(12) United States Patent (10) Patent No.: US 9,670,521 B2 Grabstein Et Al

US0096.70521B2

(12) United States Patent (10) Patent No.: US 9,670,521 B2 Grabstein et al. (45) Date of Patent: Jun. 6, 2017

(54) AMINO ACID DERVATIVES 271/22 (2013.01); C07K 14/43595 (2013.01); C07K 16/244 (2013.01); C07K 16/248 (71) Applicant: MEDIMMUNE LIMITED, Cambridge (2013.01); C07K 16/2863 (2013.01); C07K (GB) 16/32 (2013.01); C07K 16/40 (2013.01); C07K 16/46 (2013.01); C07K 17/08 (2013.01): CI2N (72) Inventors: Kenneth H. Grabstein, Mercer Island, 9/93 (2013.01); C12N 15/85 (2013.01); C07K WA (US); Michael Van Brunt, 2317/14 (2013.01); C07K 231 7/24 (2013.01); Covington, WA (US); Marcello C07K 23.17/31 (2013.01); C07K 2317/40 Marelli, Seattle, WA (US) (2013.01); C07K 2317/51 (2013.01); C12Y 601/01025 (2013.01) (73) Assignee: MEDIMMUNE LIMITED, Cambridge (58) Field of Classification Search (GB) CPC. C07C 233/49; C07C 233/56; C07C 235/74; C07C 247/04; C07C 271/12: C07C (*) Notice: Subject to any disclaimer, the term of this 27 1/20; C07C 271/22 patent is extended or adjusted under 35 See application file for complete search history. U.S.C. 154(b) by 17 days. (56) References Cited (21) Appl. No.: 14/430,412 U.S. PATENT DOCUMENTS PCT Fed: Sep. 24, 2013 3,711,458 A 1/1973 Olofson (22) 4,512,979 A 4, 1985 Patchett 5,216,023 A 6/1993 Literati et al. (86) PCT No.: PCT/EP2013/069888 8, 168,407 B2 5/2012 Yokoyama et al. S 371 (c)(1), 2010/0304431 A1 12/2010 Yokoyama et al. (2) Date: Mar. 23, 2015 FOREIGN PATENT DOCUMENTS

(87) PCT Pub. No.: WO2O14/044873 EP 1911840 A1 4/2008 GB 2470770 A 8, 2010 PCT Pub. Date: Mar. 27, 2014 WO 200404 1752 A2 5, 2004 WO 2011044255 * 4, 2011 (65) Prior Publication Data WO 20110442.55 A1 4, 2011 WO 2011087810 A1 T/2011 US 2015/025 1994 A1 Sep. 10, 2015 WO 2012032181 A2 3, 2012 WO 2012O387O6 A1 3, 2012 WO 2014/036492 A1 3, 2014 Related U.S. Application Data (60) Provisional application No. 61/862.495, filed on Aug. OTHER PUBLICATIONS 5, 2013, provisional application No. 61/705,116, filed AXup et al., “Synthesis of site-specific antibody-drug conjugates on Sep. 24, 2012. using unnatural amino acids”. P.N.A.S. 109(40), (2012) 16101 16106. (51) Int. C. Dose et al., “Single nucleotide specific detection of DNA by native C07C 233/49 (2006.01) chemical ligation of fluorescence labelled PNA-probes'. Bioorg. CD7C 233/56 (2006.01) Med. Chem., 16, (2008) 65-77. Hancock et al., “Expanding the Genetic Code of Yeast for Incor C07C 235/74 (2006.01) poration of Diverse Unnatural Amino Acids via a Pyrrolysyl-tRNA CD7C 247/04 (2006.01) Synthetase/tRNA Pair”. J. Am. Chern. Soc., 132, (2010), 14819 C07C 27L/12 (2006.01) 14824. C07C 27L/20 (2006.01) Mukai et al., “Addling L-lysine derivatives to the genetic code of C07C 27L/22 (2006.01) mammalian cells with engineered pyrrolysyl-tRNA synthetases'. C07K 6/24 (2006.01) Biochemical and Biophysical Research Communications, 371 C07K 6/32 (2006.01) (2008), 818-822. C07K I6/46 (2006.01) Vrabel et al., “Optimization of the posttranslational click modifi C07K 7/08 (2006.01) cation of proteins”. Collect. Czech. Chern. Commun. 76 (9), (2011) C07K 6/40 (2006.01) 1089-1101. CI2N 9/00 (2006.01) (Continued) CI2N 15/85 (2006.01) CI2P 2 1/02 (2006.01) Primary Examiner — Paul A Zucker A6 IK 47/48 (2006.01) Assistant Examiner — Mark Luderer C07K I4/435 (2006.01) (74) Attorney, Agent, or Firm — Glance Law Group: C07K 6/28 (2006.01) Melissa Pytel (52) U.S. C. (57) ABSTRACT CPC ...... CI2P21/02 (2013.01); A61K 47/48.569 There are provided amino acid derivatives of formula Vand (2013.01); C07C 233/49 (2013.01); C07C VI as defined herein which are pyrrolysine analogs for use 233/56 (2013.01); C07C 235/74 (2013.01); in bioconjugation processes. C07C 247/04 (2013.01); C07C 27.1/12 (2013.01); C07C 271/20 (2013.01); C07C 13 Claims, 2 Drawing Sheets US 9,670.521 B2 Page 2

(56) References Cited Nishino et al., “Tandem enzymatic relsolution yielding L-alpha aminoalkanedioic acid omega-esters'. Chem. Pharm. Bull., 44(1), 212-214 (1996). OTHER PUBLICATIONS Noda et al., “Modified benzyloxycarbonyl groups for protection of epsilon-amino group of lysine'. Bull. Chem. Soc. Japan, 43(6), Yasobu et al., “Design, Synthesis and Antitumor Activity of 1883-1885 (1970). Plass et al., “Genetically encoded copper-free click chemistry”. 4-Halocolchicines and their Pro-drugs Activated by Cathepsin B”. Angewandte Chemie, Int Edition, 50(17), 3878-3881 (2011). ACS Med. Chem. Lett. 2, (2011) 348-352. Popovitz-Biro et al., “A new series of amphiphilic molecules form Coward et al., “Analogs of S-adenosylhomocysteine as potential ing stable Z-type (polar) Langmuir-Blodgett films”. 112(7), 2498 inhibitors of biological transmethylation. Synthesis and biological 2506 (1990). activity of homocysteine derivatives bridged to adenine'. J. Med. Spanton et al. “Chemical defence and self-defence: Biochemical Chem., 15(4), 381-384 (1972). transformations of contact insecticides produced by Soldier ter Erickson et al. “Use of chlorinated benzyloxycarbonyl protecting mites”, Tetrahedron, 38(13), 1921-1930 (1982). groups to eliminate N. epsilon-branching at lysine during Solid Theodoropoulos, “Synthesis of epsilon-peptides of lysine”. J. Org. phase peptide synthesis”, J.A.C.S., 95(11), 3757-3763 (1973). Chem. 23(1), 140, (1958). Jermyn “Carbobenzoxy derivatives of S-aminoalkyl-L-cysteines”. Xin Li et al., “N6-(2-(R)-propargylglycyl)lysine as a clickable Australian Journal of Chemistry, 19(10), 1999-2000 (1966). pyrrolysine mimic', Chemistry—An Asian Journal, 5(8), 1765 Jie Li et al: "Ligand-free palladium-mediated site-specific protein 1769 (2010). labelling inside gram-negative bacterial pathogens'. J.A.C.S., Zhang et al., “Mechanism of inactivation of neuronal nitric oxide 135(19), 7330-7338, (2013). synthase by Nomega-allyl-L-arginine”. J.A.C.S., 119 (45), 10888 Kimbonguila et al., “Allylic protection of thiols and cysteine: I: The 10902 (1997). allyloxycarbonylaminomethyl group”. Tetrahedron, 55(22), 6931 Sun et al., “Vitamin K epoxide reductase significantly improves 6944 (1999). carboxylation in a cell line overexpressing factor X. Blood, Ledger et al., “The use of sequestering agents in the preparation of 106(12):3811-3813 (2005). epsilon-acyl-L-lysine and delta acyl-L-ornithine derivatives'. International Search Report mailed Nov. 14, 2013, in PCT/EP2013/ Australian Journal of Chemistry, 18(6), 933-935, (1965). 069887 (5 pages). Lindley “The preparation of compounds related to S-2-aminoethyl International Search Report mailed Jan. 3, 2014, in PCT/EP2013/ L-cysteine” Australian Journal of Chemistry 12(2), 296-298, 069888 (4 pages). (1959). International Search Report mailed Jul. 4, 2015, in PCT/IB2014/ Matsui, "Studies on acylase activity and microorganisms. XXIV. 002505 (8 pages). Properties of delta-ornithine acylase: 5-N-acylomithine amidohydrolase”, Chem. Pharm. Bull., 15(10), 1586-1596 (1967). * cited by examiner U.S. Patent Jun. 6, 2017 Sheet 1 of 2 US 9,670,521 B2

Figure 1.

