Functionalization of tripodal scaffold molecules on solid support

Article abstract of DOI:10.1002/ejoc.200800259

A versatile synthetic approach is described for the functionalization of tripodal scaffold molecules on solid support. Intrinsic problems related to the attachment of tripodal scaffolds to a resin (for example mono- vs. polyadducts and intramolecular cyclizations) are studied and solutions are provided. The synthetic approach relies on the use of specific protecting groups in the critical steps of the synthesis. Importantly, however, protecting groups on the scaffold molecule are never used, which significantly facilitates scaffold variation, for instance in combinatorial studies. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800259

FULL PAPER
DOI: 10.1002/ejoc.200800259
Functionalization of Tripodal Scaffold Molecules on Solid Support
Cristian Guarise,[a] Giovanni Zaupa,[a] Leonard J. Prins,*[a] and Paolo Scrimin*[a]
Keywords: Tripodal structures / Multivalency / Solid-phase synthesis / Scaffold molecules / Oligopeptides
A versatile synthetic approach is described for the function-
alization of tripodal scaffold molecules on solid support. In-
trinsic problems related to the attachment of tripodal scaf-
folds to a resin (for example mono- vs. polyadducts and intra-
molecular cyclizations) are studied and solutions are pro-
vided. The synthetic approach relies on the use of specific
protecting groups in the critical steps of the synthesis. Im-
portantly, however, protecting groups on the scaffold mole-
cule are never used, which significantly facilitates scaffold
variation, for instance in combinatorial studies.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
the preparation of large combinatorial libraries of tripodal
structures.
Introduction
Tripodal molecular structures are increasingly applied in
the fields of catalysis,[1] recognition,[2] sensing,[3] and biomi-
metics.[4] The interest is driven by the observation that func-
tional groups, such as acids/bases, metal-binding sites, H-
bond donors/acceptors, oriented towards a central cavity
enable selective substrate recognition,[5] and also enzyme-
like catalysis[6] via a cooperative interplay between the func-
tional groups. Alternatively, the divergent orientation of the
head groups enables binding to large objects, such as pro-
tein and even cell surfaces, via multipoint interactions, re-
sulting in remarkably strong complexes.[7]
Efforts in this direction generally involve the use of scaf-
fold molecules with an AB3-functionalization pattern with
A the chemical group used for anchoring the scaffold to
resin and B the anchoring points for scaffold functionaliza-
tion carrying orthogonally compatible protecting groups
(Figure 1, top).[8] For instance, impressive libraries of tripo-
Having molecular weights typically ranging from 0.5–
3 kDa, tripodal structures belong to a distinct class of com-
pounds intermediating between small molecules and macro-
molecular objects like dendrimers and polymers. As such,
syntheses of tripodal structures are often accompanied with
specific challenges and pitfalls, which require the develop-
ment of new synthetic methodology. Generally, tripodal
structures are synthesized in solution following a one-com-
pound-one synthesis scheme, which often requires optimis-
ation steps for each different structure synthesized.
In addition, whereas C3-symmetrical structures can be
prepared in one step via a threefold introduction of the
functional group on the scaffold, the introduction of dif-
ferent functions requires orthogonally compatible protect-
ing groups on the scaffolds’ anchoring points. For these ap-
plications, scaffold functionalization on solid support offers
significant advantages. Solid-phase synthesis allows reac-
tions to be driven to completeness using an excess of rea-
gents, offers a relatively easy purification, and also permits
Figure 1. Examples of AB3-type scaffolds with orthogonally com-
patible functional groups on the anchoring points (top) and il-
lustrative examples of amine terminated A3-type scaffolds (bot-
tom). The synthesis of the scaffolds can be found in the following
references: I[8f], II,[8a] III,[8h] IV,[8g] V,[12c] VII,[11c] VIII,[11a] IX.[11b]
Scaffolds VI [tris(2-aminoethyl)amine], and X (triazacyclononane)
are commercially available compounds.
[a] Department of Chemical Sciences and ITM-CNR, Padova sec-
tion, University of Padova,
Via Marzolo 1, 35131 Padova, Italy
Fax: +39-049-8275239
E-mail: leonard.prins@unipd.it
paolo.scrimin@unipd.it
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dal receptors containing up to 27000 members have been
Currently we are working on the development of meth-
prepared and screened for binding affinity for FeIII[8f] and odologies that eliminate these issues, thus allowing the func-
small peptides[8c] showing the high potential of this ap- tionalization of a wide variety of scaffolds on solid-support
proach. However, two drawbacks can be identified related without using protecting groups. Recently, we have shown
to the use of AB3-type scaffolds. Firstly, only a limited that the catalytic activity of heterofunctionalized tripodal
number of synthetically easily accessible AB3-type scaffolds structures can be determined directly from small mixtures,
is available. Consequently, there is hardly any possibility of simply obtained in a one-pot synthesis without the use of
scaffold variation, which is essential in order to explore a any protecting group.[9] In this proof-of-principle study only
larger chemical space. Secondly, the extensive use of orthog- a minimal four-component library was studied, with the ex-
onal protecting groups requires a demanding synthesis of pansion towards larger libraries hampered by the limita-
the appropriately protected AB3-scaffold and, subsequently, tions of solution-phase chemistry. Therefore we are now de-
a series of deprotection–coupling steps to give the final veloping synthetic methodology that allows the functionali-
product.
