Vitamin B12 Suitably Tailored for Disulfide-Based Conjugation

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Vitamin B12 Suitably Tailored for Disulfide-Based Conjugation Aleksandra Wierzba, Monika Wojciechowska, Joanna Trylska, and Dorota Gryko Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00599 • Publication Date (Web): 22 Dec 2015 Downloaded from http://pubs.acs.org on December 27, 2015

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Bioconjugate Chemistry

Vitamin B12 Conjugation

Suitably

Tailored

for

Disulfide-Based

Aleksandra Wierzba,† Monika Wojciechowska,‡ Joanna Trylska,*,‡ Dorota Gryko*,† †

Institute of Organic Chemistry Polish Academy of Sciences, M. Kasprzaka 44/52, 01-224 Warsaw, Poland ‡

Centre of New Technologies, University of Warsaw, S. Banacha 2c, 02-097 Warsaw, Poland

ABSTRACT: Vitamin B12 has been proposed as a natural vector for in vivo delivery of biologically active compounds. Most synthetic methodologies leading to vitamin B12 conjugates involved functionalization at the 5’ position via either carbamate-based linkages or using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) leading to stable conjugates that are not cleaved within the cell. We have developed a novel vitamin B12 derivative suitably tailored for disulfide-based conjugation that can undergo cleavage in the presence of glutathione (GSH) - the most abundant thiol in mammalian cells. This active compound is simple to prepare and allows for direct disulfide-based attachment of therapeutic cargos. INTRODUCTION One of the crucial issues in drug development is the delivery of an active compound to its site of action.1 Recently, vitamin B12 (B12, cobalamin, Cbl, 1) has been studied as a potential drug or imaging-agent transporter due to its unique dietary uptake pathway and its constant demand in dividing cells.2,3 In order for B12 (1) to act as a carrier, its structure must be modified to allow selective coupling of biologically active compounds and at the same time high affinity to transport proteins: HC (haptocorrin), IF (intrinsic factor) and TCII (transcobalamin II) must be retained.3a,4 Though promising, the strategy has not been fully exploited yet. To date, numerous vitamin B12 conjugates, mainly with peptides, were prepared and studied.3a,3d,5 It was revealed that although all are metabolically stable in vivo, not all are recognized by transport proteins. Only those conjugated at b-, d-, and e- amides at the periphery of the corrin ring, the phosphate moiety, the primary hydroxyl group at the 5’ position, and the central Co (III) ion retain cobalamin’s binding ability of trafficking proteins are: (Figure 1).3a 1 ACS Paragon Plus Environment


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Figure 1. Possible conjugation sites in vitamin B12. Certainly, the most popular and exploited conjugable position is the 5’-hydroxyl group. Russel-Jones et al. found that activation of the hydroxyl group by 1,1’-carbonyldiimidazole (CDI) or 1,1’-carbonyldi-(1,2,4-triazole) (CDT) and subsequent reaction with an amine led to 5’-carbamate derivatives.6 Furthermore, Doyle developed selective, but low-yielding, oxidation of the hydroxyl group to the carboxylic acid that allowed direct conjugation with amines.7 Our group successfully prepared ‘clickable vitamin B12’ via sequential mesylation of the hydroxyl group and substitution reaction with NaN3, giving the respective azide reactive towards alkynes in the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC).8,9 Alternative positions on B12 (1) are also available, such as the cobalt center but unattractive anhydrous and anaerobic conditions must be used during preparation and the isolated conjugates tend to be extremely light sensitive.10 Also, amide groups at the periphery of the macrocycle could be functionalized but the required partial hydrolysis led to a mixture of products.11 On the other hand disulfides have been widely studied as responsive linkers in the development of drug-carriers and prodrugs due to the naturally occurring difference between the extra- and intra-cellular redox environment triggering drug releases at a targeted location.12 For example, their potential was utilized in preparation of antibody-drug conjugates,13 for delivery of nucleic acids,14 in creating reduction-sensitive polymers,15 and nanocarriers for drug delivery.16 It was proved that this strategy exhibits high therapeutic performance due to the advantages of the S-S bond over others in terms of biocompatibility, stability in blood, and cleavage by specific enzymes.17 2 ACS Paragon Plus Environment

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In vitamin B12 chemistry disulfide based motifs were involved in the preparation of conjugates possessing thiol moiety installed in the middle of a linker attached to the epropionamide group.18 A similar linker was also used for the preparation of B12-protein conjugates at the 5’ position.19 The hydroxyl group was initially reacted with an active glutaric acid derivative and then coupled with a linker containing the disulfide bond installed. However, for bioconjugates, the nature of a linker between a biologically important molecule and a transporter dictates the degree of successful delivery and its outcome. Most, if not all, cobalamin conjugates prepared so far are based on ester,20 amide,7,21 and carbamate bonds6 which are not cleavable inside cells, therefore, it would be advantageous to have an access to conjugates that are joined via labile bonds. The reversible nature of the S-S bond is certainly attractive but at the same time synthetically challenging especially when unsymmetrical disulfides are desired. There are two major approaches towards their synthesis: a) thioalkylation or thiolysis of S-containing compounds and b) oxidative coupling of thiols and their derivatives.22 For conjugation of two different molecules, the introduction of thiol groups into both constitutes is required unless endogenous thiols such as cysteines are present. To this end, we envisaged that the introduction of a group reactive towards thiols directly at 5’ position would allow for the direct S-S-bond formation between vitamin B12 (1) and a compound of interest independently of a linker used, an approach that to the best of our knowledge has never been proposed before. The proposed methodology combines assets associated with the possibility of reductive cleavage of the S-S bond and the use of a unique vitamin B12 delivery pathway, very promising in the context of drug delivery. RESULTS AND DISCUSSION Selective and high-yielding functionalizations of B12 (1) with simple purification methods are highly desirable, however the complexity of cobalamin’s structure makes them extremely challenging. One approach in thiol synthesis concerns the reaction of alkylating agent with nucleophilic sulfur species followed by hydrolysis or reduction.23 We hypothesized that the use of recently developed cobalamin mesylate (B12-5’-OMs, 2) in nucleophilic substitution with a thiolate anion should provide the desired thiol derivative (B12-5’-SH, 4, Table1). Treatment of B12-5’-OMs (2) with NaSH, regardless of the conditions applied, led either to a complex mixture of products or to the recovery of the starting material 2.23 Other sulfur nucleophiles (AcSK, NH4SCN, KSCN) gave similar results.24 Luckily, alkylation of 3 ACS Paragon Plus Environment


