Facilitated Synthesis of Heterofunctional Glycopolypeptides

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Facilitated Synthesis of Heterofunctional Glycopolypeptides Kai-Steffen Krannig, Afroditi Doriti, and Helmut Schlaad* Max Planck Institute of Colloids and Interfaces, Department of Colloid Chemistry, Research Campus Golm, 14424 Potsdam, Germany S Supporting Information *

S

functionalization step, to yield heterofunctional glycopolypeptides. Such polypeptide samples would not be accessible by (co)functionalization of polyallylglycine for reason for poor solubility in organic solvents or water.18,20,29 The NCAs of DL- and L-AGly (1(DL) and 1(L), respectively) were synthesized from the commercial amino acids and triphosgene (Fuchs-Farthing method) in tetrahydrofuran (THF) solution, using α-pinene as HCl scavenger.19 The products were isolated in yields of 50−60% after multiple precipitations from heptane and characterized by 1H NMR spectroscopy and melting point analysis (see Supporting Information). Glycosylation of AGly NCA (→ glyco-NCA 3(DL) and 3(L)) is expected to occur by radical addition of 1-thio-β-Dglucopyranose-2,3,4,6-tetraacetate (AcGlcSH, 2) (or any other, more complex 1-thio-sugar) to the exocyclic vinyl group, as outlined in Scheme 1. However, the thiol may also act as nucleophile and add to the heterocylic ring (at C5), ultimately initiating polymerization of the NCA.30 This pathway could be eliminated if the radical addition proceeds much faster than the nucleophilic ring-opening and is quantitative. Three test reactions (A-C) were conducted using AGly NCA 1 and various under-stoichiometric amounts of thiol 2, i.e. [SH]0 < [CC]0 (Table 1). Benzophenone (0.2

ynthetic polypeptides are promising materials with high potential in key biomedical and biotechnological applications such as tissue engineering, drug delivery, or as polymer therapeutics.1−4 Recently, glycosylated polypeptides attracted enormous attention due to their close relationship to natural glycoproteins fulfilling numerous tasks in the human body, ranging from cellular recognition, adhesion, and lubrication to cancer cell metastasis and infection of pathogens.5 Their synthesis, structural characteristics, self-assembly behavior, and ability to selectively interact with biological material have lately been highlighted in some excellent reviews, manifesting the increasing importance of this class of materials.2,6−8 But although significant progress has been made,5 the preparation of well-defined glycopolypeptides is still a challenging task for synthetic polymer chemists. Recent efforts include the polymerization of glycosylated amino acid N-carboxyanhydrides (glyco-NCAs)9−16 as well as the postpolymerization functionalization of ready-made polypeptides carrying appropriate functional groups in the side chains.17−28 However, most of these approaches require multiple steps and tedious purification protocols and are usually labor- and timeconsuming processes. Here, we report a facilitated synthesis of glycopolypeptides by in situ glycosylation and polymerization of 2-aminopent-4enoic acid (allylglycine, AGly) NCA, combining radical thiol− ene photochemistry and nucleophilic ring-opening polymerization (ROP) (Scheme 1). The prepared glycopolypeptides contain a predetermined amount of sugar and remaining vinyl groups, which are susceptible to additional functional groups, e.g. sugar, carboxylic acid, amine, thiol, etc., through a second

Table 1. Preparation of Glyco-NCA 3 by Partial Thiol−Ene Glycosylation of AGly NCA 1 with AcGlcSH 2 (According to Scheme 1) reacn A B C

Scheme 1. One-Pot Partial Glycosylation and Copolymerization of AGly NCAa

NCA 1 L-AGly DL-AGly L-AGly

feed [1]0/[2]0

product [1]/[3]a

1.0/0.5 1.0/0.6 1.0/0.9

0.5/0.5 0.4/0.6 0.1/0.9

a1

H NMR analysis (400.1 MHz, THF-d8), see Figure 1 and Supporting Information.

equiv) was used as a photoinitiator in a rather large amount to ensure a sufficiently high radical concentration and fast addition rate. Photoadditions were carried out in 0.15 M deuterated THF solutions under irradiation with two 26 W energy-saving bulbs (Exo Terra ReptiGlo 5.0, λ = 290−690 nm, filtered through glass wall of reaction vessel) for ∼45 min. The reactions A-C were monitored by in situ 1H NMR and FT-IR spectroscopy (Supporting Information). The 1H NMR spectrum of the isolated (carefully precipitated) product mixture 1(L)/3(L) from reaction C is exemplarily shown in

a

Reagents and conditions: (a) thiol−ene photoaddition, benzophenone, hν, THF, room temperature, 45 min (y < x, Ac = acetyl); (b) nucleophilic ROP, 1-hexylamine, THF/DMF, room temperature, 7 d. © 2014 American Chemical Society

Received: February 20, 2014 Published: March 19, 2014 2536

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Note

Figure 1. The success of the radical thiol−ene addition is confirmed by the decrease of the alkene signal at δ 5.8 ppm.

