On-resin synthesis of novel arginine-isostere peptides bearing substituted amidine headgroups

Article abstract of DOI:10.1002/psc.1412

A methodology is presented for the facile synthesis of Arg-containing peptides modified at the guanidine headgroup as substituted amidine cores. This process allows for the iterative construction of these Arg isosteres while the peptide is being built out on the solid support, providing a high potential for diversity in substitution pattern in the resulting peptide. A series of N-Pmc-substituted thioamides were condensed with deprotected δ-N Orn-bearing peptides while attached to the solid support using Mukaiyama's reagent as coupling reagent, yielding isosteric Arg-containing analogs. Peptides were cleaved using trimethylsilyl trifluoromethanesulfonate/TFA and analyzed in their crude form in order to illustrate the amenability of this process toward production of peptide isolates in high crude purity. Arg-containing peptides having a single Arg isostere were utilized to show the general utility of this approach as well as a multiple-Arg-containing construct, illustrating the amenability of this method toward stepwise construction of differently substituted amidine headgroups within the same peptide.

Full text of DOI:10.1002/psc.1412

Research Article
Received: 21 June 2011
Revised: 2 August 2011
Accepted: 11 August 2011
Published online in Wiley Online Library: 27 October 2011
(wileyonlinelibrary.com) DOI 10.1002/psc.1412
On-resin synthesis of novel arginine-isostere
peptides bearing substituted amidine
headgroups
Stevenson Flemer Jr. and José S. Madalengoitia*
A methodology is presented for the facile synthesis of Arg-containing peptides modified at the guanidine headgroup as
substituted amidine cores. This process allows for the iterative construction of these Arg isosteres while the peptide is being
built out on the solid support, providing a high potential for diversity in substitution pattern in the resulting peptide. A series
of N-Pmc-substituted thioamides were condensed with deprotected d-N Orn-bearing peptides while attached to the solid
support using Mukaiyama’s reagent as coupling reagent, yielding isosteric Arg-containing analogs. Peptides were cleaved
using trimethylsilyl trifluoromethanesulfonate/TFA and analyzed in their crude form in order to illustrate the amenability of
this process toward production of peptide isolates in high crude purity. Arg-containing peptides having a single Arg isostere
were utilized to show the general utility of this approach as well as a multiple-Arg-containing construct, illustrating the ame-
nability of this method toward stepwise construction of differently substituted amidine headgroups within the same peptide.
Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.
Keywords: amidine; arginine; arginine analogs; solid-phase synthesis; combinatorial chemistry
generated toward the maximization of its binding efficiency
and salt-bridge-forming potential in chemically altered analogs.
Introduction
Protein–protein interactions form the basis of most enzymatic
and signal transduction pathways in biological systems. Similarly,
small peptides are frequently the preferred binding substrates for
a diverse range of signaling cascades and hormone interactions
[1]. Binding specificity in these cases is an exquisitely tuned
process based primarily upon interactions between the amino
acid side chains of the binding partners, normally mediated by
protein tertiary structure. Of the four major types of tertiary
structure-maintaining vectors in the structural stabilization of
proteins, salt bridge interactions between cationic and anionic
side chains provide a crucial staple in preserving active three-
dimensional conformation as well as maximizing binding efficiency
between partners.
Arginine is a vital and ubiquitous amino acid residue, essen-
tial to many crucial biochemical functions. The uniquely basic
character of the Arg side chain guanidine headgroup keeps it
protonated and positively charged at physiological pH, allowing
it to participate in ion pairings and salt bridge interactions
crucial to binding and recognition events in cell signaling
processes. Native Arg is the preferred substrate for nitric oxide
synthase (NOS) in the production of NO for various cellular
and neuronal processes [2]. As a component residue in various
small peptide sequences and enzyme active sites, the Arg side
chain mediates critical interactions and is heavily represented
in natural substrates of trypsin-like serine proteases [3] and
those involved in the blood coagulation cascade. Additionally,
polyarginine-containing peptides have synergistic cell membrane-
permeating abilities that have been routinely utilized as a peptidyl
carrier module for drug delivery [4].
Isosteric replacement of Arg in biologically relevant peptides
has been of value in improving efficiency of the substrate as well
as in SAR studies of enzyme-binding pockets [5]. Moreover,
carefully designed structural analogs of native Arg have provided
entry into NOS inhibitory constructs [6]. The extent of Arg
diversification described in the literature is immense and has
been well described in various reviews [7,8]. Structural diversifica-
tion of Arg, whether in its discrete form or incorporated into a
peptide sequence, can be approached in one of three possible
manners: (i) N or C diversification in order to elongate or cap
these termini [9,10], (ii) alteration within the three-carbon
extension of its side chain to introduce constraint [11] or structural
adjustment [12], or (iii) diversification of the guanidine headgroup
to alter its basicity [13] and/or form [14,15].