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thiourea with B12-5’-OMs (2) in EtOH at 50 ˚C furnished thiouronium salt 3, though in a low yield (Table 1, entry 1).25 Optimization of the reaction conditions revealed that an increase in reaction’s temperature to 80 ˚C led to an increase in the yield (entry 5). Furthermore, the use of a higher excess of thiourea and prolongation of the reaction time to 18 h allowed to obtain compound 3 in 88% yield (entry 6). The reaction could be also conducted in water though more by-products were formed (entry 9). Table 1. Optimization of the synthesis of B12-5’-SC(NH2)NH2+ (3).a

Entry 1c

Thiourea Time Conversionb Yieldb [equiv.] [h] [%] [%] 1.5 18 23 23

2d

1.5

18

49

45

3d

1.5

32

53

45

4d

1.5

42

55

47

5d

3.0

8

73

70

6d

3.0

18

94

88

7

d

4.5

8

77

71

8

d

4.5

18

91

84

e

4.5

6

82

62

9 a

General conditions: substrate 2 (21 µmol), EtOH (150 µL); b Based on HPLC analyses; c Reaction was performed at 50 ˚C; d Reaction was performed at 80 ˚C; e Reaction was performed in 150 µL of H2O at 100 ˚C. Following the isolation of thiouronium salt 3, the synthesis of B12-5’-SH (4) by basic hydrolysis was investigated (Scheme 1). Utilizing the standard procedure with NaOH as well as with other bases (K2CO3, NaHCO3, AcONa, NaSMe) a complex mixture of products was obtained lacking the expected B12-5’-SH (4).25 Instead, according to ESI-MS, a dimeric vitamin B12 derivative (presumably B12-5’-SS-5’-B12, m/z [M + 2Na]2+ 1392.5239) was formed regardless of the conditions used. However, in the presence of MeNH2 evidence of compound B12-5’-SH (4) was observed although isolation of the pure product was hampered by its continuous conversion into the dimer (Scheme 1). 4 ACS Paragon Plus Environment

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Scheme 1. Hydrolysis of thiouronium salt 3.

General conditions: Substrate 3 (3.54 µmol), MeNH2 (10 equiv), EtOH (200 µL), rt. Regardless of the conditions used the unwanted dimer prevailed.26 Similar results were observed for the reaction in the presence of EtNH2 while other amines: n-BuNH2 and ethanolamine did not promote hydrolysis of thiouronium salt 3. Therefore, to prevent undesired dimer formation, we decided to trap the reactive intermediate formed upon treatment with MeNH2 with L-cysteine as a model thiol. The reaction gave product 5a in 96% yield after 4 h (Scheme 2). Similar results were obtained for glutathione (product 5b).27 Disulfide 5 formed efficiently when pure thiouronium salt 3 was used as a starting material. However, purification of salt 3 required the addition of TFA (unspecified byproducts formed), as the chromatography performed without the acid gave pure 3 (>99%) but with drastically diminished yield. Scheme 2. Direct synthesis of disulfides 5a and 5b.

General conditions: Substrate 3 (21.2 µmol), MeNH2 (10 equiv.), H2O (1.2 mL), rt, 4 h. To avoid problematic purification steps, an alternative methodology involving preparation of activated pyridyl disulfide 6 and its subsequent reaction with a thiol was examined (Table 2). Such approach is usually more efficient as 2-mercaptopyridine (7) acts as a ‘trap’ for reactive thiolate anion, formed upon the reaction with MeNH2, and resulting pyridyl disulfide retains reactivity towards thiols.28 Salt 3 was reacted with 2-mercaptopyridine (7) in the presence of MeNH2 (10 equiv.) in H2O giving product 6 in 99% yield within 30 min. (entry 4). A decrease in amount of 2-mercaptopyridine (1 equiv.), as well as the use of other solvents (DMSO,

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DMF, EtOH) furnished product in high yields. Gratifyingly, the synthesis of B12-5’-SSPy (6) did not require pure thiouronium salt 3 (see experimental section). Table 2. Optimization of B12-pyridyl disulfide (B12-5’-SSPy, 6).a

Entry 1

Solvent Time Conv.b 7 [h] [%] [equiv.] 5 H2 O 1 >99

2

3

H2 O

0.5

>99

3

2

H2 O

0.5

>99

4

1

H2 O

0.5

>99

a

General conditions: Substrate 3 (3.54 µmol), MeNH2 (10 equiv.), solvent (200 µL), rt. Based on HPLC analyses.

b

To further optimize our process, a large scale (300 mg of vitamin B12, 1) synthesis of compound 6 was investigated (Scheme 3). Synthesis and purification of intermediates B12-5’OMs (2) and B12-5’-SC(NH2)NH2+ (3) required only precipitation and quick filtration through RP gel (see experimental section). The three step synthesis of B12-pyridyl disulfide was efficient affording desired product 6 in 60% (Scheme 3). Since both steps, the synthesis of compounds 3 and 6 could be carried out in water, one flask two step synthesis starting from B12-5’-OMs (2) was performed giving compound 6 in 30% . Scheme 3. Three step synthesis of B12-pyridyl disulfide (6).

The structure of B12-5’-SSPy (6) was unambiguously confirmed by X-ray analysis (Figure 2.). B12-pyridyl disulfide was crystallized from water and yielded single crystals in orthorhombic space group P212121, the usual for B12 derivatives. The upward folding of the corrin ring was 6 ACS Paragon Plus Environment

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determined to be 13.1˚ vs. 18.0˚ for cobalamin. The upper β axial ligand-Co bond was found to be 1.870 Å vs. 1.861 Å in Cbl, while α axial ligand-Co bond was determined as 2.029 Å vs. 2.011 Å. Some differences between bond lengths were also observed for the distance between R4–R5 (1.508 Å vs. 1.527 Å) and R3–O2P (1.415 Å vs. 1.409 Å). The distance between R5 and sulfur atom was determined to be 1.810 Å and S-S bond length was found to be 2.030 Å. The position of the pyridine ring suggests π-π interactions between the N,Ndimetylbenzimidazole ring present in cobalamin structure (dihedral angle between rings44.7˚). In such conformation, sulfur atom at the position 5’ is elegantly exposed to nucleophilic attack making it very reactive towards thiols.

Figure 2. X-ray structure of B12-pyridyl disulfide (B12-5’-SSPy, 6). As with intermediate 3, the reactivity of B12-R5’SSPy (6) was tested with L-cysteine (Table 3). Regardless of a solvent used (DMF, DMSO, EtOH, MeOH, or H2O) the reaction yielded desired disulfide 5a in high. The broad range of possible solvents allows for the reaction with both hydrophobic and hydrophilic thiols. Table 3. Optimization studies - disulfide 5a formation.a

Entry

L-cysteine

1

[equiv.] 5

2

5

Time Conv.b Solvent [h] [%] DMF 1(2) 78(99) DMSO

1


99


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

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3

5

EtOH

1

99

4

5

MeOH

1(2)

83(98)

5

5

H2O

0.5

99

6

2

H2O

0.5

99

7

1

H2O

0.5

94

General conditions: substrate 6 (3.4 µmol), solvent (0.4 mL), rt. b Based on HPLC analyses.

