Aldolase-Catalyzed Asymmetric Synthesis of N-Heterocycles by Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes

Article abstract of DOI:10.1002/adsc.201801530

Nitrogen heterocycles are structural motifs found in many bioactive natural products and of utmost importance in pharmaceutical drug development. In this work, a stereoselective synthesis of functionalized N-heterocycles was accomplished in two steps, com

Full text of DOI:10.1002/adsc.201801530

Title: Aldolase-Catalyzed Asymmetric Synthesis of N-Heterocycles by
Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes
Authors: Raquel Roldan, Karel Hernández, Jesús Joglar, Jordi Bujons,
Teodor Parella, Wolf-Dieter Fessner, and Pere Clapés
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201801530
Link to VoR: http://dx.doi.org/10.1002/adsc.201801530

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
UPDATE
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Aldolase-Catalyzed Asymmetric Synthesis of N-Heterocycles by
Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes
Raquel Roldana, Karel Hernandeza, Jesús Joglara, Jordi Bujonsa, Teodor Parellab, Wolf-
Dieter Fessnerc, and Pere Clapésa,*
a
Instituto de Química Avanzada de Cataluña IQAC-CSIC. Jordi Girona 18–26, 08034 Barcelona, Spain. Phone
+34934006127, Fax: +34932045904, email: pere.clapes@iqac.csic.es.
b
Servei de Ressonància Magnètica Nuclear. Universitat Autònoma de Barcelona, Bellaterra, Spain.
c
Institut für Organische Chemie und Biochemie, Technische Universität Darmstadt, Petersenstraße 22, D-64287
Darmstadt. Germany.
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
Abstract. Nitrogen heterocycles are structural motifs found
in many bioactive natural products and of utmost importance
in pharmaceutical drug development. In this work, a
stereoselective synthesis of functionalized N-heterocycles
was accomplished in two steps, comprising the biocatalytic
aldol addition of ethanal and simple aliphatic ketones such as
propanone, butanone, 3-pentanone, cyclobutanone, and
cyclopentanone to N-Cbz-protected aminoaldehydes using
engineered variants of D-fructose-6-phosphate aldolase from
Escherichia coli (FSA) or 2-deoxy-D-ribose-5-phosphate
aldolase from Thermotoga maritima (DERATma) as catalysts.
FSA catalyzed most of the additions of ketones while
DERATma was restricted to ethanal and propanone.
Subsequent treatment with hydrogen in the presence of
palladium over charcoal, yielded low-level oxygenated N-
heterocyclic derivatives of piperidine, pyrrolidine and N-
bicyclic structures bearing fused cyclobutane and
cyclopentane rings, with stereoselectivities of 96–98 ee and
97:3 dr in isolated yields ranging from 35 to79%.
Keywords: Biocatalysis; Aldol reaction; Aldolases;
Nitrogen heterocycles; Reductive amination
available to prepare these compounds is extensive,
Introduction
developing
new
stereoselective
synthetic
methodology is essential yet challenging.[1a,3]
Nitrogen heterocycles are structural motifs frequently
found in bioactive natural products and
pharmaceuticals, being an area of constant
development owing to their importance in drug
discovery.[1] The ring type, e.g. piperidine,
pyrrolidine, pyrrolizidine, N-bicyclics, and the
structure, position and stereochemistry of the
substituents, play a fundamental role in modulating
their biological activity.[1b,2] Methods for the total
synthesis of a broad structural diversity of N-
heterocycles, either as building blocks or as final
compounds for biological testing, are thus in high
demand.[1a] Although the number of chemical routes
Biocatalytic C–C bond forming reactions by means of
carboligases is highly attractive because of
unparalleled high efficiency and stereoselectivity,
avoidance of unnecessary functional group protection
and use of mild reaction conditions. Among them,
aldolases have proven their remarkable synthetic
utility in the preparation of different types of highly
oxygenated N-heterocycles, usually termed as
iminosugars or azasugars.[4]
Recent synthetic developments of D-fructose-6-
phosphate aldolase from E. coli (FSA, EC 4.1.2.–)[5]
showed that FSA D6X variants, where X = A, L, N,
Q, S, T, E, and H, and double variants combined with
1
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
A165G, L107A, L163A and T26X (X: I, A, L and V)
mutations, are affective catalysts for homo and
crossed aldol additions of simple aliphatic ketones
and aldehydes leading to the preparation of chiral
intermediates and deoxysugars.[6] Hence, these FSA
variants can offer new synthetic options toward the
preparation of iminosugar derivatives having a low
degree of hydroxylation. Likewise, 2-deoxy-D-ribose-
5-phosphate aldolase (DERA, EC 4.1.2.4) has been
recognized as a powerful aldolase for the synthesis of
deoxysugars and iminosugars.[7] Wild-type DERA
was unique among the aldolases in that it uses simple
aliphatic aldehydes both as nucleophilic and
electrophilic aldol components and therefore is a
qualified catalyst for the proposed synthesis.
adduct formation. Electrophilic components chosen
were prochiral substrates 2a and 2b, which avoid
complications arising from kinetic enantiomer
selectivity, and pure enantiomers of chiral substrate
2c. The latter contains a chiral center of defined
absolute configuration that acts as a reference in the
determination of the overall relative configuration of
the products.
Aldol additions of nucleophiles 1a-f to electrophiles
N-Cbz-3-aminopropanal
(2a)
and
N-Cbz-2-
aminoethanal (2b) were catalyzed by several FSA
variants (Figure 2). Variants with different amino
acids substituting for D6 were found to be active for
various nucleophile-electrophile combinations, some
of them yielding good to excellent product formation.
Interestingly, FSA A165G was also efficient for the
additions of 1b-c, e-f to 2a and 2b, and a quintuple
We have investigated the systematic chemoenzymatic
preparation
of
low-level
functionalized
two-steps
N-heterocyclic compounds along
a
FSA
mutant
(FSA
qm:
FSA
strategy: The first step consists of an aldol addition of
ethanal (1a) and simple linear (1b-d) or cyclic
aliphatic ketone (1e-g) to an N-Cbz-protected amino
aldehyde 2a-c (Figure 1) catalyzed by FSA variants
and DERA from Thermotoga maritima (DERATma).
In a second step, the aldol adducts obtained are
transformed into the corresponding N-heterocycles
via intramolecular reductive amination by using H2–
Pd/C.
L107Y/A129G/R134S/A165G/S166G) for addition
of 1a to 2a. In contrast, FSA A165G and all variants
that include the native D6 residue had been found
inactive when hydroxyaldehyde derivatives were
employed as electrophiles.[6b] Hence, N-Cbz-
aminoaldehydes appear to establish different, and
potentially stronger, interactions with the FSA
substrate binding site through the carbamate and/or
aromatic moieties, which likely render them
electrophiles superior to hydroxyaldehydes.[4d,8a]
3
This is also consistent with the fact that previously
developed FSA A165G was more efficient than the
wild-type in aldol additions of hydroxyacetone (HA)
and dihydroxyacetone (DHA) to 2a and, particularly,
to 2b.[8a] Likewise, wild-type FSA also converted 1a-
c and 1f using D,L-glyceraldehyde-3-phosphate, the
best electrophilic substrate known to date for wild-
type FSA.[6a] None of the FSA variants assayed
catalyzed the addition of 1a-g to either enantiomer of
N-Cbz-alaninal (2c). This was quite surprising since
FSA A165G can catalyze the addition of HA and
DHA to both S-2c and R-2c.[8a] Cyclohexanone (1g)
was not accepted as a substrate, probably due to steric
incompatibility.
Figure 1. Panel of nucleophiles 1a-g and electrophiles 2a-
c assayed for enzymatic cross-aldolization.
In comparison, DERATma was also active for the aldol
addition of ethanal (1a) and propanone (1b) to 2a and
to R- and S-2c, whereas 2b only reacted with 1b
(Figure 2). In stark contrast to all FSA catalysts,
DERATma were highly active with both enantiomers
of the -substituted amino aldehydes 2c. On the other
hand, DERATma did not catalyze additions with any of
the ketones 1c-g to any of the electrophiles 2a-c. This
is consistent with the observation that the affinity of
DERA for ketones is significantly lower than for
ethanal (1a), which is its natural nucleophilic
substrate.[9]
Results and Discussion
Screening experiments
We studied the aldol addition of simple aliphatic
nucleophiles 1a-g (concentration range 100 mM to
2.6 M (15% v/v), see SI for optimization analysis) to
electrophiles 2a-c (80-100 mM) using FSA variants
and wild-type DERATma as catalysts (Figure 2) in
small-scale preparative synthetic conversions. FSA
catalysts were selected among those found to be
efficient toward the nucleophiles
1
and
N-Cbz-aminoaldehydes 2.[4c,6b,8] The screening was
performed by direct HPLC monitoring of aldol
2
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
Figure 2. Results of conversion to aldol adduct from the screening of FSA variants and DERATma as catalysts for the aldol
addition of nucleophiles 1a-g to electrophiles 2a-c. Conversions (mol %) at 24 h were determined by HPLC analysis using
the external standard methodology. Conditions: [1a] = 100 mM; [1b] = 2.6 M (15% v/v), [1c] = 0.60 M, [1d] = 0.47 M,
[1e] = 0.68 M, [1f] = 0.56 M, and [1g] = 0.48 M (5% v/v; in 1c-g), [2] = 80 mM, except for the addition if 1a to R-2c and
S-2c catalyzed by DERATma where [1a] = [2c] = 100 mM. Reactions were conducted in aqueous 50 mM triethanolamine
buffer pH 8 at 25 °C. FSA qm = FSA quintuple mutant = FSA L107Y/A129G/R134S/A165G/S166G. A complete list of
all FSA variants assayed can be found in SI.
Preparative
synthesis
and
product
formed in both cases, which is consistent with the
stereochemical outcome expected for FSA catalysis.
FSA variants D6N and D6T were the most effective
catalysts for the aldol addition of 1b to 2a, whereas
FSA A165G was the best for the reaction between 1b
and 2b (Figure 2), furnishing 3b and 15b in 85% and
35% isolated yields, respectively, and with high
enantioselectivity (96%-98% ee) as determined by
HPLC on chiral stationary phase (Table 1 entry 3 and
Table 2 entry 1). DERATma catalyst gave identically
configured aldol adducts in 47% and 30% isolated
yields, with 84% and 90% ee, respectively (Table 1
entry 4 and Table 2 entry 2). No self-addition of 2b
characterization
Addition of 1a to N-Cbz-3-aminopropanal (2a) was
catalyzed by FSA D6L, D6N, D6E/A165G, FSA qm
and DERATma. FSA qm and DERATma catalysis
yielded the cyclic N-Cbz-protected hemiaminal 3a
with trans-configured hydroxyl groups at C2:C4
positions in 45% and 25% isolated yields,
respectively (Table 1, entry 1 and 2). The lower yield
of 3a obtained with DERATma is most likely due to
consumption of 1a in the competing enzymatic
trimerization of 1a leading to a stable trideoxyhexose
(i.e., 3R,5R-dihydroxyhexanal; see Scheme 3).[10] On
the other hand, neither trimerization reaction nor self-
aldol addition of 1a was detected with FSA qm
variant.[6b] Single addition of 1a to 2a was exclusively
obtained with both FSA qm and DERATma catalysts,
and no consecutive double addition product was
observed, neither upon application of an excess of
ethanal nor upon continuous addition of 1a using a
syringe pump. In the self-addition of 1a and the
crossed addition of 1a to 2a DERA is reported to
show strict R-stereoselectivity for the newly formed
chiral centers.[10-11] Therefore, 3a was assigned a
(2S,4R)-configuration. Identical NMR spectra were
recorded for the adduct obtained by catalysis of FSA
qm, and their matching optical rotation (Table 1)
verified that the identical product (2S,4R)-3a was
was observed in the reactions catalyzed by DERATma
.
Again, a single addition product was exclusively
obtained in the aldol reaction of 1b with 2a and 2b
with FSA D6N, FSA A165G and DERATma catalysts.
