Building Blocks for Introducing Post-translational Modified Amino Acids

The Novabiochem^® product line has always been at the forefront of the development for methods for the synthesis of peptides containing PTMs, being the first company to provide effective building blocks for synthesis of phosphorylated peptides, and sulfotyrosine and methylated arginine containing peptides by Fmoc chemistry. Here, we provide an overview of our available reagents, together with recommendations and details of their use.

This topic has been recently reviewed by Dr. John Offer and Novabiochem^® scientists.¹

Amino acid	PTM	side chain	Recommended building block	Product No.
Arg	Me		Fmoc-Arg(Me,Pbf)-OH	852105
Arg	s-diMe		Fmoc-SDMA(Boc)2-ONa	852310
Arg	a-diMe		Fmoc-ADMA(Pbf)-OH	852107
Asn	AcNH-β-Glc		Fmoc-Asn(Ac3AcNH-β-Glc)-OH	852135
Lys	Me		Fmoc-Lys(Me,Boc)-OH · HCl	852106
Lys	diMe		Fmoc-Lys(Me)2-OH	852111
Lys	triMe		Fmoc-Lys(Me)3Cl-OH	852112
Ser	PO3H2		Fmoc-Ser(PO(OBzl)OH)-OH Fmoc-D-Ser(PO(OBzl)OH)-OH	852069 852244
Ser	AcNH-β-Glc		Fmoc-Ser(Ac3AcNH-β-Glc)-OH	852349
Ser	AcNH-α-Gal		Fmoc-Ser(Ac3AcNH-α-Gal)-OH	852136
Thr	PO3H2		Fmoc-Thr(PO(OBzl)OH)-OH Fmoc-D-Thr(PO(OBzl)OH)-OH	852070 852245
Thr	AcNH-β-Glc		Fmoc-Thr(Ac3AcNH-β-Glc)-OH	852350
Thr	AcNH-α-Gal		Fmoc-Thr(Ac3AcNH-α-Gal)-OH	852229
Tyr	PO3H2		Fmoc-Tyr(PO(OBzl)OH)-OH Fmoc-D-Tyr(PO(OBzl)OH)-OH	852071 852246
Tyr	SO3H		Fmoc-Tyr(SO3nP)-OH	852347

Phosphorylation

Tyr

1234

For step-wise synthesis of phosphotyrosine containing peptides, the three most commonly used derivatives are Fmoc-Tyr(PO(OBzl)OH)-OH (1),² Fmoc-Tyr(PO₃H₂)-OH (2),³ and Fmoc-Tyr(PO(NMe₂)₂)-OH (3).⁴ Each has its merits and limations. Fmoc-Tyr(PO(OBzl)OH)-OH is the most popular, despite the partially protected phosphate causing issues during coupling and chain extension. It is best incorporated using uronium-based coupling reagents as HBTU or HATU ⁵ (Method 1). Increasing the excess of DIPEA used with the uronium reagent from the typical two to three-fold excess has been found to be beneficial.⁶ The partially protected phosphate can form salts with piperidine during Fmoc removal.⁷ Acylation of this piperidine results in a reduction in the excess of activated amino acid present in the coupling reaction. Whilst this does not pose a significant problem when the peptide contains only one phosphoamino acid, it can lead to serious difficulties with incomplete coupling reactions with peptides containing numerous phosphorylated residues. Fortunately, this problem can be overcome by simply increasing the excess of coupling reagents used or by exchanging the piperidine counterion for a tertiary amine (Method 2). The benzyl side-chain protecting groups are normally removed in 1-2 hours during the course of the standard 95% TFA. Benzylation of sensitive residues such as Tyr, Cys during the cleavage reaction has occasionally been observed. In such cases, the use of EDT in the cleavage mixture should minimize this side reaction.

Method 1: Coupling protocol for phosphoamino acid building blocks

1. Dissolve Fmoc-Aaa(PO(OBzl)OH)-OH (5 eq.^a), TBTU (5 eq.^a) and HOBt (5 eq.^a) in the minimum volume of DMF.
2. Add DIPEA (15 eq.^a) to mixture, mix and add immediately to Fmoc-deblocked peptide resin.
3. Allow to couple for 1-2h.
4. Check completeness of reaction with the Kaiser or TNBS test.  Wash resin and repeat reaction if necessary.