Subsequently, a set of thiols including glutathione, 2-methylbenzyl mercaptan, biotin derivative with sulfhydryl group, hexapeptide Cys-Phe-Phe-Phe-Lys-Lys-NH2, and peptide nucleic acid (PNA) mixed-sequence oligomer terminated with L-cysteine (full structure given in supporting information) were reacted with B12-pyridyl disulfide (Scheme 4). Irrespectively of a structural complexity of a thiol employed, the reaction proceeded with very high conversion giving exclusively desired products 5 a-f. Unquestionable advantage of this reaction is that it does not require any additives to proceed thus facilitating purification of final products. Scheme 4. Synthesis of B12 disulfide-based conjugates.


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a

PNA is a synthetic analogue of nucleic acids in which N-(2-aminoethyl)-glycine units are

connected via amide bonds. Purine and pyrimidine nucleic bases are attached to the backbone by methylene carbonyl linkages. The crucial issue in the application of the proposed approach is the reduction triggered release of a compound attached. Since glutathione (GSH) is the most abundant thiol-source in mammalian cells, and is responsible for maintaining the equilibrium between formation and reduction of disulfide bonds, we have used it as a reducing agent.29 To evaluate the S-S bond stability disulfide 5e was treated with GSH solutions (10 equiv. at four different concentrations 2.5, 5 and 10 mM). The reaction was monitored using HPLC analysis for 6 hours and samples were tested at 1 hour intervals and after 24 h (for details see SI). As expected, the reaction led to disulfides 5b and 9 (Scheme 5). Additionally, product B12-5’-SH (4) was formed but it converted into 5b as time progressed (in all cases after 24 h, see SI). The reaction rate depended on the GSH concentration, after 1 h almost full conversion of substrate 9 ACS Paragon Plus Environment


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5e (94 %) was observed for reactions carried in 5 and 10 mM GSH solution. Reactions performed at lower GSH concentration required longer reaction time, 75% and 81 % of conversion was observed for 1 mM and 2.5 mM solutions respectively after 24 h. The same conditions were applied for the reduction of disulfide 5c, and after 48 h almost full conversion (95%) was observed in 5 mM GSH solution. (for more details see SI). This confirms our theory that cobalamin based disulfides are prone to reduction in the presence of glutathione and therefore one can assume that they would be reduced inside cells (cGSH = 1 – 5 mM), allowing for the release of a conjugated compound. Scheme 5. Reduction of disulfide 5e.

General conditions: substrate 5e (1 µmol), GSH (10 equiv., c = 1 – 10 mM ) in phosphate buffer pH = 7, (c = 50 mM), rt CONCLUSIONS In summary, we have developed a three-step synthesis of vitamin B12 derivative, highly reactive towards thiols. This compound allows for the preparation of vitamin B12 conjugates with various thiols. To the best of our knowledge this is the first time when a cleavable type connection was used to prepare conjugates in which compound with sulfhydryl functionality can be directly attached to cobalamin at 5’ position via the disulfide bond. This approach gives access to cleavable compounds, an attractive alternative to stable cobalamin derivatives prepared via coupling leading to carbamate-based linkages or via azide-alkyne cycloaddition reactions. Thus we created a tool that can serve as a delivery vehicle allowing for direct cleavable disulfide-based conjugation between vitamin B12 and various thiols. EXPERIMENTAL SECTION General information: Commercially available reagents and solvents were used as received. Fmoc protected Lysine was obtained from Novabiochem and cysteine was obtained from Sigma-Aldrich. Fmoc-XAL PEG PS resin for the PNA synthesis was obtained from Merck. Fmoc/Bhoc-protected PNA monomers were purchased from Panagene. Nα-Fmoc protected L10 ACS Paragon Plus Environment

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amino acids used for peptide synthesis were obtained from Novabiochem (Fmoc-Lys(Bhoc)OH) and Sigma-Aldrich (Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH). Rink-amide resin (TentaGel S RAM resin) for the peptide synthesis was obtained from Sigma-Aldrich. 1H and

13

C NMR

spectra were recorded at rt on Bruker 500 MHz or Varian 600 MHz spectrometers with the residual solvent peak as an internal standard. Data are reported as follows: chemical shift, peak multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constants (Hz) and number of protons. UV-visible spectra were recorded on a Jenway 7315 Spectrophotometer. High resolution ESI mass spectra were recorded on a Mariner and SYNAPT spectrometer. All reactions and product purities were monitored using RP HPLC techniques. Preparative chromatography was performed using C18- reversed phase silica gel 90 Å (Sigma-Aldrich) with redistilled water and HPLC grade MeCN as eluents. HPLC measurement conditions: column, Eurospher II 100-5, C18, 250 mm × 4.6 mm with a precolumn; detection, UV/vis, pressure, 10 MPa; temperature, 30 ˚C; wavelengths, flow rates and HPLC methods are given below. General procedure for synthesis of B12-5’-OMs (2). Vitamin B12 (1, 300 mg, 0.22 mmol) was dissolved in NMP (1.1 mL), and then a solution of MsCl (84 mg, 0.73 mmol) in NMP (450 µL) and DIPEA (450 µL, 2.60 mmol) were added in three portions at 1 h intervals. Note that, each portion of MsCl solution in NMP was freshly prepared directly before adding, and added simultaneously with base. After addition of the third portion the reaction mixture was stirred for an additional hour. It was then poured into Et2O (200 mL) and the resulting precipitate was filtered through cotton wool. The precipitate was washed with Et2O (3 x 50 mL) and AcOEt (3 x 50 mL) and then removed with MeOH and concentrated in vacuo. After drying the resulting solid was dissolved in water, loaded onto RP gel and flushed with water (3 x 100 mL). The product was then eluted using H2O/MeOH (50% v/v). The solvent was concentrated in vacuo yielding a red powder (309 mg), yield: 98%. The spectral data matched that in the literature. General procedure for synthesis of B12-5’-SC(NH2+)NH2 (3). B12-5’-OMs 2 (300 mg, 0.20 mmol) and thiourea (48 mg, 0.63 mmol) were dissolved in EtOH (1.5 mL) and refluxed in an oil bath for 16 h. The solvent was evaporated to dryness, dissolved in H2O and loaded onto an RP column. By-products were removed by washing with water (3 x 100 mL), followed by the product using H2O/MeOH (50% v/v) and H2O + 0.025% TFA/MeOH (50% v/v). The product was subsequently evaporated in vacuo, and the resulting red solid dried and used in further reactions. An analytical sample was additionally purified using RP chromatography, 11 ACS Paragon Plus Environment


