Sequence-Defined Macrocycles for Understanding and Controlling the Build-up of Hierarchical Order in Self-Assembled 2D Arrays

Article abstract of DOI:10.1021/jacs.9b06410

Anfinsen's dogma that sequence dictates structure is fundamental to understanding the activity and assembly of proteins. This idea has been applied to all manner of oligomers but not to the behavior of cyclic oligomers, aka macrocycles. We do this here by providing the first proofs that sequence controls the hierarchical assembly of nonbiological macrocycles, in this case, at graphite surfaces. To design macrocycles with one (AAA), two (AAB), or three (ABC) different carbazole units, we needed to subvert the synthetic preferences for one-pot macrocyclizations. We developed a new stepwise synthesis with sequence-defined targets made in 11, 17, and 22 steps with 25, 10, and 5% yields, respectively. The linear build up of primary sequence (1°) also enabled a thermal Huisgen cycloaddition to proceed regioselectively for the first time using geometric control. The resulting macrocycles are planar (2° structure) and form H-bonded dimers (3°) at surfaces. Primary sequences encoded into the suite of tricarb macrocycles were shown by scanning-tunneling microscopy (STM) to impact the next levels of supramolecular ordering (4°) and 2D crystalline polymorphs (5°) at solution-graphite interfaces. STM imaging of an AAB macrocycle revealed the formation of a new gap phase that was inaccessible using only C₃-symmetric macrocycles. STM imaging of two additional sequence-controlled macrocycles (AAD, ABE) allowed us to identify the factors driving the formation of this new polymorph. This demonstration of how sequence controls the hierarchical patterning of macrocycles raises the importance of stepwise syntheses relative to one-pot macrocyclizations to offer new approaches for greater understanding and control of hierarchical assembly.

Full text of DOI:10.1021/jacs.9b06410

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Article
Sequence-defined Macrocycles for Understanding and Controlling
the Build-up of Hierarchical Order in Self-assembled 2D Arrays
James R Dobscha, Henry D Castillo, Yan Li, Rachel E Fadler, Rose D Taylor,
Andrew A Brown, Colleen Q Trainor, Steven L. Tait, and Amar H Flood
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b06410 • Publication Date (Web): 10 Sep 2019
Downloaded from pubs.acs.org on September 11, 2019
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Sequence-defined Macrocycles for Understanding and Controlling
the Build-up of Hierarchical Order in Self-assembled 2D Arrays
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a,
a,
a
a
a†
¶
¶
James R. Dobscha , Henry D. Castillo , Yan Li , Rachel E. Fadler , Rose D. Taylor , Andrew
A. Browna‡, Colleen Q. Trainora§, Steven L. Tait*a, Amar H. Flood*a
a
Molecular Materials Design Laboratory, Department of Chemistry, Indiana University, 800 E.
Kirkwood Avenue, Bloomington, IN 47405, USA
¶ These authors contributed equally to this work.
†
Current address: Department of Chemistry, College of Wooster, 1189 Beall Avenue, Wooster,
OH 44691, USA
‡
Current address: Department of Chemistry, Louisiana Tech University, 1 Adams Boulevard,
Ruston, LA 71272, USA
§ Current address: Department of Chemistry, Hillsdale College, 33 E. College Street, Hillsdale, MI
49242, USA
Email: aflood@indiana.edu; tait@indiana.edu
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Abstract
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Anfinsen’s dogma that sequence dictates structure is fundamental to understanding the activity
and assembly of proteins. This idea has been applied to all manner of oligomers but not to the
behavior of cyclic oligomers, aka macrocycles. We do this here by providing the first proofs that
sequence controls the hierarchical assembly of non-biological macrocycles, in this case, at graphite
surfaces. To design macrocycles with one (AAA), two (AAB), or three (ABC) different carbazole
units, we needed to subvert the synthetic preferences for one-pot macrocyclizations. We developed
a new step-wise synthesis with sequence-defined targets made in 11, 17, and 22 steps with 25, 10,
and 5% yields, respectively. The linear build-up of primary sequence (1°) enabled a thermal
Huisgen cycloaddition to proceed regioselectively for the first time using geometric control.
Resulting macrocycles are planar (2° structure) and form H-bonded dimers (3°) at surfaces.
Primary sequences encoded into the suite of tricarb macrocycles were shown by scanning-
tunneling microscopy (STM) to impact the next levels of supramolecular ordering (4°) and 2D
crystalline polymorphs (5°) at solution-graphite interfaces. STM imaging of an AAB macrocycle
revealed the formation of a new gap phase that was inaccessible using only C3-symmetric
macrocycles. STM imaging of two additional sequence-controlled macrocycles (AAD, ABE)
allowed us to identify the factors driving the formation of this new polymorph. This demonstration
of how sequence controls the hierarchical patterning of macrocycles raises the importance of
stepwise syntheses relative to one-pot macrocyclizations to offer new approaches for greater
understanding and control of hierarchical assembly.
