Method of quantifying peptide-derivative libraries using phage display

Abstract

The present application provides a method of quantifying an amount of a derivatized peptide displayed on a phage by phage display, the method comprising: providing a phage containing a target peptide thereon; reacting the phage containing the target peptide with a first reagent to derivatize the target peptide to form a derivatized peptide, reacting the derivatized peptide with a capture agent comprising a detection marker, thereby incorporating the detection marker within the derivatized peptide; and determining an amount of the detection marker, thereby quantifying the amount of the derivatized peptide dis-played on the phage. A kit comprising a capture agent compris-ing a detection marker for quantifying the phage displayed derivatized peptides is also provided.

Claims

1. A method of quantifying derivatized peptides displayed on a phage by phage display, the method comprising: providing a phage containing a target peptide displayed thereon; reacting the phage containing the target peptide with a first chemical reagent to derivatize the peptide to form a derivatized peptide, reacting the derivatized peptide with a capture agent comprising a detection marker, thereby incorporating the detection marker within the derivatized peptide; and determining an amount of the detection marker, thereby quantifying the amount of the derivatized peptide displayed on the phage; reacting the derivatized peptide with a second reagent in a second reaction which eliminates the detection marker, and determining an amount of the detection marker after the second reaction, thereby quantifying the amount of the derivatized peptide after the second reaction.

2. The method of claim 1, wherein the detection marker is biotin, fluorescein or mannose.

3. The method of claim 1, wherein the target peptide is a peptide comprising an N-terminal amino acid.

4. The method of claim 3, wherein the N-terminal amino acid is serine, threonine or cysteine.

5. The method of claim 1, wherein the derivatized peptide is a glycopeptide or a macrocyclic peptide.

6. The method of claim 1, wherein the first reagent is NaIO4, iodoacetamide, or (tricarboxyethyl)phosphine (iTCEP).

7. The method of claim 6, wherein the concentration of NaIO4 is 20-500 μM.

8. The method of claim 7, wherein the concentration of NaIO4 is 20 or 500 μM.

9. The method of claim 1, wherein the capture agent is biotin-iodoacetamide (BIA), biotin-ARP, mannose-hydroxylamine, or chloroacetamide.

10. The method of claim 9, wherein the reaction time of the capture agent with the derivatized peptide is 1-4 hours.

11. The method of claim 4 wherein the target peptide is oxidized to form a derivatized peptide comprising an aldehyde group.

12. A method of quantifying derivatized peptide displayed on a phage by phage display comprising the steps of: providing a phage containing a target peptide displayed thereon; in a first reaction, reacting the phage containing the target peptide with a first chemical reagent to derivatize the peptide to form a first derivatized peptide having a first functional group (F1), in a second reaction, reacting the first derivatized peptide with a first capture agent (CA1) to react F1 to a second functional group (F2), wherein the first capture agent is coupled to a first detection marker, reacting the derivatized peptides with a second capture agent (CA2) coupled to a second detection marker, wherein CA2 is reactive with F1 and the second detection marker is different from the first detection marker, determining an amount of the first detection marker to quantify the amount of the derivatized peptide bearing F2, and determining an amount of the second detection marker to quantify the amount of derivatized peptide bearing F1; wherein the first chemical reagent oxidizes an N-terminal amino acid to produce an aldehyde moiety, and the second reagent converts the aldehyde moiety to an oxime.

13. The method of claim 12 wherein the first chemical reagent reduces disulfide bonds and CA1 alkylates the peptides.

14. A method of selecting for a first genetically encoded library of first derivatized peptides displayed on a phage (A) or a second genetically encoded library of second derivatized peptides displayed on a phage (B), comprising the steps of: providing phages containing a target peptide displayed thereon; producing a library of intermediate derivatized peptides from the target peptides with a chemical reagent; reacting the intermediate derivatized peptides with a capture agent coupled with a detection marker in a reaction which, if complete, results in the first derivatized peptides displaying the detection marker, and if incomplete, results in the second derivatized peptides not displaying the detection marker, isolating the first derivatized peptides using the detection marker; wherein the capture agent coupled with a detection marker comprises .sup.−C≡N.sup.+—biotin, which reacts with the aldehyde moiety to produce a macrocylic peptide, if complete.

15. The method of claim 14 wherein the target peptides are reacted to produce intermediate derivatized peptides having terminal aldehyde moieties.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) For a better understanding of the present invention, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

(2) FIG. 1 shows an example of optimization and quantification of chemical reactions on phage during oxidation of the N-terminal amino acids displayed on phage.

(3) FIG. 2 shows profiles of the effect of oxidation-quencher on reaction efficiency.

