Developing Equipotent Teixobactin Analogues against Drug-Resistant Bacteria and Discovering a Hydrophobic Interaction between Lipid II and Teixobactin

Article abstract of DOI:10.1021/acs.jmedchem.7b01241

Teixobactin, targeting lipid II, represents a new class of antibiotics with novel structures and has excellent activity against Gram-positive pathogens. We developed a new convergent method to synthesize a series of teixobactin analogues and explored structure-activity relationships. We obtained equipotent and simplified teixobactin analogues, replacing the l-allo-enduracididine with lysine, substituting oxygen to nitrogen on threonine, and adding a phenyl group on the d-phenylalanine. On the basis of the antibacterial activities that resulted from corresponding modifications of the d-phenylalanine, we propose a hydrophobic interaction between lipid II and the N-terminal of teixobactin analogues, which we map out with our analogue 35. Finally, a representative analogue from our series showed high efficiency in a mouse model of Streptococcus pneumoniae septicemia.

Full text of DOI:10.1021/acs.jmedchem.7b01241

Subscriber access provided by Chalmers Library
Developing Equipotent Teixobactin Analogues
Against Drug-Resistant Bacteria and Discovering A
Hydrophobic Interaction Between Lipid II and Teixobactin
Yu Zong, Xiuyun Sun, Hongying Gao, Kirsten J. Meyer, Kim Lewis, and Yu Rao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01241 • Publication Date (Web): 09 Apr 2018
Downloaded from http://pubs.acs.org on April 9, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Developing Equipotent Teixobactin Analogues
Against Drug-Resistant Bacteria and Discovering
A Hydrophobic Interaction Between Lipid II and
Teixobactin
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
Yu Zong1‡, Xiuyun Sun1, 2‡, Hongying Gao1,2, Kirsten J. Meyer3, Kim Lewis3, Yu Rao1*
1MOE Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences, MOE Key
Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University,
Beijing 100084, P.R. China.
2Tsinghua-Peking Center for Life Sciences, Haidian Dist., Beijing 100084, China.
3Antimicrobial Discovery Center, Northeastern University, Department of Biology, Boston,
Massachusetts 02115, USA.
KEYWORDS. Teixobactin, Antibacterial, Convergent Synthetic Route, Structure-Activity
Relationship.
ACS Paragon Plus Environment
1

Journal of Medicinal Chemistry
Page 2 of 37
1
2
3
4
5
6
7
8
9
ABSTRACT. Teixobactin, targeting lipid II, represents a new class of antibiotics with novel
structures and has excellent activity against Gram-positive pathogens. We developed a new
convergent method to synthesize a series of teixobactin analogues and explored structure-activity
relationships. We obtained equipotent and simplified teixobactin analogues, replacing the L-allo-
enduracididine with lysine, substituting oxygen to nitrogen on threonine, and adding a phenyl
group on the D-phenylalanine. Based on the antibacterial activities that resulted from
corresponding modifications of the D-phenylalanine, we propose a hydrophobic interaction
between lipid II and the N-terminal of teixobactin analogues, which we map out with our analogue
35. Finally, a representative analogue from our series showed high efficiency in a mouse model of
Streptococcus pneumoniae septicemia.
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
INTRODUCTION.
In 2014, the World Health Organization declared that the post-antibiotic era - a time in which
people could die from ordinary infections and minor injuries - could begin this century. The
emergence of multidrug resistant bacterial pathogens has created an urgent need for the
development of effective antibiotics against those drug-resistant strains to prevent this era from
arising1-12. Novel antibacterial lead structures are essential to guarantee future therapeutic efficacy.
Recently, Lewis13 and coworkers reported a new antibiotic named teixobactin, which targets lipid
II14-16. Teixobactin demonstrated excellent activity against Gram-positive pathogens, such as
MRSA, vancomycin-resistant enterococci and even against M. tuberculosis. Importantly, it is
difficult for pathogens to develop resistance against teixobactin as teixobactin binds to a lipid
substrate of an enzyme, rather than the mutable protein enzyme itself. This new antibiotic filled
the discovery void and has generated excitement. Up to date, the Payne group reported the first
ACS Paragon Plus Environment
2

Page 3 of 37
Journal of Medicinal Chemistry
1
2
3
4
total synthesis of teixobactin17. Simultaneously, the Li group accomplished an elegant total
5
6
synthesis of teixobactin using Ser/Thr ligation18, 19. Nowick20-22, Su23, Albericio24, 25, Singh26-28
,
7
8
9
and other groups29, 30 reported the synthesis of teixobactin analogues using SPPS (solid phase
peptide synthesis). However, to the best of our knowledge, just Singh group reported two
analogues Leu10-teixobactin and Ile10-teixobactin with identical potency to teixobactin recently31.
Simultaneously, teixobactin analogues were reported based on Leu10-teixobactin and Ile10-
teixobactin32. In this regard, the exploration of SAR of teixobactin will be invaluable to develop
new-generation teixobactin-based antibiotics for potential clinical applications.
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
Considering relatively complicated structure of L-allo-enduracididine and six hydrophobic
amino acids, undeniably, synthetic teixobactin has a few limitations including gelation in PBS due
to poor solubility and high cost due to tedious synthesis of L-allo-enduracididine33, which have
hindered teixobactin’s clinical application.Therefore, our group wished to hunt for analogues,
which had simplified structures but exhibited comparable potency and stronger hydrophilicity to
teixobactin. From the aspect of synthetic strategy, compared with the fully solid phase peptide
synthesis (SPPS), we envisioned a highly convergent and efficient synthesis method to obtain
diverse analogues of teixobactin.
RESULTS AND DISCUSSION.
At the beginning of our investigation, we planned to replace L-allo-enduracididine with natural
amino acids, such as Arg or Lys, to simplify the structure of analogues and synthetic route. These
strategies were applied in teixobactin analogues synthesis previously. As shown in Scheme 1, two
additional major synthetic challenges were addressed based on our retrosynthetic analysis, which
included: 1) site of macrolactamization, and 2) condensation of linear and cyclic peptide motifs.
ACS Paragon Plus Environment
3

