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Bioconjugation through Mesitylene Thiol Alkylation Fernando Albericio, Ivan Tomillero, Gema Perez-Chacon, Beatriz Somovilla-Crespo, Francisco Sanchez-Madrid, Juan M Dominguez, Carmen Cuevas, Juan M Zapata, and Hortensia Rodriguez Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00828 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018
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Bioconjugate Chemistry
Bioconjugation through Mesitylene Thiol Alkylation Iván Ramos-Tomillero,†,‡ Gema Perez-Chacon,§ Beatriz Somovilla-Crespo,|| Francisco Sanchez-Madrid,|| Juan Manuel Domínguez,◊ Carmen Cuevas,◊ Juan Manuel Zapata,*,§ Hortensia Rodríguez,*,†, ↓ Fernando Albericio.*,†,‡, ∆ , ↕ †
Institute for Research in Biomedicine, 08028-Barcelona, Spain; ‡Department of Organic Chemistry, University of Barcelona, 08028-Barcelona, Spain; §Instituto de Investigaciones Biomedicas “Alberto Sols”, CSIC-UAM, 28029-Madrid, Spain; ||Servicio de Inmunología, Instituto de Investigación Sanitaria Hospital de la Princesa, 28006-Madrid, Spain; ◊Pharmamar, ↓School of Chemical Science and Engineering, Yachay Tech, Yachay City of Knowledge, 100650-Urcuqui, Ecuador; ∆CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, 08028-Barcelona, Spain; ↕School of Chemistry, University of KwaZulu-Natal, 4001-Durban, South Africa.
*Juan M. Zapata:
[email protected] *Hortensia María Rodríguez:
[email protected] *Fernando Albericio:
[email protected] Abstract The design and generation of complex multifunctional macromolecular structures by bioconjugation is a hot topic due to increasing interest conjugates with therapeutic applications. In this regard, the development of efficient, selective, and safe conjugation methods is a major objective. In this report, we describe the use of the bis(bromomethyl)benzene scaffold as a linker for bioconjugation with special emphasis in antibody conjugation. We first performed the monothioalkylation of 1,3,5-tris(bromomethyl)benzene, which rendered the reactive dibromotrimethylbenzyl derivatives to be used in thiol bis-alkylation. Next, we introduced either into a bis(Cys)-containing peptide and also with anti-CD4 and CD13 monoclonal antibodies, previously subjected to partial reduction of disulphide bonds, into the linker. Mass spectrometry, UV-Vis spectra and SDS-PAGE experiments revealed that this bis-alkylating agent for bioconjugation preserved both antibody integrity and antibodyantigen binding affinity, as assessed by flow cytometry. Taken together, our results show that the mesitylene scaffold is a suitable linker for thiol-based bioconjugation reactions. This linker could be applicable in a near future for the preparation of antibody drug conjugates. Introduction Drug discovery efforts are being channelled into finding efficient, selective, and stable pharmaceutical agents with minimum side effects. Due to the biological activity of peptides, proteins and antibodies, therapies based on these biomolecules have become the strategies of choice to treat a wide range of diseases.1,2 The chemical modification of these biomolecules can enhance their biological properties and strengthen their selectivity.3–6 The approval of new chemically modified biomolecules by the FDA in recent years7 underscores the increasing scientific interest and potential of this field.8 In nature, chemical modifications occur in a wide variety of amino acid side chains. Thus, the challenge has been to mimic these kinds of natural modification. Regioselectivity in ACS Paragon Plus Environment
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bioconjugation is achieved through a variety of reactive groups, such as amines, thiols, alcohols and even carboxylic acids.6 Several methods involve bioconjugation9 through Lys, Asp/Glu, and Ser/Thr, but the random distribution of these amino acids over the biomolecule structure leads to heterogeneous conjugations and, as a result, a broad number of chemical species. Given this observation, Cys, which is generally found as a disulphide bond and requires reduction of a disulfide bond to generate the active thiol group,10,11 has been successfully used for regioselective bioconjugation in peptides, proteins and antibodies. Thus, Cys emerges as an amino acid of choice to perform site-specific conjugations and, consequently, to generate homogeneous conjugates with improved stability and therapeutic index. Accordingly, strategies using Cys have been specifically applied for the development of antibody-drug conjugates (ADCs).12–16 The most widely used method for Cys conjugation is the well-known thiol group alkylation via Michael addition over the double bond of a maleimide ring.17,18 However, the undesired retro-Michael reaction can lead into the loss of conjugated linker in the plasma.16,19 In order to avoid this side reaction, alkylating agents such as haloacetyl derivatives or alkyl halides are also used for this purpose.20,21 Most Cys-conjugation procedures involve the thiol monoalkylation of the residue; however, as mentioned above, in nature Cys residues are often found as disulfide bonds, where they play a key role in the structural conformation of proteins. Although bis-alkylating agents have been widely used for peptide cyclization and labelling,22–38 only a few examples of bis-alkylation of a previously reduced pair of Cys residues of an antibody using a bis-sulfone linker and bromomaleimides have been described in the literature.39–43 Thus new chemical entities able to preserve the native structure of the antibodies after bioconjugation are needed. Hartman and co-workers extended the use of 1,3,5-tris(bromomethyl)benzene (TBMB) as a peptide stapler and also as a linker for carboxylic acids.44 They described the monoalkylation of TBMB using various kinds of carboxylic acid in basic conditions, yielding functionalized dibromobenzyl derivatives, which allowed simultaneous cyclization and peptide labelling in response to bis-Cys containing peptides. This remarkable simultaneous approach, together with the use of dibromobenzyl derivatives as linkers,45,46 inspired us to broaden it using more complex bis-Cys-containing molecules such as antibodies. The chemical stability of the thioether bond to the benzylic scaffold makes the former a suitable linker for a variety of bioconjugation reactions, including those aimed to produce ADCs. To this end, here we describe a novel antibody(Ab)-based bioconjugation strategy (Figure 1), which comprises previous mono-functionalization of TBMB using thiol derivatives as potential cargo of the TBMB scaffold, and subsequent conjugation to peptides or Abs. This strategy involves the reduction of a disulfide bond in the antibody in order to release two thiol groups, which finally react with the mono-functionalized dibromobenzyl derivative to yield the desired trimethylbenzyl-mediated conjugate.
