But-2-ene-1,4-diamine and But-2-ene-1,4-diol as Donors for Thermodynamically Favored Transaminase- and Alcohol Dehydrogenase-Catalyzed Processes

Article abstract of DOI:10.1002/adsc.201501066

Both cis- and trans-but-2-ene-1,4-diamines have been prepared and efficiently applied as sacrificial cosubstrates in enzymatic transamination reactions. The best results were obtained with the cis-diamine. The thermodynamic equilibrium of the stereoselective transamination process is shifted to the amine formation due to tautomerization of 5H-pyrrole into 1H-pyrrole, achieving high conversions (78–99%) and enantiomeric excess (up to >99%) by using a small excess of the amine donor. Furthermore, when the reaction proceeded, a strong coloration was observed due to polymerization of 1H-pyrrole. A structurally related compound, cis-but-2-ene-1,4-diol, has been utilized as cosubstrate in different alcohol dehydrogenase (ADH)-mediated bioreductions. In this case, high conversions (91–99%) were observed due to a lactonization process. Both strategies are convenient from both synthetic and atom economy points of view in the production of valuable optically active products. (Figure presented.).

Full text of DOI:10.1002/adsc.201501066

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DOI: 10.1002/adsc.201501066
Very Important Publication
But-2-ene-1,4-diamine and But-2-ene-1,4-diol as Donors for
Thermodynamically Favored Transaminase- and Alcohol
Dehydrogenase-Catalyzed Processes
Lía Martínez-Montero,a Vicente Gotor,a Vicente Gotor-Fernµndez,a,*
and Ivµn Lavanderaa,*
a
Departamento de Química Orgµnica e Inorgµnica, Instituto Universitario de Biotecnología de Asturias, University of
Oviedo, C/Juliµn Clavería 8, 33006 Oviedo, Spain
Fax: (+34)-985-103446; phone: (+34)-985-103454 (V.G.-F.) or (+34)-985-103452 (I.L.); e-mail: vicgotfer@uniovi.es or
lavanderaivan@uniovi.es
Received: November 15, 2015; Revised: February 3, 2016; Published online: March 17, 2016
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201501066.
Abstract: Both cis- and trans-but-2-ene-1,4-diamines role. A structurally related compound, cis-but-2-ene-
have been prepared and efficiently applied as sacrifi- 1,4-diol, has been utilized as cosubstrate in different
cial cosubstrates in enzymatic transamination reac- alcohol dehydrogenase (ADH)-mediated bioreduc-
tions. The best results were obtained with the cis-di- tions. In this case, high conversions (91–99%) were
amine. The thermodynamic equilibrium of the ste- observed due to a lactonization process. Both strat-
reoselective transamination process is shifted to the egies are convenient from both synthetic and atom
amine formation due to tautomerization of 5H-pyr- economy points of view in the production of valuable
role into 1H-pyrrole, achieving high conversions (78– optically active products.
99%) and enantiomeric excess (up to >99%) by
using a small excess of the amine donor. Further-
more, when the reaction proceeded, a strong colora- Keywords: alcohol dehydrogenases; atom economy;
tion was observed due to polymerization of 1H-pyr- biocatalysis; cofactor recycling; transaminases
Introduction
For ADHs, among the different existing methodol-
ogies, the use of a cheap sacrificial alcohol such as 2-
Transaminase (TA)- and alcohol dehydrogenase propanol has been commonly described in a ꢀcoupled-
(ADH)-catalyzed transformations are prominent bio- substrateꢁ approach, generally obtaining excellent re-
catalytic examples at academic and industrial levels to sults.[6] For TAs, a similar methodology can be suc-
obtain enantiopure target molecules.[1] Both biocata- cessfully applied using an auxiliary amine donor such
lyst types can act over carbonyl compounds to accom- as isopropylamine.[7] Unfortunately, these cosubstrates
plish desymmetrization processes (ideally in quantita- must be utilized in a huge molar excess regarding the
tive yield and selectivity), affording (chiral) amines[2] carbonyl compound to adequately force the reaction
and alcohols,[3] respectively. Their mechanisms are dif- into the reduced derivatives. This obviously leads to
ferent, but they have as a common feature the neces- a poorer atom economy of the process.
