Epimerization of peptide nucleic acids analogs during solid-phase synthesis: Optimization of the coupling conditions for increasing the optical purity

Article abstract of DOI:10.1039/b104146k

Peptide nucleic acid (PNA) analogs based on N^α-(thymin-1-ylacetyl)ornithine were previously shown to form triplexes with complementary RNA. In order to obtain optically pure compounds for hybridization experiments, chiral monomers based on D- or L-ornithine, N^δ-Fmoc-N^α-(thymin-1-ylacetyl)ornithine 2 and N^δ-Fmoc-N^α (uracil-1-ylacetyl)ornithine 3 were synthesized either by a one-step or by a simple three-step procedure starting from N^δ-protected ornithine; the latter procedure led to enantiomerically pure products. Oligomerization of 2 and 3 was carried out either in solution, or by solid-phase peptide synthesis (SPPS) on an MBHA-Rink amide resin. The oligomers turned out to contain large amounts of epimerization products, especially those obtained by SPPS. Therefore, we examined carefully the parameters which may be involved in epimerization: the nature of the coupling reagent, of the base, and the addition mode. Coupling of the monomer L-3 was performed under various conditions. Lower racemization was found to occur when using (7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU) as coupling agent and 2,4,6-trimethylpyridine (sym-collidine, TMP) as base, without preactivation, leading to a residual 4% of the D-enantiomer. By applying a procedure based on the stepwise addition of the base the D-enantiomer content was reduced to less than 1%. Using this procedure, a decamer of L-3 was synthesized, which was shown to contain less than 2% of the D-ornithine derivative.

Full text of DOI:10.1039/b104146k

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Epimerization of peptide nucleic acids analogs during solid-phase
synthesis: optimization of the coupling conditions for increasing
the optical purity
1
Roberto Corradini,a Stefano Sforza,a Arnaldo Dossena,a Gerardo Palla,a Raniero Rocchi,b
Ferdinando Filira,b Flavia Nastric and Rosangela Marchelli*a
a Dipartimento di Chimica Organica e Industriale, University of Parma,
Parco Area delle Scienze 17/A, I-43100 Parma, Italy
b Dipartimento di Chimica Organica, Università di Padova, Via Marzolo 1, I-35131 Padova,
Italy
c Dipartimento di Chimica, Università “Federico II” di Napoli, Via Cinzia, 45, I-80126 Napoli,
Italy
Received (in Cambridge, UK) 4th May 2001, Accepted 17th August 2001
First published as an Advance Article on the web 1st October 2001
Peptide nucleic acid (PNA) analogs based on N α-(thymin-1-ylacetyl)ornithine were previously shown to form
triplexes with complementary RNA. In order to obtain optically pure compounds for hybridization experiments,
chiral monomers based on - or -ornithine, N δ-Fmoc-N α-(thymin-1-ylacetyl)ornithine 2 and N δ-Fmoc-N α-
(uracil-1-ylacetyl)ornithine 3 were synthesized either by a one-step or by a simple three-step procedure starting
from N δ-protected ornithine; the latter procedure led to enantiomerically pure products. Oligomerization of 2
and 3 was carried out either in solution, or by solid-phase peptide synthesis (SPPS) on an MBHA-Rink amide
resin. The oligomers turned out to contain large amounts of epimerization products, especially those obtained by
SPPS. Therefore, we examined carefully the parameters which may be involved in epimerization: the nature of the
coupling reagent, of the base, and the addition mode. Coupling of the monomer L-3 was performed under various
conditions. Lower racemization was found to occur when using (7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) as coupling agent and 2,4,6-trimethylpyridine (sym-collidine, TMP) as base, without
preactivation, leading to a residual 4% of the -enantiomer. By applying a procedure based on the stepwise addition
of the base the -enantiomer content was reduced to less than 1%. Using this procedure, a decamer of L-3 was
synthesized, which was shown to contain less than 2% of the -ornithine derivative.
Introduction
Optical purity and methods for preventing epimerization are
essential features for peptide synthesis, since the presence of
diastereomeric impurities can considerably lower the yield and
purity of products, and since biological activity is strictly
related to stereochemistry.1 The problem is particularly import-
ant in peptide synthesis carried out in solution, and could be a
serious problem in the case of coupling with N-methyl amino
Chart 1
acids,2 in segment coupling,3 in convergent solid-phase peptide
synthesis (CSPPS),4 or in the synthesis of conformationally
constrained cyclic peptides.5 By carefully choosing the coupling
reagents, the base, and the reaction conditions, epimerization
can be reduced to very small levels, as shown in several studies
by Carpino and co-workers.6
(Chart 1a, R = H). They were shown to form hybrids with com-
plementary DNA or RNA strands of remarkable affinity and
selectivity,9 and are currently used as very powerful tools in
molecular biology for the detection of defined DNA sequences
or mutations,10 and for the inhibition11 and regulation of gene
expression.12 PNAs are also promising as antisense drugs.13,14
PNA oligomers can be easily obtained on a milligram-to-
gram scale from monomeric units by solid-phase peptide
synthesis (SPPS),15 using standard procedures on automatic
instruments. Since their discovery in 1991, many studies have
been published describing modified PNA, with the aim of
characterizing the best structural features for DNA complex-
ation.16,17 Among these, particular attention has been given to
chiral PNA analogs,18 since chirality could, in principle, induce
some degree of pre-organization (helicity)19,20 and thus favor
the interaction with helically structured DNA. Furthermore,
chirality has been used as a tool to increase sequence selec-
tivity.21 Preferential complexation of DNA by PNA derived
Optical purity is a crucial point also for oligonucleotide
analogs, since stereochemically impure products may contain
many diastereoisomers, each interacting with complementary
DNA or RNA with different affinity. For example, methyl-
phosphonate or phosphorothioate oligonucleotides, which have
been used as antisense drugs, are normally synthesized as mix-
tures of 2n diastereoisomers (n = number of monomeric units),
and it was shown that their binding properties strongly depend
upon the absolute configuration of the phosphorus stereogenic
center.7
Peptide nucleic acids are DNA mimics, first introduced by
Nielsen, Buchardt, Berg and Egholm,8 in which the sugar-
phosphate backbone was replaced by a polyamide chain com-
posed of aminoethylglycine covalently linked to DNA bases
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This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b104146k

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from - or -amino acids could also shed some light on the
origin of homochirality in nature,22 since PNAs can act as
templates for RNA synthesis.23
Chiral PNAs have been synthesized by replacement of the
aminoethylglycine unit with - or -aminoethyl amino acids
(Chart 1a)24,25 or by more drastic changes in the original
design.26–29 However, we have recently demonstrated that chiral
PNA monomers can undergo epimerization during SPPS.30
Some of the proposed chiral modifications have also shown
DNA-complexation properties comparable with, or in some
cases better than, those of achiral PNAs.31 A substantial
modification of PNA was introduced by Nielsen and collabor-
ators,32 and by others,33 who used thymine-containing homo-
chiral oligomers derived from -ornithine (Chart 1, type b).
