Synthesis and In Vivo PET Imaging of Hyaluronan Conjugates of

Article pubs.acs.org/bc

Synthesis and In Vivo PET Imaging of Hyaluronan Conjugates of Oligonucleotides Satish Jadhav,† Meeri Kak̈ ela,̈ ‡ Jussi Mak̈ ila,̈ ‡,§ Max Kiugel,‡ Heidi Liljenbac̈ k,‡,∥ Jenni Virta,‡ Paï vi Poijar̈ vi-Virta,† Tiina Laitala-Leinonen,‡ Ville Kytö,⊥ Sirpa Jalkanen,# Antti Saraste,‡,⊥,¶ Anne Roivainen,‡,∥ Harri Lönnberg,† and Pasi Virta*,† †

Department of Chemistry, University of Turku, FI-20014 Turku, Finland Turku PET Centre and ⊥Heart Center, University of Turku and Turku University Hospital, FI-20520 Turku, Finland § Department of Cell Biology and Anatomy, ∥Turku Center for Disease Modeling, #MediCity Research Laboratory and Department of Medical Microbiology and Immunology, and ¶Institute of Clinical Medicine, University of Turku, FI-20520 Turku, Finland ‡

S Supporting Information *

ABSTRACT: Synthesis for 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated oligonucleotide hyaluronan (HA) tetra- and hexasaccharide conjugates is described. A solid-supported technique is used to introduce NOTAchelator into the 3′-terminus of oligonucleotides and a copper-free strain promoted azide alkyne cycloaddition (SPAAC) to HA/ oligonucleotide conjugation. Protecting group manipulation, required for the HA-moieties, is carried out after the SPAACconjugation. Positron emission tomography (PET) is used (1) in the whole-body distribution kinetic studies of the conjugates in healthy rats and (2) to show the potential of hyaluronan-induced targeting of oligonucleotides into the infarcted area of rats with myocardial infarction.

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INTRODUCTION Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide composed of a repeating disaccharide unit of D-glucuronic acid and N-acetyl-D-glucosamine (Figure 1). It is essential for proper

play an important role in cell migration, tumorigenesis, metastasis, and regulation of immune responses.5 However, all CD44-expressing cell types do not bind HA. The CD44 is composed of many transmembrane glycoproteins with extensive molecular heterogeneity. The biological functions of CD44 are based on HA-binding phenotype and three different states may be distinguished with respect to HA binding: nonbinding, nonbinding unless activated by physiological stimuli, and constitutively binding.6 An up-regulation of CD44 occurs in many cancers of epithelial origin.7 Furthermore, the CD44 structures in cancer cells are more diverse and they usually show enhanced HA binding compared to that of normal cells.5 Under inflammatory conditions, in turn, the binding of CD44 to HA on endothelial cells is induced on T lymphocytes and monocytes after

Figure 1. Structure of hyaluronan.

cell growth, organ structural stability, and tissue organization and is involved in a variety of biological processes including cell differentiation, phagocytosis, angiogenesis, and some pathological conditions, e.g., inflammation and cancer.1,2 HA is also recognized as a pharmacologically active signaling molecule and a variety of cell types respond to HA of different sizes.3 Interaction between HA (length of 6−8 monosaccharides4) and CD44 (a major cell-surface receptor for HA) has been found to © 2015 American Chemical Society

Special Issue: Molecular Imaging Probe Chemistry Received: September 2, 2015 Revised: October 27, 2015 Published: October 30, 2015 391

DOI: 10.1021/acs.bioconjchem.5b00477 Bioconjugate Chem. 2016, 27, 391−403

Article

Bioconjugate Chemistry Scheme 1a

a

Conditions: (i) TMSOTf, DCM, 0 °C, (ii) Et3N·3HF, THF, r.t., (iii) Cl3CCN, DBU, 0 °C, (iv) NH2NH2·H2O, AcOH, pyridine, r.t.

Scheme 2a

a

Conditions: (i) TMSOTf, DCM, 0 °C, (ii) NH2NH2·H2O, AcOH, pyridine, r.t., (iii) p-TSA, MeOH, r.t.

of the oligonucleotide phosphorothioates was tested in a CD44-expressing cell line. In the present study, in vivo targeting of 68Ga-labeled 1,4,7triazacyclononane-1,4,7-triacetic acid (NOTA)-chelated oligonucleotide HA-conjugates, 68Ga-NOTA-HA-hexasaccharide-T6 18, 68Ga-NOTA-HA-hexasaccharide-anti-miR-15b 19, and 68 Ga-NOTA-HA-tetrasaccharide-anti-miR-15b 20, (anti-miR15b = a 22-mer 2′-O-methyl oligoribonucleotide: 5′-UGU AAA CCA UGA UGU GCU GCU A-3′) in rats were studied using PET. Recent studies suggest that inhibition of miR-15b protects against cardiac ischemic injury.21 The major challenge for successful drug (i.e., anti-miR-15b) action is to ensure efficient strategies for cell-specific targeting and cellular delivery. Conjugation to hyaluronic acid may meet these

activation by antigen and stimulation by inflammatory agents, respectively.8 The existing HA-CD44 interaction may hence offer a potential way to use HA conjugates of certain drugs in therapeutic applications.9−15 Enrichment of HA conjugate of potential therapeutic oligonucleotides (miRNA, siRNA, and antisense oligonucleotides)16−19 on the CD44 expressing cell surface could lead to enhanced internalization by endocytosis and eventually also to increased concentration in the cytoplasm. We have previously studied clustered HA disaccharides as mediators of cellular delivery of antisense phosphorothioate oligonucleotides through receptor-mediated endocytosis.20 The influence of the conjugated HA-clusters on the cellular uptake 392


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Bioconjugate Chemistry Scheme 3a

Conditions: (i) 11 or 12 + 9, 10 or 15 (2.0 equiv) in MeCN-H2O (1:9, v/v), overnight at r.t., (ii) 0.1 mol L−1 aq NaOH, 3 h at 55 °C, (iii) conc. aq NH3, 5 days at 55 °C, (iv) Ac2O, Et3N, H2O, CH3CN, 6 h at r.t., (v) conc. aq NH3, overnight at 55 °C. RP HPLC: An analytical RP-column (C-18, 250 × 5 mm, 5 μm), a gradient elution 0−40% MeCN in 0.1 mol L−1 aqueous Et3NH+AcO− in 25 min, λ at 260 nm. Notation: anti-miR-15b = a 22mer 2′-O-methyl oligoribonucleotide: 5′-UGU AAA CCA UGA UGU GCU GCU A-3′. a

challenges via the CD44/HA-binding mechanism related to inflammatory conditions. The conjugates (18−20) were first intravenously administered in healthy rats and the whole-body distribution kinetics were studied by dynamic in vivo PET imaging. Uptake of the conjugates with HA hexasaccharide (18 and 19) was then studied in more detail in the infarcted area of rats with myocardial infarction (infarction-to-remote ratio was evaluated by digital autoradiography). For the synthesis of the

conjugates, our recently described solid-supported technique22,23 was utilized to introduce NOTA-chelator into the 3′terminus of oligonucleotides (anti-miR-15b and T6). A copperfree strain-promoted azide alkyne cycloaddition (SPAAC)24 was then used to HA/oligonucleotide conjugation into the 5′terminus. It may be emphasized that the complex protecting group manipulation [viz., (1) hydrolysis of the benzoyl protections and methyl esters of the glucuronic acid units, 393


