Incorporation of a Doubly Functionalized Synthetic Amino Acid into

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Incorporation of a doubly functionalized synthetic amino acid into proteins for creating chemical and light-induced conjugates Atsushi Yamaguchi, Takayoshi Matsuda, Kazumasa Ohtake, Tatsuo Yanagisawa, Shigeyuki Yokoyama, Yoshihisa Fujiwara, Takayoshi Watanabe, Takahiro Hohsaka, and Kensaku Sakamoto Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00602 • Publication Date (Web): 01 Dec 2015 Downloaded from http://pubs.acs.org on December 5, 2015

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Bioconjugate Chemistry

Incorporation of a doubly functionalized synthetic amino acid into proteins for creating chemical and light-induced conjugates Atsushi Yamaguchi†, ‡, ††, Takayoshi Matsuda†, ‡, Kazumasa Ohtake†, Tatsuo Yanagisawa§, Shigeyuki Yokoyama§, Yoshihisa Fujiwara⊥, Takayoshi Watanabe††, Takahiro Hohsaka*, ††, and Kensaku Sakamoto*, †, ‡ †

Division of Structural and Synthetic Biology and ‡Molecular Network Control Project,

RIKEN Center for Life Science Technologies, §RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, 7-7-15 Saitoasagi, Ibaraki, Osaka 567-0085, and

⊥Shinsei

Chemical Company Ltd.,

††

School of Materials Science, Japan

Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan. *

To whom correspondence and requests may be addressed. Telephone: +81-45-503-9459;

Fax: +81-45-503-9458; E-mail: [email protected] (K. S.), and Telephone: +81-761-51-1681; Fax: +81-761-51-1149; E-mail: [email protected] (T. H.)

1 ACS Paragon Plus Environment


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Abstract Z-lysine (ZLys) is a lysine derivative with a benzyloxycarbonyl group linked to the ɛ-nitrogen. It has been genetically encoded with the UAG stop codon, using the pair of an engineered variant of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl. In the present study, we designed a novel Z-lysine derivative (AmAzZLys), which is doubly functionalized with amino and azido substituents at the meta positions of the benzyl moiety, and demonstrated its applicability for creating protein conjugates. AmAzZLys was incorporated into proteins in Escherichia coli, by using the ZLys-specific PylRS variant. AmAzZLys was then site-specifically incorporated into a camelid single-domain antibody specific to the epidermal growth factor receptor (EGFR). A one-pot reaction demonstrated that the phenyl amine and azide were efficiently linked to the 5 kDa polyethylene glycol and a fluorescent probe, respectively, through specific bio-orthogonal chemistry. The antibody was then tested for the ability to form a photo-crosslink between its phenylazide moiety and the antigen, while the amino group on the same ring was used for chemical labeling. When incorporated at a selected position in the antibody and exposed to 365 nm light, AmAzZLys formed a covalent bond with the EGFR ectodomain, with the phenylamine moiety labeled fluorescently prior to the reaction. The present results illuminated the versatility of the ZLys scaffold, which can accommodate multiple reactive groups useful for protein conjugation. Introduction The incorporation of designer amino acids into proteins has facilitated the conjugation of proteins with drugs, chemical compounds, peptides, polyethylene glycol, and other protein molecules1―3. Such amino acids are functionalized with various chemical groups, such as α-keto, azido, and alkynyl groups, and then serve as a “chemical handle” for site-selective protein modification through bio-orthogonal chemistry. Their incorporation relies on the development of the specific pairs of aminoacyl-tRNA synthetase (aaRS) and tRNA variants, which translate particular base sequences into the amino acids. The UAG stop codon, as well as base quadruplets or “four-letter” codons4, has been used for the site-specific incorporation of non-natural amino acids into peptides and proteins. However, few


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synthetic amino acids possessing two or more reactive groups have been incorporated into proteins, despite their clear advantage in creating double conjugates of proteins. In contrast, the simultaneous incorporation of two or more amino acids with different bio-orthogonally reactive groups is laborious, and the yields of the protein products are small5. An ethynyl derivative of pyrrolysine was previously incorporated into proteins, by using the pair of the wild-type pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl in Escherichia coli6. This amino acid has an ethynyl substituent on the pyrrol ring as a reactive group, and the imine nitrogen of the ring can be used as a second site for conjugation. Thus, a protein with this amino acid could be linked with two different payloads. This report prompted us to seek a versatile scaffold of a synthetic amino acid that would be useful for creating double conjugates of proteins. PylRS, occurring in archaea and some bacteria, directly charges tRNAPyl with pyrrolysine, the 22nd amino acid, and thus incorporates it into proteins7. Its amino-acid binding pocket, which is large and lined predominantly with hydrophobic residues8―10, has been modified to recognize various types of non-natural amino acids11. We considered Nɛ-benzyloxycarbonyl-L-lysine (ZLys) (Scheme 1) to be suitable for our aim, because the benzyl moiety can be functionalized with more than one reactive group. A ZLys derivative with one reactive group, Nɛ-(o-azidobenzyloxycarbonyl)-L-lysine (oAzZLys), has been used to create protein conjugates12. In addition, a reactive group at the benzyl moiety can be linked with a long side chain, to maintain some distance from the protein surface and avoid steric hindrance. Photo-crosslinking between interacting proteins was thus facilitated, by utilizing a ZLys derivative with the benzyl moiety bearing a photo-reactive diazirinyl group13. ZLys and its derivatives can be incorporated into proteins by using the variant of Methanosarcina mazei PylRS, with Ala and Phe in place of Tyr at positions 306 and 384, respectively11―14, although no doubly functionalized derivative was previously available. In the present study, we designed a ZLys derivative with amino and azido groups in the benzyl moiety (AmAzZLys). The phenylazide undergoes either alkyne-azide cycloaddition or a photo-induced reaction, while the phenylamine can undergo reductive alkylation under particular conditions that prevent the reactions of other amino groups in the protein. The designed amino acid was incorporated into the variable domain of the camelid heavy-chain



