Simultaneous and Traceless Ligation of Peptide Fragments on DNA

Subscriber access provided by EKU Libraries

Article

Simultaneous and Traceless Ligation of Peptide Fragments on DNA Scaffold Gosuke Hayashi, Masafumi Yanase, Yu Nakatsuka, and Akimitsu Okamoto Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01655 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 26, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31 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

Biomacromolecules

Simultaneous and Traceless Ligation of Peptide Fragments on DNA Scaffold

Gosuke Hayashi,†,⊥ Masafumi Yanase,†,⊥ Yu Nakatsuka,† and Akimitsu Okamoto*,†,‡ †Department

of Chemistry and Biotechnology, The University of Tokyo, 7-3-1 Hongo,

Bunkyo-ku, Tokyo 113-8656, Japan ‡Research

Center for Advanced Science and Technology, The University of Tokyo, 4-6-1

Komaba, Meguro-ku, Tokyo 153-8904, Japan ⊥These

authors contributed equally to this work.

ABSTRACT Peptide ligation is an indispensable step in the chemical synthesis of target peptides and proteins that are difficult to synthesize at once by a solid-phase synthesis. The ligation reaction is generally conducted with two peptide fragments at a high aqueous concentration to increase the reaction rate; however, this often causes unpredictable aggregation

and

precipitation

of

starting

or

resulting

peptides

due

to

their

hydrophobicities. Here, we have developed a novel peptide ligation strategy harnessing the two intrinsic characteristics of oligodeoxynucleotides (ODNs), i.e., their hydrophilicity and hybridization ability, which allowed an increase in the water solubility of peptides and the reaction kinetics due to the proximity effect, respectively. Peptide–ODN conjugates cleavable to regenerate native peptide sequences were synthesized using

ACS Paragon Plus Environment

Biomacromolecules 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

Page 2 of 31

novel lysine derivatives containing conjugation handles and photolabile linkers, via solidphase

peptide

synthesis

and

subsequent

conjugation

to

15-mer

ODNs.

Two

complementary conjugates were applied to carbodiimide-mediated peptide ligation on a DNA scaffold and the subsequent DNA removal was conducted by photoirradiation in a traceless fashion. This DNA scaffold-assisted ligation resulted in a significant acceleration of the reaction kinetics and enabled ligation of a hydrophobic peptide at a micromolar concentration. Based on this chemistry, a simultaneous ligation of three different peptide fragments on two different DNA scaffolds has been conducted for the first time.


Page 3 of 31 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

Biomacromolecules

INTRODUCTION A key reaction in chemical protein synthesis, which has permitted generation of various full-length

proteins

consisting

of

native

amino-acid

sequences

with

site-specific

modifications,1–5 is peptide ligation to form a new amide bond between two peptide fragments in aqueous solution. Because the ligation reaction inevitably competes with hydrolysis, more than a millimolar concentration of the reactant peptides is generally used for efficient amide-bond formation. However, the water solubility of peptides is dependent on their amino-acid composition, higher-order structure, and intermolecular self-interaction. Therefore, hydrophobic peptides cannot always be applied to the aqueous ligation reaction due to their aggregation or precipitation.6–8 To overcome this problem, several strategies utilizing additives such as organic solvent and detergent9–11 or introducing an isopeptide bond12,13 have been developed so far. In particular, the use of a solubilizing tag is one of the promising strategies to synthesize hydrophobic peptides and proteins such as membrane proteins. Although initial tags had been attached to peptide termini,14–17 which cannot retain the solubility enhancement after peptide ligation, semipermanent tags that can be cleaved by external stimuli have been developed by several groups recently.18–24 Given that the synthesis of peptide fragments consisting of more than 100 amino acids in a single solid-phase peptide synthesis (SPPS) procedure is not feasible,25 the ligation reaction must be repeated to synthesize various natural proteins of interest. However, the repeated purification and isolation of ligated peptides are not only timeconsuming and laborious but also cause a significant decrease in the overall yield, which prevents the chemical synthesis of large proteins. To address this issue, one-pot sequential ligation of more than two peptide fragments has been developed by utilizing mild protection/deprotection chemistry.26 However, development of an alternative concept is required to accelerate the chemical synthesis of a greater variety of proteins. Nucleic acids have recently been used as a template or scaffold for various chemical and biochemical reactions.27,28 In fact, natural translation machinery consisting of ribosome, mRNA, aminoacyl-tRNAs, and other translation factors achieves protein



synthesis in a considerably diluted condition (micromolar range) by utilizing nucleic acid interactions represented by mRNA–tRNA interaction within the ribosome. DNA-templated organic synthesis (DTS) adopts nature’s approach, harnessing an increased effective molarity obtained through DNA hybridization to enable various coupling reactions in the relatively lower concentration (nM to M) than conventional organic synthesis (mM to M).29 Several DNA or peptide nucleic acid hybridization (PNA)-assisted amide-bondforming reactions have been reported so far using nucleophile–electrophile pairs such as two different amino acids,30–34 peptide–oligonucleotide,35 and peptide–peptide.36 However, the generated products in these research cannot be recovered because of the noncleavable linker between product and nucleic acids, although PNA-assisted peptide-side chain coupling strategy, in which the generated isopeptide can be recovered by the removal of the PNA scaffold, has been reported more recently.37 Here, we report a peptide ligation strategy, in which photocleavable peptide– oligodeoxynucleotide (ODN) conjugates were employed. Peptide bond formation rapidly proceeds in highly diluted condition due to the hybridization of ODNs, and the ligated peptides can be released as a native peptide sequence after photoirradiation (Figure 1). Furthermore, we demonstrated the simultaneous ligation of three peptide fragments by using the DNA-programmed peptide ligation strategy.