It could be that On the basis of the stereochemical
outcome of both enzymes in examples with non-
natural substrates,[4c,4e,10] 3b has (R)- and 15b has (S)-
configuration. Intramolecular reductive amination of
(R)-3b was stereoselective, furnishing 5 with cis-
stereochemistry as expected from previous works.[12]
On the other hand, (S)-15b gave a mixture of both
cis- and trans-configured pyrrolidines 17 and 18,
respectively.
The addition of butanone (1c) to 2a could be
catalyzed by FSA D6S, D6T and D6E, and addition
to 2b by D6T and A165G variants, generally with
good conversions (Figure 2). The preparative reaction
3
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
between 1c and 2a using FSA D6E produced two
regioisomeric adducts, one was arising from the
attack of the α-methylene carbon of 1c (i.e., C3
attack) and the other one from the attack of the α-
methyl carbon (i.e., C1 attack), with a C1:C3 ratio of
13:87. Chromatographic separation of the product
isomers from the reaction mixture proved difficult
and only allowed enrichment, furnishing a major
fraction (48%) containing a 3c’/4c’ diasteromer
mixture in a 86:14 ratio, originating exclusively from
C3 attack (Table 1, entry 5) as inferred from the
NMR analysis of the derived N-heterocycles 6 and 7,
and a second fraction (1%) consisting of a mixture of
aldol regioisomers from C1:C3 attack in a 56:43
molar ratio, which could not be further resolved
(Scheme 1). On the other hand, D6T and A165G FSA
variants were identified as the best catalysts for the
addition of 1c to 2b (Figure 2). Similar as above, the
preparative reaction using FSA A165G furnished a
C1:C3 regioisomeric mixture in 13:87 ratio, which
upon chromatographic separation yielded a major
fraction (28%) containing a 88:12 mixture of
diastereomers 15c’/16c’ arising from C3 attack
(Table 2, entry 3), and a second fraction (2.8%) as a
68:32 mixture of C1:C3 aldol regioisomers (Scheme
1). This is in contrast to results observed for the aldol
addition of 1c to L-G3P using FSA D6H as catalyst,
where exclusively C1 attack was observed.[6a]
Table 1. Preparative scale FSA- and DERA-catalyzed aldol additions of 1a-f to N-Cbz-3-aminopropanal (2a).
Aldol
addition
er or dr,
%
Reductive
Aldol adduct
formed,a),
%
Yield,b)
%
amination
Entry Biocatalyst
Nu
Product
product,c)
dr
–
–
5
e
1
2
3
4
5
6
7
FSA qm
3ad
85
77
94
70
78
71
56
45
25
85
47
48
35
46
–
1a
1a
1b
1b
1c
1d
1e
e
DERATma
FSA D6N
DERATma
FSA D6E
FSA D6E
FSA D6L
3ad
–
98:2f
3b
3b
3c’/4c’
3d
92:8f
–
86:14g
>97:3h,i
80:20 i
6:7 86:14
8
9
3e/4e
10:11:12:13:14
8
FSA A165G
3f/4f
75
65
67:33 i
1f
15:11:15:22:37
a)
b)
c)
Values determined by HPLC analysis of preparative scale reactions. Isolated yield of aldol adduct. Diastereomer
d)
e)
ratio determined by NMR. Aldol adduct isolated as cyclic hemiaminal in equilibrium with its open form. er not
determined; optical rotation: FSA qm []D20= + 8.5 (c = 2.1 in MeOH); DERATma []D20 = + 4.6 (c = 0.69 in MeOH). f) er
determined by chiral HPLC, referenced to a racemic sample prepared by chemical synthesis (see SI). g) Inferred from the
h)
i)
reduction amination products 6:7. No other diastereomers were detected by NMR analysis. dr of the aldol adduct
determined by NMR analysis. FSA qm: FSA quintuple mutant = FSA L107Y/A129G/R134S/A165G/S166G. Nu:
Nucleophile. Green asterisk indicates the stereogenic center formed after reductive amination.
4
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
electrophile, yielding the (R)-configuration at C-
next to the carbonyl group (Scheme 2).[6b,6c] The
addition of 1e to 2b (Table 2, entry 5), catalyzed by
FSA A165G, furnished only diastereomer 15e
indicating a preferential attack at the si-face of the
electrophilic carbonyl. On the contrary, the addition
of 1e to 2a was less selective, furnishing an anti:syn-
mixture of diastereomers 3e/4e in a 80:20 ratio,
which indicates an unexpectedly high 20% chance of
unconventional attack at the re-face of the
electrophilic carbonyl group. Nevertheless, upon
reductive amination of this mixture, only product 9
was obtained from 3e but no N-bicyclic product
corresponding to 4e was detectable by NMR analysis,
possibly due to strain imposed upon cyclization
(Table 1, entry 7) (Scheme 2).
Scheme 1. Product distribution in the aldol addition of 1c
to 2a and 2b catalyzed by FSA D6E and A165G variants.
Additions of 1f to 2a-b gave mixtures of anti:syn
adducts with dr of 66:34 for 3f/4f and 50:50 for
15f/16f, respectively (Table 1 entry 8 and Table 2
entry 6). The occurrence of diastereoisomers
observed for some of the aldol additions studied is
plausibly explained by an imperfect orientation of the
electrophilic C=O relative to the enzyme-enamine
complex, thereby generating epimers at C-β to the
carbonyl group of the nucleophiles. However, it is
Consistent with observations for butanone addition to
L-G3P, i.e. si-face attack of the FSA-nucleophile
enamine complex to the C=O si-face of the
electrophile, the major diastereomers 3c’/15c’
obtained with electrophiles 2a and 2b correspond to
the expected D-threo (i.e. syn) configuration.[6a] The
minor diastereomers 4c’/16c’ arise from an inverted
orientation of the C=O electrophile exposing its re-
face to the FSA-Nu complex.[4d,12b] Intramolecular
reductive amination of the 3c’/4c’ mixture gave the
even more conceivable that
a
spontaneous
isomerization of the tertiary stereocenter at C- next
to the carbonyl in the cyclic aldol adduct may occur
under the reaction conditions (aq buffer at pH 7.5-8.
Isomerizations of this kind, which effectively lead to
mirror image compounds of the imperfect aldol
diastereomers, have been reported previously for
aldol additions of cyclopentanone to L-G3P and upon
addition of cyclopentanone enolate to Boc-N-
phenylalaninal in aqueous buffered solutions.[6a,16]
Reductive amination of 15f/16f furnished the N-
bicyclic derivatives 27/28 (Table 2, entry 6),
respectively, with identical configuration of the new
stereogenic center formed. Reductive amination of
mixture 3f/4f was not stereoselective and yielded a
mixture of four diastereoisomers 10-13 (Table 1,
entry 8). In addition, an unexpected dehydroxylated
product 14 was also detected by NMR analysis as the
major component, which was probably formed during
the Cbz removal/reductive amination step in the
presence of the Pd catalyst by formal H2O
elimination/hydrogenation.
Addition of ethanal (1a) and propanone (1b) to
individual enantiomers of (S)-2c and (R)-2c by
DERATma catalysis furnished the corresponding aldol
adducts in 20%-79% isolated yields (Table 3). The
lower isolated yields obtained for the addition
products from 1a (1 eq) were mainly caused by
difficulties in separating (3R,5R)-dihydroxyhexanal
(present in its lactol form), which simultaneously
formed by enzymatic self-aldolization of 1a as a side
reaction. When a mild excess of 1a was employed
(1.25 eq) to achieve complete conversion of 2c to 29,
no subsequent addition was observed (tandem
addition to 2c) but the formation of the lactol
increased thus complicating its removal.
corresponding
N-heterocycles
6
and
7
stereoselectively as directed by the relative
orientation of the C4 hydroxyl group (Table 1, entry
5).[13] From the 15c’/16c’ mixture four N-pyrrolidine
heterocycles 19-22 were generated as a consequence
of the lack of stereoselectivity in the reductive
amination step with each of the diastereoisomers, as
typical for imine reductions in 5-membered ring
systems (Table 2 entry 3).
3-Pentanone (1d) was also tolerated as a nucleophilic
substrate by different FSA variants, with D6T and
D6E being the most efficient catalysts (Figure 2). The
D6E catalyzed addition of 1d to 2a furnished only the
syn diastereomer 3d (Table 1, entry 6), while FSA
D6T catalyzed addition to 2b yielded a syn:anti
mixture of 15d/16d in a 87:13 ratio (Table 2, entry 4).
Considering the stereochemical outcome of FSA in
previous reactions, 3d has (4S,5R)-configuration, 15d
should be (4S,5S), and 16d (4R,5S). Reductive
amination of 3d furnished the N-heterocycle 8 with
good diastereoselectivity, while from 15d/16d four
diasteroisomers 23-26 were identified, among which
the major 23 (79%) and the minor 26 (8%) showed
the C2 configuration predicted for Pd catalysis.[13-14]
Cyclobutanone (1e) and cyclopentanone (1f) were
also converted in the presence of 2a and 2b
electrophiles. FSA variants D6L, D6Q and,
particularly, A165G were the most efficient (Figure
2). As reported previously,[6a,6b] the cyclic ketones 1e
and 1f imposed an E-configured FSA K85-enamine
nucleophile complex, contrary to the Z-configuration
invariably observed with acyclic nucleophiles such as
hydroxyethanal, hydroxyacetone or dihydroxyacetone,
and other aliphatic ketone nucleophiles.[4d,6c,15] The
structural and mechanistic features invariably impose
an attack of the nucleophile from its si-face to the
5
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
Table 2. Preparative scale FSA- and DERA-catalyzed aldol additions of 1b-f to N-Cbz-2-aminoethanal (2b).
Reductive
Aldol adduct
formed,a)
%
Isolated
yield,b)
%
Aldol
addition
er or dr,%
amination
products,c)
dr
Entry Biocatalyst
Nu
Aldol adduct
17:18
70:30
–
1
2
3
FSA A165G
DERATma
95
85
60
35
30
28
99:1d
95:5d
88:12
1b
1b
1c
15b
15b
19:20:21:22
80:8:8:4
23:24:25:26
79:8:5:8
FSA A165G
15c’/16c’
4
5
6
FSA D6T
15d:16d
15e
47
87
50
30
37
38
87:13
1d
1e
1f
g
FSA A165G
FSA A165G
>95:5e
50:50e, f
–
27:28
33:67
15f:16f
a) Values obtained at preparative scale and determined by HPLC. b) Isolated yield of aldol adduct. c) dr: diasteromeric ratio
d)
determined by NMR. er determined by chiral HPLC, a racemic sample was obtained by non-stereoselective chemical
e)
f)
methods (see SI). dr of the aldol adduct determined by NMR. The 50:50 diastereomeric mixture obtained in the aldol
addition was purified by column chromatography. Two fractions were obtained: one of pure 15f and another one of a
15f:16f 33:67 mixture in order to characterize both N-heterocycles. The reductive amination was carried out on the 15f:16f
33:67 mixture. g) The reductive amination produced a complex mixture of products, which could not be assigned to any
structure. Nu: Nucleophile. Green asterisk indicates the stereogenic center formed after reductive amination.
Scheme 3. DERATma-catalyzed aldol addition of 1a to S-2c
with an excess of 1a leading to the formation of 3R,5R-
dihydroxyhexanal and formation of 37 during reductive
amination.
Scheme 2. Product distribution in the aldol addition of 1e
to 2a and 2b catalyzed by FSA D6E and A165G variants.
The chiral center of (S)-2c and (R)-2c was used as
reference to assign the absolute stereochemistry of
Consequently, during the reductive amination
the pyrrolidine derivatives 31-36 and infer the
procedure the imino moiety of the formed 29
stereochemical outcome of the biocatalytic aldol
underwent a subsequent intermolecular reductive
addition and reductive amination. The aldol addition
amination with the aldehyde group of the remaining
of 1a to (S)-2c and (R)-2c was fully stereoselective
lactol, yielding the corresponding conjugate 37
furnishing (4S,5S)-29 and (4S,5S)-29, respectively,
(Scheme 3).
consistent with previous reports.[17] A similar
situation was found upon addition of 1b to (S)-2c. On
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
the other hand, (R)-2c gave
a
mixture of
methylene carbon with a C1:C3 regioisomer ratio of
17:83 and an 86:14 syn:anti dr for the C3-regiosomer.
