Efficient enzymatic routes for the synthesis of new eight-membered cyclic β-amino acid and β-lactam enantiomers

Article abstract of DOI:10.3390/molecules22122211

Efficient enzymatic resolutions are reported for the preparation of new eight-membered ring-fused enantiomeric β-amino acids [(1R, 2S)-9 and (1S, 2R)-9] and β-lactams [(1S, 8R)-3, (1R, 8S)-3 (1S, 8R)-4 and (1R, 8S)-7], through asymmetric acylation of (±)-4 (E > 100) or enantioselective hydrolysis (E > 200) of the corresponding inactivated (±)-3 or activated (±)-4 β-lactams, catalyzed by PSIM or CAL-B in an organic solvent. CAL-B-catalyzed ring cleavage of (±)-6 (E > 200) resulted in the unreacted (1S, 8R)-6, potential intermediate for the synthesis of enantiomeric anatoxin-a. The best strategies, in view of E, reaction rate and product yields, which underline the importance of substrate engineering, are highlighted.

Full text of DOI:10.3390/molecules22122211

molecules
Article
Efficient Enzymatic Routes for the Synthesis of
New Eight-Membered Cyclic β-Amino Acid and
β-Lactam Enantiomers
Eniko˝ Forró 1,*, Loránd Kiss 1, Judit Árva 1 and Ferenc Fülöp 1,2,
*
ID
1
Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eötvös u. 6, Hungary;
kiss.lorand@pharm.u-szeged.hu (L.K.); Arva.Judit@pharm.u-szeged.hu (J.A.)
Research Group of Stereochemistry of the Hungarian Academy of Sciences, University of Szeged,
H-6720 Szeged, Eötvös u. 6, Hungary
2
*
Correspondence: forro.eniko@pharm.u-szeged.hu (E.F.); fulop@pharm.u-szeged.hu (F.F.);
Tel.: +36-62-544-964 (E.F.); +36-62-545-564 (F.F.)
Received: 3 November 2017; Accepted: 8 December 2017; Published: 13 December 2017
Abstract: Efficient enzymatic resolutions are reported for the preparation of new eight-membered
ring-fused enantiomeric
(1S,8R)- and (1R,8S)-
hydrolysis (E > 200) of the corresponding inactivated (
PSIM or CAL-B in an organic solvent. CAL-B-catalyzed ring cleavage of (
the unreacted (1S,8R)- , potential intermediate for the synthesis of enantiomeric anatoxin-a. The best
β
-amino acids [(1R,2S)-
], through asymmetric acylation of (
)- or activated (
9
and (1S,2R)-
9
±
] and
β
-lactams [(1S,8R)-
(E > 100) or enantioselective
)-4 β-lactams, catalyzed by
)- (E > 200) resulted in
3, (1R,8S)-3
4
7
)-
4
±
3
±
±
6
6
strategies, in view of E, reaction rate and product yields, which underline the importance of substrate
engineering, are highlighted.
Keywords: anatoxin-a; β-Amino acid; enzyme catalysis; β-Lactam; traceless activating group
1. Introduction
Chiral
properties and their use in diverse syntheses. For example, (1R,2S)-2-aminocyclopentanecarboxylic acid
(cispentacin) [ ], the simplest, naturally occurring carbocyclic -amino acid, is an antifungal antibiotic,
but can also be found in the structures of some natural products, e.g., amipurimycin [ ]. Its methylene
derivative, (1R,2S)-2-amino-4-methylenecyclopentanecarboxylic acid (icofungipen, PLD-118) [ ],
in turn, is active in vitro against Candida species. Enantiomeric eight-membered ring-fused -lactams
(1R,8S)- and (1S,8R)-9-azabicyclo[6.2.0]dec-4-en-10-one are potential key intermediates [ ] in the
syntheses of anatoxin-a [ ]. It is a neurotoxic alkaloid, one of the most toxic of the cyanobacterial toxins,
but a potent and stereospecific agonist at nicotinic acetylcholine receptors [ ]. A relatively large
number of publications deal with its isolation from strains of Anabaena flos aqua, a freshwater blue-green
alga, but synthetic methods [ 10], also for the preparation of new anatoxin-a homologues [11] have
also been described. Cyclic -amino acids can serve as building blocks for the synthesis of modified
peptides with increased activity and stability [12] and with well-defined three-dimensional structures
(foldamers) (e.g., -peptides with possible antibiotic activity) similar to those of natural peptides [13 14].
β-amino acids and β-lactams are important compounds because of their pharmacological
1
β
2
3
β
4
5
5–7
8–
β
β
,
Additionally, the alkene functionality in molecules is amenable to a range of transformations. Cyclic
β-amino acids can also be used in heterocyclic [15,16] and combinatorial [17] chemistry.
In addition to conventional resolution methods for the preparation of enantiomeric
β-amino acids
and
β
-lactams, enzymatic strategies have also been described [18 20]. Our research group has also
–
devised a number of efficient enzymatic kinetic and sequential kinetic resolution processes (acylations,
deacylations and hydrolyses). Most of these methods have been reviewed [21–23].