zation of A3-type scaffolds on solid support.[10] A wide vari-
Scheme 1. Schematic presentation of the protocol for the synthesis of compounds 1-(Asp-FG)3 on Wang resin.[10]
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Functionalization of Tripodal Scaffold Molecules
ety of A3-type scaffolds are commercially available or can products can be completely suppressed by switching from a
be synthesized via short synthetic routes (Figure 1, bot- Wang resin to a DHPP resin.
tom).[8a,8f–8h,11,12c] This means that the use of A3-type scaf- – Alternative for the Asp residue. The use of Wang resin
folds significantly enhances the freedom of scaffold-varia- results in the introduction of carboxylic acids in the final
tion with respect to those of type AB3. The use of A3-type structure (originating from the Asp residues). For several
scaffolds in solid-phase synthesis requires a conceptually applications this may be undesirable. It is shown how the
different synthetic strategy since the chemical handle B is same strategy can be applied on a 2-chlorotrityl resin, which
absent. As a result of the absence of synthetic methodology results in the incorporation of an amino group in the final
this type of scaffold has rarely been used in solid-phase syn- product.
thesis. A notable exception in this respect is the work of
The solution of these problems transforms our procedure
Anslyn and co-workers, which strongly relies on the use of in a very versatile protocol for the homo- or heterofunction-
the 1,3,5-tris(aminoethyl)-2,4,6-triethylbenzene scaffold 1a, alization of A3-type scaffolds.
for the development of sensing arrays on solid support.[12]
In another isolated example small peptide trimers were pre-
Results and Discussion
pared in modest yield on resin via threefold coupling to a
1,3,5-trisubstituted benzene-based scaffold.[13] However, de-
spite the potential of A3-type scaffolds, a systematic study
that addresses the functionalization of A3-type scaffolds on
solid support is so far completely absent.
Synthesis of Heterofunctionalized Scaffold Molecules
We have shown before that the addition of scaffold mole-
cule 1a to Mmt-Asp(OWang)-OH resulted in the formation
Our previously reported approach was based on the ad- of three products 1a-[Asp(OWang)-Mmt]x (with x = 1–3)
dition of A3-type scaffold molecules 1 containing multiple on the resin, evidenced (HPLC, see Figure 2, a) by the fact
amino groups, to Wang resin previously functionalized with that cleavage from the resin gives compounds 1a-(Asp-
an Asp residue via its side-chain (Scheme 1).[10] This re- NH2)x (with x = 1–3) in a 48:35:17 ratio, respectively. This
sulted in scaffolds linked to the resin one, two, or even three was contrary to the observations by Anslyn and co-workers
times. Key step in the procedure was the subsequent reac- who in a much related synthetic protocol exclusively ob-
tion of the remaining amino groups of the scaffold with served the monocoupling of the identical scaffold 1a.[12d]
Fmoc-Asp(OtBu)-OH, restoring C3-symmetry and yielding We hypothesized that this difference might be related to the
three terminal amino groups accessible for further function- difference in spacer length between the scaffold and the
alization. The generality of this approach was illustrated by resin. In order to verify this, we incremented the distance
the functionalization of three structurally diverse scaffold between the resin and the Asp residue inserting either a Gly
molecules 1a–c with a variety of different functional groups residue or a GlyPro dimer at a constant resin loading of
FG. Advantages of this approach are its simplicity and the 0.44 mmol/g. It was observed that the insertion of one Gly
freedom to functionalize any desirable scaffold molecule, residue was already sufficient to cause the quantitative
without the use of protecting groups or special functionali- mono-coupling of scaffold molecule 1a to the resin. In fact,
zation patterns. On the other hand, we identified the follow- resin cleavage after the addition of the scaffold gave com-
ing limitations/problems that are addressed in this paper:
pound 1a-[Asp(Gly-OH)-NH2] as the exclusive product as
– Synthesis of heterofunctionalized scaffold molecules. Al- evidenced by HPLC-MS and ESI-MS (Figure 2, b); com-
though C3-symmetrical structures currently find a large pound 1a-[Asp(Gly-OH)-NH2]2, the di-coupled adduct, was
interest in the fields of recognition and catalysis, for a series not observed at all. Analogously, when the GlyPro dimer
of applications the ability to differentiate between the func- was used as spacer, exclusively the mono-coupled product
tional groups is essential. Here we demonstrate how one of 1a-[Asp(ProGly-OH)-NH2] was observed.
