Solid-phase and solution-phase syntheses of oligomeric guanidines bearing peptide side chains

Article abstract of DOI:10.1021/jo051226t

Synthetic strategies for preparing N,N′-bridged oligomeric guanidines bearing peptide side chains both on solid support and in solution are presented. Monomers are prepared from common α-amino acids and therefore contain conventionally protected peptide s

Full text of DOI:10.1021/jo051226t

Solid-Phase and Solution-Phase Syntheses of Oligomeric
Guanidines Bearing Peptide Side Chains
Zhongsheng Zhang and Erkang Fan*
Department of Biochemistry, Biomolecular Structure Center, University of Washington,
Seattle, Washington 98195
erkang@u.washington.edu
Received June 15, 2005
Synthetic strategies for preparing N,N′-bridged oligomeric guanidines bearing peptide side chains
both on solid support and in solution are presented. Monomers are prepared from common R-amino
acids and therefore contain conventionally protected peptide side chains. The side chains include
alkyl, aromatic, hydroxyl, amino, carboxylic acid, and amide functional groups. Oligomer elongation
utilizes acid-sensitive sulfonyl activated thiourea through the formation of carbodiimide intermedi-
ate. With proper preparation of monomers, synthesis of oligomer can be performed in two directions
(equivalent to N to C terminal or C to N terminal in a peptide sequence) with excellent efficiency.
Introduction
Peptides are an important class of compounds for
biomedical studies, such as inhibitors or agonists of
protein function. Synthetic peptide libraries are also the
first examples used to demonstrate the power of combi-
natorial chemistry.1-3 However, for therapeutic develop-
ment, peptides are often unfavorable. One main issue is
the generally poor bioavailability of peptides due to facile
degradation by enzymes and/or inadequate cell mem-
brane permeability. To overcome the drawbacks of pep-
tides, the development of peptidomimetics based on
oligomers with alternative unnatural backbones while
preserving the common side chains has attracted much
attention in recent years (Figure 1). Many different types
of unnatural bio-oligomers have been studied in connec-
tion with peptidomimetics and are the subjects of recent
reviews.4-9 Examples of representative unnatural bio-
FIGURE 1. Oligomeric structures of a peptide, some un-
natural bio-oligomers, and an oligomeric guanidine.
oligomers include â-peptides,10 peptoids,11 ureapeptoids,12
oligocarbamates,13 oligoureas,14-17 azatides,18 and aza-â3-
* To whom correspondence should be addressed. Phone: (206)-685-
7048. Fax: (206)-685-7002.
(1) Geysen, H. M.; Meloen, R. H.; Barteling, S. J. Proc. Natl. Acad.
Sci. U.S.A. 1984, 81, 3998-4002.
(2) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmier-
ski, W. M.; Knapp, R. J. Nature 1991, 354, 82-84.
(3) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley,
C. T.; Cuervo, J. H. Nature 1991, 354, 84-86.
(4) Soth, M. J.; Nowick, J. S. Curr. Opin. Chem. Biol. 1997, 1, 120-
129.
(5) Schneider, S. E.; Anslyn, E. V. In Advances in Supramolecular
Chemistry; Gokel, G. W., Ed.; 1999; Vol. 5, pp 55-120.
10.1021/jo051226t CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/01/2005
J. Org. Chem. 2005, 70, 8801-8810
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Zhang and Fan
peptides.19 However, there are limitations to the current
unnatural oligomeric backbones. For example, if a highly
hydrophobic peptide sequence is to be mimicked, com-
pounds built on most of the current unnatural backbones
will likely suffer from very poor aqueous solubility and/
or severe random aggregation in aqueous solution, which
is of concern for library screening of inhibitors.20 We
sought to synthesize unnatural oligomers built on guani-
dine backbones (Figure 1), which may offer a significant
advantage in terms of aqueous solubility due to the high
pKa of alkyl- or aryl-substituted guanidine units.21 In
addition, because of the charged guanidine group under
physiological conditions, oligomeric guanidines may pos-
sess unique properties for application in biomedical
research. For example, these oligomers may be used in
gene therapy to deliver oligonuceotides with negatively
charged backbones. In designing protein ligands, the
oligomer backbone may interact with target proteins by
electrostatic interactions and/or hydrogen bonds. Oligo-
meric guanidines have also been shown to be resistant
to certain enzyme degradations.22 All of these properties
make oligomeric guanidines a very important comple-
ment to the existing unnatural bio-oligomers.
substantial limitations on yields, difficulties in deprotec-
tion, or limited choices of side chains.27-29 Despite those
reports, none of the above examples include guanidine
oligomers that contain common peptide side chains. In
addition, conditions similar to those that work well for
replacing the phosphate backbones in oligonucleotides
with guanidines did not produce desirable results in the
synthesis of peptidomimetics on guanidine backbones.28
In a previous communication, we have successfully
demonstrated the first efficient solid-phase synthesis of
N,N′-bridged oligomeric guanidines bearing simple pep-
tide like side chains (Figure 2).30 Our method utilized Pbf-
activated thiourea for oligomer elongation and azide as
a mask for the backbone amine (Pbf: 2,2,4,6,7-penta-
methyldihydrobenzofuran-5-sulfonyl). The direction of
synthesis (direction A in Figure 2) corresponds to the
N-terminal to C-terminal direction in a peptide sequence.
In this paper, we present detailed synthetic studies of
two new sequences of oligomeric guanidine (1 and 2 in
Figure 2) that expand the side-chain functional groups
to include neutral hydrophilic, acidic, basic, as well as
secondary amine groups. In addition, the preparation of
monomers has been improved. The syntheses of 1 and 2
are accomplished both on solid support and in solution,
but with different directions for oligomer elongation
(direction A for 1 and direction B for 2, Figure 2).
Synthesis direction B is important for the successful
preparation of 2 that contains a secondary-amine mono-
mer derived from proline. The advantages and limitations
in monomer and oligomer syntheses are discussed.
In contrast to a large number of synthetic routes for
variously substituted single guanidine units, synthetic
methods for oligomeric guanidines are of very limited
choice. For example, the N,N′-disubstituted guanidine
moiety has been incorporated in oligomeric form to
replace the diphosphate backbone in various forms of
oligonucleotides.23-26 Oligomeric guanidine synthesis has
also been demonstrated by two other groups, but with
Results and Discussion
(6) Kirshenbaum, K.; Zuckermann, R. N.; Dill, K. A. Curr. Opin.
Struct. Biol. 1999, 9, 530-535.
Direction A, Synthesis of 1. The overall synthetic
strategy is identical to that described in our previous
communication for solid-phase synthesis,30 with some
modifications in the procedure to make it more efficient
as will be discussed below. The required monomers for
the synthesis of 1 were prepared as shown in Scheme 1.
The Fmoc-protected amino alcohols 4 were from com-
mercial sources or converted from the corresponding
Fmoc-protected amino acids 3 by reacting first with
isobutyl chloroformate, followed by reduction with
NaBH4.31 Fmoc-protected amino alcohols were trans-
formed into azide compounds 5 under Mitsunobu condi-
tions in good yields as described by Boeijen et al.17 The
Fmoc groups in 5 were removed, and the resulting free
amino groups were reacted with Pbf-NCS32 to form
monomers 6. This route is improved from our previously
reported method of monomer synthesis where the forma-
tion of Pbf-thiourea was accomplished in two steps
involving the use of strong nucleophilic conditions (an-
(7) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. Rev. 2001,
101, 3219-3232.
(8) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S.
Chem. Rev. 2001, 101, 3893-4011.
(9) Oh, K.; Jeong, K. S.; Moore, J. S. Nature 2001, 414, 889-893.
