One-pot, regioselective synthesis of substituted arylglycines for kinetic resolution by penicillin G acylase

Article abstract of DOI:10.1002/adsc.200800203

Amido-alkylation of electron-rich arenes with phenylacetamide and glyoxylic acid offers an in-expensive route to a large variety of N-phenylacetylated arylglycines that are suited for immediate enzymatic resolution by penicillin G acylase. When performed under mild conditions at 5 °C in acetic acid/HCl, this simple one-pot operation resulted in the formation of single regioisomers only (≥98%). Subsequent kinetic resolution of the amino acid derivatives by penicillin G acylase at pH 8.0 occurred generally with E values >100 and thus furnished free (S)-configurated arylglycines with high enantiomeric purity. The corresponding enantiopure (R)-substrates, easily separable by a phase-selective extraction process, provided the corresponding (R)-enantiomers upon conventional hydrolysis. This one-pot, two-step procedure for arylglycine synthesis, resolution and work-up requires a minimum of equipment and grants rapid access to both enantiopure (S)- and (R)-antipodes of non-natural α-amino acids in small-to large-scale quantities.

Full text of DOI:10.1002/adsc.200800203

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DOI: 10.1002/adsc.200800203
One-Pot, Regioselective Synthesis of Substituted Arylglycines for
Kinetic Resolution by Penicillin G Acylase
Peter Grundmanna and Wolf-Dieter Fessnera,*
a
Department of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, Petersenstr. 22,
64287 Darmstadt, Germany
Fax : (+49)-6151-16-6636; e-mail: fessner@tu-darmstadt.de
Received: April 2, 2008; Revised: May 29, 2008; Published online: July 10, 2008
Dedicated to Chi-Huey Wong on the occasion of his 60th birthday
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: Amido-alkylation of electron-rich arenes strates, easily separable by a phase-selective extrac-
with phenylacetamide and glyoxylic acid offers an in- tion process, provided the corresponding (R)-enan-
expensive route to a large variety of N-phenylacety- tiomers upon conventional hydrolysis. This one-pot,
lated arylglycines that are suited for immediate enzy- two-step procedure for arylglycine synthesis, resolu-
matic resolution by penicillin G acylase. When per- tion and work-up requires a minimum of equipment
formed under mild conditions at 58C in acetic acid/ and grants rapid access to both enantiopure (S)- and
HCl, this simple one-pot operation resulted in the (R)-antipodes of non-natural a-amino acids in small-
formation of single regioisomers only (ꢀ98%). Sub- to large-scale quantities.
sequent kinetic resolution of the amino acid deriva-
tives by penicillin G acylase at pH 8.0 occurred gen-
erally with E values>100 and thus furnished free Keywords: amino acids; antibiotics; enzyme cataly-
(S)-configurated arylglycines with high enantiomeric sis; p-hydroxyphenylglycine; kinetic resolution; peni-
purity. The corresponding enantiopure (R)-sub- cillin G acylase
Introduction
are important intermediates in the commercial pro-
duction of semisynthetic b-lactam antibiotics, the (R)-
b-Lactam antibiotics, following the discovery of peni- configurated d-enantiomers of phenylglycine (ampi-
cillin by Fleming in 1929,[1] have revolutionised che- cillin, cefachlor) and p-hydroxyphenylglycine (1a)
motherapy against pathogenic micro-organisms. The (amoxicillin, cefadroxil) being the predominant repre-
rapid spread of bacterial resistance to natural antibi- sentatives (Figure 1). According to WHO data, in
otics accessible by fermentation has stimulated the 2000 ampicillin and amoxicillin together accounted
development of semisynthetic b-lactam derivatives, for almost half of the 45,000 metric tons of b-lactam
which offer a broader spectrum of activity for the antibiotics produced globally.[2,3] Apart from a few iso-
treatment of a wider range of infections. Arylglycines lated examples, all clinically important semisynthetic
Figure 1. Examples of the most important semisynthetic b-lactam antibiotics.
