Spin system assignment of homo-o-phenylene ethynylene oligomers

Article abstract of DOI:10.1021/jo061175f

We previously reported the synthesis and solution characterization of short o-phenylene ethynylene (oPE) foldamers. Proton correlation techniques are not adequate for NMR assignment in these compounds as the ethynylene linkers interrupt proton connectivit

Full text of DOI:10.1021/jo061175f

Spin System Assignment of Homo-o-Phenylene Ethynylene
Oligomers
Morris M. Slutsky, Ticora V. Jones, and Gregory N. Tew*
Polymer Science & Engineering Department, UniVersity of Massachusetts, Amherst, Massachusetts 01003
tew@mail.pse.umass.edu
ReceiVed June 8, 2006
We previously reported the synthesis and solution characterization of short o-phenylene ethynylene (oPE)
foldamers. Proton correlation techniques are not adequate for NMR assignment in these compounds as
the ethynylene linkers interrupt proton connectivity. In order to facilitate structural characterization and
more fully harness the power of NMR, it is necessary to know the sequence of spin systems along the
molecular backbone. For example, spin system assignment is required to unambiguously assign NOE
correlations for structural determination of folded forms in solution. Therefore, we developed a method
to assign the aromatic spin systems in these compounds using HMBC experiments. This has been performed
for tetrameric (Es ), pentameric (Es ), and hexameric (Es ) oligomers and is expected to prove useful for
4 5 6
this class of foldamers in general. The proton assignments obtained by this technique have been useful
toward confirming the previous hypotheses of helical folding in oPE systems.
Introduction
contrast, the regioisomeric o-phenylene ethynylene (oPE)
5
,25-27
backbone has been studied in far less detail.
We recently
Abiotic molecules designed to fold into a desired conforma-
tion in solution, known as foldamers, have been the subject of
intense study in recent years.1-4 A primary motivation for this
research is to examine simple motifs that mimic the folded
structures of biological macromolecules in order to decipher
fundamental requirements leading to well-defined conformations
in solution. Oligomeric and polymeric m-phenylene ethynylene
reported a series of short oPE oligomers that showed evidence
of helical folding by NMR measurements of chemical shift and
NOE interactions for the first time.
27
(
10) Masu, H.; Sakai, M.; Kishikawa, K.; Yamamoto, M.; Yamaguchi,
K.; Kohmoto, S. J. Org. Chem. 2005, 70, 1423-1431.
11) Matsuda, K.; Stone, M. T.; Moore, J. S. J. Am. Chem. Soc. 2002,
24, 11836-11837.
12) Prest, P. J.; Prince, R. B.; Moore, J. S. J. Am. Chem. Soc. 1999,
(
1
(
PE) foldamers have been widely synthesized and characterized
(
5-19
20-24
by many experimental
and computational
methods. In
121, 5933-5939.
(
(
13) Ray, C. R.; Moore, J. S. AdV. Polym. Sci. 2005, 177, 91-149.
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(1) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S.
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2005, 16, 189-194.
Chem. ReV. 2001, 101, 3893-4011.
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2) Cheng, R. P. Curr. Opin. Struct. Biol. 2004, 14, 512-520.
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2423-2427.
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(17) Arnt, L.; Tew, G. N. Langmuir 2003, 19, 2404-2408.
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Phys. Chem. B 2006, 110, 3527-3532. .
1
619-1627.
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13518.
Soc. 1999, 121, 2643-2644.
(
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7) Hecht, S.; Khan, A. Angew. Chem., Int. Ed. 2003, 42, 6021-6024.
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9) Lahiri, S.; Thompson, J. L.; Moore, J. S. J. Am. Chem. Soc. 2000,
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22, 11315-11319.
10.1021/jo061175f CCC: $37.00 © 2007 American Chemical Society
3
42
J. Org. Chem. 2007, 72, 342-347
Published on Web 12/19/2006

Homo-o-phenylene Ethynylene Oligomers
FIGURE 1. Structures of Es
4
, Es
5
, and Es
6
oligomers. The rings are labeled consecutively starting from the Si terminus. R ) (CH
2 2 3 3
CH O) CH .
In order to fully interpret NMR measurements of these oPE
oligomers, a means of accurately assigning the proton resonances
and spin systems along the aromatic backbone is necessary as
these backbones consist of identical aromatic units, each with
1
1
FIGURE 2. (Left) Structures Es
chemical shift. (Inset) Spectrum of Es
aggregation. When the sample is diluted, the peaks shift downfield, further supporting aggregation. Letter labels indicate splitting pattern of each
4
, Es
5
, and Es
6
6
, with final H assignments. (Right) Aromatic H regions of Es
4
, Es
5
, and Es
6
, labeled in order of
at 1.25 mM, lacking the small peaks visible in the more concentrated spectrum which are attributed to
protonsA denotes a wide (≈8.4 Hz) doublet, B denotes a narrow (≈2.5 Hz) doublet, and C denotes a doublet of doublets (J
1
≈ 2.5 Hz, J ≈ 8.4
2
Hz). Splitting patterns unambiguously identify ring position of each proton.
J. Org. Chem, Vol. 72, No. 2, 2007 343