They showed no affinity for DNA, while forming triplexes with
RNA. Recently it was demonstrated that these products, when
synthesized by common SPPS procedures exhibit a large
amount of epimerization,34 and a synthesis of optically pure
PNA oligomers of this type has been described, based on a
modified procedure using submonomer synthesis with orni-
thine protected with orthogonal groups (Boc for Nα and Fmoc
for Nδ), although the number of coupling steps was doubled.35
The optically pure oligomers showed increased affinity for
complementary DNA and RNA. Therefore, the synthesis of
these ornithine PNAs can be regarded as one of the most chal-
lenging models for the study of epimerization as a function of
the coupling conditions.
Fig. 1 Enantiomeric analysis by GC of the decamers (a): L-9 and (b):
D-9 after hydrolysis with 6 M HCl and derivatization with HCl–propan-
2-ol and trifluoroacetic anhydride. Chiral phase Phe-3-O-TA;
temperature program: 120 ЊC (3 min), 120–160 ЊC (4 ЊC minϪ1), 160 ЊC
final isotherm; detector: FID.
In this paper we report the synthesis of thymine and uracil
PNA oligomers based on - and -ornithine both in solution
and in the solid phase, with an extensive study of the enantio-
meric composition of the products as a function of the
coupling conditions: the nature of the coupling reagent, of
the base, and the coupling protocol were investigated. The
enantiomeric analysis was based on the previous experience
developed by some of us in the field of chiral recognition, in
particular of amino acids,36–38 and of PNAs30 by chromato-
graphic methods. The aim is to develop a synthetic method
for extensively reducing the epimerization in the coupling of
ornithine PNA monomers.
Chart 2
oligomers failed, due to the low solubility of the products and
low coupling yields.
Results and discussion
The decamers - and -(ThyOrn)10GlyNH2 8 and - and
-(UrOrn)10GlyNH2 9 were prepared by standard SPPS on an
MBHA-Rink amide resin, using HBTU–diisopropylethylamine
(DIEA) with 5 min preactivation (see Experimental section).
This resulted in extensively epimerized products (ee = 10% for L-
8, 6% for D-8, 34% for L-9 and 28% for D-9). The gas chromato-
grams obtained for the decamers L-9 and D-9 are reported as an
example in Fig. 1.
In order to establish which step of the peptide synthesis was
responsible for the very high epimerization observed during the
synthesis of the decamers, we analyzed the enantiomeric com-
position of ornithine after each step of the solid-phase syn-
thesis of the dimer L-10 under the same conditions used for the
synthesis of decamers. The results showed that epimerization
occurred in the coupling step of the first chiral monomer to the
solid phase to an extent comparable to that observed for the
decamers (ee = 20%), and was not substantially increased by
either the capping or the deprotection steps. After the introduc-
tion of the second chiral monomer, the overall enantiomeric
composition was almost the same. We therefore concluded that
each step led to the same extent of epimerization during the
coupling reaction. As a consequence, the synthesis of the
decamers by standard SPPS should give rise to 1024 (= 210)
diastereoisomers.
Synthesis of chiral monomers and oligomers
Fmoc-protected T- (2) and U-containing (3) monomers were
synthesized by treating N δ-Fmoc-ornithine with 1-(carboxy-
methyl)thymine (CMT) or 1-(carboxymethyl)uracil (CMU)39
N-hydroxysuccinimidyl (OSu) esters (Scheme 1, route a), with-
out protection of the carboxy group. The overall yields
obtained with this strategy were excellent (70–80% based on
ornithine), although the optical purity was not completely pre-
served (ee = 92–98%). Therefore, we protected the carboxylic
function of N δ-Fmoc-ornithine as its methyl ester before treat-
ing it with CMT and CMU, using 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethlyluronium tetrafluoroborate (TBTU) as
coupling agent, and removed the methyl group by mild
hydrolysis using barium hydroxide in THF–water in the last
step (Scheme 1, route b). In this way optically pure monomers
2 and 3 were obtained (ee > 98%).
The oligomers synthesized in the present study (7–10) are
reported in Chart 2.
The pentamers - and -(ThyOrn)5GlyOEt L- and D-7 (Chart
2) were synthesized in solution using 2-(benzotriazol-l-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)–
Et3N and precipitation after each step (see Experimental
section). The two pentamers were obtained in satisfactory
yields, but with a low ee (68%). Epimerization was measured
either on a tetraamidic chiral stationary phase (Phe-3-O-TA)
column prepared in our laboratories,40,41 or on a commercial
Chirasil-Val column.42 However, attempts to prepare longer
Effect of the coupling conditions on epimerization
Both monomers 2 and 3, as well as other chiral PNAs, are N-
alkanoyl amino acids and therefore, similarly to N-acetyl amino
J. Chem. Soc., Perkin Trans. 1, 2001, 2690–2696
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Table 1 Effect of the reaction conditions on epimerization during the coupling of the monomer -3 to the MBHA-Rink amide resin
Coupling agent
Base
Protocol
Average yielda (%)
%-Enantiomer (std. dev.)b
HBTU
HBTU
HATU
HATU
HATU
HATU
HATU
HATU
DIEA
DIEA
DIEA
DIEA
DIEA
TMP
a
b
a
b
c
a
b
c
>99
48.5 (0.3)
22.3 (2.1)
44.6 (0.5)
17.0 (2.7)
6.2 (0.6)
8.7 (1.1)
3.9 (0.9)
1.0 (0.3)
95
>99
97
99
98
98
92
TMP
TMP
a Measured by the absorption, at 301 nm, of the piperidine–dibenzofulvene adduct obtained after deprotection (referred to that obtained in the
deprotection of the Fmoc-protected MBHA-Rink amide resin). b Measured by chiral analysis using GC-MS with Chirasil-Val column, single ion
detection at 166 amu.
