Experimental section, Chapter 2 , Nahid Fatema

Experimental Section
application of enzymes in transformations

1. Chapter -II Experimental Section
1.Introduction:
Application of biocatalysts, especially enzymes, in organic transformations has
nowadays turned out to be a popular choice.
Their ability to be flexible and exhibit effective Regio- and chemo-selectivity in mild
reaction conditions aligns with the principles of green chemistry, which has led to
their application in various fields.
Baker's Yeast (Saccharomyces cerevisiae), which had been at the centre stage of the
bakery industry for centuries, is one such biocatalyst, which generates metabolites in
situ and these metabolites catalyse various chemical transformations.
Despite being a microorganism, it is nontoxic in nature and has been known to
catalyse various functional group conversions. (Singh, N.G. et.al).
With Reference to this Bakers' yeast tends to produce many enzymes during the
fermentation period. Lipase, being one of them, is believed to be the enzyme
responsible for enhancing our one-pot transformation process. Lipase is composed of
diverse amino acids namely, histidine, serine, aspartic acid, etc. The histidine NH
proton has been reported to be responsible for enhancing the electrophilic character of
carbonyl carbon of benzaldehyde. After thorough consideration of the above
statements, we have proposed a mechanism for the preparation of (3a-l) and is shown
in Figure 1.
Figure 1. Tentative mechanism for the Baker’s Yeast catalyzed Synthesis of α, β Unsaturated
‐
imines or chalcone-imines (3a-l).
Reports also reveal the use of Baker's Yeast for the synthesis of dihydropyridyl
compounds via Hantzsch reaction etc. (Lee, J.H., et al.). In this report Hantsch 1,4-

2. dihydropyridines are prepared by fermenting bakers’ yeast with alkyl acetoacetate
and ammonium acetate.

Figure 2. Preparation of Hantsch 1,4-dihydropyridines by fermenting bakers’ yeast with alkyl
acetoacetate and ammonium acetate.
The application of a cheap biocatalyst, Baker's Yeast, towards quick, water mediated,
chemoselective, oxidative hydroxylation of aryl/heteroarylboronic acids and
arylboronate esters at room temperature, in a metal and ligand free condition, without
the addition of external base or acid. (Lima et al) Baker's Yeast is a fairly stable
substrate, bearing considerably good shelf life. Commercially available Baker's Yeast
is very cheap and is required in very less amount (5
 mg per mmol of arylboronic acid).
Thus, economic viability, easy availability of catalyst and ease of handling the
reaction, make this an efficient and facile methodology for the synthesis of diversified
phenols.
Figure 3. A schematic diagram of oxidative hydroxylation of aryl/heteroarylboronic acids and
arylboronate esters at room temperature
The use of baker’s yeast as biocatalyst because it is readily available, the experimental
procedures is very easy and the versatility of its as it uses in terms of efficiency and
stereoselectivity. Santaniello, E.; et al.

3. α, β Unsaturated imines or conjugated imines are important compounds or
‐
intermediates and are used in the synthesis of nitrogen containing heterocyclic
‐
compounds, agrochemicals and pharmaceuticals. Suman K. Saha et al
Figure 4. Enantioselective reactions of α,β-unsaturated imines by using synthetic methodologies
for synthesizing various nitrogen-containing heterocycles that contain four to six-membered
rings. The synthesis of rarely found seven-, eight- and nine-membered nitrogen-containing
heterocycles have also been reported. (Suman K. Saha et al).
α, β Unsaturated imines or conjugated imines provide a lot of significant cyclic as
‐
well as acyclic products through diverse reactivity with a versatile family of
compounds. Suman K. Saha et al , C. Solé et al and A. D. J. Calow et al.
In the literature α, β-unsaturated imines can be prepared by heat elimination of imino-
alcohol skeleton, allyl cation-mediated Schmidt between azides and alcohols aza-
Wittig reaction of α-ketophosphonates and trimethyl phosphazenes, Dehydrogenative
coupling between primary allylic alcohol and a primary amine. Suman K. Saha et al
Recently Sc (OTf)3-catalyzed annulations reactions of Vinyl Diazoacetates with in
situ formation of Indole-Derived α, β Unsaturated imines
‐
for the preparation of
Cyclopenta[b]indoles have been reported. Qin Jiang et al