^aRelative to resin substitution.

Method 2: Phosphate counterion exchange

1. Wash resin with DMF (2 x).
2. Wash resin twice with DMF containing DIPEA (20 eq.)^a and TFA (18 eq.^a).
3. Wash resin with DMF (2 x).

^aRelative to phosphate content of resin.

Fmoc-Tyr(PO₃H₂)-OH is the simplest and most cost effective derivative for the introduction of phosphotyrosine by Fmoc synthesis.³ As the phosphate group is not protected, there are no issues with protecting group removal. However, like Fmoc-Tyr(PO(OBzl)OH)-OH, the lack of phosphate protection does lead to problems during its introduction and that of subsequent residues. Coupling is generally very sluggish and is best achieved using HATU in conjunction with at least three equivalents of DIPEA. Furthermore, pyrophosphate formation has been observed in peptides containing adjacent Tyr(PO₃H₂) residues.^{8, 9}

In contrast to Fmoc-Tyr(PO₃H₂)-OH and Fmoc- Tyr(PO(OBzl)OH)-OH, which have potentially reactive acid functionalities in their side-chains, the phosphate group in Fmoc-Tyr(PO(NMe₂)₂)-OH ⁴ is fully protected. This confers not only improved solubility properties on the compound but, more importantly, avoids the side reactions associated with the use of the fomer. Fmoc-Tyr(PO(NMe₂)₂)-OH can be introduced using any of the standard coupling methods, such as PyBOP^®/DIPEA, TBTU/DIPEA and DIPCDI/HOBt. Regeneration of phosphotyrosine from the phosphodiamidate is effected by acid catalyzed hydrolysis.⁷ Detachment of the peptide from the resin and deprotection of side-chains is first carried out in the usual manner with TFA containing the appropriate scavengers. 10 % by volume of water is then added to the cleavage solution and the mixture left to stand overnight, during which time hydrolysis of the phosphodiamidate takes place (Method 3).

Method 3: Deprotection of Tyr(PO(NMe₂)₂)

1. Treat peptidyl resin with TFA/TIS/water (95:2.5:2.5) for 3 h. After this time check removal of standard protecting groups by removing a small small of solution and analyzing by HPLC.
2. Add 10% v/v of water to the mixture and leave to react overnight. Work-up cleavage reaction in normal manner.

Fmoc-Phe(CF₂PO₃H₂)-OH (4) (Fmoc-F₂Pmp-OH) is used to incorporate the stable phosphotyrosine isostere, F₂Pmp.¹⁰ F₂Pmp is an excellent mimic of pTyr as it has a pKa2 of 5.71 so is fully ionized at neutral pH, and the methylene fluorine atoms can undergo H-bonding. Peptides substituted with F₂Pmp exhibit high binding affinities to SH2 domains.¹¹ 1000-Fold enhancements in affinities of F₂Pmp-containing peptide compared with those containing the non-fluorinated Pmp have been observed in assays against PTPs.¹² The use of the Fmoc-F₂Pmp-OH, which lacks side-chain protection, for the introduction of F₂Pmp residues eliminates the need for the harsh conditions required to remove the ethyl group usually employed for phosphonate protection.¹³ However, lack of phosphate protection does result in sluggish coupling of Fmoc-F₂Pmp-OH and the subsequent amino acid derivative. Overnight coupling with HATU and three equivalents of DIPEA appears to be effective.

Ser/Thr

56

For the step-wise synthesis of phosphoserine and threonine containing peptides, the only option is to use the monobenzyl derivatives, Fmoc-Ser(PO(OBzl)OH)-OH (5) ^{14, 2, 15} and Fmoc-Thr(PO(OBzl)-OH ^{2, 15} (6), since fully protected phosphate triesters of these amino acids undergo β-elimination when treated with piperidine. As with Fmoc-Tyr(PO(OBzl)OH)-OH, the side-chain phosphate group is only partially protected in these derivatives, so special consideration must be given to the conditions employed for their introduction. Best results are obtained using uronium-based coupling reagents in the presence of a three-fold excess of DIPEA.

