Microwave-Facilitated SPOT-Synthesis of Antibacterial Dipeptoids

Article abstract of DOI:10.1021/acscombsci.7b00096

With microwave irradiation, the submonomer synthesis of dipeptoids on functionalized cellulose can be accelerated with good yields and purity. Optimization provided a library of 96 dipeptoids. From these, 29 compounds were found with an antibacterial activity against MRSA at a concentration of 25 μM. Large nonpolar residues, such as undecylamine and dehydroabiethylamine, are the key components engendering the observed antibacterial activity of these peptoids.

Full text of DOI:10.1021/acscombsci.7b00096

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Article
Microwave-Facilitated SPOT-Synthesis of Antibacterial Dipeptoids
Anne Schneider, Daniel Fritz, Joseph Kurt Vasquez,
Sidonie Vollrath, Helen E. Blackwell, and Prof. Stefan Brase
ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.7b00096 • Publication Date (Web): 16 Oct 2017
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Microwave-Facilitated SPOT-Synthesis of Antibacterial Dipeptoids.
Anne C. Schneider,a Daniel Fritz,a Joseph K. Vasquez,b Sidonie B. L. Vollrath,a Helen E. Black-
9
wellb,* and Stefan Bräsea,c,*
.
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a) Institute of Organic Chemistry, Karlsruhe Institute of Technology, Fritz-Haber-Weg 6, 76131 Karlsruhe,
Germany.
b) Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, Wisconsin
53706, USA.
c) Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1,
76344 Eggenstein-Leopoldshafen, Germany.
KEYWORDS. Peptoids, antibacterial, ampetoids, antimicrobials, MRSA, Staphylococcus aureus, SPOT-synthesis, cel-
lulose, peptidomimetics, combinatorial library.
ABSTRACT: With microwave irradiation, the submonomer synthesis of dipeptoids on functionalized cellulose can be
accelerated with good yields and purity. Optimization provided a library of 96 dipeptoids. From these, 29 compounds
were found with an antibacterial activity against MRSA at a concentration of 25 µM. Large nonpolar residues, such as un-
decylamine and dehydroabiethylamine, are the key components engendering the observed antibacterial activity of these
peptoids.
active structures had a combination of cationic groups
and hydrophobic side chains. Further study of minimal
Introduction
peptoids as antibacterial agents has slowed. In view of
their straightforward synthesis and the dire need for new
anti-infective agents, more research into minimal pep-
toids is needed to examine their efficacy as antibacterial
compounds.
The rapidly increasing resistance of bacteria to current-
ly available antibiotics will be one of the main problems
in the treatment of bacterial infections in the future.
More than 70% of infections in US hospitals are already
resistant to at least one of the conventional antibacterial
drugs.1-2 Antibacterial agents, including peptides (AMPs)
and their corresponding peptidomimetics have been
known since the 1920s.3-4 Recent advances in the field of
medicinal chemistry are leading to the fact that the modi-
fication of existing antibiotics and discovery of novel an-
tibiotics classes is the focus of science today.
SPOT-Synthesis
Combinatorics is a well known approach in chemical
biology and medical chemistry research.10 Solid phase
synthesis is one of the most frequent strategies for prepa-
ration of combinatorial libraries, including the synthesis
of peptoid libraries.11-12 However, the conventional poly-
meric supports (i.e. insoluble resin beads) have some dis-
advantages. These hydrophobic polymers are often ex-
pensive, fragile, difficult to manipulate and often incom-
patible with biological assays performed directly on the
carrier. The disadvantages of conventional solid phases
has motivated the development of new synthesis and
screening platforms for combinatorial chemistry.
Helical, amphiphilic N-alkyl glycine oligomers, or “pep-
toids”, were described by Barron et al. to imitate the effect
of the natural antimicrobial peptide magainin. The au-
thors succeeded in detecting the activity of these "am-
petoids" (antimicrobial peptoid oligomers) against various
pathogenic bacteria, as well as investigating their function
depending on their structure.6
However, a trend is emerging towards the study of
smaller “minimal” peptoid structures, which can be easily
and quickly investigated using combinatorial solid-phase
chemistry. The first minimal peptoids with efficacious
antibacterial activities were found by screening a library
containing 845 trimeric peptoids. A particularly im-
portant aspect was the introduction of a side chain with a
terminal amino function, which had the greatest effect in
the middle position.7-8 Similar results were also obtained
through the screening of a library with 10,000 trimers.9 All
Frank et al. solved some of these carrier problems by
development of the SPOT-synthesis technique13 that was
originally used in peptide synthesis.14 The advantages of
the SPOT-synthesis is based on the local addressed syn-
thesis of small molecules/peptides using planar cellulose
as a solid support, in order to form matrices of
molecules.15 In contrast to the polystyrene resins usually
applied in solid-phase synthesis, the hydrophilic cellulosic
membranes are easy to manipulate during the synthesis
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and washing steps and have surprisingly high mechanical
Results and Discussion
stability.16 In addition, the hydrophilic cellulose is com-
patible with a range of biological screening methods di-
rectly on the carrier. The introduction of heating systems
such as drying ovens and the unconventional microwave
irradiation have also significantly reduced long reaction
times.17 Hence, the SPOT-synthesis provides a robust
method for the synthesis and screening of large, parallel
libraries of a range of molecules, including peptoids.18-19
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Preparation of the Cellulose Support
For SPOT-synthesis, Whatman 1 CHR cellulose chroma-
tography paper was modified using the optimized method
of Blackwell et al.20 To activate cellulose, the primary al-
cohols of support were directly tosylated. The diamine 3
was introduced as a “spacer” element prior to coupling of
the Rink-Amide linker, using a combination of N,N'-
diisopropylcarbodiimide
(DIC)
and
N-
9
Herein, we report the microwave-assisted SPOT-
synthesis of dipeptoids on functionalized cellulose as a
carrier and its optimization using microwaves. Such new
combinatorial method easily provided a library of dipep-
toids in high purity. This library was subsequently tested
for its antibacterial activity against methicillin-resistant
Staphylococcus aureus strains (MRSA), and several active
compounds were identified. Re-synthesis and testing of
selected active agents served to validate their biological
activity, and our method overall.
