UPDATES DOI: 10.1002/adsc.201801530 1 2 3 4 Aldolase-Catalyzed Asymmetric Synthesis of N-Heterocycles by 5 Addition of Simple Aliphatic Nucleophiles to Aminoaldehydes 6 7 8 Raquel Roldán,a Karel Hernández,a Jesús Joglar,a Jordi Bujons,a Teodor Parella,b 9 Wolf-Dieter Fessner,c and Pere Clapésa,* 10 a 11 Dept. Biological Chemistry. Instituto de Quı́mica Avanzada de Cataluña IQAC-CSIC, Jordi Girona 18–26, 08034 Barcelona, Spain 12 Phone + 34934006127 13 Fax: + 34932045904 14 E-mail: pere.clapes@iqac.csic.es 15 b Servei de Ressonncia Magntica Nuclear. Universitat Autnoma de Barcelona, Bellaterra, Spain 16 c Institut fr Organische Chemie und Biochemie, Technische Universitt Darmstadt, Petersenstraße 22, D-64287 Darmstadt, 17 Germany 18 19 Manuscript received: November 13, 2018; Revised manuscript received: January 22, 2019; 20 Version of record online: February 15, 2019 21 22 Supporting information for this article is available on the WWW under https://doi.org/10.1002/adsc.201801530 23 24  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. 25 This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, 26 which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial 27 and no modifications or adaptations are made. 28 29 30 Abstract: Nitrogen heterocycles are structural mo- 31 Introduction tifs found in many bioactive natural products and 32 of utmost importance in pharmaceutical drug 33 development. In this work, a stereoselective syn- Nitrogen heterocycles are structural motifs frequently 34 thesis of functionalized N-heterocycles was accom- found in bioactive natural products and pharmaceut- 35 plished in two steps, comprising the biocatalytic icals, being an area of constant development owing to 36 aldol addition of ethanal and simple aliphatic their importance in drug discovery.[1] The ring type, 37 ketones such as propanone, butanone, 3-pentanone, e. g. piperidine, pyrrolidine, pyrrolizidine, N-bicyclics, 38 cyclobutanone, and cyclopentanone to N-Cbz-pro- and the structure, position and stereochemistry of the 39 tected aminoaldehydes using engineered variants of substituents, play a fundamental role in modulating 40 d-fructose-6-phosphate aldolase from Escherichia their biological activity.[1b,2] Methods for the total 41 coli (FSA) or 2-deoxy-d-ribose-5-phosphate aldo- synthesis of a broad structural diversity of N-hetero- 42 lase from Thermotoga maritima (DERATma) as cycles, either as building blocks or as final compounds 43 catalysts. FSA catalyzed most of the additions of for biological testing, are thus in high demand.[1a] 44 ketones while DERATma was restricted to ethanal Although the number of chemical routes available to 45 and propanone. Subsequent treatment with hydro- prepare these compounds is extensive, developing new 46 gen in the presence of palladium over charcoal, stereoselective synthetic methodology is essential yet 47 yielded low-level oxygenated N-heterocyclic deriv- challenging.[1a,3] 48 atives of piperidine, pyrrolidine and N-bicyclic Biocatalytic C C bond forming reactions by means 49 structures bearing fused cyclobutane and cyclo- of carboligases is highly attractive because of unparal- 50 pentane rings, with stereoselectivities of 96–98 ee leled high efficiency and stereoselectivity, avoidance 51 and 97:3 dr in isolated yields ranging from 35 to of unnecessary functional group protection and use of 52 79%. mild reaction conditions. Among them, aldolases have 53 proven their remarkable synthetic utility in the 54 Keywords: Biocatalysis; Aldol reaction; Aldolases; preparation of different types of highly oxygenated N- 55 Nitrogen heterocycles; Reductive amination heterocycles, usually termed as iminosugars or azasu- 56 gars.[4] 57 Adv. Synth. Catal. 2019, 361, 2673 – 2687 2673  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 Recent synthetic developments of d-fructose-6- Electrophilic components chosen were prochiral sub- 2 phosphate aldolase from E. coli (FSA, EC 4.1.2.-)[5] strates 2 a and 2 b, which avoid complications arising 3 showed that FSA D6X variants (X = A, L, N, Q, S, T, from kinetic enantiomer selectivity, and pure enan- 4 E, and H), and double variants combined with A165G, tiomers of chiral substrate 2 c. The latter contains a 5 L107A, L163A and T26X (X: I, A, L or V) mutations, chiral center of defined absolute configuration that 6 are effective catalysts for homo and crossed aldol acts as a reference in the determination of the overall 7 additions of simple aliphatic ketones and aldehydes relative configuration of the products. 8 leading to the preparation of chiral intermediates and Aldol additions of nucleophiles 1 a–f to electro- 9 deoxysugars.[6] Hence, these FSA variants can offer philes N-Cbz-3-aminopropanal (2 a) and N-Cbz-2- 10 new synthetic options toward the preparation of aminoethanal (2 b) were catalyzed by several FSA 11 iminosugar derivatives having a low degree of hydrox- variants (Figure 2). Variants with different amino acids 12 ylation. Likewise, 2-deoxy-d-ribose-5-phosphate aldo- substituting for D6 were found to be active for various 13 lase (DERA, EC 4.1.2.4) has been recognized as a nucleophile-electrophile combinations, some of them 14 powerful aldolase for the synthesis of deoxysugars and yielding good to excellent product formation. Interest- 15 iminosugars.[7] Wild-type DERA was unique among ingly, FSA A165G was also efficient for the additions 16 the aldolases in that it uses simple aliphatic aldehydes of 1 b–c, e–f to 2 a and 2 b, and a quintuple FSA 17 both as nucleophilic and electrophilic aldol compo- mutant (FSA qm: FSA L107Y/A129G/R134S/A165G/ 18 nents and therefore is a qualified catalyst for the S166G) for addition of 1 a to 2 a. In contrast, FSA 19 proposed synthesis. A165G and all variants that include the native D6 20 We have investigated the systematic chemoenzy- residue had been found inactive when hydroxyalde- 21 matic preparation of low-level functionalized N-heter- hyde derivatives were employed as electrophiles.[6b] 22 ocyclic compounds along a two-steps strategy: The Hence, N-Cbz-aminoaldehydes appear to establish 23 first step consists of an aldol addition of ethanal (1 a) different, and potentially stronger, interactions with 24 and simple linear (1 b–d) or cyclic aliphatic ketone the FSA substrate binding site through the carbamate 25 (1 e–g) to an N-Cbz-protected amino aldehyde 2 a–c and/or aromatic moieties, which likely render them 26 (Figure 1) catalyzed by FSA variants and DERA from electrophiles superior to hydroxyaldehydes.[4d,8a] This is 27 Thermotoga maritima (DERATma). In a second step, also consistent with the fact that previously developed 28 the aldol adducts obtained are transformed into the FSA A165G was more efficient than the wild-type in 29 corresponding N-heterocycles via intramolecular re- aldol additions of hydroxyacetone (HA) and dihy- 30 ductive amination by using H2 Pd/C. droxyacetone (DHA) to 2 a and, particularly, to 2 b.[8a] 31 Likewise, wild-type FSA also converted 1 a–c and 1 f 32 using d,l-glyceraldehyde-3-phosphate, the best elec- 33 trophilic substrate known to date for wild-type 34 FSA.[6a] None of the FSA variants assayed catalyzed 35 the addition of 1 a–g to either enantiomer of N-Cbz- 36 alaninal (2 c). This was quite surprising since FSA 37 A165G can catalyze the addition of HA and DHA to 38 both S-2 c and R-2 c.[8a] Cyclohexanone (1 g) was not 39 accepted as a substrate, probably due to steric 40 hindrance. 41 Figure 1. Panel of nucleophiles 1 a–g and electrophiles 2 a–c In comparison, DERATma was also active for the 42 assayed for enzymatic cross-aldolization. aldol addition of ethanal (1 a) and propanone (1 b) to 43 2 a and to R- and S-2 c, whereas 2 b only reacted with 44 1 b (Figure 2). In stark contrast to all FSA catalysts, 45 Results and Discussion DERATma was highly active with both enantiomers of 46 the a-substituted amino aldehydes 2 c. On the other Screening Experiments 47 hand, DERATma did not catalyze additions with any of 48 We studied the aldol addition of simple aliphatic the ketones 1 c–g to any of the electrophiles 2 a–c. This 49 nucleophiles 1 a–g (concentration range 100 mM to is consistent with the observation that the affinity of 50 2.6 M (15% v/v), see SI for optimization analysis) to DERA for ketones is significantly lower than for 51 electrophiles 2 a–c (80–100 mM) using FSA variants ethanal (1 a), which is its natural nucleophilic sub- 52 and wild-type DERATma as catalysts (Figure 2) in strate.[9] 53 small-scale preparative synthetic conversions. FSA 54 catalysts were selected among those found to be Preparative Synthesis and Product Characterization 55 efficient toward the nucleophiles 1 and N-Cbz-amino- 56 aldehydes 2.[4c,6b,8] The screening was performed by Addition of 1 a to N-Cbz-3-aminopropanal (2 a) was 57 direct HPLC monitoring of aldol adduct formation. catalyzed by FSA D6L, D6N, D6E/A165G, FSA qm Adv. Synth. Catal. 