PEGylated Heavy Chain

heavy Chain

Light Chain

g U.S. Patent Jun. 6, 2017 Sheet 2 of 2 US 9,670,521 B2

Figure 2. US 9,670,521 B2 1. 2 AMNO ACID DERVATIVES studies, see e.g. Yanagisawa et al., focused on mutations within the PyIRS enzyme in order to accommodate analogs CROSS REFERENCE TO RELATED in which the N6 substituent were an aromatic ring within the APPLICATIONS binding pocket pyrrolysine. Others, for instance Nguyen et al (also in WO2010/139948), and Lietal (also in WO2011/ This application is a national stage application under 35 044255) focused on identification of pyrrolysine analogs U.S.C. S371 (c) of PCT Application No. PCT/EP2013/ which do not carry a bulky N6 substituent, with the result of 069888, entitled “AMINO ACID DERIVATIVES, filed on obtaining simpler analogs which would be simple to Syn Sep. 24, 2013, which claims priority to U.S. Provisional thesize and interact with native pylRS/tRNApyl pairs. Fur Patent Application Nos. 61/705,116, filed Sep. 24, 2012 and 10 thermore, Chin et al developed two analogs with terminal 61/862.495, filed Aug. 5, 2013, the entire disclosures of alkyne and azide groups, amenable to use for protein label which are incorporated by reference herein in their entirety. ing via copper catalyzed click chemistry (CUAAC). REFERENCE TO SEQUENCE LISTING There remains a need to develop further pyrrolysine SUBMITTED ELECTRONICALLY 15 analogs. Whilst pyrrolysine analogs made thus far have been restricted to those evolved from a lysine backbone, the The content of the electronically submitted sequence present inventors have generated pyrrolysine analogs suc listing in ASCII text file entitled Sequence Listing..txt and cessfully incorporated into proteins with native pylRS/ having a size of 38 kilobytes filed with the application is tRNApyl pairs starting from a variety of amino acid struc incorporated herein by reference in its entirety. tures. INTRODUCTION SUMMARY OF THE INVENTION Pyrrolysine is a natural amino acid, the only one that is According to the invention there are provided pyrrolysine authentically specified by an amber codon. It uses a 21st 25 analogues of formulae V and VII as described herein. aminoacyl-tRNA synthetase (PylRS), naturally evolved to There is also provided a mutant protein containing as be orthogonal to all other amino acids and tRNAs. Blight et non-natural amino acid one or more (e.g. one) pyrrolysine Al., 2004 showed that PyIRS and its counterpart tRNA analogues of formulae V and VII as described herein. (tRNApyl) can incorporate pyrrolysine at amber codons in There is also provided an antibody containing as non E. coli. They also showed that the wt PyIRS is naturally 30 natural amino acid one or more (e.g. one) pyrrolysine promiscuous and can incorporate analogs of lysine. analogues of formulae V and VII as described herein in each Yokoyama et al (EP1911840) demonstrated that the heavy and/or light chain. PyIRS/tRNApyl system is orthogonal in eukaryotic cells There is also provided a mutant protein or antibody as and showed the incorporation of several non natural amino aforesaid which is conjugated via the one or more (e.g. one) acids (nnAAS) into a target proteins encoded by amber 35 non-natural amino acids to one or more (e.g. one) moieties codons in bacterial cells. These authors also identified key selected from proteins, cytotoxic agents, drugs and poly amino acid residues in pylRS that form the amino acid CS. binding pocket and function in selecting pyrrolysine over There is also provided use of a pyrrolysine analogue as other canonical amino acids. Mutations at this site generated aforesaid in the manufacture of a mutant protein e.g. anti mutants able the recognize and aminoacylate the tRNApyl 40 body containing one or more non-natural amino acids. with AZZ-lys (Yanagisawa 2008). Amino acid analogs described in the present invention are This orthogonality extends to bacteria and eukaryotic new and useful and have the merit of being straightforward cells. to prepare, in being readily incorporated into proteins (typi PyIRS is a naturally promiscuous synthetase that has cally without loss of bioactivity when used appropriately) naturally evolved to exclude lysine, but will incorporate 45 and in providing useful means for bioconjugation. lysine analogs without mutation, including azides, alkynes and alkenes, (Yanagisawa et al., 2008; Neumann et al. 2008: BRIEF DESCRIPTION OF THE FIGURES Mukai et al., 2008; Nguyen et al., 2009). The basis of this specificity is dependent on hydrophobic interactions FIG. 1. PEGylation of azide containing monoclonal anti between amino acid residues of the pylRS binding pocket 50 bodies. Lane 1: Untreated Antibody, Lane 2: Antibody with with the pyrrole ring of pyrrolysine that stabilizes and pyrrolysine analog Formula V.1 incorporated into heavy correctly positions the amino acid in the active site of the chain and subjected to PEGylation conditions; Lane 3: synthetase (Kavran et al., 2007). This RS/tRNA pair has Antibody with pyrrolysine analog Formula VI.1 incorpo been introduced via transient transfection into bacterial, rated into heavy chain and subjected to PEGylation condi yeast and mammalian cells and shown to be effective for 55 tions. incorporation of a number of non-natural amino acids into FIG. 2. HIC chromatogram of a 4D5-Auristatin F anti target proteins. body drug conjugate with the antibody originally containing For instance, EP 1911840 demonstrates incorporation of the pyrrolysine analog Formula VI.1, incorporated into the N-e-boc-Lysine into a target protein in E. coli cells. heavy chain. Pyrrolysine analogs, defined as amino acid derivatives 60 recognized by either native or genetically evolved PylRS BRIEF DESCRIPTION OF THE SEQUENCES OF and incorporated into proteins at amber codon sites, have THE SEQUENCE LISTING been disclosed in the past few years and reviewed, for instance, by Feckner et. al (Fekner, Li, & Chan, 2010) and SEQ ID No. 1: PyIRS Methanosarcina mazei WI nucleo Liu et al. Analogs bearing functional groups or post trans 65 tide sequence lational modifications have been site-specifically incorpo SEQ ID No 2: PyIRS Methanosarcina mazei WT amino rated into proteins using pylRS-tRNApyl systems. Several acid sequence US 9,670,521 B2 3 4 SEQ ID No. 3: PyIRS Methanosarcina mazei, Y384F typically be 5 to 10. Exemplary groups include cyclopente mutant nucleotide sequence nyl, 3-methyl-cyclopropenyl and cyclohexenyl. SEQ ID No 4: PyIRS Methanosarcina mazei, Y384F The term "heterocyclyl refers to a cycloalkyl or mutant amino acid sequence cycloalkenyl moiety in which the ring contains one or more SEQ ID No 5: tRNApyl Methanosarcina mazei (e.g. one, two or three, such as one or two, especially one) SEQ ID No 6: U6 snRNA Promoter heteroatom selected from O, N and S. Examples include SEQ ID No 7: U6-tRNApyl construct aZetidine, pyrrolidine, piperidine, piperazine, N-methylpip SEQ ID No 8: GFP nucleotide sequence erazine, morpholine and thiomorpholine. SEQ ID No 9: GFP amino add sequence The term “aryl” refers to an aromatic ring structure that SEQ ID No 10: GFPY40 nucleotide sequence 10 can be part of a linkage or part of a Substituent. Aryl moieties SEQ ID No 11: GFPY40 amino add sequence may contain one ring (e.g. phenyl) or two rings (e.g. SEQ ID No 12: anti-Her2 (4D5) gamma nucleotide naphthyl). Aryl groups may be substituted e.g. by one or Sequence more (e.g. one or two, such as one) Substituent selected from SEQ ID No 13: anti-Her2 (4D5) gamma amino acid alkyl, alkenyl, alkynyl, fluoroalkyl, halogen, alkoxy, nitro Sequence 15 and cyano. An exemplary aryl is phenyl. SEQ ID No. 14: anti-Her2 (4D5) gamma Kazamber The term "heteroaryl refers to a heteroaromatic ring nucleotide sequence structure that can be part of a linkage or part of a Substituent. SEQ ID No. 15: anti-Her2 (4D5) gamma Kazamber The heteroaromatic ring may contain 1-4 (more usually 1-3 amino acid sequence e.g. one or two) heteroatoms selected from O, N and S. SEQ ID No 16: anti-Her2 (4D5)Kappa nucleotide Heteroaryl moieties may contain one ring or two rings. Sequence Example groups containing one 6 membered ring include SEQ ID No 17: anti-Her2 (4D5)Kappa amino add pyridine and pyrimidine. Example groups containing one 5 Sequence membered ring include pyrrole, furan, thiophene, oxazole, thiazole, diazole, thiadiazole and tetrazole. Heteroaryl moi DETAILED DESCRIPTION OF THE 25 eties that contain two rings may contain heteroatoms in one INVENTION or both rings. Examples include quinoline and isoquinoline. Heteroaryl groups may be substituted e.g. by one or more Definitions (e.g. one or two. Such as one) Substituent selected from alkyl, alkenyl, alkynyl, fluoroalkyl, halogen, alkoxy, nitro and The term "amide' refers to a C(=O)—NH linkage. 30 cyano. The term “carbamate” refers to a —O C(=O)—NH The term “aromatic halide” refers to an aromatic ring linkage. (typically phenyl) which is substituted by at least one (e.g. The term “ester” refers to a —C C(=O)—O C link one) halo group Such as fluorine, chloride, bromide or age iodine. Said aromatic ring may contain further Substituents The term “alkyl refers to an aliphatic linkage or sub 35 e.g. those mentioned for aryl. stituent, typically containing 1-6 e.g. 1-4 carbon atoms and The term “azide” and “azido” refers to a N=N(+)—N(-) can be straight chain or branched. Examples include methyl, functional group. ethyl, n-propyl, i-propyl. n-butyl and t-butyl. The term "cycloalkyne” refers to a cyclic arrangement of The term “alkoxy' refers to the group —O-alkyl. carbon atoms (typically 6-9 membered, especially 8-9 mem The term “alkenyl”, “alkene' or “olefin” refers to an 40 bered) which includes a carbon-carbon triple bond captured aliphatic linkage or Substituent, typically containing 2-6 e.g. in the ring structure. Examples include cyclooctyne and 2-4 carbon atoms and can be straight chain or branched and cyclononyne. A further example is benzyne. Cycloalkyne which is unsaturated in respect of containing at least one groups may containing branching. The total number of C=C moiety. Examples include ethenyl, propen-1-yl, pro carbon atoms will typically be 6 to 12 e.g. 6 to 10. pen-2-yl, and 2-methyl-propen-2-yl. An alkenyl group may 45 The term “ketone' refers to a C C(=O)—C linkage. be optionally Substituted e.g. by one or more (e.g. 1) The term "pyrrolysine analog means an amino acid Substituents such as halogen (e.g. Cl) or an ether group (e.g. derivative recognized by either native or genetically evolved —O—C alkyl) although Suitably it is not substituted. PyIRS and incorporated into proteins at an amber codon site. The term “alkynyl' or “alkyne” refers to an aliphatic The expression “the side chain of one of the 20 natural linkage or Substituent, typically containing 2-6 e.g. 2-4 50 amino acids’ refers to the group R in the formula HOOC carbon atoms and can be straight chain or branched and CHR NH, relating to the 20 natural amino acids known by which is unsaturated in respect of containing at least one their single letter codes A, C, D, E, F, G, H, I, K, L, M, N, C=C moiety. Examples include —C=CH and —C=C P, Q, R, S, T, V, W and Y. Either L or S stereochemistry (or CH. An alkynyl group may be optionally substituted e.g. by a mixture thereof) is intended, although L. Stereochemistry is one or more (e.g. 1) Substituents such as halogen (e.g. Cl) or 55 preferred. an ether group (e.g. —O—C alkyl) although suitably it is The present invention discloses pyrrolysine analogs. not substituted. Some pyrrolysine analogs of the present invention have The term “cycloalkyl refers to an alicyclic and unsatu the structure of Formula V: rated compound typically containing 3 to 8 cyclic carbon atoms. Cycloalkyl groups may containing branching. The 60 total number of carbon atoms will typically be 3 to 10. Formula V Exemplary groups include cyclopropyl, cyclobutyl, cyclo pentyl, 3-methyl-cyclopropyl and cyclohexyl. The term "cycloalkenyl refers to an alicyclic compound typically containing 5 to 8 cyclic carbon atoms and contain 65 ing at last one C=C moiety. Cycloalkenyl groups may containing branching. The total number of carbon atoms will US 9,670,521 B2 5 6 wherein (2S)-2-amino-6-(pent-4-enamido)hexanoic acid Z-bond, CH, CH-NH, CH-OH, NH, O, S or CH NH; Formula V.5 b is 0 or an integer 1-7; and O FG=azide, alkene, alkyne, ketone, ester, aryl O cycloalkyne. --~~ N. In formulae V when FG represents aryl, an example is HO s H r aromatic halide e.g. 4-halo phenyl such as 4-iodo phenyl. 10 Moiety Z(CH),FG may, for example, represent CO-aryl e.g. CO-phenyl or —COalkyl e.g. —COMe. Exemplary (S)-2-amino-6((2-oxo-2-phenylacetamide)hexanoic acid compounds of formula V are the following: (2S)-2-amino-6-(2-azidoethoxy)carbonyl aminohexanoic acid 15 Formula V.6 O