(10) Seebach, D.; Overhand, M.; Kuhnle, F. N. M.; Martinoni, B.;
Oberer, L.; Hommel, U.; Widmer, H. Helv. Chim. Acta 1996, 79, 913-
941.
(11) Zuckermann, R. N.; Kerr, J. M.; Kent, S. B. H.; Moos, W. H. J.
Am. Chem. Soc. 1992, 114, 10646-10647.
(12) Kruijtzer, J. A. W.; Lefeber, D. J.; Liskamp, R. M. J. Tetrahe-
dron Lett. 1997, 38, 5335-5338.
(13) Cho, C. Y.; Moran, E. J.; Cherry, S. R.; Stephans, J. C.; Fodor,
S. P. A.; Adams, C. L.; Sundaram, A.; Jacobs, J. W.; Schultz, P. G.
Science 1993, 261, 1303-1305.
(14) Burgess, K.; Linthicum, D. S.; Shin, H. W. Angew. Chem., Int.
Ed. Engl. 1995, 34, 907-909.
(15) Kim, J. M.; Bi, Y. Z.; Paikoff, S. J.; Schultz, P. G. Tetrahedron
Lett. 1996, 37, 5305-5308.
(16) Burgess, K.; Ibarzo, J.; Linthicum, D. S.; Russell, D. H.; Shin,
H.; Shitangkoon, A.; Totani, R.; Zhang, A. J. J. Am. Chem. Soc. 1997,
119, 1556-1564.
(17) Boeijen, A.; van Ameijde, J.; Liskamp, R. M. J. J. Org. Chem.
2001, 66, 8454-8462.
(18) Han, H.; Janda, K. D. J. Am. Chem. Soc. 1996, 118, 2539-
2544.
(19) Cheguillaume, A.; Salaun, A.; Sinbandhit, S.; Potel, M.; Gall,
P.; Baudy-Floc’h, M.; Le Grel, P. J. Org. Chem. 2001, 66, 4923-4929.
(20) Seidler, J.; McGovern, S. L.; Doman, T. N.; Shoichet, B. K. J.
Med. Chem. 2003, 46, 4477-4486.
(26) Linkletter, B. A.; Bruice, T. C. Bioorg. Med. Chem. 2000, 8,
1893-1901.
(27) Tanatani, A.; Kagechika, H.; Azumaya, I.; Fukutomi, R.; Ito,
Y.; Yamaguchi, K.; Shudo, K. Tetrahedron Lett. 1997, 38, 4425-4428.
(28) Schneider, S. E.; Bishop, P. A.; Salazar, M. A.; Bishop, O. A.;
Anslyn, E. V. Tetrahedron 1998, 54, 15063-15086.
(29) Tanatani, A.; Yamaguchi, K.; Azumaya, I.; Fukutomi, R.; Shudo,
K.; Kagechika, H. J. Am. Chem. Soc. 1998, 120, 6433-6442.
(30) Zhang, Z.; Carter, T.; Fan, E. Tetrahedron Lett. 2003, 44, 3063-
3066.
(21) Jenks, W. P.; Regenstein, J. In Handbook of Biochemistry.
Selected Data for Molecular Biology; Sober, H. A., Ed.; CRC Press:
Cleveland, 1985.
(22) Barawkar, D. A.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 11047-11052.
(23) Dempcy, R. O.; Luo, J.; Bruice, T. C. Proc. Natl. Acad. Sci.
U.S.A. 1996, 93, 4326-4330.
(24) Linkletter, B. A.; Bruice, T. C. Bioorg. Med. Chem. Lett. 1998,
8, 1285-1290.
(31) Rodriguez, M.; Llinares, M.; Doulut, S.; Heitz, A.; Martinez, J.
Tetrahedron Lett. 1991, 32, 923-926.
(32) Zhang, Z.; Pickens, J. C.; Hol, W. G. J.; Fan, E. Org. Lett. 2004,
6, 1377-1380.
(25) Linkletter, B. A.; Szabo, I. E.; Bruice, T. C. J. Am. Chem. Soc.
1999, 121, 3888-3896.
8802 J. Org. Chem., Vol. 70, No. 22, 2005

Syntheses of Oligomeric Guanidines
FIGURE 2. Previously reported oligomeric guanidine bearing peptide side chains and compounds (1 and 2) studied in this paper.
Synthesis directions A (equivalent to N to C terminal in a peptide) and B (equivalent to C to N terminal in a peptide) are defined
as shown.
SCHEME 1. Preparation of Monomer 6 for Synthesis of Oligomers in Direction A
ionic form of Pbf-NH2).30 During this transformation, the
protocol for Fmoc removal is also worth noting. In
conventional solid-phase synthesis, the Fmoc group can
be removed by 20% piperidine in DMF. However, using
similar conditions for monomer synthesis in solution was
inconvenient because the excess piperidine required to
deprotect the Fmoc group was itself difficult to be
separated from the resulting amine product. In large-
scale monomer synthesis, the same problem was found
for TBAF in THF, which we used in our previous report.30
Application of resin-bound piperidine to remove the Fmoc
group is costly and also inefficient in our hands. We found
that DBU was a good choice to remove Fmoc in solution-
phase synthesis,33,34 especially using ethyl acetate as the
solvent. Only 1.5 equiv of DBU was required, and Fmoc
removal was finished in minutes. The small excess of
DBU can be washed away by water. The dibenzofulvene
released from the Fmoc group stayed in the organic
solution. The resulting amine was used directly in the
preparation of monomer 6 without further purification.
Apparently, the dibenzofulvene did not form a significant
amount of adduct with the liberated amine to interfere
with the reaction and was easily separated from the final
products 6.
Synthesis of oligomer 1 in direction A either on solid
support or in the solution phase is shown in Scheme 2.
In the solid-phase protocol, Rink amide resin was reacted
with monomer 6a to form the first guanidine unit, which
is different from our earlier investigation that used the
modified Wang resin to yield an amine in the final
product. All of the guanidinylation steps on the solid
support employed 5 equiv of monomers 6 in anhydrous
(33) Chang, C.-D.; Waki, M.; Ahmad, M.; Meienhofer, J.; Lundell,
E. O.; Huag, J. D. Int. J. Pept. Protein Res. 1980, 15, 59.
(34) SheppeckII, J. E.; Kar, H.; Hong, H. Tetrahedron Lett. 2000,
41, 5329-5333.
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Zhang and Fan
SCHEME 2. Synthesis of 1 in Direction A on Solid Support or in Solution
CH2Cl2 in the presence of EDC. The reaction was allowed
to proceed at room temperature for 12 h. After each
elongation, the azido group at the terminal was reduced
to amine using the SnCl2/PhSH/DIPEA complex in
anhydrous THF.35-37 Cycles of guanidinylation and azide
reduction were performed. Cleavage of the final product
in salt form was achieved using TFA with TIS (triiso-
propylsilane) and water as scavengers. The overall
isolated yield of 1 as TFA salt after HPLC purification
was 66%, representing an average of >90% yield for each
oligomer elongation cycle.
The anchor for the solution-phase synthesis of 1 was
2,4-dimethoxybenzylamine (DMB-NH2), which provided
an amino group for guanidinylation with 6a. The DMB
group can be removed in the final step with other acid-
labile protecting groups when exposed to TFA as shown
in Scheme 2. The guanidinylated intermediate after each
elongation step was purified by silica gel chromatogra-
phy, eluting with ethyl acetate and hexanes. Even though
such purification required extra work when compared to
the solid phase route, the advantage is that only 1 equiv
of monomer 6 was required in solution-phase synthesis
and that the total reaction time was also shorter (4 h
reaction for each guanidinylation step). The azido group
was also reduced with the SnCl2/PhSH/DIPEA complex
in anhydrous THF, and the excess reducing agent was
removed by washing with 2 M NaOH.37 Oligomer 1 in
fully protected form (Scheme 2) prior to TFA treatment
was obtained in 60% overall isolated yield, which is
similar to the solid-phase protocol for the final product
despite the need for extra purification of intermediates.
