Application of the Logic of Cysteine-Free Native Chemical Ligation to the Synthesis of Human Parathyroid Hormone (Hpth)

Application of the logic of cysteine-free native chemical ligation to the synthesis of Human Parathyroid Hormone (hPTH)

Shiying Shanga, Zhongping Tana, and Samuel J. Danishefskya,b,1

aBio-Organic Chemistry Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065; and bDepartment of Chemistry, Columbia University, 3000 Broadway, New York, NY 10027

Contributed by Samuel J. Danishefsky, February 24, 2011 (sent for review December 13, 2010)

The power of chemical synthesis of large cysteine-free polypep- stable forms of hPTH, where “pharmacolability” is attenuated tides has been significantly enhanced through the use of nonpro- without undercutting biological activity, would be of great inter- teogenic constructs which bear strategically placed thiol groups, est (26). It is also of interest to interrogate the consequences enabling native chemical ligation. Central to these much expanded of employing nonproteogenic inserts (27). While this goal can capabilities is the specific, radical-induced, metal-free dethiolation, be accomplished by cleverly designed recombinant methods, che- which can be accomplished in aqueous medium. mical synthesis could well be more convenient for servicing the initial production of probe structures for such structure-activity alanine ligation ∣ desulfurization ∣ leucine ligation ∣ peptide ∣ relationship (SAR) evaluations (28, 29). Previously, the chemical valine ligation synthesis of hPTH required either the solid phase synthesis of an 84-mer-long peptide or the assembly of fully protected peptide he chemical synthesis of proteins offers the potential of segments. Such methods are not ideal for the generation of ana- Tsolving a multitude of problems in biomedical sciences (1). logs (30–33). It was recognized that enhancement of the power of Chemical synthesis can exert great control on protein composi- chemical synthesis for such objectives, including that of hPTH tion. Moreover, chemical synthesis can facilitate the creation of itself, could be accomplished by extending the reach of the under- CHEMISTRY new proteins with desirable properties. Historically, the chemical lying elegant concept of NCL (4–17). To this end, we report a preparation of biotherapeutic proteins and their analogs has pleasing and encouraging example wherein these recent findings relied on the use of the powerful cysteine-based native chemical have been pooled such that the molecule hPTH can be conveni- ligation (NCL) method of Kent and associates (2, 3). However, ently assembled from small synthetic peptide fragments. This given the relative scarcity of cysteine residues in nature, a clear technology would enable access to more proteins through chemi- impetus arises for the realization of new NCL capabilities (4–17). cal synthesis. Anticipating this type of problem, our laboratory (9, 11, 14, 17) As a model for what would have to be accomplished in a typical and others (5–7, 10) have developed strategies by which to ac- protein synthesis, we chose to use the generally preferred conver- complish ligation at a range of noncysteine amino acid residues. gent strategy to prepare hPTH (Fig. 2), although a more efficient The logic of our approach is outlined in Fig. 1. The N-terminal synthesis might be achieved with other disconnection patterns. peptide, D, to be ligated is equipped with an N-terminal sulfur- The primary structure of hPTH is shown in Fig. 2. On the basis bearing amino acid surrogate, A, itself prepared by synthesis of its amino acid sequence, we decided to assemble the hPTH and coupled, by a suitable method, to the peptide B. In this polypeptide chain from four approximately equal sized frag- way, N-terminal ligation candidate D is ready for coupling with ments, hPTH (1–23) 1, hPTH (24–38) 2, hPTH (39–59) 3, and C-terminal acyl donating peptide C (usually a thioester). Ligation hPTH (60–84) 4. The peptide fragments contain 23, 15, 21 resi- product E can be maintained as such, thereby affording a speci- dues, and 25 amino acid residues, respectively, which are acces- fically placed thiol group in a nonnatural context. Theoretically, sible through solid phase peptide synthesis (34, 35). These de-thiolation of E will produce F wherein the "R" group had been fragments were to be joined at the sites of three of the most abun- governed by the synthetically derived A. In principle, the strategy dant amino acids present in hPTH, i.e., Leu24, Ala39, and shown in Fig. 1 is of universal scope. Val60 (Fig. 2). Studies using a variety of model peptides have demonstrated Results and Discussion the fragment-coupling capability of the cysteine-free strategy. Here, in this paper, we seek to examine the general applicability The synthesis of hPTH is shown in Fig. 3. The fully protected of this strategy to the total synthesis of cysteine-poor proteins. peptides to be ligated were synthesized by Fmoc chemistry on Human Parathyroid Hormone (hPTH) is chosen to serve as a a 0.05 mmol scale. The preleucine and prevaline surrogates model molecule because of its representativeness in terms of were attached to the N termini of the fully protected peptides by amino acid composition and its therapeutic value. hPTH is a 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexa- biological messenger that is secreted by the parathyroid glands fluorophosphate (HATU) coupling (17). The peptide fragments, as a peptide containing 84 amino acids (18, 19). Upon binding bearing C-terminal thioesters, were prepared from the fully to its receptor, hPTH can enhance the concentration of calcium protected peptides using the 1-ethyl-3-(3-dimethylaminopropyl) (Ca2þ) in the blood (20). Because of their important physiological carbodiimide (EDCI)-mediated amide formation reaction under – the nonepimerizing conditions developed by Sakakibara et al. roles, hPTH and one of its fragments, hPTH (1 34), are now A given (by subcutaneous injection) for the treatment of hypopar- (Fig. 3 ) (36). Kinetically controlled ligation (37) at leucine of athyroidism and osteoporosis in men, as well as for postmenopau- sal women at high risk of fracture (21–23). Not unlike most other Author contributions: S.S., Z.T., and S.J.D. designed research; S.S. and Z.T. performed hormone drugs, the recombinant hPTH therapeutics have very research; S.S., Z.T., and S.J.D. analyzed data; and S.S., Z.T., and S.J.D. wrote the paper. short half-lives in the human body and need to be administered The authors declare no conflict of interest. at least once a day (24, 25). The need for continuous daily sub- 1To whom correspondence should be addressed. E-mail: [email protected]. cutaneous injection is an obvious disadvantage which serves to This article contains supporting information online at www.pnas.org/lookup/suppl/ compromise use of the hormone. Clearly, the production of more doi:10.1073/pnas.1103118108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103118108 PNAS Early Edition ∣ 1of4 Downloaded by guest on September 27, 2021 Fig. 1. Ligation at noncysteine amino acid residues. Key: (A) A single amino acid residue A has been added to peptide B to make the N-terminal ligation partner D;(B) Merger of C-terminal peptide C and N-terminal peptide D.