2019, 361, 2673 – 2687 2674  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Figure 2. Results of conversion to aldol adduct from the screening of FSA variants and DERATma as catalysts for the aldol 23 addition of nucleophiles 1 a–g to electrophiles 2 a–c. Conversions (mol %) at 24 h were determined by HPLC analysis using 24 the external standard methodology. Conditions: [1 a] = 100 mM; [1 b] = 2.6 M (15% v/v), [1 c] = 0.60 M, [1 d] = 0.47 M, [1 e] = 25 0.68 M, [1 f] = 0.56 M, and [1 g] = 0.48 M (5% v/v; in 1 c–g), [2] = 80 mM, except for the addition if 1 a to R-2 c and S-2 c 26 catalyzed by DERATma where [1 a] = [2 c] = 100 mM. Reactions were conducted in aqueous 50 mM triethanolamine buffer 27 pH 8 at 25 8C. FSA qm = FSA quintuple mutant = FSA L107Y/A129G/R134 S/A165G/S166G. A complete list of all FSA 28 variants assayed can be found in SI. 29 30 31 and DERATma. FSA qm and DERATma catalysis FSA variants D6N and D6T were the most 32 yielded the cyclic N-Cbz-protected hemiaminal 3 a effective catalysts for the aldol addition of 1 b to 2 a, 33 with trans-configured hydroxyl groups at C2:C4 posi- whereas FSA A165G was the best for the reaction 34 tions in 45% and 25% isolated yields, respectively between 1 b and 2 b (Figure 2), furnishing 3 b and 15 b 35 (Table 1, entry 1 and 2). The lower yield of 3 a in 85% and 35% isolated yields, respectively, and with 36 obtained with DERATma is most likely due to con- high enantioselectivity (96%–98% ee) as determined 37 sumption of 1 a in the competing enzymatic trimeriza- by HPLC on chiral stationary phase (Table 1 entry 3 38 tion of 1 a leading to a stable trideoxyhexose (i. e., and Table 2 entry 1). DERATma catalyst gave identi- 39 3R,5R-dihydroxyhexanal; see Scheme 3).[10] On the cally configured aldol adducts in 47% and 30% 40 other hand, neither trimerization reaction nor self- isolated yields, with 84% and 90% ee, respectively 41 aldol addition of 1 a was detected with FSA qm (Table 1 entry 4 and Table 2 entry 2). No self-addition 42 variant.[6b] Single addition of 1 a to 2 a was exclusively of 2 b was observed in the reactions catalyzed by 43 obtained with both FSA qm and DERATma catalysts, DERATma. Again, a single addition product was 44 and no consecutive double addition product was exclusively obtained in the aldol reaction of 1 b with 45 observed, neither upon application of an excess of 2 a and 2 b with FSA D6N, FSA A165G and DERATma 46 ethanal nor upon continuous addition of 1 a using a catalysts. On the basis of the stereochemical outcome 47 syringe pump. In the self-addition of 1 a and the of both enzymes in examples with non-natural sub- 48 crossed addition of 1 a to 2 a DERA is reported to strates,[4c,e,10] 3 b has (R)- and 15 b has (S)-configura- 49 show strict R-stereoselectivity for the newly formed tion. Intramolecular reductive amination of (R)-3 b 50 chiral centers.[10–11] Therefore, 3 a was assigned a was stereoselective, furnishing 5 with cis-stereochemis- 51 (2S,4R)-configuration. Identical NMR spectra were try as expected from previous works.[12] On the other 52 recorded for the adduct obtained by catalysis of FSA hand, (S)-15 b gave a mixture of both cis- and trans- 53 qm, and their matching optical rotation (Table 1) configured pyrrolidines 17 and 18, respectively. 54 verified that the identical product (2S,4R)-3 a was The addition of butanone (1 c) to 2 a could be 55 formed in both cases, which is consistent with the catalyzed by FSA D6S, D6T and D6E, and addition to 56 stereochemical outcome expected for FSA catalysis. 2 b by D6T and A165G variants, generally with good 57 conversions (Figure 2). The preparative reaction be- Adv. Synth. Catal. 2019, 361, 2673 – 2687 2675  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 Table 1. Preparative scale FSA- and DERA-catalyzed aldol additions of 1 a–f to N-Cbz-3-aminopropanal (2 a). 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Entry Biocatalyst Nu Product Aldol adduct Yield,[b] Aldol addition Reductive amination 17 formed,[a] % er or dr, product,[c] 18 % % dr 19 20 1 FSA qm 1a 3 a[d] 85 45 –[e] – 21 2 DERATma 1a 3 a[d] 77 25 –[e] – 22 3 FSA D6 N 1b 3b 94 85 98:2[f] 5 4 DERATma 1b 3b 70 47 92:8[f] – 23 5 FSA D6E 1c 3 c’/4 c’ 78 48 86:14[g] 6:7 86:14 24 6 FSA D6E 1d 3d 71 35 > 97:3[h,i] 8 25 7 FSA D6L 1e 3 e/4 e 56 46 80:20[i] 9 26 8 FSA A165G 1f 3 f/4 f 75 65 67:33[i] 10:11:12:13:14 27 15:11:15:22:37 28 [a] 29 Values determined by HPLC analysis of preparative scale reactions. [b] Isolated yield of aldol adduct. 30 [c] Diastereomer ratio determined by NMR. 31 [d] Aldol adduct isolated as cyclic hemiaminal in equilibrium with its open form. 32 [e] er not determined; optical rotation: FSA qm [a]D20 = + 8.5 (c = 2.1 in MeOH); DERATma [a]D20 = + 4.6 (c = 0.69 in MeOH). 33 [f] er determined by chiral HPLC, referenced to a racemic sample prepared by chemical synthesis (see SI). 34 [g] Inferred from the reductive amination products 6:7. 35 [h] No other diastereomers were detected by NMR analysis. [i] 36 dr of the aldol adduct determined by NMR analysis. FSA qm = FSA L107Y/A129G/R134S/A165G/S166G. 37 Nu: Nucleophile. Green asterisk indicates the stereogenic center formed after reductive amination. 38 39 40 tween 1 c and 2 a using FSA D6E produced two 41 regioisomeric adducts, one was arising from the attack 42 of the a-methylene carbon of 1 c (i. e., C3 attack) and 43 the other one from the attack of the a-methyl carbon 44 (i. e., C1 attack), with a C1:C3 ratio of 13:87. 45 Chromatographic separation of the product isomers 46 from the reaction mixture proved difficult and only 47 allowed enrichment, furnishing a major fraction 48 (48%) containing a 3 c’/4 c’ diasteromer mixture in a 49 86:14 ratio, originating exclusively from C3 attack 50 (Table 1, entry 5) as inferred from the NMR analysis 51 of the derived N-heterocycles 6 and 7, and a second Scheme 1. Product distribution in the aldol addition of 1 c to 52 fraction (1%) consisting of a mixture of aldol 2 a and 2 b catalyzed by FSA D6E and A165G variants. 53 regioisomers from C1:C3 attack in a 56:43 molar ratio, 54 which could not be further resolved (Scheme 1). On 55 the other hand, D6T and A165G FSA variants were reaction using FSA A165G furnished a C1:C3 regioi- 56 identified as the best catalysts for the addition of 1 c to someric mixture in 13:87 ratio, which upon chromato- 57 2 b (Figure 2). Similar as above, the preparative graphic separation yielded a major fraction (28%) Adv. Synth. Catal. 2019, 361, 2673 – 2687 2676  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 Table 2. Preparative scale FSA- and DERA-catalyzed aldol additions of 1 b–f to N-Cbz-2-aminoethanal (2 b). 2 3 4 5 6 7 8 9 10 11 12 13 14 Entry Biocatalyst Nu Aldol adduct Aldol adduct Isolated yield,[b] Aldol addition Reductive amination 15 formed,[a] % er or dr,% products,[c] 16 % dr 17 18 1 FSA A165G 1b 15 b 95 35 99:1[d] 17:18 19 70:30 20 2 DERATma 1b 15 b 85 30 95:5[d] – 21 3 FSA A165G 1c 15 c’/16 c’ 60 28 88:12 19:20:21:22 80:8:8:4 22 4 FSA D6T 1d 15 d:16 d 47 30 87:13 23:24:25:26 23 79:8:5:8 24 5 FSA A165G 1e 15 e 87 37 > 95:5[e] –[g] 25 6 FSA A165G 1f 15 f:16 f 50 38 50:50[e,f] 27:28 26 33:67 27 [a] 28 Values obtained at preparative scale and determined by HPLC. [b] 29 Isolated yield of aldol adduct. [c] dr: diasteromeric ratio determined by NMR. 30 [d] er determined by chiral HPLC, a racemic sample was obtained by non-stereoselective chemical methods (see SI). 31 [e] dr of the aldol adduct determined by NMR. 32 [f] The 50:50 diastereomeric mixture obtained in the aldol addition was purified by column chromatography. Two fractions were 33 obtained: one of pure 15 f and another one of a 15 f:16 f 33:67 mixture in order to characterize both N-heterocycles. The 34 reductive amination was carried out on the 15 f:16 f 33:67 mixture. 35 [g] The reductive amination produced a complex mixture of products, which could not be assigned to any structure. 36 Nu: Nucleophile. Green asterisk indicates the stereogenic center formed after reductive amination. 37 38 39 containing a 88:12 mixture of diastereomers 15 c’/16 c’ (Table 1, entry 5).[13] From the 15 c’/16 c’ mixture four 40 arising from C3 attack (Table 2, entry 3), and a second N-pyrrolidine heterocycles 19–22 were generated as a 41 fraction (2.8%) as a 68:32 mixture of C1:C3 aldol consequence of the lack of stereoselectivity in the 42 regioisomers (Scheme 1). This is in contrast to results reductive amination step with each of the diaster- 43 observed for the aldol addition of 1 c to l-G3P using eoisomers, as typical for imine reductions in five- 44 FSA D6H as catalyst, where exclusively C1 attack was membered ring systems (Table 2 entry 3). 45 observed.[6a] 3-Pentanone (1 d) was also tolerated as a nucleo- 46 Consistent with observations for butanone addition philic substrate by different FSA variants, with D6T 47 to l-G3P, i. e. si-face attack of the FSA-nucleophile and D6E being the most efficient catalysts (Figure 2). 