On the solution structure of PHB: Preparation and NMR analysis of isotopically labeled oligo[(R)-3-hydroxybutanoic acids] (OHBs)

Article abstract of DOI:10.1002/1522-2675(20010613)84:6<1821::AID-HLCA1821>3.0.CO;2-C

While the chain conformation of poly- and oligo[(R)-3-hydroxybutanoate] (PHB, OHB) is known to be 2₁-and 3₁-helical in stretched fibers and in the crystalline state, respectively (Fig. 2), the structure in solution is unknown. To be

Full text of DOI:10.1002/1522-2675(20010613)84:6<1821::AID-HLCA1821>3.0.CO;2-C

Helvetica Chimica Acta ± Vol. 84 (2001)
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On the Solution Structure of PHB: Preparation and NMR Analysis of
Isotopically Labeled Oligo[(R)-3-hydroxybutanoic Acids] (OHBs)
by Peter Waser1), Magnus Rueping2), and Dieter Seebach*)
Laboratorium für Organische Chemie der Eidgenössischen Technischen Hochschule, ETH-Zentrum,
Universitätstrasse 16, CH-8092 Zürich
and
Elke Duchardt3) and Harald Schwalbe*4))
Department of Chemistry, Francis Bitter Magnet Laboratory, Massachusetts, Institute of Technology,
170 Albany Street, Cambridge, MA 02139, USA
Dedicated to Professor Edgar Heilbronner with best wishes on the occasion of his 80th birthday
While the chain conformation of poly- and oligo[(R)-3-hydroxybutanoate] (PHB, OHB) is known to be 21-
and 31-helical in stretched fibers and in the crystalline state, respectively (Fig. 2), the structure in solution is
unknown. To be able to determine the NMR-solution structure, specifically labeled linear oligomers have been
prepared: a 16-mer consisting of alternating pairs of fully 13C-labeled and non-labeled residues (1) and a 20-mer
containing an O-13CH(13CH2D)-13CHDSi-13CO residue in position 9 (from the O-terminus) and a fully 13C-
labeled residue in position 12 (2), both with (t-Bu)Ph2Si protection at the O- and Bn protection at the C-
terminus. The labeled (R)-3-hydroxybutanoic acid building blocks were prepared by Noyori hydrogenation of
the ethyl ester of fully 13C-labeled acetoacetic acid, and the D-atoms were incorporated by D2/Pd-C reduction of
a previously reported dibromo-1,3-dioxinone 8 (Scheme 1). The oligomers were obtained by a series of fragment
couplings (Schemes 2 and 3). 600-MHz NMR COSY, HSQC, ROESY, and cross-correlated relaxation
measurements (Figs. 4 ± 6, 9, and 12, and Tables 1 ± 3) at different temperatures and interpretations thereof led
to assignments of all resonances, including those from the diastereotopic C(2)H2 protons, and to determination
of the conformationally averaged dihedral angles f2 and f3 (Figs. 2, 7, and 8) in the chain of the oligoester. The
conclusions are: all but five or six terminal residues adopt the same conformation; the 21 helix is not the
predominant secondary structure; the structure of the HB chain is averaged, even at ±308. Our investigation
confirms the high flexibility of the polyester chain, a property that has been deduced previously from biological
studies of PHB in membranes, in ion channels, and as appendage of proteins.
Introduction. ± The biopolymer poly[(R)-3-hydroxybutanoic acid] (PHB) is a
microbial storage material of high molecular weight (ca. 106 Da [1]). Short-chain
derivatives (ca. 12 Â 103 Da, corresponding to 150 residues) have been shown to form,
or be part of, ion channels through planar or liposomal phospholipid bilayers and cell
walls [2]. The actual structure of the PHB that causes the ion-transporting effect is
unknown. Several models have been proposed for the ion channels or pores involving
PHB [3] (Fig. 1). These are derived from modeling [4] or from the known PHB
structures in stretched fibers [5], in lamellar crystallites [6], and in single crystals of
1
)
)
)
)
Part of Dissertation Nr. 13552 of P. W., ETH-Zürich, 2000.
Part of the projected Ph. D. thesis of M. R., ETH-Zürich.
Part of the projected Ph. D. thesis of E. D., MIT, Cambridge, USA.
Correspondence author for the NMR part. We acknowledge stimulating discussions with Prof.
C. Griesinger.
2
3
4

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cyclic oligomers [7] (Fig. 2). Since the phospholipid bilayer may be considered a two-
dimensional nonpolar solvent covered on both sides by a layer of charged, polar groups,
the structure of PHB in solution must be elucidated. We have recently reported the CD
spectra of oligo(hydroxybutanoic acids) (OHBs), containing 8, 16, and 32 HB units in
homogeneous solution, and in a bilayer consisting of racemic phospholipids [8]. The
observed Cotton effects near 210 nm are an indication for the presence of chiral
secondary structures in the ensemble of molecules ± on the very short time scale of UV
spectroscopy. Also, FRET measurements with doubly fluorescence-labeled OHBs of
various chain lengths in 10 6 m solutions provide distances between the termini, which
are compatible with folded conformations of the intermediate chains [8], again on the
short time scale of UV/VIS spectroscopy5).
Fig. 1. Proposed structures of ion channels consisting of or containing short HB chains. Hairpins of HB 32mers
(in a 21-helical conformation of the chain) have been proposed to form aggregates for ion transport through
phospholipid bilayers by a single-channel (a) or by a pore mechanism (b) [3][8]. Calcium polyphosphate (chain
lengths ca. 75 units) complexes with PHB (ca. 150 units) form Ca-specific channels for which structures c and d
have been proposed [4][3].
It would be desirable to determine the NMR-solution structure of PHB chains ± if
the highly flexible polyester chain has a preferred conformation at all on the rather long
time scale of this spectroscopy. Qualitatively, the NMR spectra of cyclic and linear
OHBs [10] look very similar, exhibiting only one set of average signals for all the
residues. Thus, the cyclic hexamer, a 24-membered ring, that exists in crystals in a
1
folded and twisted conformation resembling the number 8 (Fig. 3) has an H-NMR
spectrum with only one set of sharp signals from the CH(CH3)CH2 protons (Fig. 3).
This means that the molecule has C6 geometry on the NMR time scale.
To obtain more detailed information about the conformation of linear HB
oligomers by NMR spectroscopy in solution, we have now prepared the alternatingly
5
)
For review articles covering the chemistry, structure and biology of PHB, see [9].

Helvetica Chimica Acta ± Vol. 84 (2001)
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Fig. 2. Models for the structure of PHB and OHB chains. The 21 helix (a) has been determined by fiber X-ray
diffraction of PHB [5], the 31 helix (b) is constructed from single helix turns seen in crystal structures of cyclic
oligomers of HB. Torsion angles (f1 f4) and corresponding Newman projections of both preferred HB-
conformations from crystal structures of macrolides [7].

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Fig. 3. 1H-NMR Spectrum of the cyclic HB-hexamer (hexolide). One set of CH(CH3)CH2 resonances indicates
that the compound has a C6 symmetry on the NMR time scale, while it is twisted and folded (as shown) in the
solid state.
13C-labeled 16-mer 1 and the specifically 2H- and 13C-labeled 20-mer 2, hoping that the
magnetic labels would enable us to determine the local angles of the HB residues (from
coupling constants and cross-correlated relaxation rates) and the overall conformation
of the chain (via ROE intensities). The positions with an asterix in the formulae 1 and 2
are 13C-atoms.

