Field of the Invention

The present invention is an improved method of making sequenced chemical compounds through the application of laser printing technology to light directed spatially addressed parallel chemical synthesis.

Background of the Invention

Chemicals of sequenced units, such as proteins which are composed of sequences of amino acids may be produced by solid phase chemistry wherein chem¬ ical units attached to a solid support are reacted with chemicals that attach other selected units to the units attached to the solid support to form unit chain compounds. Such two unit compounds may then be treated with the same or other chemicals repeatedly to form any predetermined number of units of sequenced compounds.

By providing photolabile protecting groups to the exposed units and sequentially unmasking cer¬ tain areas of such units by light irradiation and exposing such unprotected areas to a reactive chem¬ ical disposed to attach a desired unit to the

exposed units it is possible to create two unit chains in a confined area of the support. By re¬ peating such steps in the same or other areas of the support and repeating varying chemical treat- ments as set forth above it may be readily seen that numerous differing chemical chain compounds may be effected in a confined space on a single suppor .

This technology is of particular significance in the biological production of antibodies with se¬ quences of amino acid units that will exhibit a high level of binding and specificity for a pro¬ tein. The binding of an antibody to a specific antigen is critically important in biological systems, since the specificity and strength of the antibodyantigen interaction is crucial to the oper¬ ation of the immune system. The specificity of an antibody for a protein has been traced to highly variable segments in the antibody chain generally consisting of 6 to 10 amino acid sequences. It has been found that antibody segments containing one highly variable sequence, called a domain, exhibit high levels of binding and specificity for a pro¬ tein ("Single-Domain Antibodies Promise New Chemi- cal. Medical Applications" C&E News, November 20, 1989) .

The determination of the set of polypeptides that bind specifically to any given protein would be of obvious use in a variety of therapeutic and diagnostic treatments. Brute force determination of the sequences has been thought of as all but im¬ possible. There are 20 common amino acids, so the number of possible 6 member polypeptides is 20 ⁶ , or 64 million.

A recent development has shown promise in determining these sequences, through the synthesis of large numbers of peptides bound to a solid sup¬ port. This method combines techniques of solid phase peptide synthesis, photolabile protecting groups, and photolithography to achieve light directed, spatially addressable parallel chemical synthesis. The method works as follows. A solid support (typically a glass microscope slide) is reacted with aminopropyltriethoxysilane to form amine groups on its surface. These groups are then reacted with an amino acid whose amine function is protected by a photolabile protecting group. The surface is then masked, for example, the surface is everywhere covered except for a rectangular strip exposing 5% of exposed surface. The surface is then irradiated with UV light, activating only 5% of the exposed surface. The surface is then re¬ acted with an amino acid whose amine function is also protected by a photolabile group; only the activated portion of the surface reacts with the amino acid. The masking, irradiation, and re¬ action step is repeated, with a different area exposed and a different amino acid reacted with the surface. After 20 steps, 20 parallel strips exist on the surface, each strip containing a different amino acid bound to the surface.

This process is then repeated, but with masks rotated to a position perpendicular to those used in the first masking sequence. At the end of the process all possible two member polypeptides have been synthesized on the solid support. Each polypeptide combination is located in squares formed from the intersection of the strips. The above procedure may be repeated to achieve any

number of differing amino chain combinations in such intersecting squares.

This method has been used to prepare 1,024 different polypeptides on a square centimeter of surface. Utilizing the method of the present in¬ vention the potential exists for the synthesis of millions of polypeptides bound on a solid support. This method has also been used to synthesize dif¬ ferent oligonucleotides on a solid support.

Summary of the Invention

The present invention is the discovery that significantly improved deprotection of photolabile protected chemical units attached to a solid sup- port can be achieved by scanning such units with ultra violet laser beams. Higher density yields of synthesis sites at less time are obtained over con¬ ventional irradiation techniques. It is also pos¬ sible to employ programmable deprotection schemes when utilizing such beams by applying laser print¬ ing techniques. Also, the use of laser beams and technology offers a much simpler and convenient means for deprotecting such reactants over the con¬ ventional masking and exposure techniques. It is accordingly an object of the present invention to provide a laser beam to deprotect photolabile protected reactive chemical units attached to a solid support.

It is also an object of the present invention to scan photolabile protected reactive chemical units attached to a flat solid support with a UV laser beam.

A further object of the present invention is to apply programmable deprotection schemes to photolabile protected chemically reactive units

attached to a support through the use of laser copy technology.

A still further object of the present inven¬ tion is to provide a means for determining the set of polypeptides that bind specifically to a given protein through the synthesis of large numbers of peptides bound to a solid support.

Brief Description of the Drawing The figure is a schematic representation of a laser engine which may be used to accomplish the method of the present invention.