hydroxysuccinimide (HO-Su) as an activator. Thereafter,
the support was treated to blanket acetylation to cap any
free amino groups in order to avoid undesired side reac-
tions ( Scheme 1).
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The loading level of support 5 was quantified as 400 -
1100 nmol/cm2 by measuring the Fmoc-deprotection rate.
The linker of support 5 was deprotected immediately pri-
or to use by treatment with 20% piperidine in DMF.
Scheme 1. Preparation of functionalized cellulose support 6 and linker installation.
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SPOT-Synthesis Optimization for Peptoids
First, the free amine on the support was acylated by an
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9
active ester 17 or an activated haloacetic acid, such as
bromoacetic acid 11 or cloroacetic acid 12, with N,N-
diisopropylcarbodiimide (DIC). 2,4-Dinitrophenyl bro-
moacetic acid ester (17) was synthesized from bromoace-
tic bromide (10) and 2,4-dinitrophenol (16), as shown in
Scheme 3. In the synthesis of 17, dry solvents as well as
dry starting material 16 was found to play a crucial role.
Under dry conditions, the product 17 can be easily puri-
fied by crystallization.
In order to optimize the reaction conditions for peptoid
synthesis on cellulose, different methods were evaluated.
Our studies were initiated through the synthesis and
study of a small set of dipeptoids (7 - 10, Figure 1). This
test set was prepared using the standard submonomer
peptoid synthesis approach21 in which we varied several
reaction conditions, shown in Scheme 2. These submon-
omer syntheses all included microwave heating to accel-
erate each step, to expedite library generation.
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Thereafter we introduced frequently used primary
amines (13) in peptoid chemistry such as pentan-1-amine,
tryptamine,
3,3-diphenylpropylamine
and
3-
methoxypropan-1-amine, without changing their se-
quence in the peptoids backbone, to ensure reproducibil-
ity of the syntheses. Mono Boc-protected diamines22 were
used to avoid side reactions. The synthetic cycle of acety-
lation/amination was repeated to obtain dipeptoids.
Cleavage from the support was carried out under TFA
vapor in desiccator for 30 min, using the method of
Blackwell et al.20 The yields and purities of the synthe-
sized dipeptoids 7 - 10 (Figure 1) were determined using
HPLC and LCMS measurements, and the eight synthesis
methods were systematically compared (Table 1).
The methods using bromoacetic acid (11) or chloroacetic
acid (12) in DMF and microwave (entries 6 - 7, Table 1) did
not provide satisfying purities. An overview of the best
methods is given in
Table 2.
Scheme 3. Synthesis of active ester 17.
Figure 1. Dipeptoids 7 - 10 used for SPOT-synthesis optimi-
zation.
O
R´
H2N
R
X
solv.
N
13
NH2
reagents, solv.
MW / r.t
MW / r.t.
x = Br 11
Cl 12
6
O
H
N
R
N
R´
14
Scheme 2. General SPOT-synthesis of peptoids. R, R´ =
amine residues (see Figure 3).
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Table 1. Summary of the conditions for the SPOT- The use of the ester 17 in combination with NMP and
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7
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synthesis.
microwave irradiation does not appear to be particularly
advantageous for the purities of peptoids 7 - 9 (entry 4).
Ester 17, on the other hand, reached similarly good yields
(entry 1) at room temperature. These methods, described
in entries 1 and 3, are not as fast and simple compared to
carrying out the method with DIC and bromoacetic acid
(11) in NMP and microwave irradiation (entry 2). In addi-
tion, the ester 17 must first be prepared, while all other
starting materials are commercially available. Hence, the
method with DIC and bromoacetic acid (11), 1-methyl-2-
pyrrolidinone (NMP) as solvent and microwave irradia-
tion (entry 2, Table 1) was selected for further work.
