text:
Patent application title: MICROARRAYS OF TAGGED COMBINATORIAL TRIAZINE LIBRARIES
Young-Tae Chang (Singapore, SG)
Young-Tae Chang (Singapore, SG)
Ho-Sang Moon (Gyeonggi-Do, KR)
New York University, NYU Medical Center, Department of Industrial Liaison
IPC8 Class: AC40B3004FI
USPC Class:
506 9
Class name: Combinatorial chemistry technology: method, library, apparatus method of screening a library by measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Publication date: 2009-11-19
Patent application number: 20090286693
Sign up to receive free email alerts when patent applications with chosen keywords are published SIGN UP
Inventors list
Agents list
Assignees list
List by place
Classification tree browser
Top 100 Inventors
Top 100 Agents
Top 100 Assignees
Usenet FAQ Index
Other FAQs
Patent application title: MICROARRAYS OF TAGGED COMBINATORIAL TRIAZINE LIBRARIES
Young-Tae CHANG
Ho-Sang Moon
New York University, NYU Medical Center, Department of Industrial Liaison
Origin: WASHINGTON, DC US
IPC8 Class: AC40B3004FI
USPC Class:
506 9
Patent application number: 20090286693
Triazine linkers can be used to prepare universal small molecule chips for
functional proteomics and sensors. These triazine linker compounds are
prepared by making a first building block by adding a first amine by
reductive amination of triazine, making a second building block by adding
a second amine to cyanuric chloride, and combining the first and second
building blocks by aminating the first building block onto one of the
chloride positions of the second building block. These triazine linkers
are then linked to a substrate for determining binding affinity of
1. A high density chip comprising a surface onto which are linked tagged
combinatorial trisubstituted triazine libraries, said triazine libraries.
2. The chip according to claim 1 wherein the triazines are linked to the surface with 2,2'-[1,2-ethanediyl-bus(oxy)]bismethanamine.
3. The chip according to claim 1 wherein the triazines are selected from compounds of the following formula: ##STR00072## wherein R1 is selected from the group consisting of ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## wherein R2 is selected from the group consisting of NH2, CH3(C═O)NH-- and CH5(C═O)NH.
4. The chip according to claim 1 wherein the triazines are selected from compounds of the following formula: ##STR00078## wherein R1 is a C1-C14 alcohol group directly bound to the triazine ring via an oxygen atom or a C1-C14 amino group directly bound to the triazine ring via a nitrogen atom, and R2 is a C1-C14 alkyl amine directly bound to the triazine ring via a nitrogen atom.
5. The chip according to claim 1 wherein the triazines are selected from the group consisting of: ##STR00079## ##STR00080##
6. The chip according to claim 1 wherein the surface is a glass slide.
7. The chip according to claim 1 wherein the amino end of the linker is connected to an activated functional group on the surface of the chip.
8. The chip according to claim 6 wherein the activated functional group is selected from the group consisting of isocyanate, isothiocyanate, and acyl imidazole.
9. A method for determining the binding affinity of proteins to a plurality of molecules comprising incubating a high density small molecule ship according to claim 1 with a plurality of labeled proteins and analyzing the labels to determine which molecules have affinity for which proteins.
10. The method according to claim 8 wherein the label is a fluorescent label.
[0001]The present application is a continuation in part of Ser. No. 10/267,044, filed Oct. 9, 2002, which claims priority from non-provisional application Ser. No. 60/339,294, filed Dec. 12, 2001, the entire contents of each of which are hereby incorporated by reference.
[0002]The present invention relates to microarrays containing tagged triazine libraries which can be used as universal small molecule chips for functional proteomics and sensors.
[0003]The Human Genome Project provided a huge amount of sequence data for dozens of thousands of genes. Elucidating the function of each gene (so-called functional genomics) is the next step in the challenge of understanding human genetics1. Conventionally, geneticists have investigated the function of unknown genes by comparing normal phenotypes with knock-out or over-expression of the target gene, based on the assumption that the phenotypic difference is closely related to the function of the target gene. Recent developments in RNAi2 and antisense techniques3 have make it possible to temporarily turn off given gene expression by targeting mRNA rather than the DNA genome itself.
[0004]A novel approach using chemical library screening to find an interesting phenotypic change by targeting specific gene products, that is, proteins, has emerged as an alternative tactic; this is called chemical genetics4. In chemical genetics, one chemical compound may specifically inhibit or activate one target protein (for purposes of illustration, called "protein A"). Thus, the compound is equivalent to the gene knock-out or over-expression of the corresponding gene A, as in conventional genetics.
[0005]Combinatorial library techniques5 facilitate the synthesis of many molecules. These techniques can be combined with high throughput screening (HTS) to screen many compounds to discover a novel, small molecule in the first step of chemical genetics study. Once one finds an intriguing small molecule, here referred to as "molecule A", that induces a novel phenotype in cells or in an embryonic system, the next step is to identify the target protein and the biochemical pathways involved. An affinity matrix on bead or a tagged molecule (photoaffinity, chemical affinity, biotin or fluorescence) obtained by modifying molecule A, is commonly used for identifying the target protein. The target can be fished out by binding affinity of the proteins to the immobilized molecule, followed by separation on gel and sequencing by tandem mass spectrometry (MS-MS) technique. As the affinity matrix isolation usually gives multiple proteins, including non-specific binders, it is best to compare the gel results with those of control matrices side by side. Desirable control matrices will be obtained from structurally similar, molecules to molecule A which are inactive. The proteins that bind only to the active affinity matrix, without binding to the control matrices, are promising target candidates. The candidate proteins are then purified and screened in vitro with molecule A to confirm that the isolated protein is truly protein A.
[0006]As a whole, successful chemical genetics work will identify a novel gene product (i.e., protein A), and its on or off switch, small molecule pairs. By analyzing the phenotype change, the function of protein A, which is the expression product of gene A, will be discerned. At the same time, the identified small molecule key, molecule A, is a useful biochemical tool to regulate the pathway of protein A, and may be a promising drug candidate as well.
[0007]Unfortunately, the current approach of chemical genetics intrinsically contains a very difficult step, that of modifying molecule A into an affinity molecule. In order to add a linker to molecule A without adversely affecting its activity, a thorough structure-activity relationship (SAR) study of molecule A is required to find a proper site for linker addition. This site is probably a site of molecule A exposed to the solvent direction from a binding pocket in protein A. This procedure is, in many cases, extremely cumbersome, and sometimes is even completely impossible.
[0008]It is an object of the present invention to provide tagged combinatorial triazine libraries that can be used for chemical genetics.
[0009]It is another object of the present invention to provide an improved method for chemical genetics.
[0010]It is a further object of the present invention to synthesize linker libraries by combinatorial methods for screening in phenotypic assays.
[0011]The present invention comprises a method for chemical genetics using library molecules carrying a linker (LL: library with linker) from the first step of the procedure. In this method, LL is synthesized by combinatorial methods and screened in phenotypic assays. The selected active compounds are directly linked to resin beads or to a tagging moiety without further SAR study using the already existing linker. Eliminating the requirement for structure-activity relationship determination dramatically accelerates the connection of function screening to the affinity matrix step. This reduces the assay time from months to days, making the chemical genetics approach much more practical and powerful than it has been heretofore.
[0012]FIG. 1 shows examples of triazine-linker compounds.
[0013]FIG. 2 shows a conventional synthetic pathway of triazine library by solution chemistry.
[0014]FIG. 3 shows an orthogonal solid phase synthesis pathway for the triazine library with linker according to the present invention.
[0015]FIG. 4 illustrates synthesis of building blocks according to the present invention.
[0016]FIG. 5 shows syntheses of triazine compound with linker.
[0017]FIG. 6 illustrates agarose bead synthesis of the triazine derivatives of the present invention.
[0018]FIG. 7 shows NHS-derivatized slides with 2688 triazine compounds spotted in duplicates and probed with human IgG-Cy3.
[0019]Triazine is used as the linker library scaffold. Triazines are used because they are structurally similar to purine and pyrimidine, and they have demonstrated their biological potentials in many applications. In particular, triazines have many drug-like properties, including molecular weight of less than 500, cLogP of less than 5, etc. As the triazine scaffold has three-fold symmetry, the modification is also highly flexible and able to generate diversity. Furthermore, the starting material, triazine trichloride, and all of the required building blocks, which are amines, are relatively inexpensive. Because if its ease of manipulation and the low price of the starting material, triazine has elicited much interest as an ideal scaffold for a combinatorial library, resulting in several triazine libraries having been published in the literature7. However, all of the reported library synthesis procedures, both for solid and solution phase chemistry, are based on sequential aminations using the reactivity differences of the three reaction sites. This is shown in FIG. 2, the conventional synthetic pathway of a triazine library by solution chemistry.
[0020]In this conventional method, the first substitution occurs at low temperatures while the second and third reactions require subsequently higher temperatures. This stepwise amination approach, however, is difficult to generalize for nucleophiles having differing reactivities. Thus, they may generate many byproducts together with the desired product. Substituted cyanuric dichloride moiety was loaded onto a TGlinker-functionalized resin, whereas previously, a linker mono-substituted (the linker as the first substituent) cyanuric dichloride was loaded onto the resin as the first step in the solid-phase synthesis (Scheme 1). This TG-linker-functionalized resin allows for rapid library diversification through simple splitting of the resin. As a consequence of the altered scheme, the second and more important improvement is the addition of primary alcohols to the library building block palette that were unattainable with our previous approach. Primary alcohols may only be efficiently and cleanly added to the cyanuric chloride scaffold as the first (of three) substitutions. This is due to the drastic decrease in reactivity seen with substituted cyanuric chloride analogues. Introducing an alcohol moiety as the first substituent, thus forming a building block II which can be subsequently loaded to a TG-linker-functionalized resin, is a very useful addition to our chemical toolbox and allows for N versus O atom substitution comparisons with hit compounds in later studies. The general tagged linker strategy is advantageous for a number of additional reasons. The basic linker used in all.
[0021]Scheme 1. General Synthetic Scheme for Construction of TG Triazine Library B Uttamchandani et al. Journal of Combinatorial Chemistry cases, 2,2-[1,2-ethanediyl-bis(oxy)]bisethanamine, is commercially available and affordable and is easily monoprotected (N-Boc) in one step. Compound cleavage from the resin and linker deprotection is accomplished simultaneously in one step. The linker provides a sufficient space between the compounds and the microarray surface, at the same time allowing for greater conformational flexibility in the immobilized compounds. Furthermore, its hydrophilic character may provide a more protein-friendly environment during subsequent microarray screening processes. Last, the amino functional group allows for facile small-molecule immobilization and for a rapid transition to further downstream studies, such as affinity matrix pull-down experiments, without the need for any hit compound modification.
[0022]The compounds were spotted, in duplicates, as an SMM on a modified glass substrate derived from standard microscope slides in a deterministic fashion that ensures immediate high-fidelity locus-based identification (Scheme 2). In total, 5376 spots corresponding to 2688 triazine-based library compounds were printed; 1152 of those were TG compounds and were synthesized as reported herein, and 1536 compounds were synthesized as reported previously by our group. In addition, we included in our arrayed grids a dye reference to not only validate the slide derivatization process, but also appropriately home in the software in the subsequent data acquisition.
