Peptide synthesis chemistry and modifications

Peptide synthesis chemistry and modifications Peptides and proteins exhibit the largest structural and functional variation of all classes of biologically active macromolecules. Biological functions as diverse as sexual maturation and reproduction, enzyme inhibition, blood pressure regulation, glucose metabolism, thermal control, analgesia and learning and memory are now thought to be regulated by peptides. Peptide synthesis chemistry Synthetic peptides are valuable tools in analysis of naturally occurring peptides or proteins. Since Emil Fischer s pioneering work in the early 1900 s, synthesis methods have improved continually especially with Merrifield s development of solid-phase syntheses. Besides the classical synthesis in solution, solid-support synthesis is now the most widely used method to prepare synthetic peptides. The advantages of solid-support synthesis are its speed, versatility, ease of automation and low costs. owever, peptide chemistry still remains a difficult and exacting science. Solid-phase synthesis is usually carried out as follows: 1. loading of -terminal amino acid to resin (not shown below) 2. deprotection: removal of -terminal protecting group (PG) at amino residue 3. activation of next amino acid at carboxy residue 4. coupling reaction 5. start synthesis cycle 2-4 again or cleave fully-synthesised peptide off resin PG PG activation deprotection etc. PG coupling PG removal 1

Protecting groups (PGs) ne of the demanding parts in peptide synthesis is the necessity to block those functional groups that must not participate in peptide bond formation. Such protecting groups are needed for all specific side chain functions of DA-encoded amino acids (p.e. the amino group of the amino acid that lends its activated carboxyl group to the coupling reaction and for the carboxyl group of the amino acid that will be acylated in its amine group). In order to elongate the resulting dipeptide, one protecting group has to be removed. This has to be done under such conditions that the peptide bond itself is not harmed and transient protecting groups still stay on, too. Solid phase synthesis requires two types of protecting groups: a) Transient protecting groups for amino groups that form the peptide bond. b) Permanent protecting groups for functional groups within the amino acid side chains. These PGs have to be stable enough to sustain chemicals used to remove transient PGs. Transient PGs for amino acids Such PGs should be easily removable but still stable enough to survive the conditions of coupling reactions and other manipulations. Two commonly used amino protecting groups are: deprotection t-boc (t-butoxycarbonyl) under mild acidic conditions with TFA (50% TFA in DM) advantage stable towards catalytic hydrogenation, can be used with Z group for side chain protection disadvantage final workup with F necessary Fmoc (9-fluorenylmethyloxycarbonyl) mild basic, non-hydrolytic conditions with primary or secondary amines: 20% piperidine in DMF deprotection does not affect amide or t-butyl protected side-chain esters, stable towards tertiary amines 3 tboc F Fmoc 3 3 TFA 3 3 3 TFA Piperidine Permanent PGs for side chains Since the different side chains of the DA-encoded amino acids encompass the majority of the common functional groups in organic chemistry, several different types of side chain protecting groups are required for peptide synthesis. These PGs are usually based on the Bzl or tbu group. Amino acids with alcohols or carboxylic acids in the side chain can be protected either as Bzl ethers or as Bzl or cex esters. Another alternative is protection as tbu ethers or esters. ther types of functional groups (amino group of Lys, thiol group of ys, imidazole of is or guanidino group of Arg) may require other special protecting groups. ertain amino acids contain functional groups in their side chains that can cause unwanted side reactions if not protected but even with the semi-permanent protection, the situation is far from satisfactory with certain amino acids. 2

