part 2, section 2 biological and chemical reduction of...
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2.2.1 Introduction
Hydantoins (imidazolidine-2,4-diones) serve as important building blocks for
enantioselective amino acid synthesis because enantiomerically pure amino acids can
be prepared from these by dynamic kinetic racemic resolution.102-104 Hydantoins and
their bi- and tricyclic derivatives represent an important class of biologically active
molecules that have broad medicinal (anticancer, anticovulsant, antimuscarinic,
antiulcer, and antiarrythmic) and agrochemical (herbicidal and fungicidal)
applications.105-110 Hydantoin and some of their derivatives are structural units found
in the naturally occurring substances of marine organisms, and in bacteria. Figure 5
shows some of the natural products containing hydantoin moiety.
H~N~ NH
H B
3'- Deimino-3'-oxoaplysinopsin (27) (+)- Hydantocidin (28) (E)Axinohydantoin (29)
Naamidinene A (30) Phenytoin (31) (-) - Deltoin (32)
Mukanadin B (33) Midpacamide (34)
Figure 5: Natural products containing hydantoin moiety
128
Part 2, Scetion 2, Introduction
Examples for many alkaloids extracted from sponges or corals which contain a
hydantoin moiety (shown below) are the well-known aplysinopsins (27) with
cytotoxic properties, axinohydantoins (29) from Axinella, Hymeniacidon and
Stylotella species inhibiting protein kinase C, naamidinene A (30), a dehydro
hydantoin derivative from the genus Leucetta, and mukanadin B (33) from Agelas
species.111-117 Hydantocidin (28) is a spiro nucleoside from Streptomyces
hygroscopicus, which possesses herbicidal and plant growth regulatory activity due to
the inhibition of adenylsuccinate synthetase.118-
122
2.2.2 Solution phase synthesis of hydantoins and their derivatives
Numerous hydantoin syntheses, both in the solution phase and on solid
supports, have been reported in the literature. There are several approaches to
hydantoins starting from different building blocks. The most important principles of
hydantoin construction are shown in Figure 6.
Hydantoins can be formed from (a) ureas and carbonyl compounds, one of the
methods being Biltz synthesis, (b) According to the Bucherer-Bergs method, N-1 and
N-3 unsubstituted hydantoins can be generated by the reaction of a carbonyl
compound with inorganic cyanide and introducing a second nitrogen and a carbonyl
unit by ammonium carbonate, (c) the Read-type reaction of amino acids (esters) with
inorganic iso(thio)cyanates gives hydantoins with an unsubstituted N-3 position, (d)
the use of alkyl or aryl iso(thio )cyanates results in substitution at nitrogen N-3, (e)
amino amides already contain four ring atoms, and an introduced C-1 unit can
complete the hydantoin ring and (f) unsubstituted hydantoins are generated when a
halogen amides are reacted with inorganic iso(thio)cyanates.
A few number of solution phase synthesis for the preparations of hydantoin
libraries have appeared in the literature. The synthesis of small-molecule libraries in
solution is less popular than the solid-phase methodology, although the former offers
advantages e.g. ease of scaleup and does not require a potentially redundant handle
c: . l'nk 123 124 Al . . f . c: . h b 1or resm 1 age. ' so, vast maJonty o orgamc trans1ormatwns ave yet to e
optimized for the solid phase. The main advantage of solid-phase synthesis lies in
ease of product purification. However, this can often be conveniently accomplished
129
Part 2, Scetion 2, Introduction
for reactions in solution by liquid-liquid partitioning or the use of scavengers to
remove undesired material. 125-127
~ ~ I
/
X H
(a) (b) (c)
~ m I
: 12 H R
(d) (e) (f)
Figure 6: Synthetic strategies and building blocks in hydantoin formation
From monocarbonyl compounds or carbon dioxide and ureas
In an attempt to examine to what extent substituted hydantoins can be made
directly from simple, inexpensive starting materials, a one-pot synthesis of 5-, 3,5-,
and 1 ,3,5-substituted hydantoins that is based on the carbonylation of aldehydes in the
presence of urea derivatives has been described. 128 Scheme 9 shows the palladium
based carbonylation. To what extent sulfonamide, urethanes and urea derivatives can
be used as amide components in the carbonylation of aldehydes with amides
(amidocarbonylation) in the presence of a palladium catalyst has been studied.129•130
The conversion of cyclohexanecarbaldehyde with the respective amide component
served as a model reaction. In order to demonstrate the utility of this reaction, the
amidocarbonylation of cyclohexanecarbaldehyde was examined with a variety of
substituted urea derivatives (Scheme 9). Indeed, free urea gave very good selectivities
(90%) for the hydantoin 35 when water-absorbing agents such as triethyl
orthoformate or acetic anhydride were used. Free N-carbamoyl amino acid (36) can
be prepared in good yields by simply combining one equivalent of water with the
corresponding hydantoin.131-135 Hydantoin 37 was obtained in very good yield (86%).
130
Part 2, Scetion 2, Introduction
The reaction with symmetrical dimethylurea led to the 5-cyclohexyl-1 ,3-
dimethylhydantoin (38) in comparably high yield (80%).
Scheme 9
~ ")-N.cn, n,c),Ncn~ HN~-cH3 z H H H H3?~-cH3
0 :JL 0 37 38
86% :··0<:·------·------------------ --~-------··: 80%
0 i c i 0 ~ : : ~
H2N NH 2 i ~ + i H2N NHz CH(OEt)j'··----------!P.d),-Br-.,.H------------· H
20
~H 0
35 90%
35 + 36 25% 45%
~H HN~NH2
0
36
55%
In the course of conducting a structure-activity relationship (SAR) study,
hydantoin 40 has been prepared by cyclizing (R)-amino amide 39 in the presence of
1, 1-carbonyldiimidazole (CDI) (Scheme 1 0). But the cyclization proceeded with
complete racemization.
Scheme 10
39 40
Initially, the base i.e. triethylamine was thought to be responsible for
racemization, so the reaction was tried with CDI in the absence of base, some
racemization still occurred, reducing the enantiomeric excess to 70%. In addition, the
reaction was sluggish and only gave the hydantoin product in 50% yield. Triphosgene,
131
Part 2, Scetion 2, Introduction
was then selected as a coupling reagent. 136 This resulted in cyclization of 39 to give
enantiomerically pure hydantoin 40.