[0023]The present process solves the problem of byproducts using a straightforward synthetic pathway that can be used for the general preparation of a trisubstituted triazine library. The present process does not use selective amination, which requires careful monitoring of the reaction and purification steps. Instead, the present process uses three different kinds of building blocks to construct the library. The first amine (linker) is loaded onto an acid-labile aldehyde resin substrate such as by reductive amination mono- or di-methoxybenzaldehyde resins. The second amine is then added to cyanuric chloride to form a building bock with the dichlorotriazine core structure. These two building blocks are then combined by amination of the first building block onto one of the chloride positions of the second building block. Any sequential over-amination on the other chloride position is efficiently suppressed by physical segregation from any other amine available on the solid support. The third building block, which can be a primary or secondary amine, then reacts with the last chloride position to produce the trisubstituted triazine. Since all reactions are orthogonal to each other, no further purification is required after cleavage of the final compound, as shown in FIG. 3. Using this established synthetic scheme, a linker was introduced in the trisubstituted triazine library to synthesize thousands of library linker compounds in amounts of about 1-2 mg.
Syntheses of Building Blocks
[0024]To a solution of 100 mg (0.543 mmole) cyanuric chloride, purchased from A cross Chemical Company, USA, and 0.05 ml DIEA, purchased from Aldrich Chemical Company, USA, in 5 ml anhydrous THF, purchased from Aldrich Chemical Company, USA, was added each amine or alcohol reagent (0.652 mmol, or 1.2 eq) at 0° C. The reaction mixture was stirred for 30 minutes at 0° C. After TLC checking, the reaction mixture was filtered and the solvent removed in vacuo. The compounds were purified by column chromatography. Each compound was identified by LC-MS (Agilent 1100 model). This scheme is shown in FIG. 4, and the identification of the building blocks is shown in Table 1.
TABLE-US-00001 TABLE 1 Identification of Building Blocks (A1-Y1) The products were identified LC-MS (Agilent 1100 model) Comp. Mass ID (m + 1) A1 235 B1 205 C1 219 D1 359 E1 299 F1 207 G1 273 H1 235 11 233 J1 289 K1 221 L1 269 M1 255 N1 256 O1 249 P1 315 Q1 241 R1 291 S1 285 T1 242 U1 206 V1 208 W1 332 X1 222 Y1 180
Syntheses of Triazine Library with Linker
[0025]To a solution of 1.0 g (1.1 mmole) PAL®-aldehyde resin, purchased from Midwest Bio-Tech, USA, was added 1.5 g (3.5 mmole) of Boc-linker (2-[2-amino-ethoxy-ethoxyethyl]-carbamic tert-butyl ester) in 50 ml anhydrous THF containing 10 ml of acetic acid at room temperature. The reaction mixture was stirred for one minute at room temperature and then 1.63 g (7.7 mmole, 7 eq) sodium triacetoxyborohydride was added. The reaction mixture was stirred for twelve hours and filtered. The resin was washed three times with DMF, three times with dichloromethane, three times with methanol, and three times with dichloromethane.
[0026]The next step was performed by general solid phase synthesis. To a solution of 1.0 g resin and 1 ml DIEA in 50 ml anhydrous THF at room temperature, amino-mono-substituted triazine compounds of a mono-alkoxy-substituted triazine (4 eq) was added. The reaction mixture was stirred for two hours at 60° C. and filtered. The resin was washed three times with DMF, three times with dichloromethane, three times with methanol, and three times with dichloromethane.
[0027]The final coupling step was performed by general solid phase synthesis. To the resin (10 mg) and 0.1 ml DIEA in 0.7 ml NMP was added 4 eq of each amine. The reaction mixture was stirred for two hours at 120° C. and filtered. The resin was washed three times with DMF, three times with dichloromethane, three times with methanol, and three times with dichloromethane. Resin cleavage was conducted using 10% trifluoroacetic acid in dichloromethane for 30 minutes at room temperature, after which the resin was washed with dichloromethane. The products were identified using LC-MS ((Agilent 1100 model).
[0028]FIG. 5 illustrates syntheses of triazine compounds with linker. In this Figure, the reagents are: [0029]a. 2-[2-amino-ethoxy-ethoxymethyl]-carbamic tert-butyl ester, 2% acetic acid in DMF, room temperature, one hour [0030]b. sodium triacetoxyborobutyride, room temperature, for twelve hours [0031]c. 2,4-dichloro-6-morpholine-4-yl-[1,3,5]-triazine, DIEA, at 60° C. for two hours [0032]d. cyclopentylamine or benzylamine, DIEA, at 120° C. for two hours [0033]e. 10% trifluoroacetic acid in dichloromethane for 30 minutes
[0034]FIG. 1 illustrates examples of triazine-linker compounds. These examples are for purposes of illustration only, and are not intended to be limiting of the invention.
[0035]Table 2 illustrates compounds synthesized by the method of the present invention which were identified by LC-MS (Agilent 1100 model).
TABLE-US-00002 TABLE 2 Identification of Synthesized Compounds (with LC-MS). The products were identified LC-MS (Agilent 1100 model). R1 R2 A B C D E F G H I J K L M 0 347 317 331 471 411 319 385 347 345 401 333 381 367 1 433 403 417 557 497 405 471 433 431 487 419 467 453 2 502 472 486 626 566 474 540 502 500 556 488 536 522 3 486 456 470 610 550 458 524 486 484 540 472 520 506 4 368 338 352 492 432 340 406 368 366 422 354 402 388 5 422 392 406 546 486 394 460 422 420 476 408 456 442 6 444 414 428 568 508 416 482 444 442 498 430 478 464 7 419 389 403 543 483 391 457 419 417 473 405 453 439 8 419 389 403 543 483 391 457 419 417 473 405 453 439 9 436 406 420 560 500 408 474 436 434 490 422 470 456 10 522 492 506 646 586 494 560 522 520 576 508 556 542 11 418 388 402 542 482 390 456 418 416 472 404 452 438 12 497 467 481 621 561 469 535 497 495 551 483 531 517 13 384 354 368 508 448 356 422 384 382 438 370 418 404 14 440 410 424 564 504 412 478 440 438 494 426 474 460 15 384 354 368 508 448 356 422 384 382 438 370 418 404 16 474 444 458 598 538 446 512 474 472 528 460 508 494 17 452 422 436 576 516 424 490 452 450 506 438 486 472 18 382 352 366 506 446 354 420 382 380 436 368 416 402 19 424 394 408 548 488 396 462 424 422 478 410 458 444 20 424 394 408 548 488 396 462 424 422 478 410 458 444 21 410 380 394 534 474 382 448 410 408 464 396 444 430 22 438 408 422 562 502 410 476 438 436 492 424 472 458 23 396 366 380 520 460 368 434 396 394 450 382 430 416 24 508 478 492 632 572 480 546 508 506 562 494 542 528 25 478 448 462 602 542 450 516 478 476 532 464 512 498 26 478 448 462 602 542 450 516 478 476 532 464 512 498 27 398 368 382 522 462 370 436 398 396 452 384 432 418 28 436 406 420 560 500 408 474 436 434 490 422 470 456 29 436 406 420 560 500 408 474 436 434 490 422 470 456 30 436 406 420 560 500 408 474 436 434 490 422 470 456 31 398 368 382 522 462 370 436 398 396 452 384 432 418 32 370 340 354 494 434 342 408 370 368 424 356 404 390 33 448 418 432 572 512 420 486 448 446 502 434 482 468 34 448 418 432 572 512 420 486 448 446 502 434 482 468 35 462 432 446 586 526 434 500 462 460 516 448 496 482 36 432 402 416 556 496 404 470 432 430 486 418 466 452 37 432 402 416 556 496 404 470 432 430 486 418 466 452 38 424 394 408 548 488 396 462 424 422 478 410 458 444 39 424 394 408 548 488 396 462 424 422 478 410 458 444 40 424 394 408 548 488 396 462 424 422 478 410 458 444 41 398 368 382 522 462 370 436 398 396 452 384 432 418 42 518 488 502 642 582 490 556 518 516 572 504 552 538 43 440 410 424 564 504 412 478 440 438 494 426 474 460 44 432 402 416 556 496 404 470 432 430 486 418 466 452 45 396 366 380 520 460 368 434 396 394 450 382 430 416 46 462 432 446 586 526 434 500 462 460 516 448 496 482 47 383 353 367 507 447 355 421 383 381 437 369 417 403 R1 R2 N O P Q R S T U V W X Y 0 368 361 427 353 403 397 354 318 320 444 334 292 1 454 447 513 439 489 483 440 404 406 530 420 378 2 523 516 582 508 558 552 509 473 475 599 489 447 3 507 500 566 492 542 536 493 457 459 583 473 431 4 389 382 448 374 424 418 375 339 341 465 355 313 5 443 436 502 428 478 472 429 393 395 519 409 367 6 465 458 524 450 500 494 451 415 417 541 431 389 7 440 433 499 425 475 469 426 390 392 516 406 364 8 440 433 499 425 475 469 426 390 392 516 406 364 9 457 450 516 442 492 486 443 407 409 533 423 381 10 543 536 602 528 578 572 529 493 495 619 509 467 11 439 432 498 424 474 468 425 389 391 515 405 363 12 518 511 577 503 553 547 504 468 470 594 484 442 13 405 398 464 390 440 434 391 355 357 481 371 329 14 461 454 520 446 496 490 447 411 413 537 427 385 15 405 398 464 390 440 434 391 355 357 481 371 329 16 495 488 554 480 530 524 481 445 447 571 461 419 17 473 466 532 458 508 502 459 423 425 549 439 397 18 403 396 462 388 438 432 389 353 355 479 369 327 19 445 438 504 430 480 474 431 395 397 521 411 369 20 445 438 504 430 480 474 431 395 397 521 411 369 21 431 424 490 416 466 460 417 381 383 507 397 355 22 459 452 518 444 494 488 445 409 411 535 425 383 23 417 410 476 402 452 446 403 367 369 493 383 341 24 529 522 588 514 564 558 515 479 481 605 495 453 25 499 492 558 484 534 528 485 449 451 575 465 423 26 499 492 558 484 534 528 485 449 451 575 465 423 27 419 412 478 404 454 448 405 369 371 495 385 343 28 457 450 516 442 492 486 443 407 409 533 423 381 29 457 450 516 442 492 486 443 407 409 533 423 381 30 457 450 516 442 492 486 443 407 409 533 423 381 31 419 412 478 404 454 448 405 369 371 495 385 343 32 391 384 450 376 426 420 377 341 343 467 357 315 33 469 462 528 454 504 498 455 419 421 545 435 393 34 469 462 528 454 504 498 455 419 421 545 435 393 35 483 476 542 468 518 512 469 433 435 559 449 407 36 453 446 512 438 488 482 439 403 405 529 419 377 37 453 446 512 438 488 482 439 403 405 529 419 377 38 445 438 504 430 480 474 431 395 397 521 411 369 39 445 438 504 430 480 474 431 395 397 521 411 369 40 445 438 504 430 480 474 431 395 397 521 411 369 41 419 412 478 404 454 448 405 369 371 495 385 343 42 539 532 598 524 574 568 525 489 491 615 505 463 43 461 454 520 446 496 490 447 411 413 537 427 385 44 453 446 512 438 488 482 439 403 405 529 419 377 45 417 410 476 402 452 446 403 367 369 493 383 341 46 483 476 542 468 518 512 469 433 435 559 449 407 47 404 397 463 389 439 433 390 354 356 480 370 328
[0036]Table 3 illustrates structures of R1 groups in the triazine compounds produced according to the present invention. These structures are for purposes of illustration only, and not for limitation.