Molecules 2017, 22, 2211; doi:10.3390/molecules22122211
www.mdpi.com/journal/molecules

Molecules 2017, 22, 2211
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A primary aim of this work was to devise adequate enzymatic strategies for the
preparation of valuable new enantiomeric eight-membered carbocyclic -lactam and -amino
acid derivatives. In view of earlier results on enzymatic acylation of N-hydroxymethyl
eight-membered carbocyclic -lactams [ 24], we first planned to carry out enzymatic acylation of
N-hydroymethyl-9-azabicyclo[6.2.0]dec-6-en-10-one [( )- ] (Figure 1). The extensive investigations
of the lipase-catalyzed ring cleavage of unactivated [25 27] and activated [28 -lactams then
suggested the possibility of the lipase-catalyzed enantioselective ring cleavage of racemic
unactivated 9-azabicyclo[6.2.0]dec-6-en-10-one [( )- ] and activated ( )- and N-hydroxymethyl
9-azabicyclo[6.2.0]dec-4-en-10-one [( )- ]. A systematic comparison of the efficiency of these methods,
β
β
β
4,
±
4
–
] β
±
3
±
4
±
6
with regard to E, reaction rate and yield for the products, was also intended.
Figure 1. Substrates (±)-3, (±)-4 and (±)-6 in the enzymatic strategies planned.
2. Results and Discussion
2.1. Synthesis of β-lactams (±)-3–(±)-6
Lactams (
±
)-
3
and (
±
)-
5
were synthesised from cyclooctadiene
1
or
2
by the addition of
30].
chlorosulfonyl isocyanate (CSI), according to a slightly modified literature procedure (Scheme 1) [29
,
Then product lactams were reacted with paraformaldehyde under sonication to form N-hydroxymethyl
β-lactams (±)-4 and (±)-6 [4].
Scheme 1. Synthesis of (±)-3–(±)-6.
2.2. Lipase-Catalyzed O-acylation of (±)-4
On the basis of the earlier results on the enzymatic resolution of N-hydroxymethyl
9-azabicyclo[6.2.0]dec-4-en-10-one [
first the acylation of ( )- (Scheme 2) was carried out with vinyl butyrate (VB) in the presence of PSIM
(Burkholderia cepacia) in diisopropyl ether (iPr2O) at −15 ◦C (Table 1, entry 1).
4] and N-hydroxymethyl 9-azabicyclo[6.2.0]decane-4-en-10-one [24],
±
4
Scheme 2. Lipase-catalyzed O-acylation of (±)-4.

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Table 1. Enzyme-catalyzed acylation of (±)-4 a.
b
b
Enzyme
(30 mg mL
Acyl Donor
(Equiv)
Temp.
( C)
R. Time
(Min)
Conv.
(%)
ees
eep
Entry
Solvent
E
−1
◦
)
(%)
(%)
1
2
3
4
PSIM
PSIM
PSIM
PSIM
VB (2)
VB (2)
VB (2)
iPr2O
iPr2O
iPr2O
iPr2O
-15
2-3
30
120
60
10
10
10
25
44
43
46
51
45
50
76
70
77
87
77
91
96
94
90
84
96
92
112
67
44
32
114
VB (10)
VB (10) + Et3N +
30
5
PSIM
iPr2O
30
Na2SO4
76
2,2,2-Trifluoroethyl-
butyrate(10)
VA (10)
6
PSIM
iPr2O
30
20
49
83
86
34
7
8
9
PSIM
PSIM
PSIM
AK c
iPr2O
iPr2O
iPr2O
iPr2O
iPr2O
iPr2O
iPr2O
iPr2O
30
30
30
30
30
30
30
30
30
30
30
10
240
10
20
240
10
50
120
10
5
60
50
16
52
49
25
29
67
20
51
49
49
81
12
87
74
14
12
2
23
87
89
86
82
62
80
78
42
29
1
91
85
93
89
25
5
25
17
28
2
EtOAc(10)
Ac2O (10)
VB (10)
VB (10)
VB (10)
VB (10)
VB (10)
VB (10)
VB (10)
10
11
12
14
14
15
16
17
AY c
CAL-A c
CAL-B
PPL
1
26
34
82
47
PSIM
PSIM
PSIM
tBuOMe
Toluene
Acetone
VB (10)
a
0.015 M substrate; b According to GC (Experimental Section); c Contains 20% (w/w) of lipase adsorbed on Celite
in the presence of sucrose.
When the acylation was performed at higher temperatures, the reaction rate increased with the
concomitant decrease in E (entries 2 and 3 vs. 1). As the amount of VB was increased from 2 to
10 equiv., the reaction rate increased while E apparently decreased (entry 4 vs. 3). The addition of a
catalytic amount of Et3N and Na2SO4 resulted in a clear increase in E (entry 5 vs. 4).
To further increase the E values, acyl donors, such as 2,2,2-trifluoroethyl butyrate, vinyl acetate
(VA), ethyl acetate (EtOAc) and acetic anhydride (Ac2O) were tested (entries 6–9). Unfortunately, none
of the acyl donors tested exerted any beneficial influence on the reaction course. Several solvents have
also been tested. When iPr2O was replaced by tBuOMe, practically no change was observed in the
reaction course (entry 15 vs. 4). The same high E but somewhat lower reaction rate was observed in
acetone (entry 17), while the best E and fastest reaction were detected in toluene (entry 16). Finally the
environmentally less harmful iPr2O was selected as solvent.
In addition to lipase PSIM, several enzymes, such as lipase AK (Pseudomonas fluorescens), lipase
AY (Candida rugosa), CAL-A (lipase A from Candida antarctica), CAL-B (lipase B from Candida antarctica)
and PPL (porcine pancreatic lipase) have been investigated (entries 10–14). However, in terms of E and
reaction rate, none of them proved to be better than PSIM.