4. Figure 5. Typical procedure for cyclization.
Biological point of view the α, β Unsaturated imines are exhibiting, antimicrobial,
‐
Anti-oxidant Activity and anti-corrosion activities. Raut A et al, Alwan et al, and
Natalia Guranova et al.
Result and discussion:
Thus, in view of the importance of selective synthesis of α, β Unsaturated imines or
‐
chalcone-imines there is a immense need for the chemo-selective synthesis of α, β
‐
Unsaturated imines or chalcone-imines. In continuation of our ongoing efforts for the
successful application of baker’s yeast as biocatalysts in the synthesis of biological activity
compounds by various functions group transformation Carlos MP et al and multi-
component reaction (unpublished results), we wish to report our efforts for the chemo-
selective synthesis of α, β Unsaturated imines or chalcone-imines.
‐
While working on the baker’s yeast mediated synthesis we were trying to
prepare β‐amino carbonyl compounds via the addition amine to chalcone using
baker’s yeast as catalyst in ethanol our earlier reported conditions for the Schiff
base formation.
The reaction mixture shows the consumption of stating material by TLC. To our
surprise after purification when we have taken the carried out the mass analysis
in the mass spectra we did not got the desire product peak i.e., 301.15, instead
of that we got the peak of 284.12 which is corresponding to the imine M
+1
(
283.14+1) peak. (Scheme-1)

5. Scheme-1: Efforts fort the reaction of Chalcone with aniline for the formation of
b-aminoketone by using Baker’s Yeast as catalyzed

After obtaining this result then we try to optimize the condition to synthesize
the α,β‐Unsaturated imines or chalcone-imines we started to investigate the
effect of the solvent. The results are summarized in the Table-1.

6. Table-1: Baker’s Yeast catalyzed synthesis of α, β‐Unsaturated imines or
chalcone-imines: Optimization for the compound 3a
From the results of Table-1 it is inferred that the reaction in the solvents such as
Benzene (entry-1), Toluene (entry-2), DCM (entry-3), EtOAc (entry-4) and THF
(entry-5) were not proceeded as no product formation observed for 12 h. When
Ethanol is used as solvent the reaction is proceeded and the progress of the
reaction monitored by at different intervals of times (entry-6-11).
The best results were obtained when the reaction mixture were shaking for 30h
in Ethanol as used solvent and BY as biocatalyst to give the product 3a in 88%
yield.
To check the effect of the BY as biocatalyst, when the reaction is carried in the
absence of BY the reaction did not proceeded even after 18 h of time.
The after getting optimized condition in hands the generality of the reaction is
checked by taking the substituted chalcone (1a-b) and substituted anilines (2a-l)
(Scheme-2).
S. No Solvent Catalyst
Baker’s
Yeast (BY)
Reaction
condition
(stirring)
Yield %
1 Benzene BY 12 h NR
2 Toluene BY 12 h NR
3 DCM BY 12 h NR
4 EtOAc BY 12 h NR
5 THF BY 12 h NR
6 Ethanol BY 6 h NR
7 Ethanol BY 12 h 10
8 Ethanol BY 18 h 30
9 Ethanol BY 24h 50
10 Ethanol BY 30h 70
11 Ethanol BY 36h 88
12 Ethanol Without BY 18 h NR