the functional groups of scaffold molecules 1 can be differ-
entiated from the other two without using any prior (par- that the second coupling between scaffold and resin bears
tial) protection of the amino groups on 1. resemblance of an “intramolecular” reaction, leading to the
These results can be rationalized by taking into account
– Position of the Asp residue. In the original procedure, the formation of a cycle (with the resin constituting part of that
Asp residue acts as a spacer between the scaffold and the cycle). Studies of intramolecular ring closure reactions have
desired functional group. This can pose a problem espe- shown that the effectiveness of the intramolecular reaction
cially in cases where one is looking for synergetic effects is inversely related to the size of the ring.[14] Thus, increas-
between the functional groups for instance in (biomimetic) ing the “ring size” by inserting the Gly- or GlyPro spacer
catalysis. We show that the protocol can be modified in or- disfavors the intramolecular reaction and consequently di-
der to freely choose the position of the Asp residue, en- minishes the amount of di- or tricoupled scaffold molecules.
abling the possibility to directly connect the functional Presumably, the number of bonds formed between scaffold
groups to the scaffold molecule.
and resin is also inversely related to the resin loading. At
– Yield optimisation. It was noticed that the original pro- low resin loadings the formation of multiple bonds between
cedure in some cases gave rise to the formation of signifi- scaffold and resin is less likely because of the larger distal
cant amounts of side products (in some cases up to 20– separation of anchoring points on the resin. However,
30%), especially when the flexible scaffold molecules 1b or working with reduced resin loadings is highly unfavorable
1c were used. Here we show that the formation of these side because of the obvious decrease in yield per gram of resin
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Figure 2. (a) The three products formed after addition of scaffold 1a to Mmt-Asp(OWang)-OH and (b) the product formed after the
addition of 1a to Mmt-Asp(Gly-OWang)-OH. The HPLC chromatograms show the composition of the crude mixture after cleavage.
Peaks were assigned using ESI-MS. The peak marked with an asterisk is a low-molecular weight impurity structurally not related to the
scaffold molecules.
and therefore we did not consider this to be a direction with initial NǞC growth on resin (Scheme 2).[15] As a first step,
practical application.
the OAll protecting group of the resin bound Asp residue 2
The ability to couple scaffolds 1 using exclusively one of was removed using standard reductive conditions and the
the three amines is very important, since it allows one arm resulting carboxylic acid activated with PyBOP. Next, an
to be functionalized in a diverse manner with respect to the OAll-protected His residue [H2N-His(Boc)-OAll] was cou-
other two (for an example see later). The sites that can be pled via the free NH2 group to give 3. After subsequent
selectively functionalized are indicated in Figure 2 (b) with deprotection of the His-OAll group and activation, scaffold
a square and circles, respectively. It should be emphasized molecule 1a was added. Subsequently, adduct 4 was symme-
that no prior protection of any of the amines of scaffold 1a trized in two cycles using His and Asp, respectively, in the
is required. Anslyn and co-workers have exploited this for regular CǞN direction using Fmoc-based chemistry. At
the preparation of a combinatorial library on Tentagel resin this point, on a small amount of resin both the Mmt- and
by using two sites for the insertion of (identical) tripeptides Fmoc-protecting groups were cleaved, the terminal amines
and the remaining site for the insertion of a fluorophore acetylated and the products cleaved from resin. The HPLC
that served as a reporter unit to indicate binding.[12d]
chromatogram of the cleavage mixture (Figure 3, a) showed
the clean formation of compound 1a-(HisAsp-NHAc)3 as
confirmed by ESI-MS. One of the critical issues of NǞC
growth on resin is the possibility of amino acid racemiza-
The Position of the Asp Residue
An obvious consequence of our synthetic approach is the tion upon activation of the carboxylic acids. However, the
presence of one or more Asp residues in the final com- NMR spectrum of compound 1a-(HisAsp-NHAc)3 shows
pounds, positioned as spacers between the scaffold 1 and the exclusive presence of a C3-symmetrical species indicat-
the functional groups FG (Scheme 1). This may not be an ing that racemization has only occurred to a negligible ex-
ideal situation, especially in case one is interested in syner- tent (if not at all). This is in line with literature reports in
getic interactions between the functional groups. The inser- which similar coupling conditions were used.[15b]
tion of spacers in those cases should be avoided, as they
Symmetrization of the scaffold resulted in 5, in which
increase the distance between the functional groups. There- one Asp residue has the Mmt-protecting group, whilst the
fore, we developed a synthetic procedure that allows control other two Asp residues have an Fmoc-protecting group. As
over the position of the Asp residue, which relies on an discussed before, the orthogonality of these protecting
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Functionalization of Tripodal Scaffold Molecules
Scheme 2. Synthetic protocol for controlling the position of the Asp residue; i) Pd0(PPh3)4, PhSiH3, CH2Cl2, ii) PyBOP, DIEA, DMF/
CH2Cl2, iii) H2N-His(Boc)-OAll, DIEA, DMF, NMP, iv) 1a, DIEA, DMF/CH2Cl2, v) Fmoc-Asp(OtBu)-OH, PyBOP, DIEA, DMF,
NMP, vi) piperidine 20% in NMP, vii) acetic acid, TFE, CH2Cl2, viii) acetic anhydride, DIEA, CH2Cl2, ix) TFA (95%).
groups can be used to selectively functionalize one arm with
respect to the other. This was demonstrated by removing
the Mmt group under slightly acidic conditions and coup-
ling of a new Asp residue. Cleavage from resin after acety-
lation of the terminal amines resulted in the formation of
compound 1a-(HisAspAsp-NHAc)(HisAsp-NHAc)2 in rea-
sonable yield and purity as observed from the HPLC chro-
matogram of the crude cleavage mixture (Figure 3, b).