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Introduction
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Sequence information is foundational to nature’s structural and functional complexity. We
know that the primary sequence of amino acids controls protein folding1-2 and that the residues
displayed on a protein’s exterior surface directs their hierarchical assembly into biological
nanostructures.3-5 This bottom-up control of molecular architectures has long motivated interest in
establishing correlations between sequence information and structure in natural systems, e.g.,
DNA6-8 and proteins,9-11 and it is now being explored in artificial systems like sequence-controlled
polymers12-19 and sequence-defined foldamers.20-27 Biomimetic sequence control is an exciting
new challenge for artificial systems. To help bridge that gap, structure-property relationships are
being explored with DNA origami28-33 and de novo protein design.34-37 For example, Ulijn has
leveraged the diversity of 20 amino acids in tripeptides to identify those that fold up and provide
the right 3D display of side chains to enhance their self-assembly.38-41 Cyclic molecules offer
superior control over the 3D display of functional groups as a result of a smaller set of
conformations. In this way, for example, Kirshenbaum and Logan designed sequence-specific
cyclic peptoids to bind and disrupt protein-protein interfaces.42 Use of these approaches with non-
biological macrocycles are not known. We explore the use of primary sequences (1°, Figure 1a)
encoded into macrocycles (Figure 1c-e) with planar secondary structures (2°) that are shape
persistent in order to limit conformations (Figure 1b), and develop new syntheses using elementary
chemistries and high building block versatility to make three-component sequences (AAA, AAB,
and ABC-type). We show how sequence controls the build-up of hierarchical structures on
graphite surfaces into dimers (3°), local superstructures (4°), and then polymorphs (5°).
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Figure 1. (a) The primary (1°) structure of a tricarb macrocycle in which A, B, and C are three
carbazole monomers linked by triazole (Tz) units. (b) The tricarb macrocycle adopts a planar
secondary (2°) structure. (c) The tertiary (3°) structure adopted on graphite surfaces involves
macrocycle dimers stabilized by lateral H-bonding between CH donors and N-atom acceptors,
forming a H-bonding array at the inter-macrocycle seam. (d) The propagation of side-on
interactions between macrocycles leads to the generation of quaternary (4°) superstructures on
graphite surfaces; 6-membered rosettes are preferred. (e) The patterning of rosettes across the
surface results in the formation of crystalline 2D polymorphs.
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Many macrocycles are composed of repeating monomers and, thus, sequence information
is latent to their structure. To lay a foundation for exploring sequence information, however,
synthetic control over sequence must be achieved. Examples of synthetically accessible
macrocycles intrinsically encoded with sequence information have been known since the total
synthesis of chlorophyll A.43-44 Macrocycles in which we can identify sequences ex post facto have
been reported, e.g., the sequence of non-symmetric calixarenes have been shown to influence guest
recognition,45-47 the performance of supramolecular catalysts,48-50 as well as intramolecular
reactivity51 but sequence-level information was never addressed explicitly. Sequence has also been
indirectly investigated in heteromacrocycles, defined as those macrocycles that do not have a
regularly repeating backbone. These include expanded porphyrins,52-59 amide-based macrocycles
that form nanopores and transport ions,60 and a coumarin-containing triamide macrocycle used as
a sensor.61 Use of a flexible heteromacrocycle that was explicitly encoded with a three-unit
sequence to enable unidirectional motion in a catenane,62 showcases the potential benefits of this
type of structural control. However, explicit investigation of the role of sequence on the
hierarchical ordering of the many macrocycles capable of self-association63-68 is unexplored.
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Motivation for sequence-controlled macrocycle syntheses must overcome the traditional
priorities of an expedient synthesis. Shape-persistent macrocycles are made using either
convergent one-pot reactions or multistep pathways. Step-wise routes are tenable but often longer
and have traditionally been given less value relative to one-pot methods. This valuation is seen in
developments of one-pot preparations of macrocycles after their step-wise syntheses.69-73 With the
one-pot approach, the macrocycles offer greater scalability and, with just one repeating monomer,
have more straightforward structure-property relationships. These benefits are achieved at the
expense of diversity; only a limited set of interactions can be encoded in the macrocycle. Non-
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symmetric macrocycles are sometimes made in one-pot, e.g., porphyrins,74-76 but statistical product
distributions (Figure 2a) limit yields and impede isolation. These one-pot reactions also fail to
control the sequence. One approach to circumventing these limitations has been to incorporate
orthogonal dynamic covalent chemistry into the one-pot synthesis of heteromacrocycles.77-78
While this method minimizes side products and allows for the expedient synthesis of
multicomponent heteromacrocycles, it still does not offer site-specific control over sequence.
Thus, inverting synthetic priorities while elevating the importance of sequence remains the best
approach for controlling the build-up of hierarchical order. The payoff for the synthetic investment
comes when a correlation can be made between the primary sequence and the resulting higher
levels of structural order (1° → 2° → 3° → 4° → 5°) to control the final architecture.
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Figure 2. Syntheses of a three-component macrocycle by means of either (a) a statistical one-pot
method in which the desired macrocycle (*) is produced along with a statistical distribution of nine
additional macrocycles or (b) a step-wise method in which the desired macrocycle is made over
more steps in a targeted manner.