Cyclobutanone and cyclopentanone imposed an E
configured FSA K85-enamine nucleophile complex,
therefore under FSA catalysis the anti adducts were
mainly formed. This was unequivocally established
with cyclobutanone, though inconclusive for
cyclopentanone presumably because of spontaneous
isomerization at the tertiary C- stereocenter to the
carbonyl in aqueous media at pH 7.8. DERATma
catalyzed the addition of ethanal and propanone to
both enantiomers of N-Cbz-alaninal, reactions
unattainable by any of FSA variants screened. The
additions were fully diastereoselective except for
addition of propanone to S-2c. Thus, FSA and DERA
catalysts seem to be highly complementary tools,
where FSA variants offer greater flexibility with
structural variations in the nucleophile reactants, and
DERA is superior for the conversion of sterically
more demanding electrophiles.
diasteroisomers (4S,5R)-29:(4R,5R)-30 in a 77:23 dr,
indicative of a loss of enantiofacial selectivity during
the electrophile approach. Reductive amination of
(4S,5S)-29 gave a mixture of the N-heterocycles
33/34, epimeric at C2, while from the mixture of
(4S,5R)-29:(4R,5R)-30 the pyrolidines 35/36 resulted,
in which the new configuration was controlled by the
hydroxyl group at C4, in agreement with previous
reports.[12c,13]
Conclusion
FSA variants and DERATma are able to catalyze aldol
additions of ethanal (1a) and simple linear (1b-d) and
cyclic (1e-f) aliphatic ketones to N-protected
aminoaldehydes
(2a-c),
affording
diversely
functionalized N-heterocycles. FSA variants with
different residues at position D6 are active for various
nucleophile-electrophile combinations, though the
optimal catalyst depends on both the nucleophile and
electrophile structure. Such unprecedented novel
activity was also hidden in the previously reported
variant FSA A165G, which was again particularly
active towards N-Cbz-2-aminoethanal (2b). Hence,
the effectivity of a FSA catalyst toward a nucleophile
strongly depends upon the quality of the electrophilic
component. Aldol additions of ethanal (1a) to 2a and
propanone (1b) to 2a and 2b were fully
stereoselective, yielding (R)-configured aldol adducts
identical to those obtained using DERATma catalysis.
Butanone gave preferentially an addition at the
Stereochemical outcome of reductive amination
reactions followed a general pattern identical to that
observed previously in the synthesis of other
iminosugars.[4c,13] The straightforward two-step
synthetic
approach
comprising
biocatalytic
carboligation and chemical reductive amination
described in this work foresees new avenues in the
synthesis of N-heterocycles, allowing the preparation
of new derivatives that otherwise are difficult to
prepare by the use of existing chemical
methodologies.
Table 3. Preparative scale DERATma-catalyzed aldol addition of 1a and 1b to (S)- and (R)-N-Cbz-2-alaninal (S-2c and R-
2c)
Aldol adduct
formed,a)
%
Reductive amination
Yield,b)
%
dr,c)
%
Nu
E
Aldol adduct
product,d)
dr
31
32
S-2c 85e
R-2c 80e
S-2c 81
R-2c 91
34f
20f
79
(4S,5S)-29
(4R,5S)-29
(4S,5S)-29
(4S,5R)-29:(4R,5R)-30
>95:5
>95:5
>95:5
77:23
1a
1a
1b
1b
33:34 56:44
35:36 77:23
78
a)Determined by HPLC. b)Isolated yield of aldol adduct. c)dr: inferred from the reductive amination products. d)dr:
diasteromeric ratio assessed by NMR. e) [1a] = [E] = 100 mM. Several purification column chromatography runs had to
f)
be performed to remove the trimerization product from enzymatic double addition of 1a.Table Footnote. Nu = Nucleophile.
E = Electrophile. Green asterisk indicates the stereogenic center formed after reductive amination.
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
methanol (1 mL) and the samples were measured at 25 °C
with a 0.1 dm cell.
Experimental Section
Materials. Synthetic oligonucleotides were purchased
from Eurofins MWG Operon. Phosphoglucose isomerase
from baker’s yeast (S. cerevisiae), glucose-6-phosphate
dehydrogenase from baker's yeast (S. cerevisiae),
D,L-glyceraldehyde 3-phosphate (G3P), nicotinamide
adenine dinucleotide phosphate (NADP+), antibiotics,
Chiral HPLC analyses. Chiral HPLC analyses were
performed on a CHIRALPACK® IC, 5 µm, 4.6×250 mm
column from Daicel (Illkirch, France). Samples (10 µL)
were injected and eluted with hexane:isopropanol (85:15
(v/v)) for 40 min, flow rate 1 mL·min–1, detection at 209
nm.
acrylamide-bisacrylamide,
buffer
aminoethanal dimethyl
R-2-amino-1-propanol,
components,
propanolamine,
S-2-amino-1-propanol,
acetal,
Preparation
of
N-Cbz-aminoaldehydes.
The
N-(benzyloxycarbonyloxy)succinimide, ethanal, acetone,
butanone, 3-pentanone, cyclobutanone, cyclopentanone,
cyclohexanone were purchased from Sigma-Aldrich.
Culture media components for bacteria were from
Pronadisa. Milli-Q grade water was used for analytical and
preparative HPLC, buffer preparations and other assay
solutions were obtained from an AriumTM Pro Ultrapure
Water Purification System (Sartorius Stedim Biotech). All
the other solvents used were of analytical grade.
2-Deoxy-D-ribose-5-phosphate aldolase from Thermotoga
maritima (DERATma) as cell free crude extract was a
generous gift from Prozomix Ltd (activity 0.056 U mg–1
lyophilized powder); 1 U is defined as the amount of
protein, which catalyzes the cleavage of 1 μmol of 2-
deoxy-D-ribose-5-phosphate (Dr5P) per minute at [Dr5P] =
0.7 μM, 50 mM triethanolamine buffer pH 7.5 and 30 °C.
N-Cbz-aminoaldehydes were synthesized from the
corresponding aminoalcohols using the procedures
described in previous publications.[13] The amino group of
the
aminoalcohols
was
first
protected
with
benzyloxycarbonyl (Cbz) group, followed by oxidation of
the alcohol function using 2-iodoxybenzoic acid (IBX)
with a yield between 90-95%. In most instances, a simple
workup followed by a rough crystallization or precipitation
provided the N-Cbz-aminoaldehydes in a form pure
enough to be used in the enzymatic aldol reactions.
Screening of the enzymatic reactions
General procedure for aldol addition of ethanal and
ketones to N-Cbz-aminoaldehydes.
Reactions were conducted in 96 well plates of 1 mL of
capacity. Total reaction volume was 300 µL.
N-Cbz-aminoaldehydes 2a-c (80 mM final concentration)
was dissolved in propanone (1b) (45 µL). For the other
ketones (1c-g), 2a-c (80 mM final concentration) was
dissolved in the corresponding ketone (15 µL). For ethanal
(1a): to 2a-c (80 mM final concentration), 1a (30 µL of a 1
M stock solution) was added. For all of them: 50 mM
triethanolamine buffer pH 8.0 (15 µL of a 1 M stock
solution) was then added. The reaction was started by
adding the lyophilized FSA variant preparation (3 mg·mL-1,
1 mg dissolved in 240 µL, 270 µL or 255 µL of plain
water) or DERATma (1.5 mg, 0.084 U dissolved in 240 µL,
270 µL or 255 µL of plain water). Reaction mixtures were
shaken (1500 rpm) at 25 °C. Reaction monitoring by
HPLC: samples (50 µL) were diluted in MeOH (450 µL)
and after centrifugation were injected to the HPLC system.
FSA catalysts. The single FSA variants A165G, FSA
L107A, FSA L163A, D6A, D6L, D6N, D6Q, D6S, D6T,
D6E, D6H, the double variants FSA D6H/A165G, FSA
D6H/L107A, FSA D6H/L163A, FSA D6E/A165G, FSA
D6E/L107A, FSA D6E/L163A, FSA D6L/A165G, FSA
D6L/L107A, FSA D6L/L163A were obtained using the
procedure described.[8] The FSA multiple variants FSA
L107Y/A129G/A165G/S166G,
L107Y/A129G/R134S/A165G/S166G,
L107Y/A129G/R134E/A165G/S166G,
L107Y/A129G/R134H/A165G/S166G,
L107Y/A129G/R134P/A165G/S166G
FSA
FSA
FSA
FSA
and
L107Y/A129G/R134V/A165G/S166G were obtained
following the mutagenesis and protein expression and
purification procedures described in our previous
publications.[4c] Activity values are shown in Table S1.[8]
Methods. HPLC analyses. HPLC analyses were
performed on an X-BridgeTM C18, 5 µm, 4.6×250 mm
column from Waters (Milford, USA). Samples (30 µL)
were injected and eluted with the following conditions:
solvent system (A) aqueous trifluoroacetic acid (TFA)
(0.1% (v/v) and (B): TFA (0.095% (v/v)) in CH3CN/H2O
(4:1), gradient elution from 10-100 %B in 30 min, flow
rate 1 mL·min–1, detection at 215 nm, column temperature
30 °C. The amount of aldol adduct product was quantified
from the peak areas using and external standard
methodology. Samples were withdrawn (50 µL) from the
reaction medium, centrifuged, diluted with methanol (450
µL) and analyzed with HPLC.
Scale up of aldol addition of ketones (1b-g) to
N-Cbz-aminoaldehydes usingFSA catalysis
General procedure: The reaction was conducted in a 50
mL screw capped conical-bottom polypropylene tubes
(Falcon tubes). To an FSA catalyst solution (3 mg mL–1) in
plain water (16 mL or 18 mL), 1 M triethanolamine buffer
pH
8
(1 mL) was added. To this solution,
N-Cbz-aminoaldehydes 2a-b (80 mM) dissolved in the
ketones (3 mL of 1b or 1 mL of 1c-g) was added. Total
reaction volume was 20 mL. Reaction mixtures were
shaken (1000 rpm) at 25 °C for 24 h. After that, the
reaction was stopped with MeOH (30 mL) to precipitate
the enzyme. Then, the mixture was filtered through Celite,
the MeOH was evaporated and the aqueous residue was
lyophilized, purified characterized and submitted to
reductive amination.
1
NMR analysis. High field H and 13C nuclear magnetic
resonance (NMR) analyses were carried out using an
AVANCE 500 BRUKER spectrometer equipped with a
high-sensitive CryoProbe for [D2]H2O and [D4]MeOH
solutions. Full characterization of the described
compounds was performed using typical gradient-
enhanced 2D experiments: COSY, NOESY, HSQC and
HMBC, recorded under routine conditions. When possible,
NOE data were obtained from selective 1D NOESY
versions using a single pulsed-field-gradient echo as a
selective excitation method and a mixing time of 500 ms.
Intramolecular reductive amination
General procedure: The aldol adduct (3 mM final
concentration) was dissolved in H2O:MeOH 1:1 (70 mL),
then Pd/C (60 mg) was added. The mixture was shaken
under H2 (2.5 atm) overnight at room temperature. After
that, the reaction was filtered and the solvent was
evaporated. Products were characterized without any
further purification.
1
Routine, H (400-500 MHz) and 13C (101 MHz) NMR
spectra of compounds were recorded with Varian Mercury-
400 and Varian Anova-500 spectrometers.
Determination of the specific optical rotations ([α]D25).