TABLE-US-00003 TABLE 3 Structures of R1 Group. R1 Structure A ##STR00001## B ##STR00002## C ##STR00003## D ##STR00004## E ##STR00005## F ##STR00006## G ##STR00007## H ##STR00008## I ##STR00009## J ##STR00010## K ##STR00011## L ##STR00012## M ##STR00013## N ##STR00014## O ##STR00015## P ##STR00016## Q ##STR00017## R ##STR00018## S ##STR00019## T ##STR00020## U ##STR00021## V ##STR00022## W ##STR00023## X ##STR00024## Y CH3OH
TABLE-US-00004 TABLE 3 Structures of R2 Group. R2 Structure 0 Cl 1 ##STR00025## 2 ##STR00026## 3 ##STR00027## 4 ##STR00028## 5 ##STR00029## 6 ##STR00030## 7 ##STR00031## 8 ##STR00032## 9 ##STR00033## 10 ##STR00034## 11 ##STR00035## 12 ##STR00036## 13 ##STR00037## 14 ##STR00038## 15 ##STR00039## 16 ##STR00040## 17 ##STR00041## 18 ##STR00042## 19 ##STR00043## 20 ##STR00044## 21 ##STR00045## 22 ##STR00046## 23 ##STR00047## 24 ##STR00048## 25 ##STR00049## 26 ##STR00050## 27 ##STR00051## 28 ##STR00052## 29 ##STR00053## 30 ##STR00054## 31 ##STR00055## 32 ##STR00056## 33 ##STR00057## 34 ##STR00058## 35 ##STR00059## 36 ##STR00060## 37 ##STR00061## 38 ##STR00062## 39 ##STR00063## 40 ##STR00064## 41 ##STR00065## 42 ##STR00066## 43 ##STR00067## 44 ##STR00068## 45 ##STR00069## 46 ##STR00070## 47 ##STR00071##
[0037]Generally, R1 may be a C1-14 alcohol or amino group, a C1-14 alkyl group, phenyl substituted with at least one of F, Cl, methoxy, ethoxy, trifluoromethyl, or C1-6 alkyl; or benzyl substituted with at least one of F, Cl, methoxy, ethoxy, trifluoromethyl, or C1-6 alkyl. R2 may be a C1-14 amino group a C1-14 alkyl group, phenyl substituted with at least one of F, Cl, methoxy, ethoxy, trifluoromethyl, or C1-6 alkyl; or benzyl substituted with at least one of F, Cl, methoxy, ethoxy, trifluoromethyl, or C1-6 alkyl.
Agarose Bead Synthesis
[0038]In a 1 ml syringe cartridge (Ppcartridge with 20 m PE frit), 1 ml of Reacti-Gel 6X in acetone (purchased from Pierce), 10 ml of crosslinked agarose, 45-165 mm, >50 mmole/ml gel was added and 2 mL×10.1 M K2CO3 Reacti-Gel 6X in a 3 mL syringe cartridge was suspended with 1 mL of 0.1 M K2CO3. To this was added 100 mL (50 mM) in DMSO) triazine-linker compound with amine. The coupling buffer was removed and Tris buffer was added to block any excess reactive groups. The reaction mixture was washed twice with 10 mL H2O and twice with 10 mL PBS.
Application of Triazine Linker Library and Affinity Matrices
[0039]The triazine linker library molecules can be used in a variety of phenotypic assays to find interesting small molecules and their binding proteins in an expeditious way. These assays include Zebrafish embryo development, morphological changes in S-pombi, membrane potential sensing in cell systems, phenotypic screening in C-elenas, muscle regeneration in newt, tumorigenesis in brain cells, apoptosis and differentiation of cancer cells, cell migration and anti-angiogenesis. The active compounds are classified depending upon their ability to induce unique morphological changes, and these are then used for affinity matrix work.
[0040]Selected linker library molecules are loaded onto activated agarose beads via their amino-end linkers as described above. These affinity matrix beads are incubated with cell or tissue extract, and found proteins run on gel. The found proteins are analyzed using MS-MS sequencing after in-gel digestion to give the peptide sequences of the target protein.
[0041]The linker library molecules can be used for making a high density small molecule chip. Thousands of linker library molecules are immobilized on a glass slide by a spotting method, which can add hundreds to thousands or molecules to a slide. The amino end of the linker is connected to an activated functional group on the slide, such as isocyanate, isothiocyanate, or acyl imidazole. Fluorescent labeled proteins with different dyes are incubated with the slide. A scanner analyzes the color to give the absolute and relative binding affinity of different proteins on each compound. For example, no color means there is no activity with any kind of proteins. A strong mixed color means that the compounds are non-specifically active with multiple proteins. Exclusively stained compounds, with a singe color, indicate a selective bind of the relevant protein. Using this technique, thousands of small molecules can be tested in a shot time using a small amount of protein. In this approach, limited numbers of purified proteins compete with each other in the presence of multiple small molecules. This approach is analogous to DNA microarray technology, which has been important in advances in functional genomics. Although there have been some reports of protein chips 8, at yet no small molecule library chip has been demonstrated. Therefore, the small molecule chips of the present invention will offer totally new techniques in the field of chemical genetics, which will expand the study of the entire genome.
[0042]Immunoglobulins have seen numerous applications spanning immunoassays, diagnostics, and immunotherapeutics.1c The production of immunoglobulins, for example, valuable humanized variants, for therapeutic applications requires stringent purification measures before being administered as approved drugs. However high molecular weight ligands, such as staphylococcal protein A and streptococcal protein G, are unfavorable for medicinal applications for their potential pyogenic effect as well as for other problems, including low biological stability, leakage from solid support, and difficulty in large-scale production and purification, contributing to high overall cost.10 Recent literature has shown that triazines may prove useful small-molecule ligand alternatives to IgG. For example, Li et al. used compute raided molecular modeling to successfully identify triazene analogues that bind to IgG with affinity constants of 105-106 M-1.1c We thus hypothesized valuable potentials in screening human IgG against our arrays not only as proof of our overall concept but also in the discovery of efficacious ligands with direct relevance to industry.
[0043]Human IgG was fluorescently labeled with Cy3-NHS to allow for sensitive visualization of small molecule-IgG interactions on the array. Spotted slides alone, without incubation with labeled IgG, were also scanned to ensure that the fluorescence did not originate from the spotted compounds themselves. The resulting scans were typical to that seen in FIG. 1. Cases in which only one of the two duplicate spots displayed a substantive signal were dismissed as artifacts, and only hits that corroborated well in repeated experiments were deemed true positives. Three of the strongest hits on the array, based on intensity, were chosen for further validation, namely AMD10, AMD3, and K28. A faint positive, K42, and a negative, APF29, were also used as comparative benchmarks. In separate control experiments, other fluorescently labeled proteins (unrelated to human IgG) were used to screen against the same slide: none of the hits (e.g. AMD10, AMD3, K28, and K42) showed any positive binding, indicating that their binding toward human IgG is, indeed, highly specific. The spot intensities of these molecules are given in Table 1, with the background subtracted accordingly.
[0044]Dissociation constants were determined for each of the compounds selected using SPR on a Biacore X system with BiaEvaluation software. Competitive binding experiments were performed with differentially proportioned mixtures of a small molecule and protein A on a CM-5 chip immobilized with human IgG (see Supporting Information) (Table 1), which also ensures the small molecule binding to the same Scheme 2. General Experimental Scheme a (a) Directed immobilization of triazine libraries to generate a high-density microarray. (B) Incubation with a fluorescently labeled protein. (C) Removal of the unbound protein through washing. (D) Detection with instantaneous deconvolution of positive hits. (E) Assessing efficacy of hits using SPR. An averaged dissociation constant of 1.25×10-9 M was obtained for protein A with IgG alone. Further assessments made by passing 2.5 iM of a small molecule against the IgG surface were also performed to give measurable association levels that correlate with binding affinities (Table 1). Immobilizing the small molecules and applying IgG in the solution phase obtained equivalent binding measurements; however, by employing the competitive binding method described, a single chip surface may be used for screening against multiple potential small-molecule ligands, economizing the process. A φ2 value of <10 was obtained for all Kd measurements, denoting good statistical validity of the results obtained.
[0045]All three of the strong hits defined by the microarray screening were shown to give significant dissociation constants in the micromolar region with IgG. This relationship was further confirmed with a strong increase in response units (RU) when these three molecules were passed across an IgG-derivatized surface. AMD10, AMD3, and K28 gave the strongest results with the lowest Kd values of 4.35×10-6, 2.02×10-6, and 2.02×10-6 M, respectively. These values were more than an order of magnitude lower than that of the secondary binder, K42, which was only weakly positive on the microarray screen. Expectedly, the negative control gave the weakest binding signals. These results further validate that tagging of the target protein with the dye for array applications did not perturb its binding properties with the small molecules. It is also interesting to note that the dissociation constant (e.g., Kd), as well as Koff (Supporting Information), of the hits as determined by SPR correlated well with their fluorescence intensity obtained from the microarray screening, with tight-binding compounds consistently giving stronger fluorescence spots. Overall, the dissociation constants obtained from the best hits identified in our experiments compare favorably with what was reported previously with other triazine-based small molecules.1c
[0046]The present process provides a high-throughput screening system to detect small-molecule ligands for virtually any target and have shown its efficacy in discerning targets of a model protein, human IgG. The SMM used libraries of tagged triazine compounds, one of which is a novel library possessing a high degree of diversity and synthetic versatility. The tagged libraries intrinsically factor the linker in the screen, thus eliminating potential false negatives and increasing throughput. Further, we have developed a fully addressable microarray containing a few thousand compounds, with each compound, once being displayed as a positive, becoming immediately identifiable.
[0047]FIG. 7. NHS-derivatized slides with 2688 triazine compounds spotted in duplicates and probed with human IgG-Cy3. The actual-sized array is enclosed in a blue box, with blow-ups describing the loci and the corresponding molecules that were selected for further assessments. (a-c) Correlated with strongly positive molecules, producing spot intensities at least two times that of the background. An intermediate (d) and a negative control (e) were also picked for comparative assessment. The reference control (f) is shown, and four sets of the Cy3-NH2 dye were printed at the ends of the grids.