From a-dicarbonyl compounds and ureas
Here, Biltz synthesis is employed to generate hydantoin moiety containing
pharmaceutically important products. Phenytoin and phenytoin derivatives have been
synthesized by irradiating an alkaline mixture of (thio)ureas and benzils in DMSO
with 750 W microwave pulses (Scheme 11).137'138 A solvent-free microwave-assisted
synthesis of disubstituted hydantoins and thiohydantoins is also reported in
literature. 139
Scheme 11
41
H R'GrNlNH2 X=O,S
42
PPE _. MW R~~ I ~ )::::/
~X H
43
Arylglyoxals 41 when reacted with phenylurea or phenylthiourea 42 and
polyphosphoric ester as reaction mediator produced the corresponding disubstituted
hydantoin/thiohydantoin, 43. Biltz synthesis occurs by pinacol-pinacolone
rearrangement. There are other reactions between a-dicarbonyl compounds and ureas
building hydantoin derivatives which deviate from the mechanism of the Biltz
synthesis. Ishii et.al. have illustrated the condensation of oxalyl chloride with
monosubstituted ureas to form 2,4,5-trioxoimidazolidines, which represent substituted
parabanic acids. 140 Ring opening of a carbamoylizatin derivative by urea gave the
oxalylurea analogue, which could be cyclized in two different mechanisms: (i) first
generating the quinazolin-2-one unit and followed by fonnation of the hydantoin ring
under acidic conditions or (ii) first forming the hydantoin moiety and followed by
generation of the quinazolin-2-one ring using primary amines.
132
Part 2, Scetion 2, Introduction
Methods based on the Bucherer-Bergs synthesis
The Bucherer-Bergs synthesis is a practical and suitable route to synthesize
hydantoins. The synthesis involves the reaction of carbonyl compounds with
potassium cyanide and ammonium carbonate. The aldose reductase inhibitor, sorbinil
(46), has been prepared from benzopyranone (44) as shown below (Scheme 12). 141
Scheme 12
KCN, (NH4hC~ C2H 50H, H 20
~NH ~NH F~ F'CQN ~ 1. Brucin
~ --------~~ ~ ,~ 2. HCI
44 45 46
The synthesis of PET ligands for tumor detection via hydantoins has been
reported in literature.142 Ultrasonication accelerates hydantoin formation using the
Bucherer-Bergs reaction. 143 a-Amino nitrites have been reacted with carbon dioxide to
give the disubstituted ureas which underwent cyclization in water at room temperature
followed by hydrolyzation of the imino compounds to the corresponding
h d . 144-145 y antoms.
Methods based on the Read synthesis
A frequently applied method for preparation of (thio) hydantoins is the Read
synthesis. 146-147 During their efforts to obtain silicon-containing hydantoins 49, Smith
eta!. treated silylated amino acids 47 with potassium cyanate in pyridine followed by
acid cyclization as shown (Scheme 13). 148
Scheme 13
49
Access to the 5-methylenehydantoin has been achieved by conversion of
cystine via a double Read synthesis and cleavage of the dimer under standard
alkylation conditions. 149
133
Part 2, Scetion 2, Introduction
From amino acids or esters and isocyanates
Hydantoins can be prepared by treatment of a-amino acids with aryl or alkyl
isocyanates via the intermediate ureido acids. Esters or amides of a-amino acids and
even peptides can also act as starting materials. The Edman degradation has been
varied that heterocyclic modification of the N-terminus of a peptide takes place.150
Thus, the thiourea formed from the amino acid and the aryl isocyanate was subjected
to dehydrothiolation reaction and subsequent trapping of the intermediate
carbodiimide by the adjacent amide nitrogen resulting in a small library of 2-
iminohydantoins. The intermediate ureido acids are reported to undergo both acid
catalyzed and base catalyzed cyclizations. 151-156
From amino acid amides and carbonic acid derivatives
Hydantoins can also be obtained from amino acid amide. 157 Coupling Boc
protected amino acids to primary amines and subsequent deprotection affords the
desired amino acid amides, which can be cyclized with carbonyldiimidazole (CDI).
This cyclization strategy has been used in solid-phase synthesis.
Miscellaneous conversions of carboxamides
Cyclopropane dicarboxylic acid derivatives undergo Hofmann rearrangement
to form 1 ,3-unsubstituted hydantoins (Scheme 14). 158
Scheme 14
50
~ONHBr
~ONHBr
51
Conversions of other heterocyclic compounds to hydantoins
Conversion reactions from three-membered rings
~ H
52
1,5-Disubstituted hydantoins 55 could be prepared from reacting aziridinones
53 with NH2CN and treatment of the formed iminohydantoins 54 with HN02
(Scheme15).159
134
Part 2, Scetion 2, Introduction
Scheme 15
55
Conversion reactions from other five-membered rings
Transformations of other five-membered rings to hydantoins are important for
the synthesis of naturally occurring compounds with a hydantoin moiety.
Pyrroloazepinones containing a 2-imidazolone substituent have been synthesized and
oxidized by three equivalents of bromine to afford the axinohydantoin derivatives 57
and 58 as shown in (Scheme 16). 115
Scheme 16
56
Acolr B NaOAc
45%
57
+
Ring contraction reactions from six-membered rings
B
35%
58
Ring contraction reactions from six-membered nngs to hydantoins started
from pyrimidine derivatives, such as barbiturates. A photochemical conversion of 5-
allyl( ethyl)-1-methyl-5-phenylbarbituric acid to 5-allyl( ethyl)-3-methyl-5-
phenylhydantoin has been described, the reactions involved the loss of carbon
monoxide. Another approach is based on the new aminobarbituric acid-hydantoin
rearrangement.160-161 First, diethyl acetamidomalonates were treated with ureas and
the intermediate 5-acetaminobarbituric acids 59 undergoes rearrangement to yield 5,
5-disubstituted hydantoins 60 in a one-pot synthesis as shown (Scheme 17).
135
Scheme 17
59
Part 2, Scetion 2, Introduction
OR
R~ H
60
t
An efficient one-pot procedure for the preparation of hydantoins and
thiohydantoins is depicted in Scheme 18 (X =0, S).162 The synthesis begins with
imine formation between an (R)-amino acid ester 61 and an aldehyde 62, followed by
in situ reduction by sodium triacetoxyborohydride to secondary amine 63. An
isothiocyanate 64 is then added to give a thiourea intermediate 65, which cyclizes to
the thiohydantoin 66. This sequence occurs in nearly quantitative yields with
stoichiometric reagent quantities.
