Fragrance release from the surface of branched poly(amide)s

Article abstract of DOI:10.3390/10010081

Enzymes are powerful tools in organic synthesis that are able to catalyse a wide variety of selective chemical transformations under mild and environmentally friendly conditions. Enzymes such as the lipases have also found applications in the synthesis and degradation of polymeric materials. However, the use of these natural catalysts in the synthesis and the post-synthetic modification of dendrimers and hyperbranched molecules is an application of chemistry yet to be explored extensively. In this study the use of two hydrolytic enzymes, a lipase from Candida cylindracea and a cutinase from Fusarium solani pisii, were investigated in the selective cleavage of ester groups situated on the peripheral layer of two families of branched polyamides. These branched polyamides were conjugated to simple fragrances citronellol and L-menthol via ester linkages. Hydrolysis of the ester linkage between the fragrances and the branched polyamide support was carried out in aqueous buffered systems at slightly basic pH values under the optimum operative conditions for the enzymes used. These preliminary qualitative investigations revealed that partial cleavage of the ester functionalities from the branched polyamide support had occurred. However, the ability of the enzymes to interact with the substrates decreased considerably as the branching density, the rigidity of the structure and the bulkiness of the polyamide-fragrance conjugates increased.

Full text of DOI:10.3390/10010081

Molecules 2005, 10, 81-97
molecules
ISSN 1420-3049
http://www.mdpi.org
Fragrance Release from the Surface of Branched Poly(amide)s
1
1
2
2
Francesca Aulenta , Michael G.B. Drew , Alison Foster , Wayne Hayes 1, *, Steven Rannard ,
David W. Thornthwaite 2 and Tristan G.A. Youngs 1
1
School of Chemistry, The University of Reading, Whiteknights, Reading, RG6 6AD, UK
Unilever Research and Development, Port Sunlight Laboratory, Quarry Road East, Bebington,
2
Wirral, CH63 3JW, UK
* Author to whom correspondence should be addressed; e-mail: w.c.hayes@reading.ac.uk.
Received: 14 May 2004 / Accepted: 29 June 2004 / Published: 31 January 2005
Abstract: Enzymes are powerful tools in organic synthesis that are able to catalyse a
wide variety of selective chemical transformations under mild and environmentally
friendly conditions. Enzymes such as the lipases have also found applications in the
synthesis and degradation of polymeric materials. However, the use of these natural
catalysts in the synthesis and the post-synthetic modification of dendrimers and
hyperbranched molecules is an application of chemistry yet to be explored extensively. In
this study the use of two hydrolytic enzymes, a lipase from Candida cylindracea and a
cutinase from Fusarium solani pisii, were investigated in the selective cleavage of ester
groups situated on the peripheral layer of two families of branched polyamides. These
branched polyamides were conjugated to simple fragrances citronellol and L-menthol via
ester linkages. Hydrolysis of the ester linkage between the fragrances and the branched
polyamide support was carried out in aqueous buffered systems at slightly basic pH
values under the optimum operative conditions for the enzymes used. These preliminary
qualitative investigations revealed that partial cleavage of the ester functionalities from
the branched polyamide support had occurred. However, the ability of the enzymes to
interact with the substrates decreased considerably as the branching density, the rigidity
of the structure and the bulkiness of the polyamide-fragrance conjugates increased.
Keywords: Poly(amide)s, Fragrance, Hydrolysis, HPLC, MALDI-TOF Mass
Spectrometry