EXPERIMENTAL SECTION Synthesis of peptide-DNA conjugates 1) Conjugation by SPAAC To an eppendrof, 2.0 M TEAA buffer (final concentration was 0.1 M, pH 7.0) was added with DBCO-modified oligonucleotide (1.0 mM, 1 eq.) and Kaz-containing peptide (2.0 mM, 2 eq.) and agitated overnight. Purification of the peptide-ODN conjugate was by RP-HPLC with a linear gradient of 1 to 45% of acetonitrile in 0.1 M TEAA buffer (pH


Page 4 of 31

Page 5 of 31 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

Biomacromolecules

7.0) over 30 min. The conjugate product was collected and lyophilized. The desired product was characterized by MALDI-MS. 2) Conjugation by CuAAC To a solution of azide-modified ODN (final concentration; 100 µM, 1.0 eq.) and alkynepeptide (100 µM, 1.0 eq.) in sodium phosphate buffer (0.1 M, pH 7.0), pre-chelate solution of CuSO4 (250 µM, 2.5 eq.) and THPTA (1.25 mM, 12.5 eq.) and aminoguanidine･HCl solution (5.0 mM, 50 eq.) were added. Sodium ascorbate solution (5.0 mM, 50 eq.) was then added to start the reaction. Reaction mixture was stirred for 2 hours, purified by RP-HPLC and freeze-dried to obtain pure product. The desired product was characterized by MALDI-MS. 3) Conjugation with 2 DNA in one-pot by continuous SPAAC and CuAAC To a sodium phosphate buffer (1.0 M, pH 7.0), DBCO-modified DNA (1 mM, 1.0 eq.) and azide-alkyne-peptide (1 mM, 1.0 eq.) were added and allowed to react for 2 hours at room temperature. After starting material was consumed by RP-HPLC monitoring, pre-chelate solution of CuSO4 (0.25mM, 2.5 eq) and THPTA (1.25 mM, 12.5 eq.) and aminoguanidine･HCl solution (5.0 mM, 50 eq.), azide-modified ODN (100 µM, 1.0 eq) were added. Then, water was added to make the final concentration of peptide and ODN 100 µM.Sodium ascorbate solution (5.0 mM, 50 eq.) was then added to start the reaction. Reaction mixture was stirred for 2 hours, purified by RP-HPLC and freeze-dried to obtain pure product. The desired product was characterized by ESI-MS. Photolysis of peptide-DNA conjugates Photolysis reactions were performed with 365 nm dual intensity transiluminator (Funakoshi) using a glass vial with 11 mm diameter and 50 mm height (continuous, non-pulsed). 100 µl of 10% TFA was added to the peptide-ODN conjugate (1.0 µM) in 1.0 mL of ligation buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), 0.1 M sodium chloride, 0.02 M pyridine, pH 5.5) and the sample solution was UV-irradiated by UV-transiluminator for different time span. The reaction was analyzed by RP-HLC



with a gradient of 10 to 55% of B over 30 min to calculate the conversion yield based on calculations of the HPLC integrates. Peptide ligation on DNA scaffold Various factors such as the concentration of EDC･HCl or peptide-ODN conjugate, the composition of buffer, additive and pH were differed in each experiment. In a typical ligation, stocks of 400 mM EDC ･ HCl (Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and 400 mM MES buffer containing 80 mM pyridine and 400 mM NaCl (pH 5.5) were prepared. The P1-ODN1 and the P2-ODN1c were added to a final concentration of 1.0 µM and 1.5 µM, respectively in 1.0 mL of MES buffer (final concentration; 100 mM, pH 5.5) containing 100 mM NaCl, 20 mM pyridine and 100 mM EDC･HCl and incubated at room temperature. At each time point, 0.1 by volume of 10% TFA aqueous solution was added to quench the reaction. Afterwards, the sample solution was UV-irradiated for 60 min by UV-transilluminator and analyzed by RP-HLC with a gradient of 5 to 55% of B over 25 min to calculate the conversion yield based on calculations of the HPLC integrates. The % yields were calculated according to the following equation: Yield = [AreaP1-X*/(AreaP1-X* + AreaP1*+EDC + AreaP1*)] × 100. Simultaneous ligation of three peptide fragments Stocks of 400 mM EDC･HCl and 400 mM MES buffer containing 80 mM pyridine and 400 mM NaCl (pH 5.5) were prepared. P1-ODN1, ODN1c-P12-ODN2, and P13-ODN2c were added to a final concentration of 1.0 µM, 1.5 µM and 2.0 µM, respectively in 1.0 mL of MES buffer (final concentration; 100 mM, pH 5.5) containing 100 mM NaCl, 20 mM pyridine and 100 mM EDC･HCl and incubated at room temperature. At each time point, 0.1 by volume of 10% TFA aqueous solution was added to quench the reaction. Afterwards, the sample solution was UV-irradiated for 60 min by UV-transilluminator and analyzed by RP-HLC with a gradient of 5 to 55% of B over 25 min to calculate the conversion yield based on calculations of the HPLC integrates. The % yields were calculated according to the following equation: Yield = [AreaP1-12-13*/(AreaP1-12-13* + AreaP112*

+ AreaP1-13*+ AreaP1*+EDC + AreaP1*)] × 100.


