Online monitoring of peptide synthesis reactions

Abstract

A method and apparatus for online UV monitoring of an automated synthesis reaction. The apparatus includes a module having a fluid cell, the operating parameters of which remain substantially constant not only during the monitoring process but also during the time between subsequent processes. The module includes a separable housing structure that accommodates a UV light source and photodetector integrated with a fluid cell that is substantially unaffected by temperature. A portion of the cell is formed within a cell chassis and defined by a slot complemented by an inlet and an outlet, the inlet and outlet being sized so that no bubbles and / or stagnant fluid are present in the cell during the time the liquid reactants to be measured are delivered to the cell through the inlet. The method is configured to determine the progress and completion of the reaction, as well as modifications to the reaction time and the number of reaction iterations, in real time.

Description

Technical Field

[0001] Cross - reference to Related Applications This international patent application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 592,378, filed on October 23, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention generally relates to the process of monitoring chemical reactions during automated synthesis, and more specifically to systems and methods for online monitoring, for example, the fluorenylmethyloxycarbonyl (Fmoc) deprotection reaction during peptide synthesis, and / or the coupling reaction during peptide synthesis (the latter is a reaction in which two or more reactants are involved in coupling two amino acids, that is, the unprotected amine of one amino acid reacts with the unprotected carboxylic acid group of the other amino acid to form a peptide bond), and to systems and methods for online monitoring of such reactions.

Background Art

[0003] Biologically - derived products are often synthesized by automated synthesis. For example, the production of natural and artificial peptides and proteins is generally carried out using automated equipment for solid - phase peptide synthesis (SPPS). The general principle of SPPS is one cycle of repeating coupling and deprotection. Essentially, the free N - terminal amine of the peptide bound to the solid - phase support is coupled to the carboxyl - terminal of a single N - terminal - protected amino acid. This newly coupled amino acid is then deprotected to expose a new N - terminal amine to which further protected amino acids can be coupled.

[0004] Assuming that the final yield of the synthesized product depends on the yield of each step in the synthesis process, the coupling of amino acids in SPPS must be highly optimized. Since the scale of deprotection is a critical parameter in SPPS, in many cases, deprotection must be repeated until "completion." That is, so much deprotection must be performed that further iterations are likely to be futile. Therefore, various methods have been developed to monitor the scale of deprotection completion.

[0005] For example, Fmoc deprotection has been monitored using conductivity assays. However, for other reasons as well, it has been found that sensitivity to conductive impurities, in particular, can lead to unnecessary repetition of the deprotection reaction, resulting in time overruns, reagent consumption, and reduced yield.

[0006] A more sensitive method of deprotection monitoring requires measuring the adsorption amount of dibenzofluben-piperidine adducts formed during the deprotection reaction using ultraviolet (UV) light at a wavelength of 365 nm. Unfortunately, this method also has a significant drawback: it is associated with unnatural readings caused by the undesirable adsorption of other reagents (e.g., triazole-based coupling reagents). Subsequently, it was found to be more advantageous to measure the adsorption amount of deprotection reagents and / or adducts using UV light at a wavelength of 301 nm.

[0007] Some known apparatuses and methods for UV monitoring at 301 nm require the use of a flow cell, UV light source, and detector located outside the synthesizer, and therefore rely on moving the liquid reagent from the reactor to the detector's flow cell at the end of the deprotection reaction. This necessitates excessive chemical rinsing between UV measurements to clean the tubing and remove bubbles. Furthermore, the flow rate through the cell can affect the measurement accuracy, and waiting times before and after readings must be observed to ensure stable values. Due to the excessive use of reagents and the time spent, this method becomes uneconomical at scales exceeding 1.0 mmol. Moreover, since UV measurements are performed offline (immediately after the end of the deprotection reaction cycle), the length of the deprotection reaction cannot be adjusted in real time, resulting in unnecessarily long reaction times.

[0008] Another solution is described in U.S. Patent No. 8,535,947 (the disclosure of which is incorporated herein by reference), in which an embodiment of a UV monitoring device is located proximal to the reaction vessel (where the deprotection reaction occurs) and incorporated into a conduit immediately below the reaction vessel. There, the reaction fluid is moved a short distance from the reaction vessel to the UV monitoring system at specified time intervals, measured for the progress and completion of deprotection, and exchanged within the reaction vessel. While this proximity to the reaction vessel improved some of the existing shortcomings of measurements performed by prior art systems, the cell or chamber containing the reagent (target fluid) to be measured by UV light is constructed as a specific tube, and as a result of this configuration, it is not possible to utilize a precise and time-invariant path length for optical monitoring, and furthermore, the optical properties of the chamber degrade over time.

[0009] Therefore, online monitoring systems and methods are still needed to produce high-quality, high-yield synthetic products more reliably and repeatedly. [Overview of the project] [Means for solving the problem]

[0010] The deprotection and / or coupling monitoring system and method of the present invention requires a series of flow cells, a UV light source, and a detector, which perform UV measurements during the deprotection and / or coupling reaction steps to enable real-time correction of the deprotection / coupling time and number of iterations (for the purposes of this disclosure and the appended claims, the real-time performance of the system is understood as performance targeting the operational deadline from a given event to the system's response to that event. For example, real-time extraction of optical information from the photodetector system (e.g., irradiance and / or phase of light arriving from a target scene) may be triggered by the user and performed concurrently or concurrently with the interruption of the photoimaging process when such information was recorded). This is achieved by integrating the UV light monitoring system within the automated synthesizer itself, positioned proximal to the reaction vessel in which the reaction occurs. Thus, the reaction fluid is located only a short distance from the reaction vessel, thereby leaving most of the liquid in the vessel in preparation for the reaction. Furthermore, since the UV flow cells are aligned with the conduit through which the rinsing reagents for the reactor would normally flow, no special rinsing is required.

