Mimicking of Arginine by Functionalized Nω-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples

Article

pubs.acs.org/jmc

Mimicking of Arginine by Functionalized N^ω‑Carbamoylated Arginine

As a New Broadly Applicable Approach to Labeled Bioactive

Peptides: High Aﬃnity Angiotensin, Neuropeptide Y, Neuropeptide

FF, and Neurotensin Receptor Ligands As Examples

Max Keller,*^,†Kilian K. Kuhn,^†Jurgen Einsiedel,^‡Harald Hubner,^‡Sabrina Biselli,^†Catherine Mollereau,^§

̈

David Wiﬂing,^†Jaroslava Svobodova,^†Gunther Bernhardt,^†Chiara Cabrele,^∥

́

̈

Patrick M. L. Vanderheyden,^⊥Peter Gmeiner,^‡and Armin Buschauer^†

^†Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, Universitatsstrasse 31, D-93053 Regensburg,

̈

Germany

^‡Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, D-91052 Erlangen,

Germany

^§Institut de Pharmacologie et Biologie Structurale, CNRS/IPBS, 205 route de Narbonne, 31077 Toulouse cedex 5, France

^∥Department of Molecular Biology, University of Salzburg, Billrothstrasse 11, A-5020 Salzburg, Austria

^⊥Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

S

* Supporting Information

ABSTRACT: Derivatization of biologically active peptides by

conjugation with ﬂuorophores or radionuclide-bearing moi-

eties is an eﬀective and commonly used approach to prepare

molecular tools and diagnostic agents. Whereas lysine, cysteine,

and N-terminal amino acids have been mostly used for peptide

conjugation, we describe a new, widely applicable approach to

peptide conjugation based on the nonclassical bioisosteric

replacement of the guanidine group in arginine by a func-

tionalized carbamoylguanidine moiety. Four arginine-containing

peptide receptor ligands (angiotensin II, neurotensin(8−13), an

analogue of the C-terminal pentapeptide of neuropeptide Y, and

a neuropeptide FF analogue) were subject of this proof-of-concept study. The N^ω-carbamoylated arginines, bearing spacers with a

terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized

chemically stable peptide derivatives showed high receptor aﬃnities with K_ivalues in the low nanomolar range, even when bulky

ﬂuorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

devoid of lysine and cysteine residues but contain arginine.

Moreover, arginine-containing chemically modiﬁed peptides

such as selective or metabolically stable neurotensin NTS₂

receptor ligands,²²⁻²⁴NPFF analogues (e.g., DTyr¹,NMePhe³]-

NPFF and EYF (Figure 1B)),^25,26C-terminal neuropeptide Y

(NPY) analogues,²⁷⁻²⁹integrin binding RGD peptides,³⁰⁻³²

cyclic pentapeptides addressing the CXCR₄chemokine

receptor,^33,34opioid receptor ligands,³⁵and peptidic melano-

cortin receptor ligands³⁶⁻³⁸were described. Generally, the

strongly basic guanidine groups (pK_a12−13) in arginine-

containing peptides are considered to undergo key interactions

with acidic amino acids of the respective receptor. In

combination with the low chemical reactivity of the positively

charged moiety,¹⁷this may be the reason why the development

1. INTRODUCTION

Conjugation of biologically active peptides, e.g., to ﬂuorophores,

radionuclide-bearing moieties, or surfaces, is widely applied to

prepare pharmacological tools, diagnostic agents, or drug

delivery systems.¹⁻¹⁶Proteinogenic amino acids, allowing site-

speciﬁc and eﬃcient conjugation of peptides, are lysine (ε-amino

group), cysteine (sulfhydryl group), and uncapped N-terminal

amino acids (α-amino group). Among them, lysine is most

commonly used due to the high reactivity of the basic (pK_a≈ 10)

ε-amino group in its nonionized form allowing various reactions,

i.e., acylation, alkylation, arylation, and amidination.¹⁷However,

retained aﬃnity of the modiﬁed peptide to the respective bio-

logical target cannot be taken for granted.

Numerous biologically active endogenous or truncated

peptides such as angiotensin II (AngII), bombesin, bradykinin,

gonadoliberin, vasopressin, neuropeptide FF (NPFF), pancreatic

polypeptide,^18,19neurotensin(8−13),²⁰and kisspeptin-13,²¹are

Received: September 25, 2015

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Figure 1. Concept of the present study applied to selected arginine-containing peptides. (A) Bioisosteric replacement (in a nonclassical sense⁵¹) of the

guanidine group in arginine by a carbamoylguanidine moiety bearing an amino-functionalized residue enables a versatile conjugation of arginine-

containing peptides. (B) Arginine-containing peptidic receptor ligands, which were subject of the proof-of-concept study. ^aBalasubramaniam et al.²⁸

of labeling strategies based on the derivatization of the guanidine

group in arginine has been neglected. Previously, we demon-

strated that acylation of guanidine groups in nonpeptide ligands

of G-protein-coupled receptors (GPCRs), e.g., histamine and

NPY receptors (see Supporting Information, Figure S1),³⁹⁻⁴³

results in retained aﬃnities regardless of basicity reduced by

4−5 orders of magnitude. Acylation of the guanidine group of

arginine residues in bioactive peptides was recently reported for

leuprolide, a gonadotropin-releasing hormone receptor agonist,

and microcystin, a cyanobacterial toxin, to introduce, e.g., a

ﬂuorescence label or a biotin tag.⁴⁴⁻⁴⁶However, the acyl-

guanidine moiety was shown to be unstable in aqueous solu-

tion at pH > 4.^42,47−49By contrast, carbamoylation instead

of acylation of the guanidine group resulted in chemically

stable histamine and NPY receptor ligands with retained

aﬃnity.^42,48,49

This prompted us to explore if the guanidine-carbamoylguanidine

bioisosteric approach is applicable to arginine replacement in

biologically active peptides by incorporating amino-function-

alized N^ω-carbamoylated arginine (“arginysine”) enabling

versatile conjugation and labeling by analogy to methodologies

applied to lysine (Figure 1A). In the present study, we report on a

proof-of-concept covering the peptide ligands of subtypes of four

GPCR families: AngII, neurotensin, NPY, and NPFF receptors

(in the following encoded by color shading in yellow, blue, green,

and gray, respectively). The selected parent ligands, comprising

one, two, or four arginine residues in diﬀerent positions, were

AngII, NT(8−13), the dimeric peptide 1, a derivative of the

reported NPY Y₄receptor agonist 2,²⁸and EYF,^26,50an analogue

of NPFF (Figure 1B). Appropriately modiﬁed arginine building

blocks were synthesized and used in standard solid phase peptide

synthesis (SPPS) (Figure 2). The prepared peptide derivatives,

including new radio- and ﬂuorescence labeled molecular tools,

were characterized in binding and functional assays at the

receptors of interest.

2. RESULTS

N^ω-Carbamoylated Arginine Building Blocks for SPPS.

N^α-Fmoc protected, N^ω-carbamoylated arginine derivatives

(10, 11, 16), bearing Boc-protected terminal amino groups,

were prepared to be incorporated into peptides using standard

Fmoc strategy SPPS (Scheme 1, Figure 2A). Whereas compounds

10 and 16 contain an aliphatic tetramethylene spacer in the

N^ω-substituent, compound 11 comprises a dioxaoctamethylene

spacer. Unlike 10 and 11, which were Boc-protected at the

guanidine group, the respective position in 16 was unprotected

(Scheme 1, Figure 2A). The convenient synthesis of arginine

building blocks 10, 11, and 16, which were obtained in 99%

chemical purity, is shown in Scheme 1: Fmoc-Orn(Boc)-OH (3)

was esteriﬁed with benzylic alcohol to give the ornithine

derivative 4,⁵²which was treated with TFA to obtain amine

5.⁵³Guanidinylation of 5 using various S-methyl-isothiourea

derivatives (6,⁴²7,⁵⁴or 14) aﬀorded the intermediates 8, 9,

and 15, which were converted to the arginine building blocks 10,

11, and 16 by hydrogenolytic cleavage of the benzyl ester

(Scheme 1).

To investigate the suitability of 10, 11, and 16 (Scheme 1,

Figure 2A) for SPPS, the model peptide H-Tyr-(N^ω-carbamoyl)-

Arg-Leu-NH₂(17) was synthesized on a Rink amide resin using

the arginine building blocks 11 and 16 (Figure 2B). The coupling

of 11 was performed under various conditions. When coupling

was performed at 22 °C, the incorporation of 11 was incom-

plete (giving the side product H-Tyr-Leu-NH₂), irrespective of

both coupling reagents (HBTU, PyBOP) and repeated cou-

pling procedure. Peptide 17 was obtained in high yield when

anhydrous DMF was used and the temperature increased to

35 °C. Unlike 11, building block 16 was inappropriate for SPPS

due to the formation of lactam 18 by an intramolecular ring

closure upon activation of the carboxylic group with HBTU,

showing that protection of the guanidine group as in 10 and 11 is

indispensible (Figure 2C).

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overall yield. In NT(8−13), either Arg⁸or Arg⁹was replaced by a

N^ω-substituted arginine (analogues 25, 26, and 31), and in

peptide 1, arginine in position 2 was replaced (33). The amino-

functionalized peptides 19, 20, 25, 26, 31, 33, and 35 were

propionylated to obtain the propionamide derivatives 21, 22, 27,

28, 32, 34, and 36 (Figure 3, Supporting Information, Figure S2).

To explore if bulkier moieties than propionyl are tolerated with

respect to receptor binding, a chromene or a cyanine dye was

attached to give the ﬂuorescently labeled AngII and NT(8−13)

derivatives 23, 24, 29, and 30 (Figure 3, for ﬂuorescence spectra

and quantum yields, see Supporting Information, Figures S19

and S20, Table S1). The ﬂuorescence properties of the ﬂuoro-

phores were comparable to those of the labeled peptides, as

demonstrated for compounds 24 and 30 (Supporting Informa-

tion, Figure S20, Table S1). For the synthesis of the peptide

derivatives (1, 19−36), see Supporting Information (Figure S2)

and Experimental Section.

Stability of the Peptides in Buﬀer and under Assay

Conditions. The chemical stability of the synthesized N^ω-

carbamoylated peptide derivatives was assessed for the AngII

derivatives 19−21, 23, and 24, the NT(8−13) analogues 25, 27,

29, and 30, and the EYF derivatives 35 and 36 in phosphate

buﬀer (pH 7.0, 21 °C) over periods of 24 or 48 h. All peptides

proved to be stable (21, 27, Figure 4A; 19, 20, 23−25, 29, 30, 35,

and 36, Supporting Information, Figures S3 −S11). The “carba”

analogue of the NT(8−13) derivative 25, containing an acyl-

guanidine instead of the carbamoylguanidine moiety (compound

44, Supporting Information, Figure S16) was cleaved, giving the

parent compound NT(8−13) devoid of the N^ω-acyl substituent

(Supporting Information, Figures S17 and S18). This is con-

sistent with the reported instability of previously reported non-

peptide NPY Y₁and histamine H₂receptor ligands containing an

acylguanidine moiety.^42,47−49Additionally, the stabilities of

AngII and derivatives 21 and 22 as well as of NT(8−13) and

analogues 27 and 28 were investigated under assay conditions in

the presence of CHO-hAT₁and HT-29 cells, respectively, over a

period of up to 6 h. Under these conditions, a small additional

peak, amounting to <5.5% of the respective initial peak area of

the intact peptide, was detected by HPLC at 220 nm for both the

parent and the modiﬁed peptides (Figure 4B; Supporting

Information, Figures S12 −S15). This demonstrates that degra-

dation of the peptides during saturation and competition binding

experiments (incubation period of 2 h) is negligible.

Figure 2. N^ω-Carbamoylated arginine building blocks for SPPS.

(A) Structure of the synthesized arginine building blocks 10, 11, and 16.

(B) Incorporation of 11 into a model peptide via SPPS. Reagents and

conditions: (a) Fmoc-strategy SPPS using 5 equiv of Fmoc-aa, HBTU/

HOBt (4.9/5 equiv), and DIPEA (10 equiv); solvent, DMF/NMP (8:2 v/

v), rt, 2 × 60 min (coupling procedure repeated once); Fmoc-deprotection,

20% piperidine in DMF/NMP (8:2 v/v). (b) Varying conditions (see

inserted table); solvent, (DMF/NMP 8:2 v/v) using either DMF for

peptide synthesis packed under nitrogen (coupling at 22 °C) or anhydrous

DMF over molecular sieve (coupling at 35 °C), Fmoc-deprotection as

under (a); (c) triﬂuoroacetic acid (TFA)/H₂O (95:5 v/v), rt, 2 h.

(C) Formation of lactam 18 from arginine derivative 16. Reagents and

conditions: (d) HBTU/HOBt (1/1 equiv), DIPEA (2 equiv), DMF/NMP

(8:2 v/v), rt, >90% conversion after 5 min.

Functional Studies with Analogues of AngII and NT(8−

13). The potencies of the AngII analogues 19−22 (AT₁R) and

the NT(8−13) derivatives 25 and 27 (NTS₁R) were investigated

in comparison with the respective parent peptide in whole cell

functional assays by measuring intracellular calcium (Ca²⁺)

mobilization (Figure 5). In an aequorin Ca²⁺assay performed

with CHO-hAT₁-AEQ-G_α16cells, the AngII analogues 19−22

were full agonists exhibiting potencies (pEC₅₀) comparable to

that of AngII (Figure 5A). Likewise, in a fura-2 Ca²⁺assay per-

formed with hNTS₁receptor expressing HT-29 colon carcinoma

cells, the NT(8−13) analogues 25 and 27 proved to be equi-

potent and equi-eﬀective compared to NT(8−13) (Figure 5B).

Preparation of the Tritiated Peptides [³H]21 and [³H]

27. The tritiated AngII derivative [³H]21 and the tritiated

NT(8−13) analogue [³H]27 were prepared by treating an excess

of the precursor peptides 19 and 25, respectively, with

commercially available succinimidyl [³H]propionate ([³H]37)

(Figure 6A). [³H]21 and [³H]27 were obtained in high radio-

chemical yields and radiochemical purities (Figure 6B) and

Incorporation of N^ω-Carbamoylated Arginines into

Peptides. The parent peptides in the present study were the

octapeptide AngII with arginine in position 2, the hexapeptide

NT(8−13) containing two arginines in positions 8 and 9,

the dimeric pentapeptide 1 comprising four arginines, and the

undecapeptide EYF, a derivative of NPFF, with arginine in

position 10 close to the C-terminus (Figure 1B). Instead of the

previously reported Y₄R agonist 2, an analogue of the C-terminal

pentapeptide of NPY containing two chiral centers in the linker

moiety,²⁸we synthesized analogue 1 containing an unsubstituted

suberyl linker (Figure 1B, Supporting Information, Figure S2).

Incorporation of the arginine building blocks 10 and 11 into

the parent peptides using Fmoc strategy SPPS yielded the

peptide analogues 19, 20, 25, 26, 31, 33, and 35 bearing amino-

functionalized carbamoyl residues at the guanidine group

of arginine (Figure 3). Remarkably, the synthesis of the EYF

derivative 35, which required the subsequent coupling of nine

amino acids after the incorporation of 10, was obtained in 34%

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a

Scheme 1. Synthesis of the N^ω-Carbamoylated Arginine Building Blocks 10, 11, and 16

a

Reagents and conditions: (a) DCC, DMAP, CH₂Cl₂, 0 °C to rt, 20 h, 94%; (b) TFA/CH₂Cl₂(1:5 v/v), rt, 3 h, 98%; (c) HgCl₂, DIPEA, rt, 1.5 h,

63% (8), 76% (9); (d) 10% Pd/C, H₂, 2-propanol, rt, 3−4.5 h, 76% (10), 81% (11); (e) triphosgene, DIPEA, CH₂Cl₂, 34%; (f) HgCl₂, DIPEA, rt,

3 h, 67%; (g) 10% Pd/C, H₂, MeOH, rt, 60 min, 46%.

Figure 3. Structures of the synthesized analogues of AngII, NT(8−13), 1, and EYF, containing N^ω-substituted arginines (peptides 19−36).

proved to be stable upon storage at −20 °C (Supporting

Information, Figure S21).

the AngII analogue [³H]21 at live CHO cells expressing the

human AT₁R revealed a dissociation constant (K_d) of 0.94 nM

(Figure 7A, Table 1A). The number of speciﬁc binding sites per

cell amounted to approximately 20,000 and was stable over at

Receptor Binding Studies with the Radiolabeled

Peptides [³H]21 and [³H]27. Saturation binding studies with

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Figure 4. Chemical stability of the AngII derivative 21 and the NT(8−13) derivative 27 investigated in PBS pH 7.0 at 21 °C (A) and under assay

conditions in the presence of CHO-hAT₁and HT-29 cells, respectively (B). Shown are the chromatograms of the reversed-phase HPLC analysis after

incubation for 48 h (A) or 6 h (B). A small additional peak (marked with red arrow; t_R7.3 min in case of 21 and 7.4 min in case of 27), corresponding to

<2% and <4% of the respective initial peak areas of 21 and 27, respectively, was detected (220 nm) after 2 and 6 h of incubation.

least 45 passages. The K_dvalue of [³H]21 determined at rat

mesangial cells natively expressing the AT₁receptor subtype,

revealing high NTS₁R aﬃnity with K_dvalues of 0.51 and 0.58 nM,

respectively (Table 1B). The numbers of receptors per cell were

approximately 45,000 and 300,000, respectively. To investigate if

the NTS₂receptor subtype is expressed by HT-29 cells, too,

saturation binding of the nonselective radioligand [³H]27 (K_iof

the “cold” form (27) at the NTS₂R: 2.5 nM, Table 1B) was

performed in the presence of an excess of the highly selective

NTS₂R ligand 45²³(structure, see Supporting Information,

Figure S29). The B_maxwas the same in the presence and in the

absence of 45 (Figure 7A), suggesting, in contrast to a report

in the literature,⁵⁵that the NTS₂receptor is not expressed by

amounted to 1.5

0.12 nM (mean

SEM from ﬁve inde-

pendent experiments) with a B_maxcorresponding to approxi-

mately 25,000 sites per cell (a representative saturation isotherm

is shown in the Supporting Information, Figure S25A).

Saturation binding studies with the tritiated NT(8−13)

analogue [³H]27 were performed at human HT-29 colon

carcinoma cells natively expressing the NTS₁receptor subtype

(Figure 7A) and at CHO cells stably transfected with the human

NTS₁receptor gene (Supporting Information, Figure S26),

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Figure 5. Agonistic eﬀect of the AngII derivatives 19−22 and the

NT(8−13) derivatives 25 and 27 on the mobilization of intracellular

Ca²⁺. (A) Concentration−response curves (CRCs) of AngII and 19−22

from an aequorin assay on CHO-hAT₁-AEQ-G_α16cells. Mean values

SEM from ≥2 independent experiments (performed in triplicate).