Molecules 2005, 10
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Introduction
The majority of the enzymes operate at physiological temperatures, in neutral aqueous media, and
in the absence of substrate functional group protection [1]. Furthermore, in many cases enzymes
display chemo-, regio- and enantioselectivity, and possess high turnover values, thereby rendering
these natural catalysts very appealing, especially in the pharmaceutical and agrochemical industries
[2]. The use of enzymes as catalysts has also been exploited in areas such as asymmetric synthesis [3].
Esterases, lipases and proteases – all hydrolytic biocatalysts, have been employed extensively in the
preparation of enantiopure compounds from racemic mixtures, prochiral and meso compounds [4].
However, enzymes are also used frequently in the protection/deprotection of sugars and peptides,
where selective masking of α-amino groups, carboxy groups and various side chains represent the most
important problems in peptide chemistry [5]. Lipases are a class of enzymes employed frequently in
organic synthesis and catalyse the hydrolysis of esters in aqueous medium and the esterification of
carboxylic acids in organic solvents [6]. Numerous lipases have also been used frequently in the
synthesis, transformation and biodegradation studies of polymeric materials [7].
A notable example of enzyme-catalysed polymeric degradation has been described by Hayashi and
co-workers. The lipase from Pseudomonas fluorescence was used in the in vitro degradation of
poly(hexano-6-lactone) (PLC) fibres [8]. Seebach and co-workers reported subsequently the enzyme-
promoted degradation of ester moieties from a dendritic surface [9] and this approach has great
potential in material science and drug delivery. Indeed, the use of enzymes for the selective post-
synthetic modification of hyperbranched molecules is still a branch of chemistry yet to be exploited
fully.
The enzyme mediated selective hydrolysis of ester functionalities on the external layers of simple
linear and branched polyamides architectures (see 1-8 in Figure 1) is described in this paper. Two
different ester types were conjugated onto the surfaces of the polyamides – either esters derived from a
primary alcohol, citronellol or from a secondary alcohol, L-menthol. It was hoped that the use of
different esters would lead to different hydrolytic profiles and thus provide information upon the
enzyme selectivity and specificity. In order to modify selectively the outer layer of the hyperbranched
molecules, it was decided to employ branched block-copolymers featuring polyamide functionalities in
the interior and ester linkages on the periphery. It was then envisaged to use hydrolytic enzymes that
are selective towards ester-type bonds, such as lipases and cutinases. The hydrolytic cleavage of the
ester bonds was performed under the optimum operative conditions for the enzymes – generally
aqueous buffered systems at almost neutral pH and the alcohol release profiles were monitored by
reverse phase high performance liquid chromatography (RP-HPLC), gas chromatography (GC) and
MALDI-TOF mass spectrometry. Mass spectrometry was employed in order to identify possible
degradation products and to investigate the hydrolytic processes. The nomenclature used in this report
to describe the dendrons and dendrimers was first proposed by Fréchet [10], whereby the nature and
the number of peripheral functional groups are followed by the generation number (generally reported
in squared brackets) and finally by the nature of functional group at the focal point or at the core. The
perfume molecules resident on the outer shell of the dendrons/dendrimers are described using the
abbreviations C and M for citronellol and L-menthol, respectively.

Molecules 2005, 10
Figure 1. Linear and Branched Polyamides 1-8 Bearing Ester Functionalities on the
83
External Surface.
O
O
O
O
O
O
O
O
O
O
N
H
N
H
N
H
N
O
H
O
C2-[G-0]2-C
1
M2-[G-0]2-C
2
O
O
O
O
O
H
N
O
H
N
O
O
NH
N
O
O
O
O
O
NH
N
O
HN
O
NH
O
O
O
C3-[G-0]3-N
3
M3-[G-0]3-N
4
O
O
O
NH
NH
O
O
O
O
O
O
O
O
O
HN
HN
HN
HN
NH
NH
O
O
O
O
N
N
N
N
H
O
O
H
H
O
O
H
O
O
O
O
O
O
O
O
M4-[G-1]2-C
6
C4-[G-1]2-C
5
O
O
O
O
O
O
H
N
O
O
O
O
HN
H
N
NH
O
O
O
HN
O
O
HN
O
O
N
N
O
O
O
NH
O
O
N
H
HN
HN
H
HN
O
HN
O
NH
O
O
O
H
N
N
O
O
O
O
NH
O
O
O
NH
O
HN
O
O
O
O
O
O
O
C6-[G-1]3-N
7
M6-[G-1]3-N
8
Results and Discussion
Synthesis
The linear and branched polyamides 1-8 bearing the ester functionalities on the external surfaces
were synthesised in a convergent [11] fashion starting from the corresponding primary or secondary

Molecules 2005, 10
84
alcohols, respectively [12]. Succinic anhydride was ring opened under basic conditions [13] using
citronellol and L-menthol, leading to the production of the carboxyester derivatives 9 as a white solid
(in 95% yield) and 10 as a transparent oil (in 90% yield), respectively. The ester-carboxylic acid
derivatives 9 and 10 were then coupled to the ‘core’ amine units such as 1,7-diaminoheptane or tris-(2-
ethylamino)amine to afford the desired polyamides 1-4 featuring an increasing number of ester
moieties (Scheme 1).
Scheme 1. Synthesis of simple and branched polyamides (1-4)
H2N
NH2
H
N
H
N
O
O
RO
OR
O
O
EDCI, HOAt,
NMM, DMF
R = Citronellol 1
= L-Menthol 2
O
OH
RO
O
O
R = Citronellol 9
= L-Menthol 10
RO
O
NH
NH
O
O
OR
N
N
O
H
H2N
NH2
RO
N
O
H2N
R = Citronellol 3
= L-Menthol 4
In order to increase the number of ester groups on the core units described above, the cinnamic
acid derivative 3,5-diaminocinnamic acid 11 [12] was used as a branching moiety in the construction
of the branched polyamides. The selection of the convergent approach [11] to the desired branched
polyamides via selective coupling between the orthogonal amino moieties of 3,5-diaminocinnamic
acid 11 and the carboxylic acid functionalities of the L-menthol and citronellol esters 9 and 10
necessitated the use of protecting group chemistries. Therefore, the allyl derivative of 3,5-diamino-
cinnamic acid 12 was synthesised in four steps from the commercially available 3,5-dinitrobenzoic
acid 13 (Scheme 2) in good yield.
Scheme 2. Synthesis of 3,5-diaminocinnamic acid allyl ester (12)
O
O
CHO
NO2
(C2H5O)2P(O)CH2CO2allyl
O2N
O2N
PCC
O B
3
1/3
NaH, THF, ∆
THF, ∆
O2N
O2N
NO2
SnCl2,
THF/H2O 50:50, ∆
O
OH
H3B-S(CH3)2
THF, ∆
H2N
NH2
11
O
O
CO2H
NO2
O2N
13
H2N
NH2
12