TABLE-US-00005 TABLE 4 Microarray and SPR Results Obtained with Five Selected Triazines array signal Small (fluorescence molecule units) Kd/M o2 Kd/M χ2 AMD10 179 (++) 4.35 × 10-6 3.42 AMD03 185 (++) 2.02 × 10-6 2.32 K28 143 (++) 2.54 × 10-6 0.917 K42 65 (+) 6.02 × 10-5 2.19 APF29 <10 (-) 1.51 × 10-4 4.02
solely by its position within the grid without the need for additional assessment. The IgG ligands discovered herein may soon find potential applications in the large-scale purification of immunoglobulins and would be useful alternatives to existing protein A-based isolation and purification systems. Studies are underway to establish the utility of the hits in this respect as well as work to identify further triazine ligands for other candidate proteins and DNA targets.
Experimental Section
[0048]Materials Used. Unless otherwise noted, materials and solvents were obtained from commercial suppliers (Acros and Aldrich) and were used without further purifications. PAL-aldehyde (4-formyl-3,5-dimethoxyphenoxymethyl) resin from Midwest Bio-Tech (Catalogue No. 20840, Lot no. SY03470, loading level 1.10 mmol/g) was used for the generation of library compounds. Building block II compounds, made by solution phase chemistry, were purified by flash column chromatography on Sorbent Technologies silica gel, 60 Å (63-200 mesh). TLC was performed on SAI F254 precoated silica gel plates (250-im layer thickness). All library products were identified by an LC-MS at 250 nm (Agilent Technology, HP1100) using a C18 column (20×4.0 mm) with a gradient of 5-95% CH3CN--H2O (containing 0.1% acetic acid) as an eluent over 4 min.
[0049]Preparation of Triazine Libraries. The parallel syntheses of triazine libraries, excluding the TG library reported herein, and the synthesis of Boc-linker (2-[2-aminoethoxyethoxyethyl]carbamic tert-butyl ester), were previously published.
[0050]Preparation of TG Libraries. General Procedure for Coupling of the Linker onto the Resin (Scheme 1). To a solution of PAL-aldehyde resin (1.0 g, 1.1 mmol) was added Boc-linker (2-[2-aminoethoxyethoxyethyl]carbamic tert-butyl ester) (1.36 g, 5.5 mmol, 5 equiv) in THF (50 mL, containing 2% of acetic acid) at room temperature. The reaction mixture was stirred for 1 h at room temperature, followed by the addition of sodium triacetoxyborohydride (1.63 g, 7.7 mmol, 7 equiv). The reaction mixture was stirred for 12 h and filtered. The resin was washed with DMF (3 times), dichloromethane (3 times), methanol (3 times), and dichloromethane (3 times). The resin was dried in vacuo.
[0051]General Procedure for Building Block I. Cyanuric trichloride (1 equiv) was dissolved in THF with DIEA (10 equiv) at 0° C. The desired amine (1.2 equiv) in THF was added dropwise. For addition of alcohols to cyanuric chloride, the same reaction conditions were followed, except 2.5 equiv of K2CO3 was used instead of DIEA. The reaction mixture was stirred and monitored by TLC. Reaction time was 45 min to 1 h. A solid precipitate slowly formed. Upon completion of the reaction, the reaction mixture was quickly filtered through a plug of flash silica and washed with EA.
[0052]The filtrate was evaporated in vacuo. The resulting products were purified using flash column chromatography (particle size 32-63 mm) and characterized by LC-MS.
[0053]General Procedure for Coupling Building Block I with the Resin. Building block I (0.44 mmol) was added to the resin (0.11 mmol) in DIEA (1 mL) and anhydrous THF (10 mL) at room temperature. The reaction mixture was heated to 60° C. for 3 h and filtered. The resin was washed with DMF (5 times); alternatively with dichloromethane and methanol (5 times); and finally, with dichloromethane (5 times). The resin was dried in vacuo.
[0054]General Procedure for the Final Amination on the Resin and Product Cleavage Reaction. Desired amines (4 equiv) were added to the resin (10 mg), coupled with building block I and Boc linker, in DIEA (8 iL) and 1 mL of NMP/n-BuOH (1:1). The reaction mixture was heated to 120° C. for 3 h. The resin was washed with DMF (5 times); alternatively with dichloromethane and methanol (5 times); and finally, with dichloromethane (5 times). The resin was dried in vacuo. The product cleavage reaction was performed using 10% trifluoroacetic acid (TFA) in dichloromethane (1 mL) for 30 min at room temperature and washed with dichloromethane (0.5 mL). The purity and identity of all the products were monitored by LC-MS at 250 nm (Agilent 1100 model); more than 90% of compounds demonstrated >90% purity.
[0055]AMD03: ESI-MS (M+H)+calcd, 580.4; found, 581.6.
[0056]AMD10: ESI-MS (M+H)+calcd, 540.4; found, 541.5.
[0057]TGK28: ESI-MS (M+H)+calcd, 421.3; found, 422.5.
[0058]TGK42: ESI-MS (M+H)+calcd, 503.3; found, 504.5.
[0059]APF29: ESI-MS (M+H)+calcd, 578.3; found, 579.5.
[0060]Preparation and Analysis of Small-Molecule Arrays. Preparation of Slides Activated with N-Hydroxysuccinimide Esters. 2 Briefly, 25 mm×75 mm slides (Fisher Scientific) were cleaned in piranha solution (sulfuric acid/hydrogen peroxide, 7:3). An amine functionality was incorporated onto the slides by silanization using a solution of 3% (aminopropyl)triethoxysilane in 2% water and 95% ethanol. After 1-2 h of soaking, the slides were washed with ethanol and cured at 150° C. for at least 2 h. Subsequently, the amine slides were incubated in a solution of 180 mM succinic anhydride in DMF for 30 min thereafter were transferred to a boiling water bath for 2 min. The slides were washed again in ethanol and dried under a stream of nitrogen. The carboxylic acid moieties now created on the slide surface were activated with a solution of 100 mM of TBTU (O-(benzotriazol-1-yl)-N,N,N,N-tetramethyluronium tetrafluoroborate), 200 mM DIEA, and 100 mM N-hydroxysuccinimide in DMF, thus generating the NHS-derivatized slides.
[0061]The slides once generated were stored in a desiccator at -20° C. and used within 3 months.
[0062]Microarray Printing. By individually weighing the solid compounds, triazine stock solutions were prepared to 2.5 mM in DMF, and 40-iL preparations were distributed across seven 384-well plates to give a total of 2688 distinct and pure compounds. Slides were spotted on an ESI SMA arrayer (Ontario, Canada) with the printhead installed with eight ArrayIt Chipmaker 7 Microspotting pins (Telechem, U.S.A.). Printing was performed in duplicate, and the pins were washed and sonicated in ethanol between samples. A repotting blotting process was also performed on plain slides to ensure spot uniformity. An additional solution of 0.2 mM Cy3-NH2 11 reference was spotted at the ends of the grids using a final eighth plate.
[0063]After spotting, the slides were allowed to sit for at least 12 h in situ and then were quenched by washing in an 1% ethanolamine (in DMF) bath for 2 h. After rinsing with Journal of Combinatorial Chemistry Discovery of Small-Molecule Ligands of Human IgG E ethanol and drying, the arrayed slides were stored in a desiccator at 4° C. The slides were stable for extended periods and, when required, were simply brought to room temperature.
[0064]Protein Screening with Labeled Human IgG. Proteins were tagged with Cy3-NHS by incubating 5 iL of 20 mM Cy3-NHS (Amersham Biosciences, U.K.) with 200 ig of human IgG (Calbiochem, U.S.A.) in a sodium bicarbonate buffer at pH 8. After 1 h of incubation, the labeled protein was separated from the free dye by a NAP-5 Sephadex G-50 column (Amersham Biosciences, U.K.). Before incubation with the labeled protein, the slides were preblocked to remove any nonspecific binding by soaking in 1% BSA in PBS for 1 h. After a brief rinsing with water, the slides were incubated with the labeled IgG.
[0065]A 1000-fold dilution of the above protein preparation was used as the incubation solution in a PBS buffer containing 1% BSA with the small-molecule arrays using the cover slip method for 30 min in a humid incubation chamber. Excess IgG was then removed by washing with distilled water. The background was further reduced using repeated washes with PBST. Control screening experiments were performed with unrelated, fluorescently labeled proteins to ensure spots identified from the IgG experiment were highly specific.
[0066]Slide Scanning and Analysis. Slides were scanned on an ArrayWoRx scanner (Applied Precision, U.S.A.). Excel sheets were prepared to assign various compounds to specific numberings that could be readily tallied with reference numbers generated by the program. The ArrayWoRx software allows generation of reference files in which the spotting arrangement and the program overlay the spots on the results obtained, allowing compounds to be assigned in a rational fashion to every position. The software also provides large-scale analysis of hits, which rapidly analyzes the entire array, further enhancing throughput.
[0067]Surface Plasmon Resonance (SPR) Determination of Dissociation Constants. Maintenance. SPR measurements were made on a Biacore X system (Biacore AB, Uppsala, Sweden). Various maintenance steps were performed to ensure that the instrument was kept in good working conditions. The integrated flow cell was washed, sanitized, and maintained using standard cleansing reagents on a weekly basis. Calibration checks were performed quarterly to ensure that the signal was of good quality, and the instrument was kept separate from other equipments to prevent interference. When not in use, the system was docked with a spare chip and flushed with water at a low flow rate of 5 iL/min to prevent clogging. The system was primed at least twice before use or for the purpose of initiating a new buffer type. A desorb process was performed every 2 days during periods of active use to remove proteins or other contaminating compounds that may have accumulated within the flow cell.
[0068]Procedures. Various approaches were conceived to assess the Kd values of the interactions. One successful method immobilized the small molecule on the CM-5 sensor chips and ran them through varying concentrations of IgG. This was found to be a suitable but costly method, because it required multiple chips for analyzing the binding interactions of different small molecules. We conceived that it would be easier to immobilize IgG on the surface and apply differing proportioned mixtures of the "hit" small molecule and protein A (Amersham Biosciences, U.K.). The Heterogeneous Analyte Module of the BiaEvaluation software using this method worked efficiently in providing the required Kd values of the small molecules with IgG, allowing a single chip to be used repeatedly to assess different binding constants. Checks showed that up to 200 injections could be delivered on a single chip with negligible loss in signal output, with a regeneration buffer of dilute HCl, pH 2 (used throughout). Additionally, protein A was found not to bind to any of the small molecules that were tested. The presence of DMF in our small-molecule preparation was problematic in SPR measurements, because it perturbed the refractive index of the buffer, causing anomalous results. In our case, we overcame this problem by using a reference flow cell, thereby negating the effect of differing refractive indexes of the sample and buffer during sample introduction.
[0069]Immobilizing Samples on CM-5 Chips. The standard protocol supplied by the manufacturers was employed. The system was set to 25° C. and equilibrated with degassed HBS buffer (comprising 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005 v/v P20 surfactant). One flow cell was activated with 1:1 NHS/EDC for 8 min with a flow rate of 5 iL/min, while the other was kept as a reference.
[0070]After coupling IgG to give an immobilization increase of 5000 RU, the surface was quenched using 1 M ethanolamine, pH 9, for 7 min.