Yield Optimisation
As mentioned earlier, products using our original pro-
cedure were in some cases obtained in a modest yield (down
to 50%), especially when the flexible scaffolds 1b (Tren) or
1c (Tacn) were used. The HPLC chromatograms after cleav-
age showed significant amounts of side products, with
masses corresponding to the desired products minus 18 or
36 amu, signifying the loss of one or two water molecules.
Other side products, for instance lacking one or more
amino acid residues were never observed, leading to the
conclusion that the symmetrization and functionalization
steps occur cleanly and quantitatively. Given the character-
istic masses of the side products, we hypothesized that these
should result from an intramolecular reaction involving the
Figure 3. (a) The HPLC trace of compound 1a-(HisAsp-NHAc)3
ester bonds connecting the Asp residues to the Wang resin.
obtained from the crude cleavage mixture. (b) The HPLC trace of
Likely candidates as nucleophiles were either the terminal
amino groups (after Fmoc deprotection) or the amides, re-
compound 1a-(HisAspAsp-NHAc)-(HisAsp-NHAc)2 obtained
from the crude cleavage mixture.
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Scheme 3. (a) Preparation of the DHPP resin via coupling of 4-(1Ј,1Ј-dimethyl-1Ј-hydroxypropyl)phenoxyacetyl to Merrifield resin; i) KI,
DMF. (b) Synthesis of compound 1a-(Asp-NH2) on DHPP resin; i) Fmoc-Asp(F)-OAll, DIEA, DMF/CH2Cl2, ii) piperidine 20% in
DMF, iii) Mmt-Cl, DIEA, CH2Cl2, iv) Pd0(PPh3)4, PhSiH3, CH2Cl2, v) PyBOP, DIEA, DMF/CH2Cl2, vi) 1a, DIEA, DMF/CH2Cl2, vii)
acido acetico, TFE, CH2Cl2, viii) TFA (95%).
sulting in diketopiperazine or aspartimide formation, ditional advantage of suppressing the formation of doubly
respectively, both well-known side products in solid-phase and triply connected scaffolds (at similar resin loadings:
peptide synthesis. Considering the fact that formation of 0.32 mmol/g for the DHPP resin vs. 0.44 mmol/g for the
side products was also observed when the scaffolds were Wang resin). In order to test whether the formation of side
functionalized with diBOC-protected TACN (which were products is suppressed on DHPP resin, the synthesis of 1c-
removed upon cleavage from resin, implying that free ter- (AspArg-NH2)3 was repeated. The synthesis of this com-
minal amines were never present) we ruled out the first pos- pound on Wang resin gave the product with a modest pu-
sibility and postulated that the side products were the result rity of 70% based on analysis of the HPLC chromatogram
of aspartimide formation. Only this hypothesis was compat- and large amounts of a side product with a difference in
ible with the formation of products incorporating all amino mass of –18 amu were detected.[10] On DHPP resin, how-
acids, but lacking fragments corresponding to one or two ever, the same compound was obtained with a much im-
water molecules. Since the scaffolds are connected to the
resin via one, two, or three ester bonds (see before) at maxi-
mum two cyclizations can be observed. An eventual third
cyclization would lead to a complete cleavage from the resin
and removal during the washing steps. In addition, the ob-
servation that the extent of side product formation strongly
depends on the type of scaffold used, made us conclude that
cyclization does not occur within one of the three branches.
In such a case, the amount of side product would have been
independent of the type of scaffold used.
So, since side product formation most presumably in-
volves a nucleophilic attack on the benzyl ester connecting
Asp to the resin, we reasoned that the use of an ester less
prone to nucleophilic attack should improve the yield.
Therefore, we decided to use the DHPP [4-(1Ј,1Ј-dimethyl-
1Ј-hydroxypropyl)phenoxyacetyl] handle 7 for attaching the
Asp residue as a tert-butyl ester.[16] Generally, the DHPP
handle is connected to Merrifield resin via the formation of
a benzyl ester (which resists TFA treatment) and then ex-
poses a tert-butyl alcohol for further resin functionalization
(Scheme 3, a). The attachment of Asp to DHPP requires
harsher coupling conditions compared to Wang resin. Ac-
cordingly, Fmoc-Asp(OH)-OAll was converted into its acyl
fluoride using cyanuric fluoride and reacted with the DHPP
resin at 50 °C for 1 night.[17] In order to verify our synthetic
protocol on DHPP resin, first the well-performing scaffold
1a was coupled to 9 using the same procedure as described
before (Scheme 3, b). We were satisfied to observe that after
sample cleavage from the resin, exclusively the mono adduct
1a-(Asp-NH2) was observed in the HPLC chromatogram
after cleaving (a) 1a-(Asp-NH2) and (b) 1c-(Asp-Arg-NH2)3 from
(Figure 4, a), indicating that the DHPP linker has the ad- DHPP resin.