Page 6 of 31

Page 7 of 31 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

Biomacromolecules

RESULTS AND DISCUSSION Design and Synthesis of Fmoc–Lys Derivative with Photolabile Linker. Efficient and orthogonal conjugation/deconjugation chemistries are necessary to achieve the idea of DNA hybridization-assisted peptide ligation and following peptide recovery. For the peptide–ODN conjugation, we chose the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction, which is widely used for bioorthogonal conjugation of proteins and nucleic acids in in vitro and in vivo experiments.38–40 For the deconjugation chemistry, which

needs

to

regenerate

a

native

peptide

sequence,

photouncaging

of

nitroveratryloxycarbonyl (NVOC)-based amino group protection was adopted due to the high efficiency and orthogonality.41–43 As a linker molecule meeting the above requirements, we designed an Fmoc–lysine derivative 1, named Fmoc–Kaz, (Scheme 1), which can be used in standard Fmoc SPPS. Fmoc–Kaz contains an azide moiety at the terminus, which works as a conjugation handle for SPAAC, and an NVOC linker cleavable to regenerate the amino group of the lysine residue after photoirradiation. The synthetic route of Fmoc– Kaz is shown in Scheme 1 and S1. An azide-containing NVOC derivative 2 was synthesized via four steps with a 23% yield from 4-hydroxy-3-methoxyacetophenone as a starting material (Scheme S1). The hydroxyl group at the benzylic position of 2 was activated by N,N-disuccinimidyl carbonate to generate a N-hydroxysuccinimide (NHS) carbonate 3, and then Fmoc–Lys–OH was coupled to afford the target compound 1 in 62% yield in two steps.

Synthesis and Properties of Peptide–ODN Conjugates. Two different peptide–ODN conjugates,

whose

ODN

sequences

were

complementary

to

each

other,

were

synthesized through Kaz-containing peptide synthesis in the solid phase and the



Page 8 of 31

subsequent SPAAC conjugation of ODNs in the solution phase (Figure 2A). Peptides P1 and P2, which contain Kaz at the C- and N-terminal sides, respectively, were prepared through standard Fmoc–SPPS procedures using Wang and Rink amide-resin, respectively. At the N-terminus of P1, a rhodamine B derivative synthesized according to the previous report (Scheme S2)44 was directly introduced on the resin. This fluorophore is expected to enhance the sensitivity of small-scale reaction monitoring. After TFA cleavage from resin, crude peptides were purified by HPLC and identified by MALDI-TOF MS (Figure 2B and S1A). We also prepared dibenzocyclooctyne (DBCO)containing ODNs, ODN1, and ODN1c, through coupling between amino-modified ODN and

DBCO-NHS

ester

(Scheme

S3,

Figure

2C,

and

S1B).

To

construct

the

photocleavable peptide–ODN conjugates, SPAAC reactions between Kaz-containing peptides and DBCO-modified ODNs were conducted in 0.1 M triethylammonium acetate (TEAA) buffer (pH 7.0). In the presence of an equivalent amount of P2 and ODN1c, the reaction proceeded quantitatively within 10 min at room temperature to produce a peptide–ODN conjugate, P2–ODN1c (Figure 2D). We also obtained a conjugate P1– ODN1 in the same procedure (Figure S1C). To investigate the efficiency of ODN removal from peptide–ODN conjugates, P1–ODN1 was exposed to irradiation by 365 nm UV light at room temperature. We then analyzed the photolyzed product by HPLC to monitor absorbance at 564 nm derived from rhodamine B. While 2-min irradiation provided a new peak at a later retention time compared with that of P1–ODN1, 10-min irradiation completely consumed the starting material (Figure 2E). MALDI-TOF MS identified the obtained product as photouncaged peptide P1*, which has a native lysine residue.

Ligation of Two Peptide Fragments on the DNA Scaffold. With two complementary conjugates in hand, P1–ODN1 and P2–ODN1c, we investigated the DNA-assisted peptide ligation

by

using

water-soluble

carbodiimide,

EDC

･

HCl

(ethyl-3-[3-

dimethylaminopropyl]carbodiimide hydrochloride) as a carboxyl-activating agent (Figure 3A). After the amide coupling reaction, the scaffold ODNs were removed by