(4) FIG. 3 shows an assessment of the kinetics of the oxidation of the N-terminal Ser on phage.

(5) FIG. 4 shows exemplary yields of oxidation to aldehyde and oxime formation.

(6) FIG. 5 shows a schematic for selecting the subset of a library reactive in oxidation and coupling.

(7) FIG. 6 illustrates the synthesis of genetically-encoded chemical libraries of glycopeptides (A) or macrocyclic peptides (B).

(8) FIG. 7 illustrates kinetics of panning and amplification of peptide derivatives synthesized via oxidation-oxime ligation.

(9) FIG. 8 illustrates a reaction showing optimization of the synthesis of peptide derivatives via reduction of disulfides and alkylation of cysteines.

(10) FIG. 9 illustrates schematics and results from analysis of kinetics of reduction and alkylation.

(11) FIG. 10 shows a scheme and results of optimization of the linker coupling and a cyclization reaction on clonal phage.

(12) FIG. 11 shows a scheme for optimization of a cyclization reactions on library phage and differences in reactivity between clonal population of phage and library of phage.

(13) FIG. 12 illustrates a scheme for the generation of a light responsive library using cyclization with a light responsive linker.

(14) FIG. 13 illustrates results of panning a library cyclized with unnatural light-sensitive linker.

(15) FIG. 14 illustrates the quantification of reactions, described in FIG. 1-7, using other biochemical methods.

(16) FIG. 15 illustrates the detection of the marker with linker using SDS-page gel electrophoresis.

(17) FIG. 16 illustrates an optimization of the capture of derivatized peptides by biotin/streptavidin interactions.

(18) FIG. 17 illustrates an optimization of various aldehyde reaction on phage

(19) FIG. 18 illustrates an example of a step-wise cyclization reaction

DETAILED DESCRIPTION

Definitions

(20) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

(21) As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

(22) The term “comprising” as used herein will be understood to mean that the list following is non-exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and/or ingredient(s) as appropriate.

(23) The term “assay” is used herein to refer to a test to qualitatively or quantitatively detect the presence of a substance in a sample.

(24) The term “biological sample” is used herein to refer to both animal and human body fluids, excreta and tissues obtained from a living or dead organism. The term “body fluid”, as used herein includes a naturally occurring and/or secreted and/or excreted and/or discharged fluid and/or wash fluid from the surface or inside the bodies of a human or an animal and includes, but is not limited to: saliva, sputum, serum, plasma, blood, pharyngeal, nasal/nasal pharyngeal and sinus secretions, urine, mucous, feces, chyme, vomit, gastric juices, pancreatic juices, semen/sperm, cerebral spinal fluid, products of lactation or menstruation, egg yolk, amniotic fluid, aqueous humour, vitreous humour, cervical secretions, vaginal fluid/secretions, bone marrow aspirates, pleural fluid, sweat, pus, tears, lymph and bronchial or lung lavage.

(25) The terms “body tissue” or “tissue”, as used herein, refer to an aggregate of cells usually of a particular kind together with their intercellular substance that form one of the structural materials of a plant or an animal and that in animals include, for example, connective tissue, epithelium, mucosal membrane, muscle tissue, placental tissue, and tissue from liver, intestines, spleen, kidney, brain, heart, nerve tissue, and the like. Samples of body tissue can be obtained from living humans or animals by a variety of non-limiting methods, such as fine needle aspirates, scrapings or biopsy tissue or from the remains of dead humans or animals. The term “tissue” can be used to refer to naturally occurring tissue or synthetic tissue.

(26) Biological samples can also include pre-processed foodstuffs including plants, samples of meats and processed foods, as well as swab samples from environmental sources such as food processing facilities, hospitals, water, soil, and air. Other biological sample types include isolates/fractions/concentrates of blood (e.g. platelets, red blood cells, white blood cells or leukocytes), including umbilical cord blood or placental blood, bone marrow, aspirates, fine needle organ or lesion aspirates, cervical samples, cultured cells, body swabs, or body smears.

(27) The terms “dormant bacteriophage” and “dormant phage” are used interchangeably to refer to bacteriophage that is non-infective, but that will become infective following activation.

(28) As used herein, the term “reporter gene” refers to a construct comprising a coding sequence attached to heterologous promoter or enhancer elements and whose expression product can be assayed easily and quantifiably when the construct is introduced into bacteria, for example through infection by bacteriophage carrying the reporter gene.

(29) As used herein, a “target peptide” is a peptide of interest displayed on the surface of phage that is to be derivatized and assayed. As would be understood, the surface of a phage can display a plurality of peptides; a target peptide, thus, is one of the plurality of displayed peptides which is targeted for derivatization and assaying.