In solid-phase synthesis this becomes even more acute, because acylating reagents have to be used in excess to force the reaction to completion. The most commonly used protection strategies are the tboc/bzl and Fmoc/tBu methods. strategy tboc/bzl Fmoc/tBu amino protection with tboc Fmoc side chain protection with Bzl or cex, either soft acid labile or tbu, ideally base stable and TFA stable and thallium/tfa labile or weak acid labile soft acid stable The following table displays a summary of special protection strategies for different amino acids: tboc/bzl Amino Protecting omment acid group Asn Gln unprotected use of active ester or D/BT or BTU Amino acid Arg Fmoc/tBu Protecting group Mtr Pmc Pbf Asp Glu Bzl Asn Gln Trt Tmob Ser, Thr Bzl Trp tboc Tyr Bzl Met unprotected 2-Br.Z 2,6-di-l-Bzl Lys 2-l-Z Asp Glu tbu Arg Tos Mts Mbs Ser, Thr, Tyr tbu is Trp Met ys Tos Bum Bom Trt Dnp unprotected For unprotected Met() tbu MeBzl Mob Acm TFA labile, protection for one cycle stable to F requires thiolytic cleavage prior to F Lys is ys tboc Trt omment Mtr requires long deprotection time removable TFA/TIS Acm removal by I 2, g(ac) 2 StBu reduction with thiols and phosphines Trt TFA/TIS 3

oupling For many years the most popular acylating agents have been carbodiimides. D (dicyclohexylcarbodiimide) mediated couplings have been used primarily in solid-phase synthesis. The activation step of D-mediated coupling reactions is the formation of an -acylisourea intermediate of the carboxylic acid, which is favoured by nonpolar solvents, such ac DM. This intermediate can either - react directly with resin-bound amines to form the desired amide - or it reacts with further acids to yield the symmetric anhydride - or it reacts with another agent, such as Bt (1-hydroxybenzotriazole) to form a secondary acylating reagent. ertain side reactions of carbodiimides have led to the examination of other acylating agents. Two of the more popular reagents are BP and BTU, both of which require activating bases. The various preactivated tboc or Fmoc amino acid species formed by these reagents are: - symmetric anhydrides (carbodiimides, BP, BTU) - acid chlorides (thionyl chloride) - and esters. For solid-phase synthesis, a large excess of acylating reagent (3-4 molar excess of resin-bound amine) is recommended. Thus, under a normal and routine single coupling reaction, reaction rates of >99% are usually observed. To ensure that the coupling reaction goes beyond that limit, a second coupling reaction with a different solvent is used. Since DMF is a better solvent of peptide resins, a mixed mode double-coupling procedure i.e. symmetric anhydride in DM (1 st coupling) and D/Bt or symmetric anhydride in DMF (2 nd coupling), can be useful for kinetics of coupling and for accommodation to the varying solvating properties of the growing peptide chain. Since a rapid coupling reaction is crucial for the Fmoc/tBu strategy to prevent cleavage side reactions of Fmoc by resin-bound amines, coupling by symmetric anhydride in DMF is often chosen. Some amino acids like is and Arg require special handling due to their proneness to certain side reactions as Fmoc amino acids are expensive and coupling reactions with symmetric anhydrides have to be performed with excess reagents, active esters are chosen as alternatives. Two useful active esters are Bt and dhbt (oxobenzotriazine) either as preactivated forms or generated in-situ. 4