Scheme 18
H Rz-CHO (62) RtryR1
NHz Na(OAc))BH
61
66
136
RJtyR, NH......,...R2
63
I RrNCX(64) fEt3N
65
Part 2, Scetion 2, Introduction
2.2.3 Solid-phase syntheses
Solid-phase synthesis of structurally diverse, non-peptidic heterocycles
bearing one or more nitrogen atoms, in particular, the synthesis of small organic
molecules which have improved pharmacological properties over peptides is a major
focal point in search of leads utilizing automated high-throughput screening (HIS).
The hydantoin scaffold is quiet often selected as it provides a chemically tractable
molecular framework. Cyclization and cleavage (involved in the solid phase
synthesis) from the resin typically occurrs in two ways: (i) by cyclo-elimination, i.e.
cyclization of the acyclic resin-bound compound and spontaneous autocleavage and
(ii) by performing cyclization and cleavage in separate steps. The reactions are
divided into two groups.
Cyclo-elimination release strategies
(i) Acid-catalyzed cyclizations
DeWitt et.al. reported synthesis of hydantoins on a solid support in 1993
using both a combinatorial and automated approach.163 A resin-bound amino acid
(linked to the resin through C-terminal ester functionality) was linked with a variety
of substituted isocyanates to generate the urea precursor. The desired products were
cyclized and spontaneously cleaved with strong acid at elevated temperatures.
Apart from polystyrene Wang resin used by Dewitt et.al., other polymers for
the acid-catalyzed cyclo-elimination release of hydantoins have also been used eg 2-
polystyrylsulfonyl ethanol support and high-loading radiation grafted polymers. 164-165
(ii) Base-catalyzed cyclizations
Analogous to the synthetic route employed by DeWitt et al. for the acidic
cyclo-elimination, Kim et al. applied milder, basic cleavage conditions using neat
diisopropylamine at room temperature.166 This method is fast (less than 1 h),
convenient, mild, and affords high yield and purity.
Separate cyclization and cleavage steps
(i) Cyclizations induced by carbonyldiimidazole or phosgene derivatives
Primary or secondary amine functionalities of amino acids have been treated
with carbonyldiimidazole or triphosgene to fonn intermediate isocyanates which then
underwent a ring closure reaction to yield the corresponding hydantoins. 167 Cleavage
137
Part 2, Scetion 2, Introduction
of the obtained di- or trisubstituted hydantoins resulted from treatment of the resin
with HF /anisole in a separate step as shown (Scheme 19). Instead of triphosgene, use
of diphosgene in solid-phase hydantoin synthesis has also been reported.168
Scheme 19
~R, H R, W'
' N)fNH2 Triphoge"i, '}N NH H 0 orCDI H lr
-~O N NH Boc removal and acylati~ ~ )( HF/ anisole 0
BocNH BocNH
HN
~Rz 67 68 69
(ii) Other separate cyclization and cleavage steps
Attaching aldehydes to solid support, e.g. a 5-hydroxymethylfurfural template
and reacting them stepwise with an amino acid, NaBH3CN and an isocyanate leading
to the formation of the hydantoin ring after treatment with a base has been reported.169
Release from the resin was performed with TF A. Heine et al. introduced a spot
hydantoin synthesis on cellulose membranes.170 Acid treatment led to the cyclization
of ureas to hydantoins. Depending on the linker type chosen, simultaneous cleavage
occurred or release from a photo-linker was achieved by irradiation.
2.2.4 Reactivity of hydantoins and their derivatives
Hydrolysis of hydantoins
Hydrolysis of hydantoins can be performed either in an acidic or basic
medium. C-5 substituted hydantoin derivatives are of synthetic utility as precursors to
a-amino acids. The hydrolytic degradation proceeds through the intermediacy of
ureido acids. This can be performed by biocatalytic conversion, e.g. using microbial
or plant hydantoinases to produce ureido acids. The further transformation to amino
acids can then be catalyzed by other enzymes or acids. 171-172 The formation of amino
acids from hydantoins can be achieved non-enzymatically also. Rare unnatural amino
acids can be prepared from hydantoins both under acidic or basic conditions.
Aminobicyclo[2.2.1 ]heptane dicarboxylic acids have been prepared from
spirohydantoins by acidic hydrolysis. 173
138
Part 2, Scetion 2, Introduction
N-Alkylations with electrophilic reagents
N-unsubstituted hydantoins can easily be monoalkylated at the imide nitrogen
in position 3, whereas substitution of both nitrogens in one step requires much harder
conditions. Alkylation at amide N-1 could be done after first protecting the N-3.174-175
N-3 alkylation of hydantoins is a commonly applied reaction to modify the core
scaffold.
N-Alkylations by Mitsunobu coupling
Hydaritoins have been shown to react with 4-nitrophenyl alcohol or 5-ethyl
alcohol trytamine derivative under Mitsunobu conditions (Scheme 20). Similarly,
Mitsunobu couplings have also been shown in the synthesis of novel P2X receptor 0 176 antagomsts.
Scheme 20
HN-f ~H
70 71
Aldol-type reactions
Hydantoins having a free methylene group in the C-5 position can be
condensed with aldehydes resulting in C-5-unsaturated compounds. Synthesis of the
aplysinopsin derivative is shown (Scheme 21 ).
Scheme 21
or+ H
CH3 _/fJ N\ Pyridine,Reflux
)rN'cH, 73 74 75
139
Part 2, Scetion 2, Introduction
Cycloaddition reactions of hydantoins
Diels-Alder reaction of a 5-methylene hydantoin (R=(S)-1-phenylethyl) acting
as dienophile with cyclopentadiene acting as diene has been reported (Scheme 22). 177
Scheme 22
76 77
Other reactions of hydantoins
l.Lewis acid, -70 °C, 1 h
2. 0°,3 h, DCM
78
5,5-Disubstituted hydantoins have been shown to react with 3-
(dimethylamino)-2,2-dimethyl-2H-azirine to give 4H-imidazoles in a very complex
ring transformation reaction (Scheme 23).
Scheme 23
79 80 81
Complexation of hydantoins with metal ions
Interactions ofhydantoins with metal ions, such as copper(II) (Zwikker test) or
cobalt(Il) (Parri test) are widely used in colour reactions for identification.