Journal of Medicinal Chemistry
Page 4 of 37
1
2
3
4
5
6
7
8
9
For the first, the relatively less sterically hindered site of Thr-Ala was selected to overcome ring
strain and realize the cyclization. For the second, teixobactin analogues could be synthesized from
a cyclic pentapeptide and a linear hexapeptide (‘5+6’). A hybridized synthesis strategy combining
solution-phase synthesis with solid-phase synthesis was employed to obtain teixobactin analogues.
Following this design, the cylic and linear fragment were constructed via solution-phase and solid-
phase synthesis respectively.
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
Scheme 1 Retrosynthetic Analysis of Teixobactin Analogues
In the initial stage, the preparation of the cyclic pentapeptide started with Boc-D-Thr-OBn.
Firstly, Boc was removed under 50% TFA in DCM, followed by coupling with Boc-Ser(tBu)-OH
to yield the dipeptide Boc-Ser(tBu)-D-Thr-OBn. Then the dipeptide was coupled with Fmoc-IIe-
OH using EDCI/DMAP, followed by removal of Fmoc with 33% Et2NH to provide compound 3
in 69% yield. Starting from Boc-Arg(Pbf)-OH, removal of Boc and esterification were
accomplished simultaneously under 2 M HCl in MeOH. Without purification, the intermediate
was reacted with Cbz-Ala-OH to give dipeptide Cbz-Ala-Arg(Pbf)-OH, followed by hydrolysis of
ester bond to access compound 5 in 86% yield for 3 steps. Next, ‘3+2’ coupling between 3 and 5
was achieved in 80% yield. Subsequently, the two protecting groups (Cbz, Bn) were removed
under reductant conditions (Pd(OH)2/C, H2). The final cyclization was achieved with
PyAOP/HOAt/DIEA17, 24 in an isolated yield of 66%.
ACS Paragon Plus Environment
4

Page 5 of 37
Journal of Medicinal Chemistry
1
2
3
4
Scheme 2 Synthesis of Precursory Linear Pentapeptide
5
6
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
Synthesis of Linear Precursor. Fmoc-L-Ile-OH was first attached to 2-Cl-Trt resin in the
presence of DIEA in DCM, followed by addition of MeOH for capping the unreacted Cl group on
the resin. The peptide-bond formation was carried out using Fmoc-protected amino acids (3 equiv.)
and HATU/DIEA (3:6 equiv.) in DMF for 50 minutes. Finally, the linear peptide 8 was cleaved
from the resin with TFE/DCM (1:4) (Scheme 3).
ACS Paragon Plus Environment
5

Journal of Medicinal Chemistry
Page 6 of 37
1
2
3
4
Scheme 3 Synthesis of Linear Precursor
5
6
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
Scheme 4 Synthesis of Teixobactin Lactone Analogues via ‘5+6’ Strategy
With the cyclic pentapeptide and linear hexapeptide in hand, desired compound was obtained
in the presence of DEPBT/DIEA34 with 76% isolated yield. Final global deprotection of the peptide
was carried out with TFA-H2O-TIPS (95:2.5:2.5) in 1 h. The crude peptide was purified by semi-
preparative reversed-phase high-performance liquid chromatography (HPLC) to furnish
compound 9 in 12% isolated yield from starting material. (Scheme 4).
ACS Paragon Plus Environment
6

Page 7 of 37
Journal of Medicinal Chemistry
1
2
3
4
Scheme 5 Synthesis of Teixobactin Lactam Analogue Via ‘ 5+6 ’ Strategy
5
6
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
An alternative pathway was developed to synthesize analogue 35 with a lactam scaffold.
Starting material 10 was synthesized from D-allo-threonine according to the literature35. The azide
ACS Paragon Plus Environment
7

Journal of Medicinal Chemistry
Page 8 of 37
1
2
3
4
5
6
7
8
9
group was reduced by trimethyl phosphine to an amine, which was protected with FmocOSu to
get compound 11. After treatment with 4 M HCl in dioxane, compound 11 was coupled with Boc-
Ser(tBu)-OH to form intermediate 12. Along with removal of the benzyl group under reductant
conditions (Pd(OH)2/C, H2), the dipeptide was immobilized onto the 2-Cl resin with the aid of
DIEA to generate intermediate 13. Subsequently, Fmoc-Ile-OH, Fmoc-Lys(Alloc)-OH and Fmoc-
Ala-OH was linked onto the resin under standard conditions to form the linear precursor for
cyclization. After cleavage from the resin under mild acidic conditions, the intermediate was
treated with HATU/HOAT/DIEA35 at the concentration of 2 mM to accomplish the lactam with
42% isolated yield from compound 12. Compound 15 was synthesized via a similar ‘5+6’ strategy.
After removal of Alloc group under Pd(PPh3)4/ 1,3-dimethylbarbituric conditions, all the other
acid-labile protecting groups were cleaved with the aid of TFA-H2O-TIPS (95:2.5:2.5) to provide
desired lactam analogue 35 with 2.6% isolated yield from starting material 10. (Scheme 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
ACS Paragon Plus Environment
8

Page 9 of 37
Journal of Medicinal Chemistry
1
2
3
4
Scheme 6 MIC (µg/mL) for MRSA (BAA-1695）
5
6
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
ACS Paragon Plus Environment
9

Journal of Medicinal Chemistry
Page 10 of 37
1
2
3
4
5
6
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
Biological Tests and SAR Study. Based on our newly developed synthetic route, a series of
analogues were synthesized, and we evaluated their antibacterial activity against MRSA BAA-
1695 (Scheme 6). Firstly, compound 9 showed modest inhibition with MIC 2 µg/mL. The linear
(compound 16) and cyclic fragments (compound 17) of compound 9 were assessed to determine
which motif was essential. However, 16 and 17 showed no inhibition at all with MIC greater than
32 µg/mL. Previously, Su group23 has demonstrated that the methyl group on N-Me-D-
phenylalanine was not essential, so a series of analogues that lacked the methyl group were
synthesized. To explore the minimum effective structure and increase solubility, the last
hydrophobic N-Me-D-phenylalanine was removed to form decapeptide 18. However, it exhibited
no inhibition either. Given that the peptide could not be shortened, hydrophilic functional groups
such as morpholine were linked on the N-terminal to improve the solubility. Unfortunately,
ACS Paragon Plus Environment
10

Page 11 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compound 19 with morpholine was still ineffective. Furthermore, a series of compounds were
synthesized linking hydrophilic groups to side chains of compound 9 to improve aqueous solubility.
Firstly, D-Phe was replaced with D-Tyr to form compound 20. Galactose was installed on the
hydroxyl group of D-Tyr to obtain compound 21. To our disappointment, the MIC of compounds
20 and 21 was greater than 32 µg/mL. The simple addition of the hydroxyl group between D-Tyr
on compound 20 and D-Phe on compound 9 resulted in 10-fold-potency loss. It was speculated
that steric hindrance and negative charge may account for this phenomenon. The hydroxyl group
was substituted by an amine to access compound 22. It exhibited no inhibition at all. Interestingly,
when instead of a hydroxyl a methyl group was added to D-Phe of compound 9, the MIC of
compound 23 was 2 µg/ml. The above results indicated that hydrophobic substitution of D-Phe
would increase antibacterial activity while hydrophilic groups would decrease activity
dramatically. So we speculated that there exists a hydrophobic interaction between lipid II and the
N-terminal of teixobactin. To verify our hypothesis, compound 24, possessing a phenyl group
extended from the side chain of phenylalanine to enhance hydrophobic interaction, was prepared.
Surprisingly, the potency was increased 2-fold compared with 23. It confirmed the existence of
hydrophobic interactions at the N-terminal of teixobactin. On the other end of the molecule, when
the Arg was replaced by Lys, the potency was better than compound 9. Combining the biphenyl
and Arg to Lys modification, compound 26 was obtained. It demonstrated very strong potency
(MIC was as low as 0.5 µg/mL), which was comparable to teixobactin. Next, compounds 27 - 30
were synthesized to modify the phenylalanine, which showed similar potency to compound 26.
Then serine was modified by phosphoric acid (compound 31) to further improve solubility.
Unfortunately, although solubility was improved, it lost activity. We further confirmed that
compound 31 was not a good prodrug via an in vivo study. D-Gln was modified by N, N-
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
ACS Paragon Plus Environment
11

Journal of Medicinal Chemistry
Page 12 of 37
1
2
3
4
5
6
7
8
9
dimethylethane-1,2-diamine to get compound 32, and was replaced by D-Orn and D-Arg to get
compound 33 and 34 respectively. However, MIC of these three compounds showed 4-fold loss
of potency. According to previous studies, the cyclic tetrapeptide may interact with
pyrophosphoric acid20. Hence, the hydroxyl group in the Thr was replaced with amine to access
compound 35 with lactam scaffold. Compound 35 showed 2-fold better potency than compound
26. Considering this result, we speculate that when the oxygen is replaced by nitrogen, it may
improve the interaction between the cyclic tetrapeptide and the lipid pyrophosphate. Finally,
compounds 26 and 35 were selected for further antibacterial study. Compared with teixobactin
against different pathogenic microorganisms, they showed similar potency to teixobactin in MRSA,
MSSA, VRE, Streptococcus pneumoniae, and Streptococcus pyogenes (Table 1).
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
Table 1 Activity of Compound 26, 35 Against Pathogenic Microorganisms MIC (μ g/mL)
From our structure-activity relationship study, we drew the following conclusions. Firstly, L-
allo-enduracididine could be replaced by simplified Lys. Secondly, N-terminal could not be
shortened or elongated with hydrophilic groups. Previously, Nowick confirmed that the N-terminal
linear pentapeptide could be replaced by a lipid chain. Other than demethylation, acetylation and
dimethylation of amine in phenylalanine at N-terminal induced inactivation. The amine group and
ACS Paragon Plus Environment
12

Page 13 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
phenyl ring may interact with phosphate and lipid chain of membrane respectively. Thus, we
speculated that the N-terminal phenylalanine played an essential role in anchoring on the
membrane. Thirdly, when the hydrophobic effect was strengthened, stronger antibacterial potency
emerged. It further confirmed our hypothesis that hydrophobic interactions exist between lipid II
and the N terminal of the analogues. Lastly, when the lactone was substituted by lactam, it
increased activity, perhaps through the binding affinity to pyrophosphate (Scheme 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
Scheme 7 Structure-Activity Relationship of Teixobactin Analogues
The mechanism of action of compounds 26 and 35 was compared to teixobactin through lipid
II antagonization and binding assays13. Addition of lipid II eliminated the activity of compounds
26 and 35 against S. aureus at the same ratios that antagonized teixobactin. For all three compounds,
lipid II was more effective at antagonizing activity than undecaprenyl-phosphate (C55-P) (Figure
1A, Supplementary Figure 1). Likewise, as observed with teixobactin, compound 35 prevented
in vitro butanol extraction of lipid II but not C55-P. Compound 26 did not completely prevent lipid
II extraction at the ratios tested(lipid II: compound 26/1:4), perhaps reflecting its 2-fold lower
potency than teixobactin and compound 35 and weaker binding to the lipid pyrophosphate (Figure
ACS Paragon Plus Environment
13

Journal of Medicinal Chemistry
Page 14 of 37
1
2
3
4
5
6
7
8
9
1B, Supplementary Figure 2). Both compounds have comparable mechanism of action to
teixobactin. Compared with compound 35, compound 26 could be available in large scale easily,
so compound 26 was selected to conduct the ITC and in vivo study.
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
Figure 1. Lipid II Antagonization and Binding.
Considering the bad solubility of lipid II stemming from the long lipid chain, Park’s nucleotide
(UDP-MurNAc-L-Ala-γ-D-Glu-meso-Dap-D-Ala-D-Ala) was selected as the target to validate
binding. Using ITC, we evaluated the binding affinity of compound 26 to Park’s nucleotide36
which was isolated from Bacillus subtilis. Compound 26 bound to Park’s nucleotide with a Kd of
23 µM. The loss of long lipid chain could account for more than 10-fold binding affinity decrease
(Figure 2). Based on all of our results, we propose a pattern diagram of the interaction between
lipid II and compound 35 (Figure 3).
ACS Paragon Plus Environment
14

Page 15 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
In Vivo Efficacy. Finally, compound 26 was evaluated with an in vivo study. An animal
efficacy study was performed in a mouse sepsis protection model13. Mice were infected
intraperitoneally with Streptococcus pneumonia at a dose that leads to 90% death. B6 female mice
were infected with 0.2 ml of bacterial suspension (1×104 c.f.u. per mouse). An hour post-infection,
compound 26 was introduced i.v. at single doses ranging from 1 to 5 mg per kg. Vancomycin
which was selected as the positive-control drug and was introduced i.v. at a dose of 5 mg per kg.
As expected, compound 26 revealed excellent results within 48 hours after infection. The survival
rate of all animals was 100%. Then B6 female mice were infected with a higher inoculum of
Streptococcus pneumoniae suspension (5×104 c.f.u. per mouse, 0.2 mL). Animals were dosed 1 h
post infection, and blood c.f.u was evaluated at 12, 18, and 22 h. Compound 26 caused over a 6
log10 reduction of c.f.u. in blood at 22 h post infection.. (Figure 4, Supplementary Figure 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
Figure 2 ITC of Park’s Nucleotide ＋ Compound 26
ACS Paragon Plus Environment
15

Journal of Medicinal Chemistry
Page 16 of 37
1
2
3
4
Figure 3 Pattern Diagram of the Interaction of Lipid II and Compound 35
5
6
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
ACS Paragon Plus Environment
16

Page 17 of 37
Journal of Medicinal Chemistry
1
2
3
4
Figure 4 Compound 26 Efficacy in Mouse Models of Infection.
5
6
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
CONCLUSION
Compared with the linear synthesis strategy, we have developed a new highly convergent
method (‘5+6’ strategy) to prepare diverse teixobactin analogues to explore the SAR more
efficiently. Based on our study, we drew the following conclusions: Firstly, L-allo-enduracididine
could be replaced by more simplified lysine. Secondly, there may exist a hydrophobic interaction
between lipid II and the N terminal of teixobactin analogues. Properly increasing the hydrophobic
groups was beneficial to the interaction. We proposed that the biphenyl group could anchor to the
membrane and the free amine of N-terminal interacts with the phosphate of the lipid bilayer.
Thirdly, the increased potency of the lactam analog was perhaps due to the additional amide bond
contributing to the binding affinity to the pyrophosphate of lipid II. The most potent compound,
35, demonstrated comparable potency to teixobactin in various Gram-positive species. Finally,
ACS Paragon Plus Environment
17

Journal of Medicinal Chemistry
Page 18 of 37
1
2
3
4
compound 26 showed effectiveness in in vivo studies. Further studies on increasing solubility and
5
6
analyzing pharmacokinetic properties are ongoing in our lab.
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
EXPERIMENTAL SECTION
Materials. All commercial materials (Alfa Aesar, Aladdin, J&K Chemical LTD.) were used
without further purification. All solvents were analytical grade. The 1H NMR spectra were
recorded on a Bruker 400 MHz spectrometer. Low-resolution mass spectral analyses were
performed with a Waters AQUITY UPLCTM/MS. Analytical HPLC was performed on a
SHIMADZU system, using a Vydac 218TP C18 column (5 µm, 4.6×250 mm). Semi-preparative
HPLC was performed on a SHIMADZU system, using a Vydac 218TP C18 column (10 µm,
10×250 mm, A column), Dr. Maisch GmbH Reprosil 100 C18 column (5 µm, 10×250 mm, B
column). Buffer A: 0.1% TFA in acetonitrile; buffer B: 0.1% TFA in H2O. Analytical TLC was
performed on Yantai Chemical Industry Research Institute silica gel 60 F254 plates and flash
column chromatography was performed on Qingdao Haiyang Chemical Co. Ltd silica gel 60 (200-
300 mesh). The rotavapor was BUCHI’s Rotavapor R-3. MicroCal iTC200 system (GE
Healthcare). Mice was from Beijing Vital River Laboratory Animal Technology Co., Ltd..
Synthesis of compound 2 Compound 1 (2.874 g, 9.3 mmol) was dissolved in 15 mL 50% TFA in
DCM. The resulting solution was stirred at room temperature for 30 min to remove the Boc group.
The TFA was blown off under a stream of condensed air. Boc-Ser(tBu)-OH (2.613 g, 10 mmol),
HCTU (4.13 g, 10 mmol), and DIEA (3.63 mL, 22 mmol) were mixed in 50 mL anhydrous DCM.
The resulting solution was added to the above TFA salt. The reaction mixture was stirred at room
temperature for 3 h. After completion, the reaction mixture was diluted with DCM (250 mL) and
ACS Paragon Plus Environment
18

Page 19 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
washed with 1N HCl (100 mL x 3) and brine (100 mL x 1). The organic phase was dried over
Na2SO4, concentrated under vacuum and purified by flash column chromatography on silica gel
(hexane/EA, 4:1) to give compound 2 (3.573 g, 85%). 1H NMR (400 MHz, CDCl3) δ (ppm) 7.40-
7.30 (m, 5H), 7.26-7.20 (m, 1H), 5.44 (s, 1H), 5.25-5.15 (m, 2H), 4.65 (d, J = 8.76 Hz, 1H), 4.35-
4.25 (m, 1H), 4.22 (s, 1H), 3.80-3.70 (m, 1H), 3.43 (t, J = 8.00 Hz, 1H), 1.45 (s, 9H), 1.19 (d, J =
6.40 Hz, 3H), 1.18 (s, 9H). LRMS (ESI) calculated for C23H36N2O7 [M + H]+ 453.25, found 453.34.
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
Synthesis of compound 3 Fmoc-Ile-OH (0.424 g, 1.2 mmol,), EDCI (0.23 g, 1.2 mmol), DMAP
(18.3 mg, 0.15 mmol) was dissolved in 10 mL DCM and the mixture was stirred for 20 min. Then
compound 2 (0.452 g, 1 mmol) was added to the solution and stirred at room temperature for 12
h. The reaction mixture was washed with 1 N HCl (10 mL x 1) and brine (10 mL x1). The organic
phase was dried over Na2SO4, concentrated under vacuum and purified by flash column
chromatography on silica gel (EA/PE, 1:1) to give intermediate. Then the intermediate was
dissolved in 10 mL of a mixture of MeCN/diethylamine (2/1, v/v) and stirred at room temperature
for 10 min to remove the Fmoc group. The reaction mixture was concentrated under vacuum and
purified by flash column chromatography on silica gel (DCM/MeOH, 20:1) to give the free amine
3 (0.389 g, 69% over 2 steps). 1H NMR (400 MHz, CDCl3) δ (ppm) 7.36-7.30 (m, 5H), 7.26-7.20
(m, 1H), 5.52-5.46 (m, 1H), 5.46 (s, 1H), 5.15 (d, J = 12.0 Hz, 1H), 5.04 (d, J = 12.0 Hz, 1H), 4.86
(dd, J = 9.2 Hz, J = 2.0 Hz, 1H), 4.24 (s, 1H), 3.79 (s, 1H), 3.44 (t, J = 8.0 Hz, 1H), 3.18 (d, J =
5.2 Hz,1H), 1.70-1.60 (m, 1H), 1.45(s, 9H), 1.40-1.10 (m, 7H) 1.19 (s, 9H); 0.89-0.86 (m, 6H).
LRMS (ESI) calculated for C29H47N3O8 [M + H]+ 566.34, found 566.62.
Synthesis of compound 5 Boc-Arg(Pbf)-OH (10 mmol, 5.27 g) was dissolved in 2 M HCl of 15
mL MeOH and stirred for 5 hours at room temperature. After completion, the solvent was removed
and dissolved in ethyl acetate. Then the solution was washed with aqueous saturated NaHCO3 and
ACS Paragon Plus Environment
19

Journal of Medicinal Chemistry
Page 20 of 37
1
2
3
4
5
6
7
8
9
brine, dried over anhydrous Na2SO4 and concentrated for next step without further purification.
Cbz-Ala-OH (2.232 g, 10 mmol), HATU (3.8 g, 10 mmol) and DIEA (1.815 ml, 11 mmol) were
mixed in 30 mL DMF:DCM (2:8). Then the intermediate was added to the above solution. The
reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with
DCM (250 mL), washed with 1N HCl (100 mL x 3) and brine (100 mL x 1). The organic phase
was dried over Na2SO4, concentrated under vacuum and purified by flash column chromatography
on silica gel (DCM/MeOH, 20:1) to give intermediate. Then it was dissolved in 30 mL THF/H2O
(3:1). LiOH (0.38 g, 15.84 mmol) was added to the above solution and stirred at room temperature
for 5 min. After completion, the mixture was acidified to pH 3 with 10 % citric acid and extracted
with EA (100 mL x 3). The combined organic phase was dried over Na2SO4 and concentrated
under vacuum to give compound 5 without further purification (5.425 g, 86% over 3 steps). 1H
NMR (400 MHz, CDCl3) δ (ppm) 7.56 (s, 1H), 7.28-7.20 (m, 5H), 7.10-6.90 (s, 1H), 6.80-6.20
(m, 3H) ,6.13 (s, 1H), 5.04 (d, J = 12.0 Hz, 1H), 4.95 (d, J = 12.0 Hz, 1H), 4.53-4.40 (m, 1H),
4.38-4.28 (m, 1H), 3.20-3.10 (m, 2H), 2.90 (s, 2H), 2.51 (s, 3H), 2.45 (s, 3H), 2.04 (s, 3H), 1.96-
1.65 (m, 2H), 1.65-1.50 (m, 2H), 1.43 (s, 6H), 1.33 (d, J = 6.56 Hz, 3H). LRMS (ESI) calculated
for C30H41N5O8S [M + H]+ 632.27, found 632.88.
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
Synthesis of compound 5s Followed the synthesis of compound 5 to obtain compound 5s. 1H
NMR (400 MHz, DMSO-d6) δ (ppm) 8.02 (d, J = 7.56 Hz, 1H),7.88 (d, J = 7.48 Hz, 2H), 7.68
(d, J = 7.4 Hz, 2H), 7.43-7.39 (m, 3H), 7.34-7.30 (m, 7H), 5.0 (s, 2H), 4.30-4.27 (m, 2H), 4.21-
4.03 (m, 3H), 2.9-3.0 (m, 2H), 1.73-1.62 (m, 1H), 1.61-1.5 (m, 1H), 1.42-1.35 (m, 2H), 1.35-1.24
(m, 2H), 1.20-1.19 (d, J = 7.12 Hz, 3H). LRMS (ESI) calculated for C32H35N3O7 [M + H]+ 574.25,
found 574.83.
ACS Paragon Plus Environment
20

Page 21 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Synthesis of compound 6 Compound 5 (0.379 g, 0.6 mmol) and compound 3 (0.283 g, 0.5 mmol)
were mixed in 6 mL DCM/DMF(9:1). DEPBT (0.24 g, 0.6 mmol) and DIEA (119 µl, 0.72 mmol)
were added at 0 ℃. Then the reaction was stirred at room tempreture over night. The reaction
mixture was washed with 1 N HCl (10 mL x 1) and brine (10 mL x 1). The organic phase was
dried over Na2SO4, concentrated under vacuum and purified by flash column chromatography on
silica gel (DCM/MeOH, 20:1) to give compound 6 (0.471 g, 80 %). 1H NMR (400 MHz, DMSO-
d6) δ (ppm) 8.14 (d, J = 9.04 Hz, 1H), 7.06 (d, J = 7.80 Hz, 1H), 7.88 (d, J = 7.88 Hz, 1H), 7.47
(d, J = 7.20 Hz, 1H), 7.35-7.31 (m, 10H), 6.82-6.38 (m, 3H), 5.36-5.34 (m, 1H), 5.13-4.96 (m, 4H),
4.81 (d, J = 9.08 Hz, 1H), 4.33-4.32 (m, 1H), 4.25-4.21 (m, 2H), 4.08-4.04 (m, 1H), 3.47 (d, J =
5.76 Hz, 2H), 3.05 (m, 2H), 2.94 (s, 2H), 2.47 (s, 3H), 2.42 (s, 3H), 1.99 (s, 3H), 1.78 (m, 1H),
1.66 (m, 1H), 1.51-1.37 (m, 18H), 1.20-0.95 (m, 18H), 0.81-0.76 (m, 6H). LRMS (ESI) calculated
for C59H86N8O15S [M + H]+ 1179.59, found 1179.62.
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
Synthesis of compound 6s The procedure followed the synthesis of compound 6. LRMS (ESI)
calculated for C61H80N6O14 [M + H]+ 1121.57, found 1121.97.
Synthesis of compound 7 To a solution of compound 6 (0.118 g, 0.1 mmol) in MeOH was added
Pd(OH)2/C (20% w/w, 23 mg), and the reaction mixture was stirred at room tempreture under H2
(1 atm) for 30 min. Then the reaction mixture was filtrated and concentrated in vacuum. To a
solution of the residue and DIEA (132μ L, 0.8 mmol) in mixed solvents of DCM/DMF (50 mL,
9:1, 0.002 M) was added PyAOP (0.208 g, 0.4 mmol), HOAT (55 mg, 0.4 mmol) at 0 ℃, and then
the resulting reaction mixture was stirred at rt for 24 h. After completion, the reaction mixture was
washed with 1N HCl (100 mL x 3) and brine (100 mL x1). The organic phase was dried over
Na2SO4, concentrated under vacuum and purified by flash column chromatography on silica gel
(DCM/MeOH, 20:1) to give compound 7 (62 mg, 66 %). 1H NMR (400 MHz, DMSO-d6) δ (ppm)
ACS Paragon Plus Environment
21

Journal of Medicinal Chemistry
Page 22 of 37
1
2
3
4
5
6
7
8
9
8.24 (m, 1H), 7.87-7.86 (m, 2H), 7.71 (m, 1H), 7.23 (m, 1H), 6.38 (m, 1H), 5.30-5.29 (m, 1H),
4.64 (d, J = 9.48 Hz, 1H), 4.26-4.24 (m, 1H), 4.17 (m, 1H), 4.09-3.96 (m, 3H), 3.58-3.52 (m, 2H),
3.05 (m, 2H), 2.96 (s, 3H), 2.47 (s, 3H), 2.42 (s, 3H), 2.00 (s, 3H), 1.68 (m, 3H), 1.40-1.34 (m,
21H), 1.16-1.11 (m, 13H), 0.84-0.79 (m, 6H). LRMS (ESI) calculated d for C44H72N8O12 [M + H]+
937.50, found 937.61.
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
Synthesis of compound 7s The procedure followed the synthesis of compound 7. 1H NMR (400
MHz, CD3OD-d4) δ (ppm) 7.78 (d, J = 7.6 Hz, 2H), 7.63 (d, J = 7.2 Hz, 2H), 7.38 (t, J = 7.6 Hz,
2H), 7.30 (t, J = 7.2 Hz, 2H), 5.60-5.58 (m, 1H), 4.45 (t, J = 8.0 Hz, 1H), 4.32-4.30 (m, 1H), 4.24
(d, J = 8.0 Hz, 1H), 4.18-4.06 (m, 4H), 3.66-3.61 (m, 1H), 1.85-1.68 (m, 3H), 1.54-1.43 (m, 18H),
1.29 (d, J = 6.8 Hz, 4H), 1.27-1.16 (m, 11H), 0.90-0.86 (m, 6H). LRMS (ESI) calculated for
C46H66N6O11 [M + H]+878.48, found 880.18.
Synthesis of compound 8 0.2 g of 2-Cl-Trt resin (0.5 mmol/g) was placed in a 10 mL
polypropylene syringe. The resin was activated with DCM/DMF (1:1), washed with DMF (2 ×10
mL, 1 min) and DCM (2 × 10 mL, 1 min) followed by addition of the first amino acid Fmoc-L-
Ile-OH (0.177 g, 0.5 mmol) and DIEA (165 μ l, 1 mmol) in 2 ml DCM/DMF (1:1) and shacked
for 4 h. Then the unreacted Cl group was blocked with MeOH/DIEA/DMF (1:1:8) for 5 minutes.
Next, the resin was washed with DMF and DCM. Then, Fmoc removal was achieved by 20 %
piperidine in DMF (2 × 10 mL, 15 min). The next amino acids were added by using the following
coupling condition: For coupling conditions: Fmoc-AA-OH/HATU/DIEA (3:3:6) in 1 mL DMF
for 50 min. The following amino acids were coupled one by one: Fmoc-D-allo-Ile-OH, Fmoc-D-
Gln(Trt)-OH, Boc-Ser(tBu)-OH, Fmoc-Ile-OH, Boc-NMe-D-Phe-OH. For Fmoc removal: 20 %
piperidine in DMF (2 × 10 mL, 15 min). Washing: DMF (2 × 10 mL, 1 min), DCM (2 × 10 mL, 1
min) and DMF (2 × 10 mL, 1min). Boc-NMe-D-Phe-OH. At last, the resin was washed with 20%
ACS Paragon Plus Environment
22

Page 23 of 37
Journal of Medicinal Chemistry
1
2
3
4
TFE (in DCM) for 4 h, and then washed with dry DCM (3 x). The filtrates were combined and
5
6
concentrated under reduced pressure to afford compound 8 (0.107 g, 95% ).
7
8
9
Synthesis of compound 9 Compound 7 (0.033 mmol, 31 mg) was dissolved in 3 ml 4 M HCl in
dioxane and stirred for 15 min. when the reaction finished, the protecting group Boc was removed
completely, then the mixture was diluted with EA and washed with aqueous saturated NaHCO3.
The organic phase was dried over Na2SO4 and concentrated under vacuum without purification.
Then the intermediate and compound 8 (0.03 mmol, 33 mg) were dissolved in THF/DMF (2 mL).
DEPBT (0.033 mmol, 10 mg) and DIEA (6 μ L, 0.033 mmol) were added at 0 ℃ and stirred at
room tempreture overnight. The mixture was diluted with EtOAc (10 mL) and washed with 1M
HCl (2 x 5 mL), aqueous saturated NaHCO3 (2 x 5 mL), and brine (1 x 5 mL). Then the organic
layer was dried with Na2SO4, filtered and concentrated. The product was purified by silica gel
column with DCM/MeOH (20:1) to give intermediate S9 (45 mg, 76%). Intermediate S9 (35 mg)
was treated with 5 ml TFA/H2O/TIPS (v/v/v = 95:2.5:2.5) for 1 hour and the reaction was
monitored by LC-MS. After completion, the reaction mixture was concentrated under low
temperature, following by the addition of cold diethyl ether to precipitate the crude product. The
crude product was purified by preparative HPLC (5-60% CH3CN/H2O over 30 min) to afford
compound 9 (15 mg, 51% ) 1H NMR (400 MHz, MeOH-d4) δ (ppm) 7.40-7.25 (m, 5H), 5.61-
5.52 (m, 1H), 4.53 (t, J = 8.60 Hz, 1H), 4.50-4.28 (m, 4H), 4.28-4.10 (m, 4H), 4.05 (d, J = 7.52
Hz, 1H), 4.00-3.88 (m, 2H), 3.88-3.78 (m, 2H), 3.33-3.28 (m, 3H), 3.18-3.08 (m, 1H), 2.68 (s, 3H),
2.38-2.30 (m, 2H), 2.20-2.10 (m, 1H), 2.10-1.95 (m, 2H), 1.94-1.76 (m, 4H), 1.76-1.45 (m, 9H),
1.32-1.10 (m, 8H), 1.02-0.72 (m, 25H). LRMS (ESI) calculated for C58H97N15O15 [M+H+]:
1244.73, found:1244.69
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
ACS Paragon Plus Environment
23

Journal of Medicinal Chemistry
Page 24 of 37
1
2
3
4
5
6
7
8
9
Synthesis of compound 11 Compound 10 (33.5 mg, 0.1 mmol) was dissolved in THF (4 mL) and
H2O (0.4 mL) and kept at 0 oC, followed by addition of PMe3 (1.0 M in THF, 0.3 mL, 0.3 mmol).
After that, the reaction was warmed to room temperature and stirred for another 5 h. The solvent
was removed in vacuo to afford the crude primary amine, which was dissolved in dioxane (3 mL)
and H2O (1 mL), followed by addition of NaHCO3 (33.6 mg, 0.4 mmol) and FmocOSu (67.4 mg,
0.2 mmol). The reaction was stirred at room temperature overnight. After that, the reaction mixture
was evaporated in vacuo and extracted with ethyl acetate (3 × 15 mL), the combined organic phases
were washed with brine, dried with MgSO4, filtered and concentrated in vacuo. The residue was
purified by silica gel column chromatography to afford compound 11 (31.8 mg, 60 % for two
steps). 1H NMR (400 MHz, CDCl3) δ (ppm) 7.77 (d, J = 7.4 Hz, 2H), 7.58 (d, J = 5.2 Hz, 2H),
7.45-7.28 (m, 9H), 5.47 (s, 1H), 5.21-5.11 (m, 2H), 5.02 (s, 1H), 4.48-4.21 (m, 4H), 4.21-4.10 (m,
1H), 1.45 (s, 9H), 1.21 (d, J = 6.52 Hz, 3H). LRMS (ESI) calculated for C31H34N2O6 [M + H]+
531.24, found 531.70.
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
Synthesis of compound 12 Compound 11 (0.06 mmol, 31.8 mg) was dissolved in 3 mL 4 M HCl
in dioxane and stirred for 15 min. when the reaction finished, the protecting group Boc was
removed completely, then the mixture was diluted with EA and washed with aqueous saturated
NaHCO3. The organic phase was dried over Na2SO4 and concentrated under vacuum without
purification to get intermediate. Then Boc-Ser(tBu)-OH (0.07 mmol, 18.3 mg), HATU (0.07 mmol,
26.6 mg), and DIEA (0.08 mmol, 14 μ L) were dissolved in 2 mL of DCM/DMF (9:1). The
intermediate was added and stirred at room temperature for 1h. The mixture was diluted with DCM
(10 mL) and washed with 1M HCl (2 x 5 mL), aqueous saturated NaHCO3 (2 x 5 mL), and brine
(1 x 5 mL). Then the organic layer was dried with Na2SO4, filtered and concentrated. The product
was purified by silica gel column with DCM/MeOH (20:1) to give compound 12 (34 mg, 84%).
ACS Paragon Plus Environment
24

Page 25 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1H NMR (400 MHz, CDCl3) δ (ppm) 7.75 (d, J = 7.52 Hz, 2H), 7.60-7.52 (m, 2H), 7.45-7.28 (m,
10 H), 5.45 (s, 1H), 5.21-5.11 (m, 2H), 5.02 (s, 1H), 4.74 (d, J = 8.72 Hz, J = 0.84 Hz, 1H), 4.40-
4.30 (m, 2H), 4.25-4.15 (m, 2H), 3.78 (s, 1H), 3.42 (d, J = 8.92 Hz, J = 1.76 Hz, 1H), 1.45 (s, 9H),
1.20-1.10 (m, 12 H). LRMS (ESI) calculated for C31H34N2O6 [M + H]+ 674.34, found 674.97.
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
Synthesis of compound 13 To a solution of compound 12 (34 mg, 0.05 mmol) in MeOH was
added Pd(OH)2/C (20% w/w, 7 mg), and the reaction mixture was stirred at rt under H2 (1 atm) for
20 min. Then the reaction mixture was filtrated and concentrated in vacuum to get the intermediate.
0.1 g of 2-Cl-Trt resin (0.5 mmol/g) was placed in a 10 mL polypropylene syringe. The resin was
activated with DCM/DMF (1:1), washed with DMF (2 ×10 mL, 1 min) and DCM (2 × 10 mL, 1
min) followed by addition of the intermediate and DIEA (82 μ L, 0.5 mmol) in 2 ml DCM/DMF
(1:1) and shacked for 4 h. Then the unreacted Cl group was blocked with MeOH/DIEA/DMF
(1:1:8) for 5 min to get intermediate 13.
Synthesis of compound 14 Fmoc-Lys(alloc)-OH, Fmoc-Ala-OH were linked onto the resin by
using the following conditions in sequence. For coupling conditions: Fmoc-AA-OH/HATU/DIEA
(3:3:6) in 2 mL DMF for 50 min. For Fmoc removal: 20 % piperidine in DMF (2 × 10 mL, 5 min).
Washing: DMF (2 × 10 mL, 1 min), DCM (2 × 10 mL, 1 min) and DMF (2 × 10 mL, 1min). Then
the free pentapeptide was cleaved from the resin and the solvent was evaporated. The following
procedure complied with the synthesis of compound 7. Isolated yield from compound 14 was 42%.
1H NMR (400 MHz, MeOD-d4) δ (ppm) 5.95-5.88 (m, 1H), 5.28 (d, J = 17.2 Hz, 1H), 5.17 (d, J
= 10.4 Hz, 1H), 4.58-4.50 (m, 4H), 4.18-4.15 (m, 2H), 4.04-4.00 (m, 1H), 3.76 (m, 1H), 3.65 (m,
1H), 3.15 (m, 2H), 1.90-1.88 (m, 3H), 1.56-1.54 (m, 3H), 1.47 (s, 9H), 1.41-1.24 (m, 5H), 1.21-
1.17 (m, 12H), 0.91-0.89 (m, 6H). LRMS (ESI) calculated for C31H34N2O6 [M + H]+ 740.45, found
741.09.
ACS Paragon Plus Environment
25

Journal of Medicinal Chemistry
Page 26 of 37
1
2
3
4
5
6
7
8
9
General Procedure for synthesis of substituted phenylalanine via Suzuki-Miyaura
Couplings37 Boc-4-iodophenylalanine (1 equiv), arylboronic acid (1.5 equiv), sodium carbonate
(3 equiv), palladium acetate (0.05 equiv), and tri-o-tolylphosphine (0.1 equiv) were added to a
degassed mixture of dimethoxyethane (6 mL/mmol of amino acid) and water (1 mL/mmol of
amino acid). The reaction mixture was kept under argon and heated to 80 °C for 4-6 h. After being
cooled to room temperature, the solvent was evaporated under reduced pressure and diluted with
ethyl acetate and washed with 1M HCl. At last the solvents were removed. The products were
purified using flash chromatography using mixtures of DCM and MeOH as eluent. The isolated
yields were 50-80 %.
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
Synthesis of compound 15 The synthetic procedure complied with the synthesis of compound 9.
General procedure to synthesize compound 16-24 The synthetic procedure of compound 16-24
complied with the synthesis of compound 9.
General procedure to synthesize compound 25-34 The synthetic procedure of compound 25-34
complied with the synthesis of compound 9. Fmoc group was removed under 33% diethylamine
in MeCN for 15 min and the solvent was evaporated. At last, deprotection was conducted with
TFA/H2O/TIPS (v/v/v = 95:2.5:2.5) for 1 h.
Synthesis of compound 35 Compound 15 (19.2 mg, 0.026 mmol) was dissolved in the DCM.
Pd(PPh3)4 (6 mg, 0.005 mmol), 1,3-Dimethylbarbituric acid (8 mg, 0.052 mmol) was added and
the mixture was stirred at RT for 1 hour. Then the solvent was evaporated. At last, deprotection
was conducted with TFA/H2O/TIPS (v/v/v = 95:2.5:2.5) for 1h.
Compound 9,16-35 were purified by semiprep HPLC, all compounds synthesized showed a
purity >95% by HPLC (See SI for chromatograms).
ACS Paragon Plus Environment
26

Page 27 of 37
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1H NMR for Compound 24 (400 MHz, MeOH-d4) δ (ppm) 7.65-7.597 (m, 4H), 7.44 (t, J = 7.84
Hz, 2H), 7.38-7.32 (m, 3H), 5.60-5.50 (m, 1H), 4.53-4.47 (m, 1H), 4.43-4.25 (m, 4H), 4.21-4.06
(m, 5H), 3.96-3.89 (m, 2H), 3.89-3.75 (m, 2H), 3.30-3.17 (m, 4H), 2.32 (t, J = 7.44 Hz, 2H), 2.185-
2.05 (m, 1H), 2.05-1.921 (m, 2H), 1.92-1.75 (m, 4H), 1.71-1.55 (m, 4H), 1.55-1.45 (m, 5H), 1.32-
1.10 (m, 8H), 0.97-0.70 (m, 23H).
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
1H NMR for compound 25 (400 MHz, MeOH-d4) δ (ppm) 7.40-7.22 (m, 5H), 5.58-5.52 (m, 1H),
4.50 (t, J = 8.60 Hz, 1H), 4.45-4.35 (m, 2H), 4.35-4.28 (m, 2H), 4.28-4.18 (m, 2H), 4.18-4.08 (m,
2H), 4.05 (d, J = 7.24 Hz, 1H), 3.98-3.90 (m, 2H), 3.86-3.72 (m, 2H), 4.28-3.18 (m, 1H), 3.18-
3.05 (m, 1H), 2.94 (t, J = 7.66 Hz, 1H), 2.68 (s, 3H), 2.32 (t, J = 7.64 Hz, 2H), 2.15-2.08 (m, 1H),
2.03-1.93 (m, 2H), 1.90-1.78 (m, 4H), 1.78-1.68 (m, 2H), 1.68-1.60 (m, 2H), 1.60-1.40 (m, 6H),
1.38-1.10 (m, 9H), 1.10-0.80 (m, 19H), 0.80-0.77 (m, 6H).
1H NMR for compound 26 (400 MHz, MeOH-d4) δ (ppm) 7.64-7.59 (m, 4H), 7.44 (t, J = 7.36
Hz, 2H), 7.38-7.35 (m, 3H), 5.55-5.54 (m, 1H), 4.52 (t, J = 8.60 Hz, 1H), 4.43-4.31 (m, 5H), 4.24-
4.11 (m, 4H), 4.19-4.09 (m, 2H), 3.93-3.67 (m, 4H), 3.24-3.17 (m, 4H), 2.32 (t, J = 8.16 Hz, 2H),
2.18-2.02 (m, 1H), 2.02-1.91 (m, 2H), 1.91-1.71 (m, 4H), 1.69-1.58 (m, 4H), 1.56-1.48 (m, 5H),
1.28-1.12 (m, 8H), 0.97-0.82 (m, 25H).
1H NMR for compound 35 (400 MHz, MeOH-d4) δ (ppm) 7.64-7.59 (m, 4H), 7.44 (t, J = 7.36
Hz, 2H), 7.40-7.30 (m, 3H), 4.65-4.55 (m, 2H), 4.55-4.48 (m, 1H), 4.45-4.20 (m, 6H), 4.13-4.03
(m, 2H), 4.03-3.75 (m, 5H), 3.18 (d, J = 7.84 Hz, 2H), 2.92 (t, J = 7.32 Hz, 2H), 2.33 (t, J = 7.36
Hz, 2H), 2.20-2.05 (m, 2H), 2.05-1.60 (m, 10H), 1.60-1.35 (m, 8H), 1.28-1.10 (m, 8H), 1.00-0.85
(m, 16H), 0.79 (d, J = 6.84 Hz, 7H), 0.78-0.71 (m, 3H).
ACS Paragon Plus Environment
27

Journal of Medicinal Chemistry
Page 28 of 37
1
2
3
4
5
6
7
8
9
Minimum Inhibitory Concentration13 (MIC) MIC was determined by broth microdilution
according to CLSI guidelines. The test medium for most species was cation adjusted Mueller-
Hinton Broth (MHB). The physical state of all analogues was amorphous, in the form of
trifluoroacetates. Analogues were prepared to 10 mg/mL in DMSO, then they were diluted with
MHB. The same test medium was supplemented with 3% lysed horse blood (Cleveland Scientific,
Bath, OH) for growing Streptococci. All test media were supplemented with 0.002% polysorbate
80 to prevent drug binding to plastic surfaces, and cell concentration was adjusted to approximately
5×105 cells per ml. After 20 h of incubation at 37oC, the MIC was defined as the lowest
concentration of antibiotic with no visible growth. The assay was done three times to confirm
results.
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
Antagonization assays13 Compound 26, compound 35, or teixobactin, each at 4x or 8x the broth
MIC concentration, were mixed with lipid II or undecaprenyl-phosphate (C55-P) in MHII broth
(with 0.05% tween-80). Lipid II and C55-P (purified by Tanja Schneider) were added in 2x, or
0.5x molar excess to the test compound. After incubation at rt for 30 min, compound and lipid
mixtures were transferred to a round-bottom 96-well plate and exponential S. aureus ATCC 29213
added to a final OD600 of 0.001. Plates were incubated overnight at 37 °C, and then examined by
eye and by spectrophotometer plate reader to determine growth of S. aureus. The assay was done
three times for compound 35 and two times for compound 26 to confirm results.
Lipid thin layer chromatography13 In vitro complex formation of compounds with lipid II or
C55-P was analyzed as described. In 50 mM Tris/HCl pH 7.5, 1 nmol of lipid II or C55-P was
incubated with 1, 2, 3, or 4 nmol of test compound (26, 35 and teixobactin) for 30 min at rt. An
equal volume of pyridinium acetate (6 M pH 4.2)/butanol (1:2 v/v) was added, vortexed for 30 s,
and centrifuged at high speed for 30 s. The organic phase was collected, and the extraction repeated.
ACS Paragon Plus Environment
28

Page 29 of 37
Journal of Medicinal Chemistry
1
2
3
4
The pooled organic fraction was washed with butanol-saturated water, and then loaded onto a silica
5
6
TLC plate (Sigma, 02599). TLC was run with
a
mobile phase of
7
8
9
chloroform/methanol/water/ammonia (88:48:10:1 v/v/v/v) and stained with phosphomolybdic
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
acid. The assay was done two times to confirm results.
ITC UDP-MurNAc-L-Ala-D-Glu-meso-Dap-D-Ala-D-Ala (6, Park’s nucleotide) was isolated
36
from Bacillus subtilis according to the method of Holtje . The titration was performed using
MicroCal ITC200 system (GE Healthcare) at 25 °C. Lipid II analogue was made to 0.375 mM
solution with co-solvent (DMSO:H2O/85:15), compound 26 was prepared with the same co-
solvent to 0.025 mM solution. The lipid II analogue was titrated into compound 26 (0.025 mM).
The resultant ITC curves were processed using the Origin 7.0 software. The assay was done two
times to confirm results.
In vivo study13. All animal experiments were done under the guidelines of Laboratory Animal
Research Center, Tsinghua University and used an approved animal protocol (16-RY2, PI, Yu
Rao). Mice source：Beijing Vital River Laboratory Animal Technology Co., Ltd.. Age: 6-8 weeks.
Sex: female. Species: C57BL/6JCnc. Strain of animals: C57BL/6J, The Jackson laboratory
(https://www.jax.org/strain/000664). The physical state of compound 26 was amorphous. It was
used in the form of trifluoroacetates. Compound 26 and vancomycin were dissolved in a mixture
solvent (PBS:Cremophor-EL:DMSO/94:5:1).
1) Compound 26 was tested against Streptococcus pneumoniae D39 in a mouse septicemia
protection assay. C57BL/6J female mice (6-8 week) were infected with 0.2 mL of bacterial
suspension (1×104 c.f.u. per mouse, 6 mice per group) via intraperitoneal injection, a concentration
that achieves at least 90% mortality within 48 h after infection. At one hour post-infection, female
ACS Paragon Plus Environment
29