[0011] The apparatus provided by embodiments of the present invention includes a substrate of an optically opaque material and a hollow space, the hollow space being limited by first and second substantially flat walls formed within such a substrate and first and second plates of an optically transparent material, which are positioned to span the top and bottom of these two walls, respectively, and substantially connect their top and bottom portions. The first and second walls include corresponding first and second trenches, these trenches being defined on the surface of the respective walls and connecting the top and bottom portions of such walls. The apparatus includes inlet and outlet passages formed within the substrate along at least one of the first and second plates and fluidly connected to each other via the hollow space. In particular, the apparatus is configured to perform photometric measurements of a target fluid placed between the first and second trenches in an automated peptide synthesizer. In at least one embodiment, the first and second trenches are substantially parallel and opposite to each other, and in a cross-section of the substrate, an aperture is defined at least partially by the combination of the first and second trenches, formed across the hollow space in a plane substantially parallel to at least one of the first and second plates, the dimensions of which are greater than the separation distance between the flat portions of the substantially flat walls of the first and second. Alternatively or additionally, and in substantially all implementations of the apparatus, to prevent the formation of bubbles in the hollow space when the target fluid is delivered to the hollow section through the inlet passage, the inlet passage may be configured to always have a portion with cross-sectional dimensions smaller than the separation distance between the first and second walls, and / or the outlet passage may be configured to always have a portion with cross-sectional dimensions smaller than the separation distance between the first and second walls. Optionally, all implementations of the apparatus may be,

[0012] The apparatus may include a light source configured to deliver light along axes substantially parallel to the first and second trenches, and a photodetector positioned to receive light delivered through the first and second plates, through a hollow space, and through first and second apertures formed in the first and second material layers, respectively, which are separated from each other by the hollow space (at least in the latter case, the first and second material layers may constitute a sealing gasket). Alternatively or additionally, and in substantially all mounting configurations of the apparatus, the substrate and the first and second plates may be configured to ensure that the hollow space remains substantially unaffected by temperature fluctuations, and that the transmittance of light of the target UV wavelength by each of the first and second plates exceeds 90%.

[0013] Embodiments of the present invention also provide a method for real-time monitoring of the deprotection reaction of an Fmoc protecting group. Such a method deals with a process for monitoring the deprotection reaction of an Fmoc protecting group in an automated peptide synthesizer, including one embodiment of the apparatus described above and a reaction vessel. Monitoring is achieved by at least (a) drawing a fluid containing dibenzoflubene and associated adducts and by-products from the reaction vessel through an inlet passage into a hollow space; (b) irradiating the fluid in the hollow space with UV light having a wavelength of about 301 nm, delivered along the first and second trenches between the first and second trenches from the first plate to the second plate; (c) obtaining optical data representing the absorption of UV light by the fluid and determining the rate of the deprotection reaction by calculating a specified number of floating average readings; and (d) returning the fluid to the reaction vessel via an outlet passage unless the change in the floating average value within a user-specified time no longer exceeds a pre-selected value, indicating that the reaction is complete. In one embodiment of the method, the step of drawing the fluid from the reaction vessel is performed during the deprotection reaction. In at least one embodiment, method steps (a) to (d) may be performed at specified time intervals during the deprotection reaction. Alternatively or additionally, some particular embodiments of the Method may further include a step of extending the time of the deprotection reaction based on an extension time calculated from optical data recorded during acquisition of step (c), and / or a step of repeating the deprotection reaction based on optical data recorded during acquisition of step (c), wherein the optical data is used to calculate the degree of completion of the deprotection reaction, and / or a step of extending the time of a subsequent amino acid or monomer coupling reaction based on the time of the deprotection reaction. Furthermore, one particular embodiment may include a step of providing a visually perceptible output representing the optical data during the synthesis process (optionally, the visually perceptible output may include a graphical synthesis summary and / or a graphical representation of individual deprotection reactions).Alternatively or additionally, substantially all implementations of the method may further include a step of preventing the formation of bubbles in the hollow space during the step of drawing in the fluid, which is done at least to some extent by passing the fluid through a first portion of the inlet passage distal to the hollow space and having a first cross-sectional dimension, and then passing the fluid through a second portion of the inlet passage proximal to the hollow space and having a second cross-sectional dimension (where the second cross-sectional dimension is always smaller than the minimum separation distance between the first and second substantially flat walls, and the first cross-sectional dimension is larger than the second cross-sectional dimension). If such conditions are met, the step of returning the fluid to the reaction vessel includes passing the fluid through a first portion of the outlet passage that is proximal to the hollow space and has a third cross-sectional dimension, and then passing the fluid through a second portion of the outlet passage that is distal to the hollow space and has a fourth cross-sectional dimension (where the third cross-sectional dimension is always greater than the minimum separation distance between the first and second substantially flat walls, and the fourth cross-sectional dimension is greater than the third cross-sectional dimension). Alternatively or additionally, and in substantially all implementations of the method, the step of irradiating the fluid may be configured to include passing the fluid in the hollow space across a beam of UV light having a cross-sectional dimension greater than the separation distance between the opposing flat portions of the first and second substantially flat walls. Optionally, the synthesizer includes a UV light source and a UV photodetector within the synthesizer.