(30) The term “capture agent” as used herein refers to a compound that reacts with a particular functional group on a phage-displayed peptide. The capture agent comprises a reactive moiety that reacts with the desired functional group, and a detection marker which is used to quantify the binding of the capture agent with the peptide. “Detection marker”, thus, refers to a molecule in association with the moiety to form the capture agent, and that is capable of being assayed when the capture agent reacts with and is attached to the desired functional group. Exemplary capture agents include those comprising reactive moieties that react with terminal aldehyde groups on the phage-displayed peptides, for example. Exemplary detection markers include biotin, fluorescein and mannose.

(31) As used herein, “derivatization” in the context of a “derivatized phage molecule” or “derivatized peptide” refers to the modification of a target peptide residue presented on the surface of a phage, prior to reaction with a capture agent. The modification typically involves reaction of a particular functional group presented on the target peptide, such as the terminal peptide residue, thus forming a derivatized phage molecule, such as a derivatized peptide. This derivatized peptide is then detected by and reacted with the capture agent.

Phage Display Quantification Process

(32) The quantification process as described herein generally employs two types of phage: peptide-displaying phage “P” and control peptide-free phage “C”. P contains an insert in its genome which encodes and displays a peptide with its coat proteins. P can also represent a library of sequences (P.sub.lib). The present method is designed such that no change in procedure is necessary when characterizing reaction on one phage P or a library of phage P.sub.lib.

(33) The control phage C is typically genetically similar to P, but lacks inserts in the phage genome and, thus, a lack of DNA sequence encoding peptides in the coat protein region. It is desired to be able to perform independent quantification of number of P and C phage particles in the same solution.

(34) Methods known in the art can be used to differentiate P and C phages. For example:

(35) i) P containing LacZ, while C is LacZ-free. The plaques corresponding to P and C are distinguishable by color on plates containing colorimetric reagent X-gal. Any suitable enzyme other than LacZ can be used, and visualized using known means, such as with a fluorescence substrate.

(36) ii) P containing genes encoding GFP, RFP or other fluorescent reporters in the genome, while C is reporter-free. The reporter does not have to be physically linked to any proteins of the phage; it simply can only be expressed in the host cell. The plaques corresponding to P and C can be distinguished by color or fluorescence on plates as described previously.

(37) iii) P containing antibiotic resistance gene in the phage genome, while C is resistance-free. The plaques corresponding to P and C can be distinguished by plating the mixture of phage on two plates, with and without the antibiotic. Alternatively, P and C can carry resistance for two types of antibiotics and visualized separately.

(38) iv) P comprises a sequence encoding a short peptide sequence such as FLAG recognized by a specific ligand, such as anti-FLAG antibody, while C is sequence-free. P and C phage can be distinguished by transferring the colonies onto nitrocellulose paper and probing with the antibody or other ligand.

(39) v) A mixture of P and C can be lysed to isolate DNA and subject to quantification of specific DNA sequence by real-time PCR or other quantitative methods. Primers for PCR are designed for insert region would recognize DNA of only P but not C.

(40) The characterization of the mixture of P and C is beneficial for determining the success of the phage display process. Alternatively, P and C can be modified chemically and quantified in two separate solutions. For simplicity, in all subsequent discussions, examples of the present method as described herein are based on the presence of reporter Xgal in P but not in C. This method allows for rapid quantification of P and C by counting plaques of specific color (blue for P and white for C) in a plaque-forming assay.

Analysis and Quantification of Phage Displayed Peptide Derivatives

(41) Characterization of phage libraries can be a challenge, because each library member is often present in mixture as a single molecule (single clone). The present method allows for quantification of reaction yields in multi-step reactions on complex mixtures of clones (for example, up to 10.sup.9 different peptides) using a series of capture agents for each reaction step. The method as described herein employs any known substrate molecule and detection technology that can be conducted on amino-acid sequences.

(42) Quantification and characterization of the chemical reactions in peptide libraries has been lacking. Existing approaches for quantitative characterization by spectroscopic methods (mass-spectrometry, elemental analysis) and biochemical methods (gel electrophoresis, Western blot, etc) are typically only applicable to large clonal population or phage and viruses (e.g., 10.sup.10-10.sup.13 particles). They cannot detect modifications which occur in a population that contains millions of different phage clones, with each clone present in low amount (e.g., 1-100 copies), The present method provides optimized reaction conditions for assaying derivatized peptide libraries.