Figure 4. HPLC chromatograms of the crude mixtures obtained
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Functionalization of Tripodal Scaffold Molecules
proved purity of 95% as evidenced by the HPLC chromato- for deprotecting OAll), and strongly acidic conditions (resin
gram obtained after cleavage from resin (Figure 4, b).
cleavage). This excluded a large variety of protecting groups,
except for those based on trityl. On Wang resin the only
protecting group permitting a clean synthesis was the Mmt-
protecting group, which is cleaved under very mild acidic
Lysine as an Alternative for the Asp Residue
A direct consequence of functionalizing the scaffolds 1 conditions (a 1:2:6 mixture of acetic acid/TFE/DCM),
on either Wang or DHPP resin is the introduction of one which does not cause cleavage of the products from resin.
or more Asp residues in the final structure, and, conse-
Synthesis on 2-Cl-trityl-resin liberates compounds with a
quently, a corresponding number of carboxylic acids. This terminal NH2 group after cleavage. Intramolecular cycliza-
may be undesirable, when an acidic function (or a nega- tion reactions as observed when using Wang resin are not
tively charged carboxylate at neutral pH) negatively inter- likely to occur, because of the bulkiness of the trityl group.
feres with the supposed activity of the functionalized scaf- For obvious reasons, the Mmt-protecting group or any
fold (e.g. metal binding, anion receptor, etc.). In order to other acid sensible protecting group has to be rigorously
widen the scope of our approach, we therefore decided to excluded. Therefore, we were constricted to search for an
explore whether also other amino acids then Asp can be alternative amino protecting group, which now required
used as linkers. Since the nature of the functional group is stability against nucleophiles/bases, reductive conditions,
strictly related to the type of resin, we developed an analo- strong and weak acidic conditions. This excludes most of
gous synthetic scheme for 2-Cl-trityl resin. Compounds are the commonly used protecting groups.[18] Eventually, we
cleaved from this resin as an amine, which is highly comple- obtained excellent results using the Teoc (trimethylsilyl-
mentary to the carboxylic acid residue.
ethylcarbamate) protecting group. This protecting group is
The chemical bond connecting the scaffold to the resin stable under all requested conditions and is generally re-
determines which kind of orthogonal protecting groups can moved by the strongly nucleophilic F– ion (TBAF, TASF,
be used. In this respect, the protecting group on the amino etc.) On Wang resin this protecting group cannot be used
acid used for coupling the scaffold is highly demanding in because, contrary to the trityl-resin, quantitative cleavage
our protocol. During our initial attempts on Wang resin we from Wang resin is observed during the deprotection step.
kept an Fmoc group on the Asp residue, but found rather So, instead of changing the Fmoc group on Fmoc-
quickly that this group was not compatible with the coup- Asn(Rink amide)-OAll by Mmt, the Teoc group was intro-
ling step with the scaffold. After coupling to Asp, the re- duced via reaction with TeocONP at 50 °C for 6 hours. Fol-
maining free amines of the scaffold caused a rapid deprotec- lowing the synthetic protocol given in Scheme 4, we synthe-
tion of the Fmoc group with concomitant irreversible trans- sized compound 1b-(LysPhe-NHAc)3 in good yield (80%)
fer of the 9-fluorenylmethyl group. A wide variety of side and purity as evidenced by the HPLC chromatogram and
products was observed. Consequently, the protecting group ESI-MS spectra (Scheme 4).
for the Asp amino group required stability against nucleo-
philes/bases (the scaffold), reductive conditions (required
Scheme 4. Synthesis of compound 1b-(LysPhe-NHAc)3 on 2-Cl-trityl resin; i) Fmoc-Lys-OAll.TFA, DIPEA, CH2Cl2; ii) piperidine 20%
in DMF; iii) Teoc-ONp, DIPEA, CH2Cl2, 50 °C; iv) Pd(PPh3)4, PhSiH3, CH2Cl2; v) Tren, PyBOP, NMM, DMF/CH2Cl2; vi) Fmoc-
Lys(Boc)-OH, PyBOP, NMM, DMF/CH2Cl2; vii) TBAF, DCM, 50 °C; viii) N-Ac-Phe-OH, HBTU, HOBT, NMM, DMF/CH2Cl2; ix)
TFA, CH2Cl2, TIPS, H2O. The ESI MS spectrum of the crude cleavage mixture is shown.
