Casuarine stereoisomers from achiral substrates: Chemoenzymatic synthesis and inhibitory properties

Article abstract of DOI:10.1021/jo500991p

A straightforward chemoenzymatic synthesis of four uncovered casuarine stereoisomers is described. The strategy consists of l-fuculose-1-phosphate aldolase F131A-variant-catalyzed aldol addition of dihydroxyacetone phosphate to aldehyde derivatives of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) and its enantiomer (LAB) and subsequent one-pot catalytic deprotection-reductive amination. DAB and LAB were obtained from dihydroxyacetone and aminoethanol using d-fructose-6-phosphate aldolase and l-rhamnulose-1-phosphate aldolase catalysts, respectively. The new ent-3-epi-casuarine is a strong inhibitor of α-d-glucosidase from rice and of rat intestinal sucrase.

Full text of DOI:10.1021/jo500991p

Note
pubs.acs.org/joc
Casuarine Stereoisomers from Achiral Substrates: Chemoenzymatic
Synthesis and Inhibitory Properties
Alda Lisa Concia,† Livia Gomez,† Teodor Parella,‡ Jesus Joglar,† and Pere Clapes*,†
́
́
́
†Biotransformation and Bioactive Molecules Group, Departamento de Química Biolog
Química Avanzada de Cataluna, IQAC−CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
́
ica y Modelizacion
́
Molecular, Instituto de
̃
‡Servei de Ressonan
̀
cia Magnet
̀
ica Nuclear, Departament de Química, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain
̀
S
* Supporting Information
ABSTRACT: A straightforward chemoenzymatic synthesis of
four uncovered casuarine stereoisomers is described. The
strategy consists of L-fuculose-1-phosphate aldolase F131A-
variant-catalyzed aldol addition of dihydroxyacetone phosphate
to aldehyde derivatives of 1,4-dideoxy-1,4-imino-D-arabinitol
(DAB) and its enantiomer (LAB) and subsequent one-pot
catalytic deprotection−reductive amination. DAB and LAB
were obtained from dihydroxyacetone and aminoethanol using D-fructose-6-phosphate aldolase and L-rhamnulose-1-phosphate
aldolase catalysts, respectively. The new ent-3-epi-casuarine is a strong inhibitor of α-D-glucosidase from rice and of rat intestinal
sucrase.
asuarine (1,2,6,7-tetrahydroxy-3-hydroxymethylpyrrolizi-
dine, 1; Figure 1) is a pyrrolizidine-type sugar mimic
Because of these potentially useful biological activities,
synthetic efforts have been devoted to the preparation of
casuarines and their stereoisomers.3,8,12−18 The presence of six
stereogenic centers is a challenge for their synthesis, which
usually involves elaborated chiral starting materials and
cumbersome protection−deprotection schemes that lead to
moderate stereoselection and global yields.15,16 Therefore, it is
regarded as significant to devise new routes that provide
concise access to casuarine stereoisomers.
During our ongoing project on the chemoenzymatic
synthesis of iminocyclitols, we developed a procedure for the
preparation of polyhydroxylated pyrrolizidines of the hyacin-
thacine and alexine types that may successfully compete with
those already described.15,19−21 Our methodology is based on
aldol addition reactions of dihydroxyacetone phosphate
(DHAP) to N-Cbz-pyrrolidinecarbaldehyde derivatives cata-
lyzed by L-rhamnulose-1-phosphate aldolase (RhuA) and
L‑fuculose-1-phosphate aldolase F131A variant (FucAF131A),
both from Escherichia coli.22−24 Inspired by the potential
versatility of this approach, we herein explore the chemo-
enzymatic synthesis of highly polyhydroxylated pyrrolizidines of
the casuarine type using multistep aldol addition reactions
catalyzed by D-fructose-6-phosphate aldolase (FSA) and the
DHAP-dependent aldolases FucA and RhuA.