[0014] Embodiments of the present invention further include a method for manufacturing embodiments of the apparatus described above by at least certain steps of assembling a chassis assembly. The assembly process may include (i) forming a slot having substantially parallel walls that penetrate an optically opaque substrate; (ii) fabricating an inlet passage and an outlet passage that are fluidly connected to each other through the slot, wherein the corresponding dimensions of each of the inlet passage and the outlet passage at their respective intersections with the slot are always smaller than the width of the slot, defined by the separation distance between the substantially parallel walls; (iii) placing first and second plates of an optically transparent material into the respective recesses of the substrate to completely cover the slot; and (iv) pressing the first and second plates onto the surfaces of the respective recesses to form a cell defined by the slot and the surfaces of the first and second plates (where the cell is fluidly sealed at the junction between the first and second plates and the optically opaque substrate). Optionally, in one embodiment of the Method, the step of forming a slot includes forming spatially opposing, parallel trenches in walls parallel to each other. Alternatively or additionally, and in substantially all implementations of the Method, the step of manufacturing an inlet passage and an outlet passage may include forming a portion of each such passage distal to the slot, wherein the cross-sectional dimensions of such portion are greater than the dimensions of the corresponding passage at the intersection of the passage and the slot, and / or further include the step of fixing a chassis assembly between the first and second parts of the housing of the apparatus (in this case, the first part of the housing may house a light source directed to deliver a light beam substantially across an optically opaque substrate through the first and second plates and the slot, and the second part of the housing may house a photodetector system arranged to receive the light beam coming through the first and second plates and the slot). Optionally, and in substantially all implementations of the Method, the step of forming a slot may include a thermal expansion coefficient of 10 -5The steps may include forming slots in an optically opaque substrate made of a material not exceeding a certain value, and the steps of arranging the first and second plates may include using such first and second plates made of fused silica or quartz glass.

[0015] Embodiments of this method further provide the use of the above-described apparatus embodiments for performing photometric measurements of a target fluid within an automated peptide synthesizer system. In one example, such photometric measurements include real-time monitoring of the deprotection reaction of an Fmoc protecting group.

[0016] Embodiments of the present invention further provide a computer-assisted method for passing fluid within an automated peptide synthesizer between the reaction vessel and the waste container of the synthesizer. Such a method includes at least the steps of: (a) a computer initiates drawing a fluid containing a deprotection reagent from a reaction vessel through an inlet passage formed in an optically opaque substrate to a slot formed in an optically opaque plate of a photometric measuring device in a synthesizer; (b) a computer activates the transmission of UV light through the fluid to a photodetector of the photometric measuring device by irradiating the fluid in the slot with UV light; (c) a computer determines the rate of the deprotection reaction in the fluid by obtaining optical data from the photodetector representing the absorption of UV light by the fluid in the slot and calculating a floating average of a specified number of detector readings; and (d) if the computer determines that the deprotection reaction is complete because the change in the floating average value calculated over a user-specified time exceeds a pre-selected value, it returns the fluid to the reaction vessel; otherwise, it sends the fluid through an outlet passage formed in an optically opaque substrate to a waste container fluidly separated from the reaction vessel by the photometric measuring device. Here, each of the inlet and outlet passages has a corresponding first cross-sectional dimension in the portion of them proximal to the slot and a second cross-sectional dimension in the portion of them distal to the slot, wherein the second cross-sectional dimension is always smaller than the minimum separation distance between the first and second walls, and the first cross-sectional dimension is larger than the second cross-sectional dimension. In at least one implementation of such a method, the starting step includes starting to draw a fluid containing dibenzoflubene and associated adducts and by-products into a hollow space, the hollow space being defined by first and second walls formed in an optically opaque substrate and first and second flat and parallel plates of an optically transparent material positioned across the top of the first wall and the top of the second wall and configured to connect the first wall and the second wall substantially leak-free (where the slot is defined by the fact that the flat portions of the first and second walls are substantially parallel to each other).In virtually all implementations of such computer-aided methods, the computer initiates a step of drawing into the deprotection reaction, and / or the method further includes a step of computer-aided repetition of the deprotection reaction in a synthesizer based on optical data recorded during the acquisition of step (c) (the optical data is used to calculate the degree of completion of the deprotection reaction). Alternatively or additionally, virtually all implementations of the method may include a step of computer-aided extension of the time of the subsequent amino acid or monomer coupling reaction in the synthesizer, based on the time of the deprotection reaction.

[0017] Embodiments of this method also provide a computer program product that is encoded in a non-temporary computer-readable medium and is usable by a programmable computer processor that communicates with a peptide autosynthesizer, the computer program product comprising computer-readable program code that, when executed, causes the programmable processor to implement any of the embodiments of the computer implementation method described above.

[0018] Further features and advantages of the present invention will become apparent when the following detailed description of specific preferred embodiments is read in conjunction with the accompanying drawings.

[0019] The present invention will be better understood by referring to the following detailed description of specific embodiments in conjunction with the drawings. The drawings are as follows.

[0020] In general, similar elements or components in different drawings may be referenced by similar reference numerals or labels, and / or, in order to adequately promote the conciseness, clarity, and understanding of the drawing, the size and relative scale of each element in the drawing may be set differently from the actual size. For the same reason, not all elements in one drawing necessarily have to be shown in another drawing. [Brief explanation of the drawing]

[0021] [Figure 1A] ~ [Figure 1B] Respectively shown are an exploded perspective view and an assembled perspective view of an embodiment of the photometric device of the present invention, configured at least for UV monitoring of deprotection and / or coupling. This embodiment is shown as including a top housing assembly, a chassis assembly, and a bottom housing assembly. [Figure 2A] to even [Figure 2C] Additional cross-sectional views of the embodiment of FIGS. 1A and 1B are shown. The insertion view of FIG. 2A shows the internal contents of the chassis assembly in more detail. The positions of the cross-sections of FIGS. 2A and 2B are shown in FIG. 2C. [Figure 3A] to even [Figure 3C] Further additional cross-sectional views of the embodiment of the photometric device of FIGS. 1A and 1B are shown. The positions of the cross-sections of FIGS. 3A and 3B are shown in FIG. 3C. [Figure 4A] to even [Figure 4B] A schematic view of an embodiment of a part of the chassis assembly of the device of FIGS. 1A and 1B is shown. [Figure 5A] to even [Figure 5C] A view and a structural explanation of the cap plate of the chassis assembly of FIGS. 4A and 4B are shown. [Figure 6A] to even [Figure 6B] An embodiment of a flow cell chassis part of the chassis assembly of FIGS. 4A and 4B and related information are shown. [Figure 7] A general system for deprotection monitoring using UV light, utilizing an embodiment of a current photometric device, is schematically shown. [Figure 8] A flowchart of an embodiment of a method for UV monitoring of a deprotection reaction according to the concept of the present invention is shown.