Scheme 1
acids or to dipeptides, are more liable to racemization than are
the carbamate-protected amino acids commonly used in
SPPS.43 In a previous work on ornithine PNA, van Boom and
co-workers utilized a model reaction in solution to test the
effect of various coupling agents (HBTU, BOP, CF3NO2Py-
BOP, DCC–HOBt) on the epimerization. The yields were found
to be low (75–80%) and racemization rather high (10–25%).35
In preliminary experiments, the monomers D- and L-3 were
directly coupled to the MBHA-Rink amide resin using HBTU
and DIEA, then cleaved and analyzed as reported above. In
agreement with the literature when using dipeptides as syn-
thons,3 it was shown that racemization could be reduced by
decreasing the amount of base, and by eliminating the pre-
activation step before coupling (results not shown). Attempts to
decrease the equivalents of the base under the 1 : 1 ratio (based
on monomer) did not give satisfactory results, as indicated by
At least three tests were performed for each coupling
condition, and yields were measured on the basis of the
dibenzofulvene–piperidine adduct absorption (301 nm) after
deprotection.47 In all protocols, 4 equivalents of the monomer,
of the coupling agent, and of the base (based on the resin active
sites) were used. The results are reported in Table 1.
First it can be noted that for each protocol the use of HATU
instead of HBTU led to a lower degree of epimerization, and
even better results were obtained by substituting DIEA with
collidine (TMP). The coupling protocol was found to be
important also.
In protocol a the monomer L-3, the coupling agent, and the
base in 1 : 1 : 1 proportions were mixed together and stored at rt
for 5 min before being introduced into the reactor and stirred
for 30 min. These conditions were chosen in order to reproduce
those used in solid-phase synthesis; a large degree of racemiz-
ation was observed, as a consequence of the action of the base
on the monomer in its activated form. Accordingly, using
HATU as coupling agent, the stronger base DIEA gave rise to
larger quantites of the -isomer (44.6%) than when using the
weaker base TMP (8.7%).
3
positive Kaiser tests. Mixed solvents, such as DMF–CH2Cl2
could not be used in this case, due to the lack of solubility of
the monomers 2 and 3 in CH2Cl2. The addition of 1-hydroxy-
benzotriazole (HOBt) was avoided, since it has been reported to
increase epimerization.3
Using the same test reaction we then evaluated the effect
of the coupling agent, of the base used for neutralization, and
of the addition mode of the base on the epimerization of
the monomer L-3. According to Carpino et al. the use of the
coupling agent HATU44,45 and of the base sym-collidine (2,4,6-
trimethylpyridine, TMP)46 is a convenient combination for
obtaining high yields and for preventing epimerization. We
therefore focused our attention on these conditions.
In protocol b the monomer L-3 and the coupling agent in a
1 : 1 ratio were mixed and stored at rt for 5 min. The mix-
ture was then introduced into the reactor and the base was
immediately added. The results obtained by this protocol
improved, clearly showing that the preactivation step is critical
and should be avoided, though in some cases this can lead to
lower yields. When using this protocol, a competition occurs
between acylation and racemization of the active ester, and,
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Conclusions
In the present work we have demonstrated that ornithine-based
chiral PNA analogs undergo extensive epimerization which is
strongly dependent on the coupling conditions. This issue has
not been addressed as much as for ordinary peptide synthesis,
since the first PNAs described were achiral. It is therefore very
important to carefully determine the enantiomeric purity of
chiral PNAs before performing hybridization experiments with
nucleic acids. By carefully examining the effect of several
parameters on the epimerization rate of ornithine-based
monomers we found that the stepwise addition of a weak base
(TMP), together with the use of HATU, dramatically decreases
epimerization during the coupling steps, by favoring the
acyl nucleophilic substitution versus proton abstraction. The
method reported herein is particularly suitable not only for
chiral PNA analogs, but also for peptide couplings with amino
acids or segments that present severe epimerization problems.
The protocol developed allowed us to synthesize decamers
containing either - or -ornithine of high optical purity, which
will give more clear-cut results about the effect of chirality on
the stability and conformational properties of complexes
formed by these molecules with complementary RNA or DNA.
Preliminary hybridization results obtained with these enantio-
merically pure compounds are very promising.
Fig.
2
a) MALDI-TOF and b) GC-MS enantiomeric analysis
(column: Chirasil-Val; temperature program: 120 ЊC (1 min), 120–175
ЊC, 4 ЊC minϪ1, 175 ЊC final isotherm) of the decamer L-9 synthesized by
protocol c in combination with HATU and TMP.
also in this case, the use of the weaker base TMP gave the best
result (3.9% of -enantiomer), with only a little loss of the
coupling efficiency (98% as compared with 99% with DIEA).
However, the degree of epimerization was still not optimal.
Protocol c was designed according to the following consider-
ations: by comparing the results of protocols a and b, it was
clear that the base should be added as late as possible, in order
to prevent the formation of the active ester in the presence of an
excess of base. Therefore, it is better to add the base in sub-
sequent steps during the coupling reaction, since this provides
only the quantity required for the neutralization of the HOBt
once the coupling reaction has occurred. Accordingly, we
devised a protocol based on the stepwise addition of the base:
the monomer and the coupling agent were mixed in a 1 : 1 ratio
and then introduced into the reactor; then 1/4 of the total
amount of base (hence in a 1 : 1 ratio with the resin active sites)
was immediately added, to initiate the reaction; the other three
(1/4) portions of the base were added stepwise, every 15 min.
The effect of this procedure is remarkable if one compares the
results obtained using HATU and DIEA for protocols b and c:
the -enantiomer content was lowered from 17% to 6.2%, with-
out loss of coupling efficiency. By using this protocol, in com-
bination with the use of HATU and TMP, we were able to
obtain coupling reactions with the formation of only 1% of the
-enantiomer (less than 1/40 of that obtained with previous
procedures), within a reasonable reaction time (1 h). The yields
obtained with this method, though lower than those obtained
with protocol b, were still high (92%), thus indicating that the
partial reaction of the coupling agents with the amino groups
of the growing peptide to give the guanidinium derivatives is
only a minor problem in this type of coupling.
By using these reaction conditions (HATU, TMP and proto-
col c), we synthesized the decamer -(UrOrn)10GlyNH2 L-9,
which was purified by HPLC and analyzed by MALDI-TOF
(Fig. 2a). A small amount of the decamer, hydrolyzed and
analyzed as reported above, showed the presence of 1.7% of
-ornithine (Fig. 2b). Unfortunately, it was not possible to
compare the optical purity obtained by us with that previously
obtained by van Boom and co-workers,35 since the enantiomeric
composition of their T10 oligomers was not reported. The
slightly higher degree of epimerization, as compared with that
observed during the monomer coupling with the same method,
and the lower coupling yields (average 83%) could indicate that
during the synthesis of homochiral oligomers coupling could
be not equally easy for all monomers, leading to better com-
petition by the epimerization reaction. However, by using
HATU, TMP, and protocol c, it was possible to decrease the
-monomer content from 34% to 1.7%, providing a product of
high optical purity.