Entry Solv. Coupling reagent
Conditions
1
NMP ester 17
r.t.;
acylation 15 min;
amination 45 min
MW;
2
NMP DIC/bromoacetic
acid (11)
9
acylation 30 s,
500 W;
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amination 90 s,
550 W
Identification of Suitable Amines for Peptoid Libraries
3
NMP DIC/bromoacetic
acid (11)
r.t.;
With the optimized method for microwave-assisted
SPOT-synthesis in hand, a combinatorial library of dipep-
toids was synthesized. However, prior to library synthesis,
the amines that were suitable for such a library had to be
identified. The amines should react well under the chosen
conditions and provide the expected peptoids in high
purity, since they would be used directly for biological
tests without further purification.
acylation 15 min;
amination 45 min
MW;
4
NMP ester 17
acylation 30 s,
500 W;
amination 90 s,
550 W
5
6
7
8
DMF ester 17
MW;
The most promising amines were determined by SPOT-
synthesis of two test libraries. In the first library, 3,3-
diphenylpropylamine (29) was incorporated as the first
amine in the peptoid synthesis and the amines 20 - 53
(Error! Reference source not found.) were varied in the
second position. In the second library, amines 20 - 53
were varied in the first position while 29 was used as the
second amine (Figure 2). The test libraries would thus
report which amines can be installed in both positions of
the dipeptoid scaffold.
acylation 30 s,
500 W;
amination 90 s
550 W
DMF DIC/bromoacetic
acid (11)
MW;
acylation 30 s,
500 W;
amination 90 s
550 W
After synthesis, the dipeptoids were cleaved from the
membrane in TFA vapor and analyzed by HPLC and
LCMS to determine their purities and confirm their exist-
ence. With the amines 20 – 37, purities of more than 70%
were achieved in both dipeptoid libraries. Therefore, they
were selected for the final SPOT-synthesis dipeptoid li-
brary. The secondary amines 35, 36, 50 and 52 were se-
lected for use only in the second position of the peptoids,
as in the first position they did not undergo high yielding
acylation reactions. The amines 34 and tryptamine (24)
were not incorporated into the first position since their
additional amino functional groups could be acylated as a
side reaction.
DMF DIC/chloroacetic
acid (17)
MW;
acylation 30 s,
500 W;
amination 90 s
550 W
DMF DIC/chloroacetic
acid (12)
r.t.;
acylation 15 min;
amination 45 min
Table 2. Comparison of the purities of the SPOT-
synthesis achieved by different methods. Purities were
determined by HPLC (218 nm).
7
8
9
10
[%]
77
80
70
4
Entry Conditions
[%]
89
[%]
23
47
45
23
61
[%]
60
51
1
ester 17/NMP/r.t.
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3
4
5
11/DIC/NMP/MW 80
11/DIC/NMP/r.t. 71
71
ester 17/NMP/MW 80
ester 17/DMF/MW 81
12
Figure 2. General description of synthesized peptoid librar-
ies. R groups derived from amines 20 - 53.
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Figure 3. Amines used in the SPOT-synthesis of the test libraries. Amines 38 - 53 did not provide good purity and were not used
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in further testing.
Peptoids with the amines 38 - 53 did not achieve the
minimum purity of 70%. These amines had several draw-
backs: they reacted with themselves under those condi-
tions (e.g. 44 and 45), they were volatile (e.g. 46 - 49), not
sufficiently nucleophilic (e.g. 39 - 41 and 43), sterically
hindered (e.g. 40, 47, 49 and 50) or did not survive the
cleavage conditions (e.g. 42). Therefore, they were not
used for the final SPOT-synthesis of the dipeptoid library.
studies above) could be combined to generate a 96-
membered library (structures are shown in Table 4). Spe-
cial focus was made on ensuring a high structural diversi-
ty. Amines 23, 27, 29, 30, 32, 50 and 53 have already been
incorporated into tripeptoids that display antibacterial
activity.7-9 Accordingly, we examined them in the current
study to determine whether they continue to contribute
to antibacterial activity in further minimized dipeptoids.
The synthesis was carried out on a freshly prepared cel-
lulose membrane 5. To obtain a representative overview
of the purity, 30 selected representatives (Table 4) were
analyzed by means of HPLC and MALDI-TOF after cleav-
age of the 96 synthesized dipeptoids.
Synthesis of the Final Dipeptoid Library
With the amine screening data completed, a final 96-
membered dipeptoid library was designed, with the final
aim of being suitable for testing for antibacterial effects.
The synthesis was designed in a way that the particularly
reactive amines 20 - 37 (as revealed via out optimization
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Of the dipeptoids tested, ten (55, 57, 58, 62, 64, 94, 96,
97, 105 and 107) could be obtained with a purity of more
than 80%. Purities above 70% were measured for peptoids
7, 81, 86, 88, 116, 135, 146, and 148. For the remaining
compounds, the purity was less than 70% or could not be
determined unequivocally since superimposing peaks
prevented product classification.
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To be able to interpret the results of the antibacterial
tests, the corresponding concentrations of the tested
compounds must be known. To determine the yield of the
synthesized peptoids, the vacuum-dried peptoids were
dissolved in acetonitrile and the concentrations were de-
termined using the absorption integral at 218 nm. A dilu-
tion series of peptoid 7 was used as a reference.
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Antibacterial Tests
The synthesized peptoids were tested for their antibac-
terial activity against MRSA strain ATCC 33591. A first
series of experiments was carried out with a concentra-
tion of 50 μ
M
in DMSO. Peptoids that displayed activity at
in DMSO.