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antibody or VHH antibody15. Various types of antibody fragments, including VHH, single-chain variable fragments (scFv), antigen-binding fragments (Fab), and diabodies, have been developed with hopes for their future medical and diagnostic applications16―19. An efficient conjugation method is pivotal, because these fragments are expected to be primarily used in the forms of conjugates with drugs, chemicals, peptides, and proteins. Scheme 1

Results and discussion Design of a doubly-functionalized ZLys derivative and its incorporation into a VHH antibody For the design of a bi-functional ZLys derivative, we considered the following two points. First, a previously reported ZLys derivative (oAzZLys) had an azido group attached to the ortho position of the benzyl moiety, and thus the reactive group was placed in the narrow space between the benzene ring and the aliphatic chain (Scheme 1). Secondly, since ZLys includes a benzyl carbamate structure, an electron-donating group attached to the para or ortho position of the benzyl moiety can break the amide bond by either 1, 6- or 1, 4-benzyl elimination20. Therefore, we attached the azido and amino groups to the meta positions of the benzyl moiety of ZLys, thus creating Nɛ-(3-amino-5-azidobenzyloxycarbonyl)-L-lysine (AmAzZLys) (Scheme 1). We also synthesized Nɛ-(m-azidobenzyloxycarbonyl)-L-lysine (mAzZLys), for comparison with oAzZLys.


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Two PylRS variants were examined for their ability to incorporate these ZLys derivatives into proteins. The first variant was M. mazei PylRS, possessing the aforementioned Tyr306Ala and Tyr384Phe substitutions, together with two auxiliary mutations (Arg61Lys and Gly131Glu)21. The other was M. barkeri PylRS with the Tyr271Ala substitution22, which corresponds to Tyr306Ala in the first variant. The ZLys derivatives were incorporated into the VHH antibody, 7D12, specific to the human epidermal growth factor receptor (EGFR)23, in place of Thr14, a residue located opposite from the antigen binding site (Figure 1A). The gene encoding the VHH(7D12) antibody had the amber codon at position 14, and a hexahistidine sequence at the C-terminus. The rich growth medium used for expression was supplemented with the ZLys derivatives, each at a final concentration of 1 mM. As shown in Figure 1B, both enzymes incorporated mAzZLys more efficiently than oAzZLys in the E. coli BL21-Gold(DE3) strain. The yields of VHH(mAzZLys) were nearly 50% of that of the wild-type VHH. By contrast, the yield of VHH(AmAzZLys) was significantly higher with the M. mazei PylRS variant than the M. barkeri PylRS variant, and the higher yield was almost equal to that of VHH(mAzZLys). By using the M. mazei variant, the purified VHH variants containing oAzZLys, mAzZLys, and AmAzZLys were obtained in yields of 26, 32, and 53 mg/l, respectively, while the yield of the wild-type antibody was 105 mg/l (Figure 1C). Although the yield of the VHH with AmAzZLys thus appeared to be a half of that of the wild-type VHH, we noticed that the existence of the vector pCDF, used for expressing PylRS and tRNAPyl, reduced the expression level of the wild-type molecule to the half (Supporting Figure S1). Therefore, the achieved yield of the variant was almost equal to that of the wild-type counterpart. The recognition of bulky AmAzZLys probably relies on the enlarged binding pocket of PylRS with the Tyr-to-Ala substitution. The reported variant of M. mazei PylRS with Tyr306Gly24 is probably similarly useful. The incorporation of mAzZLys and AmAzZLys was confirmed by mass spectrometry (MALDI-ToF MS). The trypsin-digested peptides (residues 4―19) containing mAzZLys and AmAzZLys were observed as peaks 2 and 3, respectively (Figure 1D). The observed masses of the peptides indicated that trypsin did not cleave these peptides on the C-terminal sides of the ZLys derivatives. In addition to peak 3, the peptide with lysine in place of



