Quantitative enzymatic protection of D-amino acid methyl esters by exploiting 'relaxed' enantioselectivity of penicillin-G amidase in organic solvent

Article abstract of DOI:10.1016/j.tetlet.2004.10.153

A quantitative method for enzymatic protection of esters of various d-amino acids in organic solvent is reported. The lower enantioselectivity displayed by penicillin-G amidase (PGA) from E. coli in organic solvent has been exploited for developing a facile, fast and quantitative method for protection of esters of various d-amino acids via N-acylation. The feasibility of the deprotection of the acylated products was also demonstrated by employing PGA from two different sources in aqueous media. Experimental results are in agreement with previous calculations based on in silico models of the enzyme active site.

Full text of DOI:10.1016/j.tetlet.2004.10.153

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 9649–9652
Quantitative enzymatic protection of D-amino acid
methyl esters by exploiting ÔrelaxedÕ enantioselectivity
of penicillin-G amidase in organic solvent
Chiara Carboni,a,b Peter J. L. M. Quaedflieg,b Quirinus B. Broxterman,b
Paolo Lindaa and Lucia Gardossia,*
a
`
Dipartimento di Scienze Farmaceutiche, Universita di Trieste, Piazzale Europa 1, 34127 Trieste, Italy
bDSM Research, Life Sciences—Advanced Synthesis, Catalysis and Development, PO Box 18, 6160 MD Geleen, The Netherlands
Received 6August 2004; revised 26October 2004; accepted 27 October 2004
Available online 11 November 2004
Abstract—The lower enantioselectivity displayed by penicillin-G amidase (PGA) from E. coli in organic solvent has been exploited
for developing a facile, fast and quantitative method for protection of esters of various D-amino acids via N-acylation. The feasibility
of the deprotection of the acylated products was also demonstrated by employing PGA from two different sources in aqueous media.
Experimental results are in agreement with previous calculations based on in silico models of the enzyme active site.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The application of biocatalysts in peptide chemistry of-
fers several important advantages since stereo-, regio-
and chemo-selective reactions take place under very
mild conditions, so that the use of toxic and expensive
chemicals for activation and (de-)protection are corre-
spondingly reduced as well as operational hazards.1,2
The methodology has not been fully exploited for possi-
ble synthesis of a number of biologically important pep-
tides containing D-amino acids or other nonproteogenic
amino acid derivatives. Antibiotic peptides, synthetic
peptides of enhanced hormonal or neuronal activity
and many prodrugs used in chemotherapy often contain
D-amino acid residues.3 Recombinant DNA technology
is generally limited to the production of peptides con-
taining only L-amino acids as, in general, they have been
approached by protease catalysis.1
deprotection of the amino function in aqueous solution
at ambient temperature without affecting other bonds or
reactive groups.
The 3-D structure of PGA is well known and crystallo-
graphic data are accessible6,7 which indicate how the enz-
yme presents a highly selective acylic sub-site for the
phenylacetic group and an aminic sub-site characterised
by a quite wide tolerance for substrates. The aminic sub-
site, however, is responsible for enantio-discrimination,8
so that the enzyme has been largely applied also in resol-
ution of aliphatic alcohols,9 amines10,11 and especially
amino acid derivatives.12 In the case of amino acids
and their derivatives, the L-enantiomer is the favourite
substrate, both in acylation reaction13 and in the hydro-
lysis of the corresponding N-acylated compounds.14
Protection and deprotection strategies are one of the
crucial factors in determining the successful accomplish-
ment of any peptide synthesis. Penicillin-G amidase
(PGA) has become one of the enzymes of choice for pro-
tection/deprotection of amino groups2,4,5 due to its high
selectivity towards the phenylacetic group enabling the
For instance, an enantioselectivity ratio (E value) of 280
was reported for the PGA mediated hydrolysis of N-
phenylacetyl-AlaOMe and an E value of >1000 for
N-phenylacetyl-PhGly.14 Furthermore, it was demon-
strated that D-TyrOEt is acylated only with unaccept-
ably slow rate (7% after 44h) even in the presence of
an organic co-solvent.15 Due to this strict L-enantioselecti-
vity of PGA in aqueous media the N-protection of D-ami-
no acids derivatives by PGA has never been disclosed.
Keywords: D-Amino acids; Penicillin-G amidase; Protection of amino
groups; Organic solvent; Biocatalysis.