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The hierarchical ordering of molecules can be readily seen at surfaces using scanning
tunneling microscopy (STM). The resulting real-space images produced by STM (e.g., Figure 1e)
enable the detailed study of the hierarchical assembly of macrocycles79-81 but sequence information
is infrequently investigated. Sequence is expected to impact molecular ordering at surfaces
primarily by altering intermolecular contacts.82 Packing relies on molecular shape and edge-
directed interactions between macrocycles, which are intimately connected to the sequence of
noncovalent interactions encoded into the macrocycle.83 Despite the >2000 studies of molecular
assembly at solid-solution interfaces,84 only a few incorporate sequence information. In one case,
the precise geometric substitution of a porphyrin was used to control assemblies formed on gold
surfaces.85 Porphyrins with two nitrile groups displayed in either a linear (180°) or perpendicular
(90°) manner form either a line or square shaped assemblies, respectively. In another instance,
rectangular, tetrasubstituted dehydro[24]annulene macrocycles were shown to adopt unique
packing patterns depending on the arrangement of substituents either along the wider or narrower
edge of the macrocycle.86 Sequence programming has been explored in linear peptides to define a
2D pattern that builds up hierarchically into either linear or hexagonal lattices.87 Thus, and despite
these supramolecular assemblies showing a connection between latent sequence information and
their surface patterns, the role of sequence in the hierarchical assembly of macrocycles needs to
be investigated.
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We describe a high-yielding synthetic pathway to sequence-defined macrocycles called
tricarbazole triazolophanes (tricarb)67, 88 and the use of sequence to identify complementary
driving forces controlling their self-assembly into 2D superstructures. The synthetic pathway
developed herein was used to create four new tricarb macrocycles with A-Tz-A-Tz-A-Tz, A-Tz-
A-Tz-B-Tz, and A-Tz-B-Tz-C-Tz type sequences (Figure 3a,b; hereafter abbreviated to AAA,
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AAB, and ABC-type). To establish the viability of the method, we first prepared the AAA-type
and C3-symmetric macrocycle TC-10 (Figure 3c) for comparison to the product of a previously
described one-pot reaction.67 This step-wise pathway also afforded us the opportunity to then
showcase the regioselectivity of a thermal Huisgen cycloaddition reaction by using covalent
control instead of by supramolecular control seen previously.89-91 The new stepwise method allows
us to prepare sequence-defined macrocycles of lower symmetry comprising either AAB-type (TC-
6618 and TC-66HEG, Figure 3c) or ABC-type (TC-610MeCy, Figure 3c) structures with
uniquely substituted carbazole building blocks (6 = hexyl, –C6H13; 10 = decyl, –C10H21; 18 =
octadecyl, –C18H37; HEG = hexaethylene glycol monomethyl ether, –(CH2CH2O)6CH3; MeCy =
methylcyclohexyl, –CH2–C6H11). The power and efficiency with which the step-wise pathway can
be utilized to encode sequence information within a tricarb macrocycle allowed us to probe the
role of sequence on surface assembly. Macrocycle TC-6618 was observed to form two new
surface polymorphs not seen with the totally symmetric TC-10 macrocycle;67 a gap phase and a
disordered phase. To identify the driving forces underpinning these new phases we leveraged the
stepwise synthesis to make AAB-type macrocycle TC-66HEG and ABC-type TC-610MeCy
(Figure 3c). Assembly studies then showed that the newly characterized gap phase arises as a
consequence of the information encoded within the substituents of the TC-6618 macrocycle, the
long octadecyl chain, and not as a result of lower molecular symmetry. These studies verify that
sequence can control the hierarchical patterning of shape-persistent macrocycles on planar graphite
surfaces and that new polymorphs can be produced only when using non-symmetric compounds.
These findings help elevate the role of high-yielding stepwise syntheses in investigating and
controlling how sequence information programs the build-up of hierarchical structure.
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Figure 3. (a) Primary (1°) sequence of the tricarb macrocycles synthesized in this work: AAA,
BBB, CCC, AAC, AAD, ABE. (b) Formulas and color-coded letters of the sequence elements
used in tricarb macrocycles. (c) Chemical structures of all the synthesized tricarb macrocycles
overlaid onto their cartoon representations.
Results and Discussion
Convergent One-pot Synthesis of Self-assembling Macrocycles for Benchmarking
The C3-symmetric AAA-type tricarb macrocycles studied in this work, TC-6 (AAA),88
TC-10 (BBB),67 and TC-18 (CCC), were synthesized from difunctional building block 7 (Scheme
1) substituted with the corresponding alkyl chains (see 7a-c). Novel tricarb macrocycle TC-18 was
synthesized following a previously developed seven-step pathway.88 An octadecyl (–C18H37) chain
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was substituted on the bridgehead nitrogen of carbazole in the first step (2c) followed by
introduction of the azide and alkyne functional groups at the 3 and 6 positions of carbazole over
five steps to give the difunctional octadecyl-building block 7c. Carbazole 7c was then subjected to
a one-pot copper-catalyzed azide-alkyne cycloaddition (CuAAC) to give tricarb macrocycle TC-
18 in 68% yield. This sequence and the yields are typical of the one-pot pathway.67, 88
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Scheme 1. Synthetic sequence leading to the one-pot preparation of C3-symmetric macrocycles
(TC-6, TC-10, TC-18) from the azide-alkyne building block 7 bearing the appropriate alkyl chain.