48 enamine complex to the C=O si-face of the electro- The D6E catalyzed addition of 1 d to 2 a furnished 49 phile, the major diastereomers 3 c’/15 c’ obtained with only the syn diastereomer 3 d (Table 1, entry 6), while 50 electrophiles 2 a and 2 b correspond to the expected d- FSA D6T catalyzed addition to 2 b yielded a syn:anti 51 threo (i. e. syn) configuration.[6a] The minor diaster- mixture of 15 d/16 d in a 87:13 ratio (Table 2, entry 4). 52 eomers 4 c’/16 c’ arise from an inverted orientation of Considering the stereochemical outcome of FSA in 53 the C=O electrophile exposing its re-face to the previous reactions, 3 d has (4S,5R)-configuration, 15 d 54 FSA Nu complex.[4d,12b] Intramolecular reductive ami- should be (4S,5S), and 16 d (4R,5S). Reductive amina- 55 nation of the 3 c’/4 c’ mixture gave the corresponding tion of 3 d furnished the N-heterocycle 8 with good 56 N-heterocycles 6 and 7 stereoselectively as directed by diastereoselectivity, while from 15 d/16 d four diaster- 57 the relative orientation of the C4 hydroxyl group oisomers 23–26 were identified, among which the Adv. Synth. Catal. 2019, 361, 2673 – 2687 2677  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 major 23 (79%) and the minor 26 (8%) showed the stereocenter at C-a next to the carbonyl in the cyclic 2 C2 configuration predicted for Pd catalysis.[13–14] aldol adduct may occur under the reaction conditions 3 Cyclobutanone (1 e) and cyclopentanone (1 f) were (aq buffer at pH 7.5–8). Isomerizations of this kind, 4 also converted in the presence of 2 a and 2 b electro- which effectively lead to mirror image compounds of 5 philes. FSA variants D6L, D6Q and, particularly, the imperfect aldol diastereomers, have been reported 6 A165G were the most efficient (Figure 2). As reported previously for aldol additions of cyclopentanone to l- 7 previously,[6a,b] the cyclic ketones 1 e and 1 f imposed G3P and upon addition of cyclopentanone enolate to 8 an E-configured FSA K85-enamine nucleophile com- Boc-N-phenylalaninal in aqueous buffered solution- 9 plex, contrary to the Z-configuration invariably ob- s.[6a,16] 10 served with acyclic nucleophiles such as hydroxyetha- Reductive amination of 15 f/16 f furnished the N- 11 nal, hydroxyacetone or dihydroxyacetone, and other bicyclic derivatives 27/28 (Table 2, entry 6), respec- 12 aliphatic ketone nucleophiles.[4d,6c,15] The structural and tively, with identical configuration of the new stereo- 13 mechanistic features invariably impose an attack of genic center formed. Reductive amination of mixture 14 the nucleophile from its si-face to the electrophile, 3 f/4 f was not stereoselective and yielded a mixture of 15 yielding the (R)-configuration at C-a next to the four diastereoisomers 10–13 (Table 1, entry 8). In 16 carbonyl group (Scheme 2).[6b,c] The addition of 1 e to addition, an unexpected dehydroxylated product 14 17 2 b (Table 2, entry 5), catalyzed by FSA A165G, was also detected by NMR analysis as the major 18 furnished only diastereomer 15 e indicating a prefer- component, which was probably formed during the 19 ential attack at the si-face of the electrophilic Cbz removal/reductive amination step in the presence 20 carbonyl. On the contrary, the addition of 1 e to 2 a of the Pd catalyst by formal H2O elimination/hydro- 21 was less selective, furnishing an anti:syn-mixture of genation. 22 diastereomers 3 e/4 e in a 80:20 ratio, which indicates Addition of ethanal (1 a) and propanone (1 b) to 23 an unexpectedly high 20% chance of unconventional individual enantiomers of (S)-2 c and (R)-2 c by 24 attack at the re-face of the electrophilic carbonyl DERATma catalysis furnished the corresponding aldol 25 group. Nevertheless, upon reductive amination of this adducts in 20%–79% isolated yields (Table 3). The 26 mixture, only product 9 was obtained from 3 e but no lower isolated yields obtained for the addition prod- 27 N-bicyclic product corresponding to 4 e was detectable ucts from 1 a (1 eq) were mainly caused by difficulties 28 by NMR analysis, possibly due to strain imposed upon in separating (3R,5R)-dihydroxyhexanal (present in its 29 cyclization (Table 1, entry 7) (Scheme 2). lactol form), which simultaneously formed by enzy- 30 matic self-aldolization of 1 a as a side reaction. When 31 a mild excess of 1 a was employed (1.25 eq) to achieve 32 complete conversion of 2 c to 29, no subsequent 33 addition was observed (tandem addition to 2 c) but the 34 formation of the lactol increased, thus complicating its 35 removal. 36 Consequently, during the hydrogenation procedure 37 the imino moiety of the formed 29 underwent a 38 subsequent intermolecular reductive amination with 39 the aldehyde group of the remaining lactol, yielding 40 the corresponding conjugate 37 (Scheme 3). 41 42 43 44 Scheme 2. Product distribution in the aldol addition of 1 e to 45 2 a and 2 b catalyzed by FSA D6E and A165G variants. 46 47 48 Additions of 1 f to 2 a–b gave mixtures of anti:syn 49 adducts with dr of 66:34 for 3 f/4 f and 50:50 for 15 f/ 50 16 f, respectively (Table 1 entry 8 and Table 2 entry 6). Scheme 3. DERATma-catalyzed aldol addition of 1 a to S-2 c 51 The occurrence of diastereoisomers observed for some with an excess of 1 a leading to the formation of 3R,5R- 52 of the aldol additions studied is plausibly explained by dihydroxyhexanal and formation of 37 during reductive 53 an imperfect orientation of the electrophilic C=O amination. 54 relative to the enzyme-enamine complex, thereby 55 generating epimers at C-b to the carbonyl group of 56 the nucleophiles. However, it is even more conceiv- The chiral center of (S)-2 c and (R)-2 c was used as 57 able that a spontaneous isomerization of the tertiary reference to assign the absolute stereochemistry of the Adv. Synth. Catal. 2019, 361, 2673 – 2687 2678  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 Table 3. Preparative scale DERATma-catalyzed aldol addition of 1 a and 1 b to (S)- and (R)-N-Cbz-2-alaninal (S-2 c and R-2 c). 2 3 4 5 6 7 8 9 10 11 12 13 14 Nu E Aldol adduct Yield,[b]% Aldol adduct dr,[c] Reductive amination 15 formed,[a] % product,[d] 16 % dr 17 1a S-2 c 85[e] 34[f] (4S,5S)-29 > 95:5 31 18 1a R-2 c 80[e] 20[f] (4R,5S)-29 > 95:5 32 19 1b S-2 c 81 79 (4S,5S)-29 > 95:5 33:34 56:44 20 1b R-2 c 91 78 (4S,5R)-29:(4R,5R)-30 77:23 35:36 77:23 21 [a] 22 Determined by HPLC. [b] 23 Isolated yield of aldol adduct. [c] 24 dr: inferred from the reductive amination products. [d] dr: diasteromeric ratio assessed by NMR. 25 [e] [1 a] = [E] = 100 mM. 26 [f] Several purification column chromatography runs had to be performed to remove the trimerization product from enzymatic 27 double addition of 1 a. 28 Nu = Nucleophile. E = Electrophile. Green asterisk indicates the stereogenic center formed after reductive amination. 29 30 31 32 pyrrolidine derivatives 31–36 and infer the stereo- pends on both the nucleophile and electrophile 33 chemical outcome of the biocatalytic aldol addition structure. Such unprecedented novel activity was also 34 and reductive amination. The aldol addition of 1 a to hidden in the previously reported variant FSA A165G, 35 (S)-2 c and (R)-2 c was fully stereoselective furnishing which was again particularly active towards N-Cbz-2- 36 (4S,5S)-29 and (4S,5S)-29, respectively, consistent with aminoethanal (2 b). Hence, the effectivity of a FSA 37 previous reports.[17] A similar situation was found catalyst toward a nucleophile strongly depends upon 38 upon addition of 1 b to (S)-2 c. On the other hand, the quality of the electrophilic component. Aldol 39 (R)-2 c gave a mixture of diasteroisomers (4S,5R)- additions of ethanal (1 a) to 2 a and propanone (1 b) to 40 29:(4R,5R)-30 in a 77:23 dr, indicative of a loss of 2 a and 2 b were fully stereoselective, yielding (R)- 41 enantiofacial selectivity during the electrophile ap- configured aldol adducts identical to those obtained 42 proach. Reductive amination of (4S,5S)-29 gave a using DERATma catalysis. Butanone gave preferen- 43 mixture of the N-heterocycles 33/34, epimeric at C2, tially an addition at the methylene carbon with a 44 while from the mixture of (4S,5R)-29:(4R,5R)-30 the C1:C3 regioisomer ratio of 17:83 and an 86:14 syn:anti 45 pyrolidines 35/36 resulted, in which the new config- dr for the C3-regiosomer. Cyclobutanone and cyclo- 46 uration was controlled by the hydroxyl group at C4, in pentanone imposed an E configured FSA K85- 47 agreement with previous reports.