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Cyclization of RGD peptides by Suzuki-Miyaura cross-coupling Isabell Kemker, Christian Schnepel, David Christopher C. Schröder, Antoine Marion, and Norbert Sewald J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00360 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 16, 2019

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Journal of Medicinal Chemistry

Cyclization of RGD peptides by Suzuki-Miyaura cross-coupling

Isabell Kemker,¶ Christian Schnepel,¶ David C. Schröder,¶ Antoine Marion,§ Norbert Sewald¶,* ¶Organic

and Bioorganic Chemistry, Bielefeld University, Department of Chemistry,

Universitätsstraße 25, 33615 Bielefeld, Germany §Department

of Chemistry, Middle East Technical University, 06800 Ankara, Turkey

KEYWORDS. halotryptophan, RGD peptides, integrins, on-resin Suzuki-Miyaura crosscoupling, side chain-to-tail cyclization, conformational analysis

ABSTRACT. Halogenated

L-

or

D-tryptophan

obtained by biocatalytic halogenation was

incorporated into RGD peptides together with a variety of alkyl or aryl boronic acids. SuzukiMiyaura cross-coupling either in solution or on-resin results in side chain-to-tail-cyclized RGD peptides, e.g. with biaryl moieties, providing a new dimension of structure-activity relationships. An array of RGD peptides differing in macrocycle size, presence of

D-amino

acid, N-

methylation, or connectivity between the indole moiety and the boronic acid showed that in particular connectivity exhibits a major impact on affinities towards integrins, e.g. αVβ3. Structure-activity relationship studies yielded peptides with affinities towards αVβ3 in the low

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nanomolar range, good selectivity as wells as high plasma stability. Structural characteristics of representative molecules have been investigated by molecular dynamics simulations which allowed understanding the observed activity differences.

Introduction Integrins recognize and bind RGD sequences that naturally occur in extracellular matrix (ECM) proteins, e.g. vitronectin or fibronectin, which play a crucial role in the bidirectional cell signaling. Targeting integrin-ECM interaction is an appealing strategy for medical applications in the field of cancer treatment1, radio therapy2 or surface coating of implants.3 Hence, a multitude of ligands binding αVβ3,4;

5

α5β1,6 or more recently αVβ67;

8

and αVβ89 have been

developed. The integrin subtypes αVβ3 and α5β1 are known to be up-regulated in tumor tissues and are required for angiogenesis although their role remains ambiguous.10 The highly potent αVβ3 ligand Cilengitide, c(RGDf(NMe)V), had been developed for the treatment of glioblastoma. Promising preclinical results demonstrated an inhibition of angiogenesis in models,11 but Cilengitide failed in phase III as the progression-free survival and overall survival did not turn out to be superior to standard treatment. One additional major concern was that low doses of Cilengitide can enhance angiogenesis, which actually favors cancer growth.12 This questioned not only the integrity of preclinical models for rational drug design13 but also demanded improving pharmacological properties, e.g. oral bioavailability as the short half-life led to greatly varying plasma concentrations during administration.14; 15 However, the synthesis of active and specific ligands can help to overcome the lack of knowledge regarding the problems when targeting integrins. The pioneering work of Kessler et al. established rational drug design based on conformation-dependent recognition leading to an αVβ3 receptor model.5;

16

Peptide


2

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cyclization is an efficient approach to increase affinity and selectivity, as the degree of freedom is reduced compared to linear peptides.17; 18 Further restraints as turn-inducing amino acids or Nmethylation help to favor a discrete conformation, while the latter also increases metabolic stability and bioavailability.4 Spatial screening of c(RGDFV), where one amino acid is substituted by its

D-enantiomer

inducing βII’-turns showcases how affinity depends on the

orientation of side chains. Combined with other biochemical data, a αVβ3 receptor model evolved stating that the position after the RGD sequence should be occupied by a hydrophobic amino acid, while the next position can accommodate variable residues without compromising activity.5 Results and Discussion Our interest in RGD peptides19–22 prompted us to utilize RGD peptides as a benchmark system for side chain-to-tail cyclization by Suzuki-Miyaura cross-coupling (SMC) in peptides, which gives access to biaryl motifs. SMC relies on the reaction of a halogenated arene with a boronic acid and has been demonstrated on protected and unprotected peptides in solution23 or on solidphase.24 Only recently, the incorporation of bromotryptophan in pentapeptides that were cyclized by on-resin SMC to give access to cyclic and bicyclic peptides, was published.25 Chemical halogenation of tryptophan lacks selectivity and requires elemental bromine or chlorine and high temperatures. Owing to their benign reaction conditions and high selectivity, halogenases have emerged as useful biocatalysts for C-H functionalization.26 Flavin-dependent tryptophan halogenases (FDH) only require halide salts, oxygen and FADH2 as cofactor for regioselective halogenation. The reaction involves hypohalous acid (HO-X) as the halogenating agent.27–29 A remarkable feature of these enzymes is their strict regioselectivity, which allows for halogenation at electronically disfavored position C5 (PyrH), C6 (Thal) or C7 (RebH) of the indole moiety.


3


Page 4 of 49

Enzymatic synthesis of preparative quantities of halogenated tryptophan is mainly hampered by low enzymatic stability and insufficient kinetic parameters. It was shown that the immobilization utilizing cross-linked enzyme aggregates (CLEAs) gives access to L-7-bromotryptophan on gram scale, which paved the way for applications in the field of peptide chemistry.30 The introduction of bromotryptophan isomers in RGD peptide analogs allows using the bromoarene together with an intramolecular boronic acid for cyclization by SMC. The SMC is compatible with many functional groups and accepts a versatility of starting materials.31 The success of the SMC also comprises a wide range of applications, for instance bioorthogonal reactions, labeling, site-selective and late-stage modification and, importantly, the resulting biaryl motifs possess a huge potential to improve biological activity and expand the portfolio in drug development.32–34 It has previously been shown that cross-coupling of unprotected halotryptophans in aqueous media gives fluorescent aryl tryptophans in high yields.35 Encouraged by these findings a sequential one-pot synthesis for the cross-coupling after biocatalytic halogenation without intermediary purification was developed by us.36 Furthermore, the formation of fluorescent aryl tryptophans was utilized to establish a halogenase activity assay capable to monitor halogenase turnover by fluorescence readout in a microtiter plate that is applicable for high-throughput screening of mutant libraries.37 Biochemistry. For preparative synthesis of halotryptophans, combiCLEAs of the halogenase RebH, flavin reductase PrnF, and alcohol dehydrogenase (ADH) allow gram-scale synthesis of L-7-bromotryptophan.30

The halogenase Thal giving 6-bromotryptophan provides similar

conversions whereas PyrH is less active (not shown). Previous studies on RGD peptides revealed that

D-amino

acids are crucial building blocks to induce turn structures that can increase

activity.17; 18 Hence, the enzymatic synthesis of D-configured bromotryptophan was attempted.


4

Page 5 of 49

RebH is known to accept

D-Trp

in low concentrations but without reaching full conversion

(Figure 1).30 By reducing the amount of iso-propanol (i-PrOH) to 0.5% the conversion for D-7BrTrp (1) reached 90% which made synthetic applications feasible. The lower amount of i-PrOH may lead to enhanced enzymatic stability. With Thal almost quantitative conversion for D-6BrTrp (2) was attained, whereas with PyrH preparative scales for

D-5-BrTrp

(3) could not be

achieved. A simple desalting step using a C18-column and subsequent purification by RP-HPLC gives the desired product. Just recently, an alternative approach combining biocatalytic halogenation with Trp synthase and

L-amino

acid oxidase RebO-induced dynamic

stereoinversion extended the preparative scope of D-configured halotryptopthans enabling the synthesis of D-fluoro- or iodotryptophan, which are not accessible with halogenases.38 Figure 1. Optimized conditions for the halogenation of D-Trp using combiCLEAs.a D-6Br-Trp

NH2

OH

O NAD+

OH

NADH+H+

4d

3d 2d

1d

ADH

e

FAD PrnF

tim

O

FADH2

on

NH2

D-Trp

H N

Br

c ti

H N

re a

combiCLEAs (halogenase, PrnF, ADH) 30 mM NaBr, O2, i-PrOH, pH 7.4, 25 °C

A (280 nm)

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


0d

Enzyme

0

O

OH

D-Trp

2

4

6

8

10

tr / min

[mM] i-PrOH [%] d Conv. RP-HPLC [%] Yield [%]

Product

RebH

0.5

5

5

63

30 b

D-7Br-Trp

(1)

RebH

0.75

5

5

49

37 b

D-7Br-Trp

(1)

RebH

1

0.5

5

90

72 b

D-7Br-Trp

(1)

Thal

1

0.5

4

99

quant.c

D-6Br-Trp

(2)


5


PyrH a

1

0.5

4

Page 6 of 49

n.i. d

9

D-5Br-Trp

(3)

Reaction conditions: 100 µM NAD+, 1 µM FAD, 15 mM Na2HPO4, 30 mM NaBr, i-PrOH in a

final volume of 1 L H2O, pH adjusted with H3PO4;

b

after desalting over C18-column and

preparative RP-HPLC; c after desalting over C18-column; d n.i.: not isolated.