Specific optical rotations were measured with a Perkin
Elmer Model 341 (Überlingen, Germany) polarimeter (Na
lamp, 589 nm). The products (520 mg) were dissolved in
Product description
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
(R)-N-Cbz-6-amino-4-hydroxyhexan-2-one (3b): This
compound was obtained following the procedure described
above. N-Cbz-3-aminopropanal (2a) (331.2 mg, 1.6 mmol)
and FSA D6N were used. After 24 h an additional amount
of FSA D6N (3 mg ml–1) was added and the reaction was
shaken (1000 rpm) at 25 °C for 24 h. The product was
purified by silica gel column chromatography and eluted
with a step gradient of hexane:AcOEt from 1:0 to 3:7,
yielding 266.2 mg (85%) of 3b. []D20= + 10.74 (c = 2.1 in
MeOH); HPLC: 10 to 100% of B in 30 min, tR = 16 min;
Chiral HPCL: tR = 25 min (racemic sample: tR(R) = 25 min,
regioisomers I:II. []D20= + 18.5 (c = 0.9 in MeOH);
HPLC: 10 to 100% of B in 30 min, tR = 19 min;
Regioisomer II: 1H NMR (400 MHz, CD3OD) δ 7.59-7.17
(m, 5H), 5.07 (s, 2H), 4.09 (dt, J = 8.2, 4.0 Hz, 1H), 3.24
(q, J = 7.0, 6.4 Hz, 2H), 2.59-2.54 (m, 2H), 2.49 (qd, J =
7.3, 1.8 Hz, 2H), 1.76-1.60 (m, 2H), 1.01 (t, 3H). 13C NMR
(101 MHz, CD3OD) δ 212.7, 159.0, 138.5, 129.2, 128.9,
128.8, 66.2, 65.3, 49.1, 36.8, 36.1, 34.6, 6.43. The
intramolecular
reductive
amination
reaction
of
regioisomer I produced two diastereoisomers in a 87:13
rate. Major: (2R,3R,4R)-2,3-dimethylpiperidin-4-ol (6):
1H NMR (400 MHz, CD3OD) δ 3.36-3.22 (m, 2H), 2.96 (td,
J = 13.3, 3.1 Hz, 1H), 2.81 (dq, J = 10.7, 6.5 Hz, 1H), 2.05
(ddt, J = 13.7, 5.2, 2.8 Hz, 1H), 1.71-1.56 (m, 1H), 1.44-
1.32 (m, 1H), 1.30 (d, J = 6.5 Hz, 3H), 1.06 (d, J = 6.5 Hz,
3H). 13C NMR (101 MHz, CD3OD) δ 70.6, 56.3, 43.6, 42.3,
1
tR(S) = 30 min); H NMR (400 MHz, CD3OD) δ 7.45-7.18
(m, 5H), 5.07 (s, 2H), 4.19-3.99 (m, 1H), 3.23 (t, J = 7.0
Hz, 2H), 2.59 (d, J = 6.4 Hz, 2H), 2.15 (s, 3H), 1.76-1.47
(m, 2H). 13C NMR (101 MHz, CD3OD) δ 210.4, 159.0,
138.4, 129.4, 128.9, 128.8, 67.4, 66.5, 51.7, 38.6, 38.2,
30.6. The intramolecular reductive amination reaction
31.6,
16.3,
12.4.
Minor.
(2S,3R,4S)-2,3-
1
produced: (2R,4R)-2-methylpiperidin-4-ol (5): H NMR
dimethylpiperidin-4-ol (7): 1H NMR (500 MHz, CD3OD)
δ 3.83 (dt, J = 10.2, 4.4 Hz, 1H), 3.32-3.28 (m, 2H), 3.24
(d, J = 12.0 Hz, 1H), 2.91-2.85 (m, 1H), 2.05-2.02 (m, 1H),
1.77-1.66 (m, 2H), 1.25 (d, J = 6.8 Hz, 3H), 0.94 (d, J =
7.2 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 68.3, 53.8,
42.7, 37.7, 26.6, 15.1, 4.8. ESI-TOF: calculated for
[M+H]+ C7H16NO: 130.1231, found 130.1236.
(400 MHz, CD3OD) δ 3.77 (tt, J = 11.0, 4.5 Hz, 1H), 3.41-
3.33 (m, 1H), 3.25-3.14 (m, 1H), 2.97 (td, J = 13.4, 3.0 Hz,
1H), 2.09 (tdd, J = 13.3, 4.7, 2.5 Hz, 2H), 1.55 (tdd, J =
13.7, 11.0, 4.5 Hz, 1H), 1.39 (dd, J = 13.1, 11.3 Hz, 1H),
1.32 (d, J = 6.6 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ
65.2, 51.4, 42.3, 39.5, 30.9, 18.0. ESI-TOF: calculated for
[M+H]+ C6H14NO: 116.1075, found 116.1077.
Aldol addition of 1c to 2b. Regioisomer I (arising from
the attack of the methylene carbon, C3-attack): (3S,4S)-N-
Cbz-5-amino-4-hydroxy-3-methylpentan-2-one (mixture
of diasteromers 15c’:16c’). This mixture was obtained
following the procedure described above. N-Cbz-glycinal
(2b) (1.6 mmol), butanone (1c) and FSA A165G were used.
The product was purified by silica gel column
chromatography and eluted with a step gradient of
hexane:AcOEt from 1:0 to 3:7, yielding 119.2 mg (28%) of
product. []D20= + 13.0 (c = 0.78 in MeOH); HPLC:10 to
100 % of B in 30 min, tR = 17 min; Only one diasteromer
(S)-N-Cbz-5-amino-4-hydroxypentan-2-one (15b): This
compound was obtained following the procedure described
above. N-Cbz-glycinal (2b) (310.4 mg, 1.6 mmol) and
FSA A165G were used. The product was purified by silica
gel column chromatography and eluted with a step gradient
of Hexane:AcOEt from 1:0 to 3:7, yielding 114.8 mg
(29%) of 15b. [α]D20= + 3.4 (c = 1.9 in MeOH); HPLC:10
to 100 % of B in 30 min, tR = 15 min; Chiral HPCL: tR =
31 min (racemic sample: tR(R) = 31 min, tR(S) = 28 min);
1H NMR (400 MHz, CD3OD) δ 7.34 (d, J = 6.0 Hz, 5H),
5.08 (s, 2H), 4.18-4.04 (m, 1H), 3.16 (dd, J = 5.8, 1.4 Hz,
2H), 2.64-2.52 (m, 2H), 2.15 (s, 3H). 13C NMR (101 MHz,
CD3OD) δ 209.9, 159.1, 138.3, 129.4, 129.0, 128.8, 126.3,
68.0, 67.5, 48.4, 47.4, 30.6. The intramolecular reductive
amination reaction produced two diastereoisomers in a
70:30 ratio. Major: (3S,5R)-5-methylpyrrolidin-3-ol (17).
1H NMR (400 MHz, CD3OD) δ 4.41-4.31 (m, 1H), 3.22 (p,
J = 7.0, 7.0, 6.9, 6.9 Hz, 1H), 2.92 (dd, J = 12.2, 1.5 Hz,
1H), 2.87 (dd, J = 12.2, 4.9 Hz, 1H), 2.27 (ddd, J = 13.5,
7.8, 6.5 Hz, 1H), 1.32 (dddd, J = 13.6, 7.8, 3.7, 1.2 Hz,
1H) , 1.27 (d, J = 6.5 Hz, 3H). 13C NMR (101 MHz,
CD3OD) δ 71.8, 54.5, 54.0, 42.1, 19.3. Minor: (3S,5S)-5-
methylpyrrolidin-3-ol (18). 1H NMR (400 MHz, CD3OD)
δ 4.41-4.29 (m, 1H), 3.45 (dp, J = 10.0, 6.5, 6.5, 6.4, 6.4
1
was detected by NMR. H NMR (400 MHz, CD3OD) δ
7.38-7.25 (m, 5H), 5.08 (s, 2H), 3.99 (q, J = 5.8 Hz, 1H),
3.16 (qd, J = 13.9, 6.1 Hz, 2H), 2.65 (dd, J = 7.3, 5.6 Hz,
1H), 2.16 (s, 3H), 1.10 (d, J = 7.0 Hz, 3H). 13C NMR (101
MHz, CD3OD) δ 213.6, (C=O carbamate and Cipso not
detected), 129.4, 129.0, 128.9, 71.6, 67.5, 51.2, 45.8, 29.0,
11.0. Regioisomer II (C1-attack): N-Cbz-6-amino-5-
hydroxyhexan-3-one. The product was purified by
preparative HPLC and eluted with a step gradient of
H2O:CH3CN from 100:0 to 50:50, flow rate 1 mL min–1
and detection at 215 nm, yielding 12 mg of a 68:32
mixture of regioisomers I:II. []D20= + 0.15 (c = 0.8 in
MeOH); HPLC:10 to 100% of B in 30 min, tR = 18 min;
Regioisomer II: 1H NMR (400 MHz, CD3OD) δ 7.45-7.09
Hz, 1H), 3.27 (dd, J = 12.1, 5.2 Hz, 1H), 2.83-2.74 (m, 1H), (m, 5H), 5.05 (s, 2H), 4.09 (dt, J = 11.6, 5.9 Hz, 1H), 3.17-
1.92 (ddt, J = 13.4, 6.2, 1.4, 1.4 Hz, 1H), 1.48 (ddd, J =
13.4, 10.0, 5.8 Hz, 1H), 1.20 (d, J = 6.5 Hz, 3H). 13C NMR
(101 MHz, CD3OD) δ 71.5, 54.0, 52.9, 42.8, 18.3. ESI-
TOF: calculated for [M+H]+ C5H12NO: 102.0919, found
102.0210.
3.10 (m, 2H), 2.54-2.50 (m, 2H), 2.46 (q, J = 7.3 Hz, 2H),
0.98 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ
(C=O carbamate and carbonyl and Cipso not detected),
128.0, 127.6, 127.4, 66.6, 66.1, 46.3, 46.0, 36.0, 6.4. The
intramolecular
reductive
amination
reaction
of
regioisomer I produced four diasteroisomers in 80:8:8:4
ratio. Major (80%). (3S,4R,5R)-4,5-dimethylpyrrolidin-
3-ol (19): 1H NMR (500 MHz, CD3OD) δ 3.99 (td, J = 6.6,
4.9 Hz, 1H), 3.40 (dd, J = 12.1, 6.9 Hz, 1H), 3.14 (dq, J =
9.7, 6.6 Hz, 1H), 3.06-2.99 (m, 1H), 1.79-1.69 (m, 1H),
1.39 (d, J = 6.6 Hz, 3H), 1.11 (d, J = 6.9 Hz, 3H). 13C
NMR (101 MHz, CD3OD) δ 75.2, 60.4, 50.1, 47.5, 15.3,
13.3. Minor (8%) (3S,4R,5S)-4,5-dimethylpyrrolidin-3-ol
Aldol addition of 1c to 2a. Regioisomer I (arising from the
attack of the methylene carbon, C3-attack): N-Cbz-6-
Amino-4-hydroxy-3-methylhexan-2-one (mixture of
diasteromers 3c’:4c’). This mixture was obtained following
the procedure described above. N-Cbz-3-aminopropanal
(2a) (1.6 mmol), butanone (1c) and FSA D6E were used.
The product was purified by silica gel column
chromatography and eluted with a step gradient of
hexane:AcOEt from 1:0 to 3:7, yielding 213 mg (48%) of
product. []D20= + 33.5 (c = 2.1 in MeOH); HPLC: 10 to
100 % of B in 30 min, tR = 18 min. Only one diasteromer
1
(20): H NMR (500 MHz, CD3OD) δ 4.15 (dt, J = 4.5, 2.0
Hz, 1H), 3.98-3.90 (m, 1H), 3.53-3.46 (m, 1H), 3.11-3.06
(m, 1H), 2.26-2.19 (m, 1H), 1.31 (d, J = 7.0 Hz, 3H), 0.92
(d, J = 7.5 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 75.6,
56.7, 50.5, 44.1, 11.6, 9.9. Minor (8%) (3R,4R,5R)-4,5-
dimethylpyrrolidin-3-ol (21): 1H NMR (500 MHz,
CD3OD) δ 4.24 (t, J = 4.1 Hz, 1H), 3.43-3.40 (m, 1H),
3.38-3.33 (m, 1H), 3.02-2.97 (m, 1H), 1.88-1.77 (m, 1H),
1
was detected by NMR. H NMR (400 MHz, CD3OD) δ
7.50-7.19 (m, 5H), 5.05 (s, 2H), 3.90 (dt, J = 9.2, 4.5 Hz,
1H), 3.26-3.15 (m, 2H), 2.67-2.53 (m, 1H), 2.14 (s, 3H),
1.57 (tdd, J = 15.2, 7.7, 5.1 Hz, 2H), 1.06 (d, J = 7.0 Hz,
3H). 13C NMR (101 MHz, CD3OD) δ 214.0, 158.2, 137.1,
128.7, 128.3, 128.1, 69.0, 65.9, 52.3, 37.3, 34.7, 28.2, 9.8.