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antibiotics containing optically active side chains are zymes have been well characterised by X-ray crystal
derived from arylglycine (e.g., the ureidopenicillins).[2] structure determination, including those of substrate-
While d-phenylglycine is available on a technical liganded complexes.[20]
scale from benzaldehyde by conventional Strecker
Stimulated by a program to investigate the synthet-
synthesis, resolution with camphorsulphonic acid and ic potential of GA and PGA for asymmetric synthesis
subsequent racemisation of the distomer,[4] (R)-p-hy- and protecting group chemistry,[8] we were interested
droxyphenylglycine [(R)-1a] cannot be prepared by to study the potential of PGA and GA for the resolu-
this route in a cost-competitive way, because p-hy- tion of phenylacetamide and glutaric monoamide de-
droxybenzaldehyde is too expensive.
rivatives of arylglycines and have investigated an op-
More elegantly, (R)-1a is produced by an enzyme- portunity to prepare suitable amino acid derivatives
mediated, dynamic kinetic resolution of 5-(4-hydroxy- by direct amido-alkylation of arenes. Here we report
phenyl)-hydantoin (p-HPH) formed by Mannich-type that enantiopure arylglycines can be produced from
condensation of phenol (2a), glyoxylic acid (3) and readily available bulk chemicals in a simple one-pot
urea.[4] Although a variety of processes (Kaneka, Re- operation with high regioselectivity, and resolved into
cordati, etc.) have been patented, the fundamental both (S)- and (R)-antipodes of high enantiomeric
benefits and drawbacks are the same: Assisted by purity by highly efficient enzyme-mediated kinetic
spontaneous racemisation of the substrate p-HPH, resolution.
two consecutive biocatalytic steps involving (R)-selec-
tive hydantoinases and N-carbamoylases furnish the
enantiopure aminoacid (R)-1a in nearly quantitative Results and Discussion
yield;[5] the major drawbacks are due to an incom-
plete selectivity in the formation of p-HPH by amido- In the industrial hydantoinase process phenol is trans-
alkylation of phenol where a significant amount of re- formed by amido-alkylation with 3 and urea to give
gioisomeric 5-(2-hydroxyphenyl)-hydantoin (o-HPH) racemic arylhydantoins for subsequent resolution to
is also produced and large amounts of mineral acid arylglycines. By analogy, the amido-alkylation of (het-
are needed.[6,7] The moderate regioselectivity of the ero)arenes using a combination of 3 and simple car-
arene substitution results from the elevated tempera- boxylic acid amides under acidic conditions has been
tures applied to maximise the space-time yield. The shown to yield N-acylated aromatic a-amino acids
synthesis of enantiopure arylglycines is currently fur- (Scheme 1).[21] In the course of the reaction, N-acyl
ther limited, particularly on a smaller than industrial
scale, by the lack of suitable (S)-selective hydantoin-
ACHTREUNGases and N-carbamoylases towards the complementa-
ry enantiomeric (S)-arylglycines, or by the need to in-
troduce additional protecting groups to ease kinetic
resolution or subsequent coupling steps.
For an alternative approach, aminopeptidases and
aminoamidases are most prominent amongst the en-
zymes applied for the kinetic resolution of amino
acids.[9] Industrial penicillin G acylase (PGA; EC
3.5.1.11), which is used for the production of b-lactam
intermediates, has a rather narrow specificity for the
phenylacetyl moiety, but accepts a broad spectrum of
phenylacylated substrates and therefore has been ex-
plored extensively in organic synthesis. PGA has been
Scheme 1. Formation of N-acylated arylglycines by amido-
alkylation of arenes.
