Preparation of free and of specifically protected oligo[β-malic acids] for enzymatic degradation studies

Article abstract of DOI:10.1002/1099-0690(200004)2000:7<1207::AID-EJOC1207>3.0.CO;2-2

The polyanionic poly[β-(S)-malic acids] (β-PMA) occur in slime molds (myxomycetes), black yeasts and other fungi and are involved in DNA replication. In order to be able to study the cleavage mechanism of β-PMA hydrolases, we have synthesized cyclic and linear oligomers of malic acid (β-OMA) consisting of up to eight residues. To this end, fragments with three different protecting groups were prepared, with allyl ester groups on the C-terminus, TBDPS groups at the O-terminus, and benzyl ester groups at the side chains (Schemes 2, 3, 7). Selective deprotection and fragment coupling (COCl₂/C₅H₅N/CH₂Cl₂/-75 °C) gave dimers, tetramers, and octamers, either fully protected or specifically protected at the O- or C- terminus or at the side chain acid groups, and also fully deprotected oligoacids (Schemes 3-7). The new compounds were fully characterized (R(f), IR, ¹H- and ¹³C-NMR spectroscopy, mass spectrometry and elemental analysis or high-resolution electrospray mass spectrometry). Enzymatic degradation experiments with the previously prepared cyclo-tetramer, the unprotected, and the O- or C-terminally protected samples of linear β-OMAs show that the enzyme from Physarum polycephalum is an exo-hydrolase cleaving the chain from the O-terminus.

Full text of DOI:10.1002/1099-0690(200004)2000:7<1207::AID-EJOC1207>3.0.CO;2-2

FULL PAPER
Preparation of Free and of Specifically Protected Oligo[β-Malic Acids] for
Enzymatic Degradation Studies
Christoph M. Krell[a] and Dieter Seebach*[a]
Keywords: Poly[β-(S)-malic acid] / Biopolyesters / Oligomers / Protecting groups / Allyl ester / Hydrolase
The polyanionic poly[β-(S)-malic acids] (β-PMA) occur in
slime molds (myxomycetes), black yeasts and other fungi and
are involved in DNA replication. In order to be able to study
the cleavage mechanism of β-PMA hydrolases, we have syn-
thesized cyclic and linear oligomers of malic acid (β-OMA)
consisting of up to eight residues. To this end, fragments with
three different protecting groups were prepared, with allyl
ester groups on the C-terminus, TBDPS groups at the O-ter-
octamers, either fully protected or specifically protected at
the O- or C-terminus or at the side chain acid groups, and
also fully deprotected oligoacids (Schemes 3–7). The new
compounds were fully characterized (Rf, IR, 1H- and 13C-
NMR spectroscopy, mass spectrometry and elemental ana-
lysis or high-resolution electrospray mass spectrometry). En-
zymatic degradation experiments with the previously pre-
pared cyclo-tetramer, the unprotected, and the O- or C-ter-
minus, and benzyl ester groups at the side chains (Schemes minally protected samples of linear β-OMAs show that the
2, 3, 7). Selective deprotection and fragment coupling
(COCl2/C5H5N/CH2Cl2/–75 °C) gave dimers, tetramers, and
enzyme from Physarum polycephalum is an exo-hydrolase
cleaving the chain from the O-terminus.
Introduction
β-PMA hydrolyzes spontaneously at room temperature in a
buffered aqueous solution of pH 7.5.[11] However, when the
side chain carboxyl groups are esterified, the material ap-
pears to be stable under these conditions.[12]
Polyhydroxyalkanoates (PHA) represent a class of biopo-
lymers whose importance for living organisms was only re-
cognized in the last decades.[1,2] The most prominent mem-
ber is poly[(R)-3-hydroxybutyric acid] (PHB), an apolar,
water-insoluble material of high molecular weight, which
was discovered in bacteria, where it accumulates as a car-
bon and energy storage substance (sPHB) in the form of
inclusion bodies.[3] Initially, PHB and related PHAs have
attracted much interest, both, as chiral synthetic starting
materials[4] and as biocompatible and biodegradable mate-
rials.[1,5] However, in the last fifteen years PHB was also
detected in many eukaryotic organisms, and its low-molecu-
lar-weight form cPHB (complexing PHB) was found to be
involved in calcium ion transport across membranes.[6,7]
Furthermore, low-molecular-weight PHB has proven to be
a valuable material for investigating the role of any kind
of PHB in nature. Well-defined synthetic oligomers of 3-
hydroxybutanoic (OHBs) and higher alkanoic acids
(OHAs) have been most useful in the study of ion transport
and enzyme stereoselectivity, as they permit the unambigu-
ous identification of the (in some cases critical) size of an
oligomer/polymer fragment.[2,7,8]
A natural polymer consisting of malic acid was first isol-
ated from Penicillium cyclopium, thirty years ago.[13] Al-
though this substance is an inhibitor of acid proteases such
as pepsin, it was not characterized with respect to its consti-
tution. In 1989 Holler and co-workers reported that
poly[(S)-malic acid], isolated from plasmodia of the slime
mold Physarum polycephalum, acts as an endogenous inhib-
itor of DNA polymerase α.[14] Its constitution corresponds
to β-PMA.[15] The biopolyester of molecular mass 4–
50 kDa was found in the cytoplasm and in nuclei, and is
secreted into the culture medium.[16] However, highest con-
centrations were measured in the nuclei, (100 mg/mL) as
the polyanion strongly interacts with the cationic nuclear
proteins. It was proposed that β-PMA has the function of
a storage and carrier molecule (e.g. for DNA polymerases)
and is therefore assumed to be involved in DNA replica-
tion.[17] The organisms most efficient in producing β-PMA
are the black yeasts Aureobasidium sp.[18] and A. pullul-
ans,[19] from which up to 61 g/l and 9.8 g/l culture medium
were isolated, respectively. The polymer from these species
was reported to be branched, and its molecular mass is 5–
9 kDa. Recently, more β-PMA producing fungi and
myxomycetes were discovered, among them Cladosporium
cladosporioides and Corollospora fusca.[20] Until now, all
known β-PMA producing organisms are eukaryotes.
In contrast to PHB, the structurally related poly[β-(S)-
malic acid] (β-PMA) is an acidic, highly water-soluble poly-
ester which was artificially prepared before it was disco-
vered in nature.[9] The potential application of this material
as drug carrier or part of a prodrug was extensively studied
due to the non-toxicity of malic acid, a constituent of the
citric acid cycle.[10] In this context it has been shown that
Unlike the PHAs, the enzymatic degradation of which
has been studied thoroughly,[5] not much is known about
depolymerases that degrade β-PMA. Two enzymes have
been isolated and characterized up to now. The first hy-
drolase, a 68 kDa glycoprotein, was found in the cytoplasm
and culture medium of P. polycephalum.[21] It hydrolyzes β-
PMA specifically by cleaving single (S)-malic acid units in
[a]
Laboratorium für Organische Chemie der Eidgenössischen
Technischen Hochschule, ETH Zentrum,
Universitätstrasse 16, 8092 Zürich, Switzerland
Fax: (internat.) ϩ 41-1/632-1144
E-mail: seebach@org.chem.ethz.ch
Eur. J. Org. Chem. 2000, 1207Ϫ1218
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0407Ϫ1207 $ 17.50ϩ.50/0
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C. M. Krell, D. Seebach
FULL PAPER
an exolytic manner (pH optimum 3.5). Studies on a syn- formation of previously unreported isomeric monobenzyl
thetic polymer derivative suggested that hydrolysis starts at ester could thus be reduced from 5% to 3%, but not com-
the O-terminus.[22] Only recently, Steinbüchel and co- pletely suppressed. This isomer shows a singlet at δ ϭ 5.16
workers reported on a specific bacterial β-PMA hydrolase in the 1H-NMR spectrum as the only distinct signal. In
(43 kDa, pH optimum 8.1) isolated from the outer mem- view of the synthesis of long oligomer chains, the isomeric
brane of Comamonas acidovorans.[23] It was shown that both purity of the starting material is of great importance. How-
enzymes are neither metallo nor serine esterases, and that ever, the removal of the remaining impurity by recrystalliza-
they are distinct from PHB depolymerases.
tion of the low-melting solid 2 (all previous reports[27] de-
All the dissimilarities between β-PMA and the PHAs scribe the compound as an oil) failed, and final purification
make this highly functionalized biopolymer appealing for was therefore performed in the following steps.
further investigations. We have described the preparation of
cyclic oligomers of (S)-malic acid as model compounds for
β-PMA.[24] For structural and enzymatic studies, we have
now developed a synthetic route providing access to pure,
specifically modifiable and protected, linear oligomers of
the type shown in Scheme 1.
Scheme 2. Preparation of the monomers 3 and 4 by specific protec-
tion of malic acid (1)
Scheme 1. Linear oligomers of 3-hydroxyalkanoic acids and of
malic acid
Hydroxy acid 2 was further protected by alkylation of its
carboxylate group with allyl bromide in the presence of 10
mol-% of tetrabutylammonium bromide, giving alcohol 3 in
91% yield after a reaction time of only 4 h. Using standard
DCC/DMAP esterification[28] with five equivalents of allyl
alcohol, a yield of only 40% of 3 was obtained, accompan-
Results and Discussion
Synthesis of Fully Deprotected Oligomers of Malic Acid
Bearing in mind β-PMA’s sensitivity to water as well as ied by a considerable amount of dimeric material. On the
its hygroscopic nature, a synthetic strategy was devised, other hand, a short route, other than silylation of 3 followed
which involves the liberation of the side chain carboxyl by allyl ester cleavage, was desirable for the preparation of
groups under non-aqueous conditions as the final step. Fur- acid 4. Hence, 2 was allowed to react with 1.3 equivalents of
thermore, the strategy involves orthogonal protection of tert-butylchlorodiphenylsilane, giving a 3:2 mixture of the
both terminal functionalities, enabling both efficient chain desired acid 4 and its TBDPS ester derivative (doubly silyl-
elongation by segment coupling, and specific modification ated product). As the TBDPS ester does not decompose
of the termini. The protecting group chosen for the side during aqueous workup,[29] the crude mixture was treated
chain is the cleanly cleavable benzyl (Bn) ester. The allyl with aqueous TFA for 2 h, affording acid 4 in 80% yield
ester[25] was selected as second carboxylic acid protecting after column chromatography. A small amount of this
group and the tert-butyldiphenylsilyl (TBDPS) group[26] product was converted into methyl ester 5. Comparison by
was chosen to mask the O-terminus in a manner that per- HPLC with a sample of rac-5 (the enantiomers of which
mits acid chloride coupling as method for the backbone were separated on a Chiralcel OD column; for details see
ester formation.
the Experimental Section) confirmed acid 4 to be enantiom-
Following known procedures,[27] malic acid 1 was con- erically pure.
verted into the trifluoroacetate of its cyclic anhydride which
For the preparation of the oligomers, acid chloride coup-
was subsequently treated with benzyl alcohol to give the ling was used under the conditions that were successful in
monobenzyl ester 2 as the main regio isomer in high yield the synthesis of OHBs containing up to 128 units.[30] Treat-
(Scheme 2). In contrast to the published procedures, the ment of acid 4 with oxalyl chloride gave the corresponding
ring-opening was not carried out at room temperature, but oily acid chloride, which (like all acid chlorides mentioned
between –25 °C and 0 °C in CH2Cl2, conditions under in this work) had to be dried thoroughly under high va-
which the intermediate anhydride forms a suspension. The cuum prior to its use, in order to completely remove excess
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Free and of Specifically Protected Oligo[β-Malic Acids]
FULL PAPER
Scheme 4. Preparation of tetramer 13, involving transformation of
the terminal allyl ester in 9 to a benzyl ester
Tetramer 9 was therefore subjected to the conditions used
most successfully to cleave the allyl ester in dimer 6. How-
ever, the desired acid 10 (Scheme 4) was isolated in yields
of only 12% to 16% due to fast decomposition under the
basic reaction conditions, probably a result of intramolecu-
lar attack of backbone esters by the carboxylate anion. A
study on a comparable model substrate, using neutral and
weakly basic nucleophiles, revealed N-methylaniline and
imidazole to be mild alternatives to morpholine. Indeed,
when using 10 equivalents[35] of imidazole in the presence
of 8 mol-% of Pd catalyst, deprotection occurred cleanly in
less than one hour, giving acid 10 in an excellent yield. The
liberated carboxylic acid was then treated with a solution
of phenyldiazomethane,[36,37] yielding tetramer 11, which
was O-terminally deprotected to afford alcohol 12. Hydro-
genolysis of all five benzyl ester groups occurred smoothly,
and gave tetramer 13 in an overall yield of 46% (initially
2.2%). A residual amount of 1.5 equivalents of 1,4-dioxane
was detected after thorough drying under vacuum for sev-
eral days[38] (this is probably due to the product’s highly
polar nature).