Page 9 of 31 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

Biomacromolecules

photoirradiation and reacted peptides were analyzed by HPLC. In the presence of 50 mM EDC in 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing 1.0 M NaCl (pH 6.0), P1–ODN1 (1.0 M) and P2–ODN1c (1.5 M) were reacted at room temperature. The sample solution was quenched at different time points by mixing with 10% TFA and then exposed under 365 nm irradiation for 60 min. HPLC analysis of the released peptides revealed that the peak of unligated peptide P1* almost disappeared within 3 h and, instead, a new peak derived from ligated peptide P1–2* emerged (Figure 3B). It is notable that, in addition to the ligated peptide, we also identified an EDC adduct of unligated P1 (P1*+EDC). Because this by-product was isolable and stable in water, we reasoned that P1*+EDC was not an activated carboxylic acid (i.e., O-acylisourea), but an imide structure (i.e., N-acylurea), which was generated via an O–N acyl shift (Scheme S5).45 The time-course analysis with three different EDC concentrations (20, 50, and 100 mM) showed that the higher concentration of EDC accelerated the reaction kinetics, but the conversion yields of each reaction converged to the same level after 24 h (Figure 3C and S2). We

then

optimized

the

reaction

conditions

such

as

pH,

stoichiometry,

salt

concentration, and additives. The conversion ratio of ligation mixtures with different pHs was compared after 30 min reaction by analytical HPLC, indicating that an acidic environment is preferable to promote the reaction (Figure 3D and S3). This is consistent with the previous result showing that protonation of EDC accelerates the O-acylisourea formation, which is the rate-limiting step of EDC-mediated amide-bond formation.46 The peptide ligation in the absence and presence of different amounts of P2–ODN1c demonstrated that the yields of P1–2* plateau at 1 equiv of P2–ODN1c and an excess amount of the substrate did not affect the yield (Figure S4). High salt concentration was also shown to cause a negative effect on the ligation efficiency (Figure S5). Interestingly, when we added 4-dimethylaminopyridine, imidazole, or pyridine as the additives in the reaction mixture, pyridine showed a strong effect in reducing the production of P1*+EDC, whereas imidazole and DMAP did not show such effect and rather enhanced the EDC adduct formation (Figure 3E and S6). This suppressive effect



of pyridine on O–N acyl shift is consistent with a previous study, although the mechanism is still unknown.47 The effect of the DNA scaffold on reaction kinetics was then investigated using the conjugate pair (P1–ODN1 and P2–ODN1c) and a nonconjugate pair (P1 and P2). We estimated the initial rate of reaction by measuring the yield of P1–2* after 2 min of reaction. The pair of P1–ODN1 (1.0 M) and P2–ODN1c (1.5 M) generated the ligation product in 17% yield (first order rate constant, kapp = 0.0014 s-1), while the nonconjugate pair generated no product in the same concentration and less amount of the product (7% yield) even in 500-fold higher concentration (Figure S7). The higher production efficiency of the DNA-templated system in even lower concentration indicated that the effective molarity of the ligation reaction was increased significantly by the proximity effect provided by the DNA scaffold. To estimate the sequence dependence of the reaction rate and yield at the ligation site, we designed several Kaz-containing peptides as a counterpart of P1 (Table 1). Peptides P3 to P6 have a different single amino acid at the N-terminus (Pro, Val, Phe, or Gln, respectively) compared with Gly in P2, whereas P7 to P10 have different numbers of Gly residues at the N-terminus. Synthesis, purification, and identification of each peptide and ODN1c-conjugate were performed according to the procedure described above (Figures S8 and S9). DNA-directed ligation reactions between P1– ODN1 and PX–ODN1c (X = 3–10) were monitored by HPLC (Figures S10 and S11), and the calculated initial rate constants and the yields at 3 h are summarized in Table 1. When we investigated the N-terminal amino-acid dependence by using P2 to P6, the reaction rates were similar except for P3, although Val (P4) and Gln (P6) showed relatively slow kinetics among all the tested peptides. N-terminal Pro residue showed approximately 10-fold slower rate than Gly, probably due to the secondary amine group, but comparable yield at the 3 h time point. Although increased numbers of Gly residues at the N-terminus tended to show decreased reaction rates, the differences were negligible.


Page 10 of 31

Page 11 of 31 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

Biomacromolecules

ODN attached to a peptide works not only as a scaffold to increase the effective molarity of the reactive groups but also as a solubilizing tag. A hydrophobic peptide P11 was designed and synthesized (Figure 4A) and then conjugated with ODN1c to generate P11–ODN1c (Figure 4B). While the saturation concentration of P11 was lower than 100 M, that of P11–ODN1c increased dramatically, showing the function of the solubilizing tag. Then, DNA-directed ligation between P1–ODN1 and P11–ODN1c was analyzed by HPLC monitoring. The kapp value of the ligation was 0.00067 s–1 and the yield of ligated P1–11* at 3 h was 75%, which is comparable to those of other peptides tested in Table 1. The series of peptide ligation experiments using DNA scaffold suggest that the reaction rate of this rapid DNA-directed peptide ligation is dominantly determined by the hybridization of the DNA scaffold and the effect of amino-acid structure, while the distance between reaction sites or peptide hydrophobicity is ignorable. The DNA scaffold-assisted peptide ligation shown above is the first demonstration that ODN works not only as “solubilizing tag” to increase the water solubility of the hydrophobic peptide but also as “scaffold” to accelerate ligation reaction between two different native oligopeptides by a hybridization-driven proximity effect. Because the removal of ODNs proceeds quickly and quantitatively, the native peptide sequence can be efficiently recovered after the ligation reaction.