(B) CRCs of NT(8−13), 25, and 27 from a fura-2 assay on HT-29 colon

carcinoma cells. Mean values SEM from ≥3 independent experiments

(performed in singlet).

Figure 6. Preparation and HPLC analysis of the tritiated peptides [³H]

21 and [³H]27. (A) [³H]propionylation of the precursor peptides 19

and 25, yielding the radiolabeled peptides [³H]21 and [³H]27.

(B) Radiochemical purity of [³H]21 and [³H]27 determined by RP-HPLC

analysis using radiometric detection (column: Luna C18(2), 3 μm, 150 mm ×

4.6 mm; gradient: 0−20 min, acetonitrile + 0.04% TFA/0.05% aq TFA

14:86−30:70; 20−25 min, 30:70−95:5; 25−32 min, 95:5) (see Experimental

Section). Retention times: 19.4 and 17.4 min, respectively.

respectively, with K_ivalues in the picomolar or one-digit nano-

molar range (Table 1A,B). Competition binding curves of the

peptides 19, 21, 23, 25, 27, 29, and 32 are shown in Figure 7B.

The K_ivalues of the AngII derivative 21 and NT(8−13) analogue

27 were in excellent agreement with the K_dvalues of their triti-

ated analogues [³H]21 and [³H]27, respectively (Table 1A,B).

Binding data of reported AT₁and NTS₁receptor ligands (AT₁:

AngII, candesartan, and losartan; NTS₁: NT(8−13) and 46⁶²

(SR142948A, structure see Supporting Information, Figure S29),

determined with [³H]21 or [³H]27, were in good accordance

with reported data (see Table 1A,B). In addition to NTS₁

receptor binding, aﬃnities of the NT(8−13) analogues 25 and

27 were determined at the NTS₂receptor subtype. K_ivalues were

comparable to that of the parent peptide NT(8−13) (Table 1B).

The Y₄R ligand 1 and its N^ω-carbamoylated derivatives 33 and

34 showed comparable aﬃnities with K_ivalues in the range of

1.5−7.4 nM (Table 1C). The N^ω-carbamoylated EYF analogues

35 and 36 exhibited K_ivalues of 27 and 41 nM, respectively

(Table 1D).

HT-29 cells. This is supported by Western blot analysis as shown

in Figure 8. Unspeciﬁc binding of both radioligands, [³H]21 and

[³H]27, was very low, that is, <10% of total binding at

concentrations corresponding to the K_dvalues (Figure 7A,

Supporting Information, Figures S25A and S26).

Receptor association and dissociation kinetics of [³H]21 and

[³H]27 were determined at CHO-hAT₁-AEQ-G_α16and HT-29

cells, respectively (results shown in the Supporting Information,

Figures S23, S24, and Tables S2, S3). Speciﬁc binding of [³H]21

and [³H]27 was reversible, although mild acid treatment (acid

stripping), which is considered as the method of choice to

investigate internalization of peptidic membrane receptor

agonists,⁵⁶⁻⁵⁸revealed a high degree of internalization of the

radioligands (Supporting Information, Figures S27 and S28).

This phenomenon can be explained by receptor recycling

and ligand externalization as previously described for the AT₁R

and [¹²⁵I]AngII.⁵⁹The suitability of [³H]27 for the labeling

of neurotensin receptors in tissues was demonstrated by

autoradiography with sections of a HT-29 tumor xenograft

(Figure 7C).

3. DISCUSSION

Competition Binding Studies at AT₁, NTS₁, NTS₂, Y₄,

and NPFF₂Receptors. K_ivalues of the new peptide analogues

19−36 were determined by competition binding at intact cells

(AT₁R, NTS₁R, Y₄R), cell membranes (NPFF₂R), or cell

homogenates (NTS₂R) using the radioligands [³H]21 (AT₁R),

[³H]27 (NTS₁R), [³H]NT(8−13)⁶⁰(NTS₂R), and [³H]EYF⁵⁰

(NPFF₂R) or the ﬂuorescently labeled peptide S0586-[K⁴]hPP⁶¹

(Y₄R). All AngII analogues (19−24) and all NT(8−13)

derivatives (25−32) exhibited high AT₁R and NTS₁R aﬃnities,

The concept of bioisosteric replacement of the guanidine group

in arginine by a carbamoylguanidine moiety was applied to obtain

amino-functionalized N^ω-carbamoylated arginine (Figure 1A),

enabling radio and ﬂuorescence labeling of peptides, as demon-

strated for AngII, NT(8−13), peptide 1, and EYF, ligands of AT₁,

NTS₁, NTS₂, NPY Y₄, and NPFF₂receptors:

AngII Derivatives. The octapeptide AngII contains one

arginine, which is essential for AT₁receptor binding of AngII, as

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Figure 7. Binding and displacement of the radioligands [³H]21 and [³H]27 at the AT₁R or the NTS₁R. (A) Saturation binding with [³H]21 at

CHO-hAT₁-AEQ-G_α16cells and with [³H]27 at HT-29 cells: saturation isotherm, unspeciﬁc binding, and Scatchard transformation. Error bars of

“speciﬁc binding” and error bars in the Scatchard plot represent propagated errors calculated according to the Gaussian law of errors. Data of “unspeciﬁc

binding” represent the mean SEM (n = 3). Dashed line: experiment performed in the presence of the selective NTS₂R ligand 45²³(structure, see

Supporting Information, Figure S29) (200-fold excess compared to [³H]27). (B) Displacement of [³H]21 (c = 2 nM) by AngII, 19, 21, 23, and

candesartan (structure see Supporting Information, Figure S29) at the hAT₁receptor, and displacement of [³H]27 (c = 1 nM) by NT(8−13), 25, 27, 29,

32, and 46 (structure, see Supporting Information, Figure S29) at the NTS₁R. Mean values SEM from ≥3 independent experiments (performed in

triplicate). (C) Autoradiography of a HT-29 colon carcinoma xenograft (ﬁrst in vivo passage) using the tritiated NTS₁/NTS₂receptor ligand [³H]27.

For histological micrographs at higher magniﬁcation, see Supporting Information, Figure S30.

becomes obvious from the AT₁R aﬃnities of [DesAsp¹]AngII

(AngIII) and [DesAsp¹,desArg²]AngII (AngIV): whereas AngIII

exhibits almost the same AT₁R aﬃnity as AngII, AngIV, devoid of

arginine, is by ca. 3 orders of magnitude less potent than AngII.⁶⁶

The replacement of arginine in AngII by a carbamoylated arginine

bearing an amino group (19, 20), a propionamide moiety (21,

22), or bulky ﬂuorophores (23, 24) was well tolerated, resulting in

derivatives with high AT₁R aﬃnities (K_iof 19−24: 0.71 to 13 nM)

and high agonistic potencies (EC₅₀of 19−22: 1.1 to 2.4 nM). The

radiolabeled AngII analogue [³H]21, characterized by saturation

binding studies and kinetic binding experiments, represents a

valuable high aﬃnity molecular tool (K_d0.94 nM (human AT₁R),

1.5 nM (rat AT₁R)), which can be economically prepared from the

precursor peptide 19 and commercially available succinimidyl

[³H]propionate.

both arginines with acidic amino acids of the receptor protein

(Supporting Information, Figure S31).^68,69Remarkably, in the

present study, replacement of either arginine in NT(8−13) by a

N^ω-carbamoylated arginine aﬀorded derivatives with high

NTS₁R aﬃnities, irrespective of a conjugation to space ﬁlling

ﬂuorophores (K_iof 25−32 (HT-29 cells): 0.26 to 4.1 nM). The

NT(8−13) derivative [³H]27, conveniently prepared from

precursor peptide 25, represents a high aﬃnity radioligand

(K_d(NTS₁R, HT-29 cells) 0.51 nM) to study the expression

of neurotensin receptors in cells and tissues and to deter-

mine binding constants by competition binding. The high

aﬃnity ﬂuorescently labeled NT(8−13) derivatives 29 and 30

(K_i(NTS₁R, HT-29 cells) 1.7 and 4.1 nM, respectively) can be

used, e.g., in ﬂow cytometric binding studies or to investigate

receptor traﬃcking.

NT(8−13) Analogues. In NT(8−13), both Arg⁸and Arg⁹

contribute to receptor binding as demonstrated by alanine

replacement.^23,67This is in accordance with X-ray data of the rat

NTS₁R in complex with NT(8−13), proving the interaction of

Analogues of the NPY Y₄Receptor Ligand 1. In case of

the homodimeric peptidic ligand 1, one of the four arginine

residues was replaced. The carbamoylated analogues of 1,

the amine 33, and the propionamide 34 had Y₄R aﬃnities

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Table 1. Receptor Binding Data (K_i) of the Parent Peptides AngII, NT(8−13), 1, EYF, and Their Analogues Containing a

N^ω-Substituted Arginine (19−36), and K_dValues of the Radioligands [³H]21 and [³H]27 As Well As AT₁and NTS₁Receptor

Agonist Potencies (EC₅₀) of Selected Peptides

(A)

hAT₁receptor

a

b

EC₅₀[nM]

K_i[nM]

K_d[nM]

AngII

0.52 0.024

1.4 0.18

2.1 0.31

1.1 0.21

0.24 0.019 (lit. K_i0.16 nM⁶³

)

19

2.1 0.33

20

2.8 0.13

21

[³H]21

0.71 0.053

c

0.94 0.021

22

2.4 0.18

3.1 0.14

9.0 0.040

13 0.57

23

nd

24

Candesartan

Losartan

3.5 0.48 (lit. IC₅₀0.69 nM⁵⁷

)

12 0.50 (lit. K_i17 nM⁶⁴

)

(B)

hNTS₁receptor

hNTS₂R receptor

d

e

f

g

EC₅₀[nM]

K_i[nM]

K_d[nM]

K_i[nM]

NT(8−13)

25

1.4 0.25

1.7 0.51

nd

0.14 0.010 (lit. K_i0.24 nM²³)

1.2 0.17

0.47 0.049

0.69 0.052

0.63 0.030

0.74 0.040

26

0.26 0.010

27

[³H]27

2.0 0.23

0.47 0.085

2.5 0.15

h

i

0.51 0.040 0.58 0.069

nd

28

nd

0.41 0.047

1.7 0.031

4.1 0.17

29

30

7.9 1.7

31

0.36 0.077

0.59 0.092

1.1 0.096 (lit. K_i1.0 nM⁶⁵

)

nd

32

1.7 0.32

46

(C)

hY₄receptor

(D)

hNPFF₂receptor

j

k

K_i[nM]

hPP

1

0.50 0.068

7.4 2.4

hNPFF

1.2 0.42

EYF

[³H]EYF⁵⁰

0.53 0.18

33

34

1.5 0.14

3.6 0.28

(lit. K_d0.54 nM⁵⁰

27 7.5

)

35

36

41 1.5

a

b

Half-maximally eﬀective concentration determined in an aequorin Ca²⁺assay on CHO-hAT₁-AEQ-G_α16cells. Mean SEM (n ≥ 2). Dissociation

c

constant determined by competition binding with [³H]21 (c = 2 nM) at CHO-hAT₁-AEQ-G_α16cells. Mean SEM (n ≥ 3). Dissociation constant

d

determined by saturation binding at CHO-hAT₁-AEQ-G_α16cells. Mean SEM (n = 4). Determined in a fura-2 Ca²⁺assay at human HT-29 colon

e

carcinoma cells. Mean SEM (n ≥ 3). Determined by competition binding with [³H]27 (c = 1 nM) at HT-29 cells. Mean SEM (n ≥ 3).

f

g

Determined by competition binding with [³H]27 (c = 1 nM) at CHO-hNTS₁cells. Mean SEM (n ≥ 5). Determined by competition binding

h

with [³H]NT(8−13) (c = 0.5 nM) at HEK-hNTS₂cell homogenates. Mean SEM (n ≥ 2). Determined by saturation binding at HT-29 cells.

i

j

Mean SEM (n = 4). Determined by saturation binding at CHO-hNTS₁cells. Mean SEM (n = 4). Determined by competition binding with the

k

ﬂuorescent Y₄R ligand S0586-[K⁴]hPP (c = 10 nM) at CHO-hY₄-G_qi5-mtAEQ cells. Mean SEM (n ≥ 2). Determined by competition binding

with [³H]EYF (c = 0.35 nM) at CHO-hNPFF₂cell membranes. Mean SEM (n = 2). n represents the number of independent experiments

comparable to that of the parent peptide 1 (K_i(Y₄R) 7.4 nM (1),

1.5 nM (33), and 3.6 nM (34)). Peptide 34 represents a potential

radioligand, which can be prepared by [³H]propionylation from

the precursor peptide 33 by analogy with the protocols for the

synthesis of [³H]21 and [³H]27.

EYF Derivatives. The undecapeptide EYF, containing one

arginine in position 10, close to the C-terminus, is an analogue of

the RFamide peptide NPFF with a preference for the NPFF₂

receptor.^26,50In these peptides, the arginine residue is of

utmost relevance, as becomes obvious from the replacement

by alanine, resulting in a more than 10,000-fold decrease in

receptor aﬃnity.⁷⁰N^ω-carbamoylation of the arginine in EYF

(K_i(NPFF₂R) 0.53 nM) resulted in a by far less pronounced

decrease in aﬃnity (K_i(NPFF₂R) 27 nM (35), 41 nM (36)).

However, unlike the retained or even improved binding data of

the modiﬁed angiotensin, neurotensin, and NPY receptor

ligands, the reduced NPFF₂R binding aﬃnities of 35 and 36

compared to the parent peptide recall that the success of the

presented approach, as for any labeling strategy, depends on the

peculiarities of the individual ligand−receptor interactions. Such

diﬀerences between structure−activity relationships may be

exploited by the arginine-carbamoylarginine replacement with

respect to increasing receptor subtype selectivity and speciﬁcity,

as recently demonstrated for the discrimination of nonpeptide

argininamide-type antagonists between NPY Y₁and NPFF

receptors.⁷¹

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NPY Y₁and Y₂, and the vasopressin V₁and V₂receptors (see

GPCRDB⁷⁴).

4. CONCLUSION

The presented concept harbors a high potential for the

development of peptide-based pharmacological tools and is of

interest for the development of radiopharmaceuticals, as

numerous peptide-binding receptors have been suggested as

biomarkers and targets for cancer diagnosis and treatment,

respectively.^{4,7,9,13,15,75−77}

5. EXPERIMENTAL SECTION

Materials. If not otherwise stated, solvents, reagents, and buﬀer

components were of analytical grade purchased from commercial

suppliers. Technical grade solvents (ethyl acetate, light petroleum

(40−60 °C), and dichloromethane) were distilled before use.

Acetonitrile and MeOH for HPLC (gradient grade) were obtained

from Merck (Darmstadt, Germany). 2-Propanol (HPLC grade) for

hydrogenations was obtained from Carl Roth (Karlsruhe, Germany).

N,N-Diisopropylethylamine (99%) was from ABCR (Karlsruhe,

Germany). Anhydrous DMF over molecular sieve (>99.8%), triﬂuoro-

acetic acid (ReagentPlus, 99%), phenol, triisopropylsilane (99%),

1,1,1,3,3,3-hexaﬂuoro-2-propanol (HFIP) (>99%), Triton X-100,

amine 13, the ﬂuorescent dye Fluorescence Red Mega 480 succinimidyl

ester (38), and suberic acid monomethyl ester (42) were purchased

from Sigma-Aldrich (Deisenhofen, Germany). HBTU, PyBOP, and

Fmoc-Orn(Boc)-OH (3) were from Iris Biotech (Marktredwitz,

Germany). The succinimidyl ester 39 (S2197) of the ﬂuorescent dye

S0223 was purchased from FEW Chemicals (Wolfen, Germany). HOBt

hydrate, NMP (for peptide synthesis, nitrogen ﬂushed), and DMF (for

peptide synthesis, packed under nitrogen, code D/3848/PB17) were

obtained from Acros Organics/Fisher Scientiﬁc (Nidderau, Germany).

Bovine serum albumin (BSA) was purchased from Serva (Heidelberg,

Germany). Fura-2 AM and Pluronic F-127 were obtained from

Calbiochem/Merck Biosciences (Beeston, UK). AngII was obtained

from Bachem (Bubendorf, Switzerland). Coelenterazin h was obtained

from Biotrend (Cologne, Germany). Human pancreatic polypeptide

(hPP),⁷⁸compounds 6,⁴²7,⁵⁴and S-methylisothiourea hydroiodide⁴³

were prepared according to previously reported procedures. The

syntheses of NT(8−13),⁶⁰S0586-[K⁴]hPP,⁶¹and compound 41⁴¹were

described previously. EYF was prepared by SPPS according to the

general procedure (see below). Millipore water was used throughout for

the preparation of buﬀers and HPLC eluents. The 1.5 or 2 mL

Figure 8. Immunoblot analysis of homogenates (12 μg protein) of

HEK-hNTS₂(1), CHO-hNTS₁(2), and HT-29 colon carcinoma (3)

cells using a polyclonal NTS₂R speciﬁc antibody. An immunopositive

band was only detected in case of the homogenate of the NTS₂receptor

expressing control cells (1). M = molecular weight standard.

The incorporation of amino-functionalized N^ω-carbamoylated

arginines into peptides containing arginine either close to the

N- or the C-terminus (Figures 1B and 3) was conveniently

achieved by standard Fmoc SPPS using appropriately protected

amino-functionalized arginine building blocks (10 and 11). In

contrast to both nonpeptidic^42,47−49and peptidic (Supporting

Information, Figures S17 and S18) receptor ligands containing

an acylguanidine moiety, the N^ω-carbamoylated peptides

presented in this study proved to be stable in buﬀer and in

the presence of cells (Figure 4 and Supporting Information,

Figures S3 −S15). Therefore, misinterpretation of binding and

functional assays due to the release of the bioactive parent

compounds can be precluded.

We demonstrated that the synthesized (precursor) peptides,

bearing a primary amine functionality at the modiﬁed arginine

side chain, can be conjugated and labeled in a versatile manner by

analogy with the procedures established for lysine. Notably,

whereas acylation of the ε-amino group in lysine is accompanied

by loss of basicity, the strong basicity of the guanidine group in

arginine is reduced but not abolished by N^ω-carbamoylation

(pK_avalue of the carbamoylguanidine moiety: 7−8).⁷²This

guarantees a suﬃcient portion of the carbamoylguanidine

group to be positively charged at physiological pH, a prerequisite

for retained aﬃnity, as arginine residues of biologically

active peptides typically interact with acidic amino acids of the

receptor.

The high receptor aﬃnities of the ﬂuorescently labeled

analogues of AngII (23, 24) and NT(8−13) (29, 30) revealed

that even the attachment of bulky moieties to arginine is

tolerated. This can be explained by the fact that acidic residues of

peptide-binding receptors, preferentially interacting with argi-

nine residues of the peptide, are mainly located in regions of

the receptor facing the extracellular space. This assumption is

supported by the crystal structures of the rat NTS₁^68,69and the

human AT₁receptor⁷³(Supporting Information, Figures S31

and S32) by a homology model of the NPY Y₄receptor

(Supporting Information, Figure S33) as well as reported models

of the AngII AT₂, apelin, bradykinin B₁and B₂, bombesin BB₁

and BB₂, melanocortin MC₃, neurotensin NTS₂, NPFF₁, NPFF₂,

polypropylene reaction vessels with screw cap from Sud-Laborbedarf

̈

(Gauting, Germany) (in the following referred to as “reaction vessel

with screw cap”) were used for the preparation and storage of stock

solutions, for small scale reactions (e.g., the synthesis of radiolabeled and

ﬂuorescently labeled peptides) and for the investigation of chemical

stabilities. The 1.5 or 2 mL polypropylene reaction vessels from Sarstedt

(Numbrecht, Germany) (in the following referred to as “reaction

̈

vessel”) were used to dilute compounds, e.g., for assays and HPLC

analyses.

General Experimental Conditions. Thin layer chromatography

was performed on Merck silica gel 60 F₂₅₄TLC aluminum plates. If not

otherwise stated Silica Gel 60 (40−63 μm, Merck) was used for column

chromatography. In the case of the puriﬁcation of acid sensitive com-

pounds (Boc protecting group (except for 10, 11 and 16)), the

stationary phase was conditioned with the respective solvent containing

0.2% triethylamine. Melting points were determined with a Buchi 510

̈

apparatus (Buchi, Essen, Germany) and are uncorrected. Speciﬁc optical

̈

rotations at 589 nm (Na-D line) were measured on a polarimeter P8000-

T equipped with an electronic Peltier thermostat PT31 (A. KRUSS

̈

Optronic, Hamburg, Germany) using a microcuvette (layer thickness,

100 mm; volume, 1 mL; thermostat-controlled at 20 °C). IR spectra

were measured with a NICOLET 380 FT-IR spectrophotometer

(Thermo Electron Corporation). NMR spectra were recorded on a

Bruker Avance 300 instrument (7.05 T, ¹H 300 MHz; ¹³C 75 MHz), a

Bruker Avance 400 instrument (9.40 T, ¹H 400 MHz; ¹³C 100 MHz) or

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1

a Bruker Avance 600 instrument with cryogenic probe (14.1 T, H

600 MHz; ¹³C 150 MHz) (Bruker, Karlsruhe, Germany). Elemental

analysis was performed with a Vario MICRO Cube elemental analyzer

(Elementar Analysensysteme, Hanau, Germany). Low-resolution mass

spectrometry (MS) was performed on a Finnigan ThermoQuest TSQ

7000 instrument (Thermo Finnigan, San Jose, CA) equipped with an

electrospray ionization (ESI) source. High-resolution mass spectrom-

etry (HRMS) analysis was performed on an Agilent 6540 UHD

Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa

Clara, CA) using an ESI source. Preparative HPLC was performed with

a system from Knauer (Berlin, Germany) consisting of two K-1800

pumps and a K-2001 detector. A Gemini-NX C18, 5 μm, 250 mm ×

21 mm (Phenomenex, Aschaﬀenburg, Germany), a Kinetex-XB C18,

5 μm, 250 mm × 21 mm (Phenomenex), or a YMC-Actus Triart C8,

5 μm, 250 mm × 20 mm (YMC Europe, Dinslaken, Germany) served as

RP-columns at ﬂow rates of 22 (Gemini-NX) or 20 mL/min (Kinetex-

XB, YMC-Actus Triart). Mixtures of 0.1% aqueous TFA and acetonitrile

(or MeOH) were used as mobile phase, and a detection wavelength of

220 nm was used throughout. The solvent from collected fractions was

removed by lyophilization using a Christ alpha 2−4 LD lyophilization

apparatus equipped with a vacuubrand RZ 6 rotary vane vacuum pump.

Analytical HPLC analysis of compounds 10, 11, 16, 19−32, 35, 36, 44,

and EYF (concentrations between 70 and 120 μM) was performed with

a system from Agilent Technologies composed of a 1290 Inﬁnity binary

pump equipped with a degasser, a 1290 Inﬁnity autosampler, a 1290

Inﬁnity thermostated column compartment, a 1260 Inﬁnity diode array

detector, and a 1260 Inﬁnity ﬂuorescence detector. A Kinetex-XB C18,

2.6 μm, 100 mm × 3 mm (Phenomenex), served as stationary phase at a

ﬂow rate of 0.6 mL/min. Mixtures of acetonitrile (A) and 0.04% aq TFA

(B) were used as mobile phase. The following linear gradients were

applied. Compounds 10, 11, and 16: 0−20 min, A/B 20:80−95:5;

20−25 min, 95:5. Compounds 19−22, 25−28, 31, 32, 35, 36, and 44:

0−12 min, A/B 10:90−30:70; 12−16 min, 30:70−95:5; 16−20 min,

95:5. Compounds 23, 24, 29, and 30: 0−15 min, A/B 15:85−42:58;

15−18 min, 42:58−95:5; 18−23 min, 95:5. The oven temperature was

25 °C, and the injection volume was 20 μL. Detection was performed at

220 nm throughout and additionally at 500 nm (23, 29) or 630 nm

(24, 30). Analytical HPLC analysis of compounds 1, 33, and 34

(concentration: 100 μM) was performed on a system from Merck-

Hitachi composed of a L-6200-A pump, an AS-2000A autosampler, a

L-4000A UV detector, and a D-6000 interface. A Kinetex-XB C18, 5 μm,

250 mm × 4.6 mm (Phenomenex) served as stationary phase at a ﬂow

rate of 0.8 mL/min. Mixtures of acetonitrile (A) and 0.1% aq TFA (B)

were used as mobile phase. The following linear gradient was applied:

0−25 min, A/B 10:90−40:60; 25−35 min, 60:40−95:5; 35−45 min,

95:5. Detection was performed at 220 nm, the oven temperature was

30 °C, and the injection volume was 50 μL.

Fmoc-Trp(Boc)-OH, Fmoc-Tyr(tBu)-OH, and Fmoc-Val-OH) and the

solid supports Fmoc-Rink amide AM polystyrene (PS) resin, H-Phe-2-

ClTrt PS resin, and H-Leu-2-ClTrt PS resin were purchased from Merck

Chemicals (Schwalbach am Taunus, Germany). The Sieber PS resin was

obtained from Iris Biotech. DMF/NMP (8:2 v/v) was used as solvent

throughout (ca. 2.2 mL per 1 mmol Fmoc-aa). Standard amino

acids (5-fold excess) were preactivated with HBTU/HOBt/DIPEA

(4.9/5/10 equiv) for 5 min and added to the resin. The vessel was shaken

at rt for 60 min. The coupling procedure was repeated in case of standard

amino acids. The coupling of the arginine building blocks 10 and 11

(3-fold excess, preactivated with HBTU/HOBt/DIPEA (2.95/3/6 equiv)

for 5 min) was performed at 35 °C for 16 h (“single” coupling) using

anhydrous DMF. After completed coupling of a Fmoc-aa the resin was

washed with solvent (4×) followed by Fmoc deprotection with 20%

piperidine in DMF/NMP (8:2 v/v) (rt, 2 × 10 min; after incorporation of

10 or 11, 2 × 5 min) and thorough washing of the resin with solvent (6×).

After the last coupling and Fmoc deprotection step, the resin was washed

with solvent (4×) and CH₂Cl₂(6×) (in caseof 2-ClTrt resinsCH₂Cl₂was

treated with K₂CO₃). Peptides synthesized at the Rink aminde AM resin

(17, 35) were cleaved oﬀ with TFA/water (95:5 v/v) at rt for 2 h (17) or

with TFA/phenolum liquefactum/water/triisopropylsilane (95:2.4:1.6:

1 v/v/v/v) at rt for 2.5 h (35). The resin was separated by ﬁltration and

washed three times with MeCN/water (40:60 v/v) (2 mL each). All

ﬁltrates were collected and combined in a round-bottom ﬂask containing

water (120 mL), and the mixture was lyophilized to obtain the crude

peptide, which was puriﬁed by preparative HPLC. Peptides synthesized at

the 2-ClTrt resin (19, 20, 25, 26, 31) were cleaved oﬀ with CH₂Cl₂/HFIP

(3:1 v/v) (rt, 2 × 20 min). The resin was separated by ﬁltration and

washed once with CH₂Cl₂/HFIP (3:1) (1 mL). The ﬁltrates were

combined, the volatiles were evaporated and the residue taken up in

CH₂Cl₂(20 mL), followed by evaporation of the solvent. TFA/water

(95:5 v/v) (1−2 mL) was added, and the mixture was incubated at rt for

2 h for peptides without Arg(Pbf) or 3.5 h for peptides containing

Arg(Pbf). Water (120 mL) was added followed by lyophilization and

puriﬁcation of the crude peptide by preparative HPLC. Peptides

synthesized at the Sieber resin (33, 40, 43) were cleaved oﬀ with

CH₂Cl₂/TFA (97:3 v/v) (rt, 10 × 6 min). The resin was removed by

ﬁltration. All ﬁltrates were collected and combined in a round-bottom

ﬂask containing water (10 times the volume of the ﬁltrate). The organic

solvent was evaporated, and the aqueous phase was lyophilized followed

by side chain deprotection (33) or puriﬁcation by preparative HPLC

(40 and 43).

Chemistry: Synthesis Protocols and Analytical Data of

Compounds 1, 10, 11, 16, and 19−36. Octanedioyl-bis(tyrosyl-

arginyl-leucyl-arginyl-tyrosinamide) Tetrakis(hydrotriﬂuoroacetate)

(1). Compound 40 (20 mg, 13.3 μmol) was dissolved in anhydrous

DMF/DIPEA (99:1 v/v) (600 μL). Compound 41 (1.96 mg, 5.3 μmol)

was added, and the mixture was stirred at 35 °C for 16 h. Water (10 mL)

was added and the protected intermediate extracted with CH₂Cl₂(2 ×

10 mL). The combined extracts were evaporated and the residue dried

in vacuo. TFA/water (95:5 v/v) (2 mL) was added, and the mixture was

stirred at rt for 2.5 h. Water (100 mL) was added, followed by

lyophilization. The product was puriﬁed by preparative HPLC (column,

Kinetex-XB C18 250 mm × 21 mm; gradient, 0−20 min; MeCN/0.1%

aq TFA 14:86−52:48 (ﬂow rate: 15 instead 20 mL/min); t_R= 11 min).

Lyophilization of the eluate aﬀorded 1 as a white ﬂuﬀy solid (5.7 mg,

51%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.83 (d, 6H, J = 6.5 Hz), 0.87

(d, 6H, J = 6.5 Hz), 1.03 (br s, 4H), 1.30 (br s, 4H), 1.37−1.56 (m, 16H),

1.56−1.73 (m, 6H), 1.95−2.05 (m, 4H), 2.57−2.65 (m, 2H), 2.68−2.75

(m, 2H), 2.81−2.89 (m, 4H), 3.02−3.13 (m, 8H), 4.17−4.24 (m, 2H),

4.24−4.36 (m, 6H), 4.39−4.46 (m, 2H), 6.59−6.65 (m, 8H), ca. 6.9 (br

s, 8H), 6.95−6.99 (m, 4H), 6.99−7.03 (m, 4H), 7.05 (s, 2H), ca. 7.3 (br

s, 8H), 7.36 (s, 2H), 7.49−7.57 (m, 4H), 7.74 (d, 2H, J = 7.9 Hz), 7.85−

7.94 (m, 4H), 8.03 (d, 2H, J = 7.7 Hz), 8.17 (d, 2H, J = 7.8 Hz), 9.16 (s,

2H), 9.17 (s, 2H). ¹³C NMR (150 MHz, DMSO-d₆): δ 21.4 (2 carb),

23.1 (2 carb), 24.1 (2 carb), 24.9 (2 carb), 25.0 (2 carb), 25.1 (2 carb),

28.4 (2 carb), 28.9 (2 carb), 29.0 (2 carb), 35.2 (2 carb), 36.6 (2 carb),

36.8 (2 carb), 40.4 (4 carb), 40.7 (2 carb), 50.9 (2 carb), 52.1 (2 carb),

52.2 (2 carb), 53.9 (2 carb), 54.1 (2 carb), 114.8 (4 carb), 114.9 (4 carb),

127.5 (2 quat carb), 127.9 (2 quat carb), 129.9 (4 carb), 130.0 (4 carb),

Compound Characterization. New synthetic organic compounds

were characterized by ¹H and ¹³C NMR spectroscopy, HRMS, optical

rotation (if applicable), and melting point (if applicable). In addition,

the key compounds 10, 11, and 16 were characterized by 2D-NMR

spectroscopy (¹H-COSY, HSQC, HMBC), RP-HPLC analysis, IR

spectroscopy, and elemental (combustion) analysis. New peptides (fully

deprotected), obtained after SPPS and cleavage from the solid support,

were characterized by ¹H and ¹³C NMR, 2D-NMR (¹H-COSY, HSQC,

HMBC, TOCSY), HRMS, and RP-HPLC analysis. Propionylated deriv-

1

atives of these peptides were characterized by H NMR, HRMS, and

RP-HPLC analysis. Fluorescently labeled derivatives were characterized

by HRMS and RP-HPLC analysis.

The purity of ﬁnal compounds, determined by RP-HPLC (220 nm),

was ≥96% throughout. Elemental analysis data (10, 11, and 16) fulﬁlled

the requirement of less than 0.4% deviation.

General Procedure for SPPS. Peptides were synthesized by

manual SPPS applying the Fmoc strategy. First, 5 mL BD Discardit II

syringes (Becton Dickinson, Heidelberg, Germany) equipped with a

35 μm polyethylene frit (Roland Vetter Laborbedarf OHG, Ammerbuch,

Germany) served as reaction vessels. Protected standard ^L-amino acids

(Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gln-

(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH,

Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH,

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155.7 (2 quat carb), 155.8 (2 quat carb), 155.6 (2 quat carb), 155.7

(2 quat carb), 170.8 (2 quat carb), 171.1 (2 quat carb), 171.8 (2 quat

carb), 172.0 (2 quat carb), 172.3 (2 quat carb), 172.7 (2 quat carb).

HRMS (ESI): m/z [M + 4H]⁴⁺calcd for [C₈₀H₁₂₆N₂₄O₁₆]⁴⁺419.7440,

found 419.7452. RP-HPLC (220 nm): 98% (t_R= 20.2 min, k = 6.0).

C₈₀H₁₂₂N₂₄O₁₆·C₈H₄F₁₂O₈(1676.01 + 456.09).

2975, 2935, 2870, 1715, 1635, 1605, 1515, 1450, 1415, 1365, 1250,

1150 cm⁻¹. HRMS (ESI): m/z [M + H]⁺calcd for [C₃₈H₅₅N₆O₁₁]⁺

771.3923, found 771.3933. Anal. Calcd for C₃₈H₅₄N₆O₁₁·H₂O: C 57.85,

H 7.15, N 10.65. Found: C 58.14, H 6.94, N 10.59. RP-HPLC (220 nm):

99% (t_R= 9.8 min, k = 11.9). C₃₈H₅₄N₆O₁₁·H₂O (770.87 + 18.02).

N^ω-[N-(4-tert-Butoxycarbonylaminobutyl)aminocarbonyl]-N^α-

ﬂuoren-9-ylmethoxycarbonyl-^L-arginine Dihydrate (16). In a two-

necked round-bottom ﬂask, under an atmosphere of argon, a 10% Pd/C

catalyst (160 mg) was added to a solution of 15 (1.45 g, 1.74 mmol) in

MeOH, and a slow stream of hydrogen was passed through a glass tube

into the vigorously stirred suspension for 60 min. The catalyst was

removed by ﬁltration, and the solvent was evaporated. The product was

puriﬁed by column chromatography (eluent, CH₂Cl₂/MeOH 20:1 to

3:1 (column packed in CH₂Cl₂/MeOH/AcOH 200:10:1)). The eluate

was concentrated under reduced pressure, water (200 mL) was added,

and the mixture was lyophilized. Relyophilization after uptake in

MeCN/water 60:40 (100 mL) and evaporation of the major amount of

MeCN at 30 °C aﬀorded the product as a white solid (0.515 g, 46%).

TLC (CH₂Cl₂/MeOH 5:1 v/v): R_f= ca. 0.4. [α]²_D⁰3.4 (0.032 M in

N^ω-tert-Butoxycarbonyl-N^ω′-[N-(4-tert-butoxycarbonylaminobutyl)-

aminocarbonyl]-N^α-ﬂuoren-9-ylmethoxycarbonyl-L-arginine Monohy-

drate (10). In a two-necked 100 mL round-bottom ﬂask, under an

atmosphere of argon, a 10% Pd/C catalyst (300 mg) was added to a

solution of 8 (2.05 g, 2.56 mmol) and acetic acid (8 μL) in 2-propanol

(30 mL), and a slow stream of hydrogen was passed through a glass tube

into the vigorously stirred suspension for 4.5 h (additional catalyst was

added after 2.5 h (150 mg) and 3.5 h (100 mg)). The main part of the

catalyst was removed by ﬁltration through a pad of cotton wool,

followed by a ﬁltration using a 25 mm syringe ﬁlter (Nylon, 0.45 μm,

PP housing). Water (300 mL) was added to the ﬁltrate, and the mixture

was lyophilized. The product was puriﬁed by column chromatography

(Merck silica gel Geduran 60 (0.063−0.200 mm), eluent, MeCN/EtOH

40:1 to 10:1; upon appearance of the product in the eluate, the eluent

was changed from MeCN/EtOH 10:1 to MeCN/EtOH/AcOH

1000:125:1). After completion of elution, the eluate was immediately

diluted with water (600 mL) and lyophilized. Product 10 was obtained

as a white powder (1.42 g, 75%) and stored at −20 °C. TLC (MeCN/

EtOH/AcOH 100:10:1 v/v/v): R_f= 0.6. [α]²_D⁰(0.037 M in MeCN/H₂O

1

EtOH). H NMR (600 MHz, DMSO-d₆): δ 1.29−1.41 (m, 13H),

1.46−1.57 (m, 2H), 1.57−1.65 (m, 1H), 1.65−1.89 (m, 1H), 2.81−2.93

(m, 2H), 3.03 (br s, 2H), 3.19 (br s, 2H), 3.70−3.89 (m, 1H), 4.17−4.33

(m, 3H), 6.76 (t, 1H, J = 5.2 Hz), 7.07 (br s, 1H), 7.24−7.34 (m, 2H),

7.36−7.58 (m, 3H), 7.64−7.75 (m, 2H), 7.86−7.91 (m, 2H), 8.39 (br s,

2H), 9.45 (br s, 1H), 13.6 (br s, 1H). ¹³C NMR (150 MHz, MeOH-d₄):

δ 25.7, 27.9, 28.2, 28.8, 30.9, 40.4, 41.0, 41.8, 48.5, 57.2, 67.6, 79.8, 120.9,

126.23, 126.26, 128.1, 128.8, 142.57, 142.61, 145.2, 145.4, 155.9, 156.1,

158.3, 158.5, 180.3. IR (KBr): 3350, 2975, 2940, 1690, 1610, 1540,

1450, 1400, 1250, 1170 cm⁻¹. HRMS (ESI): m/z [M + H]⁺calcd

for [C₃₁H₄₃N₆O₇]⁺611.3188, found 611.3198. Anal. Calcd for

C₃₁H₄₂N₆O₇·H₄O₂: C 57.57, H 7.17, N 12.99. Found: C 57.78, H

6.87, N 12.82. RP-HPLC (220 nm): 99% (t_R= 7.2 min, k = 8.5).

C₃₁H₄₂N₆O₇·H₄O₂(610.70 + 36.03).

1

7:3 v/v). H NMR (600 MHz, DMSO-d₆) (two major rotamers with

1

a ratio of approximately 1:1 were evident in the H and ¹³C NMR

spectra): δ 1.30−1.39 (m, 13H), 1.40 (s, 4.5H), 1.44 (s, 4.5H), 1.46−

1.64 (m, 3H), 1.68−1.77 (m, 1H), 2.83−2.92 (m, 2H), 2.92−2.98

(m, 1H), 2.98−3.05 (m, 1H), 3.20−3.30 (m, 2H), 3.86 (br s, 1H), 4.19−

4.27 (m, 2H), 4.27−4.34 (m, 1H), 6.75 (t, 0.5H, J = 5.5 Hz), 6.78 (t,

0.5H, J = 5.3 Hz), 6.96 (t, 0.5H, J = 5.6 Hz), 7.24 (br s, 1H), 7.29−7.34

(m, 2H), 7.37−7.43 (m, 2H), 7.67−7.73 (m 2H), 7.83−7.91 (m, 2.5H),

8.14 (br s, 0.5H), 9.16 (t, 0.5H, J = 5.1 Hz), 11.06 (s, 0.5H), 12.52 (s,

0.5H), 12.8 (br s, 0.5H). ¹³C NMR (150 MHz, DMSO-d₆): δ 25.5, 26.3,

26.86, 26.91, 27.0, 27.7, 28.1, 28.3, 29.0, 38.8, 39.0, 39.6, 39.8, 46.7, 54.3,

65.4, 77.28, 77.33, 81.9, 120.1, 125.2, 127.0, 127.6, 140.7, 143.8, 143.9,

152.4, 153.0, 154.6, 155.6, 155.8, 156.4, 162.6, 164.3, 174.3. IR (KBr):

3340, 3065, 2975, 2935, 2870, 1715, 1635, 1605, 1515, 1450, 1415,

1365, 1250, 1150 cm⁻¹. HRMS (ESI): m/z [M + H]⁺calcd for

[C₃₆H₅₁N₆O₉]⁺711.3712, found 711.3729. Anal. Calcd for

C₃₆H₅₀N₆O₉·H₂O: C 59.33, H 7.19, N 11.53. Found: C 59.14, H

6.83, N 11.70. RP-HPLC (220 nm): 99% (t_R= 10.0 min, k = 12.2).

C₃₆H₅₀N₆O₉·H₂O (710.82 + 18.02).

H-Asp-{N^ω-[N-(4-aminobutyl)aminocarbonyl]}Arg-Val-Tyr-Ile-His-

Pro-Phe-OH Tetrakis(hydrotriﬂuoroacetate) (19). The peptide was

synthesized according to the general procedure using a H-Phe-2-ClTrt

resin (loading: 0.73 mmol/g). Puriﬁcation by preparative HPLC was

performed with a YMC-Actus Triart C8 250 mm × 20 mm (gradient,

0−30 min; MeCN/0.1% aq TFA 10:90−35:65; t_R= 22 min), and the

product fraction still containing byproduct was rechromatographed on a

Gemini-NX C18 250 mm × 21 mm (gradient, 0−30 min; MeOH/0.1%

aq TFA 17:83−57:43, t_R= 25 min). Lyophilization of the eluate aﬀorded

peptide 19 as a white ﬂuﬀy solid (47.9 mg, 45%). ¹H NMR (600 MHz,

DMSO-d₆): δ 0.71−0.81 (m, 12H), 1.00−1.09 (m, 1H), 1.31−1.39 (m,

1H), 1.44−1.59 (m, 7H), 1.60−1.68 (m, 2H), 1.75−1.88 (m, 3H),

1.89−1.97 (m, 1H), 1.99−2.06 (m, 1H), 2.60−2.68 (m, 2H), 2.75−2.84

(m, 4H), 2.89−2.96 (m, 2H), 2.99−3.07 (m, 2H), 3.08−3.14 (m, 2H),

3.18−3.27 (m, 2H), 3.45−3.50 (m, 1H), 3.61−3.67 (m, 1H), 4.09−4.16

(m, 2H), 4.18 (dd, 1H, J = 8.6, 6.6 Hz), 4.34−4.45 (m, 3H), 4.47−4.53

(m, 1H), 4.74−4.80 (m, 1H), 6.58−6.63 (m, 2H), 6.99−7.03 (m, 2H),

7.17−7.22 (m, 1H), 7.23−7.29 (m, 4H), 7.37 (s, 1H), 7.61 (br s, 1H),

7.80 (br s, 4H), 7.85 (d, 1H, J = 8.5 Hz), 8.03 (d, 1H, J = 8.0 Hz), 8.19

(br s, 3H), 8.28 (d, 1H, J = 7.7 Hz), 8.31 (d, 1H, J = 7.5 Hz), 8.46 (br s,

2H), 8.60 (d, 1H, J = 7.8 Hz), 8.93 (s, 1H), 9.04 (br s, 1H), 9.21 (br s,

1H), 10.7 (br s, 1H), 12.9 (br s, 2H), 14.3 (br s, 2H). ¹³C NMR (150

MHz, DMSO-d₆): δ 10.8, 15.2, 17.7, 19.2, 24.2, 24.3, 24.4, 24.6, 26.0,

26.3, 29.0, 29.2, 30.9, 35.5, 36.5, 36.57, 36.62, 38.5, 38.6, 40.5, 46.9, 48.8,

49.6, 52.5, 53.6, 53.9, 56.7, 57.2, 59.3, 114.8, 117.1 (q, J = 299 Hz)

(TFA), 117.3, 126.4, 127.7, 128.2, 128.8, 129.3, 130.0, 133.7, 137.3,

153.8, 153.9, 155.7, 158.5 (q, J = 32 Hz) (TFA), 167.8, 168.1, 170.5,

170.6, 170.8, 170.9, 171.1, 171.8, 172.6. HRMS (ESI): m/z [M + 2H]²⁺

calcd for [C₅₅H₈₃N₁₅O₁₃]²⁺580.8142, found 580.8146. RP-HPLC

(220 nm): 99% (t_R= 7.3 min, k = 8.6). C₅₅H₈₁N₁₅O₁₃·C₈H₄F₁₂O₈

(1160.3 + 456.09).

N^ω-tert-Butoxycarbonyl-N^ω′-[N-(8-tert-butoxycarbonylamino-

3,6-dioxaoctyl)aminocarbonyl]-N^α-ﬂuoren-9-ylmethoxycarbonyl-L-

arginine Monohydrate (11). Compound 11 was prepared from 9

(2.32 g, 2.69 mmol) using the procedure for the preparation of 10.

Added portions of catalyst: 400 mg (at ﬁrst), 200 mg (after 1 h), and

100 mg (after 2 h). The conversion was complete after 3 h. Column

chromatography was performed as described for 10. Lyophilization of

the eluate aﬀorded the product as a white solid, which contained about

40 mol % of acetic acid. For removal of the acetic acid, lyophilization was

repeated three times after uptake in MeCN/water 40:60 (250 mL).

Compound 11 was obtained as a white powder (1.72 g, 81%). TLC

(MeCN/EtOH/AcOH 100:10:1 v/v/v): R_f= 0.5. [α]²_D⁰3.1 (0.021 M in

1

MeCN/H₂O 7:3 v/v). H NMR (600 MHz, DMSO-d₆) (two major

rotamers with a ratio of approximately 1:1 were evident in the ¹H and

¹³C NMR spectra): δ 1.36 (s, 9H), 1.40 (s, 4.5H), 1.45 (s, 4.5H), 1.48−

1.66 (m, 3H), 1.68−1.77 (m, 1H), 3.00−3.09 (m, 2H), 3.10−3.16 (m,

1H), 3.17−3.20 (m, 1H), 3.21−3.30 (m, 2H), 3.33−3.53 (m, 8H),

3.90−3.98 (m, 1H), 4.19−4.25 (m, 2H), 4.25−4.35 (m, 1H), 6.74

(t, 1H, J = 5.2 Hz), 6.87 (t, 0.5H, J = 5.6 Hz), 7.29−7.35 (m, 2H), 7.37−

7.43 (m, 2H), 7.54 (br s, 1H), 7.69−7.75 (m 2H), 7.86−7.90 (m, 2H),

7.92 (t, 0.5H, J = 5.2 Hz), 8.28 (br s, 0.5H), 9.13 (t, 0.5H, J = 5.0 Hz),

11.06 (s, 0.5H), 12.43 (s, 0.5H), 12.6 (br s, 1H). ¹³C NMR (150 MHz,

DMSO-d₆): δ 25.5, 25.6, 27.7, 28.1, 28.2, 28.4, 39.1, 39.6 (two carbon

nuclei), 46.7, 53.8, 65.6, 68.9, 69.17, 69.20, 69.46, 69.52, 77.4, 77.6, 82.0,

120.1, 125.3, 127.1, 127.6, 140.71, 140.72, 143.8, 143.9, 152.4, 153.2,

154.7, 155.6, 156.0, 156.3, 162.5, 164.4, 174.0. IR (KBr): 3340, 3065,

H-Asp-{N^ω-[N-(8-amino-3,6-dioxaoctyl)aminocarbonyl]}Arg-Val-

Tyr-Ile-His-Pro-Phe-OH Tetrakis(hydrotriﬂuoroacetate) (20). The

peptide was synthesized according to the general procedure using a

H-Phe-2-ClTrt resin (loading: 0.73 mmol/g). Puriﬁcation by pre-

parative HPLC was performed with a YMC-Actus Triart C8 250 mm ×

20 mm (gradient, 0−24 min; MeCN/0.1% aq TFA 10:90−30:70;

K

DOI: 10.1021/acs.jmedchem.5b01495

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

t_R= 23 min), and the product fraction still containing byproduct was

rechromatographed on a Gemini-NX C18 250 mm × 21 mm (gradient,

0−30 min; MeOH/0.1% aq TFA 17:83−57:43; t_R= 25 min).

Lyophilization of the eluate aﬀorded peptide 20 as a white ﬂuﬀy solid

(m, 1H), 4.74−4.80 (m, 1H), 6.57−6.63 (m, 2H), 6.98−7.04 (m, 2H),

7.17−7.22 (m, 1H), 7.23−7.28 (m, 4H), 7.37 (s, 1H), 7.51 (t, 1H, J =

4.9 Hz), 7.75−7.87 (m, 3H), 8.04 (d, 1H, J = 8.1 Hz), 8.19 (br s, 3H),

8.30 (d, 1H, J = 7.8 Hz), 8.32 (d, 1H, J = 7.7 Hz), 8.43 (br s, 2H), 8.58 (d,

1H, J = 7.9 Hz), 8.94 (br s, 1H), 9.00 (br s, 1H), 9.16 (br s, 1H), 10.1

(br s, 1H), 12.8 (br s, 1H), 13.0 (br s, 1H), 14.1 (br s, 2H). HRMS

(ESI): m/z [M + 2H]²⁺calcd for [C₆₀H₉₁N₁₅O₁₆]²⁺638.8379, found

638.8375. C₆₀H₈₉N₁₅O₁₆. RP-HPLC (220 nm): 99% (t_R= 8.9 min, k =

10.7). C₆H₃F₉O₆(1276.4 + 342.07).

1

(66.4 mg, 26%). H NMR (600 MHz, DMSO-d₆): δ 0.71−0.81 (m,

12H), 1.00−1.09 (m, 1H), 1.31−1.39 (m, 1H), 1.44−1.59 (m, 3H),

1.60−1.68 (m, 2H), 1.75−1.88 (m, 3H), 1.89−1.97 (m, 1H), 1.99−2.07

(m, 1H), 2.60−2.68 (m, 2H), 2.76−2.83 (m, 2H), 2.89−3.00 (m, 4H),

3.00−3.08 (m, 2H), 3.18−3.30 (m, 4H), 3.43−3.51 (m, 3H), 3.53−3.61

(m, 6H), 3.61−3.67 (m, 1H), 4.10−4.16 (m, 2H), 4.18 (dd, 1H, J = 8.6,

6.6 Hz), 4.34−4.40 (m, 2H), 4.40−4.45 (m, 1H), 4.47−4.53 (m, 1H),

4.74−4.80 (m, 1H), 6.58−6.63 (m, 2H), 6.98−7.03 (m, 2H), 7.17−7.22

(m, 1H), 7.23−7.28 (m, 4H), 7.37 (s, 1H), 7.48 (br s, 1H), 7.74−7.96

(m, 5H), 8.03 (d, 1H, J = 8.1 Hz), 8.19 (br s, 3H), 8.28 (d, 1H, J =

7.7 Hz), 8.31 (d, 1H, J = 7.5 Hz), 8.47 (br s, 2H), 8.60 (d, 1H, J =

7.7 Hz), 8.95 (s, 1H), 9.06 (br s, 1H), 9.21 (br s, 1H), 10.7 (br s, 1H),

12.8 (br s, 2H), 14.2 (br s, 2H). ¹³C NMR (150 MHz, DMSO-d₆): δ

10.8, 15.2, 17.7, 19.2, 24.2, 24.3, 24.6, 26.3, 29.0, 29.2, 30.9, 35.5, 36.5,

36.57, 36.61, 38.5, 39.1, 40.5, 46.9, 48.8, 49.6, 52.5, 53.6, 53.9, 56.7, 57.2,

59.3, 66.7, 68.7, 69.4, 69.6, 114.8, 117.0 (q, J = 299 Hz) (TFA), 117.3,

126.4, 127.7, 128.2, 128.8, 129.3, 130.0, 133.7, 137.3, 153.7, 153.9, 155.7,

158.5 (q, J = 32 Hz) (TFA), 167.8, 168.1, 170.5, 170.6, 170.8, 170.9,

171.1, 171.8, 172.6. HRMS (ESI): m/z [M + 2H]²⁺calcd for

[C₅₇H₈₇N₁₅O₁₅]²⁺610.8248, found 610.8254. RP-HPLC (220 nm):

99% (t_R= 7.4 min, k = 8.7). C₅₇H₈₅N₁₅O₁₅·C₈H₄F₁₂O₈(1220.4 +

456.09).

H-Asp-{N^ω-[N-(4-{N-[6-(6-{2-[7-(diethylamino)-2-oxo-2H-chro-

men-3-yl]vinyl}-3-sulfonato-pyridin-1-ium-1-yl)hexanoyl]amino}-

butyl)aminocarbonyl]}Arg-Val-Tyr-Ile-His-Pro-Phe-OH Tris-

(hydrotriﬂuoroacetate) (23). A solution of Fluorescence Red Mega

480 succinimidyl ester (38) (1 mg, 1.63 μmol) in anhydrous DMF

(15 μL) was added to a solution of 19 (6.4 mg, 3.96 μmol), DIPEA

(7 mg, 54 μmol), and K₂CO₃(4 mg, 40 μmol) in DMF/water (4:6 v/v)

(100 μL) in a 1.5 mL reaction vessel with screw cap, and the vessel was

shaken at rt in the dark for 30 min. Then 10% aq TFA (70 μL) was

added, and the product was puriﬁed by preparative HPLC (column,

Kinetex 250 mm × 21 mm; gradient, 0−25 min; MeCN/0.1% aq TFA

10:90−40:60; t_R= 24.5 min). Lyophilization of the eluate yielded 23 as a

dark-red solid (0.48 mg, 15%). HRMS (ESI): m/z [M + 3H]³⁺calcd for

[C₈₁H₁₁₂N₁₇O₁₉S]³⁺553.2685, found 553.2692. RP-HPLC (220 and

500 nm): 98% (t_R= 10.4 min, k = 12.7). C₈₁H₁₀₉N₁₇O₁₉S·C₆H₃F₉O₆

(1656.90 + 342.07).

H-Asp-[N^ω-(N-{4-[N-(6-{3,3-dimethyl-2-[5-(1,3,3-trimethyl-3H-

indol-1-ium-2-yl)penta-2,4-dien-1-ylidene]indolin-1-yl}hexanoyl)-

amino]butyl}aminocarbonyl)]Arg-Val-Tyr-Ile-His-Pro-Phe-OH Tri-

ﬂuoroacetate Tris(hydrotriﬂuoroacetate) (24). Compound 24 was

prepared from 19 (6.4 mg, 3.96 μmol) and 39 (1.7 mg, 2.57 μmol)

following the procedure for the preparation of 23. DIPEA: 7 mg, 54 μmol.

K₂CO₃: 4 mg, 40 μmol. The product was puriﬁed by preparative HPLC

(column, Kinetex 250 mm × 21 mm; gradient, 0−22 min; MeCN/0.1%

aq TFA 10:90−37:63; 22−40 min, 37:63−45:55; t_R= 33 min).

Lyophilization of the eluate aﬀorded 24 as a dark-blue solid (1.3 mg,

25%). HRMS (ESI): m/z [M + 2H]³⁺calcd for [C₈₇H₁₂₀N₁₇O₁₄]³⁺

H-Asp-{N^ω-[N-(4-propanoylaminobutyl)aminocarbonyl]}Arg-Val-

Tyr-Ile-His-Pro-Phe-OH Tris(hydrotriﬂuoroacetate) (21). The reaction

was performed in a 2 mL reaction vessel with screw cap equipped with a

magnetic microstirrer. DIPEA (7.6 mg, 58.8 μmol) and K₂CO₃(8.4 mg,

83.9 μmol) were added to a solution of peptide 19 (11.3 mg, 6.94 μmol)

in DMF/water (1:1 v/v) (400 μL), followed by the addition of

succinimidyl propionate (37) (0.72 mg, 4.2 μmol) dissolved in DMF

(40 μL) (added as 10 μL portions with a time lag of 5 min). Then 60 min

after continued stirring, 10% aq TFA (100 μL) was added. The product

was puriﬁed by preparative HPLC (column, Gemini-NX C18 250 mm ×

21 mm; gradient, 0−35 min; MeCN/0.1% aq TFA 10:90−37:63; t_R=

23 min). Lyophilization of the eluate aﬀorded 21 as a white ﬂuﬀy solid

(5.0 mg, 77%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.72−0.82 (m, 12H),

0.98 (t, 3H, J = 7.6 Hz), 1.01−1.09 (m, 1H), 1.31−1.44 (m, 5H), 1.44−

1.57 (m, 3H), 1.64 (br s, 2H), 1.75−1.88 (m, 3H), 1.90−1.97 (m, 1H),

2.00−2.08 (m, 3H), 2.61−2.67 (m, 2H), 2.75−2.83 (m, 2H), 2.89−2.96

(m, 2H), 2.99−3.06 (m, 4H), 3.06−3.12 (m, 2H), 3.22 (br s, 2H), 3.43−

3.49 (m, 1H), 3.61−3.68 (m, 1H), 4.08−4.17 (m, 2H), 4.19 (dd, 1H, J =

8.7, 6.6 Hz), 4.34−4.45 (m, 3H), 4.47−4.53 (m, 1H), 4.74−4.80 (m,

1H), 6.58−6.63 (m, 2H), 6.99−7.04 (m, 2H), 7.18−7.22 (m, 1H),

7.23−7.28 (m, 4H), 7.37 (s, 1H), 7.51 (t, 1H, J = 4.9 Hz), 7.74 (t, 1H, J =

5.5 Hz), 7.81 (d, 1H, J = 9.1 Hz), 7.84 (d, 1H, J = 8.3 Hz), 8.04 (d, 1H,

J = 8.1 Hz), 8.16 (br s, 3H), 8.28−8.34 (m, 2H), 8.39 (br s, 2H), 8.58 (d,

1H, J = 7.8 Hz), 8.94 (br s, 1H), 8.98 (br s, 1H), 9.16 (br s, 1H), 10.0

(br s, 1H), 12.7 (br s, 1H), 13.0 (br s, 1H), 14.1 (br s, 2H). HRMS

(ESI): m/z [M + 2H]²⁺calcd for [C₅₈H₈₇N₁₅O₁₄]²⁺608.8273, found

608.8281. RP-HPLC (220 nm): 99% (t_R= 9.1 min, k = 11.0).

C₅₈H₈₅N₁₅O₁₄·C₆H₃F₉O₆(1216.4 + 342.07).

542.3061, found 542.3066. RP-HPLC (220 and 630 nm): 98% (t_R

13.9 min, k = 17.3). C₈₇H₁₁₈N₁₇O₁₄·C₈H₃F₁₂O₈(1625.02 + 455.09).

=

H-{N^ω-[N-(4-aminobutyl)aminocarbonyl]}Arg-Arg-Pro-Tyr-Ile-

Leu-OH Tetrakis(hydrotriﬂuoroacetate) (25). The peptide was

synthesized according to the general procedure using a H-Leu-2-ClTrt

resin (loading: 0.81 mmol/g). Puriﬁcation by preparative HPLC

was performed with a Gemini-NX C18 250 mm × 21 mm (gradient,

0−35 min; MeCN/0.1% aq TFA 8:92−41:59; t_R= 19 min).

Lyophilization of the eluate aﬀorded 25 as a white ﬂuﬀy solid (72.6

mg, 59%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.80 (t, 3H, J = 7.4 Hz),

0.82−0.86 (m, 6H), 0.90 (d, 3H, J = 6.6 Hz), 1.00−1.10 (m, 1H), 1.36−

1.44 (m, 1H), 1.44−1.65 (m, 12H), 1.65−1.76 (m, 4H), 1.78−1.87 (m,

3H), 1.94−2.04 (m, 1H), 2.68 (dd, 1H, J = 14.0, 8.2 Hz), 2.75−2.83 (m,

2H), 2.83−2.90 (m, 1H), 3.03−3.15 (m, 4H), 3.26 (br s, 2H), 3.53−

3.63 (m, 2H), 3.80−3.86 (m, 1H), 4.18−4.24 (m, 2H), 4.32−4.37 (m,

1H), 4.39−4.44 (m, 1H), 4.46−4.53 (m, 1H), 6.58−6.63 (m, 2H),

6.96−7.01 (m, 2H), ca. 7.0 (br s, 2H), ca. 7.4 (br s, 2H), 7.59 (br s, 1H),

7.72−7.84 (m, 5H), 7.88 (d, 1H, J = 7.9 Hz), 8.14−8.25 (m, 4H), 8.49

(br s, 2H), 8.68 (d, 1H, J = 7.4 Hz), 9.09 (br s, 1H), 9.21 (br s, 1H), 10.7

(br s, 1H), 12.5 (br s, 1H). ¹³C NMR (150 MHz, DMSO-d₆): δ 10.9,

15.1, 21.2, 22.9, 23.5, 24.1, 24.2, 24.3, 24.4, 24.6, 26.0, 28.2, 28.3, 29.1,

36.4, 37.1, 38.5, 38.6, 39.8, 40.2, 40.5, 46.8, 50.1, 50.4, 51.6, 54.1, 56.4,

59.2, 114.8, 116.9 (q, J = 298 Hz) (TFA), 127.6, 130.1, 153.9 (2 quat

carb), 155.8, 156.9, 158.7 (q, J = 32 Hz) (TFA), 168.3, 169.2, 170.7,

170.9, 171.2, 173.8. HRMS (ESI): m/z [M + 2H]²⁺calcd for

[C₄₃H₇₆N₁₄O₉]²⁺466.2954, found 466.2956. RP-HPLC (220 nm):

96% (t_R= 5.7 min, k = 6.5). C₄₃H₇₄N₁₄O₉·C₈H₄F₁₂O₈(931.14 +

456.09).

H-Asp-{N^ω-[N-(8-propanoylamino-3,6-dioxaoctyl)aminocarbonyl]}-

Arg-Val-Tyr-Ile-His-Pro-Phe-OH Tris(hydrotriﬂuoroacetate) (22).

Compound 22 was prepared from 20 (16.85 mg, 10.05 μmol) and 37

(0.9 mg, 5.26 μmol) following the procedure for the preparation of 21.

DIPEA: 15.6 mg, 121 μmol. K₂CO₃: 10 mg, 99 μmol. The product was

puriﬁed by preparative HPLC (column, Gemini-NX C18 250 mm ×

21 mm; gradient, 0−35 min; MeCN/0.1% aq TFA 10:90−34:66; t_R=

27 min). Lyophilization of the eluate aﬀorded 22 as a white ﬂuﬀy solid

(7.4 mg, 87%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.72−0.82 (m, 12H),

0.97 (t, 3H, J = 7.6 Hz), 1.01−1.10 (m, 1H), 1.31−1.39 (m, 1H), 1.43−

1.58 (m, 3H), 1.60−1.68 (m, 2H), 1.76−1.88 (m, 3H), 1.90−1.97 (m,

1H), 2.00−2.09 (m, 3H), 2.61−2.67 (m, 2H), 2.75−2.83 (m, 2H),

2.88−2.96 (m, 2H), 2.99−3.08 (m, 2H), 3.16−3.29 (m, 6H), 3.39 (t,

2H, J = 6.1 Hz), 3.43−3.49 (m, 3H), 3.49−3.54 (m, 4H), 3.62−3.67 (m,

1H), 4.11 (d, 1H, J = 6.9 Hz), 4.15 (t, 1H, J = 8.0 Hz), 4.18 (dd, 1H,

J = 8.7, 6.7 Hz), 4.34−4.41 (m, 2H), 4.41−4.45 (m, 1H), 4.47−4.53

H-{N^ω-[N-(8-amino-3,6-dioxaoctyl)aminocarbonyl]}Arg-Arg-Pro-

Tyr-Ile-Leu-OH Tetrakis(hydrotriﬂuoroacetate) (26). The peptide was

synthesized according to the general procedure using a H-Leu-2-ClTrt

resin (loading: 0.81 mmol/g). Puriﬁcation by preparative HPLC

was performed with a Gemini-NX C18 250 mm × 21 mm (gradient,

0−35 min; MeCN/0.1% aq TFA 8:92−41:59; t_R= 19 min). Lyophilization

of the eluate aﬀorded 26 as a white ﬂuﬀy solid (76.5 mg, 59%). ¹H NMR

L

DOI: 10.1021/acs.jmedchem.5b01495

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

(600 MHz, DMSO-d₆):δ 0.80 (t, 3H, J = 7.5 Hz), 0.82−0.86 (m, 6H), 0.90

(d, 3H, J = 6.6 Hz), 1.00−1.10 (m, 1H), 1.36−1.46 (m, 1H), 1.46−1.66

(m, 8H), 1.66−1.76 (m, 4H), 1.77−1.86 (m, 3H), 1.94−2.04 (m, 1H),

2.68 (dd, 1H, J = 14.0, 8.2 Hz), 2.81−2.93 (m, 1H), 2.94−3.00 (m, 2H),

3.03−3.15 (m, 2H), 3.20−3.31 (m, 4H), 3.47 (t, 2H, J = 5.6 Hz), 3.53−

3.62 (m, 8H), 3.80−3.86 (m, 1H), 4.18−4.25 (m, 2H), 4.32−4.37 (m,

1H), 4.39−4.44 (m, 1H), 4.46−4.53 (m, 1H), 6.58−6.63 (m, 2H), 6.96−

7.01 (m, 2H), ca. 7.0 (br s, 2H), ca. 7.4 (br s, 2H), 7.46 (br s, 1H), 7.72−

7.78 (m, 2H), 7.78−7.96 (m, 4H), 8.13−8.27 (m, 4H), 8.51 (br s, 2H),

8.68 (d, 1H, J = 7.4 Hz), 9.11 (br s, 1H), 9.21 (br s, 1H), 10.7 (br s, 1H),

12.5 (br s, 1H). ¹³C NMR (150 MHz, DMSO-d₆): δ 10.9, 15.1, 21.2, 22.8,

23.5, 24.1, 24.2, 24.3, 24.6, 28.2, 28.3, 29.1, 36.4, 37.1, 38.5, 39.1, 39.8, 40.2,

40.5, 46.8, 50.1, 50.4, 51.6, 54.1, 56.4, 59.2, 66.7, 68.7, 69.4, 69.6, 114.8,

116.9 (q, J = 298 Hz) (TFA), 127.6, 130.1, 153.7, 153.9, 155.8, 156.9, 158.7

(q, J = 32 Hz) (TFA), 168.3, 169.2, 170.7, 170.9, 171.2, 173.8. HRMS

(ESI): m/z [M + 2H]²⁺calcd for [C₄₅H₈₀N₁₄O₁₁]²⁺496.3060, found

496.3065. RP-HPLC (220 nm): 98% (t_R= 5.9 min, k = 6.8).

C₄₅H₇₈N₁₄O₁₁·C₈H₄F₁₂O₈(991.19 + 456.09).

(6.7 mg, 4.8 μmol) and 38 (1 mg, 1.63 μmol) following the procedure

for the preparation of 23. DIPEA: 7 mg, 54 μmol. K₂CO₃: 4 mg,

40 μmol. The product was puriﬁed by preparative HPLC (conditions as

for 23; t_R= 25 min). Lyophilization of the eluate aﬀorded 29 as a

dark-red solid (0.38 mg, 13%). HRMS (ESI): m/z [M + 3H]³⁺calcd for

[C₆₉H₁₀₅N₁₆O₁₅S]³⁺476.5883, found 476.5891. RP-HPLC (220 and

500 nm): 99% (t_R= 10.3 min, k = 12.6). C₆₉H₁₀₂N₁₆O₁₅S·C₆H₃F₉O₆

(1427.71 + 342.07).

H-[N^ω-(N-{4-[N-(6-{3,3-dimethyl-2-[5-(1,3,3-trimethyl-3H-indol-1-

ium-2-yl)penta-2,4-dien-1-ylidene]indolin-1-yl}hexanoyl)amino]-

butyl}aminocarbonyl)]Arg-Arg-Pro-Tyr-Ile-Leu-OH Triﬂuoroacetate

Tris(hydrotriﬂuoroacetate) (30). Compound 30 was prepared from

25 (8.4 mg, 6.06 μmol) and 39 (1.7 mg, 2.57 μmol) following the

procedure for the preparation of 23. DIPEA: 7 mg, 54 μmol. K₂CO₃:

4 mg, 40 μmol. The product was puriﬁed by preparative HPLC (column,

Kinetex 250 mm × 21 mm; gradient, 0−23 min; MeCN/0.1% aq TFA

10:90−38:62; 23−40 min, 38:62−46:54; t_R= 33 min). Lyophilization of

the eluate aﬀorded 30 as a dark-blue solid (1.4 mg, 28%). HRMS (ESI):

m/z [M+2H]³⁺calcd for [C₇₅H₁₁₃N₁₆O₁₀]³⁺465.9603, found 465.9611.

RP-HPLC (220 and 630 nm): 98% (t_R= 13.9 min, k = 17.3).

C₇₅H₁₁₁N₁₆O₁₀·C₈H₃F₁₂O₈(1396.78 + 455.09).

H-{N^ω-[N-(4-propanoylaminobutyl)aminocarbonyl]}Arg-Arg-Pro-

Tyr-Ile-Leu-OH Tris(hydrotriﬂuoroacetate) (27). Compound 27 was

prepared from 25 (14.2 mg, 10.22 μmol) and 37 (1.06 mg, 6.2 μmol)

following the procedure for the preparation of 21. DIPEA: 10.6 mg,

82 μmol. K₂CO₃: 10 mg, 99 μmol. The product was puriﬁed by

preparative HPLC (column, Gemini-NX C18 250 mm × 21 mm;

gradient, 0−35 min; MeCN/0.1% aq TFA 10:90−37:63; t_R= 23 min).

Lyophilization of the eluate aﬀorded 27 as a white ﬂuﬀy solid (7.1 mg,

86%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.80 (t, 3H, J = 7.4 Hz), 0.82−

0.86 (m, 6H), 0.90 (d, 3H, J = 6.6 Hz), 0.98 (t, 3H, J = 7.6 Hz), 1.01−

1.09 (m, 1H), 1.34−1.46 (m, 5H), 1.46−1.65 (m, 8H), 1.65−1.75 (m,

4H), 1.78−1.87 (m, 3H), 1.96−2.02 (m, 1H), 2.05 (q, 2H, J = 7.6 Hz),

2.67 (dd, 1H, J = 14.1, 8.2 Hz), 2.83−2.90 (m, 1H), 3.00−3.05 (m, 2H),

3.05−3.14 (m, 4H), 3.25 (br s, 2H), 3.53−3.63 (m, 2H), 3.78−3.84 (m,

1H), 4.18−4.25 (m, 2H), 4.32−4.38 (m, 1H), 4.39−4.45 (m, 1H),

4.47−4.54 (m, 1H), 6.58−6.63 (m, 2H), ca. 6.9 (br s, 2H), 6.96−7.02

(m, 2H), ca. 7.3 (br s, 2H), 7.50 (t, 1H, J = 5.1 Hz), 7.58 (t, 1H, J =

5.4 Hz), 7.71−7.78 (m, 2H), 7.90 (d, 1H, J = 7.9 Hz), 8.07−8.18 (m,

3H), 8.20 (d, 1H, J = 7.8 Hz), 8.42 (br s, 2H), 8.67 (d, 1H, J = 7.5 Hz),

9.06 (br s, 1H), 9.17 (br s, 1H), 10.1 (br s, 1H), 12.5 (br s, 1H). HRMS

(ESI): m/z [M + 2H]²⁺calcd for [C₄₆H₈₀N₁₄O₁₀]²⁺494.3085, found

494.3092. RP-HPLC (220 nm): 99% (t_R= 8.2 min, k = 9.8).

C₄₆H₇₈N₁₄O₁₀·C₆H₃F₉O₆(987.20 + 342.07).

H-Arg-{N^ω-[N-(8-amino-3,6-dioxaoctyl)aminocarbonyl]}Arg-Pro-

Tyr-Ile-Leu-OH Tetrakis(hydrotriﬂuoroacetate) (31). The peptide was

synthesized according to the general procedure using a H-Leu-2-ClTrt

resin (loading: 0.81 mmol/g). Puriﬁcation by preparative HPLC

was performed with a Gemini-NX C18 250 mm × 21 mm (gradient,

0−35 min; MeCN/0.1% aq TFA 8:92−41:59; t_R= 19 min). Lyophilization

of the eluate aﬀorded 31 as a white ﬂuﬀy solid (48.2 mg, 37%). ¹H NMR

(600MHz, DMSO-d₆): δ 0.79 (t, 3H, J = 7.4 Hz), 0.82−0.86 (m, 6H), 0.90

(d, 3H, J = 6.6 Hz), 0.99−1.09 (m, 1H), 1.36−1.45 (m, 1H), 1.45−1.48

(m, 5H), 1.48−1.77 (m, 7H), 1.77−1.87 (m, 3H), 1.94−2.04 (m, 1H),

2.68 (dd, 1H, J = 14.0, 8.2 Hz), 2.81−2.92 (m, 1H), 2.94−3.00 (m, 2H),

3.06−3.14 (m, 2H), 3.17−3.31 (m, 4H), 3.46 (t, 2H, J = 5.6 Hz), 3.53−

3.62 (m, 8H), 3.78−3.85 (m, 1H), 4.18−4.25 (m, 2H), 4.31−4.37 (m,

1H), 4.39−4.45 (m, 1H), 4.48−4.55 (m, 1H), 6.58−6.63 (m, 2H), 6.96−

7.01 (m, 2H), ca. 7.1 (br s, 2H), ca. 7.4 (br s, 2H), 7.46 (br s, 1H), 7.75 (d,

1H, J = 8.9 Hz), 7.80 (t, 1H, J = 5.8 Hz), 7.81−7.98 (m, 4H), 8.12−8.26

(m, 4H), 8.52 (br s, 2H), 8.69 (d, 1H, J = 7.4 Hz), 9.08 (br s, 1H), 9.21 (br

s, 1H), 10.7 (br s, 1H), 12.5 (br s, 1H). ¹³C NMR (150 MHz, DMSO-d₆):

δ 10.9, 15.1, 21.2, 22.8, 24.1 (3 carb), 24.2, 24.3, 28.2, 28.4, 29.1, 36.4, 37.1,

38.6, 39.1, 39.8, 40.1, 40.6, 46.8, 50.1, 50.4, 51.7, 54.1, 56.3, 59.2, 66.7, 68.7,

69.4, 69.6, 114.8, 116.8 (q, J = 297 Hz) (TFA), 127.6, 130.1, 153.7, 153.9,

155.8, 156.8, 158.7 (q, J = 32 Hz) (TFA), 168.3, 169.2, 170.7, 170.9, 171.2,

173.8. HRMS (ESI): m/z [M + 2H]²⁺calcd for [C₄₅H₈₀N₁₄O₁₁]²⁺

496.3060, found 496.3063. RP-HPLC (220 nm): 99% (t_R= 6.3 min,

k = 7.3). C₄₅H₇₈N₁₄O₁₁·C₈H₄F₁₂O₈(991.19 + 456.09).

H-{N^ω-[N-(8-propanoylamino-3,6-dioxaoctyl)aminocarbonyl]}-

Arg-Arg-Pro-Tyr-Ile-Leu-OH Tris(hydrotriﬂuoroacetate) (28). Com-

pound 28 was prepared from 26 (12.9 mg, 8.91 μmol) and 37 (1.1 mg,

6.43 μmol) using the procedure for the preparation of 21, but DMF/

water (4:6 v/v) (200 μL) was used as solvent. DIPEA: 9.0 mg, 70 μmol.

K₂CO₃: 8.6 mg, 85 μmol. The product was puriﬁed by preparative

HPLC (column, Gemini-NX C18 250 mm × 21 mm; gradient,

0−35 min; MeCN/0.1% aqTFA 8:92−43:57; t_R= 21 min). Lyophilization

of the eluate aﬀorded 28 as a white ﬂuﬀy solid (7.7 mg, 86%). ¹H NMR

(600 MHz, DMSO-d₆):δ 0.80 (t, 3H, J = 7.4 Hz), 0.82−0.86 (m, 6H), 0.90

(d, 3H, J = 6.6 Hz), 0.97 (t, 3H, J = 7.6 Hz), 1.00−1.09 (m, 1H), 1.37−1.46

(m, 1H), 1.46−1.65 (m, 8H), 1.65−1.76 (m, 4H), 1.78−1.88 (m, 3H),

1.96−2.04 (m, 1H), 2.06 (q, 2H, J = 7.6 Hz), 2.67 (dd, 1H, J = 14.1,

8.2 Hz), 2.83−2.90 (m, 1H), 3.04−3.15 (m, 2H), 3.16−3.21 (m, 2H),

3.21−3.31 (m, 4H), 3.39 (t, 2H, J = 6.1 Hz), 3.46 (t, 2H, J = 5.4 Hz), 3.49−

3.54 (m, 4H), 3.54−3.63 (m, 2H), 3.78−3.84 (m, 1H), 4.19−4.25 (m,

2H), 4.32−4.37 (m, 1H), 4.39−4.44 (m, 1H), 4.47−4.54 (m, 1H), 6.58−

6.63 (m, 2H), ca. 6.9 (br s, 2H), 6.97−7.03 (m, 2H), ca. 7.3 (br s, 2H), 7.49

(t, 1H, J = 5.4 Hz), 7.57 (t, 1H, J = 5.4 Hz), 7.75 (d, 1H, J = 8.9 Hz), 7.79

(t, 1H, J = 5.4 Hz), 7.90 (d, 1H, J = 7.9 Hz), 8.08−8.18 (m, 3H), 8.20 (d,

1H, J = 7.7 Hz), 8.44 (br s, 2H), 8.67 (d, 1H, J = 7.4 Hz), 9.07 (br s, 1H),

9.17 (br s, 1H), 10.1 (br s, 1H), 12.5 (br s, 1H). HRMS (ESI): m/z [M +

2H]²⁺calcd for [C₄₈H₈₄N₁₄O₁₂]²⁺524.3191, found 524.3196. RP-HPLC

(220 nm): 99% (t_R= 8.2 min, k = 9.8). C₄₈H₈₂N₁₄O₁₂·C₆H₃F₉O₆

(1047.3 + 342.07).

H-Arg-{N^ω-[N-(8-propanoylamino-3,6-dioxaoctyl)-

aminocarbonyl]}Arg-Pro-Tyr-Ile-Leu-OH Tris(hydrotriﬂuoroacetate)

(32). Compound 32 was prepared from 31 (12.5 mg, 8.64 μmol) and 37

(1.05 mg, 6.13 μmol) using the procedure for the preparation of 21, but

DMF/water (4:6 v/v) (200 μL) was used as solvent. DIPEA: 9.0 mg,

70 μmol. K₂CO₃: 8.6 mg, 85 μmol. The product was puriﬁed by

preparative HPLC (column, Gemini-NX C18 250 mm × 21 mm;

gradient, 0−35 min; MeCN/0.1% aq TFA 8:92−43:57; t_R= 21 min).

Lyophilization of the eluate aﬀorded 32 as a white ﬂuﬀy solid (7.6 mg,

89%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.80 (t, 3H, J = 7.4 Hz), 0.82−

0.86 (m, 6H), 0.90 (d, 3H, J = 6.6 Hz), 0.97 (t, 3H, J = 7.6 Hz), 1.01−

1.09 (m, 1H), 1.36−1.44 (m, 1H), 1.44−1.58 (m, 5H), 1.58−1.77 (m,

7H), 1.78−1.88 (m, 3H), 1.96−2.04 (m, 1H), 2.06 (q, 2H, J = 7.6 Hz),

2.67 (dd, 1H, J = 14.0, 8.1 Hz), 2.83−2.90 (m, 1H), 3.06−3.13 (m, 2H),

3.16−3.20 (m, 2H), 3.21−3.30 (m, 4H), 3.39 (t, 2H, J = 6.1 Hz), 3.46 (t,

2H, J = 5.3 Hz), 3.49−3.54 (m, 4H), 3.54−3.62 (m, 2H), 3.77−3.83 (m,

1H), 4.19−4.25 (m, 2H), 4.32−4.37 (m, 1H), 4.40−4.46 (m, 1H),

4.49−4.54 (m, 1H), 6.58−6.63 (m, 2H), 6.96−7.02 (m, 2H), ca. 7.0 (br

s, 2H), ca. 7.3 (br s, 2H), 7.51 (br s, 1H), 7.58 (t, 1H, J = 5.9 Hz), 7.75 (d,

1H, J = 9.0 Hz), 7.79 (t, 1H, J = 5.1 Hz), 7.89 (d, 1H, J = 7.5 Hz), 8.07−

8.16 (m, 3H), 8.19 (d, 1H, J = 7.7 Hz), 8.44 (br s, 2H), 8.67 (d, 1H, J =

7.5 Hz), 9.02 (br s, 1H), 9.17 (br s, 1H), 9.96 (br s, 1H), 12.5 (br s, 1H).

HRMS (ESI): m/z [M + 2H]²⁺calcd for [C₄₈H₈₄N₁₄O₁₂]²⁺524.3191,

H-{N^ω-[N-(4-{N-[6-(6-{2-[7-(diethylamino)-2-oxo-2H-chromen-3-

yl]vinyl}-3-sulfonato-pyridin-1-ium-1-yl)hexanoyl]amino}butyl)-

a m i n oc a r bo n y l] } A rg - A r g - P r o - T y r - Il e - L e u - O H Tr i s -

(hydrotriﬂuoroacetate) (29). The compound was prepared from 25

M

DOI: 10.1021/acs.jmedchem.5b01495

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

found 524.3199. RP-HPLC (220 nm): 99% (t_R= 8.3 min, k = 9.9).

C₄₈H₈₂N₁₄O₁₂·C₆H₃F₉O₆(1047.3 + 342.07).

Lyophilization of the eluate aﬀorded 35 as a white ﬂuﬀy solid (58 mg,

34%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.84 (d, 3H, J = 6.5 Hz), 0.87

(d, 3H, J = 6.6 Hz), 1.14−1.22 (m, 6H), 1.34−1.57 (m, 9H), 1.57−1.67

(m, 2H), 1.70−1.90 (m, 5H), 1.90−1.96 (m, 2H), 2.00−2.07 (m, 1H),

2.09−2.17 (m, 2H), 2.32−2.38 (m, 2H), 2.60−2.67 (m, 1H), 2.70−2.85

(m, 3H), 2.92−3.06 (m, 3H), 3.08−3.15 (m, 3H), 3.15−3.22 (m, 2H),

3.49−3.56 (m, 2H), 3.56−3.65 (m, 2H), 3.72−3.79 (m, 1H), 4.09−4.14

(m, 1H), 4.14−4.19 (m, 1H), 4.22−4.27 (m, 1H), 4.27−4.37 (m, 3H),

4.37−4.43 (m, 1H), 4.45−4.53 (m, 2H), 4.59−4.65 (m, 1H), 6.61−6.66

(m, 2H), 6.84 (s, 1H), 6.93−6.98 (m, 1H), 7.02−7.09 (m, 3H), 7.13 (s,

1H), 7.15−7.19 (m, 2H), 7.19−7.26 (m, 4H), 7.28 (s, 1H), 7.30−7.35

(m, 2H), 7.58 (d, 1H, J = 7.9 Hz), 7.61 (br s, 1H), 7.75 (br s, 3H), 7.81−

7.87 (m, 2H), 7.95 (d, 1H, J = 7.3 Hz), 7.98 (d, 1H, J = 8.2 Hz), 8.01 (d,

1H, J = 7.0 Hz), 8.02−8.09 (m, 4H), 8.19 (d, 1H, J = 7.0 Hz), 8.37 (d,

1H, J = 7.7 Hz), 8.44 (br s, 2H), 8.48 (d, 1H, J = 7.9 Hz), 8.97 (br s, 1H),

9.21 (br s, 1H), 10.4 (br s, 1H), 10.7 (s, 1H), 12.3 (br s, 1H). ¹³C NMR

(150 MHz, DMSO-d₆): δ 16.7, 17.9, 21.4, 23.2, 24.1, 24.3, 24.4, 24.5,

26.0, 26.6, 27.2, 27.6, 28.7, 28.9, 29.0, 31.4, 36.5, 37.4, 38.5, 38.6, 40.4,

40.5, 46.3, 46.7, 47.9, 51.0, 51.4, 52.4, 52.8, 53.4, 53.8, 54.6, 54.8, 59.7,

61.7, 109.9, 111.2, 114.9, 115.6 (TFA), 117.6 (TFA), 118.2, 118.4,

120.8, 123.5, 126.2, 127.4, 127.6, 128.0, 129.1, 130.1, 136.0, 137.8, 153.7,

153.9, 155.9, 158.4 (q, J = 33 Hz) (TFA), 168.2, 170.0, 170.9 (2 quat

carb), 171.0, 171.4, 171.6, 171.7, 171.9, 172.8, 173.6, 174.1. HRMS

(ESI): m/z [M + 2H]²⁺calcd for [C₇₀H₁₀₃N₁₉O₁₇]²⁺740.8884, found

740.8890. RP-HPLC (220 nm): 99% (t_R= 8.9 min, k = 10.7).

C₇₀H₁₀₁N₁₉O₁₇·C₆H₃F₉O₆(1480.7 + 342.07).

At Position Arg²N^ω-[N-(4-Aminobutyl)aminocarbonyl]-monosub-

stituted Octanedioyl Bis(tyrosyl-arginyl-leucyl-arginyl-tyrosinamide)

Pentakis(hydrotriﬂuoroacetate) (33). Compound 33 was synthesized

on a Sieber resin (loading: 0.61 mmol/g). Fmoc-amino acids and

arginine building block 10 were coupled according to the general

procedure in the order Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH,

Fmoc-Leu-OH, 10, and Fmoc-Tyr(tBu)-OH followed by the coupling

of building block 43 (50 mg, 32 μmol) using HBTU/HOBt/DIPEA

(1/1/2 equiv to 43) and anhydrous DMF (800 μL) as solvent (35 °C,

16 h). The peptide was cleaved from the resin according to the general

procedure. The residue obtained after lyophilization was dissolved in

TFA/water (95:5 v/v) (2 mL), and the mixture was stirred at rt for 2.5 h

and poured into ice-cold diethyl ether (15 mL), causing the precipitation

of the crude product, which was separated from the organic solvent by

centrifugation and decantation. Puriﬁcation by preparative HPLC

(column, Kinetex-XB C18 250 mm × 21 mm; gradient, 0−25 min;

MeCN/0.1% aq TFA 13:87−43:57 (ﬂow rate: 15 instead 20 mL/min);

t_R= 16 min) aﬀorded 33 as a white ﬂuﬀy solid (22 mg, 41%). ¹H NMR

(600 MHz, DMSO-d₆): δ 0.83 (d, 6H, J = 6.5 Hz), 0.87 (d, 6H, J =

6.5 Hz), 1.03 (br s, 4H), 1.30 (br s, 4H), 1.36−1.57 (m, 20H), 1.57−

1.75 (m, 6H), 1.94−2.06 (m, 4H), 2.57−2.65 (m, 2H), 2.68−2.75 (m,

2H), 2.75−2.82 (m, 2H), 2.82−2.90 (m, 4H), 3.02−3.14 (m, 8H), 3.24

(br s, 2H), 4.17−4.23 (m, 2H), 4.24−4.36 (m, 6H), 4.39−4.46 (m, 2H),

6.59−6.65 (m, 8H), ca. 6.95 (br s, 6H), 6.95−6.99 (m, 4H), 6.99−7.03

(m, 4H), 7.05 (s, 2H), ca. 7.3 (br s, 6H), 7.33−7.39 (m, 2H), 7.52−7.63

(m, 4H), 7.67−7.79 (m, 4H), 7.84−7.95 (m, 4H), 7.99−8.08 (m, 2H),

8.17 (d, 1H, J = 7.8 Hz), 8.20 (d, 1H, J = 7.8 Hz), 8.45 (br s, 2H), 9.00 (s,

1H), 9.17 (br s, 4H), 10.38 (s, 1H). ¹³C NMR (150 MHz, DMSO-d₆): δ

21.4 (2 carb), 23.1, 23.1, 24.1 (2 carb), 24.4, 24.9 (3 carb), 25.0, 25.1

(2 carb), 26.0, 28.4 (2 carb), 28.9, 29.0 (3 carb), 35.2 (2 carb), 36.6

(2 carb), 36.8 (2 carb), 38.5, 38.6, 40.4 (3 carb), 40.5, 40.7 (2 carb), 51.0

(2 carb), 50.1 (2 carb), 52.26, 52.29, 53.89, 53.92, 54.1, 54.2, 114.8

(4 carb), 114.9 (4 carb), 115.6 (TFA), 117.6 (TFA), 127.5 (2 quat carb),

127.89, 127.91, 130.02 (4 carb), 130.04 (4 carb), 153.7, 153.8, 155.75

(2 quat carb), 155.82 (2 quat carb), 156.71 (2 quat carb), 156.73, 158.5

(q, J = 33 Hz) (TFA), 170.81, 170.84, 171.08, 171.13, 171.8, 171.9,

172.0 (2 quat carb), 172.31, 172.34, 172.7 (2 quat carb). HRMS (ESI):

m/z [M + 4H]⁴⁺calcd for [C₈₅H₁₃₆N₂₆O₁₇]⁴⁺448.2639, found

448.2652. RP-HPLC (220 nm): 96%, (t_R= 19.3 min, k = 5.7).

C₈₅H₁₃₂N₂₆O₁₇·C₁₀H₅F₁₅O₁₀(1790.16 + 570.11).

H-Glu-Tyr-Trp-Ser-Leu-Ala-Ala-Pro-Gln-{N^ω-[N-(4-

propanoylaminobutyl)aminocarbonyl]}Arg-Phe-NH₂Tris-

(hydrotriﬂuoroacetate) (36). Compound 36 was prepared from 35

(13.8 mg, 7.57 μmol) and 37 (0.91 mg, 5.32 μmol) using the procedure

for the preparation of 21, but DMF (160 μL) was used as solvent and

K₂CO₃was omitted. DIPEA: 12 mg, 92.9 μmol. The product was

puriﬁed by preparative HPLC (column, Kinetex-XB C18 250 mm ×

21 mm; gradient, 0−30 min; MeCN/0.1% aq TFA 15:85−38:62; t_R=

21.5 min). Lyophilization of the eluate aﬀorded 36 as a white ﬂuﬀy solid

(7.8 mg, 83%). ¹H NMR (600 MHz, DMSO-d₆): δ (ppm) 0.84 (d, 3H,

J = 6.5 Hz), 0.87 (d, 3H, J = 6.6 Hz), 0.98 (t, 3H, J = 7.6 Hz), 1.15−1.21

(m, 6H), 1.35−1.46 (m, 7H), 1.46−1.54 (m, 2H), 1.56−1.67 (m, 2H),

1.70−1.90 (m, 5H), 1.90−1.96 (m, 2H), 2.00−2.08 (m, 3H), 2.09−2.16

(m, 2H), 2.32−2.38 (m, 2H), 2.61−2.67 (m, 1H), 2.78−2.84 (m, 1H),

2.92−3.06 (m, 5H), 3.06−3.15 (m, 3H), 3.15−3.21 (m, 2H), 3.50−3.55

(m, 2H), 3.56−3.63 (m, 2H), 3.72−3.77 (m, 1H), 4.09−4.15 (m, 1H),

4.15−4.21 (m, 1H), 4.23−4.27 (m, 1H), 4.27−4.36 (m, 3H), 4.38−4.44

(m, 1H), 4.45−4.53 (m, 2H), 4.60−4.65 (m, 1H), 5.04 (br s, 1H), 6.61−

6.66 (m, 2H), 6.84 (s, 1H), 6.94−6.98 (m, 1H), 7.02−7.09 (m, 3H),

7.13 (s, 1H), 7.14−7.19 (m, 2H), 7.19−7.26 (m, 4H), 7.27 (s, 1H), 7.31

(d, 1H, J = 8.1 Hz), 7.33 (s, 1H), 7.49 (br s, 1H), 7.59 (d, 1H, J = 7.8 Hz),

7.73 (t, 1H, J = 5.2 Hz), 7.81−7.87 (m, 2H), 7.95 (d, 1H, J = 7.3 Hz),

7.97 (d, 1H, J = 8.5 Hz), 7.98−8.07 (m, 5H), 8.16 (d, 1H, J = 7.1 Hz),

8.36 (d, 1H, J = 7.6 Hz), 8.38 (br s, 2H), 8.46 (d, 1H, J = 7.7 Hz), 8.86

(br s, 1H), 9.18 (br s, 1H), 9.58 (br s, 1H), 10.7 (br s, 1H), 12.3 (br s,

1H). HRMS (ESI): m/z [M + 2H]²⁺calcd for [C₇₃H₁₀₇N₁₉O₁₈]²⁺

768.9015, found 768.9024. RP-HPLC (220 nm): 99% (t_R= 10.8 min,

k = 13.2). C₇₃H₁₀₅N₁₉O₁₈·C₄H₂F₆O₄(1536.7 + 228.05).

At Position Arg²N^ω-[N-(4-Propanoylaminobutyl)aminocarbonyl]-

monosubstituted Octanedioyl Bis(tyrosyl-arginyl-leucyl-arginyl-ty-

rosinamide) Tetrakis(hydrotriﬂuoroacetate) (34). Compound 33 (10

mg, 4.24 μmol) was dissolved in anhydrous DMF/DIPEA (99:1 v/v)

(600 μL). Succinimidyl propionate (37) (0.8 mg, 4.7 μmol) was added,

and the mixture was stirred at rt for 2 h. Then 0.2% aq TFA (9 mL) was

added, and the product was puriﬁed by preparative HPLC (column,

Kinetex-XB C18 250 mm × 21 mm; gradient, 0−25 min; MeCN/0.1%

aq TFA 12:88−43:57 (ﬂow rate: 15 instead 20 mL/min); t_R= 18 min).

Lyophilization of the eluate aﬀorded 34 as a white ﬂuﬀy solid (5.7 mg,

58%). ¹H NMR (600 MHz, DMSO-d₆): δ 0.83 (d, 6H, J = 6.5 Hz), 0.87

(d, 6H, J = 6.6 Hz), 0.98 (t, 3H, 7.7 Hz), 1.03 (br s, 4H), 1.30 (br s, 4H),

1.34−1.57 (m, 20H), 1.57−1.75 (m, 6H), 1.94−2.07 (m, 6H), 2.57−

2.64 (m, 2H), 2.69−2.75 (m, 2H), 2.82−2.88 (m, 4H), 2.99−3.13 (m,

10H), 3.23 (br s, 2H), 4.17−4.24 (m, 2H), 4.24−4.36 (m, 6H), 4.40−

4.46 (m, 2H), 6.59−6.65 (m, 8H), ca. 6.85 (br s, 6H), 6.95−6.99 (m,

4H), 6.99−7.03 (m, 4H), 7.05 (s, 2H), ca. 7.3 (br s, 6H), 7.37 (s, 2H),

7.46−7.53 (m, 4H), 7.69−7.77 (m, 3H), 7.84−7.93 (m, 4H), 8.03 (m,

2H), 8.18 (m, 2H), 8.40 (br s, 2H), 8.96 (s, 1H), 9.16 (s, 4H), 9.87 (s,

1H). HRMS (ESI): m/z [M + 4H]⁴⁺calcd for [C₈₈H₁₄₀N₂₆O₁₈]⁴⁺

462.2704, found 462.2717. RP-HPLC (220 nm): 96%, (t_R= 21.1 min,

k = 6.3). C₈₈H₁₃₆N₂₆O₁₈·C₈H₄F₁₂O₈(1846.22 + 456.09).

Preparation of [³H]21 and [³H]27. The radiolabeled peptides [³H]

21 and [³H]27 were prepared by [³H]propionylation of the precursor

peptides 19 and 25, respectively. A solution of succinimidyl [³H]-

propionate (speciﬁc activity: 80 Ci/mmol, purchased from American

Radiolabeled Chemicals Inc. (St. Louis, MO) via Hartman Analytics

(Braunschweig, Germany)) (3.2 mCi, 6.8 μg, 40 nmol, and 4.8 mCi,

10.3 μg, 60 nmol, respectively) in EtOAc (0.4 and 0.6 mL, respectively)

was transferred from the delivered ampule into a 1.5 mL reaction vessel

with screw cap, and the solvent was removed in a vacuum concentrator

(about 30 min at ca. 30 °C). Immediately, a solution of the precursor

peptide (19, 0.8 mg, 495 nmol; 25, 0.7 mg, 505 nmol) and DIPEA

(0.41 mg, 0.55 μL, 3.2 μmol) in DMF/250 mM bicarbonate buﬀer pH

9.4 (1:1 v/v) (60 μL) was added and the vessel was shaken at rt for 3 h.

The mixture was acidiﬁed by the addition of 2% aq TFA (80 μL)

followed by the addition of acetonitrile/water (10:90 v/v) (80 μL).

[³H]21 and [³H]27 were puriﬁed using a HPLC system from Waters

H-Glu-Tyr-Trp-Ser-Leu-Ala-Ala-Pro-Gln-{N^ω-[N-(4-aminobutyl)-

aminocarbonyl]}Arg-Phe-NH₂Tris(hydrotriﬂuoroacetate) (35). The

peptide was synthesized according to the general procedure using a Rink

amide resin (loading: 0.73 mmol/g). Puriﬁcation by preparative HPLC

was performed with a Kinetex-XB C18 250 mm × 21 mm (gradient,

0−30 min; MeCN/0.1% aq TFA 15:85−38:62; t_R= 18.5 min).

N

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(Eschborn, Germany) consisting of two pumps 510, a pump control

module, a 486 UV/vis detector, and a Flow-one beta series A-500

radiodetector (Packard, Meriden, CT). A YMC-Triart C18, 5 μm,

250 mm × 6.0 mm (YMC Europe) served as stationary phase at a ﬂow

rate of 1.4 mL/min. Mixtures of acetonitrile supplemented with 0.04%

TFA (A) and 0.05% aq TFA (B) were used as mobile phase. The

following linear gradient was applied: 0−20 min, A/B 10:90−37:63;

20−22 min, 37:63−95:5; 22−32 min, 95:5. For each radiolabeled

peptide two HPLC runs (UV detection: 225 nm; no radiometric

detection) were performed. The products (t_R= 19.9 and 19.2 min,

respectively) were collected in 2 mL reaction vessels with screw cap by

combining the two eluates of each product in one vessel. The volumes of

the combined product fractions were reduced by evaporation to 300 and

600 μL, respectively, and ethanol containing 200 μM TFA ([³H]21,

200 μL; [³H]27, 400 μL) was added. The solutions were transferred into

3 mL borosilicate glass vials with conical bottom (Wheaton NextGen

3 mL V-vials). The 2 mL reaction vessels were rinsed twice with EtOH/

water (40:60 v/v), and the washings were transferred to the 3 mL glass

vials to reach volumes of the tentative stocks of 1270 μL ([³H]21) or

2120 μL ([³H]27)). For the quantiﬁcation of the radioligands, a four-

point calibration was performed with 21 and 27 (0.3, 0.75, 1.5, and

2.5 μM; injection volume, 100 μL; UV detection, 225 nm) using the

above-mentioned HPLC system from Waters and a Luna C18(2), 3 μm,

150 mm × 4.6 mm (Phenomenex) as stationary phase at a ﬂow rate of

0.8 mL/min. Mixtures of acetonitrile supplemented with 0.04% TFA

(A) and 0.05% aq TFA (B) were used as mobile phase. The following

linear gradient was applied: 0−20 min, A/B 14:86−30:70; 20−25 min,

30:70−95:5; 25−32 min, 95:5 (retention times: 18.9 and 17.3 min,

respectively). Aliquots ([³H]21, 4 μL; [³H]27, 4.43 μL) of the

tentative stock solutions were added to acetonitrile/0.05% aq TFA

(14:86 v/v) (126 and 125.6 μL, respectively), 100 μL of the resulting

solutions were analyzed by HPLC, and ﬁve times 3 μL were counted in

3 mL of scintillator (Rotiszint eco plus) with a LS 6500 liquid

scintillation counter (Beckmann-Coulter, Munich, Germany). These

analyses were performed twice. The molarities of the tentative stock

solutions were calculated from the mean of the peak areas and the linear

calibration curve obtained from the peak areas of the standards. To

determine the radiochemical purity and to proof the identity, a 0.5 μM

solution of the radiolabeled peptide (100 μL), spiked with the “cold”

analogue 21 or 27 (20 μM), was analyzed by RP-HPLC using the

column, ﬂow rate, gradient, and UV detection as for the quantiﬁcation

and additionally radiometric detection (ﬂow rate of the liquid scintillator

(acetonitrile/Rotiszint eco plus (Carl Roth) 15:85 v/v): 4.0 mL/min).

The radiochemical purities amounted to 99% and 98%, respectively (see

Figure 6 and Figure S21A (Supporting Information)). This analysis was

repeated after 12 months of storage at −20 °C (see Supporting

Information, Figure S21B). Calculated speciﬁc activity: 1.74 TBq/mmol

(47.01 Ci/mmol). The activity concentration was adjusted to 27.75

MBq/mL (0.75 mCi/mL) by addition of EtOH/water (40:60 v/v)

yielding a molarity of 15.9 μM (ﬁnal volumes: [³H]21, 2.528 mL;

[³H]27, 4.156 mL). Storage conditions: −20 °C. Chemical yields

(tris(hydrotriﬂuoroacetate) of [³H]21 and [³H]27): 62.6 μg, 40.2 nmol,

and 87.8 μg, 66.1 nmol, respectively. Radiochemical yields: [³H]21,

1.896 mCi (70.16 MBq), 59%; [³H]27, 3.117 mCi (115.3 MBq), 65%.

Investigation of the Chemical Stability. The chemical stability of

peptides 19−21, 23−25, 27, 29, 30, 35, and 36 was investigated in PBS

at 21 °C. The incubation of compounds 19−21, 25, 27, 35, and 36 was

started by addition of a 5 mM solution in EtOH/20 mM HCl (40:60 v/v)

(8.8 μL) to 2 mM autoclaved PBS pH 7.0 (211.2 μL) to give a ﬁnal

concentration of 200 μM. After 0, 4, 24, and 48 h an aliquot (40 μL) was

taken and diluted with acetonitrile/1% aq TFA (2:8 v/v) (40 μL).

An aliquot (20 μL) of the resulting solution (pH < 3) was analyzed

by RP-HPLC (analytical HPLC system and conditions see general

experimental conditions).

the resulting solution was analyzed by RP-HPLC (HPLC conditions see

General Experimental Conditions).

The stability of the NT(8−13) derivative 44 was investigated as

described above for 19−21, 25, 27, 35 and 36 in PBS pH 7.0 and,

additionally, in a 10 mM autoclaved phosphate buﬀer pH 8.0 (results

shown in Supporting Information, Figure S18). The 5 mM stock

solution of 44 was prepared in MeCN/0.05% aq TFA (1:1 v/v).

Peptide Stability under Assay Conditions. The stability under

assay conditions was investigated for AngII and derivatives 21 and 22 in

the presence of CHO-hAT₁cells, and for NT(8−13) and derivatives 27

and 28 in the presence of HT-29 cells at 22 °C using the same binding

buﬀer (Dulbecco’s PBS pH 7.4 supplemented with 1% BSA and 100 μg/

mL bacitracin) as for radioligand binding studies performed with [³H]

21 and [³H]27 (see below). One day prior to the experiment, cells were

seeded into Primaria 24-well plates (Corning Life Sciences, Oneonta,

NY). On the day of the experiment, cell conﬂuency was 90−100%. The

culture medium was removed by suction, the cells were washed with PBS

(500 μL), and binding buﬀer (250 μL) containing AngII, NT(8−13), 21,

22, 27, or 28 (50 μM) was added. The plates were gently shaken during

incubation at 22 °C for 30 s (designated 0 h), 2 or 6 h. After incubation,

200 μL were taken from the wells, transferred into 1.5 mL reaction

vessels and cooled on ice for 5−10 min. Acetonitrile (cooled to −18 °C)

(470 μL) was added carefully. To precipitate protein, the reaction vessels

were allowed to warm up to rt under vortexing for 5 min. The vessels were

centrifuged at 16,000 g at 10 °C for 10 min and 640 μL of the supernatants

were transferred into 1.5 mL reaction vessels containing 15 μL of 10% aq

TFA. The solvent was removed in a vacuum concentrator (Speed-Vac

Plus SC110A Savant, Thermo Electron Corporation) at 40 °C and the dry

residue was redissolved in90 μL ofacetonitrile/0.05%aqTFA (10:90 v/v)

under vortexing (2 min) and sonication (8 min). The solutions were

ﬁltered with 0.2-μm syringe ﬁlters (4 mm, regenerated cellulose,

Phenomenex), and 20 μL of the ﬁltrates were analyzed by RP-HPLC

(HPLC conditions see General Experimental Conditions; additionally to

the UV detection at 220 nm ﬂuorescence detection was performed at

ex./em. 275/305 nm). The experiments were performed in triplicate. The

recoveries oftheinvestigated compounds(mean values rangingfrom76 to

86%) were fairly constant.

Fluorescence Spectroscopy and Determination of Quantum

Yields. Fluorescence quantum yields of the ﬂuorescently labeled

peptides 23, 24, 29, 30 and reference compound 47 were determined in

PBS (pH 7.4) and PBS containing 1% BSA with a Cary Eclipse spectro-

ﬂuorimeter and a Cary 100 UV/vis photometer (Varian Inc., Mulgrave,

Victoria, Australia) according to a previously described procedure using

acryl cuvettes (10 mm × 10 mm, Sarstedt, ref. 67.755) and cresyl violet

perchlorate as quantum yield standard.⁴²The following concentrations,

at which the absorbances at the excitation wavelengths were between

0.09 and 0.15, were used: 23, 29: 3 μM in PBS and 2.5 μM in PBS + 1%

BSA; 24, 30, 47: 1.6 μM in PBS and 1.3 μM in PBS + 1% BSA. Emission

spectra were recorded at the slit adjustments (ex./em.) 10/5 nm and

10/10 nm, and the quantum yields obtained for these slit combinations

were averaged.

Cell Culture. All cell lines were maintained in 25 or 75 cm²culture

ﬂasks (Sarstedt) in a water saturated atmosphere (95% water, 5% CO₂)

at 37 °C. CHO-hAT₁-G_α16-mtAEQ cells^79,80were cultured in Ham’s

F12 medium (Sigma) supplemented with 10% fetal calf serum

(Biochrom, Berlin, Germany), G418 (Biochrom) (1000 μg/mL) and

zeocin (InvivoGen, San Diego, CA) (250 μg/mL). Rat mesangial cells

(kindly provided by Prof. Dr. A. Kurtz, Department of Physiology,

University of Regensburg)⁸¹were maintained in antibiotic-free RPMI-

1640 medium (Sigma) containing 10% fetal calf serum (FCS) and

bovine insulin (Sigma insulin I-5500) (660 U/L). HT-29 colon

carcinoma cells (ATCC no. HTB-38) were cultured in antibiotic-free

McCoy’s 5A medium supplemented with 5% FCS. CHO-hNTS₁cells²³

were maintained in DMEM/HAM’s F12 medium (1:1) supplemented

with 10% FCS and hygromycin B (250 μg/mL). CHO-hY₄-G_qi5-mtAEQ

cells were grown as previously reported.⁶

The incubation of compounds 23, 24, 29 and 30 was started by

addition of a 1 mM solution in DMSO (15.6 μL) (23, 24) or DMSO/

water (20:80 v/v) (19.5 μL) (29, 30) to 2 mM PBS (114.4 and 110.5 μL,

respectively) to give ﬁnal concentrations of 120 and 150 μM, respec-

tively. After 0, 3, 6, and 24 h an aliquot (30 μL) was taken and diluted

with acetonitrile/1% aq TFA (2:8 v/v) (30 μL). An aliquot (20 μL) of

Immunoblotting. HT-29, HEK-hNTS₂and CHO-hNTS₁cells

were grown in 177 cm²cell culture dishes (150 × 20 mm, Sarstedt) to

80% conﬂuency. Cells were rinsed twice with ice-cold PBS and scraped

from the dishes in a 50 mM Tris buﬀer pH 7.4 supplemented with 1 mM

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EDTA and protease inhibitors (SIGMAFAST Protease Inhibitor

cocktail tablets, Sigma), in the following referred to as “Tris buﬀer”

(20 mL per cell culture dish). After centrifugation (400 g, 5 min, at 4 °C)

the cells were resuspended in Tris buﬀer (20 mL per culture dish) and

homogenized using a Ultra-Turrax (IKA Werk, Staufen, Germany)

(20,000 rpm, 5 × 5 s) under cooling in an ice bath followed by

centrifugation (50,000g, 15 min, at 4 °C). The pellets were resuspended

in Tris buﬀer (0.5 mL per culture dish) and treated with a Potter-

Elvehjem homogenizer. The protein concentration was determined

by the Bradford method (BioRad, Munich, Germany) and adjusted to

1600 μg/mL for each cell homogenate. Fifteen μL of cell homogenate

were diluted with 15 μL of sample buﬀer pH 8 (water (27 mL), 1 M Tris

in water (1.5 mL), glycine (6 mL), urea (28.8 g), SDS (1.5 g),

dithiothreitol (1.8 g), bromophenol blue (6 mg)), and the mixtures were

incubated under shaking at 30 °C for 1 h. Samples (15 μL) were loaded

onto SDS gradient gels (12 wells, Novex 8−16% Tris-Glycine Gel,

Fisher Scientiﬁc). SDS-PAGE was performed at 130 V, (ca. 1.5 h, buﬀer:

Tris (3 g), glycine (14.4 g), SDS (1 g) ad 1 L of water, pH 8.3). A ready-

to-use stained protein standard (Precision Plus Protein Dual Color

Standard (BioRad) (6 μL) served as molecular weight marker. All

samples were run in duplicate. The separated proteins were transferred

to a nitrocellulose membrane (pore size 0.2 μm, Peqlab Biotechnologie,

Erlangen, Germany) at 200 mA for 45 min (blotting buﬀer: Tris (0.3 g),

glycine (1.4 g) ad 100 mL of water/MeOH (8:2 v/v)) followed by

blocking with 5% skimmed milk powder in Tris-buﬀered saline (20 mM

Tris and 150 mM NaCl in water, pH 7.6) containing 1% Tween-20

(in the following referred to as “TBST”) at rt for 1.5 h. The membrane

was cut in half. One half of the membrane was incubated in TBST + 5%

milk powder (7 mL), containing the primary antibody (Anti-

Neurotensin Receptor 2 Antibody (AB15134), Merck Chemicals,

Darmstadt, Germany) at a concentration of 1.0 μg/mL, under rotation

at 4 °C overnight, the second half was treated in the same manner, but

the primary antibody was omitted. The membrane was washed with

TBST (5 × 5 min, rt) followed by incubation in TBST + 5% milk

powder, containing the secondary antibody (donkey antirabbit IgG-HRP

(sc-2313), Santa Cruz Biotechnology, Dallas, TX) at a concnetration of

0.04 μg/mL, under shaking at rt in the dark for 1 h, washing with TBST

(6 × 5 min, rt), and incubation with the substrate (Clarity Western ECl

Substrate, BioRad) at rt for 5 min. Bioluminescence was detected with a

ChemiDoc MP Imaging System (BioRad). No bands were detectable in

the absence of primary antibody (data not shown).

Dulbecco’s PBS with Ca²⁺and Mg²⁺(1.8 mM CaCl₂, 2.68 mM KCl,

1.47 mM KH₂PO₄, 3.98 mM MgSO₄, 136.9 mM NaCl and 8.06 mM

Na₂HPO₄) supplemented with 1% BSA and 100 μg/mL of the protease

inhibitor bacitracin (Serva) was used as binding buﬀer. PBS was used for

the washing steps (prior to the incubation at rt and after incubation ice-

cold). Unspeciﬁc binding was determined in the presence of AngII (500-

fold excess to [³H]21). The incubation period was 2 h. The conﬂuency

of the cells was at least 90% on the day of the experiment.

