Sialyl Lewisx analogs based on a quinic acid scaffold as the fucose mimic

Article abstract of DOI:10.1016/j.bmcl.2005.05.004

(-)-Quinic acid was used as a starting material for the preparation of sialyl Lewis^x mimetics in order to target E-selectin. Spatial orientation of the hydroxyl groups of quinic acid could mimic the L-fucose ones. Introduction of a side chain ending with a carboxylic acid was effected to replace the sialic acid interaction at the carbohydrate recognition domain. A first series of derivatives, incorporating amino acids linked to quinic acid, were tested for their affinity and found to interact with E-selectin with IC₅₀ within the millimolar range.

Full text of DOI:10.1016/j.bmcl.2005.05.004

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3224–3228
Sialyl Lewisx analogs based on a quinic acid scaffold
as the fucose mimic
Christian Girard,* Jennifer Dourlat, Aline Savarin, Christine Surcin, Stefanie Leue,
*
Virginie Escriou, Celine Largeau, Jean Herscovici and Daniel Scherman
´
´ ´
Laboratoire de Pharmacologie Chimique & Genetique (UMR 8151 CNRS/U 640 INSERM),
´
Ecole Nationale Superieure de Chimie de Paris, 11 rue P.&M. Curie, 75005 Paris, France
Received 6 January 2005; revised 1 March 2005; accepted 2 May 2005
Abstract—(À)-Quinic acid was used as a starting material for the preparation of sialyl Lewisx mimetics in order to target E-selectin.
Spatial orientation of the hydroxyl groups of quinic acid could mimic the L-fucose ones. Introduction of a side chain ending with a
carboxylic acid was effected to replace the sialic acid interaction at the carbohydrate recognition domain. A first series of derivatives,
incorporating amino acids linked to quinic acid, were tested for their affinity and found to interact with E-selectin with IC50 within
the millimolar range.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
terminal tetrasaccharidic appendage of a leukocytesÕ gly-
copeptide (Fig. 1). The exact conformation of sLex and
its interaction with E-selectin have been revealed by
X-ray analysis of crystals of the soluble tetrasaccharide
bound to the protein.3
Carbohydrate binding proteins (lectins) are molecules
involved in many important biological processes, rang-
ing from leukocytesÕ recruitment at inflammatory sites
to mechanisms in cancer development, as well as glyco-
protein levels, control in the cell and the circulation, and
other cellular interactions related to immune response.1
Due to the importance of sLex and E/P-selectinsÕ interac-
tion, and the possible applications to inhibit or use this
adhesion for targeting, several articles related to the
synthesis and use of sLex and its analogs have been
published in the past few years.4
Within this class of cellular receptors, selectins (E, P,
and L) are calcium-dependent lectins (C-type) involved
in the events related to the action of bloodstream cells.
E- and P-selectins are usually only transiently expressed
on the vascular endothelium after the prime inflamma-
tory response. They are responsible for the interaction
between endothelial cells and leukocytes leading to their
rolling, as the first step needed for extravasation. These
molecules are also overexpressed, especially E- and P-
selectins, during some pathological processes such as
chronic inflammatory diseases, neoangiogenesis, and
may be a way cancer cells can metastasize by adhesion
to the endothelium.2
Most of the analogs synthesized so far were of saccha-
ridic nature, with some modifications. More recent
For E-selectin, the natural ligand by which such interac-
tions are possible is the sialyl Lewisx epitope (sLex), a
Keywords: E-selectin; P-selectin; Sialyl Lewisx; Mimics; Quinic acid.
*
Corresponding authors. Tel.: +33 1 44 27 67 22; fax: +33 1 43 25 79
75 (C.G.); e-mail: christian-girard@enscp.fr
Figure 1. Sialyl Lewisx tetrasaccharide structure at E-selectin CRD.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.004

C. Girard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3224–3228
3225
Figure 2. Minimum pharmacophores (colored groups) and structural
analogies between sialyl Lewisx and quinic acid derivatives.
examples have shown that simpler structures can be as
efficient as the previously reported mimics, with a gained
ease for their preparation.5 A number of these analogs
have shown an equal or much higher activity than sialyl
Lewisx, proving their value as inhibitors or targeting
ligands. However, there is still some room for new sLex
mimic, especially structures that can be easily generated,
with the possibility to access larger molecular diversity.