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Figure 5. Schematic representation of the versatility of the synthetic procedure.
corded using a Shimadzu LC-10AT dual pump system and a Shim-
adzu SPD-10A UV–Vis detector. HR ESI-mass spectra were ob-
Conclusions
In this article we have presented a versatile synthetic pro-
cedure for the functionalization of tripodal scaffold mole-
cules on solid support. The general type of molecules that
are accessible using our approach is summarized in Fig-
ure 5. The type of solid support determines whether the fi-
nal compound is terminated with an amino or carboxylic
acid group. A first peptide fragment can be inserted before
the branching point by conventional peptide chemistry and
tained using
equipped with a TOF analyzer.
a Perspective Biosystem Mariner spectrometer
General Synthetic Procedures
Substitution of the Fmoc Group for Mmt (on Wang and DHPP
resin): The Fmoc group on Fmoc-Asp(OWang)-OAll or Fmoc-Asp-
(ODHPP)-OAll was removed using a solution of 20% piperidine
in NMP (2ϫ10 min). After washing with DMF (2ϫ) and CH2Cl2
(2ϫ), 4-methoxytriphenylchloromethane (Mmt-Cl, 6 equiv.) and
after the branching point by peptide growth in the NǞC N,N-diisopropylethylamine (DIEA, 15 equiv.) were added in
CH2Cl2 and the mixture was agitated for 2 h.
direction. Subsequently, any C3-symmetrical scaffold con-
taining NH2 terminal groups can be attached exclusively
on one position introducing the advantage of a controlled
subsequent functionalization of the scaffold with two dif-
ferent moieties. Alternatively, C3-symmetrical tripodal scaf-
folds can be prepared by inserting the peptide, branching
point, and identical functional group on the remaining two
positions of the scaffold. It should be emphasized that there
are no restrictions on the structure of either one of the sub-
components, including the scaffold, which illustrates the
versatility of the methodology. Our studies have shown that
the coupling of tripodal scaffold molecules to a resin can
give rise to very specific problems, such as polyadducts, in-
tramolecular cyclizations, etc. The purity of the products is
strongly determined by the choice of resin and the protect-
ing group on the branching unit.
Substitution of the Fmoc Group for Teoc (on Cl-Tritylresin): The
Fmoc group on Fmoc-Lys(Cl-Trit)-OAll was removed using a solu-
tion of 20% piperidine in DMF (2ϫ10 min). The Teoc group was
introduced by the reaction of the resin with Teoc-ONp [2-(trimeth-
ylsilyl)ethyl-4-nitrophenyl carbonate, 3.5 equiv.] in CH2Cl2/DIPEA,
9:1 for 3 h at 50 °C. The mixture was then cooled down and the
reaction continued overnight at room temperature. The completion
of the reaction was confirmed by the Kaiser test.
Removal of the Allyl Group and Activation: The allyl group was
removed under reductive conditions as reported.[19] Tetrakis-
(triphenylphosphane)palladium(0) (0.2 equiv.) and phenylsilane
(15 equiv.) in CH2Cl2 were added and the mixture was stirred for
2 h under an inert N2 atmosphere. After the washing steps [CH2Cl2
(2ϫ), NMP/DIEA = 4:1 (2ϫ20 min), NMP (2ϫ) and CH2Cl2
(2ϫ)], the carboxylic acid was activated by adding PyBOP
(5 equiv.) and N-methylmorpholine (10 equiv.) in DMF/CH2Cl2 =
1:1 for 1 hr.
It is our expectation that the synthetic procedures de-
scribed here will be very useful for the synthesis and screen-
ing of multivalent, heterofunctionalized structures for appli-
cation in the fields of (bio)recognition and catalysis.
Removal of the Mmt-Protecting Group (on Wang and DHPP resin):
The resin was washed with CH2Cl2 (3ϫ) and treated with a mixture
of acetic acid/trifluoroethanol/CH2Cl2 = 1:2:6 for 1 h and washed
again with CH2Cl2 (3ϫ).
Removal of the Teoc-protecting group (on Cl-tritylresin). The
Teoc-protecting group was removed by treatment of the resin sus-
pended in DCM with 10 equiv. of tetrabutylammonium fluoride
(1  in THF) for 4 h at 50 °C (3ϫ).
Experimental Section
General: All starting materials, solvents, and resins were obtained
from commercial sources and used without further purification.
Commercially available resins with a loading of 0.93 mmol/g Attachment of the DHPP Linker to Merrifield Resin: 4-(1Ј,1Ј-Di-
(Wang), 1.1 mmol/g (Merrifield), and 1.0 mmol/g (2-Cl-Trityl) were
used. Standard resin loading protocols were used for Wang and 2-
Cl-tritylresin. Solid-phase synthesis was performed using standard
Fmoc-based peptide chemistry (either using HBTU/HOBT or
PyBOP activation in the presence of DIPEA) and the specific pro-
cedures reported below.[10] The DHPP linker[16b] was synthesized
according to a literature procedure. Characterization data for 1c-
(AspArg-NH2)3 were identical to those already reported.[10] 1H
NMR spectra were recorded on a Bruker AC-300 (300.13 MHz)
spectrometer operating at 301 K. HPLC chromatograms were re-
methyl-1Ј-hydroxypropyl)phenoxyacetyl (DHPP) (4 equiv.) was dis-
solved in EtOH (2 ml/mmol) and water (0.5 ml/mmol), after which
the pH was adjusted to 7 by adding Cs2CO3. The solution was
evaporated to dryness and after subsequent adding of dioxane the
evaporation was repeated. The Cs+ salt of DHPP was added to a
suspension of Merrifield resin (0.055 mmol end groups) in DMF
for 12 hours at 50 °C in the presence of KI (0.1 equiv.) as catalyst.
