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Synthesis, In Vitro Evaluation, and In Vivo Metabolism of Fluor/ Quencher Compounds Containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3) Karen E. Linder,* Edmund Metcalfe, Palaniappa Nanjappan, Thangavel Arunachalam, Kimberly Ramos, Tina Marie Skedzielewski, Edmund R. Marinelli, Michael F. Tweedle,† Adrian D. Nunn, and Rolf E. Swenson Ernst Felder Laboratories, Bracco Research USA, 305 College Road East, Princeton, New Jersey 08540, United States

bS Supporting Information ABSTRACT: Protease-cleavable peptides containing a suitable fluor/quencher (Fl/Q) pair are optically dark until cleaved by their target protease, generating fluorescence. This approach has been used with many Fl/Q pairs, but little has been reported with IRDye 800CW, a popular near-infrared (NIR) fluor. We explored the use of the azo-bond-containing Black Hole Quencher 3 (BHQ-3) as a quencher for IRDye 800CW and found that IRDye 800CW/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Cleavage of IRDye 800CW-PLGLK(BHQ-3)AR-NH2 (8) and its D-arginine (Darg) analogue (9) by matrix metalloproteinases (MMPs) in vitro yielded the expected cleavage fragments. In vivo, extensive metabolism was found. Significant decomposition of a “non-cleavable” control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane)-BHQ-3 (10) was evident in plasma of normal mice by 3 min post injection. The major metabolite showed a m/z and UV/vis spectrum consistent with azo bond cleavage in the BHQ-3 moiety. Preparation of an authentic standard of this metabolite (11) confirmed the assignment. Although the IRDye 800CW/BHQ-3 constructs showed efficient contact quenching prior to enzymatic cleavage, BHQ-3 should be used with caution in vivo, due to instability of its azo bond.

’ INTRODUCTION In vivo optical imaging using compounds that fluoresce in the near-infrared (NIR) region ∼750900 nm is a useful research tool. This spectral region is marked by both low autofluorescence and absorbance minima for water, hemoglobin, and lipids. Use of NIR fluors such as Indocyanine Green (ICG) or IRDye 800CW facilitates both access of the excitation light to the fluorophore and escape of emitted fluorescence from the animal to the detector, due to the significant tissue penetration depths attainable.1,2 NIR fluorescent probes fall into three main classes. The first are nonspecific agents, often consisting of only a fluorophore itself. These have been used to visualize the lymphatic system3 and to image pathology in areas where exit from the vasculature is enhanced due to leakage, such as tumors4,5 or arthritis.6,7 Here, contrast is obtained due to enhanced extravasation and/or delayed clearance from abnormal tissue. The second type of probe is a targeted agent labeled with a fluorophore. These agents are also permanently fluorescent and range in size from small molecules and peptides to antibodies and nanoparticles.8 With such compounds, sufficient clearance from blood and nontarget tissues is required to detect localization in the target compounds. Fluorophore-containing constructs of the third type are designed to become fluorescent only after proteolytic cleavage.9,10 r 2011 American Chemical Society

Such “smart probes” or “activatable probes” either contain a multiplicity of self-quenching fluors or contain a nonfluorescent quencher that “quenches” fluorescence in the intact construct (Figure 1). After cleavage, the fluorophore and quencher are separated, resulting in an increased fluorescent signal. Such compounds are optically dark before cleavage, so background signal from parent compound does not interfere with signal at the target. This is an obvious advantage that stands in contrast to typical fluorescently labeled compounds, where both the parent compound and its metabolites are fluorescent. Many activatable probes are labeled with a fluor and quencher that allegedly undergo fluorescence resonance energy transfer (FRET), wherein the emission of the fluor falls within the absorbance envelope of the quencher. Such an approach has long been used for the detection of protease activity1113 in vitro. Enzyme activity in cells or biological fluids can be readily measured in a functional assay, based on the fluorescence generated following proteolytic cleavage of a synthetic fluor/quencher peptide that contains a substrate for the enzyme of interest. Received: October 16, 2010 Revised: May 31, 2011 Published: June 04, 2011 1287

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Bioconjugate Chemistry

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and metabolism of 8 and 9 and a “noncleavable” control with a PEG linker (IRDye 800CW-Ttda-(BHQ-3) (10), along with synthesis and characterization of selected analogues and metabolites.

’ EXPERIMENTAL PROCEDURES Synthesis of Fluor-Quencher Constructs and Intermediates. Solvents were E. Merck Omni grade. N,N-DimethylformaFigure 1. Schematic for protease-cleavable activatable probes. Fluorescence is quenched until proteolytic cleavage, which separates the fluor and quencher, generating fluorescence.