Detailed Description of the Invention Particularly desirable means for controlling the spatial coordinates of a laser beam is accom¬ plished by the apparatus depicted by the drawing. A UV beam source 10 generates ultra violet beam 12 which reflects from the surfaces of a rotating ir- ror 16 and onto the surface of a glass support 18 that is seated on a precision translator 19.

The surface of glass support 18 has been provided with attached chemical units (not shown) that have been provided with photolabile protecting groups. Mirror 16 is many sided (octagonal etc.) being composed of multiple flat surfaces (illu¬ strated at 16a, 16b etc.). Each such surface upon rotation is disposed to reflect laser beam 12 onto the surface of support 18 through focusing lens 20, curvature compensating lens 22 and reflecting mir¬ ror 24 when facing the beam. By rotating the mir¬ ror 16 about its axis 17, as each flat surface 16a, 16b, etc. sequentially faces beam 12, the beam is swept across the surface of support 18 and its coating of photolabile protected units deprotecting

such units along the scan lines 23. By incre¬ mentally advancing the support 18 in the direction of the arrow 25 between each scan by each of the mirrors during rotation a series of scan lines 23 (deprotected areas) is achieved. The result is a support having multiple scan lines of deprotected chemical units which, may then be reacted with chemicals to add new optionally different chemical units which may also be protected by photolabile groups. Individual scan lines may be selectively rescanned with beam 12 for subsequent additions of varying chemical units to provide controlled yields of chain compounds of unlimited differing chain units. Further, support 18 may be rotated and rescan¬ ned with beam 12 to provide intersecting scan areas and multiple chain units as described above in respect to the conventional masking techniques.

Preferred control of the spatial coordinates of the laser beam deprotection of the units is achieved by employing a shutter (not shown) to block the beam surface when desired. A resulting pulsed beam enables one to build grids with points or restricted areas of deprotection substantially increasing the number of polypeptides that can be created on a single substrata or in a space of a few square micrometers.

Although adequate pulsating laser beam gener¬ ating diodes have not been developed for UV lasers, should such a diode be developed it would be ideal for the present application. Any means for puls¬ ating beam 12 would be useful for this application. Alternatively, beam 12 may be a fixed beam laser and support 18 can be moved under the beam to effect the scan lines 23. This would be most effective by using a precision optical translator

19 (illustrative only) to incrementally move sup¬ port 18. In this embodiment it would also be help¬ ful to pulse the fixed UV laser beam.

It is also possible to combine the mirror scanning embodiment of the drawing with the pre¬ cision optical translator technique, for example by scanning with one such technique in one dimension on the support and in a perpendicular direction with the other. The term ultraviolet (UV) laser beam as used in this specification means lasers that emit in the UV at wavelengths preferable above 3200 Angstroms. Examples of lasers that emit in this region are Cadminum lasers (3250A) , Nitrogen lasers (3370A) , and Xenon Fluoride lasers (3520A) . Satisfactory lasers are those sold by LiCONiX Company of 3281 Scott Blvd. Santa Clara, California, 95054 such as HeCd Model No. 3207. Other models sold by this company are also satisfactory. A description of the technical details of these lasers may be found in U.S. Patent Nos. 4,704,583 and 4,746,201.

A satisfactory precision translator is one manufactured by Oriel of 250 Long Beach Blvd., P.O. Box 872, Stratford, Ct. 06497, Model 16127 Standard Translator with 25 mm Motor Mike Drive.

The spinning mirror laser device depicted by the drawing is an adaptation of a laser printer such as is described and shown in the article "Lights1 Camera1 Print It! Laser Printer Technology Explained" by Alfred Poor (PC Magazine, November 14, 1989, pp. 168-169).

The term "unit" as it is used in this specifi¬ cation shall mean chemical molecules or subdivi¬ sions thereof including radicals and reactive groups such as amino acids that may be linked

together with other such molecules or subdivisions thereof to form chain compounds such as proteins.

The photolabile protecting groups may be any such group known in the art for use in conjunction with the chain compound being constructed. For example, in conjunction with the described poly¬ peptides, amino groups at the ends of linkers attached to a glass substrata may be reacted with nitroveratrioxycarbonyl (NVOC) a photoremovable protection group. The use of such compounds are described in a Research Article in the publication entitled "Light-Directed, Spatially Addressable Parallel Chemical Synthesis" by S. Fodor, et. al., Science, 251, 767 (1991) . As an example of the method of the present invention a LiCONiX UV laser Model 3207N may be used to provide a continuous 3250 A UV laser beam to photoprotected amino acid units attached to a glass support. The components of this device are those illustrated in the drawing. Radiation of wavelength longer than 3200 A will not affect even the most light sensitive amino acid, tryptophan. The rotating mirror and detector (UV enhanced silicon photodiode detector) are linked to a controller 28 through a detector 26 (available from H&N Instruments) to control the scan rate. Focus¬ ing of the beam is accomplished using fused silica laser lenses ("Best Form", Oriel) capable of pro¬ ducing spot sizes on the order of a few micro- meters. The support 18 consists of a glass microscope slide contained in a small bath which is attached to a precision drive manual translator (Oriel) with 0.02 urn resolution. A liquid chromatograph (Pharmacia "FPLC system") is pro- grammed to react the glass support with the required solutions. The entire apparatus is