sity of a second molecule (cofactor) to accomplish
Recently, the application of cosubstrates that can
these transformations.[4] These coenzymes, nicotin- greatly reduce the equivalents utilized in biocatalyzed
amide [NAD(P)H] for ADHs and pyridoxal 5’-phos- transformations mediated by ADHs and TAs, is gain-
phate (PLP) for TAs, must usually be employed in ing more relevance. Hence, the use of diols such as
catalytic amounts due to their high costs and inhibi- 1,4-butanediol[8] or 1,6-hexanediol[9] has allowed ꢀsub-
tion issues. To afford this, excellent cofactor recycling strate-coupledꢁ redox processes in close to stoichio-
strategies that can overcome the use of stoichiometric metric relation regarding the target substrate. This is
quantities of the coenzyme together with a driving of due to the thermodynamically favored ring closure of
the thermodynamic equilibrium of the process have the w-hydroxy aldehyde intermediate, thus forming
been described.[5]
the corresponding hemiacetal, which in a subsequent
oxidative step renders the final lactone co-product
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But-2-ene-1,4-diamine and But-2-ene-1,4-diol as Donors
Scheme 1. Strategies to drive the thermodynamic equilibria in ꢀcoupled-substrateꢁ approaches: a) ADH-catalyzed reductions
via lactonization; b) TA-catalyzed aminations via aromatization; c) and d) ADH- and TA-catalyzed processes using cis-1,4-
but-2-ene-diol and cis-1,4-but-2-ene-diamine, respectively (this study).
(Scheme 1a). On the other hand, a cyclic amine[10] or pound of interest. With this in mind, diamines cis-2
a diamine[11] has been described as amine donor that and 1,4-butanediamine 3 were envisioned as perfect
can highly displace the equilibria in TA-catalyzed pro- candidates. Moreover, in the case of cis-2, after cycli-
tocols due to ring aromatization (Scheme 1b). Howev- zation to 5H-pyrrole, it could tautomerize into 1H-
er, the high cost of these tailored amines and difficul- pyrrole (Scheme 1d).
ties in separating the excess of the donors from the
products make interesting the study of other alterna-
tives.
Herein, we have studied the application of two re-
Results and Discussion
lated compounds, cis-but-2-ene-1,4-diol (cis-1) and While diamine 3 is commercially available, we synthe-
cis-but-2-ene-1,4-diamine (cis-2), as cosubstrates in sized cis-2. As a first approach, we used diol cis-1 as
ADH- and TA-mediated transformations, respectively starting material.[12] Different conditions such as Mit-
(Scheme 1c and d). Thus, after optimization of the re- sunobu reaction or alcohol activation through O-tosy-
action conditions, we demonstrate that these deriva- lation were tried, but unfortunately these protocols
tives can be employed at much lower levels than con- did not work out. Hence, we employed a slightly
ventional cosubstrates to obtain enantioenriched alco- modified method proposed by Delcros and co-work-
hol and amine products through enzyme-catalyzed re- ers,[13] starting from commercially accessible cis-1,4-di-
actions, with high purity after a simple extraction.
chlorobut-2-ene (cis-4, Scheme 2). In a first step, both
Based on previously described protocols that make halogen atoms were substituted by azides using a typi-
use of designed cosubstrates to favor TA-mediated cal nucleophilic substitution protocol and, in a second
processes,[10,11] we envisaged that simple 1,4-diamines step, the reduction of diazido compound cis-5 was car-
could be used for this purpose. Once the w-amino al- ried out under Staudinger conditions with triphenyl-
dehyde intermediates were formed, they could intra- phosphine, followed by acidic hydrolysis affording cis-
molecularly cyclize, thus providing the driving force 2 as dichlorohydrate salt. During this synthetic path-
that could transform quantitatively the carbonyl com- way an alkene isomerization into the trans-diamine
Scheme 2. Synthetic pathway to obtain diamines cis-2 and trans-2.
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Lía Martínez-Montero et al.
