Solid-phase synthesis of amidine-substituted phenylbenzimidazoles and incorporation of this DNA binding and recognition motif into amino acid and peptide conjugates

Article abstract of DOI:10.1007/s00726-014-1691-5

Amidine-substituted phenylbenzimidazoles are well-established DNA-binding structural motifs that have contributed to the development of diverse classes of DNA-targeted agents; this ring system not only assists in increasing the overall DNA affinity of an agent, but can also influence its site selectivity. Seeking a means to conveniently exploit these attributes, a protocol for the on-resin synthesis of amino acid- and peptide-phenylbenzimidazole-Amidine conjugates was developed to facilitate installation of phenylbenzimidazole- Amidines into peptide chains during the course of standard solid-phase syntheses. Building from a resin-bound amino acid or peptide on Rink amide resin, 4-formyl benzoic acid was coupled to the resin-bound free amine followed by introduction of 3,4-diamino-N′-hydroxybenzimidamide (in the presence of 1,4-benzoquinone) to construct the benzimidazole heterocycle. Finally, the resin-bound N′-hydroxybenzimidamide functionality was reduced to an amidine via 1 M SnCl₂·2H₂O in DMF prior to resin cleavage to release final product. This procedure permits the straightforward synthesis of amino acids or peptides that are N-terminally capped by a phenylbenzimidazole-Amidine ring system. Employing this protocol, a series of amino acid-phenylbenzimidazole-Amidine (Xaa-R) conjugates was synthesized as well as dipeptide conjugates of the general form Xaa-Gly-R (where R is the phenylbenzimidazole-Amidine and Xaa is any amino acid).

Full text of DOI:10.1007/s00726-014-1691-5

Amino Acids
DOI 10.1007/s00726-014-1691-5
ORIGINAL ARTICLE
Solid-phase synthesis of amidine-substituted phenylbenzimidazoles
and incorporation of this DNA binding and recognition
motif into amino acid and peptide conjugates
•
Matthew L. Garner Taxiarchis M. Georgiadis
•
•
Jessica Bo Li Tianxiu Wang Eric C. Long
•
Received: 22 November 2013 / Accepted: 31 January 2014
Ó Springer-Verlag Wien 2014
Abstract Amidine-substituted phenylbenzimidazoles are
well-established DNA-binding structural motifs that have
contributed to the development of diverse classes of DNA-
targeted agents; this ring system not only assists in
increasing the overall DNA affinity of an agent, but can
also influence its site selectivity. Seeking a means to con-
veniently exploit these attributes, a protocol for the on-
resin synthesis of amino acid- and peptide-phenylbenzi-
midazole-amidine conjugates was developed to facilitate
installation of phenylbenzimidazole-amidines into peptide
chains during the course of standard solid-phase syntheses.
Building from a resin-bound amino acid or peptide on Rink
amide resin, 4-formyl benzoic acid was coupled to the
resin-bound free amine followed by introduction of 3,4-
diamino-N0-hydroxybenzimidamide (in the presence of 1,4-
benzoquinone) to construct the benzimidazole heterocycle.
Finally, the resin-bound N0-hydroxybenzimidamide func-
tionality was reduced to an amidine via 1 M SnCl2ꢀ2H2O in
DMF prior to resin cleavage to release final product. This
procedure permits the straightforward synthesis of amino
acids or peptides that are N-terminally capped by a phe-
nylbenzimidazole-amidine ring system. Employing this
protocol, a series of amino acid–phenylbenzimidazole-
amidine (Xaa-R) conjugates was synthesized as well as
dipeptide conjugates of the general form Xaa-Gly-R
(where R is the phenylbenzimidazole-amidine and Xaa is
any amino acid).
Keywords DNA binding ꢀ Benzimidazole ꢀ Amidine ꢀ
Amino acids ꢀ Solid-phase synthesis
Introduction
The development and investigation of biologically active
compounds that target DNA through equilibrium binding
continue to be active areas of pursuit (Soeiro et al. 2013;
Wei et al. 2013; Yang et al. 2013; Glass et al. 2010;
Nguyen et al. 2009; Long et al. 2009; Tse and Boger 2004;
Dervan and Edelson 2003; Neidle 2001). Among organic
structural elements that have assisted in the development of
such agents, the benzimidazole heterocycle has proven to
be a versatile DNA recognition and anchoring moiety
(Viger and Dervan 2006; Ismail et al. 2005; Briehn et al.