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MeCN/H2O (gradually from 0 to 8% v/v) giving compound 3 as red crystals. 1H NMR (500 MHz, CD3OD) δ 7.25 (s, 1H), 7.07 (s, 1H), 6.59 (s, 1H), 6.31 (d, J = 2.8 Hz, 1H), 6.07 (s, 1H), 4.51 (d, J = 8.3 Hz, 1H), 4.37 – 4.27 (m, 2H), 4.19 – 4.05 (m, 3H), 3.72 – 3.61 (m, 3H), 3.20 (d, J = 10.4 Hz, 1H), 2.89 – 2.81 (m, 1H), 2.81 – 2.73 (m, 1H), 2.68 (s, 3H), 2.68 – 2.60 (m, 7H) 2.58 (d, J = 3.6 Hz, 6H), 2.48 – 2.42 (m, 2H), 2.39 – 2.32 (m, 2H), 2.28 (d, J = 5.8 Hz, 6H), 2.09 – 1.96 (m, 5H), 1.89 (s, 3H), 1.87 – 1.76 (m, 4H), 1.46 (s, 3H), 1.36 (s, 6H), 1.25 (d, J = 6.3 Hz, 3H), 1.20 (s, 3H), 0.40 (s, 3H).

13

C NMR (CD3OD, 126 MHz) δ 181.7,

180.1, 177.6, 177.3, 176.8, 175.8, 175.5, 175.3, 174.6, 174.5, 174.4, 172.4, 167.1, 166.9, 143.1, 138.3, 135.8, 134.1, 131.3, 117.9, 112.4, 108.9, 105.0, 95.7, 88.5, 86.5, 81.7, 76.3, 76.1, 74.1, 70.6, 60.3, 57.6, 56.9, 55.5, 52.4, 49.8, 48.4, 47.0, 43.9, 43.0, 40.1, 39.5, 36.2, 35.6, 34.6, 33.9, 33.5, 32.4, 32.2, 29.2, 27.7, 27.3, 20.9, 20.4, 20.3, 19.9, 17.5, 17.0, 16.4, 16.1. UV/vis (H2O) λmax (nm) (ε, L mol−1 cm−1) 550 (6.4 × 103), 521 (5.8 × 103), 361 (2.0 × 104), 277 (1.3 × 104), 221 (4.0 × 104). HRMS (ESI) m/z [M + Na]2+ calcd for C64H91CoN16O13PSCoNa

718.2814,

found

718.2811

Anal.

calcd

for

C65H94N16O16PS2Co·7H2O: C, 47.73; H, 6.66; N, 13.70. Found: C, 47.61; H, 6.56; N, 13.66. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 8.95 min. Synthesis of B12-5’-SSPy (6). B12-5’-SC(NH2+)NH2 3 (300 mg, 0.21 mmol) and 2mercaptopyridine (47 mg, 0.42 mmol) were dissolved in H2O (12 mL) and stirred at room temperature. A 2 M solution of MeNH2 (1 mL, 2.12 mmol) in MeOH (1.0 mL, 2.1 mmol) was added dropwise to the reaction mixture via syringe. The mixture was stirred for 2 h, and then evaporated to dryness. The resulting solid was dissolved in MeOH (2 mL), precipitated with Et2O and centrifuged. The red solid after drying was dissolved in water, loaded onto a RP column an eluted gradually with MeCN/H2O (from 10 to 15% v/v). Fractions containing desired product 6 were collected and concentrated in vacuo yielding red crystals. 1H NMR (500 MHz, CD3OD) δ 8.33 (d, J = 4.0 Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.76 (td, J = 7.9, 1.7 Hz, 1H), 7.27 (s, 1H), 7.13 – 7.08 (m, 2H), 6.59 (s, 1H), 6.25 (d, J = 2.9 Hz, 1H), 6.06 (s, 1H), 4.77 – 4.73 (m, 1H), 4.50 (d, J = 8.6 Hz, 1H), 4.44 – 4.38 (m, 1H), 4.19 (t, J = 3.4 Hz, 1H), 4.16 – 4.11 (m, 2H), 3.66 – 3.56 (m, 3H), 3.40 – 3.34 (m, 1H), 3.25 (d, J = 10.9 Hz, 1H), 2.89 – 2.83 (m, 1H), 2.79 (dd, J = 13.9, 9.5 Hz, 1H), 2.69 – 2.42 (m, 11H), 2.60 (s, 3H), 2.58 (s, 3H), 2.39 – 2.32 (m, 2H), 2.29 (s, 6H), 2.24 – 2.18 (m, 1H), 2.15 – 2.11 (m, 1H), 2.07 (d, J = 13.9 Hz, 1H), 2.05 – 1.97 (m, 2H), 1.90 (s, 3H), 1.86 – 1.74 (m, 4H), 1.44 (s, 3H), 1.36 (s, 3H), 1.35(s, 3H), 1.17 (s, 3H), 1.13 (d, J = 6.3 Hz, 3H), 0.43 (s, 3H). 13C NMR (CD3OD, 126 MHz) δ 181.6, 180.2, 177.6, 177.3, 176.9, 176.7, 175.6, 175.3, 174.6, 173.8, 167.1, 166.9, 12 ACS Paragon Plus Environment

