Design, Synthesis, and Bioactivation of O-Glycosylated Prodrugs of

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Design, Synthesis, and Bioactivation of O-Glycosylated Prodrugs of the Natural Nitric Oxide-Precursor N-Hydroxy-L-Arginine #

Felix-A. Litty, Julia Gudd, Ulrich Girreser, Bernd Clement, and Dennis Schade J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00810 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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Journal of Medicinal Chemistry

Design, Synthesis, and Bioactivation of O-Glycosylated Prodrugs of the Natural Nitric Oxide-Precursor Nω-Hydroxy-L-Arginine Felix-A. Litty‡1, Julia Gudd‡1, Ulrich Girreser1, Bernd Clement1, Dennis Schade*,2 ‡ both authors contributed equally *

corresponding author

1

Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, Christian-

Albrechts-University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany 2

TU Dortmund, Department of Chemistry & Chemical Biology, Otto-Hahn-Straße 6, 44227

Dortmund

ACS Paragon Plus Environment


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ABSTRACT

Naturally occurring Nω-hydroxy-L-arginine (NOHA, 1) is the best substrate of NO synthases (NOS). The development of stable and bioavailable prodrugs would provide a pharmacologically valuable strategy for the treatment of cardiovascular diseases that are associated with endothelial dysfunction. To improve NOHAs drug-like properties, we demonstrate that O-substitution by (glycosylic) acetal formation greatly increased the chemical stability of the hydroxyguanidine moiety and provided a non-toxic group which could be easily bioactivated by glycosidases. A straightforward synthetic concept was devised and afforded a series of diversely substituted prodrugs by O-conjugation of the hydroxyguanidine moiety with different monosaccharides. Systematic exploration of their bioactivation profile revealed that glucose-based prodrugs were more efficiently bioactivated than their galactose counterparts. NOS-dependent cytosolic NO release was quantified by automated fluorescence microscopy in a cell-based assay with murine macrophages. Glucose-based prodrugs performed particularly well and delivered cellular NO levels comparable to 1 demonstrating proof-of-concept.


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INTRODUCTION Cardiovascular diseases (CVDs) account for almost 40% mortality in the Western world, representing the major cause of death.1 It is well-recognized that low endogenous nitric oxide (NO) levels are closely linked to the pathology of a variety of CVDs, with endothelial dysfunction and atherosclerosis representing hallmarks of such disease states.2,3 However, NO is involved in a plethora of physiological and pathophysiological processes.4,5 Therefore, it is highly desirable to rather selectively affect pathophysiologically altered processes of NO generation and NO availability, thereby providing a safe and efficient therapy. As a consequence, a great deal of research has focused in the past decades on pharmacological modulation of the predominant enzymes involved in the regulation of NO levels.6 The nitric oxide synthases (NOSs) represent the key enzymes of NO formation as they catalyze the five-electron oxidation of

L-arginine

to

L-citrulline

and NO via Nω-hydroxy-L-arginine (NOHA, 1) as a catalytic

intermediate (Figure 1).7 Although NOSs are attractive targets, their active sites have demonstrated very little promiscuity, and to date only few alternative substrates have been identified.8 In addition to representing the best substrate for NOSs, 1 mainly releases NO only where it is ultimately needed, i.e. at the NOS-site-of-action, as opposed to other NO donors (e.g., nitrates). Furthermore, 1 is a potent inhibitor of arginases (Figure 1).9 These enzymes catalyze the conversion of L-arginine to L-ornithine, and thus, can potentially deplete L-arginine as a substrate for NOS-mediated NO generation.10 An increased expression and activity of arginase is indeed closely linked to atherosclerotic changes and endothelial dysfunction.11 Hence, 1 exhibits a unique dual and selective mode of action for potential therapeutic applications in several CVDs.



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Figure 1. Nω-Hydroxy-L-arginine (1, NOHA) acts as a key intermediate of NOS catalysis and is a potent inhibitor of arginases; CYP, cytochrome P450; L-arg, L-arginine; L-cit, L-citrulline; L-orn, L-ornithine;

NO, nitric oxide; NOHA, Nω-hydroxy-L-arginine; NOS, nitric oxide

synthases. However, direct application of 1 is limited by its poor drug-like properties. In our previous work we pursued different strategies to overcome its chemical and metabolic liabilities.6,12 We demonstrated that O- and N-substitution of the N-hydroxyguanidine moiety greatly increased hydrolytic and oxidative stability.12 Unfortunately, in vitro experiments later revealed that all Nsubstituted prodrugs (i.e., carbamates) were not bioactivated which in turn would prevent 1 of being accepted as a NOS substrate for the desired release of NO (data not shown). Building on our observation that sole O-substitution efficiently protects from oxidation and hydrolytic degradation, we next aimed at designing carbamate-free derivatives. Moreover, simple O-alkyl prodrugs were supposed to be avoided as they required involving CYP enzymes in the bioactivation cascade, thereby increasing risks for drug-drug interactions.13


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Here, we introduce a concept that afforded chemically stable prodrugs of 1 which are taken up by cells and bioactivated in a monooxygenase-independent fashion while releasing degradation products that are uncritical from a toxicological point of view. Specifically, O-acetalic incorporation of the labile N-hydroxyguanidine group into monosaccharides (i.e., galactose and glucose) furnished stable prodrug candidates that are bioactivated by glycosidases (Figure 2).

Figure 2. Illustration of a novel prodrug concept for 1 by O-glycosylation.



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RESULTS AND DISCUSSION Synthesis of O-glycosylated prodrugs of 1 Within our long-standing efforts in designing various Nδ- and Nω-substituted L-arginines we have utilized several synthetic strategies.12,14–16 A number of O-alkylated and N-acylated derivatives of 1 were already accessible by reaction of carbamoylated thioureas with the desired hydroxylamine in the presence of a desulfurizing agent (i.e., EDCI). This strategy only furnished prodrugs that were N-carbamoylated (e.g., Eoc or Cbz group) which required harsh conditions for their removal. Thus, for the herein described novel carbamate-free prodrug candidates, an orthogonal protecting group was needed that would allow efficient synthesis of the N-hydroxyguanidine group but also ensure mild cleavage conditions that were compatible with the carbohydrate conjugate and α-amino acid protecting groups. We reasoned that the Alloc protecting group could fulfill these requirements by a nucleophilic attack of a palladium(0) catalyst to afford π-allyl palladium complexes which can then be trapped by mild nucleophiles such as 5,5-dimethylcyclohexane-1,3-dione (dimedone). For a series of ethyl ester prodrugs (i.e., 3, 5, 6, 8), protected L-ornithine 12 was prepared in two steps from commercially available 10. First, 10 was treated with phthalic anhydride in tetrachloroethylene and then esterified under Steglich conditions with 4-DMAP, EtOH and EDCI.17,18 The Alloc group could then be introduced by removing the phthalimide with hydrazine and directly reacting the intermediate ornithine ethyl ester with freshly prepared allyloxycarbonyl isothiocyanate (AllocNCS, 13)19,20 to furnish thiourea 14b in moderate yield (40%). Attempts to isolate the intermediate failed largely due to rapid cyclization to a δ-lactam. For the desired free α-amino acid prodrug candidates (i.e., 2, 4, 7, 9), Nα-Boc-L-ornithine tertbutyl ester 15 was reacted with 13 to obtain thiourea 14a in excellent yield (94%).


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O H2N

O

O

i OH NHBoc

N

N

OH NHBoc O

10

O

O ii

OEt NHBoc

O

11

12

R1 = tBu or Et iii-iv R2 =

AcOOAc

O

O

Gal AcO

OAc

Alloc

O Bu NHBoc

OAc

AllocNCS (13)

N H

15

O

Glc AcO AcO

S

iv t

H2N

*

OAc

AllocNCS (13) O

OR1 NHBoc 14a: R1 = tBu 14b: R1 = Et N H

* R2

v R3

= R2 O

O

Gal HO

OH

*

OH HO HO

N

H2N

H2N

ix (2 and 4) O- Na+

R3

N

N H

vi-vii

N

H2N

7 Gal 9 Glc

2 3

R3 O

O OR4

N H

NH2

R2

R4

Gal Gal

H Et

OR1 NHBoc

17a-b: R1 = tBu 18a-b: R1 = Et

x 2 HCl

NH2

O N H

viii

O N H

NHBoc

Alloc

19a-b: R1 = tBu 20a-b: R1 = Et

*

R2 O N

vi OR1

N H

O OH

R2 O

O

R4 = H or Et

R3 O

NH2

16

HOOH

Glc

O

4 5

H2N

x 2 HCl N

O N H

OEt NH2

R2

R4

R3

Glc Glc

H Et

6 Gal 8 Glc

Scheme 1. Synthetic routes to diverse O-glycosylated prodrugs of 1. (i) phthalic anhydride, C2Cl4, 100 °C, 6 h (61%); (ii) EtOH (5 equiv.), 4-DMAP (40 mol-%), EDCI, DCM, r.t., 24 h (54%); (iii) N2H4×H2O (15 equiv.), DCM/EtOH 1:1, r.t., 2.5 h; (iv) allyloxycarbonyl isothiocyanate (13), DCM, 0 °C–r.t., 2 h (94%, 14a and 40% for 14b in one pot, 2-steps); (v) DIPEA, EDCI, DCM, 48–72 h, r.t. (73–93%); (vi) Pd(0)[Ph3P]4, dimedone (8 equiv.), THF, r.t., 30 min (73–90%); (vii) acetyl chloride, EtOH abs., argon, 0 °C–r.t., 48 h (82–93%); (viii) 1. HCl(g) for 20 min, Et2O abs., argon, -10 °C, 2. 4–8 °C overnight (75–97%); (ix) NaOMe, MeOH, 0 °C–r.t., 2.5 h (90%–quant.).



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Next, the fully protected N-hydroxyguanidine prodrug precursors 17a-b and 18a-b were obtained in good yields (73–93%) by reacting thioureas 14a-b with the desired peracetyl protected 1-aminooxysugars 16 (R2 = Gal, Glc),21,22 DIPEA and EDCI in dichloromethane. Importantly, chemoselective cleavage of Alloc was achieved in good yields (73–90%) with Pd(0)[P(Ph)3]4 and dimedone under mild conditions in THF without compromising the other protecting groups or the O-acetalic function.23 In view of the lability of the β-glycosylic bond towards acidic conditions, final deprotection of the α-amino acid moiety represented a crucial step but was achieved under water-free conditions with gaseous HCl in Et2O. The desired prodrugs 2-5 precipitated at 4–8 °C overnight as dihydrochloride salts (determined by quantitative

35

Cl NMR spectroscopy, see Supporting

Information). Prodrugs 6 and 8 were obtained from 18a-b by in situ generation of HCl in absolute EtOH which ensured retaining the α-carboxylic ester while all acetyl groups could be removed via re-esterification. The preparation of fully deprotected prodrug candidates 7 and 9 was achieved from 2 and 4 using sodium methoxide in MeOH (Scheme 1).24 In previous work we found that 1 and other N-hydroxyguanidines only existed in the oxime-type structure and not in the hydroxylamine-type tautomer.25,26 Here, we additionally examined the cis-trans (E/Z) isomerism of several prodrugs and synthetic precursors (i.e., 18a) by 1H, 1H-NOESY and

15

N NMR. The data revealed that in all analyzed compounds the sugar

moiety was located in trans- (E) position to the α−amino acid (for detailed spectroscopic data see Supporting Information).