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85
The first generation polyamide dendrons C2-[G-1]-CO2allyl (14) and M2-[G-1]-CO2allyl (15) were
constructed in excellent yields (90-98%) using either 1-ethyl-3-(3’-dimethylaminopropyl)carbodiimide
hydrochloride (EDCI) and 1–hydroxy-7-azabenzotriazole (HOAt) or benzotriazole-1-yl-oxy-tris-
(dimethylamino)-phosphonium hexafluorophosphate (BOP) coupling reagents (Scheme 3) [14].
Cleavage of the allyl ester at the dendrons’ focal point required very mild deprotection conditions
(either Pd(OAc)2, Pd(dba)2, Pd(PPh3)4 in conjunction with ammonium formate) in order to prevent
detrimental decomposition of the terpene ester linkages and to afford acceptable yields (64-74%) of the
desired carboxylic acid derivatives 16 and 17.
Scheme 3. Synthesis of the first generation polyamide dendrons featuring either
citronellol or L-menthol at the peripheral surface.
O
OR
HN
HN
O
BOP, DiPEA,
CH3CN
O
O
O
O
RO
+
OH
O
O
O
H2N
NH2
OR
O
R = Citronellol 9
= L-Menthol 10
12
R = Citronellol 14
= L-Menthol 15
Pd(OAc)2/HCO2NH4, PPh3,
THF
or
Pd(dba)2/HCO2NH4, PPh3,
THF
O
O
OR
OR
HN
O
O
HO
O
HN
R = Citronellol 16
= L-Menthol 17
The dendrimers were constructed from [G-0] to [G-2] by coupling each dendron to either
bifunctional or trifunctional central core units. Synthesis of the first generation dendrimers was carried
out initially using EDCI/HOAt as the coupling reagent system, but the yields were very poor. Use of
BOP afforded M4-[G-1]2-C (6) as a white solid in excellent yield (96%) and M6-[G-1]3-N (8) as pale
yellow solids in moderate yield (48%) over a prolonged reaction time (48 hours). The BOP reagent
was also used in the synthesis of C4-[G-1]2-C (5) and C6-[G-1]3-N (7). These dendrimers were
obtained after purification by column chromatography as pale yellow solids in acceptable yields (98%
and 28%, respectively), in agreement with the results obtained for the series of L-menthol first
generation dendrimers (Scheme 4).

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Scheme 4. Synthesis of the branched polyamides 5-8.
O
O
NH
NH
RO
OR
OR
H2N
NH2
O
O
HN
HN
C4-[G-1]2-C 5
M4-[G-1]2-C 6
O
O
N
H
N
O
O
H
O OH
RO
O
O
BOP, DiPEA
CH3CN
O
O
OR
N
N
O
O
H
O
H
OR
OR
NH
OR
O
O
R = Citronellol 16
= L-Menthol 17
NH
O
O
RO
HN
HN
HN
HN
O
O
O
C6-[G-1]3-N 7
M6-[G-1]3-N 8
N
HN
O
O
NH
H2N
O
OR
NH2
O
N
RO
O
NH
OR
O
H2N
O
Hydrolysis Studies
Preliminary studies of alcohol release under enzymatic hydrolysis were conducted on the ester
groups situated on the peripheral layer of the linear and branched polyamides 1-8 with lipase from
Candida cylindracea in an aqueous solution buffered to pH 7.2 [15]. The sampling was carried out
over variable times (between 40 and 96 hours) and the alcohol released from the peripheral surface of
the polyamides was extracted from the aqueous phases with an organic solvent (CHCl3) and its
concentration monitored by RP-HPLC or GC analyses. The extracts were also analysed by MALDI-
TOF mass spectrometry, from which it was possible to identify several of the major hydrolytic
products. For example, when C2-[G-0]2-C (1) was subjected to the hydrolysis in the presence of the
lipase an increase in the concentration of citronellol (∼15%) was exhibited. However, a similar trend
was also observed during the hydrolysis of 1 carried out without the lipase. This result may suggest
that a slow hydrolytic process occurred as a consequence of the slightly basic conditions of the
solution, and the enzyme did not participate in the catalysis of the process. However, the MALDI-TOF
mass spectra of the samples extracted from the reaction mixtures did not reveal the presence of species
that could be attributable to products of the hydrolysis. Therefore, it was not possible to confirm the
occurrence of the hydrolytic process in this case.
In contrast, unambiguous results were obtained from the hydrolysis of M2-[G-0]2-C (2), where
cleavage of the ester linkages was not observed. This result was not unexpected since compound 2
features esters that are derived from a secondary alcohol, and hence more difficult to cleave either
under basic conditions or by enzymatic hydrolysis.
More interesting results were obtained from the hydrolysis studies of C3-[G-0]3-N (3). This
compound was characterised by the presence of three citronellol moieties bound to the aliphatic
polyamide structure via a succinate spacer. Furthermore, a tertiary amine functionality was present in
the core unit of the molecule. When 3 was subjected to the hydrolytic conditions in the presence of