2003). These attributes are due to the overall size, shape,
and heteroatom positioning of a benzimidazole ring that
promote its steric compatibility with DNA minor groove
binding and hydrogen bonding (Briehn et al. 2003; Neidle
2001). Indeed, Hoechst 33258, a well-studied agent that
contains a bis-benzimidazole core, targets the DNA minor
groove and has provided an important paradigm for the
understanding of benzimidazole–DNA interactions as well
as small molecule recognition of the DNA minor groove in
general (Neidle 2001).
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-014-1691-5) contains supplementary
material, which is available to authorized users.
Given the above, benzimidazoles have added signifi-
cantly to the repertoire of heterocycles investigated in the
development of DNA-binding polyamides, peptides, and
other agents (Briehn et al. 2003; Viger and Dervan 2006;
Behrens et al. 2001; Farahat et al. 2011; Ismail et al. 2005;
Goodwin et al. 2006; Tanious et al. 2007). In particular,
M. L. Garner ꢀ T. M. Georgiadis ꢀ J. B. Li ꢀ T. Wang ꢀ
E. C. Long (&)
Department of Chemistry and Chemical Biology, Indiana
University-Purdue University Indianapolis (IUPUI), 402 N.
Blackford St., Indianapolis, IN 46202, USA
e-mail: eclong@iupui.edu
123

M. L. Garner et al.
and the straightforward generation of amino acids or pep-
tides that are N-terminally capped with this moiety.
Accordingly, we demonstrate the on-resin synthesis of the
above and the syntheses of model end-capped amino acids
and dipeptides. While developed and demonstrated here for
amino acid and peptide modifications, these same proce-
dures should permit the end capping of any reactive termini
compatible with similar solid-phase syntheses.
Fig. 1 General structure of a resin-bound amino acid (Xaa, n = 1) or
peptide (n [ 1) N-terminally capped with an amidine-substituted
phenylbenzimidazole
phenylbenzimidazole-amidines, cationic species under
physiological conditions, have led to a host of biologically
active antiparasitic agents that target the DNA minor
groove (Soeiro et al. 2013; Goodwin et al. 2006; Tanious
et al. 2007). Indeed, aromatic diamidines in general con-
taining the phenylbenzimidazole-amidine structural unit
have attracted considerable interest due to their antimi-
crobial activities that are believed derived from DNA
binding (Soeiro et al. 2013). The combined effect of the
benzimidazole ring and its capacity for minor groove
hydrogen bonding, along with the cationic amidine moiety,
makes these structural units interesting candidates for the
further development of DNA minor groove binding agents
based on peptide chemistry.
Materials and methods
All chemicals and organic solvents were of reagent grade,
purchased from Sigma-Aldrich or Fisher, and used without
further purification. Protected amino acids were purchased
from Bachem, and Rink amide resins (loading: 66 mmol/g)
were purchased from EMD Millipore. 1H NMR spectra
were obtained using a 500-MHz Bruker Avance III NMR
spectrometer with a 5-mm broadband probe. LC/MS
analyses were performed using an Agilent 1100 series
HPLC with a binary pump, auto-sampler, and diode array
detector outfitted with a C18 column (150 9 4.6 mm;
5-micron particle size); the MS component was an Agilent
1946D mass spectrometry detector equipped with electro-
spray ionization.
Given the attributes of phenylbenzimidazole-amidines
and with an eye towards the expedited generation of DNA-
targeted agents through combinatorial strategies, we sought
to develop a convenient on-resin route through which to
modify natural or unnatural amino acids and peptides with
this structural element or to N-terminally ‘‘cap’’ peptides to
augment their DNA-binding activities (Fig. 1). These goals
complement prior reported efforts in which: (1) the entire
aryl bis-benzimidazole ‘‘core’’ element of Hoechst 33285
was redesigned for inclusion in solid-phase peptide syn-
theses (Behrens et al. 2001; Bunkenborg et al. 2002), and
(2) strategies for the synthesis of unnatural amino acids and
compound libraries bearing derivatized benzimidazole side
chains (Koprowska-Ratajska et al. 2009; Vourloumis et al.
2003). Differing from these efforts, however, we have
developed a solid-phase protocol for installing benzimi-
dazoles that includes an amidine moiety of crucial impor-
tance to effective DNA binding and biological activity
(Soeiro et al. 2013). Additionally, this strategy focuses on
inclusion of single benzimidazole rings. A single ring
would be expected to influence to a far less extent the DNA
recognition and binding properties of a final product, per-
haps working synergistically with other structural elements
present in the total structure. Indeed, a complete Hoechst-
like bis-benzimidazole structure appears to dominate the
DNA recognition of peptides in which it has been included
(Behrens et al. 2001).