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2. Synthesis of the Isotopically Labeled OHBs. ± We have previously reported
the synthesis of linear oligomers of HB up to a chain-length of 128 residues by
fragment coupling [11]. The syntheses started from (R)-3-hydroxybutanoic
acid, as obtained from the commercial PHB. The fragments were protected
orthogonally by (t-Bu)Ph2Si on the O-terminus and by CH2Ph on the C-terminus, so
that selectively mono-protected intermediates (t-Bu)Ph2Si (OCHMeCH2CO)n OH
and H (OCHMe CH2 CO)n OBn are readily available for coupling ((COCl2)
activation/pyridine in CH2Cl2, with warming from 788). Thus, the specifically labeled
fragments had to be prepared and coupled to give the desired OHBs 1 and 2. To this
end (Scheme 1), commercial ethyl 3-oxo-butanoate 3*, which was labeled ꢀ99% on
1
each of the four butanoate C-atoms (by H-NMR analysis) was hydrogenated under
Noyori conditions [12] ((M)-BINAP/RuBr2 catalyst/H2 [13]) to give the labeled (R)-3-
hydroxybutanoate 4* of 99.3% enantiomer purity in 96% yield. Protection of the OH
group and conversion of the ethyl to the benzyl ester 5* was achieved in an overall yield
of 77% (from the commercial acetoacetate 3*, on a 6-g scale). Debenzylation (H2/Pd-C)
of half of the material ( ! 6*) and desilylation (HF ´ pyridine) of the other half ( ! 7*),
with subsequent coupling gave the labeled O-silyl- and C-benzyl-protected ꢀdimerꢁ
Scheme 1. Preparation of the (R)-3-Hydroxybutanoic Acid Derivatives 6*, 7*, and 9 for the Assembly of
Labeled OHBs
a) [Ru((M)-BINAP)Br2], 85 atm H2, EtOH, b) TBDPSCI, DMAP, DMF; KOH, EtOH; BnBr, Na2CO3,
DMF. c) H2, Pd/C in MeOH. d) HF-pyridine, CH2Cl2. e) 1n KOH; Me3SiOTf, Et3N, Me3CCHO; NBS,
AlBN, CCl4. f) D2, C6H6, Pd/C; H2, Pd/C; Dowex, MeOH/H2O; BnBr, Na2CO3, DMF. Abbreviations: AlBN ˆ
2,2'-azo[isobutyronitril]. BINAP ˆ 2,2'-Bis(diphenylphosphino)-1,1'-binaphthalene, TBDPS ˆ (t-Bu)Ph2Si,
DMAP ˆ 4-(Dimethylamino)pyridine, Bn ˆ PhCH2, TBDMS ˆ (t-Bu)Me2Si, Tf ˆ trifluoromethylsulfonyl,
NBS ˆ N-bromosuccinimide.

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Scheme 2. Preparation of the 13C-Labeled Oligomers 13 and 14 (containing four and eight HB units,
respectively), Intermediates on the Way to the Hexadecamer 1 with Specific Labeling on Eight Positions
10* (80%), ready to be channeled into fragment-coupling processes (Schemes 2
and 3).
2
The H- and 13C-labeled hydroxy-butanoate 9 was prepared through the dibromo-
dioxinone 8. This compound ± unlabeled ± had been used by us previously as an
intermediate on the way to the 2-(tert-butyl)-6-methyl-1,3-dioxin-4-one [14]; Br/D-
exchange (D2/Pd-C in C6H6) and hydrogenation from the face trans to the t-Bu group
[15] gave the desired specifically labeled compound 9 in an overall yield of ca. 12%
from ester 4*, in eight steps, the poorest one being the bromination with NBS [16],

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Scheme 3. Principle of Fragment Coupling for OHB Synthesis and Intermediates 15 ± 18 on the Way to the
Icosamer 2. Fully (silyl and benzyl) protected oligomers are converted to the O-Si-protected oligo-acids A or
hydroxy oligo-esters B. These are coupled to give the larger, fully protected oligomers C.
which gave only 24% of purified 8. Perhaps not surprisingly, there was some over-
deuteration in the Br/D exchange step, placing more than one D into the Me group at
C(4) of the ester 9 (degree of deuteration 0.42 instead of 0.33). On the other hand, the
hydrogenation of the CˆC bond was surprisingly clean and diastereoselective: the

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ester 9 was of 91% enantiomer purity and contained 99% of D at C(2) in the Si-
position6).
The subsequent assembly of the target molecules 1 and 2 was achieved by applying
the previously published fragment coupling [11], which is schematically outlined in
Scheme 3. Thus, the nonlabeled O-TBDPS-protected hydroxy acid 6 and benzyl ester 7
were coupled to the ꢀdimerꢁ 10, O- and C-terminal deprotection of 10 and 10* ( ! 12,
11*), respectively, and, again, coupling gave the ꢀtetramerꢁ 13 with two labeled and two
nonlabeled HB units (Scheme 2). Two rounds of fragment coupling (Scheme 3) with
the (HB)4 derivatives 13 ( ! 14 ! 1) gave the protected target hexadeca[(R)-3-
hydroxybutanoate] 1 with alternating pairs of 13C-labeled and unlabeled units.
For the assembly of the specifically 2H- and 13C-labeled icosa(hydroxybutanoate) 2,
we used the building blocks 15, containing eight HB units [11], and the protected
ꢀdimerꢁ 10 [11]. Following the fragment-coupling scheme, 10 was debenzylated ( ! 11)
and combined with the labeled hydroxy ester 7* to give the ꢀtrimerꢁ 16, which, in turn,
was debenzylated and connected with the O-desilylated 8-mer derivative 15 to give 18,
containing 11 HB units. On the other end, the debenzylated 8-mer derivative 15 was
2
elongated with the H- and 13C-labeled benzyl 3-hydroxybutanoate 9 to give the
ꢀnonamerꢁ 17. Ester formation between the C-terminal hydroxy acid residue of
debenzylated 17 and the O-terminal residue of desilylated 18, finally, provided the
desired 20-mer derivative 2 (Scheme 3).
3. NMR Analysis: Spectral Assignment. ± The 1H-1D and the COSY spectra of the
16-mer 1 are shown in Fig. 4 together with the spectral assignment. The resonances of
all repetition units overlap except for three 12C- and three 13C-labeled units. Because of
ROE signals to aromatic protons, the unit comprising the C(2)H2 protons at ca. 4 ppm
is assigned to be the O-terminal residue 1. This unit shows C(4)H3,C(2)H2 cross-peaks
to the non-bulk 13C-labeled unit that is resonating close to bulk-frequency, which is,
therefore, assigned to be the subsequent O-terminal residue 2. The third resolved 13C-
labeled unit that also gives rise to C(2)H2 resonances at ca. 4 ppm is of minor intensity
and does not show any interresidual cross peaks.
Assignment of the non-bulk 12C units, which resonate close to the bulk resonances,
cannot be performed due to missing interresidual cross-peaks. However, it is likely that
they are the two C-terminal units and the first 12C unit adjacent to the O-terminus.
The extent of signal overlap in the 20-mer is about the same as in the 16-mer.
However, the 13C-HSQC (Fig. 5) shows that the two 13C-labeled units can be
discriminated because of the primary isotope shift of C(4) and C(2), as well as the
secondary isotope shift of the remaining C(2)H2 and C(4)H3 protons in the deuterated
unit (CHD) relative to the respective resonances in the nondeuterated unit (CH2). In
addition, the difference in multiplet structure of the C(4)H3 and the C(2)H2 cross-peaks
of the two units in the indirect detected dimension in the 13C- and 2H-coupled spectrum
further resolves the spectrum. For instance, the C(3)H,C(2)H2 cross-peaks are split by
1
1
two times J(C,H) in the indirect detected dimension, due to the J(C,H) coupling to two
protons during this time period. The C(2)HD,C(3)H cross-peak, in contrast, is split by
6
)
For a detailed analysis by 1H- and 13C-NMR spectroscopy and high-resolution mass spectrometry, we refer
the reader to the dissertation of P. W. ). See also the NMR part of this paper.
1

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Fig. 4. 1H-1D and 1H-DQF-COSY spectra of the 16-mer 1 without carbon decoupling in t1 and t2. The spectral
assignment is given in the 1D; 12C-bound protons are indicated with an asterisk*, for the resonances originating
1
from 13C-bound protons, the splitting due to the J(C,H) coupling is indicated.