The final product 1 as TFA salt was obtained by cleaving
all protecting groups in one step with TFA/TIS/H2O,
followed by ether wash. The yield was 91% from protected
1 (HPLC purity 97%).
mation of the thiourea moiety into carbodiimide.28 In the
preparation of monomers of Pbf-thiourea 6, as shown in
Scheme 1, compounds derived from most common amino
acids can provide a primary amine after Fmoc removal.
The subsequent reaction with Pbf-NCS will lead to Pbf-
thiourea that can later be transformed into carbodiimide
by EDC during oligomer synthesis. However, when
proline is used for monomer synthesis, removal of Fmoc
will produce a secondary amine, which cannot later be
used for guanidine synthesis through activated thiourea
followed by carbodiimide formation.38 To develop a syn-
thetic route compatible to the monomer derived from
proline, one may envision that intermediate 5 (Scheme
1) can provide a primary amine after azide reduction.
This primary amine can then serve to prepare Pbf-
thiourea and be used in oligomer synthesis. In this
strategy, the amino group used for Pbf-thiourea forma-
tion is derived from the carboxyl end of the starting
amino acid, which is “reversed” as compared to monomers
6. As a consequence, synthesis of oligomers compatible
to proline-based monomer will elongate from a different
direction (direction B in Figure 2).
If the azido function in compound 5 is turned into Pbf-
thiourea, we now face the question of whether to keep
or replace the Fmoc protection group of the remaining
amine. The reason is that during oligomer elongation,
the nucleophilic nitrogen atom of the FmocNH moiety
may react with the formed carbodiimide intermediate to
produce undesirable cyclic product.28,39 While this com-
peting side reaction is not a concern for proline-based
monomer due to Fmoc-protected secondary amine, other
linear monomers may suffer. To probe the protection
needed for the amino group, we prepared ethylenedi-
amine-based monomers (glycine equivalent) bearing dif-
ferent protecting groups and tested guanidinylation
reactions on solid support (Scheme 3).
Direction B, Synthesis of 2. The commonly accepted
mechanism for the activation of thiourea derivatives with
EDC during guanidine synthesis involves the transfor-
We first tested the Fmoc-protected monomer 8, which
was synthesized by reaction of mono-Fmoc-protected
ethylenediamine with Pbf-NCS. The trityl resin-bound
(35) Kim, J.-M.; Bi, Y.; Paikoff, S. J.; Schultz, P. G. Tetrahedron
Lett. 1996, 37, 5305-5308.
(36) Kick, E. K.; Ellman, J. A. J. Med. Chem. 1995, 38, 1427-1430.
(37) Bartra, M.; Romea, P.; Urpi, F.; Vilarrasa, J. Tetrahedron 1990,
46, 587-594.
(38) Linton, B. R.; Carr, A. J.; Orner, B. P.; Hamilton, A. D. J. Org.
Chem. 2000, 65, 1566-1568.
(39) Evindar, G.; Batey, R. A. Org. Lett. 2003, 5, 1201-1204.
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Syntheses of Oligomeric Guanidines
SCHEME 3. Probing Monomers with Different
N-Protections during Guanidinylation
SCHEME 4. Confirmation of Cyclic Guanidine
Products
protected amine. We hoped that the Dde will also reduce
the intramolecular cyclic guanidine formation. The syn-
thesis of Pbf-thiourea-activated Dde-protected ethylene-
diamine 12 is shown in Scheme 3. The mono-Boc pro-
tected ethylenediamine was reacted with DdeOH to form
Boc- and Dde-doubly protected ethylenediamine 10. After
Boc removal, reaction with Pbf-NCS gave the desired
monomer 12 for testing. During the guanidinylation test
with trityl-resin bound 1,3-diaminopropane (Scheme 3),
it was found that no cyclic guanidine was formed in the
reaction solution. However, the Dde migration to the
resin-bound 1,3-diaminopropane was a severe problem,
which masked ∼86% of the amino group to be guanidi-
nylated. It is known that Dde could undergo both intra-
and intermolecular N to N migration in peptide synthe-
sis, resulting in the scrambling of the lysine group within
a peptide chain.41 However, it was unexpected that the
intermolecular migration was so severe in the solid-phase
guanidinylation test. Usually, such migration in peptide
synthesis can be overcome by replacing the Dde with the
more steric ivDde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-
ylidene)-3-methylbutyl) group.42 Therefore, we prepared
ivDde-protected monomer 13 as shown in Scheme 3. In
the guanidinylation test, no ivDde migration was found.
Unfortunately, intramolecular cyclic guanidine formation
of 19 was serious, and only 8% of guanidinylation
occurred. It is plausible that, compared to Dde, the much
higher steric strain in ivDde could favor the intramo-
lecular cyclization for releasing the strain. The cyclization
product 19 was found as two isomers in equilibrium.
LC-MS analysis showed two peaks in the ratio of 4:1
with identical mass. These two isomers can be separated
by preparative reverse phase HPLC and can also rees-
tablish equilibrium with each other in solution. To
confirm that 19 is ivDde-protected cyclic guanidine, the
mixture of isomers 19 was treated with 2% hydrazine in
DMF to remove the ivDde. N-Pbf cyclic guanidine 20 was
formed as shown in Scheme 4. Compound 20 thus
prepared is identical to the sample obtained from 15 by
removal of the Fmoc protecting group.
1,3-diaminopropane was used to test the guanidinylation
reaction. The guanidinylation reaction was carried out
with 5 equiv of 8 and EDC in CH2Cl2 at room tempera-
ture overnight. As expected, in the reaction solution, the
majority (>90%) of 8 was cyclized into 15. Guanidinyla-
tion only took place on 29% of resin-bound propylenedi-
amine (Scheme 3), while the rest of the resin-bound
amine remained unchanged (see the Experimental Sec-
tion).
We then chose 1-(4,4-dimethyl-2,6-dioxocyclohex-1-
ylidene)ethyl (Dde) to replace Fmoc as the amino protect-
ing group. The Dde group is widely used in solid-phase
peptide synthesis to orthogonally protect primary amine
and can be easily removed with 2% hydrazine in DMF.40
Both the vinylogous amide moiety and the steric hin-
drance reduce the nucleophilic property of the Dde-
Given the difficulties in using Fmoc, Dde, or ivDde for
the protection of backbone primary amines during guanid-
inylation reaction, we decided to prepare monomers
similar to those used in direction A synthesis where an
azide group is used as amine mask. This requires us to
convert the Fmoc protected amino group to an azido
group in the monomer. Fortunately, direct conversion of
an amine to azide using triflyl azide is possible, and this
transformation can be achieved efficiently and consis-
tently with Cu2+ catalysis, as demonstrated initially in
carbohydrate chemistry.43 This Cu2+-catalyzed conversion
(41) Augustyns, K.; Kraas, W.; Jung, G. J. Pept. Res. 1998, 51, 127-
133.
(42) Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft,
B. W.; Chan, W. C. Tetrahedron Lett. 1998, 39, 1603-1606.
(43) Alper, P. B.; Hung, S.-C.; Wong, C.-H. Tetrahedron Lett. 1996,
37, 6029-6032.
(40) Bloomberg, G. B.; Askin, D.; Gargaro, A. R.; Tanner, M. J. A.
Tetrahedron Lett. 1993, 34, 4709-4712.