Structural diversification of the guanidine headgroup of Arg
offers the most profitable means of maximizing binding potential
or interaction efficacy because of the highest potential for
stuctural diversity. One of the means of carrying this out is
through the installation of an alkyl or aryl substituent at one or
more of the guanidine nitrogens. There has been a fair degree
of interest and research activity in this area recently, evidenced
by multiple publications by our research group [16,17] as well as
those of Fan [18] and Martin [19,20]. All of these research efforts
were based upon a similar synthetic design: the nucleophilic attack
a
a
*
Correspondence to: José S. Madalengoitia, Department of Chemistry, A232
Cook Physical Sciences Building, 82 University Place, University of Vermont,
Burlington, VT 05405, USA. E-mail: jmadalen@uvm.edu
Because of the high level of relevance and importance imbued
upon this amino acid residue, a great deal of interest has been
Department of Chemistry, University of Vermont, A232 Cook Physical Sciences
Building, 82 University Place, Burlington, VT, 05405, USA
J. Pept. Sci. 2012; 18: 30–36
Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

SOLID-PHASE SYNTHESIS OF AMIDINE-TEMPLATED ARGININES
of an Orn amine upon an activated N-arylsulfonyl-N′-substituted thio-
urea under gentle 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDCI)-mediated conditions to afford the corresponding substi-
tuted Arg adduct, sulfonyl-protected at its guanidine moiety.
Syntheses that resulted in the formation of properly protected
Arg adducts suitable for solid phase peptide synthesis [17,20]
allowed for insertion of these substituted derivatives into a
growing synthetic peptide.
The underlying base structure of the guanidino functionality
itself can similarly be altered to exist as a structural mimic of
the native architecture. Although there are examples of Arg
mimics whose guanidine headgroup takes a completely different
isoform such as that of a borate functionality [21] or a sulfamoyl-
type structure [22], the amidine core motif has emerged as highly
representative of alternative guanidine architecture in a number
of Arg isosteres reported in the literature. In particular, the aceta-
midine moiety is quite prevalent as a guanidinyl isoform as stuc-
tural analogs bearing this basic motif have been found to act as
very effective inhibitors of NOS [23–25].
thiourea transfer agent under EDCI-mediated conditions to afford
the Pmc-protected Arg properly substituted at the guanidine
core (Figure 1(A)). We found this synthetic route to be extremely
convenient and highly desirable because of its high potential
for diversification and its amenability toward a completely solid-
supported synthesis. This specific guanidinylation methodology
was expanded in a subsequent publication to include solution syn-
theses of substituted amidines in which the scope and the limita-
tions of the method were carefully explored [32]. This analogous
synthetic route utilized N-Pmc-substituted thioamides rather than
thioureas as the amidinylation agents. The primary salient feature
which emerged from this effort was the necessity to apply a differ-
ent thiophile (Mukaiyama’s reagent) to mediate the smooth con-
version to amidine core structure on the model systems studied.
It was perhaps logical that our curiosities led us to apply this meth-
odology to the solid support, particularly as a tool to bring about
the iterative formation of amidine-functionalized guanidine cores
on Arg-containing peptide systems (Figure 1(B)).
In this effort, we report a systematic assay of known thiophiles
identified in our previous report to be effective at mediating ami-
dine formation between an amine and an N-Pmc thioamide
transfer agent as to their effectiveness when applied to a d-amino
group of a resin-bound Orn as the amine partner in the amidiny-
lation process. We then apply these optimized conditions toward
the condensation of an array of differently substituted N-Pmc
thioamide transfer agents and an Orn-bearing test peptide in
order to explore the scope and the limitations of the solid-
supported reaction. Finally, the utility of this methodology is
highlighted in the solid-phase iterative construction of a peptide
bearing three Arg residues, all differentially functionalized as
their corresponding substituted amidine headgroups. Similarly
to our previous work describing the stepwise solid-supported
synthesis of a substituted Arg-containing peptide [17], we believe
that this is the first description of the iterative formation of Arg-
containing peptides bearing amidine functionalization as the
point of diversification.