Several types of dual-labeled fluor/quencher oligonucleotide probes (e.g., “molecular beacons”) have also been developed14,15 for the in vitro or in vivo monitoring of DNA and RNA-related processes such as polymerase chain reactions hybridization, sequencing, and ligation. Activatable fluor/quencher probes have been used successfully for imaging or in vitro studies with peptide-containing constructs cleaved by cathepsins,1620 MMPs,2124 and caspases.9,2527 The cyanine dye Cy5.5 [excitation/emission (Ex/Em), 660/ 710 nm] is frequently used as a fluor in such constructs. Reported studies on molecules labeled with IRDye 800CW28 are somewhat limited,3,2934,36 and to our knowledge, no studies on FRET-based compounds containing IRDye 800CW (Ex/Em: 778/789 nm) have been reported to date. This is likely due to the fact that until recently35 no commercial quenchers with the proper spectral characteristics to quench the dye via FRET have been available. An effective quencher for IRDye 800CW would be of significant interest, as it has been reported that an anti-EGF antibody labeled with IRDye 800CW showed significantly reduced background from autofluorescence relative to its Cy5.5 analogue, leading to an enhanced tumor to background ratio.36 On the basis of its effectiveness as a quencher in other systems, we decided to evaluate the effectiveness of the “dark” quencher known as Black Hole Quencher 3 (BHQ-3)37 to quench IRDye 800CW, despite the fact that it does not show the spectral overlap needed for FRET (Figure 8a). BHQ-3 is widely used in DNA duplex probes, where a fluorescent emitter is on one strand and an acceptor is on the complementary one,38 and in activatable probes to quench Cy5.5, Cy5, fluorescein, and pheophorbide.26,39 To probe the quenching capabilities of this combination, we have prepared an IRDye 800CW/BHQ-3 analogue of the wellknown matrix metalloproteinase (MMP)-cleavable sequence Mca-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2, first identified by Knight et al.,40 where Mca is 7-methoxycoumarin and Dnp is the quencher 2,4-dinitrophenyl. This compound is commercially available for the detection of MMPs in vitro, but does not have the proper spectral properties for in vivo imaging. An imaging agent for the detection of MMP activity in vivo would be of interest, as this family of highly homologous proteases collectively cleave most, if not all, of the constituents of the extracellular matrix (ECM) and have been implicated in the growth and metastasis of tumors41,42 and in inflammatory processes such as arthritis.43 The studies described here show that IRDye 800CW-PLGLK(BHQ-3)-AR-NH2 (8) and its D-arginine analogue (9) are effective substrates for MMPs and that IRDye 800CW/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for FRET. This manuscript describes the synthesis, spectral characterization, in vitro cleavage by MMPs, and in vivo pharmacokinetics

mide (DMF) was purchased from Pharmco Products Inc. and was peptide synthesis grade or low water/amine-free Biotech grade quality. Piperidine (sequencing grade, redistilled 99þ%) and trifluoroacetic acid (TFA, spectrophotometric grade or sequencing grade) were purchased from Sigma-Aldrich Corporation or Fluka. N,N0 -Diisopropylcarbodiimide (DIC), N,N-diisopropylethylamine (DIEA), O-(N-succinimidyl)-N,N,N0 ,N0 -bis(tetramethylene) uronium hexafluorophosphate (HSPyU), and triisopropylsilane (TIPS) were purchased from Sigma-Aldrich. The 4,7,10-trioxatridecane-1,13-diamine (Ttda), Fmoc-protected amino acids, O-(benzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium hexafluorophosphate (HBTU), and N-hydroxybenzotriazole (HOBt) were purchased from Novabiochem. Black hole quencher 3 carboxylic acid succinimidyl ester (BHQ3-OSu) was purchased from Biosearch Technologies, Inc. and IRDye 800CW-NHS ester (NHS = N-hydroxysuccinimide) was purchased from LI-COR Biosciences. HPLC and MS. Analytical HPLC data were obtained using a Shimadzu LC-10AT VP dual-pump gradient system using a Waters XTerra MS-C18 column (4.6 50 mm, 5 μ, 120 Å, 3 mL/min flow rate) and linear gradient elution using H2O/(0.1% TFA) and CH3CN/(0.1% TFA) as solvents A and B, respectively. Compounds were detected at 220 and 254 nm (deuterium lamp) or at 701 and 790 nm (tungsten lamp). Preparative HPLC was conducted on a Shimadzu LC-8A dual-pump gradient system equipped with a SPD-10AV UV detector and a Waters XTerra Prep MS-C18 column (19 300 mm, 10 μ, 120 Å) using gradient elution with H2O/(0.1% TFA) and CH3CN/ (0.1% TFA) as solvents A and B, respectively, and a flow rate of 40 mL/min unless otherwise stated. Generally, the crude peptide solution was loaded onto the column using a third pump. Reaction mixture solvents were eluted from the column at low organic phase composition, followed by linear gradient elution with increasing percentages of solvent B. Mass spectral data were obtained in-house on an Agilent LCMSD (1100) mass spectrometer using API-ES in - ion mode. MS results and HPLC purity were used to monitor the completeness of reactions, and to select appropriate fractions during HPLC purification. Solid-Phase Peptide Synthesis of Peptides 1 and 2. Fmocprotected linear peptides Fmoc-Pro-Leu-Gly-Leu-Lys-Ala-XNH2, X = Arg (1) and D-arginine (2) were synthesized on a 0.25 mmol scale with an ABI 433A Peptide Synthesizer employing FastMoc protocols, Fmoc-Pal-Peg-PS, or NovaSyn TG Sieber resin (0.2 mmol/g), Fmoc-protected amino acids, and HBTU-mediated HOBt ester activation. The general methods used for preparation were described previously.44 Compounds were analyzed using gradient elution from 20% to 65% B over 15 min. Detection: UV at 230 and 254 nm. Both 1 and 2 had a retention time tR = 6.29 min. MS: m/z = 975.2 [MþH]þ; 487.5 [Mþ2H]2þ/2. Preparation of BHQ3-Containing Peptides 3 and 4. (BHQ3)-OSu (5 mg, 0.0063 mmol) was added to a solution of peptide 1288