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Chemical stability We performed a systematic analysis on the stability profile of the new prodrugs using several O-galactosyl derivatives as representative examples. The primary aim was to investigate whether the novel O,O-acetal consisting of an N-hydroxyguanidine and a monosaccharide represents a stability-determining structural moiety. Hence, prodrug 7, with a free α-amino acid and the deprotected galactosyl residue, was tested under various conditions. As shown in Figure 3, this prodrug was highly stable at pH 2.0, 7.4 and 9.0 over 24 hours at 37 °C. This result suggested a surprisingly high chemical stability of these novel carbohydrate conjugates even under acidic conditions. Moreover, our previous work revealed that 1 is very labile at basic pH but can be stabilized by O-alkylation.12 Since prodrug 7 is also very stable at pH 9 it seems that the same effect can also be achieved by O-acetalic substitution with carbohydrates. Furthermore, we addressed which carboxylic esters are more susceptible to chemical hydrolysis by focusing on the simple ethyl ester 6 as well as the per-acetylated galactosyl prodrug 2. Interestingly, the ethyl ester in 6 was much faster hydrolyzed at pH 9 and pH 7.4 (t1/2 = ca. 10–12 hours) compared to the acetyl group(s) in 2 (t1/2 > 24 h) likely due to neighboring effects of the α-amino group and/or for steric reasons. It should be noted that a slow chemical hydrolysis of the different esters is welcome in view of a slow and sustained release of the active drug 1 (= bioactivation). Overall, all three prodrug candidates exhibited a sufficient stability profile allowing the conduction of various in vitro experiments. These compounds would also be well-suited for any in vivo application, even for an ultimately desired peroral application.



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Figure 3. Chemical stability of the O-galactosyl prodrugs 2, 6 and 7. Prodrugs (250 µM) were incubated at 37 °C in potassium phosphate buffer (10 mM) and analyzed by HPLC. Each data point represents mean ± SD (n = 3). In vitro bioactivation Since our initially developed N-carbamate-containing prodrugs of 1 were not bioactivated, it was essential to investigate the release of 1 from the herein disclosed carbamate-free prodrug series. Depending on the respective prodrug candidate, the required bioactivation consists of ester as well as acetal hydrolysis as depicted in Figure 4a for several galactose conjugates (i.e., 2, 3, 6, 7). Glucose-conjugated prodrugs of 1 were expected to be similarly bioactivated by the action of β-glucosidase instead of β-galactosidase. To systematically evaluate the proposed steps of bioactivation along with substrate specificity, a selection of distinct galactose- (2, 6 and 7) and glucose-based (4 and 9) prodrugs were tested with various enzyme sources (Figure 4b-d). First, it was addressed whether a per-acetylated galactose would still be accepted by

β-galactosidase. In fact, prodrug 2 did not show any turnover by β-galactosidase in contrast to the unsubstituted galactose conjugate 7 (Figure 4b, left). This result turned out to be in sharp


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contrast to the substrate specificity of β-glucosidase which efficiently catalyzed the conversion of both the per-acetylated glucose prodrug 4 and the unsubstituted prodrug 9 (Figure 4d). Moreover, β-glucosidase accepted not only glucose-based prodrugs as substrates but also cleaved off the galactosyl residue from prodrug 7 at comparable turnover rates (Figure 4d). This broad substrate specificity is well-documented in the literature and seems to also account for the herein introduced compounds.27,28 Next, a stepwise bioactivation was simulated by incubating ethyl ester prodrug 6 together with carboxylesterase and β-galactosidase (Figure 4b, right). In accordance with the proposed steps of bioactivation, intermediately formed prodrug 7 was detected in addition to the actual drug 1. However, 7 was formed at much lower rates than 1 suggesting that β-galactosidase already converted the ethyl ester 6 to a high extent (see Supporting Information). Furthermore, the simultaneous release of D-galactose as a cleavage product upon bioactivation of 7 was verified and quantified using a colorimetric enzyme-based assay (Figure 4c).



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Figure 4. In vitro bioactivation of O-glycosyl prodrugs of 1. a) Proposed steps of bioactivation and metabolic intermediates as exemplified for galactose-based prodrugs 2, 3, 6 and 7, b) HPLCbased quantification of 1 after incubation of prodrugs 2, 6 or 7 (500 µM) with: 25 U/mL

β-galactosidase (left), 33 U/mL β-galactosidase and 133 U/mL carboxylesterase (right), c) Colorimetric quantification of

D-galactose

after incubation of prodrug 7 (10 mM) with

β-galactosidase (59 U/mL) d) HPLC-based quantification of 1 after incubation of prodrugs 4 or 9 (500 µM) with 33 U/mL β-glucosidase. Each data point represents mean ± SD (n = 2-3).


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In vitro bioactivation studies were complemented by also quantifying the generation of NO from a one-pot biochemical activation cascade employing carboxylesterase, β-galactosidase and NOS catalysis (see Supporting Information for details). The galactose prodrugs series was examined in this set-up (i.e., 3, 4, 6, 7). Interestingly, only the deacetylated prodrugs 6 and 7 were able to release NO to a similar extent as 1 (Supporting Information Figure S3). The data are in accordance with the above described findings that β-galactosidase does not accept per-acetylated carbohydrate prodrugs. Therefore, a rate-limiting bioactivation step for these prodrugs is the chemical and enzymatic hydrolytic cleavage of the acetyl esters. Taken together, all tested prodrugs were sufficiently bioactivated by carboxylesterase and glycosidases with sustained release of 1. The data suggested that glucose-based prodrug candidates might be more rapidly and more efficiently bioactivated under in vivo conditions since β-glucosidase appeared to exhibit broader substrate specificity.

Cellular uptake and NO release After demonstrating sufficient chemical stability of the new O-glycosyl prodrugs of 1 as well as their bioactivation in vitro, the next step was to evaluate their uptake and bioactivation in living cells. To address this question, an image-based cellular assay was devised that quantifies the in-cell generation of NO by automated fluorescence microscopy. We used 4-amino-5methylamino-2‘,7‘difluorofluorescein diacetate (DAF-FM-DA) as a non-fluorescent, membranepermeable compound that is activated by cellular esterases to form DAF-FM. In the presence of oxygen, DAF-FM reacts with NO to form a fluorescent benzotriazole (DAF-FM-T) (Figure 5a).29,30



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Several imaging assays have been described in the literature using DAF-FM-DA as a probe to detect NO but most of them investigate signal transduction pathways that affect NOS expression. As a consequence, relatively large changes in NO production are typically detected and quantified.31,32 However, Melisi et al. were able to quantify the intracellular release of NO from carboxyl ester-galactose conjugates of D- and L-arginine.31 In a similar fashion, we established a reliable method for quantifying intracellular NO by fluorescence microscopy in mediumthroughput (i.e., 96-well plate-format) for best possible comparability between different groups of

treatment.

Briefly,

murine

macrophages

(J744A.1

cells)

were

stimulated

with

lipopolysaccharide for 24 hours to induce iNOS expression which ensured sufficient levels of NOS for later catalysis of the intracellularly released substrate 1 (Figure 5a). Next, cells were incubated with prodrug candidates for 2.5 hours and then treated with DAF-FM-DA and Hoechst 33342 before automated image acquisition (Figure 5b, representative images). No signs of toxicity were observed at concentrations up to 1 mM as judged by cell morphology (i.e., cell shape, blebbing, apoptosis) and their proliferative capacity (i.e., cell numbers) compared to vehicle treated controls. About 1000 cells per well/condition (six images per well) were captured providing a high validity of the addressed biological question. NO-dependent in-cell fluorescence was normalized to total cell number and vehicle control (= DPBS buffer). Another important control was to verify that the quantified NO signals were due to NOS-dependent bioactivation. Thus, all prodrugs and 1 were also incubated together with the NOS inhibitor nitroarginine methyl ester (L-NAME). As summarized in Figure 5c, co-incubation with L-NAME reduced all quantified NO signals which were significant for the galactosyl prodrug 3 and all prodrugs from the glucosyl series (4, 5, 8, 9).


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On a technical note, we were not able to establish a robust assay with a decent dynamic range in cellular systems that would more authentically recapitulate the pathology to be targeted, i.e. endothelial dysfunction in endothelial cell lines (HUVEC cells, EA.hy926). 1 (and also arginine) did not yield sufficient intracellular NO for image-based quantification under various conditions, such as increasing NOS expression/activity by transient overexpression, treatment with acetylcholine,

bradykinine,

calcium

ionophores

and/or

through

tetrahydrobiopterin

supplementation.

Figure 5. Image-based quantification of cellular NO release by O-glycosyl prodrugs of 1; a) Schematic illustration of prodrug uptake, bioactivation, in-cell release of NO and its detection in



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the form of DAF-FM-T; b) Representative fluorescence microscopy images of 1-treated J744A.1 macrophages after Hoechst staining at 377/447 nm (upper panel, DAPI channel) and 472/520 nm (lower panel, GFP channel), yellow bar = 100 µm; c) Average intracellular fluorescence intensity of prodrug-treated cells, data normalized to cell number and NO-release by 1 (= 100%), shown as differences (∆) to buffer control, mean ± SEM. Left: galactosyl conjugates (n = 9), right: glucosyl conjugates (n = 6). * = significant difference to buffer control with p < 0.05. Compounds 1-9 and L-NAME were applied at a final concentration of 1 mM. Interestingly, all glucose conjugates showed a superior behavior regarding intracellular NO release compared to the galactose conjugates (Figure 5c). Only prodrug 3 from the galactosyl series achieved approx. 75% of the effect of 1 as the “endogenous” physiological source of NO. In contrast, all prodrugs from the glucosyl series (i.e., 4, 5, 8, 9) achieved a similar level of NO release compared to 1. For both series there seems to be a tendency of increased NO generation with higher substituted prodrugs. Concluding from these results, O-glycosyl prodrugs of choice for in vitro and in vivo pharmacology applications are derivatives from the glucosyl series. None of these prodrug candidates are able to generate higher cellular NO levels compared to 1. In general, it might be difficult to achieve a higher cellular uptake than that driven by amino acid transporters. Therefore, an excellent outcome of these studies is that all O-glucosyl prodrugs are at least similarly well absorbed as 1, even with a protected (4, 5) or unprotected sugar moiety (8, 9) and independent of a free α-amino acid (9) or when the α-carboxy group is esterified (8). For instance, in the case of 8 and 9 it is possible that glucose transporters served as alternate uptake routes and eventually compensated for the lack of amino acid transporter-mediated cellular uptake.