N Formula W.1 HO --~- 5 H O

(S)-2-amino-6((2-oxo-2-propanamide)hexanoic acid 5. H O 25 Formula V.7 (2S)-2-amino-6-(prop-2-yn-1-yloxy)carbonyl O O

aminohexanoic acid N HO--~- 30 s H O

Formula W.2 and

O 35 (2S)-2-amino-6-(2-azidoacetamido)hexanoic acid --~~ oué Formula V.8 orn-inSH, O 40 (2S)-2-amino-6-(prop-2-en-1-yloxy)carbonyl aminohexanoic acid Alternative pyrrolysine analogs Suitable for use as non natural amino acids in the present invention have the struc 45 ture of Formula VI:

Formula V.3 Formula VI O 50 /\ / --~~~~ in HN ()\ H/, -- ()H/, 5 H O

55 wherein (2S)-2-amino-6-(3-azidopropanamido)hexanoic acid Z=CH, CH NH, CH-OH, NH, O or S; FG-azide, alkene, alkyne, ketone, ester, aryl or cycloalkyne; and b an integer 1-4. 60 In formulae VI when FG represents aryl, an example is Formula V.4 aromatic halide e.g. 4-halo phenyl such as 4-iodo phenyl. Suitably Z represents NH. In another embodiment it represents CH. b may, for example, represent 1 or 2. 5. H O 65 FG may, for example, represent —N, —CH=CH, - C=CH, -COCH, COOCH phenyl substituted by halogen or cyclooctyne. US 9,670,521 B2 7 8 Exemplary compounds of Formula VI are: VI are easily modified to incorporate a variety of useful (2S)-2-amino-6-(2-azidoethyl)carbamoyloxy)-hexanoic functional groups which can be used for site selective post acid translational modification. Alkynes and alkenes are readily incorporated. The pyrrolysine analogs disclosed herein can 5 be made using various methods. The reaction conditions can Formula VI.1 generally be determined by one of the ordinary skill in the O art. Formula V analogs are readily prepared by the addition of --~~ O 10 HO r N-1SN an activated carbonyl group, such as a chloroformate, acti NH, O vated carboxylic acid ester, isocyanate, activated carbonate or sulfonyl halide to a mono-protected diamino substrate of (2S)-2-amino-6-(prop-2-yn-1-yl)carbamoyl type 1, in which the C-amino group is protected by a oxy} hexanoic acid 15 protecting group (“PG') such as a Boc, Cbz, TFA, Acetyl or Fmoc group (see Scheme 1). The coupled product 3 can undergo further modifications, such as the displacement of Formula VI.2 halides with an azido nucleophile to install the desired O functionality. Otherwise, the intermediate 3 is deprotected to remove the C-amino acid masking group to afford the HO --~~O Rué desired Formula V analog. NH, O 25 Scheme 1:

Formula VI.3 Activated --z / FG O 30 H H A H --~~ O 2 HO Y N-1s Hoc- NH2 an coupling w activated carbonyl NH, O NH H Such as chloroformate, isocyanate Y 4 or activated carboxy acid 35 PG and 1 (2S)-2-amino-6-(prop-2-en-1-yl)carbamoyl 1) Additional oxy} hexanoic acid modificiations f In structures of formulae V and VI, when FG represents 40 necessary eg) alkene, it suitably represents —CH=CH or —CH=CH Halide CH, preferably -CH=CH. displacement In structures of formulae V and VI, when FG represents Hoc- N-C-7 FG 2) withFinal N3 alkyne, it suitably represents —C=CH or —C=C CH, NH H 4 H H ) rection preferably -C=CH. 45 PG In structures of formulae V and VI, when FG represents 3 ketone, it suitably represents —C(=O)—CH or —C(=O)—CH, CH, preferably —C(=O)—CH. In structures of formula VI, when FG represents ketone it / \ / may, for example, represent —C(=O)-aryl e.g. —C(=O)- 50 HOC - – C-Z FG phenyl. NH2 H 4 H H In structures of formulae V and VI, when FG represents ester, it suitably represents —C(=O)—Oalkyl e.g. 4 —C(=O)—Omethyl. 55 In structures of formulae V and VI, when FG represents aromatic halide, it suitably represents phenyl substituted by Formula VI analogs were prepared by conjugation of halogen, especially iodine (e.g. 4-iodo-phenyl). hydroxyl amino acids 9 to substrates with activated carbo In structures of formulae V and VI, when FG represents nyls such as carboxylic acid ester, isocyanate, acid chlorides, cycloalkyne, it suitably represents cyclooctyne, e.g. 60 activated carbonates or sulfonyl halides. The coupled prod cyclooct-4.5-yne. uct 11 can undergo further modifications, such as the instal Advantageously, the nnAAs of formulas V and VI of the lation of the azide functional group by displacement of present invention have been shown to have good incorpo leaving groups such as halides or activated alcohols. The ration as demonstrated by GFP assay. Formula VI.1 had a 65 desired amino acid analog 12 is obtained by final deprotec similar level of translational competency to Formula V.1 in tion to remove the C-amino acid masking group. Protecting the GFP assay incorporation assay. Both the Formula V and groups may be used as per Scheme 1. See Scheme 2: US 9,670,521 B2 10 the ribosome and the nnAA released to form a peptide bond with the growing polypeptide chain. / Recombinant proteins modified to incorporate a pyrroly Activated -- Z C FG sine analog of the invention include all recombinant proteins amenable to site specific post translational modifications, e.g. therapeutic proteins, for instance cytokines, antibodies | | | 10 HOC-C C O-H and antibody derivatives (such as Fab fragments, or single amino coupling w activated carbonyl chain antibodies, e.g. single chain variable fragments NH H 4 Such as chloroformate, isocyanate PG or activated carboxy acid (Scfvs)), peptides, enzymes, fusion proteins, decoy recep 10 tors, protein vaccines, protein hormones, e.g. insulin, growth 9 factors, (e.g. human growth hormone. hCH, hCGCSF, hFSH, 1) Additional hHCG). Further proteins modifiable with pyrrolysine ana modificiations logs of the invention include diagnostic labels, imaging f necessary eg) reagents. Halide 15 Suitably, proteins may be modified site specifically to displacement incorporate one or more than one innAA (pyrrolysine analog) Hoc- O-C-Z FG with N3 of the invention. For instance, an antibody may incorporate 2) Final a nnAA of the invention at the heavy chain, or at the light PG NH H 4 H retion chain, or at both light and heavy chain. Site Specific Conjugation of Proteins with Incorporated 11 Non-Natural Amino Acids Proteins having incorporated pyrrolysine analogs of the / \ . ( present invention may be used for the preparation of func HOC - O-C-Z FG tionalized protein conjugates. Molecules that may be con NH2 H 4 H 25 jugated to proteins having incorporated non-natural amino acids include (i) other proteins, e.g. antibodies especially 12 monoclonal antibodies; (ii) polymers e.g. PEG groups or other groups that may cause halflife extension in the system; (iv) cytotoxic agents e.g. Auristatin F; and (V) drug moieties Incorporation of Non-Natural Amino Acid into Proteins e.g. doxorubicin and moieties containing radioactive iso The pyrrolysine analogs disclosed herein can be incorpo topes. Moreover these modified proteins can be conjugated rated into recombinant proteins. In particular, site specific to drugs or nucleotides for targeted delivery of these potent incorporation of the analog into a recombinant protein can compounds. be achieved through amber Suppression, wherein a nonsense More details of certain embodiments are given below in (amber) codon is inserted within the nucleotide sequence 35 the discussion of antibody drug conjugates. encoding the recombinant protein, at a site where the pyr Pyrrolysine analogs may conveniently contain a unique rolysine analog is to be inserted. The mutated nucleotide chemical group permitting conjugation in a targeted fashion sequence, along with one or more plasmids encoding the without risk of side reaction with other amino acids. For PAS and tRNApyl are inserted into a cell of a cell free example non-natural amino acids may contain azide or expression system. 40 alkyne groups permitting reaction with a molecule to be The host cell may be a eukaryotic cell line which is conjugated which contains a corresponding alkyne or azide transformed with a vector comprising a DNA construct as group using the Huisgen 1,3-dipolar cycloaddition reaction. aforesaid. Preferred conjugation chemistries of the invention include Alternatively, a cell-free expression system is provided, reactions which are orthogonal to the natural twenty amino wherein a synthesis reaction lysate obtained from a host cell 45 acids. Such reactions do not interact or cause side reactions comprises at least one component required for the synthesis with the native 20 amino acids, they are specific to the of polypeptides. The synthesis reaction lysate is obtained functional groups associated with the reaction. Suitably the from bacterial or eukaryotic cells. Preferably, the synthesis necessary functional groups are incorporated into the target reaction lysate is obtained from eukaryotic cells, more protein via the pyrrolysine analogs of the present invention. 50 Further, said reactions proceed under conditions which preferably, from rabbit reticulocytes or wheat germ. are not destructive to the protein, for instance aqueous Preferably, the cell-free expression system is capable of Solvents, with a pH range which is acceptable to the protein expressing WT PyIRS and tRNApyl of the present inven and maintains its solubility, at a temperature which does not tion, wherein tRNApyl is introduced into the cells used to lead to deleterious effects upon the protein. obtain the synthesis reaction lysate with DNA constructs of 55 Increasing the stability of the attachment moiety between the invention. the protein and the linker can be advantageous. Conven Cell-free expression systems suitable for use in the pres tional methods conjugate to the thiol groups of cysteine by ent invention are described for instance in WO201008110, reaction with a maleimide forming a thiol ether. The thiol WO2010081111, WO2010083148, incorporated in their ether can undergo the reverse reaction releasing the linker entirety herein by reference. 60 drug derivative from the antibody. In an embodiment of the When the pyrrolysine analog is added to the cell or invention, the conjugation chemistry employed between an expression system, said analog is incorporated in the recom azide and an alkyne results in an aromatic triazole which is binant protein at the specified position. The nnAA and the significantly more stable, and not as prone to reversibility. tRNApyl are bound by the pyIRS and the tRNApyl is In addition, the product of the reaction, the linkage subsequently aminoacylated with the nnAA. This tRNApyl 65 between protein and payload, ought to be stable, equal to or containing an amber anticodon is released into the cytosol greater than the stability associated with conventional link where in response to an amber stop codon can interact with ages (amide, thiol ether). Though not an impediment to US 9,670,521 B2 11 12 conjugation, it is often advantageous if the conjugation tive groups, said nnAAS can react with each other to reactions can be done under native conditions, as this will generate an intramolecular link. eliminate an extra refolding processing step. In an embodiment, conjugation chemistry of the invention Preferred chemical conjugations for production of conju is used for preparing an antibody drug conjugate. The gates of the invention include: a 3+2 alkyne-azide cycload conjugation chemistry may also be used to assemble anti dition, 3+2 dipolar cycloaddition, Husigen 3+2 cycloaddi body-protein conjugates, protein conjugates Such as bispe tion, Copper promoted azide-alkyne cycloaddition cifics composed of antibody fragments. The conjugation (CuAAC), Ruthenium promoted azide alkyne cycloaddition chemistry may also be used to conjugate polymer bond drug (RAAC), metal promoted azide alkyne cycloaddition conjugates to targeting agents such antibodies and antibody (MAAC), and strain promoted azide alkyne cycloaddition 10 (SPAAC), palladium based couplings including the Heck fragments. The conjugation chemistry can also be used to reaction, Sonogashira reaction Suzuki reaction Stille cou attach polymers such as PEG to proteins to manipulate pling Hiyama/Denmark reaction olefin metathesis Diels pharmacokinetic properties. alder reaction carbonyl condensation with hydrazine, PEG Moieties hydrazide, alkoxy amine or hydroxylamine; strain promoted 15 Target proteins may be conjugated to PEG moieties. PEG cycloadditions with nitriles and nitrile oxides; electron pro moieties may be incorporated into antibody drug conjugates. moted cycloaddition; fragment extrusion cycloaddition; alk The PEG moiety may typically have a molecular weight ene cycloaddition followed by a B-elimination reaction. ranging between 0.5 kDa and 40 kDa e.g. 5 kDa and 40 kDa. According to one preferred embodiment, the incorporated More preferably, the PEG moiety may have a molecular amino acid contains an azide or an alkyne group and the weight of around 20 kDa. In addition, the PEG moieties can process of chemical modification comprises reacting said have a molecular weight range from 100-2000 Da. PEG azide or alkyne group with a reagent comprising an alkyne moieties may be straight chain or branched or multi armed or azide group. The envisaged reaction is a Huisgen 1.3- The PEG moieties can be functionalized with terminal dipolar cycloaddition reaction which leads to production of alkynes, azides, cyanides, cycloalkynes, alkenes, aryl a triazole linkage. The reagent comprising an alkyne or azide 25 halides. The PEG can be functionalized in such as way as to group may be a protein (e.g. an antibody) or a cytotoxic be monofunctional, homobifunctional, heterobifunctional, agent or a drug or a Substance Suitable for half life extension and multi-homofunctional. (e.g. a PEG group) which carries an alkyne or azide group Antibody Drug Conjugates (ADCs) optionally via a linker. Pyrrolysine analogs according to the invention are par Optionally, the Huisgen 1,3-dipolar cycloaddition reac 30 ticularly useful for production of Antibody Drug Conjugates tion can be performed in the presence of Cu(I) catalysis. (recombinant antibody covalently bound by a synthetic Preferably, copper catalyzed cycloaddition reactions are linker to a given drug, typically a cytotoxic drug, or else a carried at room temperature, in aqueous solution in presence protein or a PEG group) which are homogeneous nature, in of cysteine and tris(1-benzyl-1H-1,2,3-triazol-4-yl)methyl which the number of drugs (or other conjugated molecule) amine (TBTA). Alternatively, the copper catalyzed cycload 35 per antibody and position of those drugs upon the antibody dition reactions are carried out from 4° C. to 50° C. in are explicitly controlled, whereby monoclonal antibodies aqueous Solution in the presence of Sodium ascorbate and containing incorporated non-natural amino acids are tris(3-hydroxypropyltriazolylmethyl)amine (THPTA). The obtained and site specifically conjugated to a linker carrying reactions can also be carried out in mixed aqueous/organic a drug moiety (or other conjugated molecule) through solution with the organic component consisting of DMSO, 40 orthogonal chemistry. DMF, methanol, ethanol, t-butanol, trifluoroethanol, propyl ADCs obtained with pyrrolysine analogs of the present ene glycol, ethylene glycol and hexylene glycol. invention may be manufactured following methods includ In a variant reaction, the incorporated amino acid contains ing the following steps: an azide or an alkene group and the process of chemical 1. Introducing into a stable cell line of the invention one modification comprises reacting said azide or alkene group 45 or more plasmids carrying the DNA sequence coding for a with a reagent comprising an alkene or azide group. The full length antibody, whereby a stop codon is introduced at reagent comprising an alkene or azide group may be a specific positions within the sequence protein (eg an antibody) or a toxin or a Substance Suitable for 2. Purify the modified antibody with the pyrrolysine half life extension (eg a PEG group) which carries an alkyne analog (nnAA) installed at desired position(s). or alkene group optionally via a linker. 