50 μ were subsequently tested again at 25 μ
M
M
The effects of the peptoids on bacterial growth were
measured via simple absorbance assays in microtiter
plates. In brief, 2 µL of a 5 m
was added to 200 μL of bacteria in Luria-Bertani (LB)
growth medium to generate 50 μ solutions. After 18 h
M solution of the dipeptoids
M
incubation at 37 °C with shaking at 200 rpm, the absorp-
tion of each well at 595 nm wavelength was measured.
Figure 4 shows the measured absorptions of the pep-
toids. With this test, 31 peptoids could be found that have
an antibacterial effect at a 50 μ
M concentration. A struc-
ture-function pattern is especially striking. On closer ex-
amination of the active peptoids, the two particularly hy-
drophobic amines, undecylamine (21) and dehydroabie-
tylamine (DHAA, 32) were incorporated as side chains in
all of the active substances. Compounds which contained
neither of the two amines 21 and 32 showed no antibacte-
rial effect.
Figure 4. Antibacterial assay with MRSA and 50 μ
M of pep-
toids 7 and 65 - 148. The bars correspond to absorption at
595 nm. Lower values indicate more potent antibacterial
effect.
With the assumption that the amines 21 and 32 cause
the actual activity of the peptoids, it is reasonable to con-
clude that homo dipeptoids containing these amines as
side chains at both positions, must be particularly active.
However, homo dipeptoid 68 carrying side chain 32 twice
showed no antibacterial activity. Homo dipeptoid 61, with
two undecylamine (21) side chains, did show an antibac-
terial effect. Activity against MRSA by the peptoids 60
and 69 consisting of both amines 21 and 32 was also de-
tected. In most dipeptoids with either variation as a side
chain (21 or 32, respectively) antibacterial effects were
detected (e.g. 7 and 76, 54 and 62, 55 and 63, 56 and 64, 57
and 65, etc.). All dipeptoids containing DHAA (32) as a
side chain were found as active compounds, whereas
some analogous peptoids with undecylamine (21) were
inactive (e.g., 91 was active and 92 inactive, 115 active and
116 inactive, 131 active and 132 inactive, etc.).
Since 50 μ
al substances, a further series of tests with the concentra-
tion of 25 μ was performed. In this experiment, only the
substances that already showed an antibacterial effect at
50 μ were tested. The measured absorption is plotted in
Figure 5.
M is still a high concentration for antibacteri-
M
M
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Table 3. MIC values for peptoids and control compounds
1
in LB and TSB media
2
3
Compound
LB (µM)
TSB (µM)
4
5
54
10
-
56
50
10
-
6
7
8
9
10
11
60
-
62
10
12.5
20
-
64
15
Figure 5. Antibacterial test with MRSA and a peptoid con-
centration of 25 μ . The bars correspond to the absorption at
595 nm
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20
20
-
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60
M
DHAA
25
-
.
undecylamine
In this experiment, it became clear that almost all ac-
tive substances that already had an antibacterial effect at
a concentration of 50 μM, retained this activity at 25 μM.
The only exceptions were the dipeptoids 91 and 139.
While in the case of peptoid 139, a small antibacterial ef-
fect could be seen, no activity was observed at 91. The two
peptoids 91 and 139 with dehydroabietylamine (32) as a
To ensure that the observed antibacterial effect for this
series of active peptoids was not already produced by
amines 21 and 32 alone, these amines were also tested.
However, no significant antibacterial effect could be de-
tected at these concentrations for undecylamine, the ac-
tivity of DHAA was less than for all compounds in LB ex-
cept 56, and DHAA is less active than the two compounds
with antimicrobial activity in TSB, compounds 62 and 64
side chain lost activity at a concentration of 25 μ
noteworthy that the dipeptoids 92 and 140 with undecyl-
amine (21) as a side chain were already inactive at 50 μ
M. It is
M
,
though having similar sequence structure. All other di-
peptoids still had an antibacterial effect, regardless of
which side chains were incorporated.
(Figure
2S,
A subset of six representative dipeptoids was chosen for
re-synthesis and re-screening to validate the library (See
SI, Figure 2S). Authentic samples of the selected dipep-
toids were synthesized and highly purified using RP-
HPLC, lyophilized, and diluted to make 5 m
M DMSO
stock solutions. From the DMSO stocks, dilutions were
prepared and the compounds were screened for antimi-
crobial activity at concentrations of 50, 25, 20, 15, 12.5, 10,
and 5.0 µM as described above in LB medium as well as
TSB (tryptic soy broth) medium, as TSB is a common me-
dium for growth assays and antimicrobial susceptibility
testing with S. aureus.23-26 We used a MRSA strain (ATCC
33591) for all of these assays (see Methods). The results
validate our preliminary assays of the SPOT library, ex-
cept that compound 56 did not inhibit growth at 25 µ
LB medium (Table 3). In LB, several of the peptoids show
inhibition of growth at concentrations as low as 10 µ . In
TSB, only two peptoids show strong inhibition. 64 has an
MIC of 20 µ , while 62 has an MIC of 12.5 µ , based on
the collected data.
M in
M
M
M
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Table 3).
In future studies, these results need to be confirmed via
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3
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5
6
7
8
the re-synthesis of all the active substances (on larger
scale and in a quantifiable manner), and the effects of the
active substances investigated with other bacteria. Never-
theless, these initial results for this dipeptoid library are
compelling and suggestive of the minimized dipeptoid
scaffold as a useful structural class for further study as
antibacterials.
