Design of antimicrobially active small amphiphilic peptide dendrimers

Article abstract of DOI:10.3390/molecules14103881

Novel polyfunctional small amphiphilic peptide dendrimers characterized by incorporation of a new core compounds - tris-amino acids or tetrakis-amino alcohols that originated from a series of basic amino acids - were efficiently synthesized. These new cor

Full text of DOI:10.3390/molecules14103881

Molecules 2009, 14, 3881-3905; doi:10.3390/molecules14103881
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Design of Antimicrobially Active Small Amphiphilic Peptide
Dendrimers
Piotr Polcyn 1, Margarita Jurczak 1, Aleksandra Rajnisz 2, Jolanta Solecka 2 and
Zofia Urbanczyk-Lipkowska 1,*
1
Institute of Organic Chemistry PAS, Kasprzaka Str. 44/52 01-224 Warsaw, Poland;
E-Mails: polcynp@icho.edu.pl (P.P.); rita@icho.edu.pl (M.J.)
2
National Institute of Public Health – PZH, Chocimska Str. 24, 00-791 Warsaw, Poland;
E-Mails: olarajnisz@yahoo.co.uk (A.R.); jsolecka@pzh.gov.pl (J.S.)
* Author to whom correspondence should be addressed; E-Mail: ocryst@icho.edu.pl;
Tel.: +48 22 3432207.
Received: 31 August 2009; in revised form: 22 September 2009 / Accepted: 27 September 2009 /
Published: 29 September 2009
Abstract: Novel polyfunctional small amphiphilic peptide dendrimers characterized by
incorporation of a new core compounds – tris-amino acids or tetrakis-amino alcohols that
originated from a series of basic amino acids – were efficiently synthesized. These new
core elements yielded molecules with multiple branching and (+5)/(+6) charge at the 1-st
dendrimer generation. Dendrimers exhibited significant antimicrobial potency against
Gram(+) and Gram(-) strains involving also multiresistant reference strains (S. aureus
ATCC 43300 and E. coli ATCC BAA-198). In addition, high activity against fungi from
the Candida genus was detected. More charged and more hydrophobic peptide dendrimers
expressed hemolytic properties.
Keywords: dendrimeric peptides; antimicrobial; antibiotic resistance
1. Introduction
The rapid emergence of multi-drug resistance against conventional antibiotics is one of the greatest
challenges of modern medical science. In places like health care units, the food industry, dental or
implant materials that are confronted every day with microbial invasion new microbicides and

Molecules 2009, 14
3882
disinfectants are an urgent need. Public awareness of the slow discovery of novel antibiotics practiced
by pharmaceutical companies during last decades has recently prompted a vigorous search for
complementary and alternative antimicrobial medicines characterized by new structure(s) and acting
on new cellular targets [1]. Nanomedicine is one of the very promising research directions in designing
novel multifunctional materials that offer better therapeutic properties in many areas of medicine,
including the treatment of microbial infections [2].
Dendrimers - synthetic macromolecules of nanoscopic dimensions built from several layers of
branches located around a central core [3-5] are among several groups of macromolecules that are
considered as promising nanopharmaceuticals with high potential. The multivalent nature of these
compounds, their unambiguous composition, reliability and versatility of their synthesis, make this
type of carriers well-suited in various applications for diagnostic purposes, [6] protein mimetics, [7]
antiviral agents, [8,9] vaccines, [10,11] versatile prospective drugs [12,13] and gene delivery vehicles
[14]. The problem of designing antimicrobial compounds emerged in the early days of dendrimer
chemistry. Cationic dendrimers with terminal ammonium groups originated from PAMAM [15], PPI
[16], or carbosilane [17] dendrimers have been shown to affect the integrity of negatively charged
bacterial membranes resulting in high bacteriostatic and bactericidal potency. PAMAM dendrimers
have been used as carriers of quinolone [18] and penicillin V [19] antibiotics with improved
bioavailability or as containers for biocidal silver nanoparticles [20].
Natural antimicrobial peptides (AMPs) constitute another groups of macromolecules that are
considered a good source of new strategies and new nanopharmaceuticals [21]. Their structural
diversity and mechanism of action (modification of the membrane permeability), which differs from
that of the conventional antibiotics, makes them an interesting alternative to classic antibiotics. The
majority of natural AMPs contain a 10-50 amino acids sequence that includes several basic amino
acids (Lys, Arg) and up to 60% of these with lipophilic side chains (Phe, Tyr, Ala, etc.). Such a
composition provides a positive charge and enables formation of amphipatic structure(s) essential for
interactions with microbial membranes.
Intensive research on economically feasible smaller analogs has allowed the introduction of several
compounds into clinical trials [1,22]. Based on these natural compounds Tam and co-workers designed
lysine (Lys) dendrimers that have been used as carriers of two to eight copies of tetra- or octapeptide
fragments of the antimicrobial peptide tachyplesin. The obtained dendrimers expressed high potency
against broad range of microorganisms while being at the same concentration less toxic than natural
precursors [23].
An interesting novel application of dendrimers designed to act as new microbial targets has been
reviewed recently by Rojo and Deldgado [24 and references cited therein]. These authors summarized
the work of several groups focused on the synthesis of dendrimers carrying multiple copies of
polysaccharides or lipopolysaccharides [25] and their intended use as infection preventing anti-
adhesive agents.
According to our concept of the “non-sequential” pharmacophore [26,27] dendrimer trees can be
used not only as carriers of antimicrobial fragments, but also for de novo design of positively charged
amphiphilic molecules. This approach allowed for the successful synthesis of a novel class of low
molecular mass amphiphilic dendrimeric peptides (tetra- to octapeptides) having considerable
antimicrobial activity against Gram(+) and less activity against Gram(-) bacteria with structure-

Molecules 2009, 14
3883
dependent cytotoxicity [28,29]. According to DSC studies, these cationic amphiphilic molecules based
on the Lys(Lys)2 scaffold interact stronger with anionic dimirystoyl phosphatidyl glycerol (DMPG)-
prepared model multilamelar vesicles that emulate the properties of microbial membranes than with
vesicles prepared from neutral phospholipids [30].
These studies confirmed that their selectivity is shifted towards negatively charged microbial ones
rather than towards neutral human cell membranes. Although it is generally acknowledged that the
structural complexity of biological membranes is a source of the selective response of microorganisms
towards antibiotics the last thirty years of intensive research has provided no specific rules relating
structure and biological activity of natural peptides. In the case of macromolecular dendrimeric
compounds the efficiency of their interactions with heterogenic negatively charged microbial
membranes depends on the number and distribution of the charged and hydrophobic groups
(polyvalency) and on their structural flexibility, i.e. adaptability of the dendrimer to the dynamic
structure of the membrane.
Here we present the synthesis and spectroscopic data for two groups of cationic, amphiphilic
peptide dendrimers which utilize novel multibranched scaffolds of amino acid origin. Use of these new
building blocks as a core element yielded first generation peptide dendrimers characterized by high
charge (+5 or +6) and high polyvalency.
The antimicrobial activity of these multicharged small cationic dendrimers against both Gram(+),
Gram(-) bacteria and fungi from the Candida genus was studied and results were compared with the
activity of the previously studied less charged and less branched analogs [28]. The obtained results
demonstrate that the new small but highly charged, more branched amphiphilic peptide dendrimers
express better properties: along with high activity against Gram(+) bacteria, they show significant
activity against Gram(-) bacteria and fungi, including antibiotic resistant strains. Hemotoxicity towards
human red blood cells for more hydrophobic compounds was detected, however.
2. Results and Discussion
2.1. Synthesis
Preparation of the new cores involved cyanoethylation of the respective basic amino acids
(Scheme 1), followed by reduction of the nitriles to polyamines. Michael addition of acrylonitrile to
polyamines (ethylene- or propylenediamine) and subsequent reduction of nitriles to yield amines is one
of the classic routes in the dendrimer chemistry [31]. Condensation of acrylonitrile with several α-
amino acids in aqueous solution in the presence of alkali salts, yielding mono-, di-, and tricyanoethyl
derivatives was studied by McKinney [32,33].
Synthesis of a tris-N-cyanoethyl derivative of lysine in low yield (22.3%) was reported by Riehm
and Scheraga without isolation and identification, however, of the second product – the corresponding
tetrakis-N-cyanoethyl derivative [34]. In the case of basic amino acids, reaction with acrylonitrile gave
a mixture of tris- and tetrakis-N-cyanoethyl derivatives with the ratio depending on the reaction
conditions (Scheme 1). For example, the bis-N-cyanoethyl derivative 11 was initially observed as an
impurity. However, when the reaction was performed at 0 °C in methanol, using four equivalents of
acrylonitrile per amino group, the N,N-bis(cyanoethyl)-α,β-diaminopropionic acid 11 was obtained
exclusively. Under the same conditions, the other three amino acids yielded tris-cyanoethyl derivatives

Molecules 2009, 14
3884
as the major products. Most of the reduction procedures of the multiple nitrile groups involve catalytic
hydrogenation with the use of the Raney-Ni or Co [35,36]. Unexpectedly, there was a pronounced
difference in reactivity between tris- and tetrakis-N-cyanoethyl derivatives.
Scheme 1. Synthesis of N-cyanoethylated derivatives of 1.
NC
CN
CO2H
N
CN
(CH2)n
N
H
CO2H
NH2
2a-d
i
H2N
(CH2)n
NC
CN
1a: n = 1
1b: n = 2
1c: n = 3
1d: n = 4
CO2H
N
CN
(CH2)n
N
3a-d
CN
CO2H
CO2H
.
HCl
ii
.
H
N
HCl
H2N
CN
NH2
N
H
NC
11
1a
Reagents: (i) 1. 6 eq CH2=CH-CN per amino acid molecule, 1 eq NaOH, H2O-THF; 2. 1 eq HCl;
(ii) 1. 4 eq CH2=CH-CN, 2 eq NaOH, MeOH, 24 h, 0-24 °C; 2. 2 eq HCl.
While catalytic reduction of the tris-N-cyanoethyl amino acids 2 in MeOH using Raney-Ni in a Parr
hydrogenation apparatus gave the respective N-propylamino derivatives 4 in almost quantitative yields
(Scheme 2), reduction of tetrakis-N-cyanoethyl derivatives 3 under the above conditions left 90% of
the original substrate unconverted. Finally, a family of compounds 5 was obtained using three
equivalents of 10 M borane dimethylsulfide complex per nitrile group with extention of the reaction
time to 24-48 h (Scheme 2) [37].
The above procedures provide a rapid access to new templates for preparation of branched peptides
or other dendrimers.
The synthesis of the lysine-functionalized first generation dendrimers 7e-h from the N-protected
trimeric scaffolds 4e-h started with coupling with benzylamine, yielding the amides 6e-h. Then the
amino group deprotection was followed by coupling with (2-Cl-Z)Lys(Boc) to afford 7e-h in 67-71%
overall yield (Scheme 3).

Molecules 2009, 14
3885
Scheme 2. Reduction of N-cyanoethylated derivatives 2 and 3.
NHR
NHR
NC
CN
CO2H
CO2H
NR
N
CN
N
(CH2)n
i
(CH2)n
N
H
NHR
2a-d
4a - d, R = H
4e - h, R = CBz
.
CO2H
HCl
H
N
CO2H
HCl
CN
N
NC
NR
NHR
H
NHR
NR
11
12: R = H
13: R = CBz
NH2
NH2
NC
CN
CO2H
N
ii
.
6 HCl
CH2OH
N
N
CN
(CH2)n
N
(CH2)n
5a-d
NH2
3a-d
CN
NH2
Reagents: (i) 1. H2/Raney-Ni, 90 psi, 1 eq NaOH, MeOH, 14-16 h; 2. CBz-Cl, Na2CO3,
H2O:dioxane 1:1 (ii) 1. 10M SMe2, 3 eq/CN, reflux THF, 24-48 h; 2. HCl/MeOH.
Scheme 3. Synthesis of the dendrimers 7e-h.
HN
CO2H
(CH2)n
Z
ZNH
N
(CH2)n
Z
O
ZNH
N
i
N
N
ZNH
ZNH
NHZ
NHZ
4e: n = 1
4f: n = 2
4g: n = 3
4h: n = 4
6e: n = 1
6f: n = 2
6g: n = 3
6h: n = 4
Cl
O
NH2
HN
O
(CH2)n
N
H
N
O
HN
ii
H2N
H
N
HN
NH
.
5HCl
H2N
Cl
O
O
O
O
Cl
HN
7e: n = 1
7f: n = 2
7g: n = 3
7h: n = 4
O
O
Reagents: (i) benzylamine, DCC, HOBt, THF (ii) 1. H2/10% Pd/C, MeOH; 2. (2-Cl-Z)Lys(Boc),
DCC, HOSu, DMF; 3. TFA/CH2Cl2 3h; 4. HCl/AcOEt.