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Bioconjugate Chemistry 1 or 2 (10 mg, 0.0068 mmol) in dry DMF (0.3 mL), followed by DIEA (10 mg, 0.077 mmol). The mixture was stirred for 4 h at room temperature (r.t.) in a foil-covered flask to protect from light, then diluted to 10 mL with dry DMF. Piperidine (0.2 mL) was added, and the mixture was stirred for 30 min to remove the Fmoc group. The reaction mixture was diluted to 50 mL with water and loaded onto a preparative HPLC column. After the sample was applied to the column, the eluant was ramped to 20% B over 1 min, followed by a linear gradient to 45% B over 50 min. Fractions (15 mL) were manually collected using absorbance at 700 nm as an indicator of product elution. Product-containing fractions were combined and freezedried to afford 5 to 7 mg (4050% yield) of the desired BHQ-3-containing peptide (>95% purity). Compounds were analyzed by HPLC using gradient elution from 25% to 55% B over 30 min. Detection: 701 and 790 nm. Both 3 and 4 had a tR = 6.23 min. MS: m/z = 1282.7 [MþH]þ; 641.5 [Mþ2H]2þ/2. Addition of IRDye 800CW to Form 8 and 9. IRDye 800CWNHS ester (3 mg, 0.0026 mmol) was added to a solution of the peptide-(BHQ-3) conjugate 3 or 4 (3 mg, 0.0017 mmol) in dry DMF (0.3 mL), followed by DIEA (10 mg, 0.077 mmol). The solution was stirred for 8 h at r.t., protected from light. After completion of the coupling reaction with IRDye 800CW NHS ester, the reaction mixture was diluted to 20 mL with water, loaded onto the preparative HPLC column, purified, and freezedried. Isolated 8 and 9 were analyzed by HPLC using gradient elution from 15% to 45% B over 20 min. Detection: 701 and 790 nm. Yield: 23 mg (3040%). Purity: >98%, tR = 6.27 min, MS: m/z = 2268.8 [MþH]þ; 1134.0 [Mþ2H]2þ/2; 756.4 [Mþ3H]3þ/3. 1-N-(BHQ-3)-13-amino-4,7,10-trioxatridecane (H-TtdaBHQ-3) (5). BHQ-3 NHS ester (17 mg, 0.026 mmol) was added to a solution of 1-N-trityl-4,7,10-trioxatridecane-13-amine (Ttda, 10 mg, 0.022 mmol) and DIEA (10 mg, 0.038 mmol) in CH2Cl2 (1 mL) and stirred for 3 h at r.t. Volatiles were removed on a rotary evaporator, and the paste obtained was treated with a mixture of CH2Cl2/TFA/TIPS (5 mL, 90:5:5) and stirred for 2 h to remove the trityl group. After evaporation of volatiles, the paste was redissolved in H2O/CH3CN (5 mL, 1:1), purified by preparative HPLC (flow rate = 40 mL/min) and freezedried. Isolated 5 eluted at 6.28 min using gradient elution from 25% to 55% B over 30 min. Detection: 701 and 790 nm. Yield: 9 mg (46%). Purity: >95%. MS: m/z = 750 [MþH]þ; 375.8 [Mþ2H]/2. IRDye 800CW-Ttda-BHQ-3 (10). PEG-containing fluor/quencher construct 10 was prepared by coupling IRDye 800CW NHS ester (8 mg, 0.007 mmol) to 5 (5 mg, 0.007 mmol), following the general procedure described for 8 and 9 above. The resulting solid was analyzed using gradient elution from 25% to 65% B over 15 min. Detection: 701 and 790 nm. It was isolated as a fluffy dark bluegreen solid in 40% yield (5 mg). Purity: >98%. tR = 6.60 min, MS: m/z = 1733.2 [MH]; 1155.2 [2M3H]/3; 865.6 [M2H]/2. N-t-Butyloxycarbonyl-1,4-phenylenediamine (14). Di-t-butyldicarbonate (6.2 g, 28 mmol) was added to a solution of 1,4phenylenediamine 3 2HCl (13) (5 g, 28 mmol) and K2CO3 (8.0 g, 58 mmol) in dioxanewater (2:1, 100 mL). The mixture was stirred at r.t. for 4 h, volatiles were removed on a rotary evaporator, and the product was added to water (50 mL) to precipitate the product. The solid was filtered, washed with water (2 50 mL), and dried to yield 14 as a colorless solid (7.5 g, 78%). 1H NMR (500 MHz, CDCl3) δ 1.52 (s, 9H, C4H9), 6.22 (bs, 1H, NH) and 7.17, 7.34 (2 d, 4H, ArH). MS: m/z = 208 [MþH]þ.

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Figure 2. Synthesis of IRDye 800CW-PLGLK(BHQ-3)-AR-NH2 (8). a) (BHQ-3)-OSu, DIEA, NMP, DMF, RT, 4 h; b) Piperidine, NMP, RT, 30 min; c) IRDye800CW NHS ester, DIEA, DMF, RT, 8 h.