Table 1. Amination of ketones 6a–19a using cis-2 (3 equiv.) as amine donor.[a]
Entry
Ketone
Enzyme
c [%][b]
ee [%][c]
1
2
3
4
5
6
7
8
6a
6a
7a
7a
8a
8a
9a
10a
11a
12a
12a
13a
14a
15a
16a
17a
18a
19a
ATA-033
TA-P1-G06
ATA-033
TA-P1-G06
ATA-025
TA-P1-A06
ATA-012
ATA-033
ATA-024
ATA-025
TA-P1-A06
ATA-033
TA-P1-A06
TA-P1-A06
TA-P1-A06
ATA-113
TA-P1-A06
ATA-256
96
97
98
99
99
99
96
79
82
93
93
98
78
99
92
80
82
89
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (S)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
99 (R)
>99 (R)
>99 (S)
92 (S)
95 (S)
82 (S)
9
10
11
12
13
14
15
16
17
18
88 (S)
26 (S)
[a]
For reaction conditions, see the Supporting Information.
Measured by GC analysis.
Measured by chiral GC or HPLC analysis.
[b]
[c]
(trans-2) was observed, therefore recrystallization in
a methanol/diethyl ether (1:1) mixture was per-
formed, providing the desired compound with high
cis/trans selectivity (approx. 95:5). To check the effect
of the double bond stereochemistry, diamine trans-2
was also synthesized following the same reaction
route (Scheme 2).
In a first set of experiments, three different ketones
(6a–8a, see Table 1) were used as suitable substrates
with two commercially available transaminases
(Figure 1). Thus, a small excess of the amine donor
(1.5 equivalents) was applied. Gladly, TAs could
accept diamine cis-2 as donor, affording the corre-
sponding amines 6b–8b with high to excellent conver-
Figure 1. Effect of the amine donor in TA-catalyzed reac-
sions after 48 h at 308C. Although in a minor extent, tions using 1.5 equiv. of cis-2, trans-2, and 3.
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But-2-ene-1,4-diamine and But-2-ene-1,4-diol as Donors
trans-2 could also provide moderate to high conver- ieve a reversible 1,4-addition.[16] In our case, PLP
sions, and the saturated diamine 3 appeared as a poor could act as the isomerization agent.
substrate for these enzymes. These results illustrate
Subsequently, cis-2 was applied as amine donor in
the relevance of the presence and stereochemistry of phosphate buffer and in the presence (2.5% v/v) of di-
the double bond in the structure of the diamine, methyl sulfoxide (DMSO) as solubilizing agent with
which is the key to provide the ring aromatization.
ketones 6a–19a (Table 1). This selection was made
Interestingly, when the reaction proceeded with cis- based on a previous study performed in our group[17]
2 and trans-2, the formation of black colored precipi- due to the relevance of the obtained amines as pre-
tates was observed. This can be ascribed to the poly- cursors of interesting derivatives.[18] Aromatic (en-
merization of the pyrrole by-product resulting in the tries 1–9), heteroaromatic (entries 10–12), and aliphat-
formation of polypyrrole.[14] In fact, when we incubat- ic substrates (entries 13–17) could be transformed
ed pyrrole in the reaction medium in the absence of into the corresponding enantioenriched amines with
enzyme and PLP, we did not observe coloration, but excellent conversions and enantiomeric excess. Inter-
when we accomplished the same experiment adding estingly, TAs with opposite stereopreference can work
only PLP, a quick formation of the black polymers under these conditions, therefore providing both anti-
was detected. It seems that PLP mediates the poly- podes for some of the selected examples. ATA-025
merization of pyrrole acting as an acidic catalyst. This and ATA-033 showed the best results in terms of R-
phenomena is also in agreement with that previously selectivity, and TA-P1-A06 and TA-P1-G06 were in
described by Turner and co-workers,[11] where the iso- general the best candidates to obtain the S-enantio-
indole co-product (Scheme 1b) polymerized under mers. We also carried out a study on a cyclic ketone
similar conditions affording intense colored deriva- such as 1-indanone (19a, see the Supporting Informa-
tives. As in their case, this observation can potentially tion). We were pleased to observe that meanwhile 1-
lead to a sensitive and operationally simple colorimet- indanone was slightly converted for most of the en-
ric assay to identify TA activity.[11,15]
zymes tested using isopropylamine as amine donor, in
The formation of pyrrole was also demonstrated by some cases conversions dramatically increased using
NMR experiments by performing the enzymatic reac- cis-2 to afford the corresponding amine with modest
tions in deuterium oxide (see the Supporting Informa- enantiomeric excess (entry 18).
