The N-Hydroxymethyl Group as a Traceless Activating Group for the CAL-B-Catalysed Ring Cleavage of β-Lactams: A Type of Two-Step Cascade Reaction

Article abstract of DOI:10.1002/ejoc.201600234

An efficient enzymatic two-step cascade procedure has been devised for rapid access to diverse amino acids from N-hydroxymethyl-β-lactams; representative amino acids include the antifungal agent cispentacin, intermediates for the taxol side-chain, and assorted cathepsin inhibitors. When CAL-B-catalysed hydrolyses of racemic N-hydroxymethyl-β-lactams were performed with H₂O (0.5 equiv.) in iPr₂O at 60 °C, relatively quick (vs. non-activated counterparts) and enantioselective (E > 200) ring cleavage reactions took place. As the ring-opened amino acids formed, the hydroxymethyl group, as a traceless activating group, underwent spontaneous in situ degradation. Consequently, the desired β-amino acid and unreacted N-hydroxymethyl-β-lactam enantiomers (ee > 95 %) were formed. The formation of polymers, induced by liberation of formaldehyde, was successfully restricted by the addition of benzylamine as a capture agent, to the enzymatic reactions. An efficient enzymatic two-step cascade procedure was devised for CAL-B-catalysed hydrolysis of racemic N-hydroxymethyl-β-lactams. Conditions in which the hydroxymethyl group serves as a traceless activating group (E > 200), giving desired β-amino acid along with unreacted starting lactam enantiomers (ee > 95 %) were identified; polymerization was controlled by addition benzylamine addition.

Full text of DOI:10.1002/ejoc.201600234

DOI: 10.1002/ejoc.201600234
Full Paper
Traceless Activation
The N-Hydroxymethyl Group as a Traceless Activating Group for
the CAL-B-Catalysed Ring Cleavage of ꢀ-Lactams: A Type of
Two-Step Cascade Reaction
Enikő Forró,*[a] Zsolt Galla,[a] and Ferenc Fülöp*[a,b]
Abstract: An efficient enzymatic two-step cascade procedure (E > 200) ring cleavage reactions took place. As the ring-opened
has been devised for rapid access to diverse amino acids from amino acids formed, the hydroxymethyl group, as a traceless
N-hydroxymethyl-ꢀ-lactams; representative amino acids include activating group, underwent spontaneous in situ degradation.
the antifungal agent cispentacin, intermediates for the taxol Consequently, the desired ꢀ-amino acid and unreacted N-
side-chain, and assorted cathepsin inhibitors. When CAL-B-cata- hydroxymethyl-ꢀ-lactam enantiomers (ee > 95 %) were formed.
lysed hydrolyses of racemic N-hydroxymethyl-ꢀ-lactams were The formation of polymers, induced by liberation of formalde-
performed with H2O (0.5 equiv.) in iPr2O at 60 °C, relatively hyde, was successfully restricted by the addition of benzyl-
quick (vs. non-activated counterparts) and enantioselective amine as a capture agent, to the enzymatic reactions.
1. Introduction
amipurimycin.[4b,5] Underscoring the pharmacological impor-
tance of ꢀ-lactams, it is noteworthy that (1R,2R,5S,6S)-3-azatri-
cyclo[4.2.1.02.5]non-7-en-4-one [(1R,2R,5S,6S)-8] is used for the
preparation of CEP-28122, an anaplastic lymphoma kinase in-
hibitor.[6]
Chiral ꢀ-lactams, ꢀ-amino acids, and their derivatives are of
great interest from both pharmaceutical and chemical perspec-
tives.[1] Acyclic ꢀ-amino acids have been recognized as an im-
portant class of compounds in the design and synthesis of po-
tential pharmaceutical drugs; for example, they can serve as
building blocks for medicinally important molecules such as
taxol and its analogue taxotère, currently considered to be
among the most efficacious drugs in cancer chemotherapy.[2]
For instance, the (2R,3S)-3-phenylisoserine [(2R,3S)-10] side-
chain is essential for the antitumour activity of taxol. Another
example, (R)-3-amino-3-(o-tolyl)propanoic acid [(R)-12] has
been identified as the enantiomer preferred for assembly of
novel ꢀ-amino acid derivatives that inhibit cathepsin.[3] Some
carbocyclic ꢀ-amino acids, such as the simplest carbocyclic ꢀ-
amino acid, (1R,2S)-2-aminocyclopentanecarboxylic acid (cis-
pentacin) [(1R,2S)-14], or its methylene derivative (1R,2S)-2-
amino-4-methylenecyclopentanecarboxylic acid (icofungipen,
PLD-118) display biological activity,[4] but are also present in