Although the significant number of synthetic routes into dis-
crete amidines have been reviewed elsewhere [26,27], amidine-
based isologs of Arg have been prepared as discrete compounds
through a variety of different means, including various iterations
of standard Pinner syntheses of nitrile substrates [28,29], conden-
sation of alkyl acetimidates with the d-amine of appropriately
functionalized Orn precursors [30,31], and treatment of Orn
d-thioamides with ammonia [24]. Overwhelmingly, the final
product in these syntheses has been the discrete Arg isologs
themselves, without their incorporation into larger peptides.
Indeed, the vast majority of synthetic research in this arena
has involved the construction of discrete NOS inhibitory Arg
substrates. As such, this research effort signifies the first time
in which Arg isosteres bearing functionalized amidine architec-
ture have been incorporated into peptides constructed on the
solid support and represents a major step forward in the itera-
tive construction of widely diversified Arg isolog-containing
peptides.
Our prior efforts in this area involved practical approaches to-
ward the construction of Arg-containing peptides bearing diversifi-
cation at the guanidine nitrogen [17]. This methodology was
shown to be very effective toward the iterative synthesis of guani-
dinyl-substituted Arg peptides in a stepwise manner while on the
solid support. In this procedure, a resin-bound Orn residue was
Mtt deprotected while on the solid support followed by guanidiny-
lation via treatment with an excess of N-Pmc-N′-substituted
Materials and Methods
Materials
Pmc chloride was purchased from Omegachem (Saint-Romuald,
QC, Canada). Fmoc-Leu-Wang resin (0.47 mmol/g) and Fmoc-Orn
(Boc)-OH were purchased from Novabiochem (San Diego, CA,
USA). PEG-PS resin (0.21 mmol/g) was purchased from Peptides
Figure 1. (A) Previously studied synthetic route toward synthesis of on-resin Arg analogs from Orn precursors. (B) Similar proposed synthesis into anal-
ogous amidine-based isologs of Arg from Orn precursors.
J. Pept. Sci. 2012; 18: 30–36 Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

FLEMER JR. AND MADALENGOITIA
International (Louisville, KY, USA). 2-(7-Aza-1H-benzotriazole-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate HATU was pur-
chased from Oakwood Products (Jackson Hole, WY, USA). All other
standard Fmoc amino acids and O-benzotriazole-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate (HBTU) were purchased
from RS Synthesis (Louisville, KY, USA). DMF, HPLC-grade acetoni-
trile, and TFA were purchased from Fisher Scientific (Pittsburgh,
PA, USA). Mukaiyama’s reagent, trimethylsilyl trifluoromethanesul-
fonate (TMSOTf), and all other reagents were purchased from
Sigma-Aldrich (Milwaukee, WI, USA).
agent and Mukaiyama’s reagent (4 equiv ea. with regard to resin
loading) are dissolved in 2 ml 1 : 1 DCM/DMF buffered to 0.2 M
NMM and allowed to incubate for 5 min. This solution is then ap-
plied to the resin, and the slurry is allowed to agitate gently for
4 h. Following the incubation period, the resin is washed thor-
oughly with DMF and DCM. Cleavage of peptides from their
resins was accomplished through treatment of the resin with
94 : 5 TFA/TMSOTf for 2 h. Following filtration of the resin, the
cleavage supernatant was evaporated to one-tenth its original
volume by blowing air over it, followed by precipitation of the
crude peptide by the addition of cold anhydrous ether.
Peptide Syntheses
All peptides were manually synthesized on Wang resin (0.51 mmol/g)
on a 40mmol scale via Fmoc protocol. Double coupling using 1 : 3
HATU/HBTU activation was employed for peptide elongation. A
typical single coupling procedure is as follows: 20% piperidine/
DMF (2 Â 10 min); DMF wash (6Â 30 s); 3 equiv Fmoc amino acid
and 1 : 3 HATU/HBTU in 0.2M NMM/DMF (2 Â 30min); and DMF
washes (3Â 30 s). Following the last coupling and subsequent
Fmoc deprotection, the a-N-terminal amine was Boc protected
via incubation of the peptide resin with Boc2O (10 equiv) in 5 ml
0.1 M NMM in 1 : 1 DMF/DCM for 40 min.
Results and Discussion
Our retrofitted approach toward carrying out this solution-phase
chemistry on the solid support utilized the identical N-Pmc sub-
stituted thioamides as in our previous report but with a resin-
bound Orn side-chain playing the role of the amine partner. As
such, an assortment of N-Pmc thioamides was constructed as
previously described [32] to explore the scope and limitations
of this methodology (Table 1), using a variation on the method
of Walter [33], which we had further optimized. Briefly, Pmc iso-
thiocyanate 1 was treated with a variety of Grignard reagents
to afford a series of N-Pmc-substituted thioamides 2a–2i. As illus-
trated in Table 1, a variety of substitution patterns were chosen,
which encompassed a diversity of alkyl and aryl frameworks.