7. Table:3. The spectral data .

8. Compound Yield M.P. Mass
FTIR (KBr,
νmax, cm–1)
1H-NMR (400 MHz) δ
ppm
3a
88% 189-191ºC
(m.f.:C21H17N):
calculated:283.14
found:284.1(M+)
3049 (Ar, C-H),
1608 (C=N),
1530,
1490(C=C);
7.42-7.43 (dd, 3H), 7.49-
7.59 (m, 7H), 7.64- 7.67
(dd, 2H), 7.71-7.80 (tt, 2H),
7.80-7.84 (d, 1H), 8.01-8.04
(dt, 2H)
3b
85%
95-197 ºC
(m.f.:C21H16ClN):
calculated:317.10
found: 317.18
(M+)
3037 (Ar, C-H),
1607 (C=N),
1527, 1478
(C=C);
7.42-7.43 (dd, 3H), 7.49-
7.59 (m, 5H), 7.64-7.67 (dd,
2H), 7.71-7.730 (tt, 2H),
7.84 (s, 1H), 8.01 (s, 1H),
8.02-8.03 (t, 2H)
3c
86%
176-178 ºC
(m.f.:C21H16FN):
calculated:301.13
found: 302.10
(M+1).
3020 (Ar, C-H),
1594 (C=N),
1530, 1492
(C=C);
7.40-7.43 (dd, 3H), 7.47-
7.53 (m, 6H), 7.59-7.64 (dd,
2H), 7.70-7.73 (tt, 2H),
7.73-7.80 (d, 1H), 7.84-8.03
(dd, 2H);
3d
84%206-208 ºC
(m.f.:C21H16BrN)
calculated:361.05
found: 380.07
(M+1 + H2O).
3038 (Ar, C-H),
1608 (C=N),
1320, 1512
(C=C)
6.54-6.56 (d, 1H), 7.09-
7.13 (t, 2H), 7.21-7.23 (d,
1H), 7.44-7.59 (m, 5H),
7.62-7.65 (dd, 3H), 7.71-
7.73 (dd, 2H), 8.00-8.02
(dd, 2H)
3e
90%179-182 ºC
(m.f.: C22H19N):
calculated:297.15
found: 320.17
(M+ + sodium
salt)
3021 (Ar, C-H),
1604 (C=N),
1508, 1465(C=C)
7.09-7.13 (t, 2H), 7.44-7.59
(m, 7H), 7.62-7.66 (tt, 4H),
7.71-7.29 (dd, 3H), 7.73-
7.76 (d, 1H), 7.80-8.02 (t,
2H), 2.12 (s,3H)
3f
92%185-187 ºC

(m.f.:C22H19NO):
calculated:313.15
found: 341.19
(M+2 + MeOH
adduct).
3045 (Ar,CH),
1610 (C=N),
1621, 1553
(C=C)
6.90-6.92(d, 1H), 7.06- 7.09
(tt, 2H), 7.11-7.26 (tt, 1H),
7.44-7.60 (m, 12H), 7.62-
7.74 (m, 4H), 7.78-8.01 (dd,
2H), 3.94 (s,3H)
3g
90%182-184 ºC
(m.f.: C21H16FN):
calculated:301.13
found: 302.16
(M+ ).
3073 (Ar, CH)
1600, (C=N),
1509, (C=C)
7.41-7.43(tt, 3H), 7.49-7.59
(m, 6H), 7.61-7.66 (dd, 2H),
7.70-7.73 (tt, 2H), 7.80-7.84
(d, 1H), 8.01-8.03 (dd, 2H)
3h
88%193-195 ºC
(m.f.:C21H15ClF):
calculated:335.09
found: 336.07
(M+1).
3086 (Ar-C-H),
1589 (C=N),
1522 1432 (C=C)
7.42- 7.44 (tt, 3H), 7.49-
7.59 (m, 6H), 7.61-7.73 (tt,
3H), 7.80-7.84 (d, 1H),
8.01-8.03 (dd, 2H)
3i
89%187-189 ºC
(m.f.:C21H15F2
N)
calculated:319.12
found: 320.14
(M+1).
3090 (Ar-C-H),
1585 (C=N),
1516 1428 (C=C)
7.09-7.13 (t, 2H), 7.48-7.59
(m, 5H), 7.62-7.65 (tt, 3H),
7.71-7.73 (dd, 2H), 7.76-
7.80 (d, 1H), 8.00-8.02 (dd,
2H)
3j
88%177-179 ºC
Mass(m.f.:C21HB
FN):
calculated:379.04
found: 380.07
(M+1).
3044 (Ar-C-H),
1592 (C=N),
1506 1434 (C=C)
7.09- 7.13 (t, 2H), 7.44-7.59
(m, 6H), 7.62-7.65 (tt, 3H),
7.71-7.73 (dd, 1H), 7.76-
7.80 (d, 1H), 8.00-8.02 (dd,
2H)
3k
(m.f.: C22H18FN):
7.09-7.14 (t, 2H), 7.44-7.54