Platinum(II) complexes with 5-methyl-5-phenylhydantoin (83) have been synthesized
and found to be effective in cytotoxicity tests (Scheme 24).178
Scheme 24
r-=\. ~NH AgN03 ~N~o -c-is---[P-t(_N_H.;_3_h_C-112~] H
82 83
140
Part 2, Scetion 2, Results and discussion
Among other transition metal complexes with hydantoin ligands iron (II),
Nickel (II), copper(ll) and gold (I) complexes have been synthesized and
characterized.179-182 Moreover, the complexations of 5,5-diphenylhydantoin or
hydantoin itself with silver (1), zinc (II), and cadmium (II) ions or with antimony (V)
and mercuric (II) ions have also been reported.
2.2.5 Present work: Results and Discussion
The objective of the present work was to study the biological and chemical
reduction of the carbonyl group of hydantoin derivatives. Hydantoin derivatives are
important industrial intermediates especially for the preparation of natural and non
natural amino acids. Synthesis of hydantoin derivatives is well established (Section
2.2.2). Conversion of hydantoins to corresponding carbamyl derivative or a-amino
acids by enzymatic and chemo-enzymatic methods has been well documented
(Section 2.2.4). But, till date, no report has appeared in literature that documents the
study of enzymatic reduction of carbonyl function of hydantoin derivatives. We
envisaged that the reduction of carbonyl group of a suitably substituted hydantoin
could lead to a quick entry into optically active cyclic, heterocyclic and alicyclic 1 ,2-
diamines as described in Scheme 25.
Scheme 25
84
H2N~NH2 ----~-~
X H 0'Y COOH
Jl =~tN NR~ Xn, H
Y OOH
85 86
R~o R2 =protecting groups X= CH2, n = I or 2 Y= CH2 or heteroatom
87
Moreover, oxidation-reduction cycles could lead to optically active 1 ,2·
aminoalcohols 92 and a-amino aldehydes 90 (Scheme 26). In addition, an alternate
route for the preparation of a-amino acids 94 is also feasible.
141
Scheme 26
0
HN)lNH
RKO 88
Reduction
Part 2, Scetion 2, Results and discussion
0 Jl HN)lNH :::;;:=~::: HN NH2 Reductiolj. ~ ~CHO
If "oH If 89
94
90
! Oxid•tion
0
HN)lNH2
r_cooH 93
0
HN)lNH2
rCH20H
91
!
We initiated our work with the study of chemical and enzymatic reduction of
the carbonyl group of 1,3-dibenzyl-5-methylhydantoin (96) and 3-benzyl-5-
methylhydantoin (97). Compounds 96 and 97 were prepared by benzylation of 5-
methylhydantoin (95) (Scheme 27). 5-methylhydantoin (95) was prepared by a two
step literature method starting from racemic alanine. 183 Hydantoin 95 was purified by
crystallization and characterized by 1HNMR and IR spectral data. In NMR, the 5-
methyl group of 95 resonated as a doublet at 8 1.39 (J=6.6 Hz), while the quartet for
the methine proton appeared at 8 4.15 (J=6.6 Hz). IR spectra showed bands for
carbonyl stretching at 1720 and 1740 cm-1•
Benzylation of 95 with 2.5 equivalent of benzyl bromide in the presence of
potassium carbonate in DMF produced dibenzyl derivative, 96 and monobenzyl
derivative, 97 in 9:1 ratio (Scheme 27a).
Scheme 27
HNJl.NH
0 0
DMF,K2C03 Jl. Jl. (a) K Benzyl bromide (2.5 eq) ~N N:'O + HN N:'O
H3C 0 overnight stirring at rt H3CHO H3CHO h
95 96 97 96/97 = 9:1
HNJl.NH
0 0
DMF,K2C03 Jl. Jl.
(b) K Benzyl bromide (1.1 eq) ~N N:'O + HN N:'O
H3C 0 overnight stirring at rt H3CHO H3CHO h
95 96 97 96/97 = 2:8
142
Part 2, Section 2, Results and discussion
To obtain monobenzyl derivative as maJor compound, the reaction was
repeated under same conditions using 1.1 equivalent of benzyl bromide. Under these
conditions 96 and 97 were obtained in the ratio of2:8 (Scheme 27b).
The compounds 96 and 97 were separated by flash chromatography (ethyl
acetate/hexane, 1 :3). The faster moving fraction in the flash chromatography was
identified as N,N-dibenzyl-5-methylimidazolidine-2,4-dione (1,3-dibenzyl-5-
methylhydantoin, 96) based on 1 HNMR spectral data. Doublet for the -CH3 protons at
C-5 appeared at 8 1.27 (1=7.1 Hz) while the -CH proton appeared as quartet at 8 3.68
(1=7.1 Hz). An interesting observation made in the 1HNMR spectrum of this product
was that the methylene protons of the benzyl group not only at N-1, but also at N-3
showed geminal coupling and appeared as separate doublets because of the presence
of an asymmetric carbon centre at C-5. The protons of the methylene group present on
the N-1 appeared as two doublets (1=15.2 Hz) at 8 4.07 and 8 4.88 while the protons
of the methylene group present on N-3 appeared as doublets at (1=18 Hz) 8 4.62 and 8
4.56. The aromatic protons appeared as multiplets at 8 7.2-7.4.
The slow moving fraction obtained from the flash chromatography was
identified as N-benzyl-5-methylimidazolidine-2,4-dione (3-benzyl-5-
methylhydantoin, 97) on the basis of 1 HNMR spectral data. Doublet for the -CH3
protons at C-5 appeared at 8 1.01 (1=6.6 Hz) while the methine proton appeared as
quartet at 8 3.75 (1=6.6 Hz). The methylene protons ofthe benzyl group appeared as a
singlet at 8 4.63. The ureido proton appeared at 8 6.3 as a singlet while the aromatic
protons appeared at 8 7.18-7.28.
2.2.5.1 Sodium borohydride reduction of 1,3-dibenzyl-5-methylhydantoin (96)
Initially sodium borohydride reduction of dibenzyl derivative, 96 was studied
(i) to understand the behaviour of carbonyl group at position 4 and (ii) to obtain
standard samples of alcohols, if reduction occurs. Moreover, the behaviour of 4-
carbonyl group in borohydride reduction would have implications for the enzymatic
reduction since borohydride and NAD(P)H reductions exhibit several similarities.