Ethyl 4-N-(4-t-Boc-aminophenyl)-butyrate (15). Ethyl 4-bromobutyrate (2.25 g, 11.5 mmol) was added to a solution of 14 (2 g, 9.6 mmol) and powdered anhydrous K2CO3 (2.7 g, 19.3 mmol) in dry DMF (20 mL) and heated at 60 °C for 4 h. The reaction mixture was then poured into ice-cooled water (100 mL), stirred, and filtered. The collected ethyl 4-N-(4-t-Boc-aminophenyl)butyrate 15 (Boc = tert-Butyloxycarbonyl) was isolated as a colorless solid, washed with water (3 50 mL), and dried. Yield: 2.8 g (90%). MS: m/z = 323 [MþH]þ. 4-N-(4-t-Boc-aminophenyl)-4-N-methylbutyric Acid (16). NaH (60% dispersion in mineral oil, 0.08 g, 2.0 mmol) was added to a solution of 15 (0.5 g, 1.56 mmol) in dry DMF (2 mL) and the mixture was stirred at r.t. for 30 min under N2. Methyl iodide (0.25 g, 1.76 mmol) was added and the mixture was stirred for 4 h. LiOH (0.2 g, 8.3 mmol) in watermethanol (1:1, 1 mL) was added, and the reaction mixture was stirred for 2 h. After completion of the hydrolysis, the reaction mixture was diluted with H2O to 5 mL and loaded onto the preparative HPLC column. After the sample solution was applied to the column, the HPLC system was ramped to 10% B over 1 min, followed by a linear gradient from 10% to 35% B over 50 min. Fractions (15 mL) were collected using UV at 230 nm as an indicator of product elution. Product-containing fractions of >95% purity were combined and freezedried to afford 0.25 g (52% yield) of the desired 4-N-(4-t-Boc-aminophenyl)-4-N-methylbutyric acid 16. 1H NMR (500 MHz, CDCl3) δ 1.53 (s, 9H, C4H9), 1.79 (m, 2H, CH2CH2CH2), 2.38 (t, 2H, CH2COOH), 3.18 (s, 3H, CH3), 3.61 (t, 2H, NCH2) and 7.25, 7.54 (2 d, 4H, ArH). MS: m/z = 309 [MþH]þ. 1-N-(4-N-(4-t-Boc-aminophenyl)-4-N-methylbutyroyl)-13amino-4,7,10-trioxatridecane (H-Ttda-BocApmba4) (Apmba = 4-N-(4-Aminophenyl)-4-N-methylbutyric acid) (6). HSPyU (150 mg, 0.36 mmol) was added to a solution of 16 (100 mg, 0.32 mmol) in CH2Cl2 (2 mL), followed by DIEA (100 mg, 0.77 mmol), and stirred for 30 min at r.t. 1-N-Trityl-4,7,10trioxatridecane-13-amine (Ttda, 100 mg, 0.22 mmol) was added, and the reaction mixture was stirred for an additional 3 h. Volatiles 1289



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Figure 3. Synthesis of “noncleavable control” IRDye 800CW-Ttda-(BHQ-3) (10) and its azo-bond cleavage product IRDye 800CW-TtdaApmba4 (11). a) BHQ-3 NHS ester (1.2 eq.), NMP, DIEA, RT, 4 h; b) 4-N-(4-t-Boc-aminophenyl)-4-N-methylbutyric acid (1.2 eq.), HSPyU (1.2 eq.), DIEA (4 eq.), CH2Cl2, RT, 4 h; c) CH2Cl2:TFA:TIPS (95:2:3), RT, 30 min; d) IRDye800CW NHS ester (1 eq.), DIEA (3 eq.), CH2Cl2, RT. 4 h; e) CH2Cl2:TFA:TIPS (50:45:5), RT, 30 min.

Table 1. Summary of Fluorogenic IRDye800CW/BHQ-3 Constructs, Intermediates, And Metabolites Synthesized compound

Figure 4. Synthesis of 4-N-(4-t-Boc-aminophenyl)-4-N-methylbutyric acid, 4-t-Boc Apmba4 (16). a) Di-t-butyldicarbonate (1.1 eq.), K2CO3 (2 eq.), Dioxane-water (1:1), RT, 4 h; b) Ethyl 4-bromobutyrate (1.2 eq.), K2CO3 (2 eq.), NMP, 60 °C, 6 h; c) CH3I (1.2 eq.), NaH (1.1 eq.). NMP, RT, 6 h; d) LiOH (4 eq.), CH3OH:H2O (1:1), RT, 4 h.

peptide sequence

MW

1

Fmoc-Pro-Leu-Gly-Leu-Lys-Ala-Arg-NH2

2

Fmoc-Pro-Leu-Gly-Leu-Lys-Ala-Darg-NH2

3 4

H-Pro-Leu-Gly-Leu-Lys(BHQ-3)-Ala-Arg-NH2 H-Pro-Leu-Gly-Leu-Lys(BHQ-3)-Ala-Darg-NH2

5

H-Ttda-(BHQ-3)

6

H-Ttda-BocApmba4

7

H-Pro-Leu-Gly-Leu-Lys(Boc-Apmba4)-Darg-NH2

1042

8

IRDye800CW-Pro-Leu-Gly-Leu-Lys

2268

975 975 1283 1283 750 511

(BHQ-3)-Ala-Arg-NH2

were removed on a rotary evaporator, and the paste obtained was treated with a mixture of CH2Cl2/TFA/TIPS (5 mL, 95:2:3) and stirred for 2 h at r.t. to remove the trityl group. After evaporation of the volatiles, the paste was redissolved in H2O/CH3CN (5 mL, 1:1) and loaded onto a preparative HPLC column that had been pre-equilibrated with 5% B. After the sample was applied to the column, the eluant was ramped to 20% B over 1 min, followed by a linear gradient to 45% B over 50 min. Fractions of >95% purity were pooled and freezedried to afford 6 in 55% yield (62 mg). tR = 6.47 min using gradient elution from 15% to 35% B over 20 min. MS: m/z = 510.8 [MþH]þ; 256.2 [Mþ2H/2]. IRDye 800CW-Ttda-Apmba4 (11). IRDye 800CW-NHS ester (5 mg, 0.0045 mmol) was added to a solution of 6 (10 mg, 0.02 mmol) in CH2Cl2 (0.5 mL), followed by DIEA (10 mg, 0.077 mmol). The solution was stirred for 4 h at r.t., protected from light. After completion of the coupling reaction, volatiles were removed on a rotary evaporator and the paste obtained was treated with a mixture of CH2Cl2/TFA/TIPS (5 mL, 95:2:3) and