General solid-phase synthetic procedures
All ambient temperature solid-phase syntheses were per-
formed manually in fritted SPE column reservoirs (15 mL)
outfitted with Teflon stopcocks and septa and agitated on
an orbital shaker. Manual solid-phase syntheses carried out
at elevated temperatures were performed in glass scintil-
lation vials (40 mL) containing a stir bar and placed in a
reaction block fitted to a hot plate stirrer with temperature
control. Weighed portions of Rink amide resin were dis-
tributed to each reaction vessel and swollen in 1:1
DCM:DMF for 30 min to 1 h. Fmoc deprotection was
performed by shaking resin, or resin-amino acids and resin-
peptides, in 1:4 (v/v) piperidine: DMF for 30 min at room
temperature; deprotections were monitored via Kaiser test
(Stewart and Young 1984). Amino acid couplings to
deprotected portions of Rink amide resin and the genera-
tion of resin-bound Gly-Xaa dipeptides prior to phe-
nylbenzimidazole-amidine assembly were carried out using
standard manual peptide synthesis protocols (0.25 g Rink
amide resin, 0.825 mmol protected amino acid, and equi-
molar equivalents of HOBt and DIC in 15 mL DMF)
(Stewart and Young 1984; Chan and White 2000). Cou-
plings were carried out at room temperature for 1.5 h and
verified by Kaiser test. After completion of each coupling
step, reactants were drained from reaction vessels and
resins or resin-peptides were washed with DMF
Herein, we describe a solid-phase protocol that permits
the on-resin assembly of phenylbenzimidazole-amidines
123

Solid-phase synthesis of amidine-substituted phenylbenzimidazoles
(3 9 5 mL), methanol (3 9 5 mL), DCM (3 9 5 mL),
and diethyl ether (3 9 5 mL). Solvents were removed by
filtration using a vacuum system. All reactions were per-
formed under nitrogen.
1.5 h. Resins were removed by filtration and rinsed with
DCM. Filtrates containing products were collected in
weighed vials, and evaporated with a stream of nitrogen
until a small volume remained, which was then precipitated
into cold ether. The resulting solid precipitate was col-
lected by filtration, dissolved in methanol and flushed
through a silica column with methanol (Biotage flash col-
umn cartridge containing 1 g silica gel) to remove bulk
impurities. Products were purified by HPLC using C18
reverse-phase chromatography (A: H2O, 0.1 % TFA; B:
ACN, 0.1 % TFA. Gradient: 0 min, 80 % A; 2 min, 80 %
A; 20 min, 20 % A; 25 min, 0 % A; 30 min, 0 % A;
32 min, 80 % A; 35 min, 80 % A). All products were
Synthesis of 3,4-diamino-N0-hydroxybenzimidamide
(1)
3,4-Diaminobenzonitrile (7.5 mmol, 1.0 g) in ethanol
(20 mL) was mixed with 20 equivalents of 50 % aqueous
hydroxylamine (150 mmol, 4.95 g) and refluxed for 5 h.
Upon completion, the ethanol was evaporated under
reduced pressure. The product was purified via column
chromatography [EtOAc:MeOH (90:10)] and confirmed
1
confirmed via MS and H NMR (Online Resource 1).
1
1
The generalized procedure described above led to 2
when 4-carboxybenzaldehyde was coupled directly to Rink
amide resin; and compound series 3a–p and series 4a–t
when a single, C-terminal amino acid, Xaa, or Gly-Xaa
dipeptide, respectively, was present on the resin prior to the
coupling of 4-carboxybenzaldehyde. At the synthesis scale
employed throughout these studies (0.25 g Rink amide
resin; 0.66 mmol/g), *50–100 mg of final product was
obtained per reaction (see Online Resource 1 for individual
gram yields); at this time, there is no reason to suspect that
difficulties will be encountered upon scale-up.
via LC/MS and H NMR. (Yield: 1.06 g, 85 %); H NMR
(DMSO-d6); d 4.45 (s, 2H), 4.63 (s, 2H), 5.38 (s, 2H), 6.46
(d, J = 8 Hz, 1H), 6.71 (dd, J = 8.5 Hz, 1H), 6.85 (s, 1H),
9.11 (s, 1H); LC/MS calcd. for [M ? H]?: C7H10N4O
167.1, found 167.3.