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CONCLUSIONS Since 1 represents the best known substrate for NOSs it ensures an NO release as close to physiological conditions as possible, thereby minimizing unwanted biological effects of the pleiotropic signaling molecule NO. Moreover, 1 exhibits potent arginase inhibiting properties, thus representing a unique pharmacological profile for the treatment of cardiovascular diseases that are associated with impaired NO availability. In an attempt to overcome 1’s problematic physicochemical

properties,

a

prodrug

strategy

was

devised

that

stabilized

the

N-hydroxyguanidine group via O-glycosylation. As the preparation of N-carbamate-free prodrugs was a key requirement, Alloc was employed as an orthogonal protecting group that allowed efficient preparation of the substituted Nhydroxyguanidines and warranted cleavage without compromising the O-glycosylic bond or the other protecting groups. The newly introduced O-glycosyl prodrugs were exceptionally stable, even at pH 2. In vitro experiments revealed that glucose-based prodrugs are more efficiently bioactivated than their galactose counterparts, mainly taking advantage of the broader substrate specificity of β-glucosidases. Moreover, proof-of-concept could be shown in a cell-based system with murine macrophages employing a medium-throughput imaging assay. Especially the glucose-based prodrugs performed very well and led to cellular NO levels comparable to 1 indicating a likewise efficient cellular uptake and sufficient bioactivation. In summary, we introduced a novel prodrug concept for N-hydroxyguanidines as exemplified with the physiological NO precursor 1. The presented approach successfully tackled several shortcomings that limit widespread therapeutic application of 1 for CVDs that are associated with endothelial dysfunction. In particular, glucose conjugates (i.e., 4, 5, 8, 9) represent attractive agents as they are not only chemically stable but also enable a slow, prolonged and sustained



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release of 1 while being efficiently taken up by cells. As a function of the degree of substitution, a more or less pronounced chemical retardation can eventually be achieved depending on the desired onset of action and/or required routes of administration. Finally, it should be stressed that the described prodrug approach holds promise as a general strategy for stabilization and chemical retardation of prodrugs for guanidino group-containing drug candidates.


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EXPERIMENTAL General Melting points were uncorrected. 1H (300 MHz),

13

C (75 MHz),

15

N (30 MHz) and

35

Cl (29

MHz) NMR spectra were recorded on a Bruker Avance III, 300, spectrometer at 298 K equipped with Bruker Topspin 2.1 Software. Chemical shifts (δ values) were quoted in ppm relative to TMS as an internal standard, 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (TPS) as an external standard for experiments in D2O or alternatively, relative to the solvent residual signal. Spectra interpretation was performed by first order analysis. Signal assignments were obtained by the help of 1H, 1H-COSY and

13

C-HSQC and -HMBC spectra. Quantification of Cl- by 35Cl

NMR was verified by a three-point calibration with NaCl in D2O. Low resolution mass spectra were recorded using a Bruker amazon SL system with LC coupling, electrospray ionization, in the positive mode. Recordings of exact mass spectra were performed by the Department of Physical Chemistry, Christian-Albrechts-University of Kiel, on a 7.05 Tesla Bruker APEX III FT-ICR mass spectrometer in ESI-positive mode; substances were dissolved in EtOH and diluted with a solvent mixture containing MeOH, H2O and formic acid (49.9/49.9/0.2) to a concentration of about 100 pmol/µL. Elemental analyses were performed on a CHNS analysator (HEKAtech GmbH) by the Department of Inorganic Chemistry, Christian-Albrechts-University of Kiel. IR spectra were recorded on a Shimadzu IRAffinity-1S FTIR spectrometer equipped with MIRacle 10 Single Reflection ATR Accessory. Reactions were monitored by TLC on precoated silica gel plates (SiO2, 60, F254). All compounds could be detected by either UV detection or by ninhydrine spray or Ce(IV)-sulfate staining and heating to 120 °C. Purification of synthesized compounds was carried out on a CombiFlash Rf, version 1.8.2, flash-chromatography apparatus using Interchim PF-30SIHP-JP/12G or 40G or PF-15SIHP/12G silica gel columns. Reverse phase



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chromatography was performed by column chromatography using silica gel 60 silanized (0.0630.200 mm, Merck). All starting materials were commercially available and used without further purification. Nα-(tert-Butyloxycarbonyl)-L-ornithine (CAS Registry Number 21887-64-9) 10 and Nα-(tert-Butyloxycarbonyl)-L-ornithine-tert-butylester (CAS Registry Number 214629-97-7) 15 was purchased from Bachem. All solvents were distilled and dried according to standard procedures.

Chemical synthesis Nα-(tert-Butyloxycarbonyl)-Nδ-phthalimido-L-ornithine

(11).17

Nα-(tert-

Butyloxycarbonyl)-L-ornithine 10 (1.9 g, 8.4 mmol) and phthalic anhydride (2.2 g, 15 mmol) were suspended in 20 mL of chloroform and 150 mL of tetrachloroethylene. This suspension was heated for 2 h at 60 °C and additional 6 h at 100 °C. The clear reaction mixture was concentrated in vacuo to afford the crude product. Purification was carried out by flash column chromatography (silica gel, CH2Cl2-MeOH, 0-5%). Yield: 1.9 g (61%) of a white crystalline solid. mp. 126 °C. TLC: Rf = 0.54 (silica gel, CH2Cl2/MeOH, 9:1). The spectroscopic data were in agreement with those reported. 1H NMR (300 MHz, CDCl3): δ/ppm = 1.43 (s, 9 H, C(CH3)3), 1.65-2.00 (m, 4H, β,γ-CH2), 3.72 (t, 3J = 6.8 Hz, 2H, δ-CH2), 4.14-4.37 (m, 1H, α-CH ), 5.16 (d, 3

J = 8.2 Hz, 1H, NH), 7.68-7.75 (m, 2H, ArH), 7.80-7.86 (m, 2H, ArH), 9.36 (br s, 1H, OH).

13

C NMR (75 MHz, CDCl3): δ/ppm = 37.4 (δ-CH2), 29.8 (β-CH2), 28.3 (C(CH3)3), 24.8 (γ-

CH2), 53.0 (α-CH), 80.3 (C(CH3)3), 123.3 (2 × ArCH), 132.0 (2 × ArC), 134.0 (2 × ArCH), 155.6 (CO-Boc), 168.4 (2 × CO-Pht), 176.5 (COOH). IR (ATR): /cm-1 = 2976, 1771, 1697, 1514, 1396, 1366, 1159, 1047, 718. MS (ESI): m/z = 725 [2 × M + H]+, 625 [2 × M – C4H8 –


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CO2 + H]+, 363 [M + H]+, 263 [M – C4H8 – CO2 + H]+. Anal. calcd. for C18H22N2O6 (362.38): C 59.66, H 6.12, N 7.73; found: C 59.21, H 6.15, N 7.58. Nα-(tert-Butyloxycarbonyl)-Nδ-phthalimido-L-ornithine ethyl ester (12). 2.2 g (6.0 mmol) of the protected ornithine 11 was dissolved in 30 mL of dry DCM. To this solution 0.3 g DMAP (2.4 mmol, 40 mol-%), 1.8 mL of dry EtOH (30 mmol) and 1.2 g (6.3 mmol) EDCI were added. The reaction mixture was stirred magnetically for 24 h at r.t. and subsequently diluted with 25 mL of DCM and washed with 25 mL of 1% HCl, water and brine, each. The organic phase was dried with Na2SO4 and concentrated in vacuo. Further purification was performed by flash column chromatography (silica gel, cyclohexane-EtAc, 0-30%). Yield: 1.3 g (54%) of a white solid. mp. 82 °C. TLC: Rf = 0.40 (cyclohexane/EtAc, 2:1). 1H NMR (300 MHz, CDCl3): δ /ppm = 1.26 (t, 3J = 7.2 Hz, 3H, CH2-CH3), 1.43 (s, 9H, C(CH3)3), 1.62-1.94 (m, 4H, β, γ-CH2), 3.71 (t, 3J = 6.8 Hz, 2H, δ-CH2), 4.18 (q,3J = 7.2 Hz, 2H, CH2-CH3), 4.24-4.35 (m, 1H, α-CH ), 5.06 ( br d, 3J = 8.1 Hz, 1H, NH), 7.68-7.75 (m, 2H, ArH), 7.81-7.87 (m, 2H, ArH).

13

C NMR (75

MHz, CDCl3): δ/ppm = 14.2 (CH2-CH3), 24.7 (γ-CH2), 28.3 (C(CH3)3), 30.2 (β-CH2), 37.5 (δCH2), 53.2 (α-CH), 61.4 (CH2-CH3), 79.9 (C(CH3)3), 123.3 (2 × ArCH), 132.1 (2 × ArC), 134.0 (2 × ArCH), 155.4 (CO-Boc), 168.4 (2 × CO-Pht), 172.5 (COOEt). MS (ESI): m/z = 413 [M + Na]+, 291 [M – C4H8 – CO2 + H]+. Anal. Calcd for C20H26N2O6 (390.44): C 61.53, H 6.71, N 7.17. Found: C 61.73, H 7.32, N 6.94. Nα-(tert-butyloxycarbonyl)-Nω-allyloxycarbonyl-L-thiocitrulline tert-butyl ester (14a). 2.3 g (7.0 mmol) of Nα-Boc-L-ornithine tert-butylester hydrochloride 15 were dissolved in 250 mL of dry DCM. The solution was cooled to 0 °C, 1.1 mL (8.0 mmol) TEA were added and 40 mL of a 0.5 M solution (in dry DCM) of 13 (20 mmol) dropwise over 30 min. The reaction mixture was stirred for 2 h while the solution was being warmed up to r.t. The mixture was



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concentrated in a vacuum to approx. one third of the original volume and washed with 30 mL of 1% HCl, water and brine, each. The organic phase was dried with Na2SO4 and concentrated in a vacuum. The thiourea 14a was at this point already pure by > 96% (1H NMR) and further purified by flash column chromatography (silica gel, cyclohexane/EtAc, 0-30%). Yield: 2.9 g of a colourless oil (94%). TLC: Rf = 0.44 (cyclohexane/EtAc, 3:1). 1H NMR (300 MHz, CDCl3):

δ/ppm = 1.47, 1.50 (2 × s, 2 × 9H, 2 × C(CH3)3), 1.65-1.76 (m, 4H, β, γ-CH2), 3.67 (pseudo q, 2H, δ-CH2), 4.14-4.27 (m, 1H, α-CH), 4.65 (dt, 3J = 5.7, 1.4 Hz, 2H, CH2=CH-CH2), 5.10 (d, 3J = 7.9 Hz, 1H, NH-Boc), 5.29-5.40 (m, 2H, CH2=CH-CH2), 5.90 (ddt, 3J = 17.2, 10.4, 5.7 Hz, 1H, CH=CH2), 8.11 (br s, 1H, NH), 9.66 (br s, 1H, NH-CH2). 13C NMR (75 MHz, CDCl3): δ/ppm = 24.9 (γ-CH2), 28.0, 28.3 (2 × C(CH3)3), 30.3 (β-CH2), 45.1 (δ-CH2), 53.5 (α-CH), 67.0 (O-CH2), 79.8, 82.2 (2 × C(CH3)3), 119.5 (CH=CH2), 130.9 (CH=CH2), 152.4 (CO-Alloc), 155.3 (COBoc), 171.5 (COOtBu), 179.2 (C=S). IR (ATR): /cm-1 = 3292, 2976, 1711, 1520, 1366, 1219, 1152, 1028, 773. MS (ESI): m/z = 454 [M + Na]+, 432 [M + H]+, 376 [M – 2 × C4H8 + H]+, 276 [M – 2 × C4H8 −CO2 + H]+. Anal. Calcd for C19H33N3O6S · 2 H2O (467.59): C 48.81, H 7.98, N 8.99. Found: C 49.03, H 7.90, N 8.59. Nα-(tert-butyloxycarbonyl)-Nω-allyloxycarbonyl-L-thiocitrulline ethyl ester (14b).