Slutsky et al.
demonstrated earlier on tetrameric oPE foldamers.27 However,
as the oligomers become larger, distinguishing the aromatic
protons in the molecular backbone becomes more challenging.
Classical methods of assigning the primary sequence of peptides
and proteins via local NOE interactions between amide and
28
alpha protons have been adapted to peptidomimetic and related
2
9,30
foldamers.
However, these methods do not suffice for
foldamers with backbones containing a series of tertiary carbon
atoms. In the case of oPE oligomers, the aromatic spin systems
are spaced too far apart to be linked by sequential NOE
interactions. Heteronuclear multiple bond correlation (HMBC)
1
13
spectroscopy may be used to correlate H resonances to
C
resonances that are separated by multiple bonds and was
expected to allow assignment of oPE foldamer backbones.
Recent work demonstrated the use of HMBC to assign
backbone protons in quinoline and pyridine-derived oligomers.31
These foldamers are also composed of aromatic backbones with
many tertiary carbon atoms, and although connected by amide
linkages, standard NOE connectivity methods could not be used.
HMBC methods, using the amide linkage to bridge spin systems,
allowed precise assignment of the aromatic rings along the
backbone. In the oPE oligomers described here, it was unclear
if HMBC methods would be able to bridge the longer couplings
across the carbon-carbon triple bonds. A few examples are
documented in the literature where HMBC couplings spanning
FIGURE 3. Ester, side chain, and aromatic 13C signals for Es
and Es oligomers. Red carbon atoms indicate the type of peak(s) in
each region.
4 5
, Es ,
6
32
five or even six bonds, including across carbon-carbon triple
3
3,34
bonds,
have been used for structural assignment. For oPE,
HMBC couplings across a minimum of four bonds would be
required to establish spin system connectivity leading to
determination of the primary sequence. Therefore, it seemed
reasonable that HMBC methods would prove useful.
The ester-substituted oPE oligomers under investigation in
this report are shown in Figure 1, and the rings are labeled
consecutively starting from the Si terminus. The tetramer,
denoted as Es4, has previously shown evidence of a helical
structure in solution by chemical shift measurements, which
indicated ring stacking, and ROESY (rotating-frame Overhauser
effect spectroscopy) interactions between the terminal N3Et2 and
27
Si(CH3)3 groups. Reasonable assignments of primary sequence
1
could be made for Es4 based on H chemical shifts of model
compounds and monomeric, dimeric, and trimeric precursors.
However, this method of assignment becomes difficult or
impossible with increasing oligomer length. This report confirms
our assignments for Es4 and provides the full assignment of
the aromatic protons for Es5 and Es6. Assignments of the
(
(
(
24) Lee, O. S.; Saven, J. G. J. Phys. Chem. B 2004, 108, 11988-11994.
25) Blatchly, R. A.; Tew, G. N. J. Org. Chem. 2003, 68, 8780-8785.
26) Jones, T. V.; Blatchly, R. A.; Tew, G. N. Org. Lett. 2003, 5, 3297-
3
299.
(27) Jones, T. V.; Slutsky, M. M.; Laos, R.; de Greef, T. F. A.; Tew,
G. N. J. Am. Chem. Soc. 2005, 127, 17235-17240.
(28) Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley: New
York, 1986.
FIGURE 4. Assignment of all 13C acetylene signals in Es
4
5
, Es , and
6
Es oligomers, labeled in order of chemical shift. Each acetylenic carbon
(
7.
29) Seebach, D.; Hook, D. F.; Glattli, A. Biopolymers 2006, 84, 23-
is referred to either as â to proton B or â to proton A as shown on the
3
3
8
structure of Es
in Es
4
. The internal acetylene carbons, for example, R to R
(30) Seebach, D.; Mathad, R. I.; Kimmerlin, T.; Mahajan, Y. R.;
4
, separate into two regions with those â to proton B between 97
Bindschadler, P.; Rueping, M.; Jaun, B.; Hilty, C.; Etezady-Esfarjani, T.
and 94 ppm and those â to proton A between 94 and 92 ppm.
HelV. Chim. Acta 2005, 88, 1969-1982.
(31) Dolain, C.; Grelard, A.; Laguerre, M.; Jiang, H.; Maurizot, V.; Huc,
I. Chem.sEur. J. 2005, 11, 6135-6144.
32) Araya-Maturana, R.; Delgado-Castro, T.; Cardona, W.; Weiss-Lopez,
B. E. Curr. Org. Chem. 2001, 5, 253-263.
33) Cavin, A.; Potterat, O.; Wolfender, J. L.; Hostettmann, K.; Dyat-
myko, W. J. Nat. Prod. 1998, 61, 1497-1501.
34) Zgoda, J. R.; Freyer, A. J.; Killmer, L. B.; Porter, J. R. J. Nat. Prod.
2001, 64, 1348-1349.
protons of similar chemical shift. This decoding of the primary
sequence along the backbone is required to more fully harness
the power of NMR. For example, through-space NOE correla-
tions become extremely powerful when they can be assigned
to specific protons on the molecular backbone. This was
(
(
(
344 J. Org. Chem., Vol. 72, No. 2, 2007