Scheme 3. Formation and selective deprotection of 6, followed by
segment coupling of the dimer moieties 7 and 8
reagent (Scheme 3). Coupling with alcohol 3 at reduced
temperature occurred upon addition of pyridine, yielding
dimer 6, the first oligomer in the synthesis that bears all
three different protecting groups.[31]
Selective cleavage of the silyl ether 6 by HF–pyridine
complex[32] (which was chosen to prevent ester cleavage that
would have occurred with the basic TBAF) gave alcohol 7
in high yield. Using the conditions of Kunz and co-
workers,[33] the C-terminal carboxylic acid group was liber-
ated by Pd0-catalyzed allyl transfer to morpholine, furnish-
ing acid 8 in reproducibly excellent yields. At least 3 mol-%
of the purchased catalyst was necessary for the reaction to
take place within a reasonable period of time. Having both
moieties in hand, acid 8 was activated as before and coupled
with the alcohol segment 7 under carefully defined condi-
tions to obtain the fully protected tetramer 9. In the case
of the OHBs, it had been shown that carboxylic acids carry-
ing an acyloxy substituent in the 3-position are prone to
β-elimination after activation.[30] This destructive reaction,
which not only gives rise to elimination products but also
to oligomers containing one unit less, could be suppressed
by keeping the temperature below –45 °C.[34]
More straightforward was the preparation of the corres-
ponding dimer 17 (Scheme 5). Dibenzyl malate 14[39] was
acylated with 4 by the DCC/DMAP method,[28] giving
dimer 15, which was deprotected as above to yield pure 17.
The chromatograms of the hydroxypenta- and -triacids 13
and 17 are shown in Figure 1.
On the way to a completely deprotected oligomer, tetra-
mer 9 was first O- then C-terminally deprotected by means
of the methods used before. However, we did not succeed
in separating Ph3PϭO, formed from the Pd catalyst during
Preparation of Specifically Protected Oligomers
The substrates that had been used before to study the
workup, neither by silica gel nor by reversed-phase (RP) specificity of the β-PMA hydrolase were low-molecular-
column chromatography. Debenzylation of the crude mat- weight polymers carrying at one or the other terminus a
erial would have made an RP-HPLC purification step inev- fluorescent label or a positively charged triethylammonium
itable, furnishing the product in a hydrous and thus un- group.[22] In the course of this work, we intended to access
stable form (cf. introduction). In order to avoid this amphi- enzyme substrates that are chemically and sterically as
philic, resinous intermediate, it was intended to transform closely related to β-PMA as possible. Thus, the require-
the allyl ester into a benzyl ester prior to silyl ether cleavage ments were: (1) large enough molecular weight to be a
and debenzylation.
‘‘real’’ substrate, (2) monodisperse, (3) easy to identify and
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C. M. Krell, D. Seebach
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Scheme 5. Preparation of dimer 17
Scheme 6. Synthesis of terminally protected tetramers 21 and 22
(pKa ϭ 3.6),[18] and the first constant of malic acid
(pKa ϭ 3.4).
The C-terminally protected substrate 22 was directly pre-
pared from 18 by simultaneous hydrogenation of the double
bond and hydrogenolysis of the benzyl ester groups. Satura-
tion of the allyl group was accompanied by 3.5% of hydro-
genolytic ester cleavage, a known side reaction[41] (the tetra-
mer 13, thereby formed, was detected and quantified by
RP-HPLC, see below). Mass-spectrometric analysis of the
protected tetramers 9–12 and 18–20 by the MALDI-TOF
method was best suited to verify the monodispersity of the
compounds. For the acids 13, 21, and 22, electrospray mass
spectrometry (ESI-MS; both ion modes) was appropriate.
In order to be able to determine kind and size of oligo-
mers produced during enzymatic degradation of the sub-
strates, as well as to check the uniform composition of the
prepared compounds, RP-HPLC conditions were de-
veloped that permit the reproducible separation of the fully
deprotected (13 and 17) and terminally protected (21 and
22) oligomers (Figure 1). For that purpose, a single linear
gradient (phosphate buffer of pH ϭ 2.4[22]/MeCN) was
used (cf. Experimental Section). The purities determined
with this method were Ͼ 99% (dimer 17), 99% (tetramer
13), 97.5% (acetate 21), and 93.5% (propyl ester 22). In the
case of ester 22, we chose to keep the product in the rela-
tively stable, water-free form and, therefore, no preparative
purification was performed.
Figure 1. RP-HPLC traces of free (13 and 17) and terminally pro-
tected (21 and 22) acids (C18 column; absorbance at 220 nm; linear
gradient: cf. Experimental Section)
quantify, and (4) same polarity and solubility as β-PMA.
Therefore, tetramers that are modified as shown in
Scheme 1 were prepared.
The common precursor of the two products 21 and 22
with a free CO2H and a free OH group, respectively, is alco-
hol 18, which was obtained by cleavage of the silyl ether
group in 9 (Scheme 6). In order to prepare the O-terminally
protected substrate, 18 was first acetylated to yield 19.
Again, Pd0-catalyzed allyl ester cleavage using imidazole as
allyl acceptor proceeded cleanly and afforded acid 20 in a
yield of 78%.[40] Finally, the five benzyl ester groups were
cleaved hydrogenolytically to give 21, the first of the two
terminally modified substrates. Tetramer 21 was subjected
to a pKa determination procedure, in order to learn about
the dependence of dissociation on the degree of depro-
tonation. However, only one value (pKa ϭ 3.46) could be
measured as the other pKa values are too close and there-
Preparation of an Octamer and Tests of Other Coupling
Methods
Undoubtedly, octamers or larger oligomers are interest-
fore could not be distinguished. The measured pKa value is ing targets as their molecular weight approach that of the
in agreement with that determined for β-PMA from A. sp. biopolyester more closely. Moreover, such compounds are
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Free and of Specifically Protected Oligo[β-Malic Acids]
FULL PAPER
appealing for structural studies in solution and in the solid the benzyl ester next to the silyl protecting group is cleaved
state. Thus, octamer 23 was prepared in the same manner more slowly than the others. In order to explore the syn-
as tetramer 9, by coupling of acid 10 with alcohol 18 thetic usefulness of this rate difference, dimer 6 was hydro-
(Scheme 7). However, 10 did partially not survive the condi- genated, with careful monitoring of the progress of the reac-
tions of acid chloride formation, and as a result, by-prod- tion (Scheme 9). After 4.5 h, the reaction was stopped and
ucts were formed that were separable only with great diffi- the components of the product mixture were separated by
culty (see the low yield). Therefore, alternative coupling column chromatography. Acid 26 was isolated in analytic-
methods were tested on comparable, simpler substrates.
ally pure form in 56% yield. Obviously, the carboxylate
group near the C-terminus and next to an acyloxy group is
a better ‘‘leaving group’’ (more acidic) than the one near
the O-terminus and next to the OTBDPS group. It is known
that oxygen-bound benzyl groups are hydrogenolized faster
the better negative charge is stabilized in the ‘‘leaving
group’’.[45] However, the bulky TBDPS group might also
influence the reaction by rendering access to the catalyst
surface more difficult.
Scheme 7. Preparation of the octamer 23 by acid chloride coupling
Whereas DCC/DMAP esterification[28] led to decomposi-
tion, no conversion of the starting material was observed
with the 2,2Ј-dipyridyl disulfide method[42] in the presence
of CuBr2,[43] not even in boiling MeCN. More promising
were attempts with the BOP-Cl reagent,[44] after replacing
Et3N by NMM. However, when the tetramer segments 10
and 18 were allowed to react under these conditions, the
fumaric acid ester 24 was isolated as the main product, and
no octamer 23 was detected. The formation of 24 may be
rationalized in the following way (see Scheme 8): As a result
of carboxylic acid activation, the acidity of the protons in
α-position is increased to an extent which causes elimina-
tion to be much faster than coupling; subsequently, the ac-
tivated fumaric acid intermediate acylates the free hydroxy
group of the oligoester to form a product such as 24. Re-
placement of NMM by pyridine resulted in no conversion.
These observations suggest that a procedure that permits
the preparation of larger oligomers of this type in good
yields has to work in the absence of stronger bases.
Scheme 9. Preferred cleavage of one of the benzyl ester groups in 6
Enzymatic Degradation Studies
The oligomers 13, 17, 21, and 22, and the cyclo-tetramer
(see ref.[24]) shown in Figure 2 were subjected to enzymatic
degradation by the extracellular β-PMA hydrolase from P.