C
Figure 1. Naturally occurring casuarine (1), 3-epi-casuarine (2), and 6-
epi-casuarine (3).
that was first isolated from Casuarina equisetifolia (Casuar-
inaceae) and Eugenia jambolana (Myrtaceae).1−3 Bark extracts
of C. equisetifolia have been claimed to be useful for treatment
of diarrhea and colic and have been prescribed for the
treatment of cancer in Western Samoa. Infusions prepared from
E. jambolana and Eugenia uniflora were used in natural
Paraguayan and Indian medicine as antidiarrheics, diuretics,
antirheumatics, antifebriles, and antidiabetics.4−7 Casuarine
possesses six adjacent stereogenic centers, and therefore, 64
stereoisomers are possible. Two diastereomers were isolated
from natural sources, namely, 3-epi-casuarine (2) from the
shrub Myrtus communis L. (Myrtle)8 and 6-epi-casuarine (3)
from E. uniflora (Figure 1).4
Casuarine (1) is a potent and specific inhibitor of
α‑glucosidase from rice (IC50 = 1.2 μM), amyloglucosidase
from Aspergillus niger (IC50 = 0.7 μM), rat intestinal maltase
(IC50 = 0.7 μM), and isomaltase (IC50 = 3.9 μM).9 It has
recently been reported that casuarine inhibits human maltase-
glucoamylase (MGAM)10 more strongly than acarbose, a
tetrasaccharide analogue currently on the market as an
antidiabetic drug (Glucobay, Precose).11
As shown in the retrosynthetic analysis (Scheme 1), we
envisaged that casuarines (i.e., 1,2,6,7-tetrahydroxy-3-hydrox-
ymethylpyrrolizidines) could be derived by two asymmetric
aldol additions of dihydroxyacetone (DHA) and reductive
amination reactions starting from a conveniently protected
aminoethanal (e.g., N-Cbz-aminoethanal).
Received: May 6, 2014
Published: May 8, 2014
© 2014 American Chemical Society
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Note
of the aldehydes allow the unequivocal determination of the
stereochemistries of the molecules. Thus, ent-3-epi-casuarine
(7), ent-casuarine (ent-1), 2-epi-casuarine (8), and 2,3-epi-
casuarine (9) were obtained.
Scheme 1. Retrosynthetic Analysis of Casuarine-Type
Iminocyclitols: Enzymatic Aldol Addition and Catalytic
Reductive Amination
From the configurations of the stereocenters of the casuarine
stereoisomers thus obtained, it can be inferred that the aldol
addition of DHAP to 4 and ent-4 was highly stereoselective.
The stereochemical outcome depended on the aldehyde: 4 gave
the syn-(1′S,2′R)-configured aldol adduct, while ent-4 gave the
anti-(1′R,2′R)-configured adduct, typical of L-fuculose-1-
phosphate, the natural FucA substrate (Scheme 2). This
stereochemical outcome appears to be general, as judged for the
examples reported in previous works.23,24 On the other hand,
the reductive amination was not stereoselective, and mixtures of
epimers at C3 were obtained. The reactions were scaled up to
5.2−6.0 mmol under the screening assay conditions, except for
the enzyme content (i.e., 10 mg of protein/mL for 1.2 mmol
scale vs 4 mg/mL for 5.2−6 mmol scale). Each pair of
diastereoisomers, 7 + ent-1 and 8 + 9, was separated by ion-
exchange chromatography. Purification procedures of the aldol
adduct and the final compounds were not optimized.
Intriguingly, in the screening experiments conducted at the
1.2 mmol scale, the aldol addition reaction of DHAP to 4
followed by dephosphorylation and reductive amination
furnished three casuarine stereoisomers: 7, ent-1, and a minor
one (∼8%) identified as (1R,2S,3R,6R,7R,7aS)-1,2,6,7-tetrahy-
droxy-3-hydroxymethylpyrrolizidine (ent-1,3-epi-casuarine)
(compound 10; see the Supporting Information), which was
not recovered from the purification. This compound arose from
the aldol adduct epimer of 5 at C1′ [i.e., (R)-C1′] as a result of
the attack of the DHAP-FucAF131A complex to the Re face of 4.