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AmAzZLys was observed, as peak 4. This peptide was probably produced by the degradation of AmAzZLys into lysine during the MS analysis, rather than the mis-incorporation of lysine into the peptide, because peptides with lysine are cleaved by trypsin. Taken together with the observation that the variant antibody was not synthesized in the absence of the ZLys derivatives (Figure 1B), the MS analyses unambiguously indicated that no other amino acid than the ZLys derivative was incorporated at position 14. To examine if the antigen binding ability of the VHH antibody was affected by the incorporation of the ZLys derivatives, the dissociation constants (KD) of the VHH variants containing oAzZLys, mAzZLys, and AmAzZLys with the EGFR ectodomain were determined by surface plasmon resonance measurements. The KD value of the wild-type antibody was 12.5 nM, which is similar to the reported values (10.4 and 25.7 nM) obtained by a cell-based assay25. All of the VHH variants also exhibited similar values to that of the wild-type molecule (Table 1), confirming that the ZLys derivatives were safely incorporated into the antibody without impairing the antigen binding.


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Figure 1. The incorporation of ZLys derivatives with phenylazide into a VHH antibody. (A) Part of the crystal structure of EGFR domain III bound to VHH(7D12) (PDB code: 4KRL). The threonine residue at position 14 of the antibody is depicted by balls and sticks (orange). (B) The yields of the VHH(7D12) variants containing the indicated amino acid at position 14 are shown, relative to the average yield of the wild-type VHH antibody (wt). “oA”, “mA”, and “AA” represent oAzZLys, mAzZLys, and AmAzZLys, respectively. “Mm” and “Mb” represent the M. mazei and M. barkeri PylRS variants, respectively. Error bars indicate standard deviations (n = 3). (C) The purified VHH antibodies with the indicated ZLys derivatives, obtained by using the M. mazei PylRS variant, were analyzed by SDS-PAGE. The yields were measured by a BCA assay. (D) The MS analyses of the trypsin-digested peptides of the VHH variants. The calculated and observed masses are tabulated in the table. The masses of mAzZLys and AmAzZLys were calculated, taking into account the fact that phenylazide degrades into phenylamine at a preparative step before the MS analysis and also during the analysis, due to the ultraviolet laser of the MALDI-ToF MS. Table 1. The binding kinetics of the VHH(7D12) variants with the ZLys derivatives at position 14 determined by surface plasmon resonance Kon (105 M-1s-1) a

Koff (10-4 s-1) a

KD (nM) a

wild-type

4.9 ± 0.4

60.9 ± 5.4

12.5 ± 0.2

oAzZLys

4.1 ± 0.1

57.3 ± 0.4

14.1 ± 0.3

mAzZLys

5.2 ± 0.5

67.1 ± 4.8

13.0 ± 0.3

AmAzZLys

4.7 ± 0.2

53.2 ± 3.0

11.5 ± 0.2

VHH(7D12)

a

The standard deviations were obtained from three independent measurements.

One-pot chemical reaction of the antibody variant containing AmAzZLys We first assessed the advantage of attaching an azido group to the meta position of the benzyl moiety of ZLys, in comparison with the ortho position. The efficiency of a labeling reaction using the strain-promoted alkyne-azide cycloaddition (SPAAC)26 was compared



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between p-azido-L-phenylalanine (AzPhe)27 (Scheme 1), oAzZLys12, mAzZLys, and AmAzZLys. The VHH variants with these amino acids at position 14 were mixed with a ten-fold molar excess of the dibenzocyclooctyne-functionalized Alexa Fluor 647 dye, and the products were separated by hydrophobic-interaction chromatography for quantification. The analyses indicated that mAzZLys and AmAzZLys reacted faster than oAzZLys and AzPhe, while all of them eventually generated similar levels of the labeled products after 24 hours (Figure 2 and Table 2). This finding showed that attaching an azido group to the meta position of the benzyl moiety of ZLys confers an advantage regarding the initial reaction rate, rather than the final amount of the labeled product.

Figure 2. Time course of labeling reactions of the azido-containing amino acids. The yields of the labeled products were calculated, based on the peak areas in HIC-HPLC. The standard deviations were obtained from three independent experiments. Table 2. Final levels of the labeled products of the VHH variants containing the indicated amino acids VHH(7D12) wild-type

Labeling rate (%)a 0±0

AzPhe

88.5 ± 1.3

oAzZLys

93.0 ± 1.1

mAzZLys

96.8 ± 1.6

AmAzZLys

90.9 ± 0.8

a



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Next, we tested the phenylamine of AmAzZLys for another conjugation site. Since aromatic amines have lower pKa values (~5) due to the delocalized lone pair of the nitrogen, the phenylamine of AmAzZLys is partially deprotonated and maintains nucleophilicity under weakly acidic conditions, whereas the amino group of lysine and the N-terminal amino group are mostly protonated, and thus hardly reactive under the same conditions. Therefore, the amino group of AmAzZLys reacts preferably. To examine the feasibility of our