Molecules 2005, 10
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lipase from Candida cylindracea progressive increase in the concentration of citronellol (~50%) was
revealed by the RP-HPLC analysis. Although a certain degree of ester cleavage was also observed in
the absence of enzyme (ca. 25 %), in this case a significant difference between the enzymatic
hydrolysis and that performed without the enzyme was evident. MALDI-TOF mass spectrometric
analysis of the samples during the hydrolysis study revealed important information about the
hydrolytic process. In both reactions (the hydrolysis in the presence and absence of the enzyme) the
appearance of two peaks corresponding to the loss of one molecule of citronellol (MH+=704 Da) and
of two citronellol units (MH+=548 Da, or as the MK+=589 Da), was observed, which suggested
formation of cyclic imides (Figure 2). The peak at 704 Da increased throughout the reaction, while the
peak corresponding to the loss of two molecules of citronellol (592 Da) appeared at the initial stage of
the hydrolytic process, but its relative intensity did not increase considerably as the hydrolysis
proceeded. However, from the mass spectrometric analysis, it was not possible to obtain any
correlation between peak intensities and concentration of the species present, therefore, it was only
possible to suggest that the main hydrolytic pathway involved the release of a single molecule of
citronellol per molecule of starting material C3-[G-0]3-N (3), and formation of a cyclic succinimide
followed by subsequent release from the monoimide species. The other peaks detected in the spectra
were attributed to the matrix used (4-hydroxy-α-cyanocinnamic acid), its aggregates and fragments, as
confirmed by the spectrum obtained by the analysis of the matrix on its own recorded under identical
analytical conditions. Formation of succinimides from succinates under neutral and basic conditions
are well-documented in the literature [16]. For example, Matsumoto et al. [17] have reported recently
the use of succinyl ester derivatives as an easily hydrolysable spacer for the conjugation of a new class
of prodrug form of an HIV protease inhibitor. The esters of succinamic acids are known to undergo
rapid hydrolysis via succinimide formation under mild alkaline conditions [18].
Figure 2. MALDI-TOF Mass Spectra of the hydrolysis of C3-[G-0]3-N (3) recorded at
(a) time = 0 and (b) after 65 Hours.
860.44
O
O
O
NH
N
NH
O
HN
O
O
O
O
O
a) Hydrolysis of 3 at pH = 7.2, in the presence of Lipase from Candida cylindracea, t = 0 hours.

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O
O
O
O
O
O
O
8608.5680.58
O
O
O
O
O O
HN HNNH NH
O
O
O
O
O
O
N
N
O
N
N
O
NH NH
O
O
OH OH
O
O
N
N
O
O
O
O
NH NH
O
O
O
O
O O
HN HN
7057.0065.06
O
O
O
O
HN HNNH NH
O
O
O
O
O
O
N
O
O
O
O
O
N
N
NNH NH
O
O
O
O
N
HN HN
O
O
N
O
O O
N
O
N
N
N
N
O
O
O
O
O
OHO OH
N
O
O
O
O
N
NN
OO
N
6066.3006.30
5925.2942.24
O
O
3923.5972.57
b) Hydrolysis of 3 at pH = 7.2, in the presence of Lipase from Candida cylindracea, t = 65 hours
Recently, the use of lipase from Candida cylindracea to generate amide bonds in organic solvents
from the corresponding esters has been reported by Gotor and co-workers [19]. Furthermore, the lipase
from Pseudomonas cepacia has been employed in the enzymatic hydrazinolysis of diesters and the
synthesis of N-aminosuccinimide derivatives in organic solvents [20]. The principle of ring closure
with consequent elimination of an alcohol as a leaving group has also been embraced for the
development of controlled release of fragrance alcohols from poly(propyleneimine) dendrimers [21].
In this case, fragrance release was based upon proton abstraction from a carbamoyl moiety by a basic
buffer system. The intermediate nucleophile generated then underwent an intramolecular ring closure
on the neighbouring ester moiety to release the desired fragrance and generate a cyclic imide by-
product. The cyclisation occurred rapidly in slightly alkaline conditions and represented the driving
force for the efficient release of even tertiary alcohols. In the last example, the efficiency of the
cyclisation was promoted by the presence of a rigid benzene ring that held the two reactive
components in close proximity with the conformation most appropriate to afford efficient imide ring
closure.
In the current study, the linker used to conjugate the alcohol unit to the dendritic surface was a
flexible succinate chain. Therefore, it is possible to assume that the pH-promoted cyclisation was not
favoured as in the study reported by Hermann and co-workers [21], and occurred at a slower rate. This
explanation does not contradict the different hydrolytic profiles exhibited by C3-[G-0]3-N (3) in the
presence and absence of the lipase. It is proposed that in this case (hydrolysis of 3 in the buffered
system) the enzyme simply accelerated a process that was already occurring. It is also proposed that
the presence of the tertiary amine at the core unit of the molecule participated in the de-protonation of
the amide, hence promoting the cyclisation even in the absence of the enzyme. It was noticeable that
the ring closing process did not occur in the case of C2-[G-0]2-C (1), which was characterised by the
same succinyl ester moieties, no central tertiary amine and a longer alkyl chain as the core unit.