Molecules 2009, 14
3886
Synthesis of the tetra-functional branched compound using the hexahydrochloride of 5c as a starting
material was complicated by the fact that 5a-d are practically insoluble in the commonly used organic
solvents. Eventually, they were dissolved in hot DMF (50-60 °C) with addition of a large excess of
triethylamine. In this case, coupling with 2,4,5-trichlorophenyl ester of (Boc)Phe was carried out for 7
days affording the compound 9a-d in 54% yield (Scheme 4).
Scheme 4. Synthesis of the dendrimers 10a-d.
NH2
NH2
Boc-Phe-HN
i
CH2OH
N
CH2OH
N
(CH2)n
NH-Phe-Boc
N
N
NH2
(CH2)n
6 HCl
.
Boc-Phe-HN
NH-Phe-Boc
NH2
9a: n = 1
9b: n = 2
9c: n = 3
9d: n = 4
5a: n = 1
5b: n = 2
5c: n = 3
5d: n = 4
ii
Cl
O
O
O
H
N
O
10a: n = 1
10b: n = 2
10c: n = 3
10d: n = 4
N
H
HN
.
6 HCl
H2N
CH2OH
n
N
N
H
N
NH
O
O
O
NH
HN
O
O
O
O
N
HN
O
O
H
N
H
HN
Cl
Cl
N
H
O
O
O
NH2
NH2
NH2
Cl
Reagents: (i) 4.4 eq (Boc)Phe-2,4,5-trichlorophenyl ester, N(Et)3, DMF, 7 days (ii) 1. TFA/CH2Cl2,
3h; 2. 2-Cl-Z-Lys-Boc, DCC, HOSu, N(Et)3, DMF, 24 h; 3. TFA/CH2Cl2 3h; 4. HCl/AcOEt.
2.1. Antimicrobial and hemolytic activities
The results of in vitro antimicrobial activity of the amphiphilic dendrimers 7e-h, 10a-d and two
reference compounds 5c-d assayed against two Gram(+) strains, six Gram(-) strains, and three strains
from the Candida genus are shown in Table 1. Due to application in the synthesis 3- or 4-arm cores the
families of compounds 7e-h vs. 10a-d are structurally different regarding the relative distribution of
cationic and lipophilic groups and net charge. In the compounds 7e-h, 3-arm cores are N-terminated
with lysine hydrochloride residues substituted at N with 2-chlorobenzyloxycarbonyl- residue (2-Cl-Z)

Molecules 2009, 14
3887
and with a benzyl group located at the C-end (Scheme 3). In the compounds 10a-d, all four arms of the
core are elongated with phenylalanine residues (Phe) that are followed by substituted lysine residues
similar to those above (Scheme 4). Within each family, compounds have the same net charge - (+5) or
(+6) and differ in the number of methylene groups in the core.
Table 1. Minimal inhibitory concentrations (MICs) of studied dendrimers against
Gram(+), Gram(-) and fungi and two reference compounds 5c and 5d (M). a
Compound
7e
7f
7g
7h
10a
10b
10c
10d
Lys
(+)6
3.4
5c
5d
Lys
(+)6
221
DAP c DAB d Orn
Lys
(+)5
21.6
DAP DAB Orn
(+)6
1.7
Orn
(+)6
113
Scaffold/charge b
(+)5
2.8
(+)5
11.7
(+)5
10.9
(+)6
1.3
(+)6
27.2
S. aureus
ATCC 25923
S. aureus
11.8
24.3
11
10.3
48
10.9
21.8
2.7
21.6
86
3.4
30.2
3.4
7.3
47
13.6
54
1.7
14
-
-
-
-
-
-
-
-
-
-
ATCC 43300
P. aeruginosa
ATCC 27853
B. bronchiseptica
ATCC 4617
A. baumani
ATCC 19606
K. pneumoniae
ATCC 13882
E.coli
ATCC 25922
E. coli
ATCC BAA-198
C. albicans
ATCC 90028
C. krusei
ATCC 6258
C. parapsilosis
11
5.4
>86
>86
86
3.4
13.8
>55
7.3
6.9
6.9
6.9
6.9
6.8
1.7
3.4
>54
6.8
3.4
6.8
3.4
3.4
>89
>89
11.8
>88
>88
44
>88
>88
48
5.4
13.8
>55
7.3
27.3
54
87
21.8
10.9
10.9
5.4
27.3
13.6
54
>226 >221
>89
>89
44
43
6.9
-
-
-
-
-
-
-
-
>86
43
6.8
6.8
27.3
54
88
89
5.4
86
6.8
ATCC 22019
a
MICs of the reference compounds: Penicillin G against S. aureus ATCC 25923 – 6.6 (M);
polymyxin B against E. coli ATCC 25922 and P. auruginosa ATCC 27853 – 0.55 (M);
amphotericin B against C. krusei ATCC 6258 – 1.1 M; indolicidin against S. aureus
b
ATCC 25923 – 2.1 (M) and E. coli ATCC 25922 – 4.2 (M); charge assigned by elementary
analysis from Cl content; c diaminopropionic acid core; d diaminobutyric acid core.
Dendrimers 7e,f,h characterized by the MIC values in the range 2.8-21.6 M displayed less than
30% hemotoxicity up to a concentration 1,200 M (extrapolated), whereas dendrimer 7g, exhibited
32% toxicity at a concentration of 400 M (Figure 1). Compounds of the second series 10a,b,c
characterized by MICs in the 1.3-54 M range displayed 30% hemolysis at 400, 150 and 600 M
concentrations, respectively (Figure 2). Although hemotoxicity of the most potent compound 10d
characterized by the MIC values in the range 1.7-14 M reaches 25% in the region of antimicrobial
activity. From this group of compounds the best antimicrobial activity against Candida genus and low

Molecules 2009, 14
3888
hemolytic effect correspond to dendrimer 7g built around the smallest diaminopropionic acid core
(DAP), and dendrimer 10a, that presents a similar antimicrobial profile and significantly lower
hemotoxicity than compound 10d.
Figure 1. Hemolytic activity of the three-arm compounds 7e-h.
100
90
80
70
60
50
40
30
20
10
0
7e
7f
7g
7h
10
30
150
400
600
1200
2400
concentration of dendrimers []
Figure 2. Hemolytic activity of the four-arm compounds 10a-d.
100
90
80
70
60
50
40
30
20
10
0
10a
10b
10c
10d
10
30
150
400
600
1200

2400
concentration of dendrimers [
]
Selectivity of molecule-membrane interactions has been the main concern of numerous studies on
biomedical applications of both multi-charged antimicrobial peptides [39,40] and dendrimers
(PAMAM, PPI, etc.) [41,42]. Multiple positive charge and amphiphatic structure are proposed as a
source of higher affinity of natural peptides to microbial versus human membranes [22]. In case of the
title peptide dendrimers, the charge comes from the protonated amino groups of lysines (3 or 4) and
two protonated ternary N-atoms from the core. Accumulation of positively charged groups in relatively
small branched molecules should promote extended “carpet-like” conformations, facilitating multiple
electrostatic interactions with microbial membranes composed mainly from acidic phospholipids
(phosphatidylglycerol and cardiolipin). In contrast, outer leaflets of mammalian membranes are mainly

Molecules 2009, 14
3889
composed from zwitterionic phospholipids (phosphatidylcholine and sphingomyelin) with only minor
contribution of negatively charged species. Low impact of cationic compounds on red blood cells is
regarded as an indication of a compound’s selectivity. In the present case, differences in hemolytic
properties are observed within compounds belonging to the same series and even more pronounced
between structurally different 7e-h and 10a-d series. Four-arm dendrimers 10a-d, [(+6) charge]
containing four additional hydrophobic side chains from phenylalanine residues expressed higher
antimicrobial potency associated however, with enhancement of hemolytic properties. Similarly,
recent data on cationic, amphiphilic polymethacrylates revealed that lower mole fraction of
hydrophobic groups is one of the important factors in design of potent, non-hemolytic antimicrobial
polymers [43].
The presented data show that designing more charged amphiphilic peptide dendrimers resulted in
better antimicrobial properties. It appears however, that an increase of charge from (+5) to (+6) is not
sufficient for significant enhancement of antimicrobial potency between 7e-h vs. 10a-d series and for
shift in antimicrobial profile. Both, antimicrobial activity and hemolytic properties of dendrimeric
peptides depend also on distribution of charged and lipophylic groups, i.e. on structural factors. Unlike
many other dendrimer types, dendrimeric peptides constructed from, or terminated with basic amino
acids allow to distribute on the surface independently two different types of residues – e.g. charged
and lipophilic. Their number, relative sizes and orientations may be varied providing an opportunity to
obtain dendrimers with good antimicrobial activity and low toxicity.
3. Experimental
3.1. General
All solvents and reactants were of analytical grade and were used without further purification. Mass
spectra were recorded with a Mariner ESI time-of-flight mass spectrometer (PerSeptive Biosystems)
for the samples prepared in MeOH. Proton and carbon NMR spectra were recorded using a Bruker
Avance spectrometer at 500 or 400 MHz, respectively, using deuterated solvents and TMS as an
internal standard. Chemical shifts are reported as  values in parts per million and coupling constants
are given in hertz. The optical rotations were measured with JASCO J-1020 digital polarimeter.
Melting points were recorded on a Köfler hot-stage apparatus and are uncorrected. Thin layer
chromatography (TLC) was performed on aluminum sheets with silica gel 60 F254 from Merck.
Column chromatography (CC) was carried out using silica gel (230-400 mesh) from Merck or
Sephadex LH20. The TLC spots were visualized by treatment with 1% alcoholic solutions of
ninhydrin and heating.
3.2. Antifungal susceptibility testing
Yeasts: Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019 and Candida krusei
ATCC 6258 were cultivated onto Sabouraud dextrose agar (Difco) during 24 hrs at 35 °C.

Molecules 2009, 14
3890
Medium: RPMI – 1640 (Sigma) supplemented with 0.2% (wt/vol) glucose, 0.3 g/L L-glutamine,
0.0053 g/L phenol red, buffered with morpholinopropanesulfonic acid (MOPS; Sigma) at a final
concentration of 0.165 M and adjusted to pH 7.0 was prepared.
Reference Method: The broth microdilution susceptibility test was performed as described in Clinical
and Laboratory Standards Institute (CLSI) reference method M27 – A2 (1) [44]. A series of the
twofold dendrimers and amphotericin B dilutions in DMSO were diluted 1:49 with RPMI. Aliquots
(100 µL0 were dispensed into microdilution sterile plates (Mar-Four). Then, yeast inoculum (100 µL)
containing 1 × 103 to 5 × 103 CFU/mL was added to each well. The final concentration of dendrimers
ranged from 128 to 2 µg/mL [or 105 to 1.6 M for an average molecular mass for 7 series (1,213) and
61 to 0.9 M for an average molecular mass for 10 series (2,103)], and amphotericin B from 1 to
8 µg/mL (1.1 to 8.1 M, respectively), all in twofold dilution steps. The plates were incubated at 35 °C
and read after 24 and 48 hrs. MIC’s (Minimal Inhibitory Concentration) was defined as the lowest
drug concentration that reduced growth by 100%.
3.3. Antibacterial susceptibility testing
Bacteria. Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia
coli ATCC 25922 and Staphylococcus aureus ATCC 43300 were cultivated on tryptone - soy agar
(TSA; Oxoid). Klebsiella pneumoniaeATCC 13882, Bordetella bronchiseptica ATCC 4617,
Acinetobacter baumannii ATCC 19606 were cultivated onto nutrient agar (Difco). Escherichia coli
ATCC BAA-198 was cultivated on TSA with ceftazidime (10 µg/mL or 15.7 M; Sigma). All strains
were incubated during 24 hrs at 37 °C.
Medium. Mueller – Hinton Broth (Oxoid) was supplemented with cations: 12.5 mg Mg ++/L and
25 mg Ca++/L. The pH of the medium after sterilization is between 7.2 and 7.4 [cation–adjusted
Mueller – Hinton Broth” (CAMHB)].
Reference method. Broth microdilution susceptibility test was performed as described in Committee
Laboratory Standards (CLSI) reference method M7-A7 (2) [45]. A series of the twofold dendrimers
dilutions in DMSO and twofold polymyxin B and penicillin G dilutions in CAMHB were diluted 1: 94
with CAMHB. Aliquots (95 µL) were dispensed into microdilution sterile plates (Mar-Four). Then,
bacteria inoculum (5 µL) containing 5 × 104 CFU/mL were added. The final concentration of
dendrimers ranged from 128 to 2 µg/mL [or 105 to 1.6 M for an average molecular mass for 7 series
(1213) and 61 to 0.9 M for an average molecular mass for 10 series(2,103)], polymyxin B and
penicillin G from 8 – 0.15 µg /mL (or 5.8 to 0.1 M and 21.5 to 0.4 M, respectively for polymyxin B
and penicillin G), all in twofold dilution steps. The plates were incubated at 35 °C and read after 18 or
24 hours depending on bacterial strain. MICs (Minimal Inhibitory Concentration) was defined as the
lowest drug concentration that reduced growth by 100%.

