Site-Selective Synthesis of Insulin Azides and Bioconjugates

Apr 4, 2019 - Chemistry Capabilities for Accelerating Therapeutics, Merck & Co., Inc. , Kenilworth ... (RHI) was developed via diazo-transfer chemistr...
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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Site-Selective Synthesis of Insulin Azides and Bioconjugates Sobhana Babu Boga,† Shane W. Krska,† Songnian Lin,† Dmitri Pissarnitski,† Lin Yan,† Ahmet Kekec,† Weijuan Tang,‡ Nicholas A. Pierson,‡ Christopher A. Strulson,‡ Eric Streckfuss,§ Xiaohong Zhu,† Xiaoping Zhang,⊥ Terri Kelly,⊥ and Craig A. Parish*,† †

Chemistry Capabilities for Accelerating Therapeutics, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States Analytical Research & Development, Merck & Co., Inc., Rahway, New Jersey 07065, United States § Discovery Chemistry, Merck & Co., Inc., West Point, Pennsylvania 19486, United States ⊥ Screening, Target and Compound Profiling, Merck & Co., Inc., Kenilworth, New Jersey 07033, United States Downloaded via UNIV AUTONOMA DE COAHUILA on April 4, 2019 at 22:26:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: A synthetic method to access novel azido-insulin analogs directly from recombinant human insulin (RHI) was developed via diazo-transfer chemistry using imidazole-1-sulfonyl azide. Systematic optimization of reaction conditions led to site-selective azidation of amino acids B1-phenylalanine and B29-lysine present in RHI. Subsequently, the azido-insulin analogs were used in azide−alkyne [3 + 2] cycloaddition reactions to synthesize a diverse array of triazole-based RHI bioconjugates that were found to be potent human insulin receptor binders. The utility of this method was further demonstrated by the concise and controlled synthesis of a heterotrisubstituted insulin conjugate.

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in applying diazo transfer chemistry for site-selective azidation of recombinant human insulin (RHI, 1). Direct, selective generation of an insulin azide intermediate would provide rapid access to the synthesis of conjugates by reaction with this bio-orthogonal functional handle. Accordingly, this approach would enable a broad range of biochemical applications, such as modulating insulin ligand−receptor binding interactions. Treatment of 1 with excess ISA (4 equiv) in H2O:MeOH (9:1 v/v) using aqueous NaHCO3 as base with catalytic amounts of CuSO4·H2O at ambient temperature for 6 h led to the formation of tris-azido RHI 2 (Table 1, entry 2). The reaction proceeded in quantitative conversion and resulted in 72% isolated yield of 2 after preparative HPLC. A1B1B29 trisazido RHI was evaluated in a human insulin receptor (hIR) binding assay and was only 22-fold less potent when compared to the native ligand 1 (IC50(2) = 5.3 nM; IC50(1) = 0.24 nM; see the Supporting Information for all hIR binding data). Subsequent copper(I)-catalyzed 1,3-dipolar [3 + 2] cycloaddition between 2 and excess phenylacetylene proceeded

ost-translational modification of native peptides via the site-selective chemical functionalization of amino acids is of great current interest for biological and therapeutic applications.1−3 Despite many advances in this field, siteselective modification of amino groups found either at the peptide N-terminus or on lysine side chains (ε-NH2) remains a daunting challenge.4−8 Direct conversion of an amine to an azide via diazo-transfer chemistry was reported by GoddardBorger et al. using the diazo transfer reagent (DTR) imidazole1-sulfonyl azide (ISA).9 More recent reports have described improvements to the stability and safety of this reagent, as well as its use with protein substrates for bioconjugation applications.10−12 The azide functional group introduced by this chemistry is attractive for a number of reasons, including its small size, which should result in minimal perturbation of the overall structure and function of the biomolecule, as well as its distinct bio-orthogonal reactivity.13−16 Insulin is a natural polypeptide hormone widely studied and used extensively for treating diabetes mellitus, accounting for >50% of current therapies for this chronic disease.17,18 Many insulin analogues, e.g., lispro and glargine, have been generated by recombinant DNA technology in order to modulate their pharmacokinetic and pharmacodynamic properties, thereby reducing the risk of hypoglycemia.19,20 We became interested © XXXX American Chemical Society

Received: January 27, 2019 Revised: March 15, 2019

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DOI: 10.1021/acs.bioconjchem.9b00069 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Table 1. Synthesis of Azido Insulins and Phenyl Triazole− Insulin Conjugates and Their Binding Affinities to the Human Insulin Receptor (hIR)

Figure 2. Role of pH and copper(II) additive on the selective azidation of RHI (1) with ISA·HCl. Values reported were determined by HPLC UV area percentages (diode array 210−400 nm). For additional experimental details, see the Supporting Information.