Figure 3. Coupling between quinic acid acetate 1 and some amino
acids by HOBt ester activation (Method A) to obtain structures 2a–7a.
The use of standard peptide coupling procedures (NHS
or HOBt, EDC, and Et3N) gave the desired compounds.
However, the reactions were found to be quite slow (3–6
days) and needed supplementary purification steps to
remove the by-products. The yields using this method
(Method A, Table 1) ranged from low to good (39–
76%).
Our approach is based on structural analogies with sLex
active conformation and the minimum pharmacophores
needed to select the basic mimic structure (Fig. 2). With-
in the interactions implicated, it seems that the most
important features for the recognition are the hydroxyls
borne by L-fucose to chelate the calcium ion, as well as
the carboxyl group of sialic acid. Hydroxyls on galac-
tose also seem to play a role, maybe at a lesser extent,
while N-acetylglucosamine merely acts as a scaffold.6
To improve both yields and the ease of isolation, we
decided to use a mild method that we had previously
published for the preparation of esters and amides, via
the formation of acyl chloride in the presence of a scav-
enging resin (Fig. 4).9 Follow-up of the reaction (NMR)
of the peracetate 1 has shown that formation of the acyl
chloride was complete in 4 h.10 Two coupling conditions
were then used and compared to prepare the quinyl-
aminoester derivatives (2a–9a).
Among the chiral natural products available, a carbocy-
clic molecule, D-(À)-quinic acid or (1R,3R,4S,5R)-
1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid, has
the required axial–equatorial–equatorial 1,2,3-triol pat-
tern that could mimic the fucose part.7 Furthermore,
the carboxylic acid onto the cyclohexane ring can serve
as an anchoring point to introduce various CO2H-end-
ing side chains, which have been chosen to probe car-
boxyl interactions with protein residues within or near
the CRD.8
In Method B (Table 1), the acyl chloride was reacted
onto amino ester salts in the presence of Et3N as a base.
The reactions were conducted overnight (14 h) in order
to insure complete coupling. An acido-basic aqueous
2. Synthesis of quinic acid derivatives
Table 1. Formation of quinyl-amino ester derivatives (2a–9a)
Compound
Method Aa
yield (%)
Method Bb
yield (%)
Method Cc
yield (%)
A first series of derivatives was prepared by coupling the
quinic acid counterpart with amino acids. We selected
the peracetylated quinic acid 1 as the starting material
in order to graft amino acids (Fig. 3). The amino acid
derivatives were selected to provide a range of substitu-
tions between quinic acid and the terminal carboxyl. Es-
ters of glycine (2a), aminobutyric (3a), and hexanoic (4a)
acids were used to increase the length between both
parts of the mimics. Benzyl glutamate (5a) was selected
to place a supplementary carboxyl group in the spacer
region. Alanine (6a) and isoleucine (7a) provided alkyl
groups, while threonine (8a) and serine (9a) esters fur-
nished the hydroxylated ones.
2a
3a
4a
5a
6a
7a
8a
9a
76
39
76
64
77
46
—
75
65
80
72
87
70
55
60
55
80
84
86
91
91
88
96
86
a EDC/HOBt coupling/aqueous work-up/purification.
b Acyl chloride/EtN3 as a base/aqueous work-up.
c Acyl chloride/Amberlyst A-21 as a base/Amberlyst A-15 as scavenger/
evaporation.

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C. Girard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3224–3228
Figure 4. The reaction between quinic acid peracetate 1 and amino acids, via the acyl chloride formation (Methods B and C), to prepare compounds
2a–9a.
Figure 5. Synthesis of 2b–9b using scavenging resins.
work-up gave access to compounds 2a–9a in fair to
good yield (55–80%).
The synthesis of these derivatives was made even simpler
by using the last tested conditions (Method C, Table 1)
based on the scavenging resinsÕ use. The overnight reac-
tion between the acyl chloride of peracetate 1 and the
amino ester salts was cleanly effected using Amberlyst
A-21 as the base, which serves to deprotonate the amino
ester salt and trap the produced hydrogen chloride. The
reaction mixture was filtered and contained only the de-
sired products and the excess amino ester. Amberlyst
A-15 was then added to remove the unreacted amino
ester without the need for any other workup procedures
(Fig. 5).