Homo-o-phenylene Ethynylene Oligomers
FIGURE 5. Assignment of TMS and N
acetylene carbons in Es ssimilar peaks are observed in Es
carbon and nearby ring protons in Es , Es , and Es , respectively.
3
Et
2
termini for Es
4
, Es
5
, and Es
6
. (A) HMBC interaction between the Si(CH
3
)
3
protons and neighboring
Et
4
5
and Es
6
as well. (B, C, and D) HMBC correlations between the aromatic C-N
3
2
4
5
6
aromatic 1H protons in Es4, Es5, and Es6 oligomers were
performed by a combination of 1D, COSY, and HMBC
measurements. Splitting patterns of each 1D signal were used
to identify ring position, COSY measurements were taken to
identify spin systems, and in order to assign the primary
sequence of spin systems, HMBC measurements were taken.
of the concentration and these additional peaks were not large
enough to interfere with structural assignment, this concentrated
sample was used for data collection. Assignment of A, B, and
C to individual spin systems or aromatic rings was achieved
using COSY. In the case of Es6, COSY data was confirmed by
the observation of HMBC correlations between ester carbons
and aromatic ring protons. This enabled each set of A, B, and
C protons, connected to an individual ring, to be grouped
together, although it did not provide any sequence order along
the backbone.
1
3
Shorter-range HMBC couplings were used to assign the
C
signals for acetylenic carbons adjacent to each ring, and
afterward, longer range HMBC couplings across the carbon-
carbon triple bonds were used to place the rings in order.
13C signals, shown in Figures 3 and 4, could be identified as
ester, aromatic, or acetylenic by chemical shift, and each type
is highlighted by color in the chemical structure. Signals from
the two end groups had unique chemical shifts that aided in
structure determination. The aromatic carbon connected to the
N3Et2 group at the N terminus has a chemical shift at roughly
Results and Discussion
1
1
The aromatic H signals from 1D H NMR were labeled first
according to their splitting pattern on the ring as A, B, or C.
This assignment is consistent with our previous nomenclature
and retained here for clarity. Next, the individual signals in the
spectra were numbered according to their chemical shift order
as shown in Figure 2. This follows conventional assignment
procedures and is not associated with ring numbers along the
backbone, as can be seen with Es4 in which the Si-terminal ring
1
56 ppm which falls between that of the ester carbons and the
other aromatic carbons, as shown in Figure 3, while the Si-
CH3)3 carbons at the Si terminus of the oligomer provide a
(
strong signal close to 0 ppm.
3
4
4
contains A , B , and C . On examining Figure 2c some smaller
peaks are observed in the aromatic region of the Es6 sample
which are due to aggregation at high concentration and not an
impurity, as the spectrum of a more dilute Es6 sample is shown
in the inset, and HPLC analysis showed good (>95%) purity.
As the time required for COSY and HMBC acquisition at a
given resolution and S/N is inversely proportional to the square
The acetylenic carbons between 105 and 90 ppm were labeled
1-
8
1- 10
1- 12
according to chemical shift order, from R R , R R , or R R
in Es4, Es5, and Es6 respectively, as shown in Figure 4. Within
the acetylenic carbon region, there appears to be three groups
of signals, the two unique carbons located on the terminal
acetylene (shown in blue) and then two regions with several
J. Org. Chem, Vol. 72, No. 2, 2007 345