High Performance Liquid Chromatography
HPLC analysis was carried out on a Shimadzu analytical HPLC sys-
tem with LC-10AD pumps, SPD-10A UV-vis detector, and SCL-10A
controller using a SymmetryW C18 À5 mm column from Waters
(4.6 Â 150 mm). Aqueous and organic phases were 0.1% TFA in
water (buffer A) and 0.1% TFA in HPLC-grade acetonitrile (buffer
B), respectively. Beginning with 100% buffer A, a 1.4 ml/min gra-
dient elution increase of 1% buffer B/min for 50 min was used for
all peptide chromatograms. Peptides were detected at both 214
and 254 nm. Preparative HPLC purification was carried out on a
Shimadzu preparatory HPLC system utilizing LC-8A pumps, an
SPD-10A UV-vis detector, and an SCL-10A controller. A Waters
Symmetry Prep C18 preparatory column (7 mm pore size,
1900 Â 150 mm) was utilized in these separations. Beginning with
100% buffer A, a 17 ml/min gradient elution increase of 1% buffer
B/min for 50 min was used for all preparative chromatograms.
Table 1. Substitution pattern on N-Pmc thioamides 2 used in this study.
Entry
R
2a
2b
2c
2d
2e
Mass Spectrometry
MALDI–TOF MS spectra were obtained on a Voyager DE Pro instru-
ment under positive ionization and in reflectron mode. All samples
were run using a matrix of 10 mg/ml 2,5-dihydroxybenzoic acid,
vacuum-dried from a solution of 1 : 1 H2O/ACN buffered to 0.1% TFA.
2f
Synthesis of N-Pmc-N′-Substituted Thioamide Transfer
Agents
2g
All N-Pmc-substituted thioamides 2a–2i were prepared accord-
ing to a previously established procedure [32].
2h
2i
General Solid-Phase Amidinylation Protocol
Resin-bound peptide of 40 mmol containing a d-N-Mtt Orn resi-
due is swelled in DCM followed by sequential treatments with
1% TFA/DCM (5 min each) until all traces of yellow color is absent
from supernatant. Resin is then washed with DCM (2 Â 2 ml), DMF
(2 Â 2 ml), 0.4 M NMM/DMF (2 Â 2 ml), and DMF (2 Â 2 ml). In a
separate reaction vessel, N-Pmc-N′-substituted thioamide transfer
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SOLID-PHASE SYNTHESIS OF AMIDINE-TEMPLATED ARGININES
As we were attempting to synthesize amidines in analogy to
our previous reports [32], we needed to carefully reevaluate our
synthetic strategy in order to ensure its applicability to conditions
of SPPS. As such, we decided to assay the solid phase applicabil-
ity of this approach over multiple thiophile candidates in order to
optimize the reaction conditions toward SPPS. In our previous
work, we had found that the EDCI thiophile, which had worked
so efficiently at mediating analogous guanidinylation reactions,
was virtually ineffective when applied to analogous amidinyla-
tion protocol. We subsequently found that HATU, PyBOP, and 2-
chloro-1-methyl-pyridinium iodide (Mukaiyama’s reagent) acted
as superb thiophile mediators of this reaction in solution. As such,
a comprehensive assay was designed in which these four candi-
dates were tested as to their ability to carry out solid-phase ami-
dinylation between a peptide test sequence bearing an Orn
amino side chain and two N-Pmc-substituted thioamides, bearing
representative alkyl and aryl groups (Figure 2).