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Bioconjugate Chemistry

released from the support by a two-step cleavage protocol, viz., treatment with aq. NaOH (to hydrolyze the carboxylic methyl esters) followed by conventional ammonolysis.22 The conjugates (11, 12) were purified by semipreparative RP HPLC and their identity was verified by ESI-MS. 3-Azidopropyl hexasaccharide 9 and tetrasaccharide 10 were conjugated to cyclooctyne functionalized oligonucleotides 11 and 12 by copper-free strain promoted azide alkyne cycloaddition (SPAAC) in aq MeCN, which according to RP HPLC analysis (after global deprotection) proceeded virtually quantitatively. The protecting groups of the sugar moieties were removed in two steps: the methyl ester protections of the carboxy functions were first removed by brief hydrolysis in aq NaOH and the trichloroacetyl groups from the amino functions by 5d ammonolysis at 55 °C. The structure of the fully deprotected conjugates (13, 14) was verified by RP HPLC and ESI-MS. Peracetylated maltohexose (15) was conjugated similarly as a 3-azidopropyl glycoside, but the conjugate was deprotected by overnight ammonolysis at 55 °C to obtain 21. To acetylate the amino groups of the glucosamine units, the conjugates were repeatedly treated with a mixture of Et3N and Ac2O in aq MeCN. The completion of the acetylation was verified by HPLC and ESI-MS. The acetylation was not, however, selective for aliphatic amines but the nucleobase amino groups were also acetylated (16, 17). That is why the conjugates had to be once again treated with concentrated ammonia, which gave the desired N-acetylated glucosamine products (18, 19, and 20). The conjugates were purified by RP HPLC and their identity was verified by ESI-MS (Table 1).

(2) ammonolysis of the trichloroacetyl groups, and (3) selective N-acetylation of the exposed glucosamine units, Scheme 3] was successfully carried out after the SPAACconjugation. Ga68−NOTA-maltohexose anti-miR-15b 21, 68GaNOTA-anti-miR-15b 22, and 68Ga-NOTA-T6 23 were also prepared and used as reference compounds (together with 68 Ga-NOTA 24) in the whole-body distribution studies.

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RESULTS AND DISCUSSION Synthesis of 3-Azidopropyl Tetra- and Hexa-Saccharide Precursors of Hyaluronan Conjugates. Hyaluronan hexasaccharide has been previously synthesized by several slightly different strategies.25−29 In addition, hyaluronan oligomers have been successfully assembled from dimeric building blocks on a solid support.30 The solution phase methods fall in two categories: either the glucuronic acid unit is introduced as a carboxy ester,26,27,29 or the disarming carboxy function is generated by oxidation after assembly of the hexasaccharide.25,28 The amino function has been protected with the trichloroacetyl,26,27 phthaloyl,25,28 or 2,2,2-trichloroethoxycarbonyl29 group. A thioglycoside25−27,29 or trichloroacetimidate28 method has been applied to the assembly of the hexasaccharide from disaccharide donors. Based on these previous studies, we prepared the 1-O-(3-azidopropyl) derivative of hyaluronan hexasaccharide as depicted in Schemes 1 and 2. Of the starting monosaccharides, (methyl 2,3-di-Obenzoyl-4-O-levulinoyl-β-D-glucopyranosyluronate) trichloroacetimidate (1) was synthesized according to Bindschädler et al.31 and tert-butyldimethylsilyl 4,6-O-benzylidene-2-deoxy-2trichloroacetamido-β-D-glucopyranoside (2) according to Palmacci and Seeberger32 (Scheme 1). Trimethylsilyl triflate promoted glycosidation of 1 with 2 in DCM gave tertbutyldimethylsilyl (methyl 2,3-di-O-benzoyl-4-O-levulinoyl-β-Dglucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2-deoxy-2trichloroacetamido-β-D-glucopyranoside (3) that was then converted to trichloroacetimidate glycosyl donor 4 by desilylation with Et3N·3HF in THF and subsequent treatment with Cl3CCN in the presence of DBU. The next step, glycosidation with 3-azidopropanol, turned out to be unexpectedly difficult. The 3-azidopropyl glycoside 5 was obtained in only 40% yield. The levulinoyl protection was finally removed with hydrazinium acetate in pyridine (6). Trichloroacetimidate donor 4 was glycosidated with 3azidopropyl disaccharide acceptor 6 by trimethylsilyl triflate activation in DCM (Scheme 2) to give tetrasaccharide 7 (33%). The levulinoyl group of 7 was removed and tetrasaccharide 8 was used as an acceptor in the next glycosylation reaction with 4. The crude hexasaccharide obtained was subjected to acidolytic removal (p-TSA in MeOH) of the benzylidene protections and purified by RP HPLC (9, overall yield 16%, calculated from 8). The benzylidene protections of tetrasaccharide 7 were removed in similar manner to give 10 (74%). The partially protected hexasaccharide (9) and tetrasaccharide (10) were used for the conjugations to cyclooctyne-modified oligonucleotides. Preparation of Hyaluronan Conjugates of NOTAFunctionalized Oligonucleotides. A 22-mer 2′-O-methyl anti-miR-15b sequence and T6 were assembled on a NOTA modified CPG support22 by the phosphoramidite chemistry. A cyclooctynyl group was introduced to the 5′-terminus by an additional coupling cycle with 2-(bicyclo[6.1.0]non-4-yn-9yl)ethyl 2-cyanoethyl N,N-diisopropylphosphoramidite (BCN phosphoramidite) (Scheme 3). The oligonucleotides were

Table 1 entry

conjugate

calculated molecular mass

observed molecular massa

1 2 3 4

18 19 20 21

3755.9 9306.6 8927.3 9141.5

3755.4 9306.5 8927.8 9142.2

a

Observed monoisotopic masses are calculated from [M-nH]n− and from the corresponding potassium and sodium adducts.

It may be emphasized that the reaction conditions required for the HA moieties (18−20) could be monitored by RP HPLC and ESI-MS throughout the synthesis. This was the main reason for the postsynthetic protecting group manipulation. Once the conditions have been optimized, conjugation between the prefabricated units (i.e., HA hexa- and tetrasaccharide) may, however, be the method of choice. Preparation of reference compounds 22 and 23 (i.e., oligonucleotides released from the supports prior to the coupling of BCN phosphoramidite) has been described previously.22 PET Studies. 68Ga-Radiochemistry. Each of the conjugates was subjected to radiolabeling with 68Ga as described previously.22,23 According to the radio-HPLC analysis, the radiochemical purities of the conjugates were 96 ± 0.21% (18), 94 ± 1.5% (19), 92% (20), 89% (21), 99% (22), 95 ± 1.7% (23), and 98 ± 0.049% (24). The retention times were 8.9 ± 0.042 min (18), 9.7 ± 0.071 min (19), 10 min (20), 10 min (21), 8.3 min (22), 5.4 ± 0.035 min (23), and 16 ± 0.015 min (24). Representative radio-HPLC chromatogram is shown in Figure 2. The specific radioactivity was 4.7 ± 2.1 MBq/nmol (average for 18−24) at the end of syntheses. 394


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Whole-Body Distribution Kinetics in Healthy Rats. Biodistribution of intravenously injected 68Ga-conjugates varied notably with the structure (Figures 3−5). The highest radioactivity concentration was observed in urinary bladder, kidneys, liver, intestine, spleen, and bone marrow. The distribution was, however, more dependent on the oligonucleotide sequence than on the conjugated sugar moiety. As seen, the uptake in kidney, bone marrow, salivary gland, and liver of conjugates with anti-miR-15b structure (19−22) was significantly higher (kidney: P ≤ 0.021, bone marrow: P ≤ 0.0044, salivary gland: P ≤ 0.00048, liver: P ≤ 0.049) compared to that of conjugates with T6 (18 and 23) (cf. especially the uptakes of 18 vs 19 and 22 vs 23). Among the anti-miR-15b conjugates (19−22), the sugar moiety showed some variation to the biodistribution: The highest bone marrow uptake was seen with 19 (i.e., the conjugate with HA hexasaccharide), whereas the

Figure 2. Example (68Ga-NOTA-HA-anti-miR-15b, 19) of radioHPLC chromatograms.