the structures of certain natural products such as the antibiotic
A relatively large number of syntheses, including enzymatic
routes, have been developed for the preparation of such enan-
tiomeric ꢀ-amino acids and ꢀ-lactams[7] and new synthetic ap-
proaches continuously appear. As examples, very efficient CAL-
B-catalysed kinetic (E > 200) and sequential kinetic strategies
(ee products > 98 %) for the preparation of the above-men-
tioned enantiomeric ꢀ-amino acids and ꢀ-lactam have been de-
vised;[8] ring-opening of the corresponding non-activated race-
mic ꢀ-lactams has been central to these approaches. Ring-
opening of N-activated ꢀ-lactams has also been analysed. For
instance, Kanerva et al. examined the effects of N-substitution
with electron-withdrawing groups such as N-acetyl, N-chloro-
acetyl and N-tert-butoxycarbonyl on the lipase-catalysed ring-
opening reaction of racemic 4-phenylazetidin-2-one with MeOH
in dry organic solvents. These efforts revealed that the ring-
opening reaction is sensitive to the structure of the nitrogen-
protecting group.[9] Very recent synthetic efforts involving 1,3-
oxazinan-6-one generation through the use of N-activated ꢀ-
lactams [10] have highlighted the utmost importance of activat-
ing groups (sulfonyl-based groups, amide or tert-butoxy-
carbonyl, with different electron-withdrawing abilities) in dictat-
ing amide bond activation towards hydrolytic scission.
[a] University of Szeged, Institute of Pharmaceutical Chemistry,
Eötvös u. 6, 6720 Szeged, Hungary
E-mail: Forro.Eniko@pharm.u-szeged.hu
galla.zsolt@pharm.u-szeged.hu, fulop@pharm.u-szeged.hu
http://www2.pharm.u-szeged.hu/gyki/index.php/en/staff/
182-prof-forro-eniko
http://www2.pharm.u-szeged.hu/gyki/index.php/en/staff/214-galla-zsolt
http://www2.pharm.u-szeged.hu/gyki/index.php/en/staff/
179-prof-ferenc-fulop
Encouraged by the results generated using traceless activat-
ing groups, such as the carboxylic acid in a three-step synthesis
of ( )-pregabalin[11] or the cyano group in a five-step synthesis
of ( )-isoretronecanol,[12] we set out in the present work to find
an activating group (R) for CAL-B-catalysed ring cleavage of N-
activated ꢀ-lactams ( )-1–4 which, after reaction, might un-
[b] MTA-SZTE Stereochemistry Research Group, Hungarian Academy of
Sciences,
Eötvös u. 6, 6720 Szeged, Hungary
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201600234.
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dergo traceless removal, furnishing the desired ꢀ-amino acids bined with a chemical reaction to furnish a domino sequence,
(Scheme 1). Transformations of the enantiomeric N-activated ꢀ- are known in the literature.[8c,17] The recent two-step cascade
lactams into desired inactivated ꢀ-lactams and ꢀ-amino acid reaction of (3S*,4R*)-( )-1 consists of two consecutive transfor-
hydrochlorides were also planned.
mations, without any "intervention", and requires a fast initial
enantioselective (E > 200) enzymatic step [cleavage of the
lactam at N1–C2 with formation of (2R,3S)-9] and a second
chemical step [spontaneous removal of the hydroxymethyl
group of (2R,3S)-9 with formation of (2R,3S)-10]. The absolute
configurations were verified by comparing the [α] values of the
ring-opened amino acid with literature data (Experimental Sec-
tion).
Scheme 1. CAL-B-catalysed ring cleavage of racemic N-activated ꢀ-lactams 1–
4.
2. Results and Discussion
Racemic N-hydroxymethylated ꢀ-lactams (3S*,4R*)-( )-1, ( )-2,
( )-3 and ( )-4 were prepared from the corresponding lactams
( )-5–8, synthesized using literature methods,[11] with para-
formaldehyde under sonication (Scheme 2).[12]
Scheme 3. CAL-B-catalysed ring cleavage of racemic (3S*,4R*)-1.
Table 1. CAL-B-catalysed ring-opening of ( )-1–4 and ( )-13–16.
Conv.[a]
[%]
eeS
[%]
eeP
[%]
[b]
[b]
Entry
Racemate
Reaction time
[h]
1
2
3
4
5
6
7
8
9
10
11
12
13
14
(3S*,4R*)-1[c]
(3S*,4R*)-5[c]
(3S*,4R*)-1[c]
(3S*,4R*)-1[e]
(3S*,4R*)-5[e]
( )-2[e]
0.5
1
0.5
1
48
42
83[d]
50
42
45
38
38
49
17
17
50
11
10
90
72
90
98
72
82
62
60
96
20
20
98
12
11
99
99
99
99
99
99
99
99
99
99
99
99
99
99
Scheme 2. Preparation of racemic N-hydroxymethyl ꢀ-lactams 1–4.