Next, the resin was washed with MeOH/DMF/DIEA (1:1:0.1)
(4ϫ), 20% piperidine in DMF (2ϫ10 min), DMF (2ϫ), MeOH
(2ϫ) and dried under vacuum.
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 3559–3568

Functionalization of Tripodal Scaffold Molecules
Attachment of Fmoc-Asp(F)-OAll to DHPP resin. Fmoc-Asp(F)-
OAll (5 equiv.) was added to DHPP resin in DMF in the presence
of DIEA (15 equiv.) and the mixture was agitated for about 10 h
(overnight) at room temperature.
50% in 20 min, UV detection 226 nm]: 19.9 min (100%). 1H NMR
(CD3OD 250 MHz): δ = 0.89 (m, 6 H), 1.45 (m, 6 H), 1.68 (m, 6
H), 1.96 (d, 9 H), 2.92 (m, 12 H), 3.41 (m, 6 H), 3.58 (m, 6 H),
4.09–4.24 (m, 3 H), 4.39–4.53 (m, 3 H), 7.28 (m, 15 H) ppm. ESI-
MS [M + H]+ requires 1098.6750, found 1098.6742.
Synthesis and Characterization
Abbreviations
Fmoc-Asp(F)-OAll: Fmoc-Asp(OH)-OAll (0.19 g, 0.48 mmol) is
dissolved in CH2Cl2 (5 mL) and cooled down to 0 °C, after which
cyanuric fluoride (76 µL, 0.74 mmol) and pyridine (0.1 mL,
1.4 mmol) are added. After stirring for 1.5 h the reaction mixture
is quickly extracted with H2O (3ϫ) and dried on Na2SO4, after
which the solvent is evaporated under reduced pressure and dried
under high vacuum. The product is obtained as a white solid
(0.17 g, 91%). 1H NMR (CDCl3, 250 MHz): δ = 3.16 (m, 2 H),
4.23 (t, J = 6.8 Hz, 1 H), 4.43 (d, J = 6.9 Hz, 1 H), 4.67 (dd, J =
5.5, J = 14.8 Hz, 2 H), 5.29 (d, J = 10.2 Hz, 2 H), 5.38 (s, 1 H),
5.72 (d, J = 7.2 Hz, 1 H), 5.88 (dd, J = 5.5, J = 11.3 Hz, 1 H), 7.37
(td, J = 7.2 Hz, 4 H), 7.58 (d, J = 7.3 Hz, 2 H), 7.77 (d, J = 7.5 Hz,
2 H) ppm. 13C NMR (CDCl3, 63 MHz): δ = 47.02, 49.80, 66.99,
67.36, 119.63, 120.01, 124.97, 127.05, 127.78, 130.86, 141.28,
143.45 ppm. ESI MS [M + H+] requires 398.1, found 398.2.
ONBS: 2-nitrophenylsulfonyl, Fmoc: (9-fluorenyl)methoxycar-
bonyl, Aloc: allyloxycarbonyl, Dde: 1-(4,4-dimethyl-2,6-dioxocy-
clohexylidene)ethyl, Mmt: 4-methoxyphenyldiphenylmethyl, Teoc:
trimethylsilylethylcarbamate, OAll: allyloxy, Boc: tert-butoxycar-
bonyl, OtBu: tert-butoxy, PyBOP: [(benzotriazole-1-yl)oxy]tripyr-
rolidinophosphonium hexafluorophosphate, HOBT: 1-hydroxy-
benzotriazole, HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetrameth-
yluronium hexafluorophosphate, DIEA: N,N-diisopropylethyl-
amine, DMF: dimethylformamide, NMP: N-methylpyrrolidone,
TFE: 2,2,2-trifluoroethanol, TFA: trifluoroacetic acid, Tren: tris(2-
aminomethyl)amine, Tacn: triazacyclononane, DHPP: 4-(1Ј,1Ј-di-
methyl-1Ј-hydroxypropyl) phenoxyacetyl, Wang resin: p-benzyl-
oxybenzyl alcohol resin, Merrifield resin: chlormethylstyrene-divi-
nylbenzene resin, Rink amide: 4-(2Ј,4Ј-dimethoxyphenyl)-Fmoc-
aminomethyl-phenoxy-acetamido-norleucyl aminomethyl resin,
TBAF: tetrabutylammonium fluoride, NMM: N-methylmorph-
oline, TIPS: triisopropylsilane, TASF: tris(dimethylamino)sulfo-
nium difluorotrimethylsilanide.