9. Scheme-2: Baker’s Yeast catalyzed Synthesis of α, β‐Unsaturated imines or
chalcone-imines (3a-l).
The table-2 represents the structures of the compounds prepared along with
their corresponding yields. Al the products were characterized with their M.P.,
Mass and 1H-NMR spectra which are in agreement with their structure.
Table-2: Structures of the products obtained from Baker’s Yeast catalyzed
one pot Betti reaction (3a-l) with isolated yields*
3a
Aniline
3b
4-chloroaniline
3c
4-Fluoroaniline
3d
4-Bromoaniline
3e
p-toulidine
3f
p-Anisidine
3g
Aniline
3h
4-chloroaniline
3i
4-Fluoroaniline
3j
4-Bromoaniline
3k
p-toulidine
3l
p-Anisidine
2. Experimental:

10. 2.1General Experimental Information
Melting points were determined with a hot-plate microscope apparatus and are
uncorrected. The
1
H-NMR spectra were recorded on Varian-400NMR spectrometer at
400MHz using CDCl3 and TMS as solvent and internal standard respectively. DIP
Mass Spectrum was recorded on Agilent- G6160 A infinity lab LC/MSD/IQ mass
spectrometer.
All the solvents were distilled and dried before use. The chemicals purchased from
commercial vendors and were used without any further purification. The reactions
were monitored by using TLC on Silica gel 60 plates, with a typical ratio of petroleum
ether: ethyl acetate (7:3) as mobile phase.
2.2 General Procedure for the synthesis of Imino-chalcones (3a-l)
A reaction mixture was prepared by mixing Schiff base (1) (1 mmol), substituted
anilines (2) (1 mmol) in 10 mL of ethanol.
500mg baker’s yeast was added in the reaction mixture. The reaction mixture was
kept on stirring on orbital shaker for 36 h.
After completion of the reaction as indicated by TLC (ethyl acetate: hexane 30:70,
v/v), the reaction mixtures were filtered through celite to remove the BY.
The residue was washed with ethanol. To the filtrate 20 mL water was added and the
contents were extracted with ethyl acetate (2X30ml).
The combined organic layers were collected and sodium sulphate was added to
remove the moisture. The organic layer was evaporated to get the pure crude product.
The obtained products were recrystallized with ethanol to get the pure imino-
chalcones (4a-l) in good to excellent yield.
All the compounds were characterized by their melting points, mass and
1
H-NMR
spectral analysis. The spectral data are given in Table-3 and the spectra are included
in supporting information file.
Conclusion
Thus in conclusion we have developed a very simple, efficient, chemo-selective
and sustainable bio-catalytic method for the synthesis of α, β‐Unsaturated

11. imines or chalcone-imines (3a-l) by using Baker’s yeast. When, the chalcones
(1a-b) is reacted with the substituted anilines (2a-f) in the presence of Baker’s
yeast as a whole cell biocatalyst at ambient temperature in ethanol as a green
solvent. The reaction procedure is easy to follow and takes place at room
temperature (25-28 C). The commercially available Baker’s Yeast is very cheap
◦
economically viable, ease of handling the reaction, make this method as an
efficient and facile chemo-selective methodology for the synthesis of α,β‐
Unsaturated imines or chalcone-imines.