Solid-phase peptide synthesis (SPPS) SPPS consists of three distinct steps: - chain assembly on a resin - simultaneous or sequential cleavage and deprotection of the resin-bound, fully protected chain - purification and characterisation of the target peptide. Various chemical strategies exist for chain assembly and cleavage/deprotection methods, but purification and characterisation are more or less invariant. The two currently most popular SPPS strategies utilise either the acid labile tboc or the base labile Fmoc protecting groups. Each method involves fundamentally different amino acid side chain protection as well as cleavage/deprotection. The principles of stepwise solid-phase synthesis were first described by Merrifield: - a tboc amino acid (-terminal amino acid of peptide) was covalently attached to an insoluble polymeric support (the resin) - the tboc group was removed by TFA, the free amino terminus neutralised by TEA - the next amino acid (with protected amino group) was activated and reacted with the resinbound amino acid to an amino-protected dipeptide - DM (dichloromethane) or DMF (dimethylformamide) served as primary solvent for deprotection, coupling and washing - Excess reagents and by-products were removed by simple filtration and washing. - the amino protecting group was removed and chain elongation continued with the 3 rd and subsequent protected amino acids - after the target-protected peptide had been built, all side chain protecting groups were removed and the anchoring bond between peptide and resin was cleaved (F or TFMSA) Despite the use of optimised chemistry, not all peptides can be made with equal ease by SPPS. Some amino acid sequences are more difficult to produce than others. In general, difficulties are rather sequence-dependent than related to specific amino acid residues and they show a strong dependency on solvents used for the coupling reaction. Deprotection of peptides hemical synthesis of peptides whether by solution or SPPS requires a method to release the desired peptide from its protecting groups. Acidolysis remains the most popular amongst the synthetic strategies to cleave peptides from both resin and protecting groups. Acidolytic deprotection is a major component in the overall scheme of SPPS approaches. The first scheme the tboc/bzl strategy uses the principle of differential acid lability, whereas the second approach the Fmoc/tBu strategy offers promising potential and a great selection for the acid labile side chain protecting groups due to the wide range between basic and acidic cleavage. Despite their usefulness for chemical synthesis of peptides, strong acids as deprotecting reagents have led to many serious side reactions and to a loss of product invariably resulting from one common origin: the modification of peptides by carbocations generated during the cleavage reaction. Efforts have been made to reduce the carbocation formation during deprotection and to understand the fundamental mechanism of non-aqueous acid cleavage reactions. 5

Purification of peptides Purification strategies are usually based on a combination of separation methods which exploit the physiochemical aspects of peptides or proteins, i.e.: - their size - their charge - their hydrophobicity. Among the various purification techniques can be found: - size-exclusion chromatography - IE (ion-exchange chromatography) - partition chromatography - PL (high-performance liquid chromatography) Thermo Electron uses P-PL (reverse phase PL) as it is the most versatile and most widely used PL method. It is called reverse phase, because it behaves in the opposite way to normal phase chromatography: - The stationary phase is silica chemically bound with alkylsilyl compounds resulting in a non-polar, hydrophobic surface. - Solute retention is mainly due to hydrophobic interactions between the solutes and the hydrocarbonaceous stationary phase surface. Polar mobile phases, usually water mixed with methanol, acetonitrile and/or other water miscible organic solvents, are used for elution. - Solutes are eluted in order of decreasing polarity (increasing hydrophobicity). Increasing the polar component of the mobile phase increases retention of the solute. Under identical PL conditions the retention of solutes increases proportionately with the carbon chain length of bound groups (18, 8, 4 PL columns). In general, the more polar (less hydrophobic) compounds are best separated with mobile phases of lower organic content, as they are more suited to the analysis of these compounds. The longer chain hydrocarbon phases interact best with mobile phases of higher organic content and are, therefore, best suited to non-polar, hydrophobic solutes. 6

Modifications of peptides Peptides themselves can already inherit biological activity that can be observed in basic medical and biological research. With the completion of UG its becoming more and more interesting to characterise gene functions and the functions of their encoded proteins. Thus, researchers have growing need for synthetic peptides and depending on their experimental design, modified peptides as well. Among common standard modifications of peptides you can find: - D-amino acids - unnatural amino acids (6-Aminocaproic acid, Aminobutyric acid, itrulline, orleucine, etc.) - eavy amino acids (labelled with 13 and/or 15 ) - cyclisation - phosphorylation or sulfurylation (at Ser, Tyr, Thr) - biotinylation - conjugation to carrier proteins (BSA, KL, VA) - branching of peptides (MAPs multiple antigenic peptides) In general, there are some standard peptide moieties accessible to be modified: - -terminal amino group - amino group of Lys 2 - thiol group of ys - hydroxyl groups of Ser, Thr, Tyr - guanidine group of Arg - -terminal carboxyl group S Amino group modifications (-terminus or via Lys side chain) All modifications carrying amine-reactive functional groups can be used. Among the most commonly used ones, you can find: - activated esters - isothiocyanates - carboxylic acids Standard modifications that are coupled via amino groups are: - biotin - different dyes - bifunctional linkers - different acetylating groups Thiol group modifications (via ys side chain) All modifications carrying thiol-reactive functional groups can be used. Among the most commonly used ones, you can find: - iodoacetamides - maleimides - alkyl halides Standard modifications that are coupled via thiol groups are: - different dyes - KL or BSA 2 7