*
Here we report, for the first time, on the PGA catalysed
acylation of a series of D-amino acids esters in organic
Corresponding author. Tel.: +39 040558310; fax: +39 04052572;
e-mail: gardossi@units.it
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.10.153

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C. Carboni et al. / Tetrahedron Letters 45 (2004) 9649–9652
O
R
O
OCH3
OCH3
R
PGA-450
NH
+
OCH3
O
organic solvent
O
NH2
Scheme 1. N-acylation of D-amino acids esters catalysed by penicillin-G amidase.
solvent. The ability of PGA to accept the unfavoured D-
enantiomer is related to a modification of the enantiose-
lectivity of PGA in the presence of nonaqueous media.
The use of an organic medium in enzymatically cata-
lysed acylations not only causes a ÔrelaxationÕ of enzyme
enantioselectivity but also prevents undesirable hydro-
lytic side reactions, so that equimolar amounts of acylat-
ing agents can be used. As a matter of fact, acylation in
aqueous media requires the use of an excess of one of the
reactants or extraction or precipitation of the prod-
uct11,12,15 to reverse the equilibrium towards synthesis.
Although the presence of water is essential for preserv-
ing the activity of PGA, we have earlier demonstrated
that PGA is active in various organic solvents as long
as the water activity (aw) of the reaction system is main-
tained above 0.44,16,17(Scheme 1).
with 1.0equiv of methyl phenylacetate in the presence
of immobilised PGA-450 (2g/mmol substrate); the
activity of the enzyme was assured by controlling the
water activity (aw = 0.73–0.77) of the medium accord-
ing to methods previously described.17 As evident from
Table 1, both aliphatic and aromatic substrates
were quantitatively converted in a fairly short time
frame.21,22
The complete conversion and the absence of side prod-
ucts allowed for the immediate isolation of the pure
N-acylated D-amino acids methyl esters (in isolated
yields ranging from 75% to 95%) upon removing of
the enzyme by filtration and evaporation of the solvent.
At the values of water activity employed for the enzy-
matic acylation (aw = 0.73–0.77) no hydrolytic reaction
was observed.
In our previous study18 concerning PGA mediated acyl-
ations in toluene we demonstrated that PGA is suffi-
ciently L-enantioselective in this medium to resolve
racemic PhGlyOMe with ee > 98%. However, kinetic
measurements carried out on the separate enantiomers
showed that the acylation rate for the D-enantiomer
was not negligible, being 0.25lmolhÀ1 UÀ1 as compared
to 4.58lmolhÀ1 UÀ1 determined for the L-enantiomer.
More evidently, in another study concerning PGA enan-
tioselectivity in toluene we reported an ee as low as 24%
in the resolution of D,L-AlaOMe.8
It must be noted that previous studies in aqueous media
reported no reactivity of PGA towards D-Val.13 Fur-
thermore, in the specific case of the unnatural amino
acid PhGly, of which the L-enantiomer is accepted by
PGA both as nucleophile and as acyl donor, it was pre-
viously demonstrated that the D-enantiomer was
accepted as acyl donor in aqueous media but not at all
as nucleophile.23 This is also the reason why during
the PGA mediated synthesis of the antibiotics Cepha-
lexin and Ampicillin, wherein D-PhGly esters or amides
are used as acylating agents, the corresponding D,D-
dipeptides are not formed whatsoever.
These results, when compared to the data obtained by
others in aqueous or partly aqueous media, prompted
us to investigate the generality of this ÔrelaxedÕ L-enan-
tioselectivity of PGA in organic solvent. Several studies
reported the variation or even inversion of enzyme selec-
tivity upon changing the reaction medium. According to
most authors the influence of the reaction medium on
enzyme selectivity can be ascribed to three different fac-
tors: (i) the alteration of the enzyme–substrate recogni-
Table 1. Protection of various D-amino acids methyl esters catalysed
by (immobilised) PGA-450 in toluene or dichloromethane at
aw = 0.73–0.77 using methyl phenylacetate as acyl donor
Entrya Substrate
Solvent
T
Time for
(ꢁC) complete
conversion (h)
tion process due to
a variation in the enzyme
conformation; (ii) the binding of solvent molecules in-
side the active site and (iii) altered energetics of substrate
solvation.19
1
2
3
4
5
6
7
8
D-SerOMe
D-ValOMe
D-AlaOMe
D-LeuOMe
D-TyrOMe
D-PheOMe
Dichloromethane 30
24
Toluene
Toluene
Toluene
Toluene
Toluene
30
30
30
40
30
30
50
24
24
48
24
72
72
24b
We first investigated whether D-amino acids methyl es-
ters20 could be smoothly and quantitatively acylated
using immobilised PGA (PGA-450) and methyl phenyl-
acetate as the acyl donor.
