Letters in Peptide Science, 7: 107–112, 2000.
`KLUWER/ESCOM
`© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
`
`107
`
`Base-induced side reactions in Fmoc-solid phase peptide synthesis:
`Minimization by use of piperazine as N!-deprotection reagent"
`
`John D. Wade" ", Marc N. Mathieu, Mary Macris & Geoffrey W. Tregear
`Howard Florey Institute, University of Melbourne, Parkville, VIC 3052, Australia
`
`Received 1 November 1999; Accepted 24 November 1999
`
`Key words: aspartimide formation, base-induced side reaction, Fmoc-solid phase peptide synthesis, N!-
`deprotection reagent, piperazine
`
`Summary
`Base-induced aspartimide (cyclic imide) and subsequent base adduct formation in the Fmoc-solid phase synthesis
`of sensitive sequences are serious side reactions that are difficult to both anticipate and control. The effect
`of extended treatment of piperazine as N!-Fmoc deprotection reagent on two sensitive peptide sequences was
`examined. For comparison, other bases were also investigated, including piperidine, 1-hydroxypiperidine, tet-
`rabutylammonium fluoride, and 1,8-diazabicyclo[5.4.0]undec-7-ene. The results showed that all bases induced
`varying degrees of both aspartimide and, in some cases, base adduct formation, although piperazine caused the
`least side reaction. Use of N-(2-hydroxy-4-methoxybenzyl) peptide backbone amide protection was confirmed to
`confer complete protection against side reaction. In the absence of such protection, for all bases, the use of 1-
`hydroxybenzotriazole as additive had some, but not complete, beneficial effect in further reducing side reaction.
`Best results were obtained with piperazine containing 0.1M 1-hydroxybenzotriazole indicating that this reagent
`merits serious consideration for N!-deprotection in the Fmoc-solid phase synthesis of base-sensitive sequences.
`A further advantage of this reagent is that it causes little racemisation of resin-bound C-terminal cysteine, an
`occasionally serious base-mediated problem in Fmoc-solid phase assembly.
`
`Introduction
`
`formation is a long-
`Aspartimide (cyclic imide)
`recognized side reaction that can occur both during
`solid phase peptide synthesis (SPPS) and storage of
`peptides, and may be either acid- or base-catalyzed
`[1]. Numerous studies on the mechanism of the re-
`action have shown it to be dependent on the nature
`of the acid or base, and the residue adjoining the
`carboxyl of the aspartate as well as the side chain pro-
`tecting group used [1]. Imide formation was originally
`thought not to occur in Fmoc-SPPS. However, several
`recent studies have shown it to be a significant side
`reaction and one that is highly sequence and conform-
`ation dependent [2–4]. The problem is not confined
`
`" A preliminary account of this work was presented at the 25th
`European Peptide Symposium, Budapest, Hungary, 1998.
`"" To whom correspondence should be addressed. E-mail:
`j.wade@hfi.unimelb.edu.au
`
`exclusively to Asp-X sequences, for there has also
`been a report of Asn-X cyclization [5]. An additional
`side reaction now known to be associated with sensit-
`ive Asp-X sequences is subsequent modification of the
`imide by nucleophilic base to produce a base adduct
`(Figure 1). Several palliative measures for controlling
`imide and adduct formation have been recommended.
`These include addition to the N!-Fmoc deprotection
`reagent of choice, piperidine, of agents such as 1-
`hydroxybenzotriazole (HOBt), but none completely
`suppress side reaction [2,6,7]. Aspartyl side chain pro-
`tecting groups other than the commonly employed
`tert-butyl ester have also been reported to give im-
`proved yields of !-aspartyl peptides through increased
`steric hindrance. These include 1-adamantyl and "-3-
`methylpent-3-yl esters [2,8]. However, these are either
`not entirely compatible with Fmoc-SPS or commer-
`cially unavailable. The sole effective preventive meas-
`ure to date is the use of Asp-X amide bond protec-
`
`MYLAN EXHIBIT - 1023
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`