tion for more details). Surprisingly, when we studied
It has been previously described that similar cosub-
the transformation using trans-2 as amine donor, the strates can also favor these processes from a kinetic
presence of pyrrole was also observed in the reaction point of view regarding conventional donors.[8c] In this
crude. This fact can explain why in the presence of case, the initial rates of TA-catalyzed reactions using
this diamine the aspect of the medium was similar. If isopropylamine, cis-2, and trans-2 were measured. We
pyrrole is formed, at some point there must be a cis/ found that isopropylamine reacted faster than both di-
trans isomerization of the double bond, otherwise cy- amines (especially than the trans isomer), leading to
clization would not be possible. When we incubated the highest initial rate (see the Supporting Informa-
trans-2 in the reaction medium without any ketone tion). This fact shows that these transformations are
substrate, isomerization was not observed. Therefore, mainly thermodynamically (but not kinetically)
we speculate that the trans-w-amino aldehyde inter- driven.
mediate formed after deamination of trans-2 must be
The use of this diamine is particularly advanta-
the species which undergoes this process (Scheme 3). geous, since after a simple extraction protocol, it is
This is not surprising as it has been described that possible to obtain the desired amines with high purity,
similar a,b-unsaturated aldehydes can isomerize in as diamine cis-2 is completely soluble in water and
the presence of pyridine derivatives, which can ach- the excess of this compound remains in the aqueous
phase even after extraction under basic conditions.
Moreover, polymers coming from pyrrole can be
easily removed by centrifugation.
As a proof, we applied this transformation concept
on a preparative scale. Thus, 100 mg of ketones 7a
and 8a were transformed into the corresponding (R)-
amines (>99% ee) using ATA-033 and cis-2
(1.5 equiv.) in 74–83% yield after 48 h of reaction.
Finally, we have compared the conventional meth-
odology using an excess of isopropylamine, with ther-
modynamically favored strategies employed for TA-
catalyzed reactions, calculating the theoretical value
of waste generated according to the reaction equa-
tion.[19] To perform this, we have used the EATOS
Scheme 3. Transformation of diamine trans-2 into pyrrole
through the isomerization of the trans-w-amino aldehyde in-
termediate.
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Table 2. Quantity of waste generated per kg of amine (6b)
synthesized depending on the amine donor.[a]
Entry
Amine donor
Molar
Waste
excess[b]
(kgkgÀ1 product)
1
2
3
4
IPA[c]
40
17
cyclic amine[d]
diamine[e]
cis-2
1.05
1.1
1.5
1.04
1.07
0.92
[a]
Calculated using the EATOS program. Quantitative con-
version was assumed.
Regarding the ketone substrate.
Isopropylamine.
[b]
[c]
[d]
Amine donor described by Berglund and co-workers (see
Scheme 1b).[10]
Figure 2. Effect of the hydrogen donor in ADH-catalyzed
reactions using 0.5 equiv. (for HLADH) or 3 equiv. (for
ADH-T) of cis-1, EtOH (HLADH) and 2-PrOH (ADH-T).
[e]
Amine donor described by Turner and co-workers (see
Scheme 1b).[11]
program,[20] assuming a cosubstrate molar excess as
described by the corresponding authors,[10,11] and of the excellent conversions attained using a small
a quantitative conversion of a model ketone (6a, excess of these cosubstrates. Furthermore, in the case
Table 2). As can be seen, our methodology (entry 4) of the diamine cis-2, a strong coloration of the enzy-
was perfectly comparable to similar previously de- matic transformation was observed when the reaction
scribed strategies (entries 2 and 3), while greatly im- proceeded due to the pyrrole polymerization. The
proving the standard conditions using isopropylamine excess of both cosubstrates and coproducts formed
(entry 1). The method presented here upgrades the can be easily removed from the products of interest,
previous ones as the coproduct obtained (pyrrole), making these compounds highly appealing for ADH-
displays a substantially lower molecular weight.