polycephalum by Holler and Gasslmaier.[46] The C-termin-
ally protected tetramer 22 was thus readily hydrolyzed to
give fragments that were detected by RP-HPLC. In con-
trast, neither the acetate 21 nor the cyclic oligomer was de-
graded at all. This is compatible with the proposal[22] that
enzymatic hydrolysis starts at the O-terminus and that the
enzyme is an exo-hydrolase.[21] Another observation is, that
not only tetramer 13 but also dimer 17 is degraded. Obvi-
ously, two monomer units are sufficient for substrate recog-
nition. The quantitative investigation of the degradation
rate, which allows for determination of KM and Vmax, is in
progress. Complete and detailed results will be published
elsewhere.[46]
Scheme 8. β-Elimination prior to acylation in the case of carboxylic
acid activation in the presence of a base
Selective Hydrogenolysis of Benzyl Esters near the C- and
the TBDPS-Protected O-Terminus
Conclusion
During hydrogenolytic debenzylation of the TBDPS-pro-
Starting from (S)-malic acid, differently protected mono-
tected tetramer 9 (not described here), it was observed that mers (3 and 4) were prepared in such a manner that con-
Eur. J. Org. Chem. 2000, 1207Ϫ1218
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C. M. Krell, D. Seebach
FULL PAPER
under vacuum (0.1–0.01 mbar) at 45 °C (oligomers containing four
monomeric units during t10 d) and/or under h.v. (high vacuum: 5
ϫ 10–6 mbar; Balzers TPG 251; oligomers with free carboxyl
groups). Solvent content of the oligoacids was determined by 1H
NMR. – Reagents: TFA and TFAA (Solvay), TBDPSCl (FMC and
ABCR), Pd(PPh3)4 (Aldrich), imidazole (BASF). All other reagents
[47]
[36]
were purchased from Fluka. CH2N2 and PhCHN2 were pre-
pared according to the literature. – Thin layer chromatography
(TLC): Merck silica gel 60 F254 (0.25 mm on glass). Detection by
UV light or dipping into a solution of phosphomolybdic acid hy-
drate (2.5 g), cerium(IV) sulfate tetrahydrate (1 g) and conc. H2SO4
(6 mL) in water (94 mL), followed by heating. – Flash chromato-
graphy (FC): Fluka silica gel 60 (40–63 µm); at ca. 0.3 bar. – Nor-
mal-phase HPLC: Knauer HPLC system (pump 64), Daicel
Chiralcel OD (250 ϫ 4.6 mm) column, detection at 254 nm. Eluent:
tBuOMe/hexane 1:6, flow rate of 2 mL/min. – RP-HPLC: Knauer
HPLC system (pump WellChrom K-1000 Maxi-Star), Macherey–
Nagel Nucleosil 100–5 C18 (250 ϫ 4 mm) column, detection at
220 nm. Linear gradient of A (10 m sodium phosphate buffer,
pH ϭ 2.4) and B (MeCN) at a flow rate of 1 mL/min. 2 to 10% B
in 12 min, 10 to 20% B in 13 min. t0 ϭ 2.3 min. – M.p.: Büchi 510;
uncorrected values. – Optical rotations: Perkin–Elmer 241 polari-
meter (10-cm, 1-mL cell). – IR: Perkin–Elmer 1600 (FT). – NMR:
Varian Gemini 300 (1H: 300 MHz, 13C: 75 MHz), Bruker AMX
400 (1H: 400 MHz, 13C: 100 MHz), AMX 500 and DRX 500 (1H:
500 MHz, 13C: 125 MHz); chemical shifts (δ) in ppm, relative to
Me4Si as internal standard. Carbon multiplicities were assigned by
DEPT techniques. – MS: VG TRIBRID (EI; 70 eV), VG ZAB2-
SEQ (FAB; 3-nitrobenzyl alcohol), Finnigan MAT TSQ 7000 (ESI;
MeOH solutions), Bruker REFLEX (MALDI-TOF). – HRMS:
VG ZAB2-SEQ (FAB), Finnigan New Star Dual Cell FT/MS (ESI;
ICR, 7 T), Bruker APEX II FTMS (ESI; ICR, 4.7 T) – Elemental
analyses and pKa determination: Microanalytical Laboratory of the
Laboratorium für Organische Chemie, ETH Zürich.
Figure 2. Enzymatic degradation of oligomers 13, 17, 21, and 22,
and of a cyclic tetramer (an X-ray crystal structure of the tetra-
benzyl ester[24] of this tetramer was determined; the coordinates can
be provided at request to the correspondence author)
struction of oligomers of up to eight units (24) was possible
by segment coupling. Acid chloride coupling proved to be
the most reliable method for backbone ester formation and
can, so far, be replaced by a condensation reagent only in
the case of the dimer. In order to avoid destruction of the
larger oligomers during Pd0-catalyzed allyl ester cleavage,
milder deprotection conditions had to be elaborated, and
were found by replacing the widely used allyl acceptor mor-
pholine by imidazole. It was shown that simultaneous hy-
drogenolytic cleavage of up to five benzyl ester groups per-
mits the final deprotection of the water-sensitive products
under non-aqueous conditions. By choosing orthogonal
protecting groups for the termini, specific modification of
the oligomers was possible. The compounds prepared in
this way (21 and 22) proved to be useful for the determina-
tion of the substrate specificity of the extracellular β-PMA
hydrolase of P. polycephalum, confirming that the enzyme
hydrolyzes in an exolytic manner, starting from the O-ter-
minus. Furthermore, it was shown that the oligomers can
be cleanly separated by RP-HPLC, which may be advant-
ageous for the study of β-PMA hydrolysis and detection.
General Procedure for the Preparation of Acid Chlorides (GP 1):
The acid (1 equiv.) was dissolved in CH2Cl2 (0.25  solution) in a
flask, equipped with a bubble trap, and oxalyl chloride (1.5 equiv.)
was added at room temp. After 30 min, DMF (0.5–1 drop/mmol
acid) was added, and the mixture was stirred at room temp. over
night. The volatile components were removed under reduced pres-
sure ( 30 °C), and the resulting yellowish oil was dried under h.v.
for 2–3 d. The acid chlorides were used for couplings without fur-
ther purification. Compared with the corresponding carboxylic
acid, the signals of the CH2COCl protons exhibit a downfield shift
1
of ca. 0.5 ppm in the H-NMR spectrum.
General Procedure for the Coupling of Acid Chlorides with the Cor-
responding Alcohols (GP 2): A solution of the thoroughly dried,
crude acid chloride (1 equiv.) in CH2Cl2 was cooled to –78 °C, and
a solution of the alcohol (1 equiv.) in CH2Cl2 was added. After
10 min, pyridine (1.5 equiv.), diluted with the same volume of
CH2Cl2, was added dropwise to the stirred solution over the given
time period. The mixture was allowed to reach room temp. within
14–20 h. After that, no more starting material could be detected by
TLC, in general. The mixture was diluted with Et2O (double the
volume of CH2Cl2) and washed with 1  HCl (2 ϫ), sat. NaHCO3,
and sat. NaCl solutions, dried (MgSO4), and the solvent evapor-
ated. The crude oligomer was purified by FC and dried under va-
cuum.
Experimental Section
General: BnOH ϭ benzyl alcohol, BOP-Cl ϭ bis(2-oxo-3-oxazolid-
inyl)phosphinic chloride, DCC ϭ 1,3-dicyclohexylcarbodiimide,
DMAP ϭ 4-(dimethylamino)pyridine, NMM ϭ N-methylmorpho-
line, TBDPSCl ϭ tert-butylchlorodiphenylsilane, TFA ϭ trifluo-
roacetic acid, TFAA ϭ trifluoroacetic anhydride. – All reactions,
requiring water-free conditions, were carried out under Ar in oven-
dried glassware (140 °C, for at least 12 h), which was allowed to
cool in a desiccator. Solvents for reactions were of reagent-grade
General Procedure for the Cleavage of tBuPh2Si Ethers (GP 3): A
solution of the tBuPh2Si ether (1 equiv.) in CH2Cl2 in a polyethyl-
ene bottle was cooled to 0 °C and a solution of 70% HF in pyridine
(10–20 equiv. HF) was added. The mixture was vigorously stirred
˚
quality and stored over activated 4-A molecular sieves. THF used
for Pd-catalyzed reactions was freshly distilled (Na/benzophenone;
under Ar). Samples for analytical purposes were thoroughly dried
1212
Eur. J. Org. Chem. 2000, 1207Ϫ1218

Free and of Specifically Protected Oligo[β-Malic Acids]
FULL PAPER
for 15 min (emulsion) and afterwards was allowed to warm to room
temp. Stirring was continued for 12–22 h, and the progress of the
reaction was monitored by TLC. The reaction time was kept min-
trated. Purification by FC (Et2O/pentane 1:1) gave 3 (18.0 g, 92%)
as a colorless, oily liquid. – Rf (Et2O/pentane 1:1) ϭ 0.28. –
t
[α]rD ϭ –20.0 (c ϭ 1.0, CHCl3). – IR (CHCl3): 3538w, 1738s,
imal. The mixture was poured into water and Et2O or CH2Cl2 was 1498w, 1456w, 1423w, 1378m, 1177s, 1104m, 1039w, 986m, 938w. –
added. The organic layer was separated, washed with water (3 ϫ),
sat. NaHCO3, and sat. NaCl solutions, dried (MgSO4) and concen-
trated. The resulting oil was purified by FC and dried under va-
cuum.
1H NMR (300 MHz, CDCl3): δ ϭ 2.83 and 2.88 [AB of ABX,
JAB ϭ 16.5, JAX ϭ 6.2, JBX ϭ 4.4 Hz, 2 H, C(O)CH2], 3.19 (d, J ϭ
5.3 Hz, 1 H, OH), 4.53 (X of ABX, 1 H, HOCH), 4.55–4.58 (m,
2 H, CH2CHϭCH2), 5.20–5.33 (m, 2 H, CHϭCH2), 5.22 (s, 2 H,
PhCH2), 5.79–5.92 (m, 1 H, CHϭCH2), 7.32–7.38 (m, 5 H, arom.
H). – 13C NMR (75 MHz, CDCl3): δ ϭ 38.50 (t), 65.58 (t), 67.26
(d), 67.68 (t), 118.71 (t), 128.49 (s), 128.69 (s), 128.72 (d), 131.75
(d), 135.05 (s), 170.22 (s), 173.32 (s). – MS (EI): m/z (%) ϭ 264 (4)
Mϩ, 140 (2), 129 (46), 107 (25), 91 (100), 87 (15). – C14H16O5
(264.28): calcd. C 63.63, H 6.10; found C 63.61, H 6.30.
General Procedure for Hydrogenations (GP 4): Glassware intended
for use in hydrogenation reactions was carefully rinsed with 2 
HCl solution, deionized water, acetone, and CH2Cl2 and sub-
sequently oven-dried. The benzyl ester was dissolved in the solvent
and a catalytic amount of 10% Pd/C was added. The apparatus
was evacuated and flushed with H2 (3 ϫ), and the mixture was
stirred under H2 (balloon) for 12–22 h. The suspension was filtered
through Celite and the filtrate concentrated under reduced pressure
followed by thorough drying (up to three weeks) under vacuum
and under h.v. However, the solvent never could be completely re-
moved from the oligoacids.
(3S)-4-Benzyloxy-3-(tert-butyldiphenylsilyloxy)-4-oxobutanoic Acid
(4): Acid 2 (897 mg, 4.0 mmol) and DMAP (1.22 g, 10 mmol) were
dissolved in DMF (12 mL) and TBDPSCl (1.33 mL, 5.2 mmol) was
added over 15 min. After stirring the mixture at room temp. for
28 h, it was concentrated (at ca. 1 mbar; 35 °C) to a quarter of its
initial volume. The residue was dissolved in Et2O (60 mL), washed
with 10% aq. citric acid solution (2 ϫ 10 mL), 5% NaHCO3 solu-
tion (8 mL), water, and sat. NaCl solution. Evaporation of the solv-
ent gave an oil which was dissolved in THF/H2O 7:1 (16 mL). The
solution was cooled to 0 °C, treated with TFA (0.77 mL, 10 mmol),
and stirred at room temp. for 2 h before Et2O (60 mL) and water
(10 mL) were added. The aqueous phase was separated and dis-
carded. The organic phase was washed as described above, dried
(MgSO4) and concentrated. Purification by FC (Et2O/pentane 1:2)
gave 4 (1.47 g, 80%) as a colorless oil which solidified upon
standing at 5 °C. – M.p. 70.5–71.5 °C. – Rf (Et2O/pentane 1:2) ϭ
(3S)-4-Benzyloxy-3-hydroxy-4-oxobutanoic Acid (2): (S)-Malic acid
(40.2 g, 0.30 mol; pre-dried under vacuum for 12 h) was cooled to
0 °C and TFAA (101 mL, 0.72 mol) was added in portions over
10 min. The suspension was stirred at 0 °C for 3 h. After 90 min,
it had turned into a colorless solution which again became cloudy
at the end. The volatile components were removed under reduced
pressure at 0 °C. The resulting white solid was thoroughly dried
under vacuum and CH2Cl2 (150 mL) was added. The stirred sus-
pension was cooled to –25 °C and BnOH (77.7 mL, 0.75 mol; dis-
tilled over CaO) was added over 15 min. The mixture was allowed
to warm slowly to 0 °C, and stirring was continued at this temper-
ature. After 18 h, the colorless solution was concentrated by rotary
evaporation. The resulting residue was dissolved in Et2O (100 mL),
stirred and sat. NaHCO3 solution (300 mL) was added. The mix-
ture was diluted with Et2O (300 mL) and the aqueous phase separ-
ated. The ether phase was extracted with sat. NaHCO3 solution
(2 ϫ 300 mL). The combined aqueous extracts were washed with
Et2O (5 ϫ 300 mL), cooled to 0 °C, acidified to pH ϭ 2 with 6
HCl solution and extracted with Et2O (6 ϫ 300 mL). After washing
the combined organic layers with sat. NaCl solution and drying
(MgSO4), the solvent was evaporated and 2 (62.4 g, 93%) was ob-
tained as a colorless oil which solidified upon standing at 5 °C.