`

`108
`
`Figure 1. Pathway of base-mediated aspartimide and adduct formation. For non-nucleophilic bases such as DBU and TBAF, base adduct
`formation does not occur.
`
`tion with the N-(2-hydroxy-4-methoxybenzyl) (Hmb)
`group [9–11]. Such protection is not always practical
`as not all amino acid derivatives are commercially
`available.
`More recently, it has been shown that certain other
`bases, including piperazine and 1-hydroxypiperidine,
`are much less detrimental than piperidine in causing
`side reactions [7], although a thorough evaluation of
`their effectiveness was not undertaken. In our labor-
`atory, we have recently carried out the solid phase
`synthesis of a number of peptides that possess poten-
`tial aspartimide formation sites. These include the B-
`chain of ovine Leydig cell insulin-like peptide, which
`contains an Asp-Gly sequence at its C-terminus [12],
`and a C-terminal segment of rat relaxin C-peptide [13].
`As part of an exercise to minimise or prevent imide
`formation during the assembly of these peptides, we
`undertook a closer examination of the effect of pro-
`longed treatment of two known aspartimide-sensitive
`
`peptides with piperazine as an alternative base for
`N!-Fmoc deprotection. This also allowed a compar-
`ison with the more commonly employed piperidine for
`extent of side reaction.
`
`Materials and methods
`
`Amino acid derivatives were purchased from Auspep
`(Melbourne, Australia) with the exception of bis-
`Fmoc(Hmb)-Gly-OPfp that was obtained from Nova-
`biochem (Läufelfingen, Switzerland). All other syn-
`thesis reagents and chemicals were of the highest
`grade available. Continuous flow solid phase synthesis
`of the two test peptides, I and II, was carried out as
`previously described [12]. For one peptide (I), syn-
`thesis was also repeated using bis-Fmoc(Hmb)-Gly-
`OPfp. After each assembly, aliquots of the peptide-
`resins were treated separately for 24 h with base
`
`

`

`109
`
`Figure 2. RP-HPLC of the cleavage products following 24 h base treatment of I (left column) and II (right column) with 20% piperidine/DMF
`(a and b), 6% piperazine/DMF (c and d), and 6% piperazine/0.1M HOBt/DMF (e and f). Peptide peak identification was by MALDI-TOF MS.
`For example, left column, panel c, found MH# values: Peaks " and !, 762.0; I, 693.9; Ic, 675.9. Expected MH# values: target peptide I, 693.8;
`cyclic imide, 675.8; piperazide, 762.0. Note: The symbols !, ", D-! and D-" refer to corresponding base adducts including the D-isomers. Ic
`and IIc refer to the cyclic imide of I and II. The peak denoted " could not be conclusively identified. For all chromatography, the detection
`wavelength was 214 nm.
`
`solution in DMF. Bases examined were piperidine
`(20% v/v solution in DMF), piperazine (6% w/v),
`1-hydroxypiperidine (20% v/v), tetrabutylammonium
`fluoride (0.02M) and 1,8-diazabicyclo[5.4.0]undec-7-
`ene (2% v/v) with concentrations being those re-
`commended for routine solid phase peptide synthesis.
`
`After washing and drying, these were then cleaved
`and deprotected by treatment with 95% TFA in the
`presence of appropriate scavengers. Each peptide was
`then subjected to analytical RP-HPLC on a Vydac C18
`column (Hesperia, U.S.A.) using a linear gradient of
`acetonitrile. Buffer A was 0.1% aqueous TFA and B
`
`