[12c,13] enamine nucleophile complex, therefore under FSA 48 catalysis the anti adducts were mainly formed. This 49 was unequivocally established with cyclobutanone, 50 Conclusions though inconclusive for cyclopentanone presumably 51 FSA variants and DERATma are able to catalyze aldol because of spontaneous isomerization at the tertiary 52 additions of ethanal (1 a) and simple linear (1 b–d) and C-a stereocenter to the carbonyl in aqueous media at 53 cyclic (1 e–f) aliphatic ketones to N-protected amino- pH 7.8. DERATma catalyzed the addition of ethanal 54 aldehydes (2 a–c), affording diversely functionalized and propanone to both enantiomers of N-Cbz-alani- 55 N-heterocycles. FSA variants with different residues at nal, reactions unattainable by any of the FSA variants 56 position D6 are active for various nucleophile-electro- screened. The additions were fully diastereoselective 57 phile combinations, though the optimal catalyst de- except for addition of propanone to S-2 c. Thus, FSA Adv. Synth. Catal. 2019, 361, 2673 – 2687 2679  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 and DERA catalysts seem to be highly complemen- eluted with the following conditions: solvent system (A) 2 tary tools, where FSA variants offer greater flexibility aqueous trifluoroacetic acid (TFA) (0.1% (v/v) and (B): TFA 3 with structural variations in the nucleophile reactants, (0.095% (v/v)) in CH3CN/H2O (4:1), gradient elution from 4 and DERA is superior for the conversion of sterically 10–100%B in 30 min, flow rate 1 mL · min 1, detection at 5 more demanding electrophiles. 215 nm, column temperature 30 8C. The amount of aldol adduct product was quantified from the peak areas using and 6 Stereochemical outcome of reductive amination external standard methodology. Samples were withdrawn 7 reactions followed a general pattern identical to that (50 mL) from the reaction medium, centrifuged, diluted with 8 observed previously in the synthesis of other iminosu- methanol (450 mL) and analyzed with HPLC. 9 gars.[4c,13] The straightforward two-step synthetic ap- 10 proach comprising biocatalytic carboligation and NMR analysis. High field 1H and 13C nuclear magnetic 11 chemical reductive amination described in this work resonance (NMR) analyses were carried out using an 12 foresees new avenues in the synthesis of N-hetero- AVANCE 500 BRUKER spectrometer equipped with a high-sensitive CryoProbe for [D2]H2O and [D4]MeOH 13 cycles, allowing the preparation of new derivatives solutions. Full characterization of the described compounds 14 that otherwise are difficult to prepare by the use of was performed using typical gradient-enhanced 2D experi- 15 existing chemical methodologies. ments: COSY, NOESY, HSQC and HMBC, recorded under 16 routine conditions. When possible, NOE data were obtained 17 from selective 1D NOESY versions using a single pulsed- 18 Experimental Section field-gradient echo as a selective excitation method and a 19 Materials. Synthetic oligonucleotides were purchased from mixing time of 500 ms. Routine, 1H (400–500 MHz) and 13C 20 Eurofins MWG Operon. Phosphoglucose isomerase from (101 MHz) NMR spectra of compounds were recorded with 21 baker’s yeast (S. cerevisiae), glucose-6-phosphate dehydro- Varian Mercury-400 and Varian Anova-500 spectrometers. 22 genase from baker‘s yeast (S. cerevisiae), d,l-glyceraldehyde 3-phosphate (G3P), nicotinamide adenine dinucleotide Determination of the specific optical rotations ([a]D25). 23 Specific optical rotations were measured with a Perkin Elmer phosphate (NADP +), antibiotics, acrylamide-bisacrylamide, 24 Model 341 (Überlingen, Germany) polarimeter (Na lamp, buffer components, propanolamine, aminoethanal dimethyl 25 589 nm). The products (5–20 mg) were dissolved in methanol acetal, S-2-amino-1-propanol, R-2-amino-1-propanol, N- 26 (benzyloxycarbonyloxy)succinimide, ethanal, acetone, buta- (1 mL) and the samples were measured at 25 8C with a 27 none, 3-pentanone, cyclobutanone, cyclopentanone, cyclo- 0.1 dm cell. 28 hexanone were purchased from Sigma-Aldrich. Culture Chiral HPLC analyses. Chiral HPLC analyses were per- 29 media components for bacteria were from Pronadisa. Milli-Q formed on a CHIRALPACK IC, 5 mm, 4.6 3 250 mm column 30 grade water was used for analytical and preparative HPLC, from Daicel (Illkirch, France). Samples (10 mL) were injected 31 buffer preparations and other assay solutions were obtained and eluted with hexane:isopropanol (85:15 (v/v)) for 40 min, 32 from an AriumTM Pro Ultrapure Water Purification System flow rate 1 mL · min 1, detection at 209 nm. 33 (Sartorius Stedim Biotech). All the other solvents used were of analytical grade. 2-Deoxy-d-ribose-5-phosphate aldolase Preparation of N-Cbz-aminoaldehydes. The N-Cbz-amino- 34 from Thermotoga maritima (DERATma) as cell free crude aldehydes were synthesized from the corresponding amino- 35 extract was a generous gift from Prozomix Ltd (activity alcohols using the procedures described in previous publica- 36 tions.[13] The amino group of the aminoalcohols was first 0.056 U mg 1 lyophilized powder); 1 U is defined as the 37 protected with benzyloxycarbonyl (Cbz) group, followed by amount of protein that catalyzes the cleavage of 1 mmol of 2- 38 oxidation of the alcohol function using 2-iodoxybenzoic acid deoxy-d-ribose-5-phosphate (Dr5P) per minute at [Dr5P] = 39 0.7 mM, 50 mM triethanolamine buffer pH 7.5 and 30 8C. (IBX) with a yield between 90–95%. In most instances, a 40 simple workup followed by a rough crystallization or 41 FSA catalysts. The single FSA variants A165G, FSA L107A, precipitation provided the N-Cbz-aminoaldehydes in a form 42 FSA L163A, D6A, D6L, D6N, D6Q, D6S, D6T, D6E, D6H, pure enough to be used in the enzymatic aldol reactions. 43 the double variants FSA D6H/A165G, FSA D6H/L107A, FSA D6H/L163A, FSA D6E/A165G, FSA D6E/L107A, FSA 44 Screening of the Enzymatic Reactions D6E/L163A, FSA D6L/A165G, FSA D6L/L107A, FSA 45 D6L/L163A were obtained using the procedure described.[8] 46 General procedure for aldol addition of ethanal and ketones The FSA multiple variants FSA L107Y/A129G/A165G/ 47 to N-Cbz-aminoaldehydes. S166G, L107Y/A129G/R134S/A165G/S166G, FSA L107Y/ 48 A129G/R134E/A165G/S166G, FSA L107Y/A129G/R134H/ Reactions were conducted in 96 well plates of 1 mL of 49 A165G/S166G, FSA L107Y/A129G/R134P/A165G/S166G capacity. Total reaction volume was 300 mL. N-Cbz-amino- 50 and FSA L107Y/A129G/R134V/A165G/S166G were ob- aldehydes 2 a–c (80 mM final concentration) was dissolved in 51 tained following the mutagenesis and protein expression and propanone (1 b) (45 mL). For the other ketones (1 c–g), 2 a–c 52 purification procedures described in our previous publica- (80 mM final concentration) was dissolved in the correspond- 53 tions.[4c] Activity values are shown in Table S1.[8] ing ketone (15 mL). For ethanal (1 a): to 2 a–c (80 mM final 54 concentration), 1 a (30 mL of a 1 M stock solution) was Methods. HPLC analyses. HPLC analyses were performed 55 added. For all of them: 50 mM triethanolamine buffer pH 8.0 on an X-BridgeTM C18, 5 mm, 4.6 3 250 mm column from (15 mL of a 1 M stock solution) was then added. The reaction 56 Waters (Milford, USA). Samples (30 mL) were injected and was started by adding the lyophilized FSA variant prepara- 57 Adv. Synth. Catal. 2019, 361, 2673 – 2687 2680  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 tion (3 mg · mL 1, 1 mg dissolved in 240 mL, 270 mL or 255 mL (S)-N-Cbz-5-amino-4-hydroxypentan-2-one (15 b): This com- 2 of plain water) or DERATma (1.5 mg, 0.084 U dissolved in pound was obtained following the procedure described 3 240 mL, 270 mL or 255 mL of plain water). Reaction mixtures above. N-Cbz-glycinal (2 b) (310.4 mg, 1.6 mmol) and FSA 4 were shaken (1500 rpm) at 25 8C. Reaction monitoring by A165G were used. The product was purified by silica gel 5 HPLC: samples (50 mL) were diluted in MeOH (450 mL) and column chromatography and eluted with a step gradient of after centrifugation were injected to the HPLC system. Hexane:AcOEt from 1:0 to 3:7, yielding 114.8 mg (29%) of 6 15 b. [a]D20 = + 3.4 (c = 1.9 in MeOH); HPLC:10 to 100% of 7 B in 30 min, tR = 15 min; Chiral HPCL: tR = 31 min (racemic 8 Scale up of Aldol Addition of Ketones (1 b–g) to N- sample: tR(R) = 31 min, tR(S) = 28 min); 1H NMR (400 MHz, 9 Cbz-aminoaldehydes using FSA Catalysis CD3OD) d 7.34 (d, J = 6.0 Hz, 5H), 5.08 (s, 2H), 4.18–4.04 10 (m, 1H), 3.16 (dd, J = 5.8, 1.4 Hz, 2H), 2.64–2.52 (m, 2H), General procedure: The reaction was conducted in a 50 mL 11 2.15 (s, 3H). 13C NMR (101 MHz, CD3OD) d 209.9, 159.1, screw capped conical-bottom polypropylene tubes (Falcon 12 tubes). To an FSA catalyst solution (3 mg mL 1) in plain 138.3, 129.4, 129.0, 128.8, 126.3, 68.0, 67.5, 48.4, 47.4, 30.6. 13 water (16 mL or 18 mL), 1 M triethanolamine buffer pH 8 The intramolecular reductive amination reaction produced 14 (1 mL) was added. To this solution, N-Cbz-aminoaldehydes two diastereoisomers in a 70:30 ratio. Major: (3S,5R)-5- 15 2 a–b (80 mM) dissolved in the ketones (3 mL of 1 b or 1 mL methylpyrrolidin-3-ol (17). 1H NMR (400 MHz, CD3OD) 16 of 1 c–g) was added. Total reaction volume was 20 mL. d 4.41–4.31 (m, 1H), 3.22 (p, J = 7.0, 7.0, 6.9, 6.9 Hz, 1H), 2.92 17 Reaction mixtures were shaken (1000 rpm) at 25 8C for 24 h. (dd, J = 12.2, 1.5 Hz, 1H), 2.87 (dd, J = 12.2, 4.9 Hz, 1H), 2.27 18 After that, the reaction was stopped with MeOH (30 mL) to (ddd, J = 13.5, 7.8, 6.5 Hz, 1H), 1.32 (dddd, J = 13.6, 7.8, 3.7, precipitate the enzyme. Then, the mixture was filtered 1.2 Hz, 1H), 1.27 (d, J = 6.5 Hz, 3H). 