A single-Arg-containing corazonin test peptide sequence [34]
was chosen as a representative model system upon which to
carry out the thiophile assay. Initially prepared on Wang resin
as its Mtt-protected Orn analog, Orn d-N Mtt deprotection was
carried out via treatment of the resin with 1% TFA/DCM. Follow-
ing this, separate aliquots of the resin were incubated for 4 h with
a fourfold excess of two N-Pmc-substituted thioamides along
It was found that solid-supported amidinylation efficiency
tracked similarly to that of previously studied solution-based con-
densations. EDCI was found to be completely ineffective as a con-
densation reagent and was immediately excluded as a candidate
as determined by its complete inability to mediate any on-resin
amidinylation. PyBOP, while offering acceptable amidinylation
abilities for alkyl-substituted thioamide 2a, was found to be
completely ineffective at mediating the condensation of phe-
nyl-substituted thioamide 2f as shown by recovery of unreacted
Orn-corazonin peptide. This limitation was observed previously
in solution-phase amidinylations, and for this reason PyBOP
was similarly disregarded as a thiophile candidtate. Gratifyingly,
both HATU and Mukaiyama’s reagent mediated both alkyl and
aryl amidinylations completely and efficiently [32]. Although these
two candidates emerged as optimal thiophiles for peptide-based
amidinylations, Mukaiyama’s reagent was chosen as the most
favorable candidate for further study because of the known pro-
pensity of HATU to act as its own undesired guanidinylation
‘capping’ agent when used in excess over resin-bound primary
amines [35].
Having identified the optimal thiophile for further study of
amidinylation reactions on the solid support, the scope and lim-
itations of the methodology were then probed on a different test
peptide system. An angiotensin-based peptide sequence [36]
bearing a single Mtt-protected Orn residue at the 2-position
was utilized to probe the on-resin amidinylation with all thioa-
mides given in Table 1 (Figure 3). The Mtt deprotection and ami-
dinylation reaction were carried out identically as before using
Mukaiyama’s reagent as thiophile, and by following peptide
cleavage, the crude peptides were analyzed by analytical HPLC
and MALDI.
with
a different thiophile reagent noted earlier. Methyl-
substituted 2a and phenyl-substituted 2f were chosen as repre-
sentative thioamides because of their minimal substitution pat-
tern, offering the least steric hindrance toward condensation and
allowing the process to be mediated solely by choice of thiophile
while minimizing other potential mitigating steric factors. Final
deprotection/cleavage of the test peptide was carried out using
a 5% TMSOTf/TFA cocktail. We had earlier discovered that, in
contrast to the facile removal of Pmc protection from a guanidine
framework using TFA and minimal scavengers, similar Pmc
deprotection of analogous amidine systems could never be driven
to completion [32]. There was therefore an absolute requirement
for TMSOTf as an added active component to the deprotection
mixture. Treatment of the peptide resins with this cocktail for 2 h
allowed for isolation of the crude Corazonin analogs, which were
submitted to HPLC and MALDI analyses.
The results of the amidinylation substituent survey offered
some interesting insight into the utility of this methodology
when applied to the solid phase. Overall, it was evident that the
majority of the amidinylations were successful, and these exam-
ples afforded crude isolates which exhibited reasonable yield
and purity in their crude form, each showing a major peak attrib-
utable to the desired peptide product (see chromatograms,
Figure 3). The amidinylation profile illustrated a wide tolerance
for substitution pattern on the thioamide partner, allowing a
Figure 2. Selective amidination assay on a resin-bound corazonin (ZTFQYSRGWN) peptide template to afford acetamidyl and benzamidyl Arg analog
from an Orn-substituted precursor. HATU and Mukaiyama’s reagent were found to be the most effective thiophiles in these initial on-resin test systems,
with Mukaiyama’s reagent ultimately selected as the most optimal candidate.
J. Pept. Sci. 2012; 18: 30–36 Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

FLEMER JR. AND MADALENGOITIA
Figure 3. Amidination series on NRVYIHPFHL angiotensin template using Mukaiyama’s reagent as the optimized choice of thiophile. A representative
series of alkyl-, aryl-, and heterocycle-substituted amidine systems were prepared as Arg headgroup architectural mimics. Crude HPLC chromatograms
of each diversified peptide isolate illustrates the generally good purity of peptide product from this methodology.
diverse range of alkyl, aryl, and heteroaryl architecture to be
co-opted into the final products.
amidinylated product, affording only the Orn precursor peptide
upon cleavage. This was indicative of a reaction pathway which
precluded amidinylation. It was clear that an alternate mechanis-
tic route was being undertaken as evidenced by the reaction so-
lution taking on a deep purple coloration immediately following
addition of Mukaiyama’s reagent to the dissolved 2e thioamide.
None of the other amidinylation reactions took on this coloration
at any time during their incubation period. This result was per-
haps not unexpected as the identical observations were recorded
Perhaps more revealing than the solid-phase amidinylation
reactions that were successful, the instances in which the con-
densations were ineffective offered important information as to
the limitations of the process and provided the first examples
of instances in which the solid-phase reaction results differed in
scope from the previously studied solution phase condensations.