Regioisomer II (C1 attack): (R)-N-Cbz-7-amino-5-
hydroxyheptan-3-one: The product was purified by
preparative HPLC and eluted with a step gradient of
H2O:CH3CN from 1:0 to 1:1, flow rate 1 mL min–1 and
detection at 215 nm, yielding 25 mg of a mixture 44:56 of
1.35 (d, J = 7.1 Hz, 3H), 1.05 (d, J = 2.6 Hz, 3H). 13
C
NMR (101 MHz, CD3OD) δ 71.8, 58.6, 52.1, 45.2, 13.7,
8.8. Minor (4%) (3R,4R,5S)-4,5-dimethylpyrrolidin-3-ol
1
(22): H NMR (500 MHz, CD3OD) δ 4.30 (td, J = 4.7, 2.6
Hz, 1H), 3.84-3.75 (m, 1H), 3.35-3.27 (m, 1H), 3.19-3.14
(m, 1H), 2.35-2.30 (m, 1H), 1.33 (d, J = 10.8 Hz, 3H), 1.05
(d, J = 2.6 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 71.8,
9
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
57.2, 51.5, 40.0, 13.8, 7.1. ESI-TOF: calculated for
[M+H]+ C6H14NO: 116.1075, found 116.1078.
Cbz-3-aminopropanal (2a) (1.6 mmol), cyclobutanone (1e)
and FSA D6L were used. The mixture was purified by
silica gel column chromatography and eluted with a step
gradient of hexane:AcOEt from 1:0 to 3:7, yielding 201.8
mg (46%) of the mixture. []D20= + 36.49 (c = 1.82 in
MeOH); HPLC:10 to 100 % of B in 30 min, tR = 17 min.
Major 3e (80%): 1H NMR (400 MHz, CD3OD) δ 7.41-7.25
(m, 5H), 5.06 (s, 2H), 3.73 (dt, J = 9.0, 4.4 Hz, 1H), 3.47-
3.35 (m, 1H), 3.23 (t, J = 6.9 Hz, 2H), 3.03-2.78 (m, 2H),
2.09 (qd, J = 10.3, 5.9 Hz, 1H), 1.96 (td, J = 10.6, 5.2 Hz,
1H), 1.86-1.63 (m, 2H). 13C NMR (101 MHz, CD3OD) δ
212.3, 159.0, 138.4, 129.4, 129.0, 128.8, 69.1, 67.4, 66.9,
(4S,5R)-N-Cbz-7-amino-5-hydroxy-4-methylheptan-3-
one (3d). This compound was obtained following the
procedure described above. N-Cbz-3-aminopropanal (2a)
(1.6 mmol), 3-pentanone (1d) and FSA D6E were used.
The product was purified by silica gel column
chromatography and eluted with a step gradient of
hexane:AcOEt from 1:0 to 3:7, yielding 164.1 mg (35%) of
3d. []D20= + 36.5 (c = 2.3 in MeOH); HPLC: 10 to 100%
of B in 30 min, tR = 20 min; 1H NMR (400 MHz, CD3OD)
δ 7.45-7.17 (m, 5H), 5.05 (s, 2H), 3.83 (ddd, J = 9.4, 5.6,
3.7 Hz, 1H), 3.20 (hept, J = 7.2 Hz, 2H), 2.62 (p, J = 6.8
Hz, 1H), 2.52 (qd, J = 7.2, 2.0 Hz, 2H), 1.67-1.39 (m, 2H),
1.08 (d, J = 7.0 Hz, 3H), 0.99 (t, J = 7.2 Hz, 3H). 13C NMR
(101 MHz, CD3OD) δ 215.2, 157.6, 137.0, 128.0, 127.5,
127.3, 69.4, 65.9, 51.5, 37.5, 34.7, 34.5, 10.4, 6.5. The
intramolecular reductive amination reaction produced
1
45.7, 38.7, 36.1, 14.4. Minor 4e (20%): H NMR (400
MHz, CD3OD) δ 7.41- 7.25 (m, 5H), 5.06 (s, 2H), 3.94-
3.87 (m, 1H), 3.21-3.10 (m, 1H), 3.22-3.15 (m, 2H), 3.01-
2.80 (m, 2H), 2.14-1.90 (m, 2H), 1.71-1.52 (m, 2H). 13C
NMR (101 MHz, CD3OD) δ 212.3, 159.0, 138.4, 129.4,
129.0, 128.8, 67.2, 67.1, 66.8, 45.9, 38.5, 36.5, 13.5. The
intramolecular reductive amination reaction produced
(1R,2R,6R)-1,2,6-trimethylbicyclo[4.2.0]octane (9) as the
1
(2R,3R,4R)-2-ethyl-3-methylpiperidin-4-ol (8). H NMR
1
solely product. H NMR (400 MHz, CD3OD) δ 3.77 (dt, J
(400 MHz, CD3OD) δ 3.27-3.25 (m, 2H), 2.87 (td, J = 13.1,
2.9 Hz, 1H), 2.56 (ddd, J = 10.6, 7.4, 3.3 Hz, 1H), 2.02
(ddt, J = 13.3, 5.1, 2.8 Hz, 1H), 1.85 (dqd, J = 15.2, 7.6,
2.8 Hz, 1H), 1.66-1.49 (m, 2H), 1.38 (tq, J = 10.2, 6.5 Hz,
1H), 1.05 (d, J = 6.5 Hz, 3H), 1.00 (t, J = 7.6 Hz, 3H). 13C
NMR (101 MHz, CD3OD) δ 73.1, 62.9, 44.5, 43.3, 34.0,
25.3, 13.8, 9.3. ESI-TOF: calculated for [M+H]+ C8H18NO:
144.1388, found 144.1385.
= 10.7, 5.7 Hz, 1H), 3.63-3.55 (m, 1H), 3.17 (dt, J = 13.2,
4.2 Hz, 1H), 2.74 (t, J = 7.1 Hz, 1H), 2.62 (ddd, J = 13.7,
12.0, 2.9 Hz, 1H), 2.34-2.19 (m, 2H), 1.96-1.78 (m, 2H),
1.78-1.63 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 64.2,
52.9, 40.3, 37.3, 28.1, 24.4, 18.4. ESI-TOF: calculated for
[M+H]+ C7H14NO: 128.1075, found 128.1070.
(R)-2-((S)-N-Cbz-2-amino-1-hydroxyethyl)cyclobutan-
1-one (15e). This compound was obtained following the
procedure described above. N-Cbz-glycinal (2b) (1.6
mmol), cyclobutanone (1e) and FSA A165G were used.
The product was purified by silica gel column
chromatography and eluted with a step gradient of
hexane:AcOEt from 1:0 to 3:7, yielding 157.8 mg (37%) of
the title compound. HPLC: 10 to 100% of B in 30 min, tR =
16 min; 1H NMR (400 MHz, CD3OD) δ 7.39-7.21 (m, 5H),
5.05 (s, 2H), 3.77 (dt, J = 7.2, 4.9 Hz, 1H), 3.48-3.36 (m,
1H), 3.25 (dd, J = 10.6, 6.0 Hz, 2H), 2.88-2.82 (m, 2H),
2.13-1.94 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 210.4,
157.6, 139.9, 128.0, 127.5, 127.4, 69.1, 66.1, 63.1, 44.3,
12.8. The reductive amination gave a complex NMR
spectrum, which could not be resolved and that probably
indicate a mixture of side products.
N-Cbz-6-amino-5-hydroxy-4-methyl-hexan-3-one
(mixture of diasteromers 15d (4S,5S):16d (4R,5S). This
mixture was obtained following the procedure described
above. N-Cbz-glycinal (2b) (1.6 mmol), 3-pentanone and
FSA D6T were used. The product was purified by silica
gel column chromatography and eluted with a step gradient
of hexane:AcOEt from 1:0 to 3:7, yielding 134.4 mg
(30 %) of product. []D20 = + 12.3 (c = 1.26 in MeOH);
HPLC:10 to 100 % of B in 30 min, tR= 19 min. Only one
1
diasteromer was detected by NMR. H NMR (400 MHz,
CD3OD) δ 7.44-7.16 (m, 5H), 5.19-5.02 (m, 2H), 3.92 (q, J
= 5.6 Hz, 1H), 3.16-3.06 (m, 2H), 2.64 (dt, J = 12.9, 6.8
Hz, 1H), 2.51 (q, J = 7.2 Hz, 2H), 1.08 (d, J = 7.0 Hz, 3H),
0.97 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ
214.6, 157.6, 136.9, 128.0, 127.5, 127.4, 70.5, 66.1, 49.0,
44.4, 34.3, 10.3, 6.5. The intramolecular reductive
amination reaction produced four diasteoisomers in ≈
79:8:5:8 rate. Major (79%): (3S,4R,5R)-5-ethyl-4-
methylpyrrolidin-3-ol (23). 1H NMR (500 MHz, CD3OD)
δ 3.97 (q, J = 5.3 Hz, 1H), 3.36-3.30 (m, 1H), 3.02 (dd, J =
12.0, 4.5 Hz, 1H), 2.91 (tdd, J = 8.7, 5.1, 1.5 Hz, 1H),
1.91-1.75 (m, 2H), 1.72-1.61 (m, 1H), 1.10 (d, J = 7.0 Hz,
3H), 1.03 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz,
CD3OD) δ 75.7, 66.7, 50.3, 46.3, 25.1, 14.5, 9.9. Minor
(8%): (3S,4R,5S)-5-ethyl-4-methylpyrrolidin-3-ol (24).
1H NMR (400 MHz, CD3OD) 1H NMR (500 MHz,
CD3OD) δ 4.24 (t, J = 4.1 Hz, 1H), 3.41-3.34 (m, 1H),
3.17-3.12 (m, 2H), 1.89-1.82 (m, 2H), 1.54-1.43 (m, 1H),
(R)-2-((R)-N-Cbz-3-amino-1-
hydroxypropyl)cyclopentan-1-one (3f) and (R)-2-((S)-N-
Cbz-3-amino-1-hydroxypropyl)cyclopentan-1-one (4f).
These compounds were obtained following the procedure
described above as a mixture of 3f:4f in a 67:33 ratio. N-
Cbz-3-aminopropanal (2a) (1.6 mmol), cyclopentanone
(1f) and FSA A165G were used. The mixturet was purified
by silica gel column chromatography and eluted with a
step gradient of hexane:AcOEt from 1:0 to 3:7, yielding
303.5 mg (65%) of a mixture 3f:4f in a 67:33 ratio. HPLC:
10 to 100% of B in 30 min, tR = 18 and 19 min. Major
(67%) (3f): 1H NMR (400 MHz, CD3OD) δ 7.41-7.17 (m,
1.10-1.09 (m, 3H), 1.02-0.98 (m, 3H). 13C NMR (101 MHz, 5H), 5.06 (s, 2H), 3.78 (ddd, J = 9.1, 5.4, 3.4 Hz, 1H),
CD3OD) δ 72.0, 64.8, 52.0, 43.6, 23.6, 15.5, 10.1. Minor
(5%): (3R,4R,5S)-5-ethyl-4-methylpyrrolidin-3-ol (25):
1H NMR (500 MHz, CD3OD) δ 4.14 (d, J = 4.5 Hz, 1H),
3.73-3.66 (m, 3H), 3.49-3.44 (m, 2H), 3.11-3.07 (m, 1H),
2.32-2.24 (m, 1H), 1.71-1.65 (m, 2H), 1.04 (d, J = 7.5 Hz,
3H), 0.88 (d, J = 7.5 Hz, 3H). 13C NMR (101 MHz,
CD3OD) δ 75.7, 62.7, 50.6, 43.0, 22.1, 9.9, 9.5. Minor
(8%): (3R,4R,5R)-5-ethyl-4-methylpyrrolidin-3-ol (26):
1H NMR (500 MHz, CD3OD) δ 4.35 (q, J = 5.7 Hz, 1H),
3.47-3.43 (m, 1H), 3.27-3.23 (m, 1H), 3.08-3.04 (m, 1H),
2.38 (td, J = 7.4, 5.0 Hz, 1H), 1.74-1.66 (m, 1H), 1.06 (d, J
= 5.1 Hz, 19H), 0.97 (d, J = 5.2 Hz, 2H). 13C NMR (101
MHz, CD3OD) δ 71.3, 63.1, 49.7, 39.2, 25.9, 9.9, 6.7. ESI-
TOF: calculated for [M+H]+ C7H16NO: 130.1231, found
130.1234.