The reduction of 1 ,3-dibenzyl-5-methylhydantoin (96) was done in the
presence of 0.5 eq. of sodium borohydride in ethanol at 0 °C (Scheme 28). Progress of
the reaction was monitored by TLC and the the products were identified by 1 HNMR
spectroscopy. The chemical reduction resulted in the formation of two diastereomeric
alcohols, 98 and 99, in approximately 1:1 ratio, structures of which were confirmed
143
Part 2, Section 2, Results and discussion
by 1HNMR of the crude product. Methyl protons at C-5 for the two diastereomers
appeared as doublet at 8 1.26 (J=6.6 Hz) and 8 1.02 (J=6.6 Hz). Doublet of quartet for
C-5 proton for the two diastereomers resonated at 8 3.25 (J=6.6 and 7.1 Hz) and 8
3.18 (J=6.6 and 8.0 Hz). The doublet for the single proton at C-4 was observed at 8
4.28 (J=6.9 Hz) and 8 4.27 (J=7.8 Hz). The -OH protons appeared as broad singlet at
8 3.27 and 8 1.72. The doublets for the methylene protons at N-1 for the diastereomers
appeared at 8 4.13 (J=6.9 Hz) and 8 4.62 (J=6.9 Hz) and 8 4.10 (J=7.1 Hz) and 8 4.60
(J=7.1 Hz) and the doublets for the methylene protons at N-3 were observed at 8 4.7
(J=6.9 Hz) and 8 4.87 (J=6.9 Hz) and 8 4.4 (J=7.2 Hz) and 8 4.54 (J=7.2 Hz). The
aromatic protons were observed as multiplet at 8 7.0-7.35. The crude product also
showed the presence of trace amount of starting material 96.
Scheme 28
Jl:tCJ N\. __ _l 1 NaBHiEthanol ~ h ooc
H3C 0
0 0
~~'U) + 0~)~-u) H3C OH H3C OH
96 98 99
Crude product was subjected to flash chromatography over silica-gel (ethyl
acetate/hexane, 3 :7). The following fractions were isolated.
Fraction 1: The fastest moving fraction corresponded with the unreacted starting
material, 1,3-dibenzyl-5-methyl-imidazolidine-2,4-dione (96), confirmed by 1HNMR
spectral data.
Fraction 2: The second fraction was assigned the structure, 1,3-dibenzyl-5-methyl-
4,5-dihydroimidazol-2-one (100) based on 1HNMR spectral data, which showed the
methyl protons as a singlet at 8 1.86. The olefinic proton appeared as a singlet at 8
5.77. The methylene protons of the benzyl groups at N-1 and N-3 resonated at 8 4.67
and 8 4.74 each as a singlet while the aromatic protons were observed as multiplet at 8
7.0-7.35. This product was not present in the crude mixture, suggesting that a silica
gel mediated dehydration of alcohol has occurred on the column.
Fraction 3: The slowest moving fraction was a mixture of diastereomeric alcohols,
the 1HNMR of which has been described above.
144
Part 2, Section 2, Results and discussion
The diastereomeric alcohols were very labile and prone to undergo
dehydration over silica-gel. Doping the silica-gel with triethylamine did not prevent
dehydration. The compound was unstable even at 4 oc and dehydrated within 48 h of
storage, presumably due to the presence of trace amounts of acetic acid left after ethyl
acetate evaporation (Scheme 29).
An interesting observation made during the course of this work was that when
a solution of sample in CDCh was scanned for 1 HNMR after standing for about two
h, one of the diastereomers (slow moving compound on TLC) almost completely
disappeared in favour of the olefin 100, leaving behind the second diastereomer
(faster moving compound on TLC)
Scheme 29
Jl C(:N N:)) H3C)---lz,"OH
98/99
Silica gel chromatography
or Storage at 4°C for 48 h
100
Based on this observation, the faster movmg compound on TLC was
tentatively assigned cis-structure, 99. Its 1HNMR spectral data (abstracted from the
crude mixture, which contained starting material, olefin and the alcohol) 1s giVen
below:
Doublet for the -CH3 appeared at 8 1.26 (1=6.6 Hz) and the doublet of quartet
for C-5 proton was resonated at 8 3.25 (1=6.6 Hz and 7.1 Hz). The doublet for the
proton at C-4 was observed at 8 4.28 (1=6.9 Hz). The -OH group at C-4 appeared at 8
3.27 as a broad singlet. The doublets for the methylene protons at N-1 appeared at 8
4.13 (1=6.9 Hz) and 8 4.62 (1=6.9 Hz) and the doublets for the methylene protons at
N-3 were observed at 8 4.7 (1=6.9 Hz) and 8 4.87 (1=6.9 Hz). The aromatic protons
appeared as multiplet at 8 7.21-7.35.
Manual subtraction of the peaks of tentatively assigned cis-alcohol from the
mixture of diastereomeric alcohols produced the spectral data for the trans-alcohol,
98. Its 1HNMR spectral data is given below:
145
Part 2, Section 2, Results and discussion
Doublet for the C-5 proton appeared at () 1.02 (1=6.6 Hz) and the doublet of
quartet for the C-5 proton was seen at() 3.18 (1=6.6 Hz and 8.0 Hz). The doublet for
the single proton at C-4 was observed at () 4.27 (1=7.8 Hz). The -OH group at C-4
appeared at () 1. 72 as a broad singlet. In this case also, the methylene protons of the
benzyl groups at N-1 and N-3 showed geminal coupling and appeared as separate
doublets. The doublets for the methylene protons at N-1 appeared at & 4.10 (1=7 .1 Hz)
and () 4.60 (1=7.1 Hz) and the doublets for the methylene protons at N-3 were
observed at() 4.4 (1=7.2 Hz) and() 4.54 (1=7.2 Hz). The aromatic protons appeared as
multiplet at () 7.17-7 .26.
2.2.5.2 Enzymatic reduction of 1,3-dibenzyl-5-methylhydantoin (96)
(i) Strain selection
Purified cultures of more than 300 bacterial and 200 fungal strains isolated
from different soil samples were screened for their ability to reduce the carbonyl
functionality of hydantoins and their benzyl derivatives. Microbial cells (0.5 g) were
suspended in 20 mM phosphate buffer, pH 7.5 (5 mL) and 5 mg of 1,3-dibenzyl-5-
methylhydantoin was added from a stock solution in DMSO to the cell suspension
and the contents were incubated at 30 oc (fungal) or 37 oc (bacterial) at 200 rpm.
Progress of the reaction was monitored by TLC using samples of, 98, 99 and olefin
100 obtained by the borohydride reduction of96 as standards. An aliquot of 1 mL was
withdrawn from the reaction mixture and extracted with ethyl acetate (1 mL). The
organic layer was used for TLC. Two strains, one bacterium and one fungal, were
able to reduce the carbonyl of the substrate 96.
