57fa63ed6d54c6f83a1693ab388d3df8 catalysts_a2021v11n8p973.pdf 62a6b5f20e2ea8a47c0dc3d22a71d456b7631010 catalysts_a2021v11n8p973.pdf e663c33031e2902fc0e44696009860e621a1503d322f81c68b56b3da6f28fbee catalysts_a2021v11n8p973.pdf Title: Chiral Synthesis of 3-Amino-1-phenylbutane by a Multi-Enzymatic Cascade System Subject: Asymmetric synthesis of chiral amines from prochiral ketones using transaminases is an attractive biocatalytic strategy. Nevertheless, it is hampered by its unfavorable thermodynamic equilibrium. In the present work, an insitu by-product removal strategy was applied for the synthesis of 3-amino-1-phenylbutane (3-APB) by coupling a transaminase with a pyruvate decarboxylase (PDC), which does not require the use of any expensive additional cofactor. Using this strategy, the pyruvate obtained in the transamination reaction is transformed by PDC into acetaldehyde and CO2 which are of high volatility. Two different transaminases from Chromobacterium violaceum (CviTA) and Vibrio fluvialis (VflTA) were characterized to find out the appropriate pH conditions. In both cases, the addition of PDC dramatically enhanced 3-APB synthesis. Afterwards, different reaction conditions were tested to improve reaction conversion and yield. It was concluded that 30 C and a 20-fold alanine excess lead to the best process metrics. Under the mentioned conditions, yields higher than 60% were reached with nearly 90% selectivity using both CviTA and VflTA. Moreover, high stereoselectivity for (S)-3-APB was obtained and ee of around 90% was achieved in both cases. For the first time, the asymmetric synthesis of 3-APB using PDC as by-product removal system using CviTA is reported. Keywords: chiral amine; transaminase; pyruvate decarboxylase (PDC); multi-enzymatic system Author: Natàlia Alcover, Gregorio Álvaro and Marina Guillén Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Aug 16 08:14:21 2021 CEST ModDate: Mon Aug 16 08:19:10 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1680787 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- YRSCBV+URWPalladioL-Ital Type 1 Custom yes yes no 10 0 PLDSPO+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 SSXAQU+URWPalladioL-Roma Type 1 Custom yes yes no 19 0 XVSDDF+CMSY10 Type 1 Builtin yes yes no 23 0 KGFXSR+EURM10 Type 1 Builtin yes yes no 27 0 MKXUQA+VnURWPalladioL-Bold Type 1 Custom yes yes no 31 0 FPSJLQ+VnURWPalladioL Type 1 Custom yes yes no 36 0 CGDLTM+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 44 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 61 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 65 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 69 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 83 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 89 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 95 0 KBZJTA+MSAM10 Type 1 Builtin yes yes no 106 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 112 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 118 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 124 0 FNTSBS+TimesNewRomanPSMT CID TrueType Identity-H yes yes yes 130 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 142 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 148 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 154 0 FNTSBS+Calibri CID TrueType Identity-H yes yes yes 160 0 TGDCLJ+stmary9 Type 1 Builtin yes yes no 170 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 176 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 182 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 188 0 FNTSBS+Wingdings-Regular CID TrueType Identity-H yes yes yes 194 0 FNTSBS+Wingdings3 CID TrueType Identity-H yes yes yes 200 0 FNTSBS+Wingdings2 CID TrueType Identity-H