investigated, e.g., for kinetic resolutions of alcohols[10] hemiaminals are formed which, upon protonation and
and amines,[11] including a-amino acid,[12] b-amino loss of water, give rise to resonance-stabilised carbo-
acid,[13] and g-amino acid derivatives,[14] stereospecific cations that will react with electron-rich arene nucleo-
peptide synthesis,[15] or protecting group operations.[16] philes. By this sequence, N-protected arylglycines
Less attention has so far been paid to PGAꢁs capabili- become accessible in a facile one-pot/two-step synthe-
ty to enantioselectively hydrolyse N-phenylacet- sis. Research activities so far mainly focussed on the
ACHTREUNG
amides of free a-amino acids.[17] For applications of amides of acetic, chloroacetic and acrylic acid, as well
the related industrial glutaryl acylase (GA; EC as on ethyl carbamate.[22] Usually, product mixtures
3.5.1.93), there are few studies only; the enzyme has are obtained with good regioselectivity.[21,22]
recently been found to hydrolyse a variety of amides
In essence, the choice of the acyl moiety in the
and esters enantioselectively,[18] but data concerning starting amide determines the type of N-protection in
the stereoselectivity towards N-acylated amino acids the arylglycine product, and thus the type of appropri-
are scarce.[19] Representatives for both type of en- ate amidase biocatalysts for potential racemate reso-
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One-Pot, Regioselective Synthesis of Substituted Arylglycines
lution, including their respective configurational selec- (Table 1). Highly oxygenated components (e.g., 2e/g)
tivity.[23] Using phenylacetamide (4) or glutaric acid gave dark reaction mixtures with loss of material,
monoamide (5) as starting materials for the formation probably from oxidative side reactions. Initially, reac-
of the respective acyl hemiaminals (6), this procedure tions were run at room temperature, and products
should yield immediate substrates for biocatalytic res- were isolated after solvent removal by simple washing
olution by PGA or GA. In addition, the nature of the the solid remainder with water and ether, except for
acyl substituent will contribute to the efficacy of thymol (2f) where unreacted arene had to be re-
work-up conditions. It was anticipated that for both moved by thorough extraction. The regioselectivity of
reactants the acylated starting material and hydro- the aromatic substitution (2a–h) could be further en-
lysed product should be readily separable by pH-se- hanced by reducing the temperature to 58C. At even
lective phase-switching separation, and that the aro- lower temperatures the reaction mixture becomes
matic nature of the phenylacetyl, or the polar carbox- highly viscous and finally solidifies, prohibiting the
ylate of the glutaryl residues should also facilitate homogeneous dispersion of the acid catalyst. Keeping
product purification by crystallisation.
the temperature of the reaction mixture close to 58C
The N-acyl hemiaminals were prepared by reacting resulted in most cases in the formation of a single re-
the hydrate of 3 with the respective amide in acetic gioisomer only (>99%), as confirmed by HPLC anal-
acid at 508C. Although phenylacetamide (4) is avail- ysis of the crude reaction mixtures and by NMR anal-
able commercially, it is easily prepared by partial hy- ysis of the isolated solids. Only 2a yielded detectable
drolysis of inexpensive benzyl cyanide.[24] The mono- amounts of the ortho-adduct (o-HPG, ~2%), which is
ACHTREUNG
electron-rich arenes were included as benzene and
less reactive derivatives require more drastic condi-
In comparison, the industrially operated hydantoin-
AHCTREaUNG se process results in moderate regioselectivity only
tions that cause a low regioselectivity.[21,22] The cata- with p-HPH/o-HPH ratios not better than 7.2,[7]
lyst of choice proved to be gaseous hydrogen chloride which is due to the need for elevated reaction temper-
as it can easily be removed after the reaction by ap- atures (>408C). The unwanted isomer has to be re-
plying reduced pressure.