The solution was finally refiltered and evaporated to
give access to products 2a–9a in good yield (80–96%),
but, most of all, in their pure form and without the need
for any further purification.
Figure 6. Deprotection of compounds 2a–9a to obtain the mimics
2b–9b.
Deprotection of the synthesized products was done by
saponification, followed by aqueous TFA treatment
for the tert-butyl ether derivatives (Fig. 6). Fully depro-
tected mimics 2b–9b were obtained in yields ranging
from 90% to quantitative.
analog. The binding, which is relevant to the natural
interaction between the selectin and the cells, leads to
adhesion of HL-60 to the coated protein. The ability
of the mimic to displace the interaction of the selectin
(E or P) and HL-60 was monitored, and % of inhibition
and IC50 were evaluated (Table 2).
The resulting compounds were lyophilized and all ob-
tained as white water soluble powders.
For the control experiments, lacto-sialyl Lewisx showed
IC50 values within the awaited range: of 0.7 (E) and
12 mM (P). Quinic acid itself does not have any affinity
towards E- and P-selectins up to 100 mM.
3. Bioassay of quinic acid based mimics
The compounds were then tested in a competition assay
for human recombinant E- and P-selectins coated to
wells and using a cell-based assay, as previously de-
scribed.11 HL-60 cells expressing the selectin ligand (sia-
lyl Lewisx) were added together with the synthetic
Regarding E-selectin, all the analogs have shown mod-
erate affinity. Inhibitions ranged from 30 to 46% at

C. Girard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3224–3228
3227
Table 2. Inhibition (at 50 mM) and IC50 for E- and P-selectins against HL-60 cells for lacto-sialyl Lewisx, quinic acid and compounds 2b–9b
Compound
E-selectin inhibition
at 50 mM, %a
E-selectin inhibition
IC50, mMa
P-selectin inhibition
at 50 mM, %a
P-selectin inhibition
IC50, mMa
Lacto-sLex
Quinic acid
n.m.
n.a.
0.7 ( 0.1)
n.a.
>50
n.m.
n.a.
12 ( 2)
n.a.
>50
2b
3b
4b
5b
6b
7b
8b
9b
38 ( 2)
40 ( 8)
31 ( 2)
43 ( 2)
30 ( 2)
44 ( 2)
n.m.
38 ( 2)
83 ( 2)
14 ( 1)
86 ( 2)
8 ( 1)
92 ( 2)
n.m.
>50
>50
40 ( 2)
>50
>50
>50
21 ( 1)
>50
>50
19 ( 1)
n.m.
19.5 ( 0.5)
n.m.
>50
46 ( 1)
88 ( 2)
a All essays were performed in triplicate; standard deviation is given in parentheses; n.m. = not measured; n.a. = not active up to 100 mM. Lacto-
sLex = [a-Neu5Ac-(2,3)-b-D-Gal-(1,4)(a-Fuc-(1,3))-D-Glc].
50 mM (IC50 P 50 mM). However, the quinic acid
derivatives seemed to be able to prevent the fixation of
sLex to the selectin, whereas quinic acid alone did not,
showing both the validity of the selected structures
and the importance of the side chain on these
compounds.
ligand sialyl Lewisx. Further results and studies in this
area will be reported in due course.
