Synthetic glycopeptides from the E-selectin ligand 1 with varied sialyl Lewisx structure as cell-adhesion inhibitors of E-selectin

Article abstract of DOI:10.1002/anie.200604442

(Chemical Equation Presented) Cooperation is key: Peptide and saccharide portions cooperate in sialyl Lewis^x glycopeptides and their mimetics in the fucose and sialic acid parts (see formula), resulting in up to greater than 100-fold stronger b

Full text of DOI:10.1002/anie.200604442

Communications
DOI: 10.1002/anie.200604442
Better than Nature
Synthetic Glycopeptides from the E-Selectin Ligand 1 with Varied
Sialyl Lewisx Structure as Cell-Adhesion Inhibitors of E-Selectin
Christian Filser, Danuta Kowalczyk, Claire Jones, Martin K. Wild, Ute Ipe, Dietmar Vestweber,
and Horst Kunz*
Dedicated to Professor Klaus Müllen on the occasion of his 60th birthday
During the recruitment and direction of leucocytes into
inflamed tissues, carbohydrate-recognizing receptors P- and
E-selectin play decisive roles at the beginning of a cell-
adhesion cascade.[1] Inhibition of E-selectin would be a great
benefit in the therapy of acute or chronic inflammation[2,3] or
in blocking tumor metastasis.[4] The tetrasaccharide sialyl
Lewisx (1) was identified as the ligand of P- and E-selectin[1]
agents. The biological half-time of the ligands is expected to
increase by exchanging l-fucose for d-arabinopyranose[5a,11c]
or l-galactose,[15] which do not occur in mammals. The same
should be true for substitution of sialic acid by its mimic (S)-
cyclohexyl lactic acid introduced by Ernst and co-workers.[16]
During the synthesis of such a set of sialyl Lewisx
glycopeptide selectin ligands, problems mainly caused by
the acid lability of the fucoside bond should be solved.
Because of this acid lability it has not been possible so far to
and, therefore, serves as the lead structure for the develop-
ment of selectin inhibitors.[1e–h] Binding studies with variants
of 1,[5–7] transfer NOE NMR experiments,[8] and X-ray
structure analysis[9] have shown that the hydroxy groups of
fucose,[5] the 4- and 6-hydroxy groups[6] of galactose, and the
carboxy function of sialic acid[7,9] are essential for an effective
binding to E-selectin. The glucosamine residue is necessary
for an optimal orientation of fucose and galactose.[10]
By experiments with synthetic sialyl Lewisx glycopeptides
it was revealed that affinity and selectivity of binding to P-
and E-selectin depend upon the peptide part of these
compounds.[11] In the natural ligand E-selectin ligand 1
(ESL-1)[12] 1 is presented at different positions. The amino
acid sequence 672–681 2 of ESL-1 containing one of five
potential N-glycosylation sites[13] is highly conserved in E-
selectin ligands from different species (MG160/rat/human;
CFR/chicken).[14] A glycopeptide with this sequence had
shown an increased affinity to E-selectin.[11c] Therefore, this
peptide is considered a favorable anchor structure for
systematically varied sialyl Lewisx ligands.
681
G672N LTELESED
2
*
Saccharide structure variants, especially in the fucose and
sialic acid portions, would be valuable as anti-inflammatory
[*] Dr. C. Filser, D. Kowalczyk, Prof. Dr. H. Kunz
Institut für Organische Chemie
Johannes Gutenberg-Universität Mainz
Duesbergweg 10–14, 55128 Mainz (Germany)
Fax : (+49)6131-392-4786
Scheme 1. a) Trimethylsilyltrifluoromethanesulfonate, CH2Cl2, molecu-
lar sieve (4 Š), ꢀ508C!208C, 70%; b) 1. [IrI(cod)(PMePh2)2]PF6, THF,
H2; 2. 4-toluenesulfonic acid, MeOH, 85%; c) CuBr2, nBu4NBr, molec-
ular sieve (4 Š), CH2Cl2/DMF (1:1); d) NaOMe, MeOH; e) MeSBr,
AgOTf, molecular sieve (4 Š), CH2Cl2/CH3CN (1:2), ꢀ508C!ꢀ308C;
f) Ac2O, dimethylaminopyridine (DMAP), pyridine; g) Bu2SnO, MeOH,
reflux; h) Ac2O, DMAP, pyridine. cod=cycloocta-1,5-diene.