However, ent-1,3-epi-casuarine was not detected in the NMR
spectra of the reaction performed under the 5.2−6 mmol scale
conditions. It is likely that the aldol adduct precursor of ent-1,3-
epi-casuarine was a thermodynamically controlled product that
could appear as a consequence of the higher enzyme
concentration used in the 1.2 mmol scale reactions.
The aldol additions of DHA to N-Cbz-aminoethanal
catalyzed by FSA and RhuA to furnish 1,4-dideoxy-1,4-imino-
D-arabinitol (DAB) and its enantiomer (LAB), respectively, and
their transformation into aldehydes 4 and ent-4 (Scheme 2) was
described in a previous work.25 Hence, the aldol additions of
DHAP to 4 and ent-4 were investigated here using wild-type
FucA, the FucAF131A variant, and wild-type RhuA from E. coli
(Scheme 2).
The biocatalyst screening assays (i.e., 1.2 mmol scale)
revealed that only FucAF131A tolerated both 4 and ent-4 with 90
and 70% conversion to 5 and 6, respectively. This is consistent
with the results obtained in previous works, confirming the
significant ability of FucAF131A to accept constrained substrate
acceptors.23,24 Substitution of alanine for phenylalanine at
position 131 in wild-type FucA creates the necessary space to
accommodate bulky aldehyde acceptors.23,24 Also noticeable is
the fact that RhuA, which accepts a broad structural variation of
substrates,22,26 did not tolerate 4 and ent-4. It is likely that the
sterically constrained structures of 4 and ent-4 direct the
hydroxyl groups toward hydrophobic areas of the enzyme active
site, disfavoring the binding.
Adducts 5 and 6 were treated with acid phosphatase and
purified by HPLC in ca. 30−36% isolated yield. The reductive
aminations of 5 and 6 with H2 and Pd/C catalyst rendered four
casuarine stereoisomers. As ascertained in previous synthetic
reports, the attack of the FucAF131A-bound nucleophile to the
aldehyde always occurs from the Re face, rendering the (R)-C2′
configuration (Scheme 2).23,24,27−31 Moreover, the specific
absolute configurations introduced by the stereogenic centers
Scheme 2. Chemoenzymatic Synthesis of Four Stereoisomers of Casuarine
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Note
solution) were added under vigorous agitation. Finally, FucAF131A (400
mg, 4 mg/mL of reaction mixture) was added, and the solution was
mixed again. The reaction mixture was placed on a horizontal shaking
bath (100 rpm) at a constant temperature of 4 °C. The reactions were
monitored by HPLC until the peak of the aldol adduct was constant
with time (24 h, 90% conversion). The enzymatic reactions were
stopped by addition of MeOH (100 mL), and the precipitated enzyme
was removed by centrifugation (3000 rpm, 15 min). Then the
methanol was evaporated, and the aqueous solution was washed with
ethyl acetate (4 × 100 mL) to remove the unreacted 4. The aqueous
layer was collected and the pH adjusted to 5 with HCl (0.5 M), and
the remaining ethyl acetate dissolved in the aqueous layer was
removed under reduced pressure.
Dephosphorylation. The aqueous solution was diluted with pH 5
citrate buffer (25 mL of a 1 M solution) and H2O to a final volume of
500 mL. Acid phosphatase (572 units) was then added. The reaction
was monitored by HPLC until no starting material was detected,
typically 24 h. The crude reaction mixture was loaded onto a glass
column (5 × 30 cm) packed with Amberlite XAD 16 (Rohm and
Haas) stationary phase, previously equilibrated with H2O. The column
was washed with H2O (2 × 600 mL) first, and the product was then
eluted with 7:3 EtOH/H2O (1750 mL, taking fractions of 250 mL).
Fractions containing 5 were collected. EtOH was removed in vacuum,
and the aqueous residue was lyophilized.