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Zhang and Fan
SCHEME 5. Preparation of Monomers for Synthesis of Oligomers in Direction B
retains the stereochemistry of the amino group to be
transformed and has been adapted to peptide synthe-
sis.44,45 Based on this conversion, we successfully pre-
pared azide masked monomers for Direction B oligomer
synthesis as shown in Scheme 5. First, amination of
Fmoc-protected amino alcohol 4 with phthalimide pro-
ceeded smoothly under the Mitsunobu conditions as
reported by the Burgess group16 to give 21 in 83-94%
yield. After the Fmoc was removed by DBU in ethyl
acetate as performed for compounds 5, the resulting
amines from 21 were converted to azide using the
condition reported by the Wong group.43 The azido
compounds 22 were obtained in 72-88% yield when we
used 3-fold of triflyl azide in the reaction instead of the
2-fold as reported by the Wong group. Hydrazinolysis of
22 and reaction with Pbf-NCS gave the desired mono-
mers 23 in ∼90% yield. For proline-based monomer, the
Fmoc-protected amino alcohol 24 was first turned into
azide 25. Then the azide was reduced and converted to
Pbf-thiourea. Monomer 26 retained the Fmoc protecting
group.
To investigate the efficiency of monomer 26 in oligomer
synthesis, it was tested with a small amount of Fmoc-
protected Rink amide resin, and the Fmoc-removal/
guanidinylation cycle was performed four times. Cleavage
of the product from the resin gave crude 27 (shown in
Figure 3), which was subjected to LC-MS analysis. The
result (Figure 3) indicated that monomer 26 can ef-
ficiently afford the desired oligomer 27 with no major side
reactions.
With the monomers 23 and 26 in hand, synthesis of
oligomer 2 in direction B, as shown in Scheme 6, was
straightforward both on the solid support and in solution,
using conditions very similar to those in the direction A
synthesis of oligomer 1. The exceptions in conditions are
that after guanidinylation with the proline-based mono-
mer 26, the Fmoc protection was removed using either
piperidine in DMF (solid phase) or DBU in ethyl acetate
(solution phase). Again, similar to the direction A syn-
thesis of 1, high yield was achieved in either the solid-
phase or the solution-phase protocol of Direction B
synthesis. The final overall isolated yield of 2 as TFA salt
FIGURE 3. LC-MS analysis of crude oligomer 27 with four units of proline-based monomer 26. Top: LC trace monitored at 260
nm. Bottom: MS spectrum (m/z) of the main peak at 10.2 min, showing (M + H)+ of 27 at 740.2 and (M + 2H)2+ at 370.8.
8806 J. Org. Chem., Vol. 70, No. 22, 2005

Syntheses of Oligomeric Guanidines
SCHEME 6. Synthesis of 2 in Direction B on Solid Support or in Solution
Glutamate-Based Monomer 6a. This monomer was syn-
from solid phase synthesis was 60%, representing >90%
yield in each guanidinylation and deprotection cycle. In
solution synthesis, the yield of fully protected 2 prior to
TFA treatment was 56%, also indicating >90% yield in
each elongation step despite the required chromato-
graphic purification after each guanidinylation reaction.
thesized according to the general procedure on 1.6 mmol scale.
Yield: 714 mg (85%). Mp: 43 °C. Rf ) 0.29 (EtOAc/hexanes,
1/4). 1H NMR (500 MHz, CDCl3): δ ) 1.42 (s, 9H), 1.47 (s,
6H), 1.65-1.73 (m, 1H), 1.87-2.05 (m, 1H), 2.06-2.11 (m, 5H),
2.52 (s, 3H), 2.59 (s, 3H), 2.99 (s, 2H), 3.41-3.49 (m, 2H), 4.47-
4.54 (m, 1H), 7.77 (d, J ) 8.5 Hz, 1H), 8.46 (s, 1H). 13C NMR
(125 MHz, CDCl3): δ ) 12.4, 17.5, 19.2, 27.1, 28.0, 28.4, 31.3,
42.8, 53.5, 54.0, 80.7, 87.5, 118.8, 125.9, 126.6, 134.4, 140.0,
160.9, 171.5, 178.2. HRMS (ESI-TOF): m/z calcd 526.2152 (M
+ H)+, found 526.2160; calcd 548.1972 (M + Na)+, found
548.1969.
Conclusions
Two sequences of oligomeric guanidines bearing pep-
tide side chains (1 and 2) were synthesized efficiently
either on solid support or in solution. Monomers were
derived from common R-amino acids, containing a Pbf-
thiourea moiety for elongation of the guanidine backbone
and an azide moiety as the mask of backbone primary
amine. Oligomer chain elongation can be performed in
two opposite directions (Figure 2), one of which is
compatible to monomers containing backbone secondary
amines, such as the proline-based monomer. In the two
guanidine oligomers 1 and 2 reported here, the side
chains include most common peptide side chain func-
tionalities. The wide range of side chain compatibility will
be a significant advantage of our synthetic strategy for
the generation of chemical libraries of short oligomeric
guanidines, which may be implemented conveniently
using our solid-phase protocols.
Solid-Phase Synthesis of Oligomer 1. A 100 mg sample
of Rink amide MBHA resin (theoretical loading: 0.64 mmol/
g) in a polypropylene column was swelled in 5 mL of DMF for
30 min. The Fmoc group was removed by the treatment of
resin with 5 mL of 20% piperidine in DMF for 10 min twice.
Guanidinylation was performed with 5 equiv of monomer 6a
(168 mg, 0.32 mmol) in 4 mL of anhydrous CH2Cl2 in the
presence of DIPEA (0.111 mL, 0.64 mmol) and EDC (62 mg,
0.32 mmol). The reaction was allowed to proceed for 12 h at
rt. After being washed with CH2Cl2, the resin was capped with
acetic anhydride (0.061 mL, 0.64 mmol) and DIPEA (0.223 mL,
1.28 mmol) in 5 mL of CH2Cl2 for 20 min. After washing with
CH2Cl2 and THF, a fresh solution of SnCl2/PhSH/DIPEA (0.32
mmol:1.28 mmol:1.60 mmol or 61 mg:0.13 mL:0.28 mL) in
anhydrous THF (3 mL) was added at room temperature and
the mixture was rotated for 40 min. This reduction was
repeated once. The resin was then washed with 6 mL of THF
five times. The loading was determined to be 0.51 mmol/g by
monitoring Fmoc release after capping a small amount of resin
with FmocOSu overnight. The final yield of 1 was based on
this loading. The above monoguanidinylated resin was sub-
jected to four more cycles of guanidinylation with, respectively,
0.255 mmol of monomer 6b (145 mg), 6c (143 mg), 6d (116
mg), and 6e (177 mg) and azide reduction as described above
except that no reduction was performed for the last monomer
6e. The above resin-bound-protected 1 was cleaved by treat-
ment with 7 mL of TFA/TIS/H2O (94:3:3) overnight. After
solvent removal, the residue was washed with ether. The
residue was then dissolved in 0.1% TFA water and acetonitrile.
The product was purified by reversed-phase HPLC using a
ZOBAX extended-C18 column (Agilent) with 0.1% TFA water
and acetonitrile as solvents. Lyophilization gave 51 mg of
Experimental Section
Monomer 6 for Direction A Synthesis. General Pro-
cedure. Fmoc-protected amino azide 5 (2.0 mmol) was dis-
solved in ethyl acetate (80 mL), and DBU (0.457 mL, 3.0 mmol)
was added. The mixture was stirred at room temperature for
5-20 min until the Fmoc was completely removed as moni-
tored by TLC. The organic solution was washed with 60 mL
of H2O and saturated NaCl and then dried over anhydrous
Na2SO4. The solution was filtered, and Pbf-NCS (2.0 mmol)
was added dropwise at room temperature. After 2 h of stirring,
the mixture was purified by silica gel chromatography eluting
with EtOAc/hexanes, and monomer 6 was obtained as a white
solid.