Solid-phase syntheses of phenylbenzimidazole-
amidines and amino acid/peptide conjugates
Portions of deprotected Rink amide resin (0.25 g,
0.66 mmol/g) [or equivalent amounts of Fmoc deprotected
resin-bound amino acid (Xaa) or Gly-Xaa dipeptide] were
coupled to 4-carboxybenzaldehyde (0.8250 mmol,
123.8 mg) in the presence of HOBt (0.8250 mmol,
111.5 mg), and DIC (0.8250 mmol, 104.1 mg) in 15 mL
DMF (15 mL). The resulting mixtures were agitated on an
orbital shaker at room temperature for 1.5 h and comple-
tion of each coupling was confirmed by Kaiser test. Upon
resin washing with DMF (3 9 5 mL), methanol
(3 9 5 mL), DCM (3 9 5 mL), and diethyl ether
(3 9 5 mL), 3,4-diamino-N0-hydroxybenzimidamide, 1,
Results and discussion
The goal of the work described here was to develop a
straightforward means to incorporate an N-terminal phe-
nylbenzimidazole-amidine moiety into resin-bound amino
acids or peptides. While reports exist in the literature that
describe the on-resin generation of simple benzimidazoles
and benzimidazole derivatives (Sun and Yan 1998; Ko-
prowska-Ratajska et al. 2009; Vourloumis et al. 2003),
these examples lead to the generation of benzimidazoles,
unnatural amino acids bearing benzimidazole side chains,
or independent, low-molecular weight species not intended
for further conjugation. Thus, to the best of our knowledge,
this is the first report of the assembly of the phenylbenzi-
midazole-amidine structural unit of established DNA-
binding ability directly to a resin or resin-bound amino acid
or peptide via a main-chain peptide linkage. As noted
earlier, the main efforts of this work are directed towards
the eventual generation of DNA-targeted agents given the
promising biological activities demonstrated by com-
pounds containing phenylbenzimidazole-amidines. How-
ever, the widespread use of benzimidazole heterocycles in
medicinal chemistry (Bansal and Silakari 2012) makes it
clear that this work could similarly impact the development
of compounds for other biological purposes and targets.
(1.650 mmol,
274.2 mg)
and
1,4-benzoquinone
(0.165 mmol, 17.8 mg) were dissolved in DMF (15 mL)
and coupled to the resin-bound aldehyde for 5 h at 60 °C
using an IKA-Werke magnetic stirrer/heat block. The
above procedures led to a resin- or amino acid/peptide-
bound equivalent of phenyl-benzimidazole containing an
N0-hydroxybenzimidamide substituent. To effect final
conversion of the resin-bound N0-hydroxybenzimidamide
moiety to an amidine, SnCl2ꢀ2H2O (2.475 mmol,
558.5 mg) was dissolved in DMF (2.5 mL), introduced into
the reaction vessel, and allowed to react. Reaction condi-
tions were maintained using an IKA-Werke magnetic
stirrer/heat block at 80 °C over a period of 40 h with fresh
additions of SnCl2 solution (500 mg in 2.5 mL DMF)
being added to the reaction mixture every 4 h (excluding
overnight hours). Final products were cleaved from resin
supports via shaking in 1:1 solutions of TFA:DCM for
123

M. L. Garner et al.
Fig. 2 Generalized scheme for the on-resin syntheses of amidine-substituted phenylbenzimidazole 2 or amino acids (3a–p) or peptides (4a–t)
N-terminally capped with an amidine-substituted phenylbenzimidazole (Xaan represents any amino acid of n number of residues)
Solid-phase synthesis of phenylbenzimidazole-
amidines
would otherwise occur with a C-terminal carboxylate at
physiological pH. The presence of a negative charge would
diminish the DNA-binding capacity of a final product due
to electrostatic repulsion.
The initial target in our development of a solid-phase
synthesis protocol for phenylbenzimidazole-amidines was
2 (Fig. 2). In this simplest case, the phenylbenzimidazole-
amidine was assembled directly on resin and permitted
development of the synthesis procedure in the absence of a
C-terminal amino acid or peptide link to the solid phase.
Throughout these syntheses, Rink amide resins were
employed due to the nature of the ultimate intended pur-
pose of the final compounds: DNA binding. Upon final
product cleavage from the solid support, Rink amide resins
release C-terminal amide functionalities rather than car-
boxylic acids. The presence of a C-terminal amide within
the final compound thus eliminates the negative charge that
To initiate the synthesis of 2, 1,4-carboxybenzaldehyde
was coupled directly to deprotected Rink amide resin using
conditions typical for the coupling of amino acids to solid
supports. It was determined that the most efficient means to
drive this coupling to completion involved initiating a
second coupling after an initial 1.5 h of reaction time;
longer initial coupling times and increased amounts of
initially added carboxybenzaldehyde failed to decrease the
reaction times necessary for complete coupling.
