多肽合成仪操作手册

特别说明 此资料来自豆丁网(http://www.docin.com/) 您现在所看到的文档是使用下载器所生成的文档 此文档的原件位于 感谢您的支持 抱米花 http://blog.sina.com.cn/lotusbaob http://www.docin.com/p-57613894.html Table of Contents Specifications and Pre-Installation requirements Page 2 Chapter 1 Introduction 1.1 Introduction to solid phase peptide synthesis 1.2 General descriptions of Hardware 1.3 Introduction to Synthesizer Software 1.4 Safety A丛寸 A斗 A" 丁 叮31i1 且 1i Chapter 2 Synthesizer 2.1 Components 2.2 Detailed Description 17 17 Chapter 3 ßasic Operations 3.1 Detailed description of Synthesizer Functions 18 Chapter 4 Software 4.1 Software overview 4.2 Detailed description of Software 臼nction 4.3 Administrator / User / Passwords AY 气，马 OO --句，&竹马 Chapter 5 Using Pre-Installed Synthesizer Protocols 5.1 U sing Pre-Installed Synthesizer Protocols 29 Chapter 6 Maintenance 6.1 Before each Synthesis 6.2 Periodic Maintenance 6.3 Shutdown 喃自 EA 唱··且咽 'EA 句3 句3 句3 Chapter 7 Troubleshooting 7.1 Malfunction description 7.2 Mal臼nction chart 32 32 Appendix 1 Parts List 34 Appendix 2 矶Ta町anty 37 Appendix 3 Full Service Warranty 38 Appendix 4 Metering Level Calibration 39 Appendix 4 Flow Schematic 40 Specjficabons and P re IDstaJJabon Requjrern e口ts GENERAL SPECIFICATIONS Dimensions Synthesizer Box . Clearances: Each side Front Also required: Storage space for nitrogen tank Space for computer Ambient operating temperature range Reservoir capacity: 1 Solvent reservoir 3 Solvent reservoir 2 Solvent reservoir 2 Solvent reservoir 24 Amino acid reservoirs 2 Metering vessels (max.) 2 Transfer vessels (max.) Nitrogen requirements: Purity Maximum dew point Regulator Compressed air: Regulator Intemal regulator: Solvent lines Electrical requirements: Line power source Computer system requirements: Computer Operating system SPACE REQUIREMENTS 30"Hx 39"Wx30"D 12 inches 30 inches 15-40 0 C 5 Liter 2 Liter 1 Liter 500mL 50ml each 250ml each 250ml each >99.5% -60 0 C 20 psi (1 45 kPa) 70 psi (500 kPa) 7 psi (50 kPa) 120V, 6A or 220V 3A Pentium or bet1er Win 98 or higher The instrument is designed to fit into the space 6 白 wide and 3 ft deep. 2 Chapter 1 Introduction 1.1 Introduction to Solid-Phase Peptide Synthesis Thepu甲ose ofthis chapter is two-fold. First, a briefhistorical perspective on the development and most common applications of solid-phase peptide synthesis will enable the user to best apply the two most widely-used synthetic strategies Boc/Benzyl and Fmocl Butyl chemistries to his/her projects. Second, a description ofthe recommended chemistries for coupling and deprotection cycles is provided, with ‘helpful hints' so that newcomers to peptide science will hopefully avoid some ofthe obstacles which often lead to expensive mistakes and/or poor synthesis yields. This point cannot be overemphasized 一 . The flexibility ofthe CSPEP/TRIPEP software packages is designed to give the user virtually limitless freedom in the customization of existing protocols. However, this is not without an important . The successful stepwise synthesis of even a 10- residue peptide embodies the sum ofbetween 30 and 50 independent chemical reactions which, by absolute necessity, proceed to completion. While this should not deter the user from experimenting with new chemistries/methodologies, it is important that such experimentation be performed in accord with sound chemical principles and hands-on experience. 1. A Brief Historical Perspective The chemistry ofpeptide synthesis first developed in the early 1900's by Emil Fischer- arguably marks the birth of organic synthesis as we know it today. Whereas the lionshare of organic synthesis continues to be performed by solution-phase methods, . ., with each independent chemical reaction followed by a purification step and characterization of the resulting synthetic intermediate, two peculiarities of peptide chemistry spurred the development of a more efficient synthetic strategy. In contrast to most total synthesis efforts, the synthesis of peptides at least until the final deprotection step - is an iterative process, with α-amino (α) deprotection and amide couplings performed in succession until the desired full-length target peptide is obtained. In addition, most peptides of biological interest are grossly insoluble in most organic solvents irrespective of side-chain protection tactics. The net result of these two characteristic features of peptides was that the first sixty-odd years of peptide synthesis forged little ground until the landmark work in the late 1950's ofR.B. 