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Bioconjugate Chemistry
R
S
S
S
1) TCEP (1 eq.)
Oxytocin (4)
5
R
R
2) S
S
S S
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Br Br 3c,e,g,h Br Base
Anti-CD4 Anti-CD13
Conj 1 Anti-CD4 + 3h R=H
Br
Br
Anti-CD4: Conj 1-3 Anti-CD13: Conj 4-6
R-SH
Conj 3 Anti-CD4 + 3g R=
Conj 2 Anti-CD4 + 3c R= S
S
O Fmoc
N H
OH
FITC
O
N H
O
H N
N H
NH2
O
O HO
Conj 4 Anti-CD13 + 3h R=H
Conj 6 Anti-CD13 + 3e R=
Conj 5 Anti-CD13 + 3c R=
S
O
S Fmoc
N H
N H
OH O
O
A
NH2 O
O
Figure 1. Conjugation of bromobenzyl derivatives to Anti-CD4 and Anti-CD13 antibodies.
The relatively low abundance of Cys in peptides and proteins confers this methodology an advantage since conjugation can be restricted to a particular Cys in the protein. In the case of Abs subjected to this bioconjugation reaction, the stable dibenzylic dithioether can replace the native interchain disulphide bond of the Ab. Given that disulphide bonds are sensitive to agents such as reduced glutathione, which is present in our body,47 the introduction of the mesytilene scaffold confers additional stability to the peptide, protein or Ab due to the robustness of the thioether bond.9 Specifically, we tested the cargo bioconjugation through dibromobenzyl derivatives into a peptide –reduced Oxytocin– and monoclonal antibodies HP2/6 Anti-CD4 and TEA1/8 AntiCD13. Moreover, we examined whether this bioconjugation strategy affects Ab-antigen binding affinity, a feature that is crucial for Ab function and efficacy.
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Results and discussion The optimization of the thiol monoalkylation of TBMB using various thiol derivatives (phenolic, benzylic or alkylic thiols) was tackled (SI). Afterwards, the bioconjugation step was studied using the dibromobenzyl derivatives previously obtained. Monoalkylation studies and linker-cargo preparation As a starting point, a range of conditions (number of equivalents, solvent, reaction time, and temperature) were studied for the monoalkylation of TBMB with phenolic, benzylic or alkylic thiols in the presence of DIEA, to render the corresponding dibromobenzyl derivatives (SI). These were further used for bioconjugation. Some thiol derivatives were commercially available (2a,b,f) while others were previously synthesized for the purpose of this study (2ce,g) (see SI). Thus, in addition to the simple phenyl (2a), we used benzyl (2b), and FmocCys-OH (2f) as well as some Cys-bearing FITC (2d), 7-methoxycoumarin (2e) or short peptides such as reduced glutathione (Gln) (2f), and a FITC-labelled tripeptide FITC-β-AlaCys-Asp-NH2 (2g). Table 1-SI shows the conditions used for testing the monoalkylation reaction, the conversion ratio detected by HPLC or UPLC in function of the reaction time, the temperature, and the solvent. The monoalkylation reaction was performed using TBMB (1.0–1.5 eq.) and the corresponding thiol (1 eq.) in the presence of diisopropylethylamine (DIEA). Our results revealed the aliphatic thiols (2c-g) as the best choice in terms of reaction conversion, but also because, unlike aromatic thiols, they did not produce an unpleasant smell. Accordingly, monoalkylation of less polar thiols (entries 1-13) gave better results than more polar substrates (entries 14-16). For long reaction times, polyalkylation products appeared in the reaction mixture, thereby hindering the isolation of the desired monoalkylate derivative (see SI). Moreover, the average of the reaction conversions corroborated that bimolecular nucleophilic substitution (SN2) rates were increased in polar solvents (DCM < MeCN < DMF). Although the syntheses of dibromoxylene derivatives were challenging, we obtained the amount of 3c, 3e and 3g required to carry out the bioconjugation experiments. A rigorous control of the reaction conditions (e.g., reaction time, temperature, and solvents) was needed to obtain the monoaddition products and to minimizing the undesired polyalkylation. This is exacerbated due to the high reactivity of the thiol group in response to the halobenzylic compounds. In addition, product isolation