(ii) Strain identification
The fungal strain was a known strain, previously isolated in our laboratory for
the enantioselective reduction of ethyl 4-chloro-3-oxobutanoate. The strain has been
previously identified as Penicillium funiculosum and has been assigned accession
number MTCC 5246. 184
The bacterial strain, designated as B4W is a new isolate from the soil samples
collected from Lothal, Gujrat. The strain has been identified as Bacillus pumilis based
on morphological, biochemical characterization (Table 1) and full 16s rDNA
sequence. Strain has been assigned accession number MTCC B6033.
146
Part 2, Section 2, Results and discussion
Table 1: Summary of the results of the morphological, biochemical and physiological tests conducted on strain B4W
Morphological Characteristics Rods, Gram's positive, Motile, Opaque colonies, Motile, Endospore forming.
Biochemical and Physiological Tests
1. Growth on MacConkey agar 16. Acid production from a) lactose fermentor (-) carbohydrates b) non lactose fermentor (+) Adonitol (-)
2. Indole test (-) Arabinose (+) 3. Methyl red test (+) Cellobiose (+) 4. Voges Proskauer test (+) Dextrose (+) 5. Citrate utilization (+) Fructose (+) 6. Casein hydrolysis (+) Galactose (-) 7. Starch hydrolysis (+/-) Inositol (-) 8. Urea hydrolysis (-) Lactose (-) 9. Nitrate reduction (+/-) Maltose (-) I 0. H2S production (-) Malibiose (-) 11. Oxidase test (+) Raffinose (-) 12. Catalase test (+) Sialcin (+) 13. Oxidation/fermentation (-) Sorbitol (-) 14. Gelatin liquefaction (+) Sucrose (+) 15. Arginine dihydroxylase (+) Trehalose (-)
17. Growth temp. 15-42 °C 18. Growth pH 5.7-11.0 19. NaCl tolerance 2.5-10%
16 s rDNA Analysis
The chromosomal DNA of strain B4W was isolated according to the procedure
described by Rainey et a/. 185 The 16S rRNA gene was amplified with primers 8-27f (5'
AGAGTTTGATCCTGGCTCAG-3') and 1500r (5'AGAAAGGAGGTGATCCAGGC-
3'). The amplified DNA fragment was separated on 1 % agarose gel, eluted from the
gel and purified using QIAquick gel extraction kit (Qiagen, Germany). The purified
PCR product was sequenced with four forward and three reverse primers namely 8-27f
(5'AGAGTTTGATCCTGGCTCAG-3'), 357f (5'-CTCCTACGGGAGGCAGCAG-'),
704f5'-TAGCGGTGAAATGCGTAGA-3'), 1114f( 5'- GCAACGAGCGCAACC-3' ),
685r (5'-TCTACGCATTTCACCGCTAC-3'),1110r (5'-GGGTTGCGCTCGTTG-3')
and 1500r (5'-GAAAGGAGGTGATCCAGGC-3'), respectively (Escherichia coli
numbering system). The rDNA sequence was determined by the dideoxy chain
termination method using the Big-Dye terminator kit using ABI 310 Genetic Analyzer
(Applied Biosystems, USA). The 16S rDNA sequence of the strain B4W generated in
this work (1352 bases; Figure 7) was aligned with the 16S rDNA sequence of other
closely related members of the genus Bacillus. A sequence similarity search was done
147
Part 2, Section 2, Results and discussion
using GenBank BLASTN .186 Sequences of closely related taxa were retrieved; aligned
using Clustal X programme187 and the alignment was manually corrected. For the
neighbour-joining analysis, 188 the distances between the sequences were calculated
using Kimura's two-parameter model.189 Bootstrap analysis was performed to assess the
confidence limits ofthe branching.190 The results are summarized in Figure 8.
TGCANTCGAGCGGANAGAAGGGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGT AACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGAGCTAATACCGGATAGTTCCTTGAACCGCAT GGTTCAAGGATGAAAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTG AGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAG ACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGC AACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCAAGAG TAACTGCTTGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAA TACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGA TGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAG TGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCT GGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACG CCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCAC TCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGA GCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAG ATAGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATG TTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTNAGTTGGGCACTCTAAGG TGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGC TACACACGTGCTACAATGGACAGAACAAAGGGCTGCGAGACCGCAAGGTTTAGCCAATCCCACAAATCT GTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAG CATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC
Figure 7: 1352 bases generated by the 16S rDNA sequence of the strain B4W
0.01 Bacillus licheniformis NCOO 1772 T (X60623)
acillus atrophaeus NCIMB 12899T (X60607)
99 Bacillus amylo/iquefaciens A TCC 23350T (X60605)
51 Bacillus pumilus NCOO 1766T (X60637)
100 Strain B4W
50 '----------Bacillus cereus lAM I2605T (016273)
.-----------Bacillus coagulans JCM 2257T (078313)
'--------Bacillus badius ATCC 14574T (X77790)
,------Bacilus circulans lAM 12462 T (078312)
'-------Bacillus flrmus JCM 2512 T (078314)
'-----------Bacillus inso/itus OSM5T (X60642)
Figure 8: Neighbour-joining tree based on 16S rDNA (1352 bases) sequences showing the phylogenetic relationship between strain B4W and other closely related species of the genus Bacillus. Bootstrap values (expressed as percentage of 1000 replications) greater than 50% are given at the nodes. Bar, 1 %sequence variation.
148
Part 2, Section 2, Results and discussion
In a parallel programme on screemng of microorganisms for vanous
biocatalytic activities in our laboratory, the strain B4W has also shown excellent
sulphide oxidation activity. Characterization of enzyme and its application in organic
synthesis is the subject matter of another thesis submitted to JNU, New Delhi (Shefali
Sangar, 2007).
2.2.5.3 Bacillus pumilis catalyzed reduction of 1,3-dibenzyl-5-methylhydantoin
(96)
Cells of Bacillus pumilis (5.0 g) were washed with phosphate buffer, pH 7.5
and resuspended in 20 mM phosphate buffer (pH 7.5, 50 mL). 50 mg of 1,3-dibenzyl-
5-methylhydantoin (96) was added from a DMSO stock solution to the cell
suspension and the contents were incubated at 3 7 °C at 200 rpm. Progress of the
reaction was monitored by TLC for which an aliquot of 5 mL was withdrawn from the
reaction mixture and extracted with ethyl acetate (5 mL). The organic layer was used
for TLC. Final work up of the reaction mixture involved extraction of the reaction
mixture with ethyl acetate (2x40 mL). The organic layer was separated, washed twice
with brine solution, dried over sodium sulphate and evaporated under reduced
pressure. Structures of the products were confirmed by 1HNMR spectroscopy.
