Penicillin G amidase-catalysed hydrolysis of phenylacetic hydrazides on a solid phase: A new route to enzyme-cleavable linkers

Article abstract of DOI:10.1002/adsc.200505038

A novel catalytic property of penicillin G amidase (PGA) is described. Unexpectedly, the enzyme can hydrolyse hydrazide bonds with good efficiency, and in solution the enzyme shows a selectivity that is similar to phenylacetamides. The hydrolysis of phenylacetic hydrazides releases hydrazine, but no inhibition due to the formation of such reactive compounds was observed. This novel catalytic property was assayed also on a solid phase as a pioneering route for the design of enzyme-cleavable linkers and masked scavengers for ketones. On a solid phase a phenylacetic hydrazide compound was chemically synthesised on PEGA₁₉₀₀ and PEGA⁺ (two co-polymers of acrylamide and ethylene glycol) and the efficiency of PGA in the release of phenylacetic acid depended on the diffusion of the protein inside the polymer. On PEGA⁺ the enzyme, as previously described, shows a good diffusion due to an improved electrostatic interaction with PGA thus achieving good hydrolytic conversions.

Full text of DOI:10.1002/adsc.200505038

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Penicillin G Amidase-Catalysed Hydrolysis of Phenylacetic
Hydrazides on a Solid Phase: A New Route to
Enzyme-Cleavable Linkers
Alessandra Basso,a Cynthia Ebert,a Lucia Gardossi,a,* Paolo Linda,a
Thao Tran Phuong,b Mingzhao Zhu,b Ludger Wessjohannb,*
a
`
Laboratory of Applied and Computational Biocatalysis, Dipartimento di Scienze Farmaceutiche, Universita degli Studi,
Piazzale Europa 1, 34127, Trieste, Italy
Phone: (þ39)-040-558-3110, Fax: (þ39)-040-52572, e-mail: gardossi@units.it
Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany
Phone: (þ49)-345-5582-1301, Fax: (þ49)-345-5582-1309, e-mail: wessjohann@ipb-halle.de
b
Received: January 23, 2005; Accepted: March 29, 2005
Abstract: A novel catalytic property of penicillin G
amidase (PGA) is described. Unexpectedly, the en-
zyme can hydrolyse hydrazide bonds with good effi-
ciency, and in solution the enzyme shows a selectivity
that is similar to phenylacetamides. The hydrolysis of
phenylacetic hydrazides releases hydrazine, but no
inhibition due to the formation of such reactive com-
pounds was observed. This novel catalytic property
was assayed also on a solid phase as a pioneering
route for the design of enzyme-cleavable linkers
and masked scavengers for ketones. On a solid phase
a phenylacetic hydrazide compound was chemically
synthesised on PEGA1900 and PEGAþ (two co-poly-
mers of acrylamide and ethylene glycol) and the ef-
ficiency of PGA in the release of phenylacetic acid
depended on the diffusion of the protein inside the
polymer. On PEGAþ the enzyme, as previously de-
scribed, shows a good diffusion due to an improved
electrostatic interaction with PGA thus achieving
good hydrolytic conversions.
Such conditions often restrict the application of linkers.
Acid-sensitive linkers are not suitable for acid-labile
compounds, such as carbohydrates. Specific linkers
have, therefore, been developed for acid-labile com-
pounds, such as silyl ether linkages, thioether linkages
and ester linkages. However, they have the disadvantage
that they are quite labile to common chemical reagents.
Also, almost all linkers available are unsuitable for mul-
tifunctional compounds such as complex natural prod-
ucts with sensitivity to several orthogonal cleavage
methods.
In organic synthesis enzymatic methods in many cases
have opened up advantageous alternatives to such clas-
sical chemical techniques since enzyme-catalysed trans-
formations typically proceed under very mild conditions
(pH and temperature) and with pronounced chemo-, re-
gio- and stereoselectivity.[2]
In particular, enzymatic transformations have ena-
bled the establishment of alternative protecting group
techniques. Therefore enzymatic transformations that
may be employed for the removal of protecting groups
in solution, in principle, also may open up alternative op-
portunities to release compounds from polymeric sup-
ports.