Simultaneous Ligation of Three Peptide Fragments on Dual DNA Scaffolds. We hypothesized that if three peptide fragments (e.g., peptides A, B, and C) are aligned on two different DNA scaffolds, simultaneous and site-selective ligation of those peptides could occur to produce a site-specifically linked peptide A–B–C. To demonstrate this idea, a conjugate bearing two different ODNs at the N- and C-terminus region must be prepared to connect both N- and C-terminus conjugates. The synthesis of such a dual ODN conjugate requires an additional conjugation chemistry that should be orthogonal to the first conjugation of SPAAC. We decided to use copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC)48 as the orthogonal chemistry. Therefore, we designed another Fmoc–Lys derivative 4, named Fmoc–Kal (Figure 5A), which contains an alkyne moiety



at the terminus instead of the azide moiety of Fmoc–Kaz. Fmoc–Kal was synthesized via the same six-step procedure as Fmoc–Kaz except for the use of propargylamine instead of azidopropylamine in the amide formation reaction (Scheme S3). By using the manual Fmoc SPPS, we synthesized a peptide P12, which contains both Kaz and Kal at the N- and C-terminal regions, respectively (Figure 5A). P12 was purified by HPLC and identified by MALDI-TOF MS in the conventional procedure (Figure S12A). As the conjugation counterpart, a 5-azide-modified ODN2 was synthesized using an aminomodified ODN and NHS-activated 2-azidoacetic acid (Scheme S3), then purified by HPLC and identified by MALDI-TOF MS (Figure S12B). To synthesize the conjugate modified with two different ODNs, one-pot tandem click chemistry consisting of first SPAAC and subsequently CuAAC was conducted using P12, ODN1c, and ODN2 (Figure 5B and 5C). The obtained conjugate ODN1c–P12– ODN2 was purified by HPLC and identified by MALDI-TOF MS (Figure S12C). P13– ODN2c, which has a complementary sequence against ODN2, was also synthesized with Kal-containing peptide P13 and 3-azide-modified ODN2c through the CuAAC reaction (Figure S13). We examined the simultaneous ligation of three peptide fragments on two DNA scaffold by using three peptide–ODN conjugates of P1–ODN1, ODN1c–P12–ODN2, and P13– ODN2c (Figure 5D). These conjugates were dissolved in MES buffer (pH 5.5) containing 20 mM pyridine and 100 mM sodium chloride and reacted in the presence of 100 mM EDC at room temperature. The reaction mixture was quenched after 3 h and exposed under UV light irradiation to produce native peptide sequences. HPLC analysis of the reaction mixture revealed the formation of the full-length peptide, P1–12–13* in 32% yield (Figure 5E). A middle fragment-skipped peptide, P1–13* was also generated in a similar yield, although fragment-overlapped peptides were not observed. As negative controls, samples without ODN1c–P12–ODN2 or DNA scaffolds (i.e., the use of P1, P12, and P13 instead of the conjugates) were also tested and resulted in no ligated product formation (Figure S14A).


Page 12 of 31

Page 13 of 31 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

Biomacromolecules

To investigate the molecular mechanism for the generation of both P1–12–13* and P1– 13* in similar yields, we analyzed the reaction kinetics. The coupling rate between P1 and P12 was similar to that between P1 and P13 (Figure S14B), suggesting that there is almost no difference in the collision frequency between C-terminal carboxylic acid of P1 and both N-terminal amino groups of P12 and P13, although the extended distance between P1 and P12 (100 atoms) is shorter than that between P1 and P13 (250 atoms) (Figure S15). This paradox might be explained by the long and flexible linker moiety located between each peptide and ODN. It is possible that free and random molecular motion of the ternary conjugate caused by the long and flexible linker could provide similar 3-D distance from C-terminus of P1 to N-terminus of both P12 and P13. Although the linker length and flexibility should be improved to increase the site specificity of the ligation reaction, it is notable that this is the first example demonstrating the simultaneous ligation of three different peptide fragments.

CONCLUSION The novel peptide ligation strategy employing a DNA scaffold has contributed to the drastic acceleration of the ligation reaction in highly diluted condition as well as enhancement of the water solubility of peptide fragments. The photolabile linkers between peptides and ODNs enabled facile and efficient recovery of ligated peptides after photoirradiation. Given the high affinity of complementary ODNs, it is possible to conduct a ligation reaction in more dilute conditions. The first simultaneous multiple peptide ligation was also conducted, although the site specificity between two reaction points was not sufficient. We believe that after several improvements, this fusional approach combining chemical protein synthesis with DNA technology could be an alternative approach for synthesizing “difficult proteins” such as large or aggregationprone proteins.



Page 14 of 31

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at DOI: xx.xxxx/xxxxxx.

Synthetic procedures, NMR spectra (1H and

13C

NMR), preparation of

modified ODNs, HPLC traces and MALDI spectra for sample preparations and peptide ligation reactions.

AUTHOR INFORMATION Corresponding Author [email protected] ORCID 0000-0002-7418-6237 Author Contributions


Page 15 of 31 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

Biomacromolecules

⊥These

authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (A) 15H02190 and 18H03931, Grant-in-Aid for Scientific Research (C) 18K05313, and Grant-in-Aid for Young Scientists (B) 25870186.



Figure 1. Peptide ligation on DNA scaffold using photocleavable peptide– ODN conjugates and following DNA removal by photoirradiation.


Page 16 of 31

Page 17 of 31 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

Biomacromolecules

Scheme 1. Synthesis of Fmoc-Kaz (1).