double-reaction

scheme

(Figure

3A),

formaldehyde

and

azadibenzocyclooctyne-PEG4-tetramethylrhodamine (DBCO-TAMRA) were mixed with the antibody variant with AmAzZLys. Formaldehyde was added to the reaction first, for the reaction with the phenylamine in the presence of 2-picoline-borane at pH 4. The reaction-terminating agent for the reductive alkylation was then added to the reaction, together with the cyclooctyne-functionalized compound, which underwent SPAAC with the azido group. The wild-type and variant antibodies were analyzed by mass spectrometry, after the double reaction was performed. The trypsin digestion of the variant molecule generated the peptide containing AmAzZLys, which was di-methylated by formaldehyde at the amino group and also conjugated with TAMRA at the azido group (the calculated m/z value, [M+] = 2713.3; the observed m/z value, [M+] = 2713.7) (Figure 3B). Modified peptides were not detected for the wild-type molecule. This result showed that AmAzZLys can undergo two distinct reactions on the same benzene ring, and become doubly conjugated in a one-pot manner. We then replaced the formaldehyde with the aldehyde linked to 5 kDa polyethylene glycol (PEG5k) in the conjugation reaction. The antibody variant with AmAzZLys exhibited an upward shift on an SDS-PAGE gel, and emitted fluorescence (Figure 3C); the observed change in the molecular weight roughly corresponded to one molecule of PEG5k. The phenylamine thus reacted with one molecule of PEG5k, probably because the bulkiness of PEG5k prevented another molecule from reacting with the same amino group. The wild-type antibody did not show any changes after the reaction. The yield of the doubly modified antibody was estimated as 79.2% of the total amount of the antibody subjected to the reaction, based on the analytical cation-exchange chromatography profile.



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This efficiency, in spite of presence of two bulky compounds to react with the same benzene ring, confirmed that the incorporation of AmAzZLys is a useful approach for creating chemical conjugates of proteins with two different payloads.

Figure 3. One-pot synthesis of doubly modified VHH antibody. (A) Illustration of our approach for creating double conjugates in a one-pot reaction. (B) The MALDI-ToF MS analyses of the trypsin digests of the wild-type and variant antibodies after the conjugation reaction. (C) The wild-type and variant antibodies were analyzed after the double conjugation reaction by SDS-PAGE with Coomassie Brilliant Blue (CBB) staining (left) and fluorescence imaging (right). The excitation and emission wavelengths were 520 and 575 nm, respectively.


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Chemical and light-induced conjugation of the antibody variant containing AmAzZLys A phenylazide undergoes an ultraviolet light-induced reaction and forms a covalent bond with a chemical group in its vicinity. This reaction might be applied to create conjugates between interacting proteins. A notable advantage is that it does not require the incorporation of a particular non-natural chemical group into the other protein, as a UV-activated phenylazide can react with common nucleophilic group, such as an amino group28. The azido group of AmAzZLys might be used for photo-crosslinking, while the amino group is used for chemical conjugation. We first identified the ideal incorporation sites for AmAzZLys to achieve covalent bonding between the antibody variant and EGFR. Nine sites were selected, based on the crystal structure of the complex between the VHH antibody (7D12) and the EGFR domain III29 (PDB code: 4KRL). The Cα atoms of all of the selected residues are located within 15 Å from the nearest atoms of the receptor (Figure 4A). Ser7 and Thr14, which are far from the antigen binding site, were also selected for comparison. The photo-linked products between the antibody variant and the EGFR ectodomain were formed in vitro, when Ile93, Thr107, and Leu108 were replaced with AmAzZLys (Figure 4B). Interestingly, the crosslinkable sites identified for the azido-containing amino acids (AzPhe, mAzZLys, and oAzZLys) were different from those for AmAzZLys, and also varied between the first three amino acids. This finding indicated that the length of the amino acid, the position of the azido group, and the co-existence of another functional group on the benzene ring can affect the locations of the suitable crosslinking sites. As expected, all of the sites that were found to be crosslinkable were on the side of the antibody facing the receptor in the crystal structure. The affinities of the antibody variants with AmAzZLys at positions 93, 107, and 108 were determined by surface plasmon resonance measurements. Only the incorporation of AmAzZLys at position 107 reduced the affinity for the antigen (Table 3). We decided to use the position-108 variant for further experiments, since the variant gave the best yield of the crosslinked product and exhibited a better (tenfold higher) affinity for EGFR than the wild-type molecule. Before proceeding to photo-conjugation experiments, we conducted preliminary experiments to identify which



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feature of AmAzZLys at position 108 was responsible for the affinity improvement. The variants with ZLys, mAzZLys, and AmAzZLys at this position exhibited similar KD values, between 1 and 2 nM, which suggests that neither of the functional groups on the benzene ring were relevant. The original leucine residue at position 108 is located at the interface between the VHH antibody and the antigen, with its side chain extended toward the antigen (Figure 4A). ZLys, bulkier than Leu, possibly hampers the antibody-antigen interaction, but stabilizes their complex by filling a space between the molecules; this hypothesis apparently explains the slower Kon and even slower Koff of the variant.