yes yes yes 206 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 222 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 228 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 234 0 FNTSBS+Wingdings-Regular CID TrueType Identity-H yes yes yes 240 0 FNTSBS+Wingdings3 CID TrueType Identity-H yes yes yes 246 0 FNTSBS+Wingdings2 CID TrueType Identity-H yes yes yes 252 0 FNTSBS+PalatinoLinotype-Bold CID TrueType Identity-H yes yes yes 265 0 FNTSBS+PalatinoLinotype-Roman CID TrueType Identity-H yes yes yes 271 0 FNTSBS+PalatinoLinotype-Italic CID TrueType Identity-H yes yes yes 277 0 FNTSBS+Wingdings-Regular CID TrueType Identity-H yes yes yes 283 0 FNTSBS+Wingdings3 CID TrueType Identity-H yes yes yes 289 0 FNTSBS+Wingdings2 CID TrueType Identity-H yes yes yes 295 0 FNTSBS+PalatinoLinotype-BoldItalic CID TrueType Identity-H yes yes yes 301 0 JHYTSG+CMR10 Type 1 Builtin yes yes no 322 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-05-27 03:28:45 CEST RepresentationInformation: catalysts_a2021v11n8p973.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 11:58:18 CEST Size: 1680787 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 500 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Enzyme Characterization Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Transaminase Testing Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Synthesis of 3-APB Catalyzed by TA and PDC Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Effect of Temperature on 3-APB Synthesis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Effect of Amine Donor Concentration on 3-APB Synthesis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Enantiomeric Excess Determination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Chemicals and Enzymes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Transaminase Activity Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Pyruvate Decarboxylase Activity Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Influence of pH on Enzyme Activity and Stability Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Transaminase Screening Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Asymmetric Synthesis of APB with Transaminase and Pyruvate Decarboxylase Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Temperature and Amine Donor Concentration Effect on 3-APB Synthesis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Quantification of 3-APB and 4-PB Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Enantiomeric Excess Determination Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Info: Title: Chiral Synthesis of 3-Amino-1-phenylbutane by a Multi-Enzymatic Cascade System Author: Natàlia Alcover, Gregorio Álvaro and Marina Guillén Subject: Asymmetric synthesis of chiral amines from prochiral ketones using transaminases is an attractive biocatalytic strategy. Nevertheless, it is hampered by its unfavorable thermodynamic equilibrium. In the present work, an insitu by-product removal strategy was applied for the synthesis of 3-amino-1-phenylbutane (3-APB) by coupling a transaminase with a pyruvate decarboxylase (PDC), which does not require the use of any expensive additional cofactor. Using this strategy, the pyruvate obtained in the transamination reaction is transformed by PDC into acetaldehyde and CO2 which are of high volatility. Two different transaminases from Chromobacterium violaceum (CviTA) and Vibrio fluvialis (VflTA) were characterized to find out the appropriate pH conditions. In both cases, the addition of PDC dramatically enhanced 3-APB synthesis. Afterwards, different reaction conditions were tested to improve reaction conversion and yield. It was concluded that 30 C and a 20-fold alanine excess lead to the best