The Bacillus pumilis catalyzed reduction of 1,3-dibenzyl-5-methylhydantoin
(96) resulted in the exclusive formation of one diastereomer of 1,3-dibenzyl-4-
hydroxy-5-methylhydantoin. On TLC the alcohol corresponded with the slow moving
diastereomer obtained in sodium borohydride reduction and was therefore assigned
trans-structure 98 (Scheme 30).
Scheme 30
96
Bacillus pumilis Phosphate buffer, pH 7.t;
16 h
149
98
100
Part 2, Section 2, Results and discussion
The 1HNMR spectral data of the biocatalyzed product corresponded with the 1HNMR spectral data assigned to the trans diastereomer in borohydride reduction. No
olefin was produced under the reaction conditions. But storage of the pure sample
after evaporation of ethyl acetate resulted in dehydration to alkene 100, the 1HNMR
spectra of which corresponded with the spectra of alkene obtained in borohydride
reduction.
2.2.5.4 Sodium borohydride reduction of 3-benzyl-5-methylhydantoin (97)
Next we investigated the reduction of 3-benzyl-5-methylhydantoin (97) with
sodium borohydride. The reaction was done in absolute ethanol at 0 °C. But to our
surprise, desired alcohols could not be obtained by the chemical reduction of 3-
benzyl-5-methylhydantoin (97) even after many attempts. Instead, complete reduction
of the carbonyl functionality at C-4 occurred tb form 3-benzyl-5-methyl
imidazolidine-2-one (101; Scheme 31).
Scheme 31
N aBH4/Ethanol
0°C
101
Structure of the product 101 was confirmed by 1HNMR spectroscopy in which
doublet for the -CH3 appeared at 8 1.01 (1=6.6 Hz) and the methine proton was seen
as a multiplet at 8 3.75. The two methylene protons at C-4 showed germinal coupling
because of the presence of an asymmetric centre and thus each proton appeared as
doublet of a doublet at 8 3.45 (1=10.8 Hz, 3.9 Hz) and 8 3.33 (1=10.8 Hz, 6.9 Hz).
The methylene protons of the benzyl group were observed as a singlet at 8 4.63. The
aromatic protons appeared as multiplet at 8 7.18-7 .28. The -NH proton appeared as a
broad singlet at 8 5.3.
The results may be explained as shown in Scheme 32. Sodium borohydride
reduction of 97 may initially produce 102, which undergoes rapid dehydration to
produce olefin 103a, which inturn exists in ene-amine tautomeric forms. The imine
form 103b is further reduced with sodium borohydride to give the product 101.
150
Part 2, Section 2, Results and discussion
Overall, it is an interesting result as carbonyl group has been reduced to methylene
group with sodium borohydride under very mild conditions.
Scheme 32
97
NaBHiEthanol
0°C
102 103a
2.2.5.5 Bacillus pumilis catalyzed reduction of 3-benzyl-5-methylhydantoin (97)
The bacterium was grown in rich medium comprising of peptone (0.5%), meat
extract (0.2%), yeast extract (0.1 %) and NaCl (0.5%) for 22 hr. The cells were
separated by centrifugation and washed with phosphate buffer (20 mM, pH 7 .5). Cells
(5.0 g) were suspended in 20 mM phosphate buffer (pH 7.5, 50 mL) and 50 mg of 3-
benzyl-5-methylhydantoin was added from a stock solution in DMSO to the cell
suspension and the contents were incubated at 3 7 oc at 200 rpm. Progress of the
reaction was monitored by TLC (ethyl acetate/hexane, 2:3) for which an aliquot of 5
mL was withdrawn from the reaction mixture and extracted with ethyl acetate (5 mL).
The organic layer was used for TLC. Final work up of the reaction mixture involved
extraction of the reaction mixture with ethyl acetate (2 x 40 mL). The organic layer
was separated, washed twice with brine solution, dried over sodium sulphate and
evaporated under reduced pressure. To our surprise, in this case also complete
reduction of the substrate took place and the product that we could isolate from the
reaction was only 3-benzyl-5-methylimidazolidine-2-one (101; Scheme 33). 1HNMR
of the product is in agreement with the assigned structure and corresponded with the
product obtained by sodium borohydride reduction.
151
Scheme 33
Bacillus pumilis ., Phosphate buffer, pH 7.5;
18h
Part 2, Section 2, Results and discussion
97 101
2.2.5.6 Sodium borohydride reduction of methyl 2-{(1,3-dibenzyl-2,5-
dioxoimidazolidin-4-yl)methylthio }acetate (1 04)
Substrate, 104 was selected as the next example because its reduction followed
by cyclization would lead to the synthesis of an advanced intermediate of biotin
(Scheme 34).
Scheme 34
reduction
104
Steps
106
~Steps Biotin
First, the reduction of 104 with sodium borohydride was attempted. The
reaction was done in absolute ethanol at 0 oc. Progress of the reaction was monitored
by TLC. Complete consumption of the starting material occurred with the formation
of a mixture of three compounds, which were separated by flash chromatography over
silica-gel (Scheme 35).
Scheme 35
100
+ --.... _OH Hs- ......., 107
152
Part 2, Section 2, Results and discussion
Fraction 1: The fastest moving compound showed the absence of -SCH2COOCH3
residue in the 1HNMR. Instead a methyl group was observed as a singlet at() 1.86. In
addition, a singlet appeared in the olefinic region at () 5.76. The benzylic protons
appeared as two singlets at () 4.67 and () 4.74. The aromatic protons resonated as a
multiplet at() 6.9-7.4. The 1HNMR spectral data was indistinguishable from the olefin
100, obtained during the reduction of 1 ,3-dibenzyl-5-methyl hydantoin with sodium
borohydride.