`

`110
`
`was 0.1% TFA in acetonitrile. For peptide I, the gradi-
`ent used was 10–40% B over 30 min; for peptide II,
`the gradient was 5–60% B over 30 min. Peaks were
`collected and mixed with !-cyano-4-hydroxycinnamic
`acid as matrix and analyzed by matrix-assisted laser
`desorption ionization time of flight mass spectrometry
`(MALDI-TOF MS) using a Bruker (Bremen, Ger-
`many) Biflex instrument in the linear mode at 19.5
`kV.
`
`To determine the effect of base on racemization
`of C-terminal cysteine, aliquots of Fmoc-Cys (Trt)-
`hydroxymethylphenoxy acetyl–Pepsyn K were treated
`as previously described [14]. However, resolution and
`quantitation of D- and L-cysteic acid following de-
`rivatization with Marfey’s reagent was achieved by
`RP-HPLC on a Phenomenex Luna C18 column (5 #,
`250 ! 2 mm, Torrance, U.S.A.) also as previously
`described [15].
`
`Results and discussion
`
`Several side reactions have been reported to occur dur-
`ing Fmoc-solid phase peptide synthesis. These include
`aspartimide formation [1], C-terminal racemization
`[16], C-terminal cysteine conversion to a dehydration
`product [17], and diketopiperazine formation [18].
`All of these are base-mediated and raise questions
`as to the general suitability of the long time base of
`choice, piperidine, which, as an unhindered second-
`ary amine, elicits the fastest rate of N!-deprotection
`[19]. Although the Fmoc group is labile to other or-
`ganic bases including primary and secondary amines
`[20, 21], apart from isolated reports, comparatively
`little work has been carried out on the effectiveness of
`alternative bases to piperidine in Fmoc-SPS. In addi-
`tion to these bases, fluoride ion has also been shown
`to effect smooth removal of the Fmoc group [22].
`Additionally, previous work in this laboratory demon-
`strated the usefulness of the tertiary amidine, DBU, as
`N!-deprotection reagent [14]. It is particularly benefi-
`cial in cases of slow, hindered Fmoc group removal
`in so-called ‘difficult’ syntheses [23, 24]. However,
`more experience has since shown this base to also
`be capable of causing significant aspartimide form-
`ation as well as converting resin-bound C-terminal
`cysteine to dehydroalanine [17]. More recently it was
`shown that piperazine and 1-hydroxypiperidine were
`much less detrimental in causing imide formation dur-
`ing the solid phase synthesis of aspartimide-sensitive
`sequences [7]. We sought to confirm this and to com-
`
`Table 1. Extent of target peptide remaining after 24 h
`treatment with base
`
`Conditions
`
`% Target producta
`Peptide I
`Peptide II
`
`15.8
`Piperidine
`n.db
`Piperidine + HOBt
`99.5
`Piperidine + Hmb
`58.3
`Piperazine
`74.9
`Piperazine + HOBt
`99.1
`Piperazine + Hmb
`58.8
`1-Hydroxypiperidine
`99.2
`1-Hydroxypiperidine + Hmb
`n.dc
`TBAF
`99.2
`TBAF + Hmb
`n.dc
`DBU
`89.7
`DBU + Hmb
`a Determined by HPLC peak area integration.
`bNot experimentally determined.
`c Not determined. Substantial (greater than 80%) "-peptide
`could not be adequately resolved from !-peptide.
`
`30.8
`63.4
`n.db
`70.5
`89.7
`n.db
`70.3
`n.db
`20
`n.db
`11
`n.db
`
`pare their effectiveness with other more commonly
`employed bases including piperidine.
`The test peptides used were the hexapeptide frag-
`ment derived from the scorpion toxin II, VKDGYI
`[25], I, and the sequence LTEDNK [7], II. Both have
`been reported to be exceptionally susceptible to im-
`ide formation, both during synthesis and on prolonged
`treatment with base. Thus these serve as useful models
`for assessing the effectiveness of alternative bases to
`reduce the level of this side reaction. In the first in-
`stance, I and II were treated with piperidine solution
`for 24 h at room temperature and the resulting cleaved
`products were analyzed by RP-HPLC. Identification
`of side products was by MALDI-TOF MS and an es-
`timate of side reaction was provided by RP-HPLC
`peak area quantitation. The results confirmed earlier
`work that these peptides are prone to considerable
`modification by this base (Figure 2a, Table 1). Repeat
`experiments with fresh aliquots of peptide-resins were
`then carried out with the four remaining bases and the
`extent of side reaction was determined.
`MALDI-TOF MS analysis of each peak showed
`that significant side product formation occurred fol-
`lowing extended treatment with each base in DMF.
`The results also showed the side reaction profile to
`be markedly different for each peptide. While both
`peptides I and II showed a high propensity for adduct
`formation with nucleophilic bases (compare Figure 2a
`with 2b), the level and diversity was much greater
`
`