[0071]Determination of Association Levels of Small Molecule with IgG. Small-molecule preparations of 2.5 iM were passed through the IgG-activated flow cell with the reference automatically negating bulk effects. The flow rate used was 30 iL/min using PBS buffer, and 50 iL of each small molecule was applied over 1 min. The increase in response units observed directly tallied with small molecules that actively bound to the IgG-activated surface, thus providing a semiquantitative method to intercompare putative binders.
[0072]Determination of Dissociation Constants of Small Molecule with IgG. Twenty-five-microliter preparations of the small-molecule analytes (250 nM, 1 iM, 2 iM, 5 iM) were premixed with an equal volume of 238 nM of protein A and injected to the flow cell. The flow rate used was 30 iL/min with degassed PBS buffer. The results were entered into the BiaEvaluation module where the Heterogeneous Analyte Module was applied to obtain the binding and association constants required. Again, the reference cell was used to eliminate any bulk effects arising from the differing buffer composition.
[0073]Thus the use of microarrays of tagged combinatorial triazine libraries dramatically accelerates chemical genetics techniques by connecting phenotypic assay and affinity matrix work without any delay, rather than requiring months to years of SAR work. This powerful technique will revolutionize the study of the genome and will open a new field of chemical proteomics. Combining the binding protein data with a phenotype index will serve as a general reference of chemical knock-out. The present invention makes it possible to identify novel protein targets for drug development as well as drug candidates.
[0074]The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.
[0075]1. Dhand, R. Ed., "Nature insight: Functional Genomics", Nature, 2000, 405, 819-867. [0076]2. (a) RNA interference: listening to the sound of silence, Zamore, P. D. Nat. Struct. Biol. 2001, 8, 746-750. (b) RNAi and brain function: was McConnell on the right track, Smalheiser, N. R.; Manev, H.; Costa, E. Trends Neurosci. 2001, 24, 216-218. (c) Gene silencing by double-stranded RNA, Carthew, R. W. Curr. Opin. Cell Biol. 2001, 13, 244-248. (d) A conserved mechanism for post-transcriptional gene silencing?, Maine, E. M. Genome Biol. 2000, 1, 1018. (e) High-throughput reverse genetics: RNAi screens in Caenorhabditis elegans, Bargmann, C. I. Genome Biol. 2001, 2, 1005. (f) Genome-wide RNAi, Barstead, R. Curr. Opin. Chem. Biol. 2001, 5, 63-66. [0077]3. (a) Morpholino antisense oligomers: design, preparation, and properties, Summerton, J.; Weller, D. Antisense Nucl. Acid Develop. 1997, 7, 187-195. (b) Morpholino antisense oligomers: the case for an RNase H-independent structural type, Summerton, J. Biochim. Biophys. Acta 1999, 1489, 141-158. (c) RNA-based silencing strategies in plants, Matzke, M. A.; Matzke, A. J.; Pruss, G. J.; Vance, V. B. Curr. Opin. Genet. Dev. 2001, 11, 221-227. [0078]4. (a) Chemical genetics resulting from a passion of synthetic organic chemistry, Schreiber, S. L. Bioorg. Med. Chem. 1998, 6, 1127-1152. [0079]5. (a) Combinatorial Chemistry (Methods in Enzymology); Abelson, J. N. Ed.; Academic Press: New York, 1996. (b) Combinatorial Libraries-synthesis, screening and application potential; Cortese R. Ed.; Walter de Gruyter: Berlin, 1996. (c) Molecular Diversity and Combinatorial Chemistry: Libraries and Drug Discovery; Chaiken, I. M. and Janda, K. D. Ed.; American Chemical Society: New York, 1996. (d) Combinatorial Chemistry and Molecular Diversity in Drug Discovery; Gordon, E. M., Kerwin, J. F. Eds.; Wiley & Sons: New York, 1997. (e) Chemistry Synthesis and Application; Wilson, S. R., Czarnik, A. W. Eds.; Wiley & Sons: New York, 1997. (f) Combinatorial Peptide and Nonpeptide Libraried (a Handbook); Jung, G. Ed.; Wiley & Sons: New York, 1997. (g) A Practical Guide to Combinatorial Chemistry; Czarnik, A., DeWitt, S. Eds.; American Chemical Society, 1998. (h) Bunin, B. A. The Combinatorial Index; Academic Press: New York, 1998. (i) Terrett, N. K. Combinatorial Chemistry; Oxford University Press: Oxford, 1998. (j) Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries; Obrecht, D., Villalgordo, J. M. Eds.; Pergamon: Netherlands, 1998. (k) Combinatorial Chemistry. Synthesis, Analysis, Screening; Jung G. Ed.; Wiley-VCH: Weinheim, 1999. (l) Dorwald, F. Z. Organic Synthesis on Solid Phase; Wiley-VCH: New York, 2000. (m) Seneci, P. Solid-Phase Synthesis and Combinatorial Technologies; Wiley & Sons: New York, 2000. [0080]6. (a) Screening for novel antimicrobials from encoded combinatorial libraries by using a two-dimensional agar format, Silen J. L; Lu A. T.; Solas D. W.; Gore M. A.; MacLean D.; Shah N. H.; Coffin J. M.; Ehinderwala N. S.; Wang Y.; Tsutsui K. T.; Look G. C.; Campbell D. A.; Hale R. L.; Navre M.; DeLuca-Flaherty C. R. Antimicrob. Agents Chemother. 1998, 42, 1447-1453. (b) A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand, Teng, S. F.; Sproule, K.; Hussain, A.; Lowe, C. R. J. Mol. Recognit. 1999, 12, 67-75. (c) Design, synthesis and evaluation of biomimetic affinity ligands for elastases, Filippusson, H.; Erlendsson, L. S.; Lowe, C. R. J. Mol. Recognit. 2000, 13, 370-381. [0081]7. (a) Incorporation of carbohydrates and peptides into large triazine-based screening libraries using automated parallel synthesis, Gustafson, G. R.; Baldino, C. M.; O'Donnel, M. E.; Sheldon, A. Tarsa, R. J.; Verni, C. J.; Coffen, D. L. Tetrahedron 1998, 54, 4051-4065. (b) Spatially Addressed Synthesis of Amino- and Amino-Oxy-Substituted 1,3,5-Triazine Arrays on Polymeric Membranes, Scharn, D.; Wenschuh, H.; Reineke, U.; Schneider-Mergener, J.; Germeroth, L. J. Comb. Chem. 2000, 2, 361-369. (c) Solution- and solid-phase synthesis of combinatorial libraries of trisubstituted 1,3,5-triazines, Masquelin, T.; Meunier, N.; Gerber, F.; Rosse, G. Heterocycles 1998, 48, 2489-2505. (d) Libraries of N-alkylamino heterocycles from nucleophilic aromatic substitution with purification by solid supported liquid extraction, Johnson, C. R.; Zhang, B.; Fantauzzi, P.; Hocker, M.; Yager, K. M. Tetrahedron 1998, 54, 4097-4106. (e) Library generation through successive substitution of trichlorotriazine, Stankova, M.; Lebl, M. Mol. Diversity. 1996, 2, 75-80. [0082]8. Printing proteins as microarrays for high-throughput function determination, MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. [0083]9. ArQule: Incorporation of Carbohydrates and Peptides into Large Triazine-Based Screening Libraries Using Automated Parallel Synthesis. Gustafson, G. R.; Baldino, C. M.; O'Donnel, M. E.; Sheldon, A. Tarsa, R. J.; Verni, C. J.; Coffen, D. L. Tetrahedron 1998, 54, 4051-4065. [0084]10. Selectide Corporation: Library generation through successive substitution of trichlorotriazine. Stankova, M.; Lebl, M. Mol. Diversity. 1996, 2, 75-80. [0085]11. Arris Pharmaceutical: Johnson, Charles R.; Zhang, Birong; Fantauzzi, Pascal; Hocker, Michael; Yager, Kraig M. Libraries of N-alkylamino heterocycles from nucleophilic aromatic substitution with purification by solid supported liquid extraction. Tetrahedron (1998), 54(16), 4097-4106. CODEN: TETRAB ISSN:0040-4020. CAN 128:308415 AN 1998:233899 [0086]12. Abbott: Hajduk, Philip J.; Dinges, Juergen; Schkeryantz, Jeffrey M.; Janowick, David; Kaminski, Michele; Tufano, Michael; Augeri, David J.; Petros, Andrew; Nienaber, Vicki; Zhong, Ping; Hammond, Rachel; Coen, Michael; Beutel, Bruce; Katz, Leonard; Fesik, Stephen W. Novel Inhibitors of Erm Methyltransferases from NMR and Parallel Synthesis. J. Med. Chem. (1999), 42(19), 3852-3859. CODEN: JMCMAR ISSN:0022-2623. CAN 131:308768 AN 1999:567005 [0087]13. Humboldt-Universitaet: Scharn, Dirk; Wenschuh, Holger; Reineke, Ulrich; Schneider-Mergener, Jens; Germeroth, Lothar. Spatially Addressed Synthesis of Amino- and Amino-Oxy-Substituted 1,3,5-Triazine Arrays on Polymeric Membranes. J. Comb. Chem. (2000), 2(4), 361-369. CODEN: JCCHFF ISSN:1520-4766. CAN 133:135605 AN 2000:355907 [0088]14. Hoffmann-La Roche: Masquelin, Thierry; Meunier, Nathalie; Gerber, Fernand; Rosse, Gerard. Solution- and solid-phase synthesis of combinatorial libraries of trisubstituted 1,3,5-triazines. Heterocycles (1998), 48(12), 2489-2505. CODEN: HTCYAM ISSN:0385-5414. CAN 130:196625 AN 1999:50090 [0089]15. Affymax: Silen J L; Lu A T; Solas D W; Gore M A; MacLean D; Shah N H; Coffin J M; Bhinderwala N S; Wang Y; Tsutsui K T; Look G C; Campbell D A; Hale R L; Navre M; DeLuca-Flaherty C R Screening for novel antimicrobials from encoded combinatorial libraries by using a two-dimensional agar format. ANTIMICROBIAL AGENTS AND CHEMOTHERAPY (1998 June), 42(6), 1447-53. Journal code: 6HK. ISSN:0066-4804. DN 98287588 PubMed ID 9624492 AN 1998287588 [0090]16. U. Cambridge: Teng, Su Fern; Sproule, Kenny; Hussain, Abid; Lowe, Christopher R. A strategy for the generation of biomimetic ligands for affinity chromatography. Combinatorial synthesis and biological evaluation of an IgG binding ligand. J. Mol. Recognit. (1999), 12(1), 67-75. CODEN: JMORE4 ISSN:0952-3499. CAN 131:30755 AN 1999:224014 [0091]17. U. Iceland: Filippusson, Horour; Erlendsson, Lyour S.; Lowe, Christopher R. Design, synthesis and evaluation of biomimetic affinity ligands for elastases. J. Mol. Recognit. (2000), 13(6), 370-381, 3 Plates. CODEN: JMORE4 ISSN:0952-3499. CAN 134:174701 AN 2000:857136 [0092]18. Closest in terms of biological activity and structure: Abbott 1: Henkin, Jack; Davidson, Donald J.; Sheppard, George S.; Woods, Keith W.; McCroskey, Richard W. Preparation of triazine-2,4-diamines as angiogenesis inhibitors. PCT Int. Appl. (1999), 66 pp. CODEN: PIXXD2 WO 9931088 A1 19990624 CAN 131:58855 AN 1999:404953 [0093]19. Abbott 2: Shock, Richard U. 2-[2-(5-Nitrofuryl)]-4,6-diamino-s-triazine. US 2885400 19590505 CAN 53:94898 AN 1959:94898 [0094]20. ArQule: Coffen, David L.; Hogan, Joseph C., Jr. Synthesis and use of biased arrays. PCT Int. Appl. (1998), 53 pp. CODEN: PIXXD2 WO 9846551 A1 19981022 CAN 129:302216 AN 1998:706192 [0095]21. Trustees of Boston University: Panek, James S.; Zhu, Bin. Synthesis of aromatic compounds by Diels-Alder reaction on solid support. PCT Int. Appl. (1998), 26 pp. CODEN: PIXXD2 WO 9816508 A2 19980423 CAN 128:308494 AN 1998:251157 [0096]22. ISIS Pharmaceuticals, Inc.: Cook, P. Dan; An, Haoyun. Preparation of Compounds or Combinatorial Libraries of compounds having a plurality of nitrogenous substituents. PCT Int. Appl. (1998), 187 pp. CODEN: PIXXD2 WO 9805961 A1 19980212 CAN 128:180338 AN 1998:112497 [0097]23. Hoffman-La Roche: Huber, Ulrich. Silanyl-triazines as light screening compositions. Eur. Pat. Appl. (1999), 26 pp. CODEN: EPXXDW EP 933376 A2 19990804 CAN 131:130123 AN 1999:505797
User Contributions:comments("1"); ?> comment_form("1"); ?>
Inventors list
Agents list
Assignees list
List by place
Classification tree browser
Top 100 Inventors
Top 100 Agents
Top 100 Assignees
Usenet FAQ Index
Other FAQs
Patent applications by Ho-Sang Moon, Gyeonggi-Do KR
Patent applications by Young-Tae Chang, Singapore SG
Patent applications in class By measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Patent applications in all subclasses By measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
User Contributions:
Comment about this patent or add new information about this topic:
|People who visited this patent also read:
|Patent application number
|Adaptive Content Selection
|Dynamic Interface to Read Database Through Remote Procedure Call
|Management Device, Management Method, And Recording Medium


text: I've been in communication with our travel agent, Thomson holidays welfare team, and the hotel we're going to, over many weeks trying to find out if its possible to get a tiny amount of extra baggage allowance to take GF foods with me. Thomson wanted to know the weight of the food. I figured I needed to know if the hotel would be willing to prepare it first (long life bread, pasta, possibly pizza) first and its taken weeks to find out. Finally got a nice email from the hotel in Turkey saying yes, we're willing to prepare your food. Calculated about 2kg for bread, pasta and pizza, and emailed Thomson's Welfare team. Got the reply today. Sorry, the cabin baggage cannot be more that the 5kg allowance, and any food you take must be in its original, unopened packaging. Not very accommodating are they? I could put the stuff in my hold baggage but it will surely get mashed to a paste. And what about the pizza I usually take on flights to stave off the hunger? How can I put the topping on it and cook it while it is still in its original unopened packaging? Whoever said that travelling abroad was easy?