was tricky since the overall yields decreased drastically due to decomposition (see SI). Bioconjugation With the dibromoxylenes derivatives 3c, 3e and 3g in hand, various assays were tested to perform the bioconjugation using a peptide and Abs. As proof of concept, the bioconjugation was assayed with a dithiol-containing model peptide. For this purpose, the reduced form of the natural hormone oxytocin (4) was prepared by automatic microwave-assisted solid-phase peptide synthesis (SPPS) (see SI). The thiol of Cys protected with the S-tetrahydropyran (Thp) group previously developed in our group was introduced into the SPPS.48 Thp minimizes Cys racemization during coupling and is removed during global deprotection with trifluoroacetic (TFA) cocktail. The conjugation between 4 and the dibromobenzyl derivative 3c was performed in basic NaHCO3 buffer (pH = 8) at room temperature. The conjugation ACS Paragon Plus Environment
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Bioconjugate Chemistry
reaction took place rapidly and was completed after 15 min, as demonstrated by the absence of 4 (Figure 2). These results prompted us to use the dibromobenzyl system for conjugation to Abs. Fmoc-Cys-OH S
S
S
H Cys
Cys Pro Leu Gly NH2
Tyr
Asn Ile Gln Fmoc-Cys-OH S
SH
Br
SH
Br
H Cys Tyr Ile Gln Asn Cys Pro Leu Gly NH2
Figure 2. HPLC chromatograms of the starting materials 3c and reduced Oxytocin (4), and the bioconjugation product 5.
The monoclonal antibodies (mAbs) used for bioconjugation were the anti-CD4 HP2/6 and the anti-CD13 TEA1/8. Both CD4 and CD13 are targets for therapeutic intervention.49–51 Indeed, a humanized anti-CD4 mAb, Zanolimumab, has shown promising results in the treatment of reumathoid arthritis, psoriasis, Sezary syndrome and against T cell lymphomas.49,52 To perform the corresponding bioconjugation with the dibromoxylene system, a previous disulfide reduction step of the mAbs was needed to release reactive thiol groups. To study the behavior of bromobenzyl derivatives in response to two or more reactive thiols, an anti-CD4 solution was treated with a range of amounts of TCEP (1 eq., 3 eq. and an excess >100 eq. for conjugates 2a, 2b and 2c respectively). After disulfide reduction, the dibromobenzyl derivative 3c was added to each mAb solution (for experimental details see SI). Then the conjugates (Conj2a-c) were purified, and characterized by SDS-PAGE (see SI). Using SDSPAGE in non-reducing conditions, the entire mAb band (≈150 kDa) was observed with the naked antibody and with the conjugate analyzed which was reduced using equimolar amount of TCEP and conjugated with the dibromobenzyl derivative 3c (Conj2a). However, when the amount of TCEP was higher than that of 3c (more than 1 eq.) new bands appeared in the electrophoresis gel (see SI). The SDS-PAGE under reduced conditions showed that the reduction step affected the integrity of the conjugate. As expected, the more equivalents of TCEP used, the greater the degree of Ab reduction. The SDS-PAGE analysis indicated that with 1 and 3 eq. of TCEP (Conj2a and 2b), the bands corresponded to the light and heavy chains (25 and 50 kDa, respectively). In contrast, when using excess of TCEP (Conj2c), more than one band corresponding to the light chain was observed, thereby indicating that these conditions affected the structure of the conjugate. Due to the low molecular weight of 3c ACS Paragon Plus Environment
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[(459.6 g/mol) corresponding to C27H25NO4S2+] in the SDS-PAGE analysis, the differences between the conjugate and the naked antibody were minimal, thus hindering the analysis of the results by this technique. Having determined the optimal conditions to reduce disulfide bonds without affecting the integrity of the Ab, we next prepared a set of conjugates. To this end, the dibromobenzyl derivatives 3c, 3e and 3g and also the commercially available m-dibromoxylene (3h) were conjugated to the previously disulfide-reduced Anti-CD4 and Anti-CD13 Abs by adding the dibromobenzyl compounds (from 1 eq. to an excess) dissolved in DMSO (