Isomeric purity according to 1H NMR: 97%. – M.p. 39.5–41.0
t
0.08. – [α]rD ϭ –49.8 (c ϭ 1.0, CHCl3). – IR (CHCl3): 2933m,
2860m, 1730s, 1472w, 1427m, 1392w, 1362w, 1287m, 1175m, 1113s,
998w, 956w. – 1H NMR (300 MHz, CDCl3): δ ϭ 1.04 (s, 9 H,
Me3C), 2.77 and 2.79 (AB of ABX, JAB ϭ 16.2, JAX ϭ 5.2, JBX
ϭ
6.3 Hz, 2 H, C(O)CH2), 4.56 (X of ABX, 1 H, SiOCH), 4.92 and
5.00 (AB, JAB ϭ 12.1 Hz, 2 H, PhCH2), 7.14–7.44 (m, 11 H, arom.
H), 7.59–7.67 (m, 4 H, arom. H). – 13C NMR (75 MHz, CDCl3):
δ ϭ 19.20 (s), 26.64 (q), 39.50 (t), 66.86 (t), 69.35 (d), 127.62 (d),
127.75 (d), 128.37 (d), 128.53 (d), 129.93 (d), 130.03 (d), 132.67 (s),
132.73 (s), 135.26 (s), 135.99 (d), 136.05 (d), 171.48 (s), 175.85 (s). –
MS (FAB): m/z (%) ϭ 485 (100) [M ϩ Na]ϩ, 475 (14), 455 (12),
405 (95), 385 (98), 199 (11), 197 (16), 135 (14), 91 (55). –
C27H30O5Si (462.62): calcd. C 70.10, H 6.54; found C 70.16,
H 6.41.
t
°C. – [α]rD ϭ –15.9 (c ϭ 1.0, CHCl3). – IR (CHCl3): 3526w, 1739s,
1498w, 1456w, 1409w, 1263m, 1105m, 1039w, 954w, 908w. –
1H NMR (300 MHz, CDCl3): δ ϭ 2.84 and 2.91 [AB of ABX,
JAB ϭ 16.8, JAX ϭ 6.5, JBX ϭ 4.1 Hz, 2 H, C(O)CH2], 4.54 (X of
ABX, 1 H, HOCH), 5.23 (s, 2 H, PhCH2), 7.31–7.39 (m, 5 H, arom.
H). The spectrum of the other isomer differs as follows: δ ϭ 5.16
(s, 2 H, PhCH2). – 13C NMR (75 MHz, CDCl3): δ ϭ 38.27 (t),
67.03 (d), 67.86 (t), 128.51 (d), 128.75 (d), 134.92 (s), 173.19 (s),
176.11 (s). – MS (FAB): m/z (%) ϭ 449 (8) [2 M ϩ H]ϩ, 247 (8)
[M ϩ Na]ϩ, 225 (40) [M ϩ H]ϩ, 107 (19), 91 (100).
1-Benzyl 4-Methyl (2S)-2-(tert-Butyldiphenylsilyloxy)butanedioate
(5): Acid 4 (140 mg, 0.30 mmol) was dissolved in Et2O (4 mL) and
treated with an ethereal solution of diazomethane until the color
of the solution remained yellow. After 12 h, the solvent was evapor-
ated and the residue purified by FC (Et2O/pentane 1:4) to give 5
(129 mg, 89%) as a colorless oil. – Rf (Et2O/pentane 1:4) ϭ 0.28. –
t
HPLC: tr ϭ 14.2 min. – [α]rD ϭ –51.6 (c ϭ 1.0, CHCl3). – IR
(CHCl3): 2955w, 1738s, 1428w, 1364w, 1167m, 1113s. – 1H NMR
(300 MHz, CDCl3): δ ϭ 1.04 (s, 9 H, Me3C), 2.74 and 2.78 [AB of
ABX, JAB ϭ 15.6, JAX ϭ 5.4, JBX ϭ 6.4 Hz, 2 H, C(O)CH2], 3.56
4-Allyl 1-Benzyl (2S)-2-Hydroxybutanedioate (3): Ground K2CO3
(12.3 g, 89.2 mmol) and tetrabutyl ammonium bromide (2.40 g,
7.43 mmol) were suspended in DMF (150 mL), and acid 2 (16.7 g, (s, 3 H, Me), 4.59 (X of ABX, 1 H, SiOCH), 4.92 and 4.99 (AB,
74.3 mmol) and allyl bromide (7.55 mL, 89.2 mmol) were added.
The mixture was stirred at room temp. After 4 h, it was concen-
trated (at ca. 1 mbar; 35 °C) to a quarter of its initial volume and
Et2O (250 mL), 5% aq. citric acid solution (150 mL) and water
(50 mL) were added. The aqueous phase was separated and ex-
tracted with Et2O. The combined organic phases were washed with
JAB ϭ 12.1 Hz, 2 H, PhCH2), 7.14–7.44 (m, 11 H, arom. H), 7.59–
7.68 (m, 4 H, arom. H). – 13C NMR (75 MHz, CDCl3): δ ϭ 19.31
(s), 26.70 (q), 39.86 (t), 51.66 (q), 66.71 (t), 69.67 (d), 127.49 (d),
127.58 (d), 128.21 (d), 128.38 (d), 128.42 (d), 129.77 (d), 129.85 (d),
132.70 (s), 132.74 (s), 135.25 (s), 135.83 (d), 135.91 (d), 170.37 (s),
171.46 (s). – MS (FAB): m/z (%) ϭ 476 (Ͻ 1) Mϩ, 419 (100), 399
5% NaHCO3 and sat. NaCl solutions, dried (MgSO4), and concen- (47), 391 (14), 341 (20), 283 (11), 251 (11), 199 (11), 197 (18), 135
Eur. J. Org. Chem. 2000, 1207Ϫ1218 1213

C. M. Krell, D. Seebach
FULL PAPER
(24), 91 (62). – C28H32O5Si (476.64): calcd. C 70.56, H 6.77; found
C 70.49, H 6.72.
40.6 (c ϭ 1.0, CHCl3). – IR (CHCl3): 3036w, 2934w, 2859w, 1749s,
1602w, 1456w, 1428w, 1362w, 1281m, 1162s, 1114m, 956w. 1H NMR
(300 MHz, CDCl3): δ ϭ 1.03 (s, 9 H, Me3C), 2.77–2.92 [m, 4 H,
1-Benzyl 4-Methyl 2-(tert-Butyldiphenylsilyloxy)butanedioate (rac-
5): Prepared, starting from rac-malic acid, in a similar manner as
5. White solid. – M.p. 46.5–48.5 °C. – HPLC: tr(R) ϭ 10.3 min,
tr(S) ϭ 14.2 min. – The spectroscopic data were in accordance with
those of 5.
C(O)CH2], 4.54–4.58 (m, 1 H, SiOCH), 4.91 and 4.96 (AB, JAB
ϭ
12.1 Hz, 2 H, PhCH2), 5.15 (s, 2 H, PhCH2), 5.45–5.49 (m, 1 H,
OCHCH2), 7.12–7.44 (m, 16 H, arom. H), 7.59–7.67 (m, 4 H, arom.
H). – 13C NMR (75 MHz, CDCl3): δ ϭ 19.18 (s), 26.62 (q), 35.49
(t), 39.34 (t), 66.79 (t), 67.47 (t), 68.14 (d), 69.20 (d), 127.60 (d),
127.75 (d), 128.28 (d), 128.32 (d), 128.51 (d), 128.56 (d), 128.69 (d),
129.89 (d), 130.02 (d), 132.72 (s), 132.78 (s), 135.06 (s), 135.34 (s),
136.00 (d), 136.03 (d), 168.36 (s), 169.05 (s), 171.40 (s), 174.02 (s). –
MS (FAB): m/z (%) ϭ 692 (9) [M ϩ Na]ϩ, 197 (14), 181 (19), 135
(8), 133 (38), 105 (15), 91 (100). – C38H40O9Si (668.81): calcd.
C 68.24, H 6.03; found C 68.05, H 5.91.
4-Allyl 1-Benzyl (2S)-2-[(3S)-3-Benzyloxycarbonyl-3-(tert-butyldi-
phenylsilyloxy)propanoyloxy]butanedioate (6): According to GP 1,
acid 4 (4.16 g, 9.0 mmol) was converted into the acid chloride
which was dissolved in CH2Cl2 (25 mL) and coupled according to
GP 2 with 3 (2.38 g, 9.0 mmol) in CH2Cl2 (25 mL) upon addition
of the pyridine solution within 45 min. FC (Et2O/pentane 2:5)
yielded 6 (5.96 g, 93%) as a colorless, viscous oil. – Rf (Et2O/pent-
Protected Tetramer 9: According to GP 1, acid
8 (1.90 g,
t
ane 2:5) ϭ 0.25. – [α]rD ϭ –38.5 (c ϭ 1.0, CHCl3). – IR (CHCl3):
2.84 mmol) was converted into the acid chloride which was dis-
solved in CH2Cl2 (7.5 mL) and coupled according to GP 2 with 7
(1.34 g, 2.84 mmol) in CH2Cl2 (7.5 mL) upon addition of the pyrid-
ine solution within 30 min. FC (Et2O/pentane 3:4) yielded 9 (2.71 g,
85%) as a colorless, highly viscous glass. – Rf (Et2O/pentane 1:1) ϭ
2954w, 2859w, 1746s, 1602w, 1456w, 1428w, 1380w, 1278m, 1161s,
1114m, 986w. – 1H NMR (300 MHz, CDCl3): δ ϭ 1.03 (s, 9 H,
Me3C), 2.78–2.92 (m, 4 H, C(O)CH2), 4.50–4.58 (m, 3 H,
CH2CHϭCH2, SiOCH), 4.90 and 4.96 (AB, JAB ϭ 12.1 Hz, 2 H,
PhCH2), 5.15 (d, J ϭ 1.5 Hz, 2 H, PhCH2), 5.18–5.31 (m, 2 H,
CHϭCH2), 5.46–5.51 (m, 1 H, OCHCH2), 5.78–5.91 (m, 1 H,
CHϭCH2), 7.12–7.44 (m, 16 H, arom. H), 7.59–7.67 (m, 4 H,
arom. H). – 13C NMR (75 MHz, CDCl3): δ ϭ 19.31 (s), 26.74 (q),
35.96 (t), 39.43 (t), 65.71 (t), 66.76 (t), 67.39 (t), 68.46 (d), 69.22
(d), 118.62 (t), 127.50 (d), 127.63 (d), 128.20 (d), 128.39 (d), 128.42
(d), 128.55 (d), 129.75 (d), 129.88 (d), 131.64 (d), 132.65 (s), 132.70
(s), 135.04 (s), 135.27 (s), 135.88 (d), 135.93 (d), 168.30 (s), 168.48
(s), 168.88 (s), 171.13 (s). – MS (FAB): m/z (%) ϭ 731 (2) [M ϩ
Na]ϩ, 651 (49), 631 (33), 309 (100), 297 (64), 277 (68), 197 (33),
181 (56), 135 (41). – C41H44O9Si (708.88): calcd. C 69.47, H 6.26;
found C 69.47, H 6.22.