1
15
15
15
22
22
22
19
19
19
A recent observation that the lactam ring of N-hydroxy-
( )-6[c]
methyl-cis-3-acetoxy-4-phenylazetidin-2-one
opened during CAL-B (30 mg mL–1)-catalysed O-acetylation
(0.015
substrate, 6 equiv. of vinyl-acetate, iPr2O, 50 °C),[14]
was
partially
( )-6[e]
( )-3[e]
( )-7[c]
M
( )-7[e]
combined with results on the CAL-B-catalysed ring cleavage of
inactivated ꢀ-lactams,[7a,7e] suggested the possibility of CAL-B-
catalysed ring-opening of (3S*,4R*)-( )-1. It is noteworthy that
only one of the earlier works dealing with the acylation of N-
hydroxymethyl-ꢀ-lactams[8d,15] noted the use of CAL-B
(30 mg mL–1) as an optimum catalyst for the acylation of 9-
azabicyclo[6.2.0]dec-4-en-10-one (1 equiv. of vinyl-butyrate,
acetone or iPr2O).[15d] However, no ring-opening was observed
during butyrylation, probably due to the low reaction tempera-
( )-4[e]
( )-8[c]
( )-8[e]
[a] Calculated from eeS and eeP. [b] According to GC (Experimental Section).
[c] 0.015 substrate, 0.5 equiv. of H2O, iPr2O, 60 °C. [d] Determined by using
M
n-heptadecane as internal standard. [e] 0.015
1 equiv. of benzylamine, iPr2O, 60 °C.
M substrate, 0.5 equiv. of H2O,
Comparisons of the ring-opening reaction rates of N-
ture of –15 °C, which we envisioned, might not support forma- hydroxymethyl lactam (3S*,4R*)-( )-1 and the corresponding in-
tion of the optimum hydrolytic conformation of the enzyme.[16] activated lactam ( )-5 under the same conditions clearly dem-
In view of the earlier noted results, CAL-B-catalysed ring-open- onstrated the beneficial accelerator effect of the activating
ing of (3S*,4R*)-( )-1 was first carried out in iPr2O at 60 °C, with group (Table 1, Entry 1 vs. Entry 2). The preparative-scale ring-
0.5 equiv. of added H2O (Scheme 3) (Table 1, Entry 1). To our opening of (3S*,4R*)-1 furnished products with ee > 99 %, but
surprise, not only did highly enantioselective (E > 200) ring- the poor yield for unreacted (3S,4R)-1 (14 %) revealed that
opening of (3S*,4R*)-( )-1 occur, but so too did in situ degrada- formaldehyde-induced polymerization of the lactam might be
tion of the N-hydroxymethyl group of the ring-opened amino a consideration. In order to follow the progress of the reaction
acid. Examples of two-step enzymatic transformations, where precisely, and to investigate possible side-reactions, n-hepta-
the reaction sequence is triggered by a biocatalyst, and is com- decane was added to the reaction as an internal standard
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Table 2. CAL-B-catalysed preparative-scale resolutions of (3S*,4R*)-( )-1,[a] ( )-2,[b] ( )-3[c] and ( )-4.[d]
Reaction
time
[h]
Product enantiomer
Unreacted enantiomer
Yield
[%]
Isomer
ee[e]
[α]D25
H2O
Yield
[%]
Isomer
ee[e]
[α]D25
[%]
[%]
CHCl3
(3S*,4R*)-( )-1
( )-2
3
48
47
46
49
(2R,3S)-10
(R)-12
(1R,2S)-14
99
99
99
99
–7.1[f]
+25[h]
–9.0[j]
–12.4[l]
42
47
45
48
(3S,4R)-1
(S)-2
(1S,5R)-3
(1R,2R,5S,6)-4
95
97
99
99
–225[g]
–275[i]
24
48
19
( )-3
( )-4
–35.7[k]
+82.1[m]
(1R,2R,3S,4S)-16
[a] 0.78 mmol substrate, 0.039
M
, 0.5 equiv. of H2O, 1 equiv. of benzylamine, 600 mg of CAL-B (30 mg mL–1), iPr2O, 60 °C. [b] 0.79 mmol substrate, 0.039
, 0.5 equiv. of H2O, 1 equiv. of
, 0.5 equiv. of H2O, 1 equiv. of benzylamine, 300 mg of CAL-B
M,
0.5 equiv. of H2O, 1 equiv. of benzylamine, 600 mg of CAL-B (30 mg mL–1), iPr2O, 60 °C. [c] 0.35 mmol substrate, 0.05
M
benzylamine, 210 mg of CAL-B (30 mg mL–1), iPr2O, 60 °C. [d] 0.49 mmol substrate, 0.049
M
(30 mg mL–1), iPr2O, 60 °C. [e] Determined by GC (Experimental Section). [f] c = 0.34. [g] c = 0.51. [h] c = 0.15. [i] c = 0.30. [j] c = 0.34. [k] c = 0.40. [l] c = 0.32.