Keywords: hydrazine; hydrolysis; penicillin G ami-
dase; phenylacetic hydrazide; solid phase biocatalysis
To this extent, penicillin G amidase, which is highly
specific for phenylacetic groups, is an enzyme of first
The success of solid phase synthesis lies on the choice of choice.[3] During the last years much effort has been
a suitable support and an appropriate linker. Solid phase made for the design of penicillin G amidase-cleavable
synthesis involves the use of linkers between the com- linkers.[4]
pounds and the solid supports which must be stable dur-
Some years ago, Waldmann proposed a phenyl hydra-
ing the reactions. These linkers have to be cleavable as zide as an enzyme-labile protecting group.[5] Using a ty-
desired, usually at the end of the synthetic sequence, rosinase, it was possible in solution to convert the chemi-
with high selectivity and in good yield, without affecting cally stable hydrazide precursor into a labile intermedi-
the structures of the products that are released from the ate, thus releasing the free carboxylic acid. In other ex-
polymeric support. In this way molecules can be assem- amples, amidases from different strains of Rhodococcus
bled, modified and finally released through the cleavage were used to synthesise hydrazides.[6]
of the linker.
Our idea was to create a linker where the high nucle-
Previously, linkers have mostly been cleaved by classi- ophilicity of the reactive hydrazine is masked with a phe-
cal chemical methods, for instance, using strong acids.[1] nylacetic group. After the cleavage by penicillin G ami-
Adv. Synth. Catal. 2005, 347, 963–966
DOI: 10.1002/adsc.200505038
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Alessandra Basso et al.
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dase the free, released hydrazine could react and release the released hydrazine in the range of the reaction
a target compound that can also be located far from this time considered. However, it must be underlined that
enzymatic site of cleavage. However, the design of such the enzyme might undergo covalent modifications
an enzyme-cleavable linker would be totally useless if whose effect would become apparent only after a
the enzyme is not able to cleave hydrazide or hydrazone much longer reaction time.
bonds and no examples of such selectivity have been re-
ported for penicillin G amidase so far.
Here we report the first example of penicillin G ami-
Starting from these encouraging results, the hydrolysis
of phenylacetic hydrazides was assayed on solid phase.
Recently, the use of PEGA resins (co-polymers of eth-
dase selectivity towards hydrazide bonds both in solu- ylene glycol and acrylamide) functionalised with hydra-
tion and on a solid phase. First attempts were focused zides, as new scavenger for the reversible linking of alde-
on the hydrolysis of commercial phenylacetic hydrazide hydes has been reported.[8] The same synthetic proce-
in solution. PGA catalysed the hydrolysis of phenylace- dure was used for the synthesis of compound 5 on
tic hydrazide with an initial rate of hydrolysis that was PEGA1900 (5a) and PEGAþ (5b) (Scheme 2). PEGAþ,
only seven times lower than that of the corresponding produced by Polymer Laboratories by introduction of
phenylacetamide. The hydrolytic reaction was complete charged monomers in the PEGA1900 structure was dem-
within 2 h. The hydrolysis of phenylacetic hydrazide re- onstrated to improve PGA accessibility on the solid sup-
sults in the formation of the very reactive hydrazine that, port due to electrostatic interactions.[9,10] Penicillin G
in principle, could react with carbonyl groups of the en- amidase that is negatively charged at pH 8.0 is attracted
zyme causing damage.[7] Thus, the same hydrolytic reac- by the positively charged polymer so that conversions
tion was assayed also in the presence of acetone in order are improved.
to capture the released hydrazine through formation of
Benzaldehyde was successfully coupled on PEGA1900
the corresponding hydrazone (Scheme 1). Our results and PEGAþ leading to the imine compounds 2a, b
showed no measurable effect of the released hydrazine that, after reduction in the presence of sodium triacet-
on the enzyme activity since the initial rates did not oxyborohydride to give 3a, b, were reacted with 1,4-di-
change upon addition of acetone. Hydrolytic reactions (bromomethyl)benzene to give 4a, b. The final hydra-
went to completion regardless of the presence of ace- zide 5a, b was prepared by reaction with phenylacetic
tone.
In both cases, after the complete hydrolysis of phenyl- as demonstrated by determination of the final loading.
acetic hydrazide, fresh substrate was added and initial PGA-catalysed hydrolysis of compounds 5a, b was
hydrazide. All the initial amino groups were acylated,
rates measured. No difference was observed, thus indi- then assayed using hydrolytic procedures already re-
cating that the enzymatic activity was not affected by ported.[9,10] Enzymatic hydrolysis on PEGA1900 (5a)
Scheme 1. Formation of hydrazine after hydrolysis by PGA and reaction with acetone with formation of hydrazone.