Fraction 2: This fraction also showed the absence of -SCH2COOCH3 moiety, but
showed the presence methyl protons at C-5 as doublet at () 1.26 (1=6.6 Hz) and() 1.02
(1=6.6 Hz). Doublet of quartet for C-5 proton resonated at() 3.25 (1=6.6 and 7.1 Hz)
and() 3.18 (1=6.6 and 8.0 Hz). The doublet for the single proton at C-4 was observed
at () 4.28 (1=6.9 Hz) and () 4.27 (1=7.8 Hz). The -OH protons appeared as broad
singlet at() 3.27 and() 1.72. The doublets for the methylene protons at N-1 appeared
at() 4.13 (1=6.9 Hz) and() 4.62 (1=6.9 Hz) and() 4.10 (1=7.1 Hz) and() 4.60 (1=7.1
Hz) and the doublets for the methylene protons at N-3 were observed at() 4.7 (1=6.9
Hz) and() 4.87 (1=6.9 Hz) and() 4.4 (1=7.2 Hz) and() 4.54 (1=7.2 Hz). The aromatic
protons were observed as multiplet at () 7.0-7.35. The 1 HNMR spectral data of the
compound was indistinguishable from the diastereomeric alcohols 98 and 99 obtained
from the reduction of 1 ,3-dibenzyl-5-methyl hydantoin with sodium borohydride.
Fraction 3: This was identified as 2-hydroxyethanethiol, 107.
The desired alcohol, 105 was however, not obtained. The formation of
products may be explained as shown in Scheme 36. In the first step, elimination of the
thiol moiety, -SCH2COOCH3 may occur to produce 1,3-dibenzyl-5-
methylenehydantoin (108). Michael type reduction of 108 would produce 1,3-
dibenzyl-5-methylhydantoin (96), which is then further reduced to diastereomeric
alcohols 98/99. The dehydration of 98 over silica-gel would produce olefin 100 as
described earlier (Section 2.2.5 .1 ).
153
Scheme 36
100
~)lN~ ~ Co V -HSCH2COOCH\.
s-n0.....CH3
104 0
dehydration
(Silica-gel)
Jl ~NN~ ~ K~--~ OH .
98/99
Part 2, Section 2, Results and discussion
108
96
2.2.5.7 Bacillus pumilis catalyzed reduction of methyl 2-{(1,3-dibenzyl-2,5-
dioxoimidazolidin-4-yl)methylthio }acetate (1 04)
Microbial reduction of 104 with Bacillus pumilis was studied next. The
reaction conditions for the biocatalyzed reduction were same as described before. The
biocatalyzed reduction proceeded in a manner similar to the chemical reduction,
except that only one diastereomer of alcohol, trans-1 ,3-dibenzyl-4-hydroxy-5-
methylhydantoin (98) was obtained along with the 4,5-dihydro product (100) (Scheme
37). The structure of the products was confirmed by 1HNMR spectral data. The
products were same as those obtained during Bacillus pumilis catalyzed reduction of
1 ,3-dibenzyl-5-methylhydantoin (96).
Scheme 37
Jl v.N N:'O~ 0 Bacillus pumilis
0 h Phosphate bufftr, ~ pH7.0;
s II CH3 14 h
0 104 98 100
The reduction of substrate 104 was also studied with Penicillium funiculosum.
The fungus was grown in rich medium comprising of peptone (0.2%), KH2P04
(0.2%), yeast extract (0.2%) and 50% glucose (0.2%). The cells were harvested by
centrifugation after 45 hand washed with phosphate buffer (20 mM, pH 7.0). Cells
(10.0 g) were resuspended in 20 mM phosphate buffer (pH 7.5, 50 mL) and 50 mg of
104 was added. The contents were incubated at 30 °C at 200 rpm in an orbital shaker.
154
Part 2, Section 2, Results and discussion
TLC of the crude product showed the presence of two compounds. The slower
moving compound was identified as trans-1 ,3-dibenzyl-4-hydroxy-5-methylhydantoin
(98) based on 1 HNMR and TLC of the crude product. The faster moving compound
was isolated by preparative TLC. Its 1HNMR spectral data showed the presence of an
exo-methylene group which appeared as two singlets at 8 5.1 and 8 5.35. 1HNMR
spectral data was in agreement with the structure of 1,3-dibenzyl-5-methylene
hydantoin (108; Scheme 38).
Scheme 38
108
Thus, sodium borohydride reduction and the biocatalyzed reduction of
substrate 104 also followed similar pathway. The isolation of 5-methylene hydantoin
derivative 108 supports the proposed mechanism as shown in Scheme 36.
2.2.5.8 Sodium borohydride and Bacillus pumilis catalyzed reduction of 1,3-
dibenzyl-5-phenylhydantoin (109)
1,3-dibenzyl-5-phenylhydantoin (109) was synthesized according to literature
method as described in experimental section.183 First, the reduction of 1,3-dibenzyl-5-
phenylhydantoin (109) was performed with sodium borohydride in absolute ethanol
at 0 oc. However, no reduction occurred even after prolonged reaction time, refluxing
the reaction mixture and using excess of sodium borohydride also failed to reduce
109. Only the starting material was recovered unchanged.
Biocatalyzed reduction was studied next. Bacillus pumilis was suspended in
phosphate buffer (pH 7.0, 20 mM, 50 mL). 109 (50 mg) was added to the cell
suspension and the contents incubated at 37 oc for 24 h. No reaction was observed
and only the starting material was recovered unchanged. A close analysis of the 1HNMR of 1,3-dibenzyl-5-phenylhydantoin (109) revealed that this compound
primarily exists in the enol form (110). The methylene protons of the benzyl group
appeared as two singlets at 8 3.06 and 8 3.44. The singlet for C-5 proton could not be
observed at its predicted place; instead a singlet corresponding to enol appeared at 8
155
Part 2, Section 2, Results and discussion
8.24. The aromatic protons were observed between 8 7.17-7.59 as multiplet. The
resistance of 1,3-dibenzyl-5-phenylhydantoin (109) to undergo reduction with sodium
borohydride or biocatalyst may thus be attributed to the existence of the compound in
the enol form 110.
109 110
2.2.6 Conclusion
In conclusion, we have shown that borohydride and biocatalyzed reduction of
hydantoin derivatives follows exactly the same path, only difference being that
whereas biocatalyzed reduction is highly stereoselective and results in the production
of only one diastereomer, the borohydride reduction is non stereoselective and
produces a diastereomeric mixture of alcohols. Interesting results were obtained in
borohydride or biocatalyzed reduction of 3-benzyl-5-methylhydantoin (97). Under
both the conditions, carbonyl was completely reduced to methylene. 1 ,3-dibenzyl-5-
phenylhydantoin (109) failed to undergo reduction with borohydride or biocatalyst,
presumably due to the occurance of this compound in predominantly enol form, (110).
156