To

a

stirred solution of 541 mg (1.4 mmol) of the protected ornithine 12 in 20 mL of a mixture of DCM/EtOH 1:1 was added 1 mL (20 mmol, 15 eq.) hydrazine hydrate (98%) . After 2.5 h precipitated phthalhydrazide was filtered off and the residue was washed three times with 5 mL of DCM. The filtrate was mixed with 15 mL of saturated NaHCO3 solution and shaken. The phases were separated, and the aqueous phase was extracted twice with 10 mL of DCM. Finally, the pooled organic phases were washed with 15 mL of brine, dried with Na2SO4 and chilled on ice to 0 °C. To this pre-cooled solution was added dropwise during about 30 min 5 mL of a 0.5


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M stock solution of 13 (in DCM). The reaction mixture was stirred for additional 2 h at r.t. The solvent was removed in vacuo and the greasy product purified by flash column chromatography (silica gel, cyclohexane/EtAc, 0-25%). Yield: 224 mg of a colourless oil (40%). TLC: Rf = 0.33 (cyclohexane/EtAc, 3:1). 1H NMR (300 MHz, CDCl3): δ/ppm = 1.29 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.45 (s, 9H, C(CH3)3), 1.63-1.96 (m, 4H, β, γ-CH2), 3.68 (pseudo q, 2H, δ-CH2), 4.21 (q, 3J = 7.2 Hz, 2H, CH2CH3), 4.26-4.38 (m, 1H, α-CH), 4.65 (ddd, 3J = 5.8, 4J = 1.7, 1.4 Hz, 2H, CH2=CH-CH2), 5.10 (d, 3J = 8.1 Hz, 1H, NH-Boc), 5.31 (ddd, 2J = 2.7, 3Jcis = 10.5, 4J = 1.2 Hz, 1H, CH2=CH-CH2), 5.37 (ddd, 2J = 3.4, 3Jtrans = 17.2, 4J = 1.4 Hz, 1H, CH2=CH-CH2) 5.90 (ddt, 3

J = 17.2, 10.5, 5.7 Hz, 1H, CH=CH2), 8.25 (br s, 1H, NH), 9.67 (br s, 1H, NH-CH2). 13C NMR

(75 MHz, CDCl3): δ/ppm = 14.2 (CH2CH3), 24.3 (γ-CH2), 28.3 (C(CH3)3), 30. (β-CH2), 45.0 (δCH2), 53.1 (α-CH), 61.5 (CH2CH3), 67.0 (O-CH2), 80.0 (2 × C(CH3)3), 119.5 (CH=CH2), 130.9 (CH=CH2), 152.5 (CO-Alloc), 155.4 (CO-Boc), 172.4 (COOEt), 179.2 (C=S). IR (ATR): /cm-1 = 3364, 3173, 2980, 1746, 1715, 1545, 1520, 1242, 1201, 1155, 1030, 671. MS (ESI): m/z = 426 [M + Na]+, 404 [M + H]+, 348 [M – C4H8 + H]+, 304 [M – C4H8 − CO2 + H]+. Anal. Calcd for C17H29N3O6S · 1.2 H2O (425.12): C 48.03, H 7.45, N 9.88 S 7.54. Found: C 47.94, H 6.60, N 9.70, S 7.56. N-Allyloxycarbonylisothiocyanate (13) Compound 13 was prepared as described previously with allyl chloroformate (50 mmol) as reagent.19,20 Yield: 5.9 g of a bright yellow oil (83%). TLC: Rf = 0.83 (cyclohexane/EtAc, 4:1). 1H NMR (300 MHz, CDCl3): δ/ppm = 4.67 (ddd, 3J = 5.9, 4J =1.5, 1.1 Hz, 2H, CH2=CH-CH2), 5.34 (ddd, 2J = 2.5 Hz, 3Jcis = 10.4 Hz, 4J = 1.1 Hz, 1H, CH2=CH-CH2), 5.40 (ddd, 2J = 3.0 Hz, 3Jtrans = 17.1 Hz, 4J = 1.4 Hz, 1H, CH2=CH-CH2), 5.93 (ddt, 3J = 17.2, 10.4, 5.7 Hz, 1H, CH=CH2). 13C NMR (75 MHz, CDCl3): δ/ppm = 69.4 (O-CH2), 120.2 (CH=CH2), 130.3 (CH=CH2), 147.1 (N=C=S), 149.9 (C=O).



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1-Aminooxy-2,3,4,6-tetra-O-acetyl-β -D-galactopyranose (16, Gal) Galactose derivative 16 was prepared as described previously.12,22 Yield (6 mmol batch): 2.1 g of a white foamy solid (96%; lit.: 67%). mp. 50 °C. TLC: Rf = 0.39 (cyclohexane/EtAc, 3:1). 1H NMR (300 MHz, CDCl3): δ/ppm = 1.99, 2.06, 2.08, 2.16 (4 × s, 4 × 3H, 4 × COCH3), 3.98 (td, 3J = 6.7, 1.2 Hz, 1H, 5'-CH), 4.12-4.23 (m, 2H, 6'-CH2), 4.69 (d, 3J = 8.3 Hz, 1H, 1'-CH), 5.05 (dd, 3J = 10.4, 3.5 Hz, 1H, 3'-CH2), 5.26 (dd, 3J = 10.4, 8.3 Hz, 1H, 2'-CH), 5.40 (dd, 3J = 3.5, 1.1 Hz, 1H, 4'-CH), 5.83 (br s, 2H, NH2). 13C NMR (75 MHz, CDCl3): δ/ppm = 20.5, 20.6, 20.7, 20.8 (4 × COCH3), 61.0 (6'-CH2), 67.0 (4'-CH), 67.3 (2'-CH), 70.7 (5'-CH), 71.0 (3'-CH), 103.9 (1'-CH), 169.7, 170.1, 170.2, 170.4 (4 × COCH3). MS (ESI): m/z = 364 [M + H)+, 331 [C14H19O9]+. 1-Aminooxy-2,3,4,6-tetra-O-acetyl-β -D-glucopyranose (16, Glc) Glucose derivative 16 was prepared as described previously.21,22 Yield (3.8 mmol batch): 1.2 g of a white solid (88%). mp. 123 °C. TLC: Rf = 0.83 (cyclohexane/EtAc, 4:1). 1H NMR (300 MHz, CDCl3): δ/ppm = 2.01, 2.03, 2.07, 2.10 (4 × s, 4 × 3H, 4 × COCH3), 3.75 (ddd, 3J = 10.0, 4.5, 2.3 Hz, 1H, 5'-CH), 4.17 (dd, 2J = 12.2, 3J = 2.4 Hz, 1H, 6'a-CH2), 4.30 (dd, 2J= 12.2, 3J = 4.5 Hz, 1H, 6'b-CH2),4.72 (d, 3J = 8.3 Hz, 1H, 1'-CH), 5.07 (dd, 3J = 9.5, 2.1 Hz, 1H, 4'-CH), 5.10 (dd, 3J = 9.5, 3.4 Hz, 1H, 2'CH), 5.23 (pseudo t, 3J = 9.4 Hz, 1H, 3'-CH), 5.84 (br s, 2H, NH2). 13C NMR (75 MHz, CDCl3):

δ/ppm = 20.6, 20.7, 20.8 (4 × COCH3), 61.8 (6'-CH2), 68.2 (4'-CH), 69.6 (2'-CH), 71.8 (5'-CH), 72.9 (3'-CH), 103.4 (1'-CH), 169.4, 169.5, 170.2, 170.7 (4 × COCH3). MS (ESI): m/z = 364 [M + H)+, 331 [C14H19O9]+. General procedure for the preparation of fully protected NOHA prodrug precursors (17a-b, 18a-b). 3 mmol of thiourea 14a-b, 3.4 mmol of 16 (Gal or Glc) and 3.4 mmol DIPEA were dissolved in 30 mL of dry DCM. The batch was cooled to 0 °C, 3.4 mmol EDCI were added and the mixture magnetically stirred for 72 h at r.t. The solution was diluted with approx.