`

`Table 2. Time course analysis of
`% D-enantiomer formation in solid
`phase
`bound
`S-trityl-protected
`cysteine
`exposed
`to
`6%
`piperazine/0.1M HOBt
`
`Time (h)
`
`% D-cysteic acid
`
`0
`1
`5
`24
`
`0.8
`1.2
`1.5
`3.9
`
`for I than II. This confirmed the high degree of se-
`quence dependence for base-mediated side reaction.
`For peptide I, when piperazine was used as base at a
`concentration of 6% (w/v) in DMF (its limited solubil-
`ity prevents higher concentrations from being used), in
`addition to the cyclic imide, two additional products
`were identified as base adducts, the corresponding !-
`and "-piperazides (Figure 2c). The overall modifica-
`tion of 42% of the peptide corresponds to 0.3% per
`standard 10 min piperazine/DMF cycle (Table 2). Ad-
`dition of 0.1M HOBt to base [6] significantly reduced
`– but did not completely abolish – imide and adduct
`formation (Figure 2e). This is in contrast to the find-
`ings of Dölling et al. [7] who observed an apparent
`elimination of side reaction. For comparison, the ef-
`fect of three other bases, 1-hydroxypiperidine, TBAF
`and DBU, was also examined on the test peptides.
`The former base caused an extent of side reaction that
`was equivalent to that caused by piperazine. In con-
`trast, TBAF and DBU caused substantial production
`of "-peptide in addition to imide but, because of their
`low nucleophilicity, no adduct formation (Table 1).
`Addition of HOBt to all three bases had a benefi-
`cial effect, with that for 1-hydroxypiperidine being
`approximately equivalent to that for piperazine. A
`similar observation was made for all bases using pep-
`tide II as model (Figures 2d and 2f, Table 1). Side
`reaction formation in peptide I by each base was com-
`pletely suppressed by use of Hmb backbone amide
`protection (Table 1).
`The results show that none of the bases used in
`this study for N!-Fmoc deprotection can completely
`prevent aspartimide formation in sensitive sequences,
`although piperazine containing 0.1M HOBt caused the
`lowest degree of side reaction. It must also be said
`that the two test peptides used in this study are ex-
`ceptionally labile to modification and that the great
`majority of solid phase syntheses of Asp-X-containing
`
`111
`
`peptides are unlikely to encounter side reaction of
`this severity. However, its weaker basicity [19] sug-
`gests that
`the effectiveness of piperazine in other
`aspects of SPPS such as complete N!-deprotection
`during ‘difficult’ syntheses needs to be carefully as-
`sessed. It was, however, evaluated for any beneficial
`effect against one serious piperidine-mediated side re-
`action, that of racemization of resin-bound C-terminal
`hydroxymethyl-linked cysteine, which occurs to an
`extent of nearly 30% after 24 h of treatment [16]. Re-
`markably, piperazine containing 0.1M HOBt caused
`less than 5% racemization after 24 h (Table 2), sug-
`gesting this to be a useful alternative to the use of
`chlorotrityl-based resins [26] or the synthesis of pep-
`tide amides for the Fmoc solid phase assembly of
`C-terminal cysteine-containing peptides.
`
`Conclusions
`
`In the absence of prior information regarding the sus-
`ceptibility of a new peptide sequence to modification
`during Fmoc-solid phase synthesis, it is recommen-
`ded that – where feasible – Asp-X pairs be routinely
`protected with the Hmb moiety. Should this not be
`possible, then piperazine containing 0.1M HOBt is a
`practical and effective alternative.
`
`Acknowledgements
`
`The work was supported by an Institute Block Grant
`Reg Key Number 983001 from the NHMRC of Aus-
`tralia. We thank Jelle Lahnstein of Nucleic Acid and
`Protein Chemistry Unit, ARC SRC for Basic and Ap-
`plied Plant Molecular Biology, Department of Plant
`Science, Waite Campus, The University of Adelaide,
`for the cysteine racemization analyses.
`
`References
`
`1. Bodanszky, M., Principles of Peptide Synthesis, Springer-
`Verlag, Berlin, 1984.
`2. Lauer, J.L., Fields, C.G. and Fields, G.B., Lett. Pept. Sci., 1
`(1994) 197.
`3. Dölling, M., Beyermann, M., Haenel, J., Kernchen, F., Krause,
`E., Franke, P., Brudel, M. and Bienert, M., In Epton, R.
`(Ed.) Innovation and Perspectives in Solid Phase Synthesis,
`Proceedings of the 3rd Solid Phase Synthesis Symposium,
`Mayflower Worldwide, Birmingham, 1994, pp. 489–492.
`4. Dölling, M., Beyermann, M., Haenel, J., Kernchen, F., Krause,
`E., Franke, P., Brudel, M. and Bienert, M., J. Chem. Soc.,
`Chem. Commun., (1994) 853.
`
`