Embodiments for Carrying Out the Invention

[0022] For an overview of embodiments of UV monitoring methods for deprotection and / or coupling reactions employed in the prior art, see U.S. Patent No. 8,535,947. The following disclosure deals with embodiments of UV measuring devices thoughtfully configured to be used to implement such methods in place of the methods described in the '947 patent. In fact, the use of UV light transmission tubes in the '947 patent measuring system has been found to have fairly critical operational drawbacks and deficiencies, one of which stems from the fact that the same UV transmission tube is used as the measuring cell of the UV measuring device and, at the same time, as a channel for delivering deprotection reagents to the measuring cell. As a result of such a configuration, on the one hand, if the UV transmission tube outside the housing of the UV measuring device of the system deteriorates or has other problems, it is also necessary to disassemble the measuring device in order to replace the measuring cell portion of the measuring device. On the other hand, the amount of deprotection reagent in the measuring cell of the device of the entire system of the '947 patent is, of course, determined by the fairly large and not necessarily user-controlled inner diameter of such UV transmission tubes, and as a result, monitoring chemical reactions using the prior art device consumes an unduly large amount of reagent. Furthermore, experiments have revealed that the nearly geometrically uniform nature of the UV-transmitting tubes used in prior art devices makes them prone to the formation and / or trapping of bubbles and / or (depending on the inner diameter of the UV-transmitting tube) non-flowing fluids within the measurement chamber space of the device. Their presence inevitably leads to unpredictable errors in photometry performed using this device. Moreover, due to the properties of the flexible material used in the manufacture of the UV-transmitting tubes, the volume of the measurement cell determined by such tubes is sensitive to temperature fluctuations and reacts quickly. Meanwhile, the transmittance of light of the target UV wavelength through the walls of the UV-transmitting tube changes over time as the tube degrades, inevitably leading to unpredictable inaccuracies in the measurement results.

[0023] The structure of the photometric (or measuring) apparatus proposed and discussed below avoids and / or prevents all of these drawbacks and deficiencies, which is achieved by significantly reducing the size of the flow cell or flow chamber (operatively corresponding to the measuring cell of the '947 patent), and further by the specific geometric shape of the flow cell and the inlet / outlet portions of such flow cells. In particular, the inlet / outlet of the flow cell is further configured to allow induction channels and / or discharge channels (e.g., flexible tubes) for the delivery of deprotection reagents to be attached to the flow cell of the proposed apparatus.

[0024] One embodiment of the structural module 100 of the UV measuring device is shown in various representations in Figures 1A, 1B, 2A, 2B, 2C, 3A, 3B, and 3C. Module 100 is reversibly disassembled and is a separable combination of a chassis assembly 104 and an overall housing structure including upper (topmost) and lower (bottom) housing parts or assemblies 108A, 108B (as will be readily apparent to those skilled in the art, the spatial orientation of each part constituting module 100 may be changed, for example, horizontally, in which case the chassis assembly 104 is rotated 90 degrees and sandwiched between the housing parts 108A, 108B from the left and right. Alternatively, it may be changed to any other applicable orientation. Here, the vertical spatial coordination of each part of module 100 is shown as an example).

[0025] Referring to Figure 2A and the corresponding insert, a portion of the overall housing structure (shown here as the upper housing portion 108A) is configured to support a light source 110 (here a UV light LED source), and the light source 110 is oriented so that the beam of light generated by the light source 100 passes almost freely and without obstruction through an aperture 118 (see Figures 2A, 6A) formed throughout the chassis assembly toward a photodetector system (here shown as a photodetector 114) located on the opposite side, which is appropriately fixed to the opposite portion of the housing (here the bottom housing portion 108B). At least the operation of the light source 110 and the photodetector system may be controlled by electronic circuitry (e.g., a processor (not shown)) appropriately programmed to initiate a photometric process and correct the optical data from the photodetector system.

[0026] Referring to Figures 2A, 4A, 4B, and 6A, the chassis assembly 104 includes an optically nearly opaque substrate 404 (also referred to herein without distinction as the UV flow cell chassis) thoughtfully dimensionally defined to define through-slots / channels 122. Such slots / channels 122 form a portion of the fluid chamber space that houses the target fluid, which is subject to photometric measurements performed across the aperture 118 using the light source 110 and detector 114 when module 100 is in operation. Naturally, the through-aperture 118 (which is optionally molded as a cylindrical channel) (its cross-sectional dimensions are larger than those of the slots 122) defines corresponding trenches or channels on the corresponding faces of the two substantially flat, opposing walls of the slots 122. The substrate (flow cell chassis) 404 is mounted with recesses 124 formed on each of its surfaces (for example, in Figures 6A and 6B, only one recess 124 is clearly visible on the upper side of the substrate 404, and the other recess 124 is formed on the opposite side (lower side in the figures) of the plate 404). These recesses 124 are thoughtfully sized to receive and accommodate optically transparent windows or plates 408 (made of fused silica or quartz glass in one example), and when the plate 408 is placed within the recesses 124 and works in a sealing manner with the recesses 124, it defines the range by limiting the slot 122 from both sides (approximately parallel), thereby defining the flow chamber and the corresponding space, and the target fluid passes through the flow chamber (along the axis crossing the axis connecting the LED 110 and the photodetector 114) for photometric / spectrophotometric measurement.

[0027] Referring again to Figures 4A and 4B, the process of fluidly sealing the fluid chamber of the chassis assembly 104 to be virtually leak-free can be achieved as at least one implementation using an optically transparent window or plate 408 and a membrane gasket 412 (if present, this membrane gasket 412 may be configured to include a through slot approximately the same dimensions as the slot 122) positioned between the plate 408 and the bottom of the corresponding recess 124, and / or a sealing gasket 416 that covers the plate 408. In this case, the stack of components 416, 408, and 412 is placed within the recess 124 and compressed by fastening means (e.g., bolts (shown) or clamps (not shown), and caps (or cap plates) 420, etc.) to form a fluid seal surrounding the space of the slot 122, thereby completing the fluid chamber of the chassis assembly 104. The same work is performed on the opposite side of the flow cell chassis 404 (in this case, the lower side, as shown in Figure 4A). An example of an embodiment of the cap plate 420 and its structure are described in Figures 5A, 5B, and 5C. During operation, light from the light source 110 is delivered to the detector 114 by passing through the central apertures of the upper and lower cap plates 420, through both light windows, and through the space of the fluid chamber.