50 3. React a cytotoxin-linker derivative modified to include The site specific conjugations between the incorporated a functional group complimentary to the nnAA installed in nnAA and the target payload can be done with fully folded the antibody with the modified antibody containing a proteins such as antibodies, antibody fragments, and cytok complementary reactive group through an orthogonal chem ines. Alternatively, the conjugation can be done on dena istry tured proteins in the presence of denaturants such as sodium 55 4. Purify the resulting ADC dodecylsulfate and urea. The copper catalyzed azide alkyne Thus, the present invention also provides ADCs whereby addition can be done in the presence of denaturants and the antibody component has been modified to incorporate reducing agents such as dithiothreitol and 2-mercaptoetha non natural amino acids bearing a unique reactive functional nol. group at desired positions, whereby such functional group When more than one innAA is incorporated into a target 60 allows conjugation to a drug moiety (or protein or PEG protein (e.g. an antibody), the chemical modification may be group). the same or different. For example if two nnAAs are In an embodiment the present invention provides an incorporated, one may be modified to be conjugated to a antibody conjugate comprising an anti-Her-2 antibody drug moiety and one may be modified to be conjugated to a which is conjugated to one or more moieties (e.g. one, two, PEG moiety. 65 three or four, preferably one or two, especially one) selected Conveniently, upon incorporation of more than one innAA from protein, drug and PEG moieties via linkers comprising of the invention bearing different but complementary reac a triazole moiety. US 9,670,521 B2 13 14 In particular, the triazole moiety may be formed by Maytansine: Lewis-Phillips G. D. Cancer Res., 63, 9280 reaction of an azide or alkyne moiety in the side chain of a 9290, 2008. Bouganin: MacDonald GC, et. al., J. Immuno non-natural amino acid incorporated into the sequence of the therapy, 32 574-84, 2009. anti-Her-2 antibody and an alkyne or azide moiety attached Most suitably the drug moiety is a moiety selected from to the protein, drug or PEG moiety. a doxorubicin, paclitaxel and auristatin moiety. In one embodiment, the triazole moiety is formed by Salts reaction of an azide or alkyne moiety in the side chain of a Pyrrolysine analogues described herein may optionally be non-natural amino acid incorporated into the sequence of the employed in the form of a salt. Any Such salts form an aspect anti-Her-2 antibody and an alkyne or azide moiety attached of the invention. Salts of carboxylic acids may include salts to the protein, drug or PEG moiety under conditions of Cu(I) 10 formed with Group 1 and Group 2 metals, especially soluble catalysis. salts such as Sodium and potassium salts. Salts of amines Cu(I) catalysis is accomplished by using either a native may include salts formed with weak and strong acids. Such Cu(I) source such as copper iodide, copper bromide, copper as HCl, HBr or acetic acid. chloride, copper thiolate, copper cyanide. The Cu(I) species 15 INCORPORATION BY REFERENCE can also be generated in situ by using a copper (II) source and a reducing agent. The copper (II) source can be copper All references cited herein, including patents, patent Sulfate, copper (II) chloride, or copper acetate. The reducing applications, papers, text books and the like, and the refer agent can be sodium ascorbate, dithiothreitol, TCEP b-mer ences cited therein, to the extent that they are not already, are captoethanol, hydrazine, hydroxylamine, Sodium bisulfite, hereby incorporated herein by reference in their entirety. cystamine and cysteine. Suitably, Cu(I) catalyzed cycloaddition are carried out in EXAMPLES presence of ligands to stabilize the Cu(I) species present at the start of the reaction or generated in situ by reduction of Example 1. Preparation of Formula V and VI a Cu(II) source Such as Sodium sulfate with sodium ascor 25 Analogs bate, including TBTA, THPTA, phenanthroline derivatives, pyridylmethanimine derivatives, diethylenetriamine, bipyri Preparation of (2S)-2-amino-6-(pent-4-enamido) dine derivatives, TMEDA, N,N-bis(2-pyridylmethyl)amine hexanoic acid (Formula V.5) (BPMA) derivatives.N.N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) derivatives, trialkylamines such as 30 triethylamine, diisopropyl ethylamine, HEPES and MES. In one embodiment a copper azide alkyne cycloaddition is used for the conjugation. Suitably, the reaction utilizes a cytotoxic agent such as auristatin, amanitin, taxol or doxo rubicin bearing a terminal alkyne. Further, the reaction 35 employs a copper source such as copper Sulfate, copper O acetate, copper iodide or copper bromide; a reducing agents Such as Sodium ascorbate, hydrazine, hydroxylamine, --~~ --TFA sodium bisulfite, dithiothreitol, cysteine, b-mercaptoethanol: 40 HO ^-n a copper chelating ligand Such as Tris(1-benzyl-1H-1,2,3- O triazol-4-yl)methylamine (TBTA) or Tris(3-hydroxypropy ltriazolylmethyl)amine (THPTA). Suitably, the reaction may be performed at 4-50° C. Suitably, the reaction time ranges from 0.5 to 48 hrs. In an alternative embodiment, a strain 45 promoted azide alkyne cycloaddition is used for conjuga tion. Suitably, the reaction utilizes a dye, a PEG polymer, or cytotoxic agent such as auristatin bearing a cycloocytne In a 25 mL roundbottomed flask was placed N-Boc group. Suitably, the reaction is allowed to incubate at room Lysine (500 mg, 2.0 mmol) suspended in dioxane (10 mL). temperature for 0.5-48 h. 50 1M KCO was added (5 mL) and the solution was cooled Drug Moieties to OC. 4-pentenoyl chloride (224 uI, 2.0 mmol) in dioxane Drug moieties of the present invention, such as cytotoxin (2 mL) was added slowly. The solution was allowed to stir drug moieties, include Small molecules, natural products, at 0C for 1 h and then at room temperature for 4 h. The synthetically derived drugs, proteins such as immunotoxins, Solution was transferred to a extraction funnel and parti and radionuclides. 55 tioned between water and ether. The organic layer was In an embodiment, the drug moiety is an auristatin moiety removed and the aqueous layer made acidic (pH-2) with eg auristatin or a derivative thereof Such as monomethyl citric acid. The aqueous layer was extracted with ethyl auristatin E (MMAE)(Vedotin) or monomethyl auristatin F acetate (3x50 mL), the organic layers combined and dried (MMAF), Auristatin F (AF), amanitin, Paclitaxel and doxo over sodium sulfate, filtered and concentrated. The resulting rubicin. 60 residue was carried forward into the next step. Other drug moieties include maytansine, paclitaxel, doxo The crude N-Boc-e-N-4-pentenoyl amide-lysine was rubicin and immunotoxins such as exotoxin or bouganin as placed in a 50 mL round bottomed flask with acetonitrile (5 well as radionuclides such as Iodine-131, Yttrium-90, mL) and TFA (2 mL) and magnetically stirred for 2 h. The Samarium-135, and Strontium-89 which may also be incor mixture was concentrated. The solution was treated with porated into organic molecules. (see for instance: MMAE: 65 toluene (10 mL) and concentrated (2x) and acetonitrile (10 Senter, P E, et al, BLOOD, 102, 1458-1465. MMAF: mL) and concentrated (2x). The residue was dried overnight Senter, P E, et. al., Bioconj. Chem. 2006, 17, 114-124. under vacuum. The residue was taken up in MeCH and US 9,670,521 B2 15 precipitated with methyl-t-butyl ether. The viscous oil was -continued isolated by centrifugation, the Supernatant was disposed. O Analytical MS: m/z (ES+) expected 229.2 (M--H)+... found H 230.3. HO--~~ N n NaN Preparation of Preparation of (S)-2-amino-6((2-oxo NHBoc O 2-phenylacetamide)hexanoic acid (Formula V.6) O --~~ NH HipTFA 10 HO ^s. O NHBoc O 1. DCCNHS, DCMDMF O OH 2. O --~~ NH --~~ NH2 HO rs, O HO 15 NH, O NHBoc DCMDMF In a 25 mL roundbottomed flask was placed N-Boc O O Lysine (500 mg, 2.0 mmol) suspended in dioxane (5 mL). H HCI N -e- Saturated NaHCO was added (2 mL) and the solution was OH cooled to 0°C. Bromoacetyl chloride (169 uL 2.0 mmol) in dioxane (2 mL) was added slowly. The solution was allowed O NHBOc to stir at 0C for 1 h and then at room temperature for 4 h. O O The solution was transferred to a extraction funnel and H 25 partitioned between water and ether. The organic layer was N removed and the aqueous layer made acidic (pH-2) with citric acid. The aqueous layer was extracted with ethyl ~~~ acetate (3x50 mL), the organic layers combined and dried O NH2 over sodium sulfate, filtered and concentrated. The resulting 30 residue was carried forward into the next step. In a 50 mL round bottomed flask was placed the crude In a 50 mL round bottomed flask with magnetic stirrer N-Boc-e-2-bromoacetyl-lysine (740 mg, 2.0 mmol) in diox was dissolved pyruvic acid (3.5 g. 23.3 mmol) in a 2:1 ane (10 mL). To this was added a solution of sodium azide mixture of dichloromethane and DMF (20 mL). To this (10 mL, 1M). The solution was stirred at 60° C. overnight. mixture was added DCC (5.7g, 27.6 mmol) and NHS (3.2 The mixture was partitioned between citric acid (1M, 50 g, 27.6 mmol). The mixture was heated to 50 C for 30 min 35 mL) and ethyl acetate (100 mL). The organic layer was with stirring. The solution was allowed to cool and then retained, and the aqueous layer extracted 3 additional times. added through a filter to a suspension of N-Boc-Lysine (5.2 The organic layers were combined, dried over sodium Sulfate and concentrated to an oil. g, 21.2 mmol) in DMF (20 mL) in a separate 100 mL round The crude N-Boc-e-2-azido-acetyl-lysine was dissolved bottomed flask with magnetic stirrer. Triethyl amine (8.8 40 in acetonitrile (10 mL) and TFA (2 mL) was added. The mL, 63.6 mmol) was added after addition of the activated mixture was stirred for 2 h and then concentrated. The ester, and the mixture was stirred overnight. The mixture solution was treated with toluene (10 mL) and concentrated was partitioned between ethyl acetate and citric acid. The (2x) and acetonitrile (10 mL) and concentrated (2x). The layers were separated and the aqueous layer was extracted 4 residue was dried overnight under vacuum. The residue was times with ethyl acetate. The organic layers were combined, 45 taken up in MeOH and precipitated with methyl-t-butyl dried over Sodium sulfate and concentrated. The resulting ether. The viscous oil was isolated by centrifugation, the residue was further purified by flash chromatography to supernatant was disposed. Analytical MS: m/z (ES+) afford the final N-Boc lysine derivative as an oil. expected 229.1 (M+H)+. found 230.2. In a 100 mL round bottomed flask was placed the keto Preparation of Hydroxy-Norleucine Derivatives N-Boc lysine derivative (4g, 10.6 mmol) in acetonitrile (50 50 mL). To this was added a solution of hydrochloric acid (15 (Formula VI. 1 and Formula VI.2) mL, 4N in dioxane). The solution was stirred for 2 h and concentrated. Final purification by flash chromatography afforded the target amino acid. Analytical MS: m/z (ES+) expected 278.1 (M+H)+. found 279.2. 55 Preparation of (2S)-2-amino-6-(2-azidoacetamido) hexanoic acid (Formula V.8)

