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(12) United States Patent (10) Patent No.: US 7,696,328 B2 Nanduri Et Al

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(12) United States Patent (10) Patent No.: US 7,696,328 B2 Nanduri Et Al US007696328B2 (12) United States Patent (10) Patent No.: US 7,696,328 B2 Nanduri et al. (45) Date of Patent: Apr. 13, 2010 (54) N-CARBOBENZYLOXY (56) References Cited (N-CBZ)-DEPROTECTING ENZYME AND U.S. PATENT DOCUMENTS USES THEREFOR 5,508,272 A 4, 1996 Rob (75) Inventors: Venkata B. Nanduri, East Brunswick, 5,552,397 A 9/1996 Karanewsky et al. NJ (US); Ramesh N. Patel, Bridgewater, NJ (US); Steven L. Goldberg, Basking Ridge, NJ (US); Robert M. Johnston, FOREIGN PATENT DOCUMENTS Whitehouse Station, NJ (US) WO WOOOf 472O7 8, 2000 (73) Assignee: Bristol-Myers Squibb Company, WO WOO2,53724 12/2001 Princeton, NJ (US) (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 29 days. Pohl et al., Tetrahedron Letters, vol. 36, No. 17. (1995) pp. 2963 2966. (21) Appl. No.: 12/150,544 Theodoridis, G., Tetrahedron 56 (2000) pp. 2239-2358. Waldmann et al., 277 Chemical Reviews, American Chemical Soci (22) Filed: Apr. 28, 2008 ety, 94 (Jun. 1994) No. 4, pp. 91 1-937. Matsumura, E. et al., “A Novel Enzyme N'-Benzyloxycarbonyl (65) Prior Publication Data amino acid urethane hydrolase II' from Lactobacillus fermenti 36 ATCC9338' Chem. Pharm. Bull., vol. 33(1), pp. 408-411 (1985). US 2009/O104670 A1 Apr. 23, 2009 Green and Wuts, Protective Groups. In Organic Synthesis, John Wiley & Sons, NY, 1991. pp. 307-348. Related U.S. Application Data Jeyaraj et al., Tetrahedron Letters 42 (2001) pp. 835-837. (60) Division of application No. 1 1/502.704, filed on Aug. Pohl et al., J. Am. Chem. Soc. 119, (1997) pp. 6702-6710. 10, 2006, now Pat. No. 7,416,876, which is a division Kappes et al., Carbohydrate Research 305 (1998) pp. 341-349. of application No. 10/453,418, filed on Jun. 3, 2003, Primary Examiner—Dong Jiang now Pat. No. 7,119,188, which is a continuation-in Assistant Examiner Sandra Wegert part of application No. 10/017,711, filed on Dec. 14, (74) Attorney, Agent, or Firm Nikki L. Parlet 2001, now Pat. No. 6,828,119. (57) ABSTRACT (60) Provisional application No. 60/259,715, filed on Jan. 4, 2001. This invention relates to isolated or recombinant N-carboben (51) Int. Cl. Zyloxy-deprotecting enzyme polypeptides that catalyze the C07K 6/00 (2006.01) removal of carbobenzyloxy from carbobenzyloxy-protected amino acids and alcohols. Also related are isolated nucleic C07K 6/40 (2006.01) acids encoding N-carbobenzyloxy-deprotecting enzyme C07K I6/46 (2006.01) polypeptides thereof, as well as vectors and host cells com CO7K (4/OO (2006.01) prising these nucleic acids. The invention also relates to meth CO7K 5/OO (2006.01) ods of obtaining isolated nucleic acids, polypeptides, and (52) U.S. Cl. ............ 530/387.9; 530/388.1:530/388.26; antibodies, and methods of using the polypeptides in various 530/391.1; 530/350, 530/300 reactions for industrial or pharmaceutical applications. (58) Field of Classification Search ....................... None See application file for complete search history. 3 Claims, No Drawings US 7,696,328 B2 1. 2 N-CARBOBENZYLOXY variants, modifications, and fragments thereof. N-carboben (N-CBZ)-DEPROTECTING ENZYME AND Zyloxy-deprotecting enzyme is herein also referred to as USES THEREFOR N-CBZ-deprotecting enzyme. An N-CBZ-deprotecting enzyme polypeptide has been isolated from S. paucimobilis. This application is a divisional application of U.S. appli- 5 It is also an object of the invention to provide isolated cation Ser. No. 1 1/502.704 filed on Aug. 10, 2006, issued as N-CBZ-deprotecting enzyme polynucleotides, e.g., DNA U.S. Pat. No. 7,416,876, which is a divisional application of and RNA molecules, comprising nucleotide sequences U.S. application Ser. No. 10/453,418 filed on Jun. 3, 2003, encoding N-CBZ-deprotecting enzyme polypeptides and now issued as U.S. Pat. No. 7,119,188, which is a CIP of U.S. application Ser. No. 10/017,711, filed Dec. 14, 2001, now 10 complementary sequences thereof, as well as nucleic acid issued as U.S. Pat. No. 6,828,119, which claims the benefit of variants, modifications, fragments thereof. U.S. Provisional Application Ser. No. 60/259,715, filed Jan. It is a further object of the present invention to provide 4, 2001. Both U.S. application Ser. No. 10/017,711 and U.S. nucleic acid probes and primers, as well as vectors and host Provisional Application Ser. No. 60/259,715 are incorporated cells, comprising polynucleotides of the invention. herein by reference in their entirety. 