(43) The present method can also be used for the improvement of yield in chemical reactions conducted on derivatized peptide libraries. In a collection of many peptides, certain peptides undergo a specific type of the reaction faster than others. For example, cyclization can occur faster in peptides of specific conformation, while substitution or elimination can occur in peptides of specific steric and electronic properties. Although the selection of individual peptide sequences with unique reactivity has been described previously (Barbas, et al., U.S. patent application Ser. No. 08/573,415, filed Dec 15, 1995; Eldridge, G. M., Weiss, G. A. (2011), Hydrazide reactive peptide tags for site-specific protein labeling. Bioconjugate Chem. 22: 2143-2153), the present invention allows for the optimization of multi-step reactions on peptides to generate a large library (10.sup.3-10.sup.8) of non-peptidic structures in high yield. Although these libraries are non-peptidic in nature, they are amplifiable quantitatively just like parent peptides. Their identities are genetically-encoded and can be deduced from the original peptidic starting materials.

(44) One advantage of the present method is serial optimization of the two-step modification of phage libraries. In one example, quantification of oxidation of N-terminal serine (Ser) and threonine (Thr) electrophilic addition to aldehyde is performed. In another example, quantification of site-selective reduction or disulphide and its alkylation by light-sensitive linker is shown. However, it would be understood that other functional groups, on other amino acids, whether terminal or internal within the displayed peptide, may be contemplated. The examples shown herein illustrate only certain embodiments of the present method.

(45) A library of phage-displayed peptide-derivatives generated with the help of a quantification approach contains a well-defined number of ligands of defined structure. In standard phage display, rounds of panning and amplification discover new peptides; in a library of peptide-derivatives rounds of modification, panning and amplification discover new peptide-derivatives.

(46) To illustrate the above described system and method, an exemplary panning of peptide-derivatives is shown on model proteins targets, such as streptavidin (for binding to biotin) and Concanavalin A (for binding to mannose). However, as would be readily understood that other protein targets using other capture agents and detection markers can be used.

(47) The phage concentrations [P].sub.0, [C].sub.0, [P].sub.b4, [C].sub.b4, [P].sub.af and [C].sub.af measured in the present method provide information about yield and specificity of the reaction (initial step 1), as well as its interference with viability of phage. Serial variation of the reaction conditions can be then performed to identify the best outcome in those parameters.

(48) The present method can be applied to other multi-step reactions. For example, multi-step reactions (Rxn1, Rxn2, etc.) can employ different reaction conditions. Rxn2 converts functional group F1 from Rxn1 to another functional group F2. To characterize Rxn2, a mixture of phage from reaction 1 is exposed to appropriate reagents (Reag2. 1 Reag 2.2, etc). After an appropriate time, the reaction is terminated and the yield concentration of group F2 is determined using capture agent 2 (CA2), which contains a group reactive with F2. Exposure to capture agent CA1, reactive to original group F1, can be used to quantify the amount of unreacted groups F1. For example, quantification of the reaction of aldehyde with hydroxylamine can be done by counting phage with unreacted aldehyde groups, using aldehyde reactive capture agent. Further, quantification of the reaction of thiols (quantification of unreacted thiols) can be done using thiol-specific capture agent. Also, quantification of the cyclization can be done using chloroacetamide reactive capture agent. Examples of these reactions are described herein.

(49) Thus, quantification and optimization of a N-step reaction can be done using N capture agents to quantify the yield at each step. Changing the condition at each step optimizes the yield at each step.

(50) The capture agents (CA1, CA2, etc., described above for quantification) can serve another purpose. They can be used to select members of the library that are prone to undergo a particular reaction more effectively. This selection can be applied when reactions are conducted on mixture of phage that display library of peptides P.sup.lib. By running reaction on P.sup.lib and C in specific conditions, the yield can be quantified using capture agent CA1. Unreacted peptides can be separated from those that are reacted. A sub-library containing derivatized peptides can be amplified to give a pure subpopulation of phage that contain modified ligands only. The present method provides several examples of this, including: (1) modification of a random library to select a sub-library with N-amino acids reactive to oxidation; and (2) modification of a random library of 7 amino acids flanked by two cysteines to select sub-library that can undergo efficient cyclization with unnatural linker.

Calculations of Yield and Viability

(51) Yield of the specific reaction is determined as: Y.sup.s=100%*[P].sub.af/[P].sub.b4

(52) Yield of the non-specific reaction is determined as: Y.sup.n=100%*[C].sub.af/[C].sub.b4.

(53) Yield of the reaction that occurs specifically on a peptide, as opposed to any other protein segment of the phage, is determined by comparing Y.sup.s and Y.sup.n. Ideally, the Y.sup.n is zero, while Y.sup.s is close to 100%.