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Table 4. Dipeptoids synthesized on cellulose, their activity against MRSA as well as their analyzed retention times, puri-
ties and masses.
1
2
3
4
5
6
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
Purity
[%]
Antibac.
50 µ
Antibac.
25 µ
Dipeptoid Structure
-time
[min]
M
M
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
237: M+
428.6
7
32.2
71
(+)
(+)
2 Na/2
54
-
-
395.6
-
(+)
(+)
55
31.5
92
411.5
412.3
(+)
(+)
56
57
-
-
355.6
375.6
-
(+)
(+)
(–)
(+)
31.5
86
376.3
58
34.8
85
479.7
480.2
(+)
(+)
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 10 of 27
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
Dipeptoid Structure
50 µM
25 µM
[min]
4
5
6
7
8
9
59
-
-
-
-
-
-
403.6
553.9
439.7
-
-
-
(+)
(+)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
60
(+)
(+)
61
(+)
(+)
62
63
35.0
91
509.8
525.6
510.2
(+)
(+)
(+)
(+)
-
-
-
64
33.3
91
469.7
489.7
471.2
(+)
(+)
(+)
(+)
65
-
-
-
ACS Paragon Plus Environment

Page 11 of 27
ACS Combinatorial Science
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
Dipeptoid Structure
50 µM
25 µM
[min]
4
5
6
7
8
66
-
-
593.8
-
(+)
(+)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
67
-
-
517.8
-
(+)
(–)
(+)
(+)
68
-
-
668.0
-
-
69
-
-
553.9
-
(+)
70
-
-
-
-
384.5
-
-
(–)
(–)
-
-
71
400.4
72
-
-
344.5
-
(–)
-
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 12 of 27
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
73
-
-
364.4
-
(–)
-
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
257: M
+ 2
Na/2
74
28.0
64
468.6
(–)
-
75
-
-
392.5
-
(–)
-
294: M
+ 2
Na/2
76
34.9
54
542.8
(+)
(+)
77
78
-
-
-
-
444.6
460.5
-
-
(–)
(–)
-
-
79
-
-
404.5
-
(–)
-
ACS Paragon Plus Environment

Page 13 of 27
ACS Combinatorial Science
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
8
9
80
18.5
55
424.5
528.7
425.2
(–)
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
81
23.6
74
529.1
(–)
-
82
-
-
452.6
-
(–)
-
83
-
-
602.9
-
(+)
(+)
84
-
-
-
-
488.7
329.4
-
-
(+)
(+)
85
(–)
-
86
17.1
70
345.3
346.1
(–)
-
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 14 of 27
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
87
-
-
289.4
-
(–)
-
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
88
17.2
71
309.4
310.1
(–)
-
89
-
-
413.5
-
(–)
-
90
-
-
337.4
-
(–)
-
91
-
-
487.7
-
(+)
(–)
92
-
-
373.5
-
(–)
-
ACS Paragon Plus Environment

Page 15 of 27
ACS Combinatorial Science
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
8
9
93
94
-
-
323.5
339.4
-
(–)
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
21.5
98
340.2
(–)
-
95
96
-
-
283.4
303.4
-
(–)
(–)
-
-
21.8
91
304.2
97
27.0
98
407.6
408.2
(–)
-
98
-
-
331.5
-
(–)
-
99
-
-
481.7
-
(+)
(+)
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 16 of 27
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
50 µ
Antibac.
25 µ
Dipeptoid Structure
M
M
[min]
4
5
6
7
8
100
-
-
367.6
-
(+)
(+)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
101
-
-
423.6
-
(–)
-
102
-
-
439.5
-
(–)
-
103
-
-
383.5
-
(–)
-
104
-
-
403.5
-
(–)
-
105
24.0
93
507.6
508.2
(–)
-
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Page 17 of 27
ACS Combinatorial Science
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
50 µ
Antibac.
25 µ
Dipeptoid Structure
M
M
[min]
4
5
6
7
8
9
106
107
108
-
-
431.5
581.8
467.7
-
(–)
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
29.7
82
582.2
(+)
(+)
-
-
-
(+)
(+)
109
110
-
-
-
-
321.4
337.3
-
-
(–)
(–)
-
-
111
21.0
36 + 411) 281.3
282.1
(–)
-
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 18 of 27
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
8
9
112
-
-
-
-
301.3
-
-
(–)
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
113
405.5
(–)
-
114
27.0
26
329.4
479.7
365.5
330.1
(–)
-
115
-
-
-
(+)
(+)
116
30.5
72
366.2
(–)
-
2)
-
117
118
19.8
47 + 271) 370.5
(–)
(–)
-
-
36 + 19
386.4
+ 241)
2)
-
15.2
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Page 19 of 27
ACS Combinatorial Science
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
8
119
-
-
330.5
-
(–)
-
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
32 + 18
+ 241)
2)
120
16.0
350.5
-
(–)
-
121
-
-
454.6
-
(–)
-
122
-
-
378.5
-
(–)
-
123
-
-
528.8
-
(+)
(+)
124
-
-
414.6
-
(+)
(+)
125
-
-
325.5
-
(–)
-
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 20 of 27
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
126
19.8
54 + 39
341.4
342.13)
(–)
-
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
127
128
-
-
285.4
305.4
-
(–)
(–)
-
-
19.7
52 + 38
306.23)
129
-
-
409.5
-
(–)
-
130
-
-
333.4
-
(–)
-
131
-
-
483.7
-
(+)
(+)
132
-
-
369.5
-
(–)
-
ACS Paragon Plus Environment

Page 21 of 27
ACS Combinatorial Science
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
Antibac.