2.3. Lipase-Catalyzed Ring Cleavage of (±)-3–(±)-6
In view of the results on the lipase-catalyzed ring cleavage of carbocyclic
the ring opening of ( )- was attempted with 1 equiv. of H2O in the presence of CAL-B (lipase B
from Candida antarctica) in iPr2O at 60 ◦C (Scheme 3; Table 2, entry 1). When the ring cleavage of the
regioisomeric 9-azabicyclo[6.2.0]dec-4-en-10-one [( )- ] was performed under the same conditions,
β-lactams [25–27],
±
3
±
5
a similar fast reaction and the same high ee values (>98%) were observed (entry 2).
In further studies, we have probed our very recent results found about the ring cleavage
of specially activated lactams [28], where the activating group underwent to a traceless, in situ
degradation. Accordingly, the ring cleavage of (
CAL-B and benzylamine to capture formaldehyde in iPr2O at 60 C (Scheme 4, Table 2, entry 3).
±
)-
4
was attempted with H2O in the presence of
◦
Excellent ee (>99%) characterized formed amino acid (1R,2S)-
cleavage of regioisomeric ( )- was also carried out under the same conditions (entry 4), and the same
high ee (>98%) for amino acid (1R,2S)-10 and unreacted lactam (1S,8R)- , potential intermediate in the
9 at a conversion close to 50%. The ring
±
6
6

Molecules 2017, 22, 2211
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synthesis of enantiomeric anatoxin-a was observed. In good accordance with the earlier observation
that the hydroxymethyl group activates the ring cleavage of lactams, racemic and underwent ring
4
6
cleavage much faster than their corresponding inactivated counterparts (entry 3 vs. 1 and 4 vs. 2).
Scheme 3. Lipase-catalyzed ring cleavage of (±)-3 and (±)-5.
Table 2. CAL-B-catalyzed ring cleavage of racemic 3 a, 4 b, 5 a and 6 b.
c
e
d
Entry
Substrate
R. Time (h)
Conv. (%)
eep (%)
ees (%)
1
2
3
4
(±)-3
(±)-5
(±)-4
(±)-6
5
5
3
3
43
46
49
50
75
84
96
>99
>99
>99
98
>99
0.015 M substrate, 30 mg mL−1 CAL-B, 0.5 equiv. of H2O, iPr2O, 60 ◦C; b 0.015 M substrate, 30 mg mL−1 CAL-B,
a
0.5 equiv. of H2O, 1 equiv. of benzylamine, iPr2O, 60 ◦C; Calculated from eeS and eeP; According to GC
c
d
(Experimental Section); e Determined by using GC, after double derivatization (DD) [31].
Scheme 4. Lipase-catalyzed two-step transformation of (±)-4 and (±)-6.
On the basis of the preliminary results, preparative-scale resolutions of racemic 3, 4 and 6 were
performed under the optimized conditions (footnotes to Table 3). The results are reported in Table 3
and Experimental Section.
Table 3. Preparative-scale resolutions.
Product
Unreacted substrate
[α]2D5
(EtOH)
a
Reaction R. Time Conv.
Yield
(%)
eeP
(%)
Yield
(%)
eeS
Isomer
[α]2D5
Isomer
Partner
(Min)
(%)
(%)
(±)-3 b
(±)-4 g
(±)-4 j
(±)-6 j
−17 d
+39.2 h
−17.1 d
+24.9 k
−140.6 f
−142.4 i
−140.4 f
−28.7 l
H2O
VB
H2O
H2O
330
10
180
180
50
51
49
50
48
46
47
47
(1R,2S)-9
(1R,8S)-7
(1R,2S)-9
(1R,2S)-10
99 c
94 e
49
44
48
46
(1S,8R)-3
(1S,8R)-4
(1S,8R)-4
(1S,8R)-6
99 e
96 e
98 e
99 e
>99 c
99 c
a Calculated from eeS and eeP; b 0.015 M substrate, 30 mg mL−1 CAL-B, 0.5 equiv. of H2O, iPr2O, 60 ◦C; c Determined
d
f
by GC after DD (Experimental Section); c = 0.35; H2O; e Determined by GC (Experimental Section); c = 0.5;
g
0.015 M substrate, 30 mg mL−1 PSIM, 10 equiv. of VB, catalytic amount of Et3N and Na2SO4, iPr2O 30 ◦C;
h
c = 0.35; EtOH; i c = 0.45; j 0.015 M substrate, 30 mg mL−1 CAL-B, 0.5 equiv. of H2O, 1 equiv. of benzylamine,
iPr2O, 60 ◦C; k c = 0.3; H2O; l c = 0.5.

Molecules 2017, 22, 2211
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2.4. Further Transformations
Hydrolysis of enantiomeric
3,
4,
6
and
7
with 18% aqueous HCl (Schemes 2–4) gave the
corresponding hydrochloride salts 9·HCl and 10·HCl (ee > 97%), while treatment of enantiomeric
with NH4OH/MeOH (Scheme 2) resulted in -lactams (1R,8S)- and (1S,8R)- (ee 95%). Catalytic
reduction of enantiomeric lactams and amino acids with H2 or cyclohexene as a hydrogen donor
and using palladium-on-carbon furnished saturated lactams (1R,8S)- and (1S,8R)- , and amino acids
4 and
7
β
3
3
≥
3
9
8
8
(1R,2S)-11 and (1S,2R)-11, respectively (Schemes 2 and 3), without a drop in ee (>96%). Physical data
on the enantiomers prepared are reported in Table 4 and Experimental Section.