N-terminal Ser(PO(OBzl)OH) residues can undergo β-elimination during piperidine-mediated Fmoc removal under microwave conditons. This problem could be eliminated by replacing piperidine with cyclohexylamine just for this step.¹⁶

Sulfation

Tyr

7

It is believed that up to 1% of all protein tyrosine residues in eukaryotes may be sulfated.¹⁷ Sulfation is thought to be involved in the modulation of the extracellular protein-protein interactions of secreted and transmembrane proteins. It is also an essential requirement for maintaining the biological activity of a number of peptide hormones such as gastrin II, cholecystokinin, and caerulein.

One of the principal hurdles to studying tyrosine sulfation is the difficulty in obtaining site-specifically sulfated peptides because tyrosine sulfate esters are rapidly degraded in acid and fragment during mass spectrometry, making their synthesis and characterisation highly problematic. With the introduction by Novabiochem^® of Fmoc-Tyr(SO₃nP)-OH (7),¹⁸ the synthesis of sulfotyrosine-containing peptides is now routine. Fmoc-Tyr(SO₃nP)-OH, can be coupled using any standard coupling method. Furthermore, the derivative has excellent solubility in DMF or NMP, faciliting its use in automated synthesizers without modification of existing protocols. The nP group is stable to piperidine and TFA, so the nP group requires removal post-cleavage with either sodium azide or ammonium acetate.¹⁹

Extended cleavage times should be avoided as some loss of the nP group can occur during prolonged contact with TFA. Ideally, the progress of the cleavage reaction should be followed by sampling of the reaction and analysis by HPLC. Cleavage times of 1.5 hours generally result in minimal loss of the nP group. Peptides containing Tyr(SO₃nP) are more strongly retained on HPLC columns than the corresponding Tyr and Tyr(SO₃-) peptides. They can also be detected by positive mode ES-MS and MALDI-MS.

nP sulfate esters are stable to piperidine but are readily cleaved using small powerful nucleophiles such as azide or cyanide. For peptides the reaction is most conveniently conducted in DMSO or DMF at 50 °C. Overnight treatment is usually sufficient to effect complete reaction. Following reaction, removal of excess azide can be effected by SPE or HPLC, using non-acidic buffers. For some peptides, the nP group can be removed by simply dissolving the peptide in 2 M ammonium acetate and warming the mixture at 37 °C overnight.

Glycosylation

Ser/Thr

For the Fmoc SPPS of O-glycosylated peptides, Novabiochem^® offers Fmoc-Ser(Ac₃AcNH-α-Gal)-OH, Fmoc-Thr(Ac₃AcNH-α-Gal)-OH, Fmoc-Ser(Ac₃AcNH-β-Glc)-OH and Fmoc-Thr(Ac₃AcNH-β-Glc)-OH. O-linked glycosides based on 2-acetamido-2-deoxy-α-D-galactopyranosyl (AcNH-α-Gal) are found in wide variety of biologically important glycoproteins such as mucin, the tumor-associated Tn-antigen, and gp120 from HIV²⁰. Modification of serine or threonine residues with 2-acetamido-2-deoxy-β-D-glucopyranosyl (AcNH-β-Glc) on the other hand is more akin to phosphorylation.²¹ The addition and removal of O-AcNH-β-Glc is a dynamic process controled by a transferase, UDPGlcNAc polypeptide transferase (OGT), and removed by a β-N-acetylglucos-aminidase. Only a single sugar is added the carbohydrate is not further extended. O-AcNH-β-Glc glycosylation processes are thought to be involved in transcription, signal transduction, apoptosis, and glucose homeostasis.

Asn

N-linked glycosides all contain a mannotriosido-di-N-acetyl-chitobiose core linked via a b(1→Nβ) linkage to Asn. Novabiochem offers the glycopeptide precursor, Fmoc-Asn(Ac₃AcNH-β-Glc)-OH ²² in which a single N-acetylglucosamine moiety is attached to the side chain of Asn.