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1
only J(C,H) , since the C couples to one H only. Further splittings and additional
relaxation effects arise due to the presence of the D. A distinct coupling pattern is
observed for the three different C(4)HnDm isotopomers shown with the relevant 1D
slices from the 13C-HSQC spectrum in Fig. 6.
Assignment of the Diastereotopic CH2(2) Protons. To determine f2, the two
diastereotopic C(2)H2 protons have to be stereospecifically assigned. In the case of the
20-mer, one of the 13C-labeled units is stereospecifically deuterated at the C(2)HSi-
position. Since the remaining C(2)H proton resonates at an upfield chemical shift, this
proton is C(2)HRe, and the downfield proton is C(2)HSi. To stereospecifically assign the
C(2)H2 protons (independently of this ꢀsynthetic informationꢁ), vicinal coupling
constants have been measured and interpreted for the 16-mer 1 and for the 20-mer 2.
The three possible conformations (ap, ( )- and (‡)-sc) around the f2 are shown in
Fig. 7 together with the characteristic relative sizes of homo- and heteronuclear
coupling constants.
3
3
Since both J(C(4),C(2)H)-coupling constants are close to zero and J(C(1),C(3)H) is also
3
3
small, while J(C(2)H,C(3)Down is larger than J(C(2)H,C(3)HUp in the bulk units for both
)
)
compounds (Table 1), the predominant conformation around the f2 angle is ( )-sc. In
agreement with the diastereospecific synthesis, this leads to the following assignment:
C(2)HDown is C(2)HSi, and C(2)HUp is C(2)HRe. This stereospecific assignment is
opposite to that reported by Doi and co-workers for dimers and trimers of HB (no
stereospecific assignment has been performed in this investigation) [17].
Table 1. Homo- and Heteronuclear Coupling Constants in the Resolved Units of the 16-Mer 1 and the 20-Mer 2.
n.d. ˆ not determined. The data were determined from HCCH-E.COSY experiments [20].
Parameter
16-Mer
Bulk
20-Mer
CH2
Terminal
residue 1
Terminal
residue 2
CHD
3J(C(3)H,C(2)HDown,Si
)
7.3 Hz
5.7 Hz
2.6 Hz
0.6 Hz
2.7 Hz
6.4 Hz
6.9 Hz
n.d.
1.5 Hz
2.8 Hz
7.2 Hz
6.7 Hz
n.d.
n.d.
2.6 Hz
7.4 Hz
5.5 Hz
0.1 Hz
0.4 Hz
3.2 Hz
n.d.
5.6 Hz
n.d.
0.4 Hz
n.d.
3J(C(3)H,C(2)HUp,Re
)
3J(C(2)HDown,Si,C(4))
3J(C(2)HUp,Re,C(4))
3J(C(3)H,C(1))
For the two 13C-labeled O-terminal units of the 16-mer, the assignment of the
chemical shifts of the diastereotopic C(2)H2 protons is identical, although the relative
size of the 3J(C(2)H,C(3)H)-coupling constants is reversed.
Conformational Analysis: Backbone Angle f2. In both helices, the torsion angle f2
is close to the ( )-sc conformation ( 62.48 for a 21 and 52.18 for a 31 helix). The
3J(C(2)H,C(3)H)-coupling constants (Table 1) show that the conformation around f2 is
averaged for both compounds 1 and 2. The populations of the different rotamers are
given in Table 2. A detailed description of the coupling-constant analysis is given in the
caption of Table 2.
In all of the bulk-residues, the ( )-sc-conformation is populated by ca. 55%. The
highest possible helix content is, therefore, ca. 0.55n, with n being the number of
residues involved in the helix (e.g., for 5 3-HB-units, the possible helix content is ca.

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Fig. 6. 1D Slices of the 13C-HSQC spectrum depicted in Fig. 5 at the frequency of the different C(4)HmDn
isotopomers (isotopomeric ratio determined in monomers). The multiplet patterns are indicated. Populations of
the different species are given. *: 13C-Natural abundance signal from nonlabeled units; #: Partial overlap of
signals from different isotopomers; §: Second component of the multiplet weakened due to differential
relaxation.

Helvetica Chimica Acta ± Vol. 84 (2001)
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3
3
Fig. 7. The three staggered conformations around f2. Relative sizes of the J(C(2)H,C(3)H) and J(C,H)-coupling
constants are indicated for each conformation. Bold, large ꢀ ‡ ꢁ ˆ large relative coupling constant, small ꢀ ‡ ꢁ ˆ
small relative coupling constant.
5%). The conformation of the two O-terminal units (residues 1 and 2) is also averaged,
with residue 1 being predominantly in the ap-conformation. Residue 2 has a larger
population of the ( )-sc-conformation (54.9%).
Conformational Analysis: Backbone Angle f3. The angle f3 between C(2) and C(1)
is the only backbone angle, in which the two proposed secondary structures differ
significantly (Fig. 2). Due to the lack of neighboring ꢀNMR-activeꢁ nuclei, this angle
cannot be analyzed via vicinal coupling constants. However, it can be determined from
the interpretation of dipole,dipole-CSA cross-correlated relaxation rates between the
C(2),C(2)H dipole(s) and the chemical-shift anisotropy (CSA) of the ester CˆO group
DD;CSA
Cꢁ2†;Cꢁ2†H;Cꢁ1†
G
, the value of which depends on the projection angles of the CH-dipole
vector(s) onto the different components of the fully anisotropic C(1) chemical-shift
tensor as follows [18]:
IDDQIUZQ
IDZQIUDQ
1
H tcf(sx,sy,sz) ˆ ln
T
h wcgcg
2p r3CH
DD;CSA
Cꢁ2†Cꢁ2†H;Cꢁ1†
G
ˆ 4/15
(1)

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wC ˆ Carbon Lamor frequency
gC ˆ Carbon magnetogyric ratio
gH ˆ Proton magnetogyric ratio
rHC ˆ C(2) C(2)H Bond length
tC ˆ Rotational correlation time of the molecule
2
f(sx,sy,sz) ˆ 0.5 [sx(3cos2qx 1) ‡ sy(3cos2qy 1) ‡ sz(3cos qz 1)]
q ˆ Angle between the dipole vector and the respective axis of the CSA-tensor.
sx, sy, sz ˆ Principal values of CSA Tensor
U;D
ZQ;DQ
I
ˆ Intensity of upfield (U)/downfield (D) multiplet component in the
ZQ/DQ spectrum
Tˆ Relaxation delay
The CH-dipole vector(s) are oriented along the CH bond(s). The size of the
principle values and the orientation of the ester CˆO chemical-shift tensor is known
DD;CSA
Cꢁ2†Cꢁ2†H;Cꢁ1†
from model compounds [19]. The dependence of G
on f3 is given in Fig. 8 for
the two 13C-labeled residues. The angle dependence of GCDꢁD2†;CCSꢁ2A†H;Cꢁ1† for the deuterated
unit is shifted by 608 relative to the one of the nondeuterated unit, since, in the first case,
the relaxation rate depends on the projection angle between the bond vector of the
remaining C(2)H proton and the CSA tensor components, while, in the second case, the
two C(2),C(2)H vectors are averaged at the position of the vector product. While the
rates have the same sign and are of comparable size for both the nondeuterated and the
deuterated units, in a 31 helix, they have opposite signs and are of different size in a 21
helix (Fig. 8).
Table 2. 3J(C(3)H,C(2)H)-Coupling Constants and Resulting Populations of the Three Staggered Conformations
Re
around f2. The populations have been calculated with the Pachler-analysis equations: 3J(C(3)H,C(2)H ˆ POGJOG
‡
)
Si
PTJT ‡ PGJG, 3J(C(3)H,C(2)H ˆ PTJOG ‡ PGJT ‡ POGJG and 1 ˆ PT ‡ PG ‡ POG. JT, JO and JOG can be obtained from
)
the generalized Karplus equation:
3J(H,H) ˆ 13.7cos2f 0.73cosf ‡ SiDci[0.56 2.47cos2(zif ‡ 16.9 j Dci j )].
In this equation, f is the dihedral angle between the two protons, z is the orientation factor of substituent i, Dci is the
electronegativity difference between substituent i and the respective proton. Dcc is calculated between the protons and
the two other substituents bound to the same C-atom. Not only the direct substituents ia are considered but also the
atoms bonded to the ia substituents (ib). Thus, Dci is determined according to:
Dci ˆ Dci,a 0.14SDci,b (O:Dci ˆ 1.3, C:Dci ˆ 0.4, H:Dci ˆ 0).
The coupling constants are 11.63 Hz for f ˆ 1808(ap), 3.55 Hz for 608 ((‡)-sc) and 1.97 Hz for 608 (( )-sc),
respectively, for both C(2)H protons.
Compound
16-Mer
Repetition unit
Bulk
Coupling constant
Coupling [Hz]
ap [%]
(
)-sc [%]
(‡)-sc [%]
9.2
3J(C(2)HSi,C(3)H)
3J(C(2)HRe,C(3)H)
3J(C(2)HSi,C(3)H)
3J(C(2)HRe,C(3)H)
3J(C(2)HSi,C(3)H)
3J(C(2)HRe,C(3)H)
7.3
5.7
6.4
6.9
7.2
6.7
35.4
53.7
45.1
54.9
terminal
residue 1
terminal
residue 2
50.3
49.7
4.6
4.6
20-Mer
CH2
3J(C(2)HSi,C(3)H)
3J(C(2)HRe,C(3)H)
n.d.
7.4
5.5
n.d.
5.6
35.4
n.d.
54.6
n.d.
10
CHD
n.d.
3J(C(2)HRe,C(3)H)