subset: commoncrawl


text:
Here is a Post I did for the class last time it was taught. Issues related to 3D printing are, we will discover, updated a bit in 2017, but the challenges remain. So here is a primer on the kinds of notions we'll be exploring in class, in this case related to 3D printing.
There are a lot of sides to 3D printing archaeology...
Ever since Sustainable Archaeology obtained a 3D printer, I have been struggling with very conflicting thoughts on this capacity we have. On the one hand, I'm dazzled by the technology and think it pretty darn cool to print out objects to size or scaled... or even reassembled. On the other hand, the ability to print a 3D model - to "adjust" or improve on the original artifact - feels a bit like a line being crossed, raising for me a host of questions about the limits of dissemination, who should be getting a say on what can and can't get printed, the obvious "let's make some money from these" sentiments that people seem too quickly to rush towards, etc. On the other hand, I find we are asked all the time by Descendant groups asking for prints of artifacts so they can have a copy, it can be displayed in a Council office, they have a copy of an object repatriated and reburied, or a desire to have a printed copy of an important object in a ceremony. So I appreciate that this is a complex topic.
One recurring strand of these musings has been an uncertainty I have over the increasing tendency for Museums and cultural institutions to widely make 3D models of objects in their holdings accessible online. Clearly part of the impulse to provide wide access and appreciation of the heritage, and at least in some cases embracing the principles of Open Access, I was intrigued by a post I stumbled across recently that talked about how the British Museum has decided to allow people to download and print 3D models of select artifacts from its holdings.
As the post notes, other Museums are also following suit. Of course, the Smithsonian is all about digitizing millions of its holdings, and you can print a bust of Abraham Lincoln... or Barak Obama, to your heart's content! This is part of a broader trend of Museums to digitize art and collections, and make them accessible and even inviting people to be creative in repurposing the images. Generally, the sentiment, reflected in comments such as by Nina Simon, is that this is a good thing, creating access where there was none before. And there are plenty of examples of why printing is good for heritage science.
The British Museum is providing access to these models through Sketchfab, Which is an upload and share site for all kinds of 3D models... you can come across artifacts, sites and other heritage features available as 3D models, some done very well, some less so. More notable is Threeding, a web page that purports among other things, to preserve the past by allowing you access to high resolution 3D models of various types of heritage objects, including a few Egyptian objects. Apparently Threeding is Now partnering with Artec Group to expand its offerings of heritage objects.
So here's the thing. The British Museum, Smithsonian, and others scan objects that are clearly a "global" cultural heritage, mostly of other nations and peoples, making that accessible to be consumed and repurposed by anyone anywhere. Indeed, the objects the British Museum has put up so far are all not of English heritage, and speaks to the legacy of the global harvesting of heritage objects over that last few centuries by the British colonial empire. Over at Threeding, a perhaps more insidious feature is charging a fee for 3D models (Roman gravestones for 15-25 dollars a pop... Aztec figure free!). My radar starts going off when I see these trends, as I wonder what the implications are for things like appropriation and Intellectual Property (IP). Certainly the broader implications of how 3D printing can really mess with IP and copyright is a very hot topic for discussion, from Slate Magazine and Zdnet, to a slew of thought pieces from law firms, such as here, and even the Financial Times sees in 3D printing the emergence of the next Napster challenge to copyright laws (see also 3Dprint). Of course, this makes makes me wonder if IP lawyers aren't chomping at the prospect for the "business" this will generate?!
But what of 3D printing cultural heritage? Well the idea is that the "good" of preservation is at least one strand driving concerns to ensure digitization, and even printing, for archaeology - and for museums - continues, but the ethics are complicated. And the logics behind copyrighting 3D prints a dog's breakfast of contradictory thought, as seen here, and here. But, as Threeding makes clear, making money is going to drive justification for providing access too. This all plays into a broader question of the intellectual property of archaeological objects... cuz now Indigenous images, forms of ceramic decoration, structures, and, really, anything physical that is archaeology, and also someone's or everyone's heritage, can be continually re-purposed, not just as images but as objects too.
So of course I have no answers here, just musings! And I will no doubt be musing about these ideas in class, and picking your brains for your thoughts, too. And hey, kicking around these ideas will also help shape what I say writing on this topic, thus requiring me to need to cite you all to ensure I don't misappropriate our collective brainstorming! So let me know what you think!


text:
New Models
Top 100
Our Picks
Live Cams
Live Cams
Site Navigation
Discount Memberships
Advanced Search
Model Search
Male Porn Stars
Paysite Galleries
Suggest A Model
Barelist Exclusives
Barelist Articles
Barelist Blog
Barelist Cams
Barelist Interviews
Barelist Babes Galleries
Barelist Top 25 Babes
Browse Models
Select Models...
Browse Pornstars
Browse Centerfolds
Browse Male Porn Stars
Browse Teens
Browse Mature
Browse Asians
Browse Latinas
Browse Ebony
Browse Caucasians
Browse Big Tits
Browse Small Tits
Browse Blondes
Browse Brunettes
Browse Black Hair
Browse Redheads
Names Starting With "A"
Names Starting With "B"
Names Starting With "C"
Names Starting With "D"
Names Starting With "E"
Names Starting With "F"
Names Starting With "G"
Names Starting With "H"
Names Starting With "I"
Names Starting With "J"
Names Starting With "K"
Names Starting With "L"
Names Starting With "M"
Names Starting With "N"
Names Starting With "O"
Names Starting With "P"
Names Starting With "Q"
Names Starting With "R"
Names Starting With "S"
Names Starting With "T"
Names Starting With "U"
Names Starting With "V"
Names Starting With "W"
Names Starting With "X"
Names Starting With "Y"
Names Starting With "Z"
Barelist Offers
Social Networks
Twitter - Follow Us
Pinterest - Follow Us
Facebook - Like Us
tumblr. - Follow Us
Instagram - Follow Us
Google+ - Add Us
MySpace Account
YouTube Videos
Browse Categories
Picture Galleries
Video Galleries
Big Boob Galleries
Small Boob Galleries
Blonde Galleries
Brunette Galleries
Redhead Galleries
Hardcore Galleries
Lesbian Galleries
Teen Galleries
Asian Galleries
Ebony Galleries
Latina Galleries
Bookmark Us
Mobile Site
RSS Feed
Featured Gallery
Featured Model
Alexis Texas Interview For Barelist
Adult industry superstar Alexis Texas spends a little time with Barelist
Feb 15, 2012 - Posted By: Peter
We are extremely excited to have had the honor of interviewing adult industry superstar Alexis Texas. Alexis has always been one of our favorite porn stars, and our visitors agree with Alexis' amazing all time rank of 4th most popular model on Barelist. That's an amazing accomplishment seeing that there are over 10,000 models featured on Barelist.
Alexis has always been known for her amazing ass, but we always thought that Alexis is the total package... with amazing good looks and a stunning body. Alexis also does just as well in glamour photo shoots as she does in full on hardcore scenes, we just love seeing models with such a broad range. Alexis has been on all of the top adult sites including
Naughty America
, and has been honored by all the major industry awards including AVN and FAME. We would really like to thank Alexis for taking the time out of her busy schedule to do our interview, and please keep checking her
profile page
for all of the newest Alexis Texas picture and video galleries. Please also take some time to visit her personal site :
Alexis Texas Official Site
What was your first job?
My first job was at a nursing home as a personal care assistant
How and when did you get into the adult industry?
I got into the business in 2006 when I was approached by Shane's world who was doing a line called college amateur tour.
How does your family feel about your decision to be an adult model?
My family respects and is supportive of the decision I made to become an adult model.
If you had not joined the adult industry, what do you think you would be doing now?
Being a nurse
Do you get recognized in real life?
Do you prefer video or photo shoots?
I like them both for obvious different reasons ;)
Where is the most exotic location you have shot at?
What are your favorite and least favorite parts of your body?