D-PhGlyOMe Toluene
D-PheOMe Toluene
a Reaction conditions: 1mL of organic solvent, 200mg PGA-450,
100lmol of D-amino acids methyl ester, 100lmol of methyl
phenylacetate.
To this end a structurally versatile series (Table 1) of
aliphatic and aromatic D-amino acids methyl esters
(0.1M solution in dry organic solvent) were reacted
b Another portion of fresh enzyme (200mg) was added after 6h.

C. Carboni et al. / Tetrahedron Letters 45 (2004) 9649–9652
9651
The results in Table 1 also clearly indicate that the acyl-
Acknowledgements
ation of the aromatic D-amino acids methyl esters is
slower. This is in agreement with the structural basis
of PGA enantio-discrimination which was reported in
our previous study.8 Computational investigation of
the PGA active site demonstrated that in the case of
the bulkier aromatic amino acids the stabilising effect
of H-bonds between the acyl group and the PGA aminic
sub-site effectively occurs only with the L-enantiomer,
thus inducing enantio-discrimination. This is not the
case with the smaller aliphatic amino acids, where both
enantiomers can be accommodated inside the aminic
sub-site so to acquire a conformation able to engage sta-
bilising H-bonds with the active site. Thus, these theo-
retical calculations excellently explain the much lower
reactivity of the aromatic D-amino acids compared to
the aliphatic ones.
We are grateful to Boehringer–Mannheim for the gener-
ous gift of PGA-450. We further greatly acknowledge
Professor Cynthia Ebert and Dr. Alessandra Basso for
useful discussions and Mr. Luca Boscarol for the prep-
aration of the D-amino acids methyl esters.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet-
let.2004.10.153. Materials for chemical synthesis; meth-
ods for enzyme dehydration as also reported in Ref.
17; procedure for the determination of enzyme activity
as also described in Ref. 17; procedure for the determin-
ation of the water activity as also described in Ref. 17;
chemical synthesis of the D-amino acids methyl esters
starting from the corresponding D-amino acids, as also
described in Ref. 20; preparation of the free amines
starting from the corresponding hydrochloride salts.
The difference in activities cannot be ascribed simply to
solubility differences of the substrates since, for instance,
D-SerOMe, which is the most polar and least soluble
among the substrates considered, is completely acylated
in 24h even in dichloromethane, which had been demon-
strated to be a less efficient solvent than toluene.17 Final-
ly, early studies on PGA had already demonstrated that
the lower reactivity of the D-enantiomer was not ascrib-
able to an inhibitory effect on PGA but only to kinetic
factors.15
References and notes
1. Jakubke, H. D.; Kuhl, P.; Ko¨nnecke, A. Angew. Chem.,
Int. Ed. Engl. 1985, 24, 85–93.
2. (a) Waldmann, H.; Sebastian, D. Chem. Rev. 1994, 911–
937, and references cited therein; (b) Fuganti, C.; Gras-
selli, P.; Casati, P. Tetrahedron Lett. 1986, 27, 3191–3194.
3. Kreil, G. Annu. Rev. Biochem. 1997, 66, 337–345, and
references cited therein.
It should be noted that in the case of D-TyrOMe and
D-PheOMe the higher enantioselectivity of PGA for the
L-enantiomers can be partially overcome by speeding
up the enzymatic acylation through a temperature incre-
ase to 40 or 50ꢁC, respectively.
4. Basso, A.; Biffi, S.; De Martin, L.; Ebert, C.; Gardossi, L.;
Linda, P. Croat. Chem. Acta 2001, 74, 757–762.
5. Didzˇiapetris, R.; Drabnig, B.; Schellenberger, V.;
Secondly, we investigated whether N-phenylacetyl-D-
amino acids methyl esters could also be smoothly and
quantitatively deprotected in aqueous solution using
immobilised PGA from both E. coli (PGA-450) and A.
faecalis. This latter amidase was selected because of
the very high specific activity of its immobilised form
(1390U/g compared to 155U/g of PGA-450) in order
to increase the very low reaction rates which were ex-
pected for the hydrolytic reactions in aqueous medium.
Furthermore, recent computational studies pointed out
some important structural differences at the level of
the aminic sub-site which induce to expect a wider sub-
strate tolerance.24 For the substrates tested (N-phenyl-
acetyl-D-AlaOMe and N-phenylacetyl-D-PheOMe) the
deprotection in buffered aqueous solution was complete
within 24h and 48h, respectively, demonstrating the
practical potential of the N-phenylacetyl protecting
group for D-amino acids, for instance in peptide
synthesis.25
ˇ
Jakubke, H.-D.; Svedas, V. K. FEBS 1991, 287, 31–33.