and TA-catalyzed transformations, enhancing their
Likewise, diol cis-1, previously tried as synthetic possible applications from both synthetic and atom
precursor of diamine cis-2, was envisioned as a suita- economy points of view. Thus, the amount of waste
ble cosubstrate for ADH-mediated bioreductions in generated due to the amine donor employed, was
a similar way as shown by Hollmann and co-workers comparable with current existing transaminase-cata-
with the saturated derivatives.[8,9]
lyzed methods to shift the reaction equilibrium to-
After a first enzymatic screening, we observed that wards the desired product, improving the traditional
horse liver ADH (HLADH) and Thermoanaerobacter methodology using an excess of isopropylamine.
sp. ADH (ADH-T) overexpressed in E. coli, were
able to accept this diol as hydrogen donor. Then, it
was demonstrated that cis-1 can drive the equilibrium
into the reduction mode with higher efficiency than
the usual donors such as EtOH (for HLADH) or 2-
PrOH (for ADH-T), since by simple addition of an
equimolar or a slight excess amount of the diol, excel-
lent conversions (and ee for 11c, 21c and 22c) into the
corresponding alcohols were achieved (Figure 2). The
formation of the unsaturated lactone furan-2(5H)-one
coproduct was confirmed by GC analysis (see the
Supporting Information).
Experimental Section
Synthesis of cis/trans-But-2-ene-1,4-diamine
Dihydrochloride (cis/trans-2)
Diazide intermediates cis/trans-5 were obtained from the re-
spective cis/trans-4 using a nucleophilic substitution reaction.
Dichlorinated compound cis/trans-4 (4.75 mmol, 0.5 mL)
was added to a stirred solution of NaN3 (42.7 mmol, 2.77 g)
in DMF (7 mL) and the magnetic stirring was continued
until disappearance of the starting material (30–45 min).
Then, the reaction was quenched by addition of water
(10 mL) and extracted with EtOAc (3”15 mL). The organic
layer was dried with Na2SO4, filtered off and concentrated.
The residue was purified by flash column chromatography
(20% EtOAc/hexane) on silica gel to remove the DMF still
remaining after the extraction, leading to the corresponding
diazide compounds cis/trans-5 as orange oil in quantitative
yields.
Conclusions
Herein we have shown the use of two related com-
pounds, diamine cis-2 and diol cis-1, in order to drive
the equilibrium of TA- and ADH-catalyzed processes
by using a ꢀcoupled-substrateꢁ approach. A tautomeri-
zation of co-product 5H-pyrrole into 1H-pyrrole and
The desired diamines were prepared as dihydrochloride
salts through the reduction of the previous diazides cis/
a lactonization reaction, respectively, are responsible trans-5 and subsequent acidification. To a solution of cis/
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But-2-ene-1,4-diamine and But-2-ene-1,4-diol as Donors
trans-5 (4.70 mmol, 649 mg) in dry THF (9 mL), a solution
of Ph3P (9.45 mmol, 2.48 g) in dry THF (6 mL) was added.
This mixture was stirred at room temperature during the
first hour. After this time, the reaction mixture was heated
at 508C for 16 h. Then, the hydrolysis was carried out by the
addition of an aqueous HCl solution 6M (1 mL), maintain-
ing the reaction at 508C during one additional hour. Finally,
water (3 mL) and concentrated HCl (500 mL) were added
and THF was removed under vacuum. To the obtained resi-
due, water (10 mL) was added, and the aqueous layer was
extracted with EtOAc (5”10 mL). The water was removed
under reduced pressure (508C), affording a brown crude.
The brown crude, obtained by evaporating the aqueous
layer, was recrystallized from a mixture of MeOH-Et2O (1/
1, v/v), leading to the desired diamine dihydrohloride cis-2
as a white solid (yield: 60%), with a small amount of the un-
General Procedure for Alcohol Dehydrogenase-
Catalyzed Reductions using cis-But-2-ene-1,4-diol
(cis-1) as Cosubstrate
For E. coli/HLADH: In a 1.5-mL Eppendorf vial, aldehyde
20a or 21a (0.022 mmol, 32 mM) was dissolved in phosphate
buffer (700 mL, 50 mM, pH 8), containing NADH (1 mM)
and cis-1 (0.5 equiv., 16 mM) as cosubstrate. Finally E. coli/
HLADH (1.5 mg) was added. The reaction was shaken at
308C and 250 rpm for 22 h and extracted with EtOAc (2”
0.5 mL). The organic layer was separated by centrifugation
(2 min, 13000 rpm) and dried over Na2SO4. Conversions and
enantiomeric excess (for 21c) were determined by GC and
HPLC, respectively (see the Supporting Information,
Table S4). The presence of furan-2(5H)-one in the reaction
media was confirmed by GC analysis.