Table 4. Physical data on enantiomers prepared.
[α]2D5
Entry
Enantiomers
ee (%)
1
2
3
4
5
6
7
8
9
(1R,8S) 3 from (1R,8S)-7
(1S,8R) 3 from (1S,8R)-4
(1R,8S) 8 from (1R,8S)-3
95
96
98
96
99
99
98
97
99
99
+147 (c = 0.5; EtOH)
−148.7 (c = 0.4; EtOH)
+17.7 (c = 0.5; CHCl3)
−17.1 (c = 0.5; CHCl3)
+19.6 (c = 0.5; H2O)
+19.6 (c = 0.6; H2O)
−17.3 (c = 0.35; H2O)
−15.0 (c = 0.5; H2O)
+19.2 (c = 0.4; H2O)
−19 (c = 0.33; H2O)
(1S,8R) 8 from (1S,8R)-3
(1S,2R)-9·HCl from (1S,8R) 3
(1S,2R)-9·HCl from (1S,8R) 4
(1R,2S)-9·HCl from (1R,8S)-7
(1S,2R)-10·HCl from (1S,8R)-6
(1R,2S)-11 from (1R,2S)-9
(1S,2R)-11 from (1S,2R)-9
10
2.5. Absolute Configurations
Absolute configurations were determined by comparing the [
compounds with literature data. Specifically, [ ] values of enantiomeric compounds
3 and 4), obtained from enantiomeric compounds and (Scheme 2), were compared with the
literature data of (1S,8R)- 18 (c = 0.5; CHCl3)} [25]. In a
-azabicyclo[6.2.0]decan-10-one {[α]D25
similar way, the [ ] values of enantiomeric compounds 11 (Table 4, entries 9 and 10), obtained
α
] values of the enantiomeric
α
8
(Table 4, entries
4
7
9
=
−
α
from enantiomeric compounds 9·HCl (Scheme 3), were compared with the literature data for
(1R,2S)-2-aminocyclooctane-1-carboxylic acid {[α]2D5 = +17.8 (c = 0.4; H2O)} [25]. Thus, the (1R,8S)
configuration is determined for ester
is assigned to unreacted alcohol
cleavage of either inactivated lactams
9
and the corresponding lactam (
and the corresponding lactam (
and or activated lactams and 6
3
3
), and the (1S,8R) configuration
). The CAL-B-catalyzed ring
afforded the corresponding
4
3
5
4
amino acids with (1R,2S) absolute configuration I.
3. Experimental Section
3.1. Materials and Methods
CAL-B (lipase B from Candida antarctica), produced by the submerged fermentation of a genetically
modified Aspergillus oryzae microorganism and adsorbed on a macroporous resin (Catalogue no.
L4777), and porcine pancreatic lipase (PPL) were from Sigma-Aldrich (St. Louis, MO, USA). CAL-A
(lipase A from Candida antarctica) was purchased from Roche Diagnostics Corporation. Lipase PSIM
(Burkholderia cepacia, immobilized on diatomaceous earth) was a kind gift from Amano Enzyme
Europe Ltd. (Suqian, China) Lipase AK (Pseudomonas fluorescens) was from Amano Pharmaceuticals,
and lipase AY (Candida rugosa) was from Fluka. The solvents were of the highest analytical
grade. Melting points were determined with a Kofler apparatus. NMR spectra were recorded
on a Bruker DRX 400 spectrometer. Optical rotations were measured with a Perkin-Elmer 341
polarimeter. Elemental analyses were performed with a Perkin-Elmer CHNS-2400 Ser II Elemental
Analyzer. The syntheses of racemic N-hydroxymethyl 9-azabicyclo[6.2.0]dec-6-en-10-one [(±)-5] and
N-hydroxymethyl 9-azabicyclo[6.2.0]dec-4-en-10-one [( )-6] were performed according to our earlier
±
method [4].

Molecules 2017, 22, 2211
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3.2. Typical Small-Scale Enzymatic Experiments
Racemic
(30 mg mL−1). The tested acyl donor (2 or 10 equiv.) in acylations or H2O (1 equiv.) in hydrolyses was
added. The mixture was shaken at
15 ◦C, 2–3 ◦C, 30 ◦C or 60 ◦C. The progress of the reaction was
followed by taking samples from the reaction mixture at intervals and analysing them by using a gas
chromatograph equipped with a chiral column. The ee values for the unreacted -lactam enantiomers
were determined directly on a Chromopack Chiralsil-Dex CB column [retention times are given in min;
β-lactam in an organic solvent (0.05 M solution, 1 mL) was added to the lipase tested
−
β
◦
◦
◦
140 C for 25 min
140 ◦C for 7 min
or on a Chirasil-L-Val column, [140 ◦C for 7 min
(1S,8R)-4: 14.39 (antipode: 14.61); (1S,8R)-6: 14.40 (antipode: 14.87); (1R,8S)-7: 14.67 (antipode: 14.36)].