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When the hydrolysis of M3-[G-0]3-N (4) was investigated, release of the secondary alcohol was not
observed either in the presence or absence of the enzyme. This result revealed: – i) the lack of
interactions between the enzyme and the substrate, which may be a consequence of the rigidity of the
structure of the alcohol moieties, and the presence of esters of a secondary alcohol and ii) the low
tendency of these esters to undergo ring closing under neutral conditions, as a consequence of the poor
ability of secondary alcohols to behave as leaving group under mildly basic conditions.
In order to examine the hydrolysis characteristics of molecules C3-[G-0]3-N (3) and M3-[G-0]3-N
(4) further, preliminary computer modelling studies were performed on single ‘arms’ of the
polyamides, (3a and 4a, respectively – see Figure 3). Structures were built within the Cerius2 software
[22] and geometry optimised at the HF/6-31G(d) level using Gaussian98 [23]. For each system two
geometries were studied with different values of the O=C–O–R torsion angle φ. The lowest-energy
conformation was found with φ = 0° (denoted cis) while the high-energy conformer has φ = 180°
(denoted trans). Firstly, it was noted that the energy differences between the cis and trans conformers
for 3a and 4a were identical (8.52 kcal mol–1). In contrast, the corresponding hydrolysis product in
which either the citronellol or L-menthol components have been removed to afford the corresponding
carboxylic acid, the energy difference between conformers was 6.24 kcal mol–1. The rotational energy
profile of φ is similar for all three structures – the alcohol leaving group does not significantly affect
the energy of the different conformers of a given molecule, thus steric repulsions between the alcohols
and the remainder of the molecule are small. Plots of the electron density for the cis conformers of 3a
and 4a (Figure 3) reveal that there is minimal difference in the local density around the carboxy group.
While the isopropyl substituent of the cyclohexane ring in 4a-cis would appear to occlude the reactive
carbonyl centre, the approach angle onto the C=O group will be perpendicular to the C=O vector and
thus not close to the bulky ester group. Based upon this preliminary evidence, it appears that the
observed lack of hydrolysis of 4 cannot not attributed to a predominant steric effect, and is most likely
related to the poor leaving ability of the alcohol functionality. Indeed, one calculated difference
between 3a and 4a are the partial atomic charges of the carbon atom of the COOR group, with values
of –0.38 and –0.34 e, respectively. While this difference is only a small distinction, it is likely that the
cause is the increased positive inductive effect offered by the leaving group in 4a cf. that of 3a, which
promotes an increased negative (more positive) charge on the adjacent atoms.
Figure 3 Plots of the total electron density of the cis conformers of 3a and 4a, drawn
using the Molekel [24] software.
3a
4a

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A study of the ester cleavage from the first generation polyamide dendrimers 5-8 was conducted by
GC analysis. Although sample preparation was carried out under similar conditions used for the
branched molecules 1-4, the chloroform solution resultant from the extraction of the aqueous mother
liquor was injected directly into the GC column. This procedure eliminated one step in the sample
preparation and decreased any potential error associated with this process. Furthermore, the analyses
employed an internal standard to minimise the errors related with the sampling and injection processes.
However, under the same hydrolytic conditions used in the other cases examined, release of the
citronellol from C6-[G-1]3-N (7) was not observed. This result was rationalised as a consequence of
two major factors: increased bulkiness of the dendritic structure which may have inhibited the access
of the molecule into the enzyme active site, and also increased rigidity of the system introduced by the
presence of the cinnamate moieties. Furthermore, it was speculated that the increased rigidity might
also render the deprotonation process of the amide unit more difficult to achieve and consequently
reduce the ability of intramolecular nucleophilic addition/elimination that was observed in the case of
C3-[G-0]3-N (3) under mild basic conditions. Since the lipase from Candida cylindracia was found to
catalyse the cleavage of the ester moieties only from C3-[G-0]3-N 3, it was decided to investigate
another enzyme which belonged to the class of hydrolytic enzymes referred to as cutinases. The
enzyme used was a solid-supported cutinase from Fusarium solani pisii, used commonly in detergent
products. The release profiles for the series of citronellol-based dendrimers obtained under the
optimum conditions for this enzyme, were very surprising. For example, C2-[G-0]2-C 1 underwent
cleavage of the ester moieties (∼80%) within the first 40 hours of hydrolysis. However, a decrease in
concentration of citronellol was observed after this period of time, thereby indicating the occurrence of
possible side reactions involving the alcohol (Figure 4).
Figure 4. HPLC Traces of the Hydrolysis of 1 in the presence of a Cutinase, recorded at
Different Times During the Reactions.
1
Citronellol
t = 76 h
t = 0 h
5.00
1.00
2.00
3.00
4.00
t (min)