t
0.27. – [α]rD ϭ –31.8 (c ϭ 1.0, CHCl3). – IR (CHCl3): 3038w,
2950w, 2860w, 1751s, 1602w, 1498w, 1456w, 1380w, 1278m, 1161s,
1113m, 984w. – 1H NMR (400 MHz, CDCl3): δ ϭ 1.03 (s, 9 H,
Me3C), 2.80–2.98 [m, 8 H, C(O)CH2], 4.52–4.55 (m, 2 H, CH2CHϭ
CH2) 4.56–4.59 (m, 1 H, SiOCH), 4.89 and 4.95 (AB, JAB
ϭ
12.2 Hz, 2 H, PhCH2), 5.09–5.18 (m, 6 H, PhCH2), 5.18–5.29 (m,
2 H, CHϭCH2), 5.46–5.53 (m, 3 H, OCHCH2), 5.78–5.88 (m, 1 H,
CHϭCH2), 7.11–7.41 (m, 26 H, arom. H), 7.59–7.67 (m, 4 H,
arom. H). – 13C NMR (100 MHz, CDCl3): δ ϭ 19.34 (s), 26.77 (q),
35.47 (t), 35.49 (t), 35.81 (t), 39.29 (t), 65.78 (t), 66.73 (t), 67.40 (t),
67.57 (t), 68.16 (d), 68.40 (d), 68.74 (d), 69.27 (d), 118.74 (t), 127.51
(d), 127.64 (d), 128.16 (d), 128.25 (d), 128.26 (d), 128.30 (d), 128.40
(d), 128.42 (d), 128.54 (d), 128.58 (d), 128.64 (d), 129.74 (d), 129.87
(d), 131.61 (d), 132.75 (s), 132.83 (s), 134.95 (s), 135.13 (s), 135.39
(s), 135.94 (d), 135.98 (d), 167.82 (s), 167.90 (s), 167.91 (s), 168.09
(s), 168.12 (s), 168.45 (s), 169.00 (s), 171.21 (s). – MS (MALDI):
m/z ϭ 1144 [M ϩ Na]ϩ. – C63H64O17Si (1121.27): calcd. C 67.49,
H 5.75; found C 67.49, H 5.71.
4-Allyl 1-Benzyl (2S)-2-[(3S)-3-Benzyloxycarbonyl-3-hydroxypro-
panoyloxy]butanedioate (7): According to GP 3, dimer 6 (2.71 g,
3.83 mmol) was dissolved in CH2Cl2 (10 mL) and treated with 70%
HF in pyridine (0.99 mL, 38.3 mmol) for 14 h. FC (Et2O/pentane
1:1) yielded 7 (1.71 g, 95%) as a colorless oil. – Rf (Et2O/pentane
t
1:1) ϭ 0.13. – [α]rD ϭ –26.2 (c ϭ 1.2, CHCl3). – IR (CHCl3):
3541w, 3011w, 2953w, 1744s, 1498w, 1456w, 1379m, 1273s, 1166s,
1106m, 1058w, 986m. – 1H NMR (300 MHz, CDCl3): δ ϭ 2.84–
3.01 [m, 4 H, C(O)CH2], 3.28 (d, J ϭ 5.9 Hz, 1 H, OH), 4.52–4.56
(m, 1 H, HOCH), 4.56–4.59 (m, 2 H, CH2CHϭCH2), 5.13–5.24 (m,
4 H, PhCH2), 5.24–5.33 (m, 2 H, CHϭCH2), 5.53–5.57 (m, 1 H,
OCHCH2), 5.80–5.93 (m, 1 H, CHϭCH2), 7.30–7.40 (m, 10 H,
arom. H). – 13C NMR (75 MHz, CDCl3): δ ϭ 35.70 (t), 38.55 (t),
65.82 (t), 67.20 (d), 67.62 (t), 67.71 (t), 68.52 (d), 118.87 (t), 128.40
(d), 128.58 (d), 128.66 (d), 128.74 (d), 131.67 (d), 134.97 (s), 135.08
(s), 168.49 (s), 168.79 (s), 169.36 (s), 172.94 (s). – MS (EI): m/z
(%) ϭ 471 (3) [M ϩ H]ϩ, 273 (19), 245 (52), 181 (22), 175 (12), 131
(12), 129 (22), 107 (11), 91 (100). – C25H26O9 (470.47): calcd.
C 63.82, H 5.57; found C 63.67, H 5.53.
Tetramer Acid 10: Allyl ester 9 (4.25 g, 3.79 mmol) was dissolved in
THF (38 mL) and Pd(PPh3)4 (350 mg, 0.303 mmol) and imidazole
(2.58 g, 37.9 mmol) were added. The pale yellow solution was
stirred at room temp. for 1 h and then concentrated under reduced
pressure (30 °C) to half of the initial volume. The solution was
diluted with CH2Cl2 (200 mL), washed with 0.5  HCl (3 ϫ) and
half-sat. NaCl solutions, dried (Na2SO4) and concentrated. The re-
sulting residue was dissolved in Et2O (10 mL), filtered through Cel-
ite and purified by FC (Et2O/hexane 1:1, 2% AcOH) to yield acid
10 (3.87 g, 95%) as a yellowish, highly viscous glass. – Rf (Et2O/
t
hexane 1:1, 2% AcOH) ϭ 0.14. – [α]rD ϭ –33.1 (c ϭ 1.0, CHCl3). –
IR (CHCl3): 3036w, 2950w, 1752s, 1456w, 1428w, 1381w, 1278w,
1
1163m, 1113w, 960w. – H NMR (400 MHz, CDCl3): δ ϭ 1.02 (s,
(3S)-4-Benzyloxy-3-[(3S)-3-benzyloxycarbonyl-3-(tert-butyldi-
phenylsilyloxy)propanoyloxy]butanoic Acid (8): Dimer 6 (2.45 g,
9 H, Me3C), 2.77–2.96 [m, 8 H, C(O)CH2], 4.54–4.57 (m, 1 H, Si-
OCH), 4.91 and 4.96 (AB, JAB ϭ 12.1 Hz, 2 H, PhCH2), 5.08–5.23
3.45 mmol) was dissolved in THF (35 mL) and Pd(PPh3)4 (225 mg, (m, 6 H, PhCH2), 5.43–5.50 (m, 2 H, OCHCH2), 5.52–5.56 (m,
0.195 mmol) and morpholine (3.0 mL, 34.5 mmol) were added. 1 H, OCHCH2), 7.11–7.41 (m, 26 H, arom. H), 7.58–7.65 (m, 4 H,
After stirring the solution at room temp. for 2 h, the THF was arom. H). – 13C NMR (100 MHz, CDCl3): δ ϭ 19.31 (s), 26.75 (q),
removed under reduced pressure (35 °C). The residue was dissolved 35.36 (t), 35.58 (t), 35.62 (t), 39.28 (t), 66.98 (t), 67.61 (t), 68.39
in CH2Cl2, washed with 1  HCl solution (3 ϫ), dried (MgSO4) (d), 68.59 (d), 68.67 (d), 69.24 (d), 127.54 (d), 127.68 (d), 128.24
and concentrated. The resulting oil was dissolved in Et2O (5 mL), (d), 128.29 (d), 128.31 (d), 128.42 (d), 128.46 (d), 128.55 (d), 128.59
filtered through Celite and the solution concentrated. Purification (d), 128.65 (d), 128.66 (d), 129.05 (d), 129.80 (d), 129.93 (d), 132.65
by FC (Et2O/pentane 4:1) yielded acid 8 (2.15 g, 93%) as a highly
(s), 132.71 (s), 134.89 (s), 134.91 (s), 134.98 (s), 135.21 (s), 135.92
(d), 135.95 (d), 167.78 (s), 167.80 (s), 167.82 (s), 168.01 (s), 168.54
t
viscous, yellow oil. – Rf (Et2O/pentane 4:1) ϭ 0.16. – [α]rD ϭ –
1214
Eur. J. Org. Chem. 2000, 1207Ϫ1218

Free and of Specifically Protected Oligo[β-Malic Acids]
FULL PAPER
(s), 169.16 (s), 171.38 (s), 171.63 (s). – MS (MALDI): m/z ϭ 1126 (2.31 g, 5.0 mmol), dibenzyl (S)-malate (14) and DMAP (61 mg,
[M – H ϩ 2 Na]ϩ, 1120 [M ϩ K]ϩ, 1104 [M ϩ Na]ϩ. – C60H60O17Si 0.5 mmol) in CH2Cl2 (50 mL) was cooled to 0 °C and DCC (1.14 g,
(1081.21): calcd. C 66.65, H 5.59; found C 66.81, H 5.82.
5.5 mmol) was added. The mixture was stirred at 0 °C for 6 h and
was then filtered twice through Celite. The filtrate was diluted with
Et2O (100 mL), washed with 0.5  HCl (3 ϫ), sat. NaHCO3, and
sat. NaCl solutions, dried (MgSO4), and concentrated under re-
duced pressure. Purification by FC (Et2O/pentane 1:3) yielded
dimer 15 (3.18 g, 84%) as a colorless oil. – Rf (Et2O/pentane 1:3) ϭ
Protected Tetramer 11: Acid 10 (1.10 g, 1.01 mmol) was dissolved
in CH2Cl2 (50 mL) and treated with an ethereal solution of phenyl-
diazomethane (48 mL, ca. 0.03 ). After stirring the solution at
room temp. for 14 h, it was diluted with CH2Cl2, washed with 5%
aq. citric acid and sat. NaCl solutions, dried (MgSO4) and concen-
trated under reduced pressure. Purification by FC (Et2O/pentane
3:4) gave 11 (1.10 g, 93%) as a yellowish, viscous oil. – Rf (Et2O/
t
0.20. – [α]rD ϭ –34.5 (c ϭ 1.0, CHCl3). – IR (CHCl3): 3040w,
2956w, 1747s, 1456w, 1428w, 1361w, 1281w, 1161m, 1114m. –
1H NMR (400 MHz, CDCl3): δ ϭ 1.03 (s, 9 H, Me3C), 2.77–2.92
[m, 4 H, C(O)CH2], 4.55–4.58 (m, 1 H, SiOCH), 4.90 and 4.96 (AB,
JAB ϭ 12.1 Hz, 2 H, PhCH2), 5.08 (s, 2 H, PhCH2), 5.10 (s, 2 H,
PhCH2), 5.47–5.50 (m, 1 H, OCHCH2), 7.12–7.42 (m, 21 H, arom.