Figure 3. Radioactivity concentration at 60 min after intravenous injection of 68Ga-conjugates 18−24 as measured ex vivo by gamma counting of excised tissues of healthy rats. Results are expressed as standardized uptake value (SUV, mean ± SD). 395


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Figure 4. Representative whole-body coronal PET images of healthy rats intravenously injected with 68Ga-conjugates 18−24. Images represent average radioactivity concentration at 0 to 60 min after injection. 68Ga-radioactivity is distributed especially to heart (Hr), liver (Lv), kidneys (Kd), and urinary bladder (Bl). The intestine (In) uptake is observed with 19 and bone epiphyses (Ep) uptake with 21 and 22. SUV, standardized uptake value.

respectively (Figure 6E). Ex vivo gamma counting further confirmed the increased uptake of 68Ga-NOTA-HA-T6 (18) in the infarcted left ventricle (SUV 0.183 ± 0.003) compared to sham-operated control (0.100 ± 0.009, P = 0.006; Table 2). Immunohistochemistry showed CD44 positive cells within the infarcted area (Figure 6C).

highest salivary gland uptake was seen with 21 (i.e., the conjugate with maltohexose). A notable bone epiphyses (Ep) uptake was additionally observed with 21 (Figure 4). The other important observations were as follows: The intestine (full) uptake of 18 was significantly higher compared to 19−23 (P ≤ 0.018). The blood uptake of 22 was the highest. The lowest kidney uptake and the highest urine excretion were seen with 18. The urine uptake of 18 was significantly higher compared to that of all the other conjugates (P ≤ 0.031). The lung uptake of 18 was significantly lower compared to all other conjugates (19−23, P ≤ 0.010) and to NOTA-68Ga-complex alone. It may be emphasized that in vivo PET imaging cannot discriminate if the heart radioactivity is coming from myocardium or blood. The seemingly high heart signal, e.g., with compound 19 reflects the slow blood clearance. More detailed information about the accumulation in the infarction site was provided by digital autoradiography of heart cryosections (cf., accumulation of 18 and 19 in rats with myocardial infarction below). Rats with Myocardial Infarction. Representative autoradiograph (18 68Ga-NOTA-HA-T6), hematoxylin-eosin, and CD44 immunohistohemical staining of the heart left ventricle cross section in a rat with myocardial infarction are shown in Figure 6A−C. An increased uptake of 18 (68Ga-NOTA-HA-T6) into the infarcted myocardium was observed as compared with the remote noninfarcted areas and myocardium of sham-operated rats (Figure 6D−F). The infarction-to-remote ratio of 18 (68Ga-NOTA-HA-T6) and 19 (68Ga-NOTA-HA-anti-miR-15b) were 4.2 ± 0.75 (P = 0.008) and 1.1 ± 0.4 (P = 0.016),

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CONCLUSION Ga-NOTA-chelated oligonucleotide−HA conjugates [viz., 68 Ga-NOTA-HA-hexasaccharide-T6 (18), 68G-NOTA-HA-hexasaccharide-anti-miR 15b (19), and 68Ga-NOTA-HA-tetrasaccharide-anti-miR-15b (20)] were synthesized using a solidsupported technique to introduce NOTA-chelator into the 3′terminus and a copper-free strain promoted azide alkyne cycloaddition (SPAAC) to HA/oligonucleotide conjugation. A protecting group manipulation, required for the HA-moieties, was successfully carried out after the SPAAC-conjugation (i.e., in the presence of oligonucleotide sequence T6 and anti-miR15b). The whole-body biodistribution of the conjugates in healthy rats varied more with the oligonucleotide structure (anti-miR 15b vs T6) than with the conjugated sugar moiety (HA hexasaccharide, HA tetrasaccharide, maltohexose, or without sugar). The uptake in kidney, bone marrow, salivary gland, and liver of conjugates with anti-miR-15b structure (19−22) was significantly higher compared to that of conjugates with T6. Among the anti-miR-15b conjugates (19−22), conjugate with HA hexasaccharide (19) showed the highest bone marrow 68

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Figure 5. Time−activity curves of the distribution kinetics in healthy rats. SUV, standardized uptake value.

the length, sequence, and plausible backbone-modification) for the targeting.

uptake. HA hexasaccharide additionally increased the uptakes of oligonucleotides (T6 and anti-miR-15b, 18 and 19) into infarcted myocardium (with CD44 positive cells) as compared with the remote noninfarcted areas and myocardium of shamoperated rats. This uptake was, unfortunately, also strongly dependent on the oligonucleotide structure33 which diluted the role of HA in targeting of anti-miR-15b (19). The infarction-toremote ratios of 68Ga-NOTA-HA hexasaccharide-T6 (18) and 68 Ga-NOTA-HA hexasaccharide-anti-miR-15b (19) were 4.2 ± 0.75 (P = 0.008) and 1.1 ± 0.4 (P = 0.016), respectively. The observed infarction-to-remote ratio with 18 may be ascribed to the HA-CD44 interaction, demonstrating the potential of HA for the targeting of oligonucleotides into infarcted myocardium. Further studies are, however, required to evaluate the importance of the oligonucleotide structure (considering, e.g.,

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EXPERIMENTAL PROCEDURES

General Methods. The NMR spectra were recorded at 400 and 500 MHz. The chemical shifts are given in ppm from internal TMS. The mass spectra were recorded using a MS (ESI-TOF) spectrometer. RP HPLC analysis and purification of the oligonucleotides were performed using a Thermo ODS Hypersil C18 (150 × 4.6 mm, 5 μm) analytical column and a Phenomenex Oligo-RP C18 (250 × 10 mm, 5 μm) semipreparative column with a gradient elution from 0% to 40% MeCN in aqueous 0.1 mol L−1 Et3NH+AcO−. 1.0 mL min−1 (analytical) and 3.0 mL min−1 (semipreparative) flow rates and detection wavelength at 260 nm were used. 68Ga was 397


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Figure 6. 18 (68Ga-NOTA-HA-T6) and 19 (68Ga-NOTA-HA-hexasaccharide-anti-miR-15b) in rat myocardial infarction. (A) Autoradiograph showing focally increased uptake of 68Ga-NOTA-HA-T6 in the anterolateral wall (arrow) of the left ventricle at 7 days after coronary occlusion and (B) the same section stained with hematoxylin-eosin that shows an infarcted area in the anterolateral wall (arrow). (C) High-magnification photomicrograph of immunohistochemical staining shows CD44 positive cells (arrows) in the infarcted area (scale bar 50 μm). Radioactivity concentration expressed as (D) photostimulated luminescence per square millimeter (PSL/mm2) and (E) infarction-to-remote ratio in myocardial autoradiographs shows higher 18 (68Ga-NOTA-HA-T6) uptake in the infarcted than in the remote noninfarcted area or in the myocardium of shamoperated rats. (F) Time−activity curves of the distribution kinetics in rats (heart) with myocardial infarction. SUV, standardized uptake value.