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In contrast, when the hydrolysis was carried out in the absence of the enzyme, significant release of
alcohol was not detected. MALDI-TOF mass spectrometry did not reveal significant information
regarding the hydrolytic pathway, although a decrease in intensity of the peaks corresponding to the
starting material was observed with the proceeding of the reaction. Unfortunately, citronellol was not
detectable under the conditions used in the mass spectrometric analysis, and also was not observed in
the spectra of standard samples. Furthermore, analysis of negative ions via MALDI-TOF MS was not
conducted, therefore, possible negatively charged species resulted from the hydrolysis were not
observed. From the results obtained for the hydrolysis of 1, it was concluded that the hydrolysis of the
ester moieties was enzyme-catalysed although it occurred without formation of the cyclic imide. The
presence of the enzyme also appeared to influence much more significantly the hydrolytic process in
comparison to the other enzyme tested (lipase) thus indicating more favourable interactions between
the substrate and the enzyme active site. Similar behaviour was also observed in the case of the
citronellol derived first generation polyamide dendrimers 5 and 7.
Figure 5. GC Chromatograms of the Hydrolysis of C4-[G-1]2-C (5) in the Presence of
Cutinase, Recorded after 0, 30, 44 and 72 Hours.
DMSO
L-Menthol
t = 0 h
T = 30
L-Menthol
DMSO
Citronellol
Citronellol
L-Menthol
T = 44 H
T = 72 H
L-Menthol
DMSO
DMSO
Citronellol
Citronellol
The analysis of the hydrolytic products was carried out using GC, in which each sample was
spiked with L-menthol as the internal standard. When the hydrolysis was performed in the presence of
a lipase, cleavage of the ester groups was not detected. However, improved results were obtained in
the presence of the cutinase, whereas a small percentage of citronellol was released from the dendritic

Molecules 2005, 10
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structures (∼15-25%). The dendrimer C4-[G-1]2-C (5) exhibited release of citronellol over a period of
40 hours after which decrease in the concentration of citronellol was observed in a similar fashion as
described above (Figure 5). Furthermore, the hydrolysis carried out in the absence of enzyme did not
exhibit relevant release of citronellol, thus indicating that the ester cleavage was promoted by the
enzyme rather than dictated by the reaction conditions. MALDI-TOF mass spectrometric analysis in
this case also did not prove to be informative: – the almost complete absence of the peak
corresponding to the dendritic starting material was observed from the beginning of the hydrolysis.
This result was attributed to the experimental procedure required for the preparation of the analytical
samples. Since the enzyme was solid supported, each sample was subjected to filtration and extraction
with an organic solvent prior to analysis. However, the lack of the signal corresponding to the dendritic
species in the MALDI-TOF mass spectrometric analysis led to the hypothesis that the dendrimers were
absorbed onto the solid support and not eluted in the organic solvent used in the extraction step.
The polyamide dendrimer C6-[G-1]3-N (7) also exhibited similar behaviour in the presence of the
cutinase, although the increase in concentration of citronellol was very moderate (∼15%). This
moderate hydrolytic process – as in the case of the hydrolysis carried out in the presence of a lipase –
may be attributable to the increased rigidity of the structure C6-[G-1]3-N (7) when compared with
C4-[G-1]2-C (5). The dendrimer C6-[G-1]3-N (7) was characterised by a tetrafunctional core with three
arms possessing only two carbon chains. In contrast, C4-[G-1]2-C (5) possessed a bifunctional core
with longer alkyl chains (seven methylenic residues), which imparted a higher degree of flexibility to
the structure. Furthermore, 7 was characterised by the presence of three rigid polyamide dendrons
around the core unit, with considerable increase of the bulkiness of the molecules.
The series of dendrimers bearing L-menthol esters at the periphery were also subjected to the
hydrolytic conditions in the presence of the cutinase. However, as in the case of the hydrolysis in the
presence of lipase, these molecules did not exhibit any evidence of alcohol release, confirming the
selectivity of these classes of hydrolases towards esters of primary alcohols.
Conclusions
Following the pioneering work of Seebach and co-workers [9], the post-synthetic modification of
dendritic surfaces was investigated by mean of two hydrolytic enzymes, a lipase and a cutinase. From
the investigations carried out with the aid of analytical techniques such as RP-HPLC, GC and MALDI-
TOF mass spectroscopy emerged the ability of cutinase to efficiently interact with the linear
polyamides 1 and hence catalyse the alcohol release (up to 80% release). However, increasing the
degree of branching and reducing the flexibility of the polyamides resulted in a significant reduction in
the catalytic activity, probably dictated by the steric hindrance of these large branched molecules.
The lipase proved to be a less efficient catalyst for these branched substrates, since cleavage of the
ester was only observed in the case of the polyamide C3-[G-0]3-N (3). However, this polyamide
exhibited ester cleavage in a smaller extent also in the absence of the enzyme, via a neighbouring
group participation effect, whereby the alcohol was released as a consequence of an intramolecular
nucleophilic addition/elimination reaction. This process has been described in the literature [18], and
occurs even under mild basic conditions. In this specific case, it was suggested that the tertiary amine
in the core unit of the molecule acted as a base catalysing the deprotonation of the amide and hence the
formation of the cyclic imide. The enzymatic ester cleavage of L-menthol esters was unsuccessful in