moved in an additional step by selective crystallisa-
From phenylacetamide, the respective arylglycines tion of p-HPH. Other preparative methods leading to
(7a–h) were obtained in moderate to excellent yields 5-arylhydantoins, like the Bucherer–Bergs synthesis[25]
Table 1. Preparation of N-acylated arylglycines by amido-alkylation of arenes.[a]
Entry
Acylamide
Arene
Arylglycine
Regioselectivity
Yield [%]
1
2
3
4
5
6
7
8
9
phenylacetamide (4)
4
4
4
4
4
4
4
2a
2b
2c
2d
2e
2f
2g
2h
2c
7a
7b
7c
7d
7e
7f
7g
7h
8c
98%[b]
>99%
>99%
>99%
>99%
>99%
>99%
>99%
>99%
63
39
86
61
36
73
32
88
9
glutarylmonoamide (5)
[a]
One-pot, two-step reactions were performed, first by condensation of acylamide with glyoxylic acid in acetic acid at
508C, then arenes were reacted at 58C using HCl gas as catalyst.
2% ortho-isomer
[b]
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Peter Grundmann and Wolf-Dieter Fessner
or amido-alkylation with 5-bromohydantoin,[6] are tive to intramolecular attack by the terminal carboxyl-
hampered by requiring expensive or highly toxic start- ate group. No further efforts were invested for opti-
ing materials (e.g., substituted carbaldehydes, HCN or misation.
bromine).
For a determination of the acylase enantioselectivi-
From a corresponding series of reactions with re- ty in the racemate resolution of selected N-acylated
agent 5 expected to yield N-glutaryl-arylglycines, only arylglycines (Scheme 3, Table 2) the influence of the
the 3,4-dimethoxyphenyl derivative 8c could be isolat- reaction medium was also investigated as, like for
ed from the reaction mixture, however, in a disap- many enzymes, PGA acitivity and selectivity have
pointingly low yield (Scheme 2). Presumably, this in- been shown to be remarkably influenced by the addi-
efficiency is due to the instability of the N-glutaryl- tion of small amounts of organic cosolvent.[26] The in-
ACHTREUNGamidoglycolic acid (6) intermediate under the acidic fluence of organic cosolvents on the enantioselectivity
reaction conditions, as the amide bond may be sensi- of enzymes is thought to originate mainly from two
Scheme 2. Inefficient formation of N-glutaryl-arylglycines.
Scheme 3. Kinetic resolution of N-acylated arylglycines.
Table 2. PGA mediated hydrolysis with/without cosolvent.[a]
Compound
Enzyme
PGA
Medium
Conversion [%]
eeS [%]
E
7a
H2O
5% MeOH
H2O
5% MeOH
H2O
5% MeOH
H2O
5% MeOH
phosphate buffer
buffer + 5% MeOH
49.6ꢁ0.3
50.9ꢁ0.3
50.7ꢁ0.3
50.2ꢁ0.3
50.4ꢁ0.2
50.5ꢁ0.2
50.3ꢁ0.2
49.8ꢁ0.2
50.6ꢁ0.2
48.4ꢁ0.2
95.5ꢁ0.1
95.1ꢁ0.1
96.1ꢁ0.1
96.1ꢁ0.1
97.1ꢁ0.1
97.8ꢁ0.1
98.2ꢁ0.1
97.6ꢁ0.1
90.3ꢁ0.1
84.0ꢁ0.1
>100 (256)
87
7b
7c
7d
8c
PGA
PGA
PGA
GA
>100 (117)
>100 (166)
>100 (187)
>100 (214)
>100 (313)
>100 (547)
49
48
[a]
Data based on HPLC analysis; average values from three independent determinations each. PGA reactions performed at
room temperature, GA reaction at 378C using purified soluble enzymes. Absolute configurations of the products are re-
lated to an authentic sample of (S)-1a.