Experimental
1
Melting points: Electrothermal apparatus. H and 13C NMR
spectra: Bruker AC 100 and AC 300; J-values are given in Hz.
Mass spectra: Finnigan spectrometer Mat SSQ 710. Optical
rotation: Autopol polarimeter, Rudolph Research. [α]D-Values
are given in 10Ϫ1 deg cm2 gϪ1. UV measurements: UVIKON 941
double-beam spectrophotometer. CD spectra: JASCO J-715
spectropolarimeter. All the commercially available reagents
were used without purification. DMF used in the synthesis and
in the coupling rections was distilled under vacuum and stored
on molecular sieves. 1-(Carboxymethyl)thymine (CMT) and
1-(carboxymethyl)uracil (CMU) were synthesized as reported
in the literature.37
N ꢀ-Fmoc-ornithine 1
- or -Ornithine hydrochloride (0.909 g, 5.39 mmol) and cop-
per() acetate (0.538 g, 2.69 mmol) were dissolved in 16 mL of
10% aq. sodium carbonate, and the solution was vigorously
stirred for 45 minutes. To the stirred solution were added 180
mL of water and 200 mL of 1,4-dioxane, followed by slow add-
ition of a solution of Fmoc-succinimidyl carbonate (2 g, 5.93
mmol) in 80 mL of 1,4-dioxane. After 45 minutes the reaction
was acidified with 6 M HCl, then extracted twice with diethyl
ether and twice with ethyl acetate. The aqueous solution was
treated with H2S (CAUTION!) for 15 minutes and stirred for
about 1 hour. After filtration to remove CuS, the solution was
concentrated under vacuum, and the pH was adjusted to 5.5
with 1 M NaOH. The solution was stored at 5 ЊC overnight,
then the precipitate was filtered off, and dried under vacuum
over P2O5 to yield 1.83 g of the title compound as a white solid.
L-1 (96%) mp 140 ЊC (decomp.) (from water); [α]2D5 ϩ1.25 (c 1
in DMF). D-1 (96%) mp 140 ЊC (decomp.) (from water); [α]2D5
Ϫ1.25 (c 1 in DMF) (Found: C, 64.20; H, 6.16; N, 7.45. Calc.
for C20H22N2O4ؒH2O: C, 64.50; H, 6.50; N, 7.52%); νmax (KBr)/
cmϪ1 3332 (NH), 3500–2500br (NH3ϩ), 2956 (CH), 1690 (C᎐O),
᎐
1596 (asymm. COOϪ), 1538 (NH), 1410 (symm. COOϪ), 1263
(C-O); δH [300 MHz; DMSO d6 (2% TFA)] 1.40–1.60 (2H,
m, CHα-CH2-CH2-CH2-NH), 1.70–1.90 (2H, m, CHα-CH2-
CH2-CH2-NH), 3.00 (2H, q, J 5.6, CHα-CH2-CH2-CH2-NH),
3.90–4.00 (1H, m, CHα-CH2-CH2-CH2-NH), 4.20–4.30 (1H, m,
CH-CH2 Fmoc), 4.30–4.40 (2H, m, CH-CH2 Fmoc), 7.30 (2H,
t, J 7.4, aromatic C-H Fmoc), 7.30–7.40 (1H, br m, Fmoc-N-
H), 7.41 (2H, t, J 7.4 aromatic C-H Fmoc), 7.68 (2H, d, J 7.3,
J. Chem. Soc., Perkin Trans. 1, 2001, 2690–2696
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aromatic C-H Fmoc), 7.88 (2H, d, J 7.4, aromatic C-H Fmoc),
8.25 (3H, br s, NH3ϩ); δC [75 MHz; DMSO-d6 (2% TFA),
assignments based on DEPT] 25.2 (CH2), 25.6 (CH2), 39.8
(CH2), 46.9 (CH), 51.9 (CH), 65.4 (CH2), 120.2 (CHArom), 125.2
(CHArom), 127.2 (CHArom), 127.7 (CHArom), 140.9 (Cquat. arom.),
L-6 (76%) mp 159–161 ЊC (from water); [α]2D0 Ϫ5.9 (c 1 in
DMF). D-6 (76%) mp 155–157 ЊC (from water); [α]2D0 ϩ5.6 (c 1 in
DMF) (Found: C, 60.78; H, 5.76; N, 10.96. Calc. for
C27H28N4O7ؒ¹H2O: C, 61.24; H, 5.51; N, 10.58%); νmax (KBr)/
¯
²
cmϪ13316 (NH), 3066 (CH), 2966 (CH), 1683 (C᎐O), 1550
᎐
144.0 (Cquat.
), 156.3 (C᎐O), 171.1 (C᎐O); m/z (CI) 355
(NH), 1266 (C-O); δH (300 MHz; DMSO-d6) 1.30–1.80 (4 H, m,
CHα-CH2-CH2-CH2-NH), 2.90–3.10 (2H, m, CHα-CH2-CH2-
CH2-NH), 3.62 (3H, s, CH3O), 4.10–4.30 (4 H, m, CH-CH2
᎐
᎐
arom.
(MHϩ, traces), 207 (20%), 179 (90), 178 (100), 115 (20), 81 (55),
79 (100), 63 (100), 62 (80).
Fmoc ϩ CH ), 4.39 (2H, s, CH -C᎐O), 5.55 (1 H, d, J 7.5, CH
᎐
α
2
N ꢀ-Fmoc-ornithine methyl ester hydrochloride 4
uracil), 7.27 (1H, s br, NH Fmoc), 7.32 (2H, t, J 7.3, aromatic
CH Fmoc), 7.40 (2H, t, J 7.2, aromatic CH Fmoc), 7.51 (1H, d,
J 7.5, CH uracil), 7.67 (2H, d, J 7.2, aromatic CH Fmoc), 7.86
(2H, d, J 7.3, aromatic CH Fmoc), 8.61 (1H, d, J 6.6, NH
amide), 11.26 (1H, s, NH uracil); δC (75 MHz; DMSO-d6,
assignments based on DEPT) 25.7 (CH2), 28.3 (CH2), 39.6
(CH2), 46.7 (CH), 49.1 (CH2), 51.8 (CH), 51.9 (CH3), 65.2
(CH2), 100.4 [CHUr(5)] 120.0 (CHArom.), 125.1 (CHArom.), 127.0
(CHArom.), 127.6 (CHArom.), 140.7 (Cquat. arom), 143.8 (Cquat. arom),
Into a suspension of Nδ-Fmoc-ornithine (1.5 g, 4.2 mmol) in
100 mL of methanol, cooled with an ice-bath, was bubbled
gaseous HCl (CAUTION!) for 20 min. The mixture was then
allowed to attain room temperature during 3 h, and the solvent
was evaporated off. Methanol was added and evaporated thrice
in order to eliminate excess of HCl. The crude product was
dissolved in MeOH and recrystallized by addition of diethyl
ether (1.48 g).