Figure 4. UV-induced conjugation between the VHH variant and EGFR. (A) Part of the crystal structure of VHH(7D12) with EGFR. The residues replaced by azido-containing amino acids are depicted by orange sticks. The binding interface of EGFR is shown in green. (B) Western blotting analysis of the photo-linked products (XL), detected using the anti-His-tag antibody. The EGFR ectodomain was C-terminally tagged with a decahistidine sequence.


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Table 3. The yields and affinities for the antigen of VHH(7D12) variants yield (mg/l)

Kon (105 M-1s-1) a

Koff (10-4 s-1) a

KD (nM) a

93AmAzZLys

1.6

4.1 ± 0.8

37.7 ± 5.5

9.3 ± 0.7

107AmAzZLys

3.2

1.6 ± 0.3

64.1 ± 5.1

41.6 ± 4.4

108AmAzZLys

41.3

14.1 ± 1.1

23.1 ± 0.8

1.6 ± 0.1

VHH(7D12)

a


We then examined if AmAzZLys can serve simultaneously as a photo-crosslinker and a chemical reaction site. 5(6)-TAMRA-X-C7-aldehyde was conjugated with the phenylamine of AmAzZLys at position 108 of the antibody through reductive alkylation, and the labeled antibody was then exposed to UV light in the presence of EGFR (Figure 5A). The labeled antibody formed the crosslinked product almost as efficiently as the non-labeled molecule (left gel, Figure 5B), showing that the bulky TAMRA dye, linked via a spacer to the benzene ring, did not hamper the photo-reaction of the azido group on the same ring to form a covalent bridge with EGFR. The light-induced conjugate was clearly detected in the fluorescent image (right gel, Figure 5B). A fluorescent band with a similar size to that of the ectodomain of the receptor was also detected. This band probably appeared because, after the crosslinked product was formed, the benzyl carbamate linkage in the TAMRA-modified AmAzZLys residue was subsequently disrupted by UV-irradiation, thus separating the antibody from the receptor and leaving the fluorescent dye on the receptor. Since the benzyloxycarbonyl group is originally a leaving group to protect the ε-amino group of lysine, the degree of this undesirable separation of a photo-conjugate might be reduced by changing the carbamate linkage to other types, such as a urea linkage30. Further study will help to improve the yield of photo-conjugated products. In the present study, we showed that AmAzZLys is useful for linking a protein molecule with two different payloads at a defined site. The alkyne-azide cycloaddition and the reductive alkylation efficiently occurred on the same benzene ring, whereas the light-induced conjugation involving the azido group showed relatively low productivity. However, the photo-conjugation has an advantage in that the payload molecule, either a



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peptide/protein or a small ligand, does need not to possess any particular reactive group to be covalently linked with the carrier protein. ZLys has been functionalized with various chemical groups11―14, and our results showed that it provides a versatile scaffold for doubly functionalized amino acids. Nɛ-(3-Nitro-5-azidobenzyloxycarbonyl)-L-lysine (AzNiZLys) (Scheme 1), a ZLys derivative with the combination of nitro and azido substituents, can also be incorporated into proteins (Supporting Figure S2), although an analysis of its utility is beyond the scope of the present study. More combinations of functional groups might be appended to the ZLys scaffold, to create various bi-functional designer amino acids.

Figure 5. Chemical and light-induced conjugation of the antibody variant with AmAzZLys at position 108. (A) Illustration of the chemical and light-induced conjugation on the same benzene moiety of AmAzZLys. VHH and the EGFR domain III are colored blue and green, respectively. Position 108 is marked in orange. (B) SDS-PAGE analysis of


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the labeled and crosslinked complex. The gel was stained with CBB (left) and subjected to fluorescence imaging (right). The excitation and emission wavelengths were 520 and 575 nm, respectively.



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Experimental Procedures Non-natural amino acids AzPhe was purchased from Watanabe Chemical Industries (Japan). oAzZLys was from Shinsei Chemical Company. The syntheses of mAzZLys and AmAzZLys are described in the Supporting Information. Plasmids The kanamycin resistance gene in the vector pET26b(+) (Novagen) was replaced by the ampicillin resistance gene from pET21b(+) (Novagen), to generate the vector pET26b(AmpR). The VHH(7D12) gene with a C-terminal hexahistidine sequence was commercially synthesized, and cloned downstream of the pelB leader sequence of pET26b(AmpR), to create pET-VHH. Amber mutations were introduced into the VHH(7D12) gene with a PrimeSTAR mutagenesis kit (Takara Bio Inc.). To construct the expression vector for PylRS variants and tRNAPyl, the glnSˊ promoter and rrnC terminator were replaced with the T5/LacO promoter and the lambda t0 terminator, respectively, in the previously reported pCDF-derived plasmid, pCDF-Mm, to express the M. mazei PylRS variant with Tyr384Phe and tRNAPyl from M. mazei (MmtRNAPyl)10. The PylRS gene was further mutated to have Arg61Lys, Gly131Glu, and Tyr306Ala, one of the two copies of the MmtRNAPyl gene was removed, and the lpp promoter for expressing the tRNA gene was replaced with the lppP5 promoter32, to create pCDF-Mm2. To express the M. barkeri PylRS variant (MbPylRS) and tRNAPyl from M. barkeri (MbtRNAPyl), the genes encoding the corresponding M. mazei molecules were replaced with their M. barkeri counterparts in pCDF-Mm2, and then the Tyr271Ala mutation was introduced into the MbPylRS gene, to create pCDF-Mb. The DNA fragment bearing the AzPhe-specific tyrosyl-tRNA synthetase variant and the amber suppressor tRNATyr, both from Methanocaldococcus jannaschii and under the control of the glnSˊ and lpp promoters, respectively, from the previously reported vector (pAzFA133), was cloned downstream of the lacI gene of a kanamycin-resistant vector derived from the vector pCDF-1b (Novagen), by replacing the streptomycin resistance gene with the kanamycin resistance gene. The resulting plasmid was designated as pCDF-Az.