65 In a 100 mL round bottomed flask with magnetic stirring was placed N-Boc-Hydroxyl Norleucine (1 g, 4.1 mmol) and acetonitrile (50 mL). The mixture was cooled to 0°C. and US 9,670,521 B2 17 p-nitrophenylchloroformate (97.9 mg, 4.9 mmol) and Pyri -continued dine (2 mL) was added and the mixture stirred overnight. The mixture was concentrated and purified by flash chro matography. (Silica, DCM/MeOH gradient). --~~ O NH "So.NaN3,DMSO S Dioxane

HN OC O ACN HN 1N1 N Ben-N-BH

10 In a 100 mL round bottomed flask with magnetic stirring O was placed 2-N-Boc-ethylbromide (1 g, 4.4 mmol) in 25 mL of dioxane. To this was added a solution of sodium azide HO --~~ O r N-1 ns (1M, 22.2 mmol). The solution was stirred at 60 Covernight. NH, O The mixture was partitioned between water and ethyl 15 acetate. The ethyl acetate layer was retained and the aqueous layer was extracted with ethyl acetate three additional times. Step 1: In a 4 mL vial with magnetic stirrer was placed The organic layers were combined, dried over sodium Boc-N-6-hydroxynorleucine (50 mg, 1 eq) and DMF (1 Sulfate and concentrated to an oil. mL). To this was added 2-chloroethyl isocyanate (17.3 mg, The oil was taken up in acetonitrile (35 mL) and HCL in 1.0 eq) and pyridine (32.3 uL., 2 eq). The vial was capped dioxane was added (4M, 10 mL). The mixture was stirred for and allowed to stir for 5 h. The solution was transferred to two hours and concentrated under vacuum. a extraction funnel, diluted with ethylacetate and 100 mM Preparation of (2S)-2-amino-6-(2-azidoethyl)car citric acid. The mixture shaken and the layers separated. The bamoyloxy)-hexanoic acid (Formula VI.1) aqueous layer was extracted with ethyl acetate two addi

In a 50 mL round bottomed flask was placed the N-Boc 40 tional times. The organic layers combined, washed with 5% Norleucine p-nitrophenyl carbonate (503 mg, 1.2 mmol) in lithium chloride, dried with sodium sulfate, filtered and dioxane (10 mL). To this was added a solution of the amino-azide (105 mg, 1.2 mmol) in dioxane (5 mL) and concentrated. The product was identified by mass spectrom pyridine (1 mL). The solution was stirred overnight. The etry and taken forward into the next step directly. mixture was partitioned between ethyl acetate and 500 mM 45 Step 2: In a 4 mL vial with magnetic stirrer was placed the citric acid. The ethyl acetate layer was retained and the crude chloro derivative from above and DMSO (1 mL). aqueous layer was extracted with ethyl acetate three addi tional times. The organic layers were combined, dried over Sodium azide (130 mg, 5 eq) and pyridine (32.3 ul. 2 eq) Sodium Sulfate and concentrated to an oil. The oil was were added to the mixture and the vial was capped. The further purified by flash chromatography. 50 mixture was stirred overnight at 60° C. The mixture was The isolated Boc-protected amino acid was taken up in transferred to an extraction funnel and diluted with 100 mM acetonitrile (15 mL) and treated with HCl in dioxane (4M, 5 mL). The mixture was stirred for two hours and concen citric acid and ethyl acetate. The mixture was shaken and the trated under vacuum. layers separated. The aqueous layer was extracted with ethyl acetate two additional times. The organic layers combined, Alternative Preparation of (2S)-2-amino-6-(2-azi 55 doethyl)carbamoyloxylhexanoic acid, Formula washed with 5% lithium chloride, dried with sodium sulfate, VI.1 filtered and concentrated. The product was carried on to the next step.

60 Final Step: In a 20 mL vial was placed the crude Boc protected amino acid and acetonitrile (2 mL). To this was added a solution of hydrochloric acid in dioxane (4N, 2.5 mL). The solution was stirred for 2 hand then concentrated

65 under reduced pressure. The mixture was lyophilized to a semi solid and used in translational testing. Analytical MS: m/z (ES+) expected 259.1 (M+H)+. found 260.2. US 9,670,521 B2 19 20 Preparation of (2S)-2-amino-6-(prop-2-yn-1-yl) carbamoyloxy)-hexanoic acid (Formula VI.2)