[m] c = 0.97.
(Table 1, Entry 3). Next, to restrict possible undesired side-reac-
An attempt was also made to improve the enzyme:substrate
tions, benzylamine (1 equiv.) was added to the reaction; its role mass ratio (from 10:1 to 4:1) in the case of ( )-1. Notably, a
was to serve as a nucleophilic capture agent capable of con- similarly good result (E > 200) was obtained, but a longer reac-
suming formaldehyde generated in situ (Table 1, Entry 4). A tion time was required [conv.: 50 % after 3 h vs. 1 h (Table 1,
faster ring-opening reaction, lacking any evidence of side-reac- Entry 4)].
tions appeared to take place (Table 1, Entry 4 vs. Entry 2). We
hypothesized that this observation results from the fact that
the enzyme surface was no longer coated, and thus inhibited,
by the products of a putative polymerization reaction. Finally,
no catalytic activity attributable to benzylamine was evident;
comparisons of the results of ring cleavage reactions with
(3S*,4R*)-5 in the presence and absence of benzylamine under-
score this fact (Table 1, Entry 5 vs. Entry 2).
On the basis of the preliminary results, preparative-scale res-
olutions of racemic 1–4 were performed under the optimized
conditions; the results are reported in Table 2 and the Exp. Sec-
tion.
As further transformations, the ring-opening of ꢀ-lactams
(3S,4R)-1, (S)-2, (1S,5R)-3 and (1R,2R,5S,6S)-4 with 18 % aqueous
HCl resulted in enantiomeric ꢀ-amino acid hydrochlorides
(Scheme 5). The N-deprotection reactions of ꢀ-lactam enantio-
mers from the hydroxymethylated counterparts (with no drop
in ee) with KMnO4 in a mixture of acetone and H2O[18] or
NH4OH and MeOH[15d] are well known in the literature. Thus,
treatment of N-hydroxymethyl-ꢀ-lactam (1R,2R,5S,6S)-4 with
NH4OH/MeOH afforded (1R,2R,5S,6S)-8 as an established inter-
mediate en route to CEP-28122 (see Scheme 5 and Exp. Sect.).
In view of the excellent results, the ring cleavage reactions
of racemic 2–4 were also investigated in the presence of , under
the same conditions (Scheme 4 and Table 1).
Scheme 5. Transformations of the enantiomers.
Scheme 4. CAL-B-catalysed ring cleavage of racemic 2–4.
3. Conclusions
Highly enantioselective (E > 200) reactions accompanied in
each case by in situ degradation of the N-hydroxymethyl group The CAL-B-catalysed two-step cascade procedure ensures a
of the ring-opened amino acids were observed (Table 1, Entries highly efficient strategy for rapid access to diverse amino acids
6, 9 and 12). As compared with the results obtained for the ring- from N-hydroxymethyl-ꢀ-lactams. The method was successfully
opening reactions of the corresponding inactivated lactams ( )- applied to the synthesis of cispentacin and intermediates for
6–8, there was again evidence of the beneficial accelerator ef- the taxol side-chain, cathepsin inhibitors and CEP-28122. When
fect of the activating group (Table 1, Entries 6, 9 and 12 vs. the CAL-B-catalysed hydrolyses of racemic N-hydroxymethyl-ꢀ-
Entries 7, 10 and 13). The presence of benzylamine in the enzy- lactams were performed with H2O (0.5 equiv.) in iPr2O at 60 °C,
matic ring cleavage reactions of ( )-6–8 was investigated as enantioselective (E > 200) ring cleavage reactions took place.
well. As was the case for reactions with ( )-5, no catalytic activa- As the ring-opened amino acids formed, the N-hydroxymethyl
tion attributable to benzylamine could not be detected groups underwent spontaneous degradation, and the desired
(Table 1, Entries 8, 11 and 14 vs. Entries 7, 10 and 13).