Slutsky et al.
FIGURE 6. Short (crossing two carbon-carbon bonds) HMBC
interactions between A and B protons of Es and acetylene carbons
FIGURE 7. Short (crossing two carbon-carbon bonds) HMBC
4
6
interactions between A and B protons of Es and acetylene carbons
adjacent to each ring are shown in black arrows on the structure and
connected by lines on the spectrum. Weaker cross-peaks are labeled
with numbered green arrows on the structure and correspond to the
number on the spectrum. These indicate medium-range (crossing three
carbon-carbon bonds) HMBC interactions between A and B protons
of each ring and acetylene carbons adjacent to each ring.
adjacent to each ring, shown in black arrows on the structure and
connected by lines on the spectrum. Weaker, unlabeled cross-peaks
indicate medium-range (crossing three carbon-carbon bonds) HMBC
interactions between A and B protons of each ring and the adjacent
acetylene carbons.
couplings from this aromatic carbon are also observedsone that
2
3
4
5
carbon signals between 97 and 94 ppm (shown in red) and 94-
could indicate C , B , or B and another that could indicate B ,
3
4
4
4
3
9
2 ppm (shown in green).
C , or C . COSY data assigns A , B , and C to a single spin
system which combined with the HMBC signals allows definite
assignment of these protons to ring 5.
On the basis of the number of signals in each of these two
groups, we speculated that they might be the acetylenic carbons
â to the B proton on the adjacent ring (red) and the acetylenic
carbons â to the A proton on the adjacent ring (green), as shown
for Es4 in Figure 4. This was confirmed after complete
assignment of the structures, resulting in the observation that
the chemical shifts of acetylenic carbons â to protons at position
B on the ring are well separated from those â to protons at
position A.
Short-range HMBC couplings, defined here as spanning two
carbon-carbon bonds, from A and B protons on each ring were
used to assign the acetylene carbons â to each of these protons
as shown in Figures 6 and 7 and Supporting Information Figure
S14. Medium-range couplings, defined here as spanning three
carbon-carbon bonds but not crossing a triple bond, were used
to cross-check these assignments. For example, six weaker
signals are also shown in Figure 6. These HMBC signals
correspond to couplings between A and B protons and the other
acetylene carbon attached to the ring. The HMBC spectrum for
Es6 shown in Figure 7 shows very similar features with 10 short-
and 10 medium-range couplings. Figure S14 shows the HMBC
spectrum for Es5, which was collected with a different pulse
sequence due to an upgrade of the NMR facility. This pulse
program was optimized for longer range couplings and did not
always show the short and medium range with significantly
different intensities as observed for Es4 and Es6 in Figures 6
and 7. Nonetheless, unambiguous assignment can still be made
using the knowledge gained from assigning Es4 and Es6,
followed by cross checking with long-range HMBC couplings
across the carbon-carbon triple bond. Another cross check is
that these assignments are consistent with the separation of the
acetylene carbons into two groups. With each set of acetylene
carbons assigned to the spin systems with which they are
connected, it only remains to make the connections between
ring systems across the triple bond.
With the 1D H and 13C spectra completed, as well as COSY
spectra, HMBC experiments were performed to assign the
primary sequence. Starting with the unique acetylenic carbon
signals at the Si terminus of Es4, the Si(CH3)3-acetylene
assignment was confirmed as shown in Figure 5a. These
terminal acetylene carbons were used to assign the protons in
1
1
4
ring 1. A strong HMBC signal between R and B along with
2
3
1
weaker signals between R and A as well as between R and
3
A confirmed the assignment. At the other end of the Es4
3
3
4
molecule the ring 4 protons, B , C , and A , were assigned based
on HMBC couplings to the ring carbon bonded to the N3Et2
group, as shown in Figure 5b. Similar procedures were used
for assignment of Es5 and Es6. During assignment of rings 5
and 6 in Es5 and Es6, respectively, some ambiguity in HMBC
1
signals was observed due to H overlap, as shown in Figure 5c
and 5d; however, this was easily resolved using the COSY data
to select the set of A, B, and C protons from a single spin
system. For example, in Es5, a clear HMBC signal between the
4
aromatic carbon bonded to N3Et2 and A is seen. Two other
346 J. Org. Chem., Vol. 72, No. 2, 2007