13C NMR (101 MHz, 19  through Celite , the MeOH was evaporated and the aqueous CD3OD) d 71.8, 54.5, 54.0, 42.1, 19.3. Minor: (3S,5S)-5- 20 residue was lyophilized, purified characterized and submitted methylpyrrolidin-3-ol (18). 1H NMR (400 MHz, CD3OD) 21 d 4.41–4.29 (m, 1H), 3.45 (dp, J = 10.0, 6.5, 6.5, 6.4, 6.4 Hz, 22 to reductive amination. 1H), 3.27 (dd, J = 12.1, 5.2 Hz, 1H), 2.83–2.74 (m, 1H), 1.92 23 (ddt, J = 13.4, 6.2, 1.4, 1.4 Hz, 1H), 1.48 (ddd, J = 13.4, 10.0, 24 Intramolecular Reductive Amination 5.8 Hz, 1H), 1.20 (d, J = 6.5 Hz, 3H). 13C NMR (101 MHz, 25 CD3OD) d 71.5, 54.0, 52.9, 42.8, 18.3. ESI-TOF: calculated General procedure: The aldol adduct (3 mM final concen- 26 for [M + H] + C5H12NO: 102.0919, found 102.0210. tration) was dissolved in H2O:MeOH 1:1 (70 mL), then Pd/C 27 (60 mg) was added. The mixture was shaken under H2 Aldol addition of 1 c to 2 a. Regioisomer I (arising from the 28 (2.5 atm) overnight at room temperature. After that, the attack of the methylene carbon, C3-attack): N-Cbz-6-Amino- 29 reaction was filtered and the solvent was evaporated. 4-hydroxy-3-methylhexan-2-one (mixture of diasteromers 30 Products were characterized without any further purification. 3 c’:4 c’). This mixture was obtained following the procedure 31 described above. N-Cbz-3-aminopropanal (2 a) (1.6 mmol), 32 butanone (1 c) and FSA D6E were used. The product was 33 Product Description purified by silica gel column chromatography and eluted 34 (R)-N-Cbz-6-amino-4-hydroxyhexan-2-one (3 b): This com- with a step gradient of hexane:AcOEt from 1:0 to 3:7, 35 pound was obtained following the procedure described yielding 213 mg (48%) of product. [a]D20 = + 33.5 (c = 2.1 in 36 above. N-Cbz-3-aminopropanal (2 a) (331.2 mg, 1.6 mmol) MeOH); HPLC: 10 to 100% of B in 30 min, tR = 18 min. Only 37 and FSA D6N were used. After 24 h an additional amount of one diasteromer was detected by NMR. 1H NMR (400 MHz, FSA D6N (3 mg ml 1) was added and the reaction was CD3OD) d 7.50–7.19 (m, 5H), 5.05 (s, 2H), 3.90 (dt, J = 9.2, 38 shaken (1000 rpm) at 25 8C for 24 h. The product was 4.5 Hz, 1H), 3.26–3.15 (m, 2H), 2.67–2.53 (m, 1H), 2.14 (s, 39 purified by silica gel column chromatography and eluted 3H), 1.57 (tdd, J = 15.2, 7.7, 5.1 Hz, 2H), 1.06 (d, J = 7.0 Hz, 40 with a step gradient of hexane:AcOEt from 1:0 to 3:7, 3H). 13C NMR (101 MHz, CD3OD) d 214.0, 158.2, 137.1, 41 128.7, 128.3, 128.1, 69.0, 65.9, 52.3, 37.3, 34.7, 28.2, 9.8. yielding 266.2 mg (85%) of 3 b. [a]D20 = + 10.74 (c = 2.1 in 42 Regioisomer II (C1 attack): (R)-N-Cbz-7-amino-5-hydroxy- MeOH); HPLC: 10 to 100% of B in 30 min, tR = 16 min; 43 heptan-3-one: The product was purified by preparative Chiral HPCL: tR = 25 min (racemic sample: tR(R) = 25 min, 44 tR(S) = 30 min); 1H NMR (400 MHz, CD3OD) d 7.45–7.18 HPLC and eluted with a step gradient of H2O:CH3CN from 45 (m, 5H), 5.07 (s, 2H), 4.19–3.99 (m, 1H), 3.23 (t, J = 7.0 Hz, 1:0 to 1:1, flow rate 1 mL min 1 and detection at 215 nm, 46 2H), 2.59 (d, J = 6.4 Hz, 2H), 2.15 (s, 3H), 1.76–1.47 (m, 2H). yielding 25 mg of a mixture 44:56 of regioisomers I:II. 47 13 C NMR (101 MHz, CD3OD) d 210.4, 159.0, 138.4, 129.4, [a]D20 = + 18.5 (c = 0.9 in MeOH); HPLC: 10 to 100% of B in 48 128.9, 128.8, 67.4, 66.5, 51.7, 38.6, 38.2, 30.6. The intra- 30 min, tR = 19 min; Regioisomer II: 1H NMR (400 MHz, 49 molecular reductive amination reaction produced: (2R,4R)- CD3OD) d 7.59–7.17 (m, 5H), 5.07 (s, 2H), 4.09 (dt, J = 8.2, 50 2-methylpiperidin-4-ol (5): 1H NMR (400 MHz, CD3OD) 4.0 Hz, 1H), 3.24 (q, J = 7.0, 6.4 Hz, 2H), 2.59–2.54 (m, 2H), d 3.77 (tt, J = 11.0, 4.5 Hz, 1H), 3.41–3.33 (m, 1H), 3.25–3.14 2.49 (qd, J = 7.3, 1.8 Hz, 2H), 1.76–1.60 (m, 2H), 1.01 (t, 3H). 51 13 (m, 1H), 2.97 (td, J = 13.4, 3.0 Hz, 1H), 2.09 (tdd, J = 13.3, C NMR (101 MHz, CD3OD) d 212.7, 159.0, 138.5, 129.2, 52 4.7, 2.5 Hz, 2H), 1.55 (tdd, J = 13.7, 11.0, 4.5 Hz, 1H), 1.39 128.9, 128.8, 66.2, 65.3, 49.1, 36.8, 36.1, 34.6, 6.43. The 53 intramolecular reductive amination reaction of regioisomer I (dd, J = 13.1, 11.3 Hz, 1H), 1.32 (d, J = 6.6 Hz, 3H). 13C NMR 54 produced two diastereoisomers in a 87:13 rate. Major: (101 MHz, CD3OD) d 65.2, 51.4, 42.3, 39.5, 30.9, 18.0. ESI- 55 (2R,3R,4R)-2,3-dimethylpiperidin-4-ol (6): 1 H NMR TOF: calculated for [M + H] + C6H14NO: 116.1075, found 56 116.1077. (400 MHz, CD3OD) d 3.36–3.22 (m, 2H), 2.96 (td, J = 13.3, 57 Adv. Synth. Catal. 2019, 361, 2673 – 2687 2681  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 3.1 Hz, 1H), 2.81 (dq, J = 10.7, 6.5 Hz, 1H), 2.05 (ddt, J = 2.35–2.30 (m, 1H), 1.33 (d, J = 10.8 Hz, 3H), 1.05 (d, J = 2 13.7, 5.2, 2.8 Hz, 1H), 1.71–1.56 (m, 1H), 1.44–1.32 (m, 1H), 2.6 Hz, 3H). 13C NMR (101 MHz, CD3OD) d 71.8, 57.2, 51.5, 3 1.30 (d, J = 6.5 Hz, 3H), 1.06 (d, J = 6.5 Hz, 3H). 13C NMR 40.0, 13.8, 7.1. ESI-TOF: calculated for [M + H] + C6H14NO: 4 (101 MHz, CD3OD) d 70.6, 56.3, 43.6, 42.3, 31.6, 16.3, 12.4. 116.1075, found 116.1078. 5 Minor. (2S,3R,4S)-2,3-dimethylpiperidin-4-ol (7): 1H NMR (4S,5R)-N-Cbz-7-amino-5-hydroxy-4-methylheptan-3-one (500 MHz, CD3OD) d 3.83 (dt, J = 10.2, 4.4 Hz, 1H), 3.32– 6 (3 d). This compound was obtained following the procedure 3.28 (m, 2H), 3.24 (d, J = 12.0 Hz, 1H), 2.91–2.85 (m, 1H), 7 described above. N-Cbz-3-aminopropanal (2 a) (1.6 mmol), 2.05–2.02 (m, 1H), 1.77–1.66 (m, 2H), 1.25 (d, J = 6.8 Hz, 8 3-pentanone (1 d) and FSA D6E were used. The product was 3H), 0.94 (d, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CD3OD) 9 purified by silica gel column chromatography and eluted d 68.3, 53.8, 42.7, 37.7, 26.6, 15.1, 4.8. ESI-TOF: calculated 10 with a step gradient of hexane:AcOEt from 1:0 to 3:7, for [M + H] + C7H16NO: 130.1231, found 130.1236. 11 yielding 164.1 mg (35%) of 3 d. [a]D20 = + 36.5 (c = 2.3 in 12 Aldol addition of 1 c to 2 b. Regioisomer I (arising from the MeOH); HPLC: 10 to 100% of B in 30 min, tR = 20 min; 1H attack of the methylene carbon, C3-attack): (3S,4S)-N-Cbz- NMR (400 MHz, CD3OD) d 7.45–7.17 (m, 5H), 5.05 (s, 2H), 13 5-amino-4-hydroxy-3-methylpentan-2-one (mixture of dia- 3.83 (ddd, J = 9.4, 5.6, 3.7 Hz, 1H), 3.20 (hept, J = 7.2 Hz, 14 steromers 15 c’:16 c’). This mixture was obtained following 2H), 2.62 (p, J = 6.8 Hz, 1H), 2.52 (qd, J = 7.2, 2.0 Hz, 2H), 15 the procedure described above. N-Cbz-glycinal (2 b) 1.67–1.39 (m, 2H), 1.08 (d, J = 7.0 Hz, 3H), 0.99 (t, J = 7.2 Hz, 16 (1.6 mmol), butanone (1 c) and FSA A165G were used. The 3H). 13C NMR (101 MHz, CD3OD) d 215.2, 157.6, 137.0, 17 product was purified by silica gel column chromatography 128.0, 127.5, 127.3, 69.4, 65.9, 51.5, 37.5, 34.7, 34.5, 10.4, 6.5. 18 and eluted with a step gradient of hexane:AcOEt from 1:0 to The intramolecular reductive amination reaction produced 19 3:7, yielding 119.2 mg (28%) of product. [a]D20 = + 13.0 (c = (2R,3R,4R)-2-ethyl-3-methylpiperidin-4-ol (8). 1H NMR 20 0.78 in MeOH); HPLC:10 to 100% of B in 30 min, tR = (400 MHz, CD3OD) d 3.27–3.25 (m, 2H), 2.87 (td, J = 13.1, 21 17 min; Only one diasteromer was detected by NMR. 1H 2.9 Hz, 1H), 2.56 (ddd, J = 10.6, 7.4, 3.3 Hz, 1H), 2.02 (ddt, 22 NMR (400 MHz, CD3OD) d 7.38–7.25 (m, 5H), 5.08 (s, 2H), J = 13.3, 5.1, 2.8 Hz, 1H), 1.85 (dqd, J = 15.2, 7.6, 2.8 Hz, 1H), 23 3.99 (q, J = 5.8 Hz, 1H), 3.16 (qd, J = 13.9, 6.1 Hz, 2H), 2.65 1.66–1.49 (m, 2H), 1.38 (tq, J = 10.2, 6.5 Hz, 1H), 1.05 (d, J = 24 (dd, J = 7.3, 5.6 Hz, 1H), 2.16 (s, 3H), 1.10 (d, J = 7.0 Hz, 3H). 6.5 Hz, 3H), 1.00 (t, J = 7.6 Hz, 3H). 13C NMR (101 MHz, 13 C NMR (101 MHz, CD3OD) d 213.6, (C=O carbamate and CD3OD) d 73.1, 62.9, 44.5, 43.3, 34.0, 25.3, 13.8, 9.3. ESI- 25 Cipso not detected), 129.4, 129.0, 128.9, 71.6, 67.5, 51.2, 45.8, TOF: calculated for [M + H] + C8H18NO: 144.1388, found 26 29.0, 11.0. Regioisomer II (C1-attack): N-Cbz-6-amino-5- 144.1385. 27 hydroxyhexan-3-one. The product was purified by prepara- 28 N-Cbz-6-amino-5-hydroxy-4-methyl-hexan-3-one (mixture of tive HPLC and eluted with a step gradient of H2O:CH3CN 29 diasteromers 15 d (4S,5S):16 d (4R,5S). This mixture was from 100:0 to 50:50, flow rate 1 mL min 1 and detection at 30 obtained following the procedure described above. N-Cbz- 215 nm, yielding 12 mg of a 68:32 mixture of regioisomers glycinal (2 b) (1.6 mmol), 3-pentanone and FSA D6T were 31 I:II. [a]D20 = + 0.15 (c = 0.8 in MeOH); HPLC:10 to 100% of used. The product was purified by silica gel column 32 B in 30 min, tR = 18 min; Regioisomer II: 1H NMR (400 MHz, chromatography and eluted with a step gradient of hexa- 33 CD3OD) d 7.45–7.09 (m, 5H), 5.05 (s, 2H), 4.09 (dt, J = 11.6, ne:AcOEt from 1:0 to 3:7, yielding 134.4 mg (30%) of 34 5.9 Hz, 1H), 3.17–3.10 (m, 2H), 2.54–2.50 (m, 2H), 2.46 (q, product. [a]D20 = + 12.3 (c = 1.26 in MeOH); HPLC:10 to 35 J = 7.3 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, 100% of B in 30 min, tR = 19 min. Only one diasteromer was 36 CD3OD) d (C=O carbamate and carbonyl and Cipso not detected by NMR. 1H NMR (400 MHz, CD3OD) d 7.44–7.16 37 detected), 128.0, 127.6, 127.4, 66.6, 66.1, 46.3, 46.0, 36.0, 6.4. (m, 5H), 5.19–5.02 (m, 2H), 3.92 (q, J = 5.6 Hz, 1H), 3.16– The intramolecular reductive amination reaction of re- 38 3.06 (m, 2H), 2.64 (dt, J = 12.9, 6.8 Hz, 1H), 2.51 (q, J = gioisomer I produced four diasteroisomers in 80:8:8:4 ratio. 