Molecules 2009, 14
3891
3.4. Hemolysis assay
Dendrimer induced hemolysis was observed as previously reported [18]. Briefly, serum free human
red blood cells obtained from the Institute of Hematology and Transfusion Medicine in Warsaw, were
suspended in phosphate buffered saline (PBS, pH 7.4). Prepared suspension of 1% hematocrit was
incubated with serial concentration of dendrimers for 30 min at 23 °C. After centrifugation (1,000 rpm,
5 min) the absorbance of supernatant was measured at 540 nm (Jasco 630, Japan). A value of 100%
hemolysis was determined by incubation of erythrocytes with double-distilled water (30 min at 23o). In
a control experiment, cells were incubated in buffer without peptide and absorbance at 540 nm value
was used as a blank.
A - absorbance of the samples incubated with dendrimers; Ab – absorbance of the blank samples; A100%
- absorbance of the reference; Ac - absorbance of red blood cells in PBS, hematocrit 1%.
3.5. Synthesis
3.5.1. Substrate preparation
The monohydrochlorides of L-,β-diaminopropionic acid (1a) and L-α,-diaminobutyric acid (1b)
were obtained according to the Rao protocol as white crystals, with the correct melting points,
elemental analysis and optical rotation values: α[D]28,5°C = +25 ± 1º and +24 ± 1º (C=2 in 1M HCl) for
1a and 1b, respectively, corresponding to the literature data [46].
To a stirred aqueous solution containing 0.1 mol of a basic amino acid and 0.1 mol of NaOH, an
excess of acrylonitrile (0.6 mol) and THF (30-40 mL) was added. The mixture was stirred for 48 h at
room temperature and then refluxed for 3 h. To a cold reaction mixture, 0.1 mol of conc. HCl was
added and left in refrigerator overnight. The crystalline product was separated and washed with several
portions of cold MeOH-H2O (1:1). The washings were collected and evaporated to dryness. Then
acetone (150 mL) was added to the oily residue and filtered. Two crops of precipitate were combined
and recrystallized from hot water or MeOH-H2O (7:3) giving the tris-N-cyanoethylated amino acids
2a-d as white crystals in 49.5-69.2% yield.
The acetone filtrate was evaporated to dryness and purified by flash chromatography, eluting with
an 8:2 mixture of ethyl acetate and hexane plus 3% of methanol, yielding the tetrakis-N-
cyanoethylated derivatives 3a-d as amber-colored gums (except Nα,Nβ-tetrakis(cyanoethyl)-L-α,β-
diaminopropionic acid, which slowly crystallized to give a white solid) in 21.1-28.6% yield. Both the
yields and the 2:3 ratio depend on the reaction temperature. The yield of 3 can reach 50% when the
reaction is performed in a boiling mixture of water-methanol (1:1) overnight.
N,N,N’-tris(cyanoethyl)-L-α,β-diaminopropionic acid (2a): Yield: 13.0 g (49.5%); mp 178-180 °C
1
(dec.); ESI MS 264 (M+1), 286 (M+23); H-NMR (D2O)  2.74 (m, 4H, CH2CN), 2.97 (m, 2H,
CH2CN), 2.99 (dd, J = 3.96 Hz, 1H, CHa), 3.05 [m, 4H, N-(CH2)2], 3.15 (dd, J = 3.96 Hz, 1H,
CHb), 3.6 (t, J = 6.96 Hz, 2H, NHα-CH2), 3.9 (dd, J = 3.96 Hz, 1H, αCH); 13C-NMR  15.2 (CH2-
CN), 15.4 (βCH2-CN), 42.7 (NHα-CH2), 48.8 [N-(CH2)2], 53.2 (C), 61.1 (αC), 117.6 (αCN), 121.0

Molecules 2009, 14
3892
(CN), 170.9 (COOH); Anal. Calcd for C12H17O2N5: C, 54.7; H, 6.5; N, 26.6. Found: C, 54.53; H,
6.42; N, 26.44; [D]26.2 °C = +24.3  1 (c = 2 in 1M HCl).
N,N,N’-tris(cyanoethyl)-L-α,-diaminobutyric acid (2b): Yield: 17.3 g (62.6%); mp 170-172 °C; ESI
1
MS 278 (M+1), 300 (M+23); H-NMR (D2O)  2.09 (q, J = 6.55 Hz, 2H, βCH2), 2.73 (m, 6H,
CH2CN), 2.93 [m, 4H, N-(CH2)2], 3.03 (t, J = 6.85 Hz, 2H, NHα-CH2), 3.46 (t, J = 6.85 Hz, 2H,
CH2), 3.84 (t, J = 6.08 Hz, 1H, αCH); 13C-NMR  15.5 (CH2-CN), 15.5 (CH2-CN), 26.9 (βC), 42.7
(NHα-CH2), 48.2 [N-(CH2)2], 50.0 (C), 62.4 (αC), 117.9 (αCN), 121.2 (CN), 172.9 (COOH); Anal.
Calcd for C13H19O2N5: C, 56.30; H, 6.90; N, 25.25. Found: C, 56.05; H, 6.80; N, 25.20; [D]27.8C
+24.2  1 (c = 2 in 1M HCl).
=
N,N,N’-tris(cyanoethyl)-L-ornithine (2c): Yield: 18.4 g (63.3%); mp 192-194 °C; ESI MS 292 (M+1),
314 (M+23); 1H-NMR (D2O)  1.51, 1.92 (2m, 4H, CH2, βCH2), 2.62 (t, J = 7.28 Hz, 2H, αCH2CN),
2.69 (t, J = 6.65 Hz, 4H, CH2CN), 2.91 [t, J = 6.73 Hz, 4H, N-(CH2)2], 3.02 (t, 2H, NHα-CH2), 3.42
(m, 2H, CH2), 3.72 (dd, J = 4.95 Hz, 1H, αCH); 13C-NMR  15.3 (CH2-CN), 15.7 (CH2-CN), 22.0
(C), 27.6 (βC), 42.3 (NHα-CH2), 48.5 [Nδ-(CH2)2], 52.3 (C), 62.9 (αC), 117.9 (αCN), 121.1 (CN),
173.4 (COOH); Anal. Calcd for C14H21O2N5: C, 57.71; H, 7.26; N, 24.03. Found: C, 57.52; H, 7.28; N,
23.85; [D]27.5C = +24.5  1 (c = 2 in 1M HCl).
N,N,N’-tris(cyanoethyl)-L-lysine (2d): Yield: 21.1 g (69.2%); mp 214-216 °C; ESI MS 306 (M+1), 328
(M+23); 1H-NMR (DMSO)  1.30-1.38 (bm, 4H, CH2, CH2), 1.50-1.55 (2m, 2H, βCH2), 2.45 (t, J =
7.05 Hz, 2H, CH2), 2.55 (m, 6H, CH2CN), 2.73 [t, J = 6.76 Hz, 4H, Nε-(CH2)2], 2.65, 2.81 (2m, 2H,
NHα-CH2), 3.15 (t, J = 6.42 Hz, 1H, αCH); 13C-NMR  15.5 (CH2-CN), 17.7 (CH2-CN), 22.8 (C),
26.5 (C), 32.0 (βC), 42.9 (NHα-CH2), 48.4 [Nε-(CH2)2], 52.3 (C), 60.2 (αC), 119.7 (αCN), 119.9
(CN), 175.2 (COOH); Anal. Calcd for C15H23O2N5: C, 58.99; H, 7.59; N, 22.9. Found: C, 58.9; H,
7.47; N, 22.89; [D]22C = +23  1 (c = 2 in 1M HCl).
N,N’,N,N’-tetrakis(cyanoethyl)-L-α,β-diaminopropionic acid (3a): Yield: 9.05
g
(28.6%);
mp 126-128 °C; ESI MS 317 (M+1), 339 (M+23); 1H-NMR (DMSO)  2.57-2.67 (bm, 9H, CH2CN,
CHa), 2.81-2.98 [bm, 9H, N-(CH2)2, CHb], 3.48 (dd, J = 6.24 Hz, 1H, αCH), 12.64 (s, 1H, COOH);
13C-NMR  15.3, 17.4 (CH2-CN, CH2-CN), 47.1, 48.6 [Nα-(CH2)2, N-(CH2)2], 53.3 (C), 61.7
(αC), 119.68, 119.72 (αCN, CN), 172.9 (COOH); Anal. Calcd for C15H20O2N6: C, 56.95; H, 6.37; N,
26.56. Found: C, 56.99; H, 6.37; N, 26.67; [D]29.1C = -71 (1, c=2 in acetone). Monocrystals of
rac-3a prepared in a separate procedure were subjected to X-ray analysis [31].
N,N’,N,N’-tetrakis(cyanoethyl)-L-α,-diaminobutyric acid (3b): Yield: 7.1 g (21.1%); semisolid; ESI
MS 353 (M+23); 1H-NMR (DMSO)  1.61 (m, 2H, βCH2), 2.47 (t, J=6.95 Hz, 2H, CH2), 2.58 [m,
8H, CH2CN), 2.75 [m, 8H, N-(CH2)2], 3.67 (t, J = 6.00 Hz, 1H, αCH), 11.55 (s, 1H, COOH); 13C-NMR
 15.6, 18.0 (CH2-CN, CH2-CN), 31.1 (βC), 47.8, 48.4 [Nα-(CH2)2, N-(CH2)2], 51.6 (C), 65.1
(αC), 119.1, 120.0 (αCN, CN), 174.6 (COOH); Anal. Calcd for C16H22O2N6: C, 58.16; H, 6.71; N,
25.43. Found: C, 57.95; H, 6.62; N, 25.23; [D]29.1C = -64 (1, c = 2 in acetone).