9


10

(BHQ-3)-Ala-Darg-NH2 IRDye800CW-Ttda-(BHQ-3)

1735

11

IRDye800CW-Ttda-Apmba4

1397

12


1928

2268

(Apmba4)-Ala-Darg-NH2

stirred for 2 h to remove the trityl group. After evaporation of the volatiles, the paste was redissolved in H2O/CH3CN (5 mL, 1:1) and purified by preparative HPLC as described for 8 and 9. Fractions of >95% purity were combined and freezedried to afford 2.8 mg (44%) of dark blue IRDye 800CW-Ttda-Apmba4 (11) (tR = 6.98 min using a linear gradient of 2060% B over 20 min). Detection: 701 and 790 nm. MS: 1396.7 [MH]; 696.5 [M2H]/2. H-Pro-Leu-Gly-Leu-Lys(t-Boc-Apmba4)-Ala-D-arginine-NH2 (7). HSPyU (40 mg, 0.10 mmol) was added to a solution of 16 1290



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Table 2. Results from MMP Cleavage Assaya peptide without enzyme peptides IRDye800CW-PLGLK- (BHQ-3)-AR-NH2 (8)

MMP-1

T = 0 (60) min

max RFU*

IRDye800CW-Ttda-BHQ-3 (10) a

MMP-3

MMP-9

slope Y-int. slope Y-int. slope Y-int. slope Y-int.

MMP-13 slope

Y-int.

28 (22)

1755 ( 105 0.42 2.84 1.18 3.44 0.91 2.04 2.77 3.89

6.72

2.98

28 (25)

0.28 3.47 1.60 3.51 0.76 2.33 2.41 2.85

6.66

4.84

0.23 2.98 1.27 3.58 0.57 3.00 2.38 3.56

6.72

4.39

6.70

5.65

26 (23) IRDye800CW-PLGLK- (BHQ-3)-A-r-NH2 (9)

MMP-2

70 (63) 119 (120)

1574 121 (no cleavage

121 (no cleavage)

Int. = intercept. Max RFU = maximum fluorescence intensity (observed after 60 min at 37 °C in the presence of MMP-13).

Figure 5. In vitro cleavage of IRDye 800CW-PLGLK(BHQ-3)ARNH2 (8) by MMP-2, -9, and -13 at r.t. (∼23 °C). Cleavage is fastest with MMP-13.

(25 mg, 0.08 mmol) in CH2Cl2 (2 mL), followed by DIEA (50 mg, 0.38 mmol). The mixture was stirred for 30 min at r.t., FmocPro-Leu-Gly-Leu-Lys-Ala-D-arginine-NH2 2 (95 mg, 0.10 mmol) was added, and the reaction was stirred for an additional 3 h. The reaction mixture was diluted to 10 mL with dry DMF, piperidine (0.2 mL) was added, and the mixture was stirred for 30 min. After removal of the Fmoc group was completed, the reaction mixture was diluted with water to 50 mL and loaded onto the preparative HPLC column. Purification was performed as described for 3 and 4, using UV at 230 nm as an indicator of product elution. Productcontaining fractions of >95% purity were combined and freezedried. By HPLC using gradient elution from 15% to 35% B over 20, the isolated peptide 7 had a tR = 9.98 min. MS: m/z = 1043.2 [MþH]þ; 522.4 [Mþ2H]2þ/2. IRDye 800CW-Pro-Leu-Gly-Leu-Lys(Apmba4)-Ala-D-arginine-NH2 (12). Compound 12, an authentic standard for the azo-bond cleavage product of 9 was prepared by coupling the t-Boc-Apmba4-peptide containing compound 7 (5 mg, 0.005 mmol) with IRDye 800CW-NHS ester (5 mg, 0.0046 mmol), following the procedure described for 8 and 9. 12 was isolated as a bluegreen solid in 33% yield (3 mg). By HPLC using gradient elution from 20% to 60% B over 20 min, the compound had a tR = 10.91 min. Detection: 701 and 790 nm. MS: m/z = 1926.4 [MH]; 1283.8 [2M2H]2/3; 962.4 [M3H]3/2. In Vitro Cleavage Studies with MMPs. Fluor/quenchercontaining peptides were tested in an in vitro assay to determine their relative rates of cleavage by MMPs. MMPs (AnaSpec) were stored in 50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2 (pH 7.5) buffer at 80 °C until use. APMA (aminomethylphenyl mercuric acetate) was purchased from Sigma. The assay was performed in 96-well plates (NUNC MaxiSorb black plate) on a fluorescence plate reader (BioTek-Synergy2), using excitation and emission