Molecules 2005, 10
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all of the cases examined, thus indicating the selectivity of these hydrolytic enzymes towards esters
derived from primary alcohols under these conditions.
Acknowledgements
F.A. would like to thank Unilever Research and Development for a postgraduate studentship. The
authors would like to acknowledge EPSRC (GR/M91884) and SAI Ltd for the funding provided for
the MALDI-TOF MS facilities at the University of Reading. W.H. would like to thank EPSRC
(GR/M42824) for providing funds for the HPLC equipment used in this study.
Experimental
General
Reagents were purchased from the Aldrich Chemical Company, Acros Organics and Lancaster and
were used as received without purification. Solvents were used as supplied, with the exception of the
following: – tetrahydrofuran (THF) was distilled under nitrogen from sodium and benzophenone,
dichloromethane (CH2Cl2) was distilled under nitrogen from calcium hydride. Solvents of analytical
and HPLC-grade were purchased from Fisher Scientific and used as received without purification.
Lipase from Candida cylindracea (943 unitmg-1) was purchased from Aldrich and used without
purification, while cutinase (1% active enzyme protein) from Fusarium solani pisii was supplied by
Unilever Research and Development, Port Sunlight, UK. Succinic acid mono-(5-isopropyl-2-methyl-
cyclohexyl) ester (9) was prepared according to the method reported by Jabloner and Dunbar [13b].
The synthesis of the polyamide compounds described in this paper has been reported elsewhere [12].
MALDI-TOF mass spectra were obtained on a SAI LT3 LaserTof MALDI-TOF mass spectrometer
using α-cyano-4-hydroxycinnamic acid as the matrix. A typical sample preparation is described as
follows: 3 µL of a solution of the analyte in THF (1-10 mgmL-1) was combined with 10-20 µL of the
freshly prepared matrix (0.1 or 0.2 M in THF) in a mini-vial, and from the mixture was taken a 2 µL
aliquot which was transferred carefully onto a sample plate and left to air dry prior to analysis.
Reverse-phase high performance liquid chromatographic analyses (RP-HPLC) were carried out using a
Perkin-Elmer Series 410 LC pump in conjunction with a refractive index detector Polymer
Laboratories PL-RI 800 and interfaced with PL Caliber® LC/GC version 4 software (Polymer
Laboratories, Ltd). Each sample (20 µL) was dissolved in CH3CN and injected (20 µL) onto a
Phenonomex Luna C18 (2), 100 × 4.6 mm column (3 µm beads) equipped with a pre-column Hichrom
C18 (5 µm), and temperature controlled at 40 °C. The flow rate of the mobile phase (CH3CN) was 1
mLmin-1. Gas chromatographic (GC) analyses were carried out using a Perkin-Elmer Autosystem XL
gas chromatograph in conjunction with a flame ionisation detector (FID) and interfaced with
TotalChrom™ workstation chromatography software (Perkin-Elmer Instruments). Each sample
(dissolved in CHCl3) was injected (10 µL) onto a Supelcowax 10 column, 30 × 0.25 mm ID, 0.25 µm
film, using helium as gas carrier at a pressure of 16 PSI. The temperature of the column was held at 50
°C for the first two minutes, then was increased to 180 °C with a ramp of 2.5 °Cmin-1 and finally held
at 180 °C for 5 minutes.