Helvetica Chimica Acta ± Vol. 84 (2001)
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DD;CSA
Cꢁ2†Cꢁ2†H;Cꢁ1†
Fig. 8. The dependence of G
on f3. Solid line: CHD unit, dashed line: CH2 unit. The theoretical
values for a 31 and a 21 helix are indicated. The values on the y-axis are arbitrary.
DD;CSA
Cꢁ2†Cꢁ2†H;Cꢁ1†
The ZQ and the DQ spectra for the 16-mer are shown in Fig. 9. The G
values for the 16-mer bulk units are positive. In the 20-mer, the rates for the
nondeuterated as well as for the deuterated unit are also positive, as can be inferred
from the relative intensities of the multiplet components. This excludes the 21 helix as
predominant conformation, whereas the 31 helix (f3 ˆ 1518) is still possible.
Conformational Analysis: ROE Data. ± The design of the selectively labeled 20-mer
2 offers the opportunity to obtain unit-specific information due to the possibility of
spectral editing for the multiplicity of CHn groups using a 13CHx-edited ROESY pulse
sequence. Thus, the C(2)HD group can be separated from the C(2)H2 group, and the
different C(4)HnDm isotopomers can be separated from each other. The goal of this
experiment is a comparative analysis of cross-peak intensities of the two 13C-labeled
units in order to probe the presence of a 31-helix (Table 3).
Table 3. Characteristic Interresidual H,H Distances in the 21 (i, i ‡ 2 distances) and the 31-Helix (i, i ‡ 3).
i,i ‡ 2-Distances [Š]
i,i ‡ 3-Distances [Š]
C(4)H
C(3)H
C(4)H
C(3)H
C(2)HRe
C(2)HSi
3.2
4.8
5.2
6.7
5.3
6.7
4.6
3.2

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A schematic presentation of the different cross-peak patterns of these two units in the
13CHx-edited ROESY spectrum is given in Fig. 10. The corresponding spectra recorded
at 208 are shown in Fig. 11.
Fig. 10. Scheme of the multiplet pattern of the C(2)H2,C(3)H and the C(2)H2,C(4)H3 cross-peaks in the CHx-
filtered ROESY spectrum for the nondeuterated and the deuterated 13C-labeled unit of the 20-mer 2. Corresponding
regions of the CHx-filtered ROESY spectra are shown in Fig. 11
Relative intensities of the interresidual ROE signals extracted from the CHx-
filtered ROESY spectra recorded at 208 and 278 are collected for both labeled units
in Table 4. No significant difference in any cross-peak intensity was observed for
C(2)HSi and C(2)HRe signals, hence, they can both be regarded as C(2)H2. If either one
of the helices would be the predominant conformation of the 20-mer, differential
effects should be detectable between the two C(2)H2 protons. All of the interresidual
ROE signals appear between 13C-labeled units and the 12C units. No interresidual
ROEs between the 13C-labeled units were detected, although C(2)H2 to C(2)H2i ‡ 3
and C(4)H3i ‡ 3 signals could potentially be resolved due to a primary or secondary
isotope shift. However, neither of these ROE signals is to be expected in a 31 helix (all
of these distances are 5.3 Š or larger). The indicative C(2)H2,C(3)Hi ‡ 3 cross-peak
for a 31 helix cannot be resolved from the intraresidual cross-peak. However, signal-
intensity differences between the nondeuterated and the deuterated units for this cross-
peak have been observed. While there is a strong 13C(2)H2,12C(3)H ROE for the

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Table 4. Interresidual ROE Signals of the 20-Mer 2 from the CHx-Filtered ROESY Spectra at 268 and at 308.
Relative ROE intensities are indicated by the number of ꢀ ‡ ꢁ, n.d. ˆ not determined.
Proton-ID
Proton-ID
Signal intensity at
208
278
Undeuterated unit
Deuterated unit
C(2)H
C(2)H
C(4)H
C(4)H
12C(4)H
12C(3)H
12C(3)H
12C(2)H
‡‡
‡
‡‡‡
‡‡‡
‡‡‡
n.d.
‡‡
n.d.
C(2)HRe
C(2)HRe
C(4)H
12C(4)H
12C(3)H
12C(3)H
‡
‡
‡
nondeuterated unit, the corresponding 13C(2)HRe,12C(3)H cross-peak is very weak for
the deuterated unit (Fig. 11). Also, relative to the respective intraresidual
13C(2)H2,13C(3)H signals, the signal of the deuterated unit is weaker than that of the
nondeuterated one. In agreement with the presence of a 31 helix, this indicates that the
deuterated unit shows a C(2)H2,C(3)Hi ‡ 3 contact to the undeuterated unit, while the
nondeuterated unit shows the same contact to a 12C unit. If a 31 helix were the
predominant conformation, then only the two C(2)H2,C(3)H cross-peaks would have
to be of different intensity, since in this conformation the C(2)HRe,C(3)Hi ‡ 3 distance
is 4.6 Š while C(2)HSi and C(3)Hi ‡ 3 are only 3.2 Š apart from each other.
Conformational Analysis: Temperature Dependence. Some of the interresidual
signals become more intense with decreasing temperature (Table 4), suggesting a less-
averaged conformation at lower temperature. This is most pronounced for the
C(2)H2,C(3)H ROE in the nondeuterated unit. Also, the same ROE intensifies slightly
in the nondeuterated unit. In contrast, the intensity of other interresidual signals does
not change.
1H-1D Spectra recorded at different temperatures (Fig. 12) show that 3J(C(2)H,C(3)H)
-
coupling constants do not change significantly with temperature, which ± in
combination with the inconsistent temperature dependence of some ROE signals ±
leads us to assume that there can be no pronounced temperature dependence of the
conformation.
4. Conclusions. ± In conclusion, we synthesized and investigated the structures of
two specifically labeled linear oligo(hydroxybutanoates) by NMR spectroscopy in
3
3
solution. Due to similarity in J(H,H) and J(C,H)-coupling constants, we conclude that
both compounds adopt the same conformation. In these compounds, all but five to six
terminal residues adopt the same conformation. The labeling scheme of the 20-mer
made a unit-specific conformational analysis within the PHB chain possible. All
acquired NMR parameters show that the conformation is averaged. According to the
3
analysis of homonuclear J(C(2)H,C(3)H)-coupling constants the structure of both
compounds is strongly averaged, with neither of the helices contributing significantly
to the conformational ensemble. Interresidual-distance information from ROE data
confirms unit-specifically that the structure of the 20-mer is averaged, and that neither
DD;CSA
Cꢁ2†Cꢁ2†H;Cꢁ1†
one of the two helices is the predominant conformation. The analysis of G

Helvetica Chimica Acta ± Vol. 84 (2001)
1841
Fig. 12. 1D Spectra of the 20-mer 2 at 268 and at 308
excludes the 21 helix as the possible conformation. The averaged conformation is
maintained even at temperatures as low as 308. The extremely high flexibility of the
PHB backbone on the NMR time scale is in line with conclusions about its biological
role as ion-channel component and as appendage of proteins where it has never been
structurally identified, for instance, by diffraction methods [9].
Experimental Part
General. Ethyl 3-oxobutanoate 3* was purchased from CAMPRO Scientific, Germany. All other reagents
were used as received from Fluka or Senn. All labeled compounds 1 ± 2, 4* ± 7*, 9, 10*, 11*, 13, 14, 16 ± 18 were
synthesized as described for the unlabeled isomers [11]. Compounds 6, 7, 10, and 12 were prepared according to
1
the procedure in [11]. IR Spectra: Perkin-Elmer 1600-FT spectrophotometer. H-NMR: Bruker AMX-II-500
(500 MHz), AMX-400 (400 MHz), ARX-300 (300 MHz), or Varian Gem-200 (200 MHz) spectrometer.
13C-NMR: Bruker AMX-II-500 (125 MHz), AMX-400 (100 MHz), ARX-300 (75 MHz), or Varian Gem-200
(50 MHz) spectrometer; in CDCl3 unless other specified. All 2D measurements have been performed on Bruker
DRX-600 spectrometers in the Francis Bitter Magnet Laboratory and the Department of Chemistry at M.I.T. For
both compounds, the experiments have been recorded using 10-mg samples in 500 ml CDCl3. Different
HC(C)H-E.COSY experiments [20] have been performed to obtain homo- and heteronuclear 3J-coupling
constants. For the deuterated 20-mer, a CHx-edited ROESY was recorded to separate CHn moieties, where n is
either even or odd, resulting in a separation of ROE signals from the D-labeled and the unlabeled residue. To
investigate the overall conformation of the 20-mer in CDCl3 and the temp. dependence of the conformation,
CHx-filtered ROESY spectra were taken at 208 and 278. All other experiments were carried out at 208. MS:
VG ZAB2-SEQ for LSI (FAB) with 3-nitrobenzyl alcohol as matrix; and Bruker Reflex MALDI-TOF
spectrometer for MALDI-TOF.
Ethyl (R)-3-Hydroxy[1,2,3,4-13C4]butanoate (4*). 1H-NMR (300 MHz): 4.19 (m, 1J(H,13C) ˆ 144.6,
CHOH); 4.18 (2dq, 3J(H,H) ˆ 7.2, 3J(H,13C) ˆ 3.3, J ˆ 0.9, MeCH2); 3.03 (br. s OH); 2.45 (m, 1J(13C,H) ˆ
127.2, CH2); 1.28 (td, 3J(H,H) ˆ 7.2, J ˆ 0.9, MeCH2); 1.23 (qd, 1J(13C,H) ˆ 141.9, 2J(13C,H) ˆ 141.9, 2J(13C,H) ˆ
5.1, 3J(13C,H) ˆ 0.9, Me). 13C-NMR (300 MHz): 173.25, (ddd, J ˆ 228.3, 19.5, 9.9, C(4)); 64.40 (t, J ˆ 150.6,
C(3)); 60.78 (MeCH2); 42.85 (dd, J ˆ 145.5, 223.2, C(2)); 22.47 (dd, J ˆ 155.1, 19.5, C(4)); 14.24 (MeCH2).