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161.5, 150.3, 143.3, 139.1, 138.3, 135.7, 133.9, 131.4, 122.3, 121.8, 117.9, 112.6, 108.8, 105.2, 95.6, 88.2, 86.4, 81.7, 77.0, 76.4, 73.6, 70.5, 60.3, 57.7, 57.0, 55.1, 52.5, 49.5, 48.4, 46.7, 44.0, 43.0, 40.1, 36.2, 34.8, 33.2, 33.2, 32.6, 32.3, 32.2, 28.7, 27.5, 27.4, 20.9, 20.6, 20.4, 20.3, 20.2, 20.2, 20.0, 17.5, 17.2, 16.4, 16.2. UV/vis (H2O) λmax (nm) (ε, L mol−1 cm−1) 551 (7.0 × 103), 522 (6.2 × 103), 361 (2.2 × 104), 278 (1.7 × 104), 222 (4.3 × 104). HRMS (ESI) m/z [M + 2Na]2+ calcd for C68H91N15O13PS2CoNa2 762.7608, found 762.7584. Anal. calcd for C68H91N15O13PS2Co ·7H2O: C, 50.83; H, 6.59; N, 13.08. Found: C, 50.86; H, 6.62; N, 13.01. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 11.03 min. General procedure for synthesis of B12-thiol conjugates from B12-5’-SC(NH2+)NH2 3. B125’-SC(NH2+)NH2 (3, 20 mg, 14 µmol) and thiol (28 µmol) were dissolved in H2O (0.8 mL) and stirred at room temperature. 2M solution of MeNH2 in MeOH (71 µL, 140 µmol) was added dropwise to the reaction mixture. The reaction was monitored by HPLC. When full consumption of a substrate was observed (usually after 2 - 4 h), the solvent was evaporated in vacuo. The resulting solid was subsequently dissolved in H2O, loaded onto a RP column (20 cm3), washed with H2O followed by H2O/MeOH (50% v/v). Analytical samples were additionally purified using RP gradient chromatography with a mixture of MeCN/H2O as an eluent. General procedure for synthesis of conjugates 5a-d from B12-5’-SSPy 6. B12-5’-SSPy (6, 30 mg, 20 µmol) and a thiol (41 µmol) were dissolved in H2O (1.2 mL) and stirred at room temperature (synthesis of 6c was carried in DMF). Reactions were monitored using HPLC. When full conversion was observed (usually after 1 h), the solvent was evaporated in vacuo (the crude reaction mixture with 6c was precipitated with AcOEt and then centrifuged). The crude solid was subsequently dissolved in MeOH (1 mL), precipitated with Et2O and then centrifuged. The dried solid was then dissolved in H2O, loaded onto a RP column (20 cm3) and washed with H2O The product was removed using H2O/MeOH (50% v/v). Analytical samples were purified using RP gradient column chromatography. Compound 5a. Red powder (28 mg), yield: 96%. Sample for analysis was purified by RP column chromatography, with MeCN/H2O (gradually from 10 to 15% v/v). 1H NMR (500 MHz, CD3OD) δ 7.27 (s, 1H), 7.11 (s, 1H), 6.57 (s, 1H), 6.35 (s, 1H), 6.04 (s, 1H), 4.48 (d, J = 7.7 Hz, 1H), 4.33 (s, 2H), 4.20 (s, 1H), 4.12 (d, J = 11.4 Hz, 1H), 3.89 (s, 1H), 3.68 (d, J = 13.7 Hz, 1H), 3.61 (dd, J = 10.7, 5.2 Hz, 1H), 3.51 – 3.42 (m, 2H), 3.28 – 3.25 (m, 2H), 3.16 – 3.08 (m, 1H), 2.90 – 2.78 (m, 2H), 2.66 – 2.56 (m, 7H), 2.62 (s, 3H), 2.57 (s, 3H), 2.54 – 2.32 (m, 7H), 2.27 (s, 3H), 2.26 (s, 3H), 2.20 – 1.98 (m, 6H), 1.93 – 1.79 (m, 4H), 1.88 (s, 13 ACS Paragon Plus Environment


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3H), 1.45 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 1.30 – 1.27 (m, 1H), 1.18 – 1.12 (m, 1H), 1.18 (s, 3H), 0.45 (s, 3H). 13C NMR (CD3OD, 126 MHz) δ 181.6, 180.1, 177.6, 177.3, 177.1, 176.6, 175.6, 175.3, 174.6, 174.2, 167.2, 166.9, 143.3, 138.3, 135.6, 133.8, 131.5, 117.8, 112.8, 108.8, 105.2, 95.7, 88.3, 86.4, 81.3, 76.8, 76.4, 73.7, 70.6, 66.9, 60.4, 57.7, 57.0, 55.3, 54.9, 52.6, 46.8, 44.0, 43.0, 40.1, 36.2, 35.0, 33.6, 33.1, 32.9, 32.4, 32.3, 28.9, 27.4, 20.9, 20.5, 20.3, 19.9, 17.5, 17.1, 16.4, 16.2, 15.4. UV/vis (H2O) λmax (nm) (ε, L mol−1 cm−1) 548 (6.9 × 103), 522 (6.3 × 103), 361 (2.0 × 104), 277 (1.5 × 104), 221 (3.9 × 104). HRMS (ESI) m/z [M + Na + H]2+ calcd for C66H94N15O15PS2CoNa 756.7726, found 759.7719. Anal. calcd for C66H93CoN15O15PS2 ·9H2O: C, 47.96; H, 6.77; N, 12.71. Found: C, 47.91; H, 6.86; N, 12.40. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 10.02 min. Compound 5b. Red powder (32 mg), yield: 95%. Sample for analysis was purified by RP column chromatography gradually with MeCN/H2O (gradually from 5 to 10% v/v). 1H NMR (500 MHz, CD3OD) δ 7.30 (s, 1H), 7.12 (s, 1H), 6.57 (s, 1H), 6.36 (s, 1H), 6.03 (s, 1H), 4.48 (d, J = 8.5 Hz, 1H), 4.41 – 4.35 (m, 1H), 4.34 – 4.30 (m, 1H), 4.21 (s, 1H), 4.12 (d, J = 11.4 Hz, 1H), 3.79 (s, 2H), 3.72 – 3.67 (m, 2H), 3.62 (dd, J = 10.5, 5.2 Hz, 1H), 3.54 – 3.52 (m, 1H), 3.40 – 3.37 (m, 1H), 3.25 – 3.23 (m, 1H), 2.97 – 2.92 (m, 1H), 2.87 – 2.78 (m, 2H), 2.68 – 2.56 (m, 5H), 2.61 (s, 3H), 2.57 (s, 3H), 2.54 – 2.32 (m, 8H), 2.28 (s, 6H), 2.21 – 1.98 (m, 8H), 1.92 – 1.78 (m, 4H), 1.89 (s, 3H), 1.73 – 1.66 (m, 1H), 1.45 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 1.32 – 1.28 (m, 2H), 1.23 (d, J = 6.0 Hz, 3H), 1.19 (s, 3H), 1.16 – 1.10 (m, 1H) 0.45 (s, 3H).

