.[291Filtration of the reaction mixture through Sephadex provided the sialylated saccharides,
which were characterized by ‘H N M R spectroscopy and
MALDI-TOF mass spectometry. Finally fucose was introduced
with guanosine 5’-diphospho-fucose ( G D P - ~ U C )as
[ ~the
~ ]fucose
donor and fucosyl transferase VI[331in a cacodylate buffer at
p H 6.5, again in the presence of CIAP. Thus the desired compounds 8 and 9 could be obtained in 6-20 mg amounts, which
was sufficient for the ensuing biological assays.[341
Verlag~~esellsrimfi
mbH, 0-49451 Weinheim,1996
+
0570-0833/96/3S16-r%~3
$15.00 .25;0
1813
COMMUNICATIONS
470
-
OH
HO
h
m
2
42 0
In
-
6
370-
320-r
r-
270-
220-
k
0
N
t
1
(D
m
m
N
rnm
0
a
l
'r-
N
0
(D
k
in
(D
N
0
Q
In
m
m
170-
m
m
(0
: N
0
r-8
N
N
0
2.0 --
1500
1600
1700
1BOO
I
1900
mlz
I
2000
I
2100
I
2200
1
2300
-1
2400
__t
Fig. 1. MALDI-TOF mass spectrum of the reaction mixture from the fucosylation of 6 ( n = 4): matrix: 0.1 M 2,4,6-trihydroxyacetophenone
in ethanol.
All IC,, values have been normalized to one SLe" unit. Although the divalent compounds 8 show varying binding affinities, their overall affinity to E-selectin is not significantly enhanced over that of the monovalent SLe", which was attached
p-glycosidically to the aglycon (N-acetylated, 0-tevt-butyl protected aspartic acid). In contrast, the affinity of the trimeric
compound 9 (n = 4) to E-selectin is four times higher than that
of the monomer and almost nine times higher than that of the
corresponding dimer 8 (Table 1).
For monitoring the progress of the formation of these complex glycopeptides MALDI-TOF mass spectrometry proved to
be an indispensable analytical t 0 0 1 . ~Figure
~ ~ ~ 1 shows a spectrum of a small sample drawn from the incubation mixture from
Table 1. IC,, values of the octa- (8) and dodecasaccharides (9) [34]
Compound
Ic50
SLe' aglycon
8(n=l)
8 (n = 2 )
8 (n = 3)
8 (n = 4)
8 (n = 5 )
8 (n = 6)
0.60
0.46
0.64
1.20
0.47
8 (n =7)
0.80
0.48
9 (n
9 (n
0.14
0.39
= 4)
=
1814
5)
[mMI
c> VCH Verlugsgesellschafi mbH, 0-69451 Weinheim, 1996
the fucosylation of compound 6 (n = 4). All final compounds
were additionally characterized by 'H NMR spectro~copy.[~"'
In Figure 1 peaks 2-4 arise from nonfucosylated starting material ( M = 1756.7) with Na', 2 N a + , and 3Na' ions. Peaks 5-7
correspond to monofucosylated intermediates ( M = 1902.8) associated similarly with Na+ ions. Peaks 9-10 arise from the
desired difucosylated final compound ( M = 2047.0), again associated with Na+ and 2 N a + ions. The incubation was terminated when no peaks corresponding to either the starting material or intermediates were detected. The final fucosylated
glycopeptides were purified by filtration through Sephadex.
MALDI-TOF mass spectrometry can also be used to check the
purity of the end products.
Our method, which relies on a set of accessible transferases
and activated donor sugars, allows the rapid assembly of oligovalent SLe" conjugates. The rigidity of the peptide backbone
(+ presentation of the SLe" moieties) and the individual distances of the SLe" moieties are controlled by the proper choice
of amino acid monomers. SLe" analogues of higher "valency"
can be formed sequentially by known peptide chemistry.