H), 7.58–7.67 (m, 4 H, arom. H). – 13C NMR (100 MHz, CDCl3):
δ ϭ 19.34 (s), 26.77 (q), 36.06 (t), 39.45 (t), 66.78 (t), 66.89 (t),
67.39 (t), 68.48 (d), 69.26 (d), 127.53 (d), 127.66 (d), 128.22 (d),
128.37 (d), 128.42 (d), 128.45 (d), 128.56 (d), 128.57 (d), 128.79 (d),
129.91 (d), 132.71 (s), 132.73 (s), 135.06 (s), 135.32 (s), 135.41 (s),
135.91 (d), 135.97 (d), 168.30 (s), 168.68 (s), 168.90 (s), 171.15 (s). –
MS (FAB): m/z (%) ϭ 781 (14) [M ϩ Na]ϩ, 771 (31), 701 (64), 681
(59), 567 (29), 547 (28), 309 (49), 297 (22), 277 (28), 271 (42), 197
(28), 181 (100), 135 (54), 105 (20). – C45H46O9Si (758.94): calcd.
C 71.22, H 6.11; found C 71.27, H 6.30.
t
pentane 1:1) ϭ 0.31. – [α]rD ϭ –29.9 (c ϭ 1.1, CHCl3). – IR
(CHCl3): 3036w, 2956w, 1752s, 1498w, 1456w, 1381w, 1280m, 1161s,
1113m, 964w. – 1H NMR (500 MHz, CDCl3): δ ϭ 1.03 (s, 9 H,
Me3C), 2.80–2.94 [m, 8 H, C(O)CH2], 4.56–4.58 (m, 1 H, SiOCH),
4.88 and 4.95 (AB, JAB ϭ 12.2 Hz, 2 H, PhCH2), 5.05–5.15 (m,
8 H, PhCH2), 5.46–5.53 (m, 3 H, OCHCH2), 7.11–7.40 (m, 31 H,
arom. H), 7.59–7.66 (m, 4 H, arom. H). – 13C NMR (125 MHz,
CDCl3): δ ϭ 19.33 (s), 26.77 (q), 35.44 (t), 35.88 (t), 39.28 (t), 66.71
(t), 66.94 (t), 67.38 (t), 67.55 (t), 68.16 (d), 68.37 (d), 68.74 (d),
69.26 (d), 127.49 (d), 127.63 (d), 128.14 (d), 128.23 (d), 128.25 (d),
128.30 (d), 128.38 (d), 128.41 (d), 128.43 (d), 128.52 (d), 128.56 (d),
128.58 (d), 128.61 (d), 128.63 (d), 129.73 (d), 129.86 (d), 132.74 (s),
132.82 (s), 134.91 (s), 134.94 (s), 135.11 (s), 135.33 (s), 135.38 (s),
135.93 (d), 135.97 (d), 167.79 (s), 167.87 (s), 167.90 (s), 168.04 (s),
168.10 (s), 168.59 (s), 168.98 (s), 171.19 (s). – MS (MALDI): m/z ϭ
1210 [M ϩ K]ϩ, 1194 [M ϩ Na]ϩ. – C67H66O17Si (1171.33): calcd.
C 68.70, H 5.68; found C 68.85, H 5.80.
Dibenzyl (2S)-2-[(3S)-3-Benzyloxycarbonyl-3-hydroxypropanoyloxy]-
butanedioate (16): According to GP 3, dimer 15 (2.13 g, 2.8 mmol)
was dissolved in CH2Cl2 (10 mL) and treated with 70% HF in pyr-
idine (0.73 mL, 28 mmol) for 15 h. FC (Et2O/hexane 2:1) yielding
16 (1.37 g, 94%) as a colorless oil. – Rf (Et2O/hexane 2:1) ϭ 0.24. –
Tetramer Alcohol 12: According to GP 3, tetramer 11 (447 mg,
0.382 mmol) was dissolved in CH2Cl2 (3 mL) and treated with 70%
HF in pyridine (0.20 mL, 7.63 mmol) for 17 h. FC (Et2O/CH2Cl2
1:20) yielded 12 (314 mg, 89%) as a colorless oil. – Rf (Et2O/CH2Cl2
t
[α]rD ϭ –23.2 (c ϭ 1.0, CHCl3). – IR (CHCl3): 3536w, 1745s,
1498w, 1456w, 1379w, 1265m, 1165s, 1105w, 1056w, 965w. –
1H NMR (400 MHz, CDCl3): δ ϭ 2.80–2.96 [m, 4 H, C(O)CH2],
3.28 (d, J ϭ 5.5 Hz, 1 H, OH), 4.50–4.54 (m, 1 H, HOCH), 5.09–
5.23 (m, 6 H, PhCH2), 5.53–5.56 (m, 1 H, OCHCH2), 7.25–7.38
(m, 15 H, arom. H). – 13C NMR (100 MHz, CDCl3): δ ϭ 35.89 (t),
38.63 (t), 67.03 (t), 67.22 (d), 67.64 (t), 67.72 (t), 68.57 (d), 128.32
(d), 128.44 (d), 128.46 (d), 128.51 (d), 128.57 (d), 128.59 (d), 128.61
(d), 128.63 (d), 128.65 (d), 134.86 (s), 135.02 (s), 135.30 (s), 168.31
(s), 168.79 (s), 169.18 (s), 172.76 (s). – MS (FAB): m/z (%) ϭ 1041
(4) [2 M ϩ H]ϩ, 543 (14) [M ϩ Na]ϩ, 521 (36) [M ϩ H]ϩ, 271 (36),
225 (24), 181 (100), 106 (12). – C29H28O9 (520.53): calcd. C 66.92,
H 5.42; found C 67.02, H 5.19.
t
1:20) ϭ 0.10. – [α]rD ϭ –20.6 (c ϭ 1.0, CHCl3). – IR (CHCl3):
3541w, 2959w, 1750s, 1498w, 1456w, 1379w, 1358w, 1266m, 1164s,
1105w, 1052w, 961w. – 1H NMR (400 MHz, CDCl3): δ ϭ 2.82–2.98
[m, 8 H, C(O)CH2], 3.37 (d, J ϭ 6.0 Hz, 1 H, OH), 4.51–4.56 (m,
1 H, HOCH), 5.04–5.24 (m, 10 H, PhCH2), 5.51–5.57 (m, 3 H,
OCHCH2), 7.24–7.38 (m, 25 H, arom. H). – 13C NMR (100 MHz,
CDCl3): δ ϭ 35.41 (t), 35.87 (t), 38.67 (t), 66.98 (t), 67.28 (d), 67.60
(t), 67.66 (t), 67.69 (t), 68.27 (d), 68.44 (d), 68.78 (d), 128.27 (d),
128.35 (d), 128.40 (d), 128.46 (d), 128.53 (d), 128.56 (d), 128.60 (d),
128.63 (d), 128.67 (d), 134.86 (s), 134.89 (s), 134.90 (s), 135.12 (s),
135.31 (s), 167.87 (s), 167.94 (s), 168.01 (s), 168.09 (s), 168.17 (s),
168.67 (s), 169.23 (s), 172.76 (s). – MS (MALDI): m/z ϭ 956 [M
ϩ Na]ϩ. – C51H48O17 (932.93): calcd. C 65.66, H 5.19; found
C 65.46, H 5.15.
(2S)-2-[(3S)-3-Carboxy-3-hydroxypropanoyloxy]butanedioic
Acid
(17): Dimer 16 (66 mg, 0.127 mmol) was deprotected in dioxane
(2 mL) according to GP 4 in the presence of 10% Pd/C (7 mg) to
yield the oligoacid 17 (42 mg, containing 0.9 equiv. dioxane, quant.)
as a colorless, hygroscopic glass. – RP-HPLC: tr ϭ 4.1 min, purity
Free Tetramer 13: Tetramer 12 (49 mg, 0.053 mmol) was depro-
tected in dioxane (2 mL) according to GP 4 in the presence of 10%
Pd/C (5 mg) to yield the oligoacid 13 (38 mg, containing 1.5 equiv.
dioxane, quant.) as a colorless, hygroscopic glass. – RP-HPLC: tr ϭ
9.6 min, purity 99%. – IR (neat): 3479m, 2960m, 2605w, 1959w,
1738s, 1641w, 1377m, 1234m, 1171s, 1048m, 955w. – 1H NMR
(500 MHz, CD3OD): δ ϭ 2.75–3.08 [m, 8 H, C(O)CH2], 4.52–4.55
(m, 1 H, HOCH), 4.88 (br. s, 6 H, 5 ϫ CO2H, HOCH), 5.44–5.49
(m, 3 H, OCHCH2) – 13C NMR (125 MHz, CD3OD): δ ϭ 36.68
(t), 36.70 (t), 36.73 (t), 39.96 (t), 68.15 (d), 69.72 (d), 69.95 (d),
70.38 (d), 170.05 (s), 170.07 (s), 171.20 (s), 171.58 (s), 171.94 (s),
171.97 (s), 172.76 (s), 176.14 (s). – MS (ESI–): m/z (%) ϭ 481 (100)
[M – H]–, 365 (18), 347 (17), 249 (39), 133 (22); (ESIϩ): m/z (%) ϭ
505 (100) [M ϩ Na]ϩ. – HRMS (ESI–, 7 T): m/z (C16H17O17–, [M –
H]–) calcd. 481.0471; found 481.0485.
t
Ͼ 99%. – [α]rD ϭ –13.3 (c ϭ 0.50, H2O; calcd. for solvent free
compound). – IR (neat): 3415m, 3108m, 2969m, 2615w, 1737s,
1
1635w, 1404m, 1376m, 1173s, 1047m, 952w. – H NMR (400 MHz,
CD3OD): δ ϭ 2.72–2.95 [m, 4 H, C(O)CH2], 4.51–4.54 (m, 1 H,
HOCH), 5.01 (br. s, 4 H, 3 ϫ CO2H, HOCH), 5.42–5.45 (m, 1 H,
OCHCH2). – 13C NMR (100 MHz, CD3OD): δ ϭ 36.85 (t), 39.99
(t), 68.19 (d), 70.19 (d), 171.28 (s), 172.32 (s), 172.85 (s), 176.15
(s). – MS (FAB): m/z (%) ϭ 501 (8) [2 M ϩ H]ϩ, 273 (100) [M ϩ
Na]ϩ, 251 (48) [M ϩ H]ϩ. – HRMS (FAB): m/z (C8H10NaO9ϩ, [M
ϩ Na]ϩ) calcd. 273.0217; found 273.0219.