Molecules 2009, 14
3893
N,N’,N,N’-tetrakis(cyanoethyl)-L-ornithine (3c): Yield: 8.2 g (23.6%); semisolid; ESI MS 345 (M+1),
1
367 (M+23); H-NMR (CDCl3)  1.40, 1.54, 1.68 (3m, 4H, CH2, βCH2), 2.50 (m, 2H, CH2), 2.59
[m, 8H, CH2CN), 2.76, 2.88 [2m, 8H, N-(CH2)2], 3.34 (dd, J = 4.45 Hz, 1H, αCH); 13C-NMR  15.5,
17.4 (CH2-CN, CH2-CN), 23.5 (C), 27.1 (βC), 46.9, 48.4 [Nα-(CH2)2, N-(CH2)2], 51.9 (C), 62.5
(αC), 119.86, 119.91 (αCN, CN), 174.2 (COOH); Anal. Calcd for C17H24O2N6: C, 59.28; H, 7.02; N,
24.40. Found: C, 59.04; H, 6.96; N, 24.19; [D]29.1C = -65.2 (1, c = 2 in acetone).
N,N’,N,N’-tetrakis(cyanoethyl)-L-lysine (3d): Yield: 8.5 g (23.8%); semisolid; ESI MS 359 (M+1),
1
381 (M+23); H-NMR (CDCl3)  1.52, 1.68, 1.85 (3m, 6H, CH2, CH2, βCH2), 2.52 (m, 10H,
CH2CN, CH2), 2.85, 3.04 [2m, 8H, N-(CH2)2], 3.38 (dd, J = 5.34 Hz, 1H, αCH); 13C-NMR  16.9,
18.4 (CH2-CN, CH2-CN), 24.1 (C), 26.9 (C), 29.6 (βC), 47.5, 49.5 [Nα-(CH2)2, Nε-(CH2)2], 52.9
(C), 63.4 (αC), 118.77, 118.84 (αCN, CN), 177.2 (COOH); Anal. Calcd for C18H26O2N6: C, 60.3; H,
7.3; N, 23.4. Found: C, 60.08; H, 7.26; N, 23.15; [D]28.6C = -63 (1, c = 2 in acetone).
3.5.2. Synthesis of N,N-bis(cyanoethyl)-L-α,β-diaminopropionic acid (11)
L-,β-Diaminopropionic acid hydrochloride (14.05 g, 0.1 mol) was suspended in MeOH (100 mL),
followed by slow addition of NaOH (8 g) in MeOH (100 mL). The reaction mixture was cooled to
0 °C in an ice-water bath. Then CH2=CH-CN (26.33 mL, 0.4 mol) was added in small portions and
vigorous stirring was continued overnight, followed by addition of concentrated HCl (16.9 mL,
0.2 mol). The resulting white precipitate was collected on a filter and recrystallized from a MeOH-H2O
mixture (1:1, v/v) to give 16.9 g (66.92%) of 11. Yield: 16.9g (66.9%); mp 180-182 °C (dec.); ESI MS
1
211 (M+1), 233 (M+23), 265 (M+23+32); H-NMR (D2O)  3.03, 3.07 (2t, J = 6.74 Hz, 4H, ,
CH2CN), 3.45-3.65 (bm, 6H, N-CH2), 4.06 (dd, J = 5.50 Hz, 1H, αCH); 13C-NMR  15.3, 15.8 (,
βCH2-CN), 42.74, 43.78 (, NH-CH2-), 45.8 (C), 56.4 (αC), 117.7, 118.0 (, CN), 170.2 (COOH);
Anal. Calcd for C9H15O2N4Cl: C, 43.82; H, 6.12; N, 22.71; Cl, 14.37. Found: C, 43.66; H, 6.20; N,
22.55; Cl, 14.41; [D]24.5C = +24.2  1 (c = 2 in 1 M HCl).
3.5.3. General procedure for the catalytic reduction of the nitriles 2a-d to the tris-N-propylamino
derivatives 4a-d and the synthesis of the Z-protected derivatives 4e-h
To a methanolic solution (150 mL) containing a tris-N-cyanoethylated derivative of basic amino
acid 2a-d (5 g, ca. 15 mmol) and NaOH (0.64 g, 16 mmol), Raney-Ni suspension (5 g) was added. The
resulting mixture was agitated at room temperature for 14-16 h under 90 psi (6 bar) of H2 pressure.
The progress of the reaction was monitored by TLC using 1% ninhydrin in ethanol. After completion
of the reaction, the catalyst was separated by filtration on Celite and the remaining solution was
evaporated to dryness giving the compounds 4a-d as white hygroscopic oils in almost quantitative
yields. The crude products were used for the next step without further purification.
N,N,N’-tris(3-aminopropyl)-L-α,β-diaminopropionic acid (4a): Yield: ~100%; ESI MS 298 (M+23),
1
330 (M+23+32); H-NMR (D2O)  1.50-1.70 (m, 6H, C-CH2-C), 2.50-2.70 (bm, 14H, N-CH2), 3.15
13
(dd, J = 5.65 Hz, 1H, αCH), 3.34 (s, 6H, NH2); C-NMR  28.3 (βCH2-CH2NH2), 28.9 (CH2-

Molecules 2009, 14
3894
CH2NH2), 38.9 (αCH2NH2), 39.3 (βCH2NH2), 44.9 (NHα-CH2), 49.1 [Nβ-(CH2)2], 62.0 (αC), 181.8
(COO-).
N,N,N’-tris(3-aminopropyl)-L-α,-diaminobutyric acid (4b): Yield: ~100%; ESI MS 344 (M+23+32);
1H-NMR (D2O)  1.55-1.75 (bm, 8H, C-CH2-C), 2.40-2.80 (bm, 14H, N-CH2), 2.98 (dd, J = 5.33 Hz,
1H, αCH), 3.35 (s, 6H, NH2); 13C-NMR  28.8 (γCH2-CH2NH2), 29.0 (CH2-CH2NH2), 32.0 (βC),
38.9 (αCH2NH2), 39.4 (γCH2NH2), 45.1 (NHα-CH2), 49.1 [Nγ-(CH2)2], 50.9 (γC), 62.67 (αC), 182.3
(COO-).
1
N,N,N’-tris(3-aminopropyl)-L-ornithine (4c): Yield: ~100%; ESI MS 326 (M+23); H-NMR (D2O) 
1.39 (m, 4H, β,γCH2), 1.53 (m, 6H, C-CH2-C), 2.30-2.60 (bm, 14H, N-CH2), 2.95 (dd, J = 6.02 Hz,
1H, αCH), 3.28 (s, 6H, NH2); 13C-NMR  21.5 (CH2-CH2NH2), 28.1 (CH2-CH2NH2), 30.7 (C),
31.6 (βC), 38.4 (αCH2NH2), 38.9 (δCH2NH2), 44.6 (NHα-CH2), 48.6 [Nδ-(CH2)2], 50.4 (δC), 63.4 (αC),
182.3 (COO-).
1
N,N,N’-tris(3-aminopropyl)-L-lysine (4d): Yield: ~100%; ESI MS 318 (M+1); H-NMR (D2O)  1.50
(m, 2H, CH2), 1.81 (m, 2H, CH2), 2.06 (m, 2H, βCH2), 2.15 (m, 6H, C-CH2-C), 3.10 (t, J = 7.7 Hz,
6H, CH2NH2), 3.20-3.30 (bm, 8H, Nε-CH2, NHα-CH2), 3.34 (s, 6H, NH2), 3.97 (dd, J = 5.09 Hz, 1H,
13
αCH); C-NMR  21.5 (CH2-CH2NH2), 21.7 (CH2-CH2NH2), 22.9(C), 23.9 (C), 28.6 (βC), 36.6
(CH2NH2), 36.7 (αCH2NH2), 43.9 (NHα-CH2), 50.1 [Nε-(CH2)2], 52.7 (C), 60.7 (αC), 181.5 (COO-).
+
N,N-bis(3-aminopropyl)-L-α,β-diaminopropionic acid (12): Yield: ~100%; ESI MS 272 (M+NH4 +
2H2O), 273 (M+Na++MeOH), 275 (M+K++H2O); 1H-NMR (D2O)  1.60 (m, 4H, C-CH2-C), 2.50-2.75
(bm, 10H, βCH2, N-CH2), 3.19 (t, J = 6.46 Hz, 1H, αCH), 3.35 (s, 6H, NH); 13C=NMR  31.9, 32.1 (,
βCH2-CH2NH2), 38.9 (α, βCH2NH2), 45.4, 46.4 (NH-CH2), 51.4 (βCH2), 63.2 (αC), 181.44 (COO-).
3.5.4. General synthesis of the Z-protected derivatives 4e-h
To the crude products from the previous reaction (8.5-9.5 g, ca. 15 mmol, 100%), a dioxane-water
mixture (1:1, v/v, 100 mL) was added, followed by addition of benzyloxycarbonyl chloride (12 mL,
85.7 mmol). The pH value of the mixture was maintained between 9 and 10 by adding 1M NaOH (88
mL). The reaction mixture was cooled in an ice-water bath; after addition of substrates, it was allowed
to warm to room temperature and was stirred overnight. Then the aqueous phase was washed three
times with diethyl ether (50 mL; the ether extracts were discarded), acidified using 10% citric acid,
and extracted three times with chloroform (100 mL). The CHCl3 layers were combined, dried over
Na2SO4 overnight and concentrated in vacuo. The residue was purified by flash chromatography
(CHCl3:MeOH 8:1) to give 4e-h (58.7-69%) as pale colored gums.
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-α,β-diaminopropionic acid (4e):
1
Yield: 9.05g (58.7%); yellow gum; ESI MS 826 (esterification in MeOH in the spectrometer!); H-
NMR (DMSO, 350K)  1.60-1.80 (m, 6H, C-CH2-C), 2.80-3.30 (bm, 12H, N-CH2), 3.60 (m, 2H,
βCH2), 4.30 (m, 1H, αCH), 5.04 (s, 8H, CH2Ar), 7.29 (m, 20H, Ar-H); 13C-NMR  28.5, 29.5 (C-CH2-
C), 39.79, 39.81 (CH2NH), 43.7 (Nα-CH2), 49.6 [Nβ-(CH2)2], 51.8 (βC), 59.1 (αC), 65.4, 66.8 (CH2-

Molecules 2009, 14
3895
Ar), 127.7, 127.8, 128.3, 137.0 (CAr), 155.1, 156.4 (O-CO-N-), 172.4 (COOH); [D]26.6C
-10.30  0.5 (c=2 in acetone).
=
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-α,-diaminobutyric acid (4f):
Yield: 10.25 g (69%); colorless gum; ESI MS 839 (M’ + 1, esterification in MeOH in the
+
1
spectrometer!), 916 (M + C7H7 ); H-NMR (DMSO, 350K)  1.49-1.66 (bm, 8H, C-CH2-C), 3.0-3.6
(bm, 15H, N-CH2) 4.15 (m, 1H, αCH), 5.02 (s, 8H, CH2-Ar), 7.33 (bs, 20H, Ar-H); 13C-NMR  25.6,
26.1 (C-CH2-C), 28.5 (βC), 38.5 (CH2NH), 45.0 (NHα-CH2), 50.7 [Nγ-(CH2)2], 51.0 (γC), 60.1 (αC)
64.8, 66.0 (CH2-Ar), 126.8, 127.0, 127.8, 136.9 (CAr), 155.6 (O-CO-N-), 172.3 (COOH); [D]26.3C
-10.1  0.5 (c = 2 in acetone).
=
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-ornithine (4g):Yield: 8.16 g
(56.6%); pale yellow gum; ESI MS 840 (M+1), 854 (M’ + 1, esterification in MeOH in the
1
spectrometer!); H-NMR (DMSO, 350K)  1.48, 2.31 (2m, 10H, C-CH2-C, β, CH2), 2.90-3.80 (bm,
14H, N-CH2), 4.24 (m, 1H, αCH), 5.02, 5.18 (s, 8H, CH2Ar), 7.31 (m, 20H, Ar-H); 13C-NMR  23.2
(C), 26.8 (C-CH2-C), 29.4 (βC), 38.7 (CH2NH), 45.1 (Nα-CH2), 50.8 [Nδ-(CH2)2], 52.8 (δC), 59.8
(αC), 65.1, 66.3 (CH2-Ar), 127.8, 128.3, 137.3 (CAr), 155.4, 156.0 (O-CO-N-), 171.5 (COOH);
[D]26.5C = -10.12  0.5 (c = 2 in acetone).
Nα-benzyloxycarbonyl-N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-lysine (4h): Yield: 8.50 g
+
+
(62.27%); yellow gum; ESI MS 854 (M+1), 944 (M + C7H7 ), 1034 (M-1+2C7H7 ); 1H-NMR (DMSO,
350K)  1.15-2.0 (bm, 12H, β, γ, CH2, C-CH2-C), 2.50, 3.10 (2 bm, 14H, NCH2), 4.10 (m, 1H, αCH),
4.97, 5.03 (bs, 8H, CH2Ar), 7.29 (bm, 20H, Ar-H); 13C-NMR  22.6 (γC), 28.6 (C), 28.8 (C-CH2-C),
29.8 (βC), 37.6, 38.5 (CH2NH), 44.5 (Nα-CH2), 50.6 [Nε-(CH2)2], 52.2 (C), 58.4 (αC), 66.3, 66.9
(CH2-Ar), 127.9, 128.3, 136.7, 136.8 (CAr), 156.6, 157.1 (O-CO-N-), 172.7 (COOH); [D]23.6C
-10.33  0.5 (c = 2 in acetone).
=
N,Nβ-bis(benzyloxycarbonyl)-Nα,Nβ-bis(benzyloxycarbonyl-3-aminopropyl)-L-α,β-diaminopropionic
1
acid (13): Yield: 11.1g (61.83%); amber-colored gum; ESI MS 777 (M+23, CHCl3); H-NMR
(DMSO, 350K)  1.60-1.70 (m, 4H, C-CH2-C), 2.96 (m, 4H, CH2NH), 3.20, 3.30 (2 m, 4H, N-CH2),
13
3.70 (m, 2H, βCH2), 4.25 (m, 1H, αCH), 5.04 (s, 8H, CH2Ar), 7.29 (m, 20H, Ar-H); C-NMR  28.7
(C-CH2-C), 38.8 (CH2NH), 45.8 (NH-CH2), 47.1 (βC), 59.2 (αC), 64.9, 66.0 (CH2-Ar), 128.1, 128.7,
137.79, 137.82 (CAr), 155.7, 156.5 (O-CO-N-), 171.39 (COOH); [D]24.5C = -52.50  1 (c = 2 in
acetone).
3.5.5. General procedure for the reduction of the nitriles to the corresponding tetrakis-N-propylamino
derivatives 5a-d
To a derivative 3a-d (5 g, ca. 15 mmol) dissolved in dry THF (250 mL), 10 M solution of
BH3 × SMe2 (Aldrich, 15.8 mL) were added. The reaction mixture was heated to reflux and stirred
vigorously overnight. After cooling to room temperature, 1M HCl in MeOH (94.8 mL) and additional
MeOH (100 mL) were added. The mixture was evaporated in vacuo, the oily residue was treated three