[0028] Referring again to Figures 2A, 2B, 4A, and 4B, the fluid inlets and outlets 128A and 128B connected to the chamber space of the chassis assembly 104 form operationally interchangeable inlets and outlets. In particular, the inlets / outlets 128A and 128B have a structure in which the cross-sectional dimensions (when viewed in a plane approximately perpendicular to the axes of the inlets / outlets) are not constant along their axes. Specifically, as shown in the insert in Figure 2A, the portions 132A and 132B of each inlet / outlet path 128A and 128B that are proximal to the through-slot 122, connected to the through-slot 122, and coupled to the through-slot 122 have a corresponding cross-sectional size that is smaller than the cross-sectional size of the portion outside the corresponding inlet / outlet path (distal to the slot 122). Furthermore, the cross-sectional dimensions of the proximal portions 132A and 132B of the inlet / outlet 128A and 128B are preferably smaller than the minimum cross-sectional dimensions of the slot 122 (and therefore the minimum separation distance between the first and second walls), because this creates an operationally favorable situation, namely, the formation and / or capture of bubbles and / or non-flowing fluid in the space of the flow chamber (when the target fluid is delivered into / through the flow chamber), which is achieved, at least to some extent, by restricting the flow of the target fluid at the junction between the inlet path and the slot 122, and / or the junction between the output path and the slot 122. Based on the cross-sectional views in Figures 3A, 3B, and 3C, it is possible to compare the relative cross-sectional dimensions of portions 132A and 132B with the relative cross-sectional dimensions of the slot 122 and the aperture 118.

[0029] In general, one implementation of the photometric apparatus of the present invention has a flow chamber (flow cell, cuvette unit) which is structured to allow the repeated and volumetrically uniform operation of filling-spatial fixation-measurement-cleaning-reuse without substantially forming bubbles in the flow chamber, and at the same time provides a spatially / geometrically restricted target fluid (sample aliquot) such that the path length of light traversing the flow chamber (flow cell) installed in the photometric apparatus is substantially constant (e.g., time-constant).

[0030] In at least one implementation, the operating principle of the entire apparatus including module 100 may remain substantially the same as that described in U.S. Patent No. 8,535,947. Figure 7 schematically shows an embodiment 700 for this purpose, which is an embodiment of an entire system (automatic peptide synthesizer) for real-time monitoring and control of a deprotection reaction using UV light, and is structured to include a built-in (i.e., integrated into system 700) monitoring device 100. The reaction vessel (RV) 710 contains the deprotection reagent 712. The deprotection reagent 712 is delivered to the photometric measuring device 100 by being pushed (e.g., by the pressure generated by nitrogen) at specified time intervals. Optionally, and depending on the particular geometric shape of system 700, such delivery may be carried out via a lead channel 717A located proximal to the reaction vessel 710. The lead channel is connected to an inlet 128A, if present. Optionally, but preferably, the lead channel 717A, if present, is positioned directly below the reaction vessel 710 in the synthesizer, thereby retaining a portion of the deprotection reagent 712 in the reaction vessel even after it has been delivered to the fluid chamber of the chassis assembly and during measurement. Immediately after measurement is complete, the deprotection reagent is returned to the reaction vessel 710, as shown in the flow diagram of Figure 8, to prepare for further reaction with the solid-phase reactants, or sent to the waste area 720 if the reaction has reached a completion point or another stop point.

[0031] Next, we consider application examples of a photometric device constructed according to the concept of the present invention, and methods requiring such a photometric device. Figure 8 shows the corresponding logic flow diagram. Between each delivery of the deprotection solution, the deprotection solution is pushed down to the UV sensor of the photometric device every 10 seconds, as shown in the figure, or at other intervals selected by the user. The rate of reaction is determined by calculating the floating average of a specified number of existing readings. When the change in the floating average no longer exceeds a pre-selected delta, the deprotection chemical has been depleted and is no longer effective for the process, and the fluid is sent to a waste location and a new deprotection solution is delivered. As will be readily understood by those skilled in the art, one particular implementation of a method for real-time monitoring of the deprotection reaction of an Fmoc protecting group in an automated peptide synthesizer (equipped with the photometric device described above) generally includes a process of drawing a fluid (in one example, a fluid containing dibenzoflubene and associated adducts and by-products) from the reaction vessel of the synthesizer into the hollow space of the photometric device through an inlet passage formed in the flow cell chassis of the photometric device, and then irradiating such fluid in the hollow space with selected UV light delivered along first and second trenches formed inside the flow slots of the flow cell chassis and passing between them, and through a plate of optically transparent material. Next, a process is carried out to acquire optical data representing the absorption of such UV light by the fluid and to determine the rate of the deprotection reaction (for example, by calculating a floating average of a specified number of readings), using a photodetector separated from the UV light source by the flow cell chassis and two plates of optically transparent material, and a processor that works in conjunction with such photodetector. Finally, as soon as the data acquisition process and the process of determining the rate of the deprotection reaction are completed, the fluid is returned to the reaction vessel through the outlet passage in the flow chassis of the photometric device (unless the change in the floating-average value no longer exceeds a pre-selected value during the user-specified time, indicating the completion of the reaction). Optionally, the time of the deprotection reaction may be extended based on values ​​calculated from the optical data recorded during the acquisition process.