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30 mL of DCM and washed with 30 mL of 1% HCl, water and brine, each. The organic phase was dried with Na2SO4 and evaporated to dryness. Purification of the crude products was carried out by flash column chromatography (silica gel, cyclohexane/EtAc, 0-30%). Nα-(tert-Butyloxycarbonyl)-Nω-allyloxycarbonyl-Nω'-(2,3,4,6-tetra-O-acetyl-

β-D-galactopyranos-1-yloxy)-L-arginine tert-butyl ester (17a). Yield (3 mmol batch): 1.74 g of a white foamy solid (76%). mp. 78 °C. TLC: Rf = 0.49 (cyclohexane/EtAc, 1:1). 1H NMR (300 MHz, CDCl3): δ/ppm =1.44, 1.47 (2 × s, 2 × 9H, 2 × C(CH3)3), 1.54-1.90 (m, 4H, β, γ-CH2), 2.00, 2.04, 2.07, 2.16 (4 × s, 4 × 3H, 4 × COCH3), 3.10 (pseudo q, 2H, δ-CH2), 3.98 (td, 3J = 6.9, 1.0 Hz, 1H, 5'-CH), 4.12-4.23 (m, 3H, 6'-CH2, α-CH ), 4.63 (dt, 3J = 5.6, 1.4 Hz, 2H, CH2=CHCH2), 4.87 (d, 3J = 8.4 Hz, 1H, 1'-CH), 5.05-5.12 (m, 2H, 3'-CH2, NH-Boc), 5.23-5.43 (m, 4H, 2', 4'-CH, CH2=CH-CH2), 5.93 (ddt, 3J = 17.2, 10.4, 5.7 Hz, 1H, CH=CH2), 6.45 (br t, 3J = 5.2, 1H, NH-CH2), 7.76 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ/ppm = 20.66, 20.69, 20.7, 20.9 (4 × COCH3), 24.8 (δ-CH2), 28.0, 28.3 (2 × C(CH3)3), 30.3 (β-CH2), 40.5 (δ-CH2), 53.7 (αCH), 61.0 (6'-CH2), 66.5 (O-CH2), 66.9, 68.0, 70.5, 70.9 (4', 2', 5', 3'-CH), 79.7, 81.9 (2 × C(CH3)3), 102.5 (1'-CH), 118.9 (CH=CH2), 131.5 (CH=CH2), 150.3 (C=N), 152.8 (CO-Alloc), 155.4 (CO-Boc), 170.07, 170.13, 170.3, 170.25, 170.34 (4 × COCH3), 171.7 (COOtBu). MS (ESI): m/z = 783 [M + Na]+, 761 [M + H]+. IR (ATR): 3450, 2980, 1732, 1645, 1366, 1215, 1151, 1043, 772 cm-1. Anal. Calcd for C33H52N4O16 (760.79): C 52.80, H 6.86, N 7.24. Found: C 52.34, H 6.67, N 7.28. Nα-(tert-Butyloxycarbonyl)-Nω-allyloxycarbonyl-Nω'-(2,3,4,6-tetra-O-acetyl-

β-D-glucopyranos-1-yloxy)-L-arginine tert-butyl ester (17b). Yield (0.9 mmol batch): 548 mg of a white foamy solid (81%). TLC: Rf = 0.42 (cyclohexane/EtAc, 1:1). 1H NMR (300 MHz, CDCl3): δ/ppm = 1.30 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.44, 1.46 (2 × s, 2 × 9H, 2 × C(CH3)3),



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1.55-1.90 (m, 4H, β, γ-CH2), 2.02, 2.03, 2.08, 2.09 (4 × s, 4 × 3H, 4 × COCH3), 3.02-3.16 (m, 2H, δ-CH2), 3.77 (ddd, 3J = 10.3, 4.2, 2.3 Hz, 1H, 5'-CH), 4.04-4.23 (m, 4H, 6'a-CH2, α-CH, CH2CH3 ), 4.33 (dd, 2J = 12.5, 3J = 4.3 Hz, 1H, 6'b-CH2), 4.89 (d, 3J = 8.2 Hz, 1H, 1'-CH), 5.025.18 (m, 3H, 2', 4'-CH, NH-Boc), 5.25 (dd, 3J = 9.4, 7.9 Hz, 1H, 3'-CH), 6.50 (br t, 3J = 5.1 Hz, 1H, NH-CH2), 7.63 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3): δ/ppm = 14.3 (CH2CH3), 20.61, 20.63, 20.7, 20.8, (4 × COCH3), 24.9 (γ-CH2), 28.0, 28.3 (2 × C(CH3)3), 30.3 (β-CH2), 40.5 (δ CH2), 53.7 (α-CH), 61.8 (6'-CH2), 62.2 (CH2CH3), 68.3, 70.2, 71.7, 72.9 (4', 2', 5', 3'-CH), 79.3, 81.9 (2 × C(CH3)3), 102.0 (1'-CH), 150.7 (C=N), 153.1 (CO-Eoc), 155.4 (CO-Boc), 169.5, 169.8, 170.2, 170.7 (4 × COCH3), 171.7 (COOtBu). MS (ESI): m/z = 771 [M + Na]+, 749 [M + H]+. IR (ATR): /cm-1 = 1740, 1717, 1645, 1506, 1464, 1366, 1213, 1151, 1034, 905, 773. Anal. Calcd for C32H52N4O16 (748.78): C 51.33, H 7.00, N 7.48. Found: C 51.35, H 7.26, N 7.22. Nα-(tert-Butyloxycarbonyl)-Nω-allyloxycarbonyl-Nω'-(2,3,4,6-tetra-O-acetyl-

β-D-galactopyranos-1-yloxy)-L-arginine ethyl ester (18a). Yield (1.0 mmol batch): 653 mg of a white foamy solid (93%). mp. 65 °C. TLC: Rf = 0.31 (cyclohexane/EtAc, 1:1). 1H NMR (300 MHz, CDCl3): δ/ppm = 1.28 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.45 (s, 9H, C(CH3)3), 1.59-1.94 (m, 4H, β, γ-CH2), 2.00, 2.04, 2.11, 2.16 (4 × s, 4 × 3H, 4 × COCH3), 3.10 (pseudo q, 2H, δ-CH2), 3.99 (td, 3J = 6.9, 1.0 Hz, 1H, 5'-CH), 4.07-4.34 (m, 5H, 6'-CH2, α-CH, CH2CH3 ), 4.66 (dt, 3J = 5.6, 1.4 Hz, 2H, CH2=CH-CH2), 4.87 (d, 3J = 8.2 Hz, 1H, 1'-CH), 5.03-5.13 (m, 2H, 3'-CH, NHBoc), 5.22-5.43 (m, 4H, 2', 4'-CH, CH2=CH-CH2), 5.92 (ddt, 3J = 17.2, 10.4, 5.7 Hz, 1H, CH=CH2), 6.44 (br t, 3J = 5.2, 1H, NH-CH2), 7.76 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3):

δ/ppm =14.2 (CH2CH3), 20.57, 20.64, 20.67, 20.94 (4 × COCH3), 25.0 (γ-CH2), 28.3 (C(CH3)3), 30.0 (β-CH2), 41.1 (δ-CH2), 53.2 (α-CH), 60.9 (6'-CH2), 61.4 (CH2CH3), 66.8 (4'-CH), 67.0 (OCH2), 67.6, 68.4, 70.7 (2', 5', 3'-CH), 79.7 (C(CH3)3), 103.1 (1'-CH), 119.4 (CH=CH2), 131.5


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(CH=CH2), 150.1 (C=N), 152.8 (CO-Alloc), 155.4 (CO-Boc), 169.96, 170.14, 170.26, 170.31 (4 × COCH3), 171.5 (COOEt). MS (ESI): m/z = 755 [M + Na]+, 733 [M + H]+. IR (ATR): /cm-1 = 3400, 1744, 1651, 1506, 1367, 1215, 1161, 1047, 910, 772. Anal. Calcd for C31H48N4O16 · 1.6 H2O (761.57): C 48.89, H 6.78, N 7.36. Found: C 48.64, H 6.98, N 6.88. Nα-(tert-Butyloxycarbonyl)-Nω-allyloxycarbonyl-Nω'-(2,3,4,6-tetra-O-acetyl-

β-D-glucopyranos-1-yloxy)-L-arginine ethyl ester (18b). Yield (0.56 mmol batch): 300 mg of a white foamy solid (73%). TLC: Rf = 0.31 (cyclohexane/EtAc, 1:1). 1H NMR (300 MHz, CDCl3):

δ/ppm = 1.28 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.45 (s, 9H, C(CH3)3), 1.55-1.91 (m, 4H, β, γ-CH2), 2.02, 2.03, 2.06, 2.07 (4 × s, 4 × 3H, 4 × COCH3), 3.05-3.15 (m, 2H, δ-CH2), 3.77 (ddd, 3J = 10.2, 4.5, 2.3 Hz, 1H, 5'-CH), 4.10-4.36 (m, 5H, 6'-CH2, α-CH, CH2CH3 ), 4.62 (dt, 3J = 5.7, 1.7 Hz, 2H, CH2=CH-CH2), 4.89 (d, 3J = 8.2 Hz, 1H, 1'-CH), 5.06-5.16 (m, 3H, 2', 4'-CH, NH-Boc), 5.22-5.41 (m, 3H, 3'-CH, CH2=CH-CH2), 5.92 (ddt, 3J = 17.2, 10.4, 5.7 Hz, 1H, CH=CH2), 6.45 (br t, 3J = 5.3, 1H, NH-CH2), 7.72 (br s, 1H, NH).

13

C NMR (75 MHz, CDCl3): δ/ppm = 14.2

(CH2CH3), 20.61, 20.63, 20.7, 20.9, (4 × COCH3), 25.0 (γ-CH2), 28.3 (C(CH3)3), 30.1 (β-CH2), 40.4 (δ-CH2), 53.3 (α-CH), 61.8 (CH2CH3), 61.9 (6'-CH2), 66.6 (O-CH2), 68.3, 70.2, 71.7, 72.8 (4', 2', 5', 3'-CH), 79.9 (C(CH3)3), 102.0 (1'-CH), 119.0 (CH=CH2), 131.4 (CH=CH2), 150.5 (C=N), 152.8 (CO-Alloc), 155.4 (CO-Boc), 169.5, 169.9, 170.2, 170.7 (4 × COCH3), 172.6 (COOEt). MS (ESI): m/z = 755 [M + Na]+, 733 [M + H]+. IR (ATR): /cm-1 = 3450, 1738, 1717, 1645, 1456, 1366, 1213, 1161, 1034, 908, 772. Anal. Calcd for C31H48N4O16 (732.74): C 50.82, H 6.60, N 7.65. Found: C 50.78, H 6.74, N 8.08. General procedure for the preparation of carbamate-free NOHA prodrug precursors (19a-b, 20a-b). 1.0 mmol of the completely protected compound 17a-b or 18a-b, 8 mmol (8 equiv.) dimedone and 0.1 mmol (10 mol-%) Pd(0)[Ph3P]4 were dissolved in 20 mL of THF and



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the reaction mixture was stirred for 30 min at r.t. The solvent was removed under reduced pressure and the solid residue dissolved in 30 mL of EtAc. Solid dimedone was filtered off and the organic phase was washed three times with 10 mL of diluted aqueous NH3-solution (3%), once with 10 mL of water and brine. The organic phase was dried with Na2SO4 and concentrated in a vacuum. The crude product was further purified by flash column chromatography (silica gel, DCM/MeOH, 0-5% for about 25 min). Nα-(tert-Butyloxycarbonyl)-Nω-(2,3,4,6-tetra-O-acetyl-β -D-galactopyranos-1-yloxy)-Larginine tert-butyl ester (19a). Yield: 588 mg of a light yellow solid (90%). mp. 95 °C. TLC: Rf = 0.48 (DCM/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ/ppm =1.44, 1.46 (2 × s, 2 × 9H, 2 ×C(CH3)3), 1.53-1.83 (m, 4H, β, γ-CH2), 1.99, 2.03, 2.07, 2.18 (4 × s, 4 × 3H, 4 × COCH3), 2.983.06 (m, 2H, δ-CH2), 3.59 (br s,1H, NH-CH2), 3.98 (td, 3J = 6.9, 1.0 Hz, 1H, 5'-CH), 4.10-4.24 (m, 3H, 6'-CH2, α-CH), 4.33 (br s, 2H, NH2), 4.87 (d, 3J = 8.3 Hz, 1H, 1'-CH), 5.03-5.30 (m, 3H, 2', 3'-CH, NH-Boc), 5.40 (dd, 3J = 3.5, 1.0 Hz, 1H, 4'-CH). 13C NMR (75 MHz, CDCl3): δ/ppm = 20.6, 20.7, 21.0, (4 × COCH3), 25.4 (γ-CH2), 28.0, 28.4 (2 × C(CH3)3), 30.8 (β-CH2), 41.5 (δ CH2), 53.4 (α-CH), 61.0 (6'-CH2), 66.9, 68.1, 70.4, 71.0 (4', 2', 5', 3'-CH), 79.7, 82.1 (2 × C(CH3)3), 102.5 (1'-CH), 155.6 (C=N), 156.6 (CO-Boc), 170.1, 170.3, 170.4 (4 × COCH3), 171.7 (COOtBu).