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One-Pot, Regioselective Synthesis of Substituted Arylglycines
distinct effects.[27] Firstly, water acts as a molecular lu- the aqueous phase in high yield by evaporation, fol-
bricant allowing for a high degree of conformational lowed by extraction of the solid remainder using boil-
flexibility; addition of cosolvent rigidifies the enzyme ing EtOH and recrystallisation. The enantiopure free
structure by conserving hydrogen bonds. This reduces (R)-1a was obtained from the EtOAc extraction
the ability of complex formation between enzyme and phase by acidic hydrolysis with 1M HCl and subse-
the less favoured enantiomer, resulting in an im- quent recrystallisation from 1-propanol.
proved enantioselectivity. The second effect concerns
Due to the unsatisfactory results in the amido-alky-
the desolvation when a substrate traverses from hy- lation with 5, an investigation of the GAꢁs resolving
drophilic reaction medium to the more hydrophobic capability was only of general interest. The effect of
active site; a less polar medium lowers the barrier be- organic cosolvents on GA enantioselectivity has so
tween bulk phase and the enzymeꢁs catalytic centre, far only been investigated for the hydrolysis of ester,
resulting in a higher contribution of the velocity of but not amide functionalities.[29] We found that, in
enzyme-substrate complex formation relative to the contrast to PGA, addition of small concentration of
total reaction velocity.
organic cosolvent to the reaction medium had no sig-
For PGA reactions with 7a–d, water and addition nificant effect on the kinetic enantiomer differentia-
of 5% MeOH were chosen, while the pH was kept tion of 8c by GA. The enantioselectivity found is ac-
constant at the enzymeꢁs optimum of pH 8.0 by auto- ceptable, but clearly inferior to that of PGA, for the
mated titration with 0.1M Na2CO3. Due to the limited generation of optically pure amino acids.
quantity of 8c and the small scale, hydrolytic reactions
with GA were carried out in 0.1M sodium phosphate
buffer for convenience and compared to solvent in- Conclusions
cluding 5% MeOH. For PGA-mediated hydrolysis,
conversion and enantiomeric excess were determined The amido-alkylation of aromatic hydrocarbons with
simultaneously by HPLC analysis on lyophilised sam- phenylacetamide and glyoxylic acid offers a facile and
ples by relating the peaks obtained for phenylacetic inexpensive route to a large variety of arylglycines.
acid and for both enantiomers of the respective N- Low temperatures yield almost exclusively one regio-
phenylacetyl-arylglycine to calibration charts record- isomer and work-up procedures are in most cases re-
ed independently. For the GA-mediated hydrolysis of duced to a simple washing protocol. Kinetic resolu-
8c conversion and ee were determined from separate tion of the free amino acids with PGA, a readily
analyses. The enantiomer selectivity E was calculated available, robust biocatalyst, furnishes the phenylace-
from the conversion (c) and ee values of the substrate tylated (R)-7a–d and free (S)-1a–d amino acids, re-
as determined by HPLC analysis.[28] E is defined as spectively, with high enantiomeric purity; substrate
and product can easily be separated by a phase-selec-
tive extraction process. This result is of special inter-
est with respect to the fact that the synthesis of (S)-ar-
ln½ð1ÀcÞð1ÀeeSފ
E=ln½ð1ÀcÞ<ð1þee ފ
.
S
All PGA resolutions gave high enantioselectivity ylglycines by the hydantoinase process has not yet
ratios that allow an efficient access to both enantio- been implemented on an industrial scale. By adding a
pure antipodes of aryl glycines. PGA enantioselectivi- suitable N-acylamino acid racemase[9c,30] to the resolu-
ty clearly increases with the steric bulk of the aryl tion process even quantitative yields would be feasi-
substituent, i.e., 2,4-dimethoxyphenyl>3,4-dimeth- ble by dynamic kinetic resolution. The corresponding
ACHTREUNGoxyphenyl>thienyl. Remarkably, the uncharged aryl- (R)-enantiomers can easily be obtained by subsequent
glycines (1b–d) were resolved with higher E values acidic or enzymatic hydrolysis of the corresponding
with 5% MeOH as a cosolvent, in good accordance phenylacetylamides. Our method of arylglycine syn-
with prior observations on amine resolutions.[26] Reso- thesis, resolution and work-up procedure requires a
lution of the (p-hydroxyphenyl)-glycine derivative minimum of equipment and therefore grants rapid
(7a) using PGA proved to be more efficient without access to enantiopure non-natural a-amino acids in
cosolvent, which may be due to the fact that the phe- small- to large-scale quantities.