assembled on a standard optical platform with vibration control (Kinetics Vibration Table) . The protocol for peptide synthesis is similar to the procedures governed by .Fodor, et. al. (Science, 251, 767 [1991]), as follows:

1. The glass slide is a inated by dipping it in a solution of 0.1% aminopropyltriethoxysilane in 95% ethanol, and curing at 110° C for 20 minutes. Alternatively, the solution may be methanol con- taining 0.1% each of aminopropyltriethoxysilane, a wetting agent (such as Pluronic L122, manufactured by BASF Corp.), water, and acetic acid, followed by curing at 140°C for ten minutes.

2. Free amino sites are reacted with an in situ generated hydroxybenzotriazole (HOBt) ester of a nitroveratryloxycarabonyl (NVOC) or 2-nitrobenzyl- oxycarbonyl (NBOC) amino-protected amino acid, such as NVOC-Leucine. These are formed by mixing 0.25 mmol of the NVOC or NBOC amino acid with 37 mg HOBt, 111 mg of benzotriazolyl-n-oxytris(dimethyl- amino) phosphonium hexafluorophosphate (BOP) , and 86 ul of diisopropylethyla ine (DIEA) in 2.5 ml of DMF. The coupling takes 2 hours at room temper¬ ature. 3. The slide is washed with dimethylforma ide

(DMF) and methylene chloride. Supports about 0.1 mm high are placed around the edges of the slide, and a UV optical grade fused quartz window is placed on these supports. A solution of, 1% semi- carbazide hydrochloride in a UV transparent solvent such as methanol or ethylene glycol is then in¬ jected onto the slide, filling the space between the slide and quartz window. _. A solution of semi- carbazide hydrochloride is necessary to make the subsequent photochemical reaction quantitative [see

"Photoremovable Protecting Groups in Organic Syn¬ thesis", V. Pillai, Synthesis, 1 (1980), and A. Patchornik, et al., JACS, 92, 6333, (1970)]. The fused quartz window serves to guarantee a constant, optically reproducible quantity of solvent present during the subsequent deprotection step.

4. Deprotection is accomplished by scanning the laser described above over the programmed sites.

5. Steps 2-4 are repeated as required. In step 2 other NBOC or NOVC-amino acids are reacted as desired.

6. The entire surface is scanned at the end of the synthesis to photolyze the remaining NVOC or NBOC groups. The first layer of immobilized amino acid serves to introduce a photolabile species into the array and is not varied. All other positions in the immobilized peptide can be varied.

A shutter may be added to generate spots and the glass support may be rotated to produce a grid. A complete automation of the apparatus involv¬ ing using a detector 26 and a central controller 28 to control the LC apparatus (pumps, valves) as well as the optical apparatus may be employed. A significant advantage of the method of the present invention is the ability to completely automate the synthesis of immobilized peptide arrays. Automation can be efficiently accomplished using a H & N Instruments controller. The H & N Instruments controller is based on the Mororola 68HC11 microcontroller with New Micros MaxForth. This microcontroller has 5 digital I/O 8 bit ports, 2 serial communication ports, 5 timers, 512 bytes of EEPROM, 256 bytes of RAM, and a Forth kernel in 8K of on chip ROM. These features are

contained in a single 52 pin chip. H & N Instru¬ ments has designed circuit modules that combine with this chip to provide additional memory (EPROM or RAM) , high resolution analog I/O, and control of 4 floppy disk drives. These modules are combined on a custom designed circuit boards, designed in- house using state-of-the-art CAD programs. The tools available - in this hardware library are sufficient to implement most automation tasks. The H & N Instruments controller is programmed in Forth for real time data collection and instru¬ ment control. Forth is an extensible high level programming language. It is easy to add options to the language. Every program written becomes an operation that can be used in the same manner as the operations that are native to the language. Every operation is a word which can be used along with other operations or words which can be used to define new words. Words or operations can be executed immediately by typing the word followed by a carriage return. The use of words to execute operations encourage modular programming. If the word itself describes the operation it performs, then it can be nearly self-documenting. Several words can be used to define program modules which then become words themselves-leading to a final program which is really a series of stacked program modules.