Molecules 2009, 14
3896
times with 50 mL of MeOH and evaporated to dryness, to give 8.5-9 g (ca. 100%) of a white, very
hygroscopic foam. The elemental analysis shows a mixture of penta- and hexahydrochlorides of 5a-d.
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,β-diaminopropanol (5a): Yield: 8.9g (~100%); ESI MS 319
(M+1); 1H-NMR (D2O)  1.90-2.10 (m, 8H, C-CH2-C), 3.00-3.70 (3m, 20H, N-CH2, CH2OH), 3.90
(2m, 1H, αCH); 13C-NMR  26.7, 29.3 (C-CH2-C, C-CH2-C), 37.6, 38.0 (CH2NH2), 48.7, 57.8 [Nα-
(CH2)2, N-(CH2)2], 58.34 (C), 59.2 (αC), 61.7 (CH2OH); [D]26.5 C = +15.6 (1, c = 2 in 1M HCl).
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,-diaminobutanol (5b): Yield: 8.8g (~100%); ESI MS 353
(M+23); 1H-NMR (D2O)  1.60-2.2 (bm, 10H, CH2, C-CH2C), 2.80-3.60 (bm, 20H, N-CH2, CH2OH),
13
3.87 (m, 1H, αCH); C-NMR  21.7, 22.9 (C-CH2-C), 28.8 (C), 36.5, 36.6 (CH2NH2), 49.0, 50.1
[Nα-(CH2)2, Nγ-(CH2)2], 52.1 (γC), 57.35 (αC), 61.9 (CH2OH); [D]29.1C = +12.7 (1, c=2 in 1M
HCl).
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,-diaminopentanol (5c): Yield: 8.7g (~100%); ESI MS 347
1
(M+1); H-NMR (CDCl3)  1.55, 1.80, 2.10 (3m, 12H, β, CH2, C-CH2-C), 2.90-3.40 (bm, 20H, N-
CH2, CH2OH), 3.95 (2m, 1H, αCH); 13C-NMR  21.8 (C-CH2-C), 22.9 (C), 27.9 (βC), 36.6, 36.7
(CH2NH2), 50.0 [α, N-(CH2)2], 52.9 (C), 57.8 (αC), 61.6 (CH2OH); [D]29.1C = +12.3 (1, c=2 in
1M HCl).
N,N’,N,N’-tetrakis(3-aminopropyl)-L-α,-diaminohexanol (5d): Yield: 8.9 g (~100%); ESI MS 361
1
(M+1), 383 (M+23); H-NMR (CDCl3)  1.50-2.30 (3m, 14H, β, , CH2, C-CH2-C), 2.90-3.40 (bm,
20H, N-CH2, CH2OH), 3.95 (m, 1H, αCH); 13C-NMR  22.0 (C-CH2-C), 23.3 (C), 23.9 (C), 26.3
(βC), 36.9, 37.3 (CH2NH2), 49.2, 50.21 [α,  N-(CH2)2], 53.2 (C), 58.9 (αC), 61.8 (CH2OH);
[D]28.6C = +13.2  1, (c = 2 in 1M HCl).
3.5.6. Preparation of dendrimeric compounds 7e-7h
To benzylamine (3.21 g, 3.3 mL = 30 mmol) dissolved in dry THF (15 mL), 4g (2.55 g, 3 mmol),
HOBt (0.46 g, 3 mmol) and DCC (0.63 g, 3 mmol) were added, the mixture was stirred for 24 h and
the solvent was evaporated in vacuo. The residue was dissolved in CHCl3 (50 mL) and washed
consecutively with 10% Na2CO3, H2O, 1% citric acid and saturated NaCl solution, then dried over
Na2SO4 and evaporated to dryness: yield 2.07 g (74.3%) of the amide 6g as a light yellow gum.
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-diaminoalanine benzylamide (6e): C51H60O9N6,
1
Yield: 1,92g (65.3%); yellow gum; ESI MS 901 (M+H+), 923 (M+Na+); H-NMR (DMSO, 350K) 
1.66-1.85 (m, 6H, C-CH2-C), 2.79-3.38 (bm, 12H, N-CH2), 3.63 (m, 2H, βCH2), 4.23 (s, 2H, Ar-CH2-
NH), 4.3 (m, 1H, αCH), 5.05, 5.09 (2s, 8H, CH2-Ar), 7.2-7.3 (bm, 25H, Ar-H); 13C-NMR  28.6, 29.1
(C-CH2-C), 40.1 (CH2NH), 42.3 (Ar-CH2-NH), 43.7 (Nα-CH2), 49.5 [Nβ-(CH2)2], 51.1 (βC), 59.6 (αC),
65.1, 66.3 (CH2-Ar), 127.7, 127.8, 128.2 (CHAr), 136.6, 136.9 (CAr), 155.0, 156.2 (O-CO-N-), 173.1
(CONH).

Molecules 2009, 14
3897
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-diaminobutyric benzylamide (6f): Yield 2.15 g
1
(70.1%); pale yellow gum; ESI MS 915 (M+H+), 937 (M+Na+); H-NMR (DMSO, 350K)  1.45-1.7
(3m, 8H, βCH2, C-CH2-C), 2.9-3.5 (bm, 14H, N-CH2), 4.2 (s, 2H, Ar-CH2-NH), 4.28 (m, 1H, αCH),
13
5.07, 5.11 (2s, 8H, CH2-Ar), 7.2-7.3 (bm, 25H, Ar-H). C-NMR  25.5, 26.4 (C-CH2-C), 29.1 (βC),
38.5 (CH2NH), 42.4 (Ar-CH2-NH), 44.5 (NHα-CH2), 50.3 [Nγ-(CH2)2], 51.1 (γC), 59.8 (αC), 65.0, 66.1
(CH2-Ar), 126.9, 127.0, 127.8, 128.0 (CHAr), 136.8, 137.0 (CAr), 155.3, 156.2 (O-CO-N-), 173.0
(CONH).
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-ornithine benzylamide (6g): Yield: 74.3%;
C53H64O9N6, pale yellow gum; ESI MS 929 (M+1), 951 (M+23), 967 (M+39), 1,019 (M + 91 =
+
C7H7 ); 1H-NMR (DMSO, 350K)  1.40 - 2.30 (2m, 10H, C-CH2-C, β, CH2,), 2.85-3.70 (bm, 14H, N-
CH2), 4.22 (s, 2H, Ar-CH2-NH), 4.31 (m, 1H, αCH), 5.05, 5.20 (s, 8H, CH2Ar), 7.29 (m, 25H, Ar-H);
13C- NMR  23.2 (C), 26.3 (C-CH2-C), 29.6 (βC), 38.6 (CH2NH), 42.2 (Ar-CH2-NH), 44.2 (Nα-CH2),
50.6 [Nδ-(CH2)2], 52.6 (δC), 59.7 (αC), 65.0, 66.3 (OCH2-Ar), 127.6, 127.7, 128.3, 137.3 (CAr), 155.2,
156.1 (O-CO-N-), 171.3 (CONH).
N,N,N’-tris(benzyloxycarbonyl-3-aminopropyl)-L-lysine benzylamide (6h): Yield: 78.6%; C54H66O9N6,
+
yellow gum; ESI MS 943 (M+1), 965 (M+23), 981 (M+39) 1,033 (M+91 = C7H7 ); 1H-NMR (DMSO,
350K)  1.2-2.1 (bm, 12H, β, γ, CH2, C-CH2-C), 2.52, 3.12 (2 bm, 14H, NCH2), 4.19 (m, 1H, αCH),
4.26 (s, 2H, Ar-CH2-NH), 5.03, 5.1 (bs, 8H, CH2Ar), 7.15-7.3 (bm, 25H, Ar-H); 13C-NMR  22.7(γC),
28.4 (C), 28.5 (C-CH2-C), 29.4 (βC), 37.4, 38.1 (CH2NH), 42.1 (Ar-CH2-NH), 43.9 (Nα-CH2), 49.9
[Nε-(CH2)2], 52.1 (C), 58.5 (αC), 66.4, 66.8 (OCH2-Ar), 127.9, 128.4, 136.8, 136.9 (CAr), 156.6,
156.9 (O-CO-N-), 171.9 (CONH).
A) 6g (1.5 g, 1.6 mmol) dissolved in MeOH (25 mL) was stirred for 24 h with 10% Pd/C (150 mg)
under an atmospheric pressure of H2. Then the catalyst was separated on Celite® and washed with
MeOH. The collected methanol fractions were evaporated to dryness yielding 0.59 g (93.1%) of
deprotected 6g as a white waxy solid. To the solution of deprotected 6g in DMF (25 mL), (2-Cl-
Z)Lys(Boc) (2.04 g, 4.95 mmol), HOSu (0.57 g, 4.95 mmol) and DCC (1.02 g, 4.95 mmol) were
added. The mixture was stirred for 24 h (until disappearance of free amino groups in the ninhydrin
test), filtered and evaporated in vacuo. The residue was dissolved in CHCl3 (50 mL) and washed
with 10% Na2CO3, H2O, 1% citric acid, dried over Na2SO4 overnight and evaporated to dryness.
The residue was purified by flash chromatography (CHCl3-MeOH 8:1) to give 1.7 g (71.3%) of
Boc-protected 7g as a dark yellow resin.
B) Boc-protected 7g (0.5 g, 0.32mmol) was dissolved in CH2Cl2 (5 mL) and trifluoroacetic acid (TFA,
5 mL) was added. The reaction mixture was stirred at room temperature for 3 h. Then the reaction
mixture was evaporated in vacuo, the residue was dissolved in ethyl acetate (5 mL) and evaporated
(3 times) and then in diethyl ether (5 mL) and evaporated (twice) – to remove all remaining
trifluoroacetic acid. Trifluoroacetate ions were replaced by chlorides by dissolving the oily residue
in HCl-saturated ethyl acetate and evaporation in vacuo (four times) to give 470 mg (99%) of 7g
hexahydrochloride as a yellow glassy gum.