The enzymatic methodology is not limited to the formation of
the natural SLe" structure.[371It has been shown recently that
nonnatural substrates (donors and acceptors) are recognized by
glycosyl transferases, and glycosides are produced with high
regio- and stereo~electivity!~~~
The breadth of substrates
amenable to glycosyl transferase catalysis is not yet fully investigated. Even pseudo-sugars can be synthesized efficiently with
OS70-O833j96j3Sl6-lsl4S 15.00+ .2S/O
Angew. Chem. Int. Ed. Engl. 1996, 35, No 16
the help of glycosyl transferases in a stereospecific manner without protecting group manipulations.138*391 Thus SLe' mimeti c ~ , ' ~some
']
of them showing surprisingly high affinities to E-sel e ~ t i n , ' ~may
' ~ be accessible easily.
Received; December 7, 1995
Revised version: March 4. 1996 [28621 IE]
German version: Angew Chem. 1996, 108, 1949-1952
Keywords: glycopeptides
trometry . sialyl Lewis'
-
glycosyl transferases
-
mass spec-
[I] J. 8 . Lowe in Molecular Glycobiology (Eds.: M. Fukuda. 0. Hindsgaul). IRLPress, 1994, p. 163, and references therein.
[2] M. B. Lawrence. T. A Springer, Cell 1991. 65, 859.
[3] K. Ley, P. Gaethgens, C. Fennie, M. S. Singer, L. A. Lasky, S. D. Rosen. Blood
1991. 77.2553
[4] L. A. Lasky. .4nn. Rev. Biochem. 1995.64, 113.
[5] C. R. Bertozzi, Chem Eiol. 1995. 2, 703.
[6] M. Edwards. Curr. Opin. Therup. Put. 1991, 1617.
[7] R. M. Nelson, S. Dolich. A. Aruffo, 0. Cecconi. M. P. Bevilaqud, J Clin.
Invest. 1993. Yl. 1157.
[8] T. Feizi. Curr Opin. Struct. Eiol. 1993, 3. 701.
[9] J. K. Welply. S. 2. Abbas, P. Scudder, J. L. Keene, K. Broschat, S. Casnocha,
C. Gorka. C. Steiniger. S. C Howard. .I.J. Schmuke, M Grdneto, J. M. Rotseart. J D. Manger. G. s. Jacob, Glycobiology 1994, 4, 259
[lo] G Kretzschmar, U. Sprengard, H. Kunz. E. Bartnik, W. Schmidt, A. Topfer,
B. Horsch. M. KrduSe, D. Seiffge. Tetrahedron 1995. 51, 13015.
[I 11 After submission of this paper the synthesis of a trivalent SLeXconjugate on a
cyclopeptide template was published: U. Sprengard, M. Schudok, W Schmidt,
G. Kretzschmar. H. Kunz. Angew Chem. 1996. 108, 359, Angew Chem. Int.
Ed Engl. 1996. 35. 321.
[12] S. A. DeFrees. W Kosch. W Way. J. C. Paulson, S. Sabesan, R. R. Halcomb,
D.-H. Huang, Y. Ichikawa. C.-H. Wong, J Am. Chem. Soc. 1995, 117. 66.
[13] H. Benz. Synthesis 1994. 337.
[I41 C. Unverzdgt. S. Kelm, J. C. Paulson, Carbohjdr. Res. 1*4, 251. 285.
(151 C:H
Wong. R. R. Halcomb, Y. Ichikawa. T. Kajimoto, Angetc. Chem. 1995.
107. 569. Anyew. Chem. lnt. Ed. Engl. 1995. 34, 521
[16] 0. Hindsgaul. Sem. Cell E d 1991, 2, 319.
[17] K. von dem Bruch. H. Kunz. Angen.. Chem 1994, 106, 87; Angew. Chern. lnt.
Ed. 1994. 33, 101.
[18] The cDNA sequences of a large number of transferases are deposited and cell
clones that express the desired transferase can often he obtained; see M. C .
Field. L. J. Wainwright, Glyobrologj 1995, 5,463.