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Benzyl (R)-3-[(tert-Butyl)diphenylsilyloxy][1,2,3,4-13C4]butanoate (5*). 1H-NMR (300 MHz): 7.69 ± 7.63
(m, Ph); 7.45 ± 7.27 (m, Ph); 5.06 (dd, 2J ˆ 12.3, 3J(H,13C(1)) ˆ 3.0, 1 H, PhCH2); 4.99 (dd, 2J ˆ 12.3,
3J(H,13C(1)) ˆ 3.0, 1 H, PhCH2); 4.31 (m, 1J(H,13C(3)) ˆ 145.5, CH); 2.60 (d, J ˆ 4.2, 1J(H,13C(2)) ˆ 130.5,
CH); 2.44 (m, J ˆ 3.3, 1J(H,13C(2)) ˆ 127.8, CH); 1.10 (qd, J ˆ 4.8, 1J(H,13C(4)) ˆ 126.6, 13C(4)H3); 1.02 (s, t-Bu).
13C-NMR (300 MHz): 171.54 (dd, 1J(C(1),C(2)) ˆ 228.3, 2J ˆ 9.9, C(1)); 145.45 (dd, J ˆ 165.0, 281.8, PhCH2);
136.17 (d, J ˆ 9.9, PhSi); 134.36 (d, J ˆ 92.1, PhSi); 129.88 (d, J ˆ 24.3, PhSi); 128.79 (s, PhCH2); 128.4 8 (d, J ˆ
24.3, PhCH2); 127.80 (d, J ˆ 19.5, PhSi); 123.24 (d, J ˆ 281.5, PhSi); 122.24 (d, J ˆ 281.5, PhSi); 67.02 (t,
1J(C(3),C(2)) ˆ 1J(C(3),C(4)) ˆ 155.4, C(3)); 44.74 (dd, 1J(C(2),C(3)) ˆ 155.4, 1J(C(2),C(1)) ˆ 228.3, C(2));
26.97 (s, Me3C); 23.70 (d, 1J(C(4),C(3)) ˆ 155.4, C(4)); 18.34 (d, J ˆ 33.9, PhSi); 17.79 (d, J ˆ 29.1, PhSi).
Benzyl (R)-3-Hydroxy-[1,2,3,4-13C4]butanoate (7*). 1H-NMR (300 MHz): 7.73 ± 7.66 (m, Ph); 7.48 ± 7.32
1
1
(m, Bn); 5.15 (d, J ˆ 3.3, PhCH2); 4.21 (m, J(13C,H) ˆ 144.9, J ˆ 2.1, CH); 2.92 (s, OH); 2.50 (m, J(13C,H) ˆ
130.5, J ˆ 3.0, J ˆ 1.5, CH2); 1.22 (qd, J(13C,H) ˆ 126.0, J ˆ 4.5, 3 H, MeCH); 1.09 (d, J ˆ 1.2, t-Bu). 13C-NMR
1
(300 MHz): 173.04 (dd, J ˆ 19.5, 1J ˆ 233.2, C(1)); 146 ± 122 (PhCH2, PhSi); 64.41 (t, 1J(C(3),C(4)) ˆ 155.4,
C(3)); 42.90 (dd, 1J ˆ 150.6, 1J ˆ 228.3, C(2)); 26.0 (s, Me3C); 22.49 (dd, 1J(C(4),C(3)) ˆ 155.4, J ˆ 19.5, C(4));
18.30 (d, J ˆ 33.9, PhSi); 17.75 (d, J ˆ 29.1, PhSi).
(R)-3-[(tert-Butyl)diphenylsilyloxy][1,2,3,4-13C4]butanoic Acid (6*). H-NMR (300 MHz): 7.70 ± 7.65 (m,
1
Ph); 7.46 ± 7.33 (m, Ph); 4.26 (dm, 1J(13C,H) ˆ 145.8, H C(3)); 2.52 (m, 1J(13C,H) ˆ 130.2, J ˆ 4.8, J ˆ 5.4, 1 H,
CH2); 2.46 (m, 1J(13C,H) ˆ 127.8, 1 H, CH2); 1.13 (qd, 1J(13C,H) ˆ 126.3, J ˆ 4.8, J ˆ 5.4, 3 H-C(4)); 1.03 (s, t-Bu).
13C-NMR (300 MHz); 177.23 (dd, J(C(1),C(2)) ˆ 213.7, J ˆ 9.6, C(1)); 136.02 (s, PhSi); 133.86 (d, J ˆ 150.34,
1
PhSi); 129.90 (d, J ˆ 29.1, PhSi); 127.78 (d, J ˆ 24.0, PhSi); 66.84 (t, 1J(C(3),C(4)) ˆ 1J(C(3),C(2)) ˆ 150.6,
C(3)); 44.26 (dd, 1J(C(2),C(3)) ˆ 150.6, 1J(C(2),C(1) ˆ 213.7, C(2)); 35.13 (dd, J ˆ 135.6, J ˆ 218.2, PhSi); 27.03
(s, PhSi); 23.54 (d, J ˆ 155.4, C(4)); 18.62 (dd, J ˆ 155, 296.2, PhSi); 13.78 (dd, J ˆ 14.4, 135.9, PhSi).
(2R)-5-Bromo-6-(bromo[13C1]methyl)-2-(tert-butyl)-4-oxo[4,5,6-13C3]-[1,3,5]-dioxin (8). IR (CHCl3):
2981m, 2967m, 2910w, 2876w, 1702s, 1547s, 1484m, 1463w, 1418w, 1406m, 1370m, 1308s, 1275w, 1154m,
1
3
1121m, 1074s, 1048w, 988w, 962m, 926w. H-NMR (300.08 MHz): 5.16 (t, J(H,C) ˆ 1.8, H C(2)); 4.30 (dddd,
J ˆ 157.5, 10.8, 2.7, 1.8, 1 H, BrCH2); 4.09 (dddd, J ˆ 154.8, 10.8, 5.7, 4.8, 1 H, BrCH2); 1.08 (s, t-Bu). 13C-NMR
(300.06 MHz): 166.09 (dd, J ˆ 335.2, 233.2, C(4)); 158.92 (d, J ˆ 310.6, C(5)); 107.55 (s, C(2)); 93.94 (ddd, J ˆ
330.1, 310.6, 19.5, C(6)); 34.66 (s, Me); 25.39 (ddd, J ˆ 228.0, 19.2, 9.6, Br13CH2); 23.90 (s, Me3C). EI-MS 336.0