filters with 780 ( 20 and 820 ( 20 nm cutoff values, respectively. Peptide stock solutions (500 μM) in DMSO were diluted to 10 μM using assay buffer, just prior to assay. APMA solutions (10 mM) were prepared in water. MMPs (supplied at a concentration of 1 μg in 100 μL) were activated on the day of the study with 1 mM APMA just prior to assay, using incubation/activation times based on the manufacturer recommendations. As an example, MMP-13 (1 μg in 100 μL) was diluted to 450 μL in MMP assay buffer, mixed with APMA (10 mM, 50 μL), and incubated at 37 °C for 45 min. It was then cooled in ice (4 °C), diluted to 1.25 mL in assay buffer to give a final enzyme concentration of 0.8 μg/mL, and stored in ice until use. Each IRDye800-containing compound (50 μL of a 10 μM solution, ∼500 pmol/well) was added to duplicate wells in a 96-well plate, followed by 50 μL of assay buffer. Various activated MMPs were added to the wells (50 μL, 0.8 μg/mL, 40 ng/well), providing a peptide to enzyme ratio of about 1000:1. The plate was incubated at r.t. (∼23 °C) for up to 90 min. The progress of the enzymatic cleavage was determined by measuring the relative increase in fluorescence (RFU) using the plate reader. A parallel experiment was carried out at 37 °C to determine the maximum RFU emitted by the fully cleaved IRDye 800CW dye-containing fragment from each peptide tested. Data were normalized and plotted as % cleaved, rather than as relative fluorescence units (RFU), to allow comparison between different compounds with different final fluorescence values. The normalized data were plotted as % maximum vs time. From this, the initial slope and the Y-intercept were determined to obtain the rate of cleavage and the background fluorescence of the fluorogenic peptide under study. LC/MS Analysis of In Vitro Cleavage Products. Following MMP cleavage studies with 8 or 9, a 100 μL aliquot of the test well was analyzed by LC/MS to establish identity of the cleavage fragments formed. In Vivo Metabolism Studies. Animal studies with 8, 9, and 10 were conducted in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals as well as institutional guidelines. Subjects (male Balb/cJ mice) were restrained in a Decapicone restrainer (Braintree Scientific). Test compound (40 nmol, 0.1 mL) was administered via the lateral tail vein. Retro-orbital or saphenous blood samples (1550 μL) were collected from conscious animals at 3, 8, 20, 40, and 60 min into heparinized microvettes (Sarstedt Microvette CB300) and placed on ice immediately. Within 10 min of collection, each sample was centrifuged at 4 °C for 15 min at 1000 g to remove the cellular fraction. Proteins were precipitated from a known 1291



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Figure 6. Time course for in vivo metabolism of IRDye 800CW-PLGLK(BHQ-3)AR-NH2 (8) in mouse plasma. Over time, 8 is metabolized to a number of products. Peaks from tR = 710 min contain spectral features for IRDye 800CW only; no BHQ-3 absorbance peak is observed.

Figure 7. Time course for metabolism of IRDye 800CW-Ttda-(BHQ-3) in mouse plasma. Within 3 min, significant metabolism is evident, as shown by the rapid conversion of 10 at tR of 12.65 min to a fluorescent metabolite with tR of 10 min. By 20 min, almost no 10 remains. The primary metabolite coelutes with compound 11.

volume of plasma using an equal volume of 50% EtOH, followed by a 3 volume of icecold CH3CN, and centrifugation

for 20 min at 20 000g at 4 °C. The supernates were taken to dryness (Speedvac), resuspended in 30% MeOH, and analyzed 1292



Figure 8. (A) Normalized absorbance spectrum of BHQ-3-O-succinimidyl ester (blue) and the fluorescence emission spectrum of IRDye 800CW in water (red). The spectral characteristics of the IRDye 800CW/BHQ-3 fluor/quencher pair are not suitable for quenching by FRET. (B) Normalized absorbance spectra of BHQ-3-O-succinimidyl ester (blue), IRDye 800CW (red), and IRDye 800CW-Ttda-(BHQ-3) 10 (green) in water. Conjugation of the quencher and fluor via a PEGtype linker causes a significant shift in the absorbance maxima associated with the quencher and fluor, suggestive of contact quenching.

by LC-MS at pH 6.8 on a Phenomenex Luna C8(2) column eluted with a gradient of 0100% B over 20 min, where A = H2O, and B = 1:1 CH3CN/MeOH, both A and B containing 1 g/ LNH4OAc. Urine was collected from subjects following sacrifice by cervical dislocation, chilled on ice immediately, and treated as described for plasma. In Vivo Metabolism Studies and Plasma Recovery. Compounds 8 and 10 were dissolved in DMSO to a concentration of 400 μM. For 10, a second standard was prepared at 80 μM by diluting the 400 μM standard 1/5 in DMSO. These stock solutions were diluted to 1 μM, 20 μM, and 400 μM using mouse plasma (Lampire Biological Laboratories, Inc. or Innovative Research, Na Citrate anticoagulant). Samples were chilled on ice immediately, and then processed, extracted, resuspended, and HPLC analyzed in an identical fashion to that used for the in vivo metabolism samples above.