The ee values for the -amino acids produced were determined on a Chirasil-L-Val column, after
→
190 C (temperature rise 20 C
190 ◦C (temperature rise 10 ◦C min−1; 140 kPa, (1S,8R)-
190 ◦C (temperature rise 10 ◦C/min; 140 kPa),
·
min−1; 140 kPa, (1S,8R)-
3
: 27.74 (antipode: 27.56);
→
5: 12.55 (antipode: 12.12)]
→
β
double derivatization with (i) CH2N2 [31] (caution! derivatization with diazomethane should be performed
under a well-ventilated hood); (ii) Ac2O in the presence of 4-dimethylaminopyridine and pyridine
[120 ◦C for 7 min
→
190 ◦C (temperature rise 10 ◦C min−1; 140 kPa, (1R,2S)-9: 12,41 (antipode: 12.95);
(1R,2S)-10: 12.88 (antipode: 14.06)] and after double derivatization with (i) CH2N2 and (ii) (PropCO)2O
in the presence of 4-dimethylaminopyridine and pyridine [140 ◦C for 10 min
rise 10 ◦C min−1; 140 kPa; (1R,2S)-11: 17.58 (antipode: 17.78)].
→
190 ◦C (temperature
3.3. Synthesis of Racemic 9-Azabicyclo[6.2.0]dec-6-en-10-one [(±)-3]
A solution of CSI (2.28 mL, 26.2 mmol) in CH2Cl2 (25 mL) was added dropwise to a stirred
◦
solution of 1,3-cyclooctadiene (3 mL, 26.2 mmol) in CH2Cl2 (25 mL) at 0 C. The reaction mixture
was stirred at room temperature for 21 h, and the resulting liquid was then added dropwise to a
vigorously stirred solution of Na2SO3 (0.33 g) and K2CO3 (7.12 g) in H2O (50 mL). After stirring the
mixture for 3.5 h, the combined organic layers were dried (Na2SO4) and, after filtration, concentrated.
The resulting white solid, racemic 3 (3.41 g, 86%), was recrystallized from iPr2O (m.p. 103–106 ◦C).
The 1H-NMR (400 MHz, CDCl3, 25 ◦C, TMS)
3.34–3.38 (1H, dd, J = 5.34, 12.26 Hz, H-1), 4.57–4.58 (1H, m, H-8), 5.41–5.79 (2H, m, CHCH), 6.01 (1H,
δ
(ppm) data for (
±
)-
3
: 1.46–2.11 (8H, m, 4
×
CH2),
bs, NH). Anal. calcd for C9H14NO: C, 71.49; H, 8.67; N, 9.26; Anal. found: C, 71. 30; H, 8.52; N, 9.23.
3.4. Synthesis of Racemic N-Hydroymethyl-9-azabicyclo[6.2.0]dec-6-en-10-one [(±)-4]
Racemic 3 (3 g, 19.84 mmol) was dissolved in THF (35 mL) followed by adding paraformaldehyde
(0.6 g, 19.84 mmol), K2CO3 (0.17g, 1.19 mmol) and H2O (1.1 mL). The solution was sonicated for 4 h,
the solvent was evaporated off and the residue was dissolved in ethyl acetate (50 mL). The solution
was dried (Na2SO4) and then concentrated. The residue was recrystallised from iPr2O to afford (
±
)-
4
as a white crystalline product (3.2 g, 89%; m.p. 81–84 ◦C).
◦
The 1H-NMR (400 MHz, CD3OD, 25 C, TMS)
δ
(ppm) data for (
±
)-
4: 1.45–2.09 (8H, m, 4
×
CH2),
3.30–3.32 (1H, dd, J = 1.89. 5.52 Hz, H-1), 3.33-3.39 (1H, s, OH), 4.60–4.64 (1H, dd, J = 7.78, 11,58 Hz,
CH2OH), 4.69–4.71 (1H, m, H-8), 4.85–4.89 (1H, dd, J = 6.47, 11,66 Hz, CH2OH), 5.57–5.60 (2H, m,
CHCH). Anal. calcd. for C10H15NO2: C, 66.27; H, 8.34; N, 7.73; found: C, 66.19; H, 8.44; N, 7.76.
3.5. Gram-Scale Resolution of N-hydroxymethyl-9-azabicyclo[6.2.0]dec-6-en-10-one [(
±
)-4] through Acylation
Racemic
4
(1 g, 5.51 mmol) was dissolved in iPr2O (40 mL). Lipase PSIM (1.2 g, 30 mg mL−1) then
VB (6.29 mL◦, 55.1 mmol), Et3N (a few drops) and Na2SO4 (0.2 g) were added and the mixture was
shaken at 30 C. After 10 min, the enzyme was filtered off at 51% conversion and iPr2O was evaporated.
The residue was chromatographed on silica (EtOAc:n-hexane 7:3) affording unreacted (1S,8R)-
44%; [
25 = –142.4 (c = 0.45, EtOH); m.p. 79–83 ◦C (recrystallized from iPr2O); ee = 96%] and ester
(1R,8S)-7 (0.63 g, 46%; [α]2D5 = +39.2 (c = 35, EtOH); ee = 94%) as a pale-yellow oil.
4 [0.44 g,
α
]
D

Molecules 2017, 22, 2211
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◦
The 1H-NMR (400 MHz, CDCl3, 25 C, TMS)
δ
(ppm) data for (1R,8S)-
7: 0.93–0.97 (3H, t, J = 7.4 Hz,
CH3), 1.41–1.44 (2H, m, CH2CH2CH3), 1.57–2.33 (10H, m, CH2CH2CH3 and 4
×
CH2 from ring),
3.31–3.32 (1H, m, H-1), 4.59–4.60 (1H, dd, J = 1.58, 3.88 Hz, H-8), 5.15–5.18 (1H, d, J = 11.36 Hz,
CH2OCOPr), 5.20–5.23 (1H, d, J = 11.36 CH2OCOPr), 5.49–5.81 (2H, m, CHCH). Anal. calcd. for
C14H21NO3: C, 66.91; H, 8.42; N, 5.57; found: C, 66.83; H, 8.60; N, 5.42.