Chemistry. Peptide Synthesis. The Fmoc/tBu strategy was applied to obtain the linear peptides by solid-phase peptide synthesis (SPPS). N-Terminal protection of bromotryptophan was introduced using FmocOSu to give Fmoc-D-Br-Trp-OH (4a-b) and Fmoc-L-Br-Trp-OH,25 resp., which were loaded on Rink Amide resin. Alkyl or aryl boronic acids (5a-f) for SMC on-resin or in solution were attached as the final residue to the N-terminus. N-Methylation of amino acids was performed on-resin by activating the N-terminus with 2-nitrobenzenesulfonyl chloride (oNBS) prior to alkylating with dimethyl sulfate (DMS).39 Peptides were cleaved from the resin with a mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIPS) and water with removal of protecting groups. The reversed peptide was synthesized on 2-chlorotrityl chloride resin (CTC) and was cyclized between Gly and Asp to avoid epimerization. To retain protecting groups the peptide was cleaved ten times with 1% TFA in DCM. The peptide was cyclized under high dilution using HATU and HOAt and the protecting groups were removed with a mixture of TFA, TIPS and water. The crude peptides were purified using RP-HPLC. Side chain-to-tail cyclization by Suzuki-Miyaura cross-coupling. The SMC was carried out to effect side chain-to-tail cyclization between bromotryptophan and the boronic acid. At first, intramolecular cross-couplings in solution starting from linear precursor peptides that were cleaved from resin and purified by RP-HPLC (peptides 6 - 10) were investigated (Scheme 1). Optimization studies with peptide 6 were conducted on analytical scales (1 µmol). As Na2PdCl4


6

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together with water soluble sSPhos (sodium 2’-dicyclohexylphosphino-2,6-dimethoxy-1,1’biphenyl-3-sulfonate hydrate) as phosphine ligand yielded excellent conversions for crosscouplings of free bromotryptophan with different arylboronic acids in solutions, this methodology was employed for peptide cyclization.36 Under high dilution conditions (0.2 mM), which is favorable to avoid intermolecular SMC, no product was detectable by MALDI-TOFMS analysis. Increasing the substrate concentration to 2.0 mM led to significant conversion without formation of undesired dimer (see Supporting Information, Figure S1). Further improvements were obtained by increasing the temperature to 100 °C and the addition of a chaotropic salt like guanidinium hydrochloride (GdnHCl, see Supporting Information, Figure S23). It is supposed that the latter disrupts secondary structures detrimental to cross-coupling and thus allowing a smoother cyclization.40 Unfortunately, some starting material decomposes by deborylation. This common side reaction could, however, not be suppressed by using degassed solvents under inert conditions.41 Although the optimized reaction conditions showed only minor formation of side products, on-resin SMC offers important advantages, e.g. a yield-lowering purification step can be avoided. Therefore, conditions by Afonso et al. were adapted for onresin cross-coupling with Pd2(dba)3, potassium fluoride and a mixture of 1,2-dimethoxyethane (DME)/EtOH/H2O.42 The reaction was monitored by MALDI-TOF-MS or LC-MS for all cyclic peptides (11 - 24) and finally no starting material or side product could be detected. In general, the on-resin reaction proceeded smoothly with full conversion of the linear peptide and without significant side products (see Supporting Information, Figure S4). Scheme 1. Synthesis of RGD peptides and side chain-to-tail cyclization on-resin and in solution by SMC.


7


Biohalogenation

Solid-phase peptide synthesis Br

Br

NH OH

H 2N *

NH

NH

a) H 2N *

O

O

O

*

N * R3 O

R2

d-e)

b-c) Xaa1

O

H N

R1

OH

O

Xaa2 Xaa3 R3

2

O

Xaa4

3

N

NH2

Xaa4 NH2

7

HN

N H

*

R3

NH2

21 - 24

SMC in solution

Xaa1-5 RGDfV RGDf RGD RGDf RGD RGDF RGD RGDF RGDF RGD

N

RGDF

11 - 20

6 - 10

No. 6 7 8 9 10 11 12 13 14 15

O

O

O

6

N H

7

N

*

4

O

6

R3

3

*

4

Xaa1 Xaa2 Xaa3

2

Xaa5

d-e)

n

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 8 of 49

SMC on-resin

* L L L L L D D D D D

R3 H H H H H H H H Me Me

Connection 7-4 7-4 7-4 7-3 7-3 7-4 7-4 6-4 7-4 7-4

No. 16 17 18 19 20 21 22 23 24

Xaa1-5/ n RGDf RGD RGDF RGDF RGDF 5 4 3 2

* L L D D D D D D D

R3 Me Me Me Me Me Me Me Me Me

Connection 7-4 7-4 6-4 7-3 7-2

aReagents

and conditions: (a) combiCLEAs (halogenase-PrnF-ADH), NAD+, FAD, Na2HPO4, NaBr, i-PrOH, 1 L water, pH 7.4, O2,; (b) TFA/H2O/TIPS; (c) Na2PdCl4, sSPhos, K3PO4, GdnHCl, water/dioxane (3:1), 100 °C, µwave, 0.5 h; (d) Pd2(dba)3, sSPhos, KF, DME/EtOH/H2O (9:9:1), 120 °C, µwave, 0.5 h; (e) TFA/H2O/TIPS. R1 = alkyl or aryl boronic acid, R3 = H or CH3.

The LC-MS and 1H-NMR analyses revealed for most peptides broadened signals or double peaks, which was observed for both biaryl- and alkyl-aryl-cyclized peptides and was attributed to conformers or diastereomers (see Supporting Information). In many 1H-NMR spectra more than


8

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one signal was found for the indole-NH, which is a good probe due to a pronounced downfield shift compared to other signals. For peptide 17 no isomers were visible in the 1H-NMR spectrum whereas peptide 16 that is elongated by one

D-Phe

shows two signals for the indole-NH.

Temperature studies in DMF-d7 from 248 – 328 K did not show any change of integrals which hints toward the presence of diastereomers (see Supporting Information Figure, S5). It has been reported previously that tryptophans involved in cyclic biaryls, e.g. in complestatin, are known to form atropisomers caused by the indole moiety.43 Accordingly, molecular modeling studies were performed to clarify these observations (vide infra). Additionally, peptide 19 was synthesized in a reverse manner starting from a suitably protected biaryl with a final backbone cyclization to examine whether this phenomenon is only caused by SMC cyclization (Scheme 2). For this approach, SMC between compound 25 and 3-(tert-butoxycarbonyl)phenylboronic acid was initially performed in solution. Bromotryptophan had to be converted into the corresponding amide to maintain structural analogy as the peptides were synthesized on Rink Amide resin. After removal of the Boc and tert-butyl protecting groups the N-terminus was Fmoc-protected (27) and this biaryl building block was coupled to the peptide precursor. After cleavage from the resin, cyclization of the linear peptide was carried out using HATU and HOAt under high dilution conditions leading to quantitative conversion. Purified peptide 28 led to the same features in the 1H-NMR spectrum concerning the indole-NH shift as peptide 19 that had been obtained by on-resin cyclization (see Supporting Information Figure, S6). This indicates that the formation of isomers is not conditioned by SMC but a consequence of different, stable isomers/conformers. For further validation molecular dynamics simulations were applied to elucidate the role of conformers and atropisomers. It is further important to note that peptides 19 and 28 do not significantly differ in affinity (see Table 1).


9


Page 10 of 49

Scheme 2. Reversed synthesis of peptide 19.a

Br

H N

Br

H N

a)

b)

NH2 O

OH

O

OH f-g)

HN O

tBu

O

d-e)

H N

HN Boc

OH

H N Boc

NH2 26

25

O H N

O

c)

NH2

1

O

O H N

O

NH2

H N R(Pbf)G-OH

O NH h-i)

H N

Fmoc N FD(tBu)-H NH2 O

O H 2N

NH2

O

NH N

O

NH NH HN

H N

NH2 NH

O

O O OH

27 28: 19%

aReagents

and conditions: (a) combiCLEA (RebH, ADH, PrnF), NAD+, FAD, Na2HPO4, NaBr, i-PrOH, 1 L water, pH 7.4, O2; (b) (1) SOCl2, MeOH 0 °C  rt, 36 h; (2) Et3N, Boc2O, MeOH, rt, 24 h; (3) NH4OH, MeOH, rt, 36 h; (c) Na2PdCl4, sSPhos, K3PO4, water/dioxane, 100 °C, 1 h, Ar; (d) TFA/DCM/thioanisole/TIPS/EDT/H2O, rt, 3 h; (e) FmocCl, Na2CO3, water/dioxane, 0 °C  rt, 1 h, f) HATU, HOAt, DIEA, rt; g) TBTU, DIEA, rt; h) HATU, HOAt, DIEA, rt; i) TFA/H2O/TIPS.

Biological Evaluation The binding affinity towards αVβ3 and α5β1 was determined in an ELISA-like assay with isolated integrins44;

45

and cell-adhesion19 assays with human melanoma cancer cells (WM115)

overexpressing αVβ3 to establish structure-activity relationships. An array of side chain-to-tail cyclized RGD peptides was synthesized which differ in length, presence and position of D-amino acid or N-methylation, connectivity and alkyl or aryl moieties attached to the indole. In contrast to lead peptide c(RGDfV), Phe and Val were substituted by the hydrophobic biaryl moiety


10

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obtained by cross-coupling between Br-Trp and a boronic acid. Among the first generation of peptides (7 - 13) the best affinity towards αVβ3 is observed with hexapeptide 11 (counting the aryl moiety as an amino acid). Spatial screening studies revealed a favorable stabilization of turn-structures upon incorporation of

D-amino

acids for the lead peptide.17 Although the

conformation for the cross-coupled peptides could not be elucidated, D-configured tryptophan resulted in overall increased affinities. Reports about the conformation of c(RGDfV) demonstrate a tight γ turn around Gly whereas for c(RGDFv) the position of the γ turn was shifted to Asp resulting in an opposing orientation of the Arg and Asp side chains and decreased affinity.17; 18 However, in our case switching the position of the

D-amino

acid from Trp (11) to Phe (7)

decreases affinity. Interestingly, the connectivity of the indole and phenyl moiety seems to have a major impact on the affinity as observed by the low affinity of peptides 9 and 10. The connectivity can be influenced by the choice of the boronic acid (ortho, meta, para) and the position of the bromide in the indole using different halogenases (C6, C7). The second generation of peptides was Nmethylated because this has been reported to increase oral bioavailability, conformational restriction and metabolic stability.4 With peptide 11 as the first lead structure a small array containing N-methylated RGD peptides was synthesized. For comparison, peptide 11 was Nmethylated to give peptide 14 which shows αVβ3 affinity comparable to 11 but lacks selectivity over α5β1. However, hexapeptide 14 displays higher affinity towards αVβ3 than pentapeptide 15. Regarding the connectivity it was concluded from the first results that a 7-3 connection might be promising. Indeed, peptide 19 displays the highest affinity towards αVβ3 in this set as shown by both cell adhesion assay (8.9 µM) and ELISA (5.4 nM). Especially the selectivity over α5β1 for peptide 19 or 11 is a major improvement compared to Cilengitide and meets the request of a


11


highly discriminating ligand. Another interesting approach was to create a small set of compounds with alkyl boronic acids with a chain length of three to six C-atoms. The affinity for αVβ3 increases with a shorter alkyl chain, most likely due to the decreased conformational flexibility. The lack of affinity in cell-adhesion assays, e.g. for peptide 24, might derive from inhomogeneous integrin expression levels which is a drawback in cell-based tests. For a selection of important derivatives affinity towards platelet integrin αIIbβ3 revealed no or very low affinity avoiding potential bleeding issues due to deficient platelet aggregation. Table 1. IC50 values determined by a cell-free method using isolated integrins and WM115 cells overexpressing αVβ3. O

Xaa1 Xaa2 Xaa3 R3

2 3

N

NH2

*

4

O

Xaa4

n

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 49

HN

O

6

* NH2

R3

RGDF

N H

7

N

O

7 - 20

21 - 24

Xaa1-4 / n

*

R3

Connection

IC50 αVβ3 [nM]

IC50 α5β1 [nM]

IC50 αIIbβ3 [nM]

IC50 WM115 [µM]

7

RGDf

L

H

7-4

579

5669

n.d.b

> 500

8

RGD

L

H

7-4

491

3660

n.d.b

> 500

9

RGDf

L

H

7-3

65

983

n.d.b

> 500

10

RGD

L

H

7-3

19

8029

n.d.b

> 500

11

RGDF

D

H

7-4

17.7±2.6

9287±2577

> 10000

51±9.4

12

RGD

D

H

7-4

41.3±6.9

5944±2253

> 10000

> 500

13

RGDF

D

H

6-4

59.2±5.5

> 10000

n.d.b

310±37

No.


12

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14

RGDF

D

Me

7-4

23.0±4.6

835±263

> 10000

11.1±2.2

15

RGD

D

Me

7-4

537±229

>10000

n.d.b

> 500

16

RGDf

L

Me

7-4

95.2±28.9

2161±530

n.d.b

> 500

17

RGD

L

Me

7-4

239±52

>10000

n.d.b

> 500

18

RGDF

D

Me

6-4

14.9±3.3

2722±475

> 10000

244±48

19

RGDF

D

Me

7-3

5.4±0.6

547±87

5804±541

9.4±1.3

20

RGDF

D

Me

7-2

64.6±7.7

1.92±0.4

n.d.b

158±36

21

5

D

Me

38.9±10.2

419±122

n.d.b

> 500

22

4

D

Me

36.2±10.0

563±159

n.d.b

> 500

23

3

D

Me

34.0±2.4

154±10

n.d.b

> 500

24

2

D

Me

12.8±0.8

325±21

5028±644

250±39

28a

RGDF

D

Me

13.9±1.4

n.d.b

n.d.b

n.d.b

29

c(RGDf(NMe)V)

0.54±0.02

15.4±4.0

n.d.b

n.d.b

30

c(RGDfV)

n.d.b

n.d.b

n.d.b

0.75±0.05

31

Tirofiban

n.d.b

n.d.b

1.3±0.2

n.d.b

7-3

asynthesized

in reversed order compared to peptide 19; b n.d.: not determined. When sufficient amounts of peptide were available cell-free ELISA was performed twice in duplicates otherwise only in duplicates and cell-adhesion assays were performed twice with four technical replicates for active compounds.

Plasma stability in human plasma was investigated with one representative peptide to evaluate degradation and metabolic stability (see Supporting Information, Figure S7). For peptide 19 good stability in human plasma (t1/2 > 24 h) is observed, whereas procaine as a positive control (t1/2 = 0.5 h) is rapidly degraded. Molecular Modeling. Further examination of spatial structures by molecular modeling was necessary as experimental data suggested the formation of isomers or conformers. While the spectrum of compound 17 allows peak assignment corresponding to a single conformation,


13


Page 14 of 49

multiple sets of signals for compounds 14 and 19 point towards the coexistence of different conformations. Extensive molecular dynamics (MD, Amber16/AmberTools17) simulations in explicit solvent (i.e., DMSO and water) at 300 K as well as at 350 K were performed to assess the convergence of conformational sampling, to gain insights into the structural preferences of these three compounds, and to further rationalize their observed activity towards integrins. The last 100 ns of four parallel MD simulations were gathered for each system and analyzed by means of a two-step structural clustering based i) on the root-mean-square-deviation (RMSD) of backbone atoms only and ii) on the RMSD of all non-hydrogen atoms in the molecule (see Supporting Information, Figure S8-10). In the representative structure of all three compounds obtained from MD simulations in DMSO at 300 K the polar side chains fold towards the core of the cyclic peptide to minimize their contact with the apolar solvent (Figure 2). This results in an interaction between the carboxylate group of Asp and the guanidinium group of Arg that remains stable along the dynamics. Only one conformation of compound 17 was significantly populated (i.e., greater than 10%) in the simulation in DMSO. Clustering based on backbone RMSD for this molecule resulted in two clusters with populations of 43% and 35%, for which the representative structures differed only by a 180˚ rotation of the phenyl group of the linker about its principal axis (see Supporting Information, Figure S11). Since the carbon atoms of this phenyl ring are pairwise nondistinguishable, the two geometries are strictly identical and result in a single conformation with population of 78%. In contrast, compound 14 is characterized by two conformations with populations of 60% as well as 39% and differing in the orientation of the peptide bond between the Arg and Gly amino acids of the RGD motif. This difference results in the loss of a hydrogen bond interaction between the amide NH group of the mentioned peptide bond and the


14

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carboxylate group of the Asp residue in the second conformation. Similarly, three conformations of compound 19 were found to be stable throughout the trajectory in DMSO, with populations of 64%, 18% and 14%, respectively. The former two conformations are atropisomers and differ in the relative orientation of the phenyl and indol moieties in the linker of the cyclic molecule (Figure 2). The third conformation did not show any significant difference compared to the main one (see Supporting Information, Figure S12). For all three compounds, the simulations at 350 K yielded a clustering analysis nearly identical to that performed at 300 K, with slightly different populations of the clusters (see Supporting Information). Even though the loss of hydrogen bond in compound 14 and the presence of atropisomers in compound 19 contribute to minor structural changes, the relative population of the two resulting conformers for these molecules is significant enough to be also detected by NMR spectroscopy thus explain multiple signal sets. In contrast, the single conformation identified for compound 17 correlates with the correspondingly neat spectrum recorded experimentally.

Figure 2. 1H-NMR spectra (left panel) and predicted representative structures as obtained from molecular dynamics simulations at 300 K (right panel) of compounds 17, 14 and 19 in DMSO.


15


Page 16 of 49

The first and second most populated conformations of 14 and 19 are depicted with carbon atoms shown in gray and pink, respectively. Relative populations are reported for each conformation based on backbone RMSD clustering. All other conformations of compound 17 have populations lower than 8%. Backbone RMSD of the two structures are reported for compound 14 and 19. The dotted-delimited areas indicate the main differences between the conformations. Only relevant hydrogen atoms are represented for clarity. Similar MD studies were also performed in water to get a closer insight into the conformational properties in a more biologically relevant context. This would be comparable to the conditions of the affinity assays as well as the desired application. As illustrated in Figure 3, the polar side chains of Arg and Asp amino acids in the RGD moiety of the three cyclic peptides are separated in water and are individually well solvated. The average distance between the carbon atoms of the guanidinium and carboxylate groups (R-D distance), as obtained from the last 100 ns of four independent MD runs for each molecule at 300 K, is: 7.5  2.5 Å, 9.4  1.9 Å, and 9.3  2.8 Å, for compounds 17, 14 and 19, respectively. Structural clustering revealed only one, highly populated conformation for compound 17 in water, with a population of 99%. In contrast, compounds 14 and 19 are characterized by one particular main conformation with a population of 42% and 52%, respectively, and more than 10 other low-occupied conformations (1-8%). In the crystal structure of αVβ3 by Xiong et al. (PDB: 1L5G),46 the co-crystallized cyclic RGD peptide presents an extended conformation of the polar side chains with an R-D distance of 13.7 Å. This spatial separation between the guanidinium and carboxylate groups is crucial for receptor binding since both groups interact with distinct binding pockets. Analysis of the average R-D distance as obtained from MD simulations in water indicates that such a separation between the Arg and Asp side chains is rather unlikely for compound 17, while the target distance falls


16

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near the range of observed values for compounds 14 and 19. Comparison of the representative structures for the three molecules showed that none of the geometries obtained adopt the exact conformation that Xiong et al. identified for their active RGD containing ligand (see Supporting Information, Figure S13). However, the rigidity of compound 17 compared to the relative flexibility of compounds 14 and 19 indicates that the latter two are more prone adopting a conformation that favorably interacts with the ligand-binding cavity of the integrin. The structural features, as revealed from MD simulations, correlate with the measured activity of the three compounds and explain the poor performance of compound 17 as a ligand for αVβ3. As the intrinsic flexibility of 17 is limited because of its rigid structure, a favorable interaction with the integrin binding cavity is therefore not possible.


17


Page 18 of 49

Figure 3. Representative structure for the main conformation of compounds 17, 14, and 19 in water as obtained from molecular dynamics simulations at 300 K. The relative population of the conformation based on backbone RMSD is reported for all three molecules. The average R-D distances are measured from the carbon atom of the Arg guanidinium groups to the carbon atom of the Asp carboxylate group, and reported with their corresponding standard deviations. Only relevant hydrogen atoms are represented for clarity as well as water molecules within 3 Å of the Asp or Arg side chain with corresponding hydrogen bond interactions depicted with dashed lines. Conclusion Selective and active ligands for αVβ3 remain a crucial target in medicinal chemistry and represent a good benchmark system to explore the influence of an intramolecular side chain-to-tail cyclization of RGD peptides. Enzymatic halogenation delivers bromotryptophan which provides a powerful handle for late-stage modifications by means of cross-coupling. For this approach preparative amounts of L- and D-bromotryptophan are required that was initially hampered by low conversion of D-tryptophan and optimized to nearly full conversion by reducing the excess of co-substrate i-PrOH for halogenases RebH and Thal. Robust methods for cyclization between an N-terminal boronic acid and the indole ring of bromotryptophan are established, capable of cross-coupling in solution and on-resin. Particularly, cyclization on-resin simplifies the reaction setup; giving higher overall yields while typical side products, as deborylation, are diminished. Experimental data and extensive MD simulations for three representative molecules reveal the occurrence of stable, distinct conformers or atropisomers.


18

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In vitro affinity assays using isolated integrins and cell adhesion assays show improved αVβ3 affinities for peptides containing a D-amino acid at the position of the former bromotryptophan whereas N-methylation plays an ambiguous role. In particular, the connectivity between the indole and aromatic moiety has a major impact on both affinity and selectivity. Notably, a 7-3 connection is superior leading to peptide 19 with low nanomolar αVβ3 affinity in this array. Introducing non-canonical halotryptophans, generated by biocatalysis, into RGD peptides as a benchmark system delivers not only active ligands but a powerful platform for side chain-to-tail cyclizations utilizing cross-coupling reactions. This further expands Nature’s toolkit of unusual biaryl systems which gain considerable attention as pharmaceutical building blocks. Experimental Section General Procedures. All solvents and chemicals were used as purchased without further purification. Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH and Fmoc-Phe-OH were purchased from Iris Biotech GmbH. Aromatic boronic acids were purchased from Sigma Aldrich or Alfa Aesar. 2-Boronatobenzoic acid (Sigma Aldrich) led to insufficient coupling and was synthesized as described elsewhere.47 Moisture- and air-sensitive reactions were conducted in oven-dried glassware under argon. The water used for reactions, work-ups and purifications was purified by a Millipore Milli-Q biocel device. If necessary, DCM was freshly distilled from CaH2 and THF from sodium/benzophenone. All final compounds were analyzed by analytical RPHPLC to confirm a purity of ≤ 95% purity (220 nm). Analytical RP-HPLC was performed on a Thermo Scientific Accela 600 equipped with a UV6000 LP detector, a P-4000 pump, a Hypersil Gold C18 column (3 µm column, 150×2.1 mm). Analytical LC-MS and determination of ESI-HRMS was performed on an Agilent 6220 TOF-


19


Page 20 of 49

MS (Agilent Technologies) with a dual ESI-source operating with a spray voltage of 2.5 kV, 1200 HPLC system with autosampler, degasser, binary pump, column oven, diode array detector and a Hypersil Gold C18 column (3 µm, 150×2.1 mm). Nitrogen was generated by a nitrogen generator NGM 11 and served as nebulizer and dry gas. External calibration was performed with ESI-L Tuning Mix (Agilent Technologies). MALDI-TOF/TOF-MS was conducted with an Ultraflex (Bruker Daltonik) operated in reflectron positive mode using an LTB nitrogen laser MNL200 (337 nm, 50 Hz, 1000 shots/spectrum, maximum resolution: 20000) using DHB (2,5dihydroxybenzoic acid) or CHCA (α-cyano-4-hydroxycinnamic acid) as matrix. Samples were dissolved in mixtures of acetonitrile and water and calibration was conducted with PEG 4001200. FlexControl 3.0 and FlexAnalysis 3.4 (Bruker Daltonik) were used for recording and processing. Preparative HPLC was performed using a Merck-Hitachi LaChrom HPLC consisting of interface D-7000, pump L-7150, detector L-7420 and a Hypersil Gold C18 column (1.9 µm, 250×21.2 mm) or a Hypersil Gold C18 column (7 µm, 250×10.0 mm). NMR spectra were recorded on a Bruker DRX-500, Avance III 500 (1H: 500 MHz,

13C:

126 MHz) or Avance 600

(1H: 600 MHz, 13C: 151 MHz) spectrometer. Chemical shifts are given in parts per million (ppm) relative to tetramethylsilane, residual solvent peaks for 1H and

13C

were used as internal

standard. For monitoring reaction progress with TLC, silica gel 60 coated aluminum sheets with F254 fluorescence indicator were used with solvent mixtures specified in the corresponding experiment. Spots were visualized using UV light (254 or 366 nm) or KMnO4 staining solution. Column chromatography was performed with silica gel 60 (Merck, 40-63 µm). Enzymatic halogenation. Expression and purification of halogenases RebH, Thal and PyrH and cofactor regenerating enzymes PrnF and ADH were conducted according to our previously published procedures48; 26 as well as the formation of combiCLEAs.30 E. coli cells from 1.5 L


20

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culture medium containing overexpressed halogenases (RebH, Uniprot ID: Q8HKZ8; Thal, UniprotID: A1E280 or PyrH, Uniprot: A4D0H5) were suspended in Na2HPO4 buffer (100 mM, pH 7.4) and lysed three times by French press. The lysate was spun down (4200×g, 4 °C, 30 min) and the crude supernatant was transferred into a fresh tube. After addition of PrnF (2.5 U/mL), ADH (1 U/mL) and finely ground ammonium sulfate (16.2 g) the mixture was incubated for 1 h at 4 °C in a tube rotator. For crosslinking glutardialdehyde (RebH: 0.5%, Thal: 1.0%, PyrH:1.25% w/v) was added and the mixture was further incubated for 2 h at 4 °C. Next, combiCLEAs were centrifuged (4200×g, 4 °C, 30 min) and washed three times thoroughly with Na2HPO4 buffer (100 mM, pH 7.4, 30 mL; 4200×g, 4 °C, 30 min). To a solution of Trp (1 mM), NaBr (30 mM), Na2HPO4 (15 mM), NAD+ (100 µM), FADH (1 µM) and i-PrOH (0.5% v/v) in water (995 mL) with a pH of 7.4 adjusted by H3PO4 the combiCLEAs were added. For optimal conversion the combiCLEAs must be finely suspended. The combiCLEAs were shaken for 3 to 5 days until the reaction was complete or conversion stopped. The combiCLEAs were removed by centrifugation (4200×g, 4 °C, 30 min) and the solvent was evaporated. The residue was dissolved in water/0.1% TFA and desalted using a manual C18 reversed-phase column (25 g, 400-220 mesh). For equilibration and desalting water/0.1% TFA (200 mL) was used whereas for eluting CH3CN/0.1% TFA was used (300 mL). Subsequently, the solvent was evaporated, lyophilized and if necessary purified using RP-HPLC (method 1). D-7Br-Trp

(1). The TFA-salt was isolated as a slightly yellowish solid after desalting and

purification by RP-HPLC (285 mg, 0.72 mmol, 72%). C11H11BrN2O2; 1H-NMR (500 MHz, DMSO-d6): δ = 11.28 (s, 1H, Ind-NH), 8.17 (s, 2H, CαNH2), 7.58 (d, 1H, 3J = 7.9 Hz, Ind-H4), 7.32 (d, 1H, 3J = 7.5 Hz, Ind-H6), 7.30 (d, 1H, 3J = 2.3 Hz, Ind-H2), 6.97 (t, 1H, 3J = 7.7 Hz, Ind-H5), 4.14 (dd, 1H, 3J = 6.1 Hz, 3J = 6.1 Hz, CαH), 3.30 - 3.21 (m, 2H, CβH).


21


D-6Br-Trp

Page 22 of 49

(2). The TFA-salt was isolated as a slightly yellowish solid after desalting (397 mg,

1.00 mmol, quant.). C11H11BrN2O2; 1H-NMR (500 MHz, DMSO-d6): δ = 11.22 (s, 1H, Ind-NH), 8.20 (s, 2H, CαNH2), 7.56 (d, 1H, 4J = 1.7 Hz, Ind-H7), 7.52 (d, 1H, 3J = 8.4 Hz, Ind-H4), 7.25 (d, 1H, 3J = 2.4 Hz, Ind-H2), 7.14 (dd, 1H, 3J = 8.4 Hz, 4J = 1.7 Hz, Ind-H5), 4.15 (dd, 1H, 3J = 6.1 Hz, 3J = 6.1 Hz, CαH), 3.26 - 3.24 (m, 2H, CβH). Fmoc-protection of halotryptophan. FmocOSu (1.1 equiv, 1.29 mmol) was added to a solution of the brominated Trp derivative (1 equiv, 1.17 mmol) and NaHCO3 (3 equiv, 3.51 mmol) in CH3CN/H2O (8 mL, 6:4) and stirred at rt. The pH was kept between 8-9 with 1 M NaOH and the reaction progress was monitored by RP-HPLC until completion. The solvent was evaporated under reduced pressure, the residue dissolved in EtOAc/H2O (1:1, 20 mL) and extracted with EtOAc (3×20 mL). The combined organic layers were washed with 5% aq. KHSO4 (20 mL), the organic layer dried over MgSO4 and the solvent evaporated. The crude was purified by RPHPLC (method 2) and lyophilized yielding Fmoc-Br-Trp as a white solid. Fmoc-D-7Br-Trp (4a). The compound was isolated as a colorless solid (458 mg, 0.91 mmol, 78%). 1H-NMR (600 MHz, DMSO-d6): δ = 12.74 (bs, 1H, COOH), 11.11 (s, 1H, Ind-NH), 7.88 (d, 2H, 3J = 7.6 Hz, Fmoc-H4, Fmoc-H5), 7.73 (d, 1H, 3J = 8.2 Hz, CαNH), 7.66 - 7.59 (m, 3H, Fmoc-H1, Fmoc-H8, Ind-H6), 7.40 (q, 2H, 3J = 7.0 Hz, Fmoc-H3, Fmoc-H6), 7.34 - 7.24 (m, 4H, Fmoc-H2, Fmoc-H7, Ind-H2, Ind-H4), 6.95 (t, 1H, 3J = 7.7 Hz, Ind-H5), 4.27 - 4.15 (m, 4H, CαH, Fmoc-H9, Fmoc-CH2), 3.20 (dd, 1H, 2J = 14.7 Hz, 3J = 4.6 Hz, CβH1), 3.03 (dd, 1H, 2J = 14.7 Hz, 3J = 9.8 Hz, CβH2). 13C-NMR (126 MHz, DMSO-d6): δ = 173.5, 155.9, 143.7, 143.7, 140.6, 134.3, 128.9, 127.6, 127.0, 125.2, 125.1, 123.4, 120.0, 119.9, 117.8, 111.7, 65.6, 54.8, 46.5, 26.8. HRMS: m/z calculated for C26H21BrN2O4+H+ [M+H+] = 505.07574. Found: 505.07510.


22

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Fmoc-D-6Br-Trp (4b). The compound was isolated as a colorless solid (scale: 1.00 mmol, 359 mg, 0.71 mmol, 71%). 1H-NMR (600 MHz, DMSO-d6): δ = 12.71 (bs, 1H, COOH), 11.00 (s, 1H, Ind-NH), 7.88 (d, 2H, 3J = 7.6 Hz, Fmoc-H4, Fmoc-H5), 7.70 (d, 1H, 3J = 8.3 Hz, CαNH), 7.64 (t, 2H, 3J = 8.3 Hz, Fmoc-H1, Fmoc-H8), 7.57 - 7.50 (m, 2H), 7.41 (q, 2H, 3J = 7.1 Hz, Fmoc-H3, Fmoc-H6), 7.32 - 7.26 (m, 2H), 7.20 (d, 1H, 3J = 2.2 Hz, Ind-H2), 7.10 (dd, 1H, 3J = 8.5 Hz, 5J = 1.6 Hz), 4.23 - 4.15 (m, 4H, CαH, Fmoc-H9, Fmoc-CH2), 3.17 (dd, 1H, 2J = 14.6 Hz, 3J = 4.4 Hz, CβH1), 3.01 (dd, 1H, 3J = 14.6 Hz, 2J = 9.9 Hz, CβH2).

13C-NMR

(126

MHz, DMSO-d6): δ = 173.5, 155.9, 143.7, 143.7, 140.6, 136.9, 127.6, 127.0, 126.2, 125.2, 124.7, 121.2, 120.0, 113.9, 113.7, 110.6, 65.6, 54.9, 46.5, 26.6. HRMS: m/z calculated for C26H21BrN2O4+H+ [M+H+] = 505.07574. Found: 505.07748. Peptide Syntheses: General Procedures. Peptide synthesis was conducted according to the Fmoc/tBu-strategy in a plastic syringe fitted with a polypropylene porous disk at rt. Washing steps were performed after each reaction with DMF (5×) or NMP (5×) with 10 mL/g resin. Solvents and soluble reagents were removed by suction. During couplings the resin was shaken with a horizontal shaker. SPPS was conducted on scales between 0.1-0.5 mmol using 10 mL/g resin for reactions. Reaction control was performed by MALDI-TOF-MS or LC-MS. Loading of Rink Amide resin. For on-resin side chain-to-tail cyclization Rink Amide resin was used. The resin (0.5 mmol/g) was swollen in DMF (10 mL/g) for 15 min. The resin was deprotected twice with 20% piperidine/DMF, 0.1 M 1-hydroxybenzotriazole (HOBt) for 20 min and

washed.

Fmoc-Br-Trp-OH

(1 equiv)

and

O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-

tetramethyluronium-hexafluorophosphate (HATU) (1.3 equiv) were dissolved in DMF, combined, diisopropylethylamine (DIEA) (3 equiv) was added and the mixture was shaken for 30 sec. This solution was added to the resin and incubated for 3 h at rt. Afterwards the resin was


23


Page 24 of 49

filtered, washed and capping was carried out twice using Ac2O (10 equiv) and pyridine (10 equiv) in DMF. The resin was dried in a desiccator over night or washed with Et2O and dried under vacuum (50 mbar). The loading capacity ranged between 95-100%. Loading of CTC resin. For peptides cyclized via the backbone CTC resin was used. CTC (1.0 mmol/g) was swollen under argon in dry DCM (10 g/mL) for 15 min. Fmoc-Gly-OH (1 equiv) dissolved in dry DCM and DIEA (10 equiv) was added within 5 min to the resin. After overlaying the resin with argon, it was shaken for 3 h at rt. For capping MeOH (2 mL/g) was added to the resin and incubated for further 10 min. After filtration, washing and drying the resin loading was estimated 96%. Manual SPPS. For Fmoc-deprotection the resin was treated twice with a solution of 20% piperidine/DMF, 0.1 M HOBt for 20 min. Coupling steps were repeated twice. Fmoc-Xaa-OH (5 equiv) and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetrafluoroborate (TBTU) (5 equiv) were dissolved in DMF, combined and DIEA (10 equiv) was added. After inverting the solution for 0.5 min, it was added to the resin and incubated for 15 min. For Fmoc-Br-Trp-OH and compound 26 (1.1 equiv) coupling was carried out only once in NMP with HATU (1.3 equiv), 1-hydroxy-7-azabenzotriazole (HOAt) (1.3 equiv) and DIEA (3 equiv) for 3 h. A subsequent capping was conducted twice with Ac2O (10 equiv) and pyridine (10 equiv) in DMF for 20 min. Coupling of aromatic boronobenzoic acid (5 equiv) was performed twice using TBTU (5 equiv) and DIEA (10 equiv) in NMP for 20 min. Coupling of alkyl boronobenzoid acid (1.3 equiv) was performed once using TBTU (1.3 equiv) and DIEA (3 equiv) in DMF for 3 h. On-resin N-Methylation. The Fmoc-group was deprotected and the resin washed with NMP. All steps were repeated twice. For the NBS-protection a freshly prepared solution of o-NBS (4


24

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equiv) with 2,4,6-collidine (10 equiv) in NMP was incubated with the resin for 15 min. Then a solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (3 equiv) and DMS (10 equiv) in NMP was incubated with the resin for 15 min. The completion of the N-methylation was monitored by RP-HPLC. To cleave the o-NBS group 2-mercaptoethanol (10 equiv) and DBU (5 equiv) in NMP were incubated with the resin for 15 min. The next Fmoc-Xaa-OH (3 equiv) was attached using HATU (3 equiv), HOAt (3 equiv) and DIEA (6 equiv) in NMP for 3 h.39 Cleavage of peptides from CTC resin. Peptides to be cyclized in solution were cleaved by treating 10× for 5 min with a mixture of 1% TFA in DCM. The cleavage solution was filtered into excess of i-PrOH (100 mL) and the combined fractions were evaporated under reduce pressure. The crude peptide was purified by RP-HPLC (method 1). General synthesis of alkyl boronic acids. General procedure for borylation. A Schlenk tube charged with CuI (0.2 equiv), bis(pinacolato)diboron (2 equiv) and MeOLi (3 equiv) was evacuated and filled with argon (3×). DMF (0.5 mL) was added under argon and the suspension was heated at 60 °C for 15 min and cooled to 40 °C for 20 min. A solution of tert-butyl 5-bromopentanoate (1 equiv, 0.56 mmol) or tert-butyl 6-bromohexanoate (1 equiv, 1.54 mmol) in DMF (0.5 mL) was added under argon and was stirred at 40 °C for 24 h. The suspension was diluted with EtOAc, filtered through celite, concentrated and purified by silica column chromatography (EtOAc/petroleum spirit, 10:90) giving a brown oil. 49 tert-Butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pentanoate (5a). Yield: 48%, 76 mg, 0.27 mmol. C15H29BO4.


25


tert-Butyl 6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)hexanoate

Page 26 of 49

(5b).

Yield:

53%,

243 mg, 0.82 mmol. C16H31BO4. General procedure fo tBu-deprotection. For the tBu-deprotection compound 5a or 5b (0.420.24 mmol) was taken up in a mixture of DCM/TFA (4 mL, 1:1, v/v) and stirred at rt for 2 h. The solvent was concentrated in vacuo to give the deprotected compounds 5c and 5d as a colorless oil. 5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pentanoic acid (5c). Yield: 88%, 48.2 mg, 0.21 mmol. C11H21BO4;

1H-NMR

(500 MHz, CDCl3): δ = 2.32 (t, 2H,

3J

= 7.5 Hz,

BCH2CH2CH2CH2C), 1.62 (p, 2H, 3J = 7.5 Hz, BCH2CH2CH2CH2C), 1.44 (p, 2H, 3J = 7.8 Hz, BCH2CH2CH2CH2C), 1.22 (s, 12H, (C(CH3)2), 0.77 (t, 2H, 3J = 7.9 Hz, BCH2CH2CH2CH2C). 13C-NMR

(126 MHz, CDCl3): δ = 180.1, 83.1, 34.3, 27.3, 24.8, 23.6.

6-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)hexanoic acid (5d). Yield: 99%, 197 mg, 0.81 mmol. C12H23BO4;

1H-NMR

(500 MHz, CDCl3): δ = 2.34 (t, 2H,

3J

= 7.5 Hz,

BCH2CH2CH2CH2CH2C), 1.63 (p, 2H, 3J = 7.5 Hz, BCH2CH2CH2CH2CH2C), 1.43 (p, 2H, 3J = 7.6 Hz, BCH2CH2CH2CH2CH2C), 1.39 - 1.29 (m, 2H, BCH2CH2CH2CH2CH2C), 1.24 (s, 12H, (C(CH3)2), 0.78 (t, 2H, 3J = 7.7 Hz, BCH2CH2CH2CH2CH2C). 13C-NMR (126 MHz, CDCl3): δ = 179.4, 83.1, 33.9, 31.8, 24.9, 24.6, 23.7. 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)propanoic acid (5e). An oven dried flask was charged with CuCl (0.03 equiv, 0.10 mmol), NaOtBu (0.09 equiv, 0.30 mmol) and bis[(2diphenylphosphino)phenyl] ether (DPEPhos, 0.03 equiv, 0.10 mmol), evacuated and flushed with argon (3×). Dry THF (5 mL) was added and the suspension was stirred for 0.5 h at rt. Then bis(pinacolato)diboron (1.05 equiv, 3.62 mmol) in dry THF (4 mL) was added dropwise under


26

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argon and the suspension was stirred for another 0.5 h at rt. Finally, tert-butyl acrylate (1.0 equiv, 3.44 mmol) and MeOH (0.5 mL) were added and the suspension was stirred for 3 h at rt. After filtrating over celite and washing with EtOAc, the solvent was evaporated under reduced pressure and the crude product purified using silica column chromatography (EtOAc/petroleum spirit, 10:90). The compound was obtained as a colorless oil (705 mg, 3.37 mmol, 98%).50 For the tBu-deprotecting the ester (1 equiv, 3.37 mmol) was stirred in a mixture of TFA/DCM (1:1, v/v, 4 mL) for 2 h at rt and subsequently concentrated to give the final compound as a colorless oil (593 mg, 3.19 mmol, 95%). C9H17BO4; 1H-NMR (500 MHz, CDCl3): δ = 2.51 (t, 2H, 3J = 7.4 Hz, BCH2CH2C), 1.24 (s, 12H, (C(CH3)2), 1.03 (t, 2H, 3J = 7.4 Hz, BCH2CH2C). 13C-NMR (126 MHz, CDCl3): δ = 180.1, 83.5, 28.6, 24.8. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)butanoic acid (5f). A flask was charged with tert-butanol (1.5 equiv, 15.6 mmol), triethylamine (1 equiv, 10.4 mmol) and pentan (20 mL) and cooled to 0 °C. trans-Crotonyl chloride (1 equiv, 10.4 mmol) was added dropwise within 10 min giving a yellow suspension with brown precipitate. The cooling was removed and the suspension stirred for 3 h at rt. Subsequently, saturated aq. NaHCO3 (10 mL) followed by water (25 mL) was added and the mixture extracted with Et2O/pentane (10:90, 3×20 mL). The combined organic layers were dried with MgSO4, concentrated carefully and the crude purified using silica column chromatography (pentane/EtOAc, 98:2) giving tert-butyl but-3-enoate as a colorless oil (860 mg, 6.05 mmol, 58%).51 For the borylation, an oven dried flask equipped with 1,2bis(diphenylphosphino)ethane (0.03 equiv, 50 µmol) and bis(1,5-cyclooctadiene)diiridium(I) dichloride (0.008 equiv, 13 µmol) was evacuated and flushed with argon (3×). After the addition of dry DCM (3 mL) the suspension was cooled to 0 °C and tert-butyl but-3-enoate (1.0 equiv, 1.65 mmol) was added. Then pinacolborane (1.5 equiv, 2.48 mmol) was added within 5 min and


27


Page 28 of 49

the suspension was stirred over night at rt. Upon the addition of water (5 mL) gas evolution was visible and the layers were extracted with EtOAc (3×30 mL). The combined organic layers were dried with MgSO4, concentrated and purified using silica column chromatography (petroleum spirit/EtOAc, 95:5) giving tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)butanoate as a colorless oil (338 mg, 1.25 mmol, 76%).52 For the tBu-deprotecting the latter (1 equiv, 1.25 mmol) was stirred in a mixture of TFA/DCM (4 mL, 1:1, v/v) for 3 h at rt and subsequently concentrated to give the final compound as a colorless oil (248 mg, 1.16 mmol, 93%). C10H19BO4; 1H-NMR (500 MHz, CDCl3): δ = 2.40 (t, 2H, 3J = 7.5 Hz, BCH2CH2CH2C), 1.76 (p, 2H, 3J = 7.6 Hz, BCH2CH2CH2C), 1.26 (s, 12H, (C(CH3)2), 0.86 (t, 2H, 3J = 7.8 Hz, BCH2CH2CH2C). 13C-NMR (126 MHz, CDCl3): δ = 180.4, 83.6, 36.1, 24.8, 19.3. General procedure for the side chain-to-tail cyclization in solution. A glass tube was charged with the linear peptide precursor on-resin (1 equiv), K3PO4 (5 equiv), GdnHCl (1 mM) and solved in water/dioxane (3:1, v/v) with a final peptide concentration of 2 mM. sSPhos (15 mol%) and Na2PdCl4 (5 mol%) were preactivated at 40 °C for 10 min, then added to the tube and the sealed tube was heated in a microwave at 100 °C for 0.5 h. The crude was purified by preparative RP-HPLC (method 1) and lyophilized to give a white solid. The cyclization in solution was conducted on scales between 1-6 µmol. Peptide Formula

m/zcalc

m/zfound

Yielda [%]

6

C44H52N10O10 880.4100 880.4100 10

7

C39H44N10O8

781.3416 781.3416 24

8

C30H35N9O7

634.2732 634.2732 22

9

C39H44N10O8

781.3416 781.3416 10

10

C30H35N9O7

634.2732 634.2732 3


28

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aoverall

yield after peptide synthesis and SMC.

General procedure for the side chain-to-tail cyclization on-resin. A Schlenk tube was charged with the linear peptide precursor of 11 - 24 (1 equiv), Pd2(dba)3 (20 mol%), sSPhos (40 mol%) and KF (4 equiv), evacuated and flushed with argon (3×). A degassed solution of DME/EtOH/H2O (9:9:1, v/v/v, 2-3 mL) was added under argon and the mixture was heated using a microwave at 120 °C for 0.5 h. Subsequently, the resin was washed with DMF (6×), EtOH (6×) and Et2O (6×). The cyclic peptide was cleaved from the resin with a mixture of TFA/H2O/TIPS (95:2.5:2.5 v/v/v) for 2×3 h each. The solvent was evaporated under reduced pressure, the residue dissolved in CH3CN/H2O and lyophilized. The crude peptide was purified using preparative RP-HPLC (method 1, 4). The on-resin cyclization was conducted in scales between 20-150 µmol.42 Peptide Formula

m/zcalc

m/zfound

Yielda [%]

11

C39H44N10O8 781.3416 781.3416 22

12

C30H35N9O7

13

C39H44N10O8 781.3416 781.3415 8

14

C40H46N10O8 795.3572 795.3577 36

15

C31H37N9O7

16

C40H46N10O8 795.3572 795.3577 77

17

C31H37N9O7

18

C40H46N10O8 795.3572 795.3555 26

19

C40H46N10O8 795.3572 795.3587 41

20

C40H46N10O8 795.3572 795.3568 53

21

C39H52N10O8 789.4042 789.4063 38

22

C38H50N10O8 775.3885 775.3900 21

634.2732 634.2736 11

648.2888 648.2876 18

648.2888 648.2912 84


29


aoverall

Page 30 of 49

23

C37H48N10O8 761.3698 761.3729 36

24

C36H46N10O8 747.3572 747.3565 33

yield after peptide synthesis and SMC.

((5S,11S,14S)-14-carbamoyl-5-(3-guanidinopropyl)-13-methyl-3,6,9,12-tetraoxo-4,7,10,13tetraaza-11H-1(7,3)-indola-2(1,4)-benzenacyclopentadecaphane-11-yl)acetic acid (17). 1HNMR (500 MHz, DMSO-d6): δ = 12.50 (bs, 1H, D-COOH), 9.98 (s, 1H, Ind-NH), 7.80 (d, 1H, 3J

= 9.1 Hz, D-CαNH), 7.74 (d, 1H, 3J = 5.0 Hz, R-CαNH), 7.56 (d, 1H, 3J = 7.9 Hz, Ind-H6),

7.53 (t, 1H, 3J = 5.8 Hz, R-Cδ-NH) , 7.50 (d, 2H, 3J = 7.2 Hz, Phe), 7.35 - 7.33 (m, 3H, Phe, Gdn-NH), 7.16 (d, 2H, 3J = 6.6 Hz, Ind-H4), 7.14 (d, 1H, 3J = 8.0 Hz, G-CαNH), 7.05 (t, 1H, 3J = 8.2 Hz, Ind-H5), 7.02 (d, 1H, 3J = 2.4 Hz, Ind-H2), 6.94 (s, 1H), 5.20 (dd, 1H, 2J = 12.4 Hz, 3J = 4.2 Hz, W-CαH), 4.88 (dd, 1H, 3J = 7.5 Hz, 3J = 7.5 Hz, D-CαH), 4.05 - 4.02 (m, 1H, R-CαH), 3.52 (dd, 1H, 2J = 18.3 Hz, 3J = 8.0 Hz, G-CαH1), 3.40 - 3.37 (m, 1H, W-CβH1), 3.16 - 3.10 (m, 2H, R-CδH2), 3.08 (s, 3H, W-N(CH3)), 2.98 (dd, 1H, 2J = 14.6 Hz, 3J = 12.7 Hz, W-CβH2), 2.65 (dd, 1H, 2J = 16.9 Hz, 3J = 7.4 Hz, D-CβH1), 2.26 - 2.18 (m, 2H, D-CβH2, G-CαH2), 1.73 - 1.66 (m, 1H, R-CγH1), 1.62 - 1.55 (m, 1H, R-CβH1), 1.52 - 1.45 (m, 2H, R-CγH2, R-CβH2). 13C-NMR (151 MHz, DMSO-d6): δ = 173.2, 172.7, 172.3, 172.0, 166.0, 157.1, 139.4, 134.6, 134.2, 128.6, 127.9, 127.6, 125.6, 124.6, 119.3, 118.2, 110.3, 57.4, 56.8, 43.7, 41.8, 41.0, 37.1, 31.2, 29.7, 25.9, 23.5. 7-Bromo-Nα-(tert-butoxycarbonyl)-D-tryptophanamide (25). Thionyl chloride (4 equiv, 1.8 mmol) was added dropwise to a solution of

D-Trp(7Br)

(1 equiv, 0.45 mmol) in MeOH

(3 mL) at 0 °C and stirred at rt until after 36 h the starting material was consumed. The solvent was evaporated under reduced pressure and the residue was suspended in Et2O and filtrated to give the methyl ester as a white solid (110 mg, 0.33 mmol). The latter was suspended in MeOH


30

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(5 mL) and Boc2O (1.5 equiv, 0.5 mmol) as well as Et3N (1.5 equiv, 0.5 mmol) were added and stirred at rt overnight. The solvent was evaporated under reduced pressure, the residue dissolved in DCM (20 mL) and extracted with 1 M HCl (3×40 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo (149 mg, 0.37 mmol). The latter was dissolved in MeOH (4 mL) and a solution of ammonium hydroxide (25%, 11 mL) was added. The suspension was stirred for 36 h at rt and extracted with DCM (3×20 mL), dried over MgSO4 and concentrated in vacuo to give compound 25 (92 mg, 0.24 mmol, 53%) as a white solid.53 C16H20BrN3O3; 1H-NMR (500 MHz, DMSO-d6): δ = 11.03 (s, 1H, Ind-NH), 7.64 (d, 1H, 3J = 7.9 Hz, Ind-H), 7.39 (bs, 1H, CONH1), 7.28 (d, 1H, 3J = 7.5 Hz, Ind-H), 7.20 (bs, 1H, CαNH), 7.01 (bs, 1H, CONH2), 6.94 (t, 1H, 3J = 7.7 Hz, Ind-H), 6.71 (d, 1H, 3J = 8.4 Hz, Ind-H), 4.15 4.10 (m, 1H, CαH), 3.06 (dd, 1H, 2J = 14.6 Hz, 3J = 4.6 Hz, CβH1), 2.89 (dd, 1H, 2J = 14.6 Hz, 3J = 9.4 Hz, CβH2), 1.30 (s, 9 H, C(CH3)3). (R)-Nα-tert-Butoxycarbonyl-6-(3-(tert-butoxycarbonyl)phenyl)tryptophanamide

(26).

A

sealed tube was charged with compound 25 (1 equiv, 0.24 mmol), tert-butyl-3-boronobenzoate (2 equiv, 0.48 mmol) and K3PO4 (5 equiv, 1.2 mmol), evacuated and flushed with argon (3×) and dissolved in H2O/dioxane (1:1, v/v, 2.5 mL). sSPhos (15 mol%, 0.04 mmol) and Na2PdCl4 (5 mol%, 0.01 mmol) were preactivated for 10 min at 37 °C. The suspension was added to the tube and heated in a thermoblock for 0.5 h at 100 °C. The crude was acidified with 1 M HCl (pH 2-3), extracted with DCM (3×20 mL) and the combined organic fractions were dried over MgSO4. The solvent was evaporated under reduced pressure and the crude biaryl was purified with silica column chromatography (DCM/MeOH, 99:2) to give a brown oil (109 mg, 0.23 mmol, 96%).


31


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(R)-Nα-Fmoc-6-(3-carboxyphenyl)tryptophanamide (27). Compound 26 was stirred with a mixture of TFA/DCM/thioanisole/TIPS/EDT/H2O (3 mL, 60:26:5:3:3:3, v/v/v/v/v/v) for 3 h at rt. The solvent was coevaporated with toluene, lyophilized and purified using RP-HPLC to give a brown solid (62 mg, 0.14 mmol, 61%). The residue was dissolved in 10% aq. Na2CO3 and dioxane (1:1, v/v) and cooled to 0 °C. FmocCl (1.1 equiv, 0.15 mmol) was added dropwise and allowed to reach rt. After 1 h the reaction was complete, and the mixture was extracted with EtOAc (3×20 mL). The combined organic fractions were re-extracted with 10% aq. Na2CO3 (4×20 mL) and the combined aqueous fractions were acidified to pH 2 using conc. HCl. The aqueous phase was extracted with EtOAc (3×40 mL) and the combined organic layers were dried over MgSO4, the solvent evaporated under reduced pressure and dried under high-vacuum to obtain compound 27 as a colorless oil (85 mg, 0.16 mmol, 61%). C33H27N3O5; 1H-NMR (600 MHz, DMSO-d6): δ = 12.80 (s, 1H, D-COOH), 10.69 (s, 1H, Ind-NH), 8.18 (t, 1H, 4J = 1.7 Hz, Phe-H), 7.99 (d, 1H, 3J = 7.7 Hz, Phe-H), 7.88 - 7.82 (m, 3H, Phe-H, Fmoc-H), 7.75 7.69 (m, 1H, Ind-H), 7.63 (m, 3H, Phe-H, Fmoc-H), 7.39 (q, 2H, 3J = 7.0 Hz, Fmoc-H), 7.30 (bs, 1H, CONH1), 7.27 - 7.20 (m, 4H, Ind-H2, Fmoc-H, CαNH), 7.15 - 7.09 (m, 2H, Ind-H), 6.95 (s, 1H, CONH2), 4.35 - 431 (m, 1H, CαH), 4.26 - 4.14 (m, 3H, Fmoc-H9, Fmoc-CH2), 3.22 (dd, 1H, 2J

= 14.6 Hz, 3J = 4.8 Hz, CβH1), 3.05 (dd, 1H, 2J = 14.6 Hz, 3J = 9.2 Hz, CβH2).

13C-NMR

(151 MHz, DMSO-d6): δ = 174.3, 167.7, 156.3, 144.2, 144.2, 141.0, 139.6, 133.6, 133.0, 131.8, 129.6, 129.5, 128.8, 128.4, 128.0, 127.5, 125.8, 125.7, 125.3, 124.6, 121.6, 120.5, 119.4, 118.9, 111.5, 66.1, 55.8, 47.0, 28.2. ((5S,11S,14S,17R)-14-benzyl-17-carbamoyl-5-(3-guanidinopropyl)-16-methyl-3,6,9,12,15pentaoxo-4,7,10,13,16-pentaaza-11H-1(7,3)-indola-2(1,4)-benzenacyclooctadecaphane-11yl)acetic acid (28). The linear peptide was cleaved from CTC resin by treating it 10× for 5 min


32

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with a mixture of 1% TFA in DCM. The cleavage solution was filtered into excess of i-PrOH (100 mL) and the combined fractions were evaporated under reduce pressure. The crude peptide was purified by RP-HPLC (method 3) and lyophilized to obtain a white solid (3%, 37 mg, 0.03 mmol). A flask was charged with DMF, DIEA (3 equiv, 0.09 mmol) and HATU (0.1 equiv, 3 µmol) and HOAt (0.1 equiv, 3 µmol). Two syringes containing the linear peptide (1 equiv, 0.03 mmol) and separately HATU (1.5 equiv, 0.05 mmol) and HOAt (1.5 equiv, 0.05 mmol) were added dropwise into the flask with the help of a dual syringe pump (1.25 mL/h) reaching a final concentration of 5 mM for the peptide. When the addition was finished, the syringes were rinsed with DMF and the solution stirred for another 1 h. The solvent was evaporated under reduced pressure and the protection groups were cleaved using a mixture of TFA/TIPS/H2O (95:2.5:2.5, v/v/v, 3 mL) for 3 h at rt. The solvent was co-evaporated with toluene, the crude was lyophilized and purified using RP-HPLC (method 1) to obtain compound 28 (5.2 mg, 6 µmol, 19%) as a white solid. HRMS: m/z calculated for C40H46N10O8+H+ [M+H+] = 795.3572. Found: 795.3584. Integrin Binding Assay. An ELISA-like assay using isolated integrins45 and cell-adhesion assays with WM-115 cells was performed to determine activity. Cilengitide (αVβ3: 0.54 nM, α5β1: 15.4 nM) and Tirofiban (αIIbβ3: 1.2 nM) were used as references. All wells of flat-bottom 96-well Immuno Plates (BRAND) were coated overnight at 4 °C with 100 µL protein (1) in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Each well was then washed with PBS-T-buffer (phosphate-buffered saline/Tween20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween20, pH 7.4; 3x200 µL) and blocked for 1 h at room temperature with TS-B-buffer (Tris-saline/bovine serum albumin (BSA) buffer, 150 µL/well, 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA).


33


Page 34 of 49

Meanwhile, a dilution series of the compounds and internal standard was prepared in an extra plate in 1:5 dilution steps using TS-B-buffer. After washing the assay plate with PBS-T (3×200 µL), 50 µl of the dilution series was transferred to each well from B-G. For a negative control well A was filled with 100 µl TS-B-buffer and for a positive control well H was filled with 50 µl TS-B-buffer. 50 µL of human integrin (2) in TS-B-buffer were transferred to wells HB and incubated for 1 h at rt. After washing the plates (3×200 µL) with PBS-T buffer, 100 µL primary antibody (3) was added to all wells and incubated for 1 h at rt. The plate was washed (3×200 µL) with PBS-T buffer and 100 µL of secondary antibody (4) was added to all wells and incubated for 1 h at rt. The plate was washed (3×200 µL) with PBS-T buffer and 50 µL SeramunBlau Fast (Seramun Diagnostic GmbH, Heidesee) was added to all wells. The development was stopped with 3 M H2SO4 (50 µL/well) when a blue color gradient from well A to H was visible (αVβ3: ~1 min, α5β1: < 1 min, αIIbβ3: 2-3 min). The absorbance was measured with a plate reader at 450 nm (Tecan, Infinite M200). The resulting curves were analyzed with OriginPro 2017G with the inflection point describing the IC50 value. Each compound was tested in duplicates (7 - 10) or, if enough compound was available, twice in duplicates (11 - 28) and referenced to the internal standard. αVβ3

α5β1

αIIbβ3

(1) 1.0 µg/mL human

0.5 µg/mL human

10 µg/mL human

vitronectin, R&D

fibronectin, R&D

fibrinogen, Merck Millipore

2.0 µg/mL, human α5β1

5.0 µg/mL, human integrin

integrin, R&D

αIIbβ3, Merck Millipore

1.0 µg/mL, mouse anti-

2.0 µg/mL, mouse anti-

human CD49e, BD

human CD41b, BD

(2) 2.0 µg/mL, human αvβ3 integrin, R&D (3) 2.0 µg/mL, mouse antihuman CD51/61, BD


34

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Bioscience (4) 1.0 µg/mL, anti-mouse IgGPOD goat, Sigma Aldrich