3.27-3.12 (m, 2H), 2.35-2.21 (m, 2H), 2.18-2.09 (m, 3H),
1.83-1.71 (m, 3H), 1.70-1.61 (m, 1H). 13C NMR (101 MHz,
CD3OD) δ 221.4, 157.5, 137.0, 128.0, 127.5, 127.3, 69.1,
66.1, 53.7, 38.7, 37.4, 25.8, 22.6, 20.2. Minor (33%) (4f):
1H NMR (400 MHz, CD3OD) δ 7.41-7.17 (m, 5H), 5.06 (s,
2H), 4.06 (ddd, J = 8.4, 4.7, 3.2 Hz, 1H), 3.28-3.16 (m,
2H), 2.28-2.18 (m, 1H), 2.18-2.09 (m, 5H), 1.83-1.71 (m,
1H), 1.70-1.61 (m, 2H). 13C NMR (101 MHz, CD3OD) δ
220.9, 157.5, 137.0, 128.0, 127.5, 127.3, 66.1, 66.8, 55.2,
38.9, 38.5, 36.3, 24.0, 21.5. The intramolecular reductive
amination reaction produced four diasteroisomers
including
a
dehydroxylated analogue (14) in
a
15:11:15:22:37 ratio. Owing to the signal overlapping the
some chemical shifts identified as multiplets are given as
the middle point rather than as range. Selected signals of
the NMR spectra. Detailed assignment in Figure (S15).
Minor
10
(15%):
(4R,4aS,7aR)-octahydro-1H-
(R)-2-((R)-N-Cbz-3-amino-1-
cyclopenta[b]pyridin-4-ol (10). 1H NMR (500 MHz,
MeOD) δ 4.19 (dt, J = 10.6, 5.2 Hz, 1H), 3.64 (m, 1H),
3.36 (m, 1H), 3.02 (m, 1H), 2.49 (m, 1H), 2.19-1.59 (m,
6H). 13C NMR (101 MHz, CD3OD) δ 65.5, 59.0, 44.6, 42.3.
hydroxypropyl)cyclobutan-1-one (3e) and (R)-2-((S)-N-
Cbz-3-amino-1-hydroxypropyl)cyclobutan-1-one (4e):
These compounds were obtained following the procedure
described above as a mixture of 3e:4e in a 80:20 ratio. N-
10
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
Minor
11
(11%):
(4R,4aS,7aS)-octahydro-1H-
solution in plain water (18.9 mL), 1 M triethanolamine
buffer, pH 8 (1 mL) was added. To this solution, N-Cbz-3-
aminopropanal (331.2 mg, 80 mM) and ethanal (100 mM,
112 µL) were added. Total reaction volume was 20 mL.
Reaction mixtures were shaken (1000 rpm) at 25 °C for 24
h. After that, the reaction was stopped with MeOH (30
mL) to precipitate the enzyme, and the mixture was filtered
through Celite. MeOH was evaporated and the reaction
was lyophilized. The product was purified by silica gel
column chromatography and eluted with a step gradient of
hexane:AcOEt from 1:0 to 3:7, yielding 191.3 mg (47%) of
3a. []D20= + 8.51 (c = 2.1 in MeOH); HPLC: 10 to 100%
of B in 30 min, tR = 14.5 min; 1H NMR (400 MHz,
CD3OD) δ 7.59-7.24 (m, 5H), 5.58-5.47 (m, 1H), 5.22-4.99
(m, 2H), 4.11-4.06 (m, 1H), 4.03 (tt, J = 11.3, 4.5 Hz, 1H),
3.12-2.98 (m, 1H), 2.24-2.13 (m, 1H), 2.02-1.92 (m, 1H),
1.94-1.84 (m, 1H), 1.49-1.42 (m, 1H), 1.42-1.33 (m, 1H).
13C NMR (101 MHz, CD3OD) δ 155.7, 136.5, 128.2, 127.8,
cyclopenta[b]pyridin-4-ol (11). 1H NMR (500 MHz,
MeOD) δ 3.99 (td, J = 4.7, 3.1 Hz, 1H), 3.77 (m, 1H), 3.32
(m, 1H), 3.17 (dt, J = 12.8, 4.4 Hz, 1H), 2.22 (m, 1H),
2.19-1.59 (m, 7H). 13C NMR (101 MHz, CD3OD) δ 63.2,
55.4, 45.4, 37.9. Minor 12 (15%): (4S,4aS,7aS)-
1
octahydro-1H-cyclopenta[b]pyridin-4-ol (12). H NMR
(500 MHz, MeOD) δ 3.62 (m, 1H), 3.50 (m, 1H), 3.08 (dd,
J = 13.6, 3.5 Hz, 1H), 2.90 (td, J = 11.3, 7.1 Hz, 1H), 2.17
(m, 1H), 2.19-1.59 (m, 8H). 13C NMR (101 MHz, CD3OD)
δ 70.5, 59.9, 49.7, 45.0. Minor 13 (22%): (4S,4aS,7aR)-
1
octahydro-1H-cyclopenta[b]pyridin-4-ol (13). H NMR
(500 MHz, MeOD) δ 3.48 (m, 1H), 3.47 (m, 1H), 3.01 (m,
1H), 2.78 (td, J = 11.2, 7.1 Hz, 2H), 2.14 (m, 1H), 2.19-
1.59 (m, 8H). 13C NMR (101 MHz, CD3OD) δ 61.4, 57.9,
43.9, 42.1. Major 14 (37%) dehydroxylated analogue:
1
(4aR,7aR)-octahydro-1H-cyclopenta[b]pyridine (14): H
NMR (500 MHz, MeOD) δ 3.58 (ddd, J = 7.9, 5.4, 2.9 Hz,
4H), 3.27 (t, J = 3.7 Hz, 2H), 3.01 (m, 1H), 2.35-2.25 (m,
1H), 2.11 (m, 1H), 1.79 (m, 2H), 1.85 (m, 2H), 1.71 (m,
1H), 1.76 (m, 2H). 13C NMR (101 MHz, CD3OD) δ 57.8,
42.3, 37.1, 28.2, 26.2, 22.4, 20.5, 17.8. ESI-TOF:
calculated for [M+H]+ C8H16NO: 142.1231, found
142.1238.
1
127.5, 83.0, 67.1, 63.4, 38.7, 37.0, 35.1, 31.2. Rotamer: H
NMR (400 MHz, CD3OD) δ 7.59-7.24 (m, 5H), 5.49-5.43
(m, 1H), 5.16-5.08 (m, 2H), 4.03-3.99 (m, 1H), 3.97-3.91
(m, 1H), 3.45-3.38 (m, 1H), 2.12-2.04 (m, 1H), 1.86 (dt, J
= 14.7, 3.9 Hz, 1H), 1.81-1.70 (m, 2H). 13C NMR (101
MHz, CD3OD) δ 155.4, 136.5, 128.2, 127.8, 127.5, 82.5,
67.1, 60.0, 35.2, 33.0, 31.3. ESI-TOF: calculated for
[M+H]+ C13H18NO4: 252.1235, found 252.1231.
(R)-2-((S)-N-Cbz-2-amino-1-hydroxyethyl)cyclopentan-
1-one
(15f)
and
(R)-2-((R)-N-Cbz-2-amino-1-
hydroxyethyl)cyclopentan-1-one (16f). These compounds
were obtained following the procedure described above as
a mixture of 15f:16f in a 50:50 ratio; HPLC: 10 to 100% of
B in 30 min, tR = 17 and 18 min, respectively. N-Cbz-
glycinal (2b) (1.6 mmol), cyclopentanone (1f) and FSA
A165G were used. The product was purified by silica gel
column chromatography and eluted with a step gradient of
hexane:AcOEt from 1:0 to 3:7, yielding two fraction pools:
fraction pool 1 containing 15f pure (53 mg,) and a fraction
pool 2 as a mixture of 15f:16f in a 1:2 ratio (117.2 mg),
total 170.2 mg (38 %) of product. Fraction pool 1 (R)-2-
((S)-N-Cbz-2-amino-1-hydroxyethyl)cyclopentan-1-one
Scale up of aldol additions of propanone (1b) to 2a-c
catalyzed by DERATma
General procedure: The reactions were conducted in a 50
mL screw capped conical-bottom polypropylene tubes. To
a DERATma solution (3 mg mL–1) in plain water (17 mL), 1
M triethanolamine buffer, pH 8 (1 mL) was added. To this
solution, the aldehyde (2a-c) (80 mM) dissolved in
propanone (1b) (2 mL) was added. Total reaction volume
was 20 mL. Reaction mixtures were shaken (1000 rpm) at
25 °C for 24 h. After that, the reaction was stopped with
MeOH (30 mL) to precipitate the enzyme. Then, the
mixture was filtered through Celite, the MeOH was
evaporated and the aqueous residue was lyophilized ad
submitted to reductive amination. Reductive amination:
identical procedure as described above.
1
(15f). H NMR (400 MHz, CD3OD) δ 7.38-7.24 (m, 5H),
5.07 (s, 2H), 4.09 (td, J = 6.9, 3.0 Hz, 1H), 3.23 (dd, J =
13.7, 6.2 Hz, 1H), 3.14 (dd, J = 13.7, 7.2 Hz, 1H), 2.33-
2.15 (m, 2H), 2.15-1.92 (m, 4H), 1.83-1.70 (m, 1H). 13C
NMR (101 MHz, CD3OD) δ 222.2, 159.0, 138.4, 129.4,
129.0, 128.9, 69.6, 67.5, 53.2, 46.3, 39.8, 23.7, 21.7.