15

N NMR (30 MHz, 25 °C, 0.2% MeNO2 in CDCl3 as external standard):

δ/ppm = -328.4 (NH2), -321.2 (NH-CH2), -294.8 (NH-Boc), the nitrogen of the oxime moiety was not observed. IR (ATR): /cm-1 = 3450, 2950, 1744, 1714, 1645, 1516, 1366, 1217, 1152, 1043, 908. MS (ESI): m/z = 677 [M + H]+. HRMS (ESI) m/z for C29H48N4O14 [M + H]+: 677.3239, found: 677.3219. Nα-(tert-Butyloxycarbonyl)-Nω-(2,3,4,6-tetra-O-acetyl-β -D-glucopyranos-1-yloxy)-Larginine tert-butyl ester (19b). Yield: 582 mg of a light yellow solid (89%). mp. 94 °C. TLC:


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Rf = 0.31 (DCM/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ/ppm = 1.44, 1.47 (2 × s, 2 × 9H, 2 × C(CH3)3), 1.51-1.87 (m, 4H, β, γ-CH2), 2.01, 2.02, 2.06, 2.07 (4 × s, 4 × 3H, 4 × COCH3), 2.96-3.23 (m, 2H, δ-CH2), 3.70 (br s,1H, NH-CH2), 3.77 (ddd, 3J = 10.1, 4.2, 2.4 Hz, 1H, 5'-CH), 4.07-4.22 (m, 2H, 6'a-CH2, α-CH), 4.27-4.45 (m, 3H, 6'b-CH2, NH2), 4.88 (d, 3J = 8.6 Hz, 1H, 1'-CH), 5.03-5.29 (m, 4H, 2', 3', 4'-CH, NH-Boc). 13C NMR (75 MHz, CDCl3): δ/ppm = 20.62, 20.65, 20.8, 20.9 (4 × COCH3), 25.6 (γ-CH2), 28.0, 28.4 (2 × C(CH3)3), 30.7 (β-CH2), 41.2 (δCH2), 53.0 (α-CH), 61.8 (6'-CH2), 68.3, 70.3, 71.6, 72.9 (4', 2', 5', 3'-CH), 80.0, 82.2 (2 × C(CH3)3), 102.1 (1'-CH), 155.7 (C=N), 156.8 (CO-Boc), 169.5, 169.9, 170.2, 170.8 (4 × COCH3), 171.7 (COOtBu). IR (ATR): /cm-1 = 1744, 1714, 1643, 1639, 1520, 1366, 1215, 1161, 1112, 1096, 1034, 908. MS (ESI): m/z = 677 [M + H]+. HRMS (ESI) m/z for C29H48N4O14 [M + H]+: 677.3239, found: 677.3223. Nα-(tert-Butyloxycarbonyl)-Nω-(2,3,4,6-tetra-O-acetyl-β -D-galactopyranos-1-yloxy)-Larginine ethyl ester (20a). Yield (0.40 mmol batch): 220 mg of a light yellow solid (73%). TLC: Rf = 0.55 (DCM/MeOH, 95:5). 1H NMR (300 MHz, CDCl3): δ/ppm = 1.29 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.44 (s, 9H, C(CH3)3), 1.56-1.91 (m, 4H, β, γ-CH2), 1.99, 2.04, 2.09, 2.15 (4 × s, 4 × 3H, 4 × COCH3), 3.04-3.29 (m, 2H, δ-CH2), 4.00 (td, 3J = 6.9, 1.0 Hz, 1H, 5'-CH), 4.12-4.35 (m, 5H, 6'-CH2, α-CH, CH2CH3 ), 4.73 (br s, 2H, NH2), 4.87 (d, 3J = 8.3 Hz, 1H, 1'-CH), 5.07 (dd, 3J = 10.5, 3.4 Hz, 1H, 3'-CH), 5.20-5.31 (m, 2H, 2'-CH, NH-Boc), 5.41 (dd, 3J = 3.4, 1.0 Hz, 1H, 4'-CH). 13C NMR (75 MHz, CDCl3): δ/ppm = 14.2 (CH2CH3), 20.60, 20.66, 20.7, 21.0, (4 × COCH3), 25.6 (γ-CH2), 28.3 (C(CH3)3), 30.5 (β-CH2), 41.2 (δ-CH2), 52,7 (α-CH), 60.9 (6'-CH2), 61.6 (CH2CH3), 66.9 (4'-CH), 67.9, 70.7, 70.8 (2', 5', 3'-CH), 80.3 (C(CH3)3), 103.0 (1'-CH), 155.9 (C=N),157.1 (CO-Boc), 170.1, 170.2, 170.3 (4 × COCH3), 172.5 (COOEt). IR (ATR): /cm-1 = 1748, 1713, 1368, 1215, 1161, 1055, 955, 743. MS (ESI): m/z = 649 [M + H]+.



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Anal. Calcd for C27H44N4O14 · 1,4 H2O (673.89): C 48.12, H 7.00, N 8.31. Found: C 48.28, H 7.14, N 7.55. Nα-(tert-Butyloxycarbonyl)-Nω-(2,3,4,6-tetra-O-acetyl-β -D-glucopyranos-1-yloxy)-Larginine ethyl ester (20b). Yield (0.33 mmol batch): 180 mg of a light yellow solid (84%). TLC: Rf = 0.52 (DCM/MeOH, 9:1). 1H NMR (300 MHz, CDCl3): δ/ppm =1.28 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.44 (s, 9H, C(CH3)3), 1.52-1.90 (m, 4H, β, γ-CH2), 2.01, 2.02, 2.06, 2.07 (4 × s, 4 × 3H, 4 × COCH3), 3.02-3.27 (m, 2H, δ-CH2), 3.55 (br s, 1H, NH-CH2), 3.77 (ddd, 3J = 10.2, 4.6, 2.4 Hz, 1H, 5'-CH), 4.10-4.37 (m, 7H, 6'-CH2, α-CH, CH2CH3, NH2 ), 4.90 (d, 3J = 8.4 Hz, 1H, 1'-CH), 5.02-5.29 (m, 4H, 2', 3', 4'-CH, NH-Boc). 13C NMR (75 MHz, CDCl3): δ/ppm = 14.2 (CH2CH3), 20.62, 20.65, 20.8, 20.9, (4 × COCH3), 25.6 (γ-CH2), 28.3 (C(CH3)3), 30.6 (β-CH2), 41.4 (δ-CH2), 53.0 (α-CH), 61.5 (CH2CH3), 61.9 (6'-CH2), 68.4, 70.4, 71.6, 73.0 (4', 2', 5', 3'CH), 80.1 (C(CH3)3), 102.1 (1'-CH), 155.7 (C=N), 156.6 (CO-Boc), 169.5, 169.9, 170.2, 170.8 (4 × COCH3), 172.6 (COOEt). MS (ESI): m/z = 671 [M + Na]+, 649 [M + H]+. HRMS (ESI) m/z for C27H44N4O14: 649.2927 found: 649.2948. Nω-(2,3,4,6-Tetra-O-acetyl-β -D-galactopyranos-1-yloxy)-L-arginine dihydrochloride (2). In a triple-necked flask was dissolved under argon 1.23 mg (1.8 mmol) of the carefully dried carbamate-free compound 19a in approx. 30 mL of absolute Et2O. The solution was stirred for 30 min at -10 °C. Subsequently gaseous HCl was carefully bubbled through the solution under argon for 20 min. The reaction mixture was stirred for additional 2 h at 0 °C and subsequently stored for 24 h in the refrigerator. The solvent was carefully removed in a vacuum and the white to light yellow solid dissolved in approx. 30 mL of MeOH. The solution was chilled on ice and silica gel was added. This silica gel containing mixture was evaporated to dryness and further purified by flash column chromatography (silica gel, DCM/MeOH, 0-20%). Product containing


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fractions were pooled and evaporated to dryness. Yield: 0.93 g of a white, hygroscopic powder (86%). TLC: Rf = 0.65 (DCM/MeOH, 1:1). 1H NMR (300 MHz, D2O): δ/ppm = 1.54-1.98 (m, 4H, β,γ-CH2), 1.99, 2.06, 2.11, 2.20 (4 × s, 4 × 3H, 4 × COCH3), 3.30 (t, 3J = 6.8 Hz, 2H, δCH2), 3.95 (t, 3J = 6.2 Hz, 1H, 5'-CH), 4.16-4.31 (m, 3H, 6'-CH2, α-CH ), 5.17 (d, 3J = 7.9 Hz, 1H, 1'-CH), 5.26 (dd, 3J = 10.0, 3.2 Hz, 1H, 3'-CH2), 5.34 (dd, 3J = 10.1, 7.9 Hz, 1H, 2'-CH2), 5.47 (d, 3J = 3.1 Hz, 1H, 4'-CH2).