fragment 1 thioester and fragment 2 alkylthioester was carried alyst generated 12 in 63% yield (38). Not surprisingly, the mod- out over 9h at pH 7.5, to afford peptide 9 in 59% yield. The ified NCL methods are operative even in the presence of internal reaction of fragments 3 and 4 was carried out in pH 7.5 guanidine (side chain) thiols (2). Our aqueous, metal-free desulfurization buffer for 9 h to provide peptide 10. After ligation was completed, method, applied to 12, was completed in 2 h and yielded the the thiazolidine function in peptide 10 was converted into the final full-length hPTH (9). Purification by HPLC provided pure required N-terminal cysteine by treatment with methoxylami- hPTH in 86% yield. ne•HCl at pH 4.0 (86% yield over two steps, Fig. 3B). Following Not surprisingly, the fully synthetic noncysteine containing these syntheses, ligation of peptide 9 thioester and 11 in the hormone folded spontaneously under native conditions (39–41). presence of 200 mM 4-mercaptophenylacetic acid (MPAA) cat- Circular dichroism (CD) measurements from 190 to 250 nm

Fig. 2. Retrosynthetic analysis for the preparation of Human Parathyroid Hormone (hPTH) by the native chemical ligation/desulfurization strategy. The desired full-length hPTH was prepared by noncysteine native chemical ligation of 1, 2, 3, and 4 followed by desulfurization reaction.