孔1errifield at Rockefeller University. During the course ofhis Ph.D. work, Merrifield (a biochemist) proposed an entirely new paradigm in organic synthesis. As is often the case when an outsider looks into an insular field 3 and pursues a tack that is anathema to the existing experts in the field, Merrifield's idea of performing all synthetic manipulations using the C-terminus of the target peptide linked to an insoluble solid support was met with much criticism. However, it was not long before the chemistry of solid-phase peptide synthesis (SPPS) was honed to a point where traditional solution-phase methodologies were no match with regard to speed and versatility. The original "Merrifield" version of SPPS 1 more accurately referred to as Boc/Benzyl chemistry - was roughly finalized in the late 1960s, and employs a system for manipulation of all protecting groups (Scheme 1). In this strategy, the α-amino -butoxycarbonyl (Boc) protection is removed with TF A, while side-chain protections and the peptide-resin anchorage (the linker) require much harsher acidic conditions for cleavage. This final step is accomplished using liquid HF, a much stronger acid than TFA (acidity functions of -11 and 0.1 , respectively). Scheme 1 depicts the manner in which Boc/Benzyl SPPS simplifies all of the reactions involved in peptide synthesis by allowing for purification filtration, so that excess reagents can be employed and removed by simple washing. It is important to emphasize that the chemistry of SPPS is not fundamentally different from that used in solution-phase peptide synthesis. The sole chemical distinction between solution- and solid-phase peptide synthesis is that the . This difference notwithstanding, all side-chain and αprotecting groups, as well as coupling chemistries, employed in solid-phase synthesis have been successfully applied in solution-phase synthesis, and vice-versa, with few exceptions. During the 1970s several groups were actively developing milder methods for SPPS that avoided the use of liquid HF as for the final deprotection/cleavage reagent. While a variety of milder graduated acid lability systems were devised, the method that rose to general applicability was the system ofFmoc/ Bu chemistry. This strategy - developed by R.C. Sheppard at Cambridge University differs from Boc/Benzyl chemistry in that the side-chain and α protecting groups are removed under conditions that leave the other class entirely intact. In Fmoc/tBu chemistry, a mild base - usually piperidine (pKa 11.1) - is employed for iterative α 9-f1uorenylmethoxycarbonyl (Fmoc) deprotection, while global side-chain deprotection/cleavage is accomplished with TF A (Scheme 2). It must be emphasized that the more traditional Boc/Benzyl and Fmoc/ Bu chemistries, while differing in chemical minutiae, are fundamentally the same process. In both cases, the target peptide chain is assembled in a stepwise fashion fromα- and side-chain protected amino acids. In both cases, the ‘ transient'αamino protection is employed solely during chain elongation (the coupling reaction) and then removed for the subsequent coupling reaction. Last1y, in both cases, the final step - global side-chain deprotection and cleavage of the peptide-resin anchorage - is accomplished by acidolysis, and the target peptide isolated by trituration from ether and purified by reversed-phase high performance liquid chromatography (RP-HPLC). 1 In fact, the first variant of SPPS developed by Merrifield employed a different, orthogonal chemistry, which employed extremely harsh acid and base treatments. The merits and general applicability of TF A-mediatedα- deblocking and HF-mediated global deprotection/c1eavage (Boc/Benzyl chemistry) were ear1y recognized and arose to prominence as the standard "Merrifield" chemistry for SPPS. 4 During the first 20 years or so of SPPS (1 970-1990), most laboratories in academia and industry performed Boc/Benzyl chemistry. Only when the comparatively ‘younger' Fmoc/tBu chemistry had matured - with appropriate side-chain protections and its unique side reactions circumvented - in the late 1980s did it begin to overtake Boc/Benzyl chemistry for routine SPPS needs. It is the author's experience that Boc/Benzyl chemistry is , while Fmoc/ Bu chemistry is (and therefore well-suited to the newcomer to SPPS), II. Boc and Fmoc SPPS Compared There is unfortunately a pervasive mentality that Boc and Fmoc chemistry are fundamentally different, but this is not the case. • In both Boc and Fmoc chemistry， αprotection is achieved using a mildly electron- withdrawing carbamate. In both cases, the side-chain protections are based on ethers, esters, carbamates, carboxamides, and sulfonamides. In both cases, carboxyl activation is accomplished using the same activating reagents. And in both cases, the scissile peptide-resin anchorage is cleaved in acid, leaving the desired C-terminal functionality (usually an acid or amide). The principal chemical minutiae differentiating the two strategies lie in the method for iterative α amino deprotection and the degree of acid stability of the side-chain and peptide-resin linkages. The reader willlikely notice this high degree of architectural similarity from Schemes 1 a and 2a. At present, Boc chemistry continues to hold an