5. H

In a 50 mL round bottomed flask was placed the N-Boc trated. The product was identified by mass spectrometry and Norleucine p-nitrophenyl carbonate (337 mg, 0.8 mmol) in taken forward into the next step directly. dioxane (10 mL). To this was added a solution of the In a 20 mL vial was placed the crude hydroxyl leucine amino-azide (135 mg, 2.4 mmol) in dioxane (5 mL). The allyl carbamate derivative in acetonitrile (2 mL). To this was Solution was stirred overnight. The mixture was partitioned added a solution of hydrochloric acid in dioxane (4N, 2.5 mL). The solution was stirred for 2 hand then concentrated between ethyl acetate and 500 mM citric acid. The ethyl under reduced pressure. The mixture was lyophilized to a acetate layer was retained and the aqueous layer was 25 semi Solid and used in translational testing. The product was extracted with ethyl acetate three additional times. The confirmed by mass spectrometry. Additional purification organic layers were combined, dried over Sodium sulfate and could be done with ion exchange chromatography concentrated to an oil. The oil was further purified by flash (DOWEX-50). Analytical MS: m/z (ES+) expected 230.1 chromatography. (M+H)+. found 231.2. The isolated Boc-protected amino acid was taken up in 30 acetonitrile (15 mL) and treated with HCl in dioxane (4M, Example 2. Translational Testing of Novel 5 mL). The mixture was stirred for two hours and concen Pyrrolysine Analogs as nnAAS with a GFP Assay trated under vacuum An in vitro cell based assay was developed to assess the compatibility of the pylRS/tRNA pair and the pyrrolysine Preparation of (2S)-2-amino-6-(prop-2-en-1-yl) 35 analogs of the present invention (nnAAS) by and the effi carbamoylaminohexanoic acid, Formula VI.3 ciency of nnAAS integration into a target protein. For this, HEK293 cells stably expressing pylRS (3H7) were tran siently transfected with plasmids for the expression of tRNApyl and a reporter construct encoding GFPY40 (con 40 taining amber codon in place of tyrosine at amino acid residue number 40 (where 1 is the initiator methionine)) using standard transfection protocols. Transfected cells were incubated with nnAAs at 2 mM for 2-3 days GFP production was analyzed qualitatively by visual inspection under the 45 microscope. The GFP fluorescence was quantified by flow cytometry using an Accuri flow cytometer and the geometric mean of the fluorescent cells determined. This cell based assay was used to determine whether the different innAAs were suitable substrates for the pyIRS and 50 allowed its translation into a target protein. Cells expressing the PyIRS/tRNApyl pair and containing a vector encoding the GFPY40 reporter gene were incubated in the presence of the nnAAs. nnAAs that are readily utilized by the PyRS/ tRNApyl pair support the translation of the nnAA into the 55 amber site of GFP and allow read-through of the gene producing full length GFP (fluorescent protein). The fluo rescence intensity of the cells depends on the efficiency of In a 4 mL vial with magnetic stirrer was placed Boc-N- nnAA incorporation. Thus, nnAAS that are poorly utilized 6-hydroxynorleucine (50 mg, 1 eq) and DMF (1.5 mL). To produce weakly fluorescent or non-fluorescing cells. Micro this was added allyl isocyanate (18.0 uL. 1.0 eq) and 60 scopic observation identified a number of nnAAs usable by pyridine (32.3 uL., 2 eq). The vial was capped and allowed the pyIRS (Table 1, Positive GFP). Furthermore, the relative to stir for 4 h. The solution was transferred to an extraction expression levels in each sample was compared to those funnel, diluted with ethylacetate and 100 mM citric acid. generated by substrates known to be efficiently utilized by The mixture shaken and the layers separated. The aqueous pyIRS. Formula V.1 (MFI=931,289), Formula V.2 (MFI=1, layer was extracted with ethyl acetate two additional times. 65 676.250) and Formula V.3 (MFI=2,250,000) (see Table 1) The organic layers were combined, washed with 5% lithium supported high levels of GFP expression with a geometric chloride, dried with sodium sulfate, filtered and concen Ca. US 9,670,521 B2 21 22 Analog Formulae VI. 1 and VI.3 and of the present inven to generate IgG containing the nnAAS described above. The tion were found by the inventors to be incorporated in the cells were grown to a density of 1-2x10 cells/mL in Excel GFP reporter gene and yield green cells under the experi DHFR-medium (Sigma-Aldrich) and nnAA added to culture mental conditions used. Among these, the analog of Formula to a final concentration of 1 mM. Cells were incubated for VI.1 supported high levels of GFP expression (MFI904206) 5 days and IgG purified from the growth medium. Super and represents an analogue that is efficiently utilized by the natants were harvested and Subjected to centrifugation to pyIRS/tRNA pair under the experimental conditions tested collect suspended cells and other debris. The supernatant (see Table 2). was then filtered through a 0.22 um filter to remove any particulate material prior to application to a chromatography TABLE 1. 10 column. The filtered supernatant was applied to a 1 mL-5 mL prepacked HiTrap protein A Sepharose at 1-5 mL/min Formula V analog GFP results flow rate using an AKTA chromatography system. The For- Positive bound material and resin were washed with PBS to remove mula IUPAC Name GFP MFI 15 loosely bound proteins and the bound material eluted with V.1 (2S)-2-amino-6-(2- Yes 93.1289 100 mM glycine (pH 3.0) at a flow rate of 1 mL/min. Peak azidoethoxy)carbonyl)aminohexanoic acid fractions containing the target protein were neutralized with V.2 (2S)-2-amino-6-(prop-2-yn-1- Yes 16762SO yloxy)carbonyl)aminohexanoic acid 0.1 fraction volumes of 1M Tris-HCl (pH8.0). All constructs V.3 (2S)-2-amino-6-(prop-2-en-1- Yes 22SOOOO were dialyzed to PBS at 4° C. for 16 hours into the final yloxy)carbonyl)aminohexanoic acid phosphate buffer. The antibody with Formula VI.1 as nnAA incorporated into both of its heavy chains at position 274 was called "4D5-2AZAb-HC274-(2S)-2-amino-6-(2-azi TABLE 2 doethyl)carbamoyloxy-hexanoic acid”. Formula VI analog GFP results 25 PEGylation of 4D5-2AZAb-HC274-(2S)-2-amino-6- For- Positive {(2-azidoethyl)carbamoyloxy)-hexanoic acid mula IUPAC Name GFP Assay MFI VI.1 (2S)-2-amino-6-(2- Yes 9042O6 In a 200 uL PCR tube was placed phosphate buffer (5 uL. azidoethyl)carbamoyloxy)-hexanoic acid VI.3 (2S)-2-amino-6-(prop-2-en-1- Yes 30 500 mM, pH=7.4). A solution of 4D5-2AZAb-HC274-(2S)- yl)carbamoyloxy)-hexanoic acid 2-amino-6-(2-azidoethyl)carbamoyloxy)-hexanoic acid (Formula VI.1). (10 ul, 0.55 mg/mL) was added followed Construction and Expression of Anti-Her2 Antibody by a solution of 20KPEG cyclooctyne (3.3, 60 mg/mL). The A full length anti-Her2 antibody containing two non 35 Solution was mixed vigorously on a Vortexer. The mixture natural amino acids (one in each heavy chain) (4D5-2AZab) was allowed to stand overnight. The mixture was diluted to was expressed in mammalian cells. AnnAA, containing an 200 ul, and applied to Protein-A magnetic beads. The azide moiety, was incorporated at the selected sites and mixture was vortexed and allowed to rotate to mix the beads purified by affinity chromatography using either protein A for 90 min. The beads were immobilized and the run through resin (GE Healthcare) or by IgSelect (GE Healthcare, 40 material disposed. The beads were washed with PBS (2x) 17096901). The purified material was then concentrated and Subjected to a conjugation reaction. and then Suspended in reducing gel buffer. Vortexed and then An antibody directed to the extracellular domain of Her2/ heated to 95 C for 3 min. The suspension was loaded directly neu was generated by cloning the variable regions of both onto an SDS-PAGE gel. Commassie staining of the SDS the heavy and light chains of the mouse antibody 4D5 into 45 PAGE gel indicated the selective PEGylation of the Heavy vectors containing genes encoding human IgG. The variable chain (FIG. 1, Lane 3). regions of 4D5 were generated by gene synthesis using overlapping oligomers and cloned into the human IgG1 Conjugation of 4D5-2AZAb-HC274-(2S)-2-amino frameworks encoded by pFUSE-CHIg-hG1 (IgG1 heavy 6-(2-azidoethyl)carbamoyloxy)-hexanoic acid chain; gamma) and pFUSE-CHLIg-hK (light chain; kappa; 50 Invivogen) to generate a mouse-human hybrid. Amber with Fluoroscene dye by SPAAC codons were introduced into the heavy chain (gamma) at positions K274 by site directed mutagenesis. Clones con In a 200 uL PCR tube was placed phosphate buffer (65uIL, taining the amber codon were identified by DNA sequenc 50 mM, pH=7.4). A solution of 4D5-2AZAb-HC274-(2S)- ing. To generate an integrating construct the promoters and 55 2-amino-6-(2-azidoethyl)carbamoyloxy)-hexanoic acid ORF for the heavy chain was amplified by PCR and cloned (30 uI, 0.55 mg/mL) was added followed by a solution by restriction enzyme digestion and ligation into pCptivec DMCO-Fluor 488 cyclooctyne (5.4, 5 mM in DMSO, click (Life Technologies). The light chain and a single copy of the chemistry tools). The Solution was mixed vigorously on a tRNA were joined by two step PCR method using overlap vortexer. The mixture was allowed to stand for 24 h. The ping oligomers and cloned into available sites into the 60 pOptivec plasmid containing the heavy chain. The construct mixture was analyzed by HIC chromatography (Tosoh TSK was then transfected into a CHO cell line containing the gel Butyl NPR with a gradient of 1M Sodium sulfate to pyIRS/tRNA pair and stably transfected cell lines showing phosphate buffer) showing the conjugation had occurred and high expression of the IgG selected. This represents a second resulted in a mixture wherein conjugation had occurred at example of a cell line stably expressing a mAb containing a 65 one or two sites (generally referred to as DAR1 and DAR2 nnAA indicating that the process has wide applicability for species; wherein DAR is defined as drug-to-antibody ratio) the use in the expression of mAbs. This cell line was utilized (FIG. 2).

US 9,670,521 B2 29 30 neering of Pyrrolysyl-tRNA Synthetase to Genetically thetase reveals the pre- and post-aminoacyl-tRNA syn Encode Ne-(o-Azidobenzyloxycarbonyl)lysine for Site thesis conformational states of the adenylate and amino Specific Protein Modification. Chemistry and Biology, 15, acyl moieties and an asparagine residue in the catalytic 1187-1197. site. Acta Crystallographica Section D, D69, 5-15. Yanagisawa, T., Sumida, T., Ishii, R., & Yokoyama, S. 5 All patents and patent applications referred to herein are (2013). A novel crystal fom of pyrrolysyl-tRNA syn incorporated by reference in their entirety.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 17

<21 Os SEQ ID NO 1 &211s LENGTH: 1365 212s. TYPE : DNA <213> ORGANISM: Methanosarcina mazei