arboxyl group modification (-terminus) All modifications carrying carboxy-reactive functional groups can be used. Among the most commonly used ones, you can find: - amino groups - amines - bifunctional aminolinkers Standard modifications that are coupled via carboxy groups are: - amides - chromophores Single modifications Amino acids D-amino acids Amino acids carrying four different groups on their α- atom (i.e. asymmetric atom, or *) are chiral substances. These α-amino acids can be found in respective L- and D-forms (enantiomers): 2 2 L-amino acid D-amino acid The predominant form in natural proteins is the L-form. As some enzyme classes are enantioselective, i.e. they can distinguish between L- and D-forms and specifically accept only one of the two forms as substrate, this enantioselectivity makes D-amino acids a valuable tool in medicine (e.g. in peptide antibiotics) and enzyme assays. eavy Amino Acids In contrast to standard amino acids composed of 12 and 14 atoms, heavy amino acids can be substituted with 13 and/or 15 atoms. These heavy amino acids are non-radioactive, but 1 Da heavier than the standard amino acids. This molecular weight difference makes them useful tools for quantitative analysis of peptides by Mass Spectrometry (MS) and ucleic Magnetic esonance Spectroscopy (M) e.g. for determination of protein structure and dynamics. Among Thermo s standard heavy amino acids you can find: amino acid (aa) heavy isotope difference to standard aa L-Ala U- 13 3, 98%; 15, 95% + 4 Da L-Leu U- 13 6, 98%; 15, 95% + 7 Da L-Ile U- 13 6, 98%; 15, 95% + 7 Da L-Phe U- 13 9, 98%; 15, 95% + 10 Da L-Pro U- 13 5, 98%; 15, 95% + 6 Da L-Val U- 13 5, 98%; 15, 95% + 6 Da 8

Unnatural amino acids, special amino acids and protecting groups In contrast to the 20 natural amino acids (or proteinogenic), these amino acids are not encoded by the Universal Genetic ode usually they can be found in nature as metabolic products, especially in plants and bacteria. Some examples can be found below: 2 2 2 2 itrulline rnithine Thermo Electron s range of special amino acids comprises the following molecules. Please inquire if you do not find your desired amino acid listed. ε-acetyl-lysine 2 3 β-alanine (3-amino-propionic acid) is the only natural occurring β-amino acid, present e.g in panthothenic acid. 2 Aminobenzoic acid 2 6-Aminocaproic acid (Aca, 6-Aminohexanoic acid) This amino acid is often used as a linker to increase the distance between the peptide and an additional modification, e.g. a fluorescent dye. 2 9

Aminobutyric acid (Abu) γ-aminobutyric acid (or GABA) is an inhibitory transmitter of the central nerval system. It enhances permeability of postsynaptic membranes for chloride ions, thus leading to hyperpolarisation and consequently to an increase of the membrane s activation potential. 2 itrulline is a metabolic reagent in the urea metabolism pathway of many terrestric vertebrates. In this pathway, unwanted ammonia is being detoxified and eliminated. 3 2 2 ysteine, Acm (Acetamidomethyl) protected this specially protected ys is used to selectively form disulfide bridges. 2 S 3 Dimethyl-Lysine 2 3 3 ydroxy-proline (yp) is present almost exclusively in structural proteins (e.g. collagens or connective tissues in plant cell walls or mammals). It is formed during a posttranslational modification of proline in cells. 10

Mercaptopropionic acid S Methyl-Lysine 2 3 3-itro-Tyrosine 2 2 orleucine (le = 2-amino hexanoic acid) 2 pyro-glutamic acid (Pyr) is a common -terminal amino acid modification in many biologically active peptides (hormones). 3 Z (arbobenzoxyl) special protecting group for -terminus. 11