Step-wise Synthesis of Sequence-defined Tricarb Macrocycles
The design of a step-wise synthesis of the tricarb macrocycle scaffold relies on the
progressive growth of an oligomer in a controlled and high yielding manner to establish the
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primary sequence. Examining the possible synthetic pathways as based on the various preparations
of building blocks 1–7 led us to a step-wise sequence that takes advantage of a protection-
deprotection scheme to control the growth of the macrocyclic precursors (Scheme 2). In this
approach, amine groups and trimethylsilyl (TMS) protected alkyne moieties were employed as
precursors to reactive azide and alkyne groups, respectively. The amine-substituted carbazoles
(Reaction 4) can be converted easily to azides in excellent yields,92 while TMS groups (Reactions
2 and 5) can be quantitatively deprotected with mild base. Furthermore, neither the amine nor
TMS-protected alkynyl groups were anticipated to undergo side-reactions during the proposed
triazole-forming steps (Reactions 1 and 3), making them ideal protecting groups for the efficient
coupling of building blocks. Satisfyingly, a carbazole building block functionalized with an amine
and TMS-protected alkynyl group displayed no reactivity under the CuAAC conditions employed
herein.
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The viability of the proposed step-wise synthesis was confirmed using the previously
established TC-10 macrocycle67 as the target. TMS-protected carbazole 7b-TMS was made in six
steps while amine-substituted carbazole 8b was made by subjecting 5b to Sonogashira coupling
with TMS-acetylene followed by base deprotection. These two carbazoles (7b-TMS and 8b) were
then used to form crescent dimer-1010 (Step I, Scheme 2) using CuAAC conditions followed by
base deprotection. This dimer was then subjected to another CuAAC (Step II, Scheme 2) with
carbazole 7b-TMS to grow the oligomer and produce the amine-substituted precursor of crescent
trimer-101010. The amino group of the trimer intermediate was converted to an azide and the
alkyne moiety was activated by base deprotection to give crescent trimer-101010. This trimer was
closed by a third CuAAC reaction to afford TC-10 in 80% yield (Step III, Scheme 2).
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Scheme 2. General stepwise synthesis of sequence-defined tricarb macrocycles. (Step I) TMS-
protected carbazoles (7a-TMS, 7b-TMS, or 7e-TMS) are coupled with amine-substituted
carbazoles (8a or 8b) and, after deprotection, give the corresponding crescent dimer. (Step II) The
crescent dimer is coupled with an additional TMS-protected carbazole building block (7b-TMS
to 7d-TMS). (Step III) Following installation of the azide and deprotection of the alkyne, the
crescent trimer can be closed to produce the tricarb macrocycle. Reactions 1 and 3: CuSO4 /
NaAsc / TBTA / 2:1:1 THF:EtOH:H2O / 55 °C / Ar. Reactions 2 and 5: K2CO3 / 1:1 MeOH:THF.
Reaction 4: TsOH / NaNO2 / NaN3 / THF / 0 °C.
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To verify the build-up of the oligomer’s primary sequence and the higher symmetry upon
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macrocyclization, we recorded the H NMR spectra of all of the key intermediates along the
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synthetic pathway (Figure 4). We observe the aromatic region becoming more complex at each
stage up until the final ring-closing reaction. The addition of each building block is expected, and
found, to introduce seven new aromatic resonances: one from the newly formed triazole ring and
six peaks from the additional carbazole. Consistently, crescent dimer-1010 displays thirteen
aromatic proton resonances (Figure 4b) while crescent trimer-101010 displays twenty (Figure 4c).
This signature then simplifies to seven signals following macrocyclization (Figure 4d). This
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simplified H NMR spectrum is identical to the one for the TC-10 macrocycle formed by the
original one-pot reaction.67
We can compare the efficiencies of the two synthetic approaches. The one-pot method is
expedient and high-yielding. Starting from carbazole, macrocycle TC-10 is made from a single
building block in just seven total steps with an overall yield of 40%. In comparison, the stepwise
pathway requires six steps to synthesize azido building block 7b-TMS from carbazole (55% yield
over six steps), one subsequent step to synthesize alkynyl building block 8b from compound 5b
(95% yield), two coupling reactions and one deprotection step to reach crescent trimer-101010
(63% yield over three steps), and a final coupling reaction to give macrocycle TC-10 in an overall
yield of 25% for the eleven total steps. While the overall yield of the stepwise route (25%, 11
steps) is lower than the convergent one-pot synthesis (40%, 8 steps), this is the only way to
expediently make tricarb macrocycles of any sequence (Figure 2). Thus, the benefit of this
approach is sequence control.
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Figure 4. The aromatic region of the 1H NMR spectra of the key intermediates along the step-wise
pathway (500 MHz, 298 K, CDCl3).
Next, we highlight the versatility of the stepwise synthesis by preparing broken symmetry
macrocycles. The first target was an AAB-styled macrocycle with two hexyl side chains (–C6H13)
and one octadecyl chain (–C18H37). This specific combination was selected for the purposes of
investigating surface assembly (vide infra). Satisfyingly, tricarb macrocycle TC-6618 (AAC) was
synthesized using our generalized three-step pathway (Scheme 2) from the appropriate building
blocks. Hexyl (–C6H13) substituted monomers 7a-TMS and 8a were used to form crescent dimer-
66 over two steps with an overall yield of 84%. Crescent dimer-66 was then combined with
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octadecyl (–C18H37) substituted building block 7c-TMS to give crescent trimer-6618 over three
steps with an overall yield of 74%. The crescent trimer-6618 was then closed to give macrocycle
TC-6618 with a 75% yield for this final step (12% overall yield over 20 steps starting from
carbazole).