enantiomeric ꢀ-amino acid and unreacted N-hydroxymethyl-ꢀ-
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Racemic N-Hydroxymethyl-exo-3-azatricyclo[4.2.1.02.5]non-7-
en-4-one [( )-4]: Via the above procedure, the reaction of racemic
( )-8 (1 g, 7.39 mmol), paraformaldehyde (0.22 g, 8.13 mmol equiv.),
K2CO3 (0.6 g) and H2O (0.39 mL) in THF (10 mL) afforded a white
crystalline product, ( )-4 (0.84 g), 69 %, m.p. 77–78 °C (recrystallized
from Et2O) after 5 h. The 1H NMR (400 MHz, CDCl3, 25 °C, TMS) data
for ( )-4: δ = 1.63–1.69 (d, 1 H, J = 9.84 Hz, CHAHB); 1.71–1.77 (d, 1
H, J = 9.88 Hz, CHAHB); 2.95–2.98 (br. s, 1 H, CH); 3.00–3.04 (m, 1 H,
H–2); 3.07–3.10 (br. s, 1 H, CH); 3.64–3.67 (d, 1 H, J = 3.96 Hz, H-3);
4.67–4.76 (dd, 2 H, J = 11.41, J = 15.00 Hz, CH2OH); 6.11–6.29 (m, 2
H, CH=CH) ppm. 13C NMR (100.62 MHz, CDCl3): δ = 39.70, 41.26,
42.95, 57.20, 64.65, 135.82, 138.90, 170.21 ppm. C9H11NO2 (165.2):
calcd. C 65.44, H 6.71, N 8.48; found C 65.51, H 6.59, N 8.43.
lactam enantiomers (ee > 95 %) were formed. Polymerization
side reactions induced by liberation of formaldehyde were suc-
cessfully restricted by the addition of benzylamine as a nucleo-
philic capture agent in the enzymatic reaction mixtures. Impor-
tantly, these studies underscore the importance of the hydroxy-
methyl group as a traceless activating group for the CAL-B-cata-
lysed ring cleavage of ꢀ-lactams.
Experimental Section
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), was from Sigma–Aldrich. The solvents
were of the highest analytical grade.
Preparative-Scale Hydrolysis of Racemic N-Hydroxymethyl-cis-
3-hydroxy-4-phenylazetidin-2-one [(3S*,4R*)-( )-1]: (3S*,4R*)-
( )-1 (150 mg, 0.78 mmol) was dissolved in iPr2O (20 mL). CAL-B
Racemic N-hydroxymethyl-ꢀ-lactams 1–4 were prepared from read- (600 mg, 30 mg mL–1), benzylamine (85 μL, 0.78 mmol.) and H2O
ily available racemic lactams 5–8[8] with paraformaldehyde under
(7 μL, 0.39 mmol) were added and the mixture was shaken in an
incubator shaker at 60 °C for 180 min. The reaction was stopped by
filtering off the enzyme at 49 % overall conversion. The solvent was
evaporated off, and the residue was subjected to column chroma-
tography (EtOAc:n-hexane 1:1). The resulting ꢀ-lactam (3S,4R)-1
crystallized out (63 mg), 42 %; recrystallized from Et2O {[α]D25 = –225
(c = 0.51 in CHCl3), m.p. 115–117 °C; ee = 95 %}. The filtered enzyme
was washed with distilled H2O (3 × 15 mL), and the H2O was evapo-
rated off, yielding crystalline ꢀ-amino acid (2R,3S)-10 {76 mg, 48 %.