to 0 °C under nitrogen and TMSOTf (28 μL, 0.15 mmol) was slowly added. The mixture was stirred at 0 °C for 45 min under nitrogen, neutralized by addition of triethylamine (100 μL), filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (25% EtOAc in petroleum ether) to yield 1.35 g (81%) of the desired disaccharide 3 as white solid. 1 H NMR (500 MHz, CDCl3): δ 0.04 (s, 3H), 0.07 (s, 3H), 0.83 (s, 9H), 2.01(s, 3H), 2.34 (m, 1H), 2.50 (m, 3H), 3.28 (m, 1H), 3.55 (ddd, 1H, J = 9.9 Hz, 9.8 Hz and 4.9 Hz), 3.67 (s, 3H), 3.83 (dd, 1H, J = 10.3 Hz and 10.2 Hz), 3.89 (dd, 1H, J = 10.3 Hz and 9.2 Hz), 3.91 (d, 1H, J = 10.1 Hz), 4.26 (dd, 1H, J = 4.6 Hz, 11.0 Hz and 4.6 Hz), 4.67 (dd, 1H, J = 9.5 Hz and 9.2 Hz), 5.03 (d, 1H, J = 7.9 Hz), 5.29 (d, 1H, J = 6.7 Hz), 5.37 (dd, 1H, J = 9.6 Hz and 5.0 Hz), 5.41 (dd, 1H, J = 8.0 Hz and 2.8 Hz), 5.51 (s, 1H), 5.56 (dd, 1H, J = 9.6 Hz and 9.5 Hz), 6.93 (d, 1H, J = 6.6 Hz), 7.29−7.38 (m, 11H), 7.82−7.87 (m, 4H); 13C NMR (125 MHz, CDCl3): δ −5.1, −4.3, 17.8, 25.6,

obtained in the form of [68Ga]Cl3 from a 68Ge/68Ga generator (Eckert & Ziegler, Valencia, California, USA). (Methyl 2,3-di-O-Benzoyl-4-O-levulinoyl-β-Dglucopyranosyluronate)trichloroacetimidate (1). This was prepared as described in the literature.31 The 1H and 13C NMR spectra were consistent with those reported. tert-Butyldimethylsilyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (2). This was prepared as described in the literature.32 The 1H and 13C NMR spectra were consistent with those reported. tert-Butyldimethylsilyl(methyl 2,3-di-O-benzoyl-4-O-levulinoyl-β-D-glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (3). Glycosyl donor 1 (1.34 g, 2.03 mmol) and glycosyl acceptor 2 (0.86 g, 1.62 mmol) were dried by repeated coevaporations with toluene and dissolved in dry DCM (18 mL) containing 4 Å molecular sieves. The mixture was cooled 398


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Table 2. Ex Vivo Biodistribution of 18 and 19 at 70 min after Intravenous Injection in Rats Studied at 7 Days after Myocardial Infarction or in Sham-Operation, and in Healthy Controlsa compound 18 tissue Adrenal gland BAT Blood Blood cells Bone Bone marrow Bonewbm Brain Heart left ventricle Intestine (empty) Intestine (full) Kidney Liver Lung Pancreas Plasma Salivary glands Skeletal muscle Skin Spleen Urine WAT

MI (n = 2) 0.10 0.086 0.23 0.18 0.055 0.043 0.043 0.013 0.18 0.47 1.5 3.0 0.41 0.19 0.080 0.40 0.083 0.037 0.20 0.29 480 0.048

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.027 0.0018 0.023 0.042 0.0075 0.037 0.0035 0.0024 0.0031 0.12 1.2 0.26 0.0041 0.0028 0.063 0.070 0.0062 0.0052 0.0024 0.070 130 0.014

compound 19

sham (n = 2) 0.079 0.058 0.19 0.17 0.051 0.069 0.032 0.014 0.10 0.32 0.42 2.9 0.41 0.17 0.071 0.36 0.071 0.031 0.14 0.26 260 0.030

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

healthy (n = 4)

0.018 0.0072 0.0027 0.012 0.0012 0.0075 0.0014 0.0047 0.0088 0.13 0.47 0.77 0.078 0.0075 0.0039 0.0078 0.0035 0.0012 0.0012 0.069 29 0.0050

0.071 0.063 0.19 0.14 0.035 0.053 0.032 0.0092 0.040 0.46 1.1 1.9 0.46 0.12 0.063 0.31 0.060 0.033 0.11 0.27 240 0.035

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.011 0.020 0.012 0.012 0.0082 0.014 0.011 0.00099 0.0068 0.22 0.42 0.23 0.040 0.020 0.018 0.027 0.0059 0.011 0.0082 0.032 120 0.031

MI (n = 4) 0.18 0.20 0.30 0.24 0.56 1.2 0.40 0.014 0.25 0.30 0.35 82 1.9 0.34 0.35 0.53 0.48 0.085 0.32 0.49 32 0.099

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

healthy (n = 4)

0.013 0.040 0.053 0.058 0.075 0.30 0.14 0.0079 0.015 0.065 0.18 2.4 0.26 0.067 0.070 0.12 0.083 0.013 0.028 0.085 3.4 0.024

0.17 0.15 0.17 0.090 0.35 1.5 0.12 0.011 0.16 0.37 0.20 160 1.7 0.31 0.38 0.37 0.43 0.056 0.34 0.66 21 0.047

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0063 0.017 0.011 0.031 0.053 0.20 0.038 0.0074 0.011 0.051 0.011 11 0.077 0.030 0.051 0.032 0.035 0.0060 0.028 0.12 2.3 0.013

a MI: rat with myocardial infarction; Sham: sham-operated rat; BAT: brown adipose tissue; WAT: white adipose tissue; Bonewbm: bone without bone marrow. Results are expressed as standardized uptake values (mean ± SD with two significant figures).

TOF): m/z calcd. for C43H40Cl6N2NaO16 [M + Na] 1073.0407, found 1073.0407. 3-Azidopropyl(methyl 2,3-di-O-benzoyl-4-O-levulinoyl-βD -glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2deoxy-2-trichloroacetamido-β-D-glucopyranoside (5). Glycosyl donor 4 and 3-azidopropanol were dried following the procedure described above for the synthesis of 3. The glycosidation (4, 0.47 g, 0.45 mmol and 3-azidopropanol, 0.46 g, 4.54 mmol) was then carried out in dry DCM (6.0 mL) under nitrogen at 0 °C using TMSOTf (4.1 μL, 22 μmol) as a Lewis acid. The reaction was stirred for 30 min under nitrogen, neutralized by addition of triethylamine (100 μL), filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (40% EtOAc in toluene) to yield 0.18 g (40%) of the product (5) as white foam. 1H NMR (400 MHz, CDCl3): δ 1.79 (m, 2H), 2.02 (s, 3H), 2.30−2.59 (m, 4H), 3.33 (m, 2H), 3.40 (m, 1H), 3.56 (m, 2H), 3.64 (s, 3H), 3.77−3.92 (m, 4H), 4.32 (dd, 1H, J = 10.5 Hz and 4.9 Hz), 4.66 (dd, 1H, J = 9.3 Hz, both), 5.04 (d, 1H, J = 7.7 Hz), 5.07 (d, 1H, J = 8.2 Hz), 5.39 (dd, 1H, J = 9.7 Hz and 9.6 Hz), 5.44 (m, 1H), 5.52 (s, 1H), 5.57 (dd, 1H, J = 9.5 Hz, both), 7.27−7.33 (m, 3H), 7.37−7.40 (m, 4H), 7.53−7.49 (m, 4H), 7.81−7.87 (m, 4H); 13 C NMR (100 MHz, CDCl3) δ 27.6, 29.0, 29.6, 37.5, 48.0, 52.9, 59.0, 66.1, 66.9, 68.6, 69.4, 71.9, 72.0, 72.6, 76.3, 80.0, 91.9, 98.8, 99.6, 101.4, 126.0, 128.3, 128.4, 128.7, 128.8, 129.2, 129.8, 130.0, 133.38, 133.42, 137.1, 162.1, 164.9, 165.6, 167.2, 171.2, 205.6; HRMS (ESI-TOF): m/z calcd. for C44H44Cl3N4O16 [M-H]− 989.1818, found 989.1817. 3-Azidopropyl(methyl 2,3-di-O-benzoyl-β-D-glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (6). To a solution of 5 (0.31 g, 0.31 mmol) in a mixture pyridine (2.87 mL) and acetic acid (0.71 mL), hydrazine monohydrate (104 μL, 2.16 mmol) was