39 7.2 Hz, 2H), 1.08 (d, J = 7.0 Hz, 3H), 0.97 (t, J = 7.2 Hz, 3H). Major (80%). (3S,4R,5R)-4,5-dimethylpyrrolidin-3-ol (19): 13 40 1 C NMR (101 MHz, CD3OD) d 214.6, 157.6, 136.9, 128.0, H NMR (500 MHz, CD3OD) d 3.99 (td, J = 6.6, 4.9 Hz, 1H), 41 127.5, 127.4, 70.5, 66.1, 49.0, 44.4, 34.3, 10.3, 6.5. The 3.40 (dd, J = 12.1, 6.9 Hz, 1H), 3.14 (dq, J = 9.7, 6.6 Hz, 1H), 42 intramolecular reductive amination reaction produced four 3.06–2.99 (m, 1H), 1.79–1.69 (m, 1H), 1.39 (d, J = 6.6 Hz, 43 diasteoisomers in  79:8:5:8 rate. Major (79%): (3S,4R,5R)- 3H), 1.11 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CD3OD) 44 5-ethyl-4-methylpyrrolidin-3-ol (23). 1H NMR (500 MHz, d 75.2, 60.4, 50.1, 47.5, 15.3, 13.3. Minor (8%) (3S,4R,5S)-4,5- CD3OD) d 3.97 (q, J = 5.3 Hz, 1H), 3.36–3.30 (m, 1H), 3.02 45 dimethylpyrrolidin-3-ol (20): 1H NMR (500 MHz, CD3OD) (dd, J = 12.0, 4.5 Hz, 1H), 2.91 (tdd, J = 8.7, 5.1, 1.5 Hz, 1H), 46 d 4.15 (dt, J = 4.5, 2.0 Hz, 1H), 3.98–3.90 (m, 1H), 3.53–3.46 1.91–1.75 (m, 2H), 1.72–1.61 (m, 1H), 1.10 (d, J = 7.0 Hz, 47 (m, 1H), 3.11–3.06 (m, 1H), 2.26–2.19 (m, 1H), 1.31 (d, J = 3H), 1.03 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, CD3OD) 48 7.0 Hz, 3H), 0.92 (d, J = 7.5 Hz, 3H). 13C NMR (101 MHz, d 75.7, 66.7, 50.3, 46.3, 25.1, 14.5, 9.9. Minor (8%): 49 CD3OD) d 75.6, 56.7, 50.5, 44.1, 11.6, 9.9. Minor (8%) (3S,4R,5S)-5-ethyl-4-methylpyrrolidin-3-ol (24). 1H NMR 50 (3R,4R,5R)-4,5-dimethylpyrrolidin-3-ol (21): 1H NMR (400 MHz, CD3OD) 1H NMR (500 MHz, CD3OD) d 4.24 (t, (500 MHz, CD3OD) d 4.24 (t, J = 4.1 Hz, 1H), 3.43–3.40 (m, 51 J = 4.1 Hz, 1H), 3.41–3.34 (m, 1H), 3.17–3.12 (m, 2H), 1.89– 1H), 3.38–3.33 (m, 1H), 3.02–2.97 (m, 1H), 1.88–1.77 (m, 52 1.82 (m, 2H), 1.54–1.43 (m, 1H), 1.10–1.09 (m, 3H), 1.02–0.98 1H), 1.35 (d, J = 7.1 Hz, 3H), 1.05 (d, J = 2.6 Hz, 3H). 13C 53 (m, 3H). 13C NMR (101 MHz, CD3OD) d 72.0, 64.8, 52.0, NMR (101 MHz, CD3OD) d 71.8, 58.6, 52.1, 45.2, 13.7, 8.8. 54 43.6, 23.6, 15.5, 10.1. Minor (5%): (3R,4R,5S)-5-ethyl-4- Minor (4%) (3R,4R,5S)-4,5-dimethylpyrrolidin-3-ol (22): 1H 55 methylpyrrolidin-3-ol (25): 1H NMR (500 MHz, CD3OD) NMR (500 MHz, CD3OD) d 4.30 (td, J = 4.7, 2.6 Hz, 1H), 56 d 4.14 (d, J = 4.5 Hz, 1H), 3.73–3.66 (m, 3H), 3.49–3.44 (m, 3.84–3.75 (m, 1H), 3.35–3.27 (m, 1H), 3.19–3.14 (m, 1H), 57 2H), 3.11–3.07 (m, 1H), 2.32–2.24 (m, 1H), 1.71–1.65 (m, Adv. Synth. Catal. 2019, 361, 2673 – 2687 2682  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 2H), 1.04 (d, J = 7.5 Hz, 3H), 0.88 (d, J = 7.5 Hz, 3H). 13C following the procedure described above as a mixture of 2 NMR (101 MHz, CD3OD) d 75.7, 62.7, 50.6, 43.0, 22.1, 9.9, 3 f:4 f in a 67:33 ratio. N-Cbz-3-aminopropanal (2 a) 3 9.5. Minor (8%): (3R,4R,5R)-5-ethyl-4-methylpyrrolidin-3- (1.6 mmol), cyclopentanone (1 f) and FSA A165G were used. 4 ol (26): 1H NMR (500 MHz, CD3OD) d 4.35 (q, J = 5.7 Hz, The mixturet was purified by silica gel column chromatog- 5 1H), 3.47–3.43 (m, 1H), 3.27–3.23 (m, 1H), 3.08–3.04 (m, raphy and eluted with a step gradient of hexane:AcOEt from 1H), 2.38 (td, J = 7.4, 5.0 Hz, 1H), 1.74–1.66 (m, 1H), 1.06 (d, 1:0 to 3:7, yielding 303.5 mg (65%) of a mixture 3 f:4 f in a 6 J = 5.1 Hz, 19H), 0.97 (d, J = 5.2 Hz, 2H). 13C NMR 67:33 ratio. HPLC: 10 to 100% of B in 30 min, tR = 18 and 7 (101 MHz, CD3OD) d 71.3, 63.1, 49.7, 39.2, 25.9, 9.9, 6.7. 19 min. Major (67%) (3 f): 1H NMR (400 MHz, CD3OD) 8 ESI-TOF: calculated for [M + H] + C7H16NO: 130.1231, found d 7.41–7.17 (m, 5H), 5.06 (s, 2H), 3.78 (ddd, J = 9.1, 5.4, 9 130.1234. 3.4 Hz, 1H), 3.27–3.12 (m, 2H), 2.35–2.21 (m, 2H), 2.18–2.09 10 (m, 3H), 1.83–1.71 (m, 3H), 1.70–1.61 (m, 1H). 13C NMR 11 (R)-2-((R)-N-Cbz-3-amino-1-hydroxypropyl)cyclobutan-1- (101 MHz, CD3OD) d 221.4, 157.5, 137.0, 128.0, 127.5, 127.3, 12 one (3 e) and (R)-2-((S)-N-Cbz-3-amino-1-hydroxypropyl) 69.1, 66.1, 53.7, 38.7, 37.4, 25.8, 22.6, 20.2. Minor (33%) (4 f): cyclobutan-1-one (4 e): These compounds were obtained 1 13 H NMR (400 MHz, CD3OD) d 7.41–7.17 (m, 5H), 5.06 (s, following the procedure described above as a mixture of 14 2H), 4.06 (ddd, J = 8.4, 4.7, 3.2 Hz, 1H), 3.28–3.16 (m, 2H), 3 e:4 e in a 80:20 ratio. N-Cbz-3-aminopropanal (2 a) 15 2.28–2.18 (m, 1H), 2.18–2.09 (m, 5H), 1.83–1.71 (m, 1H), (1.6 mmol), cyclobutanone (1 e) and FSA D6L were used. 16 1.70–1.61 (m, 2H). 13C NMR (101 MHz, CD3OD) d 220.9, The mixture was purified by silica gel column chromatog- 17 157.5, 137.0, 128.0, 127.5, 127.3, 66.1, 66.8, 55.2, 38.9, 38.5, raphy and eluted with a step gradient of hexane:AcOEt from 36.3, 24.0, 21.5. The intramolecular reductive amination 18 1:0 to 3:7, yielding 201.8 mg (46%) of the mixture. [a]D20 = + reaction produced four diasteroisomers including a dehy- 19 36.49 (c = 1.82 in MeOH); HPLC:10 to 100% of B in 30 min, droxylated analogue (14) in a 15:11:15:22:37 ratio. Owing to 20 tR = 17 min. Major 3 e (80%): 1H NMR (400 MHz, CD3OD) the signal overlapping, some chemical shifts identified as 21 d 7.41–7.25 (m, 5H), 5.06 (s, 2H), 3.73 (dt, J = 9.0, 4.4 Hz, multiplets are given as the middle point rather than as range. 22 1H), 3.47–3.35 (m, 1H), 3.23 (t, J = 6.9 Hz, 2H), 3.03–2.78 (m, Selected signals of the NMR spectra. Detailed assignment in 23 2H), 2.09 (qd, J = 10.3, 5.9 Hz, 1H), 1.96 (td, J = 10.6, 5.2 Hz, Figure S15. Minor 10 (15%): (4R,4aS,7aR)-octahydro-1H- 1H), 1.86–1.63 (m, 2H). 13C NMR (101 MHz, CD3OD) 24 cyclopenta[b]pyridin-4-ol (10). 1H NMR (500 MHz, MeOD) d 212.3, 159.0, 138.4, 129.4, 129.0, 128.8, 69.1, 67.4, 66.9, 45.7, 25 d 4.19 (dt, J = 10.6, 5.2 Hz, 1H), 3.64 (m, 1H), 3.36 (m, 1H), 38.7, 36.1, 14.4. Minor 4 e (20%): 1H NMR (400 MHz, 26 3.02 (m, 1H), 2.49 (m, 1H), 2.19–1.59 (m, 6H). 13C NMR CD3OD) d 7.41–7.25 (m, 5H), 5.06 (s, 2H), 3.94–3.87 (m, 27 (101 MHz, CD3OD) d 65.5, 59.0, 44.6, 42.3. Minor 11 (11%): 1H), 3.21–3.10 (m, 1H), 3.22–3.15 (m, 2H), 3.01–2.80 (m, 28 (4R,4aS,7aS)-octahydro-1H-cyclopenta[b]pyridin-4-ol (11). 2H), 2.14–1.90 (m, 2H), 1.71–1.52 (m, 2H). 13C NMR 1 29 H NMR (500 MHz, MeOD) d 3.99 (td, J = 4.7, 3.1 Hz, 1H), (101 MHz, CD3OD) d 212.3, 159.0, 138.4, 129.4, 129.0, 128.8, 30 3.77 (m, 1H), 3.32 (m, 1H), 3.17 (dt, J = 12.8, 4.4 Hz, 1H), 67.2, 67.1, 66.8, 45.9, 38.5, 36.5, 13.5. The intramolecular 2.22 (m, 1H), 2.19–1.59 (m, 7H). 13C NMR (101 MHz, 31 reductive amination reaction produced (1R,2R,6R)-1,2,6- CD3OD) d 63.2, 55.4, 45.4, 37.9. Minor 12 (15%): 32 trimethylbicyclo[4.2.0]octane (9) as the solely product. 1H (4S,4aS,7aS)-octahydro-1H-cyclopenta[b]pyridin-4-ol (12). 33 NMR (400 MHz, CD3OD) d 3.77 (dt, J = 10.7, 5.7 Hz, 1H), 1 H NMR (500 MHz, MeOD) d 3.62 (m, 1H), 3.50 (m, 1H), 34 3.63–3.55 (m, 1H), 3.17 (dt, J = 13.2, 4.2 Hz, 1H), 2.74 (t, J = 3.08 (dd, J = 13.6, 3.5 Hz, 1H), 2.90 (td, J = 11.3, 7.1 Hz, 1H), 35 7.1 Hz, 1H), 2.62 (ddd, J = 13.7, 12.0, 2.9 Hz, 1H), 2.34–2.19 2.17 (m, 1H), 2.19–1.59 (m, 8H). 13C NMR (101 MHz, 36 (m, 2H), 1.96–1.78 (m, 2H), 1.78–1.63 (m, 2H). 13C NMR CD3OD) d 70.5, 59.9, 49.7, 45.0. Minor 13 (22%): 37 (101 MHz, CD3OD) d 64.2, 52.9, 40.3, 37.3, 28.1, 24.4, 18.4. (4S,4aS,7aR)-octahydro-1H-cyclopenta[b]pyridin-4-ol (13). ESI-TOF: calculated for [M + H] + C7H14NO: 128.1075, found 1 38 H NMR (500 MHz, MeOD) d 3.48 (m, 1H), 3.47 (m, 1H), 128.1070. 39 3.01 (m, 1H), 2.78 (td, J = 11.2, 7.1 Hz, 2H), 2.14 (m, 1H), 40 (R)-2-((S)-N-Cbz-2-amino-1-hydroxyethyl)cyclobutan-1-one 2.19–1.59 (m, 8H). 13C NMR (101 MHz, CD3OD) d 61.4, 41 (15 e). This compound was obtained following the procedure 57.9, 43.9, 42.1. Major 14 (37%) dehydroxylated analogue: 42 described above. N-Cbz-glycinal (2 b) (1.6 mmol), cyclobuta- (4aR,7aR)-octahydro-1H-cyclopenta[b]pyridine (14): 1H 43 none (1 e) and FSA A165G were used. The product was NMR (500 MHz, MeOD) d 3.58 (ddd, J = 7.9, 5.4, 2.9 Hz, purified by silica gel column chromatography and eluted 4H), 3.27 (t, J = 3.7 Hz, 2H), 3.01 (m, 1H), 2.35–2.25 (m, 1H), 44 with a step gradient of hexane:AcOEt from 1:0 to 3:7, 2.11 (m, 1H), 1.79 (m, 2H), 1.85 (m, 2H), 1.71 (m, 1H), 1.76 45 yielding 157.8 mg (37%) of the title compound. HPLC: 10 to (m, 2H). 13C NMR (101 MHz, CD3OD) d 57.8, 42.3, 37.1, 46 28.2, 26.2, 22.4, 20.5, 17.8. ESI-TOF: calculated for [M + H] + 100% of B in 30 min, tR = 16 min; 1H NMR (400 MHz, 47 CD3OD) d 7.39–7.21 (m, 5H), 5.05 (s, 2H), 3.77 (dt, J = 7.2, C8H16NO: 142.1231, found 142.1238. 48 4.9 Hz, 1H), 3.48–3.36 (m, 1H), 3.25 (dd, J = 10.6, 6.0 Hz, 49 (R)-2-((S)-N-Cbz-2-amino-1-hydroxyethyl)cyclopentan-1- 2H), 2.88–2.82 (m, 2H), 2.13–1.94 (m, 2H). 