[α]D25 = –7.1 (c = 0.34 in H2O), ref.[8c] = –7.2 (c = 0.34 in H2O), ee >
98 %, m.p. 255–257 °C (recrystallized from H2O/acetone), ref.[8c]
252–255 °C; ee = 99 %}. The 1H NMR (400 MHz, CD3OD, 25 °C, TMS)
and 13C NMR (100.62 MHz, CD3OD) data for (3S,4R)-1 were similar
to those for (3S*,4R*)-( )-1. C10H11NO3 (193.2): calcd. C 62.17, H 5.47,
sonication.[8d,13.14,15b]
In a typical small-scale enzymatic experiment, racemic substrate
(0.015 or 0.03
M solution) in iPr2O (1 mL) was added to CAL-B
(30 mg mL–1). Benzylamine (0 or 1 equiv.) and H2O (0.5 equiv.) were
next added. The mixture was shaken at 60 °C. The progress of the
reaction was followed by taking samples from the reaction mixture
at intervals and analysing them by GC on a Chrompack Chirasil-Dex
CB column (25 m) [190 °C; 140 kPa; retention times (min), (3S,4R)-
1: 16.64 (antipode: 15.07); (S)-2: 5.44 (antipode: 5.26); 140 °C for
4 min → 190 °C (temperature rise 20 °C min–1); 140 kPa; retention
times (min), (1S,5R)-3: 5.33 (antipode: 5.14); (1R,2R, 5S,6S)-4: 7.04
(antipode: 6.96)]. The ee values for the ꢀ-amino acids prepared were
determined by a GC method[19] using Chrompack Chirasil-Dex CB
1
N 7.25; found C 62.23, H 5.45, N 7.29. The H NMR (400 MHz, D2O)
or L
-Val column after a simple and rapid double derivatization[19]
data for (2R,3S)-10:
δ = 4.32–4.36 [d, 1 H, J = 5.88 Hz,
with (i) CH2N2 (Caution! Reactions with diazomethane should be
performed under a well-working hood!), and (ii) Ac2O in the pres-
ence of 4-dimethylaminopyridine and pyridine. Precise GC condi-
tions: [Chrompack Chirasil-Dex CB; 140 °C for 7 min → 190 °C (tem-
perature rise 10 °C min–1); 100 kPa; retention times (min), (2R,3S)-
CH(OH)(COOH)]; 4.55–4.59 (d, 1 H, J = 5.88 Hz, CHNH2); 7.43–7.55
(m, 5 H, Ar). C9H11NO3 (181.2): calcd. C 59.66, H 6.12, N 7.73; found
C 59.75, H 6.09, N 7.72.
Preparative-Scale Hydrolysis of Racemic N-Hydroxymethyl-4-(o-
tolyl)azetidin-2-one [( )-2]: Via the procedure described above,
the reaction of racemic ( )-2 (150 mg, 0.78 mmol), benzylamine
(85 μL, 0.78 mmol.) and H2O (7 μL, 0.39 mmol) in iPr2O (20 mL) in
the presence of CAL-B (600 mg, 30 mg mL–1) at 60 °C afforded
amino acid (R)-12 {66 mg, 47 %. [α]D25 = +25 (c = 0.15 in H2O),
ref.[8d] = +24.5 (c = 0.38 in H2O), ee = 95 %; ee = 99 %, m.p. 240–
242 °C (recrystallized from H2O/acetone), ref.[8d] 236–237 °C} and
unreacted (S)-2, {70 mg, 47 %. [α]D25 = –275 (c = 0.30 in CHCl3),
ref.[8d] = –292.7 (c = 0.32 in CHCl3), ee > 99 %; ee = 97 %, m.p. 64–
66 °C (crystallized out from Et2O), ref.[8d] 66–68 °C} after 24 h. The
1H NMR (400 MHz, CDCl3, 25 °C, TMS) data for (S)-2 were similar to
those for ( )-2: δ = 2.33 (s, 3 H, CH3); 2.70–2.77 (dd, 1 H, J = 2.72,
J = 14.84 Hz, CHAHB); 3.36–3.44 (dd, 1 H, J = 5.48, J = 14.84 Hz,
CHAHB); 4.27–4.35 (d, 1 H, J = 11.40 Hz, CHN overlapping with br. s,
1 H, OH); 5.06–5.14 (m, 2 H, CH2OH); 7.15–7.34 (m, 4 H, Ar) ppm.
C11H13NO2 (191.2): calcd. C 69.09, H 6.85, N 7.32; found C 69.10, H
10: 18.99 (antipode: 18.70); L-Val; 150 °C; 100 kPa; retention times
(min), (R)-12: 39.50 (antipode: 40.90); 140 °C for 7 min → 190 °C
(temperature rise 10 °C min–1); 100 kPa; retention times (min),
(1R,2S)-14: 8.12 (antipode: 8.40); (1R,2R,3S,4S)-16: 10.10 (antipode:
10.65)].
Optical rotations were measured with a Perkin–Elmer 341 polarime-
ter. 1H and 13C NMR spectra were recorded with a Bruker Avance
DRX 400 spectrometer. Melting points were determined with a Ko-
fler apparatus.