13

C NMR (CD3OD, 126 MHz) δ 181.6, 180.2, 177.6, 177.3, 176.9, 175.6, 175.3,

174.6, 174.0, 173.2, 173.0, 167.1, 166.9, 143.3, 138.3, 135.6, 133.8, 131.5, 117.8, 112.7, 108.8, 105.2, 95.7, 88.8, 86.4, 81.1, 76.6, 76.4, 73.7, 71.5, 70.5, 60.4, 57.7, 57.0, 55.8, 55.2, 53.9, 52.6, 49.8, 46.7, 44.0, 43.2, 43.03, 42.3, 41.6, 40.2, 36.2, 35.0, 33.6, 33.0, 32.3, 30.7, 29.2, 27.8, 27.4, 20.9, 20.4, 19.9, 17.5, 17.1, 16.4, 16.2, 14.4. UV/vis (H2O) λmax (nm) (ε, L mol−1 cm−1) 548 (7.9 × 103), 520 (6.9 × 103), 361 (2.4 × 104), 278 (1.4 × 104), 221 (4.4 × 104); HRMS (ESI) m/z [M + Na]2+ calcd for C73H103N17O19PS2CoNa2 860.7956, found 860.7942. Anal. calcd for C73H103N17O19PS2Co·8H2O: C, 48.15; H, 6.59; N, 13.08. Found: C, 48.20; H, 6.86; N, 12.91. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 9.30 min. Compound 5c. Red crystals (29 mg), yield: 97%. Sample for analysis was purified by RP column chromatography gradually with MeCN/H2O (from 10 to 20% v/v). 1H NMR (500 MHz, CD3OD) δ 7.29 – 7.21 (m, 2H), 7.16 – 7.09 (m, 4H), 6.58 (s, 1H), 6.27 (s, 1H), 6.06 (s, 1H), 4.74 – 4.69 (m, 1H), 4.49 (d, J = 8.6 Hz, 1H), 4.41 – 4.31 (m, 2H), 4.20 (s, 1H), 4.12 (d, 14 ACS Paragon Plus Environment

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J = 11.4 Hz, 1H), 4.02 – 3.98 (m, 2H), 3.70 (d, J = 13.8 Hz, 1H), 3.62 (dd, J = 9.8, 4.7 Hz, 1H), 3.42 (d, J = 12.8 Hz, 1H), 3.12 (dd, J = 14.4, 5.8 Hz, 1H), 2.90 – 2.84 (m, 2H), 2.67 – 2.19 (m, 15H), 2.59 (s, 3H), 2.57 (s, 3H), 2.33 (s, 3H), 2.28 (s, 3H), 2.27 (s, 3H), 2.16 – 1.95 (m, 4H), 1.88 (s, 3H) 1.92 – 1.80 (m, 2H), 1.77 – 1.70 (m, 1H), 1.43 (s, 3H), 1.37 (s, 3H), 1.36 (s, 3H), 1.25 (d, J = 6.1 Hz, 3H), 1.17 (s, 3H), 1.16 – 1.11 (m, 1H), 0.44 (s, 3H).13C NMR (126 MHz, CD3OD) δ 181.6, 180.2, 177.7, 177.3, 177.0, 176.7, 175.6, 175.5, 175.3, 174.6, 173.9, 167.1, 166.9, 143.3, 138.3, 138.0, 136.3, 135.7, 133.9, 131.5, 131.5, 128.8, 127.0, 117.9, 112.6, 108.7, 105.3, 95.6, 88.2, 86.4, 81.8, 77.4, 76.4, 73.7, 70.7, 60.3, 57.7, 57.0, 54.9, 52.5, 48.4, 46.6, 43.9, 43.1, 41.95, 40.1, 36.2, 35.0, 33.2, 33.1, 32.6, 32.3, 32.2, 28.7, 27.5, 27.4, 20.9, 20.6, 20.5, 20.2, 19.9, 19.5, 17.5, 17.2, 16.4, 16.2. UV/vis (H2O) λmax (nm) (ε, L mol−1 cm−1) 546 (5.7 × 103), 520 (5.0 × 103), 361 (1.8 × 104), 276 (1.2 × 104), 221 (3.6 × 104. HRMS (ESI) m/z [M + Na]+ calcd for C71H96N14O13PS2CoNa 1529.5690, found 1529.5680. Anal. calcd for C71H96N14O13PS2Co·8H2O: C, 51.63; H, 6.83; N, 11.87. Found: C, 51.46; H, 6.78; N, 11.82. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 12.48 min. Compound 5d. Red powder (18 mg), yield: 55%. Sample for analysis was purified by RP column chromatography gradually with MeCN/H2O (from 10 to 15% v/v). 1H NMR (500 MHz, CD3OD) δ 7.29 (s, 1H), 7.14 (s, 1H), 6.59 (s, 1H), 6.30 (d, J = 2.8 Hz, 1H), 6.05 (s, 1H), 4.76 – 4.72 (m, 1H), 4.51 – 4.46 (m, 2H), 4.40 – 4.35 (m, 1H), 4.31 (dd, J = 7.7, 4.4 Hz, 2H), 4.22 – 4.18 (m, 1H), 4.12 (d, J = 11.3 Hz, 1H), 3.70 (d, J = 13.9 Hz, 1H), 3.62 (dd, J = 10.6, 5.2 Hz, 1H), 3.57 – 3.46 (m, 3H), 3.24 – 3.18 (m, 1H), 3.14 (dd, J = 14.4, 5.7 Hz, 1H), 2.95 – 2.80 (m, 5H), 2.69 (d, J = 12.7 Hz, 2H), 2.61 (s, 3H), 2.57 (s, 3H), 2.65 – 2.32 (m, 12H), 2.28 (s, 3H), 2.27 (s, 3H), 2.24 (t, J = 7.4 Hz, 3H), 2.15 – 2.07 (m, 2H), 2.04 – 1.96 (m, 2H), 1.89 (s, 3H), 1.86 – 1.57 (m, 8H), 1.49 – 1.42 (m, 2H), 1.45 (s, 3H), 1.35 – 1.31 (m, 1H), 1.37 (s, 3H), 1.36 (s, 3H), 1.25 (d, J = 6.3 Hz, 3H), 1.18 (s, 3H), 0.44 (s, 3H). 13C NMR (126 MHz, CD3OD) δ 181.6, 180.2, 177.7, 177.3, 177.0, 176.7, 174.6, 173.9, 167.2, 166.9, 166.1, 143.3, 138.3, 135.7, 133.9, 131.4, 117.9, 112.6, 110.1, 108.7, 105.2, 100.7, 95.7, 88.3, 86.5, 81.8, 76.4, 73.9, 70.7, 63.4, 61.6, 60.3, 57.7, 57.0, 55.1, 52.5, 49.6, 48.4, 46.6, 44.0, 43.0, 42.8, 41.1, 40.1, 39.5, 38.5, 36.8, 36.2, 35.1, 33.4, 33.0, 32.7, 32.4, 32.3, 29.7, 29.5, 28.7, 27.4, 26.8, 20.9, 20.5, 20.3, 19.9, 17.5, 17.2, 16.4, 16.2. UV/vis (H2O) λmax (nm) (ε, L mol−1 cm−1) 548 (7.8 × 103), 520 (6.9 × 103), 361 (2.4 × 103), 278 (1.4 × 104), 221 (4.4 × 104). HRMS (ESI) m/z [M + 2Na]2+ calcd for C75H107N17O15PS3CoNa2 858.8074, found 858.8058 Anal. calcd for C75H107CoN17O15PS3 ·10H2O: C, 48.61; H, 6.91; N, 12.85. Found: C, 48.44; 15 ACS Paragon Plus Environment