(54) The viability of the phage over the course of the reaction is determined as V=100%*[P].sub.b4/[P].sub.0. Ideally, the viability of the reaction should be maintained close to 100%, and optimization of the length of reaction, and concentration of reagents, or their presentation can be used to maximize V. This can result in an increase of the yield with a decrease in viability, as shown herein (see, for example, in FIGS. 1 and 3).

(55) The ability to quantify the yield allows for rapid selection of optimal reaction conditions, which maximizes yield and specificity and minimizes interference with viability. Parameters that can be varied to optimize the yield and specificity include: reagent concentration and reaction time, and the presence of a catalyst. These parameters are exemplified herein.

(56) To gain a better understanding of the invention described herein, the following examples are set forth. It should be understood that these examples are for illustrative purposes only. Therefore, they should not limit the scope of this invention in any way.

EXAMPLES

Example 1: Two-Step Modification of Phage Libraries

(57) The synthesis of peptide derivatives is a sequence of reactions (for example, labelled “Rxn1”, “Rxn2”, etc.). In reaction 1, a mixture of peptide-displaying phage [P] and control peptide-free phage [C] in known concentration [P].sub.0 and [C].sub.0 are mixed with appropriate reagents (“Reag 1.1”, “Reag 1.2”, etc). After an appropriate time, the reaction is terminated and a capture agent 1 (CA1) is added. The capture agent typically contains a complimentary reactive group and, for example, biotin. The solution is then mixed with capture support (such as a magnetic bead with a biotin-binding protein streptavidin). Phage that has undergone the Rxn1 successfully is captured on the support. The numbers of phage before capture ([P].sub.b4 and [C].sub.b4) and after capture ([P].sub.af and [C].sub.af) are recorded.

(58) FIG. 1 shows optimization and quantification of chemical reactions on phage during oxidation of the N-terminal amino acids displayed on phage. Rxn1 comprises the oxidation of an N-terminal serine residue by Reag1.1=NaIO.sub.4, which produces an aldehyde moiety. Capture agent CA1 is aldehyde-reactive biotin-hydroxylamine. A phage or library of phage that presents peptide with N-terminal Ser or Thr can be oxidized to aldehyde. The amount of the aldehyde and specificity of the oxidation can be assessed using an aldehyde-reactive capture agent (2). A phage is then captured using streptavidin-coated magnetic beads. Quantification of the number of phage before and after capture can be used to calculate the yield of the reaction. The concentration of reagents and timing of reactions was used to maximize the yield.

(59) FIG. 1B illustrates a profile of the yield and specificity of the reaction in different reaction conditions. Reactions were performed on mixture of phage clones: (1) phage displaying a peptide with N-terminal Ser (“SVEK phage”) and (2) phage containing N-terminal Ala (“WT”). Two phage clones were characterized independently because SVEK contained LacZ reporter and formed blue plaques, while WT formed white plaques. Identical characterization can be performed using a library of clones that contain N-terminal Ser.

(60) FIG. 1C illustrates a profile of the viability of the phage in individual reaction conditions. The method can be used to rapidly profile multiple conditions to select those that maximize the yield of specific reaction on SVEK phage, minimizes non-specific reaction on WT phage, minimizes reaction time, and maximizes the viability of the phage (see boxed values). As demonstrated herein, the ideal concentration of NaIO.sub.4 in Rxn1 is 20-500 μM, more particularly 20 μM or 500 μM.

(61) The concentration of biotin-ARP was also optimized to avoid non-specific binding. Ideally, the concentration of biotin-ARP is about 0.2-5 mM, typically 0.2 mM, 1 mM or 5 mM. The ideal reaction time (Rxn 2) was found to be 1-4 hours, typically 1, 2, 3 or 4 hours incubation time. The addition of an analine catalyst was found to favourably promote the reactions.

(62) FIG. 2 shows a profile of the effect of an oxidation-quencher on reaction efficiency. The oxidation of N-terminal Ser on phage can be terminated by a variety of reducing agents. The use of some reducing agents (thio vs. sulfide) can dramatically improve the yield. Thiol-based reducing agents provide similar yields, but glutathione has the least negative impact on phage viability.

(63) In FIG. 3, the oxidation of N-terminal Ser presented on phage was “fine tuned” to produce an optimal yield of captured phage. All reactions were conducted with 10.sup.11 pfu/mL of phage. After each oxidation, the reaction was terminated with 4-methoxybenzenethiol; the phage was exposed to 1 mM biotin-hydroxylamine for 1 h, diluted 1,000,000-fold and captured on Strep-MB. The resultant kinetic curves demonstrate that yield of the reaction in these conditions cannot be increased beyond 90% regardless of oxidant concentration or reaction time (top two panels). At extended reaction time or high oxidant concentrations, a decrease in viability take place (lower panels). Side reactions, including modification of WT phage, are minimal in these reaction regimes.