5. Typical experimental procedures
5.1. N-[(1R,3R,4S,5R)-1,3,4,5-tetraacetoxycyclohexane-
1-carboxyl]glycine methyl ester (2a)
The effect is even more important while testing the compe-
tition with P-selectin. It seems that the analogs had a bet-
ter affinity for this selectin with inhibition at 50 mM
starting at 14% and going up to 88%. Chain length influ-
ence can be evaluated while looking at 2b–4b. Only the
compound with a 4-carbon chain (3b), derived from
GABA, possesses an IC50 at 40 mM. The derivatives with
2- (2b) and 6-carbon (4b) chains, however, have IC50
much higher than 50 mM. Introduction of a second car-
boxylic acid function, while examining 3b and 5b, shows
that the affinity is even better, IC50 falling from 40 (3b)
to 21 mM (5b). Interestingly, analogs incorporating iso-
leucine (7b) and serine (9b) had affinities in the same range
(19 mM). These results show that the basis for the prepa-
ration of analogs is once again valid. The higher affinities
for P-selectin can be due to the slight modification of the
geometry of the P-selectin CRD, which is anyhow highly
analogous to the E-selectin one.6
To a solution of 360 mg (1 mmol) of (1R,3R,4S,5R)-
1,3,4,5-tetraacetoxycyclohexane-1-carboxylic acid (1)12
in dry CH2Cl2 (10 mL) containing dry Amberlyst A-21
(500 mg, 2.4 mmol) was added under a nitrogen atmo-
sphere SOCl2 (145 lL, 2 mmol). The mixture was gently
stirred for 4 h at room temperature. The supernatant
was separated from A-21 using syringe transfer to another
flask and the volatiles were removed in vacuo. The result-
ing acyl chloride (off-white solid) was redissolved in dry
CH2Cl2 (10 mL) and added dropwise to a suspension of
glycine methyl ester hydrochloride (151 mg, 1.2 mmol)
and A-21 (625 mg, 3 mmol) in dry CH2Cl2 (30 mL). The
mixture was stirred overnight (14 h) and filtered. Amber-
lyst A-15 (300 mg, 1.2 mmol) was added to the solution,
which was stirred for 30 min before being filtrated and
evaporated under vacuum. The product 2a was isolated
as a white solid (344 mg, 80%).
In view of the interesting results gathered here, the
search for more active analogs based on a quinic acid
scaffold has to continue in order to find better candi-
dates with an activity at least equivalent to the one of
the natural ligand: sialyl Lewisx.
Mp = 171 °C. [a]D = À30.5 (c 1, CHCl3). FT-IR (KBr):
m = 3370 (NH), 2986, 2955, 2837 (CH), 1742 (C@O
ester) and 1675 (NHC@O) cmÀ1. RMN-1H (300 MHz,
CDCl3): d = 1.88 (m, 1H), 1.97 (s, 3H), 2.01 (s, 3H),
2.05 (s, 3H), 2.16 (s, 3H), 2.43 (dd, 1H, J = 3.3,
16.2 Hz), 2.53 (m, 1H), 2.84 (m, 1H), 3.73 (s, 3H), 3.98
(ddd, 1H, J = 5.3, 9.9, 18.3 Hz), 4.97 (dd, 1H, J = 3.6,
10.2 Hz), 5.41 (m, 1H), 5.57 (dd, 1H, J = 3.3, 6.9 Hz),
and 6.47 (t, 1H, NH, J = 5.1 Hz) ppm. RMN-13C
(75 MHz, CDCl3): d = 20.6, 20.9, 21.6, 30.9, 38.1, 41.3,
52.4, 66.6, 68.0, 71.9, 81.2, 169.5, 169.6, 169.9, 170.0,
and 170.4 ppm. Elemental analysis: Calcd for
C18H25NO11: C, 50.12; H, 5.84; N, 3.25%. Found: C,
50.29; H, 5.37; N, 3.16%.
4. Conclusion
We presented in this letter the preparation of analogs
based on quinic acid to mimic the sialyl Lewisx epitope.
The synthesis of these derivatives was made simple and
efficient by the use of scavenging resin approaches, giv-
ing access to products without the need for cumbersome
purification techniques. The analogs were tested for
their affinity toward markers expressed during inflam-
mation and other diseases, such as vascular endothelium
E- and P-selectins. It was found that the compounds
possessed moderate affinity for both selectins. However,
they were more efficient in inhibiting the fixation be-
tween P-selectin and HL-60 cells expressing the natural
5.2. N-[(1R,3R,4S,5R)-1,3,4,5-tetrahydroxycyclohexane-
1-carboxyl]glycine (2b)
The protected mimic 2a (84 mg) was dissolved in THF
(2 mL), cooled in an ice bath, and NaOH 2 N (2 mL)

3228
C. Girard et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3224–3228
was added. The solution was stirred for 14 h at room
temperature. The mixture was treated with prewashed
(MeOH and then THF) Amberlite IR-120(H+) until
pH 3, filtered, and evaporated under reduced pressure.