process metrics. Under the mentioned conditions, yields higher than 60% were reached with nearly 90% selectivity using both CviTA and VflTA. Moreover, high stereoselectivity for (S)-3-APB was obtained and ee of around 90% was achieved in both cases. For the first time, the asymmetric synthesis of 3-APB using PDC as by-product removal system using CviTA is reported. Keywords: chiral amine; transaminase; pyruvate decarboxylase (PDC); multi-enzymatic system Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Aug 16 08:14:21 CEST 2021 ModDate: Mon Aug 16 08:19:10 CEST 2021 ID: 0xf40733ed53660d84b1c8b0970d5770a1, 0xf40733ed53660d84b1c8b0970d5770a1 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: JHYTSG+CMR10 FontSubset: true FirstChar: 61 LastChar: 61 FontDescriptor: FontName: JHYTSG+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true Font: BaseFont: SSXAQU+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: SSXAQU+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: FPSJLQ+VnURWPalladioL FontSubset: true FirstChar: 129 LastChar: 225 FontDescriptor: FontName: FPSJLQ+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XVSDDF+CMSY10 FontSubset: true FirstChar: 0 LastChar: 48 FontDescriptor: FontName: XVSDDF+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: YRSCBV+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: YRSCBV+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: KBZJTA+MSAM10 FontSubset: true FirstChar: 4 LastChar: 125 FontDescriptor: FontName: KBZJTA+MSAM10 Flags: Symbolic FontBBox: 8, -463, 1331, 1003 FontFile: true Font: BaseFont: TGDCLJ+stmary9 FontSubset: true FirstChar: 31 LastChar: 97 FontDescriptor: FontName: TGDCLJ+stmary9 Flags: Symbolic FontBBox: -128, -2956, 1083, 823 FontFile: true Font: BaseFont: KGFXSR+EURM10 FontSubset: true FirstChar: 11 LastChar: 34 FontDescriptor: FontName: KGFXSR+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: PLDSPO+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: PLDSPO+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MKXUQA+VnURWPalladioL-Bold FontSubset: true FirstChar: 129 LastChar: 178 FontDescriptor: FontName: MKXUQA+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 164, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 107, 358, 154, 367 Annotation: Subtype: Link Rect: 35, 346, 132, 355 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 384, 268, 391, 277 ActionDest: 315 Annotation: Subtype: Link Rect: 394, 268, 401, 277 ActionDest: 315 Annotation: Subtype: Link Rect: 403, 231, 410, 240 ActionDest: 315 Annotation: Subtype: Link Rect: 413, 231, 425, 240 ActionDest: 319 Annotation: Subtype: Link Rect: 222, 181, 229, 189 ActionDest: 319 Annotation: Subtype: Link Rect: 230, 180, 236, 189 ActionDest: 319 Annotation: Subtype: Link Rect: 237, 181, 249, 189 ActionDest: 319 Annotation: Subtype: Link Rect: 252, 180, 264, 189 ActionDest: 319 Annotation: Subtype: Link Rect: 502, 92, 514, 102 ActionDest: 319 Annotation: Subtype: Link Rect: 514, 92, 526, 102 ActionDest: 319 Annotation: Subtype: Link Rect: 363, 67, 374, 76 ActionDest: 319 Annotation: Subtype: Link Rect: 115, 17, 255, 28 Annotation: Subtype: Link Rect: 404, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 423, 87, 442 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 204, 712, 216, 721 ActionDest: 319 Annotation: Subtype: Link Rect: 216, 711, 228, 721 ActionDest: 319 Annotation: Subtype: Link Rect: 452, 699, 464, 708 ActionDest: 319 Annotation: Subtype: Link Rect: 464, 699, 476, 708 ActionDest: 319 Annotation: Subtype: Link Rect: 224, 674, 236, 683 ActionDest: 319 Annotation: Subtype: Link Rect: 237, 674, 249, 683 ActionDest: 319 Annotation: Subtype: Link Rect: 249, 674, 261, 683 ActionDest: 319 Annotation: Subtype: Link