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When we evaluated the 1H NMR spectrum of macrocycle TC-6618 (Figure 5a, black), and
despite the fact that this is a lower symmetry macrocycle, it displayed only seven aromatic protons
typical of the higher-symmetry macrocycles (Figure 3a). This higher apparent symmetry was also
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present in the aromatic region of the C-NMR spectrum.93 Only the alkyl region (10–40 ppm,
Figure 5b, black) showed unique resonances arising from both hexyl and octadecyl side-chains.
Figure 5. (a) The aromatic regions of the 1H NMR spectra (1 mM / 500 MHz / CDCl3 / 298 K) of
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TC-6618, TC-18, and TC-6 show identical chemical shifts. (b) The C NMR spectra (1 mM /
125 MHz / CDCl3 / 298 K) of TC-6618, TC-18, and TC-6 can only be distinguished by the unique
peaks from the alkyl-chain substituents. 13C NMR peaks arising from octadecyl chains are marked
in red and peaks arising from hexyl chains are marked in blue.
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We extended the success of the stepwise synthesis to a second two-component, AAB-type
macrocycle. TC-66HEG (AAD) was made in four steps totaling 45% yield (6% yield over 20
steps starting from carbazole) using the hexaethylene glycol substituted monomer 7d-TMS
together with crescent dimer-66, which was a common intermediate in the preparation of
macrocycle TC-6618. Much like the AAC macrocycle TC-6618, the 1H NMR spectrum of TC-
66HEG only displayed seven aromatic protons.93 This finding suggests that the use of simple
functional groups on the carbazole building blocks in these AAB-type tricarb macrocycles have
little influence on the symmetry of the macrocycle’s core. This outcome is consistent with the fact
that the carbazole substituents are directed outward from the macrocyclic core.
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Figure 6. Aromatic region of the 1H NMR spectrum of TC-610MeCy (0.5 mM / 600 MHz / CDCl3
/ 298 K).
To accentuate further the versatility of the stepwise synthesis, we prepared the ABC-type
macrocycle TC-610MeCy (ABE). The methylcyclohexyl (–CH2–C6H11, MeCy) substituted
monomer 7e-TMS was reacted with hexyl substituted monomer 8a to give crescent dimer-6MeCy
in 84% yield over two steps. Crescent dimer-6MeCy and decyl monomer 7b-TMS were then
combined to give crescent trimer-610MeCy in a 70% yield over three steps. Crescent trimer-
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610MeCy was then closed to give macrocycle TC-610MeCy in 75% yield (5% yield over 24 steps
starting from carbazole). Only macrocycle TC-610MeCy showed the expected lower symmetry
in the 1H NMR spectrum with splitting in the aromatic resonances (Figure 6).
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The overall yields of the sequence-controlled tricarb macrocycles (5-25%) are reminiscent
of the overall yields obtained in the statistical synthesis of multiply-substituted porphyrin
macrocycles (<10%).94-97 Isolation and purification of porphyrins benefits from the variety of
visible colors associated with this class of molecule. Consequently, the ability to separate various
products from within a complex mixture of similar species (Figure 2a) often offsets the lack of
control over the distribution of products. However, the ability to isolate a specific target does not
necessarily guarantee access to any specific target. For tricarb, and many other macrocycles, they
are not highly colored. Thus, the controlled and stepwise build-up of sequence is the only way to
expediently synthesize and isolate AAB and ABC-type tricarb macrocycles with any arbitrary
sequence.
Macrocycle formation by Regioselective Thermal Huisgen
One of the structural features of the pre-macrocyclic crescent trimers are their nearby
placement (Figure 7b) of the terminal azide and alkyne groups for reaction. The spatial proximity
and orientation of these two reactive functionalities suggested to us that macrocyclization could
proceed in a regioselective way by a thermal Huisgen cycloaddition. Under thermal conditions,
Huisgen cycloaddition reactions typically give a mixture of 1,4- and 1,5-triazole regioisomers
(Figure 7a).98 We hypothesized that the rigidity of the crescent backbone would geometrically
inhibit formation of the 1,5-adduct. Satisfyingly, heating the tridecyl-substituted crescent trimer-
101010 at 100 °C for four days produced tricarb macrocycle TC-10 in 65% yield (Figure 7b).
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Similar regioselective thermal Huisgen cycloadditions reactions have been carried out using
different strategies to effect spatial control in the solid state,99-102 as well as in solution, by using
components encapsulated in a resorcinarene capsule90 or a cucurbit[6]uril.89, 91 However, to the
best of our knowledge, this is the first example of a thermal Huisgen reaction to selectively produce
the 1,4-triazole adduct as a result of geometric control.