Scheme 2. a) Benzaldehyde, HC(OMe)3, room temperature, 24 hours; b) NaB(OAc)3H, MeOH, 1% AcOH (v/v), room tem-
perature, 48 hours; c) 1,4-di(bromomethyl)benzene, DIPEA, DMF, room temperature, 48 hours; (d) phenylacetic hydrazide,
DMF, room temperature, 48 hours.
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308C, 250 rpm. Initial rates were calculated by withdrawing
samples and analysing with RP-HPLC (60% H2O, 40%
MeCN, 0.1% TFA in both phases). Reactions with acetone
were performed by adding 0.2 mmol of acetone (or acetophe-
none). v0 phenylacetamide: 7.9 mmol/min; v0 phenylacetic hy-
drazide: 1.2 mmol/min. Blanks were run of all experiments
and the results were negative.
Hydrolyses of compounds 5a, b were performed as describ-
ed before.[9,10]
Synthesis of Compound 5a (PEGA1900) and 5b
(PEGAþ)
Figure 1. Comparison of the influence of polymer charge and
buffer concentration on the cleavage of polymer bound phe-
nylacetic hydrazide.
The synthesis is similar to that described in detail in ref.[8] Thus,
PEGA1900 and PEGAþ were reacted with benzaldehyde in the
presence of trimethyl orthoformate for 24 h and room temper-
ature to give 2a, b. Compound 2a, b was reduced with sodium
triacetoxyborohydride in the presence of methanol/acetic
acid (1% v/v) at room temperature for 48 h. After washing
the resins 3a, b, they were reacted with di(bromomethyl)ben-
zene and diisopropylethylamine in DMF for 48 h at room tem-
perature. Products 5a, b were obtained by reacting 4a, b with
phenylacetohydrazide in DMF at room temperature for 48 h.
and PEGAþ (5b), in 0.1 M Kpi buffer, lead to the forma-
tion of 0.033 and 0.063 mmol/g dry of phenylacetic acid,
corresponding to conversions of 22% and 32%, respec-
tively (Figure 1). Conversions were determined at the
equilibrium, after 24 h reaction: the equilibrium posi-
tion was confirmed by the addition of fresh enzyme
that caused no additional observed hydrolysis. As in
the case of hydrolysis of phenylacetic hydrazide in solu-
tion, it was also verified that the release of phenylacetic
acid did not cause any shift in the pH of the medium,
even when reactions were performed at 0.01 M buffer
concentration.
As demonstrated before, the use of positively charged
polymers (PEGAþ) in diluted buffer can improve the
accessibility of penicillin G amidase.[9] Indeed, when
the hydrolysis of PEGAþ derived compound 5b was per-
formed in 0.01 M Kpi buffer, the release of phenylacetic
acid increased to almost 60%, whereas with PEGA1900
the conversion was unacceptably low (20%).
Loading Determination
The corresponding hydrazide of compound 4a (1 g of wet resin
corresponding to about 0.1 g dry) was reacted with FMOC-
chloride (9-fluorenylmethyl chloroformate; 20 equivs.,
0.3 mmol), DIPEA (diisopropylethylamine, 20 equivs.) in
10 mL dry DMF. The reaction mixture was stirred for 30 mi-
nutes, then a second coupling occurred. The loading was deter-
mined by cleavage of FMOC group in the presence of piperi-
dine[9] taking into account the double acylation on nitrogens
of the hydrazide group. PEGA1900: 0.15 mmol/g, PEGAþ:
0.2 mmol/g.
This novel catalytic property of PGA offers undoubt-
ed potential to organic chemists: highly reactive hydra-
zines can be masked by a stable phenylacetic hydrazide
bond that can be selectively cleaved through enzymatic
hydrolysis. We are currently investigating the steric re-
quirements of different hydrazide substrates for PGA
with the aid of computational methods and an experi-
mental approach. Information will be exploited for the
rational design of specific linker structures that can be
cleaved by PGA.
Acknowledgements
The authors gratefully acknowledge financial support from the
EU (COMBIOCAT project). Thanks are due to Polymer Lab-
oratories for providingPEGA resinas part of this project(http://
www.polymerlaboratories.com).
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