6. Duggleby, H. J.; Tolley, S. P.; Hill, C. P.; Dodson, E. J.;
Dodson, G.; Moody, P. C. E. Nature 1995, 373, 264–268.
7. McVey, C. E.; Walsh, M. A.; Dodson, G. G.; Wilson, K.
S.; Brannigan, J. A. J. Mol. Biol. 2001, 313, 139–150.
8. Basso, A.; Braiuca, P.; Clementi, S.; Ebert, C.; Gardossi,
L.; Linda, P. J. Mol. Catal. B: Enzym. 2002, 19–20, 423–
430.
9. Waldmann, H. Tetrahedron Lett. 1989, 30, 3057–3058.
10. Rossi, D.; Calcagni, A.; Romeo, A. J. Org. Chem. 1979,
44, 2222–2225.
11. Zmijewski, M.; Briggs, B. S.; Thompson, A. R.; Wright, I.
G. Tetrahedron Lett. 1991, 32, 1621–1622.
12. (a) Bossi, A.; Cretich, M.; Righetti, P. G. Biotechnol.
Bioeng. 1998, 60, 454–461; (b) Fernandez-Lafuente, R.;
Rosell, C. M.; Guisan, J. M. Enzyme Microb. Technol.
1998, 22, 583–587.
ˇ
13. Didzˇiapetris, R.; Svedas, V. Biomed. Biochim. Acta 1991,
10/11, 237–242.
ˇ
14. Svedas, V. K.; Savchenko, M. V.; Beltser, A. I.; Gurunda,
D. F. Ann. N. Y. Acad. Sci. 1996, 799, 659–669.
15. Pessina, A.; Luthi, P.; Luisi, P. L. Helv. Chim. Acta 1988,
71, 631–641.
16. Basso, A.; De Martin, L.; Ebert, C.; Gardossi, L.; Linda,
P. J. Mol. Catal. B: Enzym. 1999, 6, 437–445.
17. Basso, A.; De Martin, L.; Ebert, C.; Gardossi, L.; Linda,
P.; Zlatev, V. J. Mol. Catal. B: Enzym. 2001, 11, 851–855.
18. Basso, A.; Braiuca, P.; De Martin, L.; Ebert, C.; Gardossi,
L.; Linda, P. Tetrahedron: Asymmetry 2000, 11, 1789–
1796.
In conclusion, a fast, efficient and versatile method was
developed for the protection of D-amino acids esters by
exploiting the lower enantioselectivity displayed by
PGA in organic media. The procedure is of practical
importance due to the absence of hydrolytic side prod-
ucts and the easy work-up. The results obtained are in
agreement with previously reported theoretical
calculations.

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C. Carboni et al. / Tetrahedron Letters 45 (2004) 9649–9652
19. Ottolina, G.; Riva, S. In Methods in Non-aqueous Enzy-
mology; Gupta, M. N., Ed.; Birkhauser: Basel, 2000; pp
133–145.
20. (a) Quaedflieg, P. J. L. M.; Boesten, W. H. J. Process for
Esterification of Amino acids and Peptides. Eur. Patent
Appl. EP977726, 1998; (b) Basso, A.; De Martin, L.;
Ebert, C.; Gardossi, L.; Linda, P. J. Mol. Catal. B: Enzym.
2001, 16, 73–80.
21. Materials and methods: PGA-450 was a generous gift of
Boehringer–Mannheim. It consists of penicillin-G amidase
from E. coli covalently immobilised on a polymer the
chemical nature of which is not disclosed by the producer.
The PGA-450 used in the present study had a water
content of 62wt% (determined by Karl–Fischer titration)
and an activity, before partial dehydration, of 155U/g
corresponding to 408U/gdry (please refer to supplementary
data and Ref. 17). For the N-acylation reactions, the
PGA-450 was partially dehydrated before use with the aid
of Celite R-640ꢂ as previously described.17 The PGA from
A. faecalis, covalently immobilised on a polymer support
the chemical nature of which is not disclosed, was from
DSM Anti-Infectives (Delft, The Netherlands). The enzy-
matic activity was 1390U/g. 1H NMR spectra were
(2H, d, –CH2Ph), 3.6(3H, s, –OCH 3), 4.5 (1H, m,
–CHNH), 7–7.4 (10H, m, 2 · Ph), 8.6(1H, d, –NHCO).