Figure 2. Synthesis and properties of photocleavable peptide–ODN conjugates. (A) Synthetic procedures for peptide–ODN conjugates, P1-ODN1 and P2ODN1c. (B) HPLC trace (564 nm in the linear gradient with water/acetonitrile containing 0.1% TFA) and MALDI-TOF mass spectrum of purified P1. (C) HPLC trace (260 nm in the linear gradient with TEAA buffer/acetonitrile) and MALDI-TOF mass spectrum of purified ODN1. (D) HPLC traces (260 nm in the linear gradient with TEAA buffer/acetonitrile) of the reaction mixture or the purified product obtained from SPAAC between P2 and ODN1c and


Page 18 of 31

Page 19 of 31 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

Biomacromolecules

MALDI-TOF mass spectrum of purified P2-ODN1c. (E) HPLC traces (564 nm in the linear gradient with TEAA buffer (pH 7.0)/acetonitrile) for photolysis of P1-ODN1 and MALDI-TOF mass spectrum of cleaved peptide P1*. HPLC traces and MALDI-TOF mass spectra are shown in blue and red, respectively.



Figure 3. EDC-mediated peptide ligation using P1-ODN1 and P2-ODN1c. (A) Reaction scheme for DNA-assisted peptide ligation and subsequent DNA removal by photoirradiation. (B) HPLC traces (564 nm in the linear gradient with water/acetonitrile containing 0.1% TFA) for photolyzed ligation mixture. (C) Time-course reaction profiles of P1*, P1-2*, and P1*+EDC with different EDC concentrations (20, 50 and 100 mM). (D) pH dependence of reaction profiles in 30 min. (E) Effect of additives on reaction yields. DMAP, Im, and Py indicate 4-dimethylaminopyridine, imidazole, and pyridine, respectively.


Page 20 of 31

Page 21 of 31 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

Biomacromolecules

Table 1. Reaction rate constant and yield of DNA-assisted ligation for tested sequences with different N-terminal amino-acids or different numbers of Nterminal Gly residues.

P2 P3 P4 P5 P6 P7 P8 P9 P10

Sequences H-GKazNSGRA-NH2 H-PKazNSGRA-NH2 H-VKazNSGRA-NH2 H-FKazNSGRA-NH2 H-QKazNSGRA-NH2 H-KazNSGRA-NH2 H-GGKazNSGRA-NH2 H-GGGGGKazNSGRA-NH2 H-GGGGGGGGGGKazNSGRA-NH2


kapp (s-1) 1.4 ×10-3 1.4 ×10-4 5.0 ×10-4 1.1 ×10-3 7.5 ×10-4 1.1 ×10-3 1.3 ×10-3 7.8 ×10-4 7.0 ×10-4

Yields (%) 79 65 74 83 74 69 78 67 71


Figure 4. Synthesis and ligation of hydrophobic peptide P11. (A) HPLC trace (220 nm in the linear gradient with water/acetonitrile containing 0.1% TFA) and MALDI-TOF mass spectrum of purified P11. (B) HPLC trace (260 nm in


Page 22 of 31

Page 23 of 31 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

Biomacromolecules

the linear gradient with TEAA buffer/acetonitrile) and MALDI-TOF mass spectrum of purified P11-ODN1c. (C) HPLC traces (564 nm in the linear gradient with water/acetonitrile containing 0.1% TFA) for photolyzed ligation mixtures containing P1-ODN1 and P11-ODN1c. All of the HPLC traces and MALDI-TOF mass spectra were shown in blue and red, respectively.



Figure 5. Simultaneous ligation of three peptide fragments on two DNA scaffolds. (A) Chemical structure of Fmoc-Kal and synthetic scheme of azideand alkyne-bearing peptide, P12. (B) Synthetic scheme of ODN1c-P12-ODN2 through tandem click chemistry. (C) HPLC traces (260 nm in the linear gradient with TEAA buffer/acetonitrile) for the one-pot tandem SPAAC and subsequent CuAAC. (D) Reaction scheme of simultaneous three fragment ligation using three peptide-ODN conjugates, P1-ODN1, ODN1c-P12-ODN2, and P13-ODN2c. (E) HPLC traces (564 nm in the linear gradient with water/acetonitrile containing 0.1% TFA) for photolyzed ligation mixtures of


Page 24 of 31

Page 25 of 31 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

Biomacromolecules

simultaneous three fragments ligation and MALDI-TOF mass spectrum of P112-13*.



REFERENCES (1) Bode, J. W. Chemical Protein Synthesis with the α-Ketoacid-Hydroxylamine Ligation.

Acc. Chem. Res. 2017, 50, 2104–2115. (2) Bondalapati, S.; Jbara, M.; Brik, A. Expanding the chemical toolbox for the synthesis of large and uniquely modified proteins. Nat. Chem. 2016, 8, 407–418. (3) Dawson, P. E.; Kent S. B. H. Synthesis of native proteins by chemical ligation. Annu.