Tetramer Alcohol 18: According to GP 3, tetramer 9 (5.61 g,
5.0 mmol) was dissolved in CH2Cl2 (25 mL) and treated with 70%
HF in pyridine (1.95 mL, 75 mmol) for 22 h. FC (Et2O/hexane 2:1)
Dibenzyl
(2S)-2-[(3S)-3-Benzyloxycarbonyl-3-(tert-butyldiphenyl-
yielded 18 (3.85 g, 87%) as a colorless oil. – Rf (Et2O/hexane 2:1) ϭ
t
silyloxy)propanoyloxy]butanedioate (15): A solution of acid 4 0.15. – [α]rD ϭ –21.7 (c ϭ 1.1, CHCl3). – IR (CHCl3): 3530w,
Eur. J. Org. Chem. 2000, 1207Ϫ1218
1215

C. M. Krell, D. Seebach
FULL PAPER
3037w, 2954w, 1750s, 1602w, 1498w, 1456w, 1379w, 1275m, 1166s,
O-Terminally Protected Tetramer 21: Acid 20 (308 mg, 0.348 mmol)
1105w, 1059w, 984w. – 1H NMR (400 MHz, CDCl3): δ ϭ 2.82–2.99 was deprotected in dioxane (8 mL) according to GP 4 in the pres-
[m, 8 H, C(O)CH2], 3.35 (d, J ϭ 6.0 Hz, 1 H, OH), 4.52–4.56 (m, ence of 10% Pd/C (30 mg) to yield the oligoacid 21 (249 mg, con-
3 H, HOCH, CH2CHϭCH2), 5.08–5.29 (m, 10 H, CHϭCH2, 4 ϫ taining 1.9 equiv. dioxane, quant.) as a colorless, hygroscopic
PhCH2), 5.52–5.58 (m, 3 H, OCHCH2), 5.78–5.88 (m, 1 H, CHϭ
glass. – RP-HPLC: tr ϭ 15.1 min, purity 97.5%. – pKa (25 °C):
t
CH2) 7.26–7.36 (m, 20 H, arom. H). – 13C NMR (100 MHz, 3.46. – [α]rD ϭ –17.3 (c ϭ 0.49, H2O; calcd. for solvent free com-
CDCl3): δ ϭ 35.44 (t), 35.47 (t), 35.80 (t), 38.68 (t), 65.80 (t), 67.30 pound). – IR (neat): 3479m, 2940m, 2595w, 1959w, 1740s, 1641w,
(d), 67.59 (t), 67.65 (t), 67.68 (t), 68.28 (d), 68.47 (d), 68.79 (d), 1377m, 1236m, 1169s, 1051m, 949w.
–
1H NMR (400 MHz,
118.75 (t), 128.26 (d), 128.33 (d), 128.45 (d), 128.52 (d), 128.55 (d),
128.58 (d), 128.64 (d), 128.66 (d), 131.61 (d), 134.88 (s), 134.93 (s),
135.15 (s), 167.87 (s), 167.94 (s), 167.99 (s), 168.11 (s), 168.17 (s),
CD3OD): δ ϭ 2.09 (s, 3 H, Me), 2.80–3.13 [m, 8 H, C(O)CH2],
4.95 (br. s, 5 H, CO2H), 5.41–5.51 (m, 4 H, OCHCH2) – 13C NMR
(100 MHz, CD3OD): δ ϭ 20.58 (q), 36.72 (t), 36.75 (t), 36.84 (t),
168.49 (s), 169.21 (s), 172.75 (s). – MS (MALDI): m/z ϭ 922 [M 69.59 (d), 69.93 (d), 69.97 (d), 70.41 (d), 170.04 (s), 170.06 (s),
ϩ K]ϩ, 906 [M ϩ Na]ϩ. – C47H46O17 (882.87): calcd. C 63.94,
H 5.25; found C 63.97, H 5.39.
170.17 (s), 171.55 (s), 171.64 (s), 171.91 (s), 171.98 (s), 172.19 (s),
172.78 (s). – MS (ESI–): m/z (%) ϭ 523 (100) [M – H]–, 365 (21),
249 (14); (ESIϩ): m/z (%) ϭ 547 (100) [M ϩ Na]ϩ. – HRMS
(ESI–, 4.7 T): m/z (C18H19O18–, [M – H]–) calcd. 523.0577; found
523.0573.
Tetramer Acetate 19: A solution of compound 18 (646 mg,
0.73 mmol) in CH2Cl2 (8 mL) was cooled to –10 °C and pyridine
(0.59 mL) was added. Acetyl chloride (0.078 mL, 1.1 mmol) was
added over 10 min before the solution was stirred at –10 °C for
45 min. The reaction mixture was poured on ice and extracted with
CH2Cl2 (3 ϫ). The organic layer was washed with 5% aq. citric
acid, sat. NaHCO3, and half sat. NaCl solutions, dried (Na2SO4),
and concentrated under reduced pressure. Purification by FC
(Et2O/CH2Cl2 3:97) yielded acetate 19 (654 mg, 97%) as a colorless
C-Terminally Protected Tetramer 22: Tetramer 18 (2.22 g,
2.51 mmol) was deprotected in dioxane (60 mL) according to GP
4 in the presence of 10% Pd/C (200 mg) to yield the oligoacid 22
(1.61 g, containing 1.5 equiv. dioxane, 98%) as a colorless, hygro-
scopic glass. – RP-HPLC: tr ϭ 24.2 min, purity 93.5% (3.5% of
tetramer 13). – IR (neat): 3468m, 2967w, 2600w, 1738s, 1639w,
1381w, 1173s, 1056m. – 1H NMR (400 MHz, CD3OD): δ ϭ 0.95
(t, J ϭ 7.4 Hz, 3 H, MeCH2), 1.66 (tq, J ϭ 6.6, 7.4 Hz, 2 H,
MeCH2), 2.74–3.08 [m, 8 H, C(O)CH2], 4.09 (t, J ϭ 6.6 Hz, 2 H,
CH2CH2O) 4.52–4.55 (m, 1 H, HOCH), 4.88 (br. s, 5 H, 4 ϫ
CO2H, HOCH), 5.44–5.51 (m, 3 H, OCHCH2) – 13C NMR
(100 MHz, CD3OD): δ ϭ 10.72 (q), 22.99 (t), 36.73 (t), 36.77 (t),
37.01 (t), 39.99 (t), 67.86 (t), 68.17 (d), 69.74 (d), 69.97 (d), 70.25
(d), 169.99 (s), 170.05 (s), 171.12 (s), 171.21 (s), 171.56 (s), 171.76
(s), 171.95 (s), 176.17 (s). – MS (ESI–): m/z (%) ϭ 523 (100) [M –
H]–, 407 (14), 389 (10), 291 (38), 249 (11); (ESIϩ): m/z (%) ϭ 547
[M ϩ Na]ϩ. – HRMS (ESI–, 4.7 T): m/z (C16H17O17–, [M – H]–)
calcd. 523.0941; found 523.0932.
t
oil. – Rf (Et2O/CH2Cl2 3:97) ϭ 0.23. – [α]rD ϭ –19.1 (c ϭ 1.0,
CHCl3). – IR (CHCl3): 3032w, 2952w, 1751s, 1499w, 1456w, 1378w,
1
1281w, 1166m, 1053w, 986w. – H NMR (500 MHz, CDCl3): δ ϭ
2.07 (s, 3 H, Me), 2.86–3.01 (m, 8 H, C(O)CH2), 4.53–4.55 (m, 2 H,
CH2CHϭCH2), 5.10–5.18 (m, 8 H, PhCH2), 5.19–5.29 (m, 2 H,
CHϭCH2) 5.50–5.57 (m, 4 H, OCHCH2), 5.79–5.87 (m, 1 H, CHϭ
CH2) 7.27–7.36 (m, 20 H, arom. H). – 13C NMR (125 MHz,
CDCl3): δ ϭ 20.44 (q), 35.42 (t), 35.46 (t), 35.57 (t), 35.76 (t), 65.76
(t), 67.40 (t), 67.55 (t), 67.57 (t), 67.63 (t), 67.95 (d), 68.38 (d), 68.41
(d), 68.74 (d), 118.73 (t), 128.18 (d), 128.22 (d), 128.24 (d), 128.29
(d), 128.36 (d), 128.43 (d), 128.51 (d), 128.54 (d), 128.57 (d), 128.58
(d), 128.62 (d), 128.64 (d), 131.58 (d), 134.86 (s), 134.89 (s), 134.93
(s), 135.07 (s), 167.76 (s), 167.83 (s), 167.92 (s), 168.05 (s), 168.11
(s), 168.42 (s), 168.53 (s), 169.93 (s). – MS (MALDI): m/z ϭ 948
[M ϩ Na]ϩ. – C49H48O18 (924.91): calcd. C 63.63, H 5.23; found
C 63.61, H 5.13.
Octamer 23: According to GP 1, acid 10 (162 mg, 0.150 mmol) was
converted into the acid chloride which was dissolved in CH2Cl2
(1.1 mL) and coupled according to GP 2 with 18 (132 mg,
0.150 mmol) in CH2Cl2 (0.5 mL) upon addition of the pyridine so-
lution within 40 min. Two FC (Et2O/CH2Cl2 1:30; first Et2O/hex-
ane 2:1, then Et2O/CH2Cl2 1:1) and recrystallization from boiling
(iPr)2O yielded 23 (39 mg, 13%) as a white solid. – M.p. 79.5–80.0
Tetramer Acid 20: Allyl ester 19 (651 mg, 0.704 mmol) was dis-
solved in THF (7 mL) and Pd(PPh3)4 (65 mg, 0.056 mmol) and im-
idazole (477 mg, 7.0 mmol) were added. The pale yellow solution
was stirred at room temp. for 2 h and then concentrated under re-
duced pressure (30 °C) to half of the initial volume. The solution
t
°C. – Rf (Et2O/CH2Cl2 1:30) ϭ 0.19. – [α]rD ϭ –20.2 (c ϭ 0.20,
was diluted with CH2Cl2 (30 mL), washed with 1  HCl (3 ϫ) and CHCl3). – IR (CHCl3): 3036w, 1752s, 1602w, 1498w, 1456w, 1377w,
1
half sat. NaCl solutions, dried (Na2SO4), and concentrated. The
resulting residue was dissolved in Et2O (5 mL), filtered through
Celite and purified by two FC (Et2O/hexane 2:1, 2% AcOH; Et2O/
toluene 1:5, 2% AcOH) to yield acid 20 (488 mg, 78%) as a yellow-
1280w, 1161m, 1112w, 1059w. – H NMR (500 MHz, CDCl3): δ ϭ
1.02 (s, 9 H, Me3C), 2.79–2.97 [m, 16 H, C(O)CH2], 4.52–4.53 (m,
2 H, CH2CHϭCH2) 4.55–4.57 (m, 1 H, SiOCH), 4.87 and 4.94
(AB, JAB ϭ 12.2 Hz, 2 H, PhCH2), 5.08–5.17 (m, 14 H, PhCH2),
ish, highly viscous glass. – Rf (Et2O/hexane 2:1, 2% AcOH) ϭ 0.15; 5.18–5.28 (m, 2 H, CHϭCH2), 5.45–5.47 (m, 1 H, OCHCH2), 5.49–
t
Rf (Et2O/toluene 1:5, 2% AcOH) ϭ 0.17. – [α]rD ϭ –21.4 (c ϭ 1.0,
5.53 (m, 6 H, OCHCH2), 5.78–5.86 (m, 1 H, CHϭCH2), 7.11–7.13
CHCl3). – IR (CHCl3): 3032w, 1752s, 1600w, 1498w, 1376w, 1165m, (m, 2 H, arom. H), 7.24–7.40 (m, 44 H, arom. H), 7.59–7.66 (m, 4
1
1056w. H NMR (400 MHz, CDCl3): δ ϭ 2.07 (s, 3 H, Me), 2.80–
H, arom. H). – 13C NMR (125 MHz, CDCl3): δ ϭ 19.34 (s), 26.78
3.02 [m, 8 H, C(O)CH2], 5.10–5.19 (m, 8 H, PhCH2), 5.50–5.60 (m, (q), 35.37 (t), 35.42 (t), 35.47 (t), 35.78 (t), 39.28 (t), 65.78 (t), 66.71
4 H, OCHCH2), 5.6–6.6 (br. s, 1 H, CO2H), 7.24–7.36 (m, 20 H, (t), 67.35 (t), 67.47 (t), 67.53 (t), 67.54 (t), 67.59 (t), 67.64 (t), 68.21
arom. H). – 13C NMR (100 MHz, CDCl3): δ ϭ 20.46 (q), 35.38 (t), (d), 68.42 (d), 68.76 (d), 69.28 (d), 118.76 (t), 127.50 (d), 127.64
35.44 (t), 35.58 (t), 35.61 (t), 67.57 (t), 67.65 (t), 67.68 (t), 67.72 (t),
(d), 128.14 (d), 128.22 (d), 128.24 (d), 128.27 (d), 128.31 (d), 128.36
68.00 (d), 68.57 (d), 68.63 (d), 128.23 (d), 128.26 (d), 128.32 (d), (d), 128.39 (d), 128.43 (d), 128.44 (d), 128.48 (d), 128.49 (d), 128.53
128.51 (d), 128.60 (d), 128.62 (d), 128.66 (d), 134.83 (s), 134.85 (s),
134.97 (s), 167.78 (s), 167.82 (s), 167.83 (s), 167.98 (s), 168.24 (s),
(d), 128.57 (d), 128.59 (d), 128.61 (d), 128.62 (d), 128.64 (d), 128.66
(d), 129.73 (d), 129.86 (d), 131.61 (d), 132.77 (s), 132.86 (s), 134.89
168.29 (s), 168.72 (s), 170.22 (s), 172.08 (s). – MS (MALDI): m/z ϭ (s), 134.92 (s), 134.95 (s), 134.99 (s), 135.01 (s), 135.05 (s), 135.18
929 [M – H ϩ 2 Na]ϩ, 907 [M ϩ Na]ϩ. – C46H44O18 (884.84): (s), 135.42 (s), 135.94 (d), 135.99 (d), 167.81 (s), 167.84 (s), 167.86
calcd. C 62.44, H 5.01; found C 62.20, H 5.08.