Compounds 7, ent-1, 8, and 9 were assayed as inhibitors
against a panel of commercial glycosidases and rat intestinal
disaccharide hydrolases. The compounds were found to be
inhibitors of α-D-glucosidase from baker’s yeast and rice and
α‑rhamnosidase from Penicillium decumbens (Table 1).
Table 1. Activities (IC50, μM) of the Synthesized Casuarine
a
Stereoisomers against Commercial Glycosidases; For
Comparison, the Activities of DAB and LAB Iminosugars
Are Included
α-glucosidase
(baker’s yeast)
α-glucosidase
α-rhamnosidase
(P. decumbens)
product
(rice)
7
250 71
297 44
320 19
530 138
0.32 0.03
1.9 0.2
7.9 5.2
261 47
680 193
94 32
ent-1
b
8
9
51
8
−
b
111
61
3
−
c
b
DAB
7
−
d
LAB
0.05 0.2
56
5
a
Data are means of triplicate experiments
standard error of the
b
c
mean (SE). No inhibition. DAB = 1,4-dideoxy-1,4-imino-D-
arabinitol. LAB is the enantiomer of DAB.
d
Removal of the Cbz Group and Reductive Amination. Pd/C (600
mg) was added to a solution of 5 (1.23 g) in 4:1 H2O/MeOH (200
mL). The reaction mixture was shaken under H2 (50 psi) overnight at
rt, and then the catalyst was filtered off. The solvent was evaporated
under reduced pressure, and the residue was lyophilized, furnishing a
mixture of 7 and ent-1 (400 mg, 33% yield). Compounds 7 and ent-1
were separated and purified by ion-exchange chromatography (see
below).
Synthesis of (1R,2S,3R,6S,7S,7aR)-1,2,6,7-Tetrahydroxy-3-
hydroxymethylpyrrolizidine (2-epi-Casuarine, 8) and
(1R,2S,3S,6S,7S,7aR)-1,2,6,7-Tetrahydroxy-3-hydroxymethyl-
pyrrolizidine (2,3-epi-Casuarine, 9). The same procedure was
followed starting with N-Cbz-(2R,3S,4S)-3,4-dihydroxypyrrolidine-2-
carbaldehyde (ent-4) (1.4 g, 5.2 mmol). The enzymatic aldol addition
provided 70% conversion after 24 h. The crude unphosphorylated
aldol adduct 6 (1.1 g) gave a mixture of diasteromers 8 and 9 (380 mg,
36% yield).
ent-3-epi-Casuarine (7) was a strong and selective inhibitor of
α-D-glucosidase from rice. Moreover, 7 was also a strong
inhibitor of rat intestinal sucrase (IC50 = 3.5 0.6 μM) and
moderate against rat intestinal maltase (IC50 = 39 13 μM).
The rest of compounds showed only moderate to weak
inhibitory activities. ent-Casuarine (ent-1) was a moderate
inhibitor of L-rhamnosidase but with lower inhibitory potency
against α-glucosidase from rice.9 Compounds 7, ent-1, 8, and
9 were inactive against β-D-glucosidase from sweet almonds,
β‑D-galactosidase bovine liver, α‑D-mannosidase from jack
beans, α‑L-fucosidase from bovine kidney, and in front of rat
intestinal lactase and threalase.
In summary, we have developed a concise chemoenzymatic
method for the synthesis of four uncovered casuarine
stereosiomers. The method consists of two key enzymatic
aldol addition reactions to control four stereogenic centers, two
of them being controlled by catalytic reductive amination. This
approach did not require protection of the hydroxyl groups,
minimizing the intermediate chemical transformations and the
purification steps. The reductive amination was not stereo-
selective and thus generated two epimers, which also conferred
structural diversity. The new compound ent-3-epi-casuarine (7)
was found to be a strong and selective inhibitor of
α-D-glucosidase from rice and of rat intestinal sucrase.