1
product 1 as TFA salt (66%). H NMR (500 MHz, D2O): δ )
0.87 (d, J ) 6.5 Hz, 3H), 0.92 (d, J ) 6.5 Hz, 3H), 1.22-1.32
(m, 1H), 1.33-1.99 (m, 10H), 2.16-2.80 (m, 4H), 2.90-4.20
(m, 19H), 6.86 (d, J ) 8.5 Hz, 2H), 7.14 (d, J ) 8.5 Hz, 2H).
(44) Lundquist, J. T.; Pelletier, J. C. Org. Lett. 2001, 3, 781-783.
(45) Rijkers, D. T. S.; van Vugt, H. H. R.; Jacobs, H. J. F.; Liskamp,
R. M. J. Tetrahedron Lett. 2002, 43, 3657-3660.
J. Org. Chem, Vol. 70, No. 22, 2005 8807

Zhang and Fan
MS (ESI-ion trap): m/z 416.3 (M + 2H)2+, 831.2 (M + H)+,
945.1 (M + H + TFA)+.
UV (260 nm) intensities of the Fmoc moiety in the released
products corresponding to capped 1,3-diaminopropane and
resin-bound 14 were determined to be in a 2.4:1 ratio,
indicating the degree of guanidinylation at ∼29%. The com-
binations of filtration were subjected to silica gel chromatog-
raphy to obtain a white solid 15 in 94% yield (240 mg). Mp:
104 °C dec. Rf ) 0.49 (MeOH/CH2Cl2, 3%). 1H NMR (500 MHz,
CDCl3): δ ) 1.40 (s, 6H), 2.02 (s, 3H), 2.53 (s, 3H), 2.60 (s,
3H), 2.83 (s, 2H), 3.67 (d, J ) 8.3 Hz, 2H), 3.92 (d, J ) 8.3 Hz,
2H), 4.24 (t, J ) 7.5 Hz, 1H), 4.41 (d, J ) 7.5 Hz, 2H), 7.10 (t,
J ) 7.5 Hz, 2H), 7.35 (t, J ) 7.5 Hz, 2H), 7.54 (d, J ) 7.5 Hz,
2H), 7.66 (s, 1H), 7.72 (d, J ) 7.5 Hz, 2H). 13C NMR (125 MHz,
CDCl3): δ ) 12.4, 17.9, 19.3, 28.5, 39.7, 43.0, 43.7, 46.4, 66.3,
86.4, 117.5, 119.8, 124.7, 125.4, 127.0, 127.7, 131.4, 133.0,
139.0, 141.1, 143.3, 151.3, 152.9, 159.2. HRMS (ESI-TOF): m/z
calcd 560.2219 (M + H)+, found 560.2208.
Solution-Phase Synthesis of Oligomer 1. 2,4-Dimeth-
oxybenzylamine (0.035 mL, 0.225 mmol) and monomer 6a (118
mg, 0.225 mmol) were dissolved in 10 mL of anhydrous CH2-
Cl2. EDC (43 mg, 0.225 mmol) and DIPEA (0.08 mL, 0.45
mmol) in 5 mL of anhydrous CH2Cl2 were added. The solution
was stirred at rt for 4 h. After the solvent was removed, the
residue was purified by silica gel chromatography eluting with
EtOAc/hexanes to give the monoguanidinylated compound.
The monoguanidinylated compound was subjected to reduction
with a fresh solution of SnCl2/PhSH/DIPEA (0.45 mmol:1.8
mmol:2.25 mmol or 86 mg:0.18 mL:0.39 mL) in anhydrous
THF (4 mL). The clear yellow solution was stirred at rt for 1
h under N2 atmosphere. After the solvent was removed, the
residue was dissolved in 20 mL of CH2Cl2, washed with 20
mL of 2 N NaOH twice and 20 mL of saturated NaCl, and
dried over Na2SO4. The above reduced monoguanidinylated
compound was subjected to four more cycles of guanidinylation
with, respectively, 0.225 mmol of monomer 6b (128 mg), 6c
(126 mg), 6d (102 mg), and 6e (157 mg) and azide reduction
as described above except that no reduction was performed
for the last monomer 6e. After the solvent was removed, the
residue was purified by silica gel chromatography eluting with
3% methanol in CH2Cl2 to give 362 mg of protected 1 as a white
solid (60%). The protected 1 was treated with 10 mL of TFA/
TIS/H2O (94:3:3) overnight. After solvent removal, the residue
was washed with ether. The residue was then dissolved in
0.1% TFA water and acetonitrile and its purity checked by
LC-MS: purity 97% based on UV absorbance at 230 nm.
Lyophilization gave 192 mg of product 1 as TFA salt (91%).
N-[2-(Fmoc-amino)ethyl]-N′-Pbf-thiourea 8. Mono-Fmoc-
protected ethylenediamine hydrochloride 7 (610 mg, 1.92
mmol) was suspended in anhydrous CH2Cl2 (30 mL). DIPEA
(0.40 mL, 2.30 mmol) was added at room temperature. After
the solution became clear, Pbf-NCS (1.92 mmol) in 10 mL of
anhydrous CH2Cl2 was added dropwise. After the mixture was
stirred at room temperature for 2 h, the solvent was removed
under reduced pressure. The crude product was purified on
silica gel eluting with MeOH/CH2Cl2, and monomer 8 was
obtained as a white solid (968 mg, 85%). Mp: 88 °C dec. Rf )
0.58 (MeOH/CH2Cl2, 5%). 1H NMR (500 MHz, CDCl3): δ )
1.41 (s, 6H), 2.05 (s, 3H), 2.39 (s, 3H), 2.56 (s, 3H), 2.84 (s,
2H), 3.26 (m, 2H), 3.73 (m, 2H), 4.21 (m, 1H), 4.45 (d, J ) 6.5
Hz, 2H), 4.88 (m, 1H), 7.30-7.33 (m, 2H), 7.38-7.41 (m, 2H),
7.58 (d, J ) 7.5 Hz, 2H), 7.76 (d, J ) 7.5 Hz, 2H), 8.12 (m,
1H), 8.39 (br, 1H). 13C NMR (125 MHz, CDCl3): δ ) 12.4, 17.5,
19.2, 28.4, 40.0, 42.6, 45.1, 47.1, 66.6, 87.5, 118.8, 119.9, 125.0,
125.7, 126.6, 127.1, 127.7, 134.4, 140.1, 141.3, 143.7, 156.5,
160.9, 179.2. HRMS (ESI-TOF): m/z calcd 594.2091 (M + H)+,
found 594.2088.