[0032] In a non-limiting example of using a photometric device in an automated peptide synthesizer, one of three UV monitoring modes may be selected: (1) basic monitoring mode (measuring the degree of deprotection but without adjusting or otherwise modifying the synthesis reaction), (2) feedback-based deprotection monitoring mode (measuring the degree of deprotection and using the data to control the deprotection reaction time and number of iterations), and (3) feedback-based deprotection and coupling monitoring mode (measuring the degree of deprotection and using the data to control the deprotection reaction time and number of iterations and appropriately extend the coupling time).

[0033] Accordingly, an online UV light source and detector are provided, which are complemented by a suitable processor and configured to record data at specified time intervals during the deprotection reaction as well as at the end of the deprotection reaction, thereby providing an automatic means to correct the deprotection time and number of iterations based on the process shown in the flowchart of Figure 8. Experimentally, it was revealed that the reproducibility and consistency of the measurement results obtained using module 100 are ultimately independent of temperature fluctuations, and the transmittance of light of the target UV wavelength by each of the first and second plates exceeds 90%, thereby ensuring the predictability and controllability of the measurements.

[0034] Those skilled in the art who are interested in this disclosure will now readily see the novel and inventive aspects of the apparatus and method of the present invention. In addition to the advantages described above, it is possible to use real-time data collected during synthesis using a programmable processor (not shown for brevity of description) (working operationally with embodiments of the present invention) to generate a visually perceptible representation (e.g., graphs and / or images, at least in some cases, displayed on a monitor) of individual deprotection and / or coupling reactions, or an overall summary of the synthesis, concurrently with the synthesis process. Furthermore, various reaction steps during and after the synthesis can be revealed.

[0035] For the purposes of this disclosure and the appended claims, the use of “substantially,” “approximately,” “about,” and similar phrases when referring to the descriptors of values, elements, properties, or features presented is intended to emphasize that the referenced values, elements, properties, or features will, in practice, still be considered as stated by those skilled in the art, although not necessarily strictly literally. When applied to descriptors of specific properties or qualities, these phrases mean “mostly,” “mainly,” “considerably,” “by and large,” “essentially,” “to great or significant extent,” and “largely but not necessarily wholly the same,” for example, to appropriately indicate approximations and to describe a particular property or descriptor in such a way that its scope is understood by those skilled in the art. In one specific example, when the terms “approximately,” “substantially,” and “about” are used in relation to numerical values, they represent a range of ±20% of a given value, more preferably ±10%, even more preferably ±5%, and most preferably ±2%. As a non-limiting example, two values ​​being “substantially equal” means that the difference between the two values ​​may be within ±20% of the values ​​themselves, preferably within ±10% of the values ​​themselves, more preferably within ±5% of the values ​​themselves, and even more preferably within ±2% or less of the values ​​themselves. Using these terms to describe a selected characteristic or concept does not imply or provide any basis for ambiguity or for imposing any numerical limitations on a particular characteristic or descriptor.As those skilled in the art will understand, any actual deviation of the exact value or characteristic of such a value, element, or attribute from what is so stated will fall within a numerical range defined by the typical experimental measurement error when using measurement methods accepted in the art for such purposes, and may vary within that range.

[0036] Throughout this specification, any reference to “one embodiment,” “an embodiment,” “a related embodiment,” or similar phrases means that a particular feature, structure, or characteristic described in relation to the referenced “embodiment” is included in at least one embodiment of the present invention. Therefore, throughout this specification, the phrases “in one embodiment,” “in an embodiment,” and similar phrases may all refer to the same embodiment, but not all do so. Naturally, no part of this disclosure, either in itself or in possible relation to the drawings, fully describes all features of the present invention. While embodiments are described in this specification to enable a clear and concise description, the embodiments may be combined or separated in various ways, as intended and, naturally, without departing from the scope of the invention. In particular, naturally, all features described herein are applicable to substantially all aspects of the present invention.

[0037] In addition, while the features of the present invention are described in this disclosure with reference to the corresponding drawings (where, wherever possible, similar reference numerals represent the same or similar elements), structural elements are generally not drawn to the correct scale, and certain components are drawn larger than others for emphasis and understanding. Naturally, no single drawing supports a complete description of all features of the present invention. In other words, a given drawing broadly illustrates only some of the features of the present invention, and broadly does not illustrate all of them. A given drawing, and the relevant parts of this disclosure including descriptions that refer to such drawings, generally do not include all elements of a particular drawing, or all features that could be shown in that drawing, at least to simplify the given drawing and description, and to direct the description to the elements characterized in that drawing. As those skilled in the art will understand, the present invention may, in some cases, be implemented without one or more of a particular feature, element, component, structure, detail, or characteristic, or using other methods, components, materials, etc. Accordingly, specific details of one embodiment of the present invention do not necessarily have to be shown in each drawing illustrating such an implementation, but it may be implied that such details are present in the drawings unless otherwise intended in the context of the description. In other cases, well-known structures, details, materials, or operations do not need to be shown or described in detail in the given drawings, so as not to obscure the described embodiments of one embodiment of the present invention. Furthermore, individual features, structures, or properties of the present invention described may be combined in any suitable manner in one or more other embodiments.

[0038] The "real-time" performance of a system is defined or understood as performance that targets the operational deadline from a given event to the system's response to that event. For example, real-time extraction of contact information (such as optical data) from a photodetector may be triggered by a user or a programmable processor and may be performed concurrently or concurrently with interruptions to the process of acquiring such data.

[0039] Naturally, regardless of whether there is a specific reference to the use of a processor in this disclosure, implementations of the methods of the present invention may include, and preferably include, a processor controlled by instructions stored in memory. The memory may be random access memory (RAM), read-only memory (ROM), flash memory, or any other memory, or a combination thereof, suitable for storing control software or other instructions and data. As will be readily apparent to those skilled in the art, instructions or programs defining the functions of the present invention may be delivered to the processor in various forms, including, but not limited to, information permanently stored in non-writable storage media (e.g., read-only memory devices (ROM, etc.) within a computer, or devices readable by computer input / output devices (CD-ROM or DVD discs, etc.), information modifiable in writable storage media (e.g., floppy disks, removable flash memory, and hard drives), or information transported to the computer via communication media, including wired or wireless computer networks. Furthermore, while the present invention may be implemented in software form, the functions necessary for implementing the present invention may, optionally or alternatively, be implemented in part or in whole using firmware components and / or hardware components, for example, combinational logic, application-specific integrated circuits (ASICs), field-programmable gate arrays (FPGAs), or other hardware, or any combination of hardware components, software components, and / or firmware components.