146.6 [CHUr(6))], 150.9 (C᎐O), 156.2 (C᎐O), 162.4 (C᎐O), 163.9
L-4 (91%) mp 124 ЊC (from Et2O); [α]2D0 ϩ 10.5 (c 1 in MeOH).
D-4 (91%) mp 120–122 ЊC (from Et2O); [α]2D0 Ϫ10.6 (c 1 in
MeOH) (Found: C, 60.00; H, 6.46; N, 6.69. Calc. for
C21H24N2O4ؒH2O: C, 59.64; H, 6.43; N, 6.62%); νmax (KBr)/cmϪ1
᎐
᎐
᎐
(C᎐O), 167.1 (C᎐O), 172.2 (C᎐O); m/z (ESI) 521 (MHϩ, 100%).
᎐
᎐
᎐
N ꢀ-Fmoc-Nꢁ-(thymin-1-ylacetyl)ornithine 2 and N ꢀ-Fmoc-Nꢁ-
(uracil-1-ylacetyl)ornithine 3
3339 (NH), 3200–2500br (NH3ϩ), 2954 (CH), 1746 (C᎐O), 1693
᎐
(C᎐O), 1535 (NH), 1450 (CH), 1266 (C-O), 1149 (C-O); δ (400
᎐
H
Route a. CMT (0.275 g, 1.5 mmol) or CMU (0.255 g, 1.5
mmol) and N-hydroxysuccinimide (0.170 g, 1.5 mmol) were
dissolved in the minimum amount of DMF. The solution
was cooled to 0 ЊC and DCC (0.310 g, 1.5 mmol) was added.
After 15 minutes of stirring, the solution was allowed to warm
at room temperature. After 5 hours the DCU was filtered
off, washed with DMF, and N δ-Fmoc-Orn 1 was added. The
solution was stirred for 24 h, then was concentrated under
vacuum. Water was added and the solution was stored at 5 ЊC.
After several hours the precipitate was filtered off, washed with
water, redissolved in DMF, and precipitated with diethyl ether
to yield the pure product as a white solid (2: 0.550 g. 3: 0.555 g.
Average yield 70%).
MHz; DMSO-d6) 1.40–1.60 (2H, m, CHα-CH2-CH2-CH2-NH),
1.70–1.90 (2H, m, CHα-CH2-CH2-CH2-NH), 3.00 (2H, q,
J 6.4, CHα-CH2-CH2-CH2-NH), 3.74 (3H, s, OCH3), 4.00–4.10
(1H, m, CHα-CH2-CH2-CH2-NH), 4.21 (1H, t, J 6.8, CH-CH2
Fmoc), 4.30 (2H, d, J 6.8, CH-CH2 Fmoc), 7.34 (2H, t, J 7.4,
aromatic CH Fmoc), 7.37 (1H, t, J 5.8, NH-Fmoc), 7.42 (2H, t,
J 7.4, aromatic CH Fmoc) 7.69 (2H, d, J 7.4, aromatic CH
Fmoc), 7.90 (2H, d, J 7.4, aromatic CH Fmoc), 8.53 (3H, br s,
NH3ϩ); δC (75 MHz; DMSO-d6, assignments based on DEPT)
24.9 (CH2), 27.4 (CH2), 39.5 (CH2), 46.7 (CH), 51.7 (CH), 52.7
(CH3), 65.3 (CH2), 120.1 (CHArom.), 125.1 (CHArom.), 127.0
(CHArom.), 127.6 (CHArom.), 140.7 (Cquat. arom), 143.9 (Cquat. arom),
156.1 (C᎐O), 169.8 (C᎐O); m/z (CI) 369 (MHϩ, 15%), 207 (12),
᎐
᎐
179 (100), 165 (7), 113 (10), 79 (10).
Route b. To a suspension of 1.2 mmol of the methyl ester 5
(or 6) in 39 mL of THF was added a solution of Ba(OH)2ؒ8H2O
(568 mg, 1.8 mmol) in water (39 mL). After 15 minutes the
reaction was complete, as monitored by TLC (silica gel; eluent:
methanol–dichloromethane 1 : 9). The pH was adjusted to 2
with HCl, the solvent was evaporated off, and the residue was
dissolved in the minimum amount of DMF and precipitated
with water. The solid was filtered off, dried under vacuum,
redissolved in DMF (6 mL), filtered on a sintered glass funnel
to eliminate insoluble residues, and dried under vacuum over
P2O5. Yields: 2: 0.813 g (99%). 3: 0.752 g (99%). Gas chromato-
graphic analysis (see below) showed the presence of 0.1–0.6%
of the minor isomer.
N ꢀ-Fmoc-Nꢁ-(thymin-1-ylacetyl)ornithine methyl ester 5 and
N ꢀ-Fmoc-Nꢁ-(uracil-1-ylacetyl)ornithine methyl ester 6
Fmoc-ornithine methyl ester hydrochloride (500 mg, 1.23 mmol)
and CMT (0.251 g, 1.36 mmol) or CMU (0.255 g, 1.50 mmol)
were dissolved in DMF (6 mL). TBTU (655 mg, 2.04 mmol)
was added at room temperature, and DIEA was gradually
added until basic pH (about 8) (total addition: 711 µL). After
two hours under stirring at room temperature, the DMF was
partly evaporated off and water was added to precipitate the
product (5: 0.582 g, 6: 0.503 g).