Guanidinylation of Resin-Bound 1,3-Diaminopropane
with 8. A 400 mg sample of trityl chloride resin (theoretical
loading: 1.20 mmol/g) in a polypropylene column was swelled
in 5 mL of CH2Cl2. 1,3-Diaminopropane (2.0 mL, 24.0 mmol)
in 6 mL of CH2Cl2 was added to the resin suspension. The
mixture was rotated for 2 h and then quenched with 2 mL of
methanol. The resin was washed with CH2Cl2 to give the resin-
bound 1,3-diaminopropane (loading 1.02 mmol/g, as deter-
mined by derivatization with Fmoc-OSu and monitoring the
Fmoc release). A 100 mg sample of resin-bound 1,3-diamino-
propane in 3 mL of anhydrous CH2Cl2 was mixed with 8 (302
mg, 0.51 mmol) and DIPEA (89 µL, 0.51 mmol). Then EDC
(98 mg, 0.51 mmol) and DIPEA (89 µL, 0.51 mmol) in 2 mL of
anhydrous CH2Cl2 were added. The mixture was rotated at rt
overnight. The resin was filtered and washed with CH2Cl2. The
filtrates were combined. The resin was further reacted with
Fmoc-OSu (172 mg, 0.51 mmol) and DIPEA (0.18 mL, 1.02
mmol) in CH2Cl2 overnight to cap the unreacted resin-bound
1,3-diaminopropane. The resin was subsequently treated with
TFA/H2O/TIS (94:3:3) for 2 h. After the solvent was removed,
the residue was subjected to LC-MS analysis. The relative
N-[2-(Dde-amino)ethyl]-N′-Pbf-thiourea 12. DdeOH
(0.276 g, 1.51 mmol) in 10 mL of anhydrous CH2Cl2 was added
into a solution of mono-Boc-protected ethylenediamine 9 (0.20
mL, 1.26 mmol) in 10 mL of anhydrous CH2Cl2. After 8 h of
stirring at room temperature under N2, the solvent was
removed and the residue was purified by silica gel chroma-
tography. N-Boc-N′-Dde-ethylenediamine 10 was obtained as
a white solid (0.376 g, 92%). Mp: 123 °C. Rf ) 0.25 (MeOH/
1
CH2Cl2, 1/20). H NMR (500 MHz, CDCl3): δ ) 0.99 (s, 6H),
1.40 (s, 9H), 2.32 (s, 4H), 2.53 (s, 3H), 3.32-3.33 (m, 2H), 3.53-
3.54 (m, 2H), 5.06 (br, 1H). 13C NMR (125 MHz, CDCl3): δ )
17.8, 28.18, 28.26, 30.0, 40.1, 43.0, 52.8, 79.9, 108.1, 155.8,
174.1, 198.1. HRMS (ESI-TOF): m/z calcd 325.2127 (M + H)+,
found 325.2130. The diprotected amine 10 (0.310 g, 0.96 mmol)
was treated with 5 mL of TFA/CH2Cl2 (1:1) for 15 min, and
then the solvent was removed under vacuum. The residue was
dissolved in anhydrous CH2Cl2 (20 mL), and DIPEA was added
dropwise to neutralize the remaining trace amount of TFA.
Subsequently, DIPEA (0.26 mL, 1.50 mmol) and Pbf-NCS (0.96
mmol) in 5 mL of CH2Cl2 was added dropwise. After 2 h of
stirring at rt, the product was purified by silica gel chroma-
tography eluting with MeOH/CH2Cl2. Compound 12 was
obtained as a white solid (0.462 g, 90%). Mp: 77 °C dec. Rf )
1
0.27 (MeOH/CH2Cl2, 1/20). H NMR (500 MHz, CDCl3): δ )
1.01 (s, 6H), 1.47 (s, 6H), 2.10 (s, 3H), 2.35 (s, 4H), 2.46 (s,
3H), 2.53 (s, 3H), 2.55 (s, 3H), 2.97 (s, 2H), 3.64 (m, 2H), 3.80
(m, 2H), 8.26 (t, J ) 5.5 Hz, 1H), 8.89 (br, 1H). 13C NMR (125
MHz, CDCl3): δ ) 12.5, 17.5, 17.8, 19.2, 28.2, 28.5, 30.0, 41.1,
42.8, 44.3, 52.7, 87.5, 108.2, 118.6, 125.7, 126.7, 134.5, 140.2,
161.0, 174.3, 179.7. HRMS (ESI-TOF): m/z calcd 536.2247 (M
+ H)+, found 536.2251.
Guanidinylation of Resin-Bound 1,3-Diaminopropane
with 12. The guanidinylation step was similar to those
performed with 8, but instead using 0.51 mmol of 12. The resin
was filtered and washed with CH2Cl2 and then was treated
with TFA/H2O/TIS (94:3:3) for 2 h and filtered. After the
solvent was removed, the residue was subjected to LC-MS
analysis. The relative UV (230 nm) intensities of the released
products corresponding to resin-bound 16 and 17 were ob-
tained. The ratio indicated that the degree of guanidinylation
was 14% of resin-bound 1,3-diaminopropane. In a separate
experiment, 50 mg of 12 (0.093 mmol) was mixed with EDC
(18 mg, 0.093 mmol) and DIPEA (0.064 mL, 0.186 mmol) in
10 mL of anhydrous CH2Cl2 and stirred at rt overnight. No
cyclization product was found in the solution.
N-[2-(ivDde-amino)ethyl]-N′-Pbf-thiourea 13. To a solu-
tion of mono-Boc-protected ethylenediamine 9 (0.20 mL, 1.26
mmol) in 30 mL of ethanol was added ivDdeOH (0.552 mL,
2.52 mmol). After being refluxed overnight under N2, the
solution was removed and the residue was purified by silica
gel chromatography eluting with MeOH/CH2Cl2. Diprotected
amine 11 was obtained as an oil (0.401 g, 87%). Rf ) 0.59
(MeOH/CH2Cl2, 5%). 1H NMR (500 MHz, CDCl3): δ ) 0.86 (d,
J ) 7.0 Hz, 6H), 0.91 (s, 6H), 1.33 (s, 9H), 1.83 (m, 1H), 2.24
(s, 4H), 2.90 (br, 2H), 3.26 (m, 2H), 3.51 (m, 2H), 5.34 (br, 1H).
13C NMR (125 MHz, CDCl3): δ ) 22.3, 28.0, 28.1, 28.8, 29.6,
8808 J. Org. Chem., Vol. 70, No. 22, 2005

Syntheses of Oligomeric Guanidines
36.9, 40.2, 43.1, 52.8, 79.5, 107.0, 155.7, 176.9. HRMS (ESI-
TOF): m/z calcd 367.2597 (M + H)+, found 367.2591. Com-
pound 11 (0.151 g, 0.412 mmol) was treated with 3 mL of TFA/
CH2Cl2 (1:1) for 15 min, and the solvent was removed under
vacuum. The residue was dissolved in anhydrous CH2Cl2 (10
mL), and DIPEA was added dropwise to neutralize the
remaining trace amount of TFA. Subsequently, DIPEA (0.14
mL, 0.824 mmol) and Pbf-NCS (0.412 mmol) in CH2Cl2 (3 mL)
was added dropwise. After 4 h of stirring at rt, the product
was purified by silica gel chromatography eluting with EtOAc/
hexanes. compound 13 was obtained as a white solid (0.209 g,
88%). Mp: 45 °C dec. Rf ) 0.60 (MeOH/CH2Cl2, 3%). 1H NMR
(300 MHz, CDCl3): δ ) 0.94 (d, J ) 6.9 Hz, 6H), 1.01 (s, 6H),
1.46 (s, 6H), 1.84-1.95 (m, 1H), 2.12 (s, 3H), 2.36 (s, 4H), 2.46
(s, 3H), 2.53 (s, 3H), 2.96-3.00 (m, 4H), 3.64-3.70 (m, 2H),
stirred overnight at rt and was concentrated under vacuum.
After silica gel chromatography eluting with EtOAc/hexane,
compound 21 was obtained as a white solid.
Glutamate-Based Derivative 21a. This compound was
synthesized according to the general procedure on a 1.88 mmol
scale. Yield: 903 mg (89%). Mp: 64 °C. Rf ) 0.71 (EtOAc/
1
hexanes, 1/1). H NMR (500 MHz, CDCl3): δ ) 1.48 (s, 9H),
1.83 (m, 1H), 1.93 (m, 1H), 2.39-2.42 (m, 2H), 3.80 (d, J ) 6.5
Hz, 2H), 4.01 (m, 1H), 4.11 (m, 2H), 4.24-4.25 (m, 1H), 5.37
(d, J ) 9.0 Hz, 1H), 7.24-7.30 (m, 2H), 7.37-7.38 (m, 2H),
7.50-7.51 (m, 2H), 7.55-7.57 (m, 2H),7.72-7.76 (m, 4H). 13
C
NMR (125 MHz, CDCl3): δ ) 27.7, 27.9, 31.9, 41.8, 47.0, 50.6,
66.6, 80.4, 119.7, 123.16, 123.20, 125.0, 125.1, 126.8, 127.4,
131.7, 133.8, 133.9, 140.95, 141.02, 143.6, 144.0, 156.1, 168.3,
172.3. HRMS (ESI-TOF): m/z calcd 541.2339 (M + H)+, found
541.2332; calcd 563.2158 (M + Na)+, found 563.2153.