1191 J. E. Heidlas. K. J. Williams, G. M. Whitesides. Act.. Chem. Res. 1992.25, 307.
[20] Y. Ichikawa. .1. L. Liu. G:J. Shen. C.-H. Wong. J Am. Chem. Sac. 1991, 113,
6300
[21] T. Inazu, K. Kobayashi. Synlett 1993, 869.
[22] P. Schultheiss-Reimann, H. Kunz, Angel%. Chem. 1983, 95, 64; Angew Chem.
Ed. Int. Engl 1983.22, 62; Angen. Chem. Suppi. 1983, 39.
[23] D . Seghal, I. K. Vijay, Anal. Eiochem. 1994, 218, 87.
1241 S. Chen. J. Xu. Teirahedron Lei!. 1992. 33. 647.
[25] Compounds 1 -5 were obtained easily in pure form ('H NMR spectroscopy) in
gram amounts by silica gel chromatography. Compounds 6 and 7 were obtained in pure form in 100--250mg amounts by chromatography over RP-18gel .
[26] J. E. Heidlas. W. J. Lees. G. M. Whitesides. J Org. Chem. 1992, 57. 152; commercially available from Sigma (U-4500)
[27] Sigma G-5507.
[28] Boehringer No. 108146.
[29] M. M. Palcic, Meth0d.s Enqmol 1994. 230, 300.
[30] M Kittelmann. T. Klein, U. Kragl. C. Wandrey, 0. Ghisalba, Appl. Microbiol.
Eiotechnol. 1995. 44, 59.
[31] S. Gosselin. M. Alhussaini. M. Streiff. K. Takabayashi. M. M. Palcic, Anal.
Eiorhrm. 1994. 220, 92.
1321 U B. Gokhale. 0 . Hindsgaul, M. M. Palcic. Can. J Chem. 1990, 68. 1063;
gram amounts of this donor and different derivatives thereof are easily prepared chemoenzymatically: R. Ohrlein, G. Baisch, unpublished.
[33] Soluble fucosyl transferase V1 was obtained from the supernatant of a highly
productive, trdnsfected C H O cell line: A. Katopodis, B. Bowen, unpublished.
[34] The inhibition constants (lC5J were obtained from a static assay with recombinant human E-selectin. The assays were performed by Dr. J. Magnani
(GlycoTech Corp., 14915 Broschart Road, Rockville, M D 20850. USA).
1351 This very sensitive and mild method was originally developed to analyze
oligonucleotides (U. Pieles, W. Zurcher, M. Schir. H. E. Moser. Nucteic Acids
Re.?. 1993.21. 3191). Wecould apply this method t o analyze complex glycopeptides in a reliable manner.
Angeu-. Chrrn. 1111.Ed. Engl. 1996, 35, No. 16
[36] Selected ' H N M R data (D,O, 500 MHz): 8 (n = 4): 6 = 1 27 (d, J =7.0 Hz,
6H. 6-H(fuc)). 1.54 and 1.68 (each m and 2 H , O,CCH,CH,CH,CH,N). 1.76
(t. J =11 Hz. 2 H , 3-Ha,(sia)). 1.94 and 1.96 (each s and 3H. NHAc), 2.00 (s.
9H. NHAc), 2.25 (m. 2 H , O,CCH,CH,CH,CH,N). 2.75 (m. 6 H , 3-He,(sia)
and D-H(asn)). 3.15 (m, 2 H , O,CCH,CH,CH,CH,N). 4.49 (d, J =7.5 Hz,
1 H,l-H(gaI)),4.51 (d. J = 7 S H z , l H , l-H(gal)),4.60(m.2H.z-H(asn)),5.14
(m. 4H. I-H(fuc) and 1-H"(g1cNAc)). 9 (n = 4): 6 = 1.31 (d. J =7.0 Hz. 9H.
6-H(fuc)). 1.68 ( m , 8 H , O,CCH,CH,CH,CH,N). 1 9 4 (t. J = 1 1 Hz. 3H.