Upon verification of complete coupling of the carb-
oxybenzaldehyde to the solid support, the assembly of the
benzimidazole ring was achieved through the introduction
123

Solid-phase synthesis of amidine-substituted phenylbenzimidazoles
of 3,4-diamino-N0-hydroxybenzimidamide (1). In our
hands, reactions with this intermediate came to completion
readily when 10 equivalents were allowed to react with the
resin-linked aldehyde for 5 h at 60 °C. Using fewer
equivalents of 1 led to incomplete coupling within this
timeframe and room temperature reactions required up to
24 h to come to completion.
Table 1 Series 3 Xaa-phenylbenzimidazole-amidine conjugates and
yields
Compound R (Xaa) Yield (%) Compound R (Xaa) Yield
(%)
3a
3b
3c
3d
3e
3f
3g
3h
3i
Gly
Ala
Val
Leu
Ile
88
74
81
76
73
73
79
64
77
79
3k
3l
Gln
Tyr
Lys
Arg
Asp
Glu
Cys
His
Ser
73
61
64
58
78
72
0a
0a
0a
0a
3 m
3n
3o
3p
–
Having now formed a resin-bound benzimidazole bearing
an N0-hydroxylated benzimidamide, the final step involved
reduction of this N-hydroxylated substituent to the final
desired amidine. This reduction was achieved through
treatment of the resin with SnCl2ꢀ2H2O in DMF at 80 °C for
Met
Pro
Phe
Thr
Asn
–
¨
40 h (Dorwald 2002). To increase yields, aliquots of fresh
–
SnCl2 were added to the reaction mixture every 4 h. Initial
attempts at this conversion using a single addition of SnCl2
led to thickening of the reaction mixture and no desired
product. Similarly, increasing the concentration and/or vol-
ume of the initially added SnCl2 did not alleviate this prob-
lem and usually resulted in\25 % yield of desired product.
An exploration of shortened reaction times also led to
incomplete conversion to the amidine. Thus, 40 h was
determined to be optimal. Eventually, additional aliquots
added at 4-h intervals throughout the day, as described
above, were discovered to prevent resin thickening and led to
the desired product upon resin cleavage. Final resin cleavage
generated typical product yields 76 %.
3j
–
Trp
a
Final product not observed
4, bearing a C-terminal peptide containing a variable amino
acid connected via a Gly linker (Gly-Xaa) to the phe-
nylbenzimidazole-amidine moiety. This latter model sys-
tem was explored not only as a peptide mimic, but also to
determine if varied amino acid side chains influenced the
coupling of 4-carboxybenzaldehyde to resin-bound
peptide.
Synthesis of series 3 followed the same route described
for the synthesis of 2 upon coupling of the 4-carboxy-
benzaldehyde to a Rink amide resin bearing a deprotected
amino acid. As shown in Table 1, acceptable yields for 16
out of the 20 naturally occurring amino acids were
obtained. However, despite numerous attempts, final
desired products derived from Rink-amide bearing Cys,
His, Ser, and Trp were not obtained and appeared to fail
during the course of the SnCl2ꢀ2H2O reduction step; all
steps involving these amino acids prior to SnCl2ꢀ2H2O
reduction were completed and analyses of resin-bound
intermediates by LC/MS showed the expected masses
confirming the presence of the initial resin-amino acids and
their respective 4-carboxybenzaldehyde and N0-hydroxyl-
ated diaminobenzimidamide products (Fig. 2). The reason
for the lack of reactivity for these four amino acids is not
clear. However, based on the results described below for
Xaa-Gly dipeptides leading to 4 that permitted inclusion of
all 20 naturally occurring amino acids, it is likely that
sterics and not chemical reactivity influenced the outcome.