Figure 2. (a) 1H NMR spectrum (400.1 MHz, TFA-d) of glycopolypeptide P1. (b) SEC-RI traces (eluent, NMP + 0.5 wt % LiBr, 70 °C; stationary phase, PSS-GRAM; Ve = elution volume) of glycopolypeptides P1−P4. Figure 1. 1H NMR spectrum (400.1 MHz, THF-d8) of the product mixture 1(L)/3(L) from reaction C after multiple precipitations from heptane (removing residual traces of photoinitiator) and isolation.

distribution (dispersity index, Đ ∼ 1.2) (Figure 2b and Table 2). Very similar results are obtained for P1(DL) and P2(L). P3 has virtually the same composition as P1 (or P2) but a higher degree of polymerization (DPn = 55, 38 glycosylated and 17 nonglycosylated units), however, considerably lower than the targeted one (90) (at complete conversion of NCA). Nevertheless, the dispersity of P3 is low (Đ ∼ 1.2). A polypeptide with a targeted degree of glycosylation of 50% was also prepared (→P4). P4 was found to have the expected DPn and low dispersity (Table 2) but the fraction of glycosylated units was just 0.37 instead of 0.5, confirming that the NCA 1 exhibits a higher reactivity than 3 and is preferentially incorporated into the growing polypeptide chain. After all, this one-pot approach allows the synthesis of welldefined glycopolypeptides with good control over composition, chain length, and dispersity, however within certain limits regarding chain length and comonomer sequence. The AGly units in the partially glycosylated copolypeptides are still susceptible to postpolymerization modification via thiol−ene photochemistry (Scheme 2). Exemplarily, P1 was reacted with three different ω-functional thiols, i.e. 3mercaptopropionic acid (4 → P1T1), N-acetylcysteamine (5 → P1T2), and thioacetic acid (6 → P1T3), to introduce carboxyl, amine, and thiol functionalities, respectively (which in natural proteins are presented by aspartic or glutamic acid, lysine, and cysteine units). Solutions of P1, benzophenone (0.1 equiv), and respective thiol 4, 5, or 6 ([SH]0/[CC]0 = 1.5) in THF were exposed to UV light at room temperature for 16 h. The products P1T13 were purified by dialysis and isolated by freeze-drying; results are summarized in Table 3. The addition of 4 to the P1 double bonds (9 AGly units per chain) was found to be quantitative, as indicated by the lack of 1H NMR signals at δ 5.8 ppm (Figure 3a). The integration of all signals is consistent with the expected structure in Figure 3a, i.e. P1T1 carries 19 glucose and 9 carboxyl side chains (DPn = 28). SEC (Figure 3b) shows a monomodal peak of P1T1, similar to that of P1, suggesting that the molar mass distribution of the polypeptide was not affected during thiol−ene reaction. For P1T2 and P1T3, 1H NMR (Supporting Information) suggests that 4/9 and 8/9 of AGly units, respectively, were functionalized with thiol. The molar mass distributions were monomodal with low dispersity (Table 3, Supporting Information). Hence, it is possible to prepare heterofunctional polypeptides by a sequential combination of one-pot glycosylation/ROP and

Also the coupling pattern of the anomeric proton of the carbohydrate at 4.7 ppm has changed, from triplet to doublet, as well as the splitting of the signal of the α-CH (C4) of the NCA at 4.4 ppm (Figure 1, inset). Notably, the signal of the αCH of 3 is shifted by 0.1 ppm to higher field and appears well separated from that of 1, allowing to quantify the ratio [1]/[3]. Compositions of the product mixtures 1/3 are in excellent agreement with the respective feed ratios, indicating that thiol− ene additions always went to completion (Table 1). Furthermore, 13C NMR analysis (Supporting Information) reveals the carbonyl signals of the NCA at δ 176 ppm (C2) and 150 ppm (C5) but no thioester signal, expected at 200−170 ppm, suggesting the absence of nucleophilic addition reaction and preservation of the NCA functionality. (A quantitatively glycosylated AGly NCA may be prepared when the thio-sugar is used in excess, requiring an additional purification step.12) Encouraged by these results, we attempted the copolymerization of the as-prepared NCA mixture 1/3, i.e. without further purification, using a primary amine initiator. For this, a mixture of 1(DL) or 1(L) (1 equiv), 2 (0.8 equiv), and benzophenone (0.2 equiv) in THF was irradiated with UV light for 45 min. The crude mixture was afterward diluted with dry N,Ndimethylformamide (DMF, final concentration: 5 wt % with respect to AGly) and 1-hexylamine was added. The mixture was then stirred for 7 days at 25 °C under reduced pressure (∼0.5 mbar; to remove CO2, Scheme 1). Three polymers were prepared with monomer-to-initiator ratios [NCA]0/[NH2]0 = 30 (→P1 and P2) and 90 (→P3). The products were precipitated multiple times from isopropanol and collected by centrifugation (isolated yields ∼80%). The chemical compositions and chain lengths of the polymers were determined by 1H NMR spectroscopy (Figure 2a and Supporting Information). P1 was found to have a number-average degree of polymerization (DPn) of 28, very close to the targeted value of 30. The ratio of glycosylated (19) to nonglycosylated (9) glycine units was 0.7:0.3, slightly deviating from the expected value of 0.8:0.2, indicating that there was a preference for the addition of the sterically less hindered, nonglycosylated NCA. Hence, the copolypeptides should have a gradient rather than random structure. Analysis by size exclusion chromatography (SEC) reveals a monomodal and rather narrow molar mass 2537