Benzyl-substituted thioamide 2e gave none of the corresponding
Figure 4. Amidine-diversified cGMP-dependent PKG inhibitor (RTGRRNAI) analog illustrating the potential for iterative installation of amidine diversity
while peptide is being built on solid support.
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SOLID-PHASE SYNTHESIS OF AMIDINE-TEMPLATED ARGININES
in previously reported solution phase amidinylation attempts us-
ing 2e as the thioamide condensation partner [32]. Indeed, it was
shown that this coloration phenomenon was independent of the
presence or absence of added amine partner to the mixture,
which would explain the unreactivity of the resin-bound Orn
amine and its subsequent isolation as the lone identifiable prod-
uct upon peptide cleavage and workup.
The failure of thioamide 2e to condense with the resin-bound
Orn did illustrate an important limitation to the general method-
ology. Another limitation was highlighted by the failure of thioa-
mide 2h to fully condense with the test peptide amine to yield
the expected p-trifluoromethylphenyl-substituted amidinyl Arg
angiotensin. Analytical HPLC of the crude peptide mixture
showed two approximately equal peaks, one of which corre-
sponded to the desired peptide but another corresponded to
an unknown M-75 Da component following careful MALDI analy-
sis of the two peaks. Because the two components’ retention
times were less than 0.5 min apart, purification of the desired
product would have been virtually impossible. Further, one of
the highlights of this methodology is its ability to produce crude
peptide isolates in which the desired product is of reasonable pu-
rity. As a result of the failure of thioamides 2e and 2h to produce
acceptable pure product profiles, these were excluded from entry
into Figure 3.
Having shown the ability of this amidinylation methodology to
be applied directly toward the construction of Arg isosteres on
the solid support, we desired to illustrate the applicability of this
process toward the construction of multiple Arg-containing pep-
tides. Moreover, we desired to carry out a model synthesis using
a stepwise approach similarly to the iterative installation of sub-
stituted Arg residues into model peptides as previously described
by us [17]. As a final testament to the utility of this methodology,
a cGMP-dependent PKG inhibitor peptide template bearing
multiple Arg residues was chosen [5] to carry out the iterative
installation of sequential amidinations (Figure 4). The separate
introduction of three different amidine transfer agents (2a, 2c,
and 2f) to Orn side chains as the peptide was being built out on
the solid support was achieved in high crude purity. Orthogonally
disposed Fmoc/Mtt-protected Orn residues were coupled and
selectively side chain deprotected as previously described.
Amidinylation was then carried out in a routine fashion before
continuing the amide backbone extension of the peptide. This
iterative approach was ultimately repeated twice more within the
sequence to afford the triply amidinylated cGMP-dependent PKG
inhibitor analog in >90% purity.
The optimized conditions allowed for the facile and routine
condensation of a wide diversity of N-Pmc thioamides with
the resin-bound Orn side chain amine on various model peptide
systems. It was found that this methodology tolerates a
wide spectrum of thioamide architecture, giving rise to many
different alkyl-substituted, aryl-substituted, and heterocycle-
substituted Arg isosteres bearing amidine core headgroups.
This process is further amenable to the sequential construction
of multiple Arg-containing isosteric systems through the ability
to build out the peptide chain concurrent with the installation
of amidine headgroups in a sequential iterative fashion. Given
the ease with which these amidination reactions are able to
be carried out on-resin, this methodology has high synthetic
potential with prospective applications toward highly diversi-
fied peptide library systems and finely tuned Arg-containing
peptide targets.
Acknowledgements
Financial support for this project was provided by CHE-041283
and instrumentation grant CHE-0821501 from the NSF.
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Choi-Sledeski YM, Green D, Cha D, Spada AP, Cheney DL, Mason JS,
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Conclusion
Similar to our previously published solution amidinylation
accounts, the results of the initial thiophile assay on solid-
supported Orn d-amine partners upheld the previous discovery
that, although EDCI may be the coupling agent of choice for
guanidinylation reactions of resin-bound amines and thiourea
transfer agents, it was a very poor choice for the analogous ami-
dination reactions of amines and thioamide transfer agents. It
was shown that three thiophiles (PyBOP, HATU, and Mukaiyama’s
reagent) outperformed EDCI in the mediation of these solution-
condensation reactions. It was further discovered that only
Mukaiyama’s reagent and HATU were able to afford complete ami-
dination between most thioamide transfer agents studied and a
resin-bound amine.
J. Pept. Sci. 2012; 18: 30–36 Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jpepsci

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