The 1H-NMR (400 MHz, CDCl3, 25 ◦C, TMS)
δ (ppm) data for (1S,8R)-4 are similar to those for
(±)-4. Anal. found: C, 66.39; H, 8.24; N, 7.73.
3.6. Gram-Scale Resolution of 9-azabicyclo[6.2.0]dec-6-en-10-one [(±)-3] through Hydrolysis
Racemic
(0.5 g, 3.3 mmol) was dissolved in iPr2O (20 mL). CAL-B (0.6 g, 30 mg mL−1) and
H2O (29.7
L, 1.65 mmol) were added, and the mixture was shaken in an incubator shaker at 60 ◦C
for 5.5 h. The reaction was stopped by filtering off the enzyme at 50% conversion. The solvent was
evaporated off and the residue crystallized to give (1S,8R)- 140.6 (c = 0.5;
[240 mg, 48%; [α]D25
EtOH); m.p. 151–153 ◦C recrystallized from iPr2O; ee = 99%]. The filtered-off enzyme was washed
3
µ
3
=
−
with distilled water (3
×
15 mL), and the water was evaporated off, yielding crystalline β-amino acid
(1R,2S)-9 [268 mg, 48%; [α]2D5 = −17 (c = 0.35; H2O); 266–288 ◦C with sublimation (recrystallized from
H2O/acetone); ee = 99%].
The 1H-NMR (400 MHz, CD3OD, 25 C, TMS)
δ
(ppm) data for (1S,8R)-
3
were similar to those for
CH2) 2.86–2.90
◦
(±)-3. Anal. found: C, 71.33; H, 8.57; N, 9.10.
The 1H-NMR (400 MHz, D2O)
δ
(ppm) data for (1R,2S)-
9
: 1.45–2.24 (8H, m, 4
×
(1H, m, H-1) 4.42–4.45 (1H, m, H-2) 5.71–6.03 (2H, m, CHCH). Anal. calcd. for C9H15NO2: C, 63.88; H,
8.93; N, 8.28; found: C, 63.99; H, 8.84; N, 8.28.
3.7. Gram-Scale Resolution of N-hydroxymethyl 9-azabicyclo[6.2.0]dec-6-en-10-one [( )-4] through Hydrolysis
±
( )-4 (250 mg, 1.37 mmol) was dissolved in iPr2O (10 mL) followed by the addition of CAL-B
±
(300 mg, 30 mg mL−1), benzylamine (150◦ µL, 1.37 mmol) and H2O (12.3
µL, 0.68 mmol) and by shaking
the mixture in an incubator shaker at 60 C for 3 h. The reaction was stopped by filtering off the enzyme
at 49% conversion. After solvent evapo◦ration the residue (1S,8R)-
4 crystallized out [120 mg, 48%;
140.4 (c = 0.5; EtOH), m.p. 80–83 C (recrystallized from iPr2O), ee = 98%]. The filtered enzyme
[α]2D5
=
−
was washed with distilled H2O (3
×
15 mL), and crystalline
β-amino acid (1R,2S)-9 was isolated after
evaporation of H2O. [109 mg, 47%; [α]D25
H2O/acetone), ee > 99%].
=
−
17.1 (c = 0.35; H2O), m.p. 264–265 ◦C (recrystallised from
The 1H-NMR (400 MHz, CD3OD, 25 C, TMS)
δ
(ppm) data for (1S,8R)-
4
were similar to those for
◦
(±)-4. Anal. found: C, 66.18; H, 8.35; N, 7.73.
The 1H-NMR (400 MHz, D2O) data
δ
(ppm) for (1R,2S)-
9
from ( )-
±
4 were similar to those for
(1R,2S)-9 from (±)-3 (4.4.). Anal. found: C, 63.95; H, 9.01; N, 8.25.
3.8. Gram-Scale Resolution of N-hydroxymethyl 9-azabicyclo[6.2.0]dec-4-en-10-one [(
±
)-6] through Hydrolysis
Via the procedure described above, the reaction of racemic 6 (250 mg, 1.37 mmol), benzylamine
(150 µL, 1.37 mmol) and H2O (12.3 µL, 0.68 mmol) in iPr2O (10 mL) in the presence of CAL-B (300 mg,
30 mg mL−1) at 60 C afforded amino acid (1R,2S)-10 [108 mg, 47%; [α]D25 = +24.9 (c = 0.3; H2O),
◦
◦
lit. [26] = +23.9 (c = 0.3; H2O), ee = 95%; m.p.: 264–265 C (recrystallized from H2O/acetone), lit.
[26] 218–220 C; ee = 99%] and unreacted (1S,8R)-
28.5 (c = 1; MeOH), lit. [4] =
n-hexane), lit. [4] 48–49 ◦C; ee = 99%] after 3 h.