Fraction pool 2 15f:16f 33:67 ratio. NMR of 16f: (R)-2-
((R)-N-Cbz-2-amino-1-hydroxyethyl)cyclopentan-1-one
(R)-N-Cbz-6-amino-4-hydroxyhexan-2-one (3b): This
compound was obtained following the procedure described
above. N-Cbz-3-aminopropanal (2a) (331.2 mg, 1.6 mmol)
was used. After 24 h additional DERATma (3 mg ml–1) was
added and the reaction was shaken (1000 rpm) at 25°C for
24 h (70% product formed by HPLC). The product was
purified by silica gel column chromatography and eluted
with a step gradient of hexane:AcOEt from 1:0 to 3:7,
1
(16f): H NMR (400 MHz, CD3OD) δ 7.42-7.16 (m, 5H),
5.07 (s, 2H), 3.83 (q, J = 5.5 Hz, 1H), 3.38-3.29 (m, 2H),
2.32-1.98 (m, 5H), 1.91-1.71 (m, 2H). 13C NMR (101 MHz,
CD3OD) δ 222.5, 138.5, 129.4, 128.8, 70.8, 67.5, 52.6,
45.7, 40.1, 27.5, 21.8. The intramolecular reductive
amination reaction of the fraction pool 2 (33:67 15f:16f
ratio) produced two diastereoisomers the corresponding to
the aldol adducts 27 and 28, respectively. From aldol
adduct 15f: (3S,3aS,6aR)-octahydrocyclopenta[b]pyrrol-
3-ol (27): 1H NMR (500 MHz, CD3OD) δ 4.35 (dt, J = 6.7,
3.5 Hz, 1H), 3.99 (ddd, J = 9.0, 7.2, 3.3 Hz, 1H), 3.15 (d, J
= 3.5 Hz, 2H), 2.81 (tdd, J = 9.3, 6.4, 3.6 Hz, 1H), 2.00-
1.92 (m, 1H), 1.93-1.86 (m, 2H), 1.89-1.75 (m, 2H), 1.67-
1.62 (m, 1H). 13C NMR (101 MHz, CD3OD) δ 70.6, 64.6,
53.9, 47.1, 30.8, 26.0, 24.9. From aldol adduct (16f):
(3R,3aS,6aR)-octahydrocyclopenta[b]pyrrol-3-ol (28):
1H NMR (500 MHz, CD3OD) δ 4.19-4.12 (m, 2H), 3.20-
3.15 (m, 1H), 3.09 (dt, J = 12.1, 1.6 Hz, 1H), 2.65 (ddd, J
= 9.6, 7.9, 6.3 Hz, 1H), 1.99-1.92 (m, 1H), 1.93-1.87 (m,
1H), 1.69-1.67 (m, 1H), 1.68-1.61 (m, 1H), 1.57-1.51 (m,
1H), 1.40 (dq, J = 13.3, 6.7 Hz, 1H). 13C NMR (101 MHz,
CD3OD) δ 75.6, 63.0, 51.7, 51.6, 31.7, 29.1, 24.9. ESI-
TOF: calculated for [M+H]+ C7H14NO: 128.1075, found
128.1079.
20
yielding 198.6 mg (47%) of product. []D = + 15.8 (c =
0.39 in MeOH); HPLC: 10 to 100% of B in 30 min, tR = 16
min; Chiral HPCL: tR = 23 min (racemic sample: tR(R) =
23 min, tR(S) = 27 min). NMR spectral data was identical
to that obtained by FSA D6N catalysis (see above).
(S)-N-Cbz-5-amino-4-hydroxypentan-2-one (15b): This
compound was obtained following the procedure described
above. N-Cbz-glycinal (2b) (310.4 mg, 1.6 mmol) was
used (85% 15b formed). The product was purified by silica
gel column chromatography and eluted with a step gradient
of hexane:AcOEt from 1:0 to 3:7, yielding 120.2 mg (30%)
20
of product. []D = + 2.2 (c = 0.92 in MeOH); HPLC: 10
to 100% of B in 30 min, tR = 15 min; Chiral HPCL: tR = 25
min (racemic sample: tR(R) = 25 min, tR(S) = 23 min).
NMR spectra were identical to that obtained by FSA
A165G catalysis (see above).
(4S,5S)-N-Cbz-5-amino-4-hydroxyhexan-2-one (4S,5S-
29): This compound was obtained following the procedure
described above. (S)-N-Cbz-Alaninal (S-2c) (331.2 mg, 1.6
mmol), propanone (2b) (2 mL) and DERATma (in 17 mL of
plain water) were used (81% (4S,5S)-29 formed). The
product was purified by silica gel column chromatography
and eluted with a step gradient of hexane:AcOEt from 1:0
to 3:7, yielding 336.9 mg (79%) of product. []D20 = – 23.8
Scale up of aldol addition of ethanal (1a) to Cbz-3-
aminopropanal (2a)
(2S,4R)-N-Cbz-piperidine-2,4-diol (3a): The reaction was
conducted in a 50 mL screw capped conical-bottom
polypropylene
tubes.
To
a
FSA
L107Y/A129G/R134S/A165G/S166G (3 mg mL–1)
11
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
(c = 2.6 in MeOH); HPLC: 10 to 100 % of B in 30 min, tR
reaction) were added. Reaction mixtures were shaken
(1000 rpm) at 25 °C for 24 h (98% aldol product formed as
1
= 16 min; H NMR (400 MHz, CD3OD) δ 7.46-7.14 (m,
5H), 5.05 (d, J = 4.8 Hz, 2H), 4.00 (td, J = 6.5, 3.2 Hz, 1H), determined by HPLC). After that, the reaction was stopped
3.66 (qd, J = 6.8, 3.1 Hz, 1H), 2.56-2.50 (m, 2H), 2.10 (s,
3H), 1.13 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz,
CD3OD) δ 208.7, 157.1, 137.0, 128.0, 127.5, 127.4, 69.4,
66.0, 50.3, 46.5, 29.2, 15.8. The intramolecular reductive
amination reaction produced two diasteroisomers in a
56:44 ratio. (2S,3S,5S)-2,5-dimethylpyrrolidin-3-ol (33)
(56%) 1H NMR (400 MHz, CD3OD) δ 4.09 (td, J = 4.0, 2.0
Hz, 1H), 3.20 (dt, J = 8.7, 6.6 Hz, 1H), 2.98 (qd, J = 6.7,
3.9 Hz, 2H), 2.41-2.27 (m, 2H), 1.39 (ddd, J = 14.1, 6.8,
2.2 Hz, 2H), 1.27 (d, J = 6.5 Hz, 4H), 1.21 (d, J = 5.4 Hz,
with MeOH (30 mL) to precipitate the enzyme, and the
mixture was filtered through Celite. MeOH was evaporated
and the reaction was lyophilized. The product was purified
by silica gel column chromatography and eluted with a
step gradient of hexane:AcOEt: 100:0 (200 mL) 80:20
(200 mL), 70:30 (200 mL), 60:40 (200 mL) and 50:50 (700
mL), yielding 171.2 mg (34%) of aldol adduct. The
reductive amination was conducted as follows: the aldol
adduct (171.2 mg) was dissolved in MeOH (400 mL), then
Pd (10%)/C with 50% humidity (200 mg) was added. The
9H). 13C NMR (101 MHz, CD3OD) δ 73.1, 59.9, 53.0, 42.4, mixture was shaken under H2 (2.5 atm) overnight at room
19.6, 11.7. (2S,3S,5R)-2,5-dimethylpyrrolidin-3-ol (34)
temperature. After that, the reaction was filtered and the
solvent was evaporated. Product (51.2 mg) was
1
(44%) H NMR (400 MHz, CD3OD) δ 4.14-4.10 (m, 1H),
1
3.66 (dp, J = 9.1, 6.8 Hz, 1H), 2.07 (ddd, J = 13.6, 7.1, 1.3
Hz, 1H), 1.65 (ddd, J = 13.6, 9.0, 4.5 Hz, 1H), 1.21 (d, J =
5.7 Hz, 7H), 1.20 (d, J = 5.4 Hz, 6H). 13C NMR (101 MHz,
CD3OD) δ 73.1, 58.1, 52.0, 42.3, 19.5, 11.8. ESI-TOF:
calculated for [M+H]+ C6H14NO: 116.1075, found
116.1076.
characterized without any further purification. H NMR
(400 MHz, CD3OD) δ 4.25 (t, J = 3.1 Hz, 1H), 3.48 (tt, J =
2x6.7, 2x3.4 Hz, 1H), 3.46 – 3.34 (m, 1H), 2.26 – 2.13 (m,
1H), 2.09 – 2.00 (m, 1H), 1.38 (d, J = 6.7 Hz, 2H). 13C
NMR (101 MHz, CD3OD) δ 72.1, 61.6, 44.0, 34.1, 11.7.
(2R,3S)-2-methylpyrrolidin-3-ol (32). This compound
was synthetized following the procedure described for
(2S,3S)-2-methylpyrrolidin-3-ol (31). In this case the
80% aldol product formation was obtained as determined
by HPLC. The product was purified by silica gel column
chromatography and eluted with a step gradient of
hexane:AcOEt: 100:0 (200 mL) 80:20 (200 mL), 70:30
(200 mL), 60:40 (200 mL) and 50:50 (750 mL). To remove
the product from enzymatic double addition of 1a, two
purification runs were needed to obtain 101.0 mg (20%) of
aldol adduct. The reductive amination was conducted as
follows: the aldol adduct (40.0 mg) was dissolved in
MeOH (100 mL), then Pd (10%)/C with 50% humidity (40
mg) was added. The mixture was shaken under H2 (2.5
atm) overnight at room temperature. After that, the
reaction was filtered and the solvent was evaporated.
Product (51.2 mg) was characterized without any further
N-Cbz-5-amino-4-hydroxyhexan-2-one
(mixture of
diasteromers (4S,5R)-29:(4R,5R)-30 in a 77:23 ratio) This
mixture was obtained following the procedure described
above. (R)-N-Cbz-alaninal (R-2c) (331.2 mg, 1.6 mmol),
acetone (2 mL) and DERATma (in 17 mL of plain water)
were used (91% mixture formed). The mixture was
purified by silica gel column chromatography and eluted
with a step gradient of hexane:AcOEt from 1:0 to 3:7,
20
yielding 330.1 mg (78%) of product. []D = + 2.8 (c =
0.7 in MeOH). HPLC: 10 to 100% of B in 30 min, tR = 16
min. Only one diasteromer was detected by NMR. 1H
NMR (400 MHz, CD3OD) δ 7.35-7.19 (m, 5H), 5.05 (s,
2H), 3.94 (ddd, J = 7.9, 6.1, 4.8 Hz, 1H), 3.56 (p, J = 6.7
Hz, 1H), 2.55 (dd, J = 7.8, 3.9 Hz, 2H), 2.12 (s, 3H), 1.12
(d, J = 6.8 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 208.8,
156.9, 136.9, 128.0, 127.5, 127.4, 70.2, 66.0, 51.1, 46.9,
29.2, 14.8. Another diasteromer was identified after the
reduction amination reaction. The intramolecular reductive
amination reaction produced two diastereoisomers in a
77:23 ratio. (2R,3S,5R)-2,5-Dimethylpyrrolidin-3-ol (35)
(77%).1H NMR (400 MHz, CD3OD) δ 3.80 (td, J = 6.5, 5.2
Hz, 1H), 3.35 (dt, J = 14.1, 7.1 Hz, 1H), 3.04 (qd, J = 6.6,
5.1 Hz, 1H), 2.31 (dt, J = 13.0, 7.1 Hz, 1H), 1.35 (ddd, J =
13.1, 7.7, 6.3 Hz, 1H), 1.21 (d, J = 6.5 Hz, 4H), 1.12 (d, J
= 6.7 Hz, 3H). 13C NMR (101 MHz, CD3OD) δ 77.8, 60.1,
1
purification. H NMR (400 MHz, CD3OD) δ 4.13 (dt, J =
5.4, 3.4, 3.4 Hz, 1H), 3.53 (ddt, J = 10.3, 7.0, 2x3.2 Hz,
1H), 3.45 – 3.37 (m, 2H), 2.27 (dtd, J = 14.1, 2x9.0, 5.4 Hz,
2H), 2.03 – 1.93 (m, 2H), 1.31 (d, J = 7.0 Hz, 2H). 13C
NMR (101 MHz, CD3OD) δ 76.1, 63.4, 43.7, 32.3, 15.6.
Organocatalytic reactions:
N-Benzyloxycarbonyl-(E)-6-aminohex-4-en-2-one:
N-Cbz-3-aminopropanal (0.4 mmol, 82.8 mg) was
dissolved in DMF (4 mL). Acetone (20%, 1 mL) and (d,l)-
proline (0.08 mmol, 9.21 mg) were added. Total reaction
volume was 5 mL. Reaction mixture was stirred at 25°C
for 4 hours. The solvent was evaporated and the product
was purified by silica gel column chromatography and
eluted with a step gradient of hexane:AcOEt from 1:0 to
1:1, yielding 80 mg (80%) of product. 1H NMR (400 MHz,
CD3OD) δ 7.43-7.23 (m, 5H), 6.85 (dt, J = 16.0, 7.1 Hz,
1H), 6.06 (dt, J = 16.2, 1.5 Hz, 2H), 5.04 (s, 2H), 3.36-3.05
(m, 2H), 2.41 (qd, J = 6.7, 1.5 Hz, 1H), 2.19 (s, 3H).13C
NMR (101 MHz, CD3OD) δ 199.8, 157.4, 146.1, 132.1,
128.0, 127.5, 127.3, 65.9, 38.9, 32.8, 25.2.