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Figure 7. (a) General mechanism in which (top) 1,5 and (bottom) 1,4 regioisomers can form in
thermal Huisgen’s azide-alkyne cycloaddition. (b) Molecular mechanics modelling (MMFF94)
showing the orientation of the terminal azido and alkynyl functionalities within a tricarbazole
crescent and the reaction conditions for the formation of TC-10 under thermal conditions.
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Hierarchical Assembly of Macrocycles on Graphite Surfaces
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Our sequence-defined synthesis provides the ability to control the substituents on the
exterior of the tricarb macrocycles. A critical next step in valorizing this stepwise synthesis is to
investigate and verify that the sequence of the substituted carbazoles are important for directing
the build-up of hierarchical order (Figure 1). Specifically, we investigate how these substituents
influence the macrocycle’s patterns of 2D assembly into superstructures (4°) and polymorphs (5°)
on flat surfaces. We ultimately found that being able to pre-determine the substituents gave us
insights that were not possible when using only C3-symmetric macrocycles.
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Previous work67 has shown that the symmetric TC-10 macrocycle forms two related but
different 2D crystalline polymorphs on highly oriented pyrolytic graphite (HOPG): honeycomb
(Figure 1e, top) and flower (Figure 1e, bottom) surface morphologies.67 These morphologies are
built up by the side-on packing of macrocycles into rosettes, which constitute a preferred
quaternary (4°) structure. The rosettes define a central pore in which are disposed some of the
decyl chains from the six constituent macrocycles. Rosette fusion results in the high-density
honeycomb phase. An alternative arrangement of this quaternary structure produces the flower
phase. Instead of being fused, rosettes are spaced regularly apart as mediated by co-adsorbed
solvent molecules.67 The flower phase is present at low concentrations (75 μM) and metastable at
higher concentrations where the honeycomb dominates. At room temperature, the initial flower
phase is replaced by the honeycomb phase in minutes at 100 μM and days at 5 μM. With the
packing relying on the lateral H-bonding between macrocycles (Figure 1c), the three decyl chains
of TC-10 were assumed67 to play a subservient role in controlling the hierarchical structure of
these two polymorphs. We sought to investigate this assumption by altering the relative lengths of
the alkyl chains.
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Figure 8. Models of the (a) flower and (c) honeycomb phases formed by (b) TC-18. High-
resolution STM images of TC-18 at the TCB/graphite interface showing the (d) flower phase (5
μM, It = 0.3 nA, Vsample = –0.8 V), (e) mixed phases (75 μM, It = 0.03 nA, Vsample = –0.4 V), and (f)
honeycomb phase (100 μM, It = 0.03 nA, Vsample = –0.4 V; unit cell: a = b = 2.9 ± 0.1 nm).
The easiest way to test this idea is to lengthen all alkyl chains in a symmetric manner and
examine the distribution of flower and honeycomb phases (Figure 8a-c). To this end, we
synthesized (Scheme 1) the AAA-type tricarb macrocycle TC-18 bearing three long octadecyl
(–C18H37, Figure 8b). We anticipated that the octadecyl chains would display an increased affinity
for the graphite surface leading to their enhanced adsorption relative to the decyl chains of TC-10.
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Consequently, we would expect preferential stabilization of a flower-like phase with an expanded
unit cell relative to TC-10. Contrary to expectation, when a solution of TC-18 dissolved in the
non-volatile solvent 1,2,4-trichlorobenzene (TCB) was deposited onto an HOPG surface and
subsequently imaged using STM, we saw both the honeycomb and flower phases as a function of
concentration (Figure 8d-f). We were also able to observe bright features within the STM images
of TC-18 attributed to the kind of multilayer stacking seen previously with TC-10.93 However, the
observation of these stacking events was rare and so we have elected to focus on 2D self-assembly
in this work. At low concentrations, below 75 μM, the flower phase (Figure 8d) is observed to be
the dominant packing pattern on the surface. At moderate concentrations, between 75 and 100 μM,
a mixture of the flower and honeycomb phases are seen (Figure 8e). Finally, at high concentrations,
above 100 μM, the TC-18 macrocycle predominantly forms the honeycomb phase (Figure 8f). The
flower phase formed by TC-18 was also observed to be metastable with the honeycomb phase at
room temperature. At low concentrations, the flower-phase is supplanted by the honeycomb phase
over the course of hours. At high concentrations, this transformation occurs faster and is completed
within minutes. This behavior is identical to the 2D self-assembly of the TC-10 macrocycle. The
inability to influence the relative stability of the flower phase simply by symmetrically lengthening
all the alkyl chains motivated designs of sequence-defined tricarb macrocycles.
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Sequence-controlled Macrocycles Direct Emergence of New Surface Superstructures (4°) and
Polymorphs (5°)
Our observations that macrocycle TC-6 only formed the honeycomb phase at
concentrations at which macrocycles TC-10 and TC-18 were observed to form the flower phase
led us to hypothesize that a balance between three factors led to the formation and metastability of
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the flower phase: the lateral interactions between adsorbed macrocycles; the adsorption affinity
of the side chains for the surface; and the steric interactions between side chains sequestered within
the rosette pore. As a means to precisely investigate the interactions that direct surface patterning,
we designed a two-component macrocycle that displays properties complementary to the packing
demands of the flower phase, macrocycle TC-6618 (AAC, Figure 9a). Macrocycle TC-6618
employs an octadecyl (–C18H37) chain to drive surface interactions that are stronger than decyl
chains (–C10H21), while hexyl (–C6H13) chains were chosen to maximize the disparity in surface
adsorption strength relative to the octadecyl chain, to minimize, while producing a macrocycle
where the total number of carbons contained within the sum of alkyl chains was equivalent to TC-
10. Given the predispositions of these constituent alkyl chains, we expected the flower polymorph
to be preferred: we anticipated the long surface-adsorbing octadecyl chains would be directed
outwards from the rosettes to disfavor the honeycomb. The smaller hexyl chains were expected to
play secondary structure-directing roles, and would either be adsorbed inside the rosette pores or
backfolded into solution to ultimately favor the flower phase.