Molecules 2009, 14
3898
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-diaminoalanine
benzylamide
pentahydrochloride (7e): C61H87O10N12Cl3·5HCl; Yield 0.99g (97.5%); ESI MS 627 (M+2H+)2+ - base
1
peak, 647 (M+H++Na++H2O)2+, 1,253 (M+H+); H-NMR (DMSO, 350K) 1.1 – 2.1 (4bm, 24H, C-
CH2-C, β, γ,δCH2 G-1 Lys), 2.6-3.5 (3 bm, 20H, αNH-CH2, βN-(CH2)2, CH2-NH core, εCH2 G-1 Lys),
3.53 (m, 1H, αCH core), 4.18 (m, 3H, αCH G-1 Lys), 4.30 (Ar-CH2-NH), 5.11, 5.18 (2bs, 6H, Ar-
13
CH2O- from 2-Cl-Z), 7.2-7.4 (bm; 17H, Ar-H); C-NMR  22.2 (γC G-1 Lys), 25.1 (C-CH2-C core),
29.7 (C G-1 Lys), 31.0 (βC G-1 Lys), 38.5 (C G-1 Lys), 40.1 (CH2NH core), 43.4 (ArCH2-NH), 44.6
(Nα-CH2 core), 50.9 [Nβ-(CH2)2 core], 52.7 (βC core), 55.4 (αC G-1 Lys), 59.5 (αC core Lys), 62.1,
62.3 (Ar-CH2-O), 126.5, 126.6, 127.4, 127.8, 128.7, 129.0, 129.1, 129.2 (CHAr), 132.0 (CAr-Cl), 133.7
(CAr-CH2-NH), 134.1 (CAr-CH2-O), 155.6, 156.7 (O-CO-NH-), 171.3 (CONH G-1 Lys), 172.2 (CONH
core); Anal. Calcd for C61H87O10N12Cl3·5HCl, (12 days/P2O5): C, 51.0; H, 6.45; N, 11.70; Cl, 19.73.
Found: C, 50.67; H, 6.51; N, 11.43; Cl, 19.5.
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-diaminobutyro
benzylamide
pentahydrochloride (7f): C62H89O10N12Cl3·5HCl; Yield 1.0g (98.3%); hygroscopic yellow gum; ESI
1
MS 634 (M+2H+)2+- base peak, 654 (M+H++Na++H2O)2+, 1,267 (M+H+); H-NMR (DMSO, 350K) 
1.1 – 2.1 (bm, 26H, C-CH2-C, βCH2 core and β, γ,δCH2 G-1 Lys), 2.5-3.55 (2 bm, 20H, αNH-CH2, γ
N-(CH2)2, CH2-NH core, εCH2 G-1 Lys), 3.68 (m, 1H, αCH core), 4.15 (m, 3H, αCH G-1 Lys), 4.28
(Ar-CH2-NH), 5.09, 5.15 (2bs, 6H, Ar-CH2O- grup 2-Cl-Z), 7.2-7.4 (bm; 17H, Ar-H); 13C-NMR 
22.1 (γC G-1 Lys), 25.3 (C-CH2-C core), 29.1 (βC core), 29.6 (C G-1 Lys), 31.1 (βC core Lys), 38.2
(C G-1 Lys), 39.9 (CH2NH core), 43.2 (ArCH2-NH), 44.9 (Nα-CH2 core), 50.8 [Nγ-(CH2)2 core], 53.1
(γC core), 55.3 (αC G-1 Lys), 59.7 (αC core), 62.2, 62.4 (Ar-CH2-O), 126.5, 126.7, 127.3, 127.8,
128.7, 129.0, 129.1, 129.2 (CHAr), 131.9 (CAr-Cl), 133.8 (CAr-CH2-NH), 134.0 (CAr-CH2-O), 155.7,
156.8 (O-CO-NH-), 171.4 (CONH G-1 Lys), 172.2 (CONH core); Anal. Calcd for
C62H89O10N12Cl3·5HCl, (12 days/P2O5): C, 51.3; H, 6.52; N, 11.58; Cl, 19.54. Found: C, 50.96; H,
6.61; N, 11.31; Cl, 19.42.
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-ornithine benzylamide penta-
hydrochloride (7g): Yield: 99%; C63H91O10N12Cl3·5HCl, hygroscopic, glass-like gum; ESI MS 641
(M+2)2+, 1,281 (M+1), 1,299 (M+18+1); 1H-NMR (DMSO, 350K)  1.3 – 2.2 (bm, 28H, core β, γCH2,
C-CH2-C, G1 β, γ,δCH2), 2.4-3.3 (bm, 20H, core αNH-CH2, δN-(CH2)2, CH2-NH, G1 εCH2), 3.60 (m,
1H, core αCH), 4.19 (m, 3H, G1 αCH), 4.38 (Ar-CH2-NH), 5.17 (m, 6H, G1 Ar-CH2O-), 7.2-7.4 (bm;
17H, Ar-H); 13C-NMR  22.4 (G1 γC), 23.1 (core C), 27.6 (core C-CH2-C), 29.3 (G1 C), 29.5 (core
βC), 32.3(G1 βC), 36.3 (core CH2NH), 39.8, 40.0 (G1 C), 43.2 (ArCH2-NH), 44.1 (core Nα-CH2),
49.7 [core Nδ-(CH2)2], 51.6 (core δC), 55.0, 55.2 (G1 αC), 58.8 (core αC), 66.5, 66.8 (Ar-CH2-O),
126.67, 126.7, 126.8, 127.6, 128.5, 128.9, 129.0, 129.3 (CHAr), 132.8 (G1 CAr-Cl), 133.9 (CAr-CH2-
NH), 134.4 (G1 CAr-CH2-O), 155.8, 156.0 (G1 O-CO-N-), 174.0 (core CONH), 175.2 (G1 CONH);
Anal. Calcd for C63H91O10N12Cl3·5HCl: C, 51.65; H, 6.26; N, 11.47; Cl, 19.35. Found: C, 51.43; H,
6.45; N, 10.31; Cl, 19.05.
N,N,N’-tris[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-3-aminopropyl]-L-lysine benzylamide penta-
hydrochloride (7h): Yield: 99%; C64H93O10N12Cl3·5HCl, hygroscopic, glassy solid; ESI MS 648

Molecules 2009, 14
3899
1
(M+2)2+, 659 (M+1+23)2+ 1,295 (M+1), 1,313 (M+18+1); H-NMR (DMSO, 350K)  1.1–2.1 (bm,
30H, C-CH2-C, core and G1 β, γ,δCH2), 2.6-3.5 (2 bm, 20H, core αNH-CH2, εN-(CH2)2, CH2-NH, G1
εCH2), 3.65 (m, 1H, core αCH), 4.05 (m, 3H, G1 αCH), 4.33 (Ar-CH2-NH), 5.12 (m, 6H, G1 Ar-
CH2O-), 7.2-7.4 (bm; 17H, Ar-H); 13C-NMR  21.9 (G1 γC), 22.1 (core C), 24.5 (core δC) 25.6 (core
C-CH2-C), 28.8 (core βC), 29.8 (G1 C), 31.0 (G1 βC), 38.2 (G1 C), 39.7 (core CH2NH), 43.1
(ArCH2-NH), 45.3 (core Nα-CH2), 51.4 [core Nε-(CH2)2], 53.6 (core εC), 55.6 (G1 αC), 59.9 (core αC),
62.4, 62.5 (Ar-CH2-O), 126.5, 126.7, 127.1, 127.8, 128.7, 129.0, 129.1, 129.2 (CHAr), 131.8 (G1 CAr-
Cl), 133.9 (CAr-CH2-NH), 134.0 (G1 CAr-CH2-O), 156.7, 157.0 (G1 O-CO-N-), 171.5 (G1 CONH),
172.0 (core CONH); Anal. Calcd for C64H93O10N12Cl3*5HCl: C, 51.9; H, 6.67; N, 11.36; Cl, 19.17.
Found: C, 51.63; H, 6.8; N, 11.19; Cl, 18.93.
3.5.7. Preparation of the dendrimeric compounds 9a-d
5c (450 mg, 0.72 mmol) was suspended in DMF (15 mL) with addition of N(Et)3 (2.40 mL,
17.28 mmol) and stirred at 40 °C until all of 5c was dissolved. Then (Boc)-L-Phe-2,4,5-trichlorophenyl
ester 1.76 g (3.96 mmol, 5.5 eq) was added and the reaction mixture was stirred at room temperature
for 7 days until the complete disappearance of free amino groups in the ninhydrin test. The solution
was evaporated in vacuo, the residue was dissolved in CHCl3 (50 mL) and washed with 20% K2CO3
and saturated NaCl solution, dried over Na2SO4 overnight, filtered and evaporated to dryness. The
residue was purified by flash chromatography (CHCl3-MeOH, 8:1) to give 520 mg of 9c (54%) as a
white foam.
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,-diaminopropanol (9a): Yield 2.76g
(66%); C71H106O13N10; white foam; ESI MS 654 (M+2H+)2+, 1,307 (M+H+); 1H-NMR (DMSO, 350K)
1.2-1.3 (m, 36H, Boc C-CH3), 1.47 (m, 8H, C-CH2-C), 2.33 (m, 4H, Nβ-CH2), 2.39 – 2.43 (m, 6H,
βCH2, Nα-CH2), 2.83, 2.93 (dd, J 5.3, 8.5 Hz, 8H, Ar-CH2), 3.18 (m, 8H, CH2-NH), 3.42 (m, 2H,
CH2OH), 3.86 (m, 1H, αCH core), 4.23 (m, 4H, αCH Phe), 7.2 (m; 20H, Ar-H). 13C-NMR  21.2 (C-
CH2-C), 28.2 (Boc C-CH3), 36.8, 36.9 (α, βCH2NH core), 50.0 [α, βN-(CH2)2], 52.8 (βC), 58.4 (αC),
62.0 (CH2OH), 77.7 (Boc C-CH3), 125.2, 125.6, 127.6, 127.8, 128.7, 128.8 (CHAr), 137.6, 138.5 (CAr),
154.4, 154.8 (O-CO-NH), 172.2 (CONH Phe).
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,-diaminobutanol (9b): Yield 3.9 g (71%);
1
C72H108O13N10; ESI MS 662 (M+2H+)2+, 1,321 (M+H+); H-NMR (DMSO, 350K) 1.2-1.3 (m, 36H,
Boc C-CH3), 1.46 (m, 10H, βCH2, C-CH2-C), 2.35 (m, 6H, Nγ-CH2), 2.38 (m, 4H, Nα-CH2), 2.82, 2.94
(dd, J 5.3, 8.5 Hz, 8H, Ar-CH2), 3.15 (m, 8H, CH2-NH), 3.39 (m, 2H, CH2OH), 3.96 (m, 1H, αCH
core), 4.22 (m, 4H, αCH Phe), 7.2 (m; 20H, Ar-H); 13C-NMR  21.6 (C-CH2-C), 27.8 – 28.4 (βC, Boc
C-CH3), 36.6, 36.7 (α, γCH2NH core), 50.1 [α, γN-(CH2)2], 52.6 (γC), 57.7 (αC), 61.7 (CH2OH), 77.8
(Boc C-CH3), 125.2, 125.7, 127.6, 127.7, 128.6, 128.8 (CHAr), 137.7, 138.6 (CAr), 154.3, 154.5 (O-
CO-NH), 171.3 (CONH Phe).
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,-diaminopentanol (9c): Yield: 54%;
1
C73H110O13N10, white foam; ESI MS 1,335 (M+1); H-NMR (350K, DMSO)  1.25-1.35 (m, 36H, C-