Scheme 1. Plausible Diazo Transfer Reaction Mechanisms Involving Cu-Mediated and Direct Nucleophilic Displacement for the Azidation of Amines in Peptides

a

Isolated yields after preparative HPLC. b4 equiv of ISA used. c1 equiv of ISA used. See the Supporting Information for complete experimental details and compound characterization.

azidate A1-glycine, B1-phenylalanine, and B29-lysine amino acid residues present in RHI and thereby access single-site bioconjugates. Treatment of RHI (1) with ISA (1 equiv) under otherwise identical conditions as those described earlier gave a stochastic mixture of mono-, bis-, and tris-azido RHI products with no apparent site-selectivity. HPLC-MS analysis of the crude reaction mixture revealed it consisted of three RHI monoazides, comprising approximately 35% of the total mass balance according to UV area %, three bis-azides (24%) and triazide 2 (4%), along with 20% of unreacted RHI and other unidentified minor products (Supporting Information, Figure S1). Although the reaction resulted in multiple products, it was quite amenable to scale-up (3 g scale). Subsequent preparative HPLC purification targeting the isolation of the three monoazides led to pure samples of A1-glycine azido RHI 4, B29lysine azido RHI 5, and B1-phenylalanine azido RHI 6 in 3%, 32%, and 2% isolated yield, respectively (Table 1, entries 4− 6).29 With purified samples of all three mono-azides 4, 5, and 6, along with tris-azido RHI 2, in hand, the site of azidation was unequivocally confirmed by endoproteinase Glu-C digestion of each peptide followed by HRMS analysis.30 The peptide digests generated by Glu-C treatment were separated by UPLC, and characteristic fragments were used to identify the

Figure 1. Endoproteinase Glu-C digestion fragments for the azidation products of RHI (1).

smoothly to give tris(1-phenyl-1,2,3-triazole) RHI 3 in excellent yield (86%; Table 1, entry 3). Compound 3 displayed an IC50 of 21 nM in the hIR binding assay, about a 90-fold loss in potency, and was unable to fully displace the competitive ligand (∼30% of maximum; see the Supporting Information). Having shown that the diazo transfer chemistry with RHI was clean and high-yielding, we were interested in the opportunity to further develop this methodology to site-selectively monoB

DOI: 10.1021/acs.bioconjchem.9b00069 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry

Cleavage of insulin by endoproteinase Glu-C provides six potential peptide fragments (in red) after DTT disulfide reduction. Without DTT treatment, only four fragments, F1− F5, F2−F6, F3, and F4 (in black), are observed due to the disulfide linkages. HRMS analysis is used to confirm the identity of the peptide azide fragments (without DTT treatment). Conversion of amine to azide results in a mass shift of the corresponding fragment equal to +2N/−2H (+26), confirming the site of modification. The three mono-azido insulins, A1-glycine-N3 RHI 4, B29lysine-N3 RHI 5, and B1-phenylalanine-N3 RHI 6, maintained strong binding to the human insulin receptor with IC50 values of 3.8 nM, 1.2 nM, and 4.7 nM, respectively (Table 1, entries 4−6). From these data, the conversion of the three amine residues to each corresponding azide did have an impact on the ability of the modified insulins to bind to its receptor, decreasing binding potency by approximately 5- to 10-fold. In general, the decrease of hIR binding potency for the azide RHI derivatives compared to the parent insulin highlights the importance of these amines in the binding interaction.21,22 This impact can be attributed to a number of factors, including the change in net charge from positive to neutral, the loss of two H-bond donors, and an increase in molecular size when each amine is converted to the corresponding azide. Van Hest et al. have demonstrated that the site selective introduction of azides into proteins can be achieved via diazo transfer under metal-free and pH-controlled conditions.12,23 Since 1 has three amino acids with free amines, each with a distinct measured pKa value, viz., A1-glycine (α-NH2, pKa 8.4) on the A-chain and B1-phenylalanine (α-NH2, pKa 7.1) and B29-lysine (ε-NH2, pKa 11.1) on the B-chain,24 a selective chemical modification strategy would be particularly amenable to reaction conditions under pH control. Thus, we conducted a systematic screen of diazo transfer as a function of reaction pH ranging from 5 to 10 both in the presence and absence of Cu(II), looking to identify conditions for site-selective azidation of each amine functional group (Figure 2). The solubility of 1 in aqueous buffers at pH 5−7 was low and only modestly better at pH > 8. A minimal amount (5%) of dimethylacetamide (DMAc) was added as cosolvent to improve this solubility, while maintaining as much of the aqueous nature of the reaction conditions as possible. As shown in Figure 2, no reactivity at pH 5 and 6 was observed for RHI with ISA·HCl either in the presence or absence of Cu(II); this was attributed to complete protonation of all three amino groups in the peptide. This result can be rationalized by considering plausible reaction mechanisms for diazo transfer (Scheme 1), which involve formation of presumed tetrazene intermediate 11 in the presence of Cu(II) or intermediate 12 in the absence of Cu(II), followed by collapse to form azide product 13 and the imidazole sulfonamide 14 byproduct. Both of these mechanisms require a nucleophilic amine in order to proceed. At a slightly higher pH (pH 7), 1 showed some reactivity with ISA·HCl only in the presence of Cu(II) with modest, but selective, formation of the B1-mono-azide 6 (14%). Interestingly, as the pH was further increased (pH 8), the reactivity of 1 significantly increased, and high selectivity for reaction at B1 was maintained. In this case, selective azidation to form primarily B1-phenylalanine azide RHI 6 (28%) was observed in the absence of Cu(II). Repeating these conditions on a larger scale (100 mg) followed by purification using preparative reversed phase chromatography led to the isolation