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We were surprised to observe two new phases and a total of three phases from tricarb
macrocycle TC-6618 (Figure 9). At low concentrations (2.5 μM), a new phase emerges that
displays a darker line in the STM imaging that has the appearance of a gap (Figure 9b) in the more
typical 2D surface packing of the macrocycles. At 25 μM, domains of honeycomb appear to co-
exist with the gap phase. This honeycomb has an identical unit cell (a = b = 2.9 ± 0.1 nm) to the
honeycomb phase of TC-10.67 As concentration is further increased, the honeycomb’s coverage
grows, and above 200 μM, gap is not observed on the surface (Figure 9c). At high concentrations
(≥ 200 μM) a new state (Figure 9d) with high-density and reduced order is observed to co-exist
with honeycomb. The gap phase formed by the TC-6618 macrocycle was also found to be
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metastable with respect to the honeycomb phase, with the transition from gap to honeycomb slowly
occurring over the course of 24 hours from the initial scan at room temperature.
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Figure 9. High-resolution STM images of (a) TC-6618 at the TCB/graphite interface showing (b)
gap phase (25 μM, It = 0.15 nA, Vsample = –1 V), (c) honeycomb (unit cell: a = b = 2.9 ± 0.1 nm;
100 μM, It = 0.55 nA, Vsample = –1 V), and (d) non-ordered packing states (300 μM, It = 0.5 nA,
Vsample = –0.8 V).
The gap polymorph present at low concentrations of the TC-6618 macrocycle is
characterized by STM imaging (Figure 10) to have two types of macrocycle-bearing rows. The
imaging (Figure 10a) and the corresponding model (Figure 10c) show honeycomb-like rows of
tricarb macrocycles that propagate in one dimension and which are separated by low contrast
features, the “gap”. The honeycomb-like (Figure 10c, purple) rows retain the privileged quaternary
structure of six-macrocycle rosettes, but unlike the honeycomb phase, these rosettes are fused with
two neighbors instead of three. This number of intermolecular contacts in the honeycomb rows is
between that of the honeycomb (all macrocycles have three contacts) and flower phase (two
contacts). This phase also displays high-contrast zig-zag rows (Figure 10c, green). Along these
zig-zag rows, the macrocycles do not form rosettes. Instead, each macrocycle only makes lateral
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contacts with two neighbors indicative of a new quaternary structure. Transitions between zig-zag
and honeycomb rows are also seen as line defects (Figure 10c, black dot) within the 2D lattice.
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Figure 10. (a) High-resolution STM imaging of the gap phase formed by (b) TC-6618. (c) Model
of the gap phase showing the zig zag (green), honeycomb (purple), and the line defect transition
between the two structures (black dot).
Molecular modelling of the possible packing structures that could account for the darker
low-contrast gap region suggests that one or two of the long octadecyl chains are adsorbed within
the interstitial space. In addition, the shorter hexyl chains of the macrocycle are believed to be
backfolded into solution or adsorbed to the surface either inside the rosette pores or into the gap
spaces. Based on these ideas, the emergence of a new self-assembled structure from the TC-6618
macrocycle suggests that the length of the alkyl chains in sequence-controlled macrocycles can be
tuned to play a more distinct role in surface self-assembly on graphite. Alternatively, the new gap
phase may instead arise simply as a consequence of the lowered symmetry.
To investigate the role of molecular symmetry on the 2D packing of tricarb macrocycles
and to highlight the utility of the sequence-defined synthesis, a three-component ABC macrocycle
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was designed. Three relatively low surface-affinity substituents were selected as a means to
differentiate the role that symmetry plays in directing the 2D surface-assembly of tricarb
macrocycles from the effect of long surface-adsorbing alkyl chains. A methylcyclohexyl group
was chosen for its weak adsorption to graphite,103 while hexyl and decyl chains were chosen
because their respective C3-symmetric macrocycles (TC-6 and TC-10) form packing phases in
which the alkyl chains do not play a significant role in self-assembly: As noted above, macrocycle
TC-10 only forms flower and honeycomb phases,67 while TC-6 forms honeycomb and a non-
ordered phase.93 The observation that both TC-6 and TC-6618 form the non-ordered packing
phases suggests that this phase originates from the low affinity hexyl chains and not as a result of
decreased macrocycle symmetry.