3-Ha,(sia)), 2.15 (s, 9 H , NHAc). 2.20 (s. 12H. NH.4c). 2.46 (m. 4 H ,
O,CCH,CH,CH,CH,N), 2.92(m. 9H. 3-H,,(sia) and P-Hcdsn)), 3.48 (m.4H.
O,CCH,CH,CH,CH,N). 4.71 (d. J = 7 . 5 Hz, 3H. I-H(ga1)). 4.79 (m, 3H.
z-H(asn)), 5.25 (m. 6H. l-H(fuc) and I-H(glcNAc)).
[37] Recently we could show that all three glycosyl transferases used in this study
readily accepted a number of nonnatural substrates in vitro: G. Baisch, R.
Ohrlein, B. Ernst, Eiorg. Med. Chrm Lett. 1996.6.749; G . Baisch, R. Ohrlein,
B. Emst, M. Streiff, ibid.,6,755; G. Baisch. R. Ohrlein. B Ernst, A. Katopodis
rbid., 6. 759.
[38] For a recent review, see: M. M. Palcic. 0. Hindsgaul, Trends GIj<o.sci. Gljcotechnol. 1996,8, 37.
[39] L. Yu, R. Cabrera. J. Ramirez, V. A. Malinoskii, K. Brew. P. G. Wdng, Tetrahedron Lett. 1995.36,2897; C.-H. Wong, Y. Ichikawa, T. Krdch, C. GautheronLeNarvor. D. P. Dumas, G . C. Look, J Am. Chrm. So?. 1991, 113,8137; R.
Ohrlein. unpublished.
[40] N. M. Allanson. A. H. Davidson, C. D. Floyd, F. M. Martin, Tetrahedron
Asjmmelrj 1994.5,2061; A. Topfer, G. Kretzschmar, E. Bartnik. Tetrahedron
Lett. 1995. 36, 9161
Molecular Recognition Analyzed by EPR,
ENDOR, and NMR Spectroscopy**
Martin Jager and Hartmut B. Stegmann*
The formation of complexes between model receptors and
substrates is known as molecular recognition.['] Since natural
phenomena like transport, catalysis, and regulation are based
on it, extraordinary interest in intermolecular interactions has
been aroused. The formation equilibrium and the conformation
of all the components of these lock-and-key interdependencies
are dynamic systems.
We therefore decided to study these kinds of systems with
nuclear magnetic resonance spectroscopy ('H N M R ; 250 MHz)
in parallel with electron paramagnetic (EPR; 10 GHz) and electron nuclear double resonance (ENDOR) spectroscopy. The
difference in time resolution of the methods provides complementary information. The receptor A (Scheme 1) proved to be
suitable for this investigation because of its converging functional groups, providing a cylinder-lock-like cavity with specific
affinity to purine and benzimidazole moieties.''] To introduce a
part of the molecule that can be converted into a paramagnetic
state, we modified these heterocycles with suitable phenol-type
substituents in sites where they d o not hinder the intermolecular
interactions. Hence the resulting compounds 1 , 2 . 3could still be
recognized as substrates. One-electron oxidation of the phenols
1-3 leads to the paramagnetic complexes A1'- A3'.
The spectra of both the diamagnetic and paramagnetic associates depend on concentration and temperature. Since the number of N M R signals remains constant, the chemical shifts repre[*I Prof. Dr. H. B. Stegmann, DipLChem. M. Jager
Institut fur Organische Chemie der Universitlt
Auf der Morgenstelle 18, D-72076 Tuhingen (Germany)
Fax: Int. code +(7071)29-5246
e-mail : stegmann(6 uni-tuebingen.de
r * ] This work was supported by the Fonds der Chemischen Industrie and the
Deutsche Forschungsgemeinschaft within the Grdduiertenkolleg"Analytische
Chemie" (scholarship for M. J.). We thank Dr. H.-J. Egelhaaf and DipLChem.
B. Lehr for carrying out the optical investigations.
VCH Verlagsgeseil.schaft mhH. 0.69451 Wernheim, 1996
0570-083319613516-1815$ f5.00f .25jO
1815
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