Single Modifications - standard modifications Biotin Biotin (or vitamin ) is a small biologically active molecule with a molecular weight of 244,31 Da. It acts as a co-enzyme in living cells. With its highly specific affinity towards streptavidin, it is used in various biotechnology assays for quality and quantity testing. S Farnesyl is a potential substrate to study demethylase activity in enzyme assays. 3 3 3 3 Formic acid (Formyl) Myristic acid (Myristoyl) Palmitic acid (Palmitoyl) Phosphorylation of Ser, Thr and Tyr is one of the more common modifications of amino acids in nature. Many hormones can adapt the activity of specific enzymes by increasing their phosphorylation state of Ser or Thr residues. Growth factors (like insulin) can trigger phosphorylation of Tyr. The phosphate groups on these amino acids can be quickly removed, thus Ser, Thr and Tyr function as molecular switches during regulation of cellular processes (e.g. cancer proliferation). 2 2 2 - P 3-3 P 3 P 3 - Stearic acid (Stearyl) 12

Succinic acid (Succinyl) Sulfurylation at Ser, Thr and Tyr is another modifcation of amino acids in nature. Activity of many enzymes depends on the oxidation state of S-groups in these residues. 2 2 2 S 3 - - 3 S 3 S 3-13

Single Modifications - dyes and quenchers AM (7-Amino-4-methyl-coumarin) UV-excitable dye, used in enzyme assays using cuvettes or flow cytometry. 2 3 oefficient AM 353 nm 422 nm 19.000 Dabcyl Dabcyl is a non-fluorescent dye predominantly used as a quencher for other fluorophores (esp. Fluorescein type dyes, EDAS..). If Dabcyl is coupled to a peptide in close proximity to a fluorophore, it absorbs the emitted light of the fluorophore. Enlarging this distance (i.e. by enzymatic cleavage of the peptide) results in excitation of the fluorophore with an emission signal that can be detected. (Me) 2 oefficient Dabcyl 453 nm none 32.000 Dansyl Dansyl is also used as a fluorophore quencher. Unlike Dabcyl, it inherits own fluorescence and thus might not be as useful for highly sensitive assays. 3 3 S 3 oefficient Dansyl 335 nm 526 nm 4.600 14

2,4-Dinitrophenyl (DP) is a non-fluorescent dye that can be used as a fluorophore quencher (see Dabcyl for more details). 2 oefficient DP 348 nm none 18.000 2 DP-Lysine is a non-fluorescent dye that can be used as a fluorophore quencher (see Dabcyl for more details). 2 2 2 oefficient DP-Lysine 348 nm none 18.000 EDAS (5-((2-aminoethyl)amino)napthalene-1-sulfonic acid) a commonly used dye in FET (fluorescence resonance energy transfer) peptides in combination with Dabcyl as quencher. 2 S 3 oefficient EDAS 335 nm 493 nm 5.900 15

Fluorescein the commonly used fluorescent dye in confocal laser-scanning microscopy and flow cytometry applications. oefficient Fluorescein 495 nm 520 nm 83.000 BD (7-nitrobenz-2-oxa-1, 3-diazole) a fluorescent dye, used for amine modification. 2 oefficient BD 486 nm 543 nm 27.000 p-itro-aniline a chromogen used as colorimetric enzyme substrate in many standard enzyme assays in cuvettes. 2 2 Dye Excitation maximum Emission maximum p-itro-aniline 410 nm none 16

hodamine B represents one among a numerous range of rhodamine dyes, used in fluorescence assays. 2 5 2 5 5 2 2 5 oefficient hodamine B 550 nm 580 nm 90.000 Tamra the most commonly used rhodamine dye in fluorescence assays. 3 3 3 + 3 oefficient Tamra 544 nm 576 nm 90.000 17