13C NMR 50 one (15 f) and (R)-2-((R)-N-Cbz-2-amino-1-hydroxyethyl) (101 MHz, CD3OD) d 210.4, 157.6, 139.9, 128.0, 127.5, 127.4, cyclopentan-1-one (16 f). These compounds were obtained 51 69.1, 66.1, 63.1, 44.3, 12.8. The reductive amination gave a following the procedure described above as a mixture of 52 complex NMR spectrum, which could not be resolved and 15 f:16 f in a 50:50 ratio; HPLC: 10 to 100% of B in 30 min, 53 that probably indicate a mixture of side products. tR = 17 and 18 min, respectively. N-Cbz-glycinal (2 b) 54 (R)-2-((R)-N-Cbz-3-amino-1-hydroxypropyl)cyclopentan-1- (1.6 mmol), cyclopentanone (1 f) and FSA A165G were used. 55 one (3 f) and (R)-2-((S)-N-Cbz-3-amino-1-hydroxypropyl) The product was purified by silica gel column chromatog- 56 cyclopentan-1-one (4 f). These compounds were obtained raphy and eluted with a step gradient of hexane:AcOEt from 57 Adv. Synth. Catal. 2019, 361, 2673 – 2687 2683  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 1:0 to 3:7, yielding two fraction pools: fraction pool 1 5.16–5.08 (m, 2H), 4.03–3.99 (m, 1H), 3.97–3.91 (m, 1H), 2 containing 15 f pure (53 mg) and a fraction pool 2 as a 3.45–3.38 (m, 1H), 2.12–2.04 (m, 1H), 1.86 (dt, J = 14.7, 3 mixture of 15 f:16 f in a 1:2 ratio (117.2 mg), total 170.2 mg 3.9 Hz, 1H), 1.81–1.70 (m, 2H). 13C NMR (101 MHz, 4 (38%) of product. Fraction pool 1 (R)-2-((S)-N-Cbz-2- CD3OD) d 155.4, 136.5, 128.2, 127.8, 127.5, 82.5, 67.1, 60.0, 5 amino-1-hydroxyethyl)cyclopentan-1-one (15 f). 1H NMR 35.2, 33.0, 31.3. ESI-TOF: calculated for [M + H] + (400 MHz, CD3OD) d 7.38–7.24 (m, 5H), 5.07 (s, 2H), 4.09 C13H18NO4: 252.1235, found 252.1231. 6 (td, J = 6.9, 3.0 Hz, 1H), 3.23 (dd, J = 13.7, 6.2 Hz, 1H), 3.14 7 (dd, J = 13.7, 7.2 Hz, 1H), 2.33–2.15 (m, 2H), 2.15–1.92 (m, 8 4H), 1.83–1.70 (m, 1H). 13C NMR (101 MHz, CD3OD) Scale up of Aldol Additions of Propanone (1 b) to 2 a– 9 d 222.2, 159.0, 138.4, 129.4, 129.0, 128.9, 69.6, 67.5, 53.2, 46.3, c Catalyzed by DERATma 10 39.8, 23.7, 21.7. Fraction pool 2 15 f:16 f 33:67 ratio. NMR of General procedure: The reactions were conducted in 50 mL 11 16 f: (R)-2-((R)-N-Cbz-2-amino-1-hydroxyethyl)cyclopentan- screw capped conical-bottom polypropylene tubes. To a 12 1-one (16 f): 1H NMR (400 MHz, CD3OD) d 7.42-7.16 (m, DERATma solution (3 mg ·mL 1) in plain water (17 mL), 1 M 13 5H), 5.07 (s, 2H), 3.83 (q, J = 5.5 Hz, 1H), 3.38–3.29 (m, 2H), triethanolamine buffer, pH 8 (1 mL) was added. To this 14 2.32–1.98 (m, 5H), 1.91–1.71 (m, 2H). 13C NMR (101 MHz, solution, the aldehyde (2 a–c) (80 mM) dissolved in prop- 15 CD3OD) d 222.5, 138.5, 129.4, 128.8, 70.8, 67.5, 52.6, 45.7, anone (1 b) (2 mL) was added. Total reaction volume was 16 40.1, 27.5, 21.8. The intramolecular reductive amination 20 mL. Reaction mixtures were shaken (1000 rpm) at 25 8C 17 reaction of the fraction pool 2 (33:67 15 f:16 f ratio) produced for 24 h. After that, the reaction was stopped with MeOH 18 two diastereoisomers the corresponding to the aldol adducts (30 mL) to precipitate the enzyme. Then, the mixture was 27 and 28, respectively. From aldol adduct 15 f: (3S,3aS,6aR)-  filtered through Celite , the MeOH was evaporated and the 19 octahydrocyclopenta[b]pyrrol-3-ol (27): 1H NMR (500 MHz, aqueous residue was lyophilized ad submitted to reductive 20 CD3OD) d 4.35 (dt, J = 6.7, 3.5 Hz, 1H), 3.99 (ddd, J = 9.0, amination (identical procedure as described above). 21 7.2, 3.3 Hz, 1H), 3.15 (d, J = 3.5 Hz, 2H), 2.81 (tdd, J = 9.3, 22 6.4, 3.6 Hz, 1H), 2.00–1.92 (m, 1H), 1.93–1.86 (m, 2H), 1.89– (R)-N-Cbz-6-amino-4-hydroxyhexan-2-one (3 b): This com- 23 1.75 (m, 2H), 1.67–1.62 (m, 1H). 13C NMR (101 MHz, pound was obtained following the procedure described 24 CD3OD) d 70.6, 64.6, 53.9, 47.1, 30.8, 26.0, 24.9. From aldol above. N-Cbz-3-aminopropanal (2 a) (331.2 mg, 1.6 mmol) 25 adduct (16 f): (3R,3aS,6aR)-octahydrocyclopenta[b]pyrrol-3- was used. After 24 h additional DERATma (3 mg·ml 1) was 26 ol (28): 1H NMR (500 MHz, CD3OD) d 4.19–4.12 (m, 2H), added and the reaction was shaken (1000 rpm) at 25 8C for 27 3.20–3.15 (m, 1H), 3.09 (dt, J = 12.1, 1.6 Hz, 1H), 2.65 (ddd, 24 h (70% product formed by HPLC). The product was 28 J = 9.6, 7.9, 6.3 Hz, 1H), 1.99–1.92 (m, 1H), 1.93–1.87 (m, purified by silica gel column chromatography and eluted 29 1H), 1.69–1.67 (m, 1H), 1.68–1.61 (m, 1H), 1.57–1.51 (m, with a step gradient of hexane:AcOEt from 1:0 to 3:7, 1H), 1.40 (dq, J = 13.3, 6.7 Hz, 1H). 13C NMR (101 MHz, yielding 198.6 mg (47%) of product. [a]D20 = + 15.8 (c = 0.39 30 CD3OD) d 75.6, 63.0, 51.7, 51.6, 31.7, 29.1, 24.9. ESI-TOF: in MeOH); HPLC: 10 to 100% of B in 30 min, tR = 16 min; 31 calculated for [M + H] + C7H14NO: 128.1075, found 128.1079. Chiral HPCL: tR = 23 min (racemic sample: tR(R) = 23 min, 32 tR(S) = 27 min). NMR spectral data was identical to that 33 obtained by FSA D6N catalysis (see above). 34 Scale up of Aldol Addition of Ethanal (1 a) to Cbz- 35 3-aminopropanal (2 a) (S)-N-Cbz-5-amino-4-hydroxypentan-2-one (15 b): This com- pound was obtained following the procedure described 36 (2S,4R)-N-Cbz-piperidine-2,4-diol (3 a): The reaction was above. N-Cbz-glycinal (2 b) (310.4 mg, 1.6 mmol) was used 37 conducted in a 50 mL screw capped conical-bottom poly- (85% 15 b formed). The product was purified by silica gel 38 propylene tube. To a FSA L107Y/A129G/R134S/A165G/ column chromatography and eluted with a step gradient of 39 S166G (3 mg ·mL 1) solution in plain water (18.9 mL), 1 M hexane:AcOEt from 1:0 to 3:7, yielding 120.2 mg (30%) of 40 triethanolamine buffer, pH 8 (1 mL) was added. To this product. [a]D20 = + 2.2 (c = 0.92 in MeOH); HPLC: 10 to 41 solution, N-Cbz-3-aminopropanal (331.2 mg, 80 mM) and 100% of B in 30 min, tR = 15 min; Chiral HPCL: tR = 25 min 42 ethanal (100 mM, 112 mL) were added. Total reaction volume (racemic sample: tR(R) = 25 min, tR(S) = 23 min). NMR spec- 43 was 20 mL. Reaction mixtures were shaken (1000 rpm) at tra were identical to that obtained by FSA A165G catalysis 44 25 8C for 24 h. After that, the reaction was stopped with (see above). 45 MeOH (30 mL) to precipitate the enzyme, and the mixture  was filtered through Celite . MeOH was evaporated and the (4S,5S)-N-Cbz-5-amino-4-hydroxyhexan-2-one (4S,5S-29): 46 This compound was obtained following the procedure 47 reaction was lyophilized. The product was purified by silica gel column chromatography and eluted with a step gradient described above. (S)-N-Cbz-Alaninal (S-2 c) (331.2 mg, 48 1.6 mmol), propanone (2 b) (2 mL) and DERATma (in 17 mL of hexane:AcOEt from 1:0 to 3:7, yielding 191.3 mg (47%) of 49 of plain water) were used (81% (4S,5S)-29 formed). The 3 a. [a]D20 = + 8.51 (c = 2.1 in MeOH); HPLC: 10 to 100% of 50 B in 30 min, tR = 14.5 min; 1H NMR (400 MHz, CD3OD) product was purified by silica gel column chromatography 51 d 7.59–7.24 (m, 5H), 5.58–5.47 (m, 1H), 5.22–4.99 (m, 2H), and eluted with a step gradient of hexane:AcOEt from 1:0 to 52 4.11–4.06 (m, 1H), 4.03 (tt, J = 11.3, 4.5 Hz, 1H), 3.12–2.98 3:7, yielding 336.9 mg (79%) of product. [a]D20 = 23.8 (c = 53 (m, 1H), 2.24–2.13 (m, 1H), 2.02–1.92 (m, 1H), 1.94–1.84 (m, 2.6 in MeOH); HPLC: 10 to 100% of B in 30 min, tR = 54 1H), 1.49–1.42 (m, 1H), 1.42–1.33 (m, 1H). 13C NMR 16 min; 1H NMR (400 MHz, CD3OD) d 7.46–7.14 (m, 5H), 55 (101 MHz, CD3OD) d 155.7, 136.5, 128.2, 127.8, 127.5, 83.0, 5.05 (d, J = 4.8 Hz, 2H), 4.00 (td, J = 6.5, 3.2 Hz, 1H), 3.66 67.1, 63.4, 38.7, 37.0, 35.1, 31.2. Rotamer: 1H NMR (qd, J = 6.8, 3.1 Hz, 1H), 2.56–2.50 (m, 2H), 2.10 (s, 3H), 1.13 56 (400 MHz, CD3OD) d 7.59–7.24 (m, 5H), 5.49–5.43 (m, 1H), (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CD3OD) d 208.7, 57 Adv. Synth. Catal. 2019, 361, 2673 – 2687 2684  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 157.1, 137.0, 128.0, 127.5, 127.4, 69.4, 66.0, 50.3, 46.5, 29.2, (2S,3S)-2-methylpyrrolidin-3-ol (31). The reaction was con- 2 15.8. The intramolecular reductive amination reaction pro- ducted in a 20 mL screw capped conical-bottom polypropy- 3 duced two diasteroisomers in a 56:44 ratio. (2S,3S,5S)-2,5- lene tube. To a DERATma (60 mg, 3 mg ·mL 1) solution in 4 dimethylpyrrolidin-3-ol (33) (56%) 1H NMR (400 MHz, plain water (18.0 mL), 1 M triethanolamine buffer, pH 8 5 CD3OD) d 4.09 (td, J = 4.0, 2.0 Hz, 1H), 3.20 (dt, J = 8.7, (1.0 mL) was added. To this solution, (S)-N-Cbz-alaninal (S- 6.6 Hz, 1H), 2.98 (qd, J = 6.7, 3.9 Hz, 2H), 2.41–2.27 (m, 2H), 2 c) (414.0 mg, 2.0 mmol, 100 mM in the reaction) and 6 1.39 (ddd, J = 14.1, 6.8, 2.2 Hz, 2H), 1.27 (d, J = 6.5 Hz, 4H), ethanal (1 a) (120 mL, 2 mmol, 100 mM in the reaction) were 7 1.21 (d, J = 5.4 Hz, 9H). 13C NMR (101 MHz, CD3OD) added. The reaction mixture was shaken (1000 rpm) at 25 8C 8 d 73.1, 59.9, 53.0, 42.4, 19.6, 11.7. (2S,3S,5R)-2,5-dimethylpyr- for 24 h (98% aldol product formed as determined by 9 rolidin-3-ol (34) (44%) 1H NMR (400 MHz, CD3OD) d 4.14– HPLC). After that, the reaction was stopped with MeOH 10 4.10 (m, 1H), 3.66 (dp, J = 9.1, 6.8 Hz, 1H), 2.07 (ddd, J = (30 mL) to precipitate the enzyme, and the mixture was  11 13.6, 7.1, 1.3 Hz, 1H), 1.65 (ddd, J = 13.6, 9.0, 4.5 Hz, 1H), filtered through Celite . MeOH was evaporated and the 12 1.21 (d, J = 5.7 Hz, 7H), 1.20 (d, J = 5.4 Hz, 6H). 