All N- and O-linked oligosaccharide amino acid derivatives above are compatible with standard Fmoc SPPS protocols. Owing to the high cost of these reagents, their introduction is best carried out manually using small reagent excesses and the coupling checked using the TNBS or Kaiser tests. Any standard coupling method can be used for this purpose. Following chain assembly, removal of the acetyl protecting groups can be carried out by treatment of the peptidyl resin with methanolic ammonia²³ or sodium methoxide/DMF/methanol,²⁴ or treatment of the cleaved peptide with catalytic sodium methoxide in methanol.²⁵

Methylation

Arg, Lys

Post-translational methylation of arginine and lysine residues is now recognized as an important control mechanism for the regulation of protein expression in eukaryotes.^{26, 27} Most of the possible methylated derivatives have been found to occur in vivo, including mono-, di- and trimethylated lysine, and mono and dimethylarginine, both asymmetric and symmetric. Such modifications are thought to be involved in cell proliferation, signal transduction, protein transport and transcription activation. Novabiochem^® offers a comprehensive range of side-chain N-methylated amino acids bearing standard Fmoc-compatible protecting groups. For the incorporation of the most common arginine modifications, monomethyl arginine and symmetric (SAMA) and asymmetric dimethylarginine (ADMA), Novabiochem offers Fmoc- Arg(Me,Pbf)-OH, Fmoc-SAMA(Boc)₂-ONa and Fmoc-ADMA(Pbf)-OH. These derivatives can be coupled using standard activation methods, and the Pbf and Boc groups are cleaved with standard TFA cleavage cocktails. Fmoc-Lys(Me,Boc)-OH, Fmoc-Lys(Me)₂-OH ·HCl and Fmoc-Lys(Me₃Cl)-OH are available for the introduction of monomethyl, dimethyl and trimethyl lysine, respectively. All are compatible with standard Fmoc SPPS protocols. However, the side chain of dimethyllysine can promote Fmoc cleavage so short single coupling should be used for its introduction and the following amino acid in the peptide chain.

References

1. Behrendt R, White P, Offer J. 2016. Advances in Fmoc solid?phase peptide synthesis. J. Pept. Sci.. 22(1):4-27. https://doi.org/10.1002/psc.2836

2. White "W&JB, Beythien J. 1996. Innovations & Perspectives in Solid Phase Synthesis & Combinatorial Libraries, 4th International Symposium”, R. Epton (Ed.),; Mayflower Scientific Ltd., Birmingham, p. 557.

3. Ottinger J. 1993. Synthesis of Phosphotyrosine-Containing Peptides and Their Use as Substrates for Protein Tyrosine Phosphatases. Biochemistry. 324354.

4. Chao H, Leiting B, Reiss PD, Burkhardt AL, Klimas CE, Bolen JB, Matsueda GR. 1995. Synthesis and Application of Fmoc-O-[Bis(dimethylamino)phosphono]tyrosine, a Versatile Protected Phosphotyrosine Equivalent. J. Org. Chem.. 60(24):7710-7711. https://doi.org/10.1021/jo00129a001

5. Perich JW, Ede? NJ, Eagle S, Bray AM. 1999. 6(2/3):91-97. https://doi.org/10.1023/a:1008823924135

6. White P. 2001. Poster 159 presented at the 17th American Peptide Symposium.

7. R P. 2001. Peptides 2000, Proc. 26th European Peptide Symposium, J. Martinez & J.-A. Fehrentz (Eds). Éditions EDK, Paris p. 263.

8. Garc?a-Echeverr?a C. 1995. Potential pyrophosphate formation upon use of N ?-Fmoc-Tyr(PO3H2)-OH in solid-phase peptide synthesis. Lett Pept Sci. 2(2):93-98. https://doi.org/10.1007/bf00128503

9. AE O. 1996. Intramolecular pyrophosphate formation during N alpha-9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis of peptides containing adjacent phosphotyrosine residue. Pept. Res. 9223.