Molecules 2009, 14
3900
CH3, Boc), 1.48 (m, 12H, core β, CH2, C-CH2-C), 2.34 (m, 6H, core N-CH2), 2.40 (m, 4H, core Nα-
CH2), 2.80, 2.97 (dd, J = 5.26, 8.55 Hz, 8H, G1 Ar-CH2), 3.10 (m, 8H, core CH2-NH), 3.41 (m, 2H,
core CH2OH), 3.98 (m, 1H, core αCH), 4.20 (m, 4H, G1 αCH), 7.19 (m; 20H, G1 Ar-H). 13C-NMR 
26.5 (C,  core C-CH2-C), 28.5 (βC, αC-CH2-C), 36.7, 36.8 (α,  CH2NH), 37.7 (G1 Ar-CH2), 47.5,
50.9 [core α, N-(CH2)2], 53.6 (core C), 55.5, 55.7 (core and G1 αC), 61.1 (core CH2OH), 77.7 [O-C-
(CH3)3], 125.2, 125.6, 127.5, 127.2, 128.7, 128.9 (G1 CHAr), 137.6, 138.6 (G1 CAr), 154.3, 154.5 (G1
O-CO-NH), 170.7 (G1 CO-NH).
N,N',N,N’-tetrakis(Boc-L-phenylalanyl-3-aminopropyl)-L-α,ε-diaminohexanol (9d): Yield: 73.5%;
1
C74H112O13N10, white foam; ESI MS 675 (M+2)2+, 1,349 (M+1)+; H-NMR (350K, DMSO)  1.24-
1.36 (m, 36H, C-CH3, Boc), 1.43 (m, 14H, core β, , δCH2, C-CH2-C), 2.31 (m, 6H, core Nε-CH2),
2.42 (m, 4H, core Nα-CH2), 2.79, 2.95 (dd, J = 5.27, 8.54 Hz, 8H, G1 Ar-CH2), 3.15 (m, 8H, core CH2-
NH), 3.43 (m, 2H, core CH2OH), 3.99 (m, 1H, core αCH), 4.22 (m, 4H, G1 αCH), 7.20 (m; 20H, G1
Ar-H). 13C-NMR  24.1 (core γC), 26.6 (core δC, ε C-CH2-C), 28.5 (core βC, αC-CH2-C), 36.7, 36.8
(core α, ε CH2NH), 37.7 (G1 Ar-CH2-CH), 47.8, 51.0 [core α, εN-(CH2)2], 53.8 (core εC), 54.9, 55.1
(core and G1 αC), 61.1 (core CH2OH), 77.8 [O-C-(CH3)3], 125.2, 125.5, 127.2, 128.7, 129.0 (G1
CHAr), 137.7, 138.7 (G1 CAr), 154.7, 154.7 (G1 O-CO-NH), 170.6 (G1 CO-NH).
3.5.8. Preparation of the dendrimeric compound 10c
A) 9c (0.5 g, 0.375 mmol) was dissolved in CH2Cl2 (5 mL) and TFA (5 mL) was added. The reaction
mixture was stirred at room temperature for 3 h. Then the reaction mixture was evaporated in
vacuo, the residue was dissolved in ethyl acetate (5 mL) and evaporated (three times) and then in
diethyl ether (5 mL) and evaporated (twice) – to remove all remaining trifluoroacetic acid. Dark-
orange oil (0,4g) was used for the next step without purification.
B) (2-Cl-Z)L-Lys(Boc) (0.68 g, 1.65 mmol) and N-hydroxysuccinimide (HOSu, 0.19 g 1.66 mmol)
was dissolved in THF (10 mL) followed by addition of DCC (0.34 g, 1.66 mmol). The solution was
stirred for 0.5 h and added to the solution of Boc-deprotected 9c and N(Et)3 (1 mL, 7.13 mmol) in
DMF (5 mL). The final solution was stirred for 48 h at room temperature, then DCU was filtered
off and the solvents was evaporated in vacuo. The residue was dissolved in CHCl3 (50 mL) and
washed with 10% Na2CO3 and saturated NaCl solution (twice), dried over Na2SO4 overnight,
filtered and evaporated to dryness. The residue was purified by flash chromatography
(CHCl3:MeOH, 15:1) to give 0.729 g of Boc protected 10c (77.1%) as a white foam.
C) Boc-10c (360 mg, 0.14 mmol) was deprotected according to procedure A). Trifluoroacetate ions
were replaced by chlorides by dissolving the oily residue in HCl-saturated ethyl acetate and
evaporation in vacuo (four times) to give 330 mg of 10c hexahydrochloride (99%) as a white,
hygroscopic foam.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,-
diaminopropanol hexahydrochloride (10a): Yield 1.0 g (98%); C107H142O17N18Cl4·6HCl; pale yellow
foam; ESI MS 1,046 (M+2H+)2+, 1,057 (M+H++Na+)2+, 2,105 (M+H+); 1H-NMR (DMSO, 298K)  1–
2 (bm, 32H, γ,β,δCH2 G-1 Lys, C-CH2-C core arms), 2.6-3.7 (4 bm, 37H, CH2-NH core, εCH2 G-1 Lys
i βCH2 core, α, βN-(CH2)2 core, Ar-CH2-CH Phe, CH2OH & αCH core), 4.1, 4.6 (2 bm, 8H, αCH Lys

Molecules 2009, 14
3901
& Phe, respectively), 5.16 (4s, 8H, Ar-CH2 from 2-Cl-Z), 7.2 – 7.45 (m; 36H, Ar-H); 13C-NMR  22.1,
22.3 (γC G-1 Lys), 23.4 (α, γC-CH2-C core), 26.6 (δC G-1 Lys), 28.7 (βC core), 30.3, 31.1 (βC G-1
Lys), 36.3, 36.5 (α, βCH2NH core), 37.7 (Ar-CH2-CH), 38.3, 38.6 (εCH2NH2 G-1 Lys), 48.8, 49.7 [α,
βN-(CH2)2 core], 52.3 (βC core), 54.2, 55.3 (αC Phe and Lys, respectively), 58.8 (αC core), 60.3
(CH2OH core) 62.7, 62.8 (Ar-CH2-O), 126.3, 127.2, 128.2, 129.1, 129.3, 129.6, 129.7, 129.8 (CHAr),
132.1, 133.2 (Car-Cl), 134.3, 134.4 (Car-CH2 Phe), 136.9 (Car-CH2O), 155.4, 155.5 (O-CO-NH), 171.3,
172.4 (CONH Phe and L-Lys, respectively); Anal. Calcd for C107H142O17N18Cl4·6HCl: C, 55.6; H,
6.45; N, 10.9; Cl, 15.33. Found: C, 55.38; H, 6.63; N, 10.58; Cl, 15.02.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,-
diaminobutanol hexahydrochloride (10b): Yield 1.01 g (99%); C108H144O17N18Cl4·6HCl, white
hygroscopic foam; ESI MS 1,053 (M+2H+)2+, 1,064 (M+H++Na+)2+, 2,105 (M+H+); 1H-NMR (DMSO,
298K)  1 – 2 (bm, 34H, βCH2 core i γ,β,δCH2 G-1 Lys, C-CH2-C core arms), 2.65-3.75 (3 bm, 37H,
CH2-NH core, εCH2 G-1 Lys & γCH2 core, α, γN-(CH2)2 core, Ar-CH2-CH Phe, CH2OH and αCH
core), 4.15, 4.57 (2 bm, 8H, αCH Lys and Phe, respectively), 5.11 (4s, 8H, Ar-CH2 from 2-Cl-Z), 7.2–
13
7.45 (m; 36H, Ar-H). C-NMR  22.1, 22.2 (γC G-L-Lys), 23.5 (α, δC-CH2-C core), 26.5 (δC G-1
Lys), 28.8 (βC core), 30.2, 31.1 (βC G-1 Lys), 36.2, 36.3 (α, γCH2NH core), 37.6 (Ar-CH2-CH), 38.4,
38.5 (εCH2NH2 G-1 Lys), 48.9, 49.7 [α, γN-(CH2)2 core], 52.0 (γC core), 54.1, 55.1 (αC Phe & Lys,
respectively), 58.4 (αC core), 60.1 (CH2OH core) 62.6, 62.8 (Ar-CH2-O), 126.2, 127.3, 128.1, 129.1,
129.3, 129.5, 129.7, 129.8 (CHAr), 132.2, 133.3 (Car-Cl), 134.2, 134.3 (Car-CH2 Phe), 137.0 (Car-
CH2O), 155.3, 155.6 (O-CO-NH), 171.2, 172.6 (CONH Phe and Lys, respectively); Anal. Calcd for
C108H144O17N18Cl4*6HCl: C, 55.74; H, 6.5; N, 10.83; Cl, 15.23. Found: C, 55.5; H, 6.7; N, 10.51; Cl,
14.94.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,-
diaminopentanol hexahydrochloride (10c): Yield: 99%; C109H146O17N18Cl4*6HCl, white hygroscopic
1
foam; ESI MS 1,051 (M-18+2)2+, 1,060 (M+2)2+, 1,071 (M+1+23)2+, 2,119 (M+1)+; H-NMR (298K,
DMSO); NMR:  1 – 2 (bm, 36H, core γ, βCH2, G2 γ,β,δCH2, core C-CH2-C), 2.7-3.8 (3 bm, 37H,
core CH2-NH, G2 εCH2, core δCH2, core α, δN-(CH2)2, G1 Ar-CH2-CH, core CH2OH and αCH), 4.1,
4.55 (2 bm, 8H, G1 and G2 αCH), 5.15 (m, 8H, G2 Ar-CH2), 7.25 (m; 36H, Ar-H), 8.15 (bm, 21H, N-
H). 13C=NMR  22.2, 22.3 (G2 γC), 23.2 (core α, δC-CH2-C), 23.9 (core γC), 26.4 (G2 δC), 28.4 (core
βC), 30.3, 31.0 (G2 βC), 35.9, 36.2 (core α, δ CH2NH), 37.7 (G1 Ar-CH2-CH), 38.3, 38.6 (G2
εCH2NH2), 48.9, 49.6 [core α, δN-(CH2)2], 52.1 (core δC), 54.1, 55.0 (G1 and G2 αC), 57.9 (core αC),
60.3 (core CH2OH) 62.7, 62.9 (G2 CH2Ar), 126.2, 127.3, 128.0, 129.1, 129.4, 129.6, 129.7, 129.7
(CHAr), 132.1, 133.3 (G2 Car-Cl), 134.3, 134.4 (G1 Car-CH2), 136.9 (G2 Car-CH2), 155.2, 155.7 (G2 O-
CO-NH), 171.3, 172.8 (G1 and G2 CO-NH); Anal. Calcd for C109H146O17N18Cl4·6HCl: C, 55.92; H,
6.54; N, 10.77; Cl, 15.14. Found: C, 55.68; H, 6.65; N, 10.43; Cl, 14.96.
N,N',N,N’-tetrakis[(Nα-2-chlorobenzyloxycarbonyl)-L-lysil-L-phenylalanyl-3-aminopropyl]-L-α,ε-
diaminohexanol hexahydrochloride (10d): Yield: 99%; C110H148O17N18Cl4*6HCl, white hygroscopic
1
foam; ESI MS 1,058 (M-18+2)2+, 1,067 (M+2)2+, 1,078 (M+1+23)2+, 2,133 (M+1)+; H-NMR (298K,
DMSO)  1–2 (bm, 38H, core and G2 γ,β,δCH2, core C-CH2-C), 2.6-3.8 (3 bm, 37H, core CH2-NH,