The residue is dissolved in UHQ water (5 mL), filtered
on a syringe filter (0.45 l), and lyophilized. The product
2b was isolated as a white fluffy hygroscopic solid
(44 mg, 90%).
Acknowledgments
The authors acknowledge the contribution of state
organisms for the financing of this research (CNRS, IN-
SERM, and MENRT).
References and notes
1. (a) Davis, S. S. Trends Biotechnol. 1997, 15, 217; (b) Lis,
H.; Sharon, N. Chem. Rev. 1998, 98, 637.
[a]D = À37.9 (c 1, H2O). FT-IR (KBr): m = 3375 (NH),
3164 (OH/COOH), 2950, 2843 (CH), 1731 (C@O acid)
2. (a) Bevilacqua, M. P.; Stenglin, S.; Gimbrone, M. A., Jr.;
Seed, B. Science 1989, 243, 1160; (b) Rosen, S. D.; Bertozzi,
C. R. Curr. Opin. Cell Biol. 1994, 6, 663; (c) McEver, R.
Curr. Opin. Immunol. 1994, 6, 75; (d) Stewart, A. O.; Bhatia,
P. A.; McCarty, C. M.; Patel, M. V.; Staeger, M. A.;
Andersen, D. L.; Gunawardana, I. W.; Melcher, L. M.;
Zhu, G. D.; Boyd, S. A.; Fry, D. G.; Cool, B. L.; Kifle, L.;
Lartey, K.; Marsh, K. C.; Kempf-Grote, A. J.; Kilgannon,
P.; Wisdom, W.; Meyer, J.; Gallantin, W. M.; Okasinski, G.
F. J. Med. Chem. 2001, 44, 988.
3. (a) Blanck, O.; Iobst, S. T.; Gabel, C.; Drickamer, K. J.
Biol. Chem. 1996, 271, 7289; (b) Somers, W. S.; Tang, J.;
Shaw, G. D.; Camphausen, R. T. Cell 2000, 103, 467.
4. (a) Simanek, E. E.; MacGarvey, G. J.; Jablonowski, J. A.;
Wong, C.-H. Chem. Rev. 1998, 98, 833; (b) Kaila, N.;
Thomas, B. E. Med. Chem. Rev. 2002, 22, 566.
5. (a) De Vleeschauwer, M.; Vaillancourt, M.; Goudreau, N.;
Guindon, Y.; Gravel, D. Bioorg. Med. Chem. Lett. 2001, 11,
1109; (b) Tsukida, T.; Hiramatsu, Y.; Tsujishita, H.; Kiyoi,
T.; Yoshida, M.; Kurokawa, K.; Moriyama, H.; Ohmoto,
H.; Wada, Y.; Saito, T.; Kondo, H. J. Med. Chem. 1997, 40,
3534; (c) Hiramatsu, Y.; Moriyama, H.; Kiyoi, T.; Tsukida,
T.; Inoue, Y.; Kondo, H. J. Med. Chem. 1998, 41, 2302; (d)
Hiramatsu, Y.; Tsukida, T.; Nakai, Y.; Inoue, Y.; Kondo,
H. J. Med. Chem. 2000, 43, 1476.
and 1651 (NHC@O) cmÀ1
.
RMN-1H (300 MHz,
CD3OD): d = 1.97 (m, 4H), 3.40 (dd, 1H, J = 3.0,
9.0 Hz), 3.92 (s, 2H), 4.01 (ddd, 1H, J = 4.8, 9.2,
10.8 Hz), and 4.14 (q, 1H, J = 3.0 Hz) ppm. RMN-13C
(75 MHz, CDCl3): d = 37.5, 41.0, 41.8, 66.9, 70.9, 75.8,
76.6, 173.2, and 176.0 ppm.
5.3. E-Selectin binding inhibition measurements with
HL-60 cells
Inhibition tests were conducted according to a published
procedure11 and at room temperature unless otherwise
stated. Human recombinant E- or P-selectin (R&D Sys-
tems Europe, Lille) was coated onto 96-well plates
(Immulon 2, Dynatech) using 50 lL of a 3 lg mLÀ1
solution (150 ng per well) of the selectin in DPBS buffer
(DulbeccoÕs phosphate-buffered saline) for 3 h. The wells
were washed three times with 200 lL DPBS buffer con-
taining 1% of bovine serum albumin (DPBS/1% BSA),
and then treated with 200 lL of the same solution for
1 h to block the uncoated surfaces.