PhAc-D-PhGlyOMe: 3.55 (2H, s, –CH2Ph), 3.6(3H, s,
–OCH3), 3.7 (1H, d, –CH–), 7–7.5 (10H, m, 2 · Ph),
9.0 (1H, d, –NH–). PhAc-D-LeuOMe: 0.9 (6H, m,
–CH(CH3)2, 1.3 (1H, m, –CH(CH3)2), 1.5 (2H,
m, CH2CH(CH3)2), 3.45 (2H, d, –CH2Ph), 4.2 (1H, m,
–CHNH), 7.35 (5H, m, –Ph), 8.5 (1H, d, –NHCO). PhAc-
D-SerOMe: 3.25 (2H, m, –CH2OH), 3.5 (2H, d,
–CH2Ph), 3.65 (3H, s, –OCH3), 4.4 (1H, dd, –CHNH),
7.35 (5H, m, –Ph), 8.4 (1H, d, –NHCO). PhAc-D-
ThrOMe: 1.1 (3H, d, –CH3), 3.6(3H, s, –OCH 3), 3.8
(2H, s, –CH2Ph), 4.3 (1H, t, –CH), 4.5 (1H, m, –CHOH)
7–7.5 (5 H, m, Ph), 8.0 (1H, s, –OH), 8.5 (1H, d, –NH–).
PhAc-D-TyrOMe: 2.78 (2H, m, –CH2Ph), 3.45 (2H, d,
–CH2Ph), 3.6(3H, s, –OCH 3), 4.4 (1H, m, –CHNH), 6.45–
7.4 (9H, m, 2 · Ph), 8.45 (1H, d, –NHCO), 9.2 (1H, s, –OH).
ˇ
23. van Langen, L. M.; van Rantwijk, F.; Svedas, V. K.;
Sheldon, R. A. Tetrahedron: Asymmetry 2000, 11, 1077–
1083.
24. Braiuca, P.; Ebert, C.; Fischer, L.; Gardossi, L.; Linda, P.
Chem. Biochem. 2003, 4, 615–622.
25. PGA catalysed hydrolysis of PhAc-D-AlaOMe and PhAc-D-
PheOMe: 200mg of PGA-450 (from E. coli) or 200mg of
PGA from A. faecalis were added to a suspension of the
N-acylated D-amino acids methyl ester (100mg) in 5mL aq
sodium phosphate buffer (50mM, pH = 8.0) at 30ꢁC. The
reactions were monitored by TLC (eluent: 1-butanol/acetic
acid/ethyl acetate/water 1:1:1:1 (v/v/v/v)). For PhAc-D-
AlaOMe the deprotection reaction was complete after 24h
using both enzymes (product detectable already after
10min with PGA from A. faecalis and after 1h with PGA
from E. coli). After 24h, the reaction mixtures were
filtrated and the filtrates evaporated in vacuo and analysed
by NMR confirming the complete deprotection into D-
AlaOMe. In the case of PhAc-D-PheOMe the solubility in
water was considerably lower, so that an emulsion was
obtained and the deprotection reaction took longer time.
After 24h both reactions were not complete and a new
portion of the enzymes (200mg) was added and the
temperature increased to 40ꢁC. After 24h of further
stirring the deprotections were complete as was also
confirmed by NMR.
recorded at 200MHz using
spectrometer.
a Varian–Gemini 200
22. N-acylation of D-amino acids methyl esters using PGA-450:
PGA-450 (200mg) was suspended in toluene or CH2Cl2
(1mL) and the system was equilibrated for 24h
(aw = 0.73–0.77) at 30ꢁC. Subsequently, the D-amino acids
methyl ester (100lmol of free base) and methyl phenyl-
acetate (100lmol) were added. The course of the reactions
was monitored with TLC (eluent: EtOAc/n-hexane 7:3 (v/
v)). After full conversion of the D-amino acids methyl ester
and methyl phenylacetate to the N-phenylacetyl-D-amino
acids methyl esters (see Table 1), the enzyme was filtered
off and the organic solvent evaporated in vacuo. 1H NMR
(DMSO-d6), d (ppm): PhAc-D-ValOMe: 0.9 (6H, m,
–CH(CH3)2), 2.0 (1H, m, –CH(CH3)2), 3.5 (2H, d, –CH2
Ph), 3.6(3H, s, –OCH 3), 4.2 (1H, dd, –CHNH), 7.35 (5H,
m, –Ph), 8.4 (1H, d, –NHCO). PhAc-D-AlaOMe: 1.3 (3H,
d, –CH3), 3.4 (2H, d, –CH2Ph), 3.6(3H, s, –OCH ), 4.3
3
(1H, dd, –CHNH), 7.25 (5H, m, –Ph), 8.55 (1H, d,
–NHCO). PhAc-D-PheOMe: 3.0 (2H, m, –CH2Ph), 3.4