(1.0), 335.0 (4.7), 334.0 (15.7), 332.0 (9.0), 332.0 (29.6), 331.0 (4.9), 330.0 (14.9), 290.0 (3.2), 288.9 (5.9), 286.9
(3.1), 255.0 (1.2), 254.1 (3.0), 253.1 (2.2), 252.1 (3.0), 251.0 (2.2), 249.9 (1.6), 248.9 (37.6), 247.9 (50.6), 246.9
(77.5), 245.9 (100), 244.9 (40.1), 243.9 (50.4), 242.9 (1.7), 238.0 (12.9), 237.0 (1.0), 236.0 (12.4), 219.9 (2.2), 217.9
(4.7), 215.9 (2.3), 196.0 (4.0), 194.0 (3.8), 176.9 (4.1), 174.9 (8.4), 172.9 (4.5), 169.0 (8.3), 168.0 (23.3), 167.0
(10.3), 166.0 (23.8), 165.0 (1.7), 151.9 (23.5), 149.9 (24.6), 149.0 (1.1), 138.0 (4.3), 136.0 (4.2), 124.9 (3.6), 123.9
(4.5), 122.9 (4.6), 121.9 (6.4), 109.0 (7.4), 107.0 (7.4), 87.1 (2.7), 86.1 (15.4), 85.1 (4.6), 71.1 (3.8), 69.1 (3.7), 57.1
(17.5), 41.1 (2.1).
Benzyl (2S,3R)-[1,2,3,4-13C4,2,4-2H2]butanoate (9). IR (CHCl3): 3672w, 3539m, 3081w, 3067w, 2962m,
2889w, 2454w, 2212w, 1951w, 1873w, 1810w, 1678s, 1604w, 1498m, 1455m, 1375m, 1299m, 1265m, 1160s, 1080m,
1
2
1
1018m, 914m, 820w. H-NMR (300.08 MHz): 5.16 (d, J ˆ 3.3, PhCH2); 4.21 (m, J ˆ 143.7, H C(3)); 2.89 (s,
OH); 2.52 (m, 1J ˆ 126.6, J ˆ 2.7, H C(2)); 1.22 (m, 1J ˆ 126.0, 2 H C(4)). 13C-NMR (300.08 MHz): 172.90
(dd, J ˆ 223.3, 19.5, C(1)); 135.74; 128.83; 128.60; 128.46; 66.67; 64.34 (t, J ˆ 150.3, C(3)); 42.61 (ddt, J ˆ 155.4,
77.7, 155.4, C(2)); 22.57 ± 21.77 (dd, dq, dh, 2J ˆ 19.5, J ˆ 160.2, J ˆ 77.7, J ˆ 77.7, C(4)). D-NMR (400 MHz): 2.477
(d, J ˆ 128.8, D C(2)); 1.217 (d, J ˆ 125.8, D C(4)). EI-MS: 203.2 (2.2), 202.2 (5.7), 201.2 (8.7), 200.1 (7.3),
199.1 (3.1), 181.1 (4.0), 173.2 (2.4), 172.2 (5.9), 171.1 (6.9), 170.1 (4.3), 155.1 (3.1), 154.1 (6.1), 152.1 (5.0), 110.1
(1.9), 109.1 (8.3), 108.1 (60.5), 107.1 (52.2), 106.1 (9.7), 105.1 (24.4), 93.1 (2.1), 92.1 (11.6), 91.1 (100), 90.1 (7.8),
89.1 (4.7), 79.1 (7.8), 78.1 (2.2), 77.1 (5.8), 65.1 (4.3).
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]bis[(R)-oxy(3-[13C1]methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl]
(10*). 1H-NMR (300 MHz): 7.70 ± 7.63 (m, Ph); 7.42 ± 7.30 (m, Ph); 5.22 (d, 1J(H C(3),CH) ˆ 151.5, J ˆ 2.4,
CH); 5.08 (d, J ˆ 3.0, PhCH2); 4.24 (d, 1J(H,C(3)) ˆ 145.5, J ˆ 3.3, CH); 2.64 (d, 1J(H,C(2)) ˆ 130.5, 1 H, CH2);
2.51 (d, 1J(H,C(2)) ˆ 128.7, 1 H, CH2); 2.45 (d, 1J(H,C(2)) ˆ 129.6, 1 H, CH2); 2.32 (d, 1J(H,C(2)) ˆ 128.7, 1 H,
CH2); 1.22 (qdd, 1JH,C(4)) ˆ 127.2, J ˆ 4.8, J ˆ 1.5, C(4)H3); 1.09 (qdd, 1J(H,C(4)) ˆ 126.0, J ˆ 4.8, J ˆ 1.5,
C(4)H3); 1.03 (s, t-Bu). 13C-NMR (300 MHz): 170.70 (d, 1J(C(1),C(2)) ˆ 228.0, C(1)); 170.33 (dd,
1J(C(1),C(2)) ˆ 228.0, J ˆ 9.6, C(1)); 136.10 (d, J ˆ 9.6, PhSi); 134.39 (d, J ˆ 97.2, PhSi); 129.89 (d, J ˆ 19.5,
PhSi); 128.84 (s, PhSi); 128.55 (d, J ˆ 9.9, PhSi); 127.80 (d, J ˆ 19.5, PhSi); 67.39 (t, 1J(C(3),C(2) ˆ
1J(C(3),C(4)) ˆ 160.2, C(3)); 66.85 (t, 1J(C(3),C(2)) ˆ 1J(C(3),C(4)) ˆ 160.2, C(3)); 44.71 (dd, J ˆ 155.4,
1
228.3, C(2)); 40.82 (dd, J ˆ 155.4, 228.3, C(2)); 26.98 (s, PhSi); 23.44 (d, JCH,C(4)) ˆ 155.4, C(4)); 19.83 (dd,
2
1J(H,C(4)) ˆ 155.4, J(C(4),H) ˆ 9.6, C(4)).