3.76-3.82 (m, 2H), 8.27 (t, J ) 6.0 Hz, 1H), 8.93 (br, 1H). 13
C
NMR (125 MHz, CDCl3): δ ) 12.4, 17.5, 19.2, 22.4, 28.1, 28.5,
29.0, 29.8, 37.0, 41.3, 42.4, 44.4, 52.9, 87.4, 107.4, 118.6, 125.6,
126.8, 134.5, 140.1, 160.9, 177.4, 179.7. HRMS (ESI-TOF): m/z
calcd 578.2722 (M + H)+, found 578.2730.
Azido N-Phthaloyl Derivative 22. General Procedure.
The azide transfer reaction utilized the reported method for
carbohydrates in the presence of Cu2+ 43
. Triflyl azide prepara-
tion: A solution of sodium azide (1.158 g, 17.81 mmol) was
dissolved in distilled H2O (2.9 mL) with CH2Cl2 (4.9 mL) and
cooled on an ice bath. Triflyl anhydride (1.018 g, 3.6 mmol)
was added slowly over 5 min while stirring continued for 2 h.
The mixture was placed in a separatory funnel, and the
CH2Cl2 phase was collected. The aqueous portion was extracted
with 3 mL of CH2Cl2 twice. The organic fractions, containing
the triflyl azide, were pooled, washed once with saturated
Na2CO3, and used without further purification. Compound 21
(1.2 mmol) was dissolved in ethyl acetate (30 mL), and DBU
(0.229 mL, 1.5 mmol) was added. The mixture was stirred at
room temperature for about 10 min until Fmoc was completely
removed as indicated by TLC. The solution was washed with
40 mL of H2O. The solvent was removed, and the residue was
combined with CuSO4 pentahydrate (1.84 mg), distilled H2O
(3.92 mL), and CH3OH (11.8 mL). The trifle azide in CH2Cl2
was added, and the mixture was stirred at rt overnight.
Subsequently, the solvent was removed, and the residue was
purified by silica gel chromatography eluting with EtOAc/
hexanes to give compound 22.
Guanidinylation of Resin-Bound 1,3-Diaminopropane
with 13. The guanidinylation step was similar to those
performed with 8, but instead using 0.51 mmol of 13. The resin
was filtered and washed with CH2Cl2. The filtrates were
combined. A small amount of the resin was cleaved with TFA/
H2O/TIS (94:3:3) for 2 h. After the solvent was removed, the
residue was subjected to LC-MS analysis, which indicated the
presence of guanidinylated product corresponding to the
release of resin-bound 18. No ivDde modified 1,3-ethylenedi-
amine was detected, indicating the absence of intermolecular
migration of the ivDde group. Then the remaining resin was
exposed to 2% hydrazine in DMF for 30 min to remove ivDde
in resin-bound 18. After being washed with DMF and
CH2Cl2, the resin was reacted with Fmoc-OSu (172 mg, 0.51
mmol) and DIPEA (0.18 mL, 1.02 mmol) in CH2Cl2 overnight
to cap the unreacted resin-bound 1,3-diaminopropane and the
free terminal amine of resin-bound guanidine product. The
Fmoc-capped resin was treated with TFA/H2O/TIS (94:3:3) for
2 h. After the solvent was removed, the residue was subjected
to LC-MS analysis. The relative UV (260 nm) intensities of
mono Fmoc-protected 1,3-diaminopropane and the derivative
from resin-bound 18 were obtained to indicate the degree of
guanidinylation at 8%. The combined filtrates were subjected
to silica gel chromatography to obtain a white solid 19 as
isomers in 85% yield (235 mg). Rf ) 0.27 (MeOH/CH2Cl2, 3%).
HRMS (ESI-TOF): m/z calcd 544.2845 (M + H)+, found
544.2840.
Glutamate-Based Derivative 22a. This compound was
synthesized according to the general procedure on 1.2 mmol
scale. Yield: 313 mg (80%). Colorless oil. Rf ) 0.51 (EtOAc/
1
hexanes, 1/2). H NMR (500 MHz, CDCl3): δ ) 1.41 (s, 9H),
1.70-1.77 (m, 1H), 1.87-1.94 (m, 1H), 2.33-2.45 (m, 2H),
3.67-3.70 (m, 1H), 3.75-3.83 (m, 2H), 7.70-7.71 (m, 2H),
7.82-7.84 (m, 2H). 13C NMR (125 MHz, CDCl3): δ ) 27.4, 27.9,
31.6, 41.1, 59.8, 80.7, 123.4, 131.7, 134.1, 167.9, 171.5. HRMS
(ESI-TOF): m/z calcd 345.1563 (M + H)+, found 345.1566;
calcd 367.1382 (M + Na)+, found 367.1383.
N-Pbf-N′,N′′-ethyleneguanidine 20. Compound 20 can be
formed from isomers of 19 or from 15. (a) A 200 mg portion of
19 (0.368 mmol) was added to 20 mL of 2% hydrazine in DMF.
The solution was stirred at rt for 2 h. The solvent was removed,
and the residue was washed with water. After silica gel
chromatography eluting with ethyl acetate/hexanes, a light
yellow solid 20 was obtained (112 mg, 90%). (b) A 200 mg
portion of 15 (0.358 mmol) was dissolved in 30 mL of ethyl
acetate, and DBU (0.107 mL, 0.716 mmol) was added. The
solution was stirred at rt for 15 min and washed with 20 mL
of water. After silica gel chromatography purification eluting
with ethyl acetate/hexanes, a light yellow solid 20 was obtained
Monomer 23 for Direction B Synthesis. General Pro-
cedure. A flask with reflux condenser was charged with
compound 22 (1.0 mmol), followed by 40 mL of EtOH and N2H4
hydrate (0.49 mL, 10 mmol). The mixture was refluxed
overnight under N2 atmosphere, cooled, and concentrated. The
residue was dissolved in CH2Cl2 (30 mL), washed with 20
mL of water twice and saturated NaCl once, and dried over
Na2SO4. The solution was filtered, and Pbf-NCS (1.0 mmol)
in 10 mL of CH2Cl2 was added dropwise at room temperature.
After 2 h of stirring, the solvent was removed under vacuum
and the residue was purified by silica gel chromatography
eluting with EtOAc/hexanes to give monomer 23.
1
(115 mg, 95%). Mp: 214 °C. Rf ) 0.32 (EtOAc). H NMR (500
MHz, CDCl3): δ ) 1.45 (s, 6H), 2.08 (s, 3H), 2.49 (s, 3H), 2.54
(s, 3H), 2.94 (s, 2H), 3.51 (s, 4H), 6.57 (m, 2H). 13C NMR (125
MHz, CDCl3): δ ) 12.4, 17.7, 19.2, 28.5, 41.8, 43.2, 86.4, 117.5,
124.6, 132.2, 132.5, 138.5, 158.8, 160.5. HRMS (ESI-TOF): m/z
calcd 338.1538 (M + H)+, found 338.1544.