`

`112
`
`5. Ede, N.J., Chen, W., McCluskey, J., Jackson, D.C. and Purcell,
`A.W., Biomed. Pept. Protein Nucl. Acids, 1 (1995) 231.
`6. Martinez, J. and Bodanszky, M., Int. J. Pept. Protein Res., 12
`(1978) 277.
`7. Dölling, R., Beyermann, M., Haenel, J., Kernchen, F.,
`Krause, E., Franke, P., Brudel, M. and Bienert, M., In Maia,
`H.L.S. (Ed.) Peptides 1994, Proceedings of the 23rd European
`Peptide Symposium, ESCOM, Leiden, 1995, pp. 244–245.
`8. Karlstrom, A. and Unden, A., Tetrahedron Letts., 37 (1996)
`4243.
`9. Quibell, M., Owen, D., Packman, L.C. and Johnson, T., J.
`Chem. Soc., Chem. Commun., (1994) 2343.
`10. Urge, L. and Otvos Jr., L., Lett. Pept. Sci., 1 (1994) 207.
`11. Offer, J., Quibell, M. and Johnson, T., J. Chem. Soc., Perkin
`Trans. 1, (1996) 175.
`12. Dawson, N.F., Macris, M., Tan, Y.-Y., Summers, R.J., Tregear,
`G.W. and Wade, J.D., J. Pept. Res., 53 (1999) 542.
`13. Wade, J.D., Dawson, N.F., Macris, M., Mathieu, M., Lin,
`F., Tan, Y.-Y., Summers, R.J. and Tregear, G.W., In Hu, X.,
`Wang, R. and Tam, J.P. (Eds.), Peptides – Biology and Chem-
`istry, Proceedings of the 1998 Chinese Peptide Symposium,
`Kluwer, Dordrecht, 2000, pp. 81–84.
`14. Wade, J.D., Bedford, J., Sheppard, R.C. and Tregear, G.W.,
`Pept. Res., 4 (1991) 194.
`15. Kochhar, S. and Christen, P., Anal. Biochem., 178 (1989) 17.
`
`16. Atherton, E., Hardy, P.D., Harris, D.E. and Matthews, B.H., In
`Giralt, E. and Andreu, D. (Eds.), Peptides 1990, Proceedings
`of the 21st European Peptide Symposium, ESCOM, Leiden,
`1991, pp. 243–244.
`17. Lukszo, J., Patterson, D., Albericio, F. and Kates, S.A., Lett.
`Pept. Sci., 3 (1996) 157.
`18. Pedroso, E., Grandas, A., de las Heras, X., Eritja, R. and
`Giralt, E., Tetrahedron Lett., 27 (1986) 743.
`19. Atherton, E. and Sheppard, R.C., In Udenfriend, S. and Mei-
`enhofer, J. (Eds.) The Peptides. Analysis, Synthesis, Biology,
`Vol. 9, Academic Press, New York, NY, 1987, pp. 1–38.
`20. Carpino, L.A., Acc. Chem. Res., 20 (1987) 401.
`21. Fields, G.B. and Noble, R.L., Int. J. Pept. Protein Res., 35
`(1990) 161.
`22. Ueki, M. and Amemiya, M., Tetrahedron Lett., 28 (1987)
`6617.
`23. Kates, S.A., Solé, N.A., Beyermann, M., Barany, G. and
`Albericio, F., Pept. Res., 9 (1996) 106.
`24. Pegoraro, S., Vigano, S., Rovero, P., Revoltella, R., Bicci-
`ato, S., Bagno, A., Dettin, M. and Di Bello, C., In Maia,
`H.L.S. (Ed.) Peptides 1994, Proceedings of the 23rd European
`Peptide Symposium, ESCOM, Leiden, 1995, pp. 254–255.
`25. Nicolás, E., Pedroso, E. and Giralt, E., Tetrahedron Lett., 30
`(1989) 497.
`26. Fujiwara, Y., Akaji, K. and Kiso, Y., Chem. Pharm. Bull., 42
`(1994) 724.
`
`