25 µ
Dipeptoid Structure
50 µM
M
[min]
4
5
6
7
8
9
133
134
-
-
-
-
391.5
-
-
(–)
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
407.4
(–)
-
135
26.5
77
475.6
476.0
(–)
-
136
-
-
371.4
-
(–)
-
137
32.3
67
549.7
550.1
(–)
-
138
-
-
399.5
-
(–)
-
139
23.9
67
335.4
336.3
(+)
(–)
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 22 of 27
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
50 µ
Antibac.
25 µ
Dipeptoid Structure
M
M
[min]
4
5
6
7
8
140
-
-
435.6
-
(–)
-
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
141
142
-
-
-
-
375.5
391.4
-
-
(–)
(–)
-
-
143
144
-
-
-
-
335.4
355.4
-
-
(–)
(–)
-
-
145
-
-
459.6
-
(–)
-
146
24.9
78
383.5
384.2
(–)
-
ACS Paragon Plus Environment

Page 23 of 27
ACS Combinatorial Science
Ret.
Calc.
mass
Found
mass
[g/mol] [M+H]+
1
2
3
Purity
-time
[%]
Antibac.
50 µ
Antibac.
25 µ
Dipeptoid Structure
M
M
[min]
4
5
6
7
8
9
147
-
-
533.7
-
(+)
(+)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
148
32.2
75
419.6
420.3
(+)
(+)
1) Fractions were combined; 2) fractions were not collected; 3) mass was not found in both fractions.
formed with a Vydac protein & peptide C18-column
(10 μm, 22 mm × 250 mm) with a flow rate of 9 mL/min.
An acetonitrile gradient in water with 0.1% TFA was used
as eluent. The purity was calculated by integration of the
signals at 218 nm. Furthermore, preparative reverse-phase
HPLC was performed via a Shimadzu HPLC system with
two LC-8A pumps, a CBM-10A system controller, a SIL-
10A autoinjector, a FRC10A fraction collector and a SPD-
10A vp UV/Vis detector using a Vydac protein & peptide
C18 column (10 μm, 22 mm × 250 mm). The flow rates var-
ied between 10 - 24 mL/min. The method (gradient, tem-
perature and flow rate) was adjusted to each sample. Mi-
crowave reactions of dipeptoids on cellulose supports
were carried out in a Milestone MicroSYNTH Labstation
multimodal microwave synthesis reactor equipped with a
continuous power source (1000 W max.). For reaction
control, the instrument was interfaced with an Ethos Mi-
croSYNTH Lab PC running EasyWave reaction monitor-
ing software. Microwave reaction of dipeptoids on resins
in fritted plastic syringes (Multisyntech GmbH) were car-
ried out in a single mode CEM Discover LabMate micro-
wave operated with CEM’s SynergyTM software. This in-
strument works with a constantly focused power source
(0–300 W). Irradiation can be adjusted via Power- or
Temperature Control. The temperature was monitored
with an optical fiber at atmospheric pressure. Solid phase
synthesis of the comparison dipeptoid was performed
with Rink Amide aminomethyl polystyrene resin (from
Merck-Novabiochem) as solid support. Resin loading was
given as 0.61 mmol/g. All materials were purchased from
Experimental Procedures
General Methods
NMR spectra were recorded at 25 °C on a Bruker AC 250
[250 MHz (1H)], Bruker AC+ 300 [300 MHz (1H) and 75
MHz (13C)] and a Bruker AM 400 [100 MHz (13C)] spec-
trometer. Chemical shifts are reported in parts per million
(ppm) on the δ scale from an internal standard (TMS).
Matrix-assisted laser desorption ionization time of flight
(MALDI-TOF) mass spectra of the peptoids were record-
ed on a Bruker Biflex IV or Bruker REXLEX II spectrome-
ter with a pulsed ultraviolet nitrogen laser (200 µJ at
337 nm) and a time-of-flight mass analyzer with a 125 cm
linear flight path. For every spectrum the samples were
shot between 100 and 300 times with a repetition rate of
1 - 3 Hz. The software used for recording and processing
the spectra was XACQ Version 4.0.4 and XMASS_TOF
Version 5.1.0. The samples were spotted on a Bruker
Standard stainless steel target with 386 spots. As matrix
2,5-dihydroxybenzoic acid (DHB) as saturated solutions
in 50% acetonitrile in water with 0.1% trifluoroacetic acid
(TFA) was used. The crystallisation was effected on air at
room temperature. The abbreviation used for the proto-
nated molecule ion is [M+H]+. Reverse-phase HPLC was
performed in a Shimadzu HPLC system (LC-10AT vp
pump, SCL-10A vp controller, FCV-10ALvp low pressure
gradient unit, SPD-M10A vp UV/Vis diode array detector
(DAD)). For analytical measurements, a Restek Premier
C18 column (5 μm, 4.6 mm × 250 mm) with a flow rate of
1 mL/min was used. Preparative purifications were per-
ACS Paragon Plus Environment

ACS Combinatorial Science
Page 24 of 27
commercial sources and used without further purifica- 18 by 12 cm 96-spot grid), and the sheet was heated at
1
2
3
4
5
6
7
8
tion.