Homo-o-phenylene Ethynylene Oligomers
In all oligomers, at least one HMBC coupling across every triple
bond was present, ensuring that no assignment was dependent
upon any single HMBC coupling.
Conclusions
HMBC, COSY, 1D H, and 1D 13C NMR methods provided
the necessary information to assign the primary sequence of
three oPE oligomers. These results confirm the use of HMBC
to assign the sequence of homo-oligomers that contain an
abundance of tertiary carbon centers. In addition to establishing
the full assignment of the aromatic protons, HMBC revealed
that these oligomers have directionality along the backbone that
remains resolved even in the hexamer, likely due to the electron-
withdrawing effect of the ester group. This is clearly seen in
the acetylene carbons in which those â to A protons (the
N-terminus side of the ring) are located upfield compared to
the acetylene carbons â to B protons (the Si-terminus side of
the ring).
1
This total assignment of the aromatic protons has proven
invaluable for interpretation of chemical shift and NOE data
collected during studies to determine the solution conformation
of these foldamers. In particular, investigation of solvent- and
temperature-dependent folding of Es5 and Es6, analogous to our
FIGURE 8. Long-range HMBC interactions across carbon-carbon
4
triple bonds between aromatic protons of Es and acetylene carbons
are shown in black arrows on the structure and connected by lines on
the spectrum.
27
previously published work with Es4, has been greatly assisted.
A particular point of interest is ring 3 of Es5. In a polar solvent
Using long-range interactions across the triple bond, it was
possible to connect each spin system with its neighbors such
that unambiguous assignment of the entire primary sequence
was obtained. Figure 8 and Supporting Information Figures S15
and S16 contain the long-range HMBC spectra used for
assignment of Es4, Es5, and Es6 respectively. For example, in
Figure 8 the seven clear long-range interactions that were used
to assign Es4 are shown. As rings 1 and 4 of Es4 have already
been assigned from the end groups, the remaining task is to
such as the CD CN used in these measurements a helical
conformation with three rings per turn may be expected. In this
3
conformation ring 3 would be the one ring remaining unstacked
with any other and therefore not subject to the resulting upfield
shifting as observed for the other rings. It is only by this HMBC
assignment that the protons on ring 3 can be assigned with
accuracy. Thus, the fact that only protons on ring 3 remain
unshifted upon folding supports the helical conformation. It is
expected that similar techniques will allow assignment of
aromatic protons in other PE oligomers with various side chains
and end groups.
5
4
assign rings 2 and 3. HMBC couplings between R and B as
5
3
well as between R and A allow the assignment that ring 2 is
3
2
connected to ring 1. HMBC couplings between R and A as
8
3
8
4
well as between R and B and R and A allow the assignment
that ring 3 is connected to ring 4. Connections between ring 2
and ring 3 are indicated by HMBC couplings between R and
Acknowledgment. We thank L. Charles Dickinson for
valuable assistance with NMR instrumentation. T.V.J. thanks
the Ford Foundation for financial support. We thank the NSF
for financial support (NSF CAREER CHE-0449663). G.N.T
thanks the ARO and ONR Young Investigator programs in
addition to the PECASE program, 3M Nontenured faculty grant,
and Dupont Young Faculty Award for generous support. We
thank Professor Rich A. Blatchly for many helpful discussions.
7
1
4
1
B as well as between R and A . These were used to confirm
the assignments of the aromatic protons of Es4. Following this
procedure, working from the terminal rings inward, full assign-
ment of the aromatic protons of Es5 and Es6 was made.
The assignment of Es4 is extremely easy to make, as three
or four HMBC couplings across each triple bond are present,
and clear differentiation of signal strengths based on distance
is shown. The signal-to-noise ratio was not as high in the case
of Es6, but weak signals were readily distinguished from noise
Supporting Information Available: Experimental Section;
synthetic procedures for all oligomers reported here; tables of all
observed HMBC and COSY interactions used for assignment;
1
by the characteristic shape from their H splitting pattern. More
detail is provided in the Supporting Information. HMBC
interactions in Es5 showed a high signal-to-noise ratio but due
to the use of a long-range pulse program did not display a clear
dependence on distance. Furthermore, some extremely long-
range interactions were visible in the spectra of Es5, which made
assignment more difficult. However, there are only six possible
primary sequences after the relatively easy assignment of rings
HPLC and NMR measurements used for confirmation of Es
HMBC data for Es in ester-carbon aromatic-hydrogen region, used
to confirm COSY assignments for Es ; COSY data for all oligomers;
HMBC data for Es -6, R1-R2-carbon aromatic-proton region.
HMBC for Es -6 acetylene-carbon aromatic-proton region; details
6
purity;
6
6
4
4
and discussion for assignment of long-range HMBC peaks. This
materialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org.
1
and 5 by end-group interactions, and only one potential
assignment of rings 2-4 adequately explained all observations.
JO061175F
J. Org. Chem, Vol. 72, No. 2, 2007 347