Helvetica Chimica Acta ± Vol. 84 (2001)
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a-Hydroxy-w-[(tert-butyl)diphenylsilyloxy]bis[(R)-oxy(3-[13C1]methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl]
(11*). 1H-NMR (300.08 MHz): 7.68 ± 7.66 (m, Ph); 7.45 ± 7.34 (m, Ph); 5.18 (m, 1J ˆ 151.2, CHCH2COOH); 4.24
1
1
1
(m, J ˆ 144.9, SiOCH); 2.70 ± 2.27 (m, CH2); 1.38 (m, J ˆ 39.6, J ˆ 4.2, Me); 1.03 (s, t-Bu); 0.95 (m, J ˆ 41.1,
J ˆ 4.2, SiOCHMe). 13C-NMR (300.08 MHz): 176.20 (d, 1J ˆ 223.2, COO); 170.69 (d, 1J ˆ 228.1, COO); 135;
134.38; 134.00; 129.84; 129.76; 127.75; 127.67; 67.38 (t, J ˆ 77.2, OCH); 66.86 (t, J ˆ 77.7, OCH); 44.77 (dd, 1J ˆ
228.4, J ˆ 155.4, CH2COOH); 40.52 (dd, 1J ˆ 223.3, J ˆ 160.2, SiOCHCH2); 27.05; 23.54 (d, 1J ˆ 155.4, CH3);
19.91 (d, 1J ˆ 155.1, SiOCHMe).
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]{bis[(R)-oxy(3-methyl-1-oxopropane-1,3-diyl)]bis[(R)-oxy(3-
[13C1]methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl)]} (13). Ir (CHCl3): 3072m, 2864m, 2932m, 2858m, 1959w, 1895w,
1736s, 1690s, 1587w, 1456m, 1427m, 1382m, 1368m, 1301m, 1290m, 1176s, 1110s, 1106s, 1060s, 994m, 910w, 822m,
1
613m. H-NMR (300.06 MHz): 7.69 ± 7.63 (m, Ph); 7.45 ± 7.28 (m, Ph); 5.29 (m, J ˆ 6.6, J ˆ 1.8, CH); 5.21 (m,
CH); 5.18 (m, 1J ˆ 151.2, J ˆ 2.4, 13CH); 5.12 (s, PhCH2); 4.26 (m, 1J ˆ 145.2, J ˆ 3.0, SiOCH); 2.68 (dd, 2J ˆ 15.6,
2
3
2
3
3J ˆ 7.5, 1 H, HReCH); 2.53 (dd, J ˆ 15.6, J ˆ 5.4, 1 H, HCHSi); 2.52 (dd, J ˆ 15.6, J ˆ 7.5, 1 H, HReCH); 2.37
(dd, 2J ˆ 15.6, 3J ˆ 6.3, HCHSi); 2.85 ± 2.09 (m, 13CH2); 1.27 (d, 3J ˆ 6.3, Me); 1.21 (dm, 1J ˆ 127.2, J ˆ 4.5, 13CH3);
1.21 (d, 3J ˆ 6.3, Me); 1.11 (m, 1J ˆ 126.3, 13CH3); 1.03 (s, t-Bu). 13C-NMR (300.06 MHz): 170.50 (d, 1J ˆ 228.3,
13COO); 169.37 (dd, 1J ˆ 228.3, 3J ˆ 9.6, SiOCHCH2COO); 136.00; 135.87; 134.43; 134.10; 129.82; 129.76;
128.77; 128.53; 127.75; 127.67; 67.38 (t, 1J ˆ 140.7, O13CH); 66.86 (t, 1J ˆ 145.8, SiOCH); 44.74 (dd, 1J ˆ 228.0,
2J ˆ 155.1, 13CH2); 41.03 (dd, 1J ˆ 233.1, 2J ˆ 160.2, SiOCHCH2); 27.08; 23.59 (d, 1J ˆ 155.4, 13CH3); 20.00; 19.90
(d, 1J ˆ 155.1, SiOCHMe); 19.90. MALDI-TOF-MS: 717.6 (0.91), 720.6 (2.9), 721.6 (55.28), 722.6 (24.97), 723.6
(11.58), 724.7 (3.52), 725.6 (0.79).
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]bis{bis[(R)-oxy(3-methyl-1-oxopropane-1,3-diyl)]bis[(R)-oxy-
(3-[13C1]methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl)]} (14). IR (CHCl3): 3011m, 2969m, 2932m, 2859m, 1736s,
1690s, 1456m, 1428m, 1383m, 1369m, 1299m, 1290m, 1173s, 1105s, 1159s, 1037w, 993m, 976m, 8.22m. 1H-NMR
(300.06 MHz): 7.68 ± 7.65 (m, Ph); 7.45 ± 7.30 (m, Ph); 5.24 (m, 1J ˆ 151.5, J ˆ 3.9, 2, 13CH); 5.36 ± 5.16 (m, J ˆ 6.3,
4 CH); 5.18 (m, 1J ˆ 151.5, 1 13CH); 5.12 (s, PhCH2); 4.25 (m, 1J ˆ 145.5, SiOCH); 2.88 ± 2.09 (m, 13CH2); 2.72 ±
2.36 (m, CH2); 1.28 (m, 1J ˆ 127.2, 13CH3); 1.21 (m, 1J ˆ 127.2, 1 13CH3); 1.11 (m, 1J ˆ 126.3, SiOCHMe); 1.28 (d,
3J ˆ 6.0, 1 Me); 1.26 (d, 3J ˆ 6.0, 2 Me); 1.24 (d, 3J ˆ 5.7, 1 Me); 1.03 (s, t-Bu). 13C-NMR (300.06 MHz): 170.47 (d,
1J ˆ 233.1, 13COO); 169.3 (d, 1J ˆ 228.0, 2 13COO); 169.29 (d, 1J ˆ 233.1, 1 13COO); 169.29 (COO); 136.00;
1
1
134.45; 134.12; 129.84; 129.76; 128.77; 128.51; 127.75; 127.69; 67.77 (t, J ˆ 160.2, 2 O13CH); 67.35 (t, J ˆ 150.3,
1 O13CH); 66.88 (t, 1J ˆ 155.4, SiOC); 44.75 (dd, 1J ˆ 228.0, 1J ˆ 155.1, 1 13CH2); 41.03 (dd, 1J ˆ 228.3, 1J ˆ 155.4,
1 13CH2); 40.96 (dd, 1J ˆ 2 13CH2); 27.10; 23.59 (d, 1J ˆ 155.4, 13CH3); 19.91 (d, 1J ˆ 155.1, 3 13CH3); 19.95; 19.35.
MS (MALDI): 1047.5, 1048.5 (5.51), 1049.5 (30), 1050.1 (7.09), 1051.1 (100.00), 1052.0 (53.44), 1053.1 (30.4),
1054.0 (11.1), 1055.0 (3.60).
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]tetrakis{bis[(R)-oxy(3-methyl-1-oxopropane-1,3-diyl)]bis[(R)-
oxy(3-[13C1]methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl)]} (1). IR (CHCl3): 2978m, 2933m, 1736s, 1692s, 1456m,
1427m, 1383m, 1370m, 1301m, 1291s, 1169s, 1104m, 1058m, 971m, 823w. 1H-NMR (500.13 MHz): 7.68 ± 7.65 (m,
Ph); 7.42 ± 7.34 (m, Ph); 5.25 (m, 1J ˆ 152.8, 13CH); 5.33 ± 5.21 (m, CH); 5.18 (m, 1J ˆ 152.7, 1 13CH); 5.12 (s,
1
1
PhCH2); 4.26 (m, J ˆ 145.3, SiOCH); 2.73 ± 2.31 (m, 13CH2); 2.68 ± 2.40 (m, CH2); 1.27 (m, J ˆ 127.5, J ˆ 4.1,
13CH3); 1.281 (d, 3J ˆ 6.2, 1 Me); 1.27 (d, 3J ˆ 6.3, Me); 1.26 (d, 3J ˆ 6.3, Me); 1.245 (d, 3J ˆ 6.3, 1 Me); 1.243 (d,
3J ˆ 6.3, 1 Me); 1.21 (m, 1J ˆ 127.4, J ˆ 4.5, 13CH3); 1.11 (m, 1J ˆ 126.2, J ˆ 4.5, SiOCHMe); 1.03 (s, t-Bu).
13C-NMR (500 MHz): 170.30 (d, 1J ˆ 228.5, 13COO); 169.90 (COO); 169.19 (dd, J ˆ 230.5, 10.5, 13COO); 169.17
(COO); 169.14 (COO); 169.14 (dd, J ˆ 231.5, 8.5, 13COO); 169.13 (COO); 135.83; 135.81; 135.72; 134.28;
133.94; 129.66; 129.59; 128.60; 128.35; 128.345; 127.59; 127.51; 67.60 (t, 1J ˆ 157.1, 67.76; 67.70; 67.17 (td, 1J ˆ
156.6, J ˆ 8.7, O13CH); 66.70 (t, 1J ˆ 155.5, O13CH); 66.48; 44.58 (dd, 1J ˆ 228.4, 1J ˆ 154.5, 13CH2); 41.03 (Me);
40.86 (ddd, 1J ˆ 231.1, 1J ˆ 157.0, 2J ˆ 5.9, 13CH2); 40.78 (dd, 1J ˆ 231.5, J ˆ 157.3, 13CH3); 40.64 (Me); 40.56
(Me); 27.0; 23.41 (ddd, 1J ˆ 155.7, 2J ˆ 8.4, 3J ˆ 4.6, 13CH3); 19.81 (Me). MALDI-TOF-MS: 1751.4 (3.6), 1752.4
(2.7), 1753.3 (3.0), 1754.3 (19.3), 1755.3 (100.00), 1756.3 (67.5), 1757.3 (49.7), 1758.3 (23.0), 1759.3 (12.0),
1760.3 (3.3).
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]{[(R)-oxy(3-(13C1)methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl)]-
bis[(R)-oxy(3-methyl-1-oxopropane-1,3-diyl)]} (16). 1H-NMR (300.08 MHz): 7.69 ± 7.65 (m, Ph); 7.45 ± 7.29 (m,
Ph); 5.27 (m, J ˆ 148.2, OCHCH2CO2Bn); 5.15 (m, J ˆ 6.3, CH); 5.11 (d, J ˆ 3.6, PhCH2); 4.26 (m, J ˆ 6.3,
SiOCH); 2.65 (m, J ˆ 130.2, CHReHCOOBn); 2.51 (m, J ˆ 128.4, J ˆ 3.3, CHHSiCOOBn); 2.53 (dd, 2J ˆ 6.9,
3J ˆ 5.1, CHReH); 2.48 (dd, 2J ˆ 6.9, 3J ˆ 3.0, CHHSi); 2.38 (dd, 2J ˆ 6.6, CHReH); 2.33 (dd, 2J ˆ 6.9, 3J ˆ 3.6,
CHHSi); 1.24 (m, J ˆ 127.5, 1,5, Me); 1.19 (d, J ˆ 6.3, Me); 1.11 (d, J ˆ 5.7, SiOCHMe); 1.03 (s, t-Bu). 13C-NMR
(300.08 MHz): 170.10 (dd, J ˆ 232.9, 9.9, COOBn); 135.99; 134.12; 129.85; 129.78; 128.78; 128.55; 127.78; 127.69;