13

C NMR (75 MHz, D2O): δ/ppm = 21.8, 22.0, 22.1, (4 ×

COCH3), 25.5 (γ-CH2), 29.0 (β-CH2), 42.6 (δ -CH2), 55.0 (α-CH), 63.7 (6'-CH2), 68.5, 69.7, 73.1, 73.2 (4',2',5',3'-CH), 104.5 (1'-CH), 160.1 (C=N), 174.4, 174.5, 174.8, 174.9 (4 × COCH3), 175.3 (COOH). 35Cl NMR (29 MHz, D2O, NaCl) calcd. for C20H34Cl2N4O12: Cl 11.41, found: Cl 10.88 (recovery: 95%). IR (ATR): /cm-1 = 3224 (broad), 1740, 1651, 1634, 1433, 1368, 1167, 1123, 1057, 955, 910. MS (ESI): m/z = 521 [M + H]+, 479 [M – C2H2O + H]+, 331 [C14H19O9]+, 191 [C6H14N4O3 + H]+. HRMS (ESI) m/z for C20H32N4O12 [M + H]+: 521.2089, found: 521.2078. Nω-(2,3,4,6-Tetra-O-acetyl-β -D-galactopyranos-1-yloxy)-L-arginine ethyl ester dihydrochloride (3). Compound 3 was prepared in analogy to compound 2 with the protected precursor 20a as reagent. Yield (0.55 mmol batch): 206 mg of a white, hygroscopic powder (60%). mp. 88 °C TLC: Rf = 0.76 (DCM/MeOH, 2:1). 1H NMR (300 MHz, D2O): δ/ppm = 1.27 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.56-1.86 (m, 2H, γ-CH2), 1.90-2.04 (m, 2H, β-CH2), 1.99, 2.06, 2.11, 2.20 (4 × s, 4 × 3H, 4 × COCH3), 3.26 (t, 3J = 6.9 Hz , 2H, δ-CH2), 4.14 (t, 3J = 6.2 Hz, 1H, 5'-CH), 4.19-4.34 (m, 5H, 6'-CH2, α-CH, CH2CH3), 5.15 (d, 3J = 7.8 Hz, 1H, 1'-CH), 5.25 (dd, 3J = 10.2, 3.2 Hz, 1H, 3'-CH2), 5.34 (dd, 3J = 10.2, 7.8 Hz, 1H, 2'-CH2), 5.47 (d, 3J = 3.2 Hz, 1H, 4'CH2).

13

C NMR (75 MHz, D2O): δ/ppm = 15.1 (CH2CH3), 21.82, 21.83, 22.0, 22.1, (4 ×

COCH3), 25.5 (γ-CH2), 28.8 (β-CH2), 42.3 (δ -CH2), 54.3 (α-CH), 63.4 (6'-CH2), 65.5 (CH2CH3), 68.6, 69.7, 73.08, 73.11 (4',2',5',3'-CH), 104.3 (1'-CH), 160.9 (C=N), 171.6 (COOEt),



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174.4, 174.8, 174.9, 175.3 (4 × COCH3). IR (ATR): /cm-1 = 3400 (broad), 2950, 1740, 1670, 1369, 1213, 1167, 1121, 1057, 955, 908, 858. MS (ESI): m/z = 549 [M + H]+, 507 [M – C2H2O + H]+, 331 [C14H19O9]+ , 219 [C8H18N4O3]+. HRMS (ESI) m/z for C22H36N4O12 [M + H]+: 549.2403, found: 549.2398. Nω-(2,3,4,6-Tetra-O-acetyl-β -D-glucopyranos-1-yloxy)-L-arginine dihydrochloride (4). Compound 7 was prepared in analogy to compound 2 with the protected precursor 19b as reagent. Yield (0.86 mmol batch): 491 mg of a white, hygroscopic powder (97%). mp. 130 °C TLC: Rf = 0.45 (DCM/MeOH, 1:1). 1H NMR (300 MHz, D2O): δ/ppm = 1.59-1.82 (m, 2H, γCH2), 1.83-1.99 (m, 2H, β-CH2), 2.02, 2.05, 2.08 (4 × s, 4 × 3H, 4 × COCH3), 3.28 (t, 3J = 6.7 Hz , 2H, δ-CH2), 3.98 (t, 3J = 6.3 Hz, 1H, α-CH ), 4.07 (ddd, 3J = 10.3, 4.6, 2.5 Hz, 1H, 5'-CH), 4.25 (dd, 2J = 12.8, 3J =2.5 Hz, 1H, 6'a-CH2), 4.32 (dd, 2J = 12.8, 3J = 4.1 Hz, 1H, 6'b-CH2), 5.04-5.25 (m, 3H, 1', 2', 4'-CH), 5.26 (pseudo t, 3J = 8.9 Hz, 1H, 3'-CH2). 13C NMR (75 MHz, D2O): δ/ppm = 21.88, 21.89, 21.90, 21.98 (4 × COCH3), 25.4 (γ-CH2), 28.9 (β-CH2), 42.5 (δ CH2), 54.7 (α-CH), 63.5 (6'-CH2), 69.8, 70.6, 73.6, 74.9 (4',2',5',3'-CH), 104.1 (1'-CH), 160.8 (C=N), 174.1, 174.4, 174.6, 174.9 (4 × COCH3), 175.4 (COOH). MS (ESI): m/z = 521 [M + H]+, 479 [M – C2H2O + H]+, 331 [C14H19O9]+ , 191 [C6H14N4O3 + H]+. HRMS (ESI) m/z for C20H32N4O12 M + H]+: 521.2090, found: 521.2088. Nω-(2,3,4,6-Tetra-O-acetyl-β -D-glucopyranos-1-yloxy)-L-arginine ethyl ester dihydrochloride (5). Compound 5 was prepared in analogy to compound 2 with the protected precursor 20b as reagent. Yield (0.40 mmol batch): 182 mg of a white, hygroscopic powder (75%). mp. 82 °C TLC: Rf = 0.78 (DCM/MeOH, 2:1). 1H NMR (300 MHz, D2O): δ/ppm = 1.28 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.58-1.86 (m, 2H, γ-CH2), 1.88-2.05 (m, 2H, β-CH2), 2.03, 2.07, 2.10 (4 × s, 4 × 3H, 4 × COCH3), 3.29 (t, 3J = 6.9 Hz , 2H, δ-CH2), 4.09 (ddd, 3J = 10.6, 4.2, 2.5


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Hz, 1H, 5'-CH), 4.14 (t, 3J = 6.2 Hz, 1H, α-CH), 4.24-4.37 (m, 4H, 6'-CH2, CH2CH3), 5.12 (t, 3J = 9.6 Hz, 1H, 4'-CH), 5.22 (dd, 3J = 16.4, 8.3 Hz, 1H, 2'-CH2), 5.23 (d, 3J = 8.3 Hz, 1H, 1'-CH), 5.39 (t, 3J = 8.5 Hz, 1H, 3'-CH2).

13

C NMR (75 MHz, D2O): δ/ppm = 15.1 (CH2CH3), 21.88,

21.89, 21.99, 22.00 (4 × COCH3), 25.4 (γ-CH2), 28.7 (β-CH2), 42.4 (δ -CH2), 54.3 (α-CH), 63.6 (6'-CH2), 65.5 (CH2CH3), 69.8, 70.5, 73.6, 74.9 (4',2',5',3'-CH), 103.9 (1'-CH), 160.8 (C=N), 171.6 (COOEt), 174.4, 174.6, 174.8, 175.4 (4 × COCH3). IR (ATR): /cm-1 = 3400 (broad), 1740, 1670, 1368, 1211, 1148, 1069, 1034, 907, 856. MS (ESI): m/z = 549 [M + H]+, 507 [M – C2H2O + H]+, 331 [C14H19O9]+ , 219 [C8H18N4O3]+. HRMS (ESI) m/z for C22H36N4O12 [M + H]+: 549.2403, found: 549.2398. Nω-(β-D-Galactopyranos-1-yloxy)-L-arginine ethyl ester dihydrochloride (6). In a well dried round-bottomed flask was mixed 6 mL of dry EtOH under argon with 2 mL (28 mmol) of acetyl chloride. This mixture was stirred for 15 min at 0 °C and subsequently 200 mg (0.3 mmol) of the carefully dried compound 19a was added. The reaction was stirred for additional 48 h at r.t. and finally the solvent was removed in vacuo. The white solid was taken up with 1-2 mL of aq. bidest. and purified by RP-18 column chromatography (eluent: aq. bidest, isocratic). Product containing fractions were pooled and concentrated in a vacuum at 30 °C to a residual volume of about 1 mL. The remaining water was removed in a high vacuum to give 6 as a white, hygroscopic solid. Yield: 111 mg of a white, hygroscopic powder (82%). mp. 99 °C TLC: Rf = 0.30 (i-PrOH/H2O, 2:1). 1H NMR (300 MHz, D2O): δ/ppm = 1. 26 (t, 3J = 7.2 Hz, 3H, CH2CH3), 1.60-2.08 (m, 4H, β, γ-CH2), 3.31 (t, 3J= 6.7 Hz , 2H, δ-CH2), 3.63-3.79 (m, 5H, 2',4', 5'-CH, 6'CH2), 3.91 (d, 3J = 1.7 Hz, 1H, 3'-CH), 4.13 (t, 3J= 6.4 Hz, 1H, α-CH), 4.28 (q, 3J = 7.2 Hz, 2H, CH2CH3), 4.69 (d, 3J = 8.0 Hz, 1H, 1'-CH2). 13C NMR (75 MHz, D2O): δ/ppm = 15.1 (CH2CH3), 25.4 (γ-CH2), 28.7 (β-CH2), 42.4 (δ -CH2), 54.3 (α-CH), 62.8 (6'-CH2), 65.5 (CH2CH3), 70.2,



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70.6, 74.4, 77.5 (4',2',5',3'-CH), 108.4 (1'-CH), 158.5 (C=N), 171.7 (COOEt). IR (ATR): /cm-1 = 3200 (broad), 2936, 1738, 1667, 1639, 1472, 1381, 1227, 1140, 1057, 1016, 858, 779. MS (ESI): m/z = 381 [M + H]+, 353 [M – C2H4 + H]+, 219 [C8H18N4O3 + H]+ , 191 [C6H14N4O3 + H]+. HRMS (ESI) m/z for C14H28N4O8 [M + H]+: 381.1980, found: 381.1980. Nω-(β-D-Glucopyranos-1-yloxy)-L-arginine ethyl ester dihydrochloride (8). Compound 8 was prepared in analogy to compound 6 with the protected precursor 19b as reagent. Yield (0.31 mmol batch): 185 mg of a white, hygroscopic powder (93%). mp. 86 °C TLC: Rf = 0.31 (DCM/MeOH, 2:1). 1H NMR (300 MHz, D2O): δ/ppm = 1. 29 (t, 3J = 7.2 Hz, 3 H, CH2CH3), 1.62-1.89, (m, 2H, γ-CH2), 1.90-2.08 (m, 2H, β-CH2), 3.26-3.37 (m,2H, δ-CH2), 3.40-3.67 (m, 4H, 2', 3', 4', 5'-CH), 3.75 (dd, 2J = 12.4, 3J =5.1 Hz, 1H, 6'a-CH2), 3.90 (dd, 2J = 12.8, 3J = 1.9 Hz, 1H, 6'b-CH2), 4.15 (t, 3J = 6.5 Hz, 1H, α-CH), 4.30 (q, 3J = 7.2 Hz, 2H, CH2CH3), 4.77 (d, 3J = 7.7 Hz, 1H, 1'-CH2). 13C NMR (75 MHz, D2O): δ/ppm = 15.1 (CH2CH3), 25.4 (γ-CH2), 28.8 (β-CH2), 42.4 (δ-CH2), 54.4 (α-CH), 62.3 (6'-CH2), 65.5 (CH2CH3), 70.9, 72.9, 77.4, 78.2 (4',2',5',3'-CH), 107.9 (1'-CH), 160.4 (C=N), 171.7 (COOEt).