Purification by Ion-Exchange Chromatography. The mixture
of diasteromers 7 and ent-1 (400 mg) was separated by ion-exchange
chromatography on an FPLC system, and the fractions were analyzed
by NMR. A glass column (450 mm × 25 mm) was packed with
+
stationary phase (CM-Sepharose CL-6B in NH4 form) into a final
bed volume of 220 mL. The flow rate was 4 mL/min. The CM-
+
Sepharose-NH4 was washed initially with H2O, and then 7 + ent-1
(100 mg) dissolved in water adjusted to pH 7 with HCOOH (1 M)
was loaded onto the column. Minor colored impurities were washed
away with H2O (440 mL, 2 bed volumes). The retained compounds 7
and ent-1 were eluted with 320 and 240 mL, respectively, of aqueous
10 mM NH4OH. The process was repeated (i.e., four runs) until all of
the crude material was consumed. Pure fractions were pooled and
lyophilized, affording 7 (65 mg, 16% from the mixture) and ent-1 (80
mg, 20% from the mixture). The diasteromers 8 and 9 were separated
and purified using the same procedure as above. The 8 + 9 mixture
(380 mg) was loaded onto the column packed with CM-Shepharose-
EXPERIMENTAL SECTION
■
Methods. N-Cbz-(2S,3R,4R)- and -(2R,3S,4S)-3,4-dihydroxypyrro-
lidine-2-carbaldehyde (4 and ent-4, respectively) were prepared as
previously described by our group.25 After the oxidation step with IBX,
the organic phase containing the aldehyde was washed with 5% (w/v)
aqueous NaHCO3, and the ethyl acetate was removed under reduced
pressure.
+
NH4 . After the column was washed with plain water, the retained
compounds were eluted isocratically with aqueous 10 mM NH4OH.
The procedure was repeated (i.e., four runs) until all of the crude
material was consumed, affording 8 (96 mg, 25% from the crude
mixture) and 9 (80 mg, 21% from the crude mixture). The products
were lyophilized at pH 5.
(1S,2S,3R,6R,7R,7aS)-1,2,6,7-Tetrahydroxy-3-hydroxyme-
thylpyrrolizidine (ent-3-epi-Casuarine, 7). [α]2D2 −5.9 (c 0.85,
MeOH) [lit.15 of the enantiomer [α]D251 +2 (c 0.04, H2O); lit.8 of the
enantiomer [α]2D3 +5.7 (c 0.5, H2O)]. H NMR (400 MHz, D2O): δ
4.51 (s, 1H, H-1), 4.38 (d, J = 2.3 Hz, 1H, H-2), 4.28 (m, 2H, H-7, H-
6), 4.10 (dd, J = 7.1, 3.8 Hz, 2H, H-8, H-8′), 4.03 (m, 1H, H-3), 3.82
Synthesis of (1S,2S,3R,6R,7R,7aS)-1,2,6,7-Tetrahydroxy-3-
hydroxymethylpyrrolizidine (ent-3-epi-Casuarine, 7) and
(1S,2S,3S,6R,7R,7aS)-1,2,6,7-Tetrahydroxy-3-hydroxymethyl-
pyrrolizidine (ent-Casuarine, ent-1). Enzymatic Aldol Addition.
Reactions (100 mL total volume) were carried out in 250 mL
Erlenmeyer flasks. N-Cbz-(2S,3R,4R)-3,4-dihydroxypyrrolidine-2-car-
baldehyde (4) (1.6 g, 6 mmol) was dissolved in DMF (20 mL) and
cooled to 0 °C. A freshly prepared solution of DHAP at pH 6.9 (62
mL of a 97.7 mM solution, 6 mmol), KCl (2.5 mL of a 200 mM
solution), and triethanolamine (TEA) buffer (5 mL of a 1 M, pH 6.7
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The Journal of Organic Chemistry
Note
(d, J = 8.1 Hz, 1H, H-7a), 3.72 (dd, J = 10.9, 6.2 Hz, 1H, H-5), 3.58 (t,
J = 10.7 Hz, 1H, H-5′). 13C NMR (101 MHz, D2O): δ 76.7 (C-1),
76.7 (C-2), 75.9 (C-7), 75.7 (C-7a), 72.8 (C-6), 67.4 (C-3), 55.0 (C-
8), 50.4 (C-5). HRMS (ESI-TOF) m/z: [M + H]+ calcd for
(7) Mentreddy, S. R. J. Sci. Food Agric. 2007, 87, 743.