Fav is my boobs I think that the are overlooked by my big booty ;)
Who is your favorite adult star (past or present)?
What is your favorite sex toy?
Do you exercise on a regular basis? What kind?
Nope the only exercise I get is from sex and dancing !!
Where did you go on your last vacation (not work related)?
Do you have any pets?
Yes 2 dogs kimbo and Suri
What is the last book you read?
Girlvert by Ashley blue
Do you have a guilty pleasure TV show? Song?
General hospital I've been watching it for years ;)
What is your dream car?
What is your favorite alcoholic beverage?
Peach ciroc
Do you have a hobby that would surprise most people?
I like playing basketball
Do you have any special talents?
I can play the flute
What's the best way for your fans to contact you?
My twitter
What are the best sites to see your work?
Image Courtesy of Holly Randall
Alexis Texas is represented by LADirect Models
Return to Barelist Interviews...
Visitor Comments:
Your Name
Alexis is just an amazing woman here, Her face, her ass, her legs, her pussy... She is a Woman of dreams!! And simply a stunningly beautiful woman....
Posted by: Elias on 5/29/2012 2:10:27 PM
Copyright © 2009-2018 Barelist.com. All Rights Reserved.
All models, actors, actresses and other persons that appear in any visual portrayal of actual or simulated sexually explicit conduct appearing on, or otherwise contained in, this Website were over the age of eighteen (18) years at the time the visual image was produced. Records required for all depictions of actual sexually explicit conduct by Title 18 U.S.C. 2257 are on file with the appropriate Records Custodian.
18 U.S.C.2257 Statement
Contact Us


text:
Preschool is back in full swing and the 2013-2014 school year is off to a
We've been having a blast in preschool so far this year. My students just finished up a unit about apples and letter A - they really LOVED learning about and, of course, eating apples! Here are a few pictures from our apple unit.
Apple Printing - we made these nifty little handles out of our apples which let us actually eat more of the apples than the way I used to do it! The kids really thought the 'handle' was great!
Letter A Tape & Sticker projects - great fine motor practice for early letter learners! Just tape the letter on a surface using painters taper or colored masking tape. Provide tiny stickers for placing along the lines of the letter. When students are finished, tape over top of their work with packing tape, peel entire letter off and place on a piece of colored paper to display. Great artifact for portfolio! I usually put the letter on our table, but students love doing this work on other surfaces like the windows, doors or walls.
|Painters Tape & Sticker Letter Practice
Letter A hunt... huddle up and GO! Color, cut and tape these great bracelets around little wrists for instant interest in letters! I've had students pointing out letter A in the environment for days without any additional prompting.
|Alphabet Bracelets can be found in my TpT store!
We also enjoyed using some of the games and worksheets form my Apple Roll, Count & Graph early literacy and math pack.
Now back to the hustle and bustle - off to pick up my own toddler, finish dinner, walk the dog, do the laundry and of course grade papers and come up with a master plan for tomorrow's activities! A teacher's work is never done...


text:
"Breakfast with Lucian," Geordie Greig's juicy, eye-popping book about Lucian Freud, the notoriously priapic painter best known for raw portraits that stripped his sitters bare in every sense, doesn't pretend to be objective or comprehensive. Greig offers a fond but by no means whitewashed account of how Freud's spectacularly messy life relates to his extraordinary body of work as "the greatest realist figurative painter of the twentieth century."
A grandson of Sigmund Freud, the budding artist escaped from Nazi Germany to England with his family when he was 10, in 1933. Greig writes, "Just like Sigmund, it was Lucian's business to get people to sit on beds or couches, and to reveal more about themselves than perhaps they wished to show."
Two years after the artist's death at 88, Greig, an arts journalist and editor who worked his way into Freud's confidence in the last 10 years of his life, reveals more about him than this adamantly private man might have wished to show. "Breakfast with Lucian" is at once broader in scope and more personal than art critic Martin Gaylord's fascinating 2010 book "Man with a Blue Scarf: On Sitting for a Portrait by Lucian Freud."
Bookshelf: Sign up for our email newsletter
Greig makes the case that the closely connected obsessions that drove Freud were painting and sex, and he analyzes his art and "mind-boggling merry-go-round of liaisons" with equal finesse and fervor. That said, he reminds us that "In trying to understand the complexity that is Lucian Freud, it is necessary to stay focused on his art. The pictures tell who he slept with and spent time with. He mostly kept the names hidden but the paintings don't lie."
Freud got away with what Greig calls his "blatantly selfish life" by essentially charming the pants off people and then keeping his scores of lovers — and the many children they produced — hidden from one another. Freud dated 17-year-olds when he was 16 and continued to bed 17-year-olds well into his 70s. Many were younger than his daughters. Indeed, some were the daughters of former lovers or the girlfriends of the sons of former lovers.
"This was musical beds on a grand and anarchic scale," Greig writes, adding that Freud was "a relentless and somewhat cruel lover" but also "one of life's eels, always wriggling away from any and every grasp."
While not all of his models had sex with him, nearly all of his lovers modeled for him. The process was both an excruciating test of endurance and a path to further intimacy — and, as it turned out, immortality in oil. Greig explains: "It was never easy. Paint for Lucian was 'pain' with a 't' on the end, as one of his sitters noted."
Along with Freud's sexual profligacy and self-destructive passion for gambling, Greig captures the intensity of the artist's ambition and drive, his exacting work ethic and his numerous "splintered" friendships, including with fellow artist Francis Bacon. Greig's own friendship with Freud provides access to the chaos and squalor of his home and studio — littered with used brushes, flicked paint splotches and the carcasses of half-eaten dinners. His portrait comes alive with descriptions of Freud's "ferret-thin figure," "shabby-chic style," penchant for silk scarves, nougat candy, wads of cash and hair-raising drives in his brown Bentley.
Freud's outrageous, egregious behavior comes through more clearly than his oft-cited charm and magnetism. And although Greig surely deserves some sort of patience award for untangling Freud's snarled web of lovers, the bodies pile up not just alarmingly but a smidge tediously. (A notable exception is Guinness heiress Lady Caroline Blackwood, Freud's second and last wife, whose story Greig manages to follow all the way to her deathbed because of his relationship with her daughter, Ivana.)
The book becomes even more interesting when it turns to Freud's offspring — 14 of whom he acknowledged, borne by six mothers between 1948 and 1984. An appended family tree helps keep them straight, including the three born to three mothers in 1961. Greig says the numbers are no doubt higher, perhaps as great as 40.
Six daughters and one son posed naked for their father. "Journalists had a field day trying to explain the Freudian significance of children in their teens or early adulthood stripping bare for their father," Greig writes. Yet he contends that, although Freud's children craved more of him than he gave, "they all fell under his spell" and most "loved him unreservedly, despite all his obvious failures and complications as a father."
When he died in 2011, several had never met one another. All 14 were left equal portions of his staggering 96 million pounds estate.
Some may argue that paintings like "Hotel Bedroom," "Naked Man with Rat" and "And the Bridegroom" require neither explanations nor back-stories to convey their power. But Greig's book, a rare case in which the text and illustrations are equally gripping, brings into sharp focus this bold iconoclast who "pushed boundaries, artistic as well as sexual." Even better, it makes us look more closely and deeply — and see more.
McAlpin reviews books regularly for NPR.org and the Washington Post and writes the Reading in Common column for the Barnes & Noble Review.
Breakfast With Lucian
The Astounding Life and Outrageous Times of Britain's Great Modern Painter
Geordie Greig
Farrar, Straus & Giroux, 260 pp, $30


text:
Scaffolding Party Makeover
This young beauty Mary is for a vacation in Marmaris, Turkey. She loves nature, sea and the sunshine but she also aware of fashion, style and look. Today there will be a big party on the beach and Mary will attend that too. Help her to get a complete makeover and make her look best ever. Be little bit flexible when doing her make up so that she look elegant and sizzling too.
Tags: dressupkiss.com, dressupkiss, Scaffolding, party girls, makeover, makeup, blush


text:
NEW YORK (Reuters) - Private equity investors have found another way to get into the growing U.S. exchange traded fund (ETF) market: investing in firms that manage ETF portfolios.
Seven years ago, venture capitalists clamored to invest in start-up ETFs - investment funds that are traded much like stocks. But with over 1,460 ETFs now available in the U.S., the landscape is crowded. That has led to a growing interest in ETF model portfolio firms - the companies that manage a portfolio made up solely of ETFs.
The model portfolio invests in a selection of ETFs, rather than individual securities. On the other end, a retail investor gets exposure to a basket of index ETFs. The portfolio manager actively manages that basket and can switch in and out of ETFs.
FTV Capital, Aquiline Capital Partners LLC, Century Capital and Grail Partners are among the private equity firms actively looking to acquire or make investments in such firms.
Model ETF portfolios have become more popular as people turn away from traditional mutual funds in search of less expensive and more transparent options, said Andrew Gogerty, an ETF managed portfolio strategist at Morningstar.
Unlike mutual funds, which often pay brokerage firms to sell their portfolios at-cost to investors, ETF portfolios only charge investors management fees, which means they are cheaper and there are fewer potential conflicts of interest, Gogerty said.
Equity mutual funds saw $2.5 billion in outflows in the first quarter of this year while equity ETFs saw $22.6 billion in new money, according to Lipper.
BlackRock's iShares ETF business keeps one of the most comprehensive lists of model ETF portfolios available today. Called the iShares Connect Program, it includes the majority of providers in the U.S., with more than 200 strategies run by 104 managers with a total of $46 billion in assets.
The firm projects the ETF model portfolio market to hit $120 billion by the end of 2015. Venture capitalists say they expect model ETF portfolios to become mainstream in the next few years.
"The big-hit sensation of the mid-2000s was anything ETF," said Liz Nesvold, managing partner of Silver Lane Advisors, a New York-based investment banking firm that is representing a number of model ETF portfolio providers in discussions with potential investors. "Now the focus is on how to use the ETFs."
Charles Schwab Corp. (SCHW.N) put ETF model portfolios on the map for private equity and retail investors in 2010 when it acquired Windward Investment Management, a $3.8 billion ETF model portfolio provider. Then, in June 2011, TA Associates bought a 54 percent stake in ETF model portfolio provider Stadion Money Management LLC. Terms were not disclosed.
Not long after, Morningstar began ranking these portfolios, leading more private equity investors to take notice. Those investors say there a number of trends that will contribute to the growth of the ETF portfolio market.
Several years of volatile markets have created a new appreciation among investors for tactical allocations - where managers can jump from one asset class to another to generate returns - experts said. ETF model managers can do this while mutual funds managers usually cannot.
Later this year new regulations require investors and employers to be shown the fees they pay for their 401(k) investments. That could pave the way for cheaper ETF models to gain traction in retirement plans.
"We think ETF model portfolios will stack up very well versus target-date mutual funds, which we think have not been good for most investors," said Chris Lalonde, a principal at Century Capital Management, a Boston-based private equity firm that hopes to invest in a model portfolio provider this year.
Most target-date funds only include portfolios managed by the fund's parent company, Lalonde said. ETF model providers are not bound to use specific ETFs and are therefore likely to perform better, he said.