50.8,
41.4,
20.5,
16.3.
(2R,3R,5S)-2,5-
1
dimethylpyrrolidin-3-ol (36) (23%). H NMR (400 MHz,
CD3OD) δ 4.09 (ddd, J = 6.1, 3.9, 2.2 Hz, 1H), 3.20 (dt, J
= 8.7, 6.7, 6.7 Hz, 1H), 2.98 (tt, J = 6.7, 6.7, 3.3, 3.3 Hz,
1H), 2.41-2.31 (m, 4H), 1.44-1.35 (m, 1H), 1.27 (d, J = 6.6
Hz, 3H), 1.20 (d, J = 5.6 Hz, 3H). 13C NMR (101 MHz,
CD3OD) δ 73.1, 59.9, 42.4, 19.9, 11.8. ESI-TOF:
calculated for [M+H]+ C6H14NO: 116.1075, found
116.1072.
Scale up of aldol additions of ethanal (1a) to 2a-c
catalyzed by DERATma
(2S,4R)-N-Cbz-piperidine-2,4-diol (3a). This compound
was synthetized following the procedure described above
catalyzed by FSA variant. In this case DERATma (3 mg mL–
1) was used and 77% aldol product formation by HPLC
was obtained, yielding 99.3 mg (25%) of pure product.
[]D20 = + 4.6 (c = 0.69 in MeOH); HPLC: 10 to 100 % of
B in 30 min, tR = 14.5 min. NMR spectra was identical to
Preparation of racemic aldol adducts
Lithium diisopropylamine LDA reactions: General
procedure.[18] Lithium diisopropylamine (LDA) (700 µL of
a 2 M stock solution in THF) was added to anhydrous THF
(8 mL) and cooled down to – 78 °C. Then, propanone (1b)
(100 µL) was added and stirred for 20 minutes. After that,
the aldehyde, dissolved in anhydrous THF (2 mL), was
added. The reaction was stirred for 2 hours at – 78 °C, and
then left to warm up to room temperature. When the
reaction mixture was at room temperature, NaHCO3 (20
mL of 10% aqueous solution) was added. The product was
extracted by AcOEt (3 x 15 mL), the organic layers pooled,
dried over anhydrous Na2SO4, and the solvent was
evaporated to dryness.
that
obtained
by
FSA
(see
L107Y/A129G/R134S/A165G/S166G
above).
catalysis
(2S,3S)-2-methylpyrrolidin-3-ol (31). The reaction was
conducted in a 20 mL screw capped conical-bottom
polypropylene tubes. To a DERATma (60 mg, 3 mg mL–1)
solution in plain water (18.0 mL), 1 M triethanolamine
buffer, pH 8 (1.0 mL) was added. To this solution, (S)-N-
Cbz-alaninal (S-2c) (414.0 mg, 2.0 mmol, 100 mM in the
reaction) and ethanal (1a) (120 μL, 2 mmol, 100 mM in the
12
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
rac-N-Cbz-6-amino-4-hydroxyhexan-2-one
(rac-3b):
2015, 357, 1787-1807; d) A. Szekrenyi, A. Soler, X.
Garrabou, C. Guerard-Helaine, T. Parella, J. Joglar, M.
Lemaire, J. Bujons, P. Clapés, Chem. Eur. J. 2014, 20,
12572-12583; e) A. Soler, X. Garrabou, K. Hernández,
M. L. Gutiérrez, E. Busto, J. Bujons, T. Parella, J.
Joglar, P. Clapés, Adv. Synth. Catal. 2014, 356, 3007-
3024; f) P. Laborda, F. J. Sayago, C. Cativiela, T.
Parella, J. Joglar, P. Clapés, Org. Lett. 2014, 16, 1422-
1425; g) A. L. Concia, L. Gómez, T. Parella, J. Joglar,
P. Clapés, J. Org. Chem. 2014, 79 5386–5389.
N-Cbz-3-aminopropanal (2a) (209 mg, 1 mmol) was used.
The product was purified by silica gel column
chromatography and eluted with a step gradient of
hexane:AcOEt from 1:0 to 3:7, yielding 130.6 mg (49 %)
of the title compound. The NMR spectra was
indistinguishable from the one obtained enzymatically (see
above).
rac-N-Cbz-6-amino-4-hydroxypentan-2-one (rac-15b):
N-Cbz-glycinal (2b) (196 mg, 1 mmol) was used. The
product was purified by silica gel column chromatography
and eluted with a step gradient of hexane:AcOEt from 1:0
to 3:7, yielding 88.2 mg (35 %) of the title compound. The
NMR spectra was indistinguishable from the one obtained
enzymatically (see above).
[5] M. Schürmann, G. A. Sprenger, J. Biol. Chem. 2001,
276, 11055-11061.
[6] a) R. Roldán, I. Sanchez-Moreno, T. Scheidt, V.
Hélaine, M. Lemaire, T. Parella, P. Clapés, W.-D.
Fessner, C. Guérard-Hélaine, Chem. Eur. J. 2017, 23,
5005-5009; b) R. Roldán, K. Hernandez, J. Joglar, J.
Bujons, T. Parella, I. Sánchez-Moreno, V. Hélaine, M.
Lemaire, C. Guérard-Hélaine, W.-D. Fessner, P. Clapés,
ACS Catal. 2018, 8, 8804-8809; c) S. Junker, R.
Roldan, H.-J. Joosten, P. Clapés, W.-D. Fessner, Angew.
Chem. Int. Ed. 2018, 57, 10153-10157.
Acknowledgements
This project has received funding from the European Union’s
Horizon 2020 research and innovation program under grant
agreement No 635595 (CarbaZymes), the Ministerio de
Economía y Competitividad (MINECO), the Fondo Europeo de
Desarrollo Regional (FEDER) (grant CTQ2015-63563-R to P.C.
and CTQ2015-64436-P to T.P.), Programación Conjunta
Internacional PCI2018-092937 and COST action CM1303
Systems Biocatalysis.
[7] H. J. M. Gijsen, L. Qiao, W. Fitz, C.-H. Wong, Chem.
Rev. 1996, 96, 443-473.
[8] a) M. Gutierrez, T. Parella, J. Joglar, J. Bujons, P.
Clapés, Chem. Commun. 2011, 47, 5762-5764; b) D.
Güclü, A. Szekrenyi, X. Garrabou, M. Kickstein, S.
Junker, P. Clapés, W.-D. Fessner, ACS Catal. 2016, 6,
1848-1852.
References
[1] a) E. Marcantoni, M. Petrini, Adv. Synth. Catal. 2016,
358, 3657-3682; b) P. R. Lazzara, K. P. Fitzpatrick, C.
C. Eichman, Chem. Eur. J. 2016, 22, 16779-16782; c)
F. Lovering, J. Bikker, C. Humblet, J. Med. Chem.
2009, 52, 6752-6756; d) M. Rubiralta, E. Giralt, A.
Diez, Piperidine. Structure, Preparation, Reactivity,
and Synthetic Applications of Piperidine and its
Derivatives, Vol. 40, Wiley-VCH Verlag GmbH & Co.
KGaA, Amsterdam 1992; e) M. J. Schneider, in
Alkaloids: Chemical and Biological Perspectives, Vol.
Volume 10 (Ed.: S. W. Pelletier), Pergamon, 1996, pp.
155-299.
[9] L. Chen, D. P. Dumas, C.-H. Wong, J. Am. Chem. Soc.
1992, 114, 741-748.
[10] H. Sakuraba, K. Yoneda, K. Yoshihara, K. Satoh, R.
Kawakami, Y. Uto, H. Tsuge, K. Takahashi, H. Hori, T.
Ohshima, Appl. Environ. Microbiol. 2007, 73, 7427-
7434.
[11] a) N. Ran, L. Zhao, Z. Chen, J. Tao, Green Chem.
2008, 10, 361-372; b) S. Hu, J. Tao, Z. Xie,
WO2006134482A1, 2006.
[12] a) A. L. Concia, C. Lozano, J. A. Castillo, T. Parella,
J. Joglar, P. Clapés, Chem. Eur. J. 2009, 15, 3808-
3816; b) J. A. Castillo, J. Calveras, J. Casas, M. Mitjans,
M. P. Vinardell, T. Parella, T. Inoue, G. A. Sprenger, J.
Joglar, P. Clapés, Org. Lett. 2006, 8, 6067-6070; c) L.
Espelt, J. Bujons, T. Parella, J. Calveras, J. Joglar, A.
Delgado, P. Clapés, Chem. Eur. J. 2005, 11, 1392-
1401; d) L. Espelt, T. Parella, J. Bujons, C. Solans, J.
Joglar, A. Delgado, P. Clapés, Chem. Eur. J. 2003, 9,
4887-4899.
[2] a) C. Saotome, Y. Kanie, O. Kanie, C.-H. Wong,
Bioorgan Med Chem 2000, 8, 2249-2261; b) J.
Bolleddula, K. DeMent, J. P. Driscoll, P. Worboys, P. J.
Brassil, D. L. Bourdet, Drug Metabolism Reviews 2014,
46, 379-419; c) O. O. Grygorenko, M. Kurkunov, I. A.
Levandovskiy, A. V. Tymtsunik, Synthesis 2018, 50,
1973-1978; d) C. Wang, Z. Lu, Org. Lett. 2017, 19,
5888-5891.
[3] a) F. Zhang, Y. Peng, Y. Gong, Chirality 2008, 20,
805-811; b) S. R. Chemler, D. A. Copeland, Top.
Heterocycl. Chem. 2013, 32, 39-75; c) S. Rougnon
Glasson, J.-L. Canet, Y. Troin, Tetrahedron Lett. 2000,
41, 9797-9802; d) H. Peng, E. Wei, J. Wang, Y. Zhang,
L. Cheng, H. Ma, Z. Deng, X. Qu, ACS Chem. Biol.
2016, 11, 3278-3283.
[13] J. Calveras, M. Egido-Gabás, L. Gómez, J. Casas, T.
Parella, J. Joglar, J. Bujons, P. Clapés, Chem. Eur. J.
2009, 15, 7310-7328.
[14] T. Kajimoto, L. Chen, K. K. C. Liu, C.-H. Wong, J.
Am. Chem. Soc. 1991, 113, 6678-6680.
[4] a) S. Takayama, G. J. McGarvey, C.-H. Wong, Annu.
Rev. Microbiol. 1997, 51, 285-310; b) L. J. Whalen, C.-
H. Wong, Aldrichimica Acta 2006, 39, 63-71; c) A.
Soler, M. L. Gutiérrez, J. Bujons, T. Parella, C.
Minguillon, J. Joglar, P. Clapés, Adv. Synth. Catal.
[15] A. Szekrenyi, X. Garrabou, T. Parella, J. Joglar, J.
Bujons, P. Clapés, Nat. Chem. 2015, 7, 724-729.
[16] M. T. Konieczny, P. H. Toma, M. Cushman, J. Org.
Chem. 1993, 58, 4619-4624.
13
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
[17] C.-H. Wong, R. L. Halcomb, Y. Ichikawa, T.
Kajimoto, Angew. Chem. Int. Ed. Engl. 1995, 34, 412-
432.
[18] P. A. Wender, M. F. T. Koehler, M. Sendzik, Org.
Lett. 2003, 5, 4549-4552.
14
This article is protected by copyright. All rights reserved.

10.1002/adsc.201801530
Advanced Synthesis & Catalysis
UPDATE
Aldolase-Catalyzed Asymmetric Synthesis of N-
Heterocycles by Addition of Simple Aliphatic
Nucleophiles to Aminoaldehydes
Adv. Synth. Catal. Year, Volume, Page – Page
Raquel Roldan, Karel Hernandez, Jesús Joglar,
Jordi Bujons, Teodor Parella, Wolf-Dieter Fessner,
and Pere Clapés*
15
This article is protected by copyright. All rights reserved.