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Synthesis of VHH(7D12) variants E. coli BL21-Gold(DE3) cells (Agilent Technologies) were transformed with the pET-VHH plasmid and its derivatives with amber mutations in the VHH gene, together with pCDF-Mm2, pCDF-Mb, or pCDF-Az. The transformed cells were cultured in 5 ml of 2×YT auto-induction medium, containing the indicated synthetic amino acid at a final concentration of 1 mM, at 30ºC for 24 h. The compositions of the supplements for the auto-induction medium were based on Studier’s report34. The cells were harvested by centrifugation, and then lysed using the Bugbuster Master Mix reagent (Merck Millipore). The supernatant of the centrifuged lysate was mixed with cOmplete His-Tag Purification resin (Roche), which was then washed in 50 mM HEPES-K buffer (pH 7.8), and the antibody was eluted in the buffer containing 100 mM imidazole. The concentration of the antibody in the obtained solution was estimated by measuring the optical density at 280 nm. For larger scale preparation, the transformed cells were cultured in 20 ml of the 2×YT auto-induction medium. The supernatant of the cell lysate was mixed with Ni-Sepharose 6 Fast Flow resin (GE Healthcare), which was washed with 50 mM HEPES-K buffer (pH 7.8), and eluted with the buffer containing 500 mM imidazole. The buffer of the obtained solution was changed to 50 mM MES buffer (pH 5.6) with a Zeba Spin Desalting column (7K MWCO, 2 ml) (Thermo Scientific), and then the antibody was further purified using a HiTrap SP HP column (GE Healthcare). The column retaining the protein was washed with the MES buffer, and then subjected to elution in the MES buffer containing 0.2 to 0.5 M NaCl. The fractions corresponding to 0.3 M and 0.4 M NaCl were collected and desalted against HBS buffer [20 mM HEPES-Na (pH 7.3) 150 mM NaCl] with an Amicon Ultra-4 3K filter unit (Merck Millipore). The antibody molecules thus purified were fractionated by SDS-PAGE and stained with the SimplyBlue SafeStain reagent (Life Technologies). The concentration of the antibody in the solution was determined using the BCA protein assay Kit (Reducing Agent Compatible) (Thermo Scientific). Purification of EGFR ectodomain The EGFR ectodomain (amino acids 1―643), with a C-terminal TEV protease cleavage



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site, a FLAG-tag, and a His10-tag, was purified from the culture supernatant of HEK293GnT1 cells overproducing it. The molecule was obtained from the supernatant by liquid chromatography on a HisTrap HP column (GE Healthcare), using an ÄKTA prime chromatography system (GE Healthcare), with an imidazole concentration gradient from 20 to 500 mM in 20 mM Tris-HCl (pH 7.5) buffer, containing 500 mM NaCl. The purified EGFR ectodomain was dialyzed against the Tris buffer containing 500 mM NaCl and 20 mM imidazole, and then subjected to 2 rounds of digestion by TEV protease (20 µg/ml) at 4ºC for 17 h, to cleave the C-terminal tags. The cleaved tags were removed by applying the digest to a HisTrap HP column, using the ÄKTA prime system. The flow-through fraction was collected and then concentrated using an Amicon Ultra (10K) filter unit. To remove the sugar chain, the EGFR ectodomain was treated with 60 kU of endoglycosidase H (New England Biolabs) at 37ºC for 5 h, and then with an additional 30 kU of the enzyme for 2 h. After dialysis against 20 mM Tris-HCl (pH 8.5) buffer containing 10 mM NaCl, the EGFR ectodomain was purified by anion exchange chromatography on the ÄKTA prime system with a HiTrap Q column (GE Healthcare), using 20 mM Tris-HCl (pH 8.5) buffer containing 10 mM NaCl (buffer A) and 20 mM Tris-HCl (pH 8.5) buffer containing 500 mM NaCl (buffer B), with a gradient from 0 to 25% B. The fractions containing the eluted EGFR ectodomain were collected and purified by size exclusion chromatography on a Hiload 16/600 Superdex 200 column (GE Healthcare), using an ÄKTA crystal chromatography system (GE Healthcare) with 20 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl. Mass spectrometry The disulfide bond in the antibody was cleaved by reduction in 10 mM dithiothreitol (DTT) at 95ºC for 5 min, and then carbamidomethylated by 10 mM iodoacetamide/50 mM ammonium bicarbonate at 25ºC for 20 min. A 10 µg portion of the sample was digested by 200 ng of Trypsin/Lys-C Mix, Mass Spec Grade (Promega), at 37ºC overnight. The digested sample was desalted using a SPE C-tip (C18) (Nikkyo Technos, Japan), and eluted with an 80% acetonitrile solution containing 0.1% trifluoroacetic acid (TFA). After evaporating the acetonitrile, the sample solution was mixed with equal volumes of an