Grail Partners LLC, chief executive Don Putnam said the firm is looking at ETF model portfolio providers even though in April 2011, it sold its own actively-managed ETF business after it failed to gain enough assets. Putnam said he believes there is more growth potential in the ETF model portfolio business today.
One challenge for venture capital firms is determining whether model ETF portfolio providers have enough resources in place to sell their products.
Some providers have a sales force. Others have partnered with a firm that sells the portfolios for them. But many have neither or maintain only a small sales force, making it hard to gain assets, experts said.
"You can have a great product but if you don't understand distribution you will have $100 million in assets and low profitability," said Ben Cukier, a partner at private equity firm FTV Capital, which invests in a number of ETF providers.
Discerning a model ETF firm's performance can also be tricky. Many providers are new and others haven't been in business long enough to discuss anything more than hypothetical historical performance.
Most firms said they will not consider investing in a provider that does not have at least three or five years of actual performance. That has limited the options and led some private equity firms to stay on the sideline - for now.
"The firms are so small that we would not invest in these at such an early stage," said Jeffrey Lovell, a managing director at Lovell Minnick Partners. "But it will stay on our radar."
(This story has been refiled to specify firms are all private equity firms)
Reporting by Jessica Toonkel, editing by Jennifer Merritt, desking by Andrew Hay


text:
And here we are on another Friday and if you're anything like me you're wondering what happened to your week, or planning what to do over the weekend…… and possibly craving some major amounts of chocolate. Fear not, I've got that part covered!
So I was wondering recently if our late fall/early winter months were going to be slow this year. They're the months that my mind still thinks of as summer since I grew up in the Northern Hemisphere, but now where I live they're just consecutive months of cold and wet. That was still a pretty dumb thing to do on my part though because now, of course, they are already filling up with plans. For example, there is another missions team planning a trip soon. If any of you have somehow managed to follow along with all my shenanigans since last year (doubtful) then you may remember me telling you about the previous team. Some of our really close friends here in Chile are missionaries and they sort of act as the home base for when their church is planning a mission trip down here. Last year my family welcomed the group that came to use our house for their services a few times and of course there was food involved. It was a crazy few weeks of planning, visiting, and tons of cooking, but so much fun too.
This year we've offered to keep as many from the team as need a place to stay, so I'm pretty positive I'll be playing bed and breakfast for those two weeks. That may not sound like fun to most people but I'm crazy enough to be super psyched about it. There are sure to be a ton of hilarious moments and planty laughing with so many people under one roof, not to mention it means more people around to feed, which is where I come in. My planning gears are already shifting into fifth!
Oh, and sidenote: The gear reference reminded me, I have been trying to force myself to face mastering a stick shift vehicle because there aren't many automatics here. I am going to be completely honest and admit that it terrifies me! I can't seem to get the initial concept to work with my hands and feet. The last time I was trying a few months ago, well let's just say I didn't do so great, but I am determined to get it down. I got my license driving an automatic and I'm stinking twenty years old now! I want to drive. In fact, that's why I'm telling you guys, to keep me motivated. You see, because I keep wanting to chicken out every time I try to plan a practice and I figured if I tell you then you can help make me brave. Ughh, why is it so frustratingly difficult to master the coordination?
Okay, but like we already acknowledged, it's Friday so I should probably hurry my butt up and tell you about these deep dark chocolate cookies that I'm sharing today! They are not only filled with the wonderful magic of spice and chocolate together, but I also made some homemade java chips which are basically a mocha version of chocolate chips and they are really simple.
The recipe makes more java chips than you'll use for one batch of these but I figured you guys won't have any trouble finding a use for the extra! I mean come on, it is the weekend! Also, since I'm a people pleaser when it comes to food, I just wanted you to know this if you are gawking at these and thinking they look delicious but you're not into spice and/or coffee with your chocolate (I'll try not to judge). You can make them hold-the-mexican or hold-the-Jo (skip the spices or java chips altogether). It'll make these simply fudgy double chocolate chip cookies but if you must do this, don't forget to add plenty of extra chocolate chips! Happy Friday peeps!
- 1/4 cup coconut oil
- 1/4 teaspoon salt
- 1/4 cup dairy free semi-sweet chocolate
- 1/2 tablespoon instant espresso powder
- 2 tablespoons cocoa powder
- 1/2 teaspoon vanilla extract
- 1 cup powdered sugar
- 1/2 cup 1 stick Miyoko's "butter" (or any dairy free butter), cut into pieces
- 2 tablespoons full-fat coconut milk
- 1/4 cup applesauce or pumpkin puree
- 1 cup sugar
- 1/3 cup cocoa powder
- 1 teaspoon baking soda
- 1/2 teaspoon salt
- 2 teaspoons vanilla extract
- 1 2/3 cups all purpose flour
- 1 teaspoon cinnamon
- 1/2 teaspoon chili powder
- 1/4 teaspoon paprika
- 1/8 teaspoon cayenne pepper
- 1/2 cup dairy free semi-sweet chocolate chips or chunks, divided
- Melt coconut oil over medium low heat. Reduce heat to low and add salt and chocolate and stir until melted evenly. Set aside for a moment and combine the espresso powder, cocoa powder, and powdered sugar in the bowl of a stand mixer. Scrape the contents of your saucepan into the bowl and mix on low until combined then add vanilla and mix well, You should have a thick and shiny looking chocolate "dough". Now line a tray or cutting board with parchment and press into a semi-even layer (you can use a rolling pin if you prefer.) Cover with plastic wrap and place in freezer for at least 30 minutes while you make the cookie dough.
- Combine the butter and sugar in the bowl of a stand mixer and beat on medium high speed until creamed. Stop and scrape sides of bowl, then add the coconut milk, vanilla, soda, baking powder, salt, and cocoa. Mix in the applesauce or pumpkin puree until combined. Mix in flour and spices until dough is smooth. Cover bowl and place in fridge for to chill as long as possible while you cut your java chips.
- Preheat oven to 350 degrees F (180 C) and line 2 cookie sheets with parchment. Remove the java chip sheet from your freezer and use a sharp knife to cut it into small squares. Pour most of the chips you just cut along with about 1/4 cup dark chocolate into the bowl of dough and mix just until combined to avoid smashing your chips. You'll use the rest of your java and dark chocolate chips for the tops.
- Roll the dough into 1 1/2 tablespoon sized balls and place them about 1 inch apart on prepared cookie sheets. Press a few of your reserved chips into the tops of dough balls and bake for 7 to 9 minutes, just until the edges start to look done (you want the middle to stay a little fudgy). Remove and cool for as long as you can stand the urge to devour them.
Since the java chips are homemade they might melt and burn easily on your tray but if you're having this problem try placing the dough balls in the freezer for at least an hour prior to baking.


text:
Netflix's Stranger Things was one of 2016's BIGGEST shows. Set in 1984, Season 2 is undoubtedly one of the most anticipated television events of the year and Netflix just uploaded the trailer for it. Half way through, an iconic voice fills your ears...
I was waiting for an MJ reference or wink since the first season. I hope the show includes Thriller in one of the episodes of this second season, not just the trailer or at least mention Michel in some way or another. I can't wait to see it!
Caleb McLaughlin (Lucas) posted this pic on his Twitter account last year though. LOVE his cool tee so much!
Loved season one so much! Can't wait for this and an MJ nod is great! It's not the 80's without MJ!
Like I keep saying MICHAEL IS LOVED.
I was just watching the trailer and I was thinking to myself, "Those organ chords sound... familiar. Can't place it though." Then when Vincent Prince's rap kicked in I said out loud, "whaaaaaaaaaaaaaaaat?" :lol:
Awesome surprise!
It's so cool how the trailers only 3-4 hours old and it's already at almost a million views. I was reading the comments for the trailer on Reddit and there's lots of praise for it's use of Thriller! I'm really digging what they did to the song too. A Redditor posted a breakdown of how it differs from the album version we're all accustomed to:
I went back and listened to the original Thriller, and the trailer may not be using a 'cover' by some other artist, but the synths when it starts are not from the original song. It's a really interesting remix they've done - it's like they wrote almost 1:40 of Thriller-like atmospheric music and gradually slide into the real song.
At 0:58, the atmospherics that start have the same notes & chords as the organ part from Thriller, but they're on new instruments. Similarly, at 1:10 - 1:20, that frenetic mid-range part and the low rumbling bass part are the synth and bass parts from the original, but they're being done on 2017 instruments.
The Vincent Price laugh at 1:20 is obviously original... but when he starts his "Darkness falls" monologue, the backing still isn't the real thing. It's synced up like the real song, though. At 1:36, right after the first MJ vocals, you hear a pipe organ, and that definitely is from the original -- this is where the mix starts to become the real song.
By 1:53 it's mostly the original, but there are a few things that have still been added to punch up the mix! At 2:09 a lot of the modern parts have dropped out and it's like 90% the original... but they've still chopped a bunch of the 1980s synthesized werewolves & screams out, and trimmed it down (or built it from parts). [Credit to Jurph]
I love all the changes they made to it.
It was done so perfectly! Amazing use of an amazing song
Great. Fits perfectly. Loved Stranger Things.
Maybe this has something to do with the Thriller rerelease this year?!
Makes sense as the second season takes place around the time Thriller was at it's peak and the short film was just released.
I haven't seen any of season 1, so I don't know how much if anything (in terms of characters, story etc) is carried over to season 2. However, these little screen caps from the trailer of Season 2 reminded me of MJ:
1. Toy monkey on the shelf in pic 1
2. Fair isle sweater ( reminds me of the Australian knitted jacket)
3. The dustbin (and Oscar the grouch)
Last edited by myosotis; 23-07-2017 at 12:51 PM.
'We may not change the world in one day but we still can change some things today, in our small way.'[/SIZE][/SIZE]
The trailer has only been out for 14 hours and it already has 23,100,000+ views on Facebook alone... combine that with the other versions and you realize that in just the past 14 hours, 20+ million people have heard Thriller... woah
And many people praised the use of Thriller calling it brilliant and the best use of Thriller ever. It was indeed brilliant and spot on since this new alien creature Will and the rest of the gang have to face looks more terrifying than the previous one. No homage to the 80s movies and pop culture would've been completed without Michael Jackson which is Stranger Things. It certainly put a smile on my face! ^_^
I have loved the first season, can't wait for the new one Hope they'll mention Michael also in the series or something.


text:
Reverse Plank with Tricep Dip
This week's #MondayMove is 'an amazing move for the triceps, shoulders and core using just your bodyweight', says Anya Lahiri, trainer at Barry's Bootcamp.
Get into a reverse plank position with your hands under your shoulders and facing backwards
Balance on your heels and bend your knees slightly
Bend your elbows and dip down as far as you can go and then push back up so your arms are fully extended. Try and keep your elbows as narrow as possible
Repeat 10 times (3 sets)
If you are more advanced you can raise alternate legs in the air
Anya is wearing Nike Pro 7.5cm Tiger Shorts, £23. Nike Pro Classic Bra, £22. Nike G87 Training Vest, £26. Nike Zoom Agility Training Shoes, £100.