Rev. Biochem. 2000, 69, 923–960. (4) Hojo H. Recent progress in the chemical synthesis of proteins. Curr. Opin. Struct. Biol. 2014, 26, 16–23. (5) Malins L. R.; Payne R. J. Recent extensions to native chemical ligation for the chemical synthesis of peptides and proteins. Curr. Opin. Chem. Biol. 2014, 22, 70–78. (6) Olschewski, D.; Becker, C. F. W. Chemical synthesis and semisynthesis of membrane proteins. Mol. Biosyst. 2008, 4, 733–740. (7) Paradis-Bas, M.; Tulla-Puche, J.; Albericio, F. The road to the synthesis of "difficult peptides". Chem. Soc. Rev. 2016, 45, 631–654. (8) Zuo, C.; Tang, S.; Zheng, J. S. Chemical synthesis and biophysical applications of membrane proteins. J. Pept. Sci. 2015, 21, 540–549. (9) Clayton, D.; Shapovalov, G.; Maurer, J. A.; Dougherty, D. A.; Lester, H. A.; Kochendoerfer, G. G. Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis. Proc. Natl.

Acad. Sci. USA 2004, 101, 4764–4769. (10) Dittmann, M.; Sauermann, J.; Seidel, R.; Zimmermann W.; Engelhard, M. Native chemical ligation of hydrophobic peptides in organic solvents. J. Pept. Sci. 2010, 16, 558– 562. (11) Kochendoerfer, G. G.; Salom, D.; Lear, J. D.; Wilk-Orescan, R.; Kent, S. B. H.; DeGrado, W. F. Total chemical synthesis of the integral membrane protein influenza A virus M2: role of its C-terminal domain in tetramer assembly. Biochemistry 1999, 38, 11905–11913. (12) Hojo, H.; Tanaka, H.; Hagiwara, M.; Asahina, Y.; Ueki, A.; Katayama, H.; Nakahara, Y.; Yoneshige, A.; Matsuda, J.; Ito, Y.; Nakahara, Y. Chemoenzymatic synthesis of


Page 26 of 31

Page 27 of 31 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

Biomacromolecules

hydrophobic glycoprotein: synthesis of saposin C carrying complex-type carbohydrate. J. Org.

Chem. 2012, 77, 9437–9446. (13) Sohma, Y.; Sasaki, M.; Hayashi, Y.; Kimura, T.; Kiso, Y. Novel and efficient synthesis of difficult sequence-containing peptides through O-N intramolecular acyl migration reaction of O-acyl isopeptides. Chem. Commun. 2004, 124–125. (14) Bondalapati, S.; Eid, E.; Mali, S. M.; Wolberger, C.; Brik, A. Total chemical synthesis of SUMO-2-Lys63-linked diubiquitin hybrid chains assisted by removable solubilizing tags.

Chem. Sci. 2017, 8, 4027–4034. (15) Johnson, E. C. B.; Kent, S. B. H. Towards the total chemical synthesis of integral membrane proteins: a general method for the synthesis of hydrophobic peptide-thioester building blocks. Tetrahedron Lett. 2007, 48, 1795–1799. (16) Johnson, E. C. B.; Malito, E.; Shen, Y. Q.; Rich, D.; Tang, W. J.; Kent, S. B. H. Modular total chemical synthesis of a human immunodeficiency virus type 1 protease. J.

Am. Chem. Soc. 2007, 129, 11480–11490. (17) Sato, T.; Saito, Y.; Aimoto, S. Synthesis of the C-terminal region of opioid receptor like 1 in an SDS micelle by the native chemical ligation: effect of thiol additive and SDS concentration on ligation efficiency. J. Pept. Sci. 2005, 11, 410–416. (18) Hossain, M. A.; Belgi, A.; Lin, F.; Zhang, S. D.; Shabanpoor, F.; Chan, L.; Belyea, C.; Truong, H. T.; Blair, A. R.; Andrikopoulos, S.; Tregear, G. W.; Wade, J. D. Use of a temporary "solubilizing" peptide tag for the Fmoc solid-phase synthesis of human insulin glargine via use of regioselective disulfide bond formation. Bioconjugate Chem. 2009, 20, 1390–1396. (19) Huang, Y. C.; Li, Y. M.; Chen, Y.; Pan, M.; Li, Y. T.; Yu, L.; Guo, Q. X.; Liu, L. Synthesis of autophagosomal marker protein LC3-II under detergent-free conditions. Angew.

Chem., Int. Ed. 2013, 52, 4858–4862. (20) Jacobsen, M. T.; Petersen, M. E.; Ye, X.; Galibert, M.; Lorimer, G. H.; Aucagne, V.; Kay, M. S. A Helping Hand to Overcome Solubility Challenges in Chemical Protein Synthesis.

J. Am. Chem. Soc. 2016, 138, 11775–11782.



Page 28 of 31

(21) Maity, S. K.; Mann, G.; Jbara, M.; Laps, S.; Kamnesky, G.; Brik, A. Palladium-Assisted Removal of a Solubilizing Tag from a Cys Side Chain To Facilitate Peptide and Protein Synthesis. Org. Lett. 2016, 18, 3026–3029. (22) Tan, Z. P.; Shang, S. Y.; Danishefsky, S. J. Rational development of a strategy for modifying the aggregatibility of proteins. Proc. Natl. Acad. Sci. USA 2011, 108, 4297–4302. (23) Tsuda, S.; Mochizuki, M.; Ishiba, H.; Yoshizawa-Kumagaye, K.; Nishio, H.; Oishi, S.; Yoshiya,

T. Easy-to-Attach/Detach

Solubilizing-Tag-Aided

Chemical

Synthesis

of

an

Aggregative Capsid Protein. Angew. Chem., Int. Ed. 2018, 57, 2105–2109. (24) Zheng, J. S.; He, Y.; Zuo, C.; Cai, X. Y.; Tang, S.; Wang, Z. P. A.; Zhang, L. H.; Tian, C. L.; Liu, L. Robust Chemical Synthesis of Membrane Proteins through a General Method of Removable Backbone Modification. J. Am. Chem. Soc. 2016, 138, 3553–3561. (25) Behrendt, R.; White, P.; Offer, J. Advances in Fmoc solid-phase peptide synthesis. J.