L-5 (87%) mp 164–166 ЊC (from water); [α]2D0 Ϫ5.8 (c 1 in
DMF). D-5 (87%) mp 163–165 ЊC (from water); [α]2D0 ϩ5.0 (c 1 in
DMF) (Found: C, 62.60; H, 5.67; N, 10.21. Calc for
C28H30N4O7: C, 62.91; H, 5.66; N, 10.48%); νmax (KBr)/cmϪ1
L-2 mp 240 ЊC (decomp.), (from Et2O); [α]2D0 Ϫ4.1 (c 1 in
DMF). D-2 mp 240 ЊC (decomp.), (from Et2O); [α]2D0 ϩ4.0 (c 1 in
DMF) (Found: C, 59.10; H, 5.78; N, 10.39. Calc. for
C27H28N4O7ؒ1.5 H2O: C, 59.23; H, 5.70; N, 10.23%); νmax (KBr)/
cmϪ1 3600–2800br (OH), 3324 (NH), 3283 (NH), 3063 (CH),
3330 (NH), 3070 (CH), 2950–2920 (CH), 1680 (C᎐O), 1550 (δ
᎐
NH), 1230 (C-O); δH (300 MHz; DMSO-d6) 1.40–1.80 (4H, m,
CHα-CH2-CH2-CH2-NH), 1.74 (3H, s, CH3 thymine), 2.90–3.00
(m, 2 H, CH2-NH), 3.63 (3H, s, CH3O), 4.20–4.40 (4H, m, CH-
CH2 Fmoc ϩ CHα), 4.36 (2H, s, CH2 acetyl linker), 7.25–7.35
(3H, m, aromatic CH Fmoc ϩ NH-Fmoc), 7.35–7.45 (3H, m,
CH thymine ϩ aromatic CH Fmoc ), 7.68 (2H, d, J 7.3,
aromatic CH Fmoc), 7.88 (2H, d, J 7.4, aromatic CH Fmoc),
8.60 (1H, d, J 7.4, NH amide), 11.24 (1H, s, NH thymine);
δC (75 MHz; DMSO-d6, assignments based on DEPT) 11.9
(CH3), 25.8 (CH2), 28.4 (CH2), 39.7 (CH2), 46.8 (CH), 48.9
(CH2), 51.78 (CH), 51.83 (CH3), 65.3 (CH2), 107.9 [CThy(5)],
120.1 (CHArom. Fmoc), 125.1 (CHArom. Fmoc), 127.1 (CHArom. Fmoc),
127.6 (CHArom. Fmoc), 140.7 (Cquat. arom), 142.4 [CThy(6)], 143.9
2950 (CH), 1698 (C᎐O), 1674 (C᎐O), 1538 (NH), 1264 (C-O);
᎐
᎐
δH (300 MHz; DMSO-d6) 1.40–1.80 (4H, m, CHα-CH2-CH2-
CH2-NH), 1.74 (3H, s, CH3 thymine), 2.90–3.10 (2H, m, CH2-
NH), 4.10–4.30 (4H, m, CH-CH2 Fmoc ϩ CHα), 4.34 (2H, s,
CH2 acetyl linker), 7.25–7.35 (1H, m, NH-Fmoc), 7.33 (2H, t, J
7.3, aromatic CH Fmoc), 7.41 (1H, s, CH thymine), 7.41 (2H, t,
J 7.3, aromatic CH Fmoc), 7.68 (2H, d, J 7.3, aromatic CH
Fmoc), 7.88 (2H, d, J 7.4, aromatic CH Fmoc), 8.46 (1H, d,
J 7.7, NH amide), 11.2 (1H, s, NH thymine); δC (75 MHz;
DMSO-d6, assignments based on DEPT) 12.0 (CH3), 26.0
(CH2), 28.8 (CH2), 40.0 (CH2), 46.9 (CH), 49.1 (CH2), 52.1
(CH), 65.4 (CH2), 108.0 [Cquat Thy(5)], 120.2 (CHArom. Fmoc), 125.3
(CHArom. Fmoc), 127.2 (CHArom. Fmoc), 127.7 (CHArom. Fmoc), 140.9
(Cquat. arom), 151.0 (C᎐O), 156.2 (C᎐), 164.5 (C᎐O), 167.2 (C᎐O),
᎐
᎐
᎐
᎐
172.2 (C᎐O); m/z (ESI) 535 (MHϩ), 557 (MNaϩ).
(Cquat. arom), 142.5 [CHThy(6)], 144.1 (Cquat.
), 151.1 (C᎐O),
᎐
arom
᎐
2694
J. Chem. Soc., Perkin Trans. 1, 2001, 2690–2696

View Article Online
156.3 (C᎐O), 164.6 (C᎐O), 167.1 (C᎐O), 173.4 (C᎐O); m/z (CI)
at rt; 4) capping with a solution of acetic anhydride (4.7%) and
᎐
᎐
᎐
᎐
521 (MHϩ, 1%), 281 (4), 207 (8), 179 (100), 165 (8), 115 (4).
L-3 mp 200 ЊC (from Et2O); [α]2D0 Ϫ3.4 (c 1 in DMF). D-3 mp
200 ЊC (from Et2O); [α]2D0 ϩ3.4 (c 1 in DMF) (Found: C, 61.37;
H, 5.38; N, 11.05. Calc. for C26H26N4O7: C, 61.65; H, 5.17; N,
11.06%); νmax (KBr)/cmϪ1 3286 (NH), 3063 (CH), 2951 (CH),
pyridine (4%) in DMF (1 min). All steps were repeated twice
and the resin was washed five times with 500 µL of DMF after
each step. Cleavage from the resin was achieved by treatment
with a TFA–water 95 : 5 mixture for 4 h. The products were
then precipitated with diethyl ether, centrifuged, and washed
four times with diethyl ether [yield: 8 63 mg (88%), 9 63 mg
(92%)]. The crude product was purified by HPLC using a
reversed-phase RP-18 column Spherisorb 10 ODS (30 × 1 cm),
by gradient elution (eluent A: water; eluent B: 0.05% TFA in
acetonitrile, gradient from 90% A to 100% B).
1670 (C᎐O), 1538 (NH), 1256 (C-O); δH (300 MHz; DMSO-d6)
᎐
1.40–1.80 (4H, m, CHα-CH2-CH2-CH2-NH), 2.98 (2H, q, J 5.7,
CH2-NH), 4.20–4.30 (4H, m, CH-CH2 Fmoc ϩ CHα), 4.38 (2H,
s, CH -C᎐O), 5.54 (1H, d, J 7.8, CH uracil), 7.30–7.40 (1H, m,
᎐
2
NH-Fmoc), 7.33 (2H, t, J 6.7, aromatic CH Fmoc), 7.41 (2H, t,
J 7.3, aromatic CH Fmoc), 7.53 (1H, d, J 7.8, CH uracil), 7.68
(2H, d, J 7.3, aromatic CH Fmoc), 7.88 (2H, d, J 7.4, aromatic
CH Fmoc), 8.47 (1H, d, J 7.7, NH amide), 11.25 (1H, s, NH
uracil); δC (75 MHz; DMSO-d6, assignments based on DEPT)
25.9 (CH2), 28.6 (CH2), 39.8 (CH2), 46.8 (CH), 49.1 (CH2), 51.8
(CH), 65.2 (CH2), 100.4 [CHUracil(5)], 120.1 (CHArom. Fmoc), 125.1
(CHArom. Fmoc), 127.0 (CHArom. Fmoc), 127.6 (CHArom. Fmoc), 140.7
8 MALDI-TOF: Calc.: M, 2876.9. Found: MHϩ, 2875.
9 MALDI-TOF: Calc.: M, 2736.6. Found: MHϩ, 2741.
Analysis of epimerization during solid-phase peptide synthesis
Analysis of steps. The steps reported above for the decamer
synthesis were repeated on 50 mg of MBHA-Rink amide resin
with Fmoc-Gly, and then twice with L-3 to obtain the dimer 10.