The 1H-NMR (400 MHz, D2O)
(ppm) data for (1R,2S)-10: 1.81–2.58 (8H, m, 4
◦
6
, [115 mg, 46%; [α]D25
=
−
◦ 28.7 (c = 0.5; EtOH),
[α]2D5
=
−
−
27.0 (c = 1; MeOH), ee = 97%; m.p. 48–49 C (recrystallized from
δ
×
CH2) 2.79 (1H,
m, H-1) 3.70–3.73 (1H, m, H-2) 5.72–5.73 (2H, m, CHCH). Anal. calcd. for C9H15NO2: C, 63.88; H, 8.93;
N, 8.28; found: C, 63.59; H, 8.88; N, 8.18.

Molecules 2017, 22, 2211
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The 1H-NMR (400 MHz, CDCl3, 25 ◦C, TMS) data
: 1.86–2.21 and 2.37–2.44 (8H, m, 4 CH2), 3.28–3.30 (1H, m, H-1), 3.71 (1H, bs, OH), 3.93–3.96
(1H, m, H-8), 4.45–4.48 (1H, d, J = 11.6 Hz, CH2OH), 4.73–4.79 (1H, d, J = 11.6 Hz, CH2OH), 5.67–5.71
(2H, m, CHCH). Anal. calcd. for C10H15NO2: C, 66.27; H, 8.34; N, 7.73; found: C, 66.10; H, 8.28; N, 7.66.
δ
(ppm) for (1S,8R)-
6
were similar to those for
(
±
)-
6
×
3.9. Synthesis of (1R,8S)-8 and (1S,8R)-8 through (1R,8S)-3 and (1S,8R)-3 from β-lactams (1S,8R)-4
and (1R,8S)-7
Ester (1R,8S)-7 (100 mg, 0.66 mmol) was dissolved in MeOH (10 mL), NH4OH (1 mL) was added
and the mixture was stirred at room temperature for 6 h. The solvent was evaporated off, the residue
chromatographed on silica (EtOAc:n-hexane 7:3) providing white crystals of (1R,8S)-
3
[48 mg, 80%;
[59 mg, 71%;
and (1R,8S)- were
[α]2D5 = +147 (c = 0.5; EtOH); m.p. 152–153 ◦C (recrystallised from iPr2O); ee = 95%].
Similarly, (1S,8R)-4 (100 mg, 0.55 mmol) afforded white crystals of (1S,8R)-3
[α]2D5 = −148.7 (c = 0.4; EtOH); m.p. 150–153 ◦C (recrystallised from iPr2O); ee = 96%].
1
◦
The H-NMR (400 MHz, CD3OD, 25 C, TMS)
δ
(ppm) data for (1S,8R)-
3
3
similar to those for (
C, 71.55; H, 8.67; N, 9.12.
Palladium-on-carbon (100 mg) was added to (1R,8S)-
in a mixture of MeOH (10 mL) and cyclohexene (1 mL). The mixture was treated at reflux temperature
±
)-3. Anal. found for (1S,8R)-3: C, 71.45; H, 8.57; N, 9.22. Anal. found for (1R,8S)-3:
3
or (1S,8R)-3 (100 mg, 0.66 mmol) dissolved
for 3 h, and then the catalyst wa◦s filtered off. After evaporation, (1R,8S)-
8
[51 mg, 51%; [α]2D5 = +17.7
[47 mg, 47%;
(c = 0.5; CHCl3); m.p. 104–106 C (recrystallized from iPr2O); ee = 98%] or (1S,8R)-
8
[α]2D5
=
−
17.1 (c = 0.5; CHCl3), lit. [25] [α]D25
=
−
18 (c = 0.5; CHCl3)]; m.p. 105–107 ◦C, lit. [25] m.p.
108–112 ◦C; ee = 96%] was obtained as white crystalline product.
The 1H-NMR (400 MHz, CD3OD, 25 C, TMS)
(1R,8S)- : 1.30–2.09 (12H, m, 6xCH2), 3.03–3.06 (1H, m, H-1), 3.65–3.69 (1H, m, H-8), 5.89 (1H, bs, NH).
Anal. found for C9H15NO, (1S,8R)- : C, 70.48; H, 9.76; N, 9.14 and for (1R,8S)- : C, 70.51; H, 9.82; N, 9.08.
δ (ppm) data for (1S,8R)-8 were similar to those for
◦
8
8
8
3.10. Preparation of (1R,2S)-11 and (1S,2R)-11
Palladium-on-carbon (60 mg) was added to enantiomer (1R,2S)-9 or (1S,2R)-9 (100 mg) dissolved
in MeOH (20 mL) and H2 was bubbled through the system at RT for 6 h. The catalyst was then
filtered off and after evaporation white crystalline enantiomers of 2-aminocyclooctane-1-carboxylic
acid (1R,◦2S)-11 [69.1 mg, 68%; [α]D25 = +19.2 (c = 0.4; H2O), lit. [25] [α]D25 = +17.8 (c = 0.4; H2O); m.p.
◦
250–252 C (recrystallized from H2O/acetone), lit. [25] m.p◦. 245–248 C; ee = 99%] and (1S,2R)-11
[76.2 mg, 75%; [α]D25
=
−
19 (c = 0.33; H2O); m.p. 241–245 C (recrystallized from H2O/acetone);
ee = 99%] were isolated.
