Biocatalytic Transamination of Aldolase‐Derived 3‐Hydroxy Ketones

Abstract Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi‐enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3‐amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)‐ and (S)‐selective TAs, both diastereomers of the 1,3‐amino alcohol motif were accessible. A two‐step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products.


Synthesis of racemic 3a-c
The procedure was adapted from Roldán et al. [1] Lithium diisopropylamide (LDA) (1400 μL of a 1.0 M stock solution in hexane) was added to anhydrous THF (8 mL) in a heat dried Schlenk tube and the solution was cooled down to -78 ºC.Then acetone (100 μL, 2 mmol) was added and stirred for 20 minutes.After that, the electrophile (1a-c) (150 μL, 1 mmol) dissolved in anhydrous THF (2 mL) was added.The reaction was stirred for 2 hours at -78°C.After that the reaction was left to warm up at room temperature and NaHCO3 (20 mL of a saturated solution) was added.The product was extracted with EtOAc (3 x 15 mL), the organic phases were pooled, dried with anhydrous Na2SO4 and evaporated to dryness.Conversion: 60%.The products were used without purification as standards for CSP-HPLC analysis.

Derivatization of GC-MS samples
Aqueous samples (150 μl) were withdrawn and NaOH (10% v/v, 10 M in water) was added followed by extraction with ethyl acetate (150 μl).The organic phase was dried over anhydrous Na2SO4, centrifuged and subjected to derivatization in glass vials.For this, N-Methylbis(trifluoroacetamide) (30% v/v, MBTFA) was added and the sample was agitated for 45 min at 60°C prior to injection.

HPLC-UV Calibration
Table S3.Calibration data of the substrates with HPLC-UV.

Identification of relative configurations
The relative configurations and thus the absolute configuration of the amine products was identified with the scalar coupling constants of the diastereotopic H-atoms at C3. [2] The diastereotopic C3 H-atoms split into a doublet of doublet of doublets, which can be seen in an excerpt of the 1 H-NMR spectrum of product anti-6a (Figure S18).The dihedral angle betweenthe coupled protons H2 and H4 determines the coupling constants.As the hydroxy-and amino-groups strongly hinder the free rotation, the presence of one major relative configuration can be safely assumed.The diastereotopic H-atoms show a geminal coupling constant of J = The strong coupling of the signals of the H-3 protons is also confirmed by a pure shift spectrum, [3] (Figure S19c) which yields only one broad signal.

Figure S3 .
Figure S3.Products obtained by transamination of 3b with an (S)-or (R)-TA.In case of (a) TA-166 was used, in case of (b) AtTA.In case of (a) syn-and (b) anti-diastereomer of derivatized 1,3-amino alcohol 6b.

14. 3
Hz, whereas the vicinal coupling constants depend on the relative configurations of the molecule.In anti-6a, the diastereotopic protons each have a vicinal proton coupling partner in cis-and trans-configurations.The trans vicinal coupling yields a J value of 8.6 Hz, while the cis coupling constants are 3.1 and 3.5 Hz.In syn-6a the chemical shifts of the two diastereotopic protons H-3 are almost identical.Even at 700 MHz they are only barely separated yielding a strongly coupled signal, which does not allow a straightforward extraction of coupling constants.However, the signals can be simulated with the proper coupling constants.FigureS19shows the experimental multiplet of the H-3 protons in a) and the calculated one in b).For the calculated spectrum the following coupling constants and chemical shifts were used:  ( 1 H) H-3 proR: 1.515 ppm  ( 1 H) H-3 proS: 1.525 ppm, 2 J (H3proR-H3proS): 14.2 Hz, 3 J (H2-H3proR): 10.0 Hz, 3 J (H2-H3proS): 3.0 Hz, 3 J (H4-H3 proR): 6.6 Hz, 3 J (H4-H3proS): 3.0 Hz.

Figure S19 .
Figure S19.a) Experimental multiplet of the H-3 proton shifts in syn-6a at 700 MHz.b) Calculated multiplet of the H-3 protons shifts in syn-6a.c) Pure shift spectrum of H-3 proton shifts in syn-6a.

Table S2 .
Synthesis of aldol products using EcFSA variants.

1.3. Determination of transaminase activity Figure S2. Expemplary
initial rate plot of Arthrobacter sp.TA lyophilized whole cell preparation with model substrate