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Table 2. Characteristics of the Glycopolypeptides P1−P4 Prepared by Partial Thiol−Ene Glycosylation of AGly NCA 1 with AcGlcSH 2 Followed by Primary Amine-Initiated Ring-Opening Polymerization of the NCA Mixture 1/3 (According to Scheme 1) feed [1]0/[2]0

[NCA]0/[NH2]0a

yieldb (%)

composition [1]/[3]

DPnc,d

Mnc,e (g mol−1)

Mnapp f,g (g mol−1)

Đapp g,h

1.0/0.8 1.0/0.8 1.0/0.8 1.0/0.5

30 30 90 30

80 80 78 62

0.32/0.68 0.29/0.71 0.31/0.69 0.63/0.37

28 27 55 24

9800 9540 19300 7800

8500 6700 13000 5300

1.22 1.29 1.22 1.19

P1(DL) P2(L) P3(DL) P4(DL)

a Monomer-to-initiator ratio (NCA/1-hexylamine). bDetermined by gravimetry. cDetermined by 1H-NMR analysis (400.1 MHz, TFA-d) (see Figure 2 and Supporting Information). dAverage number of glycine units. eNumber-average molar mass. fApparent number-average molar mass. g Determined by SEC. Calibration: PMMA. hApparent dispersity index.

limited by the accessibility of reactive sites and reactivity of thiol.31 In summary, we described a one-pot modular strategy for the preparation of complex glyco(co)polypeptides carrying multiple functionalities, based on the partial functionalization and copolymerization of a single amino acid (allylglycine) NCA without needing to apply tedious synthesis and purification protocols. Current work is devoted to the production of glycopolypeptides carrying more complex sugars or oligosaccharides and to the studies of secondary structures and stimuliresponsive aggregation behavior in aqueous solution (deprotected samples).

Scheme 2. Modification of the Partially Glycosylated Copolypeptide P1 with Thiol R−SHa

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a Reagents and conditions: (c) benzophenone, hν, THF, room temperature, 16 h.

S Supporting Information *

Table 3. Characteristics of the Modified Glycopolypeptides P1T1−3 Prepared by the Addition of Thiols 4−6 to P1 (according to Scheme 2)

P1 P1T1 P1T2 P1T3

R−SH

composition [Glc]/[R]/[CC]a,b

Mnb,c (g mol−1)

Đapp d

− 4 5 6

19/0/9 19/9/0 19/4/5 19/8/1

9800 10280 10600 10100

1.22 1.26 1.27 1.24

ASSOCIATED CONTENT

Experimental procedures, 1H NMR, 13C NMR, and FT-IR spectra of NCA monomers and copolypeptides (also SEC traces). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(H.S.) E-mail: [email protected].

a

Number of repeating units carrying glucose (Glc, from thiol 2), functional groups R (from thiols 4−6) and double bonds (AGly). b Determined by 1H NMR analysis (400.1 MHz, TFA-d). cNumberaverage molar mass. dApparent dispersity index, determined by SEC, calibration: PMMA.

Notes

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS The authors would like to thank Felix Wojcik, Nora Fiedler, Jessica Brandt, Marlies Gräwert, and Olaf Niemeyer for their valuable contributions to this work. Financial support was given by the Max Planck Society and the German Research Foundation (within the IUPAC Transnational Pilot Call in Polymer Chemistry).

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REFERENCES

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Figure 3. (a) 1H NMR spectrum (400.1 MHz, TFA-d) of the carboxylated glycopolypeptide P1T1. (b) SEC-RI traces (eluent, NMP + 0.5 wt % LiBr, 70 °C; stationary phase, PSS-GRAM; Ve = elution volume) of P1T1 and corresponding precursor P1.

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