Molecules 2005, 10
94
Phosphate Buffer Preparation
0.01 M Phosphate buffer, pH 7.2 (100.00 mL) was prepared by combining 0.20 M sodium
phosphate monobasic solution (8.40 mL) – (stock solution A), – 0.20 M sodium phosphate dibasic
solution (21.60 mL) – (stock solution B) – and distilled water (60.00 mL). Stock solution A was
obtained by dissolving Na2HPO4 (13.90 g, 0.10 mol) in distilled water (500.00 mL). The stock solution
B was obtained dissolving NaH2PO4·12H2O (71.63 g, 0.20 mol) in distilled water (1000.00 mL).
0.10 M Phosphate buffer, pH 7.8 (200.00 mL) was prepared by combining 0.2 M sodium phosphate
monobasic solution (8.50 mL) – (stock solution A), – 0.2 M sodium phosphate dibasic solution (91.50
mL) – (stock solution B) – and distilled water (100.00 mL). Stock solution A was obtained dissolving
Na2HPO4 (2.40 g, 0.02 mol) in distilled water (100.00 mL). The stock solution B was obtained
dissolving NaH2PO4·12H2O (7.16 g, 0.02 mol) in distilled water (100.00 mL).
Citronellol standards for RP-HPLC analyses:
Known aliquots of citronellol were weighed and transferred into clean, dry volumetric flasks (25
mL). CH3CN was added to volume in order to obtain final concentrations of 0.1, 0.5, 1.0, 2.0, 5.0 and
10.0 mgmL-1. The peak areas obtained from the RP-HPLC analyses were then plotted against the
concentration in order to obtain a linear correlation between the two variables. The optimum
correlation was obtained by using Equation 1:
Equation 1. Linear Correlation Between Peak Areas and Concentrations.
y = 0.0498X (R2 = 0.9207),
where
× is the peak area
y is the concentration.
Using this equation the unknown concentration of samples of citronellol were determined.
Citronellol standards for GC analyses:
Known aliquots of citronellol were weighed and transferred into clean, dry volumetric flasks (25
mL). A solution of L-menthol in CHCl3 (10.0 mgmL-1) was added to obtain a final concentration of 1.0
mgmL-1. CHCl3 was then added to the volumetric flask up to the required volume in order to obtain
final concentrations of 0.01, 0.05, 0.10, 0.50, 1.00 and 2.00 mgmL-1. The peak areas obtained from the
GC traces were then plotted against the concentration and the correlation between the two variables
was calculated according to the method of the internal normalisation, where the detector response
factor (DRF value)25 is given by Equation 2:
Equation 2. The Detector Response Factor.



AC CM






DRF =
= 1.1373
AM CC





Molecules 2005, 10
95
where:
AC is the peak area of the citronellol;
AM is the peak area of the internal standard, L-menthol;
CM is the concentration of the internal standard, L-menthol and
CC is the concentration of citronellol.
Using this equation the unknown concentration of samples of citronellol were determined.
General Procedure for the Hydrolysis Studies in 0.01 mM Phosphate Buffer, pH= 7.2 at 37 °C in the
Presence of Lipase from Candida Cylindracea: C2-[G-0]2-C (1).
The hydrolysis was conducted in a carousel reactor holding 12 sealed vials equipped with a
temperature-controlling probe set at 37 °C. Into each vial was placed a solution of the enzyme (4.90
mL). The enzyme solution was prepared as follows: 24.0 mg of lipase from Candida cylindracea were
transferred carefully into a 100 mL volumetric flask and the phosphate buffer was added up to the
desired volume. The resultant suspension was subjected to ultrasound for 10 minutes prior to use. The
enzyme suspension was left equilibrating at 37 °C for 2 hours prior the addition of a solution of
C2-[G-0]2-C (1) in CH3CN (0.10 mL), obtained by dissolving 0.13 g of 1 in CH3CN (1.30 mL). After
set periods of time the content of each vial was extracted with CHCl3 (2 × 5.00 mL). The solvent was
then removed under reduced pressure, the residue dissolved in CH3CN (0.50 mL) and the solution
analysed by RP-HPLC chromatography and MALDI-TOF mass spectrometry.
Hydrolysis studies in 0. 1 mM phosphate buffer, pH= 7.8 at 37 °C in the presence of Cutinase from
Fusarium Solani pisii – typical procedure: C2-[G-0]2-C (1).
Into each vial were placed: the enzyme (1% active enzyme protein) (0.10 g) and the phosphate
buffer (2.45 mL). The vials were then kept at 37 °C for a period of 2 hours prior to addition of a
solution of C2-[G-0]2-C (1) in CH3CN (50 µL), obtained by dissolving 1 (0.10 g) in CH3CN (1.00 mL).
After set intervals of time the content of each vial was filtered on a sintered funnel, washed with
CHCl3 (6.00 mL) and then with distilled water (3.00 mL). The organic phase was separated (2.50 mL)
and the solvent removed under reduced pressure. The residue was dissolved in CH3CN (0.30 mL) and
analysed by RP-HPLC chromatography, GC chromatography and MALDI-TOF mass spectrometry.
Note: The hydrolysis studies of compounds M2-[G-0]2-C (2), C4-[G-1]2-C (5), M4-[G-1]2-C (6) and
C6-[G-1]3-N (7) were carried according to the procedures described above. ‘Blank’ hydrolysis assays
were carried out in exactly the same manner but in these cases the phosphate buffer added to each vial
did not contain the enzyme.
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