13C NMR reaction was lyophilized. The product was purified by silica 13 (101 MHz, CD3OD) d 73.1, 58.1, 52.0, 42.3, 19.5, 11.8. ESI- gel column chromatography and eluted with a step gradient 14 TOF: calculated for [M + H] + C6H14NO: 116.1075, found of hexane:AcOEt: 100:0 (200 mL) 80:20 (200 mL), 70:30 15 116.1076. (200 mL), 60:40 (200 mL) and 50:50 (700 mL), yielding 16 171.2 mg (34%) of aldol adduct. The reductive amination N-Cbz-5-amino-4-hydroxyhexan-2-one (mixture of dia- 17 was conducted as follows: the aldol adduct (171.2 mg) was steromers (4S,5R)-29:(4R,5R)-30 in a 77:23 ratio) This 18 dissolved in MeOH (400 mL), then Pd (10%)/C with 50% mixture was obtained following the procedure described humidity (200 mg) was added. The mixture was shaken under 19 above. (R)-N-Cbz-alaninal (R-2 c) (331.2 mg, 1.6 mmol), H2 (2.5 atm) overnight at room temperature. After that, the 20 acetone (2 mL) and DERATma (in 17 mL of plain water) reaction was filtered and the solvent was evaporated. 21 were used (91% mixture formed). The mixture was purified Product (51.2 mg) was characterized without any further 22 by silica gel column chromatography and eluted with a step purification. 1H NMR (400 MHz, CD3OD) d 4.25 (t, J = 23 gradient of hexane:AcOEt from 1:0 to 3:7, yielding 330.1 mg 3.1 Hz, 1H), 3.48 (tt, J = 2 3 6.7, 2 3 3.4 Hz, 1H), 3.46–3.34 (m, 24 (78%) of product. [a]D20 = + 2.8 (c = 0.7 in MeOH). HPLC: 1H), 2.26–2.13 (m, 1H), 2.09–2.00 (m, 1H), 1.38 (d, J = 10 to 100% of B in 30 min, tR = 16 min. Only one diasteromer 25 6.7 Hz, 2H). 13C NMR (101 MHz, CD3OD) d 72.1, 61.6, 44.0, was detected by NMR. 1H NMR (400 MHz, CD3OD) d 7.35– 26 34.1, 11.7. 7.19 (m, 5H), 5.05 (s, 2H), 3.94 (ddd, J = 7.9, 6.1, 4.8 Hz, 1H), 27 3.56 (p, J = 6.7 Hz, 1H), 2.55 (dd, J = 7.8, 3.9 Hz, 2H), 2.12 (s, (2R,3S)-2-methylpyrrolidin-3-ol (32). This compound was 28 3H), 1.12 (d, J = 6.8 Hz, 3H). 13C NMR (101 MHz, CD3OD) synthetized following the procedure described for (2S,3S)-2- 29 d 208.8, 156.9, 136.9, 128.0, 127.5, 127.4, 70.2, 66.0, 51.1, 46.9, methylpyrrolidin-3-ol (31). In this case the 80% aldol 30 29.2, 14.8. Another diasteromer was identified after the product formation was obtained as determined by HPLC. 31 reduction amination reaction. The intramolecular reductive The product was purified by silica gel column chromatog- 32 amination reaction produced two diastereoisomers in a 77:23 raphy and eluted with a step gradient of hexane:AcOEt: 33 ratio. (2R,3S,5R)-2,5-Dimethylpyrrolidin-3-ol (35) (77%).1H 100:0 (200 mL) 80:20 (200 mL), 70:30 (200 mL), 60:40 34 NMR (400 MHz, CD3OD) d 3.80 (td, J = 6.5, 5.2 Hz, 1H), (200 mL) and 50:50 (750 mL). To remove the product from 35 3.35 (dt, J = 14.1, 7.1 Hz, 1H), 3.04 (qd, J = 6.6, 5.1 Hz, 1H), enzymatic double addition of 1 a, two purification runs were 36 2.31 (dt, J = 13.0, 7.1 Hz, 1H), 1.35 (ddd, J = 13.1, 7.7, 6.3 Hz, needed to obtain 101.0 mg (20%) of aldol adduct. The 37 1H), 1.21 (d, J = 6.5 Hz, 4H), 1.12 (d, J = 6.7 Hz, 3H). 13C reductive amination was conducted as follows: the aldol NMR (101 MHz, CD3OD) d 77.8, 60.1, 50.8, 41.4, 20.5, 16.3. adduct (40.0 mg) was dissolved in MeOH (100 mL), then Pd 38 (2R,3R,5S)-2,5-dimethylpyrrolidin-3-ol (36) (23%). 1H NMR (10%)/C with 50% humidity (40 mg) was added. The mixture 39 (400 MHz, CD3OD) d 4.09 (ddd, J = 6.1, 3.9, 2.2 Hz, 1H), was shaken under H2 (2.5 atm) overnight at room temper- 40 3.20 (dt, J = 8.7, 6.7, 6.7 Hz, 1H), 2.98 (tt, J = 6.7, 6.7, 3.3, ature. After that, the reaction was filtered and the solvent 41 3.3 Hz, 1H), 2.41–2.31 (m, 4H), 1.44–1.35 (m, 1H), 1.27 (d, was evaporated. Product (51.2 mg) was characterized without 42 J = 6.6 Hz, 3H), 1.20 (d, J = 5.6 Hz, 3H). 13C NMR (101 MHz, any further purification. 1H NMR (400 MHz, CD3OD) d 4.13 43 CD3OD) d 73.1, 59.9, 42.4, 19.9, 11.8. ESI-TOF: calculated (dt, J = 5.4, 3.4, 3.4 Hz, 1H), 3.53 (ddt, J = 10.3, 7.0, 2 3 44 for [M + H] + C6H14NO: 116.1075, found 116.1072. 3.2 Hz, 1H), 3.45–3.37 (m, 2H), 2.27 (dtd, J = 14.1, 2 3 9.0, 45 5.4 Hz, 2H), 2.03–1.93 (m, 2H), 1.31 (d, J = 7.0 Hz, 2H). 13C 46 NMR (101 MHz, CD3OD) d 76.1, 63.4, 43.7, 32.3, 15.6. Scale up of Aldol Additions of Ethanal (1 a) to 2 a–c 47 Catalyzed by DERATma 48 Organocatalytic Reactions: 49 (2S,4R)-N-Cbz-piperidine-2,4-diol (3 a). This compound was 50 synthetized following the procedure described above cata- N-Benzyloxycarbonyl-(E)-6-aminohex-4-en-2-one: N-Cbz-3- 51 lyzed by FSA variant. In this case DERATma (3 mg ·mL 1) aminopropanal (0.4 mmol, 82.8 mg) was dissolved in DMF 52 was used and 77% aldol product formation by HPLC was (4 mL). Acetone (20%, 1 mL) and (d,l)-proline (0.08 mmol, 53 obtained, yielding 99.3 mg (25%) of pure product. [a]D20 = + 9.21 mg) were added. Total reaction volume was 5 mL. 4.6 (c = 0.69 in MeOH); HPLC: 10 to 100% of B in 30 min, Reaction mixture was stirred at 25 8C for 4 hours. The solvent 54 tR = 14.5 min. NMR spectra was identical to that obtained by was evaporated and the product was purified by silica gel 55 FSA L107Y/A129G/R134S/A165G/S166G catalysis (see column chromatography and eluted with a step gradient of 56 hexane:AcOEt from 1:0 to 1:1, yielding 80 mg (80%) of above). 57 Adv. Synth. Catal. 2019, 361, 2673 – 2687 2685  2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA UPDATES asc.wiley-vch.de 1 product. 1H NMR (400 MHz, CD3OD) d 7.43–7.23 (m, 5H), and Biological Perspectives, Vol. Volume 10 (Ed.: S. W. 2 6.85 (dt, J = 16.0, 7.1 Hz, 1H), 6.06 (dt, J = 16.2, 1.5 Hz, 2H), Pelletier), Pergamon, 1996, pp. 155–299. 3 5.04 (s, 2H), 3.36–3.05 (m, 2H), 2.41 (qd, J = 6.7, 1.5 Hz, 1H), [2] a) C. Saotome, Y. Kanie, O. Kanie, C.-H. Wong, Bioorg. 4 2.19 (s, 3H).13C NMR (101 MHz, CD3OD) d 199.8, 157.4, Med. Chem. 2000, 8, 2249–2261; b) J. Bolleddula, K. 5 146.1, 132.1, 128.0, 127.5, 127.3, 65.9, 38.9, 32.8, 25.2. DeMent, J. P. Driscoll, P. Worboys, P. J. Brassil, D. L. Bourdet, Drug Metab. Rev. 2014, 46, 379–419; c) O. O. 6 Grygorenko, M. Kurkunov, I. A. Levandovskiy, A. V. 7 Preparation of Racemic Aldol Adducts Tymtsunik, Synthesis 2018, 50, 1973–1978; d) C. Wang, 8 Z. Lu, Org. Lett. 2017, 19, 5888–5891. 9 Lithium diisopropylamine reactions: General procedure.[18] [3] a) F. Zhang, Y. Peng, Y. Gong, Chirality 2008, 20, 805– 10 Lithium diisopropylamine (700 mL of a 2 M stock solution in 811; b) S. R. Chemler, D. A. Copeland, Top. Heterocycl. 11 THF) was added to anhydrous THF (8 mL) and cooled down Chem. 2013, 32, 39–75; c) S. Rougnon Glasson, J.-L. to 78 8C. Then, propanone (1 b) (100 mL) was added and 12 Canet, Y. Troin, Tetrahedron Lett. 2000, 41, 9797–9802; stirred for 20 minutes. After that, the aldehyde, dissolved in 13 d) H. Peng, E. Wei, J. Wang, Y. Zhang, L. Cheng, H. anhydrous THF (2 mL), was added. The reaction was stirred 14 for 2 hours at 78 8C, and then left to warm up to room Ma, Z. Deng, X. Qu, ACS Chem. Biol. 2016, 11, 3278– 15 temperature. When the reaction mixture was at room 3283. 16 temperature, NaHCO3 (20 mL of 10% aqueous solution) was [4] a) S. Takayama, G. J. McGarvey, C.-H. Wong, Annu. 17 added. The product was extracted by AcOEt (3 3 15 mL), the Rev. Microbiol. 1997, 51, 285–310; b) L. J. Whalen, C.- 18 organic layers pooled, dried over anhydrous Na2SO4, and the H. Wong, Aldrichimica Acta 2006, 39, 63–71; c) A. 19 solvent was evaporated to dryness. Soler, M. L. Guti rrez, J. Bujons, T. Parella, C. Minguil- 20 lon, J. Joglar, P. Clap s, Adv. Synth. Catal. 2015, 357, rac-N-Cbz-6-amino-4-hydroxyhexan-2-one (rac-3 b): N-Cbz- 1787–1807; d) A. Szekrenyi, A. Soler, X. Garrabou, C. 21 3-aminopropanal (2 a) (209 mg, 1 mmol) was used. The Guerard-Helaine, T. Parella, J. Joglar, M. Lemaire, J. 22 product was purified by silica gel column chromatography Bujons, P. Clap s, Chem. Eur. J. 2014, 20, 12572–12583; 23 and eluted with a step gradient of hexane:AcOEt from 1:0 to e) A. Soler, X. Garrabou, K. Hern ndez, M. L. Guti r- 24 3:7, yielding 130.6 mg (49%) of the title compound. The rez, E. Busto, J. Bujons, T. Parella, J. Joglar, P. Clap s, 25 NMR spectra was indistinguishable from the one obtained Adv. Synth. Catal. 2014, 356, 3007–3024; f) P. Laborda, 26 enzymatically (see above). F. J. Sayago, C. Cativiela, T. Parella, J. Joglar, P. Clap s, 27 Org. Lett. 2014, 16, 1422–1425; g) A. L. Concia, L. rac-N-Cbz-6-amino-4-hydroxypentan-2-one (rac-15 b): N- 28 Cbz-glycinal (2 b) (196 mg, 1 mmol) was used. The product G mez, T. Parella, J. Joglar, P. Clap s, J. Org. Chem. 29 was purified by silica gel column chromatography and eluted 2014, 79 5386–5389. 30 with a step gradient of hexane:AcOEt from 1:0 to 3:7, [5] M. Schrmann, G. A. Sprenger, J. Biol. 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