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2006, 17, 2702.
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Nash, R. J.; Fleet, G. W. J.; Wormald, M. R.; Kizu, H.; Ikeda, K.;
Asano, N. Tetrahedron: Asymmetry 2003, 14, 325.
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Re, D.; Soldaini, G.; Vogel, P.; Bello, C.; Goti, A.; Gratteri, P. Eur. J.
Org. Chem. 2010, 5574.
+
C8H16NO5 206.1022, found 206.1011.
(1S,2S,3S,6R,7R,7aS)-1,2,6,7-Tetrahydroxy-3-hydroxyme-
thylpyrrolizidine (ent-Casuarine, ent-1). [α]2D2 −13.5 (c 1.0, H2O)
[lit.16 of the enantiomer [α]D23 +18.1 (c 1.0, H2O); lit.1 of the
enantiomer [α]2D4 +16.9 (c 0.8, H2O)]. H NMR (400 MHz, D2O) δ
1
4.52 (s, 1H, H-7), 4.50 (d, J = 2.1 Hz, 1H, H-6), 4.41 (t, J = 8.6 Hz,
1H, H-1), 4.01 (m, 2H, H-2, H-8), 3.89 (dd, J = 13.1, 5.3 Hz, 1H, H-
8′), 3.76 (m, 2H, H-5, H-7a), 3.64 (ddd, J = 10.3, 5.2, 3.1 Hz, 1H, H-
3), 3.55 (d, J = 13.1 Hz, 1H, H-5′). 13C NMR (101 MHz, D2O): δ
76.7 (C-6), 76.5 (C-7), 75.3 (C-1), 75.0 (C-7a), 73.2 (C-2), 71.0 (C-
3), 58.6 (C-5), 56.8 (C-8). HRMS (ESI-TOF) m/z: [M + H]+ calcd
(11) Cardona, F.; Parmeggiani, C.; Faggi, E.; Bonaccini, C.; Gratteri,
P.; Sim, L.; Gloster, T. M.; Roberts, S.; Davies, G. J.; Rose, D. R.; Goti,
A. Chem.Eur. J. 2009, 15, 1627.
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Elbein, A. D.; Fleet, G. W. J. Tetrahedron Lett. 1997, 38, 5869.
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6527.
+
for C8H16NO5 206.1022, found 206.1011.
(1R,2S,3R,6S,7S,7aR)-1,2,6,7-Tetrahydroxy-3-hydroxyme-
thylpyrrolizidine (2-epi-Casuarine, 8). [α]2D2 −20.8 (c 1.2, MeOH).
1H NMR (400 MHz, D2O): δ 4.37 (dd, J = 8.8, 4.0 Hz, 1H, H-1), 4.26
(15) Ritthiwigrom, T.; Nash, R. J.; Pyne, S. G. Tetrahedron 2010, 66,
9340.
(16) Ritthiwigrom, T.; Willis, A. C.; Pyne, S. G. J. Org. Chem. 2010,
75, 815.
(m, 2H, H-2, H-6), 4.21 (t, J = 3.0 Hz, 1H, H-7), 3.85 (dd, J = 11.3,
7.2 Hz, 1H, H-8), 3.71 (dd, J = 11.2, 6.4 Hz, 1H, H-8′), 3.31 (m, 3H,
H-3, H-5, H-7a), 2.94 (dd, J = 12.3, 3.7 Hz, 1H, H-5′). 13C NMR (101
MHz, D2O): δ 78.5 (C-7), 77.9 (C-6), 74.9 (C-1), 73.6 (C-7a), 73.0
(C-2), 70.3 (C-3), 59.7 (C-8), 57.9 (C-5). HRMS (ESI-TOF) m/z:
(17) Parmeggiani, C.; Cardona, F.; Giusti, L.; Reissig, H.-U.; Goti, A.