[0040] While the present invention has been described through the non-limiting specific embodiments described above, as will be understood by those skilled in the art, the exemplary embodiments may be modified or altered as long as they do not deviate from the inventive concept disclosed herein. The embodiments of this disclosure may also be combined in ways not mentioned above. Therefore, the present invention should not be considered to be limited to the disclosed embodiments.

Claims

1. It is a device, A substrate made of an optically opaque material, It is a hollow space, A first substantially flat wall formed within the substrate, the first wall including a first trench, the first trench defined on the surface of the first wall and connecting the top of the first wall to the bottom of the first wall, A second substantially flat wall formed within the substrate, the second wall includes a second trench, the second trench is defined on the surface of the second wall and connects the top of the second wall to the bottom of the second wall, and the second wall is substantially parallel to the first wall, A first plate made of an optically transparent material is positioned across the uppermost part of the first wall and the uppermost part of the second wall, and connects the uppermost part of the first wall and the uppermost part of the second wall in a substantially leak-free manner. A second plate of the optically transparent material is positioned across the bottom of the first wall and the bottom of the second wall, and connects the bottom of the first wall and the bottom of the second wall in a substantially leak-free manner. The hollow space is limited by, Along at least one of the first and second plates, an inlet passage and an outlet passage are formed in the substrate and are fluidly connected to each other via the hollow space, Includes, The apparatus is configured to perform photometric measurements of a target fluid placed between the first and second trenches within an automated peptide synthesizer. Device.

2. The apparatus according to claim 1, wherein the first trench and the second trench are substantially parallel and opposite to each other, and in a cross-section of the substrate formed across the hollow space in a plane substantially parallel to at least one of the first and second plates, an aperture is defined at least in part by the combination of the first and second trenches, the dimensions of which are greater than the distance between the flat portions of the first and second substantially flat walls.

3. The apparatus according to any one of claims 1 to 2, wherein the inlet passage always has a portion whose cross-sectional dimensions are smaller than the separation distance between the first and second walls, in order to prevent the formation of bubbles in the hollow space when the target fluid is delivered to the hollow section through the inlet passage.

4. The apparatus according to claim 3, wherein the exit passage always has a portion whose cross-sectional dimensions are smaller than the separation distance between the first and second walls.

5. A light source configured to deliver light along an axis substantially parallel to the first and second trenches, A photodetector is arranged to receive light delivered through the first and second plates, through the hollow space, and through first and second apertures formed in the first and second material layers, respectively, which are separated from each other by the hollow space. The apparatus according to any one of claims 1 to 4, further comprising:

6. The apparatus according to claim 5, wherein the first and second material layers are configured as a sealing gasket.

7. The apparatus according to any one of claims 1 to 6, wherein the substrate and the first and second plates are configured to ensure that the hollow space remains largely unaffected by temperature fluctuations, and that the transmittance of light of the target UV wavelength by each of the first and second plates exceeds 90%.

8. A method for real-time monitoring of the deprotection reaction of an Fmoc protecting group, wherein the method is: An automated peptide synthesizer comprising the apparatus and reaction vessel according to any one of claims 1 to 7, comprising the step of monitoring the deprotection reaction of the Fmoc protecting group, wherein the monitoring step comprises at least: (a) drawing a fluid containing dibenzoflubene and related adducts and by-products from the reaction vessel through the inlet passage into the hollow space, (b) Irradiating the fluid in the hollow space with UV light having a wavelength of about 301 nm, delivered along the first and second trenches and between the first and second trenches from the first plate to the second plate, (c) Obtain optical data representing the absorption of UV light by the fluid and determine the rate of the deprotection reaction by calculating the floating average of a specified number of readings. (d) unless the change in the floating average value within the time specified by the user has not exceeded a pre-selected value, indicating that the reaction is complete, the step of returning the fluid to the reaction vessel via the outlet passage, It will be carried out by method.

9. The method according to claim 8, wherein the step of drawing fluid from the reaction vessel is performed during the deprotection reaction.

10. The method according to any one of claims 8 to 9, wherein steps (a) to (d) are carried out at specified time intervals during the deprotection reaction.

11. The method according to any one of claims 8 to 10, further comprising the step of extending the time of the deprotection reaction based on an extension time calculated from the optical data recorded during the acquisition of step (c).

12. The method according to any one of claims 8 to 11, further comprising the step of repeating the deprotection reaction based on the optical data recorded during the acquisition of step (c), wherein the optical data is used to calculate the degree of completion of the deprotection reaction.

13. The method according to any one of claims 8 to 12, comprising the step of extending the time of an amino acid or monomer coupling reaction following the deprotection reaction based on the time of the deprotection reaction.

14. The method according to claim 13, further comprising the step of providing a visually perceptible output representing the optical data during the synthesis process.

15. The method according to claim 14, wherein the visually perceptible output includes a graphical composite summary and / or a graphical representation of individual deprotection reactions.

16. The step further includes preventing the formation of bubbles in the hollow space during the step of drawing in the fluid, the preventing step being performed at least to some extent by passing the fluid through a first portion of the inlet passage distal to the hollow space and having a first cross-sectional dimension, and then passing the fluid through a second portion of the inlet passage proximal to the hollow space and having a second cross-sectional dimension. The second cross-sectional dimension is always smaller than the minimum separation distance between the first and second substantially flat walls. The first cross-sectional dimension is larger than the second cross-sectional dimension. The method according to any one of claims 8 to 15.