1
The H-NMR (400 MHz, D2O) data
δ (ppm) for (1R,2S)-11 are similar to those for (1S,2R)-11:
1.50–1.92 (12H, m, 6xCH2) 2.78–2.79 (1H, m, H-1) 3.59–3.63 (1H, m, H-2). Anal. calcd. for C9H17NO2:
C, 63.14; H, 10.01; N, 8.18; found for (1R,2S)-11: C, 63.20; H, 10.18; N, 8.02; found for (1S,2R)-11: C,
63.14; H, 9.91; N, 8.29.
3.11. Acidic Hydrolyses to β-amino Acid Hydrochlorides
When the enantiomeric lactam at issue (0.2 mmol) was treated with 18% aqueous HCl (5 mL) at
reflux temperature, the desired amino acid hydrochloride was obtained, as follows:
(1R,2S)-9·HCl [37 mg, 92%; [α]D25
=
−
17.3 (c = 0.35; H2O); m.p. 218–220 ◦C (recrystallised from
EtOH/Et2O); ee = 98%] from 50 mg (1R,8S)-7.
(1S,2R)-9·HCl [35 mg, 86%; [α]2D5 = +19.6 (c = 0.5; H2O); m.p. 214–218 C (recrystallised from
EtOH/Et2O); ee = 99%] from 30 mg (1S,8R) 3.
◦
(1S,2R)-9·HCl [31 mg, 76%; [α]2D5 = +19.6 (c = 0.6; H2O); m.p. 219–220 C (recrystallised from
◦
EtOH/Et2O); ee = 99%] from 36 mg (1S,8R) 4.
(1S,2R)-10·HCl [38 mg, 93%; [α]D25
=
−
15.0 (c = 0.5; H2O), lit. [25] [α]D25
=
−
15.9 (c = 0.3; H2O;
m.p. 200–204 ◦C (recrystallised from EtOH/Et2O ee = 97%] from 36 mg (1S,8R)-6.

Molecules 2017, 22, 2211
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The 1H-NMR (400 MHz, D2O) data
δ
(ppm) for (1R,2S)-9·HCl are similar to those for (1S,2R)-9·HCl:
1.52–2.31 (8H, m, 4 × CH2) 3.21–3.24 (1H, m, H-1) 4.55–4.58 (1H, m, H-2) 5.76–6.14 (2H, m, CHCH).
1
The H-NMR (400 MHz, D2O) data
δ
(ppm) for (1S,2R)-10·HCl: 1.64–2.33 (8H, m, 4
×
CH2)
2.91–2.98 (1H, m, H-1) 3.66–3.69 (1H, m, H-2) 5.56–5.63 (2H, m, CHCH).
Anal. calcd. for C9H16ClNO2: C, 52.56; H, 7.84; N, 6.81; found for (1R,2S)-9·HCl: C, 52.50; H, 7.68;
N, 6.80; found for (1S,2R)-9·HCl from (1S,8R) : C, 52.68; H, 7.64; N, 6.78; found for (1S,2R)-9·HCl from
3
(1S,8R)-4: C, 52.56; H, 7.66; N, 6.95; found for (1S,2R)-10·HCl: C, 52.47; H, 7.77; N, 6.62.
4. Conclusions
A highly efficient CAL-B (lipase B from Candida antarctica)-catalyzed ring-cleavage reaction
◦
(E > 200) of 9-azabicyclo[6.2.0]dec-6-en-10-one [(
new eight-membered ring-fused -lactam (1S,8R)-
more efficient two-step transformation was carried out for the ring cleavage of activated lactams
±
)-
3
3
)] with 1 equiv. of H2O in iPr2O at 60 C resulted
β
and
β-amino acid (1R,2S)-
9
(ee
≥
99%). Even
(
±
)◦-
4
with 1 equiv. of H2O, in the presence of CAL-B using 1 equiv. of benzylamine in iPr2O at
)- , carried out under the same conditions resulted
, potential intermediate for the synthesis of enantiomeric anatoxin-a. These results
60 C. The ring cleavage of regioisomeric (
unreacted (1S,8R)-
±
6
6
underline the importance of substrate engineering, since faster reactions were clearly detected when
activated lactams vs. their inactivated counterparts were reacted. Advantages of these reactions are
the spontaneous degradation of N-hydroxymethyl groups and easy product separation by organic
solvent–H2O extraction.
An indirect enzymatic method, in view of the synthesis of enantiomeric
β-amino acids, has also
been devised, through a relatively fast acylation of N-hydroxymethyl 9-azabicyclo[6.2.0]dec-6-en-10-one
[( )-4)] with 10 equiv. of VB mediated by lipase PSIM (Burkholderia cepacia) using a catalytic amount of
±
Et3N and Na2SO4 in iPr2O at 30 ◦C (E = 114). This route is somewhat less efficient and, in fact, it is a
longer procedure to prepare amino acid enantiomers. However, it ensures the simultaneous preparation
of new β-lactam enantiomers [(1S,8R)-4 and (1R,8S)-7, and (1S,8R)-3 and (1R,8S)-3, respectively].
Acknowledgments: The authors are grateful to the Hungarian Research Foundation (OTKA No K 71938
and K115731) for financial support.
GINOP-2.3.2-15-2016-00014.
This research was supported by EU-funded Hungarian grant
Author Contributions: Eniko˝ Forró and Ferenc Fülöp conceived and designed the experiments. Loránd Kiss and
Judit Árva performed the experiments. Eniko˝ Forró analyzed the data and wrote the paper. All authors read and
approved the final manuscript. Ferenc Fülöp revised and edited the manuscript.
Conflicts of Interest: There are no conflict to declare.
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©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).