E-mail: hokunz@uni-mainz.de
Dr. C. Jones, Priv.-Doz. Dr. M. K. Wild, U. Ipe, Prof. Dr. D. Vestweber
Max-Planck-Institut für Molekulare Biomedizin
Röntgenstrasse 20, 48149 Münster (Germany)
2108
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Chemie
apply common acid-labile anchors
and side-chain protecting groups in
solid-phase syntheses of such glyco-
peptides.[11c,17] Benzyl protecting
groups, on the other hand, have
disadvantages owing to their final
removal and serious side reactions
(for example, aspartimide forma-
tion). Herein, we describe the syn-
thesis of sialyl Lewisx asparagine
building blocks which can generally
be used in solid-phase syntheses on
acid-labile anchor groups according
to Fmoc strategy.
The azido group served as the
anomeric protecting group in glucos-
amine building block 3 in the syn-
thesis of sialyl Lewisx glycopeptides
and mimetics.[11] Not the fucose but
the b-galactosyl residue as its tri-
chloroacetimidate 4[18,19] was first
introduced to furnish the lactosa-
mine backbone 5 of all envisioned
sialyl Lewisx variants (Scheme 1). In
this way, the steric hindrance at the
4-position of the acceptor and the
acid-labile
fucoside
can
be
avoided.[11]
After catalytic removal of the
allyl ether in 5,[20] l-fucose 7,[21] d-
arabinose 8,[11c] and l-galactose 9
building blocks were introduced
under in situ anomerization condi-
tions.[22,23] After removal of the O-
acetyl groups, trisaccharides 10–12
were sialylated with sialic acid xan-
thate 13.[24] Thus, tetrasaccharides
14–16, which vary in the fucose
part, were obtained after acetylation
of the hydroxy groups. Regio- and
stereoselective sialylation was ach-
ieved by activation of the xanthate
13 with methylsulfenyl triflate,[25]
thereby exploiting the nitrile
Scheme 2. a) Raney nickel, pH 7.0, isopropyl alcohol/H2O (9:1), H2; b) TBTU, HOBt, DMF, iPr2NEt,
pH 6.5–7.5; c) Pd/C (10%), H2, MeOH/AcOH (95:5); d) Ac2O, DMAP, pyridine; e) TFA/CH2Cl2
(1:2), triethylsilane; f) FmocOSu, CH2Cl2, iPr2NEt, pH 9.0–9.5. TBTU=benzotriazol-1-yltetramethyl-
uronium tetrafluoroborate; HOBt=1-hydroxybenzotriazole; TFA=trifluoroacetic acid; FmocO-
Su=N-(fluorenylmethoxycarbonyloxy) succinimide.
effect.[26] From Lewisx azides 10–12, tetrasaccharide mimetics
17–19 were obtained according to the stannylene method-
ology[27] by reaction with the cyclohexyl lactic acid triflate
derivative 20[1g,28] and subsequent acetylation.
For this purpose benzyl ethers were hydrogenolyzed over
palladium on carbon (10%), and the resulting free hydroxy
groups were acetylated to stabilize the glycosidic bonds.[29]
Then, Boc and tert-butyl protecting groups were cleaved with
trifluoroacetic acid without affecting the glycoside bonds.
Selective N-acylation with FmocOSu[33] gave Fmoc(sialyl
Lewisx)asparagine 28 and variants 29–33. Pure b-anomers of
28 and 31–33 were isolated after preparative reverse-phase
HPLC, and those of compounds 29 and 30 were isolated after
flash chromatography[34] and were used in the solid-phase
syntheses.
Reduction of the glycosyl azides 14–19 through hydro-
genation over neutral-washed Raney nickel[29] gave glycosyl
amines, which immediately were coupled with tert-butyloxy-
carbonyl (Boc)[30] asparagin acid tert-butyl ester 21[31] to
furnish the protected glycosyl amino acids 22–27[32]
(Scheme 2). Analytical samples of pure b-anomers were
isolated by using HPLC. Separation of the anomers by flash
chromatography was only possible for 24. However, separa-
tion through preparative reverse-phase HPLC was not
necessary because an exchange of protecting groups was
carried out to increase the acid stability of the compounds.
The automated syntheses were conducted according to
the Fmoc strategy using TentaGel resin[35] loaded with
aspartic acid (Scheme 3). Activation of Fmoc amino acids
was performed with HBTU[36] and HOBt,[37] and activation of
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Communications
of 0.21 mm, which is lower by a factor of
13 than that of sialyl Lewisx 1 as the
standard. Substitution of enzyme-labile
fucose by l-galactoses that are more
stable in mammals (36) caused almost no
loss of affinity (IC50 = 0.26 mm). In the
case of 35, in which l-fucose is substi-
tuted by d-arabinose, inhibition is
decreased by a factor of two (IC50
=
0.59 mm). Exchange of sialic acid (in
34–36) by (S)-cyclohexyl lactic acid in
glycopeptides 37–39 decreased the IC50
values by an additional factor of ten (37:
IC50 = 20; 38: IC50 = 56; 39: IC50 =
25 mm). The glycopeptide 39 inhibits the
adhesion between the murine neutro-
phile cell line 32Dcl3 expressed by E-
selectin ligand[42] and E-selectin more
than 100-fold stronger than sialyl-Lewisx
(1), although 39 contains the metabol-
ically more stable l-galactose instead of
fucose, which usually is thought to be
essential.[5,8,9]
These properties are of interest for
the development of practically applica-
ble inhibitors of inflammatory processes
and tumor metastasis. In the course of
this work, a more economic synthesis for
sialyl Lewisx amino acids was developed
by forming the common lactosamine
backbone first. By means of an exchange
of benzyl for acetyl protecting groups
sufficiently acid-stable building blocks
were synthesized that are generally
applicable in solid-phase syntheses by
the Fmoc strategy.