Following the generation of the series 3 single amino
acid bearing phenylbenzimidazole-amidines, a second
dipeptide-based series (4) of conjugates was pursued
(Table 2). In this series of reactions, we sought to deter-
mine the impact of the presence of a resin-bound dipeptide
equipped uniformly with a flexible Gly linker on the gen-
eration of peptides N-capped with the phenylbenzimida-
zole-amidine. Synthesis of the dipeptide conjugates
proceeded through the same protocol employed in the
It is noteworthy that during the development of the
above procedure attempts were made to incorporate ami-
dines into resin-bound benzimidazoles via: (1) the Pinner
conversion of an on-resin nitrile substituent (see Parrish
et al. 1978 for an example); (2) the catalytic hydrogenation
of an on-resin N-hydroxy benzimidamide via Pd on char-
coal (Ismail et al. 2004, 2005); and (3) the conversion of an
on-resin nitrile to an N-hydroxybenzimidamide via
hydroxylamine and subsequent treatment with SnCl2ꢀ2H2O
¨
(Dorwald 2002). However, none of these procedures
proved to be successful. In particular, in the latter case, we
found that the on-resin benzimidazole decomposed during
resin treatment with hydroxylamine. Thus, in light of
reports indicating that SnCl2ꢀ2H2O reacted to convert
aromatic nitro compounds to amines on solid support
¨
(Dorwald 2002), the use of SnCl2ꢀ2H2O was employed
successfully in conjunction with 1 prepared in solution.
Solid-phase syntheses of amino acid and peptide
conjugates of phenylbenzimidazole-amidine
Having developed an on-resin protocol for the generation
of phenylbenzimidzole-amidines, the ability to react with
resin-bound amino acids and peptides was explored. Two
series of amino acid-bearing phenylbenzimidazole-ami-
dines were synthesized: one series bearing a single C-ter-
minal naturally occurring amino acid (Xaa), 3, and another,
123

M. L. Garner et al.
that the complete phenylbenzimidazole-amidine structural
unit can be assembled on resin and also as an amino acid or
peptide conjugate. In the latter case, uniform coupling to a
resin-peptide terminating with a Gly ‘‘linker’’ leading to a
phenylbenzimidazole-amidine derivative proceeded with-
out difficulty. Thus, this work enables the inclusion of a
unique structural unit into established peptide synthesis
protocols and could permit the expedited or combinatorial
generation of phenylbenzimidazole-amidine derivatives for
biological testing. While beyond the scope and intention of
the work described herein, we plan to report the impact of
the incorporation of this moiety into DNA binding agents
in due course.1
Table 2 Series 4 Xaa-Gly-phenylbenzimidazole-amidine conjugates
and yields
Compound
Xaa
Yield (%)
Compound
Xaa
Yield (%)
4a
4b
4c
4d
4e
4f
Gly
Ala
Val
Leu
Ile
93
82
87
85
91
90
91
77
74
59
4k
4l
Thr
Asn
Gln
Tyr
Cys
Lys
Arg
His
Asp
Glu
89
69
73
65
44
94
78
61
85
71
4m
4n
4o
4p
4q
4r
4s
Met
Pro
Phe
Trp
Ser
4g
4h
4i
4j
4t
Acknowledgments We gratefully acknowledge financial support
from an IUPUI Research Support Funds Grant (to ECL). The authors
would also like to thank Prof. Marty O’Donnell (IUPUI) for com-
ments on the manuscript during its preparation.
generation of 2 and 3. After the initial coupling of all 20
naturally occurring amino acids to the Rink amide resin,
capping with Gly, and deprotection, 4-carboxybenzalde-
hyde was coupled to each terminal Gly residue. Subse-
quently, the phenyl-benzimidazole core was assembled
along with reduction of the resin-bound N0-hydroxylated
benzimidamide to an amidine. With each dipeptide resin,
the N-terminal capping procedure leading to Xaa-Gly-
phenylbenzimidazole-amidine proceeded smoothly to final
product (Table 2). All 20 naturally occurring amino acid–
Gly dipeptides in this instance led to final products (4).
This outcome reinforces the notion that the inability to
directly produce some of the amino acid (Cys, His, Ser, and
Trp) conjugates in series 3 is likely due to inhibition of the
reduction step through some means other than the direct
reactivity with these particular protected side chains.
The results described above indicate that the procedures
employed here can lead to the straightforward on-resin
generation of amino acids and dipeptides that are N-ter-
Conflict of interest The authors declare that they have no conflict
of interest.
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1
While the model compounds synthesized during the course of this
work (2, 3, and 4) were not intended or designed purposefully with
the targeting of DNA in mind, we nonetheless carried out preliminary
tests of their DNA binding—these studies indicated that systems
possessing the phenylbenzimidazole-amidine moiety exhibited sig-
nificantly enhanced DNA binding. A full account of these activities
within rationally designed peptide sequences will be reported in due
course.
Conclusions
The foregoing work has described a straightforward means
to incorporate an established DNA binding motif into on-
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