edge over Fmoc/tBu chemistry from an economic standpoint with regard to the cost of the necessary protected amino acid derivatives and solvents. In addition, longer peptides rich in ß-sheet structure tend to more accessible by Boc chemistry owing to the powerful disaggregating properties of TF A, used during iterative α­ deprotection steps. This feature - the lower incidence of failed syntheses - continues to be a potent driving force for the continued popularity ofBoc chemistry. Finally, the synthesis of C- terminal α-carboxythioesters is generally regarded to be best accomplished by Boc chemistry. While several methods have been reported to allow the synthesis of peptide thioesters by Fmoc chemistry, such methods have significant drawbacks and have been only sparsely validated. As peptide thioesters are important synthons for the total chemical synthesis of proteins and bioconjugates, the ease with which these can be prepared by Boc chemistry 臼rther substantiates its continued popularity. The convenience ofFmoc/tBu chemistry (vis-a-vis the absence ofHF) and its facile amenability to parallel synthesis and cleavage without specialized apparatus are attractive features in industry and academia alike, and are largely responsible for its rise in popularity in recent years. Aside from these practical considerations, Fmoc chemistry is ideally suited to certain side-chain modifications, which are less conveniently installed by Boc chemistry. For instance, the elaboration of synthetic peptide targets with sugars (glycopeptides), sulfates (sulfopeptides), and phosphates (phosphopeptides) is easiest accomplished by Fmoc chemistry. The reason for this admonition is two-fold: first, Fmoc chemistry allows for more dimensions of 5 orthogonality than Boc chemistry, so site-specific modifications ofthe resin-bound peptide are easier to perform. Second, the milder nature of TF A cleavage/ deprotection allows acid-labile modifications (e.g. , pendant sugars) to survive intact. O H R1/Nγ 、OH R2 .. R2 N=C=N N 一­ C 一一+M川U川 N ，N飞Dicyclohexylcarbodiimide (DIC) ' R2 NH O H R1/ N丫、N一巴旦旦旦 R2 … a auv 川uuv' VA NHMH 叶 、JIlt\01 川 {/mmu NHOar- D N N H2N一巴旦旦旦 Scheme 3. Mechanism for amide coupling via O-acylurea activation Overall, Boc and Fmoc chemistry can be used equally well for the vast majority of synthetic peptide targets, despite the fact that the chemical peculiarities of specific modified peptides may contraindicate the use of one chemical strategy versus the other. Suffice it to say that the selection of a SPPS strategy for a given peptide target is due as much to the personal experience of the user and expertise available to himlher than chemical considerations III. Coupling Methods i. -Acylureas The oldest form of widely used carboxyl activation in stepwise SPPS is the -acylurea intermediate (Scheme 3). While classical acylation chemistries such as the acid chloride and mixed anhydride are in theory viable for stepwise SPPS, significant levels of side reactions attend the use of such reactive intermediates, and were early recognized to contraindicate their use in the synthesis of targets of any degree of complexity and size. The -acylurea species is readily formed in organic solvent in the absence of base; as this addition reaction is typically faster in nonpolar - rather than polar - solvents, the -acylurea is usually formed in DCM. If desired, solvent can be removed in vacuo so that the acylating species can be resolubilized in another solvent such as DMF. Although an improvement over its earlier ancestors with regard to attenuated reactivity, the -acylurea bears some significant drawbacks. First, an intramolecular • acyl transfer easily allows the reactive -acylurea to rearrange to the inert -acylurea. This species does not pose any specific problems for stepwise SPPS other than the consumption of the active 6 acy1ating species prior to amide coup1ing. Second, the high reactivity of these species allows for an unacceptab1e 1eve1 of racemization at the αposition， presumab1y through the formation of an O H RrNY 、OH R2 O H R1/ N \f 、0 N=C=N N 一一 户U一一 + M川U川 O H RJNγ 、OH R2 .. R2 .. 1,3-Diisopropylcarbodiimide (DIC) 叫 /少 』 O H R1/ N O f让人N O H R川γ 、OH R2 H2N一巴里坦E Scheme 4. Mechanism for amide coupling via symmetric anhydride activation oxazo1one intermediate. These considerations - both stemming from the recognized high reactivity of the -acy1urea intermediate - early motivated the use of additives which wou1d afford a more stab1e, a1beit still reactive, acy1ating species. ii. Symmetric Anhydrides The use of symmetric anhydrides in p1ace of -acy1ureas hera1ded a quantum 1eap in the efficiency and yie1ds of SPPS. The reader will notice that symmetric anhydrides are formed a two-step reaction sequence in which the initia1 -acy1urea reacts with another mo1ecu1e of the same α-protected amino acid, 1iberating a urea by-product a10ng with a