nolic OH group is deprotonated at pH 8.0, the mole-
The corresponding reaction pathway using glutaric
cule thus bearing an additional negative charge. Com- monoamide as starting material is apparently ham-
pounds 7e–h have not been tested in this study but pered by the intermediateꢁs instability and resulted in
can be assumed to be amenable to kinetic differentia- low yields. Also, GA seems to be a less powerful cata-
tion with similar efficiency.
lyst for the resolution of arylglycines than PGA.
In a preparative experiment performed on a gram-
scale, enzymatic hydrolysis of 7a was stopped by
acidification, after which unreacted 7a and phenylace-
tic acid were removed by selective extraction with
EtOAc. Enantiopure free (S)-1a was recovered from
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MeCN=95:5, flow rate 1.2 mLminÀ1, l=254 nm (tR: 1c=
3.4 min; 8c=4.8 min). The ee was determined on a Chiracel
IB column as described for the N-phenylacetyl arylglycines
(tR: 8c=19.7, 21.8 min).
Experimental Section
General Procedure for the Synthesis of N-Phenyl-
acetyl-arylglycines
A 50% aqueous solution of glyoxylic acid (3, 37.00 g, 0.25
mol) was concentrated to 80% acid content under reduced
pressure. The syrup was dissolved in acetic acid (60 mL),
and phenylacetamide (4; 33.80 g, 0.25 mol) was added to the
solution. The mixture was kept at 508C for 5 h, then cooled
to 58C and the aromatic component added. A gentle stream
of dry hydrogen chloride was fed into the solution with stir-
ring for 1 h, making sure that the temperature did not
exceed 108C. The mixture was kept at 58C for 16 h, then
the volatile compounds were removed under reduced pres-
sure by gradually rising the temperature to 708C. The re-
mainder was triturated in cold water. The solid was collect-
ed by filtration and thoroughly washed with cold water,
Et2O and dried under vacuum.
Preparative Scale Kinetic Resolution of Racemic
N-Phenylacetyl-(4-hydroxyphenyl)-glycine
Racemic 7a (1.00 g, 3.5 mmol) was dissolved in H2O
(20 mL), the pH adjusted to 8.0 and the reaction started by
addition of PGA (200 mL, 228 U). The pH was kept constant
by automatic titration of 0.1M Na2CO3. After 20 min the
pH was adjusted to 3.0 with 2.0M HCl, the denatured
enzyme removed by filtration and the total volume adjusted
to 50 mL with H2O. The solution was extracted with EtOAc
(3”50 mL), the organic phases combined, dried over
Na2SO4, and the solvent removed under reduced pressure.
The composition of the obtained solid (0.71 g) was exam-
ined by HPLC and found to be phenylacetic acid and pure
(R)-N-phenylacetyl-d-(4-hydroxyphenyl)-glycine
(R)-7a.
The free (R)-amino acid was obtained by hydrolysing the
solid with 1M HCl (50 mL) at 508C for 10 h. The solution
was evaporated to dryness and the remainder recrystallised
from 1-propanol to afford enantiopure (R)-1a; yield: 0.27 g
(93%).
Free (S)-1a was recovered from aqueous phase after the
extractions by evaporation, and extraction of the solid re-
mainder with EtOH under reflux (3”25 mL). The liquid ex-
tracts were combined and evaporated to yield 0.26 g (S)-1a
with 91% ee (89%). Recrystallisation from 1-propanol yield-
ed enantiopure (S)-1a.