For E. coli/ADH-T: In a 1.5-mL Eppendorf vial, the cor-
responding ketone 11a or 22a (0.017 mmol, 34 mM) was dis-
solved in Tris·HCl buffer (500 mL, 50 mM, pH 7.5), contain-
ing NADPH (1 mM) and cis-1 (102 mM, 3 equiv.) as cosub-
strate. Finally E. coli/ADH-T (15 mg) was added. The reac-
tion mixture was shaken at 308C and 250 rpm for 24 h and
then extracted with EtOAc (2”0.5 mL). The organic layer
was separated by centrifugation (2 min, 13000 rpm) and
dried over Na2SO4. Conversions and enantiomeric excess
were determined by GC analysis (see the Supporting Infor-
mation, Table S5). The presence of furan-2(5H)-one in the
reaction media was confirmed by GC analysis.
1
wished isomer trans-2, just observed in the H NMR spectra
(ratio cis/trans: 95:5) or to diamine trans-2 in high purity as
a white solid (yield: 64%).
General Procedure for Enzymatic Transamination
Reactions using cis-2 as Amine Donor
In a 1.5-mL Eppendorf vial, substrate 6a–19a (5 mM,
12.5 mL from 200 mM stock in DMSO), was dissolved in
phosphate buffer (500 mL, 100 mM, pH 7.5) containing PLP
(2 mM) and cis-2 (7.5 mM, 1.5 equiv. or 15 mM, 3 equiv.). Fi-
nally, the commercially available transaminase (2 mg) was
added. The initial slightly yellow reaction mixture was
shaken at 308C and 250 rpm for 48 h (observing after this
time that the reaction media was completely black with the
appearance of a solid at the bottom of the vial), and then
stopped by the addition of an aqueous 10M NaOH solution
(250 mL). Then, the mixture was extracted with EtOAc (2”
0.5 mL), the organic layer was separated by centrifugation
(2 min, 13000 rpm) and dried over Na2SO4. Conversions
were determined by GC and enantiomeric excess by chiral
GC or HPLC (see Table 1).
Acknowledgements
We thank Prof. Martina Pohl (Forschungszentrum Jülich
GmbH) and Prof. Wolfgang Kroutil (TU Graz) for the gen-
erous donation of HLADH and ADH-T plasmids, respec-
tively. Financial support from MICINN (Project CTQ2013-
44153-P) and the Principado de Asturias (Project FC-15-
GRUPIN14-002) are gratefully acknowledged. L. M.-M.
thanks the Principado de Asturias for her predoctoral fellow-
ship Severo Ochoa.
Scaling-Up Reactions
In an Erlenmeyer flask (100 mL), ketone 7a or 8a (25 mM,
100 mg) was dissolved in phosphate buffer (100 mM,
pH 7.5) containing PLP (2 mM), MeCN (2.5% v/v) as organ-
ic cosolvent and cis-2 (37.5 mM, 1.5 equiv.) as amine donor. References
Finally, ATA-033 (75 mg) was added. Then, the initial slight-
ly yellowish reaction was shaken at 308C and 250 rpm for
48 h. After that time, the reaction media was completely
black. The conversion was determined by taking a sample
(200 mL) of the reaction mixture which was basified with an
aqueous NaOH solution 10M (100 mL) and extracted as
shown above, leading to 78% and 89% conversions for 7a
and 8a, respectively (measured by GC). At this point, the
solution was centrifuged at 4,900 rpm for 7 min. The super-
natant was decanted, acidified with an aqueous HCl solution
3M (5 mL) and extracted with Et2O (3”25 mL) discarding
the organic layer, in order to remove the small amount of
the starting material. Then, the aqueous phase was basified
with an aqueous NaOH solution 10M (8 mL) and extracted
with Et2O (3”35 mL). The organic layers were combined,
dried over Na2SO4 and the solvent was evaporated under re-
duced pressure, providing (R)-7b (yield: 74%, >99% ee)
and (R)-8b (yield: 83%, >99% ee).
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Adv. Synth. Catal. 2016, 358, 1618 – 1624