500 W in the microwave oven for 30 s. Spotting and heat-
ing in the microwave oven were repeated once. Subse-
quently, the acylated support was washed as described
above.
Functionalization of Cellulose
For the synthesis of the dipeptoid library, Whatman 1
CHR chromatography paper (20 × 20 cm) was used. The
spots were marked with a pencil.
Step 3: Amination: A 1
M solution of amine in NMP was
prepared. From this solution, 3 μL were spotted onto the
support (using a different amine at each localized spot).
The support was heated at 550 W in the microwave oven
for 90 s. Spotting and heating by microwave were repeat-
ed once. Subsequently, the aminated supported was
washed as described above.
First, the paper 1 was shaken in 20% trifluoroacetic acid
(TFA) in dichloromethane for 20 min in a covered glass
bowl. Subsequently, the cellulose was washed.27 In this
washing procedure, the sheet of cellulose was placed in a
glass dish and covered with solvent. The filled bowl was
gently swirled for 5 min. The solvent was decanted and
the glass bowl with the cellulose was filled directly with
next solvent. The solvents used for washing were as fol-
lows: DMF, ethanol/methanol (2 ×), dichloromethane.
The support was then dried under an air stream.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Steps 2) and 3) were repeated again for the coupling of
the second amine, to generate each dipeptoid.
Thereafter, the support was dried under a stream of air.
The individual spots were punched out and placed into
separate glass vials. These vials were loosely capped and
placed in a desiccator that also contained an open flask
containing about 10 mL of TFA. The desiccator was evac-
uated for 10 min to generate a TFA vapor atmosphere, and
then allowed to stand in the TFA atmosphere for a further
50 min. The desiccator was then aerated for 15 min, and
1 mL of acetonitrile was then added to each of the vials.
The mixture was shaken for 15 min. The cellulose spots
were removed from the solution with a clean pair of twee-
zers, and the solvent was removed in vacuo. The resulting
residue was dissolved in acetonitrile for HPLC tests and
dissolved in DMSO for antibacterial tests.
For tosylation, the sheet was shaken in
2 M p-
toluenesulfonyl chloride solution in pyridine for 20 min.
The tosylated cellulose 2 was washed again as described
before. The tosylated cellulose 2 was placed in a glass dish
pre-heated to 80 °C with 75 mL of spacer 3 and covered.
After 40 minutes at 80 °C in the oven, the sheet was
washed as mentioned before with following solvents:
DMF, ethanol, 2
M sodium hydroxide solution, water, eth-
anol (2 ×) and dichloromethane. The support was then
dried under an air stream.27 This washing step was used
during the submonomer method.
A solution of 243 mg (450 μmol) of Fmoc-Rink linker,
52 mg (0.450 mmol) of HO-Su and 70 μL (57 mg of 0.450
mmol) of DIC in 1.0 mL of DMF was vortexed briefly and
then allowed to stand for 30 min. The precipitated white
solid was filtered off, and 6 µL of the filtrate was spotted
onto the cellulose. The support was then placed in the
oven at 80 °C for 30 min, after which it was washed as
described above. To protect the unreacted amines, the
support was then shaken in a glass dish with a solution of
20 mL of acetic anhydride and 10 mL of pyridine in 70 mL
of DMF for 20 min at room temperature and washed as
described above.
Microwave-Assisted Solid-Phase Synthesis via the Sumon-
omer Method with Bromoacetic Acid.
1.00 equiv. of dry Rink-Amide aminomethyl resin was
swollen in a plastic fritted-syringe with twice its volume
of DMF for 2 h. Then the solvent is removed. For Fmoc
deprotection the resin is treated with a solution of 20%
piperidine in DMF (approx. 0.5 – 1 mL/100 mg resin) for
5 min at room temperature. The solvent is filtered off and
the procedure is repeated two more times. Afterwards the
resin is washed with DMF (× 3) and peptide synthesis
grade DMF (× 1).
Step 1: Coupling of Bromoacetic Acid: The solvent is re-
The loading of support 5 was determined via standard
Fmoc quantification. Two spots of 5 were punched out
and placed into separate vial. A 1 mL aliquot of 4% 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) was then added in
DMF to each vial and vortexed for 30 s. After 15 min, the
vials were vortexed again for 30 s. From these solutions,
100 μL were removed and diluted with 2 mL of DMF. The
absorbance at 296 nm of these dilutions was determined,
and the loading of the support 5 was calculated.
moved from the resin. A 1.2
M solution of bromoacetic
acid (8.00 equiv.) and DIC (8.00 equiv.) in peptide syn-
thesis grade DMF is added to the resin. The mixture is
stirred at 35 ºC under microwave irradiation (max. power:
40 W, open vessel, optical fiber temperature control) for
1 min. Then the resin is washed with DMF (× 4) and pep-
tide synthesis grade DMF (× 1).