27.7, 29.6, 37.6, 52.8, 61.6, 66.1, 68.7, 69.3, 71.9, 72.1, 72.5, 76.0, 79.8, 91.9, 93.8, 99.5, 101.4, 126.1, 128.37, 128.41, 128.7, 128.9, 129.1, 129.8, 129.9, 133.3, 137.1, 161.8, 164.9, 165.5, 167.0, 171.1, 205.6; HRMS (ESI-TOF): m/z calcd. for C47H54Cl3NNaO16Si [M + Na] 1044.2175, found 1044.2170. (Methyl 2,3-di-O-benzoyl-4-O-levulinoyl-β-D-glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl trichloroacetimidate (4). Et3N· 3HF (0.83 mL, 5.08 mmol) was added to a solution of disaccharide 3 (1.30 g, 1.27 mmol) in THF (15 mL). The reaction mixture was stirred overnight at ambient temperature, evaporated to dryness, and the residue was purified by silica gel chromatography (40% EtOAc in toluene). The resulted hemiacetal (1.06 g, 1.17 mmol, white foam) was dissolved in a mixture of DCM (6 mL) and Cl3CCN (10 mL), the mixture was cooled to 0 °C, and DBU (35 μL, 0.23 mmol) was added. The reaction mixture was stirred for 30 min, evaporated to dryness, and the residue was purified by silica gel chromatography (50% EtOAc in toluene) to yield 1.12 g (83%, overall from 3) of 4 as white foam. 1H NMR (500 MHz, CDCl3): δ 2.03 (s, 3H), 2.37 (m, 1H), 2.54 (m, 3H), 3.60 (m, 1H), 3.63 (s, 3H), 3.72 (dd, 1H, J = 10.4 Hz and 8.7 Hz), 3.94 (dd, 1H, J = 7.6 Hz, both), 4.10 (d, 1H, J = 10.0 Hz), 4.15 (dd, 1H, J = 7.6 Hz and 3.6 Hz), 4.23 (dd, 1H, J = 7.7 Hz and 3.5 Hz), 4.38 (dd, 1H, J = 10.6 Hz and 5.2 Hz), 5.08 (d, 1H, J = 8.0 Hz), 5.42 (dd, 1H, J = 9.8 Hz and 9.7 Hz), 5.51 (dd, 1H, J = 9.7 Hz and 8.0 Hz), 5.58 (s, 3H), 5.70 (dd, 1H, J = 9.6 Hz, both), 6.13 (d, 1H, J = 7.7 Hz), 7.33−7.41 (m, 7H), 7.45−7.53 (m, 4H), 7.88−7.90 (m, 2H), 7.94−7.97 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 27.6, 29.6, 37.6, 52.9, 63.3, 68.4, 68.6, 69.7, 71.6, 72.4, 72.6, 77.2, 78.2, 81.0, 86.2, 101.1, 101.4, 105.3, 126.0, 128.3, 128.4, 128.8, 129.0, 129.2, 129.90, 129.94, 133.37, 133.44, 136.8, 162.4, 163.2, 165.0, 165.5, 166.9, 171.2, 205.6; HRMS (ESI399


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10.4 Hz and 10.2 Hz), 3.21 (d, J = 3.3 Hz), 3.30−3.37 (m, 4H), 3.40−3.48 (m, 2H), 3.50−3.60 (m, 2H), 3.65 (s, 3H), 3.71 (s, 3H), 3.71−3.83 (m, 4H), 3.90−3.96 (m, 2H), 4.11 (ddd, J = 9.5 Hz, 9.2 Hz and 3.0 Hz), 4.31−4.37 (m, 2H), 4.44 (dd, 1H, J = 9.4 Hz, both), 4.57 (dd, 1H, J = 9.5 Hz and 9.4 Hz), 4.94 (d, 1H, J = 7.2), 4.99 (d, 1H, J = 8.2), 5.06 (d, 1H, J = 7.0 Hz), 5.11 (d, 1H, J = 8.3 Hz), 5.17 (s, 1H), 5.32−5.39 (m, 3H), 5.48 (dd, 1H, J = 9.3 Hz, both), 5.57 (s, 1H), 6.70 (d, 1H, J = 7.6 Hz), 6.94 (d, J = 7.4 Hz), 7.31−7.58 (m, 22H), 7.88−7.96 (m, 8H). 13C (125 MHz, CDCl3): δ 29.0, 48.0, 52.7, 53.0, 58.4, 58.8, 65.9, 66.2, 66.8, 67.7, 68.6, 70.2, 71.7, 72.2, 72.5, 73.8, 74.1, 75.1, 75.6, 76.0, 76.2, 79.4, 79.7, 92.1, 92.2, 98.6, 99.4, 99.6, 99.9, 101.15, 101.20, 125.9, 126.0, 128.32, 128.35, 128.39, 128.45, 128.86, 128.99, 129.0, 129.2, 129.80, 129.87, 129.90, 129.96, 133.26, 133.29, 133.33, 133.4, 137.0, 137.1, 161.4, 162.0, 165.0, 165.2, 165.3, 166.4, 168.2, 169.0; MS (ESI-TOF): m/z calcd for C75H70Cl6N5O27 [M - H]− 1682.24, found 1682.27. 3-Azidopropyl(methyl 2,3-di-O-benzoyl-4-O-levulinoyl-βD-glucopyranosyluronate)-(1 → 3)-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1 → 4)-(methyl 2,3-di-O-benzoylβ-D-glucopyranosyluronate)-(1 → 3)-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1 → 4)-(methyl 2,3-di-O-benzoyl-β-D-glucopyranosyluronate)-(1 → 3)-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (9). Glycosyl donor 4 and acceptor 8 were dried, following the procedure described above for the synthesis of 3. The glycosidation (4, 0.48 g, 0.46 mmol and 8, 0.20 g, 0.12 mmol) was then carried out in dry DCM (3.0 mL) under nitrogen at 0 °C using TMSOTf (4.2 μL, 23 μmol) as a Lewis acid. The reaction mixture was stirred for 1 h under nitrogen, quenched by addition of triethylamine (100 μL), filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (25% EtOAc in petroleum ether) to yield 0.13 g of crude white solid, which, in addition to the desired fully protected hexasaccharide, contained traces of the glycosyl donor 4. The crude product (50 mg) was dissolved in MeOH (5.0 mL) and a catalytic amount of p-TSA was added. The mixture was stirred overnight at ambient temperature, neutralized by addition of triethylamine, and evaporated to dryness. The residue was dissolved in a small amount of DMF and purified by a semipreparative RP HPLC (C-18, 250 × 10 mm, 5 μm) using a gradient elution from 25% to 80% MeCN in 0.1 mol L−1 aqueous Et3NH+AcO− in 25 min (9 eluted with tR = 23 min, λ = 220 nm; flow rate 3.0 mL min−1). The product fractions were combined and lyophilized to yield 18 mg of the product (9) as white powder (contained also traces of dimethyl acetal of the levulinoyl group; overall yield 16% from 8). 1H NMR (500 MHz, CDCl3): δ 1.78 (m, 2H), 2.04 (s, 3H), 2.34−2.65 (m, 4H), 3.03 (m, 1H), 3.11 (m, 1H), 3.17−3.23 (m, 3H), 3.27−3.36 (m, 6H), 3.43 (m, 1H), 3.48−3.60 (m, 4H), 3.73 (s, 3H), 3.75 (s, 3H), 3.76 (s, 3H), 3.82−3.97 (m, 3H), 4.35−4.09 (m, 8H), 4.80 (d, 1H, J = 7.1 Hz), 4.83−4.87 (m, 2H), 4.99 (d, 1H, J = 8.4 Hz), 5.01 (d, 1H, J = 8.3 Hz), 5.30 (dd, 1H, J = 7.3 Hz and 7.2 Hz), 5.34−5.43 (m, 3H), 5.50 (dd, 1H, J = 8.0 Hz, both), 5.54 (dd, 1H, J = 7.9 Hz and 7.7 Hz), 5.65 (dd, 1H, J = 9.4 Hz, both), 6.83 (b, 1H, NH), 6.99 (b, 2H), 7.33−7.43 (m, 12H), 7.46−7.57 (m, 6H), 7.85−7.94 (m, 12H); 13C (125 MHz, CDCl3): δ 27.5, 29.0, 29.6, 37.5, 46.2, 48.0, 53.38, 53.45, 57.8, 58.1, 58.2, 62.5, 62.6, 62.7, 66.6, 70.1, 70.26, 70.30, 71.7, 71.81, 71.84, 71.88, 71.92, 72.1, 72.2, 73.9, 74.1, 74.9, 75.3, 75.8, 75.9, 81.8, 82.4, 82.5, 98.1, 98.7, 99.0, 100.3, 100.8, 128.46, 128.53, 128.6, 128.7, 129.0, 129.6, 129.8, 130.0, 130.1, 133.56, 133.61,