Determination of Conversion and Enantioselectivity
Hydrolytic reactions were performed at room temperature
using a Titroline Alpha automatic titrator (Schott) in the
pH-Stat mode. Spontaneous chemical hydrolysis was evalu-
ated by monitoring the pH of the reaction solutions 10 min
prior to enzyme addition and was found to be negligible.
PGA catalysis: the substrate (200 mg) was dissolved in
H2O (or in an aqueous solution containing 5% of MeOH;
total volume 20 mL), and reactions were started by addition
of 25 mL (1140 U/mL) of PGA suspension. Reaction solu-
tions were gently stirred at room temperature in an open
flask maintaining the pH constant at 8.0 by automated addi-
tion of 0.1M Na2CO3. Samples of 200 mL were withdrawn
after 30 min, mixed with acetonitrile (400 mL) to stop the en-
zymatic hydrolysis, and the solution was lyophilised immedi-
ately.
Supporting Information
Full details of methods and materials as well as the charac-
terisation data for new compounds are available as Support-
ing Information.
GA catalysis: the substrate (20 mg) was dissolved in 0.1M
sodium phosphate buffer (with/without 5% MeOH; total
volume 2 mL), and reactions were started by addition of
10 mL (368 U/mL) of GA suspension. Reaction solutions
were shaken at 600 rpm and 378C in an Eppendorf Thermo-
mixer. Samples (200 mL) were withdrawn after 15 min,
mixed with acetonitrile (400 mL), and the solution was
lyophilised immediately.
Acknowledgements
We gratefully acknowledge Dr. Peter Rasor from Roche Di-
agnostics (Germany) for generous gifts of penicillin G ami-
dase and glutaryl acylase.
HPLC analysis: lyophilised samples were dissolved by ad-
dition of EtOH (100 mL), TFA (2 mL), CHCl3 (200 mL) and
hexane (400 mL), centrifuged at 12,000 ” g for 1 min and References
submitted to HPLC analysis. For the N-phenylacetyl-arylgly-
cines the conversion and ee were determined simultaneously
[1] A. Fleming, Br. J. Exp. Pathol. 1929, 10, 226–236.
by using a Chiracel IB column (250”4.6 mm), eluent
[2] A. Bruggink, P. D. Roy, in: Synthesis of b-Lactam Anti-
biotics, (Ed.: A. Bruggink), Kluwer, Dordrecht, 2001,
pp 12–54.
[3] R. P. Elander, Appl. Microbiol. Biotechnol. 2003, 61,
385–392.
[4] M. A. Wegman, M. H. A. Janssen, F. van Rantwijk,
R. A. Sheldon, Adv. Synth. Catal. 2001, 343, 559–576.
[5] K. Yokozeki, K. Kubota, Agric. Biol. Chem. 1987, 51,
721–728.
hexane-CHCl3-EtOH-TFA=66:30:4:0.1,
flow
rate
1.0 mLminÀ1, l=265 nm [tR: 7a=19.8 (R)-enantiomer, 21.5
(S)-enantiomer; o-hydroxyphenylglycine=6.3, 6.6; 7b=6.7,
7.2; 7c=8.9, 9.5; 7d=6.5, 7.8; phenylacetic acid=4.9 min].
The degree of conversion was determined by comparing the
ratio of the peaks obtained for phenylacetic acid to the sum
of both enantiomers of N-phenylacetylarylglycines 7a–d
with the aid of calibration charts.
Conversions of the N-glutarylacylated arylglycine was de-
termined by using a thermostated (308C) Partisil 5C8
column (250”4.6 mm), eluent 0.1M NH4OAc (pH 5.2)-
[6] C. Cativiela, J. M. Fraile, J. I. Garcia, G. Lafuente, J. A.
Mayoral, R. Tahir, A. Pallares, J. Catal. 2004, 226, 192–
196.
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