Molecules 2009, 14
3902
core and G2 εCH2, core α, εN-(CH2)2, G1 Ar-CH2-CH, core CH2OH, core αCH), 4.0, 4.49 (2 bm, 8H,
G1 and G2 αCH), 5.10 (m, 8H, G2 Ar-CH2), 7.20 (m; 36H, G1 and G2 Ar-H), 8.11–8.7 (3 bm, 21H,
13
N-H); C-NMR  22.2, 22.3 (G2 γC), 23.0 (core α, εC-CH2-C), 24.4 (core γC), 25.2 (core δC), 26.3,
26.4 (G2 δC), 28.6 (core βC), 30.0, 31.2 (G2 βC), 36.0, 36.2 (core α, ε CH2NH), 37.7 (G1 Ar-CH2-
CH), 38.3, 38.4 (G2 εCH2NH2), 48.5, 49.8 [core α, εN-(CH2)2], 51.3 (core εC), 53.7, 54.0, 54.7 (G1
and G2 αC), 57.4 (core αC), 60.4 (core CH2OH) 62.8, 62.9 (G2 CH2Ar), 126.2, 127.2, 127.9, 129.2,
129.5, 129.6, 129.7 (G1 and G2 CHAr), 132.1, 133.2 (G2 Car-Cl), 134.2, 134.3 (G1 Car-CH2), 137.6
(G2 Car-CH2), 155.6, 155.8 (G2 O-CO-NH), 171.0, 171.4, 172.7 (G1 and G2 CO-NH); Anal. Calcd for
C110H148O17N18Cl4*6HCl: C, 56.1; H, 6.6; N, 10.7; Cl, 15.05. Found: C, 55.65; H, 6.76; N, 10.35 Cl,
14.91.
4. Conclusions
Two series of new low molecular weight amphiphilic peptide dendrimers were efficiently
synthesized and characterized. They contain novel core elements - basic tris-amino acids 3 and
tetrakis-amino alcohols 4. Their application in the synthesis of amphiphilic peptide dendrimers yielded
molecules with (+5)/(+6) charge and multiple distribution of cationic and lipophylic groups in the 1st
dendrimer generation. These cationic dendrimeric peptides were significantly more potent against
Gram(+) and Gram(-) bacteria and fungi from C. albicans genus, with single micromolar MICs, than
previously designed derivatives built around a Lys(Lys)2 core. Interestingly, for the first time high
activity of the dendrimeric species against antibiotics resistant MRSA ATCC 43300 and ESBL ATCC
BAA-198 pathogens was detected. Both, higher antimicrobial potency and higher hemolytic properties
are associated with higher charge and hydrophobicity of the present dendrimers. The high potency and
broad range of activity of the new and previously described compounds evidences rationality of
searching for simple, economically attractive antimicrobial compounds in the class of low molecular
weight peptide dendrimers.
Acknowledgements
Financial support from the Ministry of Science and Higher Education of Poland, grant No 3T09B
115 28 and EC STREP program NORMOLIFE (CT-2006-037733) are acknowledged.
References and Notes
1. Coates, A.R.M.; Hu, Y. Novel approaches to developing new antibiotics for bacterial infections.
British J. Pharm. 2007, 152, 1147-1154.
2. Yacoby, I.; Benhar, I. Antibacterial nanomedicine. Nanomedicine 2008, 3, 329-341.
3. Boas, U.; Heegaard, P.M.H. Dendrimers in drug research. Chem. Soc. Rev. 2004, 33, 43-63.
4. Tomalia, D.A.; Frechet, J.M.J. Introduction to dendrimers and dendritic polymers. Prog. Polym.
Sci. 2005, 30, 217-219.
5. Tomalia, D.A.; Frechet, J.M.J. Discovery of dendrimers and dendritic polymers: A brief historical
perspective. J. Polym. Sci. A Polym. Chem. 2002, 40, 2719-2728.

Molecules 2009, 14
3903
6. Stiriba, S.E.; Frey, H.; Haag, R. Dendritic polymers in biomedical applications: From potential to
clinical use in diagnostics and therapy. Angew. Chem. Int. Ed. 2002, 41, 1329-1334.
7. Goodman, M.; Cai, W.B.; Kinberger, G.A. The new science of protein mimetics. Macromol.
Symposia 2003, 201, 223-236.
8. Gong, E.; Matthews, B.; McCarthy, T.; Chu, J.H.; Holan, G.; Raff, J.; Sacks, S. Evaluation of
dendrimer SPL7013, a lead microbicide candidate against herpes simplex viruses. Antivir. Res.
2005, 68, 139-146.
9. Landers, J.J.; Cao, Z.Y.; Lee, I.; Piehler, L.T.; Myc, P.P.; Myc, A.; Hamouda, T.; Galecki, A.T.;
Baker, J.R. Prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymer. J.
Infect. Dis. 2002, 186, 1222-1230.
10. Chaves, F.; Calvo, J.C.; Carvajal, C.; Rivera, Z.; Ramirez, L.; Pinto, M.; Trujillo, M.; Guzman, F.;
Patarroyo, M.E. Synthesis, isolation and characterization of Plasmodium falciparum antigenic
tetrabranched peptide dendrimers obtained by thiazolidine linkage. J. Pept. Res. 2001, 58,
307-316.
11. Sadler, K.J.; Tam, P. Peptide dendrimers: Applications and synthesis. Rev. Mol. Biotechnol. 2001,
90, 195-229.
12. Svenson, S.; Tomalia, D.A. Dendrimers in biomedical applications—reflections on the field. Adv.
Drug Deliv. Rev. 2005, 57, 2106-2129.
13. Dufes, C.; Uchegbu, I.F.A.; Schatzlein, G. Dendrimers in Gene Delivery. Adv. Drug Deliv. Rev.
2005, 57, 2177-2202.
14. Braun, C.S.; Vetro, J.A.; Tomalia, D.A.; Koe, G.S.; Koe, J.G.; Middaugh, C.R. Structure/function
relationships of polyamidoamine/DNA dendrimers as gene delivery vehicles. J. Pharm. Sci. 2005,
94, 423-436.
15. Calabretta, M.K.; Kumar, A.; McDermott, A.M.; Cai, C.Z. Antibacterial activities of
poly(amidoamine) dendrimers terminated with amino and poly(ethylene glycol) groups.
Biomacromolecules 2007, 8, 1807-1811.
16. Czen, C.Z.; Cooper, S.L. Interactions between dendrimer biocides and bacterial membranes.
Biomaterials 2002, 23, 3359-3368.
17. Ortega, P.; Copa-Patiño, J.L.; Muñoz-Fernandez, M.A.; Soliveri, J.; Gomez, R.; de la Mata, F.J.
Amine and ammonium functionalized chloromethylsilane-ended dendrimers. Antimicrobial
activity studies. Org. Biomol. Chem. 2008, 6, 3264-3269.
18. Cheng, Y.Y.; Qu, H.; Ma, M.L.; Xu, Z.H.; Xu, P.; Fang, Y.J.; Xu T.W. Polyamidoamine
(PAMAM) dendrimers as biocompatible carriers of quinolone antimicrobials: An in vitro study.
Eur. J. Med. Chem. 2007, 42, 1032-1038.
19. Yung, H.; Lopina, S.T. Penicillin V—Conjugated PEG-PAMAM star polymers. J. Biomat. Sci.-
Polym. 2003, 14, 1043-1056.
20. Balogh, L.; Swanson, D.R.; Tomalia, D.A.; Hagnauer, G.L;. McManus, A.T. Dendrimer-silver
complexes and nanocomposites as antimicrobial agents. Nano Lett. 2001, 1, 18-21.
21. Hancock, R.E.W.; Patrzykat, A. Clinical development of cationic antimicrobial peptides: From
natural to novel antibiotics. Curr. Drug Targets Infect. Disord. 2002, 2, 79-83.
22. Marr, A.K.; Gooderham, W.J.; Hancock, R.E.W. Antibacterial peptides for therapeutic use:
Obstacles and realistic outlook. Curr. Opin. Pharm. 2006, 6, 468-472.

Molecules 2009, 14
3904
23. Tam, J.P.; Lu, Y.A.; Yang, J.L. Antimicrobial dendrimeric peptides. Eur. J. Biochem. 2002, 269,
923-932.
24. Rojo, J.; Delgado, R. Dendrimers and dendritic polymers as anti-infective agents: New
antimicrobial strategies for therapeutic drugs. Antiinf. Agents Med. Chem. 2007, 6, 151-174.
25. Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature
2007, 449, 819-826.
26. Janiszewska, J.; Swieton, J.; Misicka, A.; Lipkowski, A.W.; Urbanczyk-Lipkowska, Z. Small
peptide dendrimers with antimicrobial properties. Peptides 2002, 568-567.
27. Janiszewska, J.; Urbanczyk-Lipkowska, Z. Amphiphilic dendrimeric peptides as model non-
sequential pharmacophores with antimicrobial properties. J. Mol. Microbiol. Biotechn. 2007, 13,
220-225.
28. Janiszewska, J.; Swieton, J.; Lipkowski, A.W.; Urbanczyk-Lipkowska, Z. Low molecular mass
peptide dendrimers that express antimicrobial properties. Bioorg. Med. Chem. Lett. 2003, 13,
3711-3713.
29. Klajnert, B.; Janiszewska, J.; Urbanczyk-Lipkowska, Z.; Bryszewska, M.; Shcharbin, D.;
Labieniec, M. Biological properties of low molecular mass peptide dendrimers. Int. J. Pharm.
2006, 309, 208-217.
30. Klajnert, B.; Janiszewska, J.; Urbanczyk-Lipkowska, Z.; Bryszewska, A.; Epand, R.M. DSC
studies on interactions between low molecular mass peptide dendrimers and model lipid
membranes. Int. J. Pharm. 2006, 327, 145-152.
31. Buhleyer, E.; Wehner, W.; Voegtle, F. Cascade “and non-skid-chain-like” syntheses of molecular
cavity topologies. Synthesis 1978, 155-158.
32. McKinney, L.L.; Uhing, E.H.; Setzkorn, E.A.; Cowan, J.C. Cyanoethylation of -amino acids. I.
Monocyanoethyl derivatives. J. Am. Chem. Soc. 1950, 72, 2599-2603.
33. McKinney, L.L.; Uhing, E.H.; Setzkorn, E.A.; Cowan, J.C. Cyanoethylation of alpha amino acids,
II. Dicyanoethyl and tricyanoethyl derivatives. J. Am. Chem. Soc. 1951, 73, 1641-1652.
34. Riehm, J.P.; Scheraga, H.A. Structural studies of ribonuclease. XX. Acrylonitrile. A reagent for
blocking the amino groups of lysine residues in ribonuclease. Biochemistry 1966, 5, 93-99.
35. Rylander, P.N. Hydrogenation Methods; Academic Press: London, UK, 1985.
36. de Brabander-van den Berg, E.M.M.; Meijer, E.W. Poly(propylene imine) Dendrimers: Large-
Scale Synthesis via Heterogeneously Catalyzed Hydrogenation. Angew. Chem. Int. Ed. Engl.
1993, 9, 1308-1311.
37. Brown, H.C.; Choi, Y.M.; Narasimhan, S. Selective reductions. 29. A simple technique to achieve
an enhanced rate of reduction of representative organic compounds by borane-dimethyl sulfide.
J.O.C. 1982, 47, 3153-3163.
38. Hancock, R.W.E. Cationic peptides: Effectors in innate immunity and novel antimicrobials.
Lancet Infect. Dis. 2001, 1, 156-164.
39. Dawson, R.M.; Liu, C.Q. Properties and applications of antimicrobial peptides in biodefense
against biological warfare threat agents. Critical Rev. Microbiol. 2008, 34, 89-107.
40. Conlon, J.M.; Al-Ghaferi, N.; Abraham, B.; Leprince, J. Strategies for development of naturally
occurring antimicrobial peptides into therapeutically valuable anti-infective agents. Methods
2007, 42, 349-357.

Molecules 2009, 14
3905
41. Fisher, D.; Li, Y. Ahlenmeyer, B.; Krieglstein, J.; Kissel, T. In vitro cytotoxicity testing of
polycations: Influence of polymer structure on cell viability and hemolysis. Biomaterials 2003,
24, 1121-1131.
42. Malik, N.; Wiwattanapatapee, R.; Klopsch, R.; Lorentz, K.; Frey. H.; Weener, J.W.; Meijer, E.W.;
Paulus, W.; Duncan, R. Dendrimers: Relationship between structure and biocompatibility in vitro,
and preliminary studies on the biodistribution of 125I-labelled PAMAM dendrimers in vivo. J.
Control. Release 2000, 65, 133-148.
43. Kuroda, K.; Caputo, G.A.; DeGrado, W.F. The role of hydrophobicity in the antimicrobial and
hemolytic activities of polymethacrylate derivatives. Chem. Eur. J. 2009, 15, 1123-1133.
44. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved
Standard, 2nd ed.; M27-A2; CLSI: Wayne, PA, USA, 2002.
45. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow
Aerobically; M07-A7, M100-S17; CLSI: Wayne, Pa, USA, 2006.
46. Rao, S.L.N. Chemical synthesis of N-β-oxalyl-l-α,β-diaminopropionic acid and optical specificity
in its neurotoxic action. Biochemistry 1975, 14, 23-31.
Sample Availability: Samples of the compounds 4e-h are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).