Bioscience

Bioscience

2.0 µg/mL, anti-mouse IgG-

1.0 µg/mL, anti-mouse IgG-

POD goat, Sigma Aldrich

POD goat, Sigma Aldrich

Cell culture. The epithelial melanoma cell line WM-115 (ATCC) overexpressing αVβ3 was cultivated in MEM supplemented with 10% fetal bovine serum and gentamycin (50 µg/mL). Cells were cultivated at 37 °C and 5.3% (v/v) CO2 and divided every 3-5 days in a ratio of 1:3. Cell adhesion assay.19 Flat-bottom MaxiSorp Nunc 96-wells were coated over night with human or recombinant vitronectin (1 µg/mL, 100 µL/well) at 4 °C. To this 100 µl of blocking solution containing fatty acid free milk powder in PBS (5% w/v) was added for 1 h at 4 °C. The suspension was discarded and fresh blocking solution (200 µL/well) was added. Meanwhile, the WM-115 cells were washed with MEM-medium and detached using Trypsin-EDTA (0.05%/0.02% in D-PBS) for 5 min at 37 °C. After addition of MEM-medium the cells were centrifuged (130×g, 6 min) and resuspended in medium containing fluorescein diacetate (1.5 mg/mL) to a cell density of 5×105 cells/mL for 0.5 h at 37 °C in the dark under shaking. After washing the cells twice with medium cells were incubated with CaCl2 and MgCl2 (20 mM) at 0 °C for 0.5 h. In the meantime, a dilution series of the compound starting from 500 µM in 1:1 dilution steps with PBS buffer was prepared. If the compound was not soluble in PBS, it was diluted from a DMSO stem solution with a final DMSO concentration lower than 2%. 12 tubes were filled with 240 µL of the dilution series and one tube was filled with 240 µL of medium as a positive control. To all tubes, 240 µL cell suspension was added and the tubes were incubated at 37 °C for 0.5 h in the dark. The blocking solution in the 96-well plate was discarded and the plate washed with medium (200 µL). The preincubated cell suspension was added to the plate


35


Page 36 of 49

(4×100 µL/well) and incubated at 37 °C for 1 h in the dark. After washing the plate with medium (3×100 µL), medium was added as a final step to all wells (100 µL/well) and the fluorescence was measured with a plate reader (λex: 485 nm, λem: 514 nm, Tecan Infinite M200). The resulting curves were analyzed using OriginPro 2017G. All measurements were carried out twice with four technical replicates for active compounds and once with four technical replicates for less active compounds. Plasma stability assay. Compounds and internal standards were chosen to be dissolved in a DMSO concentration that never exceeds 1% in the final assay. The stem solution was added to human citrate plasma (pH 7.5) at 37 °C to give final concentrations for peptide 19 of 500 µM and for procaine of 250 µM. The plasma was incubated for 24 h at 37 °C and 400 rpm. At 0, 1, 3, 6, 8 and 24 h 100 µL of plasma was withdrawn and precipitated with 400 µL of cold quenching solution consisting of CH3CN with a final concentration of 50 µM warfarin as an internal standard. The samples were stored at 4 °C and centrifugated (4200×g, 4 °C, 30 min). The supernatant was analyzed by LC-MS (injection volume: 20 µL). The degradation was determined by comparing the peak area of the UV signal of the internal standard with the tested compound. Molecular modeling. Molecular dynamics (MD) simulations and corresponding analysis were performed using the Amber16/AmberTools17 modeling suite.54 MD runs were performed using the CUDA accelerated version of pmemd as implemented in Amber16. Quantum mechanical (QM) calculations for the derivation of molecular mechanics (MM) parameters were performed with Orca (version 4.0.1.2).55 MM parameters for the Trp derivative linker were based on the general Amber force field (GAFF)56 and ff14SB57 parameters were used for standard amino acids. Atomic partial charges


36

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of the new residues were obtained by following the RESP procedure58 using multiple conformations and orientations of the molecules. Electrostatic potential grids were calculated at the HF/6-31G* level of theory, and conformations of the Trp derivative backbone were selected according to the minima identified on the conformational potential energy surface of an Nmethylated D-Ala dipeptide calculated at the RI-MP2/cc-pVTZ//HF-6-31G* level and reported in Figure S14. Specific torsion parameters were derived for an indol-phenyl model to ensure proper description of the conformational space of this moiety in the cyclic molecules considered in this work. The parameterization was based on our previously reported protocol59 to fit potential energy surfaces computed at the RI-MP2/cc-pVTZ//HF/6-31G* level. The reference and final surfaces are depicted in Figure S15 for the para- and meta-substituted variants of the indolphenyl linker. All parameters derived in this work are available as Supplementary Information. The same simulation protocol was applied to all systems. Starting conformations of the solutes were selected from clustering analysis of 100 simulated annealing MD runs in gas phase and further solvated in a truncated octahedron-shaped box of DMSO60 or TIP4Pew water61 with a buffer region of 12.0 Å using the tleap program of AmberTools17. After minimization and gradual heat up to 300 K (or 350 K), each simulation box was simulated for four parallel production runs of 200 ns each in the NPT ensemble, using the same starting point but different initial velocities. The average density of the simulation boxes during production runs was 1.12 g cm-3 and 1.06 g cm-3 in DMSO, and 1.00 g cm-3 and 0.99 g cm-3 in water, at 300 and 350 K, respectively. Heat up was performed in seven consecutive steps, first in the NVT ensemble with temperature ramps of 0-10, 10-50, and 50-100 K, for 10, 10 and 20 ps, respectively. Following simulations were performed in the NPT ensemble at 100K for 50 ps, and with a temperature ramp of 100-200 and 200-300 K (or 200-350 K) for 100 and 200 ps,


37


Page 38 of 49

respectively. A final short equilibration step was performed for 500 ps at 300 K (or 350 K). All MD simulations used a time step of 2 fs and periodic boundary conditions. Long range electrostatic interactions were calculated using the particle-mesh Ewald scheme as implemented in Amber16, and the SHAKE algorithm was applied to constrain all hydrogen-containing bonds. Temperature and pressure were controlled with Langevin dynamics (collision frequency of 2.0 ps−1) and isotropic position scaling, respectively. Trajectory frames were saved every 20 ps for all production runs. Analysis of the trajectories was performed using the cpptraj program of AmberTools17. Selection of atom for backbone RMSD included all non-hydrogen atoms of the Trp derivative except the C-terminal amide group, all non-hydrogen atoms of the phenyl moiety of the linker except the carbonyl oxygen, as well as the carbonyl carbon, the -carbon, and the amide nitrogen atoms of the standard amino acids. Structural clustering was performed in two steps by combining frames separated by 100 ps within the last 100 ns of all four independent simulations for each system. First the structures were clustered based on backbone RMSD with a hierarchical agglomerative algorithm and minimum distance of 0.4 Å between clusters (i.e., the epsilon keyword of cpptraj). In a second step, each backbone-based cluster was re-analyzed using the same clustering algorithm, based on all non-hydrogen atoms RMSD and an epsilon value of 1.0 Å, to filter the most probable conformation of the whole molecule, for a given conformation of the backbone. The structures reported in this work are the representative of the main cluster of each backbone cluster and the populations correspond to the backbone clustering.

ASSOCIATED CONTENT


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Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Analytical HPLC and mass spectrometry (ESI-MS, MALDI-TOF), 1H-NMR spectra, plasma stability, molecular modeling, PDB files and further experimental data (PDF) Molecular Formula Strings (CVS)

AUTHOR INFORMATION Corresponding Author Phone: +49-521-1062051. E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT Financial support by Deutsche Forschungsgemeinschaft (SE 609/16-1) is gratefully acknowledged. We thank Dr. F. Reichart and Prof. Dr. H. Kessler with assistance on the ELISAtype affinity assay and M. Pohl, F. Hüsers and H. Gruß for support on synthesis. High Performance Computing resources were partially provided by the EXPLOR centre hosted by the University de Lorraine. Computational chemistry was supported by the BMBF-funded de.NBI Cloud within the German Network for Bioinformatics Infrastructure (de.NBI) (031A537B, 031A533A, 031A538A, 031A533B, 031A535A, 031A537C, 031A534A, 031A532B). The


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authors gratefully thank the de.NBI Cloud Team for the access to the de.NBI Cloud as major computational resource for this project. ABBREVIATIONS ADH, alcohol dehydrogenase; BSA, bovine serum albumin; CHCA, α-cyano-4-hydroxycinnamic acid; CLEAs, cross-linked enzyme aggregates; CTC, 2-chlorotrityl chloride; 7Br-Trp, 7bromotryptophan; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DHB, 2,5-dihydroxybenzoic acid; DIEA, diisopropylethylamine; DME, 1,2-dimethoxyethane; DMS, dimethyl sulfate; DPEPhos, bis[(2-diphenylphosphino)phenyl] ether; E. coli, Escherichia coli; EDT, 1,2-ethanedithiol; FDH, flavin dependent halogenase; FmocOSu, N-(9-fluorenylmethoxycarbonyloxy)succinimide; GAFF, general Amber force field; GdnHCl, guanidinium hydrochloride; HATU, O-(7azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorphosphate; HOAt, 1-hydroxy-7azabenzotriazole; HOBt, 1-hydroxybenzotriazole; HO-X, hypohalous acid; i-PrOH, isopropanol;MEM, minimum essential media; MM, molecular mechanics; n.d., not determined; n.i., not isolated; o-NBS, ortho-nitrobenzenesulfonyl chloride; Pbf, 2,2,4,6,7pentamethyldihydrobenzofuran-5-sulfonyl; PBS-T-buffer, phosphate-buffered saline/Tween20; PEG, polyethylene glycol; QM, quantum mechanical; RGD, Arg-Gly-Asp; SMC, SuzukiMiyaura cross-coupling; SPPS, solid phase peptide synthesis; sSPhos, sodium 2′dicyclohexylphosphino-2,6-dimethoxy-1,1′-biphenyl-3-sulfonate hydrate; t1/2, half-life; TBTU, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetrafluorborate; tBu, tert-butyl; TIPS, triisopropylsilane; TS-B-buffer, Tris-saline-BSA buffer; µwave, microwave irradiation. REFERENCES 1

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Table of Contents graphic Suzuki-Miyaura Cross-Coupling

Biohalogenation

combiCLEAs (halogenase, PrnF, ADH) 30 mM NaBr, O2, i-PrOH, pH 7.4, 25 °C

H N NH2 O

OH

FADH2

Br

H N

B(OH)2

O

Br HN

NH2

FAD PrnF O

R(Pbf)GD(tBu)F

OH O

NAD+ NADH+H+ cofactor regeneration

1) Pd2(dba)3, sSPhos, KF, DME/EtOH/H2O (9:9:1), 120 °C, µwave, 0.5 h 2) TFA/H2O/TIPS

O HN

N O

N

RGDF 19

IC50 V3 = 5.4 nM IC50 51 = 547 nM


49

NH2

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