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STM imaging of the sequence-defined TC-610MeCy macrocycle at the solution-graphite
interface revealed the formation of only two packing polymorphs: honeycomb and, not
surprisingly given the poor-surface interacting hexyl and methyl cyclohexyl substituents, the non-
ordered state (Figure 11). The gap phase was not observed. At the lower concentrations (≤ 2.5 μM)
needed to form the gap phase, we instead see the TC-610MeCy macrocycle forming the
honeycomb phase. At 100 μM, domains of the non-ordered phase co-exist with honeycomb. As
concentration is increased further to 1 mM, the surface coverage of the non-ordered phase grows
such that it is the only one observed. The absence of a gap phase suggests that its emergence in
TC-6618 arises from driving forces beyond the non-symmetric display of functional groups.
Rather, the surface affinity and local interactions involving the long octadecyl chain are also key
for accessing this packing morphology. Additionally, the non-ordered phase is a kinetically
trapped state and can be converted to the honeycomb phase by solvent annealing with toluene.93
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We acknowledge that a more detailed investigation of the factors that give rise to the non-ordered
packing phase is warranted and will be further discussed in a later work.
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Figure 11. (a) Macrocycle TC-610MeCy. High-resolution STM images of TC-610MeCy at the
TCB/graphite interface showing (b) honeycomb ordering (unit cell: a = b = 2.9 ± 0.1 nm 300 μM,
It = 0.13 nA, Vsample = –0.8 V), (c) the coexistence of honeycomb and non-ordered phases (300
μM, It = 0.6 nA, Vsample = –0.5 V), and (d) the non-ordered packing state (300 μM, It = 1.1 nA,
Vsample = –0.6 V).
To provide an independent test of the hypothesis that surface interactions encoded by the
long octadecyl chain direct the gap phase, we designed macrocycle TC-66HEG (Figure 12a) with
an isosteric hexaethylene glycol (HEG) chain in place of the octadecyl chain. The chains are
similar in length (nineteen and eighteen heavy atoms, respectively), however, glycol chains encode
for minimal graphite surface affinity.104 We anticipated that the low surface-affinity chain would
stop formation of the gap phase at low concentrations. Gratifyingly, the gap phase is not observed
when solutions of TC-66HEG are deposited onto HOPG. Instead, macrocycle TC-66HEG only
forms the honeycomb phase (Figure 12b) at those concentrations where TC-6618 forms the gap
phase (2.5 to 50 μM). This observation is consistent with the idea that the surface interactions of
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the octadecyl chains lead to their adsorption into the gap region (Figure 12c). By contrast the
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Figure 12. (a) Macrocycle TC-66HEG. (b) STM images of TC-66HEG at the TCB/graphite
interface with honeycomb ordering (10 μM, It = 0.15 nA, Vsample = –0.7 V). (c) Models of the
octadecyl chains (green) of TC-6618 surface adsorbed to form the gap and (d) HEG chains (red)
of TC-66HEG directed away into solution leading to the formation of the honeycomb phase.
Conclusions
We have shown that the primary sequence (1º) of sequence-controlled macrocycles
encodes for the hierarchical build-up of ordering on graphite surfaces. This control is expressed at
the 4º level of organization in the many-molecule superstructures (rosettes and zig-zag rows) and
at 5° levels of the surface polymorphs (flower-honeycomb, gap-honeycomb-disorder, honeycomb-
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disorder). This work was investigated with tricarb macrocycles bearing either one (AAA TC-6,
BBB TC-10, CCC TC-18), two (AAC TC-6618, AAD TC-66HEG), or three (ABE TC-610
MeCy) different substituents made in 11-25 total steps. The design of these macrocycles required
the development of a step-wise synthesis, thus inverting the typical synthetic predilection for one-
pot macrocyclizations. Along the way, the final macrocycle-forming reaction of the step-wise
route was carried out regioselectively for the first time in a geometrically-controlled Huisgen
cycloaddition under thermal conditions without intermolecular ordering. We found that the AAC
tricarb macrocycle with one octadecyl and two hexyl chains assembled into the new gap phase.
Only by making an AAD and ABE macrocycle were we able to show that the gap phase arises
from the local interactions generated upon the adsorption of the octadecyl chain and not from the
chain’s bulk or the macrocycle’s lowered symmetry. We note that in some cases, significant
changes to the peripheral functional groups do not have a noticeable impact on the packing
structure. For example, TC-10 and TC-18 produce similar packing structures, in spite of nearly
doubling the peripheral alkyl length. It seems that, in this and other cases, the adjustments to the
peripheral units do not shift the balance away from other intermolecular interactions. This study
provides the framework necessary to shift the perceived importance of synthesis from one-pot
macrocyclizations to stepwise methods to enable sequence to control the build-up of hierarchical
structure of macrocycles. Ultimately, this shift in perspective offers the potential for greater control
and a deeper understanding of hierarchical assembly.
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Supporting Information
General Experimental Procedures, Synthetic Schemes, and Spectra
Additional STM Images and Characterization
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Acknowledgements
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JRD, YL, SLT, and AHF thank the National Science Foundation (DMR 1533988) for support;
HDC thanks Indiana University and the Arts and Sciences President’s Diversity Initiative
Fellowship for support; REF, RDT, AAB, and CQT thank the National Science Foundation (CHE
1460720) for support at Indiana University’s Research Experiences for Undergraduates (REU) in
Materials Chemistry, Nanoscience, and Assembly.
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