Chem.Eur. J. 2013, 19, 10595.
+
[M + H]+ calcd for C8H16NO5 206.1022, found 206.1007.
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P.-Q. J. Org. Chem. 2011, 76, 4952.
(1R,2S,3S,6S,7S,7aR)-1,2,6,7-Tetrahydroxy-3-hydroxyme-
thylpyrrolizidine (2,3-epi-Casuarine, 9). [α]2D2 −12.8 (c 0.7, H2O).
1H NMR (400 MHz, D2O): δ 4.23 (m, 1H, H-1), 4.16 (m, 2H, H-2,
(19) Liu, W.-J.; Ye, J.-L.; Huang, P.-Q. Org. Biomol. Chem. 2010, 8,
2085.
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H-6), 3.92 (m, 3H, H-7, H-8, H-8′), 3.35 (m, 3H, H-3, H-5, H-7a),
3.00 (t, J = 9.4 Hz, 1H, H-5′). 13C NMR (101 MHz, D2O): δ 77.8 (C-
7), 74.4 (C-6), 74.3 (C-7a), 73.0 (C-1), 70.2 (C-2), 64.5 (C-3), 58.2
(C-8), 50.0 (C-5). HRMS (ESI-TOF) m/z: [M + H]+ calcd for
(22) Calveras, J.; Casas, J.; Parella, T.; Joglar, J.; Clapes
Catal. 2007, 349, 1661.
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Clapes
, P. Chem.Eur. J. 2010, 16, 10691.
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Clapes, P. Org. Lett. 2014, 16, 1422.
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Cardona, P. J.; Joglar, J.; Clapes, P. Org. Biomol. Chem. 2013, 11, 2005.
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Joglar, J.; Bujons, J.; Clapes
́
, P. Adv. Synth.
+
C8H16NO5 206.1022, found 206.1007.
ASSOCIATED CONTENT
́
■
S
* Supporting Information
1
́
Materials, experimental biological activities, and copies of H
and 13C NMR, COSY, and HSQC spectra for all compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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, P. Chem.Eur. J. 2009, 15, 7310.
(27) Gefflaut, T.; Blonski, C.; Perie, J.; Willson, M. Prog. Biophys.
AUTHOR INFORMATION
Corresponding Author
*E-mail: pere.clapes@iqac.csic.es.
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Mol. Biol. 1995, 63, 301.
(28) Fessner, W.-D.; Sinerius, G.; Schneider, A.; Dreyer, M.; Schulz,
G. E.; Badia, J.; Aguilar, J. Angew. Chem., Int. Ed. Engl. 1991, 30, 555.
(29) Espelt, L.; Parella, T.; Bujons, J.; Solans, C.; Joglar, J.; Delgado,
Notes
A.; Clapes
(30) Espelt, L.; Bujons, J.; Parella, T.; Calveras, J.; Joglar, J.; Delgado,
A.; Clapes
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, P. Chem.Eur. J. 2003, 9, 4887.
The authors declare no competing financial interest.
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, P. Chem.Eur. J. 2005, 11, 1392.
ACKNOWLEDGMENTS
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(31) Dalby, A.; Dauter, Z.; Littlechild, J. A. Protein Sci. 1999, 8, 291.
This work was supported by the Spanish MINECO
(CTQ2012-31605 and CTQ2012-32436), the Generalitat de
Catalunya (2009 SGR 00281), and ERA-IB MICINN
(PIM2010EEI-00607) (EIB.10.012. MicroTechEnz-EIB, www.
fkit.unizg.hr/miten).
NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published ASAP on May 14, 2014. Text
describing adducts 5 and 6 of Scheme 2 was corrected. The
revised paper was reposted on May 16, 2014.
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