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Fig. 3. Synthesis of Human Parathyroid Hormone. Key: (A) H-Trp-SPh, EDCI, HOOBt, DIEA, DMSO, 3 h; (B)TFA∶TIS∶H2O(95∶2.5∶2.5), 45 min; (C) Boc-Leu(SSMe)- OH, HATU, DIEA, DMSO, 1 h; (D) TFE∶AcOH∶CH2Cl2 (8∶1∶1), 2 h; (E) H-Gly-SCH2CH2CO2Et, EDCI, HOOBt, DIEA, DMSO, 1 h; (F) H-Leu-SPh, EDCI, HOOBt, DIEA, DMSO, 2 h; (G) Boc-Val(SSMe)-OH, HATU, DIEA, DMSO, 1 h; (H)6MGn•HCl, 100 mM Na2HPO4, and 50 mM TCEP, pH 7.5, 9 h; (I) MeONH2 • HCl, pH 4, 2.5 h; (J)6M Gn•HCl, 300 mM Na2HPO4, 200 mM MPAA, and 20 mM TCEP, pH 7.9; and (K) VA-044, tBu-SH, TCEP, H2O, MeCN, 37 °C, 2 h.

served to demonstrate the folding of the fully synthetic polypep- In summary, we have described herein a rather interesting tide (Fig. 4). instance of extending the scope of native chemical ligation to the Remarkably, the hPTH prepared by laboratory synthesis convergent synthesis of a highly active, substantially sized poly- derived from our chemistry is significantly more pure than is the peptide, hPTH. Our method brings together readily purifiable recombinant hPTH obtained as a reference sample. As chemical peptide segments using the underlying logic of S → N acyl trans- synthesis develops further, it may well emerge as a credible alter- fer inherent in NCL with the feature that the target of synthesis “ “ native to biologic type methods, where there is a particularly contains no cysteine residues. high priority for enhancing homogeneity.

Fig. 4. Unnormalized CD spectra of hPTH. Nadirs at 208 and 222 nm are characteristic of α-helical structures. Key: (A) CD comparison of the synthetic and recombinant PTH at concentration of 14 μM; (B) CD spectra of synthetic PTH at concentration of 14 μMand7μM.

Shang et al. PNAS Early Edition ∣ 3of4 Downloaded by guest on September 27, 2021 The chemistry described above is, with some modification on 2695 Separations Module and a Waters 996 Photodiode Array Detector a case by case basis, being extended to the synthesis of hPTH equipped with Varian Microsorb columns at a flow rate of 0.2 mL∕ min. analogs, hoping to discover systems of enhanced potency and The ultra performance liquid chromatography (UPLC)-MS analysis of the pharmacostability. The results of these efforts will be described peptide was performed using a Waters Acquity™ UPLC system equipped in due course. We anticipate that the synthesis concepts demon- with Acquity UPLC® BEH columns at a flow rate of 0.3 mL∕ min. The mobile strated above will find broad application, and are conducting phase for HPLC analysis and separation was a mixture of 0.05% TFA (vol∕vol) experiments toward such ends. in water (solvent A)/0.04% TFA in acetonitrile (solvent B). All the ligation and desulfurization reactions were carried out as described previously (17). Materials and Methods A detailed description of materials and methods is given in SI Appendix: All commercial reagents and solvents were used without further purification. Materials and Methods Automated peptide synthesis was performed on an Applied Biosystems Pioneer continuous flow peptide synthesizer using commercially available ACKNOWLEDGMENTS. We thank Prof. David Elizer and Dr. Khurshida Shahidul- and synthetic building blocks. The deblock solution was a mixture of 100/ lah (Weill Cornell) for their assistance with the CD measurements. We thank 5/5 of dimethylformamide/piperidine/1,8-Diazabicyclo[5.4.0]undec-7-ene. All Dr. Barney Yoo (Memorial Sloan-Kettering Cancer Center Organic Synthesis the peptide thioesters were prepared as previously described (17). The Core facility) and Rebecca Lambert for valuable discussions. We also thank peptide side chain cleavage solution was a mixture of 95/2.5/2.5 of tri- Dr. George Sukenick, Hui Fang, and Sylvi Rusli of Sloan-Kettering Institute’s fluoroacetic acid (TFA)/Triisopropylsilane ðTISÞ∕H2O. The purification of the NMR core facility for mass spectral and NMR assistance and Laura Wilson for peptides was achieved using a Ranin HPLC solvent delivery system equipped assistance with the preparation of the manuscript. Support for this research with a Rainin UV-1 detector and Varian Dynamax using Varian Microsorb was provided by the National Institute of Health (CA28824 to S.J.D.). We columns at a flow rate of 16.0 mL∕ min. The liquid chromatography-mass dedicate this paper to the memory of Dr. Shoshanah Trachtenberg Frackman spectrometry (LC-MS) analysis of the peptide was performed using a Waters for her work in biomedical ethics.

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