1216
(s), 167.91 (s), 167.94 (s), 167.95 (s), 168.00 (s), 168.07 (s), 168.16
Eur. J. Org. Chem. 2000, 1207Ϫ1218

Free and of Specifically Protected Oligo[β-Malic Acids]
FULL PAPER
(s), 168.44 (s), 169.03 (s), 171.21 (s). – MS (MALDI): m/z ϭ 1969
[M ϩ Na]ϩ. – C107H104O33Si (1946.06): calcd. C 66.04, H 5.39;
found C 65.59, H 5.27.
(d), 127.49 (d), 127.63 (d), 128.20 (d), 128.38 (d), 128.47 (d), 129.74
(d), 129.90 (d), 132.55 (s), 132.66 (s), 135.14 (s), 135.87 (d), 135.90
(d), 169.05 (s), 169.43 (s), 171.41 (s), 173.80 (s). – MS (FAB): m/z
(%) ϭ 659 (2) [M ϩ K]ϩ, 643 (12) [M ϩ Na]ϩ, 225 (12), 197 (100),
195 (14), 183 (33), 181 (50), 165 (13), 137 (10), 135 (25), 104 (20). –
C34H40O9Si (620.77): calcd. C 65.79, H 6.49; found C 65.65,
H 6.63.
Tetramer 24: Acid 10 (216 mg, 0.20 mmol) and alcohol 18 (185 mg,
0.21 mmol) were dissolved in CH2Cl2 (1 mL) and NMM (42 mg,
0.42 mmol) and BOP-Cl (53 mg, 0.21 mmol) were added. The sus-
pension was stirred at room temp. for 27 h and a 5% NaHCO3
solution (10 mL) and CH2Cl2 were added. The organic layer was
separated, washed with 0.5  HCl (2 ϫ), 5% NaHCO3 (2 ϫ), and
half-sat. NaCl solutions, dried (MgSO4) and concentrated. FC
(Et2O/CH2Cl2 1:50) yielded 24 (125 mg, 58%) as a colorless oil. –
(2S)-4-[(1S)-1-Benzyloxycarbonyl-2-propoxycarbonylethoxy]-2-(tert-
butyldiphenylsilyloxy)-4-oxobutanoic Acid (27): Colorless oil. – Rf
1
(tBuOMe/hexane 4:1) ϭ 0.48. – H NMR (300 MHz, CDCl3): δ ϭ
0.90 (t, J ϭ 7.5 Hz, 3 H, MeCH2), 1.10 (s, 9 H, Me3C), 1.54–1.64
(m, 2 H, MeCH2), 2.74–2.95 [m, 4 H, C(O)CH2], 4.03 (t, J ϭ
6.7 Hz, 2 H, CH2CH2O), 4.43–4.47 (m, 1 H, SiOCH), 5.15–5.20
(m, 2 H, PhCH2), 5.47–5.51 (m, 1 H, OCHCH2), 7.30–7.50 (m,
11 H, arom. H), 7.62–7.70 (m, 4 H, arom. H). – MS (FAB): m/z
(%) ϭ 643 (100) [M ϩ Na]ϩ, 563 (32), 543 (24), 309 (41), 297 (32),
277 (54), 269 (20), 199 (76), 197 (79), 183 (23), 181 (27), 165 (21),
139 (22), 135 (95), 105 (25).
t
Rf (Et2O/CH2Cl2 1:50) ϭ 0.13. – [α]rD ϭ –16.7 (c ϭ 1.0, CHCl3). –
IR (CHCl3): 2954w, 1751s, 1498w, 1455w, 1379w, 1262m, 1166s,
1057w, 979w. – 1H NMR (400 MHz, CDCl3): δ ϭ 2.82–3.07 [m,
8 H, C(O)CH2], 4.52–4.54 (m, 2 H, CH2CHϭCH2), 5.07–5.29 (m,
12 H, CHϭCH2, 4 ϫ PhCH2), 5.50–5.57 (m, 3 H, OCHCH2), 5.61–
5.65 (m, 1 H, OCHCH2), 5.76–5.88 (m, 1 H, CHϭCH2), 6.90 and
6.94 (AB, JAB ϭ 15.8 Hz, 2 H, C(O)CHϭCH), 7.25–7.39 (m, 25 H,
arom. H). – 13C NMR (100 MHz, CDCl3): δ ϭ 35.44 (t), 35.76 (t),
65.78 (t), 67.08 (t), 67.57 (t), 67.59 (t), 67.63 (t), 67.64 (t), 68.40
(d), 68.49 (d), 68.74 (d), 118.74 (t), 128.25 (d), 128.27 (d), 128.31
(d), 128.34 (d), 128.50 (d), 128.52 (d), 128.53 (d), 128.56 (d), 128.58
(d), 128.62 (d), 128.65 (d), 131.59 (d), 132.65 (d), 134.57 (d), 134.87
(s), 134.90 (s), 135.20 (s), 163.76 (s), 164.49 (s), 167.80 (s), 167.81
(s), 167.84 (s), 167.86 (s), 167.94 (s), 167.96 (s), 168.07 (s), 168.45
(s). – MS (MALDI): m/z ϭ 1110 [M ϩ K]ϩ, 1094 [M ϩ Na]ϩ. –
C58H54O20 (1071.05): calcd. C 65.04, H 5.08; found C 65.10,
H 4.88.
(2S)-2-(tert-Butyldiphenylsilyloxy)-4-[(1S)-1-carboxy-2-propoxy-
carbonylethoxy]-4-oxobutanoic Acid (28): Colorless oil. – Rf (tBu-
OMe/hexane 4:1) ϭ 0.03. – 1H NMR (300 MHz, CDCl3): δ ϭ 0.92
(t, J ϭ 7.2 Hz, 3 H, MeCH2), 1.08 (s, 9 H, Me3C), 1.60–1.67 (m,
2 H, MeCH2), 2.73–2.96 [m, 4 H, C(O)CH2], 4.07 (t, J ϭ 6.7 Hz,
2 H, CH2CH2O), 4.48–4.52 (m, 1 H, SiOCH), 5.45–5.49 (m, 1 H,
OCHCH2), 7.35–7.48 (m, 6 H, arom. H), 7.64–7.68 (m, 4 H, arom.
H). – MS (FAB): m/z (%) ϭ 530 (3) Mϩ, 352 (45), 332 (12), 289
(12), 257 (16), 239 (19), 199 (100), 197 (30), 177 (10), 165 (13).
Hydrogenation of the Fully Protected Dimer 6: Dimer 6 (213 mg,
0.30 mmol) was dissolved in dioxane (5 mL) and 10% Pd/C (21 mg)
was added. The apparatus was evacuated and flushed with H2
(3 ϫ), and the mixture was stirred under H2 (balloon) while follow-
ing the progress of the reaction by TLC. The reaction was stopped
after 4.5 h and the suspension was filtered through Celite. The fil-
trate was concentrated under reduced pressure and the components
separated by FC (tBuOMe/hexane 3:1 to 5:1, 2% AcOH) yielding
25 (33 mg, 16%), 26 (105 mg, 56%), 27 (10 mg, 5%) and 28
(36 mg, 23%).
Acknowledgments
We thank Mr. D. Bierbaum and Mr. A. Hugenmatter for technical
assistance. Dr. W. Amrein, ETH Zürich, the Bruker Daltonik
GmbH, Bremen, and the Finnigan Corp., Madison, USA, are
thanked for high-resolution ESI-MS measurements of some of the
oligomers described herein. We gratefully acknowledge the dona-
tion of reagents: BASF, Ludwigshafen (imidazole); FMC Corp.,
Bessemer City, USA (TBDPSCl); Solvay, Hannover (TFA and
TFAA). Permission by Professor E. Holler (Universität Regens-
burg) for allowing us to mention results of hitherto unpublished
enzymatic measurements is greatly appreciated.
1-Benzyl 4-Propyl (2S)-2-[(3S)-3-Benzyloxycarbonyl-3-(tert-butyldi-
phenylsilyloxy)propanoyloxy]butanedioate (25): Colorless oil. – Rf
1
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0.88 (t, J ϭ 7.5 Hz, 3 H, MeCH2), 1.04 (s, 9 H, Me3C), 1.52–1.62
(m, 2 H, MeCH2), 2.80–2.86 [m, 4 H, C(O)CH2], 3.98–4.03 (m,
2 H, CH2CH2O), 4.56–4.59 (m, 1 H, SiOCH), 4.90 and 4.96 (AB,
JAB ϭ 12.2 Hz, 2 H, PhCH2), 5.15 (s, 2 H, PhCH2), 5.48–5.50 (m,
1 H, OCHCH2), 7.13–7.43 (m, 16 H, arom. H), 7.60–7.67 (m, 4 H,
arom. H). – MS (FAB): m/z (%) ϭ 734 (9) [M ϩ Na]ϩ, 654 (21),
634 (12), 309 (93), 297 (67), 277 (65), 269 (28), 227 (23), 225 (34),
197 (100), 181 (100), 135 (76), 105 (24).
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(t), 26.70 (q), 35.96 (t), 39.35 (t), 66.79 (t), 66.92 (t), 68.55 (d), 69.28
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1217

C. M. Krell, D. Seebach
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[O99561]
1218
Eur. J. Org. Chem. 2000, 1207Ϫ1218