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H, 6.93; N, 12.64. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 11.07 min. Synthesis of compound 5e. B12-SSPy (6, 30 mg, 20 µmol) and Cys-Phe-Phe-Phe-Lys-LysNH2 (15 mg, 18 µmol) were dissolved in H2O (1.2 mL) and stirred at room temperature. The reaction was monitored by HPLC. When full conversion of a substrate was achieved (1-2 h) the solvent was evaporated in vacuo. The resulting solid was subsequently dissolved in MeOH (1 mL), precipitated with Et2O and centrifuged. The dried product was subsequently purified by RP column chromatography gradually with MeCN/H2O (from 10 to 15% v/v) giving a red powder (30 mg), yield: 75%. 1H NMR (600 MHz, CD3OD) δ 7.29 – 7.14 (m, 15H), 7.07 (s, 1H), 6.59 (s, 1H), 6.30 (s, 1H), 6.08 (s, 1H), 4.74 (s, 2H), 4.67 – 4.64 (m, 1H), 4.62 – 4.59 (m, 1H), 4.55 – 4.49 (m, 2H), 4.37 – 4.29 (m, 4H), 4.26 (s, 1H), 4.22 (s, 1H), 4.12 (d, J = 10.3 Hz, 1H), 3.83 (d, J = 13.2 Hz, 2H), 3.69 (t, J = 7.4 Hz, 2H), 3.64 (s, 1H), 3.60 (dd, J = 14.1, 7.0 Hz, 1H), 3.52 – 3.44 (m, 2H), 3.25 (dd, J = 14.3, 5.0 Hz, 2H), 3.21 – 3.14 (m, 3H), 3.07 (dd, J = 14.0, 5.4 Hz, 2H), 3.03 – 2.76 (m, 9H), 2.63 (s, 3H), 2.59 (s, 3H), 2.51 – 2.31 (m, 4H), 2.29 (s, 3H), 2.27 (s, 3H), 2.14 – 2.05 (m, 3H), 2.04 – 1.95 (m, 3H), 1.90 (s, 3H), 1.87 – 1.64 (m, 9H), 1.46 (s, 3H), 1.54 – 1.39 (m, 6H), 1.38 (s, 3H), 1.34 (s, 3H), 1.39 – 1.24 (m, 3H), 1.29 (s, 3H), 1.20 – 1.12 (m, 3H), 0.90 (t, J = 7.1 Hz, 1H), 0.42 (s, 3H),

13

C

NMR (151 MHz, CD3OD) δ 181.7, 179.9, 177.5, 177.3, 177.2, 176.8, 176.5, 175.8, 175.5, 175.3, 174.4, 173.6, 173.3, 173.2, 172.9, 169.1, 167.2, 166.9, 142.9, 138.4, 138.3, 138.3, 135.7, 134.0, 131.4, 130.4, 130.3, 130.1, 129.6, 129.5, 127.9, 127.8, 117.9, 112.5, 108.7, 105.1, 95.7, 88.6, 86.4, 81.9, 77.2, 76.4, 74.0, 70.5, 60.5, 58.3, 57.6, 56.8, 56.3, 56.0, 55.2, 54.6, 54.2, 52.4, 52.3, 47.0, 43.0, 41.9, 40.5, 39.9, 38.6, 38.5, 36.2, 34.6, 34.4, 34.1, 33.5, 33.1, 32.6, 32.3, 32.2, 30.7, 29.7, 28.9, 28.1, 27.7, 27.3, 26.1, 23.8, 23.7, 20.9, 20.6, 20.5, 20.3, 19.9, 18.4, 17.5, 17.3, 16.4, 16.0, 14.4. MALDI-TOF m/z [M - CN]+ calcd for C104H145CoN22O19PS2 2161.47, found 2161.43. UV/vis (H2O) λmax (nm) (ε, L mol−1 cm−1) 540 (8.3 × 103), 520 (7.3 × 103), 361 (2.6 × 103), 275 (1.7 × 104), 219 (5.6 × 104); tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min): 10.13 min. Synthesis of compound 5f. B12-SSPy (6, 1.9 mg, 1.3 µmol) and Cys-PNA-Lys-NH2 (2.6 mg, 0.65 µmol) were dissolved in H2O (200 µL). The mixture was stirred at room temperature for 2 h. The product was purified by semi-preparative RP-HPLC on Knauer C18 column (8×250 mm, 5 µm particle size) using the SYKAM System. A mixture of MeCN + 0.05% TFA and H2O + 0.05% TFA was used as an eluent. MS MALDI-TOF m/z [M - CN]+ calcd for C222H295CoN88O61PS2 5326.38, found 5327.78. tR (RP-HPLC, from 0% MeCN 0.05% + 16 ACS Paragon Plus Environment

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TFA/H2O + 0.05% TFA to 50% MeCN + 0.05% TFA/H2O + 0.05% TFA in 30 min.; flow rate 1.5 mL/min; λ = 267 nm): 14.7 min. Conversion = 99%. General procedure for disulfide bond reduction: GSH (3.08 mg, 10 µmol) was dissolved in phosphate buffer (c = 50 mM). The volume of a buffer for 1, 2.5, 5, 10 mM GSH solution was 10, 4, 2, 1 mL, respectively. Compound 5e (2.19 mg, 1 µmol) was dissolved in GSH solution and the reaction was monitored by RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min. X-ray structure determination: The data has been collected at 100 K using the P13 Macromolecular Crystallography beamline at the PETRA III Synchrotron in Hamburg (Research Centre of the Helmholtz Association) at a wavelength of 0.68880 Å, and processed with XDS. The structure was solved by direct methods using SHELXS program and was refined by full-matrix least-squares on F2 using program SHELXL. All non-hydrogen atoms were refined with anisotropic displacement parameters. The hydrogen atoms were introduced at geometrically idealized coordinates with fixed isotropic displacement parameters. Formula: C68H127.53Co1N15O31.27P1S2: Unit Cell Parameters: a = 16.015(3), b = 23.333(5), c = 23.480(5) Å, space group P212121, V = 8774(3) Å3, Z = 4, F(000) = 3859, ρcalcd=1.370 g/cm3; µ = 0.323 mm-1; θmax = 35.632˚, 41014 unique reflections. Refinement converged at R1 = 0.0631, wR2 = 0.1448 for all data and 1141 parameters, 8 restraints (R1 = 0.0560, wR2 = 0.1388 for 37444 reflections with Io>2σ(Io)). The goodness-of-fit on F2 was 1.110. A weighting scheme w = [σ 2(F2o + (0.0649P)2 + 4.5094P]-1 in which P = (F2o + 2 F2c)/3 was used in the final stage of refinement. The residual electron density was + 1.045/-0.749 e Å-3. CCDC 1414143 contains the supplementary crystallographic data for this paper. These data can