Rect: 264, 674, 276, 683 ActionDest: 319 Annotation: Subtype: Link Rect: 408, 649, 420, 658 ActionDest: 319 Annotation: Subtype: Link Rect: 420, 649, 432, 658 ActionDest: 319 Annotation: Subtype: Link Rect: 433, 649, 445, 658 ActionDest: 319 Annotation: Subtype: Link Rect: 365, 624, 377, 633 ActionDest: 319 Annotation: Subtype: Link Rect: 377, 624, 389, 633 ActionDest: 319 Annotation: Subtype: Link Rect: 452, 536, 464, 545 ActionDest: 319 Annotation: Subtype: Link Rect: 478, 498, 485, 507 ActionDest: 319 Annotation: Subtype: Link Rect: 485, 498, 497, 507 ActionDest: 319 Annotation: Subtype: Link Rect: 498, 498, 510, 507 ActionDest: 319 Annotation: Subtype: Link Rect: 292, 460, 304, 469 ActionDest: 319 Annotation: Subtype: Link Rect: 304, 461, 316, 469 ActionDest: 319 Annotation: Subtype: Link Rect: 247, 395, 254, 407 ActionDest: 78 Annotation: Subtype: Link Rect: 229, 73, 236, 85 ActionDest: 103 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 359, 722, 371, 731 ActionDest: 319 Annotation: Subtype: Link Rect: 371, 722, 383, 731 ActionDest: 319 Annotation: Subtype: Link Rect: 389, 710, 401, 719 ActionDest: 319 Annotation: Subtype: Link Rect: 498, 356, 510, 365 ActionDest: 319 Annotation: Subtype: Link Rect: 513, 340, 520, 352 ActionDest: 137 Annotation: Subtype: Link Rect: 396, 293, 408, 302 ActionDest: 319 Annotation: Subtype: Link Rect: 409, 293, 421, 302 ActionDest: 319 Annotation: Subtype: Link Rect: 396, 255, 408, 264 ActionDest: 319 Annotation: Subtype: Link Rect: 256, 115, 263, 126 ActionDest: 137 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 197, 172, 204, 184 ActionDest: 167 Annotation: Subtype: Link Rect: 455, 110, 462, 123 ActionDest: 167 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 443, 331, 455, 340 ActionDest: 319 Annotation: Subtype: Link Rect: 285, 306, 297, 315 ActionDest: 319 Annotation: Subtype: Link Rect: 312, 290, 319, 303 ActionDest: 214 Annotation: Subtype: Link Rect: 267, 190, 274, 202 ActionDest: 214 Annotation: Subtype: Link Rect: 225, 130, 237, 139 ActionDest: 319 Annotation: Subtype: Link Rect: 311, 117, 323, 126 ActionDest: 319 Annotation: Subtype: Link Rect: 424, 80, 436, 89 ActionDest: 319 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 513, 722, 525, 731 ActionDest: 326 Annotation: Subtype: Link Rect: 466, 475, 473, 487 ActionDest: 217 Annotation: Subtype: Link Rect: 487, 387, 494, 399 ActionDest: 217 Annotation: Subtype: Link Rect: 355, 277, 367, 286 ActionDest: 326 Annotation: Subtype: Link Rect: 288, 212, 295, 224 ActionDest: 217 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 285, 187, 297, 196 ActionDest: 319 Annotation: Subtype: Link Rect: 298, 187, 309, 196 ActionDest: 319 Annotation: Subtype: Link Rect: 310, 187, 322, 196 ActionDest: 326 Annotation: Subtype: Link Rect: 413, 175, 420, 184 ActionDest: 315 Annotation: Subtype: Link Rect: 420, 175, 432, 184 ActionDest: 319 Annotation: Subtype: Link Rect: 433, 175, 445, 184 ActionDest: 319 Annotation: Subtype: Link Rect: 445, 175, 457, 184 ActionDest: 319 Annotation: Subtype: Link Rect: 458, 175, 470, 184 ActionDest: 326 Annotation: Subtype: Link Rect: 452, 162, 464, 171 ActionDest: 319 Annotation: Subtype: Link Rect: 353, 137, 365, 146 ActionDest: 319 Annotation: Subtype: Link Rect: 217, 84, 224, 96 ActionDest: 260 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 410, 720, 417, 732 ActionDest: 260 Annotation: Subtype: Link Rect: 362, 694, 369, 706 ActionDest: 260 Annotation: Subtype: Link Rect: 341, 659, 353, 669 ActionDest: 319 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 435, 634, 447, 643 ActionDest: 319 Annotation: Subtype: Link Rect: 199, 596, 211, 605 ActionDest: 319 Annotation: Subtype: Link Rect: 495, 571, 507, 580 ActionDest: 326 Annotation: Subtype: Link Rect: 463, 445, 475, 454 ActionDest: 326 Page: Sequence: 10 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 424, 592, 560, 603 Annotation: Subtype: Link Rect: 166, 580, 272, 591 Annotation: Subtype: Link Rect: 339, 544, 346, 555 ActionDest: 167 Annotation: Subtype: Link Rect: 231, 532, 238, 543 ActionDest: 167 Annotation: Subtype: Link Rect: 511, 532, 517, 543 ActionDest: 167 Annotation: Subtype: Link Rect: 387, 520, 394, 531 ActionDest: 167 Annotation: Subtype: Link Rect: 231, 508, 237, 519 ActionDest: 217 Annotation: Subtype: Link Rect: 472, 508, 478, 519 ActionDest: 217 Annotation: Subtype: Link Rect: 313, 496, 319, 507 ActionDest: 217 Annotation: Subtype: Link Rect: 547, 496, 553, 507 ActionDest: 217 Annotation: Subtype: Link Rect: 385, 484, 392, 495 ActionDest: 217 Annotation: Subtype: Link Rect: 229, 472, 236, 483 ActionDest: 217 Annotation: Subtype: Link Rect: 469, 472, 476, 483 ActionDest: 260 Annotation: Subtype: Link Rect: 311, 460, 317, 471 ActionDest: 260 Annotation: Subtype: Link Rect: 547, 460, 553, 471 ActionDest: 260 Annotation: Subtype: Link Rect: 387, 448, 394, 459 ActionDest: 260 Annotation: Subtype: Link Rect: 229, 436, 236, 447 ActionDest: 260 Annotation: Subtype: Link Rect: 470, 436, 476, 447 ActionDest: 260 Annotation: Subtype: Link Rect: 408, 158, 445, 169 Annotation: Subtype: Link Rect: 498, 146, 536, 157 Annotation: Subtype: Link Rect: 367, 123, 405, 134 Annotation: Subtype: Link Rect: 343, 100, 380, 111 Annotation: Subtype: Link Rect: 345, 77, 383, 88 Annotation: Subtype: Link Rect: 389, 77, 425, 88 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 95, 722, 133, 732 Annotation: Subtype: Link Rect: 232, 698, 269, 709 Annotation: Subtype: Link Rect: 140, 676, 178, 686 Annotation: Subtype: Link Rect: 333, 652, 371, 663 Annotation: Subtype: Link Rect: 155, 630, 193, 640 Annotation: Subtype: Link Rect: 240, 607, 277, 617 Annotation: Subtype: Link Rect: 109, 584, 146, 594 Annotation: Subtype: Link Rect: 108, 549, 146, 559 Annotation: Subtype: Link Rect: 152, 549, 188, 559 Annotation: Subtype: Link Rect: 405, 525, 442, 536 Annotation: Subtype: Link Rect: 448, 525, 484, 536 Annotation: Subtype: Link Rect: 482, 514, 520, 525 Annotation: Subtype: Link Rect: 343, 479, 381, 490 Annotation: Subtype: Link Rect: 170, 457, 207, 467 Annotation: Subtype: Link Rect: 213, 457, 249, 467 Annotation: Subtype: Link Rect: 494, 445, 532, 456 Annotation: Subtype: Link Rect: 104, 423, 141, 433 Annotation: Subtype: Link Rect: 180, 400, 217, 410 Annotation: Subtype: Link Rect: 275, 376, 312, 387 Annotation: Subtype: Link Rect: 200, 354, 238, 364 Annotation: Subtype: Link Rect: 441, 330, 478, 341 Annotation: Subtype: Link Rect: 209, 307, 246, 318 Annotation: Subtype: Link Rect: 253, 307, 289, 318 Annotation: Subtype: Link Rect: 301, 284, 339, 295 Annotation: Subtype: Link Rect: 408, 261, 445, 272 Annotation: Subtype: Link Rect: 452, 261, 488, 272 Annotation: Subtype: Link Rect: 494, 249, 532, 260 Annotation: Subtype: Link Rect: 269, 226, 307, 237 Annotation: Subtype: Link Rect: 228, 192, 266, 203 Annotation: Subtype: Link Rect: 272, 192, 308, 203 Annotation: Subtype: Link Rect: 519, 169, 557, 180 Annotation: Subtype: Link Rect: 59, 158, 95, 168 Annotation: Subtype: Link Rect: 358, 134, 395, 145 Annotation: Subtype: Link Rect: 127, 112, 164, 122 Annotation: Subtype: Link Rect: 104, 77, 141, 88 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 302, 722, 339, 732 Annotation: Subtype: Link Rect: 407, 698, 444, 709 Annotation: Subtype: Link Rect: 328, 675, 366, 686 Annotation: Subtype: Link Rect: 427, 652, 464, 663 Checksum: 46b183fa Type: CRC32 Checksum: 57fa63ed6d54c6f83a1693ab388d3df8 Type: MD5 Checksum: 62a6b5f20e2ea8a47c0dc3d22a71d456b7631010 Type: SHA-1