17. The step of returning the fluid to the reaction vessel includes passing the fluid through a first portion of the outlet passage that is proximal to the hollow space and has a third cross-sectional dimension, and then passing the fluid through a second portion of the outlet passage that is distal to the hollow space and has a fourth cross-sectional dimension, The third cross-sectional dimension is always greater than the minimum separation distance between the first and second substantially flat walls. The cross-sectional dimension of the fourth is larger than the cross-sectional dimension of the third. The method according to claim 16.

18. The method according to any one of claims 8 to 17, wherein the step of irradiating the fluid includes causing the fluid in the hollow space to be traversed by a beam of UV light having a cross-sectional dimension greater than the separation distance between the opposing flat portions of the first and second substantially flat walls.

19. The method according to any one of claims 8 to 17, wherein the synthesizer includes the UV light source and the UV photodetector within the synthesizer.

20. A method comprising the step of manufacturing an apparatus according to any one of claims 1 to 7, wherein the step of manufacturing the apparatus comprises at least: This is carried out by the step of assembling the chassis assembly, and the said assembly step is, (i) The step of forming a slot having substantially parallel walls that penetrate an optically opaque substrate, (ii) A step of manufacturing an inlet passage and an outlet passage that are fluidly connected to each other through the slot, wherein the corresponding dimensions of each of the inlet passage and the outlet passage at their corresponding intersections with the slot are always smaller than the width of the slot, defined by the distance between the substantially parallel walls, (iii) The step of placing first and second plates of optically transparent material into the respective recesses of the substrate in order to completely cover the slot, (iv) Pressing the first and second plates against the surfaces of the respective recesses to form a cell defined by the slot and the surfaces of the first and second plates, wherein the cell is fluidly sealed at the joint between the first and second plates and the optically opaque substrate, including, method.

21. The method according to claim 20, wherein the step of forming the slots includes the step of forming spatially opposing, parallel trenches in the walls parallel to each other.

22. The method according to any one of claims 20 to 21, wherein the step of manufacturing an entrance passage and an exit passage includes the step of forming a portion of each of the passages distal to the slot, wherein the cross-sectional dimensions of such portion are greater than the dimensions of the corresponding passage at the intersection of the passage and the slot.

23. Steps to fix the chassis assembly between the first and second parts of the housing of the device. It further includes, The first portion of the housing houses the first and second plates and a light source oriented to deliver a light beam substantially across the optically opaque substrate through the slots, The method according to any one of claims 20 to 22, wherein the second portion of the housing houses a photodetection system arranged to receive the light beam that has passed through the first and second plates and the slot.

24. The step of forming the slot is performed when the coefficient of thermal expansion is 10 －５ The method according to any one of claims 20 to 23, comprising the step of forming the slots in an optically opaque substrate made of a material not exceeding a certain value, wherein the step of arranging the first and second plates comprises the step of using the first and second plates made of fused silica or quartz glass.

25. Use of the apparatus according to any one of claims 1 to 7 for performing photometric measurements of a target fluid within an automated peptide synthesizer system.

26. The use according to claim 25, wherein the photometric measurement includes real-time monitoring of the deprotection reaction of the Fmoc protecting group.

27. A computer-aided method for passing a fluid in an automated peptide synthesizer between the reaction vessel of the synthesizer and the waste container of the synthesizer, (a) The computer initiates drawing the fluid containing the deprotection reagent from the reaction vessel through an inlet passage formed in the optically opaque substrate to a slot formed in the optically opaque plate of the photometric measuring device of the synthesizer, (b) The computer activates the process of irradiating the fluid in the slot with UV light and transmitting the UV light that has passed through the fluid to the photodetector of the photometric measuring device, (c) The computer performs the steps of obtaining optical data from the photodetector representing the absorption of UV light by the fluid in the slot, and calculating the floating average of a specified number of detector readings to determine the rate at which the deprotection reaction proceeds in the fluid, (d) If the computer determines that the deprotection reaction is complete because the change in the floating average value calculated during the user-specified time exceeds a pre-selected value, the fluid is returned to the reaction vessel; otherwise, the fluid is sent through an outlet passage formed in the optically opaque substrate to the waste container, which is fluidly separated from the reaction vessel by the photometric measuring device. Includes, Each of the aforementioned entrance passage and exit passage has a corresponding first cross-sectional dimension in the portion of the passage proximal to the slot and a second cross-sectional dimension in the portion of the passage distal to the slot, wherein the second cross-sectional dimension is always smaller than the minimum separation distance between the first and second walls, and the first cross-sectional dimension is larger than the second cross-sectional dimension. method.

28. The aforementioned initiation step is, The step includes initiating the drawing of the fluid containing dibenzoflubene and related adducts and by-products into a hollow space, the hollow space being The first and second walls formed within the optically opaque substrate, The first and second flat and parallel plates of optically transparent material are positioned across the top of the first wall and the top of the second wall, and are configured to connect the first wall and the second wall in a substantially leak-free manner. It is limited by, The slot is defined by the fact that the flat portions of the first and second walls are substantially parallel to each other. The computer implementation method according to claim 27.

29. The computer implementation method according to any one of claims 27 to 28, wherein the computer initiates the retraction step during the deprotection reaction.

30. A step in which the computer initiates the repeating step, wherein the optical data recorded during the acquisition of step (c) is used to calculate the degree of completion of the deprotection reaction, and the optical data is used to calculate the degree of completion of the deprotection reaction. A computer implementation method according to any one of claims 27 to 29, further comprising:

31. The computer-assisted method according to any one of claims 27 to 30, comprising the step of using the computer to extend the time of a coupling reaction of an amino acid or monomer in the synthesizer, which follows the deprotection reaction, based on the time of the deprotection reaction.

32. A computer program product encoded in a non-temporary computer-readable medium and usable by a programmable computer processor communicating with a peptide autosynthesizer, the computer program product comprising computer-readable program code that, when executed, causes the programmable processor to carry out the method according to any one of claims 27 to 31.

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