After each step involving the chiral monomer, one-sixth of the
resin was separated and treated with a TFA–water 95 : 5 mix-
ture for 4 h. These solutions were dried under vacuum, and the
residue was treated as reported in the following section for GC
chiral analysis.
(Cquat. arom), 143.9 (CHquat arom), 146.6 [CUracil(6)], 150.9 (C᎐O),
᎐
156.1 (C᎐O), 163.8 (C᎐O), 166.8 (C᎐O), 173.1 (C᎐O); m/z (CI)
᎐
᎐
᎐
᎐
507 (MHϩ, traces), 266 (10%), 207 (20), 196 (25), 179 (100).
H-(L-ThyOrn)5GlyOEt 7
The pentamer was obtained by homogeneous peptide synthesis,
through deprotection–coupling cycles. Typical procedures for
each step will be reported.
Monomer coupling. In a typical experiment 10 mg of the
MBHA-Rink amide resin (corresponding to 5 µeq. of reactive
sites) were used for the evaluation of racemization during
monomer coupling. After treatment with 30% piperidine in
DMF, the coupling reaction was carried out with the monomer
L-3 using the following protocols.
Protocol a. The monomer L-3 (10 mg, 20 µmol) and the
coupling agent (HBTU or HATU, 20 µmol) were dissolved in
100 µL of DMF; then 20 µL of a 1 M solution of the base
(DIEA or TMP) in DMF were added. The mixture was stored
at rt for 5 min before being introduced into the reactor, and was
then stirred for 30 min.
Protocol b. The monomer L-3 (10 mg, 20 µmol) and the
coupling agent (HBTU or HATU, 20 µmol), were dissolved in
100 µL of DMF and stored at rt for 5 min; then it was intro-
duced into the reactor. Immediately, 20 µL of a 1 M solution of
the base (DIEA or TMP) were added, and the reaction mixture
was stirred for 30 min.
Protocol c. The monomer L-3 (10 mg, 20 µmol) and the
coupling agent (HBTU or HATU, 20 µmol), were dissolved in
100 µL of DMF and stored at rt for 5 min; then it was intro-
duced into the reactor. Immediately 5 µL of a 1 M solution of
the base (DIEA or TMP) were added, to start the reaction, and
the reaction mixture was stirred. Three other 5 µL portions of
the 1 M solution of the base (DIEA or TMP) were added, one
every 15 min. The overall reaction time was 1 h. After each
reaction, the ornithine monomer was deprotected with 30%
piperidine. The absorbance of the resulting solution was
measured at 301 nm, and compared with that obtained by the
deprotection of the resin, in order to calculate the yield. The
monomer was then cleaved from the resin as reported above,
and subjected to chiral analysis.
First coupling with glycine ethyl ester. D- or L-2 (0.120 g,
0.23 mmol) was dissolved in the minimum amount of
DMF. The solution was cooled to 0 ЊC and GlyOEtؒHCl
(0.04 g, 0.28 mmol) and Et3N (87 µL, 0.28 mmol) were added.
After 15 minutes the solution was allowed to warm to room
temperature and HBTU (0.11 g, 0.28 mmol) was added. After
2 hours, a few mL of water were added to quench the reaction
and precipitate the product, which was then purified by pre-
parative HPLC (C18 column, eluent MeOH–water 72 : 28) to
yield 0.11 g of N δ-Fmoc-N α-(thymin-1-ylacetyl)ornithylglycine
ethyl ester as a white solid (80%)
Deprotection. The compound obtained from the coupling
reaction (for the first step, Fmoc--ThyOrn-GlyOEt) was dis-
solved in 15% piperidine–DMF solution, and stirred at rt for 10
minutes. The solution was evaporated to dryness. The residue
was taken up in MeOH–Et2O and isolated by centrifugation.
Typical yield >99%.
Coupling. The compound obtained from the deprotection
reaction was dissolved in the minimum amount of DMF.
Fmoc--ThyOrn (1 eq.), HBTU (1 eq.) and Et3N (1 eq.) were
added. The solution was stirred at rt for 3 hours, then evapor-
ated to dryness. The residue was taken up in MeOH–Et2O and
isolated by centrifugation. Yields for each coupling step were as
follows: 1) 72%, 2) 83%, 3) 70%, 4) 76%.
7. MALDI-TOF: Calc. M, 1504.6. Found: MHϩ, 1506.
Solid-phase synthesis of decamers H-(ThyOrn)10-Gly-NH2 8 and
H-(UrOrn)10-Gly-NH2 9
Synthesis of optically pure L-9. This was carried out by using
standard coupling (HBTU, DIEA with 5 min preactivation) for
the first glycine residue, and protocol c for all ornithine mono-
mers. Yields for each step were: 1(gly): 95%; 2) 93%; 3): 82%; 4)
>99%; 5) 63%; 6) 82%; 7) 78%; 8) >99%; 9) 71%; 10) 71%.
The synthesis of 8 was carried out on a Shimadzu PSSM-8
peptide synthesizer and that of 9 manually in a glass reactor
using the same Fmoc protocol. An MBHA-Rink amide resin
(50 mg, 0.5 m eq. gϪ1) was used with the following steps for each
monomeric unit: 1) deprotection with 30% piperidine in DMF
(500 µL; 8 min); 2) preactivation in the first step was obtained
by adding to Fmoc-Gly (29.1 mg, 98 µmol) and TBTU
(31.5 mg, 98 µmol) a 1 M solution of DIEA in DMF (147 µL,
147 µmol), a 0.5 M solution of HOBt in DMF (196 µL,
98 µmol) and mixing for 5 min at room temperature; in the
following cycles the monomer 2 (51 mg, 98 µmol) or 3 (49.6 mg,
98 µmol) was used instead of Fmoc-Gly; 3) coupling for 30 min
Enantiomeric analysis by GC. Hydrolysis of the monomers
and of the products obtained by SPPS (typical sample: 2 mg)
was performed in a 2 mL solution of 6 M HCl for 6 h at 100 ЊC.