added. The reaction mixture was stirred for 15 min at ambient temperature, diluted with EtOAc, and washed with 0.1 mol L−1 aq HCl, saturated aq NaHCO3, and brine. The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (40% EtOAc in toluene) to yield 0.21 g (75%) of the product (6) as white foam. 1H NMR (500 MHz, CDCl3): δ 1.80 (m, 2H), 3.21 (d, 1H, J = 3.1 Hz), 3.32 (m, 2H), 3.39 (m, 1H), 3.55 (m, 2H), 3.77 (s, 3H), 3.79−3.92 (m, 4H), 4.12 (m, 1H), 4.34 (dd, 1H, J = 10.5 Hz and 4.9 Hz), 4.63 (dd, 1H, J = 9.6 Hz and 9.1 Hz), 5.03 (d, 1H, J = 7.1 Hz), 5.05 (d, 1H, J = 8.1 Hz), 5.37−5.43 (m, 2H), 5.55 (s, 1H), 6.92 (d,1H, J = 7.1 Hz), 7.29−7.39 (m, 7H), 7.44−7.50 (m, 4H), 7.86−7.90 (m, 4H); 13C (125 MHz, CDCl3) δ 29.0, 48.0, 52.9, 59.1, 66.1, 66.9, 68.7, 70.3, 71.6, 74.1, 75.0, 76.1, 79.8, 91.9, 99.0, 99.6, 101.6, 126.0, 128.37, 128.43, 128.44, 128.86, 128.91, 129.3, 129.88, 129.91, 133.9, 137.0, 162.0, 165.1, 166.4, 169.2; HRMS (ESI-TOF): m/z calcd. for C39H38Cl3N4O14 [M-H]− 891.1450, found 891.1442. 3-Azidopropyl(methyl 2,3-di-O-benzoyl-4-O-levulinoyl-βD-glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1 → 4)(methyl 2,3-di-O-benzoyl-β-D-glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (7). Glycosyl donor 4 and acceptor 6 were dried, following the procedure described for the synthesis of 3 above. The glycosidation between (4, 0.42 g, 0.40 mmol) and 6 (0.31 g, 0.35 mmol) was then carried out in dry DCM (3.0 mL) under nitrogen at −30 °C using TMSOTf (16 μL, 87 μmol) as a Lewis acid. The reaction mixture was stirred for 1.5 h under nitrogen, neutralized by addition of triethylamine (100 μL), filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (30% EtOAc in toluene) to yield 0.20 g (33%) of the product (7) as white foam. 1H NMR (500 MHz, CDCl3): δ 1.79 (m, 2H), 2.01 (s, 3H), 2.33 (m, 1H), 2.43−2.53 (m, 3H), 3.23−3.33 (m, 4H), 3.37−3.42 (m, 2H), 3.48−3.58 (m, 2H), 3.61 (s, 3H), 3.66 (s, 3H), 3.67 (m, 1H), 3.75−3.80 (m, 3H), 3.85 (d, 1H, J = 9.3 Hz), 3.90 (m, 1H), 4.30 (dd, 1H, J = 9.2 Hz, both), 4.33 (dd, 1H, J = 10.7 Hz and 4.9 Hz), 4.44 (dd, 1 H, J = 9.6 Hz and 9.0 Hz), 4.54 (dd, 1H, J = 9.4 Hz, both), 4.91 (d, 1H, J = 7.7 Hz), 4.99 (d, 1H, J = 8.2 Hz), 5.01 (d, 1H, J = 7.1 Hz), 5.10 (1H, J = 7.8 Hz), 5.10 (s, 1H), 5.30− 5.35 (m, 3H), 5.44 (dd, 1H, J = 8.8 Hz, both), 5.48 (dd, 1H, J = 9.5 Hz and 9.4 Hz), 5.54 (s, 1H), 6.71 (d, 1H, J = 7.5 Hz), 6.93 (d, 1H, J = 7.3 Hz), 7.14−7.18 (m, 3H), 7.24−7.27 (m, 7H), 7.30−7.34 (m, 14H), 7.38−7.56 (m, 9H), 7.80−7.93 (m, 8H); 13 C (125 MHz, CDCl3): δ 27.6, 29.0, 29.6, 37.5, 48.0, 52.8, 53.0, 58.6, 58.9, 65.8, 66.2, 66.9, 67.7, 68.6, 69.4, 71.9, 72.16, 72.18, 72.5, 72.6, 73.8, 75.4, 76.14, 76.17, 77.2, 79.5, 79.7, 92.04, 92.08, 98.3, 99.3, 99.6, 99.9, 101.2, 125.9, 126.0, 128.3, 128.4, 128.7, 128.8, 128.9, 129.0, 129.1, 129.8, 129.9, 130.0, 133.30, 133.34, 133.4, 136.9, 137.1, 161.4, 161.9, 164.8, 165.2, 165.2, 165.5, 166.9, 168.1, 171.0, 205.6; MS (ESI-TOF): m/z calcd. for C80H76Cl6N5O29 [M−H]− 1780.28, found 1780.26. 3-Azidopropyl(methyl 2,3-di-O-benzoyl-β-D-glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1 → 4)- (methyl 2,3-di-Obenzoyl-β-D-glucopyranosyluronate)-(1 → 3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (8). The levulinoyl group was removed from 7 as described above for the conversion of 5 to 6. Notations: 0.12 g (0.067 mmol) of 7 yielded 0.088 g (78%) of 8 as white solid, 30% EtOAc in toluene was used for the silica gel purification. 1H NMR (500 MHz, CDCl3): δ 1.77 (m, 2H), 2.49 (dd, 1H, J = 400