Forth is also a stacked oriented language. It uses post fix notation much like an HP calculator. The stack is extensively used and is especially useful for passing parameters from one part of a program to another. The stack eliminates the need for formal parameter of local variable declara- tions.

Forth is an interactive language. A word can be executed immediately after it is defined by typing the word followed by a carriage return. This feature is convenient for debugging and testing. A new word can easily be tested and modified until it works as desired. This is especially useful for- debugging hardware control and I/O interface-programs.

H & N Instruments has a modular software library with routines for most common instrumen¬ tation and control tasks. It is analogous and complimentary to the hardware design library. Routines exist for floppy disk based data collec¬ tion, temperature programming and control, analog voltage input and output, various timing functions, and a sophisticated user interface for inputing experiment and control parameters. These routines are programmed in Forth and are easily applied to the automation of the instant laser method. As an example of the present invention utilizing the procedures set forth above, alter¬ nating rows of Gly (G) and γ-aminobutyric acid - Gly (GABA-G) are synthesized. The surface is incubated with rabbit anti-GABA antibody (available from Sigma Chemicals) which binds to GABA groups on the surface. A second incubation with gold con¬ jugated goat anti-rabbit IGG is used to detect bound rabbit antibody. Development of the slide with silver stain yields alternating rows of light and dark bands, corresponding to bound Gly and GABA-Gly.

As another example (not yet completed) of the present invention utilizing the procedures set forth above alternating rows of H-N-Tyr-Gly- Gly- Phe-Leu (YGGFL) and iϊ-N-Pro-Gly-Gly-Phe-Leu (PGGFL)

are synthesized. The surface is reacted with mouse monoclonal antibody to β-endorphin (3E7-Accurate Chemical and Scientific Corp) which binds YGGFL and YGGFM. A second incubation with fluorescein- labeled goat antibody to mouse (Accurate Chemical and Scientific Corp) is used to detect bound 3E7. A high degree of binding of 3E7 to YGGFL rows only will demonstrate the fidelity of synthesis.

As a further example (not yet completed) of the present invention utilizing the procedures set forth above alternating rows of ff ₂ N-Phe-Arg-Leu-

Pro-Leu(FRLPL) , B ₂ N-Leu-Leu-Leu-Pro—Leu(LLLPL) , and H ₂ N-Phe-Arg-Met-Phe-Leu (FRMFL) are synthesized.

The surface is reacted with fluorescein labled polyclonal antibody to FRLPL (Peninsula Labora¬ tory) . This antibody binds FRLPL with a cross reactivity of 22% to FRMF. A high degree of binding of the antibody to FRLPL rows, weaker binding to FRMFL rows, and no binding to LLLPL rows will demonstrate the fidelity of synthesis.

Publications other than those mentioned above that may be helpful in understanding the invention are:

"Binding activities of a repertoire of single immunoglobulin variable domains secreted from

Escherichia coli" E.S. Ward, et.al., Nature, Vol. 341, 544 (1989) ; "Peptides, Structure and Function", edited by CM. Deber, V.J. Hruby, and K.D. Kopple, Pierce Chem- ical Co., Rockford, IL, (1985);

"Searching for Peptide Ligands with an Epitope Library" J.K. Scott and G.P. Smith, Science, Vol. 249, 386 (1990); "Random Peptide Libraries: A Source of Specific Protein Binding Molecules" J.J. Devlin, L.C.

Panganiban, and P.E. Devlin, Science, Vol. 249, 404 (1990) ; "Laser Recording And Image-Quality Evaluation" K. Minoura, et.al., SPIE, Vol. 1254, 24 (1990); "A laser scanning optical system for high- resolution laser printer" T. Maruyama, et.al. , SPIE, Vol. 1254, 54 (1990); "Immunogenicity of Synthetic Peptides Corre¬ sponding to Flexible and 4Antibody-accessible Segments of Mouse Lactate Dehydrogenase (LDH) -C ₄ ^* "

H. Hogrefe, P. Kau aya, and E. Goldberg, J. Biol. Chem. , Vol. 264, 10513 (1989); "Peptide engineering of protein topographic determinants for vaccines" P. Kaumaya, et.al., in "Peptides 1990", ESCOM Science B.V. (1991); and "Synthesis and Biophysical Characterization of Engineered Topographic Immunogenic Determinants with αα Topology" P. Kaumaya, et.al., Biochem¬ istry, 29, 13 (1990) . Having thus described the apparatus and pro¬ cedural steps for carrying out the invention, it will be clear to those having ordinary skill in the art, that various modifications may be made in the apparatus and the procedural steps without depart- ing from the inventive concept. It is not intended that the words used in the specification to de¬ scribe the invention, nor the drawings illustrating the same, be limiting on the invention. Rather it is intended that the invention be- limited only by the scope of the appended claims.