6. Sialyl Lewisx binds to both E- and P-selectins. There is a
strong homology between both selectin CRDs. There is
only a slight modification in the conformation due to
minor modifications of amino acids next to the CRDs (see
Ref. 3b). This results, however, in a change in the spatial
disposition of some amino acids (Tyr48 and Tyr94, as an
example) implicated in interactions with the Neu5Ac
residue of sLex.
7. (a) Quinic acid has been used to mimic mannose, see:
Angyalosi, G.; Grandjean, C.; Lamirand, M.; Auriault, C.;
Gras-Masse, H.; Melnyk, O. Bioorg. Med. Chem. Lett.
2002, 12, 2723, and references cited therein. Quinic-style
derivatives have also been tested for their affinity toward C-
type lectins; (b) Schuster, M. C.; Mann, D. A.; Buchholz, T.
J.; Johnson, K. M.; Thomas, W. D.; Kiessling, L. L. Org.
Lett. 2003, 5, 1407; (c) Yuri, M.; Miyauchi, H. Jpn Kokai
Tokkyo Koho JP 11246503 A2, 1999.
8. Some analogs can have an equivalent activity to the one of
sLex without the COOH binding to the exact residue
implicated in binding Neu5Ac. As an example, Gravel,
Guindon et al. analogs of carboxylate bind to Tyr94 (near
CH2OH of Gal), instead of Arg97 (binding Neu5Ac-
COOH).5a Furthermore, both enantiomers of their analog
were found to equally bind to E-selectin, suggesting that
other interactions may be involved.
The blocking solution was removed and 40 lL of
inhibitor solutions, prepared from a 10 mM DBPS
stock solution diluted in HanksÕ solution containing
20 mM Hepes (pH 7.2–7.4), 0.2% glucose, and 1%
BSA, was added, followed immediately by 20 lL of a
105 HL-60 cell suspension. After 15 min incubation
time, the solution was removed and the wells were
washed three times by 200 lL HanksÕ solution 20 mM
Hepes (pH 7.2–7.4), 0.2% glucose, 1% BSA, and
1 mM CaCl2.
Lysis buffer was then added (50 lL citric acid 24 mM,
dibasic sodium phosphate 51 mM, and 0.1% Nonidet
P-40) and the plates were shaken for 5 min. Myeloper-
oxidase liberated during the lysis process was then de-
tected by addition of o-phenylenediamine as a substrate
(50 lL citric acid 24 mM, dibasic sodium phosphate
51 mM, 0.1% o-phenylenediamine, and 0.03% hydro-
gen peroxide). After 1 h, the reaction was stopped by
addition of 40 lL of 4 N sulfuric acid, and the absor-
bance of the solution was measured at 492 nm. The
inhibition percentage was calculated by comparison
to the absorbance of a positive control in which only
HL-60 was added (no inhibitor). Results are summa-
rized in Table 1.
´
9. Girard, C.; Tranchant, I.; Niore, P.-A.; Herscovici, J.
Synlett 2000, 1577.
10. Grewe, R.; Haendler, H. Liebigs Ann. Chem. 1962, 113.
11. DeFrees, S. A.; Kosch, W.; Way, W.; Paulson, J. C.;
Sabesan, S.; Halcomb, R. L.; Huang, D.-H.; Ichikawa, Y.;
Wong, C.-H. J. Am. Chem. Soc. 1995, 117, 66.
12. Guenin, R.; Schneider, C. H. Helv. Chim. Acta 1983, 66,
1101.
Sialyl Lewisx [a-Neu5Ac-(2,3)-b-D-Gal-(1,4) (a-Fuc-
(1,3))-D-GlcNAc] and ‘‘lacto-sLex’’ [a-Neu5Ac-(2,3)-b-
D-Gal-(1,4)(a-Fuc-(1,3))-D-Glc] tetrasaccharides used
as controls were purchased from Sigma.