Racemic
N-Hydroxymethyl-cis-3-hydroxy-4-phenylazetidin-2-
one [(3S*,4R*)-( )-1]: Racemic ( )-5 (1.2 g, 7.34 mmol) was dis-
solved in THF (10 mL). Paraformaldehyde (0.22 g, 8.04 mmol equiv.),
K2CO3 (0.6 g) and H2O (0.39 mL) were added. The solution was
sonicated for 5 h. The solvent was evaporated off and the residue
was dissolved in EtOAc (20 mL). The solution was dried (Na2SO4)
and then concentrated. The residue was crystallized from iPr2O to
afford a white crystalline product, ( )-4 (0.93 g), 66 %, m.p. 119–
120 °C (recrystallized from Et2O). The 1H NMR (400 MHz, CD3OD,
25 °C, TMS) data for (3S*,4R*)-( )-1: δ = 3.27–3.32 (m, 1 H, OH);
4.30–4.36 (d, 1 H, J = 11.28 Hz, CH2OH); 4.91–4.97 (d, 1 H, J =
1
6.86, N 7.31. The H NMR (400 MHz, D2O) data for (R)-12: δ = 2.47
(s, 3 H, CH3); 2.80–2.96 (m, 2 H, CH2); 4.97–5.02 (dd, 1 H, J = 6.3 Hz;
J = 8.2 Hz, CH); 7.35–7.52 (m, 4 H, Ar). C10H13NO2 (179.2): calcd. C
67.02, H 7.31, N 7.82; found C 66.92, H 7.29, N 7.86.
11.24 Hz, CH2OH); 4.97–5.00 (d, 1 H, J = 4.88 Hz, CHCO); 5.02–5.07 Preparative-Scale Hydrolysis of Racemic N-Hydroxymethyl-6-
(d, 1 H, J = 4.88 Hz, CHNH); 7.28–7.40 (m, 5 H, Ar) ppm. 13C NMR
azabicyclo[3.2.0]heptan-7-one [( )-3]: Via the procedure de-
(100.62 MHz, CD3OD): δ = 60.93, 61.63, 77.05, 126.73, 126.90, 133.48, scribed above, the reaction of racemic ( )-3 (50 mg, 0.35 mmol),
169.00 ppm. C10H11NO3 (193.2): calcd. C 62.17, H 5.47, N 7.25; found
C 62.29, H 5.50, N 7.22.
benzylamine (39 μL, 0.35 mmol.) and H2O (3 μL, 0.17 mmol) in iPr2O
(7 mL) in the presence of CAL-B (210 mg, 30 mg mL–1) at 60 °C
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afforded amino acid (1R,2S)-14 {21 mg, 46 %. [α]D25 = –9 (c = 0.34 in (2S,3R)-10·HCl, (S)-12·HCl, (1S,2R)-14·HCl, (1S,2S,3R,4R)-16·HCl and
H2O), ref.[8a] = –9.1 (c = 0.50 in H2O), ee = 99 %, m.p. 207–212 °C
lactam (1R,2R,5S,6S)-8 are presented in the Supporting Information.
(recrystallized from H2O/acetone), ref.[8a] 206–211 °C} and unreacted
(1S,5R)-3, {22.5 mg, 45 %. [α]D25 = –35.7 (c = 0.40 in CHCl3), ref.[13]
=
Acknowledgments
–32.4 (c = 1.00 in CHCl3), ee = 98 %; ee = 99 %; a pale-yellow oil}
after 48 h. The 1H NMR (400 MHz, CDCl3, 25 °C, TMS) data for
(1S,5R)-3 were similar to those for ( )-3: δ = 1.28–2.11 (m, 6 H, 3 ×
CH2); 3.43–3.49 (dd, 1 H, J = 3.68 Hz; 8.16 Hz, H–1); 4.16–4.21 (t, 1
H, J = 4.12 Hz, H–5); 4.51–4.56 (d, 1 H, J = 11.56 Hz, CH2OH); 4.65–
4.72 (d, 1 H, J = 11.56 Hz, CH2OH) ppm. C7H11NO2 (141.2): calcd. C
59.56, H 7.84, N 9.92; found C 59.59, H 7.82, N 9.89. The 1H NMR
(400 MHz, D2O) data for (1R,2S)-14: δ = 1.74–2.23 (m, 6 H, 3 × CH2);
2.86–2.99 (m, 1 H, H-1); 3.76–3.84 (m, 1 H, H-2). C6H11NO2 (129.2):
calcd. C 55.80, H 8.58, N 10.84; found C 55.78, H 8.60, N 10.84.
The authors acknowledge financial support by the Hungarian
Scientific Research Fund (OTKA) (grant numbers K-108943 and
K115731).