<4 OOs SEQUENCE: 1

atggataaaa aacCactaaa. cactctgata totgcaa.ccg ggct Ctggat gtc.caggacc 60

ggaacaattic ataaaataaa. acaccacgaa gt ct citcgaa gcaaaatcta tattgaaatg 12O

gCatgcggag accaccittgt tgtaaacaac to Caggagca gcaggactgc alaga.gc.gctic 18O

aggcaccaca aatacaggaa gacctgcaaa cgctgcaggg titt.cggatga ggat ct caat 24 O

aagttcc to a caaaggcaaa cgalagaccag acaa.gcgtaa aagttcaaggt cgtttctgcc 3 OO

cctaccagaa cgaaaaaggc aatgccaaaa to cqttgcga gaggcc.cgaa acct cittgag 360

aatacagaag cggcacaggc to aacct tot ggat.ctaaat titt cacctgc gataccggitt 42O

to cacccaag agt cagttt c tgtc.ccggca totgtttcaa CatCalatat C aagcatttct 48O

acaggagcaa. ctgcatcc.gc actggtaaaa gggaatacga acco Cattac atccatgtct 54 O

gcc cctdttic aggcaagtgc cc cc.gcactt acgaagagcc agactgacag gcttgaagtic 6 OO ctgttaalacc caaaagatga gattt coctd aatticcggca agcc titt cag ggagcttgag 660

to cqaattgc totct cqcag aaaaaaagac Ctgcagcaga totacgcgga agaaagggag 72O

aattatctgg ggaaactica gcgtgaaatt accaggttct ttgttggacag gggttittctg 78O

gaaataaaat cc ccgatcct gatcc ct citt gagtatat cq aaaggatggg cattgataat 84 O

gat accgaac tittcaaaa.ca gatct tcagg gttgacaaga acttctgcct galgacc catg 9 OO

cittgctic cala acctitta Cala ctacctg.cgc aagcttgaca gggccCtgcc tgat coaata 96.O

aaaatttittg aaataggcCC atgct acaga aaagagt cc.g acggcaaaga acacct cqaa 102O

gagtttacca tgctgaactt Ctgccagatg ggat.cgggat gcacacggga aaatcttgaa 108O

agcataatta cggactitcct galaccacctg ggaattgatt tdaagat.cgt. aggcgatt CC 114 O

tgcatggtct atggggatac cc ttgatgta atgcacggag acctggaact tt cotctgca 12 OO

gtagt cqqac ccataccgct tgaccgggaa tgggg tattg at aaac cctd gataggggca 1260

togaacgc.ct totaaaggtt aaac acgact ttaaaaatat Caagagagct 132O

gcaaggit cog agtct tact a taacgggatt totaccalacc tgtaa 1365

<21 Os SEQ ID NO 2 &211s LENGTH: 45.4 212s. TYPE : PRT <213> ORGANISM: Methanosarcina mazei

<4 OOs SEQUENCE: 2 Met Asp Llys Llys Pro Lieu. Asn. Thir Lieu. Ile Ser Ala Thr Gly Lieu. Trp 1. 5 15 Met Ser Arg Thr Gly Thr Ile His Lys Ile Llys His His Glu Val Ser 25 3 O Arg Ser Lys Ile Tyr Ile Glu Met Ala Cys Gly Asp His Lieu Val Val US 9,670,521 B2 31 32 - Continued

35 4 O 45

Asn Asn Ser Arg Ser Ser Arg Thir Ala Arg Ala Lell Arg His His SO 55 6 O

Tyr Arg Thir Lys Arg Arg Wall Ser Asp Glu Asp Luell 65 70

Phe Luell Thir Lys Ala Asn Glu Asp Glin Thir Ser Wall Wall 85 90 95

Wall Wall Ser Ala Pro Thir Arg Thir Lys Ala Met Pro Lys Ser Wall 105 11 O

Ala Arg Ala Pro Pro Lell Glu Asn Thir Glu Ala Ala Glin Ala Glin 115 12 O 125

Pro Ser Gly Ser Phe Ser Pro Ala Ile Pro Wall Ser Thir Glin Glu 13 O 135 14 O

Ser Wall Ser Wall Pro Ala Ser Wall Ser Thir Ser Ile Ser Ser Ile Ser 145 150 155 160

Thir Gly Ala Thir Ala Ser Ala Luell Wall Lys Gly Asn Thir Asn Pro Ile 1.65 17s

Thir Ser Met Ser Ala Pro Wall Glin Ala Ser Ala Pro Ala Luell Thir 18O 185 19 O

Ser Glin Thir Asp Arg Lell Glu Wall Luell Luell ASn Pro Lys Asp Glu Ile 195

Ser Luell Asn Ser Gly Pro Phe Arg Glu Luell Glu Ser Glu Luell Luell 21 O 215

Ser Arg Arg Asp Lell Glin Glin Ile Tyr Ala Glu Glu Arg Glu 225 23 O 235 24 O

Asn Luell Gly Lys Lell Glu Arg Glu Ile Thir Arg Phe Phe Wall Asp 245 250 255

Arg Gly Phe Luell Glu Ile Ser Pro Ile Luell Ile Pro Luell Glu 26 O 265 27 O

Ile Glu Arg Met Gly Ile Asp Asn Asp Thir Glu Lell Ser Glin Ile 28O 285

Phe Arg Wall Asp Asn Phe Luell Arg Pro Met Lell Ala Pro Asn 29 O 295 3 OO

Lell Asn Lell Arg Luell Asp Arg Ala Lell Pro Asp Pro Ile 3. OS 310 315 32O

Ile Phe Glu Ile Gly Pro Arg Glu Ser Asp Gly 3.25 330 335

Glu His Luell Glu Glu Phe Thir Met Luell Asn Phe Glin Met Gly Ser 34 O 345 35. O

Gly Thir Arg Glu Asn Lell Glu Ser Ile Ile Thir Asp Phe Luell Asn 355 360 365

His Luell Gly Ile Asp Phe Lys Ile Wall Gly Asp Ser Met Wall 37 O 375 38O

Gly Asp Thir Luell Asp Wall Met His Gly Asp Luell Glu Lell Ser Ser Ala 385 390 395 4 OO

Wall Wall Gly Pro Ile Pro Lell Asp Arg Glu Trp Gly Ile Asp Lys Pro 4 OS 41O 415

Trp Ile Gly Ala Gly Phe Gly Luell Glu Arg Luell Lell Wall His 425 43 O

Asp Phe Lys Asn Ile Arg Ala Ala Arg Ser Glu Ser Asn 435 44 O 445

Gly Ile Ser Thir Asn Lell 450 US 9,670,521 B2 33 34 - Continued

SEQ ID NO 3 LENGTH: 1365 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic sequence: PylRS Methanosarcina mazei Y384F mutant nucleotide sequence SEQUENCE: 3 atggataaaa aaccactaaa cactctgata t ctdcaa.ccg ggctctggat gtcCagg acc 6 O ggaacaattic ataaaataaa acaccacgaa gtct citcqaa gcaaaatcta tattgaaatg 12 O gcatgcggag accaccittgt ttaaacaac to Caggagca gCagg actgc aagagcgctic 18O aggcaccaca aatacaggaa gacctgcaaa cqctgcaggg titt.cgatga ggat.ct caat 24 O aagttcct ca caaaggcaaa Caagaccag acaag.cgitaa aagtcaaggt cgtttctgcc 3OO

Cctaccagaa caaaaaggc aatgccaaaa t cc.gttgcga gagcc.ccgaa acct cittgag 360 aatacagaag cqgcacaggc ticaac cittct ggat.ctaaat titt cacctgc gat accggitt tccacccaag agt cagtttctgtc.ccggca totgtttcaa Cat Calatat C aag catttct acaggagcaa. Ctgcatcc.gc actggtaaaa gggaatacga acco cattac atc catgtct 54 O gcc cct gttc aggcaa.gtgc ccc.cgcactt acgaagagcc agactgacag gcttgaagtic ctgttaaacc caaaagatga gattt coctd aattic cqgca agc ctitt cag ggagcttgag 660 tccgaattgc tict Ctc.gcag aaaaaaagac Ctgcagoaga tctacgcgga agaaagggag 72 O aattatctgg galaact.cga gCdtgaaatt acc aggttct ttgttggaCag gggttittctg gaaataaaat coccgatcct gatcc ct citt gagtatat cq aaaggatggg cattgataat 84 O gataccgaac tittcaaaa.ca gat ctitcagg gttgacaaga actitctgcct gag acccatg 9 OO cittgct coaa acctttacaa citacctg.cgc aagcttgaca gggcc ctgcc tgatccaata 96.O aaaatttittgaaataggc cc atgct acaga aaagagt cc.g acggcaaaga acacct cqaa gagtttacca totgaactt Ctgcc agatg ggat.cgggat gcacacggga aaatcttgaa 108 O agcataatta cqgact tcct gaaccacctg ggaattgatt t caagat cqt aggcgatt CC 114 O tgcatggit ct ttggggatac Ccttgatgta atgcacggag acctggaact titcct ctdca 12 OO gtag toggac C cataccgct taccgggala tiggg tattg ataaaccctg gataggggca 126 O ggtttcgggc ticgaacgc.ct tctaaaggitt aaacacgact ttaaaaatat Caagaga.gct 132O gcaaggtocq agt ct tacta taacgggatt totaccalacc tgtaa 1365

SEQ ID NO 4 LENGTH: 45.4 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic sequence: PylRS Methanosarcina mazei Y384F mutant amino acid sequence

SEQUENCE: 4 Met Asp Llys Llys Pro Lieu. Asn. Thir Lieu. Ile Ser Ala Thr Gly Lieu. Trp 1. 15

Met Ser Arg Thr Gly Thr Ile His Lys Ile Llys His His Glu. Wall Ser 2O 25 3O

Arg Ser Lys Ile Tyr Ile Glu Met Ala Cys Gly Asp His Lieu Wall Wall 35 4 O 45

Asn Asn. Ser Arg Ser Ser Arg Thr Ala Arg Ala Lieu. Arg His His Lys US 9,670,521 B2 35 36 - Continued

SO 55 6 O

Tyr Arg Thir Lys Arg Arg Wall Ser Asp Glu Asp Luell Asn 65 70