Pept. Sci. 2016, 22, 4–27. (26) Raibaut, L.; Ollivier, N.; Melnyk, O. Sequential native peptide ligation strategies for total chemical protein synthesis. Chem. Soc. Rev. 2012, 41, 7001–7015. (27) Hong, F.; Zhang, F.; Liu Y.; Yan, H. DNA Origami: Scaffolds for Creating Higher Order Structures. Chem. Rev. 2017, 117, 12584–12640. (28) Michaelis, J.; Roloff, A.; Seitz, O. Amplification by nucleic acid-templated reactions.

Org. Biomol. Chem. 2014, 12, 2821–2833. (29) Li, X. Y.; Liu, D. R. DNA-templated organic synthesis: nature's strategy for controlling chemical reactivity applied to synthetic molecules. Angew. Chem., Int. Ed. 2004, 43, 4848– 4870. (30) Erben, A.; Grossmann, T. N.; Seitz, O. DNA-triggered synthesis and bioactivity of proapoptotic peptides. Angew. Chem., Int. Ed. 2011, 50, 2828–2832. (31) Ficht, S.; Mattes, A.; Seitz, O. Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates. J. Am. Chem. Soc. 2004, 126, 9970–9981. (32) Gartner, Z. J.; Kanan, M. W.; Liu, D. R. Expanding the reaction scope of DNAtemplated synthesis. Angew. Chem., Int. Ed. 2002, 41, 1796–1800. (33) Li, Y. Z.; Zhao, P.; Zhang, M. D.; Zhao, X. Y.; Li, X. Y. Multistep DNA-templated synthesis using a universal template. J. Am. Chem. Soc. 2013, 135, 17727–17730.


Page 29 of 31 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

Biomacromolecules

(34) Sayers, J.; Payne, R. J.; Winssinger, N. Peptide nucleic acid-templated selenocystineselenoester ligation enables rapid miRNA detection. Chem. Sci. 2018, 9, 896–903. (35) Bruick, R. K.; Dawson, P. E.; Kent, S. B.; Usman, N.; Joyce, G. F. Template-directed ligation of peptides to oligonucleotides. Chem. Biol. 1996, 3, 49–56. (36) Di Pisa, M.; Hauser, A.; Seitz, O. Maximizing Output in RNA-Programmed PeptidylTransfer Reactions. Chembiochem 2017, 18, 872–879. (37) Middel, S.; Panse, C. H.; Nawratil, S.; Diederichsen, U. Native Chemical Ligation Directed by Photocleavable Peptide Nucleic Acid (PNA) Templates. Chembiochem 2017, 18, 2328–2332. (38) Dommerholt, J.; Rutjes, F. P. J. T.; van Delft, F. L. Strain-Promoted 1,3-Dipolar Cycloaddition of Cycloalkynes and Organic Azides. Topics Curr. Chem. 2016, 374, 16. (39) McKay, S.; Finn, M. G. Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 2014, 21, 1075–1101. (40) Tang, W.; Becker, M. L. "Click" reactions: a versatile toolbox for the synthesis of peptide-conjugates. Chem. Soc. Rev. 2014, 43, 7013–7039. (41) Hansen, M. J.; Velema, W. A.; Lerch, M. M.; Szymanski, W.; Feringa, B. L. Wavelength-selective cleavage of photoprotecting groups: strategies and applications in dynamic systems. Chem. Soc. Rev. 2015, 44, 3358–3377. (42) Klan, P.; Solomek, T.; Bochet, C. G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy. Chem. Rev. 2013, 113, 119–191. (43) Mayer, G.; Heckel, A. Biologically active molecules with a "light switch". Angew.

Chem., Int. Ed. 2006, 45, 4900–4921. (44) Nguyen, T.; Francis, M. B. Practical synthetic route to functionalized rhodamine dyes.

Org. Lett. 2003, 5, 3245–3248. (45) Nakajima, N.; Ikada, Y. Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media. Bioconjugate Chem. 1995, 6, 123–130. (46) Chan, L. C.; Cox, B. G. Kinetics of amide formation through carbodiimide/Nhydroxybenzotriazole (HOBt) couplings. J. Org. Chem. 2007, 72, 8863–8869.



(47) Benoiton, N. L.; Lee, Y. C.; Chen, F. M. F. Identification and suppression of decomposition during carbodiimide-mediated reactions of Boc-amino acids with phenols, hydroxylamines and amino acid ester hydrochlorides. Int. J. Pept. Prot. Res. 1993, 41, 587– 594. (48) Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Analysis and optimization of coppercatalyzed azide-alkyne cycloaddition for bioconjugation. Angew. Chem., Int. Ed. 2009, 48, 9879–9883.


Page 30 of 31

Page 31 of 31 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

Biomacromolecules

TOC


Simultaneous and Traceless Ligation of Peptide Fragments on DNA

Recommend Documents