The solution was then dried under vacuum, and the residue was
treated with 4 mL of 2 M HCl in propan-2-ol and heated in a
screw-cap test-tube (90 ЊC for 1 h); after evaporation of the
J. Chem. Soc., Perkin Trans. 1, 2001, 2690–2696
2695

View Article Online
13 K. L. Dueholm and P. E. Nielsen, New J. Chem., 1997, 21, 19.
mixture, the sample was treated with 0.3 mL of trifluoroacetic
anhydride and 2 mL of CH2Cl2, and heated in a screw-cap
test-tube (60 ЊC for 1 h); the solution was evaporated and the
residue was redissolved in 0.5 mL of CH2Cl2.
14 H. Knudsen and P. E. Nielsen, Anti-Cancer Drugs, 1997, 8, 113.
15 E. Bayer, Angew. Chem., Int. Ed. Engl., 1991, 30, 113.
16 B. Hyrup, M. Egholm, P. E. Nielsen, P. Wittung, B. Nordén and
O. Buchardt, J. Am. Chem. Soc., 1994, 116, 7964.
Chiral GC analysis was performed on a Phe-3-O-TA column
(20 m; ID 0.3 mm; film thickness 0.2 µm), on a DANI 3900
instrument (detector FID), with the following temperature pro-
gram: 120 ЊC for 3 min, 120–160 ЊC (3 ЊC minϪ1), 160 ЊC final
isotherm; the retention times were tD 20 min, and tL 21 min.
Identification of the peaks was performed by injection of
standard samples of - and -ornithine derivatives, and con-
firmed by GC-MS. Standard deviation of /( ϩ ) or /( ϩ
)% for this chromatographic system was 1%.
Chiral GC-MS analyses were performed on a Hewlett-
Packard HP 6890 instrument, equipped with a 5973 Mass
Selective Detector (quadrupole analyzer), on a Chirasil-Val
(25 m, ID 0.25 mm; film thickness 0.12 µm), in scan mode for
the identification of peaks, and in SIM mode (m/z 166, 306,
355) for quantitation (in order to achieve best signal-to-noise
ratio). The temperature program was the following: 120 ЊC for
1 min, 120–175 ЊC, (4 ЊC minϪ1), 175 ЊC final isotherm. Reten-
tion times were tD 14.8 min for -ornithine and tF 15.5 min for
-ornithine. Standard deviation of /( ϩ ) or /( ϩ )% for
this chromatographic system was 0.2%, with a detection limit
of 0.1%.
17 B. Hyrup, M. Egholm, M. Rolland, P. E. Nielsen, R. H. Berg and
O. Buchardt, J. Chem. Soc., Chem. Commun., 1993, 518.
18 K. L. Dueholm, K. H. Petersen, D. K. Jensen, M. Egholm, P. E.
Nielsen and O. Buchardt, Bioorg. Med. Chem. Lett., 1994, 4, 1077.
19 P. Wittung, P. E. Nielsen, O. Buchardt, M. Egholm and B. Nordén,
Nature, 1994, 368, 561.
20 S. Sforza, G. Haaima, R. Marchelli and P. E. Nielsen, Eur. J. Org.
Chem., 1999, 197.
21 S. Sforza, R. Corradini, S. Ghirardi, A. Dossena and R. Marchelli,
Eur. J. Org. Chem., 2000, 2905.
22 J. L. Bada, Nature, 1995, 374, 594.
23 C. Bohler, P. E. Nielsen and L. E. Orgel, Nature, 1995, 376, 578.
24 G. Haaima, A. Lohse, O. Buchardt and P. E. Nielsen, Angew. Chem.,
Int. Ed. Engl., 1996, 35, 1939.
25 A. Puschl, S. Sforza, G. Haaima, O. Dahl and P. E. Nielsen,
Tetrahedron Lett., 1998, 39, 4707.
26 G. Lowe and T. Vilaivan, J. Chem. Soc., Perkin Trans. 1, 1997,
539; G. Lowe and T. Vilaivan, J. Chem. Soc., Perkin Trans. 1, 1997,
547.
27 P. Lagriffoule, P. Wittung, M. Eriksson, K. Jensen, B. Nordèn,
O. Buchardt and P. E. Nielsen, Chem. Eur. J., 1997, 3, 912.
28 Y. S. Tsantrizos, J. F. Lunetta, M. Boyd, L. D. Fader and M. C.
Wilson, J. Org. Chem., 1997, 62, 5451.
29 A. Pushl, T. Tedeschi and P. E. Nielsen, Org. Lett., 2000, 2, 4161.
30 R. Corradini, G. L. Di Silvestro, S. Sforza, G. Palla, A. Dossena,
P. E. Nielsen and R. Marchelli, Tetrahedron: Asymmetry, 1999, 10,
2063.
31 S. Jordan, C. Schwemler, W. Kosch, A. Kretschmer, U. Stropp,
E. Schwenner and B. Mielke, Bioorg. Med. Chem. Lett., 1997, 7, 687.
32 K. H. Petersen, O. Buchardt and P. E. Nielsen, Bioorg. Med. Chem.
Lett., 1996, 6, 793.
33 E. Lioy and H. Kessler, Liebigs Ann. Chem., 1996, 201.
34 S. Sforza, Tesi di Dottorato, Università di Parma, Italy, 1997.
35 A. C. van der Laan, I. van Amsterdam, G. I. Tesser, J. H. van Boom
and E. Kuyl-Yeheskiely, Nucleosides Nucleotides, 1998, 17, 219.
36 R. Corradini, A. Dossena, G. Galaverna, R. Marchelli and G. Palla,
Chiral Recognition by Chromatographic Methods in Seminars in
Organic Synthesis, Polo Editoriale Chimico, 1994, 4, pp. 79–103.
37 I. Gandolfi, G. Palla, L. Delprato, F. De Nisco, R. Marchelli and
C. Salvadori, J. Food Sci., 1992, 57, 377.
Acknowledgements
This work was supported by a grant from CNR (Consiglio
Nazionale delle Ricerche, Rome, Target Project ‘Biotech-
nology’). We wish to thank Luigia Mazzucco and Alessandra
Marchesi for their helpful contribution to this work.
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