’ RESULTS Synthesis of IRDye800/BHQ-3 Fluorogenic Constructs. IRDye 800CW-PLGLK(BHQ-3)AR-NH2 (8) and a “noncleavable” control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane [Ttda])-BHQ-3 (10) were prepared from the corresponding

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precursors, Fmoc-PLGLKAR-NH2, and Trt-Ttda linker-NH2 by coupling first with (BHQ-3)-O-Succinimide, followed by coupling to IRDye 800CW-NHS, after removal of the Fmoc/Trt protecting group. The synthesis of 8 is outlined in Figure 2. Its D-arginine analogue 9 and a des-Arg analogue (data not shown) were similarly prepared. The synthetic scheme used to prepare 10 is shown in Figure 3. Synthesis of Azo-Bond-Cleaved Metabolites. Metabolism studies in mice (vide infra) demonstrated that the azo bond of the BHQ-3 quencher in 10 was readily cleaved in vivo. An authentic sample of metabolite IRDye 800CW-Ttda-Apmba4 (11) was prepared by the reaction of (H-Ttda-Boc-Apmba4) 6 [Apmba = 4-N-(4-Aminophenyl)-4-N-methylbutyric acid)] with IRDye 800CW-NHS ester, followed by removal of the trityl group. The synthesis of the Apmba4 precursor 16 used to prepare 6 is shown in Figure 4. 16 was also used in the preparation of IRDye 800CW-Pro-Leu-Gly-Leu-Lys(Apmba4)-Ala-D-arginine-NH2 (12), an authentic standard of the azo-bond cleavage product of 9. All compounds prepared had a purity of >95% by HPLC and displayed mass spectral and UV/vis data consistent with their proposed formulation. Table 1 summarizes the compounds prepared. In Vitro MMP Cleavage Assay. Summary results from the fluorogenic cleavage assay with various matrix metalloproteases (MMPs) are shown in Table 2. Almost complete (>98%) selfquenching was found for 8 until it was cleaved by MMP-13, causing fluorescence to rise from 28 ( 1 relative fluorescence units (RFU) at t = 0 to 1755 ( 105 after 60 min (n = 3), a 56-fold increase in Fl with enzyme treatment. Similar results were observed for D-arginine analogue 9. For 8, slower cleavage was seen with other MMPs (Figure 5). Under similar conditions, 10 had values of 119 and 121 RFU in the absence of MMPs at t = 0 and 60 min, revealing less complete quenching; no cleavage was noted when treated with MMP-13 in vitro (Supporting Information Figure 1). Analysis of well contents by LC/MS after MMP cleavage revealed that the expected cleavage fragments IRDye 800CW-PLG-OH and NH2-LK(BHQ-3)-AX (X = R, r) were formed from 8 and 9. In Vivo Metabolism. As shown in Figure 6, following administration of 8 to normal mice, rapid metabolism was observed in mouse plasma in vivo. A major metabolite at tR = 11.5 min was identified as the dearginated compound IRDye 800CW-PLGLK(BHQ-3)A-OH, by comparison to an authentic standard (data not shown). Administration of 9, which contains D-arginine in the place of the N-terminus Arg of 8 prevented the formation of the dearginated metabolite, but polar metabolites eluting between 8 and 10 min were still formed. This finding indicates that these polar products did not form as a result of exopeptidase activity. Using LC/MS, many of the metabolites were identified (Figure 9), based on their spectral characteristics (Supplementary Figure 2) and molecular weights, and/or by comparison to authentic standards. IRDye 800CW-PLGOH, the same product formed in the in vitro MMP assay, was a major component found in vivo in both plasma and urine. This suggests that at least one of the MMPs found in normal mice can cleave 8 and 9. Surprisingly, “noncleavable” control 10, which contains only a PEG spacer between IRDye 800CW and BHQ-3, was very rapidly metabolized (Figure 7). LC/MS analysis of mouse plasma extracts demonstrated that the key metabolite formed was 11, the azo-bond cleavage product of the BHQ-3 moiety in 10. HPLC traces comparing the retention times of authentic standards of 10 and 11 to those of the products found in mouse plasma at 3 min post administration (Supporting Information 1293



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Figure 9. Metabolites formed from IRDye 800CW-PLGLK(BHQ-3)AR-NH2 (8) in vitro and in vivo.

Figure 3) show excellent agreement between the retention time of the primary metabolite (tR = 9.7 min) and authentic standard 11. The azo bond in BHQ-3 is very rapidly cleaved in vivo. However, none of the metabolites observed contained any apparent damage to IRDye 800CW. Recovery results for 8 and 10 from mouse plasma were >90% at all spike levels tested (Supporting Information Table 1), with little degradation observed due to workup. These results demonstrate that the peaks observed in metabolism samples are due to in vivo metabolism, and not to post collection decomposition.

’ DISCUSSION The compounds described in this paper were prepared to determine how well BHQ-3 can quench fluorescence from the NIR fluorophore IRDye 800CW and whether such constructs are metabolized in normal mice. The data obtained indicate that BHQ-3 is an effective quencher for IRDye 800CW, but examination of the visible spectra of the compounds prepared indicates

that quenching is not likely due to FRET,4547 wherein excitation is transferred from a donor fluor to an acceptor molecule through dipoledipole interaction without the emission of a photon. Figure 8A shows an overlay of the fluorescence emission spectrum of IRDye 800CW and the absorption spectrum of (BHQ-3)-O-Su ester. The excitation and emission spectra show very poor overlap for the fluor and quencher, as the emission maximum for IRDye 800CW falls at 789 nm, whereas the absorption maximum for BHQ-3 is at a much shorter wavelength of 612 nm. As 8, 9, and 10 show relatively little florescence prior to cleavage, an alternate quenching mechanism to FRET must be proposed. Figure 8B compares the absorbance spectrum of BHQ-3 and IRDye 800CW when tethered together by a PEG linker in 10. The absorbance band for BHQ-3 has shifted 43 to 655 nm, and the absorbance maximum for IRDye 800CW has shifted 22 nm from 774 to 786 nm. Such shifts in absorbance maxima have been ascribed to the formation of an intramolecular ground state complex between the fluor and quencher, which leads to 1294