Keywords: Enzyme catalysis · Hydrolysis · Lactams · Amino
acids · Cascade reaction · Enantioselectivity · Traceless
activating group
Preparative-Scale Hydrolysis of Racemic N-Hydroxymethyl-exo-
3-azatricyclo[4.2.1.02.5]non-7-en-4-one [( )-4]: Via the procedure
described above, the reaction of racemic ( )-4 (80 mg, 0.48 mmol),
benzylamine (53 μL, 0.48 mmol.) and H2O (4.4 μL, 0.24 mmol) in
iPr2O (10 mL) in the presence of CAL-B (300 mg, 30 mg mL–1) at
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60 °C afforded amino acid (1R,2R,3S,4S)-16 {36 mg, 49 %. [α]D25
=
–12.4 (c = 0.32 in H2O), ref.[8b] = –12.2 (c = 0.40 in H2O), ee = 98 %;
ee = 99 %, m.p. 262–266 °C (recrystallized from H2O/acetone), ref.[8b]
> 260 °C} and unreacted (1R,2R,5S,6S)-4, [38.6 mg, 48 %. [α]D25
=
+82.1 (c = 0.97 in CHCl3); ee = 99 %; a pale-yellow oil] after 19 h.
The 1H NMR (400 MHz, CDCl3, 25 °C, TMS) and 13C NMR
(100.62 MHz, CDCl3) data for (1R,2R,5S,6S)-4 were similar to those
for ( )-4. C9H11NO2 (165.2): calcd. C 65.44, H 6.71, N 8.48; found C
65.48, H 6.81, N 8.36. The 1H NMR (400 MHz, D2O) data for
(1R,2R,3S,4S)-12: δ = 1.64–1.72 (d, 1 H, J = 9.88 Hz, CHAHB); 1.92–
1.98 (d, 1 H, J = 9.88 Hz, CHAHB); 2.54–2.60 (d, 1 H, J = 7.52 Hz, H-
2); 3.06–3.12 (br. s, 1 H, CH); 3.13–3.18 (br. s, 1 H, CH); 3.34–3.40 (d,
1 H, J = 7.60 Hz, H-3); 6.26–6.50 (m, 2 H, CH=CH). C8H11NO2 (153.2):
calcd. C 62.73, H 7.24, N 9.14; found C 62.75, H 7.22, N 9.15.
Amino Acid Hydrochlorides (2S,3R)-10·HCl, (S)-12·HCl, (1S,2R)-
14·HCl and (1S,2S,3R,4R)-16·HCl: The unreacted hydroxymethyl-
ꢀ-lactam enantiomers were dissolved one by one in 18 % HCl
(15 mL) and refluxed for 3–4 h. The solvents were evaporated off,
and the products, obtained almost quantitatively, were recrystal-
lized from EtOH and Et2O. The amino acid hydrochlorides, obtained
as white crystals, were characterized as follows:
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(2S,3R)-10·HCl: [α]D25 = +14 (c = 0.20 in 6
M
HCl), ref.[8c] = +15 (c =
0.25 in 6
M
HCl), ee > 98 %, m.p. 215–220 °C, ref.[14] 215–217 °C;
ee = 95 %.
(S)-12·HCl: [α]D25 = –6.1 (c = 0.60 in H2O), ref.[8d] = –6.4 (c = 0.55 in
H2O), ee = 99 %, m.p. 172–177 °C, ref.[8d] 185–188 °C; ee = 97 %.
(1S,2R)-14·HCl: [α]D25 = +5.1 (c = 0.50 in H2O), ref.[8a] = +5.2 (c =
0.50 in H2O), ee = 99 %, m.p. 166–169 °C, ref.[8a] 166–169 °C; ee =
99 %.
(1S,2S,3R,4R)-16·HCl: [α]D25 = +10.7 (c = 0.40 in H2O), ref.[8b] = +10.6
(c = 0.40 in H2O), ee = 99 %, m.p. 199–206 °C, ref.[8b] 196–210 °C;
ee = 99 %.
Preparation of Enantiomeric exo-3-Azatricyclo[4.2.1.02.5]non-
7-en-4-one [(1R,2R,5S,6S)-8]: The N-hydroxymethyl-ꢀ-lactam
(1R,2R,5S,6S)-4 (20 mg, 0.06 mmol) was dissolved in MeOH (2 mL).
NH4OH (2 mL) was then added and the mixture was stirred at room
temperature for 4 h. The solvent was evaporated and the remaining
residue was chromatographed on silica, and elution with ethyl acet-
ate afforded white crystals of (1R,2R,5S,6S)-8 {15 mg, 92 %. [α]D25
=
+125.5 (c = 0.55 in CHCl3), ref.[8b] = +123.7 (c = 0.50 in CHCl3) ee =
99 %, m.p. 85–87 °C (recrystallized from iPr2O), ref.[8b] 89–91 °C; ee =
99 %}. The 1H NMR and analysis data for amino acid hydrochlorides
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Received: March 1, 2016
Published Online: May 2, 2016
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim