Extra Sugar on Vancomycin: New Analogues for Combating Multidrug

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Extra sugar on vancomycin: new analogues for combating multidrugresistant Staphylococcus aureus and vancomycin-resistant Enterococci Dongliang Guan, Feifei Chen, Lun Xiong, Feng Tang, Faridoon Faridoon, Yunguang Qiu, Naixia Zhang, Likun Gong, Jian Li, Lefu Lan, and Wei Huang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01345 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017

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Journal of Medicinal Chemistry

Extra sugar on vancomycin: new analogues for combating multidrug-resistant Staphylococcus aureus and vancomycin-resistant Enterococci

Dongliang Guan1,2,†, Feifei Chen1,†, Lun Xiong1,3, Feng Tang1,2, Faridoon1, Yunguang Qiu1,2, Naixia Zhang1,2, Likun Gong1,2, Jian Li3, Lefu Lan1,2,4*, Wei Huang1,2,5* 1

CAS Key Laboratory of Receptor Research, CAS Center for Excellence

in Molecular Cell Science, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Pudong, Shanghai 201203, China 2

University of Chinese Academy of Sciences, No.19A Yuquan Road,

Beijing 100049, China 3

Shanghai Key Laboratory of New Drug Design, School of Pharmacy,

East China University of Science and Technology, Shanghai 200237, China 4

State Key Laboratory of Drug Research, Shanghai Institute of Materia

Medica, Chinese Academy of Sciences , Shanghai 201203, China. 5

Center for Bio-drug Development, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences , Shanghai 201203, China.

†

These authors contribute to this work equally.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 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

*

Corresponding authors:

Prof. Wei Huang Tel: +86-21-20231000 ext. 2517 Fax: +86-21-50807088 Email: [email protected]

Prof. Lefu Lan Tel: +86-21-50806600 Fax: +86-21-50807088 Email: [email protected]

Abstract Lipophilic substitution on vancomycin is an effective strategy for development of novel vancomycin analogues against drug-resistant bacteria by enhancing bacterial cell wall interaction. However, hydrophobic structures usually led to long elimination half-life and accumulative toxicity, therefore hydrophilic fragments were also introduced to the lipo-vancomycin to regulate their PK/PD properties. Here, we synthesized a series of new vancomycin analogues carrying


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various sugar moieties on the 7th-amino acid phenyl ring and lipophilic substitutions on vancosamine with extensive SAR analysis. The optimal analogues indicated 128-1024 fold higher activity against MSSA, VISA, and VRE compared with vancomycin. In vivo pharmacokinetics studies demonstrated the effective regulation of extra sugar motifs, which shortened the half-life and addressed the concerns of accumulative toxicity of lipo-vancomycin. This work presented an effective strategy in lipo-vancomycin derivative design by introducing extra sugars, which led to better antibiotic-like properties in enhanced efficacy, optimal PK, and lower toxicity. Keywords: vancomycin analogues, drug-resistant bacteria, VISA, VRE, sugar moiety



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Introduction Vancomycin as the first member of glycopeptide antibiotics was approved for clinical use in the 1950s1. Its structure was not fully characterized until 19832. In antibacterial history, vancomycin opened a new era to fight against

drug-resistant

Gram-positive

bacteria

including

multidrug-resistant Staphylococcus aureus (MRSA), and was rewarded as “the last resort to fight against untreatable bacterial pathogens” and “ace antibiotics”3. Recent years, due to the abuse of antimicrobial agents, drug-resistant bacterial infection has become a life-threatening problem in public health. In an estimated survey, drug-resistant bacterial infection may cause 10 million deaths annually in worldwide by 20504, 5. The emergence of vancomycin-resistant bacteria deteriorates the situation. In 1988, vancomycin-resistant enterococci (VRE) was firstly reported6, thereafter vancomycin-intermediate resistant Staphylococcus aureus (VISA) and vancomycin-resistant Staphylococcus aureus (VRSA) were discovered in 1997 and 20027-12. The spread of MRSA and vancomycin-resistant relevant pathogens brought serious concerns in outbreak of drug-resistant bacterial infection. Desire of new antibiotics has become an urgent need to combat these pathogens13.

The new-generation glycopeptide antibiotics derived from vancomycin demonstrated

improved

antibacterial

activities


against

various

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vancomycin-resistant strains, therefore providing a strategy to tackle the crisis of drug-resistant infections14-18. Three semi-synthetic vancomycin analogues, Telavancin, Dalbavancin and Oritavancin, were recently launched on the market in 2009 and 2014 for treatment of MRSA infection19-21. These analogues are also called lipoglycopeptides since their structures bear a linear lipid or a biphenyl hydrophobic appendix (Figure 1). These lipo-structures anchor the antibiotics to bacterial cell wall and dramatically enhance the antibacterial effect22. The rigid structure of chlorobiphenyl substitution could directly inhibit the transglycosylase23 and contribute to the binding with pentaglycyl bridge segments of cell wall peptidoglycan24. However, hydrophobic groups caused longer half-life and lower clearance rate in vivo that may lead to accumulative toxicity such as nephrotoxicity25,

26

. Hence, hydrophilic

groups such as extra sugar moieties (Figure 1) were usually introduced to the scaffold to optimize their pharmacokinetics and toxicity27.



2

H3C

O HO O

NH

HO

N H

OH Cl OH

O

Cl H N

O

O

H3C

O HO CH3O O O

O

H N

N H

O

HO

OH NH O O O

O

H N

HO

O

O NH

N H

Cl

H N

OHHN

OH NH

Page 6 of 92

O HO CH3O O O O

O

Cl H N

O

HO H3C

Cl OH

H N

NH O O O

HO H 3C H N

O CH3 O

H2N

H 2N OH

HO

OH

N H

Vancomycin

N H

O N

N H

O N H

NH

Telavancin

HO

O

O

N H

OH

Ac

N H

O OH OH

H N

Oritavancin

H N

NH O O O

H N

O HO

HO

O

Teicoplanin A2-1

O

O

O

OH

OH OH OH

OH

Cl

O

O O

O

N H

NH

Cl H N

O

O

N H

OH H N

O O

N H

Teicoplanin A2-2 O

NH2

H2N O

NH O O O

O O

HO HO

O

OH H N

R: N H

HO OH OH

O

Cl H N

O N H

HO OH OH R Cl

O

O

PO3H2

Cl OH

O

Cl H N

O N H

OH

H2N

O

O O

O

O

OH

HO OH OH

O

HO

OH

O

NH

HO HO

O HO CH3O O O

NH2

OH

N H

O

HN

OH

O

OH

O OH

Teicoplanin A2-3

OH OH

O OH OH

Dalbavancin

Figure 1. Structures of Vancomycin and its lipo-analogues. Extra sugar moieties or hydrophilic groups are displayed in red color.

In structures of Dalbavancin, Oritavancin, and Teicoplanin (Figure 1), extra sugar moieties such as mannose, N-acetylglucosamine, and epi-vancosamine were substituted onto different hydroxyls of the glycopeptide core and play important roles in structural optimization. These extra sugar substitutions were all derived from corresponding fermentation products28-31 by biosynthesis that hampers the diversification on various sugar substitutions for new analogue design. Telavancin carries a phosphonomethylaminomethyl group as the hydrophilic tail onto the phenyl of the 7th-amino acid via a selective Mannich reaction32. With the same reaction, direct assembly of an extra sugar glucosamine26 was previously reported, but the antibacterial activities of these analogues were significantly reduced even with a linear lipid tail on vancosamine. ACS Paragon Plus Environment

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These results implicated the systematic structure-activity relationship (SAR) investigation with various sugar moieties and hydrophobic fragments are required for this strategy. Recently, Haldar group reported16, 17

a

series

of

novel

vancomycin

“lipophilic-vancomycin-carbohydrate”

analogues

conjugation

by

employing coupling

of

different sugars onto the C-terminal carboxylic acid and introducing linear lipid groups onto vancosamine. The successful improvement of antibacterial activity against VISA and VRE demonstrated the assembly of extra sugar moieties also modulated the drug efficacy. These examples indicated that introducing the extra sugar to vancomycin by chemical modification presented a new strategy for structural optimization on vancomycin analogues. However, there are still many questions to address, such as what kind of sugar to assemble? which position on vancomycin to introduce the sugar especially when a rigid lipophilic tail like chlorobiphenyl was linked on vancosamine? how the extra sugar moieties influence the activity and pharmacokinetics? We sought to answer

these

questions

by

investigation

on

precise

SAR

of

extra-sugar-bearing vancomycin analogues.

Results Synthesis of new vancomycin analogues carrying extra saccharide motif



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In order to investigate the role of extra sugar moieties on lipo-vancomycin for PK/PD optimization, we introduced various monosaccharides and a disaccharide to the 7th-amino acid phenyl group of lipo-vancomycin via a two-step synthesis (Schemes 1 and 2). Firstly, decylaminoethyl (derived from Telavancin) and chlorobiphenylmethyl (derived from Oritavancin) were assembled onto the amino group of vancomycin by reductive amidation. Then, sugar motifs including glucose (Glc), mannose (Man), galactose (Gal), glucosamine (GlcN), mannosamine (ManN), galactosamine (GalN), and open-ring glucose (orGlc) and lactose (orLac) were attached on the 7th-amino acid resorcinol via

Mannich

reaction

decylaminoethyl-vancomycin

(compounds and

compounds

5-14

from

15-21

from

chlorobiphenylmethyl-vancomycin). In vitro anti-MRSA and anti-VISA assays suggested GalN, Gal, and orGlc were the optimal sugar structures, therefore in the second-round structural modification we chose these three saccharides to investigate SAR with various lipophilic fragments on vancosamine including linear alkyls and acyls, and rigid hydrophobic structures containing benzene rings (compound 22-46). Some of the lipo-fragments were selected from previous reports on the basis of the SAR studies25, 31. The full structures of compounds 5-46 were listed in Table 1.


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R2

HO HN

OH NH

HO HN

R2

2

H 3C

HO O

O

O NH

HO

N H

O HO CH3O O O O

O

Cl H N

O

O

N H

H3 C OH Cl OH

O

NH O O O

R2-CHO (2a-2n) or R2-COCl (2o,2p)

H N

H 2N HO

OH

OH

O HO

N H

NH

OH Cl OH

O OH

O

O

H N

N H

O O O

OH

HO

ii H N

O

R1-NH2 (4a-4k)

2m

HO

F3C

2h

4a

CHO CHO

2n

4b

CHO CHO

CHO

2i 2j

OH HO

O

HO

CHO

2p

O

OH

Cl O Cl

HO HO HO

O

4d

HO HO HO

O

4e 2l

N

NH O O O

H N

OH

5-46 4g

HO HO HO

4h

HO HO HO

NH O2 OH O NH2 OH

NH2

OH

HO OH

HO

NH 2 OH

NH2

4j

O NHAc

NH2

NH2

OH

OH

4k

OH

OH

OH

O

P

NH2

O

HO OH

N H

NH2

N H

NH2

O

HO O

HO

OH

O

HO

CHO

4f

OH

O

4i

O

2k

CHO

2f

NH2

O

4c

CHO

2e

O

H N

N H

HO

O

2o

CHO

2d

OH

OH O

HO HO HO

O

2c

OH

O

R1

O

CHO

Cl OH

H 2N

CHO

2g Si

N H

NH

3a-3p

CHO

Cl H N

O

O

HO

OH

Cl

2b

NH

OH

O

H 2N HO

N CHO Fmoc Cl

O HO CH 3O O O

O

Cl H N

O

O

1 O

2a

HO

i

OH H N

H3C

O HO CH3 O O O

OH OH

OH

Scheme 1. Synthesis of new vancomycin derivatives 5-46. Reagents and conditions: i) for synthesis of 3a-3n: (a) 2a-n, DIPEA, DMF, room temperature or 55 oC, 2-4h; (b) NaCNBH3, TFA, MeOH, room temperature, 1h (for 3a, additional treatment of 20% piperidine in DMF for 20 min); for synthesis of 3o-3p: (c) 2o-p, DIPEA, DMF, 0 oC, 2h; ii) HCHO, 4a-k, DIPEA, H2O:MeCN=1:1, -10 oC, 12h.

To understand how the C-terminus modification influences antibacterial activity of lipo-vancomycin-carbohydrate structure, we introduced dimethylaminopropylamine

(derived

from

Dalbavancin)

to

the

C-terminus carboxylic acid by coupling reaction and synthesized compound 54-56 (Scheme 3 and Table 2). We also utilized aminoglycoside antibiotics (kanamycin and amikacin), which consist of trisaccharide-like motifs, to assemble them onto vancomycin (compound 58-63) and test their antibacterial activities (see details in supporting information).



AcO AcO AcO

OAc O

AcO AcO AcO

i

NHCbz

+ HO

O

R1 AcO R2 AcO

iv

OAc R3

Cbz

R1 HO

O

4b R1=R3=OH R2=H 4c R2=R3=OH R1=H 4d R1=H R2=OH R3=NHAc

H 2N

O

O

OH

OH

v,vi

+

HO

53a

52a

O

OH OH HO O

4j H 2N

O

O

HO

HO

v,iii

+

O

NH2

N H

OH

OH

BocHN

HO

OH

NH 2

R3 4b-d

OH

HO

O

HO

NHCbz

50a,51a R1=R3=OAc R2=H 50b,51b R2=R3=OAc R1=H 50c,51c R1=H R2=OAc R3=NHAc

HO

O

ii iii R 2

O

51a-c

O

NH2

O 4a

R3

50a-c

HO

OH O

49

48

R1 AcO R2 AcO

+

HO HO HO

ii iii NH

O

SPh 47

48

OAc O

Page 10 of 92

HO

NH2

N H

OH

OH

O CbzHN

HO

O

OH

53b

52b

4k HO OH

OH

Scheme 2. Synthesis of saccharide intermediates 4a-k. Reagents and conditions: i), NIS, TfOH, MS4A, dry DCM, 0 oC to room temperature, 12h; ii), MeOH, NaOMe, room temperature, 4h; iii), H2, Pd-C, MeOH, 2h. iv), BF3Et2O (for 51a, 51b) or SnCl4 (for 51c), dry MeCN, 0 oC to room temperature, 12h; v) CH3OH, reflux, 4h; vi）2N HCl, MeOH, room temperature, 2h. Cl

OH HN H3C

HO O

O

O

HO

NH

N H

O HO CH3O O O O

O

Cl H N

O

O

N H

H3 C OH Cl OH

N

NH2

4m

OH

HO

i

H N

NH O O O

H N

O R3

O

O NH

N H

H3 C

O HO CH3O O O O

O

Cl H N

O N H

O

OH

O HO CH3O O O

Cl OH

O

O

Cl H N

O

HO

4a, 4j OH

ii

H N

NH O O O

H N

O R3

O

O NH

OH

3m

O

N H

Cl OH

OH H N

NH O O O

H N

H2 N HO

OH

N H

OH

H2N

H2N HO

Cl

OH HN

Cl

OH HN

HO

OH

OH

OH

OH

R1

54

55, 56

Scheme 3. Synthesis of vancomycin analogues 54-56. Reagents and conditions: i) DIPEA, HBTU, DMF, room temperature, 24h; ii) HCHO, 4a or 4j, DIPEA, H2O:MeCN = 1:1, -10 oC, 12h.

NMR characterization of vancomycin analogues Although chemical modification on vancomycin 7th-amino acid resorcinol via Mannich reaction has been previously reported and NMR chemical shifts of the aryl protons (Ar-Hs) of the modified position were described ACS Paragon Plus Environment

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in the literature26, 32-34, the detailed assignment of these Ar-Hs with clear 2D spectrum data was lacked and not yet reported. Herein, we presented the precise characterization with 2D NMR to confirm the assemble position via Mannich reaction (Figure 2). Ha (6.41 ppm) and Hb (6.25 ppm) of intermediate 3m were assigned based on 1H NMR, 1H-1H COSY, 1

H-13C HSQC, 1H-13C HMBC. In the HMBC spectrum (Figure 2, panel

B), Hb-Cα but no Ha-Cα correlation was observed that clearly distinguished these two protons. After Mannich reaction, although chemical shift of Hb (product 46) changed to downfield (6.56 ppm), Hb-Cα correlation was still clearly identified and the disappearance of Ha suggested it was substituted during the reaction. This is the first-time report on the precise identification of vancomycin derivatives via selective Mannich reaction with solid spectral data and is helpful for future characterization on compounds of this type.



A Ha

HO 2C

α β 7 γ HO OH δ

Hb

Hb

Ha

3m Hb

HO2 C

α β 7 γ HO OH δ Hb

N H

OH O

O HO

OH OH

46 B

3m：： Ha

46：： Hb

Hb

50 60

Hb/C α

Hb/Cα

Ha/Cß Ha/C?

Hb/Cγ

100

Hb/Cδ

120

100

Hb/Cγ Hb/Cδ

f 1 ( pp m )

80 f1 ( p p m )

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

Page 12 of 92

150

140 160 6.4

6.3 6.2 f2 (ppm)

6.1

6.6

6.5

6.4 6.3 f2 (ppm)

6.2

6.1

Figure 2. NMR characterization and signal assignment of vancomycin analogues. Panel A: 1H NMR spectra of compound 3m (upper) and 46 (bottom); Panel B: 1H-13C HMBC spectra of compound 3m (left) and 46 (right).

In vitro anti-MSSA and anti-VISA assay of vancomycin analogues and SAR analysis With these newly synthesized vancomycin analogues in hands, we sought to investigate the SAR by in vitro antibacterial assays. Firstly, we chose a MSSA (Methicillin-susceptible S. aureus) strain (Newman)35-37 and a VISA strain (Mu50)37,38 to perform the assay and measure the MIC90 values

of

our

compounds.

As

shown

in

Table

3,

for

decylaminoethyl-vancomycins (compound 5-14 and Telavancin), extra


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sugar moieties of GalN, Gal, and orGlc indicated 4-16 fold higher antibacterial activity than other sugars like Glc, GlcN, Man, ManN, orLac, etc. at a similar MIC90 level of Telavancin in both Newman and Mu50 strains. For chlorobiphenylmethyl-vancomycins (compound 15-21), GalN, Gal, and orGlc substitution also led to 8-64 fold higher activity than other sugars and the phosphonomethylaminomethyl group (derived from Telavancin) in Newman strain and 4-32 fold higher in Mu50 strain. For lipophilic structures on vancosamine (compound 22-46), decyl and biphenyl groups exhibited 8-128 fold better efficacy against MSSA and VISA than other linear or rigid hydrophobic tails (18/36/40 vs 11; 32 vs 13; 46 vs 6). For C-terminus modified compound (54-56), the MIC90 increased to 1-2 µg/mL compared with corresponding unmodified compound 16 (MIC90, 0.06 µg/mL) and 19 (MIC90, 0.06 µg/mL), which implicated the limited tolerance on C-terminus modification for biphenyl-vancomycin and suggested the assembling position of extra sugar moieties on 7th-amino acid resorcinol would be better than that on C-terminus for MSSA and VISA. In summary, the extra sugar attachment combined with suitable lipophilic modifications is an effective strategy to develop new vancomycin analogues against MSSA and VISA. The optimal compounds (18/32/36/40/46) bearing GlcN/Gal/orGlc and decyl/biphenyl groups achieved the best activities as 64-128 fold higher than vancomycin, 8-16 fold higher than Telavancin, and comparable



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activity with Oritavancin in MSSA and VISA in vitro models.

Table 3. In vitro activities of vancomycin derivatives 5-56 against MSSA and VISA* Compd.

MIC90 (µg/ml) Newman

Mu 50

5 6

1 0.5

2 2

7

4

8 9

1 1

10 11

Compd.

MIC90 (µg/ml)

Compd.

MIC90 (µg/ml)

Newman

Mu 50

Newman

Mu 50

21 22

0.5 0.5

0.5 2

37 38

0.25 0.5

0.5 2

16

23

0.12

0.5

39

2

8

4 4

24 25

4 0.12

8 0.5

40 41

0.015 0.12

0.12 0.5

8 4

32 8

26 27

1 1

2 2

42 43

2 0.5

4 2

12

0.12

0.5

28

4

8

44

0.015

0.12

13 14

0.5 0.25

1 1

29 30

4 1

8 4

45 46

0.12 0.03

0.5 0.25

15 16

2 0.06

4 0.25

31 32

1 0.06

4 0.25

54 55

1 2

1 4

17

0.03

0.12

33

0.5

1

56

1

2

18 19

0.03 0.06

0.12 0.12

34 35

32 16

64 32

Vanco. Tela.

2 0.25

8 1

20

0.25

0.5

36

0.03

0.25

Orita.

0.06

0.06

*Newman strain, vancomycin-susceptible Methicillin-susceptible S. aureus(MSSA), ATCC 590435-37. Mu 50 strain, a Healthcare associated Methicillin-resistant S. aureus(HA-MRSA) and vancomycin intermediate resistant S. aureus (VISA) strain isolated in Japan37, 38.

In vitro anti-VRE assay of vancomycin analogues and SAR analysis Next, we investigated the anti-VRE activities of selected compounds (Table 4). Five strains of three different subtypes of VRE (vanA, vanM, and vanB)39 were used for the examination. Based on the results, compounds containing the rigid hydrophobic tails demonstrated higher activities than the linear lipophilic structures in all three VRE subtypes, but extra sugar moieties and C-terminus modification indicated different influence in three subtypes. For vanA and vanM subtypes, C-terminus


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modification enhanced the anti-VRE activity in 4-8 fold (55 vs 16; 56 vs 19); but for vanB, C-terminus modification decreased 8-32 fold. The extra sugar motifs also implicated complicated influence against different VRE subtypes. For vanA, the optimal sugar of GalN/Gal (compounds 16/18/40/46) indicated similar MIC90 compared with no extra sugar compounds (17/44) and the phosphonomethylaminomethyl compound 21. For vanM, the extra sugar caused 2-4 fold decrease in activity compared with no extra sugar compounds (16/19 vs 17; 40/46 vs 44) and 8-64 fold decrease compared with phosphonomethylaminomethyl compound (16/19 vs 21). However, for vanB, the optimal sugar modification exhibited excellent activities with MIC90 at 0.06 µg/mL (compound 40 and 46) which was 1024 fold better than vancomycin, 16 fold of Telavancin, and 4-fold of Oritavancin. These data suggested VRE subtypes may have diverse drug-resistant mechanisms that caused the different response to different vancomycin analogues via various modification strategies. The systematic SAR studies would help to understand their individual mechanisms and to develop personalized treatments. After all, the optimal compounds (15/40/46/55) bearing extra sugars exhibited dramatically enhanced activities against VREs especially for vanB subtype. Table 4. In vitro activities of vancomycin derivatives against resistant Enterococci* MIC(µg/ml) Compd. vanA vanM vanB



Efm-HS-0649

Page 16 of 92

(Efm-HSvb01) 16 16 8 1 4 9 64 64 32 8 16 10 64 64 32 8 16 11 16 8 8 8 1 12 2 4 2 0.12 2 13 0.5 1 2 0.12 2 15 4 2 16 4 0.12 16 2 1 4 2 17 ≤0.06 2 2 8 4 0.25 18 4 1 16 8 1 19 2 4 2 0.12 0.25 21 2 1 8 4 0.5 32 8 8 32 16 0.5 36 8 8 32 16 1 37 1 1 4 2 40 ≤0.06 1 1 4 2 44 ≤0.06 1 0.5 2 1 1 45 2 1 8 2 46 ≤0.06 0.5 1 1 0.12 2 54 0.5 1 2 0.5 4 55 1 2 2 1 8 56 >128 >128 >128 >128 64 Vanco. 4 8 4 0.12 1 Tela. 0.25 0.25 1 0.5 0.25 Orita. *Efm-HS-0649 and Efm-HS-06188: Glycopeptide-resistant E.faecium, vanA phenotype, isolated in China39; Efm-HS-0847 and Efm-HS-08257: Glycopeptide-resistant E.faecium, vanM phenotype, isolated in China39; Efm-HS-vb01: Glycopeptide-resistant E.faecium, vanB phenotype, isolated in China. Efm-HS-06188

Efm-HS-0847

Efm-HS-08257

In vivo antibacterial assay of vancomycin analogues To test the in vivo efficacy, we established two animal models, lethal challenge assay and abscess formation assay respectively. Compounds 18, 32, 40, 44, and 46 were selected for lethal challenge assay on a mice model infected by a MRSA strain USA300 LAC40. Each group contains 15 infected mice and was treated with five selected compounds and


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control compounds of vancomycin and Telavancin via i.p. injection at a single dose of 7 mg/kg. During 10-days monitoring, the survival numbers of each group were recorded as shown in Figure 3. The survival rate of vancomycin group after ten days was 6.67% (1/15), while that of compound 18 group was 93.3% (14/15) as same as Telavancin. Treatment of compound 46 also led to a survival rate of 86.7% (13/15) and the other three compounds all indicated much better efficacy than vancomycin.

For in vivo abscess formation assay, each group contains 12 mice infected by a VISA strain (Mu50). Compounds 18, 46, vancomycin, and Telavancin were administrated to the infected mice via twice i.p. injection at 7 mg/kg dosage in 24 h interval. Five days post infection, the mice were sacrificed and the liver CFUs (colony-forming units) were counted for each mouse as shown in Figure 4. Vancomycin group did not shown significant different in liver CFU level compared with the control group. While, compound 18, 46, and Telavancin groups significantly reduced the liver CFU to a similar lower level. These results demonstrated the in vivo efficacy of our new compounds exceeding vancomycin and achieved similar activity as the launched drug Telavancin in MRSA and VISA.



100

Percent survival(%)

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

Page 18 of 92

80

Negative control Vancomycin Telavancin 18 32 40 44 46

60 40 20 0 0

1

2

3

4

5

6

7

8

9

10

Time(day)

Figure 3. Survival chart of USA300 LAC (MRSA) infected mice after treatment with vancomycin derivatives in an in vivo lethal challenge assay. All compounds and positive controls were administrated with a single dose of 7 mg/kg via i.p. injection at the first day of infection.

Figure 4. Liver CFU (colony-forming unit) counts of Mu50 (VISA and MRSA) infected mice after treatment with vancomycin derivatives in an in vivo abscess formation assay. All compounds and positive controls were administrated via twice i.p. injection at 7 mg/kg dosage. Statistical significance determined by the Mann−Whitney test (two-tailed): *p < 0.05, **p < 0.01, n.s. indicates no significant difference. Each symbol represents the value for an individual mouse. Horizontal bars indicate the observation means, and dashed lines mark the limits of detection.


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In vivo pharmacokinetic assay of vancomycin analogues Furthermore, we selected compounds 18, 32, 40, and 46 to investigate how extra sugar moieties regulate the in vivo pharmacokinetics (PK) properties

in

normal

CD-1

trifluoromethylbiphenyl-vancomycin

mice (3m)

compared and

with

control groups of

vancomycin and Telavancin (Table 5). Compound 3m indicated dramatic increase in T1/2 (~6 h) and AUC (64,194 h*ng/mL) compared with vancomycin (T1/2 0.6 h, AUC 1242 h*ng/mL) that will cause high accumulation in organs. While, our four compounds containing extra sugars reduced half-life in ~2 fold and AUC in 3-6 fold that implicated the extra sugar motifs are good fragments to enhance the in vivo clearance and address the concerns of accumulative toxicity. Moreover, comparison on their PK properties with Telavancin, these compounds exhibited comparable T1/2 and AUC values that suggested the extra sugar attachment is a good strategy to improve the drug-like features. Table 5. Pharmacokinetic properties of selected compounds* Compd.

T1/2

AUClast

AUCINF_obs

CL_obs

MRTINF_obs

VSS_obs

(h)

(h*ng/ml)

(h*ng/ml)

(mL/min/kg)

(h)

(mL/kg)

3m

6.04±0.30

64194±6049

67452±6633

1.24±0.12

6.83±0.24

509±39

18

3.81±0.31

12112±2483

12238±2486

7.0±1.38

4.18±0.34

1754±380

32

3.72±0.70

10666±2063

13450±2725

6.4±1.39

4.79±0.24

1826±334

40

3.45±1.26

20105±1714

24345±2804

3.45±0.40

4.41±1.23

900±180

46

2.94±1.39

16631±4960

20047±5489

4.42±1.42

4.21±2.08

1077±449

Vanco.

0.60±0.21

1242±335

1271±347

68.7±18

0.79±0.26

3076±175

Tela.

1.13±0.13

17143±5611

17304±5745

5.1±1.5

1.71±0.13

520±121

*T1/2: half-life; AUClast: area under the concentration-time curve up to last sampling time; AUCINF_obs:area under the concentration-time curve up to infinity time; CL_obs: clearance;



MRTINF_obs: mean retention time up to infinity time; VSS_obs:volume of distribution at steady

state

Cytotoxicity assay of vancomycin analogues To evaluate the toxicity of our compounds, we performed cytotoxicity assays of compound 16 and 46 on human renal proximal tubule epithelial cells (HK-2) and human liver cell line (HL-7702). As shown in Figure 5, compound 16 and 46 indicated significant lower toxicity in HL-7702 compared with vancomycin and Telavancin at three tested concentrations. On HK-2 cells, these two compounds exhibited similar effect in cell viability as Telavancin and slight reduced toxicity compared with vancomycin. These data demonstrated the advantage in safety of our selected compounds.

B

cell viability % 120

16 46 Telavancin Vancomycin

100

cell viability % 120

80

80

60

60

10 0

(µM)

50

10 0

40

50

40

16 46 Telavancin Vancomycin

100

10

A

10

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

Page 20 of 92

(µM)

Figure 5. Cytotoxicity assays of selected compounds. Panel A: cell viability test on HL-7702 cells (human liver cells); Panel B: cell viability test on HK-2 cells (human renal proximal tubule epithelial cells).

Interaction of vancomycin analogue 46 with bacterial peptidoglycan precursor peptide Vancomycin binds to the peptidoglycan precursor peptide Lys-dAla-dAla ACS Paragon Plus Environment

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on bacterial cell wall therefore breaks the cell wall biosynthesis and kills the bacteria34, 41-43. To test whether the attached extra sugar is involved in this peptide ligand recognition, we investigated the interaction of compound

46

with

the

peptidoglycan

precursor

tripeptide

Ac2Lys-dAla-dAla by NMR measurement. Constant-time 1H-13C HSQC NMR analysis was performed for the solo compound 46 and after adding the precursor tripeptide. The changes on chemical shifts represented the corresponding signals were involved in the interaction. Figure 6 summarized all the changed 1H-13C correlation signals observed in this assay. The signals of x1, x2, x3, x5, V1, and V5 significantly changed or disappeared which means these positions on original vancomycin structure interacted with the target tripeptide ligand. Interestingly, we also observed a signal change of Gal C3/H3 or C5/H5 (these two signals are difficult to distinguish from each other) that suggested the Gal structure of 46 might also contributed the interaction with the tripeptide. This results explained a possible mechanism of extra sugar moiety with enhanced interaction with the bacterial cell wall.

Then, we used the molecular modeling program Maestro (Schrȍdinger) to investigate the binding mode of 46 and the tripeptide. Molecular modeling was performed starting with X-ray crystal structure of vancomycin cocrystallized with the precursor tripeptide ligand (PDB



code 1FVM43). Vancomycin was replaced with 46 then the complex was optimized via energy minimalization to give a rational binding model (Figure S1 in Supporting Information). In this model, an H-bond interaction of Gal motif with tripeptide was observed. Combined the modeling result and NMR data, it is very likely the extra sugar moiety participated the bacterial cell wall binding therefore enhanced the antibacterial activity. This mechanism is important and helpful for the future designs on novel vancomycin derivatives against drug-resistant bacteria. x1

x2

x3

62.0

39

f1 ( pp m)

f1 ( pp m)

60

62.5

63.0

40

f1 ( pp m)

59

41 63.5

4.10

4.05 f2 (ppm)

4.00

3.95

4. 2 5

4.20 4.15 f2 (ppm)

57.5

x5

59.0

4.5

4.4

4.3 f2 (ppm)

96

V1

58.0

58.5

4.10

4.2

4.1

V5

63

f1 ( pp m)

4.15

97

f1 ( p p m )

4.20

64

98 65

59.5 99

60.0

4.65

4.60

4.55 f2 (ppm)

4.50

5.35

4.45

5.30

5.25 5.20 f2 (ppm)

5.15

5.10

4.90

4.85

46:

Gal C3/H3 Gal C5/H5

H3C

67

Gal C2/H2

68 69 70

HO O

71 72

HO

O

O NH

N H

O V1HO O O O

V5CH3 O

O

Cl H N

O

x5

N H

O

3.8

3.7 3.6 f2 (ppm)

3.5

3.4

4.65

4.60

OH Cl OH

OH H N

x2 NH

x3

O O O

H N

x1

H2N

73 3.9

4.80 4.75 4.70 f2 (ppm)

CF3

OH HN

Gal C4/H4

f1 ( pp m)

61

f1 ( pp m)

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

Page 22 of 92

HO

OH OH

OH N H

O

OH

Gal

O

HO

OH

Figure 6. Interaction of compound 46 with bacterial peptidoglycan precursor peptide ligand Ac2Lys-dAla-dAla via 1H-13C HSQC NMR measurement. The spectral signals in red represented the solo compound 46, and the signals in green represented the mixture of 46 and


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peptide ligand. The significantly changed signals of 46 after adding the peptide ligand were displayed in the selected spectral regions and assigned with their position information (x1, x2, x3, x5, V1, V5, Gal C2-5/H2-5).

Discussion and Conclusions Carbohydrate structure of vancomycin plays important roles in the in vivo activity, PK properties, and drug distribution44. Although vancomycin aglycon also indicated good in vitro antibacterial activity45, glycosylation is still indispensable for vancomycin-based drug development. Previous studies reported various strategies to replace the vancosamine/glucose with diverse sugar moities by enzymatic glycosylation44, 46 or chemical synthesis47,

48

. Recently, assembly of extra sugars on vancomycin

C-terminus16, 17 or 7th-amino acid32 presented alternative chemistry in vancomycin analogue design.

Lipidation on vancosamine is an efficient approach to prepare lipo-vancomycin derivatives and has been extensively reported19-24. Introducing lipid structures to other hydroxyl groups of vancomycin by various catalysts broadened the diversity of lipo-vancomycins49. The advantage of these lipo-analogues comes with dramatically enhanced antibacterial activities against drug resistant bacteria. Meanwhile, these compounds usually led to long half lives and brought concerns in accumulative toxicities25, 26. Recently launched glycopeptide antibiotics



(Figure 1) feature with lipophilic fragments and extra sugar motifs on vancomycin core that implicates the balance of lipophilic/hydrophilic groups is important in the structural design.

Here, we synthesized a vancomycin analogue library with various sugar motifs and lipophilic structures (Schemes 1 and 3) to understand the SAR and optimal combination of lipo-fragments and extra sugars. The in vitro antibacterial assay revealed that the GalN, Gal, and orGlc were the optimal sugar moieties than other tested sugars for better antibacterial activity. And decyl or biphenyl substitution indicated better activity than other lipo-structures when combined with the sugar attachment in vancomycin modification. The rigid lipo-vancomycin prefers the assembly of extra sugar moieties on 7th-amino acid resorcinol rather than on C-terminus for anti-MSSA and VISA activities. In vitro anti-VRE assays on three VRE subtypes indicated different response to C-terminus modified and extra sugar attached lipo-vancomycin derivatives and suggested diverse drug-resistant mechanisms of these subtypes. The optimal vancomycin derivatives exhibited excellent antibacterial activity against MSSA, VISA, and VRE with 64-1024 fold enhanced activity compared with vancomycin.

We evaluated in vivo activity of the optimal compounds on two infected


Page 24 of 92

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mice models for lethal challenge assay and abscess formation assay. The mice were infected by retro-orbital injection with MRSA or VISA strains following published literature37, 50-52. In our previous work, treatment of high-dose vancomycin (225 mg/kg within 108 h) showed an effective therapeutic effect to Mu50 infection37. In the current work, since our compounds exceeded vancomycin in in vitro assays, we lowered the dosage to 7 mg/kg for comparison. In lethal challenge experiment (Figure 3) the survival rates of compounds 18 and 46 are significantly higher than vancomycin. The abscess model is to mimic the S. aureus sepsis, a severe and life-threatening disease caused by S. aureus infection. The abscess was gradually developed, and could be most obvious between ~ 96-120 hours after infection with S.aurues53. We euthanized mice for 5 day to detect the accumulation of abscess in kidney, heart, and liver. The abscess in liver is the most sensitive to antibiotics treatment therefore provides a good model to evaluate the antibacterial efficacy. Compounds 18 and 46 indicated excellent therapeutic effect to relieve liver abscess but vancomycin did not show significant activity (Figure 4).

Pharmacokinetic data also showed that the extra sugar significantly regulates half-life and clearance rate of these analogues that proved our design strategy (Table 5). In the time-course profiles of blood drug concentration (Figure S3), all the tested compounds maintain > 1 µg/mL



Page 26 of 92

concentrations in blood after 4 h which is significantly higher than their MIC values. These optimal PK properties explained their remarkable in vivo efficacy.

The NMR binding experiment elucidated that the extra sugar motif of compound 46 is involved in D-Ala-D-Ala ligand interaction. This result suggested an interesting mechanism of 46 in enhancing bacterial cell wall attachment. One of the vancomycin-resistant mechanisms is the mutation of D-Ala-D-Ala to D-Ala-D-Lac on bacterial cell wall that reduced its binding affinity with vancomycin. Bogar group14, 15 reported a series of novel

[Ψ[CH2NH]Tpg4]

and

[Ψ[C(=NH)NH]Tpg4]

vancomycin

analogues with enhanced binding with D-Ala-D-Lac which demonstrated fantastic antibacterial activities against VREs. These works implicate the enhancement of D-Ala-D-Ala/D-Ala-D-Lac binding is a good strategy to fight against vancomycin-resistant bacteria. The extra sugar in our analogues could interact with the D-Ala-D-Ala ligand that provides a new modification strategy in vancomycin derivative design. Another possible mechanism of these vancomycin analogues with enhanced antibacterial activities may be due to the conformational change of vancomycin after modification. It is reported54 that subtle alteration on vancomycin hydroxyl groups may lead to significant change in conformation. On the other hand, the detailed structural mechanism still remains to elucidate in


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the future.

In summary, we reported here an effective strategy to introduce extra sugar moieties onto lipo-vancomycin for enhanced drug efficacy, optimal PK properties, and lower toxicity against drug-resistant bacterial including MRSA, VISA, and VRE.

Experimental Section Material and Instrumentation. All reagents and solvents were commercially purchased from Sinopharm, Bide Pharmatech Ltd. and Shanghai Titan Ltd. (Shanghai, China) and used without further purification. Analytical thin layer chromatography (TLC) was performed on TLC plates precoated with silica gel HSGF254 (200µm±30µm thickness). Analytic RP-HPLC analysis was performed on a Beijing Chuang Xin Tong Heng LC-3000 (analytic model) instrument with a C-18 column (5 µm, 4.6 x 150 mm) at 40oC. The column was eluted with a gradient of 2-90% acetonitrile containing 0.1% TFA in 30 min at a flow rate of 1 mL/min. Preparative RP-HPLC preparation was performed on a Beijing Chuang Xin Tong Heng LC-3000 (preparative model) instrument with a C-18 column (10 µm, 19 x 250 mm) at room temperature. The column was eluted with a gradient of 2-70% acetonitrile containing 0.1% TFA in 30 min at a flow rate of 10 mL/min. HPLC analysis showed that



the purity of all the final products were more than 95%. 1H-NMR, 13

C-NMR, HSQC and HMBC spectra were recorded on a BRUKER

AscendTM 400 MHz or 600 MHz instrument. Chemical shifts were assigned in ppm and coupling constants were assigned in Hz. Constant-time 1H-NMR and HSQC were recorded on AVANCE III HD BRUKER AscendTM 600 MHz instrument. All the final products were added 20µl D2O to exchange the active hydrogen of the target products when the solvent was DMSO-d6. ESI-MS spectra were measured with an Agilent 6230 LC-TOF MS spectrometer. Bacterial strains Newman36, Mu5038 and USA300 LAC40 were from laboratory stock. Efm-HS-0649, Efm-HS-06188, Efm-HS-0847, Efm-HS-0825739 and VanB(R) were obtained from Institute of antibiotics, Huashan hospital, Fudan University. Female BALB/c mice were purchased from SIPPR-BK Lab Animal Ltd. Male CD-1 mice were purchased from Shanghai Lingchang biological technology co., LTD.

Synthesis and characterization

Aminoethyl glycoside (4a-4d). Compounds 49 and 51a-51c were dissolved in methanol, and treated with NaOMe to remove acetyls, followed by hydrogenation55 to generate 4a-4d in 70-90 % yield. 4a and 4d were used directly in next step.


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Aminoethyl β-D-galactopyranoside 4b. 1

H NMR (400 MHz, D2O) δ 4.31 (d, J = 7.8 Hz, 1H), 3.94 - 3.87 (m, 1H),

3.81 (dd, J = 3.4, 1.0 Hz, 1H), 3.72 - 3.67 (m, 1H), 3.67 - 3.64 (m, 1H), 3.60 (ddd, J = 6.9, 5.0, 3.9 Hz, 1H), 3.55 (dd, J = 9.9, 3.5 Hz, 1H), 3.43 (dd, J = 9.9, 7.8 Hz, 1H), 2.94 - 2.87 (m, 2H).

2-Aminoethyl β-D-glucopyranoside 4c. 1

H NMR (400 MHz, D2O) δ 4.38 (d, J = 7.8 Hz, 1H), 3.90 (ddd, J = 10.6,

5.7, 4.5 Hz, 1H), 3.81 (dd, J = 12.3, 2.3 Hz, 1H), 3.69 (ddd, J = 10.9, 6.1, 4.6 Hz, 1H), 3.61 (dd, J = 12.3, 5.9 Hz, 1H), 3.44 - 3.33 (m, 2H), 3.29 (d, J = 9.1 Hz, 1H), 3.19 (dd, J = 9.5, 7.9 Hz, 1H), 2.89 (ddd, J = 6.3, 4.4, 1.7 Hz, 2H).

Glycolactone derivatives (4j-4k). D-gluconic acid lactone (52a, 2 g, 11.2 mmol) or lactobionolactone (52b, 2 g, 5.9 mmol) were refluxed with N-Boc-1,3-propanediamine (1.2 equiv) or N-Cbz-1,3-propanediamine (1.2 equiv) in methanol for 4 hours respectively. The solvent was removed and the residue was washed with enough ethyl acetate to get rid of excess N-Boc-1,3-propanediamine or N-Cbz-1,3-propanediamine. The corresponding products were obtained after dried under vacuum (see supporting information for 1H-NMR ACS Paragon Plus Environment


characterization of these intermediates). Then the protection groups of Boc and Cbz were removed respectively by 2N HCl and H2 hydrogenation to give 4j and 4k which were used in the next step directly.

Gluconic acid lactone derivative (4j). 1

H NMR (400 MHz, D2O) δ 4.23 (d, J = 3.4 Hz, 1H), 3.98 (s, 1H), 3.73 -

3.68 (m, 1H), 3.64 (q, J = 2.0 Hz, 2H), 3.58 - 3.52 (m, 1H), 3.27 (ddt, J = 26.9, 14.0, 7.1 Hz, 2H), 2.92 (t, J = 7.6 Hz, 2H), 1.85 - 1.75 (m, 2H).

General procedures for synthesis of compounds 5-46. Reductive amination reaction, acylation reaction and Mannich reaction were used in the synthesis of final compounds successively. First, for intermediate 3a-3n: Vancomycin hydrochloride (100 mg, 67 µmol), R2-CHO 2a-2n (2 equiv) and DIPEA (30 µl, 172µmol) were stirred in DMF (3 ml) for 2-4 hours to form Schiff base at 55°C. The reaction was monitored by analytic RP-HPLC until complete conversion. Then, NaCNBH3 (8 mg, 127 µmol) was dissolved in methanol (1 ml) and the solution was added into above reaction mixture. TFA (30 µl) was added to adjust pH to 4. The residue was stirred at room temperature for 2 hours to reduce the Schiff base C-N double bond to C-N single bond. In the case of 3a, it required deprotection of Fmoc by treatment with 20%


Page 30 of 92

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piperidine in DMF for 20 minutes before performing reductive amination. For intermediate 3o-3p: Vancomycin hydrochloride (100 mg, 67 µmol), DIPEA (30 µl, 172µmol) were stirred in DMF (3 ml) until the solution became clear. Then the mixture was cooled to 0 °C and R2-COCl 2o-2p (1 equiv) was added in small portions by three times every half hour. The residue was stirred at 0 °C for one more hour. Then the crude product was precipitated by addition of diethyl ether (50ml) and centrifuged. The supernatant was removed and the solid cake was washed with diethyl ether (30ml) once more to give the crude product. The crude was dissolved in water/acetonitrile and was purified by preparative RP-HPLC. The fractions containing target compound was combined and lyophilized to give the product as a white fluffy solid in 20-80% yield.

Mannich reaction was performed in the next step to give the final product. R1-NH2 (10 equiv) and DIPEA (800 µmol) were mixed with water (1ml) and CH3CN (1ml). The mixture was stirred until the solution was clear. Formaldehyde (37% in water, 4.5µl, 60µmol) was added, then the solution was stirred for 20 minutes at room temperature. The residue was cooled to -10 °C and the intermediates 3a-3p (20 µmol) in water (0.5ml) and CH3CN (0.5ml) was added into the solution. The reaction was monitored by analytic RP-HPLC. After 12 hours the conversion rate reached 50-80%, then the residue was treated with TFA to adjust the pH



to 2-3. The residue was subject to preparative RP-HPLC purification to give compound 5-46 as white fluffy solids in 35-95% yield.

(α α-D-mannopyranosylethyl) aminomethyl-N-decylaminoethyl vanco mycin (5). Yield 71% (26.5 mg, 14.2 µmol). R.T. = 16.188 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.56 (br s, 1H), 7.49 (d, J

= 8.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.28 (dd, J =7.2, 0.6 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H),6.84 (d, J = 8.6 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.71 (s, 1H), 5.66 (s, 1H), 5.29 (d, J = 7.6 Hz, 1H), 5.26 (s, 1H), 5.12 (m, 3H), 4.83 (s, 1H), 4.65 (s, 2H), 4.44 (s, 1H), 4.40 (s, 1H), 4.11 (br s, 3H), 3.82 (m, 1H), 3.67 - 3.56 (m, 5H), 3.38 (t, J = 9.4 Hz, 1H), 3.34 (m, 2H), 3.26 (m, 3H), 3.10 - 3.02 (m, 2H) , 2.80 (br s, 2H), 2.54 (s, 3H), 2.18 - 2.06 (m, 1H), 1.85 - 1.70 (m, 2H), 1.68 - 1.58 (m, 2H), 1.54 - 1.45 (m, 3H), 1.25-1.20 (m, 16H), 1.06 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.2 Hz, 3H), 0.85 (m, 6H). HRMS (ESI) calcd. for C87H117Cl2N11O30 [M+3H] 3+m/z 622.9193, found m/z 622.9264.

(β β-D-galactopyranosylethyl) aminomethyl-N-decylaminoethyl vanco mycin (6).


Page 32 of 92

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Yield 77% (28.7 mg, 15.4 µmol). R.T. = 16.174 min (analytical HPLC). 1


= 8.5 Hz, 1H), 7.46 (dd, J = 8.3, 1.8 Hz, 1H), 7.30 (dd, J = 8.1, 3.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.72

(m, 1H), 5.67 (s, 1H), 5.29 (d, J =

7.6 Hz, 1H), 5.25 (s, 1H), 5.14 (s, 1H), 5.11 (d, J = 10.0 Hz, 3H), 4.82 (s, 1H), 4.64 (d, J = 7.0 Hz, 1H), 4.45 (s, 1H), 4.41 (s, 1H), 4.18 - 4.09 (m, 5H), 3.96 (s, 1H), 3.84 - 3.75 (m, 1H), 3.67 (d, J = 10.6 Hz, 1H), 3.62 (d, J = 3.0 Hz, 1H), 3.40 - 3.37 (m, 1H), 3.34 - 3.23 (m, 5H), 2.83 (br s, 2H), 2.56 (s, 3H), 2.18 - 2.07 (m, 1H), 1.85 - 1.70 (m, 2H), 1.67 - 1.60 (m, 2H), 1.55 - 1.45 (m, 3H), 1.30 (s, 3H), 1.26-1.20 (m, 16H), 1.07 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (m, 6H). HRMS (ESI) calcd. for C87H117Cl2N11O30 [M+3H] 3+m/z 622.9193, found m/z 622.9115.

(β β-D-glucopyranosylethyl) aminomethyl-N-decylaminoethyl vancom ycin (7). Yield 72% (27.1 mg, 14.5 µmol). R.T. = 16.423 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) 7.78 (m, 1H), 7.59 (s, 1H), 7.49 (d, J =

8.5 Hz, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.19 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.53 (s, 1H), 5.71 (d, J = 7.1 Hz, 1H), 5.67 (s, 1H), 5.30 5.24 (m, 2H), 5.12 (br s, 1H), 5.11 - 5.07 (d, J = 12.0 Hz, 2H), 4.81 (s,



1H), 4.68 - 4.62 (m, 1H), 4.44 (d, J = 5.3 Hz, 1H), 4.40 (d, J = 5.7 Hz, 1H), 4.19 (d, J = 7.8 Hz, 1H), 4.15 (d, J = 7.6 Hz, 1H), 4.12 - 4.05 (m, 3H), 4.01 - 3.94 (m, 2H), 3.86 - 3.77 (m, 1H), 3.70 - 3.62 (m, 2H), 3.25 (d, J = 5.8 Hz, 3H), 3.14 (dd, J = 10.7, 6.9 Hz, 2H), 3.09 (s, 2H), 3.00 (dt, J = 17.4, 8.9 Hz, 2H), 2.89 (s, 2H), 2.57 (s, 3H), 2.16 - 2.06 (m, 1H), 1.90 (s, 1H), 1.81 (d, J = 12.5 Hz, 1H), 1.69 - 1.58 (m, 2H), 1.54 - 1.48 (m, 3H), 1.32 (s, 3H), 1.25-1.20 (m, 16H), 1.08 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.86 - 0.80 (m, 6H). HRMS (ESI) calcd for C87H117Cl2N11O30 [M+3H]3+m/z 622.9193, found m/z 622.8183..

(β β-D-acetylglucosaminylethyl) aminomethyl-N-decylaminoethyl van comycin (8). Yield 65% (25.0 mg, 13.1 µmol). R.T. = 16.201 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 7.79 (s, 1H), 7.48 (d, J = 8.7 Hz, 1H),

7.46(d, J = 8.7 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.22 (d, J = 8.3 Hz, 1H), 7.11 (s, 1H), 6.84 (dd, J = 8.5, 1.9 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.51 (s, 1H), 5.66 (s, 1H), 5.61 (s, 1H), 5.25 (d, J = 7.7 Hz, 1H), 5.22 (d, J = 3.8 Hz, 1H), 5.11 (s, 3H), 4.82 - 4.74 (m, 1H), 4.68 - 4.62 (m, 1H), 4.43 (s, 1H), 4.40 (s, 1H), 4.34 (d, J = 8.5 Hz, 1H), 4.16 - 4.06 (m, 5H), 3.92 3.86 (m, 1H), 3.86 - 3.79 (m, 1H), 3.71 (d, J = 10.2 Hz, 1H), 3.65 (d, J = 10.6 Hz, 1H), 3.30 - 3.21 (m, 5H), 3.18 - 3.13 (m, 2H), 3.08 - 2.96(m, 6H), 2.88 (dd, J = 9.4, 6.4 Hz, 2H), 2.76 - 2.71 (m, 1H), 2.36 (s, 3H), 1.92


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- 1.86 (m, 1H), 1.78 (s, 3H), 1.71 (d, J = 12.9 Hz, 1H), 1.59 - 1.44 (m, 5H), 1.28 (s, 3H), 1.23-1.19 (m, 16H), 1.04 (d, J = 6.3 Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H), 0.85 - 0.79 (m, 6H). HRMS (ESI) calcd. for C89H120Cl2N12O30 [M+3H] 3+m/z 636.5948, found m/z 636.5870.

Hydroxyethylaminomethyl-N-decylaminoethyl vancomycin (9). Yield 91% (30.8 mg, 18.1 µmol). R.T. = 16.450 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 7.81 (d, J = 1.9 Hz, 1H), 7.50 (dd, J =

8.3, 1.8 Hz, 1H), 7.47 (dd, J = 8.3, 1.8 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.12 (s, 1H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.70 (d, J = 7.1 Hz, 1H), 5.64 (s, 1H), 5.26 (d, J = 7.8 Hz, 1H), 5.24 (d, J = 4.0 Hz, 1H), 5.12 (s, 3H), 4.79 (br s, 1H), 4.67 - 4.63 (m, 1H), 4.45 (d, J = 4.8 Hz, 1H), 4.43 (d, J = 5.8 Hz, 1H), 4.17 - 4.07 (m, 4H), 3.70 - 3.64 (m, 3H),3.55 (d, J = 9.0 Hz, 1H) , 3.30 - 3.23 (m, 2H), 3.17 (s, 1H), 3.08 (q, J = 7.3 Hz, 2H), 3.00 - 2.94 (m, 2H), 2.94 - 2.89 (m, 2H), 2.13 (q, J = 8.0, 7.6 Hz, 0H), 1.93 - 1.85 (m, 1H), 1.72 (d, J = 13.1 Hz, 1H), 1.63 - 1.50 (m, 4H), 1.29 (s, 3H), 1.28 1.19 (m, 16H) 1.08 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.86 0.80 (m, 6H),1.05 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.87 0.82 (m, 6H). HRMS (ESI) calcd for C81H107Cl2N11O25 [M+3H]3+m/z 568.9017, found m/z 568.9059.



Mannosaminylmethyl-N-decylaminoethyl vancomycin (10) Yield 54% (19.7 mg, 10.8 µmol). R.T. = 16.265 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 7.80 (s, 1H), 7.54 - 7.41 (m, 4H), 7.32

(d, J = 8.2 Hz, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.13 (s, 1H), 6.86 (d, J = 8.2 Hz,1H), 6.78 (d, J = 8.2 Hz, 1H), 6.55 (s, 1H), 5.69 (d, J = 7.1 Hz, 1H), 5.64 (s, 1H), 5.26 (d, J = 7.6 Hz, 1H), 5.24 (d, J = 3.9 Hz, 1H), 5.17 5.07 (m, 3H), 4.78 (br s, 1H), 4.69 - 4.62 (m, 1H), 4.47 (s, 1H), 4.43 (d, J = 5.6 Hz, 1H), 4.28 (s, 1H), 4.12 (d, J = 11.7 Hz, 1H), 3.74 - 3.62 (m, 3H), 3.57 - 3.51 (m, 1H), 3.34 - 3.21 (m, 4H), 3.16 (s, 1H), 3.03 (br s, 1H), 2.93 - 2.85 (m, 3H), 2.19 - 2.10 (m, 1H), 1.90 (d, J = 13.1 Hz, 1H), 1.72 (d, J = 13.1 Hz, 1H), 1.59 - 1.50 (m, 4H), 1.41 - 1.34 (m, 1H), 1.29 (s, 3H), 1.27-1.22 (m, 16H), 1.06 (d, J = 6.3 Hz,3H), 0.89 (d, J = 6.0 Hz, 3H), 0.87 - 0.82 (m, 6H). HRMS (ESI) calcd. for C85H113Cl2N11O29 [M+2H]2+m/z 911.8619, found m/z 911.8621.

Glucosaminylmethyl-N-decylaminoethyl vancomycin (11). Yield 45% (16.6 mg, 9.1 µmol). R.T. = 16.400 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 7.79 (d, J = 4.7 Hz, 1H), 7.50 - 7.41 (m, 2H), 7.31 (d, J = 8.1 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (dd, J = 8.5, 2.2 Hz, 1H), 6.76 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.68 (d, J = 7.2 Hz, 1H), 5.63 (s, 1H), 5.48 (d, J = 3.4 Hz, 1H), 5.25 (d, J = 7.7 Hz, 1H), 5.23 (s, 1H), 5.12 - 5.07 (m, 3H), 4.76 (br s, 1H), 4.67 - 4.62 (m,


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1H), 4.48 - 4.44 (m, 1H), 4.41 (d, J = 5.5 Hz, 1H), 4.25 (s, 1H), 4.10 (s, 1H), 3.71 - 3.63 (m, 2H), 3.62 - 3.57 (m, 2H), 3.30 - 3.20 (m, 3H), 3.19 3.10 (m, 3H), 2.94 - 2.84 (m, 4H), 2.18 - 2.08 (m, 1H), 1.88 (d, J = 10.6 Hz, 1H), 1.71 (d, J = 13.1 Hz, 1H), 1.60 - 1.50 (m, 4H), 1.44 - 1.36 (m, 1H), 1.28 (s, 3H), 1.25-1.20

(m, 16H), 1.04 (d, J = 6.4 Hz, 3H), 0.89 (d,

J = 6.2 Hz, 3H), 0.86 - 0.78 (m, 6H). HRMS (ESI) calcd for C85H113Cl2N11O29 [M+3H]3+m/z 608.2439, found m/z 608.2428.

Galactosaminylmethyl-N-decylaminoethyl vancomycin (12). Yield 43% (15.7 mg, 8.6 µmol). R.T. = 16.685 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.69 (br s, 1H), 7.79 (d,

J = 8.5 Hz, 1H), 7.59 (br s, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.18 (d, J = 8.3 Hz, 1H), 7.13 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.71 (d, J = 7.5 Hz, 1H), 5.67 (s, 1H), 5.51 (d, J = 3.6 Hz, 1H), 5.28 (d, J = 4.4 Hz, 1H), 5.26 (d, J = 7.9 Hz, 1H), 5.12 (s, 1H), 5.09 (s, 2H), 4.80 (s, 1H), 4.66 (t, J = 6.8 Hz, 1H), 4.47 (s, 1H), 4.40 (t, J = 5.5 Hz, 1H), 4.23 (s, 1H), 4.00 (s, 1H), 3.84 (dd, J = 10.6, 3.0 Hz, 1H), 3.80 (t, J = 6.5 Hz, 1H), 3.75 (d, J = 3.2 Hz, 1H), 3.65 (d, J = 10.7 Hz, 1H), 3.29 - 3.21 (m, 4H), 3.20 - 3.09 (m, 4H), 3.07 - 3.00 (m, 1H), 2.91 (t, J = 7.7 Hz, 3H), 2.57 (s, 3H), 1.91 (d, J = 11.8 Hz, 1H), 1.82 (d, J = 13.0 Hz, 1H), 1.68 - 1.59 (m, 2H), 1.56 - 1.46 (m, 4H), 1.34 (s, 3H), 1.25-1.20 (m, 16H), 1.08 (d, J =



6.2 Hz, 3H), 0.90 (d, J = 5.9 Hz, 4H), 0.85 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C85H113Cl2N11O29 [M+3H]3+m/z 608.2439, found m/z 608.2521.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-dec ylaminoethyl vancomycin (13). Yield 52% (19.7 mg, 10.4 µmol). R.T. = 16.110 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.62 (s, 1H),7.79 (s, 1H),

7.60 (br s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 9.0 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 9.6 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.71 (s, 2H), 5.68 (s, 1H), 5.30 - 5.24 (m, 3H), 5.11 (s, 2H), 5.08 (s, 1H), 4.80 (s, 1H), 4.65 (d, J = 6.0 Hz, 1H), 4.45 (br s, 1H), 4.40 (d, J = 6.0 Hz, 1H), 4.10 (s, 1H), 4.06 (s, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (t, J = 2.9 Hz, 1H), 3.65 (d, J = 10.8 Hz, 1H), 3.57 - 3.54 (m, 1H), 3.36 (dd, J = 10.8, 5.1 Hz, 1H), 3.28 - 3.23 (m, 3H), 3.21 - 3.09 (m, 5H), 2.95 - 2.88 (m, 4H), 2.56 (s, 3H), 1.86 1.78 (m, 3H), 1.69 - 1.59 (m, 2H), 1.56 - 1.47 (m, 3H), 1.34 (s, 3H), 1.27-1.21 (m, 16H), 1.08 (d, J = 6.1 Hz, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.87 - 0.79 (m, 6H). HRMS (ESI) calcd for C88H120Cl2N12O30 [M+2H]2+m/z 948.3883, found m/z 948.3892.


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(Acyclic-lactobionolactone) acylaminopropylaminomethyl-N-decyla minoethyl vancomycin (14). Yield 51% (21.0 mg, 10.2 µmol). R.T. = 16.054 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.62 (s, 1H), 7.79 (s, 1H),

7.61 (s, 1H), 7.49 (d, J = 8.6 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.72 (d, J = 7.4 Hz, 1H), 5.68 (s, 1H), 5.29 (s, 1H), 5.27 (d, J = 7.7 Hz, 1H), 5.11 (s, 2H), 5.08 (s, 1H), 4.80 (s, 1H), 4.65 (d, J = 6.7 Hz, 1H), 4.45 (s, 1H), 4.41 (d, J = 5.6 Hz, 1H), 4.27 (d, J = 6.6 Hz, 1H), 4.11 (d, J = 2.3 Hz, 2H), 4.06 (s, 3H), 4.02 - 3.96 (m, 2H), 3.71 - 3.62 (m, 3H), 3.61 - 3.49 (m, 6H), 3.40 (d, J = 6.5 Hz, 1H), 3.32 - 3.28 (m, 2H), 3.26 (s, 3H), 3.22 - 3.08 (m, 6H), 3.07 2.97 (m, 1H), 2.94 - 2.88 (m, 4H), 2.79 - 2.67 (m, 1H), 2.56 (s, 3H),1.92 (d, J = 13.4 Hz, 1H), 1.86 - 1.77 (m, 3H), 1.68 - 1.61 (m, 2H), 1.55 - 1.47 (m, 3H), 1.34 (s, 3H), 1.27-1.21 (m, 16H), 1.09 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.88 - 0.82 (m, 6H). HRMS (ESI) calcd for C94H130Cl2N12O35 [M+2H]2+m/z 1029.4148, found m/z 1029.4148.

(β β-D-mannopyranosylethyl) aminomethyl-N-4’-chlorobiphenylmethy l vancomycin (15) Yield 71% (26.7 mg, 14.2 µmol). R.T. = 15.744 min (analytical HPLC). 1

H-NMR (600 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.67 (s, 1H), 7.83 (d, J =



2.0 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.58 (d, J = 1.9 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 8.3 Hz, 1H), 7.30 (d, J = 8.3 Hz, 1H), 7.19 (d, J = 8.5 Hz, 1H), 7.10 (s, 1H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.87 (d, J = 2.1 Hz, 1H), 5.65 (d, J = 7.4 Hz, 1H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.12 (d, J = 12.6 Hz, 2H), 5.07 (d, J = 2.0 Hz, 1H), 4.76 (s, 1H), 4.66 (d, J = 6.7 Hz, 1H), 4.64 (d, J = 1.7 Hz, 1H), 4.46 (s, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.19 - 4.08 (m, 3H), 4.06 - 3.99 (m, 1H), 3.98 - 3.92 (m, 1H), 3.88 - 3.80 (m, 1H), 3.69 - 3.60 (m, 3H), 3.57 (t, J = 8.5 Hz, 1H), 3.50 (dd, J = 9.0, 3.4 Hz, 1H), 3.38 (t, J = 9.4 Hz, 1H), 3.36 - 3.31 (m, 2H), 3.29 - 3.23 (m, 3H), 3.11 (d, J = 5.6 Hz, 2H), 2.73 (d, J = 15.5 Hz, 1H), 2.10 (d, J = 12.1 Hz, 1H), 1.95 - 1.86 (m, 1H), 1.82 (d, J = 13.1 Hz, 1H), 1.51 (s, 3H), 1.10 (d, J = 6.2 Hz, 3H), 0.91 (t, J = 6.6 Hz, 6H). HRMS (ESI) calcd. for C88H101Cl3N10O30[M+2H]2+m/z 942.2954, found m/z 942.2943.

(β β-D-galactopyranosylethyl) aminomethyl-N-4’-chlorobiphenylmeth yl vancomycin (16). Yield 76% (28.6 mg, 15.2 µmol). R.T. = 15.840 min (analytical HPLC). 1


7.72 (d, J = 8.1 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.2 Hz, 2H), 7.53 - 7.51 (m, 2H), 7.49 (d, J = 8.6 Hz, 1H), 7.46 (dd, J = 8.3, 1.8


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Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.51 (s, 1H), 5.72 (s, 1H), 5.70 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (s, 1H), 5.12 (s, 2H), 5.10 (s, 1H), 4.81 (s, 1H), 4.69 - 4.62 (m, 1H), 4.45 (s, 1H), 4.40 (s, 1H), 4.21 - 3.88 (m, 7H), 3.86 - 3.71 (m, 1H), 3.66 (d, J = 10.6 Hz, 1H), 3.61 (d, J = 3.0 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.53 - 3.49 (m, 2H), 3.38 3.33 (m, 2H), 3.32 - 3.22 (m, 5H), 2.52 (s, 3H), 2.17 - 2.03 (m, 2H), 1.81 (d, J = 13.1 Hz, 1H), 1.69 - 1.56 (m, 3H), 1.48 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 3H), 0.85 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C88H101Cl3N10O30 [M+2H]2+m/z 942.2954, found m/z 942.3032.

Hydroxyethylaminomethyl-N-4'-chlorobiphenylmethyl vancomycin (17). Yield 90% (31.0 mg, 18.0 µmol). R.T. = 15.920 min (analytical HPLC). 1


7.73 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.7 Hz, 1H), 7.48 - 7.45 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.53 (s, 1H), 5.75 5.69 (m, 2H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.12 (s, 2H), 5.09 (d, J = 1.9 Hz, 1H), 4.81 (s, 1H), 4.67 - 4.63 (m, 1H), 4.46 (d, J = 5.3 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.18 - 3.96 (m, 6H), 3.68 - 3.64



(m, 3H), 3.56 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 7.2 Hz, 1H), 3.31 - 3.21 (m, 4H), 2.98 - 2.93 (m, 2H), 2.57 (s, 3H), 2.10 (d, J = 9.6 Hz, 2H), 1.81 (d, J = 13.2 Hz, 1H), 1.68 - 1.66 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.1 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C82H91Cl3N10O25[M+3H]3+m/z 574.5152, found m/z 574.5170.

Galactosaminylmethyl-N-4'-chlorobiphenylmethyl vancomycin (18). Yield 42% (15.4 mg, 8.4 µmol). R.T. = 15.835 min (analytical HPLC). 1

H-NMR (600 MHz, DMSO-d6) δ 8.80 (br s, 1H), 8.72 (br s, 1H), 7.83 (s,

1H), 7.61 (br s, 1H), 7.55 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.5 Hz, 0H), 7.46 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.13 (s, 1H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 6.53 (s, 1H), 5.72 (d, J = 11.0 Hz, 2H), 5.51 (d, J = 3.6 Hz, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 5.12 (s, 1H), 5.10 (d, J = 2.0 Hz, 2H), 4.80 (s, 1H), 4.66 (q, J = 6.7 Hz, 1H), 4.50 - 4.44 (m, 1H), 4.40 (t, J = 5.8 Hz, 1H), 4.23 (s, 1H), 4.14 (d, J = 10.3 Hz, 1H), 4.04 - 3.97 (m, 3H), 3.84 (dd, J = 10.5, 3.0 Hz, 1H), 3.80 (t, J = 6.5 Hz, 1H), 3.75 (d, J = 3.2 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.42 - 3.38 (m, 1H), 3.30 - 3.21 (m, 2H), 3.15 (dd, J = 10.5, 3.5 Hz, 1H), 2.76 - 2.68 (m, 1H), 2.57 (s, 3H), 2.10 (d, J = 13.4 Hz, 1H), 1.82 (d, J = 13.2 Hz, 1H), 1.69 - 1.60 (m, 2H), 1.49 (s, 3H), 1.22 - 1.19 (m, 1H), 1.10 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J =


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6.1 Hz, 3H). HRMS (ESI) calcd for C86H97Cl3N10O29 [M+2H]2+m/z 920.2822, found m/z 920.2862.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-4'chlorobiphenylmethyl vancomycin (19). Yield 55% (21.0 mg, 11.0 µmol). R.T. = 15.747 min (analytical HPLC). 1


7.70 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.49 (dd, J = 8.3, 1.7 Hz, 1H), 7.46 (dd, J = 8.3, 1.7 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (dd, J = 8.4, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.72 (s, 1H), 5.71 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.12 (d, J = 3.8 Hz, 2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.68 - 4.61 (m, 1H), 4.44 (s, 1H), 4.40 (s, 1H), 4.10 (s, 1H), 4.05 (s, 2H), 4.02 (d, J = 6.7 Hz, 2H), 4.00 (d, J = 3.6 Hz, 2H), 3.89 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.6 Hz, 1H), 3.59 3.53 (m, 3H), 3.36 (dd, J = 10.7, 5.2 Hz, 1H), 3.30 - 3.23 (m, 3H), 3.22 3.16 (m, 1H), 3.16 - 3.10 (m, 1H), 2.95 - 2.87 (m, 2H), 2.55 (s, 3H), 2.15 - 2.05 (m, 2H), 1.87 - 1.77 (m, 3H), 1.68 - 1.59 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.7 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C89H104Cl3N11O30 [M+2H]2+m/z 956.8086, found m/z 956.8070.



(Acyclic-lactobionolactone) acylaminopropylaminomethyl-N-4'-chlor obiphenylmethyl vancomycin (20). Yield 51% (21.2 mg, 10.2 µmol). R.T. = 15.695 min (analytical HPLC). 1

H-NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.65 (s, 1H), 7.82 (d, J =

1.9 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 7.62 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.52 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.6 Hz, 1H), 7.48 - 7.44 (m, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.78 - 5.68 (m, 2H), 5.34 (d, J = 7.6 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.13 (s, 2H), 5.09 (s, 1H), 4.81 (s, 1H), 4.69 - 4.62 (m, 1H), 4.44 (d, J = 4.9 Hz, 1H), 4.41 (d, J = 5.7 Hz, 1H), 4.26 (d, J = 6.8 Hz, 1H), 4.10 (d, J = 2.3 Hz, 2H), 4.09 - 3.97 (m, 8H), 3.71 - 3.64 (m, 3H), 3.62 - 3.54 (m, 3H), 3.51 (dd, J = 11.4, 4.7 Hz, 1H), 3.39 (t, J = 6.3 Hz, 1H), 3.33 - 3.23 (m, 4H), 3.22 - 3.17 (m, 1H), 3.17 - 3.11 (m, 1H), 2.92 (t, J = 6.7 Hz, 2H), 2.57 (s, 3H), 2.10 (d, J = 11.1 Hz, 2H), 1.87 - 1.76 (m, 3H), 1.69 - 1.60 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C95H114Cl3N11O35 [M+2H]2+m/z 1037.8350, found m/z 1037.8290.

(Phosphonomethyl) aminomethyl-N-4'-chlorobiphenylmethyl vanco mycin (21).


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7.70 (d, J = 8.6 Hz, 2H), 7.46 (dd, J = 8.4, 1.9 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.13 (s, 1H), 6.79 (dd, J = 8.4, 2.0 Hz, 1H), 6.72 (d, J = 8.6 Hz, 1H), 6.44 (s, 1H), 5.75 (s, 1H), 5.63 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.28 (s, 1H), 5.14 (d, J = 8.4 Hz, 2H), 5.09 (s, 1H), 4.86 (s, 1H), 4.69 - 4.62 (m, 1H), 4.43 (s, 1H), 4.41 (s, 1H), 4.31 4.24 (m, 2H), 4.16 (s, 2H), 4.02 (s, 2H), 3.67 (d, J = 10.8 Hz, 1H), 3.56 (d, J = 8.5 Hz, 1H), 3.30 - 3.21 (m, 3H), 2.70 - 2.60 (m, 2H), 2.55 (s, 1H), 2.08 (d, J = 22.6 Hz, 2H), 1.82 (d, J = 13.1 Hz, 1H), 1.64 (s, 2H), 1.48 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.90 (d, J = 6.4 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C81H90Cl3N10O27P [M+3H]3+m/z 591.1671, found m/z 591.1677.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-no nyl vancomycin (22). Yield 52% (19.1 mg, 10.4 µmol). R.T. = 14.193 min (analytical HPLC). 1


= 8.7 Hz, 1H), 7.44 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.53 (s, 1H), 5.72 (s, 1H), 5.70 (s, 1H), 5.30 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 1.2 Hz, 1H), 5.11 (s, 2H), 5.07 (s, 1H), 4.79 (s, 1H), 4.09 (s,



1H), 4.08 - 4.03 (m, 3H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.7, 2.2 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 3.58 - 3.48 (m, 4H), 3.36 (dd, J = 10.6, 4.9 Hz, 1H), 3.28 - 3.21 (m, 3H), 3.21 - 3.17 (m, 1H), 3.17 - 3.11 (m, 1H), 2.96 - 2.88 (m, 2H), 2.74 (s, 1H), 2.68 (s, 1H), 2.54 (s, 3H), 2.15 - 2.07 (m, 1H), 1.96 (d, J = 11.9 Hz, 1H), 1.86 - 1.80 (m, 1H), 1.77 (d, J = 13.1 Hz, 1H), 1.68 - 1.61 (m, 2H), 1.55 - 1.46 (m, 3H), 1.33 (s, 3H), 1.27-1.21 (m, 14H), 1.06 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C85H113Cl2N11O30 [M+2H]2+m/z 919.8594, found m/z 919.8655.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-dec yl vancomycin (23). Yield 53% (19.6 mg, 10.6 µmol). R.T. = 14.283 min (analytical HPLC). 1


J = 1.8 Hz, 1H), 7.60 (s, 1H), 7.48 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.10 (s, 1H), 6.84 (dd, J = 8.5, 1.9 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.71 (d, J = 8.5 Hz, 1H), 5.69 (s, 1H), 5.30 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 4.2 Hz, 1H), 5.12 (s, 2H), 5.07 (d, J = 2.0 Hz, 1H), 4.80 (s, 1H), 4.62 - 4.56 (m, 1H), 4.43 (s, 1H), 4.41 (d, J = 5.9 Hz, 1H), 4.11 - 4.07 (m, 1H), 4.07 4.03 (m, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.6, 2.2 Hz, 1H), 3.65 (d, J = 10.7 Hz, 1H), 3.56 (t, J = 3.0 Hz, 1H), 3.55 - 3.53 (m, 1H),


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3.53 - 3.50 (m, 1H), 3.50 - 3.48 (m, 1H), 3.36 (dd, J = 10.7, 5.0 Hz, 1H), 3.28 - 3.23 (m, 3H), 3.21 - 3.17 (m, 1H), 3.16 - 3.11 (m, 1H), 2.95 - 2.88 (m, 2H), 2.77 - 2.72 (m, 1H), 2.70 - 2.64 (m, 1H), 2.56 (s, 3H), 2.15 2.07 (m, 1H), 1.96 (d, J = 12.0 Hz, 1H), 1.82 (dt, J = 14.0, 7.0 Hz, 1H), 1.77 (d, J = 12.8 Hz, 1H), 1.68 - 1.60 (m, 2H), 1.55 - 1.45 (m, 3H), 1.33 (s, 3H), 1.25-1.19 (m, 16H), 1.06 (d, J = 6.2 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.3 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C86H115Cl2N11O30 [M+2H]2+m/z 926.8672, found m/z 926.8674.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-4-e thylbenzyl vancomycin (24). Yield 46% (16.8 mg, 9.2 µmol). R.T. = 12.123 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 7.82 (s, 1H), 7.61 (br s,

1H), 7.48 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.30 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 8.6 Hz, 1H), 7.10 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.75 - 5.69 (m, 2H), 5.33 (d, J = 7.6 Hz, 1H), 5.28 (d, J = 2.0 Hz, 1H), 5.12 (s, 2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.63 (d, J = 6.6 Hz, 1H), 4.44 (s, 1H), 4.40 (s, 1H), 4.10 (s, 1H), 4.05 (br s, 3H), 4.00 (d, J = 3.6 Hz, 2H), 3.97 - 3.91 (m, 1H), 3.90 - 3.87 (m, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.58 - 3.52 (m, 2H), 3.50 (d, J = 6.0 Hz, 1H), 3.36 (dd, J = 10.5, 4.8 Hz, 1H), 3.30 - 3.22 (m, 3H), 3.21 - 3.16 (m, 1H), 3.16 - 3.11 (m, 1H),



2.92 (s, 2H), 2.61 (q, J = 6.0 Hz, 2H), 2.56 (s, 3H), 2.15 - 2.05 (m, 2H), 1.85 - 1.75 (m, 3H), 1.68 - 1.60 (m, 3H), 1.46 (s, 3H), 1.60 (t, J = 5.4 Hz 3H), 1.09 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C85H105Cl2N11O30 [M+2H]2+m/z 915.8281, found m/z 915.8275.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-pe ntylbenzyl vancomycin (25). Yield 47% (17.6 mg, 9.4 µmol). R.T. = 13.959 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.80 (br s, 1H), 8.66 (br s, 1H), 7.84 (s, 1H), 7.62 (br s, 2H), 7.51 (dd, J = 8.4, 1.8 Hz, 1H), 7.48 (dd, J = 8.4, 1.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 2H), 7.32 (d, J = 8.3 Hz, 1H), 7.24 (d, J = 8.4 Hz, 3H), 7.12 (s, 1H), 6.86 (dd, J = 8.5, 1.8 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.78 - 5.69 (m, 2H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.14 (s, 2H), 5.11 (s, 1H), 4.82 (s, 1H), 4.66 (q, J = 6.6 Hz, 1H), 4.46 (d, J = 3.0 Hz, 1H), 4.43 (s, 1H), 4.42 (s, 1H), 4.14 4.11 (m, 1H), 4.07 (s, 2H), 4.02 (d, J = 3.5 Hz, 1H), 3.96 (t, J = 12.0 Hz, 2H), 3.91 (t, J = 2.9 Hz, 1H), 3.68 (d, J = 10.7 Hz, 1H), 3.60 - 3.55 (m, 2H), 3.52 (d, J = 7.3 Hz, 1H), 3.38 (dd, J = 12.0, 4.0 Hz, 1H), 3.31 - 3.24 (m, 2H), 3.24 - 3.18 (m, 1H), 3.18 - 3.13 (m, 1H), 2.96 - 2.90 (m, 2H), 2.59 - 2.55 (m, 6H), 2.16 - 2.06 (m, 2H), 1.87 - 1.78 (m, 3H), 1.70 - 1.62 (m, 2H), 1.55 (p, J = 7.5 Hz, 2H), 1.48 (s, 3H), 1.33 - 1.20 (m, 6H), 1.11


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(d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.1 Hz, 3H), 0.87 (d, J = 6.2 Hz, 3H), 0.85 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C88H111Cl2N11O30 [M+2H]2+m/z 936.8515, found m/z 936.8536.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-but oxybenzyl vancomycin (26). Yield 45% (16.9 mg, 9.0 µmol). R.T. = 12.757 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.66 (br s, 1H), 7.84 (s,

1H), 7.61 (br s, 1H), 7.51 (dd, J = 8.3, 1.8 Hz, 1H), 7.48 (dd, J = 8.3, 1.8 Hz, 1H), 7.36 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.86 (dd, J = 8.5, 2.0 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.77 - 5.68 (m, 2H), 5.35 (d, J = 7.6 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 5.14 (s, 2H), 5.11 (s, 1H), 4.82 (s, 1H), 4.66 (q, J = 6.6 Hz, 1H), 4.46 (d, J = 5.4 Hz, 1H), 4.42 (s, 1H), 4.15 - 4.10 (m, 1H), 4.07 (s, 2H), 4.02 (d, J = 3.6 Hz, 1H), 3.97 (t, J = 6.5 Hz, 2H), 3.94 - 3.86 (m, 3H), 3.68 (d, J = 10.7 Hz, 1H), 3.60 - 3.55 (m, 2H), 3.40 - 3.36 (dd, J = 12.0, 4.3 Hz, 1H), 3.31 - 3.24 (m, 2H), 3.24 3.18 (m, 1H), 3.18 - 3.13 (m, 1H), 2.96 - 2.88 (m, 2H), 2.56 (s, 3H), 2.09 (d, J = 14.8 Hz, 2H), 1.87 - 1.77 (m, 3H), 1.71 - 1.64 (m, 5H), 1.47 (s, 3H), 1.42 (h, J = 7.4 Hz, 2H), 1.11 (d, J = 6.3 Hz, 3H), 0.94 - 0.90 (m, 6H), 0.87 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C87H109Cl2N11O31 [M+2H]2+m/z 937.8412, found m/z 937.8439.



(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-4(trimethylsilyl)ethynylbenzyl vancomycin (27). Yield 49% (18.8 mg, 9.9 µmol). R.T. = 14.595 min (analytical HPLC). 1


1H), 7.63 (br s, 1H), 7.57 - 7.49 (m, 3H), 7.49 - 7.42 (m, 3H), 7.32 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.86 (dd, J = 8.4, 1.9 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.55 (s, 1H), 5.77 - 5.69 (m, 2H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.2 Hz, 1H), 5.14 (s, 2H), 5.11 (d, J = 1.9 Hz, 1H), 4.82 (s, 1H), 4.66 (q, J = 6.6 Hz, 1H), 4.46 (d, J = 5.4 Hz, 1H), 4.43 (d, J = 5.7 Hz, 1H), 4.15 - 4.10 (m, 1H), 4.08 (s, 2H), 4.02 (d, J = 3.6 Hz, 1H), 4.01 - 3.97 (m, 3H), 3.91 (dd, J = 3.6, 2.3 Hz, 1H), 3.68 (d, J = 10.7 Hz, 1H), 3.60 - 3.55 (m, 2H), 3.53 (d, J = 7.5 Hz, 1H), 3.39 - 3.36 (m, 1H), 3.31 - 3.24 (m, 2H), 3.21 (p, J = 6.8 Hz, 1H), 3.16 (p, J = 6.6 Hz, 1H), 2.94 (t, J = 7.5 Hz, 2H), 2.59 (s, 3H), 2.17 - 2.07 (m, 2H), 1.89 1.78 (m, 3H), 1.70 - 1.62 (m, 2H), 1.48 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.93 (d, J = 6.1 Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H), 0.23 (s, 9H). HRMS (ESI) calcd for C88H109Cl2N11O30Si [M+2H]2+m/z 949.8322, found m/z 949.8331.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-4-e thynylbenzyl vancomycin (28).


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1H), 7.61 (s, 1H), 7.52 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.2 Hz, 3H), 7.30 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.84 (dd, J = 8.4, 1.9 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.71 (t, J = 6.8 Hz, 2H), 5.32 (d, J = 7.7 Hz, 1H), 5.28 (d, J = 4.1 Hz, 1H), 5.12 (d, J = 3.4 Hz, 2H), 5.08 (d, J = 1.9 Hz, 1H), 4.80 (s, 1H), 4.64 (d, J = 6.7 Hz, 1H), 4.44 (d, J = 5.2 Hz, 1H), 4.41 (d, J = 5.7 Hz, 1H), 4.25 - 4.19 (m, 1H), 4.08 (s, 1H), 4.06 (s, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.57 - 3.53 (m, 2H), 3.50 (d, J = 7.6 Hz, 1H), 3.36 (d, J = 7.0 Hz, 1H), 3.29 - 3.22 (m, 2H), 3.19 (p, J = 6.7 Hz, 1H), 3.13 (p, J = 13.4, 6.6 Hz, 1H), 2.92 (t, J = 7.4 Hz, 2H), 2.56 (s, 3H), 2.16 - 2.02 (m, 2H), 1.86 - 1.75 (m, 3H), 1.68 1.60 (m, 3H), 1.45 (s, 3H), 1.09 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C85H101Cl2N11O30 [M+2H]2+m/z 913.8124, found m/z 913.8131.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-co umaronemethyl vancomycin (29). Yield 39% (14.4 mg, 7.8 µmol). R.T. = 11.220 min (analytical HPLC). 1


1H), 7.61 (br s, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.46 (dd, J = 8.4, 1.7 Hz,



1H), 7.30 (d, J = 8.3 Hz, 1H), 7.29 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.7 Hz, 2H), 6.52 (s, 1H), 5.72 (s, 1H), 5.71 (s, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 3.9 Hz, 1H), 5.12 (s, 2H), 5.09 (d, J = 2.0 Hz, 1H), 4.80 (s, 1H), 4.66 - 4.60 (m, 1H), 4.53 (t, J = 8.6 Hz, 2H), 4.45 (d, J = 5.5 Hz, 1H), 4.41 (d, J = 6.2 Hz, 1H), 4.16 - 4.04 (m, 4H), 4.00 (d, J = 10.5 Hz, 1H), 3.88 (d, J = 12.0 Hz, 2H), 3.69 - 3.63 (m, 3H), 3.54 (d, J = 8.7 Hz, 1H), 3.29 - 3.22 (m, 3H), 3.15 (t, J = 8.8 Hz, 2H), 2.98 - 2.93 (m, 2H), 2.56 (s, 3H), 2.15 - 2.03 (m, 2H), 1.77 (d, J = 13.2 Hz, 1H), 1.69 - 1.59 (m, 3H), 1.54 - 1.48 (m, 1H), 1.45 (s, 3H), 1.22 - 1.20 (m, 1H), 1.09 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C85H103Cl2N11O31 [M+2H]2+m/z 827.2781, found m/z 827.2785.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-2menaphthyl vancomycin (30). Yield 48% (17.8 mg, 9.6 µmol). R.T. = 11.363 min (analytical HPLC). 1


1H), 7.96 (d, J = 8.6 Hz, 1H), 7.94 (dd, J = 8.0, 7.2 Hz, 1H), 7.90 (dd, J = 8.0, 7.2 Hz, 1H), 7.82 (s, 1H), 7.60 (br s, 1H), 7.58 - 7.53 (m, 3H), 7.49 (dd, J = 8.3, 1.8 Hz, 1H), 7.46 (dd, J = 8.3, 1.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.9 Hz,


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1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.76 - 5.67 (m, 2H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.2 Hz, 1H), 5.12 (s, 2H), 5.09 (s, 1H), 4.81 (s, 1H), 4.67 (q, J = 6.6 Hz, 1H), 4.45 (d, J = 5.7 Hz, 1H), 4.41 (d, J = 5.7 Hz, 1H), 4.15 (q, J = 12.8 Hz, 2H), 4.06 (s, 2H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (t, J = 2.9 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.59 - 3.51 (m, 3H), 3.35 (dd, J = 10.7, 6.0 Hz, 1H), 3.30 - 3.23 (m, 2H), 3.19 (p, J = 6.6 Hz, 1H), 3.14 (d, J = 6.4 Hz, 1H), 2.92 (t, J = 7.5 Hz, 2H), 2.57 (s, 3H), 2.15 2.08 (m, 2H), 1.86 - 1.77 (m, 3H), 1.68 - 1.60 (m, 2H), 1.51 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C87H103Cl2N11O30 [M+2H]2+m/z 926.8202, found m/z 926.8226.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-4-p yridylbenzyl vancomycin (31). Yield 44% (16.5 mg, 8.8 µmol). R.T. = 12.380 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 8.79 (br s, 1H), 8.72 (d, J = 6.2 Hz,

2H), 8.64 (br s, 1H), 8.22 (d, J = 7.1 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 7.82 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.62 (br s, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.5 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 8.7 Hz, 1H), 7.10 (s, 1H), 6.87 - 6.82 (m, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (d, J = 5.9 Hz, 1H), 5.73 (s, 1H), 5.72 (s, 2H), 5.34 (d, J = 7.6 Hz, 1H), 5.29 (s, 1H), 5.12 (s, 2H), 5.11 - 5.06 (m, 1H), 4.80 (s, 1H), 4.66 (d, J =



7.0 Hz, 1H), 4.49 - 4.43 (m, 1H), 4.41 (d, J = 5.6 Hz, 1H), 4.13 - 4.03 (m, 6H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.6, 2.3 Hz, 1H), 3.66 (d, J = 10.7 Hz, 1H), 3.56 (d, J = 7.0 Hz, 1H), 3.53 (dd, J = 7.0, 2.0 Hz, 0H), 3.36 (dd, J = 10.7, 5.0 Hz, 1H), 3.30 - 3.22 (m, 3H), 3.21 - 3.16 (m, 1H), 3.16 - 3.10 (m, 1H), 2.99 (t, J = 7.9 Hz, 1H), 2.93 (t, J = 7.0 Hz, 1H), 2.85 (t, J = 7.6 Hz, 1H), 2.56 (s, 3H), 2.15 - 2.06 (m, 2H), 1.97 - 1.90 (m, 1H), 1.87 - 1.75 (m, 3H), 1.69 - 1.61 (m, 2H), 1.49 (s, 3H), 1.21 (s, 1H), 1.11 (d, J = 6.0 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H), 0.85 (d, J = 5.9 Hz, 3H). HRMS (ESI) calcd for C88H104Cl2N12O30 [M+K+H]2+m/z 959.3037, found m/z 959.3402.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-4'-t rifluoromethylbiphenylmethyl vancomycin (32). Yield 49% (19.1 mg, 9.8 µmol). R.T. = 16.450 min (analytical HPLC). 1


2H), 7.83 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.80 (d, J = 8.1 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.85 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.75 - 5.69 (m, 2H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (s, 1H), 5.12 (s, 2H), 5.10 (s, 1H), 4.80 (s, 1H), 4.69 - 4.63 (m, 1H), 4.45 (s, 1H), 4.41 (s, 1H), 4.11 (s, 1H), 4.00 (d, J = 3.6 Hz, 1H), 3.89 (dd, J = 3.7, 2.2 Hz, 1H), 3.67 (d, J = 10.6 Hz, 1H),


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3.58 - 3.53 (m, 3H), 3.51 (d, J = 7.0 Hz, 1H), 3.36 (dd, J = 10.3, 4.7 Hz, 1H), 3.30 - 3.23 (m, 3H), 3.22 - 3.16 (m, 1H), 3.16 - 3.10 (m, 1H), 2.92 (s, 2H), 2.56 (s, 3H), 2.16 - 2.06 (m, 2H), 1.86 - 1.76 (m, 3H), 1.68 - 1.59 (m, 3H), 1.50 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C90H104Cl2F3N11O30 [M+2H]2+m/z 973.8218, found m/z 973.8216.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-3-c hloro-4-((2-methyl-[1,1'-biphenyl]-3-yl)methoxyl)benzyl vancomycin (33). Yield 43% (17.5 mg, 8.6 µmol). R.T. = 18.750 min (analytical HPLC). 1


7.62 (s, 1H), 7.58 (d, J = 1.9 Hz, 1H), 7.49 (d, J = 9.1 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 7.38 - 7.37 (m, 1H), 7.36 (dd, J = 8.7, 1.9 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 7.2, 1.2 Hz, 4H), 7.26 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 9.6 Hz, 1H), 7.19 (d, J = 9.6 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.77 - 5.64 (m, 2H), 5.33 (d, J = 7.6 Hz, 1H), 5.27 (d, J = 9.4 Hz, 2H), 5.12 (s, 2H), 5.09 (d, J = 2.0 Hz, 1H), 4.80 (s, 1H), 4.68 - 4.61 (m, 1H), 4.44 (d, J = 5.6 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.13 - 4.03 (m, 4H), 4.00 (d, J = 3.6 Hz, 2H), 3.93 (s, 2H), 3.89 (dd, J = 3.6, 2.3 Hz, 1H), 3.66 (d, J = 10.7



Hz, 1H), 3.58 - 3.53 (m, 2H), 3.51 (d, J = 7.3 Hz, 1H), 3.36 (dd, J = 10.7, 5.0 Hz, 1H), 3.30 - 3.22 (m, 2H), 3.22 - 3.17 (m, 1H), 3.16 - 3.10 (m, 1H), 2.92 (t, J = 7.4 Hz, 2H), 2.78 - 2.69 (m, 1H), 2.57 (s, 3H), 2.19 (s, 3H), 1.87 - 1.75 (m, 3H), 1.69 - 1.60 (m, 2H), 1.54 - 1.48 (m, 1H), 1.45 (s, 3H), 1.10 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C97H112Cl3N11O31 [M+2H]2+m/z 1016.8374, found m/z 1016.8385.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-dec anoyl vancomycin (34). Yield 47% (17.5 mg, 9.4 µmol). R.T. = 16.718 min (analytical HPLC). 1


1H), 7.47 (dd, J = 8.2, 1.8 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.85 (dd, J = 8.7, 2.1 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.66 (d, J = 7.8 Hz, 1H), 5.59 (s, 1H), 5.28 - 5.19 (m, 3H), 5.16 (s, 1H), 5.12 (s, 2H), 4.75 (br s, 1H), 4.70 - 4.62 (m, 1H), 4.47 (d, J = 5.8 Hz, 1H), 4.44 (d, J = 6.0 Hz, 1H), 4.14 - 4.08 (m, 1H), 4.08 - 4.04 (m, 2H), 4.01 (d, J = 3.5 Hz, 1H), 3.90 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 3.58 - 3.52 (m, 3H), 3.37 (dd, J = 10.3, 4.5 Hz, 1H), 3.29 - 3.25 (m, 3H),3.23 - 3.18 (m, 1H) 3.17 - 3.11 (m, 2H), 2.97 - 2.89 (m, 2H), 2.85 (s, 3H), 2.43 - 2.37 (m, 1H), 2.37 - 2.30 (m, 1H), 2.15 - 2.07 (m, 1H), 1.90 (d, J = 11.4 Hz, 1H), 1.86 -


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1.78 (m, 2H), 1.72 (d, J = 13.1 Hz, 1H), 1.59 - 1.50 (m, 4H), 1.39 (dq, J = 13.7, 6.9 Hz, 1H), 1.29 (s, 3H), 1.25-1.19 (m, 14H), 1.07 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.83 (t, J = 6.9 Hz, 3H), 0.81 (d, J = 6.0 Hz, 3H). HRMS (ESI) calcd for C86H113Cl2N11O31 [M+2H]2+m/z 933.8568, found m/z 933.8577.

(Acyclic-gluconic acid lactone) acylaminopropylaminomethyl-N-un decanoyl vancomycin (35). Yield 45% (16.7 mg, 8.9 µmol). R.T. = 17.583 min (analytical HPLC). 1


1H), 7.48 (dd, J = 8.2, 1.8 Hz, 1H), 7.45 (d, J = 8.6 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.85 (dd, J = 8.7, 2.1 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 6.53 (s, 1H), 5.66 (d, J = 7.8 Hz, 1H), 5.59 (s, 1H), 5.28 - 5.19 (m, 3H), 5.16 (s, 1H), 5.12 (s, 2H), 4.75 (br s, 1H), 4.70 - 4.62 (m, 1H), 4.47 (d, J = 5.8 Hz, 1H), 4.44 (d, J = 6.0 Hz, 1H), 4.14 - 4.08 (m, 1H), 4.08 - 4.04 (m, 2H), 4.01 (d, J = 3.5 Hz, 1H), 3.90 (t, J = 2.8 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 3.58 - 3.52 (m, 3H), 3.37 (dd, J = 10.3, 4.5 Hz, 1H), 3.29 - 3.25 (m, 3H), 3.17 - 3.11 (m, 2H), 2.97 - 2.89 (m, 2H), 2.85 (s, 3H), 2.43 - 2.37 (m, 1H), 2.37 - 2.30 (m, 1H), 2.15 - 2.07 (m, 1H), 1.90 (d, J = 11.4 Hz, 1H), 1.86 - 1.78 (m, 2H), 1.72 (d, J = 13.1 Hz, 1H), 1.59 - 1.50 (m, 4H), 1.39 (dq, J = 13.7, 6.9 Hz, 1H), 1.29 (s, 3H), 1.26-1.20 (m, 16H), 1.07 (d, J = 6.4 Hz, 3H), 0.88 (d, J =



6.5 Hz, 3H), 0.84 (t, J = 6.9 Hz, 3H), 0.81 (d, J = 6.0 Hz, 3H). HRMS (ESI) calcd for C87H115Cl2N11O31 [M+2H]2+m/z 940.8647, found m/z 940.8644.

Galactosaminylmethyl-N-decyl vancomycin (36). Yield 40% (14.2 mg, 8.0 µmol). R.T. = 14.790 min (analytical HPLC). 1


7.62 (s, 1H), 7.50 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.15 (s, 1H), 6.87 (d, J = 8.8 Hz, 1H), 6.78 (d, J = 8.0 Hz, 1H), 6.54 (s, 1H), 5.76 - 5.65 (m, 2H), 5.13 (s, 1H), 5.11 (d, J = 9.2 Hz, 1H), 4.82 (s, 1H), 4.61 (q, J = 6.8 Hz, 1H), 4.52 - 4.45 (m, 1H), 4.42 (t, J = 5.9 Hz, 1H), 4.25 (s, 1H), 4.16 - 4.11 (m, 1H), 4.02 (s, 1H), 3.86 (d, J = 10.2 Hz, 1H), 3.82 (t, J = 6.4 Hz, 1H), 3.77 (s, 0H), 3.67 (d, J = 10.8 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.52 (d, J = 9.6 Hz, 1H), 3.30 - 3.22 (m, 5H), 3.17 (d, J = 10.8 Hz, 1H), 2.79 - 2.72 (m, 2H), 2.71 - 2.65 (m, 1H), 2.58 (s, 3H), 1.98 (d, J = 11.9 Hz, 1H), 1.79 (d, J = 13.1 Hz, 1H), 1.71 - 1.62 (m, 3H), 1.58 - 1.48 (m, 3H), 1.35 (s, 3H), 1.27-1.20 (m, 16H), 1.08 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.87 (d, J = 6.2 Hz, 3H), 0.86 (t, J = 6.9 Hz, 3H). HRMS (ESI) calcd for C83H108Cl2N10O29 [M+2H]2+m/z 890.3408, found m/z 890.3413.

Galactosaminylmethyl-N-pentylbenzyl vancomycin (37).


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= 8.8 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.0 Hz, 2H), 7.20 (d, J = 8.4 Hz, 1H), 7.14 (s, 1H), 6.86 (d, J = 8.3 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.54 (s, 1H), 5.76 - 5.68 (m, 2H), 5.52 (d, J = 3.6 Hz, 1H), 5.34 (d, J = 7.6 Hz, 1H), 5.28 (s, 1H), 5.12 (s, 1H), 5.11 (d, J = 5.8 Hz, 1H), 4.81 (s, 1H), 4.65 (q, J = 6.6 Hz, 1H), 4.51 - 4.45 (m, 1H), 4.41 (t, J = 5.9 Hz, 1H), 4.24 (s, 1H), 4.03 3.98 (m, 1H), 3.94 (q, J = 12.8 Hz, 2H), 3.85 (d, J = 12.5 Hz, 1H), 3.81 (t, J = 6.4 Hz, 1H), 3.76 (d, J = 3.2 Hz, 1H), 3.67 (d, J = 10.7 Hz, 1H), 3.56 (t, J = 8.5 Hz, 1H), 3.53 - 3.49 (m, 1H), 3.30 - 3.22 (m, 2H), 3.16 (d, J = 10.0 Hz, 1H), 2.57 (s, 3H), 2.57 - 2.52 (m, 2H), 2.10 (d, J = 11.4 Hz, 1H), 1.80 (d, J = 13.2 Hz, 1H), 1.70 - 1.61 (m, 2H), 1.54 (p, J = 7.5 Hz, 2H), 1.47 (s, 3H), 1.32 - 1.19 (m, 5H), 1.10 (d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C85H104Cl2N10O29 [M+2H]2+m/z 900.3252, found m/z 900.3265.

Galactosaminylmethyl-N-4-(trimethylsilyl)ethynylbenzyl vancomycin (38). Yield 41% (15.0 mg, 8.2 µmol). R.T. = 14.599 min (analytical HPLC). 1H NMR (600 MHz, DMSO-d6) δ 8.82 (br s, 1H), 8.72 (br s, 1H), 7.84 (s,



1H), 7.63 (br s, 1H), 7.54 - 7.49 (m, 3H), 7.49 - 7.44 (m, 3H), 7.32 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.15 (s, 1H), 6.87 (d, J = 8.9 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.55 (s, 1H), 5.76 - 5.69 (m, 2H), 5.53 (s, 1H), 5.34 (d, J = 7.6 Hz, 1H), 5.29 (s, 1H), 5.16 - 5.08 (m, 3H), 4.81 (s, 1H), 4.66 (q, J = 6.8 Hz, 1H), 4.51 - 4.46 (m, 1H), 4.42 (t, J = 5.9 Hz, 1H), 4.04 - 3.97 (m, 3H), 3.85 (d, J = 10.7 Hz, 1H), 3.81 (t, J = 6.4 Hz, 1H), 3.76 (s, 1H), 3.68 (d, J = 10.8 Hz, 1H), 3.57 (t, J = 8.5 Hz, 1H), 3.53 - 3.50 (m, 1H), 3.31 - 3.23 (m, 2H), 3.17 (d, J = 11.2 Hz, 1H), 2.58 (s, 3H), 2.09 (d, J = 11.7 Hz, 1H), 1.81 (d, J = 13.2 Hz, 1H), 1.69 - 1.62 (m, 2H), 1.47 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.92 (d, J = 6.0 Hz, 3H), 0.87 (d, J = 6.1 Hz, 3H), 0.22 (s, 9H). HRMS (ESI) calcd for C85H102Cl2N10O29Si [M+2H]2+m/z 913.3058, found m/z 913.3060.

Galactosaminylmethyl-N-4-ethynylbenzyl vancomycin (39). Yield 42% (14.7 mg, 8.4 µmol). R.T. = 10.494 min (analytical HPLC). 1

H NMR (600 MHz, DMSO-d6) δ 7.85 (s, 1H), 7.63 (s, 1H), 7.56 - 7.52

(m, 3H), 7.52 - 7.45 (m, 3H), 7.33 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 0H), 7.15 (s, 0H), 6.87 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 8.7 Hz, 1H), 6.56 (s, 0H), 5.77 - 5.68 (m, 2H), 5.54 (d, J = 3.6 Hz, 1H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (d, J = 4.3 Hz, 1H), 5.14 (s, 1H), 5.13 - 5.11 (m, 1H), 4.83 (s, 1H), 4.67 (q, J = 6.6 Hz, 1H), 4.52 - 4.47 (m, 1H), 4.43 (t, J = 5.9 Hz, 1H), 4.27 (d, J = 1.9 Hz, 2H), 4.06 - 3.94 (m, 4H), 3.86 (dd, J = 10.6, 3.0


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Hz, 1H), 3.82 (t, J = 6.5 Hz, 1H), 3.77 (d, J = 3.3 Hz, 1H), 3.69 (d, J = 10.8 Hz, 1H), 3.58 (t, J = 8.5 Hz, 1H), 3.53 (dd, J = 10.7, 3.6 Hz, 1H), 3.33 - 3.23 (m, 3H), 3.17 (dd, J = 10.6, 3.4 Hz, 1H), 2.59 (s, 3H), 2.10 (d, J = 11.0 Hz, 1H), 1.82 (d, J = 13.1 Hz, 1H), 1.71 - 1.61 (m, 3H), 1.48 (s, 3H), 1.12 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C82H94Cl2N10O29 [M+2H]2+m/z 877.2860, found m/z 877.2866.

Galactosaminylmethyl-N-4'-trifluoromethylbiphenylmethyl vancom ycin (40). Yield 44% (16.5 mg, 8.8 µmol). R.T. = 16.579 min (analytical HPLC). 1


J = 8.1 Hz, 2H), 7.84 (d, J = 8.5 Hz, 3H), 7.82 (d, J = 8.4 Hz, 2H), 7.64 (s, 1H), 7.61 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.3 Hz, 1H), 7.23 (dd, J = 8.5, 3.3 Hz, 1H), 7.16 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 6.55 (s, 1H), 5.78 - 5.69 (m, 2H), 5.53 (d, J = 3.5 Hz, 1H), 5.36 (d, J = 7.7 Hz, 1H), 5.31 (s, 1H), 5.14 (s, 1H), 5.12 (d, J = 4.2 Hz, 1H), 4.83 (s, 1H), 4.68 (q, J = 6.7 Hz, 1H), 4.54 - 4.46 (m, 1H), 4.43 (t, J = 5.8 Hz, 1H), 4.25 (s, 1H), 4.19 4.12 (m, 1H), 4.08 - 4.05 (m, 2H), 4.03 (d, J = 8.6 Hz, 1H), 3.86 (d, J = 11.9 Hz, 1H), 3.82 (t, J = 6.5 Hz, 1H), 3.77 (d, J = 3.1 Hz, 1H), 3.69 (d, J = 10.5 Hz, 1H), 3.58 (t, J = 8.5 Hz, 1H), 3.52 (dd, J = 11.8, 5.6 Hz, 1H),



3.33 - 3.24 (m, 2H), 3.17 (d, J = 10.6 Hz, 1H), 2.80 - 2.71 (m, 1H), 2.59 (s, 3H), 2.19 - 2.07 (m, 2H), 1.84 (d, J = 13.2 Hz, 1H), 1.72 - 1.60 (m, 2H), 1.51 (s, 3H), 1.13 (d, J = 6.3 Hz, 3H), 0.93 (d, J = 6.0 Hz, 3H), 0.88 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C87H97Cl2F3N10O29 [M+2H]2+m/z 937.2954, found m/z 937.2962.

Hydroxyethylaminomethyl-N-nonyl vancomycin (41). Yield 80% (26.5 mg, 16.1 µmol). R.T. = 14.320 min (analytical HPLC). 1


1H), 7.59 (br s, 1H), 7.48 (d, J = 8.7 Hz, 1H), 7.45 (dd, J = 8.4, 1.8 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.11 (s, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.52 (s, 1H), 5.71 (s, 1H), 5.69 (s, 1H), 5.31 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 4.1 Hz, 1H), 5.11 (s, 2H), 5.08 (s, 1H), 4.80 (s, 1H), 4.59 (d, J = 6.6 Hz, 1H), 4.44 (d, J = 5.6 Hz, 1H), 4.40 (s, 1H), 4.17 - 4.04 (m, 4H), 3.69 - 3.59 (m, 4H), 3.54 (t, J = 8.5 Hz, 1H), 3.49 (dd, J = 7.0, 2.0 Hz, 1H), 3.25 (s, 3H), 2.94 (br s, 2H), 2.74 (s, 1H), 2.68 (s, 1H), 2.55 (s, 2H), 2.11 (dd, J = 16.4, 9.4 Hz, 1H), 1.97 (d, J = 11.6 Hz, 1H), 1.77 (d, J = 13.2 Hz, 1H), 1.69 - 1.58 (m, 3H), 1.56 - 1.44 (m, 4H), 1.33 (s, 3H), 1.25-1.20 (m, 14H), 1.06 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.3 Hz, 4H), 0.85 (d, J = 6.4 Hz, 3H), 0.83 (t, J = 7.1 Hz, 3H). HRMS (ESI) calcd for C78H100Cl2N10O25 [M+2H]2+m/z 824.3197, found m/z 824.3191.


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Hydroxyethylaminomethyl-N-4-ethylbenzyl vancomycin (42). Yield 82% (27.0 mg, 16.4 µmol). R.T. = 12.408 min (analytical HPLC). 1


1H), 7.49 (d, J = 8.3 Hz, 1H), 7.46 (dd, J = 8.3, 1.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 2H), 7.29 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 7.9 Hz, 2H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.4, 2.1 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.52 (s, 1H), 5.71 (s, 1H), 5.69 (s, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.27 (d, J = 4.3 Hz, 1H), 5.12 (s, 2H), 5.09 (d, J = 2.1 Hz, 1H), 4.81 (s, 1H), 4.66 - 4.60 (m, 1H), 4.45 (d, J = 5.4 Hz, 1H), 4.40 (s, 1H), 4.17 4.03 (m, 4H), 3.92 (q, J = 12.6 Hz, 3H), 3.65 (q, J = 5.2, 4.4 Hz, 3H), 3.54 (d, J = 8.4 Hz, 1H), 3.45 (d, J = 8.7 Hz, 0H), 3.29 - 3.21 (m, 3H), 2.97 - 2.91 (m, 2H), 2.59 (q, J = 7.6 Hz, 2H), 2.56 (s, 3H), 2.15 - 2.02 (m, 2H), 1.79 (d, J = 13.2 Hz, 1H), 1.68 - 1.61 (m, 2H), 1.46 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H), 1.09 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C78H92Cl2N10O25 [M+2H]2+m/z 820.2884, found m/z 820.2875.

Hydroxyethylaminomethyl-N-4-pyridylbenzyl vancomycin (43). Yield 81% (27.3 mg, 16.2 µmol). R.T. = 12.815 min (analytical HPLC). 1


2H), 8.64 (br s, 1H), 8.22 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 8.3 Hz, 2H),



7.82 (d, J = 1.9 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.61 (br s, 1H), 7.49 (dd, J = 8.4, 1.7 Hz, 1H), 7.46 (dd, J = 8.4, 1.7 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.72 (d, J = 9.5 Hz, 1H), 5.71 (s, 1H), 5.33 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.15 - 5.11 (m, 2H), 5.10 (s, 1H), 4.81 (s, 1H), 4.66 (q, J = 6.7 Hz, 1H), 4.45 (d, J = 5.3 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.16 - 4.04 (m, 6H), 4.03 - 3.96 (m, 1H), 3.69 - 3.63 (m, 3H), 3.56 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 7.2 Hz, 1H), 3.26 (d, J = 9.6 Hz, 2H), 2.95 (dd, J = 6.4, 4.6 Hz, 2H), 2.13 - 2.08 (m, 1H), 1.83 (d, J = 13.1 Hz, 1H), 1.68 - 1.60 (m, 2H), 1.49 (s, 3H), 1.21 (s, 1H), 1.11 (d, J = 6.1 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.85 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C81H91Cl2N11O25 [M+K+H]2+m/z 863.7630, found m/z 863.8606.

Hydroxyethylaminomethyl-N-4'-trifluoromethylbiphenylmethyl van comycin (44). Yield 80% (28.1 mg, 16.0 µmol). R.T. = 16.781 min (analytical HPLC). 1


J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.82 (s, 1H), 7.80 (d, J = 8.0 Hz, 2H), 7.62 (s, 1H), 7.59 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 10.1 Hz, 1H), 7.46 (dd, J = 8.4, 1.8 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 2.0 Hz, 1H), 6.77 (d, J = 8.5 Hz,


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1H), 6.53 (s, 1H), 5.76 - 5.69 (m, 2H), 5.34 (d, J = 7.6 Hz, 1H), 5.30 (s, 1H), 5.12 (s, 2H), 5.10 (s, 1H), 4.81 (s, 1H), 4.70 - 4.62 (m, 1H), 4.46 (d, J = 5.2 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.16 - 3.97 (m, 7H), 3.70 - 3.63 (m, 3H), 3.56 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 7.1 Hz, 1H), 3.30 - 3.22 (m, 2H), 2.96 (dd, J = 6.4, 4.7 Hz, 2H), 2.78 - 2.68 (m, 1H), 2.57 (s, 3H), 2.15 - 2.07 (m, 2H), 1.82 (d, J = 13.2 Hz, 1H), 1.68 - 1.59 (m, 2H), 1.49 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C83H91Cl2F3N10O25 [M+2H]2+m/z 878.2821, found m/z 878.2834.

Hydroxyethylaminomethyl-N-3-chloro-4-((2-methyl-[1,1'-biphenyl]-3 -yl)methoxyl) benzyl vancomycin (45). Yield 78% (28.7 mg, 15.6 µmol). R.T. = 18.310 min (analytical HPLC). 1


1H), 7.62 (s, 1H), 7.57 (d, J = 1.9 Hz, 1H), 7.49 (d, J = 9.1 Hz, 1H), 7.47 (d, J = 9.1 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.40 (dd, J = 8.7, 2.0 Hz, 1H), 7.39 - 7.37 (m, 1H), 7.36 (dd, J = 8.7, 1.9 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 7.2, 1.2 Hz, 4H), 7.26 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 9.6 Hz, 1H), 7.19 (d, J = 9.6 Hz, 1H), 7.10 (s, 1H), 6.85 (dd, J = 8.5, 1.8 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.53 (s, 1H), 5.73 (d, J = 7.3 Hz, 1H), 5.71 (s, 1H), 5.32 (d, J = 7.6 Hz, 1H), 5.29 5.27 (m, 1H), 5.26 (s, 2H), 5.12 (s, 2H), 5.09 (s, 1H), 4.80 (s, 1H), 4.66 -



4.58 (m, 1H), 4.45 (s, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.16 - 4.07 (m, 3H), 4.05 (s, 1H), 4.00 (s, 1H), 3.93 (s, 2H), 3.69 - 3.62 (m, 3H), 3.55 (t, J = 8.5 Hz, 1H), 3.51 (d, J = 8.8 Hz, 1H), 3.41 (s, 1H), 3.29 - 3.22 (m, 2H), 2.96 (t, J = 5.6 Hz, 2H), 2.56 (s, 3H), 2.19 (s, 3H), 1.78 (d, J = 13.1 Hz, 1H), 1.64 (d, J = 9.8 Hz, 3H), 1.54 - 1.48 (m, 1H), 1.45 (s, 3H), 1.10 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.2 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C90H99Cl3N10O26 [M+2H]2+m/z 921.2977, found m/z 921.2976.

(β β-D-galactopyranosylethyl) aminomethyl-N-4'-trifluoromethylbiphe nylmethyl vancomycin (46). Yield 75% (28.8 mg, 15.0 µmol). R.T. = 16.518 min (analytical HPLC). 1


J = 8.1 Hz, 2H), 7.83 (s, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H), 7.60 (d, J = 7.8 Hz, 3H), 7.50 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.12 (s, 1H), 6.86 (dd, J = 8.5, 1.8 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 5.73 (d, J = 7.6 Hz, 1H), 5.71 (s, 1H), 5.34 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.1 Hz, 1H), 5.13 (s, 2H), 5.10 (d, J = 2.0 Hz, 2H), 4.82 (s, 1H), 4.67 (q, J = 6.5 Hz, 1H), 4.46 (d, J = 5.4 Hz, 1H), 4.41 (d, J = 5.8 Hz, 1H), 4.21 4.00 (m, 7H), 3.99 - 3.93 (m, 1H), 3.83 - 3.78 (m, 1H), 3.67 (d, J = 10.6 Hz, 1H), 3.62 (d, J = 3.0 Hz, 1H), 3.57 (t, J = 8.5 Hz, 1H), 3.38 (t, J = 6.4


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Hz, 1H), 3.33 - 3.23 (m, 4H), 3.10 (t, J = 5.3 Hz, 2H), 2.58 (s, 3H), 2.12 (d, J = 10.0 Hz, 1H), 1.83 (d, J = 13.1 Hz, 1H), 1.69 - 1.60 (m, 2H), 1.50 (s, 3H), 1.11 (d, J = 6.2 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d, J = 6.1 Hz, 3H). HRMS (ESI) calcd for C89H101Cl2F3N10O30 [M+2H]2+m/z 959.3085, found m/z 959.3090.

2-(Benzyloxycarbonyl) aminoethyl-2,3,4,6-tetra-O-acetyl-α α-D-manno pyranoside (49). Phenyl-2,3,4,6-tetra-O-acetyl-1-thio-α-D-mannopyranoside (47, 1.1 g, 2.5 mmol) and N-Cbz-ethanolamine (48, 586mg, 3.0 mmol) were dissolved in anhydrous dichloromethane (20 mL) at 0 °C and activated 4A molecular sieves (2 g) were added. The mixture was stirred at 0 °C for 15 min under nitrogen protection. Trifluoromethanesulfonic acid (80 µl, 0.9 mmol) was added and the solution immediately turned to red color, and was stirred at rt overnight. The reaction was quenched with Et3N, diluted with dichloromethane, and the liquid layer was separated by filtration through Celite. The residue was washed with saturated aqueous NaHCO3 and brine successively. The organic phase was dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The

crude

product

was

isolated

by

column

chromatography

(EtOAc/petroleum ether 2:3 then 1:1) to give 49 as a colorless oil in 57% yield. 1H NMR (400 MHz, CDCl3) δ 7.37- 7.27 (m, 5H), 5.34 - 5.25 (m,



Page 68 of 92

2H), 5.24 - 5.19 (m, 2H), 5.09 (s, 2H), 4.80 (s, 1H), 4.23 (dd, J = 12.1, 5.8 Hz, 1H), 4.07 (td, J = 12.4, 4.5 Hz, 1H), 3.94 (ddd, J = 8.8, 5.7, 2.4 Hz, 1H), 3.75 (p, J = 4.5 Hz, 1H), 3.55 (ddd, J = 10.5, 6.8, 3.6 Hz, 1H), 3.48 - 3.32 (m, 2H), 2.13 (s, 3H), 2.06 (s, 4H), 2.01 (d, J = 4.7 Hz, 4H).

2-(Benzyloxycarbonyl) aminoethyl-2,3,4,6-tetra-O-acetyl-β β-D-glycosi de (51a,51b). Peracetylated

sugar

50a

or

50b

(1

g,

2.56

mmol)

and

N-Cbz-ethanolamine 48 (604 mg, 3.09 mmol) were dissolved in dry acetonitrile under a nitrogen atmosphere. The solution was cooled to 0 °C and BF3·Et2O (1.6 ml, 13.0 mmol ) was added dropwise. The reaction was stirred 30 min at 0 °C and then overnight at rt. The reaction was quenched with Et3N and concentrated in vacuo, the residue redissolved in dichloromethane then washed successively with saturated NaHCO3, water and brine. The organic layer was dried over Na2SO4, the solvent was removed under reduced pressure and the product was purified by column chromatography on silica. Column chromatography (EtOAc/petroleum ether 2:3) gave pyranoside 51a or 51b as a colorless oil in 30-50% yield.

2-(Benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-acetyl-β β-D-galacto pyranoside (51a).


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1

H NMR (400 MHz, CDCl3) δ 7.39 - 7.32 (m, 5H), 5.39 (dd, J = 3.4, 1.1

Hz, 1H), 5.21 (t, J = 3.3 Hz, 1H), 5.20 - 5.16 (m, 1H), 5.11 (s, 2H), 5.05 4.99 (m, 1H), 4.46 (d, J = 7.9 Hz, 1H), 4.18 - 4.11 (m, 2H), 3.94 - 3.87 (m, 2H), 3.70 (ddd, J = 10.7, 7.3, 3.7 Hz, 1H), 3.48 - 3.33 (m, 2H), 2.16 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.99 (s, 3H).

2-(Benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-acetyl-β β-D-glucopy ranoside (51b). 1

H NMR (400 MHz, CDCl3) δ 7.36 - 7.28 (m, 5H),5.19 (t, J = 9.5 Hz,

1H), 5.09 (s, 2H), 5.06 (t, J = 9.7 Hz, 1H), 4.97 (dd, J = 9.6, 8.0 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H), 4.23 (dd, J = 12.3, 4.9 Hz, 1H), 4.13 (dd, J = 12.0, 2.0 Hz, 1H), 3.86 (ddd, J = 10.0, 5.8, 3.9 Hz, 1H), 3.74 - 3.63 (m, 2H), 3.45 - 3.28 (m, 2H), 2.05 (s, 3H), 2.02 (s, 3H), 2.00 (s, 6H).

2-(Benzyloxycarbonyl)aminoethyl-2-acetamido-3,4,6-tri-O-acetyl-2-d eoxy-β β-D-glucopyranoside (51c). β-D-glucosamine

pentacetate

(50c,

1.0

g,

2.5

mmol)

and

N-Cbz-ethanolamine (48, 1.25 g, 6.4 mmol) were dissolved in dry acetonitrile (10 ml) under a nitrogen atmosphere. The reaction was cooled to 0 °C and SnCl4 (360 µl, 3.1 mmol) was added dropwise. The reaction was warmed to rt, heated to 75 °C, and stirred for 14 h. TLC indicated the completion of reaction, then the mixture was cooled to rt, quenched with



2 ml Et3N and concentrated in vacuum. The residue was dissolved in 50 ml dichloromethane, washed with water, saturated brine successively. The organic phase was then dried over Na2SO4 and concentrated after filtration. Column chromatography (EtOAc/petroleum ether 1:1 to 4:1) gave a white solid in 30% yield. 1H NMR (400 MHz, CDCl3) δ 7.42 7.30 (m, 5H), 5.57 (d, J = 8.8 Hz, 1H), 5.31 (s, 1H), 5.20 (t, J = 10.0 Hz, 1H), 5.15 - 5.04 (m, 3H), 4.60 (d, J = 8.3 Hz, 1H), 4.25 (dd, J = 12.3, 4.8 Hz, 1H), 4.15 (dd, J = 12.4, 2.5 Hz, 1H), 3.89 (ddd, J = 10.1, 6.0, 3.4 Hz, 1H), 3.68 (ddd, J = 9.9, 4.8, 2.4 Hz, 2H), 2.08 (s, 3H), 2.05 (d, J = 2.3 Hz, 6H), 1.91 (s, 3H). Kanamycin-Vancomycin and Amikacin-Vancomycin conjugates (54-56, 58-63). Amidation reaction were performed in the synthesis of compounds 54-56. Intermediate 3b (50 mg, 30 µmol), HBTU (21 mg, 45 µmol) and DIPEA (25 µl, 151 µmol) were dissolved in DMF (3ml), and the mixture was stirred for 15 minutes at rt. Then N,N-dimethylaminopropylamine (18 µl, 143 µmol) was added, the solution was stirred for 24 hours until analytic RP-HPLC showed the starting material was total consumed. Then the crude product was precipitated by addition of diethyl ether (30ml) and centrifuged. The supernatant was removed and the solid cake was washed with diethyl ether (15ml). The crude was dissolved in water and


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acetonitrile and subject to preparative RP-HPLC purification. The fractions containing the product was combined and lyophilized to give 54 as a white fluffy solid. Then the Mannich reaction was performed following the same procedure described above for the synthesis of 5-46 to introduce the Kanamycin and Amikacin fragments. Compounds 58-63 was synthesized directly by Mannich reaction with compounds 1, 3b, and 3m. All the products were purified by preparative RP-HPLC to afford the final compounds (55, 56, 58-63) in 30-50% yield.

N,N-Dimethyl-N-(3-aminopropyl)-(N-4'-chlorobiphenylmethyl-vanco mycin)-Carboxamide (54). Yield 30% (15.8 mg, 9.1 µmol). R.T. = 18.002 min (analytical HPLC). 1

H NMR (600 MHz, Deuterium Oxide) δ 8.71 (s, 1H), 8.54 (s, 1H), 7.84

(d, J = 1.9 Hz, 1H), 7.75 - 7.70 (m, 4H), 7.56 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.3 Hz, 2H), 7.49 - 7.45 (m, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.26 (s, 1H), 7.22 (d, J = 8.3 Hz, 1H), 6.78 (dd, J = 8.4, 1.9 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.39 (d, J = 2.3 Hz, 1H), 6.22 (d, J = 2.3 Hz, 1H), 5.78 5.73 (m, 1H), 5.62 (s, 1H), 5.36 (d, J = 7.7 Hz, 1H), 5.29 (d, J = 4.5 Hz, 1H), 5.28 (s, 1H), 5.19 (d, J = 3.9 Hz, 1H), 5.18 (s, 1H), 4.93 (s, 1H), 4.71 - 4.64 (m, 1H), 4.48 (d, J = 5.2 Hz, 1H), 4.32 (d, J = 5.2 Hz, 1H), 4.26 (s, 1H), 4.04 (q, J = 12.7 Hz, 2H), 3.97 (t, J = 7.2 Hz, 1H), 3.68 (d, J = 10.7 Hz, 1H), 3.58 (t, J = 8.5 Hz, 1H), 3.38 - 3.32 (m, 1H), 3.31 - 3.23



(m, 2H), 3.15 - 3.06 (m, 1H), 3.01 - 2.96 (m, 2H), 2.75 (s, 3H), 2.74 (s, 3H), 2.63 (s, 3H), 2.20 - 2.09 (m, 2H), 1.88 - 1.76 (m, 3H), 1.68 (dd, J = 13.2, 6.9 Hz, 1H), 1.65 - 1.60 (m, 1H), 1.56 (dd, J = 13.2, 6.5 Hz, 1H), 1.51 (s, 3H), 1.13 (d, J = 6.3 Hz, 3H), 0.90 (d, J = 6.2 Hz, 3H), 0.85 (d, J = 6.3 Hz, 3H). HRMS (ESI) calcd for C84H96Cl3N11O23 [M+2H]2+m/z 866.7951, found m/z 866.7988.

N,N-Dimethyl-N-(3-aminopropyl)-((α α-D-mannopyranosylethyl)amino methyl-N-4'-chlorobiphenylmethyl-vancomycin)-Carboxamide (55). Yield 35% (13.8 mg, 7.0 µmol). R.T. = 16.854 min (analytical HPLC). 1


7.72 (d, J = 8.3 Hz, 2H), 7.70 (d, J = 8.7 Hz, 2H), 7.57 (d, J = 8.0 Hz, 2H), 7.54 - 7.51 (m, 2H), 7.48 - 7.45 (m, 1H), 7.31 (d, J = 8.3 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.19 (s, 1H), 6.84 (dd, J = 8.7, 1.9 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.49 (s, 1H), 5.73 (d, J = 7.5 Hz, 1H), 5.71 (s, 1H), 5.39 (s, 1H), 5.35 (d, J = 7.6 Hz, 1H), 5.29 (s, 1H), 5.14 (s, 1H), 5.11 (s, 1H), 4.80 (s, 1H), 4.65 (d, J = 7.4 Hz, 1H), 4.64 (d, J = 1.7 Hz, 1H), 4.47 (s, 1H), 4.27 (d, J = 5.1 Hz, 1H), 4.20 - 3.97 (m, 7H), 3.88 - 3.80 (m, 1H), 3.69 - 3.59 (m, 4H), 3.56 (t, J = 8.5 Hz, 1H), 3.50 (dd, J = 9.0, 3.4 Hz, 1H), 3.38 (t, J = 9.4 Hz, 1H), 3.33 (dd, J = 6.5, 3.0 Hz, 1H), 3.29 - 3.22 (m, 2H), 3.12 (d, J = 19.2 Hz, 3H), 3.04 - 2.91 (m, 2H), 2.72 (d, J = 1.8 Hz, 6H), 2.57 (s, 3H), 2.15 - 2.06 (m, 2H), 1.89 - 1.78 (m, 3H), 1.69 -


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1.59 (m, 2H), 1.50 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). HRMS (ESI) calcd for C93H113Cl3N12O29 [M+2H]2+m/z 984.3479, found m/z 984.3486.

N,N-Dimethyl-N-(3-aminopropyl)-((Acyclic-gluconic acid lactone)ac ylaminopropylaminomethyl-N-4'-chlorobiphenylmethyl-vancomycin) -Carboxamide (56). Yield 40% (15.9 mg, 8.0 µmol). R.T. = 16.233 min (analytical HPLC). 1

H-NMR (600 MHz, DMSO-d6) δ 8.80 (s, 1H), 8.67 (s, 1H), 7.82 (m, 1H),

7.72 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.7 Hz, 1H), 7.48 - 7.44 (m, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.20 (s, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.77 (d, J = 8.5 Hz, 1H), 6.48 (s, 1H), 5.73 (s, 1H), 5.71 (s, 1H), 5.35 (d, J = 7.7 Hz, 1H), 5.30 (s, 2H), 5.13 (s, 1H), 5.10 (d, J = 1.9 Hz, 1H), 4.81 (s, 1H), 4.69 - 4.62 (m, 1H), 4.47 (s, 1H), 4.29 (d, J = 5.4 Hz, 1H), 4.17 (s, 1H), 4.05 (s, 2H), 4.02 (d, J = 11.3 Hz, 2H), 4.00 (d, J = 3.6 Hz, 2H), 3.88 (dd, J = 3.6, 2.3 Hz, 1H), 3.67 (d, J = 10.7 Hz, 1H), 3.59 - 3.53 (m, 2H), 3.53 - 3.49 (m, 1H), 3.36 (dd, J = 10.5, 4.9 Hz, 1H), 3.30 - 3.23 (m, 2H), 3.22 - 3.05 (m, 2H), 2.96 (t, J = 7.9 Hz, 2H), 2.90 (d, J = 7.2 Hz, 2H), 2.73 (s, 3H), 2.72 (s, 3H), 2.57 (s, 3H), 2.15 2.07 (m, 2H), 1.83 - 1.77 (m, 5H), 1.64 (d, J = 10.2 Hz, 2H), 1.50 (s, 3H), 1.11 (d, J = 6.3 Hz, 3H), 0.91 (d, J = 6.1 Hz, 3H), 0.86 (d,J = 6.1 Hz,3H).



HRMS (ESI) calcd for C94H116Cl3N13O29 [M+2H]2+m/z 998.8612, found m/z 998.8630

Biological Assays In vitro Antibacterial Activity. Minimum Inhibitory Concentration (MIC) determination56. The MIC values for all antimicrobial agents were measured by broth microdilution, using Mueller-Hinton II broth (cation-adjusted, BD 212322). Generally, compounds were dissolved with DMSO to 5.12 mg/ml as stock solutions. All samples were diluted with culture broth to 128 µg/ml as the initial concentration. Further 1:2 serial dilutions were performed by addition of culture broth to reach concentrations ranging from 128 µg/ml to 0.0625 µg/ml or lower. 150 µL of each dilution was distributed in 96-well plates, as well as sterile controls, growth controls (containing culture broth plus DMSO, without compounds) and positive controls (containing culture broth plus control antibiotics such as vancomycin, Telavancin, etc). Each test and growth control well was inoculated with 5 µL of a exponential phase bacterial suspension (about 105 CFU/well). The 96-well plates were incubated at 37ºC for 24 h. MIC values of these compounds was defined as the lowest concentration to inhibit the bacterial growth completely. All MIC values were interpreted according to recommendations of the Clinical and Laboratory Standards Institute (CLSI).


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In vivo Antibacterial Assay. Mice used in this study were obtained from SIPPR-BK Lab Animal Ltd. (http://www.slarc.org.cn) and housed under specified pathogen-free conditions. Overnight cultured Staphylococcus aureus

USA300

LAC

strain

(CA-MRSA)

or

Mu50

strain

(HA-MRSA/VISA) was transferred into fresh Tryptic Soy Broth (TSB) medium (1:100,v/v), and continued cultivating for 3h to reach the exponential growth phase. Strains were washed twice with sterile PBS buffer and suspended in the same buffer until use.

Six to eight week old female BALB/c mice anesthetized with pentobarbital sodium (80mg/kg, intraperitoneally), and were infected by retro-orbital injection with a suspension of ~ 2.35×108 colony-forming units (CFUs) of USA300 LAC (for lethal challenge) or ~ 3.8×108 CFUs of Mu50 (for abscess formation). For these two animal models, the group used 15 mice and 12 mice respectively, as indicated.

To evaluate the efficacy of selected compounds (18, 32, 40, 44, 46) and control compounds (Vancomycin, Telavancin) on the outcome of S.aureus pathogenesis, mice were received intraperitoneal injections with a signal dose of 7 mg/kg compounds (18, 32, 40, 44, 46, Vancomycin, Telavancin) for USA300 LAC infection or two doses of 7 mg/kg compounds (18, 46,



Vancomycin, Telavancin) dissolved in sterile ddH2O in 24h interval for Mu50 infection. The treatment was began 1h after infection.

For lethal challenge, numbers of dead mice caused by infection were recorded every day, and used for the creation of survival curve. For abscess formation, animals were euthanized by CO2 inhalation 5d after infection. Liver was harvested and homogenized in 1ml sterile PBS buffer (containing 0.01% tritonX-100). Homogenates were serially diluted and 10µl dilutions were spotted onto TSA agar to determine the CFU counts. Bacterial burdens within liver were quantified by counting CFU obtained from serial dilutions of the organ homogenate. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Public Health Clinical Center and were performed in accordance with the relevant guidelines and regulations.

In vivo Pharmacokinetic Assay CD-1 Mice used in this study were purchased from Shanghai Lingchang biological technology co. LTD, and the animal room environment was controlled (target conditions: temperature 18 to 29°C, relative humidity 30 to 70%). An electronic time controlled lighting system was used to provide a 12 hour light/12 hour dark cycle.


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Compound 18, 32, 40, 46, Vancomycin and Telavancin (5 mg/kg) were injected in caudal vein of the tail of fasted CD-1 mice (male, 18-22g, n = 18, group = 6), respectively. Blood samples were collected at 0.25h, 0.75h, 2h, 4h, 8h and 24h after intravenous administration. Samples were analyzed by Xevo TQ-S triple quadrupole mass spectrometer (Waters, USA) coupled with ACQUITY I-Class UPLC System (Waters, USA). The ACQUITY UPLC BEH C18 (1.7 µm, 2.0 mm × 50 mm, Waters, USA) was used for the analysis. After analyzing the concentrations of compounds, the value of T1/2, AUClast, AUCINF_obs, CL_obs, MRTINF_obs and VSS_obs was calculated from time-concentration curves in each animal using Phoenix WinNonlin (CERTARA, USA).

Cytotoxicity assays Cell viability kit CCK-8 (Cell Counting Kit-8)57, 58 was used to evaluate the cytotoxicity of newly synthesized vancomycin derivatives. Generally, 100 µl of HK-2 cells (human renal proximal tubule epithelial cells) and HL-7702 cells (human liver cells) suspension (~5000 cells/well) were distributed into 96-well plate. After overnight incubation, add 10 µl of various concentrations of 16, 46, Vancomycin, Telavancin were added to the plate well (final concentration: 10 µM, 50 µM and 100 µM), and further incubated for 72h. After that, 10 µl of CCK-8 solution were added into each well of the plate, and the whole plate were incubated at 37 °C



for 1.5h. Finally, optical density at 450 nm (OD450) was recorded using VERSMax microplate reader. To calculate the cell viability, growth controls (sterile PBS buffer without compounds) and blank controls (culture broth only, no cells) were also included. Each set was performed triplicated. Cell viability was defined as: ODC/ODC=0×100. For which, ODC represents the optical density of cells treated with different concentration of compounds, ODC=0 represents the optical density with no compounds added.

Interaction of vancomycin analogues with bacterial peptidoglycan precursor peptide The interaction of compound 46 with bacterial peptidoglycan precursor model peptide was determined by the constant-time [13C,1H]-HSQC. They were recorded on AVANCE III HD BRUKER AscendTM 600 MHz instrument. Firstly, compound 46 (2 mM) dissolved in D2O was determined at 25 oC under pH=3.1, then the corresponding model peptide ligand Ac2Lys-dAla-dAla were added to make the final concentration up to 10 mM, meanwhile maintaining the same condition with above. The solution was determined by the constant-time [13C,1H]-HSQC again.

Acknowledgments This work was supported by the National Natural Science Foundation of


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China (NNSFC, No.21372238 and 21572244), the SIMM institute fund (CASIMM0120162014 and CASIMM0120164007), and the Youth Innovation Promotion Association of CAS (Grant No. 2017328). We thank the NMR facility of SIMM for their kind helps in compound characterization and NMR binding tests.

Ancillary Information Supporting Information Available: molecular formula strings of all target compounds; binding model of 46 and ligand Ac2Lys-dAla-dAla in PDB file and NMR detection; synthesis and antibacterial assay of 58-63; synthetic procedures of intermediates and telavancin; NMR, MS, and HPLC spectra of target compounds. Corresponding Author: [email protected] for W.H. and llan@ simm.ac.cn for L.L. Abbreviations:

R.T.,

retention

time;

HBTU,

N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)-uronium hexafluorophosphate; MIC, minimum inhibitory concentration; DIPEA, N,N-diisopropylethylamine; MSSA, methicillinsensitive S. aureus; MRSA: methicillin-resistant S. aureus; VISA: vancomycin-intermediate resistant S. aureus; VRSA: vancomycin-resistant S. aureus; VRE: vancomycin-resistant enterococci; CFU, colony forming units; 2D: two-dimensional; DMF: dimethylformamide.



Page 80 of 92

References 1. McCormick, M. H.; McGuire, J.; Pittenger, G.; Pittenger, R.; Stark, W. Vancomycin, a new antibiotic. I. Chemical and biologic properties. Antibiot. Annu. 1954, 3, 606-611. 2. Harris, C. M.; Kopecka, H.; Harris, T. M. Vancomycin: structure and transformation to CDP-I. J. Am. Chem. Soc. 1983, 105, 6915-6922. 3. Swartz,

M.

N.

Hospital-acquired

infections:

diseases

with

increasingly limited therapies. Proc. Natl. Acad. Sci. USA 1994, 91, 2420-2427. 4. Shallcross, L. J.; Howard, S. J.; Fowler, T.; Davies, S. C. Tackling the threat of antimicrobial resistance: from policy to sustainable action. Phil. Trans. R. Soc. Lond B Biol. Sci. 2015, 370, 20140082. 5. Goff, D. A.; Kullar, R.; Goldstein, E. J.; Gilchrist, M.; Nathwani, D.; Cheng, A. C.; Cairns, K. A.; Escandón-Vargas, K.; Villegas, M. V.; Brink, A. A global call from five countries to collaborate in antibiotic stewardship: United we succeed, divided we might fail. Lancet Infect. Dis. 2017, 17, e56-e63. 6. Leclercq , R.; Derlot , E.; Duval , J.; Courvalin , P. Plasmid-mediated resistance to vancomycin and teicoplanin in Enterococcus faecium. New Engl. J. Med. 1988, 319, 157-161. 7. Control,

C.

f.

D.;

Prevention.

Reduced


susceptibility

of

Page 81 of 92 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


Staphylococcus aureus to vancomycin--Japan, 1996. MMWR. Morbidity and mortality weekly report 1997, 340, 493-501. 8. Smith , T. L.; Pearson , M. L.; Wilcox , K. R.; Cruz , C.; Lancaster , M. V.; Robinson-Dunn , B.; Tenover , F. C.; Zervos , M. J.; Band , J. D.; White , E.; Jarvis , W. R. Emergence of vancomycin resistance in Staphylococcus aureus. New Engl. J. Med. 1999, 340, 493-501. 9. Rotun, S. S.; McMath, V.; Schoonmaker, D. J.; Maupin, P. S.; Tenover, F. C.; Hill, B. C.; Ackman, D. M. Staphylococcus aureus with reduced susceptibility to vancomycin isolated from a patient with fatal bacteremia. Emerg. Infect. Dis. 1999, 5, 147-149. 10. Weinstein,

R.

A.;

Fridkin,

S.

K.

Vancomycin-intermediate

and-resistant Staphylococcus aureus: what the infectious disease specialist needs to know. Clin. Infect. Dis. 2001, 32, 108-115. 11. Ward, P. B.; Grabsch, E. A.; Mayall, B. C. Treatment failure due to methicillin-resistant Staphylococcus aureus (MRSA) with reduced susceptibility to vancomycin. Med. J. Aust. 2001, 175, 480-483. 12. Hageman, J. C.; Pegues, D. A.; Jepson, C.; Bell, R. L.; Guinan, M.; Ward, K. W.; Cohen, M. D.; Hindler, J. A.; Tenover, F. C.; McAllister, S. K.

Vancomycin-intermediate

Staphylococcus

aureus

in

a

home

health-care patient. Emerg. Infect. Dis. 2001, 7, 1023-1025. 13. Zhanel, G. G.; Calic, D.; Schweizer, F.; Zelenitsky, S.; Adam, H.; Lagacé-Wiens, P. R.; Rubinstein, E.; Gin, A. S.; Hoban, D. J.; Karlowsky,



Page 82 of 92

J. A. New lipoglycopeptides. Drugs 2010, 70, 859-886. 14. Okano, A.; Nakayama, A.; Schammel, A. W.; Boger, D. L. Total synthesis

of

[ Ψ [C

(=NH)NH]

Tpg4]

vancomycin

and

its

(4-Chlorobiphenyl) methyl derivative: impact of peripheral modifications on vancomycin analogues redesigned for dual d-Ala-d-Ala and d-Ala-d-Lac binding. J. Am. Chem. Soc. 2014, 136, 13522-13525. 15. Okano, A.; Isley, N. A.; Boger, D. L. Peripheral modifications of [Ψ [CH2NH] Tpg4] vancomycin with added synergistic mechanisms of action provide durable and potent antibiotics. Proc. Natl. Acad. Sci. USA 2017, 114, 5052-5061. 16. Yarlagadda, V.; Konai, M. M.; Manjunath, G. B.; Ghosh, C.; Haldar, J. Tackling

vancomycin-resistant

bacteria

with'lipophilic-vancomycin-carbohydrate conjugates'. J. Antibiot. 2015, 68, 302-312. 17. Yarlagadda, V.; Samaddar, S.; Paramanandham, K.; Shome, B. R.; Haldar, J. Membrane disruption and enhanced inhibition of cell‐wall biosynthesis: a synergistic approach to tackle vancomycin‐resistant bacteria. Angew. Chem. Int. Ed. 2015, 54, 13644-13649. 18. Yarlagadda, V.; Sarkar, P.; Samaddar, S.; Haldar, J. A vancomycin derivative with a pyrophosphate‐binding group: a strategy to combat vancomycin ‐ resistant bacteria. Angew. Chem. Int. Ed. 2016, 55, 7836-7840.


Page 83 of 92 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


19. Leonard, S. N.; Rybak, M. J. Telavancin: an antimicrobial with a multifunctional mechanism of action for the treatment of serious gram‐ positive infections. Pharmacother. J. Hum. Pharmacol. Drug Ther. 2008, 28, 458-468. 20. Smith, J. R.; Roberts, K. D.; Rybak, M. J. Dalbavancin: a novel lipoglycopeptide antibiotic with extended activity against Gram-positive infections. Infect. Dis. Ther. 2015, 4, 245-258. 21. Brade, K. D.; Rybak, J. M.; Rybak, M. J. Oritavancin: a new lipoglycopeptide antibiotic in the treatment of gram-positive infections. Infect. Dis. Ther. 2016, 5, 1-15. 22. Butler, M. S.; Hansford, K. A.; Blaskovich, M. A.; Halai, R.; Cooper, M. A. Glycopeptide antibiotics: back to the future. J. Antibiot. 2014, 67, 631-644. 23. Kahne, D.; Leimkuhler, C.; Lu, W.; Walsh, C. Glycopeptide and lipoglycopeptide antibiotics. Chem. Rev. 2005, 105, 425-448. 24. Kim, S. J.; Tanaka, K. S.; Dietrich, E.; Rafai Far, A.; Schaefer, J. Locations of the hydrophobic side chains of lipoglycopeptides bound to the peptidoglycan of Staphylococcus aureus. Biochemistry 2013, 52, 3405-3014. 25. Nagarajan, R.; Schabel, A.; Occolowitz, J.; Counter, F.; Ott, J.; Felty-Duckworth, A. Synthesis and antibacterial evaluation of N-alkyl vancomycins. J. Antibiot. 1989, 42, 63-72.



26. Leadbetter, M. R.; Adams, S. M.; Bazzini, B.; Fatheree, P. R.; Karr, D. E.; Krause, K. M.; Lam, B. M.; Linsell, M. S.; Nodwell, M. B.; Pace, J. L. Hydrophobic vancomycin derivatives with improved ADME properties. J. Antibiot. 2004, 57, 326-336. 27. Arhin, F. F.; Belley, A.; Far, A. R.; Lehoux, D.; Moeck, G.; Parr, T. R. Glycopeptides and Lipoglycopeptides. In Antibiot. Discov. Dev.; Dougherty, T.J. and Pucci, M.J., Eds.; Springer: New York; 2012; pp 301-346. 28. Parenti, F.; Beretta, G.; Berti, M.; Arioli, V. Teichomycins, new antibiotics from Actinoplanes teichomyceticus nov. sp. J. Antibiot. 1978, 31, 276-283. 29. Barna, J. C.; Williams, D. H.; Stone, D. J.; Leung, T. C.; Doddrell, D. M. Structure elucidation of the teicoplanin antibiotics. J. Am. Chem. Soc. 1984, 106, 4895-4902. 30. Nicas, T. I.; Mullen, D. L.; Flokowitsch, J. E.; Preston, D. A.; Snyder, N. J.; Zweifel, M. J.; Wilkie, S. C.; Rodriguez, M. J.; Thompson, R. C.; Cooper, R. Semisynthetic glycopeptide antibiotics derived from LY264826 active against vancomycin-resistant enterococci. Antimicrob. Agents Chemother. 1996, 40, 2194-2199. 31. Nagarajan, R. Structure-activity relationships of vancomycin-type glycopeptide antibiotics. J. Antibiot. 1993, 46, 1181-1195. 32. Pavlov, A. Y.; Lazhko, E. I.; Preobrazhenskaya, M. N. A new type of


Page 84 of 92

Page 85 of 92 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


chemical modification of glycopeptides antibiotics: aminomethylated derivatives of eremomycin and their antibacterial activity. J. Antibiot. 1997, 50, 509-513. 33.Pathak, T. P.; Miller, S. J. Site-selective bromination of vancomycin. J. Am. Chem. Soc. 2012, 134, 6120-6123. 34. Williams, D. H.; Kalman, J. R. Structural and mode of action studies on the antibiotic vancomycin. Evidence from 270-MHz proton magnetic resonance. J. Am. Chem. Soc. 1977, 99, 2768-2774. 35.Ni, S.; Wei, H.; Li, B.; Chen, F.; Liu, Y.; Chen, W.; Xu, Y.; Qiu, X.; Li, X.; Lu, Y.; Liu, W.; Hu, L.; Lin, D.; Wang, M.; Zheng, X.; Mao, F.; Zhu, J.; Lan, L.; Li, J. Novel inhibitors of staphyloxanthin virulence factor in comparison with linezolid and vancomycin versus methicilin-resistant, linezolid-resistant, and vancomycin-intermediate staphylococcus aureus infections in vivo. J. Med. Chem. 2017, 60, 8145-8159. 36. Duthie, E.; Lorenz, L. L. Staphylococcal coagulase: mode of action and antigenicity. Microbiology 1952, 6, 95-107. 37. Chen, F.; Di, H.; Wang, Y.; Cao, Q.; Xu, B.; Zhang, X.; Yang, N.; Liu, G.; Yang, C.-G.; Xu, Y. Small-molecule targeting of a diapophytoene desaturase inhibits S. aureus virulence. Nat. Chem. Biol. 2016, 12, 174-179. 38. Kuroda, M.; Ohta, T.; Uchiyama, I.; Baba, T.; Yuzawa, H.; Kobayashi, I.; Cui, L.; Oguchi, A.; Aoki, K.-i.; Nagai, Y. Whole genome sequencing



of meticillin-resistant Staphylococcus aureus. Lancet 2001, 357, 1225-1240. 39. Xu, X.; Lin, D.; Yan, G.; Ye, X.; Wu, S.; Guo, Y.; Zhu, D.; Hu, F.; Zhang, Y.; Wang, F. vanM, a new glycopeptide resistance gene cluster found in Enterococcus faecium. Antimicrob. Agents Chemother. 2010, 54, 4643-4647. 40. Wardenburg, J. B.; Schneewind, O. Vaccine protection against Staphylococcus aureus pneumonia. J. Exp. Med. 2008, 205, 287-294. 41. Perkins, H. Specificity of combination between mucopeptide precursors and vancomycin or ristocetin. Biochem. J. 1969, 111, 195-205. 42. Hash, J. H. Antibiotic mechanisms. Annu. Rev. Pharmacol. 1972, 12, 35-56. 43. Nitanai, Y.; Kikuchi, T.; Kakoi, K.; Hanamaki, S.; Fujisawa, I.; Aoki, K. Crystal structures of the complexes between vancomycin and cell-wall precursor analogs. J. Mol. Biol. 2009, 385, 1422-1432. 44. Nakayama, A.; Okano, A.; Feng, Y.; Collins, J. C.; Collins, K. C.; Walsh, C. T.; Boger, D. L. Enzymatic glycosylation of vancomycin aglycon: completion of a total synthesis of vancomycin and N-and C-terminus substituent effects of the aglycon substrate. Org. Lett. 2014, 16, 3572-3575. 45. Boger, D. L.; Miyazaki, S.; Kim, S. H.; Wu, J. H.; Castle, S. L.; Loiseleur, O.; Jin, Q. Total synthesis of the vancomycin aglycon. J. Am.


Page 86 of 92

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Chem. Soc. 1999, 121, 10004-10011. 46. Fu, X.; Albermann, C.; Jiang, J.; Liao, J.; Zhang, C.; Thorson, J. S. Antibiotic optimization via in vitro glycorandomization. Nat. Biotechnol. 2003, 21, 1467-1469. 47. Ritter, T. K.; Mong, K. K. T.; Liu, H.; Nakatani, T.; Wong, C. H. A Programmable one‐pot oligosaccharide synthesis for diversifying the sugar domains of natural products: a case study of vancomycin. Angew. Chem. Int. Ed. 2003, 42, 4657-4660. 48. Nicolaou, K.; Mitchell, H. J.; Jain, N. F.; Bando, T.; Hughes, R.; Winssinger, N.; Natarajan, S.; Koumbis, A. E. Total synthesis of vancomycin—Part 4: Attachment of the sugar moieties and completion of the synthesis. Chem- Eur. J. 1999, 5, 2648-2667. 49. Yoganathan, S.; Miller, S. J. Structure diversification of vancomycin through peptide-catalyzed, site-selective lipidation: a catalysis-based approach to combat glycopeptide-resistant pathogens. J. Med. Chem. 2015, 58, 2367-2377. 50. Skaar, E. P.; Humayun, M.; Bae, T.; DeBord, K. L.; Schneewind, O. Iron-source preference of Staphylococcus aureus infections. Science 2004, 305, 1626-1628. 51. Corbin, B. D.; Seeley, E. H.; Raab, A.; Feldmann, J.; Miller, M. R.; Torres, V. J.; Anderson, K. L.; Dattilo, B. M.; Dunman, P. M.; Gerads, R. Metal chelation and inhibition of bacterial growth in tissue abscesses.



Page 88 of 92

Science 2008, 319, 962-965. 52. Thammavongsa,

V.;

Missiakas,

D.

M.;

Schneewind,

O.

Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science 2013, 342, 863-866. 53. Thomer, L.; Schneewind, O.; Missiakas, D. Pathogenesis of Staphylococcus aureus bloodstream infections. Annual Review of Pathology: Mechanisms of Disease 2016, 11, 343-364. 54. Fowler, B.S.; Laemmerhold, K.M.; Miller, S.J. Catalytic site-selective thiocarbonylations

and

deoxygenations

of

vancomycin

reveal

hydroxyl-dependent conformational effects. J. Am. Chem. Soc. 2012, 134, 9755-9761. 55. Sardzik, R.; Noble, G. T.; Weissenborn, M. J.; Martin, A.; Webb, S. J.; Flitsch, S. L. Preparation of aminoethyl glycosides for glycoconjugation. Beilstein J. Org. Chem. 2010, 6, 699-703. 56. Wikler, M. A. Performance standards for antimicrobial susceptibility testing: eighteenth informational supplement. Clinical and Laboratory Standards Institute (CLSI): 2008, 28, 1. 57. Ishiyama, M.; Miyazono, Y.; Sasamoto, K.; Ohkura, Y.; Ueno, K. A highly water-soluble disulfonated tetrazolium salt as a chromogenic indicator for NADH as well as cell viability. Talanta 1997, 44, 1299-1305. 58. Tominaga, H.; Ishiyama, M.; Ohseto, F.; Sasamoto, K.; Hamamoto,


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T.; Suzuki, K.; Watanabe, M. A water-soluble tetrazolium salt useful for colorimetric cell viability assay. Anal. Commun. 1999, 36, 47-50.

Table 1. Molecular structures of vancomycin analogues 5-46.

Compd.

R1

R2

Compd.

5

26

6

27

7

28

8

29

9

30

10

31

11

32


R1

R2


12

33

13

34

14

35

15

36

16

37

17

38

18

39

19

40

20

41

21

42

22

43

23

44

24

45

25

46


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Table 2. Molecular structures of vancomycin analogues 54-56.

Compd.

R1

54

H

R2

R3

55 56



Table of contents hydrophobic tails 100

OH NH

2

H3C

HO O C-terminal modification HO

O

O NH

N H

O HO CH3O O O O

O

Cl H N

O

O

N H

OH Cl OH

antibaterial activity against VISA and VRE OH

H N

NH O O O

H2 N

H N

Percent survival(%)

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

Page 92 of 92

80

Negative control Vancomycin Telavancin 18 32 40 44 46

60 40 20 0 0

HO

OH

1

2

3

R

4

5

6

7

8

9

Time(day)

OH O

Extra sugar moieties

in vivo assay on infected mice


10

Extra Sugar on Vancomycin: New Analogues for Combating Multidrug

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