Bioconjugate Chemistry suppression of fluorescence. Johansson et al. have reported that the azo-aromatic dark quencher BHQ-1 appears to quench the fluor Cy3.5 primarily via static quenching, also known as contact quenching, wherein the fluor and quencher form an intramolecular complex.14,15,48,49 Such an intramolecular complex is likely to be operative in the compounds reported here. No metabolism of the IRDye 800 fluor was observed during the in vitro or in vivo studies described here. However, the results obtained with 10 suggest that BHQ-3 is not an ideal quencher for use in vivo. The formation of the fluorescent aromatic aminecontaining metabolite 11 via cleavage of the azo bond in the quencher was found to be facile. This should not be a surprising result, as there is a precedent for reductive cleavage of the azo linkage in vivo. Reductive enzymes in the liver and intestinal microbial azoreductases have long been known to catalyze the reductive cleavage of the azo linkage in dyes.50 In spite of our negative findings with the IRDye800CW-PEGBHQ-3 constructs, where we observed rapid breakdown of the azo bond of BHQ-3 in the PEG-linked construct in vivo, we have had some success with MMP cleavable BHQ-3 containing constructs, both in vitro (in assays of MMP activity in samples of human synovial fluid) and in imaging studies in vivo with arthritic mice. However, imaging results in arthritic mice in vivo were modest, presumably due to nonspecific cleavage of the quencher. Signal increases in arthritic paws were observed, suggestive of enzymatic cleavage, but the resulting target to background ratios were lower than desired. These data will be reported elsewhere. We hypothesize that, if BHQ-3 is used in vivo as a quencher in an enzyme-cleavable probe, the effectiveness of such a construct as a probe for protease activity will be affected by the rate at which such azo bond cleavage takes place, relative to probe activation of a fluor/quencher construct by cleavage at the desired cut site. In support of this hypothesis, we note that researchers who have used BHQ-3 as a quencher in vivo (using intratumoral injection or intracartilage injection, rather than systemic (tail vein) injection have had mixed results. With direct intertumoral injection, Lo et al.51 found that the fluorescent signal in vector positive tumors was only twice that of the FAP vector negative tumor line control. In contrast, Lee et al.52 found that injection of Cy5.5GPLGMRGLGK-(BHQ-3) into osteoarthritis-induced cartilage produced a strong fluorescent signal (up to 7.4 ( 1.4-fold increase relative to the normal joints. However, recent results indicate that use of FRET type fluor/ quencher constructs containing BHQ-3 under hypoxic conditions in vitro might prove problematical, based on the recent work of Kiyose et al.53 They surmised that a cyanine dye-containing (Cy5, Cy5.25, or Cy5.5)/BHQ-3 construct was reduced under hypoxic conditions, generating fluorescence. No characterization of the product was reported, but as the linker between the fluor and quencher was noncleavable, azo bond reduction of the BHQ-3 quencher was hypothesized, leading to generation of fluorescent signal due to the loss of FRET between the cyanine dye and the quencher. The group proposes use of this finding for the imaging of hypoxia. These varied results suggest that care should be taken to characterize metabolites if BHQ-3 is used in vivo.

’ CONCLUSIONS Efficient quenching of the fluorescence emissions from IRDye 800CW by BHQ-3 was found for IRDye 800CW-PLGLK(BHQ-3)-AR-NH2 8, D-arginine analogue 9, and IRDye 800CWTtda-BHQ-3 10. Since the spectral overlap for IRDye 800CW

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and BHQ-3 is not optimal for FRET quenching, contact quenching must be inferred, and appears to be a viable quenching approach for this fluor/quencher pair. The peptide backbone in 8 and 9 is readily cleaved by MMPs in vitro, generating the fluorescent IRDye 800CW-PLG-OH fragment expected, based on the known protease cut site in the starting peptide. Following in vivo administration of 8 and 9, many metabolites were observed in normal mouse plasma and urine. All observed metabolites in urine were fluorescent. Although the enzyme(s) responsible for cleavage in vivo are not known, one major fluorescent metabolite in plasma and urine corresponds to that formed by cleavage at the known MMP cut site for the peptides. A second (nonfluorescent) plasma metabolite corresponded to loss of the terminal Arg. Replacement of this residue with D-arginine halted deargination, but cleavage at the MMP cut site as well as cleavage of the azo bond in the BHQ-3 quencher were still observed. Most of the metabolites formed will contribute to background, thereby reducing the ability to specifically detect MMP activity in pathologies such as tumors or arthritis. Metabolism studies with IRDye 800CW-Ttda-BHQ-3 10 in normal mice clearly revealed the in vivo instability of the azo bond in BHQ-3 quencher. The primary metabolite observed had a molecular weight consistent with azo bond cleavage of the quencher, and coeluted with an authentic standard of the proposed metabolite. Thus, although IRDye 800CW and BHQ-3 appear to be a suitable Fl/Q pair, BHQ-3 should be used with caution in vivo.

’ ASSOCIATED CONTENT

bS

Supporting Information. Results from fluorogenic MMP cleavage assay, UV/vis spectra of the major metabolites of 8, and HPLC comparison of the metabolite of 10 in mouse plasma, and that of an authentic sample of azo-bond cleaved product 11. This material is available free of charge via the Internet at http://pubs. acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Karen E. Linder, E-mail: [email protected]. Phone:609683-0483. Present Addresses †

Department of Radiology, The Ohio State University Medical School Biomedical Research Tower, Rm. 710, 460 W. 12th Ave, Columbus, Ohio 43210, United States.

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