Glutamate-Based Monomer 23a. This monomer was
synthesized according to the general procedure on 0.90 mmol
scale. Yield: 435 mg (92%). White solid. Mp: 32 °C dec. Rf )
1
0.46 (EtOAc/hexanes, 1/2). H NMR (500 MHz, CDCl3): δ )
N-Fmoc-N′-phthaloyl Derivatives 21. General Proce-
dure. Under a nitrogen atmosphere, DEAD (0.315 mL, 2.0
mmol) was added dropwise to a cooled (0 °C) solution of PPh3
(525 mg, 2.0 mmol) in anhydrous THF (30 mL). Subsequently,
phthalimide (294 mg, 2.0 mmol) in anhydrous THF was added
dropwise with stirring. An Fmoc-protected amino alcohol 4 (2.0
mmol) was added in one portion. The reaction mixture was
1.41 (s, 9H), 1.45 (s, 3H), 1.46 (s, 3H), 1.62-1.74 (m, 2H), 2.09
(s, 3H), 2.28-2.31 (m, 2H), 2.50 (s, 3H), 2.57 (s, 3H), 2.97 (s,
2H), 3.40-3.46 (m, 1H), 3.65-3.70 (m, 1H), 3.76-3.81 (m, 1H),
8.08 (t, J ) 5.5 Hz, 1H), 8.58 (s, 1H). 13C NMR (125 MHz,
CDCl3): δ ) 12.4, 17.5, 19.1, 26.9, 28.0, 28.4, 31.3, 42.8, 48.4,
60.2, 80.8, 87.4, 118.7, 125.8, 126.6, 134.4, 140.0, 160.9, 171.5,
J. Org. Chem, Vol. 70, No. 22, 2005 8809

Zhang and Fan
178.8. HRMS (ESI-TOF): m/z calcd 526.2152 (M + H)+, found
526.2150; calcd 548.1972 (M + Na)+, found 548.1978.
using a ZOBAX Extended-C18 column (Agilent) with 0.1% TFA
water and acetonitrile as solvents. Lyophilization gave 58 mg
of product 2 as the TFA salt (60%). 1H NMR (500 MHz, D2O):
δ ) 0.87 (d, J ) 6.5 Hz, 3H), 0.92 (d, J ) 6.5 Hz, 3H), 1.22-
1.31 (m, 1H), 1.32-2.20 (m, 14H), 2.35-2.71 (m, 4H), 2.79-
4.28 (m, 24H), 6.86 (d, J ) 8.5 Hz, 2H), 7.14 (d, J ) 8.5 Hz,
Proline-Based Monomer 26. Fmoc-prolinol 24 (0.4169 g,
1.29 mmol) was converted into Fmoc-protected azide 25 (11,
0.385 g, 86%) according to the reported procedure.17 Compound
25 (0.351 g, 1.01 mmol) was dissolved in 20 mL of methanol/
CHCl3 (50:1). To this solution was added a catalytic amount
of 10% palladium on carbon. The mixture was stirred at rt
under a hydrogen balloon overnight. The solution was filtered,
and the solvent was removed. The resulting HCl salt was
dissolved in anhydrous CH2Cl2, and DIPEA (0.35 mL, 2.02
mmol) was added, followed by Pbf-NCS (1.01 mmol) in 10 mL
of CH2Cl2. After 3 h of stirring at rt, the product was purified
by flash chromatography eluting with 30% ethyl acetate in
dichloromethane to obtain the monomer 26 as a white solid
(512 mg, 80%). Mp: 55 °C dec. Rf ) 0.40 (MeOH/CH2Cl2, 1/50).
1H NMR (500 MHz, CDCl3): δ ) 1.45 (s, 6H), 1.53-1.87 (m,
4H), 2.08 (s, 3H), 2.48 (m, 3H), 2.57 (s, 3H), 2.92 (s, 2H), 3.06-
3.41 (m, 3H), 3.65-3.94 (m, 2H), 4.25 (m, 1H), 4.39-4.61 (m,
2H), 7.32 (m, 2H), 7.41 (m, 2H), 7.60 (m, 2H), 7.767 (m, 2H),
8.27 (br, 1H), 8.40 (br, 1H). 13C NMR (125 MHz, CDCl3): δ )
12.4, 17.5, 19.2, 28.4, 28.8, 42.8, 46.8, 47.2, 48.8, 57.2, 67.3,
87.3, 118.5, 119.9, 124.5, 125.1, 125.6, 127.0, 127.7, 134.4,
140.0, 141.3, 143.9, 155.5, 160.7, 178.9. HRMS (ESI-TOF): m/z
calcd 634.2409 (M + H)+, found 634.2401.
Solid-Phase Synthesis of Oligomer 2. Using 100 mg of
Rink amide MBHA resin, the solid-phase procedure was very
similar to those for oligomer 1, except that initial loading of
23a was determined to be 0.55 mmol/g and subsequent cycles
of guanidinylation and reduction was done with 0.275 mmol
of monomers 23b (156 mg), 23c (154 mg) and 23d (125 mg).
The resin was then guanidinylated with the proline-based
monomer 26 (174 mg, 0.275 mmol) in 4 mL of anhydrous
CH2Cl2 in the presence of DIPEA (0.096 mL, 0.55 mmol) and
EDC (53 mg, 0.275 mmol). The reaction was allowed to proceed
for 12 h at rt. The Fmoc group was removed by treatment with
6 mL of 20% piperidine in DMF for 15 min twice. After being
washed with DMF and CH2Cl2, the resin was guanidinylated
with 23e (191 mg, 0.275 mmol) in 4 mL of anhydrous CH2Cl2
in the presence of DIPEA (0.096 mL, 0.55 mmol) and EDC
(53 mg, 0.275 mmol) for 24 h at rt to ensure reaction
completion with the proline secondary amine. The resulting
resin-bound 2 was cleaved by treatment with 7 mL of TFA/
TIS/H2O (94:3:3) overnight. After solvent removal, the residue
was washed with ether. The residue was dissolved in 0.1% TFA
water and acetonitrile and purified by reversed-phase HPLC
2H). MS (ESI-ion trap): m/z 478.8 (M + 2H)2+
.
Solution-Phase Synthesis of Oligomer 2. The procedure
was very similar to those used for oligomer 1 starting with
0.225 mmol of DMB-NH2 and monomer 23a, followed by
further reaction cycles with 23b (128 mg), 23c (126 mg), and
23d (102 mg). The resulting intermediate was guanidinylated
with 26 (143 mg, 0.225 mmol) and purified. Then the Fmoc
group was removed by the treatment with DBU (0.035 mL,
0.225 mmol) in 20 mL of ethyl acetate. After the solution was
washed with water (15 mL) and saturated NaCl (20 mL) and
dried over Na2SO4, the solvent was removed and the residue
was dissolved in 10 mL of anhydrous CH2Cl2. Final guanidi-
nylation was carried out with monomer 23e (157 mg, 0.225
mmol) in 2 mL of anhydrous CH2Cl2 and DIPEA (0.08 mL,
0.45 mmol) and EDC (43 mg, 0.225 mmol) in 2 mL of
anhydrous CH2Cl2 at rt for 12 h to ensure completion of the
reaction with secondary amine. After the solvent was removed,
the residue was purified by silica gel chromatography eluting
with 3% methanol in CH2Cl2 to give 387 mg of fully protected
2 as a white solid (56%). LC-MS: purity >99% based on UV
absorbance at 230 nm. MS (ESI-ion trap): m/z 1537.2 (M +
2H)2+, 1025.4 (M + 3H)3+. The protected 2 was treated with
10 mL of TFA/TIS/H2O (94:3:3) overnight. After solvent
removal, the residue was washed with ether. The residue was
dissolved in 0.1% TFA water and acetonitrile and checked by
LC-MS: purity 93% based on UV absorbance at 230 nm.
Lyophilization gave 214 mg of product 2 as TFA salt (90%).
Acknowledgment. This work was supported in part
by the National Institutes of Health (Grant No. AI44954)
and by the W. M. Keck Foundation Center for Microbial
Pathogens at the University of Washington.
Supporting Information Available: Characterization
data for new compounds not listed in the Experimental Section
and 13C NMR spectra of nonoligomer compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO051226T
8810 J. Org. Chem., Vol. 70, No. 22, 2005