35

Cl NMR (29 MHz, D2O, NaCl)

calcd. for C14H30Cl2N4O8: Cl 15.64, found: Cl 13.25 (recovery: 85%). IR (ATR): /cm-1 = 3200 (broad), 1738, 1667, 1512, 1470, 1449, 1381, 1296, 1226, 1070, 1118, 856. MS (ESI): m/z = 381 [M + H]+, 353 [M – C2H4 + H]+, 219 [C8H18N4O3 + H]+ , 191 [C6H14N4O3 + H]+. HRMS (ESI) m/z for C14H28N4O8 [M + H]+: 381.1980, found: 381.1981. Sodium Nω-(β -D-Galactopyranos-1-yloxy)-L-arginine (7).

54

mg

(1

mmol)

sodium

methoxide was dissolved in 10 mL of MeOH and stirred for 30 min at 0 °C. 100 mg (0.17 mmol) of the peracetylated compound 2 was added and the reaction stirred for 15 min at 0 °C and additional 2 h at r.t. The cold solution was neutralized with diluted HCl and concentrated under reduced pressure. The white solid residue was taken up in 2 mL of aq. bidest. and purified by


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RP-18 column chromatography (eluent: aq. bidest, isocratic). Product containing fractions were pooled and concentrated in a vacuum at 30 °C to a residual volume of about 1 mL. The remaining water was removed in a high vacuum to give 7 as a white solid. Yield: 63 mg (quant.). mp. 139 °C. TLC: Rf = 0.35 (i-PrOH/H2O, 2:1). 1H NMR (300 MHz, D2O): δ/ppm = 1.55-1.94 (m, 4H, β,γ-CH2), 3.23 (t, 3J = 6.8 Hz , 2H, δ-CH2), 3.60-3.81 (m, 6H, 2', 3', 5'-CH, 6'-CH2, αCH ), 3.91 (d, 3J = 2.4 Hz, 1H, 4'-CH), 4.66 (d, 3J = 7.0 Hz, 1H, 1'-CH2). 13C NMR (75 MHz, D2O): δ/ppm = 25.8 (γ-CH2), 29.4 (β-CH2), 42.3 (δ-CH2), 56.2 (α-CH), 63.0 (6'-CH2), 70.3, 71.0, 74.5, 77.2 (4',2',5',3'-CH), 107.3 (1'-CH), 160.9 (C=N), 176.3 (COOH). IR (ATR): /cm-1 = 3250 (broad), 1585 (ν as COO-), 1454, 1408 (ν s COO-), 1346, 1261, 1141, 1071, 1018, 947, 667. MS (ESI): m/z = 375 [M + Na]+, 353 [M + H]+, 191 [C6H14N4O3 + H]+. HRMS (ESI) m/z for C12H24N4O8 [M + H]+: 353.1167, found: 353.1169. Sodium Nω-(β -D-Glucopyranos-1-yloxy)-L-arginine (9). Compound 9 was prepared in analogy to compound 7 with the protected precursor 4 as reagent. Yield (0.24 mmol batch): 81 mg of a white solid (90%). mp. 157 °C TLC: Rf = 0.41 (DCM/MeOH, 2:1). 1H NMR (300 MHz, D2O): δ/ppm = 1.55-1.81 (m, 2 H, γ-CH2), 1.82-1.96 (m, 2 H, β-CH2), 3.28 (t, 3J = 6.7 Hz , 2 H, δ-CH2), 3.37-3.55 (m, 4 H, 2', 3', 4', 5'-CH), 3.68-3.76 (m, 2 H, 6'a-CH2, α-CH ), 3.89 (pseudo d, 2

J = 12.3 Hz, 1 H, 6'b-CH2), 4.74 (d, 3J = 7.9 Hz, 1 H, 1'-CH2). 13C NMR (75 MHz, D2O): δ/ppm

= 25.6 (γ-CH2), 29.4 (β-CH2), 42.6 (δ-CH2), 56.2 (α-CH), 62.3 (6'-CH2), 70.9, 73.0, 77.5, 78.1 (4',2',5',3'-CH), 107.6 (1'-CH), 160.5 (C=N), 176.2 (COOH). IR (ATR): /cm-1 = 3200 (broad), 1582 (ν (C=O) in COO-), 1454, 1408 (ν (C=O) in COO-), 1346, 1070, 1018, 989, 895. MS (ESI): m/z = 375 [M + Na]+, 353 [M + H]+, 191 [C6H14N4O3 + H]+. HRMS (ESI) m/z for C12H24N4O8 [M + H]+: 353.1667, found: 353.1667; [M + Na]+: 375.1486 found: 375.1491.



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Chemical stability Prodrugs 2, 6 und 7 (250 µM) were incubated at 37 °C in potassium phosphate buffer (10 mM) at pH 2.0, pH 7.4 and pH 9.0, respectively. After 3, 6 and 24 h samples of 125 µL were mixed with an equal volume of potassium phosphate buffer (100 mM, pH 7.4) and analyzed by HPLC as described previously.12 Briefly, after o-PA derivatization samples were separated on NovaPak RP 18 (4 × 150 mm, 4 µm) with Security Guard Cartridges C18 (4 × 3 mm) tempered at 37 °C and detected by fluorescence (λex = 338 nm, λem = 425 nm). Retention times: 4.8 ± 1.8 (7), 6.8 ± 0.4 (1), 23.4 ± 0.5 (6), 24.6 ± 1.2 (2). For details see supplementary information. In vitro bioactivation assays The following enzymes were pre-incubated in triethanolamine buffer (50 mM, pH 7.0) at 37 °C for 3 min before starting the assay by adding the prodrug (500 µM): 25 U/mL

β-galactosidase (recombinant, Eschericha coli (E.coli)) for prodrugs 2 and 7, 33 U/mL β-galactosidase and 133 U/mL carboxylesterase (from porcine liver) for prodrugs 6, 33 U/mL β-glucosidase (recombinant, from almonds) for prodrugs 4, 7 and 9. Samples were taken and the reaction was stopped by adding an equal volume of ice-cold MeOH after 0, 15, 45, 90, and 150 min. After 5 min of agitation and centrifugation (10,000 g, 5 min) the supernatant was analyzed by HPLC (method see above). For control purpose, prodrugs without enzymes were treated identically. No degradation was observed. For the determination of released D-galactose from prodrug 1, a Roche Yellow Line test kit utilizing an UV-method for the determination of lactose and

D-galactose

in food and other

materials was used (based on NADH absorption). The incubation procedure was slightly modified as described in detail in the Supporting Information.


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Image-based measurement of cellular NO release J744.A1 (Cell Lines Service, Eppelheim, Germany) were cultivated in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 10% fetal bovine serum (FBS). 100 µL cell suspension (5 × 104 cells/mL) was plated on a 96 half well plate with µclear bottom for fluorescence microscopy (Greiner Bio-One, Frickenhausen, Germany). Beforehand, plates were pre-treated with 0.1% gelatine solution and washed with medium. 24 h after plating, iNOS expression was induced by adding LPS (1 µg/mL, E. coli 055:B5). 24 h after induction, cells were washed with incubations medium (DMEM without arginine and lysine supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 2% FBS) before substances were added. For parent solutions (10 mM), test substances were dissolved in Dulbecco's PBS with calcium and magnesium (DPBS++) and diluted with incubation medium prior to experiment (1:10, 1 mM final concentration). As a control group, wells were treated with DPBS++ diluted with incubation medium. After 150 min incubation, the supernatant was aspirated and 50 µL of dye mix containing 5 µM DAF-FM-DA (Invitrogen) und 10 µΜ Hoechst 33342 (Invitrogen) added and incubated for 15 min. To check for autofluorescence of the investigated compounds, additional wells were incubated with Hoechst 33342 only. After a washing step with 100 µL DPBS++, 100 µL DPBS++ were added for image acquisition. For each incubation condition, on the same plate three wells were treated and six images were acquired per well. For galactosyl prodrugs 3 to 7, 2 plates were acquired on different days and for glucosyl prodrugs 5 to 9, 3 plates were conducted. For image analysis, the module „Multi Wavelength Cell Scoring“ was employed and the resulting values for “All W2 Mean Cell Aver Intens” defined as the average intracellular fluorescence intensity normalized to number of cells was used (see Supporting Information for details on image acquisition and



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analysis). For evaluation of NO release from prodrugs, differences to the DPBS++ controls were calculated (∆ All W2 Mean Cell Aver Intens) and all data normalized to fluorescence signal from 1 in the same experiment (= 100%). Kruskal-Wallis analysis on variances on ranks followed by Dunn test for pairwise comparison to DPBS++ control was performed for each glycosyl series. In addition, for pairs of compound and compound in the mix with 1 mM L-NAME Student's t-test was performed. For prodrug 8, the equal variance test failed and the Mann-Whitney rank sum test was conducted instead. All statistic tests were performed using SigmaPlot 11.0.

ASSOCIATED CONTENT Supporting Information Additional details on image acquisition and analysis, NMR spectra, purity assessments and HPLC methods are available in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +49-231-7557083. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡ These authors contributed equally.


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Funding Sources Financial support by Bundesministerium für Wirtschaft und Technologie (SIGNO grant 03SHWB012). Notes The authors declare no competing financial interest. ACKNOWLEDGMENT We thank Martin Clemen for performing HRMS experiments and gratefully acknowledge the excellent technical assistance of Melissa Zietz and Sven Wichmann. ABBREVIATIONS abs, absolutized; Alloc, allyloxycarbonyl; CVD, cardiovascular disease; DAF-FM-DA, 4-amino5-methylamino-2‘,7‘difluorofluorescein

diacetate;

DAF-FM,

4-amino-5-methylamino-

2‘,7‘difluorofluorescein; DAF-FM-T, 4-amino-5-methylamino-2‘,7‘difluorofluorescein triazole; DPBS++, Dulbecco's PBS with calcium and magnesium; E. coli, Eschericha coli; EDCI, 1-ethyl3-(3-dimethylaminopropyl)carbodiimide; Amidoxime Reducing Component; hydroxy-L-arginine;

o-PA,

Eoc,

L-NAME,

ethyloxycarbonyl;

mARC,

mitochondrial

nitro-L-arginine methyl ester; NOHA, Nωo-phthaldialdehyde;

Pd(0)[Ph3P]4,

tetrakis(triphenylphosphin)palladium(0); PG, protecting group; quant, quantitative; SD, standard deviation.



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Table of Contents graphic


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Table of Contents Graphic Table of Contents graphic 247x162mm (150 x 150 DPI)


Design, Synthesis, and Bioactivation of O-Glycosylated Prodrugs of

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