15 It is yet a further object of the present invention to provide isolated N-CBZ-deprotecting enzyme polypeptides and FIELD OF THE INVENTION polypeptide fragments, variants, and modifications thereof. It is yet a further object of the present invention to provide This invention relates to a novel N-carbobenzyloxy-depro recombinant N-CBZ-deprotecting enzyme polypeptides, and tecting enzyme, isolatable from Sphingomonas paucimobilis, 20 polypeptide fragments, variants, and modifications thereof. which catalyzes the removal of carbobenzyloxy (CBZ) from It is another object of the present invention to provide N-CBZ protected amino acids and O-CBZ protected alco antibodies and antibody fragments that specifically bind to hols. The invention also relates to isolated nucleic acids com the polypeptides, or polypeptide variants, modifications, or prising nucleotide sequences which encode N-carbobenzy fragments thereof. loxy-deprotecting enzyme polypeptides, vectors and host 25 cells comprising these nucleic acids, isolated N-carbobenzy It is yet another object of the present invention to provide loxy-deprotecting enzyme polypeptides, recombinant N-car methods of using the polynucleotides, vectors, and host cells bobenzyloxy-deprotecting enzyme polypeptides, and anti of the invention to produce polypeptides of the invention, bodies that specifically bind to N-carbobenzyloxy Such as N-CBZ-deprotecting enzyme polypeptides. deprotecting enzyme polypeptides. The invention further 30 It is still another object of the present invention to provide relates to methods of obtaining isolated N-carbobenzyloxy methods of using the polypeptides of the invention, Such as deprotecting enzyme nucleic acids, isolated polypeptides, N-CBZ-deprotecting enzyme polypeptides, in enzymatic recombinant polypeptides, and antibodies, and to methods of reactions requiring the deprotection of an amino or hydroxyl producing N-carbobenzyloxy-deprotecting enzyme with the group. In various aspects, this process uses isolated polypep vectors and host cells, and to methods of using N-carboben- 35 tide, or cell-free extracts or whole cells expressing recombi Zyloxy-deprotecting enzyme in reactions required for the nant polypeptide. synthesis of industrial or pharmaceutical compounds. It is a further object of the present invention to provide methods of purifying the N-CBZ-deprotecting enzyme BACKGROUND OF THE INVENTION polypeptides, or polypeptide variants, modifications, or frag 40 ments thereof, using the disclosed antibodies or antibody Carbobenzyloxy (CBZ) group is commonly used to protect fragments. amino and hydroxide groups during organic synthesis. Other It is still another object of the present invention to provide similar "carbamate” and "carbonate' protecting groups are a method of deprotecting an amine or alcohol protected with also used to protect amino and hydroxyl groups. Chemical a group of formula deprotection is usually achieved by methods such as hydro- 45 genation with palladium catalyst. However, if other groups are present which are Susceptible to the deprotection condi tion (for example, Sulfur during hydrogenation), alternative where the substituents are as described below, the method methods of deprotection are necessary. It would be beneficial comprising: contacting the protected amine or alcohol with to have alternate methods of deprotection that would not 50 an enzyme effective to remove the protecting group; and destroy Susceptible groups. An enzymatic method of depro recovering the amine or alcohol. tection using S. paucimobilis N-carbobenzyloxy-deprotect It is also an object of the present invention to provide a ing enzyme isolated from Soil samples has been disclosed in method of isolating a bacteria producing an enzyme effective WO 02/053724, which application shares a priority claim to remove a protecting group comprising: growing prospec with this application. The enzymatic method can be con- 55 tive bacteria on a medium having a growth selective amount ducted under mild conditions (i.e., aqueous medium at room of an amine compound that is protected as above; and isolat temp and atmospheric pressure) and without the destroying ing bacteria that grow on said medium. Susceptible groups. Given the importance of the enzyme and It is still another object of the present invention to provide its relatively low presence in the host bacterium, it would be a method of resolving a desired enantiomer of an amine or useful to clone and express in a heterologous host Such as 60 alcohol linked to a chiral carbon comprising: providing a Escherichia coli to ensure a sufficient Supply for large-scale derivative of the compound in which the amine or alcohol is reactions. protected with a group of formula ArC*(R)H (CH), O— C(=O)—, contacting the protected compound with an SUMMARY OF THE INVENTION enzyme effective to remove the protecting group; and isolat 65 ing the compound or protected derivative thereof in a com It is an object of the present invention to provide novel position that is enantiomerically enriched in the desired enan N-carbobenzyloxy-deprotecting enzyme polypeptides, and tioner. US 7,696,328 B2 3 4 Additional objects and advantages afforded by the present term includes, for example, a recombinant DNA which is invention will be apparent from the detailed description and incorporated into a vector, e.g. into an autonomously repli exemplification hereinbelow. cating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate mol DETAILED DESCRIPTION OF THE INVENTION 5 ecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of The present invention relates to isolated nucleic acids that other DNA sequences.
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