(64) FIG. 4 shows a profile of the yield of a two-step reaction: oxidation to aldehyde and oxime formation, by characterizing unreacted aldehydes. In panel A, phage was exposed to optimal oxidation conditions (0.06 mM NaIO.sub.4 for 5 min) and quenched with glutathione. The phage was then exposed to mannose-hydroxylamine. The amount of unreacted aldehydes were quantified by exposure to capture agent biotin-hydroxylamine (panel B). As illustrated in the graph therein, approximately 90% of the phage is reactive towards biotin-hydroxylamine, but only 10% of phage are modifiable by biotin-hydroxylamine after 1 h exposure to 1 mM mannose-hydroxylamine. The yield of reaction with mannose-hydroxylamine is 90−10=80%. Panel D shows that 20% of the phage in a random library is reactive towards biotin-hydroxylamine after oxidation, confirming that 20% of the library contain N-terminal Ser or Thr residues. Panel E illustrates that the chemical bond on phage is stable for multiple days.

(65) FIG. 5 illustrates a method of selecting a subset of a library reactive in oxidation and coupling to determine the yield of displayed peptides comprising terminal Ser/Thr residues (“S/T-terminated sub-library) vs. library members not containing terminal Ser/Thr residues (“non-S/T-term. Library members”). In this example, a random library of phage (panel A) was exposed to optimal oxidation conditions (0.06 mM NaIO.sub.4 for 5 min) and quenched with glutathione and oxime-formation conditions (1 mM biotin-TEV-hydroxylamine). Since only 20% of the random library contains N-terminal Ser, the yield of the reaction on random naive library is ˜20%. After release of the captured phage, and re-amplification (panel C), the yield of identical reaction in identical conditions is typically higher, approaching nearly 100% yield after a few rounds of this selection. This process, thus, selects a subset of library that reacts optimally in these reaction conditions.

(66) FIG. 7 shows that a library of peptide derivatives synthesized via oxidation-oxime ligation can be used in panning and amplification. Example of panning of peptide-Mannose conjugates against a model mannose-binding target, Concanavalin A (panels A and B), and panning of the peptide library which has not been modified (panels C and D) are shown. To assess the efficiency of panning, the amount of phage washed at each step was measured (A and C). From these values and the amount of phage eluted from beads, back-calculation of the amount of phage on-bead at each washing step (B and D) determined the quantity of phage. These values follow standard first-rate off-rate kinetics. Each library has been deliberately contaminated with insert-free wild type phage, which can be quantified in the mixture, because it forms plaques of white color. By contrast, the library forms plaques of blue color. Insert-free phage serves as internal control for non-specific binding. To assess the efficiency of panning, the amount of “blue” and “white” phage washed at each step was measured. An increased number of phage at the elution step (EL) as compared to the last washing step (wash 10) indicated that the phage was recovered due to non-specific interaction. For more details about blue-white screen in phage-display panning see: R Derda, S Musah, B P Orner, J R Klim, L Li, L L Kiessling “High-throughput Discovery of Synthetic Surfaces that Support Proliferation of Pluripotent Cells” J Am. Chem. Soc. 2010, 132, 1289.

Example 2: Optimization of Cysteine (Cys)-Containing Residues

(67) FIG. 8 illustrates the optimization of synthesis of peptide derivatives via reduction and alkylation of phage displayed peptides. (A) Reactions were performed with a mixture of L-phage that displays disulfide CPARSPLEC and forms blue plaques with WT-phage that displays no peptide and forms white plaques. (B) After exposure of WT+L mixture to iTCEP and BIA, white and blue plaques after reaction (.sup.WTN and .sup.LN) and after the biotinylated phage is captured by streptavidin (.sup.WTN.sub.s and .sup.LN.sub.s) was counted. Capture of L phage (.sup.LCap) was used as measure of biotinylation. Capture of WT phage (.sup.WTCap) typically indicates non-specific reduction or alkylation; however, significant capture was not observed (see D-G). (C) Biotinylation of five pIII-displayed disulfides yields phage with 0-10 biotins (B.sub.0-B.sub.10) is shown. Reaction yield is calculated from captured fraction, (D) After exposure of WT+L mixture to iTCEP and BIA, capture of L-phage increases with the time of reduction and alkylation. (E) Captures were converted to yields using equation (C) and the data were fit to pseudo-first order kinetics. This kinetics was appropriate because [iTCEP] and [BIA]>>[phage] and the yields of reduction (F) and alkylation (G) of phage at a specific time were independent of the concentration of phage.