10. Burke T. 2006. Design and Synthesis of Phosphonodifluoromethyl Phenylalanine (F2Pmp):A Useful Phosphotyrosyl Mimetic. CTMC. 6(14):1465-1471. https://doi.org/10.2174/156802606777951091

11. Burke R. 1994. Nonhydrolyzable Phosphotyrosyl Mimetics for the Preparation of Phosphatase-Resistant SH2 Domain Inhibitors. Biochemistry. 336490-6494.

12. Burke T, Kole H, Roller P. 1994. Potent Inhibition of Insulin Receptor Dephosphorylation by a Hexamer Peptide Containing the Phosphotyrosyl Mimetic F2Pmp. Biochemical and Biophysical Research Communications. 204(1):129-134. https://doi.org/10.1006/bbrc.1994.2435

13. Szardenings AK, Gordeev MF, Patel DV. 1996. A general and convenient synthesis of novel phosphotyrosine mimetics. Tetrahedron Letters. 37(21):3635-3638. https://doi.org/10.1016/0040-4039(96)00666-1

14. Wakamiya T, Saruta K, Yasuoka J, Kusumoto S. 1994. An Efficient Procedure for Solid-Phase Synthesis of Phosphopeptides by the Fmoc Strategy. Chem. Lett.. 23(6):1099-1102. https://doi.org/10.1246/cl.1994.1099

15. Vorherr T, Bannwarth W. 1995. Phospho-serine and phospho-threonine building blocks for the synthesis of phosphorylated peptides by the Fmoc solid phase strategy. Bioorganic & Medicinal Chemistry Letters. 5(22):2661-2664. https://doi.org/10.1016/0960-894x(95)00475-9

16. Attard TJ, O?Brien-Simpson NM, Reynolds EC. 2009. Identification and Suppression of ?-Elimination Byproducts Arising from the Use of Fmoc-Ser(PO3Bzl,H)-OH in Peptide Synthesis. Int J Pept Res Ther. 15(1):69-79. https://doi.org/10.1007/s10989-008-9165-9

17. Seibert C, Sakmar TP. 2008. Toward a framework for sulfoproteomics: Synthesis and characterization of sulfotyrosine-containing peptides. Biopolymers. 90(3):459-477. https://doi.org/10.1002/bip.20821

18. Simpson LS, Widlanski TS. 2006. A Comprehensive Approach to the Synthesis of Sulfate Esters. J. Am. Chem. Soc.. 128(5):1605-1610. https://doi.org/10.1021/ja056086j

19. Simpson LS, Zhu JZ, Widlanski TS, Stone MJ. 2009. Regulation of Chemokine Recognition by Site-Specific Tyrosine Sulfation of Receptor Peptides. Chemistry & Biology. 16(2):153-161. https://doi.org/10.1016/j.chembiol.2008.12.007

20. Gabius J, S G. 2008. Glycosciences: . Status & Perspectives. John Wiley & Sons.

21. Galan MC, Benito-Alifonso D, Watt GM. 2011. Carbohydrate chemistry in drug discovery. Org. Biomol. Chem.. 9(10):3598. https://doi.org/10.1039/c0ob01017k

22. L O. 1989. Coupling strategies in solid-phase synthesis of glycopeptides. Pept. Res.. 2362.

23. Chan W, White P. 2000. Fmoc solid phase peptide synthesis. a practical approach. Oxford University Press, Oxford: 195.

24. Liu M, Barany G, Live D. 2005. Parallel solid-phase synthesis of mucin-like glycopeptides. Carbohydrate Research. 340(13):2111-2122. https://doi.org/10.1016/j.carres.2005.05.023

25. Meldal M, Book K. 1990. Pentafluorophenyl esters for temporary carboxyl group protection in solid phase synthesis of N-linked glycopeptides.. Tetrahedron Letters. 31(48):6987-6990. https://doi.org/10.1016/s0040-4039(00)97223-x

26. McBride AE, Silver PA. 2001. State of the Arg. Cell. 106(1):5-8. https://doi.org/10.1016/s0092-8674(01)00423-8

27. Kouzarides T. 2002. Histone methylation in transcriptional control. Current Opinion in Genetics & Development. 12(2):198-209. https://doi.org/10.1016/s0959-437x(02)00287-3