Step 2: Coupling of Primary Amine as Submonomer:
Next, a 1.0
M solution of the desired amine (9.00 equiv.) in
Optimized SPOT-Synthesis of Dipeptoids
peptide synthesis grade DMF is added to the resin. The
mixture is stirred at 60 ºC under microwave irradiation
(max. power: 40 W, open vessel, optical fiber temperature
control) for 30 or 45 min. Finally the resin is washed with
DMF (× 4) and peptide synthesis grade DMF (× 1).
Step 1: Deprotection: Support 5 was shaken in a solution
of 20% piperidine in DMF in an uncovered glass bowl and
then washed as mentioned before.
Step 2: Acylation: A 2
M solution of bromoacetic acid
(11) in NMP was added with the equivalent amount of
DIC. After vortexing, the precipitated solid was filtered off
with a syringe filter. This solution (6 μL) was spotted on
the support (in spatially localized positions to generate an
Step 1 and 2 were repeated until the desired chain
length. After the final displacement is done, the resin is
washed with dichloromethane (× 3). A 95% TFA solution
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ACS Combinatorial Science
completely new possibilities for the generation of peptoid
in dichloromethane is added to the resin (approx.
1
2
3
4
5
6
7
8
1 mL/100 mg resin) and shaken at room temperature for
15 min. The solution is collected, new cleavage mixture is
added to the resin and shaken for another 15 min at room
temperature. After filtering out the solution, the resin is
washed with the same volume of dichloromethane (× 3)
and methanol (× 3). The cleavage solutions and washes
are combined and the solvent is removed under reduced
pressure. The residue is taken up in approx. 5 mL of ace-
tonitrile/water 1:1 (v/v) for HPLC. Lyophilization yielded
of a fluffy white powder as pure product (>95% purity).
libraries, which can be tested directly on the carrier for
biological activity.
ASSOCIATED CONTENT
Supporting Information. The Supporting Information is
available free of charge via the Internet on the ACS Publica-
tions website at DOI: XXXX.
Synthesis and characterization data (MALDI-TOF-MS) of
comparison peptoid 7.
Absorption curve for the dilution series of the dipeptoid.
Synthesis and characterization data (1H NMR) of compound
17.
Antibacterial tests (MIC) of the re-synthesized dipeptoids in
LB and TSB medium.
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Antibacterial Tests of the Dipeptoids against MRSA
An aliquot of DMSO (~20 μL depending on the yield of
the dipeptoid) was added to the crude product of the
SPOT-synthesis, which had been cleaved and dried, to
AUTHOR INFORMATION
Corresponding Author
make 5 mM DMSO stock solutions of each dipeptoid. A
methicillin-resistant S. aureus (ATCC 33591) overnight
culture was diluted 1:250 into LB medium to obtain a ca.
107 CFU/mL culture, as verified by counting CFUs on
LB/agar plates. A 2.0 μL aliquot of each DMSO stock solu-
tion was diluted with 198 μL of the culture in a polysty-
*S.B.: Email: braese@kit.edu, Phone: +49-721-608-42902.
*H.E.B.: Email: blackwell@chem.wisc.edu, Phone: +1-608-
262-1503.
Author Contributions
rene 96-well microtiter plate, resulting in 50 μM solutions.
All authors have given approval to the final version of the
manuscript.
A positive control contained 2.0 μL DMSO and 198 μL
culture; a negative control contained 198 μL LB medium
only. Samples and controls were prepared in quadrupli-
cate. The microtiter plates were incubated for 18 h at
37 °C with orbital shaking of 200 RPM. Absorbance at
595 nm was recorded with a BioTek Synergy2 plate read-
er. The compounds exhibiting complete growth inhibition
had the same absorbance values as the negative controls
(~ 0.05). Compounds that did not exhibit growth inhibi-
tion had absorbance values similar to the absorbance of
the positive controls (about 0.7−1.5). The assays were re-
peated with TSB substituted for LB, with triplicates ob-
tained on separate days for each concentration tested.
ACKNOWLEDGMENT
The authors acknowledge the financial support of the Evan-
gelisches Studienwerk e.V. Villigst and the Karlsruhe House
of Young Scientists (KHYS). H.E.B acknowledges the Office
of Naval Research (N00014-16-1-2185) and the Burroughs
Wellcome Fund for support of this research. J.K.V. was sup-
ported in part by the UW−Madison NIH Chemistry-Biology
Interface Training Program (T32 GM008505 and the
UW−Madison Office of the Vice Chancellor for Research and
Graduate Education [with funding from the Wisconsin
Alumni Research Foundation]), and an NSF Graduate Re-
search Fellowship (DGE-1256259).
To validate the library, a subset of six dipeptoids
was chosen for screening at lower concentrations.
Authentic samples of the selected dipeptoids were
synthesized and highly purified using RP-HPLC, ly-
ophilized, and diluted to make 5 mM DMSO stock
solutions. From the DMSO stocks, dilutions were
prepared and the compounds were screened at con-
centrations of 50, 25, 20, 15, 12.5, 10, 5.0, and 2.5 μM as
described above in LB medium as well as TSB (tryptic
soy broth, Sigma) medium. No compound showed
growth inhibition of MRSA strain ATCC 33591 at con-
centrations lower than 5.0 μM.
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