Saturation and competition binding experiments with [³H]27 at

intact HT-29 colon carcinoma and CHO-NTS₁cells were performed in

tissue culture treated white 96-well plates with clear bottom (Corning

Incorporated Life Sciences, Tewksbury, MA; Corning cat. no. 3610)

using the binding buﬀer as aforementioned for [³H]21. Cells were

seeded one or 2 days prior to the experiment. On the day of the

experiment conﬂuency of the cells was at least 90% (HT-29 cells) or

approximately 10% (CHO-NTS₁cells). The culture medium was

removed by suction, the cells were washed with PBS (200 μL) and

covered with binding buﬀer (160 μL). For total binding, binding buﬀer

(20 μL) and the same volume of binding buﬀer containing the

radioligand 10-fold concentrated were added. For the determination of

unspeciﬁc binding (in the presence of NT(8−13) in 500-fold excess to

[³H]27) and displacement of [³H]27 (competition binding experi-

ments), binding buﬀer (20 μL) containing the competitor 10-fold

concentrated and binding buﬀer (20 μL) containing the radioligand

10-fold concentrated were added. The plates were gently shaken during

incubation for 2 h. After incubation, the liquid was removed by suction,

the cells were washed twice with ice-cold PBS (200 μL; washing period

<3 min) followed by the addition of lysis solution (urea (8 M), acetic

acid (3 M) and Triton-X-100 (1%) in water) (25 μL). The plates were

shaken for 30 min, liquid scintillator (Optiphase Supermix (Perki-

nElmer)) (200 μL) was added, and the plates were sealed with a

transparent sealing tape (permanent seal for microplates, PerkinElmer,

prod. no. 1450−461). The plates were turned up-side down several

times in order to achieve complete mixing of scintillator and lysis

solution. The plates were kept in the dark for at least 1 h prior to the

measurement of radioactivity (dpm) with a MicroBeta2 plate counter

(PerkinElmer).

If ﬂuorescently labeled ligands were investigated competition binding

experiments were performed in the dark.

Association and dissociation experiments with [³H]21 (intact CHO-

hAT₁-G_α16-mtAEQ cells) and [³H]27 (intact HT-29 cells) were perfor-

med as described for the saturation and competition binding

experiments (see above) using the same equipment, buﬀers, and

volumes. Unspeciﬁc binding was determined in the presence of AngII

and NT(8−13), respectively (each in 500-fold excess). For association

experiments the radioligand concentration was 3 and 1.2 nM, respectively.

The incubation of the cells with the radioligand was stopped after

diﬀerent periods of time (between 1 and 120 min). In case of dissoci-

ation experiments cells were preincubated with 8 nM [³H]21 and 2 nM

[³H]27, respectively (each for 90 min). The radioligand solution was

removed by suction, the cells were covered with binding buﬀer (250 and

200 μL, respectively) containing AngII (160 nM) and NT(8−13)

(100 nM), respectively, and the plates were gently shaken. After

diﬀerent periods of time the cells were washed with ice-cold PBS

followed by cell lysis and further processing as described above.

Saturation Binding and Acid Wash Experiments with [³H]21

and [³H]27. To estimate cell surface-bound and internalized radio-

ligand, saturation binding experiments were performed with [³H]21 and

[³H]27 at CHO-hAT₁and HT-29 cells, respectively, as described above

with the following modiﬁcation: Binding experiments with [³H]27 were

carried out in Primaria 24-well plates (Corning Life Sciences) as in case

of [³H]21 (see above). After washing twice with ice-cold PBS (500 μL)

at the end of the 2-h incubation period, the cells were washed twice with

ice-cold glycine buﬀer pH 3.0 (50 mM glycine and 125 mM NaCl in

water) for 5 min and the radioactivity of the collected and combined

washings (representing the fraction of cell surface-bound radioligand)

was counted in 10 mL of liquid scintillator (Rotiszint eco plus) with a LS

6500 liquid scintillation counter (Beckman-Coulter).

Fura-2 Calcium Assay (HT-29 Cells). The fura-2 Ca²⁺-assay with

HT-29 colon carcinoma cells was performed as described for human

erythroleukemia cells (Ca²⁺-response elicited by NPY) using a Perkin−

Elmer LS50 B spectroﬂuorimeter (PerkinElmer, Rodgau, Germany).⁸²

HT-29 cells (80−95% conﬂuency) were trypsinized, detached from the

culture ﬂask and treated according to the described protocol. The

intracellular Ca²⁺-mobilizations induced by NT(8−13), 25 and 27 were

normalized to the eﬀect elicited by 100 nM NT(8−13).

Functional Bioluminescence Assay (CHO-AT₁-G_α16-mtAEQ

cells). The aequorin assay was performed as previously described for

CHO-hY₄-G_qi5-mtAEQ cells⁶using CHO-G_α16-mtAEQ cells,⁷⁹that

were kindly donated by Dr. M. Detheux (Euroscreen s.a., Gosselies,

Belgium) and were stably transfected with the human AT₁R (designated

as CHO-AT₁-G_α16-mtAEQ cells).⁸⁰Measurements were carried out

with a GENios Pro plate reader (Tecan, Salzburg, Austria). Areas under

the curve were calculated using SigmaPlot 11.0 software (Systat

Software Inc.). Net fractional luminescence data were obtained by

subtraction of the fractional luminescence of the blanks (that is agonist

omitted, determined at least every 9 wells) from fractional luminescence

data acquired in the presence of agonist. Net data were normalized to the

eﬀect elicited by 100 nM AngII (determined in at least two triplicates per

96-well plate).

Radioligand Binding Experiments with [³H]21 (AT₁R) and [³H]

27 (NTS₁R). All radioligand binding experiments were performed at

22 2 °C. Saturation and competition binding experiments with [³H]

21 at intact CHO-AT₁-G_α16-mtAEQ and intact rat mesangium cells were

performed in Primaria 24-well plates (Corning Life Sciences) as

previously described for the NPY Y Y₁receptor radioligand [³H]UR-

MK136⁴⁸at intact SK-N-MC cells with the following modiﬁcations:

Lysis solution (200 μL) was added to the cells, the plates were

gently shaken for 30 min and the solution was transferred into 20 mL

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scintillation vials ﬁlled with 10 mL of scintillator. The wells were washed

with 200 μL of lysis solution, the washings were added to the scintillation

vials and the radioactivity, representing the fraction of intracellular

radioligand, was counted. Unspeciﬁc binding data were determined in

the presence of AngII and NT(8−13), respectively, in 500-fold excess to

the radiolabeled peptide. For each radioligand two independent

experiments were performed in triplicate giving comparable results.

Radioligand Competition Binding Assay with [³H]NT(8−13)

(NTS₂R). Radioligand competition binding experiments with

[³H]NT(8−13) were performed at HEK-hNTS₂cell homogenates as

previously reported.²³

Masson-Goldner staining (Jerusalem’s modiﬁcation) of the ﬁxed

sections was performed as previously described with the following

modiﬁcations:⁴⁰phosphoric acid-Orange G was 1:3 diluted in water and

the immersion period in this solution was 7 s. The incubation period in

0.2% light green was 5 min.

Homology Model of the Human Y₄Receptor. The homology

model of the human Y₄R was generated with the modeling suite SYBYL-

X 1.3 (Tripos Inc., St. Louis, MO) using the crystal structure of the rat

NTS₁R (PDB ID: 3ZEV) as template.⁶⁹Model generation was

essentially performed as described.⁸⁴Loops (ICL1-ICL3 and ECL1-

ECL3) were inserted nontruncated using the Loop-Search module

within SYBYL. The N-terminal sequence of 27 amino acids, not resolved

in the template, is not present. The model was provided with Amber7

FF99 charges and energy-minimized with the Amber7 FF99 force ﬁeld⁸⁵

up to a gradient of 0.01 kcal mol⁻¹Å⁻¹using a dielectric constant of 4.

Data Processing. Speciﬁc optical rotation was calculated according

to [α] = α/(c·l), for which α = measured angle of rotation, c =

concentration [g · cm⁻³], and l = length of the cuvette Dm]. Retention

(capacity) factors were calculated from retention times (t_R) according to

k = (t_R−t₀)/t₀(t₀= dead time).

Radioligand Competition Binding Assay with [³H]EYF

(NPFF₂R). Radioligand competition binding studies with [³H]EYF

were performed at CHO-hNPFF₂cell membranes as previously

reported.⁵⁰The K_dvalue of [³H]EYF⁵⁰was determined in the same

system by saturation binding and amounted to 6.9 nM. Determined

binding data of the reference peptides NPFF and EYF proved to be in

good agreement with reported data (see Table 1D).

Flow Cytometric Competition Binding Assay (Y₄R). The ﬂow

cytometric Y₄R competition binding assay on CHO-hY₄-G_qi5-mtAEQ

cells using S0586-[K⁴]hPP as ﬂuorescent ligand (c = 10 nM) was

performed as previously described on a FACSCalibur ﬂow cytometer

(Becton Dickinson), equipped with an argon laser (488 nm) and

a red diode laser (635 nm) (settings: FSC: E-1, SSC: 280 V, Fl-4:700−

750 V).⁸³The cell density in loading buﬀer was 10⁶cells/mL. The

samples were incubated in 1.5 mL reaction vessels (ref. 72.690.001,

Sarstedt) in the dark at rt for 90 min. Unspeciﬁc binding was determined

by addition of hPP at a concentration of 1 μM. Data acquisition was

stopped after counting of 10,000−15,000 gated events. The dissociation

constant (K_dvalue) of S0586-[K⁴]hPP, was determined in two inde-

pendent saturation experiments applying concentrations between

0.5 and 40 nM (nonspeciﬁc binding determined in the presence of

hPP 100-fold concentrated) and amounted to 7.5 2.1 nM, being in

good agreement with the reported K_dof 10.2 nM.⁶¹

Relative data from functional cellular assays (aequorin assay: %

fractional luminescence, fura-2 assay: % Ca²⁺response) were plotted

against log(concentration agonist) and analyzed by a four parameter

logistic equation (SigmaPlot 11.0, Systat Software Inc., Chicago, IL) to

obtain pEC₅₀values. Speciﬁc binding data (in dpm) from saturation

binding experiments with the radioligands [³H]21 and [³H]27 were

plotted against the free radioligand concentration and analyzed by an

equation describing hyperbolic binding (ligand binding - one site

saturation ﬁt, SigmaPlot 11.0) to obtain the dissociation (binding)

constant K_dand the maximum number of binding sites B_max. The free

radioligand concentration (nM) was calculated by subtracting the

amount of speciﬁcally bound radioligand (nM) (calculated from the

speciﬁcally bound radioligand in dpm, the speciﬁc activity and the

volume per well) from the total radioligand concentration. Unspeciﬁc

binding data from saturation binding experiments were ﬁtted by linear

regression. The number of binding sites per cell was calculated from the

B_max, the speciﬁc activity (Bq mol⁻¹) of the radioligand, the Avogadro

constant (mol⁻¹) and the number of cells per well. Speciﬁc binding data

from association experiments with [³H]21 and [³H]27 were analyzed by

a two-parameter equation describing an exponential rise to a maximum

(SigmaPlot 11.0) to obtain the observed association rate constant k_obs

and the maximum of speciﬁcally bound radioligand (B_eq), which was

used to calculate speciﬁcally bound radioligand (B) in %. Data from

dissociation experiments (% speciﬁcally bound radioligand (B) plotted

over time) with [³H]21 were analyzed by a four-parameter equation

describing a biphasic exponential decline (SigmaPlot 11.0) to obtain the

dissociation rate constants k_off(1) and k_off(2). Data from dissociation

experiments (% B plotted over time) with [³H]27 were analyzed by a

two-parameter equation describing a monophasic exponential decline

(SigmaPlot 11.0) to obtain k_off. The association rate constant (k_on) of

[³H]27 was calculated from k_obs, k_offand the radioligand concentration

([RL]) according to the correlation: k_on= (k_obs−k_off)/[RL]. Speciﬁc

binding data from radioligand or ﬂuorescent ligand competition binding

experiments were plotted as % (100% = bound radioligand in the

absence of competitor) over log(concentration competitor) and

analyzed by a four parameter logistic equation (SigmaPlot 11.0).

Resulting pIC₅₀values were transformed to IC₅₀values, which were

converted to the dissociation (binding) constant K_iaccording to the

Cheng−Prusoﬀ equation⁸⁶using the K_dvalue of the respective

radioligand or ﬂuorescent ligand ([³H]21: K_d0.94 nM (CHO-hAT₁

cells), [³H]27: K_d0.51 nM (HT-29 cells), [³H]EYF: K_d6.9 nM (CHO-

NPFF₂cell membranes), S0586-[K⁴]hPP: K_d7.5 nM).

Autoradiography. NMRI (nu/nu) mice were bred in the animal

facility of the University of Regensburg. Animal experiments were

approved by the local veterinary authorities (district government)

according to the European guidelines and national regulations of the

German animal protection act (notiﬁcation from 12th of May, 2011,

“Propagation of tumor cells in nude mice”, to the Regierung der

Oberpfalz, Department 621, veterinary services).

Autoradiography at HT-29 tumor cryosections using [³H]27 as

radioligand were essentially performed as previously described.⁴⁰

Subcutaneous HT-29 tumors were established in male NMRI (nu/

nu) mice by subcutaneous injection of a cell suspension in FCS-free

culture medium (100 μL containing about three million cells) or by

subcutaneous transplantation of tumor pieces. Tumors were excised,

immediately embedded in Tissue-Tek and frozen on dry ice, and were

stored at −78 °C. Cryosections (12 μm) were obtained at −15 °C with a

2800 Frigocut E freezing microtome (Reichert-Jung/Leica, Germany).

Adjacent sections were mounted on microscopic slides (Superfrost

Plus, 25 × 75 × 1 mm (Gerhard Menzel (via Thermo Scientiﬁc),

Braunschweig, Germany) and put in a chamber of 100% humidity. After

1−2 min tumor sections were either carefully covered with binding

buﬀer (D-PBS containing 0.2% BSA and 100 μg/mL bacitracin, ﬁltered

with a 0.2-μm syringe ﬁlter) (800−1000 μL) or immersed in an

alcoholic formaldehyde ﬁxative (37% (w/w) formaldehyde in water

(40 mL), 95% (v/v) ethanol (360 mL) and calcium acetate (0.2 g)) for

30 s. The incubation with radioligand was started less than 15 min after

preparation of the cryosections by careful removal of the binding buﬀer

and immediate recovering of the sections with binding buﬀer containing

[³H]27 (6 nM) (total binding) or binding buﬀer containing [³H]27

(6 nM) and NT(8−13) (1.8 μM) (nonspeciﬁc binding). The sections

were incubated in a humidity chamber at 22 °C for a period of 60 min

followed by washing in ice-cold PBS and drying over phosphorus

pentoxide according to the described protocol. The incubation period

on the tritium sensitive phosphor screen (medium size (252 mm ×

125 mm); PerkinElmer) was 16 days. For luminescence detection a

phosphorimager (Cyclone Storage Phosphor System, Packard) was

used.

Propagated errors were calculated according to the Gaussian law of

errors.

Figures of the rNTS₁and hAT₁receptor crystal structures and of the

homology model of the NPY Y₄receptor (Supporting Information,

Figures S31 −S33) were prepared with PyMOL Molecular Graphics

System, version 1.6 (Schrodinger LLC, Portland, OR).

̈

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DOI: 10.1021/acs.jmedchem.5b01495

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

competition binding experiment; MeCN, acetonitrile; NT(8−13),

neurotensin(8−13); NTS₁R, neurotensin receptortype 1;NTS₂R,

neurotensin receptor type 2; NPFF, neuropeptide FF; NPFF₂R,

NPFF receptor type 2; NPY, neuropeptide Y; PBS, phosphate

buﬀered saline; PS, polystyrene; PyBOP, 1H-benzotriazol-1-

yloxy)tripyrrolidinophosphonium hexaﬂuorophosphate; RGD,

arginine-glycine-aspartate sequence; RP-HPLC, reversed-phase

HPLC; SEM, standard error of the mean; SPPS, solid phase

peptide synthesis; Y₄R, NPY Y₄receptor

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01495.

■

S

Examples of histamine H₂and NPY Y₁receptor ligands

containing an acylguanidine moiety; scheme showing the

synthesis of peptides 1 and 19−36; chemical stability of

the peptides 19, 20, 23−25, 29, 30, 35, and 36; stability of

AngII, NT(8−13), 22, and 28 under assay conditions;

control experiments using a N^ω-acyl-Arg containing

peptide: chemical instability of the NT(8−13) derivative

44; excitation spectra, emission spectra, and ﬂuorescence

quantum yields of 23, 24, 29, 30, and 47; RP-HPLC

analysis of [³H]21 and [³H]27: identity, purity, and long-

term stability; Coomassie staining of the gel used for the

immunoblot shown in Figure 8; AT₁receptor association

and dissociation kinetics of [³H]21; NTS₁receptor

association and dissociation kinetics of [³H]27; saturation

and competition binding with the AngII derivative [³H]21 at

rat mesangial cells; saturation binding with the NT(8−13)

derivative [³H]27 at CHO-hNTS₁cells; saturation

binding and acid wash experiments with [³H]21 and

[³H]27; structures of the NTS₂R ligand 45, the NTS₁R

antagonist 46, and the AT₁R antagonists candesartan and

losartan; histology of the HT-29 xenograft shown in

Figure 7C; structures of the rat NTS₁, the human AT₁and

the modeled human NPY Y₄receptor; synthesis protocols

and analytical data of compounds 4, 5, 8, 9, 12, 14, 15, 17,

REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*Phone: (+49)941-9433329. Fax: (+49)941-9434820. E-mail:

max.keller@chemie.uni-regensburg.de.

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

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ABBREVIATIONS USED

■

AngII, angiotensin II; aq, aqueous; AT₁R, angiotensin receptor

type 1; br s, broad singlet; CH₂Cl₂, dichloromethane; 2-ClTrt, 2-

chlorotrityl; DIPEA, diisopropylethylamine; dpm, disintegra-

tions per minute; EtOAc, ethyl acetate; HBTU, O-(1H-

benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexaﬂuoro-

phosphate; HFIP, 1,1,1,3,3,3-hexaﬂuoro-2-propanol; HOBt,

1H-benzotriazol-1-ol; hPP, human pancreatic polypeptide; k,

retention (or capacity) factor (HPLC); K_d, dissociation (or

binding) constant obtained from a saturation binding experi-

ment; K_i, dissociation (or binding) constant obtained from a

R

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Mimicking of Arginine by Functionalized Nω-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples

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