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aqueous solution of 0.1% TFA and a 1:1 mixture of the α-cyano-4-hydroxycinnamic acid (CHCA) and 2, 5-dihydroxybenzoic acid (DHB) matrix solutions, and then spotted onto the CHCA-saturated acetone thin layer of a MALDI target plate. Mass spectrometry was performed in the reflector positive ion mode, using an AB SCIEX TOF/TOF5800 system. Surface plasmon resonance measurements The Kon, Koff, and KD values of the VHH(7D12) variants for the EGFR ectodomain were determined at 25ºC, using a Biacore3000 instrument (GE Healthcare). HBS-P buffer [10 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Surfactant P20] (GE Healthcare) was used as the running buffer. The EGFR ectodomain (5 µg/ml) in acetate buffer (pH 5.5) was immobilized to a surface density of approximately 3,000 resonance units on a sensor chip CM5 (GE Healthcare), through amine coupling. The VHH(7D12) variants were diluted to 6.25, 12.5, 25, 50, and 100 nM in HBS-P buffer. The diluted samples were analyzed by the single cycle kinetics method. A 30 µl portion of each diluent was sequentially injected, at 30 µl/min. The VHH/EGFR complex was dissociated for 15 sec between the injections of each diluent. The final dissociation was performed for 2 min. To regenerate the sensor chip between the measurements of different samples, 10 mM citrate buffer (pH 4) including 1 M NaCl was used. The obtained sensorgrams were fitted to the single cycle kinetics 1:1 binding model, using the BIAevaluation software (GE Healthcare). Determining the efficiency of labeling VHH(7D12) by SPAAC The antibody with an azido group (10 µM) was incubated with the Click-iT Alexa Fluor 647 DIBO Alkyne dye (Life Technologies) (100 µM), in HBS buffer containing 1% Tween 20, at RT for up to 24 h. The labeling reaction was terminated by adding 0.5% sodium azide to the reaction at the indicated times. The labeling reaction mixture (50 µl) was diluted by buffer A [20 mM phosphate buffer (pH 7.0), 2 M ammonium sulfate] and injected into a sample loop (20 µl), and the labeled antibody was separated by hydrophobic interaction chromatography on a TSKgel Butyl-NPR column (4.6 mm I. D. × 10 cm) (Tosoh, Japan), using a LaChrom HPLC system (Hitachi High-Technologies, Japan) at a flow rate of 0.8 ml/min at RT, with a gradient of buffer B [a 4:1 mixture of 20 mM phosphate buffer (pH



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7.0) and 2-propanol] from 0 to 80% in the mixture of buffers A and B in 9 min. The fluorescence of the Trp in the antibody was detected at 340 nm, with excitation at 295 nm. The Alexa Fluor 647 dye was detected by measuring the absorption at 600 nm. The labeling efficiency was calculated by the area normalization method. One-pot double chemical conjugation of VHH(7D12) The wild-type or AmAzZLys-containing VHH (20 µM) was incubated with 40 µM of either formaldehyde or methoxy-PEG-(CH2)2-CHO (NOF Corp., Japan) and 2-picoline-borane complex (Sigma-Aldrich) (1.5 mM), in 50 mM sodium citrate buffer (pH 4.0) at 4°C for 18 h. Then, 4-amino-L-phenylalanine (Watanabe Chemical Industries, Japan) (1 mM) was added to stop the alkylation on the antibody, and simultaneously, DBCO-PEG4-TAMRA (Sigma-Aldrich) (200 µM) was added for the second conjugation, and the incubation was continued for an additional 15 h. After the incubation, the reaction mixture was desalted using a Zeba Spin Desalting column (7K MWCO) (vol. 0.5 ml). The antibody incubated with formaldehyde and DBCO-TAMRA was digested by the Trypsin/Lys-C Mix, for analysis by MALDI-ToF MS. The antibody incubated with PEG and DBCO-TAMRA was analyzed by SDS-PAGE, followed by CBB staining with the SimplyBlue SafeStain reagent or fluorescence detection at 575 nm with excitation at 520 nm, using an ImageQuant LAS4000 spectrophotometer (GE Healthcare). The efficiency of the double conjugation was determined by separating the conjugate by cation exchange chromatography on a MAbPac SCX-10 column (4×250 mm) (Thermo Scientific), using the LaChrom HPLC system with buffer A [50 mM MES buffer (pH 5.6), 50 mM NaCl, 0.01 % Tween 20] as the initial buffer. The separation was performed at a flow rate of 0.8 ml/min at RT, with a gradient of the proportion of buffer B [50 mM MES buffer (pH 5.6), 500 mM NaCl, 0.01 % Tween 20] from 0 to 25 % in the mixture of buffer A and B during 60 min, and then 100 % B for 10 min. The reaction mixture (30 µl) was mixed with buffer A and injected into the sample loop (20 µl). The protein moiety of the conjugate was detected by the fluorescence of Trp at 350 nm, with excitation at 295 nm. The fluorescence of TAMRA was detected at 575 nm, with excitation at 545 nm. The modification efficiency was calculated by the area normalization method.