be

obtained

from

the

Cambridge

Crystallographic

Data

Centre

via

www.ccdc.cam.ac.uk/data_request/cif. Solid-Phase Synthesis of Cys-PNA-Lys-NH2. PNA oligomer was synthesized manually by Fmoc chemistry on 10 µmol scale using a 2.5-fold molar excess of the Fmoc/Bhoc–protected monomers and 3-fold molar excess of the Fmoc-protected amino acids and polyethylene glycol-polystyrene resin (Fmoc-XAL PEG PS resin, amine groups loading of 0.19 mmol/g, this resin has a linker which yields a C-terminal amide upon TFA cleavage of PNA.) Monomers were activated with the use of HATU, NMM and 2,6-lutidine (molar ratio: 0.7 : 1 : 1.5) mixture using the DMF/NMP (1:1, v/v) solution and coupled for 40 min. as active derivatives. A double coupling was performed. Fmoc deprotection was performed with the use of 20% piperidine in DMF (2 x 2 min). After the synthesis of the PNA backbone and 17 ACS Paragon Plus Environment


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removal of the N-terminal Fmoc group, the Fmoc-Cys(Trt) was attached to the N-terminus. All Fmoc protected amino acids (Lys and Cys) were assembled as active derivatives in a three-fold molar excess with the use of HATU with the addition of 1-hydroxy-7azabenzotriazole (HOAt) and collidine (molar ratio: 1 : 1 : 2) using the DMF/NMP (1:1, v/v) solution-coupling method for 2 hours. Deprotection of the Fmoc group from amino acids was carried out with 20% piperidine in DMF for 2 cycles (5 + 15 min). Deprotection of the protecting group and cleavage of PNA from resin was carried out with the use of a TFA/triisopropylsilane/m-cresol 95:2.5:2.5 (v/v/v) mixture (total volume = 5 ml) for 60 min. The obtained crude oligomer was lyophilized and subsequently purified by RP HPLC. Analytical and semi-preparative RP-HPLC of Cys-PNA-Lys-NH2 was performed on Knauer C18 column (4.6×250 mm, 5 µm particle size) and (8×250 mm, 5 µm particle size), respectively. The SYKAM System and a mixture of MeCN + 0.1% TFA and H2O + 0.1% TFA as an eluent were used. Cys-CATCTAGTATTTCT-Lys-NH2: MS analysis by QTOF Premier mass spectrometer m/z [M + H]+ calcd for C160H210N75O48S 3981.60 found 3982.25. tR (RP-HPLC, from 10% MeCN + 0.1% TFA/H2O + 0.1% TFA to 25% MeCN + 0.1% TFA/H2O + 0.1% TFA in 30 min; flow rate 1.5 mL/min; λ= 267 nm): 15.3 min. Solid-Phase Synthesis of peptide Cys-Phe-Phe-Phe-Lys-Lys-NH2. Peptide was synthesized manually by Fmoc chemistry on a 100 µmol scale using a 3-fold molar excess of the N-Fmocprotected amino acids and Rink-amide resin (TentaGel S RAM resin, amine groups loading of 0.24 mmol/g, this resin has a linker which yields a C-terminal amide upon TFA cleavage of the peptide). N-Fmoc protected amino acids were assembled as active derivatives in a threefold molar excess with the use of the HATU with the addition of HOAt and collidine (molar ratio: 1:1:2) using the DMF/NMP (1:1, v/v) solution-coupling method for 2 hours. Deprotection of the Fmoc group was carried out with 20% piperidine in DMF for 2 cycles (5 + 15 min). Deprotection of protecting groups (Boc from Lys and Trt from Cys) and cleavage of peptide from resin was carried out with the use of a TFA/triisopropylsilane/m-cresol 95 : 2.5 : 2.5 (v/v/v) mixture (total volume = 5 ml) for 60 min. The obtained crude oligomer was lyophilized and purified by RP-HPLC. Analytical and semi-preparative RP-HPLC of CysPNA-Lys-NH2 was performed on Knauer C18 column (4.6 × 250 mm, 5 µm particle size) and (8×250 mm, 5 µm particle size), respectively. The SYKAM System was applied and a mobile phase gradient profile: from 15% MeCN/H2O + 0.1% TFA to 50% MeCN/H2O + 0.1% TFA in 30 min; flow rate: 1.5 min, λ = 254 nm. MS MALDI-TOF m/z [M + H]+ calcd for C42H60N9O6S 818.44, found 818.41. tR (RP-HPLC, from 1% MeCN/H2O + 0.05% TFA to 70% MeCN/H2O + 0.05% TFA in 15 min, λ = 254 nm): 9.57 min. 18 ACS Paragon Plus Environment

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ABBREVIATIONS ADC – antibody drug conjugates, Bhoc – benzhydryloxycarbonyl; Boc – tertbutyloxycarbonyl; CDI – 1,1’-carbonyldiimidazole, CDT – 1,1’-carbonyldi-(1,2,4-triazole), CuAAC – copper(I)-catalyzed alkyne-azide cycloaddition, DMF – N,N-dimethylformamide; DMSO – dimethyl sulfoxide, Fmoc – 9-fluorenylmethoxycarbonyl; HATU – 2-(1H-7azabenzo-triazole-1-yl)-1,1,3,3-tetramethylouronium

hexafluorophosphate;

glutathione, HOAt – 1-hydroxy-7-azabenzotriazole; HRMS –

GSH

-

High Resolution Mass

Spectrometry, IF – intrinsic factor, MALDI-TOF – matrix assisted laser desorption/ionization - time of flight; NMM – 4-methylmorpholine; NMP – N-methyl-2-pyrrolidon; PNA – peptide nucleic acids, RP-HPLC – reverse phase high performance liquid chromatography; HP – haptocorrin, TCII – transcobalamin II, TFA – trifluoroacetic acid, tR – retention time; Trt – trityl or triphenylmethyl; Nucleic acid abbreviations in PNA sequence A – adenine, G – Guanine, T – thymine, C – cytosine. The common three-letter code for amino acids is used throughout. ASSOCIATED CONTENT Supporting Information Copies of 1H,

13

C NMR spectra and HPLC analyses are available free of charge via the

Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *[email protected] Notes The authors declare no competing financial interests. ACKNOWLEDGMENT Financial support for this work was provided by the National Science Centre, grant SYMFONIA 2014/12/W/ST5/00589. REFERENCES



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Table of Contents Graphic Vitamin B12 Suitably Tailored for Disulfide-Based Conjugation Aleksandra Wierzba, Monika Wojciechowska, Joanna Trylska,* Dorota Gryko*


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Vitamin B12 Suitably Tailored for Disulfide-Based Conjugation

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