Scheme 3. a) Piperidine/NMP (1:4); b) HBTU, HOBt, DMF, iPr2NEt; c) Ac2O, iPr2NEt, cat.
HOBt, NMP; d) HATU, HOAt, NMM, NMP; e) TFA/CH2Cl2 (1:1), TIS (2.5%), H2O (2.5%);
f) NaOMe, MeOH, pH 8.5–9.5; g) 1. NaOHaq, pH 9.5–10.5; 2. AcOHaq. HBTU=O-(benzotri-
azolyl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; HATU=O-(7-azabenzotriazolyl)-
N,N,N’,N’-tetramethyluronium hexafluorophosphate; HOAt=7-aza-1-hydroxybenzotriazole;
TIS= triisopropylsilane; NMP=N-methylpyrrolidin-2-one; NMM=N-methylmorpholine.
Received: October 30, 2006
Published online: February 13, 2007
glycosyl amino acids 28–33 with HATU[38] and HOAt.[39] The
Keywords: E-selectin inhibitors · glycopeptides ·
glycopeptides were detached from the resin by using TIS in
trifluoroacetic acid/dichloromethane (1:1). During this pro-
cedure, all tert-butyl protecting groups of the side-chain
functions were also cleaved. The O-acetyl groups of the
carbohydrate side chains were removed under ZemplØn
conditions.[40] The methyl esters of sialic acid and of cyclo-
hexyl lactic acid were saponified in aqueous sodium hydrox-
ide (pH 9.5–10.5). After neutralization with acetic acid, the
target compounds 34--39 were isolated by preparative
reverse-phase HPLC in yields of 22–41% based on the
resin-bound starting amino acid. The structures of all
glycopeptides were confirmed by high-resolution mass spec-
trometry and one- and two-dimensional NMR spectrosco-
py.[41]
.
protecting groups · sialyl Lewisx · solid-phase synthesis
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2.7 Hz, Fuc-4), 4.98 (m, 1H, GlcNAc-1), 4.95 (d, 1H, J3,4
=
=
2.7 Hz, Gal-4), 2.55 ppm (dd, 1H, Jeq,ax = 12.3 Hz, J3,4 = 3.7 Hz,
Sial-3eq); 29: d = 5.53 (d, 1H, J1,2 = 2.7 Hz, Ara-1), 5.00 (t, 1H,
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1), 2.55 ppm (dd, 1H, Jeq,ax = 12.1 Hz, J3,4 = 3.9 Hz, Sial-3eq); 30:
d = 5.67–5.63 (m, 2H, l-Gal-4, l-Gal-1), 5.17 (m, 1H, GlcNAc-
1), 4.95 (d, 1H, J3,4 = 3.5 Hz, Gal-4), 2.47 ppm (dd, 1H, Jeq,ax
12.1 Hz, J3,4 = 3.9 Hz, Sial-3eq); 31: d = 5.58 (m, 2H, Fuc-1, Gal-
4); 4.93 (m, 1H, GlcNAc-1), 4.05 ppm (dd, Jvic = 5.1 Hz, Jvic
7.4 Hz, CH-COOMe); 32: d = 5.56 (msmall, 2H, Ara-1, Gal-4),
4.98 (m, 1H, GlcNAc-1), 4.93 (dd, 1H, J1,2 = 3.1 Hz, J2,3
=
=
=
11.0 Hz, Ara-2), 4.05 ppm (dd, 1H, Jvic = 5.5 Hz, Jvic’ = 7,0 Hz,
CH-COOMe); 33: d = 5.65 (db, 1H, J3,4 = 2.7 Hz, l-Gal-4), 5.56
(db, 1H,
J1,2 = 3.1 Hz, l-Gal-1), 5.07 (m, 1H, GlcNAc-1),
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1655.5310 (1655.5287); 29: 1641.5093 (1641.5131); 30:
1713.5386 (1713.5342); 31: 1350.4924 (1350.4904); 32:
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