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tethered oligonucleotides (11 or 12, 0.1 μmol) and azidefunctionalized hyaluronan tetra- (10) and hexasaccharides (9) and maltohexose (15, 0.2 μmol, 2 equiv) was carried out in MeCN:H2O (1:9, v/v, 100 μL), overnight at 55 °C (i/Scheme 3). According to RP HPLC monitoring (after the global deprotection described below), the desired conjugates were formed quantitatively. Global Deprotection of the Conjugates. After the SPAAC conjugation, the mixtures of the protected conjugates were evaporated to dryness and subjected to global deprotection. For the hyaluronan conjugates, two-step deprotection was required (ii and iii in Scheme 3): the residues were first subjected to a treatment with 0.1 mol L−1 aq NaOH (1.0 mL) for 3 h at 55 °C (ii/Scheme 3), followed by neutralization with 1.0 mol L−1 aqueous ammonium chloride. The mixtures were evaporated to dryness and then trichloroacetyl groups were removed. For this purpose, the residues were dissolved in concentrated aqueous ammonia and the mixtures were incubated at 55 °C for 5 days (iii/Scheme 3). The completion of the reaction was verified by MS(ESI-TOF)- and RP HPLC-analysis (cf. 14 in Scheme 3) and the mixtures were evaporated to dryness. The protected maltohexose conjugate was directly subjected to a treatment with concentrated ammonia (33% aq NH3, overnight at 55 °C), which gave the desired globally deprotected conjugate (21, Scheme 3). Selective Acetylation of the Hyaluronan Conjugates. The glucosamine derivatives of the conjugates (0.1 μmol, 13 or 14, in Scheme 3) were dissolved in a mixture of MeCN and water (1:9, v/v, 500 μL). Et3N (50 μL) and acetic anhydride (25 μL) were added and the mixtures were shaken at ambient temperature for 2 h. The treatment was repeated twice (2 × 50 μL Et3N and 25 μL Ac2O) and the mixtures were evaporated to dryness. The completion of the acetylation was verified by RP HPLC and MS (ESI-TOF) spectroscopy. Acetylation of the exocyclic amino groups also took place during this treatment (cf. 17 in Scheme 3) and, therefore the conjugates were once again treated with concentrated ammonia (33% aq NH3, 5 h at 55 °C), which gave the desired Nacetylated glucosamine products (18, 19, and 20). Purification and Characterization of the Conjugates. The conjugates were purified by semipreparative RP HPLC (C-18, 250 × 10 mm, 5 μm) using a gradient elution from 0 to 40% MeCN in 0.1 mol L−1 aqueous Et3NH+AcO− in 25 min (λ = 260 nm, flow-rate 3.0 mL min; cf., chromatograms in Scheme 3). The product fractions were combined and lyophilized to white powders. Isolated yields for 18 (50%), 19 (47%), 20 (48%), and 21 (71%) were determined from the UVabsorbance at λ = 260 nm (using 11 and 12 as starting materials). The authenticity of the conjugates 18−21 was verified by MS (ESI-TOF) spectroscopy (Table 1). PET Studies. 68Ga Labeling of Conjugates 18−21. 68Ga was obtained in the form of 68GaCl3 from a 68Ge/68Ga generator (Eckert & Ziegler, CA, USA) by elution with 0.1 mol L−1 HCl. The 68GaCl3 eluate (500 μL) and sodium acetate (18 mg) were mixed to give a 0.4 mol L−1 solution with regard to sodium acetate. The pH was adjusted to approximately 3 with 2 mol L−1 HCl (47 μL). A conjugate (18−21, 18−20 nmol, as a 1 mmol L−1 aqueous solution) was added and the reaction mixture was incubated at 95 °C for 10 min. The radiochemical purity of 68Ga-chelated 18−21 was determined by a reversedphase high performance liquid chromatography coupled with an online radioactivity detector (radio-HPLC) on a μBondapak C18 column (3.9 × 150 mm, 125 Å, 10 μm; Waters, Ireland)

133.7, 205.5; MS (ESI-TOF): m/z calcd. for C95H96Cl9N6O42 [M − 2H]2− 1153.13, found 1153.15. 3-Azidopropyl (methyl 2,3-di-O-benzoyl-4-O-levulinoyl-βD-glucopyranosyluronate)-(1 → 3)-2-deoxy-2-trichloroacetamido-β-D-glucopyranosyl-(1 → 4)-(methyl 2,3-di-O-benzoylβ-D-glucopyranosyluronate)-(1 → 3)-2-deoxy-2-trichloroacetamido-β-D-glucopyranoside (10). Catalytic amount of p-TSA was added to a solution of tetrasaccharide 7 (30 mg, 16 μmol) in a mixture of DCM (1 mL) and MeOH (4 mL). The mixture was stirred for 12 h, neutralized by addition of NaHCO3 and evaporated to dryness. The residue was partitioned between water and DCM. The organic layer was separated, dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by silica gel chromatography (5% MeOH in DCM) to yield 20 mg (74.0%) of the product (10) as white solid. 1H NMR (500 MHz, CDCl3): δ 1.75 (m, 2H), 2.04 (s, 3H), 2.38 (m, 1H), 2.50−2.66 (m, 3H), 3.07 (m, 1H), 3.18−3.25 (m, 2H), 3.27−3.35 (m, 4H), 3.44 (m, 1H), 3.51−3.60 (m, 3H), 3.76 (s, 6H), 3.80 (m, 1H), 3.88−3.96 (m, 2H), 4.11 (s, 2H), 4.22−4.36 (m, 5H), 4.81 (d, 1H, J = 7.0 Hz), 4.87 (d, 1H, J = 8.3 Hz), 4.91 (d, 1H, J = 6.3 Hz), 5.05 (d, 1H, J = 8.3 Hz), 5.34−5.44 (m, 3H), 5.54 (dd, 1H, J = 8.0 Hz and 7.9 Hz), 5.65 (dd, 1H, J = 9.4 Hz and 9.3 Hz), 6.80 (b, d, 1H, J = 6.9 Hz), 6.93 (b, d, 1H, J = 6.9 Hz), 7.33−7.43 (m, 8H), 7.48−7.57 (m, 4H), 7.85−7.94 (m, 8H); 13C (125 MHz, CDCl3) δ 27.6, 29.0, 29.6, 37.5, 48.0, 53.4, 53.5, 58.1, 58.3, 62.6, 62.7, 66.6, 68.8, 70.26, 70. 30, 71.7, 71.8, 71.85, 71.89, 72.0, 73.9, 74.7, 75.3, 75.8, 81.8, 82.4, 92.0, 92.2, 98.2, 98.8, 100.3, 100.9, 128.46, 128.49, 128.60, 128.63, 128.66, 128.73, 129.1, 129.6, 129.8, 129.9, 130.0, 130.1, 133.58, 133.64, 133.8, 161.9, 162.3, 165.1, 165.3, 165.4, 165.5, 167.2, 168.2, 171.3, 205.5; MS (ESI-TOF): m/z calcd. for C66H68Cl6N5O29 [M − H]− 1604.21, found 1604.23. Preparation of Hyaluronan Conjugates of NOTAFunctionalized Oligonucleotides. Oligonucleotide Synthesis. The cyclooctyne tethered oligonucleotides [T6 and anti-miR-15b (a 22-mer 2′-O-methyl oligoribonucleotide)] bearing a NOTA group at the 3′-terminus were assembled by the phosphoramidite chemistry on a NOTA modified CPG support22 (Applied Biosystems 392 DNA/RNA synthesizer; 1.0 μmol scale). The cyclooctyne function was introduced into the 5′-terminus by an additional coupling cycle with 2(bicyclo[6.1.0]non-4-yn-9-yl)eth-1-yl 2-cyanoethyl N,N-diisopropyl phosphoramidite (BCN phosphoramidite; 0.1 mol L−1 solution in dry MeCN; coupling time of 600 s). Otherwise, standard chain assembly (with a 300 s coupling time) using benzylthiotetrazol as an activator was employed. The oligonucleotides were released from the support using our previously optimized two-step cleavage protocol:22 The supports were first treated with 0.1 mol L−1 aqueous NaOH (1.0 mL) for 3 h at 55 °C, followed by neutralization with 1.0 mol L−1 aqueous NH4+Cl− (0.11 mL), and then with concentrated ammonia (aqueous 33% NH3) for 15 h at 55 °C. The support was removed by filtration, the filtrates were evaporated to dryness, the residues were dissolved in water (500 μL), and the crude oligonucleotides (11, 12) were purified by semipreparative RP HPLC (C-18, 250 × 10 mm, 5 μm) using a gradient elution from 0% to 40% MeCN in 0.1 mol L−1 aqueous Et3NH+AcO− in 25 min, λ at 260 nm, and flowrate 3.0 mL min−1. SPAAC-Conjugation and Global Deprotection of the Conjugates. The copper-free strain promoted azide alkyne cycloaddition (SPAAC) between the purified cyclooctyne 401