Table 2. Synthesis of B29-Triazole−Insulin Bioconjugates

a R′ = H for all conjugates with the exception of strain-promoted azide−alkyne cycloaddition (SPAAC) product 19. bCopper-free SPAAC was used to generate 19.28

location of the azide modification (Figure 1). After cleavage at each of the four glutamic amino acids in RHI, four peptide fragments were observed in the absence of DTT treatment (labeled as F1−F5, F2−F6, F3, and F4; Figure 1). When the N-terminus of either of the two insulin chains (A1, B1) or the amine side chain of lysine B29 was converted to an azide, the expected exact mass was observed. A mass increase of 26 Da in the corresponding fragment confirmed the conversion of those specific amino acid residues from amine to azide (Figure 1). C

DOI: 10.1021/acs.bioconjchem.9b00069 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Article

Bioconjugate Chemistry Scheme 2. Site-Selective Sequential Bioconjugation of A1, B1, and B29 Amino Acid Residues in RHIa

(a) Imidazole-1-sulfonyl azide (1 equiv), CuSO4·H2O, pH 8 buffer, DMAc, RT, 2 d, 66%; (b) biotin propargyl alkyne (1 equiv), aqueous CuSO4· H2O (0.02 equiv), Na-ascorbic acid, DMSO, RT, 2 h, 53%; (c) MeOPEG5-NHS, Et3N, RT, 2 h, 58%.

a

With appreciable quantities of all three mono-azido RHI derivatives 4, 5, and 6 in hand, corresponding insulin−triazole bioconjugates were generated with diverse alkyne partners via copper catalyzed [3 + 2] cycloaddition reactions. Azido RHI derivatives 4, 5, and 6 were first conjugated to phenylacetylene using catalytic amounts of CuSO4·H2O and sodium ascorbate in DMSO at room temperature (Table 1, entries 7−9). These reactions proceeded smoothly to give the corresponding A1, B29, and B1 mono-phenyl triazole RHI analogues 7, 8, and 9 in 34, 76, and 58% isolated yields, respectively. Compounds 7, 8, and 9 displayed potent hIR binding with IC50 values of 1.1, 2.2, and 0.33 nM, respectively. It is interesting to note the complex behavior of these insulin conjugates, with the A1 and B1 phenyl triazoles 7 and 9 providing improved binding as compared to azido precursors 4 and 6. At the same time, there is a modest (2×) decrease in binding observed for B29 phenyl triazole 8 after conversion from B29 azide 5. Insulin variants have been extensively explored through covalent conjugation via amide coupling at the ε-amine of B29 lysine. These approaches modulate the pharmacokinetic properties of insulin, providing long acting parenterals, such as detemir and degludec.25,26 In view of this, a diverse array of B29-triazole RHI derivatives 15−21 was synthesized by cycloaddition of B29-azido RHI 5 with various alkynes (Table 2). Examples of bioconjugates that were prepared include nucleoside (15), lipid (16), biotin (17), the tyrosinelabeling moiety 4-phenyl-1,2,4-triazolidine-3,5-dione (PTAD)27 (18), mannose (20), and ferrocene (21). In addition, copper-free strain-promoted alkyne−azide cycloaddition (SPAAC)28 with a cyclooctyne was effective to generate analog 19. These bioconjugates were synthesized in moderate to excellent isolated yield (50−90%) and displayed varying degrees of potency in hIR binding (Table 2). As a final example to further demonstrate the potential applications of this synthetic approach, a trifunctionalized

Table 3. Insulin Receptor Binding of Triconjugated RHI Derivative 24 and Its Synthetic Precursors compd no.

yield (%)

IR (bind.) IC50 (nM)

1 (RHI) 22 23 24

66 53 58

0.24 1.2 0.74 6.0

of 6 in 30% yield. In the presence of Cu(II), small amounts of A1-glycine azide 4 (8%) were also observed. At pH 8, B29lysine azide 5 was observed in very small quantities (