(68) FIG. 9 shows one embodiment of tuning the kinetics of reduction and alkylation of the disulfide residues on Cys by TCEP and their subsequent alkylation by BIA. A) Alkylation of phage does not occur in the absence of reducing agent. Panel B illustrates quantification of yield of alkylation of phage clone L by BIA after reduction by TCEP. Panel C indicates reduction by TCEP is complete in <30 minutes. The total reaction time is X min. Panel D indicates optimization of alkylation time after 2 h reduction of phage. The total reaction time is X+Y min. Panel E illustrates optimization of one-pot reduction/alkylation of phage by varying (F) concentration of TCEP and (G) reduction time. As shown in Panel H, efficiency of one-pot reaction drops to 60% when phage concentration is <10.sup.9 PFU/mL due to low TCEP reduction efficiency at these concentrations.

(69) FIG. 10 illustrates one example of optimization of the linker coupling and cyclization on clonal phage and library phage. In panel A, reduction and alkylation of the disulfide peptide displayed on the phage and potential resulting by-products is shown. Exposure to (B) BIA is used to quantify the reduced disulfides or (C) unreacted thiols. Panel D illustrates that exposure to biotin-thiol (BSH) quantified non-cyclized peptides. In Panel E, the fraction of biotinylated phage decreases after incubation of phage with BSBCA. No biotinylation after exposure to BSH indicated that BSBCA coupling yielded cyclic peptide as major product. In Panel F, disappearance of biotinylated phage was fit with pseudo-first order kinetics to calculate the rate constant (k) of alkylation by BSBCA. In Panel G, two rate constants measured for two phage-displayed peptides (F1, F2) were similar to those measured using HPLC and a synthetic peptide of the same amino acid sequence (P1, P2). As shown in Panel H, mass spectrometry confirmed that a cyclic product was major product of reaction between BSBCA and peptide ACPARSPLECGGGSAETVESC(Cam)LAKS (underlined sequences is a synthetic fragment of pIII).

Example 3: Cyclization Reaction

(70) Exemplified herein is the generation of large sub-libraries, from random peptide libraries, that undergo quantitative N-terminal oxidation, and an efficient three-step cyclization via nucleophilic substitution, cyclization and rearrangement through the Ugi reaction.

(71) A selection for sub-library of sequences that can undergo complex, multi-component reactions involving multiple natural side chains. As an example, the Ugi multi-component reaction was performed. This reaction requires amine, carboxylic acid, aldehyde and isocyanide (see FIG. 6). Components in the phage library can be oxidized at the N-terminus (Ser and Thr-terminated peptides). Only some sequences can undergo efficient cyclization. Using an isocyanide with a capture agent (such as biotin), sequences that undergo efficient cyclization can be separated from those that do not. The sub-library can be used to generate a pure library of phage-displayed macrocycles.

(72) FIG. 6 illustrates one example of selecting for genetically-encoded chemical libraries of glycopeptides (A) or macrocyclic peptides (B). These peptides are derived from aldehyde-containing peptides (D, and intermediate C). The aldehyde displaying libraries can be quantitatively produced from any native phage-displayed peptide library; the yield of this reaction can be maximized as described in FIG. 5. A key aldehyde intermediate (C) leads to different libraries in a one-step synthesis. The Ugi reaction is shown: addition of .sup.−C≡N.sup.+-biotin initiates a cascade of cyclization and rearrangement to afford the complex macrocyclic peptides. Only product that undergoes complete transformation incorporates biotin within it. A sub-library, which undergoes Ugi macrocyclization, can thus be isolated and re-amplified to yield a pure, genetically encoded library of macrocycles.

(73) FIG. 11 illustrates one exemplary scheme for the optimization of library on phage. Conditions that are optimal for phage that display individual sequences do not yield the same yields on libraries containing multiple reactive peptides. Panel A illustrates one scheme of the reduction and alkylation reactions. In Panel B, the observed yield of the alkylation in the library is ˜35%—same conditions for clonal population of phage yield >80% alkylated phage (FIG. 10). In Panel C, the expected coupling in oxime modification, based on optimized conditions for clonal population of phage (FIGS. 2 and 3) is ˜80%. The observed yield in the library is 50%. Thus, it has been surprisingly found that conditions optimized for clones that display one type of peptide provide much lower yield in large libraries of clones presenting diverse peptides. Differences depend on the specific reaction and vary from subtle differences 80% (clone) in 60% in library to significant variations >99% (clone) in 50% (library).

(74) FIG. 12 shows an example of the selection of chemically modified genetically-encoded library and general strategy for the synthesis of genetically-encoded light-responsive phage libraries. The yield of each step (reduction, alkylation, cyclization) can be improved using steps described herein (FIGS. 7-9) to yield a pure library, in which every member contains light responsive linker.