symmetric anhydride as the active acy1ating agent (Scheme 4). It shou1d be noted that the formation of symmetric anhydrides is significantly faster in non-po1ar solvents such as DCM, which comes with the added benefit of allowing for visua1 monitoring ofthe activation process (as the urea ofDIC is inso1ub1e in this solvent, whereas it is solub1e in DMF). Symmetric anhydrides have tapered in popu1arity in recent years owing to the faci1ity with which other chemistries (notab1y HBTU) can be adapted to automation without the formation of any troub1esome inso1ub1e by-products. However, symmetric anhydrides remain a powerfu1 too1 in the peptide chemist's repertoire, as they are exceptionally reactive acy1ating species - far more so than HOBt esters - whi1e still being nearly impervious to racemization. Whi1e early derided as being wastefu1 of precious protected amino acid derivatives, this is 1ess of a concem today than it was in the early days of SPPS when suitab1y protected amino acids were rare commodities with corresponding1y high unit prices. Furthermore, the high reactivity of symmetric anhydrides makes them the most cost-effective chemistry for difficult acy1ations, such as those onto α­ disubstituted amino acid residues and secondary amines. Symmetric anhydrides a1so remain the 7 intermediate of choice for DMAP-catalyzed acylation of hydroxyllinkers during the coupling of the C-terminal residue to a linker-functionalized resin. R2 " • R2 N=C=N N 一­C = + M川UH 1,3-Diisopropylcarbodiimide (DIC) UHM 川 ON 、。 N 1-Hydroxybenzotriazole (HOBt) O H R(N丫 \N寸 Peptide R2 川 O N人N OH N M川 、， ,N H2N一巴堕旦旦 Scheme 5_ Mechanism for amide coupling via DIC/HOBt activation iii. HOBt esters DIC/HOBt -alkyl and -acyl hydroxylamine species were recognized in the mid-1970s as having ideal properties as additives during amide coupling reactions. While several have been popularized to varying extents, l-hydroxybenzotriazole (HOBt) is far-and-away the most widely used additive in standard amide coupling reactions. HOBt is ac阳ally a mild acid at the hydroxyl group (pKa 二 4.匀， intermediate between citric acid and acetic acids. The acidity of HOBt derives from two sources: (1) the inductive effect ofthe Nl atom, and (2) aromatic stabilization ofthe conjugate base. HOBt is a weak nucleophile, but still potent enough to be acylated by an -acylurea intermediate. Of critical importance, the HOBt ester is a stable entity, and prone to far fewer side reactions than more highly activated acylating species ( . ., acid chlorides, anhydrides, acylureas ). HOBt esters are usually formed through the -acylurea intermediate (Scheme 5). In scheme 5, the carbodiimide N ,N' diisopropy lcarbodiimide (D 1 C) is depicted rather than N ,N' - dicyclohexylcarbodiimide (DCC), as in scheme 3. Either carbodiimide may be used in principle, but DIC has superceded DCC in recent years owing to its more facile handling properties, particularly vis-à-vis the solubility ofthe resulting urea by-product. Whichever carbodiimide reagent is used, it is important to recognize that its role in the formation of an HOBt ester is solely as a stoichiometrically equivalent dehydrating reagent. 8 Several features of HOBt esters substantiate their popularity. First and foremost, the use of HOBt esters for amide couplings is operationally simple. In the case of Fmoc chemistry, there is no particular requirement for a specific order of addition or preactivation phase. This point is of critical importance. It should be mentioned that scheme 5 is an idealized representation of the preparation of HOBt esters DIC activation. In fact, the coupling ‘ cocktail' is precisely that a mixture of several acylating species, such as O-acylureas, symmetric anhydrides, and HOBt esters. The exact composition of said intermediates is dependent on the method of preactivation, solvent composition, and reagent stoichiometry. Furthermore, it is difficult to guarantee that preactivation does indeed occur to completion, so in reality a mixture of activated and unactivated species - together with residual carbodiimide - are added to the resin-bound amine. N onetheless,‘all roads lead to Rome' , and the fundamental point from a practical vantage is that provided the resin-bound α-amino 臼nctionality is a free base (not a salt), no serious side reactions at1ends the use ofDIC/HOBt to prepare HOBt esters. This is in stark contrast to Boc chemistry, where TF A deprotection of the Boc liberates the α-amine as a trifluoroacetate salt. As ammonium ions react readily with carbodiimides to yield guanidines, DIC/HOBt chemistry is contraindicated in modem Boc SPPS, which is usually performed without pre-neutralization of the resin-bound amine. The second primary feature ofHOBt esters formed DIC/HOBt is that this continues to be the gold-standard for minimal racemization amide couplings using carbamate-protected amino acids, even with p