1844
Helvetica Chimica Acta ± Vol. 84 (2001)
67.77 (t, J ˆ 160.2, CHCH2COOBn); 44.78; 41.06; 40.83 (dd, J ˆ 232.9, 160.2, CH2); 27.08; 23.62; 19.99 (dd, J ˆ
155.4, 9.9, MeCHCH2COOBn).
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]{octakis[(R)-oxy(3-methyl-1-oxopropane-1,3-di yl)][(R)-oxy(3-
[
13C1]methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl)]bis[(R)-oxy(3-methyl-1-oxopropane-1,3-diyl)]} (18). IR
(CHCl3): 2984m, 2934m, 2859w, 1738s, 1693m, 1457m, 1427m, 1382m, 1304m, 1265m, 1178s, 1134m, 1103m,
1083m, 1058m, 1005m, 997m, 976m, 822w, 612w. 1H-NMR (300.08 MHz): 7.69 ± 7.65 (m, Ph); 7.45 ± 7.30 (m, Ph);
5.24 (m, J ˆ 150.6, 13CH); 5.25 (m, J ˆ 6.0, J ˆ OCH); 5.18 (m, J ˆ 6.6, COCH); 5.12 (s, CH2Ph); 4.25 (m, J ˆ 6.3,
SiOCH); 2.6 (m, 13CHReH13COO); 2.5 (m, 13CHHSi13COO); 2.68 ± 2.35 (m, CH2); 1.24 (m, J ˆ 127.5, 3 H,
13CH3); 1.28 (d, J ˆ 6.0, 3 H, CH3); 1.26 (d, J ˆ 6.6, 18 H, 6 CH3); 1.24 (d, J ˆ 6.6, CH3); 1.21 (d, J ˆ 6.6, CH3);
1.11 (d, J ˆ 6.0, CH3); 1.03 (s, 9 H, t-Bu). 13C-NMR (300.08 MHz): 170.50; 170.10; 169.32; 169.31 (dd, J ˆ 228.4,
j ˆ 9.9, 13COO); 135.99; 135.86; 134.43; 134.09; 129.85; 129.77; 128.78; 128.54; 127.76; 127.68; 67.75; 67.69 (dt, J ˆ
155.1, J ˆ 19.5, O13CH); 44.76; 40.96 (s, CH2); 40.94 (dd, J ˆ 232.9, J ˆ 155.1, 13CH2); 27.08; 23.58; 19.93; 19.92 (d,
‡
‡
‡
‡
J ˆ 160.2, 13CH3); 19.33. MALDI-MS: 1320 (M ), 1336 ([M ‡ Na] ), 1360 [M ‡ Na ‡ K] ), 1229 [M Bn] ),
1143, 1057, 977, 971, 891, 885, 801, 715, 660, 629, 543, 440, 410, 367, 273.
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]{[(2S,3R)-oxy(3-([13C1,2H1]methyl-1-oxo-[1,2,3-13C3,2-2H1]pro-
pane-1,3-diyl)]octakis[(R)-oxy(3-methyl-1-oxopropane-1,3-diyl)]} (17). IR (CHCl3): 307w, 2985m, 2935m,
2858w, 1738s, 1694m, 1457m, 1427m, 1382m, 1305m, 1263m, 1178s, 1136m, 1103m, 1082m, 1059m, 1006m, 998m,
978m, 823w, 612w. 1H-NMR (300.08 MHz): 7.68 ± 7.65 (m, Ph); 7.44 ± 7.31 (m, Ph); 5.28 (m, 1J ˆ 151.2,
CHCDHCOO); 5.24 ± 5.19 (m, J ˆ 6.9, CH); 5.12 (d, 2J ˆ 3.0, PhCH2); 4.25 (m, J ˆ 6.3, SiOCH); 2.53 (m, 1J ˆ
128.7, CHDCOO); 2.62 ± 2.34 (m, CH2); 1.26 (m, 1J ˆ 127.5, CH0;1;2;3D0;1;2;3CHCHDCOO); 1.26 (d, J ˆ 6.6,
5 Me); 1.24 (d, J ˆ 6.3, Me); 1.21 (d, J ˆ 6.6, Me); 1.11 (d, J ˆ 6.0, SiOCHMe); 1.03 (s, t-Bu). 13C-NMR
(300.08 MHz): 170.11 (dd, J ˆ 228.1, 9.6, COOBn); 169.39; 169.34; 136.00; 134.43; 134.09; 129.85; 129.77;
128.78; 128.54; 127.76; 127.68; 67.77 (t, J ˆ 160.2, OCHCHDCOOBn); 66.88; 66.67; 66.64; 44.76; 41.04; 40.96;
40.51 (ddt, J ˆ 77.4, 155.1, 77.4, CHDCOOBn); 27.06; 23.60; 20.09 ± 19.15 (dd, dt, dq, J ˆ 9.6, J ˆ 77.7,
CH0;1;2;3D0;1;2;3). D-NMR (400 MHz): 2.672 (d, J ˆ 129.1, CHDCOOH); 1.275 (d, J ˆ 125.7, CH2D). MALDI-
‡
‡
‡
MS: 1166 ([M ‡ Na ‡ K] ), 1151 ([M ‡ Na] ), 1057, 981 ([M 2 HBn] ), 881, 807, 795, 709, 623, 549, 453, 410,
367, 281, 273.
a-Benzyl-w-[(tert-butyl)diphenylsilyloxy]{octakis[(R)-oxy(3-methyl-1-oxopropane-1,3-di yl)][(R)-oxy(3-
[13C1]methyl-1-oxo[1,2,3-13C3]propane-1,3-diyl)]bis[(R)-oxy(3-methyl-1-oxopropane-1,3-diyl)][(2S,3R)-oxy(3-
[13C1,2H0;1;2;3]methyl-1-oxo[1,2,3-13C3,2-2H1]propane-1,3-diyl)]octakis[(R)-oxy(3-methyl-1-oxopropane-1,3-di-
yl)]} (2). IR (CHCl3): 2985m, 2936m, 2859w, 1736s, 1693m, 1603w, 1457m, 1449m, 1427m, 1383m, 1305s, 1265m,
1178s, 1135m, 1102m, 1083m, 1059s, 1005w, 997m, 929w, 900w, 822w. 1H-NMR (300.08 MHz): 7.68 ± 7.65 (m, Ph);
7.42 ± 7.34 (m, Ph); 5.24 (m, 1J ˆ 152.4, O13CH); 5.25 (m, J ˆ 6.6, 0.9, CH); 5.16 (m, J ˆ 6.0, CH); 5.12 (s, PhCH2);
4.25 (m, J ˆ 6.3, SiOCH); 2.80 ± 2.24 (m, 13CH2); 2.68 ± 2.35 (m, CH2, CDHCOO); 1.26 (dm, 1J ˆ 127.5, 2 13CH3);
1.27 (d, 3J ˆ 6.0, Me); 1.267 (d, 3J ˆ 6.3, Me, CH0;1;2;3D0;1;2;3); 1.23 (d, 3J ˆ 7.2, Me); 1.21 (d, 3J ˆ 6.6, Me); 1.10 (d,
3J ˆ 5.7, SiOCHMe); 1.02 (s, t-Bu). 13C-NMR (300.08 MHz): 170.50; 170.10; 169.34; 169.34 (dd, J ˆ 233.2, 9.6);
135.98; 135.86; 134.43; 134.09; 129.85; 129.77; 128.78; 128.54; 127.76; 127.68; 67.77 (t, J ˆ 155.4); 44.76; 40.94 (dd,
J ˆ 232.9, 160.2); 40.96; 40.63 (m, J ˆ 77.7); 27.06; 23.60; 19.94; 19.93 (dd, J ˆ 155.4, 9.6); 19.93 ± 18.60 (m, J ˆ
82.5). D-NMR (400 MHz): 2.815 ± 2.426 (m, J ˆ 123.9, CHDCOO); 1.264 (d, J ˆ 126.7, CH2D). MALDI-MS:
‡
‡
‡
‡
‡
2141 ([M ‡ Na ‡ K] ), 2117 ([M ‡ K] ), 2101 ([M ‡ Na] ), 2009 ([M Bn] ), 1925 ([M 1 HB] ), 1839
‡
‡
‡
‡
‡
([M 2 HB] ), 1753 ([M 3 HB] ), 1667 ([M 4 HB] ), 1581 ([M 5 HB] ), 1495 ([M 6 HB] ), 1409
([M 7 HB] ), 1323 ([M 8 HB] ), 1587, 1501, 1415, 1329, 1243, 1232, 1155, 1063, 977, 891, 807, 801, 721, 715,
‡
‡
635, 629, 543, 453.
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Received April 20, 2001