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Photo-conjugation between VHH(7D12) and EGFR To identify the crosslinkable sites, an antibody variant (1 µM) was incubated with the His10-tagged EGFR ectodomain (40 nM) at RT for 10 min, in HBS buffer. The mixture was then exposed to ultraviolet A light at 365 nm for 30 min, while the control sample was kept in darkness for 30 min. These samples were separated by a NuPAGE electrophoresis system (Life Technologies) and detected by western blotting, using the Penta His HRP Conjugate (QIAGEN). To label the antibody with AmAzZLys at position 108, the molecule (20 µM) was incubated with 5(6)-TAMRA-X-C7-aldehyde (20 µM) and 2-picoline-borane complex (1 mM), in 50 mM sodium citrate buffer (pH 4.0) at 4ºC for 18 h. The solvent of the reaction mixture was changed to HBS containing 0.01% Tween 20, using an Amicon Ultra-4 3K filter unit. For subsequent crosslinking, the antibody (5 µM) was incubated with the EGFR ectodomain (1 µM) at RT for 10 min in HBS buffer, and then exposed to ultraviolet A light at 365 nm for 15 min, while the control samples were kept in darkness for 15 min. The antibody was then analyzed by the NuPAGE electrophoresis system, followed by CBB staining or fluorescence detection with an ImageQuant LAS4000 system.

ACKNOWLEDGMENTS We thank Ms. M. Ikeda and Dr. M. Shirouzu (RIKEN) for kindly providing the cells overexpressing the EGFR ectodomain. This work was supported in part by Grants-in-Aid for Scientific Research (B) (26291035) to K. S., Scientific Research on Innovative Areas (25102006) and Challenging Exploratory Research (15K13739) to T. H., and Young Scientists (B) (15K16561) to T. W., from the Japan Society for the Promotion of Science (JSPS). ASSOCIATED CONTENT Supporting Information Experimental procedures for the syntheses of mAzZLys, AmAzZLys, and AzNiZLys; Data for the expression of wild-type VHH(7D12) in the presence of an additional vector; Data



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for the incorporation of AzNiZLys into a protein. AUTHOR INFORMATION Corresponding Authors *Tel: +81-45-503-9459; Fax: +81-45-503-9458; E-mail: [email protected] *Tel: +81-761-51-1681; Fax: +81-761-51-1149; E-mail: [email protected] Notes The authors declare no competing financial interest. REFERENCES (1) Kim, C. H., Axup, J. Y., and Schultz, P. G. (2013) Protein conjugation with genetically encoded unnatural amino acids. Curr. Opin. Chem. Biol. 17, 412–419. (2) Lang, K., and Chin, J. W. (2014) Cellular incorporation of unnatural amino acids and bioorthogonal labeling of proteins. Chem. Rev. 114, 4764–4806. (3) Lang, K., and Chin, J. W. (2014) Bioorthogonal reactions for labeling proteins. ACS Chem. Biol. 9, 16–20. (4) Wals, K., and Ovaa, H. (2014) Unnatural amino acid incorporation in E. coli: current and future applications in the design of therapeutic proteins. Front. Chem. 2, 15. (5) Nikić, I., and Lemke, E. A. (2015) Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Curr. Opin. Chem. Biol. 28, 164–173. (6) Ehrlich, M., Gattner, M. J., Viverge, B., Bretzler, J., Eisen, D., Stadlmeier, M., Vrabel, M., and Carell, T. (2015) Orchestrating the biosynthesis of an unnatural pyrrolysine amino acid for its direct incorporation into proteins inside living cells. Chem. Eur. J. 21, 7701–7704. (7) Jiang, R., and Krzycki, J. A. (2011) Functional context, biosynthesis, and genetic encoding of pyrrolysine. Curr. Opin. Microbiol. 14, 342–349. 22 ACS Paragon Plus Environment

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Incorporation of a Doubly Functionalized Synthetic Amino Acid into

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