In addition to the above mentioned dyes, we also offer ATT and DY dyes. Please refer to the table below: Dye Alternative for Excitation maximum [nm] Emission maximum [nm] Molar Extinction oefficient AMA 353 422 19.000 ATT-425 438 486 45.000 EDAS 335 493 5.900 DY-475XL 493 514 100.000 6-Fam Alexa 487 495 520 83.000 Fluorescein Alexa 488 495 520 83.000 ATT-495 Alexa488/Fluorescein 495 522 80.000 hodamine 110 Alexa488/Fluorescein 505 527 68.000 DY-495 Alexa488/Fluorescein 503 528 70.000 DY-505 505 530 80.000 ATT-520 520 542 110.000 BD 486 543 27.000 DY-500XL 505 555 110.000 DY-485XL 485 560 50.000 ATT-532 533 560 115.000 DY-554 549 568 100.000 DY-555 547 572 100.000 DY-556 548 573 100.000 DY-547 y3 / Alexa 555 557 574 150.000 Tamra 544 576 90.000 ATT-550 554 576 120.000 hodamine B 550 580 90.000 DY-560 560 580 120.000 DY-510XL 509 590 50.000 ATT-565 561 590 120.000 DY-590 y3.5 / Texas ed 580 599 120.000 ox 576 601 82.000 Texas ed 583 603 116.000 DY-610 609 629 80.000 DY-480XL 500 630 50.000 ATT-610 605 630 150.000 ATT-590 598 634 120.000 DY-600XL 603 634 160.000 ATT-620 620 641 120.000 DY-615 621 641 200.000 DY-630 636 657 200.000 DY-632 637 657 200.000 DY-633 637 657 200.000 DY-631 637 658 200.000 ATT-635 635 659 120.000 DY-520XL 520 664 50.000 DY-636 645 671 200.000 DY-635 647 671 200.000 ATT-647 645 673 120.000 DY-647 y5 / Alexa 647 652 673 250.000 DY-650 653 674 220.000 DY-651 653 678 220.000 ATT-655 665 690 125.000 DY-673 673 696 200.000 DY-675 674 699 180.000 DY-676 674 699 180.000 ATT-680 680 702 125.000 DY-682 y5.5 / Alexa 680 691 708 140.000 DY-680 690 709 140.000 DY-690 691 714 180.000 DY-700 707 730 140.000 DY-701 706 731 140.000 DY-730 732 758 240.000 DY-732 736 759 240.000 DY-731 736 760 240.000 DY-752 Alexa 750 751 779 270.000 DY-781 783 800 170.000 DY-776 771 801 240.000 18

Single modifications modifications for antibody production KL, BSA, VA onjugates Peptide-protein conjugates are used for antibody production against peptides. Peptides alone are mostly too small to elicit a sufficient immune response, so carrier proteins containing many epitopes help to stimulate T-helper cells, which help induce the B-cell response. It is important to remember that the immune system reacts to the peptide-protein conjugate as a whole so there will always be a portion of antibodies to the peptide, the linker and the carrier protein. Among the most common carrier proteins one can find: KL (Keyhole Limpet emocyanin), a copper containing, non-heme protein found in arthropods and mollusca. It is isolated from Megathura crenulata and has a MW of 4.5 x 10 5 ~ 1.3 x 10 7 Da. KL is the most commonly selected carrier due to its higher immunogenicity compared to BSA. BSA (Bovine Serum Albumin), a plasma protein in cattle, belonging to the most stable and soluble albumins. It has a MW of 67 x 10 3 Da containing 59 lysines. About 30-35 of these primary amines are accessible for linker conjugation, which makes BSA a popular carrier protein for weak antigenic compounds. A disadvantage of BSA is, that it is used in many experiments as a blocking buffer reagent. If antisera against peptide-bsa conjugates are used in such assays, false positives can occur, because these sera also contain antibodies to BSA. VA (valbumin), a protein isolated from hen egg whites, with a MW of 45 x 10 3 Da. It is a good choice as second carrier protein to verify if antibodies are specific for the peptide alone and not the carrier protein (e.g. BSA) MAPs (Multiple Antigen Peptides) are branched peptides that can be used for direct immunisation to produce antibodies. MAPs are usually big enough to raise the immune response. The antigenic peptide of interest is being synthesised directly on the branched MAP structure. MAPs are available as MAP 4 (4 branches) or MAP 8 (8 branches) molecules: Schematic graph of a MAP 4: antigenic peptide antigenic peptide antigenic peptide antigenic peptide 19