Article

Bioconjugate Chemistry (Figure 2). The HPLC conditions were as follows: flow rate 1 mL min−1, A = 50 mmol L−1 NH4OAc, B = MeCN, C = 50 mmol L−1 H3PO4. The gradient (A/B/C): 0−17 min from 100/0/0 to 50/50/0, 17−18 min from 50/50/0 to 0/0/100, 18−25 min 0/0/100. The radio-HPLC system consists of LaChrom Instruments (Hitachi; Merck, Darmstadt, Germany) and of a Radiomatic 150TR radioisotope detector (Packard, Meriden, CT, USA). 22−24 were 68Ga labeled as previously described.22,23 Animals. All animal experiments were approved by the national Animal Experiment Board in Finland (ELLA) and the Regional State Administrative Agency for Southern Finland (ESAVI) and conducted in accordance with the relevant European Union Directive. The Sprague−Dawley rats were purchased from Harlan, The Netherlands. The animals received regular feed and tap water was offered ad libitum. Whole-Body Distribution in Healthy Rats. Healthy, mature, male Sprague−Dawley rats (n = 4 per 68Ga-compound, weight 369 ± 84 g) were anesthetized with isoflurane and intravenously injected with 19 ± 3.2 MBq (4.3 ± 0.3 nmol) of 18, 14 ± 2.0 MBq (5.8 ± 1.9 nmol) of 19, 14 ± 1.7 MBq (5.0 ± 2.1 nmol) of 20, 13 ± 2.6 MBq (4.4 ± 1.7 nmol) of 21, 13 ± 2.9 MBq (2.1 ± 0.2 nmol) of 22, 18 ± 1.1 MBq (5.6 ± 1.4 nmol) of 23, or 17 ± 0.8 MBq (2.2 ± 0.1 nmol) of 24. Whole-body distribution kinetics was evaluated by a 60 min dynamic PET imaging (High Resolution Research Tomograph, Siemens Medical Systems, Knoxville, TN, USA). Acquired data were iteratively reconstructed with the ordered-subsets expectation maximization 3D algorithm (OSEM3D). Quantitative analysis was performed by defining regions of interest (ROIs) on the main organs using Carimas 2.8 software (Turku PET Centre, Turku, Finland; http://www.turkupetcentre.fi/ carimas). The average radioactivity concentration kBq/mL in the ROI was used for further analyses. The uptake was reported as a standardized uptake value (SUV), which was calculated as the radioactivity concentration of the ROI normalized with the injected radioactivity dose and animal weight. Mean time− radioactivity curves extracted from dynamic PET images were used for presenting the biokinetics of the 68Ga-compounds. Immediately after PET imaging, radioactivity concentration of various tissue samples was measured ex vivo by gamma counter (1480 Wizard 3″, PerkinElmer/Wallac, Turku, Finland). The radioactivity concentration was decay corrected to the time of injection, normalized by injected radioactivity dose, animal weight, and the weight of tissue, and radioactivity remaining in the injection site was compensated. The results were expressed as SUV. Rats with Myocardial Infarction. Male Sprague−Dawley rats (weight 339 ± 76 g) were studied at 7 days after myocardial infarction induced by permanent ligation of left coronary artery and compared with sham-operated controls (n = 2−4 in each group).34 Rats were anesthetized with isoflurane and intravenously injected with 25 ± 4.7 MBq (4.5 ± 2.2 nmol) of 18 or 25 ± 3.8 MBq (4.7 ± 1.9 nmol) of 19. A 60 min dynamic PET imaging was acquired followed by contrast-enhanced CT with Inveon Multimodality scanner (Siemens Medical Solutions, Knoxville, TN, USA). PET data were reconstructed with the OSEM3D. Quantitative ROI analysis was performed using Carimas v 2.5 software. Immediately after PET/CT, left ventricle (without the atria or the right ventricle) and various other tissues were excised, weighed, and measured for radioactivity using a gamma counter

(Triathler 3″, Hidex, Turku, Finland or 1480 Wizard 3″, EG&G Wallac, Turku). Results were expressed as SUV, which was calculated as the radioactivity concentration (radioactivity per gram of tissue) divided by the relative injected radioactivity expressed per animal body weight. The left ventricle was frozen in isopentane and sliced into serial of 8- and 20-μm transverse cryosections from apex to base for digital autoradiography analysis, and for histology and immunohistochemistry. Radioactivity uptake in the infarcted and remote myocardium was measured as photostimulated luminescense per square millimeter (PSL/mm2) with TINA software v 2.1 (Raytest Isotopenmessgeräte, GmbH, Straubenhardt, Germany) as described previously.35 After autoradiography, sections were immunohistochemically stained with antiCD44 antibody and with hematoxylin−eosin. Immunohistochemical staining was performed with 1f1 anti-CD44 antibody.36 Statistical Analysis. All data are expressed as mean ± standard deviation (SD). Statistical analysis was done with SPSS Statistics software v 21 (IBM, NY, USA). An unpaired Student’s t test was applied for comparisons between two groups. Comparisons of three or more groups were done using ANOVA with Dunnett’s correction. P values < 0.05 were considered statistically significant.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.5b00477. NMR spectra for 3−10, RP HPLC profile, and MS (ESITOF) spectrum of 9 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: pamavi@utu.fi. Tel: +358 2 333 6777. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Academy of Finland (No. 251539, 252097, 256214, and 258814), the Erasmus Mundus Action 2, Strand 1 (EMA2) for the award of EXPERTS fellowships, Sigrid Juselius Foundation, and Finnish Foundation for Cardiovascular Research are gratefully acknowledged. PET studies were conducted within the Finnish Centre of Excellence in Cardiovascular and Metabolic Diseases supported by the Academy of Finland, University of Turku, Turku University Hospital and Åbo Akademi University. We also thank Aake Honkaniemi for assistance in PET/CT imaging.

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REFERENCES

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Synthesis and In Vivo PET Imaging of Hyaluronan Conjugates of

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