1a-[Asp(Gly-OH)-NH2]: White solid (1.6 mg, 85%) after purifica-
tion with RP-HPLC [Jupiter Proteus-C18; gradient: H2O/TFA
(0.1%)ǞCH3CN/TFA (0.1%) from 3% to 25% in 30 min, UV de-
1
tection 226 nm]: 26.7 min (100%). H NMR (CD3OD, 250 MHz):
δ = 1.1–1.3 (m, 9 H), 2.6–2.8 (m, 6 H), 3.05 (m, 2 H), 3.74 (dd, 1
H), 4.23 (s, 4 H), 4.3–4.5 (m, 4 H) ppm. 13C NMR (CD3OD,
63 MHz): δ = 15.58, 16.29, 24.08, 24.44, 24.62, 30.95, 37.98, 39.27,
51.14, 129.42, 129.86, 132.84, 133.02, 169.94, 174.34, 174.77 ppm.
ESI-MS [M + H]+ requires 422.2689, found 422.2678.
Acknowledgments
Experimental work by Dr. Andrea Levorato and financial support
from the University of Padova (CPDA054893) and Ministero
dell’Università e della Ricerca (MIUR) (PRIN2006) are gratefully
acknowledged.
1a-[Asp(ProGly-OH)-NH2]: White solid (1.8 mg, 61%) after purifi-
cation with RP-HPLC [Agilent Eclipse XDB-C18; gradient: H2O/
TFA (0.1%)ǞCH3CN/TFA (0.1%) from 5% to 27% in 30 min, UV
detection 226 nm]: 27.5 min (100%). 1H NMR (CD3OD,
250 MHz): δ = 1.1–1.3 (m, 9 H), 1.9–2.2 (m, 4 H), 2.6–2.8 (m, 6
H), 3.0 (br.s, 2 H), 3.5–3.7 (m, 2 H), 3.75 (dd, 1 H), 4.21 (s, 4 H),
4.3–4.5 (m, 5 H) ppm. 13C NMR (CD3OD, 63 MHz): δ = 15.59,
16.28, 24.06, 24.41, 24.48, 24.57, 25.64, 30.93, 37.94, 39.33, 42.25,
51.12, 61.61, 129.48, 129.89, 132.90, 133.05, 169.89, 169.94, 174.34,
174.77 ppm. ESI-MS [M + H]+ requires 519.3217, found 519.3227.
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1a-(HisAsp-NHAc)3: White solid (12 mg, 91%) after precipitation
with diethyl ether. HPLC [Agilent Eclipse XDB-C18; gradient:
H2O/TFA (0.1%)ǞCH3CN/TFA (0.1%) from 13% to 30% in
30 min, UV detection 226 nm]: 26.8 min (100%). 1H NMR
(CD3OD, 250 MHz): δ = 1.19 (t, J = 7.4 Hz, 9 H), 1.95 and 2.05
(2s, 9 H), 2.6–2.8 (m, 12 H), 3.0–3.3 (m, 6 H), 4.43 (s, 6 H), 4.5–
4.6 (m, 3 H), 4.7–4.8 (m, 3 H), 7.35 (s, 3 H), 8.80 (s, 3 H) ppm.
13C NMR (CD3OD, 63 MHz): δ = 16.72, 20.89, 22.64, 27.99, 36.73,
39.17, 51.95, 53.66, 118.64, 131.24, 131.29, 132.61, 135.14, 171.53,
173.66, 174.74, 174.04 ppm. ESI-MS [M + H]+ requires 1132.5098,
found 1132.5021.
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Brouwer, L. M. Verbeek, A. M. A. van Wageningen, R. J. M.
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1a-(HisAspAsp-NHAc)-(HisAsp-NHAc)2: White solid (14 mg,
72%) after precipitation with diethyl ether. HPLC [Agilent Eclipse
XDB-C18; gradient: H2O/TFA (0.1%)ǞCH3CN/TFA (0.1%) from
12% to 28% in 30 min, UV detection 226 nm]: 30.5 min (100%).
1H NMR (CD3OD, 250 MHz): δ = 1.19 (t, J = 7.4 Hz, 9 H), 1.9–
2.0 (m, 9 H), 2.6–2.8 (m, 14 H), 3.0–3.3 (m, 6 H), 4.42 (s, 6 H),
4.5–4.6 (m, 4 H), 4.7–4.8 (m, 3 H), 7.35 (s, 3 H), 8.80 (s, 3 H) ppm.
ESI-MS [M + H]+ requires 1247.5367, found 1247.5349.
1b-(LysPhe-NHAc)3: White solid (2.6 mg, 80%) after precipitation
from diethyl ether. HPLC [Agilent Eclipse XDB-C18; gradient:
H2O/TFA (0.1%)ǞCH3CN/TFA (0.1%) 5% for 5 min from 5% to
Eur. J. Org. Chem. 2008, 3559–3568
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
3567

C. Guarise, G. Zaupa, L. J. Prins, P. Scrimin
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Published Online: May 30, 2008
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© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2008, 3559–3568