(75) FIG. 13 shows the results of panning of a library cyclized with an unnatural light-sensitive linker. In Panel A, a scheme of panning procedure for modified library against streptavidin is shown. In Panel B, stacked bars show composition of the library before each round of the panning. Panel C shows enrichment of binding ligands after each round of panning represented as the ratio of the number of eluted bound phage to the number of phage input at each round. Panel D shows a Venn diagram representation of phage population after panning. Area is proportional to the number of binding/non-binding phage clones; data based on ELISA screening of 80 clones. As shown in Panel E, iterative irradiation of L36 and L42 did not cause a significant change in photo-switching ability of the ligands. The * shows that no complex was observed. The Ugi reaction is just one example of a modified sub-library that can be made. Other reactions may be used as applicable involving aldehyde, such as Passerini reaction, Petasis reaction, Wittig reaction, or Mukaiyama aldol reaction.

Example 4: Comparison of Present Method with Other Biochemical Methods

(76) FIG. 14 illustrates the quantification of reactions, described in FIG. 1-7, using other biochemical methods. The coupling of fluorescein was used to visualize which protein of phage the reaction occurs on. The bands of the SDS page gel, which correspond to minor protein pIII become progressively more fluorescent with increasing concentration of fluorophore (using semi-quantitative analysis). Because pIII protein is present at much lower abundance compared to other proteins on phage (e.g. pVIII is present at >1000-fold higher concentration), overall interpretation of gel is difficult. Large amounts of non-specific fluorescence is observed at the bottom of the gel; this may be associated with major coat protein pVIII, or could correspond to free fluorescein retained on the gel.

(77) FIG. 15 illustrates the detection of the marker with linker using SDS-page gel electrophoresis. Phage was reacted with fluorescein iodoacetamide (FIA) before and after the reaction with the linker. Fluorescence after the reaction with linker indicates remaining unreacted groups (ie. an indication of efficiency of the linker coupling). The gel shows that coupling at lower concentrations of BSBCA linker does not work even at higher reaction temperatures. At high concentration of linker (1 mM), the reaction proceed equally well at 55° C. and room temperature.

(78) Thus, it can be seen that the present method offers improved detection and quantification of derivatized peptides over conventional bulk methods.

Example 5: Optimization of the Capture of Derivatized Peptides by Biotin/Streptavidin Interactions

(79) FIG. 16 illustrates an optimization of the capture of derivatized peptides by biotin/streptavidin interactions. Conditions were selected under which biotinylated phage was captured by magnetic bead (MB)-streptavidin quantitatively but capture of non-biotinylated phage was zero. Additionally, capture of biotinylated phage is biotin-specific; it does not occur if the MB-streptavidin is pre-blocked with soluble biotin.

Example 6: Optimization of Various Aldehyde Reactions on Phage

(80) FIG. 17 illustrates the optimization of various aldehyde reactions on phage. In this figure, from top-to-bottom: oxime ligation of complex glycan, aminobenzylamine ligation/Dimroth rearrangement Wittig reaction. In each case, the phage of library was oxidized to form aldehyde. The amount of aldehyde before and after reaction was characterized using biotin-hydroxylamine as described in FIG. 4. “100% conversion” means that no reactive aldehyde was detected after the incubation with the appropriate reagent. Conversion of the N-terminal aldehyde to the described structure was confirmed in an independent reaction on synthetic peptides in the same conditions (pH, concentration, buffer, temperature) monitored by liquid chromatography mass-spectrometry.

(81) FIG. 18 shows an example of a step-wise cyclization reaction. In the first step, phage is modified by the linker that contains a detection marker, here biotin. In the second step, the detection marker is eliminated. Steps 1 and 2 can be quantified and selected independently. Unreacted molecules after step 1 can be discarded. Raising the pH promotes the second reaction (cyclization) and simultaneous elution of phage from beads. Only molecules reacted in step 2 can be carried to the next round. Provided example is reaction of phage-displayed aldehyde with molecule that contains a group (“group 1”) that reacts with an aldehyde in reaction condition 1 (oxime, reacts at pH=4.7) and a leaving group, such as ester that reacts in reaction condition 2 (pH>8). Group 1 includes a hydroxylamine (or aminooxy group). Other groups that undergo reaction with aldehyde at pH<8 would be suitable, Examples of such groups are stabilized phosphor ylides (Wittig reaction), aminobenzamidoximes and aminobenzylamines.

(82) All publications, patents and patent applications mentioned in this Specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent applications was specifically and individually indicated to be incorporated by reference.

(83) The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.